Manjusha Lekshmi

Isolation and characterization of Arcobacter spp. from fresh seafood and the aquatic environment.

Arcobacter is an emerging pathogen associated with foods of animal origin. Members of the genus Arcobacter are increasingly being isolated from fish, shellfish and the aquatic environment. In the present study, we analyzed fish, shellfish and water samples for the presence of Arcobacter spp. by conventional isolation as well as by direct PCR on the enrichment broth. Of 100 samples comprising of 42 finfish, 34 shellfish and 24 water samples analyzed, Arcobacter spp. was isolated from 8 (19%) finfish, 5 (14.7%) shellfish and 5 (20.8%) water samples. Arcobacter DNA was detected in 24 (24%) samples by direct PCR on the enrichment broth. Based on m-PCR specific to different Arcobacter spp. and 16S rRNA sequence analyses, majority (19) of the isolates were identified as Arcobacter butzleri, while two isolates were Arcobacter mytili. All Arcobacter butzleri isolates harbored putative virulence genes cadF, ciaB, mviN, pldA, tlyA and cj1349. The two isolates of A. mytili harbored mviN and cj1349 genes only. The study highlights emerging problem of the contamination of aquatic environment and fresh seafood with potentially pathogenic Arcobacter spp.

The Food Production Environment and the Development of Antimicrobial Resistance in Human Pathogens of Animal Origin

Food-borne pathogens are a serious human health concern worldwide, and the emergence of antibiotic-resistant food pathogens has further confounded this problem. Once-highly-efficacious antibiotics are gradually becoming ineffective against many important pathogens, resulting in severe treatment crises. Among several reasons for the development and spread of antimicrobial resistance, their overuse in animal food production systems for purposes other than treatment of infections is prominent. Many pathogens of animals are zoonotic, and therefore any development of resistance in pathogens associated with food animals can spread to humans through the food chain. Human infections by antibiotic-resistant pathogens such as Campylobacter spp., Salmonella spp., Escherichia coli and Staphylococcus aureus are increasing. Considering the human health risk due to emerging antibiotic resistance in food animal–associated bacteria, many countries have banned the use of antibiotic growth promoters and the application in animals of antibiotics critically important in human medicine. Concerted global efforts are necessary to minimize the use of antimicrobials in food animals in order to control the development of antibiotic resistance in these systems and their spread to humans via food and water.

Delivery of Omega-3 Fatty Acids into Cake Through Emulsification of Fish Oil-in-Milk and Encapsulation by Spray Drying with Added Polymers

Fortification of cakes with fish oil encapsulates was performed to enhance the consumption of health-beneficial polyunsaturated fatty acids like eicosapentaenoic acid and docosahexaenoic acid. Fish oil-in-milk emulsions prepared by ultrasonication at different amplitudes were encapsulated by spray drying using different wall materials. The oxidative stability of fish oil encapsulates was determined for 32 days at room and refrigerated temperatures. Oxidatively stable encapsulates and organoleptic quality of fortified cakes reveal that emulsification of fish oil-in-milk and encapsulation by spray drying are potential processes to produce fish oil encapsulates suitable for fortification of bakery products with omega-3 fatty acids.

Isolation of Escherichia coli harboring blaNDM-5 from fresh fish in India

Extended spectrum b-lactamase (ESBL)-producing Gramnegative bacteria have become a severe challenge to chemotherapy. The ESBLs are classified into several groups, the prominent of them being TEM, SHV, and CTX-M types. Members of the family Enterobacteriaceae, which cause diverse infections ranging from wound infection to meningitis, are the major ESBL-producing Gram-negative bacteria. Carbapenems are the antibiotics of choice against ESBL-producing bacteria, but the occurrence of carbapenem-resistant enterobacteria (CRE) producing plasmid-encoded metallo-b-lactamases with carbapenemase activity have emerged worldwide. In 2008, a new b-lactamase, the New Delhi metallo-blactamase (blaNDM-1), capable of hydrolyzing all b-lactams with the exception of aztreonam, was identified in Klebsiella pneumoniae isolated from a Swedish patient who was previously treated in India. Subsequently, the NDM-1 producing enterobacteria were isolated from different parts of the world. Studies from India and other countries have reported the occurrence of blaNDM-1 harboring bacteria in the environment. Variants of NDM-1 designated as NDM-2, NDM-4, NDM-5, NDM-6, and NDM-7 have arisen through point mutations. NDM genes are located on a large plasmid which also harbors resistance genes for several antibiotics such as b-lactams, aminoglycosides, sulfonamides, chloramphenicol, and macrolides and this plasmid is highly mobile leading to its rapid dissemination in the environment. Here, we report the isolation of an NDM-5 harboring Escherichia coli from fresh seafood sold in a retail market of Mumbai, India. We investigated the presence of NDM-producing enterobacteria in fresh seafood samples sold in retail markets of Mumbai. A total of 19 samples, which included 14 finfish and five shellfish samples, were analyzed. The bacterial isolations were made on MacConkey agar following enrichment of the sample in enterobacteria enrichment broth (EE broth and Mossel; Hi Media, Mumbai, India). Presumptive

