Occurrence of putative virulence-related genes, aatA, aggR and aaiC, of Enteroaggregative Escherichia coli (EAEC) among adults with travelers' diarrhea acquired in Guatemala and Mexico.

Enteroaggregative E. coli strains are important causes of diarrhea worldwide and are the second most important bacterial cause of travelers' diarrhea (TD). Pathogenicity of EAEC is not completely understood. We investigated the occurrence of putative virulence related genes (VRG), aatA, aggR and aaiC, in a nested case-control study of a cohort of US travelers >18 years of age, visited either Guatemala or Mexico. Fecal samples were collected between 2008 and 2012 from patients with TD from whom a HEp-2 cell adherent EAEC strain was identified (Cases) and from healthy subjects in the same locale without diarrhea from whom enteric pathogens were not isolated (Controls). Thirty-one subjects with acquired TD at destination was compared with 32 healthy controls. aaiC was the most expressed virulence related gene in 21 (67.7%) cases vs. 2 (6.3%) controls, (P < 0.000). aggR was found in 18 (58.1%) cases vs. 1 (3.1%) control, (P < 0.000). aatA in 9 (29.0%) cases vs. 1 (3.1%) control (P < 0.006). With genes combined, aaiC+aggR were seen together in 18 (58.1%) cases vs. 1 (3.1%) control (P < 0.000); aaiC+aatA were identified in 9 (29.0%) cases vs. 1 (3.1%) control (P < 0.006); aggR+aatA were present in 9 (29.0%) cases vs. 1 (3.1%) control, (P < 0.006). All three putative genes, aaiC+aggR+aatA were found in 9 (29.0%) cases vs. 1 (3.1%) control, (P < 0.006). The PCR products showed that aaiC, aggR, and aatA occurred in higher frequency and were more commonly associated with EAEC in cases of TD acquired in the two countries of study, as compared to controls. aaiC was found in all cases from Guatemala. Further research is needed to study geographic and host factors in EAEC-causing travelers' diarrhea.

Rifamycin SV-MMX® for treatment of travellers' diarrhea: equally effective as ciprofloxacin and not associated with the acquisition of multi-drug resistant bacteria.

Background: The novel oral antibiotic formulation Rifamycin SV-MMX®, with a targeted delivery to the distal small bowel and colon, was superior to placebo in treating travellers' diarrhea (TD) in a previous study. Thus, a study was designed to compare this poorly absorbed antibiotic with the systemic agent ciprofloxacin. Methods: In a randomized double-blind phase 3 study (ERASE), the efficacy and safety of Rifamycin SV-MMX® 400 mg twice daily (RIF-MMX) was compared with ciprofloxacin 500 mg twice daily in the oral treatment of TD. Overall, 835 international visitors to India, Guatemala or Ecuador with acute TD were randomized to receive a 3-day treatment with RIF-MMX (n = 420) or ciprofloxacin (n = 415). Primary endpoint was time to last unformed stool (TLUS), after which clinical cure was declared. Stools samples for microbiological evaluation were collected at the baseline visit and the end of treatment visit. Results: Median TLUS in the RIF-MMX group was 42.8 h versus 36.8 h in the ciprofloxacin group indicating non-inferiority of RIF-MMX to ciprofloxacin (P = 0.0035). Secondary efficacy endpoint results including clinical cure rate, treatment failure rate, requirement of rescue therapy as well as microbiological eradication rate confirmed those of the primary analysis indicating equal efficacy for both compounds. While patients receiving ciprofloxacin showed a significant increase of Extended Spectrum Beta Lactamase Producing-Escherichia coli (ESBL-E. Coli) colonization rates after 3-days treatment (6.9%), rates did not increase in patients receiving RIF-MMX (-0.3%). Both drugs were well-tolerated and safe. Conclusion: The novel multi-matrix formulation of the broad-spectrum, poorly absorbed antibiotic Rifamycin SV was found non-inferior to the systemic antibiotic ciprofloxacin in the oral treatment of non-dysenteric TD with the advantage of a lower risk of ESBL-E. Coli acquisition.

Colonization Factors in Enterotoxigenic Escherichia coli Strains in Travelers to Mexico, Guatemala, and India Compared with Children in Houston, Texas.

