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.

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.

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.

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.

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.

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.

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.

The effect of ionizing irradiation on the lipid biosynthesis by thymus and liver.

New Zealand rabbits, fasted for 12 hours, were subjected to 500 rads of whole-body irradiation. Analysis of thymus lipids, at various time intervals following irradiation, showed a threefold increase of triglycerides at 24 hours. Fatty acid composition of the 600 X g supernatant was not affected at 24 hours after irradiation. Lipid biosynthesis from acetate-1-14C by the thymus homogenates was increased to a small extent at 4 hours following irradiation, while the radioactivity distribution into fatty acids was not considerably affected. Contrary to the above findings, fatty acid synthesis from acetate-l-14C by the liver preparations showed a decreased incorporation between the fourth and twelfth hour following irradiation. Counting of the radioactivity of the separated fatty acids suggested that the system for synthesis of short-chain fatty acids was impaired as early as 4 hours following irradiation.

The effect of ionizing irradiation on the oxidation of palmitate-1-14C by thymus and liver.

New Zealand rabbits, fasted for 12 hours, were subjected to 500 rads of whole-body irradiation. K+-palmitate-1-14C oxidation was assayed with the 600 X g supernatant of thymus and liver homogenates, in the presence of ATP, at various time intervals from irradiation. For a period of 24 hours following irradiation, oxidation by liver preparations was not significantly affected. The rate of oxidation by thymus was decreased to less than one-third of the control value within 12 hours from irradiation and, at 24 hours, was almost completely abolished. Increased ATP concentration could increase only to a small extent the oxidation by thymus preparations of irradiated animals. Oxidation by isolated thymus mitochondria was also inhibited by irradiation. Counting of the water-soluble oxidation products of palmitate-1-14C suggests that the inhibition is not due to the impairment of the reactions of the citric acid cycle. The non-esterified fatty acid concentration of thymus was not altered at 12 hours following irradiation. Esterification of K+-palmitate-l-14C into the thymus lipids was not affected 12 hours after irradiation.

Prevalent mechanisms of resistance among common enterobacterial isolates in Greek hospitals.

The recent data concerning antibiotic resistance of the enterobacteria isolated in Greek hospitals are reviewed. A variety of mechanisms of resistance, clustered in most of the cases, was observed. Epidemics of plasmids were responsible for dissemination of third-generation cephalosporins, aminoglycosides, and trimethoprim resistance among Klebsiella pneumoniae and, to a lesser extent, Escherichia coli isolates. Stable depression of the expression of chromosomal cephalosporinase is the main cause of resistance to third-generation cephalosporins observed at high frequencies in Enterobacter spp. strains.

CTX-m-3 beta-lactamase-producing Escherichia coli from Greece.

An Escherichia coli clinical strain resistant to all beta-lactams except carbapenems was isolated in a Greek hospital. Analysis of beta-lactamase content by isoelectric focusing, PCR assays specific for various bla genes, and DNA sequencing showed that the strain produced TEM-1, a Citrobacter freundii AmpC-related cephalosporinase, and CTX-M-3. The blaCTX.M-3 gene was carried by a 120-kb plasmid that was readily transferable to a susceptible E. coli host.

Evaluation of an anti-PBP 2a slide latex agglutination test in coagulase-negative staphylococci isolated in Greek hospitals.

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 mecA-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%).

Effect of substitution of Asn for Arg-276 in the cefotaxime-hydrolyzing class A beta-lactamase CTX-M-4.

The effect of substitution of asparagine for arginine at position 276 (Ambler's numbering) on the properties of the extended-spectrum beta-lactamase CTX-M-4 was studied. Compared with CTX-M-4, the mutant beta-lactamase CTX-M-4(R276N) conferred lower levels of resistance to cefotaxime, ceftriaxone and aztreonam while the levels of resistance to penicillins and penicillin-inhibitor combinations were similar. Arg-276-->Asn substitution rendered CTX-M-4 slightly less susceptible to inhibition by clavulanate and tazobactam. It also caused a three-fold reduction in the relative rate of hydrolysis of cefotaxime. These results indicate that Arg-276 in CTX-M-type beta-lactamases may be implicated in hydrolysis of oxyimino-beta-lactams; they do not, however, support the hypothesis that Arg-276 is the functional equivalent of Arg-244 found in other class A beta-lactamases.

Substitution of Arg-244 by Cys or Ser in SHV-1 and SHV-5 beta-lactamases confers resistance to mechanism-based inhibitors and reduces catalytic efficiency of the enzymes.

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 pronounced in the R244C 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.

Characterization of In111, a class 1 integron that carries the extended-spectrum beta-lactamase gene blaIBC-1.

A class 1 integron, In111, carried by a self-transferable plasmid from an Escherichia coli clinical strain was characterized. The variable region of In111 constituted an array of gene cassettes encoding the extended-spectrum beta-lactamase IBC-1, the aminoglycoside-modifying enzymes AAC(6')-Ib and ANT(3")-Ia, dihydrofolate reductase I and a putative polypeptide (SMR-2) sharing similarity with the Qac transporters. Transcription of the gene cassettes was driven by a hybrid-type P1 promoter located in a typical 5' conserved segment (CS). The 3'CS included sulI, qacEDelta1, orf5 and orf6. In111 was bounded on the right by an inversely oriented IRt. The 5'CS was preceded by an intact IS26 element followed by an aphA1 gene.

Two novel plasmid-mediated cefotaxime-hydrolyzing beta-lactamases (CTX-M-5 and CTX-M-6) from Salmonella typhimurium.

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 beta-lactamase inhibitors. The respective bla 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 beta-lactamases of Klebsiella oxytoca.

Differentiation of Serratia marcescens and Serratia liquefaciens by tests for lipase and phospholipase production.

The production of lipase and phospolipase by certain members of the Enterobacteriaceae was examined by thin-layer chromatography of resting-cell suspensions incubated with triolein or lecithin. Most strains of Serratia marcescens produced both enzymes while most strains of Serratia liquefaciens exhibited strong lipase but only a minor phospholipase activity. Enterobacter spp. (25 strains), Klebsiella pneumoniae (20 strains), Escherichia coli (15 strains), Citrobacter freundii (7 strains) and Proteus spp. (20 strains) lacked both types of enzymic activity except for the following: three strains of Enterobacter cloacae, two of Proteus mirabilis and three of Proteus vulgaris possessed slight lipase activity; about one-half of the Enterobacter aerogenes and Enterobacter hafniae strains examined produced slight phospholipase activity. It is suggested that tests for lipase and phospholipase should be used in conjunction with those for DNAase production and sugar fermentation for the differentiation of S. marcescens and S. liquefaciens.