DNA barcoding Indian freshwater fishes.

DNA barcoding is a promising technique for species identification using a short mitochondrial DNA sequence of cytochrome c oxidase I (COI) gene. In the present study, DNA barcodes were generated from 72 species of freshwater fish covering the Orders Cypriniformes, Siluriformes, Perciformes, Synbranchiformes, and Osteoglossiformes representing 50 genera and 19 families. All the samples were collected from diverse sites except the species endemic to a particular location. Species were represented by multiple specimens in the great majority of the barcoded species. A total of 284 COI sequences were generated. After amplification and sequencing of 700 base pair fragment of COI, primers were trimmed which invariably generated a 655 base pair barcode sequence. The average Kimura two-parameter (K2P) distances within-species, genera, families, and orders were 0.40%, 9.60%, 13.10%, and 17.16%, respectively. DNA barcode discriminated congeneric species without any confusion. The study strongly validated the efficiency of COI as an ideal marker for DNA barcoding of Indian freshwater fishes.

Establishment and characterization of a continuous cell line from thymus of striped snakehead, Channa striatus (Bloch 1793).

The establishment and characterization of a continuous cell line from the thymus of air-breathing fish Channa striatus are described. The cell line, designated C. striatus thymus (CST), has been subcultured over 71 times and shows optimal growth at 28°C in Leibovitz's-15 (L-15) medium supplemented with 20% fetal bovine serum. The CST cells exhibited low plating efficiency which improved with increase in seeding density. The karyotype analysis revealed that CST cells have a normal diploid karyotype with 2n = 40. Partial amplification and sequencing of two mitochondrial genes, viz. 16S ribosomal RNA (rRNA) and cytochrome oxidase I, confirmed that the cell line originated from C. striatus. CST cells were successfully transfected indicating their potential application for expression of recombinant proteins. In immunocytochemical staining, CST cells showed characteristics of epithelial cells. These cells were sensitive to extracellular products of Vibrio cholerae MTCC 3904 as well as to heavy metal mercuric chloride. The CST cell line would be a useful tool in functional genomic studies such as RNA interference and gene knockout as well as for cytotoxicity studies.

Profile of fatal Streptococcal bacteraemia at a tertiary care Indian hospital.

Streptococcus pyogenes causes mild to acutely life-threatening diseases. Herein, we report our experience with five cases of fatal bacteraemia due to various groups of Streptococci, three of them due to Group G Streptococcus and one case each due to Group A Streptococcus and Group F Streptococcus. The peculiarity of all these cases was the rapidity of deaths occurring in these patients despite all the strains being sensitive to Penicillin. Hence, timely intervention in all suspected cases is strongly advocated. All isolates of beta-haemolytic Streptococci should be identified up till the species level and antimicrobial susceptibility be performed so that proper and early management can be done.

Beta-hemolytic streptococcal infections in trauma patients.

PURPOSE: ß-hemolytic streptococci (ßHS) causes a diverse array of human infections. The molecular epidemiology of ß-hemolytic streptococcal infections in trauma patients has not been studied. This study reports the molecular and clinical epidemiology of ß-hemolytic streptococcal infections at a level 1 trauma centre of India. METHODS: A total of 117 isolates of ßHS were recovered from clinical samples of trauma patients. The isolates were identified to species level and subjected to antimicrobial susceptibility testing. Polymerase chain reaction (PCR) assay was done to detect exotoxin virulence genes. The M protein gene (emm gene) types of GAS strains were ascertained by sequencing. RESULTS: Group A Streptococcus (GAS) was the most common isolate (64 %), followed by group G Streptococcus (23 %). A large proportion of GAS produced speB (99 %), smeZ (91 %), speF (95 %) and speG (87 %). smeZ was produced by 22 % of GGS. A total of 25 different emm types/subtypes were seen in GAS, with emm 11 being the most common. Resistance to tetracycline (69 %) and erythromycin (33 %) was commonly seen in GAS. CONCLUSIONS: ß-hemolytic streptococcal infections in Indian trauma patients are caused by GAS and non-GAS strains alike. A high diversity of emm types was seen in GAS isolates, with high macrolide and tetracycline resistance. SpeA was less commonly seen in Indian GAS isolates. There was no association between disease severity and exotoxin gene production.

DNA barcoding Indian marine fishes.

DNA barcoding has been adopted as a global bio-identification system for animals in recent years. A major national programme on DNA barcoding of fish and marine life was initiated in India by the authors during 2006 and 115 species of marine fish covering Carangids, Clupeids, Scombrids, Groupers, Sciaenids, Silverbellies, Mullids, Polynemids and Silurids representing 79 Genera and 37 Families from the Indian Ocean have been barcoded for the first time using cytochrome c oxidase I gene (COI) of the mtDNA. The species were represented by multiple specimens and a total of 397 sequences were generated. After amplification and sequencing of 707 base pair fragment of COI, primers were trimmed which invariably generated a 655 base pair barcode sequence. The average Kimura two parameter (K2P) distances within species, genera, families, orders were 0.30%, 6.60%, 9.91%, 16.00%, respectively. In addition to barcode-based species identification system, phylogenetic relationships among the species have also been attempted. The neighbour-joining tree revealed distinct clusters in concurrence with the taxonomic status of the species.

The human GABA(B1b) and GABA(B2) heterodimeric recombinant receptor shows low sensitivity to phaclofen and saclofen.

