PREVALENCE AND CHARACTERIZATION OF INTEGRONS IN MULTIDRUGRESISTANT NON-CLINICAL ENTERIC BACTERIAL ISOLATES
A Thesis
Presented to the faculty of the Department of Biological Sciences
California State University, Sacramento
Submitted in partial satisfaction of
the requirements for the degree of
MASTER OF SCIENCE
in
Biological Sciences
(Molecular and Cellular Biology)
by
Aaron Lee Avila
SPRING
2013
© 2013
Aaron Lee Avila
ALL RIGHTS RESERVED
ii
PREVALENCE AND CHARACTERIZATION OF INTEGRONS IN MULTIDRUGRESISTANT NON-CLINICAL ENTERIC BACTERIAL ISOLATES
A Thesis
by
Aaron Lee Avila
Approved by:
__________________________________, Committee Chair
Susanne W. Lindgren, Ph.D
__________________________________, Second Reader
Enid T. Gonzalez-Orta, Ph.D
__________________________________, Third Reader
Nicholas N. Ewing, Ph.D
____________________________
Date
iii
Student: Aaron Lee Avila
I certify that this student has met the requirements for format contained in the University
format manual, and that this thesis is suitable for shelving in the Library and credit is to
be awarded for the thesis.
__________________________, Graduate Coordinator
Jamie Kneitel, Ph.D
Department of Biological Sciences
iv
___________________
Date
Abstract
of
PREVALENCE AND CHARACTERIZATION OF INTEGRONS IN MULTIDRUGRESISTANT NON-CLINICAL ENTERIC BACTERIAL ISOLATES
by
Aaron Lee Avila
Antibiotic resistance in bacteria has been a concern in the medical field for almost
as long as antibiotics have been available. The last several decades have seen marked
increases in antibiotic resistance, leading to the discovery of multidrug-resistant (MDR)
bacteria, which can be resistant to several antibiotics. MDR bacteria are a major problem
in the healthcare industry, creating to numerous challenges such as reduced treatment
options, increased mortality rates, longer hospital stays, and increased costs. The
increasing dissemination of resistance genes is believed to be the result of horizontal gene
transfer via mobile genetic elements, including plasmids and transposons. Several studies
have also shown that integrons play a significant role in the spread of resistance, acting as
v
gene capture and expression mechanisms that are often associated with mobile genetic
elements. However, most of the studies investigating the role of integrons in the
dissemination of antibiotic resistance utilized bacterial samples from environmental
sources or hospitalized patients. Far fewer studies have examined the role of integrons in
the propagation of multidrug-resistance in bacteria from the lower intestinal tract of
healthy individuals. The purpose of this study was to determine whether or not integrons
play a significant role in the proliferation of multidrug-resistance in enteric bacteria
isolated from healthy, non-hospitalized adults. Attempts were also made to identify the
gene cassettes and organization of cassettes within the identified integrons. Over the
course of five years (2005-2009), a total of 92 enteric bacterial samples were collected
from students at CSUS via a rectal swab, and isolated on MacConkey agar. These
samples were isolated and subjected to a variety of antibiotics and biochemical tests to
determine antibiotic resistance profiles and species. PCR amplification of class 1 and
class 2 integrase genes (intI1 and intI2) yielded 19 (out of 84 unique samples) class 1
positive isolates, one of which was also found to be class 2 positive. Resistance to
trimethoprim/sulfamethoxazole, ampicillin, and piperacillin was found to be significantly
greater in class 1 positive isolates compared to class 1 negative isolates (P<0.05).
Resistance to two or more classes of antibiotics was also significantly higher in class 1
positive isolates compared to class 1 negative isolates. Resistance to two or more
antibiotics, regardless of class was also significantly higher in class 1 positive isolates.
PCR amplification of the variable regions of intI1 and intI2 samples yielded seven unique
vi
amplicons ranging in size from approximately 250bp to >3kbp. Subsequent sequencing
and nucleotide BLAST searches led to the identification of eight different gene cassettes
organized in six unique arrays.
_______________________, Committee Chair
Susanne W. Lindgren, Ph.D
_______________________
Date
vii
ACKNOWLEDGEMENTS
I owe the successful completion of this thesis to several people, including those
who have contributed directly to the project, as well as those who have supported me
along the way. First, I would like to thank Scott Baker for his initial work in collecting
samples and gathering raw data that was used in this study. I would also like to thank
Windy Miller and Amy Crum for their help in collecting and isolating samples. Thank
you also to Myra Rodriguez for her flexibility in always meeting my ever-changing
scheduling needs for school.
A special thank you must go out to Dr. Susanne Lindgren for inviting me to join
her lab when I was much too shy to ask myself. She offered me the opportunity to take
over this project and the freedom to choose the direction to take it. I thank her for her
support, guidance, and sense of calm when things were not going as planned. I also
would like to extend my gratitude to my committee members, Dr. Enid Gonzalez-Orta
and Dr. Nicholas Ewing for coming through and meeting deadlines with short notice.
I would like to express a warm thank you to my brother and all of my friends for
their continued support and encouragement over the years. In times of stress, they were
instrumental in helping me to relax, take a break, and enjoy life. Finally, and most
importantly, I owe a great deal of gratitude to my parents. Without their unwavering love,
steadfast support, and continuous encouragement, I never would have completed this
program. Thank you so much for helping me realize my dreams. I love you all, and I am
finally finished!
viii
TABLE OF CONTENTS
Page
Acknowledgements .................................................................................................... viii
List of Tables .................................................................................................................x
List of Figures .............................................................................................................. xi
INTRODUCTION .........................................................................................................1
MATERIALS AND METHODS ...................................................................................8
RESULTS ....................................................................................................................22
DISCUSSION ..............................................................................................................49
Appendix ......................................................................................................................58
Literature Cited ............................................................................................................76
ix
LIST OF TABLES
Tables
Page
1. Primers Used For Detection of Class 1 and Class 2 Integrases and Amplification
of Variable Regions ...................................................................................................... 16
2. Species Identifications .................................................................................................. 25
3. Susceptibility Comparisons Between Class 1 Positive and Class 1 Negative
Isolates for Each Tested Antibiotic .............................................................................. 26
4. Comparisons Between intI1-positive and intI1-negative Isolates Resistant to
Multiple Classes of Antibiotics .................................................................................... 31
5. Comparison Between intI1-positive and intI1-negative Isolates With Intermediate
or Resistant Phenotypes for Multiple Classes of Antibiotics ....................................... 34
6. Comparison Between intI1-positive and intI1-negative Isolates Resistant to
Multiple Antibiotics, Regardless of Class .................................................................... 34
7. Observed vs. Actual Amplicon and Restriction Fragment Sizes .................................. 40
8. Identified Gene Cassettes .............................................................................................. 47
x
LIST OF FIGURES
Figures
Page
1. Layout of MicroScan Gram Negative Combo Panels................................................... 11
2. Sample Biotype Number Panel Worksheet................................................................... 14
3. Relative Primer Locations............................................................................................. 18
4. Class 1 Integron Detection ............................................................................................ 27
5. Class 2 Integron Detection ............................................................................................ 29
6. Percentage of Cumulative Resistant Integrase-positive vs. Integrase-negative
Isolates for Varying Numbers of Antibiotic Classes .................................................... 32
7. Number of Resistant Integrase-positive vs. Integrase-negative Isolates for
Varying Numbers of Antibiotic Classes ...................................................................... 33
8. Percentage of Cumulative Resistant Integrase-positive vs. Integrase-negative
Isolates for Varying Numbers of Antibiotics, Regardless of Class .............................. 35
9. Number of Resistant Integrase-positive vs. Integrase-negative Isolates for
Varying Numbers of Antibiotics, Regardless of Class................................................. 36
10. Class 1 Integron Variable Regions ............................................................................. 37
11. Class 2 Integron Variable Regions ............................................................................. 39
12. Class 1 Variable Region Restriction Fragments ......................................................... 42
13. Variable Region Partial Alignment ............................................................................. 43
14. Gene Cassette Arrangements in Class 1 and Class 2 Integrons .................................. 48
xi
1
INTRODUCTION
The discovery of antibiotics in the late 1920s, and their subsequent use in treating
and preventing infections beginning in the 1940s, is undoubtedly one of the great medical
breakthroughs in the last 100 years (14, 15). In the early years of antibiotic treatment,
many scientists and doctors believed that infectious disease had been triumphed once and
for all (14). And while it is true that antibiotics have largely nullified several diseases and
infections that were once very difficult to treat, there is reason to be concerned that this
may not always be the case. Less than a decade after the first antibiotics were introduced
in medicine, evidence of bacterial strains resistant to those antibiotics began to surface
(14, 15). Shortly thereafter, scientists uncovered evidence that bacteria were not only
capable of developing resistance to one antibiotic, but to multiple antibiotics that were
also transferable to sensitive strains (14). The rise of multidrug-resistant (MDR) bacteria
is a result of unscrupulous antibiotic use in medicine and agriculture over the last several
decades (5, 12, 14, 31).
Today, MDR bacteria provide numerous challenges and problems for healthcare
providers, including increases in hospital-acquired infections, reduced treatment options,
higher morbidity and mortality rates, and healthcare cost increases due to longer hospital
stays (16, 43). MDR bacteria may be resistant to a couple of antibiotics, several classes of
antibiotics, and in some cases every antibiotic (8). Even MDR bacteria that are resistant
to only a couple of antibiotics can greatly complicate treatment. Often, such bacteria are
resistant to the primary antibiotic preferred for treatment, requiring the use of secondary
and tertiary drugs instead, which may be less effective and more toxic to the patient (8).
2
The growing problem of MDR infections is made even more concerning by the fact that
new discoveries of antimicrobial agents have been few and far between in recent years
(11, 14). Over the last five decades, only two new classes of antibiotics have reached the
market, and current information suggests that no new antibiotic classes will be introduced
in the near future (11). Without the continuous introduction of new antibiotics, as was
seen during the first 20 years of their use, the threat of a return to the pre-antibiotic era is
very real (11, 15).
Perhaps the most widely publicized strain of MDR bacteria is the much-feared
Gram-positive methicillin resistant Staphylococcus aureus (MRSA) (15, 33, 39).
However, less well-publicized MDR Gram negative bacteria are also capable of causing
serious, difficult to treat infections. The Antimicrobial Availability Task Force,
established by the Infectious Diseases Society of America, identified several particularly
problematic pathogens, one of which included extended-spectrum beta-lacatamase
(ESβL)-producing Enterobacteriaceae (e.g. Escherichia coli and Klebsiella pneumoniae)
(46). ESβLs are enzymes produced by bacteria that confer resistance to multiple
antibiotic classes, namely cephalosporins, penicillins, monobactams, and beta-lactamases.
(46). Over 500 different ESβLs have been identified, the most common belonging to the
CTX and CMY gene families (46). Infections caused by ESβL producers usually must be
treated with a carbapenem (e.g. imipenem, meropenem). Recently however, ESLproducing Gram-negatives have been identified that are also resistant to the carbapenem
class of antibiotics (46, 9). Carbapenem-resistant Enterobacteriaceae (CRE), or
“superbugs”, as the media often refers to them, produce metallo-beta-lactamases (MβLs)
3
that readily cleave most β-lactam substrates (46, 9). As with ESβLs, several MβL
enzymes have been discovered, the latest apparently originating from India, identified as
NDM-1 (53).
Although drug resistance is generally discussed with regard to pathogenic
bacteria, not all antibiotic-resistant bacteria are necessarily harmful to their host. Bacteria
comprising normal human flora in asymptomatic individuals have certainly been shown
to carry resistance to antibiotics (1, 29, 48). E. coli and K. pneumoniae may make up part
of a normal intestinal flora, where they cause no problems; however, introduction of
these strains to other areas of the body, or to other people, can cause infections such as
urinary tract infections, pneumonia, and septicemia (16, 43). These types of infections are
most common in people with weakened immune systems and individuals who are
hospitalized for other reasons (16, 43). Most often, such infections are acquired in
hospitals. The CDC has estimated that as many as 1.7 million hospital-acquired infections
result in nearly 100,000 deaths each year in the United States (39). Significant problems
arise in the treatment of these infections, especially when they are caused by MDR
bacteria.
Antibiotic-resistant organisms residing as part of a person’s intestinal flora,
whether they are pathogenic strains or not, may act as a reservoir for resistance genes that
can be transferred to other bacteria (28). Bacteria are able to transfer resistance genes
horizontally to one another through various mechanisms. The emergence of MDR
bacteria is the result of horizontal gene transfer (7, 14, 15, 40), where genetic information
is passed directly from one bacterium to another. Horizontal transfer of antibiotic
4
resistance genes occurs primarily through two different genetic elements: plasmids and
transposons (20, 7, 29, 31). Plasmids are small, circular, extrachromosomal DNA
molecules that may contain resistance genes (29). Plasmids can be transferred via a pilus
from one bacterium to another in a process called conjugation (42). The recipient
bacterium acquires all genes present on the plasmid, including resistance genes. Like
plasmids, transposons can also carry resistance genes. Transposons are genetic elements
that may be inserted into and excised from chromosomes and plasmids (20). Through
sharing of DNA via these two mechanisms, bacteria can rapidly acquire new genes that
make them immune to various antibiotics.
A third group of genetic elements that have been strongly implicated in the
emergence of MDR bacteria are called integrons (23). While integrons themselves are not
mobile elements, they are frequently associated with transposons and plasmids. Plasmidintegrated transposons carrying antibiotic resistance genes can be transferred to other
bacteria through conjugation (20, 7, 29, 31). Integrons are capture-and-expression genetic
elements that facilitate site-specific recombination of promoter-less gene cassettes into a
site that allows for the transcription of all genetic material contained in the cassettes (7,
18, 31). They consist of three main components located in the 5’ conserved region: an
integrase gene (intI), a recombination site (attI), and an active promoter (7, 18, 23, 31).
The integrase recognizes a conserved, 59-base element (actually varies in length from 57141 bases), which is found on resistance gene cassettes (7, 18, 31, 45). Upon recognition
of this conserved element, the integrase facilitates the integration of the cassette into the
integron at the attI site, just downstream of the active promoter (7, 18, 31). Any cassettes
5
that are integrated downstream of the promoter are then free to be transcribed; they may
also be rearranged or excised via the integrase, and new promoter-less resistance genes
can be integrated (7, 18, 31). Thus, integrons are essentially genetic elements capable of
integrating and expressing various rearrangeable antibiotic resistance gene cassettes that
can be readily mobilized into neighboring bacteria.
At least three classes of integrons have been identified, which are distinguished
primarily by the integrase gene. Genes contained within the 3’ conserved region also vary
between the three classes of integrons. Class 1 and class 2 integrons are the most
prevalent and best studied (2, 18). Class 3 integrons appear to be far less common, and
therefore less implicated in the spread of multidrug-resistance. Class 3 integrons have
been found in Serratia marcescens (3), Klebsiella pneumoniae (13), as well as Delftia
species (51). Class 1 integrons, on the other hand, have been found in many Gramnegative Enterobacteria, including species of Escherichia, Klebsiella, Pseudomonas,
Enterobacter, Salmonella, Proteus, Serratia, Citrobacter, and Shigella (18, 23). Integrons
are known to contain highly conserved regions at the 5’ end (which encodes the integrase
gene) as well as the 3’ end, downstream of integrated gene cassettes. The 3’ conserved
region of class 1 integrons consists of the qacΔ1 and sul1 genes, which confer resistance
to quaternary ammonium compounds and sulfonamides, respectively (23, 24). Class 2
integrons appear to be less widespread, although they have been identified in several
genera of bacteria, such as Shigella, Salmonella, and Acinetobacter (2, 10, 18, 37, 38), as
well as Escherichia, Morganella, and Aeromonas (35). Integrons are believed to a play
considerable role in the dissemination of antibiotic resistance genes within Gram-
6
negative bacteria (7, 14, 18, 31). A group of researchers recently created a database,
called the Repository of Antibiotic resistance Cassettes (RAC), which contains over 300
different promoter-less gene cassettes (47). Several of these antibiotic resistance gene
cassettes are frequently seen integrated into both class 1 and class 2 integrons, including
those granting resistance to aminoglycosides, cephalosporins, chloramphenicol,
penicillins, and trimethoprim (7).
The association between antibiotic resistance and integrons has been well
documented. Integrons have been shown to be particularly prevalent in many clinical
isolates of Gram-negative enteric bacteria. Integron frequencies in clinical samples as
high as 88% (37), and as low as 13% (36) have been found, though more common
frequencies fall in the range of 20%-60% (2, 10, 17, 25, 38, 50). There have also been
numerous studies investigating the prevalence of integrons in bacteria isolated from
sources other than humans. Such sources include wastewater treatment plants (35),
irrigation sediments (40), and animals (5, 6, 19, 21, 52). Far fewer studies have been
conducted to investigate the prevalence of multidrug-resistance in bacteria obtained from
healthy, non-hospitalized individuals. Studies that include commensal bacteria obtained
from humans often include clinical isolates (41), or a combination of animal and human
derived specimens (12, 32). One study that investigated integrons in a mixed sample set
of animal, commensal human, and clinical human isolates did find that MDR was
associated with the presence of integrons regardless of origin, indicating that a positive
correlation between MDR and commensal human isolates had been established. Another
study investigated the transfer of antibiotic resistance genes among nonpathogenic
7
Bacteroides within the human colon, but no attempt was made to identify the presence of
integrons or investigate their possible role (42).
Through an IRB-approved exemption, a collection of antibiotic-resistant enteric
bacteria from healthy CSUS students was accumulated over the course of five years.
Multidrug-resistance was observed in several of the enteric isolates. I hypothesized,
based on previous research, that the prevalence of class 1 and class 2 integrons would be
significantly greater in multidrug-resistant enteric bacteria comprising normal flora of
healthy adults than in isolates with low or no resistances. Few studies have attempted to
examine the prevalence or role of integrons in the propagation of MDR bacteria that exist
as part of the normal human intestinal flora. By determining the prevalence of integrons
within the drug-resistant samples collected, some insight may be gained into the role of
integrons in the dissemination and maintenance of multidrug-resistance factors in the
community.
8
MATERIALS AND METHODS
Sample Collection
Over the course of five years, through an IRB-approved exemption, enteric
bacterial samples were collected from undergraduate microbiology students at CSUS. As
part of a voluntary lab exercise, a self-administered sterile rectal swab was used to obtain
enteric bacteria from students. Once inoculated, swabs were rubbed over MacConkey
agar plates, and four antibiotic diffusion discs were placed on the plate. The antibiotic
discs used were ampicillin, erythromycin, tetracycline, and sulfamethoxazole/
trimethoprim. In addition, an antibiotic disc containing ciprofloxacin was also used on
one of the agar plates. Plates were then incubated for approximately 24 hours at 37ºC.
After the students had finished using the bacteria for their lab exercises, the plates were
wrapped with Parafilm and stored at 4ºC for up to three weeks.
