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K. pneumoniae is one of the leading microorganisms causing nosocomial infections among premature newborns. The ineffectiveness of immune defense, morphofunctional immaturity, length of hospitalization and invasive procedures create the prerequisites for the implementation of the infectious process in a hospital setting. The question of the reasons for the development of infection, the etiological agent of which colonizes the intestines, remains open. Purpose of the study: to evaluate the expression of CD14+CD282+, CD14+CD284+, CD14+HLA-DR+, and CD14+CD11b+ receptors on blood monocytes and the level of sIgA in coprofiltrates in premature infants with intestinal colonization by K. pneumonia with different genetic profiles. We examined 11 children with the uge gene (group 1), 20 newborns with the uge + fim genes (group 2), and 12 children with the kfu + uge + fim genes. Microbiological examination of feces included identification and antibiotic sensitivity of microorganisms. Detection of the uge, fim and kfu genes in K. pneumoniae strains was carried out by PCR. The expression level of monocyte activation markers was determined by flow cytometry. Gestational age and anthropometric parameters did not differ between newborns. Children identified with the fim gene in combination with other genes were more often discharged home with K. pneumoniae than with the uge gene. In these children, a decrease in the level of expression of CD14+CD282+, CD14+CD284+, CD14+CD11b+, CD14+HLA-DR+ receptors at birth and upon reaching postconceptional age, and a low sIgA content in coprofiltrates during 24 days of life were recorded. Thus, a decrease in the expression of CD14+CD282+, CD14+CD284+, CD14+CD282+ and CD14+HLA-DR+ receptors by blood monocytes and insufficiency of sIgA production in the large intestine determine a long period of colonization of K. pneumoniae strains with the presence of the fim gene in combination with other genes (from 15 to 180 days), as well as the possibility of Klebsiella infection occurring in subsequent periods of the child’s life. Much more often, children with the combination of genes uge + fim and kfu + uge + fim were discharged from the hospital with a diagnosis of anemia; only in these groups of children was the development of bronchopulmonary dysplasia recorded.

Background: Gut colonization with multidrug-resistant organisms (MDRO) frequently precedes infection among patients in the intensive care unit (ICU), although the dynamics of colonization are not completely understood. We performed a systematic review and meta-analysis of ICU studies which described the cumulative incidence and rates of MDRO gut acquisition. Methods: We systematically searched PubMed, Embase, and Web of Science for studies published from 2010 to 2023 reporting on gut acquisition of MDRO in the ICU. MDRO were defined as multidrug resistant non-Pseudomonas Gram-negative bacteria (NP-GN), Pseudomonas spp., and vancomycin-resistant Enterococcus (VRE). We included observational studies which obtained perianal or rectal swabs at ICU admission (within 48 h) and at one or more subsequent timepoints. Our primary outcome was the incidence rate of gut acquisition of MDRO, defined as any MDRO newly detected after ICU admission (i.e., not present at baseline) for all patient-time at risk. The study was registered with PROSPERO, CRD42023481569. Results: Of 482 studies initially identified, 14 studies with 37,305 patients met criteria for inclusion. The pooled incidence of gut acquisition of MDRO during ICU hospitalization was 5% (range: 1–43%) with a pooled incidence rate of 12.2 (95% CI 8.1–18.6) per 1000 patient-days. Median time to acquisition ranged from 4 to 26 days after ICU admission. Results were similar for NP-GN and Pseudomonas spp., with insufficient data to assess VRE. Among six studies which provided sufficient data to perform curve fitting, there was a quasi-linear increase in gut MDRO colonization of 1.41% per day which was stable through 30 days of ICU hospitalization (R2 = 0.50, p < 0.01). Conclusions: Acquisition of gut MDRO was common in the ICU and increases with days spent in ICU through 30 days of follow-up. These data may guide future interventions seeking to prevent gut acquisition of MDRO in the ICU. [ABSTRACT FROM AUTHOR]

Introduction: Extraintestinal Escherichia coli infections represent a growing public health threat, However, current studies often overlook important factors such as temporal patterns of infection, phylogenetic and clonal background, or the host gut E. coli population, despite their likely significance. Methods: In this study, we analyzed >7000 clinical E. coli isolates from patients at the Minneapolis Veterans Affairs Health Care System (2012-2019), and concurrent fecal E. coli from uninfected veterans. We assessed phylogenetic group distribution, membership in selected sequence types (STs), and subsets thereof--including the pandemic, resistance-associated ST131-H30R, and ST1193 lineages--and strain type, as defined by pulsed-field gel electrophoresis. We then analyzed these features alongside the temporal patterns of infection in individual hosts. Results: The H30R lineage emerged as the leading lineage, both overall and among fluoroquinolone-resistant isolates, with ST1193 following among fluoroquinolone-resistant isolates. Recurrences were common, occurring in 31% of subjects and 41% of episodes, and often multiple and delayed/prolonged (up to 23 episodes per subject; up to 2655d post-index). Remarkably, these recurrences typically involved the subject's index strain (63% of recurrences), even when affecting extra-urinary sites. ST131, H30R, ST1193, and fluoroquinoloneresistant strains generally caused significantly more recurrences than did other strains, despite similar recurrence intervals. ST131 strain types shifted significantly over the study period. Infection-causing strains were commonly detectable in host feces at times other than during an infection episode; the likelihood of detection varied with surveillance intensity and proximity to the infection. H30R and ST1193 were prominent causes of fecal-clinical clonal overlap. Discussion: These findings provide novel insights into the temporal and clonal characteristics of E. coli infections in veterans and support efforts to develop anti-colonization interventions. [ABSTRACT FROM AUTHOR]

The increasing prevalence of pathogenic and antimicrobial-resistant isolates of Salmonella Heidelberg (SH) represents a global concern. Consequently, novel strategies for preventing and controlling infections caused by this bacterium are needed. This study aimed to investigate the protective effects of in ovo injection with a formulation based on bacterial peptides, either alone or in combination with oligodeoxynucleotides containing cytosine-phosphodiester-guanine (CpG-ODN), against SH infection in chicks. Genomic data of SH available in public databases were analyzed to select amino acid sequences of structural proteins or those with greater relevance for intestinal colonization. These sequences were subjected to linear epitope prediction tools to identify highly immunogenic peptides. SPF eggs (n = 180) were incubated and injected via the allantoic cavity at day 18 of incubation. Results showed that in ovo injection of peptides + CpG-ODN reduced SH colonization in the caecal content during the first week post-infection, although it did not reduce overall faecal excretion throughout the study. Furthermore, CpG-ODN injection may positively affect intestinal health, as evidenced by reduced crypt depth at 21 dpi and increased villus height at 28 dpi. Levels of secretory IgA did not differ between chicks in any groups, and no detectable SH counts were found in the livers of chicks in any of the infected groups throughout the study. In conclusion, in ovo injection with peptides + CpG-ODN may help reduce caecal colonization by SH in the early stages of infection, but it does not impact total faecal excretion. Additionally, CpG-ODN injection may improve intestinal health parameters in chicks. Peptides + CpG-ODN reduced SH in caeca at the first week post-infection. Administered formulations did not reduce SH-faecal excretion. Levels of intestinal IgA were similar between all groups. CpG-ODN improved some parameters associated with chick intestinal health. [ABSTRACT FROM AUTHOR]

ABSTRACTInulin, an increasingly studied dietary fiber, alters intestinal microbiota. The aim of this study was to assess whether inulin decreases intestinal colonization by multidrug resistant E. coli and to investigate its potential mechanisms of action. Mice with amoxicillin-induced intestinal dysbiosis mice were inoculated with extended spectrum beta-lactamase producing E. coli (ESBL-E. coli). The combination of inulin and pantoprazole (IP) significantly reduced ESBL-E. coli fecal titers, whereas pantoprazole alone did not and inulin had a delayed and limited effect. Fecal microbiome was assessed using shotgun metagenomic sequencing and qPCR. The efficacy of IP was predicted by increased abundance of 74 taxa, including two species of Adlercreutzia. Preventive treatments with A. caecimuris or A. muris also reduced ESBL-E. coli fecal titers. Fecal microbiota of mice effectively treated by IP was enriched in genes involved in inulin catabolism, production of propionate and expression of beta-lactamases. They also had increased beta-lactamase activity and decreased amoxicillin concentration. These results suggest that IP act through production of propionate and degradation of amoxicillin by the microbiota. The combination of pantoprazole and inulin is a potential treatment of intestinal colonization by multidrug-resistant E. coli. The ability of prebiotics to promote propionate and/or beta-lactamase producing bacteria may be used as a screening tool to identify potential treatments of intestinal colonization by multidrug resistant Enterobacterales.

Galacto-N-biose (GNB) is an important core structure of glycan of mucin glycoproteins in the gastrointestinal (GI) mucosa. Because certain beneficial bacteria inhabiting the GI tract, such as bifidobacteria and lactic acid bacteria, harbor highly specialized GNB metabolic capabilities, GNB is considered a promising prebiotic for nourishing and manipulating beneficial bacteria in the GI tract. However, the precise interactions between GNB and beneficial bacteria and their accompanying health-promoting effects remain elusive. First, we evaluated the proliferative tendency of beneficial bacteria and their production of beneficial metabolites using gut bacterial strains. By comparing the use of GNB, glucose, and inulin as carbon sources, we found that GNB enhanced acetate production in Lacticaseibacillus casei, Lacticaseibacillus rhamnosus, Lactobacillus gasseri, and Lactobacillus johnsonii. The ability of GNB to promote acetate production was also confirmed by RNA-seq analysis, which indicated the upregulation of gene clusters that catalyze the deacetylation of N-acetylgalactosamine-6P and biosynthesize acetyl-CoA from pyruvate, both of which result in acetate production. To explore the in vivo effect of GNB in promoting acetate production, antibiotic-treated BALB/cA mice were administered with GNB with L. rhamnosus, resulting in a fecal acetate content that was 2.7-fold higher than that in mice administered with only L. rhamnosus. Moreover, 2 days after the last administration, a 3.7-fold higher amount of L. rhamnosus was detected in feces administered with GNB with L. rhamnosus than in feces administered with only L. rhamnosus. These findings strongly suggest the prebiotic potential of GNB in enhancing L. rhamnosus colonization and converting L. rhamnosus into higher acetate producers in the GI tract. IMPORTANCE Specific members of lactic acid bacteria, which are commonly used as probiotics, possess therapeutic properties that are vital for human health enhancement by producing immunomodulatory metabolites such as exopolysaccharides, short-chain fatty acids, and bacteriocins. The long residence time of probiotic lactic acid bacteria in the GI tract prolongs their beneficial health effects. Moreover, the colonization property is also desirable for the application of probiotics in mucosal vaccination to provoke a local immune response. In this study, we found that GNB could enhance the beneficial properties of intestinal lactic acid bacteria that inhabit the human GI tract, stimulating acetate production and promoting intestinal colonization. Our findings provide a rationale for the addition of GNB to lactic acid bacteria-based functional foods. This has also led to the development of therapeutics supported by more rational prebiotic and probiotic selection, leading to an improved healthy lifestyle for humans. [ABSTRACT FROM AUTHOR]

