Intergenerational protective anti-gut commensal immunoglobulin G originates in early life.

Maternal immunoglobulins of the class G (IgGs) protect offspring from enteric infection, but when, where, and how these antibodies are physiologically generated and confer protection remains enigmatic. We found that circulating IgGs in adult mice preferentially bind early-life gut commensal bacteria over their own adult gut commensal bacteria. IgG-secreting plasma cells specific for early-life gut bacteria appear in the intestine soon after weaning, where they remain into adulthood. Manipulating exposure to gut bacteria or plasma cell development before, but not after, weaning reduced IgG-secreting plasma cells targeting early-life gut bacteria throughout life. Further, the development of this anti-gut commensal IgG response coincides with the early-life interval in which goblet cell-associated antigen passages (GAPs) are present in the colon. Offspring of dams "perturbed" by B cell ablation or reduced bacterial exposure in early life were more susceptible to enteric pathogen challenge. In contrast to current concepts, protective maternal IgGs targeted translocating gut commensals in the offspring, not the enteric pathogen. These early-life events affecting anti-commensal IgG production have intergenerational effects for protection of the offspring.

Gut microbiota induces weight gain and inflammation in the gut and adipose tissue independent of manipulations in diet, genetics, and immune development.

Obesity and the metabolic syndrome are complex disorders resulting from multiple factors including genetics, diet, activity, inflammation, and gut microbes. Animal studies have identified roles for each of these, however the contribution(s) specifically attributed to the gut microbiota remain unclear, as studies have used combinations of genetically altered mice, high fat diet, and/or colonization of germ-free mice, which have an underdeveloped immune system. We investigated the role(s) of the gut microbiota driving obesity and inflammation independent of manipulations in diet and genetics in mice with fully developed immune systems. We demonstrate that the human obese gut microbiota alone was sufficient to drive weight gain, systemic, adipose tissue, and intestinal inflammation, but did not promote intestinal barrier leak. The obese microbiota induced gene expression promoting caloric uptake/harvest but was less effective at inducing genes associated with mucosal immune responses. Thus, the obese gut microbiota is sufficient to induce weight gain and inflammation.

Vancomycin-induced gut microbial dysbiosis alters enteric neuron-macrophage interactions during a critical period of postnatal development.

Vancomycin is a broad-spectrum antibiotic widely used in cases of suspected sepsis in premature neonates. While appropriate and potentially lifesaving in this setting, early-life antibiotic exposure alters the developing microbiome and is associated with an increased risk of deadly complications, including late-onset sepsis (LOS) and necrotizing enterocolitis (NEC). Recent studies show that neonatal vancomycin treatment disrupts postnatal enteric nervous system (ENS) development in mouse pups, which is in part dependent upon neuroimmune interactions. This suggests that early-life antibiotic exposure could disrupt these interactions in the neonatal gut. Notably, a subset of tissue-resident intestinal macrophages, muscularis macrophages, has been identified as important contributors to the development of postnatal ENS. We hypothesized that vancomycin-induced neonatal dysbiosis impacts postnatal ENS development through its effects on macrophages. Using a mouse model, we found that exposure to vancomycin in the first 10 days of life, but not in adult mice, resulted in an expansion of pro-inflammatory colonic macrophages by increasing the recruitment of bone-marrow-derived macrophages. Single-cell RNA sequencing of neonatal colonic macrophages revealed that early-life vancomycin exposure was associated with an increase in immature and inflammatory macrophages, consistent with an influx of circulating monocytes differentiating into macrophages. Lineage tracing confirmed that vancomycin significantly increased the non-yolk-sac-derived macrophage population. Consistent with these results, early-life vancomycin exposure did not expand the colonic macrophage population nor decrease enteric neuron density in CCR2-deficient mice. Collectively, these findings demonstrate that early-life vancomycin exposure alters macrophage number and phenotypes in distinct ways compared with vancomycin exposure in adult mice and results in altered ENS development.

