Tumor-resident Lactobacillus iners confer chemoradiation resistance through lactate-induced metabolic rewiring.

Tumor microbiota can produce active metabolites that affect cancer and immune cell signaling, metabolism, and proliferation. Here, we explore tumor and gut microbiome features that affect chemoradiation response in patients with cervical cancer using a combined approach of deep microbiome sequencing, targeted bacterial culture, and in vitro assays. We identify that an obligate L-lactate-producing lactic acid bacterium found in tumors, Lactobacillus iners, is associated with decreased survival in patients, induces chemotherapy and radiation resistance in cervical cancer cells, and leads to metabolic rewiring, or alterations in multiple metabolic pathways, in tumors. Genomically similar L-lactate-producing lactic acid bacteria commensal to other body sites are also significantly associated with survival in colorectal, lung, head and neck, and skin cancers. Our findings demonstrate that lactic acid bacteria in the tumor microenvironment can alter tumor metabolism and lactate signaling pathways, causing therapeutic resistance. Lactic acid bacteria could be promising therapeutic targets across cancer types.

Functional Genomics of Gastrointestinal Escherichia coli Isolated from Patients with Cancer and Diarrhea.

We describe the epidemiology and clinical characteristics of 29 patients with cancer and diarrhea in whom Enteroaggregative Escherichia coli (EAEC) was initially identified by GI BioFire panel multiplex. E. coli strains were successfully isolated from fecal cultures in 14 of 29 patients. Six of the 14 strains were identified as EAEC and 8 belonged to other diverse E. coli groups of unknown pathogenesis. We investigated these strains by their adherence to human intestinal organoids, cytotoxic responses, antibiotic resistance profile, full sequencing of their genomes, and annotation of their functional virulome. Interestingly, we discovered novel and enhanced adherence and aggregative patterns for several diarrheagenic pathotypes that were not previously seen when co-cultured with immortalized cell lines. EAEC isolates displayed exceptional adherence and aggregation to human colonoids compared not only to diverse GI E. coli , but also compared to prototype strains of other diarrheagenic E. coli . Some of the diverse E. coli strains that could not be classified as a conventional pathotype also showed an enhanced aggregative and cytotoxic response. Notably, we found a high carriage rate of antibiotic resistance genes in both EAEC strains and diverse GI E. coli isolates and observed a positive correlation between adherence to colonoids and the number of metal acquisition genes carried in both EAEC and the diverse E. coli strains. This work indicates that E. coli from cancer patients constitute strains of remarkable pathotypic and genomic divergence, including strains of unknown disease etiology with unique virulomes. Future studies will allow for the opportunity to re-define E. coli pathotypes with greater diagnostic accuracy and into more clinically relevant groupings.

Enteropathogenic Escherichia coli Infection in Cancer and Immunosuppressed Patients.

BACKGROUND: The role of enteropathogenic Escherichia coli (EPEC) as a cause of diarrhea in cancer and immunocompromised patients is controversial. Quantitation of fecal bacterial loads has been proposed as a method to differentiate colonized from truly infected patients. METHODS: We studied 77 adult cancer and immunosuppressed patients with diarrhea and EPEC identified in stools by FilmArray, 25 patients with pathogen-negative diarrhea, and 21 healthy adults without diarrhea. Stools were studied by quantitative polymerase chain reaction (qRT-PCR) for EPEC genes eaeA and lifA/efa-1 and strains characterized for virulence factors and adherence to human intestinal enteroids (HIEs). RESULTS: Patients with EPEC were more likely to have community-acquired diarrhea (odds ratio, 3.82 [95% confidence interval, 1.5-10.0]; P = .008) compared with pathogen-negative cases. Although EPEC was identified in 3 of 21 (14%) healthy subjects by qPCR, the bacterial burden was low compared to patients with diarrhea (≤55 vs median, 6 × 104 bacteria/mg stool; P < .001). Among EPEC patients, the bacterial burden was higher in those who were immunosuppressed (median, 6.7 × 103 vs 55 bacteria/mg; P < .001) and those with fecal lifA/ifa-1 (median, 5 × 104 vs 120 bacteria/mg; P = .015). Response to antimicrobial therapy was seen in 44 of 48 (92%) patients with EPEC as the sole pathogen. Antimicrobial resistance was common and strains exhibited distinct patterns of adherence with variable cytotoxicity when studied in HIEs. Cancer care was delayed in 13% of patients. CONCLUSIONS: Immunosuppressed cancer patients with EPEC-associated diarrhea carry high burden of EPEC with strains that are resistant to antibiotics, exhibit novel patterns of adherence when studied in HIEs, and interfere with cancer care.

