George F. Sensabaugh

Complete genome sequence of USA300, an epidemic clone of community-acquired meticillin-resistant Staphylococcus aureus

BACKGROUNDnUSA300, a clone of meticillin-resistant Staphylococcus aureus, is a major source of community-acquired infections in the USA, Canada, and Europe. Our aim was to sequence its genome and compare it with those of other strains of S aureus to try to identify genes responsible for its distinctive epidemiological and virulence properties.nnnMETHODSnWe ascertained the genome sequence of FPR3757, a multidrug resistant USA300 strain, by random shotgun sequencing, then compared it with the sequences of ten other staphylococcal strains.nnnFINDINGSnCompared with closely related S aureus, we noted that almost all of the unique genes in USA300 clustered in novel allotypes of mobile genetic elements. Some of the unique genes are involved in pathogenesis, including Panton-Valentine leucocidin and molecular variants of enterotoxin Q and K. The most striking feature of the USA300 genome is the horizontal acquisition of a novel mobile genetic element that encodes an arginine deiminase pathway and an oligopeptide permease system that could contribute to growth and survival of USA300. We did not detect this element, termed arginine catabolic mobile element (ACME), in other S aureus strains. We noted a high prevalence of ACME in S epidermidis, suggesting not only that ACME transfers into USA300 from S epidermidis, but also that this element confers a selective advantage to this ubiquitous commensal of the human skin.nnnINTERPRETATIONnUSA300 has acquired mobile genetic elements that encode resistance and virulence determinants that could enhance fitness and pathogenicity.

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

Context Researchers have recently identified multidrug-resistant USA300, a clone of community-acquired, methicillin-resistant Staphylococcus aureus (MRSA) that is resistant to multiple antibiotics. Contribution The authors demonstrate that the incidence of multidrug-resistant USA300 MRSA is highest in the areas of San Francisco where more male same-sex couples reside. The infection frequently manifests as an abscess or cellulitis in the buttocks, genitals, or perineum, and malemale sex was a risk factor. Caution Data were passively reported or retrospectively collected and are therefore subject to bias. Implication Multidrug-resistant USA300 MRSA infection is especially common among men who have sex with men. It might be sexually transmitted in this population. 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 (117). It can cause unusually severe disease, including necrotizing fasciitis, sepsis, endocarditis, and pneumonia (1823). Infections occur predominantly among healthy, community-dwelling persons who lack traditional risk factors for MRSA (9, 18, 2426). Whereas hospital-associated MRSA strains are resistant to multiple antimicrobial classes, USA300 and other community-associated MRSA strains are typically resistant to -lactams and 1 or 2 other drug classes. Older generic antimicrobials, such as clindamycin, tetracycline, or trimethoprimsulfamethoxazole, 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 -lactams, fluoroquinolones, tetracycline, macrolide, clindamycin, and mupirocin. Two of the resistance genes from this isolate, ermC and mupAwhich determine constitutive resistance to macrolides, clindamycin, and mupirocinare 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). Methods Population-Based Survey 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 physicians 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. HIV ClinicBased Study 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 patients 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 nonmultidrug-resistant USA300 infection, and 1 had a non-USA300 infection. Our estimates of risk for multidrug-resistant USA300 with malemale sex did not change meaningfully when these 7 patients with nonmultidrug-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. Community Health ClinicBased 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 10000 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 nonmultidrug-resistant USA300 infections. We genotyped the multidrug-resistant USA300 isolate of 1 clinic patient who reported malemale 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. Emergency DepartmentBased Study 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. Antimicrobial Susceptibility Testing We tested isolates for susceptibility to oxacillin, ciprofloxacin, erythromycin, tetracycline, clindamycin, trimethoprimsulfamethoxazole, 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). Molecular Analysis We genotyped isolates by using pulsed-field gel electrophoresis after SmaI-macrorestriction digest of chromosomal DNA (36), spa typing of the pol

The Arginine Catabolic Mobile Element and Staphylococcal Chromosomal Cassette mec Linkage: Convergence of Virulence and Resistance in the USA300 Clone of Methicillin-Resistant Staphylococcus aureus

The epidemic character of community-associated methicillin-resistant Staphylococcus aureus, especially the geographically widespread clone USA300, is poorly understood. USA300 isolates carry a type IV staphylococcal chromosomal cassette mec (SCCmec) element conferring beta-lactam antibiotic class resistance and a putative pathogenicity island, arginine catabolic mobile element (ACME). Physical linkage between SCCmec and ACME suggests that selection for antibiotic resistance and for pathogenicity may be interconnected. We constructed isogenic mutants containing deletions of SCCmec and ACME in a USA300 clinical isolate to determine the role played by these elements in a rabbit model of bacteremia. We found that deletion of type IV SCCmec did not affect competitive fitness, whereas deletion of ACME significantly attenuated the pathogenicity or fitness of USA300. These data are consistent with a model in which ACME enhances growth and survival of USA300, allowing for genetic hitchhiking of SCCmec. SCCmec in turn protects against exposure to beta-lactams.

