Evaluation of the TPX MRSA assay for the detection of methicillin-resistant Staphylococcus aureus

The aim of this study was to evaluate a new type of assay for the phenotypic detection of methicillin-resistant Staphylococcus aureus (MRSA). The assay is based on a point-of-care compatible two-photon excitation fluorescence detection technology (TPX). A collection of 243 epidemic MRSA isolates was tested in addition to 138 sporadic MRSA and 101 negative control strains. The assay proved to be both sensitive (97.9%) and specific (94.1%) in the identification of MRSA, with adequate positive (98.4%) and negative (92.2%) predictive values. The time required for obtaining a positive test result was less than 14 h for 99.0% of the MRSA true-positive samples. After a test run, the selectively enriched reaction mixtures may be recovered and further studied by molecular or standard phenotypic methods. The main benefits of the TPX methodology include a simple assay procedure, low reagent consumption, and a high-throughput capacity.     

Adapting spa typing for national laboratory-based surveillance of methicillin-resistant Staphylococcus aureus

Laboratory-based surveillance of methicillin-resistant Staphylococcus aureus (MRSA) monitors the baseline occurrence of different genotypes and identifies strains and transmission chains responsible for outbreaks. The consequences of substituting pulsed-field gel electrophoresis (PFGE) with spa typing as a first-line typing method were analyzed by typing 589 strains isolated between 1997 and 2006, with a focus on both short- and long-term correspondence between the PFGE and spa typing results. The study, covering these ten years, included all Finnish MRSA blood isolates and representatives of the two most prevalent MRSA strains (PFGE types FIN-4 and FIN-16) in Finland. In addition, all sporadic isolates from 2006 were included. spa typing was more expensive but approximately four times faster to perform than PFGE. Nearly 90% of FIN-4 and FIN-16 isolates showed consistent spa types, t172 and t067, respectively. spa typing predicted the PFGE result of the blood isolates by a Wallace coefficient of 0.9009, recognized internationally successful strains (t041, t067) to be common also in Finland, and identified a separate cluster of isolates, also related in time and place among the FIN-4 strains. Additional typing by another method was needed to provide adequate discrimination or to characterize isolates with a newly recognized spa type in Finland.     

Performance evaluation of a modified chromogenic medium, ChromID MRSA New, for the detection of methicillin-resistant Staphylococcus aureus from clinical specimens

A novel chromogenic medium for the detection of methicillin-resistant Staphylococcus aureus (MRSA), ChromID MRSA New, was evaluated and compared with the original ChromID MRSA agar, using 355 consecutive screening specimens from nose (120), throat (121) and perineum (114). The specimens were collected with an E-swab and inoculated within 24 hours onto both ChromID MRSA New and on ChromID MRSA. ChromID MRSA New was more sensitive than ChromID MRSA in detecting MRSA after 24 hours of incubation (94.3% versus 81.4%; p < 0.05). With the ChromID MRSA New, processing time is reduced from 48 h to 24 h and confirmation of the resistance to methicillin is redundant.     

Molecular epidemiological analysis of methicillin-resistant Staphylococcus aureus isolates from Chinese pediatric patients

To investigate the molecular epidemiological analysis of methicillin-resistant Staphylococcus aureus (MRSA) isolates from five pediatric hospitals in China. Seventy-three MRSA isolates were analyzed by a combination of different genotyping methods, including multilocus sequence typing (MLST), SCCmec and spa typing. Panton-Valentine Leukocin (PVL) gene was also detected. The prevalent strains were ST239-MRSA-III and ST1-MRSA clones in the northern region; ST239-MRSA-III, ST910-MRSA-IV and ST88-MRSA in the eastern region; and ST59-MRSA in the southern region. Only the ST910-MRSA-IV clone has been found in China until now, and it is closely related to ST30-MRSA-IV. All MRSA isolates were found to be resistant to penicillin and azithromycin, and multidrug resistance was observed. The cases of necrotic pneumonia, severe skin and subcutaneous tissue infection and lymphadenitis resulted from PVL gene-positive MRSA. There were several novel genetic types of MRSA. Antimicrobial susceptibility tests showed high resistance of many antimicrobials and multiple drugs.     

