- Email: [email protected]
Genotyping of Staphylococcus aureus isolates from diseased poultry
Veterinary Microbiology 162 (2013) 806–812
Contents lists available at SciVerse ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Genotyping of Staphylococcus aureus isolates from diseased poultry Stefan Monecke a,b,*, Antje Ruppelt a, Sarah Wendlandt c, Stefan Schwarz c, Peter Slickers b, Ralf Ehricht b, Sonia Cortez de Ja¨ckel d a
Institute for Medical Microbiology and Hygiene, Technical University of Dresden, Dresden, Germany Alere Technologies GmbH, Jena, Germany c Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut (FLI), Neustadt-Mariensee, Germany d Poultry Clinics and Laboratory Dr. Po¨ppel, Delbru¨ck, Germany b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 10 February 2012 Received in revised form 6 August 2012 Accepted 15 October 2012
To gain insight into the genomic diversity of Staphylococcus aureus associated with diseases in domestic poultry, 131 isolates from clinically ill turkeys (n = 80) and chickens (n = 51) were collected and genotyped using microarray hybridisations. MRSA isolates were subjected to spa and dru typing and their antimicrobial resistance geno- and phenotypes were determined. Most (68 out of 80) turkey isolates belonged to the clonal complex (CC) 398. Seventeen of the 80 isolates (21.2%) were MRSA. The most common MRSA type among turkeys was CC398-MRSA-V (n = 8), but CC5-MRSA-III (n = 4), CC9MRSA-IV (n = 2), CC398-MRSA-IV (n = 2) and a single CC398-MRSA with an unidentified/ truncated SCCmec element were also found. Among the chicken isolates, CC5 predominated (44 out of 51). Five of the chicken isolates were MRSA (9.8%), all belonging to CC398-MRSA-V. These data show that the current dissemination of livestock-associated MRSA also engulfs chickens and turkeys, and that MRSA surveillance among these species is warranted. ß 2012 Elsevier B.V. All rights reserved.
Keywords: Staphylococcus aureus MRSA MSSA CC398 Microarray analysis Minimum inhibitory concentrations spa typing dru typing
1. Introduction Staphylococcus aureus is a versatile bacterium which can infect or colonise a variety of different host species. While it is found in various mammals including humans, there are also several reports about its presence in both, domestic and wild birds. While multilocus sequence types (STs), such as ST692, have exclusively been observed in birds (see MLST database at http://saureus.mlst.net/), other STs are less host-specific. For instance, the most widespread and dominant S. aureus strains in poultry belong to ST5 within clonal complex (CC) 5, and appear to originate most likely from a single transmission from
* Corresponding author at: Institute for Medical Microbiology and Hygiene, Technical University of Dresden, Dresden, Germany. Tel.: +49 351 6200; fax: +49 351 458 6311. E-mail address: [email protected] (S. Monecke). 0378-1135/$ – see front matter ß 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetmic.2012.10.018
humans, where this lineage is also common, into fowl (Lowder et al., 2009). Strains that belong to CC398, especially the methicillin-resistant S. aureus CC398 that harbour a type V SCCmec element (CC398-MRSA-V), proved to be present in a wide variety of host species. They have been found not only in humans (Huijsdens et al., 2006; Krziwanek et al., 2009; Schijffelen et al., 2010), but also in pigs (Huijsdens et al., 2006; de Neeling et al., 2007), dairy cows and veal calves (Feßler et al., 2010; Vanderhaeghen et al., 2010), horses (Walther et al., 2009) and dogs (Nienhoff et al., 2009) and also in chickens (Nemati et al., 2008), chicken meat (Nemati et al., 2008; Feßler et al., 2011) as well as turkey meat (Feßler et al., 2011). To gain insight into the genomic relationships of S. aureus strains associated with diseases in domestic poultry, consecutive isolates from clinically ill chickens and turkeys were collected and genotyped using hybridisations to a previously described microarray (Monecke et al., 2011). This comprehensive genotyping approach of a
S. Monecke et al. / Veterinary Microbiology 162 (2013) 806–812
large number of independent methicillin-susceptible and methicillin-resistant S. aureus provides for the first time insight into the genomic relationships of such strains from diseased poultry. For a better comparison with MRSA from diseased pigs and cattle, but also with MRSA from poultry meat and poultry meat products, the MRSA isolates were subjected to spa and dru typing and their antimicrobial resistance geno- and phenotypes were determined. 2. Material and methods 2.1. Bacterial isolates A total of 131 S. aureus isolates were obtained from routine diagnostic work at the Poultry Clinic and Laboratory Dr. Po¨ppel, Delbru¨ck, Germany, between January 2009 and July 2011. This unit serves numerous large poultry farms mainly in the German federal state of North Rhine-Westphalia (although eight turkey isolates came from Baden-Wuerttemberg, and five chicken isolates from Lower Saxony). Fifty-one isolates originated from domestic chickens (38 broilers including four chicks, 13 layers) and 80 from domestic turkeys. Turkey isolates originated from 26 different farms, broilers from 18 and layers from 10 farms. The isolates were obtained from necropsy material from cases of invasive infections (joints, liver, heart, lungs), and only one isolate per animal was chosen for further analysis. Staphylococci were identified by Gram staining, catalase and coagulase test (rabbit plasma tube test) and by the ID 32 STAPH system (bioMe´rieux, Craponne, France). All strains were tested phenotypically for antibiotic resistance by the disc diffusion method using DIN breakpoints. Detection of oxacillin resistance was performed using oxacillin and cefoxitin disks and by screening for growth on the Brilliance MRSA-Agar (Oxoid, Wesel, Germany). 2.2. DNA microarray-based typing All isolates were characterised using the Alere StaphyType DNA microarray (Alere Technologies GmbH, Jena, Germany) covering 333 target sequences which correspond to approximately 170 distinct genes and their allelic variants. These include species markers, SCCmec, capsule and agr group typing markers, resistance genes and genes encoding exotoxins as well as adhesion factors (see Supplement 1). A full list including primer/probe sequences has been provided previously (Monecke et al., 2011). The assay was performed according to the instructions given by the manufacturer and to previous descriptions (Monecke et al., 2011). In short, S. aureus were grown on Columbia blood agar, harvested and lysed. DNA was prepared utilising spin columns or the automated EZ1 system (both by Qiagen, Hilden, Germany). DNA samples were subjected to a linear primer elongation using only one primer per target. During this step, biotin-16-dUTP was incorporated into the resulting amplicons. In a later step, these amplicons were hybridised to the microarray. After washing, horseradish-peroxidase-streptavidin was
807
added which subsequently triggered the precipitation of a dye. An image of the microarray was taken and analysed using reader and software provided by Alere Technologies GmbH (Jena, Germany). The automated comparison of the hybridisation patterns of the actual isolate to a reference database allowed determining its affiliation to clonal complexes as defined by MLST (Enright et al., 2000) and, in case of MRSA, to epidemic strains defined by MLST and SCCmec carriage (Monecke et al., 2011). PSM-mec was not covered by the array; it was detected by PCR as previously described (Monecke et al., 2012a). 2.3. Susceptibility testing and detection of resistance genes in MRSA Additional susceptibility testing of the MRSA isolates was performed by broth microdilution using custom-made microtitre plates (MCS Diagnostics, Swalmen, the Netherlands) which contained the following antimicrobial agents (with test range in parentheses) in two-fold dilution series: ampicillin (0.03–64 mg/ml), apramycin (0.03–64 mg/ml), ceftiofur (0.03–64 mg/ml), cefotaxime (0.015–32 mg/ml), cefquinome (0.015–32 mg/ml), cephalothin (0.06–128 mg/ ml), chloramphenicol (0.5–256 mg/ml), clindamycin (0.03–64 mg/ml), enrofloxacin (0.008–16 mg/ml), erythromycin (0.015–32 mg/ml), florfenicol (0.12–256 mg/ml), gentamicin (0.12–256 mg/ml), nalidixic acid (0.06– 128 mg/ml), oxacillin + 2% NaCl (0.03–16 mg/ml), penicillin G (0.015–32 mg/ml), quinupristin/dalfopristin (0.008– 16 mg/ml), spectinomycin (0.12–256 mg/ml), tetracycline (0.12–256 mg/ml), tiamulin (0.03–64 mg/ml), trimethoprim (0.06–128 mg/ml), trimethoprim/sulfamethoxazole (0.015/0.03–32/608 mg/ml), and vancomycin (0.008– 16 mg/ml). In addition, susceptibility to kanamycin (2–128 mg/ml) was tested by broth macrodilution. Susceptibility testing followed the recommendations given in the CLSI document M31-A3 (CLSI, 2008); classification of the isolates as resistant or susceptible based on the breakpoints given in the CLSI documents M31-A3 (CLSI, 2008) and M100-S21 (CLSI, 2011). S. aureus ATCC129213 served as quality control strain. As the aforementioned DNA microarray already covers a considerable number of resistance genes, specific PCR assays were only conducted for resistance genes not yet included in the DNA microarray. These are tet(L) (tetracycline resistance), dfrK (trimethoprim resistance), the linkage of tet(L)-dfrK, dfrK as part of Tn559; erm(T) (macrolide-lincosamide-streptogramin B resistance), spc (spectinomycin resistance), the linkage of erm(A)-spc, as well as vga(C), vga(D), vga(E) and lsa(C) (pleuromutilinlincosamide-streptogramin A resistance), and vga(E) as part of Tn6133. These PCR assays followed previously described protocols (Feßler et al., 2010, 2011; Hauschild et al., 2012). 2.4. spa and dru typing The spa typing was performed in accordance with the Ridom StaphType standard protocol (http://spaserver.ridom.de). A relatively novel typing method for methicillinresistant staphylococci, dru typing (Goering et al., 2008),
808
S. Monecke et al. / Veterinary Microbiology 162 (2013) 806–812
was performed as previously described (Feßler et al., 2010). The dru amplicons were sequenced and compared with the dru sequences and dru types stored in the dru typing database (http://dru-typing.org). Novel dru types detected during this study have been submitted to and in the meantime included in the dru typing database. 3. Results
dt12q (5a-2d-2d-4a-0-2d-4f-3a-2g-3b-4e-3e) with the latter three representing new dru types that have been identified for the first time during this study. One isolate carried an atypical SCCmec element that comprised mecA, ugpQ, mecI, mecR, xylR, dcs and PSM-mec but lacked recombinase (ccr) genes as well as the kdp operon. Thus it might represent a truncated SCCmec II or III element. For this isolate no spa PCR product was obtained in repeated attempts. Its dru type was dt11a.
