High prevalence of class 1 integrons in clinical isolates of methicillin‑resistant Staphylococcus aureus from India.

Introduction: Class1 integrons are one of the prevalent mechanisms of antibiotic resistance gene transfer in Gram‑negative organisms, but their prevalence and role in the spread of antibiotic resistance genes in methicillin‑resistant Staphylococcus aureus (MRSA) is unexplored. The purpose of this study was to investigate the prevalence of class 1 integrons in clinical isolates of MRSA. Materials and Methods: Total 143 MRSA isolates obtained from two different cities in India (Pune and Mumbai) were characterized by biochemical tests, and the antibiotic sensitivity was performed using the Clinical and Laboratory Standards Institute (CLSI) guidelines. The presence of class 1 integrons, sul1/qacEΔ1 region of class 1 integron and mecA gene among these isolates was determined by polymerase chain reaction (PCR). Results: All 143 isolates were mecA positive and coagulase‑positive. Overall, 71% of the MRSA isolates carried class 1 integrons; 58% (45/77) of the isolates obtained from Mumbai and 85% (56/66) of the isolates from Pune carried class 1 integrons. In all, 39% of these isolates carried sul1/qacEΔ1 region, thus confirming the association of class 1 integrons with antibiotic resistance genes. Along with ‑lactam antibiotics the MRSA isolates were resistant to several other antibiotics, with resistance to erythromycin, ciprofloxacin and trimethoprim‑sulfamethoxazole being observed in 75%, 66% and 60% of the isolates, respectively. Conclusion: To the best of our knowledge, this is the first report of class 1 integrons in MRSA isolates from India. The study provides insights into the prevalence of a novel mechanism adapted by MRSA for the propagation of antibiotic resistance genes.

Antibacterial activities of multi drug resistant Myroides odoratimimus bacteria isolated from adult flesh flies (Diptera: Sarcophagidae) are independent of metallo beta-lactamase gene / Atividades antibacterianas de Myroides odoratimimus isolada de moscas varejeiras adultas (Diptera: Sarcophagidae) são independentes do gene metalo beta lactamase

Flesh flies (Diptera: Sarcophagidae) are well known cause of myiasis and their gut bacteria have never been studied for antimicrobial activity against bacteria. Antimicrobial studies of Myroides spp. are restricted to nosocomial strains. A Gram-negative bacterium, Myroides sp., was isolated from the gut of adult flesh flies (Sarcophaga sp.) and submitted to evaluation of nutritional parameters using Biolog GN, 16S rRNA gene sequencing, susceptibility to various antimicrobials by disc diffusion method and detection of metallo â-lactamase genes (TUS/MUS). The antagonistic effects were tested on Gram-negative and Gram-positive bacteria isolated from human clinical specimens, environmental samples and insect mid gut. Bacterial species included were Aeromonas hydrophila, A. culicicola, Morganella morganii subsp. sibonii, Ochrobactrum anthropi, Weissella confusa, Escherichia coli, Ochrobactrum sp., Serratia sp., Kestersia sp., Ignatzschineria sp., Bacillus sp. The Myroides sp. strain was resistant to penicillin-G, erythromycin, streptomycin, amikacin, kanamycin, gentamycin, ampicillin, trimethoprim and tobramycin. These strain showed antibacterial action against all bacterial strains except W. confusa, Ignatzschineria sp., A. hydrophila and M. morganii subsp. sibonii. The multidrug resistance of the strain was similar to the resistance of clinical isolates, inhibiting growth of bacteria from clinical, environmental and insect gut samples. The metallo â-lactamase (TUS/MUS) genes were absent, and resistance due to these genes was ruled out, indicating involvement of other secretion machinery.

Moscas varejeiras (Diptera: Sarcophagidae) são causa conhecida de miíase e as bactérias de seus intestinos nunca foram estudadas quanto à atividade antibacteriana. Estudos antimicrobianos de Myroides spp restringem-se à cepas hospitalares. Uma bactéria Gram negativa, Myroides sp, foi isolada do intestino de moscas varejeiras adultas (Sarcophaga sp) e submetida à avaliação de parâmetros nutricionais pelo sistema BIOLOG GN, ao sequenciamento genético 16S rRNA, à sensibilidade a vários antimicrobianos pelo método de difusão de discos e à detecção dos genes de metalo beta lactamases (TUS/MUS). Os efeitos antagonistas foram testados contra bactérias Gram negativas e Gram positivas isoladas de material clínico humano, amostras ambientais e intestino do inseto. As espécies bacterianas incluíram Aeromonas hydrophila, A. culicicola, Morganella morganii subsp sibonii, Ochrobactrum anthropi, Weissella confusa, Escherichia coli, Ochrobactrum sp, Serratia sp, Kestersia sp, Ignatzschineria sp e Bacillus sp. A cepa Myroides sp foi resistente à penicilina G, eritromicina, estreptomicina, amicacina, canamicina, gentamicina, ampicilina, trimetoprim e tobramicina. Esta cepa apresentou atividade antimicrobiana contra todas as cepas exceto W.confusa, Ignatzschineria sp, A. hydrophila e M. morgani subsp sibonii. A resistência múltipla da cepa foi semelhante à de isolados clínicos, inibindo bactérias das amostras clínicas, ambientais e do intestino do inseto. Os genes de metalo beta lactamases (TUS/MUS) estavam ausentes, excluindo-se a resistência mediada por esses genes, o que indica o envolvimento de um mecanismo alternativo de secreção.

