Clonal complexes and virulence factors of Staphylococcus aureus from several cities in India

Background: Diseases from Staphylococcus aureus are a major problem in Indian hospitals and recent studies point to infiltration of community associated methicillin resistant S. aureus (CA-MRSA) into hospitals. Although CA-MRSA are genetically different from nosocomial MRSA, the distinction between the two groups is blurring as CA-MRSA are showing multidrug resistance and are endemic in many hospitals. Our survey of samples collected from Indian hospitals between 2004 and 2006 had shown mainly hospital associated methicillin resistant Staphylococcus aureus (HA-MRSA) carrying staphylococcal cassette chromosome mec (SCCmec) type III and IIIA. But S. aureus isolates collected from 2007 onwards from community and hospital settings in India have shown SCCmec type IV and V cassettes while several variations of type IV SCCmec cassettes from IVa to IVj have been found in other parts of the world. In the present study, we have collected nasal swabs from rural and urban healthy carriers and pus, blood etc from in patients from hospitals to study the distribution of SCCmec elements and sequence types (STs) in the community and hospital environment. We performed molecular characterization of all the isolates to determine their lineage and microarray of select isolates from each sequence type to analyze their toxins, virulence and immune-evasion factors. Results: Molecular analyses of 68 S. aureus isolates from in and around Bengaluru and three other Indian cities have been carried out. The chosen isolates fall into fifteen STs with all major clonal complexes (CC) present along with some minor ones. The dominant MRSA clones are ST22 and ST772 among healthy carriers and patients. We are reporting three novel clones, two methicillin sensitive S. aureus (MSSA) isolates belonging to ST291 (related to ST398 which is live stock associated), and two MRSA clones, ST1208 (CC8), and ST672 as emerging clones in this study for the first time. Sixty nine percent of isolates carry Panton-Valentine Leucocidin genes (PVL) along with many other toxins. There is more diversity of STs among methicillin sensitive S. aureus than resistant ones. Microarray analysis of isolates belonging to different STs gives an insight into major toxins, virulence factors, adhesion and immune evasion factors present among the isolates in various parts of India. Conclusions: S. aureus isolates reported in this study belong to a highly diverse group of STs and CC and we are reporting several new STs which have not been reported earlier along with factors influencing virulence and host pathogen interactions.

Differential modulation of intracellular survival of cytosolic and vacuolar pathogens by curcumin

Curcumin, a principal component of turmeric, acts as an immunomodulator regulating the host defenses in response to a diseased condition. The role of curcumin in controlling certain infectious diseases is highly controversial. It is known to alleviate symptoms of Helicobacter pylori infection and exacerbate that of Leishmania infection. We have evaluated the role of curcumin in modulating the fate of various intracellular bacterial pathogens. We show that pretreatment of macrophages with curcumin attenuates the infections caused by Shigella flexneri (clinical isolates) and Listeria monocytogenes and aggravates those caused by Salmonella enterica serovar Typhi CT18 (a clinical isolate), Salmonella enterica serovar Typhimurium, Staphylococcus aureus, and Yersinia enterocolitica. Thus, the antimicrobial nature of curcumin is not a general phenomenon. It modulated the intracellular survival of cytosolic (S. flexneri and L. monocytogenes) and vacuolar (Salmonella spp., Y. enterocolitica, and S. aureus) bacteria in distinct ways. Through colocalization experiments, we demonstrated that curcumin prevented the active phagosomal escape of cytosolic pathogens and enhanced the active inhibition of lysosomal fusion by vacuolar pathogens. A chloroquine resistance assay confirmed that curcumin retarded the escape of the cytosolic pathogens, thus reducing their inter- and intracellular spread. We have demonstrated that the membrane-stabilizing activity of curcumin is crucial for its differential effect on the virulence of the bacteria.

Development of experimental setup for study of adsorption characteristics of porous materials down to 4.2 K

The knowledge of adsorption characteristics of activated carbon (porous material) in the temperature range from 5 to 20 K is essential when used in cryosorption pumps for nuclear fusion applications. However, such experimental data are very scarce in the literature, especially below 77 K. So, an experimental system is designed and fabricated to measure the adsorption characteristics of porous materials under variable cryogenic temperatures (from 5 K to 100 K). This is based on the commercially available micropore-analyser coupled to a closed helium cycle two-stage Gifford McMahon (GM) Cryocooler, which allows the sample to be cooled to 4.2 K. The sample port is coupled to the Cryocooler through a heat switch, which isolates this port from the cold head of the Cryocooler. By this, the sample temperature can now be varied without affecting the Cryocooler. The setup enables adsorption studies in the pressure range from atmospheric down to 10(-4) Pa. The paper describes the details of the experimental setup and presents the results of adsorption studies at 77 K for activated carbon with nitrogen as adsorbate. The system integration is now completed to enable adsorption studies at 4.2 K.

