Molecular epidemiology of clinical and carrier strains of methicillin resistant Staphylococcus aureus (MRSA) in the hospital settings of north India

Background

The study was conducted between 2000 and 2003 on 750 human subjects, yielding 850 strains of staphylococci from clinical specimens (575), nasal cultures of hospitalized patients (100) and eye & nasal sources of hospital workers (50 & 125 respectively) in order to determine their epidemiology, acquisition and dissemination of resistance genes.

Methods

Organisms from clinical samples were isolated, cultured and identified as per the standard routine procedures. Susceptibility was measured by the agar diffusion method, as recommended by the Nat ional Committee for Clinical Laboratory Standards (NCCLS). The modified method of Birnboin and Takahashi was used for isolation of plasmids from staphylococci. Pulsed-field gel electrophoresis (PFGE) typing of clinical and carrier Methicillin resistant Staphylococcus aureus (MRSA) strains isolated during our study was performed as described previously.

Results

It was shown that 35.1% of Staphylococcus aureus and 22.5% of coagulase-negative staphylococcal isolates were resistant to methicillin. Highest percentage of MRSA (35.5%) was found in pus specimens (n = 151). The multiple drug resistance of all MRSA (n = 180) and Methicillin resistant Coagulase-negative Staphylococcus aureus (MRCNS) (n = 76) isolates was detected. In case of both methicillin-resistant as well as methicillin-sensitive Saphylococcal isolates zero resistance was found to vancomycin where as highest resistance was found to penicillin G followed by ampicillin. It was shown that the major reservoir of methicillin resistant staphylococci in hospitals are colonized/infected inpatients and colonized hospital workers, with carriers at risk for developing endogenous infection or transmitting infection to health care workers and patients. The results were confirmed by molecular typing using PFGE by SmaI-digestion. It was shown that the resistant markers G and T got transferred from clinical S. aureus (JS-105) to carrier S. aureus (JN-49) and the ciprofloxacin (Cf) and erythromycin (E) resistance seemed to be chromosomal mediated. In one of the experiments, plasmid pJMR1O from Staphylococcus aureus coding for ampicillin (A), gentamicin (G) and amikacin (Ak) resistance was transformed into Escherichia coli. The minimal inhibitory concentrations (MICs) for A and G were lower in E. coli than in S. aureus. However, the MIC for Ak was higher in E. coli transformants than in S. aureus.

Conclusion

There is a progressive increase in MRSA prevalence and multi-drug resistance in staphylococci. Vancomycin is still the drug of choice for MRSA infections. The major reservoir of methicillin resistant staphylococci in hospitals is colonized/infected inpatients and colonized hospital workers. Resistance transfer from staphylococci to E. coli as well as from clinical to carrier staphylococci due to antibiotic stress seemed to be an alarming threat to antimicrobial chemotherapy.     

Restoration of complement-enhanced neutralization of vaccinia virus virions by novel monoclonal antibodies raised against the vaccinia virus complement control protein

The vaccinia virus complement control protein (VCP) is secreted by infected cells and has been shown to inhibit complement activation through interactions with C3b/C4b. It contains four short consensus repeat (SCR) domains. It has been suggested that all four SCRs are required for VCP's activity. To elucidate which SCR domains are involved in abolishing complement-enhanced neutralization of vaccinia virus virions, we generated and characterized a panel of mouse monoclonal antibodies (MAbs) raised against VCP. Ten MAbs were isolated and all recognized VCP on Western blots under reducing conditions as well as native-bound VCP in a sandwich enzyme-linked immunosorbent assay. Three of the 10 MAbs (2E5, 3D1, and 3F11) inhibited VCP's abolition of complement-enhanced neutralization of vaccinia virus virions. These MAbs blocked the interaction of VCP with C3b/C4b. The seven remaining MAbs did not alter VCP function in the complement neutralization assay and recognized VCP bound to C3b/C4b. To understand MAb specificity and mode of interaction with VCP, we mapped the MAb binding regions on VCP. The seven nonblocking MAbs all bound to the first SCR of VCP. One of the blocking MAbs recognized SCR 2 while the other two recognized either SCR 4 or the junction between SCRs 3 and 4, indicating that structural elements involved in the interaction of VCP with C3b/C4b are located within SCR domains 2 and 3 and 4. These anti-VCP MAbs may have clinical significance as therapeutic inhibitors of VCP's complement control activity and may also offer a novel approach to managing vaccinia virus vaccine complications that occur from smallpox vaccination.     

