Thiosulphate Citrate Bile Salt Sucrose Research Articles

The Inhibitory Effect of Lemon Juice (Citrus limon) on Vibrio parahaemolyticus in Raw Oyster (Crassostrea virginica)

Introduction: Serving raw oysters with lemon juice is a delicacy in many restaurants in Malaysia. Oysters (Crassostrea virginica) live in the seacoast and they share the same environment as Vibrio parahaemolyticus. Consumption of raw oysters contaminated with V. parahaemolyticus can lead to severe gastroenteritis. A study was performed to determine whether lemon (Citrus limon) juice is able to inhibit the growth of V. parahaemolyticus after being inoculated in raw oysters. Methods: Frozen oysters bought from a local supplier weighing 6 g each were minced and placed in two bottles using sterile technique. Approximately 1 ml of 107 CFU of V. parahaemolyticus (ATCC strain 17802) was added and mixed in both bottles. The mixture was treated with 1 ml of lemon juice in only one of the bottles and the other bottle served as a control. At every 30 s intervals for 2 min, 1 g of the sample was taken for enumeration of viable cells onto thiosulphate citrate bile salt sucrose (TCBS). Results: After 30 s of treatment with the lemon juice, it was observed that the number of colonies in the treated samples reduced from 7 Log to 3 Log. Subsequently, no viable V. parahaemolyticus was seen. It was also observed that there were 3 Log reductions of V. parahaemolyticus after 30 s in untreated samples, however the number of colonies remained stable until the end of the experiment. Conclusion: This study therefore shows that lemon juice has some antimicrobial effect on V. parahaemolyticus in raw oysters.

16S rRNA gene sequence based identification of Vibrio spp. in shrimp and tilapia hatcheries of Bangladesh

Presence of Vibrio spp., one of the deadliest fish and shrimp pathogens in aquaculture facilities worldwide for which hatchery owners often suffer hectic economic losses, were identified in shrimp and tilapia hatcheries of Cox’s Bazar and Mymensingh, Bangladesh. Thirty seven Vibrio isolates, selected on the basis of their morphological dissimilarities in thiosulphate citrate bile salt sucrose agar (TCBS) plate, were subjected to amplified 16S ribosomal DNA restriction analysis (ARDRA) by using AluI restriction enzyme following their DNA extraction and amplification of 16S rRNA (1450 bp). From this analysis, representative isolates of 8 ARDRA groups, named as ARH 1 to ARH 8, of different band patterns were sequenced and identified as Vibrio alginolyticus, Aeromonas veronii, A. hydrophila, Vibrio vulnificus, V. cholerae, Edwardsiella hoshinae, Bacillus methylotrophicus and Aeromonas veronii, respectively. Vibrio species identified in this study are pathogenic for human and aquatic organisms, and were found only in shrimp hatchery with the dominance of V. alginolyticus. Findings of this study indicate the poor quality of water treatment and management of the hatchery. It was also observed that all the three Vibrio species were present in the Artemia rearing tank which indicates the possible source of pathogens.
 Dhaka Univ. J. Biol. Sci. 26(1): 45-58, 2017 (January)

Mismatch Amplification Mutation Assay (MAMA) PCR Reveals Altered El Tor Vibrio Cholerae O1 Circulating in Delhi, India

