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ABSTRACT “Leaky gut,” or high intestinal barrier permeability, is common in preterm newborns. The role of the microbiota in this process remains largely uncharacterized. We employed both short- and long-read sequencing of the 16S rRNA gene and metagenomes to characterize the intestinal microbiome of a longitudinal cohort of 113 preterm infants born between 240/7 and 326/7 weeks of gestation. Enabled by enhanced taxonomic resolution, we found that a significantly increased abundance of Bifidobacterium breve and a diet rich in mother’s breastmilk were associated with intestinal barrier maturation during the first week of life. We combined these factors using genome-resolved metagenomics and identified a highly specialized genetic capability of the Bifidobacterium strains to assimilate human milk oligosaccharides and host-derived glycoproteins. Our study proposes mechanistic roles of breastmilk feeding and intestinal microbial colonization in postnatal intestinal barrier maturation; these observations are critical toward advancing therapeutics to prevent and treat hyperpermeable gut-associated conditions, including necrotizing enterocolitis (NEC). IMPORTANCE Despite improvements in neonatal intensive care, necrotizing enterocolitis (NEC) remains a leading cause of morbidity and mortality. “Leaky gut,” or intestinal barrier immaturity with elevated intestinal permeability, is the proximate cause of susceptibility to NEC. Early detection and intervention to prevent leaky gut in “at-risk” preterm neonates are critical for decreasing the risk of potentially life-threatening complications like NEC. However, the complex interactions between the developing gut microbial community, nutrition, and intestinal barrier function remain largely uncharacterized. In this study, we reveal the critical role of a sufficient breastmilk feeding volume and the specialized carbohydrate metabolism capability of Bifidobacterium in the coordinated postnatal improvement of the intestinal barrier. Determining the clinical and microbial biomarkers that drive the intestinal developmental disparity will inform early detection and novel therapeutic strategies to promote appropriate intestinal barrier maturation and prevent NEC and other adverse health conditions in preterm infants.

ABSTRACT We have previously identified a crypt-specific core microbiota (CSCM) in the colons of healthy laboratory mice and related wild rodents. Here, we confirm that a CSCM also exists in the human colon and appears to be altered during colon cancer. The colonic microbiota is suggested to be involved in the development of colorectal cancer (CRC). Because the microbiota identified in fecal samples from CRC patients does not directly reflect the microbiota associated with tumor tissues themselves, we sought to characterize the bacterial communities from the crypts and associated adjacent mucosal surfaces of 58 patients (tumor and normal homologous tissue) and 9 controls with normal colonoscopy results. Here, we confirm that bacteria colonize human colonic crypts in both control and CRC tissues, and using laser-microdissected tissues and 16S rRNA gene sequencing, we further show that right and left crypt- and mucosa-associated bacterial communities are significantly different. In addition to Bacteroidetes and Firmicutes, and as with murine proximal colon crypts, environmental nonfermentative Proteobacteria are found in human colonic crypts. Fusobacterium and Bacteroides fragilis are more abundant in right-side tumors, whereas Parvimonas micra is more prevalent in left-side tumors. More precisely, Fusobacterium periodonticum is more abundant in crypts from cancerous samples in the right colon than in associated nontumoral samples from adjacent areas but not in left-side colonic samples. Future analysis of the interaction between these bacteria and the crypt epithelium, particularly intestinal stem cells, will allow deciphering of their possible oncogenic potential. IMPORTANCE Due to the huge number of bacteria constituting the human colon microbiota, alteration in the balance of its constitutive taxa (i.e., dysbiosis) is highly suspected of being involved in colorectal oncogenesis. Indeed, bacterial signatures in association with CRC have been described. These signatures may vary if bacteria are identified in feces or in association with tumor tissues. Here, we show that bacteria colonize human colonic crypts in tissues obtained from patients with CRC and with normal colonoscopy results. Aerobic nonfermentative Proteobacteria previously identified as constitutive of the crypt-specific core microbiota in murine colonic samples are similarly prevalent in human colonic crypts in combination with other anaerobic taxa. We also show that bacterial signatures characterizing the crypts of colonic tumors vary depending whether right-side or left-side tumors are analyzed.

