In vitro and in vivo activity of new strains of Bacillus subtilis against ESBL-producing Escherichia coli: an experimental study.

AIMS: The gastro-intestinal tract is a major reservoir of extended-spectrum beta-lactamase (ESBL) producing Escherichia coli. Bacillus spores may be used as probiotics to decrease digestive colonization by ESBL-E. coli. Our aim was to assess the in vitro and in vivo activity of new Bacillus strains against ESBL-E. coli. METHODS AND RESULTS: We screened the in vitro activity of 50 Bacillus strains against clinical isolates of ESBL-E. coli and selected B. subtilis strains CH311 and S3B. Both strains decreased ESBL-E. coli titers by 4 log10 CFU L-1 in an in vitro model of gut content, whereas the B. subtilis CU1 strain did not. In a murine model of intestinal colonization by ESBL-E. coli, CH311 and S3B did not decrease fecal titers of ESBL-E. coli. Ten sequences of putative antimicrobial peptides were identified in the genomes of CH311 and S3B, but not in CU1. CONCLUSIONS: Two new B. subtilis strains showed strong in vitro activity against ESBL-E. coli. SIGNIFICANCE AND IMPACT OF STUDY: Despite strong in vitro activities of new B. subtilis strains against ESBL-E. coli, intestinal colonisation was not altered by curative Bacillus treatment even if their spores proved to germinate in the gut. Thus, this work underlines the importance of in vivo experiments to identify efficient probiotics. The use of potential antimicrobial compounds identified by genome sequencing remains an attractive alternative to explore.

The functional capacity of plantaricin-producing Lactobacillus plantarum SF9C and S-layer-carrying Lactobacillus brevis SF9B to withstand gastrointestinal transit.

BACKGROUND: We evaluated the functional capacity of plantaricin-producing Lactobacillus plantarum SF9C and S-layer-carrying Lactobacillus brevis SF9B to withstand gastrointestinal transit and to compete among the gut microbiota in vivo. Considering the probiotic potential of Lb. brevis SF9B, this study aims to investigate the antibacterial activity of Lb. plantarum SF9C and their potential for in vivo colonisation in rats, which could be the basis for the investigation of their synergistic functionality. RESULTS: A plantaricin-encoding cluster was identified in Lb. plantarum SF9C, a strain which efficiently inhibited the growth of Listeria monocytogenes ATCC® 19111™ and Staphylococcus aureus 3048. Homology-based three-dimensional (3D) structures of SF9C plantaricins PlnJK and PlnEF were predicted using SWISS-MODEL workspace and the helical wheel representations of the plantaricin peptide helices were generated by HELIQUEST. Contrary to the plantaricin-producing SF9C strain, the S-layer-carrying SF9B strain excluded Escherichia coli 3014 and Salmonella enterica serovar Typhimurium FP1 from the adhesion to Caco-2 cells. Finally, PCR-DGGE analysis of the V2-V3 regions of the 16S rRNA gene confirmed the transit of the two selected lactobacilli through the gastrointestinal tract (GIT). Microbiome profiling via the Illumina MiSeq platform revealed the prevalence of Lactobacillus spp. in the gut microbiota of the Lactobacillus-treated rats, even on the 10th day after the Lactobacillus application, compared to the microbiota of the healthy and AlCl3-exposed rats before Lactobacillus treatment. CONCLUSION: The combined application of Lb. plantarum SF9C and Lb. brevis SF9B was able to influence the intestinal microbiota composition in rats, which was reflected in the increased abundance of Lactobacillus genus, but also in the altered abundances of other bacterial genera, either in the model of healthy or aberrant gut microbiota of rats. The antibacterial activity and capacity to withstand in GIT conditions contributed to the functional aspects of SF9C and SF9B strains that could be incorporated in the probiotic-containing functional foods with a possibility to positively modulate the gut microbiota composition.

Supplementation with Lactobacillus paracasei or Pediococcus pentosaceus does not prevent diarrhoea in neonatal pigs infected with Escherichia coli F18.

