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1.  Subtle genetic changes enhance virulence of methicillin resistant and sensitive Staphylococcus aureus 
BMC Microbiology  2007;7:99.
Community acquired (CA) methicillin-resistant Staphylococcus aureus (MRSA) increasingly causes disease worldwide. USA300 has emerged as the predominant clone causing superficial and invasive infections in children and adults in the USA. Epidemiological studies suggest that USA300 is more virulent than other CA-MRSA. The genetic determinants that render virulence and dominance to USA300 remain unclear.
We sequenced the genomes of two pediatric USA300 isolates: one CA-MRSA and one CA-methicillin susceptible (MSSA), isolated at Texas Children's Hospital in Houston. DNA sequencing was performed by Sanger dideoxy whole genome shotgun (WGS) and 454 Life Sciences pyrosequencing strategies. The sequence of the USA300 MRSA strain was rigorously annotated. In USA300-MRSA 2658 chromosomal open reading frames were predicted and 3.1 and 27 kilobase (kb) plasmids were identified. USA300-MSSA contained a 20 kb plasmid with some homology to the 27 kb plasmid found in USA300-MRSA. Two regions found in US300-MRSA were absent in USA300-MSSA. One of these carried the arginine deiminase operon that appears to have been acquired from S. epidermidis. The USA300 sequence was aligned with other sequenced S. aureus genomes and regions unique to USA300 MRSA were identified.
USA300-MRSA is highly similar to other MRSA strains based on whole genome alignments and gene content, indicating that the differences in pathogenesis are due to subtle changes rather than to large-scale acquisition of virulence factor genes. The USA300 Houston isolate differs from another sequenced USA300 strain isolate, derived from a patient in San Francisco, in plasmid content and a number of sequence polymorphisms. Such differences will provide new insights into the evolution of pathogens.
PMCID: PMC2222628  PMID: 17986343
2.  High Genetic Diversity among Community-Associated Staphylococcus aureus in Europe: Results from a Multicenter Study 
PLoS ONE  2012;7(4):e34768.
Several studies have addressed the epidemiology of community-associated Staphylococcus aureus (CA-SA) in Europe; nonetheless, a comprehensive perspective remains unclear. In this study, we aimed to describe the population structure of CA-SA and to shed light on the origin of methicillin-resistant S. aureus (MRSA) in this continent.
Methods and Findings
A total of 568 colonization and infection isolates, comprising both MRSA and methicillin-susceptible S. aureus (MSSA), were recovered in 16 European countries, from community and community-onset infections. The genetic background of isolates was characterized by molecular typing techniques (spa typing, pulsed-field gel electrophoresis and multilocus sequence typing) and the presence of PVL and ACME was tested by PCR. MRSA were further characterized by SCCmec typing. We found that 59% of all isolates were associated with community-associated clones. Most MRSA were related with USA300 (ST8-IVa and variants) (40%), followed by the European clone (ST80-IVc and derivatives) (28%) and the Taiwan clone (ST59-IVa and related clonal types) (15%). A total of 83% of MRSA carried Panton-Valentine leukocidin (PVL) and 14% carried the arginine catabolic mobile element (ACME). Surprisingly, we found a high genetic diversity among MRSA clonal types (ST-SCCmec), Simpson’s index of diversity = 0.852 (0.788–0.916). Specifically, about half of the isolates carried novel associations between genetic background and SCCmec. Analysis by BURP showed that some CA-MSSA and CA-MRSA isolates were highly related, suggesting a probable local acquisition/loss of SCCmec.
Our results imply that CA-MRSA origin, epidemiology and population structure in Europe is very dissimilar from that of USA.
PMCID: PMC3338755  PMID: 22558099
3.  Comparing Pulsed-Field Gel Electrophoresis with Multilocus Sequence Typing, spa Typing, Staphylococcal Cassette Chromosome mec (SCCmec) Typing, and PCR for Panton-Valentine Leukocidin, arcA, and opp3 in Methicillin-Resistant Staphylococcus aureus Isolates at a U.S. Medical Center 
Journal of Clinical Microbiology  2013;51(3):814-819.
Methicillin-resistant Staphylococcus aureus (MRSA) has become a common cause of skin infections and invasive infections in community dwellers in the United States since the late 1990s. Isolates characterized as USA300 by pulsed-field gel electrophoresis (PFGE) are the predominant strain type in these infections. USA100 and USA500 strains commonly cause health care-associated infections. We compared PFGE with a number of other methods of genotyping in a sample of 149 clinical MRSA isolates from the University of Chicago Medical Center. The 5 USA500 isolates yielded 3 spa types and 2 multilocus sequence types (MLSTs). Among the 24 USA100 isolates, 21 (88%) were of spa type t002, 19 (79%) were of ST5, 2 carried arcA and opp3, and 1 was Panton-Valentine leukocidin positive (PVL+). Among the 102 USA300 isolates, 96 (94%) were of ST8 and 94 (92%) were of spa type t008. The combination of traits that provided the best sensitivity (98%), specificity (97%), positive predictive value (PPV) (99%), and negative predictive value (NPV) (95%) for identifying USA300 isolates were the presence of the arcA gene and the presence of the PVL genes (area under the curve, 0.980; 95% confidence interval [CI], 0.955 to 1.0). PFGE did not delineate a homogeneous group of MRSA genetic backgrounds, as documented for other typing methods, particularly for USA500 and USA100 pulsotypes. Documenting the presence of arcA and PVL genes by PCR was an efficient and accurate means of identifying USA300 in a collection of MRSA isolates in which USA300 is common. None of the tested genotyping methods provided an accurate means of identifying the next most common PFGE-based backgrounds, USA100 and USA500.
