UNIVERSIDADE TÉCNICA DE LISBOA
Faculdade de Medicina Veterinária
"Antibioresistência de isolados de Enterobacteriaceae produtoras de β-lactamases de espectro
alargado, provenientes de Frangos"
“Antibioresistance of Extended-Spectrum β-Lactamase producer Enterobacteriaceae strains from
broiler samples”
Helena Cardoso de Carvalho Ferreira
CONSTITUIÇÃO DO JÚRI
Professora Doutora Maria Gabriela Lopes Veloso
Professora Doutora Maria Constança Matias Ferreira Pomba
Professor Doutor Len Lipman
Professora Doutora Maria João dos Ramos Fraqueza
ORIENTADOR
Professor Doutor Len Lipman
CO-ORIENTADOR
Professora Doutora Maria João dos Ramos Fraqueza
2008
LISBOA
UNIVERSIDADE TÉCNICA DE LISBOA
Faculdade de Medicina Veterinária
"Antibioresistência de isolados de Enterobacteriaceae produtoras de β-lactamases de espectro alargado
provenientes de Frangos"
“Antibioresistance of Extended-Spectrum β-Lactamase producer Enterobacteriaceae strains from
broiler samples”
Helena Cardoso de Carvalho Ferreira
DISSERTAÇÃO DE MESTRADO INTEGRADO EM MEDICINA VETERINÁRIA
CONSTITUIÇÃO DO JÚRI
Professora Doutora Maria Gabriela Lopes Veloso
Professora Doutora Maria Constança Matias Ferreira Pomba
Professor Doutor Len Lipman
Professora Doutora Maria João dos Ramos Fraqueza
ORIENTADOR
Professor Doutor Len Lipman
CO-ORIENTADOR
Professora Doutora Maria João dos Ramos Fraqueza
2008
LISBOA
DECLARAÇÃO RELATIVA ÀS CONDIÇÕES DE REPRODUÇÃO DA TESE
Nome Helena Cardoso de Carvalho Ferreira
Título da Dissertação:
Antibioresistência de isolados de Enterobacteriaceae produtoras de β-lactamases de espectro alargado,
provenientes de Frangos"
“Antibioresistance of Extended-Spectrum β-Lactamase producer Enterobacteriaceae strains from
broiler samples”
Orientadores:
Professor Doutor Len Lipman
Professora Doutora Maria João dos Ramos Fraqueza
Ano de conclusão 2008
Designação do Mestrado ou do ramo de conhecimento do Doutoramento
Mestrado Integrado em Medicina Veterinária
Nos exemplares das teses de doutoramento ou dissertações de mestrado entregues para a prestação de
provas na Universidade e dos quais é obrigatoriamente enviado um exemplar para depósito legal na
Biblioteca Nacional e pelo menos outro para a Biblioteca da FMV/UTL deve constar uma das
seguintes declarações:
1. É AUTORIZADA A REPRODUÇÃO INTEGRAL DESTA TESE/TRABALHO APENAS
PARA EFEITOS DE INVESTIGAÇÃO, MEDIANTE DECLARAÇÃO ESCRITA DO
INTERESSADO, QUE A TAL SE COMPROMETE.
Faculdade de Medicina Veterinária da UTL, 01/09/2008
Assinatura: ______________________________________________________________
i
To my dear mother, friends and family.
ii
Acknowledgements
I hereby thank everyone that has helped me throughout the execution of this work.
Specifically, I thank the laboratory assistants Ali Eggenkamp and Angele Timan, who were
always helpful and pacient with me. I also express my gratitude to Professor Dr.Lipman who
guided me through the experimental design, making this entire project possible and
welcoming me into his country. I would like to express my thanks to Raz Aldaibis who was
kind enough to let me accompany him to his trips to the slaughterhouse. I also would like to
give credit to the Institute of Risk Assessment Sciences (IRAS), for authorizing and financing
this project.
Finally, I would to show my deepest gratitude for all the insight, attention, care and concern
shown by Professor Dr. Maria João dos Ramos Fraqueza.
iii
“Antibioresistance of Extended-Spectrum β-Lactamase producer Enterobacteriaceae strains
from broiler samples”
Abstract
The aim of this study was to evaluate the effect of slaughterhouse contamination on the
presence of antimicrobial resistant bacterial isolates, specifically Salmonella and other
members of the Enterobacteriaceae family. Samples were collected in different points in a
poultry slaughterhouse in The Netherlands, and after quantification of Enterobacteriaceae and
isolation of Salmonella spp., antibiograms were made of the identified isolates.The following
antibiotics were tested: ampicillin, ceftiofur, trimethoprim+sulfamethoxazole, erythromycin,
tetracycline, gentamicin and enrofloxacin. The isolates that were resistant to ceftiofur were
considered ESBL positive.
Fom the total isolated strains (n=389), 57.5% were ESBL positive and 57.1% were resistant to
ceftiofur and other antibiotics. Salmonella showed a prevalence of 77.3% ESBL positive
isolates and 77.3% presenting a multiresistance profile. The combination of ceftiofur and
enrofloxacin resistance in the Salmonella isolates was present in 13.6% and 15.7% on the
total collected strains. The high number of resistant strains found shows that control should be
assured throughout the production chain in order to prevent transmission of resistant
determinants between different animals and humans as much as possible. This study also
confirms the need to further evaluate the role of food animal bacterial isolates as reservoirs for
antibiotic resistance.
Keywords: antimicrobial resistance, Enterobacteriaceae, slaughterhouse, Salmonella, ESBL
iv
"Antibioresistência de isolados de Enterobacteriaceae produtoras de β-lactamases de espectro
alargado provenientes de Frangos"
Resumo
O objectivo deste estudo foi determinar em que medida a higiene do matadouro influenciava a
produção de β-lactamases de espectro alargado (ESBL’s) em Salmonella e outras
Enterobacteriaceae. Para tal, foram colhidas amostras em vários pontos da linha de abate de
frangos de engorda (“broiler”) num matadouro na Holanda. A partir dos dados recolhidos
quantificou-se o número de Enterobacteriaceae e isolou-se Salmonella spp.. Testou-se a
resistência aos seguintes antibióticos: ampicillina, ceftiofur, trimethoprim+sulfametoxazol,
eritromicina, tetraciclina, gentamicina e enrofloxacina em todos os isolados. Assumiu-se que
os isolados resistentes a ceftiofur, eram produtores de β-lactamases de espectro alargado. Do
total de isolados recolhidos (n=389) verificou-se que 57.5% eram produtores de ESBL e que
57.1% apresentavam resistência a ceftiofur e outros antibióticos. Já os isolados de Salmonella
revelaram uma percentagem de bactérias produtoras de ESBL de 77.3% e de bactérias
multiresistentes de 77.3%. A resistência combinada de ceftiofur e enrofloxacina era de 15.7%
no total de isolados e de 13.6% nos isolados de Salmonella. O elevado número de isolados
resistentes mostrou que deve ser exercido controlo durante toda a cadeia de produção para que
seja possível prevenir a transmissão de resistência entre diferentes animais e o Homem. Este
estudo confirma a necessidade de avaliar o papel dos isolados bacterianos provenientes de
animais como reservatórios de resistência a antibióticos.
Palavras-chave: antibioresistência, Enterobacteriaceae, matadouro, Salmonella, ESBL
v
Index
Acknowledgements ..........................................................................................................................................................iii
Abstract ................................................................................................................................................................................. iv
Resumo ................................................................................................................................................................................... v
Figure Index........................................................................................................................................................................ ix
Table Index ......................................................................................................................................................................... xi
Preface.................................................................................................................................................................................... 1
Introduction ......................................................................................................................................................................... 2
I. Literature review........................................................................................................................................................... 3
1. Poultry Production....................................................................................................................................................... 3
2. Antibiotics ........................................................................................................................................................................ 4
2.1. Targets and mechanisms of action of antibiotics ......................................................................................... 5
2.2. β-lactams ....................................................................................................................................................................... 6
2.3. Antibiotic use and antimicrobial resistance................................................................................................... 7
2.4. Therapeutic uses of Antibiotic in Food Production Systems .................................................................. 9
2.5. Use of antibiotics in performance improvement ....................................................................................... 10
3. Poultry slaughter and HACCP ............................................................................................................................ 12
3.1. Total Enterobacteriaceae counts as an indicator of poultry hygiene............................................... 15
3.2. E. coli ........................................................................................................................................................................... 15
3.3. Salmonella spp......................................................................................................................................................... 16
3.4. Salmonella in poultry production.................................................................................................................... 17
4. Mechanisms for Emergence and Dissemination of Antimicrobial Resistance ................................ 18
4.1. Classification and mechanisms of antimicrobial resistance....................................................................... 18
4.2. Microbial gene evolution ....................................................................................................................................... 20
4.3. Stress-adaptation, co-selection, cross-resistance and cross-protection ................................................. 21
4.3.1. The mar operon ..................................................................................................................................................... 22
4.4. Dissemination of resistance determinants between microorganisms ...................................................... 23
4.4.1. Integrons.................................................................................................................................................................. 24
5. β-lactamases ................................................................................................................................................................. 26
5.1. Extended Spectrum β-Lactamases (ESBLs) ............................................................................................... 28
vi
5.1.1 TEM-type ESBL (class A).................................................................................................................................... 30
5.1.2. SHV-type ESBL (class A) ................................................................................................................................... 30
5.1.3. CTX-M–type ESBL (class A) ............................................................................................................................. 30
5.1.4. OXA-type ESBL (class D) .................................................................................................................................. 31
5.1.5. Association of β-lactamase genes with other antimicrobial resistance determinants and
transposons/integrons .............................................................................................................................................. 31
5.2 Plasmid-mediated AmpC enzymes (class C) ............................................................................................... 32
5.3. Carbapenemases (classes a, b, and d) ............................................................................................................ 33
6. β-lactamases produced in bacteria of animal origin ................................................................................... 34
7. Detection of resistance ............................................................................................................................................. 37
7.1.. Screening for ESBL production .......................................................................................................................... 38
7.2. Phenotypic confirmatory tests for ESBL production .................................................................................... 39
8. Monitoring of Resistance ........................................................................................................................................ 40
II. Experimental part .................................................................................................................................................... 41
1.1. Objectives .................................................................................................................................................................. 41
2. Material and methods .............................................................................................................................................. 41
2.1. Slaughterhouse caracterization........................................................................................................................ 41
2.2. Sample collecting .................................................................................................................................................... 41
2.3 Microbiological analysis ....................................................................................................................................... 44
2.3.1. Sample preparation and diluitions for microbiological analysis ................................................... 44
2.3.2. Enumeration of Enterobacteriaceae (standard method) ................................................................... 44
2.3.3. 3M™ Petrifilm™ Enterobacteriaceae (rapid method) ...................................................................... 44
2.3.4. Enumeration of E. coli ..................................................................................................................................... 45
2.3.5. Enterobacteriaceae Isolation and Identification ................................................................................... 45
2.3.6. Detection of Salmonella spp. .......................................................................................................................... 46
2.3.7. Enumeration of Extended Spectrum β-Lactamase positive Enterobacteriaceae .................... 49
2.3.8. Determination of Antibioresistence in Enterobacteriaceae isolates ............................................. 50
2.4 Statistical analysis ................................................................................................................................................... 52
3. Results ............................................................................................................................................................................ 52
3.1. Enumeration of Enterobacteriaceae .............................................................................................................. 52
vii
3.2. Enumeration of E. coli ......................................................................................................................................... 55
3.3. Detection of Salmonella spp............................................................................................................................... 56
3.4. Enterobacteriaceae Isolation and Identification....................................................................................... 57
3.5. Results of the enumeration of Extended Spectrum β-Lactamase positive Enterobacteriaceae
........................................................................................................................................................................................... 58
3.6. Determination of Antibioresistence in Enterobacteriaceae isolates ................................................. 60
3.6.1. Enterobacteriaceae isolates per sampling step ...................................................................................... 60
3.6.2. Enterobacteriaceae isolates per sampling day (flocks) ............................................................................. 63
3.6.3. Salmonella isolates per step .............................................................................................................................. 68
3.6.4. Salmonella isolates per Flocks (sampling day)........................................................................................... 70
3.7. Antimicrobial resistance to ceftiofur and other antibiotics ................................................................. 72
4. Discussion of the results .......................................................................................................................................... 73
4.1 Carcass contamination .......................................................................................................................................... 73
4.2. Antibioresistance screening ............................................................................................................................... 74
5. Conclusion .................................................................................................................................................................... 76
6. Future work ................................................................................................................................................................. 77
III. References.................................................................................................................................................................. 78
viii
Figure Index
Figure 1: Core structure of penicillins (1) and cephalosporins (2) (β-lactam ring in red). ........ 6
Figure 2: Poultry Slaughter Fluxogram for American slaughterhouses (United States
Department of Agriculture [USDA], 1999).............................................................................. 13
Figure 3: Mechanisms for antibiotic resistance (http://www.scq.ubc.ca/image-bank/, 2007) . 19
Figure 4 : Class 1 Integron (Collis et al., 1993). ...................................................................... 25
Figure 5: Crystal structure of representative ESBLs (Knox, 1995). ........................................ 29
Figure 6: Slaughterhouse layout (www.hyfoma.com, 2007).................................................... 42
Figure 7: chromID™ plates after culturing. Different colors are used for identification of
specific genera of Enterobacteriaceae ...................................................................................... 49
Figure 8: Antibiogram results for an Enterobacteriaceae isolate showing resistance to
ceftiofur, tetracycline, trimethopim+sulfa, erythromycin, enrofloxacin and ampicillin. ......... 51
Figure 9: Antibiogram results for an Enterobacteriaceae isolate showing resistance to
ceftiofur, trimethoprim+sulfa, ampicillin and intermediate resistance to erythromycin .......... 51
Figure 10 : Mean Enterobacteriaceae plate counts in log10 CFU/swab in live animal samples
(n=30) from Flocks D and E ..................................................................................................... 52
Figure 11: Mean number of Enterobacteriaceae plate counts in neck skins from poultry
carcasses, per Flocks................................................................................................................. 54
Figure 12: Percentage of Salmonella per broiler carcasses samples per day ........................... 56
Figure 13 : Percentage of Salmonella in samples from each step in the slaughter process...... 56
Figure 14: Enterobacteriaceae, E. coli and β-lactamase positive Enterobacteriaceae in poultry
carcasses of different flocks. .................................................................................................... 59
Figure 15: Percentage of resistant and intermediate Enterobacteriaceae isolates from live
animal samples for each antibiotic ........................................................................................... 60
Figure 16: Percentage of resistant and intermediate Enterobacteriaceae isolates from before
evisceration samples for each antibiotic ................................................................................... 62
Figure 17 : Percentage of resistant and intermediate Enterobacteriaceae isolates from before
chilling samples for each antibiotic .......................................................................................... 62
Figure 18: Percentage of Resistant (darker colours) and Intermediate (lighter colours)
Enterobacteriaceae isolates per antibiotic from different slaughter step samples. ................... 63
Figure 19: Percentage of Resistant (darker colours) and Intermediate (lighter colours)
Enterobacteriaceae isolates per antibiotic per sampling day. ................................................... 66
Figure 20: Percentage of Resistant and Intermediate Enterobacteriaceae isolates in broiler
carcass samples for each antibiotic........................................................................................... 68
ix
Figure 21: Percentage of Resistant and Intermediate resistant Salmonella isolates in the two
different sampling steps. ........................................................................................................... 69
Figure 22 : Percentage of Resistant and Intermediate resistant Salmonella isolates per
sampling day. ............................................................................................................................ 71
Figure 23: Percentage of Enterobacteriaceae and Salmonella isolates that were resistant to
ceftiofur, ceftiofur and other antibiotic and to ceftiofur and enrofloxacin. .............................. 72
x
Table Index
Table 1: β-Lactams used in veterinary medicine (Li et al., 2007). ............................................ 7
Table 2: Examples of antibiotics used in farm animals (Mathew, Cissel & Liamthong, 2007).
.................................................................................................................................................. 12
Table 3: Chronological reports of the β-lactamases including extended-spectrum β-lactamases
from bacteria of animal sources (Li et al., 2007) ..................................................................... 36
Table 4: Number of samples collected per sampling site, per sampling day. .......................... 43
Table 5: Types of procedures done according to the day the samples were taken. .................. 43
Table 6: Colony appearance on MacConkey agar. ................................................................... 45
Table 7: Interpretation of biochemical reactions. ..................................................................... 47
Table 8: Biochemical results for Salmonella (Global Salm-Surv, 2003) ................................. 48
Table 9: Antibiotic setpoints used ............................................................................................ 50
Table 10: Enterobacteriaceae plate counts in log10 CFU/g from broiler carcass per sampling
step. ........................................................................................................................................... 53
Table 11: Test between subjects effects for mean Enterobacteriaceae plate counts in log10
CFU/g ....................................................................................................................................... 55
Table 12: Pairwise comparisons for mean Enterobacteriaceae plate counts in log10 CFU/g
according to slaughter step were samples were collected ........................................................ 55
Table 13: E. coli plate counts in log10 CFU/g of neck skin from broiler carcasses. ................. 55
Table 14: Number of Salmonella in each sample group per sampling step and per flocks and
percentage of Salmonella isolates in each step and each Flocks A and B................................ 57
Table 15: Enterotube results for Enterobacteriaceae. ............................................................... 57
Table 16: Biochemical reaction results for the Salmonella isolates identified through
Enterotubes. .............................................................................................................................. 57
Table 17: Biochemical reaction results for Enterobacteriaceae isolates identified through
Enterotubes. .............................................................................................................................. 58
Table 18: Number of extended spectrum β-lactamase Enterobacteriaceae on carcasses (log10
CFU/g), per day (Flocks) in different slaughter steps. ............................................................. 59
Table 19: Number of resistant(R), intermediate(I) and sensitive (S) Enterobacteriaceae
isolates from live animal samples (n=77) for each antitiobiotic .............................................. 60
Table 20 : Number of Resistant, Intermediate and Sensitive Enterobacteriaceae isolates from
broiler carcasses in each sampling step .................................................................................... 61
xi
Table 21 Chi-square tests relating the variable “step” to the incidence of resistance to the
different antibiotics in all the Enterobacteriaceae isolates. ...................................................... 62
Table 22: Number of Resistant, Intermediate and Sensitive Enterobacteriaceae isolates from
poultry samples in each day (or flocks) .................................................................................... 65
Table 23: Chi-square tests relating the variable “Flocks” to the incidence of resistance to the
different antibiotics in all the Enterobacteriaceae isolates. ...................................................... 67
Table 24 : Number of Resistant, Intermediate and Sensitive Enterobacteriaceae from broiler
carcass samples. ........................................................................................................................ 68
Table 25: Number of Resistant, Intermediate resistant and sensitive Salmonella isolates to
each tested antibiotic per sampling step ................................................................................... 70
Table 26: Numbers of Resistant, Intermediate and Sensitive isolates for all the Salmonella
isolates in all sampling days. .................................................................................................... 70
Table 27: Number of Resistant, Intermediate and Sensitive Salmonella isolates for each tested
antibiotic ................................................................................................................................... 71
xii
Preface
As it was defined by the WHO consultation on “future trends held in veterinary public health”
in Teramo, Italy in 1999, Veterinary Public Health is “the sum of all contributions to the
physical, mental and social well-being of humans through an understanding and application of
veterinary science".
Antibiotic resistance is a major issue in food safety and human health as well, since the future
of medicine as we know it is at stake.
“2000 B.C.—Here, eat this root.
1000 A.D.—That root is heathen. Here, say this prayer.
1850 A.D.—That prayer is superstition. Here, drink this potion.
1920 A.D.—That potion is snake oil. Here, swallow this pill.
1945 A.D.—That pill is ineffective. Here, take this penicillin.
1955 A.D.—Oops…bugs mutated. Here, take this tetracycline.
1960–1999—39 more “oops.”Here, take this more powerful antibiotic.
2000 A.D.—The bugs have won! Here, eat this root.”
—Anonymous (WHO, 2000)
1
Introduction
The emergence of antimicrobial resistant Enterobacteriaceae represents a serious risk for
human health. One of the most important resistance patterns is of β-lactamase production and
diminished susceptibility to extended spectrum enzymes poses an even greater risk. The fact
that ceftriaxone (along with fluoroquinolones) is the therapy of choice in humans for
treatment of infections caused by E. coli and Salmonella spp. explains the concerns about the
emergence of this type of resistance. Therefore, decreased susceptibility to one or both of
these antibiotics can have severe consequences, ultimately ending in treatment failure.
The issue of antimicrobial resistance comprises both human and veterinary medicine. This
interdisciplinarity demands that an appropriate understanding of the intimate relationship
between animals and man is required. Food animals play a central role in this process.
