Chen, Katherine T. MD, MPH1*; Huard, Richard C. PhD2*; Della-Latta, Phyllis PhD, MSc2; Saiman, Lisa MD, MPH3
Methicillin-resistant Staphylococcus aureus (MRSA) infections, which were largely confined to hospitals and long-term care facilities, have emerged in the community.1 Community-associated MRSA infections, as opposed to health care–associated MRSA, occur in patients without traditional risk factors.2,3 In addition, community-associated MRSA strains have a susceptibility pattern with resistance to fewer classes of antimicrobial drugs and often contain specific virulence factors, such as the Panton-Valentine leukocidin.2,3 Methicillin-resistance in S aureus is mediated by the mecA gene, which is packaged in a mobile genetic element called the staphylococcal chromosomal cassette (SCC). The SCCmec types IV and V that appear to predominate among community-associated MRSA strains are smaller in genetic length than the SCCmec types I, II, and III that occur in health care–associated MRSA strains.4,5 Community-associated MRSA is increasingly recognized as a cause of serious skin and soft tissue infections, as well as necrotizing pneumonia and sepsis, with significant morbidity and mortality.6
The emergence of community-associated MRSA infections was first noted in children and adolescents.7–9 Since then, community-associated MRSA has been the cause of infections in a neonatal intensive care unit (NICU),10 among families (in infants, their siblings, and their mothers),11,12 as well as in pregnant and postpartum women.2,13 With respect to the report on community-associated MRSA infections in postpartum women, the outbreak was likely caused by a community-associated MRSA strain that was transmitted and acquired in the hospital.2 Nevertheless, the mechanisms and correlates of transmission of community-associated MRSA in these groups are not well understood. We hypothesized that methicillin-susceptible S aureus and MRSA strains would be present in the genital tract of pregnant women and could pose a pregnancy-related threat to the mother and/or neonate. To begin to address these issues, our objective was to determine the extent of methicillin-susceptible S aureus and MRSA colonization in a large cohort of pregnant women whose prenatal group B streptococcus (GBS) screening cultures were submitted to the clinical microbiology laboratory of a tertiary care facility.
MATERIALS AND METHODS
We conducted a prospective surveillance study for methicillin-susceptible S aureus and MRSA colonization in pregnant women who had been routinely screened for GBS colonization according to Centers for Disease Control and Prevention (CDC) guidelines for prevention of neonatal GBS disease.14 At the initiation of the study, the study team reviewed the CDC guidelines during a Department of Obstetrics and Gynecology Grand Rounds. Emphasis was placed on the importance of obtaining a GBS screening culture in selective broth media of both the lower vagina and rectum of pregnant women between 35 and 37 weeks of gestation and when at risk for preterm delivery. The GBS screening cultures submitted to the clinical microbiology laboratory between January 2005 and July 2005 from sites of care were cultured for GBS as standard of care. Sites of care for pregnant women included the Columbia University Medical Center (a tertiary care facility with an average of 4,000 live births annually), the Allen Pavilion (a community hospital with an average of 2,200 live births annually), and prenatal care clinics within a network of on-site and off-site clinics. This study was approved by the Columbia University Human Subjects Institutional Review Board. Approval was given to waive informed consent for the following reasons: consenting every pregnant woman who had a GBS screening culture would be impractical; all GBS screening cultures were de-identified before S aureus identification; and the study posed no risk to the patients.
The clinical microbiology laboratory processed all GBS screening cultures using current standard-of-care procedures for the detection of GBS.14 According to CDC guidelines, culture swabs were incubated overnight in selective broth medium (BBL Lim broth; Becton, Dickinson, and Company, Sparks, MD). An inoculum of the broth was then subcultured onto a blood agar plate (Columbia agar with 5% sheep blood; Becton, Dickinson, and Company) and inspected the next day for the growth of GBS. Lim broth is prepared from Todd Hewitt broth by the addition of colistin and nalidixic acid, at the recommended concentrations, plus yeast extract.
