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.)     

A Common Variant of Staphylococcal Cassette Chromosome mec Type IVa in Isolates from Copenhagen,Denmark, Is Not Detected by the BD GeneOhm Methicillin-Resistant Staphylococcus aureus Assay

Rapid tests for detection of methicillin-resistant Staphylococcus aureus (MRSA) carriage are important to limit the transmission of MRSA in the health care setting. We evaluated the performance of the BD GeneOhm MRSA real-time PCR assay using a diverse collection of MRSA isolates, mainly from Copenhagen, Denmark, but also including international isolates, e.g., USA100-1100. Pure cultures of 349 MRSA isolates representing variants of staphylococcal cassette chromosome mec (SCCmec) types I to V and 103 different staphylococcal protein A (spa) types were tested. In addition, 53 methicillin-susceptible Staphylococcus aureus isolates were included as negative controls. Forty-four MRSA isolates were undetectable; of these, 95% harbored SCCmec type IVa, and these included the most-common clone in Copenhagen, spa t024-sequence type 8-IVa. The false-negative MRSA isolates were tested with new primers (analyte-specific reagent [ASR] BD GeneOhm MRSA assay) supplied by Becton Dickinson (BD). The ASR BD GeneOhm MRSA assay detected 42 of the 44 isolates that were false negative in the BD GeneOhm MRSA assay. Combining the BD GeneOhm MRSA assay with the ASR BD GeneOhm MRSA assay greatly improved the results, with only two MRSA isolates being false negative. The BD GeneOhm MRSA assay alone is not adequate for MRSA detection in Copenhagen, Denmark, as more than one-third of our MRSA isolates would not be detected. We recommend that the BD GeneOhm MRSA assay be evaluated against the local MRSA diversity before being established as a standard assay, and due to the constant evolution of SCCmec cassettes, a continuous global surveillance is advisable in order to update the assay as necessary.Methicillin-resistant Staphylococcus aureus (MRSA) is a common nosocomial pathogen in countries all over the world. In recent years, community-associated MRSA (CA-MRSA) has become increasingly prevalent and has shown potential to cause health care-associated bloodstream infections (8, 26). Screening and isolation of MRSA-positive patients is essential to control the transmission of MRSA in hospitals (16, 24). However, conventional detection of MRSA by culture takes at least 48 h before a preliminary result is available, and as patients in many countries are only isolated when they are recognized as MRSA positive, the risk of having already transmitted MRSA is high. The real-time PCR BD GeneOhm MRSA assay (Becton Dickinson [BD] Diagnostics GeneOhm; San Diego, CA), formerly called IDI-MRSA, is one of a number of commercial kits for rapid MRSA detection directly from nasal swabs (7) and is based on primers developed by Huletsky et al. (18). The forward primers bind to the J3 region of the staphylococcal cassette chromosome mec (SCCmec), and the reverse primer binds in the orfX region that is specific for Staphylococcus aureus. At least seven SCCmec types are known (types I to VII) (3), and several subtypes, especially of type IV, have been described (21, 27).The BD GeneOhm MRSA assay has been tested in a number of studies (4, 5, 10, 11, 13-15, 22, 23, 25, 29-31). Most studies screened hospitalized patients, but only two studies described the SCCmec types of their MRSA isolates (15, 25). Therefore, it is possible that only a few predominant hospital clones with the same SCCmec types were tested. In Denmark, different CA-MRSA clones dominate and MRSA isolates mainly harbor SCCmec types IV (85%) and V (6%) (2). In-house testing with the Huletsky primers (18) revealed that they did not amplify a PCR fragment from our most-common MRSA clone, spa t024-sequence type 8 (ST8)-IVa. Based on this finding and with the knowledge of the high number of type IV subtypes known, we were interested in finding out whether the BD GeneOhm MRSA assay could detect MRSA isolates from a collection that included mainly CA-MRSA strains. We tested 349 MRSA isolates representing variants of SCCmec types I to V. Furthermore, we chose MRSA isolates of different staphylococcal protein A (spa) types to have a broad range of genetic backgrounds, testing the hypothesis that the same SCCmec type might have minor differences in different MRSA lineages and that these differences could be in the primer regions of the assay.     

Trends in Methicillin-Resistant Staphylococcus aureus Anovaginal Colonization in Pregnant Women in 2005 versus 2009

In 2005, the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) anovaginal colonization in pregnant women at our center (Columbia University Medical Center) was 0.5%, and MRSA-colonized women were less likely to carry group B streptococcus (GBS). In this study, our objectives were to identify changing trends in the prevalence of MRSA and methicillin-susceptible S. aureus (MSSA) anovaginal colonization in pregnant women, to assess the association between MRSA and GBS colonization, and to characterize the MRSA strains. From February to July 2009, Lim broths from GBS surveillance samples were cultured for S. aureus. MRSA strains were identified by resistance to cefoxitin and characterized by MicroScan, staphylococcal cassette chromosome mec (SCCmec) typing, pulsed-field gel electrophoresis (PFGE), spa typing, and Panton-Valentine leukocidin PCR. A total of 2,921 specimens from different patients were analyzed. The prevalences of MSSA, MRSA, and GBS colonization were 11.8%, 0.6% and 23.3%, respectively. GBS colonization was associated with S. aureus colonization (odds ratio [OR], 1.9; 95% confidence interval [95% CI], 1.5 to 2.4). The frequencies of GBS colonization were similar in MRSA-positive (34.2%) versus MRSA-negative patients (21.8%) (P = 0.4). All MRSA isolates from 2009 and 13/14 isolates from 2005 were SCCmec type IV or V, consistent with community-associated MRSA; 12/18 (2009) and 0/14 (2005) isolates were the USA300 clone. Levofloxacin resistance increased from 14.3% (2005) to 55.6% (2009) (P = 0.028). In conclusion, the prevalence of MRSA anovaginal colonization in pregnant women in New York City, NY, remained stable from 2005 to 2009, and USA300 emerged as the predominant clone with a significant increase in levofloxacin-resistant isolates.The rapid spread of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) across the United States has been associated primarily with the dissemination of a specific clone that has the pulsed-field gel electrophoresis (PFGE) pattern termed USA300 (20). CA-MRSA can cause infections in pregnant and postpartum women and outbreaks in newborn nurseries and neonatal intensive care units (NICUs) (5, 22, 26, 32, 35, 36, 38). CA-MRSA strains, including USA300, have replaced health care-associated (HA)-MRSA as the predominant strains isolated from infants in some NICUs (6, 35). CA-MRSA infections appear to be increasing in otherwise healthy neonates in the nursery (18, 38) who may acquire S. aureus from health care workers or from their mothers and other family members (19, 23, 25, 28).Vertical transmission from mothers to infants at delivery has also been proposed as a possible mechanism of acquisition of CA-MRSA (1, 2, 7, 28). S. aureus has been reported to colonize the vagina in 4 to 22% of pregnant women (2, 4, 9, 13, 14). In 2005, following an outbreak of USA300 in postpartum women at our medical center (Columbia University Medical Center), we determined that the prevalence of methicillin-susceptible S. aureus (MSSA) anovaginal colonization was 16.6% and the prevalence of MRSA colonization was 0.5% (9). Overall, 93% of MRSA strains were staphylococcal cassette chromosome mec (SCCmec) type IV or V, which is consistent with CA-MRSA (9). More recent studies conducted in other locales have suggested that the prevalence of anovaginal colonization with MRSA is increasing, with reported rates ranging from 3.5 to 10.4% (2, 13). The association of MRSA colonization with group B streptococcus (GBS) colonization is less clear; some reports have shown an increased rate of MRSA colonization in GBS-positive women (2), while others have shown a decreased rate (8, 32).Reports suggesting an increasing prevalence of MRSA among pregnant women, coupled with the recent emergence of USA300 in our NICU (6), led us to question whether the epidemiology of MRSA colonization was also changing in pregnant women in our population in New York City, NY. The objectives of this study were to determine the current prevalence of MRSA and MSSA colonization in pregnant women, to assess the association between MRSA and GBS colonization, and to use molecular methods to characterize MRSA strains from the current study and compare those with strains from our 2005 study.     

