Clonal group 5
Clonal group 5 was represented by two abundant EMRSA strains (South German and Rhine–Hesse), and by two sporadic strains. All isolates belonged to capsule type 5 and were uniform in terms of carriage of ssl/set genes and genes encoding MSCRAMMs. Allelic variants of these genes were generally identical to those of the sequenced strains Mu50 and N315. Clonal group 5 MRSA harboured the enterotoxin gene cluster egc (comprising enterotoxin genes seg, sei, sem, sen, seo and seu/y), but the isolates differed with respect to their carriage of additional toxin genes (see below) and resistance-associated genes, including SCCmec types. All isolates carried fosB (encoding a metallothiol transferase conferring fosfomycin resistance).
An SCCmecI strain from this clonal group is known as the South German Epidemic Strain (ST228, spa t001, t041 or t811). This is one of the four most common MRSA strains in Eastern Saxony. It is quite resistant, with isolates carrying the aacA–aphD, aphA, sat and qacA (encoding a multidrug efflux protein) genes. In contrast to other clonal group 5 strains, the gene for fibronectin-binding protein B (fnbB) was not detectable in this strain. The haemolysin-β (hlb) gene was always disrupted, and all isolates belonged to IEC type D . Altogether, four variants of the South German Epidemic Strain were discernible, based on the variable presence of the mercury resistance operon, ermA, mupR, dfrA, clfA (encoding clumping factor A) and bbp (bone/sialoprotein binding protein). A distinctive fifth variant of this strain (also spa t001) had a truncated egc locus, being positive only for the enterotoxin gene seo. It also lacked the lukD/E leukocidin genes. In addition to the isolate described here, three other isolates were found between 2005 and 2007, all of which were linked to a single ward in a rehabilitation facility.
A related strain is the Rhine–Hesse Epidemic Strain, also known as EMRSA-3 or New-York/Japan Clone (spa t003 or t627). Locally, this was one of the four most abundant strains. Isolates carried an SCCmecII element, which was in accord with the presence of ermA and, in most isolates, of aadD. The Rhine–Hesse EMRSA strain is related closely to the sequenced strain N315. Most isolates carried a distinctive allelic variant of sea (sea-N315, also known as sep), described originally in N315 . Enterotoxin carriage (sea-N315, sed + sej + ser) and the presence or absence of resistance genes (blaZ, aadD, aacA–aphD, aphA3, sat and tetM) allowed eight variants to be distinguished. Identification of these variants can be helpful for epidemiological purposes. For instance, seven of nine isolates that were sea-N315–, sed/j/r+, blaZ+ were epidemiologically linked, being sampled from a single hospital during 2003–2004. As the sak, scn and chp (encoding chemotaxis inhibitory protein) genes were present, Rhine–Hesse EMRSA isolates belonged either to IEC type B or IEC type F, depending on the carriage of sea-N315.
A related strain (spa t002, t067) carried an SCCmecIV element, corresponding to the internationally known ‘Paediatric clone’. This clone was rare, with only four isolates identified to date. It differed from the Rhine–Hesse EMRSA strain in lacking ermA, which is in accord with the presence of an SCCmecIV element, but three isolates were positive for aadD. Three isolates harboured sea-N315, sed, sej and ser, and two were positive for seb. The isolates belonged to IEC types B or F . PVL-positive variants of this strain, as reported in other countries, have not been found in Eastern Saxony to date.
A unique isolate belonging to clonal group 5 (t067) was obtained from a patient with severe pneumococcal pneumonia who was repatriated from the Canary Islands. This isolate yielded positive hybridisation signals with the probes for SCCmecIV, SCCmecVI, ermA, ermC, msrA, mpbBM (encoding macrolide 2′-phosphotransferase), aadD, aphA3 and sat. It belonged to IEC type E, carrying sak and scn.
Clonal group 8
MRSA strains from clonal group 8 generally gave hybridisation patterns related to those of the sequenced strains COL, USA300 and NCTC 8325. Carriage of the ssl/set genes and the genes encoding MSCRAMMs was rather homogeneous, and resembled that of these sequenced strains. With the exception of ST239 (see below), isolates belonged to capsule type 5 and lacked cna (encoding collagen-binding adhesin). Carriage of genes encoding resistance properties and exotoxins, as well as of IEC genes, was variable, thereby allowing several strains to be distinguished.
One strain from this group, known as Ancestral or Ancient MRSA, is a CC8/ST250-MRSA I strain, represented by the sequenced strain COL. No clinical isolates of the Ancient MRSA were found.
Another strain in this clonal group is PVL-positive ST8-MRSA IV, also known as USA300. Two cases of community-acquired infections with this strain have been identified to date in Eastern Saxony. Two additional cases were referred from a neighbouring county in the Federal State of Brandenburg. All four isolates carried msrA, mpbBM, aphA3 and sat, but lacked ermC, tetK and mupR, indicating a plasmid content different to that of the sequenced USA300 isolate (NC_007790, NC_007791, NC_007792, NC_007793). The hybridisation results for other genes were in accord with the published genome, including the presence of PVL genes, the ACME operon, and enterotoxin genes sek and seq.
Two cases of infection were noted with a related strain that was also ST8, spa t008 and SCCmecIV. In contrast to USA300, this strain did not carry the PVL, ACME or sek or seq genes, but harboured sea, sed, sej and ser, as well as aadD. This strain belonged to IEC type D . A follow-up isolate from one of the two patients was found to have acquired far1 (fusidic acid resistance) and tetK.
