Improved isolation and detection of toxigenic Vibrio parahaemolyticus from coastal water in Saudi Arabia using immunomagnetic enrichment

Background Vibrio parahaemolyticus is recognized globally as a cause of foodborne gastroenteritis and its widely disseminated in marine and coastal environment throughout the world. The main aim of this study was conducted to investigate the presence of toxigenic V. parahaemolyticus in costal water in the Eastern Province of Saudi Arabia by using immunomagnetic separation (IMS) in combination with chromogenic Vibrio agar medium and PCR targeting toxR gene of species level and virulence genes. Methods A total of 192 seawater samples were collected from five locations and enriched in alkaline peptone water (APW) broth. One-milliliter portion from enriched samples in APW were mixed with an immunomagnetic beads (IMB) coated with specific antibodies against V. parahaemolyticus polyvalent K antisera and separated beads with captured bacteria streaked on thiosulfate citrate bile salts sucrose (TCBS) agar and CHROMagar Vibrio (CaV) medium. Results Of the 192 examined seawater samples, 38 (19.8%) and 44 (22.9%) were positive for V. parahaemolyticus, producing green and mauve colonies on TCBS agar and CaV medium, respectively. Among 120 isolates of V. parahaemolyticus isolated in this study, 3 (2.5%) and 26 (21.7%) isolates of V. parahaemolyticus isolated without and with IMB treatment tested positive for the toxin regulatory (toxR) gene, respectively. Screening of the confirmed toxR gene-positive isolates revealed that 21 (17.5%) and 3 (2.5%) were positive for the thermostable direct hemolysin (tdh) encoding gene in strains isolated with IMB and without IMB treatment, respectively. None of the V. parahaemolyticus strains tested positive for the thermostable related hemolysin (trh) gene. In this study, we found that the CaV medium has no advantage over TCBS agar if IMB concentration treatment is used during secondary enrichment steps of environmental samples. The enterobacterial repetitive intergenic consensus (ERIC)-PCR DNA fingerprinting analysis revealed high genomic diversity, and 18 strains of V. parahaemolyticus were grouped and identified into four identical ERIC clonal group patterns. Conclusions The presented study reports the first detection of tdh producing V. parahaemolyticus in coastal water in the Eastern Province of Saudi Arabia.

Characterization of Vibrio spp. in environmental water samples collected from flood prone areas of Bangladesh and their antibiotic resistance profile

<abstract> <p>Last cholera epidemic has been recorded in Bangladesh between 1992–1993, while few sporadic localized outbreaks have been reported as recent as 2005. Serotype O1 of <italic>Vibrio cholera</italic> is considered as the principal causative agent which transmits through contaminated drinking water resulting that epidemic. Therefore, the objective of this research was to isolate <italic>V. cholera</italic> in 3 different water sources; River, pond and tube-well, in 5 different locations of Gazipur, Bangladesh, and to analyze their antibiogram study. A total of 45 water samples were randomly collected for the isolation and identification of <italic>Vibrio</italic> spp. Samples are then serially diluted in alkaline peptone water and streak on Thiosulfate Citrate Bile Salt Sucrose-TCBS agar for quantification of <italic>V</italic>. spp. For <italic>V. cholera</italic> isolation water samples were first enriched in nutrient broth at 37 °C for 16 hours followed by cultivation in selective media; TCBS agar at 37 °C for 24 hours. Yellow colonies on TCBS agar were screed as <italic>V. cholera</italic> and was confirmed by analyzing their biochemical characteristics like Catalase, Oxidase, MR, VP, Indole, Sugar fermentation. Following isolation antibiotic sensitivity test was performed on each <italic>V. cholera</italic> isolates to determine their antibiotic sensitivity profile. The results showed, out of 45 samples 12 contained <italic>V. cholera</italic>. Tube-well water has significantly lower concentration (log CFU/mL) of <italic>V</italic>. spp. than river and pond water (P &lt; 0.05). Bacterial concentration doesn't deviate (P &gt; 0.05) significantly in 5 different location the sample was collected from. All the 12 isolates were sensitive to Gentamicin and ciprofloxacin (100%), while Chloramphenicol (91.67%), Sulfamethoxazole (91.67%), Azithromycin (66.67%) showed high sensitivity. Isolates showed marginal sensitivity towards Tetracycline (33.33%), and Cephalexin (16.67%) and 100% resistance against antibiotics like Vancomycin, Penicillin, Erythromycin, and Nalidixic Acid. Based on these data we recommend using tube-well water instead of river and pond water for drinking purposes. Furthermore, we suggest selective use of sensitive antimicrobials listed here for therapeutics of cholera outbreak.</p> </abstract>