The residual Lim broth from GBS screening cultures was then processed for identification of S aureus. To derive single colonies, inoculums of approximately 10 μL of culture were streaked onto a blood agar plate and a selective differential chromogenic agar plate (BBL CHROMagar Staph aureus; Becton, Dickinson, and Company). S aureus appears on CHROMagar as mauve colonies. After a 20-hour incubation period, a single colony was picked from each individual CHROMagar plate and restreaked to a blood agar plate for further identification. If there were no mauve colonies apparent on the selective media, but there were colonies with morphologic features indicative of S aureus on the companion blood agar plate, then one of these colonies was picked and restreaked to another blood agar plate for further identification. Pure cultures of each isolate were then identified to the species level as S aureus using a S aureus-specific latex agglutination test (Staphaurex Test Latex; Remel Europe Ltd, Dartford, England) and the MicroScan Walkaway 96SI microtiter system (Dade International, Deerfield, IL).
The minimum inhibitory concentrations (MICs) of various antimicrobial agents for each S aureus isolate were determined using the MicroScan system. The MIC determinations and quality control protocols were followed in accordance with standards established by the Clinical and Laboratory Standards Institute (formerly National Committee for Clinical Laboratory Standards).15 Notably, the susceptibility of all S aureus strains with a MicroScan-derived MIC for oxacillin equal to 2, ie, one dilution below the breakpoint for resistance (4 or more), was repeated on MicroScan for confirmation.
As an added means of identifying MRSA strains, each S aureus isolate was streaked onto a CHROMagar MRSA plate (Becton, Dickinson, and Company). All MRSA strains that were identified in this study were also evaluated thereafter by several additional microbiological techniques, in parallel with MRSA control strains provided by Dr. Frank Lowy. These tests included growth on CNA (Columbia agar with 5% sheep’s blood, colistin and nalidixic acid) and Cefoxitin agar screen plates for oxacillin screen determination (each from Becton, Dickinson, and Company), as well as a specific MRSA PBP2′ latex agglutination test (Oxoid Ltd, Hants, UK). Erythromycin-inducible clindamycin resistance was assessed with a double-disk diffusion test (D-test) following Clinical and Laboratory Standards Institute testing guidelines.
All MRSA isolates and MRSA control strains were evaluated using a commercial MRSA real-time polymerase chain reaction (PCR) kit (IDI-MRSA, Infectio Diagnostic, Quebec City, Quebec, Canada), according to the manufacturer’s instructions, on a Cepheid SmartCycler instrument (Sunnyvale, CA). In addition, the SCCmec type of each MRSA isolate and MRSA control strain was determined using previously reported multiplex PCR protocols.5,16 One protocol5 identifies SCCmec types I–V and subtypes SCCmec type IV while the other16 identifies SCCmec types I–IV and subtypes SCCmec types I and III.
Community-associated MRSA isolates were defined as those that possessed the SCCmec type IV or V element. Community-associated MRSA isolates are also often susceptible to all four of the antimicrobial agents: ciprofloxacin, gentamicin, trimethoprim-sulfamethoxazole, and clindamycin. Therefore, a MRSA isolate with the above antibiotic susceptibility pattern was considered likely to be community-associated.17 Health care–associated MRSA isolates were defined as those strains that possessed the SCCmec type I, II, or III element and were usually resistant to two or more of the above antimicrobial agents.
We calculated proportions with 95% confidence intervals (CIs). The χ2 test was used to compare frequencies of categorical variables. P<.05 was considered statistically significant. We estimated prevalence odds ratios with 95% CIs for the association between GBS colonization and S aureus colonization. Analyses were performed with SPSS 13.0 (SPSS Inc, Chicago, IL).
