AMR Threat Perception Assessment of Heterotrophic Bacteria From Shrimp Aquaculture Through Epidemiological Cut off Values.

BACKGROUND: Emergence and dissemination of antibiotic resistance is one of the major risks associated with the rampant usage of antibiotics in food-producing animals including aquaculture. OBJECTIVE: To determine Epidemiological Cut-OFF (ECOFF) values of heterotrophic bacterial populations from shrimp culture environments against five different antibiotics. METHODS: In this present study, bacterial samples were isolated from Penaeus vannamei culture environment in different locations of Andhra Pradesh, which is the aquaculture hub of India. The bacterial isolates were assessed for antibiotic resistance towards five antibiotics belonging to different classes (oxytetracycline, chloramphenicol, erythromycin, ciprofloxacin, and co-trimoxazole) by the disc diffusion method. Determination of Epidemiological Cut-OFF (ECOFF) values and analysis by employing normalized resistance interpretation (NRI) was carried out. RESULTS: The most dominant bacterial populations from shrimp culture were Vibrio spp. (pathogenic bacteria) followed by Bacillus spp. (probiotic bacteria). The bacterial isolates showed highest resistance towards oxytetracycline (overall 23.38%) and in location L6 (59.4%) followed by co-trimoxazole (31.1%). ECOFF values calculated by employing NRI showed that the disc diffusion data were distributed in a normalized manner. The maximum ECOFF value was obtained for ciprofloxacin (23.32 mm), while the minimum value was observed for oxytetracycline (9.05 mm). The antibiotic resistant phenotypes showed that the majority of the heterotrophic bacterial isolates (>60%) belonged to the non-wild type phenotype and primarily towards oxytetracycline (90%). CONCLUSION: The presence of non-wild antibiotic-resistant phenotypes of heterotrophic bacterial populations (which include not only pathogenic bacteria but also probiotic bacteria) indicates that shrimp culture ponds may be a reservoir for drug-resistant bacteria and there is a greater risk associated with transmission of resistant genes across bacterial flora. HIGHLIGHTS: NRI analysis of antibiotic disc diffusion data of heterotrophic bacterial populations in shrimp aquaculture environments revealed that majority of them belonged to non-wild type (90%) paticularly to oxytetracycline in comparison to other studied antibiotics (chloramphenicol, erythromycin, ciprofloxacin and co-trimoxazole).

Microbiological Quality of Seafood Marketed in Taiwan.

Seafood is often associated with foodborne illnesses, and Vibrio parahaemolyticus is the most common pathogen implicated in outbreaks in Taiwan. In this study, the microbiological quality of 300 raw or mixed ready-to-eat (RTE) and other cooking-needed seafood samples was examined. The total aerobic and coliform counts of the RTE samples were significantly higher than those of other cooking-needed samples. On average, 55.8 and 29.7% of the RTE samples failed to meet the local microbiological standards for total aerobic (5 log CFU/g) and coliform (3 log most probable number [MPN] per g), counts respectively; the corresponding percentages for the RTE samples from Taipei City were 9.1 and 18.2%, respectively. The total aerobic and coliform counts in the RTE samples from supermarkets and chain restaurants were significantly lower than those from traditional restaurants. The Vibrio species were more frequently identified in the cooking-needed samples than in RTE samples. Low incidences of V. parahaemolyticus (1.4%), V. vulnificus (1.9%), and V. cholerae (0%) were detected in most RTE samples. High densities of V. parahaemolyticus and V. vulnificus (1,200 MPN/g) were detected in a few RTE samples, only one of which contained toxigenic (tdh(+)) V. parahaemolyticus. The results of this investigation reveal that better hygiene of seafood providers such as chain restaurants, supermarkets, and traditional restaurants in Taipei City would effectively improve the microbiological quality of the seafood. The results will facilitate the establishment of measures for controlling the risks associated with seafood in Taiwan.

Assessment of the Physicochemical Qualities and Prevalence of Escherichia coli and Vibrios in the Final Effluents of Two Wastewater Treatment Plants in South Africa: Ecological and Public Health Implications.

