Sigrid K. McAllister

Pulsed-Field Gel Electrophoresis Typing of Oxacillin-Resistant Staphylococcus aureus Isolates from the United States: Establishing a National Database

ABSTRACT Oxacillin-resistant Staphylococcus aureus (ORSA) is a virulent pathogen responsible for both health care-associated and community onset disease. We used SmaI-digested genomic DNA separated by pulsed-field gel electrophoresis (PFGE) to characterize 957 S. aureus isolates and establish a database of PFGE patterns. In addition to PFGE patterns of U.S. strains, the database contains patterns of representative epidemic-type strains from the United Kingdom, Canada, and Australia; previously described ORSA clonal-type isolates; 13 vancomycin-intermediate S. aureus (VISA) isolates, and two high-level vancomycin-resistant, vanA-positive strains (VRSA). Among the isolates from the United States, we identified eight lineages, designated as pulsed-field types (PFTs) USA100 through USA800, seven of which included both ORSA and oxacillin-susceptible S. aureus isolates. With the exception of the PFT pairs USA100 and USA800, and USA300 and USA500, each of the PFTs had a unique multilocus sequence type and spa type motif. The USA100 PFT, previously designated as the New York/Tokyo clone, was the most common PFT in the database, representing 44% of the ORSA isolates. USA100 isolates were typically multiresistant and included all but one of the U.S. VISA strains and both VRSA isolates. Multiresistant ORSA isolates from the USA200, -500, and -600 PFTs have PFGE patterns similar to those of previously described epidemic strains from Europe and Australia. The USA300 and -400 PFTs contained community isolates resistant only to β-lactam drugs and erythromycin. Noticeably absent from the U.S. database were isolates with the previously described Brazilian and EMRSA15 PFGE patterns. These data suggest that there are a limited number of ORSA genotypes present in the United States.

Changes in the Prevalence of Nasal Colonization with Staphylococcus aureus in the United States, 2001–2004

BACKGROUND Staphylococcus aureus is a common cause of infection, particularly in persons colonized by this organism. Virulent strains of methicillin-resistant S. aureus (MRSA) have emerged in the general community. METHODS A nationally representative survey of nasal colonization with S. aureus was conducted from 2001 through 2004 as part of the National Health and Nutrition Examination Survey. MRSA isolates were identified by the oxacillin disk-diffusion method. The pulsed-field gel electrophoresis (PFGE) type was determined for all MRSA isolates. A t statistic was used to compare the prevalence of colonization across biennia and across population subgroups. Cofactors independently associated with colonization were determined with backward stepwise logistic modeling. RESULTS The prevalence of colonization with S. aureus decreased from 32.4% in 2001-2002 to 28.6% in 2003-2004 (P < .01), whereas the prevalence of colonization with MRSA increased from 0.8% to 1.5% (P < .05). Colonization with MRSA was independently associated with healthcare exposure in males and with having been born in the United States, age > or =60 years, diabetes, and poverty in females. In 2003-2004, a total of 19.7% (95% confidence interval, 12.4%-28.8%) of MRSA-colonized persons carried a PFGE type associated with community transmission. CONCLUSIONS Nasal colonization with MRSA has increased in the United States, despite an overall decrease in nasal colonization with S. aureus. PFGE types associated with community transmission only partially account for the increase in MRSA colonization.

Prevalence of Staphylococcus aureus Nasal Colonization in the United States, 2001–2002

BACKGROUND Staphylococcus aureus is a common cause of disease, particularly in colonized persons. Although methicillin-resistant S. aureus (MRSA) infection has become increasingly reported, population-based S. aureus and MRSA colonization estimates are lacking. METHODS Nasal samples for S. aureus culture and sociodemographic data were obtained from 9622 persons > or = 1 year old as part of the National Health and Nutrition Examination Survey, 2001-2002. After screening for oxacillin susceptibility, MRSA and selected methicillin-susceptible S. aureus isolates were tested for antimicrobial susceptibility, pulsed-field gel electrophoresis clonal type, toxin genes (e.g., for Panton-Valentine leukocidin [PVL]), and staphylococcal cassette chromosome mec (SCCmec) type I-IV genes. RESULTS For 2001-2002, national S. aureus and MRSA colonization prevalence estimates were 32.4% (95% confidence interval [CI], 30.7%-34.1%) and 0.8% (95% CI, 0.4%-1.4%), respectively, and population estimates were 89.4 million persons (95% CI, 84.8-94.1 million persons) and 2.3 million persons (95% CI, 1.2-3.8 million persons), respectively. S. aureus colonization prevalence was highest in participants 6-11 years old. MRSA colonization was associated with age > or = 60 years and being female but not with recent health-care exposure. In unweighted analyses, the SCCmec type IV gene was more frequent in isolates from participants of younger age and of non-Hispanic black race/ethnicity; the PVL gene was present in 9 (2.4%) of 372 of isolates tested. CONCLUSIONS Many persons in the United States are colonized with S. aureus; prevalence rates differ demographically. MRSA colonization prevalence, although low nationally in 2001-2002, may vary with demographic and organism characteristics.

