Progress toward a vaccine for extraintestinal pathogenic E. coli (ExPEC) II: efficacy of a toxin-autotransporter dual antigen approach.

Extraintestinal pathogenic Escherichia coli (ExPEC) is a leading cause of worldwide morbidity and mortality, the top cause of antimicrobial-resistant (AMR) infections, and the most frequent cause of life-threatening sepsis and urinary tract infections (UTI) in adults. The development of an effective and universal vaccine is complicated by this pathogen's pan-genome, its ability to mix and match virulence factors and AMR genes via horizontal gene transfer, an inability to decipher commensal from pathogens, and its intimate association and co-evolution with mammals. Using a pan virulome analysis of >20,000 sequenced E. coli strains, we identified the secreted cytolysin α-hemolysin (HlyA) as a high priority target for vaccine exploration studies. We demonstrate that a catalytically inactive pure form of HlyA, expressed in an autologous host using its own secretion system, is highly immunogenic in a murine host, protects against several forms of ExPEC infection (including lethal bacteremia), and significantly lowers bacterial burdens in multiple organ systems. Interestingly, the combination of a previously reported autotransporter (SinH) with HlyA was notably effective, inducing near complete protection against lethal challenge, including commonly used infection strains ST73 (CFT073) and ST95 (UTI89), as well as a mixture of 10 of the most highly virulent sequence types and strains from our clinical collection. Both HlyA and HlyA-SinH combinations also afforded some protection against UTI89 colonization in a murine UTI model. These findings suggest recombinant, inactive hemolysin and/or its combination with SinH warrant investigation in the development of an E. coli vaccine against invasive disease.

Tamm-Horsfall protein augments neutrophil NETosis during urinary tract infection.

Urinary neutrophils are a hallmark of urinary tract infection (UTI), yet the mechanisms governing their activation, function, and efficacy in controlling infection remain incompletely understood. Tamm-Horsfall glycoprotein (THP), the most abundant protein in urine, uses terminal sialic acids to bind an inhibitory receptor and dampen neutrophil inflammatory responses. We hypothesized that neutrophil modulation is an integral part of THP-mediated host protection. In a UTI model, THP-deficient mice showed elevated urinary tract bacterial burdens, increased neutrophil recruitment, and more severe tissue histopathological changes compared to WT mice. Furthermore, THP-deficient mice displayed impaired urinary NETosis during UTI. To investigate the impact of THP on NETosis, we coupled in vitro fluorescence-based NET assays, proteomic analyses, and standard and imaging flow cytometry with peripheral human neutrophils. We found that THP increases proteins involved in respiratory chain, neutrophil granules, and chromatin remodeling pathways, enhances NETosis in an ROS-dependent manner, and drives NET-associated morphologic features including nuclear decondensation. These effects were observed only in the presence of a NETosis stimulus and could not be solely replicated with equivalent levels of sialic acid alone. We conclude that THP is a critical regulator of NETosis in the urinary tract, playing a key role in host defense against UTI.

Gestational diabetes augments group B Streptococcus infection by disrupting maternal immunity and the vaginal microbiota.

Group B Streptococcus (GBS) is a pervasive perinatal pathogen, yet factors driving GBS dissemination in utero are poorly defined. Gestational diabetes mellitus (GDM), a complication marked by dysregulated immunity and maternal microbial dysbiosis, increases risk for GBS perinatal disease. Using a murine GDM model of GBS colonization and perinatal transmission, we find that GDM mice display greater GBS in utero dissemination and subsequently worse neonatal outcomes. Dual-RNA sequencing reveals differential GBS adaptation to the GDM reproductive tract, including a putative glycosyltransferase (yfhO), and altered host responses. GDM immune disruptions include reduced uterine natural killer cell activation, impaired recruitment to placentae, and altered maternofetal cytokines. Lastly, we observe distinct vaginal microbial taxa associated with GDM status and GBS invasive disease status. Here, we show a model of GBS dissemination in GDM hosts that recapitulates several clinical aspects and identifies multiple host and bacterial drivers of GBS perinatal disease.

