E. O'Neill

A Novel Staphylococcus aureus Biofilm Phenotype Mediated by the Fibronectin-Binding Proteins, FnBPA and FnBPB

Device-associated infections involving biofilm remain a persistent clinical problem. We recently reported that four methicillin-resistant Staphylococcus aureus (MRSA) strains formed biofilm independently of the icaADBC-encoded exopolysaccharide. Here, we report that MRSA biofilm development was promoted under mildly acidic growth conditions triggered by the addition of glucose to the growth medium. Loss of sortase, which anchors LPXTG-containing proteins to peptidoglycan, reduced the MRSA biofilm phenotype. Furthermore introduction of mutations in fnbA and fnbB, which encode the LPXTG-anchored multifunctional fibrinogen and fibronectin-binding proteins, FnBPA and FnBPB, reduced biofilm formation by several MRSA strains. However, these mutations had no effect on biofilm formation by methicillin-sensitive S. aureus strains. FnBP-promoted biofilm occurred at the level of intercellular accumulation and not primary attachment. Mutation of fnbA or fnbB alone did not substantially affect biofilm, and expression of either gene alone from a complementing plasmid in fnbA fnbB mutants restored biofilm formation. FnBP-promoted biofilm was dependent on the integrity of SarA but not through effects on fnbA or fnbB transcription. Using plasmid constructs lacking regions of FnBPA to complement an fnbAB mutant revealed that the A domain alone and not the domain required for fibronectin binding could promote biofilm. Additionally, an A-domain N304A substitution that abolished fibrinogen binding did not affect biofilm. These data identify a novel S. aureus biofilm phenotype promoted by FnBPA and FnBPB which is apparently independent of the known ligand-binding activities of these multifunctional surface proteins.

Association between Methicillin Susceptibility and Biofilm Regulation in Staphylococcus aureus Isolates from Device-Related Infections

ABSTRACT Production of icaADBC-encoded polysaccharide intercellular adhesin, or poly-N-acetylglucosamine (PIA/PNAG), represents an important biofilm mechanism in staphylococci. We previously described a glucose-induced, ica-independent biofilm mechanism in four methicillin-resistant Staphylococcus aureus (MRSA) isolates. Here, biofilm regulation by NaCl and glucose was characterized in 114 MRSA and 98 methicillin-sensitive S. aureus (MSSA) isolates from diagnosed device-related infections. NaCl-induced biofilm development was significantly more prevalent among MSSA than MRSA isolates, and this association was independent of the isolates genetic background as assessed by spa sequence typing. Among MSSA isolates, PIA/PNAG production correlated with biofilm development in NaCl, whereas in MRSA isolates grown in NaCl or glucose, PIA/PNAG production was not detected even though icaADBC was transcribed and regulated. Glucose-induced biofilm in MRSA was ica independent and apparently mediated by a protein adhesin(s). Experiments performed with strains that were amenable to genetic manipulation revealed that deletion of icaADBC had no effect on biofilm in a further six MRSA isolates but abolished biofilm in four MSSA isolates. Mutation of sarA abolished biofilm in seven MRSA and eight MSSA isolates. In contrast, mutation of agr in 13 MRSA and 8 MSSA isolates substantially increased biofilm (more than twofold) in only 5 of 21 (23%) isolates and had no significant impact on biofilm in the remaining 16 isolates. We conclude that biofilm development in MRSA is ica independent and involves a protein adhesin(s) regulated by SarA and Agr, whereas SarA-regulated PIA/PNAG plays a more important role in MSSA biofilm development.

Methicillin resistance and the biofilm phenotype in Staphylococcus aureus.

Antibiotic resistance and biofilm-forming capacity contribute to the success of Staphylococcus aureus as a human pathogen in both healthcare and community settings. These virulence factors do not function independently of each other and the biofilm phenotype expressed by clinical isolates of S. aureus is influenced by acquisition of the methicillin resistance gene mecA. Methicillin-sensitive S. aureus (MSSA) strains commonly produce an icaADBC operon-encoded polysaccharide intercellular adhesin (PIA)-dependent biofilm. In contrast, the release of extracellular DNA (eDNA) and cell surface expression of a number of sortase-anchored proteins, and the major autolysin have been implicated in the biofilm phenotype of methicillin-resistant S. aureus (MRSA) isolates. Expression of high level methicillin resistance in a laboratory MSSA strain resulted in (i) repression of PIA-mediated biofilm production, (ii) down-regulation of the accessory gene regulator (Agr) system, and (iii) attenuation of virulence in murine sepsis and device infection models. Here we review the mechanisms of MSSA and MRSA biofilm production and the relationships between antibiotic resistance, biofilm and virulence gene regulation in S. aureus.

The effect of rapid screening for methicillin-resistant Staphylococcus aureus (MRSA) on the identification and earlier isolation of MRSA-positive patients.

