J Antimicrob Chemother 2013; 68 Suppl 1: i57 – i65
doi:10.1093/jac/dkt027
Molecular epidemiology of extended-spectrum b-lactamase-, AmpC
b-lactamase- and carbapenemase-producing Escherichia coli and
Klebsiella pneumoniae isolated from Canadian hospitals over a 5 year
period: CANWARD 2007–11
Andrew J. Denisuik1*, Philippe R. S. Lagacé-Wiens1,2, Johann D. Pitout3,4, Michael R. Mulvey1,5, Patricia J. Simner6,
Franil Tailor1, James A. Karlowsky1,7, Daryl J. Hoban1,7, Heather J. Adam1,7 and George G. Zhanel1 on behalf of the
Canadian Antimicrobial Resistance Alliance (CARA)†
1
Department of Medical Microbiology and Infectious Diseases, University of Manitoba, Winnipeg, Manitoba, Canada R3E 0J9;
Department of Microbiology, St Boniface General Hospital/Diagnostic Services of Manitoba, Winnipeg, Manitoba, Canada R2H 2A6;
3
Division of Microbiology, Calgary Laboratory Services, Calgary, Alberta, Canada T2L 2K8; 4Department of Pathology and Laboratory
Medicine, Microbiology, Immunology and Infectious Diseases, University of Calgary, Calgary, Alberta, Canada T2N 4N1; 5National
Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba, Canada R3E 3R2; 6Department of Clinical Microbiology,
Mayo Clinic, Rochester, Minnesota, USA 55905; 7Department of Clinical Microbiology, Health Sciences Centre/Diagnostic Services
of Manitoba, Winnipeg, Manitoba, Canada R3A 1R9
2
*Corresponding author. Department of Clinical Microbiology, Health Sciences Centre, MS673-820 Sherbrook Street, Winnipeg, Manitoba,
Canada R3A 1R9. Tel: +1-204-787-4684; Fax: +1-204-787-4699; E-mail: [email protected]
†Members are listed in the Acknowledgements section.
Objectives: To assess the proportion of Escherichia coli and Klebsiella pneumoniae from Canadian hospitals that
produce extended-spectrum b-lactamases (ESBLs), AmpC b-lactamases and carbapenemases, as well as to
describe the patterns of antibiotic resistance and molecular characteristics of these organisms.
Methods: Some 5451 E. coli and 1659 K. pneumoniae were collected from 2007 to 2011 inclusive as part of
the ongoing CANWARD national surveillance study. Antimicrobial susceptibility testing was performed to
detect putative ESBL, AmpC and carbapenemase producers, which were then further characterized by PCR
and sequencing to detect resistance genes. In addition, isolates were characterized by PFGE and an allelespecific PCR to detect isolates of sequence type (ST) 131.
Results: The proportion of ESBL-producing E. coli (2007, 3.4%; 2011, 7.1%), AmpC-producing E. coli (2007,
0.7%; 2011, 2.9%) and ESBL-producing K. pneumoniae (2007, 1.5%; 2011, 4.0%) among the isolates collected
increased during the study period. The majority of ESBL-producing E. coli (.95%), AmpC-producing E. coli
(.97%) and ESBL-producing K. pneumoniae (.89%) remained susceptible to colistin, amikacin, ertapenem
and meropenem. Isolates were generally unrelated by PFGE (,80% similarity); however, ST131 was identified
among 55.8% and 28.7% (P,0.001) of ESBL- and AmpC-producing E. coli, respectively. CTX-M-15 was the
dominant genotype in both ESBL-producing E. coli (66.2%) and ESBL-producing K. pneumoniae (50.0%),
while the dominant genotype in AmpC-producing E. coli was CMY-2 (55.7%). Carbapenemase production
was identified in 0.04% (n¼ 2) of E. coli and 0.06% (n¼ 1) of K. pneumoniae, all of which produced KPC-3.
Conclusions: The proportion of ESBL- and AmpC-producing E. coli and K. pneumoniae increased significantly during
the study period, while the number of carbapenemase producers remained low (,1%). Compared with AmpCproducing E. coli, ESBL-producing E. coli were significantly associated with multidrug resistance and the ST131 clone.
Keywords: ESBLs, Escherichia coli, Klebsiella pneumoniae, surveillance
Introduction
The b-lactams are a critically important class of antimicrobials
utilized worldwide for the treatment of serious hospital- and
community-acquired infections.1 Among Gram-negative organisms, b-lactamase production represents the single greatest
contributor to b-lactam resistance, including resistance to
the oxyimino-cephalosporins and carbapenems.2 Within the
# The Author 2013. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.
For Permissions, please e-mail: [email protected]
i57
Denisuik et al.
Enterobacteriaceae, oxyimino-cephalosporin resistance is largely
due to the production of extended-spectrum b-lactamases
(ESBLs) or AmpC b-lactamases (AmpC) and, in the last decade,
Enterobacteriaceae producing CTX-M-type ESBLs have increasingly become a significant cause of multidrug-resistant urinary
tract and bloodstream infections.3 The emergence of such
organisms is in large part due to the spread of a single pandemic
clone, Escherichia coli O25b:H4 sequence type (ST) 131.4 The production and transfer of ESBL enzymes in Enterobacteriaceae is
largely mediated by mobile elements, specifically conjugative
plasmids, allowing further dissemination.4
In E. coli, AmpC-mediated resistance can be the result of
plasmid-encoded AmpC b-lactamases or constitutive overexpression of the chromosomal ampC gene due to mutations within the
promoter/attenuator region.5 While acquired AmpC b-lactamases
are generally less prevalent than ESBLs, they remain clinically
important due to their ability to cause treatment failure and
their difficulty in laboratory detection.6,7 In addition, the presence
of inducible chromosomal AmpC b-lactamases in many members
of the Enterobacteriaceae presents the possibility of resistance
developing during treatment due to mutant selection.
Historically, the carbapenems have been reserved as last-line
agents in the treatment of serious or highly resistant infections.8
While both ESBL and AmpC enzymes generally lack the ability to
hydrolyse the carbapenems, reduced susceptibility or resistance
can arise when such enzymes are accompanied by decreased
outer membrane permeability. More importantly, b-lactamase
enzymes with carbapenemase activity have spread worldwide
and are beginning to compromise the use of these agents.9
Infections caused by b-lactamase-producing Enterobacteriaceae have serious implications for both public health and infection control practices. These infections are often associated
with delays in the administration of effective therapy, as
b-lactam resistance often undermines empirical treatment regimens. In patients with life-threatening infections, such as sepsis,
delayed therapy can greatly increase the risk of infection-related
mortality up to 5-fold.10 Furthermore, b-lactamase-producing
Enterobacteriaceae have demonstrated an overwhelming
ability to both disseminate and persist within the hospital and
community settings, and their frequent association with multidrug resistance (MDR) can severely limit the therapeutic
options available to clinicians.