3.1. S. aureus lineages in the different poultry species 3.3. Resistance markers in MSSA The most common lineage among turkey isolates was CC398. Of the 80 isolates from turkeys, 68 belonged to this clonal complex with 57 isolates being classified as MSSA and the remaining eleven isolates as MRSA. Among these CC398 MRSA isolates, one isolate had an irregular SCCmec element (see below). Two isolates carried SCCmec IV and eight isolates harboured SCCmec type V elements. The remaining turkey isolates belonged to CC5 (four CC5-MSSA, and four CC5-MRSA-III), CC7 and CC15 (one MSSA isolate each) as well as CC9 (two CC9-MRSAIV). Thus, a total of seventeen out of 80 turkey isolates (21.2%) were MRSA. Among the chicken isolates, CC5 predominated (44 out of 51 isolates) with all isolates being mecA-negative. Two isolates belonged to CC398MSSA while another five where assigned to CC398MRSA-V. The MRSA rate among the chicken isolates was at 9.8% (five out of 51). 3.2. Characterisation of the MRSA isolates from poultry The spa and dru types of the 22 MRSA isolates from poultry as well as their antimicrobial resistance phenoand genotypes are shown in Table 1 and in the Supplemental File. Thirteen different resistance phenotypes and 16 different resistance genotypes were detected. All 22 MRSA isolates from poultry were considered as multiresistant by their resistance to three or more classes of antimicrobial agents (Schwarz et al., 2010). Moreover, all 22 MRSA isolates were resistant to beta-lactams, tetracyclines and MLSB antibiotics. They carried the beta-lactam resistance genes mecA and blaZ, the tetracycline resistance genes tet(M), tet(K) and tet(L) as well as the MLSB resistance genes erm(A), erm(B), erm(C) and erm(T) alone or in various combinations. Resistances to other antimicrobial agents/classes of antimicrobial agents varied among the isolates. The two CC9-MRSA-IV isolates shared spa type t1430, dru type dt10a and displayed the same resistance phenoand genotypes. The four CC5-MRSA-III isolates also had the same spa (t002) and dru (dt9v) types. Although they showed the same resistance phenotype, the four isolates clustered into three resistance genotypes. All carried erm(A) and tet(M), but the additional carriage of erm(B), tet(L) and tet(K) varied. In total, 16 CC398-MRSA isolates were found. Two of the CC398 isolates belonged to CC398-MRSA-IV, spa type t011 and dru type dt10q. Twelve isolates belonged to CC398-MRSA-V. They showed either spa types t011 or t034 and yielded dru types dt6j, dt11a, dt11ap, dt7aa (5a-2d-4a0-3b-4e-3e), dt11aw (5a-2d-3f-0-2d-5b-3a-2g-3b), and
Similar to mecA, other resistance markers as well as corresponding phenotypes were less common in chicken than in turkey isolates (see Table 2 and the Supplemental File). For instance, the beta-lactamase operon was found in 11.8% of chicken and 97.5% of turkey isolates. This observation also corresponds to the low prevalence of these genes especially in CC5-MSSA. Only five out of 49 CC5-MSSA carried blaZ, while it was detected in 58 out of 59 CC398-MSSA. Similarly, tet and erm genes were also much more common in CC398 than in CC5 isolates. 3.4. Virulence factors A summary of the carriage of virulence factors is provided in Tables 1 and 2, full data in the Supplemental File. Neither the PVL genes (lukF/S-PV), nor the gene encoding the toxic shock syndrome toxin (tst1) were found in poultry isolates. The egc enterotoxin gene cluster which comprises the enterotoxin G, I, M, N, O and U genes (seg, sei, selm, seln, selo, selu) was found in 55 isolates, i.e., in all isolates that belonged to CC5 or CC9. The enterotoxin A gene sea was found in 41 isolates with 39 of them originating from turkeys and two from chickens. One seapositive isolate belonged to CC5, all others to CC398. In this CC5 isolate of chicken origin, sea was found together with sak, scn and chp. In CC398 isolates it was always accompanied by sak only. Interestingly none of the CC398-MRSA-IV and -V isolates carried sea or any other enterotoxin genes. One additional isolate from a turkey harboured a deviant allele of sea, ‘‘sea-N315’’ as known from the S. aureus N315 genome sequence (BA000018.3). However, this isolate belonged to CC7 while S. aureus N315 belonged to CC5. Further enterotoxin genes were not identified. The epidermal cell differentiation inhibitor gene edinA was found once, in a CC5 isolate from a chicken. 4. Discussion Molecular typing of S. aureus isolates from routine diagnostics of poultry revealed significant differences between chickens and turkeys and – especially in turkeys – a high percentage of MRSA. CC5-MSSA strains were commonly detected in chickens, but at a lower frequency also in turkeys. This is in accordance with previous observations (Lowder et al., 2009) indicating that CC5 is a major S. aureus lineage in poultry. An MRSA strain from this lineage was detected in turkeys. This strain, CC5MRSA-III, has previously been isolated from Korean chicken meat samples (Kwon et al., 2006) and German
Table 1 Genotyping data and antimicrobial resistance of the 22 MRSA isolates from poultry. Strain
Isolate Host designation
spa Type
dru Type
Resistance phenotypea
Resistance genotype
SCCmec-associated genes
Virulence-associated genes
CC5-MRSA-III
001-V203
Turkey
t002
dt9v
BLA, TET, MLSB, SPC, ENR
mecA, blaZ/I/R, tet(M), tet(K), erm(A), erm(B), spc
mecA, ugpQ, mecI, mecR, xylR, ccrA/B3, dcs, PSMmec (in 3/3 tested)
agr group II, capsule seg/i, selm/n/o/u type 5, sasG (egc cluster), lukF/S/hlgA, lukD/E
022-V234 025-V237 038-V313
Turkey Turkey Turkey
t002 t002 t002
dt9v dt9v dt9v
BLA, TET, MLSB, SPC, ENR BLA, TET, MLSB, SPC, ENR BLA, TET, MLSB, SPC, ENR
mecA, blaZ/I/R, tet(M), erm(A), spc mecA, blaZ/I/R, tet(M), tet(L), erm(A), spc mecA, blaZ/I/R, tet(M), erm(A), spc
067-V464
Turkey
t1430 dt10a
BLA, TET, MLSB, TMP, (KAN), ENR
mecA, blaZ/I/R, tet(L), erm(B), dfrK, aadD
mecA, delta mecR, ugpQ, ccrA/B2, dcs
seg/i, selm/n/o/u (egc cluster), lukF/S/hlgA,
agr group II, capsule type 5
075-V475
Turkey
t1430 dt10a
BLA, TET, MLSB, TMP, (KAN), ENR
mecA, blaZ/I/R, tet(L), erm(B), dfrK, aadD
CC398-MRSA-irreg./ 006-V208 truncated SCCmec
Turkey
n.a.
dt11a
BLA, TET, MLSB, TMP, SPC, TIA, SXT
mecA, blaZ/I/R, tet(M), tet(L), erm(A), erm(B), erm(C), dfrK, aadD
mecA, ugpQ, mecI, mecR, xylR, dcs, PSM-mec
sea, lukF/ S/hlgA, sak
agr group I, capsule type 5, cna
CC398-MRSA-IV
003-V205
Turkey
t011
dt10q
lukF/S/hlgA
agr group I, capsule type 5, cna
Turkey
t011
dt10q
mecA, blaZ/I/R, tet(M), erm(C), dfrK, aacA/aphD, aadD mecA, blaZ/I/R, tet(M), tet(K), tet(L), erm(B), dfrK, aacA/aphD, aadD
mecA, delta mecR, ugpQ, ccrA/B2, dcs
005-V207
BLA, TET, MLSB, TMP, GEN, KAN BLA, TET, MLSB, TMP, GEN, KAN
CC9-MRSA-IV
Other notable genes
090-V490
Chicken t011 (broiler) Turkey t034 Turkey t011
BLA, TET, MLSB, TMP, GEN, KAN, SXT dt7aa BLA, TET, MLSB, SPC dt11aw BLA, TET, MLSB, TIA, (ENR) dt11ap
S. Monecke et al. / Veterinary Microbiology 162 (2013) 806–812 809
mecA, blaZ/I/R, tet(M), tet(L), erm(T), mecA, ugpQ, ‘‘ccrAA’’ c, ccrC lukF/S/hlgA agr group I, capsule dfrK, aacA/aphD type 5, cna 002-V204 mecA, blaZ/I/R, tet(M), erm(A), spc 004-V206 mecA, blaZ/I/R, tet(M), tet(K), erm(C), vga(A)b 026-V238 Turkey t011 dt11aw BLA, TET, MLSB, TMP, mecA, blaZ/I/R, tet(M), tet(K), tet(L), KAN, TIA, (ENR) erm(C), erm(T), dfrK, aadD, vga(A)b 028-V240 Turkey t011 dt11aw BLA, TET, MLSB, TMP, mecA, blaZ/I/R, tet(M), tet(K), tet(L), SPC, (KAN), TIA, (ENR) erm(B), erm(C), dfrK, aadD, vga(A)b 034-V309 Turkey t011 dt11aw BLA, TET, MLSB, TIA, (ENR) mecA, blaZ/I/R, tet(M), tet(K), erm(C), vga(A)b 039-V314 Turkey t034 dt11a BLA, TET, MLSB, TMP, mecA, blaZ/I/R, tet(M), tet(L), erm(A), SPC, GEN, KAN, TIA erm(B), lnu(A), dfrK, spc, aacA/aphD, aadD, vga(E) 079-V479 Chicken t011 dt11ap BLA, TET, MLSB, TMP, mecA, blaZ/I/R, tet(M), tet(L), erm(T), broiler) GEN, KAN dfrK, aacA/aphD 106-V508 Chicken t011 dt11ap BLA, TET, MLSB, TMP, mecA, blaZ/I/R, tet(M), tet(L), erm(T), broiler) GEN, KAN dfrK, aacA/aphD 119-V525 Chicken t011 dt11ap BLA, TET, MLSB, TMP, mecA, blaZ/I/R, tet(M), tet(L), erm(T), broiler) GEN, KAN, SXT dfrK, aacA/aphD 133-V562 Chicken t011 dt11ap BLA, TET, MLSB, TMP, mecA, blaZ/I/R, tet(M), tet(L), erm(T), broiler) SPC, GEN, KAN, SXT dfrK, spc, aacA/aphD 134-V563 Turkey t011 dt12q BLA, TET, MLSB mecA, blaZ/I/R, tet(M), tet(K), erm(C) 135-V564 Turkey t034 dt6j BLA, TET, MLSB, TMP, mecA, blaZ/I/R, tet(M), tet(K), erm(A), SPC, TIA dfrK, spc a Abbreviations of the antimicrobial agents are as follows: BLA, b-lactams; ENR, enrofloxacin; GEN, gentamicin; KAN, kanamycin; MLSB, macrolides-lincosamides-streptogramin B antibiotics; SPC, spectinomycin; SXT, trimethoprim/sulfamethoxazole 1/19; TET, tetracycline; TIA, tiamulin; TMP, trimethoprim; antibiotics in parentheses indicate that the respective isolate was classified as intermediate to this antimicrobial agent. b vga(A) variant from strain S. aureus BM3327 (GenBank accession no. AF186237). c Gene encoding a hypothetical protein/recombinase accompanying ccrC in SCCmec V elements (GenBank: AM292304.1: 5654–7273). CC398-MRSA-V
810
