Gram-negative Commensal Bacteria Research Articles

Antimicrobial Resistance in Diverse Escherichia coli Pathotypes from Nigeria.

Escherichia coli is a gram-negative commensal bacterium living in human and animal intestines. Its pathogenic strains lead to high morbidity and mortality, which can adversely affect people by causing urinary tract infections, food poisoning, septic shock, or meningitis. Humans can contract E. coli by eating contaminated food-such as raw or undercooked raw milk, meat products, and fresh produce sold in open markets-as well as by coming into contact with contaminated settings like wastewater, municipal water, soil, and faeces. Some pathogenic strains identified in Nigeria, include Enterohemorrhagic (Verotoxigenic), Enterotoxigenic, Enteropathogenic, Enteroinvasive, and Enteroaggregative E. coli. This causes acute watery or bloody diarrhoea, stomach cramps, and vomiting. Apart from the virulence profile of E. coli, antibiotic resistance mechanisms such as the presence of blaCTX-M found in humans, animals, and environmental isolates are of great importance and require surveillance and monitoring for emerging threats in resource-limited countries. This review is aimed at understanding the underlying mechanisms of evolution and antibiotic resistance in E. coli in Nigeria and highlights the use of improving One Health approaches to combat the problem of emerging infectious diseases.

Cave Bats as Carriers of Extended Spectrum Beta-lactamase Produced by Escherichia coli from the Island of Lombok, Indonesia

Background: Escherichia coli is a Gram-negative commensal bacterium that normally resides in the digestive tract of humans and animals which is used as an indicator of the extended-spectrum beta-lactamase (ESBL) gene in an individual and can carry out gene transfer. Transmission of ESBL E. coli from wild animals has not been given much attention even though wild animals can act as vectors that spread ESBL in the environment so that it can pose a risk to public health. This research aims to identify the blaTEM gene which codes for ESBL in E. coli from bats that live in caves in Lombok, Indonesia. Methods: A total of 135 samples from bat rectal swabs were cultured on Eosin Methylene Blue Agar media and biochemical suspects were identified using the Indole, Methyl red, Voges-Proskauer, Citrate + and H2S (IMVIC) test. The E. coli bacteria obtained were tested for sensitivity using 7 classes of antibiotics, namely amoxicillin, ciprofloxacine, sulfamethoxazole/trimethoprim, tetracycline, gentamicin, cefotaxime and azithromycin. Bacteria that show multidrug resistance are subjected to PCR testing to detect the blaTEM gene. Result: Of the 135 test samples, it was found that 97 (71.85%) samples were positive for E. coli, 12 (12.37%) samples were Multidrug Resistance (MDR) and 2 (2.06%) samples had the blaTEM gene as the ESBL coding gene in bats that live in caves on the island of Lombok, Indonesia. The presence of the blaTEM gene in E. coli from bats can be indicated as a reservoir for MDR and ESBL transmission so that it can have an impact on public health.

Bacterial peptidoglycan signalling in microglia: Activation by MDP via the NF-κB/MAPK pathway

Bacterial peptidoglycan (PGN) fragments are commonly studied in the context of bacterial infections. However, PGN fragments recently gained recognition as signalling molecules from the commensal gut microbiota in the healthy host. Here we focus on the minimal bioactive PGN motif muramyl dipeptide (MDP), found in both Gram-positive and Gram-negative commensal bacteria, which signals through the Nod2 receptor. MDP from the gut microbiota translocates to the brain and is associated with changes in neurodevelopment and behaviour, yet there is limited knowledge about the underlying mechanisms. In this study we demonstrate that physiologically relevant doses of MDP induce rapid changes in microglial gene expression and lead to cytokine and chemokine secretion. In immortalised microglial (IMG) cells, C–C Motif Chemokine Ligand 5 (CCL5/RANTES) expression is acutely sensitive to the lowest physiologically prevalent dose (0.1 µg/ml) of MDP. As CCL5 plays an important role in memory formation and synaptic plasticity, microglial CCL5 might be the missing link in elucidating MDP-induced alterations in synaptic gene expression. We observed that a higher physiological dose of MDP elevates the expression of cytokines TNF-α and IL-1β, indicating a transition toward a pro-inflammatory phenotype in IMG cells, which was validated in primary microglial cultures. Furthermore, MDP induces the translocation of NF-κB subunit p65 into the nucleus, which is blocked by MAPK p38 inhibitor SB202190, suggesting that an interplay of both the NF-κB and MAPK pathways is responsible for the MDP-specific microglial phenotype. These findings underscore the significance of different MDP levels in shaping microglial function in the CNS and indicate MDP as a potential mediator for early inflammatory processes in the brain. It also positions microglia as an important target in the gut microbiota-brain-axis pathway through PGN signalling.

