Since the 1990s the CTX-M type beta-lactamases have comprised the most rapidly growing group of extended-spectrum beta-lactamases (ESBL). They have been detected increasingly in Europe, Asia, America and Africa, and CTX-M-15 has been particularly prevalent [1–3]. In Hungary, only the CTX-M-4 beta-lactamase has been found in one Salmonella serovar Typhimurium strain [4], and no more data on the occurrence and dissemination of CTX-M β-lactamases is currently available. In order to close this knowledge gap, the study presented here was initiated to characterize the nosocomial isolates of CTX-M-producing Klebsiella spp. submitted to the National Center for Epidemiology in Budapest in 2003.

The National ESBL Survey was initiated by the National Center for Epidemiology and started in June of 2002. In 2003, a total of 5,865 K. pneumoniae strains were isolated from patients attending participating Hungarian hospitals, and after preliminary antimicrobial susceptibility tests were performed, 158 presumably ESBL-producing isolates were submitted to the National Center for Epidemiology for confirmation. Seventeen of these 158 strains showed higher resistance to cefotaxime than to ceftazidime. The first strain was isolated in January 2003 at an intensive care unit in Pest County (Table 1). By the end of March, four strains with a similar resistance pattern had been isolated from patients attending the surgical ward of the same hospital. During the same year, 13 further strains were isolated from patients in surgery, urology, medical and nephrology wards from eight different hospitals across Hungary, predominantly from surgical wounds (10/17) and urine (3/17) (Table 1).

Table 1 Clinical and in vitro characterization of multidrug-resistant CTX-M-15-producing K. pneumoniae isolates (K) and E. coli J 53 transconjugants (TK)
Full size table

The isolates were identified using ATB ID 32 E (bio-Mérieux, Marcy l’ Étoile, France). The putative production of an ESBL was tested using the combined disk method ESBL SET (Mast Diagnostics, Merseyside, UK). The ESBL-producing strain K. pneumoniae ATCC 700603 was used as a control strain. The MICs were determined using the E-test according to the recommendations of the Clinical and Laboratory Standards Institute (formerly the National Committee for Clinical Laboratory Standards).

The mating experiments were carried out with E. coli K12 J53-2 RifR as the recipient strain. The transconjugants were selected on Mueller–Hinton agar supplemented with cefotaxime (4 mg/l) and rifampicin (300 mg/l). For fingerprinting analysis, plasmid DNA from transconjugants was obtained using a QIAprep Spin Miniprep kit (QIAGEN, Hilden, Germany) and digested with PstI and PvuII (Biolabs, Ipswich, New England).

Amplification of the bla CTX-M gene was carried out with all isolates and their transconjugants with primers specific for all of the known bla CTX-M genes: forward 5′-TTT GCG ATG TGC AGT ACC AGT AA-3′ and reverse 5′-CGA TAT CGT TGG TGG TGC CAT A-3′. The amplified bla CTX-M gene fragments were subsequently digested by PstI and PvuII restriction endonucleases to separate the five CTX-M groups [5]. PCR to detect the presence of aac(3)-II in transconjugants was carried out using the primers and conditions described previously [6].

DNA sequencing was performed on transconjugants using ABI prism Big Dye Terminator Cycle Sequencing Ready Reaction kit with Ampli TaqR DNA Polymerase FS (Perkin–Elmer, Branchburg, NJ, USA) and analyzed in an ABI Prism 310 Automated Sequencer (Perkin Elmer Biosystems, Foster City, CA, USA) using the primer pair specific for bla CTX-M-1: forward 5′-ATG GTT AAA AAA TCA CTG CG-3′ [1] and reverse 5′-CAG CGC TTT TGC CGT CTA AG-3′ [7]. Plasmid DNA from 17 K. pneumoniae isolates and from transconjugants was obtained using the alkaline lysis method [8], and agarose gel electrophoresis was performed in vertical agarose gels.