Materials and methods
colistin (susceptible, ≤2 mg/L; resistant, ≥4 mg/L) and tigecycline (susceptible, ≤2 mg/L; intermediate, 4 mg/L; resistant, ≥8 mg/L). MDR is
defined as resistance to at least three different antimicrobial classes
and extreme drug resistance (XDR) is defined as resistance to at least
five different antimicrobial classes, as described by Magiorakos et al.14
Putative ESBL producers were identified as any E. coli or Klebsiella
pneumoniae isolate with a ceftriaxone and/or ceftazidime MIC of
≥1 mg/L.13 All putative ESBL producers were confirmed using the CLSI
phenotypic confirmatory disc test.13 An AmpC phenotype in this study
was defined as any E. coli with a ceftriaxone and/or ceftazidime MIC of
≥1 mg/L and a cefoxitin MIC ≥32 mg/L that is ESBL negative by the
CLSI confirmatory disc test. In total, cefoxitin was tested against 4449
of 5451 E. coli isolates collected, as only 558 of 1560 E. coli in 2007
were tested against this agent. Putative carbapenemase producers
were identified as any E. coli or K. pneumoniae with an ertapenem MIC
≥0.5 mg/L.
Molecular characterization of ESBL-producing
E. coli and K. pneumoniae
All phenotypically confirmed ESBL-producing E. coli and K. pneumoniae
were screened by PCR and sequencing to detect the presence of blaSHV,
blaTEM, blaCTX-M and blaOXA variants, as previously described.15 The genotypic designation ‘unknown’ was applied to any ESBL-producing isolate
that was PCR negative for the enzyme families listed above.
Molecular characterization of AmpC-producing E. coli
Putative AmpC-producing E. coli were screened by PCR and sequencing
for the presence of blaENT, blaDHA, blaFOX and blaCIT acquired AmpC
b-lactamase enzymes, as previously described.16 Sequencing for the
detection of promoter/attenuator mutations within the chromosomal
ampC gene was carried out on any isolate that was PCR negative for
all acquired AmpC b-lactamases listed above, as previously described.17
Molecular characterization of carbapenemase-producing
E. coli and K. pneumoniae
All putative carbapenemase-producing E. coli and K. pneumoniae were
screened for the presence of blaKPC, blaIMP, blaVIM, blaIMI, blaNDM, blaGES
and blaOXA-48 by multiplex PCR, as described by Mataseje et al.18 Isolates
that were PCR positive for blaKPC were reamplified using the primers KPC-F
(5′ -TGTCACTGTATCGCCGTC-3′ ) and KPC-R (5′ -CTCAGTGCTCTACAGAAAA
CC-3′ ) to yield a 1000 bp product, as described by Yigit et al.19 Sequencing
was carried out using the primers KPC-F, KPC-R, KPC1 (5′ -ATGTCACTG
TATCGCCGTC-3′ ) and KPC2 (5′ -AATCCCTCGAGCGCGAGT-3′ ).
Bacterial isolates
Bacterial isolates were collected as part of the national, ongoing
CANWARD study from 2007 to 2011, as previously outlined by Zhanel
et al.11
The CANWARD study receives annual approval by the University of
Manitoba Research Ethics Board (H2009:059).
PFGE and ST131 detection
To determine whether clonal spread influences the dissemination of
ESBL- and AmpC-producing E. coli and K. pneumoniae in Canadian hospitals, PFGE was carried out as described by Mulvey et al.15 E. coli isolates of
ST131 were identified using an allele-specific PCR for the pabB gene.20
Antimicrobial susceptibility testing
Antimicrobial susceptibility testing was performed using the broth microdilution method in accordance with CLSI guidelines.12 Following two subcultures from frozen stock, the MICs of the antibiotics tested were
determined using in-house, custom-designed 96-well microtitre panels,
as previously described.11 MIC interpretive standards were defined by
CLSI (2012) breakpoints.13 FDA interpretative criteria were used for
i58
Statistical analysis
Statistical significance was calculated by the x2 test, binary logistic regression or Fisher’s exact test in the case of small sample sizes using
the SPSS statistics (Version 20) program (IBM Corporation). Statistical significance in this study is defined as a P value ≤0.05; any P value .0.05
has been denoted not statistically significant (NS) throughout this article.
JAC
CANWARD ESBL/AmpC/KPC 2007 – 11
GenBank accession number
One novel SHV b-lactamase was discovered as part of the study and is
designated SHV-168 (www.lahey.org/studies). The compete nucleotide
sequence of SHV-168 has been deposited in GenBank under the
accession number JX870080.
Results
Proportion of ESBL-, AmpC- and
carbapenemase-producing E. coli and
K. pneumoniae among isolates collected
The national and regional proportions of the E. coli and
K. pneumoniae isolates collected that produced ESBLs, AmpC
b-lactamases and carbapenemases from 2007 to 2011 are summarized in Table 1. Between 2007 and 2011, 27 123 bacterial isolates were collected as part of the ongoing CANWARD national
surveillance study, including a total of 5451 E. coli (2007,
1560; 2008, 1131; 2009, 1097; 2010, 1017; and 2011, 646)
and 1659 K. pneumoniae (2007, 455; 2008, 314; 2009, 356;
2010, 307; and 2011, 227), of which 8.5% (462/5451) of
E. coli and 9.9% (165/1659) of K. pneumoniae collected had
ceftriaxone and/or ceftazidime MICs of ≥1 mg/L (P¼ NS). In
total, 4.2% (231/5451) and 2.9% (48/1659) of E. coli and
K. pneumoniae, respectively, were phenotypically confirmed as
ESBL producers by the CLSI confirmatory disc test, while 2.6%
(115/4449) of E. coli demonstrated an AmpC phenotype.
The proportion of E. coli isolates that produced ESBLs was variable throughout the study period, with a significant national
increase being observed from 2007 to 2011 (2007, 3.4%; 2008,
4.9%; 2009, 4.3%; 2010, 2.9%; and 2011, 7.1%) (P,0.001).