Glaesserella parasuis serotype 5 induces pyroptosis via the RIG-I/MAVS/NLRP3 pathway in swine tracheal epithelial cells

Glaesserella parasuis (G. parasuis) is a common Gram-negative commensal bacterium in the upper respiratory tract of swine that can cause Glässer's disease under stress conditions. Pyroptosis is an important immune defence mechanism of the body that plays a crucial role in clearing pathogen infections and endogenous danger signals. This study aimed to investigate the mechanism of G. parasuis serotype 5 SQ (GPS5-SQ)-induced pyroptosis in swine tracheal epithelial cells (STECs). The results of the present study demonstrated that GPS5-SQ infection induces pyroptosis in STECs by enhancing the protein level of the N-terminal domain of gasdermin D (GSDMD-N) and activating the NOD-like receptor protein 3 (NLRP3) inflammasome. Furthermore, the levels of pyroptosis-related proteins, including GSDMD-N and cleaved caspase-1 were considerably decreased in STECs after the knockdown of retinoic acid inducible gene-I (RIG-I) and mitochondrial antiviral signaling protein (MAVS). These results indicated that GPS5-SQ might trigger pyroptosis through the activation of the RIG-I/MAVS/NLRP3 signaling pathway. More importantly, the reactive oxygen species (ROS) scavenger N-acetylcysteine (NAC) repressed the activation of the RIG-I/MAVS/NLRP3 signaling and rescued the decrease in Occludin and zonula occludens-1 (ZO-1) after GPS5-SQ infection. Overall, our findings show that GPS5-SQ can activate RIG-I/MAVS/NLRP3 signaling and destroy the integrity of the epithelial barrier by inducing ROS generation in STECs, shedding new light on G. parasuis pathogenesis.

A gut commensal bacterium promotes black soldier fly larval growth and development partly via modulation of intestinal protein metabolism.

Black solider fly larvae and the gut microbiota can recycle nutrients from various organic wastes into valuable insect biomass. We found that Citrobacter amalonaticus, a gut commensal bacterium of the insect, exerts beneficial effects on larval growth and development and that the expression of many metabolic larval genes was significantly impacted by the symbiont. To identify the larval genes involved in the host-symbiont interaction, we engineered the symbiont to produce double-strand RNA and enabled the strain to silence host genes in the larval gut environment where the interaction takes place. With this approach, we confirmed that two intestinal protease families are involved in the interaction and provided further evidence that intestinal protein metabolism plays a role in the interaction. This work expands the genetic toolkits available to study the insect functional genomics and host-symbiont interaction and provide the prospective for the future application of gut microbiota on the large-scale bioconversion.

Comprehensive Analysis of Bacteriocins Produced by the Hypermucoviscous Klebsiella pneumoniae Species Complex.

Klebsiella pneumoniae produces several kinds of bacteriocins that have antimicrobial effects against closely related species, but few studies have comprehensively reported bacteriocin distribution among the Klebsiella population. In this study, we identified bacteriocin genes in 180 K. pneumoniae species complex genomes, including 170 hypermucoviscous isolates, and investigated the antibacterial activity against 50 strains, including antimicrobial-resistant organisms, belonging to multiple species, namely, Klebsiella spp., Escherichia coli, Pseudomonas spp., Acinetobacter spp., Enterobacter cloacae, Stenotrophomonas maltophilia, Chryseobacterium indologenes, Staphylococcus aureus, Staphylococcus epidermidis, and Streptococcus mutans. Our study determined that 32.8% (59/180) of isolates carried at least one bacteriocin type. Different types of bacteriocin were usually present in different specific sequence types (STs); meanwhile, bacteriocins were not detected in certain STs. Microcin E492 was the most prevalent bacteriocin (14.4%), mostly in ST23 isolates, and displayed a wide spectrum of activity, including against Klebsiella spp., E. coli, Pseudomonas spp., and Acinetobacter spp. Cloacin-like bacteriocin was detected in 7.2% of strains, all of which were non-ST23 isolates, and exhibited inhibitory activity against closely related species, mainly Klebsiella spp. Klebicin B-like bacteriocin was detected at a rate of 9.4%, although 82.4% of these strains carried a disrupted bacteriocin gene, and an inhibitory effect could not be observed from the intact-gene-carrying isolates. Other bacteriocins, such as microcin S-like, microcin B17, and klebicin C-like, were detected at lower rates and had limited inhibitory activity. Our findings suggested that Klebsiella strains that carry different bacteriocin types may affect the composition of the surrounding bacterial community. IMPORTANCE Klebsiella pneumoniae is a Gram-negative commensal bacterium that asymptomatically colonizes human mucosal membranes, such as the intestinal tract, but it is also a leading cause of health care- and community-associated infections. Additionally, multidrug-resistant K. pneumoniae has been continuously evolving, which significantly challenges the available chemotherapeutic treatment for its infections. K. pneumoniae produces several kinds of antimicrobial peptides known as bacteriocins, which have antibacterial activity against closely related species. This work was the first comprehensive report of bacteriocin distribution among the hypermucoviscous K. pneumoniae species complex population and the inhibitory activity of each bacteriocin type against various species, including multidrug-resistant strains. Our findings provide a foundation for future studies on the K. pneumoniae species complex, including studies on the competition within the microflora and the potential applications of bacteriocins in treating multidrug-resistant bacteria.

