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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.

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.

As a widely spread Gram-negative bacteria, Klebsiella pneumoniae (KP) mainly causes acquired infections in hospitals, such as lung infections, urinary tract infections, and bloodstream infections. In recent years, the number of multidrug-resistant KP strains has increased dramatically, posing a great threat to human health. Carbapenem-resistant KP (CRKP) can be colonized in human body, especially in gastrointestinal tract, and some colonized patients can be infected during hospitalization, among which invasive operation, underlying disease, admission to intensive care unit, antibiotic use, severity of the primary disease, advanced age, operation, coma, and renal failure are common risk factors for secondary infection. Active screening and preventive measures can effectively prevent the occurrence of CRKP infection. Based on the epidemiological status, this study aims to discuss the correlation between colonization and secondary infection induced by CRKP and risk factors for their happening and provide some reference for nosocomial infection prevention and control.

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 Background To establish a method to induce Campylobacter jejuni colonization in the intestines of C57BL/6 mice through antibiotic-induced microbiome depletion. Results Fifty-four female C57BL/6 mice were divided into the normal, control, and experimental groups. The experimental group was administered intragastric cefoperazone sodium and sulbactam sodium (50 mg/mL) for 2 days; then, the experimental and control mice were intragastrically administered 200 µL C. jejuni, which was repeated once more after 2 days. Animal feces were collected, and the HipO gene of C. jejuni was detected using TaqMan qPCR from day 1 to day 14 after modeling completion. Immunofluorescence was used to detect intestinal C. jejuni colonization on day 14, and pathological changes were observed using hematoxylin and eosin staining. Additionally, 16S rDNA analyses of the intestinal contents were conducted on day 14. In the experimental group, C. jejuni was detected in the feces from days 1 to 14 on TaqMan qPCR, and immunofluorescence-labeled C. jejuni were visibly discernable in the intestinal lumen. The intestinal mucosa was generally intact and showed no significant inflammatory-cell infiltration. Diversity analysis of the colonic microbiota showed significant inter-group differences. In the experimental group, the composition of the colonic microbiota differed from that in the other 2 groups at the phylum level, and was characterized by a higher proportion of Bacteroidetes and a lower proportion of Firmicutes. Conclusions Microbiome depletion induced by cefoperazone sodium and sulbactam sodium could promote long-term colonization of C. jejuni in the intestines of mice.

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.

Streptococcus agalactiae, also known as Group B Streptococcus (GBS), is a predominant pathogen of neonatal sepsis, commonly associated with early-onset neonatal sepsis. GBS has also been associated with cases of late-onset sepsis potentially originating from the intestine. Previous findings have shown GBS can colonize the infant intestinal tract as part of the neonatal microbiota. To better understand GBS colonization dynamics in the neonatal intestine, we collected stool and milk samples from prematurely born neonates for identification of potential pathogens in the neonatal intestinal microbiota. GBS was present in approximately 10% of the cohort, and this colonization was not associated with maternal GBS status, delivery route, or gestational weight. Interestingly, we observed the relative abundance of GBS in the infant stool negatively correlated with maternal IgA concentration in matched maternal milk samples. Using a preclinical murine model of GBS infection, we report that both vertical transmission and direct oral introduction resulted in intestinal colonization of GBS; however, translocation beyond the intestine was limited. Finally, vaccination of dams prior to breeding induced strong immunoglobulin responses, including IgA responses, which were associated with reduced mortality and GBS intestinal colonization. Taken together, we show that maternal IgA may contribute to infant immunity by limiting the colonization of GBS in the intestine.

Bacterial translocation from the gut microbiota is a source of sepsis in susceptible patients. Previous work suggests that overgrowth of gut pathobionts, including Klebsiella pneumoniae, increases the risk of disseminated infection. Our data from a human dietary intervention study found that, in the absence of fiber, K. pneumoniae bloomed during microbiota recovery from antibiotic treatment. We thus hypothesized that dietary nutrients directly support or suppress colonization of this gut pathobiont in the microbiota. Consistent with our study in humans, complex carbohydrates in dietary fiber suppressed the colonization of K. pneumoniae and allowed for recovery of competing commensals in mouse models. In contrast, through ex vivo and in vivo modeling, we identified simple carbohydrates as a limiting resource for K. pneumoniae in the gut. As proof of principle, supplementation with lactulose, a nonabsorbed simple carbohydrate and an FDA-approved therapy, increased colonization of K. pneumoniae. Disruption of the intestinal epithelium led to dissemination of K. pneumoniae into the bloodstream and liver, which was prevented by dietary fiber. Our results show that dietary simple and complex carbohydrates were critical not only in the regulation of pathobiont colonization but also disseminated infection, suggesting that targeted dietary interventions may offer a preventative strategy in high-risk patients., Introduction Bacterial translocation from the gut microbiota is a significant source of sepsis in susceptible patients, including those with cirrhosis or acute myeloid leukemia or who are admitted to the [...]

