Abstract

Objectives

A study was designed to investigate the molecular epidemiology of extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae isolated in a centralized region over a 10 year period (2000–09).

Methods

Molecular characterization was done using isoelectric focusing, PCR and sequencing for blaCTX-M, blaTEM and blaSHV genes and plasmid-mediated quinolone resistance determinants. Genetic relatedness was determined with PFGE using XbaI and multilocus sequencing typing.

Results

A total of 89 patients with incident infections were identified; the majority presented with hospital-onset urinary tract infections. The absolute number of ESBL-producing isolates remained very low until 2003, increased slightly in 2004, remained stable until 2008 and then in 2009 there was an abrupt increase in the numbers of ESBL producers identified. The majority of K. pneumoniae produced CTX-M-14 and -15, and have replaced SHV-12-producing isolates since 2005. We identified four different major sequence types (STs) among 32% of isolates (i.e. ST17, ST20, and the new ST573 and ST575) and provided insight into their clinical and molecular characteristics. The ST isolates were more likely to produce community-onset infections, were associated with blaCTX-M and emerged during the latter part of the study period. ST17 produced CTX-M-15 and SHV-12, and was more likely to be positive for qnrB; ST20 produced CTX-M-14 and was positive for qnrS. The multiresistant ST575 that produced CTX-M-15 appeared in 2009.

Conclusions

Our study highlights the importance of molecular epidemiology in providing insight into the emergence, characteristics and distribution of STs among ESBL-producing K. pneumoniae.

Introduction

Extended-spectrum β-lactamases (ESBLs) have the ability to hydrolyse and cause resistance to various types of β-lactam antibiotics, including the oxyimino-cephalosporins and monobactams, but not the cephamycins and carbapenems.1 Organisms that produce ESBLs remain important causes of the failure of therapy with cephalosporins and have serious infection control consequences. A report from the Infectious Diseases Society of America listed ESBL-producing Klebsiella spp. and Escherichia coli as priority drug-resistant microbes for which new therapies are urgently needed.2

ESBLs were first described in Klebsiella pneumoniae isolated during 1983 in Europe3 and in 1989 in the USA.4 During the 1990s, K. pneumoniae with SHV and TEM types of ESBLs were recognized worldwide as major causes of hospital-acquired infections.1 Infections due to ESBL-producing K. pneumoniae most often involved the urinary and respiratory tracts, and were mainly associated with epidemic clones causing local outbreaks in the intensive care setting. The prevalence of ESBL-producing K. pneumoniae reached high rates during the mid 2000s among isolates collected in Latin America (44%), followed by Asia/Pacific (22.4%), Europe (13.3%) and North America (7.5%).5

From the late 1990s until the present, Enterobacteriaceae (mostly E. coli) producing different types of ESBLs, such as CTX-M enzymes, have emerged within the hospital and community settings as an important cause of urinary tract infections, and since the mid 2000s these enzymes have been the most prevalent ESBLs on a worldwide basis.6

The genes encoding ESBLs are usually located on plasmids that are highly mobile and often harbour resistance genes to several other unrelated classes of antimicrobials, such as the aminoglycosides and trimethoprim/sulfamethoxazole.1 Some CTX-M β-lactamases have been associated with the presence of different plasmid-mediated quinolone resistance (PMQR) determinants, including the qnr genes, encoding QnrA, QnrS and QnrB, and the production of a novel aminoglycoside-modifying enzyme, encoded by aac(6′)-Ib-cr, that has the additional ability to modify certain fluoroquinolones.7

Very limited data are available regarding the molecular epidemiology of ESBL-producing Enterobacteriaceae in large geographical areas. Previous population-based studies in Calgary had shown that E. coli was responsible for >95% of ESBL production in this region.8 We designed a study that investigated the clinical features, prevalence and molecular epidemiology of ESBL-producing K. pneumoniae isolated in Calgary during a 10 year period ranging from January 2000 to December 2009.

