SUMMARY Staphylococcus aureus, a major human pathogen, has a collection of virulence factors and the ability to acquire resistance to most antibiotics. This ability is further augmented by constant emergence of new clones, making S. aureus a “superbug.” Clinical use of methicillin has led to the appearance of methicillin-resistant S. aureus (MRSA). The past few decades have witnessed the existence of new MRSA clones. Unlike traditional MRSA residing in hospitals, the new clones can invade community settings and infect people without predisposing risk factors. This evolution continues with the buildup of the MRSA reservoir in companion and food animals. This review focuses on imparting a better understanding of MRSA evolution and its molecular characterization and epidemiology. We first describe the origin of MRSA, with emphasis on the diverse nature of staphylococcal cassette chromosome mec (SCCmec). mecA and its new homologues (mecB, mecC, and mecD), SCCmec types (13 SCCmec types have been discovered to date), and their classification criteria are discussed. The review then describes various typing methods applied to study the molecular epidemiology and evolutionary nature of MRSA. Starting with the historical methods and continuing to the advanced whole-genome approaches, typing of collections of MRSA has shed light on the origin, spread, and evolutionary pathways of MRSA clones.

Methicillin-Resistant Staphylococcus aureus: Molecular Characterization, Evolution, and Epidemiology

Outbreaks of hospital-acquired infections caused by methicillin-resistant Staphylococcus aureus are being recognized with increasing frequency in the United States. Two thirds of outbreaks have been centered in critical care units. Infected and colonized inpatients appear to be the major institutional reservoir, and transient carriage on the hands of hospital personnel appears to be the most important mechanism of serial patient-to-patient transmission. In over 85% of hospitals into which they have been introduced, methicillin-resistant strains of S. aureus have become established as endemic nosocomial pathogens. A program designed to control a widespread outbreak in a university hospital used three surveillance methods to identify the major institutional reservoir of colonized and infected inpatients. Daily clinical laboratory surveillance, monthly prospective microbiology surveys of high-risk inpatients, and the recognition of previously infected 38%, 31%, and 31% of new cases, respectively. After control measures were instituted, the prevalence (p less than 0.001) and the number of acquisitions (p less than 0.002) of methicillin-resistant S. aureus declined over a 12-month period.

Epidemiology of Nosocomial Infections Caused by Methicillin-Resistant Staphylococcus aureus

ABSTRACT Pulsed-fieldgel electrophoresis (PFGE) is the most common genotypic method used in reference and clinical laboratories for typing methicillin-resistant Staphylococcus aureus (MRSA). Many different protocols have been developed in laboratories that have extensive experience with the technique and have established national databases. However, the comparabilities of the different European PFGE protocols for MRSA and of the various national MRSA clones themselves had not been addressed until now. This multinational European Union (EU) project has established for the first time a European database of representative epidemic MRSA (EMRSA) strains and has compared them by using a new “harmonized” PFGE protocol developed by a consensus approach that has demonstrated sufficient reproducibility to allow the successful comparison of pulsed-field gels between laboratories and the tracking of strains around the EU. In-house protocols from 10 laboratories in eight European countries were compared by each center with a “gold standard” or initial harmonized protocol in which many of the parameters had been standardized. The group found that it was not important to standardize some elements of the protocol, such as the type of agarose, DNA block preparation, and plug digestion. Other elements were shown to be critical, namely, a standard gel volume and concentration of agarose, the DNA concentration in the plug, the ionic strength and volume of running buffer used, the running temperature, the voltage, and the switching times of electrophoresis. A new harmonized protocol was agreed on, further modified in a pilot study in two laboratories, and finally tested by all others. Seven laboratories' gels were found to be of sufficiently good quality to allow comparison of the strains by using a computer software program, while two gels could not be analyzed because of inadequate destaining and DNA overloading. Good-quality gels and inclusion of an internal quality control strain are essential before attempting intercenter PFGE comparisons. A number of clonally related strains have been shown to be present in multiple countries throughout Europe. The well-known Iberian clone has been demonstrated in Belgium, Finland, France, Germany, and Spain (and from the wider HARMONY collection in Portugal, Slovenia, and Sweden). Strains from the United Kingdom (EMRSA-15 and -16) have been identified in several othercountries, and other clonally related strains have also been identified. This highlights the need for closer international collaboration to monitor the spread of current epidemic strains as well as the emergence of new ones.