The microbial one-hit wonder.

Intestinal inflammation, in the absence of infection, occurs from contributions by genetics and environment. Chen et al. (2021. J. Exp. Med.https://doi.org/10.1084/jem.20210324) challenge this concept by demonstrating that a dominant transmissible dysbiotic microbial community predisposes to intestinal inflammation in absence of genetic alterations.

Synchronization of mothers and offspring promotes tolerance and limits allergy.

Allergic disorders, characterized by Th2 immune responses to environmental substances, are increasingly common in children in Western societies. Multiple studies indicate that breastfeeding, early complementary introduction of food allergens, and antibiotic avoidance in the first year of life reduces allergic outcomes in at-risk children. Why the benefit of these practices is restricted to early life is largely unknown. We identified a preweaning interval during which dietary antigens are assimilated by the colonic immune system. This interval is under maternal control via temporal changes in breast milk, coincides with an influx of naive T cells into the colon, and is followed by the development of a long-lived population of colonic peripherally derived Tregs (pTregs) that can be specific for dietary antigens encountered during this interval. Desynchronization of mothers and offspring produced durable deficits in these pTregs, impaired tolerance to dietary antigens introduced during and after this preweaning interval, and resulted in spontaneous Th2 responses. These effects could be rescued by pTregs from the periweaning colon or by Tregs generated in vitro using periweaning colonic antigen-presenting cells. These findings demonstrate that mothers and their offspring are synchronized for the development of a balanced immune system.

Maternal activation of the EGFR prevents translocation of gut-residing pathogenic Escherichia coli in a model of late-onset neonatal sepsis.

Late-onset sepsis (LOS) is a highly consequential complication of preterm birth and is defined by a positive blood culture obtained after 72 h of age. The causative bacteria can be found in patients' intestinal tracts days before dissemination, and cohort studies suggest reduced LOS risk in breastfed preterm infants through unknown mechanisms. Reduced concentrations of epidermal growth factor (EGF) of maternal origin within the intestinal tract of mice correlated to the translocation of a gut-resident human pathogen Escherichia coli, which spreads systemically and caused a rapid, fatal disease in pups. Translocation of Escherichia coli was associated with the formation of colonic goblet cell-associated antigen passages (GAPs), which translocate enteric bacteria across the intestinal epithelium. Thus, maternally derived EGF, and potentially other EGFR ligands, prevents dissemination of a gut-resident pathogen by inhibiting goblet cell-mediated bacterial translocation. Through manipulation of maternally derived EGF and alteration of the earliest gut defenses, we have developed an animal model of pathogen dissemination which recapitulates gut-origin neonatal LOS.

The effect of legume supplementation on the gut microbiota in rural Malawian infants aged 6 to 12 months.

BACKGROUND: Common bean and cowpea contain about 25% protein and 25% fiber, and are recommended as complementary foods in sub-Saharan Africa. OBJECTIVE: The objective of this study was to determine if a daily legume supplement given to Malawian infants aged 6 to 12 mo alters the 16S configuration of the fecal microbiota as read out by amplicon sequence variants (ASVs). METHODS: This study was conducted within the context of a randomized, double-blind, controlled clinical trial to assess whether cowpea or common bean supplementation reduced intestinal permeability or increased linear growth. There were 2 village clusters in which the study was conducted. Fresh stool collections were flash frozen from 236 infants at ≤6 time points. The stools were sequenced using Earth Microbiome project protocols and data were processed using Qiime and Qiita, open-source, validated software packages. α-diversity was measured using the Faith's test. The 16S configuration was characterized by determining the weighted UniFrac distances of the ASVs and comparing them using permutational multivariate ANOVA. RESULTS: Among the 1249 samples analyzed, the α-diversity of the fecal microbiome was unchanged among subjects after initiation of legume supplementation. Neither cowpea nor common bean altered the overall 16S configuration at any age. The 16S configuration differed between children with adequate and poor linear growth aged from 6 to 9 mo, but no specific ASVs differed in relative abundance. The 16S configuration differed between children with normal and abnormal intestinal permeability at 9 mo, but no specific ASVs differed in relative abundance. Among categorical characteristics of the population associated with different 16S configurations, village cluster was most pronounced. CONCLUSION: Legume supplementation in breastfed, rural African infants did not affect the structure of the gut microbial communities until the children were aged 9 mo. This trial was registered at clinicaltrials.gov as NCT02472262.