Hematopoietic stem and progenitor cells integrate microbial signals to promote post-inflammation gut tissue repair.

Bone marrow (BM)-resident hematopoietic stem and progenitor cells (HSPCs) are often activated following bacterial insults to replenish the host hemato-immune system, but how they integrate the associated tissue damage signals to initiate distal tissue repair is largely unknown. Here, we show that acute gut inflammation expands HSPCs in the BM and directs them to inflamed mesenteric lymph nodes through GM-CSFR activation for further expansion and potential differentiation into Ly6C+ /G+ myeloid cells specialized in gut tissue repair. We identified this process to be mediated by Bacteroides, a commensal gram-negative bacteria that activates innate immune signaling. These findings establish cross-organ communication between the BM and distant inflamed sites, whereby a certain subset of multipotent progenitors is specified to respond to imminent hematopoietic demands and to alleviate inflammatory symptoms.

The Effect of Intensity-Modulated Radiotherapy to the Head and Neck Region on the Oral Innate Immune Response and Oral Microbiome: A Prospective Cohort Study of Head and Neck Tumour Patients.

Neutrophils, also known as polymorphonuclear leukocytes (PMNs), form a significant component of the innate host response, and the consequence of the interaction between the oral microbiota and PMNs is a crucial determinant of oral health status. The impact of radiation therapy (RT) for head and neck tumour (HNT) treatment on the oral innate immune system, neutrophils in particular, and the oral microbiome has not been thoroughly investigated. Therefore, the objective of this study was to characterize RT-mediated changes in oral neutrophils (oPMNs) and the oral microbiome in patients undergoing RT to treat HNTs. Oral rinse samples were collected prior to, during and post-RT from HNT patients receiving RT at Dental Oncology at Princess Margaret Cancer Centre. The oPMNs counts and activation states were analysed using flow cytometry, and the oral microbiome was analysed using 16S rRNA gene sequencing. Statistically significant (p < 0.05) drops in oPMN counts and the activation states of the CD11b, CD16, CD18, CD64 and H3Cit markers from pre-RT to post-RT were observed. Moreover, exposure to RT caused a significant reduction in the relative abundance of commensal Gram-negative bacteria and increased the commensal Gram-positive microbes. Ionizing radiation for the treatment of HNTs simultaneously decreased the recruitment of oPMNs into the oral cavity and suppressed their activation state. The oral microbiome composition post-RT was altered significantly due to RT which may favour the colonization of specific microbial communities unfavourable for the long-term development of a balanced oral microbiome.

Dynamic changes in Holstein heifer circulatory stress biomarkers in response to lipopolysaccharide immune challenge

