Mammary Gland Pathology Subsequent to Acute Infection with Strong versus Weak Biofilm Forming Staphylococcus aureus Bovine Mastitis Isolates: A Pilot Study Using Non-Invasive Mouse Mastitis Model.

BACKGROUND: Biofilm formation by Staphylococcus aureus is an important virulence attribute because of its potential to induce persistent antibiotic resistance, retard phagocytosis and either attenuate or promote inflammation, depending upon the disease syndrome, in vivo. This study was undertaken to evaluate the potential significance of strength of biofilm formation by clinical bovine mastitis-associated S. aureus in mammary tissue damage by using a mouse mastitis model. METHODS: Two S. aureus strains of the same capsular phenotype with different biofilm forming strengths were used to non-invasively infect mammary glands of lactating mice. Biofilm forming potential of these strains were determined by tissue culture plate method, ica typing and virulence gene profile per detection by PCR. Delivery of the infectious dose of S. aureus was directly through the teat lactiferous duct without invasive scraping of the teat surface. Both bacteriological and histological methods were used for analysis of mammary gland pathology of mice post-infection. RESULTS: Histopathological analysis of the infected mammary glands revealed that mice inoculated with the strong biofilm forming S. aureus strain produced marked acute mastitic lesions, showing profuse infiltration predominantly with neutrophils, with evidence of necrosis in the affected mammary glands. In contrast, the damage was significantly less severe in mammary glands of mice infected with the weak biofilm-forming S. aureus strain. Although both IL-1ß and TNF-α inflammatory biomarkers were produced in infected mice, level of TNF-α produced was significantly higher (p<0.05) in mice inoculated with strong biofilm forming S. aureus than the weak biofilm forming strain. CONCLUSION: This finding suggests an important role of TNF-α in mammary gland pathology post-infection with strong biofilm-forming S. aureus in the acute mouse mastitis model, and offers an opportunity for the development of novel strategies for reduction of mammary tissue damage, with or without use of antimicrobials and/or anti-inflammatory compounds for the treatment of bovine mastitis.

In Vitro Antimicrobial Efficacy of Tobramycin Against Staphylococcus aureus Biofilms in Combination With or Without DNase I and/or Dispersin B: A Preliminary Investigation.

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.

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 α-toxin was detected in >90% of 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.

Intramammary Immunization of Pregnant Mice with Staphylococcal Protein A Reduces the Post-Challenge Mammary Gland Bacterial Load but Not Pathology.

Protein A, encoded by the spa gene, is one of the major immune evading MSCRAMM of S. aureus, demonstrated to be prevalent in a significant percentage of clinical bovine mastitis isolates in Australia. Given its' reported significance in biofilm formation and the superior performance of S. aureus biofilm versus planktonic vaccine in the mouse mastitis model, it was of interest to determine the immunogenicity and protective potential of Protein A as a potential vaccine candidate against bovine mastitis using the mouse mastitis model. Pregnant Balb/c mice were immunised with Protein A emulsified in an alum-based adjuvant by subcutaneous (s/c) or intramammary (i/mam) routes. While humoral immune response of mice post-immunization were determined using indirect ELISA, cell-mediated immune response was assessed by estimation of interferon-gamma (IFN-γ) produced by protein A-stimulated splenocyte supernatants. Protective potential of Protein A against experimental mastitis was determined by challenge of immunized versus sham-vaccinated mice by i/mam route, based upon manifestation of clinical symptoms, total bacterial load and histopathological damage to mammary glands. Significantly (p<0.05) higher levels of IgG1 isotype were produced in mice immunized by the s/c route. In contrast, significantly higher levels of the antibody isotype IgG2a were produced in mice immunized by the i/mam route (p<0.05). There was significant reduction (p<0.05) in bacterial loads of the mammary glands of mice immunized by Protein A regardless of the route of immunization, with medium level of clinical symptoms observed up to day 3 post-challenge. However, Protein A vaccine failed to protect immunized mice post-challenge with biofilm producing encapsulated S. aureus via i/mam route, regardless of the route of immunization, as measured by the level of mammary tissue damage. It was concluded that, Protein A in its' native state was apparently not a suitable candidate for inclusion in a cell-free vaccine formulation against mastitis.