Roles of 34 Virulence Genes in the Evolution of Hospital- and Community-Associated Strains of Methicillin-Resistant Staphylococcus aureus

BACKGROUNDnThe extent to which the horizontal transfer of virulence genes has contributed to the emergence of contemporary virulent strains of methicillin-resistant Staphylococcus aureus (MRSA) in hospital and community settings is poorly understood.nnnMETHODSnEpidemiologically well-characterized MRSA isolates collected over 8.5 years were genotyped and tested for the presence of 34 virulence genes.nnnRESULTSnSix strain types accounted for 88.2% of all MRSA infections. The evolution of contemporary hospital and community phenotypes within the CC8 and CC30 lineages--2 background genomes that produced historical pandemic MRSA clones--were associated with multiple horizontal acquisitions of virulence genes. The epidemic community phenotype of a CC8 strain, designated ST8:USA300, was linked to the acquisition of staphylococcal cassette chromosome (SCC)mec type IV, the genes for Panton-Valentine leukocidin (PVL), and the enterotoxin Q and K genes. Similarly, the epidemic community phenotype of a CC30 strain, ST30:USA1100, was linked to the acquisition of SCCmec type IV and the pvl genes. In contrast, the epidemic hospital phenotype of another CC30 strain, ST36:USA200, was associated with the acquisition of SCCmec type II, the enterotoxin A gene, and the toxic shock syndrome toxin 1 gene. The pvl genes appear not to be essential for the evolution OF other community-associated strains of mrsa, including ST8:USA500 and ST59:USA1000.nnnCONCLUSIONSnThe horizontal transfer of virulence genes, although infrequent, is epidemiologically associated with the emergence of new virulent strains of MRSA.

Widespread Skin and Soft-Tissue Infections Due to Two Methicillin-Resistant Staphylococcus aureus Strains Harboring the Genes for Panton-Valentine Leucocidin

ABSTRACT Infections caused by community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) are emerging as a major public health problem. CA-MRSA has been associated previously with skin and soft-tissue infection (SSTI) and with carriage of staphylococcal cassette chromosome mec (SCCmec) type IV and the Panton-Valentine leucocidin (PVL) virulence factor. To assess the clonal distribution of PVL-carrying strains and the association with SSTI in the San Francisco Bay area, we surveyed six collections of S. aureus isolates—671 isolates in all—collected between 1997 and 2002 originating from inpatient and outpatient clinical specimens and from a community-based sampling. Isolates were genotyped by pulsed-field gel electrophoresis, multilocus restriction fragment typing, and multilocus sequence typing and assayed for the PVL virulence factor. The S. aureus populations showed a high proportion of PVL-carrying strains, with frequencies ranging up to 70% in MRSA isolated from jail inmate patients and 69% in MRSA from patients receiving surgical treatment at an outpatient clinic specializing in treating SSTIs. PVL-carrying isolates were identified in nine clonal groups, but 88.5% of the PVL-carrying MRSA isolates belonged to only two clonal groups. These two clonal groups carried the SCCmec type IV resistance determinant and were more likely than other clonal groups to be recovered from SSTI sites than from other sites (P < 0.0001). There is evidence of clonal replacement over the period from 1999 to 2002, with one of these two clonal groups being supplanted by the other.

Isolation and Characterization of a Semen-Specific Protein from Human Seminal Plasma: A Potential New Marker for Semen Identification

The identification of semen is of paramount importance in the investigation of rape and other crimes involving sexual assault. The most commonly used procedures for semen identification center on the detection of sperm or the detection of prostatic acid phosphatase activity; methods involving the detection of spermine, choline, or semen antigens are less commonly employed. Unfortunately, none of these procedures is without one or more significant problems. For example, sperm will not be found in the semen of vasectomized or aspermic males; moreover, sperm are mechanically labile and their unequivocal identification in suspected semen stains is often difficult. Also, sperm are cleared from the vagina fairly rapidly and hence may not be found in postcoital vaginal washings. Thus the failure to detect sperm in suspect material by no means counterindicates semen. In the case of the acid phosphatase test, the problems are different. Acid phosphatase is not at all unique to semen or prostatic tissue; this enzyme activity is ubiquitous in nature. Moreover, there is evidence that prostatic acid phosphatase and the acid phosphatase found in normal vaginal secretions are genetically identical and that both are genetically identical to lysosomal acid phosphatase found in most tissues; therefore, the genetic basis of specificity of the acid phosphatase test is in question. The quantitative test can only be based on the extraordinarily high level of acid phosphatase activity in semen; the low levels of activity often found in postcoital vaginal washings are thus equivocal with respect to the question of semen detection. The other tests for semen identification are similarly suspect in reference to their specificity.

