Sensitivity and Specificity of an Improved Rapid Latex Agglutination Test for Identification of Methicillin-Sensitive and -Resistant Staphylococcus aureus Isolates

The performance of a second-generation rapid agglutination kit, Slidex Staph Plus (SSP; bioMérieux), was compared to those of the Slidex Staph (SS; bioMérieux), Staphaurex (SRX; Murex Diagnostics), and BBL Staphyloslide (BBL; Becton Dickinson) kits by using 508 clinical isolates composed of 150 methicillin-sensitive Staphylococcus aureus (MSSA) organisms, 154 methicillin-resistant S. aureus (MRSA) organisms, and 204 non-S. aureus Staphylococcus spp. Of the 508 isolates tested, 75% were fresh clinical isolates, with the remainder taken from five different freezer collections. All four agglutination tests had comparable sensitivities for MSSA and MRSA. However, the SS kit was significantly less specific (93.1%) than the three other tests (P > 0.05, McNemar test). These results demonstrate that the new rapid latex agglutination kit, SSP, was more specific for the identification of S. aureus than the previous version and performed comparably to the SRX and BBL kits.     

Methicillin-resistant Staphylococcus aureus nasal carriage among injection drug users: six years later

A survey in 2000 to detect methicillin-resistant Staphylococcus aureus (MRSA) colonization in Vancouver downtown east side injection drug users (IDUs) revealed an MRSA nasal colonization incidence of 7.4%. This is a follow-up study to determine the current prevalence of MRSA colonization and to further characterize the isolates and risk factors for colonization. In this point prevalence study of MRSA nasal carriage among IDUs, nasal swabs were cultured to detect S. aureus. Isolates were studied for their antimicrobial susceptibility patterns and the presence of mecA and Panton-Valentine leukocidin (PVL) genes and by pulsed-field gel electrophoresis (PFGE). S. aureus was isolated from 119 of 301 (39.5%) samples; three (2.5%) participants had both methicillin-sensitive S. aureus (MSSA) and MRSA, resulting in 122 isolates. Of these, 54.1% were MSSA and 45.9% were MRSA, with an overall MRSA rate of 18.6%. USA-300 (CMRSA-10) accounted for 75% of all MRSA isolates; 25% were USA-500 (CMRSA-5). None of the USA-500 isolates were positive for PVL; 41 (97.6%) USA-300 isolates contained PVL. One MSSA isolate, from an individual also carrying USA-300, was positive for PVL. The PFGE pattern of this MSSA isolate was related to that of the MRSA strain. The antibiograms of USA-300 compared to USA-500 isolates showed 100% versus 7.1% susceptibility to trimethoprim-sulfamethoxazole (TMP-SMX) and 54.8% versus 7.1% susceptibility to clindamycin. MRSA nasal colonization in this population has increased significantly within the last 6 years, with USA-300 replacing the previous strain. Most of these strains are PVL positive, and all are susceptible to TMP-SMX.     

Predominance of methicillin-resistant Staphylococcus aureus among pathogens causing skin and soft tissue infections in a large urban jail: risk factors and recurrence rates

In the 1990s, community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) strains emerged as pathogens outside of the health care environment. Epidemic foci of CA-MRSA infections were reported in jails and prisons, but risk factors for MRSA infection there are not known. All skin and soft tissue infections (SSTIs) cultured in the Cook County Jail in March 2004 to August 2005 were reviewed. Demographic and clinical risk factors were compared among patients with methicillin-susceptible S. aureus (MSSA) SSTIs and those with MRSA SSTIs. Antibiotic susceptibilities were recorded, and we performed multilocus sequence typing on a sample of MRSA isolates. There were 378 SSTIs from different patients requiring culture, of which 240 (63.5%) were of MRSA and 43 (11.4%) were of MSSA; 84.8% of S. aureus isolates were MRSA. MRSA- and MSSA-infected patients were similar with regard to age, gender, ethnicity, previous exposure to the jail, and comorbidities. In the 12 months prior to the index culture, MRSA patients were more likely to have received a β-lactam antibiotic (25% versus 9%; P = 0.02). Among 26 MRSA strains, 24 (92%) had the sequence type 8 (ST8) genotype. Within 6 months, 14% (95% confidence interval, 8.7% to 22.3%) of MRSA SSTI patients in the jail had a recurrent SSTI compared with 8.8% (95% confidence interval, 2.1% to 32.6%) of MSSA SSTI patients (P = 0.004). MRSA is the predominant cause of SSTIs requiring culture in the jail. Few risk factors differentiated MRSA from MSSA SSTIs, and detainee patients with MRSA SSTIs are at high risk for recurrent SSTIs.     

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.     

