Peer Reviewed Journal Articles (Inter.) (M,F&B)

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A revisited two-step microtiter plate assay: Optimization of in vitro multiplicity of infection (MOI) for Coliphage and Vibriophage
(Elsevier, 2021-08) Benala, Manikantha; Vaiyapuri, Murugadas; Sivam, Visnuvinayagam; Chalil George, Joshy; Raveendran, Karthika; George, Iris; Mothadaka, Mukteswar Prasad; Badireddy, Madhusudana Rao
A 2-step microtiter plate assay was developed to simultaneously check wide values of MOIs of bacteriophages, ranging between MOI-0.0001 and MOI-10000 in the first step and optimize the most suitable MOI (lowest quantity of phage) for inhibiting the growth of the target bacteria in the second step. The results of the first step revealed that the effective MOI of coliphage-ɸ5 for controlling the growth of antimicrobial resistant (AMR) E. coli was between 4.36 and 43.6 for E.coli-EC-3; between 38.2 and 382 for E.coli-EC-7 and between 81.5 and 815 for E. coli-EC-11. The optimum MOI of coliphage-ɸ5 determined in the second step was 17.44, 191 and 326 for controlling the growth of E.coli-EC-3; E.coli-EC-7 and E.coli-EC-11, respectively. The effective MOI of vibriophageɸLV6 for controlling luminescent Vibrio harveyi in the first step was found to be between 18.3 and 183 and the optimum MOI as determined in the second step was 79. The sequential 2-step microtiter plate method yielded faster optimization of MOI and was economical compared to the conventional flask method. The measurement of OD values at 550 nm and 600 nm showed similar trend and replicate data from 5-wells and 3-wells yielded identical pattern indicating that the measuring absorbance data in 3-replicate wells at either OD550 or OD600 is sufficient to generate quantifiable phage lysis data. The 2-step microtiter plate assay finds application in phage therapy in human health care, agriculture and animal agriculture for determining the optimum MOIs for selected bacteriophages.
Antibiotic susceptibility of staphylococci isolated from labeo rohita sold in burla fish market, orissa
(Asian Fisheries Society, 2009) Sahoo, R.S.; Sahu, J.; Prasad, M.M.
Screening of commercial sold freshwater Labeo rohita revealed that the staphylococcal count decreased from one to two log cycles/g upon washing the fish. The fish samples harboured more number of staphylococci in edible meat portion than in guts. The microscopic, biochemical and oxidative fermentations tests of the isolates by conventional and rapid detection methods revealed that they are Staphylococcus haemolyticus, S. auricularius and S. caseolyticus. When the isolates were compared with 45 different antibiotics the results varied from susceptible to resistant in comparison with American Type Culture Collection cultures. In comparison to Clinical Laboratory Standards Institute isolates both the staphylococcal test cultures were resistant to penicillin (G) and vancomycin. With other antibiotics viz., Pristinamycin, Ticarcillin/Clavulanic acid, Gatifloxacin, Clindamycin, Clarithromycin, Levofloxacin, Linezolid, Cefeprime, Erythromycin, Streptomycin, Fosfomycin, and Piperacillin/Tazobactam the results varied from intermediate to sensitive.
Antimicrobial resistance in Vibrios of shrimp aquaculture: Incidence, identification schemes, drivers and mitigation measures
(Springer, 2021-01-18) Vaiyapuri, Murugadas; Pailla, Sravya; Badireddy, Madhusudana Rao; Pillai, Devika; C. N., Ravishankar; Mothadaka, Mukteswar Prasad
Antimicrobial resistance (AMR) is a growing threat to the human, and animal population, worldwide. The antimicrobials used across all the sectors eventually pollute the aquatic environment and transform it into a hot spot for the emergence and spread of AMR. The bacteria under the genera Vibrio being ubiquitous to aquatic environment include a few species which are pathogenic to humans and aquatic animals. Despite extensive reviews available on AMR in human, terrestrial animal sectors and environment, there is a dearth of summarized information on AMR in Vibrios in shrimp aquaculture environment. In comparison to the human, and terrestrial animal health sector, a methodology for determination of AMR in vibrios associated with aquatic animals is still in its nascent stages and needs harmonization. The present review is centred on the AMR status in vibrios of shrimp farming environment and recent advances in the detection of resistance and discusses methodologies for AMR determination in vibrios and possible mitigation measures to control AMR spread.
