Search

Susceptibilities to P-lactam antibiotics of five VIM-1-producing Klebsiella pneumoniae isolates were determined by broth microdilution, Etest, disk diffusion, and the automated systems Vitek 2, Phoenix, and MicroScan. Significant discrepancies were observed in the determination of susceptibility to imipenem and meropenem. Interpretation problems by the automated systems were also noted.

Screening of Greek nontyphoid salmonellae from 2000 to 2002 yielded three extended-spectrum beta-lactamase (ESBL) -producing human isolates. Salmonella enterica serotype Brandenburg harbored a multiresistant SHV-5 gene-carrying plasmid. S. enterica serotype Blockley and S. enterica serotype Hadar harbored a TEM-52 gene-carrying plasmid. An S. enterica serotype Virchow strain producing plasmid-mediated CTX-M-32 was isolated twice from poultry end products. All ESBL plasmids were self-transferable and carried by clones currently common in Greece.

The susceptibility to beta -lactams and the beta -lactamase content of 110 Salmonella spp. blood isolates collected during 1997-98 in 19 European centers participating in the SENTRY Surveillance Program were studied. Thirty-one isolates (28%) were resistant to penicillins, due to production of TEM-1 (27 isolates), OXA-1 (three isolates) or TEM-1 + OXA-1 (one isolate). All OXA-1 producers and 10 TEM-1-producing isolates were also resistant to penicillin-clavulanic acid combinations. In the latter isolates, this phenotype was associated with increased production of TEM-1. Sixteen TEM-1-producing Salmonella Enteritidis isolates and one OXA-1-producing S. Typhimurium isolate were able to transfer beta -lactam resistance by conjugation.

Seventeen Klebsiella pneumoniae clinical isolates carrying the bla(VIM-1) metallo-beta-lactamase gene were collected in the intensive care units of three hospitals in Athens, Greece, in 2002. They exhibited various carbapenem resistance levels (Etest MICs of imipenem ranged from 4 to 32 mug/ml). All isolates gave positive results by the imipenem-EDTA synergy Etest. The isolates were classified into four main types by pulsed-field gel electrophoresis; the majority of the isolates (5 and 10 isolates) belonged to two types. The bla(VIM-1) gene cassette was part of the variable region of a class I integron that also included aac6, dhftI, and aadA. This structure was carried by transferable plasmids.

Fifty-eight imipenem-nonsusceptible (MIC greater than or equal to 8 mug/ml) Pseudomonas aeruginosa strains isolated during May 2001 in 15 Greek hospitals were studied. Thirty-six isolates derived from nine hospitals carried VIM-type metallo-beta-lactamase genes, as found by PCR. In 34 isolates, bla(VIM) was associated with class 1 integrons of various sizes. DNA sequencing indicated the presence of bla(VIM-2) gene cassettes in a variety of integron structures. Random amplified polymorphic DNA typing suggested diversity of the bla(VIM)-positive strains. Synergy between 2-mercaptoacetic acid and imipenem indicated carbapenemase activity in 26 bla(VIM)-positive strains.

Seventy-nine Klebsiella pneumoniae and 124 Escherichia coli clinical strains, isolated consecutively during August-October 2001 in two Greek hospitals, were examined for production of extended-spectrum beta-lactamases (ESBLs). Seventy-one (35%) isolates (46 K pneumoniae and 25 E. coli) were ESBL-positive by phenotypic methods. Isoelectric focusing of beta-lactamases and PCR assays for bla genes showed that SHV-5-type ESBLs were the most frequent (45 isolates, 22%) followed by CTX-M (24 isolates, 12%) and IBC (three isolates, 1.5%). The latter two ESBL types may have been established recently in this setting. (C) 2002 Elsevier Science B.V. and the International Society of Chemotherapy. All rights reserved.

Objective To study the possible distribution of metallo-beta-lactamases among nosocomial Pseudomonas isolates in a Greek hospital with a recent high prevalence of carbapenem-resistant Pseudomonas isolates. Methods All carbapenem-resistant (imipenem- and/or meropenem-resistant) (MICS > 8 mg/L) Pseudomonas non-replicate isolates recovered from clinical infections in the Microbiology Laboratory of Saint Demetrios Hospital, Thessaloniki, Greece, from April 1998 to November 2000 were studied for the presence of metallo-beta-lactamases. They were tested by a disk diffusion test, PCR analysis, and nucleotide sequencing. DNA fingerprints were obtained by pulsed-field gel electrophoresis (PFGE) of XbaI-digested chromosomal DNA. Results In total, 24 carbapenem-resistant isolates (23 P. aeruginosa and one P. putida) were recovered. The serotypes observed among the P. aeruginosa isolates were, in order of decreasing frequency, O:11 (52%), O:3 and O:12 (17% each), and O:6 (13%). PFGE grouped 17 of the P. aeruginosa isolates into four clusters, each containing from two to seven isolates while the remaining isolates exhibited unique genotypes. bla(VIM-2) was detected in the P. putida isolate and a P. aeruginosa serotype 0:3 isolate. The latter strain was genotypically distinct from other contemporaneous or older carbapenem-resistant P. aeruginosa Greek isolates. Conclusion These findings suggest that, although the prevalence of metallo-beta-lactamases is low, the integron-associated bla(VIM) genes can spread to P. aeruginosa serotypes that have not been previously associated with carbapenem resistance in our region, as well as to other pseudomonal species.

