Staphylococcal cassette chromosome mec elements from Staphylococcus intermedius group (SIG) isolates from dogs in a center for veterinary diagnostics in Brazil

Methicillin resistance in the Staphylococcus intermedius group (SIG) has emerged in small animal practice. Methicillinresistant SIG (MRSIG) members have been implicated as causes of infections in both companion animals and humans. Staphylococcal cassette chromosome mec (SCCmec) elements carry the mecA/C genes, which encode for the transpeptidase PBP2a (PBP2’) responsible for β-lactam antibiotic resistance in staphylococci. This study examined the SCCmec types of MRSIG isolates from different clinical specimens of dogs that exhibited methicillin MIC ≥ 0.5 μg/mL by an automated identification and susceptibility system in a Center for Veterinary Diagnostics in São Paulo, Brazil. Susceptibility to methicillin was determined by broth microdilution testing, and Oxoid® M.I.C.Evaluator® strips. PBP2a production was detected using a latex agglutination assay. SCCmec typing was performed according to the International Working Group on the Classification of Staphylococcal Cassette Chromosome Elements (IWG-SCC) guidelines. SCCmec type II (2A), SCCmec type III (3A), composite SCC structures consisting of a class A mec gene complex in addition to multiple ccr gene complexes, and non-typable SCCmec elements were reported in these MRSIG isolates. SCCmec type variants differing from those so far acknowledged by IWG-SCC were found, indicating new rearrangements in the genetic context of mecA in these canine MRSIG isolates.

and respiratory tracts, surgical wounds, ears and skin in companion animals, and have also been increasingly associated with non-bite-related injuries in humans ( VAN DUIJKEREN et al., 2011;DAVIS et al., 2014;SOMAYAJI et al., 2016). Identification of coagulasepositive staphylococci by MALDI-TOF MS or molecular methods has reliably allowed SIG species differentiation that have since been underreported or commonly misidentified as Staphylococcus aureus by conventional methods in clinical microbiology laboratories. Antibiotic-resistant SIG infections have raised concern in both veterinary small-animal practice and humans, in particular methicillinresistant S. pseudintermedius (MRSP) infections. MRSP is an opportunistic pathogen responsible for pyoderma, otitis, and wound infections in pets, and its transmission between animals and humans has been well documented (VAN DUIJKEREN et al., 2011;BÖRJESSON et al., 2012;BARDIAU et al., 2013;COUTO et al., 2014;GRÖNTHAL et al., 2014;MACCARTHY et al., 2015;DUIM et al., 2016;FEßLER et al., 2018;WORTHING et al., 2018).
Methicillin resistance in staphylococci occurs due to expression of the penicillin binding protein PBP2a (PBP2'), a transpeptidase which shows a slow rate of acylation by β-lactam antibiotics (FISHOVITZ et al., 2014). PBP2a is encoded by the mecA or mecC genes that are carried by mobile staphylococcal cassette chromosome mec (SCCmec) elements (HIRAMATSU et al., 2013). The SCCmec typing has long been used for understanding the epidemiology of methicillin-resistant S. aureus (MRSA) and community-associated MRSA (CA-MRSA) strains (CHAMBERS & DELEO, 2009), and it has also been useful to investigate MRSP spread that now has emerged as a zoonotic pathogen (PERRETEN et al., 2013;QUITOCO et al., 2013;MACCARTHY et al., 2015;WORTHING et al., 2018).
SCCmec type assignment has been established by the International Working Group on the Classification of Staphylococcal Cassette Chromosome Elements (IWG-SCC), and it is based on a combination of the mec gene complex (mecA-C resistance genes, mecR1 and mecI regulatory genes, and insertion sequences), and the ccr gene complex (cassette chromosome recombinases ccr) (IGW-SCC, 2009;HIRAMATSU et al., 2013). The only few SCCmec elements so far characterized in MRSP indicated an evolutionary diversification of SCCmec structure in certain clonal lineages. Understanding how the genetic context of the mec gene has evolved in SIG isolates is crucial for controlling methicillin resistance that is on the rise in veterinary and healthcare settings. Therefore, in this study we examined the methicillin resistance phenotype and SCCmec types of methicillin-resistant SIG (MRSIG) isolates from different clinical specimens of dogs from a Center for Veterinary Diagnostics in São Paulo, Brazil.