Rapid differentiation of methicillin-resistant Staphylococcus aureus and methicillin-susceptible Staphylococcus aureus from blood cultures by use of a direct cefoxitin disk diffusion test

A total of 276 blood culture bottles with Staphylococcus aureus were tested by direct cefoxitin disk diffusion testing; 105 (38.1%) had zone sizes of ≤17 mm (all 105 had methicillin-resistant S. aureus [MRSA]), 18 (6.5%) had zone sizes that measured 18 mm (17 had MRSA and 1 had methicillin-susceptible S. aureus [MSSA]), 8 (2.9%) had zone sizes that measured 19 mm (6 had MRSA and 2 had MSSA), 8 (2.9%) had zone sizes that measured 20 mm (6 had MRSA and 2 had MSSA), and 137 (49.6%) had zone sizes of ≥21 mm (all 137 had MSSA). Detection of MRSA/MSSA in blood cultures could be reported 10 to 24 h earlier for 88% of cultures with total accuracy.     

Associations between the genotypes of Staphylococcus aureus bloodstream isolates and clinical characteristics and outcomes of bacteremic patients

We investigated associations between the genotypic and phenotypic features of Staphylococcus aureus bloodstream isolates and the clinical characteristics of bacteremic patients enrolled in a phase III trial of S. aureus bacteremia and endocarditis. Isolates underwent pulsed-field gel electrophoresis, PCR for 33 putative virulence genes, and screening for heteroresistant glycopeptide intermediate S. aureus (hGISA). A total of 230 isolates (141 methicillin-susceptible S. aureus and 89 methicillin-resistant S. aureus [MRSA]) were analyzed. North American and European S. aureus isolates differed in their genotypic characteristics. Overall, 26% of the MRSA bloodstream isolates were USA 300 strains. Patients with USA 300 MRSA bacteremia were more likely to be injection drug users (61% versus 15%; P < 0.001), to have right-sided endocarditis (39% versus 9%; P = 0.002), and to be cured of right-sided endocarditis (100% versus 33%; P = 0.01) than patients with non-USA 300 MRSA bacteremia. Patients with persistent bacteremia were less likely to be infected with Panton-Valentine leukocidin gene (pvl)-constitutive MRSA (19% versus 56%; P = 0.005). Although 7 of 89 MRSA isolates (8%) exhibited the hGISA phenotype, no association with persistent bacteremia, daptomycin resistance, or bacterial genotype was observed. This study suggests that the virulence gene profiles of S. aureus bloodstream isolates from North America and Europe differ significantly. In this study of bloodstream isolates collected as part of a multinational randomized clinical trial, USA 300 and pvl-constitutive MRSA strains were associated with better clinical outcomes.     

Characterization of Staphylococcus aureus isolates from nasal cultures collected from individuals in the United States in 2001 to 2004

This study characterizes methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) isolates recovered from nasal cultures of noninstitutionalized individuals in the United States obtained in 2001 to 2004 as part of the National Health and Nutrition Examination Survey. Every tenth MSSA isolate and all MRSA isolates were typed by pulsed-field gel electrophoresis (PFGE), screened for multiple toxin genes, and tested for susceptibility to 14 antimicrobial agents. USA200, USA600, and USA900 were the predominant PFGE types among MSSA isolates in both the 2001 to 2002 and the 2003 to 2004 time periods, although they accounted for only 51.3% of 316 MSSA isolates typed in 2001 and 2002 and only 43.4% of 237 MSSA isolates typed in 2003 and 2004. In contrast, USA100, USA800, and USA700 accounted for 80.0% of the 75 MRSA isolates typed in 2001 and 2002, while USA100, USA800, and USA300 accounted for 78.4% of 134 MRSA isolates typed in 2003 and 2004. The proportion of MRSA isolates that were USA300 increased significantly from the first to the second time period (P = 0.03). Most USA200 isolates (both MSSA and MRSA) carried the gene for toxic shock syndrome toxin; however, carriage of the genes encoding Panton-Valentine leukocidin, while common among MRSA of PFGE type USA300, was rare among MSSA USA300 in both time periods. Most MSSA isolates remained susceptible to all antimicrobial agents except erythromycin (79.1 and 76.0% susceptibilities in the 2001 to 2002 and the 2003 to 2004 periods, respectively). In contrast, the proportions of MRSA isolates that were susceptible to chloramphenicol, clindamycin, and erythromycin were lower in 2003 and 2004 than in 2001 and 2002, although none of these differences was statistically significant.     

Genotypic characteristics of Staphylococcus aureus isolates from a multinational trial of complicated skin and skin structure infections