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 a New Selective Medium,BD BBL CHROMagar MRSA II,for Detection of Methicillin-Resistant Staphylococcus aureus in Different Specimens

The sensitivity of screening for methicillin-resistant Staphylococcus aureus (MRSA) can be improved by adding other specimen sites to nares. We describe an evaluation of a new selective medium, BBL CHROMagar MRSA II (CMRSAII), for its ability to detect MRSA from different specimen types. CMRSAII is a chromogenic medium which incorporates cefoxitin for the detection of MRSA. A study was performed at four clinical laboratories with the following specimens: 1,446 respiratory, 694 stool, 1,275 skin, and 948 wound specimens and 688 blood culture bottles containing Gram-positive cocci. The recovery of MRSA on traditional culture media was compared to results with CMRSAII. S. aureus was tested by cefoxitin disk diffusion. CMRSAII was interpreted as positive for MRSA at 24 h (range, 18 to 28 h) based solely on the visualization of mauve-colored colonies and at 48 h (range, 36 to 52 h) based on detection of mauve colonies with subsequent confirmation as S. aureus (by coagulase or latex agglutination testing). MRSA was recovered more frequently on CMRSAII (89.8% at 24 h and 95.6% at 48 h) than on traditional culture plates (83.1% at 24 h and 79.8% at 48 h) for all specimen types combined (P < 0.001). The percent sensitivities of CMRSAII at 24- and 48-h reads, respectively, were 85.5 and 92.4% for respiratory specimens, 87.9% and 98.3% for stool specimens, 88.4% and 96.1% for skin specimens, 92.1% and 94.6% for wound specimens, and 100% and 100% for positive blood cultures. The specificity was 99.8% for respiratory specimens and 100% for all others. In conclusion, CMRSAII is a reliable screening medium for multiple specimen types.Controlling the spread of multidrug-resistant microorganisms and especially methicillin-resistant Staphylococcus aureus (MRSA) has become a major infection control objective in the United States (4) and many European countries (3, 4, 21). A part of most programs to control the spread of MRSA is screening of patients (4, 8, 14), and screening has even become mandatory in some countries (11, 31).Traditionally, MRSA screening included mainly the culturing of naris swabs. However, it has been demonstrated that up to 35% of MRSA carriers may be colonized only from sites other than the nares, for example, the throat or the rectum (1, 2, 16).Usage of chromogenic media can improve the sensitivity and pace of MRSA detection (5, 6, 9, 10, 12, 13, 15, 17,19, 20, 22-24, 26-30); however, currently available media that have been marketed at this time are recommended only for nasal specimens.This study was designed to compare the performance of BBL CHROMagar MRSAII (CMRSAII), a chromogenic medium which incorporates cefoxitin, with traditional culture media in the recovery and identification of MRSA isolates from clinical specimens, including respiratory, lower gastrointestinal, and skin specimens as well as wound cultures and blood culture bottles with Gram-positive cocci. In addition, it was designed to determine whether CMRSAII results may be reported as presumptive or definitive with no (or one) confirmatory test at 24 and 48 h of incubation.(These data were presented in part at the 48th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, DC, 25 to 28 October 2008.)     

Prevalence of and Risk Factors for Colonization by Methicillin-Resistant Staphylococcus aureus among Adults in Community Settings in Taiwan

In order to determine the prevalence of methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) colonization among adults in community settings in Taiwan and identify its risk factors, we conducted the present study. For a 3-month period, we enrolled all adults who attended mandatory health examinations at three medical centers and signed the informed consent. Nasal swabs were taken for the isolation of S. aureus. For each MRSA isolate, we performed multilocus sequence typing, identification of the staphylococcal cassette chromosome mec, tests for the presence of the Panton-Valentine leukocidin gene, and tests for drug susceptibilities. Risk factors for MRSA colonization were determined. The results indicated that the MRSA colonization rate among adults in the community settings in Taiwan was 3.8% (119/3,098). Most MRSA isolates belonged to sequence type 59 (84.0%). Independent risk factors for MRSA colonization included the presence of household members less than 7 years old (P < 0.0001) and the use of antibiotics within the past year (P = 0.0031). Smoking appeared to be protective against MRSA colonization (P < 0.0001).Before the late 1990s, nearly all methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) infections occurred in patients with specific risk factors who were in health care facilities (31). However, the emergence of MRSA infections among previously healthy persons in community settings (without exposure to health care facilities) was noted thereafter (6, 31). Therefore, MRSA infections are now classified as health care-associated MRSA (HA-MRSA) infections and community-associated MRSA (CA-MRSA) infections (38).Strains responsible for CA-MRSA infections differ from those for HA-MRSA infections in several phenotypic and genetic features (1, 28). CA-MRSA strains carry type IV or V staphylococcal cassette chromosome mec (SCCmec) elements, are usually Panton-Valentine leukocidin (PVL) producing, and are not multidrug resistant; HA-MRSA strains carry type I, II, or III SCCmec elements, are usually not PVL producing, and are multidrug resistant (15, 22, 28).Initially, CA-MRSA infections were mostly reported in young children (36). However, as CA-MRSA infections became more common, infections were reported among people of all ages and contributed to the increase of community-associated S. aureus infections with significance (25, 29, 36). MRSA colonization is an important risk factor for subsequent MRSA infection (30), so several studies in the United States have characterized the MRSA colonization rate in a community setting (13, 16). These studies demonstrated that the nasal colonization rates among healthy children increased from 0.8% in 2001 to 9.2% in 2004 (13). The colonization rate was 0.84% among people participating in the 2001 to 2002 National Health and Nutrition Examination Survey (NHANES) (16).In Taiwan, MRSA strains of sequence type 59 (ST59), determined by multilocus sequence typing (MLST) and carrying type IV or V SCCmec elements, were recently found to be the major strains of CA-MRSA (5, 7, 27). Other studies demonstrated that these CA-MRSA strains were responsible for the rapid increase in the number of CA-MRSA infections among children and adults in Taiwan (7, 37). The MRSA colonization rates among Taiwanese children increased from 1.5% from 2001 to 2002 to 7.2% from 2005 to 2006 (18, 19). However, the MRSA colonization rate among adults in community settings in Taiwan is unclear. This study was conducted to determine the prevalence and risk factors for the colonization of MRSA among adults in community settings in Taiwan.     

Incidence,Risk Factors,and Outcomes of Panton-Valentine Leukocidin-Positive Methicillin-Susceptible Staphylococcus aureus Infections in Auckland,New Zealand

