Epidemiology and associated microbiota changes in deployed military personnel at high risk of traveler's diarrhea.

Travelers' diarrhea (TD) is the most prevalent illness encountered by deployed military personnel and has a major impact on military operations, from reduced job performance to lost duty days. Frequently, the etiology of TD is unknown and, with underreporting of cases, it is difficult to accurately assess its impact. An increasing number of ailments include an altered or aberrant gut microbiome. To better understand the relationships between long-term deployments and TD, we studied military personnel during two nine-month deployment cycles in 2015-2016 to Honduras. To collect data on the prevalence of diarrhea and impact on duty, a total of 1173 personnel completed questionnaires at the end of their deployment. 56.7% reported reduced performance and 21.1% reported lost duty days. We conducted a passive surveillance study of all cases of diarrhea reporting to the medical unit with 152 total cases and a similar pattern of etiology. Enteroaggregative E. coli (EAEC, 52/152), enterotoxigenic E. coli (ETEC, 50/152), and enteropathogenic E. coli (EPEC, 35/152) were the most prevalent pathogens detected. An active longitudinal surveillance of 67 subjects also identified diarrheagenic E. coli as the primary etiology (7/16 EPEC, 7/16 EAEC, and 6/16 ETEC). Eleven subjects were recruited into a nested longitudinal substudy to examine gut microbiome changes associated with deployment. A 16S rRNA amplicon survey of fecal samples showed differentially abundant baseline taxa for subjects who contracted TD versus those who did not, as well as detection of taxa positively associated with self-reported gastrointestinal distress. Disrupted microbiota was also qualitatively observable for weeks preceding and following the incidents of TD. These findings illustrate the complex etiology of diarrhea amongst military personnel in deployed settings and its impacts on job performance. Potential factors of resistance or susceptibility can provide a foundation for future clinical trials to evaluate prevention and treatment strategies.

Impact of insertion sequences on convergent evolution of Shigella species.

Shigella species are specialised lineages of Escherichia coli that have converged to become human-adapted and cause dysentery by invading human gut epithelial cells. Most studies of Shigella evolution have been restricted to comparisons of single representatives of each species; and population genomic studies of individual Shigella species have focused on genomic variation caused by single nucleotide variants and ignored the contribution of insertion sequences (IS) which are highly prevalent in Shigella genomes. Here, we investigate the distribution and evolutionary dynamics of IS within populations of Shigella dysenteriae Sd1, Shigella sonnei and Shigella flexneri. We find that five IS (IS1, IS2, IS4, IS600 and IS911) have undergone expansion in all Shigella species, creating substantial strain-to-strain variation within each population and contributing to convergent patterns of functional gene loss within and between species. We find that IS expansion and genome degradation are most advanced in S. dysenteriae and least advanced in S. sonnei; and using genome-scale models of metabolism we show that Shigella species display convergent loss of core E. coli metabolic capabilities, with S. sonnei and S. flexneri following a similar trajectory of metabolic streamlining to that of S. dysenteriae. This study highlights the importance of IS to the evolution of Shigella and provides a framework for the investigation of IS dynamics and metabolic reduction in other bacterial species.

Antimicrobial resistance profiles of diarrhoeagenic Escherichia coli isolated from travellers returning to the UK, 2015-2017.