The impact of bacterial genetic characteristics on the outcome of patients with Staphylococcus aureus infections is uncertain. This investigation evaluated potential associations between bacterial genotype and clinical outcome using isolates collected as part of an international phase 2 clinical trial (FAST II) evaluating telavancin for the treatment of complicated skin and skin structure infections (cSSSI). Ninety S. aureus isolates from microbiologically evaluable patients with cSSSI enrolled in the FAST II trial from 11 sites in the United States (56 isolates, or 62%) and 7 sites in South Africa (34 isolates, or 38%) were examined for staphylococcal cassette chromosome mec, agr, and the presence of 31 virulence genes and subjected to pulsed-field gel electrophoresis (PFGE). South African methicillin-susceptible S. aureus (MSSA) isolates were more likely to carry certain virulence genes, including sdrD (P = 0.01), sea (P < 0.01), and pvl (P = 0.01). All 44 (49%) methicillin-resistant S. aureus (MRSA) isolates were from the United States; 37 (84%) were strain USA 300 by PFGE. In the United States, MRSA isolates were more likely than MSSA isolates to carry genes for sdrC (P = 0.03), map/eap (P = 0.05), fnbB (P = 0.11), tst (P = 0.02), sea (P = 0.04), sed (P = 0.04), seg (P = 0.11), sej (P = 0.11), agr (P = 0.09), V8 (P = 0.06), sdrD, sdrE, eta, etb, and see (P < 0.01 for all). MRSA isolates were more often clonal than MSSA isolates by PFGE. Isolates from patients who were cured were significantly more likely to contain the pvl gene than isolates from patients that failed or had indeterminate outcomes (79/84 [94%] versus 3/6 [50%]; P = 0.01). S. aureus strains from different geographic regions have different distributions of virulence genes.     

False-negative test results in the Slidex Staph Plus (bioMérieux) agglutination test are mainly caused by spa-type t001 and t001-related strains

The relative sensitivity of commercial agglutination kits for fast identification of S. aureus is usually given to be about 98%. This reported sensitivity has sometimes been questioned. In this study, three collections of molecularly defined, single-copy strains of S. aureus were used to compare the sensitivities of agglutination-based identification and the MALDI-TOF mass spectrometry-based identification using the Biotyper 2.0 database to a molecularly defined reference method. Clinical isolates (n = 363) of methicillin-susceptible S. aureus (MSSA) and 240 clinical isolates of methicillin-resistant S. aureus (MRSA) were included. In order to rule out a predominance of local MRSA-strains, a collection of 104 pulsed-field-gel electrophoresis divergent MRSA strains were also tested. MALDI-TOF MS using Biotyper database (Bruker) identified all isolates, whereas the Slidex Staph Plus (bioMérieux) detected only 98.0% of the MSSA, 94.5% of the MRSA and only 70.1% of the MRSA of the molecularly divergent strain collection. Interestingly, strains with a false-negative test result in the agglutination methods were mostly spa-type t001 and t001 related. The MALDI-TOF MS based identification can thus be used as an alternative identification method for suspected false-negative results from the agglutination tests, especially if the local prevalence of t001 and t001 related strains is high.     

Phenotypic and Genotypic Characterization of Nosocomial Staphylococcus aureus Isolates from Trauma Patients

Staphylococcus aureus is a major cause of nosocomial infections. During the period from March 1992 to March 1994, the patients admitted to the intensive care unit of the University of Maryland Shock Trauma Center were monitored for the development of S. aureus infections. Among the 776 patients eligible for the study, 60 (7.7%) patients developed 65 incidents of nosocomial S. aureus infections. Of the clinical isolates, 43.1% possessed a polysaccharide type 5 capsule, 44.6% possessed a type 8 capsule, and the remaining 12.3% had capsules that were not typed by the type 5 or type 8 antibodies. Six antibiogram types were noted among the infection-related isolates, with the majority of the types being resistant only to penicillin and ampicillin. It was noted that the majority of cases of pneumonia were caused by relatively susceptible strains, while resistant strains were isolated from patients with bacteremia and other infections. Only 16 (6.3%) of the isolates were found to be methicillin-resistant S. aureus (MRSA). DNA fingerprinting by pulsed-field gel electrophoresis showed 36 different patterns, with characteristic patterns being found for MRSA strains and the strains with different capsular types. Clonal relationships were established, and the origins of the infection-related isolates in each patient were determined. We conclude that (i) nosocomial infection-related isolates from the shock trauma patients did not belong to a single clone, although the predominance of a methicillin-resistant genotype was noted, (ii) most infection-related S. aureus isolates were relatively susceptible to antibiotics, but a MRSA strain was endemic, and (iii) for practical purposes, the combination of the results of capsular and antibiogram typing can be used as a useful epidemiological marker.     

Molecular Types of Methicillin-Resistant Staphylococcus aureus and Methicillin-Sensitive S. aureus Strains Causing Skin and Soft Tissue Infections and Nasal Colonization,Identified in Community Health Centers in New York City

In November 2011, The Rockefeller University Center for Clinical and Translational Science (CCTS), the Laboratory of Microbiology and Infectious Diseases, and Clinical Directors Network (CDN) launched a research and learning collaborative project with six community health centers in the New York City metropolitan area to determine the nature (clonal type) of community-acquired Staphylococcus aureus strains causing skin and soft tissue infections (SSTIs). Between November 2011 and March 2013, wound and nasal samples from 129 patients with active SSTIs suspicious for S. aureus were collected and characterized by molecular typing techniques. In 63 of 129 patients, the skin wounds were infected by S. aureus: methicillin-resistant S. aureus (MRSA) was recovered from 39 wounds and methicillin-sensitive S. aureus (MSSA) was recovered from 24. Most—46 of the 63–wound isolates belonged to the CC8/Panton-Valentine leukocidin-positive (PVL⁺) group of S. aureus clone USA300: 34 of these strains were MRSA and 12 were MSSA. Of the 63 patients with S. aureus infections, 30 were also colonized by S. aureus in the nares: 16 of the colonizing isolates were MRSA, and 14 were MSSA, and the majority of the colonizing isolates belonged to the USA300 clonal group. In most cases (70%), the colonizing isolate belonged to the same clonal type as the strain involved with the infection. In three of the patients, the identity of invasive and colonizing MRSA isolates was further documented by whole-genome sequencing.     

