Relationship between Chlamydia-Ureaplasma-Mycoplasma Genital Detection with Semen Concentration and Motility among Greek Men.

One hundred and seventy two men at the State of Thessaly, Greece, inquiring semen analysis were enrolled in the study in order to investigate the incidence of Chlamydia, Ureaplasma and Mycoplasma (C-U-M) genera in respect to total sperm number (TSN), progressive motility (grades a and b) and total motility (grades a, b and c). Putative relation of C-U-M acquirement with sexual behavior was also investigated. Incidence of C-U-M among non-oligozoospermic and oligozoospermic men was similar. Νο correlation of C-U-M carriage to either oligozoospermia or asthenozoospermia was found. The tested semen parameters were negatively correlated to the age of sexual intercourse initiation and positively correlated to the number of sex partners. Early age of sexual intercourse initiation or high number of sexual partners was not statistical significantly correlated to C-U-M acquirement. Overall, TSN and motility (either progressive or total) were not influenced by the presence of C-U-M genera in a sample of Greek population undergoing semen evaluation. To distinguish the role of C-U-M in male infertility and clarify the so far controversial scarce literature, large control case studies are needed using nucleic acid amplification techniques to detect these pathogens.

Accumulation of multiple mutations in linezolid-resistant Staphylococcus epidermidis causing bloodstream infections; in silico analysis of L3 amino acid substitutions that might confer high-level linezolid resistance.

Fifty-six Staphylococcus epidermidis clinical isolates, showing high-level linezolid resistance and causing bacteremia in critically ill patients, were studied. All isolates belonged to ST22 clone and carried the T2504A and C2534T mutations in gene coding for 23SrRNA as well as the C189A, G208A, C209T and G384C missense mutations in L3 protein which resulted in Asp159Tyr, Gly152Asp and Leu94Val substitutions. Other silent mutations were also detected in genes coding for ribosomal proteins L3 and L22. In silico analysis of missense mutations showed that although L3 protein retained the sequence of secondary motifs, the tertiary structure was influenced. The observed alteration in L3 protein folding provides an indication on the putative role of L3-coding gene mutations in high-level linezolid resistance. Furthermore, linezolid pressure in health care settings where linezolid consumption is of high rates might lead to the selection of resistant mutants possessing L3 mutations that might confer high-level linezolid resistance.

Emergence of VIM-12 in Enterobacter cloacae.

Detection of the bla(VIM-12) gene within the originally described Inh12 integron in a clinical isolate of Enterobacter cloacae is reported for the first time worldwide. Integron Inh12 was carried on a conjugative plasmid of approximately 85 kb which also conferred resistance to aztreonam, likely due to AmpC production.

Characteristics of meropenem heteroresistance in Klebsiella pneumoniae carbapenemase (KPC)-producing clinical isolates of K. pneumoniae.

Meropenem heteroresistance was investigated in six apparently meropenem-susceptible, Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-KP) clinical isolates, compared with that in carbapenemase-negative, meropenem-susceptible controls. In population analyses, the KPC-KP isolates grew at meropenem concentrations of 64 to 256 microg/ml. Heteroresistant colonies had significantly elevated expression of the bla(KPC) gene compared with the native populations but did not retain heteroresistance when subcultured in drug-free media. Time-kill assays indicated that meropenem alone was not bactericidal against KPC-KP but efficiently killed the control strains.

Detection of mutations in the FemXAB protein family in oxacillin-susceptible mecA-positive Staphylococcus aureus clinical isolates.

Objectives Methicillin-resistant Staphylococcus aureus (MRSA) strains that express the mecA gene but are oxacillin susceptible (OS-MRSA; oxacillin MIC

Heteroresistance to meropenem in carbapenem-susceptible Acinetobacter baumannii.

The characteristics of carbapenem heteroresistance were studied in 14 apparently carbapenem-susceptible Acinetobacter baumannii isolates. The MICs for carbapenems were determined, and the isolates were genotyped by pulsed-field gel electrophoresis (PFGE) and sequence typing (ST). Population analysis, testing of the stability of the heteroresistant subpopulations, and time-killing assays were performed. The agar dilution MICs of both imipenem and meropenem for the native isolates ranged from 0.25 to 4 mg/liter. The isolates belonged to nine PFGE types and exhibited seven ST allelic profiles. Population analysis revealed subpopulations that grew in the presence of imipenem at concentrations of up to 8 mg/liter and meropenem at concentrations of up to 32 mg/liter. The meropenem-heteroresistant subpopulations of 11 isolates exhibited stable resistance with MICs that ranged from 16 to >32 mg/liter; their PFGE profiles were identical to those of the native isolates. Time-kill assays with meropenem revealed less pronounced killing for 10 isolates. These findings indicate that meropenem pressure can produce meropenem-heteroresistant subpopulations that might subsequently select for highly resistant strains.