Vancomycin-Resistant Staphylococcus aureus Isolate from a Patient in Pennsylvania

ABSTRACT A vancomycin-resistant Staphylococcus aureus (VRSA) isolate was obtained from a patient in Pennsylvania in September 2002. Species identification was confirmed by standard biochemical tests and analysis of 16S ribosomal DNA, gyrA, and gyrB sequences; all of the results were consistent with the S. aureus identification. The MICs of a variety of antimicrobial agents were determined by broth microdilution and macrodilution methods following National Committee for Clinical Laboratory Standards (NCCLS) guidelines. The isolate was resistant to vancomycin (MIC = 32 μg/ml), aminoglycosides, β-lactams, fluoroquinolones, macrolides, and tetracycline, but it was susceptible to linezolid, minocycline, quinupristin-dalfopristin, rifampin, teicoplanin, and trimethoprim-sulfamethoxazole. The isolate, which was originally detected by using disk diffusion and a vancomycin agar screen plate, was vancomycin susceptible by automated susceptibility testing methods. Pulsed-field gel electrophoresis (PFGE) of SmaI-digested genomic DNA indicated that the isolate belonged to the USA100 lineage (also known as the New York/Japan clone), the most common staphylococcal PFGE type found in hospitals in the United States. The VRSA isolate contained two plasmids of 120 and 4 kb and was positive for mecA and vanA by PCR amplification. The vanA sequence was identical to the vanA sequence present in Tn1546. A DNA probe for vanA hybridized to the 120-kb plasmid. This is the second VRSA isolate reported in the United States.

Severe community-acquired pneumonia due to Staphylococcus aureus, 2003-04 influenza season

S. aureus community-acquired pneumonia has been reported from 9 states.

Characterization of Staphylococcus aureus Isolates from Nasal Cultures Collected from Individuals in the United States in 2001 to 2004

ABSTRACT 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.

The emergence of decreased susceptibility to vancomycin in Staphylococcus epidermidis.

BACKGROUND Coagulase-negative staphylococci (CNS) are the major cause of nosocomial bloodstream infection. Emergence of vancomycin resistance among CNS is a serious public health concern, because CNS usually are multidrug-resistant, and glycopeptide antibiotics, among which only vancomycin is available in the United States, are the only remaining effective therapy. In this report, we describe the first bloodstream infection in the United States associated with a Staphylococcus epidermidis strain with decreased susceptibility to vancomycin. METHODS We reviewed the hospitals microbiology records for all CNS strains, reviewed the patients medical and laboratory records, and obtained all available CNS isolates with decreased susceptibility to vancomycin. Blood cultures were processed and CNS isolates identified by using standard methods; antimicrobial susceptibility was determined by using minimum inhibitory concentration (MIC) and disk-diffusion methods. Nares cultures were obtained from exposed healthcare workers (HCWs) to identify possible colonization by CNS with decreased susceptibility to vancomycin. RESULTS The bloodstream infection by an S. epidermidis strain with decreased susceptibility to vancomycin occurred in a 49-year-old woman with carcinoma. She had two blood cultures positive for CNS; both isolates were S. epidermidis. Although susceptible to vancomycin by the disk-diffusion method (16-17 mm), the isolates were intermediate by MIC (8-6 microg/mL). The patient had received an extended course of vancomycin therapy; she died of her underlying disease. No HCW was colonized by CNS with decreased susceptibility to vancomycin. CONCLUSIONS This is the first report in the United States of bloodstream infection due to S. epidermidis with decreased susceptibility to vancomycin. Contact precautions likely played a role in preventing nosocomial transmission of this strain, and disk-diffusion methods may be inadequate to detect CNS with decreased susceptibility to vancomycin.