Epidemiological characteristics of methicillin-resistant Staphylococcus aureus isolates from children with eczematous atopic dermatitis lesions

In this study, we investigated the rate of colonization of skin of children with atopic dermatitis (AD) by methicillin-resistant Staphylococcus aureus (MRSA) and characterized the isolates. Active skin lesions in pediatric AD patients were cultured with Rodac Staph (Komed, Korea). S. aureus isolates were examined for drug susceptibilities, analyzed for the eta, etb, tst, and pvl genes, and typed using agr polymorphism, pulsed-field gel electrophoresis of SmaI-restricted chromosomal DNA, and staphylococcal cassette chromosome mec (SCCmec) typing. Eighty-seven (75.4%) of 115 patients had cultivable S. aureus isolates, 16 of which (18.3%) were MRSA. All MRSA isolates were susceptible to chloramphenicol, rifampin, cotrimoxazole, and ciprofloxacin. While methicillin-susceptible S. aureus (MSSA) isolates were composed of 23 isolates of singular types and nine clusters comprising 48 isolates, MRSA isolates were typed into three clones: eight isolates of pulsotype A-agr-1-SCCmec IV, five isolates of pulsotype B-agr-3-SCCmec IIb-etb positive, and three isolates of pulsotype C-agr-3-SCCmec IV. Three SCCmec IVA MRSA isolates were tst positive, but none were positive for the pvl or eta gene. Among 71 MSSA isolates, 7 isolates were tst positive, 6 of which were pulsotype F-agr-3, and 9 of 10 agr-4 isolates were eta positive. The average ages of patients carrying MSSA, SCCmec IVA MRSA, and SCCmec IIb MRSA were 7.7 ± 4.6, 3.1 ± 1.5, and 8.2 ± 3.1 years, respectively. Among the patients carrying MRSA, two patients had been treated with oral antimicrobials, and one had been admitted to the hospital 18 months previously. In conclusion, community-acquired MRSA isolates of a few clones colonized the skin of patients with AD without risk factors for the acquisition of hospital-acquired MRSA, which suggested that the skin of children with AD may represent a significant reservoir of MRSA colonization in the community.     

Analysis of the Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrum of Staphylococcus aureus Identifies Mutations That Allow Differentiation of the Main Clonal Lineages

Nosocomial infections involving epidemic methicillin-resistant Staphylococcus aureus (MRSA) strains are a serious problem in many countries. In order to analyze outbreaks, the infectious isolates have to be typed; however, most molecular methods are expensive or labor-intensive. Here, we evaluated matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) of cell extracts for the molecular characterization of S. aureus strains. The peak patterns of 401 MRSA and methicillin-susceptible S. aureus (MSSA) strains, including clinical and laboratory strains, were analyzed. Database searches indicated the peptides that were represented by the corresponding peaks in the spectra. The identities of the peptides were confirmed by the sequencing of mutants, the expression of antisense RNA fragments that resulted in the knockdown of the peptide of interest and the concomitant loss of the signal, or tandem MALDI-TOF MS (MALDI-TOF/TOF MS). It was shown that the signals derive mainly from stress proteins and ribosomal proteins. Peak shifts that differentiate the main S. aureus clonal complexes CC5, CC22, CC8, CC45, CC30, and CC1 correlate to point mutations in the respective genes. Retrospective typing of an MRSA outbreak showed that it is possible to differentiate unrelated MSSA, MRSA, and borderline resistant S. aureus (BORSA) strains isolated from health care workers. In conclusion, this method allows for the detection of the epidemic lineages of S. aureus during species identification by MALDI-TOF MS analysis.     

Clinical and Economic Outcomes for Patients with Health Care-Associated Staphylococcus aureus Pneumonia