Materials and methods

Study population

In Calgary, the Calgary Zone, Alberta Health Services (formerly known as the Calgary Health Region) provides all publicly funded healthcare services to the 1.2 million people residing in the cities of Calgary and Airdrie and the numerous adjacent communities covering an area of 37 000 km2. Acute care is provided principally through one paediatric and three large adult hospitals. A centralized laboratory (Calgary Laboratory Services) performs the routine clinical microbiology services for general practitioners, medical specialists, community clinics and hospitals within the Calgary Zone. After a case of ESBL-producing K. pneumoniae was identified in the Calgary Region, infection control procedures were undertaken, consisting of rectal screening for possible colonized patients in the same ward/unit and the contact isolation of infected or colonized patients.

Bacterial isolates and patients

All ESBL-producing K. pneumoniae recovered from clinical specimens between 1 January 2000 and 31 December 2009 were studied. Only non-repeat isolates from true incident cases with infections were included in this study. Hospital-acquired cases were classified as patients that developed infections >48 h after admission to a healthcare centre. Community-onset cases were classified as those patients that had cultures drawn at community-based collection sites, nursing homes or within the first 2 days of admission to an acute care facility. The patients were further classified as having either community-acquired or healthcare-associated community-onset infections.9 Healthcare-associated community-onset cases were those that occurred among nursing home residents, haemodialysis patients or those individuals who were either admitted to a hospital for ≥2 days in the preceding 90 days or received care through a hospital-based clinic in the preceding 30 days.

Antimicrobial susceptibility testing

MICs were determined with the Vitek 2 instrument (Vitek AMS; bioMérieux Vitek Systems Inc., Hazelwood, MO, USA) for the following drugs: amoxicillin/clavulanic acid, piperacillin/tazobactam, ciprofloxacin, gentamicin, tobramycin, amikacin, meropenem, ertapenem and trimethoprim/sulfamethoxazole. Throughout this study, the results were interpreted using CLSI criteria for broth dilution.10 The quality-control strains used for this part of the study were E. coli ATCC 25922, E. coli ATCC 35218 and Pseudomonas aeruginosa ATCC 27853.

ESBL screening and confirmation testing

The presence of ESBLs was detected in clinical isolates of K. pneumoniae by using the 2009 CLSI criteria for ESBL screening and confirmation tests.10 Discs for ESBL confirmation tests were obtained from Oxoid Inc. (Nepean, Ontario, Canada). K. pneumoniae ATCC 700603 and E. coli ATCC 25922 were used as positive and negative controls, respectively.

β-Lactamase gene identification

Isoelectric focusing was performed on freeze–thaw extracts, as previously described.11 PCR amplification for blaCTX-M, blaOXA, blaTEM and blaSHV genes was carried out on the isolates with a GeneAmp 9700 ThermoCycler instrument (Applied Biosystems, Norwalk, CT, USA) using PCR conditions and primers as previously described.11 Automated sequencing was performed on the PCR products with the ABI Prism 3100 Genetic Analyzer (Applied Biosystems) using Sequence Analysis software. The sequences of the different amplicons were compared with each other and to homologous sequences using the Sequence Navigator software. The nucleotide and the deduced protein sequences were analysed using the software available from the Internet at the National Center of Biotechnology Information web site (http://www.ncbi.nlm.nih.gov).

PMQR determinants

The amplification of the qnrA, qnrS and qnrB genes was undertaken in all ESBL-positive isolates with multiplex PCR.12 The aac(6′)-Ib and qepA genes were amplified in a separate PCR using primers and conditions as previously described.13,14 The variant aac(6′)-Ib-cr was further identified by digestion with BstF5I (New England Biolabs, Ipswich, MA, USA).

PFGE

The genetic relatedness of the ESBL-producing K. pneumoniae isolates was examined by PFGE following the extraction of genomic DNA and digestion with XbaI using the standardized E. coli (O157:H7) protocol established by the CDC (Atlanta, GA, USA).15 The subsequent PFGE analyses were performed on a CHEF Mapper apparatus (Bio-Rad Laboratories, Hercules, CA, USA). PFGE banding patterns were analysed with BioNumerics software (Applied Maths, Kortrijk, Belgium) and the relatedness was calculated using the UPGMA algorithm with similarity of bands using the Dice coefficient. Cluster designation was based on the criteria of Tenover et al.16

Multilocus sequencing typing (MLST)

