Characterisation of a novel SCCmec VI element harbouring fusC in an emerging Staphylococcus aureus strain from the Arabian Gulf region

Fusidic acid is a steroid antibiotic known since the 1960s. It is frequently used in topical preparations, i.e., ointments, for the treatment of skin and soft tissue infections caused by Staphylococcus aureus. There is an increasing number of methicillin-resistant S. aureus (MRSA) strains that harbour plasmid-borne fusB/far1 or fusC that is localised on SCC elements. In this study we examined a series of related CC30-MRSA isolates from the Arabian Gulf countries that presented with SCCmec elements and fusC, including a variant that—to the best of our knowledge—has not yet formally been described. It consisted of a class B mec complex and ccrA/B-4 genes. The fusidic acid resistance gene fusC was present, but contrary to the previously sequenced element of HDE288, it was not accompanied by tirS. This element was identified in CC30 MRSA from Kuwait, Saudi Arabia and the United Arab Emirates that usually also harbour the Panton-Valentin leukocidin (PVL) genes. It was also identified in CC8 and ST834 isolates. In addition, further CC30 MRSA strains with other SCCmec VI elements harbouring fusC were found to circulate in the Arabian Gulf region. It can be assumed that MRSA strains with SCCmec elements that include fusC have a selective advantage in both hospital and community settings warranting a review of the use of topical antibiotics and indicating the necessity of reducing over-the-counter sale of antibiotics, including fusidic acid, without prescription.


Introduction
Within a year after of the introduction of penicillinase-resistant semi-synthetic penicillins such as methicillin, oxacillin and the first/second generation cephalosporins, methicillin-resistant Staphylococcus aureus (MRSA) was reported in the United Kingdom [1]. Beta-lactam resistance in MRSA is due to modified penicillin-binding proteins encoded by different mec genes, out of which mecA is by far the most common and most widespread [2,3]. The mecA gene is located on potentially mobile, large and complex genetic elements, known as SCCmec ("staphylococcal cassette chromosome" or "staphylococcal chromosomal cassette" harbouring mecA). In addition to mecA or mecC, SCCmec elements include ccr recombinase genes, regulatory elements and, variably, additional genes encoding resistance to other antimicrobials, such as aminoglycosides or macrolides, and to heavy metal ions [4][5][6][7][8][9][10][11]. They also might contain the gene fusC encoding fusidic acid resistance [12,13]. Fusidic acid [14,15] is a steroid antibiotic known since the 1960s. It is frequently used in topical preparations, i.e., ointments, for the treatment of skin and soft tissue infections caused by S. aureus. In some countries, intravenous preparations are licensed that are administered in combination with other antimicrobials in order to treat staphylococcal bloodstream or orthopaedic infections. Resistance in staphylococci towards fusidic acid can essentially be attributed to five different genetic causes. One is caused by random mutation under selective pressure in the ubiquitous fusA or efg gene coding for elongation factor G [16]. Similarly, point mutations in fusE, or rplF, encoding riboprotein L6 can confer resistance [17,18]. Another mechanism is related to the presence of the plasmid-borne gene fusB, also known as far1. Its gene product binds to elongation factor G (efg) and thereby protects efg from fusidic acid. Acquired resistance due to fusB/far1 is commonly observed in the community acquired MRSA strain CC80-MRSA-IV that is common in Mediterranean and Middle Eastern countries [19][20][21][22][23][24][25][26]. A similar gene, fusC (Q6GD50) is localized on SCC elements. Such an element was first sequenced in a methicillin-susceptible strain, MSSA476 (GenBank BX571857.1) [27] where it is accompanied by ccrA/B1 genes. However, there are also various SCCmec elements that comprise both, mecA and fusC, together with various combinations of ccr genes and other markers [12,13]. One of these markers is tirS, a putative virulence factor mimicking the human Toll/interleukin-1 receptor (TIR) resulting in attenuation of the inflammatory response [28]. Finally, there is a gene, fusD, that has been found in various coagulasenegative staphylococci (S. arlettae, S. cohnii, S. microti, S. pettenkoferi and S. saprophyticus) [17] but apparently not yet in S. aureus. A high consumption of fusidic acid at a population level has been shown to confer a clear selective advantage to strains carrying fusC and subsequently to their emergence and proliferation as it was well documented for New Zealand [29]. Much less is known on the situation in other geographic regions. However, a high prevalence of fusB/far1 nd fusC and/or a high prevalence of fusidic acid resistance suggest a similar effect in Middle Eastern/Arabian Gulf countries. Indeed, fusidic acid is-or was until recently-available for purchase over-the-counter without prescription there. In the United Arab Emirates (UAE), prescriptions for the purchase of antibiotics became mandatory in late 2017. In this work we examine a series of related MRSA isolates from Arabian Gulf countries that presented with SCCmec VI elements and fusC. This included a variant that-to the best of our knowledge-has not yet formally been described and therefore it was characterised in detail.

