Biological
Analysis
th
12
EMQAL
Leonor Faleiro
University of Algarve
Faculdade de Ciências e Tecnologia
Algarve Biomedical Centre-Research Institute
mfaleiro@ualg.pt
Module Description - The Purpose or Aims:
• Introduce fundamental concepts on biological analysis
including water and food microbiology.
• Introduce standard operating procedures in microbiological
quality of water and food and other biological analysis.
•
Learning Outcomes:
At the end of the module is expected that the student will be
able to:
1. Recognize and describe several microbial agents that can
cause food spoilage, poisoning and foodborne and waterborne
infection.
2. Be able to put in place standard operating procedures to
examine food and water microbiological quality.
3. Apply the appropriate measures to prevent food poisoning
and waterborne and foodborne infections.
Summary of Course Content:
• This module introduces concepts of classical and molecular
approaches to evaluate the microbiological quality of food
and water.
• The microbial growth, intrinsic and extrinsic factors in food
spoilage, poisoning and food and water infection are
explored.
• Laboratory techniques to examine food and water
microbiological quality are described and discussed.
Evaluation
• Multiple response test
• 5 - 6 questions about the content of each session.
• Duration 25-30 minutes.
• Done on Campus virtual during a Zoom section with the link
provided by the lecturer in this same location.
• The students need to have a camera on and no headphones.
Date: 28th January or 4 th February
Foodborne infections – introductory
considerations
• No universally accepted definition of ‘safe food’.
• Because it is a relative term, which is linked to determining the
acceptable level of risk to a mixed population or maybe a specific
subgroup.
• Our food is very diverse, and to ensure it is safe requires a
systematic, proactive approach of minimising contamination from
‘farm to fork’.
• However, our food supply involves international movement of
ingredients and processed products, and therefore the ‘farm’ is
much away from the ‘fork’.
Foodborne infections – introductory
considerations
• Food preservation: refrigeration and canning
• Implementation of ‘hazard analysis and critical control point’
(HACCP): the producer anticipates the possible hazards (microbial,
chemical and others) in the final product and ensures the
processing reduces or eliminates them to an acceptable level.
• Foodborne illness can be defined as diseases commonly
transmitted through food, and comprise a broad group of illnesses
caused by microbial pathogens, parasites, chemical contaminants
and biotoxins.
• Illness due to foodborne contamination is still a major cause of
morbidity and mortality.
Foodborne infections – introductory
considerations
• food safety problems are challenging, in part because they
change over time
• we have changes in our economy, and therefore lifestyle,
eating habits (ranges of food, eating at home or eating out),
and an ageing population who are more prone to infection
and are slower to recover.
• the causative agents of foodborne illnesses are also
changing, with the emergence of previously unrecognised
emergent pathogens.
The microbial world and its relationship to food
• Classification of microorganisms has been based on morphology, and
phenotypical (biochemical) properties.
• Morphological analysis considers whether the bacterium is spherical (coccoid),
filamentous, curved or rod-shaped, and if the bacterium has a Gram-negative
or Gram-positive type cell wall
The microbial world and
its relationship to food
• Black, J.G., Black, L.J. (2015)
The microbial world and its relationship to food
Black, J.G., Black, L.J. (2015)
The microbial world and its relationship to food
• The possession of certain enzymes, e.g. 𝛽-galactosidase for lactose
fermentation, has helped in identifying and defining a general group of
bacteria associated with faecal contamination that would have come
from the colon, the origin of the old term ‘coliforms’
• Biochemical and enzymatic activities enabled cultivable organisms to
be classified.
• Inevitably defining species according to biochemical (phenotyping)
traits will be problematic, as such functions represent only a small
portion of the total coding of an organism.
• DNA sequencing has enabled a fuller understanding of the genetic
capabilities of microorganisms
Black, J.G., Black, L.J. (2015)
• Inevitably as
our
knowledge of
organisms at
the genome
level
improves, so
previous
species may
be redefined.
Figure 1.1 Phylogenetic tree of organisms associated with food microbiology and the
archaea organism Pyrococcus furiosus, based upon the DNA sequence ribosomal small
subunit, constructed using ClustalW (freeware).
Forsythe, S.J., (2020)
Relatedness of microrganisms
• The relatedness of organisms used to be determined using
laboratory-based DNA–DNA hybridisation analysis.
• The DNA was extracted from two organisms, heated to separate the two
DNA strands and then they were allowed to anneal to each other and the
extent of hybridisation measured. The common standard was that
<70% hybridisation meant the organisms were not in the same
species.
