Shanmugaraj Gowrishankar

Coral-Associated Bacteria as a Promising Antibiofilm Agent against Methicillin-Resistant and -Susceptible Staphylococcus aureus Biofilms

The current study deals with the evaluation of two coral-associated bacterial (CAB) extracts to inhibit the biofilm synthesis in vitro as well as the virulence production like hemolysin and exopolysaccharide (EPS), and also to assess their ability to modify the adhesion properties, that is cell surface hydrophobicity (CSH) of methicillin-resistant (MRSA) and -susceptible Staphylococcus aureus (MSSA). Out of nine CAB screened, the ethyl acetate extract of CAB-E2 (Bacillus firmus) and CAB-E4 (Vibrio parahemolyticus) have shown excellent antibiofilm activity against S. aureus. CAB-E2 reduced the production of EPS (57–79%) and hemolysin (43–70%), which ultimately resulted in the significant inhibition of biofilms (80–87%) formed by both MRSA and MSSA. Similarly, CAB-E4 was also found to decrease the production of EPS (43–57%), hemolysin (43–57%) and biofilms (80–85%) of test pathogens. CLSM analysis also proved the antibiofilm efficacy of CAB extracts. Furthermore, the CAB extracts strongly decreased the CSH of S. aureus. Additionally, FT-IR analysis of S. aureus treated with CAB extracts evidenced the reduction in cellular components compared to their respective controls. Thus, the present study reports for the first time, B. firmus—a coral-associated bacterium, as a promising source of antibiofilm agent against the recalcitrant biofilms formed by multidrug resistant S. aureus.

Inhibitory efficacy of cyclo(L-leucyl-L-prolyl) from mangrove rhizosphere bacterium-Bacillus amyloliquefaciens (MMS-50) toward cariogenic properties of Streptococcus mutans.

Since Streptococcus mutans is the principal etiologic agent causing dental caries, by encompassing an array of unique virulence traits, emerging treatment strategies that specifically target the virulence of this pathogen may be promising as alternative approaches compared to conventional antibiotic therapy. In this perspective, we investigated chloroform extract of cell-free culture supernatant from mangrove rhizosphere bacterium Bacillus amyloliquefaciens (MMS-50) in terms of anticariogenic properties of S. mutans, without suppressing its viability. Crude chloroform extract of MMS-50 was subjected to column and high performance liquid chromatographic techniques to obtain the active fraction (AF), and MMS-50 AF was used for all further assays. GC-MS and FT-IR were carried out to identify the major components present in MMS-50 AF. Comparative gene expression analysis of some biofilm-forming and virulence genes (vicR, comDE, gtfC, and gbpB) was done by real-time PCR. Cyclo(L-leucyl-L-prolyl) was found to be the chief compound in MMS-50 AF responsible for bioactivity. The minimum and maximum inhibitory concentrations of MMS-50 AF against S. mutans were found to be 100 and 250 μg/mL, respectively. Anti-virulence assays performed using below-sub-MIC levels of MMS-50 AF (30 μg/mL) resulted in significant reduction in adherence (68%), acid production, acid tolerance, glucan synthesis (32%), biofilm formation (53.5%) and cell surface hydrophobicity, all devoid of affecting its viability. The micrographs of CLSM and SEM further confirmed the antibiofilm and anti-virulence efficacies of MMS-50 AF. Expression data showed significant reduction in expression of all studied virulence genes. Thus, the current study unveils the anticariogenic potential of cyclo(L-leucyl-L-prolyl) from B. amyloliquefaciens, as well as its suitability as a novel and alternative anticariogenic agent against dental caries.

Emergence of methicillin-resistant, vancomycin-intermediate Staphylococcus aureus among patients associated with group A Streptococcal pharyngitis infection in southern India

Beyond Staphylococcus aureus being an etiological agent for several serious clinical complications, the foot prints of S. aureus in pharyngitis infection has also been recently recognized. With due response to the fact, a prospective study was conducted between 2009 and 2010 to describe the molecular epidemiology of S. aureus in throat swabs of pharyngitis patients. A total of 63 methicillin-resistant S. aureus (MRSA) and 102 methicillin-susceptible S. aureus (MSSA) isolates were recovered from 265 throat swabs, representing a community-acquired outpatient population from Tamil Nadu, India. Molecular characterization of MRSA was done by two conventional multiplex PCR assays including Panton-Valentine leukocidin (PVL), mecA and nuc genes, and staphylococcal cassette chromosome mec (SCCmec) typing. Among 165 S. aureus isolates, methicillin resistance was observed in 38.2% (n=63), in which 69.8% (n=44/63) of the MRSA along with 55.9% (n=57/102) of MSSA harbored PVL toxin genes. SCCmec typing showed 50.8% of isolates as SCCmec V (n=32), 44.4% as SCCmec III (n=28), and 1.6% as SCCmec types I, II and IVa (n=1). Multilocus sequence typing performed for 26 selected MRSA isolates resulted in 12 different sequence types (ST), including a novel ST2129/SCCmec III, PVL-positive. Ten MRSA isolates were categorized as ST772 (38.5%)/SCCmec V, PVL-positive, and three isolates as ST368 (11.5%)/SCCmec III, PVL-negative. Though the prominent clones of ST772/SCCmec V were multidrug-susceptible worldwide, they were highly multidrug-resistant in the current study, including four clones intermediate to vancomycin. Totally, 10 (15.9%) out of 63 MRSA isolates were documented as vancomycin-intermediate S. aureus (VISA). Collectively, the present study for the first time portrayed the high prevalence of active MRSA pharyngitis infection and also emphasizes an alarming need for discrimination of pharyngeal-asymptomatic carriers of S. aureus from those with an active S. aureus pharyngitis infection.

