The development of culture-independent techniques has revolutionized our understanding of how our human cells interact with the even greater number of microbial inhabitants of our bodies. As part of this revolution, data are increasingly challenging the old dogma that in health, the lung mucosa is sterile. To understand how the lung microbiome may play a role in human health, we identified five major questions for lung microbiome research: (1) Is the lung sterile? (2) Is there a unique core microbiome in the lung? (3) How dynamic are the microbial populations? (4) How do pulmonary immune responses affect microbiome composition? and (5) Are the lungs influenced by the intestinal immune responses to the gut microbiome? From birth, we are exposed to continuous microbial challenges that shape our microbiome. In our changing environment, perturbation of the gut microbiome affects both human health and disease. With widespread antibiotic use, the ancient microbes that formerly resided within us are being lost, for example, Helicobacter pylori in the stomach. Animal models show that antibiotic exposure in early life has developmental consequences. Considering the potential effects of this altered microbiome on pulmonary responses will be critical for future investigations.
lung; microbiome; antibiotics; immune responses; inflammation
Persistent colonization by Helicobacter pylori, and especially by cagA-positive strains, has been related to several health outcomes with effects in opposite directions. Thus, it is important to evaluate its influence on both total and category-specific mortality.
We conducted prospective cohort analyses in a nationally representative sample of 9895 participants enrolled in the National Health and Nutrition Examination Survey III (NHANES III) to assess the association of H. pylori status with all-cause and cause-specific mortality. Analyses for the association of H. pylori cagA positivity with mortality were conducted in 7,384 subjects with data on H. pylori cagA status.
In older individuals (> 40.6 years of age), H. pylori was not associated with all-cause mortality (hazard ratio [HR], 1.00; 95% confidence interval [CI], 0.84–1.18). There was an inverse association of H. pylori status with stroke mortality (HR, 0.67; 95% CI, 0.44–1.08), and the inverse association was stronger for H. pylori cagA positivity, with the HR of 0.45 (95% CI, 0.27–0.75). H. pylori also was strongly positively related to gastric cancer mortality. After we adjusted p-values using the Benjamini–Hochberg false discovery rate (FDR) method to account for multiple comparisons, these associations remained, and H. pylori status was not related to other outcomes..
Our findings suggest that H. pylori has a mixed role in human health, but is not a major risk factor for all-cause mortality.
Epidemiology; Cohort studies; Mortality; Cardiovascular disease; Helicobacter pylori
Asthma, a serious health problem worldwide, is growing more common. The colonization of Helicobacter pylori, a major human indigenous (commensal) microbe that is present early in life may be relevant to childhood asthma risk.
We conducted cross-sectional analyses using data from 7,412 participants in the National Health and Nutrition Survey (NHANES) 1999–2000 to assess the association between H. pylori and childhood asthma.
H. pylori seropositivity was inversely associated with early-onset asthma (onset age < 5 years) and current asthma in children 3–13 years. Among participants 3–19 years of age, the presence of H. pylori was inversely related to ever having asthma (OR = 0.69; 95% CI = 0.45–1.06), and the inverse association with early childhood-onset (
This study is the first to report an inverse association of H. pylori with asthma in children. The findings indicate new directions for research and asthma prevention.
Helicobacter pylori; epidemiology; asthma; cross-sectional study
immunology; microbiology; gastric physiology; stomach; inflammation
A conference entitled ‘Human microbiome science: Vision for the future’ was organized in Bethesda, MD from July 24 to 26, 2013. The event brought together experts in the field of human microbiome research and aimed at providing a comprehensive overview of the state of microbiome research, but more importantly to identify and discuss gaps, challenges and opportunities in this nascent field. This report summarizes the presentations but also describes what is needed for human microbiome research to move forward and deliver medical translational applications.