Dairy cattle routinely face a variety of stressors. For example, climate change has resulted in more frequent heat stress events that increase the incidence of bacterial infections by inducing conditions like leaky gut syndrome, whereby the integrity of the intestinal epithelium is compromised allowing for luminal bacteria and their membrane components to infiltrate the host’s bloodstream resulting in systemic inflammation. Lipopolysaccharide (LPS) is a well-characterized and biologically relevant microbe-associated molecular pattern (MAMP) that makes up the outer membrane of pathogenic and commensal Gram-negative bacteria and is known to contribute to inflammatory disorders including mastitis, acidosis and septicemia. In the present study, Holstein heifers (n = 20) were randomly allocated into different treatment groups receiving saline, 100, 200 or 400 ng/kg of LPS intramuscularly to create an experimentally induced endotoxemic state. Serum was collected hourly for 8 hr and then again at 24 hr to profile changes in circulatory stress biomarkers. All LPS -challenged animals demonstrated distinct cortisol responses 2 hr post-LPS challenge, and in the 200 ng/kg and 400 ng/kg of LPS treatments cortisol concentrations remained significantly induced for up to 4 hr. Rectal temperature was significantly increased for heifers challenged with 100 and 200 ng/kg of LPS at 2 and 4 hr as compared to their pre-challenge temperature. All LPS-challenged animals demonstrated marked leukopenia and thrombocytopenia as compared to the saline control animals. A total of 8 cytokines, including TNFα, and IL-10, were found to be induced between 2 and 4 hr. Finally, we report that miR-1246, miR-223, miR-29 and miR-31 were significantly induced in animals challenged with LPS as compared to the saline controls. The present study demonstrated that the stress response in dairy heifers is dynamic and there are peak windows of time when cortisol, cytokines and also miRNA are induced, and blood cells are sequestered as part of the systemic inflammatory response. Variability in the response to LPS warrants further investigation in dairy cattle to better understand the contribution of genetics and associations between LPS-induced stress and health and performance.

Type VI secretion systems of pathogenic and commensal bacteria mediate niche occupancy in the gut.

The type VI secretion system (T6SS) is a contractile nanomachine widely distributed among pathogenic and commensal Gram-negative bacteria. The T6SS is used for inter-bacterial competition to directly kill competing species; however, its importance during bacterial infection invivo remains poorly understood. We report that the murine pathogen Citrobacter rodentium, used as a model for human pathogenic Escherichia coli, harbors two functional T6SSs. C.rodentium employs its T6SS-1 to colonize the murine gastrointestinal tract by targeting commensal Enterobacteriaceae. We identify VgrG1 as a C.rodentium T6SS antibacterial effector, which exhibits toxicity in E.coli. Conversely, commensal prey species E.coli Mt1B1 employs two T6SSs of its own to counter C.rodentium colonization. Collectively, these data demonstrate that the T6SS is a potent weapon during bacterial competition and is used by both invading pathogens and resident microbiota to fight for a niche in the hostile gut environment.

Gut Bacteria Induce Granzyme B Expression in Human Colonic ILC3s In Vitro in an IL-15-Dependent Manner.

Group 3 innate lymphoid cells (ILC3s) in the gut mucosa have long been thought to be noncytotoxic lymphocytes that are critical for homeostasis of intestinal epithelial cells through secretion of IL-22. Recent work using human tonsillar cells demonstrated that ILC3s exposed to exogenous inflammatory cytokines for a long period of time acquired expression of granzyme B, suggesting that under pathological conditions ILC3s may become cytotoxic. We hypothesized that inflammation associated with bacterial exposure might trigger granzyme B expression in gut ILC3s. To test this, we exposed human colon lamina propria mononuclear cells to a panel of enteric bacteria. We found that the Gram-negative commensal and pathogenic bacteria induced granzyme B expression in a subset of ILC3s that were distinct from IL-22-producing ILC3s. A fraction of granzyme B+ ILC3s coexpressed the cytolytic protein perforin. Granzyme B expression was mediated, in part, by IL-15 produced upon exposure to bacteria. ILC3s coexpressing all three IL-15R subunits (IL15Rα/β/γ) increased following bacterial stimulation, potentially allowing for cis presentation of IL-15 during bacterial exposure. Additionally, a large frequency of colonic myeloid dendritic cells expressed IL-15Rα, implicating myeloid dendritic cells in trans presentation of IL-15 to ILC3s. Tonsillar ILC3s minimally expressed granzyme B when exposed to the same bacteria or to rIL-15. Overall, these data establish the novel, to our knowledge, finding that human colonic ILC3s can express granzyme B in response to a subset of enteric bacteria through a process mediated by IL-15. These observations raise new questions about the multifunctional role of human gut ILC3s.