Single Nucleotide Polymorphism Analysis Indicates Genetic Distinction and Reduced Diversity of Swine-Associated Methicillin Resistant Staphylococcus aureus (MRSA) ST5 Isolates Compared to Clinical MRSA ST5 Isolates.

Livestock associated methicillin resistant S. aureus (LA-MRSA) are lineages adapted to livestock species. LA-MRSA can be transmitted to humans and public health concerns exist because livestock may be the largest MRSA reservoir outside of hospital settings. Although the predominant European (ST398) and Asian (ST9) lineages of LA-MRSA are considered livestock adapted, North American swine also harbor ST5, a globally disseminated and highly pathogenic lineage. This study applied whole genome sequencing and single nucleotide polymorphism (SNP) typing to compare the population structure and genetic relatedness between swine associated and human clinical MRSA ST5 isolates. The established high-resolution phylogenomic framework revealed that LA-MRSA and human clinical MRSA ST5 are genetically distinct. LA-MRSA isolates were found to be clonal within farms, while greater genome diversity was observed among sampled clinical MRSA ST5. Analysis of the accessory genome demonstrated that LA-MRSA ST5 isolates and clinical MRSA ST5 isolates harbor different AMR genes and virulence factors, consistent with the SNP analysis. Collectively, our data indicate LA-MRSA and clinical MRSA ST5 isolates are distinct and the swine reservoir is likely of minimal significance as a source of clinical MRSA ST5 infections.

Identification of a mouse Lactobacillus johnsonii strain with deconjugase activity against the FXR antagonist T-ß-MCA.

Bile salt hydrolase (BSH) activity against the bile acid tauro-beta-muricholic acid (T-ß-MCA) was recently reported to mediate host bile acid, glucose, and lipid homeostasis via the farnesoid X receptor (FXR) signaling pathway. An earlier study correlated decreased Lactobacillus abundance in the cecum with increased concentrations of intestinal T-ß-MCA, an FXR antagonist. While several studies have characterized BSHs in lactobacilli, deconjugation of T-ß-MCA remains poorly characterized among members of this genus, and therefore it was unclear what strain(s) were responsible for this activity. Here, a strain of L. johnsonii with robust BSH activity against T-ß-MCA in vitro was isolated from the cecum of a C57BL/6J mouse. A screening assay performed on a collection of 14 Lactobacillus strains from nine different species identified BSH substrate specificity for T-ß-MCA only in two of three L. johnsonii strains. Genomic analysis of the two strains with this BSH activity revealed the presence of three bsh genes that are homologous to bsh genes in the previously sequenced human-associated strain L. johnsonii NCC533. Heterologous expression of several bsh genes in E. coli followed by enzymatic assays revealed broad differences in substrate specificity even among closely related bsh homologs, and suggests that the phylogeny of these enzymes does not closely correlate with substrate specificity. Predictive modeling allowed us to propose a potential mechanism driving differences in BSH activity for T-ß-MCA in these homologs. Our data suggests that L. johnsonii regulates T-ß-MCA levels in the mouse intestinal environment, and that this species may play a central role in FXR signaling in the mouse.

CRISPR System Acquisition and Evolution of an Obligate Intracellular Chlamydia-Related Bacterium.