Development of functional extruded snacks by utilizing paste shrimp (Acetes spp.): process optimization and quality evaluation

BACKGROUND Functional extruded snacks were prepared using paste shrimp powder (Acetes spp.), which is rich in protein. The process variables required for the preparation of extruded snacks was optimized using response surface methodology. Extrusion temperature (130-144 °C), level of Acetes powder (100-200 g kg-1 ) and feed moisture (140-200 g kg-1 ) were selected as design variables, and expansion ratio, porosity, hardness, crispness and thiobarbituric acid reactive substance value were taken as the response variables. RESULTS Extrusion temperature significantly influenced all the response variables, while Acetes inclusion influenced all variables except porosity. Feed moisture content showed a significant quadratic effect on all responses and an interactive effect on expansion ratio and hardness. Shrimp powder incorporation increased the protein and mineral content of the final product. The extruded snack made with the combination of extrusion temperature 144.59 °C, feed moisture 178.5 g kg-1 and Acetes inclusion level 146.7 g kg-1 was found to be the best one based on sensory evaluation. CONCLUSION The study suggests that use of Acetes species for the development of extruded snacks will serve as a means of utilization of Acetes as well as being a rich source of proteins for human consumption, which would otherwise remain unexploited as a by-catch.

Multiple Antibiotic-Resistant, Extended Spectrum-β-Lactamase (ESBL)-Producing Enterobacteria in Fresh Seafood.

Members of the family Enterobacteriaceae include several human pathogens that can be acquired through contaminated food and water. In this study, the incidence of extended spectrum β-lactamase (ESBL)-producing enterobacteria was investigated in fresh seafood sold in retail markets. The ESBL-positive phenotype was detected in 169 (78.60%) isolates, with Escherichia coli being the predominant species (53), followed by Klebsiella oxytoca (27), and K. pneumoniae (23). More than 90% of the isolates were resistant to third generation cephalosporins, cefotaxime, ceftazidime, and cefpodoxime. Sixty-five percent of the isolates were resistant to the monobactam drug aztreonam, 40.82% to ertapenem, and 31.36% to meropenem. Resistance to at least five antibiotics was observed in 38.46% of the isolates. Polymerase Chain Reaction (PCR) analysis of ESBL-encoding genes detected blaCTX, blaSHV, and blaTEM genes in 76.92%, 63.3%, and 44.37% of the isolates, respectively. Multiple ESBL genes were detected in majority of the isolates. The recently discovered New Delhi metallo-β-lactamase gene (blaNDM-1) was detected in two ESBL+ isolates. Our study shows that secondary contamination of fresh seafood with enteric bacteria resistant to multiple antibiotics may implicate seafood as a potential carrier of antibiotic resistant bacteria and emphasizes an urgent need to prevent environmental contamination and dissemination of such bacteria.

Virulence genotypes and antimicrobial susceptibility patterns of Arcobacter butzleri isolated from seafood and its environment

Arcobacter butzleri is an emerging pathogen isolated from animals, food and the environment. In this study, 147 A. butzleri isolated from seafood and the coastal environment were tested for the presence of ten putative virulence genes (cadF, cj1349, ciaB, mviN, pldA, tlyA, hecA, hecB, irgA, iroE) and antimicrobial susceptibilities. Majority of the isolates harbored mviN (100%), cj1349 (97.2%), ciaB (95.9%), tlyA (91.8%), pldA (91.1%) and cadF (89.7%). Lower detection rates were observed for hecA (10.8%), hecB (19%), iroE (12.9%) and irgA (17.6%). Three A. butzleri isolates harbored all ten virulence genes. The occurrence of cj1349, ciaB, pldA, tlyA and hecA genes was significantly different (P≤0.05) among the isolates from different sources. All (100%) A. butzleri isolates were resistant to vancomycin, cephalothin, cefoxitin and sulphamethizole and susceptible to polymyxin-B, kanamycin, streptomycin, gentamicin, tetracycline and imipenem. Resistance to clinically important antibiotics such as cefotaxime (99.3%), ceftazidime (87.7%), nalidixic acid (70.7%), ampicillin (72.1%), ertapenem and amoxicillin-clavulanic acid (41.9%) was observed in A. butzleri from the environment. The isolates were highly susceptible to norfloxacin (97.9%) and colistin (97.2%), followed by ciprofloxacin (88.4%), meropenem (74.8%), chloramphenicol (72.7%) and erythromycin (69.3%). A. butzleri from different sources were not significantly different with respect to their antimicrobial susceptibility patterns. Multidrug resistance was observed in 66 (81.4%) isolates from fish, 29 (72.5%) isolates from shellfish and 17 (65.3%) isolates from coastal water. A. butzleri harboring virulence genes and resistance to multiple antibiotics found in seafood could be a potential health risk to seafood handlers and consumers. Continuous monitoring of seafood for potentially pathogenic A. butzleri is important to understand the evolution of antibiotic resistance in this emerging food pathogen and to determine the antimicrobial therapy regimen in the event of food-borne A. butzleri infections.