Community-Adapted Methicillin-Resistant Staphylococcus aureus (MRSA): Population Dynamics of an Expanding Community Reservoir of MRSA

To define methicillin-resistant Staphylococcus aureus (MRSA) reservoirs in the community and their population dynamics, we studied the molecular epidemiology of a random sample (n=490) from a collection of 2154 inpatient and outpatient MRSA isolates during a 7-year period in San Francisco. We noted a progressive replacement of type II staphylococcal chromosomal cassette (SCC)mec-bearing isolates with type IV SCCmec-bearing isolates, which coincided with >4-fold increase in methicillin resistance between 1998 and 2002. Type IV SCCmec-bearing isolates involved in the increase in methicillin resistance belonged to 4 molecular genotypes. These 4 genotypes were associated predominantly with community-onset disease, rather than hospital- or long-term-care facility-onset disease (76.9% vs. 19.4% vs. 3.7%; P=.0005), suggesting that they are not feral descendants of hospital isolates. The longitudinal results linked the dramatic increase in MRSA infections to an expanding community reservoir of MRSA genotypes with intrinsic community survival advantage.

Increasing Prevalence of Methicillin-Resistant Staphylococcus aureus Infection in California Jails

Staphylococcus aureus clinical isolates obtained from patients who were inmates of the San Francisco County jail system showed an increase in the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) from 29%, in 1997, to 74%, in 2002; 91% of the MRSA isolates carried staphylococcal chromosomal cassette mec (SCCmec) type IV. Pulsed field gel electrophoresis and multilocus sequence typing demonstrated 2 major clonal groups. One of these clonal groups is genetically indistinguishable from the strain responsible for an outbreak of MRSA in the Los Angeles County jail system in 2002.

Postcoital detection of a male-specific semen protein. Application to the investigation of rape

Identification of semen in vaginal fluid may provide documentation of sexual contact in alleged victims of rape. We describe an enzyme-linked immunosorbent assay for a semen glycoprotein of prostatic origin, designated p30. This test detects as little as 3 ng of the p30 antigen per milliliter in various body fluids. Semen from normal and vasectomized men contains high levels of p30 (mean, 1.55 mg per milliliter of seminal plasma), and urine from men contains low levels (mean, 260 ng per milliliter). However, the antigen cannot be detected in body fluids from women, including vaginal fluid and urine, suggesting that p30 may be a male-specific antigen. The p30 antigen was detectable in vaginal fluid for a mean period of 27 hours after coitus, as compared with 14 hours for prostatic acid phosphatase. Of 27 vaginal fluid samples from women who were allegedly raped in which the acid phosphatase test was negative, 7 (26 per cent) were unequivocally positive for p30 by our assay. We conclude that the assay for p30 offers a more sensitive and specific method of semen detection in rape investigation than the enzyme assay for prostatic acid phosphatase.

Population Dynamics of Nasal Strains of Methicillin-Resistant Staphylococcus aureus—and Their Relation to Community-Associated Disease Activity

BACKGROUNDnNasal carriage of methicillin-resistant Staphylococcus aureus (MRSA) plays a key role in the epidemiology and pathogenesis of disease. The purpose of this study was to determine the characteristics and dynamics of nasal strains of MRSA, as well as their relation to community-associated disease activity.nnnMETHODSnThis study is a cross-sectional survey and molecular epidemiologic analysis of nasal colonization by S. aureus in homeless and runaway youths, an underserved population at high risk for staphylococcal disease.nnnRESULTSnOf the 308 study participants, 27.6% carried S. aureus, and 6.2% carried MRSA. Subgroups of individuals with increased MRSA carriage rates were also at highest risk for community-associated MRSA infection; these subgroups included individuals with either HIV infection or AIDS, injection drug users, patients with abscesses, and those recently hospitalized. Multilocus sequence typing and pulsed-field gel electrophoresis identified 2 genotypes--ST59:P (USA1000) and ST8:S (USA300)--that accounted for 84.2% (16/19) of the MRSA isolates carried. The genotypes were distinct from nosocomial genotypes endemic in the hospital, although they originated from individuals with prior exposure to health care.nnnCONCLUSIONSnComparison of MRSA strains from asymptomatic carriers versus concurrently collected community-associated clinical strains from patients treated at local health-care facilities allowed for the identification of 3 population dynamics of nasal strains of MRSA: (1) endemic clones--for example, ST8:C and ST59:P--sustained asymptomatic carriage and infection over prolonged periods; (2) an epidemic clone, ST8:S, demonstrated enhanced capacity for rapid transmission and widespread infections; and (3) an outbreak clone, ST30:Z (USA1100), was highly infectious but exhibited poor asymptomatic transmission.