Genome sequencing unveils a novel sea enterotoxin-carrying PVL phage in staphylococcus aureus ST772 from India

Staphylococcus aureus is a major human pathogen, first recognized as a leading cause of hospital-acquired infections. Community-associated S. aureus (CA-SA) pose a greater threat due to increase in severity of infection and disease among children and healthy adults. CA-SA strains in India are genetically diverse, among which is the sequence type (ST) 772, which has now spread to Australia, Europe and Japan. Towards understanding the genetic characteristics of ST772, we obtained draft genome sequences of five relevant clinical isolates and studied the properties of their PVL-carrying prophages, whose presence is a defining hallmark of CA-SA. We show that this is a novel prophage, which carries the structural genes of the hlb-carrying prophage and includes the sea enterotoxin. This architecture probably emerged early within the ST772 lineage, at least in India. The sea gene, unique to ST772 PVL, despite having promoter sequence characteristics typical of low expression, appears to be highly expressed during early phase of growth in laboratory conditions. We speculate that this might be a consequence of its novel sequence context. The crippled nature of the hlb-converting prophage in ST772. suggests that widespread mobility of the sea enterotoxin might be a selective force behind its `transfer' to the PVL prophage. Wild type ST772 strains induced strong proliferative responses as well as high cytotoxic activity against neutrophils, likely mediated by superantigen SEA and the PVL toxin respectively. Both proliferation and cytotoxicity were markedly reduced in a cured ST772 strain indicating the impact of the phage on virulence. The presence of SEA alongside he genes for the immune system-modulating PVL toxin may contribute to the success and virulence of ST772.

Hydrolysis of aromatic beta-glucosides by non-pathogenic bacteria confers a chemical weapon against predators

Bacteria present in natural environments such as soil have evolved multiple strategies to escape predation. We report that natural isolates of Enterobacteriaceae that actively hydrolyze plant-derived aromatic beta-glucosides such as salicin, arbutin and esculin, are able to avoid predation by the bacteriovorous amoeba Dictyostelium discoideum and nematodes of multiple genera belonging to the family Rhabditidae. This advantage can be observed under laboratory culture conditions as well as in the soil environment. The aglycone moiety released by the hydrolysis of beta-glucosides is toxic to predators and acts via the dopaminergic receptor Dop-1 in the case of Caenorhabditis elegans. While soil isolates of nematodes belonging to the family Rhabditidae are repelled by the aglycone, laboratory strains and natural isolates of Caenorhabditis sp. are attracted to the compound, mediated by receptors that are independent of Dop-1, leading to their death. The b-glucosides-positive (Bgl(+)) bacteria that are otherwise non-pathogenic can obtain additional nutrients from the dead predators, thereby switching their role from prey to predator. This study also offers an evolutionary explanation for the retention by bacteria of `cryptic' or `silent' genetic systems such as the bgl operon.

Interspecies and intraspecies interactions in social amoebae

The stable co-existence of individuals of different genotypes and reproductive division of labour within heterogeneous groups are issues of fundamental interest from the viewpoint of evolution. Cellular slime moulds are convenient organisms in which to address both issues. Strains of a species co-occur, as do different species; social groups are often genetically heterogeneous. Intra- and interspecies 1:1 mixes of wild isolates of Dictyostelium giganteum and D.purpureum form chimaeric aggregates, following which they segregate to varying extents. Intraspecies aggregates develop in concert and give rise to chimaeric fruiting bodies that usually contain more spores (reproductives) of one component than the other. Reproductive skew and variance in the proportion of reproductives are positively correlated. Interspecies aggregates exhibit almost complete sorting; most spores in a fruiting body come from a single species. Between strains, somatic compatibility correlates weakly with sexual compatibility. It is highest within clones, lower between strains of a species and lowest between strains of different species. Trade-offs among fitness-related traits (between compatible strains), sorting out (between incompatible strains) and avoidance (between species) appear to lie behind coexistence.