The human vaginal microbiota: from clinical medicine to models to mechanisms.

The composition of the vaginal microbiota is linked to numerous reproductive health problems, including increased susceptibility to infection, pregnancy complications, and impaired vaginal tissue repair; however, the mechanisms contributing to these adverse outcomes are not yet fully defined. In this review, we highlight recent clinical advancements associating vaginal microbiome composition and function with health outcomes. Subsequently, we provide a summary of emerging models employed to identify microbe-microbe interactions contributing to vaginal health, including metagenomic sequencing, multi-omics approaches, and advances in vaginal microbiota cultivation. Last, we review new in vitro, ex vivo, and in vivo models, such as organoids and humanized microbiota murine models, used to define and mechanistically explore host-microbe interactions at the vaginal mucosa.

Vaginal microbial dynamics and pathogen colonization in a humanized microbiota mouse model.

Vaginal microbial composition is associated with differential risk of urogenital infection. Although Lactobacillus spp. are thought to confer protection against infection, the lack of in vivo models resembling the human vaginal microbiota remains a prominent barrier to mechanistic discovery. Using 16S rRNA amplicon sequencing of C57BL/6J female mice, we found that vaginal microbial composition varies within and between colonies across three vivaria. Noting vaginal microbial plasticity in conventional mice, we assessed the vaginal microbiome of humanized microbiota mice (HMbmice). Like the community structure in conventional mice, HMbmice vaginal microbiota clustered into community state types but, uniquely, HMbmice communities were frequently dominated by Lactobacillus or Enterobacteriaceae. Compared to conventional mice, HMbmice were less susceptible to uterine ascension by urogenital pathobionts group B Streptococcus (GBS) and Prevotella bivia. Although Escherichia and Lactobacillus both correlated with the absence of uterine GBS, vaginal pre-inoculation with exogenous HMbmouse-derived E. coli, but not Ligilactobacillus murinus, reduced vaginal GBS burden. Overall, HMbmice serve as a useful model to elucidate the role of endogenous microbes in conferring protection against urogenital pathogens.

Heterogeneity of the group B streptococcal type VII secretion system and influence on colonization of the female genital tract.

Type VIIb secretion systems (T7SSb) in Gram-positive bacteria facilitate physiology, interbacterial competition, and/or virulence via EssC ATPase-driven secretion of small ɑ-helical proteins and toxins. Recently, we characterized T7SSb in group B Streptococcus (GBS), a leading cause of infection in newborns and immunocompromised adults. GBS T7SS comprises four subtypes based on variation in the C-terminus of EssC and the repertoire of downstream effectors; however, the intraspecies diversity of GBS T7SS and impact on GBS-host interactions remains unknown. Bioinformatic analysis indicates that GBS T7SS loci encode subtype-specific putative effectors, which have low interspecies and inter-subtype homology but contain similar domains/motifs and therefore may serve similar functions. We further identify orphaned GBS WXG100 proteins. Functionally, we show that GBS T7SS subtype I and III strains secrete EsxA in vitro and that in subtype I strain CJB111, esxA1 appears to be differentially transcribed from the T7SS operon. Furthermore, we observe subtype-specific effects of GBS T7SS on host colonization, as CJB111 subtype I but not CNCTC 10/84 subtype III T7SS promotes GBS vaginal colonization. Finally, we observe that T7SS subtypes I and II are the predominant subtypes in clinical GBS isolates. This study highlights the potential impact of T7SS heterogeneity on host-GBS interactions.

Complete Genome Sequences of 37 Bacteria from the Human Vaginal Tract.

Microorganisms colonizing the human vaginal mucosa are associated with healthy states, as well as conditions such as bacterial vaginosis and infection-associated preterm birth. Here, we report complete genome sequences of 37 bacterial isolates from the human vaginal tract.