MLST was performed using seven conserved housekeeping genes (gapA, infB, mdh, pgi, phoE, rpoB and tonB).17 A detailed protocol of the MLST procedure, including allelic type and sequence type (ST) assignment methods, is available in the MLST databases of the Pasteur Institute (Paris, France), available at http://www.pasteur.fr/recherche/genopole/PF8/mlst/Kpneumoniae.html. New alleles and STs were submitted to the MLST web site and were approved. The MLST data are publicly available at http://www.pasteur.fr/mlst. The eBURST V3 algorithm (http://eburst.mlst.net/) was used to demonstrate phylogenetic relationships among closely related STs. Those STs that differed from the ancestor at only one of the seven MLST alleles were defined as single-locus variants (SLVs); when the ST differed at two of the seven MLST alleles they were defined as double-locus variants (DLVs). In this study, clonal complexes (CCs) were defined as a group of STs that are related to each other by SLV level. For the purpose of this study, we only included major STs if three or more isolates belonged to that specific ST.

Statistical methods

Data were analysed using Stata 11.2 software (StataCorp, College Station, TX, USA). Fishers’ exact and χ2 tests were used for comparing categorical variables among two and multiple groups, respectively. A χ2 test for trend was used to assess for an annual linear change in ESBL occurrence. P values <0.05 were deemed to represent statistical significance.

Results

Patients

During 2000–09, a total of 89 patients with incident infections due to ESBL-producing K. pneumoniae were identified (Table 1); 45 (50%) were classified as hospital-acquired, 31 (35%) as healthcare-associated community-onset and 13 (15%) as community-acquired infections. The mean patient age was 58 years (standard deviation = 25 years) and 57 (67%) of the patients were females. The majority of the patients [n = 76 (85%)] presented with urinary tract infections, 4 (5%) with intra-abdominal infections, 4 (5%) with primary sepsis, 4 (5%) with pneumonia and 1 with osteomyelitis (1%).

Table 1.

Number of K. pneumoniae, ESBL producers isolated and STs from clinical specimens in the Calgary Health Region from 2000 to 2009

Year Total no. of K. pneumoniae No. with ESBLs (%) Major STa 
2000 1440 2 (0.1%)  
2001 1120 1 (<0.1%)  
2002 1261 3 (0.2%) ST17 (n = 2) 
2003 1271 1 (<0.1%)  
2004 1251 6 (0.4%)  
2005 1375 9 (0.6%) ST20 (n = 1) 
ST575 (n = 1) 
2006 1803 15 (0.8%) ST20 (n = 5) 
ST573 (n = 1) 
ST574 (n = 1) 
2007 1970 15 (0.7%) ST20 (n = 2) 
ST17 (n = 1) 
ST336 (n = 1) 
ST573 (n = 1) 
2008 1857 13 (0.7%) ST20 (n = 3) 
ST17 (n = 2) 
ST573 (n = 1) 
2009 2023 24 (1.1%) ST17 (n = 2) 
ST575 (n = 3) 
Total 15 371 89 (0.5%) 27 (32%) 
Year Total no. of K. pneumoniae No. with ESBLs (%) Major STa 
2000 1440 2 (0.1%)  
2001 1120 1 (<0.1%)  
2002 1261 3 (0.2%) ST17 (n = 2) 
2003 1271 1 (<0.1%)  
2004 1251 6 (0.4%)  
2005 1375 9 (0.6%) ST20 (n = 1) 
ST575 (n = 1) 
2006 1803 15 (0.8%) ST20 (n = 5) 
ST573 (n = 1) 
ST574 (n = 1) 
2007 1970 15 (0.7%) ST20 (n = 2) 
ST17 (n = 1) 
ST336 (n = 1) 
ST573 (n = 1) 
2008 1857 13 (0.7%) ST20 (n = 3) 
ST17 (n = 2) 
ST573 (n = 1) 
2009 2023 24 (1.1%) ST17 (n = 2) 
ST575 (n = 3) 
Total 15 371 89 (0.5%) 27 (32%) 

aFour isolates were not viable for additional molecular characterization.

