Characterisation of S. aureus/MRSA CC1153 and review of mobile genetic elements carrying the fusidic acid resistance gene fusC

While many data on molecular epidemiology of MRSA are available for North America, Western Europe and Australia, much less is known on the distribution of MRSA clones elsewhere. Here, we describe a poorly known lineage from the Middle East, CC1153, to which several strains from humans and livestock belong. Isolates were characterised using DNA microarrays and one isolate from the United Arab Emirates was sequenced using Nanopore technology. CC1153 carries agr II and capsule type 5 genes. Enterotoxin genes are rarely present, but PVL is common. Associated spa types include t504, t903 and t13507. PVL-positive CC1153-MSSA were found in Egyptian cattle suffering from mastitis. It was also identified among humans with skin and soft tissue infections in Saudi Arabia, France and Germany. CC1153-MRSA were mainly observed in Arabian Gulf countries. Some isolates presented with a previously unknown SCCmec/SCCfus chimeric element in which a mec B complex was found together with the fusidic acid resistance gene fusC and accompanying genes including ccrA/B-1 recombinase genes. Other isolates carried SCCmec V elements that usually also included fusC. Distribution and emergence of CC1153-MRSA show the necessity of molecular characterization of MRSA that are resistant to fusidic acid. These strains pose a public health threat as they combine resistance to beta-lactams used in hospitals as well as to fusidic acid used in the community. Because of the high prevalence of fusC-positive MRSA in the Middle East, sequences and descriptions of SCC elements harbouring fusC and/or mecA are reviewed. When comparing fusC and its surrounding regions from the CC1153 strain to available published sequences, it became obvious that there are four fusC alleles and five distinct types of fusC gene complexes reminiscent to the mec complexes in SCCmec elements. Likewise, they are associated with different sets of ccrA/B recombinase genes and additional payload that might include entire mec complexes or SCCmec elements.

Nine isolates were assigned to different variants of SCCmec V or VT elements. Five of these isolates came from Kuwait, one from Riyadh, Saudi Arabia, and two from the UAE (one each from Dubai and Umm-al-Quwain) and one from an Egyptian child living in Germany. All isolates, except the oldest one (isolated in 2009 33 ) also carried the fusC gene. In two isolates, the SCCmec/fusC composite element was further characterised using a second microarray 9 assigning them to SCCmec V + fusC (rather than to SCCmec VT + fusC). One isolate (from Kuwait) yielded the same pattern (with signals for mecA, ugpQ, fusC, mvaS-SCC, Q4LAG7, ccrAA, ccrC, SCCterm3, SCCterm10) as observed www.nature.com/scientificreports/ in a possibly livestock-(i.e., camel-) associated CC15 strain from Saudi Arabia 28,34 . The other one (from Germany/ Egypt) yielded signals for mecA, ugpQ, fusC, mvaS-SCC, Q4LAG7, ccrAA, ccrC and SCCterm11 possibly indicating a difference affecting the SCCmec/orfX junction site and/or another SCCmec/fus subtype. Seven of these isolates harboured PVL genes. Enterotoxin genes and chp were not identified, but sak and scn were always present. Eight out of the nine isolates harboured fusC. All were positive for blaZ/blaI/blaR. The gentamicin resistance gene aacA-aphD was found in eight isolates, the tetracycline resistance marker tet(K) in four isolates. Seventeen CC1153-MRSA isolates belonged to a strain which, to the best of our knowledge, carried an unknown SCCmec/fusC chimeric element. Three of these isolates were investigated with the second array yielding signals with mecA, ugpQ, Delta mecR1, fusC, Q4LAG7 (MSSA476), mvaS-SCC, ccrA-1, ccrB-1 and dcs. This prompted genome sequencing of one isolate, henceforth designated M58 (see below). Isolates with the new chimeric element originated from Kuwait (n = 14), UAE (n = 2) and France (n = 1). All but one were positive for lukF/S-PV genes. Genes sak and scn were always present while enterotoxin genes were not detectable. Sixteen isolates of this strain harboured blaZ/blaI/blaR, and erm(C) was found once.
Description of the SCCmec element in M58. One of the seventeen isolates with an apparent unknown SCCmec/fusC chimeric element was subjected to genome sequencing (Nanopore) to characterise this element (see GenBank CP065857.1). Its gene content of the SCCmec/fusC element is summarised in Table 1, and Fig. 1 provides a graphical overview as well as a comparison to other, previously published, reference sequences.