Recently, a new Chlamydia-related organism, Protochlamydia naegleriophila KNic, was discovered within a Naegleria amoeba. To decipher the mechanisms at play in the modeling of genomes from the Protochlamydia genus, we sequenced the full genome of Pr. naegleriophila, which includes a 2,885,090 bp chromosome and a 145,285 bp megaplasmid. For the first time within the Chlamydiales order, we describe the presence of a clustered regularly interspaced short palindromic repeats (CRISPR) system, the immune system of bacteria, located on the chromosome. It is composed of a small CRISPR locus comprising eight repeats and associated cas-cse genes of the subtype I-E. A CRISPR locus is also present within Chlamydia sp. Diamant, another Pr. naegleriophila strain, suggesting that the CRISPR system was acquired by a common ancestor of Pr. naegleriophila, after its divergence from Pr. amoebophila. Both nucleotide bias and comparative genomics approaches identified probable horizontal gene acquisitions within two and four genomic islands in Pr. naegleriophila KNic and Diamant genomes, respectively. The plasmid encodes an F-type conjugative system highly similar to 1) that found in the Pam100G genomic island of Pr. amoebophila UWE25 chromosome, as well as on the plasmid of Rubidus massiliensis and 2) to the three genes remaining in the chromosome of Parachlamydia acanthamoebae strains. Therefore, this conjugative system was likely acquired on an ancestral plasmid before the divergence of Parachlamydiaceae Overall, this new complete Pr. naegleriophila genome sequence enables further investigation of the dynamic processes shaping the genomes of the family Parachlamydiaceae and the genus Protochlamydia.

Whole Genome Sequencing for Genomics-Guided Investigations of Escherichia coli O157:H7 Outbreaks.

Multi isolate whole genome sequencing (WGS) and typing for outbreak investigations has become a reality in the post-genomics era. We applied this technology to strains from Escherichia coli O157:H7 outbreaks. These include isolates from seven North America outbreaks, as well as multiple isolates from the same patient and from different infected individuals in the same household. Customized high-resolution bioinformatics sequence typing strategies were developed to assess the core genome and mobilome plasticity. Sequence typing was performed using an in-house single nucleotide polymorphism (SNP) discovery and validation pipeline. Discriminatory power becomes of particular importance for the investigation of isolates from outbreaks in which macrogenomic techniques such as pulse-field gel electrophoresis or multiple locus variable number tandem repeat analysis do not differentiate closely related organisms. We also characterized differences in the phage inventory, allowing us to identify plasticity among outbreak strains that is not detectable at the core genome level. Our comprehensive analysis of the mobilome identified multiple plasmids that have not previously been associated with this lineage. Applied phylogenomics approaches provide strong molecular evidence for exceptionally little heterogeneity of strains within outbreaks and demonstrate the value of intra-cluster comparisons, rather than basing the analysis on archetypal reference strains. Next generation sequencing and whole genome typing strategies provide the technological foundation for genomic epidemiology outbreak investigation utilizing its significantly higher sample throughput, cost efficiency, and phylogenetic relatedness accuracy. These phylogenomics approaches have major public health relevance in translating information from the sequence-based survey to support timely and informed countermeasures. Polymorphisms identified in this work offer robust phylogenetic signals that index both short- and long-term evolution and can complement currently employed typing schemes for outbreak ex- and inclusion, diagnostics, surveillance, and forensic studies.

Genome Sequences of Five Clinical Isolates of Klebsiella pneumoniae.

Klebsiella pneumoniae is a nosocomial pathogen of emerging importance and displays resistance to broad-spectrum antibiotics, such as carbapenems. Here, we report the genome sequences of five clinical K. pneumoniae isolates, four of which are carbapenem resistant. Carbapenem resistance is conferred by hydrolyzing class A ß-lactamases found adjacent to transposases.

Escherichia coli O157:H7 strains harbor at least three distinct sequence types of Shiga toxin 2a-converting phages.