Bacterial colonies exhibiting growth within zones of inhibition of the antibiotic
discs were streaked for isolation onto MacConkey agar plates and incubated for 24 hours
at 37ºC. To ensure purity, this process was repeated at least twice for each sample. In
some cases, more than one colony was taken from the initial plate (i.e. more than one
antibiotic-resistant sample was obtained from the same individual) if there were colonies
growing within zones of inhibition of more than one antibiotic. Once isolated, each
antibiotic-resistant bacterial isolate was grown overnight in 5 ml of lysogeny broth (LB)
in a 37ºC water bath shaking at 50 shakes per minute. Frozen stocks of each isolate were
made in duplicate by mixing 0.5 ml of overnight culture with 0.5ml of 80% glycerol in a
1.2 ml freezer vial, vortexing briefly, and placing into a -80ºC freezer. All samples were
9
collected using these methods during the fall of 2005, 2006, 2008, and 2009; no
collection was made in 2007.
Species Identification and Antibiotic Susceptibility Testing
Each antibiotic-resistant enteric isolate was subjected to a variety of biochemical
and antibiotic susceptibility tests via Dade Behring MicroScan Dried Overnight Gram
Negative Combo Panels (West Sacramento, CA). A total of three different types of
panels were used: NC 32, NC 30, and NBPC 30. NC 30 panels were used after the NC 32
panel stock was depleted, and NBPC 30 panels were used after the NC 30 panel stock
was depleted. Most samples were tested using only one of the three types of panels.
However, some samples were re-tested based on inconclusive results for the species
identification. These samples (0806, 0809, 0816B, and 0915) were re-tested on the NBPC
30 panels.
All three panels contain identical biochemical tests for species identification.
However, each panel does differ in the antibiotics it tests for and/or the concentrations of
each antibiotic. Compared to NC 32 panels, NC 30 panels contain two additional
antibiotic tests: gatifloxacin and amoxicillin/K clavulanate. NC 30 panels also test
additional concentrations of cefotetan, cephalothin, ceftriaxone, cefazolin and
piperacillin/tazobactam. NC 30 panels do not contain tests for cefotaxime, ticarcillin/K
clavulanate, moxifloxacin, or meropenem, and use fewer concentrations of amikacin and
tobramycin. NBPC 30 is a breakpoint panel, containing all of the antibiotics from NC 30
and NC 32 panels, an additional concentration of nitrofurantoin, as well as four additional
10
antibiotics: chloramphenicol, norfloxacin, cefoxitin, and tetracycline. Because it is a
breakpoint panel, NBPC 30 panels contain fewer concentrations for many of the
antibiotics—only the concentrations necessary to determine susceptibility. Figure 1
shows a diagram of each panel used, including the concentrations of all antibiotics.
Panels were inoculated using the Turbidity Standard Technique according the Dade
Behring MicroScan Dried Gram Negative Procedural Manual (34). After incubation of
the panels at 37ºC for 18 hours, the biochemical results of each panel were read manually
and interpreted as indicated by the manufacturer’s instructions. Based on the results of
the biochemical tests, a worksheet was used to generate a biotype number for each isolate
(Figure 2). The MicroScan Biotype Lookup Program (44) was used along with the
biotype number to identify the species of each isolate as well as a confidence percentage.
Minimum inhibitory concentrations (MICs) for each antibiotic were also read
manually according to the procedural manual for the panels (34). Once MICs for each
antibiotic were recorded, susceptibility was determined based on the Interpretive
Breakpoints chart included in the procedural manual (34). Each sample was assigned a
ranking of susceptible (S), intermediate (I), or resistant (R) based on their MIC for each
antibiotic.
Template DNA Preparation
Template DNA was prepared using a simple, crude preparation technique, similar to
that described by Mazel et al. (32). Frozen bacterial samples were first streaked for
11
C
G
P4
GLU
RAF
INO
URE
LYS
TDA
CIT
TAR
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NIT
K4
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SUC
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ARG
ESC
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ACE
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2/38
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Ak
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ESBL
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-b
8/4
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LOC
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2
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4
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Cft
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Tim
2
4
Mxf
2
4
8
16
Caz
2
4
8
16
Cpe
4
8
Imp
4
8
Mer
Figure 1-a. Layout of MicroScan Gram Negative Combo Panels. Negative Combo Panel
Type 32. Orange: biochemical tests used in the determination of species; Green:
biochemical tests not used; Pink: antibiotic tests, concentrations in μg/ml, abbreviations
listed in Appendix B; Yellow: putative screen for ESL production; Blue: locator for
automated panel analysis (not used).
12
C
G
P4
GLU
RAF
INO
URE
LYS
TDA
CIT
TAR
OF/G
NIT
K4
Cl4
SUC
RHA
ADO
H2S
ARG
ESC
MAL
ACE
OF/B
LOC
2/38
T/S
Fd64
SOR
ARA
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VP
ONP
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CET
DCB
2
4
8
16
Cfz
2
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Am
16
32
Ak
1
2
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32
Ctn
8
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32
64
P/T
8/4
16/8
Aug
2
4
Gat
2
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8
16
Caz
1
2
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8
Gm
8/4
16/8
A/S
2
4
Lvx
4
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32
Cax
2
4
8
To
ESBL
-a
16
64
Pi
8
16
Azt
2
4
8
16
Cpe
4
8
16
Crm
ESBL
-b
8
16
Cf
4
8
Imp
Figure 1-b. Layout of MicroScan Gram Negative Combo Panels. Negative Combo Panel
Type 30. Orange: biochemical tests used in the determination of species; Green:
biochemical tests not used; Pink: antibiotic tests, concentrations in μg/ml, abbreviations
listed in Appendix B; Yellow: putative screen for ESL production; Blue: locator for
automated panel analysis (not used).
13
C
G
P4
GLU
RAF
INO
URE
LYS
TDA
CIT
TAR
OF/G
LOC
K4
Cl4
SUC
RHA
ADO
H2S
ARG
ESC
MAL
ACE
OF/B
NIT
ESBL
-a
ESBL
-b
SOR
ARA
MEL
IND
ORN
VP
ONP
G
CET
DCB
8
16
Am
16
64
Pi
16
32
Ak
8
16
Cfz
8
32
Cft
1
2
Cp
8/4
16/8
A/S
16
64
P/T
4
8
Gm
8
16
Cf
8
16
Caz
2
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Gat
8/4
16/8
Aug
4
8
Te
4
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To
16
32
Ctn
8
32
Cax
2
4
Lvx
32
64
Fd
16
64
Tim
8
16
Azt
8
16
Cfx
8
16
Cpe
2
4
Mxf
4
8
Imp
4
8
Mer
2/38
T/S
4
8
16
Crm
8
16
C
4
8
Nxn
Figure 1-c. Layout of MicroScan Gram Negative Combo Panels. Negative Breakpoint
Combo Panel Type 30. Orange: biochemical tests used in the determination of species;
Green: biochemical tests not used; Pink: antibiotic tests, concentrations in μg/ml,
abbreviations listed in Appendix B; Yellow: putative screen for ESL production; Blue:
locator for automated panel analysis (not used).
14
Glucose Fermenter
4
+
2
+
1
GLU
RAF
INO
URE
LYS
TDA
CIT
Cl>4
TAR
OF/G
Cl>4
NIT
SUC
RHA
ADO
H2S
ARG
ESC
MAL
Cf>8
ACE
P>4
Fd>64
OXI
SOR
ARA
MEL
IND
ORN
VP
ONPG OXI
CET
K>4
To>4
4
+
2
+
1
Glucose Non-Fermenter
Identification
Figure 2. Sample Biotype Number Panel Worksheet. Only the first eight columns are used to
generate biotype numbers for glucose fermenters (100% of samples tested). Positive results in the
top row score four points, second row scores two points, and third row scores one point. Points are
added in each column to generate an eight-digit biotype number.
15
isolation on LB agar and incubated overnight at 37ºC. An isolated colony from the plate
was used to inoculate 1 ml of LB media, which was then grown overnight in a 37ºC water
bath shaking at 50 shakes per minute. The overnight culture was transferred to a sterile
1.5 ml Eppendorf tube and centrifuged at 6000 rpm for approximately 1 minute. The
supernatant was then discarded, and the pellet of bacteria was re-suspended in 0.5 ml
sterile de-ionized water. After briefly vortexing the suspension, the tubes were placed in a
100ºC water bath for 10 minutes to lyse the bacteria. The tubes were then centrifuged
again at 6000 rpm for 5 minutes to pellet cell debris. The supernatant was removed and
placed into sterile 0.5 ml tubes for use as template DNA.
PCR Detection of class 1 and class 2 Integrons
Detection of class 1 and class 2 integrons relied on amplifying a section of the
integrase gene (intI1 and intI2, respectively) via two separate PCR assays. Successful
amplification of either gene indicated the presence of an integron of the corresponding
class. Primer sets are listed in Table 1. Positive controls were used for both class 1 and
class 2 integron assays. Salmonella enterica serovar Typhimurium strain DT104 was
used as the positive control for the class 1 assay, as it is know to carry a class 1 integron
(22). For the class 2 positive control, a strain of E. coli (ATCC# 47055) was chosen
because it contains a Tn7 transposon, which is known to contain a class 2 integron (6,
12).
Class 2
Conserved
(integrase)
intI2
Class 1
Conserved
intI1
(integrase)
Target
TTATTGCTGGGATTAGGC
intI2-F
CGGGATCCCGGACGGCATGCACGATTTGTA
GATGCCATCGCAAGTACGAG
hep74
hep51
ACGGCTACCCTCTGTTATC
GTAGGGCTTATTATGCACGC
Hep59
intI2-R
TCATGGCTTGTTATGACTGT
ACATGCGTGTAAATCATCGTCG
int1-R
Hep58
GGGTCAAGGATCTGGATTTCG
Sequence (5′ to 3′)
int1-F
Primer
Variable
(~1k-5k+)
233
Variable
(~1k-5k+)
484
Amplicon
Size (bp)
59
53
56
61
Annealing
Temp (ºC)
Table 1. Primers Used For Detection of Class 1 and Class 2 Integrases and Amplification of Variable Regions.
Class 2
Class 1
Integron
Class
27, 35, 50
19, 35
49
32
Reference
16
17
All PCR reactions were performed in 50 μl volumes. The class 1 dectection assay
was composed of the following: 1.0 μl of 10mM dNTP Mix (dATP, dTTP, dCTP, dGTP0.2mM final concentration) (Promega), 5.0 μl 10x Taq Buffer Advanced (5Prime), 0.5 μl
Taq Polymerase (5U/μl) (5Prime) added after 4 minutes of denaturation, 10 μl of 2.5 μM
intI1-F primer, 10 μl of 2.5 μM intI1-R primer (0.5 μM final) (Sigma), 10.0 μl template
DNA, and 13.5 μl dH20. Negative controls were run in all assays with 10.0 μl dH20 in
place of template DNA. The cycle parameters were as follows: 7 minutes of predenaturation at 94ºC, followed by 30 cycles of 94ºC for 1 minute, 61ºC for 1 minute, and
72ºC for 1 minute, and a final elongation step of 72ºC for 8 minutes at the end.
The class 2 detection assay reaction mixture was identical to the class 1 assay
except for the following changes: 2.0 μl of 25mM MgCl2 was added, the dH20 volume
was reduced to 11.5 μl, and intI2 forward and reverse primers were used to target the
intI2 gene. The cycle parameters were as follows: 7 minutes of pre-denaturation at 94ºC,
followed by 32 cycles of 94ºC for 1 minute, 53ºC for 1 minute, and 72ºC for 45 seconds,
with a final elongation step of 72ºC for 8 minutes at the end.
PCR Amplification of Integron Variable Regions
Samples that tested positive for either class 1 or class 2 integrase were used in
separate PCR assays designed to amplify the variable region of the integron. Primers
were used to target conserved sequences on opposite sides (5′ and 3′ conserved
sequences) of the variable region of the integrons (See Figure 3 for relative primer
locations). The class 1 variable region assay was identical to the class 1 detection assay
18
A
B
Figure 3. Relative Primer Locations. A: Primer locations for amplifying section of
intI1 (class 1) and intI2 (class 2) genes in 5’-conserved region of integron; B: Primer
locations amplifying variable region between 5’-conserved region and 3’-conserved
region
19
except for the hep58 and hep59 primer pair that was used (49). The cycle parameters for
the class 1 variable assay were as follows: 5 minutes of pre-denaturation at 94ºC,
followed by 33 cycles of 1 minute at 94ºC, 45 seconds at 56ºC, and 5 minutes at 72ºC,
and a final elongation step of 5 minutes at 72ºC at the end. The class 2 variable region
assay was identical to the class 1 variable assay except for the use of a higher annealing
temperature of 59ºC and a different primer pair, hep74 and hep51, which targets
conserved regions of class 2 integrons (27, 35, 50).
Agarose Gel Electrophoresis of PCR Products
All PCR products were visualized by running 20.0 μl of PCR product mixed with
2.0 μl of loading dye on agarose gels. Products from the class 1 and class 2 integrase
detection assays were run on 2% gels, as their products were 484bp and 233bp
respectively. Products from the class 1 and class 2 variable region assays were run on 1%
gels as most of their products ranged from approximately 1kbp to >3kbp, depending on
the length of the respective integron. DNA ladders (Sigma-1kbp and Morganville
Scientific-100bp) were run on each gel. Gels ran at 90V for approximately 45 minutes
and then stained in ethidium bromide before being visualized under UV light.
Restriction Digest of Variable Region PCR Products
Variable region PCR products that appeared to be similar in size were exposed to a
restriction enzyme, AluI (BioLabs), in order to determine if the products were of the same
sequence. AluI was chosen because its recognition sequence is only four bases, thus
20
increasing the likelihood of its activity over other enzymes that target six-base sequences.
Approximately 30 μl of PCR product was mixed with 1.0 μl of 10U/ml AluI and
incubated at 37ºC for 4 hours. The products were then run on a 2% agarose gel and
visualized using the same procedures as described for PCR products. Identical sized
patterns on the gels indicated the variable regions were likely of the same sequence,
while different banding patterns on the gel would indicate different sequences. This step
was taken to help reduce the risk of needlessly sequencing multiples of identical variable
regions.
Gel Extraction, Variable Region Sequencing, and Cassette Identification
Based on the results of the restriction digest assay for variable region products of
similar size, one sample representing each unique amplicon size was chosen for
sequencing. Following PCR amplification and gel electrophoresis of variable region
products as described above, DNA bands were extracted using the QIAquick Gel
Extraction Kit (Qiagen) following the manufacturers protocol. A total of seven extracts
(six class 1 samples and one class 2) were sent to Sequetech in Mountain View, CA for
sequencing. Complete sequencing by “primer walking” was not performed due to cost.
Instead, sequencing was performed using single primer extensions from both the 5’ and
3’ conserved regions in an effort to reduce cost while sequencing as much of the template
as possible. For shorter variable regions, this was sufficient to identify all cassettes.
However, for longer products, cassettes located in the middle of the variable region could
not be identified. Sequencing data was used to conduct nucleotide searches using BLAST
in order to identify gene cassettes.
21
Nomenclature of Antibiotic-resistant Enteric Isolates
Antibiotic-resistant enteric isolates were assigned identification numbers according
to the year in which they were collected. Sample ID numbers beginning with ‘F06’
indicate samples that were collected during the fall of 2006, while ID numbers beginning
with ‘08’ or ‘09’ indicate samples that were collected during the fall of 2008 and 2009
respectively. Sample ID numbers that were collected in the fall of 2005 begin with either
‘L’ or ‘M’. Arbitrary numbers were also assigned to identify samples that were derived
from different individuals during the same collection year. These numbers, found after
the number or letter indicating the collection year, were not used to identify specific
individuals, nor were they used to track any characteristics about the individuals. In some
cases, ID numbers were also labeled with regard to the antibiotic to which they initially
exhibited resistance during the sample collection process. There are five different
antibiotic-resistance labels: SXT (sulfamethoxazole/trimethoprim), TET (tetracycline), E
(erythromycin), AMP (ampicillin), and CIP (ciprofloxacin). Finally, letters ‘A’, ‘B’, ‘C’,
‘D’, and ‘E’ found at the end of the ID number indicate multiple samples that were
collected from the same individual. For example, sample 0920A is sample number 20
collected in the fall of 2009, and was one of three isolates collected from the same
individual.
22
RESULTS
Identification of Samples and Resistance Profiles
A total of 92 antibiotic-resistant enteric bacterial samples were collected and
isolated from 66 unique healthy human donors. A probable species identification of each
of the 92 samples was made by running each sample on a MicroScan Dried Gram
Negative Panel to generate a biotype number based on the results of multiple biochemical
tests contained on the panels. Each panel consisted of three rows of biochemical tests (top
three rows on each panel, see Figure 1), not all of which were necessary for species
identification. Only the tests necessary for the identification of glucose fermenters (100%
of tested samples, n=92) were used to generate biotype numbers, indicated in Figure 1 by
the orange shaded portions, and as shown on the panel worksheets (Figure 2). Each eightdigit biotype number generated a list of probable bacteria with a rough confidence
percentage. The most probable species for each isolate was recorded. Species
identifications and antibiotic susceptibilities for all samples not labeled as ‘08xx’ or
‘09xx’ were derived from biochemical results and MIC data from previous work in the
Lindgren lab by Baker (4).
All 92 antibiotic-resistant enteric isolates were also tested against a wide range of
antibiotics of varying ranges of concentrations using the same panels that were used for
species identification (Figure 1, shaded in pink). MICs for each antibiotic were generated
based on the ability of the organism to grow at various concentrations. The MICs were
then used to determine susceptibility of the organism to each antibiotic. Not all antibiotics
contained on the panels were useful in determining susceptibility, since some of them
23
were tested only at one concentration to aid in species identification. Kanamycin,
cephalothin, penicillin, chloramphenicol, nitrofurantoin, and colistin were tested at only
one concentration for most of the samples, so susceptibility data for these antimicrobials
was incomplete. Additionally, the use of three different panels resulted in not all of the
samples being tested for the same antibiotics. In order to analyze the data appropriately,
only those antibiotics that were tested on every sample and were able to generate an MIC
were used for tabulation of results and calculations. Antibiotics that were not tested on
every sample, and therefore omitted from calculations, include the following: cefotaxime,
cefoxitin, tetracycline, ticarcillin/K clavulanate, amoxicillin/K clavulanate, gatifloxacin,
norfloxacin, moxifloxacin, and meropenem. These omissions resulted in a reduced total
of 18 antibiotics (representing seven classes) that were used to generate resistance
profiles for all samples. Complete MIC data for all 18 of these antibiotics, as well as
omitted antibiotics described above, is listed in Appendix A.
For samples obtained from the same individual, their biochemical results and MIC
results were compared to determine uniqueness. Samples derived from the same donor,
but with differing results for two or more biochemical tests, differing MICs for more than
one antibiotic, or different species identifications were deemed to be unique. The only
exceptions to these criteria were for samples 0922A/0922B and M-5-Ea/M-5-Eb because
0922B and M-5-Ea were found to contain an integron, while 0922A and M-5-Eb do not
contain an integron. In all, seven samples were determined to not be unique, and
therefore were omitted from further analysis. Finally, one more sample (L-3-E) was
removed from the analysis of the results due to failure to propagate the sample from
24
frozen storage after it had been tested on the panels, but before it could be tested for the
presence of integrons. Therefore the final number of unique antibiotic-resistant enteric
isolates that were tested for integrons was 84.
