A new multiplex PCR for easy screening of methicillin-resistant Staphylococcus aureus SCCmec types I–V

A multiplex PCR with four primer-pairs was designed to identify the five main known SCCmec types. A clear and easily discriminated band pattern was obtained for all five types. The SCCmec type was identified for 98% of 312 clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA). SCCmec type IV was by far the most common SCCmec type among both hospital- and community-acquired MRSA isolates in Denmark.     

Challenges in the identification of methicillin‐resistant Staphylococcus argenteus by routine diagnostics

Current clinical diagnostic procedures have shortcomings in the differentiation of Staphylococcus argenteus from Staphylococcus aureus. This article presents three cases of Staphylococcus argenteus obtained from clinical samples. The initial results from biochemical and molecular methods led to an incorrect identification of the isolates as methicillin‐resistant Staphylococcus aureus. Whole genome sequencing and real‐time PCR targeting the nonribosomal peptide synthetase gene led to their correct identification as methicillin‐resistant Staphylococcus argenteus. The study shows that real‐time PCR can be used to differentiate the two species in routine diagnostics. For purposes of identification based on whole genome sequencing, the MinION portable sequencer is a simple and affordable approach which could be used by many laboratories.     

Molecular characterization of Staphylococcus aureus isolates from skin and soft tissue infections samples and healthy carriers in the Central Slovenia region

Staphylococcus aureus is among the most important human pathogens. It is associated with different infections and is a major cause of skin and soft tissue infections (SSTIs). The aim of our study was to compare S. aureus isolates associated with SSTIs with isolates obtained from healthy carriers in the Central Slovenia region in terms of antimicrobial susceptibility, genetic diversity by clonal complex (CC)/sequence type, spa type, and by toxin gene profiling. In total, 274 S. aureus isolates were collected prospectively by culturing wound samples from 461 SSTI patients and nasal samples from 451 healthy carriers. We have demonstrated high heterogeneity in terms of CCs and spa type in both groups of isolates. The main clone among SSTI strains was Panton–Valentine leukocidin gene (pvl) positive CC121, whereas the main clone among carrier strains was CC45 carrying a large range of toxin genes. The main spa type in both groups was t091. Pvl was more frequently present in SSTI strains (31.2% SSTI vs 3.6% carrier strains) and staphylococcal enterotoxin C was more frequently present in carrier strains (1.6% SSTI vs 17.0% carrier strains). We have also demonstrated that methicillin‐resistant S. aureus was a rare cause (2.8%) of SSTIs in our region.     

Enhanced activity of carvacrol against biofilm of Staphylococcus aureus and Staphylococcus epidermidis in an acidic environment

Carvacrol is an antimicrobial monoterpenic phenol which occurs in many plant essential oils. The aim of this study was to investigate its activity at acidic pH on staphylococcal forming and yet established biofilms, with particular focus to improve its effectiveness on Staphylococcus epidermidis biofilm. The results showed that the subinhibitory doses (1/2, 1/4 and 1/8 MIC) of carvacrol determined a higher reduction of S. epidermidis biofilm formation than that observed at neutral pH. A potentiated inhibitory effect was also observed on established biofilm, carvacrol caused either a strong reduction of biomass (>50%) and bacteria attached to polystyrene (>7 log units). The images of scanning electron microscopy and the gas‐chromatographic analysis support these results. The development of acidic formulations containing carvacrol could be an important tool to control the staphylococcal biofilm in the medical and food environment.     

