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).

The economics of shrimp disease.

Shrimp is not only one of the world's most valuable aquaculture species, but also a species that encounter high economic losses due to diseases. Diseases are sufficiently important to influence global supply and prices for longer periods. Profitability is the driving force behind shrimp farming and high profits associated with the absence of disease largely determines where shrimp production does take place; i.e. prevalence of disease leads to geographic relocation. In this paper, a basic economic model for the impact of the disease on a shrimp farm is provided and a Monte Carlo simulation is provided to illustrate the impact of disease on economic risk. Improved technologies, knowledge, and governance are important elements utilized in the mitigation of diseases in various shrimp producing countries. Economic aspects such as profitability in the absence and presence of diseases and cost of treatment determines the global production of shrimp along with shaping technologies and production systems.

Development of a cost-efficient micro-detection slide system for the detection of multiple shrimp pathogens.

In view of the current demand for rapid detection and identification of pathogens, point-of-care testing (POCT) with fast portability, low consumption, and increased sensitivity and specificity has become more and more popular. The emerging nucleic acid isothermal amplification technology (NAIAT) has shown potential advantages in the development of rapid microbial detection. In this study, a micro-detection slide system was developed based on the NAIAT of various nucleic acids of shrimp pathogens. The system included a micro-detection slide with 48 identical detecting cells precoated with all detection reagents, except the sample template. The process of producing the micro-detection slides mainly combined super-hydrophobic/super-oleophobic and super-hydrophilic materials to obtain separated spaces for detection, and aerosol pollution was eliminated in the form of water-in-oil. The micro-detection slide system was capable of simultaneously detecting 4 groups of samples and 8 important shrimp pathogens and is a relatively low-cost, portable, and high-throughput nucleic acid (RNA and DNA) detection technology. The establishment of this technology will provide key technical support for the construction of biosecurity systems for healthy shrimp culture.

Efficacy assessment of commercially available natural products and antibiotics, commonly used for mitigation of pathogenic Vibrio outbreaks in Ecuadorian Penaeus (Litopenaeus) vannamei hatcheries.

Bacterial diseases cause high mortality in Penaeus (Litopenaeus) vannamei postlarvae. Therefore, appropriate application of efficient therapeutic products is of vital importance for disease control. This study evaluated through in vitro analyses the antimicrobial effectiveness of commercial therapeutic products used for P. vannamei bacterial diseases and antibiotics against pathogenic Vibrio strains circulating in Ecuadorian hatcheries. Twenty strains were isolated from 31 larvae samples with high bacterial counts from 10 hatcheries collected during mortality events. The strains virulence was verified through challenge tests with Artemia franciscana nauplii and P. vannamei postlarvae. Through 16S rRNA sequence analysis, strains showed a great similarity to the Vibrio sequences reported as pathogens, with 95% belonging to the Harveyi clade. Through antibiograms and minimal inhibitory concentration (MIC) in vitro tests we found that furazolidone, ciprofloxacin, chloramphenicol, norfloxacin, nalidixic acid, florfenicol, fosfomycin and enrofloxacin inhibited the growth of all or most of the strains. Less efficient antibiotics were penicillin, oxytetracycline and tetracycline. A multiple antibiotic resistance (MAR) index of 0.23 showed some level of resistance to antibiotics, with two MAR prevalent patterns (Penicillin-Oxytetracycline and Penicillin-Oxytetracycline-Tetracycline). From a total of 16 natural products (five probiotics, nine organic acids and two essential oils), only three (one probiotic, one organic acid and one essential oil) were effective to control most of the strains. Shrimp producers can apply relatively simple in vitro analyses, such as those employed in this study, to help take adequate management decisions to reduce the impact of bacterial diseases and increase profit.

Plasmid dynamics in Vibrio parahaemolyticus strains related to shrimp Acute Hepatopancreatic Necrosis Syndrome (AHPNS).