While the increasing importance of methicillin-resistant Staphylococcus aureus (MRSA) as a pathogen in health care-associated S. aureus pneumonia has been documented widely, information on the clinical and economic consequences of such infections is limited. We retrospectively identified all patients admitted to a large U.S. urban teaching hospital between January 2005 and May 2008 with pneumonia and positive blood or respiratory cultures for S. aureus within 48 h of admission. Among these patients, those with suspected health care-associated pneumonia (HCAP) were identified using established criteria (e.g., recent hospitalization, admission from nursing home, or hemodialysis). Subjects were designated as having methicillin-resistant (MRSA) or methicillin-susceptible (MSSA) HCAP, based on initial S. aureus isolates. Initial therapy was designated “appropriate” versus “inappropriate” based on the expected susceptibility of the organism to the regimen received. We identified 142 patients with evidence of S. aureus HCAP. Their mean (standard deviation [SD]) age was 64.5 (17) years. Eighty-seven patients (61%) had initial cultures that were positive for MRSA. Most (∼90%) patients received appropriate initial antibiotic therapy (86% for MRSA versus 91% for MSSA; P = 0.783). There were no significant differences between MRSA and MSSA HCAP patients in mortality (29% versus 20%, respectively), surgery for pneumonia (22% versus 20%), receipt of mechanical ventilation (60% versus 58%), or admission to the intensive care unit (79% versus 76%). Mean (SD) total charges per admission were universally high ($98,170 [$94,707] for MRSA versus $104,121 [$91,314]) for MSSA [P = 0.712]). Almost two-thirds of patients admitted to hospital with S. aureus HCAP have evidence of MRSA infection. S. aureus HCAP, irrespective of MRSA versus MSSA status, is associated with significant mortality and high health care costs, despite appropriate initial antibiotic therapy.Traditionally, infections have been categorized as either community associated or nosocomial in origin. The theory supporting this dichotomy arose from observations that pathogens causing these two types of infections were distinct. However, with the spread of health care delivery beyond the confines of acute-care hospitals, patients increasingly may present to emergency departments (ED) with infections caused by organisms such as methicillin-resistant Staphylococcus aureus (MRSA). This trend has led to the evolution of the concept of health care-associated infection (HCAI). Recent studies have validated the concept of HCAI for a number of types of infection, ranging from endocarditis to pneumonia (1, 4, 10, 12). Many such reports, however, have provided scant microbiologic information and have focused more on distinctions in patient types and risk factors for resistant infection. The situation regarding limited microbiologic data is particularly acute with respect to S. aureus. Although Fridkin and colleagues, in an assessment of the national burden of MRSA, underscored the growing prevalence of this pathogen in health care-associated pneumonia (HCAP) (3), they presented little information regarding outcomes of such infections.S. aureus in general—and MRSA in particular—remains a growing challenge for both hospitals and physicians. Good infection prevention practices necessitate isolation precautions for patients with MRSA, which has made early identification of these persons a time-sensitive endeavor. Beyond infection prevention issues, which may complicate the care of patients at risk for MRSA HCAP, patients with HCAP due to either methicillin-sensitive S. aureus (MSSA) or MRSA may consume substantial resources. Further complicating management of HCAP due to S. aureus is the shift in strain types and antimicrobial resistance implicated in pneumonia (3). The USA300 strain of MRSA, for example, may produce significant toxins and may not respond well to anti-MRSA antimicrobials that are routinely employed (11). Because of these issues, physicians require data regarding the microbiology, epidemiology, and outcomes associated with HCAP due to S. aureus (both MSSA and MRSA).To address these issues, we conducted a retrospective observational study of patients in a large urban hospital with HCAP due to culture-proven S. aureus. Our aims were to describe outcomes and resource utilization among patients with S. aureus HCAP and to understand possible differences between patients with MSSA versus MRSA pneumonia. We also sought to examine differences in outcomes and resource utilization as a function of pathogen susceptibility to vancomycin and the specific strain type involved.(Preliminary findings from this study were presented at the 48th Annual Interscience Conference on Antimicrobial Agents and Chemotherapy [ICAAC]-Infectious Diseases Society of America [IDSA] 46th Annual Meeting and the 2008 annual meeting of the American College of Chest Physicians [ACCP] [1a, 10a, 11a].)     

Spa type distribution in MRSA and MSSA bacteremias and association of spa clonal complexes with the clinical characteristics of bacteremia

The genetic distribution of invasive methicillin-susceptible (MSSA) and resistant S. aureus (MRSA) strains has to be addressed in order to target infection control strategies. A large MRSA epidemic caused by a certain MRSA strain (spa type 067) broke out in 2001 in our health district. We wanted to investigate the current spa type distribution in MRSA and MSSA bacteremias and assess the potential association of spa clonal complexes (spaCC) with the clinical characteristics of S. aureus bacteremia. One hundred nine invasive MRSA isolates and 353 invasive MSSA isolates were spa typed and grouped into clonal complexes (spaCC). Spa type distribution was compared to that of colonizing MRSA strains. Spa type and spaCC data linked to clinical information on the course of bacteremic cases was used to search for differences in virulence between strains. Spa type distribution in MRSA is less heterogenic than in MSSA. t067 dominates both in MRSA colonisations and in invasive findings. Among MSSA, no such dominating strains were found. Of spaCCs, mortality was the highest in spaCC 067 (25.6%). SpaCC 008 was more often associated with endocarditis than other CCs (22.7 vs 5.8%, p =?0.013), spaCC 2133 with skin infections (68.4 vs 36.4%, p =?0.007), and spaCC 012 with foreign body infections (25.0 vs 9.3%, p =?0.029) than other clonal complexes. A single successful strain can explain the major proportion of MRSA among S. aureus bacteremias. Certain spaCCs showed association with certain clinical characteristics. These findings suggest that S. aureus strains differ in their virulence and invasiveness.     

