Emergence of Multidrug-Resistant, Community-Associated, Methicillin-Resistant Staphylococcus aureus Clone USA300 in Men Who Have Sex with Men FREE

—The Editors

Infections caused by community-associated, methicillin-resistant Staphylococcus aureus (MRSA) have become a major public health threat. A single clone of community-associated MRSA, USA300, was not seen before 2000 but is now widely disseminated in 38 U.S. states, Canada, and 9 European Union countries (1 - 17). It can cause unusually severe disease, including necrotizing fasciitis, sepsis, endocarditis, and pneumonia (18 - 23). Infections occur predominantly among healthy, community-dwelling persons who lack traditional risk factors for MRSA ((9), (18), (24 - 26)).

Whereas hospital-associated MRSA strains are resistant to multiple antimicrobial classes, USA300 and other community-associated MRSA strains are typically resistant to β-lactams and 1 or 2 other drug classes. Older generic antimicrobials, such as clindamycin, tetracycline, or trimethoprim–sulfamethoxazole, are recommended for treating less serious community-associated MRSA infections, such as uncomplicated skin and soft tissue infections ((3), (27)). However, increased use of these antimicrobials could drive the emergence of new subclones of community-associated MRSA that are multidrug resistant. Recently, Diep and colleagues (28) described a multidrug-resistant USA300 isolate that had accumulated multiple resistance genes, rendering it resistant to β-lactams, fluoroquinolones, tetracycline, macrolide, clindamycin, and mupirocin. Two of the resistance genes from this isolate, ermC and mupA—which determine constitutive resistance to macrolides, clindamycin, and mupirocin—are carried on a large conjugative plasmid called pUSA03 (28). Researchers have identified clusters of infections due to multidrug-resistant USA300 in San Francisco and Boston (29 - 30), which could complicate disease management and contribute to development of persistent or recurrent community-associated MRSA infections (31 - 32).

We report the incidence of multidrug-resistant USA300 in San Francisco and Boston among men who have sex with men, and we describe factors associated with its spread in this high-risk population, on the basis of 4 studies: a population-based survey to estimate the incidence and spatial clustering of multidrug-resistant USA300 in San Francisco; 2 clinic-based, cross-sectional studies to identify risk factors for multidrug-resistant USA300 infection; and a post hoc analysis of multidrug-resistant USA300 isolates previously collected from emergency departments (1).

We characterized MRSA isolates previously collected in a population-based survey of MRSA infections at 9 of the 10 medical centers serving San Francisco in 2004 to 2005 (Liu C, Graber CJ, Karr M, Diep BA, Basuino L, Schwartz BS, et al. A population-based study of the incidence and molecular epidemiology of methicillin-resistant-Staphylococcus aureus disease in San Francisco, 2004-5. In preparation). The medical centers used passive surveillance for MRSA; physicians submitted cultures to laboratories for identification of pathogens when patients presented with a disease that, in the physician's opinion, required culture. The 9 medical centers operate 4368 licensed hospital beds; the medical center that did not participate in the survey operates 59 licensed hospital beds. The participating medical centers initiated routine specimen collection between February and September 2004 and collected clinical MRSA specimens from unique patients (n = 3929) for 12 consecutive months. If a specimen was submitted from a patient for whom a sample was cultured earlier in the study period, we used the first specimen. We excluded 103 isolates because they came from patient nares and did not represent active infection. Of the remaining 3826 MRSA specimens, 2495 were from patients residing in San Francisco. The 3826 MRSA specimens were stratified by the medical center of origin and then by the month of specimen collection, and we used a random-number generator to select up to 8 MRSA specimens from each stratum. The stratified random sample comprised 801 nonduplicated MRSA specimens, and of these, 532 were recovered from patients residing in San Francisco. We calculated the incidence of multidrug-resistant USA300 infection in each city ZIP code on the basis of the 532 cases, and we used 2000 U.S. Census data (33) to test the association between disease incidence estimates and the proportion of male same-sex couples living in those ZIP codes.

