Influence of corn starch based bio-active edible coating containing fumaric acid on the lipid quality and microbial shelf life of silver pomfret fish steaks stored at 4 °C.

The present study aimed at assessing the impact of addition of fumaric acid (0.5%), as an active agent, in a corn starch (2%) based edible coating, on the lipid quality and microbial shelf life of silver pomfret (Pampus argenteus) fish steaks stored at 4 °C. Treating fish steaks with FA resulted in a bacteriostatic effect leading to reduced counts of total mesophilic and psychrotrophic bacteria, H2S producing bacteria and Pseudomonas spp. The total mesophilic bacterial count of uncoated control sample exceeded the permissible limit of 7 log cfu g-1 on 6th day and had the lowest microbial shelf life. FA incorporation in the CS coating improved the microbial stability of fish steaks resulting in a shelf life of 15 days. The outcomes of the study suggest that CS based coating is beneficial in delaying lipid oxidation as displayed by the lower TBA and PV values while FA is an effective agent for further increasing the preservative action of CS coating by significantly inhibiting microbial growth as well as lipid quality deterioration, which could be exploited by the seafood industry as an active packaging component.

Virulence and intermediate resistance to high-end antibiotic (teicoplanin) among coagulase-negative staphylococci sourced from retail market fish.

This study reports the distribution of enterotoxigenic determinants among staphylococci and the susceptibility of staphylococci to various classes of antibiotics. We observed all the isolates as resistant to beta-lactam antibiotics and a few as resistant to non-beta-lactam antibiotics such as clindamycin (47.4%), erythromycin (44.7%), gentamicin (23.7%), norfloxacin (34.2%), tetracycline (26.3%), trimethoprim-sulfamethoxazole (15.8%) etc. The resistance of S. sciuri (n = 1) and S. haemolyticus (n = 1) to rifampicin and intermediate resistance of S. gallinarum (n = 2) to teicoplanin, a high-end antibiotic, are also observed in this study. The multidrug-resistance (≥ 3 classes of antibiotics) was recorded in 23 (60.5%) isolates. The virulomes such as sea, seb, seg and sei were identified predominantly in S. haemolyticus. Surprisingly, certain isolates which were phenotypically confirmed as biofilm-producers by Congo red agar (CRA) test did not harbor biofilm-associated loci. This implies the protein-mediated mechanism of biofilm formation as an alternative to polysaccharide intercellular adhesin (PIA) in staphylococci. However, icaAD locus which encodes PIA was identified in 10 (26.3%) isolates and the eno locus, encoding elastin-binding protein which can accelerate the biofilm production, is identified in all the isolates. The possession of type V SCCmec elements by the S. haemolyticus (15.8%) raised the concern about the rapid dissemination of mecA gene to other species of staphylococci including the virulent S. aureus. In short, this study acknowledges the toxigenicity of coagulase-negative staphylococci (CoNS). Through this study, surveillance of antimicrobial resistance and transference of virulomes in staphylococci is warranted.

Molecular assessment of antimicrobial resistance and virulence in multi drug resistant ESBL-producing Escherichia coli and Klebsiella pneumoniae from food fishes, Assam, India.

