First multi-locus sequence typing scheme for Arcobacter spp.

<p>Abstract</p> <p>Background</p> <p><it>Arcobacter </it>spp. are a common contaminant of food and water, and some species, primarily <it>A. butzleri </it>and <it>A. cryaerophilus</it>, have been isolated increasingly from human diarrheal stool samples. Here, we describe the first <it>Arcobacter </it>multilocus sequence typing (MLST) method for <it>A. butzleri</it>, <it>A. cryaerophilus</it>, <it>A. skirrowii, A. cibarius </it>and <it>A. thereius</it>.</p> <p>Results</p> <p>A sample set of 374 arcobacters, including 275 <it>A. butzleri</it>, 72 <it>A. cryaerophilus</it>, 15 <it>A. skirrowii </it>and 8 <it>A. cibarius </it>isolates from a wide variety of geographic locations and sources, was typed in this study. Additionally, this sample set contained four strains representing a new <it>Arcobacter </it>species, <it>A. thereius</it>. The seven loci used in the four-species <it>Arcobacter </it>MLST method are the same as those employed previously in <it>C. jejuni</it>, <it>C. coli</it>, <it>C. helveticus </it>and <it>C. fetus </it>(i.e. <it>aspA</it>, <it>atpA</it>(<it>uncA</it>), <it>glnA</it>, <it>gltA</it>, <it>glyA, pgm </it>and <it>tkt</it>). A large number of alleles were identified at each locus with the majority of isolates containing a unique sequence type. All <it>Arcobacter </it>isolates typed in this study contain two <it>glyA </it>genes, one linked to <it>lysS </it>(<it>glyA1</it>) and the other linked to <it>ada </it>(<it>glyA2</it>). <it>glyA1 </it>was incorporated into the <it>Arcobacter </it>MLST method while <it>glyA2 </it>was not because it did not increase substantially the level of discrimination.</p> <p>Conclusion</p> <p>No association of MLST alleles or sequence types with host or geographical source was observed with this sample set. Nevertheless, the large number of identified alleles and sequence types indicate that this MLST method will prove useful in both <it>Arcobacter </it>strain discrimination and in epidemiological studies of sporadic <it>Arcobacter</it>-related gastroenteritis. A new <it>Arcobacter </it>MLST database was created <url>http://pubmlst.org/arcobacter/</url>; allele and ST data generated in this study were deposited in this database and are available online.</p

Whole Genome Sequence Analysis of Salmonella Typhi Isolated in Thailand before and after the Introduction of a National Immunization Program.

Vaccines against Salmonella Typhi, the causative agent of typhoid fever, are commonly used by travellers, however, there are few examples of national immunization programs in endemic areas. There is therefore a paucity of data on the impact of typhoid immunization programs on localised populations of S. Typhi. Here we have used whole genome sequencing (WGS) to characterise 44 historical bacterial isolates collected before and after a national typhoid immunization program that was implemented in Thailand in 1977 in response to a large outbreak; the program was highly effective in reducing typhoid case numbers. Thai isolates were highly diverse, including 10 distinct phylogenetic lineages or genotypes. Novel prophage and plasmids were also detected, including examples that were previously only reported in Shigella sonnei and Escherichia coli. The majority of S. Typhi genotypes observed prior to the immunization program were not observed following it. Post-vaccine era isolates were more closely related to S. Typhi isolated from neighbouring countries than to earlier Thai isolates, providing no evidence for the local persistence of endemic S. Typhi following the national immunization program. Rather, later cases of typhoid appeared to be caused by the occasional importation of common genotypes from neighbouring Vietnam, Laos, and Cambodia. These data show the value of WGS in understanding the impacts of vaccination on pathogen populations and provide support for the proposal that large-scale typhoid immunization programs in endemic areas could result in lasting local disease elimination, although larger prospective studies are needed to test this directly

Etiology of Acute Diarrheal Disease and Antimicrobial Susceptibility Pattern in Children Younger Than 5 Years Old in Nepal

