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.

Actividad antitumoral de biopolímeros aislados de Vibrio sp. en cáncer de mama inducido en ratas / Antitumor activity of vibrio sp. isolated biopolymers in induced breast cancer in rats

RESUMEN Objetivo: Evaluar la actividad antitumoral del extracto crudo de biopolímeros aislados de la bacteria marina Vibrio sp. en cáncer de mama inducido por N-Methyl-N-nitrosourea (MNU) en ratas. Materiales y métodos: Se cultivó la bacteria marina Vibrio sp. durante siete días, luego se filtró, precipitó y concentró el sobrenadante crudo. Se administró una dosis única de MNU 50 mg/kg a 39 ratas Holtzman y fueron tratadas diariamente durante nueve semanas por vía oral: G1 (n=13): suero fisiológico 0,1 mL/100g; G2 (n=13): extracto crudo de biopolímeros de Vibrio sp. 20 mg/kg; G3 (n=13): tamoxifeno 100 mg/kg. El G4 (n=11) solo recibió suero fisiológico 0,1 mL/100g. Se valoró semanalmente el peso corporal y la aparición de tumores mamarios identificados mediante palpación; así como el examen histopatológico al final del tratamiento. Resultados: El 77% de las ratas del grupo G1 desarrollaron tumores a partir de la séptima semana en un promedio de 2,2 tumores por cada animal; en contraste al grupo tratado con el extracto crudo de biopolímeros y tamoxifeno; donde solo una rata (8%) en cada grupo desarrolló tumores y posterior a la semana nueve de la inducción (p=0,001). Los resultados histopatológicos sostienen que todos los tumores extirpados corresponden a adenocarcinoma ductal de mama con distintos patrones: sólido, papilar y quístico. Asimismo, se evidenciaron focos necróticos en el 30% de los tumores del grupo G1. Conclusión: El extracto crudo de biopolímeros aislados de Vibrio sp. presentan efecto antitumoral en cáncer de mama inducido en ratas.

ABSTRACT Objective: To evaluate the antitumor activity of the raw extract from biopolymers isolated from the Vibrio sp. marine bacteria in breast cancer induced by N-Methyl-N-nitrosourea (MNU) in rats. Materials and methods: The Vibrio sp. marine bacteria was cultured for seven days, then the raw supernatant was filtered, precipitated and concentrated. MNU was administered in a single dose of 50 mg/kg to 39 Holtzman rats and were daily treated for 9 weeks orally: G1 (n = 13): 0.1 mL/100 g of saline solution; G2 (n = 13): 20 mg/kg of raw extract from Vibrio sp. biopolymers; G3 (n = 13): 100 mg/kg of tamoxifen; G4 (n = 11) received no MNU and only 0.1 mL/100 g of saline solution. Body weight and the appearance of breast tumors identified by palpation were assessed weekly, as well as histopathological examination at the end of treatment. Results: Seventy-seven percent of the rats in the G1 group developed tumors from week 7 onwards in an average of 2.2 tumors per animal; in contrast to the group treated with the raw biopolymer extract and tamoxifen; where only one rat (8%) in each group developed tumors after week nine of induction (p = 0.001). The histopathological results support that all the removed tumors correspond to breast ductal adenocarcinoma with different patterns: solid, papillary and cystic. Likewise, necrotic foci were evidenced in 30% of the tumors of the G1 group. Conclusion: The raw extract of biopolymers isolated from Vibrio sp. present antitumor effect in breast cancer induced in rats.

Occurrence of potentially pathogenic Vibrio in oysters (Crassostrea gigas) and waters from bivalve mollusk cultivations in the South Bay of Santa Catarina

Introduction This research aimed to identify and quantify potentially pathogenic Vibrio from different cultivations of bivalve shellfish in the State of Santa Catarina, Brazil, and water regions in the South Bay, as well as correlate the incidence of these microorganisms with the physicochemical parameters of marine waters. Methods Between October 2008 and March 2009, 60 oyster and seawater samples were collected from six regions of bivalve mollusk cultivation, and these samples were submitted for Vibrio counts. Results Twenty-nine (48.3%) oyster samples were revealed to be contaminated with one or more Vibrio species. The Vibrio parahaemolyticus and Vibrio vulnificus counts in the samples ranged from < 0.5 log10 Most Probable Number (MPN) g–1 to 2.3 log10 MPN g–1 oyster and from < 0.5 log10 MPN g–1 to 2.1 log10 MPN g–1 oyster, respectively. Of the 60 seawater samples analyzed, 44 (73.3%) showed signs of contamination with one or more vibrio species. The counts of V. parahaemolyticus and V. vulnificus in the samples ranged from < 0.3 log10 MPN·100mL–1 to 1.7 log10MPN·100mL–1 seawater and from < 0.3 log10 MPN·100mL–1 to 2.0 log10 MPN·100mL–1 seawater, respectively. A positive correlation between V. vulnificus counts and the seawater temperature as well as a negative correlation between the V. parahaemolyticus counts and salinity were observed. Conclusions The results suggest the need to implement strategies to prevent vibrio diseases from being transmitted by the consumption of contaminated bivalve shellfish. .