The present study investigated the prevalence of Extended-Spectrum Beta Lactamase (ESBL) -producing E. coli and K. pneumoniae from the food fishes in retail markets in Assam, India. A total of 54 ESBL-producing E. coli and 12 K. pneumoniae isolates were recovered from 79 fish samples and were analyzed for antimicrobial resistance genes (ARGs) and virulence genes. E. coli isolates were categorized as multi drug resistant with resistance up to 12 different antibiotics with multiple antibiotic resistances (MAR) index ranging from 0.26 to 0.63. In E. coli, 100% resistance to cefotaxime along with 6% resistance to ceftazidime (third-generation cephalosporins) was observed. Moreover, 85% of the E. coli isolates were resistant to cefepime, a fourth-generation cephalosporin. K. pneumoniae showed resistance to 11 different antibiotics with MAR index value ranging from 0.21 to 0.57. All K. pneumoniae isolates showed 100% resistance to cefotaxime, 67% resistance to ceftazidime and 75% resistance to cefepime. Molecular characterization of ARGs revealed the presence of CTX-M group 1(CTX-M-15) in almost all E. coli isolates (98%, n = 53) and 100% in K. pneumoniae. A combination of uniplex and multiplex PCRs revealed fewer ARGs in E. coli isolates, with each isolate carrying 3 to 5 genes (tetA, dfrA1, sul1, sul2, qnrB, qnrS, aac(6')-Ib-cr). Majority of the E. coli were assigned to low-virulence phylogroup B1 and A while 8% of them belonged to pathogenic phylogroup D. 31 unique genetic profiles were identified for E. coli isolates by Pulsed-Field Gel Electrophoresis (PFGE) typing. K. pneumoniae isolates were highly diverse with 11 unique genetic profiles and a substantial ARG profile (blaTEM, blaSHV, blaOXA-1-like, tetA, strA, strB, dfrA1, sul1, sul2, qnrB, qnrS, aac(6')-Ib-cr, oqxA, oqxB). The frequency of ARGs ranged between 4 and 11. All K. pneumoniae isolates belonged to capsular serotype with wzi gene. Virulence gene iutA was prominent in all isolates while ybtS and kfu were confirmed in two isolates. Our findings raise concerns that fishes bought for consumption may serve as potential reservoirs of AMR genes and pose serious threat to public health. The study emphasizes the need for extensive surveillance of resistant strains in aquaculture and related settings, their in-depth analysis of population structure and transmission dynamics.

Environmental Dissemination of Multidrug Methicillin-Resistant Staphylococcus sciuri After Application of Manure from Commercial Swine Production Systems.

The deposition of manure originating from food animal farms in the environment can lead to the dissemination of antimicrobial-resistant (AMR) bacterial foodborne pathogens, thereby potentially impacting human health. The objective of our study was to determine the dissemination of multidrug methicillin-resistant Staphylococcus sciuri (MDR-MRSS) in the environment after land application of manure on commercial swine farms. A total of 400 environmental samples (40 manure and 360 soil) were collected after repeated sampling from four commercial swine farms located in North Carolina (n = 1) and Iowa (n = 3) in the United States. At each farm, we collected 10 manure and 40 soil samples (20 samples before and after 2 h of manure application) from four plots (five soil samples/plot) on day 0. Subsequently, 20 soil samples were collected on day 7, 14, and 21 from the same plots. A total of 67 (16.75%) MRSS were isolated from the 400 samples. The prevalence in soil and manure was 13.33% (48/360) and 47.5% (19/40), respectively. Prevalence was highest in the soil samples collected after 2 h of manure application on day 0 and decreased subsequently on 7, 14, and 21 days. Antimicrobial susceptibility testing was done against a panel of 12 antibiotics. A majority of S. sciuri isolates exhibited resistance against ampicillin (AMP; 95.5%), penicillin (PEN; 95.5%), clindamycin (CLI; 95.5%), cefoxitin (FOX; 92.5%), ceftiofur (XNL; 92.5%), tetracycline (TET; 86.56%), and erythromycin (ERY; 50.74%). The MDR pattern AMP FOX CLI PEN TET XNL (n = 24; 35.8%) was the most commonly observed. We detected multiple AMR genes, including mecA, aac(6'), Ie-aph(2″)Ia, tetM, tetK, mphC, ermA, ermB, and ermC. Pulsed-field gel electrophoresis clustered isolates from different sample collection days from the same farm into one group. Overall, our study identifies swine manure as an important reservoir of MDR-MRSS and highlights its dissemination in the environment upon spreading of manure.

Assessment of microbial quality of fish processing industrial effluent in bar-mouth at Bhidia landing site, Veraval, Gujarat, India.

