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Antimicrobial Agents and Chemotherapy, July 2005, p. 3066-3069, Vol. 49, No. 7
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.7.3066-3069.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Survey of Enterobacteriaceae Producing Extended-Spectrum ß-Lactamases in a Slovak Hospital: Dominance of SHV-2a and Characterization of TEM-132

Martina Zarnayová,1,2 Eliane Siebor,1 André Péchinot,1 Jean-Marie Duez,1 Helena Bujdáková,2 Roger Labia,3 and Catherine Neuwirth1*

Laboratoire de Bactériologie, Hôpital Universitaire du Bocage, BP 77908, 21079 Dijon Cedex, France,1 Department of Microbiology and Virology, Faculty of Natural Sciences, Comenius University, Bratislava, Slovakia,2 Unité FRE 2125, CNRS, UBO, MNHN, 6 rue de l'Université, 29000 Quimper, France3

Received 23 November 2004/ Returned for modification 8 February 2005/ Accepted 8 April 2005


   ABSTRACT
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Eighty-five extended-spectrum ß-lactamase-producing Enterobacteriaceae from a Slovak hospital have been studied. SHV-2a was predominant, but other variants have been detected, namely, SHV-5, SHV-12, TEM-12, TEM-15, and TEM-132, which differed from TEM-1 by amino acid substitutions R164H, E240K, and I173V and had kinetic properties similar to those of TEM-28.


   TEXT
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During the last two decades, a variety of extended-spectrum ß-lactamases (ESBLs) have emerged worldwide in the family Enterobacteriaceae (8, 12, 29, 30, 32). In Western Europe, ESBL-monitoring programs are in place in many countries (2, 13, 31, 35). Except for Poland (3, 15, 16, 17), detailed reports for Central or Eastern Europe (21, 28, 33) are more scarce. For Slovakia, such strains have been described, but the types of ESBLs were not determined (6, 10).

Seventy-one isolates of Klebsiella pneumoniae, 10 isolates of Escherichia coli, 2 isolates of Enterobacter cloacae, and 2 isolates of Citrobacter freundii were nonrepetitively collected in the University Hospital Ruzinov in Bratislava from October 1998 to September 2002. Susceptibility testing of the isolates by the disk diffusion method and MIC determinations were performed according to French recommendations (27). The ESBL production was assessed by a positive double-disk synergy test (20). The isoelectric point (pI) of the ESBLs was determined by analytical isoelectric focusing (26). The detection of the ß-lactamase activity was performed by a substrate overlaying procedure (ceftriaxone for ESBL or penicillin G for penicillinases) (24).

Analyses of total DNAs were performed by pulsed-field gel electrophoresis (PFGE) as described previously (18) with a pulse range from 5 s to 50 s at 5.4 V/cm, for 24 h with K. pneumoniae and E. cloacae and for 20 h with E. coli and C. freundii. Rifampin-resistant E. coli K-12 C600 was used as a recipient for conjugation experiments with at least one clinical isolate of K. pneumoniae representative for each PFGE type. Transconjugants were selected on Mueller-Hinton agar containing 150 µg/ml of rifampin and 2 µg/ml of ceftazidime. Plasmid extraction, PCR amplification, and sequencing of ESBL genes were performed for one isolate (or its transconjugant) of each type. Plasmid DNA extracted by an alkaline lysis method (5) was used as a template for detection of the bla gene with primers specific for blaTEM (11), blaSHV (34), or blaCTX-M (7). For cloning experiments, plasmid DNA of K. pneumoniae 1.626 was partially restricted by Sau3AI, ligated into the BamHI-digested pK18 plasmid (36), and introduced into E. coli DH5{alpha}. E. coli DH5{alpha} harboring the recombinant pK18 plasmid, designated pK132, with a 1.8-kb Sau3AI fragment, was selected on Mueller-Hinton agar containing kanamycin (30 µg/ml) and ceftazidime (2 µg/ml). The 1.8-kb cloned DNA fragment from pK132 and the PCR products were sequenced on both strands.