The final effluents of two wastewater treatment plants (WWTPs) in the Eastern Cape Province of South Africa were evaluated for their physicochemical and microbiological qualities over a period of 12 months. The physicochemical parameters assessed ranged as follows both plants. The ranges of values for the physicochemical are: pH (3.9-8.6), total dissolved solids (86.50-336.3 mg/L), electrical conductivity (13.57-52.50 mS/m), temperature (13-28 °C), nitrate (0-21.73 mg/L), nitrite (0.01-0.60 mg/L), orthophosphate (1.29-20.57 mg/L), turbidity (4.02-43.20 NTU), free chlorine (0.05-7.18 mg/L), dissolve oxygen (3.91-9.60 mg/L), biochemical oxygen demand (0.1-9.0 mg/L) and chemical oxygen demand (4.67-211 mg/L). The microbiological assessment for both WWTPs revealed the presence of E. coli in counts ranging between 0 and 1.86 × 104 CFU/100 mL and Vibrio counts ranging between 0 and 9.93 × 10³ CFU/100 mL. We conclude that these WWTPs are important point sources of pollution in surface water with potential public health and ecological risks.

Assessment of Nutrient Value and Microbiological Safety of Pomacea lineata.

Pomacea lineata is a fresh water shellfish that is utilized as food, medicinal portions, or licking remedies. The objective of this study was to evaluate the nutritional and microbiological aspects of P. lineata and spawning. The samples were collected in the Jaguaribe River (Paraíba, Brazil) during the rainy and dry seasons. The biochemical composition, mineral composition, total mesophilic bacteria (TMB), total coliforms (TC), fecal coliforms (FC), Salmonella sp., Staphylococcus aureus, and Vibrio presence were determined. Each 100 g of P. lineata presented 3.1 g of ashes; 9.4 g of proteins; 2.7 g of lipids; 0.6 g of carbohydrates; 1800.0 mg of calcium; 78.0 mg of phosphorus, and 58.4 mg of iron. Each 100 g of spawning presented 10.4 g of ashes; 4.5 g of proteins; 2.8 g of lipids; 1.3 g of carbohydrates; 3633.0 mg of calcium; 39.4 mg of phosphorus; and 4.6 mg of iron. Each 100 g of P. lineata provided 65.7 kcal and spawning 48.3 kcal. In the dry season P. lineata and spawning showed no TMB and TC, FC, S. aureus, Salmonella, or Vibrio. In the rainy season P. lineata and spawning presented increase in TMB and TC, FC, S. aureus, and Salmonella. The presence of Vibrio was not detected. P. lineata presented proteins, low lipid, and carbohydrate content, energy values close to those of lean meats, high contents of calcium and iron, and presented low contamination level with pathogenic bacteria. Since P. lineata substances may be useful in the treatment of respiratory and gastrointestinal diseases without presenting toxic or cytotoxic effects already described in the literature, it may be considered as a potential functional food.

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

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.

Multipurpose assessment for the quantification of Vibrio spp. and total bacteria in fish and seawater using multiplex real-time polymerase chain reaction.

BACKGROUND: This study describes the first multiplex real-time polymerase chain reaction assay developed, as a multipurpose assessment, for the simultaneous quantification of total bacteria and three Vibrio spp. (V. parahaemolyticus, V. vulnificus and V. anguillarum) in fish and seawater. The consumption of raw finfish as sushi or sashimi has been increasing the chance of Vibrio outbreaks in consumers. Freshness and quality of fishery products also depend on the total bacterial populations present. RESULTS: The detection sensitivity of the specific targets for the multiplex assay was 1 CFU mL⁻¹ in pure culture and seawater, and 10 CFU g⁻¹ in fish. While total bacterial counts by the multiplex assay were similar to those obtained by cultural methods, the levels of Vibrio detected by the multiplex assay were generally higher than by cultural methods of the same populations. Among the natural samples without Vibrio spp. inoculation, eight out of 10 seawater and three out of 20 fish samples were determined to contain Vibrio spp. CONCLUSION: Our data demonstrate that this multiplex assay could be useful for the rapid detection and quantification of Vibrio spp. and total bacteria as a multipurpose tool for surveillance of fish and water quality as well as diagnostic method.

Fecal pathogen pollution: sources and patterns in water and sediment samples from the upper Cook Inlet, Alaska ecosystem.

