Phage Therapy as an Alternative Treatment Modality for Resistant Staphylococcus aureus Infections.

The production and use of antibiotics increased significantly after the Second World War due to their effectiveness against bacterial infections. However, bacterial resistance also emerged and has now become an important global issue. Those most in need are typically high-risk and include individuals who experience burns and other wounds, as well as those with pulmonary infections caused by antibiotic-resistant bacteria, such as Pseudomonas aeruginosa, Acinetobacter sp, and Staphylococci. With investment to develop new antibiotics waning, finding and developing alternative therapeutic strategies to tackle this issue is imperative. One option remerging in popularity is bacteriophage (phage) therapy. This review focuses on Staphylococcus aureus and how it has developed resistance to antibiotics. It also discusses the potential of phage therapy in this setting and its appropriateness in high-risk people, such as those with cystic fibrosis, where it typically forms a biofilm.

The Role of Subinhibitory Concentrations of Daptomycin and Tigecycline in Modulating Virulence in Staphylococcus aureus.

Staphylococcus aureus (S. aureus) infections are notoriously complicated by the ability of the organism to grow in biofilms and are difficult to eradicate with antimicrobial therapy. The purpose of the current study was to clarify the influence of sub-inhibitory concentrations (sub-MICs) of daptomycin and tigecycline antibiotics on biofilm adhesion factors and exoproteins expressions by S. aureus clinical isolates. Six clinical isolates representing positive biofilm S. aureus clones (3 methicillin-sensitive S. aureus (MSSA) and 3 methicillin-resistant S. aureus (MRSA)) were grown with sub-MICs (0.5 MIC) of two antibiotics (daptomycin and tigecycline) for 12 h of incubation. RNA extracted from culture pellets was used via relative quantitative real-time-PCR (qRT-PCR) to determine expression of specific adhesion (fnbA, fnbB, clfA, clfB, fib, ebps, cna, eno) and biofilm (icaADBC) genes. To examine the effect of sub-MIC of these antibiotics on the expression of extracellular proteins, samples from the culture supernatants of six isolates were collected after 12 h of treatment with or without tigecycline in order to profile protein production via 2D gel sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2D gel-SDS-PAGE). Sub-MIC treatment of all clinical MRSA and MSSA strains with daptomycin or tigecycline dramatically induced or suppressed fnbA, fnbB, clfA, clfB, fib, ebps, cna, eno, and icaADBC gene expression. Furthermore, sub-MIC use of tigecycline significantly reduced the total number of separated protein spots across all the isolates, as well as decreasing production of certain individual proteins. Collectively, this study showed very different responses in terms of both gene expression and protein secretion across the various isolates. In addition, our results suggest that sub-MIC usage of daptomycin and tigecycline could signal virulence induction by S. aureus via the regulation of biofilm adhesion factor genes and exoproteins. If translating findings to the clinical treatment of S. aureus, the therapeutic regimen should be adapted depending on antibiotic, the virulence factor and strain type.

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.

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.

Comparative characterisation of genotypically different clones of MRSA in the production of biofilms.

The ability to adhere and produce biofilms is characteristic of enhanced virulence among isolates of methicillin-resistant Staphylococcus aureus (MRSA). The aim of the study is to find out whether these characteristics are consistently similar among isolates variations of MRSA. The study used 30 various isolates of MRSA belong to 13 spa types and 5 MLST types and determined the aggregation, the adherence, and the production of biofilms and slime for each isolate. The methods used to evaluate these characteristics were a modified Congo red agar assay (MCRA), a microtiter plate assay (MPA), high-magnification light microscopy, scanning electron microscopy (SEM), and PCR. The study found that isolates belonging to similar Spa, SCCmec, and ST types have similar abilities to produce biofilms; however, their ability to produce slime on CRA was found to be different. Moreover, isolates that have different Spa types showed high variation in their ability to produce biofilms. The results of light microscope revealed the isolates that produced strong and weak biofilms and formed similar aggregation on the glass surfaces. SEM results showed that all 30 MRSA isolates that were tested were 100% positive for biofilm formation, although to varying degrees. Further testing using PCR confirmed that 100% of the 30 isolates tested were positive for the presence of the icaADBC, fnbA, eno, ebps, clfA, and clfB genes. The prevalence of fib, cna, fnbB, and bbp in MRSA clones was 90, 93.33, 53.33, and 10%, respectively. This study indicate that differences in biofilm production capacities are caused by the differences in surface protein A (Spa) type and are not due to differences in MLST and SCCmec types.

Improved method for the isolation of RNA from bacteria refractory to disruption, including S. aureus producing biofilm.

The development of fast, reliable and inexpensive phenol protocol is described for the isolation of RNA from bacterial biofilm producers. The method was tested on Staphylococcus aureus (S. aureus) and other biofilm-producing gram-negative microorganisms and provided the highest integrity of RNA recovery in comparison to other methods reported here. In parallel experiments, bacterial lysis with Qiagen, NucleoSpin RNAII, InnuREP RNA Mini, Trizol and MasterPure RNA extraction Kits using standard protocols consistently gave low RNA yields with an absence of integrity. The boiling method presented here yielded high concentration of RNA that was free from 16S and 23S rRNA, contained 5S RNA. Higher yields due to improved biofilm bacterial cell lysis were achieved with an added hot phenol incubation step without the need for a bead mill or the enzyme. This method when used in conjunction with the Qiagen RNeasy Mini kit, RNA isolation was a success with greater integrity and contained undegraded 16S and 23S rRNA and did not require further purification. Contaminating DNA was a problem with the RNA processing samples; we used quantitative real-time PCR (RT-qPCR) to measure the recovery of RNA from bacterial biofilm cells using the method described here.