PMCID: PMC3592068  PMID: 23269731
4.  Methicillin-Resistant Staphylococcus aureus, Samoa, 2007–2008 
Emerging Infectious Diseases  2011;17(6):1023-1029.
TOC Summary: A wide range of MRSA genotypes cause wound infections.
Little is known about the epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) in most Pacific Island nations. Relatively high rates of MRSA have been reported in Polynesian people living outside the Pacific Islands. To determine the prevalence and characteristics of MRSA, we assessed wound swabs from 399 persons with skin and soft tissue infection living in Samoa. MRSA was isolated from 9% of study participants; 34 of the 196 S. aureus isolates were MRSA. Five MRSA genotypes were identified; the 3 most common were USA300, the Queensland clone, and a sequence type 1 MRSA strain that shares <85% homology with the sequence type 1 MRSA strain common in the region (WA MRSA-1). The Southwest Pacific MRSA clone was identified but accounted for only 12% of MRSA isolates. The high prevalence of MRSA in Samoa provides impetus for initiatives to improve antimicrobial drug resistance surveillance, infection control, and antimicrobial drug use in Pacific Island nations.
PMCID: PMC3358195  PMID: 21749763
Staphylococcus aureus; methicillin-resistant Staphylococcus aureus; wound infection; soft tissue infection; drug resistance; bacteria; Samoa; research
5.  Detection of a New Community Genotype Methicillin-Resistant Staphylococcus aureus Clone That Is Unrelated to the USA300 Clone and That Causes Pediatric Infections in Colombia 
Journal of Clinical Microbiology  2013;51(2):661-664.
The dissemination of a clone of community genotype methicillin-resistant Staphylococcus aureus (CG-MRSA) that is related to USA300 has been reported in Latin America. We recently detected isolates of a new clone of CG-MRSA (spa type t1635 and ACME-negative) that was genetically unrelated to the USA300 clone and that causes infections in children in Colombia. This finding indicates the appearance of a new clone of CG-MRSA in our region.
PMCID: PMC3553864  PMID: 23241375
6.  Molecular characterization of Staphylococcus aureus from outpatients in the Caribbean reveals the presence of pandemic clones 
Staphylococcus aureus infections continue to pose a global public health problem. Frequently, this epidemic is driven by the successful spread of single S. aureus clones within a geographic region, but international travel has been recognized as a potential risk factor for S. aureus infections. To study the molecular epidemiology of S. aureus infections in the Caribbean, a major international tourist destination, we collected methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) isolates from community-onset infections in the Dominican Republic (n=112) and Martinique (n=143). Isolates were characterized by a combination of pulsed-field gel electrophoresis (PFGE), spa typing, and multilocus sequence typing (MLST) typing. In Martinique, MRSA infections (n=56) were mainly caused by t304-ST8 strains (n=44), whereas MSSA isolates were derived from genetically diverse backgrounds. Among MRSA strains (n=22) from the Dominican Republic, ST5, ST30, and ST72 predominated, while ST30 t665-PVL+ (30/90) accounted for a substantial number of MSSA infections. Despite epidemiological differences in sample collections from both countries, a considerable number of MSSA infections (~10%) were caused by ST5 and ST398 isolates at each site. Further phylogenetic analysis suggests the presence of lineages shared by the two countries, followed by recent genetic diversification unique to each site. Our findings also imply the frequent import and exchange of international S. aureus strains in the Caribbean.
PMCID: PMC3226914  PMID: 21789605
7.  Multiplex PCR for Rapid Detection of Staphylococcus aureus Isolates Suspected to Represent Community-Acquired Strains▿  
Journal of Clinical Microbiology  2007;46(2):582-587.
The continuous spread of community-acquired methicillin-resistant Staphylococcus aureus (caMRSA) and the introduction of these highly virulent isolates into hospitals represent increasing threats. The timely recognition of caMRSA strains is crucial for infection control purposes. Thus, we developed a PCR-based assay for the easy and rapid determination of those caMRSA clones that currently are the most prevalent in Germany and Central Europe. This assay was able to correctly identify the majority of the isolates as caMRSA of sequence type 80 (ST80), clonal complex 1 (USA400), and ST8 (USA300). In combination with spa typing-BURP (based upon repeat pattern) analysis and resistance typing, it provides a means for the extensive characterization of suspicious isolates. Thus, this assay represents a reliable tool for monitoring the emergence and spread of different caMRSA clones. The resulting information, in combination with careful interpretation of the epidemiological records, might help to prevent the further spread of those highly virulent caMRSA clones.
