Pulsed-field gel 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,

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We analyzed a representative sample of methicillin-resistant Staphylococcus aureus (MRSA) from 11 European countries (referred to as the HARMONY collection) using three molecular typing methods used within the HARMONY group to examine their usefulness for large, multicenter MRSA surveillance networks that use these different laboratory methodologies. MRSA isolates were collected based on their prevalence in each center and their genetic diversity, assessed by pulsed-field gel electrophoresis (PFGE). PFGE groupings (<3 bands difference between patterns) were compared to those made by sequencing of the variable repeats in the protein A gene spa and clonal designations based on multilocus sequence typing (MLST), combined with PCR analysis of the staphylococcal chromosome cassette containing the mec genes involved in methicillin resistance (SCCmec). A high level of discrimination was achieved using each of the three methodologies, with discriminatory indices between 89.5% and 91.9% with overlapping 95% confidence intervals. There was also a high level of concordance of groupings made using each method. MLST/SCCmec typing distinguished 10 groups containing at least two isolates, and these correspond to the majority of nosocomial MRSA clones described in the literature. PFGE and spa typing resolved 34 and 31 subtypes, respectively, within these 10 MRSA clones, with each subtype differing only slightly from the most common pattern using each method. The HARMONY group has found that the methods used in this study differ in their availability and affordability to European centers involved in MRSA surveillance. Here, we demonstrate that the integration of such technologies is achievable, although common protocols (such as we have developed for PFGE) may also be important, as is the use of centralized Internet sites to facilitate data analysis. PFGE and spa-typing data from analysis of MRSA isolates from the many centers that have access to the relevant equipment can be compared to reference patterns/sequences, and clonal designations can be made. In the majority of cases, these will correspond to those made by the (more expensive) method of choice-MLST/SCCmec typing-and these alternative methods can therefore be used as frontline typing systems for multicenter surveillance of MRSA.Methicillin-resistant Staphylococcus aureus (MRSA) is among the most common nosocomial pathogens globally and is generally acknowledged as the most significant due to the burden of disease it causes and to the evolution and global spread of multidrug-resistant clones. MRSA isolation rates in the United States, parts of Europe, and Asia have been increasing for more than 4 decades (36), and recent figures show that in some areas Ͼ50% of S. aureus bacteremias are caused by MRSA (4, 5, 6). Emerging intermediate, and more recently high-level (vanA-encoded), vancomycin resistance (8,22) and increasing numbers of multidrug-resistant MRSA emphasize the importance of effective antimicrobial prescribing and infection control measures that can be informed...

EMRSA-15 is one of the most important strains of epidemic methicillin-resistant Staphylococcus aureus (EMRSA) found in the United Kingdom. It was originally characterized by weak lysis with phage 75 and production of enterotoxin C but not urease. Two variant strains of EMRSA-15 which show a broader phage pattern than the progenitor strain have emerged. A total of 153 recent clinical isolates representing classical EMRSA-15 (55 isolates) or these phage variants (98 isolates) were compared by SmaI macrorestriction profiles in pulsed-field gel electrophoresis (PFGE) as well as by urease and enterotoxin C production. Eight of the 98 isolates were shown to be other unrelated strains by both PFGE and their production of urease, a misidentification rate of 8% by phage typing. Seventy-one EMRSA-15 isolates were enterotoxin C negative, and the majority of these were sensitive to phage 81. Examination of PFGE profiles and Southern blotting studies suggest that the enterotoxin C gene locus is encoded on a potentially mobile DNA segment of ca. 15 kb. After elimination of the eight non-EMRSA-15 isolates, the remaining 145 were characterized by PFGE, yielding 22 profiles. All profiles were within five band differences of at least one other profile. Classical EMRSA-15 isolates showed nine PFGE profiles, with the majority of isolates (68%) in profile B1. Six of these nine PFGE profiles were unique to the classical EMRSA-15 isolates. Among the phage variants of EMRSA-15, 16 profiles were seen, but the majority of isolates (83%) fell into 1 of 4 profiles (B2, B3, B4, and B7) which correlated well with phage patterns. The most divergent PFGE profiles among the EMRSA-15 isolates had as many as 12 band differences from one another, suggesting that in examining isolates belonging to such a temporally and geographically disseminated epidemic strain, the range of PFGE profiles must be regarded as a continuum and analyzed by relating the profiles back to the most common or progenitor profile.