Diet, host gene composition, and alterations in the intestinal microbiota can contribute to obesity. In microbe-induced obesity, metabolic changes stem from primary perturbation of the microbiota, consequent to modern changes in human biology. Microbiota disruption during early development can result in syndromes of metabolic dysfunction. We focus on the pathways involved in these interactions, particularly related to energy extraction and the role of inflammation in the metabolic phenotypes. Model physiologic systems and perturbations including gastric bypass surgery, pregnancy, and hibernation provide insight into the respective roles of the critical participants.
Soil-transmitted helminths colonize more than 1.5 billion people worldwide, yet little is known about how they interact with bacterial communities in the gut microbiota. Differences in the gut microbiota between individuals living in developed and developing countries may be partly due to the presence of helminths, since they predominantly infect individuals from developing countries, such as the indigenous communities in Malaysia we examine in this work. We compared the composition and diversity of bacterial communities from the fecal microbiota of 51 people from two villages in Malaysia, of which 36 (70.6%) were infected by helminths. The 16S rRNA V4 region was sequenced at an average of nineteen thousand sequences per samples. Helminth-colonized individuals had greater species richness and number of observed OTUs with enrichment of Paraprevotellaceae, especially with Trichuris infection. We developed a new approach of combining centered log-ratio (clr) transformation for OTU relative abundances with sparse Partial Least Squares Discriminant Analysis (sPLS-DA) to enable more robust predictions of OTU interrelationships. These results suggest that helminths may have an impact on the diversity, bacterial community structure and function of the gut microbiota.
Soil-transmitted helminths are carried by large numbers of people in developing countries. These parasites live in the gut and may interact with bacterial communities in the gut, also called the gut microbiota. To determine whether there are alterations to the gut microbiota that are associated with helminth infections, we examined the types of bacteria present in fecal samples from rural Malaysians, many of whom are helminth-positive and find it likely that helminth colonization alters the gut microbiota for rural Malaysians.
It is becoming increasingly clear that our residential microbes, the key constituents in the human microbiome, are centrally involved in many aspects of our physiology. In particular, the ancient and dominant gastric bacteria Helicobacter pylori are highly interactive with human physiology. In modern times, H. pylori has been disappearing, which consequently affects the interactions between luminal bacteria and epithelial, lymphoid, and neuroendocrine cells. A growing body of evidence indicates that H. pylori protects against childhood-onset asthma, probably through the gastric recruitment of regulatory T cells. The phenomenon of disappearing ancient microbiota may be a general paradigm driving the diseases of modernity.
allergy; T cells; Helicobacter pylori; microbiota; immunity
Background. Diabetic foot infections are a leading cause of lower extremity amputations. Our study examines the microbiota of diabetic skin prior to ulcer development or infection.
Methods. In a case-control study, outpatient males were recruited at a veterans hospital. Subjects were swabbed at 4 cutaneous sites, 1 on the forearm and 3 on the foot. Quantitative polymerase chain reaction (qPCR) with primers and probes specific for bacteria, Staphylococcus species, Staphylococcus aureus, and fungi were performed on all samples. High-throughput 16S ribosomal RNA (rRNA) sequencing was performed on samples from the forearm and the plantar aspect of the foot.
Results. qPCR analysis of swab specimens from 30 diabetic subjects and 30 control subjects showed no differences in total numbers of bacteria or fungi at any sampled site. Increased log10 concentrations of Staphylococcus aureus, quantified by the number of nuc gene copies, were present in diabetic men on the plantar aspect of the foot. High-throughput 16S rRNA sequencing found that, on the foot, the microbiota in controls (n = 24) was dominated by Staphylococcus species, whereas the microbiota in diabetics (n = 23) was more diverse at the genus level. The forearm microbiota had similar diversity in diabetic and control groups.