Novel Rearrangements in the Staphylococcal Cassette Chromosome Mec Type V Elements of Indian ST772 and ST672 Methicillin Resistant Staphylococcus aureus Strains

Staphylococcus aureus is a commensal gram positive bacteria which causes severe and non severe infections in humans and livestock. In India, ST772 is a dominant and ST672 is an emerging clone of Staphylococcus aureus. Both cause serious human diseases, and carry type V SCCmec elements. The objective of this study was to characterize SCCmec type V elements of ST772 and ST672 because the usual PCR methods did not amplify all primers specific to the type. Whole genome sequencing analysis of seven ST772 and one ST672 S. aureus isolates revealed that the SCCmec elements of six of the ST772 isolates were the smallest of the extant type V elements and in addition have several other novel features. Only one ST772 isolate and the ST672 isolate carried bigger SCCmec cassettes which were composites carrying multiple ccrC genes. These cassettes had some similarities to type V SCCmec element from M013 isolate (ST59) from Taiwan in certain aspects. SCCmec elements of all Indian isolates had an inversion of the mec complex, similar to the bovine SCCmec type X. This study reveals that six out of seven ST772 S. aureus isolates have a novel type V (5C2) SCCmec element while one each of ST772 and ST672 isolates have a composite SCCmec type V element (5C2&5) formed by the integration of type V SCCmec into a MSSA carrying a SCC element, in addition to the mec gene complex inversions and extensive recombinations.

Cytotoxicity of TiO2 nanoparticles towards freshwater sediment microorganisms at low exposure concentrations

There is a persistent need to assess the effects of TiO2 nanoparticles on the aquatic ecosystem owing to their increasing usage in consumer products and risk of environmental release. The current study is focused on TiO2 nanoparticle-induced acute toxicity at sub-ppm level (<= 1 ppm) on the three different freshwater sediment bacterial isolates and their consortium under two different irradiation (visible light and dark) conditions. The consortium of the bacterial isolates was found to be less affected by the exposure to the nanoparticles compared to the individual cells. The oxidative stress contributed considerably towards the cytotoxicity under both light and dark conditions. A statistically significant increase in membrane permeability was noted under the dark conditions as compared to the light conditions. The optical and fluorescence microscopic images showed aggregation and chain formation of the bacterial cells, when exposed to the nanoparticles. The electron microscopic (SEM, TEM) observations suggested considerable damage of cells and bio-uptake of nanoparticles. The exopolysaccrides (EPS) production and biofilm formation were noted to increase in the presence of the nanoparticles, and expression of the key genes involved in biofilm formation was studied by RT-PCR. (C) 2014 Elsevier Inc. All rights reserved.

Measuring Glutathione Redox Potential of HIV-1-infected Macrophages

Redox signaling plays a crucial role in the pathogenesis of human immunodeficiency virus type-1 (HIV-1). The majority of HIV redox research relies on measuring redox stress using invasive technologies, which are unreliable and do not provide information about the contributions of subcellular compartments. A major technological leap emerges from the development of genetically encoded redox-sensitive green fluorescent proteins (roGFPs), which provide sensitive and compartment-specific insights into redox homeostasis. Here, we exploited a roGFP-based specific bioprobe of glutathione redox potential (E-GSH; Grx1-roGFP2) and measured subcellular changes in E-GSH during various phases of HIV-1 infection using U1 monocytic cells (latently infected U937 cells with HIV-1). We show that although U937 and U1 cells demonstrate significantly reduced cytosolic and mitochondrial E-GSH (approximately -310 mV), active viral replication induces substantial oxidative stress (E-GSH more than -240 mV). Furthermore, exposure to a physiologically relevant oxidant, hydrogen peroxide (H2O2), induces significant deviations in subcellular E-GSH between U937 and U1, which distinctly modulates susceptibility to apoptosis. Using Grx1-roGFP2, we demonstrate that a marginal increase of about similar to 25 mV in E-GSH is sufficient to switch HIV-1 from latency to reactivation, raising the possibility of purging HIV-1 by redox modulators without triggering detrimental changes in cellular physiology. Importantly, we show that bioactive lipids synthesized by clinical drug-resistant isolates of Mycobacterium tuberculosis reactivate HIV-1 through modulation of intracellular E-GSH. Finally, the expression analysis of U1 and patient peripheral blood mononuclear cells demonstrated a major recalibration of cellular redox homeostatic pathways during persistence and active replication of HIV.

Spray: A Multi-Modal Localization System for Stationary Sensor Network Deployment

We present a localization system that targets rapid deployment of stationary wireless sensor networks (WSN). The system uses a particle filter to fuse measurements from multiple localization modalities, such as RF ranging, neighbor information or maps, to obtain position estimations with higher accuracy than that of the individual modalities. The system isolates different modalities into separate components which can be included or excluded independently to tailor the system to a specific scenario. We show that position estimations can be improved with our system by combining multiple modalities. We evaluate the performance of the system in both an indoor and outdoor environment using combinations of five different modalities. Using two anchor nodes as reference points and combining all five modalities, we obtain RMS (Root Mean Square) estimation errors of approximately 2.5m in both cases, while using the components individually results in errors within the range of 3.5 and 9 m.