Table 2 Genotyping data and phenotypic resistance of the 109 MSSA isolates from poultry. Host
Number
SCCmec-associated genes
Resistance-associated genes
Resistance phenotypea
Virulence-associated genes
Other notable genes
CC5-MSSA
Chicken
44
None
blaZ/I/R (in 2/44), erm(A) (in 1/44), erm(C) (in 4/44), tet(K) (in 7/44), qacC (in 2/44)
AMP/AML: 5, OXA/FOX: 0, ERY: 21, TYL: 34, TIL: 4, TIA: 5, SPE: 44, ENR: 22, SXT: 2, TET: 14
agr group II, capsule type 5, sasG
CC5-MSSA
Turkey
4
None
blaZ/I/R (in 3/4), erm(A) tet(M)
agr group II, capsule type 5, sasG
CC7-MSSA
Turkey
1
None
erm(C)
sea-N315, lukF/S/hlgA, lukD/E, sak, scn
agr group I, capsule type 8
CC15-MSSA
Turkey
1
None
blaZ/I/R
lukF/S/hlgA, chp, scn
agr group II, capsule type 8, sasG
CC398-MSSA
Chicken
2
None
blaZ/I/R (in 1/2), erm(A) (in 1/2), erm(C) (in 1/2), tet(M) (in 1/2) qacC (in 1/2)
sea (in 1/29), lukF/S/hlgA, sak (in 2/2), scn (in 1/2)
agr group I, capsule type 5, cna
CC398-MSSA
Turkey
AMP/AML: 4, OXA/FOX: 0, ERY: 4, TYL: 4, TIL: 4, TIA: 1, SPE: 4, ENR: 4, SXT: 0, TET: 4 AMP/AML: 0, OXA/FOX: 0, ERY: 1, TYL: 1, TIL: 1, TIA: 0, SPE: 1, ENR: 1, SXT: 0, TET: 1 AMP/AML: 1, OXA/FOX: 0, ERY: 1, TYL: 1, TIL: 1, TIA: 1, SPE: 1, ENR: 1, SXT: 0, TET: 1 AMP/AML: 0, OXA/FOX: 0, ERY: 2, TYL: 2, TIL: 0, TIA: 0, SPE: 2, ENR: 1, SXT: 0, TET: 1 AMP/AML: 57, OXA/FOX: 0, RY: 53, TYL: 54, TIL: 40, IA: 36, SPE: 57, ENR: 49 S XT: 17, TET: 57
sea (in 1/44), seg/i, selm/n/ o/u (egc cluster), lukF/S/hlgA, lukD/E sak (in 1/44), chp (in 1/44), scn (in 3/44), edinA (in 1/44) seg/i, selm/n/o/u (egc cluster), lukF/S/hlgA, lukD/E
57
ccrA/B1 (in 1/57)
blaZ/I/R, erm(A) (in 34/57), sea (in 38/57),lukF/S/hlgA, agr group I, capsule erm(B) (in 3/57), erm(C) (in sak (in 37/57) type 5, cna 32/57), lnu(A) (in 1/57), aadD (in 11/57), tet(K) (in 5/57), tet(M) (in 56/57), qacC (in 12/59) a Numbers indicate the non-susceptible isolates. Abbreviations of the antimicrobial agents and disc contents are as follows: ampicillin (AMP, 10 mg), amoxycillin (AML, 10 mg), oxacillin (OXA, 5 mg), cefoxitin (FOX, 30 mg), erythromycin (ERY, 15 mg), tylosin (TYL, 30 mg), tilmicosin (TIL, 15 mg), tiamulin (TIA, 30 mg), spectinomycin (SPE, 10 mg), enrofloxacin (ENR, 5 mg), sulfamethoxazole/trimethoprim (SXT, 25 mg) and tetracycline (TET, 30 mg).
S. Monecke et al. / Veterinary Microbiology 162 (2013) 806–812
Strain
S. Monecke et al. / Veterinary Microbiology 162 (2013) 806–812
turkey meat products (Feßler et al., 2011). However, it was also observed among humans in KwaZulu-Natal/South Africa (Shittu et al., 2009). Other strains that were found in turkeys were CC7-MSSA and CC15-MSSA. These two lineages are common in humans (Monecke et al., 2009), so that a transmission from farmers to turkeys can be assumed. Two turkey isolates, which were MRSA, belonged to CC9. The CC9 lineage appears to be livestock-associated in a similar way as CC398. Isolates that usually carried SCCmec III, V or IX elements have mainly been described from pigs or farm workers in Asian and European countries (Cui et al., 2009; Guardabassi et al., 2009; Neela et al., 2009; Pan et al., 2009; Wagenaar et al., 2009; Vestergaard et al., 2012), and CC9-MRSA-IV was also found in turkey meat products from Germany (Feßler et al., 2011). Some CC9-MRSA of various SCCmec types, including PVLpositives, from humans without obvious livestock connection usually belong to a lineage (ST834) that is very distinct from livestock-associated CC9 strains (Nimmo and Coombs, 2008; Monecke et al., 2011, 2012b). CC398 was surprisingly common in turkeys. This includes not only the recently emerging livestock associated CC398-MRSA-V strain, but also CC398-MRSA with other SCCmec types and CC398-MSSA. CC398-MSSA have previously been observed in pigs. In a Danish study (Hasman et al., 2010), 39% of pig isolates belonged to CC398, with only one being MRSA. Given the high prevalence and diversity of CC398 S. aureus in pigs as well as in turkeys, it can hardly be determined based on epidemiological data which – if any – of either species was the original host for the CC398 lineage. A recent study on 89 whole genomes of CC398 strains (Price et al., 2012) indicated a possible emergence of this lineage in humans, followed by transmissions into turkeys and pigs. This study, as well as our observations – namely the common presence of genes from beta haemolysin-converting phages (sea, sak) in CC398 MSSA – shows a closer relationship of human isolates to the majority of CC398MSSA from turkeys than to isolates from pigs. CC398MRSA-V that do not carry these genes might secondarily have been transmitted from other livestock, such as pigs. A comparison of the CC398-MRSA isolates from diseased poultry with those from diseased pigs (Kadlec et al., 2009) and cattle suffering from mastitis (Feßler et al., 2010) revealed striking similarities not only with regard to the spa types, the dru types and the resistance pheno- and genotypes, but also with regard to the lack of major staphylococcal virulence factors. Thus, livestock-associated MRSA isolates of CC398 with similar characteristics seem to be associated with a number of different diseases in different food-producing animal species and their relative homogeneity suggests a recent expansion. In this study more isolates of CC398-MRSA-V than of CC398MRSA-IV have been found. Generally, the former strain appears to be more abundant and widespread, and it has been detected in a wider range of host species including humans, pigs, dogs, horses, cattle and poultry from various European countries, Canada, the USA, Australia and China (Huijsdens et al., 2006; Wulf et al., 2006; de Neeling et al., 2007; Monecke et al., 2007; Van Loo et al., 2007; Nemati
811
et al., 2008; Van Duijkeren et al., 2008; Krziwanek et al., 2009; Lozano et al., 2009; Nienhoff et al., 2009; Pan et al., 2009; Walther et al., 2009; Battisti et al., 2010; Potel et al., 2010; Soavi et al., 2010; Vanderhaeghen et al., 2010; Du et al., 2011; Monecke et al., 2011). CC398-MRSA-IV have been found in humans, poultry/poultry meat samples and cattle from Belgium, Germany, and The Netherlands (Ip et al., 2005; Nemati et al., 2008; Deurenberg et al., 2009; Feßler et al., 2010). Further work is needed to determine the true prevalence of MRSA among S. aureus isolates from poultry and to assess whether the S. aureus population in healthy poultry differs in terms of CC affiliation, resistance or virulence gene carriage from that of isolates from clinically ill animals or isolates from food of poultry origin. Acknowledgements The authors thank E. Mu¨ller, I. Engelmann and J. Sachtschal (Jena) and K. Meyer (Mariensee) for excellent technical assistance. We acknowledge Prof. E. Jacobs (Dresden) and E. Ermantraut (Jena) for their support. There was no external funding for this project. Stefan Monecke, Ralf Ehricht and Peter Slickers are employees of Alere Technologies GmbH, Jena, Germany. Sarah Wendlandt is supported by scholarships from the Stiftung Tiera¨rztliche Hochschule Hannover and from the Akademie fu¨r Tiergesundheit e.V..
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.vetmic.2012.10.018. References Battisti, A., Franco, A., Merialdi, G., Hasman, H., Iurescia, M., Lorenzetti, R., Feltrin, F., Zini, M., Aarestrup, F.M., 2010. Heterogeneity among methicillin-resistant Staphylococcus aureus from Italian pig finishing holdings. Vet. Microbiol. 142, 361–366. Clinical and Laboratory Standards Institute, 2008. Performance Standards for Antimicrobial Disk and Dilution Susceptibility Test for Bacteria Isolated from Animals; Approved Standard, third ed. Clinical and Laboratory Standards Institute, Wayne, PA, CLSI Document M31-A3. Clinical and Laboratory Standards Institute, 2011. Performance Standards for Antimicrobial Susceptibility Testing – Twentieth Informational Supplement. Clinical and Laboratory Standards Institute, Wayne, PA, CLSI Document M100-S21. Cui, S., Li, J., Hu, C., Jin, S., Li, F., Guo, Y., Ran, L., Ma, Y., 2009. Isolation and characterization of methicillin-resistant Staphylococcus aureus from swine and workers in China. J. Antimicrob. Chemother. 64, 680–683. de Neeling, A.J., van den Broek, M.J., Spalburg, E.C., van Santen-Verheuvel, M.G., Dam-Deisz, W.D., Boshuizen, H.C., van de Giessen, A.W., van Duijkeren, E., Huijsdens, X.W., 2007. High prevalence of methicillin reistant Staphylococcus aureus in pigs. Vet. Microbiol. 122, 366–372. Deurenberg, R.H., Nulens, E., Valvatne, H., Sebastian, S., Driessen, C., Craeghs, J., de Brauwer, E., Heising, B., Kraat, Y.J., Riebe, J., Stals, F.S., Trienekens, T.A., Scheres, J., Friedrich, A.W., van Tiel, F.H., Beisser, P.S., Stobberingh, E.E., 2009. Cross-border dissemination of methicillin-resistant Staphylococcus aureus, Euregio Meuse-Rhine region. Emerg. Infect. Dis. 15, 727–734. Du, J., Chen, C., Ding, B., Tu, J., Qin, Z., Parsons, C., Salgado, C., Cai, Q., Song, Y., Bao, Q., Zhang, L., Pan, J., Wang, L., Yu, F., 2011. Molecular characterization and antimicrobial susceptibility of nasal Staphylococcus aureus isolates from a Chinese medical college campus. PLoS ONE 6, e27328.
