Characterization and identification of novel biofilm forming antigens of Staphylococcus aureus of human origin

Treatment and prevention of Staphylococcus aureus-associated infections remains a major challenge owing to its versatility to develop persistent antibiotic resistance and produce a wide range of virulence factors including biofilm formation. Other than identifying the most significant virulence factors potentially contributing to biofilm formation, a novel biofilm-associated antigen was identified and characterized in vitro and in vivo. Potential strategies for enhancing efficacy of antibiotics against S. aureus-associated infections were also evaluated provoking recommendations

Diversity of virulence factors associated with West Australian methicillin-sensitive staphylococcus aureus isolates of human origin

An extensive array of virulence factors associated with S. aureus has contributed significantly to its success as a major nosocomial pathogen in hospitals and community causing variety of infections in affected patients. Virulence factors include immune evading capsular polysaccharides, poly-N-acetyl glucosamine, and teichoic acid in addition to damaging toxins including hemolytic toxins, enterotoxins, cytotoxins, exfoliative toxin, and microbial surface components recognizing adhesive matrix molecules (MSCRAMM). In this investigation, 31 West Australian S. aureus isolates of human origin and 6 controls were analyzed for relative distribution of virulence-associated genes using PCR and/or an immunoassay kit and MSCRAMM by PCR-based typing. Genes encoding MSCRAMM, namely, Spa, ClfA, ClfB, SdrE, SdrD, IsdA, and IsdB, were detected in >90% of isolates. Gene encoding a-toxin was detected in >90% isolates whereas genes encoding ß-toxin and SEG were detectable in 50-60% of isolates. Genes encoding toxin proteins, namely, SEA, SEB, SEC, SED, SEE, SEH, SEI, SEJ, TSST, PVL, ETA, and ETB, were detectable in >50% of isolates. Use of RAPD-PCR for determining the virulence factor-based genetic relatedness among the isolates revealed five cluster groups confirming genetic diversity among the MSSA isolates, with the greatest majority of the clinical S. aureus (84%) isolates clustering in group IIIa

Differentiation of Staphylococcus aureus and Staphylococcus epidermidis by PCR for the fibrinogen binding protein gene.

Mastitis is one of the most common and burdensome diseases afflicting dairy animals. Among other causes of mastitis, staphylococci are frequently associated with clinical and subclinical mastitis. Although Staphylococcus aureus is the predominant species involved, Staphylococcus epidermidis and other coagulase-negative staphylococci are increasingly being isolated from cases of bovine mastitis. Although Staph. aureus and Staph. epidermidis can be easily differentiated based on theirbiochemical properties, such phenotypic identification is time consuming and laborious. This study aimed to rapidly identify Staph. aureus and Staph. epidermidis. Accordingly, a multiplex PCR was developed and we found that a single gene encoding the adhesin fibrinogen binding protein could be used to identify and differentiate the two species. Consequently, a multiplex reaction combining a triplex PCR for Staph. aureus and a duplex PCR for Staph. epidermidis was standardized, first using bacterial cultures and then with pasteurized milk spiked with live organisms or DNA extracted fromthe organisms. The test could specifically detect Staph. aureus and Staph. epidermidis even in the presence of a dozen other organisms. The limit of detection for detecting Staph. aureus and Staph. epidermidis separately was 10 to 100 cfu/mL for simplex PCR and 104 cfu/mL for multiplex PCR. Conversely, the limit was 106 cfu/ mL by multiplex PCR for simultaneous detection of both the organisms when spiked into culture medium or pasteurized milk. Overnight enrichment enhanced the assay sensitivity 100-fold. The assay had a high diagnostic sensitivity and specificity. The application ofthe test was verified on 602 field isolates of staphylococci that had been characterized earlier by phenotypic methods. Importantly, 25 coagulase-negative isolates were identified as Staph. aureus by the multiplex PCR. The test could be adapted for use in clinical diagnostic laboratorie

