High-throughput low-cost nl-qPCR for enteropathogen detection: A proof-of-concept among hospitalized patients in Bangladesh.

BACKGROUND: Diarrheal disease is a leading cause of morbidity and mortality globally, especially in low- and middle-income countries. High-throughput and low-cost approaches to identify etiologic agents are needed to guide public health mitigation. Nanoliter-qPCR (nl-qPCR) is an attractive alternative to more expensive methods yet is nascent in application and without a proof-of-concept among hospitalized patients. METHODS: A census-based study was conducted among diarrheal patients admitted at two government hospitals in rural Bangladesh during a diarrheal outbreak period. DNA was extracted from stool samples and assayed by nl-qPCR for common bacterial, protozoan, and helminth enteropathogens as the primary outcome. RESULTS: A total of 961 patients were enrolled; stool samples were collected from 827 patients. Enteropathogens were detected in 69% of patient samples; More than one enteropathogen was detected in 32%. Enteropathogens most commonly detected were enteroaggregative Escherichia coli (26.0%), Shiga toxin-producing E.coli (18.3%), enterotoxigenic E. coli (15.5% heat stable toxin positive, 2.2% heat labile toxin positive), Shigella spp. (14.8%), and Vibrio cholerae (9.0%). Geospatial analysis revealed that the median number of pathogens per patient and the proportion of cases presenting with severe dehydration were greatest amongst patients residing closest to the study hospitals." CONCLUSIONS: This study demonstrates a proof-of-concept for nl-qPCR as a high-throughput low-cost method for enteropathogen detection among hospitalized patients

Exploring the mycobacteriophage metaproteome: Phage genomics as an educational platform

Bacteriophages are the most abundant forms of life in the biosphere and carry genomes characterized by high genetic diversity and mosaic architectures. The complete sequences of 30 mycobacteriophage genomes show them collectively to encode 101 tRNAs, three tmRNAs, and 3,357 proteins belonging to 1,536 "phamilies" of related sequences, and a statistical analysis predicts that these represent approximately 50% of the total number of phamilies in the mycobacteriophage population. These phamilies contain 2.19 proteins on average; more than half (774) of them contain just a single protein sequence. Only six phamilies have representatives in more than half of the 30 genomes, and only three - encoding tape-measure proteins, lysins, and minor tail proteins - are present in all 30 phages, although these phamilies are themselves highly modular, such that no single amino acid sequence element is present in all 30 mycobacteriophage genomes. Of the 1,536 phamilies, only 230 (15%) have amino acid sequence similarity to previously reported proteins, reflecting the enormous genetic diversity of the entire phage population. The abundance and diversity of phages, the simplicity of phage isolation, and the relatively small size of phage genomes support bacteriophage isolation and comparative genomic analysis as a highly suitable platform for discovery-based education. © 2006 Hatfull et al

<i>dsdA</i> Does Not Affect Colonization of the Murine Urinary Tract by <i>Escherichia coli</i> CFT073

<div><p>The urinary tract environment provides many conditions that deter colonization by microorganisms. D-serine is thought to be one of these stressors and is present at high concentrations in urine. D-serine interferes with L-serine and pantothenate metabolism and is bacteriostatic to many species. Uropathogenic <i>Escherichia coli</i> commonly possess the <i>dsdCXA</i> genetic locus, which allows them to use D-serine as a sole carbon, nitrogen, and energy source. It was previously reported that in the model UPEC strain CFT073, a <i>dsdA</i> mutant outcompetes wild type in the murine model of urinary tract infection. This “hypercolonization” was used to propose a model whereby UPEC strains sense D-serine in the urinary tract and subsequently up-regulate genes necessary for pathogenesis. Here, we show that inactivation of <i>dsdA</i> does not lead to hypercolonization. We suggest that this previously observed effect is due to an unrecognized secondary mutation in <i>rpoS</i> and that some D-serine specific effects described in other studies may be affected by the <i>rpoS</i> status of the strains used. Inactivation of <i>dsdA</i> in the original clinical isolate of CFT073 gives CFT073 Δ<i>dsdA</i> a growth defect in human urine and renders it unable to grow on minimal medium containing D-serine as the sole carbon source. However, CFT073 Δ<i>dsdA</i> is able to colonize the urinary tracts of CBA/J mice indistinguishably from wild type. These findings indicate that D-serine catabolism, though it may play role(s) during urinary tract infection, does not affect the ability of uropathogenic <i>E</i>. <i>coli</i> to colonize the murine urinary tract.</p></div

CFT073 Δ<i>dsdA</i> colonizes the murine urinary tract indistinguishably from wild type.

<p>(A) CFT073 Δ<i>dsdA</i> and CFT073 Δ<i>lacZYA</i> were co-inoculated at a 1:1 ratio into CBA/J mice (n = 17). Bacteria from bladder and kidney homogenates were enumerated on MacConkey’s medium plus lactose. Lines are drawn at the geometric mean relative competitive index (RCI). Nine mice had no detectable bacteria in their kidneys. Statistical Significance was assessed by a Wilcoxon signed-rank test relative to a hypothetical RCI of 1. (B) Manipulations were carried out as described for panel A except that single strains were used (n = 16 for each). Two mice infected with CFT073 had no detectable bacteria in their kidneys and three mice infected with CFT073 Δ<i>dsdA</i> had no detectable bacteria in their kidneys. Lines are drawn at the geometric mean CFU/organ. Statistical significance was assessed by the Mann-Whitney <i>U</i> test.</p

<i>dsdA</i>^- strains have a growth defect in human urine.