We conducted a retrospective chart review of consecutive patients (n = 183) who had MRSA cultured from an infection site from January 2004 through June 2006 at the San Francisco General Hospital (SFGH) Positive Health Program, an outpatient HIV clinic that provides specialized HIV and AIDS care in San Francisco, California. Of the 183 specimens, 83 were collected between 1 February 2004 and 31 January 2005 as part of the population-based survey; these 83 specimens represented a subset of 1014 unique MRSA isolates identified from all SFGH sites in the population survey. Using a standardized instrument, we abstracted information about patients' demographic characteristics, male homosexual behavior, HIV viral load, CD4⁺ cell count, past culture-proven MRSA infection, past clinical presentation, and site of infection from medical records. We collected information about male homosexual behavior from the patient's clinic intake form (typically administered by a social worker), in which the patient was asked for self-identification of sexual behavior. If the clinic intake form was incomplete or missing, we classified a male patient as a man who has sex with men if an anal Papanicolaou (Pap) smear was performed at any time during his history at the clinic in the absence of indications other than anal receptive intercourse (screening anal Pap smear). Eight patients had missing sexual behavior data and no history of anal Pap smears; we classified them as men who do not have sex with men. Seven of these men had non–multidrug-resistant USA300 infection, and 1 had a non-USA300 infection. Our estimates of risk for multidrug-resistant USA300 with male–male sex did not change meaningfully when these 7 patients with non–multidrug-resistant USA300 infection were reclassified as occurring in men who have sex with men or when they were excluded from analyses (data not shown).

We also compared the proportion of patients with multidrug-resistant USA300 infection in the HIV clinic with a subset of 91 MRSA cases randomly selected from the 1014 MRSA isolates identified from SFGH in the population-based study.

We conducted retrospective chart reviews of 130 consecutive patients with MRSA infection treated at Fenway Community Health, Boston, Massachusetts, from April 2004 through March 2006. Fenway Community Health is a community-based organization that provides primary care to more than 10 000 patients annually (34). Reports have noted that a large proportion of MRSA isolates recovered from skin and soft tissue infection sites of patients seen at Fenway Community Health were resistant to multiple antimicrobial agents ((30), (31)). Using the same standardized instrument developed for the SFGH HIV clinic study, we abstracted clinical data from medical records of each patient at Fenway Community Health.

Among these patients, 3 with missing data on sexual behavior and no history of screening anal Pap smears were classified as men who do not have sex with men; these 3 patients had non–multidrug-resistant USA300 infections. We genotyped the multidrug-resistant USA300 isolate of 1 clinic patient who reported male–male sex and frequent travel to and from the Castro District in San Francisco to see whether frequent travel by men who have sex with men between the East and West coasts facilitates the clonal spread of multidrug-resistant USA300.

Because the spread of multidrug-resistant USA300 is a potential public health threat, we investigated the distribution of multidrug-resistant USA300 in the general population of patients with community-associated MRSA infections. To this end, we conducted a post hoc analysis of 212 USA300 isolates collected by Moran and colleagues (1) in August 2004 from emergency departments in 11 U.S. cities.

The study was approved by the University of California, San Francisco, Committee on Human Research and the institutional review board of Fenway Community Health; the institutions waived the informed consent process because the study involved retrospective chart reviews.

We tested isolates for susceptibility to oxacillin, ciprofloxacin, erythromycin, tetracycline, clindamycin, trimethoprim–sulfamethoxazole, gentamicin, vancomycin, linezolid, and mupirocin and interpreted the results in accordance with the Clinical and Laboratory Standards Institute guidelines (35). We performed inducible clindamycin resistance (D-zone test) by using the agar disk diffusion method in accordance with Clinical and Laboratory Standards Institute guidelines (35).

We genotyped isolates by using pulsed-field gel electrophoresis after SmaI-macrorestriction digest of chromosomal DNA (36), spa typing of the polymorphic repeat regions of protein A (37), and multilocus sequence typing of fragments of 7 housekeeping genes (38). We further defined USA300 by the presence of Panton–Valentine leukocidin genes (lukF-PV and lukS-PV) and the arginine catabolic mobile element by using polymerase chain reaction assays (28). We defined multidrug-resistant USA300 by its carriage of the multiresistance conjugative plasmid pUSA03, which was assessed by using polymerase chain reaction assays and whole plasmid DNA sequencing, as described in the Appendix.

