Molecular characterization of a crustinlike antimicrobial peptide in the giant tiger shrimp, Penaeus monodon, and its expression profile in response to various immunostimulants and challenge with WSSV

A crustinlike antimicrobial peptide from the haemocytes of giant tiger shrimp, Penaeus monodon was partially characterized at the molecular level and phylogenetic analysis was performed. The partial coding sequence of 299 bp and 91 deduced amino acid residues possessed conserved cysteine residues characteristic of the shrimp crustins. Phylogenetic tree and sequence comparison clearly confirmed divergence of this crustinlike AMP from other shrimp crustins. The differential expression of the crustinlike AMP in P. monodon in response to the administration of various immunostimulants viz., two marine yeasts (Candida haemulonii S27 and Candida sake S165) and two bglucan isolates (extracted from C. haemulonii S27 and C. sake S165) were noted during the study. Responses to the application of two grampositive probiotic bacteria (Bacillus MCCB101 and Micrococcus MCCB104) were also observed. The immune profile was recorded preand postchallenge white spot syndrome virus (WSSV) by semiquantitative RTPCR. Expressions of seven WSSV genes were also observed for studying the intensity of viral infection in the experimental animals. The crustinlike AMP was found to be constitutively expressed in the animal and a significant downregulation could be noted postchallenge WSSV. Remarkable downregulation of the gene was observed in the immunostimulant fed animals prechallenge followed by a significant upregulation postchallenge WSSV. Tissuewise expression of crustinlike AMP on administration of C. haemulonii and Bacillus showed maximum transcripts in gill and intestine. The marine yeast, C. haemulonii and the probiotic bacteria, Bacillus were found to enhance the production of crustinlike AMP and confer significant protection to P. monodon against WSSV infection

Biofilm Forming Multi Drug Resistant Staphylococcus spp. Among Patients with Conjunctivitis

Biofilm forming multidrug resistant Staphylococcus spp. are major reservoirs for transmission of ophthalmic infections. They were isolated from ocular patients suffering from conjunctivitis. In this study we analyzed biofilm forming ability, antibiotic resistance profile of the Staphylococcus spp. isolated from clinical ocular patients, and their phylogenetic relationship with other community MRSA. Sixty Staphylococcus spp. strains isolated from clinical subjects were evaluated for their ability to form biofilm and express biofilm encoding ica gene. Among them 93% were slime producers and 87% were slime positive. Staphylococcus aureus and S. epidermidis were dominant strains among the isolates obtained from ocular patients. The strains also exhibited a differential biofilm formation quantitatively. Antibiotic susceptibility of the strains tested with Penicillin G, Ciprofloxacin, Ofloxacin, Methicillin, Amikacin, and Gentamicin indicated that they were resistant to more than one antibiotic. The amplicon of ica gene of strong biofilm producing S. aureus strains, obtained by polymerase chain reaction, was sequenced and their close genetic relationship with community acquired MRSA was analyzed based on phylogenetic tree. Our results indicate that they are genetically close to other community acquired MRSA

Biocompatible polyhydroxybutyrate (PHB) production by marine Vibrio azureus BTKB33 under submerged fermentation

Polyhydroxybutyrate (PHB) is known to have applications as medical implants and drug delivery carriers and is consequently in high demand. In the present study the possibilities of harnessing potential PHB-producing vibrios from marine sediments as a new source of PHB was investigated since marine environments are underexplored. Screening of polyhydroxyalkanoate (PHA)-producing vibrios from marine sediments was performed using a fluorescent plate assay followed by spectrophotometric analysis of liquid cultures. Out of 828 isolates, Vibrio sp. BTKB33 showed maximum PHA production of 0.21 g/L and PHA content of 193.33 mg/g of CDW. The strain was identified as Vibrio azureus based on phenotypic characterization and partial 16S rDNA sequence analysis. The strain also produced several industrial enzymes: amylase, caseinase, lipase, gelatinase, and DNase. The FTIR analysis of extracted PHA and its comparison with standard PHB indicated that the accumulated PHA is PHB. Bioprocess development studies for enhancing PHA production were carried out under submerged fermentation conditions. Optimal submerged fermentation conditions for enhanced intracellular accumulation of PHA production were found to be 35 °C, pH −7, 1.5 % NaCl concentration, agitation at 120 rpm, 12 h of inoculum age, 2.5 % initial inoculum concentration, and 36 h incubation along with supplementation of magnesium sulphate, glucose, and ammonium chloride. The PHA production after optimization was found to be increased to 0.48 g/L and PHA content to426.88 mg/g of CDW, indicating a 2.28-fold increase in production. Results indicated that V. azureus BTKB33 has potential for industrial production of PHB.

Molecular genetic profiling of functional genes of pearl oyster, Pinctada fucata

There are a number of genes involved in the regulation of functional process in marine bivalves. In the case of pearl oyster, some of these genes have major role in the immune/defence function and biomineralization process involved in the pearl formation in them. As secondary filter feeders, pearl oysters are exposed to various kinds of stressors like bacteria, viruses, pesticides, industrial wastes, toxic metals and petroleum derivatives, making susceptible to diseases. Environmental changes and ambient stress also affect non-specific immunity, making the organisms vulnerable to infections. These stressors can trigger various cellular responses in the animals in their efforts to counteract the ill effects of the stress on them. These include the expression of defence related genes which encode factors such as antioxidant genes, pattern recognition receptor proteins etc. One of the strategies to combat these problems is to get insight into the disease resistance genes, and use them for disease control and health management. Similarly, although it is known that formation of pearl in molluscs is mediated by specialized proteins which are in turn regulated by specific genes encoding them, there is a paucity of sufficient information on these genes.In view of the above facts, studies on the defence related and pearl forming genes of the pearl oyster assumes importance from the point of view of both sustainable fishery management and aquaculture. At present, there is total lack of sufficient knowledge on the functional genes and their expressions in the Indian pearl oyster Pinctada fucata. Hence this work was taken up to identify and characterize the defence related and pearl forming genes, and study their expression through molecular means, in the Indian pearl oyster Pinctada fucata which are economically important for aquaculture at the southeast coast of India. The present study has successfully carried out the molecular identification, characterization and expression analysis of defence related antioxidant enzyme genes and pattern recognition proteins genes which play vital role in the defence against biotic and abiotic stressors. Antioxidant enzyme genes viz., Cu/Zn superoxide dismutase (Cu/Zn SOD), glutathione peroxidise (GPX) and glutathione-S-transferase (GST) were studied. Concerted approaches using the various molecular tools like polymerase chain reaction (PCR), random amplification of cDNA ends (RACE), molecular cloning and sequencing have resulted in the identification and characterization of full length sequences (924 bp) of the Cu/Zn SOD, most important antioxidant enzyme gene. BLAST search in NCBI confirmed the identity of the gene as Cu/Zn SOD. The presence of the characteristic amino acid sequences such as copper/zinc binding residues, family signature sequences and signal peptides were found out. Multiple sequence alignment comparison and phylogenetic analysis of the nucleotide and amino acid sequences using bioinformatics tools like BioEdit,MEGA etc revealed that the sequences were found to contain regions of diversity as well as homogeneity. Close evolutionary relationship between P. fucata and other aquatic invertebrates was revealed from the phylogenetic tree constructed using SOD amino acid sequence of P. fucata and other invertebrates as well as vertebrates