Abnormal vaginal flora in symptomatic non-pregnant and pregnant women in a Greek hospital: a prospective study.

Bacterial vaginosis (BV), candidiasis, and trichomoniasis were the three established types of vaginal conditions until aerobic vaginitis (AV) was defined in the early 2000s. We sought to study the prevalence of abnormal vaginal flora (AVF) with inflammation in our hospital and to correlate it with AV. We prospectively collected vaginal smear specimens originated from symptomatic women who were examined at Iaso Obstetrics, Gynecology and Children's Hospital of Athens from April 2014 until September 2015. Amsel's criteria were used for the diagnosis of BV. The presence of leukocytes and lactobacillary grade were evaluated to classify a condition as AVF with inflammation; subsequently, bacterial cultures were performed. A total of 761 women were included. Five hundred and seventy-nine women were diagnosed with candidiasis, BV, trichomoniasis, or other types of vaginitis in which no pathogenic bacterial growth occurred in cultures. One hundred and eighty-two women (23.9 %) were diagnosed with AVF with inflammation (116 non-pregnant, 66 pregnant). Escherichia coli was the most common pathogen among these women (non-pregnant: 45.7 %, pregnant: 34.8 %). Other common pathogens were Group-B-Streptococcus (non-pregnant: 20.7 %, pregnant: 22.7 % respectively), Enterococcus faecalis (14.7 %, 18.2 %), and Klebsiella pneumoniae (6.9 %, 12.1 %). The prevalence of AVF with inflammation may be high. Since inflammation criteria were applied, most cases of BV were eliminated and the majority of cases of AVF are AV. Therefore, clinicians should include AV in the differential diagnosis of vaginitis, while microbiologists should take into account the growth of aerobic bacteria in vaginal cultures originating from women with microscopic findings of AV.

Differences in the epidemiology between paediatric and adult invasive Streptococcus pyogenes infections.

In order to investigate for possible differences between paediatric and adult invasive Streptococcus pyogenes (iGAS) infections, a total of 142 cases were identified in 17 Greek hospitals during 2003-2007, of which 96 were children and 46 adults. Bacteraemia, soft tissue infections, streptococcal toxic shock syndrome (STSS), and necrotizing fasciitis were the main clinical presentations (67·6%, 45·1%, 13·4%, and 12·0% of cases, respectively). Bacteraemia and lymphadenitis were significantly more frequent in children (P=0·019 and 0·021, respectively), whereas STSS was more frequent in adults (P=0·017). The main predisposing factors in children were varicella and streptococcal pharyngotonsillitis (25% and 19·8%, respectively), as opposed to malignancy, intravenous drug abuse and diabetes mellitus in adults (19·6%, 15·2% and 10·9%, respectively). Of the two dominant emm-types, 1 and 12 (28·2% and 8·5%, respectively), the proportion of emm-type 12 remained stable during the study period, whereas emm-type 1 rates fluctuated considerably. Strains of emm-type 1 from children were associated with erythromycin susceptibility, STSS and intensive-care-unit admission, whereas emm-type 12 isolates from adults were associated with erythromycin and clindamycin resistance. Finally, specific emm-types were detected exclusively in adults or in children. In conclusion, several clinical and epidemiological differences were detected, that could prove useful in designing age-focused strategies for prevention and treatment of iGAS infections.

Sequence of pNL194, a 79.3-kilobase IncN plasmid carrying the blaVIM-1 metallo-beta-lactamase gene in Klebsiella pneumoniae.

The nucleotide sequence of pNL194, a VIM-1-encoding plasmid, is described in this study. pNL194 (79,307 bp) comprised an IncN-characteristic segment (38,940 bp) and a mosaic structure (40,367 bp) including bla(VIM-1), aacA7, aadA1, aadA2, dfrA1, dfrA12, aphA1, strA, strB, and sul1. Tn1000 or Tn5501 insertion within fipA probably facilitated recruitment of additional mobile elements carrying resistance genes.

GES-13, a beta-lactamase variant possessing Lys-104 and Asn-170 in Pseudomonas aeruginosa.

GES-13 beta-lactamase, a novel GES variant possessing Lys-104 and Asn-170, was identified in Pseudomonas aeruginosa. bla(GES-13) was the single gene cassette of a class 1 integron probably located in the chromosome. GES-13 efficiently hydrolyzed broad-spectrum cephalosporins and aztreonam. Imipenem was a potent inhibitor of GES-13 but was not hydrolyzed at measurable rates.

