Pneumonia Due to Mycoplasma in Gnotobiotic Mice I. Pathogenicity of \u3cem\u3eMycoplasma pneumoniae\u3c/em\u3e, \u3cem\u3eMycoplasma salivarium\u3c/em\u3e, and \u3cem\u3eMycoplasma pulmonis\u3c/em\u3e for the Lungs of Conventional and Gnotobiotic Mice

Pneumonia due to mycoplasma in gnotobiotic mice. I. Pathogenicity of Mycoplasma pneumoniae, Mycoplasma salivarium, and Mycoplasma pulmonis for the lungs of conventional and gnotobiotic mice. J. Bacteriol. 92:1154–1163. 1966.—Two species of mycoplasma of human origin, Mycoplasma pneumoniae and M. salivarium, were tested for their ability to produce respiratory disease in the Ha/ICR mouse when inoculated by the intranasal route. The mouse pathogen M. pulmonis was studied as a positive control. Conventional and gnotobiotic Ha/ICR mice were employed, the latter to provide a system free from indigenous mycoplasma and bacteria. Pneumonia from which mycoplasma were isolated was produced in all groups of the conventional Ha/ICR mice, including those inoculated with sterile broth. Only M. pulmonis produced disease when inoculated intranasally into the gnotobiotic mice, and the gross and microscopic lesions resembled those described in conventional mice. The gnotobiotic mouse provided a tool to study the pathogenicity of different mycoplasma species, and indicated marked differences in host specificity that could not be clearly seen when conventional mice were used

Regional and global changes in TCR [alpha][beta] T cell repertoires in the gut are dependent upon the complexity of the enteric microflora

The repertoire of gut associated T cells is shaped by exposure to microbes, including the natural enteric microflora. Previous studies compared the repertoire of gut associated T cell populations in germ free (GF) and conventional mammals often focussing on intra-epithelial lymphocyte compartments. Using GF, conventional and monocolonised (gnotobiotic) chickens and chicken TCRbeta-repertoire analysis techniques, we determined the influence of microbial status on global and regional enteric TCRbeta repertoires. The gut of conventionally reared chickens exhibited non-Gaussian distributions of CDR3-lengths with some shared over-represented peaks in neighbouring gut segments. Sequence analysis revealed local clonal over-representation. Germ-free chickens exhibited a polyclonal, non-selected population of T cells in the spleen and in the gut. In contrast, gnotobiotic chickens exhibited a biased repertoire with shared clones evident throughout the gut. These data indicate the dramatic influence of enteric microflora complexity on the profile of TCRbeta repertoire in the gut at local and global levels

Alpha/beta and gamma interferons are induced by infection with noncytopathic bovine viral diarrhea virus in vivo

In contrast to the results of previous in vitro studies, experimental infection of calves with noncytopathic bovine viral diarrhea virus (ncpBVDV) was found to induce strong alpha/beta and gamma interferon responses in gnotobiotic animals. These responses were associated with depressed levels of transforming growth factor β (TGF-β) in serum. The results of this study indicate that the immunosuppression caused by ncpBVDV is not associated with low interferon responses or elevated levels of TGF-β

Recombinant DnaK orally administered protects axenic European sea bass against vibriosis

Vibrio anguillarum causes high mortality in European sea bass (Dicentrarchus labrax) larviculture and is a hindering factor for successful sustainable aquaculture of this commercially valuable species. Priming of the innate immune system through administration of immunostimulants has become an important approach to control disease outbreaks in marine fish larviculture. This study was conducted to evaluate immunostimulation by Escherichia coli HSP70 (DnaK) in axenic European sea bass larvae in order to protect the larvae against vibriosis. DnaK stimulates the immune response in crustaceans and juvenile fish against bacterial infections. The use of axenic fish larvae allows to study immunostimulation in the absence of an interfering microbial community. At 7 days post-hatching, larvae received a single dose of alginate encapsulated recombinant DnaK. Two non-treated control groups in which animals either received empty alginate microparticles (C1) or no alginante microparticles (C2 and C3) were included in the study. Eighteen hours later, all larvae, except the ones from group C3 (non-infected control) were challenged with V. anguillarum (10(5) CFU, bath infection). Mortality was daily recorded until 120 h post infection and at 18, 24, and 36 h post infection, larvae were sampled for expression of immune related genes. Results showed that V. anguillarum induced an immune response in axenic sea bass larvae but that the innate immune response was incapable to protect the larvae against deadly septicaemic disease. In addition, we showed that administration of alginate encapsulated DnaK to axenic European sea bass larvae at DAH7 resulted in a significant, DnaK dose dependent, upreglation of immune sensor, regulatory and effector genes. Significant upregulation of cxcr4, cas1 and especially of hep and dic was correlated with significant higher survival rates in V. anguillarum infected larvae. In the future recombinant DnaK might perhaps be used as a novel immunostimulant in sea bass larviculture

Antibiotic residues and R-plasmid selection: are in vitro methods good models?