This overall trend was generated by increases occurring from
2007 to 2008 (P ¼ NS) and, most notably, from 2010 to 2011
(P,0.001). Increases in ESBL-producing E. coli were observed in
all regions (2007 compared with 2011); however, only those
observed in the British Columbia/Alberta, Ontario and Quebec
regions were statistically significant (P ¼ 0.040, P¼ 0.009 and
P ¼ 0.018, respectively). Similarly, the overall proportion of
K. pneumoniae isolates that produced ESBLs increased significantly throughout the study period (2007, 1.5%; 2008, 3.2%;
2009, 3.4%; 2010, 3.3%; and 2011, 4.0%) (P¼ 0.047), though
on a regional scale, only the increase observed in Ontario was
found to be statistically significant (P ¼ 0.01). AmpC-producing
E. coli also demonstrated a statistically significant increase
between the first (2007) and second (2008) year of this study
(2007, 0.7%; 2008, 3.1%; 2009, 2.7%; 2010, 2.7%; and 2011,
2.9%) (P ¼ 0.004), and significant increases were observed in
the British Columbia/Alberta (2007 compared with 2009/2010)
and Ontario (2007 compared with 2008) regions (P ¼ 0.005 and
P ¼ 0.047, respectively).
Table 1. The national and regional proportion of E. coli and K. pneumoniae isolates collected from Canadian hospitals that produce ESBLs, AmpC
b-lactamases and carbapenemases
CANWARD study year: % (no. in cohort/total no. of species collected)
Cohort (n)
region
2007
2008
2009
2010
2011
7.1 (46/646)
7.1 (7/99)
3.8 (3/79)
11.0 (23/210)
4.8 (8/167)
5.5 (5/91)
ESBL E. coli (231)
national
3.4 (53/1560)
BC/AB
4.4 (12/271)
SK/MB
1.8 (5/285)
ON
6.6 (29/442)
QC
1.3 (6/456)
MAR
0.9 (1/106)
4.9
7.6
4.7
5.8
2.2
3.1
(55/1131)
(18/237)
(11/235)
(17/292)
(6/270)
(3/97)
4.3 (47/1097)
9.4 (14/149)
1.4 (2/143)
6.1 (20/328)
1.5 (5/335)
4.2 (6/142)
2.9 (30/1017)
1.9 (3/157)
2.8 (4/141)
4.6 (12/259)
1.6 (5/320)
4.3 (6/140)
AmpC E. coli (115)
national
0.7 (4/558b)
BC/AB
0.8 (1/126)
SK/MB
1.3 (1/80)
ON
0.0 (0/92)
QC
0.6 (1/154)
MAR
0.9 (1/106)
3.1
3.8
2.6
3.8
3.0
1.0
(35/1131)
(9/237)
(6/235)
(11/292)
(8/270)
(1/97)
2.7 (30/1097)
5.4 (8/149)
1.4 (2/143)
1.8 (6/328)
2.7 (9/335)
3.5 (5/142)
2.7 (27/1017)
7.6 (12/157)
2.8 (4/141)
1.9 (5/259)
0.3 (1/320)
3.6 (5/140)
2.9
1.0
5.1
2.4
3.6
3.3
ESBL K. pneumoniae (48)
national
1.5 (7/455)
BC/AB
1.3 (1/76)
SK/MB
0.0 (0/67)
ON
1.4 (2/142)
QC
3.2 (4/126)
MAR
0.0 (0/44)
3.2
3.9
0.0
5.2
2.7
0.0
(10/314)
(3/77)
(0/40)
(5/96)
(2/73)
(0/28)
3.4 (12/356)
1.9 (1/54)
0.0 (0/46)
7.9 (10/126)
0.0 (0/96)
2.9 (1/34)
3.3 (10/307)
2.9 (1/34)
4.4 (2/45)
6.3 (6/96)
1.2 (1/81)
0.0 (0/51)
4.0
3.0
0.0
6.8
4.4
0.0
2007– 11
P valuea
4.2
5.9
2.8
6.6
1.9
3.7
(231/5451)
(54/913)
(25/883)
(101/1531)
(30/1548)
(21/576)
,0.001
0.040
NS
0.009
0.018
NS
(19/646)
(1/99)
(4/79)
(5/210)
(6/167)
(3/91)
2.6
4.0
2.5
2.3
2.0
2.6
(115/4449)
(31/768)
(17/678)
(27/1181)
(25/1246)
(15/576)
0.004
0.005
NS
0.047
NS
NS
(9/227)
(1/33)
(0/24)
(5/73)
(3/68)
(0/29)
2.9
2.6
0.9
5.3
2.3
0.5
(48/1659)
(7/274)
(2/222)
(28/533)
(10/444)
(1/186)
0.047
NS
NS
0.010
NS
NS
BC, British Columbia; AB, Alberta; SK, Saskatchewan; MB, Manitoba; ON, Ontario; QC, Quebec; MAR, Maritimes (New Brunswick/Nova Scotia); NS, not
statistically significant (P.0.05).
a
P value comparing the rates of ESBL-producing E. coli, ESBL-producing K. pneumoniae and AmpC-producing E. coli from 2007 to 2011.
b
Cefoxitin was tested against 558 E. coli during CANWARD 2007.
i59
Denisuik et al.
Nationally, 0.04% (2/5451) of E. coli and 0.06% (1/1659) of
K. pneumoniae were found to produce a carbapenemase (all harbouring blaKPC). Both KPC-producing E. coli isolates were collected
from Quebec in temporally independent cases (2010 and 2011,
respectively). The one KPC-producing K. pneumoniae identified
was isolated in 2009 from a patient in Ontario.
Patient demographics
Patient demographics associated with ESBL- and AmpCproducing E. coli and K. pneumoniae are summarized in
Table 2. Overall, both ESBL- and non-ESBL-producing E. coli infections occurred more frequently in females [55.0% (127/231) and
61.4% (3203/5220), respectively]; however, no significant difference was observed between genders. ESBL-producing E. coli were
significantly more likely to be isolated from the ≥65 years old
age group [55.8% (129/231) versus 47.9% (2501/5220)]
(P ¼ 0.018) and significantly less likely to be isolated from individuals ≤17 years of age [2.6% (6/231) versus 10.9% (570/5220)]
(P ≤ 0.001) as compared with non-ESBL-producing E. coli. The incidence of ESBL-producing E. coli was highest among inpatients,
with the majority [38.5% (89/231)] being isolated from patients
on medical wards. ESBL-producing E. coli were distributed across
all specimen sources, with the majority being isolated from blood
[50.2% (116/231)] and urine [35.1% (81/231)]. Furthermore, the
frequency of ESBL-producing E. coli isolated from respiratory
Table 2. Patient demographics associated with ESBL-producing E. coli,
AmpC-producing E. coli and ESBL-producing K. pneumoniae isolated
from Canadian hospitals
Cohort: % (no. in cohort/total no. collected)
Parameter
value
ESBL E. coli
(n¼231)
AmpC E. coli
(n ¼115)
ESBL K. pneumoniae
(n¼48)
Gender
male
female
4.9 (104/2120)
3.8 (127/3331)
2.6 (46/1737)
2.5 (69/2712)
3.3 (30/906)
2.4 (18/753)
Age (years)
≤17
18– 64
≥65
1.0 (6/576)
4.3 (96/2244)
4.9 (129/2631)
2.2 (10/446)
2.6 (48/1821)
2.6 (57/2182)
3.9 (6/155)
4.0 (27/677)
1.8 (15/827)
1.9 (14/755)
2.0 (35/1716)
2.1 (4/191)
1.2 (5/433)
3.8 (16/420)
3.2 (40/1269)
4.1 (13/318)
4.0 (22/547)
3.5 (10/289)
2.4 (4/170)
2.4 (59/2413)
2.5 (40/1579)
3.7 (6/163)
3.4 (10/294)
2.7 (24/890)
3.5 (13/372)
3.8 (3/80)
2.5 (8/317)
Hospital location
clinic/office
3.3 (31/943)
emergency
3.0 (62/2081)
room
ICU
6.1 (31/506)
medical
5.8 (89/1544)
ward
surgical
4.8 (18/377)
ward
Specimen source
blood
4.2 (116/2733)
urine
3.8 (81/2141)
wound
4.0 (8/198)
respiratory
6.9 (26/379)
i60
specimens was found to be significantly greater when compared
with non-ESBL-producing E. coli [11.3% (26/231) versus 6.8%
(353/5220)] (P ¼0.009).