Vibrio parahaemolyticus is a causative agent of acute hapatopancreatic necrosis syndrome (AHPNS) which causes early mortality in white shrimp. Emergence of AHPNS has caused tremendous economic loss for aquaculture industry particularly in Asia since 2010. Previous studies reported that strains causing AHPNS harbor a 69-kb plasmid with possession of virulence genes, pirA and pirB. However, genetic variation of the 69-kb plasmid among AHPNS related strains has not been investigated. This study aimed to analyze genetic composition and diversity of the 69-kb plasmid in strains isolated from shrimps affected by AHPNS. Plasmids recovered from V. parahaemolyticus strain VPE61 which represented typical AHPNS pathogenicity, strain VP2HP which did not represent AHPNS pathogenicity but was isolated from AHPNS affected shrimp and other AHPNS V. parahaemolyticus isolates in Genbank were investigated. Protein coding genes of the 69-kb plasmid from the strain VPE61 were identical to that of AHPNS strain from Vietnam except the inverted complement 3.4-kb transposon covering pirA and pirB. The strain VP2HP possessed remarkable large 183-kb plasmid which shared similar protein coding genes to those of the 69-kb plasmid from strain VPE61. However, the 3.4-kb transposon covering pirA and pirB was absent from the 183-kb plasmid in strain VP2HP. A number of protein coding genes from the 183-kb plasmid were also detected in other AHPNS strains. In summary, this study identified a novel 183-kb plasmid that is related to AHPNS causing strains. Homologous recombination of the 69-kb AHPNS plasmid and other naturally occurring plasmids together with loss and gain of AHPNS virulence genes in V. parahaemolyticus were observed. The outcome of this research enables understanding of plasmid dynamics that possibly affect variable degrees of AHPNS pathogenicity.

Antibiotic and heavy-metal resistance of Vibrio parahaemolyticus isolated from fresh shrimps in Shanghai fish markets, China.

Vibrio parahaemolyticus is a causative agent of human serious seafood-borne gastroenteritis disease and even death. Shrimps, often eaten raw or undercooked, are an important reservoir of the bacterium. In this study, we isolated and characterized a total of 400 V. parahaemolyticus strains from commonly consumed fresh shrimps (Litopenaeus vannamei, Macrobrachium rosenbergii, Penaeus monodon, and Exopalaemon carinicauda) in Shanghai fish markets, China in 2013-2014. The results revealed an extremely low occurrence of pathogenic V. parahaemolyticus carrying two major toxic genes (tdh and trh, 0.0 and 0.5 %). However, high incidences of antibiotic resistance were observed among the strains against ampicillin (99 %), streptomycin (45.25 %), rifampicin (38.25 %), and spectinomycin (25.50 %). Approximately 24 % of the strains derived from the P. monodon sample displayed multidrug resistant (MDR) phenotypes, followed by 19, 12, and 6 % from the E. carinicauda, L. vannamei, and M. rosenbergii samples, respectively. Moreover, tolerance to heavy metals of Cr(3+) and Zn(2+) was observed in 90 antibiotic resistant strains, the majority of which also displayed resistance to Cu(2+) (93.3 %), Pb(2+) (87.8 %), and Cd(2+)(73.3 %). The pulsed-field gel electrophoresis (PFGE)-based genotyping of these strains revealed a total of 71 distinct pulsotypes, demonstrating a large degree of genomic variation among the isolates. The wide distribution of MDR and heavy-metal resistance isolates in the PFGE clusters suggested the co-existence of a number of resistant determinants in V. parahaemolyticus population in the detected samples. This study provided data in support of aquatic animal health management and food safety risk assessment in aquaculture industry.

Application of response surface methodology to optimise microbial inactivation of shrimp and conch by supercritical carbon dioxide.

BACKGROUND: Supercritical carbon dioxide (SC-CO2 ) has been shown to have a good pasteurising effect on food. However, very few research papers have investigated the possibility to exploit this treatment for solid foods, particularly for seafood. Considering the microbial safety of raw seafood consumption, the study aimed to explore the feasibility of microbial inactivation of shrimp (Metapenaeus ensis) and conch (Rapana venosa) by SC-CO2 treatment. RESULTS: Response surface methodology (RSM) models were established to predict and analyse the SC-CO2 process. A 3.69-log reduction in the total aerobic plate count (TPC) of shrimp was observed by SC-CO2 treatment at 53°C, 15 MPa for 40 min, and the logarithmic reduction in TPC of conch was 3.31 at 55°C, 14 MPa for 42 min. Sensory scores of the products achieved approximately 8 (desirable). The optimal parameters for microbial inactivation of shrimp and conch by SC-CO2 might be 55°C, 15 MPa and 40 min. CONCLUSION: SC-CO2 exerted a strong bactericidal effect on the TPC of shrimp and conch, and the products maintained good organoleptic properties. This study verified the feasibility of microbial inactivation of shrimp and conch by SC-CO2 treatment.