Introduction. Diarrhoeagenic Escherichia coli (DEC) are difficult to distinguish from non-pathogenic commensal E. coli using traditional culture methods. The implementation of PCR targeting specific virulence genes characteristic of the five DEC pathotypes, has improved the detection of DEC in faecal specimens from patients with symptoms of gastrointestinal disease.Aim. Antimicrobial resistance (AMR) profiles of 660 strains of DEC isolated between 2015 and 2017 from UK travellers reporting symptoms of gastrointestinal disease were reviewed to look for evidence of emerging AMR associated with travellers' diarrhoea.Methodology. All isolates of DEC were sequenced, and sequence type, serotype, pathotype markers and AMR profiles were derived from the genome data.Results. A travel history was provided for 54.1 % (357/660) of cases, of which 77.0 % (275/357) reported travel outside the UK within 7 days of onset of symptoms, and 23.0 % (82/357) reported no travel in that time frame. Of the 660 strains of DEC in this study, 265 (40.2 %) samples were identified as EAEC, 48 (7.3 %) as EIEC, 61 (9.2 %) were ETEC and 286 (43.3 %) were EPEC. EPEC caused the highest percentage of infections in children (40.6 %) whilst the highest proportion of cases reporting recent travel were infected with ETEC (86.1 %). There were 390/660 (59.0 %) isolates resistant to at least one antimicrobial on the panel tested (EIEC, 81.3 %; ETEC, n=65.6 %; EAEC, n=73.2 %; EPEC, 40.9 %) and 265/660 (40.2 %) were multidrug-resistant (EIEC, 33.3 %; ETEC, 32.8 %; EAEC, 56.2 %; EPEC, 28.0 %). Genes conferring resistance to the beta-lactams and fluroquinolones were highest in the EAEC pathotype, 56.6 and 60.7%, respectively.Conclusions. Increasing MDR, along with resistance to the fluroquinolones and the third-generation cephalosporins, in DEC causing travellers' diarrhoea provides further evidence for the need to restrict the use of antimicrobial agents and continuous monitoring.

A facile one-step method for cell lysis and DNA extraction of waterborne pathogens using a microchip.

Globally, waterborne organisms are the primary causative agents for the transmission of various forms of diarrheal diseases. For accurate diagnosis, molecular tools have gained considerable attention in the recent past. Molecular tools require DNA as the starting material for diagnosis, and hence, a prerequisite is the quality and integrity of DNA. To obtain high quality DNA rapidly, we have fabricated a microchip in poly(dimethyl siloxane) (PDMS) by soft lithography process. The microchip facilitated in-flow coating of chitosan on the magnetic nanoparticles, which under external mechanical vibration caused cell lysis and released DNA in the supernatant. The released DNA was captured by the nanoparticles owing to its positive charge (chitosan coating). The magnetic nanoparticle-DNA complex was then isolated from the in-flow matrix using permanent magnet, Further, removal of the cell debris, proteins, and carbohydrates was done using wash buffer. DNA extracted using the microchip was pure with absorbance (260/280) ratio of 1.77±0.04, as compared to 1.79±0.03 obtained by TRIzol method. The complete isolation of the DNA using the microchip took ~ 15min as against>2h with a TRIzol method. Six gram-negative waterborne pathogens were used to demonstrate the efficacy of the microchip based DNA extraction process. The integrity of the isolated DNA was assessed by amplifying the 16S rRNA gene using Com1 and Com2 universal primers. The presence of a band at 407bp on gel electrophoresis confirmed the amplified product. Further, the gel image was used for quantification of the amplified product using ImageJ software. Higher regression values obtained using microchip confirmed better quality and integrity of the extracted DNA as opposed to the conventional method. The lower (<2%) relative standard deviation values obtained from the data suggested that the microchip process was reproducible. The quality and integrity of the obtained DNA proved the simplicity, rapidity, and sensitivity of the microchip-assisted DNA extraction process.

Comparison of Brachyspira hyodysenteriae Isolates Recovered from Pigs in Apparently Healthy Multiplier Herds with Isolates from Herds with Swine Dysentery.