ESBL-producing K. pneumoniae were isolated more frequently
from males [62.5% (30/48)], which significantly differed from
ESBL-producing E. coli (P ¼ 0.020), but not from non-ESBLproducing K. pneumoniae [54.4% (876/1611)] (P ¼ NS). The majority of ESBL-producing K. pneumoniae were isolated from patients
18 –64 years of age [56.3% (27/48)] as compared with ESBLproducing E. coli [41.6% (96/231)] (P ¼ 0.044) and non-ESBLproducing K. pneumoniae [40.3% (650/1611)] (P ¼ 0.027), where
the majority of isolates were collected from patients ≥65 years
of age (55.8% and 50.4%, respectively). ESBL-producing
K. pneumoniae were isolated most frequently from patients on
medical wards [45.8% (22/48)] and occurred at a significantly
reduced rate in the intensive care unit (ICU) [27.1% (13/48)]
(P ¼ 0.020) and emergency room [10.4% (5/48)] (P ¼ 0.009) as
compared with ESBL-producing E. coli. As with ESBL-producing
E. coli, ESBL-producing K. pneumoniae were distributed across all
specimen sources, with the majority [50.0% (24/48)] isolated
from blood. No significant differences were observed with regard
to specimen source when compared with ESBL-producing E. coli
and non-ESBL-producing K. pneumoniae.
AmpC-producing isolates were isolated more frequently from
females [60.0% (69/115)], with no significant differences observed
compared with ESBL- and non-ESBL-non-AmpC-producing E. coli.
AmpC-producing E. coli were isolated most frequently from
patients ≥65 years of age on medical wards with bloodstream
infections. No significant demographic differences were observed
comparing AmpC-producing E. coli with non-ESBL-non-AmpCproducing E. coli. However, the frequency of AmpC-producing
E. coli infections was significantly higher in the ≤17 years old age
group compared with ESBL-producing E. coli [8.7% (10/115)
versus 2.6% (6/231)] (P¼ 0.014).
Two KPC-producing E. coli were isolated from respiratory
specimens collected from male patients aged 77 and 74 years,
respectively, located in the ICU. One KPC-producing
K. pneumoniae isolate was obtained from the blood culture of
a 67-year-old female patient located on a medical ward.
Antimicrobial susceptibility
The activities of the antimicrobials tested against ESBL- and
AmpC-producing E. coli and K. pneumoniae are summarized in
Table 3. The antibiotics with the greatest activity against the isolates in this study were amikacin, meropenem, ertapenem and
colistin. Both ESBL- and AmpC-producing E. coli also demonstrated high susceptibility (.93%) to piperacillin/tazobactam;
however, in the case of ESBL-producing K. pneumoniae, susceptibility decreased to 66.7%, with a large number (18.8%) of
isolates falling in the intermediate range. Similarly, while tigecycline demonstrated excellent activity against ESBL- and AmpCproducing E. coli (99.6% and 100% susceptibility, respectively),
ESBL-producing K. pneumoniae were significantly less likely to
be susceptible to this agent (83.3% susceptibility, P,0.001).
A multidrug-resistant phenotype was observed in 78.8% (182/231)
and 68.8% (33/48) of ESBL-producing E. coli and K. pneumoniae,
respectively (P ¼ NS), while AmpC-producing E. coli were significantly less likely to be multidrug resistant when compared with
ESBL-producing E. coli (33.9%, P,0.001). The frequency of MDR
JAC
CANWARD ESBL/AmpC/KPC 2007 – 11
Table 3. Antimicrobial activity against ESBL-producing E. coli, AmpC-producing E. coli and ESBL-producing K. pneumoniae
MIC (mg/L)
MIC interpretation
Cohort (n)
antibiotic
MIC50
MIC90
Min.
Max.