Enterotoxigenic Escherichia coli (ETEC) can be attributed to around 200 million diarrheal episodes and 380,000 deaths in the developing regions. Travelers' diarrhea occurs in 15-40% of travelers to developing regions with ETEC being the most important etiologic agent. This study aims to describe the distribution of enterotoxins and colonization factor (CF) profiles of ETEC isolates from stool samples of adult travelers acquiring diarrhea in Mexico, Guatemala, and India and a group of children with acute diarrhea in Houston, TX, between 2007 and 2012. The heat-labile/heat-stable (LT/ST) enterotoxins and CFs from 252 patients were determined using polymerase chain reaction assay. Among the 252 ETEC isolates, 15% were LT-only, 58% were ST-only, and 28% produced both LT and ST. The distribution of LT-only (12-15%) and ST-only (55-56%) isolates was similar between Latin American and Indian sites. The most prevalent CF was CS21, expressed in 65% of the isolates followed by CS6 (25%) and CS3 (17%). Among the international travelers, 64% of the ETEC isolates expressed CS21. CS21 was expressed in 46% of isolates from Latin America compared with 96% of isolates from India (P < 0.0001). CS21 was expressed in 85% isolates from Houston children. CS21 was increasingly found in ST-only (P = 0.003) and ST/LT (P = 0.026) ETEC compared with LT-only ETEC. High frequency of finding CS21 among recent isolates of ETEC over a wide geographic distribution warrants additional studies on this CF. Highly conserved CS21 is an important target for potential multivalent ETEC vaccines.

Targeting of rifamycin SV to the colon for treatment of travelers' diarrhea: a randomized, double-blind, placebo-controlled phase 3 study.

BACKGROUND: Rifamycin SV is under development for treatment of travelers' diarrhea (TD) in a new oral formulation, Rifamycin SV MMX (RIF-MMX; Santarus Inc., San Diego, CA, USA), which targets its delivery to the colon, making it a unique rifamycin drug. METHODS: This was a randomized, double-blind, phase 3 study of adult travelers to Mexico or Guatemala experiencing acute diarrhea. A total of 264 patients received RIF-MMX (2 × 200 mg twice daily for 3 days, n = 199) or placebo (n = 65) in a 3 : 1 ratio. The primary endpoint was the length of time between the administration of first dose of study drug and passage of the last unformed stool (TLUS; after which clinical cure was declared). Other endpoints included eradication of pathogens from the stools, pathogen minimum inhibitory concentration (MIC), and adverse events (AEs). RESULTS: TLUS was significantly shorter in the RIF-MMX group (median: 46.0 hours) compared with placebo (median: 68.0 hours; p = 0.0008) and a larger percentage of RIF-MMX treated patients (81.4%) achieved clinical cure compared with placebo patients (56.9%). TLUS was significantly shorter in the subgroups of patients with enteroaggregative, enterotoxigenic, or diffusely adherent Escherichia coli infections (p = 0.0035) with nonsignificant activity against invasive bacteria (p = 0.3804). Overall pathogen eradication rates were numerically higher in the RIF-MMX group (67.0%) compared with placebo (54.8%) but the difference did not reach significance (p = 0.0836). In vitro resistance to rifamycin SV was observed in some bacteria remaining after treatment of patients with RIF-MMX but was not associated with lower efficacy in them. AEs appeared to be more frequent with placebo (38.5%) than with RIF-MMX (29.6%). CONCLUSIONS: RIF-MMX shortened the duration of TD in patients with a broad range of pathogens and was well tolerated. The unique pharmacokinetic properties of the drug offer evidence that TD pathogens work at the level of the colon.

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.

Seroepidemiology of norovirus-associated travelers' diarrhea.