Klebsiella spp., an opportunistic infectious organism, is commensal in the nasal and oral cavities of humans. Recently, it has been reported that oral Klebsiella spp. ectopically colonize the intestinal tract and induce the accumulation of intestinal Th1 cells. For oral bacteria to colonize the intestinal tract, they need to compete for nutrients with indigenous intestinal bacteria. Therefore, we focused on mannose, a mucus-derived sugar, and the mannose phosphotransferase system (PTS) (ManXYZ), a mechanism for mannose uptake, in terms of their effects on intestinal colonization and immune responses to Klebsiella spp. We generated a Klebsiella manXYZ -deficient strain and investigated whether the utilization of intestinal mucus-derived sugars is associated with the growth. Furthermore, we examine the virulence of this organism in the mouse intestinal tract, especially the ability to colonize the host using competition assay. We found that Klebsiella ManXYZ is a PTS that specifically takes up mannose and glucosamine. Through ManXYZ, mannose was used for bacterial growth and the upregulated production of extracellular polymeric substances. In vivo competition assays showed that mannose metabolism promoted intestinal colonization. However, ManXYZ was not involved in Th1 and Th17 induction in the intestinal tract. The fundamental roles of ManXYZ were to ensure that mannose, which is present in the host, is made available for bacterial growth, to promote the production of extracellular polymeric substances, thus facilitating bacterial adaptation to the host environment. • ManXYZ is a PTS that specifically takes up mannose and glucosamine. • Mannose metabolism promotes intestinal colonization. • ManXYZ stimulates the production of EPS. [ABSTRACT FROM AUTHOR]

Carbapenem resistant Enterobacteriaceae (CRE) are major human pathogens because, these cause high number of difficult-to-treat infections. Allogeneic hematopoietic stem cell transplant (AHSCT) recipients are highly exposed to these type of bacteria. The aim of our study was to investigate prevalence of CRE colonization in AHSCT patients and to determine genes encoding carbapenem resistance. A retrospective study conducted between January 2015 and December 2019, involved 55 patients colonized with CRE strains. We determined the rate of antibiotic resistance according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) and the carbapenem resistance genes by PCR assays for genes encoding most frequent β-lactamases namely, blaGES, blaKPC, blaIMI, blaNDM, blaVIM, blaIMP and blaOXA-48. Eighty-one episodes of CRE colonization were recorded in 55 patients, mainly suffering from acute leukaemia (30%) and aplastic anemia (26%). History of hospitalization was noted in 80 episodes. Prior antibiotic treatment, severe neutropenia and corticosteroid therapy were respectively found in 94%, 76% and 58% of cases. Among the 55 patients, six patients (11%) developed a CRE infection. The CRE responsible for colonization were carbapenemase producers in 90% of cases. They belonged mostly to Klebsiella pneumoniae (61/81) and Escherichia coli species (10/81). Antibiotic resistance rates were 100% for ertapenem, 53% for imipenem, 42% for amikacin, 88% for ciprofloxacin and 27% for fosfomycin. Molecular study showed that blaOXA-48 gene was the most frequent (60.5%), followed by blaNDM (58%). Thirty-five (43%) strains were co-producers of carbapenemases. In our study, we report a high rate of CRE intestinal colonization in AHSCT recipients of our center. [ABSTRACT FROM AUTHOR]

Human infections caused by Pseudomonas citronellolis, an environmental bacterium, are infrequent, with only two cases related to uncommon urinary tract infections and bacteremia reported in recent years. All these cases typically occurred in elderly patients with compromised or decreased immune function. Simultaneously, the epithelial barrier disruption induced by invasive biopsy procedures or gastrointestinal disorders such as gastroenteritis provided a pathway for Pseudomonas citronellolis to infiltrate the organism. In this study, we present the first report of a case where Pseudomonas citronellolis and Escherichia coli were isolated from the inflamed appendix of a patient without underlying conditions. Compared to the Escherichia coli, Pseudomonas citronellolis has never been isolated in patients with appendicitis. We identified the species using MALDI-TOF MS and genetic sequencing. Based on our findings, we highlight the perspective that Pseudomonas citronellolis can colonize the intestines of healthy individuals and may trigger infections like appendicitis. [ABSTRACT FROM AUTHOR]

ABSTRACT Patients with hematological diseases are considered to be at high risk for intestinal colonization by carbapenem-resistant Gram-negative bacteria (CR-GNB). However, the epidemiological data regarding risk factors and molecular characteristics of intestinal colonized CR-GNB isolates in this population are insufficient in China. A multicenter case‒control study involving 4,641 adult patients with hematological diseases from 92 hospitals across China was conducted. Following culture of collected rectal swabs, mass spectrometry and antimicrobial susceptibility tests were performed to identify GNB species and CR phenotype. Risk factors were assessed through retrospective clinical information. Whole-genome sequencing was used to analyze the molecular characteristics of CR-GNB isolates. This trial is registered with ClinicalTrials.gov as NCT05002582. Our results demonstrated that among 4,641 adult patients, 10.8% had intestinal colonization by CR-GNB. Of these, 8.1% were colonized by carbapenem-resistant Enterobacterales (CRE), 2.6% were colonized by carbapenem-resistant Pseudomonas aeruginosa (CRPA), and 0.3% were colonized by carbapenem-resistant Acinetobacter baumannii (CRAB). The risk factors for CR-GNB colonization include male gender, acute leukemia, hematopoietic stem cell transplantation, β-lactam antibiotic usage, and the presence of non-perianal infections within 1 week. Compared with CRPA-colonized patients, patients using carbapenems were more likely to be colonized with CRE. NDM was the predominant carbapenemase in colonized CRE. This study revealed a high CR-GNB intestinal colonization rate among adult patients with hematological diseases in China, with CRE being the predominant one. Notably, a significant proportion of CRE exhibited metallo-β-lactamase production, indicating a concerning trend. These findings emphasize the importance of active screening for CR-GNB colonization in patients with hematological diseases.IMPORTANCECarbapenem-resistant Gram-negative bacteria (CR-GNB) has emerged as a significant threat to public health. Patients with hematological diseases are at high risk of CR-GNB infections due to their immunosuppressed state. CR-GNB colonization is an independent risk factor for subsequent infection. Understanding the risk factors and molecular characteristics of CR-GNB associated with intestinal colonization in patients with hematological diseases is crucial for empirical treatment, particularly in patients with febrile neutropenia. However, the epidemiology data are still insufficient, and our study aims to determine the intestinal colonization rate of CR-GNB, identify colonization risk factors, and analyze the molecular characteristics of colonized CR-GNB isolates.

Abstract Background Atopic dermatitis (AD) patients have high rates of colonization by Staphylococcus aureus, which has been associated with worsening of the disease. This study characterized Staphylococcus spp isolates recovered from nares and feces of pediatric patients with AD in relation to antimicrobial susceptibility, staphylococcal cassette chromosome mec (SCCmec) type, presence of pvl genes and clonality. Besides, gut bacterial community profiles were compared with those of children without AD. Results All 55 AD patients evaluated had colonization by Staphylococcus spp. Fifty-three (96.4%) patients had colonization in both clinical sites, whereas one patient each was not colonize in the nares or gut. Staphylococcus aureus was identified in the nostrils and feces of 45 (81.8%) and 39 (70.9%) patients, respectively. Methicillin-resistant Staphylococcus spp. isolates were found in 70.9% of the patients, and 24 (43.6%) had methicillin-resistant S. aureus (MRSA). S. aureus (55.6%) and S. epidermidis (26.5%) were the major species found. The prevalent lineages of S. aureus were USA800/SCCmecIV (47.6%) and USA1100/SCCmecIV (21.4%), and 61.9% of the evaluated patients had the same genotype in both sites. Additionally, gut bacterial profile of AD patients exhibits greater dissimilarity from the control group than it does among varying severities of AD. Conclusions High rates of nasal and intestinal colonization by S. aureus and methicillin-resistant staphylococci isolates were found in AD patients. Besides, gut bacterial profiles of AD patients were distinctly different from those of the control group, emphasizing the importance of monitoring S. aureus colonization and gut microbiome composition in AD patients.

Probiotics offer numerous health benefits for human beings. However, it is difficult to achieve the expected beneficial effects of oral probiotics due to the viability loss during processing, storage and digestion. Oral delivery systems are effective ways to improve the processing and storage stability, gastric acid and bile salt tolerance, and intestinal adhesion and colonization ability of probiotics. This review focuses on the state of the art of oral delivery of probiotics, and summarizes the types of probiotics commonly used in probiotic products and their functional characteristics. Specifically, this review analyzes the effects of external environmental factors such as humidity, temperature, pH and oxygen, and internal environments such as the mouth, stomach, and small intestine on the survival rate of probiotics. Furthermore, the characteristics and applications of different oral delivery systems including microcapsule, hydrogel, oleogel, nanocoating, emulsion, and nanofiber are compared. Finally, this review proposes future prospects for cost reduction, intelligent packaging, co-packaging and population experiments of probiotic delivery systems, with the aim of providing technical support for the development of probiotic formulations with high storage stability and high viability suitable for intestinal targeted delivery.