Bacteriophages for aquaculture: Are they beneficial or inimical
(Elsevier, 2015)
World fisheries production is projected at 164 million tonnes in 2020, and a major increase in the quantity of fish produced is expected to originate from aquaculture. Diseases represent a severe threat to aquaculture productivity and natural/organic strategies to combat infectious diseases are gaining new ground to address the issues of antibiotic resistance of bacteria and issues related to antibiotic residues in aquatic food animals. Phage therapy is currently considered as a viable alternative to antibiotics for treatment of bacterial infections in aquaculture systems. A cocktail of lyric phages and a synergistic combination of phages and other antimicrobials are viable options to control bacterial infections and at the same time evade phage resistance. However, lysogenic phages have the ability to transform non-virulent bacterial strains in to virulent strains which may cripple aquatic food production and also threaten food safety. The review focuses on the roles of bacteriophages and their implications for aquatic food production and food safety.
Changes of microflora in high pressure treated Indian white prawn (Fenneropenaeus indicus)
(Taylor & Francis, 2020) Ginson, J; Panda, Satyen Kumar; Kamalakanth, C. K; Bindu, J
This study was to investigate the piezotolerance and diversity indices of microflora of Indian white prawn (Fenneropenaeus indicus) after high pressure (HP)-treatment. Indian white prawns subjected to HP-treatments and its effect was studied up to species level and compared with unpressurized samples. The bacterium was identified by using bacterial identification schemes, biochemical tests and API kits (bioMérieux, Marcyl’Etoile, France). Diversity analysis was performed using PRIMER (Plymouth Routines in Multivariate Research) software v 5.2.2. The significant elimination of microflora was found to be proportional with the pressure level. In the case of spore formers, mere destruction was noticed after HP-treatment. Arthrobacter spp., Listeria grayi and Corynebacterium spp. were the most piezotolerant bacteria in HPtreated samples. Diversity indices revealed a significant reduction of microflora of Indian white prawn. The apparent reduction of microflora with pressure level was clearly evident from the diversity indices; moreover a diminished piezotolerance of Gram negative spoilage bacteria was also observed.
Characterization and phylogenitic analaysis of ecotine biosynthesis genes from bacillus halodurans
(Springer, 2008) Rajan, A.; Joseph, C.T.; Thampuran, N.; James, R.; Viswanathan, C.; Bansal, K.C.
Ectoine, a cyclic tetrahydropyrimidine (2- methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid), is a natural compound, which serves as a protective substance in many bacterial cells. In this study, the putative ectABC gene cluster from Bacillus halodurans was heterologously expressed in E. coli and the production of ectoine was con- Wrmed by HPLC analysis. The activity of the enzymes coded by the ectA, B and C genes were found to be higher in induced transgenic cells compared to the uninduced cells. Phylogenetic analysis revealed sequence identities ranging from 36–73% for ectA gene, 55–81% for ectB gene and 55–80% for ectC gene indicating that the enzymes are evolutionarily well conserved.
Characterization of V. cholerae O1 biotype El Tor serotype Ogawa possessing the ctxB gene of the classical biotype isolated from well water associated with the cholera outbreak in Kerala, South India
(IWA Publishing, 2021) P. S., Reethy; K. V., Lalitha
We investigated 22 water samples (17 well water and five pipe water – both chlorinated) and six soil samples from the surroundings of wells of the households of suspected patients from Palakkad district, Kerala (India), from where a cholera outbreak was reported during June–July 2016. A total of 25 Vibrio cholerae isolates were collected from three well water samples during a recent cholera outbreak. Biochemical and serological studies revealed that all of the isolates belonged to serogroup O1, biotype El Tor, serotype Ogawa. PCR assays confirmed the occurrence of ctxB, ctxA, hlyA, tcpA El Tor,VPI, ace, zot, ompW, rfbO1 and toxR genes in all isolates. The presence of the ctxB gene of the classical biotype in all of the El Tor isolates suggests that it is a new variant of El Tor biotype. Antibiogram profile of all V. cholerae O1 isolates revealed resistance towards five classes of antibiotics island and indicates that they were multidrug resistant. ERIC-PCR and PFGE finger prints showed the clonal relationship among the V. cholerae O1 isolates. The results of this study revealed the emergence of a new variant of El Tor biotype in the water samples from Palakkad district, from where a cholera outbreak was reported.