OBJECTIVES (1) To determine whether rapid screening with polymerase chain reaction (PCR) assays leads to the earlier isolation of patients at risk for methicillin-resistant Staphylococcus aureus (MRSA) colonization, (2) to assess compliance with routine MRSA screening protocols, (3) to confirm the diagnostic accuracy of the Xpert MRSA real-time PCR assay (Cepheid) by comparison with culture, and (4) to compare turnaround times for PCR assay results with those for culture results. DESIGN Before-and-after study conducted in a 700-bed acute tertiary care referral hospital. Study periods were (1) a 5-week period before PCR testing began, (2) a 10-week period when the PCR assay was used, and (3) a 5-week period after PCR testing was discontinued. RESULTS Among 489 at-risk patients, MRSA was isolated from 20 (33%) of 60 patients during period 1, 77 (22%) of 349 patients during period 2, and 18 (23%) of 80 patients during period 3. Twenty-two (27%) of 82 at-risk patients were not screened during period 1, compared with 40 (10%) of 389 at-risk patients not screened during period 2 (P < .001). More MRSA-positive patients were preemptively isolated during periods 1 and 3 compared with period 2 (34 [24%] of 140 vs 28 [8%] of 389; P < .001); however, more MRSA-positive patients were isolated after notification of MRSA-positive results during period 2 (47 [13%] of 349) compared with periods 1 and 3 (2 [1%] of 140; P < .001). The sensitivity, specificity, positive predictive value, and negative predictive value of the PCR assay were 95%, 97%, 82%, and 99%, respectively. The mean turnaround time from receipt of specimens in the laboratory to PCR assay result was 2.6 hours. CONCLUSIONS Rapid screening with the Xpert MRSA PCR assay facilitated compliance with screening policies and the earlier isolation of MRSA-positive patients. Discrepant results confirm that PCR testing should be used as a screening tool rather than as a diagnostic tool.

Current and Future Approaches to the Prevention and Treatment of Staphylococcal Medical Device-Related Infections

Staphylococci, in particular Staphylococcus aureus and Staphylococcus epidermidis, are a leading cause of healthcare-associated infections. Patients who have a medical device inserted are at particular risk of an infection with these organisms as staphylococci possess a wide range of immune evasion mechanisms, one of which being their ability to form biofilm. Once embedded in a biofilm, bacteria are inherently more resistant to treatment with antibiotics. Despite advances in our understanding of the pathogenesis of staphylococcal biofilm formation, medical devices colonised with biofilms frequently require removal. New and novel approaches to prevent and treat biofilm infections are urgently required. In recent years, progress has been made on approaches that include antiadhesive strategies to prevent surface adhesion or production of bacterial adhesins, dissolution of already established biofilm, targeting of biofilm matrix for degradation and interference with biofilm regulation. Several obstacles need to be overcome in the further development of these and other novel anti-biofilm agents. Most notably, although in vitro investigation has progressed over recent years, the need for biofilm models to closely mimic the in vivo situation is of paramount importance followed by controlled clinical trials. In this review we highlight the issues associated with staphylococcal colonisation of medical devices and potential new treatment options for the prevention and control of these significant infections.

An Essential Role for Coagulase in Staphylococcus aureus Biofilm Development Reveals New Therapeutic Possibilities for Device-Related Infections

High-level resistance to antimicrobial drugs is a major factor in the pathogenesis of chronic Staphylococcus aureus biofilm-associated, medical device-related infections. Antimicrobial susceptibility analysis revealed that biofilms grown for ≤ 24 hours on biomaterials conditioned with human plasma under venous shear in iron-free cell culture medium were significantly more susceptible to antistaphylococcal antibiotics. Biofilms formed under these physiologically relevant conditions were regulated by SaeRS and dependent on coagulase-catalyzed conversion of fibrinogen into fibrin. In contrast, SarA-regulated biofilms formed on uncoated polystyrene in nutrient-rich bacteriological medium were mediated by the previously characterized biofilm factors poly-N-acetyl glucosamine, fibronectin-binding proteins, or autolytic activity and were antibiotic resistant. Coagulase-mediated biofilms exhibited increased antimicrobial resistance over time (>48 hours) but were always susceptible to dispersal by the fibrinolytic enzymes plasmin or nattokinase. Biofilms recovered from infected central venous catheters in a rat model of device-related infection were dispersed by nattokinase, supporting the important role of the biofilm phenotype and identifying a potentially new therapeutic approach with antimicrobials and fibrinolytic drugs, particularly during the early stages of device-related infection.