The present study was carried out to assess the microbial quality of fish processing industries effluent at Bhidia bar-mouth, Veraval, Gujarat during April, 2012 to March 2013. The total viable bacterial count (TVBC), total Enterobacteriaceae count, E. coli count (EC), Staphylococcus aureus and Fecal Streptococcal count in effluent ranged from 3.0 x 10(-1) to 6.8 x 10(6), 9.0 x 10(1) to 2.9 x 10(4), 0 to 0. 5 x 10(4), 0 to 0. 4 x 102 and 0.3 x 10(1) to 0. 1 x 10(4) cfu.(-1)respectively. Significantly higher load of TEC, E. coli, S.aureus, Fecal Streptococci, Total coliforms and Fecal coliforms were higher during summer whereas, TVBC was higher in the month of Sept.-Oct. Furthermore, the total coliform and fecal coliform counts were found to be higher with 1400+ /100 ml MPN value throughout the year of the study, except in the month of August. Overall occurrence of pathogenic strains of E. coli, S. aureus and Fecal streptococci were 41.67%, 25.00% and 66.67% respectively during this period. The antibiogram of the isolated E. coli isolates show that almost 50% were resistant to Cefazidime/Clavulanic acid (CAC), Amoxyclav (AMC), Ciprofloxacin (CIF) and Ampicillin (AMP). The present study indicated that the effluent of fish processing industry was heavily contaminated with E. coli, S. aureus and Fecal Streptococci which confirmed improper treatment of fish processing effluent. Moreover, the precedence of antibiotic resistant E. coli may pose threat to public health safety.

Effect of chitosan based active packaging film on the keeping quality of chilled stored barracuda fish.

In the present study, active antimicrobial (AM) packaging films were prepared from chitosan (CH) incorporated with ginger (Zingiber officinale) essential oil at different concentrations (0.1, 0.2 and 0.3 % v/v) and characterized. GC-MS analysis revealed zingiberene (22.54 ± 0.13), geranial (12.34 ± 0.33), ß-sesquiphellandrene (8.14 ± 0.14), camphene (7.44 ± 0.54) and neral (5.45 ± 0.23) as the major components of essential oil extracted from ginger. Addition of ginger essential oil (GEO) improved the AM activity of the CH film against food borne pathogens, without significantly (p < 0.05) affecting the mechanical properties of the film. CH film with GEO was more effective against Gram-positive bacteria than Gram-negative bacteria and maximum antibacterial property against Staphylococcus aureus and Escherichia coli was shown by 0.3 % GEO added CH film. In a further experiment, steaks of barracuda (Sphyraena jello) fish were wrapped with the CH-GEO (0.3 %) film and stored at 2 °C for 20 days. Throughout the storage period, the total volatile basic nitrogen (TVB-N) value and total mesophilic count of fish steak wrapped with the CH-GEO film were significantly (p < 0.05) lesser than both the unwrapped control fish steak and aerobically packed fish steak in synthetic multilayer film of ethylene vinyl alcohol (EVOH) (nylon, EVOH and polyethylene). Sensorily, CH-GEO film wrapped sample was acceptable till the end of storage for 20 days compared to 12 days for unwrapped control and fish steak packed in EVOH film. The results indicate that the developed CH-GEO film is efficient in extending the storage life of fish.

Study of population genetic polymorphism and gene flow rate in Indian snow trout, Schizothorax richardsonii fish of Himalaya, India.

The genetic polymorphism and gene flow rate among the Indian snow trout fish population S. richadsonii from three different locations viz., Chirapani stream of Champawat district, Kosi and Gola river of Nainital district, Uttarakhand State, India were assessed by employing twenty numbers of Randomly Amplified Polymorphic DNA (RAPD) markers. The overall percent polymorphisms among these three populations were 14.76 with 6.56, 4.92 and 3.28 in Chirapani, Kosi and Gola river population, respectively. Chirapani population had higher proportion of polymorphic loci as compared to the Kosi and Gola. The higher value of genetic distance (0.1565) was obtained between Chirapani and Gola population and the lower value of genetic distance was observed between Chirapani and Kosi (0.1058) river population. The cluster analysis revealed that in the formation of two clusters, one consisted of Chirapani and Kosi and the other was Gola fish population. Gst estimates among these populations showed some extent of homogeneity with lower genetic differentiation rate between populations and further suggested that higher tolerance to mutation, as expected that RAPD bands, arose from both coding and non-coding DNA regions. The findings revealed that the rate of gene flow in three populations seemed very low i.e. highly conserved its genetic diversity in their natural waterbodies and indicative of little migration among populations (geographically isolated and not the possibilities man made interventions/introduction of similar kind of fish species). It is further concluded that the Chirapani, Kosi and Gola river populations of S. richardsonii were being conserved naturally in their habitat and the species actual genetic potential were being maintained (adaptation to local climatic conditions, reproduction, production traits and disease resistance trait etc) in their natural habitat.

