Clostridium difficile Modulates the Gut Microbiota by Inducing the Production of Indole, an Interkingdom Signaling and Antimicrobial Molecule.

Clostridium (Clostridioides) difficile infection (CDI) is associated with dysbiosis. C. difficile has a characteristic propensity to persist and recur 1 to 4 weeks after treatment, but the mechanism is unknown. We hypothesized that C. difficile may persist by manipulating the intestinal microenvironment, thereby hampering gut microbiota reconstitution following antibiotic-mediated dysbiosis. By screening stools from CDI patients for unique markers, a metabolite identified to be indole by mass spectrometry and Fourier transform infrared spectroscopy was identified. The average fecal indole concentration detected in CDI patients (n = 216; mean, 1,684.0 ± 84.4 µM) was significantly higher than in stools of patients with non-C. difficile diarrhea (n = 204; mean, 762.8 ± 53.8 µM). Certain intestinal bacteria, but not C. difficile, produce indole, a potent antimicrobial antioxidant. Remarkably, C. difficile induced other indole-producing gut microbes to produce increasing amounts of indole. Furthermore, a C. difficile accessory gene regulator 1 quorum sensing system mutant cannot induce indole, but complementation of the mutant strain with the wild-type gene restored its ability to induce indole production. Indole tolerance assays indicated that the amount of indole required to inhibit growth of most gut-protective bacteria was within the range detected in the CDI stools. We think that a high indole level limits the growth of beneficial indole-sensitive bacteria in the colon and alters colonization resistance and this might allow C. difficile to proliferate and persist. Together, these results reveal a unique mechanism of C. difficile persistence and provide insight into complex interactions and chemical warfare among the gut microbiota. IMPORTANCE Clostridium difficile infection is the leading cause of hospital-acquired and antibiotic-associated diarrhea worldwide. C. difficile flourishes in the colon after the diversity of the beneficial and protective gut microbiota have been altered by antibiotic therapy. C. difficile tends to persist, as does dysbiosis, encouraging recurrence a few days to weeks after treatment, and this further complicates treatment options. Here, we show that C. difficile might persist by manipulating the indigenous microbiota to produce indole, a bioactive molecule that inhibits the growth and reconstitution of the protective gut microbiota during infection. This discovery may explain a unique strategy C. difficile uses to control other bacteria in the colon and provide insight into the complex interactions and chemical warfare among the gut microbiota.

Prevalence of Clostridium difficile infections among Kenyan children with diarrhea.

BACKGROUND: Diarrhea causes significant morbidity and mortality among children worldwide. Regions most affected by diarrhea include Sub-Saharan Africa and Southeast Asia, where antibiotics are in common use and can make children more vulnerable to Clostridium difficile and pathogens that are not affected by these drugs. Indeed, C. difficile is a major diarrhea-associated pathogen and poses a significant threat to vulnerable and immunocompromised populations. Yet, little is known about the role and epidemiology of C. difficile in diarrhea-associated illness among young children. As a result, C. difficile is often neglected in regions such as Sub-Saharan Africa that are most impacted by childhood diarrhea. The purpose of this study was to establish the frequency of C. difficile in young children (<5 years) with diarrhea. METHODS: Children presenting with diarrhea at a national hospital in Kenya from 2015 to 2018 were enrolled consecutively. Following informed consent by a parent or legal guardian, stool samples were obtained from the children and demographic data were collected. The stools were examined for the presence of four common pathogens known to cause diarrhea: C. difficile, rotavirus, Cryptosporidium parvum, and Giardia lamblia. C. difficile was verified by toxigenic culture and PCR. The presence of C. parvum and/or G. lamblia was determined using the ImmunoCard STAT! Crypto/Giardia Rapid assay. Rotavirus was detected by ELISA. RESULTS: The study population comprised 157 children; 62.4% were male and 37.6% were female and their average age was 12.4 months. Of the 157 stool specimens investigated, 37.6% were positive for C. difficile, 33.8% for rotavirus, 5.1% for Cryptosporidium, and 5.1% for Giardia. PCR analysis identified at least one of the C. difficile-specific - genes (tcdA, tcdB, or tcdC). Further, 57.6% of the stools had C. difficile colonies bearing a frame-shift deletion in the tcdC gene, a mutation associated with increased toxin production. The frequency of C. difficile was 32.6% in children ≤12 months old and increased to 46.6% in children 12-24 months old. CONCLUSIONS: In Kenyan children presenting with diarrhea, C. difficile is more prevalent than rotavirus or Cryptosporidium, two leading causes of childhood diarrhea. These findings underscore the need to better understand the role of C. difficile in children with diarrhea, especially in areas with antibiotic overuse. Understanding C. difficile epidemiology and its relationship to co-infecting pathogens among African children with diarrhea will help in devising ways of reducing diarrhea-associated illness.