Pathological and Microbiological Findings in Fatal Cases of Salmonellosis in Captive Bothrops Snakes in Southern Brazil

Salmonella spp. are gram-negative commensal bacteria of vertebrates, including reptiles. Infected snakes may be asymptomatic or manifest clinical disease and death, especially after stressful events. Salmonellosis was diagnosed in 10 captive snakes from the Bothrops genus. The most frequent changes were emaciation (8/10), fibrinonecrotic or granulomatous hepatitis (8/9), fibrinonecrotic or granulomatous enterocolitis (8/9), necrotic and heterophilic myocarditis (2/10), fibrinonecrotic or granulomatous pancreatitis (2/5), fibrinoheterophilic osteomyelitis (1/10), fibrinous and pyogranulomatous pericarditis (1/10) and granulomatous splenitis (1/6). Salmonella enterica was isolated from six cases. The subspecies identified were arizonae (3/6), diarizonae (1/6) and houtenae (1/6), in addition to the serotype Typhimurium (1/6). In cases without isolation, there was immunolabelling of Salmonella spp. in intestinal (3/4), hepatic (1/4) and cardiac (1/4) lesions.

Gut Microbiota Dysbiosis and Elevated Lipopolysaccharide Serum Levels Are Associated with Venous Thromboembolism in Pediatric Patients

Background: Pediatric venous thromboembolism (VTE) is a burgeoning clinical problem. As such, there is an urgent need to identify modifiable host factors to prevent VTE and its long-term adverse effects. The gut microbiome, which can influence host physiology and is implicated in the pathogenesis of several disease states, is one such potentially modifiable host factor. Gut microbiota dysbiosis is an imbalance in the composition of the gut microbiota that often manifests as an overabundance of gram-negative bacteria. Lipopolysaccharide (LPS), the glycolipid on the outer membrane of gram-negative bacteria and a heat stable endotoxin, can cross the gut epithelial barrier into the systemic circulation, where it leads to the activation of inflammatory and coagulation pathways. The association of gut microbiota dysbiosis and VTE has not been fully explored. Objective/Hypothesis: The aims of this study were to determine if patients who develop VTE have 1) a distinct microbiota signature with an expansion of gram-negative bacteria, 2) increased systemic levels of LPS and 3) hypercoagulability as compared to controls without VTE. Methods: In this case-control study, we obtained plasma and fecal samples on newly diagnosed VTE pediatric patients and age-matched controls. Gut microbiome profiling was performed on fecal gDNA (16S rRNA sequencing, V4 region; QIIME2 software for gut microbiota taxonomic composition determination). We measured LPS levels using a sandwich ELISA assay (Cusabio) and hypercoagulability using endogenous thrombin potential (ETP) on thrombin generation assay (Calibrated automated thrombogram) on plasma samples. Results: 31 VTE patients were enrolled, of which 18 had fecal samples, and 22 had plasma samples. The median age of VTE cases was 11.0 years (range 1.0 to 19.0 years) of which 51% had underlying medical comorbidities and 48% had antibiotic exposure 30 days prior to the VTE episode. 5 controls were enrolled with a median age of 14.0 years (range 1.0 to 14.0 years). VTE cases had significant enrichment of commensal gram-negative bacteria Bacteroidetes (median 44% relative abundance compared to 21% in controls, p=0.006, Mann-Whitney) and depletion of commensal gram-positive Actinobacteria (median of 2% vs. 10% in controls, p=0.04, Mann-Whitney). Taxonomic differential abundance by linear analysis coupled with effective side measurements (LEfSe) further confirmed these findings by identifying two specific Bacteroidetes species as being enriched in patients with VTE: Bacteroides stercoris and Bacteroides vulgatus (p= 0.01 and 0.02 respectively, Kruskal-Wallis). VTE patients had significantly higher plasma levels of LPS (n=22; mean, 64.30 ng/mL; +SEM 10.17ng/mL) compared to controls (n=5; mean, 14.75ng/mL; +SEM 6.18ng/mL; p=0.03, t-test). Furthermore, VTE cases had increased ETP (n=15; mean, 1922nM.min; +SEM 144.5nM.min) compared to controls (n=5; mean, 1293nM.min; +SEM 78.11nM.min; p=0.02, t-test). Conclusion: We present the first description of elevated LPS levels, hypercoagulability, and a concomitant gut microbiota enrichment of commensal gram-negative bacteria in pediatric patients experiencing an acute VTE episode. Our findings suggest that enrichment of commensal gram-negative bacteria in pediatric patients and LPS gut translocation could play a role in hypercoagulability and the development of VTE. Disclosures Koh: Mesoblast: Consultancy; Prolacta: Consultancy; Merck: Research Funding; Novartis: Research Funding.

