Arunkumar Karunanidhi

Prevalence of Adhesion and Regulation of Biofilm-Related Genes in Different Clones of Staphylococcus aureus

Clinical information about genotypically different clones of biofilm-producing Staphylococcus aureus is largely unknown. We examined whether different clones of methicillin-sensitive and methicillin-resistant S. aureus (MSSA and MRSA) differ with respect to staphylococcal microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) in biofilm formation. The study used 60 different types of spa and determined the phenotypes, the prevalence of the 13 MSCRAMM, and biofilm genes for each clone. The current investigation was carried out using a modified Congo red agar (MCRA), a microtiter plate assay (MPA), polymerase chain reaction (PCR), and reverse transcriptase polymerase chain reaction (RT-PCR). Clones belonging to the same spa type were found to have similar properties in adheringto the polystyrene microtiter plate surface. However, their ability to produce slime on MCRA medium was different. PCR experiments showed that 60 clones of MSSA and MRSA were positive for 5 genes (out of 9 MSCRAMM genes). icaADBC genes were found to be present in all the 60 clones tested indicating a high prevalence, and these genes were equally distributed among the clones associated with MSSA and those with MRSA. The prevalence of other MSCRAMM genes among MSSA and MRSA clones was found to be variable. MRSA and MSSA gene expression (MSCRAMM and icaADBC) was confirmed by RT-PCR.

In vitro antibacterial and antibiofilm activities of chlorogenic acid against clinical isolates of Stenotrophomonas maltophilia including the trimethoprim/sulfamethoxazole resistant strain.

The in vitro antibacterial and antibiofilm activity of chlorogenic acid against clinical isolates of Stenotrophomonas maltophilia was investigated through disk diffusion, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), time-kill and biofilm assays. A total of 9 clinical S. maltophilia isolates including one isolate resistant to trimethoprim/sulfamethoxazole (TMP/SMX) were tested. The inhibition zone sizes for the isolates ranged from 17 to 29 mm, while the MIC and MBC values ranged from 8 to 16 μg mL−1 and 16 to 32 μg mL−1. Chlorogenic acid appeared to be strongly bactericidal at 4x MIC, with a 2-log reduction in viable bacteria at 10 h. In vitro antibiofilm testing showed a 4-fold reduction in biofilm viability at 4x MIC compared to 1x MIC values (0.085 < 0.397 A 490 nm) of chlorogenic acid. The data from this study support the notion that the chlorogenic acid has promising in vitro antibacterial and antibiofilm activities against S. maltophilia.

Quantitative PCR analysis of genes expressed during biofilm development of methicillin resistant Staphylococcus aureus (MRSA)

Staphylococcus aureus biofilm associated infections remains a major clinical concern in patients with indwelling devices. Quantitative real-time PCR (qPCR) can be used to investigate the pathogenic role of such biofilms. We describe qPCRs for 12 adhesion and biofilm-related genes of four S. aureus isolates which were applied during in vitro biofilm development. An endogenous control (16S rRNA) was used for signal normalization. We compared the qPCR results with structural analysis using scanning electron microscopy (SEM). The SEM studies showed different cellular products surrounding the aggregated cells at different times of biofilm formation. Using qPCR, we found that expression levels of the gene encoding fibronectin binding protein A and B and clumping factor B (fnbA/B and clfB), which involves in primary adherence of S. aureus, were significantly increased at 24h and decreased slightly and variably at 48 h when all 4 isolates were considered. The elastin binding protein (ebps) RNA expression level was significantly enhanced more than 6-fold at 24 and 48 h compared to 12h. Similar results were obtained for the intercellular adhesion biofilm required genes type C (icaC). In addition, qPCR revealed a fluctuation in expression levels at different time points of biofilm growth of other genes, indicating that different parameter modes of growth processes are operating at different times.

Genotypically Different Clones of Staphylococcus aureus Are Diverse in the Antimicrobial Susceptibility Patterns and Biofilm Formations

This study evaluated whether genotypically different clinical isolates of S. aureus have similar susceptibilities to individual antibiotics. It further aims to check the impact of biofilm on the in vitro activity of vancomycin, daptomycin, linezolid, and tigecycline against S. aureus clones. The study used a total of 60 different clinical MSSA and MRSA isolates. Susceptibilities were performed in planktonic cultures by macrobroth dilution and epsilon-test (E test) system. Biofilm production was determined using an adherent plate assay. The efficacy of antimicrobial activities against biofilms formation was checked using confocal laser scanning microscopy (CLSM). The study found that similar and different spa, MLST, and SCCmec types displayed high variation in their susceptibilities to antibiotics with tigecycline and daptomycin being the most effective. The biofilms were found resistant to high concentrations of most antibiotics tested with daptomycin being the most effective drug used in adhesive biofilms. A considerable difference exists among similar and various clone types against antibiotics tested. This variation could have contributed to the degree of virulence even within the same clonal genotype and enhanced heterogeneity in the infection potential. Thus, the development of a rapid and precise identification profile for each clone in human infections is important.

