The primary attachment to polymer surfaces followed by accumulation in multilayered cell clusters leads to biofilm production of Staphylococcus epidermidis, which is thought to contribute to virulence in biomaterialrelated infections. We purified a specific polysaccharide antigen of biofilm-producing S. epidermidis 1457 and RP62A, which was recently shown to have a function in the accumulative phase of biofilm production by mediating intercellular adhesion (D. Mack, M. Nedelmann, A. Krokotsch, A. Schwarzkopf, J. Heesemann, and R. Laufs, Infect. Immun. 62:3244-3253, 1994). Following Sephadex G-200 gel filtration, this antigen was separated by Q-Sepharose chromatography into a major polysaccharide, polysaccharide I (>80%), which did not bind to Q-Sepharose, and a minor polysaccharide, polysaccharide II (<20%), which was moderately anionic. As shown by chemical analyses and nuclear magnetic resonance spectroscopy, polysaccharide I is a linear homoglycan of at least 130 ␤-1,6-linked 2-deoxy-2-amino-D-glucopyranosyl residues. On average, 80 to 85% of them are N acetylated; the rest are non-N-acetylating and positively charged. Chain cleavage by deamination with HNO 2 revealed a more or less random distribution of the non-N-acetylated glucosaminyl residues, with some prevalence of glucosaminyl-rich sequences. Cation-exchange chromatography separated molecular species whose content of non-N-acetylated glucosaminyl residues varied between 2 and 26%. Polysaccharide II is structurally related to polysaccharide I but has a lower content of non-N-acetylated Dglucosaminyl residues and contains phosphate and ester-linked succinate, rendering it anionic. Enzyme-linked immunosorbent assay inhibition with various monosaccharides revealed the ␤-anomeric form and the acetylated amino group of the D-glucosaminyl residues as important for reactivity with the specific antiserum. The unbranched polysaccharide structure favors long-range contacts and interactions between polysaccharide strands and the cell wall and/or lectin-like proteins, leading to intercellular adhesion and biofilm accumulation. The structure of the polysaccharide is, so far, considered to be unique and, according to its function, is referred to as S. epidermidis polysaccharide intercellular adhesin (PIA).At present, coagulase-negative staphylococci, mostly Staphylococcus epidermidis, represent the most frequent cause by far of nosocomial sepsis and are the most prominent organisms responsible for infections associated with implanted biomaterials like intravascular catheters, peritoneal dialysis catheters, cerebrospinal fluid shunts, prosthetic heart valves, and prosthetic joints, resulting in substantial morbidity and mortality (25,37,54,56).By scanning electron microscopy, coagulase-negative staphylococci were shown to colonize intravascular catheters in large adherent biofilms composed of multilayered cell clusters embedded in an amorphous extracellular material, which is composed of exopolysaccharides referred to as slime or glycocalyx (13,16,28,45,51). In vitro...

The initial attachment and the accumulation of Staphylococcus epidermidis on polymer surfaces in multilayered cell clusters embedded in amorphous slime, which together lead to the plastic-adherent phenotype detected by the adherence assay used in this study, have been proposed to be major virulence factors of these bacteria. An antigen specific for plastic-adherent S. epidermidis strains was detected by an indirect immunofluorescence test using absorbed antiserum raised against the strongly plastic-adherent S. epidermidis 1457. A coagglutination assay was established, which allowed the quantitation of the antigen in bacterial extracts under different physiologic growth conditions. Expression of the antigen and of plastic adherence depended significantly on the presence of glucose in the growth medium. Parallel to increased plastic adherence, a 32- to 64-fold increase in the amount of the antigen was detected in bacterial extracts of cells grown in tryptone soya broth (TSB) compared with that in extracts of cells grown in TSB lacking glucose. A parallel time-dependent increase of plastic adherence and expression of the antigen was observed after stimulation by glucose of stationary-phase cultures of plastic-adherent S. epidermidis strains grown in TSB lacking glucose. The antigen consisted most probably of polysaccharide, because its immunologic reactivity was completely abolished by periodate oxidation but was resistant to protease digestion. A significant proportion of cells of plastic-adherent as compared with nonadherent S. epidermidis strains grown in TSB were located in large cell clusters exceeding 50 cells, which completely disintegrated after periodate oxidation of the cell preparations. Periodate oxidation of adherent bacterial films in situ led to release of the adherent cells from the plastic surface. These results strongly indicate a functional relation of the antigen to adherence of S. epidermidis to polymer surfaces, most probably by mediating intercellular adhesion of cells leading to accumulation in multilayered cell clusters.