Mycobacterium tuberculosis has diminished capacity to counteract redox stress induced by elevated levels of endogenous superoxide

Mycobacterium tuberculosis (Mtb) has evolved protective and detoxification mechanisms to maintain cytoplasmic redox balance in response to exogenous oxidative stress encountered inside host phagocytes. In contrast, little is known about the dynamic response of this pathogen to endogenous oxidative stress generated within Mtb. Using a noninvasive and specific biosensor of cytoplasmic redox state of Mtb, we for first time discovered a surprisingly high sensitivity of this pathogen to perturbation in redox homeostasis induced by elevated endogenous reactive oxygen species (ROS). We synthesized a series of hydroquinone-based small molecule ROS generators and found that ATD-3169 permeated mycobacteria to reliably enhance endogenous ROS including superoxide radicals. When Mtb strains including multidrug-resistant (MDR) and extensively drug-resistant (XDR) patient isolates were exposed to this compound, a dose-dependent, long-lasting, and irreversible oxidative shift in intramycobacterial redox potential was detected. Dynamic redox potential measurements revealed that Mtb had diminished capacity to restore cytoplasmic redox balance in comparison with Mycobacterium smegmatis (Msm), a fast growing nonpathogenic mycobacterial species. Accordingly, Mtb strains were extremely susceptible to inhibition by ATD-3169 but not Msm, suggesting a functional linkage between dynamic redox changes and survival. Microarray analysis showed major realignment of pathways involved in redox homeostasis, central metabolism, DNA repair, and cell wall lipid biosynthesis in response to ATD-3169, all consistent with enhanced endogenous ROS contributing to lethality induced by this compound. This work provides empirical evidence that the cytoplasmic redox poise of Mtb is uniquely sensitive to manipulation in steady-state endogenous ROS levels, thus revealing the importance of targeting intramycobacterial redox metabolism for controlling TB infection. (C) 2015 The Authors. Published by Elsevier Inc.

Environmental monitoring of bacterial contamination and antibiotic resistance patterns of the fecal coliforms isolated from Cauvery River, a major drinking water source in Karnataka, India

The present study focuses prudent elucidation of microbial pollution and antibiotic sensitivity profiling of the fecal coliforms isolated from River Cauvery, a major drinking water source in Karnataka, India. Water samples were collected from ten hotspots during the year 2011-2012. The physiochemical characteristics and microbial count of water samples collected from most of the hotspots exhibited greater biological oxygen demand and bacterial count especially coliforms in comparison with control samples (p <= 0.01). The antibiotic sensitivity testing was performed using 48 antibiotics against the bacterial isolates by disk-diffusion assay. The current study showed that out of 848 bacterial isolates, 93.51 % (n=793) of the isolates were found to be multidrug-resistant to most of the current generation antibiotics. Among the major isolates, 96.46 % (n=273) of the isolates were found to be multidrug-resistant to 30 antibiotics and they were identified to be Escherichia coli by 16S rDNA gene sequencing. Similarly, 93.85 % (n=107), 94.49 % (n=103), and 90.22 % (n=157) of the isolates exhibited multiple drug resistance to 32, 40, and 37 antibiotics, and they were identified to be Enterobacter cloacae, Pseudomonas trivialis, and Shigella sonnei, respectively. The molecular studies suggested the prevalence of blaTEM genes in all the four isolates and dhfr gene in Escherichia coli and Sh. sonnei. Analogously, most of the other Gram-negative bacteria were found to be multidrug-resistant and the Gram-positive bacteria, Staphylococcus spp. isolated from the water samples were found to be methicillin and vancomycin-resistant Staphylococcus aureus. This is probably the first study elucidating the bacterial pollution and antibiotic sensitivity profiling of fecal coliforms isolated from River Cauvery, Karnataka, India.