Panton-Valentine leukocidin (PVL) has been linked to invasive community-acquired methicillin-resistant Staphylococcus aureus infections. However, the association between disease and PVL-positive methicillin-susceptible Staphylococcus aureus (MSSA) has not been widely reported. We aimed to examine the epidemiology of PVL in clinical MSSA isolates from patients presenting to Auckland City Hospital. Four hundred eleven MSSA clinical isolates and 93 nasal carriage isolates were collected and tested for the presence of the lukSF-PV genes using PCR. The results were examined in light of host and disease factors. Multilocus sequence typing (MLST) was performed on a random subset of isolates to ensure that there was no single PVL-positive MSSA clone responsible for disease in Auckland. The prevalence of the lukSF-PV genes in MSSA isolates associated with disease (124/335; 37%) was not significantly different from the prevalence of the lukSF-PV genes in MSSA nasal carriage isolates (29/93; 31% [P = 0.33]). PVL-positive MSSA isolates in Auckland are genetically diverse and come from a number of different clonal complexes. PVL-positive infections peaked at between 10 and 20 years of age, with a subsequent decline. Pacific ethnicity, age, diagnosis of skin and soft tissue infection (SSTI), community-onset infection, and the need for surgical intervention were found by multivariate analysis to be independently associated with PVL-positive MSSA infection. More than one-third of MSSA infections in our patient population are caused by PVL-positive strains. Those patients with PVL-positive MSSA infection were more likely to be of Pacific ethnicity, be younger in age, have community-onset infection, have SSTI, and need surgical intervention.Staphylococcus aureus is a nasal commensal that can be detected in up to 20 to 30% of the general population, one-third of whom are persistently colonized (28). S. aureus produces a wide variety of virulence factors that contribute to its ability to colonize, invade, and evade the immune system, which includes Panton-Valentine leukocidin (PVL), a bicomponent, pore-forming toxin encoded by two contiguous genes, lukF-PV and lukS-PV. PVL can cause either neutrophil lysis or apoptosis and contributes to tissue necrosis (25). PVL has been linked to skin and soft tissue infections (SSTIs), necrotizing pneumonia, and bone and joint infections in humans (3, 11, 17). Rabbit and human leukocytes are highly sensitive to PVL-mediated leukocytosis (18), and animal studies have shown that PVL causes more-severe disease in dermonecrosis (7, 27), osteomyelitis (6), and necrotizing pneumonia models (B. A. Diep, L. Chan, and P. Tattevin, presented at the 49th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, 2009).The presence of PVL has been extensively described for methicillin-resistant S. aureus (MRSA), specifically in association with staphylococcal cassette chromosome mec (SCCmec) type IV and also SCCmec type V (4, 25). The epidemiology of PVL-positive methicillin-susceptible S. aureus (MSSA) has not been reported as extensively, and the lukSF-PV genes are not exclusively linked to the presence of the SCCmec element. In the 1950s MSSA ST80 strains, which were associated with outbreaks of SSTI, harbored the lukSF-PV genes (22). There have also been recent reports of PVL-positive MSSA causing clusters of SSTI and necrotizing pneumonia (5, 15).The vast majority of S. aureus strains in New Zealand are methicillin susceptible (MSSA); the prevalence of methicillin-resistant S. aureus (MRSA) remains low, at about 5% (12). New Zealand has a high incidence of S. aureus disease; the incidence of S. aureus bacteremia in the late 1990s was 41 cases per 100,000 adults per year (12). We aimed to examine the prevalence of the lukSF-PV genes in MSSA isolates responsible for disease and asymptomatic nasal carriage, to determine risk factors for infection with PVL-positive MSSA, and to examine the association between PVL and severity of disease.     

Detection of Methicillin-Resistant Staphylococcus aureus (MRSA) in Specimens from Various Body Sites: Performance Characteristics of the BD GeneOhm MRSA Assay,the Xpert MRSA Assay,and Broth-Enriched Culture in an Area with a Low Prevalence of MRSA Infections

Universal surveillance upon patient admission is important in reducing the transmission of methicillin-resistant Staphylococcus aureus (MRSA) and associated disease in hospitals. High costs for the health care system in conjunction with MRSA have promoted the development of rapid screening methods to detect MRSA carriers. This study compared two real-time PCR methods, the BD GeneOhm MRSA assay (BDGO) and the Xpert MRSA assay, with broth-enriched culture to define their performance characteristics and rapidity in an area with low MRSA prevalence. In total, 414 swabs from the nose and 389 swabs from the groin from 425 patients were tested. Of those 425 patients, 378 had swabs from both the nose and groin in parallel. Two hundred thirty-one and 194 patients were randomly assigned to the BDGO group and the Xpert MRSA group, respectively. In general, sensitivity, specificity, and negative predictive value (NPV) were high for the BDGO (100%, 98.5%, and 100%, respectively) and the Xpert MRSA (100%, 98.2%, and 100%, respectively), irrespective of whether or not nasal and inguinal specimens were considered alone or combined. In contrast, the positive predictive value (PPV) was lower: before the resolution of discrepant results, the PPVs for nasal and inguinal specimens alone and combined were 87.5%, 86.7%, and 82.4% for the BDGO and 91.7%, 66.7%, and 92.9% for the Xpert MRSA, respectively. After the resolution of discrepant results, PPVs were 93.8%, 93.3% and 94.1% for the BDGO and 91.7%, 88.9% and 92.9% for the Xpert MRSA, respectively. With the BDGO, 4 of 16 carriers were each identified by nasal or inguinal swabs alone, whereas in the Xpert MRSA group, 4 of 13 carriers were exclusively identified by nasal swabs and 2 of 13 were identified by inguinal swabs alone. Both PCR methods showed no significant difference in the number of discrepant results (odds ratio, 0.70 [P = 0.789]), but specimens from wounds and other body sites (axilla, vagina, and throat) produced discrepancies more often than nasal and groin specimens (odds ratios, 4.724 [P = 0.058] and 12.163 [P < 0.001], respectively). The facts that no false-negative PCR results were detected and increased PPVs were found after the resolution of discrepant results point to PCR as the actual gold standard. Since both sensitivity and NPV were exceptionally high for PCR, backup cultures may, therefore, be unnecessary in an area with low prevalence and with a preemptive isolation strategy but may still be useful for PCR-positive specimens because of the lower PPV for both methods and the possibility of susceptibility testing. The median time for analysis, including extraction, hands-on time, and actual PCR was 2 h 20 min for the Xpert MRSA versus 5 h 40 min for the BDGO. Concerning reporting time, including administration and specimen collection, the Xpert MRSA was faster than the BDGO (7 h 50 min versus 17 h).Methicillin-resistant Staphylococcus aureus (MRSA) strains have become a major concern for health care systems. Prevention of the spread of MRSA has, therefore, become a main goal in the past decade, and active screening programs have been established worldwide (4, 27). Compared to infections caused by methicillin-susceptible S. aureus (MSSA), the organism causes severe infections with increased morbidity and mortality and prolonged hospitalization (9, 17). Unlike countries facing a high prevalence of MRSA, such as the United States and Japan, the prevalence in Switzerland has remained low to date (5, 13, 21, 32). In most parts of our country, prevalence rates between 4% and 7% are observed (19). Apart from its spread in the hospital environment, MRSA carriage in our community, as well as in other countries, seems to be more prevalent than previously assumed (31, 32, 37).To facilitate the rapid detection of colonized patients, real-time PCR assays have been developed. The first method to directly detect MRSA from clinical specimens was developed by Huletsky et al. (20). The principle of this method is used in two commercially available tests, the BD GeneOhm MRSA assay (BDGO) (BD, San Diego, CA) and the Xpert MRSA assay (Cepheid, Inc., Sunnyvale, CA).Recent studies have shown that universal admission surveillance for MRSA was associated with a reduction in MRSA disease (18, 28). Likewise, Cunningham et al. have reported a reduction in MRSA transmissions in a critical care unit. The authors attributed these findings, at least partially, to the availability of rapid PCR screening tests, apart from other measures like improved hygiene measures (10). PCR screening methods are cost efficient, especially in an area of low prevalence where high-risk patients are subjected to preemptive contact isolation (6). Our facility is a 1,000-bed tertiary care teaching hospital with a known low prevalence (<5%) of MRSA colonization of patients and follows a surveillance policy similar to that of the University Hospital of Berne, Switzerland (6). As reported in other studies, this means preemptive isolation on admission of all patients who (i) came from or had traveled to countries with known high prevalence rates for MRSA, (ii) were transferred from long-term care facilities, (iii) were transferred from another health care facility, (iv) were hospitalized within the previous 6 months, and/or (v) had a history of MRSA colonization or infection (6, 8, 23). As soon as PCR is negative for MRSA, patient isolation is ended. Under these circumstances, a rapid screening method with a high negative predictive value (NPV) is desirable, because the bulk of costs emerge mainly from noncolonized patients being unnecessarily isolated. In this study, we compared two real-time PCR methods, the BDGO and Xpert MRSA assays, with broth-enriched culture to assess their performance characteristics and rapidity in an area with a low prevalence of MRSA.     