%S
%I
%R
ESBL E. coli (231)
AMC
cefazolin
cefoxitin
ceftriaxone
ceftazidime
cefepime
TZP
ertapenem
meropenem
ciprofloxacin
amikacin
gentamicin
tigecycline
SXT
colistin
8
.128
8
.64
16
8
4
≤0.06
≤0.12
.16
4
4
0.5
.8
0.5
16
.128
16
.64
.32
.32
16
0.25
≤0.12
.16
16
.32
1
.8
1
1
16
0.5
≤0.25
≤0.5
≤1
≤1
≤0.06
≤0.12
≤0.06
≤2
≤0.5
0.12
≤0.12
≤0.06
.32
.128
.32
.64
.32
.32
512
4
1
.16
.64
.32
4
.8
4
62.3
33.8
81.8
1.3
36.5
53.2
93.1
97.4
100.0
10.8
95.7
51.1
99.6
29.9
99.6
10.0
1.7
7.3
24.9
4.8
1.3
3.9
100.0
8.2
97.0
56.2
21.9
2.2
1.3
ESBL K. pneumoniae (48)
AMC
cefazolin
cefoxitin
ceftriaxone
ceftazidime
cefepime
TZP
ertapenem
meropenem
ciprofloxacin
amikacin
gentamicin
tigecycline
SXT
colistin
16
.128
8
.64
.32
8
16
0.06
≤0.12
8
≤2
2
1
.8
0.5
32
.128
.32
.64
.32
.32
256
0.5
≤0.12
.16
32
.32
4
.8
1
2
16
2
≤0.25
1
≤1
2
≤0.06
≤0.12
≤0.06
≤2
≤0.5
0.5
≤0.12
0.25
.32
.128
.32
.64
.32
.32
.512
1
0.12
.16
.64
.32
16
.8
.16
47.7
34.1
77.3
14.6
29.3
57.9
66.7
97.7
100.0
27.1
89.6
52.1
83.3
31.3
97.7
11.4
8.3
2.4
7.9
18.8
2.3
AmpC E. coli (115)
AMC
cefazolin
cefoxitin
ceftriaxone
ceftazidime
cefepime
TZP
ertapenem
meropenem
ciprofloxacin
amikacin
gentamicin
tigecycline
SXT
colistin
16
.128
.32
8
16
≤0.25
4
≤0.06
≤0.06
≤0.06
2
≤0.5
0.5
0.25
0.25
.32
.128
.32
32
.32
1
16
0.25
≤0.06
.16
4
32
1
.8
0.5
1
0.5
32
≤0.25
1
≤0.25
≤1
≤0.06
≤0.06
≤0.06
≤2
≤0.5
0.12
≤0.12
0.12
.32
.128
.32
.64
.32
.32
256
1
0.12
.16
.64
.32
2
.8
1
0.9
3.9
0.4
0.4
88.3
0.4
48.5
70.1
0.4
10.4
2.1
8.3
27.8
0.9
22.6
3.5
40.0
43.2
96.6
91.3
97.4
100.0
61.7
98.3
83.5
100.0
66.1
100.0
2.6
5.4
1.1
7.0
2.6
0.9
18.2
100.0
11.4
77.1
68.3
34.2
14.6
62.5
8.3
47.9
8.3
68.8
2.3
49.6
95.7
100.0
57.4
51.4
2.3
1.7
37.4
1.7
16.5
33.9
% S, % susceptible; % I, % intermediate; % R, % resistant; AMC, amoxicillin/clavulanic acid; TZP, piperacillin/tazobactam; SXT, trimethoprim/
sulfamethoxazole.
i61
Denisuik et al.
among ESBL-producing E. coli increased throughout the study
period, from 77.4% in 2007 to 82.6% in 2011 (P ¼ NS). Some
2.6% (6/231) of ESBL-producing E. coli and 10.4% (5/48) of
ESBL-producing K. pneumoniae demonstrated XDR, while no
AmpC-producing E. coli were extremely drug resistant.
All KPC-producing isolates demonstrated in vitro resistance
to amoxicillin/clavulanate, cefazolin, ceftriaxone, ceftazidime,
piperacillin/tazobactam, ertapenem, ciprofloxacin and trimethoprim/sulfamethoxazole, while all remained susceptible to colistin
and tigecycline. Only the one KPC-producing K. pneumoniae
isolate demonstrated in vitro resistance to meropenem according to current CLSI breakpoints (MIC 4 mg/L), with both E. coli
isolates having meropenem MICs of 1 mg/L.
Molecular characterization
The genotypic characterization of ESBL-producing E. coli and
K. pneumoniae is summarized in Tables 4 and 5. Among ESBLTable 4. The genotypic characterization of ESBL-producing E. coli
Family: % of total (n)
variant: % of total (n)
CTX-M: 94.4 (218)
CTX-M-15: 66.2 (153)
CTX-M-15
+ TEM-1
+ OXA-1
+ TEM-1, OXA-1
CTX-M-14: 19.5 (45)
CTX-M-14
+ TEM-1
+ OXA-1
CTX-M-27: 6.5 (15)
CTX-M-27
+ TEM-1
CTX-M-24: 0.9 (2)
CTX-M-24
+ TEM-1
CTX-M-3: 0.9 (2)
+ TEM-1
+ TEM-1, OXA-1
CTX-M-65: 0.4 (1)
CTX-M-65
SHV: 3.0 (7)
SHV-12: 1.7 (4)
SHV-12
+ TEM-1
SHV-2a: 1.3 (3)
SHV-2a
% of ESBL E. coli (n)
9.1 (21)
7.8 (18)
36.4 (84)
13.0 (30)
6.5 (15)
12.1 (28)
0.9 (2)
5.6 (13)
0.9 (2)
0.4 (1)
0.4 (1)
0.4 (1)
0.4 (1)
0.4 (1)
0.4 (1)
1.3 (3)
1.3 (3)
TEM: 0.4 (1)
TEM-12: 0.4 (1)
TEM-12
0.4 (1)
Unknown: 2.2 (5)
unknown
+ TEM-1
0.9 (2)
1.3 (3)
i62
Table 5. The genotypic characterization of ESBL-producing
K. pneumoniae
Genotype
CTX-M-2, SHV-11
CTX-M-3, SHV-108, TEM-1
CTX-M-14, SHV-1
CTX-M-14, SHV-11
CTX-M-14, SHV-11, TEM-1
CTX-M-15, OXA-1
CTX-M-15, SHV-1
CTX-M-15, SHV-1, OXA-1
CTX-M-15, SHV-1, TEM-1
CTX-M-15, SHV-1, TEM-1, OXA-1
CTX-M-15, SHV-5, TEM-1, OXA-1
CTX-M-15, SHV-11
CTX-M-15, SHV-11, TEM-1
CTX-M-15, SHV-11, TEM-1, OXA-1
CTX-M-15, SHV-12, TEM-1
CTX-M-15, SHV-28, OXA-1
CTX-M-15, SHV-168
CTX-M-27, SHV-11
SHV-2
SHV-2a
SHV-2a, OXA-1
SHV-12
SHV-12, TEM-1
SHV-31, TEM-1
Unknown, SHV-1
Unknown, TEM-1
% of ESBL K. pneumoniae (n)
2.1 (1)
2.1 (1)
2.1 (1)
6.3 (3)
2.1 (1)
2.1 (1)
6.3 (3)
10.4 (5)
4.2 (2)
4.2 (2)
2.1 (1)
2.1 (1)
2.1 (1)
10.4 (5)
2.1 (1)
2.1 (1)
2.1 (1)
2.1 (1)
2.1 (1)
6.3 (3)
2.1 (1)
4.2 (2)
8.3 (4)
2.1 (1)
6.3 (3)
2.1 (1)
producing E. coli and K. pneumoniae, CTX-M represented the
dominant family of enzyme identified (94.4% and 66.7%, respectively), with CTX-M-15 being the dominant variant expressed
(70.2% and 75.0%, respectively). Some 73.6% (170/231) of
ESBL-producing E. coli and 83.3% (40/48) of ESBL-producing
K. pneumoniae in this study produced multiple b-lactamases.
The narrow-spectrum b-lactamases TEM-1 and OXA-1 occurred
frequently in both ESBL-producing E. coli (37.7% and 50.6%,
respectively) and ESBL-producing K. pneumoniae (41.7% and
33.3%, respectively). In addition, 35.4% of ESBL-producing
K. pneumoniae produced SHV-1. The most common genotype
among ESBL-producing E. coli was CTX-M-15, OXA-1 [36.4%
(84/231)], while CTX-M-15, SHV-11, TEM-1, OXA-1 [10.4%
(5/48)] was the most common among ESBL-producing K. pneumoniae. Genotypic diversity was greater among ESBL-producing
K. pneumoniae, with 26 different genotypes in 48 K. pneumoniae
isolates, as compared with 20 different genotypes in 231 E. coli
isolates.
