Detection of pathogenic Vibrio species and antibiogram activity in Asian Seabass (Lates calcarifer) in Tumpat, Kelantan

@#Some of Vibrio species is well known as pathogenic bacteria in aquaculture and the marine industry. Its infection is able to generate a massive outbreak and affect the fish population, especially for net caged fish such as seabass. This study was conducted to investigate the prevalence of Vibrio spp. isolated from seabass (Lates calcarifer) in Sri Tujuh Lagoon, Tumpat, Kelantan. Then, to determine the antibiotic resistance in Vibrio isolates. Polymerase chain reaction (PCR) was used to detect Vibrio species using specific primer VR169 and VR744 with estimation base pair size band, 597 bp and further identified by sequencing. On the other hand, antibiotic susceptibility tests were continued by using 13 types of antibiotics; kanamycin (K30), chloramphenicol (C30), neomycin (N10), ampicillin (AMP10), nitrofurantoin (F300), tetracycline (TE30), streptomycin (S10), norfloxacin (NOR10), ciprofloxacin (CIP5), nalidixic acid (NA30), gentamicin (CN10), doxycycline (DO30) and sulfamethoxazole (SXT100). As a result, 14 Vibrio isolates were identified, including Vibrio fluvialis (n=6), Vibrio parahaemolyticus (n=3), Vibrio harveyi (n=2) and each isolate for Vibrio vulnificus, Vibrio alginolyticus and Vibrio spp. The results showed that all isolates were sensitive to most antibiotics except ampicillin, neomycin and streptomycin. The MAR index value was ranging from 0 to 0.31. This study demonstrates the prevalence of Vibrio spp. in seabass and the report on multidrug resistance strains that could be of concern to the fish farmers. In addition, data from this study can be further used in fish disease management plans.

Vibrio sp. ArtGut-C1, a polyhydroxybutyrate producer isolated from the gut of the aquaculture live diet Artemia (Crustacea)

BACKGROUND: Vibrio species display variable and plastic fitness strategies to survive and interact with multiple hosts, including marine aquaculture species that are severely affected by pathogenic Vibrios. The culturable Vibrio sp. strain ArtGut-C1, the focus of this study, provides new evidence of such phenotypic plasticity as it accumulates polyhydroxybutyrate (PHB), a biodegradable polymer with anti-pathogen activity, particularly in the marine larviculture phase. The strain was isolated from the gut of laboratory-reared Artemia individuals, the live diet and PHB carrier used in larviculture. Its main phenotypic properties, taxonomic status and genomic properties are reported based on the whole-genome sequencing. RESULTS: Vibrio sp. ArtGut-C1 yielded 72.6% PHB of cells' dry weight at 25 C. The genomic average nucleotide identity (ANI) shows it is closely related to V. diabolicus (ANI: 88.6%). Its genome contains 5,236,997- bp with 44.8% GC content, 3,710 protein-coding sequences, 96 RNA, 9 PHB genes functionally related to PHB metabolic pathways, and several genes linked to competing and colonizing abilities. CONCLUSIONS: This culturable PHB-accumulating Vibrio strain shows high genomic and phenotypic variability. It may be used as a natural pathogen biocontrol in the marine hatchery and as a potential cell factory for PHB production.

Prevalence of Antimicrobial Resistance Among Vibrio spp. Isolated from the Digestive Tract of Cultured Penaeus vannamei

Shrimp digestive tract microflora has been considered important, as it provides several protective and metabolic functions. Misuse of antibiotics could lead to AMR in the gut microbial community, which could be transferred to humans. The present study aimed in determining the prevalence of the digestive tract microbial community and AMR associated with them. A total of 173 isolates were collected and characterised from the digestive tract of 120 shrimps, collected from six different regions of Maharashtra and Gujarat. A total of 144 gram negative isolates comprised predominantly of Vibrio spp were isolated and characterised. Antimicrobial susceptibility pattern of the isolates against 12 different antibiotics was carried out using disk diffusion method. Most of the isolates showed resistant against beta-lactam class of antibiotics and macrolide antibiotics. Among the 144 G negative isolates, 61 (39.5%) isolates were presumptively identified as Vibrio spp, based on their growth on the specific agar plates. Biochemical characterisation of the 61 Vibrio isolates revealed the presence of 31.14% of Vibrio cholerae, 31.14% of V. parahaemolyticus, 19.67% of V. vulnificus, 9.8% of V. harveyi and 8.2% of V. alginolyticus. The antibiogram profile showed that the 40 (74.07%) isolates were resistant to ampicillin, 24 (44.4%) were resistant to cephalothin, 21 (38.8%) were to aztreonam and 17 (31.5%) were resistant against erythromycin. Prevalence of multi-drug resistance was also observed among the bacterial isolates.