<p>Bacteria were grown overnight in pooled, filter sterilized human urine. Bacteria were washed 2x in phosphate buffered saline and the OD₆₀₀ of each cell suspension was normalized to OD₆₀₀ = 1.5. Bacteria were then inoculated into fresh, pre-warmed urine to OD₆₀₀ = 0.03. Bacteria were allowed to grow for 12 hours. OD₆₀₀ (Panel A) and viable counts (Panel B) were measured at the time points indicated above. <i>dsdA</i>⁺ strains used in this analysis are CFT073, CFT073 <i>gyrA</i>_S83L, and CFT073 <i>rpoS</i>_am, and <i>dsdA</i>^- strains are CFT073 Δ<i>dsdA</i>, CFT073 <i>dsdA</i>_Δ445bp<i>gyrA</i>_S83L, and CFT073 Δ<i>dsdA rpoS</i>_am. Data points represent mean OD₆₀₀ and mean viable counts for each strain type where applicable and error bars are drawn to represent standard error of the mean (±SEM).</p

Gut Microbiota-Produced Succinate Promotes C.&nbsp;difficile Infection after Antibiotic Treatment or Motility Disturbance

Clostridium difficile is a leading cause of antibiotic-associated diarrhea. The mechanisms underlying C.&nbsp;difficile expansion after microbiota disturbance are just emerging. We assessed the gene expression profile of C.&nbsp;difficile within the intestine of gnotobiotic mice to identify genes regulated in response to either dietary or microbiota compositional changes. In the presence of the gut symbiont Bacteroides thetaiotaomicron, C.&nbsp;difficile induces a pathway that metabolizes the microbiota fermentation end-product succinate to butyrate. The low concentration of succinate present in the microbiota of conventional mice is transiently elevated upon antibiotic treatment or chemically induced intestinal motility disturbance, and C.&nbsp;difficile exploits this succinate spike to expand in the perturbed intestine. A C.&nbsp;difficile mutant compromised in succinate utilization is at a competitive disadvantage during these perturbations. Understanding the metabolic mechanisms involved in microbiota-C.&nbsp;difficile interactions may help to identify approaches for the treatment and prevention of C.&nbsp;difficile-associated diseases

High-throughput low-cost nl-qPCR for enteropathogen detection: A proof-of-concept among hospitalized patients in Bangladesh. S1 Dataset

Data collected from 961 patients with results of enteropathogen detection by Nanoliter-qPCR. This dataset supports the PLOS ONE paper, "High-throughput low-cost nl-qPCR for enteropathogen detection: A proof-of-concept among hospitalized patients in Bangladesh.

A gut bacterial pathway metabolizes aromatic amino acids into nine circulating metabolites

The human gut microbiota produces dozens of metabolites that accumulate in the bloodstream, where they can have systemic effects on the host. Although these small molecules commonly reach concentrations similar to those achieved by pharmaceutical agents, remarkably little is known about the microbial metabolic pathways that produce them. Here we use a combination of genetics and metabolic profiling to characterize a pathway from the gut symbiont Clostridium sporogenes that generates aromatic amino acid metabolites. Our results reveal that this pathway produces twelve compounds, nine of which are known to accumulate in host serum. All three aromatic amino acids (tryptophan, phenylalanine and tyrosine) serve as substrates for the pathway, and it involves branching and alternative reductases for specific intermediates. By genetically manipulating C. sporogenes, we modulate serum levels of these metabolites in gnotobiotic mice, and show that in turn this affects intestinal permeability and systemic immunity. This work has the potential to provide the basis of a systematic effort to engineer the molecular output of the gut bacterial community

Identification of Widespread Antibiotic Exposure in Patients with Cholera Correlates with Clinically Relevant Microbiota Changes

Background: A first step to combating antimicrobial resistance in enteric pathogens is to establish an objective assessment of antibiotic exposure. Our goal was to develop and evaluate a liquid chromatography-ion trap mass spectrometry (LC/MS) method to determine antibiotic exposure in patients with cholera. Methods: A priority list for targeted LC/MS was generated from medication-vendor surveys in Bangladesh. A study of patients with and those without cholera was conducted to collect and analyze paired urine and stool samples. Results: Among 845 patients, 11% (90) were Vibrio cholerae positive; among these 90 patients, analysis of stool specimens revealed ≥1 antibiotic in 86% and ≥2 antibiotics in 52%. Among 44 patients with cholera and paired urine and stool specimens, ≥1 antibiotic was detected in 98% and ≥2 antibiotics were detected in 84%, despite 55% self-reporting medication use. Compared with LC/MS, a low-cost antimicrobial detection bioassay lacked a sufficient negative predictive value (10%; 95% confidence interval, 6%-16%). Detection of guideline-recommended antibiotics in stool specimens did (for azithromycin; P =. 040) and did not (for ciprofloxacin) correlate with V. cholerae suppression. A nonrecommended antibiotic (metronidazole) was associated with decreases in anaerobes (ie, Prevotella organisms; P <. 001). Conclusion: These findings suggest that there may be no true negative control group when attempting to account for antibiotic exposure in settings like those in this study