Richard V. Goering

Characterization of a Strain of Community-Associated Methicillin-Resistant Staphylococcus aureus Widely Disseminated in the United States

ABSTRACT A highly stable strain of Staphylococcus aureus with a pulsed-field gel electrophoresis type of USA300 and multilocus sequence type 8 has been isolated from patients residing in diverse geographic regions of the United States. This strain, designated USA300-0114, is a major cause of skin and soft tissue infections among persons in community settings, including day care centers and correctional facilities, and among sports teams, Native Americans, men who have sex with men, and military recruits. The organism is typically resistant to penicillin, oxacillin, and erythromycin (the latter mediated by msrA) and carries SCCmec type IVa. This strain is variably resistant to tetracycline [mediated by tet(K)]; several recent isolates have decreased susceptibility to fluoroquinolones. S. aureus USA300-0114 harbors the genes encoding the Panton-Valentine leucocidin toxin. DNA sequence analysis of the direct repeat units within the mec determinant of 30 USA300-0114 isolates revealed differences in only a single isolate. Plasmid analysis identified a common 30-kb plasmid that hybridized with blaZ and msrA probes and a 3.1-kb cryptic plasmid. A 4.3-kb plasmid encoding tet(K) and a 2.6-kb plasmid encoding ermC were observed in a few isolates. DNA microarray analysis was used to determine the genetic loci for a series of virulence factors and genes associated with antimicrobial resistance. Comparative genomics between USA300-0114 and three other S. aureus lineages (USA100, USA400, and USA500) defined a set of USA300-0114-specific genes, which may facilitate the strains pathogenesis within diverse environments.

How to Select and Interpret Molecular Strain Typing Methods for Epidemiological Studies of Bacterial Infections: A Review for Healthcare Epidemiologists

Strain typing is an integral part of epidemiological investigations of nosocomial infections. Methods for distinguishing among bacterial strains have improved dramatically over the last 5 years, due mainly to the introduction of molecular technology. Although not all molecular techniques are equally effective for typing all organisms, pulsed-field gel electrophoresis is the technique currently favored for most nosocomial pathogens. Criteria to aid epidemiologists in interpreting results have been published. Nucleic acid amplification-based typing methods also are applicable to many organisms and can be completed within a single day, but interpretive criteria still are under debate. Strain typing cannot be used to replace a sound epidemiological investigation, but serves as a useful adjunct to such investigations.

Classification of staphylococcal cassette chromosome mec (SCCmec) : guidelines for reporting novel SCCmec elements.

Classification of staphylococcal cassette chromosome mec (SCCmec) : guidelines for reporting novel SCCmec elements.

Methicillin-resistant Staphylococcus aureus strain USA300: origin and epidemiology

Methicillin-resistant Staphylococcus aureus (MRSA) PFGE strain type USA300 (multilocus sequence type 8, clonal complex 8, staphylococcal cassette chromosome mec type IV) was first reported in the USA as a cause of skin and soft issue infection among college football players in Pennsylvania and among prisoners in Missouri in 2000. Over the next 5 years, USA300 became the predominant community-associated MRSA strain in the USA. It was the most common PFGE type recovered from skin and soft tissue infections in persons presenting to 11 emergency departments across the USA, and caused outbreaks in Native American populations, children in daycare centres, military recruits, prison inmates and among men who have sex with men. Although predominantly a cause of skin and soft issue infection, USA300 isolates also have been recovered from cases of invasive disease including bacteraemia, endocarditis, severe necrotizing pneumonia and osteomyelitis. Isolates of USA300 usually carry the genes encoding the Panton-Valentine leucocidin and the arginine catabolic mobile element, but rarely carry staphylococcal enterotoxin genes. USA300 isolates are becoming more resistant to antimicrobial agents, including erythromycin, levofloxacin, mupirocin and tetracycline, and have spread to Europe, South America and Australia. The emergence of the MRSA USA300 strain type represents a unique biological success story.

Frequent emergence and limited geographic dispersal of methicillin-resistant Staphylococcus aureus

A small number of clonal lineages dominates the global population structure of methicillin-resistant Staphylococcus aureus (MRSA), resulting in the concept that MRSA has emerged on a few occasions after penicillinase-stable β-lactam antibiotics were introduced to clinical practice, followed by intercontinental spread of individual clones. We investigated the evolutionary history of an MRSA clone (ST5) by mutation discovery at 108 loci (46 kb) within a global collection of 135 isolates. The SNPs that were ascertained define a radial phylogenetic structure within ST5 consisting of at least 5 chains of mutational steps that define geographically associated clades. These clades are not concordant with previously described groupings based on staphylococcal protein A gene (spa) typing. By mapping the number of independent imports of the staphylococcal cassette chromosome methicillin-resistance island, we also show that import has occurred on at least 23 occasions within this single sequence type and that the progeny of such recombinant strains usually are distributed locally rather than globally. These results provide strong evidence that geographical spread of MRSA over long distances and across cultural borders is a rare event compared with the frequency with which the staphylococcal cassette chromosome island has been imported.