Isolation of lactic acid bacteria from kuruma shrimp (Marsupenaeus japonicus) intestine and assessment of immunomodulatory role of a selected strain as probiotic.

Fifty-one lactic acid bacteria (LAB) strains were isolated and identified based on 16S ribosomal DNA sequence from the intestinal tracts of 142 kuruma shrimps (Marsupenaeus japonicus) collected from Kanmon Strait, Fukuoka and Tachibana Bay, Nagasaki, Japan. Cellular immunomodulatory function of 51 isolated LAB strains was assessed by measuring the level of interferon (IFN)-γ induction in mouse spleen cell culture. The strain Lactococcus lactis D1813 exhibited the highest amount of IFN-γ production and also bactericidal activity and was selected for testing its immunomodulatory role as a probiotic in kuruma shrimp. We also assessed the effect of dietary incorporation of this probiotic on resistance to Vibrio penaeicida infection in the kuruma shrimp. Our results demonstrate that probiotic L. lactis D1813-containing diet-fed (105 cfu g⁻¹) shrimps displayed a significant up-regulation of lysozyme gene expressions in the intestine and hepatopancreas. However, insignificantly higher expression of anti-lipopolysaccharide factor, super oxide dismutase, prophenoloxidase, and toll-like receptor 1 was recorded in the intestine of shrimps fed the probiotic diet. Moreover, significantly increased (P < 0.01) resistance to the bacterial pathogen in term of better post-infection survival (61.7 %) was observed in the shrimps fed with the probiotic-incorporated diet compared with the control diet-fed group (28.3 %). The present study indicates the immunomodulatory role of the LAB L. lactis D1813 on the kuruma shrimp immune system and supports its potential use as an effective probiotic in shrimp aquaculture.

Real-time assessment of the microbial quality of retail shrimp using CO2 evolution rate.

A real-time CO(2) evolution rate (CER) method together with conventional cultural and sensory techniques were utilized to determine the microbial quality and shelf life of several types of shrimp products: chloramphenicol (CAP) treated, imported farm raised, and domestic wild caught. Treatment with CAP was used to create different bacterial loads in shrimp samples to demonstrate the ability and sensitivity of the CER method for differentiating the bacterial activity in samples. Samples were divided into control (nontreated) and 0, 10, and 30 ppm of CAP treatment groups and stored at 4°C. The CER was recorded with a microrespirometer, and aerobic plate counts (APCs), olfactory sensory analyses, and pH measurements were recorded daily until spoilage occurred. The real-time CER results were highly correlated with the APCs (R(2) = 0.93) and readily distinguished the onset of spoilage in each of the treatment groups. CAP treatment at 10 and 30 ppm increased the sample shelf life by 2 and 3 days, respectively, compared with the nontreated samples. Untreated domestic wild-caught shrimp had a shelf life 1 day longer than that of the untreated imported farm-raised shrimp. No pattern of change in pH was noted throughout the storage period. When the olfactory sensory scores reached the marginally acceptable level, the mean CER was 27.23 µl/h/g and the mean APC was 5.78 log CFU/g. A cutoff CER of 25.0 µl/h/g was therefore selected to define acceptable raw shrimp. The CER method was a highly effective and sensitive real-time method for determining the microbial quality of raw shrimp.

An in vitro and in vivo assessment of the potential of Vibrio spp. as probiotics for the Pacific white shrimp, Litopenaeus vannamei.

AIMS: The objective of the work was to determine whether known strains of nonpathogenic vibrios can act as probiotics for the control of Vibrio infections in the Pacific white shrimp, Litopenaeus vannamei. METHODS AND RESULTS: Of the ten species tested, only Vibrio alginolyticus (NCIMB 1339) and Vibrio gazogenes (NCIMB 2250) showed antagonistic activity towards a panel of shrimp pathogenic vibrios. In the case of V. alginolyticus, this activity depended on the presence of live bacteria while in V. gazogenes both live and dead bacteria showed anti-Vibrio activity. Injection of shrimp with either V. alginolyticus or V. gazogenes at 3 × 10(7) or 3 × 10(5) total bacteria per shrimp resulted in mortality with higher levels in the case of V. alginolyticus (100% mortality 18 h postinjection of 3 × 10(7) bacteria). Juvenile shrimp were fed commercial diets top-coated with either chitin (an immune stimulant) or chitin + V. gazogenes. Both chitin and V. gazogenes caused a significant decline in the number of Vibrio-like bacteria in the fore and hind gut, and changes were also seen in the hepatosomatic index (a measure of digestive health) and the total number of blood cells in circulation. Analysis of mid/hindgut and faecal samples obtained using terminal restriction fragment length polymorphism showed that the gut microbiota of shrimp has limited bacterial diversity and that after 8 weeks exposure to the experimental diets there were significant changes in the microbial flora of the GI tract of shrimp as a result of the presence of V. gazogenes. CONCLUSIONS: Of the vibrios tested, V. gazogenes has potential as a probiotic for the control of bacterial diseases in shrimp. SIGNIFICANCE AND IMPACT OF THE STUDY: Overall, this study shows the promise of V. gazogenes together with chitin to improve the health and welfare of shrimp under aquaculture conditions.

