To our knowledge, these patients represent the first reported North American adults with severe community-onset MRSA pneumonia caused by strains carrying the PVL genes.

Escherichia coli K1 is a major gram-negative organism causing neonatal meningitis. E. coli K1 binding to and invasion of human brain microvascular endothelial cells (HBMEC) are a prerequisite for E. coli penetration into the central nervous system in vivo. In the present study, we showed using DNA microarray analysis that E. coli K1 associated with HBMEC expressed significantly higher levels of the fim genes compared to nonassociated bacteria. We also showed that E. coli K1 binding to and invasion of HBMEC were significantly decreased with its fimH deletion mutant and type 1 fimbria locked-off mutant, while they were significantly increased with its type 1 fimbria locked-on mutant. E. coli K1 strains associated with HBMEC were predominantly type 1 fimbria phase-on (i.e., fimbriated) bacteria. Taken together, we showed for the first time that type 1 fimbriae play an important role in E. coli K1 binding to and invasion of HBMEC and that type 1 fimbria phase-on E. coli is the major population interacting with HBMEC.The mortality and morbidity associated with neonatal gramnegative bacterial meningitis have remained significant despite advances in antimicrobial chemotherapy. This is mainly attributed to inadequate knowledge of the pathogenesis and pathophysiology of this disease. Escherichia coli K1 is the most common gram-negative bacterium that causes meningitis during the neonatal period (26).E. coli meningitis develops as a result of hematogenous spread, but it is not clear how circulating bacteria cross the blood-brain barrier. Our laboratory has successfully isolated and cultivated human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier (9, 10). We showed that E. coli invasion of HBMEC is a prerequisite for E. coli penetration into the central nervous system in vivo. However, the basis of E. coli-HBMEC interactions involved in binding to HBMEC is incompletely understood.Adherence of bacteria to their host cells is considered the initial step of the pathogenesis of infectious diseases including meningitis. To date OmpA is the only identified determinant that is involved in E. coli K 1 binding to HBMEC (8, 10). Previous reports have implied that S fimbriae might be another potential E. coli K1 factor involved in adherence to HBMEC (3,16,24,32). However, according to our recent data, S fimbriae do not play a significant role in binding of E. coli K1 to HBMEC (35).Type 1 fimbriae are filamentous surface organelles produced by E. coli and mediate mannose-sensitive adhesion of E. coli to various eukaryotic cells. In E. coli K1, type 1 fimbriae have been shown to be important for oropharyngeal colonization in a neonatal rat model (4). Type 1 fimbriae are encoded by a fim gene cluster, including at least nine genes required for their biosynthesis (20). The fimbriae are composed primarily of the major FimA protein and a small tip structure containing FimF, FimG, and FimH (12). The lectin-like adhesin, FimH, located at the tip of the fimbrial shaft is responsible for the mannosesens...

