Experimental Evaluation of Host Adaptation of Lactobacillus reuteri to Different Vertebrate Species.

The species Lactobacillus reuteri has diversified into host-specific lineages, implying a long-term association with different vertebrates. Strains from rodent lineages show specific adaptations to mice, but the processes underlying the evolution of L. reuteri in other hosts remain unknown. We administered three standardized inocula composed of strains from different host-confined lineages to mice, pigs, chickens, and humans. The ecological performance of each strain in the gastrointestinal tract of each host was determined by typing random colonies recovered from fecal samples collected over five consecutive days postadministration. Results revealed that rodent strains were predominant in mice, confirming previous findings of host adaptation. In chickens, poultry strains of the lineage VI (poultry VI) and human isolates from the same lineage (human VI) were recovered at the highest and second highest rates, respectively. Interestingly, human VI strains were virtually undetected in human feces. These findings, together with ancestral state reconstructions, indicate poultry VI and human VI strains share an evolutionary history with chickens. Genomic analysis revealed that poultry VI strains possess a large and variable accessory genome, whereas human VI strains display low genetic diversity and possess genes encoding antibiotic resistance and capsular polysaccharide synthesis, which might have allowed temporal colonization of humans. Experiments in pigs and humans did not provide evidence of host adaptation of L. reuteri to these hosts. Overall, our findings demonstrate host adaptation of L. reuteri to rodents and chickens, supporting a joint evolution of this bacterial species with several vertebrate hosts, although questions remain about its natural history in humans and pigs.IMPORTANCE Gut microbes are often hypothesized to have coevolved with their vertebrate hosts. However, the evidence is sparse and the evolutionary mechanisms have not been identified. We developed and applied an experimental approach to determine host adaptation of L. reuteri to different hosts. Our findings confirmed adaptation to rodents and provided evidence of adaptation to poultry, suggesting that L. reuteri evolved via natural selection in different hosts. By complementing phylogenetic analyses with experimental evidence, this study provides novel information about the mechanisms driving host-microbe coevolution with vertebrates and serve as a basis to inform the application of L. reuteri as a probiotic for different host species.

Optimisation of methods for bacterial skin microbiome investigation: primer selection and comparison of the 454 versus MiSeq platform.

BACKGROUND: The composition of the skin microbiome is predicted to play a role in the development of conditions such as atopic eczema and psoriasis. 16S rRNA gene sequencing allows the investigation of bacterial microbiota. A significant challenge in this field is development of cost effective high throughput methodologies for the robust interrogation of the skin microbiota, where biomass is low. Here we describe validation of methodologies for 16S rRNA (ribosomal ribonucleic acid) gene sequencing from the skin microbiome, using the Illumina MiSeq platform, the selection of primer to amplify regions for sequencing and we compare results with the current standard protocols.. METHODS: DNA was obtained from two low density mock communities of 11 diverse bacterial strains (with and without human DNA supplementation) and from swabs taken from the skin of healthy volunteers. This was amplified using primer pairs covering hypervariable regions of the 16S rRNA gene: primers 63F and 519R (V1-V3); and 347F and 803R (V3-V4). The resultant libraries were indexed for the MiSeq and Roche454 and sequenced. Both data sets were denoised, cleaned of chimeras and analysed using QIIME. RESULTS: There was no significant difference in the diversity indices at the phylum and the genus level observed between the platforms. The capture of diversity using the low density mock community samples demonstrated that the primer pair spanning the V3-V4 hypervariable region had better capture when compared to the primer pair for the V1-V3 region and was robust to spiking with human DNA. The pilot data generated using the V3-V4 region from the skin of healthy volunteers was consistent with these results, even at the genus level (Staphylococcus, Propionibacterium, Corynebacterium, Paracoccus, Micrococcus, Enhydrobacter and Deinococcus identified at similar abundances on both platforms). CONCLUSIONS: The results suggest that the bacterial community diversity captured using the V3-V4 16S rRNA hypervariable region from sequencing using the MiSeq platform is comparable to the Roche454 GS Junior platform. These findings provide evidence that the optimised method can be used in human clinical samples of low bacterial biomass such as the investigation of the skin microbiota.

Evaluation of PacBio sequencing for full-length bacterial 16S rRNA gene classification.