Pathogenic Vibrio species isolated from estuarine environments (Ceará, Brazil) - antimicrobial resistance and virulence potential profiles

ABSTRACT Detection of virulent strains associated with aquatic environment is a current concern for the management and control of human and animal health. Thus, Vibrio diversity was investigated in four estuaries from state of Ceará (Pacoti, Choró, Pirangi and Jaguaribe) followed by antimicrobial susceptibility to different antimicrobials used in aquaculture and detection of main virulence factors to human health. Isolation and identification were performed on TCBS agar (selective medium) and dichotomous key based on biochemical characteristics, respectively. Nineteen strains of genus Vibrio were catalogued. Vibrio parahaemolyticus (Choró River) and V. alginolyticus (Pacoti River) were the most abundant species in the four estuaries. All strains were submitted to disk diffusion technique (15 antimicrobials were tested). Resistance was found to: penicillin (82%), ampicillin (54%), cephalotin (7%), aztreonan (1%), gentamicin, cefotaxime and ceftriaxone (0.5%). Five pathogenic strains were chosen to verification of virulence factors. Four estuaries showed a high abundance of species. High number of tested positive strains for virulence is concerning, since some of those strains are associated to human diseases, while others are known pathogens of aquatic organisms.

Evaluación del riesgo microbiológico a Vibrio spp. en alimentos de origen marino en México / Microbial risk assessment of Vibrio spp. in seafood products in Mexico

Las enfermedades transmitidas por los alimentos son uno de los mayores problemas de salud pública que actualmente existen. La evaluación del riesgo microbiológico es un proceso utilizado para examinar los peligros ocultos en los alimentos, la probabilidad de exposición a éstos y su impacto en la salud pública. La evaluación del riesgo se realiza en cuatro fases: identificación del peligro, caracterización del peligro, evaluación de la exposición y caracterización del riesgo. De acuerdo con el proceso/resultado, las evaluaciones de riesgo microbiológico se clasifican en dos categorías: cualitativa y cuantitativa. La presente revisión pretende enmarcar la importancia de implementar estas evaluaciones en alimentos de origen marino que son consumidos crudos, fortaleciendo así el acceso a los alimentos inocuos y de buena calidad para beneficio del consumidor, y la necesidad de evaluaciones de riesgo microbiológico que hay en México.

Food-borne diseases are among the major public health problems that currently exist. Microbiological risk assessment is a process used to evaluate the hidden hazards in food, the likelihood of exposure to these hazards and their impact on public health. Risk assessment is performed in four steps: hazard identification, hazard characterization, assessment of exposure and risk characterization. According to the process/response microbial risk assessment is classified in two categories, qualitative and quantitative. The aim of this review is to underline the importance of implementing assessments in seafood that is usually consumed raw, strengthening access to good quality and safe food for the consumer's benefit and to stress the necessity of microbiological risks assessments in Mexico.

Vigilancia de brotes de enfermedades transmitidas por alimentos en Chile / Foodborne disease outbreaks surveillance in Chile

Background: Foodborne disease outbreaks are one of the main health problems globally, having an extensive impact on human welfare. The World Health Organization considers them as the main cause of morbidity and mortality in developing countries, and responsible for high levels of loss of productivity in developed countries. Aim: To describe the epidemiology of foodborne disease outbreaks according to data contained in an automated surveillance system. Method: Descriptive observational study of notified outbreaks from the surveillance system, between 2005 and 2010 in Chile. The information was based on etiology, temporal and spatial distribution, and epidemiologic description of outbreaks during this period. Results: There were 5,689 notified outbreaks. Most of them occurred during 2006 (1,106 outbreaks, rate 6.7 per 100,000 inhabitants) and 2008 (1,316 outbreaks, rate 7.9 per 100, 000 inhabitants) with an increase during summer. Fifty four percent occurred in the Metropolitan region. The group aged 15 to 44 years old, was the most affected one. Sixty four percent of the outbreaks had the food involved registered, of which fish and fishery products reached 42%. An 81% of the outbreaks did not have a precise etiologic diagnosis. Of all patients involved, 97% were outpatients, 3,2% were hospitalized patients, and 0,1% died. Only 49% of the outbreaks had information about the lack of food safety, with a 34,1% related to food handling procedures. Conclusions: Through the information on the epidemiology of foodborne diseases obtained by the Chilean surveillance system, appropriate control measures could be taken.