Background: Atopic dermatitis (AD) patients have high rates of colonization by Staphylococcus aureus, which has been associated with worsening of the disease. This study characterized Staphylococcus spp isolates recovered from nares and feces of pediatric patients with AD in relation to antimicrobial susceptibility, staphylococcal cassette chromosome mec (SCCmec) type, presence of pvl genes and clonality. Besides, gut bacterial community profiles were compared with those of children without AD. Results: All 55 AD patients evaluated had colonization by Staphylococcus spp. Fifty-three (96.4%) patients had colonization in both clinical sites, whereas one patient each was not colonize in the nares or gut. Staphylococcus aureus was identified in the nostrils and feces of 45 (81.8%) and 39 (70.9%) patients, respectively. Methicillin-resistant Staphylococcus spp. isolates were found in 70.9% of the patients, and 24 (43.6%) had methicillin-resistant S. aureus (MRSA). S. aureus (55.6%) and S. epidermidis (26.5%) were the major species found. The prevalent lineages of S. aureus were USA800/SCCmecIV (47.6%) and USA1100/SCCmecIV (21.4%), and 61.9% of the evaluated patients had the same genotype in both sites. Additionally, gut bacterial profile of AD patients exhibits greater dissimilarity from the control group than it does among varying severities of AD. Conclusions: High rates of nasal and intestinal colonization by S. aureus and methicillin-resistant staphylococci isolates were found in AD patients. Besides, gut bacterial profiles of AD patients were distinctly different from those of the control group, emphasizing the importance of monitoring S. aureus colonization and gut microbiome composition in AD patients. [ABSTRACT FROM AUTHOR]

Intestinal colonization with carbapenem-resistant Enterobacterales (CRE) has been shown as a significant risk factor for subsequent CRE infections, especially in intensive care units (ICUs). The aim of this study was to determine the prevalence of intestinal CRE colonization among ICU patients in a Chinese tertiary hospital. Fecal sample screenings for CRE were performed on ICU patients weekly. Antibiotic-susceptibility profile of CRE strains was determined using the Vitek-2 analysis system and broth microdilution method. The carbapenemases of all isolates were determined by phenotypes and genotypes. Clonal relatedness was analyzed by pulsed-field gel electrophoresis (PFGE). Whole-genome sequencing was used to identify the multilocus sequence type (ST), plasmid replicons, and insertion sequences (ISs) of isolates. The overall colonization rate of CRE was 40.4% (82/203). A total of 84 CRE strains were detected, mostly with Klebsiella pneumoniae (92.9%). Antibiotic susceptibility testing profile revealed that 84 CRE strains were resistant to most antibiotics except for tigecycline and colistin. The carbapenemase-encoding genes including blaKPC-2, blaNDM-1, and blaIMP-4 were detected, and blaKPC-2 was the predominant genotype (90.8%). A total of 9 STs were identified among 84 CRE strains, and ST11 was the most common type (83.3%). A variety of mobile genetic elements, including plasmids and ISs, were detected via online tool prediction. PFGE analysis of the 78 K. pneumoniae strains showed 8 different pulsotypes, and pulsotype A was highly prevalent. This study found that the prevalence of CRE colonization was alarmingly high in the ICU, and that effective infection control measures are urgently needed to prevent the dissemination of CRE. [ABSTRACT FROM AUTHOR]

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ABSTRACT Nitrogen is an essential element for all living organisms, including Escherichia coli. Potential nitrogen sources are abundant in the intestine, but knowledge of those used specifically by E. coli to colonize remains limited. Here, we sought to determine the specific nitrogen sources used by E. coli to colonize the streptomycin-treated mouse intestine. We began by investigating whether nitrogen is limiting in the intestine. The NtrBC two-component system upregulates approximately 100 genes in response to nitrogen limitation. We showed that NtrBC is crucial for E. coli colonization, although most genes of the NtrBC regulon are not induced, which indicates that nitrogen is not limiting in the intestine. RNA-seq identified upregulated genes in colonized E. coli involved in transport and catabolism of seven amino acids, dipeptides and tripeptides, purines, pyrimidines, urea, and ethanolamine. Competitive colonization experiments revealed that L-serine, N-acetylneuraminic acid, N-acetylglucosamine, and di- and tripeptides serve as nitrogen sources for E. coli in the intestine. Furthermore, the colonization defect of a L-serine deaminase mutant was rescued by excess nitrogen in the drinking water but not by an excess of carbon and energy, demonstrating that L-serine serves primarily as a nitrogen source. Similar rescue experiments showed that N-acetylneuraminic acid serves as both a carbon and nitrogen source. To a minor extent, aspartate and ammonia also serve as nitrogen sources. Overall, these findings demonstrate that E. coli utilizes multiple nitrogen sources for successful colonization of the mouse intestine, the most important of which is L-serine.IMPORTANCEWhile much is known about the carbon and energy sources that are used by E. coli to colonize the mammalian intestine, very little is known about the sources of nitrogen. Interrogation of colonized E. coli by RNA-seq revealed that nitrogen is not limiting, indicating an abundance of nitrogen sources in the intestine. Pathways for assimilation of nitrogen from several amino acids, dipeptides and tripeptides, purines, pyrimidines, urea, and ethanolamine were induced in mice. Competitive colonization assays confirmed that mutants lacking catabolic pathways for L-serine, N-acetylneuraminic acid, N-acetylglucosamine, and di- and tripeptides had colonization defects. Rescue experiments in mice showed that L-serine serves primarily as a nitrogen source, whereas N-acetylneuraminic acid provides both carbon and nitrogen. Of the many nitrogen assimilation mutants tested, the largest colonization defect was for an L-serine deaminase mutant, which demonstrates L-serine is the most important nitrogen source for colonized E. coli.

ABSTRACTMammalian gut microbes colonize the intestinal tract of their host and adapt to establish a microbial ecosystem. The host diet changes the nutrient profile of the intestine and has a high impact on microbiota composition. Genetic mutations in Escherichia coli, a prevalent species in the human gut, allow for adaptation to the mammalian intestine, as reported in previous studies. However, the extent of colonization fitness in the intestine elevated by genetic mutation and the effects of diet change on these mutations in E. coli are still poorly known. Here, we show that notable mutations in sugar metabolism-related genes (gatC, araC, and malI) were detected in the E. coli K-12 genome just 2 weeks after colonization in the germ-free mouse intestine. In addition to elevated fitness by deletion of gatC, as previously reported, deletion of araC and malI also elevated E. coli fitness in the murine intestine in a host diet-dependent manner. In vitro cultures of medium containing nutrients abundant in the intestine (e.g., galactose, N-acetylglucosamine, and asparagine) also showed increased E. coli fitness after deletion of the genes-of-interest associated with their metabolism. Furthermore, the host diet was found to influence the developmental trajectory of gene mutations in E. coli. Taken together, we suggest that genetic mutations in E. coli are selected in response to the intestinal environment, which facilitates efficient utilization of nutrients abundant in the intestine under laboratory conditions. Our study offers some insight into the possible adaptation mechanisms of gut microbes.IMPORTANCEThe gut microbiota is closely associated with human health and is greatly impacted by the host diet. Bacteria such as Escherichia coli live in the gut all throughout the life of a human host and adapt to the intestinal environment. Adaptive mutations in E. coli are reported to enhance fitness in the mammalian intestine, but to what extent is still poorly known. It is also unknown whether the host diet affects what genes are mutated and to what extent fitness is affected. This study suggests that genetic mutations in the E. coli K-12 strain are selected in response to the intestinal environment and facilitate efficient utilization of abundant nutrients in the germ-free mouse intestine. Our study provides a better understanding of these intestinal adaptation mechanisms of gut microbes.

Wan He,1,&ast; Changbu Wu,2,3,&ast; Guanping Chen,2– 4 Guili Zhang,2,3 Zihan Zhao,2,3 Shu’an Wen,2,3 Yuan Zhou,2,3 Xue Deng,2,3 Yu Feng,2,3 Lan-Lan Zhong,2,3 Guo-Bao Tian,2– 4 Min Dai1 1School of Laboratory Medicine, Chengdu Medical College, Chengdu, 610500, People’s Republic of China; 2Department of Immunology and Microbiology, Advanced Medical Technology Center, The First Affiliated Hospital, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, 510080, People’s Republic of China; 3Key Laboratory of Tropical Diseases Control (Sun Yat-sen University), Ministry of Education, Guangzhou, 510080, People’s Republic of China; 4Department of Immunology, School of Medicine, Sun Yat-Sen University, Shenzhen, 518107, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Min Dai, School of Laboratory Medicine, Chengdu Medical College, Chengdu, Sichuan Province, People’s Republic of China, Tel +86 28-62739128, Email daimin1015@163.com Guo-Bao Tian, Department of Immunology and Microbiology, Advanced Medical Technology Center, The First Affiliated Hospital, Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan 2nd Road, Guangzhou, 510080, People’s Republic of China, Tel/Fax +86 20 87335387, Email tiangb@mail.sysu.edu.cnBackground: The emergence and spread of hypervirulent carbapenem-resistant Klebsiella pneumoniae (hv-CRKP) is a potential epidemiological threat that needs to be monitored. However, the transmission and pathogenic characteristics of hv-CRKP in China remain unclear. We investigated the epidemiological characteristics of gut colonized hv-CRKP in a hospital in Guangdong Province, China.Methods: A total of 46 gut colonized hv-CRKP isolates were collected from Sun Yat-Sen Memorial Hospital (Guangzhou, China) from August 31st to December 31st, 2021. Minimum inhibitory concentrations (MICs) were obtained for 15 antibiotics for 46 hv-CRKP isolates. BALB/C mice infection model and mucoviscosity assay was used to evaluate the virulence of the isolates. The characteristics of genome, phylogenetic relationship and the structure of the plasmid of 46 gut colonized hv-CRKP isolates were compared with pathogenic isolates from GeneBank based on whole-genome data.Results: The hv-CRKP isolation rate of all gut colonized carbapenem-resistant Klebsiella pneumoniae was 17% (46/270), and the intestinal colonization rate of hv-CRKP was irrelevant to the sex, age, department of hospitalization, and history of antibiotic use of the host. The gut colonized hv-CRKP showed pandrug resistance and hypervirulence. The gut colonized hv-CRKP and pathogenic hv-CRKP prevalent in China were mainly ST11 hv-CRKP and had two major epidemic clades. The similarities in genomic characteristics between gut colonized hv-CRKP and pathogenic hv-CRKP were consistent. The gut colonized hv-CRKP carried an incomplete structure pK2044 virulence plasmid from hypervirulent K. pneumoniae NTUH-K2044 by analyzing the virulence plasmid structure.Conclusion: Our results suggest that the gut colonized ST11 hv-CRKP may serve as a reservoir for the clinical pathogenic ST11 HV-CRKP. It is necessary to further strengthen the monitoring of gut colonized hv-CRKP and research the potential mechanism of infection caused by gut colonized hv-CRKP.Keywords: K. pneumoniae, intestinal colonization, hypervirulent, genomic characterization, carbapenemase

ABSTRACTInflammatory bowel disease (IBD) is a broad term for a range of chronic intestinal disorders, including Crohn’s disease and ulcerative colitis. The global prevalence of IBD is rising, with over one million patients affected in the United States alone. Adherent-invasive Escherichia coli (AIEC) is a pathobiont frequently found in IBD biopsies. AIEC adhere to and invade epithelial cells, and can survive inside phagocytes in vitro. However, how AIEC contribute to IBD in vivo remains unclear. Here, we established a larval zebrafish (Danio rerio) model to study the interplay between pre-existing intestinal inflammation and AIEC colonization of the gut. We used the pro-inflammatory drug dextran sulfate sodium (DSS) to induce intestinal inflammation. This was followed by food-borne infection of larvae with AIEC using the protozoan Paramecium caudatum, a natural prey, as a vehicle. We show that AIEC more robustly colonize the zebrafish gut and are cleared slower than non-pathogenic E. coli. In addition, DSS-induced enterocolitis increases bacterial burden and decreases bacterial clearance in the larval gut. We benchmark our model against existing rodent models using two mutants deficient in the known AIEC virulence factors FimH and IbeA, which have virulence defects in both rodent and the larval zebrafish model. Finally, we show that AIEC colonization exacerbates DSS-induced enterocolitis and prevents recovery from inflammation-induced damage. In conclusion, we established a high-throughput, genetically tractable model to study AIEC-host interactions in the context of pre-existing inflammation.IMPORTANCEAlthough inflammatory bowel diseases are on the rise, what factors influence IBD risk and severity, and the underlying mechanisms remain to be fully understood. Although host genetics, microbiome, and environmental factors have all been shown to correlate with the development of IBD, cause and effect are difficult to disentangle in this context. For example, AIEC is a known pathobiont found in IBD patients, but it remains unclear if gut inflammation during IBD facilitates colonization with AIEC, or if AIEC colonization makes the host more susceptible to pro-inflammatory stimuli. It is critical to understand the mechanisms that contribute to AIEC infections in a susceptible host in order to develop successful therapeutics. Here, we show that the larval zebrafish model recapitulates key features of AIEC infections in other animal models and can be utilized to address these gaps in knowledge.

