Inflammatory cytokines and biofilm production sustain Staphylococcus aureus outgrowth and persistence: A pivotal interplay in the pathogenesis of Atopic Dermatitis

Individuals with Atopic dermatitis (AD) are highly susceptible to Staphylococcus aureus colonization. However, the mechanisms driving this process as well as the impact of S. aureus in AD pathogenesis are still incompletely understood. In this study, we analysed the role of biofilm in sustaining S. aureus chronic persistence and its impact on AD severity. Further we explored whether key inflammatory cytokines overexpressed in AD might provide a selective advantage to S. aureus. Results show that the strength of biofilm production by S. aureus correlated with the severity of the skin lesion, being significantly higher (P < 0.01) in patients with a more severe form of the disease as compared to those individuals with mild AD. Additionally, interleukin (IL)-β and interferon γ (IFN-γ), but not interleukin (IL)-6, induced a concentration-dependent increase of S. aureus growth. This effect was not observed with coagulase-negative staphylococci isolated from the skin of AD patients. These findings indicate that inflammatory cytokines such as IL1-β and IFN-γ, can selectively promote S. aureus outgrowth, thus subverting the composition of the healthy skin microbiome. Moreover, biofilm production by S. aureus plays a relevant role in further supporting chronic colonization and disease severity, while providing an increased tolerance to antimicrobials

Measuring the effect of enhanced cleaning in a UK hospital : a prospective cross-over study

Increasing hospital-acquired infections have generated much attention over the last decade. There is evidence that hygienic cleaning has a role in the control of hospital-acquired infections. This study aimed to evaluate the potential impact of one additional cleaner by using microbiological standards based on aerobic colony counts and the presence of Staphylococcus aureus including meticillin-resistant S. aureus. We introduced an additional cleaner into two matched wards from Monday to Friday, with each ward receiving enhanced cleaning for six months in a cross-over design. Ten hand-touch sites on both wards were screened weekly using standardised methods and patients were monitored for meticillin-resistant S. aureus infection throughout the year-long study. Patient and environmental meticillin-resistant S. aureus isolates were characterised using molecular methods in order to investigate temporal and clonal relationships. Enhanced cleaning was associated with a 32.5% reduction in levels of microbial contamination at handtouch sites when wards received enhanced cleaning (P < 0.0001: 95% CI 20.2%, 42.9%). Near-patient sites (lockers, overbed tables and beds) were more frequently contaminated with meticillin-resistant S. aureus/S. aureus than sites further from the patient (P = 0.065). Genotyping identified indistinguishable strains from both handtouch sites and patients. There was a 26.6% reduction in new meticillin-resistant S. aureus infections on the wards receiving extra cleaning, despite higher meticillin-resistant S. aureus patient-days and bed occupancy rates during enhanced cleaning periods (P = 0.032: 95% CI 7.7%, 92.3%). Adjusting for meticillin-resistant S. aureus patient-days and based upon nine new meticillin-resistant S. aureus infections seen during routine cleaning, we expected 13 new infections during enhanced cleaning periods rather than the four that actually occurred. Clusters of new meticillin-resistant S. aureus infections were identified 2 to 4 weeks after the cleaner left both wards. Enhanced cleaning saved the hospital £30,000 to £70,000.Introducing one extra cleaner produced a measurable effect on the clinical environment, with apparent benefit to patients regarding meticillin-resistant S. aureus infection. Molecular epidemiological methods supported the possibility that patients acquired meticillin-resistant S. aureus from environmental sources. These findings suggest that additional research is warranted to further clarify the environmental, clinical and economic impact of enhanced hygienic cleaning as a component in the control of hospital-acquired infection

Metabolic activity, urease production, antibiotic resistance and virulence in dual species biofilms of Staphylococcus epidermidis and Staphylococcus aureus

