Lateral gene transfer between obligate intracellular bacteria: evidence from the Rickettsia massiliae genome

Rickettsia massiliae is a tick-borne obligate intracellular alpha-proteobacteria causing spotted fever in humans. Here, we present the sequence of its genome, comprising a 1.3-Mb circular chromosome and a 15.3-kb plasmid. The chromosome exhibits long-range colinearity with the other Spotted Fever Group Rickettsia genomes, except for a large fragment specific to R. massiliae that contains 14 tra genes presumably involved in pilus formation and conjugal DNA transfer. We demonstrate that the tra region was acquired recently by lateral gene transfer (LGT) from a species related to Rickettsia bellii. Further analysis of the genomic sequences identifies additional candidates of LGT between Rickettsia. Our study indicates that recent LGT between obligate intracellular Rickettsia is more common than previously thought.     

Acid resistance in enteric bacteria.

Shigella species require a uniquely small inoculum for causing dysentery. One explanation for the low infective dose is that Shigella species are better able to survive the acidic conditions encountered in the stomach than are other enteric pathogens. We have tested Shigella species, Escherichia coli, and Salmonella species for the ability to survive at pH 2.5 for at least 2 h. Most isolates of Shigella and E. coli survived this treatment, whereas none of the Salmonella isolates were able to do so. The ability of Shigella species to survive at low pHs does not require the presence of the large virulence plasmid or growth at 37 degrees C but is strikingly dependent on growth phase. We have also found that Shigella isolates exposed to acid lose the ability to invade epithelial cells.     

DNA probes for studying streptothricin resistance evolution in enteric bacteria

Probes for the detection of streptothricin resistance genes have been derived from recombinant plasmids. These include the streptothricin resistance gene probe sat 1/2 derived from Tn 1826 and specific for both the sat-1 determinant of Tn 1825 and the sat-2 determinant of Tn 1826, and the probe sat D derived from and specific for the sat-1 determinant of transposon Tn 1825. A third streptothricin resistance gene probe, sat 3, represents the streptothricin resistance determinant sat-3 of the IncQ R plasmid pIE639. Hybridization studies did not reveal any sequence homology between sat-3 and the transposon-localized sat-1 and sat-2 determinants. Moreover, non of the different sat-determinants isolated from plasmids of gram negative bacteria hybridized with the analogous resistance determinant of Streptomyces noursei, which had been cloned and named nat by Krügel et al. (Gene, 1988, 62, 209-214). The sat 1/2 probe in combination with the sat D probe proved to be suitable for the identification and the differentiation of sat-1 and sat-2 determinants in different genetic environments. Streptothricin resistance genes related to those present on transposons Tn 1825 and Tn 1826 have been detected by hybridization with the probe sat 1/2 on plasmids isolated a long time ago before the application of streptothricins. The sat-3 determinant appears to be exclusively associated with the IncQ plasmid pIE639.     

Small-colony forms of enteric bacteria after exposure to aminoglycosides.

Small colony forms of enteric bacteria were isolated in vitro after exposure of near-lethal amounts of aminoglycosides. These small-colony forms were several times more resistant to all aminoglycosides than the parent clones. A small form of Escherichia coli was isolated directly from a clinical specimen, along with a more sensitive larger colony.     

New vaccines against enteric bacteria for children in less developed countries

Diarrheal diseases represent a major threat to infant survival in less developed countries. A real opportunity now exists to help alleviate this problem through the development of safe and effective multicomponent whole-bacterial cell vaccine(s) against enterotoxigenic Escherichia coli and Shigella, two important pathogens for which no licensed vaccine exists. What is preventing realization of this achievement is a lack of focus on the unique needs of children in less developed countries, along with committed and sufficient funding directed toward this goal. Live-attenuated and inactivated whole-cell vaccine candidates, some of which have languished too long, are available for testing, which, if performed in a coordinated fashion, can answer key unresolved issues concerning mucosal vaccination against enteric diseases. These candidate vaccines potentially provide a relatively simple intervention which could, if implemented, reduce the impact of these diseases upon the life and productivity of children.     

In vitro conjugative transfer of VanA vancomycin resistance betweenEnterococci andListeriae of different species

In a study designed to gain data on the in vitro transferability of vancomycin resistance from enterococci of the VanA phenotype to listeriae of different species, three clinicalEnterococcus isolates —Enterococcus faecium LS10,Enterococcus faecalis LS4, andEnterococcus faecalis A3208, all harboring a plasmid that strongly hybridized with avanA probe — were used as donors in transfer experiments. Strains of fiveListeria species were used as recipients. FromEnterococcus faecium LS10, glycopeptide resistance was transferred toListeria monocytogenes, Listeria ivanovii, andListeria welshimeri recipients, whereas no transfer occurred toListeria seeligeri orListeria innocua strains. From the twoEnterococcus faecalis isolates, no transfer occurred to anyListeria recipient. MICs of both vancomycin and teicoplanin were 256 mg/l for all transconjugants tested. Furthermore, all transconjugants harbored a plasmid that strongly hybridized with thevanA probe, withvanA consistently located in anEcoRI fragment of about 4 kb. Exposure ofListeria transconjugants to vancomycin resulted in synthesis of a membrane protein similar in size (39 kDa) to a vancomycin-induced membrane protein ofEnterococcus faecium LS10. In retransfer experiments withListeria transconjugants used as donors, glycopeptide resistance was transferred to allListeria recipients tested, including strains ofListeria innocua andListeria seeligeri, which were unable to receive the resistance fromEnterococcus faecium LS10. The frequency ofvanA transfer to listerial recipients was greater in retransfer experiments than in the primary matings. These findings suggest that thevanA resistance determinant might spread to the established pathogenListeria monocytogenes, both directly from a resistant enterococcus and through strains of nonpathogenicListeria species acting as intermediate resistance vehicles.     