Application of Molecular Techniques to the Study of Hospital Infection

SUMMARY Nosocomial infections are an important source of morbidity and mortality in hospital settings, afflicting an estimated 2 million patients in United States each year. This number represents up to 5% of hospitalized patients and results in an estimated 88,000 deaths and 4.5 billion dollars in excess health care costs. Increasingly, hospital-acquired infections with multidrug-resistant pathogens represent a major problem in patients. Understanding pathogen relatedness is essential for determining the epidemiology of nosocomial infections and aiding in the design of rational pathogen control methods. The role of pathogen typing is to determine whether epidemiologically related isolates are also genetically related. To determine molecular relatedness of isolates for epidemiologic investigation, new technologies based on DNA, or molecular analysis, are methods of choice. These DNA-based molecular methodologies include pulsed-field gel electrophoresis (PFGE), PCR-based typing methods, and multilocus sequence analysis. Establishing clonality of pathogens can aid in the identification of the source (environmental or personnel) of organisms, distinguish infectious from noninfectious strains, and distinguish relapse from reinfection. The integration of molecular typing with conventional hospital epidemiologic surveillance has been proven to be cost-effective due to the associated reduction in the number of nosocomial infections. Cost-effectiveness is maximized through the collaboration of the laboratory, through epidemiologic typing, and the infection control department during epidemiologic investigations.

Pulsed Field Gel Electrophoresis: A Review of Application and Interpretation in the Molecular Epidemiology of Infectious Disease

Over the years, approaches to the epidemiological analysis of infectious disease have undergone a remarkable evolutionary transition moving from phenotypic to molecular in nature. As discussed here, the quest for a clearer comparison of genomic relatedness between bacterial clinical isolates has involved four generations of molecular iteration. First generation plasmid analysis gave way to a second generation use of restriction enzymes and probes. This was followed by third generation pulsed field gel electrophoresis (PFGE) and PCR-based methods with movement now to fourth-generation DNA sequence-based approaches. Remarkably, despite (or perhaps because of) its more than 20-year history as a typing method, PFGE has demonstrated exceptional staying power. The reasons for this endurance as well as the pros and cons of PFGE use are examined in this review. In broad context the history and technology behind PFGE are considered. Issues commonly influencing the quality of PFGE data and its analysis are discussed. Specifics regarding the mechanics of DNA preparation, restriction-enzyme digestion, and proper conditions for electrophoresis are detailed and, most importantly for any approach to epidemiological assessment, issues regarding the analysis and interpretation of PFGE data are explored.

Colonization with penicillin-resistant Streptococcus pneumoniae in a child-care center

We obtained nasopharyngeal cultures for Streptococcus pneumoniae from 54 children ages 2 to 24 months attending an Omaha child-care center (CCC) in April 1994. Thirty-two (59%) of the 54 children were colonized with S. pneumoniae belonging to serotypes 23, 19, 6 and 11. Seventeen (53%) of the pneumococcal isolates were highly resistant to penicillin (minimal inhibitory concentration ≥2.0 mUg/ml; HR-SP) and 7 (22%) were intermediately resistant to penicillin (0.12 ≤minimal inhibitory concentration ≤ 1.0 mUg/ml; IR-SP). Within each pneumococcal capsular serotype, there were 1 to 3 DNA subtypes based on pulsed field gel electrophoresis analysis. A single pulsed field gel electrophoresis strain predominated in most CCC rooms, suggesting horizontal transmission among cohorted children. Nasopharyngeal cultures obtained 4 months later revealed similar S. pneumoniae colonization rates (28 of 52, 54%); however, only 2 (7%) of 28 isolates were HR-SP and 11 (39%) were IR-SP. Colonization with resistant pneumococci persisted after 4 months in 4 (12%) of 34 children cultured on both occasions. Antibiotic use by attendees had decreased notably between the two sampling periods, suggesting that selective pressure within the CCC might contribute to seasonal variation in colonization rates with HR-SP and IR-SP. We conclude that multiple genetic clones of penicillin-resistant pneumococci can occur simultaneously in a single CCC, especially during periods of heavy antibiotic selection pressure. However, individual clones of penicillin-resistant S. pneumoniae may be spread

Guidelines for Reporting Novel mecA Gene Homologues

Methicillin-resistant staphylococci are disseminated all over the world and are frequent causes of health care- and community-associated infections. Methicillin-resistant strains typically carry the acquired mecA gene that encodes a low-affinity penicillin-binding protein (PBP), designated PBP2a or

Epidemiologic Distribution of the Arginine Catabolic Mobile Element among Selected Methicillin-Resistant and Methicillin-Susceptible Staphylococcus aureus Isolates

ABSTRACT We tested 214 Staphylococcus aureus isolates for the arcA locus of the arginine catabolic mobile element (ACME). All USA300 SCCmec IVa isolates, but no isolates containing other SCCmec subtypes, were arcA positive. arcA was also detected in selected methicillin-susceptible USA300 and methicillin-resistant USA100 isolates. DNA sequence analysis confirmed the integration of ACME in orfX.