BACKGROUND: Noroviruses (NoVs) are the most common cause of epidemic gastroenteritis, responsible for at least 50% of all gastroenteritis outbreaks worldwide and were recently identified as a leading cause of travelers' diarrhea (TD) in US and European travelers to Mexico, Guatemala, and India. METHODS: Serum and diarrheic stool samples were collected from 75 US student travelers to Cuernavaca, Mexico, who developed TD. NoV RNA was detected in acute diarrheic stool samples using reverse transcription-polymerase chain reaction (RT-PCR). Serology assays were performed using GI.1 Norwalk virus (NV) and GII.4 Houston virus (HOV) virus-like particles (VLPs) to measure serum levels of immunoglobulin A (IgA) and IgG by dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA); serum IgM was measured by capture enzyme-linked immunosorbent assay (ELISA), and the 50% antibody-blocking titer (BT50 ) was determined by a carbohydrate-blocking assay. RESULTS: NoV infection was identified in 12 (16%; 9 GI-NoV and 3 GII-NoV) of 75 travelers by either RT-PCR or fourfold or more rise in antibody titer. Significantly more individuals had detectable preexisting IgA antibodies against HOV (62/75, 83%) than against NV (49/75, 65%) (p = 0.025) VLPs. A significant difference was observed between NV- and HOV-specific preexisting IgA antibody levels (p = 0.0037), IgG (p = 0.003), and BT50 (p = <0.0001). None of the NoV-infected TD travelers had BT50 > 200, a level that has been described previously as a possible correlate of protection. CONCLUSIONS: We found that GI-NoVs are commonly associated with TD cases identified in US adults traveling to Mexico, and seroprevalence rates and geometric mean antibody levels to a GI-NoV were lower than to a GII-NoV strain.

Efficacy and safety of a patch vaccine containing heat-labile toxin from Escherichia coli against travellers' diarrhoea: a phase 3, randomised, double-blind, placebo-controlled field trial in travellers from Europe to Mexico and Guatemala.

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) is a major cause of travellers' diarrhoea. We investigated the efficacy and safety of a skin-patch vaccine containing the pathogen's heat-labile toxin (LT) in a population of travellers to Mexico and Guatemala. METHODS: In this phase 3, randomised, double-blind, placebo-controlled field trial, healthy adults (aged 18-64 years) travelling from Germany or the UK to Mexico or Guatemala were assigned in a 1:1 ratio by a dynamic electronic randomisation system to receive transcutaneous immunisation with a patch containing 37.5 µg of ETEC LT or a placebo patch. Participants, site staff, and the investigators who did the analyses were masked to group assignment. Participants were vaccinated before travel, with two patches given 14 days apart. In the destination country, participants tracked stool output in a diary and provided stool samples for pathogen identification if diarrhoea occurred. The primary endpoint was the proportion of participants with at least one episode of moderate-to-severe diarrhoea (defined as four or more unformed stools in a 24 h period) in which either or both ETEC enterotoxins (LT and heat-stable toxin [ST]) were detected. The study is registered at ClinicalTrials.gov, number NCT00993681. FINDINGS: 2036 participants were recruited and randomly assigned between Oct 14, 2009, and Aug 13, 2010, with 1016 allocated to receive the LT patch and 1020 the placebo patch. 821 participants in the LT-patch group and 823 in the placebo group received both vaccinations and were analysed in the per-protocol population. 30 (3.7%, 95% CI 2.5-5.2) participants in the LT-patch group and 46 (5.6%, 4.1-7.4) in the placebo group had moderate or severe ETEC diarrhoea (vaccine efficacy 34.6%, -2.2 to 58.9; p=0.0621). 9333 local (ie, patch-site) adverse events (including erythema, rash, pruritus, hyperpigmentation, pain, hypopigmentation, and oedema) occurred in 943 (93%) of 1015 participants in the LT-patch group, compared with 1444 local adverse events in 574 (56%) of 1019 participants in the placebo group (p<0.0001). Serious adverse events occurred in 25 participants (14 in the LT-patch group and 11 in the placebo group), with all regarded as either unrelated or possibly related to treatment. Vaccine-induced hyperpigmentation persisted for at least 180 days after vaccination in 150 (18%) of the 849 participants who received both vaccinations and returned for final assessment in the LT-patch group, compared with none of the 842 participants in the placebo group. The vaccine was immunogenic, with a post-vaccination geometric mean titre of LT-specific serum immunoglobulin G of 3400.29, compared with 315.41 in the placebo group. INTERPRETATION: Although the LT antigen was delivered effectively by the skin patch, the vaccine did not protect travellers against diarrhoea caused by ETEC or other organisms. Future vaccines against travellers' diarrhoea might need to include several antigens against various diarrhoeal pathogens, and might need to be able to generate mucosal and higher systemic immunity.

