Antigen-43-mediated autoaggregation impairs motility in Escherichia coli

Functional interaction between bacterial surface-displayed autoaggregation proteins such as antigen 43 (Ag43) of Escherichia coli and motility organelles such as flagella has not previously been described. Here, it has been demonstrated for the first time that Ag43-mediated aggregation can inhibit bacterial motility. Ag43 overexpression produces a dominant aggregation phenotype that overrides motility in the presence of low levels of flagella. In contrast, induction of an increased flagellation state prevents Ag43-mediated aggregation. This phenomenon was observed in naturally occurring subpopulations of E coli as phase variants expressing and not expressing Ag43 revealed contrasting motility phenotypes. The effects were shown to be part of a general mechanism because other short adhesins capable of mediating autoaggregation (AIDA-I and TibA) also impaired motility. These novel insights into the function of bacterial autoaggregation proteins suggest that a balance between these two systems, i.e. autoaggregation and flagellation, influences motility.

Asymptomatic bacteriuria Escherichia coli strain 83972 carries mutations in the foc locus and is unable to express F1C fimbriae

Escherichia coli is the most common organism associated with asymptomatic bacteriuria (ABU). In contrast to uropathogenic E. coli (UPEC), which causes symptomatic urinary tract infection (UTI), very little is known about the mechanisms by which these strains colonize the urinary tract. Bacterial adhesion conferred by specific surface-associated adhesins is normally considered as a prerequisite for colonization of the urinary tract. The prototype ABU E coli strain 83972 was originally isolated from a girl who had carried it asymptomatically for 3 years. This study characterized the molecular status of one of the primary adhesion factors known to be associated with UTI, namely F1C fimbriae, encoded by the foc gene cluster. F1C fimbriae recognize receptors present in the human kidney and bladder. Expression of the foc genes was found to be up-regulated in human urine. It was also shown that although strain 83972 contains a seemingly intact foc gene cluster, F1C fimbriae are not expressed. Sequencing and genetic complementation revealed that the focD gene, encoding a component of the F1C transport and assembly system, was non-functional, explaining the inability of strain 83972 to express this adhesin. The data imply that E. coli 83972 has lost its ability to express this important colonization factor as a result of host-driven evolution. The ancestor of the strain seems to have been a pyelonephritis strain of phylogenetic group B2. Strain 83972 therefore represents an example of bacterial adaptation from pathogenicity to commensalism through virulence factor loss.

Attachment of Shiga toxigenic Escherichia coli to beef muscle and adipose tissue

Shiga toxigenic Escherichia coli (STEC) serotypes are important foodborne pathogens that cause gastrointestinal disease worldwide. An understanding of how STEC strains attach to surfaces may provide insight into the potential persistence of and contamination with STEC in food environments. The initial attachment of a selection of STEC serotypes to beef muscle and adipose tissue was evaluated for isolates grown in planktonic and sessile culture. Initial experiments were performed to determine whether attachment differed among STEC strains and between the two modes of growth. Viable counts were obtained for loosely and strongly attached cells, and the strength of attachment (S-r) was calculated. All bacterial isolates grown in sessile culture attached in higher numbers to muscle and adipose tissue than did bacteria in planktonic cultures. For all attachment assays performed, mean concentrations for loosely attached cells were consistently higher than concentrations for strongly attached cells. The mean concentrations for strongly attached bacteria for planktonic and sessile cultures were significantly higher (P < 0.05) on adipose than on muscle tissue. However, some strains of STEC, particularly those from sessile culture, did not differ in their attachment to muscle or adipose tissue. S-r values were not significantly different (P > 0.05) among STEC isolates for all assays. No correlation was found between bacterial hydrophobicity and surface charge values (previously determined) and production of surface structures, viable counts, and S-r values. STEC grown in planktonic and sessile culture seems to behave differently with respect to attachment to muscle and adipose tissue. Cells in sessile culture may have a greater potential to strongly attach to meat surfaces.

