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A 2-month survey of extended-spectrum β-lactamase (ESBL) producers was performed in a Czech hospital. Klebsiella pneumoniae produced SHV-2, -5, or -12, Escherichia coli produced CTX-M-9 or -15, and other species produced TEM-92 or -132. All K. pneumoniae and E. coli isolates belonged to sequence types (STs) or clonal complexes (CCs) spread across the world (K. pneumoniae clonal complex 11 [CC11], CC14, and sequence type 101 [ST101] and E. coli CC31, CC73, CC131, and CC405) and carried various plasmids (mainly with A/C- and FII-type replicons).
Plasmid-encoded extended-spectrum β-lactamases (ESBLs) hydrolyzing penicillins, cephalosporins, and monobactams are a major reason behind the resistance of members of the family Enterobacteriaceae to antimicrobials (4, 21, 35). Organisms with these enzymes, especially Klebsiella pneumoniae and Escherichia coli, have been disseminating worldwide over the last two decades, reaching alarming prevalence rates in some countries or larger geographic areas (5, 43). From among several ESBL types identified, enzymes of the TEM, SHV, and more recently, CTX-M families have been playing prominent roles (30, 39). Growing evidence indicates that global spread of some ESBL variants greatly depends on particular clones of the producer species and on specific types of plasmids carrying their genes (11, 12, 24, 31, 33).
In the Czech Republic, the data on ESBLs have been scarce (25, 27). However, in 2007, the rates of ESBL-producing K. pneumoniae and E. coli nosocomial invasive isolates were as high as 30.8% and 6.2%, respectively (P. Urbášková, unpublished results). This work aimed to produce a detailed analysis of ESBL-producing organisms from a large Czech hospital.
In June and July 2006, a 2-month survey was performed in the University Hospital in Plzeň, Czech Republic (1,800 beds). All nonrepeated Enterobacteriaceae isolates identified as ESBL producers were collected. Species identification was carried out by Entero test 24 (Pliva Lachema Diagnostika, Brno, Czech Republic), and ESBLs were detected by the double-disk test with modifications for the species with natural AmpC β-lactamases (15). Twenty-four isolates were identified, including 10 K. pneumoniae isolates, 9 E. coli isolates, 3 Providencia stuartii isolates, 1 Enterobacter cloacae isolate, and 1 Proteus mirabilis isolate (Table (Table1).1). All these isolates represented nosocomial contamination. Several isolates had no or unclear clinical significance (four K. pneumoniae and E. coli isolates from throat swabs that were surveillance cultures and the K. pneumoniae 1838/06 isolate from the sputum of a geriatric patient that represented colonization of the respiratory tract). Of the total number of 1,710 enterobacterial isolates recovered in the hospital over the study period (including surveillance cultures), ESBL producers represented 1.4% of the isolates, including 3.4% of K. pneumoniae isolates (293 isolates in total) and 1.2% of E. coli isolates (768 isolates in total).
The MICs of antimicrobials were determined by broth dilution as proposed by EUCAST (19). The isolates showed increased MICs of β-lactams and various patterns of resistance to other antimicrobials (Table (Table2).2). Some variations in the MICs of particular β-lactams between different strains, e.g., those of piperacillin with tazobactam for K. pneumoniae, might reflect the presence of additional resistance mechanisms in some clones of these organism. Two isolates, one K. pneumoniae isolate and one E. coli isolate, showed resistance to colistin (MIC, >64 μg/ml). Transfer of the ESBL-associated resistance was performed with E. coli A15 resistant to rifampin (rifampicin) as a recipient, as described previously (22). Transconjugants, selected with 2 μg/ml cefotaxime or ceftazidime and 128 μg/ml rifampin, were obtained for five K. pneumoniae isolates, four E. coli isolates, and the E. cloacae isolates. Along with resistance to β-lactams, they showed various combinations of resistance to other compounds (Table (Table11).