Bacterial isolates and susceptibilities

The absolute number of ESBL-producing isolates remained very low until 2003, increased slightly in 2004, remained stable until 2008 and then in 2009 there was an abrupt increase in the numbers of ESBL producers identified (Table 1). During 2000–03, 33% of cases were classified as community onset and 67% as hospital acquired; the community cases increased to 53% during 2004–08 while the hospital cases decreased to 47%. During 2009, the community and hospital cases returned to 34% and 66%, respectively. There was a significantly increasing proportion of isolates that were ESBL producers during the years of the study (P < 0.001). The rate of isolation of ESBL producers was >8-fold higher in 2009 as compared with 2000 [24/2023 (1.2%) versus 2/1440 (0.1%); P = 0.0002].

Of the 89 isolates included in this study, 39 (44%) were non-susceptible (i.e. either intermediate or resistant) to amoxicillin/clavulanic acid, 23 (26%) to piperacillin/tazobactam, 58 (65%) to trimethoprim/sulfamethoxazole, 35 (39%) to tobramycin, 24 (27%) to gentamicin, 4 (4%) to amikacin and 32 (36%) to ciprofloxacin. No resistance to meropenem or ertapenem was detected using the 2011 CLSI breakpoints. There was a significant increase in non-susceptibility to trimethoprim/sulfamethoxazole, ciprofloxacin, gentamicin and tobramycin during the latter part of the study [i.e. during 2000–04, 4/13 (31%) of K. pneumoniae isolated were non-susceptible to trimethoprim/sulfamethoxazole, 1/13 (8%) was non-susceptible to ciprofloxacin, 1/13 (8%) was non-susceptible to gentamicin and 1/13 (8%) was non-susceptible to tobramycin, and during 2005–09, 54/76 (71%) of K. pneumoniae isolated were non-susceptible to trimethoprim/sulfamethoxazole, 31/76 (41%) were non-susceptible to ciprofloxacin, 23/76 (30%) were non-susceptible to gentamicin and 34/76 (45%) were non-susceptible to tobramycin]. The increase in trimethoprim/sulfamethoxazole non-susceptibility was associated with the presence of ST20, while the increase in ciprofloxacin, gentamicin and tobramycin non-susceptibility was associated with the presence of ST575 and isolates positive for the PMQR determinants aac(6′)-Ib-cr and qnrB.

β-Lactamases and PMQR determinants

Of the 85 ESBL-producing K. pneumoniae included in this part of the study (4 isolates were not viable for additional characterization), 34 (40%) produced CTX-M-15, 20 (24%) CTX-M-14, 2 (2%) CTX-M-3 and 1 (1%) CTX-M-9, while 11 (13%) produced SHV-12, 9 (11%) SHV-2, 7 (8%) SHV-14 and 1 (1%) SHV-4. Some of the isolates also produced TEM-1 and SHV-1. Eleven (13%) of the ESBL-producing K. pneumoniae were positive for aac(6′)-Ib-cr, 12 (14%) for aac(6′)-Ib-cr and qnrB, 4 (5%) for qnrB, and 22 (26%) for qnrS. All the isolates were negative for qepA.

PFGE

PFGE identified four clusters of K. pneumoniae among 27 isolates that were designated as follows: pulsotypes A (n = 12), B (n = 8), C (n = 3) and D (n = 4). The isolates that belonged to pulsotypes A–D had >80% similar PFGE profiles among the isolates of each pulsotype. The remaining ESBL-producing isolates (n = 58) were not clonally related, i.e. exhibited <60% similar PFGE profiles and did not show patterns similar to those from pulsotypes A–D.