Establishment of two new cell lines from Bombyx mori (L.) (Lepidoptera: Bombycidae) & their susceptibility to baculoviruses.

BACKGROUND & OBJECTIVES: Lepidopteran cell cultures and baculovirus expression vector systems are becoming popular due to their potential applications in biotechnology especially for the expression of foreign proteins. Efforts were made to develop new, indigenous, cell lines from Bombyx mori larvae and pupae. METHODS: Eight to ten B. mori larvae and 10-12 pupae were surface sterilized, dissected and ovaries were removed aseptically. Ovaries were chopped finely, washed and suspended in growth medium. When the cells formed monolayers, they were subcultured and experiments were carried out. RESULTS: Two new cell lines from larval and pupal ovaries of B. mori were established in Grace's insect tissue culture medium supplemented with 20 per cent FBS (foetal bovine serum). The larval cell line consisted predominantly of epithelial-like cells (98.31%), whereas the pupal cell line had a mixed cell population of epithelial-like (71.8%) and fibroblast-like cells (27.8%). Karyology indicated a typical lepidopteran pattern in both the cell lines and had chromosome numbers ranging from 35 to 150 and 60 to 180 for larval and pupal ovaries respectively. Four-fold increase in cell number was observed in these cell lines in 7 days. Both the cell lines were found susceptible to B. mori multiple nucleopolyhedrovirus and Autographa californica multiple nucleopolyhedrovirus, but not to Helicoverpa armigera single nucleopolyhedrovirus and Spodoptera litura multiple nucleopolyhedrovirus. INTERPRETATION & CONCLUSION: These well characterized cell lines may be of immense application in biotechnology and medicine for the production of biologically active recombinant proteins to use in vaccine studies as well as in therapeutic applications.

New Helicoverpa armigera Hbn cell line from larval hemocyte for baculovirus studies.

A new cell line from the larval hemocytes of H. armigera was established in Grace's medium modified by adding lactalbumin hydrolysate and yeastolate (3.3g/l), and supplemented with fetal bovine serum (20%). The cell line was designated as NIV-HA-1195. The cell population at P-78 consisted mainly of epithelial-like cells (89.36%), fibroblast-like cells (8.31%) and giant cells (2.13%). The population doubling time was 96hr at P-8, 60hr at P-43. The chromosome number ranged from 45 to 200. The cell line is susceptible to the baculoviruses, Autographa californica nucleopolyhedrovirus (AcNPV), Spodoptera litura NPV and the homologous HaNPV. Isoenzyme profile and results of 16S rRNA heteroduplex analysis clearly indicated the species specificity of the new cell line.

Sequence analysis of mitochondrial 16S ribosomal RNA gene fragment from seven mosquito species.

Mosquitoes are vectors for the transmission of many human pathogens that include viruses, nematodes and protozoa. For the understanding of their vectorial capacity, identification of disease carrying and refractory strains is essential. Recently, molecular taxonomic techniques have been utilized for this purpose. Sequence analysis of the mitochondrial 16S rRNA gene has been used for molecular taxonomy in many insects. In this paper, we have analysed a 450 bp hypervariable region of the mitochondrial 16S rRNA gene in three major genera of mosquitoes, Aedes, Anopheles and Culex. The sequence was found to be unusually A+T rich and in substitutions the rate of transversions was higher than the transition rate. A phylogenetic tree was constructed with these sequences. An interesting feature of the sequences was a stretch of Ts that distinguished between Ae-des and Culex on the one hand, and Anopheles on the other. This is the first report of mitochondrial rRNA sequences from these medically important genera of mosquitoes.

Left handed DNA in synthetic and topologically constrained form V DNA and its implications in protein recognition.

We have investigated structural transitions in Poly(dG-dC) and Poly(dG-Me5dC) in order to understand the exact role of cations in stabilizing left-handed helical structures in specific sequences andthe biological role, if any, of these structures. From a novel temperature dependent transition it has been shown that a minor fluctuation in Na+ concentration at ambient temperature can bring about Β to Ζ transition. Forthe first time, wehave observed a novel double transition in poly(dG-Me5dC) as the Na+ concentration is gradually increased. This suggests that a minor fluctuation in Na+ concentration in conjunction with methylation may transform small stretches of CG sequences from one conformational state to another. These stretches could probably serve as sites for regulation. Supercoiled formV DNA reconstituted from pBR322 and pßG plasmids have been studied as model systems, in order to understand the nature and role of left-handed helical conformation in natural sequences. A large portion of DNA in form V, obtained by reannealing the two complementary singlestranded circles is forced to adopt left-handed double helical structure due to topological constraints (Lk = 0). Binding studies with Z-DNA specific antibody and spectroscopic studies confirm the presence of left-handed Z-structure in the pßG and pßR322 form V DNA. Cobalt hexamine chloride, which induces Z-form in Poly(dG-dC) stabilizes the Z-conformation in form V DNA even in the non-alternating purine-pyrimidine sequences. A reverse effect is observed with ethidium bromide. Interestingly, both topoisomerase I and II (from wheat germ) act effectively on form V DNA to give rise to a species having an electrophoretic mobility on agarose gel similar to that of open circular (form II) DNA. Whether this molecule is formed as a result of the left-handed helical segments of form V DNA undergoing a transition to the right-handed B-form during the topoisomerase action remains to be solved.