Of the 115 AmpC-producing E. coli, 65 (56.5%) contained
acquired AmpC b-lactamase genes, of which 64 (98.5%) produced CMY-2 and 1 (1.5%) produced FOX-5. The remaining 50
(43.5%) isolates were PCR negative for the acquired AmpC
b-lactamase genes detected in this study and were found to
contain promoter/attenuator mutations within the chromosomal ampC gene of E. coli.
PFGE was carried out in order to assess the genetic relatedness of ESBL- and AmpC-producing strains isolated from
CANWARD ESBL/AmpC/KPC 2007 – 11
Canadian hospitals. Overall, ESBL- and AmpC-producing E. coli
were generally not related by PFGE; however, in both cases,
small clusters of genetically related isolates were identified.
One clonal outbreak of AmpC-producing E. coli was identified
from an Ontario hospital in 2008. This outbreak consisted of
three isolates, all containing a 214/213 GT insertion [GTCGTT
to GTCGTGTT] within the promoter region of the chromosomal
ampC gene, as previously reported by Simner et al.21 Similarly,
one clonal outbreak of ESBL-producing E. coli was also identified
from the Ontario region in 2011, again consisting of three isolates, all of which contained a CTX-M-15, TEM-1, OXA-1 genotype. ST131 was identified among 55.8% (129/231) of
ESBL-producing E. coli as compared with 28.7% (33/115) of
AmpC-producing E. coli (P,0.001). The rate of ST131 among
ESBL-producing E. coli showed a steady, significant increase
across the study period, from a rate of 49.1% in 2007 to
71.7% in 2011 (2007, 26/53; 2008, 27/55; 2009, 25/47; 2010,
18/30; and 2011, 33/46) (P ¼ 0.020).
ESBL-producing K. pneumoniae were also generally not
related by PFGE. Analysis did, however, reveal two clusters of
interest, again from the Ontario region, suggesting possible intrahospital spread. The first cluster of isolates contained two
SHV-12 producers, both isolated from the same Ontario hospital
in 2008 and 2009; the second cluster contained four CTX-M-15
producers, all from the same Ontario hospital isolated in 2009.
All KPC-producing E. coli (n¼ 2) and K. pneumoniae (n¼ 1) produced KPC-3. The two KPC-producing E. coli isolates coexpressed
TEM-1 and were found to be genetically related (80% similarity)
by PFGE.
Discussion
E. coli was the top-ranked organism collected during the
CANWARD study, comprising 20.1% of all isolates, while K. pneumoniae ranked fifth overall and third among Gram-negative
organisms, comprising 6.1% of all isolates collected. The national
rates of ESBL-producing E. coli and K. pneumoniae from 2007 to
2011 were found to be 4.2% and 2.9%, respectively. These rates
remain lower that those reported by the SMART (2009 –10)
study, where 8.5% and 8.8% of North American E. coli and
K. pneumoniae isolates were found to produce an ESBL.22 With
respect to Canadian data, the rate of ESBL-producing E. coli is
comparable to that published by Peirano et al. 23 in a study of
E. coli blood culture isolates from the Calgary Health Region isolated between 2000 and 2010. In a similar study by the same
group,24 the rate of ESBL-producing K. pneumoniae was found
to be much lower [0.6% (89/15371)] than what is reported
here. Nationally, the proportion of E. coli and K. pneumoniae isolates collected that produced an ESBL increased significantly
during the study period, and in the case of E. coli, all regions
demonstrated significant increases with the exception of
Saskatchewan/Manitoba and the Maritimes (Nova Scotia and
New Brunswick). Although the proportion of ESBL-producing
K. pneumoniae demonstrated variability, at least in part due to
the decreased overall number of K. pneumoniae collected in
comparison with E. coli, a significant regional increase was
observed in Ontario.
We speculate that the observed increase in ESBL-producing
E. coli was largely driven by the continued success of the
JAC
O25b:H4 ST131 clone, reflected in the growing proportion of
ST131 isolates among our cohort. Similarly, Peirano et al. 23
reported a clear association between ST131 and a significant
increase in the rate of ESBL+ E. coli bloodstream infections
since 2007 in the Calgary Health Region. In addition, this clone
represents the major factor influencing the spread of CTX-M-15
in Canadian hospitals, with 102 of 153 CTX-M-15 producers
belonging to ST131. These data are consistent with reports
from the USA by Johnson et al.,25 where ST131 comprised 56%
of CTX-M-15-producing isolates.
Factors driving the observed increase in ESBL-producing
K. pneumoniae were less clear, due in part to genotypic diversity
and a lack of ST data. A recent report from Canada does,
however, indicate the absence of any one dominant ST promoting the spread of ESBL-producing K. pneumoniae, where the
major STs ST17, ST20, ST573 and ST575 comprised only 32%
of isolates.24 While a large number of ESBL-producing K. pneumoniae (50%) were found to produce CTX-M-15, it is difficult to
justify that any one successful plasmid or mobile element is currently influencing this spread, as 12 different genotypes were
identified among 24 isolates. Although indicative of a largely
polyclonal cohort, this does not rule out the possibility that one
or more dominant ST is responsible for the observed trends. For
example, in a study by Ko et al.,26 70% of ESBL-producing
K. pneumoniae from Korea belonged to ST11, while multiple
genotypes were identified within this ST, indicating several acquisition events by this clone. In order to further delineate the
molecular basis underlying the spread of ESBL-producing
K. pneumoniae in Canadian hospitals, additional investigations
are required.