812
S. Monecke et al. / Veterinary Microbiology 162 (2013) 806–812
Enright, M.C., Day, N.P.J., Davies, C.E., Peacock, S.J., Spratt, B.G., 2000. Multilocus sequence typing for characterization of methicillin-resistant and methicillin-susceptible clones of Staphylococcus aureus. J. Clin. Microbiol. 38, 1008–1015. Feßler, A., Scott, C., Kadlec, K., Ehricht, R., Monecke, S., Schwarz, S., 2010. Characterization of methicllin-resistant Staphylococcus aureus ST398 from cases of bovine mastitis. J. Antimicrob. Chemother. 65, 619–625. Feßler, A.T., Kadlec, K., Hassel, M., Hauschild, T., Eidam, C., Ehricht, R., Monecke, S., Schwarz, S., 2011. Characterization of methicillin-resistant Staphylococcus aureus isolates from food and food products of poultry origin in Germany. Appl. Environ. Microbiol. 77, 7151–7157. Goering, R.V., Morrison, D., Al-Doori, Z., Edwards, G.F., Gemmell, C.G., 2008. Usefulness of mec-associated direct repeat unit (dru) typing in the epidemiological analysis of highly clonal methicillin-resistant Staphylococcus aureus in Scotland. Clin. Microbiol. Infect. 14, 964–969. Guardabassi, L., O’Donoghue, M., Moodley, A., Ho, J., Boost, M., 2009. Novel lineage of methicillin-resistant Staphylococcus aureus, Hong Kong. Emerg. Infect. Dis. 15, 1998–2000. Hasman, H., Moodley, A., Guardabassi, L., Stegger, M., Skov, R.L., Aarestrup, F.M., 2010. spa type distribution in Staphylococcus aureus originating from pigs, cattle and poultry. Vet. Microbiol. 141, 326–331. Hauschild, T., Feßler, A.T., Kadlec, K., Billerbeck, C., Schwarz, S., 2012. Detection of the novel vga(E) gene in methicillin-resistant Staphylococcus aureus CC398 isolates from cattle and poultry. J. Antimicrob. Chemother. 67, 503–504. Huijsdens, X.W., van Dijke, B.J., Spalburg, E., van Santen-Verheuvel, M.G., Heck, M.E., Pluister, G.N., Voss, A., Wannet, W.J., de Neeling, A.J., 2006. Community-acquired MRSA and pig-farming. Ann. Clin. Microbiol. Antimircob. 5, 26. Ip, M., Yung, R.W.H., Ng, T.K., Luk, W.K., Tse, C., Hung, P., Enright, M., Lyon, D.J., 2005. Contemporary methicillin-resistant Staphylococcus aureus clones in Hong Kong. J. Clin. Microbiol. 43, 5069–5073. Kadlec, K., Ehricht, R., Monecke, S., Steinacker, U., Kaspar, H., Mankertz, J., Schwarz, S., 2009. Diversity of antimicrobial resistance pheno- and genotypes of methicillin-resistant Staphylococcus aureus ST398 from diseased swine. J. Antimicrob. Chemother. 64, 1156–1164. Krziwanek, K., Metz-Gercek, S., Mittermayer, H., 2009. Methicillin-resistant Staphylococcus aureus ST398 from human patients, upper Austria. Emerg. Infect. Dis. 15, 766–769. Kwon, N.H., Park, K.T., Jung, W.K., Youn, H.Y., Kim, S.H., Bae, W., Lim, J.Y., Kim, J.Y., Kim, J.M., Hong, S.K., Park, Y.H., 2006. Characteristics of methicillin resistant Staphylococcus aureus isolated from chicken meat and hospitalized dogs in Korea and their epidemiological relatedness. Vet. Microbiol. 117, 304–312. Lowder, B.V., Guinane, C.M., Ben Zakour, N.L., Weinert, L.A., ConwayMorris, A., Cartwright, R.A., Simpson, A.J., Rambaut, A., Nu¨bel, U., Fitzgerald, J.R., 2009. Recent human-to-poultry host jump, adaptation, and pandemic spread of Staphylococcus aureus. Proc. Natl. Acad. Sci. U.S.A. 106, 19545–19550. Lozano, C., Lopez, M., Gomez-Sanz, E., Ruiz-Larrea, F., Torres, C., Zarazaga, M., 2009. Detection of methicillin-resistant Staphylococcus aureus ST398 in food samples of animal origin in Spain. J. Antimicrob. Chemother. 64, 1325–1326. Monecke, S., Kuhnert, P., Hotzel, H., Slickers, P., Ehricht, R., 2007. Microarray-based study on virulence-associated genes and resistance determinants of Staphylococcus aureus isolates from cattle. Vet. Microbiol. 125, 128–140. Monecke, S., Luedicke, C., Slickers, P., Ehricht, R., 2009. Molecular epidemiology of Staphylococcus aureus in asymptomatic carriers. Eur. J. Clin. Microbiol. Infect. Dis. 28, 1159–1165. Monecke, S., Coombs, G., Shore, A.C., Coleman, D.C., Akpaka, P., Borg, M., Chow, H., Ip, M., Jatzwauk, L., Jonas, D., Kadlec, K., Kearns, A., Laurent, F., O’Brien, F.G., Pearson, J., Ruppelt, A., Schwarz, S., Scicluna, E., Slickers, P., Tan, H.-L., Weber, S., Ehricht, R., 2011. A field guide to pandemic, epidemic and sporadic clones of methicillin-resistant Staphylococcus aureus. PLoS ONE e17936. Monecke, S., Engelmann, I., Archambault, M., Coleman, D.C., Coombs, G.W., Cortez de Ja¨ckel, S., Pelletier-Jacques, G., Schwarz, S., Shore, A.C., Slickers, P., Ehricht, R., 2012a. Distribution of SCCmec-associated phenol-soluble modulin in staphylococci. Mol. Cell. Probes 26, 99–103. Monecke, S., Skakni, L., Hasan, R., Ruppelt, A., Ghazal, S.S., Hakawi, A., Slickers, P., Ehricht, R., 2012b. Characterisation of MRSA strains isolated from patients in a hospital in Riyadh, Kingdom of Saudi Arabia. BMC Microbiol. 12, 146. Neela, V., Mohd Zaful, A., Mariana, N.S., van Belkum, A., Liew, Y.K., Rad, E.G., 2009. Prevalence of ST9 methicillin-resistant Staphylococcus
aureus among pigs and pig handlers in Malaysia. J. Clin. Microbiol. 47, 4138–4140. Nemati, M., Hermans, K., Lippinska, U., Denis, O., Deplano, A., Struelens, M., Devriese, L.A., Pasmans, F., Haesebrouck, F., 2008. Antimicrobial resistance of old and recent Staphylococcus aureus isolates from poultry: first detection of livestock-associated methicillin-resistant strain ST398. Antimicrob. Agents Chemother. 52, 3817–3819. Nienhoff, U., Kadlec, K., Chaberny, I.F., Verspohl, J., Gerlach, G.F., Schwarz, S., Simon, D., Nolte, I., 2009. Transmission of methicillin-resistant Staphylococcus aureus strains between humans and dogs: two case reports. J. Antimicrob. Chemother. 64, 640–662. Nimmo, G.R., Coombs, G.W., 2008. Community-associated methicillinresistant Staphylococcus aureus (MRSA) in Australia. Int. J. Antimicrob. Agents 31, 401–410. Pan, A., Battisti, A., Zoncada, A., Bernieri, F., Boldini, M., Franco, A., Giorgi, M., Iurescia, M., Lorenzotti, S., MArtinotti, M., Monaci, M., Pantosti, A., 2009. Community-acquired methicillin-resistant Staphylococcus aureus ST398 infection, Italy. Emerg. Infect. Dis. 15, 845–847. ˜ lvarez-Fernandez, M., Constenla, L., A ˜ lvarez, P., Perez, S., 2010. Potel, C., A First human isolates of methicillin-resistant Staphylococcus aureus sequence type 398 in Spain. Eur. J. Clin. Microbiol. Infect. Dis. 29, 351–352. Price, L.B., Stegger, M., Hasman, H., Aziz, M., Larsen, J., Andersen, P.S., Pearson, T., Waters, A.E., Foster, J., Schupp, J., Gillec, J., Driebe, E., Liu, C.M., Laurent, F., Springer, B., Zdovc, I., Battisti, A., Franco, A., Zmudzki, J., Schwarz, S., Butaye, P., Jouy, E., Pomba, C., Porreo, M.C., Smith, T.C., Robinson, D.A., Weese, J.S., Arriola, C.O., Yu, F., Ruimy, R., Keim, P., Skov, R., Aarestrup, F.M., 2012. Staphylococcus aureus CC398: host adaptation and emergence of methicillin resistance in livestock. mBio 3, e00305–e00311. Schijffelen, M.J., Boel, C.H., van Strijp, J.A., Fluit, A.C., 2010. Whole genome analysis of a livestock-associated methicillin-resistant Staphylococcus aureus ST398 isolate from a case of human endocarditis. BMC Genomics 11, 376. Schwarz, S., Silley, P., Simjee, S., Woodford, N., van Duijkeren, E., Johnson, A.P., Gaastra, W., 2010. Assessing the antimicrobial susceptibility of bacteria obtained from animals. Vet. Microbiol. 141, 1–4. Shittu, A., Nu¨bel, U., Udo, E., Lin, J., Gaogakwe, S., 2009. Characterisation of methicillin-resistant Staphylococcus aureus isolates from hospitals in KwaZulu-Natal province, Republic of South Africa. J. Med. Microibiol. 58, 1219–1226. Soavi, L., Stellini, R., Signorini, L., Antonini, B., Pedroni, P., Zanetti, L., Milanesi, B., Pantosti, A., Matteelli, A., Pan, A., Carosi, G., 2010. Methicillin-resistant Staphylococcus aureus ST398, Italy. Emerg. Infect. Dis. 16, 346–348. Van Duijkeren, E., Ikawaty, R., Broekhuizen-Stins, M.J., Jansen, M.D., Spalburg, E.C., de Neeling, A.J., Allaart, J.G., van Nes, A., Wagenaar, J.A., Fluit, A.C., 2008. Transmission of methicillin-resistant Staphylococcus aureus strains between different kinds of pig farms. Vet. Microbiol. 126, 383–389. Van Loo, I., Huijsdens, X., Tiermersma, E., de Neeling, A., van de SandeBruinsma, N., Beaujean, D., Voss, A., Klytmans, J., 2007. Emergence of methisillin-resistant Staphylococcus aureus of animal origin in humans. Emerg. Infect. Dis. 13, 1834–1839. Vanderhaeghen, W., Cerpentier, T., Adriaensen, C., Vicca, J., Hermans, K., Butaye, P., 2010. Methicillin-resistant Staphylococcus aureus (MRSA) ST398 associated with clinical and subclinical mastitis in Belgian cows. Vet. Microbiol. 144, 166–171. Vestergaard, M., Cavaco, L.M., Sirichote, P., Unahalekhaka, A., Dangsakul, W., Svendsen, C.A., Aarestrup, F.M., Hendriksen, R.S., 2012. SCCmec type IX element in methicillin resistant Staphylococcus aureus spa type t337 (CC9) isolated from pigs and pork in Thailand. Front. Microbiol. 3, 103. Wagenaar, J.A., Yue, H., Pritchard, J., Broekhuizen-Stins, M., Huijsdens, X., Mevius, D.J., Bosch, T., van Duijkeren, E., 2009. Unexpected sequence types in livestock associated methicillin-resistant Staphylococcus aureus (MRSA): MRSA ST9 and a single locus variant of ST9 in pig farming in China. Vet. Microbiol. 139, 405–409. Walther, B., Monecke, S., Ruscher, C., Friedrich, A.W., Ehricht, R., Slickers, P., Soba, A., Wleklinski, C.G., Wieler, L.H., Lubke-Becker, A., 2009. Comparative molecular analysis substantiates a zoonotic potential of equine methicillin-resistant Staphylococcus aureus (MRSA). J. Clin. Microbiol. 47, 704–710. Wulf, M., van Nes, A., Eikelenboom-Boskamp, A., de Vries, J., Melchers, W., Klaassen, C., Voss, A., 2006. Methicillin-resistant Staphylococcus aureus in veterinary doctors and students, The Netherlands. Emerg. Infect. Dis. 12, 1939–1941.
