Microbiome Analysis of Organic and Conventional Chickens Processed Using Whole Carcass Enrichment and Rinse Methods.

Investigating the chicken microbiome is important to establish control measures for pathogens to protect consumers. This study aimed at evaluating the comparative efficiency of human pathogen detection through 16S rRNA sequencing of organic and conventional chickens processed using whole carcass enrichment (WCE) and rinse (WCR) methods. Organic and conventional whole broiler carcasses (n = 31) were vigorously shaken with 500 mL buffered peptone water (BPW). For the rinse method, a 30 mL aliquot was mixed with 30 mL of BPW. The rest of the sample, including the carcass, was used for the enrichment method. All samples were incubated at 37°C for 24 h. The samples were divided into five groups [Negative Control: only BPW without chicken (n = 5), Organic-Rinsed (n = 7), -Enriched (n = 8), Conventional-Rinsed (n = 7), and -Enriched (n = 9)]. Fifty milliliters of each sample were subjected to DNA extraction followed by 16S rRNA sequencing. Proteobacteria and Firmicutes predominated the microbiota of both conventional and organic chickens, followed by low abundances of Bacteroidetes and Fusobacterium. While the abundance of Proteobacteria and Firmicutes remained unchanged in organic chicken irrespective of the methods used, a noticeable shift in the Proteobacteria and Firmicutes ratio (59%:39% in rinsed to 38%:60% in enriched) was observed in conventional chicken. Furthermore, the choice of method did not yield any differences in Abundance-Based Coverage Estimator, and Jackknife, among conventional and organic chickens but resulted in a statistically significant difference in the Shannon, Simpson, Chao1, and phylogenetic diversity indices (p < 0.05). The relative abundance of Salmonella and Campylobacter was less than 0.1%. The results suggested the WCE method provides a broad range of information on the chicken microbiome.

Vibrio cholerae O1 El Tor strains linked to global cholera show region-specific patterns by pulsed-field gel electrophoresis.

Vibrio cholerae O1 El Tor, causative agent of the ongoing seventh cholera pandemic, is native to the aquatic environment of the Ganges Delta, Bay of Bengal (GDBB). Recent studies traced pandemic strains to the GDBB and proposed global spread of cholera had occurred via intercontinental transmission. In the research presented here, NotI-digested genomic DNA extracted from V. cholerae O1 clinical and environmental strains isolated in Bangladesh during 20042014 was analyzed by pulsed-field gel electrophoresis (PFGE). Results of cluster analysis showed 94.67% of the V. cholerae strains belonged to clade A and included the majority of clinical strains of spatio-temporal origin and representing different cholera endemic foci. The rest of the strains were estuarine, all environmental strains from Mathbaria, Bangladesh, and occurred as singletons, clustered in clades B and C, or in the small clades D and E. Cluster analysis of the Bangladeshi strains and including 157 El Tor strains from thirteen countries in Asia, Africa, and the Americas revealed 85% of the total set of strains belonged to clade A, indicating all were related, yet did not form an homogeneous cluster. Overall, 15% of the global strains comprised multiple small clades or segregated as singletons. Three sub-clades could be discerned within the major clade A, reflecting distinct lineages of V. cholerae O1 El Tor associated with cholera in Asia, Africa, and the Americas. The presence in Asia and the Americas of non-pandemic V. cholerae O1 El Tor populations differing by PFGE and from strains associated with cholera globally suggests different ecotypes are resident in distant geographies.

Draft Genome Sequences of Seven Vibrio cholerae Isolates from Adult Patients in Qatar.

We report the draft genome sequences of seven Vibrio cholerae isolates from patients. Four isolates were profiled as multilocus sequence type 69, serogroup O1, a subset of seventh-pandemic El Tor clonal isolates. Presented here are genome assemblies and evidence for major pathogenicity islands, virulence factors, and antimicrobial resistance genes.

A comparative analysis of drinking water employing metagenomics.