Association of biofilm formation and methicillin-resistance with accessory gene regulator (agr) loci in Greek Staphylococcus aureus clones.

Pathogenicity of Staphylococcus aureus is coordinated by the accessory gene regulator (agr) system. Previous studies suggested that agr Group II methicillin-resistant S. aureus (MRSA), a polymorphism that has been associated with moderate response to vancomycin, may also be related with overproduction of biofilm. In a hospital environment with endemic occurrence of MRSA, the distribution of agr groups and their association with biofilm formation was investigated. Forty-two MRSA and 32 methicillin-susceptible S. aureus (MSSA) isolates were tested and had derived from 10 genotypes and 8 clonal complexes. agr Groups I, II and IV were evenly distributed among MRSAs and MSSAs but agr Group III was not detected. agr Group II MRSAs showed significantly higher levels of biofilm production in comparison with MRSAs of the remaining agr groups as well as with all three agr groups of MSSAs. These findings suggest that agr Group II is simultaneously associated with methicillin-resistance and biofilm overproduction in a region with endemic MRSA.

Molecular epidemiology of outbreak-related pseudomonas aeruginosa strains carrying the novel variant blaVIM-17 metallo-beta-lactamase gene.

A study was designed to investigate the molecular epidemiological characteristics of multidrug-resistant outbreak-related Pseudomonas aeruginosa isolates collected in a university hospital in northern Greece. Of 29 nonreplicate P. aeruginosa isolates resistant to carbapenems and ceftazidime, 14 were positive for metallo-beta-lactamase production. PCR analyses with primers specific for bla(VIM) and bla(IMP) revealed that 13 isolates carried a novel bla(VIM-2) gene variant, designated bla(VIM-17), and only 1 isolate carried bla(VIM-2), a gene predominant among P. aeruginosa strains in Greek hospitals. Pulsed-field gel electrophoresis of XbaI-digested genomic DNAs showed a close genetic relationship for 12 of 13 bla(VIM-17)-carrying outbreak-related isolates, which were of the O11 serotype; the clonally unrelated isolate carrying bla(VIM-17) was of the O12 serotype. PCR mapping strategies for the detection of class 1 integrons and sequencing approaches revealed the presence of integrons containing one bla(VIM) cassette flanked by two aacA29 cassettes. These integrons were similar but not identical to In59 (GenBank accession number AF263519) initially described in France. All isolates carrying bla(VIM-17), regardless of their genetic profile, had an identical integron, named In59.3, indicating that although the hospital outbreak was mainly due to clonal dissemination, the horizontal transmission of the bla(VIM-17)-containing integron among P. aeruginosa isolates should also have occurred. An outbreak-related isolate and a control strain, both of which carried the bla(VIM-2) gene but which were clonally distinct, had an identical integron, named In59.2, which differed only at the level of the bla(VIM) gene from In59.3 integrons, suggesting a common ancestry. The spread of the bla(VIM-17)-containing integron in clonally unrelated P. aeruginosa isolates without any evidence of plasmid carriage is probably associated with a transposon.

Evaluation of boronic acid disk tests for differentiating KPC-possessing Klebsiella pneumoniae isolates in the clinical laboratory.

The worldwide increase in the occurrence and dissemination of KPC beta-lactamases among gram-negative pathogens makes critical the early detection of these enzymes. Boronic acid disk tests using different antibiotic substrates were evaluated for detection of KPC-possessing Klebsiella pneumoniae isolates. A total of 57 genotypically confirmed KPC-possessing K. pneumoniae isolates with varying carbapenem MICs were examined. To measure the specificity of the tests, 106 non-KPC-possessing isolates (89 K. pneumoniae and 17 Escherichia coli isolates) were randomly selected among those exhibiting reduced susceptibility to cefoxitin, expanded-spectrum cephalosporins, or carbapenems. As many as 56, 53, and 40 of the non-KPC-possessing isolates harbored extended-spectrum beta-lactamases, metallo-beta-lactamases, and plasmid-mediated AmpC beta-lactamases, respectively. By use of CLSI methodology and disks containing imipenem, meropenem, or cefepime, either alone or in combination with 400 microg of boronic acid, all 57 KPC producers gave positive results (sensitivity, 100%) whereas all 106 non-KPC producers were negative (specificity, 100%). The meropenem duplicate disk with or without boronic acid demonstrated the largest differences in inhibition zone diameters between KPC producers and non-KPC producers. By use of disks containing ertapenem, all isolates were correctly differentiated except for five AmpC producers that gave false-positive results (sensitivity, 100%; specificity, 95.3%). These practical and simple boronic acid disk tests promise to be very helpful for the accurate differentiation of KPC-possessing K. pneumoniae isolates, even in regions where different broad-spectrum beta-lactamases are widespread.