β-Lactamases were visualized by isoelectric focusing, followed by a bioassay for enzymes with cefotaxime- or ceftazidime-hydrolyzing activity (2). Eight species-specific β-lactamase profiles were observed (Table (Table1).1). The putative ESBLs had pI values of 8.2 or 7.6 in K. pneumoniae, 8.9 (mostly) or 7.9 in E. coli, and 5.9 in P. stuartii and P. mirabilis. The E. cloacae isolate produced both the pI 8.2 and 5.9 enzymes. Additionally, almost all isolates expressed β-lactamases with a pI of 5.4 (probably TEM-1), and most of the E. coli isolates had enzymes with a pI of 7.4 (probably OXA-1). The transconjugants produced ESBLs like those in the corresponding isolates, together or not with the other β-lactamases (Table (Table11).
For all but one isolate, PCR detection and sequencing of blaSHV, blaCTX-M, and blaTEM ESBL genes were performed as described previously (17). The blaCTX-M-9-like gene encoding the β-lactamase with a pI of 7.9 in E. coli 1841/06 was identified first by multiplex PCR (45) and then amplified and sequenced as proposed by Eckert et al. (16). For K. pneumoniae isolates, the PCR of blaSHV genes was performed with plasmid DNA, and for E. cloacae and P. mirabilis isolates, the blaTEM ESBL genes were cloned first in E. coli DH5α (20) using vector pHSG298 (41). In K. pneumoniae, the ESBLs with a pI of 8.2 were SHV-5 (also in E. cloacae) or SHV-12, and the enzymes with a pI of 7.6 were SHV-2 (Table (Table1).1). The β-lactamases with pIs of 8.9 and 7.9 in E. coli were identified as CTX-M-15 and CTX-M-9, respectively. The ESBLs with a pI of 5.9 were either TEM-92 (in P. stuartii) or TEM-132 (in E. cloacae and P. mirabilis). Whereas the SHV and CTX-M enzymes belonged to “cosmopolitan” ESBL types (21, 30), the TEM variants have rarely been observed so far (13, 18, 46). The presence of the ISEcp1 element in the vicinity of blaCTX-M genes in E. coli isolates was analyzed as reported previously (1). It was located 49 bp and 42 bp upstream from blaCTX-M-15 and blaCTX-M-9, respectively, demonstrating that these genes were present in widespread ISEcp1 transposition modules (3, 9, 16, 30, 33, 38).
Pulsed-field gel electrophoresis (PFGE) was performed by the method of Struelens et al. (40); DNA banding patterns were interpreted by the method of Tenover et al. (42). Four and five PFGE types were discerned among K. pneumoniae and E. coli isolates, respectively, correlating with their β-lactamase profiles (Table (Table1).1). There were two PFGE types of SHV-5-producing K. pneumoniae (types C and D) and four types of CTX-M-15-producing E. coli (types b to e). Clusters of two to four related isolates were usually specific for particular wards, indicating parallel local dissemination of several enterobacterial strains.
Thirteen K. pneumoniae and E. coli isolates of all PFGE types were subjected to multilocus sequence typing (MLST) as described previously (14, 44). Databases available at www.pasteur.fr (K. pneumoniae) and www.mlst.net (E. coli) were used for assigning sequence types (STs) and clonal complexes (CCs). The K. pneumoniae isolates represented sequence type 11 (ST11) (clonal complex 11 [CC11]; PFGE type D), ST14 (CC14; PFGE types B and C), and ST101 (PFGE type A) (Table (Table1),1), which have been observed worldwide (12, 14, 36) (www.pasteur.fr). Within the ST14 clone, the similarity between PFGE patterns B and C1/C2 was around 58.0% (Dice coefficient), and between C1 and C2, it was 93.8%, which might reflect the more recent diversification of the clone. Isolates of all these STs have been mostly recovered from humans (www.pasteur.fr); recently, ST101 bovine isolates were reported in the United States (36). The data on ESBLs in these clones have often been partial (14), not available (36), or difficult to compare because of the use of another MLST scheme (26). Recently, two Hungarian epidemic K. pneumoniae clones with CTX-M-15 were assigned to ST11 and ST15 (12), the latter one was classified in CC14 together with ST14 (14). However, in this study, the ST11 and ST14 isolates produced either SHV-2 or SHV-5.
