A comprehensive review of recent developments in the gram-negative bacterial UDP-2,3-diacylglucosamine hydrolase (LpxH) enzyme.

The emergence of multidrug resistance has provided a great challenge to treat nosocomial infections, which have become a major health threat around the globe. Lipid A (an active endotoxin component), the final product of the Raetz lipid A metabolism pathway, is a membrane anchor of lipopolysaccharide (LPS) of the gram-negative bacterial outer membrane. It shields bacterial cells and serves as a protective barrier from antibiotics, thereby eliciting host response and making it difficult to destroy. UDP-2,3-diacylglucosamine pyrophosphate hydrolase (LpxH), a crucial peripheral membrane enzyme of the Raetz pathway, turned out to be the potential target to inhibit the production of Lipid A. This review provides a comprehensive compilation of information regarding the structural and functional aspects of LpxH, as well as its analogous LpxI and LpxG. In addition, apart from by providing a broader understanding of the enzyme-inhibitor mechanism, this review facilitates the development of novel drug candidates that can inhibit the pathogenicity of the lethal bacterium.

Nervous necrosis virus titration and antigen quantitation by indirect sandwich enzyme linked immunosorbent assay.

Viral nervous necrosis (VNN) is a serious disease of marine and brackishwater fishes caused by nervous necrosis virus (NNV) resulting in up to 100% mortality in early larval and juvenile stages. Adult fish when infected are asymptomatic and spread the virus vertically to the offspring through milt and eggs. Prevention of vertical transmission of the disease is by using disease free broodstock and vaccinating the brooders. Estimation of antigen content and virus titre is essential to determine antigen/virus concentration in VNN vaccine. A monoclonal antibody based indirect sandwich ELISA was developed to quantify the NNV antigen and to estimate the virus titre by TCID50 coupled ELISA. Mouse hybridoma clones secreting monoclonal antibodies (MAb) against the capsid protein of NNV was developed and characterised. The antibodies reacted specifically with the recombinant capsid protein and purified virus in western blot. Polyclonal antibodies against NNV were used as capture antibodies and MAbs were used as detection antibodies to optimise an indirect sandwich ELISA to detect and quantify capsid protein of NNV. The developed assay had a sensitivity of 390 ng/ml and could detect the virus in clinical samples. The assay coupled with TCID50 could be used to estimate the virus titre rather than by observing the CPE which is laborious and subjective.

Trends in Monoclonal Antibody Production Using Various Bioreactor Syst.

Monoclonal antibodies are widely used as diagnostic reagents and for therapeutic purposes, and their demand is increasing extensively. To produce these proteins in sufficient quantities for commercial use, it is necessary to raise the output by scaling up the production processes. This review describes recent trends in high-density cell culture systems established for monoclonal antibody production that are excellent methods to scale up from the lab-scale cell culture. Among the reactors, hollow fiber bioreactors contribute to a major part of high-density cell culture as they can provide a tremendous amount of surface area in a small volume for cell growth. As an alternative to hollow fiber reactors, a novel disposable bioreactor has been developed, which consists of a polymer-based supermacroporous material, cryogel, as a matrix for cell growth. Packed bed systems and disposable wave bioreactors have also been introduced for high cell density culture. These developments in high-density cell culture systems have led to the monoclonal antibody production in an economically favourable manner and made monoclonal antibodies one of the dominant therapeutic and diagnostic proteins in biopharmaceutical industry.

Non-lethal screening of Asian seabass (Lates calcarifer) by monoclonal antibody based indirect enzyme linked immunosorbent assay for viral nervous necrosis.

Viral nervous necrosis (VNN) is a serious viral disease of several species of farmed and wild fishes. Adult fish are asymptomatic and become carriers of the virus when infected with nervous necrosis virus (NNV) and they transmit the virus to the offspring through eggs. ELISA is ideal for non-lethal screening of adult fish for VNN. Asian seabass (Lates calcarifer) IgM was purified using Protein A affinity column and hybridoma clones secreting monoclonal antibodies (MAb) specific to the heavy chain of IgM was developed. An Indirect ELISA using anti-seabass IgM MAb was developed by optimizing all the reagents. The assay was used to screen adult Asian seabass from grow-out farms in comparison to RT-PCR. The assay was also used to assess the immune response in Asian seabass immunized with inactivated Red-spotted grouper NNV (RGNNV). Seabass IgM on SDS-PAGE analysis revealed three heavy chain bands of size 96, 82 and 76 kDa and a single light chain of size 25 kDa. Out of 18 positive hybridoma clones, two selected clones reacted specifically with the 76 kDa heavy chain band. Out of 28 serum samples of Asian seabass from grow-out farms 26 were positive for NNV antibodies while 22 were positive by RT-PCR. Fish immunized with inactivated RGNNV showed immune response by one week post-immunization, and the peak immune response was observed four weeks post-immunization. The assay developed can be used for non-lethal screening of adult Asian seabass for VNN and to assess the immune response after vaccination.