One of the most important food animal industries is poultry production. As a source of animal
protein, poultry is both efficient and profitable. Due to the fact that poultry is consumed
worldwide, it is very important to control the quality and minimize potential health hazards.
This implies that control should be exerted upon every step of the poultry production process.
Slaughtering is one of the phases in the preparation of poultry carcasses that occurs in the
production chain from the animal to the consumer. Therefore it is important that the risk of
transmission of bacteria with antimicrobial resistance will be greatly reduced in this step.
The purpose of the present study was to evaluate the influence of poultry meat in the spread of
strains with antimicrobial resistance throughout the food chain.
In order to accomplish the aim of this work it was necessary to determine the amount of
resistant bacterial isolates throughout the slaughter process to understand the influence the
procedure has on the dissemination of this public health problem. Samples were collected at
different points in the slaughter line in order to measure the impact that contamination during
poultry slaughter has in the presence of antimicrobial resistant positive bacterial isolates.
A literature review was performed about production and slaughtering conditions, antibiotic
use and mechanisms and dissemination of antibioresistance, before the experimental part was
carried away.
2
I. Literature review
1. Poultry Production
After World War II, agriculture changed dramatically. Famine and devastation were
widespread all across Europe. With the help of foreign investment, most of the European
nations started acquiring new technologies and more advanced ways of producing food
animals were introduced. Among these were considerable improvements in animal genetics,
housing, nutrition, biosecurity, husbandry and veterinary medicine, more efficient business
practices and the emergence of economies of scale. Consequently, animal husbandry is now
perceived as a large scale industry. Everything, from the farm to the slaughterhouse and from
here to the costumer is controlled in a systematic way so that profit can be maximized. To a
large degree this has been connected to the use of antibiotics. These substances have been
used for the past fifty years, leading to a reduction in the impact of infectious disease through
treatment, prevention or control actions. They are also responsible for improving the
efficiency of feed consumption and enhancing weight gain (Gustafson & Bowen, 1997). In
this way, food safety can be assured as well as the health and welfare of these animals. The
use of antibiotics is just a part of the overall management system that has been created to
comply with the consumers’ constant demand for healthy and safe food animal products. In
modern intensive animal production systems, large groups of animals are raised together. For
instance, chickens are raised in barns that accommodate from ten thousand to twenty thousand
individuals (National Research Council [NRC], 1999). The fact that such a large number of
animals are living in a limited space leads to the emergence of physiological and
environmental stressors. In turn, these only contribute to further debilitate the already fragile
immune system these animals have. As a consequence viral and bacterial diseases can spread
from one affected animal to the entire flock in a very short period of time, leading to great
economic loss. Normally in such situations, the animal owner and the veterinary in charge of
the farm decide on medicating the entire group, rather than each animal individually (Institute
of Food Technologists [IFT], 2006).
Usually the form of administration in outbreaks is medicated water, because of the quick
response it triggers and the relative rapid intake associated with it. Another factor to be
considered is that normally sick animals have a depressed appetite but drink water normally.
For long-term prophylaxis feed medication is more efficient than medicated water. The great
advantage of these two systems is the fact that large numbers can be treated at once, without
any work safety risk associated with injecting large groups of animals (IFT, 2006).
3
In the United States, only in the year 2007 about 9.1 billion broiler chickens were produced
(USDA/NASS, 2007). In order to produce such large numbers a system has been devised in
which the entire production is controlled. The company is therefore in charge of every step,
from the breeder to the market sales. At birth, these animals are either vaccinated or given
antibiotics (such as gentamicin or ceftiofur) against opportunistic bacterial infections. In these
intensive systems, broiler chickens (typically six to eight weeks of age and between 2 kg and
4kg) are raised in pens that contain from ten to twenty thousand individuals (Lasley, 1983).
Adding drugs to drinking water and feed is the most common way of administering medicine
in broiler chickens. In the United States, antibiotics are still used as growth promoters and are
administered together with coccidiostats (NRC, 1999). The starter and growing ration include
prophylactic coccidiostat, an antibiotic growth promoter, and an arsenical compound having
both coccidiostat and growth promoting properties.
2. Antibiotics
The term antimicrobial can refer to any compound that can act against microorganisms;
including the antibiotics, the food microbial agents, sanitizers and disinfectants. Usually when
referring to the group that comprises sanitizers and disinfectants, as well as other chemical
agents, the term biocides is applied. These substances are generally broad spectrum, being
able to attack both eukaryotic and prokaryotic cells (IFT, 2006).
On the other hand, an antibiotic is a drug that can be used to treat an infectious disease in
humans, animals or plants. Antibiotics can act by inhibiting the growth or destroying the
microorganisms. Such substances can come from a natural, synthetic or semi synthetic origin.
In food animals they are often used to improve the efficiency of feed utilization apart from
their use to prevent infectious diseases. Antibiotic agents can affect the growth of bacteria in
three different ways: bacteriocidal, bacteriolytic or bacteriostatic actions.
The bacteriocidal antibiotics are those that can kill bacteria without promoting cell lysis or
rupture. The compounds in this group generally bind tightly to their cellular target and are not
removed by dilution (Lancini, Parenti & Gualberto, 1995). The bacteriolytic agents are
characterized by their ability to either inhibit cell wall sythesis or damage the cytoplasmic
membrane, killing the bacteria in this way (Lancini et al. 1995). Finally, the bacteriostatic
antibiotics are those that do not kill the bacteria but rather inhibit its growth. These antibiotics
prevent cell wall synthesis by binding to ribosomes. This binding is not tight and as a
4
consequence, when the concentration of the antibiotic is lowered, it detaches from the
ribosome and growth is resumed (Lancini et al., 1995).
Variations have been noticed when it comes to microbial sensitivity to antimicrobial agents.
Typically Gram-positive bacteria are more sensitive than their Gram-negative counterparts.
However, there are agents that are only active against Gram-negative bacteria (Greenwood,
1997).
A broad-spectrum antibiotic is generally regarded as one that can kill or inhibit the growth of
many types of bacteria. This is the preferred group when it comes to medical usage, in
contrast with narrow spectrum antibiotics, which are mostly active against only one group of
microorganisms.
2.1. Targets and mechanisms of action of antibiotics
Generally antibiotics target processes or structures that are essential for bacterial growth,
survival or both simultaneously. During this process, the eukaryotic cells in the host are not
greatly affected (Betina, 1983). The mechanism involves inhibition of a molecule that is
essential to cell multiplication, such as an enzyme or a nucleic acid. This is done by binding
to a target site in this macromolecule, creating a molecular complex that is no longer
functional (Lancini et al., 1995).
So far there have been found four identified targets: the bacterial cell wall biosynthesis
(peptidoglycan), bacterial protein synthesis (bacterial ribosomes), bacterial DNA replication
and repair (bacterial enzymes involved in DNA supercoiling) and cytoplasmic membrane
function (Walsh, 2000). There are a few antibiotics that act as competitive inhibitors by
mimicking important growth factors necessary for cell metabolism (O’Grady et al., 1997).
The major classes of antibiotics (based on chemical structure) and their mechanism of action
are (Lancini et al. 1995; Greenwood, 1995; O’Grady et al., 1997):
a) β-lactam antibiotics (penicillins and cephalosporins) - inhibit cell wall biosynthesis;
b) Glycopeptides – inhibit cell wall biosynthesis;
c) Tetracyclines – inhibit protein synthesis by binding to the 30S ribosomal subunit;
d) Aminoglycosides – inhibit protein synthesis by binding to the 30S ribosomal subunit;
e) Macrolides and lincosamides – inhibit protein synthesis by binding to the 50S
ribosomal subunit;
f) Quinolones – which inhibit DNA replication;
5
Miscellaneous (various types of chemical structure):
g) Chloramphenicol – inhibits proteins synthesis by binding to 50S ribosomal unit
h) Novobiocin - inhibits DNA gyrase
i)
Spectinomycin - inhibits protein synthesis
2.2. β-lactams
This is a antibiotic class that includes penicillin derivatives, cephalosporins, monobactams,
carbapenems, and β-lactamase inhibitors. All these compounds have in common the presence
of a β-lactam ring (Figure 1). The β-lactam antibiotics are cell wall active agents. They act by
preventing the final step in the synthesis of the bacterial cell wall. This involves the binding
of the β-lactam to a Penicillin Binding Protein (PBP), which in turn cannot cross the
peptidoglycan chains. This way the bacteria are no longer able to synthesize a stable cell wall,
leading to bacterial lysis. These substances are time-dependent bacteriocidal antibiotics.
They can range from very narrow spectrum to very broad spectrum depending on the
subgroups. For instance, penicillins and first generation cephalosporins can be used against
Gram-positive bacteria and aminopenicillins and cephalosporins are active against Gramnegative bacteria. The ones with the broadest spectrum, the third and fourth generation
cephalosporins, can inactivate both Gram-negative and Gram-positive bacteria (Prescott,
Baggot & Walker, 2000; Hornish & Kotarski, 2002; Stegemann et al., 2006).
Table 1 presents the list of β-lactams commonly used in veterinary medicine.
Figure 1: Core structure of penicillins (1) and cephalosporins (2) (β-lactam ring in red).
6
Table 1: β-Lactams used in veterinary medicine (Li et al., 2007).
Group
Drug
ampicillin, amoxicillin,
benzylpenicillin, cloxacillin,
Penicillin
hetacillin,nafcillin, penethamate
hydroiodide
Penicillin-β-lactamase inhibitor combination
amoxicillin/clavulanate
First generation cephalosporin
cefadroxil, cefapirin, cephalexin,
Third generation cephalosporin
cefovecin, cefpodoxime, ceftiofur
Fourth generation cephalosporin
cefquinome
β-lactam antibiotics play an important role in veterinary medicine, being one of the most
prescribed antibiotics in food animals in some countries (DANMAP, 2005). β-lactams can
also be used for performance improvement, at subtherapeutic levels, leading to increased
growth rate and feed efficiency in food animals.
Being limited to clinical illnesses, the extended-spectrum cephalosporins are prescription-only
medicines in veterinary medicine. The most frequently used are: cefalonium, cefoperazone,
cefquinome, ceftiofur and cefuroxime. These antibiotics can only be prescribed in the event of
serious diseases. Some examples include metritis, foot rot and mastitis in cattle, respiratory
diseases in ruminants, horses and swine, necrotic enteritis and colisepticaemia in poultry, and
septicaemia caused by E. coli in calves (Batchelor, Threlfall & Liebana, 2005).
In companion animals, β-lactam antibiotics are first-line treatments for cystitis and skin
wounds.
Since their use is so widespread, resistance to these compounds should be expected. Several
surveillance programs show clearly this trend (NARMS, 2004; DANMAP, 2005; CIPARS,
2006).
2.3. Antibiotic use and antimicrobial resistance
Since the discovery of penicillin in the 1940s, the well being of humans, animals and plants
has improved quite considerably. We have to consider that before the discovery of antibiotics
there was virtually no other way of treating infectious diseases. Research from 1920 to 1970
lead to the discovery of most the antibiotic classes we use these days. By chemically
7
modifying their structure, new antibiotic substances, with more powerful activity came into
play.
During the 1990’s a new threat began to emerge. Just when studies were no longer focusing
on finding new substances, previously susceptible organisms began to show resistance to
commonly used antibiotics. This was a situation that could clearly put at risk human health,
since this lead to increasing chances of treatment failure (Neu, 1992).
Presently, we are dealing with concerning issues regarding this amazing breakthrough. The
new era of modern medicine, heralded by the use of these chemicals brings new challenges.
The use and misuse of these substances might take to us to a setting where such substances
are no longer effective and no alternatives are available. This brings us to the issue of
selective pressure. This phenomenon can be explained by the widespread use of antibiotics in
both human and animal medicine, resulting in the specific selection of antibiotic resistant
bacteria.
There is a lack of objective data in what concerns the influence of antibiotic use in food
animals and the human health risks associated with this use (Barza & Travers, 2002). The
difficulty in obtaining direct, quantitative data has been the source of this problem (Lipsitch,
Singer & Levin, 2002). However, one must consider that the very existence of these resistant
microorganisms is in itself a risk factor. Antibiotic exposure seems to be a non-equivocal
source of this phenomenon. All the other risk factors are, in essence, the same as the ones of
acquiring any given foodborne disease (IFT, 2002). Pregnant women, immunosupressed
individuals (chemotherapy, HIV), very young and very old people (less than 5 or greater than
50) and also those that have any kind of liver or kidney damage are regarded as much more
likely to suffer the serious effects of infectious diseases. In addiction to these issues, lowered
gastric acidity or any other condition that might protect bacteria from stomach acid (ingestion
of large volumes of fluid or fatty substances) are conditions that increase the likelihood of
acquiring a foodborne disease.
Since the past 20 years, it has been recognized that the use of antibiotics increased the
prevalence of antibiotic resistant infections in those that took them (Angulo, Johnson, Tauxe
& Cohen, 2000; Anderson et al., 2003). This might be due to the disruption of the normal
microflora that protects against infection and colonization by pathogens, which increases the
potential for infection during and after the antibiotic administration period. In turn, this leads
to a lowering of the infectious dose in enteric pathogens. The trends in antimicrobial
resistance are not necessarily connected to the increase of antimicrobial resistant infections.
So for example, if there is an increase in resistance and there is a decrease in the number of
8
infections, the outcome might be that the amount of infections created by antimicrobialresistant organisms is the same.
Presently, the biggest challenge involves the definition of responsible use. It has to be the one
that can bring optimal therapeutic impact while minimizing the toxicity and the emergence of
resistance. In the event of an antimicrobial resistant foodborne disease, the loss of treatment
options and treatment failure is the most important consequence. In spite of the fact that most
Salmonella infections do not need antimicrobial treatment, it has been reported that this has
been used in 40 to 50% of the cases (Cohen & Tauxe 1986; Lee, Puhr, Maloney, Bean &
Tauxe, 1994; Glynn et al., 2004).
The reason why the use of antibiotic treatment in uncomplicated cases is contraindicated is
firstly because it does not reduce the duration or severity of the disease, possibly leading to
prolonged recovery and carrier state; additionally, it might increase the likelihood of antibiotic
resistance.
Several international organizations (FAO/WHO and OIE) have compiled a list of measures to
control antimicrobial resistance. These can be summarized into three objectives: preventing
disease which leads to a decrease in the need to antibiotic use, avoiding the use of antibiotics
if other therapies are available and finally consider the choice of antibiotics which use is not
very important in human or veterinary medicine (Codex Alimentarious Comission [CAC],
2005).
2.4. Therapeutic uses of Antibiotic in Food Production Systems
When considering food animal production antibiotics can be used in three ways. The first is
treatment, then control and finally prevention of disease (National Committee for Clinical
Laboratory Standards [NCCLS], 2002).
The first is regarded as the administration of antibiotic to cure animals with signs of clinical
disease.
Metaphylaxis or control administration of an antibiotic should be applied every time
morbidity or mortality exceeds a certain baseline norm, usually at the very beginning of the
disease onset. The reasoning behind this idea is that considering the close contact these
animals have, if there is a certain number of diseased animals there is high risk that it might
spread to the entire flock and therefore all the animals should be treated, even the ones that
show no signs of being affected yet.
9
Prevention or prophylaxis is the administration of a certain antimicrobial agent to healthy
animals that have not been exposed to the infectious agent, but that are expected to be in a
short period of time. This is generally done to the entire flock before any agent is cultured.
Often the sole presence of high risk factors for a certain disease and clinical signs in a few
individuals, combined by records of previous flock disease, might be enough to consider
administering empirical antibiotic therapy. This kind of treatment involves considerable
experience from the veterinarian and it involves pondering a great number of issues. Factors
such as animal species, susceptibility to that infectious agent, its virulence and the cost and
withdrawal times of the antibiotic, should be considered when choosing the type of treatment
to use (IFT, 2006). The diagnosis is often made retrospectively, by collecting samples from
dead animals. Sometimes, it’s also possible to analyse samples from live affected animals
within the groups. From this moment on, it’s necessary to test the infectious agent’s
susceptibility to a selected group of antibiotics, making the decision on which antimicrobial
agent to use increasingly accurate and effective. Antibiotics such as ceftiofur, enrofloxacin
and florfenicol are under veterinary prescription. These are relatively new and water-soluble
drugs and are under tighter control than drugs used for growth promotion or therapeutic use in
feed.
Extra-label use is also a possibility, but it involves a very tight client-veterinarian relationship
and strict compliance of drug withdrawal times. Extra-label use is not permitted on feed
antimicrobials.
2.5. Use of antibiotics in performance improvement
About fifty years ago, research proved that there were growth promoting effects and increased
feed conversion rates in animals that had been administered antibiotics. The effects would be
noticeable for a low level of administration and for an extended period of time (Hays, 1991).
The effect is not so significant in mature animals and this is the reason why it is mostly used
on young or growing ones. Therefore, the purpose of its use is to allow the animal producer to
increase is profit margin by reducing the feed costs and getting equally heavy animals
presented for slaughter. A great number of mechanisms have been presented (Shryock, 2000;
Gaskins, Collier & Anderson, 2002,). The growth promoting effects are mostly associated to
antibiotic inhibition of the normal flora, which in turn leads to less energy misuse by this
commensal microflora.
10
Tetracycline, tylosin and bacitracin all work by inhibiting cell wall synthesis or bacterial
protein production. It must be taken into account the fact that animal’s microflora influences
gut physiology when it comes to water uptake, immune response and nutrient availability
(Savage, 1977).
Tylosin apparently diminishes the level of activation of the intestinal immune system by
reducing the number of digestive bacteria (Collier et al., 2003). The growth promoting effects
can also be explained by the effect tylosin has on selectively contributing to the growth of
Lactobacilli. These are commensal bacteria recognized by their role in maintaining a balanced
flora. The benefit of an optimal microflora in animal production can be explained by the
exclusion effect if has on pathogens, both by antagonizing them or by competing for nutrients
in the intestinal microhabitat.
The issue of antibiotic use as growth promoters has created a great deal of concern. In the EU
it has been forbidden to administer antibiotic in food animals for this use since January 2006.
Following this trend, some large restaurant companies in the United States have decided to
take a stand and buy their supplies from antibiotic-free farms. The difference in regulations in
these two economic superpowers can lead to difficulties in assessing and controlling antibiotic
resistance. This is the subject of much debate, possibly because up to this date worldwide
figures for antibiotic usage in food production have not been available. According to the
Animal Health Institute in the USA, over 8000 tons of antimicrobials are used in food
production, corresponding to 4–25 g antibiotics/ ton of feed. Cephalosporin use for veterinary
purposes in the UK has estimated use of 3 tons in 2003 (Batchelor et al., 2005).
Table 2 shows a list of examples of antimicrobial drugs and antibiotics, by major class,
approved in the United States for animal use or human use (Compendium of Veterinary
Products [CVP], 2006; Guardabassi & Courvalin, 2006; Food and Drug Administration
[FDA], 2006). In this table, a number of antibiotics (for example, bacitracin, bambermycin,
chlortetracycline, penicillin, and virginiamycin) that are approved for use for growth
promotion and feed efficiency in broilers, turkeys and layers are identified (Institute of Food
Technologists [IFT], 2006).
It can clearly be seen that there are quite a few antibiotics that can be used in veterinary and
human medicine, simultaneously. Sometimes, they can be used for prevention and growth
promotion as well as human use.
11
Table 2: Examples of antibiotics used in farm animals (Mathew, Cissel & Liamthong, 2007).
3. Poultry slaughter and HACCP
The global society we live nowadays is increasingly aware of the importance of controlling
infectious diseases, particularly those that can be transmitted through the food chain. This
issue has been tackled in the last decades by the adoption of good practices which establish
basic principles for farming, including soil and water management, crop and animal
production, storage, processing and waste disposal. These Good Agricultural Practices (GAP),
Good Hygiene Practices (GHP) and Hazard Analysis and Critical Control Point (HACCP)
systems were created to assure that the risks associated with food animal products were
minimized. The Hazard Analysis and Critical Control Point (HACCP) system is a scientific
approach to process control. It is designed to prevent the occurrence of food safety problems
by assuring that controls are applied at any point in a food production system where
12
hazardous or critical situations could occur. Studies have shown that HACCP systems can
maintain or even improve food safety (Cates, Anderson, Karns, & Brown, 2001).
Hazards include biological, chemical, or physical contamination of food products (World
Health Organization [WHO], 1997). Figure 2 shows a typical fluxogram of a poultry
slaughter process, from the moment the live animals come from the farm to the shipping of
the finished product. This is an important tool when designing an HACCP plan, since it
enables a quick and easy review of all the steps in production.