Oxacillin resistance was examined in 258 coagulase-negative staphylococci from Greek hospitals. mecA DNA was detected in 168 isolates, which were also resistant to oxacillin by agar dilution and disk diffusion, according to the current NCCLS breakpoints. Both methods exhibited a relatively low specificity misclassifying 21 and 19 of the 90 mec4-negative isolates respectively as oxacillin resistant. In contrast, an anti-PBP 2a latex agglutination test, applied after induction by oxacillin, correctly classified 163 mecA-positive (sensitivity 97%) and 88 mecA-negative isolates (specificity 97.7%). (C) 2002 Elsevier Science Inc. All rights reserved.

A retrospective survey of the isolation rate of Enterococcus avium during the period March 1994-February 2000 conducted in Laikon General Hospital using the WHONET software, revealed a peak in the isolation rates of this species during March 1995-February 1996. The ten strains isolated during this time were studied further. No glycopeptide resistance was detected but resistance to ampicillin, ciprofloxacin, erythromycin, gentamicin (high-level) and streptomycin (high-level) was present in nine, ten, nine, three and seven of the isolates, respectively. The genes aac(6’)-Ie+aph(2”)-Ia and ant(6)-I, encoding for high-level gentamicin and streptomycin resistance, respectively, were detected only in the isolates with the corresponding phenotypes. Beta-lactamase production and haemolysis were not detected. There was evidence of ward-, floor- and building-specific distribution among the different aminoglycoside resistance phenotypes. DNA fingerprinting by PFGE grouped six of the ten isolates in a single cluster with 83% similarity, even though they expressed various resistance phenotypes. These results suggest dissemination of resistance genes among both genetically related and unrelated strains. (C) 2002 Elsevier Science B.V. and International Society of Chemotherapy. All rights reserved.

Rapid and efficient epidemiologic typing systems would be useful to monitor transmission of methicillin-resistant Staphylococcus aureus (MRSA) at both local and interregional levels. To evaluate the intralaboratory performance and interlaboratory reproducibility of three recently developed repeat-element PCR (rep-PCR) methods for the typing of MRSA, 50 MRSA strains characterized by pulsed-field gel electrophoresis (PFGE) (SmaI) analysis and epidemiological data were blindly typed by inter-IS256, 16S-23S ribosomal DNA (rDNA), and MP3 PCR in 12 laboratories in eight countries using standard reagents and protocols. Performance of typing was defined by reproducibility (R), discriminatory power (D), and agreement with PFGE analysis. Interlaboratory reproducibility of pattern and type classification was assessed visually and using gel analysis software. Each typing method shelved a different performance level in each center. In the center performing best with each method, inter-IS256 PCR typing achieved R = 100% and D = 100%; 16S-23S rDNA PCR, R = 100% and D = 82%; and MP3 PCR, R = 80% and D = 83%. Concordance between rep-PCR type and PFGE type ranged by center: 70 to 90% for inter-IS256 PCR, 44 to 57% for 16S-23S rDNA PCR, and 53 to 54% for MP3 PCR analysis. In conclusion, the performance of inter-IS256 PCR typing was similar to that of PFGE analysis in some but not all centers, whereas other rep-PCR protocols showed lower discrimination and intralaboratory reproducibility. None of these assays, however, was sufficiently reproducible for interlaboratory exchange of data.

Six distinct clones were present among Greek multidrug-resistant Salmonella enterica serotype Typhimurium phage type DT104, since isolates belonging to resistance phenotypes including the ACSSuT (ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline) core could be distinguished with respect to their pulsed-field gel electrophoresis patterns, int1 integron structures, and presence or absence of antibiotic resistance genes ant(3”)-Ia, pse-1, and tem-1.