Characterization and proteomic analysis of outer membrane vesicles from a commensal microbe, Enterobacter cloacae

Outer membrane vesicles (OMVs) are membrane-enclosed spherical entities released by gram-negative bacteria and are important for bacterial survival under stress conditions. There have been numerous studies on OMVs released by gram-negative pathogenic bacteria, but an understanding of the functions and characteristics of the OMVs produced by commensal microbes is still lacking. Enterobacter cloacae is a gram-negative commensal bacterium present in the human gut microbiome, but this organism can also function as an opportunistic pathogen. Understanding the OMV-mediated communication route between bacteria-bacteria or bacteria-host is essential for the determination of the biological functions of the commensal bacterium in the gut and delineating between benign and virulent characteristics. In this study, we have described a proteome of E. cloacae OMVs, which are membrane vesicles in a size range of 20–300 nm. Proteomic analysis showed the presence of membrane-bound proteins, including transporters, receptors, signaling molecules, and protein channels. The physical and proteomic analyses also indicate this bacterium uses two mechanisms for OMV production. This study is one of the few existing descriptions of the proteomic profile of OMVs generated by a commensal Proteobacteria, and the first report of OMVs produced by E. cloacae. SignificanceThis study prioritizes the importance of understanding the vesicular proteome of the human commensal bacterium, Enterobacter cloacae. We demonstrate for the first time that the gram-negative bacterium E. cloacae ATCC 13047 produces outer membrane vesicles (OMVs). The proteomic analysis showed enrichment of membrane-bound proteins in these vesicles. Understanding the cargo proteins of OMVs will help in exploring the physiological and functional role of these vesicles in the human microbiome and how they assist in the conversion of a bacterium from commensal to pathogen under certain conditions. While EM images reveal vesicles budding from the bacterial surface, the presence of cytoplasmic proteins and genomic DNA within the OMVs indicate that explosive cell lysis is an additional mechanism of biogenesis for these OMVs along with outer membrane blebbing. This research encourages future work on characterizing membrane vesicles produced by commensal bacterial and investigating their role in cell to cell communication.

Are all faecal bacteria detected with equal efficiency? A study using next-generation sequencing and quantitative culture of infants' faecal samples

BackgroundMany species of intestinal bacteria are present in moderate numbers in the faecal microbiota, which is dominated by obligate anaerobes. Little is known regarding the detection sensitivity of next-generation sequencing for these microbes in samples of complex microbiota. MethodsTwenty stool samples from six healthy infants, who were followed from 1 week to 1 year of age, were previously cultured quantitatively for total population counts, as well as for counts of relevant facultative bacteria and a limited selection of obligate anaerobes that are prevalent in the neonatal microbiota. The same samples were analysed by Next-generation sequencing (NGS, pyrosequencing) of the 16S rRNA gene (V1–V3 regions; average read length, 500 nucleotides; average number of reads per sample, 30,000). We used the bacterial culture data to determine the lowest bacterial populations that could be detected by NGS. Different DNA extraction kits (QIAamp DNA Stool Mini, ZR Faecal DNA MiniPrep, and PowerSoil DNA Isolation) were compared for efficacy in extracting DNA from Gram-negative and Gram-positive Type strains. ResultsNGS yielded one read per 106 CFU/g faeces of the Gram-negative commensal gut bacteria Bacteroides and Enterobacteriaceae, but only one read per 108 CFU/g faeces of Gram-positive bifidobacteria. The Gram-positive facultative bacteria Enterococcus was often undetectable by DNA-based methods despite being present at >106 CFU/g faeces. The DNA extraction kits tested varied considerably in their ability to extract DNA from bacterial samples, and showed considerably lower efficacies in extracting DNA from Gram-positive than from Gram-negative bacteria. ConclusionsNGS has lower sensitivity for detecting Gram-positive bacteria than Gram-negative bacteria, due at least in part to inefficient extraction of DNA from Gram-positive bacteria. Therefore, enzymatic lysis may enhance the yield of DNA and increase the sensitivity of NGS methods for Gram-positive bacteria, and the inclusion of positive and negative controls during DNA extraction is indicated for validation purposes. The differential extraction of DNA from bacterial samples by different DNA extraction kits may limit comparability between studies on the gut microbiota. Finally, quantitative culture methods detect certain bacteria with greater sensitivity than NGS techniques, and thus culture- and DNA-based methods can be used in tandem to define the complex composition of the gut microbiota with greater accuracy.