Whole-genome sequence analysis of Staphylococcus aureus from retail fish acknowledged the incidence of highly virulent ST672-MRSA-IVa/t1309, an emerging Indian clone, in Assam, India.

The epidemiology and toxigenicity of MRSA in the fishery environment are poorly understood. In this study, methicillin-resistant Staphylococcus aureus (MRSA) (n = 1) and methicillin-susceptible S. aureus (MSSA) (n = 2) from retail fish were subjected to comprehensive genome analysis. Here, we report the occurrence of ST672-MRSA-IV/t1309 and ST5-MSSA/t105 for the first time from India in the fishery environment. The resistome of the isolates was in concordance with their phenotypic resistance pattern. Phenotypically, the resistance profile of MSSA isolates (n = 2) was AMP-CLI-ERY-NOR-PEN. For MRSA (n = 1), it was AMP-CFZ-CLI-ERY-NOR-OXA-PEN. The antibiotic efflux genes and mutations in the antibiotic target accounted for fluoroquinolone resistance whereas methicillin resistance was conferred through possession of a mecA gene. Similarly, all three isolates carried a similar array of virulence factors. The conjugative plasmid inc18 and rep family 10 plasmids were found in two of the three isolates. This study documents the MRSA carrying SCCmec IVa elements which are the markers of community-associated MRSA (CA-MRSA). Through the possession of SCCmec IV elements, which are smaller than other types of SCCmec, MRSA can contribute to the rapid dissemination of antimicrobial resistance and virulence factors. In short, our findings highlighted that the presence of ST672-MRSA in fishery environments may pose a risk to human health.

Non-diarrhoeic pigs as source of highly virulent and multidrug-resistant non-typhoidal Salmonella.

Food-producing animals act as reservoirs of non-typhoidal Salmonella (NTS) serovars with potential food safety and public health implications. The present cross-sectional study aimed at determining the prevalence of Salmonella serotypes in non-diarrhoeic pigs and characterizing the isolates using molecular tools. Salmonella isolates (n = 22) recovered from faecal samples of 194 randomly selected pigs were characterized for virulence and antimicrobial resistance and subtyped using XbaI-PFGE. The prevalence of Salmonella in apparently healthy non-diarrhoeic pigs was 11.3% (95%CI, 4.3-19.5%), with S. Weltevreden (81.8%) and S. Enteritidis (18.2%) being the serotypes detected. Salmonella isolates harboured virulence genes such as invA (100%), stn (100%), spvR/spvC (86.3%) and fimA (22.7%). Phenotypically, isolates showed sensitivity to chloramphenicol, levofloxacin and ciprofloxacin and resistance to tetracycline and ampicillin (100%), streptomycin (86.4%), amoxicillin-clavulanate (63.6%), cefotaxime (22.7%) and ceftriaxone (9.1%). Notably, 18.2% isolates were multidrug-resistant (≥ 3 antimicrobial class) with multiple antimicrobial resistance (MAR) index of 0.56-0.67 (18.2%), 0.44 (45.5%), 0.33 (31.8%) and 0.22 (4.5%). Genotypically, isolates carried various antibiotic resistance genes: ESBL (blaTEM and blaOXA), aminoglycoside (strA, strB and aadA1), sulphonamide (sul1, sul2 and dfrA1), tetracycline (tetA and tetB) and plasmid AmpC beta-lactamase (ACC, FOX, MOX, DHA, CIT and EBC). The present investigation emphasizes the epidemiological significance of PFGE typing in the detection of emerging strains of highly virulent and multidrug-resistant S. Weltevreden and S. Enteritidis in non-diarrhoeic pigs that pose serious public health implications in the pork supply chain environment. More extensive longitudinal study is warranted to provide epidemiological links between environmental reservoirs and animal and human infections in piggery settings.