Between January 12, 2005, and July 25, 2005, 2,971 GBS screening cultures were submitted to the Clinical Microbiology Laboratory, of which 2,963 specimens were processed for S aureus, representing a capture rate of 99.7%. As GBS screening cultures were de-identified for the study, we did not know the demographics of the study population. However, from hospital databases covering the study period, 1,894 and 1,179 live births occurred at Columbia University Medical Center and at the Allen Pavilion, respectively. During the study period, the insurance status of the mothers who delivered live births at Columbia University Medical Center was 42.5% Medicaid insurance and 57.5% private insurance, and at the Allen Pavilion, 95.3% Medicaid insurance and 4.7% private insurance. In 2004, self-reported ethnic and race categories of mothers who delivered at Columbia University Medical Center were 42.5% Hispanic or Latino, 36.7% white, 12.8% black, 6.1% Asian, and 1.9% unknown; and at the Allen Pavilion, 90.0% Hispanic or Latino, 2.3% white, 6.5% black, 0.4% Asian, and 0.8% unknown.
Among the 2,963 GBS screening cultures analyzed in this study, 743 (25.1%, 95% CI 23.5–26.7%) GBS and 507 (17.1%, 95% CI 15.7–18.5%) S aureus were recovered alone or in combination. Overall, 190 GBS screening cultures were positive for both GBS and S aureus, 553 were positive for only GBS, 318 were positive for only S aureus, and 1,902 were negative for both GBS and S aureus. Group B streptococcus colonization was significantly associated with S aureus colonization (prevalence odds ratio 2.1, 95% CI 1.7–2.5, P<.001).
Among the 507 S aureus isolates, 14 (2.8%, 95% CI 1.4–4.2%) were MRSA. Five of the MRSA isolates (Isolates 2, 3, 4, 10, and 12 in Table 1) had phenotypic characteristics of community-associated MRSA because they were susceptible to ciprofloxacin, gentamicin, trimethoprim-sulfamethoxazole, and clindamycin. Three of the MRSA isolates (Isolates 1, 6, and 9) had phenotypic characteristics of health care–associated MRSA because the isolates exhibited a multidrug-resistant phenotype. Six MRSA isolates could not be categorized as health care–associated MRSA or community-associated MRSA by phenotype alone because three were resistant to ciprofloxacin (Isolates 8, 11, and 14), one resistant to gentamicin (Isolate 5), and two resistant to clindamycin (Isolates 7 and 13). Of note, 4 of the MRSA isolates (Isolates 6, 7, 9 and 13) had erythromycin-inducible clindamycin resistance as demonstrated by the D-test.
Thirteen isolates (Isolates 2 through 14) were determined to be community-associated MRSA based on SCCmec-typing. Twelve strains were found to be SCCmec type IV, and one was type V. Further subtyping of the SCCmec type IV isolates revealed that five were SCCmec type IVa, four were SCCmec type IVb, and three could not be subtyped. Inability to subtype has been reported previously, and such isolates were found to possess novel or variant SCCmec subtypes.5,16 Isolate 1 was confirmed as an health care–associated MRSA with SCCmec type II.
In this report, we describe a large prospective surveillance study of nearly 3,000 vaginal-rectal GBS screening cultures from pregnant women, which were submitted for analysis at a single clinical microbiology laboratory. To our knowledge, no previous study has assessed the prevalence of S aureus in pregnant women using GBS screening cultures. We identified S aureus in 507 vaginal-rectal cultures of pregnant women and report isolation of MRSA from 14 specimens from these anatomical sites, of which 13 are community-associated MRSA. Understanding S aureus colonization in this population and the evolution of strain virulence is of paramount importance. Humans are a natural reservoir of S aureus, and persons colonized with S aureus are at an increased risk of becoming infected with these strains.18 S aureus is a major cause of skin, soft tissue, respiratory, bone, joint, and endovascular disorders and can also cause severe life-threatening infections such as staphylococcal toxic shock syndrome.18 In obstetrics and neonatology, S aureus is recognized as a cause of abdominal wound infections, breast abscesses, and nursery outbreaks of infection.19