The first three enzymes of the pentachlorophenol (PCP) degradation pathway in Sphingobium chlorophenolicum (formerly Sphingomonas chlorophenolica) ATCC 39723 have been characterized, and the corresponding genes, pcpA, pcpB, and pcpC, have been individually cloned and sequenced. To search for new genes involved in PCP degradation and map the physical locations of the pcp genes, a 24-kb fragment containing pcpA and pcpC was completely sequenced. A putative LysR-type transcriptional regulator gene, pcpM, and a maleylacetate reductase gene, pcpE, were identified upstream of pcpA. pcpE was found to play a role in PCP degradation. pcpB was not found on the 24-kb fragment. The four gene products PcpB, PcpC, PcpA, and PcpE were responsible for the metabolism of PCP to 3-oxoadipate in ATCC 39723, and inactivational mutation of each gene disrupted the degradation pathway. The organization of the pcp genes is unusual because the four PCP-degrading genes, pcpA, pcpB, pcpC, and pcpE, were found to be located at four discrete locations. Two hypothetical LysR-type regulator genes, pcpM and pcpR, have been identified; pcpM was not required, but pcpR was essential for the induction of pcpB, pcpA, and pcpE. The coinducers of PcpR were PCP and other polychlorinated phenols. The expression of pcpC was constitutive. Thus, the organization and regulation of the genes involved in PCP degradation to 3-oxoadipate were documented.Pentachlorophenol (PCP) has been released into the environment as a wood preservative (8, 13). This compound is a major environmental pollutant due to its toxicity and recalcitrance, and it is regulated as one of the priority pollutants by the U.S. Environmental Protection Agency (16, 30). Microorganisms have been used to remove PCP from the environment (16, 17), and several aerobic PCP-degrading bacteria have been isolated from contaminated soils (7). Sphingobium chlorophenolicum (31) (formerly Sphingomonas chlorophenolica) strain ATCC 39723 is one of the bacteria capable of completely mineralizing PCP (24). The biochemistry of PCP degradation by ATCC 39723 has been extensively studied (Fig. 1). PCP 4-monooxygenase (PcpB) oxidizes PCP to 2,3,5,6-tetrachlorop-hydroquinone (TeCH) (22,35,37,38). TeCH reductive dehalogenase (PcpC) converts TeCH to 2,3,6-trichloro-p-hydroquinone and then to 2,6-dichloro-p-hydroquinone (DiCH) by reductive dechlorination (20,39,40). DiCH is subject to ring cleavage by DiCH 1,2-dioxygenase (PcpA), producing 2-chloromaleylacetate (2-CMA) (19, 33). The corresponding genes, pcpB, pcpC, and pcpA, have been individually cloned and sequenced (21,22,36). pcpB was found to be physically linked with two other putative pcp genes, pcpD and pcpR (21). pcpR is a hypothetical LysR-type regulator. Northern hybridization and enzymatic activity analysis suggest that PcpB and PcpA are PCP inducible in strain ATCC 39723 (22, 33), while PcpC is constitutively produced (20,40). However, the overall organization and regulation of PCP-degrading genes have not been reported, and the metabolic steps beyond rin...

We examined the incremental yield of stool culture (with toxin testing on isolates) versus our two-step algorithm for optimal detection of toxigenic Clostridium difficile. Per the two-step algorithm, stools were screened for C. difficile-associated glutamate dehydrogenase (GDH) antigen and, if positive, tested for toxin by a direct (stool) cell culture cytotoxicity neutralization assay (CCNA). In parallel, stools were cultured for C. difficile and tested for toxin by both indirect (isolate) CCNA and conventional PCR if the direct CCNA was negative. The "gold standard" for toxigenic C. difficile was detection of C. difficile by the GDH screen or by culture and toxin production by direct or indirect CCNA. We tested 439 specimens from 439 patients. GDH screening detected all culture-positive specimens. The sensitivity of the two-step algorithm was 77% (95% confidence interval [CI], 70 to 84%), and that of culture was 87% (95% CI, 80 to 92%). PCR results correlated completely with those of CCNA testing on isolates (29/29 positive and 32/32 negative, respectively). We conclude that GDH is an excellent screening test and that culture with isolate CCNA testing detects an additional 23% of toxigenic C. difficile missed by direct CCNA. Since culture is tedious and also detects nontoxigenic C. difficile, we conclude that culture is most useful (i) when the direct CCNA is negative but a high clinical suspicion of toxigenic C. difficile remains, (ii) in the evaluation of new diagnostic tests for toxigenic C. difficile (where the best reference standard is essential), and (iii) in epidemiologic studies (where the availability of an isolate allows for strain typing and antimicrobial susceptibility testing).Since its identification in 1978, Clostridium difficile has emerged as the predominant cause of antibiotic-associated colitis and the leading cause of diarrhea in hospitalized patients (4). Strains of C. difficile can be toxigenic or nontoxigenic; however, only toxigenic strains produce disease. The two main virulence factors are toxin A, a 308-kDa enterotoxin with some cytopathic effects (TcdA), and Toxin B (TcdB), a potent 270-kDa cytotoxin that affects various tissue cell lines in vitro and inhibits bowel motility in vivo (3, 12). The genes encoding both toxins, tcdA and tcdB, have been sequenced, and both are known to disrupt the actin cytoskeleton of intestinal epithelial cells by modifying Rho family proteins. Toxin A has long been considered more important (19,20), but an increasing number of reports of colitis due to TcdA-negative but TcdB-positive strains have now been made (5, 35). Furthermore, other virulence factors have now been identified. Recently, an increase in the frequency and severity of C. difficile-associated colitis, which is associated with a new strain that produces binary toxin (actin-specific ADP-ribosyltransferase) and is resistant to fluoroquinolones in vitro, has refocused attention on early, accurate diagnosis (2,9,23,29,36).Cell culture cytotoxicity neutralization assays (CCNA), which detect ...