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Since November 2009 carbapenemase-producing Klebsiella pneumoniae isolates have been detected in increasing numbers at the Clinical Centre University of Pécs. Molecular typing was performed for 102 clinical isolates originating from different time periods and various departments of the Clinical Centre. Pulsed-field gel electrophoresis revealed the predominance of a single clone (101/102), identified as sequence type ST15. PCR and sequencing showed the presence of blaCTX-M-15 and blaVIM-4 genes. The blaVIM-4 was located on a class 1 integron designated In238b. To our knowledge, this is the first description of a blaVIM-4 gene in the predominant CTX-M-15 extended spectrum β-lactamase-producing Hungarian Epidemic Clone/ST15.
Overuse of carbapenems due to the increasing prevalence of infections caused by third-generation cephalosporin-resistant Enterobacteriaceae has promoted the emergence of carbapenem-resistant isolates. Decreased susceptibility to carbapenems in Enterobacteriaceae can be caused by various mechanisms including production of carbapenemases (IMP, KPC, NDM, OXA-48, VIM) or production of extended spectrum β-lactamase (ESBL) or AmpC-type enzymes in conjunction with mutations of porins [1, 2]. In Hungary the first carbapenemase identified was VIM-4, located on In238b in Pseudomonas aeruginosa in 2002 . In238b was later found in Aeromonas hydrophila, Klebsiella pneumoniae sequence type 11 (ST11) and Klebsiella oxytoca [4, 5]. Beside VIM-4, production of KPC-2 by K. pneumoniae ST258 was reported in Hungary . The first carbapenemase-producing K. pneumoniae in southwest Hungary was isolated in the Clinical Centre University of Pécs in November 2009. Since then, further carbapenemase-producing isolates have been identified from various departments of the Clinical Centre. The aim of our study was to identify the carbapenemase gene and to assess the clonality.
During the study period (29 November 2009 to 31 July 2011), of the 3133 K. pneumoniae clinical isolates (originating from 1654 patients) identified in the Diagnostic Bacteriological Laboratory Clinical Centre University of Pécs, 493 (originating from 198 patients and 15 departments) were supposed to produce carbapenemase according to screening with disc diffusion and modified Hodge-test . Of these, 102 were selected for further investigation. The selection was performed so as to represent the study period, regarding time of collection and departments. Only one isolate per patient was included. Pulsed-field gel electrophoresis was performed as described previously . Multilocus sequence typing was performed for three isolates selected according to pulsed-field gel electrophoresis subclusters . Confirmation of carbapenemase production was performed using a phenotypic inhibition assay with commercially available diagnostic discs (KPC + MBL Confirmation ID Kit; Rosco, Taastrup, Denmark). The presence of different β-lactamase genes (blaSHV, blaTEM, blaCTX-M, blaKPC, blaVIM, blaIMP, blaOXA-48, blaNDM, blaDHA, blaCMY) was sought by PCR [9-14]. Digestion of blaSHV with NheI was carried out to identify Gly238Ser mutation associated with hydrolysis of third-generation cephalosporins . Sequencing of blaSHV, blaTEM and blaCTX-M was performed for three isolates . PCR mapping of integrons was conducted on ten isolates using primers described previously and sequencing was performed for one isolate . The MICs of ertapenem, imipenem and meropenem were measured with gradient test strips (Liofilchem, Roseto degli Abruzzi, Italy) and the results were interpreted according to EUCAST guidelines (The European Committee on Antimicrobial Susceptibility Testing. Breakpoint tables for interpretation of MICs and zone diameters. Version 3.1, 2013. http://www.eucast.org).
As revealed by pulsed-field gel electrophoresis all but one isolate belonged to the formerly characterized CTX-M-15-producing Hungarian Epidemic Clone (HEC). It was confirmed with multilocus sequence typing to belong to ST15. The presence of blaSHV, blaTEM, blaCTX-M and blaVIM were shown by PCR in 102, 102, 100 and 101 isolates respectively. All isolates were negative for the Gly238Ser mutation of blaSHV. The genes were identified as blaSHV-28, blaTEM-1, blaCTX-M-15 and blaVIM-4. The presence of a class 1 integron carrying aac(6')-Ib in the first gene cassette and blaVIM-4 in the second gene cassette was shown. The integron was designated as In238b according to the Integrall database (integrall.bio.ua.pt). The only VIM-negative isolate belonged to HEC and harboured blaSHV, blaTEM and blaCTX-M genes. The results of susceptibility testing and phenotypic inhibition assay for the 101 VIM-positive K. pneumoniae isolates are summarized in Tables 1 and 2.