Analysis of Fish Commonly Sold in Local Supermarkets Reveals the Presence of Pathogenic and Multidrug-Resistant Bacterial Communities.

Fish has been an important source of proteins, essential vitamins, and low saturated fats for centuries. However, improperly handled fish can expose consumers to infectious bacteria, including difficult to treat multidrug-resistant pathogens. With the goal to investigate the existence of disease-causing and antibiotic-resistant bacteria, we examined bacterial communities present on various types of fish purchased from supermarkets in Houston, Texas, USA. The bacterial communities were characterized by selective phenotypic culture methods, 16S ribosomal RNA gene sequencing, and antibiotic susceptibility testing. The results revealed the presence of different bacterial communities on the fish samples examined. The bacterial communities were not significantly different between the supermarkets sampled. The following presumptive human pathogens were isolated on the fish samples: Escherichia coli (67%), enterohemorrhagic E. coli (31%), Shigella and Salmonella species (28%), Listeria species (29%), and Staphylococcus aureus (28%). Drug sensitivity assays showed resistance to commonly prescribed antibiotics ciprofloxacin, gentamicin, and vancomycin. Out of a total of 99 E. coli samples tested, 41.4% were resistant to ciprofloxacin, whereas 33.3% were resistant to gentamicin. Of the total of 31 S. aureus isolates tested, 87% were resistant to ciprofloxacin, whereas 61.3% were resistant to vancomycin. Moreover, some of the E. coli strains were resistant to both ciprofloxacin and gentamicin (28%), whereas 49% of the S. aureus isolates were resistant to both ciprofloxacin and vancomycin. These results highlight the prevalence of antimicrobial-resistant foodborne pathogens on fish purchased from the supermarkets and underscore the risk associated with improper handling of fish.

High rate of Clostridium difficile among young adults presenting with diarrhea at two hospitals in Kenya.

BACKGROUND: Clostridium difficile infection (CDI) is the leading cause of antibiotic-associated diarrhea worldwide. As a result, the US Centers for Disease Control and Prevention have designated C. difficile as an urgent threat. Despite the global public health risk posed by CDI, little is known about its epidemiology on the African continent. This article describes the common occurrence of CDI from a cross-section of consecutively seen, randomly enrolled patients presenting with diarrhea at two major hospitals in Kenya. METHODS: Patients presenting with diarrhea at two major hospitals in Kenya from May to July 2017 were enrolled. After signing the informed consent, stool samples, demographic data, medical history, prior antibiotic use, and HIV status were obtained from the patients. C. difficile was detected and validated by toxigenic culture and PCR. RESULTS: The average age of the patients was 35.5 years (range 3-86 years); 59% were male and 41% were female. Out of 105 patient stools tested, 98 (93.3%) were positive for C. difficile by culture. PCR analysis confirmed C. difficile-specific genes, tcdA, tcdB, and tcdC, in the strains isolated from the stools. Further, 82.5% of the stools had C. difficile isolates bearing the frame-shift deletion associated with hypervirulent strains. Remarkably, 91.9% of the stools that tested positive for C. difficile came from patients under 60 years old, with 64.3% being less than 40 years of age. The majority of the patients (85%) reported over-the-counter antibiotic use in the last 30days before the hospital visit. CONCLUSIONS: Together, the results revealed an unusually high incidence of C. difficile in the stools analyzed, especially among young adults who are thought to be less vulnerable. Comprehensive research is urgently needed to examine the epidemiology, risk factors, pathogenesis, comorbidities, clinical outcomes, antibiotic susceptibility, and genetic makeup of C. difficile strains circulating on the African continent.