Changes in equine hindgut bacterial populations during oligofructose-induced laminitis

In the horse, carbohydrate overload is thought to play an integral role in the onset of laminitis by drastically altering the profile of bacterial populations in the hindgut. The objectives of this study were to develop and validate microbial ecology methods to monitor changes in bacterial populations throughout the course of experimentally induced laminitis and to identify the predominant oligofructose-utilizing organisms. Laminitis was induced in five horses by administration of oligofructose. Faecal specimens were collected at 8 h intervals from 72 h before to 72 h after the administration of oligofructose. Hindgut microbiota able to utilize oligofructose were enumerated throughout the course of the experiment using habitat-simulating medium. Isolates were collected and representatives identified by 16S rRNA gene sequencing. The majority of these isolates collected belonged to the genus Streptococcus, 91% of which were identified as being most closely related to Streptococcus infantarius ssp. coli. Furthermore, S. infantarius ssp. coli was the predominant oligofructose-utilizing organism isolated before the onset of lameness. Fluorescence in situ hybridization probes developed to specifically target the isolated Streptococcus spp. demonstrated marked population increases between 8 and 16 h post oligofructose administration. This was followed by a rapid population decline which corresponded with a sharp decline in faecal pH and subsequently lameness at 24-32 h post oligofructose administration. This research suggests that streptococci within the Streptococcus bovis/equinus complex may be involved in the series of events which precede the onset of laminitis in the horse.

Comparison of selected methods for measuring the virulence properties of Listeria spp.

The comparative ability of different methods to assess virulence of Listeria species was investigated in ten Listeria strains. All strains were initially subjected to pulsed-field gel electrophoresis analysis to determine their relatedness. Virulence characteristics were subsequently tested for by (i) determining the presence of six virulence genes by polymerase chain reaction; (ii) testing for the production of listeriolysin O, phosphatidylcholine phospholipase C, and phosphatidylinositol-specific phospholipase C; (iii) investigating the hydrophobicity of the strains; (iv) determining the strains ability to attach to, enter, and replicate within the Caco-2 cells. Variations in most of the virulence characteristics were obvious across the strains for the range of tests performed. A wide range of anomalous results among methods were apparent. In particular, the presence of virulence genes was found to be unrelated to the production of virulence-associated proteins in vitro, while virulence protein production and hydrophobicity in Listeria monocytogenes were found to be unrelated or marginally related, respectively, to the ability to invade the Caco-2 cell line. It was concluded that the methods investigated were unable to consistently and unequivocally measure the differences in the virulence properties of the strains.

Diversity of polyketide synthase genes from bacteria associated with the marine sponge Pseudoceratina clavata: culture-dependent and culture-independent approaches

Diverse ketosynthase (KS) genes were retrieved from the microbial community associated with the Great Barrier Reef sponge Pseudoceratina clavata. Bacterial isolation and metagenomic approaches were employed. Phylogenetic analysis of 16S rRNA of culturable sponge-associated bacterial communities comprised eight groups over four phyla. Ten KS domains were amplified from four genera of isolates and phylogenetics demonstrated that these KS domains were located in three clusters (actinobacterial, cyanobacterial and trans-AT type). Metagenomic DNA of the sponge microbial community was extracted to explore community KS genes by two approaches: direct amplification of KS domains and construction of fosmid libraries for KS domain screening. Five KS domains were retrieved from polymerase chain reaction (PCR) amplification using sponge metagenome DNA as template and five fosmid clones containing KS domains found using multiplex PCR screening. Analysis of selected polyketide synthase (PKS) from one fosmid showed that the PKS consists of two modules. Open reading frames located up- and downstream of the PKS displayed similarity with membrane synthesis-related proteins such as cardiolipin synthase. Metagenome approaches did not detect KS domains found in sponge isolates. All KS domains from both metagenome approaches formed a single cluster with KS domains originating from metagenomes derived from other sponge species from other geographical regions.

Evolutionary dynamics of wAu-Like Wolbachia variants in neotropical Drosophila spp.