Interspecies Interactions within the Host: the Social Network of Group B Streptococcus.

Group B Streptococcus (GBS) is a pervasive neonatal pathogen accounting for a combined half a million deaths and stillbirths annually. The most common source of fetal or neonatal GBS exposure is the maternal microbiota. GBS asymptomatically colonizes the gastrointestinal and vaginal mucosa of 1 in 5 individuals globally, although its precise role in these niches is not well understood. To prevent vertical transmission, broad-spectrum antibiotics are administered to GBS-positive mothers during labor in many countries. Although antibiotics have significantly reduced GBS early-onset neonatal disease, there are several unintended consequences, including an altered neonatal microbiota and increased risk for other microbial infections. Additionally, the incidence of late-onset GBS neonatal disease remains unaffected and has sparked an emerging hypothesis that GBS-microbe interactions in developing neonatal gut microbiota may be directly involved in this disease process. This review summarizes our current understanding of GBS interactions with other resident microbes at the mucosal surface from multiple angles, including clinical association studies, agriculture and aquaculture observations, and experimental animal model systems. We also include a comprehensive review of in vitro findings of GBS interactions with other bacterial and fungal microbes, both commensal and pathogenic, along with newly established animal models of GBS vaginal colonization and in utero or neonatal infection. Finally, we provide a perspective on emerging areas of research and current strategies to design microbe-targeting prebiotic or probiotic therapeutic intervention strategies to prevent GBS disease in vulnerable populations.

Vaginal microbial dynamics and pathogen colonization in a humanized microbiota mouse model.

Vaginal microbiota composition is associated with differential risk of urogenital infection. Although vaginal Lactobacillus spp. are thought to confer protection through acidification, bacteriocin production, and immunomodulation, lack of an in vivo model system that closely resembles the human vaginal microbiota remains a prominent barrier to mechanistic discovery. We performed 16S rRNA amplicon sequencing of wildtype C57BL/6J mice, commonly used to study pathogen colonization, and found that the vaginal microbiome composition varies highly both within and between colonies from three distinct vivaria. Because of the strong influence of environmental exposure on vaginal microbiome composition, we assessed whether a humanized microbiota mouse ( HMb mice) would model a more human-like vaginal microbiota. Similar to humans and conventional mice, HMb mice vaginal microbiota clustered into five community state types ( h mCST). Uniquely, HMb mice vaginal communities were frequently dominated by Lactobacilli or Enterobacteriaceae . Compared to genetically-matched conventional mice, HMb mice were less susceptible to uterine ascension by urogenital pathobionts group B Streptococcus (GBS) and Prevotella bivia , but no differences were observed with uropathogenic E. coli . Specifically, vaginal Enterobacteriaceae and Lactobacillus were associated with the absence of uterine GBS. Anti-GBS activity of HMb mice vaginal E. coli and L. murinus isolates, representing Enterobacteriaceae and Lactobacillus respectively, were characterized in vitro and in vivo . Although L. murinus reduced GBS growth in vitro , vaginal pre-inoculation with HMb mouse-derived E. coli , but not L. murinus , conferred protection against vaginal GBS burden. Overall, the HMb mice are an improved model to elucidate the role of endogenous microbes in conferring protection against urogenital pathogens. IMPORTANCE: An altered vaginal microbiota, typically with little to no levels of Lactobacillus , is associated with increased susceptibility to urogenital infections, although mechanisms driving this vulnerability are not fully understood. Despite known inhibitory properties of Lactobacillus against urogenital pathogens, clinical studies with Lactobacillus probiotics have shown mixed success. In this study, we characterize the impact of the vaginal microbiota on urogenital pathogen colonization using a humanized microbiota mouse model that more closely mimics the human vaginal microbiota. We found several vaginal bacterial taxa that correlated with reduced pathogen levels but showed discordant effects in pathogen inhibition between in vitro and in vivo assays. We propose that this humanized microbiota mouse platform is an improved model to describe the role of the vaginal microbiota in protection against urogenital pathogens. Furthermore, this model will be useful in testing efficacy of new probiotic strategies in the complex vaginal environment.