PMCID: PMC2238098  PMID: 18032620
8.  Rapid Change of Methicillin-Resistant Staphylococcus aureus Clones in a Chinese Tertiary Care Hospital over a 15-Year Period▿  
The incidence of methicillin-resistant Staphylococcus aureus (MRSA) has been increasing yearly at Peking Union Medical College Hospital (PUMCH). In order to understand the molecular evolution of MRSA at PUMCH, a total of 466 nonduplicate S. aureus isolates, including 302 MRSA and 164 methicillin-susceptible (MSSA) isolates recovered from 1994 to 2008 were characterized by staphylococcal cassette chromosome mec (SCCmec) typing, spa typing, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST). The 302 MRSA isolates were classified into 12 spa types and 9 sequence types (STs). During the years from 1994 to 2000, the most predominant MRSA clone was ST239-MRSA-III-spa t037. Since 2000, ST239-MRSA-III-spa t030 has rapidly replaced t037 and become the major clone. Another clone, ST5-MRSA-II-spa t002 emerged in 2002 and constantly existed at a low prevalence rate. The 164 MSSA isolates were classified into 62 spa types and 40 STs. ST398 was the most common MLST type for MSSA, followed by ST59, ST7, ST15, and ST1. Several MSSA genotypes, including ST398, ST1, ST121, and ST59, were identical to well-known epidemic community-acquired MRSA (CA-MRSA) isolates. MLST eBURST analysis revealed that the ST5, ST59, and ST965 clones coexisted in both MRSA and MSSA, which suggested that these MRSA clones might have evolved from MSSA by the acquisition of SCCmec. Two pvl-positive ST59-MRSA-IV isolates were identified as CA-MRSA according to the clinical data. Overall, our data showed that the ST239-MRSA-III-spa t037 clone was replaced by the emerging ST239-MRSA-III-spa t030 clone, indicating a rapid change of MRSA at a tertiary care hospital in China over a 15-year period.
PMCID: PMC2863666  PMID: 20176895
9.  Comparison of the DiversiLab Repetitive Element PCR System with spa Typing and Pulsed-Field Gel Electrophoresis for Clonal Characterization of Methicillin-Resistant Staphylococcus aureus▿ 
Journal of Clinical Microbiology  2011;49(4):1549-1555.
The emergence of methicillin-resistant Staphylococcus aureus (MRSA) has become an increasing problem worldwide in recent decades. Molecular typing methods have been developed to identify clonality of strains and monitor spread of MRSA. We compared a new commercially available DiversiLab (DL) repetitive element PCR system with spa typing, spa clonal cluster analysis, and pulsed-field gel electrophoresis (PFGE) in terms of discriminatory power and concordance. A collection of 106 well-defined MRSA strains from our hospital was analyzed, isolated between 1994 and 2006. In addition, we analyzed 6 USA300 strains collected in our institution. DL typing separated the 106 MRSA isolates in 10 distinct clusters and 8 singleton patterns. Clustering analysis into spa clonal complexes resulted in 3 clusters: spa-CC 067/548, spa-CC 008, and spa-CC 012. The discriminatory powers (Simpson's index of diversity) were 0.982, 0.950, 0.846, and 0.757 for PFGE, spa typing, DL typing, and spa clonal clustering, respectively. DL typing and spa clonal clustering showed the highest concordance, calculated by adjusted Rand's coefficients. The 6 USA300 isolates grouped homogeneously into distinct PFGE and DL clusters, and all belonged to spa type t008 and spa-CC 008. Among the three methods, DL proved to be rapid and easy to perform. DL typing qualifies for initial screening during outbreak investigation. However, compared to PFGE and spa typing, DL typing has limited discriminatory power and therefore should be complemented by more discriminative methods in isolates that share identical DL patterns.
PMCID: PMC3122836  PMID: 21307215
10.  Community-Associated Methicillin-Resistant Staphylococcus aureus, Iowa, USA 
Emerging Infectious Diseases  2009;15(10):1582-1589.
The proportion of invasive methicillin-resistant Staphylococcus aureus infections caused by USA300 increased in 2006.
We performed antimicrobial drug susceptibility testing and molecular typing on invasive methicillin-resistant Staphylococcus aureus (MRSA) isolates (n = 1,666) submitted to the University of Iowa Hygienic Laboratory during 1999–2006 as part of a statewide surveillance system. All USA300 and USA400 isolates were resistant to <3 non–β-lactam antimicrobial drug classes. The proportion of MRSA isolates from invasive infections that were either USA300 or USA400 increased significantly from 1999–2005 through 2006 (p<0.0001). During 2006, the incidence of invasive community-associated (CA)–MRSA infections was highest in the summer (p = 0.0004). Age <69 years was associated with an increased risk for invasive CA-MRSA infection (odds ratio [OR] 5.1, 95% confidence interval [CI] 2.06–12.64), and hospital exposure was associated with decreased risk (OR 0.07, 95% CI 0.01–0.51).