Comparison of phage-mediated and conjugative transfer of staphylococcal plasmids in vitro and in vivo

A strain of Staphylococcus aureus was constructed with which to compare transfer of resistance plasmids by the mechanisms of phage-mediated conjugation and conjugation. Transfer by each mechanism could be distinguished by the patterns of resistances transferred. Conjugation was favoured on dry absorbent surfaces, e.g., human skin, tissue and surgical gauze, whereas phage-mediated conjugation was favoured in fluids, e.g., milk and urine. The degree of hydration of the mating cells is postulated as one factor determining whether plasmids are transferred by phage-mediated conjugation or conjugation. Preliminary evidence indicates that topical creams and ointments affect the conjugative transfer of plasmids.     

Allergen gene transfer

DNA-based immunization induces a biased Th1 immune response, and offers a novel strategy for modulation of the Th2-associated response. Recent studies have provided evidence that antigen-induced allergic responses can be modulated in rodents immunized with plasmid DNAs encoding the sensitizing antigens. Further understanding of the regulatory mechanism involved and optimization of gene transfer/expression systems will assist greatly in proving the clinical utility of this process.     

Biodiversity and horizontal gene transfer in culturable bacteria isolated from activated sludge enriched in nonylphenol ethoxylates

One hundred and twenty bacterial isolates, from activated sludge of a treatment plant collecting wastes enriched in ethoxylated nonylphenols, were studied. Sixty isolates were selected on rich medium and 60 on mineral medium containing two nonylphenol ethoxylates as the sole carbon source. Analysis of biodiversity at the species level was performed by comparing the AluI restriction patterns of the 16S ribosomal DNA amplified by PCR from 120 isolates. The rDNA restriction analysis enabled us to cluster the isolates into 15 groups, five of which represented nearly 77% of the community. Phylogenetic analysis of five strains belonging to these main groups made it possible to assign four of them to the genera Acinetobacter, Aeromonas and Shewanella and one to the Proteus group. The analysis of plasmid content showed a high variability and suggested that horizontal gene transfer had taken place at the intraspecific, interspecific and intergeneric levels.     

Molecular characterization of turkey enteric reovirus S3 gene

The molecular diversity in S3 gene sequences of turkey reovirus (TRV) was determined in poult enteritis syndrome (PES)-affected and apparently healthy turkey poults. Twenty-nine TRV-positive samples (15 from PES-affected flocks and 14 from apparently healthy flocks) were tested using self-designed primers for the S3 gene. Phylogenetic analysis revealed that the TRV S3 sequences of this study clustered in clade III and formed two different groups in this clade. The avian reoviruses from duck and goose formed clade I and those from chickens formed clade II. The clade III TRV sequences had a nucleotide percent identity of 88.9 to 100% among themselves but only of 59.5 to 63.5% and 69.2 to 72.6% with clades I and II, respectively. More amino acid substitutions were present in TRVs from PES-affected flocks than in those from apparently healthy flocks using ATCC VR-818 (AY444912) as a benchmark. All TRVs of this study showed substitutions at positions 244 and 285. The impact of these changes on the virulence of the virus, if any, needs to be studied.     

Commensal enteric bacteria lipopolysaccharide impairs host defense against disseminated Candida albicans fungal infection

Commensal enteric bacteria maintain systemic immune responsiveness that protects against disseminated or localized infection in extra-intestinal tissues caused by pathogenic microbes. However, as shifts in infection susceptibility after commensal bacteria eradication have primarily been probed using viruses, the broader applicability to other pathogen types remains undefined. In sharp contrast to diminished antiviral immunity, we show commensal bacteria eradication bolsters protection against disseminated Candida albicans fungal infection. Enhanced antifungal immunity reflects more robust systemic expansion of Ly6G^hiLy6C^int neutrophils, and their mobilization into infected tissues among antibiotic-treated compared with commensal bacteria-replete control mice. Reciprocally, depletion of neutrophils from expanded levels or intestinal lipopolysaccharide reconstitution overrides the antifungal protective benefits conferred by commensal bacteria eradication. This discordance in antifungal compared with antiviral immunity highlights intrinsic differences in how commensal bacteria control responsiveness for specific immune cell subsets, because pathogen-specific CD8⁺ T cells that protect against viruses were suppressed similarly after C. albicans and influenza A virus infection. Thus, positive calibration of antiviral immunity by commensal bacteria is counterbalanced by restrained activation of other immune components that confer antifungal immunity.