Suppression of Virulence of Toxigenic Vibrio cholerae by Anethole through the Cyclic AMP (cAMP)-cAMP Receptor Protein Signaling System

Use of natural compounds as antivirulence drugs could be an alternative therapeutic approach to modify the outcome of bacterial infections, particularly in view of growing resistance to available antimicrobials. Here, we show that sub-bactericidal concentration of anethole, a component of sweet fennel seed, could suppress virulence potential in O1 El Tor biotype strains of toxigenic Vibrio cholerae, the causative agent of the ongoing 7^th cholera pandemic. The expression of cholera toxin (CT) and toxin coregulated pilus (TCP), the major virulence factors of V. cholerae, is controlled through a regulatory cascade involving activation of ToxT with synergistic coupling interaction of ToxR/ToxS with TcpP/TcpH. We present evidence that anethole inhibits in vitro expression of CT and TCP in a toxT-dependent but toxR/toxS-independent manner and through repression of tcpP/tcpH, by using bead-ELISA, western blotting and quantitative real-time RT-PCR assays. The cyclic AMP (cAMP)-cAMP receptor protein (CRP) is a well-studied global signaling system in bacterial pathogens, and this complex is known to suppress expression of tcpP/tcpH in V. cholerae. We find that anethole influences the virulence regulatory cascade by over-expressing cyaA and crp genes. Moreover, suppression of toxigenic V. cholerae-mediated fluid accumulation in ligated ileum of rabbit by anethole demonstrates its potentiality as an antivirulence drug candidate against the diseases caused by toxigenic V. cholerae. Taken altogether, these results revealing a mechanism of virulence inhibition in V. cholerae by the natural compound anethole, may have relevance in designing antivirulence compounds, particularly against multiple antibiotic resistant bacterial pathogens.     

VPAC2 receptor agonist BAY 55-9837 increases SMN protein levels and moderates disease phenotype in severe spinal muscular atrophy mouse models

Background

Spinal Muscular Atrophy (SMA) is one of the most common inherited causes of infant death and is caused by the loss of functional survival motor neuron (SMN) protein due to mutations or deletion in the SMN1 gene. One of the treatment strategies for SMA is to induce the expression of the protein from the homologous SMN2 gene, a rescuing paralog for SMA.

Methods and results

Here we demonstrate the promise of pharmacological modulation of SMN2 gene by BAY 55-9837, an agonist of the vasoactive intestinal peptide receptor 2 (VPAC2), a member of G protein coupled receptor family. Treatment with BAY 55-9837 lead to induction of SMN protein levels via activation of MAPK14 or p38 pathway in vitro. Importantly, BAY 55-9837 also ameliorated disease phenotype in severe SMA mouse models.

Conclusion

Our findings suggest the VPAC2 pathway is a potential SMA therapeutic target.     

拟南芥高亲和性K~+转运蛋白AtHAK5功能位点的鉴定

AtHAK5是拟南芥高亲和性钾转运体,其基因表达受低钾条件强烈诱导,编码蛋白在低钾条件下可以整合到质膜.生物信息学分析发现其氨基酸序列含有多处潜在的磷酸化位点.本研究假设这些位点对于AtHAK5的功能至关重要,为探讨AtHAK5的功能位点,分别将AtHAK5 cDNA和带有13种不同点突变位点的AtHAK5转化到athak5突变体中,获得14种稳定表达的转基因植株.利用athak5突变体根对Cs敏感的表型,最终确定T549A和T666A为非核心磷酸化位点.如下11个位点为AtHAK5功能必需位点:F85L,T86A,T311A,T359A,P551S,D552N,S603A,S604A,K668E,S698A和V713L.     