Bacteriophage therapy for Staphylococcus aureus bacteremia in streptozotocin-induced diabetic mice

The protective effect of bacteriophage was assessed against experimental Staphylococcus aureus lethal bacteremia in streptozotocin (STZ) induced-diabetic and non-diabetic mice. Intraperitoneal administrations of S. aureus (RCS21) of 2 × 10⁸ CFU caused lethal bacteremia in both diabetic and non-diabetic mice. A single administration of a newly isolated lytic phage strain (GRCS) significantly protected diabetic and non-diabetic mice from lethal bacteremia (survival rate 90% and 100% for diabetic and non-diabetic bacteremic groups versus 0% for saline-treated groups). Comparison of phage therapy to oxacillin treatment showed a significant decrease in RCS21 of 5 and 3 log units in diabetic and non-diabetic bacteremic mice, respectively. The same protection efficiency of phage GRCS was attained even when the treatment was delayed up to 4 h in both diabetic and non-diabetic bacteremic mice. Inoculation of mice with a high dose (10¹⁰ PFU) of phage GRCS alone produced no adverse effects attributable to the phage per se. These results suggest that phages could constitute valuable prophylaxis against S. aureus infections, especially in immunocompromised patients.     

Prevalence of nasal methicillin-resistant Staphylococcus aureus colonization in healthcare workers in a Western Australian acute care hospital

Due to a longstanding comprehensive “search and destroy policy”, methicillin-resistant Staphylococcus aureus (MRSA) is not endemic in Western Australian (WA) acute care hospitals. As the prevalence of MRSA in the community has increased, healthcare workers (HCW) are at risk of importing MRSA into hospitals. We aimed to determine the prevalence of and risk factors for nasal MRSA colonization in our HCW population. A period prevalence study was conducted at an 850-bed tertiary hospital. Basic demographics and a nasal swab were obtained. A total of 1,542 HCWs employed in our centre were screened for MRSA, of whom 3.4% (n = 52) were colonized. MRSA colonization was more common in patient care assistants (6.8%) and nurses (5.2%) than in allied health professionals (1.7%) and doctors (0.7%) (p < 0.01). Working in “high-risk” wards that cared for MRSA colonized/infected patients was the strongest risk factor for HCW MRSA colonization (p < 0.001). ST1-IV and ST78-IV (the most common community clones in the region) were the most frequently identified clones. In conclusion, MRSA colonization of HCWs occurs primarily in HCWs caring for patients colonized or infected with MRSA. Surveillance screening of HCWs should be regularly performed on wards with patients with high MRSA colonization prevalence to prevent further spread in the hospital.     

The effectiveness of methicillin-resistant Staphylococcus aureus colonisation screening in asymptomatic healthcare workers in an Irish orthopaedic unit

Methicillin-resistant Staphylococcus aureus (MRSA) infections are associated with increased mortality, costs and length of stay compared to non-MRSA infections. This observational 4-year study analyses the impact of screening and treating orthopaedic healthcare workers for MRSA colonisation. A total of 1,011 swabs were taken from 566 healthcare workers. Positive healthcare workers were treated with topical mupirocin to both anterior nares. The prevalence of MRSA colonisation on initial testing was 4.77%. The rate of positive MRSA colonisation of those tested on more than one occasion fell from 5.88% to 2.71% (p = 0.055) on subsequent screening. All healthcare workers receiving treatment were successfully cleared of colonisation; however, some required more than one course of treatment. These results show that there could be a role for screening and treating orthopaedic staff for MRSA colonisation as part of a strategy to reduce the prevalence of MRSA infections in orthopaedic units.     