ABSTRACT The mechanism(s) by which Lactobacillus-dominated cervicovaginal microbiota provide a barrier to Chlamydia trachomatis infection remain(s) unknown. Here we evaluate the impact of different Lactobacillus spp. identified via culture-independent metataxonomic analysis of C. trachomatis-infected women on C. trachomatis infection in a three-dimensional (3D) cervical epithelium model. Lactobacillus spp. that specifically produce d(−) lactic acid were associated with long-term protection against C. trachomatis infection, consistent with reduced protection associated with Lactobacillus iners, which does not produce this isoform, and with decreased epithelial cell proliferation, consistent with the observed prolonged protective effect. Transcriptomic analysis revealed that epigenetic modifications involving histone deacetylase-controlled pathways are integral to the cross talk between host and microbiota. These results highlight a fundamental mechanism whereby the cervicovaginal microbiota modulates host functions to protect against C. trachomatis infection. IMPORTANCE The vaginal microbiota is believed to protect women against Chlamydia trachomatis, the etiologic agent of the most prevalent sexually transmitted infection (STI) in developed countries. The mechanism underlying this protection has remained elusive. Here, we reveal the comprehensive strategy by which the cervicovaginal microbiota modulates host functions to protect against chlamydial infection, thereby providing a novel conceptual mechanistic understanding. Major implications of this work are that (i) the impact of the vaginal microbiota on the epithelium should be considered in future studies of chlamydial infection and other STIs and (ii) a fundamental understanding of the cervicovaginal microbiota’s role in protection against STIs may enable the development of novel microbiome-based therapeutic strategies to protect women from infection and improve vaginal and cervical health.

ABSTRACT An outbreak of cholera occurred in 1991 in Mexico, where it had not been reported for more than a century and is now endemic. Vibrio cholerae O1 prototype El Tor and classical strains coexist with altered El Tor strains (1991 to 1997). Nontoxigenic (CTX−) V. cholerae El Tor dominated toxigenic (CTX+) strains (2001 to 2003), but V. cholerae CTX+ variant El Tor was isolated during 2004 to 2008, outcompeting CTX− V. cholerae. Genomes of six Mexican V. cholerae O1 strains isolated during 1991 to 2008 were sequenced and compared with both contemporary and archived strains of V. cholerae. Three were CTX+ El Tor, two were CTX− El Tor, and the remaining strain was a CTX+ classical isolate. Whole-genome sequence analysis showed the six isolates belonged to five distinct phylogenetic clades. One CTX− isolate is ancestral to the 6th and 7th pandemic CTX+ V. cholerae isolates. The other CTX− isolate joined with CTX− non-O1/O139 isolates from Haiti and seroconverted O1 isolates from Brazil and Amazonia. One CTX+ isolate was phylogenetically placed with the sixth pandemic classical clade and the V. cholerae O395 classical reference strain. Two CTX+ El Tor isolates possessing intact Vibrio seventh pandemic island II (VSP-II) are related to hybrid El Tor isolates from Mozambique and Bangladesh. The third CTX+ El Tor isolate contained West African-South American (WASA) recombination in VSP-II and showed relatedness to isolates from Peru and Brazil. Except for one isolate, all Mexican isolates lack SXT/R391 integrative conjugative elements (ICEs) and sensitivity to selected antibiotics, with one isolate resistant to streptomycin. No isolates were related to contemporary isolates from Asia, Africa, or Haiti, indicating phylogenetic diversity. IMPORTANCE Sequencing of genomes of V. cholerae is critical if genetic changes occurring over time in the circulating population of an area of endemicity are to be understood. Although cholera outbreaks occurred rarely in Mexico prior to the 1990s, genetically diverse V. cholerae O1 strains were isolated between 1991 and 2008. Despite the lack of strong evidence, the notion that cholera was transmitted from Africa to Latin America has been proposed in the literature. In this study, we have applied whole-genome sequence analysis to a set of 124 V. cholerae strains, including six Mexican isolates, to determine their phylogenetic relationships. Phylogenetic analysis indicated the six V. cholerae O1 isolates belong to five phylogenetic clades: i.e., basal, nontoxigenic, classical, El Tor, and hybrid El Tor. Thus, the results of phylogenetic analysis, coupled with CTXϕ array and antibiotic susceptibility, do not support single-source transmission of cholera to Mexico from African countries. The association of indigenous populations of V. cholerae that has been observed in this study suggests it plays a significant role in the dynamics of cholera in Mexico.