Of the 84 unique isolates that were subsequently tested for the presence of
integrons, E. coli was the most commonly identified species comprising 76.2% (n=64) of
the samples. Other isolates included K. pneumoniae (4.8%, n=4), E. cloacae (4.8%, n=4),
K. ascorbata (4.8%, n=4), and several other species at lower frequencies (see Table 2 for
full species identification results, Appendix A for biotype numbers and confidence
levels). The average number of resistances per sample was 3.33. Resistance profiles
varied widely from sample to sample, from 13 resistances to zero resistances out of the
18 antibiotics tested for all samples. The antibiotic resisted most frequently was
ampicillin, with 55 isolates out of 84 (65.5%) growing at the highest concentration.
Piperacillin, ampicillin/sulbactam, and trimethoprim/sulfamethoxazole were also
frequently resisted (50.0%, 35.7%, and 34.5% respectively). Table 3 lists the 18
universally tested antibiotics along with susceptibility numbers.
PCR Detection of Class 1 and Class 2 Integrases
A total of 91 samples were tested for both class 1 and class 2 integrase genes via
PCR amplification. Of these samples, 84 were determined to be unique. A total of 19
(22.6%) isolates tested positive for a class 1 integron based on the amplification of 484bp
DNA fragments that matched the amplicon generated from the class 1 positive control
(Figure 4). As shown in Table 2, 15 samples were identified as E. coli, one as K.
25
Species
# Samples
Tested on
Panels
# Samples
Tested for
intI
# Unique
Samples
# Unique
Samples
Tested for
intI
Number
Unique
Samples
intI + (%)1
Escherichia coli
Klebsiella pneumoniae
Enterobacter cloacae
Kluyvera ascorbata
Escherichia fergusonii
Klebsiella oxytoca
Raoultella ornithinolytica
Salmonella sp.
Cedecea davisae
Citrobacter freundii
Enterobacter aerogenes
Total
68
5
5
4
2
2
2
1
1
1
1
92
67
5
5
4
2
2
2
1
1
1
1
91
65
4
4
4
1
2
2
1
1
1
1
85
64
4
4
4
1
2
2
1
1
1
1
84
15 (78.9)
1 (1.1)
0 (0)
1 (1.1)
0 (0)
1 (1.1)
1 (1.1)
0 (0)
0 (0)
0 (0)
0 (0)
19 (22.6)
Table 2. Species Identifications. 1 Percentage of positive unique isolates tested.
4 (21.1)
17 (89.5)
15 (78.9)
16 (84.2)
19 (100)
253 (13.3)
Piperacillin
Aztreonam
Ciprofloxacin
Levofloxacin
Imipenem
Total (Mean)
15 (0.79)
0 (0)
0 (0)
0 (0)
0 (0)
4 (21.1)
5 (26.3)
0 (0)
0 (0)
0 (0)
2 (10.5)
0 (0)
1 (5.3)
0 (0)
1 (5.3)
74 (3.9)
0 (0)
3 (15.8)
4 (21.1)
2 (10.5)
11 (57.9)
8 (42.1)
17 (89.5)
2 (10.5)
0 (0)
0 (0)
1 (5.3)
3 (15.8)
5 (26.3)
0 (0)
14 (73.7)
2 (10.5)
2 (10.5)
0 (0)
#R (%)
898 (13.8)
64 (98.5)
55 (84.6)
52 (80.0)
48 (73.8)
30 (46.1)
35 (53.8)
23 (35.4)
49 (75.4)
62 (95.4)
62 (95.4)
58 (89.2)
47 (72.3)
35 (53.8)
59 (90.8)
50 (76.9)
54 (83.1)
53 (81.5)
62 (95.4)
#S (%)
56 (0.86)
0 (0)
0 (0)
0 (0)
0 (0)
4 (6.2)
8 (12.3)
4 (6.2)
6 (9.2)
0 (0)
1 (1.5)
1 (1.5)
12 (18.5)
3 (4.6)
1 (1.5)
0 (0)
8 (12.3)
6 (9.2)
2 (3.1)
#I (%)
3 (4.6)
6 (9.2)
1 (1.5)
#R (%)
206 (3.2)
1 (1.5)
10 (15.4)
13 (20.0)
17 (26.2)
31 (47.7)
22 (33.8)
38 (58.5)
10 (15.4)
3 (4.6)
2 (3.1)
6 (9.2)
6 (9.2)
17 (26.2)
5 (7.7)
15 (23.1)
intI-negative (n=65)
N/S
N/S
N/S
N/S
N/S
<0.053
N/S
<0.042
N/S
N/S
N/S
N/S
N/S
N/S
N/S
<0.012
N/S
N/S
N/S
P-value1
Table 3. Susceptibility Comparisons Between Class 1 Positive and Class 1 Negative Isolates for Each Tested Antibiotic. S =
Susceptible; I = Intermediate; R = Resistant according to Dade Behring Procedural Manual (34). 1 N/S = Not Significant (P>0.05).
2
Significantly higher levels of resistance in intI-positive isolates. 3 Significantly lower susceptibility in intI-positive isolates
6 (31.6)
Ampicillin/Sulbactam
18 (94.7)
Cefepime
2 (10.5)
18 (94.7)
Ceftriaxone
Ampicillin
0 (0)
14 (73.7)
Cefuroxime
19 (100)
14 (73.7)
Cefazolin
17 (89.5)
18 (94.7)
Ceftazidime
Cefotetan
5 (26.3)
Trimethoprim/ Sulfamethoxazole
Piperacillin/Tazobactam
1 (5.3)
16 (84.2)
Tobramycin
1 (5.3)
16 (84.2)
Gentamicin
0 (0)
#I (%)
19 (100)
#S (%)
intI-positive (n=19)
Amikacin
Antibiotic
26
27
1
500bp
2
3
4
5
6
7

Figure 4. Class 1 Integron Detection. Post-EtBr stained agarose gel showing
484bp PCR-amplified fragments of the Class 1 integrase gene intI1. Lane 1:
positive control, Salmonella DT104; Lane 2: 100bp DNA ladder; Lane 3:
sample L-1-TET; Lane 4: L-4-TET; Lane 5: L-5-TET; Lane 6: M-16-TET;
Lane 7: negative control.
28
pneumoniae, one as Kluyvera ascorbata, and one as Raoultella ornithinolytica. Only one
sample tested positive for a class 2 integron, determined by the visualization of a 233bp
band identical to that produced by the class 2 positive control (Figure 5). Interestingly the
sample containing the class 2 integron also was one of the 19 samples that tested positive
for a class 1 integron.
Statistical Significance of Resistance and Integrons
The statistical significance of the relationship between resistance and the presence
or absence of integrons was examined in several ways. For all analyses, the Fisher Exact
Probability Test or Chi-Square Test was used to calculate P-values, with preference for
the Chi-Square Test where applicable. Calculations were made using an online program
at vassarstats.net. Based on the nature of the input data, the program determined whether
or not the Chi-Square Test could be performed. Significance was deemed to be a P-value
of <0.05. For each of the 18 tested antibiotics, the significance of the relationship
between susceptibility (resistant, intermediate, or susceptible) and presence or absence of
the gene intI1 was calculated. Resistance was significantly more prevalent in intI1positive isolates than in intI1-negative isolates for two antibiotics: trimethoprim/
sulfamethoxazole and ampicillin (Table 3). In addition, susceptibility to the antibiotic
piperacillin was found to be significantly lower in intI1-positive isolates compared to
intI1-negative isolates (Table 3).
Since there is no clear consensus on the definition of multidrug-resistance in the
literature (26), the significance of the relationship between multidrug-resistance and
29
1
300bp
200bp
2
3
4
5
6
7
8


Figure 5. Class 2 Integron Detection. Post-EtBr stained agarose gel showing
233bp PCR-amplified fragments of the Class 2 integrase gene intI2. Lane 1:
100bp DNA ladder ; Lane 2: positive control, E. coli ATCC# 47055; Lane 3:
sample F06-2-AMP; Lane 4: F06-11-AMP; Lane 5: F06-12-AMP; Lane 6:
F06-28-AMP; Lane 7: F06-30-AMP; Lane 8: negative control
30
presence or absence of an integron was calculated in three different ways. Table 4 and
Figure 6 show that a significantly greater number of intI1-positive isolates were resistant
to at least one antibiotic in two or more antibiotic classes compared to intI1-negative
isolates. Comparisons between the actual number of resistant integrase-positive and
integrase-negative isolates for each number of antibiotic classes yielded no significance
(P>0.05, See Figure 7). Inclusion of intermediate susceptibilities in the definition of
“resistant” (26), also yielded no significance between multidrug-resistance and integron
presence (Table 5). Finally, Table 6 and Figure 8 show the significance between integron
presence and the number of resistances to individual antibiotics. It was found that
resistance to two or more antibiotics, regardless of class was statistically greater in intI1positive isolates compared to intI1-negative isolates. Figure 9 shows the actual number of
resistant integrase-positive and integrase-negative isolates for each number of antibiotics,
regardless of class; no significance was found in this analysis.
PCR Amplification of Variable Regions
All 19 samples that produced positive results for the presence of the class 1
integrase gene, intI1, were further investigated by amplifying the variable region of the
integron. Analysis of EtBr-stained agarose gels yielded 6 amplicons of distinctly different
sizes: ~250bp (n=3), ~900bp (n=3), ~1100bp (n=2), ~1800bp (n=5), ~2000 (n=3), and
>3000bp (n=1). Additionally, two samples failed to produce any noticeable product, most
likely due to loss or mutation of the 3’-conserved region. The gel pictured in Figure 10
shows the various sizes (900->3kbp) of class 1 variable region amplicons.
31
# Antibiotic
Classes
≥1
≥2
≥3
≥4
≥5
Integrase Positive (n=19)
# samples
# samples not
Resistant (%)
Resistant (%)
18 (94.7)
1 (5.3)
16 (84.2)
3 (15.8)
7 (36.8)
12 (63.2)
4 (21.1)
15 (78.9)
2 (10.5)
17 (89.5)
Integrase Negative (n=65)
# samples
# samples not
Resistant (%)
Resistant (%)
52 (80.0)
13 (20.0)
36 (55.4)
29 (44.6)
17 (26.2)
48 (73.8)
9 (13.8)
56 (86.2)
2 (3.1)
63 (96.9)
P-value1
N/S
<0.03
N/S
N/S
N/S
Table 4. Comparison Between intI1-positive and intI1-negative Isolates Resistant to Multiple
Classes of Antibiotics. 1 N/S = Not Significant (P >0.05)
Figure 6. Percentage of Cumulative Resistant Integrase-positive vs. Integrase-negative Isolates
for Varying Numbers of Antibiotic Classes. Resistance to two or more antibiotic classes was
found to be significantly higher in intI1-positive isolates compared to intI1-negative isolates
(P<0.05).
32
Figure 7. Number of Resistant Integrase-positive vs. Integrase-negative Isolates for Varying
Numbers of Antibiotic Classes. No significance found for any number of antibiotic classes
(P>0.05).
33
34
# Antibiotic
Classes
≥1
≥2
≥3
≥4
Integrase Positive (n=19)
# samples Res.
# samples not
or Int. (%)
Res. or Int. (%)
18 (94.7)
1 (5.3)
16 (84.2)
3 (15.8)
8 (42.1)
11 (57.9)
4 (21.1)
15 (78.9)
Integrase Negative (n=65)
# samples Res.
# samples not
or Int. (%)
Res. or Int. (%)
56 (86.2)
9 (13.8)
43 (66.2)
22 (33.8)
24 (36.9)
41 (63.1)
14 (21.5)
51 (78.5)
P-value1
N/S
N/S
N/S
N/S
Table 5. Comparison Between intI1-positive and intI1-negative Isolates With Intermediate or
Resistant Phenotypes for Multiple Classes of Antibiotics. 1 N/S = Not Significant (P>0.05).
# of
Antibiotics
≥1
≥2
≥3
≥4
≥5
≥6
≥7
≥8
≥9
≥10
≥11
≥12
≥13
Integrase Positive (n=19)
# samples
# samples not
Resistant
Resistant
18
1
18
1
14
5
9
10
6
13
4
15
3
16
2
17
1
18
0
19
0
19
0
19
0
19
Integrase Negative (n=65)
# samples
# samples not
Resistant
Resistant
52
13
44
21
33
32
23
42
17
48
14
51
10
55
6
59
2
63
2
63
2
63
1
64
0
65
P-value1
N/S
<0.04
N/S
N/S
N/S
N/S
N/S
N/S
N/S
N/S
N/S
N/S
N/S
Table 6. Comparison Between intI1-positive and intI1-negative Isolates Resistant to Multiple
Antibiotics, Regardless of Class. 1 N/S = Not Significant (P>0.05).
Figure 8. Percentage of Cumulative Resistant Integrase-positive vs. Integrase-negative Isolates for
Varying Numbers of Antibiotics, Regardless of Class. Resistance to two or more antibiotics,
regardless of class, was found to be significantly higher in intI1-positive isolates compared to
intI1-negative isolates (P<0.05).
35
Figure 9. Number of Resistant Integrase-positive vs. Integrase-negative Isolates for Varying
Numbers of Antibiotics, Regardless of Class. No significance found for any number of antibiotics
(P>0.05).
36
37
1
2
3
4
5
6
7
8
3000bp
1500bp
1000bp
Figure 10. Class 1 Integron Variable Regions. Post-EtBr stained agarose gel showing
multiple variable region sizes. Lane 1: 100bp DNA ladder; Lane 2: positive control,
Salmonella enterica DT104 showing double integron; Lane 3: sample 0901; Lane 4: 0919;
Lane 5: L-1-TET; Lane 6: F06-12-AMP; Lane 7: 0812; Lane 8: negative control.
38
The variable region of the only detected class 2 integron (sample F06-2-AMP)
was also amplified by PCR. Analysis of the PCR product via gel electrophoresis and
subsequent staining with EtBr showed a single DNA band of approximately 2.5kb. Figure
11 shows the stained gel along with the class 2 positive control, which yielded an
amplicon of the same size.
Restriction Digest of Variable Region Amplicons
Class 1 variable region PCR products that appeared similar in size were subjected
to restriction enzyme AluI to aid in identification of unique sequences. Analysis of
restriction fragments on EtBr-stained gels yielded identical patterns for all samples within
respective size groups (250bp, 900bp, 1.1kbp, 1.8kbp, and 2kbp). The 250bp samples
produced no observable changes after exposure to AluI. This result is consistent with the
assumption that these PCR products were simply the amplified conserved regions without
any cassettes, and the fact that the conserved regions between the primers do not contain
a target site for AluI. Each of the 900bp products yielded two distinct DNA fragments,
while the 1.1kbp products yielded four distinct DNA fragments. The 1.8kbp amplicons
yielded at least five distinct bands of DNA, though some smaller fragments of similar
size may have been present and indistinguishable from each other. Likewise, the 2kbp
amplicons yielded at least seven bands, not all of which are completely distinguishable
due to multiple small fragments of similar size (See Table 7 for restriction fragment
lengths). Though the 1.8kbp and 2kbp amplicons produced restriction fragments that are
not all distinguishable from each other, it is clear that the patterns are identical within
39
1
2
3
4
5
6
7
8
3000bp
1500bp
Figure 11. Class 2 Integron Variable Regions. Post-EtBr stained agarose gel showing
~2500bp PCR-amplified variable regions. Lane 1: 100bp DNA ladder; Lane 6: positive
control, E. coli ATCC# 47055; Lane 7: sample F06-2-AMP; Lane 8: negative control.
40
Amplicon
Length
Observed (~bp)
0
Amplicon
Length
Actual (bp)
0
Restriction
Fragment LengthsObserved (~bp)
N/A
Restriction
Fragment LengthsActual (bp)
N/A
250
232
250
232
900
845
400, 500
367, 478
1100
1085
120, 280, 350, 400
114, 257, 342, 372
1800
1742
50, 220, 250, 350,
650
0901, F06-2AMP, 0813
2000
1979
1
F06-12-AMP
F06-2-AMP3
>3000
2500
Unknown
22244
Samples
0904, 0911B
M-5-Ea, F0639-AMP, M16-TET
L-1-TET,
0807, 0904
0812, 0916B
0914, 0919,
0922B, 0922C,
0805
50, 100, 1502, 200,
250, 350, 400
NT
NT
52, 57, 64, 60, 225,
261, 366, 657
9, 10, 25, 26, 76, 99,
113, 114, 119, 145,
147, 166, 248, 330,
3621
Unknown
Unknown
Table 7. Observed vs. Actual Amplicon and Restriction Fragment Sizes. Observed amplicon
and restriction fragment sizes are based on visualization of bands on Et-Br stained agarose gels
compared to a 1kb DNA ladder. Actual amplicon and fragment lengths were calculated based
on database sequences that aligned with sample sequences. N/A = not applicable; NT =
restriction fragment analysis was not tested-only one sample yielded an amplicon of this size;
Unknown = cannot be determined due to incomplete cassette identification; 1 Fragment lengths
and actual amplicon size based on possible unverified centrally located orf ; 2 Multiple
indistinguishable bands between 100 and 200bp; 3 Only class 2 positive sample; 4 Amplicon
length based on probable unverified cassette.
41
both groups and therefore likely the of the same sequence (See Figure 12 for restriction
digest gels).
Sequencing and Cassette Identification
A single representative sample from each variable region size group was
sequenced from both the 5’ and 3’ conserved ends using the same primers that were used
in the variable region PCR amplifications. While sequencing results varied and did not
reflect full coverage of the entire template, most sequencing results did produce enough
data to identify most, if not all inserted cassettes. Figure 13 shows the partial alignment
of sample 0812 with a class 1 integron from a Vibrio cholerae strain (GenBank ID:
GQ214171.1) containing cassette aadA1. Sequencing reactions yielded sufficient data to
produce an alignment with part of the gene cassette at both the 5’ and 3’ ends, with
sequence identities of 96% to 100%. Cassette identifications were further corroborated
through comparison of the restriction digest results of the amplicons compared to the
expected restriction fragment sizes. Each occurrence of the AluI target site, AGCT, was
identified in the GenBank sequences to which the sample sequences aligned, and the
expected fragment sizes were calculated (Figure 13 shows the expected AluI target sites
for the 1.1kbp amplicons). Restriction fragment sizes matched very closely for the 250bp,
900bp, 1.1kbp, and 1.8kbp variable regions, as shown in Table 7. Similarly, the expected
size of the complete amplicons were calculated and compared to estimated observed
lengths. Table 7 shows that total observed and actual amplicon lengths are also closely
matched. Together, the partial cassette alignments, identical restriction fragment patterns,
42
1
2
3
4
5
6
7
8
1
A.
2
3
4
5
B.
500bp
400bp
100bp
1
2
3
4
5
6
C.