In short, the element comprised a mec complex B, ccrA/B-1 recombinase genes and fusC, while tirS (that commonly accompanies fusC 35 ) was absent. The gene pls-SCC, which normally is part of SCCmec I, was also absent. This constellation raises the question whether the element was derived from a SCCmec I element truncated by an insertion of fusC, or if it was a mec complex B element from a SCCmec I or IV element supplemented by fusC and accompanying ccrA/B-1 recombinase genes.
The actual mecA allele was identical to one which is widespread in SCCmec IV strains including, for instance, MW2 2 but differing from the one in COL. The mec complex B was followed by some genes encoding "putative proteins" and by ccrB-1 and ccrA-1 recombinase genes, as it was also the case in SCCmec I. A closer inspection of the sequence of the genes encoding "putative proteins" and of ccrB-1 revealed differences compared to the corresponding sequences in SCCmec I. The ccrB-1 allele from SCCmec I in COL differed by 7.2% of its nucleotides while ccrA-1 was too conserved to allow a meaningful analysis. This prompted a search for possible donors of recombinase and fusC-associated genes. While fusC itself was identical to MSSA Sanger 476, GenBank BX571857.1, the surrounding region was different in both gene content (most notably, in absence of tirS) as well as in gene sequences (8.4% difference in ccrB-1). Most closely related sequences of fusC-associated genes were identified in the S. aureus CC5 strain 06BA18369, GenBank ARXY, and in the Staphylococcus hominis subsp. hominis strain NTUH-3390, GenBank KY643657.1. These strains carry Q8CU82, tarF-SCC, A9UFT0, Q9KX75, Q7A207, Q7A206, ccrB-1, ccrA-1, cch-1, DUF1413, Q83ZD5, helicase M06, Q6GD51, D3QFP0-scc, D3JCW9, fusC, tnpIS150, tnp_A8YYY6, Q4LAG7-SCCfus and yobV (for explanations and GenBank entries of the genes discussed, see Table 1 and Supplemental File 4). However, Q8CU82, tarF-SCC-1 and A9UFT0 were absent in M58. The gene encoding the putative protein Q9KX75 in M58 was virtually identical to the ones in COL and MW2 but differed from the one in 06BA18369 and NTUH-3390, GenBank KY643657.1. From Q9KX75 on downstream, however, 06BA18369 and NTUH-3390 sequences were virtually identical to the ones in M58.
The PVL prophage in M58. The sequence of the PVL prophage in the genome of M58 (CP065857.1) was identical to the one in the S. aureus CC1153 strain 3688STDY6124889, GenBank FQHT01000001.1. It was also identical to the PVL prophage in USA300-TCH1516, CP000730.1.
M58 was shown by a lateral flow assay to secrete detectable amounts of PVL. This was also the case for two other CC1153-MRSA-PseudoSCCmec [class B + fus + ccrAB1] isolates as well as for three of the CC1153-MSSA isolates.

Discussion
CC1153 and the SCCmec/fusC element in isolate M58. We describe a clonal complex of S. aureus that we identified in several Middle Eastern countries. A couple of publicly available genomes, deposited in GenBank (GenBank FQHT01000000) and/or the Short Read Archive (SAMEA2661948, SAMEA2661956, SAMEA2662240, SAMEA2662319, SAMEA2710354, SAMEA2710468, SAMEA3214613, SAMEA3448866, SAMEA3448996, SAMEA4547522, SAMN03289718) belong to it, but to the best of our knowledge, this clonal complex has not yet been reviewed. Three of these sequences originated from Thailand (GenBank FQHT01000000.1 as well as BioSamples SAMEA3448866 and SAMEA3448996), and one from the United Kingdom (SAMN03289718) while for the others, no locations were reported. An additional observation of CC1153 isolates originated from Myanmar 36 . Our isolates were collected in the greater Middle East (Egypt and Arabian Gulf countries) and Western Europe although at least one of the European cases had connections to Egypt. There are no data confirming or explaining a discontinuous distribution in the Middle East and in South-East Asia. However, the presence of millions of expatriate South-East Asians in the Gulf countries could easily explain a transmission of a S. aureus lineage into either direction.
An interesting observation is the presence of CC1153 in Egyptian bovines 37 . The detection of PVL (rather than of lukM/lukF-P83) and of haemolysin-beta-converting prophages in these isolates indicates a human provenance of these isolates so that the cattle probably served as sentinels for an unrecognised epidemiological situation among humans in the Nile Delta.