We analyzed the population-based survey data by weighting each of the 532 cases originating from patients residing San Francisco by the inverse of the probability of being included in the sample, so that the results reflect estimates for the entire population of San Francisco (39). On the basis of each patient's ZIP code of residence, we estimated the number of new cases for each San Francisco ZIP code by using the Stata subroutine svytab, which accounted for the stratified random sampling design (Stata, version 9, College Station, Texas). By using ZIP code–derived population estimates from the 2000 U.S. Census (Summary File 3 (33)), we calculated the annual incidence rates for San Francisco as a whole and for each of its 26 ZIP codes. We used Epimap (version 3.2.2, Centers for Disease Control and Prevention, Atlanta, Georgia) to display the distribution of the ZIP code–specific incidence rates, with boundary files obtained from the Family Health Outcomes Project (40). In the 2 cross-sectional studies, we used 2-sided chi-square or Fisher exact tests to evaluate associations between multidrug-resistant USA300 infections and demographic or clinical characteristics, and we used the Cochran–Mantel–Haenszel procedure to calculate unadjusted and adjusted relative risks for multidrug-resistant USA300 infection with associated 95% CIs (Stata, version 9).

Centers for Disease Control and Prevention; University of California, Berkeley and San Francisco; and Pfizer funded this study. The funding sources had no role in the study design, data collection, data analysis, data interpretation, or writing of the report.

On the basis of a stratified random sample of 532 (21%) of 2495 San Francisco residents who had a culture-proven MRSA infection that was treated at 9 of 10 medical centers serving San Francisco, we estimated the annual incidence of USA300 infection in San Francisco to be 275 cases per 100 000 persons (CI, 256 to 295 cases per 100 000 persons). The annual incidence of multidrug-resistant USA300 infection containing multiresistance conjugative plasmid pUSA03 was 26 cases per 100 000 persons (CI, 16 to 36 cases per 100 000 persons). Eight contiguous San Francisco ZIP codes had an average incidence of multidrug-resistant USA300 of 59 cases per 100 000 persons (CI, 36 to 82 cases per 100 000 persons) (Figure), compared with 4 cases per 100 000 persons (CI, 0 to 8 cases per 100 000 persons) for the other 18 ZIP codes (relative risk, 16.1 [CI, 9.8 to 26.5]). According to 2000 U.S. Census data (33), 10.3% (4896 of 47 664) of couples in the 8 ZIP codes with high incidence of multidrug-resistant USA300 were male same-sex couples, compared with 2.2% (1771 of 81 141) in the other 18 ZIP codes (P < 0.001). The Castro District (ZIP code 94114), which had the highest percentage (25.7%) of male same-sex couples of any ZIP code in the United States, had an incidence of multidrug-resistant USA300 of 170 cases per 100 000 persons (CI, 41 to 299 cases per 100 000 persons) (Figure). Taken together, the data suggest that men who have sex with men may be at increased risk for infection with multidrug-resistant USA300.

The SFGH HIV clinic is located in a ZIP code with high prevalence of multidrug-resistant USA300 (Figure). The 183 consecutive patients with MRSA infection treated at the SFGH HIV clinic were predominantly male (85%), white (54%), and men who have sex with men (69%) and had a median age of 40 years (interquartile range, 34 to 47 years). Most (n = 179) had skin and soft tissue infections that manifested as an abscess (n = 121), cellulitis (n = 17), folliculitis (n = 18), impetigo (n = 2), ulceration (n = 6), and wound infection (n = 15). The remaining 4 patients had non–multidrug-resistant USA300 or non-USA300 infection that manifested as joint infection, acute sinusitis, bloodstream infection, and pneumonia. Forty-five (25%) patients had infections involving the buttocks, genitals, or perineum (buttocks [n = 27], scrotum [n = 6], penis [n = 5], perianal area [n = 3], pubic region [n = 2], rectum [n = 1], and vulva [n = 1]).

Of the 183 MRSA cases, 170 (93%) were USA300 and 13 (7%) were non-USA300. Of the 170 USA300 cases, 30 were multidrug-resistant USA300 (16% of all MRSA cases) and 140 were non–multidrug-resistant USA300 (76% of all cases) (Table 1). In comparison, multidrug-resistant USA300 accounted for only 2 of 91 (2%) MRSA cases randomly selected from the 1014 MRSA isolates identified from throughout SFGH in the population-based study (P < 0.001). Among the 118 men who have sex with men, the proportion of USA300 and multidrug-resistant USA300 infections that involved the buttocks, genitals, or perineum was 27% (32 persons) and 11% (13 persons), respectively.