Fecal Microbiome, Metabolites, and Stem Cell Transplant Outcomes: A Single-Center Pilot Study.

BACKGROUND: Accumulating evidence suggests that the intestinal microbiome may dramatically affect the outcomes of hematopoietic stem cell transplant (HSCT) recipients. Providing 16S ribosomal RNA based microbiome characterization in a clinically actionable time frame is currently problematic. Thus, determination of microbial metabolites as surrogates for microbiome composition could offer practical biomarkers. METHODS: Longitudinal fecal specimens (n = 451) were collected from 44 patients before HSCT through 100 days after transplantation, as well as 1-time samples from healthy volunteers (n = 18) as controls. Microbiota composition was determined using 16S ribosomal RNA V4 sequencing. Fecal indole and butyrate levels were determined using liquid chromatography tandem mass spectrometry. RESULTS: Among HSCT recipients, both fecal indole and butyrate levels correlated with the Shannon diversity index at baseline (P = .02 and P = .002, respectively) and directly after transplantation (P = .006 and P < .001, respectively). Samples with high butyrate levels were enriched for Clostridiales, whereas samples containing high indole were also enriched for Bacteroidales. A lower Shannon diversity index at the time of engraftment was associated with increased incidence of acute intestinal graft-vs-host disease (iGVHD) (P = .02) and transplant-related deaths (P = .03). Although fecal metabolites were not associated with acute iGVHD or overall survival, patients contracting bloodstream infections within 30 days after transplantation had significantly lower levels of fecal butyrate (P = .03). CONCLUSIONS: Longitudinal analysis of fecal microbiome and metabolites after HSCT identified butyrate and indole as potential surrogate markers for microbial diversity and specific taxa. Further studies are needed to ascertain whether fecal metabolites can be used as biomarkers of acute iGVHD or bacteremia after HSCT.

Novel Segment- and Host-Specific Patterns of Enteroaggregative Escherichia coli Adherence to Human Intestinal Enteroids.

Enteroaggregative Escherichia coli (EAEC) is an important diarrheal pathogen and a cause of both acute and chronic diarrhea. It is a common cause of pediatric bacterial diarrhea in developing countries. Despite its discovery in 1987, the intestinal tropism of the pathogen remains unknown. Cell lines used to study EAEC adherence include the HEp-2, T-84, and Caco-2 lines, but they exhibit abnormal metabolism and large variations in gene expression. Animal models either do not faithfully manifest human clinical symptoms or are cumbersome and expensive. Using human intestinal enteroids derived from all four segments of the human intestine, we find that EAEC demonstrates aggregative adherence to duodenal and ileal enteroids, with donor-driven differences driving a sheet-like and layered pattern. This contrasts with the colon, where segment-specific tropisms yielded a mesh-like adherence pattern dominated by interconnecting filaments. Very little to no aggregative adherence to jejunal enteroids was observed, regardless of the strain or donor, in contrast to a strong duodenal association across all donors and strains. These unique patterns of intestinal segment- or donor-specific adherence, but not the overall numbers of associated bacteria, were dependent on the major subunit protein of aggregative adherence fimbriae II (AafA), implying that the morphology of adherent clusters and the overall intestinal cell association of EAEC occur by different mechanisms. Our results suggest that we must give serious consideration to inter- and intrapatient variations in what is arguably the first step in pathogenesis, that of adherence, when considering the clinical manifestation of these infections.IMPORTANCE EAEC is a leading cause of pediatric bacterial diarrhea and a common cause of diarrhea among travelers and immunocompromised individuals. Heterogeneity in EAEC strains and lack of a good model system are major roadblocks to the understanding of its pathogenesis. Utilizing human intestinal enteroids to study the adherence of EAEC, we demonstrate that unique patterns of adherence are largely driven by unidentified factors present in different intestinal segments and from different donors. These patterns are also dependent on aggregative adherence fimbriae II encoded by EAEC. These results imply that we must also consider the contribution of the host to understand the pathogenesis of EAEC-induced inflammation and diarrhea.