• Whereas now whole-genome sequencing (WGS) has led to the use of in
silico approaches, such as the average nucleotide identity (ANI) (Goris
et al. 2007) and can be undertaken using online tools, such as
http://enve-omics.ce.gatech.edu/ani
• Although a universal cut-off value to delineate bacterial species does not
exist, an ANI value of >95% ± 0.5% has been commonly used to indicate
that two strains are in the same species.
Diversity of hazards associated with food.
Biological
Chemical
Physical
Macrobiological
Veterinary residues: antibiotics, growth
stimulants
Glass
Microbiological
Plasticisers and packaging migration:
vinyl chloride, bisphenol A
Metal
Viruses
Chemical residues: pesticides (DDT),
cleaning fluids
Stones
Pathogenic bacteria
Allergens
Wood
Spore-forming
Toxic metals: lead, cadmium, arsenic, tin,
mercury
Plastic
Hepatitis A
Norovirus
Rotavirus
Bacillus cereus
Clostridium perfringens
Clostridium botulinum
Non-spore-forming
Campylobacter jejuni
Pathogenic strains of Escherichia coli
Listeria monocytogenes
Salmonella serovars
Food chemicals: preservatives,
processing aids
Parts of pests
Diversity of hazards associated with food.
Biological
Bacterial toxins
Chemical
Radiochemicals: 131I, 127Cs
Physical
Insulation material
Staphylococcus aureus
B. cereus
Shellfish toxins: domoic acid, okadaic
acid
Dioxins, polychlorinated biphenyls
(PCBs)
NSP, PSP
Prohibited substances
Parasites and protozoa
Printing inks
Bone
Fruit pits
Cryptosporidium parvum
Entamoeba histolytica
Giardia lamblia
Toxoplasma gondii
Fasciola hepatica
Taenia solium
Anisakis species
Trichinella spiralis
Mycotoxins: ochratoxin, aflatoxins,
fumonisins, patulin
NSP =
neurotoxic
shellfish
poison,
PSP = paralytic
shellfish
poisoning.
Origins of preservation of food
Food poisoning microorganisms
Food poisoning microorganisms are normally divided into two groups:
Infections; for example, Salmonella serovars, C. jejuni and pathogenic E. coli.
Intoxications; for example, Bacillus cereus, Staphylococcus aureus and
Clostridium botulinum.
The first group are bacteria that multiply in the human intestinal tract, whereas
the second group are bacteria that produce toxins either in the food or during
passage in the intestinal tract.
As a generalisation, bacterial infections cause gastroenteritis, whereas the
ingestion of a toxin causes vomiting.
Food poisoning microorganisms
Infections/ Intoxications
• This division is also very useful to help recognise the routes of food poisoning, since
bacterial infection will be due to something ingested in the previous 18–24 h, whereas vomiting
is likely to be due to preformed toxin ingested in the past few hours.
• Gastroenteritis with fever could be due to a Gram-negative infectious organism, as the host's
immune system responds to the bacterial outer membrane, which will be composed of
lipopolysaccharide (LPS);
• LPS can be very pyrogenic in some Gram-negative bacteria;
• Viral infections cause both vomiting and gastroenteritis;
• Vegetative cells are killed by heat treatment, whereas endospores (e.g., produced by B. cereus
and Clostridium perfringens) may survive and hence germinate if the food is not kept
sufficiently hot or cold.
• Another type of grouping is according to severity of illness. This approach is useful in setting
microbiological criteria (sampling plans) and risk analysis.
Sources of foodborne pathogens.
Food
Meat, poultry and eggs
Pathogen
Incidence (%)
Arcobacter species
Campylobacter jejuni
Raw chicken and turkey (45–64)
Salmonella serovars
Raw poultry (40–100), pork (3–20), eggs (0.1%)
and shellfish (16)
Staphylococcus aureusa
Raw chicken (73), pork (13–33) and beef (16)
Clostridium perfringensb
Raw pork and chicken (39–45)
Clostridium botulinum
Escherichia coli O157:H7
Raw beef, pork and poultry
Bacillus cereusb
Raw ground beef (43–63), cooked meat (22)
Listeria monocytogenes
Red meat (75), ground beef (95)
Yersinia enterocolitica
Raw pork (48–49)
Hepatitis A virus
Trichinella spiralis
Tapeworms
Sources of foodborne pathogens.