Draft genome sequence data of a T7like phage 3A_8767 isolated from wastewater of a butcher house near Palar river.

Phage 3A_8767 is a newly isolates phage from river water sample against Salmonella typhi 8767 (MTCC). The genome of the phage is linear, double stranded and 38,821 bp long in size. A total 49 functional ORF (open reading frame) were annotated and no tRNA was predicted. Phage 3A_8767 has icosahedral shaped head with stubby tail which comes under family Podoviridae, and genera T7 like virus.

Evaluation of anti-histidine-rich protein 2 monoclonal antibodies, developed by using poly (N-isopropylacrylamide) as an adjuvant for malarial diagnostic application.

OBJECTIVE: To evaluate the sensitivity and the stability of the monoclonal antibodies (Aa3c10, b10c1), against truncated Histidine-rich protein 2 (PfHRP2), developed using smart polymer, poly N-isopropylacrylamide, as adjuvant for malarial diagnostic applications in comparison with the available commercial antibodies. METHODS: Two hybridoma clones (Aa3c10, b10c1) were used for the production of ascites in BALB/c mice. Purification of monoclonal antibodies from the ascites was carried out using affinity columns. The thermal stability study of monoclonal antibodies was done by storing it at 37°C and 45°C for thirty days. The stored antibodies were analyzed using SDS-PAGE and flow-through device where the antigenantibody interaction was visualized by Protein A colloidal gold solution. Sensitivity was determined by endpoint dilution ELISA and the dissociation constant by competitive ELISA. Sensitive pair optimization was done by sandwich ELISA using biotinylated antibodies. Prototype preparation for lateral flow assay had a colloidal gold-based detection system. RESULTS: Thermal stability experiments showed that both mAbs (Aa3c10; b10c1) are stable up to thirty days at 45°C while the commercially available mAbs were stable up to fifteen days only. Compared to commercial antibodies, the mAb Aa3c10, showed the highest sensitivity in end-point titre. In sensitive pair optimization, it was observed that the mAb, b10c1, as a detector and the mAb, Aa3c10, as a capture antibody showed the highest absorbance to detect 50pg/ml PfHRP2 antigen. The prototype formulation of lateral flow assay using the mAbs (Aa3c10; b10c1) showed good reactivity with WHO panel and no false-positive results were observed with twenty clinically negative samples and five P. vivax positive samples. CONCLUSIONS: The novel monoclonal antibodies (Aa3c10, b10c1) against truncated PfHRP2, could be a strong potential candidates that can be included in making RDTs with better sensitivity and stability.

Evaluation of anti-histidine-rich protein 2 monoclonal antibodies, developed by using poly (N-isopropylacrylamide) as an adjuvant for malarial diagnostic application

@#Objective: To evaluate the sensitivity and the stability of the monoclonal antibodies (Aa3c10, b10c1), against truncated Histidine-rich protein 2 (PfHRP2), developed using smart polymer, poly N-isopropylacrylamide, as adjuvant for malarial diagnostic applications in comparison with the available commercial antibodies. Methods: Two hybridoma clones (Aa3c10, b10c1) were used for the production of ascites in BALB/c mice. Purification of monoclonal antibodies from the ascites was carried out using affinity columns. The thermal stability study of monoclonal antibodies was done by storing it at 37°C and 45°C for thirty days. The stored antibodies were analyzed using SDS-PAGE and flow-through device where the antigenantibody interaction was visualized by Protein A colloidal gold solution. Sensitivity was determined by endpoint dilution ELISA and the dissociation constant by competitive ELISA. Sensitive pair optimization was done by sandwich ELISA using biotinylated antibodies. Prototype preparation for lateral flow assay had a colloidal gold-based detection system. Results: Thermal stability experiments showed that both mAbs (Aa3c10; b10c1) are stable up to thirty days at 45°C while the commercially available mAbs were stable up to fifteen days only. Compared to commercial antibodies, the mAb Aa3c10, showed the highest sensitivity in end-point titre. In sensitive pair optimization, it was observed that the mAb, b10c1, as a detector and the mAb, Aa3c10, as a capture antibody showed the highest absorbance to detect 50pg/ml PfHRP2 antigen. The prototype formulation of lateral flow assay using the mAbs (Aa3c10; b10c1) showed good reactivity with WHO panel and no false-positive results were observed with twenty clinically negative samples and five P. vivax positive samples. Conclusions: The novel monoclonal antibodies (Aa3c10, b10c1) against truncated PfHRP2, could be a strong potential candidates that can be included in making RDTs with better sensitivity and stability.