Isolation, identification and antibiogram profile of Aeromonas hydrophila from broiler chickens in Mymensingh Sadar, Bangladesh

Investigation of Aeromonas hydrophila was conducted to assess the microbial quality of broiler chickens from July to November 2019. A total of 60 samples from 20 broiler chickens were collected from two different locations of Mymensingh Sadar: KR market, Bangladesh Agricultural University (BAU) and Shesh mor bazar (10 birds from each location). Samples included 20 skins, 20 legs and 20 breast samples from 20 broiler chickens. PCR was done for the specific detection of each isolate and finally antimicrobial susceptibility testing was performed to check sensitivity pattern of each isolate. Alkaline peptone water was used for processing and enrichment of the samples followed by inoculation onto Aeromonas selective agar supplemented with ampicillin for the isolation and identification of A. hydrophila. Out of these 60 samples, 27 isolates were confirmed as A. hydrophila through biochemical tests and PCR where 55.56% isolates were recovered from Shesh mor market and other 44.4% isolates from KR market, BAU. Source-wise analysis revealed that maximum isolates of A. hydrophila were recovered from skin (59.26 %) followed by leg (22.22 %) and breast samples (18.52 %). PCR test revealed that all 27 isolates were found carrying lip gene which is specific for A. hydrophila. Isolates of A. hydrophila were found sensitive to ciprofloxacin (92%), gentamycin (66%) and chloramphenicol (50%); intermediate against erythromycin (50%), tetracycline (50%) and imipenem (50%); resistant against co-trimoxazole (84%) and ampicillin (100%). From the present study, it was found that samples were considerably contaminated with Aeromonas hydrophila causing risks for public health. Necessary control actions should be taken in every steps of production, processing and marketing for mitigation of this contamination. Asian Australas. J. Food Saf. Secur. 2020, 4 (1), 22-30

Prevalence and genetic variability of Plesiomonas shigelloides in temperate climate surface waters of the Pannonian Plain

Plesiomonas shigelloides, a Gram-negative bacterium and the causative agent of intestinal diseases and extraintestinal infections in humans and animals, is most frequently found in aquatic environments in tropical or subtropical areas. The present study was designed to establish the prevalence and genetic variability of P. shigelloides in surface waters (lakes, rivers, ponds, inlets and canals) located in a temperate climate zone, namely the Pannonian Plain of the northern part of Serbia and southern part of Hungary. The strains were isolated directly by plating samples on inositol-brilliant green-bile agar with neutral red or phenol red as indicators. Our results indicate that phenol red effectively facilitates differentiation of P. shigelloides from other bacteria. A number of samples were enriched using alkaline peptone water broth, peptone inositolbile broth and tetrathionate broth. The recovery of the isolates was more successful with the first medium. Out of a total of 51 water samples collected from 28 different locations, 22 samples (43.1%) were found positive for P. shigelloides. Among the 37 isolated strains, 34 were from lakes (Satrinci, Ludas, Panonija, Krivaja, Pecs, Kapetanski rit, Pavlovci, Kovacsszenaja, Dobrodol, Vranjas, Borkovac, Hermann Otto, Sot, Selevrenac, Zobnatica, Palic, Orfui, Jarkovci, Conoplja) and 3 were from rivers (Danube, Sava). The strains were identified by phenotypic characteristic or by the VITEK2 system and confirmed by PCR using 23S rRNA species-specific oligos. The strains showed a high genetic variability, displaying a variety of RAPD profiles. Our results reveal for the first time a high prevalence of genetically diverse P. shigelloides populations in surface waters located in the temperate climate of central and southeastern Europe.