Prevalence of Antimicrobial Resistance Genes and Integrons in Commensal Gram-Negative Bacteria in a College Community.

Although the human intestinal microbiome has been shown to harbor antimicrobial drug resistance genes (ARGs), the prevalence of such genes in a healthy population and their impact on extraintestinal infections that occur in that community are not well established. This study sought to identify ARG prevalence and their mobile elements in the intestines of a healthy community population at a California University, and compared these genes to those previously identified among uropathogenic Escherichia coli isolated from patients with urinary tract infection from the same community. We isolated Gram-negative bacteria (GNB) from fecal samples of healthy volunteers and screened them by polymerase chain reaction for class 1 integron cassette sequences and ARGs encoding resistance against ampicillin, trimethoprim-sulfamethoxazole, gentamicin, and colistin. We found antimicrobial-resistant GNB from 83 (81%) of 102 nonredundant rectal swab samples. Seventy-four (72%) of these samples contained β-lactamase genes (blaTEM, blaSHV, blaCTX-M, blaOXA, and blaOXY), dihydrofolate reductase (DHFR) genes (dhfr-A17, dhfr-A12, dhfr-A7, dhfr-A5, dhfr-A21, dhfr-A1, dhfr-A13, and dhfr-7), and aminoglycoside resistance genes (aadA5, aadA2, aadA1, and aadB). Integron sequences were found in 37 (36%) fecal samples. These genes were found in 11 different GNB species. The high prevalence of clinically common ARGs and integrons harbored by GNB in the intestine of a healthy population suggest that human intestines may serve as a major reservoir of these mobile ARGs that appear in E. coli strains causing extraintestinal infections in the same community.

Increased Serum Immunoglobulin Responses to Gut Commensal Gram-Negative Bacteria in Unipolar Major Depression and Bipolar Disorder Type 1, Especially When Melancholia Is Present.

Major depressive disorder (MDD) is accompanied by higher serum IgM/IgA responses to LPS of Gram-negative bacteria, suggesting increased bacterial translocation and gut dysbiosis while the latter may occur in bipolar disorder (BD). There are differences between MDD and BD type 1 (BP1) and 2 (BP2) in nitro-oxidative stress biomarkers associated with leaky gut. This study examines serum IgM/IgA responses directed to LPS of 6 Gram-negative bacteria as well as IgG responses to oxidized LDL (oxLDL) in 29 BP1, 37 BP2, 44 MDD, and 30 healthy individuals. Increased IgM/IgA responses to Pseudomonas aeruginosa significantly discriminated patients with affective disorders (MDD plus BD) from controls. BP1 patients showed higher IgM responses to Morganella morganii as compared with MDD and BP2 patients. Patients with melancholia showed higher IgA responses to Citrobacter koseri as compared to controls and non-melancholic depression. The total score on the Hamilton Depression Rating Scale was significantly associated with IgA responses to C. koseri. IgG to oxLDL was significantly associated with increased bacterial translocation. In conclusion, MDD, BP1, and BP2 are accompanied by an immune response due to the increased load of LPS while these aberrations in the gut-brain axis are most pronounced in BP1 and melancholia. Activated oxidative stress pathways and autoimmune responses to oxidative specific epitopes in mood disorders may be driven by a breakdown in gut paracellular, transcellular, and/or vascular pathways. If replicated, drugs that protect the integrity of the gut barrier may offer novel therapeutic opportunities for BP1 and MDD.

The menstrual cycle may not be limited to the endometrium but also may impact gut permeability.

To examine associations between IgA responses to Gram-negative gut commensal bacteria and peri-menstrual symptoms and sex hormone levels during the menstrual cycle in women with and without premenstrual symptoms. Forty women aged 18-45 years completed the Daily Record of Severity of Problems (DRSP) during all 28 consecutive days of the menstrual cycle. We assayed, in plasma, IgA responses to six Gram-negative bacteria, that is, Hafnei alvei, Pseudomonas aeruginosa, Morganella morganii, Klebsiella pneumoniae, Pseudomonas putida and Citobacter koseri, progesterone and oestradiol at days 7, 14, 21 and 28 of the menstrual cycle. Significant changes in Δ (actual - 1 week earlier) IgA to lipopolysaccharides (LPS) of the six Gram-negative bacteria during the menstrual cycle were observed with peak IgA levels at T4 (day 28) and lows at T1 or T2 (day 7 or 14). The ΔIgA changes in H. alvei, M. Morganii, P. putida during the menstrual cycle were significantly and positively associated with changes in the total DRSP score, and severity of physio-somatic, anxiety and breast-craving, but not depressive, symptoms. The changes in IgA responses to LPS were largely predicted by changes in progesterone and steady-state levels of progesterone averaged over the luteal phase. Menstrual cycle-associated changes in IgA directed against LPS and by inference bacterial translocation may be driven by the effects of progesterone on transcellular, paracellular and vascular pathways (leaky gut) thereby contributing to the severity of physio-somatic and anxiety symptoms as well as fatigue, breast swelling and food cravings.