Contents lists available at SciVerse ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Genotyping of Staphylococcus aureus isolates from diseased poultry Stefan Monecke a,b,*, Antje Ruppelt a, Sarah Wendlandt c, Stefan Schwarz c, Peter Slickers b, Ralf Ehricht b, Sonia Cortez de Ja¨ckel d a
Institute for Medical Microbiology and Hygiene, Technical University of Dresden, Dresden, Germany Alere Technologies GmbH, Jena, Germany c Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut (FLI), Neustadt-Mariensee, Germany d Poultry Clinics and Laboratory Dr. Po¨ppel, Delbru¨ck, Germany b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 10 February 2012 Received in revised form 6 August 2012 Accepted 15 October 2012
To gain insight into the genomic diversity of Staphylococcus aureus associated with diseases in domestic poultry, 131 isolates from clinically ill turkeys (n = 80) and chickens (n = 51) were collected and genotyped using microarray hybridisations. MRSA isolates were subjected to spa and dru typing and their antimicrobial resistance geno- and phenotypes were determined. Most (68 out of 80) turkey isolates belonged to the clonal complex (CC) 398. Seventeen of the 80 isolates (21.2%) were MRSA. The most common MRSA type among turkeys was CC398-MRSA-V (n = 8), but CC5-MRSA-III (n = 4), CC9MRSA-IV (n = 2), CC398-MRSA-IV (n = 2) and a single CC398-MRSA with an unidentified/ truncated SCCmec element were also found. Among the chicken isolates, CC5 predominated (44 out of 51). Five of the chicken isolates were MRSA (9.8%), all belonging to CC398-MRSA-V. These data show that the current dissemination of livestock-associated MRSA also engulfs chickens and turkeys, and that MRSA surveillance among these species is warranted. ß 2012 Elsevier B.V. All rights reserved.
Keywords: Staphylococcus aureus MRSA MSSA CC398 Microarray analysis Minimum inhibitory concentrations spa typing dru typing
1. Introduction Staphylococcus aureus is a versatile bacterium which can infect or colonise a variety of different host species. While it is found in various mammals including humans, there are also several reports about its presence in both, domestic and wild birds. While multilocus sequence types (STs), such as ST692, have exclusively been observed in birds (see MLST database at http://saureus.mlst.net/), other STs are less host-specific. For instance, the most widespread and dominant S. aureus strains in poultry belong to ST5 within clonal complex (CC) 5, and appear to originate most likely from a single transmission from
* Corresponding author at: Institute for Medical Microbiology and Hygiene, Technical University of Dresden, Dresden, Germany. Tel.: +49 351 6200; fax: +49 351 458 6311. E-mail address: [email protected] (S. Monecke). 0378-1135/$ – see front matter ß 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetmic.2012.10.018
humans, where this lineage is also common, into fowl (Lowder et al., 2009). Strains that belong to CC398, especially the methicillin-resistant S. aureus CC398 that harbour a type V SCCmec element (CC398-MRSA-V), proved to be present in a wide variety of host species. They have been found not only in humans (Huijsdens et al., 2006; Krziwanek et al., 2009; Schijffelen et al., 2010), but also in pigs (Huijsdens et al., 2006; de Neeling et al., 2007), dairy cows and veal calves (Feßler et al., 2010; Vanderhaeghen et al., 2010), horses (Walther et al., 2009) and dogs (Nienhoff et al., 2009) and also in chickens (Nemati et al., 2008), chicken meat (Nemati et al., 2008; Feßler et al., 2011) as well as turkey meat (Feßler et al., 2011). To gain insight into the genomic relationships of S. aureus strains associated with diseases in domestic poultry, consecutive isolates from clinically ill chickens and turkeys were collected and genotyped using hybridisations to a previously described microarray (Monecke et al., 2011). This comprehensive genotyping approach of a
S. Monecke et al. / Veterinary Microbiology 162 (2013) 806–812
large number of independent methicillin-susceptible and methicillin-resistant S. aureus provides for the first time insight into the genomic relationships of such strains from diseased poultry. For a better comparison with MRSA from diseased pigs and cattle, but also with MRSA from poultry meat and poultry meat products, the MRSA isolates were subjected to spa and dru typing and their antimicrobial resistance geno- and phenotypes were determined. 2. Material and methods 2.1. Bacterial isolates A total of 131 S. aureus isolates were obtained from routine diagnostic work at the Poultry Clinic and Laboratory Dr. Po¨ppel, Delbru¨ck, Germany, between January 2009 and July 2011. This unit serves numerous large poultry farms mainly in the German federal state of North Rhine-Westphalia (although eight turkey isolates came from Baden-Wuerttemberg, and five chicken isolates from Lower Saxony). Fifty-one isolates originated from domestic chickens (38 broilers including four chicks, 13 layers) and 80 from domestic turkeys. Turkey isolates originated from 26 different farms, broilers from 18 and layers from 10 farms. The isolates were obtained from necropsy material from cases of invasive infections (joints, liver, heart, lungs), and only one isolate per animal was chosen for further analysis. Staphylococci were identified by Gram staining, catalase and coagulase test (rabbit plasma tube test) and by the ID 32 STAPH system (bioMe´rieux, Craponne, France). All strains were tested phenotypically for antibiotic resistance by the disc diffusion method using DIN breakpoints. Detection of oxacillin resistance was performed using oxacillin and cefoxitin disks and by screening for growth on the Brilliance MRSA-Agar (Oxoid, Wesel, Germany). 2.2. DNA microarray-based typing All isolates were characterised using the Alere StaphyType DNA microarray (Alere Technologies GmbH, Jena, Germany) covering 333 target sequences which correspond to approximately 170 distinct genes and their allelic variants. These include species markers, SCCmec, capsule and agr group typing markers, resistance genes and genes encoding exotoxins as well as adhesion factors (see Supplement 1). A full list including primer/probe sequences has been provided previously (Monecke et al., 2011). The assay was performed according to the instructions given by the manufacturer and to previous descriptions (Monecke et al., 2011). In short, S. aureus were grown on Columbia blood agar, harvested and lysed. DNA was prepared utilising spin columns or the automated EZ1 system (both by Qiagen, Hilden, Germany). DNA samples were subjected to a linear primer elongation using only one primer per target. During this step, biotin-16-dUTP was incorporated into the resulting amplicons. In a later step, these amplicons were hybridised to the microarray. After washing, horseradish-peroxidase-streptavidin was
807
added which subsequently triggered the precipitation of a dye. An image of the microarray was taken and analysed using reader and software provided by Alere Technologies GmbH (Jena, Germany). The automated comparison of the hybridisation patterns of the actual isolate to a reference database allowed determining its affiliation to clonal complexes as defined by MLST (Enright et al., 2000) and, in case of MRSA, to epidemic strains defined by MLST and SCCmec carriage (Monecke et al., 2011). PSM-mec was not covered by the array; it was detected by PCR as previously described (Monecke et al., 2012a). 2.3. Susceptibility testing and detection of resistance genes in MRSA Additional susceptibility testing of the MRSA isolates was performed by broth microdilution using custom-made microtitre plates (MCS Diagnostics, Swalmen, the Netherlands) which contained the following antimicrobial agents (with test range in parentheses) in two-fold dilution series: ampicillin (0.03–64 mg/ml), apramycin (0.03–64 mg/ml), ceftiofur (0.03–64 mg/ml), cefotaxime (0.015–32 mg/ml), cefquinome (0.015–32 mg/ml), cephalothin (0.06–128 mg/ ml), chloramphenicol (0.5–256 mg/ml), clindamycin (0.03–64 mg/ml), enrofloxacin (0.008–16 mg/ml), erythromycin (0.015–32 mg/ml), florfenicol (0.12–256 mg/ml), gentamicin (0.12–256 mg/ml), nalidixic acid (0.06– 128 mg/ml), oxacillin + 2% NaCl (0.03–16 mg/ml), penicillin G (0.015–32 mg/ml), quinupristin/dalfopristin (0.008– 16 mg/ml), spectinomycin (0.12–256 mg/ml), tetracycline (0.12–256 mg/ml), tiamulin (0.03–64 mg/ml), trimethoprim (0.06–128 mg/ml), trimethoprim/sulfamethoxazole (0.015/0.03–32/608 mg/ml), and vancomycin (0.008– 16 mg/ml). In addition, susceptibility to kanamycin (2–128 mg/ml) was tested by broth macrodilution. Susceptibility testing followed the recommendations given in the CLSI document M31-A3 (CLSI, 2008); classification of the isolates as resistant or susceptible based on the breakpoints given in the CLSI documents M31-A3 (CLSI, 2008) and M100-S21 (CLSI, 2011). S. aureus ATCC129213 served as quality control strain. As the aforementioned DNA microarray already covers a considerable number of resistance genes, specific PCR assays were only conducted for resistance genes not yet included in the DNA microarray. These are tet(L) (tetracycline resistance), dfrK (trimethoprim resistance), the linkage of tet(L)-dfrK, dfrK as part of Tn559; erm(T) (macrolide-lincosamide-streptogramin B resistance), spc (spectinomycin resistance), the linkage of erm(A)-spc, as well as vga(C), vga(D), vga(E) and lsa(C) (pleuromutilinlincosamide-streptogramin A resistance), and vga(E) as part of Tn6133. These PCR assays followed previously described protocols (Feßler et al., 2010, 2011; Hauschild et al., 2012). 2.4. spa and dru typing The spa typing was performed in accordance with the Ridom StaphType standard protocol (http://spaserver.ridom.de). A relatively novel typing method for methicillinresistant staphylococci, dru typing (Goering et al., 2008),
808
S. Monecke et al. / Veterinary Microbiology 162 (2013) 806–812
was performed as previously described (Feßler et al., 2010). The dru amplicons were sequenced and compared with the dru sequences and dru types stored in the dru typing database (http://dru-typing.org). Novel dru types detected during this study have been submitted to and in the meantime included in the dru typing database. 3. Results
dt12q (5a-2d-2d-4a-0-2d-4f-3a-2g-3b-4e-3e) with the latter three representing new dru types that have been identified for the first time during this study. One isolate carried an atypical SCCmec element that comprised mecA, ugpQ, mecI, mecR, xylR, dcs and PSM-mec but lacked recombinase (ccr) genes as well as the kdp operon. Thus it might represent a truncated SCCmec II or III element. For this isolate no spa PCR product was obtained in repeated attempts. Its dru type was dt11a.