In this paper, the metabolic activity in single and dual species biofilms of Staphylococcus epidermidis and Staphylococcus aureus isolates was investigated. Our results demonstrated that there was less metabolic activity in dual species biofilms compared to S. aureus biofilms. However, this was not observed if S. aureus and S. epidermidis were obtained from the same sample. The largest effect on metabolic activity was observed in biofilms of S. aureus Mu50 and S. epidermidis ET-024. A transcriptomic analysis of these dual species biofilms showed that urease genes and genes encoding proteins involved in metabolism were downregulated in comparison to monospecies biofilms. These results were subsequently confirmed by phenotypic assays. As metabolic activity is related to acid production, the pH in dual species biofilms was slightly higher compared to S. aureus Mu50 biofilms. Our results showed that S. epidermidis ET-024 in dual species biofilms inhibits metabolic activity of S. aureus Mu50, leading to less acid production. As a consequence, less urease activity is required to compensate for low pH. Importantly, this effect was biofilm-specific. Also S. aureus Mu50 genes encoding virulence-associated proteins (Spa, SpIF and Dps) were upregulated in dual species biofilms compared to monospecies biofilms and using Caenorhabditis elegans infection assays, we demonstrated that more nematodes survived when co-infected with S. epidermidis ET-024 and S. aureus mutants lacking functional spa, spIF or dps genes, compared to nematodes infected with S. epidermidis ET-024 and wild type S. aureus. Finally, S. epidermidis ET-024 genes encoding resistance to oxacillin, erythromycin and tobramycin were upregulated in dual species biofilms and increased resistance was subsequently confirmed. Our data indicate that both species in dual species biofilms of S. epidermidis and S. aureus influence each other's behavior, but additional studies are required necessary to elucidate the exact mechanism(s) involved

The distribution of plasmids that carry virulence and resistance genes in Staphylococcus aureus is lineage associated.

BACKGROUND: Staphylococcus aureus is major human and animal pathogen. Plasmids often carry resistance genes and virulence genes that can disseminate through S. aureus populations by horizontal gene transfer (HGT) mechanisms. Sequences of S. aureus plasmids in the public domain and data from multi-strain microarrays were analysed to investigate (i) the distribution of resistance genes and virulence genes on S. aureus plasmids, and (ii) the distribution of plasmids between S. aureus lineages. RESULTS: A total of 21 plasmid rep gene families, of which 13 were novel to this study, were characterised using a previously proposed classification system. 243 sequenced plasmids were assigned to 39 plasmid groups that each possessed a unique combination of rep genes. We show some resistance genes (including ermC and cat) and virulence genes (including entA, entG, entJ, entP) were associated with specific plasmid groups suggesting there are genetic pressures preventing recombination of these genes into novel plasmid groups. Whole genome microarray analysis revealed that plasmid rep, resistance and virulence genes were associated with S. aureus lineages, suggesting restriction-modification (RM) barriers to HGT of plasmids between strains exist. Conjugation transfer (tra) complex genes were rare. CONCLUSION: This study argues that genetic pressures are restraining the spread of resistance and virulence genes amongst S. aureus plasmids, and amongst S. aureus populations, delaying the emergence of fully virulent and resistant strains

Survival of Staphylococcus aureus during the manufacture and ripening of camembert cheese : a thesis presented in partial fulfilment of the requirements for the degree of Master of Food Technology, Massey University, Palmerston North, New Zealand

Staphylococcal Food Poisoning (SFP) is the third most common cause of food poisoning internationally, caused by an enterotoxin produced by Staphylococcus aureus. S. aureus contamination in dairy products, including cheese, can lead to SFP. The survivability of S. aureus during the manufacture and ripening of Camembert cheese was the focus of this study. Camembert cheeses were manufactured using pasteurized milk inoculated with one of three S. aureus strains, comprising two reference strains ATCC 4163, ATCC 9144 and one dairy strain 172 RR. Each strain was tested in triplicate. The results showed that manufacturing and ripening of Camembert cheese reduced the risk of food safety associated with contamination with S. aureus with a 1.6 to 3.1 log reduction. The largest decrease occurred following drainage, which was particularly evident in 172 RR, and coincided with the lowest pH. The combined effect of culture blend (starter and secondary flora) activity and low pH are believed to contribute to the death of S. aureus

Antibiotics with Interleukin-15 inhibition reduces joint inflammation and bone erosions but not cartilage destruction in Staphylococcus aureus-induced arthritis