PMCID: PMC2866377  PMID: 19861049
Methicillin-resistant Staphylococcus aureus infections; MRSA; community-associated MRSA; surveillance; bloodstream infection; Iowa; bacteria; Staphylococci; research
11.  Genetic Variation in Spatio-Temporal Confined USA300 Community-Associated MRSA Isolates: A Shift from Clonal Dispersion to Genetic Evolution? 
PLoS ONE  2011;6(2):e16419.
Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) are increasingly isolated, with USA300-0114 being the predominant clone in the USA. Comparative whole genome sequencing of USA300 isolates collected in 2002, 2003 and 2005 showed a limited number of single nucleotide polymorphisms and regions of difference. This suggests that USA300 has undergone rapid clonal expansion without great genomic diversification. However, whole genome comparison of CA-MRSA has been limited to isolates belonging to USA300. The aim of this study was to compare the genetic repertoire of different CA-MRSA clones with that of HA-MRSA from the USA and Europe through comparative genomic hybridization (CGH) to identify genetic clues that may explain the successful and rapid emergence of CA-MRSA.
Materials and Methods
Hierarchical clustering based on CGH of 48 MRSA isolates from the community and nosocomial infections from Europe and the USA revealed dispersed clustering of the 19 CA-MRSA isolates. This means that these 19 CA-MRSA isolates do not share a unique genetic make-up. Only the PVL genes were commonly present in all CA-MRSA isolates. However, 10 genes were variably present among 14 USA300 isolates. Most of these genes were present on mobile elements.
The genetic variation present among the 14 USA300 isolates is remarkable considering the fact that the isolates were recovered within one month and originated from a confined geographic area, suggesting continuous evolution of this clone.
PMCID: PMC3033884  PMID: 21326601
12.  Trends in Prescribing β-Lactam Antibiotics for Treatment of Community-Associated Methicillin-Resistant Staphylococcus aureus Infections▿  
Journal of Clinical Microbiology  2007;45(12):3930-3934.
Rates of prescribing of β-lactam antibiotics as initial empirical therapy for patients with skin and soft tissue infections (SSTIs) caused by molecularly and epidemiologically characterized community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) isolates were assessed over a 3-year period. A prospectively developed database was used to calculate the prevalence of CA-MRSA SSTIs from 2004 to 2006. Molecular characterization of the MRSA isolate and medical record review for assessment of initial antimicrobial therapy were performed on a subset of patients. Among 2,636 patients with S. aureus SSTIs, the prevalence of CA-MRSA was 9% in 2004, 16% in 2005, and 21% in 2006 (P < 0.0001, chi-square test for trend). Seventy-five percent of CA-MRSA isolates tested were of the USA 300 or 400 clone type. Ninety-two percent of CA-MRSA isolates tested were positive for Panton-Valentine leukocidin, of which 90% carried staphylococcal chromosomal cassette mec type IV. The rate of use of a β-lactam antibiotic as initial empirical therapy for patients with CA-MRSA SSTIs was 86%, 77%, and 60% in 2004, 2005, and 2006, respectively (P = 0.04, chi-square test for trend). Thirty percent of β-lactam-treated patients had a documented risk factor for CA-MRSA infection. The use of a β-lactam antibiotic as initial empirical therapy for CA-MRSA SSTIs has decreased significantly over the past 3 years. However, even as the prevalence of CA-MRSA SSTIs approaches 25%, the majority of patients are still receiving inactive antimicrobial therapy. Further evaluation of the outcomes associated with discordant therapy for CA-MRSA SSTIs is needed.
PMCID: PMC2168566  PMID: 17942648
13.  Rapid Multiplex PCR Assay for Identification of USA300 Community-Associated Methicillin-Resistant Staphylococcus aureus Isolates▿  
Journal of Clinical Microbiology  2006;45(1):141-146.
Recent reports have noted a discernible increase in the number of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections in patients without traditional risk factors. In the United States, the most prominent CA-MRSA strain encodes Panton-Valentine leukocidin (PVL) cytotoxin genes, belongs to pulsed field gel electrophoresis type USA300 and multilocus sequence type 8, and carries staphylococcal cassette chromosome mec (SCCmec) type IV. At present, molecular characterization of MRSA strains, such as USA300, can be time-consuming and is often beyond the technical capability of many clinical laboratories, making routine identification difficult. We analyzed the chromosomal regions flanking the SCCmec element in 44 USA300 MRSA isolates and identified a signature “AT repeat” sequence within the conserved hypothetical gene SACOL0058 located 1.4 kb downstream of the 3′ end of the J1-SCCmec chromosomal junction. Only USA300 isolates tested contained a sequence of ≥6 AT repeats in combination with PVL (e.g., related USA500 or Iberian strains had ≥6 AT repeats but were PVL negative). Using a locked nucleic acid primer specific for ≥6 AT repeats in combination with primers to detect PVL, we developed a multiplex PCR assay specific for the identification of USA300 strains. Multiplex results were 100% concordant with DNA sequencing, suggesting that the method has promise as a means of rapidly identifying USA300 isolates.
