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A gene encoding a major secreted antigen, SagA, was identified in Enterococcus faecium by screening an E. faecium genomic expression library with sera from patients with E. faecium -associated endocarditis. Recombinant SagA protein showed broad-spectrum binding to extracellular matrix (ECM) proteins, including fibrinogen, collagen type I, collagen type IV, fibronectin, and laminin. A fibrinogen-binding protein, purified from culture supernatants of an E. faecium clinical isolate, was found to match the N-terminal sequence of the predicted SagA protein and to react with the anti-SagA antibody, confirming that it was the SagA protein; this protein appeared as an 80- to 90-kDa smear on a Western blot that was sensitive to proteinase K and resistant to periodate treatment and glycoprotein staining. When overexpressed in E. faecium and Escherichia coli , the native and recombinant SagA proteins formed stable oligomers, apparently via their C-terminal domains. The SagA protein is composed of three domains: (i) a putative coiled-coil N-terminal domain that shows homology to the N-terminal domain of Streptococcus mutans SagA protein (42% similarity), previously shown to be involved in cell wall integrity and cell shape maintenance, and to the P45 protein of Listeria monocytogenes (41% similarity); (ii) a central domain containing direct repeats; and (iii) a C-terminal domain that is similar to that found in various proteins, including P45 (50% similarity) and P60 (52% similarity) of L. monocytogenes . The P45 and P60 proteins both have cell wall hydrolase activity, and the latter has also been shown to be involved in virulence, whereas cell wall hydrolase activity was not detected for SagA protein. The E. faecium sagA gene, like the S. mutans homologue, is located in a cluster of genes encoding proteins that appear to be involved in cell wall metabolism and could not be disrupted unless it was first transcomplemented, suggesting that the sagA gene is essential for E. faecium growth and may be involved in cell wall metabolism. In conclusion, the extracelluar E. faecium SagA protein is apparently essential for growth, shows broad-spectrum binding to ECM proteins, forms oligomers, and is antigenic during infection.

The first outbreak caused by vancomycin-resistant enterococci of the VanB phenotype in Poland was analyzed. It occurred in a single ward of a Warsaw hospital which is a specialized center for the treatment of hematological disorders. Between July 1999 and February 2000, 11 patients in the ward were found to be infected and/or colonized by Enterococcus faecium that was resistant in vitro to vancomycin and susceptible to teicoplanin. PCR analysis confirmed that the vancomycin-resistant E. faecium (VREM) isolates carried the vanB gene, which is responsible for the VanB phenotype. Pulsed-field gel electrophoresis (PFGE) typing revealed that the isolates belonged to four distinct PFGE types and that one of these was clearly predominant, including isolates collected from seven different patients. The isolates contained one or more copies of the vanB gene cluster of the identical, unique Dra I/ Pag I ( Bsp HI) restriction fragment length polymorphism type, which resided in either the same or different plasmid molecules or chromosomal regions. All this data suggested that the outbreak was due to both clonal spread of a single strain and horizontal transfer of resistance genes among nonrelated strains, which could be mediated by plasmids and/or by vanB gene cluster-containing transposons. The comparative analysis of vancomycin-susceptible E. faecium (VSEM) isolates collected from infections in the same ward at the time of the VREM outbreak has led to identification of a widespread VSEM strain that was possibly related to the major VREM clone. It is very likely that this endemic VSEM strain has acquired vancomycin-resistance determinants and that the acquisition occurred more than once during the outbreak.

Twenty-two vancomycin-resistant enterococcal (VRE) isolates of the VanA phenotype (21 Enterococcus faecium isolates and 1 E. faecalis isolate), representative of a large outbreak that occurred in a hospital in Gdańsk, Poland, were studied. All of the isolates demonstrated resistance to a wide variety of other antimicrobial agents in addition to glycopeptides. Several lines of evidence suggested that the outbreak most probably consisted of two epidemics that followed the independent introduction of VanA determinants into two separate hematological wards of the hospital. This hypothesis is supported by the fact that isolates recovered in these wards possessed two different polymorphs of the highly conserved DNA region encompassing the vanRSHAX genes and two distinct polymorph types of Tn1546-like transposons, which contain these genes. According to pulsed-field gel electrophoresis data, the outbreak in the adult hematology ward (HW) was highly polyclonal, which suggested a major role for the horizontal transmission of Tn1546-like elements among nonrelated strains of E. faecium and E. faecalis in this environment. On the other hand, the outbreak in the pediatric hematology ward (PHW) was most probably due to the clonal spread of two epidemic E. faecium strains, which had exchanged a plasmid carrying the Tn1546-like transposon. Restriction fragment length polymorphism studies of transposons and their insertionloci in plasmid DNA have suggested that numerous isolates from both HW and PHW contained two or more copies of Tn1546-like elements that underwent diversification due to various genetic modifications. The reported data demonstrated a very complex epidemiology of the first, and up to now the only, VanA VRE outbreak characterized in Poland.

