Influence of some environmental variables and addition of r-lysozyme on efficacy of Vibrio harveyi phage for therapy

Bacteriophage therapy is a viable proposition for controlling luminous vibriosis caused by Vibrio harveyi in shrimpaquaculture. However, environmental factors influence the growth and activity of phage and affect its efficiency incontrolling bacterial diseases. An essential problem in the use of vibrio phage as a therapeutic agent was the development ofresistance to phage attachment, rendering them resistant to the lytic action of phage. This problem could be overcome byapplying a cocktail of phages. This study aimed to evaluate the effect of salinity and pH on the phage activity and also tostudy the role of recombinant shrimp lysozyme on the performance of the V. harveyi phage. Out of three different levels ofsalinity (20, 25 and 30 ppt) and pH (6, 7 and 8) tested, optimum phage activity was observed at a salinity of 25 ppt and atneutral pH. Application of recombinant shrimp lysozyme in combination with V. harveyi phage significantly improved theactivity of phage in in vitro assay as well as in microcosm study using seawater. The application of phage along withlysozyme can be a useful approach to overcome the inability of phage to enter the bacteria and thus eliminate or reduce fish/shrimp pathogenic bacteria in aquaculture.

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) for rapid diagnosis of neonatal sepsis.

Background & objectives: The difficulties in diagnosis of neonatal sepsis are due to varied clinical presentation, low sensitivity of blood culture which is considered the gold standard and empirical antibiotic usage affecting the outcome of results. Though polymerase chain reaction (PCR) based detection of bacterial 16S rRNA gene has been reported earlier, this does not provide identification of the causative agent. In this study, we used restriction fragment length polymorphism (RFLP) of amplified 16S rRNA gene to identify the organisms involved in neonatal sepsis and compared the findings with blood culture. Methods: Blood samples from 97 neonates were evaluated for diagnosis of neonatal sepsis using BacT/Alert (automated blood culture) and PCR-RFLP. Results: Bacterial DNA was detected by 16S rRNA gene PCR in 55 cases, while BacT/Alert culture was positive in 34 cases. Staphylococcus aureus was the most common organism detected with both methods. Klebsiella spp. was isolated from four samples by culture but was detected by PCR-RFLP in five cases while Acinetobacter spp. was isolated from one case but detected in eight cases by PCR-RFLP. The sensitivity of PCR was found to be 82.3 per cent with a negative predictive value of 85.7 per cent. Eighty of the 97 neonates had prior exposure to antibiotics. Interpretation & conclusions: The results of our study demonstrate that PCR-RFLP having a rapid turnaround time may be useful for the early diagnosis of culture negative neonatal sepsis.

Serotyping & molecular characterization for study of genetic diversity among seafood associated nontyphoidal Salmonella serovars.

Background & objectives: Infections due to seafood associated Salmonella serovars are great risk to public health. Different phenotypic characteristics have been used previously for epidemiological investigation of Salmonella. Beyond the phenotypic characterization, a reliable genetic level discriminatory method is required. Therefore, this study was attempted to use different phenotypic and molecular fingerprinting methods for investigation of genetic diversity among seafood associated nontyphoidal Salmonella serovars. Methods: Fifty eight seafood associated Salmonella isolates were included in this study. All isolates were serotyped and epidemiological investigation was carried out using molecular fingerprinting methods, random amplified polymorphic DNA (RAPD) and enterobacterial repetitive intergenic consensus sequence based-PCR (ERIC-PCR) along with whole cell protein profiling using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) in our study. Results: Among the 58 Salmonella isolates, S. Weltevreden was observed to be the most predominant serovar. Typing of Salmonella serovars using RAPD and ERIC-PCR suggested the existence of a genetic diversity. Though both PCR based techniques were found to have a good discriminatory index, a better discriminatory ability was observed when the results obtained by the two techniques were combined and taken for composite analysis. Protein profiling of whole cells using SDS-PAGE demonstrated the presence of several bands with two bands of sizes 38 kDa and 46 kDa common among all 58 isolates. Interpretation & conclusions: Our study shows that use of protein profiling in combination with established typing methods such as RAPD and ERIC-PCR may provide useful information in typing of non-typhoidal Salmonella isolates associated with seafood and to develop strategies to protect public from Salmonella infections.

Use of methylation sensitive polymerase chain reaction for detection of fragile X full mutation & carrier state in males.

BACKGROUND & OBJECTIVE: Fragile X syndrome is the most common cause of inherited mental retardation. It is characterized by the progressive expansion of polymorphic (CGG) trinucleotide repeats located in the promoter region of the FMRI gene located at Xq27.3. The typical dysmorphic features that help in diagnosis are very often subtle or absent especially in pre-pubertal children. Confirmation is by molecular diagnosis based on repeat size and methylation analysis of the FMR1 gene. The present study was done to evaluate the utility of a methylation sensitive polymerase chain reaction (ms-PCR) method in the molecular diagnosis of fragile X syndrome in a select group of mentally retarded male children. METHODS: We used a methylation sensitive PCR technique, which initially modified DNA by bisulphite treatment. Two sets of PCR primers one each for methylated and unmethylated DNA sequences, were used. In full mutations, PCR specific for the methylated sequences was designed to amplify the CpG dinucleotide region upstream to the CGG repeats in clinically affected males. In healthy males and carriers, the second set of primers would amplify the unmethylated DNA sequences. The amplified PCR product size would help to differentiate between normal and premutation repeat size. RESULTS: In all, 25 blood samples collected from mentally retarded male children and five from normal controls were tested. Analysis of cases revealed one full blown mutation and one carrier state. These were further confirmed by southern blotting. INTERPRETATION & CONCLUSION: Unlike currently used methods, methylation sensitive PCR is a quick and accurate technique which could be used for the rapid screening of fragile X syndrome in mental retardation.