Background: Early detection of antimicrobial-resistant microorganisms is crucial to prevent subsequent invasive infections and contain their spread in the Neonatal Intensive Care Unit (NICU). This study aims to investigate the association between intestinal colonization (IC) by Gram-negative bacteria and the risk of bloodstream infection (BSI) in critically ill neonates. Methods: Data from the electronic medical records of 678 newborns admitted to a NICU Brazilian between 2018 and 2022 were retrospectively analyzed. Participants were monitored by the National Health Security Network. Results: Among neonates, 6.9 % had IC (56.9 % attributed to Acinetobacter baumannii); of these, 19.1 % developed BSI (66.7 % by Staphylococcus spp.). Within the A. baumannii colonization, 34.5 % occurred during an outbreak in September 2021. Colonized individuals had a longer mean length of stay (49.3 ± 26.4 days) and higher mortality rate (12.8 %) compared to non-colonized individuals (22.2 ± 16.9 days; 6.7 %, respectively). Previous use of antimicrobials and invasive devices significantly increased the risk of colonization. Colonization by drug-resistant microorganisms, along with the occurrence of BSI, was associated with increased mortality and reduced survival time. Conclusions: IC contributed to the incidence of BSI, leading to more extended hospital stays and higher mortality rates. Its early detection proved to be essential to identify an outbreak and control the spread of resistant microorganisms within the NICU.

Weijie Cao,* Jieyong Zhang,* 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*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

Abstract The mammalian gastrointestinal tract is a complex environment that hosts a diverse microbial community. To establish infection, bacterial pathogens must be able to compete with the indigenous microbiota for nutrients, as well as sense the host environment and modulate the expression of genes essential for colonization and virulence. Here, we found that enterohemorrhagic Escherichia coli (EHEC) O157:H7 imports host- and microbiota-derived L-malate using the DcuABC transporters and converts these substrates into fumarate to fuel anaerobic fumarate respiration during infection, thereby promoting its colonization of the host intestine. Moreover, L-malate is important not only for nutrient metabolism but also as a signaling molecule that activates virulence gene expression in EHEC O157:H7. The complete virulence-regulating pathway was elucidated; the DcuS/DcuR two-component system senses high L-malate levels and transduces the signal to the master virulence regulator Ler, which in turn activates locus of enterocyte effacement (LEE) genes to promote EHEC O157:H7 adherence to epithelial cells of the large intestine. Disruption of this virulence-regulating pathway by deleting either dcuS or dcuR significantly reduced colonization by EHEC O157:H7 in the infant rabbit intestinal tract; therefore, targeting these genes and altering physiological aspects of the intestinal environment may offer alternatives for EHEC infection treatment.

Oral biotherapeutics hold significant promise, but their lack of controllability and targeting poses a major challenge, particularly for intestinal bacterial biotherapeutics. In response, we have developed a nanoencapsulation approach that responds to the release of enzyme activity in the organism and activates the enzyme in situ, allowing for controlled colonization of microbes in the gut. The nano-coating comprises a two-layer structure: an inner layer of polydopamine with photothermal and adhesive properties, and an outer layer of gelatin-sodium carboxymethylcellulose, which is hydrolyzed by cellulases in the gut following photothermal interaction with dopamine. We have successfully achieved controlled colonization of a wide range of microorganisms. Furthermore, in a diabetes model, this approach has had a profound impact on regulating glucagon-like peptide-1 (GLP-1) production, β-cell physiology, and promoting insulin secretion. This nanocoating is achieved by in situ activation of cellulase without the need for genetic or targeted molecular modification, representing a new paradigm and alternative strategy for microbial therapy. It not only enables precise and controlled colonization of probiotics but also demonstrates great potential for broader application in the field of oral biotherapy. STATEMENT OF SIGNIFICANCE: We have developed a nano-encapsulation method that triggers enzyme activity in response to enzymatic activity, resulting in the controlled release and adhesion of a wide range of microorganisms in the gut. The nano coating comprises two layers: an inner layer of polydopamine with photothermal and adhesion properties, and an outer layer of a gelatin-sodium carboxymethylcellulose polymer, which can be hydrolyzed by cellulases in the intestine. Additionally, this method allows for the preparation of various microbial coatings. This approach holds significant promise for regulating GLP-1 production, the physiological function of pancreatic β-cells, and promoting insulin secretion in diabetes models., Competing Interests: Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

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.

