Detection of vancomycin susceptibility among clinical isolates of MRSA by using minimum inhibitory concentration method

Background: Staphylococcus aureus is considered as a major pathogen causing a diversity of infections including bacteremia, pneumonia, skin and soft tissue including osteoarticular infections. Since 1961, Methicillin Resistant Staphylococci aureus (MRSA) emerged has one of the major and common cause of hospital acquired infection. However, due to wide spread usage of vancomycin for MRSA infections resulted in reduced susceptibility of S. aureus to vancomycin has been identified as a serious public health concern. The aim of the study is to identify the Methicillin Resistant Staphylococcus aureus (MRSA) from various clinical samples and to detect vancomycin susceptibility by Minimum Inhibitory Concentration (MIC) method.Methods: This study was conducted over period of one year December 2013 to November 2014. Clinical samples like pus, blood, sputum, urine and cerebrospinal fluid were collected from various clinical departments in Narayana General Hospital for selective isolation of Staphylococcus aureus. A total of 100 Staphylococcal aureus isolates were isolatedby using standard laboratory procedures. MRSA were detected using Oxacillin Disc on Muller Hinton Agar with 4% NaCl. Sensitivity pattern for vancomycin (30 µg) disc and for other recommended antibiotics was determined by Kirby-Bauer’s disk diffusion method. Minimum Inhibitory Concentration (MIC) was done for vancomycin sensitive isolates by standard agar dilution method.Results: Out of 100 S. aureus isolates, all were susceptible to vancomycin (30 µg) by disk diffusion method. But, 82 isolates of MRSA were susceptible to vancomycin at the concentration of 0.5-2 μg/ml of agar. 17 isolates showed intermediate sensitivity to vancomycin, in which 13 isolates with MIC 4μg/ml and 4 isolates with MIC 8 μg/ml and one isolate was resistant to vancomycin even with MIC of 16 μg/ml.Conclusions: The present study reveals the emergence of Vancomycin Intermediate Sensitive Staphylococcus aureus (VISA) and Vancomycin Resistant Staphylococcus aureus (VRSA). Disc diffusion method should not be employed for detection of vancomycin sensitivity for MRSA stains. The major cause may be attributed to unawareness and irrational usage of broad spectrum antibiotics.

Kloniranje, ekspresija i karakterizacija paraflagelarnog gena Rod 2 bičaša Trypanosoma evansi

Paraflagellar rod is the major structural component of the trypanosomatid flagellum and is identified as a complex lattice of filaments which runs parallel to the axoneme throughout most of the flagellar length. The present study was carried out to investigate the existence of the paraflagellar rod (PFR 2) gene in Trypanosoma evansi infecting Indian cattle. Local isolates of T. evansi collected from naturally infected cow were multiplied in Wistar rats. Complementary DNA (cDNA) was synthesized from the RNA of host cell free T. evansi parasites by reverse transcription. The gel purified PCR product (PFR 2 gene of T. evansi) was cloned into the pTZ57R/T vector system. The nucleotide sequence of the PFR 2 gene of the T. evansi S.V.V.U. isolate (Accession No. KT277497) obtained in the present study revealed 100% homology with T. evansi China isolate and 99% homology with T. evansi Izatnagar and Bikaner isolates. The recombinant protein was sub-cloned into pET 32a and expressed in the BL21 (DE3) pLysS expression system. The PFR 2 gene of T. evansi S.V.V.U. isolate was further characterized by determination of its protein profile with SDS-PAGE and western blotting. Indirect ELISA was optimized for detection of the specific antibody titre against the recombinant protein of the PFR 2 gene of T. evansi. In the kinetoplastid species the PFR 2 gene is highly conserved. Therefore the PFR 2 gene was suggested as a vaccine candidate, as well as a diagnostic antigen.Paraflagelarni štapić glavna je strukturna komponenta tripanosomskog biča i dio je kompleksa filamenaza koji teku paralelno s aksonemom duž biča. Istraživanje je provedeno kako bi se ispitalo postojanje paraflagelarnog gena Rod 2 (PFR2) u bičaša Trypanosoma evansi koji invadira goveda u Indiji. Lokalni izolat T. evansi prikupljen od prirodno invadiranih krava umnožen je u Wistar štakora. Komplementarna DNA (cDNA) sintetizirana je iz RNA obrnutom transkripcijom iz stanica neinvadiranih nositelja T. evansi parazita. Pročišćeni PCR produkt (gen PFR2 bičaša T. evansi) kloniran je u vektorski sustav pTZ57R/T. Nukleotidna sekvencija gena PFR2 bičaša T. evansi, izolat S.V.V.U. (pristupni broj KT277497) dobivena u ovom istraživanju pokazala je 100 %-tnu sličnost s izolatom T. evansi China i 99 %-tnu s izolatom T. evansi Izatnagar i Bikaner. Rekombinantni protein ponovno je kloniran u sustavu pET 32a i prikazan u sustavu BL21 (DE3) pLysS. Gen PFR2 bičaša T. evansi, izolat S.V.V.U. dalje je karakteriziran određivanjem proteinskog profila metodama SDS-PAGE i Western blotting. Indirektni test ELISA optimiziran je za dokaz titra specifičnih protutijela za rekombinantni protein gena PFR2 bičaša T. evansi. U kinetoplastida gen PFR2 izrazito je očuvan. Stoga bi se gen PFR2 mogao upotrijebiti za cjepivo te kao dijagnostički antigen