The design of the HACCP plan involves the identification of effective control parameters and
the establishment of the stages of the process that we can control (Critical Control Points
[CCPs]) (ICMSF, 1991; Mortimore & Wallace, 1996; McNamara, 1997; National Advisory
Committee on Microbiological Criteria for Food, 1998).
Figure 2: Poultry Slaughter Fluxogram for American slaughterhouses (United States
Department of Agriculture [USDA], 1999).
13
There are several steps in the slaughterline that should be considered as potencial hazard
introduction source in the poultry slaughter process. A few of them are:
 Live Receiving /Live Holding – poultry may possibly contain pathogens;
 Scalding - can result in cross contamination and possible pathogen proliferation;
 Deafeathering - potential for cross-contamination at this step in the process;
 Venting/Opening /Evisceration - first point at which likelihood of significant
contamination of interior surfaces can occur and also leakage of fecal material into
body cavities can cause contamination;
 Chilling - carcass to carcass contact;
 Final Chill - fecal contamination;
Poultry carcasses usually have very high skin contamination levels (Sandrou &
Arvanitoyannis, 1999). They can present microorganisms that cause foodborne illness as well
as food spoilage. There are a series of microorganisms on the surface of the carcasses which
can be analyzed in order to indicate the microbiological quality, the level of hygiene in
production and handling, and the correct maintenance of the cold chain (Sandrou &
Arvanitoyannis, 1999).
It is therefore important to correctly apply an effective preventive system which can assure
that pathogenic microorganisms will not contaminate the surface of carcasses during sensitive
steps (such as the ones mentioned above). During the stages of slaughter, bleeding, scalding,
defeathering and evisceration, important increases of the superficial contamination of the
carcasses take place due to cross contamination and contamination from feces and
environment. After the evisceration process, the carcasses are cleaned with pressurized water
(40-180 psi). Then the carcasses are chilled using cold air (-6ºC to +2ºC) or cold water (less
than 4ºC). Any delay in the application of the cooling involves a possible microbial growth.
The rapid chilling of poultry carcasses at the end of the slaughter process is therefore a crucial
point in controlling the growth of microorganisms. The final purpose is to obtain poultry
carcasses with absence of Salmonella, complying with the Commission Regulation (EC)
1441/2007 on microbiological criteria for foodstuffs.
The presence of high numbers of bacteria can have an important role in spreading
antimicrobial
resistance
determinants,
involving
microorganisms.
14
both
pathogenic
and
commensal
3.1. Total Enterobacteriaceae counts as an indicator of poultry hygiene
The family Enterobacteriaceae comprises 30 established genera, which include bacteria such
as Salmonella spp., Escherichia spp., Shigella spp. and Yersinia spp.. Many of these show
pathogenicity towards human and animal species and quite a few of the pathogenic ones
produce toxins. A large number are commonly found in animal’s intestinal flora.
Total Enterobacteriaceae, coliforms and Escherichia coli are used as marker organisms in
foods, and the detection of specific pathogens of the family Enterobacteriaceae is widely
applied in many food control laboratories (De Boer, 1998). The testing for Enterobacteriaceae
and total bacterial counts at 30ºC are used as part of the normal analysis for poultry meat
contamination while E. coli is more specifically analyzed for the presence of pathogen
microorganisms. Normally the analysis of Enterobacteriaceae as an indicator of fecal
contamination (whether pathogenic or not) is used, indicating a mishandling in the process
(Robach, 1996; Forsythe & Hayes, 2002; Snijders and Van Knapen, 2002). Most
Enterobacteriaceae found on the surfaces of the carcasses come from feathers and fecal
contamination. Counts of these microorganisms have been used as indicators of
contamination due to unhygienic manipulation or inadequate storage (Grau, 1986; Pascual,
1992). Chickens initially show a high level of microbial contamination in their digestive tract,
as well as on their feathers, skin and paws, which comes from feces and the environment.
These microorganisms will be redistributed during the stages of the production chain. At the
same time, a cross contamination from one carcass to another can take place, as well as from
surfaces, water and poultry handlers.
3.2. E. coli
E. coli is a Gram-negative, facultative anaerobic and non-sporulating bacillus of the
Enterobacteriaceae family. Being commonly found in the lower intestine of warm-blooded
animals, most E. coli strains are harmless, but some, such as serotype O157:H7, can cause
serious food poisoning in humans.
There are five identified pathotypes: Enterotoxigenic E. coli (ETEC), Enteropathogenic E.
coli (EPEC), Enterohemorrhagic E. coli (EHEC), Enteroinvasive E. coli (EIEC),
Enteroaggregative E. coli (EAggEC). The last two are only found in humans (Torrence &
Isaacson, 2003).
Certain strains of E. coli, such as O157:H7, O121 and O104:H21, produce toxins.
15
The transmission of pathogenic E. coli is often associated with fecal-oral transmission (FDA,
2006b), farm animals and meat contaminated post-slaughter (Bach, McAllister, Vieira,
Gannon & Holley, 2002). E. coli is also considered the best and most specific indicator of
fecal contamination.
3.3. Salmonella spp.
Salmonella are Gram-negative, nonspore-forming bacilli of the Enterobacteriaceae family.
These bacteria contain three different types of antigens: somatic O, flagellar H and capsular
Vi. There are 2500 different serological types and they are differentiated based on the
agglutinating properties of those antigens (Popoff, Bockemuhl & Gheesling, 2003). The
Salmonella genus consists only of two species, S. bongori and S. enterica. In turn, S. enterica
is subdivided into six subspecies; enterica, salamae, arizonae, diarizonae, houtenae, indica.
Most of the infections in humans and warm-blooded animals (around 99%) are caused by S.
enterica subsp. I (S. enterica subsp. entericae) (Uzzau et al., 2000).The O-antigen serogroups
implicated are usually A, B, C1, C2 and D. Generally, the other serotypes in other subspecies
are found in samples from cold-blooded animals or from the environment (Humbert, 1994;
Uzzau et al., 2000).
Due to all these subdivisions, Salmonella nomenclature can be quite complex. Therefore,
often the most common serotype (Salmonella enterica subsp. entericae serotype Enteritidis) is
refered to as simply Salmonella Enteritidis (Brenner, Villar, Angulo, Tauxe & Swaminathan,
2000). Salmonella serotypes can also be divided according to their biotype and phage type
(Velge, Cloeckaert & Barrow, 2005).The biotyping is related to the differences between two
bacteria of the same serotype in relation to their biochemical activity. Phage typing is the
susceptibility that certain bacteria within the same serotype show to the lytic action of a
specific phage type (Ward, de Sa & Rowe, 1987; Varnam & Evans, 1993).
In all Salmonella serotypes, there is a large variation when it comes to host adaptation and
pathogenicity. Serotypes, such as S. Typhi and S. Paratyphi, show a high degree of adaptation
to man. They cause septicaemic typhoidic syndrome (enteric fever) and are relatively nonpathogenic to animals. On the other hand, other serotypes are highly adapted to animals, as is
the case of S. Gallinarum in poultry, not affecting human hosts (Velge et al., 2005).
However, there are a few serotypes that seem to affect a large range of hosts. This is case of
both S. Choleraesuis and S. Dublin that while being adapted to a specific host (pigs and cattle,
respectively) show great pathogenicity (Velge et al., 2005).
16
From all the Salmonella serotypes the most recognized is Salmonella Typhimurium, in both
human and animal infections. The serotype S. Enteritidis is also frequently isolated, and can
affect either humans or animals. Both serotypes lead to gastrointestinal infections that are
usually milder than the ones caused by one of the highly animal-adapted serotypes. These
ubiquitous serotypes are capable of producing typhoid-like infections in humans and mice
also being able to colonize the chicken intestine and remain totally asymptomatic (Cowden et
al., 1989).
3.4. Salmonella in poultry production
In 2006, a total of 160,649 confirmed cases of human salmonellosis were reported in the EU
(European Food Safety Authority [EFSA], 2006). Being one of the major foodborne causes of
gastroenteritis, Salmonella is frequently associated with contaminated poultry meat and is
regarded as one of the most important public health hazards (Bryan & Doyle, 1995). The
contamination of poultry or poultry meat can occur throughout the whole production chain.
Risk factors for this contamination have been recognized for each step in this process. If
effective control is to be implemented, it is required that a proper quantification of the
importance of each risk factor is obtained. Vertical transmission of S. Enteritidis and S.
Typhimurium from the parent flock to the day-old chicken leaving the hatchery has frequently
been recognized as one of the main controlling factors in programs that aim for Salmonella
eradication (Bisgaard, 1992; Cason, Cox & Bailey, 1994; Limawongpranee et al., 1999).
Even though epidemic spread has been connected to the introduction of contaminated
breeding lines in some countries (Ward et al., 2000), it is now thought that the vertical
infection route seems to be involved in a much smaller scale than horizontal transmission
(Van de Giessen, Ament & Notermans, 1994).
The following risk factors have been identified as responsible for horizontal transmission:
inadequate cleaning and disinfection of broiler rearing houses leading to contamination of the
following flock (Lahellec et al., 1986; Davies & Wray, 1996; Higgins, Malo, René-Roberge
& Gauthier, 1981; Rose et al., 1999; Rose et al., 2000), a poor level of hygiene (Henken,
Frankena, Goelema, Graat & Noordhuizen, 1992) and feed contamination (Davies et al.,
1997). Additional factors are the size of the farm (Skov, Angen, Chriél, Olsen & Bisgaard,
1999), rearing of flocks in autumn (Skov et al., 1999) and presence of litter-beetle in the
house and rodents in the farm (Baggesen, Olsen & Bisgaard, 1992; Lohren, 1994). Studies
have shown that the farm environment plays a bigger role than was initially thought (Glynn et
17
al., 1998; Davies & Breslin, 2003). One important aspect regarding the emergence of this
pathogen is the modernisation and increased intensity of poultry farming practices.
It has been proven that feed removal (one of the most frequent moulting strategies) increases
the susceptibility to S. Enteritidis infection (from 102 to 103 fold) and also leads to high levels
of excreted pathogen in faeces (Holt, 2003).
During the transport to the slaughterhouse otherwise negative flocks may become infected
(Rigby et al. 1980; Rigby et al., 1982). Cross contamination with Salmonella can also occur
during the processing that occurs in the slaughterline, according to evidences taken from the
high frequency of contaminated crates and on-site plant contamination assessment studies
(Corry, Allen, Hudson, Breslin & Davies, 2002; Olsen, Brown, Madsen & Bisgaard, 2003).
4. Mechanisms for Emergence and Dissemination of Antimicrobial Resistance
4.1. Classification and mechanisms of antimicrobial resistance
Chance has always played a part in evolution of bacteria, and the emergence of antimicrobial
resistance is one of the many aspects of this phenomenon (Courvalin, 2005). Firstly,
antimicrobial resistance should be clearly defined. When an antimicrobial treatment fails, in a
clinical context, we can assume the microorganism developed some kind of resistance. On a
laboratory perspective, a microorganism can be called resistant if its MIC (Minimal Inhibitory
Concentration) is over a certain specific threshold value, independently from the clinical
outcome of the infection. Putting it in another way, antimicrobial resistance is the ability to
thrive under conditions that would otherwise kill or impair multiplication in other members of
the strain. Even so, it should be considered that often bacteria are not affected by otherwise
effective antibiotics due to the conditions in which this substance was applied. The agent can
lose its efficacy altogether if, environmental conditions such as temperature, pH and food
composition are not suitable or if there is any sort of interaction between the agent and the
components of the medium (Davison, Low & Woolhouse 2000). The activation of otherwise
silent genes in the microorganism can also confer temporary resistance to agents bacteria were
usually sensitive to (IFT, 2006). It should also be considered that every antibiotic that is
directed towards cell wall synthesis will not be active against cells that are not growing. The
rate at which resistance occurs and what triggers it is mostly related to changes in DNA
replication, transcription and translation or to changes on the cell wall or the cell membrane.
Figure 3 shows how the processes work in a schematic way. The main strategies devised are
impaired uptake of these substances, overproduction or modification of the target sites for
18
antimicrobials, bypass of sensitive steps, absence of certain enzymes or metabolic pathways
and efflux of the antimicrobial drug itself (Russell, Furr & Maillard, 1997). Additionally,
resistance can be achieved by degradation of the drug before it reaches its active site, altering
the receptors that bind to that drug or changing membrane permeability (Dever & Dermody,
1991; Cloete, 2003).
Figure 3: Mechanisms for antibiotic resistance (http://www.scq.ubc.ca/image-bank/, 2007)
Innate or intrinsic resistance is usually controlled by the microorganism own genetic
background (Russel, 1991). In this case the microorganism is resistant to an agent without
record of previous exposure. This issue is not very problematic clinically because the
prescription of antibiotics is done considering these intrinsic resistances.
Mechanisms that create this resistance can be related to the presence of efflux mechanisms
(Gilbert & McBain, 2003), to the complexity of the cell wall or by enzymatic inactivation of
the antimicrobial agent (Russell, 2001). Gram-negative bacteria are usually more resistant to
antimicrobial action than Gram-positive ones (Russell & Chopra 1996). This is due to the
presence of a double membrane structure that prevents antibiotics, like penicillin G, from
accessing the target in the cell wall.
Other bacteria, such as Pseudomonas spp. or Corynebacterium spp. can inactivate
antimicrobial compounds by metabolizing them. For instance, these bacteria can break down
19
benzoate into succinic acid and coenzyme A rendering it harmless (Chipley, 1993). The
Mycobacterium species prevent antibiotic action by making cell walls more hydrophobic, due
to their increased lipid content.
Extrinsic or acquired resistance is related to a series of genetic changes that occur within the
bacteria. These changes can lead either to a change in the target site of the antibiotic or they
can result from the acquisition of genetic material such as plasmids and transposons
containing integron sequences (Russell & Chopra, 1996). This resistance can be acquired in
two different ways. One is through horizontal acquisition of foreign genetic information and
the other is mutation in structural or regulatory housekeeping genes (Courvalin, 2005).
Sometimes resistance can occur related to multiple mechanisms. That is the case with
tetracycline, where three different mechanisms are likely to be responsible for resistance
(Schnappinger & Hillen, 1996).
The primary resistance mechanism to β-lactam, chloramphenicol and aminogycosides is
enzymatic degradation (Bush & Sykes, 1984). In the first this is achieved by hydrolysis of the
β-lactam ring and in the case of chloramphenicol it is due to acetylation catalyzed by
chloramphenicol acetyltransferase (Dever & Dermody, 1991). In aminoglycosides the process
involves
a
great
range
of
enzymes,
namely
methylases,
acetyltransferases,
nucleotidyltransferases and phosphotransferases (Shaw, Rather, Hare & Miller, 1993; Davies
1994).
The alteration of specific receptor sites leads to impaired target recognition by the antibiotic.
The mechanism behind nalidixic acid and ciprofloxacin (both fluoroquinolones) resistance
lies in a mutation in the gyrA and gyrB genes, which are encoded target recognition proteins
(Hooper 1995; Tankovic, Perichon, Duval & Courvalin, 1996; Heddle & Maxwell 2002).
One other way of resisting the action of β-lactam is by change in membrane permeability. For
instance in E. coli, the replacement of an outer membrane porin (OmpF) by another type of
porin (OmpC) leads to resistance (Nikaido, Rosenberg & Foulds, 1983).
4.2. Microbial gene evolution
Genetics has proved to be a handy tool when it comes to understanding bacterial resistance. It
has been possible to sequence and compare a large number of microbial genomes due to
intensive research over the last years. This has shown the importance of genetic mutation in
genomic structures and their frequency rate (Perna et al., 2001; Tamas et al., 2002).
In some microorganisms, as for instance Escherichia coli and Salmonella enterica, great
genetic variability has been noticed (Ochman & Jones, 2000; Edwards, Olsen & Maloy,
20
2002). Intra-genomic recombination plays an important role in genome evolution and it has
been linked to a site-specific recombination mechanism. This feature enables the correct
processing of replicated linear chromosome ends (Kobryn & Chaconas, 2001).
Another process of increasing variability is the intergenomic exchange involving homologous
recombination. The inactivation of the mismatch pathway leads to increased chance of
mutagenesis. This pathway leads to the elimination of errors that escape the replication
proofreading process (Giraud, Radman, Matic & Taddei, 2001). However this inactivation
process plays a less significant role in genetic diversity than recombination (Spratt, Hanage &
Feil, 2001).
4.3. Stress-adaptation, co-selection, cross-resistance and cross-protection
It is possible that microorganisms that were initially resistant to one type of substance become
resistant to different antimicrobials (Yousef & Juneja, 2003). The activation of intrinsic
resistance mechanisms through exposure to sub-inhibitory concentrations of antimicrobial can
lead to decreased susceptibility to other unrelated antimicrobials. The process of co-selection
involves sequential linking of different genes (frequently on plasmids or integrons), enabling
the simultaneous transference of resistance to unrelated antimicrobials. When resistance
occurs between antimicrobials that have the same molecular targets, the process is called
cross-resistance.
Cross-protection is the process by which, adaptation to one specific antimicrobial leads to
increased resistance to other, apparently unrelated stress factor.
The production of stress response proteins in bacteria is related to exposure to sub-inhibitory
levels of stress (Yousef & Juneja, 2003). There is a multitude of situations that can lead to
transcription and translation of stress response proteins. One of the most important ones are
heat-shock proteins (HSP), these are typically regulated by RpoS or RpoH, subunits of RNA
polymerase (Jorgensen, Panaretou, Stephens & Knochel, 1996).
It is very likely that stress-adaptation leads to increased virulence, lower infectious doses and
also to increased resistance to food preservation treatments (Samelis & Sofos, 2003).
There is evidence that there is cross-resistance interaction between sanitizers or disinfectants
and antibiotics. One example is that of E. coli that by becoming resistant to thymol and
eugenol also became more resistant to chloramphenicol (Walsh et al., 2003). There has been
much debate on the influence of long-term use of sanitizers in food processing facilities and
the emergence of antibiotic resistance.
21
Many studies have tried to investigate the interactions between the use of sanitizers and the
emergence of antibiotic resistance but there have been great limitations when it comes to
choosing the methods to test resistance in these chemicals. Unlike antibiotics, sanitizers have
effect over a large range of microorganisms and their action is mostly dependent on their
concentration. Thus, hardly any conclusions can be drawn using the same methods as with
antibiotics (such as the MIC). Even so, it is very unlikely that resistance occurs directly
towards the biocide. This is due to the fact that biocides act by damaging cell wall and
membranes, leading to quick destruction of the microorganisms. Therefore, mutations that
lead to resistance in these compounds are expected to be less frequent than those that allow
for antibiotic resistance (Russell, 2003).
4.3.1. The mar operon
A very interesting situation is that of the mar (multiple antibiotic resistance) operon. This
regulator controls intrinsic resistance to different families of antibiotics and other cytotoxic
substances (Alekshun & Levy, 1999). This was the first recognized operon known to be
involved in this type of multidrug intrinsic resistance.
The mar locus of E. coli is responsible for the regulation of the acrAB efflux pump (George &
Levy, 1983; Ma et al., 1993). In E. coli, the acrAB efflux system is responsible for the
transport of tetracycline, ciprofloxacin, fluoroquinolone, β-lactams, and novobiocin as well as
ethidium bromide, acriflavine, phenylethylalcohol, sodium dodecyl sulfate, and deoxycholate
(Ma et al., 1993). It has been proved that following exposure to chloramphenicol, E. coli
O157:H7 showed decreased susceptibility to a different range of antimicrobials, showing a
link to a mutation in the mar operon (Golding & Matthews, 2004). This acr efflux system was
also present in other Enterobacteriaceae (Sukupolvi, Vaara, Helander, Viljanen & Makela,
1984; Sulavik, Gambino & Miller, 1995; Nikaido, 1998).
Studies have pointed out that the exposure of Salmonella to sublethal levels of chlorine,
sodium nitrite, sodium benzoate or acetic acid activated the mar operon, which in turn would
lead to resistance to antibiotics such as tetracycline, chloramphenicol, nalidixic acid and
ciprofloxacin (Potenski, Gandhi & Matthews, 2003). These pumps are active in a great variety
of Gram-negative bacteria and apart from the fact of being highly conserved they can also be
carried on plasmids (Nikaido, 1998). Efflux pumps can be organized into four superfamilies.
Each one of these families contains pumps responsible for the efflux of one agent along with
pumps capable of multidrug efflux resistance. The multidrug resistance pumps are able to
22
remove a wide variety of different antibiotics and disinfectants, being quite broad in terms of
specificity. These efflux pumps can be involved in both the extrusion of endogenous
metabolites or in the removal of chemotherapeutic agents. The bacteria that expressed the
efflux pumps probably started extruding metabolites and by chance this ability enabled these
bacteria to protect themselves against damaging antimicrobials. The origin of antibiotic
resistance genes is probably related to the selection of those that incorporated this strategy,
making it present in almost every genus and species of bacterium (Gilbert & Mcbain, 2003).