Diarrhea is a common cause of morbidity and mortality among children younger than 5 years in developing countries. Children from 3 to 60 months of age were recruited from two hospitals in Nepal— Bharatpur Hospital, Bharatpur, and Kanti Children’s Hospital, Kathmandu—in 2006 to 2009. Stool specimens collected from 1,200 children with acute diarrhea (cases) and 1,200 children without diarrhea (control subjects) were examined for a broad range of enteropathogens by standard microbiology, including microscopy, enzyme immunoassay for viral pathogens (adenovirus, astrovirus, and rotavirus) and protozoa (Giardia, Cryptosporidium, and Entamoeba histolytica), as well as by using reverse transcription real-time polymerase for norovirus. Antimicrobial susceptibility testing was performed using the disk diffusion method. Overall, rotavirus (22% versus 2%), norovirus (13% versus 7%), adenovirus (3% versus 0%), Shigella (6% versus 1%), enterotoxigenic Escherichia coli (8% versus 4%), Vibrio (7% versus 0%), and Aeromonas (9% versus 3%) were identified significantly more frequently in cases than control subjects. Campylobacter, Plesiomonas, Salmonella, and diarrheagenic E. coli (enteropathogenic, enteroinvasive, enteroaggregative) were identified in similar proportions in diarrheal and non-diarrheal stools. Campylobacter was resistant to second-generation quinolone drugs (ciprofloxacin and norfloxacin), whereas Vibrio and Shigella were resistant to nalidixic acid and trimethoprim/sulfamethoxazole. This study documents the important role of rotavirus and norovirus in acute diarrhea in children younger than 5 years, followed by the bacteria Shigella, enterotoxigenic E. coli, Vibrio cholera, and Aeromonas. Data on the prevalence and epidemiology of enteropathogens identify potential pathogens for public health interventions, whereas pathogen antibiotic resistance pattern data may provide guidance on choice of therapy in clinical settings.publishedVersio

Comparative Antibiotic Resistance of Diarrheal Pathogens from Vietnam and Thailand, 1996-1999

Antimicrobial resistance rates for shigella, campylobacter, nontyphoidal salmonella, and enterotoxigenic Escherichia coli were compared for Vietnam and Thailand from 1996 to 1999. Resistance to trimethoprim-sulfamethoxazole, ampicillin, chloramphenicol, and tetracycline was common. Quinolone resistance remains low in both countries, except among campylobacter and salmonella organisms in Thailand. Nalidixic acid resistance among salmonellae has more than doubled since 1995 (to 21%) in Thailand but is not yet documented in Vietnam. Resistance to quinolones correlated with resistance to azithromycin in both campylobacter and salmonella in Thailand. This report describes the first identification of this correlation and its epidemiologic importance among clinical isolates. These data illustrate the growing magnitude of antibiotic resistance and important differences between countries in Southeast Asia

Prevalence and antimicrobial resistance of non-typhoid Salmonella in military personnel, 1988-2013

Objective: To describe the spanning 25 years data for the occurrence, magnitude, and trends regarding antimicrobial resistance of non-typhoidal Salmonella (NTS) isolated from non-immune travelers to Thailand participating in joint military operations. Methods: A total of 355 NTS isolates, obtained from 2 052 fecal samples from US soldiers deployed for military maneuvers in Thailand during 1988-2013, were examined for NTS serogroup/ serotypes and tested for antimicrobial susceptibility by disk diffusion to these 10 antibiotics: ampicillin, azithromycin (AZM), ciprofloxacin, colistin, gentamicin, kanamycin, nalidixic acid, streptomycin (STR), tetracycline (TET), and trimethoprim/sulfamethoxazole. Identified AZM-resistant NTS isolates were further evaluated for their minimal inhibitory concentration by the E-test method. Results: NTS infections accounted for 17.3% (355/2 052), including 11 serogroups and 50 different serotypes. The most prevalent serogroup was Salmonella group C2-C3 (35.8%, 127/355) followed by groups B (21.1%, 75/355) and C1 (18.6%, 66/355). Identified serotypes included Salmonella hadar (n=60), Salmonella rissen (n=45), and Salmonella blockley (n=34). Among the predominate serogroups, antimicrobial resistance was consistently high against TET (76.9%, 273/355) followed by STR (40.8%, 145/355). One Salmonella senftenberg isolate demonstrated decreased ciprofloxacin susceptibility. Most isolates (94.6%) were resistant to one or more antimicrobials, and the most common multidrug resistance was TET-STR-nalidixic acid (11.5%, 41/355). Conclusions: The prevalence of NTS serotypes and the growing magnitude of antibiotic resistant bacteria isolated from deployed US military in Thailand are documented from 1988-2013. This study demonstrates the antibiotic resistance profiles, highlighting the effectiveness of AZM that is a first-line treatment for travelers to Southeast Asia. AZM-resistant NTS isolates are periodically observed over a 25- year period. Hence, the ongoing surveillance and prevalence efforts are required to monitor NTS resistant strains causing further treatment failure

Extended-spectrum β-lactamase prevalence and virulence factor characterization of enterotoxigenic Escherichia coli responsible for acute diarrhea in Nepal from 2001 to 2016

