International Clones of Klebsiella pneumoniae and Escherichia coli with Extended-Spectrum β-Lactamases in a Czech Hospital

Abstract

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K. pneumoniae and E. coli nosocomial invasive isolates were as high as 30.8% and 6.2%, respectively (P. Urbášková, unpublished results).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 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).

TABLE 1.

Clinical data, typing, β-lactamase content, and plasmid characteristics of the study isolates

Isolate	Species	Warda	Specimen	Phylogroupb	Typingc		pIs of β-lactamases found by IEFd	ESBL	Plasmid datae
					PFGE type	ST			Replicon(s)	blaOXA-1	blaTEM-1	aac(6′)-Ib-cr	Resistance in R+f
1829/06	K. pneumoniae	NICU	Catheter		A	101	8.2, 7.6, 5.4	SHV-12	FII				−
1830/06	K. pneumoniae	NICU	Throat swab		B	14	7.6, 5.4	SHV-2	A/C
1833/06	K. pneumoniae	NICU	Sputum		B	ND	7.6, 5.4	SHV-2	A/C
1835/06	K. pneumoniae	NICU	Throat swab		B	14	7.6, 5.4	SHV-2	A/C
1831/06	K. pneumoniae	NICU	Sputum		C1	14	8.2 7.6, 5.4	SHV-5	A/C				CML, TET
1832/06	K. pneumoniae	NICU	Throat swab		C1	ND	8.2, 7.6, 5.4	SHV-5	A/C				CML, TET
1834/06	K. pneumoniae	NICU	Wound swab		C1	ND	8.2, 7.6, 5.4	SHV-5	A/C				CML, TET
1838/06	K. pneumoniae	Geriatrics	Sputum		C2	14	8.2, 7.6, 5.4	SHV-5	A/C				CML, TET
1836/06	K. pneumoniae	Geriatrics	Urine		D1	11	8.2, 7.6, 5.4	SHV-5	A/C
1837/06	K. pneumoniae	Geriatrics	Urine		D2	ND	8.2, 7.6, 5.4	SHV-5	A/C
1841/06	E. coli	Urology	Urine	D	a	405	7.9, 5.4	CTX-M-9	I1				−
1839/06	E. coli	Cardiosurgery	Urine	B2	b	638	8.9, 5.4	CTX-M-15	A/C
1840/06	E. coli	Cardiosurgery	Urine	B2	b	ND	8.9, 5.4	CTX-M-15	A/C
1842/06	E. coli	Geriatrics	Urine	D	c1	393	8.9, 7.4, 5.4	CTX-M-15	FII	+	+	+	GEN
1843/06	E. coli	Geriatrics	Urine	D	c2	393	8.9, 7.4, 5.4	CTX-M-15	FII	+	+	+	GEN
1844/06	E. coli	Oncology	Urine	B2	d	131	8.9, 7.4, 5.4	CTX-M-15	FII	+	+	+	GEN, SXT
1846/06	E. coli	IMICU	Urine	B2	e1	131	8.9, 7.4, 5.4	CTX-M-15	FII, F1A, Y	+	+	+
1847/06	E. coli	IMICU	Urine	B2	e2	131	8.9, 7.4, 5.4	CTX-M-15	FII, F1A, Y	+	+	+
1848/06	E. coli	IMICU	Throat swab	B2	e1	ND	8.9, 7.4, 5.4	CTX-M-15	FII, F1A, Y	+	+	+
1849/06	E. cloacae	Hematology	Blood				8.4*, 8.2, 5.9, 5.4	SHV-12, TEM-132	FII, F1A				GEN, SXT
1850/06	P. stuartii	SICU	Wound swab		α		8.0*, 5.9	TEM-92	A/C, W
1851/06	P. stuartii	SICU	Urine		α		8.0*, 5.9	TEM-92	A/C, W
1852/06	P. stuartii	SICU	Urine		α		8.0*, 5.9	TEM-92	A/C, W
1853/06	P. mirabilis	SICU	Urine				5.9, 5.4	TEM-132	F1C, P

^aNICU, neurosurgical intensive care unit (ICU); IMICU, internal medicine ICU; SICU, surgical ICU.

^bPhylogrouping was performed only for E. coli isolates.

^cPFGE typing was not performed for single E. cloacae and P. mirabilis isolates; MLST was carried out only for selected K. pneumoniae and E. coli isolates. ND, not determined.