The influence of reduced oxygen availability on gene expression in laboratory (H37Rv) and clinical strains (S7 and S10) of Mycobacterium tuberculosis

Mycobacterium tuberculosis has the ability to persist within the host in a dormant stage. One important condition believed to contribute to dormancy is reduced access to oxygen known as hypoxia. However, the response of M. tuberculosis to such hypoxia condition is not fully characterized. Virtually all dormant models against tuberculosis tested in animals used laboratory strain H37Rv or Erdman strain. But major outbreaks of tuberculosis (TB) occur with the strains that have widely different genotypes and phenotypes compared to H37Rv. In this study, we used a custom oligonucleotide microarray to determine the overall transcriptional response of laboratory strain (H37Rv) and most prevalent clinical strains (S7 and S10) of M. tuberculosis from South India to hypoxia. Analysis of microarray results revealed that a total of 1161 genes were differentially regulated (>= 1.5 fold change) in H37Rv, among them 659 genes upregulated and 502 genes down regulated. Microarray data of clinical isolates showed that a total of 790 genes were differentially regulated in S7 among which 453 genes were upregulated and 337 down regulated. Interestingly, numerous genes were also differentially regulated in S10 (total 2805 genes) of which 1463 genes upregulated and 1342 genes down regulated during reduced oxygen condition (Wayne's model). One hundred and thirty-four genes were found common and upregulated among all three strains (H37Rv, S7, and S10) and can be targeted for drug/vaccine development against TB. (C) 2015 Published by Elsevier B.V.

An Endophytic Fungus, Talaromyces radicus, Isolated from Catharanthus roseus, Produces Vincristine and Vinblastine, Which Induce Apoptotic Cell Death

Endophytic fungi isolated from Catharanthus roseus were screened for the production of vincristine and vinblastine. Twenty-two endophytic fungi isolated from various tissues of C. roseus were characterized taxonomically by sequence analysis of the internal transcribed spacer (ITS) region of rDNA and grouped into 10 genera: Alternaria, Aspergillus, Chaetomium, Colletotrichum, Dothideomycetes, Eutypella, Eutypa, Flavodon, Fusarium and Talaromyces. The antiproliferative activity of these fungi was assayed in HeLa cells using the MTT assay. The fungal isolates Eutypella sp-CrP14, obtained from stem tissues, and Talaromyces radicus-CrP20, obtained from leaf tissues, showed the strongest antiproliferative activity, with IC50 values of 13.5 mu g/ml and 20 mu g/ml, respectively. All 22 endophytic fungi were screened for the presence of the gene encoding tryptophan decarboxylase (TDC), the key enzyme in the terpenoid indole alkaloid biosynthetic pathway, though this gene could only be amplified from T. radicus-CrP20 (NCBI GenBank accession number KC920846). The production of vincristine and vinblastine by T. radicus-CrP20 was confirmed and optimized in nine different liquid media. Good yields of vincristine (670 mu g/l) in modified M2 medium and of vinblastine (70 mu g/l) in potato dextrose broth medium were obtained. The cytotoxic activity of partially purified fungal vincristine was evaluated in different human cancer cell lines, with HeLa cells showing maximum susceptibility. The apoptosis-inducing activity of vincristine derived from this fungus was established through cell cycle analysis, loss of mitochondrial membrane potential and DNA fragmentation patterns.

First-in-Class Inhibitors of Sulfur Metabolism with Bactericidal Activity against Non-Replicating M-tuberculosis

Development of effective therapies to eradicate persistent, slowly replicating M. tuberculosis (Mtb) represents a significant challenge to controlling the global TB epidemic. To develop such therapies, it is imperative to translate information from metabolome and proteome adaptations of persistent Mtb into the drug discovery screening platforms. To this end, reductive sulfur metabolism is genetically and pharmacologically implicated in survival, pathogenesis, and redox homeostasis of persistent Mtb. Therefore, inhibitors of this pathway are expected to serve as powerful tools in its preclinical and clinical validation as a therapeutic target for eradicating persisters. Here, we establish a first functional HTS platform for identification of APS reductase (APSR) inhibitors, a critical enzyme in the assimilation of sulfate for the biosynthesis of cysteine and other essential sulfur-containing molecules. Our HTS campaign involving 38?350 compounds led to the discovery of three distinct structural classes of APSR inhibitors. A class of bioactive compounds with known pharmacology displayed potent bactericidal activity in wild-type Mtb as well as MDR and XDR clinical isolates. Top compounds showed markedly diminished potency in a conditional Delta APSR mutant, which could be restored by complementation with Mtb APSR. Furthermore, ITC studies on representative compounds provided evidence for direct engagement of the APSR target. Finally, potent APSR inhibitors significantly decreased the cellular levels of key reduced sulfur-containing metabolites and also induced an oxidative shift in mycothiol redox potential of live Mtb, thus providing functional validation of our screening data. In summary, we have identified first-in-class inhibitors of APSR that can serve as molecular probes in unraveling the links between Mtb persistence, antibiotic tolerance, and sulfate assimilation, in addition to their potential therapeutic value.