Conclusions. The feet of diabetic men had decreased populations of Staphylococcus species, increased populations of S. aureus, and increased bacterial diversity, compared with the feet of controls. These ecologic changes may affect the risk for wound infections.
microbiota; microbiome; diabetic foot; cutaneous; Staphylococcus; Staphylococcus aureus
Recent advances in next-generation DNA sequencing enable rapid high-throughput quantitation of microbial community composition in human samples, opening up a new field of microbiomics. One of the promises of this field is linking abundances of microbial taxa to phenotypic and physiological states, which can inform development of new diagnostic, personalized medicine, and forensic modalities. Prior research has demonstrated the feasibility of applying machine learning methods to perform body site and subject classification with microbiomic data. However, it is currently unknown which classifiers perform best among the many available alternatives for classification with microbiomic data.
In this work, we performed a systematic comparison of 18 major classification methods, 5 feature selection methods, and 2 accuracy metrics using 8 datasets spanning 1,802 human samples and various classification tasks: body site and subject classification and diagnosis.
We found that random forests, support vector machines, kernel ridge regression, and Bayesian logistic regression with Laplace priors are the most effective machine learning techniques for performing accurate classification from these microbiomic data.
Microbiomic data; Machine learning; Classification; Feature selection
Featured Article: Blaser MJ, Perez-Perez GI, Kleanthous H, Cover TL, Peek RM, Chyou PH, et al. Infection with Helicobacter pylori strains possessing cagA is associated with an increased risk of developing adenocarcinoma of the stomach.
Campylobacter fetus are important animal and human pathogens and the two major subspecies differ strikingly in pathogenicity. C. fetus subsp. venerealis is highly niche-adapted, mainly infecting the genital tract of cattle. C. fetus subsp. fetus has a wider host-range, colonizing the genital- and intestinal-tract of animals and humans. We report the complete genomic sequence of C. fetus subsp. venerealis 84-112 and comparisons to the genome of C. fetus subsp. fetus 82-40. Functional analysis of genes predicted to be involved in C. fetus virulence was performed. The two subspecies are highly syntenic with 92% sequence identity but C. fetus subsp. venerealis has a larger genome and an extra-chromosomal element. Aside from apparent gene transfer agents and hypothetical proteins, the unique genes in both subspecies comprise two known functional groups: lipopolysaccharide production, and type IV secretion machineries. Analyses of lipopolysaccharide-biosynthesis genes in C. fetus isolates showed linkage to particular pathotypes, and mutational inactivation demonstrated their roles in regulating virulence and host range. The comparative analysis presented here broadens knowledge of the genomic basis of C. fetus pathogenesis and host specificity. It further highlights the importance of surface-exposed structures to C. fetus pathogenicity and demonstrates how evolutionary forces optimize the fitness and host-adaptation of these pathogens.
The human skin harbors complex bacterial communities. Prior studies showing high inter-individual variation focused on subjects from developed countries. We therefore compared cutaneous bacterial communities of Amerindians in the Venezuelan Amazon with subjects in the United States. Forearm skin specimens were studied from healthy Amerindians in Platanillal village in Amazonas State, and from healthy persons in New York and Colorado. All skin sampling used similar swab/buffer techniques. Multiplexed V2-targeted 16S rRNA gene pyrosequencing yielded high quality sequences from 112 samples. The results show 20 phyla, with three (Proteobacteria, Firmicutes, Actinobacteria) predominating. US residents and Venezuelan Amerindians had significantly different forearm skin bacterial community compositions, with United States dominated by Propionibacterium. Among the Amerindians, there was a deep split based on bacterial community membership, with 30 and 42 samples, respectively, falling into each of the two groups, not associated with age, gender, or body mass index. One Amerindian group had diversity similar to the United States, but was dominated by Staphylococcus rather than Propionibacterium. The other Amerindian group was significantly more diverse and even than the US or the other Amerindian group, and featured a broad range of Proteobacteria. The results provide evidence that ethnicity, lifestyle and/or geography are associated with the structure of human cutaneous bacterial communities.
microbiome; cutaneous; microbial diversity; human; genetics
Psoriasis is a common chronic inflammatory disease of the skin. We sought to characterize and compare the cutaneous microbiota of psoriatic lesions (lesion group), unaffected contralateral skin from psoriatic patients (unaffected group), and similar skin loci in matched healthy controls (control group) in order to discern patterns that govern skin colonization and their relationship to clinical diagnosis.