Drought Stress Tolerance Mechanisms in Barley and Its Relevance to Cereals

In the changing environment, water is the major limiting factor for crop productivity throughout the world, and there is every need to generate climate-resilient crops. Since drought is a complex phenomenon, we need to dissect various mechanisms at the physiological, biochemical, and molecular levels in order to generate crop plants with better drought tolerance but without any yield penalties. Accumulated literature points out that improvement at both source and sink levels are needed to elevate final yields under water deficit conditions. Here, we summarize the current status of plant adaptation mechanisms and the strategies that we need to carve for generating drought stress-tolerant crops like barley

Characterization of a virus from pigeonpea with affinities to species in the genus Aureusvirus, family Tombusviridae

In attempts to identify the causal agent of pigeonpea sterility mosaic disease (PSMD), which is transmitted by eriophyid mites, a virus was isolated with great difficulty from some PSMD-affected pigeonpea (Cajanus cajan) plants from different locations in India. Once isolated from pigeonpea, the virus was transmitted readily by mechanical inoculation to several herbaceous species, reaching very high concentrations in some species. The virus was transmitted experimentally through soil to herbaceous test plants but not to pigeonpea. When virus particles were purified and inoculated mechanically to healthy pigeonpea, the virus induced necrosis in inoculated leaves only and did not spread systemically. Therefore, the virus is not the causal agent of PSMD. The virus has isometric particles approximately 30 nm in diameter that sediment as a single component and had a buoyant density in CsCl and Cs2SO4 of 1.34 and 1.27 g.cc-1, respectively. Purified virus particle preparations contained a single major protein of approximately 44 kDa and three RNA species of approximately 4,300, 2,700, and 1,500 nucleotides. Only the largest RNA species was infective to plants; the two smaller species were encapsidated subgenomic species of the 3′ end of the larger genomic RNA. The viral genome was sequenced and showed 93% homology to that of Pothos latent virus (PoLV), a recently described virus in the genus Aureusvirus, family Tombusviridae, and was indistinguishable from PoLV in gel double-diffusion serological tests. This virus, therefore, is regarded as a pigeonpea isolate of PoLV (PoLV-PP). In field studies in different locations in India, enzyme-linked immunosorbent assay and reverse-transcriptase polymerase chain reaction detected PoLV-PP in 10.7% of PSMD-affected and 8.1% of asymptomatic pigeonpea plants. The significance of these findings is discussed

Identification of a virus naturally infecting sorghum in India as Sugarcane streak mosaic virus