ABSTRACT Gut microbiota are increasingly being recognized as a contributing factor in the etiology of numerous diseases and as a potential determinant in the immune response to various treatments. Recent work has suggested that the suboptimal immunogenic response to vaccination in low- and middle-income countries may be associated with differences in the gut microbiome, which are known to be substantially different between Western and non-Western countries. However, insufficient consideration has been given to the characterization of non-Western microbiomes and their relationship with well-being and immunity. Humanized gnotobiotic mouse models have been used to better understand the causal associations between the gut microbiota and health outcomes but have largely been limited to the study of Western microbiota. Thus, we were interested in determining the applicability of gavage strategies used to humanize germ-free mice with Western microbiota to the humanization of germ-free mice with rural African fecal samples. Here, we assessed the impact of the number and frequency of gavages and the effect of a donor-matched diet on the colonization of Malian fecal microbiota in germ-free mice. One gavage was insufficient to provide a stable establishment of the Malian microbiome, whereas four weekly gavages resulted in a more consistent colonization of the human donor taxa. Interestingly, the donor-matched diet did not improve colonization over the fixed-formula, grain-based mouse chow. Subsequent phenotypic studies using African gut microbiota-humanized gnotobiotic mouse models will allow for a better understanding of the interaction between African gut microbiota and well-being and potentially aid in developing improved treatments for microbiota-dependent diseases in non-Western populations. IMPORTANCE There is increasing evidence that microbes residing within the intestines (gut microbiota) play important roles in the well-being of humans. Yet, there are considerable challenges in determining the specific role of gut microbiota in human diseases owing to the complexity of diverse internal and environmental factors that can contribute to diseases. Mice devoid of all microorganisms (germ-free mice) can be colonized with human stool samples to examine the specific contribution of the gut microbiota to a disease. These approaches have been primarily focused on stool samples obtained from individuals in Western countries. Thus, there is limited understanding as to whether the same methods used to colonize germ-free mice with stool from Western individuals would apply to the colonization of germ-free mice with stool from non-Western individuals. Here, we report the results from colonizing germ-free mice with stool samples of Malian children.

ObjectiveWe investigated the epidemiological surveillance of the intestinal colonization and nosocomial infection of carbapenem-resistant Enterobacteriales (CRE) isolates from inpatients, which can provide the basis for developing effective prevention.MethodsA total of 96 CRE strains were collected from 1,487 fecal samples of hospitalized children between January 2016 and June 2017, which were defined as the “CRE colonization” group. In total, 70 CRE clinical isolates were also randomly selected for the comparison analysis and defined as the “CRE infection” group. The antimicrobial susceptibility of all strains was determined by the microdilution broth method. Polymerase chain reaction (PCR) was used to analyze carbapenemase genes, plasmid typing, and integrons. Multilocus sequence typing was further used to determine clonal relatedness.ResultsIn the “CRE colonization” group, Klebsiella pneumoniae was mostly detected with a rate of 42.7% (41/96), followed by Escherichia coli (34.4%, 33/96) and Enterobacter cloacae (15.6%, 15/96). The ST11 KPC-2 producer, ST8 NDM-5 producer, and ST45 NDM-1 producer were commonly present in carbapenem-resistant K. pneumoniae (CRKPN), carbapenem-resistant E. coli (CRECO), and carbapenem-resistant E. cloacae (CRECL) isolates, respectively. In the “CRE infection” group, 70% (49/70) of strains were K. pneumoniae, with 21.4% E. cloacae (15/70) and 5.7% E. coli (4/70). The ST15 OXA-232 producer and ST48 NDM-5 producer were frequently observed in CRKPN isolates, while the majority of NDM-1-producing CRECL isolates were assigned as ST45. Phylogenetic analysis showed that partial CRE isolates from intestinal colonization and nosocomial infection were closely related, especially for ST11 KPC-2-producing CRKPN and ST45 NDM-1-producing CRECL. Furthermore, plasmid typing demonstrated that IncF and IncFIB were the most prevalent plasmids in KPC-2 producers, while IncX3/IncX2 and ColE were widely spread in NDM producer and OXA-232 producer, respectively. Then, class 1 integron intergrase intI1 was positive in 74.0% (71/96) of the “CRE colonization” group and 52.9% (37/70) of the “CRE infection” group.ConclusionThis study revealed that CRE strains from intestinal colonization and nosocomial infection showed a partial correlation in the prevalence of CRE, especially for ST11 KPC-2-producing CRKPN and ST45 NDM-1-producing CRECL. Therefore, before admission, long-term active screening of rectal colonization of CRE isolates should be emphasized.

Vibrio alginolyticus as a common pathogen infecting humans and marine animals has become a severe threat to the global mariculture industry. Using liquid chromatography and tandem-mass spectrometry (LC–MS/MS) analysis, comparative proteomics between dctP-deletion (ΔdctP) strain and wild-type V. alginolyticus HY9901 was profiled, and total of 547 differentially expressed proteins (DEPs) were identified. In these DEPs, 310 proteins were upregulated and 237 proteins were downregulated. Comparative proteomics of ΔdctP and wild-type strains identified some important regulated factors of colonization and virulent proteins. The regulated factors of bacterial colonization mainly included LuxR and ToxR (biofilm formation), flagellins (FlaB and FlrB), mannose-sensitive hemagglutinin (MshA) pilus, various substrates of the type II secretion system (EpsD, SecD and SecF) and type IV secretion system (ClpV), quorum sensing systems (LuxR homologous ValR), the virulent heat shock proteins (IbpA and YbeY), lipopolysaccharide LptE, outer membrane proteins (OmpU and TolC), glutamate synthases (GltB and GltD), oligopeptide permease (OppA) and siderophores (IrgA). In our previous study, biological characteristics (swarming motility, biofilm formation, cell adhesion and colonized ability) were significantly decreased and LD50 was significantly increased of ΔdctP compared to wide-type. Taken together, these results suggest that DctP protein plays an important role in controlling biological characteristics through DEPs in this study to finally affecting intestinal colonization and virulence. [ABSTRACT FROM AUTHOR]

Vibrio cholerae is an intestinal pathogen that can cause severe diarrheal disease. The disease has afflicted millions of people since the 19th century and has aroused global concern. The Vibrio Pathogenicity Island-2 (VPI-2) is a 57.3 kb region, VC1758–VC1809, which is present in choleragenic V. cholerae. At present, little is known about the function of VC1795 in the VPI-2 of V. cholerae. In this study, the intestinal colonization ability of the ΔVC1795 strain was significantly reduced compared to that of the wild-type strain, and the colonization ability was restored to the wild-type strain after VC1795 gene replacement. This result indicated that the VC1795 gene plays a key role in the intestinal colonization and pathogenicity of V. cholerae. Then, we explored the upstream and downstream regulation mechanisms of the VC1795 gene. Cyclic adenylate receptor protein (CRP) was identified as being located upstream of VC1795 by a DNA pull-down assay and electrophoretic mobility shift assays (EMSAs) and negatively regulating the expression of VC1795. In addition, the results of Chromatin immunoprecipitation followed by sequencing (ChIP-seq), EMSAs, and Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) indicated that VC1795 directly negatively regulates the expression of its downstream gene, VC1794. Furthermore, by using qRT-PCR, we hypothesized that VC1795 indirectly positively regulates the toxin-coregulated pilus (TCP) cluster to influence the colonization ability of V. cholerae in intestinal tracts. In short, our findings support the key regulatory role of VC1795 in bacterial pathogenesis as well as lay the groundwork for the further determination of the complex regulatory network of VC1795 in bacteria. [ABSTRACT FROM AUTHOR]

Campylobacter jejuni is an important foodborne pathogen. Despite the lack of clinical signs associated with its colonization in poultry, it has been reported to interact with the intestinal immune system. However, little is known about the interaction between C. jejuni and the chicken immune system, especially in the context of hepatic dissemination. Therefore, to follow up on our previous study showing intestinal colonization and hepatic spread of C. jejuni, cecal tonsils and liver samples were collected from these birds to determine the mRNA levels of chemokines and cytokines. Serum samples were also collected to determine serum amyloid A (SAA) concentrations and specific IgY titers. Lack of Th17 induction was observed in the cecal tonsils of only the liver-contaminated groups. This hepatic dissemination was accompanied by innate, Th1 and Th2 immune responses in livers, as well as an increase in SAA concentrations and specific IgY levels in sera. Campylobacter appears to be able to restrain the induction of the chicken gut immunity in particular conditions, possibly enhancing its hepatic dissemination and thus eliciting systemic immune responses. Although Campylobacter is often recognized as a commensal-like bacterium in chickens, it seems to modulate the gut immune system and induce systemic immunity. [ABSTRACT FROM AUTHOR]

In our previous study, we identified a metabolite of Bacillus subtilis BS-Z15 (a strain with probiotic characteristics) that could improve immunity in mice. In the present study, we examined the effects of B. subtilis BS-Z15 and its metabolites on body weight gain and the intestinal microbiota of mice. Sixty 25-day-old male Kunming white mice were selected and randomly divided into four groups: control group (A), daily saline gavage; B. subtilis-treated group (B), single gavage (1 × 109 CFU/time/animal/day); group D, 14 consecutive gavages (1 × 109 CFU/time/animal/day); and B. subtilis metabolite-treated group (E), 30 consecutive gavages (90 mg kg−1/time/animal/day). High-throughput sequencing technology was used to analyze intergroup differences in the mouse intestinal microbiota. The results showed that the three treated groups had significantly slower body weight gain compared with the control group, which lasted until the 45 days (P < 0.05), and the daily food intake of the treated mice was higher (P < 0.05). The intestinal microbiota structure of the mice in the treated groups was significantly altered compared with that in the control group, suggesting that B. subtilis BS-Z15 may regulate the weight gain of animals by affecting their intestinal bacterial composition. After stopping the gavage of B. subtilis BS-Z15, the abundance of this strain in the small intestine of the mice gradually decreased and its presence was undetectable at 45 days, indicating that B. subtilis BS-Z15 could not colonize the intestine of these mice. These findings suggest that B. subtilis BS-Z15 may regulate intestinal microbiota through its metabolites to reduce weight gain. [ABSTRACT FROM AUTHOR]

Hybrid-pathogenic Escherichia coli represent an important group of strains associated with intestinal and extraintestinal infections. Recently, we described strain UPEC-46, a uropathogenic/enteroaggregative E. coli (UPEC/EAEC) strain presenting the aggregative adherence (AA) pattern on bladder and colorectal epithelial cells mediated by aggregate-forming pili (AFP). However, the role of AFP and other uninvestigated putative fimbriae operons in UPEC-46 pathogenesis remains unclear. Thus, this study evaluated the involvement of AFP and other adhesins in uropathogenicity and intestinal colonization using different in vitro and in vivo models. The strain UPEC-46 was able to adhere and invade intestinal and urinary cell lines. A library of transposon mutants also identified the involvement of type I fimbriae (TIF) in the adherence to HeLa cells, in addition to colorectal and bladder cell lines. The streptomycin-treated mouse in vivo model also showed an increased number of bacterial counts in the colon in the presence of AFP and TIF. In the mouse model of ascending urinary tract infection (UTI), AFP was more associated with kidney colonization, while TIF appears to mediate bladder colonization. Results observed in in vivo experiments were also confirmed by electron microscopy (EM) analyses. In summary, the in vitro and in vivo analyses show a synergistic role of AFP and TIF in the adherence and colonization of intestinal and urinary epithelia. Therefore, we propose that hybrid E. coli strains carrying AFP and TIF could potentially cause intestinal and urinary tract infections in the same patient.