Population structure analyses of Staphylococcus aureus at Tygerberg Hospital,South Africa,reveals a diverse population,a high prevalence of Panton–Valentine leukocidin genes,and unique local methicillin-resistant S. aureus clones

Studies reporting on the population structure of Staphylococcus aureus in South Africa have focused only on methicillin-resistant S. aureus (MRSA). This study describes the population structure of S. aureus, including methicillin-susceptible S. aureus (MSSA) isolated from patients at Tygerberg Academic Hospital, Western Cape province. Pulsed-field gel electrophoresis (PFGE), detection of Panton–Valentine leukocidin (PVL), spa typing, multilocus sequence typing (MLST), agr typing and SCCmec typing were used to characterize strains. Of 367 non-repetitive S. aureus isolates collected over a period of 1 year, 56 (15.3%) were MRSA. Skin and soft tissue infections were the most frequent source (54.8%), followed by bone and joint (15.3%) and respiratory tract infections (7.7%). For strain typing, PFGE was the most discriminative method, and resulted in 31 pulsotypes (n = 345, 94.0%), as compared with 16 spa clonal complexes (CCs) (n = 344, 93.4%). Four MLST CCs were identified after eBURST of sequence types (STs) of selected isolates. One hundred and sixty isolates (MSSA, n = 155, 42.2%) were PVL-positive, and agr types I–IV and SCCmec types I–V were identified. Our S. aureus population consisted of genotypically diverse strains, with PVL being a common characteristic of MSSA. MSSA and MRSA isolates clustered in different clones. However, the dominant MRSA clone (ST612) also contained an MSSA isolate, and had a unique genotype. Common global epidemic MRSA clones, such as ST239-MRSA-III and ST36-MRSA-II, were identified. A local clone, ST612-MRSA-IV, was found to be the dominant MRSA clone.     

Nasal Colonization of and Clonal Transmission of Methicillin-Susceptible Staphylococcus aureus among Chinese Military Volunteers

Military facilities provide unique opportunities for studying Staphylococcus aureus nasal colonization and transmission patterns. In this cross-sectional observational study, we assessed the prevalence of S. aureus nasal colonization among Chinese military volunteers in two camps in the Beijing area. Antimicrobial resistance patterns, risk factors for colonization, and transmission patterns using pulsed-field gel electrophoresis were also evaluated. From May to July 2007, 1,044 nasal swabs were collected from military volunteers from suburban (560) and urban (484) camps. A total of 209 S. aureus isolates were recovered, of which all were methicillin susceptible. Independent factors associated with methicillin-susceptible S. aureus (MSSA) nasal colonization included younger age (odds ratio [OR] = 1.51, 95% confidence interval [95% CI] = 1.03 to 2.21, P = 0.0347), higher education (OR = 1.38, 95% CI = 1.10 to 1.73, P = 0.0056), shorter length of service (OR = 1.74, 95% CI = 1.28 to 2.36, P = 0.0004), nonsmoking (OR = 1.61, 95% CI = 1.14 to 2.28, P = 0.0069), and inactive participation in social events (OR = 2.40, 95% CI = 1.25 to 5.49, P = 0.0082). Among 209 MSSA isolates, 126 (60.3%) were determined to be epidemic and a total of 12 genotypes were identified, of which four (90 isolates [71.4%]) represented the majority of strains. Length of service and camp location were statistically related to the four major MSSA genotype clonal transmissions. Our data indicated that MSSA, not methicillin-resistant S. aureus (MRSA), nasal colonization and clonal transmission occur in healthy military volunteers in Beijing. Younger, female, nonsmoking volunteers with higher education, little or no participation in social events, and less time in service are at higher risk for nasal MSSA carriage.Staphylococcus aureus is an important cause of skin and soft tissue infections, as well as invasive infections in humans (25). Since methicillin-resistant S. aureus (MRSA) was first reported, it has become endemic in hospitals and communities around the world (10). The recent emergence of a highly virulent community-associated MRSA (CA-MRSA) and vancomycin-resistant, intermediate-resistant, or heteroresistant S. aureus further heightens public health concerns (14, 17, 37, 46). Prevention of S. aureus infection and reduction of the spread of virulent and resistant strains are therefore of great importance.On the other hand, S. aureus is a member of the commensal microflora. The anterior nares of the nose are the primary reservoirs of S. aureus colonization in humans, and many S. aureus infections occur in persons with prior nasal bacterial carriage (47). Nasal colonization is an important step in the pathogenesis of S. aureus infection and is a risk factor for acquiring nosocomial infection (22). It has been shown that 80% of nosocomial S. aureus bacteremia episodes in carriers of this bacteria were attributed to an endogenous source (44). Nosocomial S. aureus bacteremia was three times more frequent in S. aureus carriers than in noncarriers (48). Numerous studies of S. aureus nasal carriage have been carried out in various geographic regions in the United States and the Netherlands (2, 5, 7, 21, 23, 27, 28, 41). Cross-section surveys of nasal carriage prevalence and transmission mechanisms in special healthy populations are beneficial in assessing risk factors associated with S. aureus infections (2, 8, 13, 26, 32-35). Military facilities provide unique opportunities for studying S. aureus nasal colonization and transmission (11, 19, 52).In China, MRSA was shown in 63% of S. aureus isolates, among which 77% nosocomial and 43% community isolates were MRSA (49). According to a study conducted in 2005, the mean incidence of MRSA across China was over 50%, and in Shanghai, the prevalence was over 80%, contributed to by two major epidemic MRSA clones with unique geographic distribution (24, 45, 50, 51). Therefore, understanding and controlling the spread of MRSA in both hospital and community settings in China are now of paramount importance. The majority of S. aureus isolates studied in China have been limited to clinical patients, and S. aureus isolates recovered from healthy populations or those from healthy military volunteers have not been previously reported.In this study, we reported a cross-sectional observational study conducted in two military camps in the Beijing area, People''s Republic of China. The prevalence of S. aureus nasal colonization and risk factors associated with colonization were assessed. Nasal carriage S. aureus isolates were genotyped to determine potential clonal transmission in military facilities and related transmission factors.(This study was presented in part at the 109th General Meeting of the American Society for Microbiology, Philadelphia, PA, 17 to 21 May 2009.)     