Swine dysentery (SD) is a mucohaemorrhagic colitis of grower/finisher pigs classically resulting from infection by the anaerobic intestinal spirochaete Brachyspira hyodysenteriae. This study aimed to determine whether B. hyodysenteriae isolates from pigs in three healthy German multiplier herds supplying gilts to other farms differed from isolates from nine German production herds with SD. Isolates were subjected to whole genomic sequencing, and in silico multilocus sequence typing showed that those from the three multiplier herds were of previously undescribed sequence types (ST132, ST133 and ST134), with all isolates from the same herd having the same ST. All isolates were examined for the presence of 332 genes encoding predicted virulence or virulence lifestyle associated factors, and these were well conserved. Isolates from one multiplier herd were atypical in being weakly haemolytic: they had 10 amino acid substitutions in the haemolysin III protein and five in the haemolysin activation protein compared to reference strain WA1, and had a disruption in the promoter site of the hlyA gene. These changes likely contribute to the weakly haemolytic phenotype and putative lack of virulence. These same isolates also had nine base pair insertions in the iron metabolism genes bitB and bitC and lacked five of six plasmid genes that previously have been associated with colonisation. Other overall differences between isolates from the different herds were in genes from three of five outer membrane proteins, which were not found in all the isolates, and in members of a block of six plasmid genes. Isolates from three herds with SD had all six plasmid genes, while isolates lacking some of these genes were found in the three healthy herds-but also in isolates from six herds with SD. Other differences in genes of unknown function or in gene expression may contribute to variation in virulence; alternatively, superior husbandry and better general health may have made pigs in the two multiplier herds colonised by "typical" strongly haemolytic isolates less susceptible to disease expression.

Genetic analyses of GII.17 norovirus strains in diarrheal disease outbreaks from December 2014 to March 2015 in Japan reveal a novel polymerase sequence and amino acid substitutions in the capsid region.

A novel GII.P17-GII.17 variant norovirus emerged as a major cause of norovirus outbreaks from December 2014 to March 2015 in Japan. Named Hu/GII/JP/2014/GII.P17-GII.17, this variant has a newly identified GII.P17 type RNA-dependent RNA polymerase, while the capsid sequence displays amino acid substitutions around histo-blood group antigen (HBGA) binding sites. Several variants caused by mutations in the capsid region have previously been observed in the GII.4 genotype. Monitoring the GII.17 variant's geographical spread and evolution is important.

NMR-monitored titration of acid-stress bacterial chaperone HdeA reveals that Asp and Glu charge neutralization produces a loosened dimer structure in preparation for protein unfolding and chaperone activation.

HdeA is a periplasmic chaperone found in several gram-negative pathogenic bacteria that are linked to millions of cases of dysentery per year worldwide. After the protein becomes activated at low pH, it can bind to other periplasmic proteins, protecting them from aggregation when the bacteria travel through the stomach on their way to colonize the intestines. It has been argued that one of the major driving forces for HdeA activation is the protonation of aspartate and glutamate side chains. The goal for this study, therefore, was to investigate, at the atomic level, the structural impact of this charge neutralization on HdeA during the transition from near-neutral conditions to pH 3.0, in preparation for unfolding and activation of its chaperone capabilities. NMR spectroscopy was used to measure pKa values of Asp and Glu residues and monitor chemical shift changes. Measurements of R2/R1 ratios from relaxation experiments confirm that the protein maintains its dimer structure between pH 6.0 and 3.0. However, calculated correlation times and changes in amide protection from hydrogen/deuterium exchange experiments provide evidence for a loosening of the tertiary and quaternary structures of HdeA; in particular, the data indicate that the dimer structure becomes progressively weakened as the pH decreases. Taken together, these results provide insight into the process by which HdeA is primed to unfold and carry out its chaperone duties below pH 3.0, and it also demonstrates that neutralization of aspartate and glutamate residues is not likely to be the sole trigger for HdeA dissociation and unfolding.

Drug resistant Shigella flexneri in & around Dibrugarh, north-east India.

