Biofilm production has been implicated as a potential virulence factor of some Candida species responsible for catheter-related fungemia in patients receiving parenteral nutrition. We therefore compared clinical bloodstream isolates representing seven different Candida species to each other and to those from other anatomical sites for the capacity to form biofilms in glucose-containing medium. Potential associations between the capacity to form biofilms and the clinical characteristics of fungemia were also analyzed. Isolates included the following from nonneutropenic patients: 101 bloodstream isolates (35 C. parapsilosis, 30 C. albicans, 18 C. tropicalis, 8 C. glabrata, and 10 other Candida species isolates) and 259 clinical isolates from other body sites (116 C. albicans, 53 C. glabrata, 43 C. tropicalis, 17 C. parapsilosis, and 30 other Candida species isolates). Organisms were grown in Sabouraud dextrose broth (SDB) containing a final concentration of 8% glucose to induce biofilm formation, as published previously. Biofilm production was determined by both visual and spectrophotometric methods. In this medium, biofilm production by C. albicans isolates was significantly less frequent (8%) than that by non-C. albicans Candida species (61%; P < 0.0001). The overall proportion of non-C. albicans Candida species isolates from the blood that produced biofilms was significantly higher than that of non-C. albicans Candida isolates obtained from other sites (79% versus 52%; P ‫؍‬ 0.0001). Bloodstream isolates of C. parapsilosis alone were significantly more likely to be biofilm positive than were C. parapsilosis isolates from other sites (86% versus 47%; P ‫؍‬ 0.0032). Non-C. albicans Candida species, including C. parapsilosis, were more likely to be biofilm positive if isolates were derived from patients whose candidemia was central venous catheter (CVC) related (95%; P < 0.0001) and was associated with the use of total parenteral nutrition (TPN) (94%; P < 0.005). These data suggest that the capacity of Candida species isolates to produce biofilms in vitro in glucose-containing SDB may be a reflection of the pathogenic potential of these isolates to cause CVC-related fungemia in patients receiving TPN.

Many important virulence genes of pathogenic bacteria are preferentially expressed in vivo. We used the recently developed in vivo-induced antigen technology (IVIAT) to identify Vibrio vulnificus genes induced in vivo. An expression library of V. vulnificus was screened by colony blot analysis by using pooled convalescentphase serum that had been thoroughly adsorbed with in vitro-expressed V. vulnificus whole cells and lysates. Twelve clones were selected, and the sequences of the insert DNAs were analyzed. The DNA sequences showed homologies with genes encoding proteins of diverse functions: these functions included chemotaxis (a methylaccepting chemotaxis protein), signaling (a GGDEF-containing protein and a putative serine/threonine kinase), biosynthesis and metabolism (PyrH, PurH, and The expression of virulence determinants in bacteria is known to be regulated by various environmental and host factors (38). During host-parasite interactions, many novel genes that not expressed during in vitro growth have been demonstrated to be coordinately regulated or stimulated by host factors encountered in vivo (20). The usefulness of the information concerning virulence expression gained from in vitro studies is therefore incomplete in relation to in vivo bacterial pathogenesis.Vibrio vulnificus, an opportunistic pathogen, experiences a dramatic environmental change during its infection process. V. vulnificus is an estuarine bacterium that preferentially affects individuals who are heavy drinkers of alcohol and patients with underlying hepatic diseases and other immunocompromised conditions. The pathogen frequently causes fatal septicemia with a rapid progress, resulting in a mortality rate of more than 50% within a few days. The putative virulence factors of V. vulnificus reported so far include a hemolysin (15), a protease (29), phospholipase A2 (55), siderophores (53), and capsular polysaccharides (61a). We reported that the ToxRS system of V. vulnificus, a transmembrane signal-transducing transcription activator, regulated the expression of the hemolysin gene vvhA (32). The ToxRS system was reported to play an important role in regulating in vivo virulence gene expression during V. cholerae infection in a mouse model (33). However, whether the V. vulnificus ToxRS system plays an important role in regulating in vivo virulence gene expression during infection needs further study. V. vulnificus, while infecting the susceptible hosts, passes through gastric acidity, experiences an abrupt pH increase in the duodenum, receives bile secretion, invades into intestinal mucosa, and eventually enters the bloodstream where the pathogen multiplies. During this complicated infection process, V. vulnificus should be able to sense changes in the environmental parameters in the host milieu. The changing signals are likely relayed to specific genes by cognate signal transduction systems, resulting in the expression of specific virulence factors (33). Virulence factors required for in vivo survival and growth of V. vulnificus are ex...