Isolates
One CC30 MRSA isolate (RUH-32) obtained in September 2014 from a patient with septic arthritis at the King Khalid University Teaching Hospital in Riyadh, Saudi Arabia, yielded a microarray hybridisation pattern that did not match hybridisation patterns of previously known SCC elements. This isolate was subjected to whole genome sequencing. The BioSample accession number for the isolate is SAMN06925305, the master accession number of the assembled contigs is SGWB00000000.1. This investigation prompted a search for additional CC30 isolates with both, SCCmec VI elements and fusC yielding one isolate from Dubai, UAE (2018), sixteen from Kuwait (2016/2017) [30]and two from Riyadh, Saudi Arabia (2014 and 2018). All were obtained from hospitalised patients. Finally, two archived non-CC30 isolates were retrospectively found by using an array for SCCmec characterisation (see below and [9]) to harbour the same variant of a SCCmec VI element as RUH-32. One was a ST834-MRSA isolated in 2011 from a cutaneous abscess of a 3 years old child from Riyadh [31]. The other one was a CC8-MRSA that was isolated in 2017 from a hospitalised patient in Dresden, Saxony, who had a history of travel to, or migration from, the Middle East.

Microarray-based molecular characterisation
All isolates were characterised using two different microarray-based assays (Alere Technologies GmbH/Abbott, Jena, Germany) designed for S. aureus typing [32,33] and for the characterisation of SCC elements [9]. This allowed a rapid detection of species markers, virulence genes, resistance genes and SCC-related markers as well as an assignment to strains and clonal complexes. Details on DNA preparation and hybridisation procedures, as well as on probe and primer sequences and on data analysis have been described previously [9,32,33].

Genome sequencing
Whole-genome sequencing of the RUH-32 isolate was carried out by a commercial service provider using the Illumina HiSeq-2500 platform. Raw reads were deposited in the Short Read Archive under accession SRR5520614. Sequencing reads were assembled de-novo using SPAdes version 3.10.1 (http://bioinf.spbau.ru/spades). 51 Contigs were obtained with sizes larger than 200 nt and k-mer coverages greater than 10. Sequences from two reference strains, MRSA18 (European Nucleotide Archive accession number ERR108048) and 20121643 (ERR1595888), were downloaded from the European Nucleotide Archive and assembled with spades. In both cases, the complete SCC elements were found to be located on a single contig. The sequences of the SCC elements were excised from the respective contigs and then annotated.