3.1. S. aureus lineages in the different poultry species 3.3. Resistance markers in MSSA The most common lineage among turkey isolates was CC398. Of the 80 isolates from turkeys, 68 belonged to this clonal complex with 57 isolates being classified as MSSA and the remaining eleven isolates as MRSA. Among these CC398 MRSA isolates, one isolate had an irregular SCCmec element (see below). Two isolates carried SCCmec IV and eight isolates harboured SCCmec type V elements. The remaining turkey isolates belonged to CC5 (four CC5-MSSA, and four CC5-MRSA-III), CC7 and CC15 (one MSSA isolate each) as well as CC9 (two CC9-MRSAIV). Thus, a total of seventeen out of 80 turkey isolates (21.2%) were MRSA. Among the chicken isolates, CC5 predominated (44 out of 51 isolates) with all isolates being mecA-negative. Two isolates belonged to CC398MSSA while another five where assigned to CC398MRSA-V. The MRSA rate among the chicken isolates was at 9.8% (five out of 51). 3.2. Characterisation of the MRSA isolates from poultry The spa and dru types of the 22 MRSA isolates from poultry as well as their antimicrobial resistance phenoand genotypes are shown in Table 1 and in the Supplemental File. Thirteen different resistance phenotypes and 16 different resistance genotypes were detected. All 22 MRSA isolates from poultry were considered as multiresistant by their resistance to three or more classes of antimicrobial agents (Schwarz et al., 2010). Moreover, all 22 MRSA isolates were resistant to beta-lactams, tetracyclines and MLSB antibiotics. They carried the beta-lactam resistance genes mecA and blaZ, the tetracycline resistance genes tet(M), tet(K) and tet(L) as well as the MLSB resistance genes erm(A), erm(B), erm(C) and erm(T) alone or in various combinations. Resistances to other antimicrobial agents/classes of antimicrobial agents varied among the isolates. The two CC9-MRSA-IV isolates shared spa type t1430, dru type dt10a and displayed the same resistance phenoand genotypes. The four CC5-MRSA-III isolates also had the same spa (t002) and dru (dt9v) types. Although they showed the same resistance phenotype, the four isolates clustered into three resistance genotypes. All carried erm(A) and tet(M), but the additional carriage of erm(B), tet(L) and tet(K) varied. In total, 16 CC398-MRSA isolates were found. Two of the CC398 isolates belonged to CC398-MRSA-IV, spa type t011 and dru type dt10q. Twelve isolates belonged to CC398-MRSA-V. They showed either spa types t011 or t034 and yielded dru types dt6j, dt11a, dt11ap, dt7aa (5a-2d-4a0-3b-4e-3e), dt11aw (5a-2d-3f-0-2d-5b-3a-2g-3b), and
Similar to mecA, other resistance markers as well as corresponding phenotypes were less common in chicken than in turkey isolates (see Table 2 and the Supplemental File). For instance, the beta-lactamase operon was found in 11.8% of chicken and 97.5% of turkey isolates. This observation also corresponds to the low prevalence of these genes especially in CC5-MSSA. Only five out of 49 CC5-MSSA carried blaZ, while it was detected in 58 out of 59 CC398-MSSA. Similarly, tet and erm genes were also much more common in CC398 than in CC5 isolates. 3.4. Virulence factors A summary of the carriage of virulence factors is provided in Tables 1 and 2, full data in the Supplemental File. Neither the PVL genes (lukF/S-PV), nor the gene encoding the toxic shock syndrome toxin (tst1) were found in poultry isolates. The egc enterotoxin gene cluster which comprises the enterotoxin G, I, M, N, O and U genes (seg, sei, selm, seln, selo, selu) was found in 55 isolates, i.e., in all isolates that belonged to CC5 or CC9. The enterotoxin A gene sea was found in 41 isolates with 39 of them originating from turkeys and two from chickens. One seapositive isolate belonged to CC5, all others to CC398. In this CC5 isolate of chicken origin, sea was found together with sak, scn and chp. In CC398 isolates it was always accompanied by sak only. Interestingly none of the CC398-MRSA-IV and -V isolates carried sea or any other enterotoxin genes. One additional isolate from a turkey harboured a deviant allele of sea, ‘‘sea-N315’’ as known from the S. aureus N315 genome sequence (BA000018.3). However, this isolate belonged to CC7 while S. aureus N315 belonged to CC5. Further enterotoxin genes were not identified. The epidermal cell differentiation inhibitor gene edinA was found once, in a CC5 isolate from a chicken. 4. Discussion Molecular typing of S. aureus isolates from routine diagnostics of poultry revealed significant differences between chickens and turkeys and – especially in turkeys – a high percentage of MRSA. CC5-MSSA strains were commonly detected in chickens, but at a lower frequency also in turkeys. This is in accordance with previous observations (Lowder et al., 2009) indicating that CC5 is a major S. aureus lineage in poultry. An MRSA strain from this lineage was detected in turkeys. This strain, CC5MRSA-III, has previously been isolated from Korean chicken meat samples (Kwon et al., 2006) and German
Table 1 Genotyping data and antimicrobial resistance of the 22 MRSA isolates from poultry. Strain
Isolate Host designation
spa Type
dru Type
Resistance phenotypea
Resistance genotype
SCCmec-associated genes
Virulence-associated genes
CC5-MRSA-III
001-V203
Turkey
t002
dt9v
BLA, TET, MLSB, SPC, ENR
mecA, blaZ/I/R, tet(M), tet(K), erm(A), erm(B), spc
mecA, ugpQ, mecI, mecR, xylR, ccrA/B3, dcs, PSMmec (in 3/3 tested)
agr group II, capsule seg/i, selm/n/o/u type 5, sasG (egc cluster), lukF/S/hlgA, lukD/E
022-V234 025-V237 038-V313
Turkey Turkey Turkey
t002 t002 t002
dt9v dt9v dt9v
BLA, TET, MLSB, SPC, ENR BLA, TET, MLSB, SPC, ENR BLA, TET, MLSB, SPC, ENR
mecA, blaZ/I/R, tet(M), erm(A), spc mecA, blaZ/I/R, tet(M), tet(L), erm(A), spc mecA, blaZ/I/R, tet(M), erm(A), spc
067-V464
Turkey
t1430 dt10a
BLA, TET, MLSB, TMP, (KAN), ENR
mecA, blaZ/I/R, tet(L), erm(B), dfrK, aadD
mecA, delta mecR, ugpQ, ccrA/B2, dcs
seg/i, selm/n/o/u (egc cluster), lukF/S/hlgA,
agr group II, capsule type 5
075-V475
Turkey
t1430 dt10a
BLA, TET, MLSB, TMP, (KAN), ENR
mecA, blaZ/I/R, tet(L), erm(B), dfrK, aadD
CC398-MRSA-irreg./ 006-V208 truncated SCCmec
Turkey
n.a.