Clinical Application of Real-Time PCR to Screening Critically Ill and Emergency-Care Surgical Patients for Methicillin-Resistant Staphylococcus aureus: a Quantitative Analytical Study

The clinical utility of real-time PCR screening assays for methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) colonization is constrained by the predictive values of their results: as MRSA prevalence falls, the assay''s positive predictive value (PPV) drops, and a rising proportion of positive PCR assays will not be confirmed by culture. We provide a quantitative analysis of universal PCR screening of critical care and emergency surgical patients using the BD GeneOhm MRSA PCR system, involving 3,294 assays over six months. A total of 248 PCR assays (7.7%) were positive; however, 88 failed to be confirmed by culture, giving a PPV of 65%. Multivariate analysis was performed to compare PCR-positive culture-positive (P+C+) and PCR-positive culture-negative (P+C−) assays. P+C− results were positively associated with a history of methicillin-sensitive Staphylococcus aureus infection or colonization (odds ratio [OR], 3.15; 95% confidence interval [CI], 1.32 to 7.54) and high PCR thresholds of signal intensity, indicative of a low concentration of target DNA (OR, 1.19 per cycle; 95% CI, 1.11 to 1.26). P+C− results were negatively associated with a history of MRSA infection or colonization (OR, 0.19; 95% CI, 0.09 to 0.42) and male sex (OR, 0.40; 95% CI, 0.20 to 0.81). P+C+ patients were significantly more likely to have subsequent positive MRSA culture assays and microbiological evidence of clinical MRSA infection. The risk of subsequent MRSA infection in P+C− patients was not significantly different from that in case-matched PCR-negative controls. We conclude that, given the low PPV and poor correlation between a PCR-positive assay and the clinical outcome, it would be prudent to await culture confirmation before altering infection control measures on the basis of a positive PCR result.Methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) is endemic in hospitals and health care facilities in most countries of the world (5). It is frequently carried into the community on colonized, discharged patients, forming a reservoir which then returns to the health care facility when asymptomatic carriers are readmitted (9). Admitted carriers may then develop endogenous infection or become sources of nosocomial transmission to other patients. Screening for MRSA carriage on admission has therefore become a component of many infection control programs (2, 8, 26, 27, 38, 42). This facilitates targeted treatment and infection control measures for MRSA-positive patients while avoiding unnecessary isolation and treatment of noncarriers.Conventional culture-based screening may take several days to produce a result, and it is widely hypothesized that a reduced turnaround time would allow faster implementation of appropriate patient management. PCR-based MRSA screening assays have the potential to provide a result in 2 to 3 h of laboratory time and have a total turnaround time from specimen collection to ward report of approximately 20 h (1, 16, 21). The BD GeneOhm MRSA assay (previously known as IDI-MRSA; BD Diagnostics, San Diego, CA) and the Cepheid GeneXpert MRSA assay (Cepheid, Sunnyvale, CA) are two such PCR tests that detect the presence of characteristic MRSA DNA sequences bridging the SCCmec resistance cassette and the S. aureus-specific orfX open reading frame gene (18). However, concerns have been raised over false-positive and -negative results with these tests (11, 36).Previous estimates of the sensitivities, specificities, negative predictive values (NPV), and positive predictive values (PPV) of the BD IDI-MRSA test and other PCR-based assays have varied considerably (for a summary, see Table S1 in the supplemental material). In our own cluster-randomized crossover study of adult general ward patients admitted during 2006 and 2007, the overall prevalence of MRSA carriage on admission was 6.7% (4.9% of assays) and, in comparison with parallel culture screenings, the BD IDI-MRSA PCR test had a sensitivity of 88%, a specificity of 96%, an NPV of 99%, and a PPV of 55%. (21). With low carriage rates, a high NPV is to be expected, but the relatively low PPV exacerbated concerns about high rates of false positivity with this test.Our previous study did not demonstrate a difference in MRSA acquisition rates between admitted patients screened by PCR and by culture on general medical and surgical wards (21). Therefore, we now limit the use of PCR assays to the high-risk populations of patients in adult and pediatric critical care units and those admitted as surgical emergencies. In the present study, we investigate the performance of the BD GeneOhm MRSA PCR test for admission screening of these groups and consider its usefulness in guiding patient management.     

Population Structure of Staphylococcus aureus Strains Isolated from Intensive Care Unit Patients in The Netherlands over an 11-Year Period (1996 to 2006)

The genetic background and the presence of several virulence factors of Staphylococcus aureus isolates from intensive care unit (ICU) patients from 14 hospitals in The Netherlands isolated from 1996 until 2006 were investigated. In total, 936 methicillin-susceptible S. aureus (MSSA) and 7 methicillin-resistant S. aureus (MRSA) isolates were collected. The genetic background was determined by spa typing and multilocus sequence typing (MLST). The virulence determinants Panton-Valentine leukocidin (PVL), toxic shock syndrome toxin 1 (TSST-1), and collagen adhesion (CNA) were detected with real-time PCR assays. On the MRSA isolates, mobile resistance staphylococcal cassette chromosome mec (SCCmec) typing was performed. Among the MSSA isolates, 313 different spa types were observed. A genetic background common to MRSA clones, e.g., MLST clonal complex 1 (CC1), CC5, CC8, CC22, CC30, and CC45, was observed among 62% of the isolates. The remaining isolates were associated with MSSA-related MLST CCs. MLST CC1, CC25, and CC30 were continuously present, and other MLST CCs fluctuated over time. Two percent of the MSSA isolates harbored PVL, 21% had TSST-1, and 46% were positive for CNA. There were no changes in the prevalence of the virulence factors over time. Four MRSA isolates were typed as ST8-MRSA-IV (where ST is the MLST sequence type and IV is the SCCmec type), two were ST5-MRSA-II, and one was ST228-MRSA-I. All MRSA isolates were PVL, CNA, and TSST-1 negative except for the two ST5-MRSA-II isolates, which were TSST-1 positive. No changes in the S. aureus genetic background and the prevalence of the virulence factors PVL, CNA, and TSST-1 were observed in ICU patients in The Netherlands over time.Around 20% of all patients in intensive case units (ICUs) acquire an ICU-related infection as a consequence of frequent use of antibiotics and intensive treatment procedures (1, 31). Of all ICU-related infections, 25% are caused by Staphylococcus aureus (31). Knowledge of the S. aureus population structure and of the prevalence of virulence factors has been proven crucial for the investigation of the epidemiology of S. aureus throughout the world (34).Methicillin-resistant S. aureus (MRSA) clones can emerge by horizontal transfer of the staphylococcal cassette chromosome mec (SCCmec) between methicillin-resistant coagulase-negative Staphylococcus or MRSA and methicillin-susceptible S. aureus (MSSA) (51). In the event of antibiotic pressure, the MSSA isolates have a high risk of SCCmec transfer and survive. As shown in the literature, MSSA lineages with a MRSA-unrelated background may not provide a stable genomic environment for the integration of SCCmec (4, 23, 30, 32, 36, 43). SCCmec transfer has been found to be stable in MSSA with a MRSA-related genetic background, i.e., multilocus sequence typing (MLST) clonal complex 1 (CC1), CC5, CC8, CC22, CC30, and CC45 (39). The MSSA lineages with a MRSA background possess certain characteristics that favor their persistence in the host as well as the transfer between hosts.As the highest antibiotic pressure in hospitals is found in ICUs, changes in the genetic background will be the most obvious among isolates from ICU patients. However, little is known about the genetic backgrounds of ICU isolates over time, and, therefore, this study investigates the genetic background and the virulence of S. aureus isolates obtained from 1996 to 2006 from ICU patients from 14 hospitals in The Netherlands.     