The acute diarrheal disease cholera is caused by Vibrio cholerae, a major public health problem in Asia, Africa and Latin America. V. cholerae has more than 200 known serotypes but not all the strains are pathogenic. In recent years, it has been seen that the emergence of new variants of V. cholerae O1 have carried characteristics of both classical and El Tor biotypes and these variants of V. cholerae O1 are called as ‘atypical El Tor’. These strains might have evolved from El Tor variants that acquired certain characteristics from classical genome. 30 V. cholerae O1 isolates (Table 1) were revived from stock cultures maintained at Laboratory Department in Maharishi Valmiki Infectious Diseases Hospital (MVIDH), Delhi during 2012-2014. Mismatch amplification mutation assay was used for classifying the strains into prototype El Tor, hybrid, or El Tor variant biotype based on their ctxB gene. All isolates were biochemically identified as V. cholerae biotype El Tor and serologically O1 Ogawa. All isolates were amplified with classical specific primers. Cholera continues to be endemic in large number of states in the eastern, western, northern and southern parts of India and there were 38 cholera outbreak reports published in India during 2007 to 2013. These Indian outbreaks have been associated with new El Tor variant. Key-wordsV. cholerae O1, El Tor, MAMA PCR, ctxB -------------------------------------------------IJLSSR----------------------------------------------INTRODUCTION The acute diarrheal disease cholera is caused by Vibrio cholerae, a major public health problem in Asia, Africa and Latin America. According to the World Health Organisation (WHO) estimates there were 3-5 million cholera cases and 1,00,0001,20,000 deaths each year throughout the world. V. cholerae has more than 200 known serogroups but not all the strains are pathogenic. Among them, O1 and O139 antigens are highly pathogenic and acknowledged to cause epidemic and pandemic disease. The symptom of the acute cholera is rice water stools. Access this article online Quick Response Code: Website: www.ijlssr.com DOI: 10.21276/ijlssr.2016.2.6.6 Clinical manifestations of the disease range from mild symptoms such as abdominal cramps, nausea and vomiting, to more severe symptoms such as dehydration, shock and death. A devastating cholera outbreak, that had occurred in Haiti since October 2010, involved 6, 98, 893 cases and 8,540 deaths. V. cholerae serogroup O1 is classified into two biotypes, which are termed ‘classical’ and ‘El Tor’. The current seventh pandemic of cholera is caused by the El Tor biotype was originated in 1961 from Celebes Islands in Indonesia. Spread of this biotype was recorded for the first time during 1964 in India and it made its first appearance in Delhi during June, 1965. In recent years, it has been seen that the emergence of new variants of V. cholerae O1 have carried characteristics of both classical and El Tor biotypes and these variants of V. cholerae O1 are called as ‘atypical El Tor’. These strains might have evolved from El Tor variants that acquired certain characteristics from classical genome. In this study, we focused on type of CT genotype existing among V. cholerae Research Article (Open access) Int. J. Life. Sci. Scienti. Res., VOL 2, ISSUE 6 http://ijlssr.com Copyright © 2015-2016 International Journal of Life-Sciences Scientific Research Page 679 O1 isolated from the patients who were admitted in MVIDH, Delhi during 2012-2014. MATERIALS AND METHODS Collection and processing of samples 30 V. cholerae O1 isolates were revived from stock cultures were maintained at Laboratory Department in MVIDH, Delhi during 2012-2014. These samples were revived and transferred to alkaline peptone water (APW, pH-8.6) and incubated at 37°C for 4-6 hrs. After incubation, the enriched cultures were further inoculated to the thiosulphate-citrate-bile-salts-sucrose (TCBS) agar and bile salt agar (BSA, pH-8.6) (Hi-Media, Mumbai). Confirmation of V. cholerae Typical colonies appearing on TCBS/BSA were confirmed by standard biochemical tests. These isolates were further tested serologically with commercially available V. cholerae O1 polyvalent and monovalent and O139 antiserum (BD, USA and Denka Seiken, Ltd. Japan). Genomic DNA preparation Genomic DNA from each isolate was extracted using protocol described previously. PCR assays for detection of ctxB genotype Mismatch amplification mutation assay was used for classifying the strains into prototype El Tor, hybrid, or El Tor variant biotype based on their ctxB gene. In this PCR, the common forward primer for both classical and El Tor alleles was used. Two allele specific primers Re-Cla and Re-Elt were used respectively. The PCR conditions and cycles were described previously. V. cholerae O1 strains 569B (classical) and N16961 (El Tor) were used as control strains. The primers used were synthesized by Invitrogen, India and dNTPs, Taq polymerase, 10X Taq buffer used in this study were obtained from Genei, Bangalore. The primer sequences were used in this PCR is given in Table 2.

Bacteria in chicken rolls sold by fast food restaurant and their public health significance

This study determined bacterial quality of chicken rolls sold in a fast food restaurant at Bangladesh Agricultural University (BAU) campus. Fifteen chicken rolls (ten premicrowaved and five post-microwaved) were collected. Samples were inoculated into selective media, Eosin Methylene Blue (EMB) agar, Salmonella Shigella (SS) agar, Thiosulphate Citrate Bile Salts Sucrose (TCBS) agar and Mannitol Salt (MS) agar. The total viable count (TVC) and total Staphylococcal count (TSC) of pre-microwaved samples were 4.4 log CFU/g and 4.2 log CFU/g, respectively. In post-microwaved samples, the TVC and TSC were 2.7 log CFU/g and 2.6 log CFU/g, respectively. Microwave treatment significantly reduced the TVC and TSC in the chicken rolls (P<0.05). Bacteria were recovered only from samples inoculated onto MS agar. Colonies on MS agar were characteristics of Staphylococcus spp, confirmed by sugar fermentation, catalase and coagulase tests and polymerase chain reaction (PCR) assay. This study recorded coagulase negative staphylococcus (CNS) resistant to three antibiotics, ampicillin, cephalexin and vancomycin. It is suggested that chicken rolls sold in the fast food restaurant contaminated with resistant CNS might pose a public health hazard.Bangl. vet. 2015. Vol. 32, No. 1, 13-18

Cholera outbreak caused by drug resistant Vibrio cholerae serogroup O1 biotype ElTor serotype Ogawa in Nepal; a cross-sectional study.