Epidemiology of Streptococcus pneumoniae and Staphylococcus aureus colonization in healthy Venezuelan children

Streptococcus pneumoniae and Staphylococcus aureus cause significant morbidity and mortality worldwide. We investigated both the colonization and co-colonization characteristics for these pathogens among 250 healthy children from 2 to 5 years of age in Merida, Venezuela, in 2007. The prevalence of S. pneumoniae colonization, S. aureus colonization, and S. pneumoniae–S. aureus co-colonization was 28%, 56%, and 16%, respectively. Pneumococcal serotypes 6B (14%), 19F (12%), 23F (12%), 15 (9%), 6A (8%), 11 (8%), 23A (6%), and 34 (6%) were the most prevalent. Non-respiratory atopy was a risk factor for S. aureus colonization (p = 0.017). Vaccine serotypes were negatively associated with preceding respiratory infection (p = 0.02) and with S. aureus colonization (p = 0.03). We observed a high prevalence of pneumococcal resistance against trimethoprim–sulfamethoxazole (40%), erythromycin (38%), and penicillin (14%). Semi-quantitative measurement of pneumococcal colonization density showed that children with young siblings and low socioeconomic status were more densely colonized (p = 0.02 and p = 0.02, respectively). In contrast, trimethoprim–sulfamethoxazole- and multidrug-resistant-pneumococci colonized children sparsely (p = 0.03 and p = 0.01, respectively). Our data form an important basis to monitor the future impact of pneumococcal vaccination on bacterial colonization, as well as to recommend a rationalized and restrictive antimicrobial use in our community.     

Evaluation of a fourth-generation latex agglutination test for the identification of Staphylococcus aureus

 In this study, we evaluated a fourth-generation agglutination assay (Staph Plus; DiaMondiaL[DML]) for the rapid identification of Staphylococcus aureus. First, comparison with three third-generation assays (Slidex Staph Plus, bioMérieux; Staphaurex Plus, Murex Diagnostics; Pastorex Staph-Plus, Sanofi Diagnostics Pasteur) was performed on a predefined strain collection: 265 coagulase-negative staphylococci (CNS), 266 methicillin-resistant S. aureus (MRSA) and 262 methicillin-susceptible S. aureus (MSSA) strains (“strain study”). Second, patient material-derived strains (883 CNS, 847 MSSA and 135 MRSA) were tested concurrently with both the DML and Slidex assays (“daily practice study”). In the strain study, the overall sensitivity and specificity of the DML, Slidex, Staphaurex and Pastorex assays were 99.2% and 100%, 98.1% and 100%, 95.2% and 100%, and 98.2% and 98.8%, respectively. Using the respective tests, the result was indeterminate in 0.0%, 0.6%, 0.4% and 1.5% of the strains. Overall, the sensitivity of the DML and Slidex assays were comparable in both sub-studies. However, in MRSA strains, the sensitivity of the DML assay was significantly lower than the Slidex assay. The specificity of the Slidex assay was significantly higher than the DML assay. However, the percentage of indeterminate results was much higher for the Slidex than the DML assay. In conclusion, the presumptive identification of S. aureus by the DML assay proved to be equal to third-generation latex agglutination assays.     

Comparison of Austrian, Hungarian and Macedonian methicillin-resistant and methicillin-sensitive Staphylococcus aureus strains in relation to prevalence of cytotoxin genes