Using high-throughput 16S rRNA gene sequencing, we assayed the cutaneous bacterial communities of 51 matched triplets and characterized these samples using community data analysis techniques. Intragroup Unifrac β diversity revealed increasing diversity from control to unaffected to lesion specimens. Likewise, principal coordinates analysis (PCoA) revealed separation of the lesion samples from unaffected and control along the first axis, suggesting that psoriasis is a major contributor to the observed diversity. The taxonomic richness and evenness decreased in both lesion and unaffected communities compared to control. These differences are explained by the combined increased abundance of the four major skin-associated genera (Corynebacterium, Propionibacterium, Staphylococcus, and Streptococcus), which present a potentially useful predictor for clinical skin type. Psoriasis samples also showed significant univariate decreases in relative abundances and strong classification performance of Cupriavidus, Flavisolibacter, Methylobacterium, and Schlegelella genera versus controls. The cutaneous microbiota separated into two distinct clusters, which we call cutaneotypes: (1) Proteobacteria-associated microbiota, and (2) Firmicutes-associated and Actinobacteria-associated microbiota. Cutaneotype 2 is enriched in lesion specimens compared to control (odds ratio 3.52 (95% CI 1.44 to 8.98), P <0.01).
Our results indicate that psoriasis induces physiological changes both at the lesion site and at the systemic level, which select for specific differential microbiota among the assayed clinical skin types. These differences in microbial community structure in psoriasis patients are potentially of pathophysiologic and diagnostic significance.
Cutaneous microbiota; Psoriasis markers; Microbiome analysis; Cutaneotypes
The microbial communities that reside within the intestinal tract in vertebrates are complex and dynamic. In this report, we establish the utility of Caenorhabditis elegans as a model system for identifying the factors that contribute to bacterial persistence and for host control of gut luminal populations. We found that for N2 worms grown on mixed lawns of bacteria, Salmonella enterica serovar Typhimurium substantially outcompeted Escherichia coli, even when E. coli was initially present at 100-fold-higher concentrations. To address whether innate immunity affects the competition, the daf-2 and daf-16 mutants were studied; their total gut bacterial levels reflect overall capacity for colonization, but Salmonella outcompeted E. coli to an extent similar to wild-type worms. To address the role of virulence properties, Salmonella Δspi-1 Δspi-2 was used to compete with E. coli. The net differential was significantly less than that for wild-type Salmonella; thus, spi-1 spi-2 encodes C. elegans colonization factors. An E. coli strain with repeated in vivo passage had an enhanced ability to compete against an in vitro-passed E. coli strain and against Salmonella. Our data provide evidence of active competition for colonization niches in the C. elegans gut, as determined by bacterial factors and subject to in vivo selection.
Campylobacter fetus subsp. testudinum has been isolated from reptiles and humans. This Campylobacter subspecies is genetically distinct from other C. fetus subspecies. Here, we present the first whole-genome sequence for this C. fetus subspecies.
Campylobacter fetus subsp. testudinum subsp. nov. is a newly proposed subspecies of C. fetus with markers of reptile origin. We summarize epidemiologic information for 9 humans infected with this bacterium. All cases were in men, most of whom were of Asian origin. Infection might have been related to exposure to Asian foods or reptiles.
Campylobacter fetus; C. fetus subsp. fetus; C. fetus subsp. venerealis; C. fetus subsp. testudinum subsp. nov.; bacteria; reptiles; epidemiology; foodborne infections; human infections
Helicobacter pylori has diverged in parallel to its human host, leading to distinct phylogeographic populations. Recent evidence suggests that in the current human mixing in Latin America, European H. pylori (hpEurope) are increasingly dominant at the expense of Amerindian haplotypes (hspAmerind). This phenomenon might occur via DNA recombination, modulated by restriction-modification systems (RMS), in which differences in cognate recognition sites (CRS) and in active methylases will determine direction and frequency of gene flow. We hypothesized that genomes from hspAmerind strains that evolved from a small founder population have lost CRS for RMS and active methylases, promoting hpEurope’s DNA invasion. We determined the observed and expected frequencies of CRS for RMS in DNA from 7 H. pylori whole genomes and 110 multilocus sequences. We also measured the number of active methylases by resistance to in vitro digestion by 16 restriction enzymes of genomic DNA from 9 hpEurope and 9 hspAmerind strains, and determined the direction of DNA uptake in co-culture experiments of hspAmerind and hpEurope strains.