BackgroundCholera is a major cause of mortality and morbidity in underdeveloped countries including Nepal. Recently drug resistance in Vibrio cholerae has become a serious problem mainly in developing countries. The main objectives of our study were to investigate the occurrence of Vibrio cholerae in stool samples from patients with watery diarrhea and to determine the antimicrobial susceptibility patterns of V. cholerae isolates.MethodsA total of 116 stool samples from patients suffering from watery diarrhea during July to December 2012 were obtained from outbreak areas from all over Nepal. Alkaline peptone water and thiosulphate citrate bile salt sucrose agar (TCBS) were used to isolate the Vibrio cholerae. The isolates were identified with the help of colony morphology, Gram’s staining, conventional biochemical testing, serotyping and biotyping. Antimicrobial susceptibility testing was performed by determining the minimum inhibitory concentration (MIC) by agar dilution method.ResultsVibrio cholerae was isolated from 26.72 % of total samples. All isolated Vibrio cholerae were confirmed to be Vibrio cholerae serogoup O1 biotype El Tor and serotype Ogawa. All isolates were resistant to ampicillin and cotrimoxazole. Twenty nine isolates were resistant toward two different classes of antibiotics, one strain was resistant to three different classes of antibiotics and one strain was resistant to four different classes of antibiotics. According to the definition of the multidrug resistant bacteria; 6.45 % of the strains of Vibrio cholerae were found to be multidrug resistant.ConclusionsCholera due to multidrug resistant Vibrio cholerae is also possible in Nepal. According to the antimicrobial susceptibility pattern of Vibrio cholerae in our study we recommend to use any antibiotics among tetracycline, doxycycline, levofloxacin, azithromycin, chloramphenicol and ciprofloxacin for preliminary treatment of cholera in Nepal.

ECTOPARASITES AND VIBRIOS ASSOCIATED WITH FATTENING CULTURED MUD CRABS [Scylla serrata (Forsskal, 1775)] FROM PEMALANG COAST, INDONESIA

Infectious of ectoparasites and vibrios were found as a problem in fattening mud crab. The aims of this study were to determine the clinical sign of infected mud crabs and to identify the ectoparasites and generic vibrio in the fattening mud crabs from Pemalang Coast, Indonesia. Ectoparasites were collected by smear method. Whereas, 20 isolates were collected from hepatopancreas, gills, hemolymph and injured carapace of the mud crabs. Thiosulphate Citrate Bile Salt Sucrose (TCBS) medium was used to isolate suspected vibrios. The clinical signs of mud crabs infected by ectoparasites were shown through the damaged gills and the attachment of other organisms on the gills, whereas the clinical signs of mud crabs infected by vibriosis were shown through red spot on carapace and wound in carapace, claw and abdomen, followed by weak condition. It also indicated that seven ectoparasites were Ichthyobodo sp., Epistylis sp., Carchesium sp., Vorticela sp., Octolasmis sp., Lepeophtherius sp, and Copepodit sp., whilst five Vibrios were V. harveyi, V. Cholerae, V. parahaemolyticus, V. alginolyticus and V. fischeri.

새우에서 분리된 Vibrio species 동정을 위한 VITEK 2 system방법과 species-specific PCR방법 비교 평가

Vibrio is a genus of Gram-negative, curved, halophilic, and non-spore-forming bacteria. Some of the Vibrio species, such as V. cholerae and V. parahaemolyticus, often contaminate seafood products and occasionally cause human diseases when the seafood products are ingested. A total of 24 Vibrio strains were isolated from shrimp samples on Thiosulphate citrate bile salt sucrose (TCBS) media in this study. All of the 24 isolates were confirmed to belong to the genus Vibrio by using 16S rRNA gene sequence analyses. Vitek 2 system and species-specific poly- merase chain reaction (PCR) methods were used to further identify a total of 29 Vibrio strains at the species level, including the 24 shrimp Vibrio isolates and five Vibrio reference strains. The specificities of the two methods to iden- tify Vibrio strains at the species level were compared in this study. The species-specific PCR method was designed to detect five different Vibrio species, such as Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio algi- nolyticus, and Vibrio mimicus. From the 24 Vibrio shrimp isolates, the Vitek 2 system method could identify 15 (62.5%) strains as Vibrio species and 7 (29.2%) strains as non-Vibrio species, but could not identify the rest 2 (8.3%) strains. But species-specific PCR method could identify 16 (66.7%) strains as Vibrio species and could not identify the rest 8 (33.3%) strains. Among the 24 Vibrio shrimp strains, these two methods could unanimously identify 7 (7/24, 29.2%) strains (2 V. parahaemolyticus, 4 V. alginolyticus, and 1 V. mimicus). Considering that such different identifi- cation results were obtained using the two different methods in this study, identification method for Vibrio species must be carefully chosen.

Prevalence of Vibrio spp. in raw shrimps (Parapenaeus longirostris ) and performance of a chromogenic medium for the isolation of Vibrio strains