The microbiological content of drinking water traditionally is determined by employing culture-dependent methods that are unable to detect all microorganisms, especially those that are not culturable. High-throughput sequencing now makes it possible to determine the microbiome of drinking water. Thus, the natural microbiota of water and water distribution systems can now be determined more accurately and analyzed in significantly greater detail, providing comprehensive understanding of the microbial community of drinking water applicable to public health. In this study, shotgun metagenomic analysis was performed to determine the microbiological content of drinking water and to provide a preliminary assessment of tap, drinking fountain, sparkling natural mineral, and non-mineral bottled water. Predominant bacterial species detected were members of the phyla Actinobacteria and Proteobacteria, notably the genera Alishewanella, Salmonella, and Propionibacterium in non-carbonated non-mineral bottled water, Methyloversatilis and Methylibium in sparkling natural mineral water, and Mycobacterium and Afipia in tap and drinking fountain water. Fecal indicator bacteria, i.e., Escherichia coli or enterococci, were not detected in any samples examined in this study. Bacteriophages and DNA encoding a few virulence-associated factors were detected but determined to be present only at low abundance. Antibiotic resistance markers were detected only at abundance values below our threshold of confidence. DNA of opportunistic plant and animal pathogens was identified in some samples and these included bacteria (Mycobacterium spp.), protozoa (Acanthamoeba mauritaniensis and Acanthamoeba palestinensis), and fungi (Melampsora pinitorqua and Chryosporium queenslandicum). Archaeal DNA (Candidatus Nitrosoarchaeum) was detected only in sparkling natural mineral water. This preliminary study reports the complete microbiome (bacteria, viruses, fungi, and protists) of selected types of drinking water employing whole-genome high-throughput sequencing and bioinformatics. Investigation into activity and function of the organisms detected is in progress.

Vibrio cholerae O1 with Reduced Susceptibility to Ciprofloxacin and Azithromycin Isolated from a Rural Coastal Area of Bangladesh.

Cholera outbreaks occur each year in the remote coastal areas of Bangladesh and epidemiological surveillance and routine monitoring of cholera in these areas is challenging. In this study, a total of 97 Vibrio cholerae O1 isolates from Mathbaria, Bangladesh, collected during 2010 and 2014 were analyzed for phenotypic and genotypic traits, including antimicrobial susceptibility. Of the 97 isolates, 95 possessed CTX-phage mediated genes, ctxA, ace, and zot, and two lacked the cholera toxin gene, ctxA. Also both CTX+ and CTX-V. cholerae O1 isolated in this study carried rtxC, tcpAET, and hlyA. The classical cholera toxin gene, ctxB1, was detected in 87 isolates, while eight had ctxB7. Of 95 CTX+V. cholerae O1, 90 contained rstRET and 5 had rstRCL. All isolates, except two, contained SXT related integrase intSXT. Resistance to penicillin, streptomycin, nalidixic acid, sulfamethoxazole-trimethoprim, erythromycin, and tetracycline varied between the years of study period. Most importantly, 93% of the V. cholerae O1 were multidrug resistant. Six different resistance profiles were observed, with resistance to streptomycin, nalidixic acid, tetracycline, and sulfamethoxazole-trimethoprim predominant every year. Ciprofloxacin and azithromycin MIC were 0.003-0.75 and 0.19-2.00 µg/ml, respectively, indicating reduced susceptibility to these antibiotics. Sixteen of the V. cholerae O1 isolates showed higher MIC for azithromycin (≥0.5 µg/ml) and were further examined for 10 macrolide resistance genes, erm(A), erm(B), erm(C), ere(A), ere(B), mph(A), mph(B), mph(D), mef(A), and msr(A) with none testing positive for the macrolide resistance genes.

Phylogenetic Diversity of Vibrio cholerae Associated with Endemic Cholera in Mexico from 1991 to 2008.