700bp
400bp
100bp
Figure 12. Class 1 Variable Region Restriction Fragments. Post EtBr-stained agarose gels
showing DNA fragments of class 1 variable regions after exposure to AluI. A.) Lane 1: 100bp
DNA ladder; Lane 2: positive control, multiple fragments of double integron; Lanes 3-4:
fragments of 1.1kbp amplicons; Lanes 5-7: fragments of 2kbp amplicons; Lane 8: negative
control. B.) Lane 1: 100bp DNA ladder; Lane 2: positive control; Lanes 3-5: fragments of 900bp
amplicons. C.) Lanes 1, 3-6: fragments of 1.8kbp amplicons; Lane 2: 100bp DNA ladder.
937
12
997
36
1057
96
1116
1176
1236
1296
1356
1416
1476
1536
421
1596
393
1656
333
GQ214171.1
0812-5’
GQ214171.1
0812-5’
GQ214171.1
0812-5’
GQ214171.1
GQ214171.1
GQ214171.1
GQ214171.1
GQ214171.1
GQ214171.1
GQ214171.1
GQ214171.1
0812-3’
GQ214171.1
0812-3’
GQ214171.1
0812-3’
TCCCGCATTTGGTACAGCGCAGTAACCGGCAAAATCGCGCCGAAGGATGTCGCTGCCGAC
............................................................
ACGCTATGGAACTCGCCGCCCGACTGGGCTGGCGATGAGCGAAATGTAGTGCTTACGTTG
.......................G....................................
GAGGAACTCTTTGATCCGGTTCCTGAACAGGATCTATTTGAGGCGCTAAATGAAACCTTA
.......N.....A..............
CTGGCTATCTTGCTGACAAAAGCAAGAGAACATAGCGTTGCCTTGGTAGGTCCAGCGGCG
GAATGGCAGCGCAATGACATTCTTGCAGGTATCTTCGAGCCAGCCACGATCGACATTGAT
GTTGTGCACGACGACATCATTCCGTGGCGTTATCCAGCTAAGCGCGAACTGCAATTTGGA
TTGGAAACTTCGGCTTCCCCTGGAGAGAGCGAGATTCTCCGCGCTGTAGAAGTCACCATT
CTGGTTACGGTGACCGTAAGGCTTGATGAAACAACGCGGCGAGCTTTGATCAACGACCTT
GTACATTTGTACGGCTCCGCAGTGGATGGCGGCCTGAAGCCACACAGTGATATTGATTTG
ACTCAACTATCAGAGGTAGTTGGCGTCATCGAGCGCCATCTCGAACCGACGTTGCTGGCC
GCAGTCGCCCTAAAACAAAGTTAAACATCATGAG-GGAAGCGGTGATCGCCGAAGTATCG
.........................A.......NN........... 141
CGCGTTACGCCGTGGGTCGATGTTTGATGTTATGGAGCAGCAACGATGTTACGCAGCAGG
............................................................
TCATGGCTTGTTATGACTGTTTTTTTGTACAGTCTATGCCTCGGGCATCCAAGCAGCAAG
........................
1715
274
1655
334
1595
394
1535
1475
1415
1355
1295
1235
1175
1115
1056
95
996
35
43
1776
213
1836
153
1896
93
1956
33
2016
GQ214171.1
0812-3’
GQ214171.1
0812-3’
GQ214171.1
0812-3’
GQ214171.1
0812-3’
GQ214171.1
CCCTAC
2021
GCTCACAGCCAAACTATCAGGTCAAGTCTGCTTTTATTATTTTTAAGCGTGCATAATAAG
......................... 9
AGCCGACCGCGCTACGCGCGGCGGCTTAACTCCGGCGTTAGATGCACTAAGCACATAATT
.......GC....T....GC............AA..........................
CACTACGTGAAAGGCGAGATCACCAAGTCAGTTGGTAAATGATGTCTAACAATTCGTTCA
...........................GT...C..C....A...................
TATCTTGGACAAGAAGAAGATCGCTTGGCCTCCCGCGCAGATCAGTTGGAAGAATTTGTT
................................G..........................C
TGGGCAATGGAGCGCCTGCCGGCCCAGTATCAGCCCGTCATACTTGAAGCTAGACAGGCT
............................................................
2015
1955
34
1895
94
1835
154
1775
214
Figure 13. Variable Region Partial Alignment. This diagram shows the partial alignment of the variable region from
sample 0812 with a class 1 integron in Vibrio cholerae containing cassette aadA1 (GenBank ID: GQ214171.1). The
alignment shows the results of two separate sequencing reactions performed on sample 0812 from both the 5’ and 3’
conserved regions. The two primers used for amplification of the variable region and sequencing are located at the
ends of the sequence, underlined in bold. Alignments of sequencing results with GQ214171.1 are shaded. Nonshaded regions indicate no sequence coverage (middle), or poor sequence coverage (ends). Bold shaded regions
indicate sequence alignments of the gene cassette, aadA1. Sequence identity is 96% for the covered regions. The
three target sequences (AGCT) for restriction enzyme AluI are also in bold and underlined.
1716
273
GQ214171.1
0812-3’
44
45
and closely matched amplicon sizes strongly support the correct identification of gene
cassettes in the 250bp-1.8kbp amplicons.
For the longer variable region amplicons (2kb and >3kbp), identification of all
cassettes is incomplete. Based on the expected sizes of the gene cassettes identified at the
5’ and 3’ ends of the variable region of sample 0901 (total length: ~2kb), a third centrally
located cassette is likely to exist. The Genbank sequence (HM589046.1) that aligned best
with the sequencing results for sample 0901 contains a small open reading frame, orfF,
between gene cassettes dfrA12 and aadA2. Though no portion of this open reading frame
was sequenced in this study, it is possible that orfF may be present in the 2.0kbp samples
(0901, F06-2-AMP, 0813). The total expected size of the amplicon, including orfF, is
1,989bp, almost exactly the size of the observed estimated length. Furthermore, while
digestion fragments are difficult to distinguish from each other on the gels, it is possible
to make out several approximates sizes, as pictured in Figure 12 and listed in Table 7.
Using the sequence from the Genbank database to identify cleave sites for AluI, a list of
several expected fragment sizes was generated (Table 7). The expected fragment sizes do
coincide with the observed fragment sizes, lending support for the identification of the
central cassette in the 2.0kbp amplicons as orf5. For sample F06-12-AMP (total length:
>3kb), multiple attempts at sequencing from the 3’ end failed. The single cassette
identified from the 5’ end is only about 820bp in length, indicating that several additional
cassettes are likely to be present downstream.
The amplicon from class 2 positive sample, F06-2-AMP, was also too large to
identify all cassettes. While the 5’ and 3’ cassettes were determined through two solid
46
partial alignments with GenBank # AY736324.1, a third probable centrally located
cassette was unable to be directly identified. Based on the findings from other
researchers, the centrally located cassette in Tn7-bound integrons is often identified as
sat1 or sat2, which confer resistance to streptothricin (6, 21, 37). This is likely the central
cassette in both the class 2 positive control (chosen as a control because it contains a Tn7
transposon) and in the class 2 integron of sample F06-2-AMP. The total expected size of
amplicon based on the GenBank sequence above is 2,224bp, which is near the estimated
observed size.
In addition to the two putatively identified gene cassettes described above, two
main types of cassettes were determined to be present in the variable regions:
aminoglycoside-adenyltransferases, which confer resistance to aminoglycosides, and
dihydrofolate reductases, which confer resistance to trimethoprim (12). A total of four
different aminoglycoside-adenyltransferases (aadA1, aadA2, aadA5, and aadB) and four
different dihydrofolate reductases (dfrA1, dfrA7, dfrA12, and dfrA17) were identified.
Only one gene cassette (aadA1) was observed in two different amplicon groups: the
1.1kbp class 1 group and the sole 2.5kbp class 2 amplicon. Gene cassettes and their
positions relative to the conserved regions are listed in Table 8. The observed cassette
combinations, illustrated in Figure 14, have all been identified in previous studies, and
appear to be quite common configurations (6, 10, 12, 21, 37, 50).
47
Sample
0901, F06-2-AMP,
08131
0914, 0919,
0922B, 0922C,
0805
0812, 0916B
F06-12-AMP1
L-1-TET, 0807,
0904
F06-2-AMP1, 2
5’ End Cassette
3’ End Cassette
GenBank ID
dfrA12
aadA2
HM589046.1
dfrA17
aadA5
HM367091.1
aadA1
aadB
N/A
Unknown
GQ214171.1
HQ914240.1
dfrA7
N/A
FN995455.1
dfrA1
aadA1
AY736324.1
Table 8. Identified Gene Cassettes. This table shows the gene cassettes that
were identified through amplification and subsequent sequencing of class 1
and class 2 variable regions. The GenBank sequence with the greatest identity
to sample sequences is listed. N/A = only one cassette is present.
1
Incomplete cassette identification-additional cassettes between 5’ and 3’
cassettes are likely to exist. 2 Class 2 Integron.
F06-2-AMP
F06-12-AMP
0901, F06-2-AMP,
0813
0914, 0919, 0922B,
0922C, 0805
0812, 0916B
L-1-TET, 0807,
0904
M-5-Ea, M-16-TET,
F06-39-AMP
dfrA1
aadB
dfrA12
dfrA17
aadA1
dfrA7
qacEΔ1
sat1/sat2
orfF
aadA5
aadA1
ybeA
Unknown Cassettes
orf5
sul1
orf5
qacEΔ1
qacEΔ1
sul1
orf5
sul1
aadA2
qacEΔ1
qacEΔ1
sul1
qacEΔ1
ybfA
sul1
orf5
orf5
sul1
orf5
Figure 14. Gene Cassette Arrangements in Class 1 and Class 2 Integrons. Gray arrows indicate gene cassettes positively
identified through sequencing. * Black arrows indicate probable cassettes that were not identified through sequencing.
White arrows indicate genes located in conserved regions of the integrons. ** Class 2 integron.
intI2
intI1
intI1
intI1
intI1
intI1
intI1
48
49
DISCUSSION
The purpose of this study was to determine the significance of the relationship
between multidrug-resistance and the presence of integrons in enteric bacteria isolated
from healthy adults, and to identify gene cassettes within those integrons that may
contribute to such resistances. Previously, numerous studies have shown that integrons
play a considerable role in the dissemination of MDR in clinical isolates and
environmental samples. Fewer studies have aimed at investigating the role of integrons in
the propagation and maintenance of MDR phenotypes in non-clinical commensal isolates
from humans. There are probably at least two reasons for the lack of studies investigating
integrons in commensal isolates from healthy people. One reason may be due to the lack
of availability of samples. While bacteria are routinely obtained from hospitalized
patients in order to identify and treat infections, healthy people may be far less likely to
voluntarily submit to a rectal swab. Perhaps a second reason is that studying bacteria
isolated from clinical samples and from individuals with infections can easily seem like a
more pressing matter, and seem more vital to understanding the problem of the rapid
spread of multidrug-resistance. However, understanding the distribution and
dissemination of resistance factors in pathogens wreaking havoc in hospitals is only part
of a larger picture. It is possible that the general community surrounding these hospitals
is harboring resistant organisms that cause no symptoms for their host, and these bacteria
could play an important role as a reservoir for resistance genes that eventually make their
way into the clinical setting.
50
This study seized upon the opportunity to investigate a unique set of enteric
isolates that were obtained over the course of five years from healthy adults. The isolates
were subjected to numerous antibiotics and biochemical tests to determine their
resistance phenotypes and species. After identifying several bacteria that were resistant to
multiple antibiotics, it was hypothesized, based on the findings of previous studies, that
class 1 and class 2 integrons would be more prevalent in MDR bacteria than in non-MDR
bacteria. A total of 19 isolates (22.6%) tested positive for a class 1 integron. Only one
isolate was found to contain a class 2 integron, which interestingly also contained a class
1 integron.
When antibiotic resistance data was compared with the presence of integrons, the
results obtained do seem to support the stated hypothesis, depending on the exact
definition of multidrug-resistance that is used. Among the scientific community, there
seems to be no consensus on how many antibiotics or antibiotic classes constitute
multidrug-resistance. While some studies seem to classify MDR as resistance to two or
more antibiotic classes (23, 26), others claim three or more classes (48). Resistance to an
entire class of antibiotics is often taken to mean that the organism must be resistant to at
least one antibiotic (of those that are tested) within a particular class. Results pertaining
to the significance or prevalence of MDR bacteria are very dependent on which
antibiotics are tested from the various categories. Studies generally choose one or two
representative antibiotics from each class to test in order to identify MDR samples.
However, the only way to ensure an organism is susceptible to all antibiotics within a
class is to test the isolate against each antibiotic in that class. Otherwise it is probable that
51
the levels of resistance (at the class level) will be underestimated. Still, other studies
make no reference to antibiotic classes, and simply imply multidrug-resistance to mean
resistance to two or more individual drugs (30, 32). Recently a proposal was published as
a joint effort between the European Centre for Disease Prevention and Control (ECDC)
and the CDC to create standardized definitions for “multidrug-resistant”, “extensively
drug-resistant”, and “pandrug-resistant” (26). They created a unique set of antibiotic
categories for various groups of organisms, including Enterobacteriaceae, and defined
multidrug-resistance as resistance or intermediate resistance to at least one antibiotic in at
least three well-defined categories (26). This type of clear definition is needed in order to
improve the ability to compare results from the multiple studies regarding MDR bacteria.
If such a definition can be agreed upon, it would eliminate the need to test isolates against
every antibiotic, and only test those that have been deemed necessary for determining
multidrug-resistance.
The antibiotic resistance data obtained from this study was analyzed using each of
the definitions described above. In addition, complete MIC data for every isolate in this
study is available in Appendix A to allow the reader to analyze the data using a different
definition of MDR, should they choose. The results obtained do support the stated
hypothesis depending on the definition of MDR that is applied. It was found that
integron-positive isolates were statistically more prevalent in isolates that were resistant
to two or more classes of antibiotics (Table 4, Figure 6) and in isolates that were resistant
to two or more antibiotics, regardless of class (P<0.05) (Table 6, Figure 8). However, no
significant relationship between integrons and MDR was found in isolates resistant to
52
three or more antibiotic classes. When intermediate resistances were included with
resistant phenotypes, as described by Magiorakos et al. (26), no statistically significant
relationship (P>0.05) between drug resistance and integron presence was determined for
any number of classes (Table 5). Overall, the results do suggest that integrons play a
significant role in the dissemination and maintenance of MDR factors in commensal
bacteria, though not for all definitions of MDR.
The significant relationship between integrons and isolates that were resistant to
two or more antibiotics or antibiotic classes, is due to the high levels of resistance to
trimethoprim/sulfamethoxazole and ampicillin (and to a lesser extent, piperacillin)
observed in integron-positive isolates. While the combination drug trimethoprim/
sulfamethoxazole belongs to the antifolate/sulfonamide class of antibiotics, ampicillin is
member of the penicillin-class. Therefore, these two antibiotics alone account for the
significant relationship between integron presence and multidrug-resistance for at least
two of the definitions of MDR. Susceptibility to piperacillin, also a member of the
penicillin-class of antibiotics, was found to be lower in integron-positive isolates
compared to integron-negative isolates, and therefore may also be a contributing factor in
the multidrug-resistance observed in integron-containing isolates. Other studies have also
found these three antibiotics to be strongly correlated with the presence of integrons (23,
30, 50). Interestingly, these studies also found that several other antibiotics, such as
gentamicin, tobramycin, ticarcillin, and aztreonam, strongly correlated with integron
presence, while no such significance was found for the same antibiotics in our isolates.
White et al. (50) also found that chloramphenicol and tetracycline were significantly
53
associated with the presence of integrons. These two antimicrobials were not tested on all
of the samples in our study due to the use of multiple different testing panels, and
therefore were not included in the statistics. Exclusion of these antibiotics may have
resulted in an underestimation of the significance of the relationship between integrons
and MDR. It is also worth noting that the highest levels of resistance were among
integron-negative isolates, supporting the fact that although integrons may play a role in
the dissemination of resistance factors, they are by no means the only genetic factor.
The difference in the association of particular individual antibiotic resistances and
integrons between these studies (23, 30, 50) and the current study may also be attributed
to sample size. Leverstein et al. (23) investigated over 800 unique isolates, while this
study only looked at 84 unique samples. White et al. (50) and Martinez-Freijo et al. (30)
studied sample sizes of 120 and 171, respectively. The relatively small sample size used
in this study may also be attributable to the lack of significance between integron
presence and MDR for all definitions. Undoubtedly, a larger sample size would produce
more accurate statistics. The hope is to continue collecting commensal isolates from
volunteers in order to build the sample size over a period of years. The data and statistics
from previous studies, such as this one, could then be combined to hopefully improve
upon the statistics, and gain further insight into the role of integrons in the community
over a period of time. Another difference between this study and the previous studies
mentioned is of course the source from which samples were collected. While this study
focused only on commensal isolates, the studies above included clinical isolates.
54
This study also identified species through biochemical testing on dried MicroScan
testing panels. Though species identification was not paramount to this investigation, it
did yield some interesting results. Not surprisingly, the majority of integron-containing
isolates were identified as E. coli (n=15, 79%), as 76% (n=64) of the samples tested were
also E. coli (Table 2). Four other species were also found to contain a class 1 integron,
including one sample that was identified as Raoultella ornithinolytica (Sample M16TET, Appendix A). However, the biochemical testing results yielded a probability of
<85% for the identification of this isolate. Even after the organism was re-isolated and
tested again, as suggested by the biotype lookup program (44), identical results were
produced. Additional testing would need to be performed on this isolate in order to
confirm its identity. To date, no studies have shown R. ornithinolytica to contain an
integron, and based on the low probability given by the biotype lookup program, its
identity is likely inaccurate. All panels were analyzed manually, and two different
individuals interpreted the results. Baker ran and recorded panel results for a total of 32
of the unique samples (4). Though care was taken to record results as accurately as
possible based on the guidelines established by MicroScan/Dade Behring (34), the
possibility for inconsistencies and inaccuracies in the results does exist, especially
considering that more than one person performed analyses. Computer analysis of panels
would help to ensure consistency and accuracy of interpreting panel results, though
access to such equipment was not available for use in this study.
Sequencing and restriction fragment analysis of integron variable regions from
integron-containing isolates revealed eight different gene cassettes in six different arrays.
