Comparison of SCCmec, antimicrobial resistance genes and clonal lineages of Staphylococcus epidermidis and Staphylococcus haemolyticus from humans ...
Comparison of SCCmec, antimicrobial resistance genes and clonal lineages of Staphylococcus epidermidis and Staphylococcus haemolyticus from humans and companion animals Brenda A. McManus1, David C. Coleman1, Emily C. Deasy1, Gráinne I. Brennan1,2, Brian O’ Connell2,3, Stefan Monecke4,5, Ralf Ehricht4, Bernadette Leggett6, Anna C. Shore1,3 1Microbiology Research Unit, Dublin Dental University Hospital, University of Dublin, Trinity College Dublin 2, Ireland, 2National MRSA Reference Laboratory, St. James’s Hospital, James’s St., Dublin 8, Ireland, 3Department of Clinical Microbiology, School of Medicine, Trinity College Dublin, St. James’s Hospital, Dublin 8, Ireland, 4Alere Technologies GmbH, Jena, Germany, 5Institut fuer Medizinische Mikrobiologie und Hygiene, Medizinische Fakultaet Carl Gustav Carus, Fiedlerstr. 42, D-01307 Dresden, 6Pathobiology, School of Veterinary Medicine, University College Dublin, Belfield, Dublin 4, Ireland.
Introduc)on
Gene)c Relatedness
Staphylococcus epidermidis and Staphylococcus haemolyticus, are opportunistic pathogens of humans and animals. While similar methicillin-resistant Staphylococcus aureus (MRSA) strains have been found in humans and companion animals, comparative studies of other staphylococcal species from humans and companion animals are scarce. This study compared S. epidermidis (SE) and S. haemolyticus (SH) isolates from humans (Hu) with those from companion animals (CpA) (cats, dogs and horses) by characterization of SCCmec types, antimicrobial resistance genes and clonal lineages present amongst the isolates to determine if transmission occurs between the different hosts, and if SE and SH isolates are potential genetic reservoirs for more pathogenic staphylococcal species.
28427 - SCCmecIVc-Canine 23767 - SCCmecIVc Canine
2A
19829 - SCCmecIVc-Canine 13931* - SCCmecIII-Canine 31169 - SCCmecIVc-Canine 22664 - SCCmecIVc-Equine 21666 - SCCmecIVc-Canine 11513* - SCCmecIII-Canine 408562.1 - SCCmecIVc-Human BM27.1 - SCCmecIVc-Human
99
ST2 (2,1,2,2,4,1,1)
BM14.1 - SCCmecIII-Human BM12.1 - SCCmecIII-Human 25875* -NT9-Canine BM11.1 - SCCmecIVc-Human 409084.1 - SCCmecIV-Human ST87 (7,1,1,2,2,1,1) 99 401218.1 - SCCmecIV-Human 91 408979.1*- NT2-Human ST35 (2,1,2,2,4,1,1) BM33.1* - SCCmecIVc-Human ST6 (1,1,2,2,2,1,1) 66 401148.1* - SCCmecIV-Human 64 18256* - Canine ST166 (1,1,2,2,2,1,3) 408691.1* - SCCmecIV-Human ST179 (1,1,2,2,1,1,1) 12057* - NT8-Canine ST69 (1,1,8,6,2,1,1) 97 401131.1 - SCCmecIV-Human ST83 (1,2,1,2,1,1,10) 98 408192.1 - SCCmecIV-Human 88
Materials and Methods Isolates from humans (SE-Hu, n = 29, SH-Hu, n = 8) were recovered from patients attending two separate acute hospitals in Dublin, Ireland, between 2004 and 2011. Isolates from animals (SE-CpA, n = 12, SH-CpA, n = 14) were recovered mainly from wounds and infections at a veterinary hospital in Dublin, Ireland between 2004 and 2011. All isolates underwent antimicrobial susceptibility testing against the panel of 23 antimicrobial agents used in the Antibiogram Resistogram (AR) typing of MRSA according to EUCAST methodology and a combination of the interpretive criteria by EUCAST (2014), CLSI (2013) and Rossney et al., (1) was used to determine the resistance profile. Detection of antimicrobial resistance and SCCmec genes was undertaken using DNA microarray profiling (Alere, Germany). Any isolates exhibiting unusual combinations of SCCmec genes underwent additional multiplex SCCmec typing PCRs for mec complexes, ccr complexes and joining regions. Twenty isolates representative of different SCCmec types/subtypes underwent DNA microarray mecA allele identification (Alere). Multilocus sequence typing (MLST) was performed on 48 isolates (14 SE-Hu and 12 SE-CpA; eight SH-Hu and 14 SH-CpA) using species-specific MLST schemes (2, 3).