Antecedentes: Las enfermedades transmitidas por alimentos (ETA) son una importante carga de enfermedad en el mundo. La OMS las señala como la principal causa de enfermedad y muerte en países en desarrollo, mientras que en países desarrollados son responsables de altos niveles de pérdida de productividad. Objetivo: Describir epidemiológicamente los brotes de ETA chilenos de acuerdo a la información contenida en un sistema automatizado de vigilancia. Método: Estudio observacional descriptivo de los brotes notificados en el sistema de vigilancia, entre los años 2005 y 2010 en Chile. La descripción se basó en el aspecto etiológico, distribución temporal y espacial, y descripción epidemiológica de los brotes durante dicho período. Resultados: Se notificaron 5.689 brotes. La mayoría se presentó durante el 2006 (1.106 brotes, tasa 6,7 por 100.000 hab) y 2008 (1.316 brotes, tasa 7,9 por 100.000 hab) con un aumento en los meses de verano. El 54% ocurrió en la Región Metropolitana. El grupo de 15 a 44 años fue el más afectado. Del 64% que registró el alimento involucrado, pescados y productos de la pesca alcanzaron el 42%. Un 81% del total de brotes no tuvo un diagnóstico etiológico preciso. Del total de pacientes, 97% fueron ambulatorios, 3,2% se hospitalizaron, y 0,1% fallecieron. Sólo 49% de los brotes registró pérdida de inocuidad del alimento, siendo el mayor porcentaje (34,1%) atribuible al proceso de manipulación del alimento. Conclusiones: El sistema de vigilancia chileno permitió conocer el comportamiento epidemiológico de las ETA, y facilitó la adopción de medidas de control oportunas.

Raw oysters can be a risk for infections

The aims of this study were to count and identify sucrose positive and negative vibrios isolated from cultivated Crassostrea rhizophorae oysters during their growing cycle. Every month for 12 months, 10 to 18 oysters were collected for study. Collections occurred at the Center for Studies of Coastal Aquaculture (CSCA), which is associated with the Institute of Marine Science, Labomar, located in Euzebio, Ceará, Brazil. Approximately 150 oysters and their intervalvular liquor were studied. Vibrio Standard Plates Counts (SPC) from oyster meat and their intervalvular liquor varied from 25 to 59,000,000 CFU/g. For most of the 12 months of the oysters' life, it was possible to identify Vibrio parahaemolyticus. Vibrio carchariae was identified in four collections. Among other isolated species, the most important, considering public health risks, was V. vulnificus, although only one strain was confirmed. We concluded that retail purchased oysters should never be eaten raw or undercooked because many species of the genus Vibrio are known to be pathogenic to humans and live naturally on and in shellfish throughout their life cycle.

Inhibition of shrimp pathogenic vibrios by extracellular compounds from a proteolytic bacterium Pseudomonas sp. W3 / Inhibición de vibrios patógenos del camarón por compuestos extracelulares de una bacterias proteolítica Pseudomonas sp.W3

Pseudomonas sp. W3, a bacterium known to produce an extracellular alkaline protease, secreted secondary metabolites that inhibited pathogenic bacteria responsible for shrimp luminous vibriosis disease. Antivibrio compounds in the culture supernatant or culture filtrates (0.45 um and 0.22 um) of the isolate W3 were tested using an agar well diffusion method on a number of pathogenic vibrios. Vibrio harveyi PSU 2015 a pathogenic isolate was the most sensitive strain. The effectiveness of preparations from the isolate W3 against V. harveyi PSU 2015, and V. cholerae PSSCMI 0062 was in the order of culture supernatant > 0.45 um culture filtrate > 0.22 um culture filtrate. These extracellular antivibrio compounds also lysed both dead and living cells of V. harveyi PSU 2015. Results of the partial characterization tests indicated that there was some particulate antivibrio compound that was destroyed by treatment with enzymes particularly alpha-chymotrypsin, autoclaving at 121ºC for 15 min and was mostly removed by filtration through a 0.22 µm filter. Most of the inhibitory compounds were of small molecular weight able to pass through a 0.22 um filter and were resistant to treatment with various enzymes, pH values between 4-8 and temperatures up to 121ºC for 30 min. The optimum pH for the antivibrio activity in the 0.45 um culture filtrate was between pH 6-7.