Wolbachia bacteria are common intracellular symbionts of arthropods and have been extensively studied in Drosophila. Most research focuses on two Old Word hosts, Drosophila melanogaster and Drosophila simulans, and does not take into account that some of the Wolbachia associations in these species may have evolved only after their fast global expansion and after the exposure to Wolbachia of previously isolated habitats. Here we looked at Wolbachia of Neotropical Drosophila species. Seventy-one lines of 16 Neotropical Drosophild species sampled in different regions and at different time points were analyzed. Wolbachia is absent in lines of Drosophild willistoni collected before the 1970s, but more recent samples are infected with a strain designated wWiL Wolbachia is absent in all other species of the willistoni group. Polymorphic wWil-related strains were detected in some saltans group species, with D. septentriosaltans being coinfected with at least four variants. Based on wsp and ftsZ sequence data, wWil of D. willistoni is identical to wAu, a strain isolated from D. simulans, but can be discriminated when using a polymorphic minisatellite marker. In contrast to wAu, which infects both germ line and somatic tissues of D. simulans, wWil is found exclusively in the primordial germ line cells of D. willistoni embryos. We report on a pool of closely related Wolbachia strains in Neotropical Drosophila species as a potential source for the wAu strain in D. simulans. Possible evolutionary scenarios reconstructing the infection history of wAu-like Wolbachia in Neotropical Drosophild species and the Old World species D. simulans are discussed.

Genes for the majority of group A streptococcal virulence factors and extracellular surface proteins do not confer an increased propensity to cause invasive disease

Background. The factors behind the reemergence of severe, invasive group A streptococcal (GAS) diseases are unclear, but it could be caused by altered genetic endowment in these organisms. However, data from previous studies assessing the association between single genetic factors and invasive disease are often conflicting, suggesting that other, as-yet unidentified factors are necessary for the development of this class of disease. Methods. In this study, we used a targeted GAS virulence microarray containing 226 GAS genes to determine the virulence gene repertoires of 68 GAS isolates (42 associated with invasive disease and 28 associated with noninvasive disease) collected in a defined geographic location during a contiguous time period. We then employed 3 advanced machine learning methods (genetic algorithm neural network, support vector machines, and classification trees) to identify genes with an increased association with invasive disease. Results. Virulence gene profiles of individual GAS isolates varied extensively among these geographically and temporally related strains. Using genetic algorithm neural network analysis, we identified 3 genes with a marginal overrepresentation in invasive disease isolates. Significantly, 2 of these genes, ssa and mf4, encoded superantigens but were only present in a restricted set of GAS M-types. The third gene, spa, was found in variable distributions in all M-types in the study. Conclusions. Our comprehensive analysis of GAS virulence profiles provides strong evidence for the incongruent relationships among any of the 226 genes represented on the array and the overall propensity of GAS to cause invasive disease, underscoring the pathogenic complexity of these diseases, as well as the importance of multiple bacteria and/ or host factors.

Laboratory-based simulation of freezing profiles of beef trim for Escherichia coli O157 survival determinations

This study was undertaken to develop a simple laboratory-based method for simulating the freezing profiles of beef trim so that their effect on E. coli 0157 survival could be better assessed. A commercially available apparatus of the type used for freezing embryos, together with an associated temperature logger and software, was used for this purpose with a -80 degrees C freezer as a heat sink. Four typical beef trim freezing profiles, of different starting temperatures or lengths, were selected and modelled as straight lines for ease of manipulation. A further theoretical profile with an extended freezing plateau was also developed. The laboratory-based setup worked well and the modelled freezing profiles fitted closely to the original data. No change in numbers of any of the strains was apparent for the three simulated profiles of different lengths starting at 25 degrees C. Slight but significant (P < 0.05) decreases in numbers (similar to 0.2 log cfu g(-1)) of all strains were apparent for a profile starting at 12 degrees C. A theoretical version of this profile with a freezing plateau phase extended from 11 h to 17 h resulted in significant (P < 0.05) decreases in numbers (similar to 1.2 log cfu g(-1)) of all strains. Results indicated possible avenues for future research in controlling this pathogen. The method developed in this study proved a useful and cost-effective way for simulating freezing profiles of beef trim. (c) 2005 Elsevier B.V. All rights reserved.