Prevalence of Vibrio cholerae and Other Vibrios from Environmental and Seafood Sources, Tamil Nadu, India.

Aims: The aim of this study was to investigate the prevalence of diarrhoea causing human pathogen V. cholerae and other vibrios from different environmental and seafood samples in Tamil Nadu, India. Place and Duration of Study: Laboratory of Clinical Microbiology, Department of Bio- Medical Science, School of Basic Medical Sciences, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India between 2012 and 2013. Methodology: Seafood, water and plankton samples were collected at different locations of Tamil Nadu, India. All the samples were primarily enriched with alkaline peptone water (APW). 2-3 loopful of overnight cultures were streaked onto Thiosulphate Citrate Bile salt Sucrose (TCBS) agar plates. Suspected Vibrio cholerae, V. parahaemolyticus and other vibrios were picked up and identified by using standard biochemical and serological characterization and also by molecular methods. Results: Among the various samples that includes freshwater, coastal water, plankton and various seafoods, only plankton and seafood samples were found to be harbored with V. cholerae, V. parahaemolyticus and V. fluvialis. The remaining samples were negative for vibrios. All V. cholerae, V. parahaemolyticus and V. fluvialis strains possessed outer membrane protein W (ompW), thermostable direct haemolysin (tdh) and toxin regulatory protein (toxR) gene respectively. Hemolytic activity of V. cholerae exihibited different reaction isolated from seafood and plankton. The median lethal dose (LD50) of some V. cholerae strains was generally high. Conclusion: The result of the study suggested that the seafoods may act as an important reservoir of pathogenic vibrios and pose threat to human health.

Hemolytic and urease activities in vibrios isolated from fresh and frozen oysters

INTRODUCTION: The present study aimed to survey the Vibrio microbiota of oysters (Crassostrea rhizophorae) obtained from restaurants in Fortaleza, State of Ceará, Brazil, and to identify virulence factors. METHODS: The isolated vibrios were submitted to biochemical identification and were tested for hemolytic and urease activities. RESULTS: The isolated strains belonged to 13 species, with predominance of Vibrio mimicus. Of the strain isolates only from fresh samples, 20.5% and 2.8% showed hemolytic and urease activities, respectively. CONCLUSIONS: The findings support the little-publicized claim that Vibrio species other than V. parahaemolyticus and V. vulnificus can represent a health risk to public health.

Isolation and characterization of infectious Vibrio sinaloensis strainsfrom the Pacific shrimp Litopenaeus vannamei (Decapoda: Penaeidae)

Infectious diseases especially those caused by bacterial and viral pathogens are serious loss factors in shrimp farming. In this study, bacteria were isolated from the gut and hepatopancreas of stressed shrimps obtained from a commercial farm. The isolates were screened on Thiosulfate citrate bile salt sucrose (TCBS) agar plates for the selection of Vibrio species. Presumptive vibrios were characterized through tests for hemolytic and enzymatic activity, hydrophobicity, growth and molecular identification. Three experimental infections were conducted in order to confirm the pathogenicity of selected bacterial strains VHPC18, VHPC23, VHPC24 and VIC30. In the third experimental challenge the LD50 was obtained, it lasted 10 days with 10 shrimp, weighing 6.9±1.1g, per tank. The treatments in triplicate were: (1) saline solution (control group); (2) 2×10(5)CFU/shrimp; (3) 4×10(5)CFU/shrimp; (4) 2×10(6)CFU/shrimp; (5) 4×10(6)CFU/shrimp, and (6) 8×10(6)CFU/shrimp. In all challenges, water parameters measured during the experimental period remained within optimum ranges. Pathogenicity tests confirmed that the mixture of four vibrio isolates, identified as Vibrio sinaloensis, was virulent for L. vannamei. The LD50 value was 1.178×10(5)CFU/g body weight. V. sinaloensis may act as opportunistic pathogens for cultured L. vannamei. Rev. Biol. Trop. 60 (2): 567-576. Epub 2012 June 01.

