Increased sensitivity of ETEC detection in stool cultures by increasing the number of E. coli colonies tested

The cause of infection of about a third of all travelers' diarrhea patients studied is not identified. Stools of these diarrhea patients tested for known enteric pathogens are shown to be negative, and identified as pathogen negative stools. We proposed that the third of these diarrhea patients might not only include at present unknown pathogens, but also known pathogens that go undetected. Conventionally, a probability sample of five E. coli colonies are used detect enterotoxigenic E. coli (ETEC) and other diarrhea-producing E. coli from stool cultures. We compared this conventional method of using five E. coli colonies, to the use of up to twenty E. coli colonies. Testing for up to fifteen E. coli colonies detected about twice as many ETEC when compared to the detection of ETEC, testing for five E. coli colonies. When the number of E. coli colonies tested was increased from 5 to 15, the detection of ETEC increased from 19.0% to 38.8%. The sensitivity of the assay with 5 E. coli colonies was statistically significantly different to the sensitivity of the assay with 10 E. coli colonies, suggesting that for the detection of ETEC at least 10 colonies of E. coli should be tested.^

Susceptibility to travelers' diarrhea and histo-blood group antigens

Histo-blood group antigens (HBGAs) have been associated with susceptibility to enteric pathogens including noroviruses (NoVs), enterotoxigenic Escherichia coli (ETEC), Campylobacter jejuni, and Vibrio cholerae. We performed a retrospective cohort study to evaluate the relationship between traveler HBGA phenotypes and susceptibility to travelers' diarrhea (TD) and post-infectious complications. 364 travelers to Guadalajara, Mexico were followed prospectively from June 1 - September 30, 2007 and from June 7–July 28, 2008 for the development of TD and at 6 months for post-infectious irritable bowel syndrome (PIIBS). Noroviruses were detected from illness stool specimens with RT-PCR. Diarrheal stool samples were also assayed for enterotoxigenic and enteroaggregative E. coli, Salmonella species, Shigella species, Vibrio species, Campylobacter jejuni, Yersinia enterocolitica, Aeromonas species, and Plesiomonas species. Diarrheal stools were evaluated for inflammation with fecal leukocytes, mucus, and occult blood. Phenotyping for ABO and Lewis antigens with an ELISA assay and FUT2 gene PCR genotyping for secretor status were performed with saliva. 171 of 364 (47%) subjects developed TD. HBGA typing for the travelers revealed O (62.9%), A (34.6%), B (1.6%), and AB (0.8%) phenotypes. There were 7% nonsecretors and 93% secretors among the travelers. AB phenotypes were more commonly associated with Cryptosporidium species (P=0.04) and ETEC ( P=0.08) as causes of TD. AB and B phenotype individuals were more likely to experience inflammatory diarrhea, particularly mucoid diarrhea ( P=0.02). However, there were relatively few individuals with AB and B phenotypes. GI and GII NoV and Cryptosporidium species infections and PI-IBS were identified only in secretors, but these differences were not statistically significant, (P=1.00), (P=1.00), and (P=0.60), respectively. Additional studies are needed to evaluate whether AB phenotype individuals may be more susceptible to developing TD associated with Cryptosporidium species or ETEC, and whether AB and B phenotype individuals may be more likely to develop inflammatory TD. Further studies are needed to investigate whether nonsecretor travelers may be at less risk for developing infections with NoVs and Cryptosporidium species and PI-IBS.^

(Supplement) Groundwater quality in Lahore Pakistan: emphasis on Arsenic and Fluoride levels

The dataset provides detailed information on the study that was conducted in Lahore's 7 major towns. The sample was taken from 472 tubewells and analyzed for major cations and anions using APHA 2012 techniques as explained herein. Besides, E.coli determination was done to check for microbial contamination. The data includes results from PHREEQC modeling of As(III)/ As(V) species and saturation indices as well as Aquachem's computed hydrochemical water facies. The WHO (2011) and EPA standards included in Aquachem identified the parameters that where in violation. Bicarbonates dominated the groundwater types with 50.21% of the samples exceeding the EPA maximum permissible limit of 250 mg/L in drinking water. Similarly, 30.51% of the samples had TDS values greater than 500 mg/L while 85.38 % of the samples exceed 10 µg/L threshold limit value of arsenic. Also, instances of high magnesium hazard values were observed which requires constant assessment if the groundwater is used for irrigation. Higher than 50% MH values are detrimental to crops which may reduce the expected yields. The membrane filtration technique using m-Endo Agar indicated that 3.59% samples had TNC (too numerous to count) values for E.coli while 5.06% showed values higher than 0 cfu/ 100 ml acceptable value in drinking water. Any traces of E-coli in a groundwater sample indicate recent fecal contamination. Such outcomes signify presence of enteric pathogens. If the groundwater is not properly dosed with disinfectants it may cause harm to human health. It is concluded that more studies are needed and proper groundwater management implement to safeguard the lives of communities that depend solely on groundwater in the city.