Studies have shown that after sublethal exposure to some biocides, multidrug resistance can
occur (George & Levy, 1983; Levy, 2002).
4.4. Dissemination of resistance determinants between microorganisms
The persistence of antimicrobial resistant organisms involves that organisms survive, enabling
the resistant phenotype to be passed on through others. This dissemination can occur in
different ways, either through clonal spread, plasmid epidemics or transposons. All these three
can coexist, are infectious and exponential and are all associated with DNA duplication
(Courvalin, 2005).
Due to their ability to evolve rapidly, bacteria are very resourceful when it comes to exploring
new niches. Research shows that lateral gene transfer may be largely responsible for the quick
changes in ecological and pathogenic properties (Ochman, Lawrence & Groisman, 2000). The
acquisition and loss of large fraction of genetic material that included various genes could
induce genome variability rapidly. Among these horizontally transmitted DNA fragments
plasmids, genomic islands, temperate bacteriophages, as well as transposons and insertion
sequences can be included (Velge et al., 2005). It has been proven that these elements can
provide a significant advantage for the microorganism during particular circumstances
involving changes in environment (Ochman et al., 2000).
There is no certainty as to either or not these transfers of genetic material occur in nature, but
most studies support the idea that this phenomenon is widespread, (Sundin 2002; Riesenfeld,
Goodman & Handelsman, 2004) being present in a wide range of microorganisms and niches
(Riesenfeld et al., 2004). These genes appear to be present in bacteria from various sources,
often found in evolutionarily unrelated organisms (Gram-positive and Gram-negative
bacteria) (LeBlanc, Lee, Titmas, Smith & Tenover, 1988; Werner, Hildebrandt & Witte,
2001) and originating from totally different environments (Bolton, Kelley, Lee, Fedorka-Cray
&, Maurer, 1999; Sanchez, Lee, Harmon, Maurer & Doyle, 2002). The quick exchange is
23
associated with the fact that this resistance is transmitted through genetic mobile elements, the
previously mentioned plasmids, transposons and integrons. The problem that raises more
concern is that an ever increasing amount of other resistance genes present in multiple,
tandem repeats are linked in these mobile elements.
Microbial gene evolution and bacterial adaptability, which can be triggered by antibiotic use
has been proven to lead to emergence of resistant bacteria. This phenomenon and its
dissemination have been noted not only in pathogenic bacteria but also in animal and human
commensal flora (O’Brien, 2002).
There might be contamination of food animal products by these resistant endogenous
microorganisms in the same way it occurs with zoonotic bacteria. Soon after the spread of
resistance genes to antibiotics used exclusively on animals, it was found that these resistance
determinants were not only found in animal bacteria, but also on human commensal flora,
zoonotic pathogens (Salmonella) and even in strictly human pathogens (Shigella). This leads
us to the conclusion that there is ample transfer of resistance determinants between unrelated
bacteria (O’Brien, 2002).
4.4.1. Integrons
Integrons have been recently recognized as a strategy for some organisms to acquire multiple
antibiotic resistance (Stokes & Hall, 1989; Hall & Collis, 1995; Sundstrom, 1998; RoweMagnus, Guerout & Mazel, 1999; Hanau-Bercot, Podglajen, Casin & Collatz, 2002).
Integrons can be part of transposons, plasmids or chromosomal genomic islands (Summers,
2002). Most of the integrons found in clinical isolates belong to class 1 (Recchia & Hall,
1995). These integrons can include these resistance genes that are inserted into gene cassettes
through a process mediated by the integrase gene (int1) at the att1 site (Hall & Collis, 1995;
Hansson, Skold & Sundstrom, 1997; Partridge, Collins & Hall, 2002).
The integrase shown in Figure 4 produces integrons In3 and In4 by recognizing the attC
(59be) sequence in the gene cassette and mediating its excision into a circular intermediate
and/or its insertion into attC site (Collis, Grammaticopoulos, Briton, Stokes & Hall, 1993).
These class 1 integrons have been well characterized, and possess additional genes,
downstream of this attI site and their resident gene cassette(s) which include a functional
sulfonamide resistance gene, the sul1 (Stokes & Hall, 1989) and a partially deleted,
nonfunctional quaternary ammonium resistance gene, the qacE (Paulson et al., 1993). Class 1
integrons are not mobile by themselves, but they do reside on transposons and plasmids
(Liebert, Hall & Summers, 1999).
24
Figure 4 : Class 1 Integron (Collis et al., 1993).
About 50 cassettes have been recognized in encoding genes responsible for antimicrobial
resistance. Their widespread occurrence is probably related with antibiotic use in human and
veterinary medicine and their role in bacterial adaptation (Recchia & Hall, 1995; Mazel &
Davies, 1999). The emergence of multidrug resistance integrons can occur by the
accumulation of gene cassettes (Naas, Poirel, Karmin & Nordmann, 1999; Rowe-Magnus,
Guerout & Mazel, 2002).
The myriad of antibiotic resistance gene clusters found to this date can be connected to the
recombinational events and to the transfer and capture of genes that occur in integrons (Boyd,
Cloeckaert, Chaslus-Dancla & Mulvey, 2002; Doublet et al., 2003; Doublet, Weill, Fabre,
Chaslus-Dancla & Cloeckaert, 2004).
Studies where up to eight resistance cassettes have been recognized in one single integron,
can easily explain the presence of multidrug resistance in some clinical isolates (Naas et al.,
1999).
25
5. β-lactamases
Gram-negative bacteria are known to be more resistant to the attack of β-lactam antibiotics.
This is due to the fact that these bacteria are able to produce β-lactamases. Earlier, βlactamases were present but considered rare, as for instance in Staphylococcus aureus
(Medeiros, 1997). Later, as the use of antibiotics became widespread, the ability to produce
these enzymes spread to other pathogens.
Pharmaceutical researches then lead to the discovery of new β-lactam antibiotics such as the
cephamycins, the cephalosporins with an oxyimino side chain, the carbapenems and
aztreonam (a monobactam). The introduction of these new drugs subsequently forced bacteria
to develop new strategies to counter the activity of these antibiotics. Bacteria reacted then by
producing extended spectrum β-lactamases (ESBL), AmpC enzymes and carbapenemases
(Jacoby & Munoz-Price, 2005).
Three mechanisms are responsible for bacterial resistance to β-lactams: inaccessibility of the
drugs to their target (penicillin-binding proteins [PBPs]), alterations of the drug target, and/or
inactivation of the drugs by β-lactamases (Li & Nikaido, 2004; Livermore, 1995; Poole,
2004). The intrinsic resistance of Gram-negative bacteria is due to a synergenic action
between an outer membrane permeability barrier and multidrug efflux pumps (Li, Ma,
Livermore & Nikaido, 1994; Li & Nikaido, 2004). This way, any mutational or inducible
change in these mechanisms that can lead to a decreased influx or increased efflux of
antimicrobials will prevent drugs from reaching their targets. Consequently, these bacteria
will be resistant to β-lactams and other structurally unrelated antimicrobials. In spite of the
fact that these mechanisms are supposed to confer low resistance levels, in combination with
specific β-lactamases the resistance is greatly enhanced. High levels of expression of
multidrug efflux pumps have been observed in E. coli and Salmonella spp. from food animals
(Baucheron, Imberechts, Chaslus-Dancla, & Cloeckaert, 2002; Olliver, Valle, Chaslus-Dancla
& Cloeckaert, 2005).
In Gram-positive bacteria, the alterations of PBPs are the main responsible for β-lactam
resistance. The main mechanism for β-lactam resistance in Gram-negative bacteria is the
production of β–lactamases. These can be encoded in chromosome or by plasmids and their
mode of action involves the hydrolysis of the four membered β-lactam ring (Bush, Jacoby &
Medeiros, 1995; Livermore, 1995).
The β-lactamase group is quite heterogenous, so far more than 700 different ones have been
described (Perez, Endimiani, Hujer & Bonomo, 2007). Despite the fact that there is great
26
variability in their amino acid sequences, they all share a common topology comprising alphahelices and a β-pleaded sheet (Knox, 1995).
The β-lactamase enzymes are classified into four molecular classes (the Ambler classes A to
D) (Ambler, 1980) the class A, C and D enzymes are serine enzymes and the class B includes
metallo β-lactamases. It is speculated that those with a serine residue have evolved from
penicillin binding proteins, due to their similarity. This classification is based on their primary
structure.
β-lactamases can also be organized into functional groups (the Bush groups 1–4) with a
variety of subgroups, which involves their action on their specific substrate and their
responses to inhibitors (Bush et al., 1995). The role these enzymes play in β-lactam resistance
is determined by their substrate/inhibitor profile. Resistance to third generation
cephalosporins can have its origin in class C AmpC β-lactamases, class A ESBLs (such as
TEM or SHV derivatives and CTX-M family), class D OXA ESBLs or class A or class B
carbapenem hydrolyzing β-lactamases (Bradford, 2001). Even though newer β-lactams are
designed to be more resilient to β-lactamase attack, it seems that the bacterial genome
evolution never ceases to create alternatives to tackle these compounds. Considering the first
classification system, it can be said that the most common are class A and class C βlactamases (Jacoby & Munoz-Price, 2005). These two classes also have in common that they
both have serine residue at their active site, along with class D β-lactamases. Class B enzymes
are metallo β-lactamases. Initially, the class A and class D enzymes were often carried in
plasmids and the class B and C were mostly encoded by chromosomal genes (Medeiros &
Jacoby, 1986).
Most of β-lactamases reported to date have been isolated from human patients (Livermore,
1995; Bradford, 2001; Bonnet, 2004; Jacoby & Munoz-Price, 2005). Many of the newer
varieties have specifically derived from hospitalized individuals that had been subjected to βlactam therapy. Even so, a great number of β-lactamase producing bacteria have recently been
isolated in community-acquired settings (Arpin et al., 2005; Pitout, Nordmann, Laupland &
Poirel, 2005).
Salmonella resistance has been connected with several numbers of β-lactamases and
cephalosporinases (Bauernfeind et al., 1996; Hanson et al., 2002; Makanera, Arlet, Gautier &
Manai, 2003). The concern surrounding these resistance patterns stems from the fact that
fluoroquinolone, ciprofloxacin and third generation cephalosporins (namely ceftriaxone) are
currently used to treat serious Salmonella infections (Angulo et al., 2000; Fey et al., 2000).
27
5.1. Extended Spectrum β-Lactamases (ESBLs)
Generally speaking, ESBLs (extended spectrum β-lactamases) are all capable of hydrolyzing
penicillins and cephalosporins, as well as aztreonam (a monobactam). Still, ESBLs are not
active against cephamycins and carbapenems (Bush et al., 1995; Paterson & Bonomo, 2005).
An ESBL is therefore a β-lactamase that hydrolyzes oxyimino-cephalosporins and can be
inhibited by clavulanate (Li et al., 2007).
Soon after the introduction of extended-spectrum oxyimino-cephalosporins (used for the
treatment of serious bacterial infections) ESBLs were identified (Bradford, 2001). So far,
more than 200 ESBLs have been described, mostly from human Gram-negative isolates (Li et
al., 2007).
The origin of ESBLs is often related to an amino acid substitution(s) in existing β-lactamases.
The substrate and inhibitor profile of these enzymes can be significantly altered by a single or
a few residue changes (Bradford, 2001).
ESBLs belong to the Ambler class A or D and the Bush group 2be or 2d and are mainly
composed of TEM, SHV, OXA and CTX-M derivatives (Bradford, 2001; Bush et al., 1995;
Jacoby & Munoz-Price, 2005). Figure 5 shows the crystal structures of representative ESBLs.
Other class A ESBLs have been isolated but are very uncommon (Jacoby & Munoz-Price,
2005). Although these enzymes were initially rarely recovered from animal-derived bacteria,
plasmid-encoded ESBLs have been frequently isolated worldwide.
The presence of ESBLs leads to not only high-level resistance to aminopenicillins,
carboxypenicillins,
ureidopenicillins,
and
narrow-spectrum
first/second
generation
cephalosporins but also high level resistance to the third generation cephalosporins along with
variable levels of resistance to the fourth generation cephalosporins (Bonnet, 2004).
The fact that ESBLs are inhibited by β-lactamase inhibitors, such as clavulanic acid, enables
their identification through phenotypic testing. The detection of ESBLs involves a great
number of detection methods using different breakpoints.
The general consensus is that all the bacteria that are resistant to all penicillins,
cephalosporins (with the exception of cephamycins, such as cefoxitin and cefotetan) and
aztreonam should be considered ESBL producing, independently of the susceptibility tests
being used. The same applies to β-lactam/β-lactamase inhibitor combinations, as they must be
reported in every susceptibility testing method (CLSI, 2007).
Interestingly, for class A extended-spectrum β-lactamases (ESBLs) there have been
considerably less reports in food and companion animals, in contrast to what is found in
28
human isolates (Miriagou, Tassios, Legakis & Tzouvelekis, 2004; Bonnet , 2004). This is not
necessarily evidence that these ESBLs are not present, but merely that more studies need to be
done. Epidemiological studies can elucidate the role of animals as reservoirs for resistance
(Carattoli, 2008).
Figure 5: Crystal structure of representative ESBLs (Knox, 1995).
Legend: The alpha-helices are highlighted in red and β-pleated sheets are in blue. The protein data
bank number is in parenthesis
ESBL were first isolated in non-typhoidal Salmonella from hospital patients in South
America, North Africa and Eastern Europe, in 1989 (Miriagou et al., 2004; Bonnet, 2004).
None of these infections were associated with food consumption or contact with animals,
being related instead to nosocomial epidemics. The enzymes SHV-1-, TEM-1- and OXA type
β-lactamases have been frequently described in E. coli and Salmonella spp. from animals and
food of animal origin (Carattoli, 2008). Traditionally, the TEM-type and SHV-type enzymes
are the two largest families of plasmid-encoded β-lactamases found in β-lactam resistant
bacteria of human origin (Bush et al., 1995; Livermore, 1995).
29
5.1.1. TEM-type ESBL (class A)
In North and South America, the TEM-10, TEM-12 and TEM-26 are among the most
common ESBLs (Paterson et al., 2003). In ampicillin-resistant E. coli and Salmonella spp.
from animal isolates, the expression of narrow-spectrum TEM-1 or its derivatives is also
significant (Maidhof et al., 2002; Chen et al., 2004; Olesen, Hasman & Aarestrup, 2004). In
E. coli, over 90% of ESBL resistance was mediated through TEM variants (Sturenburg &
Mack, 2003). Although TEM-52 is rarely found in animal isolates, a study conducted from
2001 to 2002 in The Netherlands showed that TEM-52 was the most common ESBL in
poultry, poultry products and human patients (Hasman, Mevius, Veldman, Olesen &
Aarestrup, 2005). The fact that the recovered human and animal isolates serotypes were very
different rules out the likelihood of transmission through food consumption (Hasman et al.,
2005).
5.1.2. SHV-type ESBL (class A)
Presently, these enzymes are the most predominant in isolates from Europe and the United
States (Paterson et al., 2003; Yuan, Aucken, Hall, Pitt & Livermore, 1998). SHV-1 is very
similar to TEM-1, sharing 68 percent of its aminoacids, in addition to a similar overall
structure (Kuzin et al., 1999). Another common feature is that this group of enzymes also have
one or more aminoacid substitutions around their active site. These substitutions lead to a
great number of varieties of SHV (over 50) (Jacoby & Munoz-Price, 2005). The SHV-5 and
SHV-12 are the most common in this class (Paterson et al., 2003). The isolation of this
enzyme variant in E. coli and Salmonella isolated from poultry and pigs in different countries
points out the need to monitor its global diffusion (Brinas, Zarazaga, Saenz, Ruiz-Larrea &
Torres, 2002; Brinas, Moreno & Teshager, 2005; Hasman et al. 2005; Rankin et al., 2005;
Riano et al. 2006).
5.1.3. CTX-M–type ESBL (class A)
The name of this group was chosen to highlight the fact that these enzymes were more active
against cefotaxime than ceftazidime. These are, after the TEM and SHV families, the most
common ESBLs (Jacoby & Munoz-Price, 2005). To this date, more than 40 different enzymes
have been identified (Bonnet, 2004). The plasmids encoding for CTX-M enzymes generally
carry blaTEM genes as well as genes for resistance to aminoglycosides, chloramphenicol,
sulfonamides, trimethoprim, and tetracyclines (Bonnet, 2004; Boyd et al., 2004; Costa et al.,
30
2004). This simultaneous expression of more than one β-lactamase increases the β-lactam
resistance level, leading to greater chance of treatment failure (Brinas et al., 2003a; Teale et
al., 2005).
The ESBLs from this family have become the most frequently isolated in humans in Europe
(Livermore et al., 2007). To raise further concern, isolates of E. coli and Salmonella spp. from
food and pet animals have been shown to carry these resistance determinants. A wide range of
CTX-M enzymes have been isolated from sick and healthy animals or meat products, all over
the world (Li et al., 2007).
There is great concern for the potential risk of diffusion since CTX-M-1 has recently been
isolated from food animals in several European countries (Aarestrup et al., 2006; Meunier,
Jouy, Lazizzera, Kobisch & Madec, 2006; Girlich et al., 2007). Here it is possible to see a
clear association between antibiotic use in animal and diffusion of resistant determinants to
humans.
The CTX-M-2-positive serotype Virchow (Salmonella enterica (S.) Virchow) has emerged in
poultry in The Netherlands, Ireland and Belgium in recent years (Hasman et al., 2005;
Hopkins, Batchelor, Liebana, Deheer-Graham & Threlfall, 2006; Bertrand et al., 2006). For
this reason, surveillance of the animal reservoir is mandatory in order to control potential
diffusion to human bacterial isolates.
5.1.4. OXA-type ESBL (class D))
In this class, there have been identified twelve different ESBLs that derive from OXA-10,
OXA-1, or OXA-2 due to amino acid substituitions (Jacoby & Bush, 2005). Most of these
enzymes are resistant to inhibition by clavulanic acid. Most are especially resistant to
ceftazidime, being OXA-17 the exception, whose resistance to cefotaxime and cefepime
greater than to ceftazidime (Danel, Hall, Duke, Gur & Livermore, 1999).
5.1.5 Association of β-lactamase genes with other antimicrobial resistance determinants
and transposons/integrons
The plasmids encoding for CMY- or CTX-M usually contain multiple resistance determinants
being frequently associated with transposons/integrons (Bonnet, 2004; Boyd et al., 2004;
Giles et al., 2004; Winokur et al., 2000). In recent years, the plasmid-mediated Qnr
(quinolone resistance determinant) mechanism, protecting DNA from quinolone binding, is of
31
concern because of its frequent association with CTX-M and CMY-type enzymes inactivating
third generation cephalosporins (Rodriguez-Martinez, Poirel, Pascual & Nordmann, 2006).
Salmonella genomic islands are also characterized by the presence of integrons and
transposons. Considering the similarity in genetic organization of multidrug resistance
determinant-integron/transposon elements, it should be stated that there is significant linkage
between ESBL and fluoroquinolone resistances in Gram-negative bacteria (Li et al., 2007).
The Qnr-encoding plasmids are frequently associated with a transposon/integron carrying
simultaneously other resistance determinants which include the bla genes for β-lactamases
such as FOX-5, DHA-1, OXA-30 or SHV-7, the aad or aac gene for aminoglycoside
nucleotidyltransferase
or
acetyltransferase,
the
cat
gene
for
chloramphenicol
acetyltransferase, and the sulI gene for dihydropteroate syntase (Li, 2005).
There is increasing evidence of Qnr-mediated quinolone resistance in human isolates
(Ellington & Woodford, 2006; Li, 2005), but none of these has yet been found in animals.
This is most likely about to change, since fluoroquinolones are frequently used in food and
pet animals. It can be concluded that exposure to any drugs to which resistance is present, will
be linked to selection or enhancement of resistance to other antimicrobials. This is due to the
existence of multiple resistance determinants on a single transmissible plasmid. As a result, βlactams, aminoglycosides, amphenicols, sulfonamides or tetracyclines could be actively
involved in selecting or maintaining resistance (Li et al., 2007). The precise role of these
antimicrobials in resistance development or dissemination requires further assessment by
pharmacokinetics and pharmacodynamics studies.