Weijie Cao,&ast; Jieyong Zhang,&ast; Zhilei Bian, Li Li, Suping Zhang, Yang Qin, Dingming Wan, Zhongxing Jiang, Ran Zhang Department of Hematology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Ran Zhang; Zhongxing Jiang, Department of Hematology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450002, People’s Republic of China, Tel +86 137 8359 0246 ; +86 185 3805 3607, Fax +86 370 66295122, Email zhangran19880310@126.com; jiangzx@zzu.edu.cnPurpose: To investigate the prevalence, risk factors of intestinal carbapenem-resistant Enterobacteriaceae (CRE) colonization and bloodstream infection (BSI) caused by CRE in allogeneic hematopoietic stem cell transplantation (allo-HSCT) recipients.Methods: We analyzed the clinical data of 185 patients with hematological malignancies who underwent allo-HSCT from May 2019 to December 2021. All patients received regular CRE monitoring by rectal swab during allo-HSCT, and some CRE strains were further identified for carbapenemase phenotypes. The rates, distribution and risk factors of CRE colonization, CRE-induced BSI were analyzed.Results: CRE was detected in 44 of 185 recipients, with colonization rate of 23.8%. A total of 46 strains of CRE were isolated, including 22 Escherichia coli, 17 Klebsiella pneumoniae, three Klebsiella oxytoca, two Enterobacter hormaechei, and two other Enterobacteriaceae. Among the 19 strains identified with carbapenemase phenotypes, eight strains of E. coli produced metal β-lactamase, five K. pneumoniae produced serine carbapenemase, two K. pneumoniae produced metal β-lactamase, two K. oxytoca produced metal β-lactamase, a Citrobacter malonic acid-free produced metal β-lactamase and a Citrobacter freundii produced metal β-lactamase. In 10 patients developed with CRE-related BSI, the types and combined drug sensitivity of strains detected by rectal swab were highly consistent with blood culture. Multivariate analysis revealed that pulmonary infection, perianal infection and carbapenem application in the 3 months pre-transplant were independent risk factors for rectal CRE colonization, while rectal colonization with carbapenem-resistant K. pneumoniae (CR-KP) was an independent risk factor for CRE-induced BSI. The mortality rate within 30 days of CRE-related BSI was 50.0%, and patients receiving multi-drug therapy within 24 hours showed slightly lower mortality than that in the single-drug treatment group.Conclusion: Allo-HSCT patients with CRE-induced BSI have poor prognosis, and CR-KP rectal colonization is an independent risk factor for CRE-related BSI. Rectal swab screening during allo-HSCT could provide early warning for later CRE-induced BSI.Keywords: allogeneic hematopoietic stem cell transplantation, carbapenem-resistant enterobacteriaceae, bloodstream infection, intestinal colonization

An exceptional gut-colonizing ability may underlie the dramatic epidemiological success of the multidrug-resistant H30R subclone of Escherichia coli sequence type 131 (O25b:K+:H4). In order to inform the development of colonization-preventing measures, we studied systemic immune correlates of H30R intestinal colonization. Human volunteers' fecal samples were screened for H30R by selective culture and PCR. Subjects were assessed by enzyme immunoassay for serum levels of anti-O25 IgG (representing H30R) and anti-O6 IgG (representing non-H30 E. coli generally), initially and for up to 14 months. Whole blood was tested for the antigen-stimulated release of IFNγ, TNFα, IL-4, IL-10, and IL-17 after incubation with E. coli strains JJ1886 (H30R; O25b:K+:H4) or CFT073 (non-H30; O6:K2:H1). Three main findings were obtained. First, H30R-colonized subjects had significantly higher anti-O25 IgG levels than controls, but similar anti-O6 IgG levels, suggesting an IgG response to H30R colonization. Second, anti-O25 and anti-O6 IgG levels were stable over time. Third, H30R-colonized subjects exhibited a lower TNFα and IL-10 release than controls in response to strain JJ1886 (H30R) relative to strain CFT073 (non-H30R), consistent with TNFα hypo-responsiveness to H30R possibly predisposing to H30R colonization. Thus, H30R-colonized hosts exhibit a sustained serum anti-O25 IgG response and an underlying deficit in TNFα responsiveness to H30R that could potentially be addressed for colonization prevention. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus colonizes the anterior nares, and also the gut, particularly in infants. S. aureus is divided into lineages, termed clonal complexes (CCs), which comprise closely related sequence types (STs). While CC30 and CC45 predominate among nasal commensals, their prevalence among gut-colonizing S. aureus is unknown. Here, 67 gut commensal S. aureus strains from 49 healthy Swedish infants (aged 3 days to 12 months) were subjected to multi-locus sequence typing. The STs of these strains were related to their virulence gene profiles, time of persistence in the microbiota, and fecal population counts. Three STs predominated: ST45 (22% of the strains); ST15 (21%); and ST30 (18%). In a logistic regression, ST45 strains showed higher fecal population counts than the others, independent of virulence gene carriage. The lower fecal counts of ST15 were linked to the carriage of fib genes (encoding fibrinogen-binding proteins), while those of ST30 were linked to fib and sea (enterotoxin A) carriage. While only 11% of the ST15 and ST30 strains were acquired after 2 months of age, this was true of 53% of the ST45 strains (p = 0.008), indicating that the former may be less fit for establishment in a more mature microbiota. None of the ST45 strains was transient (persisting < 3 weeks), and persistent ST45 strains colonized for significantly longer periods than persistent strains of other STs (mean, 34 vs 22 weeks, p = 0.04). Our results suggest that ST45 strains are well-adapted for commensal gut colonization in infants, reflecting yet-unidentified traits of these strains. [ABSTRACT FROM AUTHOR]

The human gastrointestinal tract is inhabited by trillions of symbiotic bacteria that form a complex ecological community and influence human physiology. Symbiotic nutrient sharing and nutrient competition are the most studied relationships in gut commensals, whereas the interactions underlying homeostasis and community maintenance are not fully understood. Here, we provide insights into a new symbiotic relationship wherein the sharing of secreted cytoplasmic proteins, called “moonlighting proteins,” between two heterologous bacterial strains (Bifidobacterium longum and Bacteroides thetaiotaomicron) was observed to affect the adhesion of bacteria to mucins. B. longum and B. thetaiotaomicron were cocultured using a membrane-filter system, and in this system the cocultured B. thetaiotaomicron cells showed greater adhesion to mucins compared to that shown by monoculture cells. Proteomic analysis showed the presence of 13 B. longum-derived cytoplasmic proteins on the surface of B. thetaiotaomicron. Moreover, incubation of B. thetaiotaomicron with the recombinant proteins GroEL and elongation factor Tu (EF-Tu)—two well-known mucin-adhesive moonlighting proteins of B. longum—led to an increase in the adhesion of B. thetaiotaomicron to mucins, a result attributed to the localization of these proteins on the B. thetaiotaomicron cell surface. Furthermore, the recombinant EF-Tu and GroEL proteins were observed to bind to the cell surface of several other bacterial species; however, the binding was species dependent. The present findings indicate a symbiotic relationship mediated by the sharing of moonlighting proteins among specific strains of B. longum and B. thetaiotaomicron. IMPORTANCE The adhesion of intestinal bacteria to the mucus layer is an important colonization strategy in the gut environment. Generally, the bacterial adhesion process is a characteristic feature of the individual cell surface-associated adhesion factors secreted by a particular bacterium. In this study, coculture experiments between Bifidobacterium and Bacteroides show that the secreted moonlighting proteins adhere to the cell surface of coexisting bacteria and alter the adhesiveness of the bacteria to mucins. This finding indicates that the moonlighting proteins act as adhesion factors for not only homologous strains but also for coexisting heterologous strains. The presence of a coexisting bacterium in the environment can significantly alter the mucin-adhesive properties of another bacterium. The findings from this study contribute to a better understanding of the colonization properties of gut bacteria through the discovery of a new symbiotic relationship between them. [ABSTRACT FROM AUTHOR]

Clostridium perfringens type F (formerly enterotoxigenic C. perfringens type A) strains produce an enterotoxin (CPE) to cause acute cases of food poisoning and chronic nonfoodborne human gastrointestinal diseases (NFD), e.g., antibiotic-associated diarrhea (AAD). NFD strains also produce NanI sialidase, an extracellular enzyme that releases sialic acids from sialyated host macromolecules. Recent in vitro studies suggested that NanI may contribute to NFD strain intestinal colonization by enhancing the adherence of such strains to intestinal cells and promoting their bacterial growth using generated sialic acid as an energy source. The current study tested this hypothesis by developing a mouse intestinal colonization model involving clindamycin pretreatment to produce conditions mimicking those during AAD. In this model, the type F NFD strain F4969 persisted for at least 4 days in the small intestine, cecum, and colon. When clindamycin-pretreated mice were challenged by oral gavage with equivalent numbers of F4969 bacteria or its isogenic nanI null mutant, significantly lower numbers of the nanI mutant were recovered from all intestinal segments, and it was completely cleared from the small intestine by day 4. Complementation of the mutant to restore NanI production also promoted colonization. When the same nanI null mutant strain was coinoculated into the mouse model together with a nanI-producing strain, the numbers of this mutant were restored to wild-type F4969 levels in all intestinal segments. This result suggests that sialidases produced by other bacteria might also provide some support for C. perfringens intestinal colonization. Collectively, these in vivo findings identify NanI to be the first known significant contributor to chronic intestinal colonization by NFD strains.