The primary attachment to polymer surfaces followed by accumulation in multilayered cell clusters leads to production of Staphylococcus epidermidis biofilms, which are thought to contribute to virulence in biomaterialrelated infections. We isolated Tn917 transposon mutants of biofilm-producing S. epidermnidis 13-1, which were completely biofilm negative. In pulsed-field gel electrophoresis no obvious deletions of the mutants were noted. The Tn917 insertions of mutants M10 and Mll were located on different EcoRI fragments but on identical 60-kb SmaI and 17-kb BamHI chromosomal fragments. Linkage of transposon insertions of mutants M10 and Mll with the altered phenotype was demonstrated by phage transduction, whereas the several other mutants apparently represented spontaneous variants. In a primary attachment assay with polystyrene spheres, no significant difference between any of the mutants and the wild type could be detected. Cell clustering as an indication of intercellular adhesion, which is a prerequisite for accumulation in multilayered cell clusters, was not detected with any mutant. These results demonstrate that the mutants were impaired in the accumulative phase of biofilm production. Mutants Mi0 and Mll did not produce detectable amounts of a specific polysaccharide antigen (D. Mack, N. Siemssen, and R. Laufs, Infect. Immun. 60:2048-2057, 1992), whereas substantially reduced amounts of antigen were produced by the spontaneous variants. Hexosamine was determined as the major specific component of the antigen enriched by gel filtration of biofilm-producing S. epidermidis 1457 because almost no hexosamine was detected in material prepared from the isogenic biofilm-negative transductant 1457-Mll, which differentiates the antigen from other S. epidermidis polysaccharide components. Our results provide direct genetic evidence for a function of the antigen in the accumulative phase of biofilm production by S. epidermidis by mediating intercellular adhesion.

Mobilizable virulence plasmids of Yersinia enterocolitica of serotypes 0:3 and 0:9 were constructed by cointegration of a mobilizable vector into the virulence plasmids. The obtained cointegrates were mobilized into plasmidless Y. enterocolitica strains of serotypes 0:3, 0:5, 0:8, and 0:9. The transfer experiments revealed the existence of two different subgroups of plasmid-associated traits. (i) Animal virulence functions (mouse lethality and conjuctivitis provocation) were only transferable to plasmid-cured derivatives of virulent parent strains (serotypes 0:3, 0:8, and 0:9), but they were not transferable to Y. enterocolitica antigen reference strains (serotypes 0:3 and 0:8) or to a plasmidless clinical isolate of serotype 0:5. A further striking result was that a serotype 0:8 strain regained the mouse lethality trait after receipt of a plasmid from a strain not lethal to mice. These results demonstrate that plasmid-mediated animal virulence functions are not uniformly expressed within Y. enterocolitica. (ii) The second subgroup of plasmid-mediated traits (calcium dependency, surface agglutinogens, HEp-2 cell adherence, and protein release) were transferable to all Y. enterocolitica recipient strains tested (serotypes 0:3, 0:5, 0:8, and 0:9 of different origin). For the first time HEp-2 cell adherence and temperature-induced release of five major protein species are described as transferable traits.

Known and potential virulence factors of enterococcal blood culture isolates were studied using 89 Enterococcus faecalis and 24 Enterococcus faecium isolates. The prevalence of the respective factors was (Enterococcus faecalis vs. Enterococcus faecium): hemolysin 16% vs. 0%, gelatinase 55% vs. 0%, aggregation substance 63% vs. 13%, lipase 35% vs. 4%, hemagglutinin 97% vs. 0%. Deoxyribonuclease was not detected in any isolate. The study showed that hemagglutinin and lipase may represent additional virulence factors of Enterococcus faecalis but not Enterococcus faecium. The significance of these factors in the pathogenesis of enterococcal infection needs to be elucidated in further studies.