Food
Fruit and vegetables
Pathogen
Incidence (%)
C. jejuni
Mushrooms (2)
Salmonella serovar
Artichoke (12), cabbage (17), fennel (72), spinach (5)
St. aureusa
Lettuce (14), parsley (8), radish (37)
L. monocytogenes
Potatoes (27), radishes (37), bean sprouts (85), cabbage
(2), cucumber (80)
Shigella spp.
E. coli O157:H7
Celery (18) and coriander (20)
Y. enterocolitica
Vegetables (46)
Aeromonas hydrophila
Broccoli (31)
Hepatitis A virus
Norovirus
Giardia lamblia
Cryptosporidium spp.
Cl. botulinum
B. cereusb
Mycotoxins
Sources of foodborne pathogens.
Food
Pathogen
Milk and dairy products
Salmonella serovars
Incidence (%)
Y. enterocolitica
Milk (48–49)
L. monocytogenes
Soft cheese and pâté (4–5)
E. coli
C. jejuni
Shigella spp.
Hepatitis A virus
Norovirus
St. aureusa
Cl. perfringensb
B. cereusb
Mycotoxins
Powdered infant formula
Salmonella serovars,
Cronobacter species
Pasteurised milk (2–35), milk powder (15–75) cream (5–
11), ice cream (20–35)
Sources of foodborne pathogens
Food
Pathogen
Cereals, grains,
legumes and nuts
Salmonella serovars
Incidence (%)
L. monocytogenes
Shigella species
E. coli
St. aureusa
Cl. botulinumb
B. cereusb
Mycotoxinsa
Raw barley (62–100), boiled rice (10–93), fried rice (12–
86)
Sources of foodborne pathogens
Food
Pathogen
Spices
Salmonella serovars
Incidence (%)
St. aureusa
Cl. perfringensb
Cl. botulinumb
B. cereusb
Water
G. lamblia
Water (30)
Vibrio cholerae
Cronobacter spp.
Toxin not destroyed by pasteurisation.
b Spore-forming organism. Not killed by pasteurisation.
a
Incubation period and duration of common food poisoning microorganisms.
Microorganism
Aeromonas species
Bacillus cereus emetic
diarrhoeal
Campylobacter jejuni
Incubation period
Duration of illness
Unknown
1–7 days
0.5–6 hours
8–24 hours
3–5 days
1 day
1 day
2–10 days
Clostridium botulinum
12–36 hours
2 h–14 days
Clostridium perfringens
8–12 hours
1–14 days
Escherichia coli ETEC
16–72 hours
3–5 days
E. coli EPEC
16–48 hours
2–7 days
E. coli EHEC
Listeria monocytogenes
72–120 hours
2–12 days
3–70 days
Variable
Salmonella serovars
16–72 hours
2–7 days
Staphylococcus aureus
Few hours
2–3 days
Vibrio cholerae
6 hours – 3 days
1–6 days
Norovirus
24–48 hours
1–3 days
Rotavirus
24–72 hours
4–6 days
Agent
Symptoms
Heavy metal:
copper, tin, lead,
zinc
Nausea, vomiting
Fish toxins: PSP,
ciguatera and soon
Gastrointestinal and neurological symptoms
Monosodium
glutamate
Burning sensation on body, tingling, dizziness, headache,
nausea
Food allergens:
nuts, eggs, milk,
wheat
Anaphylactic shocks. Respiratory failure, rashes, nausea,
vomiting
S. aureus
Nausea, vomiting, abdominal pain
Bacillus cereus,
emetic
Nausea, vomiting, abdominal pain
Bacillus cereus,
diarrhoeal
Abdominal pain, watery diarrhoea
Clostridium
perfringens
Abdominal pain, watery diarrhoea
Salmonella
serovars
Abdominal pain, diarrhoea, chills, fever, nausea, vomiting,
loss of appetite
Clostridium
botulinum
Vertigo, blurred vision, difficulty in speaking, progressive
nervous system failure and paralysis
Streptococcus
Group A
Sore throat, fever, nausea, vomiting, rhinorrhoea, tonsillitis,
may be rash
Vibrio
parahaemolyticus
Profuse watery diarrhoea, chills, headache
Yersinia
enterocolitica
May resemble appendicitis. Gastroenteritis with diarrhoea,
and/or vomiting, fever and abdominal pain are common.
Agent
Symptoms
Vibrio cholerae
Profuse watery diarrhoea and dehydration
Shigella species
Abdominal pain, diarrhoea, stools may contain mucus and blood
Cronobacter species
Infant bacteraemia, septicaemia, necrotising enterocolitis,
meningitis
Campylobacter jejuni
Abdominal pain, diarrhoea, headache, fever, nausea, vomiting,
loss of appetite
Cyclospora
cayetanensis
Watery diarrhoea, with frequent, sometimes explosive, bowel
movements.