Three clones of E. coli, one of which was harbouring a tetracycline resistance plasmid were inoculated together into the stomach of axenic mice. Without antibiotic selective pressure, the R-Plasmid bearing strain became dominant in the faeces of mice, while the R-plasmid free strain was eliminated. When the R-plasmid bearing strain was given to mice 4 days after the inoculation with the R-plasmid free strain, it was repressed and remained at the stable level of 10(4.5) organisms per g of faeces. But a rapid spread of the R-plasmid was observed, tetracycline resistant bacteria become dominant within one day, and replace the tetracycline sensitive E. coli. The tetracycline resistance plasmid did not disadvantage the mediating strain in the gut, even in the absence of antibiotic pressure. In contrast Lebek and Egger (1983), studying the same strains in vitro, found that in a chemostat the plasmid bearing strain was overgrown by the plasmid free strain. These results strongly suggest that in vitro interactions between E. coli strains cannot be directly extrapolated to in vivo conditions. For the determination of the no-effect level of antibiotic residue on the selection of R-factor in the gut, studies should be made in vivo

Does virulence assessment of Vibrio anguillarum using sea bass (Dicentrarchus labrax) larvae correspond with genotypic and phenotypic characterization?

Background: Vibriosis is one of the most ubiquitous fish diseases caused by bacteria belonging to the genus Vibrio such as Vibrio (Listonella) anguillarum. Despite a lot of research efforts, the virulence factors and mechanism of V. anguillarum are still insufficiently known, in part because of the lack of standardized virulence assays. Methodology/Principal Findings: We investigated and compared the virulence of 15 V. anguillarum strains obtained from different hosts or non-host niches using a standardized gnotobiotic bioassay with European sea bass (Dicentrarchus labrax L.) larvae as model hosts. In addition, to assess potential relationships between virulence and genotypic and phenotypic characteristics, the strains were characterized by random amplified polymorphic DNA (RAPD) and repetitive extragenic palindromic PCR (rep-PCR) analyses, as well as by phenotypic analyses using Biolog's Phenotype MicroArray (TM) technology and some virulence factor assays. Conclusions/Significance: Virulence testing revealed ten virulent and five avirulent strains. While some relation could be established between serotype, genotype and phenotype, no relation was found between virulence and genotypic or phenotypic characteristics, illustrating the complexity of V. anguillarum virulence. Moreover, the standardized gnotobiotic system used in this study has proven its strength as a model to assess and compare the virulence of different V. anguillarum strains in vivo. In this way, the bioassay contributes to the study of mechanisms underlying virulence in V. anguillarum

An evaluation of methods to assess the effect of antimicrobial residues on the human gut flora

1. Barrier effect. Relevant models should include an anaerobic dominant flora that antagonizes minor bacterial populations such as drug resistant E. coli. 2. Anaerobes vs. aerobes. Aerobe counts are more precise and much less time consuming than anaerobe counts. Minor populations of drug resistant aerobes are sensitive markers of the ecosystem balance, and are directly relevant to the potential risk of antimicrobial residues. 3. MIC vs. plate counts. The determination of minimum inhibitory concentrations ( MIC ) of selected clones is time consuming, does not detect subdominant resistance (less than 1 %), and the MIC shift is difficult to test statistically. In contrast, direct counts of bacteria on drug supplemented media allows a rapid measure of minor resistant populations. 4. Statistics: Most published designs do not include adequate statistical evaluation. This is critical for trials made in conventional humans and animals, where data are highly variable. 5. Human trials: The lowest concentration of antibiotic tested in human volunteers (2mg oxytetracycline /d for 7d in 6 subjects) significantly increased the proportion of resistant fecal enterobacteria (P=0.05). However, the huge day-to-day and inter-individual variations of human floras make this evidence rather weak. 6. Gnotobiotic mice inoculated with human flora are living isolated models in which the effect of any antimicrobial on the human gut flora can be tested. This in vivo model does include the barrier effect of dominant anaerobes. Inter-individual and day-to-day variations of bacterial populations are lower in those mice than in humans. 7. Most resistant enterobacteria in the human gut of untreated people come from bacterial contamination of raw foods. The relative contribution of residues in selecting antibiotic resistance seems to be low when compared to bacterial contamination

Extensive horizontal gene transfer during Staphylococcus aureus co-colonization in vivo.

Staphylococcus aureus is a commensal and major pathogen of humans and animals. Comparative genomics of S. aureus populations suggests that colonization of different host species is associated with carriage of mobile genetic elements (MGE), particularly bacteriophages and plasmids capable of encoding virulence, resistance, and immune evasion pathways. Antimicrobial-resistant S. aureus of livestock are a potential zoonotic threat to human health if they adapt to colonize humans efficiently. We utilized the technique of experimental evolution and co-colonized gnotobiotic piglets with both human- and pig-associated variants of the lineage clonal complex 398, and investigated growth and genetic changes over 16 days using whole genome sequencing. The human isolate survived co-colonization on piglets more efficiently than in vitro. During co-colonization, transfer of MGE from the pig to the human isolate was detected within 4 h. Extensive and repeated transfer of two bacteriophages and three plasmids resulted in colonization with isolates carrying a wide variety of mobilomes. Whole genome sequencing of progeny bacteria revealed no acquisition of core genome polymorphisms, highlighting the importance of MGE. Staphylococcus aureus bacteriophage recombination and integration into novel sites was detected experimentally for the first time. During colonization, clones coexisted and diversified rather than a single variant dominating. Unexpectedly, each piglet carried unique populations of bacterial variants, suggesting limited transmission of bacteria between piglets once colonized. Our data show that horizontal gene transfer occurs at very high frequency in vivo and significantly higher than that detectable in vitro