Another, locally relevant, strain was the Hannover Epidemic Strain, ST8-MRSA IV, spa t009. This strain was apparently introduced to Saxony shortly after German reunification, and was common during the 1990s (L. Jatzwauk, personal communication), but no isolate has been identified since 2001. Isolates of this strain carried ermA, aacA–aphD, aphA3, sat and tetM, and were resistant to co-trimoxazole, but without harbouring dfrA. This strain belonged to IEC type D . In addition to sea, it harboured the enterotoxin genes seb, sek and seq. In contrast to other ST8-MRSA strains, but in accord with, e.g., NCTC 8325, it lacked bbp. Two variants that differed in their carriage of genes associated with SCCmec elements were distinguishable. One variant carried the mecA, ΔmecR, ugpQ, dcs, ccrA2 and ccrB2 genes, and also generated positive signals with the probes for ccrAA-85/2082 and ccrC-85/2082. The other variant was positive for mecA, ΔmecR, ugpQ and dcs, and also carried the mercury resistance operon.
Another strain belonged to ST8, spa t064 and SCCmecIV, and was possibly related to USA500 (http://spa.ridom.de/spatypes.shtml ). Three imported cases, from Ethiopia (probably community-acquired), Zimbabwe (hospital-acquired) and Mozambique (community-acquired), respectively, were found. This strain harboured seb, sek and seq, and was resistant to erythromycin, clindamycin (ermA) and rifampicin. The isolates varied in their carriage of aacA–aphD, tetM, sea, sak and scn.
One MRSA isolate, belonging to clonal group 8 (t008), carried SCCmecV and was positive for ermC and tetK, but, as with NCTC 8325, did not carry enterotoxin genes. This isolate belonged to IEC type D .
ST239-MRSA III (Vienna, Hungarian or Brazilian clones) was found to be involved in a nosocomial outbreak following the repatriation of a trauma patient from Rhodes, Greece. This strain harboured the ermC, aacA–aphD, aphA3, sat and tetM genes. It carried only the enterotoxin A gene (sea), although ST239-MRSA III may also harbour sek and seq. IEC gene carriage was in accord with IEC type D . ST239 is unique, as it can be regarded as the result of a large-scale recombination event involving the parental strains of clonal groups 8 and 30 . Indeed, alleles of ssl/set and most MSCRAMM genes were typical for ST8 strains, but the arcC allele (carbamate kinase, used in multilocus sequence typing (MLST) ), capsule type 8 (instead of type 5), spa type (t037), the allelic variant of the aureolysin (aur) gene and the presence of cna indicated the insertion of a chromosomal fragment from clonal group 30.
Clonal group 22
A clonal group 22 strain, ST22-MRSA IV, also known as the Barnim Epidemic Strain  or EMRSA-15, was one of the four most abundant MRSA strains in Eastern Saxony. In terms of hybridisation profiles, clonal group 22 was not related to clonal groups 1, 5 and 8, but shared some features with clonal group 45. The ssl/set gene patterns were atypical and showed cross-reactivity among probes for different allelic variants of a given gene. Probes for the hlgA, lukF–hlg and lukS–hlg genes regularly gave weak signals. Both of these observations could indicate the presence of, as yet, unsequenced alleles. For lukS–hlg, a specific probe was constructed, based on an ST45 sequence (GenBank EF672356). This probe yielded positive results for ST22 and ST45.
All clonal group 22 isolates harboured the egc locus, but protease genes splA, splB and splE were not detected. The capsule type was 5, and the vast majority of isolates belonged to IEC type B. Some isolates had non-truncated hlb and lacked the IEC genes. Depending on the carriage of enterotoxin genes (sec and sel), haemolysin β-converting phages, ermC and the MSCRAMM genes (fnbB, bbp), a total of eight variants could be distinguished.
ST22-MRSA IV was found among hospital isolates from apparently community-acquired infections. In particular, it was associated with diabetic foot ulcers, although this might, in part, be attributable to an outbreak of one variant (sec/l−, ermC−, IEC+) in a ward and outpatient department specialising in the care of such patients. The PVL-positive ST22-MRSA IV outbreak strain from Bavaria  has not yet been detected in Eastern Saxony.
Clonal group 45
Clonal group 45 strains resembled those of clonal group 22 in terms of atypical ssl/set and luk/hlgA gene patterns (see above). ST45 isolates also carried the egc cluster, but in contrast to ST22, belonged to capsule type 8. These isolates had a unique allele of the gene for the bone-binding protein (bbp). The sasG (biofilm production/protein G) gene was conspicuously absent from this clonal group, as in clonal groups 30 and 398.
ST45-MRSA IV, also known as the Berlin Epidemic Strain (spa t004 or t040), was one of the most abundant local MRSA strains. Ten variants of this strain could be distinguished using the microarray. The variations observed affected resistance genes aacA–aphD, aadD, ermC, dfrA, aphA3, sat and tetK, as well as a common deletion of an enterotoxin gene (seg) from the egc cluster. All except one of the isolates (which lacked IEC genes) belonged to IEC type B. A related strain, or an eleventh variant, was an ST45-MRSA IV strain, which differed in the presence of additional enterotoxin genes (sec, sel) and the absence of aphA3 plus sat. This strain was detected only once, but has also been detected in Sweden and The Netherlands (A. Matussek and F. H. J. Schuren, personal communication).
In 2007, a single isolate of an ST45-MRSA V strain was detected which was otherwise very similar to the Berlin Epidemic Strain.