In bivariate analyses, having male–male sex increased the risk for multidrug-resistant USA300 infection (relative risk, 12.8 [CI, 1.8 to 91.3]) (Table 2). Twenty-nine of the 30 patients with multidrug-resistant USA300 infection had a history of having male–male sex, consistent with the high incidence of multidrug-resistant USA300 observed in San Francisco ZIP codes with high percentages of male same-sex couples (Figure). The proportion of multidrug-resistant USA300 infections that involved the buttocks, genitals, or perineum was 30% (13 of 43 persons), whereas the proportion of multidrug-resistant USA300 infections that involved other sites was 14% (17 of 121 persons) (relative risk, 2.2 [CI, 1.1 to 4.1]). Other risk factors for infection with multidrug-resistant USA300 were past MRSA infection (relative risk, 3.2 [CI, 1.7 to 5.9]) and past use of clindamycin (relative risk, 2.8 [CI, 1.5 to 5.3]) and trimethoprim–sulfamethoxazole (relative risk, 2.2 [CI, 1.1 to 4.2]). In a multivariable analysis simultaneously adjusted for the 3 strongest predictors of developing multidrug-resistant USA300 infection, namely male–male sex, past MRSA infection, and past use of clindamycin, all 3 risk factors retained their strong association with multidrug-resistant USA300 infection (Table 3).

Of 130 patients with MRSA infection, 126 (97%) were infected with USA300 and 4 (3%) were infected with non-USA300; of the 126 USA300 infections, 60 were multidrug-resistant USA300 (46% of all MRSA cases) and 66 were non–multidrug-resistant USA300 (51% of all cases). Compared with the 170 patients with USA300 infection from the SFGH HIV clinic (Table 2), the 126 patients with USA300 infection from Fenway Community Health were more likely to be white (86% vs. 56%; P < 0.001); have male–male sex (96% vs. 69%; P < 0.001); and have infections involving the buttocks, genitals, or perineum (37% vs. 25%; P = 0.026). They were also less likely to have HIV infection (45% vs. 100%; P < 0.001) or to have used clindamycin in the preceding 12 months (1% vs. 20%; P < 0.001). The 1 Fenway Community Health patient who had used clindamycin in the preceding 12 months had multidrug-resistant USA300 infection. Among the 121 men who have sex with men in the Fenway Community Health sample, the proportions of those with USA300 and multidrug-resistant USA300 infections that involved the buttocks, genitals, or perineum were 39% (47 patients) and 18% (22 patients), respectively.

As in the SFGH HIV clinic population, having male–male sex was a risk factor for multidrug-resistant USA300 infection among patients in the Fenway Community Health sample (Table 2). All patients in this sample who had multidrug-resistant USA300 infection were men who had sex with men, and none of the more than 3000 men seen at this health center annually who did not have male–male sex had multidrug-resistant USA300 infection, suggesting the exclusive spread of the multidrug-resistant USA300 clone among men who have sex with men. Infection with HIV seemed to be a risk factor (Table 2). In a subgroup analysis involving only the 121 USA300-infected men who have sex with men, 33 of 56 (59%) who were HIV positive had multidrug-resistant USA300 infection compared with 27 of 65 (42%) who were HIV negative (relative risk, 1.4 [CI, 1.0 to 2.0]; P = 0.056). We could not calculate the adjusted relative risk for multidrug-resistant USA300 infection for men who have sex with men after controlling for the potential confounding effects of HIV infection because the 5 patients who did not have male–male sex also did not have multidrug-resistant USA300 infection. Nonetheless, these data suggest that although HIV infection is a risk factor for multidrug-resistant USA300 infection, having male–male sex is also a risk factor independent of HIV infection.

We noted a multidrug-resistant USA300 isolate from 1 patient treated at Fenway Community Health for multidrug-resistant USA300 infection who reported having male–male sex and a history of frequent travel to and from the Castro District, the epicenter of multidrug-resistant USA300 infection in San Francisco (Figure). The isolate has the pulsed-field gel electrophoresis subtype USA300-0114, as does the vast majority of multidrug-resistant USA300 isolates in San Francisco (28). The isolate also contained a 37136–base pair plasmid identical in nucleotide sequence to the multiresistance pUSA03 found in multidrug-resistant USA300 isolates from San Francisco (Appendix) (28), indicating that men who have sex with men and frequently travel between Boston and San Francisco may facilitate the clonal spread of multidrug-resistant USA300.