Toxicological assessment of Penicillium nalgiovense strains for use as starter cultures in the manufacture of dry fermented sausages.

The use of fungal starter strains in the casing of dry fermented sausages allows standardization of the manufacturing process and ensures consumer safety. Penicillium nalgiovense is normally used for this purpose. Even though this species is reported as safe with respect to the production of the most common mycotoxins, its safety may be strain specific. The aim of the present work was to assess the toxicogenic potential of nine P. nalgiovense strains isolated from dry fermented sausages that were previously suitable as starters. The strains were tested for toxicity on brine shrimp larvae and the human cell line MCF7, for mutagenicity in the Ames test, and for antibacterial activity against gram-positive and gram-negative bacteria. According to our results, several P. nalgiovense strains were positive in more than one bioassay. Therefore, it is important to use different toxicological assays when characterizing strains intended for food use. Strains S1-2 and S14-4, which belong to biotypes 6 and 5, respectively, were nontoxigenic under the conditions tested. Overall, strain S1-2 of P. nalgiovense proved to be best suited as a starter in dry fermented sausage manufacture because in addition of being nontoxicogenic it produces white conidia, which is a desirable feature.

Assessment of fluorescent-labeled bacteria for evaluation of in vivo uptake of bacteria (Vibrio spp.) by crustacean larvae.

Available methods to study crustacean digestive tract colonization by bacteria are laborious, time-consuming, and do not permit in vivo assays and observation. This paper reports on a rapid and consistent technique to apply a fluorescent label to bacteria, which can then be presented to filter-feeding crustacea such as Artemia and penaeid larvae for later in situ bacterial distribution observation. Three luminescent Vibrio spp. were stained and observed inside Artemia nauplii, shrimp zoea and mysis stages, Vibrio harveyi type strain ATCC 14126, M(1) (pathogenic) and Ea (non-pathogenic). Factors such as dye (DTAF) concentration, exposure time/temperature and sonication time were evaluated. Viability of the dye and stained bacteria were tested at 4, -20 and -70 degrees C storage temperatures for up to 81 days. Results show that 4 and -20 degrees C storage temperatures are not recommended. At -70 degrees C, both bacteria and dye are optimally preserved. Monodispersed fluorescent-labeled bacterial cells can be observed inside the digestive tract of crustacean larvae at a density of inoculation as high as 5.2 x 10(6) CFU ml(-1). After 2 to 4 h, some leaching occurs, increasing difficulty in observation, although after 24 h, it is still possible to observe monodispersed FLB inside the digestive tract of crustacean larvae. Autofluorescence may complicate observation when filter-feeding crustacean larvae are co-fed with microalgae.

Quantitative assessment of phagocytic activity of hemocytes in the prawn, Penaeus merguiensis, by flow cytometric analysis.

BACKGROUND: The blood cells of crustaceans are involved in phagocytosis of invading microorganisms, contributing to their defense mechanisms. In this study, phagocytic activity of hemocytes of the prawn, Penaeus merguiensis, was quantitated by means of flow cytometric analysis. METHOD: This study was done in vitro. Hemolymph, which was extracted from prawns, was mixed with an equal volume of anticoagulant. Heat-killed Escherichia coli prestained with propidium iodide (PI) was then added. Hemocytes were fixed at various time intervals for flow cytometric analysis. This study was supplemented with electron micrographs using transmission electron microscopy (TEM), which showed three populations of hemocytes. RESULTS: It was observed that those hemocytes that were more active engulfed and digested bacteria readily, thus having higher red fluorescence intensity. The phagocytic activity was expressed as fluorescence unit or engulfed E. coli number per hemocyte. CONCLUSIONS: With this approach, the phagocytic and cellular activity of individual hemocyte populations could be studied quantitatively.