Clinical features and molecular epidemiology of diarrheagenic Escherichia coli pathotypes identified by fecal gastrointestinal multiplex nucleic acid amplification in patients with cancer and diarrhea.

Diarrheagenic Escherichia coli (DEC) pathotypes with differing epidemiology and clinical features, are known causes of disease with worldwide occurrence. At a major cancer center in the U.S., we studied patients with cancer and diarrhea for whom a GI Biofire FilmArray multiplex GI panel (BFM) was performed. An enteropathogen was identified in 382 of 2017 (19%) samples distributed across 311 patients. Of these, 60/311(19%) were positive for DEC. Patients receiving hematopoietic stem cell transplants (HSCT) 29/60 (48%) or with a hematologic malignancy 17/60 (28%) accounted for the majority of DEC cases. Enteropathogenic E. coli (EPEC, n=35 [58%]), enteroaggregative E. coli (EAEC, n=10 [17%]) and Shiga toxin producing E. coli (STEC, n=3 [5%]) were the most common DEC identified and peaked in the summer months. Stool cultures confirmed infections in 6/10 (60%) EAEC (five typical AggR+), and EPEC was recovered in 8/35 (22%) samples (all atypical eaeA+, bfp-). DEC was identified in 22 cases (37%) that developed diarrhea >48hours after admission suggesting health care acquisition. Chronic infections were found in 2 EPEC and 1 EAEC cases that were tested at 1month or beyond with shedding that ranged from 58 to >125days. Two patients that underwent hematopoietic stem cell transplantation carried EAEC strains resistant to multiple antibiotics including fluoroquinolones and expressed extended spectrum beta lactamases. While in some instances BFM results were not verified in culture and could represent false positives, DEC pathotypes, especially EPEC and EAEC, caused chronic infections with antimicrobial-resistant strains in a subset of immunosuppressed cancer patients.

Persistent abdominal symptoms in US adults after short-term stay in Mexico.

BACKGROUND: Postinfectious irritable bowel syndrome (PI-IBS) has been reported as a complication of bacterial diarrhea including travelers' diarrhea (TD). This study assessed the role of TD among US students in Mexico in triggering the onset of persistent abdominal symptoms (PAS) and IBS. METHODS: We conducted a 6-month follow-up of a cohort of 817 US students in Mexico as short-term study to assess the frequency of PAS and IBS. Using Rome II criteria for IBS, groups of students with PAS were then categorized as PI-IBS if they met the symptom criteria for IBS or as suffering from functional abdominal disorder (FAD) if they did not meet the criteria. RESULTS: FAD and IBS were commonly found in US students 6 months after leaving Mexico. Important variables in their development were younger adult age, longer stays in Mexico and occurrence of acute diarrhea while in Mexico. Diarrhea while in Mexico occurred more commonly for those later diagnosed with FAD, 101/196 (52%), relative risk (RR) = 1.5 [confidence interval (CI) 1.2-1.8; p = 0.001]; IBS, 20/32 (63%), RR = 2.5 (CI 1.2-5.0; p = 0.007); and PAS (FAD + IBS), 121/228 (53%), RR = 1.5 (CI 1.2-1.8; p < 0.001) compared with subjects who had experienced diarrhea while in Mexico but were not diagnosed with PAS at 6 months, 227/589 (39%). Diarrhea caused by heat-labile enterotoxin-producing enterotoxigenic Escherichia coli or Providencia ssp. demonstrated a greater risk of developing PAS. CONCLUSIONS: PAS occurred commonly in a subset of younger adult travelers who stayed longer in Mexico and experienced acute diarrhea while there. Further studies with this cohort will focus on host genetic associations with the development of PAS.

