Candida parapsilosis former groups II and III have recently been established as independent species named C. orthopsilosis and C. metapsilosis, respectively. In this report, 400 isolates (290 patients) previously classified as C. parapsilosis by conventional laboratory tests were screened by BanI digestion profile analysis of the secondary alcohol dehydrogenase gene fragment and by amplification fragment length polymorphism (AFLP). Thirty-three strains collected from 13 patients were identified as C. orthopsilosis, thus giving the first retrospective evidence that C. orthopsilosis was responsible for 4.5% of the infections/colonization attributed to C. parapsilosis. AFLP was proven to unambiguously identify C. orthopsilosis at the species level and efficiently delineate intraspecific genetic relatedness. A high percentage of polymorphic AFLP bands was observed for independent isolates collected from each patient. Statistical analysis of the pairwise genetic distances and bootstrapping revealed that clonal reproduction and recombination both contribute to C. orthopsilosis genetic population structure. AFLP patterns of sequential isolates obtained from two patients demonstrated that a successful strain colonization within the same patient occurred, as revealed by strain maintenance in various body sites. No association between AFLP markers and drug resistance was observed, and none of the clinical C. orthopsilosis isolates were found to produce biofilm in vitro.

Bacillus thuringiensis is being used worldwide as a biopesticide, although increasing evidence suggests that it is emerging as an opportunistic human pathogen. While phospholipases, hemolysins, and enterotoxins are claimed to be responsible for B. thuringiensis virulence, there is no direct evidence to indicate that the flagellum-driven motility plays a role in parasite-host interactions. This report describes the characterization of a mini-Tn10 mutant of B. thuringiensis that is defective in flagellum filament assembly and in swimming and swarming motility as well as in the production of hemolysin BL and phosphatidylcholine-preferring phospholipase C. The mutant strain was determined to carry the transposon insertion in flhA, a flagellar class II gene encoding a protein of the flagellar type III export apparatus. Interestingly, the flhA mutant of B. thuringiensis synthesized flagellin but was impaired in flagellin export. Moreover, a protein similar to the anti-sigma factor FlgM that acts in regulating flagellar class III gene transcription was not detectable in B. thuringiensis, thus suggesting that the flagellar gene expression hierarchy of B. thuringiensis differs from that described for Bacillus subtilis. The flhA mutant of B. thuringiensis was also defective in the secretion of hemolysin BL and phosphatidylcholine-preferring phospholipase C, although both of these virulence factors were synthesized by the mutant. Since complementation of the mutant with a plasmid harboring the flhA gene restored swimming and swarming motility as well as secretion of toxins, the overall results indicate that motility and virulence in B. thuringiensis may be coordinately regulated by flhA, which appears to play a crucial role in the export of flagellar as well as nonflagellar proteins.Bacillus thuringiensis is a gram-positive, rod-shaped, sporeforming bacterium that is motile by peritrichous flagella. During sporulation this organism produces a variety of ␦-endotoxins (Cry proteins) that are primarily responsible for a highly specific insecticidal activity (53). For this reason, B. thuringiensis is being used presently as a biological insecticide, accounting for more than 90% of the biopesticides employed worldwide (37).Despite a long record of being safe, there is increasing public concern about the potential pathogenicity of B. thuringiensis to humans. This is due to the sporadic but sometimes severe infections caused by this organism, which include diarrheal food poisoning (34), corneal ulcer (52), cellulitis (57), and burn (13) as well as war wound (28) infections. Moreover, the pathogenicity of B. thuringiensis has been demonstrated in an animal model of infection by administering spores to mice via nasal inoculation (27,50). Similar to Bacillus cereus, B. thuringiensis produces a variety of virulence factors, which include phosphatidylcholine-preferring phospholipase C (PC-PLC) and phosphatidylinositol-specific phospholipase C (38, 40), hemolysins, and enterotoxins (1,18,21). Heierson et al. (24) isolated an avirulent...

Multilocus sequence typing (MLST) was applied to 75 Candida albicans isolates, including 2 that were expected to be identical, 48 that came from diverse geographical and clinical sources, and 15 that were sequential isolates from two patients. DNA fragments (Ϸ500 bp) of eight genes encoding housekeeping functions were sequenced, including four that have been described before for C. albicans MLST, and four new gene fragments, AAT1a, AAT1b, MPI, and ZWF1. In total, 87 polymorphic sites were found among 50 notionally different isolates, giving 46 unique sequence types, underlining the power of MLST to differentiate isolates for epidemiological studies. Additional typing information was obtained by detecting variations in size at the transcribed spacer region of the 25S rRNA gene and tests for homozygosity at the mating type-like (MTL) locus. The stability of MLST was confirmed in two sets of consecutive isolates from two patients. In each set the isolates were identical or varied by a single nucleotide. Reference strain SC5314 and a derived mutant, CAF2, gave identical MLST types. Heterozygous polymorphisms were found in at least one isolate for all but 16 (18.4%) of the variable nucleotides, and 35 (41%) of the 87 individual sequence changes generated nonsynonymous amino acids. Cloning and restriction digestion of a gene fragment containing heterozygous polymorphisms indicated that the heterozygosity was genuine and not the result of sequencing errors. Our data validate and extend previous MLST results for C. albicans, and we propose an optimized system based on sequencing eight gene fragments for routine MLST with this species.