A randomized, double-blind, pilot study of rifaximin 550 mg versus placebo in the prevention of travelers' diarrhea in Mexico during the dry season.

BACKGROUND: Rifaximin has been shown to be effective in treating and preventing travelers' diarrhea (TD) during the summer season. METHODS: The goal of this double-blinded multicenter trial was to assess the efficacy and safety of rifaximin 550 mg administered once daily for 14 days compared with placebo in the prevention of TD during the dry season in Mexico. RESULTS: There were 101 participants randomized. Overall, 25 participants developed TD during the 3 weeks of the study: 22% from the rifaximin group and 29% from the placebo group (p = 0.4). Mild diarrhea (defined as only one or two unformed stools during a 24-h period plus at least one abdominal symptoms) developed in only 3 (6%) participants taking rifaximin compared with 10 (21%) taking placebo during the first week of study (p = 0.03). No clinically significant or serious adverse events were reported. CONCLUSIONS: Antibiotic prophylaxis of TD in Mexico during the dry season needs to be further studied and its benefits weighed against the benefits of self-treatment.

Single nucleotide polymorphisms in the promoter of the gene encoding the lipopolysaccharide receptor CD14 are associated with bacterial diarrhea in US and Canadian travelers to Mexico.

BACKGROUND: Under normal conditions, the expression of CD14, which is the principal receptor for bacterial lipopolysaccharide, is down-regulated in the intestinal mucosa but increases in response to inflammatory stimuli. The aim of the present study was to investigate whether fecal CD14 levels increased in response to infection with diarrheagenic Escherichia coli and whether single nucleotide polymorphisms (SNPs) in the CD14 gene were associated with an increased susceptibility to traveler's diarrhea (TD) in US visitors to Mexico. METHODS: Six SNPs located at the promoter, exon, and untranslated regions of CD14 were typed in a prospective cohort study of 1360 visitors to Mexico at risk for TD. Stools from visitors with TD were studied for enteric pathogens by culture, colony hybridization, and polymerase chain reaction. Fecal soluble CD14 (sCD14) was measured in a subgroup of 203 adults with diarrhea and 66 healthy controls by enzyme-linked immunosorbent assay. RESULTS: The minor allele frequencies for CD14 SNPs were significantly different among the various racial and ethnic groups studied. Two SNPs in the promoter region of CD14 (-159 C > T; rs2569190 and -4191 C > T; rs5744441) were found to be associated with TD in White visitors. The -159 TT genotype was associated with a higher risk for TD (Relative risk [RR], 1.21; 95% confidence interval [CI], 1.05-1.38; P = .008), whereas individuals with the -4191 TT genotype were protected from infection (RR, 0.82; 95% CI, 0.71-0.92; P = .006). Subjects with TD excreted higher levels of fecal CD14 than did healthy controls (33,480 pg/mL vs 6178 pg/mL; P < .02). Fecal sCD14 levels were higher in stool samples from visitors with TD and the -159 TT genotype than they were in visitors with the CC/CT genotypes (P = .02), and stool samples from subjects with the -4191 CC genotype had higher fecal sCD14 levels than did stool samples from visitors with the CT/TT (P = .005) genotype. In a multivariate analysis with haplotypes constructed with the 6 SNPs studied, subjects with the haplotype containing the -159 C and the -4191 T allele were less likely to acquire TD (P = .015). CONCLUSIONS: Our study suggests that CD14 levels increase in response to bacterial diarrhea and that polymorphisms in the CD14 gene influence susceptibility to TD. Intestinal CD14 plays an important role in the innate immune response to enteric pathogens.

Campylobacter jejuni is not an important pathogen as a cause of diarrhea in US travelers to Mexico.

Campylobacter jejuni is an unusual cause of travelers' diarrhea acquired in Mexico, but previous studies have relied only on stool culture for diagnosis. We conducted a cohort study to determine if antibody seroconversion to C jejuni would better reflect the occurrence of infection acquired in Mexico. Serum IgG, IgA, and IgM antibodies to Campylobacter seroconverted in only 2 of 353 participants (0.6%). These data further support that C jejuni infection is an unusual cause of travelers' diarrhea in US visitors to Mexico.