Recently, there has been considerable debate regarding
whether it is necessary to utilize ESBL detection methods in
the case of isolates with elevated MICs to extended-spectrum
cephalosporins and the clinical relevance of such testing.27 We
feel that it is important to highlight that 1.3%, 36.5% and
53.2% of ESBL-producing E. coli and 14.6%, 29.3% and 57.9%
of ESBL-producing K. pneumoniae in this study demonstrated
in vitro susceptibility to ceftriaxone, ceftazidime and cefepime,
respectively, based on current cephalosporin breakpoints. These
data are relevant to the recent CLSI and EUCAST recommendations that lowered cephalosporin breakpoints are sufficient for
the detection of resistance genes and clinical decision making
with regard to antibiotic selection.13,28 Although the combination
of MIC-based screening criteria and phenotypic methods for the
detection of ESBL production does not represent a perfect
system, the use of such tests in a clinical setting would provide
further clarity when administering antimicrobial therapy and is
important to the implementation of proper infection control.
AmpC-producing E. coli demonstrated a significant national
increase, including regional increases in British Columbia/
Alberta and Ontario. With an overall rate of 2.6% among all
E. coli collected, AmpC-producing isolates remain a relevant
cause of antimicrobial-resistant infections in Canadian hospitals.
The molecular basis of AmpC-mediated resistance in this study
resulted from approximately equal proportions of isolates harbouring mutations within the chromosomal ampC gene and
those producing plasmid-mediated CMY-2, consistent with previous reports.21,29,30 The observed increase in the proportion of
AmpC producers among E. coli isolates collected in this study is
probably multifactorial, resulting from the clonal spread of
i63
Denisuik et al.
virulent strains and the dissemination of conjugative plasmids,
as previously described by Baudry et al.,29 as well as increased
selection pressure for chromosomal ampC hyperproducers.
The identification of carbapenemase-producing isolates was
minimal in this study and is limited by the low overall prevalence
of such organisms in Canadian hospitals combined with the
overall number of isolates collected. The first carbapenemase
producer in our cohort was received in January 2009, shortly
after KPC-producing Enterobacteriaceae first appeared in
Canadian hospitals.31 Globally, the prevalence of carbapenemaseproducing E. coli and K. pneumoniae is largely dependent on
region. From 2007 to 2009, the rate of carbapenemaseproducing E. coli and K. pneumoniae was estimated to range
between 1.8% and 2.4%, based on US, European and Latin
American data as reported by the SENTRY study.32 The national
prevalence of carbapenemase resistance among clinical Enterobacteriaceae isolates in Canada is estimated by the Canadian
Nosocomial Infection Surveillance Program (CNISP) to be significantly lower at 0.1% (59/52078), with only 10 isolates confirmed
to produce a carbapenemase enzyme from 2009 to 2010.18 If
we consider the years 2009 –11 of this study, corresponding
with the emergence of carbapenemase producers in Canadian
hospitals, the CNISP data are in agreement with findings
reported here [0.1% (3/3294)]. The detection of blaKPC-3 in all carbapenemase producers in this study is consistent with the report
by Mataseje et al.,18 where 7 of 10 carbapenemase-producing
Enterobacteriaceae isolated from Canadian hospitals produced
this variant.
In summary, we provide an update on the national prevalence and molecular epidemiology of ESBL-, AmpC- and
carbapenemase-producing E. coli and K. pneumoniae isolated
from Canadian hospitals from 2007 to 2011. Over this 5 year
period, the proportion of ESBL- and AmpC-producing E. coli and
K. pneumoniae demonstrated significant national increases,
with ESBL-producing E. coli representing the dominant group in
Canada. While the number of carbapenemase-producing Enterobacteriaceae is currently low, individual cases began to appear at
the beginning of 2009. Furthermore, through increased selection
pressure due to the rising incidence of ESBL-producing isolates
and the influence of foreign travel, it is highly likely that the
rate of carbapenemase producers in Canadian hospitals will continue to increase.33
Funding
Funding for CANWARD was provided in part by the University of Manitoba,
Health Sciences Centre (Winnipeg, Manitoba, Canada), Abbott Laboratories Ltd, Achaogen Inc., Affinium Pharmaceuticals Inc., Astellas Pharma
Canada Inc., AstraZeneca, Bayer Canada, Cerexa Inc./Forest Laboratories
Inc., Cubist Pharmaceuticals, Merck Frosst, Pfizer Canada Inc., Sunovion
Pharmaceuticals Canada Inc. and The Medicines Company.
Transparency declarations
D. J. H. and G. G. Z. have received research funding from Abbott Laboratories Ltd, Achaogen Inc., Affinium Pharmaceuticals Inc., Astellas Pharma
Canada Inc., AstraZeneca, Bayer Canada, Cerexa Inc./Forest Laboratories
Inc., Cubist Pharmaceuticals, Merck Frosst, Pfizer Canada Inc., Sunovion
Pharmaceuticals Canada Inc. and The Medicines Company. All other
authors: none to declare.
This article forms part of a Supplement sponsored by the University of
Manitoba and Diagnostic Services of Manitoba, Winnipeg, Canada.
References
1 Essack SY. The development of b-lactam antibiotics in response to the
evolution of b-lactamases. Pharm Res 2001; 18: 1391– 9.
2 Livermore DM. b-Lactamases—the threat renews. Curr Protein Pept Sci
2009; 10: 397–400.
3 Pitout JDD. Infections with extended-spectrum b-lactamase-producing
Enterobacteriaceae: changing epidemiology and drug treatment choices.
Drugs 2010; 70: 313– 33.
4 Rogers BA, Sidjabat HE, Paterson DL. Escherichia coli O25b-ST131: a
pandemic, multiresistant, community-associated strain. J Antimicrob
Chemother 2010; 66: 1 –14.
5 Jacoby GA. AmpC b-lactamases. Clin Microbiol Rev 2009; 22: 161–82.
6 Philippon A, Arlet G, Jacoby GA. Plasmid-determined AmpC-type
b-lactamases. Antimicrob Agents Chemother 2002; 46: 1 –11.
7 Peter-Getzlaff S, Polsfuss S, Poledica M et al. Detection of AmpC
b-lactamases in Escherichia coli: comparison of three phenotypic
confirmation assays and genetic analysis. J Clin Microbiol 2011; 49:
2924– 32.
8 Papp-Wallace KM, Endimiani A, Taracila MA et al. Carbapenems: past,
present, and future. Antimicrob Agents Chemother 2011; 55: 4943– 60.
9 Nordmann P, Naas T, Poirel L. Global spread of carbapenemaseproducing Enterobacteriaceae. Emerg Infect Dis 2011; 17: 1791– 8.
Acknowledgements
These data were presented in part at the Fifty-second Interscience
Conference on Antimicrobial Agents and Chemotherapy in
San Francisco, CA, USA, 2012 (Abstract no. C2-103).
The authors would like to thank the participating centres, investigators
and laboratory site staff for their continued support and cooperation.
CANWARD data can also be found at www.can-r.ca, the official web
site of the Canadian Antimicrobial Resistance Alliance (CARA).