Activating PTEN Tumor Suppressor Expression with the CRISPR/dCas9 System

PTEN expression is lost in many cancers, and even small changes in PTEN activity affect susceptibility and prognosis in a range of highly aggressive malignancies, such as melanoma and triple-negative breast cancer (TNBC). Loss of PTEN expression occurs via multiple mechanisms, including mutation, transcriptional repression and epigenetic silencing. Transcriptional repression of PTEN contributes to resistance to inhibitors used in the clinic, such as B-Raf inhibitors in BRAF mutant melanoma. We aimed to activate PTEN expression using the CRISPR system, specifically dead (d) Cas9 fused to the transactivator VP64-p65-Rta (VPR). dCas9-VPR was directed to the PTEN proximal promoter by single-guide RNAs (sgRNAs), in cancer cells that exhibited low levels of PTEN expression. The dCas9-VPR system increased PTEN expression in melanoma and TNBC cell lines, without transcriptional regulation at predicted off-target sgRNA binding sites. PTEN activation significantly repressed downstream oncogenic pathways, including AKT, mTOR, and MAPK signaling. BRAF V600E mutant melanoma cells transduced with dCas9-VPR displayed reduced migration, as well as diminished colony formation in the presence of B-Raf inhibitors, PI3K/mTOR inhibitors, and with combined PI3K/mTOR and B-Raf inhibition. CRISPR-mediated targeted activation of PTEN may provide an alternative therapeutic approach for highly aggressive cancers that are refractory to current treatments. Keywords: human PTEN protein, CRISPR/Cas systems, tumor suppressor genes, melanoma, breast neoplasm

Relative distribution of virulence-associated factors among Australian bovine staphylococcus aureus isolates: potential relevance to development of an effective bovine mastitis vaccine

Staphylococcus aureus is one of the major contagious pathogens causing bovine mastitis worldwide.1 It causes contagious mastitis resulting either clinical or subclinical mastitis with increase in the number of somatic cell count (SCC) in milk. More than $130 million is lost by the Australian dairy farmers ($A200/cow/year) every year due to poor udder health caused by mastitis resulting in reduction of milk production, increase in treatment costs, veterinary consultation fees, and number of cow culls. There are multiple pathogens that have been found to be associated with bovine mastitis in Australia.2 While the relative distribution of the different pathogens causing mastitis may differ in different regions and countries, S. aureus is one of the most significant contagious bacterial pathogens causing bovine mastitis and is of concern to public health because of its potential for transmission to humans..

In vitro antimicrobial efficacy of tobramycin against staphylococcus aureus biofilms in combination with or without DNase I and/or Dispersin B: A preliminary investigation

© Mary Ann Liebert, Inc. 2017.Staphylococcus aureus in biofilms is highly resistant to the treatment with antibiotics, to which the planktonic cells are susceptible. This is likely to be due to the biofilm creating a protective barrier that prevents antibiotics from accessing the live pathogens buried in the biofilm. S. aureus biofilms consist of an extracellular matrix comprising, but not limited to, extracellular bacterial DNA (eDNA) and poly-ß-1, 6-N-acetyl-d-glucosamine (PNAG). Our study revealed that despite inferiority of dispersin B (an enzyme that degrades PNAG) to DNase I that cleaves eDNA, in dispersing the biofilm of S. aureus, both enzymes were equally efficient in enhancing the antibacterial efficiency of tobramycin, a relatively narrow-spectrum antibiotic against infections caused by gram-positive and gram-negative pathogens, including S. aureus, used in this investigation. However, a combination of these two biofilm-degrading enzymes was found to be significantly less effective in enhancing the antimicrobial efficacy of tobramycin than the individual application of the enzymes. These findings indicate that combinations of different biofilm-degrading enzymes may compromise the antimicrobial efficacy of antibiotics and need to be carefully assessed in vitro before being used for treating medical devices or in pharmaceutical formulations for use in the treatment of chronic ear or respiratory infections

Experimental groups of mice used in the immunogenicity and protective potential trials of Protein A vaccines (n = 48).

<p>Experimental groups of mice used in the immunogenicity and protective potential trials of Protein A vaccines (n = 48).</p

Clinical signs observed in different groups of mice immunized with Protein A (Observations up to 5 days post challenge).

<p>Clinical signs observed in different groups of mice immunized with Protein A (Observations up to 5 days post challenge).</p

Detection of bacterial load and histopathological changes in the mammary glands of mice immunized with Protein A.

<p>Detection of bacterial load and histopathological changes in the mammary glands of mice immunized with Protein A.</p

IFN-γ production by splenocytes of mice immunised with Protein A and PBS by s/c and i/mam routes following stimulation with Protein A.

<p>IFN-γ production by splenocytes of mice immunised with Protein A and PBS by s/c and i/mam routes following stimulation with Protein A.</p