Prevalence of emm types 1 and 12 from invasive Streptococcus pyogenes disease in Greece--results of enhanced surveillance.

Among a total of 101 isolates from the first systematic multicentre surveillance effort concerning invasive Streptococcus pyogenes disease in Greece, conducted between 2003 and 2005 and covering 38% of the population, emm types 1 and 12 were prevalent, being responsible for 27 and nine cases, respectively. The isolates from the remaining 65 cases were assigned to 26 other emm types. Erythromycin resistance (12 isolates) was primarily mef(A)-mediated, although all emm type 1 strains were susceptible. Tetracycline resistance, due mostly to tet(M), was detected in 26 isolates. Subtyping by pulsed-field gel electrophoresis yielded 50 chromosomal fingerprints, thus discriminating further among ten of the 28 observed emm types.

Serologic prevalence of coxsackievirus group B in Greece.

Coxsackieviruses are human enteroviruses, which have been associated with myocarditis/pericarditis and sudden death. In one investigation (Spanakis N, Manolis EN, Tsakris A, Tsiodras S, Panagiotopoulos T, Saroglou G, and Legakis NJ: J Clin Pathol 2005;58:357-360), a cluster of cases of fatal myocarditis in Greece was linked to coxsackievirus B3. The information from this investigation prompted us to study serologically the prevalence of coxsackieviruses B throughout Greece. Sera were obtained from 506 healthy blood donors from various transfusion centers, covering the entire country. All sera were tested for the presence of IgG and IgM antibodies, using ELISAs with various antigenic specificities: (1) heat-denatured coxsackievirus type B1 and B5 virions, (2) a synthetic peptide from the N terminus of the VP1 protein of coxsackievirus B3, and (3) a synthetic peptide from the N terminus of the VP1 protein of coxsackievirus B4. Sera positive for IgG antibodies against coxsackieviruses B1/B5, B3, and B4 were detected in 6.7 to 21.6% of the individuals tested in the various regions of Greece. Statistical analysis revealed that the highest prevalence of IgG antibodies against coxsackieviruses B1/B5 was found in blood donors from Crete (p = 0.025), whereas the highest prevalence against coxsackievirus B4 was detected in blood donors from Athens (p = 0.01). IgM antibodies against coxsackievirus B were detected at low percentage, less than 5%, with no significant viral preference for particular geographic regions. The preference of anti-coxsackievirus IgG antibodies for particular geographic regions could be potentially related to the previously reported clustering of cases of insulin-dependent diabetes mellitus and myocarditis in Athens and Crete, respectively.

Serotypes and antimicrobial susceptibilities of 1033 pneumococci isolated from children in Greece during 2001-2004.

Pneumococci (n = 1033) isolated in the major paediatric hospitals of Athens during 2001-2004 from children with invasive infections (n = 186), non-invasive infections (n = 641) and healthy carriers (n = 206) were studied. The most prevalent serotypes were serotypes 14 (44.6%), 19F (43.5%) and 6B (22.8%) in invasive, non-invasive and carriage isolates, respectively. Among invasive isolates, the potential coverage by the seven-valent conjugate vaccine was 75.3%. Resistance rates to penicillin, amoxycillin, cefotaxime, erythromycin, co-trimoxazole, clindamycin, tetracycline and chloramphenicol were 44.6%, 2.7%, 1.2%, 43.6%, 43.5%, 12.4%, 34.7% and 5.9%, respectively. The M-phenotype accounted for 68.0% of the erythromycin-resistant isolates. All isolates were susceptible to ofloxacin.

Outbreak of multiple clones of imipenem-resistant Acinetobacter baumannii isolates expressing OXA-58 carbapenemase in an intensive care unit.

