Genetic diversity, virulence and distribution of antimicrobial resistance among Listeria monocytogenes isolated from milk, beef, and bovine farm environment.

BACKGROUND: Listeria monocytogenes is an opportunistic intracellular foodborne pathogen and is ubiquitous in nature. The occurrence of L. monocytogenes in animal production units coupled with their presence in milk, faeces, feed, water, sewage, and soil is a contributory factor for foodborne listeriosis in humans and animals. AIMS: The study was aimed to characterize genotype and serogroup of L. monocytogenes recovered from different types of samples and also to study antimicrobial patterns by phenotypic and genotypic methods. METHODS: Multiplex polymerase chain reaction (PCR) was used for the confirmation of L. monocytogenes, the identification of its serogroup and lineage, and the detection of virulence markers. Enterobacterial repetitive intergenic consensus (ERIC), and randomly amplified polymorphic DNA (RAPD)-PCR were used to characterize those isolates, and antimicrobial patterns were studied phenotypically by Kirby-Bauer method and genotypically by PCR. RESULTS: Out of the screened 474 samples (274 milk and 50 each of soil, feed, sewage, and beef), ten L. monocytogenes isolates (milk=8, soil=1, and beef=1) were confirmed by PCR targeting the hlyA gene and found to belong to the 1/2a, 3a serogroup and fall under type II lineage. Virulence potential assessment revealed that all the ten isolates harbored the iap gene while the presence of plcA and plcB genes were noticed in seven and eight isolates respectively. Six isolates from milk were found to group in the same cluster by ERIC and RAPD fingerprinting, suggesting both methods to be efficient molecular typing tools for L. monocytogenes. Genotypic characterization of antimicrobial resistance (AMR) genes revealed that seven isolates were positive for tetM, five for mefA, four for msrA, and one for lnuA genes while none of the isolates showed tetK, ermA, ermB, and lnuB genes. CONCLUSION: The presence of L. monocytogenes in bovine farm environments coupled with virulence markers, and multidrug resistance from the study area suggest a possible transmission from the environment to humans and animals which needs to be monitored regularly to ensure food safety and the well-being of animals and humans.

Conventional and molecular determination of drug resistance in Mycobacterium tuberculosis and Mycobacterium bovis isolates in cattle.

This study was aimed out to explore the presence of drug resistance among M. tuberculosis and M. bovis isolates (n = 51) of bovine origin by conventional broth microdilution method and molecular methods. By broth microdilution method, 16 isolates were found to be resistant to isoniazid, 08 isolates were resistant to pyrazinamide, 09 isolates were resistant to rifampicin and 07 isolates were found to be resistant for ethambutol. Two isolates showed resistance to rifampicin and pyrazinamide, one isolate showed resistance to pyrazinamide and ethambutol and 03 isolates showed resistance to isoniazid and ethambutol. None of isolates showed multi drug resistance (MDR). Other than the M. bovis strains, none of the other M. tuberculosis isolates showed any resistance to pyrazinamide. Molecular methods by multiplex PCR targeting katG, pncA, rpoB genes, multiplex allele specific PCR to detect mutation in embB codon 306 and sequencing showed point mutation in katG and rpoB gene. No mutation could be detected in the embB gene by multiplex allele specific PCR. The results indicates that further elaborate studies need to be carried out due to the presence of drug resistant M. tuberculosis in bovines which could be due to spill over from human in tuberculosis endemic areas making TB eradication programme more challenging.

Dermatophilus congolensis infection in sheep and goats in Delta region of Tamil Nadu.

AIM: The study was conducted to isolate and identify Dermatophilus congolensis (DC) using conventional and molecular diagnostic techniques in scab materials collected from skin infections of sheep and goats in the Delta region of Tamil Nadu. MATERIALS AND METHODS: A total of 20 scab samples collected from 18 goats and 2 sheep from Nagapattinam, Thanjavur, and Tiruvarur districts of Tamil Nadu. Smears were made from softened scab materials and stained by either Gram's or Giemsa staining. Isolation was attempted on blood agar plates, and colonies were stained by Gram's staining for morphological identification. Identification was also done by biochemical tests and confirmed by 16S rRNA polymerase chain reaction (PCR), followed by sequencing and phylogenetic analysis of the amplified product. RESULTS: The peculiar laddering arrangement of coccoid forms in stained smears prepared from scab materials revealed the presence of DC. Isolated colonies from scab materials of sheep and goats on bovine blood agar plate were small, hemolytic, rough, adherent, and bright orange-yellow in color, but some colonies were white to cream color. Gram-staining of cultured organisms revealed Gram-positive branching filaments with various disintegration stages of organisms. 16S rRNA PCR yielded 500 bp amplicon specific for DC. Sequence analysis of a sheep DC isolate showed 99-100% sequence homology with other DC isolates available in NCBI database, and phylogenetic tree showed a close cluster with DC isolates of Congo, Nigeria, and Angola of Africa. Genes for virulence factors such as serine protease and alkaline ceramidase could not be detected by PCR in any of the DC strains isolated of this study. CONCLUSION: The presence of dermatophilosis in Tamil Nadu was established from this study.

Molecular identification of Mycobacterium tuberculosis in cattle.

Bovine tuberculosis continued to be a re-emerging problem in some countries especially in endemic areas due to the fact that human and animal health surveillance system is not adopted to diagnose the infection. This crisis can be attributed due to sharing of the same habitat especially in rural areas. In the present study, a total of 148 samples were collected from cattle for isolation over a period of 3 years from cattle with and without lesions, of which 67 isolates were obtained by culture. Fifty one isolates were identified as Mycobacterium tuberculosis complex (MTBC) by IS6110 PCR of which 43 (84.3%) were identified as M. tuberculosis and 08 (15.6%) were identified as M. bovis by using 12.7kb fragment multiplex PCR. Among this, 31 isolates which were positive for IS6110 PCR were subjected to spoligotyping and revealed 28 isolates belonging to MANU1 strain of M. tuberculosis. This study clearly indicates that high prevalence of M. tuberculosis than M. bovis in bovine was identified by means of culture and by molecular methods M. tuberculosis can affect cattle producing lesion in contradiction to the earlier thoughts. This study speculates that M. tuberculosis MANU1 strain infection in cattle could be due to spill over from human or other non specific hosts in tuberculosis endemic areas. Though bovine tuberculosis due to M. tuberculosis in cattle is not considered a serious threat worldwide, in countries where human TB is endemic, M. tuberculosis infection of cattle needs to be considered.

A novel generalized design methodology and realization of Boolean operations using DNA.

The biological deoxyribonucleic acid (DNA) strand has been increasingly seen as a promising computing unit. A new algorithm is formulated in this paper to design any DNA Boolean operator with molecular beacons (MBs) as its input. Boolean operators realized using the proposed design methodology is presented. The developed operators adopt a uniform representation for logical 0 and 1 for any Boolean operator. The Boolean operators designed in this work employ only a hybridization operation at each stage. Further, this paper for the first time brings out the realization of a binary adder and subtractor using molecular beacons. Simulation results of the DNA-based binary adder and subtractor are given to validate the design.