Pathogenic E. coli
Abdominal pain, diarrhoea, stools may contain mucus and blood.
Fever may be present
Norovirus
Nausea, vomiting, diarrhoea, abdominal pain, myalgia,
headache, malaise
Listeria
monocytogenes
Fever, headache, nausea, vomiting, diarrhoea, still births,
neonatal meningitis
Hepatitis A
Malaise, anorexia, nausea, abdominal pain, jaundice, dark urine,
light-coloured stools
Epidemiological and clinical differences between
sexes and pathogens in a three-year surveillance of
acute infectious gastroenteritis in Shanghai
Seasonal distribution of infectious diarrhea.
(A) Three years of annual incidence data of acute infectious
gastroenteritis from two sentinel hospitals were aligned by
month. To better present the two peaks of the disease
epidemics, the time axis was arranged from May to December
and from the following January to April.
(B) Subjects with identified pathogens were also aligned by
month. To better present the two peaks of the viral and
bacterial gastroenteritis epidemics, the time axis was arranged
from May to December and from the following January to
April.
Lou et al. Scientific Reports volume 9, Article number: 9993 (2019)
Epidemiological and clinical differences between sexes and pathogens in a three-year
surveillance of acute infectious gastroenteritis in Shanghai
Pathogenic spectrum of acute gastroenteritis.
Identified bacteria and viruses were presented as ratios, and the proportions of bacterial or viral
coinfections were calculated separately and were not included in the individual proportion of each
pathogen.
Lou et al. Scientific Reports volume 9, Article number: 9993 (2019)
Epidemiological and clinical differences
between sexes and pathogens in a three-year
surveillance of acute infectious gastroenteritis
in Shanghai
• Ingesting unsafe food at restaurants
associated with bacterial gastroenteritis.
• Abdominal pain and fever were two
common independent symptom factors
that more frequently occurred in bacterial
gastroenteritis than in viral gastroenteritis,
regardless of sex.
•
Lou et al. Scientific Reports volume 9, Article number: 9993 (2019)
Variation in causative agent of gastroenteritis with age.
Forsythe, 2000, 2020
Causes of foodborne illness
Contributing factors
Percentagea
Factors relating to microbial
growth
Storage at ambient (room)
temperature
43
Improper cooling
32
Preparation too far in advance
of serving
41
Improper warm holding
12
Use of leftovers
5
Improper thawing and
subsequent storage
4
Extra large quantities
prepared
22
Factors relating to microbial
survival
Improper reheating
17
Inadequate cooking
13
Contributing factors
Percentagea
Factors relating to
contamination
Food workers
12
Contaminated processed
non-canned foods
19
Contaminated raw foods
7
Cross-contamination
11
Inadequate cleaning of
equipment
7
Unsafe source
5
Contaminated canned
foods
2
Percentages exceed a total of 100 since multiple
factors often contribute to foodborne illness.
a
Causes of foodborne illness
• The principal causes can be summarised as:
• inadequate control of temperature during cooking,
cooling and storage;
• inadequate personal hygiene;
• cross-contamination of raw and processed products;
• inadequate monitoring of processes.
Chronic sequelae following foodborne illness
• The potential for chronic sequelae (secondary complications)
has been recently recognised along with the variability of the
human response
• It has been estimated that chronic sequelae occur in 2–3%
of foodborne cases and may last weeks or even months.
• These sequelae may be more serious than the original illness
and result in serious long-term disability or even death
• An appreciation of the chronic sequelae is necessary to fully
appreciate the economic burden of foodborne infections.
Chronic sequelae following foodborne illness
• A major symptom of food poisoning is diarrhoea, which may
consequently lead to anorexia and malabsorption.
• Severe cases of diarrhoea may last for months or years and can be
caused by C. jejuni, Citrobacter, Enterobacter or Klebsiella enteric
infections.
• The intestinal wall permeability may be increased and absorb significant
quantities of unwanted proteins, which may induce atrophy.
• Foodborne pathogens may interact with the immune system of the host
to elude or alter the immunological process, which may subsequently
induce a chronic disease.
• In addition, genetic susceptibility of the host may predispose humans to
some types of infection.