ABSTRACT Cervicovaginal mucus (CVM) can provide a barrier that precludes HIV and other sexually transmitted virions from reaching target cells in the vaginal epithelium, thereby preventing or reducing infections. However, the barrier properties of CVM differ from woman to woman, and the causes of these variations are not yet well understood. Using high-resolution particle tracking of fluorescent HIV-1 pseudoviruses, we found that neither pH nor Nugent scores nor total lactic acid levels correlated significantly with virus trapping in unmodified CVM from diverse donors. Surprisingly, HIV-1 was generally trapped in CVM with relatively high concentrations of d-lactic acid and a Lactobacillus crispatus-dominant microbiota. In contrast, a substantial fraction of HIV-1 virions diffused rapidly through CVM with low concentrations of d-lactic acid that had a Lactobacillus iners-dominant microbiota or significant amounts of Gardnerella vaginalis, a bacterium associated with bacterial vaginosis. Our results demonstrate that the vaginal microbiota, including specific species of Lactobacillus, can alter the diffusional barrier properties of CVM against HIV and likely other sexually transmitted viruses and that these microbiota-associated changes may account in part for the elevated risks of HIV acquisition linked to bacterial vaginosis or intermediate vaginal microbiota. IMPORTANCE Variations in the vaginal microbiota, especially shifts away from Lactobacillus-dominant microbiota, are associated with differential risks of acquiring HIV or other sexually transmitted infections. However, emerging evidence suggests that Lactobacillus iners frequently colonizes women with recurring bacterial vaginosis, raising the possibility that L. iners may not be as protective as other Lactobacillus species. Our study was designed to improve understanding of how the cervicovaginal mucus barrier against HIV may vary between women along with the vaginal microbiota and led to the finding that the vaginal microbiota, including specific species of Lactobacillus, can directly alter the diffusional barrier properties of cervicovaginal mucus. This work advances our understanding of the complex barrier properties of mucus and highlights the differential protective ability of different species of Lactobacillus, with Lactobacillus crispatus and possibly other species playing a key role in protection against HIV and other sexually transmitted infections. These findings could lead to the development of novel strategies to protect women against HIV.

ABSTRACT Bacterial vaginosis (BV) is a common vaginal bacterial imbalance associated with risk for HIV and poor gynecologic and obstetric outcomes. Male circumcision reduces BV-associated bacteria on the penis and decreases BV in female partners, but the link between penile microbiota and female partner BV is not well understood. We tested the hypothesis that having a female partner with BV increases BV-associated bacteria in uncircumcised men. We characterized penile microbiota composition and density (i.e., the quantity of bacteria per swab) by broad-coverage 16S rRNA gene-based sequencing and quantitative PCR (qPCR) in 165 uncircumcised men from Rakai, Uganda. Associations between penile community state types (CSTs) and female partner's Nugent score were assessed. We found seven distinct penile CSTs of increasing density (CST1 to 7). CST1 to 3 and CST4 to 7 were the two major CST groups. CST4 to 7 had higher prevalence and abundance of BV-associated bacteria, such as Mobiluncus and Dialister, than CST1 to 3. Men with CST4 to 7 were significantly more likely to have a female partner with a high Nugent score (P = 0.03). Men with two or more extramarital partners were significantly more likely to have CST4 to 7 than men with only marital partners (CST4 to 7 prevalence ratio, 1.84; 95% confidence interval [CI], 1.16 to 2.92). Female partner Nugent BV is significantly associated with penile microbiota. Our data support the exchange of BV-associated bacteria through intercourse, which may explain BV recurrence and persistence. IMPORTANCE Bacterial vaginosis (BV) is sexually associated but not considered a sexually transmitted disease. Our findings suggest that the uncircumcised penis is an important niche for BV-associated genital anaerobes. In addition, we found a link between extramarital sexual relationships and BV-associated bacteria in men, which parallels earlier findings of the association between sexual activity and BV in women. This suggests the sexual transmissibility of BV-associated bacteria. Reducing bacterial exchange by barrier methods and managing carriage of BV-associated bacteria in men may decrease BV persistence and recurrence in women.

ABSTRACT A mechanistic understanding of the purported health benefits conferred by consumption of probiotic bacteria has been limited by our knowledge of the resident gut microbiota and its interaction with the host. Here, we detail the impact of a single-organism probiotic, Lactobacillus rhamnosus GG ATCC 53103 (LGG), on the structure and functional dynamics (gene expression) of the gut microbiota in a study of 12 healthy individuals, 65 to 80 years old. The analysis revealed that while the overall community composition was stable as assessed by 16S rRNA profiling, the transcriptional response of the gut microbiota was modulated by probiotic treatment. Comparison of transcriptional profiles based on taxonomic composition yielded three distinct transcriptome groups that displayed considerable differences in functional dynamics. The transcriptional profile of LGG in vivo was remarkably concordant across study subjects despite the considerable interindividual nature of the gut microbiota. However, we identified genes involved in flagellar motility, chemotaxis, and adhesion from Bifidobacterium and the dominant butyrate producers Roseburia and Eubacterium whose expression was increased during probiotic consumption, suggesting that LGG may promote interactions between key constituents of the microbiota and the host epithelium. These results provide evidence for the discrete functional effects imparted by a specific single-organism probiotic and challenge the prevailing notion that probiotics substantially modify the resident microbiota within nondiseased individuals in an appreciable fashion. IMPORTANCE Probiotic bacteria have been used for over a century to promote digestive health. Many individuals report that probiotics alleviate a number of digestive issues, yet little evidence links how probiotic microbes influence human health. Here, we show how the resident microbes that inhabit the healthy human gut respond to a probiotic. The well-studied probiotic Lactobacillus rhamnosus GG ATCC 53103 (LGG) was administered in a clinical trial, and a suite of measurements of the resident microbes were taken to evaluate potential changes over the course of probiotic consumption. We found that LGG transiently enriches for functions to potentially promote anti-inflammatory pathways in the resident microbes.

