RESUMEN
Clustered regularly interspaced short palindromic repeats (CRISPRs) are part of an acquired bacterial immune system that functions as a barrier to exogenous genetic elements. Since naturalized Escherichia coli are likely to encounter different genetic elements in aquatic environments compared to enteric strains, we hypothesized that such differences would be reflected within the hypervariable CRISPR alleles of these two populations. Comparison of CRISPR1 alleles from naturalized and fecal phylogroup B1 E. coli strains revealed that the alleles could be categorized into four major distinct groups (designated G6-G9), and all four allele groups were found among naturalized strains and fecal strains. The distribution of CRIPSR G6 and G8 alleles was similar among strains of both ecotypes, while naturalized strains tended to have CRISPR G7 alleles rather than G9 alleles. Since CRISPR G7 alleles were not specific to naturalized strains, they, however, would not be useful as a marker for identifying naturalized strains. Notably, CRISPR alleles from naturalized and fecal strains also had similar spacer repertoires. This indicates a shared history of encounter with mobile genetic elements and suggests that the two populations were derived from common ancestors.
Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , ADN Bacteriano/genética , Escherichia coli/genética , Variación Genética/genética , Animales , Bovinos , ADN Intergénico/genética , Escherichia coli/clasificación , Heces/microbiología , Caballos , Secuencias Repetitivas Esparcidas/genética , Tipificación de Secuencias Multilocus , Microbiología del AguaRESUMEN
Campylobacter jejuni is the most common cause of bacterium-induced gastroenteritis, and while typically self-limiting, C. jejuni infections are associated with postinfectious intestinal disorders, including flares in patients with inflammatory bowel disease and postinfectious irritable bowel syndrome (PI-IBS), via mechanisms that remain obscure. Based on the hypothesis that acute campylobacteriosis may cause pathogenic microbiota dysbiosis, we investigated whether C. jejuni may activate dormant virulence genes in noninvasive Escherichia coli and examined the epithelial pathophysiological consequences of these alterations. Microarray and quantitative real-time PCR analyses revealed that E. coli adhesin, flagellum, and hemolysin gene expression were increased when E. coli was exposed to C. jejuni-conditioned medium. Increased development of bacterial flagella upon exposure to live C. jejuni or C. jejuni-conditioned medium was observed under transmission electron microscopy. Atomic force microscopy demonstrated that the forces of bacterial adhesion to colonic T84 enterocytes, and the work required to rupture this adhesion, were significantly increased in E. coli exposed to C. jejuni-conditioned media. Finally, C. jejuni-modified E. coli disrupted TLR4 gene expression and induced proinflammatory CXCL-8 gene expression in colonic enterocytes. Together, these data suggest that exposure to live C. jejuni, and/or to its secretory-excretory products, may activate latent virulence genes in noninvasive E. coli and that these alterations may directly trigger proinflammatory signaling in intestinal epithelia. These observations shed new light on mechanisms that may contribute, at least in part, to postcampylobacteriosis inflammatory disorders.
Asunto(s)
Campylobacter jejuni/metabolismo , Medios de Cultivo Condicionados/farmacología , Enterocitos/efectos de los fármacos , Interleucina-8/inmunología , Receptor Toll-Like 4/inmunología , Campylobacter jejuni/patogenicidad , Línea Celular , Enterocitos/inmunología , Enterocitos/microbiología , Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Escherichia coli/patogenicidad , Flagelos/efectos de los fármacos , Flagelos/genética , Flagelos/metabolismo , Regulación de la Expresión Génica , Interacciones Huésped-Patógeno , Humanos , Interleucina-8/agonistas , Interleucina-8/genética , Transducción de Señal , Simbiosis , Receptor Toll-Like 4/antagonistas & inhibidores , Receptor Toll-Like 4/genética , VirulenciaRESUMEN
Structures of free-living and protozoa-associated methanogen (PAM) communities from forage-fed cattle were investigated by comparative sequence analysis of 16S rRNA and methyl coenzyme M reductase (mcrA) gene clone libraries. The free-living and protozoa-associated communities were composed of the same three genera [namely Methanobrevibacter, Methanomicrobium and rumen cluster C (RCC), which is distantly related to Thermoplasma]; however, the distribution of the methanogen genera differed between the two communities. Despite previous reports of potential bias for the degenerate mcrA primer set, the 16S rRNA (nâ=â100 clones) and mcrA (nâ=â92 clones) gene libraries exhibited a similar distribution pattern for the three methanogenic genera. RCC was more abundant in the free-living community and represented 72 and 42â% of the 16S rRNA and mrcA gene sequences, respectively, versus 54 and 13â% of the 16S rRNA and mrcA gene sequences from the PAM community, respectively. The majority of RCC sequences from the free-living and protozoa-associated communities belonged to different species-level operational taxonomic units. Methanobrevibacter species were more abundant in the PAM community and represented 42 and 79â% of clones for the 16S rRNA and mrcA gene libraries, respectively, versus 9 and 27â% of 16S rRNA and mrcA gene clones from the free-living community, respectively. Methanomicrobium species were predominantly free-living. Primers for quantitative PCR were designed to target specific methanogen groups and used to assess the effect of a high-grain diet on methanogen species composition. Switching the ruminant diet from forage to high-grain resulted in reduced protozoal diversity, along with a profound overall reduction in the relative abundance of RCC and an increase in the relative abundance of free-living Methanobrevibacter spp. It was unclear whether the reduced abundance of RCC in grain-fed animals was due to the loss of symbiotic protozoa species or due to broader changes in the rumen environment that affected both RCC and protozoa. Importantly, results from this study emphasize the need to consider the different methanogen communities when developing strategies for mitigating methane emissions in ruminants.
Asunto(s)
Biota , Metano/metabolismo , Methanobrevibacter/genética , Methanomicrobiaceae/genética , Oxidorreductasas/genética , ARN Ribosómico 16S/genética , Rumen/microbiología , Animales , Bovinos , Análisis por Conglomerados , ADN de Archaea/química , ADN de Archaea/genética , ADN Ribosómico/química , ADN Ribosómico/genética , Methanobrevibacter/metabolismo , Methanomicrobiaceae/metabolismo , Datos de Secuencia Molecular , Filogenia , Análisis de Secuencia de ADNRESUMEN
The diversity of protozoan-associated methanogens in cattle was investigated using five universal archaeal small-subunit (SSU) rRNA gene primer sets. Methanobrevibacter spp. and rumen cluster C (distantly related to Thermoplasma spp.) were predominant. Significant differences in species composition among libraries indicate that some primers used previously to characterize rumen methanogens exhibit biased amplification.