We have described the prevalence (17.1%) of S aureus colonization identified in vaginal-rectal cultures from pregnant women. Studies from the 1970s showed a prevalence of vaginal S aureus in late pregnancy of less than 5%.20,21 More recent studies from the 1990s have found that 7.5% of vaginal cultures from Japanese pregnant women22 and 8.2% of perineal swabs from British pregnant women were positive for S aureus.23 We are not certain if the GBS screening cultures were contaminated with either the skin flora of the patients or of the obstetric provider. However, the CDC recommends that GBS screening cultures include sampling from lower vagina and rectal areas only. The higher prevalence we noted in our study may be a result of additional detection of S aureus colonization from the rectal area. However, in a recent study of 3,000 healthy, nonpregnant American women, just 9% had vaginal S aureus colonization, 8% had anal S aureus colonization, and 4.4% had both vaginal and anal colonization.24 The increase in prevalence of S aureus that we noted may have also resulted from increased detection secondary to the use of a selective culture medium. The Lim broth is a medium that selects for gram-positive organisms such as GBS and S aureus. Indeed, information from previous studies on the microbiological flora of the lower female genital tract has been noted to be weakened by technical limitations, such as a failure to use appropriate enriched media.25 Therefore, in addition to an actual increase in prevalence, the novel sampling protocol used in this study may also explain an increased S aureus detection rate compared with other studies. Of note, the risk factors for S aureus colonization in pregnancy remain largely uncharacterized. One study of 600 nonpregnant women did, however, indicate that black women were twice as likely to be colonized with S aureus in the genital tract as white women.26
Interestingly, we found that GBS colonization was associated with S aureus colonization. Of note, the percentage of pregnant women colonized with GBS, as determined by GBS screening cultures, was 25.1%, which is similar to both our institutional rate of 25.0% in 2003 and to the 20–30% estimate in U.S. pregnant women.27 The reasons for this association remain unknown, but other factors associated with GBS colonization could have influenced the isolation of S aureus. In vitro, GBS has been shown to inhibit many constituents of the bacterial flora of the genital tract, with the notable exceptions of S aureus, coagulase-negative staphylococci, and gram-negative organisms.28 Similarly, S aureus was found to enhance the growth of GBS and select enterobacteriaceae.29 Further studies should explore the association between GBS and S aureus.
In our study, 14 (0.5%) of 2,963 GBS screening cultures from pregnant women were positive for MRSA. Sampling of the anterior nares has indicated that the prevalence of MRSA among community members ranges from 0.2% to 2.1%.30 One study found that 1.3% of 466 nares cultures from pregnant women in Japan were colonized with MRSA, whereas MRSA was not detected in 306 vaginal cultures in the same group.22 Therefore, nares cultures cannot be considered predictive of vaginal or rectal S aureus colonization.24
Although we found the majority of MRSA isolates to fulfill our molecular definition for community-associated MRSA, the drug susceptibility patterns did not predict either community-associated MRSA or a specific SCCmec type IV subtype. The diversity of SCCmec types and subtypes and the increasing resistance of community-associated MRSA identified in this study, and now presumably present in the community, raise concerns. Persons colonized or infected with MRSA may notably serve as a reservoir for transmission of MRSA, as well.31
The fact that we have identified MRSA colonization in prenatal vaginal-rectal cultures raises additional questions related to pregnant women and their infants’ health. Persons colonized with MRSA are at higher risk for infection by these strains,32,33 and transmission of community-associated MRSA within families has been described.11 However, screening pregnant women for MRSA is not the current standard of care. Furthermore, the associated risk of infection or transmission from vaginal or rectal MRSA colonization is not known. Although there have not been any reports confirming transmission of MRSA from mother to infant either in the intrapartum or early postpartum periods, two case reports suggest the potential for such transmission. A report from France detailed a MRSA infection in a 2-day-old infant and her mother.11 Moreover, a report from our institution described a case of transmission of MRSA among a mother and three of her quadruplets.12 In both instances, the MRSA isolate was susceptible to numerous antimicrobial agents and, thus, likely a community-associated strain. Risk determinants of infant colonization with S aureus include maternal colonization, breastfeeding, and the number of siblings.34 Thus, MRSA may prove to be an emerging threat for infection in maternal and neonatal populations that requires increased study.