This report describes the nosocomial acquisition of Candida parapsilosis candidemia by one of the six premature newborns housed in the same room of a neonatal intensive care unit at the Ospedale Santa Chiara, Pisa, Italy. The infant had progeria, a disorder characterized by retarded physical development and progressive senile degeneration. The infant, who was not found to harbor C. parapsilosis at the time of his admission to the intensive care unit, had exhibited symptomatic conjunctivitis before the onset of a severe bloodstream infection. In order to evaluate the source of infection and the route of transmission, two independent molecular typing methods were used to determine the genetic relatedness among the isolates recovered from the newborn, the inanimate hospital environment, hospital personnel, topically and intravenously administered medicaments, and indwelling catheters. Among the isolates collected, only those recovered from the hands of two nurses attending the newborns and from both the conjunctiva and the blood of the infected infant were genetically indistinguishable. Since C. parapsilosis was never recovered from indwelling catheters or from any of the drugs administered to the newborn, we concluded that (i) horizontal transmission of C. parapsilosis occurred through direct interaction between nurses and the newborn and (ii) the conjunctiva was the site through which C. parapsilosis entered the bloodstream. This finding highlights the possibility that a previous C. parapsilosis colonization and/or infection of other body sites may be a predisposing condition for subsequent C. parapsilosis hematogenous dissemination in severely ill newborns.

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In light of the need for new antifungal agents, the candidacidal activities of human lactoferrin (hLF) and synthetic peptides representing the first, hLF(1-11), and second, hLF(21-31), cationic domains of its N terminus were compared. The results revealed that hLF(1-11) was more effective in killing fluconazole-resistant Candida albicans than hLF(21-31) and much more effective than lactoferrin, as determined microbiologically and by propidium iodide (PI) staining. By using hLF(1-11) and various derivatives, it was found that the second and third residues of the N terminus of hLF(1-11) were critical for its candidacidal activity. Detailed investigation to elucidate the mechanism of action of hLF(1-11) revealed a dose-dependent release of ATP by Candida upon exposure to hLF(1-11). Our observations that sodium azide reduced the PI uptake and candidacidal activity of hLF(1-11) and that, upon exposure to hLF(1-11), the fluorescent dye rhodamine 123 first accumulated inside the mitochondria and later was released into the cytoplasm indicate that the peptide triggers the energized mitochondrion. Furthermore, oxidized ATP, which interferes with the interaction of ATP with its extracellular receptors, blocked the candidacidal action of hLF(1-11), as measured microbiologically and by PI staining. Addition of ATP (or analogues) was not a sufficient stimulus to kill C. albicans or to act synergistically with suboptimal concentrations of the peptide. The main conclusions are that the first two arginines at the N terminus of hLF are critical in the candidacidal activity of hLF(1-11) and that extracellular ATP is essential but not sufficient for the peptide to exert its candidacidal activity.

A substantial number of Bacillus species have been marketed for use in oral bacteriotherapy because of their purported ability to prevent or treat various gastrointestinal disorders. Recently, some of the Bacillus strains in Enterogermina, which is made up of aqueous suspensions of viable Bacillus spores, have been partially characterized and aligned with members of the Bacillus alcalophilus subgroup rather than with Bacillus subtilis, as previously reported. With a view toward verifying the original taxonomic position of the Enterogermina strains, we catalogued both phenotypic and genotypic traits exhibited by the four Bacillus strains isolated from the spore mixtures found in original commercial preparations dated 1975 and 1984 and commercial preparations now being propagated industrially. Analyses of physiological and biochemical traits, complete 16S rRNA gene sequences, DNA-DNA reassociation, tRNA intergenic spacer length polymorphism, single-strand conformation polymorphism of PCR-amplified spacer regions of tRNA genes, and randomly amplified polymorphic DNA led to the finding that all of the Enterogermina strains belong to a unique genospecies, which is unequivocally identified as the alkalitolerant species Bacillus clausii. Moreover, we provide evidence that in contrast to several reference strains of B. clausii, the strains constituting Enterogermina are characterized by a notable low level of intraspecific genome diversity and that each strain has remained the same for the last 25 years.

Bacillus cereus behaves as an opportunistic pathogen frequently causing gastrointestinal diseases, and it is increasingly recognized to be responsible for severe local or systemic infections. Pathogenicity of B. cereus mainly relies on the secretion of a wide array of toxins and enzymes and also on the ability to undergo swarming differentiation in response to surface-sensing. In this report, the pathogenicity exerted by B. cereus toxins is described with particular attention to the regulatory mechanisms of production and secretion of HBL, Nhe and CytK enterotoxins.