Development of anti-chloro 192 tyrosine HDL apoA-I antibodies for the immunodiagnosis of cardiovascular diseases.

High density lipoproteins (HDL) are considered cardio protective. Apolipoprotein A-I (apoA-I), a major component of HDL helps in reverse cholesterol transport, whose function is greatly affected during atherosclerosis due to oxidation by myeloperoxidase. Amino acid tyrosine residue of apoA-I at position 192 and 166 are sensitive to oxidation by myeloperoxidase resulting in the generation of chlorinated and nitrated apoA-I and they are believed to be present in atherosclerotic plaques and in circulation. These oxidized apoA-I have been suggested as potential indicator(s) of CVD risks in humans. To detect the levels of oxidized apoA-I there is a need for developing monoclonal antibodies (mAbs) with high specificity and sensitivity that could be utilized routinely in clinical immune based assays for blood plasma or for in vivo imaging. In this study, chemically chlorinated apoA-I (chlorinated 192tyrosine- apoA-I) and a short synthetic peptide, containing the corresponding chlorinated tyrosine residue, conjugated to keyhole limpet hemocyanin (KLH) carrier protein were used for immunization. Stable hybridoma clones F7D5 and G11E3 were found to be highly sensitive and reactive towards chlorinated 192tyrosine- apoA-I. Interestingly, these mAbs also displayed positive reaction with atherosclerotic plaques obtained from mouse and human biopsies. In vitro or in vivo diagnostic tests could be developed either by detecting oxidized apoA-I in human plasma or by directly imaging atheroma plaques as both mAbs were shown to stain human atheroma. The anti-chlorinated 192tyrosine- apoA-I mAbs described in this study may have a high diagnostic potential in predicting CVD risks.

Development and Characterization of Monoclonal Antibodies Against Nitro-166Tyrosine of High-Density Lipoprotein: Apolipoprotein A1.

Apolipoprotein A1 (ApoA1) of the high-density lipoprotein (HDL) plays a cardinal role in alleviating atherosclerosis in various ways. Its role in reverse cholesterol transport is preeminent. However, the ApoA1 undergoes oxidation under chronic inflammatory conditions and these oxidations are mediated by myeloperoxidase. It has been reported that the oxidation of the amino acids such as methionine, tyrosine, and tryptophan residues at specific sites of ApoA1 renders it not only dysfunctional but also proinflammatory and proatherogenic. Thus, assessing the quality of ApoA1 and, in turn, that of HDL in circulating blood can serve as an early diagnostic tool for cardiovascular diseases (CVDs). In this study, we developed monoclonal antibodies (mAbs) specific to modified ApoA1 with its tyrosine residue at the 166th position nitrated to 3-nitrotyrosine. A 20 amino acid peptide around the modification of interest was designed using an antigenicity prediction tool. The peptide was custom synthesized with ovalbumin as conjugate and used as an antigen to immunize BALB/c mice. Hybridomas were obtained by fusion of Sp2/0 mouse myeloma cells with spleen cells from the immunized mouse. A hybridoma clone 2E5B7, thus developed and characterized, was found to secrete mAb of the desired specificity and sensitivity against nitrated 166Tyrosine. The lowest concentration of the antigen that could be detected by the mAb with confidence was 15 ng. The mAb was able to detect nitrated 166Tyrosine peptide ovalbumin conjugate antigen spiked in human plasma with high specificity. The generated mAb could be potentially used in immuno-based diagnostic systems to screen the quality of HDL and in turn assess CVD risks in humans.

Diagnostic potential of monoclonal antibodies developed against C-terminal polypeptide of P. falciparum Histidine Rich Protein2 (PfHRP2) in malaria infected patients from India.