The insect endosymbiont Sodalis glossinidius utilizes a type III secretion system for cell invasion

Sodalis glossinidius is a maternally transmitted secondary endosymbiont residing intracellularly in tissues of the tsetse flies, Glossina spp. In this study, we have used Tn5 mutagenesis and a negative selection procedure to derive a S. glossinidius mutant that is incapable of invading insect cells in vitro and is aposymbiotic when microinjected into tsetse. This mutant strain harbors Tn5 integrated into a chromosomal gene sharing high sequence identity with a type III secretion system invasion gene (invC) previously identified in Salmonella enterica. With the use of degenerate PCR, we have amplified a further six Sodalis inv/spa genes sharing high sequence identity with type III secretion system genes encoded by Salmonella pathogenicity island 1. Phylogenetic reconstructions based on the inv/spa genes of Sodalis and other members of the family Enterobacteriaceae have consistently identified a well-supported clade containing Sodalis and the enteric pathogens Shigella and Salmonella. These results suggest that Sodalis may have evolved from an ancestor with a parasitic intracellular lifestyle, possibly a latter-day entomopathogen. These observations lend credence to a hypothesis suggesting that vertically transmitted mutualistic endosymbionts evolve from horizontally transmitted parasites through a parasitism–mutualism continuum.

Identification of a pathogenicity island required for Salmonella survival in host cells.

We have identified a region unique to the Salmonella typhimurium chromosome that is essential for virulence in mice. This region harbors at least three genes: two (spiA and spiB) encode products that are similar to proteins found in type III secretion systems, and a third (spiR) encodes a putative regulator. A strain with a mutation in spiA was unable to survive within macrophages but displayed wild-type levels of epithelial cell invasion. The culture supernatants of the spi mutants lacked a modified form of flagellin, which was present in the supernatant of the wild-type strain. This suggests that the Spi secretory apparatus exports a protease, or a protein that can alter the activity of a secreted protease. The "pathogenicity island" harboring the spi genes may encode the virulence determinants that set Salmonella apart from other enteric pathogens.

Relationship between evolutionary rate and cellular location among the Inv/Spa invasion proteins of Salmonella enterica.

For 21 strains of Salmonella enterica, nucleotide sequences were obtained for three invasion genes, spaO, spaP, and spaQ, of the chromosomal inv/spa complex, the products of which form a protein export system required for entry of the bacteria into nonphagocytic host cells. These genes are present in all eight subspecies of the salmonellae, and homologues occur in a variety of other bacteria, including the enteric pathogens Shigella and Yersinia, in which they are plasmid borne. Evolutionary diversification of the invasion genes among the subspecies of S. enterica has been generally similar in pattern and average rate to that of housekeeping genes. However, the range of variation in evolutionary rate among the invasion genes is unusually large, and there is a relationship between the evolutionary rate and cellular location of the invasion proteins, possibly reflecting diversifying selection on exported proteins in adaptation to variable host factors in extracellular environments. The SpaO protein, which is hypervariable in S. enterica and exhibits only 24% sequence identity with its homologues in Shigella and Yersinia, is secreted. In contrast, the membrane-associated proteins SpaP, SpaQ, and InvA are weakly polymorphic and have > 60% sequence identity with the corresponding proteins of other enteric bacteria. Acquisition of the inv/spa genes may have been a key event in the evolution of the salmonellae as pathogens, following which the invention of flagellar phase shifting facilitated niche expansion to include warm-blooded vertebrates.

Host and Environmental Correlates of Multi-Drug Resistance in Kenyan Children with Acute Bacterial Diarrhea

Thesis (Master's)--University of Washington, 2016-06

Replication of Norovirus in cell culture reveals a tropism for dendritic cells and macrophages.

Noroviruses are understudied because these important enteric pathogens have not been cultured to date. We found that the norovirus murine norovirus 1 (MNV-1) infects macrophage-like cells in vivo and replicates in cultured primary dendritic cells and macrophages. MNV-1 growth was inhibited by the interferon-alphabeta receptor and STAT-1, and was associated with extensive rearrangements of intracellular membranes. An amino acid substitution in the capsid protein of serially passaged MNV-1 was associated with virulence attenuation in vivo. This is the first report of replication of a norovirus in cell culture. The capacity of MNV-1 to replicate in a STAT-1-regulated fashion and the unexpected tropism of a norovirus for cells of the hematopoietic lineage provide important insights into norovirus biology.