Understanding the mode-of-action of Cassia auriculata via in silico and in vivo studies towards validating it as a long term therapy for type II diabetes.

ETHNOPHARMACOLOGICAL RELEVANCE Cassia auriculata (CA) is used as an antidiabetic therapy in Ayurvedic and Siddha practice. This study aimed to understand the mode-of-action of CA via combined cheminformatics and in vivo biological analysis. In particular, the effect of 10 polyphenolic constituents of CA in modulating insulin and immunoprotective pathways were studied. MATERIALS AND METHODS In silico target prediction was first employed to predict the probability of the polyphenols interacting with key protein targets related to insulin signalling, based on a model trained on known bioactivity data and chemical similarity considerations. Next, CA was investigated in in vivo studies where induced type 2 diabetic rats were treated with CA for 28 days and the expression levels of genes regulating insulin signalling pathway, glucose transporters of hepatic (GLUT2) and muscular (GLUT4) tissue, insulin receptor substrate (IRS), phosphorylated insulin receptor (AKT), gluconeogenesis (G6PC and PCK-1), along with inflammatory mediators genes (NF-κB, IL-6, IFN-γ and TNF-α) and peroxisome proliferators-activated receptor gamma (PPAR-γ) were determined by qPCR. RESULTS In silico analysis shows that several of the top 20 enriched targets predicted for the constituents of CA are involved in insulin signalling pathways e.g. PTPN1, PCK-α, AKT2, PI3K-γ. Some of the predictions were supported by scientific literature such as the prediction of PI3K for epigallocatechin gallate. Based on the in silico and in vivo findings, we hypothesized that CA may enhance glucose uptake and glucose transporter expressions via the IRS signalling pathway. This is based on AKT2 and PI3K-γ being listed in the top 20 enriched targets. In vivo analysis shows significant increase in the expression of IRS, AKT, GLUT2 and GLUT4. CA may also affect the PPAR-γ signalling pathway. This is based on the CA-treated groups showing significant activation of PPAR-γ in the liver compared to control. PPAR-γ was predicted by the in silico target prediction with high normalisation rate although it was not in the top 20 most enriched targets. CA may also be involved in the gluconeogenesis and glycogenolysis in the liver based on the downregulation of G6PC and PCK-1 genes seen in CA-treated groups. In addition, CA-treated groups also showed decreased cholesterol, triglyceride, glucose, CRP and Hb1Ac levels, and increased insulin and C-peptide levels. These findings demonstrate the insulin secretagogue and sensitizer effect of CA. CONCLUSION Based on both an in silico and in vivo analysis, we propose here that CA mediates glucose/lipid metabolism via the PI3K signalling pathway, and influence AKT thereby causing insulin secretion and insulin sensitivity in peripheral tissues. CA enhances glucose uptake and expression of glucose transporters in particular via the upregulation of GLUT2 and GLUT4. Thus, based on its ability to modulate immunometabolic pathways, CA appears as an attractive long term therapy for T2DM even at relatively low doses.

Modified DNase tube test to detect DNase activity in Stenotrophomonas maltophilia

Ubiquitous in nature, Stenotrophomonas maltophilia is a non-fermenting Gramnegative bacillus which was previously known as Bacterium bookeri, Pseudomonas maltophilia or Xanthomonas maltophilia (Palleroni & Bradbury, 1993; Denton & Kerr, 1998; Nicodemo & Paez, 2007; Brooke, 2012). Although S. maltophilia has been known for its biotechnological applicability, over the past decades, it has been increasingly associated with cystic fibrosis and nosocomial infections (Ryan et al., 2009). The ability to degrade DNA by specific DNases has identified DNase activity as a key test for the differentiation of S. maltophilia from other nonfermenters (Palleroni, 2005).