The virus associated with mosaic disease of sorghum growing around the sugarcane fields in Andhra Pradesh state, India was found to be serologically related to the Sugarcane streak mosaic virus (SStMV) and Sorghum mosaic Parbhani virus (SMPV). The reverse transcription-polymerase chain reaction (RT-PCR) of the total RNA from the enzyme-linked immunosorbent assay positive sorghum samples with the potyvirus specific degenerate primers yielded an amplicon of ∼500 bp. This amplicon sequence had a 95% identity to the SStMV-Andhra Pradesh (SStMV-AP) and SStMV-Coimbatore isolates reported to naturally infect sugarcane in India. Further confirmation was made by RT-PCR of these samples with the SStMV-AP sequence specific primers that yielded ∼1,000 bp amplicon comprising the entire coat protein and 3′ UTR of the viral genome. This amplicon sequence also had a identity of 95% at nucleotide level with the SStMV-AP sugarcane isolate, but at the CP amino acid level it had 97.8% identity. This partial sequence data confirmed the association of SStMV with the mosaic disease of sorghum in Andhra Pradesh state, India. To our knowledge, this is the first report on association of SStMV with mosaic disease of sorghum and designated as SStMV-sorghum isolat

Bud Necrosis: A Disease of Groundnut Caused by Tomato Spotted Wilt Virus

This is the first ICRISAT Information Bulletin that deals with a virus disease of groundnut. Attention is focused on bud necrosis disease, caused by tomato spotted wilt virus, because of its economic significance on three continents. Epidemics build up rapidly with little warning and cause serious losses to growers. Protocols for purification and identification of the virus are given in detail. The symptoms of the disease in groundnut are illustrated. Procedures for a simple enzyme-linked immunosorbent assay for the detection of the virus are given. The identification of the vector insects—species of Thysanoptera (thrips)—is difficult, and is still to be fully resolved. But a key is provided as an aid in identifying seven thrips species that have been implicated as vectors of tomato spotted wilt virus on groundnut. The current situation concerning management of bud necrosis disease is outlined. Suitable insecticides, cultural practices, biological control, and host-plant resistance are discussed to assist crop protection and extension workers in formulating integrated management systems appropriate to their particular situations

High Affinity Antibodies to Plasmodium falciparum Merozoite Antigens Are Associated with Protection from Malaria

Malaria kills almost 1 million people every year, but the mechanisms behind protective immunity against the disease are still largely unknown. In this study, surface plasmon resonance technology was used to evaluate the affinity (measured as k(d)) of naturally acquired antibodies to the Plasmodium falciparum antigens MSP2 and AMA1. Antibodies in serum samples from residents in endemic areas bound with higher affinities to AMA1 than to MSP2, and with higher affinities to the 3D7 allele of MSP2-3D7 than to the FC27 allele. The affinities against AMA1 and MSP2-3D7 increased with age, and were usually within similar range as the affinities for the monoclonal antibodies also examined in this study. The finding of MSP2-3D7 type parasites in the blood was associated with a tendency for higher affinity antibodies to both forms of MSP2 and AMA1, but this was significant only when analyzing antibodies against MSP2-FC27, and individuals infected with both allelic forms of MSP2 at the same time showed the highest affinities. Individuals with the highest antibody affinities for MSP2-3D7 at baseline had a prolonged time to clinical malaria during 40 weeks of follow-up, and among individuals who were parasite positive at baseline higher antibody affinities to all antigens were seen in the individuals that did not experience febrile malaria during follow up. This study contributes important information for understanding how immunity against malaria arises. The findings suggest that antibody affinity plays an important role in protection against disease, and differs between antigens. In light of this information, antibody affinity measurements would be a key assessment in future evaluation of malaria vaccine formulations

An Unilateral Rectus Sternalis Muscle: It`s Clinical Significance.

Knowledge concerning the variations of chest wall musculature is imperative for Surgeons, Radiologists and Anatomists. Rectus sternalis is a variant chest wall muscle found in the anterior thoracic wall along the side of sternum. The presence of this muscle may be mistaken for tumor on mammogram, and causes implications during mastectomy, implant reconstruction surgeries of mammary gland and may necessitate modifying the approach during mammoplasty. The early detection of this variant muscle is necessary for assessing proper dissection planes in breast surgeries and radiological examination. Hereby, we report a case of unilateral rectus sternalis muscle. The muscle originated from the fascia covering external oblique muscle inserted in to the sternum at the manubrio sternal joint; the muscle belly was 10 cm in length and 1.5 cm in width