Broad protective vaccination against systemic Escherichia coli with autotransporter antigens.

Extraintestinal pathogenic Escherichia coli (ExPEC) is the leading cause of adult life-threatening sepsis and urinary tract infections (UTI). The emergence and spread of multidrug-resistant (MDR) ExPEC strains result in a considerable amount of treatment failure and hospitalization costs, and contribute to the spread of drug resistance amongst the human microbiome. Thus, an effective vaccine against ExPEC would reduce morbidity and mortality and possibly decrease carriage in healthy or diseased populations. A comparative genomic analysis demonstrated a gene encoding an invasin-like protein, termed sinH, annotated as an autotransporter protein, shows high prevalence in various invasive ExPEC phylogroups, especially those associated with systemic bacteremia and UTI. Here, we evaluated the protective efficacy and immunogenicity of a recombinant SinH-based vaccine consisting of either domain-3 or domains-1,2, and 3 of the putative extracellular region of surface-localized SinH. Immunization of a murine host with SinH-based antigens elicited significant protection against various strains of the pandemic ExPEC sequence type 131 (ST131) as well as multiple sequence types in two distinct models of infection (colonization and bacteremia). SinH immunization also provided significant protection against ExPEC colonization in the bladder in an acute UTI model. Immunized cohorts produced significantly higher levels of vaccine-specific serum IgG and urinary IgG and IgA, findings consistent with mucosal protection. Collectively, these results demonstrate that autotransporter antigens such as SinH may constitute promising ExPEC phylogroup-specific and sequence-type effective vaccine targets that reduce E. coli colonization and virulence.

Heterogeneity of the group B streptococcal type VII secretion system and influence on colonization of the female genital tract.

Type VIIb secretion systems (T7SSb) in Gram-positive bacteria facilitate physiology, interbacterial competition, and/or virulence via EssC ATPase-driven secretion of small ɑ-helical proteins and toxins. Recently, we characterized T7SSb in group B Streptococcus (GBS), a leading cause of infection in newborns and immunocompromised adults. GBS T7SS comprises four subtypes based on variation in the C-terminus of EssC and the repertoire of downstream effectors; however, the intra-species diversity of GBS T7SS and impact on GBS-host interactions remains unknown. Bioinformatic analysis indicates that GBS T7SS loci encode subtype-specific putative effectors, which have low inter-species and inter-subtype homology but contain similar domains/motifs and therefore may serve similar functions. We further identify orphaned GBS WXG100 proteins. Functionally, we show that GBS T7SS subtype I and III strains secrete EsxA in vitro and that in subtype I strain CJB111, esxA1 appears to be differentially transcribed from the T7SS operon. Further, we observe subtype-specific effects of GBS T7SS on host colonization, as subtype I but not subtype III T7SS promotes GBS vaginal persistence. Finally, we observe that T7SS subtypes I and II are the predominant subtypes in clinical GBS isolates. This study highlights the potential impact of T7SS heterogeneity on host-GBS interactions.

Strengthening of enterococcal biofilms by Esp.