5.2. Plasmid-mediated AmpC enzymes (class C)
This class of enzymes is encoded by chromosomal genes in Gram-negative bacilli. They are
usually inducible by β-lactams. Although they are poorly expressed in E. coli and the AmpC
gene is missing in bacteria such as Salmonella spp. and Klebsiella spp., the plasmid-mediated
gene can confer the same resistance pattern as the gene-mediated one (Jacoby & MunozPrice, 2005). Over 20 different AmpC have been connected to plasmid transmission
(Philippon, Arlet & Jacoby, 2002). Many AmpC enzymes are inducible by regulatory genes,
but most of them are not. This class of enzymes is related to resistance to cephamycins and
oxyimino β-lactams. Additionally, AmpC enzymes are also resistant to inhibition by
clavulanic acid (Jacoby & Munoz-Price, 2005). These CMY β-lactamases (cephamycinases)
32
belong to the Ambler class C/the Bush group 1 and hydrolyze extended-spectrum
cephalosporins (Philippon et al., 2002). However, ESBL enzymes possess no activity against
cephamycins (such as cefoxitin) and therefore CMY enzymes cannot be considered ESBLs
(Bradford, 2001). The blaCMY genes are connected to decreased susceptibility to both
ceftiofur and ceftriaxone. The use of ceftiofur may select for cross-resistance to this last
antibiotic, which is used in human medicine.
From this group, the most relevant is CMY-2, which originates from the AmpC enzyme of
Citrobacter freundii (Batchelor et al., 2005), a plasmid encoded variant derived from the
chromosomal ampC locus (Sturenburg & Mack, 2003). CMY-2 is prevalent in extendedspectrum cephalosporin-resistant E. coli and Salmonella spp. of animal origin (Alcaine et al.,
2005; Allen & Poppe, 2002b). The emergence of this resistance in animals might be due to
the use of ceftiofur, a veterinary approved extended spectrum cephalosporin (White et al.,
2001; Hornish & Kotarski, 2002).
CMY-2-mediated cephalosporin resistance in Salmonella has been reported in several
countries throughout the world and outbreaks have often been connected to comsumption of
food animal products. This multidrug resistance phenotype included resistance to
streptomycin, sulfamethoxazole, tetracycline, ampicillin, chloramphenicol and decreased
susceptibility to extended spectrum cephalosporins (Zansky et al., 2002). Molecular and
phenotypical studies point out that this phenotype is present in very genetically diverse
strains, since cephalosporin-resistant strains show various distinct serotypes. Its diffusion can
be explained by efficient horizontal transmission by encoding plasmids, making it widely
distributed worldwide (Batchelor et al., 2005) and between the different enteric species and
genera. This hypothesis has been proven under laboratory conditions (Poppe et al., 2005)
further confirming epidemiological studies (Winokur, Vonstein, Hoffman, Uhlenhopp &
Doern, 2001). The international trade of food animals might have increased their ability to
disseminate and persist worldwide (Aarestrup, Hasman, Olsen & Sorensen, 2004; Liebana et
al., 2004).
5.3. Carbapenemases (classes a, b, and d)
This is a diverse group of enzymes. Although uncommon, there is reason for concern because
they are active against oxyimino-cephalosporins, cephamycins and carbapenems (Nordmann
& Poirel, 2002).
33
6. β-lactamases produced in bacteria of animal origin
Far fewer types of β-lactamases can be identified in animals than in human isolates. The
apparent higher prevalence of CMY and CTX-M in animals (both plasmid-transmitted
enzymes) shows the importance of the spreading and enrichment of existing resistant clones.
Prudent use of antibiotics in animals should be warranted in controlling antibiotic resistance,
considering the association of multiple resistance determinants (Peterson, 2005; McEwen,
2006).
Resistant strains show a greater potential for outbreaks and more severe and protracted
illnesses when compared to their non-resistant counterparts (Varma et al., 2005). High levels
of mortality and morbidity in calves and fatal infections in pets have been linked to
cephalosporin resistant E. coli and Salmonella (Bradford, Petersen, Fingerman & White,
1999; Brinas et al., 2003b).
Apart from this fact, animals can also act as reservoirs for resistant faecal flora. This flora can
then be transmitted to humans through the food chain, reaching the human intestinal system.
Subsequently, resistant colonizers (especially E. coli) can be responsible for urinary infection
in vulnerable patients. The existence of resistant non-pathogenic E. coli might be connected to
the transmission of resistance determinants, which can occur through the gut, by ingestion of
milk or meat products (Kruse & Sorum, 1994). There is still great uncertainty to whether this
exchange occurs directly between animal and human pathogens and the role that antibiotic
usage in veterinary medicine contributes to this problem, by providing selective pressure on
resistant bacteria.
Current studies report isolation of resistant strains from animals, but the molecular basis for
this resistance requires further investigation. This would involve a genetic characterization of
each resistant phenotype. Although there are a few undergoing studies, there is still a long
way to go, especially when comparing with research in human resistance isolates.
One common feature of these studies is that they all show an increase in the prevalence of βlactamase genes in Salmonella and E. coli and also the emergence of resistance to newer
cephalosporins (Batchelor et al., 2005).
Initially, animal studies in β-lactam resistance reported qualitative β-lactamase production,
but this trend is changing since recent research has also focused on phenotypic and genotypic
characterizations.
34
A greater understanding of the influence of β-lactam resistance in animal isolates is especially
important due to the significant role resistant bacteria play in food safety and public health (Li
et al., 2007).
For instance, figures from Canada show that ceftiofur resistance (minimal inhibitory
concentration [MIC] of 8µg/mL) in chicken-derived Escherichia coli from abattoir
surveillance increased from 16% in 2002/2003 to 25% two years later. Ceftiofur resistance in
Salmonella spp. from chickens increased from 7% in 2002/2003 to 22% in 2004. In retail
chicken meat, resistance to ceftiofur rates up to 21–34% for E. coli and 40–45% for
Salmonella spp. In food animal Salmonella spp. isolates, high rates of ceftiofur resistance (2–
20%) were also detected in 2004 (CIPARS, 2006). There is a high correlation between
ceftiofur resistance and amoxicillin-clavulanic acid (MIC 32/16 µg/mL) resistance and
cefoxitin (MIC 32 µg/mL) resistance and reduced susceptibility to ceftriaxone (MIC between
8 and <64 µg/mL) (CIPARS, 2006). This evidence suggests the presence of a common
resistance mechanism.
Ampicillin resistance (MIC 32 µg/ mL) in Salmonella spp. from poultry and swine isolates
increased from <10% in 1997 to about 15–20% in 2004 in Denmark. For E. coli in swine it
ranged between 10 and 20% during 1996–2004 but increased from 11% in 2002 to 22% in
2003 and it went as high as 32% in 2004 (DANMAP, 2005).
Ceftiofur resistance in E. coli was found in imported beef at a rate of 2.6%, but not from
domestic meats in Denmark (DANMAP, 2005). Recent data from the USA, revealed
resistance
rates
in
chicken-derived
E.
coli
to
ampicillin
of
18%,
9%
for
amoxicillin/clavulanate, 8% for cefoxitin, 5% for ceftiofur and 0,1% for ceftriaxone
(NARMS, 2004).
Salmonella isolates from food animals showed variation concerning resistance, ranging from
14–63% towards ampicillin, 3–50% to amoxicillin/clavulanate, 10–51% to cephalothin, 3–
47% to cefoxitin, 3–50% to ceftiofur and 0-0.6% to ceftriaxone (NARMS, 2004).
The emergence of resistance is supported by studies performed all over the world (White et
al., 2001; Zhao et al., 2001; Allen & Poppe, 2002a). In Table 3 a summary of all the reports
of the β-lactamases (including extended-spectrum β-lactamases) from animal sources is
shown.
35
Table 3: Chronological reports of the β-lactamases including extended-spectrum β-lactamases
from bacteria of animal sources (Li et al., 2007)
Year of the
isolates
1992
Country
Animal source
Bacterial species
β-Lactamase(s) identified
UK
Calves
TEM1 derivatives
1998
1998-1999
1998–2000
USA
USA
USA
1998–2003
UK
Japan
E. coli and
Salmonella
E. coli
CMY-2
1999–2002
Cattle
Cattle, swine
Cattle, swine, poultry
and their ground
meats
Cattle, chickens,
swine, turkey
Chickens
E. coli and
Citrobacter
freundii
Salmonella spp.
Salmonella spp.
E. coli and
Salmonella spp.
2000–2001
Spain
Chickens
E. coli
2000–2001
2000–2002
Japan
Demark
Cattle
Cattle, swine, poultry
E. coli
E. coli and
Salmonella spp.
2001
2001–2002
Greece
Poultry meat
Netherlands Poultry and its
products
Salmonella spp.
Salmonella spp.
2002
Canada
Beef, chicken, pork
2002
China
2002–2003
2003
Portugal
France
2003
2003
France
Denmark
2003
Denmark
2002
Canada
Cattle, pigs and
pigeons
Pork
Poultry and its
products
Horse meat (imported)
Quails (imported from
France)
Swine (imported from
Canada)
Beef, chicken, pork
E. coli and
Salmonella spp.
E. coli
2002
China
2003–2004
Spain
Cattle, pigs and
pigeons
Pigs, poultry
2003–2004
2004
USA
UK
Horse
Calves
CTX-M-2, CTX-M-18,
TEM-1, PSE-1; ampC
promoter mutations
identified
CMY-2, CTX-M-14,
SHV-12; ampC promoter
mutations identified
CTX-M-2
OXA, PSE, TEM-1, TEM127, TEM-128; ampC
promoter mutations
identified
CTX-M-32
ACC-1, CTX-M-2, CTXM-3, CTX-M-28, SHV-12,
TEM-1, TEM-20, TEM52, TEM-63
CMY-2
Salmonella spp.
Salmonella spp.
CTX-M-3, CTM-13, CTXM-14 and CTX-M-24
OXA-30
CTX-M-9
Salmonella spp.
Salmonella spp.
CMY-2
CTX-M-9
Salmonella spp.
CMY-2
E. coli and
Salmonella spp.
E. coli
CMY-2
Salmonella spp.
Salmonella spp.
E. coli
36
CMY-2
CMY-2
CMY-2
CTX-M-3, CTM-13, CTXM-14 and CTX-M-24
CTX-M-9, SHV-12,
TEM-1
CMY-2, SHV-12,TEM-1
CTX-M-14, TEM-35
2004
Denmark
2005
Not
specified
Not
specified
Denmark
UK
Spain
Beef (imported from
Germany)
Swine
Cattle
E. coli
TEM-52
E. coli
E. coli
Poultry, pigs and
rabbits
E. coli and
Enterobacter
cloacae
CTX-M-1
CMY-2; ampC promoter
mutations identified
CTX-M-1, CTX-M-9,
CTX-M-14, SHV-2, SHV5, TEM-52
7. Detection of resistance
Resistance can be detected in two ways: either phenotypically or genotypically.
For bacteria of clinical importance, diagnostic laboratories in the United States perform
phenotypically-based susceptibility tests according to the CLSI (Clinical Laboratory
Standards Institute). These tests include an inhibition assay that can be performed in broth or
agar discs diffusion. When applying the dilution-based growth inhibition assay it is possible
to classify bacteria as susceptible, intermediate or resistant, depending on the Minimal
Inhibitory Concentration (MIC) calculated for each bacterial isolate on a certain drug. The
establishment of various categories of resistance or breakpoints is done according to the
clinical efficiency of the drug. Previous considerations about pharmacokinetics and
pharmacodynamics and comparison with other MIC´s are factors involved in creating
breakpoints.
This is an important tool to practitioners, which in this way can have a greater chance of
prescribing a drug that does not lead to treatment failure.
The traditional way of approaching bacterial resistance is phenotypic and it involves
cultivation of bacteria followed by testing of different sets of antibiotics. This is however,
insufficient because these techniques are often highly selective and so bacteria that were not
the target for the study, less predominant strains or simply bacteria that don’t easily grow on
laboratory conditions can easily be missed (IFT, 2006).
Since the breakpoint is set considering what concentration of drug will be at the site of the
infection, a high ‘MIC’ therefore doesn’t necessary mean that a bacteria has high resistance to
that same drug. If, however the MIC is greater than the highest concentration that can be
achieved at the site (due to pharmacokinetic mechanisms) the microorganism can be
considered resistant. Various testing methods for antimicrobial susceptibility and breakpoints
have been recommended by the CLSI.
The issue at hand is that no standard for genotypic resistance methods has been used on a
routine basis in clinical laboratories. The best way to address this problem involves the use of
37
the total DNA extracted in a sample, this can be achieved by techniques such as Polymerase
Chain Reaction or DNA-DNA hybridization.
Microarrays are currently used because of the easiness to detect the activation of specific
genes involved in antimicrobial resistance (Call, Bakko, Krug & Roberts, 2003; Yu, Susa,
Knabbe, Schmid & Bachmann, 2004).
7.1. Screening for ESBL production
The Clinical Laboratory Standard Institute (CLSI), for instance has defined that the MIC for
enterobacterial resistance to ceftazidime, cefepime, ceftriaxone, cefotaxime and aztreonam is
16 µg/mL (CLSI, 2007). Numerous studies have shown that automated and diffusion disk
methods are frequently inaccurate (Tenover, Mohammed, Gorton & Dembek, 1999;
Livermore, 1995). Due to the fact that a great number of ESBL’s have setpoints below these
levels, there is a possibility that ESBL presence is undetected. Therefore numerous
institutions have been trying to put out a set of guidelines that can harmonize these standards.
The CLSI recommends both the broth diluition and the disk diffusion methods for the
screening of EBSL. For bacteria such as E. coli, K. pneumoniae and Klebsiella oxytoca with a
MIC of 8 µl/mL for cefpodoxime or a MIC of 2 µl/mL for ceftazidime, cefotaxime or
ceftriaxone or aztreonam, a specific phenotypic test for confirmation should be used. Also, the
use of more than one of these agents increases the sensitivity of the detection. The same
institution has agreed that, when using the disk diffusion method for E. coli, K. pneumoniae,
K. oxytoca and P.mirabilis, a zone inhibition lower than 22 mm for ceftazidime, 27 mm for
cefotaxime and aztreonam and 25 mm for ceftriaxone, should lead to further confirmatory
tests. For cefpodoxime, in all these bacteria the limit has to be 17 mm, except for P. mirabilis
for which it is 22 mm (CLSI, 2007).
The CLSI has not provided any recommendations concerning other Enterobacteriaceae, but
the BSAC (British Society for Antimicrobial Chemotherapy) has provided a different set of
standards. Namely that, all Enterobacteriaceae should be subjected to a confirmatory test
when they present the characteristics described below.
This way, in the case of E. coli and Klebsiella spp., confirmatory tests are mandatory when
both bacteria present a MIC of 4µl/mL or a zone inhibition of 21 mm to ceftazidime (27 mm
for the remaining species), a MIC of 2µl/mL or inhibition zone of 29 mm to cefotaxime, or a
MIC of 2µl/mL or inhibition zone of 19 mm to cefpodoxime (BSAC, 2005). The SFM
(Société Française de Microbiologie), on the other hand, suggest that all bacteria should be
38
subjected to a confirmatory test. Also, according to this committee, confirmatory tests should
be performed every time a bacterial isolate presents resistance to aminoglycosides (Comite de
L’Antibiogramme de la Société Française de Microbiologie, CASFM, 2007). This is due to
the fact that the bla gene responsible for the ESBL production is often transmitted in the same
plasmid that carries resistance determinants for aminoglycoside, tetracycline and
sulphonamide antibiotics (Bradford, 2001).
7.2. Phenotypic confirmatory tests for ESBL production
The confirmatory tests based on phenotype require the use of both ceftazidime and
cefotaxime, either or not in combination with clavulanate, according to the CLSI (2007).
Generally confirmatory tests require the use of 30 µg of ceftazidime or cefotaxime and 10 µg
of clavulanate in the case of combination disks. Discs with a combination of a cephalosporin
and clavulanate can be used reliably to test for enhancement of the zone of inhibition
compared to the individual cephalosporin. The zone diameter should increase by  5 mm or
by 50% (Med-Vet-Net, 2006). The reported sensitivity and specificity for these disks is
greater than 95% (Carter, Oakton, Warner & Livermore, 2000; Wiegand, Geiss, Mack,
Sturenburg & Seifert, 2007).
The CLSI has not given guidelines for the detection of ESBLs in other Enterobacteriaceae
such as Enterobacter spp., Morganella morganii, Providencia spp., C. freundii, and S.
marcescens or in bacteria producing AmpC enzymes. The BSAC recommends the use of
cefpirome and cefpodoxine with clavulanate as a confirmatory test in Enterobacter spp. and
C. freundii (Health Protection Agency, 2005). The SFM advises the use of cefpirome and
cefepime in a double disk diffusion assay to detect AmpC producers (CASFM, 2007).
Another alternative is the double-disc potentiation method. These are discs with a
cephalosporin (cefotaxime or ceftazidime) or aztreonam, and a disc with amoxicillinclavulanate 30 mm centre to centre. The zone of inhibition by the amoxycilin and clavulanate
is enhanced when in presence of an ESBL positive strain. The test may give false negative
results (Med-Vet-Net, 2006).
The three-dimensional extract test is yet another variant of the double disc test. In this test a
standardised inoculum is pipetted in a circular slit in the agar, 3 mm from the disc in the
centre of the plate. This way it is possible to determine the susceptibility and β-lactamase
substrate profiles. However the test is difficult and not refined for routine laboratories (MedVet-Net, 2006).
39
It is also possible to use automated tests, as for instance the Vitek (bioMérieux, France). The
basis for this test is a system that monitors growth automatically. The antibiotics to be used
are cefotaxime, ceftazidime and these in combination with clavulanate (Med-Vet-Net, 2006).
Finally, there is the Etest® (AB Biodisk, USA). In this test a stable antibiotic concentration
gradient around a test strip is applied on agar (Med-Vet-Net, 2006). The ESBL-strips combine
on the one side ceftazidime or cefotaxime with the combinations of these drugs with
clavulanate. Once more, the test is based on the reduction of ceftazidime/cefotaxime MICs in
combination with clavulanate. The Etest is reported to be more sensitive for the detection of
ESBLS than automated systems or double disc test (Med-Vet-Net, 2006). Factors affecting
the outcome of these tests are: clavulanate resistant ESBLs, lack of expression and the
simultaneous presence of chromosomally encoded β-lactamases.
8. Monitoring of Resistance
In order to assess the real environmental impact of antimicrobial resistance in human health it
is required that there is available data on the amount of transfers from environment or animal
recipients to humans, the ability of resistant bacteria to cause illness and what kind of
treatment was applied (Sanchez et al., 2002). Even though there are a few surveillance
projects across the world that measure resistance, the interpretation of these is made difficult
by the lack of standardization, because different methods and criteria are used. The literature
is also discontinued throughout time, which makes this task even harder. Even as far as
diffusion disk usage goes, there are significant differences that prevent comparisons between
studies (IFT, 2006). The same applies to other techniques, such as serial broth dilution or MIC
testing.
It is of great importance to uniform the standards and plan a worldwide approach to this
relevant problem.The European Antimicrobial Resistance Surveillance System (EARSS) is an
international network of national surveillance systems. It performs on-going surveillance of
antimicrobial susceptibility in Streptococcus pneumoniae, S. aureus, E. coli and E.
faecalis/faecium causing invasive infections in humans, and monitors variations of
antimicrobial resistance over time and from place to place (EARSS, 2004). Another
European-based surveillance network of interest is Enter-Net, an international surveillance
network for human gastrointestinal infections of Salmonella and verocytotoxin-producing E.
coli and antimicrobial resistance. Since 2007, this network has been coordinated by The
40
European Centre for Disease Prevention and Control (ECDC, 2007). In addition, Denmark
and Norway have independent surveillance systems (IFT, 2006).
II. Experimental part
1.
Antibioresistance
of
Extended-Spectrum
β-Lactamases
producer
strains
of
Enterobacteriace from broiler samples
1.1. Objectives
The aim of this work was to determine the prevalence of Enterobacteriaceae isolates
producing ESBL (Extended-Spectrum β-Lactamases) and Salmonella spp. from broilers in the
slaughterhouse and evaluate the influence of the slaughtering process in the presence of these
enzymes in the above mentioned bacterial isolates.
2. Material and methods
2.1. Slaughterhouse caracterization
Samples were collected from different intensive production poultry flocks in five distinct
sampling days, always at the same poultry slaughterhouse. This slaughterhouse had a
slaughtering capacity of about 120 thousand chickens per day. The facility was located in the
province of South Holland in the Netherlands, in an industrial complex.