Chronic sequelae following foodborne illness
• Of particular importance are:
• Guillain–Barré syndrome (C. jejuni);
• Reactive arthritis and Reiter's syndrome (Salmonella serotypes);
• Haemolytic uraemic syndrome (E. coli O157).
• Less well studied chronic sequelae are:
• Chronic gastritis due to Helicobacter pylori.
• Crohn's disease and ulcerative colitis possibly caused by Mycobacterium paratuberculosis.
• Haemolytic anaemia due to Campylobacter and Yersinia.
• Heart and vascular diseases caused by E. coli.
• Atherosclerosis following Salmonella Typhimurium infection.
• Graves' disease (autoimmune disease), which is the result of autoantibodies to the thyrotropin receptor
after Yersinia enterocolitica serotype O:3 infection.
• Severe hypothyroidism due to Giardia lamblia infection.
• Viral induction of autoimmune disorders, such as hepatitis A virus infection causing acute hepatitis with
jaundice in adults. This is probably due to molecular mimicry.
• Mycotoxins have a range of acute, subacute and chronic toxicities as some compounds are carcinogenic
and mutagenic.
Chronic
sequelae
following
foodborne
illness
Disease
Associated complication
Brucellosis
Aortitis, orchitis, meningitis, pericarditis, spondylitis
Campylobacteriosis
Arthritis, carditis, cholecystitis, colitis, endocarditis,
erythema, nodosum, Guillain–Barré syndrome, haemolytic
uraemic syndrome, meningitis, pancreatitis, septicaemia,
reactive arthritis, irritable bowel syndrome
Meningitis
Cronobacter species
E. coli (EPEC and EHEC types) infections Erythema nodosum, haemolytic uraemic syndrome,
seronegative arthopathy
Listeriosis
Meningitis, endocarditis, osteomyelitis, abortion and
stillbirth
Salmonellosis
Aortitis, cholecystitis, colitis, endocarditis, orchitis,
meningitis, myocarditis, osteomyelitis, pancreatitis, Reiter's
syndrome, rheumatoid syndromes, septicaemia, splenic
abscess, thyroiditis, irritable bowel syndrome
Erythema nodosum, haemolytic uraemic syndrome,
peripheral neuropathy, pneumonia, Reiter's syndrome,
septicaemia, splenic abscess, synovitis
Arthritis, epilepsy
Shigellosis
Taeniasis
Yersiniosis
Arthritis, cholangitis, erythema nodosum, liver and splenic
abscesses, lymphadenitis, pneumonia, pyomositis, Reiter's
syndrome, septicaemia, spondylitis, Still's disease
The size of the foodborne illness problem
The under-reporting pyramid.
The size of the foodborne illness problem
• In 2009, the European Centre for Disease Prevention and Control initiated the
‘Burden of Communicable Diseases in Europe (BCoDE)’ project to generate
evidence-based and comparable burden-of-disease estimates of infectious
diseases in Europe.
• The burden-of-disease metric used is the Disability-Adjusted Life Year (DALY),
composed of years of life lost due to premature death (YLL) and due to
disability (YLD).
•
• The DALY metric is an internationally recognised summary measure of
population health. One DALY is a health gap measure, equating to 1 year of
healthy life lost.
• Calculating DALYs facilitates comparing the relative impact of diseases
and risk factors over time.
Mangen et al. 2013 PLoS ONE 8(11): e79740. doi:10.1371/journal.pone.0079740
The size of the foodborne
illness problem
Average burden of Campylobacter spp. and Salmonella spp. in the Netherlands
(average of 2005–2007) in DALY per year, for base case and scenario analysis.
DALY are subdivided in YLL and YLD for actue illness, sequelae and total. The
95% uncertainty range is shown using error bars. Note: ‘‘Base case’’ represents a
situation where only severe GE cases are at risk to develop reactive arthritis
(ReA). ‘‘SA: ReA’’ represents the scenario analysis where all GE cases are at risk to
develop ReA.
Average burden of Campylobacter spp. (a) and Salmonella spp. (b)
in the Netherlands (average of 2005–2007) in DALY per year,
subdivided in YLL and YLD for acute illness, sequelae and total. The
95% uncertainty range is shown using error bars.
Mangen et al. 2013 PLoS ONE 8(11): e79740. doi:10.1371/journal.pone.0079740
The cost of foodborne diseases
• Economic consequences of foodborne illness.
• These costs include:
•
•
•
•
•
•
loss of income by the affected individual;
cost of health care;
loss of productivity due to absenteeism;
costs of investigation of an outbreak;
loss of income due to closure of businesses;
loss of sales when consumers avoid particular products.