Epidemic methicillin-resistant Staphylococcus aureus 16 (EMRSA-16) and EMRSA-15 are the two most important and prevalent EMRSA strains found in the United Kingdom and have also been found in a number of European countries and the United States. We describe for the first time the spread of an EMRSA strain (EMRSA-16) from its point of origin in one hospital to the surrounding hospitals and regions over the following 2 years. In the first 18 months after its original appearance, 136 hospitals referred EMRSA-16 isolates for typing, and interhospital and intraregional spread were reported: it was more prevalent in males between 60 and 80 years old and was isolated from sputum and throat more often than EMRSA-15. Important characteristics, e.g., carriage of the enterotoxin A (sea) and toxic shock syndrome toxin (tst) genes and production of urease, are described. Phage-variant strains of EMRSA-16 which share some of the characteristics of the classical strain, including toxin carriage and urease production, emerged, but without genotypic investigations, their relationship could only be inferred. A total of 129 clinical isolates from 52 hospitals, collected between March 1998 and April 1999 and representing classical EMRSA-16 (49 isolates) or phage variants (80 isolates), were compared by phage typing, pulsed-field gel electrophoresis (PFGE) following SmaI macrorestriction, antimicrobial susceptibility testing, urease production, and PCR detection of toxin gene carriage. PFGE analysis revealed 29 profiles, A1 to A29, with A1 representing the prototypic strain, NCTC 13143. All other profiles differed from A1 by 1 to 6 bands, but some differed from each other by up to 10 bands.

An extensive outbreak in a hospital on the south coast of England in 2000, involving a multi-resistant strain of methicillin-resistant Staphylococcus aureus (MRSA) phenotypically similar to a strain periodically seen in several hospitals in that region since 1996, prompted a study to characterize the strain and determine the extent of its spread. Sixty-nine isolates with related phage patterns obtained between 1997 and 2000 from 19 hospitals were selected for study. Of these, 55 isolates had an identical PFGE profile (designated F1), and eight shared five other PFGE profiles (designated F2-F6), which differed from that of F1 by no more than three bands and were considered related. Six isolates had PFGE profiles that differed from F1-F6 by 9-18 bands and were considered to be distinct strains. The 63 isolates of profiles F1-F6 were considered to comprise a single strain and were phenotypically identical, being urease positive and resistant to multiple antibiotics including methicillin, ciprofloxacin, erythromycin, fusidic acid, rifampicin, gentamicin, kanamycin, neomycin, streptomycin and tetracycline, with or without high- or low-level mupirocin resistance. Borderline resistance to teicoplanin was also commonly noted. All but one of these 63 isolates contained the gene sea, with five also carrying the genes seg and sei, and two carrying tst. The isolates of this strain had been referred from 13 different hospitals, seven of which were on or near the south coast, four from London, one from the Midlands and one from the north of England. The isolates were thus considered to comprise an epidemic MRSA (EMRSA) strain, which has been designated EMRSA-17. The six non-EMRSA-17 isolates identified in the study were sensitive to fusidic acid and rifampicin, and came from six geographically diverse hospitals including three in Northern Ireland.

The 7 and 13-valent pneumococcal conjugate vaccines (PCVs) have reduced the incidence of invasive pneumococcal disease (IPD) in children in many countries. The objective of this work was to assess the impact of PCVs and potential herd-protection in older adults in Ireland. IPD notification and typing data from adults ⩾65 years of age from July 2007 to June 2016 was assessed using national surveillance data. There was a 94% reduction in PCV7 serotypes from 2007-2008 to 2015-2016, incidence rate ratio (IRR 0·05, P < 0·0001). However, there was no decline in the additional PCV13 (PCV13-7) serotypes over the same period (IRR 0·90) nor in comparison with the pre-PCV13 period 2009-2010 (IRR 0·92). The incidence of serotypes in the 23-valent pneumococcal polysaccharide vaccine only (PPV23-PCV13) and non-vaccine types (NVTs) increased significantly (IRR 2·17, P = 0·0002 and IRR 3·43, P = 0·0001 respectively). Consequently, the overall IPD incidence rate in adults has remained relatively unchanged (from 28·66/100 000 to 28·88/100 000, IRR 1·01, P = 0·9477). Serotype 19A and NVTs were mainly responsible for penicillin resistance in recent years. The decline of PCV7 serotypes indicate that the introduction of PCV7 resulted in herd-protection for adults. However, increases in PPV23-PCV13 and NVTs suggest that changes in vaccination strategy amongst older adults are needed to build on the success of PCVs in children.

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