Members of the Canadian Antimicrobial Resistance
Alliance (CARA)
The CARA principal members include George G. Zhanel, Daryl J. Hoban,
Heather J. Adam, James A. Karlowsky, Melanie R. Baxter, Kimberly
A. Nichol, Philippe R. S. Lagacé-Wiens and Andrew Walkty.
i64
10 Kumar A, Ellis P, Arabi Y et al. Initiation of inappropriate antimicrobial
therapy results in a fivefold reduction of survival in human septic shock.
Chest 2009; 136: 1237 –48.
11 Zhanel GG, Adam HJ, Low DE et al. Antimicrobial susceptibility of
15644 pathogens from Canadian hospitals: results of the CANWARD
2007– 2009 study. Diagn Microbiol Infectious Dis 2011; 69: 291–306.
12 Clinical and Laboratory Standards Institute. Methods for Dilution
Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically—
Ninth Edition: Approved Standard M7-A9. CLSI, Wayne, PA, USA, 2012.
13 Clinical and Laboratory Standards Institute. Performance Standards
for Antimicrobial Susceptibility Testing: Twenty-second Informational
Supplement M100-S22. CLSI, Wayne, PA, USA, 2012.
14 Magiorakos AP, Srinivasan A, Carey RB et al. Multidrug-resistant,
extensively drug-resistant and pandrug-resistant bacteria: an
international expert proposal for interim standard definitions for
acquired resistance. Clin Microbiol Infect 2011; 18: 268– 81.
CANWARD ESBL/AmpC/KPC 2007 – 11
15 Mulvey MR, Bryce E, Boyd D et al. Ambler class A extended-spectrum
b-lactamase-producing Escherichia coli and Klebsiella spp. in Canadian
hospitals. Antimicrob Agents Chemother 2004; 48: 1204–14.
16 Pérez-Pérez FJ, Hanson ND. Detection of plasmid-mediated AmpC
b-lactamase genes in clinical isolates by using multiplex PCR. J Clin
Microbiol 2002; 40: 2153–62.
17 Caroff N, Espaze E, Bérard I et al. Mutations in the ampC promoter of
Escherichia coli isolates resistant to oxyimino-cephalosporins without
extended spectrum b-lactamase production. FEMS Microbiol Lett 1999; 173:
459–65.
JAC
pneumoniae over a 10 year period in Calgary, Canada. J Antimicrob
Chemother 2012; 67: 1114 –20.
25 Johnson JR, Urban C, Weissman SJ et al. Molecular epidemiological
analysis of Escherichia coli sequence type ST131 (O25:H4) and
blaCTX-M-15 among extended-spectrum-b-lactamase-producing E. coli
from the United States, 2000 to 2009. Antimicrob Agents Chemother
2012; 56: 2364– 70.
26 Ko KS, Lee JY, Baek JY et al. Predominance of an ST11
extended-spectrum b-lactamase-producing Klebsiella pneumoniae
clone causing bacteraemia and urinary tract infections in Korea. J Med
Microbiol 2010; 59: 822– 8.
18 Mataseje LF, Bryce E, Roscoe D et al. Carbapenem-resistant
Gram-negative bacilli in Canada 2009– 10: results from the Canadian
Nosocomial Infection Surveillance Program (CNISP). J Antimicrob
Chemother 2012; 67: 1359 –67.
27 Livermore DM, Andrews JM, Hawkey PM et al. Are susceptibility tests
enough, or should laboratories still seek ESBLs and carbapenemases
directly? J Antimicrob Chemother 2012; 67: 1569– 77.
19 Yigit H, Queenan AM, Anderson GJ et al. Novel carbapenem-hydrolyzing
b-lactamase, KPC-1, from a carbapenem-resistant strain of Klebsiella
pneumoniae. Antimicrob Agents Chemother 2001; 45: 1151 –61.
28 Leclercq R, Canton R, Brown DF et al. EUCAST expert rules in
antimicrobial susceptibility testing. Clin Microbiol Infect 2013; 19:
141–60.
20 Clermont O, Dhanji H, Upton M et al. Rapid detection of the
O25b-ST131 clone of Escherichia coli encompassing the CTX-M-15producing strains. J Antimicrob Chemother 2009; 64: 274–7.
21 Simner PJ, Zhanel GG, Pitout J et al. Prevalence and characterization
of extended-spectrum b-lactamase- and AmpC b-lactamase-producing
Escherichia coli: results of the CANWARD 2007– 2009 study. Diagn
Microbiol Infect Dis 2011; 69: 326–34.
22 Hoban DJ, Lascols C, Nicolle LE et al. Antimicrobial susceptibility of
Enterobacteriaceae, including molecular characterization of extendedspectrum b-lactamase-producing species, in urinary tract isolates from
hospitalized patients in North America and Europe: results from the
SMART study 2009 –2010. Diagn Microbiol Infect Dis 2012; 74: 62– 7.
23 Peirano G, van der Bij AK, Gregson DB et al. Molecular epidemiology
over an 11-year period (2000 to 2010) of extended-spectrum
b-lactamase-producing Escherichia coli causing bacteremia in a
centralized Canadian region. J Clin Microbiol 2012; 50: 294–9.
24 Peirano G, Hung King Sang J, Pitondo-Silva A et al. Molecular
epidemiology of extended-spectrum b-lactamase-producing Klebsiella
29 Baudry PJ, Mataseje L, Zhanel GG et al. Characterization of plasmids
encoding CMY-2 AmpC b-lactamases from Escherichia coli in Canadian
intensive care units. Diagn Microbiol Infect Dis 2009; 65: 379–83.
30 Mataseje LF, Neumann N, Crago B et al. Characterization of
cefoxitin-resistant Escherichia coli isolates from recreational beaches
and private drinking water in Canada between 2004 and 2006.
Antimicrob Agents Chemother 2009; 53: 3126–30.
31 Goldfarb D, Harvey SB, Jessamine K et al. Detection of
plasmid-mediated KPC-producing Klebsiella pneumoniae in Ottawa,
Canada: evidence of intrahospital transmission. J Clin Microbiol 2009; 47:
1920– 2.
32 Castanheira M, Mendes RE, Woosley LN et al. Trends in
carbapenemase-producing Escherichia coli and Klebsiella spp. from
Europe and the Americas: report from the SENTRY antimicrobial
surveillance programme (2007–09). J Antimicrob Chemother 2011; 66:
1409– 11.
33 van der Bij AK, Pitout JDD. The role of international travel in the
worldwide spread of multiresistant Enterobacteriaceae. J Antimicrob
Chemother 2012; 67: 2090 –100.
i65