Foodborne diseases
• Some numbers:
• Shiga toxin-producing Escherichia coli (STEC) infections in humans were the
third most commonly reported zoonosis in the EU and increased from 2014 to
2018
• Yersiniosis was the fourth most frequently reported zoonosis in humans in 2018
with a stable trend in 2014–2018.
• The number of reported confirmed listeriosis cases further increased in 2018,
despite Listeria rarely exceeding the EU food safety limit tested in ready-to-eat
food.
• In total, 5,146 food- and waterborne outbreaks were reported.
• Salmonella was the most commonly detected agent with S. Enteritidis causing
one in five outbreaks. Salmonella in eggs and egg products was the highest risk
agent/food pair.
EFSA and ECDC (European Food Safety Authority and European Centre for Disease Prevention and Control), 2019.
The European Union One Health 2018 Zoonoses Report. EFSA Journal 2019;17(12):5926, 276 pp.
https://doi.org/10.2903/j.efsa.2019.5926
Foodborne diseases
EFSA and ECDC (European Food Safety Authority and
European Centre for Disease Prevention and Control),
2019. The European Union One Health 2018 Zoonoses
Report. EFSA Journal 2019;17(12):5926, 276 pp.
https://doi.org/10.2903/j.efsa.2019.5926
Changes in antimicrobial resistance of foodborne pathogens Bacterial antibiotic resistance in agriculture and aquaculture
• Antibiotic resistance, or antimicrobial resistance (AMR), is a major
area of concern as the effectiveness of antibiotics to treat infections
in humans and animals is decreasing
• Each year, 33 000 people die from an infection due to bacteria
resistant to antibiotics.
• The burden of infections is comparable to that of influenza,
tuberculosis and HIV/AIDS combined.
• It is also estimated that AMR costs the EU €1.5 billion per year in
healthcare costs and productivity losses.
• Video: Antibiotic resistance evolve
Antibiotic resistance evolve
Aslam 2021 Front. Cell. Infect. Microbiol. 11:771510.
• In 2017 the European Commission adopted the EU One
Health Action Plan against AMR
Monitoring of AMR in bacteria from food-producing animals and
food is crucial to control the development of antibiotic resistance
• Promote the prudent use of antimicrobials;
• More research is needed to develop new
medicinal products, therapeutics and alternative
treatments, as well as innovative anti-infective
approaches and products for humans and animals;
• Advance the repurposing of old antimicrobials,
improving their activity and to develop new
combination therapies
• Novel, rapid and reliable diagnostics are crucial
for differentiating between bacterial and viral
infections and identifying AMR, so that the most
appropriate treatment can be given in a timely
manner. By tailoring the treatment to the nature of
the infectious pathogen and its resistance pattern,
diagnostics help reduce the unnecessary use of
antimicrobials in humans and animals.
• The release of antimicrobials into the
environment through human, animal and
manufacturing waste streams should be assessed
and
• new technologies developed to enable efficient
and rapid degradation of antimicrobials in
wastewater treatment plants, organic waste streams
or the environment
• In the agri-food sector, the links between farming
practices, animal health and AMR development
and spread need to be further investigated.
Summary of measures
Aslam 2021 Front. Cell. Infect. Microbiol. 11:771510.
Antibiotic susceptibility determination
Disk Diffusion
Also known as the Kirby-Bauer Method
• Mueller–Hinton agar plates (90 mm diameter)
are inoculated with a standardized inoculum of
the test microorganism (corresponding to 0.5
McFarland turbidity standard).
0.5
McFarland
turbidity
standard
• The disc diffusion test provides qualitative results
by categorizing the bacterial susceptibility as
susceptible intermediate or resistant, but is not
appropriate for determination of MIC (Minimum
Inhibitory Concentration).