Candida auris, an emerging multi-drug resistant fungal pathogen, causes invasive infections in humans. The factors regulating the colonization of C. auris in host niches are not well understood. In this study, we examined the effect of antibiotic-induced gut dysbiosis on C. auris intestinal colonization, dissemination, microbiome composition and the mucosal immune response. Our results indicate that mice treated with cefoperazone alone had a significant increase in C. auris intestinal colonization compared to untreated control groups. A significant increase in the dissemination of C. auris from the intestine to internal organs was observed in antibiotic-treated immunosuppressed mice. Intestinal colonization of C. auris alters the microbiome composition of antibiotic-treated mice. Relative abundance of firmicutes members mainly Clostridiales and Paenibacillus were considerably increased in the cefoperazone-treated mice infected with C. auris compared to cefoperazone-treated uninfected mice. Next, we examined the mucosal immune response of C. auris infected mice and compared the results with Candida albicans infection. The number of CD11b+ CX3CR1+ macrophages was significantly decreased in the intestine of C. auris infected mice when compared to C. albicans infection. On the other hand, both C. auris and C. albicans infected mice had a comparable increase of the number of Th17 and Th22 cells in the intestine. A significant increase in Candida-specific IgA was observed in the serum of C. auris but not in the C. albicans infected mice. Taken together, treatment with broad-spectrum antibiotic increased the colonization and dissemination of C. auris from the intestine. Furthermore, findings from this study for the first time revealed the microbiome composition, innate and adaptive cellular immune response to intestinal infection with C. auris.

ABSTRACT Extended-spectrum-β-lactamase (ESBL)-producing Enterobacteriaceae (ESBL-PE) intestinal colonization is of particular concern as it negatively impacts morbidity and is the main source of external cross-contamination in hospitalized patients. Contact isolation strategies may be caught out due to the turnaround time needed by laboratories to report intestinal colonization, during which patients may be inappropriately isolated or not isolated. Here, we developed a protocol combining enrichment by a rapid selective subculture of rectal swab medium and realization of a β-Lacta test on the obtained bacterial pellet (named the BLESSED protocol). The performances of this protocol were validated in vitro on 12 ESBL-PE strains spiked into calibrated sample suspensions and confirmed in clinical settings using 155 rectal swabs, of which 23 (reference method) and 31 (postenrichment broth culture) came from ESBL-PE carriers. In vitro, the protocol detected, with 100% sensitivity, the presence of the 12 ESBL-PE strains from 104 CFU/mL. In the clinical validation cohort, 22 out of the 23 (reference method) and 28 out of the 31 (postenrichment broth culture) ESBL-PE-positive rectal samples were accurately detected. The diagnostic performances for ESBL-PE detection, considering all ESBL-PE carriers, were 90% sensitivity, 98% specificity, an 87% positive predictive value, and a 98% negative predictive value. Our protocol is a rapid and low-cost method that can detect intestinal colonization with ESBL-PE in less than 5 h more accurately than the reference method, opening the field for further studies assessing a rapid and targeted isolation strategy applied only to patients with a positive BLESSED protocol result. IMPORTANCE To both improve the efficiency of contact isolation among ESBL-PE carriers and avoid the unnecessary isolation of noncolonized patients, we should reduce the turnaround time of ESBL screening in laboratories and improve the sensitivity of diagnostic methods. The development of rapid and low-cost methods that satisfy these two goals is a promising approach. In this study, we developed such a technique and report its good diagnostic performance, opening the door for further studies assessing a rapid and targeted isolation strategy applied in a few hours only for patients truly colonized with ESBL-producing bacteria.

Intestinal colonization is beneficial to the role of probiotics, and prebiotics can promote the adhesion and colonization of probiotics in the intestine. This study optimized the combination of complex prebiotics that could improve the growth ability and adhesion ability of Limosilactobacillus fermentum (L. fermentum) DALI02 to Caco-2 cells in vitro and determined the effect of its colonization quantity and colonization time in the immunocompromised rat model. The results showed that all five prebiotics (fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS), inulin, stachyose, and xylo-oligosaccharides (XOS)) significantly promoted the growth and adhesion of L. fermentum DALI02. It was found that 0.5% (w/w) inulin had the best growth promotion effect, and 0.5% FOS had the strongest adhesion promotion (the adhesion rate was increase by 1.75 times). In addition, 0.05% FOS, 0.20% GOS, 0.30% inulin, 0.20% stachyose, and 0.30% XOS could significantly improve the adhesion rate of L. fermentum DALI02 from 1.72% to 3.98%. After 1 w of intervention, the quantity of colonization in the fermented broth with prebiotics group was significantly higher than that in the fermented broth group. The intervention time was extended from 1 d to 4 w, and the amount of colonization of L. fermentum DALI02 in the fermented broth with prebiotics group increased significantly from 4.32 lgcopies/g to 5.12 lgcopies/g. After the intervention, the serum levels of lipopolysaccharide (LPS) and D-lactic acid in rats were significantly reduced, and the most significant was in the fermented broth with prebiotics group, with LPS and D-lactic acid levels of 74.11 pg/mL and 40.33 μmol/L, respectively. Complex prebiotics can promote the growth and adhesion of L. fermentum DALI02 and significantly increase the quantity of colonization and residence time of the strain in the intestine, which helps the restoration of intestinal barrier function and other probiotic effects. [ABSTRACT FROM AUTHOR]

Abstract Background In the pediatric population, severe Clostridioides difficile infection (CDI) sometimes occurs, but most cases are asymptomatic. The asymptomatic carriage rate in pediatric populations is reportedly higher than in the adult population. It is difficult to diagnose CDI, even if C. difficile is detected in children with diarrhea. This study aimed to evaluate the positivity rate of toxigenic C. difficile in the pediatric population with diarrhea. Methods We collected and retrospectively analyzed gastrointestinal pathogen multiplex PCR results of 960 patients to estimate the positivity rate of toxigenic C. difficile in pediatric populations aged between 0 and 18 years. Results The overall rate of C. difficile toxin B positivity was 10.1% in the stool samples. The positivity rate peaked in 1-year-old infants (29/153, 19.0%) and continually decreased thereafter. The positivity rate we observed was lower than the rates described in the literature. Remarkably, no C. difficile was detected in neonates. Antibiotic usage was inversely related to the positivity rate, especially in infants < 2 years of age. The odds ratio of antibiotics was 0.44 (95% confidence interval (CI) 0.28–0.68; P < 0.001). The presence of concomitant gastrointestinal pathogens was not associated with toxigenic C. difficile positivity. Conclusions Even though toxigenic C. difficile infection is neither an important nor a common cause of pediatric diarrhea, children can spread it to adults at risk of developing CDI. The pediatric population can act as hidden reservoirs for pathogenic strains in the community.

In the field of aquaculture, the enhancement of animal health and disease prevention is progressively being tackled using alternatives to antibiotics, including vaccines and probiotics. This study was designed to evaluate the potential of a recombinant Bacillus methylotrophicus , engineered to express the outer membrane channel protein TolC of Aeromonas hydrophila AH3 and the green fluorescent protein GFP, as an oral vaccine. Initially, the genes encoding tolC and GFP were cloned into a prokaryotic expression system, and anti -TolC mouse antiserum was generated. Subsequently, the tolC gene was subcloned into a modified pMDGFP plasmid, which was transformed into B. methylotrophicus WM-1 for protein expression. The recombinant B. methylotrophicus BmT was then administered to grass carp via co-feeding, and its efficacy as an oral vaccine was assessed. Our findings demonstrated successful expression of the 55 kDa TolC and 28 kDa GFP proteins, and the preparation of polyclonal antibodies with high specificity. The BmT exhibited stable expression of the GFP-TolC fusion protein and excellent genetic stability. Following oral immunization, significant elevations were observed in serum-specific IgM levels and the activities of acid phosphatase (ACP), alkaline phosphatase (AKP), superoxide dismutase (SOD), and lysozyme (LZM) in grass carp. Concurrently, significant upregulation of immune-related genes, including IFN-I, IL-10, IL-1β, TNF-α, and IgT, was noted in the intestines, head kidney, and spleen of the grass carp. Colonization tests further revealed that the BmT persisted in the gut of immunized fish even after a fasting period of 7 days. Notably, oral administration of BmT enhanced the survival rate of grass carp following A. hydrophila infection. These results suggest that the oral BmT vaccine developed in this study holds promise for future applications in aquaculture. • BmT expressing a GFP-TolC fusion protein was successfully constructed. • Oral administration of BmT significantly enhanced specific immune responses in grass carp. • BmT effectively colonized the gut of grass carp. • BmT provided effective immune protection against A. hydrophila infection. [ABSTRACT FROM AUTHOR]

Vibrio cholerae is an intestinal pathogen that can cause severe diarrheal disease. The disease has afflicted millions of people since the 19th century and has aroused global concern. The Vibrio Pathogenicity Island-2 (VPI-2) is a 57.3 kb region, VC1758–VC1809, which is present in choleragenic V. cholerae. At present, little is known about the function of VC1795 in the VPI-2 of V. cholerae. In this study, the intestinal colonization ability of the ΔVC1795 strain was significantly reduced compared to that of the wild-type strain, and the colonization ability was restored to the wild-type strain after VC1795 gene replacement. This result indicated that the VC1795 gene plays a key role in the intestinal colonization and pathogenicity of V. cholerae. Then, we explored the upstream and downstream regulation mechanisms of the VC1795 gene. Cyclic adenylate receptor protein (CRP) was identified as being located upstream of VC1795 by a DNA pull-down assay and electrophoretic mobility shift assays (EMSAs) and negatively regulating the expression of VC1795. In addition, the results of Chromatin immunoprecipitation followed by sequencing (ChIP-seq), EMSAs, and Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) indicated that VC1795 directly negatively regulates the expression of its downstream gene, VC1794. Furthermore, by using qRT-PCR, we hypothesized that VC1795 indirectly positively regulates the toxin-coregulated pilus (TCP) cluster to influence the colonization ability of V. cholerae in intestinal tracts. In short, our findings support the key regulatory role of VC1795 in bacterial pathogenesis as well as lay the groundwork for the further determination of the complex regulatory network of VC1795 in bacteria.