CTX-M-type beta-lactamases constitute a novel group of class A beta-lactamases with extended-spectrum properties. They are encoded by transferable plasmids and found in various enterobacteria, mostly Salmonella typhimurium, Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis. CTX-M enzymes share extensive sequence similarity with the chromosomal beta-lactamases of Klebsiella oxytoca. They efficiently hydrolyze many newer broad-spectrum oximino-beta-lactams including cefotaxime, ceftriaxone and aztreonam and are readily inhibited by tazobactam and clavulanate. CTX-M-producing enterobacteria are endemic in Latin America and in some areas of North Eastern Europe. Data on their structure, properties and epidemiology are discussed. (C) 2000 Published by Elsevier Science B.V. and International Society of Chemotherapy. All rights reserved.

In SHV-type beta-lactamases, position 276 (in Ambler’s numbering scheme) is occupied by an asparagine (Asn) residue. The effect on SHV-1 beta-lactamase and its extended-spectrum derivative SHV-5 of substituting an aspartic acid (Asp) residue for Asn276 was studied. Mutations were introduced by a PCR-based site-directed mutagenesis procedure. Wild-type SHV-1 and -5 beta-lactamases and their respective Asn276–>Asp mutants were expressed under isogenic conditions by cloning the respective bla genes into the pBCSK(+) plasmid and transforming Escherichia coli DH5 alpha. Determination of IC50 showed that SHV-1(Asn276–>Asp), compared with SHV-1, was inhibited by 8- and 8.8-fold higher concentrations of clavulanate and tazobactam respectively. Replacement of Asn276 by Asp in SHV-5 beta-lactamase caused a ten-fold increase in the IC50 of clavulanate; the increases in the IC(50)s of tazobactam and sulbactam were 10- and 5.5-fold, respectively. beta-Lactam susceptibility testing showed that both Asn276–>Asp mutant enzymes, compared with the parental beta-lactamases, conferred slightly lower levels of resistance to penicillins (amoxycillin, ticarcillin and piperacillin), cephalosporins (cephalothin and cefprozil) and some of the expanded-spectrum oxyimino beta-lactams tested (cefotaxime, ceftriaxone and aztreonam). The MICs of ceftazidime remained unaltered, while those of cefepime and cefpirome were slightly elevated in the clones producing the mutant beta-lactamases. The latter clones were also less susceptible to penicillin-inhibitor combinations. Asn276–>Asp mutation was associated with changes in the substrate profiles of SHV-1 and SHV-5 enzymes. Based on the structure of TEM-1 beta-lactamase, the potential effects of the introduced mutation on SHV-1 and SHV-5 are discussed.

All 105 non-replicate consecutive Staphylococcus aureus strains isolated in 1997 from seven Creek hospitals, were found to be susceptible to vancomycin, teicoplanin and chloramphenicol, but only five (8%) were susceptible to all 16 antibiotics tested. Forty-three (41%) isolates were methicillin-resistant, 58% homogeneously (homMRSA) and 42% heterogeneously (hetMRSA). Resistance of homMRSA strains to other antibiotics was generally high (88-100%), although only one strain was resistant to netilmicin. Resistance in hetMRSA (6-39%) or in MSSA (5-11%) was significantly lower Consequently, the majority (76%) of homMRSA were multi-drug resistant, while the dominant phenotype of hetMRSA and MSSA was resistance to penicillin (50% and 76%, respectively). Comparison of these strains with isolates from 1994 showed higher resistance rates to erythromycin among MSSA, to erythromycin and amikacin among hetMRSA and to rifampicin among homMRSA strains. (C) 1999 Elsevier Science B.V. and International Society of Chemotherapy. All rights reserved.

The conserved residue Arg-244 was substituted by the smaller uncharged amino acids Cys and Ser in SHV-1 and SHV-5 beta-lactamases by a PCR-based site-specific mutagenesis procedure. The mutant beta-lactamases displayed decreased susceptibility to clavulanate and, to a lesser extent, to tazobactam and sulbactam. As shown in comparative MIC determinations, R244C and R244S enzymes retained a residual penicillinase activity while their activity towards cephalosporins was drastically diminished. The respective SHV-5 mutants were unable to hydrolyze oxyimino-beta-lactams except aztreonam. The impaired catalytic activity of the mutant beta-lactamases was mainly due to the lowering of affinity for beta-lactam substrates. The above alterations were more mutants. These results provide information on the mode of involvement of Arg-244 in (a) inactivation by beta-lactamase inhibitors and (b) the proper positioning of beta-lactams in the active site of SHV enzymes. (C) 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V.

The effect of replacement of Met-69 by Ile or Val on the properties of the extended-spectrum beta-lactamase SHV-5 was studied. Mutant enzymes were constructed by site-specific mutagenesis and expressed under isogenic conditions in Escherichia coli DH5 alpha cells. Compared with SHV-5, the mutant beta-lactamases conferred lower levels of beta-lactam resistance and were less efficient in hydrolyzing ampicillin, cephalothin, and cefotaxime, The substitutions rendered SHV-5 less susceptible to inhibition by clavulanate, sulbactam, and tazobactam; however, the MICs of penicillin-inhibitor combinations remained similar, suggesting an attenuation of penicillinase activity.