• However, the diameter of the inhibition growth
zone is related to MIC for a particular
combination of bacteria/antimicrobial drug, and
an approximate MIC can be calculated by
comparing the inhibition zones with algorithms
Inhibition zone
Disk Diffusion
Oxoid
BBL
Disk Diffusion
Dispenser
Disk Diffusion
Individual
dispenser
The 15-15-15 minute rule
• Prepare your plates so that you:
• Use the inoculum within 15 minutes of preparation, and
never beyond 60 minutes
• Apply disks within 15 minutes of inoculating plates
• Start incubation within 15 minutes of distribution of disks
EUCAST, 2012
Disk Diffusion
The bacterial growth must be confluent and evenly spread
EUCAST, 2012
Disk Diffusion
How to Read Inhibition zone
Measure diameter of zone with caliper, ruler or automated zone reader
Use reflected light except for
§
Staphylococci and enterococci use transmitted light for possible inner colonies
Reflected Light
Transmitted Light
Disk Diffusion
Reading the inhibition zones
• Inhibition zone edges should be read at the point of complete
inhibition as judged by the naked eye
EUCAST, 2012
Disk Diffusion
ADAGIO
Automated System (Bio-Rad Laboratories, Inc.,)
Disk Diffusion
Interscience
Scan 500
Inhibition zone reader
SIRscan
automatic zone reader
Scan 1200
HD inhibition zone reader
High-performance
Scan 4000
Ultra HD inhibition zone
reader
It has been designed for the
pharmaceutical industries, medical
research and animal health. It reads 120
mm square dishes 120 mm and round
dishes up to 150 mm. Scan 4000 also
counts colonies on Petri dishes.
Disk Diffusion
Disk Diffusion
Carbapenemase-producing Enterobacteriaceae
EUCAST, 2017
• New definitions of S, I and R from 2019
• EUCAST has changed the definitions of susceptibility testing categories S, I and R
• S - Susceptible, standard dosing regimen: A microorganism is categorised as
"Susceptible, standard dosing regimen", when there is a high likelihood of therapeutic
success using a standard dosing regimen of the agent.
• I - Susceptible, increased exposure*: A microorganism is categorised as "Susceptible,
Increased exposure*" when there is a high likelihood of therapeutic success because
exposure to the agent is increased by adjusting the dosing regimen or by its
concentration at the site of infection.
• R - Resistant: A microorganism is categorised as "Resistant" when there is a high
likelihood of therapeutic failure even when there is increased exposure.
• *Exposure is a function of how the mode of administration, dose, dosing interval,
infusion time, as well as distribution and excretion of the antimicrobial agent will
influence the infecting organism at the site of infection.
Disk Diffusion
Some considerations
• Inner colonies
☛ Resistant subpopulation
☛ Mixed culture
☛ High-frequency mutants
Disk Diffusion
• Advantages
✓ Flexibility in antibiotic selection
✓ Low cost
✓ Simple to perform
✓ No special/expensive equipment is required
✓ Most established and best proven method
✓ Continues to be updated through EUCAST
✓ Qualitative results easily interpreted by clinician
Disk Diffusion
• Disadvantages
⇣Not standardized for all organisms
⇣Qualitative results only (S/I/R)
⇣Not rapid (results available in 16-24 hours)
⇣Time consuming since read and entered manually
⇣ It is often media dependent
BROTH MICRODILUTION
• Broth microdilution is the reference method for antimicrobial susceptibility
testing of rapidly growing aerobic bacteria, except for mecillinam and
fosfomycin, where agar dilution is the reference method.
• EUCAST recommends testing according to the International Standard ISO
20776-1, but with the use of MH-F broth (Mueller Hinton broth
supplemented with 5% lysed horse blood and 20 mg/L β-NAD,
www.eucast.org) for fastidious organisms.
• Results are recorded as the lowest concentration of antimicrobial agent
that inhibits visible growth of a microorganism, the Minimum Inhibitory
Concentration (MIC), expressed in mg/L or µg/mL.
BROTH MICRODILUTION
• Fastidious organisms
❑ streptococci including S. pneumoniae;
❑ H. influenzae;
❑ Moraxella catharrhalis;
❑ Listeria monocytogenes;
❑ Pasteurella spp.;
❑ Kingella kingae;
❑ Aerococcus spp.;
❑ Campylobacter spp.;
BROTH MICRODILUTION
• Microtitre trays with twofold dilutions of antibiotics
are inoculated with a
standardized inoculum of
the bacteria and incubated
under standardized
conditions.
BROTH MICRODILUTION
READING BROTH MICRODILUTION
• Results are only valid when the following criteria are met:
• Sufficient growth, i.e. obvious button or definite turbidity, in the positive
growth control.
• Pure culture
− Check for purity by subculturing from the growth-control well
immediately after inoculation onto a non-selective agar plate for
simultaneous incubation.
Correct inoculum 5 x 105 CFU/mL
− Viable colony counts can be performed by removing 10 µL from the
growth-control well or tube immediately after inoculation and diluting
in 10 mL of saline. Mix and spread 100 µL onto a non-selective agar plate.
After incubation, the number of colonies should be approximately 20-80.