Fecal pathogens are transported from a variety of sources in multi-use ecosystems such as upper Cook Inlet (CI), Alaska, which includes the state's urban center and is highly utilized by humans and animals. This study used a novel water quality testing approach to evaluate the presence and host sources of potential fecal pathogens in surface waters and sediments from aquatic ecosystems in upper CI. Matched water and sediment samples, along with effluent from a municipal wastewater treatment facility, were screened for Salmonella spp., Vibrio spp., Cryptosporidium spp., Giardia spp., and noroviruses. Additionally, Bacteroidales spp. for microbial source tracking, and the fecal indicator bacteria Enterococcus spp. as well as fecal coliforms were evaluated. Overall, Giardia and Vibrio were the most frequently detected potential pathogens, followed by Cryptosporidium and norovirus, while Salmonella was not detected. Sample month, matrix type, and recent precipitation were found to be significant environmental factors for protozoa or host-associated Bacteroidales marker detection, whereas location and water temperature were not. The relative contribution of host-associated markers to total fecal marker concentration was estimated using a Monte Carlo method, with the greatest relative contribution to the Bacteroidales marker concentration coming from human sources, while the remainder of the universal fecal host source signal was uncharacterized by available host-associated assays, consistent with wildlife fecal sources. These findings show how fecal indicator and pathogen monitoring, along with identifying contributing host sources, can provide evidence of coastal pathogen pollution and guidance as to whether to target human and/or animal sources for management.

Avaliação do sistema de cultivo integrado, a partir da reciclagem de águas residuais submetidas a tratamento primário: pesquisa de espécies dos gêneros Salmonella, Shigella, Vibrio e Aeromonas / Assessment of integrated aquaculture system from the recycling of wastewater undergoing primary treatment: search of species of Salmonella, Schigella, Vibrio and Aeromonas

Para avaliar um sistema integrado de agrucultura foram realizadas análises microbiológicas da água utilizada neste sistema e determinada a incidência e resistência antimicrobiana dos enteropatógenos no ecossistema relacionado. As amostras de água testadas apresentaram 32,9% de taxas de coliformes fecais (<- 1.600/100mL), de acordo com a OMS para piscicultura em águas residuais. Salmonella spp. foram detectadas em 14,5% das amostras. De um total de 33 cepas, 15,1% eram resistentes a um ou dois antimicrobianos testados e resistência a múltiplas drogas não foi observada. Aeromonas spp. foram identificadas em 91,6% das amostras. De um total de 416 cepas, resistência a uma classe de antimicrobianos foi observada em 66,3% e a multirresistência às drogas em 37,7%. Na avaliação da virulência dos isolados de Aeromonas hydrophila, 85,3% das cepas apresentaram Beta-hemólise nos três diferentes tipos de eritrócitos empregados e 99,1% nos eritrócitos de coelho e cavalo, sendo possível a caracterização através da PCR do gene aerA e lip, em 100% das amostras. Os resultados obtidos apontam para a relevância quanto às vantagens da implementação de um sistema integrado, disponibilizando alimentos com custo reduzido, porém este sistema necessita de um controle rígido e efetivo para que estes produtos não constituam veículos para a disseminação de doenças.

To evaluate an integrated aquaculture system, microbiological analyses of water used in this system were carried out and the incidence and antimicrobial resistance of enteropathogens were determined in the related ecosystem. Water samples tested had 32.9% of fecal coliforms rates (<-1600/100mL) in accordance with WHO for psiculture in wastewater. Salmonella spp. were detected in 14.5% of the samples. From a total of 33 strains, 15.1% were resistant to one or two antimicrobial drugs tested and multidrug-resistance was not observed. Aeromonas spp. were identified in 91.6% of the samples. From a total of 416 strains, resistance to one antimicrobial class was observed in 66.3% and multidrug-resistance in 37.7%. In relation to virulence factors of Aeromonas hydrophila, 85.3% of the strains showed beta-hemolysis in three different types of erythrocytes and 99.1% in horse and rabbit erythrocytes. It was possible to characterize by PCR assay, the genes aerA and lip in 100% of the strains. The results indicate the relevance of the benefits of implementing an integrated system, providing food with reduced cost, but this system requires a strict and effective control so that these products do not constitute a vehicle for the spread of disease.

Sensitive and rapid detection of Vibrio corallilyticus by loop-mediated isothermal amplification targeted to the alpha subunit gene of RNA polymerase.