Molecular Epidemiology and Risk Factors for Nasal Carriage of Staphylococcus aureus and Methicillin-Resistant S. aureus in Infants Attending Day Care Centers in Brazil

Investigations regarding Staphylococcus aureus carriage among Brazilian children are scarce. We evaluated the determinants of S. aureus and methicillin-resistant S. aureus (MRSA) nasal carriage in infants attending day care centers (DCCs) and the molecular features of the MRSA strains. A total of 1,192 children aged 2 months to 5 years attending 62 DCCs were screened for S. aureus and MRSA nasal carriage. MRSA isolates were characterized by pulsed-field gel electrophoresis, multilocus sequence typing, spa typing, staphylococcal cassette chromosome (SCC) mec typing and the presence of the Panton-Valentine leukocidin gene. Logistic regression was performed to determine risk factors associated with S. aureus and MRSA colonization. S. aureus and MRSA carriage were detected in 371 (31.1%) and 14 (1.2%) children, respectively. Variables found to be independently associated with an increased risk for S. aureus carriage included being older than 24 months (odds ratio [OR], 1.8; 95% confidence interval [CI], 1.3 to 2.6) and previous DCC attendance (OR, 1.5; 95% CI, 1.0 to 2.2). Having a mother with a high level of education was a protective factor for nasal colonization (OR, 0.4; 95% CI, 0.2 to 0.8). Moreover, we observed that more children carrying MRSA had younger siblings than children not colonized by MRSA. Among the 14 MRSA strains, three SCCmec types (IIIA, IV, and V) were detected, together with a multidrug-resistant dominant MRSA lineage sharing 82.7% genetic similarity with the Brazilian clone (ST239-MRSA-IIIA; ST indicates the sequence type determined by multilocus sequence typing). Although SCCmec type V was recovered from one healthy child who had been exposed to known risk factors for hospital-associated MRSA, its genetic background was compatible with community-related MRSA. Our data suggest that DCC attendees could be contributing to MRSA cross-transmission between health care and community settings.Staphylococcus aureus is an important human pathogen that causes community- and health care-associated infections worldwide in all age groups (43). Nasal colonization by S. aureus is common in children, and genetic evidence has supported a causal relationship between nasal carriers of S. aureus and methicillin-resistant S. aureus (MRSA) and invasive staphylococcal disease (8, 35, 38). In addition, children may act as vectors for spreading S. aureus and MRSA to both community and hospital environments (15). Several determinants of S. aureus carriage in healthy children have been suggested, examples of which are the number of siblings, family size, and day care center (DCC) attendance (31). DCCs constitute reservoirs of MRSA where children are at increased risk of nasal colonization by S. aureus (24, 25).The emergence and dissemination of MRSA are a global concern in both health care and community settings (13). Although MRSA has initially been recognized as a purely health care-associated pathogen, its epidemiology is changing, and it has been increasingly found in healthy individuals without conventional risk factors for MRSA acquisition (37). MRSA strains carry the mecA gene that encodes PBP2A, the central determinant of methicillin resistance, and is carried by a mobile genetic element designated staphylococcal cassette chromosome mec (SCCmec) (18). SCCmec types I, II, III, and VI have been mostly linked to health care-associated MRSA strains (HA-MRSA) while types IV and V have been commonly associated with community-associated (CA-MRSA) isolates (10, 28). Recent studies in DCCs conducted on different continents have found mainly SCCmec type II and type IV MRSA strains (15, 41).Little is known about the extent of S. aureus and MRSA carriage in children, and there are many gaps in the epidemiology and pathogenesis of CA-MRSA strains in Brazil. What is well established is that a single multidrug-resistant HA-MRSA clone (ST239-MRSA-IIIA clone; ST indicates the sequence type determined by multilocus sequence typing [MLST]) accounts for the vast majority of MRSA infections (34, 38, 42). In a previous survey of children with acute respiratory tract infections and meningitis, we found the prevalence of nasal S. aureus and MRSA to be 13.5% and 1.0%, respectively. The MRSA isolates were multidrug resistant, and all of them were classified as SCCmec type III (21). The aim of the present study was to assess the prevalence of S. aureus and MRSA nasal carriage in a large population of healthy infants and children attending DCCs and to determine the potential risk factors for its acquisition. We also describe the molecular features of MRSA strains circulating among DCCs.     

Transmission of Methicillin-Resistant Staphylococcus aureus to Household Contacts

The frequency of and risk factors for methicillin-resistant Staphylococcus aureus (MRSA) transmission from a MRSA index person to household contacts were assessed in this prospective study. Between January 2005 and December 2007, 62 newly diagnosed MRSA index persons (46 patients and 16 health care workers) and their 160 household contacts were included in the study analysis. Transmission of MRSA from an index person to household contacts occurred in nearly half of the cases (47%; n = 29). These 29 index persons together had 84 household contacts, of which two-thirds (67%; n = 56) became MRSA positive. Prolonged exposure time to MRSA at home was a significant risk factor for MRSA transmission to household contacts. In addition, MRSA colonization at least in the throat, younger age, and eczema in index persons were significantly associated with MRSA transmission; the presence of wounds was negatively associated with MRSA transmission. Furthermore, an increased number of household contacts and being the partner of a MRSA index person were household-related risk factors for MRSA acquisition from the index person. No predominant pulsed-field gel electrophoresis (PFGE) type was observed to be transmitted more frequently than other PFGE types. To date, screening household contacts and providing MRSA eradication therapy to those found positive simultaneously with the index person is not included in the “search-and-destroy” policy. We suggest including both in MRSA prevention guidelines, as this may reduce further spread of MRSA.Methicillin-resistant Staphylococcus aureus (MRSA) is currently the most prevalent antibiotic-resistant pathogen in hospitals in many parts of the world, and there are a growing number of reports describing its increasing prevalence in various community populations (10-12). MRSA is an important cause of infections, and MRSA infections are increasing in both health care centers and the community. Compared to methicillin-sensitive Staphylococcus aureus (MSSA), infections with MRSA are more difficult to treat and tend to have a poorer outcome (2, 8).Carriage of MRSA is a prerequisite for most MRSA infections and plays an important role in the dissemination of this organism within health care facilities and into the community (3, 6, 7, 9). In the Netherlands, due to the “search-and-destroy” infection control policy and a strict antibiotic policy, the number of patients colonized with MRSA is still very limited (13, 31, 34). The “Destroy” part of this policy is important, as it eliminates two out of the three known reservoirs, carriage in patients and carriage in health care workers (HCWs), whereas the third reservoir is the environment. But even in low-prevalence countries like the Netherlands, the emergence of community-acquired MRSA has caused a change in MRSA epidemiology and an increasing number of MRSA cases (13).In the past, it has been shown that carriers of Staphylococcus aureus and MRSA can be a source of transmission of these pathogens to their household contacts (5, 17, 18, 21, 26). The exact risk factors for transmission of MRSA to household contacts have not been studied properly, but close contact, the environment, or being an HCW are thought to be plausible risk factors for transmission (28, 29, 32).The contribution of transmission in households to the MRSA burden has not yet been studied, and because of lack of data and well-calculated scenarios, no evidence-based policy for this reservoir has been developed. For this reason, being a household contact of a MRSA carrier has not yet been established as a risk group for MRSA under the Dutch “search-and-destroy” policy.The aims of this study are to gain insight in the frequency of and risk factors for transmission of MRSA to household contacts and therefore into the community.(The work was presented in part at the 48th Interscience Conference on Antimicrobial Agents and Chemotherapy—Infectious Diseases Society of America [ICAAC/IDSA], 24 to 28 October 2008, Washington, DC [24a].)     

Vancomycin MIC plus Heteroresistance and Outcome of Methicillin-Resistant Staphylococcus aureus Bacteremia: Trends over 11 Years