BACKGROUND: Currently, bacterial 16S rRNA gene analyses are based on sequencing of individual variable regions of the 16S rRNA gene (Kozich, et al Appl Environ Microbiol 79:5112-5120, 2013).This short read approach can introduce biases. Thus, full-length bacterial 16S rRNA gene sequencing is needed to reduced biases. A new alternative for full-length bacterial 16S rRNA gene sequencing is offered by PacBio single molecule, real-time (SMRT) technology. The aim of our study was to validate PacBio P6 sequencing chemistry using three approaches: 1) sequencing the full-length bacterial 16S rRNA gene from a single bacterial species Staphylococcus aureus to analyze error modes and to optimize the bioinformatics pipeline; 2) sequencing the full-length bacterial 16S rRNA gene from a pool of 50 different bacterial colonies from human stool samples to compare with full-length bacterial 16S rRNA capillary sequence; and 3) sequencing the full-length bacterial 16S rRNA genes from 11 vaginal microbiome samples and compare with in silico selected bacterial 16S rRNA V1V2 gene region and with bacterial 16S rRNA V1V2 gene regions sequenced using the Illumina MiSeq. RESULTS: Our optimized bioinformatics pipeline for PacBio sequence analysis was able to achieve an error rate of 0.007% on the Staphylococcus aureus full-length 16S rRNA gene. Capillary sequencing of the full-length bacterial 16S rRNA gene from the pool of 50 colonies from stool identified 40 bacterial species of which up to 80% could be identified by PacBio full-length bacterial 16S rRNA gene sequencing. Analysis of the human vaginal microbiome using the bacterial 16S rRNA V1V2 gene region on MiSeq generated 129 operational taxonomic units (OTUs) from which 70 species could be identified. For the PacBio, 36,000 sequences from over 58,000 raw reads could be assigned to a barcode, and the in silico selected bacterial 16S rRNA V1V2 gene region generated 154 OTUs grouped into 63 species, of which 62% were shared with the MiSeq dataset. The PacBio full-length bacterial 16S rRNA gene datasets generated 261 OTUs, which were grouped into 52 species, of which 54% were shared with the MiSeq dataset. Alpha diversity index reported a higher diversity in the MiSeq dataset. CONCLUSION: The PacBio sequencing error rate is now in the same range of the previously widely used Roche 454 sequencing platform and current MiSeq platform. Species-level microbiome analysis revealed some inconsistencies between the full-length bacterial 16S rRNA gene capillary sequencing and PacBio sequencing.

Antibiotic susceptibility profile of bacilli isolated from the skin of healthy humans

Abstract In the present work, twelve bacilli were isolated from four different regions of human skin from Bela population of Nagpur district, India. The isolated bacilli were identified by their morphological, cultural and biochemical characteristics. Seven isolates were Gram negative rods, out of which five were belong to genus Pseudomonas. Three among the five Gram positive isolates were identified as Dermabactor and the remaining two Bacillus. Their antimicrobial susceptibility profile was determined by Kirby-Bauer disc diffusion method. The isolates showed resistance to several currently used broad-spectrum antibiotics. The Dermabactor genus was resistant to vancomycin, although it was earlier reported to be susceptible. Imipenem was found to be the most effective antibiotic for Pseudomonas while nalidixic acid, ampicillin and tetracycline were ineffective. Isolates of Bacillus displayed resistance to the extended spectrum antibiotics cephalosporin and ceftazidime. Imipenem, carbenicillin and ticarcillin were found to be the most effective antibiotics as all the investigated isolates were susceptible to them. Antibiotic resistance may be due to the overuse or misuse of antibiotics during the treatment, or following constant exposure to antibiotic-containing cosmetic formulations.

Draft genome sequence of Inquilinus limosus strain MP06, a multidrug-resistant clinical isolate

The bacterium, Inquilinus limosus, with its remarkable antimicrobial multiresistant profile, has increasingly been isolated in cystic fibrosis patients. We report draft genome sequence of a strain MP06, which is of considerable interest in elucidating the associated mechanisms of antibiotic resistance in this bacterium and for an insight about its persistence in airways of these patients.