1. The aim of this study was to characterize the pharmacological profile of the GABA(B1)/GABA(B2) heterodimeric receptor expressed in Chinese hamster ovary (CHO) cells. We have compared receptor binding affinity and functional activity for a series of agonists and antagonists. 2. The chimeric G-protein, G(qi5), was used to couple receptor activation to increases in intracellular calcium for functional studies on the Fluorimetric Imaging Plate Reader (FLIPR), using a stable GABA(B1)/GABA(B2)/G(qi5) CHO cell line. [(3)H]-CGP-54626 was used in radioligand binding studies in membranes prepared from the same cell line. 3. The pharmacological profile of the recombinant GABA(B1/B2) receptor was consistent with that of native GABA(B) receptors in that it was activated by GABA and baclofen and inhibited by CGP-54626A and SCH 50911. 4. Unlike native receptors, the GABA(B1)/GABA(B2)/G(qi5) response was not inhibited by high microMolar concentration of phaclofen, saclofen or CGP 35348. 5. This raises the possibility that the GABA(B1)/GABA(B2)/G(qi5) recombinant receptor may represent the previously described GABA(B) receptor subtype which is relatively resistant to inhibition by phaclofen.

The heteroduplex mobility assay (HMA) as a pre-sequencing screen for Norwalk-like viruses.

Molecular epidemiological studies of Norwalk-like viruses (NLVs), previously known as small round structured viruses (SRSVs), are dependant currently on DNA sequencing of PCR amplicons, which is expensive and time consuming. The Heteroduplex Mobility assay (HMA) was evaluated as a method for identification of PCR amplicons from the commonly circulating NLV strains without DNA sequencing. The procedure was developed for use with two reference strains, a Mexico virus-like strain (MXV-like; Hu¿NLV¿RBH¿1993¿UK) and the Grimsby virus strain (Hu¿NLV¿Gimsby¿1995¿UK), and was optimised with regards to the annealing and electrophoresis conditions and the electrophoresis gel matrix. Using the optimised conditions, amplicons of less than 90% sequence identity formed visible heteroduplexes, allowing the strains to be placed into three categories; Mexico-like, Grimsby-like and non-Mexico virus/non-Grimsby virus strains. Outbreak strains 'genotyped' previously by DNA sequencing as Mexico virus or Grimsby virus were identified correctly by the heteroduplex mobility assay. The procedure was applied prospectively to strains from 130 outbreaks occurring in the UK between 1997 and 1998. Heteroduplex mobility assay was successful on 120 (92%) strains of which 68 (57%) were GRV-like strains, three (2.5%) were Mexico virus-like strains and 49 (41%) were categorised as non- Mexico/non-Grimsby virus strains. Amplicons from 50 of the 120 strains were sequenced and there was perfect correlation between the heteroduplex mobility assay categorisation and phylogenetic analysis. HMA offers a rapid, robust and far cheaper alternative to sequencing for the identification of prevalent Norwalk-like virus genotypes for molecular epidemiological studies.

National outbreak of Salmonella senftenberg associated with infant food.

Eight cases of Salmonella senftenberg infection in infants were identified in the first half of 1995 in England, five were indistinguishable S. senftenberg strains. A case-control study showed an association between illness and consumption of one brand of baby cereal (P = 0.03). The cereal manufacturer reported isolating S. senftenberg in June 1994 from an undistributed cereal batch. Outbreak strains and the cereal strain were all plasmid-free in contrast to other human isolates of S. senftenberg in the same period. Changes in the production process were implemented to prevent further contamination. Surveillance centres should strengthen the detection and investigation of outbreaks of gastrointestinal infection in susceptible groups, especially young children. In this outbreak, the study of only five cases led to identification of the vehicle of infection. Even when few cases are reported, epidemiological investigation in conjunction with molecular typing may lead to public health action which prevents continuing or future outbreaks.

Pulsed-field electrophoretic fingerprinting of Salmonella indiana and its epidemiological applicability.

Eight Xba I-generated pulsed-field profile (PFP) types and four subtypes within one of the most common PFP types have been identified in Salmonella indiana from patients, poultry and human food in England and Wales in the three-year period from January 1994 to December 1996. Two PFP types have predominated, PFP X1 and PFP X2. Although the PFP X1 type was identified throughout the study period, the PFP X2 type was not identified until late 1995, subsequently becoming the most common PFP type in humans in the first six months of 1996 with a significant distribution in elderly patients. It is concluded that PFGE can be used in support of epidemiological investigations for the subdivision of Salm. indiana. Furthermore, as both conditions and interpretation criteria can be easily standardized, it is suggested that for many salmonella serotypes, PFGE can provide the basis for a definitive scheme of genotypic subtyping suitable for epidemiological investigations at both a national and international level.

Plasmid characterization and pulsed-field electrophoretic analysis demonstrate that ampicillin-resistant strains of Salmonella enteritidis phage type 6a are derived from Salm. enteritidis phage type 4.

Plasmid incompatibility studies have demonstrated that strains of Salmonella enteritidis phage type (PT) 6a resistant to ampicillin possess a 36 megadalton incompatibility group (Inc) X plasmid coding for resistance to ampicillin which is capable of converting strains of Salm. enteritidis belonging to PTs 1 and 4 to PT 6a, and PT 8 to PT 13. However, pulsed-field gel electrophoresis (PFGE) has demonstrated that all clinical isolates of PT 6a have a characteristic XbaI pulsed-field profile which is distinct from that of PT 1 and which can only be differentiated from that of PT 4 by the presence of plasmid-associated fragments of less than 45 kb. It is concluded that ampicillin-resistant strains of Salm. enteritidis PT 6a are derived from strains of Salm. enteritidis PT 4 by acquisition of an Inc X ampicillin resistance plasmid.