Enteric bacteria induce IFNγ and Granzyme B from human colonic Group 1 Innate Lymphoid Cells

ABSTRACT Group 1 Innate Lymphoid Cells (which include Natural Killer cells and ILC1s) aid in gut anti-bacterial defense through the production of IFNγ, which is critical for mobilizing protective responses against enteric pathogens. When intestinal epithelial barrier integrity is compromised, commensal bacteria are likely to translocate from the gut lumen into the lamina propria. Few studies have addressed the mechanisms by which commensal bacteria impact the function of gut Group 1 ILCs, especially ILC1s. Utilizing an in vitro human colonic lamina propria mononuclear cell (LPMC) model, we evaluated Group 1 ILC cytokine and cytolytic protein production in response to a panel of enteric Gram-positive and Gram-negative commensal and pathogenic bacteria. IFNγ-production by NK cells and ILC1s was significantly increased after LPMC exposure to Gram-negative commensal or pathogenic bacteria, but not after exposure to the Gram-positive bacteria commensals tested. Stimulation of IFNγ production from Group 1 ILCs was not through direct recognition of bacteria by NK cells or ILC1s, but rather required accessory cells within the LPMC population. Myeloid dendritic cells generated IL-12p70, IL-18, and IL-1β upon exposure to enteric bacteria and these cytokines contributed to Group 1 ILC production of IFNγ. Furthermore, Gram-negative commensal or pathogenic bacteria induced significant expression of Granzyme B in NK cells and ILC1s. Overall, these data demonstrate that some enteric commensal bacteria indirectly induce inflammatory cytokine production and cytolytic protein expression from human colonic Group 1 ILCs, a process which could contribute to inflammation in the setting of microbial translocation.

Commensal and Pathogenic Bacteria Indirectly Induce IL-22 but Not IFNγ Production From Human Colonic ILC3s via Multiple Mechanisms.

Innate lymphoid cells (ILCs) are a diverse family of cells that play critical roles in mucosal immunity. One subset of the ILC family, Group 3 ILCs (ILC3s), has been shown to aid in gut homeostasis through the production of IL-22. IL-22 promotes gut homeostasis through its functional effect on the epithelial barrier. When gut epithelial barrier integrity is compromised, such as in Human Immunodeficiency Virus (HIV) infection and inflammatory bowel disease (IBD), microbes from the gut lumen translocate into the lamina propria, inducing a multitude of potentially pathogenic immune responses. In murine models of bacterial infection, there is evidence that bacteria can induce pro-inflammatory IFNγ production in ILC3s. However, the impact of diverse translocating bacteria, particularly commensal bacteria, in dictating IFNγ versus IL-22 production by human gut ILC3s remains unclear. Here, we utilized an in vitro human lamina propria mononuclear cell (LPMC) model to evaluate ILC3 cytokine production in response to a panel of enteric Gram-positive and Gram-negative commensal and pathogenic bacteria and determined potential mechanisms by which these cytokine responses were induced. The percentages of IL-22-producing ILC3s, but not IFNγ-producing ILC3s, were significantly increased after LPMC exposure to both Gram-positive and Gram-negative commensal or pathogenic bacterial stimuli. Stimulation of IL-22 production from ILC3s was not through direct recognition of bacterial antigen by ILC3s, but rather required the help of accessory cells within the LPMC population. CD11c+ myeloid dendritic cells generated IL-23 and IL-1β in response to enteric bacteria and contributed to ILC3 production of IL-22. Furthermore, ligation of the natural cytotoxicity receptor NKp44 on ILC3s in response to bacteria stimulation also significantly increased the percentage of IL-22-producing ILC3s. Overall, these data demonstrate that human gut microbiota, including commensal bacteria, indirectly modulate colonic ILC3 function to induce IL-22, but additional signals are likely required to induce IFNγ production by colonic ILC3s in the setting of inflammation and microbial translocation.