Multidrug-resistant (MDR) Enterococcus faecalis are major causes of hospital-acquired infections. Numerous clinical strains of E. faecalis harbor a large pathogenicity island that encodes enterococcal surface protein (Esp), which is suggested to promote biofilm production and virulence, but this remains controversial. To resolve this issue, we characterized the Esp N-terminal region, the portion implicated in biofilm production. Small angle X-ray scattering indicated that the N-terminal region had a globular head, which consisted of two DEv-Ig domains as visualized by X-ray crystallography, followed by an extended tail. The N-terminal region was not required for biofilm production but instead significantly strengthened biofilms against mechanical or degradative disruption, greatly increasing retention of Enterococcus within biofilms. Biofilm strengthening required low pH, which resulted in Esp unfolding, aggregating, and forming amyloid-like structures. The pH threshold for biofilm strengthening depended on protein stability. A truncated fragment of the first DEv-Ig domain, plausibly generated by a host protease, was the least stable and sufficient to strengthen biofilms at pH ≤ 5.0, while the entire N-terminal region and intact Esp on the enterococcal surface was more stable and required a pH ≤ 4.3. These results suggested a virulence role of Esp in strengthening enterococcal biofilms in acidic abiotic or host environments.

Phage Resistance Accompanies Reduced Fitness of Uropathogenic Escherichia coli in the Urinary Environment.

Urinary tract infection (UTI) is among the most common infections treated worldwide each year and is caused primarily by uropathogenic Escherichia coli (UPEC). Rising rates of antibiotic resistance among uropathogens have spurred a consideration of alternative treatment strategies, such as bacteriophage (phage) therapy; however, phage-bacterial interactions within the urinary environment are poorly defined. Here, we assess the activity of two phages, namely, HP3 and ES17, against clinical UPEC isolates using in vitro and in vivo models of UTI. In both bacteriologic medium and pooled human urine, we identified phage resistance arising within the first 6 to 8 h of coincubation. Whole-genome sequencing revealed that UPEC strains resistant to HP3 and ES17 harbored mutations in genes involved in lipopolysaccharide (LPS) biosynthesis. Phage-resistant strains displayed several in vitro phenotypes, including alterations to adherence to and invasion of human bladder epithelial HTB-9 cells and increased biofilm formation in some isolates. Interestingly, these phage-resistant UPEC isolates demonstrated reduced growth in pooled human urine, which could be partially rescued by nutrient supplementation and were more sensitive to several outer membrane-targeting antibiotics than parental strains. Additionally, phage-resistant UPEC isolates were attenuated in bladder colonization in a murine UTI model. In total, our findings suggest that while resistance to phages, such as HP3 and ES17, may arise readily in the urinary environment, phage resistance is accompanied by fitness costs which may render UPEC more susceptible to host immunity or antibiotics. IMPORTANCE UTI is one of the most common causes of outpatient antibiotic use, and rising antibiotic resistance threatens the ability to control UTI unless alternative treatments are developed. Bacteriophage (phage) therapy is gaining renewed interest; however, much like with antibiotics, bacteria can readily become resistant to phages. For successful UTI treatment, we must predict how bacteria will evade killing by phage and identify the downstream consequences of phage resistance during bacterial infection. In our current study, we found that while phage-resistant bacteria quickly emerged in vitro, these bacteria were less capable of growing in human urine and colonizing the murine bladder. These results suggest that phage therapy poses a viable UTI treatment if phage resistance confers fitness costs for the uropathogen. These results have implications for developing cocktails of phage with multiple different bacterial targets, of which each is evaded only at the cost of bacterial fitness.

Human Milk Oligosaccharides Reduce Murine Group B Streptococcus Vaginal Colonization with Minimal Impact on the Vaginal Microbiota.