Deciphering foot-and-mouth disease (FMD) virus-host tropism
(2019) Singh, I.; Deb, R.; Kumar, S.; Singh, R.; Andonissamy, J.; Smita, S.; Sengar, G.S.; Kumar, R.; Ojha, K.K.; Sahoo, N.R.; Murali, S.; Chandran, R.; Nair, V.R.; Balal, S.; Mishra, D.C.; Rai, A.
The pattern of interactions between foot and mouth disease (FMD) viral protein 1 (VP1) with susceptible and resistant host integrins were deciphered. The putative effect of site-directed mutation on alteration of interaction is illustrated using predicted and validated 3D structures of VP1, mutated VP1 and integrins of Bos taurus, Gallus and Canis. Strong interactions were observed between FMDV-VP1 protein motifs at conserved tripeptide, Arg-Gly-Asp 143RGD145 and at domain 676SIPLQ680 in alphaintegrin of B. taurus. Notably, in-silico site-directed mutation in FMDV-VP1 protein led to complete loss of interaction between FMD-VP1 protein and B. taurus integrin, which confirmed the active role of arginine-glycine-aspartic acid (RGD) domain. Interestingly, in-vitro analysis demonstrates the persistence of the putative tropism site ‘SIPLQ’ in different cattle breeds undertaken. Thus, the attempt to decipher the tropism of FMDV at host receptor level interaction might be useful for future FMD control strategies through development of mimetic marker vaccines and/or host receptor manipulations
Determination of biosafety of the antibiotic oxytetracycline hydrochloride in Pangasianodon hypophthalmus
(Wiley, 2021-06) Manna, Sanjib Kumar; Bera, Asit Kumar; Das, Nilemesh; Bandopadhyay, Chinmay; Baitha, Raju; Ghadei, Sanjeev Sen; Das, Basanta Kumar; Kumar, Ashok; Ravindran, Rajisha; Krishna, Nanitha; Patil, Prasanna Kumar
Pangasianodon hypophthalmus is one of the most important aquaculture species in Southeast Asia, with a significant contribution to the world fish basket. Like other crops, the striped catfish suffers from bacterial diseases that demand the use of antibiotics. The present study has evaluated the safety of oxytetracycline, one of the approved and commonly used antibiotics, in this species. Juvenile stages of the fish (31.16 ± 1.03 g) were administered with the antibiotic in feed at the dose rate of 80–800 mg kg−1 body weight daily for 30 days, followed by 10 days of withdrawal observation. Fish health was assessed by the study of behaviour and feed intake, haematology, blood biochemistry, and histopathology. Results showed that up to 30 days of antibiotic treatment resulted in no significant toxic effects in terms of behaviour and fish mortality. However, fish suffered from reduced feed intake and hepatotoxicity evidenced by proliferative and degenerative changes of hepatocytes, and increase in AST and ALT enzyme activities, especially in 400–800 mg kg−1 doses which, however, recovered after withdrawal of the drug. Although few fish died from a bacterial infection at the lowest concentration of the drug used, fish were able to mount adaptive physiological responses best at 80 mg kg−1 fish dosage. The study establishes that in-feed administration of the antibiotic oxytetracycline @ 80 mg kg−1 fish biomass is safe for therapeutic use in P. hypophthalmus.