Two novel plasmid-mediated beta-lactamases (CTX-M-5 and CTX-M-6) produced by Salmonella typhimurium clinical strains were characterized. The enzymes exhibited a pI of 8.4, hydrolyzed oxyimino-beta-lactams and were susceptible to mechanism-based p-lactamase inhibitors. The respective bin genes were cloned and sequenced. The deduced amino acid sequences showed a high degree of homology with those of the previously described plasmid class A CTX-M-type enzymes and appeared related to the chromosomal p-lactamases of Klebsiella oxytoca. (C) 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

A comparative in vitro assessment of the inhibitory activity of clavulanate, sulbactam and tazobactam against the most frequent beta-lactamases found in enterobacteria isolated in Greek hospitals, has been performed. Tazobactam and clavulanate were potent inhibitors of TEM-1, SHV-1, SHV-2 and SHV-5 while sulbactam was less effective. In addition, tazobactam exerted a moderate inhibitory activity against AmpC-type beta-lactamases. In contrast with clavulanate, tazobactam could not cause significant induction of chromosomal cephalosporinases. Potentiation of the activity of penicillins (amoxycillin, ampicillin, ticarcillin and piperacillin), when combined with the inhibitors, has also been evaluated using a sample of recently isolated enterobacteria with beta-lactamase-mediated resistance. The results showed that the most effective among the commercially available combinations, is piperacillin/tazobactam followed by ticarcillin/clavulanate. (C) 1997 Elsevier Science B.V.

Methicillin-resistant Staphylococcus aureus (MRSA) strains isolated over a one-year period from a Greek hospital were tested for their levels of resistance to methicillin by population analysis. Heterogeneously resistant strains belonged to classes I, II, and II/III, whereas homogeneously resistant ones belonged to class IV. Strains of all classes possessed the mecA gene. Pulsed-field gel electrophoresis (PFGE) of Smal-digested genomic DNA revealed that all heterogeneously resistant strains were related. Homogeneously resistant strains were also closely related, but in a cluster distinct from the heterogeneous one. The methicillin-sensitive strains displayed a greater variety of PFGE types compared to MRSA isolates.

Previous studies have shown that there is a high incidence of resistance to cephalosporins among enterobacteria isolated in Greek hospitals. This resistance is mainly due to either the derepression of chromosomal cephalosporinases or the acquisition of plasmids coding for SHV-5 type beta-lactamase. In the present study the activity of cefpirome against a number of enterobacteria (Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Enterobacter aerogenes and Serratia marcescens) possessing the mechanisms mentioned above was examined, Cefpirome was found active against all the strains characterized by stable derepression of chromosomal class-C enzymes, The antibiotic was less potent against strains expressing SHV-5 type beta-lactamase due to its hydrolysis by the enzyme. Also cefpirome exhibited good activity against E. aerogenes strains with reduced susceptibility to imipenem, These ia vitro data suggest that cefpirome might be useful in treating infections caused by these resistant microorganisms that are frequently encountered in Greek hospitals.

Ninety Enterobacter cloacae strains isolated from 12 Greek hospitals were examined in terms of epidemiological types and resistance mechanisms. Using O serotyping 69% of the strains were assigned to a specific serotype and overall 16 different serotypes were identified. The combination of serotyping, phagetyping and biotyping efficiently discriminated most of the strains, indicating that single epidemic strains were not prevalent, although serotypes 3, 7, and group II predominated. Eight representative strains, all resistant to gentamicin, tobramycin, amikacin and netilmicin, were further examined for transferability and mechanisms of resistance. Aminoglycoside resistance was found to be transferable in most strains, and 13 R plasmids of 40-120 MDa molecular weight were detected. The enzymes detected consisted of three enzymes active against gentamicin [ANT(2”), AAC(3)-I and AAC(3)-V]; three active against tobramycin [ANT(2”), AAC(3)-V and AAC(6’)-I]; two active against netilmicin [AAC(3)-V and AAC(6’)-I]; and one active against amikacin [AAC(6’)-I]. APH(3’) and ANT (3”), which modify neomycin and streptomycin plus spectinomycin respectively, were also found. Overall up to five aminoglycoside modifying enzymes were detected on the same R plasmid, AAC(6’)-I plus ANT(2”) being the most prevalent. The high incidence of multiresistance in Enterobacter cloacae and the fact that resistance is due to enzymatic inactivation of the antibiotics, indicate that in Greece this species might act as a gene pool for the spread of resistance to other bacteria of clinical relevance.