From January to May 2014, a total of 41 SIG isolates exhibiting resistance to methicillin by BD PHOENIX Automated Microbiology System (MIC ≥ 0.5 μg/mL) were recovered from various canine specimens in a routine diagnostic laboratory in SP, Brazil (Table 1). Skin, eye and ear samples were obtained using sterile swabs, whilst urine and ascitic fluid samples were obtained by cystocentesis and abdominal puncture, respectively. Overnight cultures at 37 °C of these samples yielded colonies with a double zone of betahemolysis on blood agar (5% sheep blood), which were submitted to biochemical conventional methods used to differentiate coagulase-positive Staphylococcus species from S. aureus, such as the latex slide agglutination test (clumping factor/ protein A), the tube coagulase test, DNase production, PYR, and polymyxin B resistance (BECKER et al., 2015).
Susceptibility to methicillin was determined by broth microdilution method according to the guidelines of the Clinical and Laboratory Standards Institute -CLSI (2018). Oxacillin susceptibility breakpoints added to CLSI supplement M100 were used to predict mecA-mediated oxacillin resistance (CLSI, 2016). Oxacillin M.I.C. evaluator strips (M.I.C.E., Thermo Fisher Scientific, Basingstoke, UK) were also used for all MRSIG isolates. S. aureus subsp. aureus ATCC 29213 was used as a control for antimicrobial susceptibility testing. The product of mecA was detected using the PBP2a latex agglutination assay (Oxoid, Hampshire, United Kingdom) according to the manufacturer's instructions. S. aureus ATCC 29213 (methicillin susceptible, mecA negative), and MRSA N315 (methicillin resistant, mecA positive) were used as controls for PBP2a production.
DNA of the 41 MRSIG isolates was extracted using DNeasy blood and tissue kits (Qiagen, USA), with lysozyme (50 mg/ml) and lysostaphin (10 mg/ml) being added to the initial DNA extraction step. The SCCmec typing was performed according to the IWG-SCC guidelines. PCR assays to identify mec classes and ccr types were performed using primer sets as previously described (KONDO et al., 2007;ITO et al., 2014). Then, SCCmec types were assigned based on a combination of the type of mec gene complex (A, B, C1 and C2) and the ccr gene allotype (1 to 5). The MRSA reference strains NCTC 10442 (type I), N315 (type II), 85/2082 (type III), JCSC1978 (type IV), and MRSA clinical strains from our laboratory collection (types V, VI, VII and VIII) were used as controls. Sanger sequencing was previously carried out to confirm all PCR amplicons from the MRSA control strains.