Synergistic effects of sulbactam in multi-drug-resistant Acinetobacter baumannii

Abstract Acinetobacter baumannii is a frequently isolated etiologic agent of nosocomial infections, especially in intensive care units. With the increase in multi-drug resistance of A. baumannii isolates, finding appropriate treatment alternatives for infections caused by these bacteria has become more difficult, and available alternate treatments include the use of older antibiotics such as colistin or a combination of antibiotics. The current study aimed to evaluate the in vitro efficacy of various antibiotic combinations against multi-drug resistant A. baumannii strains. Thirty multi-drug and carbapenem resistant A. baumannii strains isolated at the Ankara Training and Research Hospital between June 2011 and June 2012 were used in the study. Antibiotic susceptibility tests and species-level identification were performed using conventional methods and the VITEK 2 system. The effects of meropenem, ciprofloxacin, amikacin, tigecycline, and colistin alone and in combination with sulbactam against the isolates were studied using Etest (bioMérieux) in Mueller-Hinton agar medium. Fractional inhibitory concentration index (FIC) was used to determine the efficacy of the various combinations. While all combinations showed a predominant indifferent effect, a synergistic effect was also observed in 4 of the 5 combinations. Synergy was demonstrated in 43% of the isolates with the meropenem-sulbactam combination, in 27% of the isolates with tigecycline-sulbactam, and in 17% of the isolates with colistin-sulbactam and amikacin-sulbactam. No synergy was detected with the sulbactam-ciprofloxacin combination and antagonism was detected only in the sulbactam-colistin combination (6.66% of the isolates). Antibiotic combinations can be used as an alternative treatment approach in multi-drug resistant A. baumannii infections.

Microbes central to human reproduction.

As studies uncover the breadth of microbes associated with human life, opportunities will emerge to manipulate and augment their functions in ways that improve health and longevity. From involvement in the complexities of reproduction and fetal/infant development, to delaying the onset of disease, and indeed countering many maladies, microbes offer hope for human well-being. Evidence is emerging to suggest that microbes may play a beneficial role in body sites traditionally viewed as being sterile. Although further evidence is required, we propose that much of medical dogma is about to change significantly through recognition and understanding of these hitherto unrecognized microbe-host interactions. A meeting of the International Scientific Association for Probiotics and Prebiotics held in Aberdeen, Scotland (June 2014), presented new views and challenged established concepts on the role of microbes in reproduction and health of the mother and infant. This article summarizes some of the main aspects of these discussions.

Fecal microbiomes of non-human primates in Western Uganda reveal species-specific communities largely resistant to habitat perturbation.

Primate gastrointestinal microbial communities are becoming increasingly appreciated for their relevance to comparative medicine and conservation, but the factors that structure primate "microbiomes" remain controversial. This study examined a community of primates in Kibale National Park, Uganda, to assess the relative importance of host species and location in structuring gastrointestinal microbiomes. Fecal samples were collected from primates in intact forest and from primates in highly disturbed forest fragments. People and livestock living nearby were also included, as was a geographically distant population of related red colobus in Kenya. A culture-free microbial community fingerprinting technique was used to analyze fecal microbiomes from 124 individual red colobus (Procolobus rufomitratus), 100 individual black-and-white colobus (Colobus guereza), 111 individual red-tailed guenons (Cercopithecus ascanius), 578 human volunteers, and 364 domestic animals, including cattle (Bos indicus and B. indicus × B. taurus crosses), goats (Caprus hircus), sheep (Ovis aries), and pigs (Sus scrofa). Microbiomes sorted strongly by host species, and forest fragmentation did not alter this pattern. Microbiomes of Kenyan red colobus sorted distinctly from microbiomes of Ugandan red colobus, but microbiomes from these two red colobus populations clustered more closely with each other than with any other species. Microbiomes from red colobus and black-and-white colobus were more differentiated than would be predicted by the phylogenetic relatedness of these two species, perhaps reflecting heretofore underappreciated differences in digestive physiology between the species. Within Kibale, social group membership influenced intra-specific variation among microbiomes. However, intra-specific variation was higher among primates in forest fragments than among primates in intact forest, perhaps reflecting the physical separation of fragments. These results suggest that, in this system, species-specific processes such as gastrointestinal physiology strongly structure microbial communities, and that primate microbiomes are relatively resistant to perturbation, even across large geographic distances or in the face of habitat disturbance.

Homo sapiens as physician and patient: a view from Darwinian medicine.

Medicine's cardinal diagnostic and therapeutic resource is the clinical encounter. Over the last two centuries and particularly over the last five decades the function of the clinical encounter has been eroded to the point of near irrelevance because of the atomized and atomizing influence of technology and microspecialization. Meanwhile, over the past five decades the exceptionalist view of Homo sapiens inherent in the social and religious traditions of the West has similarly undergone radical changes. H. sapiens is now best understood as a microecosystem integrated into a much broader ecosystem: the biosphere. That human microecosystem is composed of constituents derived from the archaeal, bacterial, and eukaryan domains via endosymbiotic, commensalistic and mutualistic interactions. This amalgamation of 100 trillion cells and viral elements is regulated by a composite genome aggregated over the 3.8 billion years of evolutionary history of organic life. No component of H. sapiens or its genome can be identified as irreducibly and exclusively human. H. sapiens' humanity is an emergent property of the microecosystem. Ironically as H. sapiens is viewed by evolutionary science in a highly integrated manner medicine approaches it as a balkanized, deaggregated entity through the eye of 150 different specialties. To effectively address the needs of H sapiens in its role as patient by the same species in its role as physician the disparate views must be harmonized. Here I review some conceptual elements that would assist a physician in addressing the needs of the patient in integrum, as a microecosystem, by the former address the latter as a historical gestalt being. The optimal way to recover the harmony between patient and physician is through a revitalization of the clinical encounter via an ecological and Darwinian epistemology.