In vitro antimicrobial susceptibility of bacterial enteropathogens isolated from international travelers to Mexico, Guatemala, and India from 2006 to 2008.

The incidence rates of travelers' diarrhea (TD) have remained high for the last 50 years. More recently, there have been increasing recommendations for self-initiated therapy and use of prophylactic drugs for TD. We last examined the in vitro susceptibilities of commonly used antibiotics against TD pathogens in 1997. We now examine 456 enteropathogens isolated from adult travelers to Mexico, India, and Guatemala with diarrhea acquired between 2006 and 2008 to determine changes in susceptibility against 10 different antimicrobials by the agar dilution method. Traditional antibiotics, such as ampicillin, trimethoprim-sulfamethoxazole, and doxycycline, continue to show high levels of resistance. Current first-line antibiotic agents, including fluoroquinolones and azithromycin, showed significantly higher MICs than in our earlier study, and MIC(90) levels were above the Clinical and Laboratory Standards Institute cutoffs for resistance. There were significant geographical differences in resistance patterns when Central America was compared with India. Entertoxigenic Escherichia coli (ETEC) isolates showed increased resistance to ciprofloxacin (P = 0.023) and levofloxacin (P = 0.0078) in India compared with Central America. Enteroaggregative E. coli (EAEC) isolates from Central America showed increased resistance to nearly all of the antibiotics tested. Compared to MICs of isolates 10 years prior, there were 4- to 10-fold increases in MIC(90) values for ceftriaxone, ciprofloxacin, levofloxacin, and azithromycin for both ETEC and EAEC. There were no significant changes in rifaximin MICs. Rising MICs over time imply the need for continuous surveillance of susceptibility patterns worldwide and geographically specific recommendations in TD therapy.

Biochemical and genetic diversity of enterotoxigenic Escherichia coli associated with diarrhea in United States students in Cuernavaca and Guadalajara, Mexico, 2004-2007.

BACKGROUND: Molecular characterization of Escherichia coli with use of the random amplified polymorphic DNA (RAPD) assay allows the determination of clonal origin and geographic clustering. METHODS: Presumed enterotoxigenic Escherichia coli (ETEC) from 213 adults with travelers' diarrhea acquired in Mexico during the summer months of 2004-2007 were studied. Biochemical testing strips determined a 7-digit fingerprint on the basis of 21 biochemical reactions. E. coli producing enterotoxin were evaluated for clonality by RAPD assay. Dendrograms were developed using Pearson correlations with 80% similarity to determine clonal groups. RESULTS: Of the presumed ETEC, 85% were confirmed to be E. coli on the basis of biochemical analysis. Other enterotoxigenic bacteria included Citrobacter species (9%) and other coliforms (all 2%). RAPD analysis with primers 1247 and 1254 determined 24 ETEC clonal groups containing 2-9 subjects each, of which 15 spanned the 4 years and 8 spanned both cities. CONCLUSIONS: Complete biochemical evaluation of E. coli-like, enterotoxigenic organisms is crucial in ETEC identification. In addition, other enterotoxigenic organisms identified should be studied further for their role in enteric disease. Travelers to Mexico are exposed to a large pool of different ETEC strains from multiple sources, with a small number of dominant types showing a widespread and persistent reservoir of infection.

Prevention of travelers' diarrhea with rifaximin in US travelers to Mexico.