Group B Streptococcus (GBS) colonizes the vaginal mucosa of a significant percentage of healthy women and is a leading cause of neonatal bacterial infections. Currently, pregnant women are screened in the last month of pregnancy, and GBS-positive women are given antibiotics during parturition to prevent bacterial transmission to the neonate. Recently, human milk oligosaccharides (HMOs) isolated from breastmilk were found to inhibit GBS growth and biofilm formation in vitro, and women that make certain HMOs are less likely to be vaginally colonized with GBS. Using in vitro human vaginal epithelial cells and a murine vaginal colonization model, we tested the impact of HMO treatment on GBS burdens and the composition of the endogenous microbiota by 16S rRNA amplicon sequencing. HMO treatment reduced GBS vaginal burdens in vivo with minimal alterations to the vaginal microbiota. HMOs displayed potent inhibitory activity against GBS in vitro, but HMO pretreatment did not alter adherence of GBS or the probiotic Lactobacillus rhamnosus to human vaginal epithelial cells. In addition, disruption of a putative GBS glycosyltransferase (Δsan_0913) rendered the bacterium largely resistant to HMO inhibition in vitro and in vivo but did not compromise its adherence, colonization, or biofilm formation in the absence of HMOs. We conclude that HMOs are a promising therapeutic bioactive to limit GBS vaginal colonization with minimal impacts on the vaginal microenvironment. IMPORTANCE During pregnancy, GBS ascension into the uterus can cause fetal infection or preterm birth. In addition, GBS exposure during labor creates a risk of serious disease in the vulnerable newborn and mother postpartum. Current recommended prophylaxis consists of administering broad-spectrum antibiotics to GBS-positive mothers during labor. Although antibiotics have significantly reduced GBS neonatal disease, there are several unintended consequences, including altered neonatal gut bacteria and increased risk for other types of infection. Innovative preventions displaying more targeted antimicrobial activity, while leaving the maternal microbiota intact, are thus appealing. Using a mouse model, we found that human milk oligosaccharides (HMOs) reduce GBS burdens without perturbing the vaginal microbiota. We conclude that HMOs are a promising alternative to antibiotics to reduce GBS neonatal disease.

Group B Streptococcus CAMP Factor Does Not Contribute to Interactions with the Vaginal Epithelium and Is Dispensable for Vaginal Colonization in Mice.

The Gram-positive pathogen group B Streptococcus (GBS) is a leading cause of neonatal bacterial infections, preterm birth, and stillbirth. Although maternal GBS vaginal colonization is a risk factor for GBS-associated adverse birth outcomes, mechanisms promoting GBS vaginal persistence are not fully defined. GBS possesses a broadly conserved small molecule, CAMP factor, that is co-hemolytic in the presence of Staphylococcus aureus sphingomyelinase C. While this co-hemolytic reaction is commonly used by clinical laboratories to identify GBS, the contribution of CAMP factor to GBS vaginal persistence is unknown. Using in vitro biofilm, adherence and invasion assays with immortalized human vaginal epithelial VK2 cells, and a mouse model of GBS vaginal colonization, we tested the contribution of CAMP factor using GBS strain COH1 and its isogenic CAMP-deficient mutant (Δcfb). We found no evidence for CAMP factor involvement in GBS biofilm formation, or adherence, invasion, or cytotoxicity toward VK2 cells in the presence or absence of S. aureus. Additionally, there was no difference in vaginal burdens or persistence between COH1 and Δcfb strains in a murine colonization model. In summary, our results using in vitro human cell lines and murine models do not support a critical role for CAMP factor in promoting GBS vaginal colonization. IMPORTANCE Group B Streptococcus (GBS) remains a pervasive pathogen for pregnant women and their newborns. Maternal screening and intrapartum antibiotic prophylaxis to GBS-positive mothers have reduced, but not eliminated GBS neonatal disease, and have not impacted GBS-associated preterm birth or stillbirth. Additionally, this antibiotic exposure is associated with adverse effects on the maternal and neonatal microbiota. Identifying key GBS factors important for maternal vaginal colonization will foster development of more targeted, alternative therapies to antibiotic treatment. Here, we investigate the contribution of a broadly conserved GBS determinant, CAMP factor, to GBS vaginal colonization and find that CAMP factor is unlikely to be a biological target to control maternal GBS colonization.

Hypoxia-Inducible Factor 1 Alpha Is Dispensable for Host Defense of Group B Streptococcus Colonization and Infection.