In the living area, poultry was unloaded, hanged manually, electrically stunned in a water bath
and bled. The animals were then taken by a conveyor belt that crossed the whole room to a
second separated area. There, the chickens were scalded in a counter current flow scalding
tank at a temperature of ±51ºC before they were mechanically plucked. The head of the
chicken was removed before the carcasses were hung over on the evisceration line. Finally,
the mechanical evisceration took place in a third room. After this, the carcasses were spraywashed and chilled (for 180 min (3 h) by a continuous flow (3.5 m/s) of cold air
(approximately –1.1°C)). During the slaughter process, only potable water was used.
2.2 Sample collecting
In Figure 6 the circles represent points in the slaughterhouse where samples were collected,
namely:
41
Live animal sampling (A)
Cloacal samples were collected from live animals in the unloading area, using a sterile swab
moistened with 10 mL Buffered Peptone Water (BPW, CM509 Oxoid, Basingstoke,
Hampshire, UK).
Before scalding (B), Before defeathering (C), Before evisceration (D), After washing and
chilling (E) and After Chilling (F) samples
The carcasses were removed from the line (with the help of an employee) just before the
scalding (B), defeathering(C), evisceration (D) and after washing/before chilling (E); and after
the chilling phase (F) using sterile gloves. The neck skin was then excised with aseptic
procedures and placed in a labelled stomacher bag. On the 03/03/2008, the whole carcass was
collected in the slaughterhouse and the neck skins were excised in the laboratory facilities.
Samples were collected on the following days: 25/11/2007, 10/12/2007, 03/03/2008,
18/03/2008 and 09/04/2008 according with the previously specified conditions. Table 4 shows
the sampling schedule including the number of collected samples per site.
Figure 6: Slaughterhouse layout (www.hyfoma.com, 2007).
42
Table 4: Number of samples collected per sampling site, per sampling day.
Sampling day 25/11/2007 10/12/2007 03/03/2008 18/03/2008 09/04/2008
Step
(Flock A)
(Flock B)
(Flock C)
(Flock D)
(Flock E)
Live animals
_
_
15
15
15
Before scalding
_
_
15
_
_
Before defeathering
_
_
15
_
_
Before evisceration
_
8
15
15
15
8
_
15
15
_
15
_
_
After washing/
chilling
before 20
After chilling
_
Several different analytical procedures were executed throughout this experiment, depending
on the sampling day. Table 5 shows the analytical procedures done on each sampling day.
Table 5: Types of procedures done according to the day the samples were taken.
25/11/2007 10/12/2007 03/03/2008 18/03/2008 09/04/2008
Sampling day
Procedure
Enumeration of
Enterobacteriaceae
(standard method)
Enumeration of
Enterobacteriaceae (quick
method)
Enumeration of E. coli
(quick method)
Detection of Salmonella
spp.
Enumeration of Extended
Spectrum β-Lactamases
(ESBL) positive
Enterobacteriaceae in
ChromIDtm agar
Isolation and
Identification of
Enterobacteriaceae
(Flock A)
(Flock B)
X
X
X
X
X
X
(Flock C)
X
(Flock D)
(Flock E)
X
X
X
X
X
X
X
X
X
43
X
2.3 Microbiological analysis
2.3.1. Sample preparation and diluitions for microbiological analysis
At the laboratory, 25 g of neck skin from each carcass were weighted and placed into a
stomacher bag. To this bag, 225 mL of Buffered Peptone Water (BPW, CM509 Oxoid,
Basingstoke, Hampshire, UK) was added, obtaining a 1:10 dilution of the initial sample. The
bag was then placed in a Stromacher machine for 2 minutes, in order to appropriately mix its
contents. From this initial suspension of 10-1 diluition other serial dilutions were made
ranging from 10-2 to 10-7 using Buffered Peptone Water (BPW, CM509 Oxoid, UK).
From the tubes containing the cloacal swabs, serial dilutions were made ranging from 10-2 to
10-7.
2.3.2. Enumeration of Enterobacteriaceae (standard method)
From the 10-2, 10-4 and 10-6 diluitions, 1mL was inoculated in a Petri dish. About 12 mL of
Violet Red Bile Glucose agar (VRBG, CM0485 Oxoid, UK) at 45ºC were then poured into
the Petri dish. The plates were then carefully shaken and left to solidify. A second layer was
added on top and the plates were left to incubate for 24 h at 37ºC according to ISO
7402:1993.
Typical colonies (pink red to purple) were counted and confirmed as oxidase-negative. Counts
were expressed as log10 CFU/g. The oxidase test was done by applying a few drops of reagent
(Pathotec® Cytochrome oxydase, Remel, Lenexa, KS) to a strip of filter paper. With a sterile
needle, a colony of bacteria was picked and streaked on the reagent-soaked filter paper. The
reagent would then undergo a chemical reaction resulting in a violet or purple color change if
the isolates were oxidase-positive. There was no colour change if the bacteria were oxidasenegative.
2.3.3. 3M™ Petrifilm™ Enterobacteriaceae (rapid method)
3M™ Petrifilm™ Enterobacteriaceae Count Plates (3M Microbiology, St. Paul, USA) were
inoculated with 1 mL from the 10-2, 10-4 and 10-6 diluitions and then left to incubate at 37º C
for 24h (method validated according to AOAC, 2006). All colonies with yellow or cream
colored zones (acid) with or without associated gas bubbles were counted as
Enterobacteriaceae. Counts were expressed as log10 CFU/g. Typical colonies were confirmed
as oxidase-negative using the procedure described above.
44
2.3.4. Enumeration of E. coli
3M™ Petrifilm™ E. coli/Coliform Count Plate (3M Microbiology, St. Paul, USA) were
inoculated with 1 mL from the 10-3, 10-5 and 10-7 diluitions and then left to incubate at 37º C
for 24h (method validated according to AOAC, 2006). All the red colonies with a blue halo
and gas were counted as E. coli. Counts were expressed as log10 CFU/g. The oxidase test was
performed on typical colonies.
2.3.5. Enterobacteriaceae isolation and identification
Pink to red or purple colonies on VRBG, yellow on Petrifilm Enterobacteriaceae and red
colonies on Petrifilm E. coli/Coliform were streaked on a selective MacConkey Agar
(CM0007 Oxoid, UK) and left to incubate for 24h at 37ºC. Table 6 shows the types of
colonies that can be distinguished on this selective media.
Table 6: Colony appearance on MacConkey agar.
Type of bacteria
Colony appeareance on MacConkey agar
Lactose fermenters (E. coli, Klebsiella spp.,
Enterobacter aerogenes, Serratia spp.)
Pink to red and may be surrounded by a pink
Non-lactose fermenters (Salmonella spp.,
Proteus spp.)
White/colourless colonies (due to peptone
zone of precipitated bile (e.g., E. coli)
use).
Pink to red and white colonies were then streaked on Tryptone Soya Agar (TSA, CM0131
Oxoid, UK) and left to incubate at 37ºC for 24h. Enterobacteriaceae isolates were inoculated
in Triple Sugar Iron (TSI Agar Slant, 221039, BD Diagnostics, USA) tubes to test for sugar
fermentation, Triptone Water tubes (TW, CM0087 Oxoid, UK) to perform the indole test and
Brilliant Green Bile Broth tubes (BB, CM0031 Oxoid, UK) to test for lactose fermentation.
The tubes were then left to incubate for 24h at 37ºC. The indole test was subsenquently
performed by adding 1mL of Kovac’s reagent to the previously incubated triptone water
tubes. The reactions were read according to the characteristics in Table 7. A few isolates
(n=25) were inoculated into Enterotubes (BD Diagnostics, USA) for biochemical
identification.
45
2.3.6. Detection of Salmonella spp..
Pre-enrichment phase
At the laboratory, 25 g from each sample were weighted and placed into a stomacher bag. To
this bag, 225 mL of Buffered Peptone Water (BPW, CM509 Oxoid, UK) was added,
obtaining a 1:10 dilution of the initial sample. The bag was then placed in a stromacher
machine for 2 minutes, in order to appropriately mix its contents. The Stomacher bag was
incubated for 18 to 24h at 37ºC performing a period of pre-enrichment for Salmonella spp.,
according to ISO 6579:2002.
The BPW swabs used for collecting cloacal samples were incubated for 18 to 24h at 37ºC
performing a period of pre-enrichment for Salmonella, according to ISO 6579:2002.
Selective enrichment phase
The next step was the inoculation of 1 mL of broth from the pre-enrichment bag in a 10 mL
tube with Muller-Kauffmann Tetrathionate-Novobiocin broth (MKTTn, CM1048 Oxoid,
UK), which was then left to incubate for 24h at 37ºC. Simultaneously, 0.1 mL of the preenrichment suspension was inoculated in 10 mL tubes with Rappaport-Vassiliadis
enrichment broth (RVS, CM866 Oxoid, UK) and incubated at 42ºC for 24h.
Isolation phase
After incubation, using a sterilized 10µL loop, an aliquot of each enrichment broth (MKTTn
and RVS) was streaked onto Brilliant Green agar (BGA; CM0329 Oxoid, UK) and Xylose
Lysine agar (XLD; CM0469 Oxoid, UK) plates. The plates were left to incubate at 37ºC for
24h. Typical colonies on BGA (red colonies surrounded by a bright red zone) and XLD (red
colonies with black centres) were then inoculated into Tryptone Soya Agar (TSA, CM0131
Oxoid, UK) and left to incubate for 24h at 37ºC. Salmonella presumed isolates were
inoculated in Triple Sugar Iron Agar (TSI Agar Slant, 221039, BD Diagnostics, USA) to test
for sugar fermentation and production of hydrogen sulfide. Urea Agar (Urea Agar Slants,
complete [Christensen] 221096, BD Diagnostics, USA) and Lysine Decarboxylase Broth
(LDC, BD Diagnostics, USA). The first was used to test for the production of urease and the
second to test for the production of lysine decarboxylase. Triptone Water tubes (TW, CM0087
Oxoid, UK) to perform the indole test were also inoculated. The tubes were then left to
incubate for 24h at 37ºC. The indole test was subsenquently performed by adding 1mL of
Kovac’s reagent to the previously incubated Triptone Water tubes. To perform the β46
galactosidase test, a ONPG disc (49940 ONPG Disks, Fluka (Sigma-Aldrich), USA) was
placed in a sterile test tube. To this tube, 0.1 ml of sterile 0.85% (w/v) sodium chloride
solution (physiological saline) was added. Each tube was inoculated with one colony using
sterile loop, emulsified and left to incubate at 35°C for 24 hours. To perform the VogesProskauer test, tubes with 3mL of MR-VP media (MR-VP Medium, 216300, Difco, USA)
were inoculated with pure cultures. The tubes were incubated for 24 hours at 37ºC. After the
appropriate incubation period, 1 mL aliquots of the medium were removed and the VogesProskauer Test was performed by adding 15 drops from the reagent A dropper (VogesProskauer Reagent A 261192, Difco/BBL, USA) and 5 drops from the reagent B dropper
(Voges-Proskauer Reagent B 261193, Difco/BBL, USA) into 1 mL of broth culture with
shaking between the addition of each reagent. The results are explained in Table 7.
A few isolates (n=6) were also inoculated into Enterotubes (BD Diagnostics, USA) for
biochemical identification.
Table 7: Interpretation of biochemical reactions.
TSI reaction
Interpretation
Alkaline slant (red), acid butt (yellow)
Glucose fermented
Acid
slant
and
butt
(medium
yellow Lactose or sucrose, or both fermented
throughout)
Alkaline slant and butt (medium entirely red)
None of the three sugars fermented
Gas bubbles in butt, medium sometimes split
Gas (CO2) produced as a by-product of
fermentation
Blackening of the butt and/or stab
Hydrogen sulfide produced
Brillian Green Bile Broth reaction
Interpretation
Production of gas collected in Durham’s tube
Lactose fermentation
Increase in turbidity
Bacterial growth
Indole reaction
Interpretation
Formation of a red ring at the surface of the Production of indole by deamination of
broth
tryptophan
Formation of a yellow ring at the surface of Negative for indole production
the broth
Urea Agar reaction
Interpretation
Change of colour to rose-pink and later to Production of urease which degrades urea to
deep cerise (change in pH)
ammonia
47
LDC broth reaction
Interpretation
Turbidity and purple colour
Presence of lysine decarboxylase enzyme
(alkaline byproducts raise the pH of the
media )
Yellow colour (due to fermentation of Negative for lysine decarboxylase
dextrose) or no change in colour
Voges-Proskauer reaction
Interpretation
Pink-to-red colour
production of acetylmethylcarbinol (acetoin),
formed from pyruvic acid in the course of
glucose fermentation
No colour change
Negative for glucose fermentation
β-galactosidase detection
Interpretation
Yellow colour
Production of o-nitrophenol (yellow)
(byproduct of lactose fermentation due to the
presence of ß-galactosidase)
Colourless/pale yellow
Negative for β-galactosidase
All the isolates that presented the typical Salmonella reactions (see Table 8) were considered
presumptive Salmonella.
Table 8: Biochemical results for Salmonella (Global Salm-Surv, 2003)
Test
Positive
or
negative
reaction
TSI glucose (acid formation)
TSI glucose (gas formation)
TSI lactose
TSI sucrose
TSI hydrogen sulfide
Urea splitting
Lysine decarboxylation
β-Galactosidase reaction
Voges-Proskauer reaction
Indole reaction
+
+
+
+
-
48
% of Salmonella
showing the
reaction
100
91.9
99.2
99.5
91.6
99
94.6
98.4
100
98.9
Serotyping
All the isolates from Flock A and Flock B (n=22) showing typical Salmonella biochemical
reactions were examined for slide-agglutination using the Salmonella O polyvalent antiserum
poly A-I + Vi (PRO-LAB Diagnostics, Toronto, Canada). In the presence of agglutination the
isolates were then subjected to agglutination with monovalent H antisera (PRO-LAB
Diagnostics, Toronto, Canada).
2.3.7. Enumeration of Extended Spectrum β-Lactamase positive Enterobacteriaceae
From the 10-2 diluition, 1mL was inoculated on the surface of ESBL selective chromogenic
agar (ESBL-Bx; bioMerieux, Marcy l’Etoile, France, renamed chromID). On the ESBL
chromogenic agar (as seen in Figure 7), colonies were counted according to the following
colour significance:
 Escherichia coli: pink to burgundy colouration of ß-glucuronidase-producing colonies.
 Klebsiella, Enterobacter, Serratia, Citrobacter (KESC): green/blue to browny-green
colouration of ß-glucosidase-producing colonies.
 Proteeae (Proteus, Providencia, Morganella): dark to light brown colouration of
deaminase-expressing strains.
Figure 7: chromID™ plates after culturing. Different colors are used for identification of
specific genera of Enterobacteriaceae
49
2.3.8. Determination of Antibioresistence in Enterobacteriaceae isolates
Typical colonies of Enterobacteriaceae were selected and inoculated into a Triptone Soya
Broth (TSB, CM0876 Oxoid, UK) and left to incubate for 24h at 37ºC.
From this suspension, a sterile loop (10µL) was used to collect an aliquot which was then
spread in the top half of the Iso-Sensitest agar plates (PO0779 Oxoid, UK). The plate was
rotated 60º and the procedure was repeated two more times, in order to equally distribute the
suspension thoughout the plate. The Neo-sensitab discs (Rosco, Denmark) were then
individually placed on top of the inoculated plate, with a minimum distance of 24 mm
between each other. The following antibiotic discs were used: ampicillin (33µg), ceftiofur
(30µg), trimethoprim+sulfamethoxazole (5.2 µg + 240µg), tetracycline (80µg), gentamycin
(40µg), erythromycin (78µg) and enrofloxacin (10µg) (NCCLS, 2002).
The inhibition areas were read after an incubation period of 24h at 37ºC.
Table 9 shows the limits (in mm) set from which each isolate was either considered Resistant,
Intermediate or Sensitive. Figures 8 and 9 show typical antibiograms.
Table 9: Antibiotic setpoints used
Classification
Antibiotic
Resistant(mm) Intermediate(mm) Sensitive(mm)
Ampicillin
<=24
>24; <27
>27
Ceftiofur
<=22
>22 ; <25
>25
Trimethoprim+Sulfamethoxazole <=22
>22; <27
>27
Erythromycin
<=22
>22, <26
>26
Tetracycline
<=24
>24; <27
>27
Gentamicin
<=24
>24; <27
>27
Enrofloxacin
<=22
>22 ; <25
>25
(Commissie Richtlijnen Gevoeligheidsbepalingen (CRG), 2000)
50
Figure 8: Antibiogram results for an Enterobacteriaceae isolate showing resistance to
ceftiofur, tetracycline, trimethopim+sulfa, erythromycin, enrofloxacin and ampicillin.
Figure 9: Antibiogram results for an Enterobacteriaceae isolate showing resistance to
ceftiofur, trimethoprim+sulfa, ampicillin and intermediate resistance to erythromycin
51
2.4 Statistical analysis
Statistical analysis was performed using SPSS (SPSS© 16.0 for Windows, 2007). The
comparison between different steps in the slaughterhouse was done by model adjustment of
an ANOVA. A Chi-square for antiobioresistance slaughterhouse steps and sampling day
(flock) variable comparison was also performed using the same tool (SPSS© 16.0 for
Windows, 2007).
3. Results
3.1. Enumeration of Enterobacteriaceae
The cloacal samples of Flocks D and E had a log10 mean of Enterobacteriaceae Colony
Forming Units (CFU) per swab of 7.81±0.58 and 8.17±0.30, respectively. The results are
shown in Figure 10.
The mean Enterobacteriaceae counts for the samples taken before evisceration (expressed in
log10 CFU/g of neck skin) were 6.18±0.47 for Flocks B (10-12-07), 7.60±0.36 for Flocks D
(18-03-08) and 8.39±0.17 for Flocks E (09-04-07).The mean of the Enterobacteriaceae counts
for all carcasses before evisceration was 7.37±0.88 log10 CFU/g.
Figure 10 : Mean Enterobacteriaceae plate counts in log10 CFU/swab in live animal samples
(n=30) from Flocks D and E
The mean Enterobacteriaceae counts for the after washing step (expressed in log10 CFU/g of
neck skin) were 6.36±0.77 for Flocks A (25-11-07), 7.01±0.27 for Flocks B (10-12-07),
52
8.00±0.24 for Flocks D (18-03-08) and 7.10±0.20 for Flocks E (09-04-07).The mean of the
Enterobacteriaceae counts for all samples taken at after washing step was 6.96±0.77 log10
CFU/g.
Table 10 shows the mean Enterobacteriaceae counts per sampling step. Means with different
letters were statistically different (p <0.05). The letters “a” and “b” between parentheses show
which Enterobacteriaceae counts were statistically different, with “a” having the highest
counts and “b” the lowest in comparison.
Statistical differences were found between Enterobacteriaceae counts from before evisceration
samples taken from Flocks B and D (letter “b” in Table 10) and between Enterobacteriaceae
counts taken from the after washing step samples in the same flocks (letter “a” in Table 10).
The after washing samples for Flocks B and D had higher Enterobacteriaceae counts. The
after washing/before chilling Enterobacteriaceae counts taken from Flocks E (letter “b” in
Table 10) were statistically different and lower than those from samples taken before
evisceration (letter “a” in Table 10). Considering all the mean Enterobacteriaceae counts per
step, the samples before evisceration show higher Enterobacteriaceae counts than those taken
after washing.
Table 10: Enterobacteriaceae plate counts in log10 CFU/g from broiler carcass per sampling
step.
Step
Sampling day
Mean
SD
Before evisceration
B(10-12-2007) 6,18 (b) ±0,47
D(18-03-2008) 7,60 (b) ±0,36
E(09-04-2008) 8,39 (a) ±0,17
Total
7,37
±0,88
After washing/before chilling A(25-11-2007) 6,36
±0,77
B(10-12-2007) 7,01 (a) ±0,27
D(18-03-2008) 8,00 (a) ±0,24
E(09-04-2008) 7,10 (b) ±0,20
Total
53
6,96
±0,77
The mean Enterobacteriaceae counts (expressed in log10 CFU/g of neck skin) were 6.36±0.77
for Flocks A (25-11-07), 6.59±0.57 for Flocks B(10-12-07), 7.75±0.37 for Flocks D (18-0308) and 7.47±0.62 for Flocks E (09-04-07) (Figure 11). The mean Enterobacteriaceae counts
for all broiler carcass samples were 7.37±0.88 log10 CFU/g. The letters “a” and “b” between
parentheses show which Enterobacteriaceae counts were statistically different. Means with
different letters were statistically different (p <0.05). There is a significant difference between
samples collected on 25-11-07 and on the 10-12-2007 (Flocks A and B) and the ones
collected in the last two days (Flocks D and E).
Figure 11: Mean number of Enterobacteriaceae plate counts in neck skins from poultry
carcasses, per Flocks.