Most of the CRS were underrepresented with consistency between whole genomes and multilocus sequences. Although neither the frequency of CRS nor the number of active methylases differ among the bacterial populations (average 8.6 ± 2.6), hspAmerind strains had a restriction profile distinct from that in hpEurope strains, with 15 recognition sites accounting for the differences. Amerindians strains also exhibited higher transformation rates than European strains, and were more susceptible to be subverted by larger DNA hpEurope-fragments than vice versa.
The geographical variation in the pattern of CRS provides evidence for ancestral differences in RMS representation and function, and the transformation findings support the hypothesis of Europeanization of the Amerindian strains in Latin America via DNA recombination.
H. pylori; Haplotypes; Restriction-Modification system; Recombination
Psoriasis is a common chronic inflammatory disease of the skin. We sought to use bacterial community abundance data to assess the feasibility of developing multivariate molecular signatures for differentiation of cutaneous psoriatic lesions, clinically unaffected contralateral skin from psoriatic patients, and similar cutaneous loci in matched healthy control subjects. Using 16S rRNA high-throughput DNA sequencing, we assayed the cutaneous microbiome for 51 such matched specimen triplets including subjects of both genders, different age groups, ethnicities and multiple body sites. None of the subjects had recently received relevant treatments or antibiotics. We found that molecular signatures for the diagnosis of psoriasis result in significant accuracy ranging from 0.75 to 0.89 AUC, depending on the classification task. We also found a significant effect of DNA sequencing and downstream analysis protocols on the accuracy of molecular signatures. Our results demonstrate that it is feasible to develop accurate molecular signatures for the diagnosis of psoriasis from microbiomic data.
We hypothesized that laparoscopic adjustable gastric banding (LAGB) reduces weight and modulates ghrelin production, but largely spares gastrointestinal endocrine function. To examine this hypothesis, we determined plasma concentrations of appetite-control, insulinotropic, and digestive hormones in relation to LAGB.
Twenty-four patients undergoing LAGB were prospectively enrolled. Body mass index (BMI) was measured and blood samples obtained at baseline and 6 and 12 months post-surgery. Plasma concentrations of leptin, acylated and total ghrelin, pancreatic polypeptide (PP), insulin, glucose-dependent insulinotropic peptide (GIP), active glucagon-like peptide-1 (GLP-1), gastrin, and pepsinogens I and II were measured using enzyme-linked immunoassays.
Median percent excess weight loss (%EWL) over 12 months was 45.7% with median BMI decreasing from 43.2 at baseline to 33.8 at 12 months post-surgery (p<0.001). Median leptin levels decreased from 19.7 ng/ml at baseline to 6.9 ng/ml at 12 months post-surgery (p<0.001). In contrast, plasma levels of acylated and total ghrelin, PP, insulin, GIP, GLP-1, gastrin, and pepsinogen I did not change in relation to surgery (p>0.05). Pepsinogen II levels were significantly lower 6 months after LAGB but returned to baseline levels by 12 months.
LAGB yielded substantial %EWL and a proportional decrease in plasma leptin. Our results support the hypothesis that LAGB works in part by suppressing the rise in ghrelin that normally accompanies weight loss. Unchanged concentrations of insulinotropic and digestive hormones suggest that gastrointestinal endocrine function is largely maintained in the long term.
Obesity; Weight loss; Bariatric surgery; Adjustable gastric banding; Ghrelin; Leptin; GIP; GLP-1