Development and characterization of an LDPE/chitosan compositeantimicrobial film for chilled fish storage
(Elsevier, 2015) Reesha, K.V.; Panda, S.K.; Bindu, J.; Varghese, T.O.
tAn antimicrobial packaging material was developed by uniformly embedding 1, 3 and 5% chitosan (w/w)in low density polyethylene matrix using maleic anhydride grafted LDPE as a compatible agent. Thematerials were mixed by compounding and blown into monolayer films via blown film extrusion. Thedeveloped films showed good barrier properties against oxygen. Characterization of the composite filmswith Fourier transform infrared spectroscopy revealed that chitosan and LDPE interacted well with eachother. Overall migration showed better release of chitosan adduct from the LDPE matrix which enhancedthe antibacterial properties of the films. The interaction between the LDPE/CS and maleic anhydridegrafted LDPE had a decreasing effect on the tensile strength and heat sealing properties. Investigationon antimicrobial properties of LDPE/CS films showed 85–100% inhibition of Escherichia coli. Efficacy ofLDPE/CS films was evaluated by using them as packaging material for chilled storage of Tilapia (Ore-ochromis mossambicus). Analysis of storage quality indices (peroxide value, free fatty acid, total volatilebase nitrogen and aerobic plate count) revealed good antibacterial property and extension of shelf life ofTilapia in the chitosan incorporated novel composite films compared to virgin LDPE film.
Dietart piperine improves haemato-immunological parameters growth profiles and resistance against Aeromonas hydrophila in Labeo rohita (Hamilton, 1822)
(2017) Nilavan, E.; Raman, R.P.; Srinivastava, S.P.; Basha, K.A.; Kumar, K.; Kumar, A.; Shukla, S.P.; Saravanan, K.; Kumar, S.
The purpose of the present study was to evaluate the effects of dietary piperine on haematoimmunological parameters, growth profile and disease resistance in Indian major carp, Labeo rohita challenged with Aeromonas hydrophila. Fish were fed with a diet containing 0 g kg-1 (T0), 0.5 g kg-1 (T1), 1.0 g kg-1 (T2), 2.0 g kg-1(T3), 4 g kg-1(T4), and 8 g kg-1 (T5) of piperine and blood of the experimental fish was collected on 14th, 28th, and 42nd for evaluation of haemato-immunological profiles and fishes were challenged with A. hydrophila. Haemato-immunological parameters at the end of 42nd day trial revealed that piperine administered through feed significantly (P<0.05) enhanced the complete blood counts, haemoglobin level, respiratory burst activity, lysozyme activity, and phagocytic activity. The percentage weight gain, feed conversion ratio and protein efficiency ratio of fishes fed with piperine were found to be significantly (P< 0.05) high compared with control. Dietary inclusion of piperine showed significantly (P<0.05) higher RPS in T3 (57.15%). The results reveal that 2 g kg-1 piperine supplemented diet has a stimulatory effect on haemato-immunological parameters along with improved growth performance and increased resistance against A. hydrophila infection in L. rohita.
Distribution and ecology of Clostridium botulinum in fish and aquatic environments of atropical region
(Academic Publications, 2000) Lalitha, K.V.; Gopakumar, K.
Draft genome sequence of a methicillin- resistant sequence type 39 staphylococcal isolate obtained from seafood
(2017) Sivaraman, G.K.; Vanik, D.; Visnuvinayagam, S.; Prasad, M.M.; Murugadas, V.; Nadella, R.K.; Ravishankar, C.N.
The draft genome sequence of a methicillin-resistant Staphylococcus aureus (MRSA) sequence type 39 (ST 39) isolate obtained from the dried ribbonfish of Gujarat, India, is reported here. Staphylococcus-specific genes were present in this MRSA isolate. The whole-genome sequence of this strain contains 2,693 proteincoding genes and 70 RNAs within the 2.82-Mb genome.
Draft genome sequence of the halophilic and highly halotolerant gammaproteobacteria strain MFB021
(American Society of Microbiology, 2014) CIFT
Effect of modified atmosphere packaging on chemical, textural, microbiological and sensory quality of seer fish (Scomberomorus commerson) steaks packaged in thermoformed trays at 0-2C
(Wiley, 2009) Yesudhason, P.; Gopal, T.K.S.; Ravishankar, C.N.; Lalitha, K.V.; Ashok Kumar, K.