Malaria, caused by Plasmodium falciparum has become a major health burden in most tropical and developing countries. P. falciparum Histidine Rich Protein2 (PfHRP2), which exhibits polymorphism, is being widely used as a diagnostic marker. Recently, we reported the development of monoclonal antibodies against conserved C-terminal 105 amino acids of PfHRP2 for malaria diagnosis. Now, in this study, the diagnostic performance of two anti-C-terminal PfHRP2 mAbs (b10c1 and Aa3c10) were evaluated with 100 blood samples from clinically identified malaria patients from seven different geographical centers in India. Sandwich ELISA, polymerase chain reaction (PCR) and statistical tools were used for the evaluation of the performance of the anti-C-terminal PfHRP2 mAb. These mAbs detected P. falciparum (mean OD value 1.525 ± 0.56) malaria with great accuracy with no cross reactivity with P. Plasmodium vivax (mean OD value 0.285 ± 0.051) and normal healthy control samples (mean OD value 0.185 ± 0.06) in Sandwich ELISA assay. The samples which were RDT negative for P. falciparum were also reactive in Sandwich ELISA with mean OD value of (1.303 ± 0.532). The amount of PfHRP2 antigen in the patients' blood sample was quantified and categorized into three distinct groups having the HRP2 antigen in high, intermediate and low amounts. The presence of Pfhrp2 gene was also confirmed by PCR analysis. The sensitivity and specificity of the mAb were found to be 95 and 96% respectively. These data strongly suggest that the anti-C-terminal PfHRP2 mAbs b10c1 and Aa3c10 have merits for improvising the existing malarial diagnostics.

Purification and characterization of an extracellular uricase from a new isolate of Sphingobacterium thalpophilum (VITPCB5).

An extracellular uricase producing bacterium (VITPCB5) was isolated from soil of the duck farm near Chidambaram, Tamilnadu, India and it was identified based on its 16S rRNA as Sphingobacterium thalpophilum. Uric acid was used as an effective inducer. The enzyme kinetics was studied using uric acid as a substrate. The Km and Vmax for the enzyme was found to be 0.28mM and 0.92µM/minml, respectively. Maximum uricase production was observed when lactose was used as a carbon source. Among the nitrogen sources tested, urea gave the maximum uricase production. The enzyme was successfully purified using a weak cation exchange convective interaction media carboxy methyl (CIM-CM) monolith column with a recovery of 79.7%±0.1 and 14.2±1.8-fold purification. The optimal reaction temperature of the enzyme was observed between 25 and 45°C. The pH optimum of the enzyme was 8.0. The enzyme activity was enhanced by copper and partially inhibited by calcium, iron, zinc and nickel ions. Treatment with ethylene diamine tetraacetic acid completely inhibited the enzyme activity. The in-gel trypsin digested peptides of 48-kDa uricase when analyzed using mass spectrometry, gave 32% sequence coverage with the uricase (30-kDa) from Cyberlindnera jadinii.

Novel monoclonal antibody against truncated C terminal region of Histidine Rich Protein2 (PfHRP2) and its utility for the specific diagnosis of malaria caused by Plasmodium falciparum.

An accurate diagnosis of malarial infection is an important element in combating this deadly disease. Malaria diagnostic test including, microscopy and other molecular tests are highly sensitive but too complex for field conditions. Rapid detection tests for P. falciparum infection using monoclonal antibodies (mAbs) against highly polymorphic PfHRP2 (Histidine Rich Protein2) are still most preferred test in field conditions, but with limitations such as specificity, and sensitivity leading to false positive and false negative results. To overcome these limitations, we carried out bioinformatics analysis PfHRP2 and PfHRP3 and found that the C-terminal region of PfHRP2 (~105 amino acids) displayed relatively lower sequence identity with PfHRP3. This C-terminal region of PfHRP2 contained unique peptide repeats and was found to be conserved in various isolates of P. falciparum. Moreover, this region was also found to be highly antigenic as predicted by antigenicity propensity scores. Thus we constructed a cDNA clone of the truncated PfHRP2 (recPfHRP2-T3) coding for C-terminal 105 amino acids and expressed it in E. coli and purified the polypeptide to homogeneity. The purified recPfHRP2-T3 was used as an antigen for development of both polyclonal and monoclonal antibody (mAb). The mAbs b10c1 and Aa3c10 developed against recPfHRP2-T3 was found to efficiently recognize recombinant PfHRP2 but not PfHRP3. In addition, the above mAbs reacted positively with spent media and serum sample of P. falciparum infection recognizing the native PfHRP2. The affinity constant of both the clones were found to be 10(9) M(-1). Quantitatively, both these clones showed ~4.4 fold higher reactivity with P. falciparum infected serum compared to serum from healthy volunteers or P. vivax infected patient samples. Thus these anti-C-terminal PfHRP2 mAbs (Aa3c10 and b10c1) display a very high potential for improvising the existing malarial diagnostic tools for detection of P. falciparum infection especially in areas where PfHRP2 polymorphism is highly prevalent.

