Miklós Füzi

Expansion and countrywide dissemination of ST11, ST15 and ST147 ciprofloxacin-resistant CTX-M-15-type β-lactamase-producing Klebsiella pneumoniae epidemic clones in Hungary in 2005—the new ‘MRSAs’?

OBJECTIVES To investigate the molecular epidemiology of ciprofloxacin-resistant CTX-M-15-producing Klebsiella pneumoniae epidemic clones (ECs) isolated from six nosocomial outbreaks and sporadic cases during 2005 in Hungary. METHODS Two hundred and eighty-one extended-spectrum beta-lactamase (ESBL)-producing K. pneumoniae clinical isolates collected from 41 centres were submitted to the National ESBL Reference Laboratory for further investigations. Of the 281 strains, 75 isolates proved to be SHV producers, whereas 6 isolates were ciprofloxacin-susceptible CTX-M-type ESBL producers. One hundred and ninety-six ciprofloxacin-resistant CTX-M-type beta-lactamase-producing isolates collected from 35 centres were subjected to macrorestriction profile analysis. Furthermore, molecular typing was performed by PCR and sequencing of several antibiotic resistance genes, plasmid profile analysis, transfer of resistance determinants and multilocus sequence typing (MLST). RESULTS PFGE revealed the existence of three genetic clusters defined as ECs, where 129 isolates belonged to the previously described Hungarian EC (HEC), 46 isolates to epidemic clone II (EC II) and 21 isolates to epidemic clone III (EC III), respectively. All isolates harboured plasmids ranging from 2.0 to 230 kb. PstI digestion of plasmid DNA from transconjugants/transformants revealed diverse restriction patterns from distinct ECs. Sequence analysis of beta-lactamase genes from 19 selected isolates detected bla(CTX-M-15) and bla(OXA-1) in strains from all three ECs and bla(TEM-1) in EC III isolates located on large plasmids. ISEcpI associated with CTX-M-15 was detected only on a 50 kb non-conjugative plasmid from EC III. MLST identified three allelic profiles: ST 15 (HEC), ST 11 (EC III) and the novel ST 147 (EC II), which correspond to the PFGE clusters, respectively. CONCLUSIONS In 2005, 97% of all CTX-M-producing K. pneumoniae isolates detected across Hungary were highly ciprofloxacin-resistant CTX-M-15 producers and represented just three stable genetic clones.

Establishing Clonal Relationships between VIM-1-Like Metallo-β-Lactamase-Producing Pseudomonas aeruginosa Strains from Four European Countries by Multilocus Sequence Typing

ABSTRACT Ten multidrug-resistant Pseudomonas aeruginosa strains producing VIM-1-like acquired metallo-β-lactamases (MBLs), isolated from four European countries (Greece, Hungary, Italy, and Sweden), were analyzed for genetic relatedness by several methodologies, including fliC sequence analysis, macrorestriction profiling of genomic DNA by pulsed-field gel electrophoresis (PFGE), random amplification of polymorphic DNA (RAPD), and multilocus sequence typing (MLST). The four approaches yielded consistent results overall but showed different resolution powers in establishing relatedness between isolates (PFGE > RAPD > MLST > fliC typing) and could usefully complement each other to address issues in the molecular epidemiology of P. aeruginosa strains producing acquired MBLs. In particular, the recently developed MLST approach was useful in revealing clonal relatedness between isolates when this was not readily apparent using RAPD and PFGE, and it suggested a common ancestry for some of the VIM-1-like MBL-positive P. aeruginosa strains currently spreading in Europe. The MBL producers belonged in three clonal complexes/burst groups (BGs). Of these, one corresponded to the previously described BG4 and included serotype O12 strains from Hungary and Sweden, while the other two were novel and included serotype O11 or nonserotypable strains from Greece, Sweden, and/or Italy. Comparison of the integrons carrying blaVIM-1-like cassettes of various isolates revealed a remarkable structural heterogeneity, suggesting the possibility that multiple independent events of acquisition of different blaVIM-containing integrons had occurred in members of the same clonal lineage, although a contribution of integrase-mediated cassette shuffling or other recombination mechanisms during the evolution of similar strains could also have played a role in determining this variability.