The effect of modified atmosphere on chemical, textural, microbiological and sensory quality of seer fish (Scomberomorus commerson) steaks stored at 0-2C for 23 days was studied. Of the chemical indicators of spoilage, significant differences (P < 0.05) in trimethylamine and thiobarbituric acid values were observed between air- and modified atmosphere (MA)-packaged samples. Seer fish under MA was acceptable on the basis of sensory and microbiological parameters even though the K value was more than 95%, indicating that K value was not a suitable quality indicator. Based on the inosine to hypoxanthine observed in this trial, seer fish was classified as hypoxanthine producers. Bacterial counts of air-packaged seer fish were always higher than MA-packaged samples. Based on sensory score and microbiological data, the shelf life of fish steaks packaged in MA 21 days whereas air-stored samples had a shelf life of 12 days only.
Effects of sodium acetate dip treatment and vacuum-packaging on chemical, microbiological, textural and sensory changes of Pearlspot (Etroplus suratensis) during chill storage
(Elsevier, 2007) Manju, S.; Jose, L.; Gopal, T.K.S.; Ravishankar, C.N.; Lalitha, K.V.
The effects of sodium acetate dip treatment, followed by vacuum-packaging, on the shelf life of beheaded, scaled and gutted Pearlspot (Etroplus suratensis) during chill storage were examined. Sodium acetate (2%, w/v) solution was used for the dip treatment. pouches (size: 15 x 22 cm) made of 12p.-polyester laminated with 300 gauge low-density polyethylene were used for packing fish. After packing, all thepacks were iced with flake ice in the ratio (1:1) fish: ice in an insulated box and were kept in a cold room maintained at 0-2 °C. The control and the treated packs were analysed periodically for chemical (pH, TBA, TMA, TVB-N), microbiological (total viable count), textural and sensory characteristics. Changes in Staphylococcus aureus, Enterobacteriacea and Feacal streptococci were determined for fresh fish and for fish samples at the time of sensory rejection. Air packed samples were found to have a shelf life of about 8 days; vacuum-packed samples were found to be acceptable up to 10 days. whereas sodium acetate-treated vacuum packed samples were found to be acceptable up to 15 days. Thus, vacuum-packaging, in combination with sodium acetate, was found to delay the spoilage, thereby significantly extending the shelf life of Pearlspot at refrigeration temperatures
Fish-borne methicillin resistant Staphylococcus haemolyticus carrying atypical staphylococcal cassette chromosome mec (SCCmec) elements
(Elsevier, 2021-03) G.K. Sivaraman; K.H. Muneeb; S. Sudha; Bibek Shome; Mark Holmes; Jennifer Cole
Prevalence of staphylococci in fish and fish products pose high risk of foodborne diseases. As far as Staphylococcus haemolyticus as a reservoir of methicillin resistance is considered, main objective of the present study was to surveil the incidence of methicillin-resistant Staphylococcus haemolyticus (MRSH) in fishes and its genotypic characterization. 79 fish samples were collected from retail markets in three pre-decided sites in Assam. The collected fish samples were subjected to microbial analysis and as result 13 MRSH could be isolated. Susceptibility of the isolates towards various classes of antibiotics was assessed by employing BD Phoenix M50 system. Polymerase chain reaction (PCR) was performed for the molecular detection of methicillin-resistance determinant. Molecular typing of the isolates was attempted by recruiting staphylococcal cassette chromosome mec (SCCmec) and pulsed-field gel electrophoresis (PFGE). Out of 79 fish samples collected from retail markets in Assam, 13 tested positive for MRSH. These isolates showed varying levels of resistance to different classes of antibiotics such as cephalosporin (100%), lincosamide (30.76%), macrolides (30.76%), aminoglycoside (53.85%), quinolones (38.46%) and sulfonamides (61.54%). BD Phoenix M50 instrument recognized a few isolates as inducible macrolide-lincosamide-streptogramin B (iMLSb) phenotype (30.76%). Remarkably, all the isolates were reported as multi-drug resistant (MDR) as they showed resistant to ≥3 classes of antibiotics. All the studied isolates were positive for mecA gene and carrying multiple SCCmec elements. PFGE cluster analysis grouped the isolates into two major clusters and seven individual lineages. In short, the current study documented the incidence of multi-drug resistant, multiple SCCmec elements carrying S. haemolyticus in fishes. Considering the fact that S. haemolyticus contributes greatly in the dissemination of methicillin resistance owing to their extreme genome plasticity, the present study admonition the necessity of continuous monitoring the S. haemolyticus contamination in food. The study also discussed role of prudent hygienic practices in flattening the AMR curves.