Vancomycin MICs (V-MIC) and the frequency of heteroresistant vancomycin-intermediate Staphylococcus aureus (hVISA) isolates are increasing among methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) isolates, but their relevance remains uncertain. We compared the V-MIC (Etest) and the frequency of hVISA (Etest macromethod) for all MRSA blood isolates saved over an 11-year span and correlated the results with the clinical outcome. We tested 489 isolates: 61, 55, 187, and 186 isolates recovered in 1996-1997, 2000, 2002-2003, and 2005-2006, respectively. The V-MICs were ≤1, 1.5, 2, and 3 μg/ml for 74 (15.1%), 355 (72.6%), 50 (10.2%), and 10 (2.1%) isolates, respectively. We detected hVISA in 0/74, 48/355 (13.5%), 15/50 (30.0%), and 8/10 (80.0%) isolates with V-MICs of ≤1, 1.5, 2, and 3 μg/ml, respectively (P < 0.001). The V-MIC distribution and the hVISA frequency were stable over the 11-year period. Most patients (89.0%) received vancomycin. The mortality rate (evaluated with 285 patients for whose isolates the trough V-MIC was ≥10 μg/ml) was comparable for patients whose isolates had V-MICs of ≤1 and 1.5 μg/ml (19.4% and 27.0%, respectively; P = 0.2) but higher for patients whose isolates had V-MICs of ≥2 μg/ml (47.6%; P = 0.03). However, the impact of V-MIC and hVISA status on mortality or persistent (≥7 days) bacteremia was not substantiated by multivariate analysis. Staphylococcal chromosome cassette mec (SCCmec) typing of 261 isolates (including all hVISA isolates) revealed that 93.0% of the hVISA isolates were SCCmec type II. These findings demonstrate that the V-MIC distribution and hVISA frequencies were stable over an 11-year span. A V-MIC of ≥2 μg/ml was associated with a higher rate of mortality by univariate analysis, but the relevance of the V-MIC and the presence of hVISA remain uncertain. A multicenter prospective randomized study by the use of standardized methods is needed to evaluate the relevance of hVISA and determine the optimal treatment of patients whose isolates have V-MICs of ≥2.0 μg/ml.The treatment of methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) bacteremia with vancomycin is often associated with a poor clinical outcome (6, 15, 28, 40). Treatment failure was reported among patients infected with isolates whose vancomycin MICs were ≥4 μg/ml (6, 9, 12, 25, 28, 42). This prompted the Clinical and Laboratory Standards Institute to lower the cutoffs for S. aureus susceptibility to ≤2 μg/ml for susceptible, 4 to 8 μg/ml for intermediate (vancomycin-intermediate S. aureus [VISA]), and 16 μg/ml for resistance (39). Within the susceptibility range, the MIC is reported to increase over time (14, 25, 35-40). This is often referred to as MIC creep (38). Additionally, isolates with heteroresistance (heteroresistant vancomycin-intermediate S. aureus [hVISA]) are emerging, and this has uncertain implications for laboratory detection and clinical management (2, 5, 15, 24, 40-42). The first isolate of hVISA to be identified was reported from Japan in 1997 (11). Since then, it has been reported worldwide at frequencies of 0 to 50% (2, 4, 6, 9, 12, 19, 20, 21, 24, 26, 27, 31, 40, 42, 44). This disparity in frequency is probably a result of its variable incidence and the different testing methodologies used. Likewise, the frequency of isolates with MICs of 1.5 to <4 μg/ml varies according to the testing method used (3, 32). The relevance of an MIC on the higher side of the susceptibility range and the presence of hVISA isolates remains uncertain (8, 19, 21). Therapeutic failure was reported in patients infected with isolates with vancomycin MICs of 2 μg/ml (6, 12, 28) and 1.5 or 1 μg/ml (25, 34, 37). Most clinical microbiology laboratories use automated testing methods that are known to underestimate the vancomycin MIC (13, 24). Additionally, most previous studies addressing the relevance of such isolates were observational and usually involved only a few patients and poorly selected controls (1, 4, 7, 9, 12, 14, 25, 35, 38, 42). At our institution, we found the frequency of hVISA isolates among isolates from patients with persistent MRSA bacteremia to be 14%; however, heteroresistance did not correlate with the mortality rate (19). In the current study, we tested all blood MRSA isolates collected over 11 years to determine whether the vancomycin MIC and the prevalence of hVISA have changed over time and to evaluate the effects of increasing vancomycin MICs and the hVISA frequency on patient outcomes.     

Clonal Complexes and Diversity of Exotoxin Gene Profiles in Methicillin-Resistant and Methicillin-Susceptible Staphylococcus aureus Isolates from Patients in a Spanish Hospital

Molecular epidemiology studies have allowed the identification of the methicillin (meticillin)-resistant (MRSA) and methicillin-susceptible (MSSA) clonal complexes (CCs) and clones of Staphylococcus aureus circulating in a Spanish hospital recently. Of 81 isolates tested, 32.1% were MRSA. Most of them carried staphylococcal cassette chromosome mec (SCCmec) IVc (88.5%) and belonged to CC5 (88.5%; multilocus sequence typing types ST125 [mainly associated with spa type t067], ST5, and ST228). A higher diversity was found among MSSA isolates (67.9%). Eighty percent shared the genetic background of major MRSA lineages (CC5 [38.2%; ST125 and ST5], CC30 [25.5%; ST30], CC45 [14.5%; ST45 and ST47], and CC8 [1.8%; ST8]), but CC12, CC15, CC51, and CC59 were also detected. Many exotoxin genes were present in each of the 81 isolates, independent of whether they were involved in sepsis (11 to 22) or other types of infections (13 to 21), and they appeared in 73 combinations. The relevant data are that (i) all isolates were positive for hemolysin and leukotoxin genes (98.8% for lukED and 25.9% for lukPV); (ii) all contained an enterotoxin gene cluster (egc with or without seu), frequently with one or more genes encoding classical enterotoxins; (iii) about half were positive for tst and 95% were positive for exfoliatin-encoding genes (eta, etb, and/or etd); and (iv) the four agr groups were detected, with agrII (55.6%) and agrIII (23.5%) being the most frequent. Taken together, results of the present study suggest a frequent acquisition and/or loss of exotoxin genes, which may be mediated by efficient intralineage transfer of mobile genetic elements and exotoxin genes therein and by eventual breakage of interlineage barriers.Staphylococcus aureus is both a commensal bacterium and an extremely versatile pathogen that causes a wide range of diseases in humans, including superficial, deep-seated, and systemic infections, as well as a variety of toxemic syndromes, such as toxic shock syndrome (TSS), staphylococcal scalded-skin syndrome (SSSS), and staphylococcal food poisoning (36). S. aureus produces a wide range of virulence factors that mediate host colonization, invasion of damaged skin and mucosa, dissemination through the body, and evasion of host defense mechanisms (8, 12). Relevant among them are a variety of exotoxins that comprise α-, β-, γ-, and δ-hemolysins, leukotoxins (the classical LukS-PV-LukF-PV Panton-Valentine leukocidin [LukPV], LukE-LukD [LukED] and LukM-LukF′-PV [LukM]), exfoliative toxins, and pyrogenic toxin superantigens, such as the staphylococcal TSS toxin (TSST-1, first referred to as SEF) and staphylococcal enterotoxins (SEs) (14, 29, 43). Five major serological types of SEs, SEA through SEE (known as classical enterotoxins, encoded by the sea to see genes, respectively) have been initially identified. However, new types of SEs and their coding genes (seg through seu) were later reported. Several SE genes (seg, sei, sem, sen, and seo) are part of an operon termed the enterotoxin gene cluster (egc), of which a variant that contains seu instead of the two pseudogenes present in the originally described egc between sei and sen has been identified (27, 33). Both TSST-1 and SEs are potent activators of T-cell populations, leading to massive proliferation and uncontrolled release of proinflammatory cytokines (14). Expression of most virulence factors in S. aureus is under the control of the agr (accessory gene regulator) locus, which encodes a two-component signaling pathway and its activating ligand, a bacterial-density-sensing peptide termed the autoinducing peptide (37). S. aureus strains can be subdivided into four major agr groups, based on polymorphisms in the amino acid sequence of the autoinducing peptide and other components of the system (26, 28). Within a given group, each strain produces a peptide that can activate the agr response in other members of the group, whereas the autoinducing peptides produced by different groups are usually mutually inhibitory (26).Apart from having pathogenic versatility, S. aureus can adapt rapidly to the selective pressure of antibiotics, with the emergence and spread of methicillin (meticillin)-resistant S. aureus (MRSA) isolates being a relevant example. Resistance to methicillin and other beta-lactam antibiotics is caused by the mecA gene, situated on a mobile genetic element, the staphylococcal cassette chromosome mec (SCCmec), which consists of the mec gene complex, the ccr gene complex, and the “junkyard” regions. Based on the variability of the differently combined components, several types of SCCmec and several variants of the types have been distinguished (21, 23, 24, 25, 30, 31, 38, 45, 51).In the present work, the techniques most commonly applied in epidemiological studies of S. aureus were used to identify the prevalent and sporadic MRSA and methicillin-susceptible S. aureus (MSSA) clones that have been causing disease in a Spanish hospital (the Hospital Universitario Central de Asturias [HUCA]) over a recent time period (2005 to 2006). These methods included pulsed-field gel electrophoresis (PFGE) of SmaI-digested genomic DNA (SmaI PFGE), S. aureus protein A gene (spa) typing, multilocus sequence typing (MLST) analysis, and SCCmec typing of MRSA (4, 9, 40, 42, 50, 52). The risk for human health posed by the accumulation of virulence genes in S. aureus (34) along with the potential application of such genes for subtyping prompted the assessment of the virulence gene repertoire of the HUCA isolates, with regard to the agr group and 30 exotoxin-encoding genes.     