BACKGROUND & OBJECTIVES: Shigella flexneri is the most common species of Shigella causing diarrhoea and dysentery in Asia including India. Multidrug resistance in Shigella species has been reported worldwide and there is rising concern regarding development of fluoroquinolone resistance. This study was undertaken to find out the resistance pattern of Sh. flexneri, the commonest shigella isolated in Dibrugarh, north east India, including detection of fluoroquinolone resistance and extended spectrum beta lactamases. METHODS: Stool samples collected from patients of diarrhoea and dysentery were tested for bacterial enteropathogens. Strains of Shigella species were confirmed by biochemical tests. Speciation was done using commercially available polyvalent antiserum. Antimicrobial susceptibility test was performed by Kirby Bauer disc diffusion method against 18 different antibiotics. Extended spectrum beta lactamase (ESBL) detection was done by disc approximation test as well as combination disc method and minimum inhibitory concentrations (MIC) of different antibiotics were also measured. RESULTS: Multidrug resistance in Sh. flexneri was found to be common (90.2%) and the commonest phenotypic multi-drug resistance profile was ampicillin-tetracycline-co-trimoxazole-nalidixic acid. High resistance to nalidixic acid was detected in 90.3 per cent isolates (MIC >240 µg/ml) and ciprofloxacin resistance was seen emerging in this region (11.2%, MIC >4 µg/ml). Present of ESBL was phenotypically confirmed in two cases. Besides the fluoroquinolones, chloramphenicol, piperacillin-tazobactum and the third generation cephalosporins were effective in 87-100 per cent of the isolates. INTERPRETATION & CONCLUSIONS: Our study showed high resistance (MIC >240 µg/ml) against nalidixic acid in Sh. flexneri isolates. Ciprofloxacin resistance is also emerging in this region. Shigellosis due to ESBL carrying Shigella can become a serious threat to public health. Guidelines for therapy should be monitored and modified based on regional reports of resistance to antimicrobial agents.

Identification of cellular calcium binding protein calmodulin as a regulator of rotavirus A infection during comparative proteomic study.

Rotavirus (RV) being the major diarrhoegenic virus causes around 527000 children death (<5 years age) worldwide. In cellular environment, viruses constantly adapt and modulate to survive and replicate while the host cell also responds to combat the situation and this results in the differential regulation of cellular proteins. To identify the virus induced differential expression of proteins, 2D-DIGE (Two-dimensional Difference Gel Electrophoresis) based proteomics was used. For this, HT-29 cells were infected with RV strain SA11 for 0 hours, 3 hours and 9 hours post infection (hpi), differentially expressed spots were excised from the gel and identified using MALDI-TOF/TOF mass spectrometry. 2D-DIGE based proteomics study identified 32 differentially modulated proteins, of which 22 were unique. Some of these were validated in HT-29 cell line and in BALB/c mice model. One of the modulated cellular proteins, calmodulin (CaM) was found to directly interact with RV protein VP6 in the presence of Ca(2+). Ca(2+)-CaM/VP6 interaction positively regulates RV propagation since both CaM inhibitor (W-7) and Ca(2+) chelator (BAPTA-AM) resulted in decreased viral titers. This study not only identifies differentially modulated cellular proteins upon infection with rotavirus in 2D-DIGE but also confirmed positive engagement of cellular Ca(2+)/CaM during viral pathogenesis.

Diarrhea as a cause of mortality in a mouse model of infectious colitis.

BACKGROUND: Comparative characterization of genome-wide transcriptional changes during infection can help elucidate the mechanisms underlying host susceptibility. In this study, transcriptional profiling of the mouse colon was carried out in two cognate lines of mice that differ in their response to Citrobacter rodentium infection; susceptible inbred FVB/N and resistant outbred Swiss Webster mice. Gene expression in the distal colon was determined prior to infection, and at four and nine days post-inoculation using a whole mouse genome Affymetrix array. RESULTS: Computational analysis identified 462 probe sets more than 2-fold differentially expressed between uninoculated resistant and susceptible mice. In response to C. rodentium infection, 5,123 probe sets were differentially expressed in one or both lines of mice. Microarray data were validated by quantitative real-time RT-PCR for 35 selected genes and were found to have a 94% concordance rate. Transcripts represented by 1,547 probe sets were differentially expressed between susceptible and resistant mice regardless of infection status, a host effect. Genes associated with transport were over-represented to a greater extent than even immune response-related genes. Electrolyte analysis revealed reduction in serum levels of chloride and sodium in susceptible animals. CONCLUSION: The results support the hypothesis that mortality in C. rodentium-infected susceptible mice is associated with impaired intestinal ion transport and development of fatal fluid loss and dehydration. These studies contribute to our understanding of the pathogenesis of C. rodentium and suggest novel strategies for the prevention and treatment of diarrhea associated with intestinal bacterial infections.