Food grade zinc oxide bulk particle composite can replace the toxic zinc oxide nano composite towards the control of pathogenic and spoilage bacteria.

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.

Draft Genome Sequence of a Methicillin-Resistant Staphylococcus aureus Isolate (Sequence Type 1) from Seafood.

The draft genome sequence of a methicillin-resistant Staphylococcus aureus (MRSA) isolate (sequence type 1 [ST 1]) from the salted dried ribbonfish from Gujarat, India, is reported here. Staphylococcus genus-specific genes were present in this MRSA isolate. The whole-genome sequence of this strain contains 2,797 protein-coding genes and 80 RNAs within the 2.85-Mb genome.

Draft Genome Sequence of a Methicillin-Resistant Sequence Type 39 Staphylococcal Isolate Obtained from Seafood.

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 protein-coding genes and 70 RNAs within the 2.82-Mb genome.

Genetics of immunocompetent traits in a synthetic broiler dam line.

Three hundred and three chicks of both sexes, from a synthetic dam line (SDL) of broiler chickens, were studied for economic traits (body weights at 4, 5 and 6 weeks of age) and immunological traits (humoral and cell mediated immune responses, and serum lysozyme concentration). The objective was to evaluate these traits and to estimate their genetic and non-genetic parameters. The humoral immune response was assessed by estimating the antibody response to sheep red blood cells using the haemagglutination (HA) test and serum IgG concentration using single radial immunodiffusion (SRID). The cell mediated immune (CMI) response was estimated as in vivo response to a mitogen (PHA-P). Serum lysozyme was measured by lysoplate assay. Least squares means for body weight at 4, 5 and 6 weeks were 684 +/- 20, 920 +/- 19 and 1205 +/- 28 g, HA titre was 6.289 +/- 0.246, CMI was 0.438 +/- 0.015 mm, lysozyme was 1.860 +/- 0.047 microg/ml and IgG was 6.287 +/- 0.194 mg/ml. There was an effect of sire on HA titre and on body weight at 4, 5 and 6 weeks of age; males were heavier than females. Heritability estimates were high for body weights but low for immunological traits. Phenotypic correlations (rp) among body weights were high and positive but were very low between body weights and most immunological traits. Among the immunological traits all rp were very low. Genetic correlations (rg) of body weights were positive and medium to high with CMI and HA and negative with serum IgG.

Studies on immunocompetence status in two turkey varieties in India.

(1) Two hundred and twenty-seven adult turkeys of both sexes, of two varieties (104 Black and 123 White) were used to evaluate their immunocompetence status and body weights. (2) Response to sheep red blood cells (SRBC) (humoral immunity) was measured by Haemagglutination (HA) test 5 days post immunisation (dpi) and expressed as log2 values. Mercaptoethanol resistant (MER) antibodies representing IgG were determined by Mercaptoethanol HA test and Mercaptoethanol sensitive (MES) antibodies, representing IgM as the difference in total HA titre and IgG. Serum lysozyme concentrations were estimated by 'Lysoplate assay' and expressed in log2 values. (3) Least squares analysis of variance revealed that the White variety had higher adult body weight (4.788 +/- 0.040 kg) than the Black (3.774 +/- 0.044 kg). Sexual dimorphism was apparent and meals were heavier than females in both varieties. The interaction effect of variety and sex on body weight was also significant. (4) Least squares means for immunological traits, namely, total anti-SRBC antibodies, MER, MES titres and serum lysozyme were 7.161 +/- 0.189, 0.801 +/- 0.071, 6.362 +/- 0.160 and 1.766 +/- 0.043 microg/ml, respectively. The Black variety had a higher MES antibody titre than the White. (5) Sex had an effect on all the immunological traits except on MER titres. Females generally had higher anti-SRBC, MER and MES titres and serum lysozyme. The variety x sex interaction effect was significant for MES titres and serum lysozyme. White males had the lowest MES titres.