In Vitro Approach for Identification of the Most Effective Agents for Antimicrobial Lock Therapy in the Treatment of Intravascular Catheter-Related Infections Caused by Staphylococcus aureus

ABSTRACT Infection of intravascular catheters by Staphylococcus aureus is a significant risk factor within the health care setting. To treat these infections and attempt salvage of an intravascular catheter, antimicrobial lock solutions (ALSs) are being increasingly used. However, the most effective ALSs against these biofilm-mediated infections have yet to be determined, and clinical practice varies greatly. The purpose of this study was to evaluate and compare the efficacies of antibiotics and antiseptics in current clinical use against biofilms produced by reference and clinical isolates of S. aureus. Static and flow biofilm assays were developed using newly described in vivo-relevant conditions to examine the effect of each agent on S. aureus within the biofilm matrix. The antibiotics daptomycin, tigecycline, and rifampin and the antiseptics ethanol and Taurolock inactivated established S. aureus biofilms, while other commonly used antistaphylococcal antibiotics and antiseptic agents were less effective. These findings were confirmed by live/dead staining of S. aureus biofilms formed and treated within a flow cell model. The results from this study demonstrate the most effective clinically used agents and their concentrations which should be used within an ALS to treat S. aureus-mediated intravascular catheter-related infections.

Use of surveillance data for prevention of healthcare-associated infection: risk adjustment and reporting dilemmas

Purpose of review Healthcare-associated or nosocomial infection (HCAI) is of increasing importance to healthcare providers and the public. Surveillance is crucial but must be adjusted for risk, especially when used for interhospital comparisons or for public reporting. Recent findings Surveillance data are increasingly adjusted for risk factors for HCAI if meaningful comparisons are made between institutions or across national boundaries. Postdischarge surveillance is important in detecting those infections that may not present to the institution in which the original procedure occurred. Caution is urged when comparing data from two sources, for example, an active surveillance program and administrative datasets. The public reporting of HCAI rates can assist in improving the quality of healthcare, but to date there is little evidence that this is happening. In the United States, a number of states have introduced mandatory reporting of HCAIs, but there is considerable variation in what data are released, how these are reported and the rigor of the validation of the dataset. Summary The consumerization of healthcare requires a response from healthcare providers to engage with the public on how, when and what risk-adjusted surveillance data to release. Information campaigns are required to ensure the public understand any publicly released data and its limitations.

Redeploying β-Lactam Antibiotics as a Novel Antivirulence Strategy for the Treatment of Methicillin-Resistant Staphylococcus aureus Infections.

Innovative approaches to the use of existing antibiotics is an important strategy in efforts to address the escalating antimicrobial resistance crisis. We report a new approach to the treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections by demonstrating that oxacillin can be used to significantly attenuate the virulence of MRSA despite the pathogen being resistant to this drug. Using mechanistic in vitro assays and in vivo models of invasive pneumonia and sepsis, we show that oxacillin-treated MRSA strains are significantly attenuated in virulence. This effect is based primarily on the oxacillin-dependent repression of the accessory gene regulator quorum-sensing system and altered cell wall architecture, which in turn lead to increased susceptibility to host killing of MRSA. Our data indicate that &bgr;-lactam antibiotics should be included in the treatment regimen as an adjunct antivirulence therapy for patients with MRSA infections. This would represent an important change to current clinical practice for treatment of MRSA infection, with the potential to significantly improve patient outcomes in a safe, cost-effective manner.

Effectiveness of podcasts as an adjunct learning strategy in teaching clinical microbiology among medical students

The educational tool of podcasting is just one way in which new technologies are being integrated into medical education. The use of podcasts in the medical curriculum provides the potential for ‘anytime, anywhere’ learning experiences. To assess the impact of podcasts as an educational tool, the Clinical Microbiology Department in the RCSI carried out a pilot study of third year medical students studying microbiology in the RCSI. The study was carried out over eleven weeks and all 270 students in the year were asked to participate. A 5-8 minute podcast was developed on individual microbiology topics e.g. antibiotic treatment of selected infections. Topics selected were those which the department felt were important to emphasise and formed an adjunct to lectures and tutorials on the topic. A quiz incorporating ten questions in a multiple choice format were devised for each podcast. Over the eleven week period the podcasts and quizzes were released at various intervals following completion of lectures and tutorials on the topic. Initially students completed a quiz on the topic one day before the release of the podcast. The quiz was then closed and the podcast on the topic released. Following release of the podcast students performed the same quiz five days later. A quiz completion rate of between 45 and 58 percent was achieved for each quiz. Analysis of the quiz results showed that the average result (out of 10) for the pre-podcast quiz was 4.2, whilst the average result for the post-podcast quiz was 5.9. This demonstrated a statistically significant difference in the quiz results pre and post release of the podcast (p-value <0.05). These results establish the importance of podcasts in complementing lectures, tutorials and e-learning to further student knowledge. Podcasting has the potential to enhance medical education by complementing current and future teaching modalities.