PMCID: PMC1828973  PMID: 17093011
14.  Characterization of a Novel Arginine Catabolic Mobile Element (ACME) and Staphylococcal Chromosomal Cassette mec Composite Island with Significant Homology to Staphylococcus epidermidis ACME Type II in Methicillin-Resistant Staphylococcus aureus Genotype ST22-MRSA-IV▿ 
The arginine catabolic mobile element (ACME) is prevalent among methicillin-resistant Staphylococcus aureus (MRSA) isolates of sequence type 8 (ST8) and staphylococcal chromosomal cassette mec (SCCmec) type IVa (USA300) (ST8-MRSA-IVa isolates), and evidence suggests that ACME enhances the ability of ST8-MRSA-IVa to grow and survive on its host. ACME has been identified in a small number of isolates belonging to other MRSA clones but is widespread among coagulase-negative staphylococci (CoNS). This study reports the first description of ACME in two distinct strains of the pandemic ST22-MRSA-IV clone. A total of 238 MRSA isolates recovered in Ireland between 1971 and 2008 were investigated for ACME using a DNA microarray. Twenty-three isolates (9.7%) were ACME positive, and all were either MRSA genotype ST8-MRSA-IVa (7/23, 30%) or MRSA genotype ST22-MRSA-IV (16/23, 70%). Whole-genome sequencing and comprehensive molecular characterization revealed the presence of a novel 46-kb ACME and staphylococcal chromosomal cassette mec (SCCmec) composite island (ACME/SCCmec-CI) in ST22-MRSA-IVh isolates (n = 15). This ACME/SCCmec-CI consists of a 12-kb DNA region previously identified in ACME type II in S. epidermidis ATCC 12228, a truncated copy of the J1 region of SCCmec type I, and a complete SCCmec type IVh element. The composite island has a novel genetic organization, with ACME located within orfX and SCCmec located downstream of ACME. One PVL locus-positive ST22-MRSA-IVa isolate carried ACME located downstream of SCCmec type IVa, as previously described in ST8-MRSA-IVa. These results suggest that ACME has been acquired by ST22-MRSA-IV on two independent occasions. At least one of these instances may have involved horizontal transfer and recombination events between MRSA and CoNS. The presence of ACME may enhance dissemination of ST22-MRSA-IV, an already successful MRSA clone.
PMCID: PMC3088263  PMID: 21343442
15.  Staphylococcus aureus Colonization Among Household Contacts of Patients With Skin Infections: Risk Factors, Strain Discordance, and Complex Ecology 
Among 350 households of patients with Staphylococcus aureus skin infections, extra-nasal S. aureus colonization was common. USA300 MRSA appeared more transmissible among household members than other S. aureus strain types. Multiple S. aureus genetic backgrounds were present in many households.
Background. The USA300 methicillin resistant Staphylococcus aureus (MRSA) genetic background has rapidly emerged as the predominant cause of community-associated S. aureus infections in the U.S. However, epidemiologic characteristics of S. aureus household transmission are poorly understood.
Methods. We performed a cross-sectional study of adults and children with S. aureus skin infections and their household contacts in Los Angeles and Chicago. Subjects were surveyed for S. aureus colonization of the nares, oropharynx, and inguinal region and risk factors for S. aureus disease. All isolates underwent genetic typing.
Results. We enrolled 1162 persons (350 index patients and 812 household members). The most common infection isolate characteristic was ST8/SCCmec IV, PVL+ MRSA (USA300) (53%). S. aureus colonized 40% (137/350) of index patients and 50% (405/812) of household contacts. A nares-only survey would have missed 48% of S. aureus and 51% of MRSA colonized persons. Sixty-five percent of households had >1 S. aureus genetic background identified and 26% of MRSA isolates in household contacts were discordant with the index patients' infecting MRSA strain type. Factors independently associated (P < .05) with the index strain type colonizing household contacts were recent skin infection, recent cephalexin use, and USA300 genetic background.
Conclusions. In our study population, USA300 MRSA appeared more transmissible among household members compared with other S. aureus genetic backgrounds. Strain distribution was complex; >1 S. aureus genetic background was present in many households. S. aureus decolonization strategies may need to address extra-nasal colonization and the consequences of eradicating S. aureus genetic backgrounds infrequently associated with infection.
PMCID: PMC3348950  PMID: 22474221
16.  The Dominant Australian Community-Acquired Methicillin-Resistant Staphylococcus aureus Clone ST93-IV [2B] Is Highly Virulent and Genetically Distinct 
PLoS ONE  2011;6(10):e25887.
Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) USA300 has spread rapidly across North America, and CA-MRSA is also increasing in Australia. However, the dominant Australian CA-MRSA strain, ST93-IV [2B] appears distantly related to USA300 despite strikingly similar clinical and epidemiological profiles. Here, we compared the virulence of a recent Australian ST93 isolate (JKD6159) to other MRSA, including USA300, and found that JKD6159 was the most virulent in a mouse skin infection model. We fully sequenced the genome of JKD6159 and confirmed that JKD6159 is a distinct clone with 7616 single nucleotide polymorphisms (SNPs) distinguishing this strain from all other S. aureus genomes. Despite its high virulence there were surprisingly few virulence determinants. However, genes encoding α-hemolysin, Panton-Valentine leukocidin (PVL) and α-type phenol soluble modulins were present. Genome comparisons revealed 32 additional CDS in JKD6159 but none appeared to encode new virulence factors, suggesting that this clone's enhanced pathogenicity could lie within subtler genome changes, such as SNPs within regulatory genes. To investigate the role of accessory genome elements in CA-MRSA epidemiology, we next sequenced three additional Australian non-ST93 CA-MRSA strains and compared them with JKD6159, 19 completed S. aureus genomes and 59 additional S. aureus genomes for which unassembled genome sequence data was publicly available (82 genomes in total). These comparisons showed that despite its distinctive genotype, JKD6159 and other CA-MRSA clones (including USA300) share a conserved repertoire of three notable accessory elements (SSCmecIV, PVL prophage, and pMW2). This study demonstrates that the genetically distinct ST93 CA-MRSA from Australia is highly virulent. Our comparisons of geographically and genetically diverse CA-MRSA genomes suggest that apparent convergent evolution in CA-MRSA may be better explained by the rapid dissemination of a highly conserved accessory genome from a common source.
PMCID: PMC3185049  PMID: 21991381
17.  Community-associated Methicillin-resistant Staphylococcus aureus Bacteremia and Endocarditis among HIV Patients: A cohort study 
BMC Infectious Diseases  2011;11:298.
HIV patients are at increased risk of development of infections and infection-associated poor health outcomes. We aimed to 1) assess the prevalence of USA300 community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) among HIV-infected patients with S. aureus bloodstream infections and. 2) determine risk factors for infective endocarditis and in-hospital mortality among patients in this population.
All adult HIV-infected patients with documented S. aureus bacteremia admitted to the University of Maryland Medical Center between January 1, 2003 and December 31, 2005 were included. CA-MRSA was defined as a USA300 MRSA isolate with the MBQBLO spa-type motif and positive for both the arginine catabolic mobile element and Panton-Valentin Leukocidin. Risk factors for S. aureus-associated infective endocarditis and mortality were determined using logistic regression to calculate odds ratios (OR) and 95% confidence intervals (CI). Potential risk factors included demographic variables, comorbid illnesses, and intravenous drug use.
Among 131 episodes of S. aureus bacteremia, 85 (66%) were MRSA of which 47 (54%) were CA-MRSA. Sixty-three patients (48%) developed endocarditis and 10 patients (8%) died in the hospital on the index admission Patients with CA-MRSA were significantly more likely to develop endocarditis (OR = 2.73, 95% CI = 1.30, 5.71). No other variables including comorbid conditions, current receipt of antiretroviral therapy, pre-culture severity of illness, or CD4 count were significantly associated with endocarditis and none were associated with in-hospital mortality.
CA-MRSA was significantly associated with an increased incidence of endocarditis in this cohort of HIV patients with MRSA bacteremia. In populations such as these, in which the prevalence of intravenous drug use and probability of endocarditis are both high, efforts must be made for early detection, which may improve treatment outcomes.
PMCID: PMC3214174  PMID: 22040268
18.  Two Distinct Clones of Methicillin-Resistant Staphylococcus aureus (MRSA) with the Same USA300 Pulsed-Field Gel Electrophoresis Profile: a Potential Pitfall for Identification of USA300 Community-Associated MRSA▿  
Journal of Clinical Microbiology  2009;47(11):3765-3768.
Analysis of methicillin-resistant Staphylococcus aureus (MRSA) characterized as USA300 by pulsed-field gel electrophoresis identified two distinct clones. One was similar to community-associated USA300 MRSA (ST8-IVa, t008, and Panton-Valentine leukocidin positive). The second (ST8-IVa, t024, and PVL negative) had different molecular characteristics and epidemiology, suggesting independent evolution. We recommend spa typing and/or PCR to discriminate between the two clones.
PMCID: PMC2772603  PMID: 19759225
19.  Identification and Characterization of the Multidrug Resistance Gene cfr in a Panton-Valentine Leukocidin-Positive Sequence Type 8 Methicillin-Resistant Staphylococcus aureus IVa (USA300) Isolate▿  
Antimicrobial Agents and Chemotherapy  2010;54(12):4978-4984.