^dIEF, isoelectric focusing. pI values in boldface type refer to β-lactamases that were produced by transconjugants, while pI values that are underlined represent enzymes that hydrolyzed cefotaxime and/or ceftazidime in the bioassay. Asterisks indicate β-lactamases that were not stained by 0.5 mM nitrocefin with 0.3 mM cloxacillin (probably natural AmpC cephalosporinases of E. cloacae and P. stuartii).

^eDetection of the bla_OXA-1, bla_TEM-1, and aac(6′)-Ib-cr genes was performed only for the CTX-M-15-producing E. coli isolates with FII replicon plasmids.

^fNon-β-lactam resistance markers in transconjugants (R⁺). Abbreviations: CML, chloramphenicol; GEN, gentamicin; SXT, co-trimoxazole; TET, tetracycline. Symbol: −, no transferable resistance to non-β-lactams was observed for these isolates.

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 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 1).

TABLE 2.

Antimicrobial susceptibility of study isolates

Isolate and/or PFGE typea	MIC(s) (μg/ml) of antimicrobialb
	AMP	AMS	PIP	TZP	CTX	CAZ	FEP	ATM	MEM	GEN	CIP	TET	SXT	CML	CST
K. pneumoniae
Type A	>64	16	>64	4	8	>16	2	>32	0.125	16	1	4	>128	8	0.5
Type B	>64	>64	>64	>64	4, 8	4, 16	1, 4	2, 4	0.125	>32	0.25, 2	4, >64	>128	>64	0.5, 2
Type C	>64	>64	>64	32, >64	4, 8	16, >16	0.5, 4	>32	0.125	>32	2, 8	>64	>128	>64	0.5, 2
Type D	>64	>64	>64	>64	4-8	>16	2, 4	>32	0.125	>32	>32	>64	>128	32, >64	0.5, >64
E. coli
Type a	>64	32	>64	4	16	4	>16	16	0.125	4	>32	>64	>128	>64	0.5
Type b	>64	32, >64	>64	8	>16	>16	>16	>32	0.125	0.5, 1	0.125, >32	1, >64	1, >128	32, 64	0.5, >64
Type c	>64	16	>64	4, 8	>16	>16	16	>32	0.125	>32	>32	4, >64	2, >128	>64	0.5
Type d	>64	16	>64	32	>16	>16	>16	>32	0.125	>32	>32	2	>128	4	0.5
Type e	>64	8, 32	>64	8	>16	16, >16	16, >16	32, >32	0.125	32, >32	>32	1, 2, >64	1, >128	1, 4	0.5
E. cloacae 1849/06	>64	>64	>64	>64	>16	>16	>16	>32	0.25	16	2	16	>128	16	0.5
P. stuartii type α	>64	4, 8	>64	8	16	8, 16	4, 8	2, 4	0.125	1, 4	>32	>64	>128	16, 32	>64
P. mirabilis 1853/06	>64	16	>64	2	>16	>16	16	>32	0.125	1	0.125	>64	>128	4	0.5

^aK. pneumoniae, E. coli and P. stuartii isolates are grouped according to PFGE types.

^bAbbreviations: AMP, ampicillin; AMS, ampicillin with sulbactam; ATM, aztreonam; CAZ, ceftazidime; CIP, ciprofloxacin; CML, chloramphenicol; CST, colistin; CTX, cefotaxime; FEP, cefepime; GEN, gentamicin; MEM, meropenem; PIP, piperacillin; SXT, co-trimoxazole; TET, tetracycline; TZP, piperacillin with tazobactam.

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 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 1).

For all but one isolate, PCR detection and sequencing of bla_SHV, bla_CTX-M, and bla_TEM ESBL genes were performed as described previously (17). The bla_CTX-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 bla_SHV genes was performed with plasmid DNA, and for E. cloacae and P. mirabilis isolates, the bla_TEM 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 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 bla_CTX-M genes in E. coli isolates was analyzed as reported previously (1). It was located 49 bp and 42 bp upstream from bla_CTX-M-15 and bla_CTX-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 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 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 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 1).

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 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 bla_SHV-2/SHV-5 genes varied in K. pneumoniae isolates of different PFGE types (B, C, and D). Links between replicon A/C and bla_SHV genes and between replicon FII and bla_SHV-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 bla_CTX-M-15 and FII-type plasmids (7, 11, 23, 24, 33). These molecules often carry also bla_OXA-1, bla_TEM-1, and aac(6′)-Ib-cr genes, which was confirmed in this work by specific PCRs (11) and sequencing (Table 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 bla_CTX-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 bla_CTX-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.