Molecular detection and antimicrobial resistance of Aeromonas from houseflies (Musca domestica) in Iran

ABSTRACT Objective. This study aimed to report the molecular detection and antimicrobial resistance of Aeromonas among houseflies (Musca domestica) in Shahrekord and Isfahan provinces of Iran. Materials and methods. Flies were caught from household kitchens, cattle farms, animal hospitals, human hospitals, slaughter house and poultry farms and put in collection separate sterile tubes. Isolation was accomplished by culture of flies in alkaline peptone water followed by identification with Aeromonas-specific Polymerase Chain Reaction (PCR). Results. Out of 600 houseflies 73 (12.2%) were infected with Aeromonas spp. Significantly higher frequencies of Aeromonas were isolated in Shahrekord province (13.0%; 39/300) than in Isfahan province (11.3%; 34/300). The recovery frequencies of the organisms were significantly lower in kitchens as compared to those in cattle farms and hospital wards which were similar. Higher proportions of infected flies were obtained during summer whereas low proportions were obtained during winter. Conclusions. It is concluded that houseflies do harbor diarrheagenic pathogens, including Aeromonas especially during summer. The carried organisms are resistant to a number of antimicrobials at different levels. Thus, future plans aimed at stemming infections caused by these organisms should take flies into account. Control efforts of infections caused by this particular bacterium should therefore take into account Musca domestica.

Toxigenic Potential of Vibrio cholerae O1and O139 Serotypes Isolated from Cases of Diarrhea in Baghdad hospitals

Cholera is most important water borne pathogen. The public health significance of a V. cholerae isolate is routinely assessed by two critical properties: the production of cholera toxin CT and the possession of either the O1 or O139 antigen, which acts as a marker of epidemic potential. The objective of this study is to detect V. cholerae serotypes directly from stools and determines their toxiginicity potential. Sixty four stool samples were collected from four hospitals in Baghdad from November 2010 to February 2011. The age of patients was ranging from two months to 12 years, 26 females and 38 males. Immunochromatographic test used for qualitative detection of O1and /or O139 serotypes was used in addition to routine culture for isolation of V. cholerae. Using specific primer cholera toxin gene, ctxA2-B, was amplified and the PCR product was detected by agarose gel electrophoresis. Out of 64 stool samples only 16 (25%) was positive. Fifeen 93.7% of these samples were positive for O1serogroup and just one 6.3% was positive for O139 serogroup. Stool sample culture on alkaline peptone water and then on TCBS agar enhance the growth of 11(17.2%) V. cholerae isolates, 10 (90.9%) were belong to O1 serotype and one 9.1% belong to O139. The results of ctxA2-B gene amplification show that, 9 (90%) out of 10 O1serotypes was positive. While the only one 100% O139 serotype was positive. As conclusion, the incidence of cholera caused by V. cholerae O1 is more than that caused by V. cholerae O139 in Baghdad hospitals. Immunochromatographic test is a rapid and sensitive test in recover V. cholerae O1 and O139 serotypes. PCR is a simple molecular tool to determine the toxigenicity of V. cholerae isolates.