Las enfermedades de etiología infecciosa, especialmente las causadas por patógenos bacterianos y virales ocasionan graves pérdidas en el cultivo de camarón blanco Litopenaeus vannamei. En este estudio se caracterizo: la actividad enzimática y hemolítica; hidrofobicidad; crecimiento e identificación molecular de vibrios aislados del intestino y hepatopancreas de camarones estresados, obtenidos de una granja comercial, en medio Agar Tiosulfato Citrato Bilis Sacarosa. Además, se realizaron tres infecciones experimentales para confirmar la patogenicidad de las cepas bacterianas seleccionadas VHPC18, VHPC23, VHPC24 y VIC30. En la tercera infección experimental se obtuvo la LD50, el reto duro 10 días, con 10 camarones por tanque con un peso de 6.9±1.1g. Los tratamientos se realizaron por triplicado: (1) solución salina (grupo control); (2) 2×10(5)UFC/camarón; (3) 4×10(5)UFC/camarón; (4) 2×10(6)UFC/camarón; (5) 4×10(6)UFC/camarón y (6) 8×10(6)UFC/camarón. En todos los retos, los parámetros del agua permanecieron dentro de los intervalos óptimos. Las pruebas de patogenicidad confirmaron que la mezcla de cuatro aislados de Vibrio, identificados como Vibrio sinaloensis, fue virulenta para L. vannamei. El valor de la LD50 fue de 1.178×10(5)UFC/g de peso corporal. Los resultados permiten establecer que las cepas de V. sinaloensis pueden actuar como patógenos oportunistas en L. vannamei cultivado.

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.

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.

Detection of marine pathogenic bacterial Vibrio species by multiplex polymerase chain reaction (PCR).

Polymerase chain reactions (PCR) were performed to accomplish quick and accurate detection of Vibrio species. The primers prepared with 16S-23S rDNA intergenic spacer (IGS) region exhibited an excellent species-specificity for Vibrio sp and detected Vibrio sp more successfully than the conventional culture method. Multiplex PCR was also fruitful not only forthe identification of the 5 standard Vibrio sp simultaneously but also for the detection of Vibrio spin the samples collected from the natural environment.

Effect of probiotics on bacterial population and health status of shrimp in culture pond ecosystem.

The artificially manufactured probiotics having beneficial bacteria, Bacillus spp. was applied regularly in a modified extensive shrimp (Penaeus monodon) culture pond, located on the bank of Vellar estuary, Parangipettai. The populations of total heterotrophic bacteria (THB), beneficial bacteria (Bacillus spp.) and pathogenic bacteria (vibrios) were monitored in water and sediment of the pond. The results were compared with a control pond, situated in the same location having same water spread area, stocking density, species managed with same technologies and optimum environmental parameters in which no probiotic was applied. The populations of THB and Bacillus spp. in the experimental pond increased and the vibrios decreased after each application of probiotics. But the result of the control pond showed an increasing trend of the populations of THB, Bacillus spp. and vibrios towards days of culture. The control pond had lower levels of THB and Bacillus spp. and higher levels of vibrios than the probiotic applied (experimental) pond. Also the probiotics maintained optimum transparency and low organic load in the experimental pond as compared to control. In general, water and sediment had almost equal number of Bacillus spp. and vibrios, but sediment had higher THB load than water. The applications of probiotics lesser pathogenic vibrios and enhance beneficial bacilli in the culture leading to improved water quality, promoted growth and survival rates and increased the health status of the shrimp without stress and disease outbreaks. Thus the application of probiotics could lead to disease-free and profitable shrimp culture operations which will be helpful for shrimp farmers as most of them are now-a-days severely affected by microbial diseases.

Detection and molecular characterization of Vibrio vulnificus from coastal waters of Malaysia.