Clinical Characteristics,Outcomes, and Microbiologic Features Associated with Methicillin-Resistant Staphylococcus aureus Bacteremia in Pediatric Patients Treated with Vancomycin

Vancomycin is the first-line therapy for methicillin-resistant Staphylococcus aureus (MRSA) bacteremia, but its efficacy in adult patients has been questioned. Less is known about the outcomes of MRSA bacteremia treated with vancomycin in pediatric patients. This study reviews the outcomes and clinical characteristics of MRSA bacteremia in children treated with vancomycin and characterizes the microbiologic and molecular features of the bloodstream isolates. A retrospective cohort study was conducted among pediatric patients with MRSA bacteremia treated with vancomycin for >5 days from 1 August 2005 to 31 May 2007 in a large tertiary care center. MRSA bloodstream isolates were characterized by antimicrobial susceptibility testing, PCR analysis of virulence genes, and Diversilab typing. Clinical records were reviewed for outcomes and comorbidities. A total of 22 pediatric patients with MRSA bacteremia were identified. Eleven cases (50.0%) were considered vancomycin treatment failures. Features significantly associated with vancomycin treatment failure were prematurity (P = 0.02) and isolates positive for Panton-Valentine leukocidin (PVL) (P = 0.008). Features typically associated with community-associated MRSA strains were identified in hospital-associated isolates. A dominant clone was not responsible for the high number of treatment failures. Further studies are needed to determine if vancomycin should be the first-line treatment for MRSA bacteremia in premature infants and for PVL-positive isolates.Staphylococcus aureus is a major cause of invasive infections in both children and adults. Methicillin-resistant S. aureus (MRSA) infections have increased substantially since the first case was reported in 1961, with a prevalence greater than 50% in certain regions of the United States (39). With this overall increase in infections, the proportion of invasive S. aureus infections has increased in the pediatric population as well, especially those with methicillin-resistant strains (12). MRSA infections have emerged in the community among people without prior hospitalization or other traditional risk factors (2, 15, 27). Community-associated (CA) MRSA strains have traditionally differed from health care-associated (HA) strains in that they are more susceptible to antibiotics, contain the staphylococcal chromosome cassette mec (SCCmec) type IV, and are characterized by the production of specific toxins such as the Panton-Valentine leukocidin (PVL) (27, 37). Recent reports have described the occurrence of characteristics typically found in CA strains in isolates causing infections that are considered HA (6, 11, 13, 25). The clinical impact of MRSA infections is significant regardless of the origin of the infection. MRSA infections in adults are responsible for increased mortality rates, longer lengths of hospital stay, and higher rates of therapeutic failure compared to methicillin-susceptible S. aureus infections (1, 18, 38).Vancomycin is generally considered the treatment of choice for most invasive MRSA infections. The outcomes of MRSA bacteremia treated with vancomycin have been well described for adult populations (23, 28). There are, however, little data on the outcomes of MRSA bacteremia in children treated with vancomycin. This report describes the outcome, factors associated with treatment failure, and microbiologic characterization of bloodstream MRSA isolates in a pediatric population treated adequately with vancomycin, defined as receiving vancomycin for at least 5 days.     

Methicillin-Resistant Staphylococcus aureus in Spain: Molecular Epidemiology and Utility of Different Typing Methods

In a point-prevalence study performed in 145 Spanish hospitals in 2006, we collected 463 isolates of Staphylococcus aureus in a single day. Of these, 135 (29.2%) were methicillin (meticillin)-resistant S. aureus (MRSA) isolates. Susceptibility testing was performed by a microdilution method, and mecA was detected by PCR. The isolates were analyzed by pulsed-field gel electrophoresis (PFGE) after SmaI digestion, staphylococcal chromosomal cassette mec (SCCmec) typing, agr typing, spa typing with BURP (based-upon-repeat-pattern) analysis, and multilocus sequence typing (MLST). The 135 MRSA isolates showed resistance to ciprofloxacin (93.3%), tobramycin (72.6%), gentamicin (20.0%), erythromycin (66.7%), and clindamycin (39.3%). Among the isolates resistant to erythromycin, 27.4% showed the M phenotype. All of the isolates were susceptible to glycopeptides. Twelve resistance patterns were found, of which four accounted for 65% of the isolates. PFGE revealed 36 different patterns, with 13 major clones (including 2 predominant clones with various antibiotypes that accounted for 52.5% of the MRSA isolates) and 23 sporadic profiles. Two genotypes were observed for the first time in Spain. SCCmec type IV accounted for 6.7% of the isolates (70.1% were type IVa, 23.9% were type IVc, 0.9% were type IVd, and 5.1% were type IVh), and SCCmec type I and SCCmec type II accounted for 7.4% and 5.2% of the isolates, respectively. One isolate was nontypeable. Only one of the isolates produced the Panton-Valentine leukocidin. The isolates presented agr type 2 (82.2%), type 1 (14.8%), and type 3 (3.0%). spa typing revealed 32 different types, the predominant ones being t067 (48.9%) and t002 (14.8%), as well as clonal complex 067 (78%) by BURP analysis. The MRSA clone of sequence type 125 and SCCmec type IV was the most prevalent throughout Spain. In our experience, PFGE, spa typing, SCCmec typing, and MLST presented good correlations for the majority of the MRSA strains; we suggest the use of spa typing and PFGE typing for epidemiological surveillance, since this combination is useful for both long-term and short-term studies.Methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) is a major cause of hospital-acquired infections worldwide (5, 25). The appearance of MRSA in the community and the potential risk of it entering hospitals are also matters of concern (29, 44). Moreover, the increasing prevalence of multidrug resistance and the emergence of isolates with intermediate or high-level vancomycin resistance emphasize the importance of the use of infection control measures (2, 49, 50). Although the rates of isolation of MRSA have been increasing throughout the world for the last few decades and in some areas the rates reach >50%, there are considerable variations in the prevalence of MRSA according to geographic area (3, 18, 21, 39, 44). In Spain, the prevalence of MRSA increased from 1.5% in 1986 to 29.2% in 2006, although it seems to have stabilized (13). Despite the worldwide increase in isolation rates, only a limited number of clones of MRSA have spread in most countries (20).Historically, the dissemination of epidemic clones such as EMRSA type 15 (EMRSA-15), EMRSA-16, the Iberian clone, and the Brazilian clone, as well as the high incidence of the community-acquired MRSA USA300 clone, has led to the increased use of molecular typing methods (11, 38, 42, 47, 53).In recent years, a variety of molecular techniques have been used for the typing of MRSA isolates. Of these, SmaI macrorestriction analysis is the “gold standard” for the analysis of the local epidemiology in the short term, spa typing in combination with BURP (based-upon-repeat-pattern) analysis has become a frontline tool for routine epidemiological typing, and multilocus sequence typing (MLST)-staphylococcal chromosomal cassette mec (SCCmec) typing is the reference method for the definition of MRSA clones (10, 34, 37, 46).The aim of the present study was to determine which clones are circulating in Spain and whether the strains have spread between hospitals by analyzing a representative sample of isolates collected in a point-prevalence study. Isolates were grouped by using pulsed-field gel electrophoresis (PFGE) and spa typing and were assigned to MRSA clones on the basis of MLST and SCCmec typing. The congruence between the different grouping methods was assessed.(This study was presented in part at the 47th Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL, 2007 [O. Cuevas, C. Marcos, P. Trincados, T. Boquete, E. Cercenado, E. Bouza, and A. Vindel; abstr. C2-148].)     