UNLABELLED: An outbreak of cholera occurred in 1991 in Mexico, where it had not been reported for more than a century and is now endemic. Vibrio cholerae O1 prototype El Tor and classical strains coexist with altered El Tor strains (1991 to 1997). Nontoxigenic (CTX(-)) V. cholerae El Tor dominated toxigenic (CTX(+)) strains (2001 to 2003), but V. cholerae CTX(+) variant El Tor was isolated during 2004 to 2008, outcompeting CTX(-) V. cholerae. Genomes of six Mexican V. cholerae O1 strains isolated during 1991 to 2008 were sequenced and compared with both contemporary and archived strains of V. cholerae. Three were CTX(+) El Tor, two were CTX(-) El Tor, and the remaining strain was a CTX(+) classical isolate. Whole-genome sequence analysis showed the six isolates belonged to five distinct phylogenetic clades. One CTX(-) isolate is ancestral to the 6th and 7th pandemic CTX(+) V. cholerae isolates. The other CTX(-) isolate joined with CTX(-) non-O1/O139 isolates from Haiti and seroconverted O1 isolates from Brazil and Amazonia. One CTX(+) isolate was phylogenetically placed with the sixth pandemic classical clade and the V. cholerae O395 classical reference strain. Two CTX(+) El Tor isolates possessing intact Vibrio seventh pandemic island II (VSP-II) are related to hybrid El Tor isolates from Mozambique and Bangladesh. The third CTX(+) El Tor isolate contained West African-South American (WASA) recombination in VSP-II and showed relatedness to isolates from Peru and Brazil. Except for one isolate, all Mexican isolates lack SXT/R391 integrative conjugative elements (ICEs) and sensitivity to selected antibiotics, with one isolate resistant to streptomycin. No isolates were related to contemporary isolates from Asia, Africa, or Haiti, indicating phylogenetic diversity. IMPORTANCE: Sequencing of genomes of V. cholerae is critical if genetic changes occurring over time in the circulating population of an area of endemicity are to be understood. Although cholera outbreaks occurred rarely in Mexico prior to the 1990s, genetically diverse V. cholerae O1 strains were isolated between 1991 and 2008. Despite the lack of strong evidence, the notion that cholera was transmitted from Africa to Latin America has been proposed in the literature. In this study, we have applied whole-genome sequence analysis to a set of 124 V. cholerae strains, including six Mexican isolates, to determine their phylogenetic relationships. Phylogenetic analysis indicated the six V. cholerae O1 isolates belong to five phylogenetic clades: i.e., basal, nontoxigenic, classical, El Tor, and hybrid El Tor. Thus, the results of phylogenetic analysis, coupled with CTXϕ array and antibiotic susceptibility, do not support single-source transmission of cholera to Mexico from African countries. The association of indigenous populations of V. cholerae that has been observed in this study suggests it plays a significant role in the dynamics of cholera in Mexico.

Hybrid Vibrio cholerae El Tor lacking SXT identified as the cause of a cholera outbreak in the Philippines.

UNLABELLED: Cholera continues to be a global threat, with high rates of morbidity and mortality. In 2011, a cholera outbreak occurred in Palawan, Philippines, affecting more than 500 people, and 20 individuals died. Vibrio cholerae O1 was confirmed as the etiological agent. Source attribution is critical in cholera outbreaks for proper management of the disease, as well as to control spread. In this study, three V. cholerae O1 isolates from a Philippines cholera outbreak were sequenced and their genomes analyzed to determine phylogenetic relatedness to V. cholerae O1 isolates from recent outbreaks of cholera elsewhere. The Philippines V. cholerae O1 isolates were determined to be V. cholerae O1 hybrid El Tor belonging to the seventh-pandemic clade. They clustered tightly, forming a monophyletic clade closely related to V. cholerae O1 hybrid El Tor from Asia and Africa. The isolates possess a unique multilocus variable-number tandem repeat analysis (MLVA) genotype (12-7-9-18-25 and 12-7-10-14-21) and lack SXT. In addition, they possess a novel 15-kb genomic island (GI-119) containing a predicted type I restriction-modification system. The CTXΦ-RS1 array of the Philippines isolates was similar to that of V. cholerae O1 MG116926, a hybrid El Tor strain isolated in Bangladesh in 1991. Overall, the data indicate that the Philippines V. cholerae O1 isolates are unique, differing from recent V. cholerae O1 isolates from Asia, Africa, and Haiti. Furthermore, the results of this study support the hypothesis that the Philippines isolates of V. cholerae O1 are indigenous and exist locally in the aquatic ecosystem of the Philippines. IMPORTANCE: Genetic characterization and phylogenomics analysis of outbreak strains have proven to be critical for probing clonal relatedness to strains isolated in different geographical regions and over time. Recently, extensive genetic analyses of V. cholerae O1 strains isolated in different countries have been done. However, genome sequences of V. cholerae O1 isolates from the Philippines have not been available for epidemiological investigation. In this study, molecular typing and phylogenetic analysis of Vibrio cholerae isolated from both clinical and environmental samples in 2011 confirmed unique genetic features of the Philippines isolates, which are helpful to understand the global epidemiology of cholera.