Abstract Background Multidrug-resistant (MDR) Gram-negative bacterial species are an increasingly dangerous public health threat, and are now endemic in many areas of South Asia. However, there are a lack of comprehensive data from many countries in this region determining historic and current MDR prevalence. Enterotoxigenic Escherichia coli (ETEC) is a leading cause of both acute infant diarrhea and traveler’s diarrhea in Nepal. The MDR prevalence and associated resistance mechanisms of ETEC isolates responsible for enteric infections in Nepal are largely unknown. Methods A total of 265 ETEC isolates were obtained from acute diarrheal samples (263/265) or patient control samples (2/265) at traveler’s clinics or regional hospitals in Nepal from 2001 to 2016. Isolates were screened for antibiotic resistance, to include extended spectrum beta-lactamase (ESBL) production, via the Microscan Automated Microbiology System. ETEC virulence factors, specifically enterotoxins and colonization factors (CFs), were detected using multiplex PCR, and prevalence in the total isolate population was compared to ESBL-positive isolates. ESBL-positive isolates were assessed using multiplex PCR for genetic markers potentially responsible for observed resistance. Results A total of 118/265 (44.5%) ETEC isolates demonstrated resistance to ≥2 antibiotics. ESBL-positive phenotypes were detected in 40/265 isolates, with isolates from 2008, 2013, 2014, and 2016 demonstrating ESBL prevalence rates of 1.5, 34.5, 31.2, and 35.0% respectively. No difference was observed in overall enterotoxin characterization between the total ETEC and ESBL-positive populations. The CFs CS2 (13.6%), CS3 (25.3%), CS6 (30.2%), and CS21 (62.6%) were the most prevalent in the total ETEC population. The ESBL-positive ETEC isolates exhibited a higher association trend with the CFs CS2 (37.5%), CS3 (35%), CS6 (42.5%), and CS21 (67.5%). The primary ESBL gene identified was bla CTX-M-15 (80%), followed by bla SHV-12 (20%) and bla CTX-M-14 (2.5%). The beta-lactamase genes bla TEM-1 (40%) and bla CMY-2 (2.5%) were also identified. It was determined that 42.5% of the ESBL-positive isolates carried multiple resistance genes. Conclusion Over 30% of ETEC isolates collected post-2013 and evaluated in this study demonstrated ESBL resistance. Persistent surveillance and characterization of enteric ETEC isolates are vital for tracking the community presence of MDR bacterial species in order to recommend effective treatment strategies and help mitigate the spread of resistant pathogens

Carbapenemase-Producing Enterobacteriaceae and Nonfermentative Bacteria, the Philippines, 2013–2016

During 2013–2016, we isolated blaNDM- and blaVIM-harboring Enterobacteriaceae and nonfermentative bacteria from patients in the Philippines. Of 130 carbapenem-resistant isolates tested, 45 were Carba NP–positive; 43 harbored blaNDM, and 2 harbored blaVIM. Multidrug-resistant microbial pathogen surveillance and antimicrobial drug stewardship are needed to prevent further spread of New Delhi metallo-β-lactamase variants

Design of a Bacteriophage Cocktail Active against Shigella Species and Testing of Its Therapeutic Potential in Galleria mellonella

Shigellosis is a leading global cause of diarrheal disease and travelers&rsquo; diarrhea now being complicated by the dissemination of antibiotic resistance, necessitating the development of alternative antibacterials such as therapeutic bacteriophages (phages). Phages with lytic activity against Shigella strains were isolated from sewage. The genomes of 32 phages were sequenced, and based on genomic comparisons belong to seven taxonomic genera: Teetrevirus, Teseptimavirus, Kayfunavirus, Tequatrovirus, Mooglevirus, Mosigvirus and Hanrivervirus. Phage host ranges were determined with a diverse panel of 95 clinical isolates of Shigella from Southeast Asia and other geographic regions, representing different species and serotypes. Three-phage mixtures were designed, with one possessing lytic activity against 89% of the strain panel. This cocktail exhibited lytic activity against 100% of S. sonnei isolates, 97.2% of S. flexneri (multiple serotypes) and 100% of S. dysenteriae serotypes 1 and 2. Another 3-phage cocktail composed of two myophages and one podophage showed both a broad host range and the ability to completely sterilize liquid culture of a model virulent strain S. flexneri 2457T. In a Galleria mellonella model of lethal infection with S. flexneri 2457T, this 3-phage cocktail provided a significant increase in survival

Blast comparison of novel phages observed in Thai S. Typhi isolates to nearest known phage sequences.

<p>(A) Novel phage STYP1 compared to Shigella sonnei phage fiAA91-ss (NC_022750). (B) Novel phage STYP2 compared to Shigella flexneri phage SfIV (NC_022749). Shaded regions indicate areas of sequence homology, intensity of shading indicates relative nucleotide similarity. Arrows represent protein coding genes (direction indicates coding strand), colored by encoded protein functions: red, DNA packaging module; orange, virion morphogenesis module; yellow, cargo genes; blue, DNA replication and lysogenic cycle maintenance; green, lysis module.</p