Glycopeptides such as vancomycin are the treatment of choice for infections due to methicillin-resistant Staphylococcus aureus. This study describes the identification of high-level vancomycin-resistant S. aureus (VRSA) isolates in a polymicrobial biofilm within an indwelling nephrostomy tube in a patient in New York. S. aureus, Enterococcus faecalis, Enterococcus faecium, Micrococcus species, Morganella morganii, and Pseudomonas aeruginosa were isolated from the biofilm. For VRSA isolates, vancomycin MICs ranged from 32 to >128 g/ml. VRSA isolates were also resistant to aminoglycosides, fluoroquinolones, macrolides, penicillin, and tetracycline but remained susceptible to chloramphenicol, linezolid, rifampin, and trimethoprim-sulfamethoxazole. The vanA gene was localized to a plasmid of ϳ100 kb in VRSA and E. faecium isolates from the biofilm. Plasmid analysis revealed that the VRSA isolate acquired the 100-kb E. faecium plasmid, which was then maintained without integration into the MRSA plasmid. The tetracycline resistance genes tet(U) and tet(S), not previously detected in S. aureus isolates, were identified in the VRSA isolates. Additional resistance elements in the VRSA isolate included a multiresistance gene cluster, ermB-aadE-sat4-aphA-3, msrA (macrolide efflux), and the bifunctional aminoglycoside resistance gene aac(6)-aph(2؆)-Ia. Multiple combinations of resistance genes among the various isolates of staphylococci and enterococci, including vanA, tet(S), and tet(U), illustrate the dynamic nature of gene acquisition and loss within and between bacterial species throughout the course of infection. The potential for interspecies transfer of antimicrobial resistance genes, including resistance to vancomycin, may be enhanced by the microenvironment of a biofilm.

In an attempt to dissect the virulence regulatory mechanism in Vibrio vulnificus, we tried to identify the V. cholerae transmembrane virulence regulator toxRS (toxRS Vc ) homologs in V. vulnificus. By comparing the sequences of toxRS of V. cholerae and V. parahaemolyticus (toxRS Vp ), we designed a degenerate primer set targeting well-conserved sequences. Using the PCR product as an authentic probe for Southern blot hybridization, a 1.6-kb BglII-HindIII fragment and a 1. Vibrio vulnificus is a halophilic estuarine bacterium that causes fatal septicemia and necrotizing wound infections. Primary septicemia occurs following ingestion of raw seafood contaminated with V. vulnificus. V. vulnificus preferentially affects persons with underlying hepatic diseases, a heavy alcohol drinking habit, and other immunocompromised conditions. Primary septicemia shows a rapidly progressing and fulminant course, which results in a high mortality rate of over 50% despite aggressive antimicrobial and supportive shock therapies (38,39,48).The optimal natural habitat of V. vulnificus is an estuary. The bacterium normally flourishes in estuarine seawater, shellfish, and plankton during warm months (8,35,50,54). V. vulnificus is concentrated in oysters and probably in other shellfish as well (21, 49). V. vulnificus opportunistically causes primary septicemia when contaminated shellfish is eaten raw by susceptible patients. This opportunist should experience a very dramatic change in environmental parameters during the infection process. Successful infection by pathogenic bacteria, in general, is established by coordinate expression of various virulence factors in vivo. Expression of virulence factors is controlled by environmental cues. Pathogenic bacteria possess elegant regulatory systems that sense and react to fluctuations in environmental parameters such as temperature, osmolarity, pH, iron concentration, CO 2 concentration, etc. (12, 27).Many pathogens employ novel signal transduction systems in regulating the virulence gene expression (12). Toxigenic V. cholerae has the toxRS system for that purpose (30). The genes toxR and toxS are clustered in an operon and encode transmembrane proteins ToxR and ToxS, respectively (10,29,32). ToxR regulates expression of multiple V. cholerae virulence factors such as the cholera toxin (ctx), toxin-coregulated pilus (tcp), and accessory colonization factor (acf) genes (9, 47). The activity of ToxR is further enhanced by ToxS, which interacts with the former protein in the periplasmic space and is thought to stabilize it (10, 36). The toxRS system seems to play universally important roles in the survival and host-microorganism interaction of Vibrio species. V. parahaemolyticus and V. fischeri also have homologs of the V. cholerae toxRS (toxRS Vc ) system (25,43). In the present study, we identified the toxRS homolog in V. vulnificus and showed the functional homology of ToxRS Vv with ToxRS Vc . We also observed that ToxR Vv regulates the production of hemolysin, the most potent exotoxin produced ...