BackgroundThe worldwide emergence and diffusion of extended-spectrum β-lactamase-K. pneumoniae (ESBL-KP) is of particular concern. Although ESBL-KP can inhabit the human gut asymptomatically, colonization with ESBL-KP is associated with an increased risk of ESBL-KP infection and mortality. In this study, we investigated the prevalence and characteristics of ESBL-KP in fecal samples from healthy persons in 12 villages in Shandong Province, China.MethodsScreening for ESBL-KP in fecal samples was performed by selective cultivation. The bacterial species were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and 16S rDNA sequence analysis. Minimum inhibitory concentrations (MICs) of 16 antibiotics were determined by the agar dilution method. Plasmid replicons, antimicrobial resistance genes and Sequence types (STs) of the isolates were determined by whole-genome sequencing (WGS). Genetic relatedness of ESBL-KP isolates was determined by the single nucleotide polymorphisms (SNP). The S1 nuclease-pulsed-field gel electrophoresis (S1-PFGE) was used to characterize the plasmids carried by ESBL-KP isolates. Conjugation assays was used to verify the transferability of blaCTX − M.ResultsESBL-KP prevalence rates increased from 12.0% in 2015 to 27.5% in 2017. The experimental results showed that 97% of isolates had multi-drug resistance. Multiple ESBL resistance genotypes were commonly detected in the isolates. STs among the ESBL-KP isolates were diverse. All 69 blaCTX−M−3-positive isolates were located on plasmids, and these genes could be transferred with plasmids between different strains. Phylogenetic analysis showed the possibility of transmission among some isolates.ConclusionThis study obtained the drug resistance patterns, the drug resistance phenotype and molecular characteristics of fecal-derived ESBL-KP in rural communities in Shandong Province, China. We report a rapid increase in occurrence of ESBL-KP among fecal samples collected from healthy rural residents of Shandong Province from 2015 to 2017. The carriage rate of multidrug-resistant bacteria in healthy residents is increasing. Thus, a need for further monitoring and possible interventions of ESBL-KP in this region is warranted.

• Commercially available double-tipped cotton swabs can be used for screening of MDRB. • Their product-specific contamination flora does not confound detection of MDRB in humans. • Further studies should clarify their status for other screenings (eg, MRSA, SARS-CoV-2). • In resource-restricted countries, cotton swabs could be a cost-saving alternative to Copan eSwabs. Rectal swabs are well-implemented screening tools for multidrug-resistant bacteria (MDRB). Since certified swabs such as the Copan eSwab system experienced a delivery bottleneck during the COVID-19 pandemic, commercially available alternatives such as commonly used double-tipped cotton swabs had to be investigated, especially considering their similarity to professional cotton swabs for microbiological purposes. Diagnostic properties of commercial cotton swabs (comparable to Q-tips) and Copan eSwabs were qualitatively compared in a prospective single-center study using microbiological standard cultures and PCR methods for the detection of multidrug-resistant Gram-negative bacteria and vancomycin-resistant enterococci (VRE). A total of 196 swab pairs were collected from 164 participants. MDRB were detected in 36 of 164 cases (22%). There were neither false-negative nor false-positive results using commercial cotton swabs. In 8 of 196 samples (4.1%) MDRB species were detected only by using cotton swabs, including vancomycin-resistant Enterococcus faecium , OXA-48 producing Escherichia coli , ESBL-producing Klebsiella pneumoniae and ESBL-producing Escherichia coli. Commercial cotton swabs turned out to be a reliable alternative to Copan eSwabs. For practical use as a screening tool, relevant storage- and manufacturer-related contamination must be ruled out beforehand. Commonly available double-tipped cotton swabs can be used for rectal MDRB screening in the event of supply shortages of certified swabs. Further studies should clarify their suitability as a sampling system for nasopharyngeal MRSA carriage or even for the molecular biological detection of SARS-CoV-2. [ABSTRACT FROM AUTHOR]

Fluoroquinolone (FQ) resistance in a major foodborne bacterial pathogen, Campylobacter jejuni, derived from cattle has recently become prevalent and poses a significant public health concern. However, the underlying factors for this increase are not entirely clear. To evaluate the effect of enrofloxacin treatment on FQ-resistance development in C. jejuni, 35 commercial calves were equally divided into five groups (Groups 1–5) and were orally inoculated with FQ-susceptible (FQ-S) C. jejuni. Eight days later, Groups 4 and 5 were challenged with Mannheimia haemolytica via a transtracheal route to induce a respiratory disease; after 8 days, Groups 2, 3, 4, and 5 were injected subcutaneously with enrofloxacin (7.5 mg/kg for Groups 2 and 4, and 12.5 mg/kg for Groups 3 and 5). Colonization levels by FQ-resistant (FQ-R) and FQ-S Campylobacter in rectal feces were determined via differential culture throughout the experiment. Before oral inoculation with C. jejuni, only five calves were naturally colonized by Campylobacter, four of which were also colonized by FQ-R C. jejuni (three in Group 1 and one in Group 3). Soon after the oral inoculation, almost all calves in the groups became stably colonized by FQ-S C. jejuni (~3–6 log10 CFU/g), except that the four calves that were pre-colonized before inoculation remained positive with both FQ-R and FQ-S C. jejuni. Following enrofloxacin administration, C. jejuni colonization declined sharply and rapidly in all treated groups to undetectable levels; however, the vast majority of the animals were recolonized by C. jejuni at comparable levels 72 h after the treatment. Notably, no FQ-R C. jejuni was detected in any of the calves that received enrofloxacin, regardless of the drug dose used or disease status of the animals. The lack of detection of FQ-R C. jejuni was likely due to the localized high concentration of the antibiotic in the intestine, which may have prevented the emergence of the FQ-R mutant. These findings indicate that single-dose enrofloxacin use in cattle poses a low risk for selection of de novo FQ-R mutants in C. jejuni. [ABSTRACT FROM AUTHOR]

Roseburia intestinalis is an anaerobic bacterium that produces butyric acid and belongs to the phylum Firmicutes. There is increasing evidence that this bacterium has positive effects on several diseases, including inflammatory bowel disease, atherosclerosis, alcoholic fatty liver, colorectal cancer, and metabolic syndrome, making it a potential "Next Generation Probiotic." We investigated the genomic characteristics, probiotic properties, cytotoxicity, oral toxicity, colonization characteristics of the bacterium, and its effect on the gut microbiota. The genome contains few genes encoding virulence factors, three clustered regularly interspaced short palindromic repeat (CRISPR) sequences, two Cas genes, no toxic biogenic amine synthesis genes, and several essential amino acid and vitamin synthesis genes. Seven prophages and 41 genomic islands were predicted. In addition to a bacteriocin (Zoocin A), the bacterium encodes four metabolic gene clusters that synthesize short-chain fatty acids and 222 carbohydrate-active enzyme modules. This bacterium is sensitive to antibiotics specified by the European Food Safety Authority, does not exhibit hemolytic or gelatinase activity, and exhibits some acid resistance. R. intestinalis adheres to intestinal epithelial cells and inhibits the invasion of certain pathogens. In vitro experiments showed that the bacterium was not cytotoxic. R. intestinalis did not affect the diversity or abundance of the gut flora. Using the fluorescent labelling method, we discovered that R. intestinalis colonizes the cecum and mucus of the colon. An oral toxicity study did not reveal any obvious adverse effects. The lethal dose (LD)50 of R. intestinalis exceeded 1.9 × 109 colony forming units (CFU)/kg, whereas the no observed adverse effect level (NOAEL) derived from this study was 1.32 × 109 CFU/kg/day for 28 days. The current research shows that, R. intestinalis is a suitable nextgeneration probiotic considering its probiotic properties and safety. [ABSTRACT FROM AUTHOR]

Thorough understanding of the initial colonization process of human intestines is important to optimize the prevention of microbiota-associated diseases, and also to further improve the current microbial therapies. In recent years, therefore, colonization of the human gut has gained renewed interest. However, due to a lack of standardization of life events that might influence this early colonization process in humans, many generally accepted insights are based on deduction and assumption. In our review, we compare knowledge on colonization in humans with research in piglets, because the intestinal tract of pigs is remarkably similar to that of humans and the early-life events are more standardized. We assess potential similarities and challenge some concepts that have been widely accepted in human microbiota research. Bacterial colonization of the human gut is characterized by successive waves in a progressive process, to a complex gut microbiota community. After re-analyzing available data from piglets, we found that the bacterial colonization process is very similar in terms of the wave sequence and functionality of each wave. Moreover, based on the piglet data, we found that, in addition to external factors such as suckling and nutrition, the bacterial community itself appears to have a major influence on the colonization success of additional bacteria in the intestine. Thus, the colonization process in piglets might rely, at least in part, on niche dependency, an ecological principle to be considered in the intestinal colonization process in humans. [ABSTRACT FROM AUTHOR]

Bacterial infections pose a significant global health threat, accounting for an estimated 7.7 million deaths. Hospital outbreaks driven by multi-drug-resistant pathogens, notably Klebsiella pneumoniae ( K. pneumoniae ), are of grave concern. This opportunistic pathogen causes pneumonia, urinary tract infections, and bacteremia, particularly in immunocompromised individuals. The rise of hypervirulent K. pneumoniae adds complexity, as it increasingly infects healthy individuals. Recent epidemiological data suggest that asymptomatic gastrointestinal carriage serves as a reservoir for infections in the same individual and allows for host-to-host transmission via the fecal-oral route. This review focuses on K. pneumoniae 's gastrointestinal colonization, delving into epidemiological evidence, current animal models, molecular colonization mechanisms, and the protective role of the resident gut microbiota. Moreover, the review sheds light on in vivo high-throughput approaches that have been crucial for identifying K. pneumoniae factors in gut colonization. This comprehensive exploration aims to enhance our understanding of K. pneumoniae gut pathogenesis, guiding future intervention and prevention strategies., Competing Interests: The authors declare no conflict of interest.

ABSTRACT Extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli can cause invasive infections in infants and immunocompromised children with high associated morbidity and mortality. The gut is a major reservoir of these strains in the community. Current dogma dictates that antimicrobial resistance is associated with a fitness cost. However, recent data show that some contemporary ESBL E. coli strains may be more “fit” compared to nonresistant E. coli strains. Here, we use whole-genome sequencing to first characterize 15 ESBL E. coli strains isolated from infants in a Pakistani community, a clinical extraintestinal pathogenic ESBL E. coli ST131 strain, and a non-ESBL commensal E. coli strain, and then use a novel animal model of early life gut colonization to assess the ability of these strains to colonize the infant mouse gut. We determined that CTX-M-15 was present in all the ESBL strains, as well as additional beta-lactamases and genes conferring resistance to multiple antibiotic classes. In the animal model, 11/16 ESBL E. coli strains had significantly higher burden of colonization at week four of life compared to commensal strains, even in the absence of selective antibiotic pressure, suggesting that these strains may have enhanced fitness despite being highly antimicrobial resistant. IMPORTANCE Antimicrobial resistance is a global public health emergency. Infants, especially preterm infants and those in the neonatal intensive care unit, immunocompromised hosts, and those with chronic illnesses are at highest risk of adverse outcomes from invasive infections with antimicrobial-resistant strains. It has long been thought that resistance is associated with a fitness cost, i.e., antimicrobial-resistant strains are not able to colonize the gut as well as nonresistant strains, and that antibiotic exposure is a key risk factor for persistent colonization with resistant strains. Here, we use a novel infant mouse model to add to the growing body of literature that some highly-resistant contemporary Escherichia coli strains can persist in the gut with a significant burden of colonization despite absence of antibiotic exposure.

