Staphylococcus aureus frequently colonizes individuals without causing disease. Approximately 30% to 40% of the population is colonized with S. aureus at any point in time, most often in the nares.1 Historically, most persons have been colonized with methicillin-sensitive S. aureus. However, methicillin-resistant S. aureus (MRSA) can also colonize individuals. Since community-associated (CA) MRSA was first reported, several studies have been conducted to determine the MRSA colonization rate among children. These studies have reported rates of MRSA colonization ranging from 0.6% to 22%.2-5 One recent study found an increase in MRSA colonization rates from 0.8% in 2001 to 9.2% in 2004.4 To evaluate the MRSA colonization rate in Minnesota elementary school children, we enrolled school children in grades kindergarten (K) through grade 3 in 7 schools during the years 2004/2005. To determine if MRSA colonization rates were increasing among school-aged children, the study was repeated during the 2006/2007 school year.
MATERIALS AND METHODS
Seven elementary schools located throughout the state of Minnesota were enrolled in the study. The schools were located near 7 sentinel hospital laboratories that report MRSA infection to the Minnesota Department of Health (MDH). Characteristics of these hospital laboratories have been described elsewhere.6 School administrators were contacted for study participation during the fall and winter of 2004/2005 and again during the fall and winter of 2006/2007. Four of the schools were located in the 7-county Minneapolis/Saint Paul metropolitan area, and 3 were located in greater Minnesota. The MDH Institutional Review Board, as well as the Research Review Committees of 4 school districts where review is required, approved the study.
Letters explaining the study, consent forms, and questionnaires were sent home from school with all children in grades K to 3. One school did not have kindergarten students, and for that school only children in grades 1 to 3 received study materials. Students who returned a consent form signed by a parent or guardian were allowed to participate in the study. Staff from MDH visited the schools, explained the nasal swabbing procedure to the students with signed consent forms, and collected nasal cultures on all students who agreed to have their nose swabbed.
Specimen Collection and Laboratory Methods
Rayon-tipped swabs (Fisher Healthcare, Houston, TX) with modified liquid Stuart's transport media were used to collect specimens. Swabs were placed in the anterior nares and rotated 3 times in each nostril. Swabs were plated onto mannitol salt agar and allowed to incubate at 37°C for 48 hours in ambient air. Bacterial colonies resembling S. aureus (yellow-pigmented colonies) underwent additional testing including Gram stain and tube coagulase (Difco Laboratories, Detroit, MI). Isolates identified as S. aureus were plated onto oxacillin screen agar containing 6 μg/mL oxacillin (BD BBL, Sparks, MD) according to published Clinical Laboratory Standards Institute methods.7
All presumptive MRSA isolates underwent antimicrobial susceptibility testing using a broth microdilution panel designed for MDH surveillance purposes (PML Microbiologicals, Wilsonville, OR). Clinical Laboratory Standards Institute breakpoints were used for all antimicrobials except mupirocin.8 A breakpoint of <4 μg/mL was used to determine susceptibility to mupirocin.9 All erythromycin-resistant, clindamycin-sensitive MRSA isolates were tested for inducible clindamycin resistance using the D test.7 All MRSA isolates underwent pulsed-field gel electrophoresis (PFGE) with Sma1 using methods described elsewhere.10 PFGE patterns were evaluated both visually and with BioNumerics software (Applied Maths, Kortrijk, Belgium) using the Dice coefficient. For patterns to be considered indistinguishable, they must visually appear identical and the DNA patterns must differ by less than 1.5% with respect to molecular weight. MRSA isolates were considered to be part of a pulsed-field type (PFT) if they had at least an 80% similarity value based on the Dice coefficient.10
Data were analyzed with SAS v. 8 (SAS Institute, Cary, NC) for Windows. Mantel-Haenzel testing was used for univariate analysis, and P values of 0.05 or less and odds ratios with confidence limits that excluded one were considered to indicate statistical significance.
Thirty-eight percent (773/2043) of eligible students returned a signed consent form and had a nasal sample taken during the 2004/2005 school year, and 29% (611/2,135) of eligible students participated during the 2006/2007 school year. There was no difference in participation rates between the grade levels in either school year. Slightly more participants were colonized with S. aureus in 2004/2005 compared with 2006/2007 (38% [296/773] vs 33% [199/611: OR 1.28; CI 1.02-1.62]). In both periods, students in grade 3 were more likely to be colonized with S. aureus than students in kindergarten through grade 2 (47% [97/207] vs 35% [199/566] in 2004/2005 [OR 1.63; CI 1.18-2.25] and 40% [70/176] vs 30% [129/435] in 2006/2007 [OR 1.57; CI 1.09-2.26]). Seven (2.4%) of the 296 S. aureus isolates obtained from students during the 2004/2005 school year were resistant to oxacillin, yielding an MRSA colonization rate of 0.9% (7/773). Three (1.5%) of the 199 S. aureus isolates obtained from students during the 2006/2007 school year were resistant to oxacillin, yielding an MRSA colonization rate of 0.5% (3/611). The difference in MRSA colonization rates was not significant.