Comparative genomics of Brachyspira pilosicoli strains: genome rearrangements, reductions and correlation of genetic compliment with phenotypic diversity.

BACKGROUND: The anaerobic spirochaete Brachyspira pilosicoli causes enteric disease in avian, porcine and human hosts, amongst others. To date, the only available genome sequence of B. pilosicoli is that of strain 95/1000, a porcine isolate. In the first intra-species genome comparison within the Brachyspira genus, we report the whole genome sequence of B. pilosicoli B2904, an avian isolate, the incomplete genome sequence of B. pilosicoli WesB, a human isolate, and the comparisons with B. pilosicoli 95/1000. We also draw on incomplete genome sequences from three other Brachyspira species. Finally we report the first application of the high-throughput Biolog phenotype screening tool on the B. pilosicoli strains for detailed comparisons between genotype and phenotype. RESULTS: Feature and sequence genome comparisons revealed a high degree of similarity between the three B. pilosicoli strains, although the genomes of B2904 and WesB were larger than that of 95/1000 (~2,765, 2.890 and 2.596 Mb, respectively). Genome rearrangements were observed which correlated largely with the positions of mobile genetic elements. Through comparison of the B2904 and WesB genomes with the 95/1000 genome, features that we propose are non-essential due to their absence from 95/1000 include a peptidase, glycine reductase complex components and transposases. Novel bacteriophages were detected in the newly-sequenced genomes, which appeared to have involvement in intra- and inter-species horizontal gene transfer. Phenotypic differences predicted from genome analysis, such as the lack of genes for glucuronate catabolism in 95/1000, were confirmed by phenotyping. CONCLUSIONS: The availability of multiple B. pilosicoli genome sequences has allowed us to demonstrate the substantial genomic variation that exists between these strains, and provides an insight into genetic events that are shaping the species. In addition, phenotype screening allowed determination of how genotypic differences translated to phenotype. Further application of such comparisons will improve understanding of the metabolic capabilities of Brachyspira species.

Drug-resistant human Staphylococcus aureus in sanctuary apes pose a threat to endangered wild ape populations.

Reintroduction of sanctuary apes to natural habitat is considered an important tool for conservation; however, reintroduction has the potential to endanger resident wild apes through the introduction of human pathogens. We found a high prevalence of drug-resistant, human-associated lineages of Staphylococcus aureus in sanctuary chimpanzees (Pan troglodytes) from Zambia and Uganda. This pathogen is associated with skin and soft tissue diseases and severe invasive infections (i.e. pneumonia and septicemia). Colonization by this bacterium is difficult to clear due to frequent recolonization. In addition to its pathogenic potential, human-related S. aureus can serve as an indicator organism for the transmission of other potential pathogens like pneumococci or mycobacteria. Plans to reintroduce sanctuary apes should be reevaluated in light of the high risk of introducing human-adapted S. aureus into wild ape populations where treatment is impossible.

Action of mouthwashes on Staphylococcus spp: isolated in the saliva of community and hospitalized individuals / Ação de anti-sépticos bucais sobre Staphylococcus spp. isolados na saliva de comunidade e de internação individual

The use of mouthwashes in critical patients has been a source of concern for health professionals due to the diverse range of products, causing uncertainty about which is the most indicated. This study aimed to assess the susceptibility of Staphylococcus spp. isolated in the saliva of individuals from the community and patients in Intensive Care Units (ICU) as to antiseptic mouthwashes. The following oral antiseptics were assessed: cetylpyridinium chloride solution, Listerine® and Neen®. Calcium alginate swab was used for saliva collection to isolate Staphylococcus spp. Microbiological processing involved growth, isolation, strain identification and determination of MID (maximum inhibitory dilution). MID was considered the greatest dilution that completely inhibited the strains. The products efficacy was analyzed by a two-factor ANOVA repeated measures and by Bonferroni adjustments in multiple comparisons, considering a significance level of α=0.05. In total, 80 strains of Staphylococcus spp. were isolated, 40 from ICU patients and 40 from community individuals. MID results revealed that cetylpyridinium chloride solution presented better results in comparison to other products, that is, 39 (97.5 percent) strains from hospital patients with MID 1:128, and 37 (92.5 percent) of individuals from the community had MID 1:64. Neen® inhibited all strains in both groups at a dilution from 1:2 to 1:4. Listerine® presented the worst MID results, 65 percent of the strains from individuals from the community and 10 percent of hospital strains were not inhibited at a dilution of 1:2.