The E. coli isolates also represented STs belonging to international clones or CCs with pathogenic strains from humans, namely, ST131 (CC131; PFGE types d and e), ST393 (CC31; PFGE type c), ST405 (CC405; PFGE type a), and ST638 (CC73; PFGE type b) (Table (Table1)1) (44) (www.mlst.net). Within ST131, the similarity between PFGE patterns d and e1/e2 was >60% (d and e1, 68.3%; d and e2, 63.0%), whereas between e1 and e2, it was 97.6%. ST131 and ST405 were recently described as E. coli clones disseminating with CTX-M-15 ESBL on a global scale (11, 29, 32, 37); however, in this study, while ST131 did indeed produce CTX-M-15, ST405 produced CTX-M-9. Phylogenetic grouping of E. coli isolates was performed by PCR as proposed by Clermont et al. (10). They all represented the more-virulent group B2 or D (Table (Table11).
PCR-based replicon typing of plasmids was performed by the method of Carattoli et al. (6), using total DNA from transconjugants or from clinical isolates when these were nonmating. Replicon profiles are shown in Table Table1.1. Replicon A/C was found in all K. pneumoniae isolates except for the single SHV-12 producer with replicon FII. Different β-lactamase profiles or mating abilities suggested that the A/C-type plasmids with blaSHV-2/SHV-5 genes varied in K. pneumoniae isolates of different PFGE types (B, C, and D). Links between replicon A/C and blaSHV genes and between replicon FII and blaSHV-12 were reported before (8, 31). The FII replicon was predominant in E. coli with CTX-M-15 ESBL and has been identified in isolates of three PFGE types (c, d, and e). The FII-type plasmids were probably specific for each of these PFGE types as shown by differences in their mating ability and transferable resistance markers. A number of reports demonstrated the strong link between blaCTX-M-15 and FII-type plasmids (7, 11, 23, 24, 33). These molecules often carry also blaOXA-1, blaTEM-1, and aac(6′)-Ib-cr genes, which was confirmed in this work by specific PCRs (11) and sequencing (Table (Table1).1). The remaining E. coli with CTX-M-15 (PFGE type b) had only replicon A/C, which has been rarely observed (31). Similarly sporadic have been I1-type plasmids with blaCTX-M-9-like genes (24, 34), as found here for E. coli producing CTX-M-9. The isolates with TEM-type ESBLs had specific replicon profiles, including the E. cloacae and P. mirabilis isolates which both expressed TEM-132. All the above data showed a diversity of plasmids, even in isolates with the same ESBL variant (SHV-5, CTX-M-15, and TEM-132), correlating well with clonal diversity. They demonstrated that plasmid transfer was not an important factor of ESBL dissemination in the hospital during the study period.
This report demonstrates that nosocomial ESBL-producing subpopulations of K. pneumoniae and E. coli may entirely consist of clones with higher dissemination potential. It confirms further the particular role of E. coli ST131 with FII-type plasmids carrying the blaCTX-M-15 gene in current ESBL epidemiology (11, 29, 32). However, by showing that other clones identified produced different ESBLs than elsewhere (11, 12), this study contributes to the growing knowledge that spread of the international K. pneumoniae and E. coli clones is not associated strictly with specific β-lactamases but that these are being acquired independently by particular clone variants in different locales (28, 37). It is possible that the E. coli clones with CTX-M-15 ESBL had been more recent introductions to the hospital's environment, while the K. pneumoniae with SHV-2 or SHV-5 ESBL had been circulating there for a longer period. This hypothesis, however, requires a comparative analysis of ESBL producers from the Plzeň hospital and the Czech Republic from before 2006, and such a study is now being conducted.
The study reported here was partially financed by research project grant MŠMT 2E08003 from the Ministry of Education, Czech Republic, and grant PBZ-MNiSW-04/I/2007 from the Polish Ministry of Science and Higher Education. It was also a part of the activities of the MOSAR integrated project (LSHP-CT-2007-037941) supported by the European Commission under the Life Science Health priority of the 6th Framework Programme (WP2 Study Team).
Published ahead of print on 26 August 2009.