55
Four of the cassettes were identified as aminoglycoside-adenyltransferases (aadA1,
aadA2, aadA5, and aadB) and four as dihydrofolate reductases (dfrA1, dfrA7, dfrA12, and
dfrA17). The dfr genes are known to confer resistance to trimethoprim, while the aad
genes confer resistance to aminoglycosides (12). These cassettes and configurations
(Figure 14) have previously been found in other studies and are commonly found in class
1 and class 2 integrons (6, 10, 12, 21, 37, 50). The presence of dfr gene cassettes in 11 of
the 19 integron-containing isolates may be attributable to the high levels of resistance to
trimethoprim/sulfamethoxazole observed. The sul1 gene within the 3’-conserved region,
which confers resistance to sulfanamides, is likely also a contributing factor to the high
levels of resistance to this combination drug. Interestingly, the presence of aad gene
cassettes did not lead to higher levels of resistance in intI-positive samples compared to
intI-negative samples. In fact, only two of the eleven (18%) isolates found to contain an
aad gene cassette (0916B and F06-12-AMP) exhibited resistance to any of the
aminoglycosides tested. The observed susceptibility to aminoglycosides despite
containing genes that confer resistance to them may be due to inefficient expression, as
the aad gene is not the first one to be transcribed in eight of the eleven isolates (Figure
14). Although resistance to ampicillin, and to some extent piperacillin (higher levels of
resistant and intermediate phenotypes), was significantly higher among intI-positive
isolates, no gene cassettes were identified in any of the samples that are known to confer
resistance to these drugs. These results suggest that although integrons may be associated
with higher levels of resistance, it may not necessarily be due to the gene cassettes they
carry. The high levels of resistance to ampicillin and piperacillin observed among
56
integron-positive isolates may be due to genetic linkage between plasmid-bound
integrons and resistance factors carried on the plasmid, outside of the integron region.
Overall, the results of this study do support the hypothesis that multidrugresistance is higher among integron-containing enteric commensal isolates from humans,
although genetic linkage between integrons and plasmids they reside on, may play a role
in conferring such high levels of resistance. These results also support the findings of
other similar studies investigating the role of integrons in the dissemination and
maintenance of resistance factors in Gram-negative enteric bacterial isolates acquired
from a variety of sources, including animals and infected patients. In this study, integron
prevalence was found to be 22.6%, which is perhaps on the lower end of the spectrum
compared to the much higher levels often found in pathogenic bacterial isolates derived
from hospitalized patients. However, considering that the isolates in this study were
commensal enteric bacteria obtained from healthy people, the lower prevalence of
integrons in this sample-set is, perhaps, not so surprising, and rather alarming. The
prevalence of integrons in commensal MDR bacteria revealed by this study does lend
support to the idea that commensal bacteria containing integrons may act as a reservoir
for resistance genes in the community. Additional insight into the role of integrons in
commensal bacteria could be gained through further sampling and testing, and continuing
to build upon the data and results that have been presented in this study. Already,
agencies such as the CDC have programs in place, such as the National Antimicrobial
Resistance Monitoring System (NARMS-http://www.cdc.gov/narms/) to track antibiotic
resistances in clinical samples. Though this study represents only a very small sample of
57
bacteria from an even smaller number of people, it does provide a rare glimpse into the
less-extensively researched and unmonitored resistance patterns of commensal enteric
bacteria. A complete understanding of how bacteria, both in the clinical and non-clinical
setting, acquire, disseminate, and harbor resistance factors will likely be needed to
combat the ever-increasing rise of MDR organisms.
58
APPENDIX
59
Appendix A: Complete antibiotic MIC data, biotype numbers, species and integron
detection results
Antibiotic Class
Antibiotic
Sample #/MICs (g/ml)
F06-12F06-28AMP
AMP
4
4
=16 (S)
4 (S)
8 (R)
1 (S)
8 (R)
1 (S)
2/38 (R)
2/38 (S)
8
8
4 (S)
32 (R)
2 (S)
16 (R)
16 (R)
16 (R)
=16 (I)
4 (S)
8 (S)
32 (R)
2 (S)
2 (S)
NT
NT
16 (S)
16 (S)
NT
NT
4
4
8 (S)
8 (S)
16 (R)
16 (R)
8/4 (S)
16/8 (R)
64 (R)
16 (S)
16 (S)
64 (R)
NT
NT
8 (S)
16 (R)
NT
NT
NT
NT
2 (R)
1 (S)
4 (R)
2 (S)
4 (R)
2 (S)
4 (S)
4 (S)
4 (S)
4 (S)
4
4
64
64
NT
NT
+
-
F06-2AMP
4
4 (S)
1 (S)
=4 (S)
2/38 (R)
8
4 (S)
2 (S)
16 (R)
=16 (I)
8 (S)
2 (S)
NT
16 (S)
NT
4
64 (R)
16 (R)
16/8 (R)
64 (R)
16 (S)
NT
16 (R)
NT
NT
1 (S)
2 (S)
2 (S)
4 (S)
4 (S)
4
64
NT
-
F06-11AMP
4
4 (S)
1 (S)
=4 (S)
2/38 (S)
8
4 (S)
2 (S)
16 (R)
4 (S)
8 (S)
2 (S)
NT
16 (S)
NT
4
64 (R)
16 (R)
8/4 (S)
64 (R)
16 (S)
NT
8 (S)
NT
NT
2 (R)
4 (R)
2 (S)
4 (S)
4 (S)
4
64
NT
-
7R; 1I
6R; 0I
8R; 0I
53117472
73017612
Species/Confidence
E. coli
<85%
E. coli
<85%
Class 1 Integron
Class 2 Integron
+
+
-
77117652
K.
ascorbata
85%
+
-
(K)**
(Ak)
Aminoglycosides
(Gm)
(To)
Antifol/Sulfa
(T/S)
(Cf)*
(Cft)***
(Caz)
(Cfz)
Cephalosporins
(Crm)
(Cax)
(Cpe)
(Cfx)***
(Ctn)
Tetracyclines
(Te)***
(P)**
(P/T)
(Am)
Penicillins
(A/S)
(Pi)
(Tim)***
(Aug)***
Monobactams
(Azt)
(Gat)***
(Nxn)***
(Cp)
Quinolones
(Lvx)
(Mxf)***
(Imp)
Carbapenems
(Mer)***
(Cl)**
Other
(Fd)*
(C)***
ESL-a Screen
ESL-b Screen
Total #
Resistant/Intermediatea
Biotype ID #
F06-30AMP
4
=16 (S)
=4 (S)
=4 (S)
2/38 (S)
8
4 (S)
2 (S)
=16 (I)
4 (S)
8 (S)
2 (S)
NT
16 (S)
NT
4
8 (S)
16 (R)
16/8 (R)
64 (R)
16 (S)
NT
16 (R)
NT
NT
1 (S)
2 (S)
2 (S)
4 (S)
4 (S)
4
64
NT
-
F06-31AMP
4
=16 (S)
=4 (S)
=8 (I)
2/38 (S)
8
4 (S)
2 (S)
4 (S)
4 (S)
8 (S)
2 (S)
NT
16 (S)
NT
4
=32 (I)
16 (R)
8/4 (S)
64 (R)
16 (S)
NT
16 (R)
NT
NT
2 (R)
4 (R)
2 (S)
4 (S)
4 (S)
4
64
NT
-
6R; 1I
4R; 1I
5R; 2I
53127442
Salmonella sp.
85%
-
73117412
77116612
E. coli
85%
E. coli
<85%
-
-
* For samples run on NC 32 panels, susceptibility to Cf was tested at only one concentration (8g/ml) for
identification purposes. For samples run on NC 32 and NC 30 panels, susceptibility to Fd was tested at only
one concentration (64g/ml) for identification purposes. Samples exhibiting a MIC for Cf of 8g/ml may
be intermediate or resistant. Samples exhibiting a MIC for Fd of 64g/ml may be susceptible or
intermediate; ** For all panels, P, Cl, and K were tested at only one concentration (4g/ml) for
identification purposes-susceptibilities could not be determined; *** Antibiotics not tested on all samples,
and not included in calculation of results; a Total number of resistant/intermediate compounds for each
isolate, only including those antibiotics that were used in the tabulation of results.
60
Appendix A Continued: Complete antibiotic MIC data, biotype numbers, species and
integron detection results
Antibiotic Class
Antibiotic
(K)**
(Ak)
Aminoglycosides
(Gm)
(To)
Antifol/Sulfa
(T/S)
(Cf)*
(Cft)***
(Caz)
(Cfz)
Cephalosporins
(Crm)
(Cax)
(Cpe)
(Cfx)***
(Ctn)
Tetracyclines
(Te)***
(P)**
(P/T)
(Am)
Penicillins
(A/S)
(Pi)
(Tim)***
(Aug)***
Monobactams
(Azt)
(Gat)***
(Nxn)***
(Cp)
Quinolones
(Lvx)
(Mxf)***
(Imp)
Carbapenems
(Mer)***
(Cl)**
Other
(Fd)*
(C)***
ESL-a Screen
ESL-b Screen
Total #
Resistant/Intermediatea
Biotype ID #
Species/Confidence
Class 1 Integron
Class 2 Integron
Sample #/MICs (g/ml)
F06-36F06-37AMP
AMP
4
4
=32 (I)
4 (S)
=8 (I)
1 (S)
=8 (I)
1 (S)
2/38 (S)
2/38 (S)
8
8
4 (S)
4 (S)
2 (S)
2 (S)
=16 (I)
4 (S)
4 (S)
4 (S)
8 (S)
8 (S)
2 (S)
2 (S)
NT
NT
16 (S)
16 (S)
NT
NT
4
4
=32 (I)
8 (S)
8 (S)
8 (S)
8/4 (S)
8/4 (S)
64 (R)
16 (S)
16 (S)
16 (S)
NT
NT
8 (S)
16 (R)
NT
NT
NT
NT
1 (S)
1 (S)
2 (S)
2 (S)
2 (S)
2 (S)
4 (S)
4 (S)
4 (S)
4 (S)
4
4
64
64
NT
NT
-
F06-32AMP
4
=16 (S)
=4 (S)
=4 (S)
2/38 (S)
8
4 (S)
2 (S)
4 (S)
4 (S)
8 (S)
2 (S)
NT
16 (S)
NT
4
=32 (I)
8 (S)
8/4 (S)
16 (S)
16 (S)
NT
8 (S)
NT
NT
1 (S)
2 (S)
2 (S)
4 (S)
4 (S)
4
64
NT
-
F06-35AMP
4
=16 (S)
=4 (S)
=4 (S)
2/38 (S)
8
4 (S)
2 (S)
4 (S)
=16 (I)
8 (S)
2 (S)
NT
16 (S)
NT
4
64 (R)
8 (S)
8/4 (S)
64 (R)
16 (S)
NT
16 (R)
NT
NT
1 (S)
2 (S)
2 (S)
4 (S)
4 (S)
4
64
NT
+
+
0R; 1I
3R; 1I
1R; 0I
77113012
E. coli
99%
-
73117412
E. coli
85%
-
53014002
E. coli
99%
-
F06-38AMP
4
4 (S)
=4 (S)
=4 (S)
2/38 (S)
8
4 (S)
=8 (S)
4 (S)
4 (S)
8 (S)
2 (S)
NT
16 (S)
NT
4
64 (R)
8 (S)
8/4 (S)
64 (R)
16 (S)
NT
16 (R)
NT
NT
1 (S)
2 (S)
2 (S)
4 (S)
4 (S)
4
64
NT
-
F06-39AMP
4
=16 (S)
=8 (I)
=4 (S)
2/38 (S)
8
4 (S)
2 (S)
4 (S)
16 (R)
8 (S)
2 (S)
NT
16 (S)
NT
4
64 (R)
8 (S)
8/4 (S)
64 (R)
16 (S)
NT
8 (S)
NT
NT
1 (S)
2 (S)
2 (S)
4 (S)
4 (S)
4
64
NT
-
1R; 5I
3R; 0I
3R; 1I
53317412
E. coli
85%
-
63117412
E. coli
85%
-
67115412
E. coli
85%
+
-
* For samples run on NC 32 panels, susceptibility to Cf was tested at only one concentration (8g/ml) for
identification purposes. For samples run on NC 32 and NC 30 panels, susceptibility to Fd was tested at only
one concentration (64g/ml) for identification purposes. Samples exhibiting a MIC for Cf of 8g/ml may
be intermediate or resistant. Samples exhibiting a MIC for Fd of 64g/ml may be susceptible or
intermediate; ** For all panels, P, Cl, and K were tested at only one concentration (4g/ml) for
identification purposes-susceptibilities could not be determined; *** Antibiotics not tested on all samples,
and not included in calculation of results; a Total number of resistant/intermediate compounds for each
isolate, only including those antibiotics that were used in the tabulation of results.
61
Appendix A Continued: Complete antibiotic MIC data, biotype numbers, species and
integron detection results
Antibiotic Class
Sample #/MICs (g/ml)
Antibiotic
(K)**
(Ak)
Aminoglycosides
(Gm)
(To)
Antifol/Sulfa
(T/S)
(Cf)*
(Cft)***
(Caz)
(Cfz)
Cephalosporins
(Crm)
(Cax)
(Cpe)
(Cfx)***
(Ctn)
Tetracyclines
(Te)***
(P)**
(P/T)
(Am)
Penicillins
(A/S)
(Pi)
(Tim)***
(Aug)***
Monobactams
(Azt)
(Gat)***
(Nxn)***
(Cp)
Quinolones
(Lvx)
(Mxf)***
(Imp)
Carbapenems
(Mer)***
(Cl)**
Other
(Fd)*
(C)***
ESL-a Screen
ESL-b Screen
Total #
Resistant/Intermediatea
Biotype ID #
Species/Confidence
Class 1 Integron
Class 2 Integron
F06-17CIP
4
=16 (S)
=8 (I)
=4 (S)
2/38 (S)
8
4 (S)
2 (S)
4 (S)
16 (R)
8 (S)
2 (S)
NT
16 (S)
NT
4
64 (R)
8 (S)
8/4 (S)
64 (R)
16 (S)
NT
16 (R)
NT
NT
2 (R)
4 (R)
4 (R)
4 (S)
4 (S)
4
64
NT
+
L-1-E
L-3-E
L-4-E
M-5-Ea
M-5-Eb
4
=16 (S)
=4 (S)
=4 (S)
2/38 (R)
8
4 (S)
2 (S)
16 (R)
=16 (I)
8 (S)
2 (S)
NT
16 (S)
NT
4
=32 (I)
16 (R)
16/8 (R)
64 (R)
16 (S)
NT
8 (S)
NT
NT
1 (S)
2 (S)
2 (S)
4 (S)
4 (S)
4
64
NT
+
4
=16 (S)
=4 (S)
=4 (S)
2/38 (S)
8
4 (S)
2 (S)
4 (S)
=16 (I)
8 (S)
2 (S)
NT
16 (S)
NT
4
8 (S)
16 (R)
16/8 (R)
16 (S)
16 (S)
NT
8 (S)
NT
NT
1 (S)
2 (S)
2 (S)
4 (S)
4 (S)
4
64
NT
-
4
=16 (S)
=4 (S)
=4 (S)
2/38 (R)
8
4 (S)
16 (R)
4 (S)
4 (S)
8 (S)
2 (S)
NT
16 (S)
NT
4
64 (R)
16 (R)
16/8 (R)
64 (R)
16 (S)
NT
8 (S)
NT
NT
2 (R)
4 (R)
4 (R)
4 (S)
4 (S)
4
64
NT
-
4
4 (S)
1 (S)
1 (S)
2/38 (S)
8
4 (S)
2 (S)
4 (S)
4 (S)
8 (S)
2 (S)
NT
16 (S)
NT
4
8 (S)
8 (S)
8/4 (S)
16 (S)
16 (S)
NT
8 (S)
NT
NT
1 (S)
2 (S)
2 (S)
4 (S)
4 (S)
4
64
NT
-
4
Inc.
=4 (S)
=4 (S)
2/38 (S)
8
4 (S)
2 (S)
4 (S)
4 (S)
8 (S)
2 (S)
NT
16 (S)
NT
4
8 (S)
8 (S)
8/4 (S)
16 (S)
16 (S)
NT
8 (S)
NT
NT
1 (S)
2 (S)
2 (S)
4 (S)
4 (S)
4
64
NT
-
6R; 1I
5R; 2I
2R; 1I
8R; 0I
0R; 0I
0R; 0I
43014002
E. coli
85%
-
73117412
E. coli
85%
-
53017412
E. coli
85%
NT
NT
53117412
E. coli
85%
-
53017612
E. coli
<85%
+
-
53017632
E. coli
<85%
-
* For samples run on NC 32 panels, susceptibility to Cf was tested at only one concentration (8g/ml) for
identification purposes. For samples run on NC 32 and NC 30 panels, susceptibility to Fd was tested at only
one concentration (64g/ml) for identification purposes. Samples exhibiting a MIC for Cf of 8g/ml may
be intermediate or resistant. Samples exhibiting a MIC for Fd of 64g/ml may be susceptible or
intermediate; ** For all panels, P, Cl, and K were tested at only one concentration (4g/ml) for
identification purposes-susceptibilities could not be determined; *** Antibiotics not tested on all samples,
and not included in calculation of results; a Total number of resistant/intermediate compounds for each
isolate, only including those antibiotics that were used in the tabulation of results.
62
Appendix A Continued: Complete antibiotic MIC data, biotype numbers, species and
integron detection results
Antibiotic Class
Antibiotic
Sample #/MICs (g/ml)
F06-28L-1-SXT
SXT
4
4
=16 (S)
4 (S)
=4 (S)
=4 (S)
=4 (S)
8 (R)
2/38 (R)
2/38 (R)
8
8
32 (R)
4 (S)
16 (R)
2 (S)
16 (R)
16 (R)
16 (R)
4 (S)
32 (R)
8 (S)
16 (R)
2 (S)
NT
NT
32 (R)
16 (S)
NT
NT
4
4
8 (S)
8 (S)
16 (R)
16 (R)
16/8 (R)
16/8 (R)
64 (R)
64 (R)
64 (R)
64 (R)
NT
NT
16 (R)
8 (S)
NT
NT
NT
NT
1 (S)
2 (R)
2 (S)
2 (S)
2 (S)
2 (S)
4 (S)
4 (S)
4 (S)
4 (S)
4
4
64
64
NT
NT
+
+
-
F06-17SXT
4
32 (R)
8 (R)
8 (R)
2/38 (S)
8
4 (S)
16 (R)
4 (S)
=16 (I)
32 (R)
=16 (I)
NT
32 (R)
NT
4
8 (S)
8 (S)
8/4 (S)
16 (S)
16 (S)
NT
16 (R)
NT
NT
2 (R)
2 (S)
4 (R)
4 (S)
4 (S)
4
64
NT
+
+
F06-21SXT
4
=16 (S)
=8 (I)
=8 (I)
2/38 (S)
8 (S)
4 (S)
2 (S)
4 (S)
4 (S)
8 (S)
2 (S)
NT
16 (S)
NT
4
8 (S)
8 (S)
8/4 (S)
16 (S)
16 (S)
NT
16 (R)
NT
NT
1 (S)
2 (S)
2 (S)
4 (S)
4 (S)
4
64
NT
-
8R; 2I
1R; 2I
11R; 0I
53016706
43016600
60000306
Species/Confidence
E. coli
<85%
E. coli
<85%
C. davisae
<85%
Class 1 Integron
Class 2 Integron
-
-
-
(K)**
(Ak)
Aminoglycosides
(Gm)
(To)
Antifol/Sulfa
(T/S)
(Cf)*
(Cft)***
(Caz)
(Cfz)
Cephalosporins
(Crm)
(Cax)
(Cpe)
(Cfx)***
(Ctn)
Tetracyclines
(Te)***
(P)**
(P/T)
(Am)
Penicillins
(A/S)
(Pi)
(Tim)***
(Aug)***
Monobactams
(Azt)
(Gat)***
(Nxn)***
(Cp)
Quinolones
(Lvx)
(Mxf)***
(Imp)
Carbapenems
(Mer)***
(Cl)**
Other
(Fd)*
(C)***
ESL-a Screen
ESL-b Screen
Total #
Resistant/Intermediatea
Biotype ID #
L-4-SXT
M-6-SXT
4
4 (S)
=4 (S)
=4 (S)
2/38 (R)
8
4 (S)
2 (S)
16 (R)
=16 (I)
8 (S)
2 (S)
NT
16 (S)
NT
4
8 (S)
16 (R)
16/8 (R)
64 (R)
16 (S)
NT
8 (S)
NT
NT
2 (R)
4 (R)
4 (R)
4 (S)
4 (S)
4
64
NT
-
4
=16 (S)
=4 (S)
=4 (S)
2/38 (S)
8
4 (S)
2 (S)
4 (S)
4 (S)
8 (S)
2 (S)
NT
16 (S)
NT
4
8 (S)
8 (S)
8/4 (S)
16 (S)
16 (S)
NT
8 (S)
NT
NT
1 (S)
2 (S)
2 (S)
4 (S)
4 (S)
4
64
NT
-
7R; 0I
7R; 1I
0R; 0I
77757752
R. ornithinolytic
<85%
-
77747372
E.
aerogenes
85%
-
53017612
E. coli
<85%
-
* For samples run on NC 32 panels, susceptibility to Cf was tested at only one concentration (8g/ml) for
identification purposes. For samples run on NC 32 and NC 30 panels, susceptibility to Fd was tested at only
one concentration (64g/ml) for identification purposes. Samples exhibiting a MIC for Cf of 8g/ml may
be intermediate or resistant. Samples exhibiting a MIC for Fd of 64g/ml may be susceptible or
intermediate; ** For all panels, P, Cl, and K were tested at only one concentration (4g/ml) for
identification purposes-susceptibilities could not be determined; *** Antibiotics not tested on all samples,
and not included in calculation of results; a Total number of resistant/intermediate compounds for each
isolate, only including those antibiotics that were used in the tabulation of results.