Infectious diarrhoea is a worldwide problem in newborns. Optimal bacterial colonisation may enhance gut maturation and protect against pathogenic bacteria after birth. We hypothesised that lactic acid bacteria (LAB) administration prevents pathogen-induced diarrhoea in formula-fed newborns. Newborn caesarean-delivered, colostrum-deprived term piglets on parenteral nutrition for the first 15 h, were used as models for sensitive newborn infants. A commercially available probiotic strain, Lactobacillus paracasei F19 (LAP, 2·6×108 colony-forming units (CFU)/kg per d) and a novel LAB isolate, Pediococcus pentosaceus (PEP, 1·3×1010 CFU/kg per d), were administered for 5 d with or without inoculation of the porcine pathogen, Escherichia coli F18 (F18, 1010 CFU/d). This resulted in six treatment groups: Controls (n 9), LAP (n 10), PEP (n 10), F18 (n 10), F18-LAP (n 10) and F18-PEP (n 10). The pathogen challenge increased diarrhoea and density of F18 in the intestinal mucosa (P<0·05). LAB supplementation further increased the diarrhoea score, relative to F18 alone (P<0·01). Intestinal structure and permeability were similar among groups, whereas brush border enzymes were affected in variable intestinal regions with decreased activities in most cases after F18 and LAB inoculation. Bacterial density in colon mucosa increased after F18 inoculation (P<0·05) but was unaffected by LAB supplementation. In colon contents, acetic and butyric acids were increased by PEP (P<0·05). The LAB used in this study failed to reduce E. coli-induced diarrhoea in sensitive newborn pigs. In vulnerable newborns there may be a delicate balance among bacterial composition and load, diet and the host. Caution may be required when administering LAB to compromised newborns suffering from enteric infections.

Late preterm birth has direct and indirect effects on infant gut microbiota development during the first six months of life.

AIM: Preterm infants display aberrant gut microbial colonisation. We investigated whether the differences in gut microbiota between late preterm and full-term infants results from prematurity or external exposures. METHODS: This study comprised 43 late preterm infants (340/7 -366/7 ) and 75 full-term infants based on faecal samples collected following birth and at two to four weeks and six months of age. We assessed clinically relevant bacteria using quantitative polymerase chain reaction. Logistic regression analyses were performed to determine whether the observed differences in gut microbiota were attributable to prematurity or perinatal exposure. RESULTS: The prevalence of bifidobacteria differed in the intestinal microbiota of the full-term and late preterm neonates. Differences in the presence of specific species were detected at the age of six months, although the microbiota alterations were most prominent following delivery. As well as prematurity, the mode of birth, intrapartum and neonatal antibiotic exposure, and the duration of breastfeeding had an additional impact on gut microbiota development. CONCLUSION: The gut microbiota composition was significantly different between late preterm and full-term infants at least six months after birth. Antibiotic exposure was common in late preterm infants and modulated gut colonisation, but preterm birth also affected gut microbiota development independently.

Impact of secreted glucanases upon the cell surface and fitness of Candida albicans during colonisation and infection.

Host recognition of the pathogen-associated molecular pattern (PAMP), ß-1,3-glucan, plays a major role in antifungal immunity. ß-1,3-glucan is an essential component of the inner cell wall of the opportunistic pathogen Candida albicans. Most ß-1,3-glucan is shielded by the outer cell wall layer of mannan fibrils, but some can become exposed at the cell surface. In response to host signals such as lactate, C. albicans shaves the exposed ß-1,3-glucan from its cell surface, thereby reducing the ability of innate immune cells to recognise and kill the fungus. We have used sets of barcoded xog1 and eng1 mutants to compare the impacts of the secreted ß-glucanases Xog1 and Eng1 upon C. albicans in vitro and in vivo. Flow cytometry of Fc-dectin-1-stained strains revealed that Eng1 plays the greater role in lactate-induced ß-1,3-glucan masking. Transmission electron microscopy and stress assays showed that neither Eng1 nor Xog1 are essential for cell wall maintenance, but the inactivation of either enzyme compromised fungal adhesion to gut and vaginal epithelial cells. Competitive barcode sequencing suggested that neither Eng1 nor Xog1 strongly influence C. albicans fitness during systemic infection or vaginal colonisation in mice. However, the deletion of XOG1 enhanced C. albicans fitness during gut colonisation. We conclude that both Eng1 and Xog1 exert subtle effects on the C. albicans cell surface that influence fungal adhesion to host cells and that affect fungal colonisation in certain host niches.