Seasonality of diarrheagenic Escherichia coli pathotypes in the US students acquiring diarrhea in Mexico.

BACKGROUND: Up to 60% of the US visitors to Mexico develop travelers' diarrhea (TD). In Mexico, rates of diarrhea have been associated with the rainy season and increase in ambient temperature. However, the seasonality of the various diarrheagenic Escherichia coli pathotypes in travelers has not been well described. OBJECTIVE: A study was undertaken to determine if ambient temperature and rainfall have an impact on the acquisition of TD due to different diarrheagenic E coli pathotypes in Mexico. METHODS: We conducted a cohort study of the US adult students traveling to Cuernavaca, Mexico, who were followed during their stay and provided a stool sample with the onset of TD. The presence of E coli was analyzed by a direct fecal multiplex polymerase chain reaction for common E coli pathotypes including enterotoxigenic, enteropathogenic, enteroinvasive, shiga toxin-producing, and enteroaggregative E coli (ETEC, EPEC, EIEC, STEC, and EAEC respectively). The presence of pathotypes was correlated with daily rainfall, average, maximum, and minimum temperatures. RESULTS: A total of 515 adults were enrolled from January 2006 to February 2007. The weekly attack rate of TD for newly arrived travelers was lower in the winter months (range 6.8%-16.3%) than in summer months (range 11.5%-25%; p = 0.05). The rate of ETEC infection increased by 7% for each degree centigrade increase in weekly ambient temperature (p = 0.003). In contrast, EPEC and EAEC were identified in similar proportions during the winter and summer seasons. CONCLUSIONS: Temperature variations in central Mexico influenced the rate of ETEC but not EAEC-associated diarrhea in the US visitors. This epidemiological finding could influence seasonal recommendations for the use of ETEC vaccines in Mexico.

A single-nucleotide polymorphism in the gene encoding osteoprotegerin, an anti-inflammatory protein produced in response to infection with diarrheagenic Escherichia coli, is associated with an increased risk of nonsecretory bacterial diarrhea in North American travelers to Mexico.

BACKGROUND: Osteoprotegerin (OPG), an immunoregulatory member of the TNF receptor superfamily, is expressed in inflamed intestinal mucosa. We investigated whether OPG is produced by intestinal epithelial cells and tested the hypothesis that single-nucleotide polymorphisms (SNPs) in the gene encoding OPG (TNFRSF11B) are associated with traveler's diarrhea (TD) among North American travelers to Mexico. METHODS: OPG concentration was measured in the supernatants of T84 cells infected with various diarrheagenic Escherichia coli pathotypes. Genotyping was performed for 4 SNPs in the OPG gene for 968 North American travelers with or without TD. Stool samples from travelers with TD were evaluated for the presence of enteric pathogens. RESULTS: T84 cells produced higher OPG levels in response to infection with various diarrheagenic E. coli pathotypes than with E. coli controls (P<.05). A SNP in the exon 1 region of the OPG gene (OPG+1181G>C) was associated with TD in white travelers who stayed in Mexico for >1 week during the summer (P=.009) and for TD due to nonsecretory pathogens (P=.001). CONCLUSIONS: Our study suggests that OPG is secreted by intestinal epithelial cells in response to enteropathogens and that a polymorphism in the OPG gene is associated with an increased susceptibility to TD.

Epidemiology of cryptosporidiosis in North American travelers to Mexico.

We studied 1,179 North American travelers who visited Mexico from 2005 to 2007. Travelers' diarrhea (TD) was reported by 521 (44%) participants. Among subjects with TD, 218 cases were examined for cryptosporidiosis by polymerase chain reaction (PCR) and enzyme-linked immunoassays (ELISA). There were 14 (6%) cases of cryptosporidiosis and 141 cases (64%) of bacterial diarrhea. Compared with bacterial diarrhea, a longer stay in Mexico was a risk factor for cryptosporidiosis. Additionally, Cryptosporidium cases passed greater number of watery stools (P < 0.05), suffered more episodes of diarrhea (P < or = 0.05), and were more likely to experience tenesmus (P < or = 0.05) compared with bacterial causes of TD. ELISA detected seven (3%) cases of Cryptosporidium, whereas PCR identified an additional seven cases (6%). Speciation by 18SrRNA sequencing showed that 13 cases were caused by C. parvum and only 1 case was caused by C. hominis. ELISA showed a sensitivity of 50% and specificity of 100% compared with PCR.