Sanger sequencing for closing the gap between the contigs
Inspection of the contigs revealed that two contigs comprised typical SCC genes and that they may be linked by an IS431-like insertion element. Primers were designed for amplifying and capillary electrophoresis (CE) sequencing of the ambiguous linker region. Primer sequences are provided in Table 1. The process included PCR amplification using a thermal cycle program with an initial denaturation temperature of 96˚C for 60s followed by 35 cycles of denaturation at 96˚C for 15s, annealing at 70˚C for 60s and extension at 72˚C for 90s. The PCR product was fractionated by gel electrophoresis on a 1.5% agarose, the band with the main amplicon was excised and purified with the QIAamp Gel Extraction Kit (Qiagen, Hilden, Germany) according to the recommendations of the manufacturer. Cycle sequencing was carried out using BigDye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems, Darmstadt, Germany) on a ABI PRISM 3130 (Applied Biosystems). The first sequence obtained was then further extended by primer walking to a final size of 1133 nt (submitted to GenBank as "lin-ker_RUH-32"). The final linker sequence (GenBank accession number MK991790) is overlapping with contig SGWB01000020.1 by 303 nt, and with contig SGWB01000002.1 by 490 nt.
These two contigs and the linker sequence were joined to a single contiguous sequence. For detailed sequence analysis, the SCC element and its flanking genes were extracted and deposited as a separate sequence entry in GenBank (accession number is MK991791). The SCC element was annotated by comparison to a database of genes which have previously been found in related SCC elements.

A novel variant of a SCCmec VI element harbouring fusC
An overview on the gene content and the order of genes in the SCCmec VI (RUH-32) element is provided in Table 2 and a graphic representation is shown in Fig 1. In short, the element consists of a class B mec complex in which a mecA BA000018 (a N315-like allele of mecA [2]) is combined with ugpQ and delta mecR1. The fusidic acid resistance gene fusC is present, tirS is absent. In sequences of RUH-32, ccrA/B-4 are present, but ccrA-4 does not yield signals in array experiments. The reasons are polymorphisms in the probe binding site of ccrA-4 in RUH-32. Several other SCCmec elements that include fusC can be identified among previously published sequences. One is present in the CC5 strain HDE288 (AF411935; [13]) and can also be identified in CC8 ("UK-EMRSA-12/13") sequences ASARH101 (SAMEA1565121), MPROS978 (SAMEA2041631) and MPROS1215 (SAMEA2663833). The SCCmec VI (RUH-32) element differed from the SCCmec VI (HDE288) element in an absence of tirS. Another difference is the presence of Q4LAG7 ("putative protein"; BX571857.1, position 55452 to 55880) in the RUH-32 sequence which was also detected by microarray. Differences to SCCmec VI element harbouring fusC of the CC8 strain MRSA18 (ERR10804/SAMEA1317993; [12]) include the presence of Q9XB68-dcs and SCCterm 3 (rather than SCCterm 7). Furthermore, MRSA18 has two copies of ccrA/B-4 genes. Strain AR466 (CP029080.1), a CC45-MRSA, has a SCCmec VI element harbouring fusC that could not be differentiated from the one in MRSA18 by array (hence, it is not shown in Table 3). However, it includes an additional hsdS/M/R (type I restriction-modification) operon and dfrA (dihydrofolate reductase), which usually is plasmid borne. Finally, there is another SCCmec VI-derived element in the CC8 strain 20121643 (ERR1595888/SAMEA3924203) that harbours fusC. It differs from RUH-32 in several markers (see Fig 1) including an absence of Q9XB68-dcs, presence of speG (spermidine N-acetyltransferase) and dfrA as well as a presence of three copies of ccrA/B-4 genes.