Vibrio na água e sedimento de viveiros de quatro fazendas de carcinicultura no estado do Ceará, Brasil / Vibrio in pond water and sediment from four shrimp farms in Ceará state, Brazil

Foram realizadas 16 coletas, oito no período chuvoso e oito no período de estio, em quatro fazendas de carcinicultura do Estado do Ceará, nos estuários dos rios Jaguaribe (fazendas A e B) e Acaraú (fazendas C e D), totalizando 32 amostras. O objetivo da pesquisa foi quantificar e identificar Vibrio spp. nas amostras de água e sedimento. Os valores máximos da Contagem Padrão em Placas (CPP) de Vibrio spp. encontrados para as amostras de água, no período chuvoso, foram de 5.103 UFC/mL est. e, para o sedimento, de 7,5.103 UFC/g est. No período de estio, a CPP máxima para água foi de 4,35.102 UFC/mL est. e de 3,55.103 UFC/g est. para as amostras de sedimento. Foram obtidos 36 isolados de Vibrio: Vibrio spp. (17), V. vulnificus B1(3); V. calviensis (2), V. cholerae (2), V. litoralis (2), V. metschnikovii (2), V. agarivorans (1), V. alginolyticus (1), V. campbellii (1), V. coralliilyticus (1), V. diazotrophicus (1), V. logei (1), V. mediterranei (1), V. vulnificus B2 (1). O conhecimento da presença de espécies, nunca anteriormente isoladas em viveiros de fazendas de carcinicultura, tais como o V. coralliilyticus, V. agarivorans, V. litoralis e V. calviensis são importantes para o monitoramento microbiológico contínuo desses ambientes.

Sixteen collections were taken, eight during rainy season and eight on the draught season in four shrimp farms in Ceará State, from Jaguaribe River (farms A and B) and Acaraú River (farms C and D) estuaries, totalizing 32 samples. The goal of the research was to identify and quantify Vibrio spp. in water and in sediment samples. The maximum Standard Plate Count (SPC) values of Vibrio spp. calculated for the rainy season, from water, was 5.103 CFU/mL est., and for the sediment 7.5.103 CFU/g est.. For the draught season, maximum counting for water was 4.35.102 CFU/mL est. and for sediment 3.55.103 CFU/g est.. Thirty six strains of Vibrio were isolated: Vibrio spp. (17), V. vulnificus B1(3), V. calviensis (2), V. cholerae (2), V. litoralis (2), V. metschnikovii (2), V. agarivorans (1), V. alginolyticus (1), V. campbellii (1), V. coralliilyticus (1), V. diazotrophicus (1), V. logei (1), V. mediterranei (1), V. vulnificus B2 (1). The isolation of species never previously isolated of shrimp farms, such as V. coralliilyticus, V. agarivorans, V. litoralis and V. calviensis are important for the continuous microbiological monitoring these environments.

Occurrence of heavy metals and antibiotic resistance in bacteria from internal organs of american bullfrog (Rana catesbeiana) raised in Malaysia

A total of 40 bacteria have been successfully isolated from internal organs of the American bullfrog (Rana catesbeiana) raised in Malaysia, namely, eight isolates of Aeromonas spp., 21 of Edwardsiella spp., six of Flavobacterium spp. and five of Vibrio spp. In terms of antibiotic susceptibility testing, each isolate was tested against 21 antibiotics, resulting in 482 (57.3 percent) cases of sensitivity and 61 (7.3 percent) cases of partial sensitivity. Meanwhile, 297 (35.4 percent) bacterial isolates were registered as resistant. The multiple antibiotic resistance (MAR) index of each bacterial species indicated that bacteria from raised bullfrogs have been exposed to tested antibiotics with results ranging from 0.27 to 0.39. Additionally, high percentages of heavy metal resistance among these isolates were observed, with values ranging from 85.0 to 100.0 percent. The current results provided us information on bacterial levels of locally farmed bullfrogs exposed to copper, cadmium, chromium as well as 21 types of antibiotics.(AU)