The shf gene of a Shigella flexneri homologue on the virulent plasmid pAA2 of enteroaggregative Escherichia coli 042 is required for firm biofilm formation.

Enteroaggregative Escherichia coli (EAEC) is an increasingly important cause of diarrhea in both developing and industrialized countries, and is characterized by strong biofilm formation on the intestinal mucosa. Sequencing of the virulent plasmid pAA2 of the prototype EAEC 042 revealed a cluster of three open reading frames (ORFs; shf, capU, and virK) ca. 93% identical to a similar cluster located in Shigella flexneri. The function of the first ORF Shf protein is not known, but the closest well-characterized homologue is the IcaB protein of Staphylococcus epidermidis, which plays a crucial role in exopolysaccharide modification in bacterial biofilm formation. To investigate the role of this cluster in the virulence of EAEC, we mutated three genes at this locus. All the mutants maintained the aggregative phenotype in the liquid phase. However, the insertional mutant of shf formed a less abundant biofilm in a microtiter plate assay than did the wild type, while the capU mutant and the virK mutant did not. The complementation of the shf mutant with this cluster restored the thick biofilm similar to that of the wild type. The shf transcriptional level decreased in the transcriptional regulator aggR mutant and was restored when the mutant was complemented with aggR. These results suggest that the shf gene is required for the firm biofilm formation of EAEC 042, and transcription of the shf gene is dependent on AggR.

[Pathogenic diagnosis of an unexplained epidemic event of infectious diarrhea].

OBJECTIVE: To determine the pathogen of an unexplained epidemic event of infectious diarrhea by laboratory diagnosis of suspected cases samples. METHODS: 28 samples from 28 suspected cases (22 fecal samples, 3 vomitus samples, 3 anus swab samples) were tested for Norovirus by RT-PCR. Sequencing and phylogenetic analysis were acomplished of 5 positive samples. RESULTS: 160 of 5694 population were ill with an attack rate of 2.81%. The peak period was 7-9, March. 14 of 28 samples were tested Norovirus positive.Sequencing and phylogenetic analysis showed Norovirus type GII/4 was the causative agent and it had highest identity (97. 9%) with epidemic strain 2006b. CONCLUSION: The epidemic event ofinfectious diarrhea were caused by GII/4 Norovirus strains.

Rapid and specific identification of 5 human pathogenic Vibrio species by multiplex polymerase chain reaction targeted to dnaJ gene.

A multiplex polymerase chain reaction (PCR) method, specifically designed for application in routine diagnostic laboratories, was developed for identifying 5 human pathogen Vibrio species: Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio mimicus, and Vibrio alginolyticus. This assay directed toward the dnaJ gene was tested on a total of 355 strains representing 13 Vibrio species and 17 non-Vibrio species. Specific PCR fragments were produced in isolates belonging to the 5 target species and were absent from all strains other than these 5 species and non-Vibrio strains, indicating a high specificity of this multiplex PCR. The multiplex PCR for the detection of Vibrio pathogens in clinical specimens was experimentally applied to spiked stool samples. Only 1 specific amplicon was observed, corresponding to the pathogen spiked into the stool sample. The detection limitation was 10(5) to 10(6) cells per milliliter stool. Our data showed that this method represented a robust tool for the specific and rapid detection of the 5 major pathogenic Vibrio species.