Non-O1/non-O139 Vibrio cholerae carrying multiple virulence factors and V. cholerae O1 in the Chesapeake Bay, Maryland.

Non-O1/non-O139 Vibrio cholerae inhabits estuarine and coastal waters globally, but its clinical significance has not been sufficiently investigated, despite the fact that it has been associated with septicemia and gastroenteritis. The emergence of virulent non-O1/non-O139 V. cholerae is consistent with the recognition of new pathogenic variants worldwide. Oyster, sediment, and water samples were collected during a vibrio surveillance program carried out from 2009 to 2012 in the Chesapeake Bay, Maryland. V. cholerae O1 was detected by a direct fluorescent-antibody (DFA) assay but was not successfully cultured, whereas 395 isolates of non-O1/non-O139 V. cholerae were confirmed by multiplex PCR and serology. Only a few of the non-O1/non-O139 V. cholerae isolates were resistant to ampicillin and/or penicillin. Most of the isolates were sensitive to all antibiotics tested, and 77 to 90% carried the El Tor variant hemolysin gene hlyAET, the actin cross-linking repeats in toxin gene rtxA, the hemagglutinin protease gene hap, and the type 6 secretion system. About 19 to 21% of the isolates carried the neuraminidase-encoding gene nanH and/or the heat-stable toxin (NAG-ST), and only 5% contained a type 3 secretion system. None of the non-O1/non-O139 V. cholerae isolates contained Vibrio pathogenicity island-associated genes. However, ctxA, ace, or zot was present in nine isolates. Fifty-five different genotypes showed up to 12 virulence factors, independent of the source of isolation, and represent the first report of both antibiotic susceptibility and virulence associated with non-O1/non-O139 V. cholerae from the Chesapeake Bay. Since these results confirm the presence of potentially pathogenic non-O1/non-O139 V. cholerae, monitoring for total V. cholerae, regardless of serotype, should be done within the context of public health.

Nontoxigenic Vibrio cholerae non-O1/O139 isolate from a case of human gastroenteritis in the U.S. Gulf Coast.

An occurrence of Vibrio cholerae non-O1/O139 gastroenteritis in the U.S. Gulf Coast is reported here. Genomic analysis revealed that the isolate lacked known virulence factors associated with the clinical outcome of a V. cholerae infection but did contain putative genomic islands and other accessory virulence factors. Many of these factors are widespread among environmental strains of V. cholerae, suggesting that there might be additional virulence factors in non-O1/O139 V. cholerae yet to be determined. Phylogenetic analysis revealed that the isolate belonged to a phyletic lineage of environmental V. cholerae isolates associated with sporadic cases of gastroenteritis in the Western Hemisphere, suggesting a need to monitor non-O1/O139 V. cholerae in the interest of public health.

Molecular analysis of Vibrio vulnificus isolated from cockles and patients in Thailand.