AIMS: A diagnostic protocol was developed for rapid detection of Vibrio corallilyticus by method of loop-mediated isothermal amplification (LAMP). METHODS AND RESULTS: For cloning and sequencing of rpo A gene of V. corallilyticus, a set of four LAMP primers were designed by targeting the rpoA gene. With Bst DNA polymerase, the reaction time and temperature were optimized for 70 min at 65 degrees C, respectively. The amplification products were detected by electrophoresis. The detection limit of V. corallilyticus by LAMP was 3.6 x 10(3) CFU ml(-1) (8 CFU per reaction), but PCR could detect up to 3.6 x 10(4) CFU ml(-1) (72 CFU per reaction). The LAMP method was ninefold more sensitive than conventional PCR. The results also indicated that the LAMP reaction was highly specific to V. corallilyticus. CONCLUSIONS: The LAMP assay was a sensitive, specific and cost-effective method for the rapid detection of V. corallilyticus. SIGNIFICANCE AND IMPACT OF THE STUDY: This LAMP method provides an important diagnostic tool for the detection of V. corallilyticus infection. It can replace laborious biochemical tests for the identification of V. corallilyticus.

Development of a haemolysin gene-based multiplex PCR for simultaneous detection of Vibrio campbellii, Vibrio harveyi and Vibrio parahaemolyticus.

AIM: To develop a haemolysin (hly) gene-based species-specific multiplex PCR for simple and rapid detection of Vibrio campbellii, V. harveyi and V. parahaemolyticus. METHODS AND RESULTS: The complete hly genes of three V. campbellii strains isolated from diseased shrimps were sequenced and species-specific PCR primers were designed based on these sequences and the registered hly gene sequences of Vibrio harveyi and Vibrio parahaemolyticus. Specificity and sensitivity of the multiplex PCR was validated with 27 V. campbellii, 16 V. harveyi, and 69 V. parahaemolyticus, 18 other Vibrio species, one Photobacterium damselae and nine other bacterial species. The detection limits of all the three target species were in between 10 and 100 cells per PCR tube. CONCLUSIONS: Specificity and sensitivity of the multiplex PCR is 100% each and sufficient to be considered as an effective tool in a prediction system to prevent potential disease outbreak by these Vibrio species. SIGNIFICANCE AND IMPACT OF THE STUDY: Because there is lack of simple, rapid and cost-effective method to differentiate these closely related V. campbellii, V. harveyi and V. parahaemolyticus species, the multiplex PCR developed in this study will be very effective in epidemiological, ecological and economical points of view.

[Etiology of off-plate syndrome in cultured larval Apostichopus japonicus at attachment stage and analysis of reservoir of pathogens].

OBJECTIVE: To clarify pathogens and sources of the off-plate syndrome at the attachment stage in the larval culture of Apostichopus japonicus, and further to find out effective medicines for this disease. METHODS: Etiological analysis was performed on larvae with typical off-plate syndrome from three larvae culture factories. Suspicious pathogens were used for artificial infection test, and were identified through morphological, physiological and biochemical tests, and 16S rDNA sequence analysis. Quantitative bacterial analysis was done on the culture systems of the three factories, including water sources, rearing water, ordure (in the pond floor), attachments and feeds. Finally, drug-sensitive tests were done against the pathogens. RESULTS: A common dominant bacterium strain was isolated from all ill larvae included in the study. Artificial infection test showed it was the causative pathogen associated with the disease, and the artificially infected sea cucumbers had same syndromes to the naturally ill ones. The bacterium was identified as Vibrio sp. Bacterial quantity of water sources was in the qualified range (<50 cfu/mL), while out of the standard range in others (> 1 x 10(5) cfu/mL). The sources of the pathogen were complicated, since pathogens were discovered in the water sources, rearing water, ordure, attachments and feeds. However, the density of causative bacteria was the highest in the feeds, middle in the attachments, and lowest in the water sources. Twelve antibiotics could inhibit growth of the pathogens. CONCLUSION: The possible pathogen for off-plate syndrome was Vibrio sp. Feeds may be the main source of the pathogen. Twelve antibiotics besides nalidixic acid could be applied for disease prevention and treatment of Apostichopus japonicus.

Microbiological profile of raw Nile water.