Group B Streptococcus (GBS) is a leading cause of neonatal morbidity and mortality, and the primary source of exposure is the maternal vagina. Intrapartum antibiotic prophylaxis for GBS-positive mothers has reduced the incidence of GBS early-onset disease, however, potential long-lasting influence of an antibiotic-altered neonatal microbiota, and the frequent clinical sequelae in survivors of invasive GBS infection, compels alternative treatment options for GBS. Here, we examined the role of transcription factor hypoxia-inducible factor 1 alpha (HIF-1α), widely recognized as a regulator of immune activation during infection, in the host response to GBS. Given the importance of endogenous HIF-1α for innate immune defense, and the potential utility of HIF-1α stabilization in promoting bacterial clearance, we hypothesized that HIF-1α could play an important role in coordinating host responses to GBS in colonization and systemic disease. Counter to our hypothesis, we found that GBS infection did not induce HIF-1α expression in vaginal epithelial cells or murine macrophages, nor did HIF-1α deficiency alter GBS colonization or pathogenesis in vivo. Furthermore, pharmacological enhancement of HIF-1α did not improve control of GBS in pathogenesis and colonization models, while displaying inhibitory effects in vaginal epithelial cytokines and immune cell killing in vitro. Taken together, we conclude that HIF-1α is not a prominent aspect of the host response to GBS colonization or invasive disease, and its pharmacological modulation is unlikely to provide significant benefit against this important neonatal pathogen.

Novel Models of Streptococcus canis Colonization and Disease Reveal Modest Contributions of M-Like (SCM) Protein.

Streptococcus canis is a common colonizing bacterium of the urogenital tract of cats and dogs that can also cause invasive disease in these animal populations and in humans. Although the virulence mechanisms of S. canis are not well-characterized, an M-like protein, SCM, has recently identified been as a potential virulence factor. SCM is a surface-associated protein that binds to host plasminogen and IgGs suggesting its possible importance in host-pathogen interactions. In this study, we developed in vitro and ex vivo blood component models and murine models of S. canis vaginal colonization, systemic infection, and dermal infection to compare the virulence potential of the zoonotic S. canis vaginal isolate G361 and its isogenic SCM-deficient mutant (G361∆scm). We found that while S. canis establishes vaginal colonization and causes invasive disease in vivo, the contribution of the SCM protein to virulence phenotypes in these models is modest. We conclude that SCM is dispensable for invasive disease in murine models and for resistance to human blood components ex vivo, but may contribute to mucosal persistence, highlighting a potential contribution to the recently appreciated genetic diversity of SCM across strains and hosts.

Evaluating Organism-Wide Changes in the Metabolome and Microbiome following a Single Dose of Antibiotic.

Antibiotics are a mainstay of modern medicine, but as they kill their target pathogen(s), they often affect the commensal microbiota. Antibiotic-induced microbiome dysbiosis is a growing research focus and health concern, often assessed via analysis of fecal samples. However, such analysis does not inform how antibiotics influence the microbiome across the whole host or how such changes subsequently alter host chemistry. In this study, we investigated the acute (1 day postadministration) and delayed (6 days postadministration) effects of a single parenteral dose of two common antibiotics, ampicillin or vancomycin, on the global metabolome and microbiome of mice across 77 different body sites from 25 different organs. The broader-spectrum agent ampicillin had the greatest impact on the microbiota in the lower gastrointestinal tract (cecum and colon), where microbial diversity is highest. In the metabolome, the greatest effects were seen 1 day posttreatment, and changes in metabolite abundances were not confined to the gut. The local abundance of ampicillin and its metabolites correlated with increased metabolome effect size and a loss of alpha diversity versus control mice. Additionally, small peptides were elevated in the lower gastrointestinal tract of mice 1 day after antibiotic treatment. While a single parenteral dose of antibiotic did not drastically alter the microbiome, nevertheless, changes in the metabolome were observed both within and outside the gut. This study provides a framework for how whole-organism -omics approaches can be employed to understand the impact of antibiotics on the entire host.IMPORTANCE We are just beginning to understand the unintended effects of antibiotics on our microbiomes and health. In this study, we aimed to define an approach by which one could obtain a comprehensive picture of (i) how antibiotics spatiotemporally impact commensal microbes throughout the gut and (ii) how these changes influence host chemistry throughout the body. We found that just a single dose of antibiotic altered host chemistry in a variety of organs and that microbiome alterations were not uniform throughout the gut. As technological advances increase the feasibility of whole-organism studies, we argue that using these approaches can provide further insight on both the wide-ranging effects of antibiotics on health and how to restore microbial communities to mitigate these effects.