BACKGROUND: Shiga toxin-producing Escherichia coli O157:H7 is a foodborne pathogen that causes severe human diseases including hemolytic uremic syndrome (HUS). The virulence factor that mediates HUS, Shiga toxin (Stx), is encoded within the genome of a lambdoid prophage. Although draft sequences are publicly available for a large number of E. coli O157:H7 strains, the high sequence similarity of stx-converting bacteriophages with other lambdoid prophages poses challenges to accurately assess the organization and plasticity among stx-converting phages due to assembly difficulties. METHODS: To further explore genome plasticity of stx-converting prophages, we enriched phage DNA from 45 ciprofloxacin-induced cultures for subsequent 454 pyrosequencing to facilitate assembly of the complete phage genomes. In total, 22 stx2a-converting phage genomes were closed. RESULTS: Comparison of the genomes distinguished nine distinct phage sequence types (PSTs) delineated by variation in obtained sequences, such as single nucleotide polymorphisms (SNPs) and insertion sequence element prevalence and location. These nine PSTs formed three distinct clusters, designated as PST1, PST2 and PST3. The PST2 cluster, identified in two clade 8 strains, was related to stx2a-converting phages previously identified in non-O157 Shiga-toxin producing E. coli (STEC) strains associated with a high incidence of HUS. The PST1 cluster contained phages related to those from E. coli O157:H7 strain Sakai (lineage I, clade 1), and PST3 contained a single phage that was distinct from the rest but most related to the phage from E. coli O157:H7 strain EC4115 (lineage I/II, clade 8). Five strains carried identical stx2a-converting phages (PST1-1) integrated at the same chromosomal locus, but these strains produced different levels of Stx2. CONCLUSION: The stx2a-converting phages of E. coli O157:H7 can be categorized into at least three phage types. Diversification within a phage type is mainly driven by IS629 and by a small number of SNPs. Polymorphisms between phage genomes may help explain differences in Stx2a production between strains, however our data indicates that genes encoded external to the phage affect toxin production as well.

Genome Sequence of Bacillus thuringiensis Strain Btm27, an Egyptian Isolate Highly Toxic to Cotton Leafworm.

Bacillus thuringiensis is a potent microbial control agent against insect pests. Here, we present the draft genome of the Egyptian strain Btm27 that shows high toxicity toward the cotton leafworm. The genome contains three insecticidal genes cry1Ac9, cry2Ab1, and vip3V that have been implicated in conferring toxicity toward lepidoptera.

Characterization of the pathogenome and phylogenomic classification of enteropathogenic Escherichia coli of the O157:non-H7 serotypes.

Escherichia coli of the O157 serogroup are comprised of a diverse collection of more than 100 O157:non-H7 serotypes that are found in the environment, animal reservoir and infected patients and some have been linked to severe outbreaks of human disease. Among these, the enteropathogenic E. coli O157:non-H7 serotypes carry virulence factors that are hallmarks of enterohemorrhagic E. coli, such as causing attaching and effacing lesions during human gastrointestinal tract infections. Given the shared virulence gene pool between O157:H7 and O157:non-H7 serotypes, our objective was to examine the prevalence of virulence traits of O157:non-H7 serotypes within and across their H-serotype and when compared to other E. coli pathovars. We sequenced six O157:non-H7 genomes complemented by four genomes from public repositories in an effort to determine their virulence state and genetic relatedness to the highly pathogenic enterohemorrhagic O157:H7 lineage and its ancestral O55:H7 serotype. Whole-genome-based phylogenomic analysis and molecular typing is indicative of a non-monophyletic origin of the heterogeneous O157:non-H7 serotypes that are only distantly related to the O157:H7 serotype. The availability of multiple genomes enables robust phylogenomic placement of these strains into their evolutionary context, and the assessment of the pathogenic potential of the O157:non-H7 strains in causing human disease.

Trafficking of Estrella lausannensis in human macrophages.