The majority of CC1153, including MRSA and MSSA, is PVL-positive harbouring the same prophage (in M58 and FQHT01000001.1) as other pandemic strains such as USA300. www.nature.com/scientificreports/ CC1153-MRSA were mostly identified in Kuwait and the UAE. The clear majority, i.e. all isolates except the oldest one 33 , harboured SCCmec/SCCfus chimeric elements and the most common variant that could be described either as SCCmec I + fusC element or as a pseudoSCCmec class B + fusC + ccrA/B-1 element was sequenced. Sequence analysis also revealed (see above and Table 1) that ccrB-1 and accompanying genes are more related to alleles from other SCCfus elements rather than to the ones from SCCmec. Thus, a description as a pseudoSCCmec class B + ccrA/B-1 + fusC element should be regarded as the correct one. The mec complex B could have been derived from either a SCCmec I or SCCmec IV element. However, the latter one was more probable based on of the MW2-like allele of mecA.
The entire region associated with fusC (encompassing Q7A207, Q7A206, ccrB-1, ccrA-1, cch-1, DUF1413, Q83ZD5, helicase M06, Q6GD51, D3QFP0-SCC, D3JCW9, fusC, tnpIS150, tnp_A8YYY6, Q4LAG7-SCCfus and yobV) could be seen as one mobile genetic element that got introduced into a CC1153-MRSA replacing Q7A207, Q7A206 and the ccr recombinase genes that previously belonged to its SCCmec element. This set of genes was also www.nature.com/scientificreports/ found in a Staphylococcus hominis subsp. hominis strain and a CC5-MSSA from Canada (06BA18369 GenBank ARXY00000000.1) as described above. Furthermore, MRSA strains from Saudi Arabia 25 (as represented by isolates CMFT492, HF569112.1 and CMFT532, GenBank HF569114.1; see Tables 2, 3, 4 and 5) also carried the same region associated with fusC (differing from the one in M58 only in minor random deletions) as part of complex chimeric SCCmec II elements. In CMFT492, this cluster was inserted between orfX and a truncated SCCmec II element that lacked the kdp locus, cstA/B/R and the transposons introducing ble/aadD and erm(A)/ant9. In CMFT532 and other strains (see Tables 2, 3, 4 and 5), the region associated with fusC was inserted between orfX and a normal SCCmec II element. In these strains, additional markers (sccterm13, Q8CU82, tarF-SCC, A9UFT0, Q9KX75) were also associated with the fusC element that were absent in M58. Furthermore, there were also strains such as FORC 090, GenBank CP029198.1 or AR466, CP029080.1 in which fusC and its immediate neighbours (Q83ZD5, helicase M06, Q6GD51, D3QFP0-SCC, D3JCW9, tnpIS150, tnp A8YYY6, Q4LAG7-SCCfus and yobV) were accompanied by other ccr recombinase genes and other genes upstream, towards orfX. This prompted us to review published sequences and to compare them with the CC1153 strain described herein to sort and to classify the different gene clusters accompanying fusC.
Review of fusC elements. When Fig. 2). These sets could be regarded as fixed gene complexes in analogy to the mec complexes A (in SCCmec II and III), B (in SCCmec I and IV) and C (in SCCmec V). Likewise, they are also associated with different sets of SCC-recombinase genes including alleles of ccrA-1/ccrB-1, ccrA-1/ccrB-3, ccrA-3/ccrB-3, ccrA-4/ccrB-4, ccrA/ ccrB1 (FORC_90) and ccrAA/ccrC. Resulting "SCCfus" elements can, besides fusC-complexes and recombinase genes, also carry additional payload including tarF (teichoic acid biosynthesis protein F), speG (spermidine N-acetyltransferase), various variants of type I restriction-modification systems or mec complexes. They also can be linked to entire SCCmec elements resulting in complex genomic islands sometimes even including multiple sets of recombinase genes. These additional components can be localized upstream (towards orfX) or downstream (see Tables 2, 3, 4, 5/Supplemental File 5).