OBJECTIVES: To investigate the resistance mechanisms and the genetic relationship of imipenem-resistant Acinetobacter baumannii isolates recovered in the intensive care unit (ICU) of a tertiary care hospital. METHODS: Imipenem-resistant A. baumannii clinical and environmental isolates were collected in the ICU of the Red Cross General Hospital, Athens, Greece between March and October 2002. The isolates were tested by Etest MBL, PCR, RT-PCR and sequencing for carbapenemase-encoding genes, PFGE and synergy experiments using meropenem and the efflux pump inhibitor carbonyl cyanide chlorophenylhydrazone. RESULTS: During the study period, 15 clinical and two environmental imipenem-resistant (MIC 8 to >128 mg/L) A. baumannii isolates were recovered. PFGE showed six different clones that included both clinical and environmental isolates. All 17 isolates were negative by Etest MBL and PCR for genes bla(IMP), bla(VIM), bla(SPM), bla(OXA-23-like) and bla(OXA-24-like). Genes bla(OXA-51-like) and bla(OXA-58-like) were amplified from 15 and 14 isolates, respectively. Sequencing of bla(OXA-51-like) amplicons identified bla(OXA-66) (nine cases) and bla(OXA-69) (six cases), whereas bla(OXA-58-like) sequences were classical bla(OXA-58). Reverse transcriptase-PCR showed that bla(OXA-51-like) genes were expressed in 12 and bla(OXA-58) in 10 isolates; in these isolates, inhibition of OXA enzymes by 200 mM of NaCl reduced carbapenem MICs by up to 4-fold. Overexpression of proton-gradient dependent efflux pumps did not contribute to carbapenem resistance in any isolate. Similarly, although AmpC expression was demonstrated in eight isolates, inhibition of AmpC with cloxacillin did not reduce the MICs of carbapenems significantly. CONCLUSIONS: These findings indicate wide dissemination of OXA-58 carbapenemase, which contributes, at least partially, to the imipenem resistance of unrelated A. baumannii isolates in our ICU.

Multicentre survey of the in-vitro activity of seven antimicrobial agents, including ertapenem, against recently isolated Gram-negative anaerobic bacteria in Greece.

The in-vitro activities of penicillin, ticarcillin-clavulanic acid, cefoxitin, imipenem, ertapenem, metronidazole and clindamycin were evaluated against 138 Gram-negative anaerobic isolates (82 Bacteroides fragilis group, 17 non-fragilis Bacteroides spp., 31 Prevotella spp., four Fusobacterium spp., two Veillonella spp., one Porphyromonas sp. and one Tissierella praeacuta) collected from six general hospitals in Athens, Greece. Overall rates of non-susceptibility (both resistant and intermediately-resistant) to penicillin and ticarcillin-clavulanic acid were 81.8% and 2.3%, respectively. The rates of non-susceptibility to cefoxitin and clindamycin were 30.3% and 31.1%, respectively, and that for metronidazole was 4.3% (four Prevotella spp. isolates, one Porphyromonas sp. isolate and one B. fragilis isolate). Only the single B. fragilis isolate was nim-positive by PCR. Only one B. fragilis isolate was resistant to both carbapenems tested, while six more Bacteroides spp. isolates were imipenem-susceptible and ertapenem-non-susceptible. The MIC range, MIC(50) and MIC(90) values were comparable for imipenem and ertapenem, although ertapenem MIC(90)s were one or two two-fold dilutions higher.

Spread of efflux pump-overexpressing, non-metallo-beta-lactamase-producing, meropenem-resistant but ceftazidime-susceptible Pseudomonas aeruginosa in a region with blaVIM endemicity.

OBJECTIVES: To investigate the resistance mechanisms of meropenem-resistant, ceftazidime-susceptible Pseudomonas aeruginosa isolates, in a clinical setting where VIM-2 or VIM-4 metallo-beta-lactamase (MBL)-producing pseudomonads are common. METHODS: During May to December 2003, 13 consecutive meropenem-resistant, ceftazidime-susceptible P. aeruginosa isolates were recovered from separate patients at the University Hospital of Larissa, Thessaly, Greece. The isolates were studied by Etest MBL, PCR for blaVIM, blaIMP and blaSPM genes and PFGE. Experiments were performed to detect synergy between meropenem or other antimicrobials and the efflux pump inhibitor carbonyl cyanide-m-chlorophenylhydrazone (CCCP). The isolates were also tested by PCR and RT-PCR for the expression of the genes mexB and mexY, which encode the efflux pumps MexAB-OprM and MexXY-OprM. RESULTS: Twelve of the isolates, belonging to six distinct PFGE types, gave negative results in the MBL Etest and lacked genes encoding MBLs but exhibited synergy between meropenem and CCCP, indicating that efflux pump activity contributed to the meropenem resistance. All 12 isolates were positive for mexB and 11 were also positive for mexY genes. RT-PCR showed that 10 and five isolates over-expressed mexB and mexY, respectively. One isolate was blaVIM-2-positive and did not show synergy with CCCP, or harbour mexB or mexY. CONCLUSIONS: In our hospital, where MBL-producing P. aeruginosa were previously prevalent, meropenem resistance due to the overexpression of efflux pumps has also now emerged. Early recognition of this resistance mechanism should allow the use of alternative beta-lactams, such as ceftazidime, which would be inactive even against phenotypically susceptible MBL producers.