BACKGROUND: Because bacterial pathogens are the primary cause of travelers' diarrhea (TD), antibiotic prophylaxis is effective in TD prevention. This study assessed the efficacy and safety of the nonsystemic antibiotic rifaximin in preventing TD in US travelers to Mexico. METHODS: Healthy adult students traveling to Mexico received rifaximin 600 mg/d or placebo for 14 days and were followed for 7 days post-treatment. Stool pattern and gastrointestinal symptoms were recorded in daily diary entries. The primary end point was prevention of TD during 14 days of treatment measured by time to first unformed stool. RESULTS: A total of 210 individuals received rifaximin (n = 106) or placebo (n = 104) and were included in the safety population. Median age was 21 years (range, 18-75 y), and the majority of participants were female (65%). Efficacy analyses were conducted in a modified intent-to-treat population of 201 patients who received rifaximin (n = 99) or placebo (n = 102). Rifaximin prophylaxis reduced risk of developing TD versus placebo (p < 0.0001). A smaller percentage of individuals who received rifaximin versus placebo developed all-cause TD (20% vs 48%, respectively; p < 0.0001) or TD requiring antibiotic therapy (14% vs 32%, respectively; p = 0.003). More individuals in the rifaximin group (76%) completed treatment without developing TD versus those in the placebo group (51%; p = 0.0004). Rifaximin provided a 58% protection rate against TD and was associated with fewer adverse events than placebo. CONCLUSIONS: Prophylactic treatment with rifaximin 600 mg/d for 14 days safely and effectively reduced the risk of developing TD in US travelers to Mexico. Rifaximin chemoprevention should be considered for TD in appropriate individuals traveling to high-risk regions.

Noroviruses as a cause of diarrhea in travelers to Guatemala, India, and Mexico.

Noroviruses (NoVs) are increasingly being recognized as an important enteric pathogen of gastroenteritis worldwide. The prevalence of NoVs as a cause of diarrhea acquired by travelers in developing countries is not well known. We examined the prevalence and importance of NoV infection in three international traveler cohorts with diarrhea acquired in three developing regions of the world, Mexico, Guatemala, and India. We also characterized the demographics and symptoms associated with NoV diarrhea in these travelers. Stool samples from 571 international travelers with diarrhea were evaluated for traditional enteropathogens. NoVs were identified using reverse transcription-PCR and probe hybridization. NoVs were identified in 10.2% of cases of travelers' diarrhea and, overall, was the second most common pathogen, following diarrheagenic Escherichia coli. The detection of NoV diarrhea significantly varied over the three study time periods in Guadalajara, Mexico, ranging from 3 of 98 (3.0%) diarrheal stools to 12 of 100 (12.0%) fecal specimens (P=0.03). The frequency of NoV diarrhea was also dependent upon the geographic region, with 17 of 100 (17.0%) travelers to Guatemala, 23 of 194 (11.9%) travelers to India, and 3 of 79 (3.8%) travelers to Mexico testing positive for NoVs from 2002 to 2003 (P=0.02). NoVs are important pathogens of travelers' diarrhea in multiple regions of the world. Significant variation in the prevalence of NoV diarrhea and in the predominant genogroup infecting travelers was demonstrated, dependent upon the specific geographic location and over time.

Microbial etiology of travelers' diarrhea in Mexico, Guatemala, and India: importance of enterotoxigenic Bacteroides fragilis and Arcobacter species.

This study examined established enteric pathogens, Arcobacter species and enterotoxigenic Bacteroides fragilis (ETBF), in 201 U.S. and European travelers with acute diarrhea acquired in Mexico, Guatemala, and India. Arcobacter butzleri and ETBF were detected in 8% and 7% of diarrhea cases, respectively.

Boty-II, a novel LTR retrotransposon in Botrytis cinerea B05.10 revealed by genomic sequence

Botrytis cinerea is a necrotrophic pathogen causing pre- and post-harvest diseases in at least 235 plant species. It manifests extraordinary genotype and phenotype variation. One of the causes of this variation is transposable elements. Two transposable elements have been discovered in this fungus, the retrotransposon (Boty), and the transposon (Flipper). In this work, two complete (Boty-II-76 and Boty-II-103) and two partial (Boty-II-95 and Boty-II-141) long terminal repeat (LTR) retrotransposons were identified by an in silico genomic sequence analysis. Boty-II-76 and Boty-II-103 contain 6439 bp nucleotides with a pair of LTRs at both ends, and an internal deduced pol gene encoding a polyprotein with reverse transcriptase and DDE integrase domains. They are flanked by 5 bp direct repeats (ACCAT, CTTTC). In Boty-II-141, two LTRs at both ends, and a partial internal pol gene encoding a protein with a DDE integrase domain were identified. In Boty-II-95, a right LTR and a partial internal pol gene encoding a protein with no conserved domains were identified. Boty-II uses a self-priming mechanism to initiate synthesis of reverse transcripts. The sequence of the presumed primer binding site for first-strand reverse transcription is 5’-TTGTACCAT-3’. The polypurine-rich sequence for plus-strand DNA synthesis is 5’-GCCTTGAGCGGGGGGTAC-3’. Fourteen Boty-II LTRs that contain 125-158 bp nucleotides and share 69.1 ~ 100 percent identities with the short inverted terminal repeats of 5 bp (TGTCA…TGACA) were discovered. Analysis of structural features and phylogeny revealed that Boty-II is a novel LTR retrotransposon. It could potentially be used as a novel molecular marker for the investigation of genetic variation in B. cinerea.