We examined microevolution in a series of Candida albicans strains isolated from patients with catheterrelated candidemia. Sixty-one isolates (29 from blood, 18 from catheters, 10 from urine, and 4 from other sites) were obtained from 15 patients who were admitted to the same hospital over a 3-year period. Isolates were analyzed by using Southern hybridization with the C1 fragment of Ca3 as a probe (C1 fingerprinting) and pulsed-field gel electrophoresis (PFGE). PFGE typing consisted of electrophoretic karyotyping (EK) and restriction endonuclease analysis of genomic DNA (REAG) by using SfiI (REAG-S) and BssHII (REAG-B). When catheter isolates were compared with blood isolates from the same patient, catheter isolates from 5 of 14 patients (36%) exhibited minor band differences (microevolution) relative to blood isolates in either C1 fingerprinting (n ‫؍‬ 4), REAG-S (n ‫؍‬ 3), or REAG-B (n ‫؍‬ 5) profiles, although they had identical EK patterns. However, the other sequential isolates from each patient, which had identical EK patterns, showed the same REAG and C1 fingerprinting patterns. Both fingerprinting methods revealed that two distinct genotypes were shared by isolates from seven patients in a neonatal intensive care unit, suggesting two nosocomial clusters. Except for two catheter isolates from the index patients of each cluster, no consecutive isolates collected from each of the two clusters showed any microevolution during the 2-or 7-month cluster periods. The findings suggest that in catheter-related candidemia, some C. albicans strains undergo microevolution during catheter colonization.Within the genus Candida, the species Candida albicans is the most common fungus isolated from humans (15) and accounts for 50 to 70% of all nosocomial bloodstream infections (BSI) resulting from Candida species (1,3,14). In recent years, it has become clear that C. albicans produces genetically altered variants at a high rate. The majority of commensal and infecting populations of C. albicans from the same individuals are clonal in origin but undergo microevolution at the site of colonization and through recurrent episodes of infection (11,21,24). These microevolutionary changes are due to genomic reorganization involving the repetitive RPS element and can be identified in clonal populations by using the Ca3 probe or the C1 fragment of the Ca3 probe (11, 16).Marco et al. (12) reported that microevolution of the colonizing strain occurs in one-third of patients with candidemia caused by C. albicans. However, the relationship between microevolution of C. albicans and subsequent BSI remains uncertain, because C. albicans commonly produces long-term colonization at various body sites, even in healthy individuals (15,24), and most candidemia arises from the patient's own endogenous flora (18,26). In the case of catheter-related BSI, the catheter may serve as the source of fungemia, as organisms proliferate on the catheter surface, from which the organism may enter the bloodstream and then spread to other sites (6). Therefor...

We assessed the genetic relatedness of sequential isolates of Candida parapsilosis during persistent or recurrent fungemia and the effect of central venous catheter (CVC) removal. Serial isolates of C. parapsilosis were obtained from 17 patients with persistent or recurrent fungemia over periods of up to 5 months. Forty-eight C. parapsilosis isolates from the blood of 17 patients were analyzed by electrophoretic karyotyping (EK) with pulsed-field gel electrophoresis (PFGE), revealing 25 different karyotypes. The strains sequentially isolated from each of seven patients whose fungemia resolved following CVC removal had the same karyotype. Two patients with fungemia that cleared without CVC removal each had two sequential isolates with different karyotypes. In six (75%) of the eight patients whose fungemia was recurrent even after CVC removal, the karyotypes of the pre-and post-CVC removal isolates were different, implying the emergence of a new strain. Overall, the sequential strains from each patient had identical karyotypes in 53% (9 of 17) of the patients and two different karyotypes in 47% (8 of 17). This study shows that EK with PFGE is useful for investigating persistent or recurrent fungemia due to C. parapsilosis and that recurrent fungemia due to C. parapsilosis is more likely caused by reinfection with a second strain.

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Candida lipolytica has rarely been reported as a human pathogen. An apparent outbreak of Candida lipolytica fungemia (n = 5 cases) occurred in a pediatric ward over a 9-week period. The five patients infected were hospitalized in three adjacent rooms and cared for by the same healthcare workers. The index patient had central venous catheter-related fungemia, whereas the second patient, who was in the adjacent single room, had transient fungemia. Three additional cases of fungemia occurred in patients with hematological disorders who shared the same room; all three patients had central venous catheters and had been receiving oral fluconazole prophylaxis (50 mg/day for more than 3 weeks) at the time of infection. In vitro susceptibility testing of the strains showed that the MIC of fluconazole for all the isolates was 32 microg/ml. Random amplified polymorphic DNA analysis provided evidence of the clonal origin of the isolates, but the source of the outbreak was not identified. All four patients with persistent fungemia were successfully treated via catheter removal or empiric amphotericin B treatment. This outbreak shows the potential for the nosocomial epidemic transmission of Candida lipolytica.