A method for fast and simple detection of major diarrhoeagenic Escherichia coli in the routine diagnostic laboratory.

A multiplex PCR was developed for the detection of the following genes characteristic of diarrhoeagenic Escherichia coli (DEC): verocytotoxins 1 (vtx1) and 2 (vtx2), characteristic of verocytotoxin-producing E. coli (VTEC); intimin (eae), found in enteropathogenic E. coli (EPEC), attaching and effacing E. coli and VTEC; heat-stable enterotoxin (estA) and heat-labile enterotoxin (eltA), characteristic of enterotoxigenic E. coli (ETEC); and invasive plasmid antigen (ipaH), characteristic of enteroinvasive E. coli (EIEC) and Shigella spp. The method allowed the simultaneous identification of all six genes in one reaction, and included a 16S rDNA internal PCR control. When applied to pure cultures from a reference strain collection, all virulence genes in 124 different DEC strains and 15 Shigella spp. were identified correctly, and there were no cross-reactions with 13 non-E. coli species. The detection limit of the method was 10(2)-10(3) DEC CFU/PCR in the presence of 10(6) non-target cells. When the multiplex PCR was tested with colonies from plate cultures of clinical stool samples, it was a faster, more sensitive, less expensive and less laborious diagnostic procedure than DNA hybridisation. When used with DNA purified from spiked stool samples (by two different commercial kits), the method had a detection limit of 10(6) CFU/mL stool sample.

Evaluating candidate agents of selective pressure for cystic fibrosis.

Cystic fibrosis is the most common lethal single-gene mutation in people of European descent, with a carrier frequency upwards of 2%. Based upon molecular research, resistances in the heterozygote to cholera and typhoid fever have been proposed to explain the persistence of the mutation. Using a population genetic model parameterized with historical demographic and epidemiological data, we show that neither cholera nor typhoid fever provided enough historical selective pressure to produce the modern incidence of cystic fibrosis. However, we demonstrate that the European tuberculosis pandemic beginning in the seventeenth century would have provided sufficient historical, geographically appropriate selective pressure under conservative assumptions. Tuberculosis has been underappreciated as a possible selective agent in producing cystic fibrosis but has clinical, molecular and now historical, geographical and epidemiological support. Implications for the future trajectory of cystic fibrosis are discussed. Our result supports the importance of novel investigations into the role of arylsulphatase B deficiency in cystic fibrosis and tuberculosis.

[Intestinal infection and irritable bowel syndrome].

OBJECTIVE: To determine whether intestinal infection plays a role on the pathogenesis of irritable bowel syndrome (IBS). METHODS: 295 patients who had no previous history of functional bowel disorder had received treatment for dysentery (n = 235) or for acute bowel infection at the hospital between April-October, 1998, were followed up for 1 - 2 years and evaluated for their subsequent bowel habits. A cohort study of 243 subjects using their siblings, husbands or wives who did not have dysentery or acute bowel infection at the same period was taken as control. Furthermore, the contents of mRNAs of IL-1alpha, IL-1beta and IL-1ra in the mucosa at the terminal ileum and recto-sigmoid junctions were determined and compared using RT-PCR method in 30 IBS patients and 12 controls. RESULTS: (1) Sixty-six (22.4%) patients were reported to have functional bowel disturbance, and 24 (8.1% total and 10.2% among cases of dysentery) developed IBS in the study group, whereas, only 7.4% had altered bowel habit and 0.8% had IBS in the control group (P < 0.01). (2) The risk of having functional bowel disturbance was higher in patients who suffered from a longer duration (> 8 d, OR = 3.5) of dysentery. (3) The IL-1beta mRNA level in the mucosa of terminal ileum and recto-sigmoid junction of IBS patients with dysentery was higher than that of controls and IBS patients without dysentery (P < 0.01). CONCLUSION: Intestinal infection plays a role on the pathogenesis of IBS through some immunological factors.