Search

ABSTRACTGenetic and antigenic characterization of 37 representative influenza A(H3N2) virus strains isolated in Greece during the 2011-2012 winter season was performed to evaluate matching of the viruses with the seasonal influenza vaccine strain A/Perth/16/2009. Hemagglutinin gene sequence analysis revealed that all Greek strains clustered within the Victoria/208 genetic clade. Furthermore, substitutions in the antigenic and glycosylation sites suggested potential antigenic drift. Our hemagglutination inhibition (HI) analysis showed that the Greek viruses were Perth/16-like; however, these viruses were characterized as Victoria/208-like when tested at the United Kingdom WHO Collaborating Centre (CC) with HI assays performed in the presence of oseltamivir, a finding consistent with the genetic characterization data. Variability in the HI test performance experienced by other European laboratories indicated that antigenic analysis of the A(H3N2) virus has limitations and, until its standardization, national influenza reference laboratories should include genetic characterization results for selection of representative viruses for detailed antigenic analysis by the WHO CCs.

ABSTRACTPhylogenetic analysis of 166 human parvovirus B19 sequences from 11 different countries attributed 91.57% to genotype 1, 5.42% to genotype 3b, and 3.01% to genotype 3a. Very similar viruses of genotype 1 circulated widely in Europe and Israel. Genotype 3b seems to show an increasing spread outside of Africa.

ABSTRACTThe presence of various numbers of EPIYA tyrosine phosphorylation motifs in the CagA protein of Helicobacter pylorihas been suggested to contribute to pathogenesis in adults. In this prospective study, we characterized H. pyloriisolates from symptomatic children, with reference to the diversity of functional EPIYA motifs in the CagA protein and vacAisotypes, and assessed the potential correlation with the histopathological manifestations of the infection. We analyzed 105 H. pyloriisolates from 98 children and determined the diversity of EPIYA motifs in CagA by amplification and sequencing of the 3' variable region of the cagAgene as well as vacAisotypes for the signal, middle, and intermediate regions. CagA phosphorylation and levels of secreted IL-8 were determined following in vitro infection of AGS gastric epithelial cells. Histopathological evaluation of H. pyloricolonization, activity, and severity of the associated gastritis was performed according to the updated Sydney criteria. EPIYA A (GLKN[ST]EPIYAKVNKKK), EPIYA B (Q[V/A]ASPEPIY[A/T]QVAKKVNAKI), and EPIYA C (RS[V/A]SPEPIYATIDDLG) motifs were detected in the ABC (46.6%) and ABCC (17.1%) combinations. No isolates harboring more than two EPIYA C motifs in CagA were found. The presence of isogenic strains with variable numbers of CagA EPIYA C motifs within the same patient was detected in seven cases. Occurrence of increasing numbers of EPIYA C motifs correlated strongly with presence of a high-vacuolation (s1 or s2/i1/m1) phenotype and age. A weak positive correlation was observed between vacuolating vacAgenotypes and presence of nodular gastritis. However, CagA- and VacA-dependent pathogenicities were not found to contribute to severity of histopathology manifestations in H. pylori-infected children.

ABSTRACTCytotoxin-associated gene A (CagA) diversity with regard to EPIYA-A, -B, -C, or -D phosphorylation motifs may play an important role in Helicobacter pyloripathogenesis, and therefore determination of these motifs in H. pyloriclinical isolates can become a useful prognostic tool. We propose a strategy for the accurate determination of CagA EPIYA motifs in clinical strains, based upon one-step PCR amplification using primers that flank the EPIYA coding region. We thus analyzed 135 H. pyloriisolates derived from 75 adults and 60 children Greek patients. A total of 34 cases were found to be EPIYA PCR negative and were consequently verified as cagAnegative by cagA-specific PCR, empty-site cagAPCR, and Western blotting. Sequencing of the remaining 101 PCR-positive amplicons confirmed that an accurate prediction of the number of EPIYA motifs on the basis of size distribution of the PCR products was feasible in all cases. Furthermore, our assay could identify closely related H. pylorisubclones within the same patient, harboring different numbers of EPIYA repeats. The prevalence of CagA proteins with three EPIYA motifs (ABC) or four EPIYA motifs (ABCC) was the same within the adult and children groups. However, CagA species with more than four EPIYA motifs were observed exclusively within adults (8.6%), suggesting that CagA-positive strains may acquire additional EPIYA-C motifs throughout adulthood. Our strategy requires no initial cagAscreening of the clinical isolates and can accurately predict the number of EPIYA repeats in single or multiple closely related subclones bearing different numbers of EPIYA motifs in their CagA, which may coexist within the same patient.