First occurrence of KPC-2-possessing Klebsiella pneumoniae in a Greek hospital and recommendation for detection with boronic acid disc tests.

OBJECTIVES: To investigate the first KPC carbapenemase-producing Klebsiella pneumoniae isolate from a Greek hospital, including phenotypic methods to aid recognition of this resistance type. METHODS: A carbapenem-resistant clinical isolate of K. pneumoniae was recovered from a hospitalized Greek patient. Detailed susceptibility testing was carried out by the agar dilution method. The isolate was screened by phenotypic and genotypic assays for the presence of various beta-lactamases. Boronic acid disc tests were performed to show the ability of these tests to detect production of the KPC enzymes. The potential for conjugal transfer of carbapenem resistance was examined by biparental matings, plasmid analysis and PCR studies. RESULTS: The isolate possessed on the same self-transferable plasmid the KPC-2 carbapenemase and the SHV-12 extended-spectrum beta-lactamase. Although the isolate did not produce an AmpC-type enzyme, the production of KPC-2 was associated with positive boronic acid disc tests using cephamycins and cefotaxime as well as cefepime and carbapenems as substrates. DISCUSSION: KPC-2-possessing K. pneumoniae clinical isolates seem to have been introduced in our region. Boronic acid disc tests using boronic acid in combination with carbapenems or cefepime may help the phenotypic detection of KPC enzymes and their distinction from plasmid-mediated AmpC enzymes.

In vitro and in vivo evaluations of oxacillin efficiency against mecA-positive oxacillin-susceptible Staphylococcus aureus.

Community-type Staphylococcus aureus strains that are positive for mecA and PBP2a but appear phenotypically susceptible to oxacillin are increasingly reported worldwide. Four S. aureus clinical isolates carrying the mecA gene with oxacillin MICs of <2 microg/ml were tested for oxacillin efficiency by population analyses and experimental thigh infections. These isolates harbored staphylococcal cassette chromosome mec type IV and belonged to two genotypes. Two of the four isolates were found by population analysis to be truly oxacillin susceptible. All four isolates exhibited significant reductions in the numbers of colonies grown after dicloxacillin treatment of experimental thigh infections, as also did a mecA-negative S. aureus control strain. These observations indicate that some of the phenotypically oxacillin susceptible mecA-positive Staphylococcus aureus isolates may be at least partially responsive to oxacillin.

Cross-transmission of multidrug-resistant Acinetobacter baumannii clonal strains causing episodes of sepsis in a trauma intensive care unit.

OBJECTIVE: To investigate the mode of transmission of imipenem-resistant Acinetobacter baumannii strains causing episodes of sepsis. SETTING: A 7-bed trauma intensive care unit (ICU) in an orthopedic hospital in Greece. DESIGN: During a 14-week period (from January 10 to April 16, 2006), clinical specimens, along with samples taken on a weekly basis from the ICU environment and from the hands of health care workers (HCWs), were prospectively tested for imipenem-resistant A. baumannii. Pulsed-field gel electrophoresis was used to study the genetic relatedness of the isolates recovered from these specimens and samples. RESULTS: During the survey, imipenem-resistant A. baumannii was identified in 14 hospitalized patients, from whom 40 multidrug-resistant and imipenem-resistant A. baumanii isolates were recovered. These pathogens caused episodes of bacteremia and sepsis in all but one of the patients and contributed to the death of 3 patients. Samples for culture were obtained from the environment and from the hands of HCWs; 29 imipenem-resistant A. baumannii isolates were recovered from the environment, and 12 from HCWs. One predominant genotype and 2 less predominant genotypes were detected among the 81 imipenem-resistant A. baumannii isolates. All 3 of these genotypes were found among patients and HCWs and were recovered from environmental samples. INTERVENTIONS: Control measures consisted of the closure of the ICU and the transfer of the patients to other units. The ICU was disinfected, and adherence to proper hand hygiene protocol was reinforced. These same clonal isolates were not recovered from clinical or environmental samples during the month after the reopening of the ICU. CONCLUSIONS: The extensive dissemination of imipenem-resistant A. baumannii clonal strains causing episodes of bacteremia and/or sepsis resulted from modes of transmission via multiple contaminated surfaces and objects and transiently colonized HCWs' hands. Closure of the ICU and its meticulous environmental decontamination led to the successful control of the outbreak.