Enzymatic characterization of Vibrio alginolyticus strains isolated from bivalves harvested at Venice Lagoon (Italy) and Guanabara Bay (Brazil) / Caracterização enzimática de cepas de Vibrio alginolyticus isoladas de bivalves coletados na Lagoa de Veneza (Itália) e Baía de Guanabara (Brasil)

The aquatic ecosystem is the natural habitat of microorganisms including Vibrio and Aeromonas genus which are pathogenic to human and animals. In the present investigation the frequency of these bacteria and the enzymatic characteristics of 34 Vibrio alginolyticus strains isolated from bivalves harvested in Venice Lagoon (Italy) and Guanabara Bay (Brazil) were carried out from November 2003 to February 2004. The mussels' samples were submitted to enrichment in Alkaline Peptone Water (APW) added with 1 percent of sodium chloride (NaCl) and APW plus 3 percent NaCl incubated at 37 ºC for 18-24h. Following the samples were streaked onto TCBS Agar (Thiossulfate Citrate Bile Sucrose Agar) and the suspected colonies were submitted to biochemical characterization. Also, the Vibrio alginolyticus strains were evaluated to collagenase, elastase and chondroitinase production. The results showed the isolation of 127 microorganisms distributed as follows: 105 Vibrio strains such as V. alginolyticus (32.4 percent), V. harveyi (19 percent) and V. parahaemolyticus (7.6 percent), 20 Aeromonas strains and two Plesiomonas shigelloides were the main pathogens isolated. We observed the production of the three enzymes from V. alginolyticus strains considered as the main virulence factors of the bacteria, especially in cases of human dermatological infection.

O ecossistema aquático é o habitat natural de microrganismos incluindo aqueles dos gêneros Vibrio e Aeromonas os quais são patogênicos para o homem e animais. Na presente investigação foi avaliada a freqüência destas bactérias e a característica enzimática de 34 cepas de Vibrio alginolyticus isoladas de bivalves coletados na Lagoa de Venice (Itália) e Baía de Guanabara (Brasil) durante o período de Novembro-2003 a Fevereiro-2004. As amostras de mexilhões foram submetidas a enriquecimento em Água Peptonada Alcalina (APA) adicionada de 1 por cento de Cloreto de Sódio (NaCl) e APA com 3 por cento de NaCl (37 ºC/18-24h). Em seguida as amostras foram semeadas em Agar TCBS (Agar Tiossulfato Citrato Bile Sacarose) e as colônias suspeitas foram submetidas à caracterização bioquímica. As cepas de Vibrio alginolyticus foram avaliadas quanto à produção das enzimas colagenase, elastase e condroitinase. Os resultados demonstraram o isolamento de 127 microrganismos assim distribuídos: 105 cepas de Vibrio das quais V. alginolyticus (32,4 por cento), V. harveyi (19 por cento) e V. parahaemolyticus (7,6 por cento), 20 cepas de Aeromonas e 2 Plesiomonas shigelloides foram os principais patógenos isolados. Observou-se a produção das três enzimas a partir de V. alginolyticus, consideradas principais fatores de virulência da bactéria, em especial em casos de infecção dermatológica humana.

Luminescent bacteria as biosensor for mercury toxicity.

Agricultural activities and human industrialization are mainly responsible for the release of heavy metals into the environment, especially the air and the water. The first step towards the effective management of water resources is the assessment of pollution levels. Biosensors for the detection of pollutants in the environment can complement analytical methods by distinguishing bioavailable from inert, unavailable forms of contaminants. A bioassay system for detecting heavy metals in water using bioluminescent bacteria, Vibrio harveyi and Vibrio fischeri has been developed, which offers the advantages of simplicity and rapidity for screening heavy metals in water sources. Bioluminescence was found to be species specific and strain specific. Mercury, zinc and copper showed definite microbial toxicity and inhibition of bioluminescence. The inhibition range for each strain of a species was standardized and its reproducibility verified. The utility of the biosensors to detect heavy metals in tap water was demonstrated with samples supplemented with Hg (II).