Development, purification, and characterization of monoclonal antibodies against recombinant histidine-rich protein 3 of Plasmodium falciparum.

In the present study, monoclonal antibodies (MAbs) against recombinant histidine-rich protein (rHRP3) were developed and assessed for their potential in detection of Plasmodium falciparum HRP3. Hybridomas were obtained by fusion of Sp2/0 mouse myeloma cells and spleen cells from the mouse immunized with purified rHRP3. Three MAbs (IgG1 isotype) specific to rHRP3 were established and characterized by enzyme-linked immunosorbent assay (ELISA) and immunoblotting for sensitivity and specificity. Purification of MAbs from hybridoma cell culture supernatant and PAbs from rabbit anti-serum were carried out using Phenylpropylamine (PPA) HyperCel(™) sorbent. The MAbs were able to detect rHRP3 and the HRP3 from P. falciparum spent medium. Sandwich ELISA was developed to quantify HRP3 in the spent medium of P. falciparum culture. The generated MAbs could be potentially used in immuno-based diagnostic systems for the detection of P. falciparum HRP.

Production and characterization of monoclonal antibodies (mAbs) against human serum albumin (HSA) for the development of an immunoaffinity system with oriented anti-HSA mAbs as immobilized ligand.

Proteins present in human serum are of immense importance in the field of biomarker discovery. But, the presence of high-abundant proteins like albumin makes the analysis more challenging because of masking effect on low-abundant proteins. Therefore, removal of albumin using highly specific monoclonal antibodies (mAbs) can potentiate the discovery of low-abundant proteins. In the present study, mAbs against human serum albumin (HSA) were developed and integrated in to an immunoaffinity based system for specific removal of albumin from the serum. Hybridomas were obtained by fusion of Sp2/0 mouse myeloma cells with spleen cells from the mouse immunized with HSA. Five clones (AHSA1-5) producing mAbs specific to HSA were established and characterized by enzyme linked immunosorbent assay (ELISA) and immunoblotting for specificity, sensitivity and affinity in terms of antigen binding. The mAbs were able to bind to both native albumin as well as its glycated isoform. Reactivity of mAbs with different mammalian sera was tested. The affinity constant of the mAbs ranged from 10(8) to 10(9)M(-1). An approach based on oriented immobilization was followed to immobilize purified anti-HSA mAbs on hydrazine activated agarose gel and the dynamic binding capacity of the column was determined.

Production of human anti-glycophorin-A monoclonal antibodies and their purification by pseudoaffinity chromatography using a convective interaction media monolithic column.

Monoclonal antibodies (MAbs) against glycophorin-A (GPA) could be used in identifying MN blood groups, detecting specific markers of erythroid differentiation, and studying parasite interactions. Large-scale production of MAbs in bioreactors demands an efficient and rapid separation technology. The present study describes the production of a human anti-GPA monoclonal antibody and its purification using a pseudo-bioaffinity L-histidine-convective interaction media (CIM) monolithic column. Hybridomas were generated by fusion of mouse myeloma cell line (Sp2/0) and spleen cells from the mouse immunized with Triton X-100 solubilized RBC membrane proteins. Hybridomas producing antibodies specific to commercial glycophorin-A were screened by indirect enzyme-linked immunosorbent assay (ELISA). The antibodies produced by the stable clones were found to be IgG1 with kappa light chain. Purification of IgG1 MAbs from the cell culture supernatant carried out with a CIM-EDA-histidine disk resulted in high specific activity with purification fold of 8.3 in the fraction eluted with MOPS buffer containing 0.2 M NaCl. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and ELISA showed that the antibodies obtained were highly pure, with high antigen-binding efficiency. The results indicate that faster separation and efficient recovery of high-purity anti-GPA MAbs could be achieved by using CIM-EDA-histidine disk.

Purification of monoclonal antibodies, IgG1, from cell culture supernatant by use of metal chelate convective interaction media monolithic columns.