In Table 11, the F value for the variable “step” is 0.013 and the significance is 0.91 (above
0.05) and therefore there are no significant differences between the samples in what concerns
the step in which they were taken. The F value for the variable “Sampling day/ Flocks” is
45.975 and the significance is 0.00, which implies that there is a statistical difference between
the samples collected during different days, corresponding to different flocks. When the
variable “step” is combined wih the variable “sampling day” (shown in the table as
“Step*Sampling day”) there are statistical differences between the samples (Sig=0.00,
F=24.56).
54
Table 11: Test between subjects effects for mean Enterobacteriaceae plate counts in log10
CFU/g
Test between subjects effects
Enterobacteriaceae classic log10 CFU/g
Step
Sampling day/ Flocks
Step*Sampling day/Flocks
df F
1 0,013
3 45,975
2 24,566
Sig.
0,91
0,00
0,00
Table 12 shows the results for pairwise comparisons done with SPSS (SPSS© 16.0 for
Windows, 2007) between different steps. In this table, the significance level shown is below
0.05 (Sig.=0.024) which indicates that there is a statistical difference between the samples
collected in the before evisceration and after washing steps.
Table 12: Pairwise comparisons for mean Enterobacteriaceae plate counts in log10 CFU/g
according to slaughter step were samples were collected
Pairwise comparisons
I
Before
evisceration
After
washing
J
After
washing
Before
evisceration
Mean
difference
(I-J)
Std.
Error
Sig.
F
0,268
0,116
0,024
-0,268
0,116
0,024
5,322
3.2. Enumeration of E. coli
The E. coli plate counts are shown in Table 13. In Flocks A carcass contamination with E.
coli was 8.66±1.67 log10 CFU/g. In Flocks B, for carcasses collected before evisceration the
E. coli plate count was 6.02±0.54 log10 CFU/g and in samples collected before chilling it was
6.81±0.26 log10 CFU/g.
Table 13: E. coli plate counts in log10 CFU/g of neck skin from broiler carcasses.
Flocks
Step
log10 CFU/g
SD
A
After washing/before chilling
8,66
±1,67
B
Before evisceration
6,02
±0,54
B
After washing/before chilling
6,81
±0,26
55
3.3. Detection of Salmonella spp.
The isolates selected by biochemical tests as presumptive Salmonella (n=210) were confirmed
by serology only in Flocks A and B. The data presented will concern only Salmonella
confirmed by serology (n=22). The identified isolates were all Salmonella serotype
Typhimurium (Group B).
Flocks A were positive for Salmonella in 55% of the total amount of broiler carcass samples
(n=20) collected after washing/before chilling. Flocks B were positive for Salmonella in
62.5% of the total amount of samples collected before evisceration (n=8) and in 25% of the
total amount of samples (n=8) collected after washing/before chilling. The carcasses sampled
from Flocks B (10-12-07) were positive for Salmonella in 43.8% of the cases. In different
sample collecting steps, the percentage of Salmonella for the samples collected before
evisceration (n=8), the percentage is 62.5% and for the samples collected after washing/before
chilling (n=28), the percentage is 52.5%. From all the collected samples (n=36), the
percentage of Salmonella positive samples was 49.4%.
Figure 12 shows the percentage of Salmonella per sample in each sampling day and in Figure
13, the percentage of positive isolates per sampling step is shown.
Table 14 shows the number of positive samples for Salmonella found on each of the carcass
sampling groups, for each step.
Figure 12: Percentage of Salmonella per
broiler carcasses samples per day
Figure 13 : Percentage of Salmonella in
samples from each step in the slaughter
process.
56
Table 14: Number of Salmonella in each sample group per sampling step and per flocks and
percentage of Salmonella isolates in each step and each Flocks A and B.
Sampling times Step
Number of samples Salmonella %
Flocks A
After washing/before chilling 20
11
55,0
Flocks B
Before evisceration
8
5
62,5
After washing/before chilling 8
2
25,0
Total
7
43,8
16
3.4. Enterobacteriaceae Isolation and Identification
The results for the 25 Enterotube tests performed are shown in Table 15. Eight different
Enterobacteriaceae strains were identified, with E. coli being the most commonly found.
Table 16 and Table 17 show the results of the biochemical tests previously described in the
same strains that were tested with the Enterotubes.
Table 15: Enterotube results for Enterobacteriaceae.
Species
Nº of identified isolates
Enterobacter cloacae
2
Serratia liquefaciens
1
Citrobacter freundii
1
Hafnia alvei
1
Pantoea agglomerans
1
Eschericia coli
12
Enterobacter aerogenes
1
Salmonella sp.subs. Cholerasuis 6
Table 16: Biochemical reaction results for the Salmonella isolates identified through
Enterotubes.
Bacterial
isolate
Salmonella
sp.subs.
Cholerasuis
TSI
Glucose
fermentation
and production
of H2O
LDC
Urea
Agar
Positive Negative
57
βgalactosidase
Negative
Indole
VP test
Negative
Negative
Table 17: Biochemical reaction results for Enterobacteriaceae isolates identified through
Enterotubes.
Bacterial isolate
TSI
Brilliant Green
Bile Broth
Eschericia coli
Sugar fermentation with production of CO2 Positive
Positive
Serratia
liquefaciens
No sugar fermentation
Negative
Citrobacter
freundii
Sugar fermentation with production of CO2 Positive
Negative
Hafnia alvei
Sugar fermentation with production of CO2 Negative
Negative
Pantoea
agglomerans
Sugar fermentation with production of CO2 Negative
Negative
Enterobacter
cloacae
Sugar fermentation with production of CO2 Negative
Negative
Enterobacter
aerogenes
Sugar fermentation with production of CO2 Negative
Negative
Negative
Indole
3.5. Results of the enumeration of Extended Spectrum β-Lactamase positive
Enterobacteriaceae
Selective media for antibioresistant Enterobacteriaceae was used in two flocks (or sampling
days) and the results for the enumeration of these bacteria are shown in Table 18.
Figure 14 compares the results of the selective media with the results from the
Enterobacteriaceae and E. coli plate counts. The ESBL positive E. coli plate counts in
carcasses before the evisceration step taken from Flocks B were 4.06±0.3 log10 CFU/g. From
the same samples, the ESBL positive Klebsiella spp. counts were 3.51±0.48 log10 CFU/g and
for ESBL positive Proteus spp. were 1.67±1.42 log10 CFU/g. Samples from carcasses of the
same flocks, taken at the after washing step, had ESBL positive E. coli counts of 3.73±0.2
log10 CFU/g, an ESBL positive Klebsiella spp. count of 3.17 ±1.22 log10 CFU/g of sample
and an ESBL positive Proteus spp. count of 1.61±1.35 log10 CFU/g.
Samples taken from live animals from Flocks E had ESBL positive E. coli counts of
3.56±0.74 log10 CFU/g, an ESBL positive Klebsiella spp. count of 1.99±1.84 log10 CFU/g and
an ESBL positive Proteus spp. count of 0.15±0.59 log10 CFU/g. The ESBL positive E. coli
plate counts in the before evisceration samples carcasses taken from Flocks E was 3.81±0.56
log10 CFU/g. From the same samples, the ESBL positive Klebsiella spp. count was 3.15±1.13
log10 CFU/g and for ESBL positive Proteus spp.the plate counts were 0.82±1.26 log10 CFU/g.
58
Samples from carcasses taken from the after washing step from the same flocks had ESBL
positive E. coli counts of 0.57±0.99 log10 CFU/g. In this sample, no ESBL positive Klebsiella
spp. or ESBL positive Proteus spp were detected by this plate count method.
Table 18: Number of extended spectrum β-lactamase Enterobacteriaceae on carcasses (log10
CFU/g), per day (Flocks) in different slaughter steps.
Flocks Step
B
B
E
E
E
Before
evisceration
After
washing
Live
animals
Before
evisceration
After
washing
E. coli SD
ESBL+
Klebsiella SD
spp.
ESBL+
Proteus SD
spp.
ESBL +
4,06 ±0,30
3,51 ±0,48
1,67 ±1,42
3,73 ±0,20
3,17 ±1,22
1,61 ±1,35
3,56 ±0,74
1,99 ±1,84
0,15 ±0,59
3,81 ±0,56
3,15 ±1,13
0,82 ±1,26
0,57 ±0,99
- -
- -
Figure 14: Enterobacteriaceae, E. coli and β-lactamase positive Enterobacteriaceae in poultry
carcasses of different flocks.
59
3.6. Determination of Antibioresistence in Enterobacteriaceae isolates
3.6.1. Enterobacteriaceae isolates per sampling step
Enterobacteriaceae isolates from cloacal samples (n=77), showed highest resistance to
erythromycin, with 97.4% resistant and 1.3% intermediate resistant isolates. Ampicillin
resistance was detected in 79.2% of the isolates (slightly lower than for the total
Enterobacteriaceae isolates). Tetracyclin and trimethopim+sulfamethoxazole had the same
resistant rates for this sampling group (68.8%). Isolates from this sampling group had
intermediate resistance to trimpethoprim+sulfamethoxazole in 2.6% of the cases. In this
sampling group, 42.9% of the isolates were resistant to enrofloxacin and 10.4% were
intermediate resistant isolates. The antibiotics with the lowest resistance incidence were
gentamicin and ceftiofur, respectively with 15.6% and with 37.7%. Intermediate resistance
incidence to these antibiotics was 9.1% for ceftiofur and 23.4% for gentamicin. Figure 15
shows these results and Table 19 presents the antibiogram results with the number of isolates
in each classification.
Figure 15: Percentage of resistant and Table 19: Number of resistant(R), intermediate(I)
intermediate Enterobacteriaceae isolates and sensitive (S) Enterobacteriaceae isolates from
from live animal samples for each antibiotic live animal samples (n=77) for each antitiobiotic
Live animal samples (n=77)
Amp Cef TrSu Ery
R 61
29
53
75
I 0
7
0
1
S 16
41
24
1
Tet
53
2
22
Gen
12
18
47
Enr
33
8
36
Considering the Enterobacteriaceae isolates from carcass samples taken before evisceration
(n=210), erythromycin is yet again the antibiotic to which less isolates are sensitive to (93.8%
resistant and 2.9% intermediate). From this sampling group 80% of the isolates present
resistance to ampicillin and 1.9% have an intermediate response to this antibiotic. Resistance
60
to trimethoprim+sulfa was present in 52.4% of the collected strains. Ceftiofur and tetracycline
show similar resistance results (63.3% and 68.1%, correspondingly), with intermediate
resistance in 4.3% of the cases for ceftiofur and in 6.7% of the cases for tetracycline.
Resistance to enrofloxacin and to gentamicin was again the lowest with an incidence of 7.6%
and 19.5%, respectively. Isolates with intermediate response were found in 22.4% of the cases
for gentamicin and 13.8% for enrofloxacin. Figure 16 shows the above mentioned
percentages. The numerical results for this sampling group are shown in Table 20.
The numerical results of isolates from carcasses collected before chilling (or after washing)
(n=49) are also presented in Table 20 and the percentages are presented in Figure 17.
Erythromycin maintains its position as the least effective antibiotic, with 93.9% of the strains
showing resistance to it and 6.1% presenting intermediate resistance. Ampicillin was the
second least effective antibiotic with 91.8% of the isolates being resistant to it and 2% of
these isolates showing intermediate resistance. Ceftiofur and tetracycline continued to show
similar resistance trends. In this sampling site, 44.9% of the recovered isolates were resistant
to ceftiofur and 42.9% were resistant to tetracycline. Intermediate resistance to ceftiofur was
present in 8.2% of these strains. For tetracycline this value was 10.2%. Like in the previously
analyzed sampling groups, the antibiotics with to which less resistance was shown were
gentamicin and enrofloxacin. The first had a resistance rate of 6.1% and for the second this
rate was 16.3%. Intermediate resistance to these antibiotics was shown in 12.2% and in 16.3%
of the cases, respectively. The incidence of resistance to trimethoprim+sulfa was 53.1% and
6.1% of them had intermediate resistance.
Table 20 : Number of Resistant, Intermediate and Sensitive Enterobacteriaceae isolates from
broiler carcasses in each sampling step
Slaughter step
Before
evisceration
(n=210)
After washing
(n=49)
Resistant
Intermediate
Sensitive
Amp
168
4
38
Cef
133
9
68
Tr+Su
110
0
100
Ery
197
6
7
Tet
143
14
53
Gen
16
47
147
Enr
41
29
140
Resistant
Intermediate
Sensitive
45
1
3
22
4
23
26
3
20
46
3
0
21
5
23
3
6
40
8
8
33
61
Figure 16: Percentage of resistant and Figure 17 : Percentage of resistant and
intermediate Enterobacteriaceae isolates from intermediate Enterobacteriaceae isolates from
before evisceration samples for each before chilling samples for each antibiotic
antibiotic
The results for the chi-square test relating to the variable “step” are shown in Table 21.
Considering that a statistically significant result has a probability of less than 0.05, all the
results bellow this value should be read as significant. Ampicillin incidence was related to the
variable “step” with a probability of 0.005 (Pearson Chi-square of 25.354). The significance
of
the
chi-square
value
for
ceftiofur
(Pearson
Chi-square=
47.613),
for
trimethoprim+sulfamethoxazole (Pearson Chi-square=71.684), tetracycline (Pearson Chisquare=43.771) and enrofloxacin (Pearson Chi-square=70.405) in relation to the “step”
variable was p<0.001. The Pearson Chi-square results for erythromycin in relation to the
variable “step” were not statistically different (Pearson Chi-square=10.507) showing a
significance value of 0.397. Gentamicin resistance incidence was also not statistically
different not depending on the step the samples were taken (Pearson Chi-square=11.806,
Sig.=0.298).
Table 21 Chi-square tests relating the variable “step” to the incidence of resistance to the
different antibiotics in all the Enterobacteriaceae isolates.
Antibiotic
Pearson Chi-Square
Amp
Cef
Tr+Su
Ery
Tet
Gen
Enr
25,354a
47,613a
71,684a
10,507a
43,771a
11,806a
70,405a
62
Asymp. Sig. (2sided)
0,005
0
0
0,397
0
0,298
0
In Figure 18, the percentage of resistant and intermediate Enterobacteriaceae isolates per
antibiotic from different slaughter step samples is shown. The bars corresponding to the
percentages of isolates with intermediate resistance have a more fading colour than the bars
corresponding to the percentages of resistant isolates. The percentages shown in Figure 18
were already discussed in the previous section.
Figure 18: Percentage of Resistant (darker colours) and Intermediate (lighter colours)
Enterobacteriaceae isolates per antibiotic from different slaughter step samples.
3.6.2 .Enterobacteriaceae isolates per sampling day (flocks)
For the Flocks A (25-11-2007), the percentage of ampicillin resistant Enterobacteriaceae
isolates was 67.7% and the incidence of intermediate resistance among the same isolates was
2.9%. In relation to ceftiofur, the percentage of resistant Enterobacteriaceae isolates was
56.8% and the incidence of intermediate resistance among the same isolates was 3.8%. The
incidence of Enterobacteriaceae isolates resistant to trimethoprim+sulfamethoxazole was
29.7%. The percentage of erythromycin resistant Enterobacteriaceae isolates was 91% and the
63
incidence of intermediate resistance among the same isolates was 3 %. The percentage of
tetracycline resistant Enterobacteriaceae isolates was 57.7% and the incidence of intermediate
resistance among the same isolates was 8.6%.
The percentage of gentamicin resistant Enterobacteriaceae isolates was 5.4% and the
incidence of intermediate resistance among the same isolates was 19%. Finally for
enrofloxacin, resistance was only present in 2.7% of the Enterobacteriaceae isolates and
intermediate resistance was shown in 8.1% of the Enterobacteriaceae isolates.
For the Flocks B (10-12-2007), the percentage of ampicillin resistant Enterobacteriaceae
isolates was 98% and the incidence of intermediate resistance among the same isolates was
1%. In relation to ceftiofur, the percentage of resistant Enterobacteriaceae isolates was 84.3%
and the incidence of intermediate resistance among the same isolates was 2.2%. The incidence
of Enterobacteriaceae isolates resistant to trimethoprim+sulfamethoxazole was 43.1%. The
percentage of erythromycin resistant Enterobacteriaceae isolates was 94.1% and the incidence
of intermediate resistance among the same isolates was 3.1 %. The percentage of tetracycline
resistant Enterobacteriaceae isolates was 45.1%. The percentage of gentamicin resistant
Enterobacteriaceae isolates was 17.6% and the incidence of intermediate resistance among the
same isolates was 36.9%. Finally for enrofloxacin, resistance was present in 39.2% of the
Enterobacteriaceae isolates and intermediate resistance was shown in 5.7% of the
Enterobacteriaceae isolates.
For the Flocks C (03-03-2008), the percentage of ampicillin resistant Enterobacteriaceae
isolates was 85.9%. In relation to ceftiofur, the percentage of resistant Enterobacteriaceae was
69.2% and the incidence of intermediate resistance among the same isolates was 6.4%. The
incidence of Enterobacteriaceae resistant to trimethoprim+sulfamethoxazole was 85.9% and
the percentage of intermediate resistance was 1%. The percentage of erythromycin resistant
Enterobacteriaceae was 100%. The percentage of tetracycline resistant Enterobacteriaceae
was 94.9% and the percentage of intermediate resistant isolates was 3%. The percentage of
gentamicin resistant Enterobacteriaceae was 5.1% and the incidence of intermediate resistance
among the same isolates was 8.8%. Finally for enrofloxacin, resistance was present in 37.1%
of the Enterobacteriaceae and intermediate resistance was shown in 23.2% of the same
isolates.
For the Flocks D (18-03-2008), the percentage of ampicillin resistant Enterobacteriaceae
isolates was 80.9% and the incidence of intermediate resistance among the same isolates was
1.1%. In relation to ceftiofur, the percentage of resistant Enterobacteriaceae isolates was
21.3% and the incidence of intermediate resistance among the same isolates was 1.7%. The
64
incidence of Enterobacteriaceae isolates resistant to trimethoprim+sulfamethoxazole was
72.3% and incidence of intermediate resistance was 1.2%. The percentage of erythromycin
resistant Enterobacteriaceae isolates was 95.7% and the incidence of intermediate resistance
among the same isolates was 1%. The percentage of tetracycline resistant Enterobacteriaceae
isolates was 55.3% and the percentage of intermediate resistance isolates was 6.6%. The
percentage of gentamicin resistant Enterobacteriaceae isolates was 2.1% and the incidence of
intermediate resistance among the same isolates was 4.2%. Finally for enrofloxacin,
resistance was present in 23.4% of the Enterobacteriaceae isolates and intermediate resistance
was shown in 10.1% of the Enterobacteriaceae isolates.
For the Flocks E (09-04-2008), the percentage of ampicillin resistant Enterobacteriaceae
isolates was 88.2%. In relation to ceftiofur, the percentage of resistant Enterobacteriaceae
isolates was 54.9% and the incidence of intermediate resistance among the same isolates was
10.9%.
The
incidence
of
Enterobacteriaceae
isolates
resistant
to
trimethoprim+sulfamethoxazole was 73.5% and incidence of intermediate resistance was 2%.
The percentage of erythromycin resistant Enterobacteriaceae isolates was 97.1% and the
incidence of intermediate resistance among the same isolates was 3%. The percentage of
tetracycline resistant Enterobacteriaceae isolates was 72.5% and the percentage of
intermediate resistance isolates was 6%.
The percentage of gentamicin resistant Enterobacteriaceae isolates was 13.7% and the
incidence of intermediate resistance among the same isolates was 26.5%. Finally for
enrofloxacin, resistance was present in 39.2% of the Enterobacteriaceae isolates and
intermediate resistance was shown in 16.8% of the same isolates.
The numerical results per sampling day are shown in Table 22 and the percentages of resistant
and intermediate resistant isolate per sampling day are presented in Figure 19.
Table 22: Number of Resistant, Intermediate and Sensitive Enterobacteriaceae isolates from
poultry samples in each day (or flocks)
Flocks
Classification Amp Cef Tr+Su Ery Tet Gen Enr
A
Resistant
75
63
33
101 64 6
3
(25-11-07)
Intermediate
3
4
0
3
9
9
Sensitive
33
44
78
7
38 84
65
21
99
B
Resistant
50
43
22
48
23 9
20
(10-12-07)
Intermediate
1
2
0
3
0
4
Sensitive
0
6
29
0
28 20
27
Resistant
67
54
67
78
74 4
29
Intermediate
0
6
1
0
3
7
20
Sensitive
11
18
10
0
1
67
29
D
Resistant
38
10
34
45
26 1
11
(18-03-08)
Intermediate
1
1
1
1
5
6
Sensitive
8
36
12
1
16 44
30
E
Resistant
90
56
75
99
74 14
40
(09-04-08)
Intermediate
0
11
2
3
6
27
17
Sensitive
12
35
25
0
22 61
45
C
(03-03-2007)
22
2
Figure 19: Percentage of Resistant (darker colours) and Intermediate (lighter colours)
Enterobacteriaceae isolates per antibiotic per sampling day.