The longest known of the fusC-complexes ("A", Table 2, see also Supplemental File 5 and Fig. 2 A second fusC-complex ("B", Table 3, see also Supplemental File 5 and Fig. 2) comprises Q6GD54, Q6GD53, tirS, Q6GD51, D3QFP0-SCC, D3JCW9 and fusC. Besides gene content, it also differs from all others in five characteristic single nucleotide polymorphisms (SNP) within the fusC gene (14A > C; 150T > G; 290G > C; 537A > T; 632T > C). This complex has apparently not yet been observed in MSSA but there are several MRSA strains harbouring it connected to various SCCmec elements. One is HDE288, as prototypical sequence the "New Paediatric" CC5-MRSA strain from Portugal 47 . Here, the fusC-complex is accompanied by ccrA/B-4 genes and a mec complex B, a combination also referred to as SCCmec VI. Another CC5 (ST149) strain, known from Malta 48 , the Middle East 14, 25 and UK 26 harbours the same fusC-complex, together with ccrA/B-3 alleles and a SCCmec IV element. It also appears, although Q6GD54 is absent, in a SCCmec I MRSA strain from France (CC5, "Geraldine Clone" 49 ).
A fourth fusC-complex ("D1", Table 5, see also Supplemental File 5 and Fig. 2) consists of Helicase M06, Q6GD51, D3QFP0-SCC, D3JCW9, fusC, sccterm03, Q6GD49, Q8CU43, Q4LAG7S-SCCfus and yobV. Its fusC gene has one characteristic SNP (309G > A). It was not yet found in MSSA, but in MRSA belonging to CC8, CC30 and ST834 30 . In these strains, it is accompanied by a mec complex B and a set of ccrA/B-4 genes. It has been found neither in any other context, nor in MSSA strains.
Finally, truncated fusC-complexes were also observed as part of a very complex composite SCCmec element in a CC779 isolate M06/0171, HE980450.1 52 and of a composite "pseudo-SCC" element (i.e., without ccr genes) in Staphylococcus hominis subsp. hominis TFGsh5-1, AB930128.1. As the third and the fourth complex, it encompasses genes for helicase M06, Q6GD51, D3QFP0-SCC, D3JCW9 and fusC; but its fusC allele indicates relation to the third one (Table 4, see also Supplemental File 5). Table 4. Staphylococcal Cassette Chromosomes with a fusC-associated complex C consisting of Q83ZD5, helicase M06, Q6GD51, D3QFP0-SCC, D3JCW9, fusC, tnpIS150, tnp A8YYY6, Q4LAG7-SCCfus and yobV. Please note that this is an abridged version; for a more detailed version, see Supplement 5. References 25,26,45,52,53 . **Split across two or more contigs so that the correct order of SCC elements and/or individual genes cannot be determined.  www.nature.com/scientificreports/ There was no obvious phenotypical correlation of fusC-complexes to fusidic acid MICs, all tested strains (Supplemental File 6) were highly resistant regardless of their actual type of fusC-complex.
Further open questions are the timeframe of the evolution of SCCfus elements as well as their geographical origins. A wide variety of fusC-positive strains has not yet been sequenced. It would be interesting to know whether additional elements exist, and whether there are MSSA strains harbouring those fusC-complexes yet observed in MRSA only. The origin and evolutionary history of tirS is another open question. This virulence factor 35 is present in two out of five fusC-complexes but to the best of our knowledge, it has never been observed in another context.
As previously discussed 30 , fusC was detected in as much as twenty-two different clonal complexes of S. aureus, CC1, CC5, CC6, CC7, CC8, CC15, CC22, CC30, CC45 [agr I], CC45 [agr IV], CC50, CC59, ST72, CC88, CC97, CC121, CC152, CC361, CC779, ST834, CC913 and CC1153 from essentially all parts of the world. This and the emergence of fusC-MRSA especially in the Middle East indicate a selective advantage associated with its presence. Fusidic acid can be administered intravenously, but this is not commonly done, and the intravenous formulation is not available everywhere. It is also used topically, as ointment for presumably staphylococcal skin conditions. Observations from New Zealand suggest a quick emergence of fusC-positive S. aureus in parallel to an increasing use of this compound 16,38 . A co-evolution of SCCmec and SCCfus elements might result in a public health hazard, as strains with composite or chimeric elements are selected for both, in the hospital by beta-lactam administration as well as in the community by topical use of fusidic acid. Thus, antibiotic stewardship and infection control measures targeting MRSA in the hospital must be accompanied by restrictions to an uncontrolled over-the-counter sale of fusidic acid in outpatient settings as well as by a prudent use in outpatient settings.