Multidimensional Proteome Profiling of Blood-Brain Barrier Perturbation by Group B Streptococcus.

Group B Streptococcus (GBS) remains the leading cause of neonatal meningitis, a disease associated with high rates of adverse neurological sequelae. The in vivo relationship between GBS and brain tissues remains poorly characterized, partly because past studies had focused on microbial rather than host processes. Additionally, the field has not capitalized on systems-level technologies to probe the host-pathogen relationship. Here, we use multiplexed quantitative proteomics to investigate the effect of GBS infection in the murine brain at various levels of tissue complexity, beginning with the whole organ and moving to brain vascular substructures. Infected whole brains showed classical signatures associated with the acute-phase response. In isolated brain microvessels, classical blood-brain barrier proteins were unaltered, but interferon signaling and leukocyte recruitment proteins were upregulated. The choroid plexus showed increases in peripheral immune cell proteins. Proteins that increased in abundance in the vasculature during GBS invasion were associated with major histocompatibility complex (MHC) class I antigen processing and endoplasmic reticulum dysfunction, a finding which correlated with altered host protein glycosylation profiles. Globally, there was low concordance between the infection proteome of whole brains and isolated vascular tissues. This report underscores the utility of unbiased, systems-scale analyses of functional tissue substructures for understanding disease.IMPORTANCE Group B Streptococcus (GBS) meningitis remains a major cause of poor health outcomes very early in life. Both the host-pathogen relationship leading to disease and the massive host response to infection contributing to these poor outcomes are orchestrated at the tissue and cell type levels. GBS meningitis is thought to result when bacteria present in the blood circumvent the selectively permeable vascular barriers that feed the brain. Additionally, tissue damage subsequent to bacterial invasion is mediated by inflammation and by immune cells from the periphery crossing the blood-brain barrier. Indeed, the vasculature plays a central role in disease processes occurring during GBS infection of the brain. Here, we employed quantitative proteomic analysis of brain vascular substructures during invasive GBS disease. We used the generated data to map molecular alterations associated with tissue perturbation, finding widespread intracellular dysfunction and punctuating the importance of investigations relegated to tissue type over the whole organ.

Developmental Immaturity of Siglec Receptor Expression on Neonatal Alveolar Macrophages Predisposes to Severe Group B Streptococcal Infection.

Streptococcus agalactiae (Group B Streptococcus, GBS) is the most common neonatal pathogen. However, the cellular and molecular mechanisms for neonatal susceptibility to GBS pneumonia and sepsis are incompletely understood. Here we optimized a mouse model of GBS pneumonia to test the role of alveolar macrophage (ΑΜΦ) maturation in host vulnerability to disease. Compared with juvenile and adult mice, neonatal mice infected with GBS had increased mortality and persistence of lung injury. In addition, neonatal mice were defective in GBS phagocytosis and killing. ΑΜΦ depletion and disruption of ΑΜΦ differentiation in Csf2-/- mice both impaired GBS clearance. AMΦ engage the heavily sialylated GBS capsule via the cell surface Siglec receptors Sn and Siglec-E. Although both newborn and adult ΑΜΦ expressed Siglec-E, newborn ΑΜΦ expressed significantly lower levels of Sn. We propose that a developmental delay in Sn expression on ΑΜΦ may prevent effective killing and clearing of GBS from the newborn lung.