Vibrios are natural inhabitants of estuarine ecosystems and some species may pose public health problem as agents of sporadic or collective food-borne infections associated with the consumption of fish or shellfish. Samples of raw shrimp (n=299), fished in coastal areas of the city of Agadir, Morocco, and collected from its fish marketplace, were examined for the presence of pathogenic vibrios. Microbiological analysis was carried out according to a protocol using thiosulphate citrate bile sucrose (TCBS) agar and CHROMagar Vibrio (CV) as selective media. Presumptive-positive colonies were identified by biochemical and species-specific PCR systems, and further tested by PCR for the presence of pathogenicity factors. The overall prevalence of Vibrio spp. in shrimps was 55·8%. Vibrio parahaemolyticus was recovered from 25 of 299 samples (8·4%), Vibrio cholerae from six samples (2%) and Vibrio alginolyticus from 161 samples (53·8%). No virulence genes were found among V.parahaemolyticus and V.cholerae isolates. The CHROMagar Vibrio plating medium was found to be more efficient than the Thiosulphate Citrate Bile salt Sucrose (TCBS) Agar in the isolation of Vibrio organisms. A significant proportion of shrimps marketed and consumed in Morocco are caught in the coastal region of the city of Agadir. This study provides interesting data of prevalence of Vibrio spp. in raw shrimps as well as better understanding of their potential virulence. It is apparent from this study that genes and primers used in multiplex PCR for identification and detection of virulence factors, can be used to monitor shrimps for the presence of potentially pathogenic strains of Vibrio cholerae and Vibrio parahaemolyticus. The results highlight the added value of using a chromogenic medium for research and isolation of pathogenic Vibrio in seafood, more specific and accurate than TCBS.

Bacterial contamination of street-vended spicy puffed-rice sold at Bangladesh Agricultural University campus

This study was undertaken to investigate the bacterial contamination of spicy puffed rice (Jhalmuri) sold by the street vendors at Bangladesh Agricultural University (BAU) campus. Fifteen spicy puffed rice samples were collected from street vendors at the Botanical Garden, Library premises, Riverside, Krishi Bishhobiddaloy (KB) High School and Veterinary Teaching Hospital compound at BAU campus. Microbial quality was assessed by total viable count (TVC), total coliform count (TCC) and total staphylococcal count (TSC). Samples were inoculated into selective media Eosin Methylene Blue (EMB) agar, Salmonella Shigella (SS) agar, Thiosulphate Citrate Bile Salts Sucrose (TCBS) agar and Mannitol Salt (MS) agar. E. coli and Staphylococcus spp. were identified from the samples. The TVC in spicy puffed rice sample ranged from log 4.5 cfu/g to log 5.4 cfu/g, TSC ranged from log 4.4 cfu/g to log 5.2 cfu/g and TCC ranged from log 1.4 cfu/g to log 4.3 cfu/g. Antibiotic sensitivity test showed that the isolates were sensitive to ciprofloxacin and gentamicin. E. coli were resistant to ampicillin, chloramphenicol and cephalexin and Staphylococcus spp. were resistant to ampicillin, cephalexin and vancomycin. Spicy puffed rice sold by the street vendors at BAU campus harboured multidrug resistant food borne bacteria which may cause public health hazard. DOI: http://dx.doi.org/10.3329/bvet.v31i1.22839 Bangl. vet. 2014. Vol. 31, No. 1, 20-26

Photobacterium damselae Infection in Yellow Tail Surgeon (zebrasoma xanthurum) of Red Sea at Hurghada, Egypt

Photobacterium damselae causes photobacteriosis of marine ornamental yellow tail surgeon (zebrasoma xanthurum) the disease appeared and spread rapidly in yellow tail surgeon in the indoor aquarium of National Institute of Oceanography and Fisheries (NIOF) at Hurghada (Egypt). The pathogen was isolated from skin lesions in the body, and internal organs namely liver,spleen and kidney of clinically diseased and moribund fish using tryptic soy agar and thio-sulphate citrate bile salt sucrose agar plates. Lethargic, off food, hemorrhagic spots on skin, skin depigmentation, and fin rot were the main clinical signs appeared on the naturally infected fish. All isolates of the bacterium constituted a homogeneous phenotypic group and were identified by morphological characterization, biochemical tests and API20E as Photobacterium damselae. The isolated strain was sensitive to Sulfamethoxazole Gentamycin, and Streptomycin.

Coliform and Vibrio cholerae Analysis of Drinking Water Collected from Cholera Outbreak Region of Bhaktapur Municipality

Water borne infections in Nepal, especially in Kathmandu valley is one the major public health problems, causing thousands of deaths every year. Among three cities in the valley, the water borne infection including cholera is most predominant in Bhaktapur district. So the study was carried out to know the microbial drinking water quality in the city and to determine the prevalence of water borne infections in the specified region of the district in 2012. Altogether eighty (two samples from a single site at different interval-2/3 days) water samples were collected from Bhaktapur Municipality, one of the most vulnerable regions for water borne diseases, following standard methods as described by APHA, 2010. All samples were transferred to Microbiology laboratory of Khwopa College, Dekocha, Bhaktapur and preceded immediately for Microbial analysis. The coliform density in the water samples were determined by Most Probable Number (MPN) method followed by microscopy, colonial morphology and biochemical characterization. Subsequently, the presence of Vibrio cholerae, a causative agent of Cholera was analyzed in the same samples by enrichment in alkaline peptone water followed by culture on Thiosulphate citrate bile-salt sucrose (TCBS) agar, a selective media for Vibrio spp. The biochemical tests were then performed to identify V. cholerae. Among eighty water samples, 87.5 percent water samples contained coliforms and half of which (45%) contained feacal coliforms, Escherichia coli and remaining 12.5 percent water samples contained no coliforms. Vibrio cholerae were isolated from four water samples (5%). The drinking water quality in the region was found to be very poor. Therefore, the people in the region were suggested to treat the drinking water by using any of physical or chemical disinfection methods prior to drinking. DOI: http://dx.doi.org/10.3126/ije.v3i3.11073 International Journal of Environment Vol.3(3) 2014: 139-145

Occurrence of Vibrio pathotypes in the final effluents of five wastewater treatment plants in Amathole and Chris Hani District Municipalities in South Africa.