ObjectiveInfections caused by Carbapenem-resistant Enterobacterales (CRE) have high treatment costs, high mortality and few effective therapeutic agents. This study aimed to determine the risk factors for progression from intestinal colonization to infection in hematological patients and the risk factors for 30-day mortality in infected patients.MethodsA retrospective case-control study was conducted in the Department of Hematology at Shandong Provincial Hospital affiliated to Shandong First Medical University from April 2018 to April 2022. Patients who developed subsequent infections were identified as the case group by electronic medical record query of patients with a positive rectal screen for CRE colonization, and patients who did not develop subsequent infections were identified as the control group by stratified random sampling. Univariate analysis and logistic regression analysis determined risk factors for developing CRE infection and risk factors for mortality in CRE-infected patients.ResultsEleven hematological patients in the study developed subsequent infections. The overall 30-day mortality rate for the 44 hematological patients in the case-control study was 11.4% (5/44). Mortality was higher in the case group than in the control group (36.5 vs. 3.0%, P = 0.0026), and septic shock was an independent risk factor for death (P = 0.024). Univariate analysis showed that risk factors for developing infections were non-steroidal immunosuppressants, serum albumin levels, and days of hospitalization. In multivariable logistic regression analysis, immunosuppressants [odds ratio (OR), 19.132; 95% confidence interval (CI), 1.349–271.420; P = 0.029] and serum albumin levels (OR, 0.817; 95% CI, 0.668–0.999; P = 0.049) were independent risk factors for developing infections.ConclusionOur findings suggest that septic shock increases mortality in CRE-infected hematological patients. Hematological patients with CRE colonization using immunosuppressive agents and reduced serum albumin are more likely to progress to CRE infection. This study may help clinicians prevent the onset of infection early and take measures to reduce mortality rates.

Finding strategies for decolonizing gut carriers of multidrug-resistant Escherichia coli (MDR-Ec) is a public-health priority. In this context, novel approaches should be validated in preclinical in vivo gut colonization models before being translated to humans. However, the use of mice presents limitations. Here, we used for the first time Zophobas morio larvae to design a new model of intestinal colonization (28-days duration, T28). Three hyperepidemic MDR-Ec producing extended-spectrum β-lactamases (ESBLs) or carbapenemases were administered via contaminated food to larvae for the first 7 days (T7): Ec-4901.28 (ST131, CTX-M-15), Ec-042 (ST410, OXA-181) and Ec-050 (ST167, NDM-5). Growth curve analyses showed that larvae became rapidly colonized with all strains (T7, ~106–7 CFU/mL), but bacterial load remained high after the removal of contaminated food only in Ec-4901.28 and Ec-042 (T28, ~103–4 CFU/mL). Moreover, larvae receiving a force-feeding treatment with INTESTI bacteriophage cocktail (on T7 and T10 via gauge needle) were decolonized by Ec-4901.28 (INTESTI-susceptible); however, Ec-042 and Ec-050 (INTESTI-resistant) did not. Initial microbiota (before administering contaminated food) was very rich of bacterial genera (e.g., Lactococcus, Enterococcus, Spiroplasma), but patterns were heterogeneous (Shannon diversity index: range 1.1–2.7) and diverse to each other (Bray–Curtis dissimilarity index ≥30%). However, when larvae were challenged with the MDR-Ec with or without administering bacteriophages the microbiota showed a non-significant reduction of the diversity during the 28-day experiments. In conclusion, the Z. morio larvae model promises to be a feasible and high-throughput approach to study novel gut decolonization strategies for MDR-Ec reducing the number of subsequent confirmatory mammalian experiments.

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.

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., Competing Interests: The authors declare no conflict of interest.

ABSTRACTGut microbiota plays a crucial role in providing colonization resistance against multi-drug resistant organisms including carbapenem-resistant Klebsiella pneumoniae (CRKP). However, the impact of different carbapenems on intestinal colonization resistance against CRKP remains poorly understood. In this study, we aimed to investigate the effects of three commonly used carbapenems (meropenem, imipenem, and ertapenem) administered at single-day doses, which are typically employed in emergency departments for managing infected patients, on CRKP gut colonization. We conducted experiments in mice by intravenously administering each carbapenem using human-simulated one-day regimens. The composition of the gut microbiota was analyzed using 16S rRNA amplicon sequencing before and after carbapenem administration. Our results revealed that all three carbapenems, when administered at a single-day dose significantly altered the abundance and diversity of the gut microbiota, leading to compromised colonization resistance against CRKP. Notably, the ertapenem and imipenem groups showed an increase in Allobaculum, Bifidobacterium, Enterobacteriaceae, while the meropenem and imipenem groups exhibited a decrease in Ruminococcaceae, S24-7, and Akkermania. Additionally, the Shannon index exhibited a negative correlation with both the number of days of CRKP colonization CFUs and the duration of bacteria shedding. Furthermore, the count of CRKP in mice after ertapenem administration on the first day and the duration of CRKP shedding were significantly lower compared to meropenem and imipenem. Predictive metabolic pathway analysis demonstrated that the three carbapenems similarly affected a range of metabolic pathways of gut microflora, including carbohydrate metabolism and vitamin B. Our findings emphasize that ertapenem, with its relatively narrow spectrum, minimizes perturbations of the gut microbiota and has a relatively less impact on gut colonization resistance against CRKP.IMPORTANCEThe intestinal colonization of carbapenem-resistant Klebsiella pneumoniae (CRKP) is an important source of clinical infection. Our research showed that even single-day dose use of carbapenems caused CRKP colonization and continuous bacterial shedding, which reminds clinical doctors to prescribe carbapenems cautiously. Whenever possible, ertapenem should be the preferred choice over other carbapenems especially when the identified or highly suspected pathogens can be effectively targeted by ertapenem.