Molecular Surveillance and Population Structure Analysis of Methicillin-Susceptible and Methicillin-Resistant Staphylococcus aureus in High-Risk Wards

In this study we report the results of analysis of 253 isolates of Staphylococcus aureus (132 methicillin [meticillin]-resistant S. aureus [MRSA] isolates and 121 methicillin-susceptible S. aureus [MSSA] isolates) from 209 patients admitted to 18 high-risk wards of six hospitals located in Florence, Italy, over an 8-month period during which a program of epidemiological surveillance of hospital-acquired infections was conducted. The majority (69%) of the 87 reported S. aureus infections were caused by MRSA. No outbreak events have been reported. All the isolates were typed by amplified fragment length polymorphism (AFLP), and AFLP profiles were analyzed in order to define similarity groups. The discriminatory power of AFLP is very high with MSSA (Simpson index of diversity [D], 95.9%), whereas its resolution capability with MRSA (D, 44.7%) is hampered by the well-known high clonality of these populations (the main MRSA group accounted for 74% of the MRSA isolates). Combining AFLP, improved by visual inspection of polymorphisms, with multiplex PCR greatly increases MRSA resolution (D, 85.5%), resolving the MRSA population to a level that is one of the highest reported in the literature. Widespread and sporadic clones of MSSA and MRSA were identified, and their diffusion in the different hospitals and wards over the surveillance period was studied. The understanding of MSSA and MRSA population structures should be the starting point for the design of a more rational surveillance program for S. aureus species, maximizing benefits and reducing the cost of infection control strategies.Surveillance of hospital-acquired infections (HAIs), as a critical part of any infection control program, is an indispensable instrument for identification of the dimensions of the problem, for early recognition of changes in infection patterns, and for monitoring of infection trends and rates. Furthermore, surveillance programs allow one to evaluate the effectiveness of interventions, reinforcing good practices and influencing key hospital staff and decision makers (3, 16).Molecular typing techniques greatly improve the quality of epidemiological information obtained by surveillance programs, allowing more-accurate differentiation of strains. Molecular typing techniques are very useful for recognizing sporadic, unrelated strains and endemic, persistent strains (1, 30) and for determining if a single strain or different unrelated strains are the cause of observed increases in the frequency of HAIs by a microbial species.Staphylococcus aureus is one of the main etiologic agents of HAIs, particularly in high-risk wards such as intensive care units (ICUs), and methicillin (meticillin)-resistant S. aureus (MRSA) strains are more frequently involved than methicillin-susceptible S. aureus (MSSA) strains (12, 35). This situation turns out to be particularly serious due to the diffusion of highly pathogenic and multidrug-resistant strains (6).The low degree of genetic variability reported for MRSA populations (30) is a major limitation to strain identification, especially when a short time period and a limited area, such as a single hospital, are monitored. Different molecular typing techniques have been used to point out minor but epidemiologically significant genetic differences between MRSA strains (7, 23, 32, 33, 34). No single technique is clearly superior to others in the resolution of MRSA populations, and a combination of two or more methods has been suggested to be the most efficacious approach (23, 34).Unlike MRSA strains, which have been the subject of several studies of virulence, pathogenesis, development of new antibiotic resistances, strain diffusion worldwide and in hospital settings, and genome analysis (5, 11, 14, 21, 28), MSSA strains, because of their susceptibility to first-line antibiotics, have only occasionally been the subject of molecular epidemiological studies in hospital settings (7, 38). Recent studies performed by multilocus sequence typing have shown a strong genetic relationship between MRSA and MSSA strains, suggesting that MRSA clones arise on multiple occasions from successful hospital MSSA clones by horizontal acquisition of the methicillin resistance (mec) gene (8).In this work, we report the results obtained from an extended molecular surveillance program for S. aureus carried out for 18 high-risk wards of six hospitals in Florence, Italy, over an 8-month period. Our aim was to study the population structure and the diffusion of the MRSA and MSSA strains that colonize and infect patients admitted to the wards under observation. With this aim, amplified fragment length polymorphism (AFLP) analysis was utilized to type MRSA and MSSA isolates, whereas multiplex PCR was used to subtype MRSA isolates falling into the same AFLP group. The Simpson index was employed to evaluate the discriminatory powers of the two molecular techniques and to analyze the structures of both the MRSA and the MSSA populations.     