We assessed the occurrence of Vibrio pathogens in the final effluents of five wastewater treatment plants (WWTPs) located in Amathole and Chris Hani District Municipalities in South Africa over a 12 months period between September 2012 and August 2013 using standard membrane filtration technique followed by cultivation on thiosulphate citrate-bile salts-sucrose (TCBS) agar. The identities of the presumptive Vibrio isolates were confirmed using polymerase chain reaction (PCR) including delineation into V. parahaemolyticus, V. vulnificus and V. fluvialis pathotypes. The counts of Vibrio spp. varied with months in all the study sites and ranged in the order of 101 and 104 CFU/100mL. Vibrio distribution also showed seasonality with high counts being obtained in autumn and spring (p < 0.05). Prevalence of Vibrio spp. among the five WWTPs also differed significantly (p < 0.05). Of the 300 isolates that were confirmed as belonging to the Vibrio genus, 29% (86) were V. fluvialis, 28% (84) were V. vulnificus and 12% (35) were V. parahaemolyticus. The isolation of Vibrio pathogens from the final effluent suggests that this pathogen is in circulation in some pockets of the population and that the WWTPs under study do not efficiently remove bacterial pathogens from the wastewater and consequently are threats to public health.

Distribution of Vibrio species in Shellfish and Water Samples Collected from the Atlantic Coastline of South-East Nigeria

Crayfish, lobster, and sea-water samples collected from five fishing islands on the Atlantic coast-Bight of Biafra (Bonny)-belonging to Ibaka Local Government Area of Akwa-Ibom State of Nigeria were bacteriologically evaluated on thiosulphate citrate bile-salt sucrose (TCBS) agar for Vibrio load and pathotypes. Mean log10 Vibrio counts of 7.64+/-2.78 cfu/g (in crayfish), 5.07+/-3.21 cfu/g (in lobster), and 3.06+/-2.27 cfu/mL (in sea-water) were obtained in rainy season (June-July) while counts in the dry season (November-December) were 6.25+/-1.93 cfu/g, 5.99+/-1.54 cfu/g, and 3.84+/-1.78 cfu/mL respectively. The physicochemical measurements (temperature, pH, and total dissolved solutes) of the sea-water did not vary significantly in the two seasons across all five islands. Vibrio species isolated were Vibrio cholerae (both O1 and non-O1 serotypes), V parahaemolyticus, V vulnificus, V mimicus, and V fluvialis. Both Ogawa and Inaba subtypes of V cholerae O1 serotype were found. In addition, the Hikojima subtype, which had not been previously reported in the region, was isolated in two samples. The results show that these Vibrio species are endemic in the area.

Molecular Characterization of the Circulating Strains of Vibrio cholerae during 2010 Cholera Outbreak in Nigeria

This study aimed at characterizing the phenotypic and toxigenic status of circulating strains of cholera during outbreaks in Nigeria, employing molecular typing techniques. Two hundred and one samples of rectal swabs, stool, vomitus, water (from the well, borehole, sachet, stream, and tap) and disinfectants (sodium hypochlorite) were collected from three states in the country. The samples were inoculated on thiosulphate-citrate bile salt-sucrose (TCBS), Cary-Blair transport medium and smeared on glass slides for direct examination. The Vibrio cholerae isolates were serotyped, biotyped, and characterized using PCR of the cytotoxin gene A (ctxA), wbeO1, and wbfO139 gene primer. Of the 201 samples screened, 96 were positive for V cholerae O1 (48%), with 69 (72%) positive for ctxA gene. The results from this study showed that the circulating strains of cholera in Nigeria were of Ogawa serotype, also observed in other outbreaks in Nigeria (1991, 1992, and 1996). However, the strains were of the Classical biotype and were mainly (72%) ctxA gene-positive. This current investigation has confirmed the production of cholera toxin by the circulating strains, and this could be harnessed for possible cholera vaccine production in Nigeria.

Microbiological and Physicochemical Assessment of Soil Contaminated with Lairage Effluent in Umuahia, Abia State, Nigeria.