63
Appendix A Continued: Complete antibiotic MIC data, biotype numbers, species and
integron detection results
Antibiotic Class
Antibiotic
(K)**
(Ak)
Aminoglycosides
(Gm)
(To)
Antifol/Sulfa
(T/S)
(Cf)*
(Cft)***
(Caz)
(Cfz)
Cephalosporins
(Crm)
(Cax)
(Cpe)
(Cfx)***
(Ctn)
Tetracyclines
(Te)***
(P)**
(P/T)
(Am)
Penicillins
(A/S)
(Pi)
(Tim)***
(Aug)***
Monobactams
(Azt)
(Gat)***
(Nxn)***
(Cp)
Quinolones
(Lvx)
(Mxf)***
(Imp)
Carbapenems
(Mer)***
(Cl)**
Other
(Fd)*
(C)***
ESL-a Screen
ESL-b Screen
Total #
Resistant/Intermediatea
Biotype ID #
Species/Confidence
Class 1 Integron
Class 2 Integron
Sample #/MICs (g/ml)
F06-28F06-31TET
TET
4
4
=32 (I)
4 (S)
=4 (S)
=8 (I)
=4 (S)
=8 (I)
2/38 (S)
2/38 (R)
8
8
=16 (I)
4 (S)
=8 (S)
2 (S)
16 (R)
4 (S)
16 (R)
4 (S)
32 (R)
8 (S)
2 (S)
2 (S)
NT
NT
16 (S)
16 (S)
NT
NT
4
4
64 (R)
64 (R)
16 (R)
16 (R)
16/8 (R)
8/4 (S)
64 (R)
16 (S)
64 (R)
16 (S)
NT
NT
16 (R)
16 (R)
NT
NT
NT
NT
1 (S)
1 (S)
2 (S)
2 (S)
2 (S)
2 (S)
4 (S)
4 (S)
4 (S)
4 (S)
4
4
64
64
NT
NT
+
+
-
F06-5TET
4
4 (S)
1 (S)
=4 (S)
2/38 (R)
8
4 (S)
2 (S)
16 (R)
=16 (I)
8 (S)
2 (S)
NT
16 (S)
NT
4
64 (R)
16 (R)
16/8 (R)
64 (R)
16 (S)
NT
16 (R)
NT
NT
1 (S)
2 (S)
2 (S)
4 (S)
4 (S)
4
64
NT
-
F06-17TET
4
=16 (S)
=8 (I)
=8 (I)
2/38 (S)
8
4 (S)
2 (S)
4 (S)
4 (S)
8 (S)
2 (S)
NT
16 (S)
NT
4
=32 (I)
8 (S)
8/4 (S)
16 (S)
16 (S)
NT
16 (R)
NT
NT
2 (R)
2 (S)
4 (R)
4 (S)
4 (S)
4
64
NT
-
7R; 1I
2R; 3I
8R; 0I
77137472
K.
ascorbata
<85%
-
73135402
53127072
C.
freundii
85%
-
E. coli
85%
-
F06-39TET
4
=16 (S)
=4 (S)
=4 (S)
2/38 (R)
8
4 (S)
2 (S)
4 (S)
=16 (I)
8 (S)
2 (S)
NT
16 (S)
NT
4
64 (R)
8 (S)
8/4 (S)
16 (S)
16 (S)
NT
8 (S)
NT
NT
1 (S)
2 (S)
2 (S)
4 (S)
4 (S)
4
64
NT
-
4
=16 (S)
=4 (S)
1 (S)
2/38 (R)
8
4 (S)
2 (S)
16 (R)
=16 (I)
8 (S)
2 (S)
NT
16 (S)
NT
4
8 (S)
16 (R)
16/8 (R)
64 (R)
16 (S)
NT
8 (S)
NT
NT
1 (S)
2 (S)
2 (S)
4 (S)
4 (S)
4
64
NT
-
4R; 3I
2R; 1I
5R; 1I
77114412
67117052
77117412
E. coli
99%
E. coli
<85%
E. coli
85%
-
-
+
-
L-1-TET
* For samples run on NC 32 panels, susceptibility to Cf was tested at only one concentration (8g/ml) for
identification purposes. For samples run on NC 32 and NC 30 panels, susceptibility to Fd was tested at only
one concentration (64g/ml) for identification purposes. Samples exhibiting a MIC for Cf of 8g/ml may
be intermediate or resistant. Samples exhibiting a MIC for Fd of 64g/ml may be susceptible or
intermediate; ** For all panels, P, Cl, and K were tested at only one concentration (4g/ml) for
identification purposes-susceptibilities could not be determined; *** Antibiotics not tested on all samples,
and not included in calculation of results; a Total number of resistant/intermediate compounds for each
isolate, only including those antibiotics that were used in the tabulation of results.
64
Appendix A Continued: Complete antibiotic MIC data, biotype numbers, species and
integron detection results
Antibiotic Class
Antibiotic
Sample #/MICs (g/ml)
M-160801
TET
4
4
=16 (S)
4 (S)
1 (S)
1 (S)
=4 (S)
8 (R)
2/38 (R)
2/38 (R)
8
8
=32 (I)
4 (S)
=16 (I)
2 (S)
4 (S)
16 (R)
4 (S)
16 (R)
=32 (I)
8 (S)
=8 (S)
2 (S)
NT
NT
16 (S)
16 (S)
NT
NT
4
4
8 (S)
8 (S)
16 (R)
16 (R)
8/4 (S)
8/4 (S)
64 (R)
64 (R)
16 (S)
16 (S)
NT
NT
8 (S)
16 (R)
NT
NT
NT
NT
2 (R)
2 (R)
2 (S)
4 (R)
=4 (I)
4 (R)
4 (S)
4 (S)
4 (S)
4 (S)
4
4
64
64
NT
NT
+
+
L-4-TETb
L-5-TET
4
4 (S)
=4 (S)
1 (S)
2/38 (S)
8
4 (S)
2 (S)
16 (R)
4 (S)
8 (S)
2 (S)
NT
16 (S)
NT
4
8 (S)
16 (R)
16/8 (R)
64 (R)
16 (S)
NT
16 (R)
NT
NT
2 (R)
4 (R)
4 (R)
4 (S)
4 (S)
4
64
NT
-
4
=16 (S)
=8 (I)
=8 (I)
2/38 (R)
8
4 (S)
2 (S)
4 (S)
4 (S)
8 (S)
2 (S)
NT
16 (S)
NT
4
=32 (I)
8 (S)
8/4 (S)
16 (S)
16 (S)
NT
16 (R)
NT
NT
1 (S)
2 (S)
2 (S)
4 (S)
4 (S)
4
64
NT
-
7R; 0I
2R; 3I
5R; 0I
63117412
77117402
Species/Confidence
E. coli
85%
E. coli
85%
Class 1 Integron
Class 2 Integron
-
-
77757776
R. ornithinolytic
<85%
+
-
(K)**
(Ak)
Aminoglycosides
(Gm)
(To)
Antifol/Sulfa
(T/S)
(Cf)*
(Cft)***
(Caz)
(Cfz)
Cephalosporins
(Crm)
(Cax)
(Cpe)
(Cfx)***
(Ctn)
Tetracyclines
(Te)***
(P)**
(P/T)
(Am)
Penicillins
(A/S)
(Pi)
(Tim)***
(Aug)***
Monobactams
(Azt)
(Gat)***
(Nxn)***
(Cp)
Quinolones
(Lvx)
(Mxf)***
(Imp)
Carbapenems
(Mer)***
(Cl)**
Other
(Fd)*
(C)***
ESL-a Screen
ESL-b Screen
Total #
Resistant/Intermediatea
Biotype ID #
0802
0803
4
4 (S)
1 (S)
1 (S)
2/38 (S)
8 (S)
4 (S)
2 (S)
=16 (I)
4 (S)
8 (S)
2 (S)
NT
16 (S)
NT
4
8 (S)
8 (S)
8/4 (S)
16 (S)
16 (S)
NT
8 (S)
NT
NT
1 (S)
2 (S)
2 (S)
4 (S)
4 (S)
4
64
NT
-
4
4 (S)
1 (S)
1 (S)
2/38 (R)
8 (S)
4 (S)
2 (S)
4 (S)
4 (S)
8 (S)
2 (S)
NT
16 (S)
NT
4
8 (S)
8 (S)
8/4 (S)
16 (S)
16 (S)
NT
8 (S)
NT
NT
1 (S)
2 (S)
2 (S)
4 (S)
4 (S)
4
64
NT
-
8R; 2I
0R; 1I
1R; 0I
53111012
53115010
53115010
E. coli
85%
E. coli
99%
E. coli
99%
-
-
-
* For samples run on NC 32 panels, susceptibility to Cf was tested at only one concentration (8g/ml) for
identification purposes. For samples run on NC 32 and NC 30 panels, susceptibility to Fd was tested at only
one concentration (64g/ml) for identification purposes. Samples exhibiting a MIC for Cf of 8g/ml may
be intermediate or resistant. Samples exhibiting a MIC for Fd of 64g/ml may be susceptible or
intermediate; ** For all panels, P, Cl, and K were tested at only one concentration (4g/ml) for
identification purposes-susceptibilities could not be determined; *** Antibiotics not tested on all samples,
and not included in calculation of results; a Total number of resistant/intermediate compounds for each
isolate, only including those antibiotics that were used in the tabulation of results. b Strikethrough text
indicates samples that were deemed not unique.
65
Appendix A Continued: Complete antibiotic MIC data, biotype numbers, species and
integron detection results
Antibiotic Class
Antibiotic
(K)**
(Ak)
Aminoglycosides
(Gm)
(To)
Antifol/Sulfa
(T/S)
(Cf)*
(Cft)***
(Caz)
(Cfz)
Cephalosporins
(Crm)
(Cax)
(Cpe)
(Cfx)***
(Ctn)
Tetracyclines
(Te)***
(P)**
(P/T)
(Am)
Penicillins
(A/S)
(Pi)
(Tim)***
(Aug)***
Monobactams
(Azt)
(Gat)***
(Nxn)***
Quinolones
(Cp)
(Lvx)
(Mxf)***
(Imp)
Carbapenems
(Mer)***
(Cl)**
Other
(Fd)*
(C)***
ESL-a Screen
ESL-b Screen
Total #
Resistant/Intermediatea
Biotype ID #
Species/Confidence
Class 1 Integron
Class 2 Integron
Sample #/MICs (g/ml)
0806
0807
4
4
4 (S)
4 (S)
8 (R)
1 (S)
=8 (I)
1 (S)
2/38 (S)
2/38 (R)
8 (S)
8 (S)
4 (S)
4 (S)
2 (S)
2 (S)
4 (S)
4 (S)
4 (S)
4 (S)
8 (S)
8 (S)
2 (S)
2 (S)
NT
8 (S)
16 (S)
16 (S)
NT
4 (S)
4
4
8 (S)
8 (S)
16 (R)
16 (R)
=16/8 (I)
8/4 (S)
=64 (I)
16 (S)
16 (S)
16 (S)
NT
8/4 (S)
8 (S)
8 (S)
NT
8 (S)
NT
4 (S)
1 (S)
1 (S)
2 (S)
2 (S)
2 (S)
2 (S)
4 (S)
4 (S)
4 (S)
4 (S)
4
4
32 (S)
64
NT
8 (S)
-
0804
4
4 (S)
1 (S)
1 (S)
2/38 (R)
8 (S)
4 (S)
2 (S)
4 (S)
4 (S)
8 (S)
2 (S)
NT
16 (S)
NT
4
8 (S)
16 (R)
=16/8 (I)
16 (S)
16 (S)
NT
8 (S)
NT
NT
1 (S)
2 (S)
2 (S)
4 (S)
4 (S)
4
64
NT
-
0805
4
4 (S)
1 (S)
=8 (I)
2/38 (R)
8
32 (R)
=16 (I)
16 (R)
16 (R)
32 (R)
=16 (I)
NT
16 (S)
NT
4
8 (S)
16 (R)
=16/8 (I)
64 (R)
16 (S)
NT
16 (R)
NT
NT
2 (R)
4 (R)
4 (R)
4 (S)
4 (S)
4
64
NT
+
+
2R; 1I
9R; 4I
2R; 3I
57114010
E. coli
99%
-
63114012
E. coli
99%
+
-
73015010
E. coli
99%
-
0808
4
4 (S)
8 (R)
=8 (I)
2/38 (S)
8
4 (S)
2 (S)
16 (R)
=16 (I)
8 (S)
2 (S)
NT
16 (S)
NT
4
8 (S)
16 (R)
=16/8 (I)
16 (S)
16 (S)
NT
8 (S)
NT
NT
1 (S)
2 (S)
2 (S)
4 (S)
4 (S)
4
64
NT
+
+
0809
4
4 (S)
8 (R)
=4 (S)
2/38 (S)
8 (S)
4 (S)
2 (S)
=8 (S)
=8 (I)
8 (S)
2 (S)
8 (S)
16 (S)
8 (R)
4
8 (S)
8 (S)
8/4 (S)
16 (S)
16 (S)
8/4 (S)
8 (S)
2 (S)
4 (S)
1 (S)
2 (S)
2 (S)
4 (S)
4 (S)
4
32 (S)
8 (S)
-
2R; 0I
3R; 3I
1R; 1I
53110010
E. coli
99%
+
-
43114012
E. coli
99%
-
77015010
E. coli
99%
-
* For samples run on NC 32 panels, susceptibility to Cf was tested at only one concentration (8g/ml) for
identification purposes. For samples run on NC 32 and NC 30 panels, susceptibility to Fd was tested at only
one concentration (64g/ml) for identification purposes. Samples exhibiting a MIC for Cf of 8g/ml may
be intermediate or resistant. Samples exhibiting a MIC for Fd of 64g/ml may be susceptible or
intermediate; ** For all panels, P, Cl, and K were tested at only one concentration (4g/ml) for
identification purposes-susceptibilities could not be determined; *** Antibiotics not tested on all samples,
and not included in calculation of results; a Total number of resistant/intermediate compounds for each
isolate, only including those antibiotics that were used in the tabulation of results.
66
Appendix A Continued: Complete antibiotic MIC data, biotype numbers, species and
integron detection results
Antibiotic Class
Antibiotic
(K)**
(Ak)
Aminoglycosides
(Gm)
(To)
Antifol/Sulfa
(T/S)
(Cf)*
(Cft)***
(Caz)
(Cfz)
Cephalosporins
(Crm)
(Cax)
(Cpe)
(Cfx)***
(Ctn)
Tetracyclines
(Te)***
(P)**
(P/T)
(Am)
Penicillins
(A/S)
(Pi)
(Tim)***
(Aug)***
Monobactams
(Azt)
(Gat)***
(Nxn)***
(Cp)
Quinolones
(Lvx)
(Mxf)***
(Imp)
Carbapenems
(Mer)***
(Cl)**
Other
(Fd)*
(C)***
ESL-a Screen
ESL-b Screen
Total #
Resistant/Intermediatea
Biotype ID #
Species/Confidence
Class 1 Integron
Class 2 Integron
Sample #/MICs (g/ml)
0811
0812
4
4
4 (S)
4 (S)
1 (S)
1 (S)
1 (S)
1 (S)
2/38 (R)
2/38 (R)
8
8
4 (S)
4 (S)
2 (S)
2 (S)
=8 (S)
4 (S)
4 (S)
4 (S)
8 (S)
8 (S)
2 (S)
2 (S)
NT
NT
16 (S)
16 (S)
NT
NT
4
4
8 (S)
8 (S)
16 (R)
16 (R)
16/8 (R)
16/8 (R)
=64 (I)
64 (R)
=64 (I)
16 (S)
NT
NT
8 (S)
8 (S)
NT
NT
NT
NT
1 (S)
1 (S)
2 (S)
2 (S)
2 (S)
2 (S)
4 (S)
4 (S)
4 (S)
4 (S)
4
4
64
64
NT
NT
-
0810A
4
4 (S)
8 (R)
1 (S)
2/38 (S)
8 (S)
4 (S)
2 (S)
4 (S)
=16 (I)
8 (S)
2 (S)
NT
16 (S)
NT
4
8 (S)
16 (R)
8/4 (S)
16 (S)
16 (S)
NT
8 (S)
NT
NT
1 (S)
2 (S)
2 (S)
4 (S)
4 (S)
4
64
NT
-
0810Bb
4
4 (S)
1 (S)
1 (S)
2/38 (S)
8 (S)
4 (S)
2 (S)
4 (S)
4 (S)
8 (S)
2 (S)
NT
16 (S)
NT
4
8 (S)
8 (S)
8/4 (S)
16 (S)
16 (S)
NT
8 (S)
NT
NT
1 (S)
2 (S)
2 (S)
4 (S)
4 (S)
4
64
NT
-
2R; 1I
0R; 0I
4R; 0I
77544370
K. pneumoniae
99%
-
77544370
K. pneumoniae
99%
-
0813
4
4 (S)
1 (S)
1 (S)
2/38 (R)
8 (S)
4 (S)
2 (S)
4 (S)
4 (S)
8 (S)
2 (S)
NT
16 (S)
NT
4
8 (S)
16 (R)
16/8 (R)
=64 (I)
16 (S)
NT
8 (S)
NT
NT
1 (S)
2 (S)
2 (S)
4 (S)
4 (S)
4
64
NT
-
0814
4
4 (S)
1 (S)
1 (S)
2/38 (R)
8 (S)
4 (S)
2 (S)
4 (S)
4 (S)
8 (S)
2 (S)
NT
16 (S)
NT
4
8 (S)
16 (R)
8/4 (S)
=64 (I)
16 (S)
NT
8 (S)
NT
NT
1 (S)
2 (S)
2 (S)
4 (S)
4 (S)
4
64
NT
-
3R; 1I
3R; 1I
2R; 1I
77114012
57115012
77114010
57114010
E. coli
99%
E. coli
99%
E. coli
99%
E. coli
99%
-
+
-
+
-
-
* For samples run on NC 32 panels, susceptibility to Cf was tested at only one concentration (8g/ml) for
identification purposes. For samples run on NC 32 and NC 30 panels, susceptibility to Fd was tested at only
one concentration (64g/ml) for identification purposes. Samples exhibiting a MIC for Cf of 8g/ml may
be intermediate or resistant. Samples exhibiting a MIC for Fd of 64g/ml may be susceptible or
intermediate; ** For all panels, P, Cl, and K were tested at only one concentration (4g/ml) for
identification purposes-susceptibilities could not be determined; *** Antibiotics not tested on all samples,
and not included in calculation of results; a Total number of resistant/intermediate compounds for each
isolate, only including those antibiotics that were used in the tabulation of results. b Strikethrough text
indicates samples that were deemed not unique.