Outbreak caused by a multidrug-resistant Klebsiella pneumoniae clone carrying blaVIM-12 in a university hospital.

From November 2006 to April 2007, nine nonrepetitive isolates of Klebsiella pneumoniae with reduced susceptibility or resistance to carbapenems were recovered from clinical specimens from separate patients hospitalized in a tertiary care hospital. The imipenem-EDTA synergy test was positive for all isolates. PCR, sequencing, and transferability experiments revealed the novel bla(VIM-12) metallo-beta-lactamase gene, which was plasmid mediated and located in a class 1 integron. Pulsed-field gel electrophoresis demonstrated a single macrorestriction pattern, indicating the clonal spread of VIM-12-producing K. pneumoniae.

Efflux system overexpression and decreased OprD contribute to the carbapenem heterogeneity in Pseudomonas aeruginosa.

Pseudomonas aeruginosa strains exhibiting a heterogeneous mode of growth against carbapenems have been described recently. This study investigated the underlying molecular mechanisms in four genetically unrelated P. aeruginosa clinical isolates that were previously characterized by population analyses as heterogeneously resistant against carbapenems. Mutant subpopulations of all four isolates had at least fourfold higher minimum inhibitory concentrations than those of native cells for imipenem and meropenem. The heterogeneous subpopulations, when compared with the respective native ones, had significantly increased transcription levels of the mexB and mexY genes (P<0.05), whereas transcription levels of the mexE gene remained unchanged. They also exhibited significantly decreased expression of the oprD gene (P<0.05) and decreased intensity of the protein band of the porin OprD. Upregulation of efflux systems, in part, and the decrease of OprD contribute to the heterogeneous growth against carbapenems in our P. aeruginosa clinical isolates.

Hidden VIM-1 metallo-beta-lactamase phenotypes among Acinetobacter baumannii clinical isolates.

A total of 87 Acinetobacter baumannii nonrepetitive consecutive clinical isolates were tested for the presence of metallo-beta-lactamases (MBLs). Results of phenotypic assays (MBL Etest, imipenem/imipenem-EDTA combined-disk test, and imipenem/EDTA double-disk synergy test) were negative in all cases, but molecular testing revealed the presence of two bla(VIM-1)-carrying isolates. One isolate had bla(VIM-1) preceded by a weak P1 promoter, and both had inactivated P2 promoters and reduced bla(VIM-1) expression, partially justifying the results revealing hidden MBL phenotypes.

Molecular cloning and biochemical characterization of VIM-12, a novel hybrid VIM-1/VIM-2 metallo-beta-lactamase from a Klebsiella pneumoniae clinical isolate, reveal atypical substrate specificity.

Metallo-beta-lactamases (MBLs) are considered an emerging family of Zn2+-dependent enzymes that significantly contribute to the resistance of many nosocomial pathogens against beta-lactam antimicrobials. Since these plasmid-encoded enzymes constitute specific molecular targets for beta-lactams, their exact mode of action is greatly important in deploying efficient anti-infective treatments and for the control of severe multi-resistant nosocomial infections, which becomes a global problem. A novel hybrid VIM-1/VIM-2-type beta-lactamase (named VIM-12) has recently been identified in a clinical isolate of Klebsiella pneumoniae in Greece. The sequence of this enzyme is highly similar with that of VIM-1 at its N-terminal region and with that of VIM-2 at its C-terminal region, raising the question of whether this sequence similarity reflects also a similar functional role. Moreover, the possible contribution of this novel beta-lactamase to the overall antibiotic resistance of this specific clinical isolate was investigated. The gene encoding VIM-12 was cloned and expressed, and the recombinant enzyme was used for detailed kinetic analysis, using a variety of beta-lactam antibiotics. VIM-12 was found to exhibit narrow substrate specificity, compared to other known beta-lactamases, limited mainly to penicillin and to a much lesser extent to imipenen. Interestingly, meropenem was found to act as a noncompetitive inhibitor of the enzyme, although the active site of VIM-12 exhibited complete conservation of residues among VIM enzymes. We conclude that VIM-12 represents a novel and unique member of the family of known metallo-beta-lactamases, exhibiting atypical substrate specificity.