52
85
BM3.1* - SCCmecIVa-Human ST81 (2,17,1,1,2,1,1) 28703* - SCCmecIVg-Feline ST125 (1,1,1,1,1,1,1)
69
0.05
401293.1-NT10- Human 408271.1 -NT10-Human
2B 98
An)microbial Resistance
26
100 90
35
80
ST1 (2,1,1,1,1,1,1)
11967 -NT14- Equine 408852.1* -NT12- Human 400977.1- NT10- Human ST8 (2,1,1,1,1,1,2) 12078-SCCmecV- Canine ST4 (2,2,1,1,1,2,2) 67 17009 -NT13- Equine 84 20283 -NT13- Equine 29531 -NT13- Equine ST2 (2,2,1,1,1,1,2) 57
70 87
% Prevalence
401144.1 -ccrAA, ccrA4-Human 400979.1 -NT10- Human 400975.1 -NT10- Human 26108 -SCCmecV- Feline 21793 -SCCmecV- Canine
60 50
99
40 30
88
20 10
31031-NT15- Canine 10709 -NT13- Equine 19488 -SCCmecV- Equine 21517 -SCCmecV- Equine
ST3 (2,2,1,1,1,1,3)
408318.1 -NT11- Human ST9 (2,2,1,1,3,1,1) 13585 -NT13- Equine ST5 (2,2,1,2,1,1,3) 25170 -SCCmecV- Equine ST6 (2,2,1,2,1,1,3) 12345- Canine ST14 (6,6,5,4,4,5,6)
0.05
S. epidermidis-Humans
S. haemolyticus-Humans
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S. epidermidis-Companion Animals
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FIG. 1 Prevalance of antimicrobial resistance genes detected amongst all isolates investigated. The aminoglycoside resistance genes aadD and aphA3 & sat, the chloramphenicol resistance gene cat and the tetracycline resistance genes tet(K) and tet(M) were more prevalent in isolates recovered from CpA. Discrepancies between the presence of a resistance gene and phenotypic susceptibility or vice versa were detected for dfrS1 (9/10 SH-CpA, 5/29 SE-Hu, 5/7 SH-Hu), erm(C) (1/29 SEHu), mupA (2/29 SE-Hu) and qacA (9/29 SE-Hu, 5/8 SH-Hu).
SCCmec typing The mecA gene was detected in 23/29 (79.3%) SE-Hu, 7/8 (87.5%) SH-Hu, 11/12 (91.7%) SE-CpA and 13/14 (92.9%) SHCpA isolates. The SCCmec type IV element predominated among both SE-Hu (12/23) and SE-CpA (7/11) isolates (Fig. 2A), and were most commonly subtype SCCmec IVc (7/12 SE-Hu, 6/7 SE-CpA) (Fig. 2A). Possible novel SCCmec types (NTs) were also identified among SE isolates (Table 1). The SCCmec V element was identified in 6/13 SH-CpA isolates but was not identified in any SH-Hu isolates (Fig. 2B). One SH-Hu isolate harbored SCCmec type VI, the remaining seven were NTs (Table 1). The arginine catabolic mobile element (ACME) was detected in 5/29 SE-Hu, 6/12 SE-CpA and 1/8 SH-Hu isolates. The mecA alleles identified amongst 20 isolates representative of different SCCmec types are shown in Table 2. SCCmec genes detected
NT (n)
S. epidermidis from humans mecA, ΔmecR1, ccrAB2, ccrC
NT1 (3)
mecA, ΔmecR1, dcs, ccrAB2, ccrAB1
NT2 (1)
mecA, ΔmecR1, dcs, kdp, ccrAB2, ccrA3, ccrB4
NT3 (1)
mecA, ΔmecR1
NT4 (1)
mecA, ΔmecR1, ccrAB2, ccrAB4
NT5 (1)
mecA, ΔmecR1, dcs, mecI, mecR1, ccrAB1
NT6 (1)
mecA, ΔmecR1, dcs, mecI, mecR1, ccrAB3, ccrB4, ccrC
NT7 (1)
S. epidermidis from companion animals mecA, ugpQ, dcs, ccrAB2
NT8 (1)
mecA, ΔmecR1, ugpQ, dcs, mecI, mecR1, xylR
NT9 (1)
S. haemolyticus from humans mecA, ccrB4
NT10 (5)
mecA, ccrAA
NT11 (1)
Genbank Accession No.