A systematic review of breast milk microbiota composition and the evidence for transfer to and colonisation of the infant gut.

The intestinal microbiota plays a major role in infant health and development. However, the role of the breastmilk microbiota in infant gut colonisation remains unclear. A systematic review was performed to evaluate the composition of the breastmilk microbiota and evidence for transfer to/colonisation of the infant gut. Searches were performed using PUBMED, OVID, LILACS and PROQUEST from inception until 18th March 2020 with a PUBMED update to December 2021. 88 full texts were evaluated before final critique based on study power, sample contamination avoidance, storage, purification process, DNA extraction/analysis, and consideration of maternal health and other potential confounders. Risk of skin contamination was reduced mainly by breast cleaning and rejecting the first milk drops. Sample storage, DNA extraction and bioinformatics varied. Several studies stored samples under conditions that may selectively impact bacterial DNA preservation, others used preculture reducing reliability. Only 15 studies, with acceptable sample size, handling, extraction, and bacterial analysis, considered transfer of bacteria to the infant. Three reported bacterial transfer from infant to breastmilk. Despite consistent evidence for the breastmilk microbiota, and recent studies using improved methods to investigate factors affecting its composition, few studies adequately considered transfer to the infant gut providing very little evidence for effective impact on gut colonisation.

Feed Safety and the Development of Poultry Intestinal Microbiota.

The first feed offered to young chicks is likely the most important meal in their life. The complex gut colonisation process is determined with early exposure and during the first days of life before the microbial community is formed. Therefore, providing access to high-quality feed and an environment enriched in the beneficial and deprived of pathogenic microorganisms during this period is critical. Feed often carries a complex microbial community that can contain major poultry pathogens and a range of chemical contaminants such as heavy metals, mycotoxins, pesticides and herbicides, which, although present in minute amounts, can have a profound effect on the development of the microbial community and have a permanent effect on bird's overall health and performance. The magnitude of their interference with gut colonisation in livestock is yet to be determined. Here, we present the animal feed quality issues that can significantly influence the microbial community development, thus severely affecting the bird's health and performance.

Impact of changes at the Candida albicans cell surface upon immunogenicity and colonisation in the gastrointestinal tract.

The immunogenicity of Candida albicans cells is influenced by changes in the exposure of microbe-associated molecular patterns (MAMPs) on the fungal cell surface. Previously, the degree of exposure on the C. albicans cell surface of the immunoinflammatory MAMP ß-(1,3)-glucan was shown to correlate inversely with colonisation levels in the gastrointestinal (GI) tract. This is important because life-threatening systemic candidiasis in critically ill patients often arises from translocation of C. albicans strains present in the patient's GI tract. Therefore, using a murine model, we have examined the impact of gut-related factors upon ß-glucan exposure and colonisation levels in the GI tract. The degree of ß-glucan exposure was examined by imaging flow cytometry of C. albicans cells taken directly from GI compartments, and compared with colonisation levels. Fungal ß-glucan exposure was lower in the cecum than the small intestine, and fungal burdens were correspondingly higher in the cecum. This inverse correlation did not hold for the large intestine. The gut fermentation acid, lactate, triggers ß-glucan masking in vitro, leading to attenuated anti-Candida immune responses. Additional fermentation acids are present in the GI tract, including acetate, propionate, and butyrate. We show that these acids also influence ß-glucan exposure on C. albicans cells in vitro and, like lactate, they influence ß-glucan exposure via Gpr1/Gpa2-mediated signalling. Significantly, C. albicans gpr1Δ gpa2Δ cells displayed elevated ß-glucan exposure in the large intestine and a corresponding decrease in fungal burden, consistent with the idea that Gpr1/Gpa2-mediated ß-glucan masking influences colonisation of this GI compartment. Finally, extracts from the murine gut and culture supernatants from the mannan grazing gut anaerobe Bacteroides thetaiotaomicron promote ß-glucan exposure at the C. albicans cell surface. Therefore, the local microbiota influences ß-glucan exposure levels directly (via mannan grazing) and indirectly (via fermentation acids), whilst ß-glucan masking appears to promote C. albicans colonisation of the murine large intestine.