Molecular characteristics of Staphylococcus aureus associated with chronic rhinosinusitis

The anterior nares have been regarded as the major carriage site of Staphylococcus aureus. From here, the organism can spread to other parts of the body where it might act as harmless commensal or cause mild to severe infections. Nasal sinuses are normally sterile, but in patients with chronic rhinosinusitis (CRS), the finding of S. aureus in maxillary sinus cultures is common. Isolates were obtained from the nares and maxillary sinus of patients with CRS and the nares of healthy controls. A significantly higher frequency of S. aureus was found in nares samples from patients (24/42) compared to controls (16/57) (p = 0.004). There is no consensus regarding whether S. aureus is a relevant pathogen in CRS. A DNA microarray was used to investigate the prevalence of S. aureus virulence genes with focus on staphylococcal enterotoxins, toxic shock syndrome toxin‐1, agr types, and cell wall‐associated proteins. The genotyping of S. aureus isolates revealed only small and non‐significant differences in gene prevalence between isolates collected from patients with CRS and those collected from healthy nasal carriers. This study provides an increased knowledge of the genetic pattern of virulence genes among S. aureus collected in CRS.     

Molecular characterization of nasal methicillin resistant Staphylococcus aureus isolates from workers of an automaker company in southeast Iran

Colonization of methicillin resistant Staphylococccus aureus (MRSA) can occur more commonly in healthy people who live in close together or are in close physical contact with each other. Having knowledge about the molecular characteristics of these strains provides considerable discernment into the epidemiology of this important microorganism. A total of 806 nasal swabs were collected from healthy workers of an automaker company in the southeast of Iran and were analyzed to detect MRSA isolates. Multilocus sequence typing (MLST), spa typing, and detection of staphylococcal cassette chromosome mec (SCCmec) were performed. The presence of genes encoding Panton‐Valentine Leukocidin (PVL) and Arginine Catabolic Mobile Element (ACME) were also investigated. Carriage rate of S. aureus was 20%. Among 10 identified MRSA, no acme was found while high prevalence of pvl (60%) was of great concern. Seven different spa types including five new ones were identified. The most frequent sequence type was the novel one; ST 3373 (n = 3), followed by each of ST22, ST88, ST859 (n = 2) and ST1955 (n = 1). MRSA isolates were clustered into two main clonal complexes; CC22 (n = 6) and CC88 (n = 4). Low genetic diversity with the dominance of CC22, SCCmecIV was found. Distribution of previously found hospital‐associated MRSA was demonstrated among our isolates.     

Presence of arginine catabolic mobile element among community‐acquired meticillin‐resistant staphylococcus aureus is linked to a specific genetic background

The prevalence of arginine catabolic mobile element (ACME) among diverse and heterogeneous community‐associated methicillin‐resistant Staphylococcus aureus community‐associated Methicillin‐resistant S. aureus (CA‐MRSA) (n = 114) in a low‐endemic area, i.e. Sweden, was investigated. Among the CA‐MRSA, represented by 47 different spa types, ACME was only found in 10 isolates with a common genetic background [t008, SCCmec type IV, Panton‐Valentine leukocidin (PVL) positive, and indistinguishable or closely related pulsed‐field gel electrophoresis (PFGE)‐patterns] corresponding to USA300. This strain does not seem to be established in our area as most of the patients contracted the CA‐MRSA abroad. Presence of ACME does not seem to be associated with colonization, long‐term carriership, or intra‐familiar transmission in a higher extent than CA‐MRSA in general.     

Ability of the GENSPEED® MRSA test kit to detect the novel mecA homologue mecC in Staphylococcus aureus

The aim of this study was to evaluate the GENSPEED^® MRSA Test Kit that in addition to the traditional MRSA gene mecA, also incorporates a probe for detection of the newly described mecA homologue mecC. So far only one commercial system is able to detect this new gene. The specific objective was to evaluate the ability of the kit to detect and separate Stapyhylococcus aureus containing either mecA, mecC or no methicillin‐resistance determinant. Ninety‐five MRSA isolates from humans were included in the test and additional mecC‐positive isolates from animals and methicillin‐sensitive S. aureus were tested. The kit demonstrated 100% sensitivity and 100% specificity compared to a standard PCR method. The kit provides the ability to perform rapid and reliable detection of both mecA‐MRSA and mecC‐MRSA.     