Molecular typing indicates an important role for two international clonal complexes in dissemination of VIM-producing Pseudomonas aeruginosa clinical isolates in Hungary

VIM metallo-beta-lactamase-producing serotype O11 or O12 Pseudomonas aeruginosa isolates infecting or colonising 19 patients from seven hospitals were reported in Hungary between 2003 and 2005. In this study we characterised VIM-producing Pseudomonas spp. clinical isolates from two novel locations in Hungary; we identified three new bla(VIM) carrying integron types and the presence of the bla(VIM-2) allele in Hungary. By applying various typing techniques, including multilocus sequence typing, we revealed an important role of two international clonal complexes, CC4 and CC11, in the dissemination of bla(VIM)-positive P. aeruginosa in hospitals in Hungary. Isolate P12-Q, a representative strain from France of the major European multiresistant P12 clone, displayed ST111 which, according to eBURST analysis, is the presently calculated founder sequence type of CC4. This is in accordance with the wide geographic distribution of the P12 clone. Our data indicate that, although the CC4 clonal complex includes serotype O1 and O6 isolates as well, it also contains the P12 clone. We characterised a P. aeruginosa nosocomial clone with a singleton sequence type (ST313), that may have acquired bla(VIM-2) and bla(VIM-4) gene cassettes from a yet unidentified local gene pool in Hungary.

Isolation of an Integron-Borne blaVIM-4 Type Metallo-β-Lactamase Gene from a Carbapenem-Resistant Pseudomonas aeruginosa Clinical Isolate in Hungary

ABSTRACT The first integron-borne metallo-β-lactamase gene was isolated in Hungary. The blaVIM-4 gene is located on a class 1 integron that also carries a novel blaOXA-like gene. The integron is harbored by a serotype O12 Pseudomonas aeruginosa strain and shows high structural similarity to integrons isolated in Greece and Poland.

Identification of the first VIM metallo-β-lactamase-producing multiresistant Aeromonas hydrophila strain

ABSTRACT A VIM metallo-β-lactamase-producing Aeromonas hydrophila strain carrying an integron-borne blaVIM-4 gene was isolated from a cirrhotic patients fecal sample in a Budapest hospital. The variable region of this integron is identical with that of a previously characterized integron from Pseudomonas aeruginosa clinical isolates in Pécs in southern Hungary.

Characterization of Extended-Spectrum β-Lactamase (TEM-52)-Producing Strains of Salmonella enterica Serovar Typhimurium with Diverse Resistance Phenotypes

ABSTRACT Two Salmonellaenterica serovar Typhimurium strains from different clonal origins, both producing an extended-spectrum β-lactamase (TEM-52), were isolated from a patient. This enzyme was encoded on a single plasmid and was found at very low levels in one strain, while being encoded on multiple plasmids and in multiple different EcoRI fragments in the other strain.

Molecular Epidemiology of VIM-4 Metallo-β-Lactamase-Producing Pseudomonas sp. Isolates in Hungary

ABSTRACT VIM metallo-β-lactamase-producing serotype O11 or O12 Pseudomonas aeruginosa isolates infecting or colonizing 19 patients from seven hospitals in Hungary were characterized between October 2003 and November 2005. Macrorestriction analysis revealed the involvement of hospitals from three different towns in northwest Hungary in an outbreak caused by VIM-4-producing P. aeruginosa.