Cytotoxin genes in 128 Austrian (AT) MSSA, 48 MRSA, 94 Hungarian (HU) MSSA, 110 MRSA and 67 Macedonian (MK) MSSA, 81 MRSA strains were examined. The presence of alfa-haemolysin gene (hla) was more common in HU MSSA strains compared to AT and MK (99%, 86%, 72%: p < 0.001). AT and MK MRSA harboured hlb genes more frequently compared to HU (60%, 62%, 33%: p < 0.001). HU and MK MRSA strains carried gamma-haemolysin gene (hlg) in higher percentage in contrast to AT (88%, 83%, 69%: p = 0.01). Haemolysin gamma-variant gene (hlgv) was more prevalent in HU MSSA compared to AT and MK (84%, 56%, 69%: p < 0.001). Panton–Valentine leukocidin genes were found only in AT, HU, MK MSSA and MK MRSA in 2.3%, 4%, 1.5% (p = 0.53) and 1% (p = 0.38), respectively. The 3-gene combination pattern comprising of hla, hlg and hld genes showed increased prevalence among AT MSSA compared to HU (27%, 11%: p < 0.001). The 4-gene pattern composed of hla, hlg, hlgv and hld genes was significantly characteristic for HU MRSA in contrast to AT and MK MRSA (56%, 12.5%, 27%: p < 0.001). Frequency of certain cytotoxin genes and combinations differed significantly in Staphylococcus aureus strains according to geographical origin and methicillin-resistance.     

In vitro antibacterial activity of porous TiO2–Ag composite layers against methicillin-resistant Staphylococcus aureus

The aim of this study was the synthesis of a porous TiO₂-Ag composite coating and assessment of its in vitro bactericidal activity against methicillin-resistant Staphylococcus aureus. The coating was produced by plasma electrolytic oxidation of Ti–6Al–7Nb medical alloy in a calcium acetate/calcium glycerophosphate electrolyte bearing Ag nanoparticles. Following oxidation, the surface of the titanium substrate was converted into the corresponding oxide (TiO₂) bearing Ca and P species from the electrolyte. In addition, Ag was detected associated with particles present in the oxide layers. The coatings revealed a porous interconnected structure with pores up to 3 μm in size, a threefold increase in roughness and improved wettability relative to the non-oxidized specimens. The composite TiO₂-Ag coating showed complete killing of methicillin-resistant S. aureus within 24 h in all culture conditions, whereas a 1000-fold increase in bacterial numbers was recorded with the ground titanium specimens and the samples oxidized in the absence of Ag nanoparticles.     

Cloning and expression in Escherichia coli and Staphylococcus aureus of the beta-lysin determinant from Staphylococcus aureus: evidence that bacteriophage conversion of beta-lysin activity is caused by insertional inactivation of the beta-lysin determinant

The beta-lysin determinant (Hlb) from Staphylococcus aureus CN6708 was cloned in Escherichia coli K-12 using the bacteriophage replacement vector lambda L47.1. The Hlb determinant was localised to a 1250 base pair DNA sequence by cloning fragments from a Hlb+ recombinant phage into the plasmid vectors pACYC184 and pBR322 in E. coli K-12, and by the subsequent construction and analysis of several sub-clones, in vitro deletion and Tn5 insertion mutations. E. coli cells harbouring Hlb+ plasmids expressed readily detectable levels of beta-lysin and sphingomyelinase activity, which were located in the cytoplasm. Two polypeptides of molecular weight 38,000 and 33,000 which were encoded by the Hlb determinant were detected in E. coli minicells, but only the 33,000 dalton protein was detected in immunoblotting experiments with specific anti-beta-lysin serum. Hybridisation analysis with probes made from the cloned Hlb determinant and from DNA of the staphylokinase-converting phage phi 13, indicated that bacteriophage conversion of S. aureus to loss of beta-lysin activity is due to insertion of phi 13 DNA into or adjacent to the beta-lysin determinant. A shuttle plasmid was used to transfer the cloned Hlb determinant into a beta-lysin negative strain of S. aureus where the wild-type chromosomal determinant was inactivated by lysogenic conversion. Beta-lysin activity was readily detected in supernatants of S. aureus harbouring the cloned determinant.     