ABSTRACT For centuries, cholera has been one of the most feared diseases. The causative agent Vibrio cholerae is a waterborne Gram-negative enteric pathogen eliciting a severe watery diarrheal disease. In October 2010, the seventh pandemic reached Haiti, a country that had not experienced cholera for more than a century. By using whole-genome sequence typing and mapping strategies of 116 serotype O1 strains from global sources, including 44 Haitian genomes, we present a detailed reconstructed evolutionary history of the seventh pandemic with a focus on the Haitian outbreak. We catalogued subtle genomic alterations at the nucleotide level in the genome core and architectural rearrangements from whole-genome map comparisons. Isolates closely related to the Haitian isolates caused several recent outbreaks in southern Asia. This study provides evidence for a single-source introduction of cholera from Nepal into Haiti followed by rapid, extensive, and continued clonal expansion. The phylogeographic patterns in both southern Asia and Haiti argue for the rapid dissemination of V. cholerae across the landscape necessitating real-time surveillance efforts to complement the whole-genome epidemiological analysis. As eradication efforts move forward, phylogeographic knowledge will be important for identifying persistent sources and monitoring success at regional levels. The results of molecular and epidemiological analyses of this outbreak suggest that an indigenous Haitian source of V. cholerae is unlikely and that an indigenous source has not contributed to the genomic evolution of this clade. IMPORTANCE In this genomic epidemiology study, we have applied high-resolution whole-genome-based sequence typing methodologies on a comprehensive set of genome sequences that have become available in the aftermath of the Haitian cholera epidemic. These sequence resources enabled us to reassess the degree of genomic heterogeneity within the Vibrio cholerae O1 serotype and to refine boundaries and evolutionary relationships. The established phylogenomic framework showed how outbreak isolates fit into the global phylogeographic patterns compared to a comprehensive globally and temporally diverse strain collection and provides strong molecular evidence that points to a nonindigenous source of the 2010 Haitian cholera outbreak and refines epidemiological standards used in outbreak investigations for outbreak inclusion/exclusion following the concept of genomic epidemiology. The generated phylogenomic data have major public health relevance in translating sequence-based information to assist in future diagnostic, epidemiological, surveillance, and forensic studies of cholera.

ABSTRACT It is not currently possible to predict the probability of whether a woman with a chlamydial genital infection will develop pelvic inflammatory disease (PID). To determine if specific biomarkers may be associated with distinct chlamydial pathotypes, we utilized two Chlamydia muridarum variants (C. muridarum Var001 [CmVar001] and CmVar004) that differ in their abilities to elicit upper genital tract pathology in a mouse model. CmVar004 has a lower growth rate in vitro and induces pathology in only 20% of C57BL/6 mouse oviducts versus 83.3% of oviducts in CmVar001-infected mice. To determine if chemokine and cytokine production within 24 h of infection is associated with the outcome of pathology, levels of 15 chemokines and cytokines were measured. CmVar004 infection induced significantly lower levels of CXCL1, CXCL2, tumor necrosis factor alpha (TNF-α), and CCL2 in comparison to CmVar001 infection with similar rRNA (rs16) levels for Chlamydiae. A combination of microRNA (miRNA) sequencing and quantitative real-time PCR (qRT-PCR) analysis of 134 inflammation-related miRNAs was performed 24 h postinfection to determine if the chemokine/cytokine responses would also be reflected in miRNA expression profiles. Interestingly, 12 miRNAs (miR-135a-5p, miR298-5p, miR142-3p, miR223-3p, miR299a-3p, miR147-3p, miR105, miR325-3p, miR132-3p, miR142-5p, miR155-5p, and miR-410-3p) were overexpressed during CmVar004 infection compared to CmVar001 infection, inversely correlating with the respective chemokine/cytokine responses. To our knowledge, this is the first report demonstrating that early biomarkers elicited in the host can differentiate between two pathological variants of chlamydiae and be predictive of upper tract disease. IMPORTANCE It is apparent that an infecting chlamydial population consists of multiple genetic variants with differing capabilities of eliciting a pathological response; thus, it may be possible to identify biomarkers specific for a given virulence pathotype. miRNAs are known to regulate genes that in turn regulate signaling pathways involved in disease pathogenesis. Importantly, miRNAs are stable and can reflect a tissue response and therefore have the potential to be biomarkers of disease severity. Currently, with respect to chlamydial infections, there is no way to predict whether an infected patient is more or less likely to develop PID. However, data presented in this study indicate that the expression of a specific miRNA profile associated with a virulent variant early in the infection course may be predictive of an increased risk of pelvic inflammatory disease, allowing more aggressive treatment before significant pathology develops.