Morin inhibits biofilm production and reduces the virulence of Listeria monocytogenes - An in vitro and in vivo approach.

The current study explores the in vitro and in vivo antibiofilm efficacy of morin against a leading foodborne pathogen-Listeria monocytogenes (LM). Minimum inhibitory concentration (MIC) of morin against LM strains was found to be 100μg/ml. The non-antibacterial effect of morin at its sub-MICs (6.25, 12.5 and 25μg/ml) was determined through growth curve and XTT assay. Morin at its sub-MICs demonstrated a significant dose dependent inhibitory efficacy against LM biofilm formation which was also evidenced through light, confocal and scanning electron microscopic analyses. However, morin failed to disperse the mature biofilm of LM even at its MIC. Our data also revealed the anti-virulence efficacy of morin, as it significantly inhibited the production of hemolysin and motility of LM. Concentration-dependent susceptibility of morin treated LM cells to normal human serum was observed. In vivo studies revealed that morin extended the lifespan of LM infected Caenorhabditis elegans by about 85%. Furthermore, the non-toxic nature and in vivo anti-adherence efficacy of morin were also ascertained through C. elegans-LM infection model. Overall, the data of the current study identifies morin as a promising antibiofilm agent and its suitability to formulate protective strategies against biofilm associated infections caused by LM.

Bacillus amyloliquefaciens-secreted cyclic dipeptide – cyclo(L-leucyl-L-prolyl) inhibits biofilm and virulence production in methicillin-resistant Staphylococcus aureus

The current study explores the inhibitory efficacy of cyclo(L-leucyl-L-prolyl) (CLP), a cyclic dipeptide from Bacillus amyloliquefaciens on the biofilm and virulence production of methicillin-resistant Staphylococcus aureus (MRSA). The minimal inhibitory concentration (MIC) and maximum bactericidal concentration (MBC) of CLP against three MRSA strains were found to be 256 and 512 μg mL−1 respectively. CLP at its sub-MICs (16, 32, 64 and 128 μg mL−1) exhibited a phenomenal dose-dependent antibiofilm activity against MRSA strains with maximum inhibitions of 85–87%. Confocal and scanning electron microscopic examinations validated the antibiofilm efficacy of CLP. In addition, CLP was proficient enough to greatly modify the surface hydrophobicity and significantly reduced the slime synthesis of MRSA. Appreciable differences noticed in the EPS constituents of CLP treated MRSA signified that the possible antibiofilm mechanism could proceed by impeding the synthesis of EPS and thereby CLP prevents biofilm assemblage and the associated virulence cascade. Interestingly, CLP displayed a prominent disruption (52–54%) on a 48 h preformed biofilm of MRSA. Data from in vivo assays using Caenorhabditis elegans unveiled the non-toxic and anti-infective efficacy of CLP. Down-regulation of all studied virulence genes affirmed the results of the phenotypic and in vivo assays. Thus, the present study exemplifies the use of CLP as a plausible alternative to conventional antibiotics in controlling biofilm associated infections of MRSA.

In Vitro and In Vivo Biofilm Characterization of Methicillin-Resistant Staphylococcus aureus from Patients Associated with Pharyngitis Infection

The present investigation was deliberately aimed at evaluating the biofilm-forming ability of 63 clinical MRSA isolates recovered from pharyngitis patients through different phenotypic assays. The molecular detection of adhesion (icaA/icaD/icaB/icaC), adhesins (fnbA/fnbB, clfA, and cna), staphylococcal accessory regulator (sarA), and α-toxin (hla) genes was done by employing polymerase chain reaction (PCR). Out of 63 isolates, 49 (77.8%) were found slime positive by the Congo red agar (CRA) method and 44 (69.8%) as biofilm positive by the quantitative microtitre plate assays. The results of MATH assay showed that most of the test pathogens are hydrophilic in nature. The molecular investigation of biofilm-associated genes revealed that 84.13% (n = 53) of isolates were found positive for icaADBC genes. The fnbA and fnbB genes were present in 49 (77.8%) and 51 (81%) MRSA isolates, respectively. In addition, 58.7% (n = 37), 73% (n = 46), and 69.8% (n = 44) of the isolates harboured the clfA, cna, and hla genes, respectively. Further, nearly 81% (n = 51) of the isolates were found positive for the gene sarA and all the ica negative isolates were also negative for the gene. Furthermore, the results of in vivo adherence assay unveiled the factual commonness in the in vitro adherence method.