Twenty-one percent (142/611) of students enrolled in the 2006/2007 school year also participated during the 2004/2005 school year. Of those students, 32% (46/142) were colonized with S. aureus during the first school swabbing, and 1 student was colonized with MRSA. Half of the students (23/46) colonized with S. aureus at the first school swabbing remained colonized with S. aureus at the second school swabbing. Ninety-six students were not colonized with S. aureus at the first school swabbing, and 72% (69/96) of those students remained noncolonized at the second school swabbing. The one student colonized with MRSA at the first school swabbing was not colonized with S. aureus at the time of the second school swabbing.
All MRSA isolates were susceptible to tetracycline, trimethoprim/sulfamethoxazole, rifampin, vancomycin, gentamicin, linezolid, and mupirocin. One 2004/2005 isolate was resistant to ciprofloxacin, and 5 isolates were resistant to erythromycin (4 from 2004/2005 and 1 from 2006/2007). All isolates were susceptible to clindamycin by broth microdilution; however, all 5 erythromycin-resistant, clindamycin-susceptible isolates demonstrated inducible clindamycin resistance by D test (Table 1).
Three (43%) of the 7 MRSA isolates from 2004/2005 and 2 (66%) of the 3 isolates from 2004/2005 had a PFGE subtype in a CA-MRSA PFT (USA400 and USA1100 in 2004/2005; USA300 and USA400 in 2006/2007). The remainder of the isolates had PFGE subtypes that were in HA-MRSA PFTs (USA100, USA200, USA800) (Table 1).10
The overall MRSA colonization rate was low in the population studied and did not increase during the 2-year period. Although MRSA colonization rates were low, the results are comparable to results of previous MRSA colonization studies in healthy children. However, we did demonstrate that a higher percentage of MRSA nasal colonization isolates was CA-MRSA strains in 2006/2007.
Our study had several limitations. The overall participation rates from the 7 schools were low during the study periods, and the colonization rates for methicillin-sensitive S. aureus and MRSA that were found may have been biased by nonparticipation. The laboratory methods used in our study may not have been sensitive enough to identify all instances of S. aureus colonization. Some researchers have reported higher S. aureus yields by using a 24-hour broth enrichment method.11 However, our S. aureus colonization rates (38% and 33%) are similar to other published studies and do not suggest that S. aureus isolation was a significant issue.1 The study population selected for our study (children in grades K-3) may not represent the ages of children most likely to have MRSA colonization. Finally, our study sought to identify MRSA nasal colonization only, and MRSA colonization may have been missed if children were colonized at nonnasal sites.
From 2004 to 2006, we noticed an increase in the number of CA-MRSA infections reported in children aged 4 to 8 years from the 7 sentinel hospitals near the enrolled schools. Fifty-two infections were reported in 2004, and 142 infections were reported in 2006. Despite an almost 300% increase in cases, we did not find an increase in MRSA colonization during the time frame studied. In addition, the proportion of CA-MRSA infections due to USA300 reported from those hospital sites increased (47% in 2004 to 74% in 2006). In comparison, 14% (1/7) of the MRSA isolates from the students in 2004/2005 were USA300, and 66% (2/3) of the MRSA isolates were USA300 in 2006/2007. Although it seemed that the USA300 colonization rates among children in the community were increasing, the total number of MRSA colonized children was too small for results to be significant. One possible explanation for the increasing rate of USA300 infections and unchanged MRSA colonization rate could be that USA300 isolates are adept at causing infections but not as successful at nasal colonization as other MRSA PFTs.
Research is needed to determine if nares is the optimal site for screening for colonization with CA-MRSA or if CA-MRSA strains are more likely to colonize nonnasal sites.
The authors would like to acknowledge the following persons who contributed to the research and preparation of this article. Minnesota Department of Health: Joanne Bartkus, John Besser, Elizabeth Boyle, Brenda Jewell, Amy Leite, Pamela LeMaster, Lindsey Lesher, Kathyrn Como-Sabetti, Richard Danila, Jane Harper, Cynthia Hickman, Allison Ishizaki, Amy Leite, Catherine Lexau, Dan Long, Anne Potts, Jean Rainbow, Maria Rubin, Terry Ristinen, Miriam Shapiro, Emily Thompson, Lori Triden, and Amy Westbrook. Local Public Health: Mary Beth Grimm, Paula Henry, and Carol Hooker. Elementary schools: Ginny Anderson, Gary Chesner, Kari Dahlquist, Jane Flower, Ann Garland, James Hess, Ann Hoxie, Frances Johnson, Angie Johnson, Thel Kocher, Tom Kusler, Fred Majeski, Charlotte Mayer, Dennis Nelson, Rebecca Patient, Berit Peterson, Joel Pourier, Marge Sanborne, Steve Schellenberg, Jerry Sparby, Nancy Stachel, Marcia Urseth, Cheryl Videen, and Thomas Watkins.
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