A summary of the results is reported in Table 1. The 71 isolates of K. pneumoniae were divided into 10 PFGE types. SHV-2a was expressed by 38 isolates of K. pneumoniae and all E. coli isolates. The transfer of blaSHV2a was obtained for representatives of four PFGE types, but two different phenotypes of resistance to aminoglycosides were observed among the transconjugants. Moreover, the plasmid content differed among K. pneumoniae isolates, except for types 2KP and 4KP, which harbored a plasmid of 150 kb. Therefore, the widespread production of SHV-2a was not a consequence of plasmid dissemination. The SHV-2a-producing isolates of K. pneumoniae were recovered in many wards, but with some particularities. For instance, the isolates from type 1KP were detected all through the survey, especially in two intensive care units and one burn unit. These strains were likely to be responsible for serious infections, because they have been isolated mostly from burn wounds, sputum, or blood. In contrast, the isolates from type 10KP were recovered mostly from urine samples in the "long stay" ward over a 4-month period. We noticed several times the concomitant dissemination of different strains simultaneously in the same ward. These observations revealed the complexity of the situation. The high number of strains harboring SHV-2a is comparable to that reported in Canadian or Korean studies (22, 29), whereas there are only a few such reports from Europe (13, 31). Several studies report the spread of CTX-M-3-producing bacteria in Poland (3, 16), and we expected a similar situation in Slovakia because of the proximity of the two countries. Surprisingly, only a single isolate in our study produced CTX-M-3, suggesting a notable epidemiological difference between these areas.


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  		TABLE 1. Characteristics of ESBL-producing isolatesa

 
Three TEM-derivatives, TEM-12, TEM-15, and TEM-132, were identified. The novel enzyme TEM-132 was produced by four K. pneumoniae isolates that also harbored three penicillinases (TEM-1, SHV-1, and the rarely detected SHV-11) and that were much more resistant to ceftazidime than to cefotaxime. The strain K. pneumoniae 1626 was retained for further studies. The ß-lactam MICs for K. pneumoniae 1626, its transconjugant (E. coli C600Tc1626), the recombinant E. coli DH5{alpha} (pK132), rifampin-resistant E. coli K12 C600, and E. coli DH5{alpha} are reported in Table 2. The coding region of the blaTEM-132 gene differed from blaTEM-1 (19) by three mutations, a G-to-A change at position 693, an A-to-G change at position 719, and a G-to-A change at position 917, according to Sutcliffe (37). blaTEM-132 was associated with promoter P3. The deduced amino acid sequence, according to Ambler (1), revealed that TEM-132 differed from TEM-1 by three substitutions, Arg-164 to His, Ile-173 to Val, and Glu-240 to Lys.


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  		TABLE 2. MICs of ß-lactams for K. pneumoniae 1626, its transconjugant (E. coli C600Tc1626), the recombinant E. coli DH5{alpha} (pK132), rifampin-resistant E. coli K12 C600, and E. coli DH5{alpha}a

 
The TEM-28 and TEM-132 ß-lactamases were purified to homogeneity according to previously described methods (4, 14, 25), and kinetic constants (Table 3) were determined by computerized microacidimetry (23). TEM-132 was characterized by a high Km value for ceftazidime, cefpirome, and cefepime. In terms of kcat/Km, TEM-132 had an efficiency comparable to that of TEM-28, and the concentrations of ß-lactamase inhibitors required to reach 50% enzyme inhibition (IC50) levels were similar for TEM-132 and TEM-28 (Table 4) (9). Thus, the Ile-173 Val substitution, not detected till now with natural TEM-type enzymes, did not play an important role for the hydrolytic properties of the enzyme. The residue Ile-173 is not conserved among class A ß-lactamases and seems not to be implicated in the hydrolysis of oxyimino-ß-lactams (38).


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  		TABLE 3. Kinetics parameters of the ß-lactamase TEM-132 compared with those of TEM-28 ß-lactamasea

 

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  		TABLE 4. IC50 values of ß-lactamase inhibitors

 
In conclusion, this work allowed us to identify the ESBLs detected in a single medical center in Slovakia and to characterize the new TEM-132. Further studies in other hospitals are necessary for comparison with our findings.

Nucleotide sequence accession number. The GenBank accession number for blaTEM-132 is AY491682.


   ACKNOWLEDGMENTS
 
Martina Zarnayova worked with a doctoral fellowship from the "Ambassade de France" in Bratislava.


   FOOTNOTES
 
* Corresponding author. Mailing address: Laboratoire de Bactériologie, Hôpital Universitaire du Bocage, BP 77908, 21079 Dijon Cedex, France. Phone: 33-3 80 29 32 60. Fax: 33-3 80 29 36 67. E-mail: catherine.neuwirth{at}chu-dijon.fr. Back


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Antimicrobial Agents and Chemotherapy, July 2005, p. 3066-3069, Vol. 49, No. 7
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.7.3066-3069.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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