Food grade zinc oxide bulk particle composite can replace the toxic zinc oxide nano composite towards the control of pathogenic and spoilage bacteria
(Oxford Academic, 2020-12-15) S. Visnuvinayagam; L. N. Murthy; U. Parvathy; A. Jeyakumari; G. K. Sivaraman; D. Karthikeyan
ZnO-Nanoparticle-Chitosan (ZnO-NP-CH) composite has potential biomedical and food applications due to its better antimicrobial activity. However, the presence of nano-metal-oxide in the composite makes the material unsuitable for any food applications. Moreover, the cost involved in the preparation of Zinc Oxide-Nano-Particle (ZnO-NP) is a major limitation for commercial food applications. Hence a suitable alternative for ZnO-NP is highly needed for food application. Since ZnO-Bulk Particles (ZnO-BP) are food grade and there is no study on the composite prepared from ZnO-Bulk Particle-Chitosan (ZnO-BP-CH), in the present study, antimicrobial activity was assessed for ZnO-BP-CH and compared with ZnO-NP-CH. Based on the study, it was observed that in the individual form of ZnO-NP possessed significantly higher antimicrobial activity than ZnO-BP. The composite form of ZnO-NP-CH and ZnO-BP-CH possessed higher antimicrobial activity than chitosan. However, no significant difference was observed between the composite forms. Hence, ZnO-BP-CH could be recommended as a suitable alternative to ZnO-NP-CH for future studies related to chitosan with ZnO composite to avoid costly nanomaterials preparation.
Functional and molecular characterization of a lipopeptide surfactant from the marine sponge-associated eubacteria bacillus licheniformis NIOT-AMKV06 of Andaman and Nicobar islands, India
(Elsevier, 2014) Lawrance, A.; Balakrishnan, M.; Joseph, T.C.; Sukumaran, D.P.; Valsalan, V.N.; Gopal, D.; Ramalingam, K.
The production of a lipopeptide surfactant from the sponge-associated eubacteria Bacillus licheniformis NIOT-AMKV06 from the Andaman and Nicobar Islands was investigated. The highest production was attained with glucose and yeast extracts as the carbon and nitrogen sources (1.789 mg mL-1), respectively. The surfactant was highly stable over a pH range of 5.0–10 and a temperature range of 20–70 0C with high NaCl concentrations. Excellent emulsification activity was exhibited by the purified surfactant with crude oil, kerosene, and diesel. A two-fold increase in surfactant production (3.0 mg mL-1) was observed using the newly formulated medium in this study. The surfactant biosynthesis gene cluster (sfp, sfpO, and srfA) from B. licheniformis NIOT-AMKV06 was heterologously expressed in Escherichia coli, and the production was increased three-fold (11.78 gL-1) over the original strain. The results confirm the potential of the surfactant for use in bioremediation of hydrocarbons in a marine environment and for enhanced oil recovery. To our knowledge, this is the first report on the ability of a hydrocarbon degrading B. licheniformis from marine sponges for the biosynthesis of a potent lipopeptide surfactant possessing characteristics of maximum stability, outstanding surfactant activity, and exceptional emulsifying capability.
Growth and toxin production by Clostridium botulinum in fish (Mugil cephalus and shrimp (Penaeus indicus) tissue homogenates stored under vacuum
(2001) Lalitha, K.V.; Gopakumar, K.
Toxin production by Clostridium botulinum types A-E in mullet and shrimp tissue homogenates stored between 4 and 30 C under vacuum for 6 weeks was evaluated. Mullet and shrimp tissue homogenates were inoculated with C. botulinum spores, then packaged under vacuum and held at 4, 10,15 and 30 C. At 30 and 15C storage, growth and toxin production by types A-E resulted in both mullet and shrimp tissue homogenates. Only type E toxin was observed in the homogenates held at 4 and 10 C. The storage temperature and the growth substrate markedly influenced the toxin titres.

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