Antivirulent Properties of Underexplored Cinnamomum tamala Essential Oil and Its Synergistic Effects with DNase against Pseudomonas aeruginosa Biofilms – An In Vitro Study

Pseudomonas aeruginosa is a nosocomial pathogen colonizing patients with chronic infectious diseases and has gained resistance to all the known broad spectrum antibiotics available today. The present study showcases the antibiofilm potential of an essential oil (EO) from an underexplored Cinnamomum species namely, C. tamala, against P. aeruginosa biofilms. Furthermore, the synergistic effects of the EO along with a commercially available DNase (DNaseI) and a DNase (MBD) isolated from a marine bacterium were explored for its antibiofilm activity. The results showed that the synergized action has maximum efficacy in inhibiting young and preformed biofilms. The synergized effect of EO and DNaseI showed 70% inhibition against matured biofilms of P. aeruginosa. The EO from C. tamala also showed quorum sensing inhibitory potential as it could inhibit the swarming motility behavior of P. aeruginosa. The synergistic action of EO and DNases offers a novel alternate therapeutic strategy for combating P. aeruginosa biofilm associated infections.

Modulation of Staphylococcus epidermidis (RP62A) extracellular polymeric layer by marine cyclic dipeptide-cyclo(l-leucyl-l-prolyl) thwarts biofilm formation

Staphylococcus epidermidis is the most frequent cause of biofilm mediated implant-associated infections. Extracellular polymeric substance (EPS) is a key component of most biofilms and in pathogens it specifically protects the entrenched-bacterial cells from antibiotics and hosts immune response, and thereby makes the infection ineradicable. Recently, the prominence of cyclic dipeptides in interfering with biofilms and the associated virulence factors of pathogens has offered an alternative to eliminate difficult-to-treat infections. Therefore, we assessed the effect of a potent antibiofilm agent cyclic dipeptide, cyclo(l-leucyl-l-prolyl) (CLP), on the EPS modification of S. epidermidis. The non-bactericidal antibiofilm efficacy of CLP against S. epidermidis was affirmed through quantitative (crystal violet and XTT assays) and qualitative (confocal and scanning electron microscopes) analyses. Notably, CLP was potent enough to reduce all the EPS components viz. polysaccharides, proteins and eDNA to a significant level. Substantial difference in the atomic composition and functionality of CLP treated EPS was evident through X-ray photoelectron spectroscopy. Furthermore, CLP dehydrated the S. epidermidis-EPS and altered the acetylated sugars as well as α-glycosidic linkage in it. The results of cyclic voltammetry (CV) indicate the decrease of total negative charge of EPS upon CLP treatment, which goes well in accordance with the decrease of eDNA. Thus, antibiofilm efficacy of CLP lies in its potency to alter the intrinsic functional groups and charge of secreted EPS.

The role of flavonoids in autoimmune diseases: Therapeutic updates

ABSTRACT Flavonoids are natural polyphenolic compounds which are included in a panoply of drugs and used to treat and/or manage human ailments such as metabolic, cardiovascular, neurological disorders and cancer. Thus, the purpose of this review is to emphasize the importance of flavonoids for the treatment of autoimmune diseases and put into the limelight of the scientific community several health‐promoting effects of flavonoids which could be beneficial for the development of novel drugs from natural products. Despite available reviews on flavonoids targeting various disease conditions, a comprehensive review of flavonoids for autoimmune diseases is still lacking. To the best of our knowledge, this is the first attempt to review the potential of flavonoids for autoimmune diseases. The structure‐activity relationship of flavonoids in this review revealed that the rearrangement and introduction of other functional groups into the basic skeleton of flavonoids might lead to the development of new drugs which will be helpful in relieving the painful symptoms of various autoimmune diseases.

Tanshinone IIA attenuates TNF-α induced PTX3 expression and monocyte adhesion to endothelial cells through the p38/NF-κB pathway

Tanshinone IIA is one of the most predominant bioactive constituents of Danshen, a traditional Chinese medicinal plant with multiple cardiovascular protective actions. Although Tanshinone IIA has been well documented for its endothelial protective efficacy, studies unveiling the mechanism and/or molecular targets for its pharmacological activity are still inadequate. In recent studies, it has been envisaged that the expression of pentraxin 3 (PTX3) was associated with atherosclerotic cardiovascular diseases (ACVD). Therefore, the current study was designed to evaluate the possible role of Tanshinone IIA in influencing the expression of PTX3 in endothelial cells and thereby prevents endothelial dysfunction. Molecular analyses through real-time PCR, western blot, and ELISA revealed that Tanshinone IIA down-regulates PTX3 gene expression as well as protein secretion in human endothelial cells in the presence or absence of TNF-α. Besides, Tanshinone IIA inhibits the adhesion of THP1 cells (a monocytic cell line) to activated-endothelial cells stimulated with TNF-α. Furthermore, mechanistic studies uncovered the role of p38 MAPK/NF-κB pathway in Tanshinone II-A mediated pharmacological effects. Thus, the present study exemplifies the manifestation of Tanshinone IIA as a plausible alternative natural remedy for ACVD by targeting PTX3.