In our post hoc analysis of 212 USA300 isolates collected in August 2004 from emergency departments in 11 U.S. cities (1), 8 isolates were constitutively resistant to clindamycin. We tested these 8 clindamycin-resistant USA300 isolates and found that only 2 carried the multidrug-resistant plasmid pUSA03. One of these isolates was recovered from an 81-year-old woman from New York City, and 1 was recovered from a 37-year-old man from Los Angeles who was identified as a man who has sex with men (Talan D. Personal communication.). These findings suggest that multidrug-resistant USA300 is presently rare in the general population.

Multidrug-resistant USA300 is the first widely disseminated, community-associated, multidrug-resistant MRSA clone. Emergence of multidrug-resistant USA300 in the community suggests that the USA300 lineage can overcome the presumed fitness cost of multidrug resistance. Data from this study suggest that multidrug-resistant USA300 has spread rapidly among men who have sex with men in San Francisco and Boston and that having male–male sex seems to be a risk factor for multidrug-resistant USA300 infection independent of HIV infection. Although the use of clindamycin and mupirocin, 2 antimicrobial agents that can select for multidrug-resistant USA300 over non–multidrug-resistant USA300 strains, may have contributed to the spread of multidrug-resistant USA300 in men who have sex with men in San Francisco (Tables 1, 2, and 3), the data also suggest that antimicrobial use is not an a priori condition for the spread of multidrug-resistant USA300 given the infrequent use of clindamycin and mupirocin among the Fenway Community Health sample.

Spread of the USA300 clone among men who have sex with men is associated with high-risk behaviors, including use of methamphetamine and other illicit drugs, sex with multiple partners, participation in a group sex party, use of the Internet for sexual contacts, skin-abrading sex, and history of sexually transmitted infections (41 - 43). The same patterns of increased sexual risk behaviors among men who have sex with men—which have resulted from changes in beliefs regarding HIV disease severity with the availability of potent antiretroviral therapy—have been driving resurgent epidemics of early syphilis, rectal gonorrhea, and new HIV infections in San Francisco, Boston, and elsewhere (44 - 46). Our findings that 27% (32 of 118) of men who have sex with men from the SFGH HIV clinic and 39% (47 of 121) of those from Fenway Community Health had infections involving the buttocks, genitals, or perineum are consistent with sexual transmission of USA300 in this population. Cook and colleagues (47) recently reported MRSA infections involving the buttocks or genitoperineal area of heterosexual partners; these community-associated MRSA infections can progress to necrotizing fasciitis of the genitoperineal region (Fournier gangrene) (48). It is not clear whether the behavior potentiating these infections among men who have sex with men is anal sex (that is, dissemination of rectal carriage of community-associated MRSA), skin-abrading sexual practices, or increased frequency of intimate skin-to-skin contact; prevention messages may therefore need to suggest caution in each of these practices.

The risk for multidrug-resistant USA300 infections in the buttocks, genitals, or perineum was 30% (13 of 43) among SFGH HIV clinic patients and 47% (22 of 47) among Fenway Community Health patients, suggesting a similar role of sexual contact in the rapid dissemination of multidrug-resistant USA300. We also found that a patient treated at Fenway Community Health for infection with multidrug-resistant USA300 who had a history of frequent travel to and from the Castro District in San Francisco had a multidrug-resistant USA300 clone identical to that isolated from San Francisco patients. Since the beginning of the AIDS epidemic, sexual contact by men who have sex with men from Boston with partners from New York City, Los Angeles, and San Francisco has been associated with the dissemination of infectious pathogens (49). Because travel history is not frequently documented in patients' charts, it is not clear to what extent contacts between men who have sex with men from Boston and San Francisco could have contributed to the clonal dissemination of multidrug-resistant USA300. However, the genotype of multidrug-resistant USA300 in the patient from Fenway Community Health and the recent marked predominance of multidrug-resistant USA300 in Boston suggest that the multidrug-resistant USA300 epidemic probably started in San Francisco and has been disseminated by the frequent cross-coastal travel of men who have sex with men. The recent emergence of multidrug-resistant, community-associated MRSA with similar antimicrobial susceptibility profiles to multidrug-resistant USA300 was recently noted among men who have sex with men in New York City (32) and Los Angeles (Miller LG, Diep BA. Unpublished data.), indicating the potential for rapid, nationwide dissemination of multidrug-resistant USA300 among men who have sex with men.