In summary, we detected an increased S aureus prevalence of 17.1% from the GBS screening cultures of 2,963 pregnant women. Furthermore, we found that 0.5% of such women carried MRSA, the majority of which were community-associated MRSA. A limitation of this study is the inability to perform multivariable prediction of methicillin-susceptible S aureus and MRSA vaginal-rectal colonization because the GBS screening cultures were de-identified and demographics not known before S aureus identification. However, future studies to assess risk factors associated with methicillin-susceptible S aureus and MRSA colonization and to delineate the molecular characteristics of such strains will improve our understanding of the evolving epidemiology of S aureus in mothers and their infants.
1. Chambers HF. The changing epidemiology of Staphylococcus aureus
? Emerg Infect Dis 2001;7:178–82.
2. Saiman L, O’Keefe M, Graham PL 3rd, Wu F, Said-Salim B, Kreiswirth B, et al. Hospital transmission of community-acquired methicillin-resistant Staphylococcus aureus
among postpartum women. Clin Infect Dis 2003;37:1313–9.
3. Weber JT. Community-associated methicillin-resistant Staphylococcus aureus
. Clin Infect Dis 2005;41 suppl:S269–72.
4. Charlebois ED, Perdreau-Remington F, Kreiswirth B, Bangsberg DR, Ciccarone D, Diep BA, et al. Origins of community strains of methicillin-resistant Staphylococcus aureus
[published erratum appears in Clin Infect Dis 2004;39:291]. Clin Infect Dis
5. Zhang K, McClure JA, Elsayed S, Louie T, Conly JM. Novel multiplex PCR assay for characterization and concomitant subtyping of staphylococcal cassette chromosome mec types I to V in methicillin-resistant Staphylococcus aureus.
J Clin Microbiol 2005;43:5026–33.
6. Zetola N, Francis JS, Nuermberger EL, Bishai WR. Community-acquired meticillin-resistant Staphylococcus aureus
: an emerging threat. Lancet Infect Dis 2005;5:275–86.
7. Herold BC, Immergluck LC, Maranan MC, Lauderdale DS, Gaskin RE, Boyle-Vavra S, et al. Community-acquired methicillin-resistant Staphylococcus aureus
in children with no identified predisposing risk. JAMA 1998;279:593–8.
8. Suggs AH, Maranan MC, Boyle-Vavra S, Daum RS. Methicillin-resistant and borderline methicillin-resistant asymptomatic Staphylococcus aureus
colonization in children without identifiable risk factors. Pediatr Infect Dis J 1999;18:410–4.
9. Centers for Disease Control and Prevention. Four pediatric deaths from community-acquired methicillin-resistant Staphylococcus aureus: Minnesota and North Dakota, 1997–1999. JAMA 1999;282:1123–5.
10. Healy CM, Hulten KG, Palazzi DL, Campbell JR, Baker CJ. Emergence of new strains of methicillin-resistant Staphylococcus aureus
in a neonatal intensive care unit. Clin Infect Dis 2004;39:1460–6.
11. L’Heriteau F, Lucet JC, Scanvic A, Bouvet E. Community-acquired methicillin-resistant Staphylococcus aureus
and familial transmission. JAMA 1999;282:1038–9.
12. Morel AS, Wu F, Della-Latta P, Cronquist A, Rubenstein D, Saiman L. Nosocomial transmission of methicillin-resistant Staphylococcus aureus
from a mother to her preterm quadruplet infants. Am J Infect Control 2002;30:170–3.
13. Laibl VR, Sheffield JS, Roberts S, McIntire DD, Trevino S, Wendel GD Jr. Clinical presentation of community-acquired methicillin-resistant Staphylococcus aureus
in pregnancy. Obstet Gynecol 2005;106:461–5.
14. Schrag S, Gorwitz R, Fultz-Butts K, Schuchat A. Prevention of perinatal group B streptococcal disease. Revised guidelines from CDC. MMWR Recomm Rep 2002 51(RR-11):1–22.
15. Clinical and Laboratory Standards Institute. Perfomance standards for antimicrobial susceptibility testing. Wayne (PA): CLSI;2005.