ABSTRACT Male circumcision reduces female-to-male HIV transmission. Hypothesized mechanisms for this protective effect include decreased HIV target cell recruitment and activation due to changes in the penis microbiome. We compared the coronal sulcus microbiota of men from a group of uncircumcised controls (n = 77) and from a circumcised intervention group (n = 79) at enrollment and year 1 follow-up in a randomized circumcision trial in Rakai, Uganda. We characterized microbiota using16S rRNA gene-based quantitative PCR (qPCR) and pyrosequencing, log response ratio (LRR), Bayesian classification, nonmetric multidimensional scaling (nMDS), and permutational multivariate analysis of variance (PerMANOVA). At baseline, men in both study arms had comparable coronal sulcus microbiota; however, by year 1, circumcision decreased the total bacterial load and reduced microbiota biodiversity. Specifically, the prevalence and absolute abundance of 12 anaerobic bacterial taxa decreased significantly in the circumcised men. While aerobic bacterial taxa also increased postcircumcision, these gains were minor. The reduction in anaerobes may partly account for the effects of circumcision on reduced HIV acquisition. IMPORTANCE The bacterial changes identified in this study may play an important role in the HIV risk reduction conferred by male circumcision. Decreasing the load of specific anaerobes could reduce HIV target cell recruitment to the foreskin. Understanding the mechanisms that underlie the benefits of male circumcision could help to identify new intervention strategies for decreasing HIV transmission, applicable to populations with high HIV prevalence where male circumcision is culturally less acceptable.

ABSTRACT Vaginal HIV microbicides offer great promise in preventing HIV transmission, but failures of phase 3 clinical trials, in which microbicide-treated subjects had an increased risk of HIV transmission, raised concerns about endpoints used to evaluate microbicide safety. A possible explanation for the increased transmission risk is that the agents shifted the vaginal bacterial community, resulting in loss of natural protection and enhanced HIV transmission susceptibility. We characterized vaginal microbiota, using pyrosequencing of bar-coded 16S rRNA gene fragments, in samples from 35 healthy, sexually abstinent female volunteer subjects (ages 18 to 50 years) with regular menses in a repeat phase 1 study of twice-daily application over 13.5 days of 1 of 3 gel products: a hydroxyethylcellulose (HEC)-based “universal” placebo (10 subjects), 6% cellulose sulfate (CS; 13 subjects), and 4% nonoxynol-9 (N-9; 12 subjects). We used mixed effects models inferred using Bayesian Markov chain Monte Carlo methods, which showed that treatment with active agents shifted the microbiota toward a community type lacking significant numbers of Lactobacillus spp. and dominated by strict anaerobes. This state of the vaginal microbiota was associated with a low or intermediate Nugent score and was not identical to bacterial vaginosis, an HIV transmission risk factor. The placebo arm contained a higher proportion of communities dominated by Lactobacillus spp., particularly L. crispatus, throughout treatment. The data suggest that molecular evaluation of microbicide effects on vaginal microbiota may be a critical endpoint that should be incorporated in early clinical assessment of microbicide candidates. IMPORTANCE Despite large prevention efforts, HIV transmission and acquisition rates remain unacceptably high. In developing countries, transmission mainly occurs through heterosexual intercourse, where women are significantly more vulnerable to infection than men. Vaginal microbicides are considered to be one of the most promising female-controlled products, in that women themselves insert the microbicides into the vagina to prevent HIV transmission during sexual intercourse. The failure of several microbicides in clinical trials has raised questions concerning the low in vivo efficacy of such anti-HIV molecules. This study was designed to gain insights into the failures of two microbicides by testing the hypothesis that the microbicides negatively affect a critical line of defense against HIV, the vaginal microbiota. The results suggest that in the early assessment of candidate microbicides, culture-independent evaluation of their effect on the vaginal microbiota should be considered and may constitute a critical endpoint.