MLST

In this study, MLST analysis revealed six different STs (ST17, ST20, ST336, ST573, ST574 and ST575). Three of them (ST573, ST574 and ST575) were described for the first time, with four novel alleles (infB56, phoE109, rpoB56 and tonB144) for the last two (Table 2). The eBURST analysis, in turn, demonstrated that ST17, ST20, ST336 and ST574 belonged to CC17. The other two, ST573 and ST575, were not associated with any CC and also did not present any genetic relationship between them (Figure 1). MLST identified 11/12 isolates of pulsotype A as ST20 (the remaining isolate was identified as ST574, which is a DLV of ST20); 7/8 isolates of pulsotype B were identified as ST17 (the remaining isolate was identified as ST336, which is an SLV of ST17). Pulsotypes C and D were identified as ST573 and ST575, respectively. The clinical features, location of acquisition, antimicrobial susceptibilities, PMQR determinants and ESBL types of CC17, ST573 and ST575 (referred to as STs) and of those that did not belong to a major ST (referred to as non-STs) are shown in Table 3. The ST isolates were more likely to cause community-onset infections, tended to be more resistant to trimethoprim/sulfamethoxazole, were more likely to be positive for qnrS and more often produced CTX-M-14. The non-ST isolates were more likely to cause hospital-associated infections, tended to be more resistant to ciprofloxacin, gentamicin and tobramycin, were more likely to be positive for aac(6′)-Ib-cr, and more often produced CTX-M-15 and SHV-2 (Table 3). ST17 (three strains isolated in 2002, 2006 and 2008) with CTX-M-15 were present in the paediatric acute care centre; ST20 (seven strains isolated in 2006, 2007 and 2008) with CTX-M-14 were present in one of the adult acute care centres. However, cases did not cluster in a specific area within Calgary or in a ward or intensive care unit within one of the acute care centres.

Table 2.

Allelic profiles of the major STs identified among ESBL-producing K. pneumoniae isolated in Calgary

Allelic profile
 
 
gapA infB mdh pgi phoE rpoB tonB ST 
17 
20 
72 336 
18 573a 
56b 574a 
109b 56b 144b 575a 
Allelic profile
 
 
gapA infB mdh pgi phoE rpoB tonB ST 
17 
20 
72 336 
18 573a 
56b 574a 
109b 56b 144b 575a 

Numbers representing each allele obtained for each respective gene studied by MLST are presented in columns 1–7; the set of alleles determines the ST, which is presented in column 8.

aNew STs.

bNew alleles found in this study.

Table 3.

Characteristics of STa and non-STb ESBL-producing K. pneumoniae isolates in the Calgary Health Region, Calgary, Canada, 2000–09

 No. of isolates (%)
 
 
Characteristic STs non-STs P value 
Location of acquisition   0.175 
 hospital 12 (44) 30 (51)  
 healthcare-associated 8 (30) 22 (38)  
 community 7 (26) 6 (10)  
Clinical presentation   0.594 
 urinary tract infection 25 (93) 47 (81)  
 intra-abdominal infection 4 (7)  
 primary sepsis 1 (4) 3 (5)  
 pneumonia 1 (4) 3 (5)  
 osteomyelitis 1 (2)  
Antimicrobial susceptibility    
 AMC NS 11 (41) 27 (47) 0.647 
 TZP NS 6 (22) 17 (29) 0.604 
 CIP NS 6 (22) 26 (45) 0.056 
 SXT NS 23 (85) 33 (57) 0.014 
 GEN NS 5 (19) 19 (33) 0.205 
 TOB NS 7 (26) 27 (47) 0.097 
 AMK NS 4 (7) 0.203 
PMQR determinants   0.030 
qnrS 12 (44) 10 (17)  
aac(6′)-Ib-cr + qnrB 4 (15) 8 (14)  
aac(6′)-Ib-cr 11 (19)  
qnrB 1 (4) 3 (5)  
Type of ESBL    
 CTX-M-14 12 (52) 8 (14) 0.005 
 CTX-M-15 9 (33) 25 (43) 0.479 
 CTX-M-3 2 (3) 1.0 
 CTX-M-9 1 (2) 1.0 
 SHV-12 3 (11) 8 (14) 1.0 
 SHV-2 9 (16) 0.52 
 SHV-14 3 (11) 4 (17) 0.674 
 SHV-4 1 (2) 1.0 
 No. of isolates (%)
 