67
Appendix A Continued: Complete antibiotic MIC data, biotype numbers, species and
integron detection results
Antibiotic Class
Antibiotic
Sample #/MICs (g/ml)
0816B
0817
4
4
4 (S)
4 (S)
1 (S)
1 (S)
1 (S)
1 (S)
2/38 (S)
2/38 (S)
8 (S)
8
4 (S)
4 (S)
2 (S)
2 (S)
4 (S)
16 (R)
4 (S)
4 (S)
8 (S)
8 (S)
2 (S)
2 (S)
NT
8 (S)
16 (S)
16 (S)
NT
4 (S)
4
4
8 (S)
8 (S)
=16 (I)
=8 (I)
=16/8 (I)
8/4 (S)
16 (S)
16 (S)
16 (S)
16 (S)
NT
8/4 (S)
8 (S)
8 (S)
NT
2 (S)
NT
4 (S)
1 (S)
1 (S)
2 (S)
2 (S)
2 (S)
2 (S)
4 (S)
4 (S)
4 (S)
4 (S)
4
4
64 (R)
64
NT
16 (R)
-
0815
4
4 (S)
1 (S)
1 (S)
2/38 (R)
8 (S)
4 (S)
2 (S)
4 (S)
4 (S)
8 (S)
2 (S)
NT
16 (S)
NT
4
8 (S)
16 (R)
8/4 (S)
=64 (I)
16 (S)
NT
8 (S)
NT
NT
1 (S)
2 (S)
2 (S)
4 (S)
4 (S)
4
64
NT
-
0816A
4
4 (S)
1 (S)
1 (S)
2/38 (S)
8 (S)
4 (S)
2 (S)
4 (S)
4 (S)
8 (S)
2 (S)
NT
16 (S)
NT
4
8 (S)
8 (S)
8/4 (S)
16 (S)
16 (S)
NT
8 (S)
NT
NT
1 (S)
2 (S)
2 (S)
4 (S)
4 (S)
4
64 (S)
NT
-
2R; 1I
0R; 0I
0R; 2I
77114010
67111010
Species/Confidence
E. coli
99%
E. coli
85%
Class 1 Integron
Class 2 Integron
-
-
77754372
K.
oxytoca
85%
-
(K)**
(Ak)
Aminoglycosides
(Gm)
(To)
Antifol/Sulfa
(T/S)
(Cf)*
(Cft)***
(Caz)
(Cfz)
Cephalosporins
(Crm)
(Cax)
(Cpe)
(Cfx)***
(Ctn)
Tetracyclines
(Te)***
(P)**
(P/T)
(Am)
Penicillins
(A/S)
(Pi)
(Tim)***
(Aug)***
Monobactams
(Azt)
(Gat)***
(Nxn)***
Quinolones
(Cp)
(Lvx)
(Mxf)***
(Imp)
Carbapenems
(Mer)***
(Cl)**
Other
(Fd)*
(C)***
ESL-a Screen
ESL-b Screen
Total #
Resistant/Intermediatea
Biotype ID #
0818A
4
4 (S)
1 (S)
1 (S)
2/38 (S)
8
=32 (I)
2 (S)
16 (R)
16 (R)
32 (R)
=8 (S)
NT
16 (S)
NT
4
8 (S)
16 (R)
16/8 (R)
64 (R)
16 (S)
NT
8 (S)
NT
NT
1 (S)
2 (S)
2 (S)
4 (S)
4 (S)
4
64
NT
+
-
0818B
4
4 (S)
1 (S)
1 (S)
2/38 (S)
8 (S)
4 (S)
2 (S)
4 (S)
4 (S)
8 (S)
2 (S)
NT
16 (S)
NT
4
8 (S)
16 (R)
=16/8 (I)
64 (R)
16 (S)
NT
8 (S)
NT
NT
2 (R)
4 (R)
4 (R)
4 (S)
4 (S)
4
64
NT
-
1R; 1I
6R; 0I
4R; 1I
77101176
67115012
73115010
E. cloacae
85%
E. coli
99%
E. coli
99%
-
-
-
* For samples run on NC 32 panels, susceptibility to Cf was tested at only one concentration (8g/ml) for
identification purposes. For samples run on NC 32 and NC 30 panels, susceptibility to Fd was tested at only
one concentration (64g/ml) for identification purposes. Samples exhibiting a MIC for Cf of 8g/ml may
be intermediate or resistant. Samples exhibiting a MIC for Fd of 64g/ml may be susceptible or
intermediate; ** For all panels, P, Cl, and K were tested at only one concentration (4g/ml) for
identification purposes-susceptibilities could not be determined; *** Antibiotics not tested on all samples,
and not included in calculation of results; a Total number of resistant/intermediate compounds for each
isolate, only including those antibiotics that were used in the tabulation of results.
68
Appendix A Continued: Complete antibiotic MIC data, biotype numbers, species and
integron detection results
Antibiotic Class
Antibiotic
(K)**
(Ak)
Aminoglycosides
(Gm)
(To)
Antifol/Sulfa
(T/S)
(Cf)*
(Cft)***
(Caz)
(Cfz)
Cephalosporins
(Crm)
(Cax)
(Cpe)
(Cfx)***
(Ctn)
Tetracyclines
(Te)***
(P)**
(P/T)
(Am)
Penicillins
(A/S)
(Pi)
(Tim)***
(Aug)***
Monobactams
(Azt)
(Gat)***
(Nxn)***
Quinolones
(Cp)
(Lvx)
(Mxf)***
(Imp)
Carbapenems
(Mer)***
(Cl)**
Other
(Fd)*
(C)***
ESL-a Screen
ESL-b Screen
Total #
Resistant/Intermediatea
Biotype ID #
0819
4
16 (S)
1(S)
2 (S)
2/38 (S)
16 (R)
NT
2 (S)
16 (R)
4 (S)
4 (S)
2 (S)
NT
4 (S)
NT
4
8 (S)
16 (R)
8/4 (S)
16 (S)
NT
16/8 (R)
8 (S)
2 (S)
NT
1 (S)
2 (S)
NT
4 (S)
NT
4
64
NT
-
0820A
4
16 (S)
1 (S)
2 (S)
2/38 (S)
16 (R)
NT
2 (S)
16 (R)
4 (S)
4 (S)
2 (S)
NT
4 (S)
NT
4
8 (S)
=16 (I)
8/4 (S)
16 (S)
NT
16/8 (R)
8 (S)
2 (S)
NT
1 (S)
2 (S)
NT
4 (S)
NT
4
64
NT
-
Sample #/MICs (g/ml)
0825
0826
4
4
16 (S)
16 (S)
1 (S)
1 (S)
2 (S)
2 (S)
2/38 (S)
2/38 (S)
16 (R)
8 (S)
NT
NT
2 (S)
2 (S)
=8 (S)
2 (S)
=8 (I)
4 (S)
4 (S)
4 (S)
2 (S)
2 (S)
NT
NT
4 (S)
4 (S)
NT
NT
4
4
8 (S)
8 (S)
16 (R)
2 (S)
=16/8 (I)
8/4 (S)
16 (S)
16 (S)
NT
NT
16/8 (R)
8/4 (S)
8 (S)
8 (S)
2 (S)
2 (S)
NT
NT
1 (S)
1 (S)
2 (S)
2 (S)
NT
NT
4 (S)
4 (S)
NT
NT
4
4
64
64
NT
NT
-
0901
4
16 (S)
1 (S)
2 (S)
2/38 (R)
8 (S)
NT
2 (S)
2 (S)
4 (S)
4 (S)
2 (S)
NT
4 (S)
NT
4
8 (S)
16 (R)
=16/8 (I)
=16 (S)
NT
8/4 (S)
8 (S)
4 (R)
NT
2 (R)
4 (R)
NT
4 (S)
NT
4
64
NT
-
0902A
4
16 (S)
1 (S)
2 (S)
2/38 (S)
8 (S)
NT
2 (S)
2 (S)
4 (S)
4 (S)
2 (S)
NT
4 (S)
NT
4
8 (S)
=16 (I)
8/4 (S)
16 (S)
NT
8/4 (S)
8 (S)
2 (S)
NT
1 (S)
2 (S)
NT
4 (S)
NT
4
64
NT
-
2R; 0I
1R; 1I
1R; 2I
0R; 0I
4R; 1I
0R; 1I
73003372
77303172
51115012
53115010
53115010
Species/Confidence
E. cloacae
85%
E. cloacae
99%
E. coli
99%
E. coli
99%
E. coli
99%
Class 1 Integron
Class 2 Integron
-
-
-
-
+
-
77744370
K. pneumoniae
99%
-
* For samples run on NC 32 panels, susceptibility to Cf was tested at only one concentration (8g/ml) for
identification purposes. For samples run on NC 32 and NC 30 panels, susceptibility to Fd was tested at only
one concentration (64g/ml) for identification purposes. Samples exhibiting a MIC for Cf of 8g/ml may
be intermediate or resistant. Samples exhibiting a MIC for Fd of 64g/ml may be susceptible or
intermediate; ** For all panels, P, Cl, and K were tested at only one concentration (4g/ml) for
identification purposes-susceptibilities could not be determined; *** Antibiotics not tested on all samples,
and not included in calculation of results; a Total number of resistant/intermediate compounds for each
isolate, only including those antibiotics that were used in the tabulation of results.
69
Appendix A Continued: Complete antibiotic MIC data, biotype numbers, species and
integron detection results
Antibiotic Class
Antibiotic
(K)**
(Ak)
Aminoglycosides
(Gm)
(To)
Antifol/Sulfa
(T/S)
(Cf)*
(Cft)***
(Caz)
(Cfz)
Cephalosporins
(Crm)
(Cax)
(Cpe)
(Cfx)***
(Ctn)
Tetracyclines
(Te)***
(P)**
(P/T)
(Am)
Penicillins
(A/S)
(Pi)
(Tim)***
(Aug)***
Monobactams
(Azt)
(Gat)***
(Nxn)***
Quinolones
(Cp)
(Lvx)
(Mxf)***
(Imp)
Carbapenems
(Mer)***
(Cl)**
Other
(Fd)*
(C)***
ESL-a Screen
ESL-b Screen
Total #
Resistant/Intermediatea
Biotype ID #
Species/Confidence
Class 1 Integron
Class 2 Integron
0902B
4
16 (S)
1 (S)
2 (S)
2/38 (S)
=16 (I)
NT
2 (S)
2 (S)
4 (S)
4 (S)
2 (S)
NT
4 (S)
NT
4
8 (S)
16 (R)
16/8 (R)
64 (R)
NT
8/4 (S)
8 (S)
2 (S)
NT
1 (S)
2 (S)
NT
4 (S)
NT
4
64
NT
-
0902Cb
4
16 (S)
1 (S)
2 (S)
2/38 (S)
=16 (I)
NT
2 (S)
2 (S)
4 (S)
4 (S)
2 (S)
NT
4 (S)
NT
4
8 (S)
16 (R)
16/8 (R)
64 (R)
NT
8/4 (S)
8 (S)
2 (S)
NT
1 (S)
2 (S)
NT
4 (S)
NT
4
64
NT
-
Sample #/MICs (g/ml)
0903A
0903B
4
4
16 (S)
16 (S)
1 (S)
1 (S)
2 (S)
2 (S)
2/38 (S)
2/38 (S)
=16 (I)
8 (S)
NT
NT
2 (S)
2 (S)
=4 (S)
2 (S)
4 (S)
4 (S)
4 (S)
4 (S)
2 (S)
2 (S)
NT
NT
4 (S)
4 (S)
NT
NT
4
4
8 (S)
8 (S)
2 (S)
16 (R)
=16/8 (I)
8/4 (S)
16 (S)
64 (R)
NT
NT
8/4 (S)
8/4 (S)
8 (S)
8 (S)
2 (S)
2 (S)
NT
NT
1 (S)
1 (S)
2 (S)
2 (S)
NT
NT
4 (S)
4 (S)
NT
NT
4
4
64
64
NT
NT
-
0904
4
16 (S)
1 (S)
2 (S)
2/38 (R)
8 (S)
NT
2 (S)
2 (S)
4 (S)
4 (S)
2 (S)
NT
4 (S)
NT
4
8 (S)
16 (R)
=16/8 (I)
64 (R)
NT
8/4 (S)
8 (S)
2 (S)
NT
1 (S)
2 (S)
NT
4 (S)
NT
4
64
NT
-
0905
4
16 (S)
1 (S)
2 (S)
2/38 (S)
=16 (I)
NT
2 (S)
=8 (S)
4 (S)
4 (S)
2 (S)
NT
4 (S)
NT
4
8 (S)
16 (R)
16/8 (R)
64 (R)
NT
=16/8 (I)
8 (S)
2 (S)
NT
1 (S)
2 (S)
NT
4 (S)
NT
4
64
NT
-
3R; 0I
3R; 0I
0R; 0I
2R; 1I
3R; 1I
3R; 0I
77315272
K.
ascorbata
85%
-
77315272
K.
ascorbata
85%
-
53115010
77115012
53015010
73115012
E. coli
99%
E. coli
99%
E. coli
85%
E. coli
99%
-
-
+
-
-
* For samples run on NC 32 panels, susceptibility to Cf was tested at only one concentration (8g/ml) for
identification purposes. For samples run on NC 32 and NC 30 panels, susceptibility to Fd was tested at only
one concentration (64g/ml) for identification purposes. Samples exhibiting a MIC for Cf of 8g/ml may
be intermediate or resistant. Samples exhibiting a MIC for Fd of 64g/ml may be susceptible or
intermediate; ** For all panels, P, Cl, and K were tested at only one concentration (4g/ml) for
identification purposes-susceptibilities could not be determined; *** Antibiotics not tested on all samples,
and not included in calculation of results; a Total number of resistant/intermediate compounds for each
isolate, only including those antibiotics that were used in the tabulation of results. b Strikethrough text
indicates samples that were deemed not unique.
70
Appendix A Continued: Complete antibiotic MIC data, biotype numbers, species and
integron detection results
Antibiotic Class
Antibiotic
(K)**
(Ak)
Aminoglycosides
(Gm)
(To)
Antifol/Sulfa
(T/S)
(Cf)*
(Cft)***
(Caz)
(Cfz)
Cephalosporins
(Crm)
(Cax)
(Cpe)
(Cfx)***
(Ctn)
Tetracyclines
(Te)***
(P)**
(P/T)
(Am)
Penicillins
(A/S)
(Pi)
(Tim)***
(Aug)***
Monobactams
(Azt)
(Gat)***
(Nxn)***
Quinolones
(Cp)
(Lvx)
(Mxf)***
(Imp)
Carbapenems
(Mer)***
(Cl)**
Other
(Fd)*
(C)***
ESL-a Screen
ESL-b Screen
Total #
Resistant/Intermediatea
Biotype ID #
0906
4
16 (S)
1 (S)
2 (S)
2/38 (S)
=16 (I)
NT
2 (S)
=4 (S)
4 (S)
4 (S)
2 (S)
NT
4 (S)
NT
4
8 (S)
16 (R)
16/8 (R)
64 (R)
NT
=16/8 (I)
8 (S)
2 (S)
NT
1 (S)
2 (S)
NT
4 (S)
NT
4
64
NT
-
0907
4
16 (S)
1 (S)
2 (S)
2/38 (S)
=16 (I)
NT
2 (S)
=4 (S)
4 (S)
4 (S)
2 (S)
NT
4 (S)
NT
4
8 (S)
16 (R)
16/8 (R)
64 (R)
NT
=16/8 (I)
8 (S)
2 (S)
NT
1 (S)
2 (S)
NT
4 (S)
NT
4
64
NT
-
Sample #/MICs (g/ml)
0908B
0908Cb
4
4
16 (S)
16 (S)
1 (S)
1 (S)
2 (S)
2 (S)
2/38 (S)
2/38 (S)
8 (S)
8 (S)
NT
NT
2 (S)
2 (S)
2 (S)
2 (S)
4 (S)
4 (S)
4 (S)
4 (S)
2 (S)
2 (S)
NT
NT
4 (S)
4 (S)
NT
NT
4
4
8 (S)
8 (S)
2 (S)
2 (S)
8/4 (S)
8/4 (S)
16 (S)
16 (S)
NT
NT
8/4 (S)
8/4 (S)
8 (S)
8 (S)
2 (S)
2 (S)
NT
NT
1 (S)
1 (S)
2 (S)
2 (S)
NT
NT
4 (S)
4 (S)
NT
NT
4
4
64
64
NT
NT
-
0909
4
16 (S)
1 (S)
2 (S)
2/38 (S)
=16 (I)
NT
2 (S)
=4 (S)
4 (S)
4 (S)
2 (S)
NT
4 (S)
NT
4
8 (S)
16 (R)
16/8 (R)
64 (R)
NT
8/4 (S)
8 (S)
2 (S)
NT
1 (S)
2 (S)
NT
4 (S)
NT
4
64
NT
-
0910
4
16 (S)
1 (S)
2 (S)
2/38 (S)
8 (S)
NT
2 (S)
2 (S)
4 (S)
4 (S)
2 (S)
NT
4 (S)
NT
4
8 (S)
2 (S)
8/4 (S)
16 (S)
NT
8/4 (S)
8 (S)
2 (S)
NT
1 (S)
2 (S)
NT
4 (S)
NT
4
64
NT
-
3R; 0I
3R; 0I
0R; 0I
0R; 0I
3R; 1I
0R; 0I
73115012
73115016
53115010
E. coli
99%
E. coli
99%
E. coli
99%
E. coli
99%
Class 1 Integron
Class 2 Integron
-
-
43315010
E.
fergusonii
85%
-
53115012
Species/Confidence
43315010
E.
fergusonii
85%
-
-
-
* For samples run on NC 32 panels, susceptibility to Cf was tested at only one concentration (8g/ml) for
identification purposes. For samples run on NC 32 and NC 30 panels, susceptibility to Fd was tested at only
one concentration (64g/ml) for identification purposes. Samples exhibiting a MIC for Cf of 8g/ml may
be intermediate or resistant. Samples exhibiting a MIC for Fd of 64g/ml may be susceptible or
intermediate; ** For all panels, P, Cl, and K were tested at only one concentration (4g/ml) for
identification purposes-susceptibilities could not be determined; *** Antibiotics not tested on all samples,
and not included in calculation of results; a Total number of resistant/intermediate compounds for each
isolate, only including those antibiotics that were used in the tabulation of results. b Strikethrough text
indicates samples that were deemed not unique.