Membrane‐active peptide PV3 efficiently eradicates multidrug‐resistant Pseudomonas aeruginosa in a mouse model of burn infection

The aim of this study was to evaluate the topical bactericidal activity of peptide PV3 against a MDR isolate of Pseudomonas aeruginosa in a mouse model of burn infection. The structural analysis of PV3 by circular dichroism spectroscopy indicated a low peptide helical content in water, whereas a high helical content was observed in the presence of the more hydrophobic 50% (v/v) trifluoroethanol/water buffer. A confocal microscopy analysis indicated that the main action of PV3 occurred at the membrane of bacteria. Peptide PV3 exhibited superior in vitro anti‐Pseudomonas activity and killing kinetics as compared with doripenem. A single dose of the topically applied peptide PV3 (4 × MBC, 120 min) was found to be sufficient to eradicate MDRP. aeruginosa in a bacterially infected mouse burn wound model, whereas doripenem (4 × MBC) failed to eradicate the initial inoculum. This indicates a potent and fast PV3‐associated bactericidal activity, contrary to doripenem. An in‐depth analysis of mouse skin by histopathology revealed that peptide PV3 (4 × MBC) did not induce any topical skin toxicity. Overall, the data strongly suggest that peptide PV3 might be a potent candidate antimicrobial agent active on antibiotic‐resistant isolates of pathogenic bacteria.     

Molecular characterization and antibiotic resistance of clinical isolates of methicillin‐resistant Staphylococcus aureus obtained from Southeast of Iran (Kerman)

Staphylococcus aureus infections, particularly infections caused by methicillin‐resistant S. aureus (MRSA) strains, are emerging as a major public health problem. The aim of this study was to determine the prevalence of MRSA, antibiotic resistance profile and staphylococcal cassette chromosome mec (SCCmec) type of MRSA isolates obtained from clinical samples. Totally, 162 S. aureus isolates were obtained from clinical samples at three university hospitals in Kerman, Iran from March 2011 to February 2012. All isolates were identified as S. aureus by phenotypic methods and confirmed by PCR amplification of the nuc gene . MRSA isolates were screened by phenotypic tests and confirmed by presence of mecA gene. The minimum inhibitory concentrations (MICs) of the MRSA isolates against antibacterial agents were determined by E‐test. All isolates were analyzed by PCR for the presence of mecA and pvl genes. SCCmec typing of MRSA isolates was performed by multiplex PCR assay. Strain typing was carried out with REP‐PCR. Using mecA gene PCR and phenotypic methods, 56.8% of the isolates were identified as MRSA. All MRSA isolates were susceptible to vancomycin and linezolid. The sensitivity of MRSA isolates to trimethoprim‐sulfamethoxazole, clindamycin, ciprofloxacin, gentamicin, and erythromycin was 70.66, 66.53, 42.4, 38.05, and 29.35%, respectively. The most frequent SCCmec types were type III (48.31%) followed by type V (19.1%), type I (16.85%), and type IV (3.37%). The pvl gene was detected in 3.08% of isolates (two MRSA and three MSSA isolates). REP‐PCR typing divided the 92 MRSA isolates into 10 distinct clusters. Our results indicate that vancomycin and linezolid are the most effective antibacterial agents against MRSA isolates and SCCmec type III is predominant in MRSA strains in this area.     

Rapid identification of Staphylococcus aureus directly from positive blood culture media using quantum dots as fluorescence probes

The subcultivation of positive blood cultures for organism identification is a time‐consuming process. We explored the use of fluorescent quantum dots (QDs) as probes for the improved detection and identification of Staphylococcus aureus directly from positive blood culture media. Immunofluorescence probes were synthesized by linking biotin‐conjugated QDs to streptavidin‐conjugated IgG molecules. This QDs‐IgG complex is capable of binding anti‐SPA antibodies which specifically bind to a cell surface protein of S. aureus. The specificity of the method was confirmed using reference strains. Among 372 positive blood cultures, 73 containing gram‐positive cocci in clusters were investigated. Relative to the standard culture method, the immunofluorescence assay showed 90.5% (19/21) sensitivity and 100% (52/52) specificity for S. aureus. These results suggest that this immunofluorescence assay allows the rapid identification of S. aureus directly from specimens.     