The staphylococcal cfr gene mediates resistance to phenicols, lincosamides, oxazolidinones, pleuromutilins, and streptogramin A, a phenotype that has been termed PhLOPSA. The cfr gene has mainly been associated with coagulase-negative staphylococcal isolates from animals, and only a few cfr-positive methicillin-resistant Staphylococcus aureus (MRSA) isolates have been described so far. This study reports the first description of a cfr-positive MRSA isolate (M05/0060) belonging to the pandemic Panton-Valentine leukocidin (PVL)-positive sequence type 8 MRSA IVa/USA300 (ST8-MRSA-IVa/USA300) clone. The cfr gene was detected in M05/0060 using a DNA microarray which was used to screen PVL-positive MRSA isolates for the presence of virulence genes, typing markers, and antimicrobial resistance genes. Antimicrobial susceptibility testing revealed that M05/0060 exhibited the cfr-associated resistance phenotype. Molecular analysis identified the presence of cfr and a second phenicol resistance gene, fexA, on a novel 45-kb conjugative plasmid, which was designated pSCFS7. Within pSCFS7, a DNA segment consisting of cfr, a truncated copy of insertion sequence IS21-558, and a region with homology to the DNA invertase gene bin3 of transposon Tn552 from Bacillus mycoides was integrated into the transposase gene tnpB of the fexA-carrying transposon Tn558. The emergence of a multidrug-resistant cfr-positive variant of ST8-MRSA-IVa/USA300 is alarming and requires ongoing surveillance. Moreover, the identification of a novel conjugative plasmid carrying the cfr gene indicates the ability of cfr to spread to other MRSA strains.
PMCID: PMC2981266  PMID: 20921317
20.  Looking toward basic science for potential drug discovery targets against community-associated MRSA 
Medicinal research reviews  2010;30(1):1-22.
The difficulties to find a conventional vaccine against S. aureus and the increasing resistance of S. aureus to many antibiotics demand the exploration of novel therapeutic options, such as by targeting virulence determinants and using specific antibodies in an antitoxin-like approach. Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) strains have recently emerged predominantly in the U.S., causing epidemic outbreaks of mostly skin and soft tissue infections, but also more dramatic and sometimes fatal diseases. CA-MRSA is now the most frequent cause of death by a single infectious agent in the U.S. The fact that at least in the U.S., CA-MRSA infections are almost entirely due to one sequence type, USA300, gives researchers a novel, unique chance to focus on one clone in their efforts to analyze pathogenesis in a clinically important S. aureus. While the molecular underpinnings of the exceptional virulence and transmissibility of USA300 are not yet well understood, recent findings indicate that increased expression of widespread virulence determinants and acquisition of mobile genetic elements have to be considered. Delineating the relative importance of virulence determinants in USA300 and other important clinical strains is a key endeavor needed to develop a potential antitoxin for CA-MRSA disease.
PMCID: PMC2798928  PMID: 19399829
Community-associated methicillin-resistant Staphylococcus aureus; antibiotic resistance; virulence
21.  Post-influenza Pneumonia Caused by the USA300 Community-Associated Methicillin-Resistant Staphylococcus aureus in Korea 
Journal of Korean Medical Science  2012;27(3):313-316.
Panton-Valentine leukocidin (PVL)-positive USA300 clone has been the most successful community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) clone spreading in North America. In contrast, PVL-negative ST72-CA-MRSA has been predominant in Korea, and there has been no report of infections by the USA300 strain except only one case report of perianal infection. Here, we describe the first case of pneumonia caused by the USA300 strain following pandemic influenza A (H1N1) in Korea. A 50-year-old man was admitted with fever and cough and chest radiograph showed pneumonic consolidation at the right lower lung zone. He received a ventilator support because of respiratory failure. PCR for pandemic influenza A (H1N1) in nasopharyngeal swab was positive, and culture of sputum and endotracheal aspirate grew MRSA. Typing of the isolate revealed that it was PVL-positive, ST 8-MRSA-SCCmec type IV. The analysis of the PFGE patterns showed that this isolate was the same pulsotype as the USA300 strain.
PMCID: PMC3286780  PMID: 22379344
Influenza, Human; Methicillin-Resistant Staphylococcus aureus; Pneumonia
22.  Molecular Evidence for Spread of Two Major Methicillin-Resistant Staphylococcus aureus Clones with a Unique Geographic Distribution in Chinese Hospitals▿  
Methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) is a serious problem worldwide. To investigate the molecular epidemiology of MRSA isolates in China, a total of 702 MRSA isolates collected from 18 teaching hospitals in 14 cities between 2005 and 2006 were characterized by antibiogram analysis, pulsed-field gel electrophoresis (PFGE), staphylococcal cassette chromosome mec (SCCmec) typing, and spa typing; and 102 isolates were selected for multilocus sequence typing (MLST). Overall, SCCmec type III was the most popular type and was found in 541 isolates (77.1%), followed by SCCmec type II (109/702; 15.5%). Twenty-four PFGE types were obtained among 395 isolates collected in 2005, and 18 spa types were obtained among 702 isolates. spa type t030, which corresponded to PFEG types A to E, constituted 52.0% (365/702) of all isolates, and isolates of this type were present in all 14 cities; spa type t037, which corresponded to PFGE types F and G, accounted for 25.5% (179/702) of all isolates, and isolates of this type were identified in 12 cities. The two spa genotypes belonged to sequence type 239 (ST239) and carried SCCmec type III. spa type t002, which included isolates of PFGE types L to T, made up 16.0% (112/702) of the isolates that belonged to ST5 and SCCmec type II, and isolates of this type were distributed in 12 cities. The distribution of spa types varied among the regions. spa type t002 was the most common in Dalian (53.4%) and Shenyang (44.4%); spa type t037 was predominant in Shanghai (74.8%), whereas spa type t030 was the most common in the other cities. Two isolates from Guangzhou that harbored SCCmec type IVa with ST59 and ST88 were identified as community-associated MRSA. The prevalence of the Panton-Valentine leukocidin gene was 2.3%. The data documented two major epidemic MRSA clones, ST239-MRSA-SCCmec type III and ST5-MRSA-SCCmec type II, with unique geographic distributions across China.
