No abstract

We investigated the molecular epidemiology of extended spectrum β-lactamase (ESBL) producing Klebsiella pneumoniae isolates derived from the teaching hospitals of University of Pécs, Pécs, Hungary in the time period [2004][2005][2006][2007][2008]. Molecular typing, antimicrobial susceptibility testing, detection of common β-lactamase genes (bla CTX-M , bla TEM and bla SHV ) and virulence associated traits (hypermucoviscosity, magA, k2a, rmpA, siderophores, type 1 and 3 fi mbria, biofi lm formation, serum resistance) were performed for 102 isolates. The results showed the presence of three major ciprofl oxacin resistant CTX-M-15 producing clones (ST15 n = 69, ST101 n = 10, and ST147 n = 9), of which ST15 was predominant and universally widespread. Considering distribution in time and place, ST101 and ST147 were detected at fewer inpatient units and within a narrower time frame, as compared to ST15. Beside major clones, eleven minor clones were identifi ed, and were shown to harbour the following β-lactamase genes: six clones carried bla CTX-M , four clones harboured bla SHV-5 and one clone possessed both bla CTX-M and ESBL type bla SHV . Among the SHV-5 producing K. pneumoniae clones a novel sequence type was found, namely ST1193, which harboured a unique infB allele. Different virulence factor content and peculiar antimicrobial susceptibility profi le were characteristic for each clone. In contrast to major clone isolates, which showed high level resistance to ciprofl oxacin, minor clone *Corresponding author; E-mail: melegh.szilvia@pte.hu 234 MELEGH et al.Acta Microbiologica et Immunologica Hungarica 62, 2015 isolates displayed signifi cantly lower MIC values for ciprofl oxacin suggesting a role for fl uoroquinolones in the dissemination of the major K. pneumoniae clones. This is the fi rst description of the CTX-M-15 producing K. pneumoniae clone ST101 in Hungary.

The interaction of laminin (Lm), a basement membrane protein abundant in the periodontium, with 66 strains of Prevotella intermedia isolated from diseased pockets, was tested in a 125I-labeled protein binding assay. The mean binding value was 28% of the total protein added. The binding significantly increased to 35% when the environmental pH decreased from 7 to 6. The Lm interaction was characterized in a highly binding (about 65%) strain, OMGS105. The binding was rapid and required about 1 min and 1-2 h for 50% and 100% equilibrium respectively. The 125I-Lm binding was maximum in the pH interval 3.0 to 6.5 and could not be displaced by unlabeled Lm or inhibited by other proteins and carbohydrates. The interaction was stable in the presence of NaCl or urea (concentrations up to 4 M) but was dissociated by > or = 1 M KSCN. The Lm-binding component was thermolabile and sensitive to proteolytic enzymes. Sodium dodecylsulfate-polyacrylamide gel electrophoresis and Western blot analysis revealed a approximately 62 kDa Lm-binding protein, both in the whole cell extract and the outer membrane preparation. Weaker binding was also observed to other proteins. These data establish the ability of P. intermedia to interact with Lm via certain cell surface proteins, a property that might contribute to the colonization of this bacterium in the periodontal pocket.

The ability of Shigelia flexneri to interact with lactoferrin (Lf) was examined with a '251-labeled proteinbinding assay. The percent binding of human lactoferrin (HLf) and bovine lactoferrin (BLf) to 45 S. flexneri strains was 19 ± 3 and 21 ± 3 (mean ± standard error of the mean), respectively. '25I-labeled HLf and BLf binding to strain M9OT reached an equilibrium within 2 h. Unlabeled HLf and BLf displaced the 251-HLf-bacteria interaction in a dose-dependent manner. The Lf-bacterium complex was uncoupled by KSCN or urea, but not by NaCl. The interaction was specific, and -4,800 HLf binding sites (affinity constant [Kal, 690 nM) or -5,700 BLf binding sites (Ka, 104 nM) per cell were estimated in strain M9OT by a Scatchard plot analysis. The native cell envelope (CE) and outer membrane (OM) did not reveal Lf-binding components in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, after being boiled, the CE and OM preparations showed three distinct horseradish peroxidase-Lf reactive bands of about 39, 22, and 16 kDa. The 39-kDa component was also reactive to a monoclonal antibody specific for porin (Pol) proteins of members of the family Enterobacteriaceae. The Lf-binding protein pattern was similar with BLf or HLf, for Crb+ and Crbstrains. The protein-Lf complex was dissociable by KSCN or urea and was stable after treatment with NaCl.Variation (loss) in the 0 chain of lipopolysaccharide (LPS) markedly enhanced the Lf-binding capacity in the isogenic rough strain SFL1070-15 compared with its smooth parent strain, SFL1070. These data establish that Lf binds to specific components in the bacterial OM; the heat-modifiable, anti-Pol-reactive, and LPS-associated properties suggested that the Lf-binding proteins are porins in S. flexneri.

Shigella sonnei colicin 7 (Scol7) is a unique bacteriocin acting only on certain dysentery-causing bacteria, like enteroinvasive Escherichia coli, S. sonnei or S. boydii. We identified a 4.2 Md plasmid (pScol7) conferring Scol7 production to the transformants. The entire plasmid was sequenced (Gene Bank Accession number AJ318075) and the structure gene of Scol7 production (sc7a) was identified. Analyzing the sequence of the plasmid revealed extensive homology to other colicin plasmids, particularly to pColE1 but only in areas not related to the bacteriocin activity gene. The similarity of the putative promoter for sc7a to the respective sequences of other colicins suggested that the production of Scol7 is under SOS regulation. Indeed, its production could be increased eightfold by mitomycin C treatment. The molecular mass of the translated polypeptide as deduced from the nucleotide sequence of sc7a (i.e. 11.2 kDa) is in good agreement with previous estimations for its subunit, but molecular filtration experiments suggest a multimeric structure of at least 50 kDa. While current data are not sufficient to predict the mode of action of Scol7, the presence of a DTLSN pentapeptide motive suggests that it could be imported to sensitive cells via the TonB transport system.