Repeated occurrence of diverse extended-spectrum beta-lactamases in minor serotypes of food-borne Salmonella enterica subsp. enterica.

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.

Coxsackievirus B3 sequences in the myocardium of fatal cases in a cluster of acute myocarditis in Greece.

AIM: The investigation of three fatal cases during a nationwide cluster of cases of an upper respiratory tract infection (URTI) associated with myocarditis and/or pericarditis in Greece in 2002. METHODS: In the three women who died, necropsies were performed and tissue sections were taken for histological examination, antigen detection by immunohistochemistry and indirect immunofluorescence assay (IFA), amplification of viral genomes by nested reverse transcription polymerase chain reaction (RT-PCR), and sequence analysis. RESULTS: All samples showed histological signs of active myocarditis. Immunohistochemistry revealed the presence of the enterovirus VP1 family of proteins and IFA revealed the presence of coxsackievirus B3 antigen. Nested RT-PCR amplified enteroviral alleles of the 5'-untranslated region which were identical to each other and to the coxsackievirus B3 sequences. CONCLUSIONS: This study provides pathological evidence of enteroviral infection among fatal myocarditis cases in a nationwide URTI cluster of cases associated with myocarditis and/or pericarditis.

Chlamydia trachomatis infections in Greece: first prevalence study using nucleic acid amplification tests.

The present retrospective study was initiated to determine the prevalence of Chlamydia trachomatis and to assess the risk factors for infection in adult women and men presenting to general practitioners, gynecologists, dermatologists, and family-planning centers in Greece. The study was carried out in four different Greek hospital centers using highly sensitive nucleic acid amplification techniques. Altogether, 16,834 women and 1,035 men were enrolled from October 1998 to April 2004. Two types of specimens were collected from each patient: cervical swabs from women, urethral swabs from men, and first-catch urine from women and men. All specimens were examined with the Cobas Amplicor C. trachomatis polymerase chain reaction assay (Roche Molecular Systems, Branchburg, NJ, USA) or the LC x C. trachomatis ligase chain reaction assay (Abbott Laboratories, Abbott Park, IL, USA). Demographic and behavioral data were collected by clinicians using a standardized questionnaire. A total of 704 (3.9%) patients were infected with C. trachomatis. The prevalence among female patients was 3.5% and that among male patients 11.2%. Among infected patients, 88% were under 30 years of age, 71% reported more than one sexual partner, and 91% reported a new sexual partner within the last year. In conclusion, the prevalence of C. trachomatis infection in Greece is low. Young age and new and multiple sexual partners within the last year were factors consistently associated with an increased risk of chlamydial infection.

Discrepancies and interpretation problems in susceptibility testing of VIM-1-producing Klebsiella pneumoniae isolates.

Susceptibilities to beta-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.

Investigation of possible cytokine induction in peripheral blood mononuclear cells by heat-stable serotypes of Campylobacter jejuni.

Several Campylobacter jejuni heat-stable (HS) serotypes have been associated with the autoimmune Guillain-Barre neurological syndrome (GBS). In order to examine the possible involvement of cytokines in this phenomenon, the levels of three pro-inflammatory cytokines (interleukin (IL)-2sRa, IL-6 and interferon (IFN)-gamma) and one anti-inflammatory cytokine (IL-10) were measured in peripheral blood mononuclear cells after induction by different C. jejuni serotypes. No differences were found for IL-6, IFN-gamma and IL-10, but the non-sialylated serotype HS:3 was associated with decreased production of IL-2sRa. The results raise the possibility that absence of sialylation might be associated with the inability to induce inflammatory factors such as cytokines.

Expanded-spectrum cephalosporin resistance in non-typhoid Salmonella.

Expanded-spectrum cephalosporins (ESCs) such as ceftriaxone, together with fluorinated quinolones, are the choice antibiotics in the treatment of invasive salmonella infections. Resistance to ESCs among non-typhoid salmonella has been recognised since the late 1980s. Currently, ESC-resistant salmonella strains are reported world-wide and in some areas their incidence is significant. Resistance is mainly due to acquisition of multi-resistant plasmids encoding a variety of extended-spectrum and AmpC-type beta-lactamases. The origins of ESC-resistant salmonellae are diverse. Exchange of resistance determinants between salmonellae and nosocomial enterobacteria seems to be frequent, at least in developing countries. Also, the use of newer beta-lactams in animal husbandry and veterinary medicine may have facilitated the spread of ESC-resistant salmonella strains in livestock.