Background: Staphylococcus aureus-induced arthritis causes rapid joint destruction, often leading to disabling joint damage despite antibiotics. We have previously shown that IL-15 inhibition without antibiotics is beneficial in S. aureus-induced arthritis. We therefore hypothesized that inhibition of IL-15, in combination with antibiotics, might represent a useful therapy that would both reduce inflammation and joint destruction, but preserve the host's ability to clear the infection. Methods: Female wildtype C57BL/6 mice were intravenously inoculated with the TSST-1-producing LS-1 strain of S. aureus with 0.8x108 S. aureus LS-1/mouse. Three days later the treatment was started consisting of cloxacillin followed by flucloxacillin, together with either anti-IL-15 antibodies (aIL-15ab) or control antibodies. Outcomes included survival, weight change, bacterial clearance, and joint damage. Results: The addition of aIL-15ab to antibiotics in S. aureus-induced arthritis reduced synovitis and bone erosions compared to controls. The number of bone-resorbing osteoclasts in the joints was reduced, whereas cartilage destruction was not significantly altered. Importantly, the combination therapy did not adversely affect the clinical outcome of S. aureus-induced arthritis, such as survival, weight change or compromise the host's ability to clear the infection. Conclusions: As the clinical outcome of S. aureus-induced arthritis was not affected, the addition of aIL-15ab to antibiotics ought to be safe. Taken together, the combination of aIL-15ab and antibiotics is a beneficial, but not optimal, treatment of S. aureus-induced arthritis as it reduces synovitis and bone erosions but has a limited effect on cartilage destruction

Staphylococcus aureus typing by digestion of protein A coding gene using Bsp143I

Background: Protein A is the virulence factors of Staphylococcus aureus rolling in its pathogenesis, and its gene is used for typing. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) with different enzymes has been used for this action. Objectives: In this study, we used Bsp143I enzyme for digestion of the gene, coding protein A (spa gene) in S. aureus. The bacteria were isolated from patients and healthy carriers in Gorgan, north of Iran. Patients and Methods: DNAs of 128 S. aureus subjects (53 from healthy carriers and 75 from patients) were extracted and amplified using specific primers of the spa gene. The product was digested by Bsp143I enzyme and its pattern was assessed by gel electrophoresis. Results: There were seven spa types among the tested S. aureus samples, among which six types differed in the repeated X region of the spa gene, but the seventh type had a deletion on one of BSP143I restriction sites. The frequency of spa types among isolated S. aureus samples as well as healthy carriers was six and five, respectively. S. aureus isolated from wounds showed the most diverse spa types (five) among clinical samples. Types 1, 2 and 4 were observed in all clinical samples, while only one case of type 3 was identified among patients, whereas this type constituted over 32% of the isolates among carriers. We found seven and four spa types among methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) isolates, respectively. Conclusions: Our results showed that typing the spa gene using PCR-RFLP using Bsp143I was an acceptable method for typing S. aureus. Furthermore, this survey showed that the types in healthy carriers and MSSA were more variable than patient and MRSA isolates, respectively. We used the Bsp143I enzyme, which was not used in any previous studies on the spa gene. The results of this study suggested that we can use PCR-RFLP of spa gene by Bsp143I for molecular typing and sequencing of S. aureus, instead of relatively expensive methods. This method is relatively rapid and inexpensive, and can be accomplished in centers with conventional molecular facilities. © 2014, Ahvaz Jundishapur University of Medical Sciences

Influence of biomaterial nanotopography on the adhesive and elastic properties of Staphylococcus aureus cells

Despite the well-known beneficial effects of biomaterial nanopatterning on host tissue integration, the influence of controlled nanoscale topography on bacterial colonisation and infection remains unknown. Therefore, the aim of the present study was to determine the nanoscale effect of surface nanopatterning on biomaterial colonisation by S. aureus, utilising AFM nanomechanics and single-cell force spectroscopy (SCFS). Nanoindentation of S. aureus bound to planar (PL) and nanopatterned (SQ) polycarbonate (PC) surfaces suggested two distinct areas of mechanical properties, consistent with a central bacterial cell surrounded by a capsullar component. Nevertheless, no differences in elastic moduli were found between bacteria bound to PL and SQ, suggesting a minor role of nanopatterning in bacterial cell elasticity. Furthermore, SCFS demonstrated increased adhesion forces and work between S. aureus and SQ surfaces at 0 s and 1 s contact times. Although WLC modelling showed similarities in contour lengths for attachment to both surfaces, Poisson analysis suggests increased short-range forces for the S. aureus–SQ interactions. In the case of S. aureus–PL, long-range forces were found to not only be dominant but also repulsive in nature, which may help explain the reduced adhesion forces observed during AFM probing. In conclusion, although surface nanopatterning does not significantly influence the elasticity of attached bacterial cells, it was found to promote the early-adhesion of S. aureus cells to the biomaterial surface