dt11a
BLA, TET, MLSB, TMP, SPC, TIA, SXT
mecA, blaZ/I/R, tet(M), tet(L), erm(A), erm(B), erm(C), dfrK, aadD
mecA, ugpQ, mecI, mecR, xylR, dcs, PSM-mec
sea, lukF/ S/hlgA, sak
agr group I, capsule type 5, cna
CC398-MRSA-IV
003-V205
Turkey
t011
dt10q
lukF/S/hlgA
agr group I, capsule type 5, cna
Turkey
t011
dt10q
mecA, blaZ/I/R, tet(M), erm(C), dfrK, aacA/aphD, aadD mecA, blaZ/I/R, tet(M), tet(K), tet(L), erm(B), dfrK, aacA/aphD, aadD
mecA, delta mecR, ugpQ, ccrA/B2, dcs
005-V207
BLA, TET, MLSB, TMP, GEN, KAN BLA, TET, MLSB, TMP, GEN, KAN
CC9-MRSA-IV
Other notable genes
090-V490
Chicken t011 (broiler) Turkey t034 Turkey t011
BLA, TET, MLSB, TMP, GEN, KAN, SXT dt7aa BLA, TET, MLSB, SPC dt11aw BLA, TET, MLSB, TIA, (ENR) dt11ap
S. Monecke et al. / Veterinary Microbiology 162 (2013) 806–812 809
mecA, blaZ/I/R, tet(M), tet(L), erm(T), mecA, ugpQ, ‘‘ccrAA’’ c, ccrC lukF/S/hlgA agr group I, capsule dfrK, aacA/aphD type 5, cna 002-V204 mecA, blaZ/I/R, tet(M), erm(A), spc 004-V206 mecA, blaZ/I/R, tet(M), tet(K), erm(C), vga(A)b 026-V238 Turkey t011 dt11aw BLA, TET, MLSB, TMP, mecA, blaZ/I/R, tet(M), tet(K), tet(L), KAN, TIA, (ENR) erm(C), erm(T), dfrK, aadD, vga(A)b 028-V240 Turkey t011 dt11aw BLA, TET, MLSB, TMP, mecA, blaZ/I/R, tet(M), tet(K), tet(L), SPC, (KAN), TIA, (ENR) erm(B), erm(C), dfrK, aadD, vga(A)b 034-V309 Turkey t011 dt11aw BLA, TET, MLSB, TIA, (ENR) mecA, blaZ/I/R, tet(M), tet(K), erm(C), vga(A)b 039-V314 Turkey t034 dt11a BLA, TET, MLSB, TMP, mecA, blaZ/I/R, tet(M), tet(L), erm(A), SPC, GEN, KAN, TIA erm(B), lnu(A), dfrK, spc, aacA/aphD, aadD, vga(E) 079-V479 Chicken t011 dt11ap BLA, TET, MLSB, TMP, mecA, blaZ/I/R, tet(M), tet(L), erm(T), broiler) GEN, KAN dfrK, aacA/aphD 106-V508 Chicken t011 dt11ap BLA, TET, MLSB, TMP, mecA, blaZ/I/R, tet(M), tet(L), erm(T), broiler) GEN, KAN dfrK, aacA/aphD 119-V525 Chicken t011 dt11ap BLA, TET, MLSB, TMP, mecA, blaZ/I/R, tet(M), tet(L), erm(T), broiler) GEN, KAN, SXT dfrK, aacA/aphD 133-V562 Chicken t011 dt11ap BLA, TET, MLSB, TMP, mecA, blaZ/I/R, tet(M), tet(L), erm(T), broiler) SPC, GEN, KAN, SXT dfrK, spc, aacA/aphD 134-V563 Turkey t011 dt12q BLA, TET, MLSB mecA, blaZ/I/R, tet(M), tet(K), erm(C) 135-V564 Turkey t034 dt6j BLA, TET, MLSB, TMP, mecA, blaZ/I/R, tet(M), tet(K), erm(A), SPC, TIA dfrK, spc a Abbreviations of the antimicrobial agents are as follows: BLA, b-lactams; ENR, enrofloxacin; GEN, gentamicin; KAN, kanamycin; MLSB, macrolides-lincosamides-streptogramin B antibiotics; SPC, spectinomycin; SXT, trimethoprim/sulfamethoxazole 1/19; TET, tetracycline; TIA, tiamulin; TMP, trimethoprim; antibiotics in parentheses indicate that the respective isolate was classified as intermediate to this antimicrobial agent. b vga(A) variant from strain S. aureus BM3327 (GenBank accession no. AF186237). c Gene encoding a hypothetical protein/recombinase accompanying ccrC in SCCmec V elements (GenBank: AM292304.1: 5654–7273). CC398-MRSA-V
810
Table 2 Genotyping data and phenotypic resistance of the 109 MSSA isolates from poultry. Host
Number
SCCmec-associated genes
Resistance-associated genes
Resistance phenotypea
Virulence-associated genes
Other notable genes
CC5-MSSA
Chicken
44
None
blaZ/I/R (in 2/44), erm(A) (in 1/44), erm(C) (in 4/44), tet(K) (in 7/44), qacC (in 2/44)
AMP/AML: 5, OXA/FOX: 0, ERY: 21, TYL: 34, TIL: 4, TIA: 5, SPE: 44, ENR: 22, SXT: 2, TET: 14
agr group II, capsule type 5, sasG
CC5-MSSA
Turkey
4
None
blaZ/I/R (in 3/4), erm(A) tet(M)
agr group II, capsule type 5, sasG
CC7-MSSA
Turkey
1
None
erm(C)
sea-N315, lukF/S/hlgA, lukD/E, sak, scn
agr group I, capsule type 8
CC15-MSSA
Turkey
1
None
blaZ/I/R
lukF/S/hlgA, chp, scn
agr group II, capsule type 8, sasG
CC398-MSSA
Chicken
2
None
blaZ/I/R (in 1/2), erm(A) (in 1/2), erm(C) (in 1/2), tet(M) (in 1/2) qacC (in 1/2)
sea (in 1/29), lukF/S/hlgA, sak (in 2/2), scn (in 1/2)
agr group I, capsule type 5, cna
CC398-MSSA
Turkey
AMP/AML: 4, OXA/FOX: 0, ERY: 4, TYL: 4, TIL: 4, TIA: 1, SPE: 4, ENR: 4, SXT: 0, TET: 4 AMP/AML: 0, OXA/FOX: 0, ERY: 1, TYL: 1, TIL: 1, TIA: 0, SPE: 1, ENR: 1, SXT: 0, TET: 1 AMP/AML: 1, OXA/FOX: 0, ERY: 1, TYL: 1, TIL: 1, TIA: 1, SPE: 1, ENR: 1, SXT: 0, TET: 1 AMP/AML: 0, OXA/FOX: 0, ERY: 2, TYL: 2, TIL: 0, TIA: 0, SPE: 2, ENR: 1, SXT: 0, TET: 1 AMP/AML: 57, OXA/FOX: 0, RY: 53, TYL: 54, TIL: 40, IA: 36, SPE: 57, ENR: 49 S XT: 17, TET: 57
sea (in 1/44), seg/i, selm/n/ o/u (egc cluster), lukF/S/hlgA, lukD/E sak (in 1/44), chp (in 1/44), scn (in 3/44), edinA (in 1/44) seg/i, selm/n/o/u (egc cluster), lukF/S/hlgA, lukD/E
57
ccrA/B1 (in 1/57)
blaZ/I/R, erm(A) (in 34/57), sea (in 38/57),lukF/S/hlgA, agr group I, capsule erm(B) (in 3/57), erm(C) (in sak (in 37/57) type 5, cna 32/57), lnu(A) (in 1/57), aadD (in 11/57), tet(K) (in 5/57), tet(M) (in 56/57), qacC (in 12/59) a Numbers indicate the non-susceptible isolates. Abbreviations of the antimicrobial agents and disc contents are as follows: ampicillin (AMP, 10 mg), amoxycillin (AML, 10 mg), oxacillin (OXA, 5 mg), cefoxitin (FOX, 30 mg), erythromycin (ERY, 15 mg), tylosin (TYL, 30 mg), tilmicosin (TIL, 15 mg), tiamulin (TIA, 30 mg), spectinomycin (SPE, 10 mg), enrofloxacin (ENR, 5 mg), sulfamethoxazole/trimethoprim (SXT, 25 mg) and tetracycline (TET, 30 mg).
S. Monecke et al. / Veterinary Microbiology 162 (2013) 806–812
Strain
S. Monecke et al. / Veterinary Microbiology 162 (2013) 806–812
turkey meat products (Feßler et al., 2011). However, it was also observed among humans in KwaZulu-Natal/South Africa (Shittu et al., 2009). Other strains that were found in turkeys were CC7-MSSA and CC15-MSSA. These two lineages are common in humans (Monecke et al., 2009), so that a transmission from farmers to turkeys can be assumed. Two turkey isolates, which were MRSA, belonged to CC9. The CC9 lineage appears to be livestock-associated in a similar way as CC398. Isolates that usually carried SCCmec III, V or IX elements have mainly been described from pigs or farm workers in Asian and European countries (Cui et al., 2009; Guardabassi et al., 2009; Neela et al., 2009; Pan et al., 2009; Wagenaar et al., 2009; Vestergaard et al., 2012), and CC9-MRSA-IV was also found in turkey meat products from Germany (Feßler et al., 2011). Some CC9-MRSA of various SCCmec types, including PVLpositives, from humans without obvious livestock connection usually belong to a lineage (ST834) that is very distinct from livestock-associated CC9 strains (Nimmo and Coombs, 2008; Monecke et al., 2011, 2012b). CC398 was surprisingly common in turkeys. This includes not only the recently emerging livestock associated CC398-MRSA-V strain, but also CC398-MRSA with other SCCmec types and CC398-MSSA. CC398-MSSA have previously been observed in pigs. In a Danish study (Hasman et al., 2010), 39% of pig isolates belonged to CC398, with only one being MRSA. Given the high prevalence and diversity of CC398 S. aureus in pigs as well as in turkeys, it can hardly be determined based on epidemiological data which – if any – of either species was the original host for the CC398 lineage. A recent study on 89 whole genomes of CC398 strains (Price et al., 2012) indicated a possible emergence of this lineage in humans, followed by transmissions into turkeys and pigs. This study, as well as our observations – namely the common presence of genes from beta haemolysin-converting phages (sea, sak) in CC398 MSSA – shows a closer relationship of human isolates to the majority of CC398MSSA from turkeys than to isolates from pigs. CC398MRSA-V that do not carry these genes might secondarily have been transmitted from other livestock, such as pigs. A comparison of the CC398-MRSA isolates from diseased poultry with those from diseased pigs (Kadlec et al., 2009) and cattle suffering from mastitis (Feßler et al., 2010) revealed striking similarities not only with regard to the spa types, the dru types and the resistance pheno- and genotypes, but also with regard to the lack of major staphylococcal virulence factors. Thus, livestock-associated MRSA isolates of CC398 with similar characteristics seem to be associated with a number of different diseases in different food-producing animal species and their relative homogeneity suggests a recent expansion. In this study more isolates of CC398-MRSA-V than of CC398MRSA-IV have been found. Generally, the former strain appears to be more abundant and widespread, and it has been detected in a wider range of host species including humans, pigs, dogs, horses, cattle and poultry from various European countries, Canada, the USA, Australia and China (Huijsdens et al., 2006; Wulf et al., 2006; de Neeling et al., 2007; Monecke et al., 2007; Van Loo et al., 2007; Nemati
811
et al., 2008; Van Duijkeren et al., 2008; Krziwanek et al., 2009; Lozano et al., 2009; Nienhoff et al., 2009; Pan et al., 2009; Walther et al., 2009; Battisti et al., 2010; Potel et al., 2010; Soavi et al., 2010; Vanderhaeghen et al., 2010; Du et al., 2011; Monecke et al., 2011). CC398-MRSA-IV have been found in humans, poultry/poultry meat samples and cattle from Belgium, Germany, and The Netherlands (Ip et al., 2005; Nemati et al., 2008; Deurenberg et al., 2009; Feßler et al., 2010). Further work is needed to determine the true prevalence of MRSA among S. aureus isolates from poultry and to assess whether the S. aureus population in healthy poultry differs in terms of CC affiliation, resistance or virulence gene carriage from that of isolates from clinically ill animals or isolates from food of poultry origin. Acknowledgements The authors thank E. Mu¨ller, I. Engelmann and J. Sachtschal (Jena) and K. Meyer (Mariensee) for excellent technical assistance. We acknowledge Prof. E. Jacobs (Dresden) and E. Ermantraut (Jena) for their support. There was no external funding for this project. Stefan Monecke, Ralf Ehricht and Peter Slickers are employees of Alere Technologies GmbH, Jena, Germany. Sarah Wendlandt is supported by scholarships from the Stiftung Tiera¨rztliche Hochschule Hannover and from the Akademie fu¨r Tiergesundheit e.V..