66
The results for the chi-square test relating to the variable “Flocks” are shown in Table 23.
Considering that a statistically significant result has a probability of less than 0.05, all the
results bellow this value should be read as significant. Differences in incidence of ampicillin
resistance were related to the variable “Flocks” with a probability of 0.000 (Pearson Chisquare of 31.443). The significance of the chi-square value for ceftiofur (Pearson Chisquare=59.730), for trimethoprim+sulfamethoxazole (Pearson Chi-square=88.393), for
tetracycline (Pearson Chi-square=59.757), for gentamicin (Pearson Chi-square=53.689) and
for enrofloxacin (Pearson Chi-square=76.200) in relation to the “Flocks” variable was
p<0.001. The Pearson Chi-square results for erythromycin in relation to the variable “Flocks”
was also statistically different (Pearson Chi-square=19.217) showing a significance value of
p<0.01.
Table 23: Chi-square tests relating the variable “Flocks” to the incidence of resistance to the
different antibiotics in all the Enterobacteriaceae isolates.
Antibiotic
Amp
Cef
Tr+Su
Ery
Tet
Gen
Enr
N of Valid Cases
389
389
389
389
389
389
389
Pearson Chi-Square
31,443a
59,730a
88,393a
19,217a
59,757a
53,689a
76,200
Asymp. Sig. (2-sided)
0
0
0
0,014
0
0
0
For the total Enterobacteriaceae isolates the incidence of ampicillin resistance was 82.3% and
of intermediate resistance was 3.3%. In the Enterobacteriaceae isolates the incidence of
resistance to ceftiofur was 57.3% and the incidence of intermediate resistance was 11.2%. The
incidence of resistance to trimethoprim+sulfamethoxazole was 60.2% and the incidence of
intermediate resistance was 1.9% in the same group of isolates. For the total
Enterobacteriaceae isolates the incidence of erythromycin resistance was 95.4% and of
intermediate resistance it was 8.8%. In the Enterobacteriaceae isolates the incidence of
resistance to tetracycline was 68.6% and the incidence of intermediate resistance was 12.1%.
The incidence of resistance to gentamicin was 8.5% and the incidence of intermediate
resistance was 21.4% in the same group of isolates. Finally, for enrofloxacin the incidence of
resistance was 26.2% in all the Enterobacteriaceae isolates, with 18.8% of the isolates
showing intermediate resistance.
In Table 24, a summary of the number of resistant, intermediate and sensitive isolates is
shown for Enterobacteriaceae. The percentage of Enterobacteriaceae is shown in Figure 20.
67
Figure 20: Percentage of Resistant and Intermediate Enterobacteriaceae isolates in broiler
carcass samples for each antibiotic
Table 24 : Number of Resistant, Intermediate and Sensitive Enterobacteriaceae from broiler
carcass samples.
Classification Amp Cef Tri+Su Ery Tet
Group
Total
Enterobacteriaceae
isolates Resistant
Gen Enr
320
223 234
371 267 33
102
Intermediate
5
25
10
23
78
59
Sensitive
64
141 151
8
99
278
228
(n=389)
4
3.6.3. Salmonella isolates per step
For the Salmonella collected before evisceration, the percentage of ampicillin resistant
Salmonella isolates was 100%. In relation to ceftiofur and to trimethoprim+sulfamethoxazole,
the percentage of resistant Salmonella isolates was 66.7%. The incidence of Salmonella
isolates resistant to erythromycin and tetracycline was 100%. Salmonella isolates collected
before evisceration were resistant to gentamicin in 33% of the cases, presenting an
intermediate resistance incidence of 50%. Finally for enrofloxacin, resistance was present in
68
50% of the Salmonella isolates and intermediate resistance was shown in 50% of the
Salmonella isolates.
For the after washing/before chilling step, the percentage of ampicillin resistant Salmonella
isolates was 100%. The percentage of resistant Salmonella isolates to ceftiofur was also
81.3% and with intermediate resistance to the same antibiotic was 6.3%. The incidence of
Salmonella isolates resistant to trimethoprim+sulfamethoxazole was 93.8%. The percentage
of erythromycin and tetracycline resistant Salmonella isolates was 100% for this sampling
step. No Salmonella isolates showed resistance to gentamicin in the samples collected after
washing/before chilling, but 6.3% of the Salmonella isolates showed intermediate resistance.
Finally for enrofloxacin, intermediate resistance was present in 18.8% of the Salmonella
isolates. In Figure 21, the percentage of resistant and intermediate isolates for each sampling
step is shown. Table 25 presents the number of Salmonella isolates that showed resistance or
intermediate resistance to each tested antibiotic per sampling step.
Figure 21: Percentage of Resistant and Intermediate resistant Salmonella isolates in the two
different sampling steps.
69
Table 25: Number of Resistant, Intermediate resistant and sensitive Salmonella isolates to
each tested antibiotic per sampling step
Amp Cef
Before evisceration
(n=6)
After washing/before
chilling
(n=16)
Resistant
Intermediate
Sensitive
Resistant
Intermediate
Sensitive
6
0
0
16
0
0
Tr+Su Ery
4
0
2
13
1
2
4
0
2
15
0
1
Tet
6
0
0
16
0
0
Gen
6
0
0
16
0
0
Enr
2
3
1
0
1
15
3
3
0
0
3
13
3.6.4. Salmonella isolates per Flocks (sampling day)
For the Flocks A (25-11-2007), the percentage of ampicillin resistant Salmonella isolates was
100%. In relation to ceftiofur, the percentage of resistant Salmonella isolates was 84.6%. The
incidence of Salmonella isolates resistant to trimethoprim+sulfamethoxazole, to erythromycin
and tetracycline was 100%. Salmonella isolates collected in Flocks A were not resistant to
gentamicin, presenting an intermediate resistance incidence of 7.7%. Finally for enrofloxacin,
resistance was present in 0% of the Salmonella isolates and intermediate resistance was
shown in 23.1% of the Salmonella isolates.
For the Flocks B (10-12-2007), the percentage of ampicillin resistant Salmonella isolates was
100%. The percentage of resistant Salmonella isolates to ceftiofur was also 100%. The
incidence of Salmonella isolates resistant to trimethoprim+sulfamethoxazole was 33.3%. The
percentage of erythromycin resistant Salmonella isolates was 100%. The percentage of
tetracycline resistant Salmonella isolates was 33.3%. The percentage of gentamicin resistant
Salmonella isolates was the same as for the previously refered antibiotic (33.3%), with 44.4%
intermediate resistant isolates. Finally for enrofloxacin, resistance was present in 44.4% of the
Salmonella isolates.
In Table 26, a summary of the number of resistant, intermediate and sensitive Salmonella
isolates per sampling day is shown.
Table 26: Numbers of Resistant, Intermediate and Sensitive isolates for all the Salmonella
isolates in all sampling days.
Flocks A
(25-11-07)
(n=13)
Flocks B
(10-12-07)
(n=9)
Amp
Resistant
13
Intermediate 0
Sensitive
0
Resistant
9
Intermediate 0
Sensitive
0
70
Cef
11
0
2
9
0
0
Tr+Su
13
0
0
3
0
6
Ery
13
0
0
9
0
0
Tet
13
0
0
3
0
6
Gen
0
1
12
3
4
2
Enr
0
3
10
4
0
5
Figure 22 : Percentage of Resistant and Intermediate resistant Salmonella isolates per
sampling day.
From all the Salmonella isolates, 100% showed resistance to ampicilin and erythromycin. In
relation to ceftiofur, 92.3% showed resistance. Resistance to trimethoprim+sulfamethoxazole
and to tetracycline was present in 66.7% of the isolates. Gentamicin resistance was present in
16.7% of the Salmonella isolates and intermediate resistance was showed in 26.1% of the
same isolates. The total isolated Salmonella showed resistance to enrofloxacin in 22.2% of the
cases and intermediate resistance was showed by 11.5% of the same isolates. Figure 22 also
shows the percentages for the total Salmonella isolates. The number of resistant, intermediate
and sensitive isolates is shown in Table 27.
Table 27: Number of Resistant, Intermediate and Sensitive Salmonella isolates for each tested
antibiotic
Amp Cef Tr+Su Ery Tet Gen Enr
Total Salmonella isolates Resistant
Intermediate
(n=22)
Sensitive
22
0
0
71
20
0
2
16
0
6
22 16
0 0
0 6
3
5
14
4
3
15
3.7. Antimicrobial resistance to ceftiofur and other antibiotics
As it can be seen in Figure 23, ceftiofur resistance was quite high for all the
Enterobacteriaceae isolates tested in this study (57.5%). Also, it can easily be noticed that
almost all the isolates that were resistant to ceftiofur were also resistant to other antibiotics
(57.1% were resistant to ceftiofur and other antibiotics).From all the ceftiofur resistant
isolates, almost a third (15.7% of the total) was also resistant to enrofloxacin.
For the Salmonella isolates the pattern was the same, with ceftiofur resistance being higher
(77.3%). Resistance to other antibiotic was present every time the isolate was resistant to
ceftiofur (77.3%). The resistance to enrofloxacin and ceftiofur combined was lower for the
Salmonella isolates (13.6%), but still showing that a high percentage of ceftiofur resistant
bacteria were also resistant to fluoroquinolones.
Figure 23: Percentage of Enterobacteriaceae and Salmonella isolates that were resistant to
ceftiofur, ceftiofur and other antibiotic and to ceftiofur and enrofloxacin.
72
4. Discussion of the results
4.1. Carcass contamination
The EC Reg. 1441/2007 does not specify the maximum Enterobacteriaceae counts for poultry
carcasses. Even so, when comparing the results of this study for the Enterobacteriaceae counts
for the after washing step (in which the lowest counts were 6.36 log10 CFU/g log) with other
authors, the results found on the present work were always higher than the values reported by
a similar study performed in Spain (Gonzalez-Miret, Escudero-Gilete & Heredia, 2006). This
clearly indicates that improvement in slaughter hygiene and a review of process controls are
mandatory in order to assure the safety of the meat products. The plate counts for E. coli were
also higher (always over 6 log10 CFU/g) than in a similar study performed in Belgium in 2008,
where E. coli counts were 4.05 and 5.24 log CFU/g for chicken carcasses (Ghafir; China;
Dierick; De Zutter & Daube, 2008).
There were significant differences (p<0.05) in the Enterobacteriaceae plate counts (Table 10)
according to slaughter step were carcass samples were collected, with the before evisceration
step showing higher mean Enterobacteriaceae plate counts in log10 CFU/g. However, there
was a wide variation in Enterobacteriaceae counts depending on the day (or flocks) the
samples were taken. In Flocks B and D, the highest counts were present in the samples taken
after washing and only in Flocks E the Enterobacteriaceae counts were higher before
evisceration.
It should be expected that the samples collected before evisceration had consistently higher
Enterobacteriaceae counts than those collected after washing, due to the high crosscontamination that can occur in the steps that precede evisceration (such as defeathering)
(Geornaras & Von Holy, 2000). According to other authors (Geornaras & Von Holy, 2000;
Gonzalez-Miret et al., 2006), the washing procedure is considered an important way of
reducing carcass contamination, resulting in lower Enterobacteriaceae counts and in this study
that was not always the case.
Significant differences were also found in relation to the Flock (sampling day), with the last
two sampling days (Flocks D and E) having higher mean Enterobacteriaceae plate counts than
the first two sampling days (Flocks A and B). These differences show that production hygiene
was insufficient throughtout the process on these specific days, probably due to deficient
control of slaughter sanitation procedures.
Salmonella isolates were detected in 62.5% of the samples taken before evisceration and in
52.5% in samples taken after washing. The decrease in the number of Salmonella isolates
73
from the before evisceration step to the after washing step maintains what was stated in the
previous section, once more emphasizing the importance of spray-washing the carcasses.
According to the most recent EFSA report (2007) the average proportion of Salmonella
positive samples in fresh broiler meat was 5.6% in EU in 2006. If this tendency is maintained
in studies to come (also by EFSA), it can be concluded that the results of present study were
higher than expected. It is very likely that these high percentages were related to deficient
hygiene practices, in which the environment leads to contamination of the carcasses with
Salmonella.
In all the tested samples (n=26) the average number of carcasses that were positive for
Salmonella in 25g was 49.4%. This high percentage of isolation in the slaughterhouse might
indicate that there has been some contamination of the slaughterhouse facilities with
Salmonella positive animals. The average percentage of Salmonella in parent-breeding flocks
for broilers in EU is 5.2% in 2006 (EFSA, 2007). The animals allegedly originated from
Germany and the Netherlands, although this fact was not confirmed. It is possible that the
animals came from Eastern European states (such as Poland) were Salmonella is more
prevalent in broiler flocks (EFSA, 2007).
Depending on the day the samples were taken, the number of Salmonella recovered isolates
varied from 55% to 43.8% in samples taken two weeks apart.
4.2. Antibioresistance screening
The data from each sampling group demonstrates that most antibiotic resistance trends were
maintained. In every sampling step, gentamicin was the most effective antibiotic and
erythromycin and ampicillin the least against Enterobacteriaceae isolates. Ceftiofur showed a
considerable variation regarding the incidence of resistance observed in isolates from cloacal
samples (37.7%) to the incidence in isolates from the before evisceration phase (63.3%).
Enrofloxacin resistance incidence also varied slightly, with 42.9% of resistant isolates from
live animal samples and only 16.3% of the isolates from the after washing/before chilling
phase. There was a smaller tetracycline resistance in the isolates collected from the after
washing/before chilling phase (42.9%), when compared to the other sampling times. Finally
for trimethoprim+sulfa resistance, the results always showed resistance levels above 50%,
with 68.8% resistant isolates in cloacal samples.
Chi-square results showed that different Flocks/sampling days lead to differences in the
antiobioresistance results of Enterobacteriaceae isolates towards all the tested antiobiotics.
74
Differences in the step where Enterobacteriaceae isolates where collected lead to different
antiobioresistance results towards all antibiotics except for erythromycin and gentamicin,
according to chi-square test results.
All the Salmonella isolates (n=22) were resistant to ampicilin and erythromycin. That was not
the case for the Enterobacteriaceae isolates as the percentage of resistant Enterobacteriaceae
to ampicillin was 82.3% and to erythromycin it was 95.4%. There was an increase in ceftiofur
resistance in Salmonella isolates when compared to the Enterobacteriaceae group (92.3% in
Salmonella compared to 57.3% in the total Enterobacteriaceae results). The incidence of
Salmonella resistance to trimethoprim/sulfamethoxazole was 66.7% and in Enterobacteriaceae
isolates resistance was shown in 60.2% of the cases. Enrofloxacin resistance was presented by
26.2% of all Enterobacteriaceae and by 22.2% of all the Salmonella isolates. Intermediate
resistance to enrofloxacin was present in 18.8% of the Enterobacteriaceae isolates and in
11.5% of the Salmonella isolates. The incidence of tetracycline resistance in Salmonella
isolates and in Enterobacteriaceae was also similar (66.7% in Salmonella compared to 68.6%
in the Enterobacteriaceae data). Gentamicin maintained as the one with the lowest rates, with
16.7% of all the Salmonella showing resistance and 26.1% of these strains having
intermediate resistance. The Salmonella isolates showed less susceptibility to this antibiotic
than the Enterobacteriaceae isolates, where resistance to gentamicin was present in 8.5% of
the isolates and intermediate resistance in 21.4% of the isolates.
The small differences (with the exception of ceftiofur) found between the two sampling
groups might be related with the small number of Salmonella isolates (n=22) that were
analysed.
A survey performed in Europe which comprised data from numerous countries, showed very
high rates of resistance towards a wide range of antibiotics (ampicillin, cefepime, cefotaxime,
ciprofloxacin,
chloramphenicol,
gentamicin,
streptomycin,
tetracycline
and
trimethoprim/sulfamethoxazole) and considerable variation between countries (Bywater et al.,
2004). In E. coli and Salmonella spp., the incidence of resistance to the older compounds such
as ampicillin and tetracycline was rather high although variable. The results of the study
performed by Bywater et al. (2004) showed that Salmonella resistance towards ampicillin was
present in 54.7% of the isolates in France and 16.1% in the Netherlands, for tetracycline
resistance the results were 52% of resistant isolates in France and 38.7% in The Netherlands.
On the other hand, resistance to newer compounds such as ciprofloxacin and cefotaxime was
low or absent. Gentamicin, although a relatively old compound, has had little use in animals
which possibly explains the lower incidence of resistance (0% in The Netherlands and 5.4%
75
in France). Even so, in France the incidence was 4.5% (E. coli) and 5.4% (Salmonella spp.).
Resistance to the combination of trimethoprim+sulfamethoxazole was present in 24% of the
Salmonella isolates from France and in 19.4% of the isolates from The Netherlands (Bywater
et al., 2004). In a study done in Denmark, ceftiofur resistance was found rarely (2.6%) and
only in imported beef, Salmonella resistance was present in poultry and swine isolates at a
much higher frequency (15-20%) (DANMAP, 2005). Overall, incidences of resistance in
Europe are lower than those found in North America (NARMS, 2004; CIPARS, 2006).
Therefore, when comparing the incidence of resistance of the isolates collected in the present
study to the results from other studies it can be noticed that the incidence of resistance was
higher than expected. This is particularly interesting when considering that the isolates were
collected in a country in Northern Europe, where typically lower incidences of resistance can
be found (Bywater et al., 2004).
5. Conclusion
The aim of this study was to evaluate the effect of slaughterhouse contamination in the
presence of antimicrobial resistant bacterial isolates, specifically Salmonella and other
members of the Enterobacteriaceae family.
The enumeration of Enterobacteriaceae from carcasses in different points in the
slaughterhouse
is
an
accurate
indication
of
contamination.
Even
though
legal
recommendations are not set for the total Enterobacteriaceae count in poultry carcasses, the
bacterial counts found in this study clearly exceed what should be expected based on other
authors. In 49.4% of the samples, collected two weeks apart from each other Salmonella spp.
was detected in 25g of sample. The EC Reg. 1441/2007 specifies that the finished product
must be totally free (absence in 25g) from Salmonella when it reaches the consumer. Even
though the samples were not taken from the post-chill carcasses it is very likely that these
same carcasses still harboured the pathogen after the chilling phase (Fries, 2002).
Therefore, it can be stated that there is a direct influence in hygiene practices in this
slaughterhouse and the quantification of such large number of Enterobacteriaceae.
The reason behind the fluctuation in the number of isolates in the slaughterhouse might be
due to several factors. The first is cross-contamination in the line, due to slaughter of infected
animals and absence of proper decontamination practices. Another point is the incorrect use
of sanitizers (insufficient concentration, exposure time) and the presence of biofilms. The
76
water used for scalding could also be a vehicle for cross-contamination, since it could have
been contaminated by infected chickens.
These heavy Enterobacteriaceae counts and detection of Salmonella explain the high
resistance pattern found in the bacterial isolates. Firstly because the contamination found in
the line shows that if a microbiological resistance determinant enters the slaughterhouse
facilities it would easily spread and diffuse to the finished product. It can also be considered
that the sanitizers used might induce resistant to these compounds and to antibiotics, since
often genetic mobile fragments are associated with these two characteristics.
Ceftiofur resistance can most likely be related to presence of an ESBL, and when in
association with resistance to other antibiotics, particularly enrofloxacin, this likelihood
increases even more. Therefore, it can be stated that the presence of ESBLs was very high
(57.5%) in the tested isolates. Multidrug resistance was present in a large number of isolates
which could indicate presence of plasmid-mediated resistance. The high number of resistant
strains found shows that control should be assured throughout the production chain in order to
prevent transmission of resistant determinants between different animals and humans as much
as possible. This study also confirms the need to further evaluate the role of food animal
bacterial isolates as reservoirs for antibiotic resistance.
6. Future work
The importance of resistance determinants derived from animals is still largely unknown.
There is a lack of research in what concerns the role of poultry-borne bacteria as vectors in
transmitting resistance, on the development of cross-resistance, and on the rates of emergence
and spread of resistance to additional drugs in common pathogens. The true impact of
antibiotic use as growth promoters is yet to be accessed, since this resistance seems to be
increasing even more, in spite of the antibiotic ban imposed in the EU. Several factors need to
be taken into account, such as poultry trade between different world countries and human
migrations. More insight is necessary on the potential links between the use of critical
antibiotics and other similar drugs in poultry production and its use in human medicine.
77
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