Table 1. MIC values and susceptibility of VIM-positive Klebsiella pneumoniae isolates (n = 101) to different antibiotics according to EUCAST breakpoints (version 3.1 2013-02-11)
|Susceptible||7 (6.9%)||57 (56.4%)||91 (90.1%)|
|Intermediate||10 (9.9%)||18 (17.8%)||8 (7.9%)|
|Resistant||84 (83.2%)||26 (25.7%)||2 (2.0%)|
|Above ECOFF||101 (100%)||61 (60.4%)||95 (94.1%)|
Table 2. Proportions of metallo-β-lactamase positivity in the phenotypic inhibition assay and mean differences between inhibition zones of meropenem and meropenem + inhibitor in relation to meropenem MIC for VIM-positive Klebsiella pneumoniae isolates (n = 101)
|Meropenem|| n ||Boronic acid||Dipicolinic acid||Cloxacillin||MBL|
|MIC (mg/L)||Mean (mm)||SD||Mean (mm)||SD||Mean (mm)||SD||positive (%)|
This study has shown that the emergence of carbapenemase-producing K. pneumoniae isolates in the Clinical Centre University of Pécs can be explained by the recent acquisition and expansion of the blaVIM-4 gene in the nationally disseminated CTX-M-15-producing K. pneumoniae HEC/ST15. To our knowledge, this is the first description of the blaVIM-4 gene in this international K. pneumoniae clone. The ciprofloxacin-resistant, CTX-M-15-producing ST15 was first described in Hungary in 2003. Since then it has become one of the dominant ESBL-producing K. pneumoniae clones not only in Hungary but also in several European and Asian countries [8, 16-18]. In238b carrying blaVIM-4 has circulated in Hungary since 2002, as indicated by continuous isolation in different species (P. aeruginosa 2002, A. hydrophila 2005, K. pneumoniae 2009, K. oxytoca 2009) [3-5]. Regarding our institution, In238b was reported earlier in P. aeruginosa and when it was acquired by HEC/ST15 a remarkable expansion of blaVIM-4 was observed . Of the 1654 patients from whom K. pneumoniae was isolated, 101 (6.1%) were proven to have VIM-producing isolates. The resistance conferred by VIM was of low level. For the majority of the isolates the MIC values of imipenem and meropenem were near the susceptibility clinical breakpoint. The low level of resistance hindered detection by phenotypic inhibition assay, as indicated by the smaller difference between inhibition zones of meropenem and meropenem + dipicolinic acid at lower meropenem MIC. This explains why 51.0% of isolates were not positive for metallo-β-lactamase production in this assay despite production of VIM being demonstrated by the modified Hodge-test. Considering the low level of resistance conferred by VIM, the usage of a modified Hodge-test and the meropenem epidemiological cut-off value proposed by EUCAST (The European Committee on Antimicrobial Susceptibility Testing. Frequently Asked Questions. updated 2012-02-12. www.eucast.org) to screen for carbapenemase production and simultaneous testing of susceptibility to the three carbapenem derivatives could be beneficial in the detection of carbapenemase production in K. pneumoniae. The emergence of VIM-4 metallo-β-lactamase in the predominant ESBL-producing ST15 K. pneumoniae clone is of great concern. It indicates the ability of this clone to successively adapt to antibiotic pressure. This fact emphasizes the importance of rational antibiotic usage, rigorous hospital hygiene measures and the need for developing newer compounds.
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The authors are thankful to Professor Levente Emődy (Department of Medical Microbiology and Immunology, Medical School, University of Pécs, Hungary) for his helpful advice and financial support (OTKA-NN-78915).
Part of this study was presented at the 40th Congress of the Hungarian Society of Clinical Microbiology and Infectious Diseases (Budapest, Hungary, 20–22 September 2012) and at the 8th International Healthcare Infection Society Conference and Federation of Infection Societies Annual Conference (Liverpool, UK, 19–21 November 2012).