CC30-MRSA-[VI+fus] strains in the Arabian Gulf region
The novel SCCmec VI (RUH-32) element described herein was not present in all CC30-MRSA- [VI+fus] isolates. Instead, they could be categorized into three distinct clusters (Table 3). Isolates of one cluster carried the SCCmec VI (RUH-32) element. They harboured the PVL genes although there was one exception. They also were positive for cadD, cadX, blaZ, linA/lnu(A) (again, with one exception), aadD, dfrG and tet(K). A second cluster carried another SCC [mec VI+fus] element that yielded the same hybridisation signals as expected for a previously sequenced element from the Portuguese CC5 strain HDE288 [13]. One major difference to SCCmec VI (RUH-32) was the presence of tirS. All isolates from this cluster were positive for the gene encoding the toxic shock syndrome toxin, tst1. A majority of them also were positive for pvl genes (nine out of twelve) and the enterotoxin gene sea (eleven out of twelve). Other markers included the copper resistance genes mco, copA2, arsenic and cadmium resistance genes arsB, cadA, cadC as well as blaZ and (in some isolates) erm(C). A third cluster, comprising of a single isolate harboured another SCCmec element consistent to one previously described from a CC8 isolate "MRSA18" [12]. The Kuwaiti CC30 isolate was PVL-positive. It lacked tirS and tst1. Heavy metal resistance genes were absent and tetK was the only antimicrobial resistance gene present in addition to mecA.

Other MRSA strains with the same SCCmec element
One PVL-negative CC8 isolate was identified in a patient in Saxony who had links to the Middle East. Based on array hybridisation results, it also carried the SCCmec VI (RUH-32) element (Table 3). This observation prompted an extensive database search yielding four more CC8-MRSA sequences with SCCmec VI  in the NCBI Short Read Archive (SAMEA2385458, SAMEA2385540, SAMEA2664046, SAMEA2664096). We also identified the same element, SCCmec VI (RUH-32) , retrospectively in a ST834 isolate (Riyadh_3497247, see Table 3).

Discussion
We describe a novel variant of a SCC element that harbours determinants for both, methicillin/beta-lactam and fusidic acid resistance. It was identified when performing DNA-microarray-based typing of clinical strains from Saudi Arabia because of a previously unseen hybridisation pattern and further characterised by sequencing. This element is related, but clearly distinguishable (see Results) from other such elements observed elsewhere as well as in the studied region. Furthermore, there is evidence for its horizontal transfer as we were able to detect it in two further, unrelated lineages of S. aureus. On a practical level, the observations suggest that emerging fusidic acid resistance might hamper its use as topical treatment for staphylococcal skin and soft tissue infections. A replacement of this substance by mupirocin is not feasible as the usability of mupirocin itself is endangered by the spread of resistance [34,35]. Other substances, such as betaisodona, polyhexanide or octenidine, should be considered. On a more theoretical level, a co-localisation of genes encoding beta-lactam (mecA) and fusidic acid (fusC) resistance on one potentially mobile genetic element is an interesting example for coalescence of two "selfish replicators" [36] for mutual advantage. A community use of easily available topical fusidic acid could thus select for mecA methicillin resistance and a hospital use of systemic beta-lactam compounds could select for fusC, conferring benefit to MRSA with such combined elements in either biotope. Thus it can be expected that such strains emerge in hospital as well as in community settings and that it will be very complicated to contain or to eradicate them once they are established in a population. , and to the best of our knowledge, they have been identified in CC5, CC7, CC8 ("UK-EMRSA-12/13"), CC22, CC30, CC45, CC97, CC152, ST834 and CC913 from Germany, United Kingdom, Portugal, Kuwait and Saudi Arabia [9,12,13,23,29,30,32,[37][38][39][40][41][42]. Observations of SCCmec elements that harbour fusC geographically cluster in Western Europe, the Middle Eastern/Arabian Gulf countries, Australia and New Zealand. Whether this is caused by a sampling bias or related to formulations and usage of fusidic acid, or both, we currently cannot tell. However, given the current patterns of travel and migration, appearance and emergence of fusidic acid resistant MRSA cannot be ruled out anywhere. This phenotypic property cannot be seen any more as a surrogate marker for the presumptive identification of PVL-positive CC80-MRSA-IV ("European"/Mediterranean clone) but it should prompt further investigation. As mentioned above, co-evolution and co-selection of resistance traits that favour resistant pathogens in hospitals as well as in the community outside pose a public health hazard. This should prompt a review of the use of topical antibiotics such as fusidic acid (or mupirocin) including restrictions to uncontrolled and unlimited over-the-counter sale of such compounds.