Materials and methods
Isolates. A list of the isolates studied is provided in Supplement 1. Isolates were selected out of various typing and epidemiological projects based on array hybridisation profiles indicative for an affiliation to CC1153.
The CC1153-MRSA from the UAE were isolated from skin and wound infections. The CC1153-MRSA from Saudi Arabia also originated from a wound infection. One CC1153-MSSA from Saudi Arabia was a nasal colonizer from a healthcare worker, the others originated from skin and soft tissue infections (with three of them being identified during an earlier study 54 ). The first CC1153-MRSA reported in Kuwait was cultured from a wound inflicted by a dog bite in 2009 33 . The other isolates were obtained between 2017 and 2020 mostly from wound infections of patients located in six hospitals. One isolate was isolated from a gynaecological swab, one from a nasal swab and one was obtained from blood culture. Egyptian isolates were identified from rural smallholder dairy cattle that showed sub-clinical mastitis, i.e., somatic cell counts > 200,000 cell/mL and positive results of California Mastitis Test. The isolates were collected from six different cows in a herd, consisting of 25 crossbred dairy cows, located at Dakahlia Governorate in the northeast of Cairo, Egypt. The milking procedure was performed manually in the examined cows, while the medical records of the farm revealed the usage of a wide spectrum of antibacterial agents 37 . French isolates (11 MSSA, 1 MRSA) had been isolated during infections (cutaneous (n = 8), respiratory (n = 3), blood culture (n = 1)) in ten different hospitals between 2010 and 2017. One German MRSA isolate was cultured from an abscess of an approximately half year-old Egyptian child whose family lives in Germany. One German MSSA isolate originated from an abscess.
PVL detection was performed on six isolates by an experimental lateral flow test 54 . Fusidic acid MICs were determined by agar gradient dilution tests with commercially available strips (01B10122 Fusidinsäure MIC Test Strip 0.016-256 µg/mL, Bestbion dx GmbH, Cologne, Germany).

Microarray-based molecular characterization.
Genotyping of all strains was performed using the S. aureus Genotyping Kit 2.0 system (Abbott [Alere Technologies GmbH, Jena, Germany]) microarray-based assay. The array covers 333 different targets related to approximately 170 different genes and their allelic variants. The list of target genes as well as sequences of probes and primers have previously been published along all relevant protocols 9,39,55 .
Staphylococcus aureus was cultivated on Colombia blood agar. The DNA extraction was performed using lytic enzymes (lysostaphin, lysozyme, RNAse) and buffer from the S. aureus Genotyping Kit 2.0 kit and Qiagen DNA extraction columns (Qiagen, Hilden, Germany) according to manufacturers' instructions. Then, a linear amplification was performed using one primer for each target sequence. During the linear multiplex-amplification, biotin-16-dUTP was incorporated into the amplicons, which were then stringently hybridised to the specific probes on the microarray. After washing steps, hybridisation was detected using streptavidin horseradish peroxidase that triggered local precipitation at those spots where amplicon was bound. Microarrays were photographed and analysed with a designated reader and software (IconoClust, Abbott [Alere Technologies]). Analysis allowed detecting presence or absence of certain genes or alleles, as well as assignment to the clonal complex, strains, and SCCmec types.
Whole-genome sequencing. Genomic DNA was isolated from an overnight culture grown at 37 °C on Columbia blood agar using a Macherey and Nagel NucleoSpin Microbial DNA kit (MACHEREY-NAGEL GmbH & Co. KG, Dueren, Germany).
The Nanopore Oxford MinION platform was used for sequencing the whole genome of the CC1153 isolate M58 from the UAE. Briefly, size selection was performed using AMPure beads in a ratio 1:1 (v/v) with the DNA sample. The DNA library was generated using the nanopore sequencing kit SQK-LSK109 and the native barcoding expansion kit EXP-NBD103 (Oxford Nanopore Technologies, Oxford, UK) according to manufacturer's instructions. The used flowcell FLO-MIN106 (R9-Version) was primed by the flow cell priming kit EXP-FLP001 (Oxford Nanopore, Oxford, UK). The protocol named "1D Native barcoding genomic DNA" was used in version

Phylogenetic analysis.
A panel of 154 non-motile, core genomic markers was selected. Inclusion criteria were presence in all S. aureus clonal complexes analysed as well as uniform length in all genomes. The used genes as well as the genome sequences considered are listed in Supplemental File 3. Sequences were concatenated and analysed using SplitsTree 4.0 56 using default settings (Supplemental File 3).