O uso de antissépticos bucais tem sido uma das preocupações dos profissionais de saúde considerando a diversidade de produtos, o que traz a insegurança sobre qual é o mais adequado. Objetivou-se avaliar a suscetibilidade de Staphylococcus spp. isolado da saliva de indivíduos adultos da comunidade e do hospital frente a antissépticos bucais. Os antissépticos avaliados foram: solução de Cloreto de Cetilpiridínio, Listerine® e Neen®. Na coleta de saliva para o isolamento do Staphylococcus spp. utilizou-se o swab de alginato de cálcio. O processamento microbiológico envolveu o crescimento, isolamento, identificação das cepas, determinação da DIM. Foi considerada DIM a maior diluição que inibiu completamente as cepas. A eficácia dos produtos foi avaliada por meio da análise de variância (ANOVA) bilateral com medidas repetidas, e pelo método de ajuste de Bonferroni nas comparações múltiplas, com um nível de significância α=0,05. Totalizou-se 80 cepas de Staphylococcus spp. sendo 40 isoladas de pacientes da UTI e 40 de indivíduos da comunidade. Comparando os resultados é possível verificar que a solução de cloreto de cetilpiridínio apresentou melhores resultados em relação aos demais produtos, ou seja, 39 (97,5 por cento) das cepas hospitalares DIM 1:128 e da comunidade 37 (92,5 por cento) DIM 1:64. Para Neen® nos dois grupos a DIM foi de 1:2 e 1:4. Listerine® apresentou o pior resultado em termos da DIM, sendo que 65 por cento das cepas da comunidade e 10 por cento hospitalar não foram inibidas na diluição 1:2.

Examination of Campylobacter jejuni putative adhesins leads to the identification of a new protein, designated FlpA, required for chicken colonization.

Campylobacter jejuni colonization of chickens is presumably dependent upon multiple surface-exposed proteins termed adhesins. Putative C. jejuni adhesins include CadF, CapA, JlpA, major outer membrane protein, PEB1, Cj1279c, and Cj1349c. We examined the genetic relatedness of 97 C. jejuni isolates recovered from human, poultry, bovine, porcine, ovine, and canine sources by multilocus sequence typing (MLST) and examined their profile of putative adhesin-encoding genes by dot blot hybridization. To assess the individual contribution of each protein in bacterium-host cell adherence, the C. jejuni genes encoding the putative adhesins were disrupted by insertional mutagenesis. The phenotype of each mutant was judged by performing in vitro cell adherence assays with chicken LMH hepatocellular carcinoma epithelial cells and in vivo colonization assays with broiler chicks. MLST analysis indicated that the C. jejuni isolates utilized in this study were genetically diverse. Dot blot hybridization revealed that the C. jejuni genes encoding the putative adhesins, with the exception of capA, were conserved among the isolates. The C. jejuni CadF, CapA, Cj1279c, and Cj1349c proteins were found to play a significant role in the bacterium's in vitro adherence to chicken epithelial cells, while CadF, PEB1, and Cj1279c were determined to play a significant role in the bacterium's in vivo colonization of broiler chicks. Collectively, the data indicate that Cj1279c is a novel adhesin. Because Cj1279c harbors fibronectin type III domains, we designated the protein FlpA, for fibronectin-like protein A.

Danish integrated antimicrobial resistance monitoring and research program.

Resistance to antimicrobial agents is an emerging problem worldwide. Awareness of the undesirable consequences of its widespread occurrence has led to the initiation of antimicrobial agent resistance monitoring programs in several countries. In 1995, Denmark was the first country to establish a systematic and continuous monitoring program of antimicrobial drug consumption and antimicrobial agent resistance in animals, food, and humans, the Danish Integrated Antimicrobial Resistance Monitoring and Research Program (DANMAP). Monitoring of antimicrobial drug resistance and a range of research activities related to DANMAP have contributed to restrictions or bans of use of several antimicrobial agents in food animals in Denmark and other European Union countries.