Accuracy of Commercial and Reference Susceptibility Testing Methods for Detecting Vancomycin-Intermediate Staphylococcus aureus

ABSTRACT We compared the results obtained with six commercial MIC test systems (Etest, MicroScan, Phoenix, Sensititre, Vitek Legacy, and Vitek 2 systems) and three reference methods (agar dilution, disk diffusion, and vancomycin [VA] agar screen [VScr]) with the results obtained by the Clinical and Laboratory Standards Institute broth microdilution (BMD) reference method for the detection of VA-intermediate Staphylococcus aureus (VISA). A total of 129 S. aureus isolates (VA MICs by previous BMD tests, ≤1 μg/ml [n = 60 strains], 2 μg/ml [n = 24], 4 μg/ml [n = 36], or 8 μg/ml [n = 9]) were selected from the Centers for Disease Control and Prevention strain collection. The results of BMD with Difco Mueller-Hinton broth were used as the standard for data analysis. Essential agreement (percent ±1 dilution) ranged from 98 to 100% for all methods except the method with the Vitek Legacy system, for which it was 90.6%. Of the six commercial MIC systems tested, the Sensititre, Vitek Legacy, and Vitek 2 systems tended to categorize VISA strains as susceptible (i.e., they undercalled resistance); the MicroScan and Phoenix systems and Etest tended to categorize susceptible strains as VISA; and the Vitek Legacy system tended to categorize VISA strains as resistant (i.e., it overcalled resistance). Disk diffusion categorized all VISA strains as susceptible. No susceptible strains (MICs ≤ 2 μg/ml) grew on the VScr, but all strains for which the VA MICs were 8 μg/ml grew on the VScr. Only 12 (33.3%) strains for which the VA MICs were 4 μg/ml grew on VScr. The differentiation of isolates for which the VA MICs were 2 or 4 μg/ml was difficult for most systems and methods, including the reference methods.

Surface Sampling Methods for Bacillus anthracis Spore Contamination

During an investigation conducted December 17–20, 2001, we collected environmental samples from a U.S. postal facility in Washington, D.C., known to be extensively contaminated with Bacillus anthracis spores. Because methods for collecting and analyzing B. anthracis spores have not yet been validated, our objective was to compare the relative effectiveness of sampling methods used for collecting spores from contaminated surfaces. Comparison of wipe, wet and dry swab, and HEPA vacuum sock samples on nonporous surfaces indicated good agreement between results with HEPA vacuum and wipe samples. However, results from HEPA vacuum sock and wipe samples agreed poorly with the swab samples. Dry swabs failed to detect spores >75% of the time they were detected by wipe and HEPA vacuum samples. Wipe samples collected after HEPA vacuum samples and HEPA vacuum samples after wipe samples indicated that neither method completely removed spores from the sampled surfaces.

Determining the significance of coagulase-negative staphylococci isolated from blood cultures at a community hospital: a role for species and strain identification.

OBJECTIVES To determine the degree to which species identification or strain relatedness assessment of successive blood culture isolates of coagulase-negative staphylococci (CNS) may improve the clinical diagnosis of bloodstream infection (BSI). SETTING 400-bed community hospital. DESIGN Prospective laboratory survey during which all CNS blood culture isolates obtained between mid-August 1996 and mid-February 1997 (study period) were saved and later identified to the species level; selected isolates were genotyped using pulsed-field gel electrophoresis at the Centers for Disease Control and Prevention (CDC). Retrospective review of medical records of 37 patients with multiple cultures positive for CNS. RESULTS During the study period, 171 patients had blood cultures positive for CNS; 130 had single positive cultures and 41 had > or =2 positive cultures. Of these 41, 23 (62%) were from patients with signs and symptoms of BSI according to CDC surveillance definitions. Species identification and strain clonality of CNS isolates from patients with > or =2 positives revealed 3 (13%) of the 23 patients did not have a consistent CNS species, and another 3 (13%) did not have a consistent genotype in the > or =2 positive cultures, suggesting that CNS from these patients probably were contaminants. Thus, species identification and strain clonality assessment reduced by 27% the number of patients with BSI diagnosed based on the presence of symptoms and > or =2 positive blood cultures. CONCLUSIONS Routine species identification and selected strain genotyping of CNS may reduce the misinterpretation of probable contaminants among patients with > or =2 positive blood cultures.