The high incidence of multidrug-resistant USA300 among men who have sex with men has major implications for empirical treatment of skin infections in these patients. Several important antimicrobial classes for treatment of MRSA infections or eradication of colonization, including clindamycin, tetracycline, and mupirocin, may not be effective in this population. Resistance to trimethoprim–sulfamethoxazole and rifampin remains rare among USA300 isolates and was not seen in multidrug-resistant USA300 in our study (Table 1). However, prophylactic antimicrobial use has selected for the emergence of trimethoprim–sulfamethoxazole resistance in subclones genetically related to the USA300 lineage in patients from San Francisco and New York City who were infected with HIV (50 - 52). Prudent use of these antimicrobial agents for suspected MRSA disease in men who have sex with men is advisable to slow the emergence of even more resistant community-associated MRSA. The pUSA03 plasmid that determines multidrug resistance in multidrug-resistant USA300 belongs to a highly promiscuous class of conjugative plasmids that could readily accept transposons encoding resistance to aminoglycosides, trimethoprim, vancomycin, and other antimicrobials, potentiating the emergence of even more resistant community-associated MRSA ((28), (53)).

Our study has limitations. Our incidence estimates for San Francisco come from a passive surveillance system and may underestimate the incidence of true infection. We relied on retrospective chart review for identification of risk factors for multidrug-resistant USA300 infection in the 2 clinic populations; because data were not collected or documented systematically, our estimates of risk may be influenced by selection, referral, documentation, or other biases. Specific sexual behaviors were not assessed or documented in clinic charts; we therefore cannot comment on the association between multidrug-resistant USA300 infection and specific male–male sexual practices. Finally, because the number of multidrug-resistant USA300 infection within risk factor subgroups was small, some of our higher estimates of risk are statistically compatible with more modest risk increases (that is, many of the CIs are wide and their lower bounds approach 1.0).

In summary, we show that multidrug-resistant USA300 has emerged as an important source of disease among men who have sex with men in 2 geographically distinct communities. The high proportion of infection involving the buttocks, genitals, and perineum suggests that community-associated MRSA may be transmitted in the setting of sexual contact among men who have sex with men. The link among USA300, multidrug-resistant USA300, and unsafe sexual risk behaviors should be evaluated further in prospective studies.

We tested for multiresistance conjugative plasmid pUSA03 by using polymerase chain reaction assays designed to detect ermC, mupA, and the conjugative gene transfer genes traE and traI(28). These assays used the following oligonucleotides: mupA–F, 5′-CTTAGTTAACTCAGCATCAG-3′; mupA–R, 5′-GGTTTGATAGCGGCTCTATGC-3′; ermC–F, 5′-GAAATCGGCTCAGGAAAAGG-3′; ermC–R, 5′-GCTATTCACTTTAGGTTTAGGATG-3′; traE-F, 5′-AACAAATGCGTACTACAGACC-3′; traE–R, 5′-CCTGCTGTTGCTGTATCC-3′; traI–F, 5′-ACCGATATGAATAACACCGTC-3′; traI–R, 5′-AAACCTTCACAAGCAATGGAC-3′.

By using whole-plasmid DNA sequencing, we found that the pUSA03 plasmid from the Fenway Community Health patient with multidrug-resistant USA300 infection who had a frequent history of travel to and from the Castro District was identical in nucleotide sequence to the multiresistance pUSA03 found in multidrug-resistant USA300 isolates from San Francisco (GenBank Accession no. CP000258) (28). In brief, we prepared plasmid DNA by using the Qiagen Plasmid Midi Kit (Qiagen, Valencia, California). We sequenced 250 ng of plasmid DNA directly with 2.0 pmol of primers by using an Applied Biosystems 96-capillary 3730xl DNA Analyzers (DNA Sequencing Facility, University of California, Berkeley, California). Primers (n = 86) were designed to provide complete sequence coverage of the prototypical pUSA03 nucleotide sequence (28); these primers are described in detail in the Appendix Table.