Contribution of flagella and motility to gut colonisation and pathogenicity of Salmonella Enteritidis in the chicken.

Salmonella Enteritidis causes fowl paratyphoid in poultry and is frequently associated to outbreaks of food-borne diseases in humans. The role of flagella and flagella-mediated motility into host-pathogen interplay is not fully understood and requires further investigation. In this study, one-day-old chickens were challenged orally with a wild-type strain Salmonella Enteritidis, a non-motile but fully flagellated (SE ΔmotB) or non-flagellated (SE ΔfliC) strain to evaluate their ability to colonise the intestine and spread systemically and also of eliciting gross and histopathological changes. SE ΔmotB and SE ΔfliC were recovered in significantly lower numbers from caecal contents in comparison with Salmonella Enteritidis at early stages of infection (3 and 5dpi). The SE ΔmotB strain, which synthesises paralysed flagella, showed poorer intestinal colonisation ability than the non-flagellated SE ΔfliC. Histopathological analyses demonstrated that the flagellated strains induced more intense lymphoid reactivity in liver, ileum and caeca. Thus, in the present study the flagellar structure and motility seemed to play a role in the early stages of the intestinal colonisation by Salmonella Enteritidis in the chicken.

Carriage prevalence of carbapenem-resistant Enterobacteriaceae in stool samples: A surveillance study.

BACKGROUND: With more people being exposed to antibiotics, intestinal microflora faces constant pressure of antibiotic selection, which has resulted in the emergence of multidrug resistant strains. This may pose a severe problem as intestinal Enterobacteriaceae members are commonly implicated in human infections. AIMS: This surveillance study was undertaken to investigate the carriage of carbapenem-resistant Enterobacteriaceae (CRE) in the gastrointestinal tract among patients attending the outpatient clinic in a tertiary care center of East Delhi, India. METHOD: We performed a prospective surveillance study to screen 242 Enterobacteriaceae isolates for carbapenemase production from the stool samples of 123 outpatients attending a tertiary care hospital in East Delhi over a four-month period. RESULTS: Twenty-four (9.9 per cent) isolates demonstrated carbapenemase activity among 242 screened Enterobacteriaceae isolates. Four stool samples had two isolates of different species, both eliciting this feature and therefore indicating presence of multiple carbapenem-resistant Enterobacteriaceae (CRE) isolates in a single sample. CONCLUSION: Screening for carriage of CRE in stools of patients undergoing elective or emergency gastrointestinal surgical procedures, with haematological malignancies taking chemotherapy, or those planned for bone marrow transplantation can guide clinicians about gut colonisation of multidrug-resistant Enterobacteriaceae as these groups of patients are at risk of possible endogenous infection.

Contribution of flagella and motility to gut colonisation and pathogenicity of Salmonella Enteritidis in the chicken

ABSTRACT Salmonella Enteritidis causes fowl paratyphoid in poultry and is frequently associated to outbreaks of food-borne diseases in humans. The role of flagella and flagella-mediated motility into host-pathogen interplay is not fully understood and requires further investigation. In this study, one-day-old chickens were challenged orally with a wild-type strain Salmonella Enteritidis, a non-motile but fully flagellated (SE ΔmotB) or non-flagellated (SE ΔfliC) strain to evaluate their ability to colonise the intestine and spread systemically and also of eliciting gross and histopathological changes. SE ΔmotB and SE ΔfliC were recovered in significantly lower numbers from caecal contents in comparison with Salmonella Enteritidis at early stages of infection (3 and 5 dpi). The SE ΔmotB strain, which synthesises paralysed flagella, showed poorer intestinal colonisation ability than the non-flagellated SE ΔfliC. Histopathological analyses demonstrated that the flagellated strains induced more intense lymphoid reactivity in liver, ileum and caeca. Thus, in the present study the flagellar structure and motility seemed to play a role in the early stages of the intestinal colonisation by Salmonella Enteritidis in the chicken.