No associations established between single nucleotide polymorphisms in human Toll‐like receptor 2 and Toll‐interacting protein and Staphylococcus aureus bloodstream infections

Staphylococcus aureus bloodstream infections (SABSI) are associated with high morbidity and mortality. The Toll‐like receptor 2 (TLR2) and Toll‐interacting protein (TOLLIP) are important in recognition and regulation of human innate immunity response to S. aureus. Single nucleotide polymorphisms (SNPs) in the TLR2 and TOLLIP encoding genes have been associated with disease, including BSI. The aim of this study was to examine potential associations between a selection of SNPs in the genes encoding TLR2 and TOLLIP, and predisposition, severity, and outcome of SABSI. All patients ≥18 years of age with at least one S. aureus positive blood culture collected from March 2011 through February 2014 at Akershus University Hospital, Lørenskog, Norway, were considered for inclusion. Patients attending elective orthopaedic surgery (total hip and knee replacements, lumbar surgery) served as a control group. The TLR2 Arg753Gln, TLR2 Pro631His, TOLLIP rs5743942, and rs5743867 polymorphisms were analysed using TaqMan SNP Genotyping Assays. A total of 209 SABSI patients and 295 controls were included. The TLR2 Arg753Gln and TLR2 Pro631His polymorphisms were infrequent with no homozygotes and <10% heterozygotes. The included TLR2 and TOLLIP polymorphisms were not associated with susceptibility to SABSI, severity, 30‐day all‐cause mortality, or SABSI caused by the clonal complex 30 (CC30) genotype.     

Protease/antiprotease network in allergy: The role of Staphylococcus aureus protease‐like proteins

Staphylococcus aureus is being recognized as a major cofactor in atopic diseases such as atopic dermatitis, chronic rhinosinusitis with nasal polyps, and asthma. The understanding of the relationship between S aureus virulence factors and the immune system is continuously improving. Although the precise mechanism of the host's immune response adaptation to the variable secretion profile of S aureus strains continues to be a matter of debate, an increasing number of studies have reported on central effects of S aureus secretome in allergy. In this review, we discuss how colonization of S aureus modulates the innate and adaptive immune response, thereby predisposing the organism to allergic sensitization and disrupting immune tolerance in the airways of patients with asthma and chronic rhinosinusitis with nasal polyps. Next, we provide a critical overview of novel concepts dealing with S aureus in the initiation and persistence of chronic rhinosinusitis with nasal polyps and asthma. The role of the S aureus serine protease‐like proteins in the initiation of a type 2 response and the contribution of the IL‐33/ST2 signaling axis in allergic responses induced by bacterial allergens are discussed.     

Predominance of staphylococcal cassette chromosome mec (SCCmec) type IV among methicillin-resistant Staphylococcus aureus (MRSA) in a Swedish county and presence of unknown SCCmec types with Panton-Valentine leukocidin genes

Methicillin-resistant Staphylococcus aureus (MRSA) is an established nosocomial pathogen, but has recently begun to appear in the community. The clones in the community may not have originated in the hospital setting, and are referred to as community-acquired MRSA (CA-MRSA). Resistance to methicillin is mediated by the gene mecA, which is carried by the mobile genetic element staphylococcal cassette chromosome mec (SCCmec). SCCmec typing (I-IV) of all clinical isolates of MRSA (n = 92) from 1987 to 2004 in Orebro County, Sweden, was performed by real-time LightCycler PCR to detect the essential genetic components mecA, mecR1, IS1272, ccrA and ccrB. Forty-one isolates harboured type IV SCCmec, of which ten could be classified further as subtype IVa, and 27 as subtype IVc. No isolates belonged to subtype IVb, but four isolates could not be subtyped, and may be examples of novel type IV SCCmec subtypes. Thirty-five MRSA isolates, assigned to six different pulsotypes by pulsed-field gel electrophoresis, did not belong to SCCmec types I-IV. The Panton-Valentine leukocidin (PVL) genes were identified in two of these pulsotypes. Only SCCmec type IV has been associated previously with the PVL toxin, but the results suggest that new PVL-positive clones with novel SCCmec types may be arising and disseminating in the community.     