 
Characteristic STs non-STs P value 
Location of acquisition   0.175 
 hospital 12 (44) 30 (51)  
 healthcare-associated 8 (30) 22 (38)  
 community 7 (26) 6 (10)  
Clinical presentation   0.594 
 urinary tract infection 25 (93) 47 (81)  
 intra-abdominal infection 4 (7)  
 primary sepsis 1 (4) 3 (5)  
 pneumonia 1 (4) 3 (5)  
 osteomyelitis 1 (2)  
Antimicrobial susceptibility    
 AMC NS 11 (41) 27 (47) 0.647 
 TZP NS 6 (22) 17 (29) 0.604 
 CIP NS 6 (22) 26 (45) 0.056 
 SXT NS 23 (85) 33 (57) 0.014 
 GEN NS 5 (19) 19 (33) 0.205 
 TOB NS 7 (26) 27 (47) 0.097 
 AMK NS 4 (7) 0.203 
PMQR determinants   0.030 
qnrS 12 (44) 10 (17)  
aac(6′)-Ib-cr + qnrB 4 (15) 8 (14)  
aac(6′)-Ib-cr 11 (19)  
qnrB 1 (4) 3 (5)  
Type of ESBL    
 CTX-M-14 12 (52) 8 (14) 0.005 
 CTX-M-15 9 (33) 25 (43) 0.479 
 CTX-M-3 2 (3) 1.0 
 CTX-M-9 1 (2) 1.0 
 SHV-12 3 (11) 8 (14) 1.0 
 SHV-2 9 (16) 0.52 
 SHV-14 3 (11) 4 (17) 0.674 
 SHV-4 1 (2) 1.0 

AMC, amoxicillin/clavulanic acid; TZP, piperacillin/tazobactam; CIP, ciprofloxacin; GEN, gentamicin; TOB, tobramycin; AMK, amikacin; SXT, trimethoprim/sulfamethoxazole; NS, non-susceptible, i.e. either intermediate or resistant.

Four isolates were not viable for molecular characterization.

aSTs: ST17, ST20, ST336, ST573, ST574 and ST575.

bNon-STs: isolates that did not belong to a major ST.

Figure 1.

eBURST population of different STs in Calgary among ESBL-producing K. pneumoniae.

Figure 1.

eBURST population of different STs in Calgary among ESBL-producing K. pneumoniae.

In this study, ST17 was more likely to produce CTX-M-15 and was more likely to be positive for qnrB, while ST20 exclusively produced CTX-M-14 and was positive for qnrS. The newly identified ST573 and ST575 did not belong to a major CC (Figure 1); isolates that belonged to ST573 produced SHV-14 and were only resistant to trimethoprim/sulfamethoxazole, while ST575 produced CTX-M-15, appeared in 2009 and was resistant to all antimicrobial agents tested except for meropenem, ertapenem and trimethoprim/sulfamethoxazole.

Discussion

In Canada, a recent report from the CANWARD surveillance programme on the prevalence of antimicrobial-resistant pathogens from Canadian hospitals showed that K. pneumoniae was the fifth most common microorganism isolated during 2006–09.18 Data from the same programme indicated that K. pneumoniae was the third most common bacterium among urinary tract isolates during 2007–09 and overall 3% were positive for ESBLs.19 Furthermore, the rate of ESBL-producing K. pneumoniae isolated from blood cultures in Canadian hospitals increased from 1.5% in 2007 to 2.6% in 2009.20

Our study investigated the molecular epidemiology and population-based surveillance of K. pneumoniae that produce ESBLs in Calgary, a centralized North American region with relatively low rates of antimicrobial resistance in Gram-negative bacteria (2010 antibiograms accessed in November 2011, http://www.calgarylabservices.com/files/LabTests/MicrobiologyNewsletters/2010_Antibiogram.pdf). Population-based surveillance studies in Calgary that previously investigated the clinical characteristics and molecular epidemiology of ESBL-producing Enterobacteriaceae showed that E. coli producing CTX-M-14 and -15 emerged during the early 2000s as important causes of community-onset urinary tract and bloodstream infections in the elderly.8,11,21 The present study showed that ESBL-producing K. pneumoniae are rare in our region, but these isolates increased during the latter part of the study period [i.e. 13/6343 (0.2%) of K. pneumoniae isolated during 2000–04 were ESBL producers as opposed to 76/9028 (0.8%) isolated during 2005–09]. The absolute number of ESBL-producing isolates remained very low until 2003, increased slightly in 2004, remained stable until 2008 and then in 2009 there was an abrupt increase in the numbers of ESBL producers identified (Table 1). A similar study from Calgary investigated the molecular epidemiology of ESBL-producing E. coli causing bacteraemia over an 11 year period (2000–10).22 The majority of E. coli produced either CTX-M-15 or CTX-M-14 and even different major STs were identified among 91% of isolates (i.e. ST10 CC, ST38, ST131, ST315, ST393, ST405 and ST648). The study also showed that ST131 with CTX-M-15 was the most common and antimicrobial-resistant ST, and the influx of a single pulsotype of ST131 was responsible for a significant increase of ESBL-producing E. coli, especially since 2007.22