Comparative studies of the immunogenicity and protective potential of biofilm vs planktonic Staphylococcus aureus vaccine against bovine mastitis using non-invasive mouse mastitis as a model system.

This study was undertaken to compare the immunogenicity and protective potential of biofilm vs planktonic Staphylococcus aureus vaccine for the prevention of mastitis using the mouse as a model system. Mice immunized with formalin-killed whole cell vaccine of S. aureus residing in a biofilm when delivered via an intramammary route produced a cell mediated immune response. Mice immunized with this biofilm vaccine showed significant reductions in colonization by S. aureus in mammary glands, severity of clinical symptoms and tissue damage in mammary glands in comparison with the mice immunized with formalin-killed whole cells of planktonic S. aureus. The planktonic vaccine administered by a subcutaneous route produced a significantly higher humoral immune response (IgG1 and IgG) than the biofilm vaccine. However, considering the host response, tissue damage, the clinical severity and colonization of S. aureus in mammary glands, the biofilm vaccine performed better in immunogenicity and protective potential when administered by the intramammary route.

Serological versus molecular typing of surface-associated immune evading polysaccharide antigens-based phenotypes of Staphylococcus aureus.

The aim of this study was to compare the performance of serological versus molecular typing methods to detect capsular polysaccharide (CP) and surface-associated polysaccharide antigen 336 phenotypes of Staphylococcus aureus isolates. Molecular typing of CP types 1, 5 and 8 was carried out using PCR, whereas serological typing of CP1, 2, 5, 8 and antigen 336 was carried out by slide agglutination using specific antisera. By genotyping, 14/31 strains were CP8 positive, 12/31 strains were CP5 and the remaining 6/31 isolates were non-typable (NT). One isolate was positive for both CP5 and CP8 by PCR, but was confirmed as CP8 type serologically. Detection of CP2 and type 336 by PCR was not possible because specific primers were either not available or non-specific. Using serotyping, 14/31 strains were CP8 positive, 11/31 CP5 positive and 2/31 positive for antigen 336. The remaining four S. aureus isolates were serologically NT. However, three of four NT and two 336-positive S. aureus isolates were encapsulated as determined by light microscopy after capsular staining. This discovery was surprising and warrants further investigations on the identification and characterization of additional capsular phenotypes prevalent among S. aureus clinical isolates. It was concluded that serological typing was a better method than molecular typing for use in epidemiological investigations based upon the distribution of surface-associated polysaccharide antigens-based phenotypes.

A rapid DNA extraction method suitable for human papillomavirus detection.

Infection with oncogenic human papillomavirus (HPV) genotypes is necessary for the development of cervical cancer. Testing for HPV DNA from liquid based cervical samples can be used as an adjunct to traditional cytological screening. In addition there are ongoing viral load, genotyping, and prevalence studies. Therefore, a sensitive DNA extraction method is needed to maximize the efficiency of HPV DNA detection. The XytXtract Tissue kit is a DNA extraction kit that is rapid and so could be useful for HPV testing, particularly in screening protocols. This study was undertaken to determine the suitability of this method for HPV detection. DNA extraction from HeLa and Caski cell lines containing HPV 18 and 16 respectively together with DNA from five liquid based cervical samples were used in a HPV PCR assay. DNA was also extracted using the QIAamp DNA mini kit (Qiagen, Hilden, Germany) as a comparison. DNA extracts were serially diluted and assayed. HPV DNA was successfully detected in cell lines and cervical samples using the XytXtract Tissue kit. In addition, the XytXtract method was found to be more sensitive than the QIAmp method as determined by a dilution series of the extracted DNA. While the XytXtract method is a closed, the QIAamp method uses a spin column with possible loss of DNA through DNA binding competition of the matrix, which could impact on the final extraction efficiency. The XytXtract is a cheap, rapid and efficient method for extracting HPV DNA from both cell lines and liquid based cervical samples.

Human methicillin-sensitive Staphylococcus aureus biofilms: potential associations with antibiotic resistance persistence and surface polysaccharide antigens.