PerR controls Mn-dependent resistance to oxidative stress in Neisseria gonorrhoeae

In previous studies it has been established that resistance to superoxide by Neisseria gonorrhoeae is dependent on the accumulation of Mn(II) ions involving the ABC transporter, MntABC. A mutant strain lacking the periplasmic binding protein component (MntC) of this transport system is hypersensitive to killing by superoxide anion. In this study the mntC mutant was found to be more sensitive to H2O2 killing than the wild-type. Analysis of regulation of MntC expression revealed that it was de-repressed under low Mn(II) conditions. The N. gonorrhoeae mntABC locus lacks the mntR repressor typically found associated with this locus in other organisms. A search for a candidate regulator of mntABC expression revealed a homologue of PerR, a Mn-dependent peroxide-responsive regulator found in Gram-positive organisms. A perR mutant expressed more MntC protein than wild-type, and expression was independent of Mn(II), consistent with a role for PerR as a repressor of mntABC expression. The PerR regulon of N. gonorrhoeae was defined by microarray analysis and includes ribosomal proteins, TonB-dependent receptors and an alcohol dehydrogenase. Both the mntC and perR mutants had reduced intracellular survival in a human cervical epithelial cell model.

Pilin glycosylation in Neisseria meningitidis occurs by a similar pathway to wzy-dependent O-antigen biosynthesis in Escherichia coli

Pili (type IV fimbriae) of Neisseria meningitidis are glycosylated by the addition of O-linked sugars. Recent work has shown that PglF, a protein with homology to O-antigen 'flippases', is required for the biosynthesis of the pilin-linked glycan and suggests pilin glycosylation occurs in a manner analogous to the wzy-dependent addition of O-antigen to the core-LPS. O-Antigen ligases are crucial in this pathway for the transfer of undecraprenol-linked sugars to the LPS-core in Gram-negative bacteria. An O-antigen ligase homologue, pglL, was identified in N. meningitidis. PglL mutants showed no change in LPS phenotypes but did show loss of pilin glycosylation, confirming PglL is essential for pilin O-linked glycosylation in N. meningitidis. (c) 2006 Elsevier Inc. All rights reserved.

Protease IV production in Pseudomonas aeruginosa from the lungs of adults with cystic fibrosis

Protease IV is important in the pathogenesis of Pseudomonas aeruginosa-induced microbial keratitis, but little is known of its role in cystic fibrosis (CF) lung infection. In this study protease IV production was examined in 43 P. aeruginosa isolates (24 non-clonal and 19 clonal) from the lungs of chronically infected adult patients attending the Royal Prince Alfred Hospital CF Clinic, Sydney, Australia. Overall, 32/43 (74 %) isolates were positive for protease IV protein by Western blotting and 22/43 (51 %) had evidence of active protease IV on gelatin zymography. Clonal strains were 1.6 times more likely than non-clonal strains to produce protease IV [18/19 (95 %) versus 14/24 (58 %), RR=1.6, CI 1.1–2.3, P=0.007] and 3 times more likely to secrete the protein [16/19 (84 %) versus 6/24 (25 %), RR=3.4, CI 1.6–6.9, P

Candidatus "Anammoxoglobus propionicus" a new propionate oxidizing species of anaerobic ammonium oxidizing bacteria

The bacteria that mediate the anaerobic oxidation of ammonium (anammox) are detected worldwide in natural and man-made ecosystems, and contribute up to 50% to the loss of inorganic nitrogen in the oceans. Two different anammox species rarely live in a single habitat, suggesting that each species has a defined but yet unknown niche. Here we describe a new anaerobic ammonium oxidizing bacterium with a defined niche: the co-oxidation of propionate and ammonium. The new anammox species was enriched in a laboratory scale bioreactor in the presence of ammonium and propionate. Interestingly, this particular anammox species could out-compete other anammox bacteria and heterotrophic denitrifiers for the oxidation of propionate in the presence of ammonium, nitrite and nitrate. We provisionally named the new species Candidatus "Anammoxoglobus propionicus".