PMCID: PMC2630620  PMID: 19029328
23.  USA300 Methicillin-Resistant Staphylococcus aureus Bacteremia and the Risk of Severe Sepsis: Is USA300 MRSA Associated with More Severe Infections? 
USA300 methicillin-resistant Staphylococcus aureus (MRSA) is increasing as a cause of severe community-associated bacteremic infections. We assessed severe sepsis in response to infection in patients with USA300 MRSA compared to non-USA300 MRSA bacteremia.
A cohort study was conducted from 1997–2008 comparing sepsis in response to infection in 271 patients with MRSA bacteremia from four VA hospitals.
Sixty-seven (25%) patients with MRSA bacteremia were USA300 MRSA; 204 (75%) were non-USA300 MRSA. The proportion of MRSA bacteremia caused by USA300 MRSA increased over time (χ2 p<0.0001). Adjusting for age and nosocomial infection, patients with USA300 MRSA bacteremia were more likely to have severe sepsis or septic shock in response to infection than patients with non-USA300 MRSA bacteremia (adjusted Relative Risk=1.82; 95% CI: 1.16–2.87; p=0.01).
This suggests that patients with USA300 MRSA are more likely to develop severe sepsis in response to their infection, which could be due to host or bacterial differences.
PMCID: PMC3118841  PMID: 21558047
24.  An Association Between Bacterial Genotype Combined With a High-Vancomycin Minimum Inhibitory Concentration and Risk of Endocarditis in Methicillin-Resistant Staphylococcus aureus Bloodstream Infection 
Methicillin-resistant Staphylococcus aureus (MRSA) CC22 isolates with reduced vancomycin susceptibility and an increased association with endocarditis have emerged on a background of population vancomycin minimum inhibitory concentration creep and a successful MRSA control program.
Introduction. Antimicrobial resistance and bacterial virulence factors may increase the risk of hematogenous complications during methicillin-resistant Staphylococcus aureus (MRSA) bloodstream infection (BSI). This study reports on the impact of increasing vancomycin minimum inhibitory concentrations (V-MICs) and MRSA clone type on risk of hematogenous complications from MRSA BSI during implementation of an effective MRSA control program.
Methods. In sum, spa typing, staphylococcal cassette chromosome mec allotyping, and vancomycin and teicoplanin MICs were performed on 821 consecutive MRSA bloodstream isolates from 1999 to 2009. Prospectively collected data, including focus of infection, were available for 695 clinically significant cases. Logistic and multinomial logistic regression was used to determine the association between clone type, vancomycin MIC (V-MIC), and focus of infection.
Results. MRSA BSIs decreased by ∼90% during the 11 years. Typing placed isolates into 3 clonal complex (CC) groups that had different population median V-MICs (CC30, 0.5 μg/mL [n = 349]; CC22, 0.75 μg/mL [n = 272]; non-CC22/30, 1.5 μg/mL [n = 199]). There was a progressive increase in the proportion of isolates with a V-MIC above baseline median in each clonal group and a disproportionate fall in the clone group with lowest median V-MIC (CC30). In contrast, there were no increases in teicoplanin MICs. High V-MIC CC22 isolates (1.5–2 μg/mL) were strongly associated with endocarditis (odds ratio, 12; 95% confidence interval, 3.72–38.9) and with a septic metastasis after catheter-related BSI (odds ratio, 106; 95% confidence interval, 12.6–883) compared with other clone type/V-MIC combinations.
Conclusions. An interaction between clone type and V-MIC can influence the risk of endocarditis associated with MRSA BSI, implying involvement of both therapeutic and host-pathogen factors.
PMCID: PMC3275756  PMID: 22186774
25.  Role of the Accessory Gene Regulator agr in Community-Associated Methicillin-Resistant Staphylococcus aureus Pathogenesis ▿ §  
Infection and Immunity  2011;79(5):1927-1935.
The molecular basis underlying the pathogenic success of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is not completely understood, but differential gene expression has been suggested to account at least in part for the high virulence of CA-MRSA strains. Here, we show that the agr gene regulatory system has a crucial role in the development of skin infections in the most prevalent CA-MRSA strain USA300. Importantly, our data indicate that this is due to discrepancies between the agr regulon of CA-MRSA and those of hospital-associated MRSA and laboratory strains. In particular, agr regulation in strain USA300 led to exceptionally strong expression of toxins and exoenzymes, upregulation of fibrinogen-binding proteins, increased capacity to bind fibrinogen, and increased expression of methicillin resistance genes. Our findings demonstrate that agr functionality is critical for CA-MRSA disease and indicate that an adaptation of the agr regulon contributed to the evolution of highly pathogenic CA-MRSA.
PMCID: PMC3088142  PMID: 21402769

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