Growth appearance
• Growth appears as turbidity or as a deposit of cells at the
bottom of the well. The appearance of growth differs
depending on the microorganism and the antimicrobial agent
tested.
• For round-bottom wells, growth will most often appear as a
button/pellet centered in the middle. For flat-bottom wells,
growth may be scattered.
• Growth in antibiotic-containing wells may differ from growth
seen in the positive growth control, even for pure cultures.
Reading MIC endpoints
• Results should be read manually. The use of a mirror may
facilitate reading.
• Read the MIC as the lowest concentration of antimicrobial
agent that completely inhibits the bacterial growth as
detected by the unaided eye.
READING BROTH MICRODILUTION
Turbidity without pellet
• Haze or turbidity without a pellet is often seen for
Pseudomonas spp. and Acinetobacter spp. This should be
regarded as growth and the endpoint read at the first well
with complete inhibition (clear broth).
Haemolysis
• For fastidious organisms tested in MH-F broth, haemolysis of
the blood can be seen. This is often accompanied by turbidity
or a deposit of growth (pellet).
• Haemolysis with turbidity or pellet should be regarded as
growth when determining endpoints.
Gram-positive cocci with bacteriostatic
antimicrobial agents
• Disregard pinpoint growth (tiny buttons) when trailing growth
occurs.
Gram-positive cocci with bacteriostatic
antimicrobial agents
• Disregard pinpoint growth (tiny buttons) when trailing growth
occurs.
Gram-negative organisms with tigecycline and
eravacycline
• Disregard pinpoint growth (tiny buttons) when trailing growth
occurs.
Trimethoprim and trimethoprim-sulfamethoxazole
• Read the MIC at the lowest concentration that inhibits ≥80 %
of growth as compared to the growth control.
Cefiderocol
• Broth microdilution MIC determination must be performed in
iron-depleted Mueller-Hinton broth and specific reading
instructions must be followed. For testing conditions,
• see http://www.eucast.org/guidance_documents/
• The MIC is read as the first well in which the reduction of
growth corresponds to a button of <1 mm or is replaced by
the presence of light haze/faint turbidity.
• The positive control should show strong growth in the form of
a button of >2 mm or heavy turbidity.
Cefiderocol
Interpretation of results
• Make sure that MIC values for relevant Quality Control strains are
within acceptable ranges before reporting results for clinical
isolates.
• See quality control criteria in EUCAST QC Tables (www.eucast.org).
• Interpret MIC values into susceptibility categories (S, I and R)
according to the current EUCAST Breakpoint Tables
(www.eucast.org).
Test
epsilon, e
e test consists of a
predefined gradient of
antibiotic
concentrations on a
plastic strip
• Allows the
determination of the
MIC value through
the gradient of
diffusion
Inhibition zone
MIC value
Test epsilon, e
• Read MIC at the point where
ellipse intersects the scale.
• If a MIC value between two twofold dilutions is seen, always
round up to the highest value.
• If the intersect differs on either
side of the strip, read the MIC as
the greater value.
Broth Microdilution & Test epsilon, e
• Advantages
- Generate quantitative results (MICs)
-Allow testing of anaerobic and fastidious species of bacteria
• Disadvantages
- Increased cost over disk
-Largely manual process
-Quantitative data may be “challenging” to interpret for some
providers
-There are some systematic biases toward higher or lower MICs
determined by the Etest when testing certain organism
antimicrobial agent combinations
Automated systems
• VITEK 2
• BD Phoenix System
• MicroScan WalkAway
System
Example of bacterial growth curves generated with the phenotypic VITEK2 system. Differences in the slope and
plateau levels of the curves generated in the presence or absence of antibiotics define whether or not a strains
will be deemed resistant, intermediary or susceptible to certain antibiotics. The layout of all current automated
systems allow for the simultaneous analysis of multiple drugs for a single bacterial species in a single diagnostic
tool.
Alex van Belkum; Wm. Michael Dunne, Jr. Pathogens: Wanted—Dead or Alive ASM Microbe Dec 2015
Advantages
Generate more rapid results
Include computerized data management
system
Automated
systems
Can set interpretation rules using software
Disadvantages
More expensive
Limited to commercially available panels
Failure to detect some inducible, subtle, or
emerging resistance mechanisms
Instrument interpretation
FACTORS
THAT
IMPACT
RESULTS
• Standardized bacterial inoculum
size
• Culture conditions (growth medium,
pH, cation concentration)
• Blood and serum supplements and
thymidine content
• Incubation conditions (atmosphere,
temperature, duration)
• Concentration of antimicrobials for
testing