71
Appendix A Continued: Complete antibiotic MIC data, biotype numbers, species and
integron detection results
Antibiotic Class
Antibiotic
(K)**
(Ak)
Aminoglycosides
(Gm)
(To)
Antifol/Sulfa
(T/S)
(Cf)*
(Cft)***
(Caz)
(Cfz)
Cephalosporins
(Crm)
(Cax)
(Cpe)
(Cfx)***
(Ctn)
Tetracyclines
(Te)***
(P)**
(P/T)
(Am)
Penicillins
(A/S)
(Pi)
(Tim)***
(Aug)***
Monobactams
(Azt)
(Gat)***
(Nxn)***
Quinolones
(Cp)
(Lvx)
(Mxf)***
(Imp)
Carbapenems
(Mer)***
(Cl)**
Other
(Fd)*
(C)***
ESL-a Screen
ESL-b Screen
Total #
Resistant/Intermediatea
Biotype ID #
Species/Confidence
Class 1 Integron
Class 2 Integron
0911A
4
16 (S)
1 (S)
2 (S)
2/38 (S)
8 (S)
NT
2 (S)
2 (S)
4 (S)
4 (S)
2 (S)
NT
4 (S)
NT
4
8 (S)
2 (S)
8/4 (S)
16 (S)
NT
8/4 (S)
8 (S)
2 (S)
NT
1 (S)
2 (S)
NT
4 (S)
NT
4
64
NT
-
0911B
4
16 (S)
1 (S)
2 (S)
2/38 (R)
16 (R)
NT
2 (S)
=8 (S)
4 (S)
4 (S)
2 (S)
NT
4 (S)
NT
4
8 (S)
16 (R)
16/8 (R)
64 (R)
NT
=16/8 (I)
8 (S)
2 (S)
NT
1 (S)
2 (S)
NT
4 (S)
NT
4
64
NT
-
Sample #/MICs (g/ml)
0912A
0912Bb
4
4
16 (S)
16 (S)
1 (S)
1 (S)
2 (S)
2 (S)
2/38 (S)
2/38 (S)
8 (S)
8 (S)
NT
NT
2 (S)
2 (S)
2 (S)
2 (S)
=8 (I)
4 (S)
4 (S)
4 (S)
2 (S)
2 (S)
NT
NT
4 (S)
4 (S)
NT
NT
4
4
8 (S)
8 (S)
=4 (S)
=4 (S)
8/4 (S)
8/4 (S)
16 (S)
16 (S)
NT
NT
8/4 (S)
8/4 (S)
8 (S)
8 (S)
2 (S)
2 (S)
NT
NT
1 (S)
1 (S)
2 (S)
2 (S)
NT
NT
4 (S)
4 (S)
NT
NT
4
4
64
64
NT
NT
-
0913
4
16 (S)
1 (S)
2 (S)
2/38 (S)
=16 (I)
NT
2 (S)
=4 (S)
4 (S)
4 (S)
2 (S)
NT
4 (S)
NT
4
8 (S)
16 (R)
16/8 (R)
64 (R)
NT
=16/8 (I)
8 (S)
2 (S)
NT
1 (S)
2 (S)
NT
4 (S)
NT
4
64
NT
-
0914
4
16 (S)
1 (S)
2 (S)
2/38 (R)
8 (S)
NT
2 (S)
2 (S)
4 (S)
4 (S)
2 (S)
NT
4 (S)
NT
4
8 (S)
16 (R)
16/8 (R)
64 (R)
NT
8/4 (S)
8 (S)
2 (S)
NT
1 (S)
2 (S)
NT
4 (S)
NT
4
64
NT
-
0R; 0I
4R; 0I
0R; 0I
0R; 1I
3R; 0I
4R; 0I
53115010
E. coli
99%
-
77115012
E. coli
99%
+
-
77115010
E. coli
99%
-
77115010
E. coli
99%
-
53115012
E. coli
99%
-
57114010
E. coli
99%
+
-
* For samples run on NC 32 panels, susceptibility to Cf was tested at only one concentration (8g/ml) for
identification purposes. For samples run on NC 32 and NC 30 panels, susceptibility to Fd was tested at only
one concentration (64g/ml) for identification purposes. Samples exhibiting a MIC for Cf of 8g/ml may
be intermediate or resistant. Samples exhibiting a MIC for Fd of 64g/ml may be susceptible or
intermediate; ** For all panels, P, Cl, and K were tested at only one concentration (4g/ml) for
identification purposes-susceptibilities could not be determined; *** Antibiotics not tested on all samples,
and not included in calculation of results; a Total number of resistant/intermediate compounds for each
isolate, only including those antibiotics that were used in the tabulation of results. b Strikethrough text
indicates samples that were deemed not unique.
72
Appendix A Continued: Complete antibiotic MIC data, biotype numbers, species and
integron detection results
Antibiotic Class
Antibiotic
(K)**
(Ak)
Aminoglycosides
(Gm)
(To)
Antifol/Sulfa
(T/S)
(Cf)*
(Cft)***
(Caz)
(Cfz)
Cephalosporins
(Crm)
(Cax)
(Cpe)
(Cfx)***
(Ctn)
Tetracyclines
(Te)***
(P)**
(P/T)
(Am)
Penicillins
(A/S)
(Pi)
(Tim)***
(Aug)***
Monobactams
(Azt)
(Gat)***
(Nxn)***
Quinolones
(Cp)
(Lvx)
(Mxf)***
(Imp)
Carbapenems
(Mer)***
(Cl)**
Other
(Fd)*
(C)***
ESL-a Screen
ESL-b Screen
Total #
Resistant/Intermediatea
Biotype ID #
Species/Confidence
Class 1 Integron
Class 2 Integron
Sample #/MICs (g/ml)
0917C
0917D
4
4
16 (S)
16 (S)
1 (S)
8 (R)
2 (S)
8 (R)
2/38 (S)
2/38 (S)
8 (S)
8 (S)
NT
NT
2 (S)
2 (S)
2 (S)
2 (S)
4 (S)
4 (S)
4 (S)
4 (S)
2 (S)
2 (S)
NT
NT
4 (S)
4 (S)
NT
NT
4
4
8 (S)
8 (S)
16 (R)
16 (R)
16/8 (R)
16/8 (R)
64 (R)
64 (R)
NT
NT
8/4 (S)
8/4 (S)
8 (S)
8 (S)
=4 (I)
4 (R)
NT
NT
2 (R)
2 (R)
4 (R)
4 (R)
NT
NT
4 (S)
4 (S)
NT
NT
4
4
64
64
NT
NT
-
0915
4
16 (S)
1 (S)
2 (S)
2/38 (S)
16 (R)
8 (S)
16 (R)
16 (R)
16 (R)
32 (R)
16 (R)
16 (R)
32 (R)
8 (R)
4
64 (R)
16 (R)
16/8 (R)
64 (R)
64 (R)
16/8 (R)
16 (R)
2 (S)
4 (S)
1 (S)
2 (S)
2 (S)
8 (R)
4(S)
4
64
8 (S)
+
+
0916B
4
16 (S)
8 (R)
2 (S)
2/38 (S)
8 (S)
NT
2 (S)
2 (S)
4 (S)
4 (S)
2 (S)
NT
4 (S)
NT
4
8 (S)
16 (R)
=16/8 (I)
=64 (I)
NT
8/4 (S)
8 (S)
2 (S)
NT
1 (S)
2 (S)
NT
4 (S)
NT
4
64
NT
-
12R; 0I
2R; 2I
5R; 0I
77115012
E. coli
99%
-
77114010
E. coli
99%
+
-
53114010
E. coli
99%
-
0917Eb
4
16 (S)
1 (S)
2 (S)
2/38 (S)
8 (S)
NT
2 (S)
2 (S)
4 (S)
4 (S)
2 (S)
NT
4 (S)
NT
4
8 (S)
16 (R)
16/8 (R)
64 (R)
NT
8/4 (S)
8 (S)
4 (R)
NT
2 (R)
4 (R)
NT
4 (S)
NT
4
64
NT
-
0919
4
16 (S)
1 (S)
2 (S)
2/38 (R)
8 (S)
NT
2 (S)
2 (S)
4 (S)
4 (S)
2 (S)
NT
4 (S)
NT
4
8 (S)
16 (R)
=16/8 (I)
16 (S)
NT
8/4 (S)
8 (S)
2 (S)
NT
1 (S)
2 (S)
NT
4 (S)
NT
4
64
NT
-
7R; 0I
5R; 0I
2R; 1I
53114010
E. coli
99%
-
53114010
E. coli
99%
-
77115010
E. coli
99%
+
-
* For samples run on NC 32 panels, susceptibility to Cf was tested at only one concentration (8g/ml) for
identification purposes. For samples run on NC 32 and NC 30 panels, susceptibility to Fd was tested at only
one concentration (64g/ml) for identification purposes. Samples exhibiting a MIC for Cf of 8g/ml may
be intermediate or resistant. Samples exhibiting a MIC for Fd of 64g/ml may be susceptible or
intermediate; ** For all panels, P, Cl, and K were tested at only one concentration (4g/ml) for
identification purposes-susceptibilities could not be determined; *** Antibiotics not tested on all samples,
and not included in calculation of results; a Total number of resistant/intermediate compounds for each
isolate, only including those antibiotics that were used in the tabulation of results. b Strikethrough text
indicates samples that were deemed not unique.
73
Appendix A Continued: Complete antibiotic MIC data, biotype numbers, species and
integron detection results
Antibiotic Class
Antibiotic
(K)**
(Ak)
Aminoglycosides
(Gm)
(To)
Antifol/Sulfa
(T/S)
(Cf)*
(Cft)***
(Caz)
(Cfz)
Cephalosporins
(Crm)
(Cax)
(Cpe)
(Cfx)***
(Ctn)
Tetracyclines
(Te)***
(P)**
(P/T)
(Am)
Penicillins
(A/S)
(Pi)
(Tim)***
(Aug)***
Monobactams
(Azt)
(Gat)***
(Nxn)***
Quinolones
(Cp)
(Lvx)
(Mxf)***
(Imp)
Carbapenems
(Mer)***
(Cl)**
Other
(Fd)*
(C)***
ESL-a Screen
ESL-b Screen
Total #
Resistant/Intermediatea
Biotype ID #
Sample #/MICs (g/ml)
0920C
0922A
4
4
16 (S)
16 (S)
4 (S)
1 (S)
4 (S)
2 (S)
2/38 (S)
2/38 (S)
=16 (I)
16 (R)
NT
8 (S)
8 (S)
2 (S)
=4 (S)
16 (R)
=16 (I)
4 (S)
8 (S)
4 (S)
8 (S)
2 (S)
NT
16 (R)
16 (S)
4 (S)
NT
8 (R)
4
4
16 (S)
8 (S)
16 (R)
16 (R)
16/8 (R)
16/8 (R)
64 (R)
64 (R)
=64 (I)
NT
=16/8 (I)
8/4 (S)
8 (S)
8 (S)
2 (S)
2 (S)
NT
4 (S)
1 (S)
1 (S)
2 (S)
2 (S)
NT
2 (S)
4 (S)
4 (S)
NT
4 (S)
4
4
32 (S)
64
NT
8 (S)
+
+
-
0920A
4
16 (S)
4 (S)
4 (S)
2/38 (S)
16 (R)
8 (S)
8 (S)
16 (R)
4 (S)
8 (S)
8 (S)
16 (R)
16 (S)
8 (R)
4
16 (S)
16 (R)
8/4 (S)
=64 (I)
=64 (I)
16/8 (R)
8 (S)
2 (S)
4 (S)
1 (S)
2 (S)
2 (S)
4 (S)
4 (S)
4
32 (S)
8 (S)
+
+
0920Bb
4
16 (S)
1 (S)
2 (S)
2/38 (S)
16 (R)
NT
2 (S)
16 (R)
4 (S)
4 (S)
2 (S)
NT
4 (S)
NT
4
8 (S)
16 (R)
8/4 (S)
16 (S)
NT
16/8 (R)
8 (S)
2 (S)
NT
1 (S)
2 (S)
NT
4 (S)
NT
4
64
NT
-
2R; 1I
2R; 0I
4R; 1I
77103172
77103172
77115012
Species/Confidence
E. cloacae
99%
E. cloacae
99%
E. coli
99%
Class 1 Integron
Class 2 Integron
-
-
-
0922B
4
16 (S)
4 (S)
4 (S)
2/38 (S)
=16 (I)
8 (S)
8 (S)
8 (S)
4 (S)
8 (S)
8 (S)
8 (S)
16 (S)
8 (R)
4
16 (S)
16 (R)
16/8 (R)
64 (R)
=64 (I)
8/4 (S)
8 (S)
2 (S)
4 (S)
1 (S)
2 (S)
2 (S)
4 (S)
4 (S)
4
32 (S)
8 (S)
-
0922C
4
16 (S)
4 (S)
4 (S)
2/38 (R)
=16 (I)
8 (S)
8 (S)
16 (R)
16 (R)
8 (S)
8 (S)
=16 (I)
16 (S)
8 (R)
4
16 (S)
16 (R)
16/8 (R)
64 (R)
16 (S)
=16/8 (I)
8 (S)
2 (S)
4 (S)
1 (S)
2 (S)
2 (S)
4 (S)
4 (S)
4
32 (S)
8 (S)
+
3R; 0I
3R; 0I
6R; 0I
77744272
K. pneumoniae
85%
-
77744272
K. pneumoniae
85%
+
-
77714272
K.
oxytoca
85%
+
-
* For samples run on NC 32 panels, susceptibility to Cf was tested at only one concentration (8g/ml) for
identification purposes. For samples run on NC 32 and NC 30 panels, susceptibility to Fd was tested at only
one concentration (64g/ml) for identification purposes. Samples exhibiting a MIC for Cf of 8g/ml may
be intermediate or resistant. Samples exhibiting a MIC for Fd of 64g/ml may be susceptible or
intermediate; ** For all panels, P, Cl, and K were tested at only one concentration (4g/ml) for
identification purposes-susceptibilities could not be determined; *** Antibiotics not tested on all samples,
and not included in calculation of results; a Total number of resistant/intermediate compounds for each
isolate, only including those antibiotics that were used in the tabulation of results. b Strikethrough text
indicates samples that were deemed not unique.
74
Appendix A Continued: Complete antibiotic MIC data, biotype numbers, species and
integron detection results
Antibiotic Class
Antibiotic
(K)**
(Ak)
Aminoglycosides
(Gm)
(To)
Antifol/Sulfa
(T/S)
(Cf)*
(Cft)***
(Caz)
(Cfz)
Cephalosporins
(Crm)
(Cax)
(Cpe)
(Cfx)***
(Ctn)
Tetracyclines
(Te)***
(P)**
(P/T)
(Am)
Penicillins
(A/S)
(Pi)
(Tim)***
(Aug)***
Monobactams
(Azt)
(Gat)***
(Nxn)***
Quinolones
(Cp)
(Lvx)
(Mxf)***
(Imp)
Carbapenems
(Mer)***
(Cl)**
Other
(Fd)*
(C)***
ESL-a Screen
ESL-b Screen
Total #
Resistant/Intermediatea
Biotype ID #
Species/Confidence
Class 1 Integron
Class 2 Integron
Sample #/MICs (g/ml)
0923
4
16 (S)
1 (S)
2 (S)
2/38 (S)
=16 (I)
NT
2 (S)
2 (S)
4 (S)
4 (S)
2 (S)
NT
4 (S)
NT
4
8 (S)
16 (R)
=16/8 (I)
64 (R)
NT
8/4 (S)
8 (S)
4 (R)
NT
2 (R)
4 (R)
NT
4 (S)
NT
4
64
NT
-
0924
4
16 (S)
4 (S)
4 (S)
2/38 (S)
8 (S)
8 (S)
8 (S)
8 (S)
4 (S)
8 (S)
8 (S)
8 (S)
16 (S)
8 (R)
4
16 (S)
16 (R)
16/8 (R)
=64 (I)
16 (S)
8/4 (S)
8 (S)
2 (S)
4 (S)
1 (S)
2 (S)
2 (S)
4 (S)
4 (S)
4
32 (S)
8 (S)
-
4R; 1I
2R; 1I
73115012
E. coli
99%
-
77115210
E. coli
<85%
+
-
* For samples run on NC 32 panels, susceptibility to Cf was tested at only one concentration (8g/ml) for
identification purposes. For samples run on NC 32 and NC 30 panels, susceptibility to Fd was tested at only
one concentration (64g/ml) for identification purposes. Samples exhibiting a MIC for Cf of 8g/ml may
be intermediate or resistant. Samples exhibiting a MIC for Fd of 64g/ml may be susceptible or
intermediate; ** For all panels, P, Cl, and K were tested at only one concentration (4g/ml) for
identification purposes-susceptibilities could not be determined; *** Antibiotics not tested on all samples,
and not included in calculation of results; a Total number of resistant/intermediate compounds for each
isolate, only including those antibiotics that were used in the tabulation of results.
75
Appendix B: Antibiotic Abbreviation Key
(Ak) Amikacin
(Am) Ampicillin
(A/S) Ampicillin/Sulbactam
(Aug) Amoxicillin/ K Clavulanate
(Azt) Aztreonam
(C) Chloramphenicol
(Cax) Ceftriaxone
(Caz) Ceftazidime
(Cf) Cephalothin
(Cft) Cefotaxime
(Cfx) Cefoxitin
(Cfz) Cefazolin
(Cl) Colistin
(Cp) Ciprofloxacin
(Cpe) Cefepime
(Crm) Cefuroxime
(Ctn) Cefotetan
(Fd) Nitrofurantoin
(Gat) Gatifloxacin
(Gm) Gentamicin
(Imp) Imipenem
(K) Kanamycin
(Lvx) Levofloxacin
(Mer) Meropenem
(Mxf) Moxifloxacin
(Nxn) Norfloxacin
(P) Penicillin
(Pi) Piperacillin
(P/T) Piperacillin/Tazobactam
(Te) Tetracycline
(Tim) Ticarcillin/K Clavulanate
(To) Tobramycin
(T/S) Trimethoprim/Sulfamethoxazole
76
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