The microbiological and physicochemical characteristics of soil contaminated with lairage effluent were investigated. A total of twenty samples were analyzed for total heterotrophic bacterial count, fungal count, Vibrio cholerae count, Salmonella-Shigella count, Escherichia coli count and coliform count. The media used were nutrient agar, potato dextrose agar, eosin methylene blue agar, Salmonella-Shigella agar, MacConkey agar, thiosulphate citrate bile-salt sucrose agar. The pour plate technique was used for the inoculation of samples. The mean total heterotrophic bacterial count for the contaminated soil ranged from 5.65 ± 0.17Log10cfu/g to 5.94 ± 0.29Log10cfu/g while the control soil was 6.85 ± 0.03Log10cfu/mL. The mean fungal count ranged from 3.04 ± 0.19Log10cfu/g to 3.64 ± 0.41Log10cfu/g for the contaminated soil and 3.38 ± 0.20Log10cfu/mL for control soil. Salmonella-Shigella mean count ranged from 2.30 ± 0.09Log10cfu/g to 2.48 ± 0.07Log10cfu/g for the contaminated soil and 2.48 ± 0.09Log10cfu/g for the control soil. Vibrio cholerae mean count for contaminated soil ranged from 2.60 ± 0.35Log10cfu/g to 3.30 ± 0.03cfu/g while the control soil was 2.30 ± 0.10Log10cfu/g. Escherichia coli mean count for contaminated soil ranged from 3.00 ± 0.22Log10cfu/g to 3.38 ± 0.15Log10cfu/g and 3.38 ± 0.05Log10cfu/g for the control soil. The coliform mean count for contaminated soil ranged from 4.53 ± 0.11Log10cfu/g to 4.66 ± 0.10Log10cfu/g and 4.40 ± 0.024Log10cfu/mL for the control soil. The mean microbial counts for the lairage effluent ranged as follows total heterotrophic bacterial count, 6.39 ± 0.10Log10cfu/mL; fungal count, 3.28 ± 0.03Log10cfu/mL; Salmonella-Shigella count, 2.60 ± 0.08Log10cfu/mL; Vibrio cholerae count, 3.23 ± 0.20Log10cfu/mL; Escherichia coli count, 3.30 ± 0.01Log10cfu/mL and coliform count, 4.74 ± 0.09Log10cfu/mL. The mean values of the physicochemical parameters for the contaminated soil were temperature, 29 ± 1.0C; pH, 5.8 ± 0.5; conductivity, 55.7 ± 3.0μS/cm, oil and grease, 2.0 ± 0.2mg/kg; total organic carbon, 1.66 ± 0.1%; phosphate, 1.95 ± 0.2mg/kg; nitrate, 0.66 ± 0.01mg/kg; sulphate, 22.5 ± 3.0mg/kg; calcium, 303 ± 20.0mg/Kg; magnesium, 92 ± 3.0mg/kg, potassium, 5.0 ± 0.5mg/kg, sodium, 0.72 ± 0.02mg/Kg. The mean values for the control soil sample were temperature, 28 ± 0.5C; pH, 6.7 ± 0.5; conductivity, 45.8 ± 2.0μS/cm; oil and grease, 1.0 ± 0.01mg/kg; total organic carbon, 0.88 ± 0.02%; phosphate, 0.57 ± 0.03mg/kg; nitrate, 0.30 ± 0.01mg/kg; sulphate, 12.74 ± 1.0mg/kg; calcium, 148 ± 10.0mg/kg; magnesium, 81± 2.0mg/kg; potassium, 2.0 ± 0.1mg/kg; sodium, ± 0.02mg/Kg. The mean values for the lairage effluent were temperature, 30 ± 2.0C; pH, 7.8 ± 1.0; conductivity, 40.8 ± 4.0μS/cm; turbidity, 72.0 ± 4.0NTU; chloride, 8.0 ± 0.5mg/L; dissolved oxygen, 1.2 ± 0.3mg/L; biochemical oxygen demand, 36.0 ± 5.0mg/L; chemical oxygen demand, 82.0 ± 10.0mg/L; total suspended solids, 63.0 ± 8.0mg/L; total dissolved solids, 24.0 ± 2.0mg/L; nitrate, 0.85 ± 0.03 mg/L; sulphate, 1.0 ± 0.05mg/L; phosphate, 2.2 ± 0.5mg/L and ammonia, 0.83 0.02mg/L. The lairage effluent from this result is responsible for the contamination of the soil.

Light‐scattering sensor for real‐time identification of Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio cholerae colonies on solid agar plate

SummaryThe three most common pathogenic species of Vibrio, Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus, are of major concerns due to increased incidence of water‐ and seafood‐related outbreaks and illness worldwide. Current methods are lengthy and require biochemical and molecular confirmation. A novel label‐free forward light‐scattering sensor was developed to detect and identify colonies of these three pathogens in real time in the presence of other vibrios in food or water samples. Vibrio colonies grown on agar plates were illuminated by a 635 nm laser beam and scatter‐image signatures were acquired using a CCD (charge‐coupled device) camera in an automated BARDOT (BActerial Rapid Detection using Optical light‐scattering Technology) system. Although a limited number of Vibrio species was tested, each produced a unique light‐scattering signature that is consistent from colony to colony. Subsequently a pattern recognition system analysing the collected light‐scatter information provided classification in 1−2 min with an accuracy of 99%. The light‐scattering signatures were unaffected by subjecting the bacteria to physiological stressors: osmotic imbalance, acid, heat and recovery from a viable but non‐culturable state. Furthermore, employing a standard sample enrichment in alkaline peptone water for 6 h followed by plating on selective thiosulphate citrate bile salts sucrose agar at 30°C for ∼ 12 h, the light‐scattering sensor successfully detected V. cholerae, V. parahaemolyticus and V. vulnificus present in oyster or water samples in 18 h even in the presence of other vibrios or other bacteria, indicating the suitability of the sensor as a powerful screening tool for pathogens on agar plates.