16. Oliveira DC, de Lencastre H. Multiplex PCR strategy for rapid identification of structural types and variants of the mec element in methicillin-resistant Staphylococcus aureus.
Antimicrob Agents Chemother 2002;46:2155–61.
17. Naimi TS, LeDell KH, Como-Sabetti K, Borchardt SM, Boxrud DJ, Etienne J, et al. Comparison of community- and health care-associated methicillin-resistant Staphylococcus aureus
infection. JAMA 2003;290:2976–84.
18. Lowy FD. Staphylococcus aureus
infections. N Engl J Med 1998;339:520–32.
19. Sweet LR, Gibbs R. Clinical microbiology of the female genital tract. In: Infectious diseases of the female genital tract. 4th ed. Philiadephia (PA): Lippincott Williams and Wilkins; 2002. p. 3–12.
20. Larsen B, Galask RP. Vaginal microbial flora: practical and theoretic relevance. Obstet Gynecol 1980;55 suppl:100S–13S.
21. Larsen B, Galask RP. Vaginal microbial flora: composition and influences of host physiology. Ann Intern Med 1982;96:926–30.
22. Mitsuda T, Arai K, Fujita S, Yokota S. Demonstration of mother-to-infant transmission of Staphylococcus aureus
by pulsed-field gel electrophoresis. Eur J Pediatr 1996;155:194–9.
23. Dancer SJ, Noble WC. Nasal, axillary, and perineal carriage of Staphylococcus aureus
among women: identification of strains producing epidermolytic toxin. J Clin Pathol 1991;44:681–4.
24. Parsonnet J, Hansmann MA, Delaney ML, Modern PA, Dubois AM, Wieland-Alter W, et al. Prevalence of toxic shock syndrome toxin 1-producing Staphylococcus aureus
and the presence of antibodies to this superantigen in menstruating women. J Clin Microbiol 2005;43:4628–34.
25. Larsen B, Monif GR. Understanding the bacterial flora of the female genital tract. Clin Infect Dis 2001;32:e69–77.
26. Linnemann Jr, CC Staneck JL, Hornstein S, Barden TP, Rauh JL, Bonventre PF, et al. The epidemiology of genital colonization with Staphylococcus aureus.
Ann Intern Me 1982 96:940–4.
27. Gibbs RS, Schrag S, Schuchat A. Perinatal infections due to group B streptococci. Obstet Gynecol 2004;104:1062–76.
28. Chaisilwattana P, Monif GRG. In vitro ability of the group B streptococci to inhibit gram-positive and gram-variable constituents of the bacterial flora of the female genital tract. Infect Dis Obstet Gynecol 1995;3:91–7.
29. Carson HM, LaPoint PG, Monif GRG. Interrelationships within the bacterial flora of the female genital tract. Infect Dis Obstet Gynecol 1997;5:305–9.
30. Salgado CD, Farr BM, Calfee DP. Community-acquired methicillin-resistant Staphylococcus aureus:
a meta-analysis of prevalence and risk factors. Clin Infect Dis 2003;36:131–9.
31. Bhalla A, Pultz NJ, Gries DM, Ray AJ, Eckstein EC, Aron DC, et al. Acquisition of nosocomial pathogens on hands after contact with environmental surfaces near hospitalized patients. Infect Control Hosp Epidemiol 2004;25:164–7.
32. Huang SS, Platt R. Risk of methicillin-resistant Staphylococcus aureus
infection after previous infection or colonization. Clin Infect Dis 2003;36:281–5.
33. Davis KA, Stewart JJ, Crouch HK, Florez CE, Hospenthal DR. Methicillin-resistant Staphylococcus aureus
(MRSA) nares colonization at hospital admission and its effect on subsequent MRSA infection. Clin Infect Dis 2004;39:776–82.
34. Peacock SJ, Justice A, Griffiths D, de Silva GD, Kantzanou MN, Crook D, et al. Determinants of acquisition and carriage of Staphylococcus aureus
in infancy. J Clin Microbiol 2003;41:5718–25.