Comparative Study of the Effects of Fluconazole and Voriconazole on Candida glabrata , Candida parapsilosis and Candida rugosa Biofilms

Infections by non-albicans Candida species are a life-threatening condition, and formation of biofilms can lead to treatment failure in a clinical setting. This study was aimed to demonstrate the in vitro antibiofilm activity of fluconazole (FLU) and voriconazole (VOR) against C. glabrata, C. parapsilosis and C. rugosa with diverse antifungal susceptibilities to FLU and VOR. The antibiofilm activities of FLU and VOR in the form of suspension as well as pre-coatings were assessed by XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] reduction assay. Morphological and intracellular changes exerted by the antifungal drugs on Candida cells were examined by scanning electron microscope (SEM) and transmission electron microscope (TEM). The results of the antibiofilm activities showed that FLU drug suspension was capable of killing C. parapsilosis and C. rugosa at minimum inhibitory concentrations (MICs) of 4× MIC FLU and 256× MIC FLU, respectively. While VOR MICs ranging from 2× to 32× were capable of killing the biofilms of all Candida spp tested. The antibiofilm activities of pre-coated FLU were able to kill the biofilms at ¼× MIC FLU and ½× MIC FLU for C. parapsilosis and C. rugosa strains, respectively. While pre-coated VOR was able to kill the biofilms, all three Candida sp at ½× MIC VOR. SEM and TEM examinations showed that FLU and VOR treatments exerted significant impact on Candida cell with various degrees of morphological changes. In conclusion, a fourfold reduction in MIC50 of FLU and VOR towards ATCC strains of C. glabrata, C. rugosa and C. rugosa clinical strain was observed in this study.

Antibacterial and Antibiofilm Activities of Nonpolar Extracts of Allium stipitatum Regel. against Multidrug Resistant Bacteria

The present study assessed the in vitro antibacterial and antibiofilm potential of hexane (ASHE) and dichloromethane (ASDE) extracts of Allium stipitatum (Persian shallot) against planktonic cells and biofilm structures of clinically significant antibiotic resistant pathogens, with a special emphasis on methicillin-sensitive Staphylococcus aureus (MSSA), methicillin-resistant S. aureus (MRSA), and emerging pathogens, Acinetobacter baumannii and Stenotrophomonas maltophilia. Antibacterial activities were determined through disk diffusion, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), time-kill kinetics, and electron microscopy. Antibiofilm activity was assessed by XTT [2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide] reduction assay and by confocal laser scanning microscopy (CLSM). The zone of inhibition ranged from 13 to 33 mm, while the MICs and MBCs ranged from 16 to 1024 μg mL−1. Both ASHE and ASDE completely eradicated overnight cultures of the test microorganisms, including antibiotic resistant strains. Time-kill studies showed that the extracts were strongly bactericidal against planktonic cultures of S. aureus, MRSA, Acinetobacter baumannii, and S. maltophilia as early as 4 hours postinoculation (hpi). ASHE and ASDE were shown to inhibit preformed biofilms of the four biofilm phenotypes tested. Our results demonstrate the potential therapeutic application of ASHE and ASDE to inhibit the growth of gram-positive and gram-negative biofilms of clinical significance and warrant further investigation of the potential of A. stipitatum bulbs against biofilm-related drug resistance.

Allium stipitatum Extract Exhibits In Vivo Antibacterial Activity against Methicillin-Resistant Staphylococcus aureus and Accelerates Burn Wound Healing in a Full-Thickness Murine Burn Model

The in vivo antibacterial and burn wound healing potency of Persian shallot bulbs (Allium stipitatum) were explored in a mice burn model infected with methicillin-resistant Staphylococcus aureus (MRSA). Hexane (ASHE) and dichloromethane (ASDE) extracts were tested. Female BALB/c mice were inflicted with third-degree thermal injury followed by infection with MRSA. ASHE and ASDE formulated with simple ointment base (SOB) at concentrations of 1%, 2%, and 5% (w/w) were topically applied to burn wounds twice a day for 20 days. Silver sulfadiazine (1%) served as drug positive control. Microbiological analysis was carried out on 1, 2, 3, 4, and 5 days postwounding (dpw) and histopathological analysis at the end of the experiment (20 dpw). Both ointments demonstrated strong antibacterial activity with complete elimination of MRSA at 48–72 h after infection. The rate of wound contraction was higher (95–100%) in mice groups treated with ASHE and ASDE ointments after 15 dpw. Histological analysis revealed significant increase (p < 0.05) in epithelialization and collagenation in treated groups. The ASHE and ASDE were found to be relatively noncytotoxic and safe to Vero cell line (383.4 μg mL−1; 390.6 μg mL−1), suggesting the extracts as safe topical antibacterial as well as promising alternatives in managing thermal injuries.