Harmonization of pulsed-field gel electrophoresis protocols for epidemiological typing of strains of methicillin-resistant Staphylococcus aureus: a single approach developed by consensus in 10 European laboratories and its applicatio

Pulsed-fieldgel electrophoresis (PFGE) is the most common genotypic method used in reference and clinical laboratories for typing methicillin-resistant Staphylococcus aureus (MRSA). Many different protocols have been developed in laboratories that have extensive experience with the technique and have established national databases. However, the comparabilities of the different European PFGE protocols for MRSA and of the various national MRSA clones themselves had not been addressed until now. This multinational European Union (EU) project has established for the first time a European database of representative epidemic MRSA (EMRSA) strains and has compared them by using a new "harmonized" PFGE protocol developed by a consensus approach that has demonstrated sufficient reproducibility to allow the successful comparison of pulsed-field gels between laboratories and the tracking of strains around the EU. In-house protocols from 10 laboratories in eight European countries were compared by each center with a "gold standard" or initial harmonized protocol in which many of the parameters had been standardized. The group found that it was not important to standardize some elements of the protocol, such as the type of agarose, DNA block preparation, and plug digestion. Other elements were shown to be critical, namely, a standard gel volume and concentration of agarose, the DNA concentration in the plug, the ionic strength and volume of running buffer used, the running temperature, the voltage, and the switching times of electrophoresis. A new harmonized protocol was agreed on, further modified in a pilot study in two laboratories, and finally tested by all others. Seven laboratories' gels were found to be of sufficiently good quality to allow comparison of the strains by using a computer software program, while two gels could not be analyzed because of inadequate destaining and DNA overloading. Good-quality gels and inclusion of an internal quality control strain are essential before attempting intercenter PFGE comparisons. A number of clonally related strains have been shown to be present in multiple countries throughout Europe. The well-known Iberian clone has been demonstrated in Belgium, Finland, France, Germany, and Spain (and from the wider HARMONY collection in Portugal, Slovenia, and Sweden). Strains from the United Kingdom (EMRSA-15 and -16) have been identified in several othercountries, and other clonally related strains have also been identified. This highlights the need for closer international collaboration to monitor the spread of current epidemic strains as well as the emergence of new ones.

https://jcm.asm.org/content/jcm/41/4/1574.full.pdf

Harmonization of pulsed-field gel electrophoresis protocols for epidemiological typing of strains of methicillin-resistant Staphylococcus aureus: a single approach developed by consensus in 10 European laboratories and its application for tracing the spread of related strains.

Bacteriophage typing (BT) (World Health Organization method) has been used at the Centers for Disease Control and Prevention for over 30 years to type isolates of Staphylococcus aureus. Since studies have shown that BT patterns have poor reproducibility and because BT fails to type a high percentage (15 to 20%) of isolates, the Centers for Disease Control and Prevention has converted from using BT to using pulsed-field gel electrophoresis (PFGE) for strain typing S. aureus. We compared the results of BT with results of PFGE for typing 300 isolates of S. aureus, including strains from several well-characterized outbreaks. Ninety-six isolates were BT group I, 19 were group II, 82 were group III, 7 were group V, and 96 were nontypeable. PFGE identified subgroups within each phage group and thus was more discriminating than BT, which identified no subgroups. PFGE was able to type all isolates and distinguish related from unrelated strains of S. aureus. Our modified, standardized PFGE methodology should enable typing laboratories to obtain rapid, reliable results in 3 to 4 days when starting with an isolated colony on agar media.

Pulsed-field gel electrophoresis as a replacement for bacteriophage typing of Staphylococcus aureus.

Standardized methods are essential if phage typing of staphylococci is to be reliable and if the results obtained in different laboratories are to be compared. This paper, prepared on behalf of the Subcommittee on Phage Typing of Staphylococcus of the Nomenclature Committee of the International Association of Microbiological Societies, gives a detailed account of methods that have been found satisfactory for propagating the phages and defines a standard testing routine by which the stability of the phage preparations can be verified. Technical details are given of methods recommended for determining the phage type of staphylococci and the interpretation of differences between the types is discussed. The need for close collaboration between the epidemiologist and the laboratory worker in the use of phage typing is emphasized.

J. E. Blair

Papers

Phage typing of staphylococci