With the increase of population on the Nile banks, a remarkable increase in industrial, agricultural, human activities, and recreational activities have also occurred. The effluents of such activities are discharged directly into the Nile or through some agricultural drains which finally discharge their wastes into the Nile. Thus, the microbiological monitoring is one of the main objectives to protect the river Nile. This study was conducted to determine the microbiological profile of river Nile at Cairo segment. Eight sites were chosen along the distance (60 Km) from El-Shobak to El-Kanater and tested for two successive years (summer 1994-spring 1996). To achieve this goal, microbiological parameters (total bacterial counts at both 22 degrees C and 37 degrees C, total coliforms, faecal coliforms, faecal streptococci, total yeasts, Candida albicans, Aeromonas hydrophila, salmonellae, total staphylococci, total vibrios and Listeria group) were evaluated. The results showed that the count of the previous parameters ranged between 1.0x10(3)-7.8x10(5) CFU/ml, 5.0x10(2)-6.4x10(5) CFU/ml, 4.9x10-1.6x10(4) MPN/100 ml, 2.0-5.2x0(3) MPN/100 ml, 2.0x10-1.4x10(3) MPN/100 ml, 1.7x10(3)-2.6x10(5) CFU/100 ml, 1.5x10(2)-1.7x10(5) CFU/100 ml, 1.0x10(3)-2.4x10(5) CFU/100 ml, 1.3x10(2)-5.1x10(3) CFU/100 ml, 1.0x10(2)-3.3x10(3) CFU/100 ml, 1.0x10(2)-3.8x10(4) CFU/100 ml and 1.0x10(2)-1.4x10(5) CFU/100 ml, respectively. The total bacterial counts and bacterial indicators (total coliforms, faecal coliforms and faecal streptococci) were detected in all samples during the period of study, and the count increased in samples collected during summer than other seasons. Also total yeasts, A. hydrophila and total staphylococci were detected in all samples with differences in count between the sites. In contrast, other microbial parameters, Candida albicans, salmonellae, total vibrios and Listeria group were not detected in some samples at some sites. According to our results it can be concluded that the river Nile is categorized as an intermediately polluted river.

Prevention of Vibrio vulnificus infections. Assessment of regulatory educational strategies.

CONTEXT: Vibrio vulnificus-related disease caused by eating contaminated raw oysters prompted California to implement regulatory educational initiatives in 1991. OBJECTIVE: To assess California guidelines and education efforts with regard to the prevention of V. vulnificus infections in Los Angeles County and to evaluate compliance with state regulations mandating posting of warning signs concerning risks of eating raw oysters. DESIGN: Review of epidemiologic investigations of V. vulnificus infections in Los Angeles County between 1993 and 1995; telephone interviews of patients or surrogates; and a survey of restaurants serving raw oysters in Los Angeles County. SETTING: General community. MAIN OUTCOME MEASURES: Prior warning of patients and posting of warning signs in restaurants. RESULTS: Recent cases of V. vulnificus infections in Los Angeles County suggest that a unique, vulnerable group (uninsured Hispanic men with viral or alcoholic liver disease) has not been reached through education efforts. Of a total of 11 cases, information regarding being warned about the hazard of eating raw shellfish was available for 8; of these, only 1 case was reported as having been warned, but he had misunderstood the preventive message. Warning signs were not posted adequately in more than 50% of restaurants surveyed and one third of these establishments had signs in English only. Warnings were never located on dining tables and rarely included in menus. CONCLUSION: A more effective strategy is required to prevent V.vulnificus infections in Los Angeles County.

Aislamiento de Vibrios Patogenos y Valoracion de la Calidad Sanitaria de Ostiones frescos cosechados en Cuba / Isolation of pathogenic Vibrios and assessment of the sanitary quality of fresh oysters grown in Cuba

Se analizaron 80 muestras de masa de ostion fresco de una zona occidental del pais. Se les realizaron a todas las muestras aislamiento y caracterizacion bioquimica de Vibrios patogenos, determinacion cuantitativa de coliformes fecales y determinacion de Salmonella. Se aislaron 234 cepas de vibrios patogenos en 77 muestras, predomino el Vibrio alginolyticus y se demostro la naturaleza autoctona de estos microorganismos en este tipo de muestra. No se aislo Salmonella en ninguna de las muestras. Quince muestras estuvieron fuera de los limites microbiologicos establecidos para el indicador coliformes fecales pero ninguno sobrepaso la cifra de 10 3 UFC/g. Se concluyo que la calidad de las muestras analizadas es satisfactoria y que los hallazgos de Vibrios patogenos no guardan relacion con los resultados satisfactorios de las otras determinaciones realizadas