ABSTRACT Currently, there are no vaccines licensed for enterotoxigenic Escherichia coli (ETEC), a leading cause of children’s diarrhea in developing countries and the most common cause of travelers’ diarrhea. A vaccine preventing ETEC bacteria from colonization at small intestines and neutralizing enterotoxin toxicity is expected to be effective against ETEC diarrhea. Protein-based multivalent vaccine candidate MecVax was demonstrated recently to induce antibodies neutralizing heat-labile toxin (LT) and heat-stable toxin (STa) enterotoxicity and inhibiting adherence of seven ETEC adhesins (CFA/I, CS1 to CS6) but also to protect against ETEC toxin-mediated clinical diarrhea in a pig challenge model. To further evaluate MecVax preclinical efficacy against ETEC colonization at small intestines, in this study, we intramuscularly immunized adult rabbits with MecVax, challenged rabbits with ETEC strain H10407 (CFA/I, LT, STa), and examined prevention of bacteria intestinal colonization. Data showed that rabbits immunized with MecVax developed antibodies to both ETEC toxins (LT, STa) and seven adhesins (CFA/I, CS1 to CS6) and had over 99.9% reduction of H10407 intestinal colonization, indicating that the broadly immunogenic ETEC vaccine candidate MecVax is protective against ETEC H10407 intestinal colonization. This study also confirmed that parenteral administration of a protein-based vaccine can prevent bacteria intestinal colonization. Protection against ETEC intestinal colonization demonstrated by this rabbit study, in conjugation with protection against ETEC enterotoxin-mediated clinical diarrhea from a previous pig challenge study, suggested that MecVax can potentially be an effective ETEC vaccine and a combined pig and rabbit challenge model can evaluate ETEC vaccine preclinical efficacy. IMPORTANCE An effective ETEC vaccine would prevent hundreds of millions of diarrhea clinical cases and save nearly 100,000 lives annually. MecVax, a protein-based injectable multivalent ETEC vaccine candidate, has been shown for the first time to induce functional antibodies against both ETEC enterotoxins (STa, LT) produced by all ETEC strains and seven ETEC adhesins (CFA/I, CS1 to CS6) expressed by ETEC strains causing a majority of ETEC diarrhea clinical cases and the moderate-to-severe cases. Moreover, MecVax was demonstrated to protect against ETEC STa or LT toxin-mediated diarrhea in a pig model. If it also protects against ETEC intestinal colonization, MecVax can be validated as an effective ETEC vaccine candidate. This adult rabbit colonization model study showed that intramuscular administration of MecVax effectively prevented intestinal colonization by H10407, perhaps the most virulent ETEC strain, affirming MecVax vaccine candidacy and accelerating vaccine development against ETEC children’s diarrhea and travelers’ diarrhea.

ABSTRACT Susceptibility to Clostridioides difficile infection (CDI) typically follows the administration of antibiotics. Patients with inflammatory bowel disease (IBD) have increased incidence of CDI, even in the absence of antibiotic treatment. However, the mechanisms underlying this susceptibility are not well understood. To explore the intersection between CDI and IBD, we recently described a mouse model where colitis triggered by the murine gut bacterium, Helicobacter hepaticus, in IL-10−/− mice led to susceptibility to C. difficile colonization without antibiotic administration. The current work disentangles the relative contributions of inflammation and gut microbiota in colonization resistance to C. difficile in this model. We show that inflammation drives changes in microbiota composition, which leads to CDI susceptibility. Decreasing inflammation with an anti-p40 monoclonal antibody promotes a shift of the microbiota back toward a colonization-resistant state. Transferring microbiota from susceptible and resistant mice to germfree animals transfers the susceptibility phenotype, supporting the primacy of the microbiota in colonization resistance. These findings shine light on the complex interactions between the host, microbiota, and C. difficile in the context of intestinal inflammation, and may form a basis for the development of strategies to prevent or treat CDI in IBD patients. IMPORTANCE Patients with inflammatory bowel disease (IBD) have an increased risk of developing C. difficile infection (CDI), even in the absence of antibiotic treatment. Yet, mechanisms regulating C. difficile colonization in IBD patients remain unclear. Here, we use an antibiotic-independent mouse model to demonstrate that intestinal inflammation alters microbiota composition to permit C. difficile colonization in mice with colitis. Notably, treating inflammation with an anti-p40 monoclonal antibody, a clinically relevant IBD therapeutic, restores microbiota-mediated colonization resistance to the pathogen. Through microbiota transfer experiments in germfree mice, we confirm that the microbiota shaped in the setting of IBD is the primary driver of susceptibility to C. diffiicile colonization. Collectively, our findings provide insight into CDI pathogenesis in the context of intestinal inflammation, which may inform methods to manage infection in IBD patients. More broadly, this work advances our understanding of mechanisms by which the host-microbiota interface modulates colonization resistance to C. difficile.

Campylobacter jejuni is an important foodborne pathogen. Despite the lack of clinical signs associated with its colonization in poultry, it has been reported to interact with the intestinal immune system. However, little is known about the interaction between C. jejuni and the chicken immune system, especially in the context of hepatic dissemination. Therefore, to follow up on our previous study showing intestinal colonization and hepatic spread of C. jejuni, cecal tonsils and liver samples were collected from these birds to determine the mRNA levels of chemokines and cytokines. Serum samples were also collected to determine serum amyloid A (SAA) concentrations and specific IgY titers. Lack of Th17 induction was observed in the cecal tonsils of only the liver-contaminated groups. This hepatic dissemination was accompanied by innate, Th1 and Th2 immune responses in livers, as well as an increase in SAA concentrations and specific IgY levels in sera. Campylobacter appears to be able to restrain the induction of the chicken gut immunity in particular conditions, possibly enhancing its hepatic dissemination and thus eliciting systemic immune responses. Although Campylobacter is often recognized as a commensal-like bacterium in chickens, it seems to modulate the gut immune system and induce systemic immunity.

Oral antibiotic treatment is often applied in animal studies in order to allow establishment of an introduced antibiotic-resistant bacterium in the gut. Here, we compared the application of streptomycin dosed orally in microcontainers to dosage through drinking water. The selective effect on a resistant bacterial strain, as well as the effects on fecal, luminal, and mucosal microbiota composition, were investigated. Three groups of rats (n = 10 per group) were orally dosed with microcontainers daily for 3 days. One of these groups (STR-M) received streptomycin-loaded microcontainers designed for release in the distal ileum, while the other two groups (controls [CTR] and STR-W) received empty microcontainers. The STR-W group was additionally dosed with streptomycin through the drinking water. A streptomycin-resistant Escherichia coli strain was orally inoculated into all animals. Three days after inoculation, the resistant E. coli was found only in the cecum and colon of animals receiving streptomycin in microcontainers but in all intestinal compartments of animals receiving streptomycin in the drinking water. 16S rRNA amplicon sequencing revealed significant changes in the fecal microbiota of both groups of streptomycin-treated animals. Investigation of the inner colonic mucus layer by confocal laser scanning microscopy and laser capture microdissection revealed no significant effect of streptomycin treatment on the mucus-inhabiting microbiota or on E. coli encroachment into the inner mucus. Streptomycin-loaded microcontainers thus enhanced proliferation of an introduced streptomycin-resistant E. coli in the cecum and colon without affecting the small intestine environment. While improvements of the drug delivery system are needed to facilitate optimal local concentration and release of streptomycin, the application of microcontainers provides new prospects for antibiotic treatment. IMPORTANCE Delivery of antibiotics in microcontainer devices designed for release at specific sites of the gut represents a novel approach which might reduce the amount of antibiotic needed to obtain a local selective effect. We propose that the application of microcontainers may have the potential to open novel opportunities for antibiotic treatment of humans and animals with fewer side effects on nontarget bacterial populations. In the current study, we therefore elucidated the effects of streptomycin, delivered in microcontainers coated with pH-sensitive lids, on the selective effect on a resistant bacterium, as well as on the surrounding intestinal microbiota in rats. [ABSTRACT FROM AUTHOR]

Background: Superior gut colonization may underlie the pandemic emergence of the resistance-associated H30 subclone of Escherichia coli sequence type 131 (ST131-H30). Little is known about the associated host and bacterial characteristics, or the comparative persistence of non-ST131 intestinal E. coli.Methods: Generic and fluoroquinolone-resistant E. coli isolates from volunteers' serial fecal samples underwent clonal analysis and extensive polymerase chain reaction (PCR)-based characterization (phylogroup, selected sequence types, virulence genes). Kaplan-Meier survival analysis and Cox proportional hazards survival analysis using penalized regression (a machine-learning method) were used to identify correlates of strain persistence.Results: Screening of 2005 subjects at the Minneapolis VA Medical Center identified 222 subjects (117 veterans, 105 human and animal household members) for longitudinal fecal surveillance. Analysis of their 585 unique-by-subject fecal E. coli strains identified multiple epidemiological, ecological, and bacterial correlates of strain persistence. ST131-H30, a strong univariable correlate of persistence, was superseded in multivariable analysis by outpatient status, fluoroquinolone resistance, and diverse (predominantly iron uptake-related) virulence genes.Conclusions: ST131-H30 exhibits exceptional intestinal persistence, possibly due to a combination of fluoroquinolone resistance and virulence factors, which may be primarily colonization factors. This identifies both likely contributors to the ST131-H30 pandemic and potential targets for interventions against it. [ABSTRACT FROM AUTHOR]

Abstract Background Searching the risk factors for carbapenem-resistant Enterobacteriaceae (CRE) infection is important in clinical practice. In the present study, we aim to investigate bacterial characteristics of colonizing strains and their correlation with subsequent CRE infection. Methods Between May 2018 and January 2019, patients hospitalized in the department of haematology and intensive care unit (ICU) were screened for CRE by rectal swabs and monitored for the outcome of infection. We identified the species and carbapenemase-encoding genes of colonizing strains and performed antimicrobial susceptibility tests and multilocus sequence typing (MLST). Risk factors for subsequent CRE infections were ascertained by univariate and multivariable analysis. Results We collected a total of 219 colonizing strains from 153 patients. Klebsiella pneumoniae was the most abundant species, and MLST analysis showed rich diversity. K. pneumoniae carbapenemase (KPC) was predominant in the infection group (72.4%). In the non-infection group, 35.4% of strains were non-carbapenemase-producing CRE (NCP-CRE), and New Delhi metallo-β-lactamase (NDM) was predominant (42.2%). The rate of high-level carbapenem resistance (minimum inhibitory concentration [MIC] ≥ 64 mg/L for meropenem and ertapenem, ≥ 32 mg/L for imipenem) was remarkably higher in the infection group than in the non-infection group (P