Molecular cloning and expression of the epidermolytic toxin A gene of Staphylococcus aureus

The gene coding for serotype A of epidermolytic (exfoliative) toxin has been cloned from Staphylococcus aureus in Escherichia coli phage lambda and plasmid vectors. The coding sequence for eta was localised by subcloning and transposon Tn5 mutagenesis experiments. The eta gene was probably expressed from its natural promoter in E. coli. The protein synthesised in E. coli was located predominantly in the periplasm. It was immunochemically indistinguishable from the toxin purified from S. aureus culture supernatants and had the same molecular weight. Furthermore, subcutaneous injection of this material caused epidermal splitting (the Nikolsky reaction) showing that it was biologically active. An eta shuttle plasmid was transformed into protoplasts of S. aureus. The level of expression of toxin in strain 8325-4 was shown to be dependent on the integrity of the agr gene which is known to be required for the expression of several exoproteins.     

High prevalence of edin-C encoding RhoA-targeting toxin in clinical isolates of Staphylococcus aureus

Staphylococcus aureus, a major causative agent of human infection, produces a large array of virulence factors, including various toxins. Among them, the host RhoA GTPase ADP-ribosylating EDIN toxins are considered as potential virulence factors. Using the polymerase chain reaction (PCR) assay, we analyzed the virulence profile of 256 S. aureus isolates from various clinical sites of infections. We developed specific primers to detect the three isoforms of edin-encoding genes. We found a prevalence of 14% (36 bacteria) of edin-encoding genes among these clinical isolates. Strikingly, we found that 90% of all edin-bearing S. aureus isolates carried the type-C allele. Both the spa types and the profile of virulence factors of these edin-positive isolates are highly variable. Notably, we show for the first time that edin-C-positive isolates were more frequently recovered from deep-seated infections than other types of infections. Our present work, thus, strongly suggests that the presence of edin-C is a risk factor of S. aureus dissemination in tissues and, thus, represents a predictive marker for a pejorative evolution of staphylococcal infections.     

Modulation of Staphylococcus aureus adhesion by biofunctional copolymers derived from polystyrene

Biomaterials-centered infections are serious complications associated with the use of implants. The infection risk of biomaterials varies between different materials and is determined by the chemical composition of materials, the host proteins and the type of bacteria. In this study we measured the initial adhesion of Staphylococcus aureus onto polystyrene derivatives containing carboxylate and sulfonate groups. Five polymers were synthesized and characterized. We studied the role of the host protein fibronectin in promoting adhesion of Staphylococcus aureus. Fibronectin adsorption was comparable on all the tested polymers (pKd=7.2±0.2) whereas bacterial adhesion was dependent on surfaces chemical compositions. Polymers substituted with sulfonate groups showed the most important inhibition of initial bacterial adhesion.     

Binding of a Staphylococcus aureus bone pathogen to type I collagen

We contrasted the collagen-binding potential of the experimental osteomyelitis pathogen, Staphylococcus aureus strain SMH, to several other strains. These included Cowan 1 (binder), Wood 46 (non-binder) and six capsular variants. These measurements were made using an ¹²⁵I-collagen binding assay. Formalin-killed S. aureus SMH strongly bound commercial type I iodinated collagen (dissociation contant, K_d = 2 × 10⁻⁹ m). The extent of binding was similar to Cowan 1. Binding was saturable and not inhibited by 100 m solutions of -glucose, -galactose, -mannose, methyl-α- -fucopyranoside, -hydroxyproline or -glycine. -lactose gave moderate inhibition of binding to collagen, and -fucose was strongly inhibitory. Trypsinized SMH did not bind collagen. None of four Ruthenium-red-staining staphylococci (encapsulated) avidly bound type I collagen. The encapsulated Smith strain, for example, did not bind to collagen but its capsule-negative variant, Smith compact, showed extensive binding. Three of five non-encapsulated S. aureus were strong collagen binders. These data suggest that the prototype bone pathogen binds to the major protein component of bone's extracellular matrix. Collagen-binding is promoted by protein adhesin(s), not capsule. The latter, in fact, appeared to interfere with this interaction. Binding was inhibited by solutions containing the simple monosaccharide, -fucose.