Implementation of a process internal control to monitor the performance of real-time PCR assays for the detection of Vibrio cholerae in water

The process control utilised in this study was an Escherichia coli (E. coli-GFP) strain carrying a chromosomally integrated copy of the green fluorescent protein (gfp) gene of the jelly fish Aequorea victoria. Application of the process control involved spiking samples with the E. coli-GFP containing cells and detecting gfp using real-time polymerase chain reaction (PCR). The process control was implemented in the context of two multiplex real-time PCR assays. The first multiplex targeted the ctxA, hlyA and gfp genes, while the second targeted the gfp, O1-rfb, and O139-rfb loci. The detection limits for both the multiplex real-time PCR assays were 20 CFU (colony forming units) per reaction. This method was evaluated using spiked and unspiked Alkaline Peptone Water (APW) enrichments of sewage, dam, catchment and tap water. The internal process control showed no substantial inhibition in the APW enriched water samples, assuring the integrity of negative results. The results from this study showed that the use of the E. coli-GFP strain as a process internal control could provide quality assurance and increase the reliable interpretation of real-time PCR results.

LAJU REDUKSI MERKURI OLEH PSEUDOMONAS DIISOLASI DARI PERAIRAN PANTAI TELUK MANADO

ABSTRACTThis study was conducted to isolate and identify Pseudomonas isolated from Manado Bay seawater and to determine the rate of mercury (Hg) reduction produced by isolated strains of Pseudomonas. A 500 ml seawater sample was collected from Tondano River and Manado Bay along the beach of the reclamation site, placed into container and taken to the laboratory for further analysis. Water samples were homogenized by hand shaking and 1 ml water sample was pipetted and then transferred to each 7 ml of Tryptic Soy Broth (pH 7.4) and Alkaline Peptone Water (pH 7.4), separately. After incubation period of 24h at 35oC, these were transferred with ose and streaked on Pseudomonas F Agar, followed by incubation at 35oC for 24h. Free growing colonies were then transferred to slant agar and kept at 4oC as stock culture. Results found that isolated Pseudomonas were chemoorganotrophic and chemoautolithotrophic, Almost all isolated Pseudomonas showed their ability in mercury ions reduction, but some of them did in mercury oxidation. Isolated chemooragnotrophic Pseudomonas tended to reduce mercury ions, but the chemoautolithotrophic one tended to oxidize it. Based on these results, it was found that the rate of Hg reduction was dependent upon the strain type.ABSTRAKTujuan penelitian ini adalah mengisolasi dan mengidentifikasi Pseudomonas dari air laut Teluk Manado serta menentukan laju reduksi merkuri (Hg) yang diproduksi oleh strain Pseudomonas terisolasi tersebut. Sampel air laut sebanyak 500 ml dikumpulkan dari Danau Tondano dan sepanjang pantai dari daerah reklamasi Teluk Manado, lalu dibawa ke laboratorium untuk dianalisa lebih lanjut. Sampel air dihomogenkan secara manual, dan 1 ml sampel air dipipet dan kemudian dipindahkan secara terpisah ke 7 ml Tryptic Soy Broth (pH 7,4) dan Alkaline Peptone Water (pH 7,4). Sete-lah masa inkubasi 24 jam pada suhu 35oC, sejumlah sampel dipindahkan ke Pseudomonas F Agar, kemudian diinkubasi pada suhu 35oC selama 24 jam. Koloni yang tumbuh bebas kemudian dipindahkan ke agar miring dan disimpan pada suhu 4oC sebagai kultur stok. Hasil penelitian menunjukkan bahwa Pseudomonas terisolasi bersifat chemoorganotrophic dan chemoautolithotrophic. Hampir semua Pseudomonas terisolasi menunjukkan kemampuan dalam penguraian ion merkuri, tetapi beberapa diantaranya dapat juga mengoksidasi merkuri. Pseudomonas yang bersifat chemooragnotrophic cenderung untuk mengurangi ion merkuri, tapi yang bersifat chemoautolithotrophic cenderung mengoksidasinya. Berdasarkan hasil tersebut dapat disimpulkan bahwa laju reduksi-Hg oleh Pseudomonas tergantung pada jenis strainnya.