Presence of Genes Encoding the Panton-Valentine Leukocidin Exotoxin Is Not the Primary Determinant of Outcome in Patients with Complicated Skin and Skin Structure Infections Due to Methicillin-Resistant Staphylococcus aureus: Results of a Multinational Trial

The role of Panton-Valentine leukocidin (PVL) in determining the severity and outcome of complicated skin and skin structure infections (cSSSI) caused by methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) is controversial. We evaluated potential associations between clinical outcome and PVL status by using MRSA isolates from patients enrolled in two large, multinational phase three clinical trials assessing telavancin for the treatment of cSSSI (the ATLAS program). MRSA isolates from microbiologically evaluable patients were genotyped by pulsed-field gel electrophoresis (PFGE) and PCR for pvl and 31 other putative virulence determinants. A single baseline pathogen of MRSA was isolated from 522 microbiologically evaluable patients (25.1%) among 2,079 randomized patients. Of these MRSA isolates, 83.2% (432/519) exhibited the USA300 PFGE genotype and 89.1% (465/522) were pvl positive. Patients with pvl-positive MRSA were more likely than those with pvl-negative MRSA to be young, to be North American, and to present with major abscesses (P < 0.001 for each). Patients were significantly more likely to be cured if they were infected with pvl-positive MRSA than if they were infected with pvl-negative MRSA (91.6% versus 80.7%; P = 0.015). This observation remained statistically significant after adjustment for presence of abscess, fever, or leukocytosis; infection size; diabetes; patient age; and study medication received. The fnbA, cna, sdrC, map-eap, sed, seg, sei, sej, SCCmec type IV, and agr group II genes were also associated with clinical response (P < 0.05). This contemporary, international study demonstrates that pvl was not the primary determinant of outcome in patients with MRSA cSSSI.Methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) is a leading cause of complicated skin and skin structure infections (cSSSI) in the United States (4, 18, 23). In many regions of the United States, the genetically distinct community-associated MRSA (CA-MRSA) clone USA300 is now the predominant cause of cSSSI (13, 18, 25). Most CA-MRSA isolates isolated from cSSSI carry pvl, the gene encoding Panton-Valentine leukocidin (PVL) (6, 19, 26). PVL is a pore-forming, bicomponent exotoxin known to induce cell death by necrosis or apoptosis (8). Infections caused by S. aureus strains carrying pvl are commonly thought to be associated with worse clinical outcome (6, 16). For example, the presence of the PVL toxin has been shown to cause necrotizing pneumonia in animal models (14) and is associated with necrotizing S. aureus pneumonia in humans (9). However, other laboratories have found that PVL is not a virulence determinant in murine models of CA-MRSA infection (1, 27). In addition, recent studies by our group have suggested that the presence of pvl was associated with a better clinical outcome in patients with cSSSI (2) or bacteremia (15) due to S. aureus. Thus, the role of PVL in determining the clinical severity and outcome of MRSA cSSSI remains unresolved.As part of two phase 3 clinical trials, we have established a large collection of contemporary, geographically diverse S. aureus isolates from a clinically well-characterized population of cSSSI patients. Using this resource, the current study sought to accomplish two objectives, (i) to validate our previous observation that the presence of pvl is not the primary determinant of clinical outcome in patients with cSSSI due to MRSA and (ii) to identify potential associations between other putative bacterial virulence genes and clinical outcome in patients with cSSSI due to MRSA.(These data were presented, in part, at the 48th Annual ICCAC/IDSA 46th Annual Meeting, 25 to 28 October 2008, Washington, DC.)     

Staphylococcal Interspersed Repeat Unit Typing of Staphylococcus aureus: Evaluation of a New Multilocus Variable-Number Tandem-Repeat Analysis Typing Method

The present study evaluates the performance of the staphylococcal interspersed repeat unit (SIRU) method applied to a diverse collection of 104 Staphylococcus aureus isolates previously characterized by pulsed-field gel electrophoresis (PFGE), spa typing, multilocus sequence typing (MLST), and staphylococcal cassette chromosome mec typing for methicillin-resistant S. aureus. The SIRU method distributed the 104 strains into 81 SIRU profiles that could be clustered into 12 groups and 29 singletons. The discriminatory power of the method at the profile level, translated by Simpson''s index of diversity (SID), was similar to that of PFGE subtyping (SID = 99.23% versus 99.85%) and slightly higher than that of spa typing (SID = 97.61%). At the group level, the SIRU SID (93.24%) was lower than that of PFGE typing (95.41%) but higher than that of MLST (SID = 91.77%). The adjusted Rand (AR) coefficient showed that SIRU typing at the group level had the highest congruence with MLST (AR = 0.5736) and with clonal complex (CC) (AR = 0.4963) but the lowest congruence with PFGE subtype (AR = 0.0242). The Wallace coefficient indicated that in the present collection, two strains with the same SIRU profile have a 100% probability of belonging to the same CC, a 90% probability of sharing the same spa type, and an 83% probability of being classified in the same sequence type. The high discriminatory power of the SIRU method, along with its apparent concordance with MLST results, makes it potentially valuable for S. aureus short-term epidemiological investigations and population dynamics as well.Staphylococcus aureus, especially methicillin-resistant S. aureus (MRSA), continues to be a major cause of health care-associated and, more recently, community-associated infections (40, 62). It is critical to have access to an accurate typing method to design cost-effective intervention and prevention strategies (45, 48, 61). A large number of molecular typing methods have been developed to assess strain relatedness for outbreak control, surveillance programs, and population structure and evolution studies (58, 61). The three most used typing methods for S. aureus have advantages and disadvantages, as follows. (i) Pulsed-field gel electrophoresis (PFGE), which is the “gold standard” typing method, has high discriminatory power and accuracy, but it is time-consuming and expensive, and the interlaboratory exchange of results is challenging. (ii) Sequence-based multilocus sequence typing (MLST) is easy to perform, and the results, given as an allelic profile, are portable and easy to exchange due to a public database available on the Internet (http://www.mlst.net), but it is expensive and not useful for local outbreak investigations. MLST is frequently combined with staphylococcal cassette chromosome mec (SCCmec) typing in order to define clonal types of MRSA (24). (iii) spa typing, a single-locus sequence typing method, is being used more frequently for S. aureus typing, and the development of a public database on the Internet (http://spaserver.ridom.de), as with MLST, ensured an international typing nomenclature and thus a great facility in exchanging typing data. By calculation of Simpson''s index of diversity (SID), it was shown that spa typing is nearly as discriminatory as PFGE (1, 25), although it takes into account a single variable region of the protein A gene.In choosing a new method, it is worth taking into consideration that PCR-based methods are commonly used in typing laboratories because of their accuracy, ease of use, low cost, and speed in retrieving results (in a few hours).Many bacterial genomes carry loci of repetitive DNA, which may contain variable repeated units among strains (43, 60). Systems based on a multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) have been used extensively for typing of clinical isolates of several bacterial species and were shown to perform well compared to other genotyping methods (43, 61). S. aureus harbors a diverse population of DNA repeats, which allowed the design of various MLVA schemes (28, 30, 39, 53, 60). Hardy et al. (36, 38) developed a MLVA scheme for S. aureus where seven novel multiple tandem repeats with a high degree of similarity in the flanking regions were identified based on the alignment of seven S. aureus sequenced genomes (strains N315, MW2, Mu50, MSSA476, MRSA252, NCTC8325, and COL). Six of these seven loci were located on intergenic regions scattered around the S. aureus genome; the remaining locus corresponds to the protein A gene, spa. The method, designated staphylococcal interspersed repeat unit (SIRU) typing, relies on PCR amplification of the seven loci of repetitive DNA, using primers specific for the flanking regions of each locus, and on the determination of the size of each amplicon, which reflects the number of repeated units present on the targeted SIRU. To each of the seven loci is attributed the respective number of DNA repeats, generating a combination of seven numbers that characterizes each strain and corresponds to the allelic profile. This allelic profile makes the SIRU method amenable to interlaboratory comparisons and database management, comparable to MLST. So far, the SIRU method has been applied to S. aureus isolates from nosocomial outbreaks in the United Kingdom and Germany, mainly MRSA isolates, and therefore to highly related strains (29, 35-37). Very recently, a single study evaluated a MLVA scheme including the SIRU typing loci and the sspA gene, using a European collection of contemporary S. aureus isolates (39).The aim of the present study was to evaluate the SIRU method with a more diverse collection of S. aureus isolates, including MRSA and methicillin-susceptible S. aureus (MSSA) isolates, from different continents, isolated throughout several decades and previously characterized by well-established typing methods (PFGE, spa typing, MLST, and SCCmec for MRSA).     

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].)