Efficient and Durable Vaccine against Intimin β of Diarrheagenic E. Coli Induced by Clay Nanoparticles

Improved strategies are urgently required to control infections with enterohemorrhagic Escherichia coli and enteropathogenic E. coli, two dominant zoonotic enteric pathogens responsible for a wide spectrum of illnesses as well as deaths of human being, with tremendous financial cost worldwide. The present study investigates the capacity of two clay nanoparticles (NPs) with opposite surface charges, namely synthetic layered double hydroxide (LDH) and hectorite (HEC) NPs as adjuvants to promote strong immune responses against the infections. Here both LDH and HEC NPs are showed to be able to carry an appreciable amount of Intimin β (1.1 and 4.4 mg per mg clay nanomaterials, respectively) and significantly facilitate antigen uptake by antigen-presenting cells. Remarkably, these clay NPs induce strong antibody and cell-mediated immune responses, which are much higher than that by the potent adjuvant, QuilA. Furthermore, these strong immune responses are well maintained for at least four months in the mouse model, during which there are no changes in histopathology of the animal organs. Collectively these data demonstrate the suitability of LDH and HEC NPs as useful adjuvants in new-generation vaccine formulations to control various infectious diseases.

Enteropatógenos y antibióticos

La gastroenteritis infecciosa continúa siendo un problema de salud pública. La etiología bacteriana es la responsable de la mayoría de los casos graves. En nuestro país, Campylobacter y Salmonella son los géneros bacterianos más prevalentes, mientras que Yersinia y Shigella son mucho menos frecuentes. La mayoría de los casos suelen ser autolimitados y, en general, el tratamiento antibiótico no está indicado, salvo en pacientes con factores de riesgo de infección grave y en shigelosis. Ciprofloxacino, cefalosporinas de tercera generación, azitromicina, ampicilina, cotrimoxazol y doxiciclina son los fármacos más recomendados. El patrón de sensibilidad de las diferentes bacterias determina la elección del tratamiento antibiótico más adecuado. El objetivo de esta revisión es analizar la situación, las novedades y la evolución de la resistencia y la multirresistencia en estos 4 enteropatógenos.

Viability of enteric bacterial pathogens in transport media

Bacterial pathogens such as enterotoxigenic Escherichia coli, Salmonella, and Campylobacter spp. are associated with up to 80% of diarrheal illness to travelers from developed countries to developing countries. In order to study acute gastrointestinal diseases, researchers from developed countries such as the United States rely on transporting clinical specimens from the developing countries to laboratories in the U.S. in transport media systems. There are few commercially available transport media systems cited in the literature or designated by transport system manufacturers for the transport of enteric bacteria. Therefore a laboratory-based study was conducted to assess three commercial available transport media systems, two gel swabs and one liquid vial, to determine the most appropriate for the maintenance and recovery of common enteric bacterial pathogens. A total of 13 bacterial enteropathogens were recovered from 25°C and 4°C storage temperatures at time points up to 21 days. The results demonstrated that the gel swab and liquid vial transport systems performed similarly for all isolates at both temperatures. All three transport media systems struggled to maintain the isolates at recoverable concentrations when stored at 4°C and it is recommended that isolates be stored at 25°C in transport media systems. Lastly, swab transport systems are recommend for transport since they are small and easy to pack, resist leakage, and are less expensive than similarly performing liquid vial transport media systems.^

Acute gastroenteritis and enteric viruses in hospitalised children in southern Brazil: aetiology, seasonality and clinical outcomes

Viral acute gastroenteritis (AG) is a significant cause of hospitalisation in children younger than five years. Group A rotavirus (RVA) is responsible for 30% of these cases. Following the introduction of RVA immunisation in Brazil in 2006, a decreased circulation of this virus has been observed. However, AG remains an important cause of hospitalisation of paediatric patients and only limited data are available regarding the role of other enteric viruses in these cases. We conducted a prospective study of paediatric patients hospitalised for AG. Stool samples were collected to investigate human adenovirus (HAdV), RVA, norovirus (NoV) and astrovirus (AstV). NoV typing was performed by nucleotide sequencing and phylogenetic analysis. From the 225 samples tested, 60 (26%) were positive for at least one viral agent. HAdV, NoV, RVA and AstV were detected in 16%, 8%, 6% and 0% of the samples, respectively. Mixed infections were found in nine patients: HAdV/RVA (5), HAdV/NoV (3) and HAdV/NoV/RVA (1). The frequency of fever and lymphocytosis was significantly higher in virus-infected patients. Phylogenetic analysis of NoV indicated that all of these viruses belonged to genotype GII.4. The significant frequency of these pathogens in patients with AG highlights the need to routinely implement laboratory investigations.