The canine MRSIG isolates of this study were carriers of SCCmec type II (2A) (10/41), SCCmec type III (3A) (2/41), and composite SCC structures consisting of a class A mec gene complex in addition to multiple ccr gene complexes as follows: types 2 and 3 (1/41); types 2, 3, and 4 (1/41); types 2, 3, and 5 (2/41); types 2, 3, 4, and 5 (2/41); types 3 and 4 (9/41) and types 3, 4, and 5 (3/41). mecA-positive MRSIG isolates carrying non-typable SCCmec elements were also reported (6/41). These SCCmec elements consisted of ccr gene complexes types 2 and 5, but classes of mec gene complex (A, B, C1 and C2) could not be identified using the primer sets described by KONDO et al. (2007) and Ito et al. (2014) (Table  1). Composite SCC structures have been reported by carrying an association of a mec gene complex with two or more ccr gene allotypes, which may result from deletions of the original J region or insertions of other IS elements (CHANCHAITHONG et al., 2015). Genetic organization of the new composite SCC structures, such as SCCmec II-III, SCCmec VII-241, SCCmec V, ψSCCmec57395, SCCmec IVg, SCCmec VT, and SCCmecKW21 has been characterized in MRSP, and consist of unusual combinations of mec gene complexes with ccr gene complexes (DESCLOUX et al., 2008;BLACK et al., 2009;SHORE et al., 2011;PERRETEN et al., 2013;CHANCHAITHONG et al., 2015;WU et al., 2015;DUIM et al., 2018). All MRSIG isolates harboring SCCmec type II (2A) also carried a ccrC gene, in addition to the ccrA2B2 gene, but the class A mec gene complex was disrupted in five of these SCCmec elements. The primer set constructed on mecA and mecI produced a PCR amplicon smaller than the expected size. Then, other primers to amplify internal fragments of mecA and its regulatory genes mecR1 and mecI were used indicating that mecR1 was disrupted. Class A mec is considered the prototype complex that contains the mecA regulatory locus. Disruption of the signal transducer protein MecR1 did not affect the mecA expression, as high oxacillin MIC levels (128 -512 μg/mL) were exhibited by most of these isolates. Transcriptional repressor MecI proteolysis, which was shown to be dependent on MecR2 instead of MecR1 (ARÊDE & OLIVEIRA, 2013), is essential for the expression of β-lactam resistance. The MRSIG isolates that carried a SCCmec type II (2A) with an intact class A mec gene complex instead showed lower oxacillin MIC's values (< 0,5 -2 μg/mL). The non-typable SCCmec elements had the same association of ccr genes (ccrC and ccrA2B2) as seen in SCCmec type II (2A), but lacked a mec gene complex structurally organized as in the classes A, B, C1 or C2. The wide variation in susceptibility to oxacillin (MIC's < 0,5 -256 μg/mL) demonstrated by these isolates might be derived from new rearrangements of the mecA-regulatory genes. MRSIG isolates carrying a SCCmec type III (3A) or SCCmec composites exhibited high oxacillin resistance (MIC's ranging from 256 to ≥ 512 μg/mL).
Heterogeneous oxacillin resistance could be detected in an SCCmec composite-carrying MRSIG isolate from urine. The mecA-positive MRSIG isolate 36 exhibited an oxacillin MIC < 0,5 μg/mL by broth microdilution testing, but subpopulations of highly resistant cells (MIC ≥ 256 μg/mL) from the MRSIG 36 culture could be identified using a M.I.C. Evaluator strip (M.I.C.E., Thermo Fisher Scientific, Basingstoke, UK). Most clinical isolates of mecApositive staphylococci express a heterogeneous oxacillin phenotype, in which most cells exhibit lowlevel oxacillin resistance while subpopulations are able to express higher oxacillin resistance levels. Heteroresistant MRSIG isolates might be unrecognized by automated identification and susceptibility systems or conventional antimicrobial susceptibility testing in clinical laboratories, leading to β-lactam therapy failures. BD PHOENIX Automated Microbiology System predicted an oxacillin MIC ≥ 0.5 μg/mL for MRSIG 36, but it could not reliably determine susceptibility to methicillin in other five mecAnegative MRSIG isolates, which exhibited MIC < 0,5 μg/mL using the oxacillin broth microdilution test.
PBP2a latex agglutination testing yielded weak positive results for the oxacillin-heteroresistant isolate 36 and all MRSIG isolates with SCCmec type II (2A) or non-typable SCCmec elements, while stronger positive reactions were observed for MRSIG isolates with SCCmec type III (3A) or SCCmec composites. No correlation between the direct PBP2a test results and the oxacillin MIC values was observed.
Further investigation must be performed to characterize the SCCmec type variants found in these canine MRSIG isolates from various clinical infections, but PCR-based SCCmec typing could indicate the formation of genetic contexts enabling horizontal transfer of mecA that differ from those so far acknowledged as new for SIG by IWG-SCC. Methicillin resistance in staphylococcal species from companion animals raises concern, as β-lactams are veterinary critically important antimicrobials, and SIG isolates have the potential for zoonotic infections.