Phage typing with 15 phages [9] and PFGE analysis with XbaI were used to ascertain the epidemiological relationship among 17 ESBL-KP strains. Clustering of PFGE profiles was performed using UPGAMA and Dice coefficient with a 1% track-length tolerance level. Pulsotypes (up to six-band differences) and subtypes (up to three-band differences) were defined following the criteria established by Tenover et al. [10]; clonal relationships were supposed if the isolates belonged to the same pulsotype.

Antimicrobial susceptibility testing (Table 1) revealed a high level of resistance to cefotaxime, gentamicin, tetracycline and ciprofloxacin and moderate resistance to ceftazidime, amoxicillin/clavulanic acid, cefepime, and netilmicin in all strains but one. The latter, strain K165/03 from Baranya county, was susceptible to gentamicin, and the aac(3)-II gene was not detected in its transconjugant.

The 544 bp fragments specific for bla CTX-M were amplified in all strains. Further digestion of PCR products with PstI and PvuII showed that all bla CTX-M genes belonged to the CTX-M-1 ESBL-group. Plasmids from CTX-M-producing strains could be divided into three profiles: 137/2.7 KB (12 isolates), 137/38/2.7 KB (1 isolate) and 137/100/38/2.7 KB (4 isolates). Only the large plasmid of 137 KB was transferred to all transconjugants, and it harbored the bla CTX-M-15, as demonstrated by sequencing, and the aac(3)-II genes (except TK165/03), as demonstrated by PCR (data not shown). Plasmid DNA from transconjugants was compared after digestion with PstI and PvuII restriction endonucleases (data not shown) and very similar restriction patterns were obtained; the gene encoding CTX-M-15 resided in the same plasmid of 137 KB in all 17 strains.

The 17 strains belong to three closely related phage types: IA26, XIA1 and XIA3 (Table 1). On the basis of these preliminary results, all strains were subjected to macrorestriction profile analysis by PFGE together with eight different SHV-producing outbreak strains previously isolated in different Hungarian neonatal intensive care units. According to cluster analysis, all CTX-M-15-producing K. pneumoniae isolates were clonally related at an 89% similarity level and were clearly different from the SHV-outbreak strains. The CTX-M-15-producing isolates were grouped into one clone represented by two subtypes with a four-band difference: N1 and N2 (Table 1). The results obtained with phage typing and PFGE were concordant and proved the existence of a strong correlation among the CTX-M-15-producing isolates.

The emergence of CTX-M-15 in K. pneumoniae in Hungary is not surprising considering its extended dissemination among different species of the family Enterobacteriaceae in Eastern Europe [1, 5]. This report represents the first description of countrywide spread of a CTX-M-15-producing KP clone in Hungary, and we propose calling it the Hungarian epidemic clone (HEC). This clone was isolated from patients at eight geographically distant hospitals in five Hungarian counties and in the city of Budapest, mainly from surgical care units and from postoperative wound infections. Interestingly, in different healthcare centers the strains were isolated at different time periods, month-by-month, during the year 2003.

In HEC, the bla CTX-M-15 gene was located on the 137 KB self-transmissible plasmid, which confers co-resistance to aminoglycosides and tetracycline. In E. coli isolates from a French geriatric hospital, the bla CTX-M-15 was located on the 120 KB conjugative plasmid [6]. In general, the CTX-M-encoding genes have been located on plasmids of different sizes between 7 and 160 KB and they are often transmissible by conjugation in vitro. These plasmids frequently carry genes for resistance to aminoglycosides, chloramphenicol, sulfonamide and tetracycline [2]. The epidemic spread of CTX-M-15-producing clones has only been reported for E. coli in the UK and France [3, 6]. The rapid dissemination of this Hungarian multidrug-resistant epidemic clone and particularly of its conjugative plasmid seriously endangers Hungarian healthcare institutions. Immediate intervention is needed for efficient eradication of this clone and continuous epidemiological monitoring is required to control its spread.