Modulation of antimicrobial efflux pumps of the major facilitator superfamily in Staphylococcus aureus

Variants of the microorganism Staphylococcus aureus which are resistant to antimicrobial agents exist as causative agents of serious infectious disease and constitute a considerable public health concern. One of the main antimicrobial resistance mechanisms harbored by S. aureus pathogens is exemplified by integral membrane transport systems that actively remove antimicrobial agents from bacteria where the cytoplasmic drug targets reside, thus allowing the bacteria to survive and grow. An important class of solute transporter proteins, called the major facilitator superfamily, includes related and homologous passive and secondary active transport systems, many of which are antimicrobial efflux pumps. Transporters of the major facilitator superfamily, which confer antimicrobial efflux and bacterial resistance in S. aureus, are good targets for development of resistance-modifying agents, such as efflux pump inhibition. Such modulatory action upon these antimicrobial efflux systems of the major facilitator superfamily in S. aureus may circumvent resistance and restore the clinical efficacy of therapy towards S. aureus infection.

Isolation of Shiga toxin-producing Escherichia coli harboring variant Shiga toxin genes from seafood

Background and Aim: Shiga toxin-producing Escherichia coli (STEC) are important pathogens of global significance. STEC are responsible for numerous food-borne outbreaks worldwide and their presence in food is a potential health hazard. The objective of the present study was to determine the incidence of STEC in fresh seafood in Mumbai, India, and to characterize STEC with respect to their virulence determinants. Materials and Methods: A total of 368 E. coli were isolated from 39 fresh seafood samples (18 finfish and 21 shellfish) using culture-based methods. The isolates were screened by polymerase chain reaction (PCR) for the genes commonly associated with STEC. The variant Shiga toxin genes were confirmed by Southern blotting and hybridization followed by DNA sequencing. Results: One or more Shiga toxins genes were detected in 61 isolates. Of 39 samples analyzed, 10 (25.64%) samples harbored STEC. Other virulence genes, namely, eaeA (coding for an intimin) and hlyA (hemolysin A) were detected in 43 and 15 seafood isolates, respectively. The variant stx1 genes from 6 isolates were sequenced, five of which were found to be stx1d variants, while one sequence varied considerably from known stx1 sequences. Southern hybridization and DNA sequence analysis suggested putative Shiga toxin variant genes (stx2) in at least 3 other isolates. Conclusion: The results of this study showed the occurrence of STEC in seafood harboring one or more Shiga toxin genes. The detection of STEC by PCR may be hampered due to the presence of variant genes such as the stx1d in STEC. This is the first report of stx1d gene in STEC isolated from Indian seafood.

Occurrence of human enterovirus in tropical fish and shellfish and their relationship with fecal indicator bacteria

Aim: Human enteroviruses in fish and shellfish are a health concern worldwide. Human infections occur due to the consumption of raw or insufficiently cooked fish or shellfish. The objective of this study was to determine the occurrence of human enteric viruses belonging to Enterovirus (EV) group in seafood in Mumbai and to correlate their occurrence with the bacterial indicators of fecal contamination. Materials and Methods: Samples of fresh fish and shellfish collected from fish landing centers and retail fish markets were analyzed by virus concentration, nucleic acid extraction, and reverse transcription-polymerase chain reaction (RT-PCR). Bacterial indicators of fecal contamination were estimated by the most probable number technique. The relationship between the presence of virus and fecal indicators was determined by statistical analysis. Results: A total of 89 samples comprising of fish, shrimps, oysters, clams, and mussels were screened in this study. EV was detected in 32 (35.95%) samples, and all the virus-positive samples belonged to bivalve molluscan group. None of the finfish and crustacean shellfish samples was positive for the enteric viruses. Clams were found to be the most contaminated with 48.4% of the samples being positive for EV. The prevalence of enteric viruses in seafood samples showed a strong positive correlation with the bacteriological indicators of fecal contamination, suggesting that fecal coliform bacteria are good indicators of EVs in tropical seafood. Conclusion: The presence of EVs in seafood is a public health hazard. Increasing level of coastal water contamination from anthropogenic sources is the primary reason for the contamination of seafood with EVs. Continuous monitoring of coastal waters and seafood for enteric viruses will help to ensure the safety of fish and shellfish for human consumption.