基于最大熵生态位模型的中华穿山甲潜在适宜生境预测

中华穿山甲（Manis pentadactyla）属于全球极度濒危物种,也是我国一级保护动物。对中华穿山甲的非法捕杀曾导致其种群数量锐减。但是,近年来相关研究报道较少,穿山甲分布状况不明,极大地制约了对该物种的有效保护。搜集了近年来国内中华穿山甲的救护记录和救护新闻,甄别出67个记录分布点,利用最大熵模型软件（MaxEnt）进行因子筛选,结果表明最冷季度降水量、人口密度、年降水量、坡度、坡向、海拔等6个环境变量是与中华穿山甲分布显著相关的影响因子。基于6个主导环境变量构建的MaxEnt模型AUC平均值为0.961±0.014,预测结果达到极好标准。刀切法（Jackknife）表明,其中最冷季度降水量、年降水量、人口密度和海拔是影响中华穿山甲分布的主要因素。中华穿山甲适宜生境（出现概率大于0.498）具有以下特点：最冷季度降水量141.22-439.46 mm,年降水量1471.67-2386.56 mm,人口密度≥390人/km²,海拔<316.98 m。该模型预测中华穿山甲在我国的潜在分布适宜区主要位于我国长江以南地区,总面积约为74.27×10⁴ km²,占国土面积的7.73%,主要集中在江西、广东、湖南和广西省,面积分别占该区域的97.58%,89.65%,76.90%和73.08%;其次是浙江、福建、台湾和安徽省。湖北、江苏、四川、云南、贵州等省份也有中华穿山甲的零星分布。湖北东南部、江苏南部、浙江西南部和福建西北部等与江西接壤的区域也是中华穿山甲的重要潜在分布适宜区。明确中华穿山甲的潜在分布适宜区,可为该物种的种群保护和栖息地管理提供科技支撑。     

In silico investigation on alkaloids of Rauwolfia serpentina as potential inhibitors of 3-hydroxy-3-methyl-glutaryl-CoA reductase

Present work aimed to investigate the in silico activity of the alkaloids of roots of Rauwolfia serpentina as inhibitors of 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR). For this purpose, the three-dimensional (3D) structure of the protein HMGCR (PDB ID: 1HW9) was downloaded from Protein Data Bank (PDB) database, as a target enzyme. The structures of twelve alkaloids from the roots of R. serpentina were selected as ligands and docked with the selected HMGCR enzyme using Molegro Virtual Docker (MVD) software. The software ‘MVD’ computes the binding (atom) energies of selected protein (enzyme) and each ligand at minimum energetic conformation state by using the PLP (Piecewise Linear Potential) scoring mechanism. Docking results of twelve tested alkaloids showed that five alkaloids including compound 1 (ajmalicine), 2 (reserpine), 3 (indobinine), 4 (yohimbine), and 5 (indobine) have displayed the highest MolDock scores and best fit within the prominent active site residues (positioned between 684 and 692 of cis-loop) of HMGCR. According to the lowest MolDock energies obtained through non-covalent interactions of alkaloids with HMGCR, these are characterized to be the potential inhibitors of HMGCR. Therefore, the alkaloids from R. serpentina can effectively suppress the cholesterol biosynthesis pathway through inhibition of HMGCR and can serve as potential lead compounds for the development of new drugs for the treatment of hyperlipidaemia.     

铅锌镉联合染毒及营养干预对大鼠血液系统的影响研究

目的：了解铅锌镉联合染毒对大鼠血液系统的影响及营养干预对其损伤的修复作用。方法：选择SPF级初断乳Wistar大鼠72只,随机分为对照组、染毒组和干预组,分别采用生理盐水、铅锌镉联合染毒液及染毒后以营养干预液灌胃28天和56天之后,检测其血液系统中五元素和血细胞的指标。结果：染毒组较对照组大鼠血铜、血锌含量高,血钙含量低于对照组,差异均有统计学意义（P〈0．05）;染毒组血铜含量高于干预组,血钙含量低于干预组,差异均有统计学意义（P〈0．05）;干预组红细胞（RBC）计数、血红蛋白（Hb）、血细胞比容（HCT）均高于染毒组,差异均有统计学意义（P〈0．05）;对照组白细胞（WBC）计数高于染毒组、干预组,差异均有统计学意义（P〈0．05）。结论：铅镉对大鼠血铜、血钙、血锌水平有影响;综合营养干预对重金属元素造成的血液系统损伤有明显的拮抗作用,对血液系统有一定的保护及修复作用。