Vibrio vulnificus can cause septicemia, wound infection and gastroenteritis. The most severe infections are related to consumption of raw or undercooked seafood. Virulence genes, biomarkers, antimicrobial resistance, and genetic relationships among V vulnificus isolated from clinical and environmental sources in Thailand have not hitherto been investigated. ViuB encoding vulnibactin siderophore was detected in 33% and 50% of clinical and environmental (cockle) V. vulnificus isolates, respectively, and capsular polysaccharide allele 1 in 67% and 75% of clinical and environmental isolates, respectively. Analysis of the 16 S rDNA gene revealed that type B was the most frequent in both clinical and environmental isolates (67%) whereas the non type-able (30%) was detected only in environmental isolates. The virulence-correlated gene (vcg) with both type C and E together was the most frequently found among the clinical (67%) and environmental (72%) isolates. Pulsed-field gel electrophoresis differentiated V vulnificus into 2 clusters; most cockle samples (83%) and all clinical isolates grouped into cluster II, indicating a possible clonal relationship between V. vulnificus isolated from patients and cockles. Only 20% of environmental isolates were resistant to ampicillin. These studies suggest that V vulnificus isolated from cockles has virulence genes similar to those in clinical isolates and thus may have the potential of causing disease.

Genetic variation of Vibrio cholerae during outbreaks, Bangladesh, 2010-2011.

Cholera remains a major public health problem. To compare the relative contribution of strains from the environment with strains isolated from patients during outbreaks, we performed multilocus variable tandem repeat analyses on samples collected during the 2010 and 2011 outbreak seasons in 2 geographically distinct areas of Bangladesh. A total of 222 environmental and clinical isolates of V. cholerae O1 were systematically collected from Chhatak and Mathbaria. In Chhatak, 75 of 79 isolates were from the same clonal complex, in which extensive differentiation was found in a temporally consistent pattern of successive mutations at single loci. A total of 59 isolates were collected from 6 persons; most isolates from 1 person differed by sequential single-locus mutations. In Mathbaria, 60 of 84 isolates represented 2 separate clonal complexes. The small number of genetic lineages in isolates from patients, compared with those from the environment, is consistent with accelerated transmission of some strains among humans during an outbreak.

Genetic characteristics of drug-resistant Vibrio cholerae O1 causing endemic cholera in Dhaka, 2006-2011.

Vibrio cholerae O1 biotype El Tor (ET), causing the seventh cholera pandemic, was recently replaced in Bangladesh by an altered ET possessing ctxB of the Classical (CL) biotype, which caused the first six cholera pandemics. In the present study, V. cholerae O1 strains associated with endemic cholera in Dhaka between 2006 and 2011 were analysed for major phenotypic and genetic characteristics. Of 54 representative V. cholerae isolates tested, all were phenotypically ET and showed uniform resistance to trimethoprim/sulfamethoxazole (SXT) and furazolidone (FR). Resistance to tetracycline (TE) and erythromycin (E) showed temporal fluctuation, varying from year to year, while all isolates were susceptible to gentamicin (CN) and ciprofloxacin (CIP). Year-wise data revealed erythromycin resistance to be 33.3 % in 2006 and 11 % in 2011, while tetracycline resistance accounted for 33, 78, 0, 100 and 27 % in 2006, 2007, 2008, 2009 and 2010, respectively; interestingly, all isolates tested were sensitive to TE in 2011, as observed in 2008. All V. cholerae isolates tested possessed genetic elements such as SXT, ctxAB, tcpA(ET), rstR(ET) and rtxC; none had IntlI (Integron I). Double mismatch amplification mutation assay (DMAMA)-PCR followed by DNA sequencing and analysis of the ctxB gene revealed a point mutation at position 58 (C→A), which has resulted in an amino acid substitution from histidine (H) to asparagine (N) at position 20 (genotype 7) since 2008. Although the multi-resistant strains having tetracycline resistance showed minor genetic divergence, V. cholerae strains were clonal, as determined by a PFGE (NotI)-based dendrogram. This study shows 2008-2010 to be the time of transition from ctxB genotype 1 to genotype 7 in V. cholerae ET causing endemic cholera in Dhaka, Bangladesh.