Bacterial species isolated from water bodies in Rivers and Bayelsa States are possible aetiological agents of diarrhoea

Background : The role of water in the transmission of diarrhoea has been established. The World Health Organization (WHO) reported that diarrhoea disease were among the 10 major causes of death in young children in 11 African, Asia and South American countries. Aim : To investigate the incidence of Salmonella and Vibrio species as aetiological agents of diarrhoea in water bodies in Rivers and Bayelsa States. Methods : A total of twenty water samples were collected from rivers and cultured for the various microorganisms.The total heterotrophic counts were determined by Standard Plate Count (SPC) method. Salmonella and Vibrio species were enumerated by an enrichment procedure in Selenite F broth and Alkaline Peptone water (APW) respectively; cultured on Bismuth sulfite agar (BSA) and Thiosulphate citrate bile salt sucrose (TCBS) agar selective for salmonella and vibrio species respectively. The total coliform counts was determined and the Most Probable Number (MPN) using the multiple tube fermentation technique. Results : The study revealed isolates with various percentage occurrences which include Vibrio (12.1%) and Salmonella (33.3%). Among the coliforms were Escherichia coli (33.3%), Klebsiella (22.2%), Enterobacter (33.3%) and Citrobacter (11.1%).MPN values for enumeration of coliforms ranged from 80/100 ml to = 16,000/100 ml of the water samples analysed. Total heterotrophic count per milliliter of the original water sample ranged between 1.0 × 106 and 3.0 ×108 cfu/ml. Conclusion : The water sources examined have proved to be potential sources of diarrhoea organisms and also could lead to disease outbreak because of the significant number of these organisms in these water bodies.

Characterization of the bacterial flora of tilapia (Oreochoromis niloticus) harvested from four lakes in the north of Cameroon

The aim of this study was to investigate the presence of Aeromonas spp., Vibrio spp. and Plesiomonas shigelloides in Cameroonian tilapia.120 samples of tilapia were collected from June to December 2005 in four localities in the north of Cameroon and were delivered to the laboratory in cool boxes. For the sample preparation to isolate the bacteria, 50 g of sample (gills, intestine and skin of individually fish) were taken using a sterile scalpel. These 50 g were homogenized in 450 ml of sterilised peptone solution. After 18 h of incubation, the homogenate sample was inoculated on thiosulphate citrate bile salt sucrose (TCBS) using an inoculation loop and incubated at 37°C for 18 to 24 h. The isolated colonies were subjected to morphological, physiological and commercially available API kits tests. In addition, the antimicrobial pattern of the identified strains to ten different antibiotics was studied using the agar disk diffusion method. The results obtained from this study show the presence of bacterial species belonging to Aeromonas spp. (n=39), Vibrio spp. (n=41) and P. shigelloides (n=14). The results of antimicrobial susceptibility show that the highest rates of resistance was found in ampicillin (100% of isolates), nitrofurantoin (more than 68% of isolates), association of trimethoprim and sulfamethoxazole (51% of isolates) and sulfamide (46 % of isolates). This work therefore highlights an important incidence of Aeromonas spp., Vibrio spp. and P. shigelloides with potential antimicrobial resistance, isolated from tilapia in four localities of Cameroon.