A mixed outbreak caused by vancomycin-resistant Enterococcus raffinosus and Enterococcus faecium carrying the vanA gene was analysed. The outbreak occurred in a large hospital in Poland and affected 27 patients, most of whom were colonised, in three wards, including the haematology unit. The E. raffinosus isolates had a high-level multiresistant phenotype and were initially misidentified as Enterococcus avium; their unambiguous identification was provided by multilocus sequence analysis. The molecular investigation demonstrated the clonal character of the E. raffinosus outbreak and the polyclonal structure of the E. faecium isolates. All of the isolates carried the same Tn1546-like element containing an IS1251-like insertion sequence, located on a c. 50-kb conjugative plasmid. One of the E. faecium clones, found previously to be endemic in the hospital, was probably the source of the plasmid. The results of the study suggest that difficulties in identification may have led to an underestimate of the importance of E. raffinosus in vancomycin-resistant enterococci (VRE) control strategies.

Vancomycin-resistant enterococci (VRE) have recently become an increasing problem in hospitals in Poland, being responsible for a growing number of nosocomial outbreaks. In this work, we have analyzed the second outbreak of VRE with the VanB phenotype to be identified in the country. It was caused by clonal dissemination of a single strain of vancomycin-resistant Enterococcus faecalis (VRES) and horizontal transmission of vancomycin resistance genes among several vancomycin-resistant Enterococcus faecium (VREM) strains. Two similar restriction fragment length polymorphism types of the vanB gene cluster characterized VRES and VREM isolates, and they both contained the same vanB2 variant of the vanB gene. Two vancomycin-susceptible E. faecium (VSEM) isolates, recovered from the same wards during the outbreak, proved to be related to certain VREM isolates and could represent endemic strains that had acquired vancomycin resistance. One VSEM and four VREM isolates, all identified in the same patient, belonged to a single clone, although they revealed remarkable diversity in terms of susceptibility, PFGE patterns, plasmid content, and number of vanB gene cluster copies. Most probably they reflected the dynamic evolution of an E. faecium strain in the course of infection of a single patient. One of the VREM isolates turned out to be resistant to teicoplanin, which coincided with the use of this antibiotic in the patient's therapy. Its vanB gene variant differed by a single mutation from that found in other isolates; however, it also lacked a large part of the vanB gene cluster, including the regulatory genes vanR B and - S B , and the vancomycin-inducible promoter P YB . Expression of the resistance genes vanH B , - B , and - X B was constitutive in the mutant, and this phenomenon was responsible for its unusual phenotype.

SummaryCaspase-mediated inflammatory cell death acts as an intrinsic defense mechanism against infection. Bacterial pathogens deploy countermeasures against inflammatory cell death, but the mechanisms by which they do this remain largely unclear. In a screen for Shigella flexneri effectors that regulate cell death during infection, we discovered that Shigella infection induced acute inflammatory, caspase-4-dependent epithelial cell death, which is counteracted by the bacterial OspC3 effector. OspC3 interacts with the caspase-4-p19 subunit and inhibits its activation by preventing caspase-4-p19 and caspase-4-p10 heterodimerization by depositing the conserved OspC3 X1-Y-X2-D-X3 motif at the putative catalytic pocket of caspase-4. Infection of guinea pigs with a Shigella ospC3-deficient mutant resulted in enhanced inflammatory cell death and associated symptoms, correlating with decreased bacterial burdens. Salmonella Typhimurium and enteropathogenic Escherichia coli infection also induced caspase-4-dependent epithelial death. These findings highlight the importance of caspase-4-dependent innate immune responses and demonstrate that Shigella delivers a caspase-4-specific inhibitor to delay epithelial cell death and promote infection.