Evaluation of the BD GeneOhm StaphSR Assay for Detection of Methicillin-Resistant and Methicillin-Susceptible Staphylococcus aureus Isolates from Spiked Positive Blood Culture Bottles

To improve the clinical outcome of Staphylococcus aureus septicemia, the early selection of appropriate antibiotic treatment is crucial. Molecular diagnostics represents an attractive approach for the rapid identification of S. aureus and the determination of its methicillin (meticillin) resistance. In direct comparison to other molecular assays (sa442 and mecA real-time PCRs) and standard laboratory procedures, we evaluated the BD GeneOhm StaphSR assay for its use in the detection of S. aureus and methicillin-resistant S. aureus (MRSA) from spiked blood culture bottles (n = 134). In the case of detecting S. aureus (n = 90; for methicillin-susceptible S. aureus, n = 45; for MRSA, n = 45), the BD GeneOhm StaphSR assay had a sensitivity and a specificity of 100% each (95% confidence intervals [CIs], 96.0 to 100% and 82.4 to 100%, respectively). For MRSA (n = 45), the test was 95.6% (95% CI, 84.9 to 99.5%) sensitive and 95.3% (95% CI, 86.9 to 99.0%) specific. Overall, five discrepant results arose with this assay due to the presence of methicillin-susceptible, revertant MRSA strains (3/45) and MRSA strains that were not detected by the BD GeneOhm StaphSR assay (2/45). Compared to other real-time PCR-based molecular approaches and to conventional standard laboratory methods, the BD GeneOhm StaphSR turned out to be an appropriate diagnostic tool for a rapid (∼1.5 h), preliminary identification of S. aureus and MRSA from blood cultures.Staphylococcus aureus septicemia is associated with high mortality rates, prolonged hospitalization, and increased costs (3, 5). The prevalence of S. aureus septicemia is increasing, primarily due to infections caused by methicillin (meticillin)-resistant S. aureus (MRSA) (20). Several studies have shown that mortality rates among patients suffering from MRSA septicemia is significantly higher than those of patients suffering from infections caused by methicillin-susceptible S. aureus (MSSA) (5, 18, 19).The early selection of an appropriate antibiotic regime for the treatment of MSSA or MRSA is crucial for the patient''s outcome (4, 14, 15). However, bacterial identification and preliminary antibiotic susceptibility testing by standard microbiological procedures still requires 24 to 48 h after growth detection by automated incubation systems. In contrast, new real-time PCR-based methods that use samples directly from positive blood culture bottles allows differentiation of MSSA, MRSA, and coagulase-negative staphylococci (CoNS) within 1.5 to 3 h (7, 12, 13, 16). Such tests promote an early appropriate antibiotic selection, thus avoiding the unnecessary use of vancomycin, and they reduce mortality, the length of hospitalization, and costs associated with bloodstream infections caused by these bacteria (3).We described recently a real-time PCR method for the detection of MSSA, MRSA, and CoNS directly from positive blood cultures; it turned out to have 100% sensitivity and 100% specificity for the detection of MSSA and MRSA (7). In this study, the differentiation between MSSA and MRSA directly from signal-positive blood cultures was achieved by the separate detection of the S. aureus-specific chromosomal fragment sa442 and the mecA gene (encoding methicillin resistance). However, since this test is not a commercialized system and does not run on a common platform like, e.g., the SmartCycler (Cepheid, Sunnyvale, CA), its widespread application is limited. Moreover, in blood cultures containing a mixture of MSSA (sa442⁺ but mecA negative) and methicillin-resistant CoNS (MR-CoNS; sa442 negative but mecA⁺), the test is prone to lead to the incorrect identification of MRSA (sa442⁺ mecA⁺).The BD GeneOhm StaphSR assay (BD Diagnostics GeneOhm, Québec, Canada) provides a rapid, simple, commercially available diagnostic test that runs on the SmartCycler for the detection of S. aureus and MRSA from nasal swabs, wounds, and blood cultures. This multiplex real-time PCR amplifies an S. aureus-specific target sequence and a specific target near the staphylococcal cassette chromosome mec (SCCmec) insertion site and the orfX junction in MRSA, thereby differentiating between MSSA and MRSA (9, 17).Using the herein-described setting, we evaluated the BD GeneOhm StaphSR assay and the PCR that detects sa442 and mecA (designated sa442-mecA-PCR) for the detection of MSSA and MRSA directly from spiked blood cultures.