Staphylococcus lugdunensis endocarditis following vasectomy – report of a case history and review of the literature

Staphylococcus lugdunensis is a coagulase‐negative Staphylococcus (CoNS), and part of the normal skin flora. The bacterium is an emerging pathogen that, unlike other CoNS, resembles coagulase‐positive Staphylococcus aureus infections in virulence, tissue destruction, and clinical course. We report a fatal case following minor surgery. The frequency of S. lugdunensis infections has probably been underestimated and under‐reported in the past as few clinical laboratories routinely identify coagulase‐negative Staphylococci.     

Comparative analysis of the virulence characteristics of epidemic methicillin–resistant Staphylococcus aureus (MRSA) strains isolated from Chinese children: ST59 MRSA highly expresses core gene–encoded toxin

This study aims to investigate the prevalence of a novel cell wall‐anchored protein gene, sasX, and to obtain information on the genetic basis for the pathogenic potential of the MRSA strains isolated from Chinese children. The molecular and virulence characteristics of the clinical strains were analyzed. Twenty‐two sequence types (STs) were obtained, with six epidemic clones ST59, ST239, ST1, ST910, ST88, and ST338 accounting for 35.8, 22, 6.6, 6.6, 5.3, and 4.1% respectively. The expression levels of hla, psmα, and RNAIII were higher in ST59 than in other STs (p < 0.05). The sasX gene was detected in 26 (10.7%) MRSA isolates. ST239‐MRSA‐SCCmecIII‐t037 (61.5%) was the predominant sasX‐positive MRSA clone. The expressions of PSMα and RNAIII were higher in sasX‐positive ST239 isolates than in sasX‐negative ST239 ones (p < 0.01). Notably, the percentage of invasive infection in infections caused by sasX‐positive ST239 MRSA was higher than that by sasX‐negative ST239 MRSA (p = 0.008). This study indicated that ST59 was the predominant clone in the MRSA isolates obtained from Chinese children and might have stronger pathogenic potential. The prevalence of the sasX gene in the MRSA isolates from children was relatively low. Furthermore, the sasX gene might be related to the expressions of PSMα and RNAIII and infection invasiveness.     

Outbreak of Methicillin‐resistant Staphylococcus aureus in a Hospital Center for Children's and Women's Health in a Swedish County

The objective of this study was to investigate a sudden increase in methicillin‐resistant Staphylococcus aureus (MRSA) cases primarily in one maternity ward at the Center for Children's and Women's Health at Linköping University Hospital, Sweden. Approximately 300 individuals including patients, their family members, and healthcare workers were screened for MRSA. The antibiotic susceptibility was tested and isolates polymerase chain reaction (PCR)‐positive for the mecA gene were spa typed. Isolates with the same antibiogram and spa type were further whole genome sequenced. Compliance to current cleaning and hygiene routines was also controlled, and environmental samples collected. The results showed that a total of 13 individuals were involved in the outbreak. It was caused by a t386 MRSA strain (ST‐1, NCBI‐accession AB505628) with additional resistance to erythromycin and clindamycin. All cases were epidemiologically connected to the index patient, who had recently emigrated from a high‐endemic area for MRSA. With improved cleaning and better compliance to basic hygiene routines, no further cases were reported. This study demonstrates how rapid an MRSA strain can disseminate in a ward with susceptible patients and insufficient cleaning and hygiene. For a better control of MRSA, clinical cultures and screening samples need to be obtained early and more extensively than according to the current recommendations.