Isolates (n)
SCCmec types (n)
GQ902038
SH-Hu (1) SH-CpA (2)
NT11 (1) SCCmec V (2)
AY786579
SE-Hu (3) SE-CpA (3) SH-Hu (3) SH-CpA (1)
NT1 (1), NT3 (1), NT5 (1) SCCmec III (1), SCCmec IV (1), NT9 (1) NT10 (2), NT12 (1) NT13 (1)
BA000018
SE-Hu (3) SE-CpA (2)
SCCmec IV (1), NT6 (1), NT7 (1) SCCmec IV (1), NT8 (1)
GU235984
SE-CpA (1)
SCCmec IV (1)
AB03731
SE-Hu (1)
NT4 (1)
TABLE 2. mecA alleles identified. The number of each staphylococcal species from each source and SCCmec types mecA, ugpQ NT13 (5) associated with each mecA allele are indicated. Each of these mecA, ugpQ, ccrB4 NT14 (1) alleles have previously been reported among both S. aureus and mecA, ugpQ, ccrA3, ccrB4 NT15 (1) coagulase-negative staphylococci (4). The AY786579 allele TABLE 1. Possible novel SCCmec types identified. predominated, being identified in 10/20 isolates examined. Possible novel types (NTs) were defined as any SCCmec types that lack, contain additional, or have unusual combinations of mec and/or ccr genes. mecA, ΔmecR, dcs, ccrAB2, ccrA1 S. haemolyticus from companion animals
FIG. 2 Neighbour-joining trees based on the concatenated polymorphisms among MLST loci of (Panel 2A) 14 SE-Hu and 12 SE-CpA isolates and (Panel 2B) eight SH-Hu and 14 SH-CpA isolates. Isolate names, SCCmec types and the host species are indicated to the left of the red brackets, and allelic profiles and corresponding sequence types (STs) are indicated to the right of the red brackets The possible novel SCCmec types (NTs) are described in Table 1. Isolates harboring ACME are marked with an asterisc (*). Isolates identified as the same ST were recovered from both Hu and CpA hosts among both SE and SH populations. The predominant ST identified among SE and SH was ST2 (nine SE-CpA, five SE-Hu) and ST1 (three SH-CpA, six SH-Hu), respectively. Overall, MLST suggests that SE and SH isolates recovered from both Hu and CpA hosts are very closely related.
Conclusions The majority of SH and SE isolates recovered from Hu and CpA harbored mecA and were methicillin resistant (MR). SCCmec IV was the predominant SCCmec type in the MR SEHu and SE-CpA isolates. In contrast, SCCmec V predominated in the MR SH-CpA isolates despite not being detected in any SH-Hu isolates. Isolates belonging to the same lineages of SE and SH are circulating among both between Hu and CpA hosts. Resistance to aminoglycosides, tetracycline and chloramphenicol was more commonly detected among SH-Cpa and SE-CpA, suggesting a genetic reservoir may exist amongst these isolates which potentially could be transmitted to humans, or to the more pathogenic species of staphylococci.
References (1) Rossney et al., 2007. J. Clin. Microbiol. 45:2554-63. (2) Thomas et al., 2007. J. Clin. Microbiol. 45:616-9. (3) Cavanagh et al., 2012. J. Microbiol. Methods 89: 159-66. (4) Monecke et al., 2012. Antimicrob. Agents Chemother. 56: 5547
NT12 (1)
Acknowledgements This study was supported by the Microbiology Research Unit, Dublin Dental University Hospital. We thank the staff at the School of Veterinary Medicine at UCD for their help with the isolate collection, and the staff at the NMRSARL for their help with the susceptibility testing.