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.

IntroductionCampylobacter jejuni stands out as one of the leading causes of bacterial enteritis. In contrast to humans, specific pathogen-free (SPF) laboratory mice display strict intestinal colonization resistance (CR) against C. jejuni, orchestrated by the specific murine intestinal microbiota, as shown by fecal microbiota transplantation (FMT) earlier.MethodsMurine infection models, comprising SPF, SAB, hma, and mma mice were employed. FMT and microbiota depletion were confirmed by culture and culture-independent analyses. Targeted metabolome analyses of fecal samples provided insights into the associated metabolomic signatures.ResultsIn comparison to hma mice, the murine intestinal microbiota of mma and SPF mice (with CR against C. jejuni) contained significantly elevated numbers of lactobacilli, and Mouse Intestinal Bacteroides, whereas numbers of enterobacteria, enterococci, and Clostridium coccoides group were reduced. Targeted metabolome analysis revealed that fecal samples from mice with CR contained increased levels of secondary bile acids and fatty acids with known antimicrobial activities, but reduced concentrations of amino acids essential for C. jejuni growth as compared to control animals without CR.DiscussionThe findings highlight the role of microbiota-mediated nutrient competition and antibacterial activities of intestinal metabolites in driving murine CR against C. jejuni. The study underscores the complex dynamics of host-microbiota-pathogen interactions and sets the stage for further investigations into the mechanisms driving CR against enteric infections.

ABSTRACT Enterococcus faecalis is a commensal bacterium of the human gastrointestinal tract that causes opportunistic infections. The E. faecalis genetic changes associated with pathogenicity, particularly gut-to-bloodstream translocation, remain poorly understood. Here, we performed a genome-wide association study (GWAS) of 736 whole-genome sequences of fecal and bloodstream E. faecalis isolates from hospitalized and nonhospitalized individuals, respectively, to identify E. faecalis genetic signatures associated with the patient’s hospitalization status and body isolation source. We found that infection by hospitalization status and extraintestinal infection are heritable traits, with ~40% and ~30% of their variation explained by E. faecalis genetics, respectively. Furthermore, a GWAS using linear mixed models did not pinpoint any clear overrepresentation of individual genetic changes by hospitalization status or body isolation source after controlling for the population structure. However, we observed elevated signals in a genomic region containing a prophage element. However, the lineages themselves and their associated virulence factors and antibiotic resistance genes showed variable frequency among blood and fecal isolates and in hospitalized and nonhospitalized individuals. Altogether, our findings indicate that E. faecalis infection by hospitalization status and body sites is partially influenced by the overall genetic background of the isolates and antibiotic resistance patterns rather than genetic variation at individual loci, which suggests a greater role of other host and environmental factors and ultimately the opportunistic pathogenic lifestyle of this versatile host generalist bacterium. IMPORTANCE Enterococcus faecalis causes life-threatening invasive hospital- and community-associated infections that are usually associated with multidrug resistance globally. Although E. faecalis infections cause opportunistic infections typically associated with antibiotic use, immunocompromised immune status, and other factors, they also possess an arsenal of virulence factors crucial for their pathogenicity. Despite this, the relative contribution of these virulence factors and other genetic changes to the pathogenicity of E. faecalis strains remain poorly understood. Here, we investigated whether specific genomic changes in the genome of E. faecalis isolates influence its pathogenicity—infection of hospitalized and nonhospitalized individuals and the propensity to cause extraintestinal infection and intestinal colonization. Our findings indicate that E. faecalis genetics partially influence the infection of hospitalized and nonhospitalized individuals and the propensity to cause extraintestinal infection, possibly due to gut-to-bloodstream translocation, highlighting the potential substantial role of host and environmental factors, including gut microbiota, on the opportunistic pathogenic lifestyle of this bacterium.

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.