A total of 57 Vibrio vulnificus isolates from coastal water were characterized for their antimicrobial resistance, plasmid profiles and were typed by the PCR-based techniques: a random amplification of polymorphic DNA (RAPD) method and the enterobacterial repetitive intergenic consensus sequence (ERIC) method. All isolates were susceptible to chloramphenicol, nalidixic acid, tetracycline and trimethoprim-sulfamethoxazole. Fifty-one isolates were resistant to one or more of the other antibiotics tested. Plasmid analysis indicated that only 18 isolates carried small plasmids of 1.6 to 16 megadaltons. Analysis of the RAPD and ERIC DNA fingerprints of the V. vulnificus isolates with Gel Compare and cluster analysis software revealed significant genetic heterogeneity among these isolates. The combination of RAPD and ERIC analysis allowed us to distinguish all isolates. Thus, the combination of the two techniques is recommended for epidemiological investigation.

Bacteriophage typing of Vibrio fluvialis.

Six stable bacteriophages of Vibrio fluvialis were isolated from 44 surface water specimens collected in Thailand and Japan. Twelve different phages types were found among 109 V. fluvialis isolated from feces of diarrheal patients and the environment. Seventy-three percent (80/109) of these 109 isolates were typable with these phages. One phage type, designated as A (1) was predominant and accounted for 43% of the V. fluvialis examined. The six bacteriophages used in this typing scheme were stable for at least during a three-month storage at 4 degrees C. This proposed bacteriophage typing scheme may be of valuable aid in tracing sources and routes of infection in outbreaks of V. fluvialis infection in man.

Numerical taxonomy of vibrio & allied organisms.

Forty four strains of Vibrio, Pasteurella, Yersinia, Aeromonas, Escherichia, Klebsiella and Enterobacter were tested for 44 characters and the overall results scored by the procedure of numerical taxonomy. The strains were clustered by the weighted pair group method with the arithmatic averaging. The V. cholerae 01 and the non-01 V. cholerae being interlinked at a very high level (86% to 97.5%) within a single cluster were considered under the same species. V.cholerae having Classic, E1 Tor and Gindha (Pfeiffer 1896, as per priority) biotypes respectively. The biotypes of V.parahaemolyticus remained clearly delineated from Vibrio and Aeromonas, and, belonged to a new separate genus. The V. parahaemolyticus biotypes were located more adjacent to Yersinia and Pasteurella than the V. cholerae cluster. V.fluvialis 1 appears as a biotype of V.parahaemolyticus, and, not a new species.

Víbrios não coléricos na rotina enterobacteriológica / Non-cholera vibrios in enterobacteriologic routine

Of 3250 diarrheal stools received for microbiologic diagnosis at a private clinical laboratory in Recife, Brazil, strains of Vibrio were isolated from 55 (1.7). The study was carried out from May 1989 through May 1991. For recovering Vibrio, fecal samples were enriched in alkaline peptone water supplemented with 2 NaCl and subcultured on thiosulfate-citrate-bile salts-sucrose agar (TCBS). Of the recovered species, V. parahaemolyticus was most commonly found (24 strains), followed by V. furnissii (15 strains), V. cholerae non-01 (6 strains), V. alginolyticus (4 strains), V. fluvialis (2 strains), and Vibrio sp. (1 strain). The low isolation rate of Vibrio raises doubts about the cost-effectiveness of the use of TCBS in the routine enterobacteriologic workup of clinical laboratories.

Evaluation of coagglutination test for serotyping of enteropathogenic bacteria.

Results of conventional agglutination and coagglutination (COA) tests for serotyping of enteropathogenic Escherichia coli (EPEC), Shigella, Salmonella and Vibrio cholerae were compared. Eighty isolates of different serotypes of EPEC falling in the Wellcome polyvalent (OK) 2, 3 and 4 antisera, showed good (4+) agglutination with COA reagent up to 1:960 dilution. Out of 50 strains of Shigella species tested, 25 of Sh. dysenteriae and 5 of Sh. sonnei gave good reaction up to 1:1920 dilution and 15 of Sh. flexneri up to 1:960 dilution in COA test. Similar reaction (4+) by 5 strains of Sh. boydii was seen up to 1:480 dilution only. All 100 isolates of different Salmonella species viz., S. paratyphi A (5), S. typhimurium (50), S. typhi (15), S. weltevreden (5) and S. senftenberg (25), when serotyped by COA, gave good reaction in 1:480 dilution except S. typhi factor 9 and dH antisera, which gave very weak reaction at 1:480 dilution and good reaction at 1:280 dilution. All seven isolates of V. cholerae gave good reaction in COA test even up to 1:1920 dilution. No cross reaction with any COA reagent was obtained in the 1206 heterologus isolates tested. Thus due to its higher efficiency, lower cost and good specificity, COA may prove to be a better method for serotyping of enteropathogens.