The development of persistent antibiotic resistance by human methicillin-sensitive Staphylococcus aureus (MSSA) strains and substantial association with poly-N-acetyl glucosamine (PNAG) in biofilms is reported in this investigation. Sixteen of 31 MSSA strains under study were found to have developed resistance to one or more antibiotics, with four strains, two of which did not produce biofilms, showing resistance to cefoxitin, undetectable by mecA amplification. Antibiotic resistance displayed by 13/14 biofilm-forming S. aureus isolates remained persistent for 4 weeks prior to reverting back to the original antibiotic susceptibility, prompting a suggestion of determining antibiograms for clinical S. aureus isolates subcultured from biofilms developed in vitro as well as planktonic subcultures prepared from the site of infection. While there was correlation of antibiotic resistance with biofilm formation confirming previous reports, this is the first time that persistence of the biofilm-associated antibiotic resistance by S. aureus as planktonic cells is reported. Among the two methods used for assessment of biofilm formation, the tissue culture plate (TCP) method revealed that almost all strains were strong or moderate biofilm producers whereas only 19/31 strains were biofilm producers using the Congo Red agar (CRA) method indicating the superiority of the TCP method in detecting biofilm producers. We also observed no association between biofilm formation and major capsule types. However, substantial, although not absolute, association of biofilm formation with PNAG was observed, warranting continued identification of additional surface-associated polysaccharide and/or protein antigens associated with biofilm formation for development of an effective vaccine against S. aureus infections regardless of capsular phenotype.

Human papillomavirus, high-grade intraepithelial neoplasia and killer immunoglogulin-like receptors: a Western Australian cohort study.

BACKGROUND: Human papillomavirus (HPV) is the causative agent in cervical cancer and HPV genotypes 16 and 18 cause the majority of these cancers. Natural killer (NK) cells destroy virally infected and tumour cells via killer immunoglobulin-like receptors (KIR) that recognize decreased MHC class I expression. These NK cells may contribute to clearance of HPV infected and/or dysplastic cells, however since KIR controls NK cell activity, KIR gene variation may determine outcome of infection. METHODS: KIR gene frequencies were compared between 147 patients with a history of high-grade cervical intraepithelial neoplasia (CIN) and a control population of 187, to determine if any KIR genes are associated with high-grade CIN. In addition a comparison was also made between cases of high grade CIN derived from 30 patients infected with HPV 16/18 and 29 patients infected with non-16/18 HPV to determine if KIR variation contributes to the disproportional carcinogenesis derived from HPV 16/18 infection. RESULTS: High-grade CIN was weakly associated with the absence of KIR2DL2 and KIR2DS2 (p = 0.046 and 0.049 respectively, OR 0.6; 95% CI 0.4 - 0.9) but this association was lost after correction for multi-gene statistical analysis. No difference in KIR gene frequencies was found between high-grade CIN caused by HPV 16/18 and non-16/18. CONCLUSION: No strong association between KIR genes, high-grade CIN and HPV genotype was found in the Western Australian population.

The persistence of biofilm-associated antibiotic resistance of Staphylococcus aureus isolated from clinical bovine mastitis cases in Australia.

The aim of this investigation was to determine the persistence of biofilm-associated antibiotic resistance developed by methicillin-sensitive Staphylococcus aureus (MSSA), of different capsular types, during biofilm formation. Because of superiority of the tissue culture plate (TCP) over the Congo Red Agar (CRA) method for measuring biofilm formation, it was used to determine the persistence of the antibiotic resistance developed by the isolates in biofilms. The antibiotic resistance was found to persist for 3-4 wk post-propagation as planktonic subcultures. Interestingly, some strains even developed resistance to vancomycin and/or teicoplanin. However, no association of either biofilm formation or persistent antibiotic resistance with the major capsular phenotype was observed. These observations highlight the potential significance of (a) determining the antibiograms of S. aureus subcultured from biofilms developed in vitro using the TCP method as well as from planktonic cultures for formulation of an optimal therapeutic strategy, and (b) continuing to identify predominant non-capsular antigens contributing to biofilm formation, regardless of the capsular phenotype for the development of an effective potentially broad-spectrum vaccine for prevention of bovine mastitis caused by S. aureus.