Characterization of a novel <i>cfr</i>/<i>fexA</i>-carrying SCC<i>mec</i> variant from a <i>Mammaliicoccus sciuri</i> isolated from henhouses

The rise in MDR MRSA poses an increasing public health threat. For example, MRSA carrying the MDR gene cfr shows resistance to linezolid, which is often considered the last-line antimicrobial agent for the treatment of serious infections caused by resistant Gram-positive bacteria in humans.1 The staphylococcal cassette chromosome mec (SCCmec) is the main mobile genetic element responsible for the spread of mecA, but also acts as a carrier of other resistance genes. For example, cfr and fexA, which are usually located in plasmids, have independently been identified in SCCmec Ⅳb and SCCmec ⅩⅤ in a few MRSA isolates.2,3 CoNS are closely associated with the origin and assembly of SCCmec, facilitating its spread in staphylococci. Among these CoNS, Mammaliicoccus sciuri is widely distributed in the environment, from farms to hospitals. In our previous study, numerous mecA-positive CoNS strains were isolated from poultry farm environments.4 Currently, we have analysed an MDR M. sciuri strain, WHA07, revealing its novel SCCmec element that mediates the co-transmission of the emergent resistant genes cfr and fexA. M. sciuri WHA07 was isolated from the bioaerosol of a henhouse in China.4 It exhibited an MDR phenotype, including resistance to oxacillin (MIC = 8 mg/L) and linezolid (MIC = 8 mg/L), and its MIC of florfenicol was 64 mg/L. The complete genome of this strain is available in GenBank under accession no. CP077960.1. Phylogenetic analysis showed that strain WHA07 was closely related to isolates from an animal in Spain and a human in China (Figure S1 and Table S1, available as Supplementary data at JAC Online). Besides mecA, resistance genes cfr, fexA, tet(M) and qacG were also found in it. The full length of the SCC element in strain WHA07 (SCCmecWHA07) is 67.8 kb, with 70 coding sequences (Table S2). It contains one class A mec gene complex (mecA2, mecR1, mecI), one type Ⅲ ccr gene complex (ccrA3B3) and another ccr gene complex (ccrA5B3) (Figure 1a). Type Ⅲ SCCmec, first discovered in Staphylococcus aureus isolate 85/2082, is composed of two SCC elements, SCCHg and SCCmec ⅢA, integrated in tandem, making it the longest SCC element.5 In our isolate, the complete structure of SCCmecWHA07 was located at the traditional integration site of SCCmec, the 3′ end of the rlmH gene. SCCmecWHA07 is also a composite SCC element, consisting of an SCCmec element and a novel SCC element (defined as SCCcfr) carrying resistance genes cfr, fexA and more (Figure 1a). Schematic comparison of the structural elements of the SCCmec Ⅲ identified in M. sciuri strain WHA07. (a) Structure of the SCCmecWHA07. The SCC elements are shown in yellow, the other resistance genes are shown in orange, and the other transposase genes are shown in green. (b) Comparison of the SCCmec element among M. sciuri WHA07 (Accession: CP077960.1), S. aureus 85/2082 (Accession: AB037671.1) and M. sciuri GDK8D55P (Accession: NZ_CP065960.1). (c) Comparison of the SCCcfr element among M. sciuri WHA07 isolates, S. pseudintermedius KM241 (Accession: AM904731.1) and S. aureus plasmid pSA737 (Accession: NC_021076.1). The SCCmec element of isolate WHA07 shows characteristics typical of type ⅢA, containing the class A mec gene complex and type Ⅲ ccr gene complex. These gene complexes share more than 99% nucleotide identity with isolate 85/2082, the reference strain for type Ⅲ SCCmec (Figure 1b). However, unlike in isolate 85/2082, SCCmecWHA07 lacks the integrated copy of plasmid pT181, which encodes tetracycline and mercury resistance genes. The region from IS431 to just before ccrA3B3 in SCCmecWHA07 is homologous to another M. sciuri strain, GDK8D55P, isolated from a duck in South China (Figure 1b). However, the GDK8D55P strain contained only a class A mec gene complex, lacking the ccr gene complex, likely the precursor of the SCCmec element.6 The novel SCCcfr element identified in isolate WHA07 differed significantly from the SCC element (SCCHg) in isolate 85/2082, and it is suggested that these two small SCC elements in type Ⅲ SCCmec developed independently.7 In SCCmecWHA07, the SCCcfr element is 38.4 kb long and carries another ccr gene complex (ccrA5B3). The hsdMSR operon, encoding a type I restriction-modification system, is located downstream of the rlmh gene. The SCCcfr element showed a characteristic multiple recombination, with ccrA5B3 and its upstream fragment behind Tn558 showing more than 90% identity with the SCCmec cassette of Staphylococcus pseudintermedius strain KM241 (Figure 1c).8 Interestingly, this SCC element contains two important resistance genes, cfr and fexA, surrounded by IS elements within a Tn558 composite transposon-like structure, suggesting that cfr and fexA may be integrated by a copy of the Tn558-like element. The cfr gene was initially detected on a multi-resistance plasmid, pSCFS1, and is responsible for the emergent linezolid resistance in staphylococci.9 In isolate WHA07, the cfr- and fexA-carrying fragment shows high similarities (99%) with plasmid pSA737 of the S. aureus 004-737X strain isolated from the USA (Figure 1c).10 Typically, cfr and fexA spread though horizontal plasmid transfer. This study provides direct evidence of integrative recombination of cfr and fexA from plasmid to mobile SCCmec element in the chromosome, suggesting potential transfer of cfr and fexA through SCCmec among staphylococci. In summary, we characterized a novel composite SCCmec ⅢA variant from a multi-resistant M. sciuri strain isolated from a henhouse. It consists of a typical SCCmec ⅢA and a newly reported SCCcfr element carrying resistance genes cfr and fexA, exhibiting multiple recombination characteristics. To the best of our knowledge, this is the first report of cfr/fexA-carrying SCCmec, suggesting the co-transmission of these emergent resistance genes through SCCmec. This work was supported by the National Key Research and Development Program of China (2022YFD1800400) and the Earmarked Fund for China Agriculture Research System (CARS-41). None to declare. Figure S1 and Tables S1 and S2 are available as Supplementary data at JAC Online.

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