Viral differences among lamivudine resistant hepatitis B (HBV) genotypes have not been yet investigated. Therefore, we analyzed the characteristics of these viral strains in vivo. Fortyone patients carrying lamivudine resistant HBV were enrolled. Twenty-six patients (63%) carried resistant HBV genotype A (group A) and 15 patients (37%) carried resistant HBV genotype D (group D). The rate of reverse transcriptase 204I mutants was significantly higher in group D (67%) compared with group A (19%), whereas rt204V mutants (81% in group A vs 33% in group D; P ‫؍‬ .006) and rt180M mutants (81% in group A vs 40% in group D, P ‫؍‬ .015) prevailed in group A. The median time of shift from rt204I to rt204V mutants was significantly shorter in group A (4 months in group A, >12 months in group D, P < .001). Additional resistance associated mutations were detected exclusively in group D (P ‫؍‬ .004). In a multivariate analysis, HBV genotype (P ‫؍‬ .039) and pretreatment serum HBV DNA (P ‫؍‬ .001) were independently associated with emerging rt204I or rt204V mutants, respectively. Serum HBV copy numbers after emergence of resistance were higher in group A (mean log 10 6.99 copies/ml; range 3-9) compared with group D (mean log 10 6.1 copies/ml; range 3.3-8; P ‫؍‬ .04). There was no difference between both groups regarding core promoter/precore mutations, viral turnover, and number of flares or disease progression during follow-up. T he emergence of drug resistant hepatitis B virus (HBV) during lamivudine treatment for chronic hepatitis B is a major problem with an incidence of 14 -36% after 1 year of treatment. [1][2][3][4] This frequency increases to 38%, 49%, and 66% after 2, 3, and 4 years of treatment, respectively. 5-7 Lamivudine resistant HBV is characterized by amino acid variations in the reverse transcriptase domain of the HBV polymerase. In particular, an exchange of the methionine within the YMDD motif by an isoleucine or a valin (rtM204I/V mutants) is associated with lamivudine resistance. Breakthrough of these drug-resistant HBV mutants leads to a viral rebound to baseline levels, 8,9 to a decrease in the rate of loss of hepatitis B e antigen (HBeAg), 10 a high rate of relapses of serum alanine transaminase (ALT) levels, 11,12 and worsening liver histology. 13 Therefore, the emergence of viral resistance is one of the critical issues in the longterm outcome of patients treated for chronic hepatitis B. On the other hand, lamivudine resistant HBV is considered to have reduced viral fitness due to less replication efficiency in vitro 14 and lower ALT levels in vivo as compared with baseline levels. 4,15,16 This led to the recommendation to continue lamivudine treatment despite the emergence of resistant variants as long as benefit to the patient is maintained. 17 Taken together, it would be useful to identify factors which are associated with a better Abbreviations: HBV, hepatitis B virus; HBeAg, hepatitis B e antigen; ALT, serum alanine transaminase; PCR, polymerase chain reaction; CP, core promoter. From the

Reptile-associated Salmonella infections are an increasing problem for humans. We have prospectively screened two breeding groups of 16 pet snakes for colonization with Salmonella species. Various serovars of S. enterica subsp. diarizonae were found in 81% of the snakes. To avoid transmission, strict hygienic precautions should be applied when reptiles are handled.

An association between adherent biofilm production on tissue culture plates and expression of a specific polysaccharide intercellular adhesion (PIA), which is functionally involved in cell clustering, was investigated for 179 Staphylococcus epidermidis isolates. Of the S. epidermidis strains, 50.8% were biofilm producers (A570 of > 0.1). There was a significant positive association between biofilm production and PIA expression: 86.8% of biofilm-producing S. epidermidis strains produced PIA as detected with a specific coagglutination assay. In contrast, 88.6% of the biofilm-negative isolates did not express PIA (P < .001). A linear association between the amount of PIA produced as detected by inhibition ELISA and the amount of biofilm produced was established for 49 S. epidermidis strains, representing a continuum from biofilm-negative to strongly biofilm-producing (r = .81, P < .001). Apparently, PIA is important for biofilm accumulation in the majority of clinical S. epidermidis isolates.