Gut physiology mediates a trade-off between adaptation to malnutrition and susceptibility to food-borne pathogens.

The animal gut plays a central role in tackling two common ecological challenges, nutrient shortage and food-borne parasites, the former by efficient digestion and nutrient absorption, the latter by acting as an immune organ and a barrier. It remains unknown whether these functions can be independently optimised by evolution, or whether they interfere with each other. We report that Drosophila melanogaster populations adapted during 160 generations of experimental evolution to chronic larval malnutrition became more susceptible to intestinal infection with the opportunistic bacterial pathogen Pseudomonas entomophila. However, they do not show suppressed immune response or higher bacterial loads. Rather, their increased susceptibility to P. entomophila is largely mediated by an elevated predisposition to loss of intestinal barrier integrity upon infection. These results may reflect a trade-off between the efficiency of nutrient extraction from poor food and the protective function of the gut, in particular its tolerance to pathogen-induced damage.

Therapeutic microbiology: characterization of Bifidobacterium strains for the treatment of enteric disorders in newborns.

Several studies support the use of probiotics for the treatment of minor gastrointestinal problems in infants. Positive effects on newborn colics have been evidenced after administration of Lactobacillus strains, whereas no studies have been reported regarding the use of bifidobacteria for this purpose. This work was therefore aimed at the characterization of Bifidobacterium strains capable of inhibiting the growth of pathogens typical of the infant gastro-intestinal tract and of coliforms isolated from colic newborns. Among the 46 Bifidobacterium strains considered, 16 showed high antimicrobial activity against potential pathogens; these strains were further characterized from a taxonomic point of view, for the presence and transferability of antibiotic resistances, for citotoxic effects and adhesion to non tumorigenic gut epithelium cell lines. Moreover, their ability to stimulate gut health by increasing the metabolic activity and the immune response of epithelial cells was also studied. The examination of all these features allowed to identify 3 B. breve strains and a B. longum subsp. longum strain as potential probiotics for the treatments of enteric disorders in newborns such as infantile colics. The formulation of a synbiotic product with an appropriate prebiotic fiber capable of supporting the growth of the selected Bifidobacterium strains was also considered in this study. In this respect the ability of the 4 selected Bifidobacterium strains to use as the sole carbon source and energy source different polisaccharide fibers was investigated The last phase of the work has been dedicated to the evaluation of the gut microbial diversity in newborns whose mothers has been subjected to antibiotic therapy a few hours before the delivery because of a Streptococcus type B infection. These newborns can represent a possible target for the probiotic strains selected in this work.

The evidence for groundwater as a source of enteric illness in developing countries: A systematic review of the literature

Groundwater constitutes approximately 30% of freshwater globally and serves as a source of drinking water in many regions. Groundwater sources are subject to contamination with human pathogens (viruses, bacteria and protozoa) from a variety of sources that can cause diarrhea and contribute to the devastating global burden of this disease. To attempt to describe the extent of this public health concern in developing countries, a systematic review of the evidence for groundwater microbially-contaminated at its source as risk factor for enteric illness under endemic (non-outbreak) conditions in these countries was conducted. Epidemiologic studies published in English language journals between January 2000 and January 2011, and meeting certain other criteria, were selected, resulting in eleven studies reviewed. Data were extracted on microbes detected (and their concentrations if reported) and on associations measured between microbial quality of, or consumption of, groundwater and enteric illness; other relevant findings are also reported. In groundwater samples, several studies found bacterial indicators of fecal contamination (total coliforms, fecal coliforms, fecal streptococci, enterococci and E. coli), all in a wide range of concentrations. Rotavirus and a number of enteropathogenic bacteria and parasites were found in stool samples from study subjects who had consumed groundwater, but no concentrations were reported. Consumption of groundwater was associated with increased risk of diarrhea, with odds ratios ranging from 1.9 to 6.1. However, limitations of the selected studies, especially potential confounding factors, limited the conclusions that could be drawn from them. These results support the contention that microbial contamination of groundwater reservoirs—including with human enteropathogens and from a variety of sources—is a reality in developing countries. While microbially-contaminated groundwaters pose risk for diarrhea, other factors are also important, including water treatment, water storage practices, consumption of other water sources, water quantity and access to it, sanitation and hygiene, housing conditions, and socio-economic status. Further understanding of the interrelationships between, and the relative contributions to disease risk of, the various sources of microbial contamination of groundwater can guide the allocation of resources to interventions with the greatest public health benefit. Several recommendations for future research, and for practitioners and policymakers, are presented.^