As we have previously noted with ESBL-producing E. coli in our region, the majority of K. pneumoniae also produced CTX-M-14, CTX-M-15 and, to a lesser extent, SHV-12, showing similar results to a worldwide study from the late 1990s that investigated ESBL-producing K. pneumoniae isolated from blood.23 However, SHV-12 was the predominant ESBL during the initial period of this study and was gradually replaced by CTX-M-14- and CTX-M-15-producing isolates [i.e. 6/9 (67%) of ESBL-producing K. pneumoniae isolated during 2000–04 produced SHV-12 as compared with 5/76 (7%) of ESBL-producing K. pneumoniae isolated during 2005–09]. It is possible that plasmids encoding CTX-M-14 and -15 were acquired from E. coli. The increase of CTX-M-15-producing K. pneumoniae has also recently been described in various countries, including Spain,24 Sweden,25 India,26 Taiwan and Korea.27

A previous study from Calgary investigated the prevalence of PMQR determinants in ciprofloxacin-resistant enterobacteria isolated during 2004 and 2007, and detected aac(6′)-Ib-cr among 2/12 K. pneumoniae isolates.28 The present study found a variety of PMQR determinants [including aac(6′)-Ib-cr, qnrB and qnrS] among 31/85 ESBL-producing isolates and highlights that PMQR determinants in K. pneumoniae isolated in Calgary are associated with the presence of blaCTX-M genes.

MLST, which uses sequence variation in a number of housekeeping genes to define STs or clones, is an excellent tool for evolutionary studies to show common ancestry lineages among bacteria.29 It has led to the definition of major STs and the recognition of successful widespread international clones, such as E. coli ST131.30 We identified four different major STs among 32% of isolates (i.e. ST17, ST20 and two new STs, ST573 and ST575). Of interest was that the ST isolates were more likely to produce community-onset infections, were associated with the presence of blaCTX-M genes and emerged during the latter part of the study period, i.e. 2/9 (22%) of ESBL-producing K. pneumoniae isolated during 2000–04 belonged to a major ST as opposed to 25/76 (33%) isolated during 2005–09.

MLST analysis using eBURST showed that ST17, ST20, ST336 and ST574 belonged to CC17. These STs have very close genetic relationships since ST20, ST336 and ST574 are SLVs of ST17 (Figure 1 and Table 2). STs belonging to CC17 were recently described among CTX-M-15-producing K. pneumoniae from Spain,31 Taiwan and Korea.27

The long-term trends in the molecular epidemiology and types of ESBLs produced by K. pneumoniae have been studied in Spain32 and Japan.33 The Spanish multicentre study investigated ESBL-producing isolates from 2006 and compared the results with a previous study conducted in 2000. The results showed a shift from SHV-type ESBLs in 2000 to CTX-M-type β-lactamases in 2006. The Japanese study investigated the types of ESBL-producing K. pneumoniae in a single hospital over a 7 year period (2003–09) and showed that the rate of ESBLs increased over the period and that the CTX-M-9 group was the most common type of ESBL identified.

To our knowledge, this is the first population-based molecular epidemiology study over a 10 year period that characterized ESBL-producing K. pneumoniae. The numbers of these bacteria were low in our centralized region, but did increase towards the latter part of the study period. This increase was associated with the emergence of certain major STs and the presence of blaCTX-M genes. Our study highlights the importance of population-based surveillance and molecular epidemiology in providing insight into the emergence, characteristics and distribution of STs among ESBL-producing K. pneumoniae.

Funding

This work was supported by a research grant from the Calgary Laboratory Services (# 73-6350). J. H. K. S. was employed through the Biomedical Technology summer student programme funded by Calgary Laboratory Services and the University of Calgary.

Transparency declarations

K. B. L. and J. D. D. P. have previously received research funds from Merck and AstraZeneca. All other authors: none to declare.

Acknowledgements

We thank Platform Genotyping of Pathogens and Public Health (Institut Pasteur) for coding MLST alleles and profiles.

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