Tetracycline-ResistantVibrio choleraeO1, Kolkata, India

To the Editor: Cholera, caused by toxigenic strains of Vibrio cholerae O1 or O139, continues to be a major cause of illness and death, particularly in developing countries. Treatment consists of early administration of rehydration therapy with appropriate oral or intravenous fluids. The World Health Organization recommends antimicrobial drug treatment for severely dehydrated patients with suspected cholera because it substantially shortens the duration of diarrhea by reducing the volume of watery stools, decreases fluid requirements, and limits transmission by decreasing fecal excretion of V. cholerae (1). The progressive increase in resistance to multiple drugs among strains causing clinical cases of cholera in developing countries is becoming a serious concern. We report the emergence of tetracycline-resistant V. cholerae O1 in a well-defined population in Kolkata, India, during 2007–2009. During a 6-year surveillance period (2004–2009), we conducted a prospective, community-based study at an impoverished urban site in Kolkata. The goals of the study were to estimate the prevalence of cholera, describe its epidemiology, and identify potential risk factors that could be addressed by public health strategies. Rectal swabs samples from patient with diarrhea were obtained, placed in Cary-Blair transport medium, and transported to the laboratory where they were processed for isolation and identification of Vibrio spp. Specimens were plated directly onto thiosulphate citrate bile salt sucrose agar (Eiken Chemical Company, Tokyo, Japan). The specimens were plated directly onto thiosulfate citrate bile salt sucrose agar. They were then incubated in alkaline peptone water (pH 8.6) for 6–8 h at 37°C and then plated onto the agar. After overnight incubation at 37°C, suspected colonies were tested biochemically and confirmed by slide agglutination with polyvalent O1 and monovalent Ogawa and Inaba antiserum (Difco Laboratories, Detroit, MI, USA). Antimicrobial drug susceptibility testing was performed by using the disk diffusion technique on Mueller-Hinton agar (Difco Laboratories) with commercial disks (Oxoid, Cambridge, UK) and appropriate control strains (2). The MIC of tetracycline was determined with 101 randomly selected strains by Etest (AB Biodisk, Solna, Sweden) following manufacturer’s instructions. During the 2004–2009 surveillance period, we isolated 809 V. cholerae O1 organisms, among which 624 (77%) were Ogawa and 185 (23%) were Inaba serotypes. The latter became the predominant serotype only in 2006. In 2007, a sudden upsurge in tetracycline resistance was noted among V. cholerae isolates, from 1% in 2004 to 76% in 2007 before decreasing to ≈50% in 2009. An increase in resistance to furazolidone and trimethoprim/sulfamethoxazole was also observed during the same period. Of the strains that were resistant to tetracycline, 99% were also resistant to furazolidone and trimethoprim/sulfamethoxazole (online Appendix Figure, www.cdc.gov/EID/content/17/3/568-appF.htm). Among the tetracycline-resistant isolates (101 randomly selected strains), 43% had high-level resistance (MIC >16 µg/mL). In addition, 57% of V. cholerae O1 organisms had reduced susceptibility (i.e., MICs ranged from 8 µg/mL to 16 µg/mL). Tetracycline is the drug of choice for treating cholera (1); however, during the 6-year period, we observed the emergence of tetracycline resistance among V. cholerae O1 isolates and a sudden upsurge in such resistance in 2007 when 76% of the isolates were resistant. Tetracycline resistance was also reported by Mhalu et al. (3), from an epidemic of cholera in Tanzania, where 76% of isolates were found to be resistant after 5 months of extensive use of this drug for treatment and prophylaxis. In a similar situation, the extensive prophylactic use of tetracycline triggered the rapid emergence and spread of tetracycline-resistant strains in Madagascar (4). Tetracycline is not used for prophylaxis in Kolkata, a known cholera-endemic area. Nonetheless, the emergence of resistant strains in our study area is not surprising because similar tetracycline-resistant strains of V. cholerae have been reported in Bangladesh (5), in Mozambique (6), and in another study of Kolkata (7). Notably, tetracycline-resistant V. cholerae O1 strains have also been responsible for major epidemics in Latin America, Tanzania, Bangladesh, and Zaire (8). In our study, resistance to tetracycline among V. cholerae O1 isolates was <10% during 2004–2006. The reasons for the sudden rise of resistant strains in 2007 and their continued persistence are still unclear. Detailed molecular studies are underway to find the explanation. Alternative drugs, such as the newer fluoroquinolones, possess excellent activity against V. cholerae O1 and O139 serogroups. However, increased resistance to newer fluoroquinolones, such as ciprofloxacin and norfloxacin, among V. cholerae strains belonging to O1 serogroup has also been reported (9). Resistance to commonly used antimicrobial drugs represents a critical public health problem because it complicates treatment and may result in longer hospital stays for patients. In addition, most of the population in developing countries cannot afford the newer and more expensive drugs. Our findings emphasize the need for continued surveillance of antimicrobial drug susceptibility patterns of V. cholerae. Providing early information on antimicrobial drug susceptibility to practitioners in affected areas will lessen the illness and death from this devastating disease.

Persistence of sucrose fermenting and nonfermenting vibrios in tissues of Manila clam species, Ruditapes philippinarum, depurated in seawater at two different temperatures

Effective post-harvest treatment to eliminate toxigenic vibrios is an important measure to reduce risk associated with seafood consumption. In the present study, we examined whether natural populations of sucrose nonfermenting (SNFV) and sucrose fermenting vibrio s (SFV) persisted in Ruditapes philippinarum, after depuration at two different temperatures. Two experiments (one in Spring and one in Summer) were performed. Clams were depurated in duplicate tanks in an open-circuit seawater system using filtration and ultraviolet treatment. Clams were sampled daily (9 days), individually processed and inoculated on thiosulphate-citrate-bile salts-sucrose (TCBS) agar plates to estimate the density of SNFV and SFV. Results showed that depuration was less effective in Summer than in Spring, for SNFV and SFV. Despite an initial lower presence in the Summer experiment, clearance of SNFV was less efficient than in Spring, with 22.2% of clams presenting SNFV on day 8 and without significant changes on day 9. When compared, daily proportion of presence of SNFV and SFV in Spring, it was observed that SFV were more frequent than SNFV on days 8 and 9. In Summer these differences were significant on days 7–9. Present study demonstrated that SNFV (involved in food poisoning) are purged, at any temperature, faster than SFV.