Metformin reduces airway glucose permeability and hyperglycaemia-induced Staphylococcus aureus load independently of effects on blood glucose

Background Diabetes is a risk factor for respiratory infection, and hyperglycaemia is associated with increased glucose in airway surface liquid and risk of Staphylococcus aureus infection. Objectives To investigate whether elevation of basolateral/blood glucose concentration promotes airway Staphylococcus aureus growth and whether pretreatment with the antidiabetic drug metformin affects this relationship. Methods Human airway epithelial cells grown at air–liquid interface (±18 h pre-treatment, 30 μM–1 mM metformin) were inoculated with 5×105 colony-forming units (CFU)/cm2 S aureus 8325-4 or JE2 or Pseudomonas aeruginosa PA01 on the apical surface and incubated for 7 h. Wild-type C57BL/6 or db/db (leptin receptor-deficient) mice, 6–10 weeks old, were treated with intraperitoneal phosphate-buffered saline or 40 mg/kg metformin for 2 days before intranasal inoculation with 1×107 CFU S aureus. Mice were culled 24 h after infection and bronchoalveolar lavage fluid collected. Results Apical S aureus growth increased with basolateral glucose concentration in an in vitro airway epithelia–bacteria co-culture model. S aureus reduced transepithelial electrical resistance (RT) and increased paracellular glucose flux. Metformin inhibited the glucose-induced growth of S aureus, increased RT and decreased glucose flux. Diabetic (db/db) mice infected with S aureus exhibited a higher bacterial load in their airways than control mice after 2 days and metformin treatment reversed this effect. Metformin did not decrease blood glucose but reduced paracellular flux across ex vivo murine tracheas. Conclusions Hyperglycaemia promotes respiratory S aureus infection, and metformin modifies glucose flux across the airway epithelium to limit hyperglycaemia-induced bacterial growth. Metformin might, therefore, be of additional benefit in the prevention and treatment of respiratory infection

Whole-genome sequencing shows that patient-to-patient transmission rarely accounts for acquisition of Staphylococcus aureus in an intensive care unit

BACKGROUND  Strategies to prevent Staphylococcus aureus infection in hospitals focus on patient-to-patient transmission. We used whole-genome sequencing to investigate the role of colonized patients as the source of new S. aureus acquisitions, and the reliability of identifying patient-to-patient transmission using the conventional approach of spa typing and overlapping patient stay. METHODS Over 14 months, all unselected patients admitted to an adult intensive care unit (ICU) were serially screened for S. aureus. All available isolates (n = 275) were spa typed and underwent whole-genome sequencing to investigate their relatedness at high resolution. RESULTS Staphylococcus aureus was carried by 185 of 1109 patients sampled within 24 hours of ICU admission (16.7%); 59 (5.3%) patients carried methicillin-resistant S. aureus (MRSA). Forty-four S. aureus (22 MRSA) acquisitions while on ICU were detected. Isolates were available for genetic analysis from 37 acquisitions. Whole-genome sequencing indicated that 7 of these 37 (18.9%) were transmissions from other colonized patients. Conventional methods (spa typing combined with overlapping patient stay) falsely identified 3 patient-to-patient transmissions (all MRSA) and failed to detect 2 acquisitions and 4 transmissions (2 MRSA). CONCLUSIONS Only a minority of S. aureus acquisitions can be explained by patient-to-patient transmission. Whole-genome sequencing provides the resolution to disprove transmission events indicated by conventional methods and also to reveal otherwise unsuspected transmission events. Whole-genome sequencing should replace conventional methods for detection of nosocomial S. aureus transmission