Monoclonal antibodies (MAbs) have diverse applications in diagnostics and therapeutics. The recent advancement in hybridoma technology for large-scale production of MAbs in bioreactors demands rapid and efficient purification methods. Conventional affinity purification systems have drawbacks of low flow rates and denaturation of antibodies owing to harsh elution conditions. Here, we attempted purification of MAbs by use of a high-throughput metal-chelate methacrylate monolithic system. Monolithic macroporous convective interaction media-iminodiacetate (CIM-IDA) disks immobilized with four different metal ions (Cu²âº, Ni²âº, Zn²âº and Co²âº) were used and evaluated for purification of anti-human serum albumin IgG1 mouse MAbs from cell culture supernatant after precipitation with 50% ammonium sulfate. Elution with 10 mM imidazole in the equilibration buffer (25 mM MMA = MOPS (Morpholino propane sulfonic acid) + MES (Morpholino ethane sulfonic acid) + Acetate + 0.5 M NaCl, pH 7.4) resulted in a purification of 25.7 ± 2.9-fold and 32.5 ± 2.6-fold in experiments done using Zn²âº and Co²âº metal ions, respectively. The highest recovery of 85.4 ± 1.0% was obtained with a CIM-IDA-Zn(II) column. SDS-PAGE, ELISA and immuno-blot showed that the antibodies recovered were pure, with high antigen-binding efficiency. Thus, metal chelate CIM monoliths could be a potential alternative to conventional systems for fast and efficient purification of MAbs from the complex cell culture supernatant.

Synechocystis MCCB 114 and 115 as putative probionts for Penaeus monodon post-larvae.

Synechocystis MCCB 114 and 115 were segregated as putative probionts for shrimp larvae from a collection of 54 cyanobacterial cultures enriched from seawater. On feeding Penaeus monodon post-larvae with the cyanobacteria, the generic diversity of the intestinal bacterial flora could be enhanced with substantial reduction or total absence of Vibrio spp. A significant difference (p < 0.001) in the percent survival of batches of post-larvae fed on the cyanobacterial cultures was observed and, on repeated challenge with V. harveyi, the relative percent survival of those batches of larvae fed on Synechocystis MCCB 114 and 115 was significantly higher. The Synechocystis MCCB 114 and 115 cultures were found to contain high levels of protein (34 to 43%), in addition to carotenoids.

Optimization of carbon and nitrogen sources and growth factors for the production of an aquaculture probiotic (Pseudomonas MCCB 103) using response surface methodology.

AIM: To develop a new medium for enhanced production of biomass of an aquaculture probiotic Pseudomonas MCCB 103 and its antagonistic phenazine compound, pyocyanin. METHODS AND RESULTS: Carbon and nitrogen sources and growth factors, such as amino acids and vitamins, were screened initially in a mineral medium for the biomass and antagonistic compound of Pseudomonas MCCB 103. The selected ingredients were further optimized using a full-factorial central composite design of the response surface methodology. The medium optimized as per the model for biomass contained mannitol (20 g l(-1)), glycerol (20 g l(-1)), sodium chloride (5 g l(-1)), urea (3.3 g l(-1)) and mineral salts solution (20 ml l(-1)), and the one optimized for the antagonistic compound contained mannitol (2 g l(-1)), glycerol (20 g l(-1)), sodium chloride (5.1 g l(-1)), urea (3.6 g l(-1)) and mineral salts solution (20 ml l(-1)). Subsequently, the model was validated experimentally with a biomass increase by 19% and fivefold increase of the antagonistic compound. CONCLUSION: Significant increase in the biomass and antagonistic compound production could be obtained in the new media. SIGNIFICANCE AND IMPACT OF THE STUDY: Media formulation and optimization are the primary steps involved in bioprocess technology, an attempt not made so far in the production of aquaculture probiotics.

A marine bacterium, Micrococcus MCCB 104, antagonistic to vibrios in prawn larval rearing systems.

A marine bacterium, Micrococcus MCCB 104, isolated from hatchery water, demonstrated extracellular antagonistic properties against Vibrio alginolyticus, V. parahaemolyticus, V. vulnificus, V. fluviallis, V. nereis, V. proteolyticus, V. mediterranei, V cholerae and Aeromonas sp., bacteria associated with Macrobrachium rosenbergii larval rearing systems. The isolate inhibited the growth of V. alginolyticus during co-culture. The antagonistic component of the extracellular product was heat-stable and insensitive to proteases, lipase, catalase and alpha-amylase. Micrococcus MCCB 104 was demonstrated to be non-pathogenic to M. rosenbergii larvae.