ABSTRACTA total of 309 Helicobacter pyloriisolates from 18 different countries were analyzed with a previously developed lectin typing system. The system was developed by using a proteolytic pretreatment to enhance the carbohydrate fraction of the sample. Four lectins from Ulex europaeus, Lotus tetragonolobus, Erythrina cristigali, and Triticum vulgariswere used to type the strains. The lectins were chosen for their specificities for sugars commonly encountered in the lipopolysaccharide of H. pylori. The isolates were received from their parent institutions as pellets of biomass and were typed at one of three centers (in Ireland, Sweden, and Estonia). All 16 possible lectin reaction patterns were observed in the study, with the isolates with the predominant pattern exhibiting reactions with all the lectins in the panel. For European patients suffering from gastritis, an association was noted between lectin reaction pattern MH4 and atrophic chronic gastritis; isolates with lectin reaction pattern MH4 were isolated from patients with atrophic chronic gastritis, whereas isolates with this pattern were not isolated from patients with chronic gastritis (P= 0.0006). In addition, statistically significant relationships were noted between the lectin reaction pattern and the associated pathology of isolates from the Swedish population. Isolates with patterns MH13 and MH16, which had low lectin reactivities, correlated with nonulcer disease (P= 0.0025 and P= 0.0002, respectively), and all four isolates from adenocarcinoma patients were characterized as possessing reaction pattern MH16. In contrast, isolates with lectin reaction patterns MH1 and MH10, which had high lectin reactivities, were associated with ulcer disease (P= 0.046 and P= 0.0022, respectively).

Summary 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 ß-lactamase. In the present study the activity of cefpirome against a number of enterobacteria (Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Enterobacter aerogenes andSerratia 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 ß-lactamase due to its hydrolysis by the enzyme. Also cefpirome exhibited good activity againstE. aerogenes strains with reduced susceptibility to imipenem. Thesein vitro data suggest that cefpirome might be useful in treating infections caused by these resistant microorganisms that are frequently encountered in Greek hospitals.

The in vitro binding of four Helicobacter pylori strains to human gastric mucin was studied with an enzyme-linked immunosorbent assay. All four strains were found to bind to purified mucin. Neuraminidase treatment and nonspecific oxidation of mucin decreased bacterial adherence to the macromolecule. Mucin preparations were also found to inhibit attachment of H. pylori to HEp-2 monolayers.

The nucleotide sequence of the gene encoding a novel cephalosporinase (LAT-1), carried by a non-self-transferable plasmid from Klebsiella pneumoniae, has been determined. It was found that the sequence shares a high degree of homology with the Citrobacter freundii OS60 ampC structural gene.

An SHV type β-lactamase frequently found in enterobacteria isolated in Greek hospitals was analyzed. The enzyme (SHV-5a) conferred resistance to ceftazidime and aztreonam. The DNA sequence of the structural gene was determined. The deduced amino acid sequence showed that positions 70–73 were occupied by the active site tetrad Ser-Thr-Phe-Lys. As in SHV-5, Ser-238 and Lys-240 were present. However, one deletion (Gly-54) and three substitutions (Arg-140 for Ala, Asn-192 for Lys and Val-193 for Leu) differentiate SHV-5a β-lactamase from SHV-5. Asn-192 and Val-193 have been reported to date only in the R974 plasmid-mediated SHV-1 β-lactamase. Hydrolysis studies with SHV-5a and SHV-5 showed that the enzymes behaved similarly. Additional evidence that they were functionally indistinguishable was provided by the similar MICs of β-lactams when the enzymes were expressed under isogenic conditions. The sequence differences, however, indicate that they are derived from different ancestors.