Enterotoxigenic Escherichia coli heat-labile toxin seroconversion in US travelers to Mexico.

BACKGROUND AND AIMS: Enterotoxigenic Escherichia coli (ETEC) is the most common bacterial pathogen isolated from travelers suffering of diarrhea. Exposure to heat-labile toxin (LT) produces a high rate of seroconversion. However, the role of LT-producing ETEC (LT-ETEC) as a cause of diarrhea is controversial. We conducted a cohort study in US students traveling to Mexico to assess the ETEC-LT seroconversion rate after natural exposure. METHODS: Participants provided a serum sample on arrival and departure and a stool sample when ill. ETEC-LT immunoglobulin G antibodies were measured by enzyme-linked immunosorbent assay, and LT-ETEC were detected by means of polymerase chain reaction done on fecal DNA. RESULTS: A total of 422 participants with a mean age of 34.5 years were followed a mean of 19.9 days; 304 were females (72.0%), and 319 (75.6%) traveled during the summer months. In total, 177 individuals (41.9%) developed travelers' diarrhea and 33.9% had LT-ETEC identified in their stools. Among individuals having an LT-ETEC strain, 74% seroconverted compared to 11% of those not having diarrhea (p < 0.0001). When analyzed with a logistic regression model, the odds of seroconversion were significantly reduced in participants not having LT-ETEC in their stool (odds ratio = 0.1, p < 0.0001) after adjusting for season, length of stay, age, gender, race, and ethnicity. CONCLUSION: In US young adults traveling to Mexico, ETEC-LT seroconversion reliably identifies individuals naturally exposed to ETEC and correlates with symptomatic illness, length and season of travel.

Human lactoferrin impairs virulence of Shigella flexneri.

Lactoferrin is a glycoprotein present in most human mucosal secretions, including human milk. Lactoferrin is bacteriostatic in low iron media and, in some settings, bactericidal. Lactoferrin impairs ability of Shigella flexneri serotype 5 strain M90T to invade HeLa cells. To determine the mechanism by which lactoferrin decreases invasiveness of Shigella organisms, its effect on the major virulence proteins responsible for bacterial uptake by host cells was evaluated. Lactoferrin induced degradation of invasion plasmid antigens IpaB and, to a lesser extent, IpaC, the key proteins responsible for bacteria-directed phagocytosis by mammalian cells. The lipid A-binding N-terminal portion of lactoferrin (residues 1-33) induces release of invasion antigens but does not induce degradation of IpaBC. Lactoferrin does not directly degrade previously released invasion plasmid antigens but works by making IpaBC susceptible to breakdown by surface-expressed protease(s).

Lactoferrin protects rabbits from Shigella flexneri-induced inflammatory enteritis.

Shigella species cause bacillary dysentery in humans by invasion, intracellular multiplication, spread to adjacent cells, and induction of brisk inflammatory responses in the intestinal epithelium. In vitro data suggest that lactoferrin, a glycoprotein present in human mucosal secretions, has a role in protection from bacterial enteric infections. We sought to determine the activity of lactoferrin in vivo, using the concentration present in human colostrum, to investigate its effect on the development of clinical and pathological evidence of inflammation in a rabbit model of enteritis. Lactoferrin protected rabbits infected with Shigella flexneri from developing inflammatory intestinal disease. Typical histological changes in ill animals included villous blunting with sloughing of epithelial cells, submucosal edema, infiltration of leukocytes, venous congestion, and hemorrhage. Lactoferrin at a concentration normally found in human colostrum blocks development of S. flexneri-induced inflammatory enteritis.