Varying fitness cost associated with resistance to fluoroquinolones governs clonal dynamic of methicillin-resistant Staphylococcus aureus

The purpose of this study was to investigate the impact of fluoroquinolone resistance on the existence and dynamic of MRSA clones. Resistance to ciprofloxacin was induced in strains of community-acquired (CA) MRSA from various sequence types and the fitness cost suffered by mutant derivatives measured in a propagation assay. In addition, the fitness of fluoroquinolone resistant health care-associated (HA) MRSA isolates from major clones prevalent in Hungary were compared with each other and with those of the CA-MRSA derivatives. The genetic background of fluoroquinolone resistance and fitness cost in CA-MRSA was investigated. The fitness cost observed in the CA-MRSA derivatives proved diverse; the derivatives of the ST30-MRSA-IV strain suffered significantly greater fitness cost than those of the ST8-MRSA-IV and ST80-MRSA-IV isolates. Strains from the New York–Japan (ST5-MRSA-II), South German (ST228-MRSA-I) and EMRSA-15 (ST22-MRSA-IV) HA-MRSA clones proved more viable than CA-MRSA derivatives with similar MIC values to ciprofloxacin and HA-MRSA strains from the Hungarian/Brazilian clone (ST239-MRSA-III). Our strains from the New York–Japan, South-German and EMRSA-15 clones seem to have a competitive edge over the tested CA-MRSA isolates in the health care setting. The greater fitness observed in our New York–Japan and South-German strains could account for the replacement by them of the Hungarian/Brazilian clone in Hungary about ten years ago. Alterations in relevant genes were detected. The Ser80 → Phe mutation in the grlA gene may have seriously compromised viability. Surprisingly silent nucleotide substitutions in the grlB gene seemed to impact fitness in derivatives of the ST30-MRSA-IV isolate.

Nationwide spread of clonally related CTX-M-15-producing multidrug-resistant Klebsiella pneumoniae strains in Hungary

Since the 1990s the CTX-M type beta-lactamases have comprised the most rapidly growing group of extendedspectrum 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-Mproducing 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). The isolates were identified using ATB ID 32 E (bioMérieux, 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 Rif 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 blaCTX-M gene was carried out with all isolates and their transconjugants with primers specific for all of the known blaCTX-M genes: forward 5′-TTT GCG ATG TGCAGTACCAGTAA-3′ and reverse 5′-CGATAT CGT TGG TGG TGC CAT A-3′. The amplified blaCTX-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 Taq DNA Polymerase FS This work was presented in summarized form at the 15th European Congress of Clinical Microbiology and Infectious Diseases in Copenhagen, Denmark, 2–5 April 2005. Abstract no. R1998.

CHARACTERISATION OF THE FIRST VIM METALLO-β-LACTAMASE-PRODUCING PSEUDOMONAS AERUGINOSA CLINICAL ISOLATE IN SERBIA

From the Central-East European region the first VIM metallo-beta-lactamase (MBL) producing Pseudomonas aeruginosa strains were published from Croatia, Poland and Hungary. The aim of this study was to assess the contribution of MBL-production to carbapenem-resistance among P. aeruginosa clinical isolates in the Military Medical Academy (MMA) in Belgrade, Serbia between August 2004 and September 2007. Only one P. aeruginosa isolate with strain number 722 proved MBL-positive that harboured a novel class 1 integron with a bla(VIM-2)-like cassette in the first position, followed by orfD, a putative gene with unknown function. Our data indicate that MBL-producing strains occur at a prevalence of less than 1% among imipenem-nonsusceptible P. aeruginosa clinical isolates in this Belgrade hospital. The newly identified VIM MBL-producing P. aeruginosa strain 722 could be assigned to serotype O11, and it was panresistant to all antimicrobials tested. The isolate displayed sequence type ST235 by multilocus sequence typing which is the founder sequence type of the previously identified international clonal complex CC11 that already contains bla(VIM)-positive isolates from Italy, Greece, Sweden, Hungary and Poland. In conclusion, this is the first report of VIM MBL-producing P. aeruginosa from Serbia and also of the occurrence of such isolates belonging to the international clonal complex CC11 in this country.