Estrella lausannensis is a new member of the Chlamydiales order. Like other Chlamydia-related bacteria, it is able to replicate in amoebae and in fish cell lines. A preliminary study investigating the pathogenic potential of Chlamydia-related bacteria found a correlation between antibody response to E. lausannensis and pneumonia in children. To further investigate the pathogenic potential of E. lausannensis, we determined its ability to grow in human macrophages and its intracellular trafficking. The replication in macrophages resulted in viable E. lausannensis; however, it caused a significant cytopathic effect. The intracellular trafficking of E. lausannensis was analyzed by determining the interaction of the Estrella-containing inclusions with various endocytic markers as well as host organelles. The E. lausannensis inclusion escaped the endocytic pathway rapidly avoiding maturation into phagolysosomes by preventing both EEA-1 and LAMP-1 accumulation. Compared to Waddlia chondrophila, another Chlamydia-related bacteria, the recruitment of mitochondria and endoplasmic reticulum was minimal for E. lausannensis inclusions. Estrella lausannensis appears to use a distinct source of nutrients and energy compared to other members of the Chlamydiales order. In conclusion, we hypothesize that E. lausannensis has a restricted growth in human macrophages, due to its reduced capacity to control programmed cell death.

Crescent and star shapes of members of the Chlamydiales order: impact of fixative methods.

Members of the Chlamydiales order all share a biphasic lifecycle alternating between small infectious particles, the elementary bodies (EBs) and larger intracellular forms able to replicate, the reticulate bodies. Whereas the classical Chlamydia usually harbours round-shaped EBs, some members of the Chlamydia-related families display crescent and star-shaped morphologies by electron microscopy. To determine the impact of fixative methods on the shape of the bacterial cells, different buffer and fixative combinations were tested on purified EBs of Criblamydia sequanensis, Estrella lausannensis, Parachlamydia acanthamoebae, and Waddlia chondrophila. A linear discriminant analysis was performed on particle metrics extracted from electron microscopy images to recognize crescent, round, star and intermediary forms. Depending on the buffer and fixatives used, a mixture of alternative shapes were observed in varying proportions with stars and crescents being more frequent in C. sequanensis and P. acanthamoebae, respectively. No tested buffer and chemical fixative preserved ideally the round shape of a majority of bacteria and other methods such as deep-freezing and cryofixation should be applied. Although crescent and star shapes could represent a fixation artifact, they certainly point towards a diverse composition and organization of membrane proteins or intracellular structures rather than being a distinct developmental stage.

Discovery of catalases in members of the Chlamydiales order.

Catalase is an important virulence factor for survival in macrophages and other phagocytic cells. In Chlamydiaceae, no catalase had been described so far. With the sequencing and annotation of the full genomes of Chlamydia-related bacteria, the presence of different catalase-encoding genes has been documented. However, their distribution in the Chlamydiales order and the functionality of these catalases remain unknown. Phylogeny of chlamydial catalases was inferred using MrBayes, maximum likelihood, and maximum parsimony algorithms, allowing the description of three clade 3 and two clade 2 catalases. Only monofunctional catalases were found (no catalase-peroxidase or Mn-catalase). All presented a conserved catalytic domain and tertiary structure. Enzymatic activity of cloned chlamydial catalases was assessed by measuring hydrogen peroxide degradation. The catalases are enzymatically active with different efficiencies. The catalase of Parachlamydia acanthamoebae is the least efficient of all (its catalytic activity was 2 logs lower than that of Pseudomonas aeruginosa). Based on the phylogenetic analysis, we hypothesize that an ancestral class 2 catalase probably was present in the common ancestor of all current Chlamydiales but was retained only in Criblamydia sequanensis and Neochlamydia hartmannellae. The catalases of class 3, present in Estrella lausannensis and Parachlamydia acanthamoebae, probably were acquired by lateral gene transfer from Rhizobiales, whereas for Waddlia chondrophila they likely originated from Legionellales or Actinomycetales. The acquisition of catalases on several occasions in the Chlamydiales suggests the importance of this enzyme for the bacteria in their host environment.