Coliform and Escherichia coli contamination of desserts served in public restaurants from Guadalajara, Mexico, and Houston, Texas.

Bacterial enteropathogens acquired from contaminated food are the principal causes of travelers' diarrhea (TD). We evaluated desserts obtained from popular restaurants in the tourist city of Guadalajara, Mexico, and Houston, Texas, to determine coliform and Escherichia coli contamination levels and presence of diarrheagenic E. coli known to be important in TD. Contamination for all organisms was seen for desserts served in Guadalajara restaurants. Desserts should be considered as potentially risky foods for development of TD among international visitors to developing regions of the world.

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.

Two LTR retrotransposon elements within the abscisic acid gene cluster in Botrytis cinerea B05.10, but not in SAS56

The plant hormone abscisic acid has huge economic potential and can be applied in agriculture and forestry for it is considered to be involved in plant resistance to stresses such as cold, heat, salinity, drought, pathogens and wounding. Now overproducing strains of Botrytis cinerea are used for biotechnological production of abscisic acid. An LTR retrotransposon, Boty-aba, and a solo LTR were identified by in silico genomic sequence analysis, and both were detected within the abscisic acid gene cluster in B. cinerea B05.10, but not in B. cinerea SAS56. Boty-aba contains a pair of LTRs and two internal genes. The LTRs and the first gene have features characteristic of Ty3/gypsy LTR retrotransposons. The second gene is a novel gene, named brtn, which encodes for a protein (named BRTN) without putative conserved domains. The impressive divergence in structure of the abscisic acid gene clusters putatively gives new clues to investigate the divergence in the abscisic acid production yields of different B. cinerea strains.

Enterotoxigenic Escherichia coli and diffusely adherent E. coli as likely causes of a proportion of pathogen-negative travelers' diarrhea--a PCR-based study.

BACKGROUND: Enteropathogens cannot be identified in 40% to 50% of subjects with travelers' diarrhea (TD). METHODS: We used polymerase chain reaction (PCR) methods to look for the presence of two bacterial causes of diarrhea in a large group of international travelers after failing to detect a pathogen by conventional tests. DNA was isolated from the diarrheal stool and subjected to PCR from 162 subjects from whom we earlier failed to identify a pathogen in a previous study and included 54 from Antigua, Guatemala, 39 from Guadalajara, Mexico, 29 from Kolkata, India, and 40 from Goa, India. Gene products for enterotoxigenic Escherichia coli (ETEC)--LT (heat-labile enterotoxin) and ST (heat-stable enterotoxin)--and diffusely adherent E. coli (DAEC), afa/dr (Afa fimbrial and Dr nonfimbrial family of adhesins), were used. RESULTS: At least one gene product was identified in diarrhea stool samples of 47 of 162 (29%) subjects. ETEC virulence genes (LT, ST) were found in 34 (21%) samples studied, with rates of occurrence ranging from 8% in Goa to 39% for the samples from Guatemala (p = 0.0006). A large number of ST-only strains explained the high ETEC rate in Guatemala. DAEC afa/dr family of adhesions was identified in between 8 and 14% of the samples. CONCLUSIONS: ETEC and DAEC were implicated in nearly one-third of the subjects initially diagnosed as pathogen negative. Direct PCR results from stools are consistent with the previous assumption that most undiagnosed TD is bacterial in nature and also highlights the potential value that PCR can add to studies designed to evaluate treatment and preventive interventions for TD, including vaccines.