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.vetmic.2012.10.018. References Battisti, A., Franco, A., Merialdi, G., Hasman, H., Iurescia, M., Lorenzetti, R., Feltrin, F., Zini, M., Aarestrup, F.M., 2010. Heterogeneity among methicillin-resistant Staphylococcus aureus from Italian pig finishing holdings. Vet. Microbiol. 142, 361–366. Clinical and Laboratory Standards Institute, 2008. Performance Standards for Antimicrobial Disk and Dilution Susceptibility Test for Bacteria Isolated from Animals; Approved Standard, third ed. Clinical and Laboratory Standards Institute, Wayne, PA, CLSI Document M31-A3. Clinical and Laboratory Standards Institute, 2011. Performance Standards for Antimicrobial Susceptibility Testing – Twentieth Informational Supplement. Clinical and Laboratory Standards Institute, Wayne, PA, CLSI Document M100-S21. Cui, S., Li, J., Hu, C., Jin, S., Li, F., Guo, Y., Ran, L., Ma, Y., 2009. Isolation and characterization of methicillin-resistant Staphylococcus aureus from swine and workers in China. J. Antimicrob. Chemother. 64, 680–683. de Neeling, A.J., van den Broek, M.J., Spalburg, E.C., van Santen-Verheuvel, M.G., Dam-Deisz, W.D., Boshuizen, H.C., van de Giessen, A.W., van Duijkeren, E., Huijsdens, X.W., 2007. High prevalence of methicillin reistant Staphylococcus aureus in pigs. Vet. Microbiol. 122, 366–372. Deurenberg, R.H., Nulens, E., Valvatne, H., Sebastian, S., Driessen, C., Craeghs, J., de Brauwer, E., Heising, B., Kraat, Y.J., Riebe, J., Stals, F.S., Trienekens, T.A., Scheres, J., Friedrich, A.W., van Tiel, F.H., Beisser, P.S., Stobberingh, E.E., 2009. Cross-border dissemination of methicillin-resistant Staphylococcus aureus, Euregio Meuse-Rhine region. Emerg. Infect. Dis. 15, 727–734. Du, J., Chen, C., Ding, B., Tu, J., Qin, Z., Parsons, C., Salgado, C., Cai, Q., Song, Y., Bao, Q., Zhang, L., Pan, J., Wang, L., Yu, F., 2011. Molecular characterization and antimicrobial susceptibility of nasal Staphylococcus aureus isolates from a Chinese medical college campus. PLoS ONE 6, e27328.
812
S. Monecke et al. / Veterinary Microbiology 162 (2013) 806–812
Enright, M.C., Day, N.P.J., Davies, C.E., Peacock, S.J., Spratt, B.G., 2000. Multilocus sequence typing for characterization of methicillin-resistant and methicillin-susceptible clones of Staphylococcus aureus. J. Clin. Microbiol. 38, 1008–1015. Feßler, A., Scott, C., Kadlec, K., Ehricht, R., Monecke, S., Schwarz, S., 2010. Characterization of methicllin-resistant Staphylococcus aureus ST398 from cases of bovine mastitis. J. Antimicrob. Chemother. 65, 619–625. Feßler, A.T., Kadlec, K., Hassel, M., Hauschild, T., Eidam, C., Ehricht, R., Monecke, S., Schwarz, S., 2011. Characterization of methicillin-resistant Staphylococcus aureus isolates from food and food products of poultry origin in Germany. Appl. Environ. Microbiol. 77, 7151–7157. Goering, R.V., Morrison, D., Al-Doori, Z., Edwards, G.F., Gemmell, C.G., 2008. Usefulness of mec-associated direct repeat unit (dru) typing in the epidemiological analysis of highly clonal methicillin-resistant Staphylococcus aureus in Scotland. Clin. Microbiol. Infect. 14, 964–969. Guardabassi, L., O’Donoghue, M., Moodley, A., Ho, J., Boost, M., 2009. Novel lineage of methicillin-resistant Staphylococcus aureus, Hong Kong. Emerg. Infect. Dis. 15, 1998–2000. Hasman, H., Moodley, A., Guardabassi, L., Stegger, M., Skov, R.L., Aarestrup, F.M., 2010. spa type distribution in Staphylococcus aureus originating from pigs, cattle and poultry. Vet. Microbiol. 141, 326–331. Hauschild, T., Feßler, A.T., Kadlec, K., Billerbeck, C., Schwarz, S., 2012. Detection of the novel vga(E) gene in methicillin-resistant Staphylococcus aureus CC398 isolates from cattle and poultry. J. Antimicrob. Chemother. 67, 503–504. Huijsdens, X.W., van Dijke, B.J., Spalburg, E., van Santen-Verheuvel, M.G., Heck, M.E., Pluister, G.N., Voss, A., Wannet, W.J., de Neeling, A.J., 2006. Community-acquired MRSA and pig-farming. Ann. Clin. Microbiol. Antimircob. 5, 26. Ip, M., Yung, R.W.H., Ng, T.K., Luk, W.K., Tse, C., Hung, P., Enright, M., Lyon, D.J., 2005. Contemporary methicillin-resistant Staphylococcus aureus clones in Hong Kong. J. Clin. Microbiol. 43, 5069–5073. Kadlec, K., Ehricht, R., Monecke, S., Steinacker, U., Kaspar, H., Mankertz, J., Schwarz, S., 2009. Diversity of antimicrobial resistance pheno- and genotypes of methicillin-resistant Staphylococcus aureus ST398 from diseased swine. J. Antimicrob. Chemother. 64, 1156–1164. Krziwanek, K., Metz-Gercek, S., Mittermayer, H., 2009. Methicillin-resistant Staphylococcus aureus ST398 from human patients, upper Austria. Emerg. Infect. Dis. 15, 766–769. Kwon, N.H., Park, K.T., Jung, W.K., Youn, H.Y., Kim, S.H., Bae, W., Lim, J.Y., Kim, J.Y., Kim, J.M., Hong, S.K., Park, Y.H., 2006. Characteristics of methicillin resistant Staphylococcus aureus isolated from chicken meat and hospitalized dogs in Korea and their epidemiological relatedness. Vet. Microbiol. 117, 304–312. Lowder, B.V., Guinane, C.M., Ben Zakour, N.L., Weinert, L.A., ConwayMorris, A., Cartwright, R.A., Simpson, A.J., Rambaut, A., Nu¨bel, U., Fitzgerald, J.R., 2009. Recent human-to-poultry host jump, adaptation, and pandemic spread of Staphylococcus aureus. Proc. Natl. Acad. Sci. U.S.A. 106, 19545–19550. Lozano, C., Lopez, M., Gomez-Sanz, E., Ruiz-Larrea, F., Torres, C., Zarazaga, M., 2009. Detection of methicillin-resistant Staphylococcus aureus ST398 in food samples of animal origin in Spain. J. Antimicrob. Chemother. 64, 1325–1326. Monecke, S., Kuhnert, P., Hotzel, H., Slickers, P., Ehricht, R., 2007. Microarray-based study on virulence-associated genes and resistance determinants of Staphylococcus aureus isolates from cattle. Vet. Microbiol. 125, 128–140. Monecke, S., Luedicke, C., Slickers, P., Ehricht, R., 2009. Molecular epidemiology of Staphylococcus aureus in asymptomatic carriers. Eur. J. Clin. Microbiol. Infect. Dis. 28, 1159–1165. Monecke, S., Coombs, G., Shore, A.C., Coleman, D.C., Akpaka, P., Borg, M., Chow, H., Ip, M., Jatzwauk, L., Jonas, D., Kadlec, K., Kearns, A., Laurent, F., O’Brien, F.G., Pearson, J., Ruppelt, A., Schwarz, S., Scicluna, E., Slickers, P., Tan, H.-L., Weber, S., Ehricht, R., 2011. A field guide to pandemic, epidemic and sporadic clones of methicillin-resistant Staphylococcus aureus. PLoS ONE e17936. Monecke, S., Engelmann, I., Archambault, M., Coleman, D.C., Coombs, G.W., Cortez de Ja¨ckel, S., Pelletier-Jacques, G., Schwarz, S., Shore, A.C., Slickers, P., Ehricht, R., 2012a. Distribution of SCCmec-associated phenol-soluble modulin in staphylococci. Mol. Cell. Probes 26, 99–103. Monecke, S., Skakni, L., Hasan, R., Ruppelt, A., Ghazal, S.S., Hakawi, A., Slickers, P., Ehricht, R., 2012b. Characterisation of MRSA strains isolated from patients in a hospital in Riyadh, Kingdom of Saudi Arabia. BMC Microbiol. 12, 146. Neela, V., Mohd Zaful, A., Mariana, N.S., van Belkum, A., Liew, Y.K., Rad, E.G., 2009. Prevalence of ST9 methicillin-resistant Staphylococcus
aureus among pigs and pig handlers in Malaysia. J. Clin. Microbiol. 47, 4138–4140. Nemati, M., Hermans, K., Lippinska, U., Denis, O., Deplano, A., Struelens, M., Devriese, L.A., Pasmans, F., Haesebrouck, F., 2008. Antimicrobial resistance of old and recent Staphylococcus aureus isolates from poultry: first detection of livestock-associated methicillin-resistant strain ST398. Antimicrob. Agents Chemother. 52, 3817–3819. Nienhoff, U., Kadlec, K., Chaberny, I.F., Verspohl, J., Gerlach, G.F., Schwarz, S., Simon, D., Nolte, I., 2009. Transmission of methicillin-resistant Staphylococcus aureus strains between humans and dogs: two case reports. J. Antimicrob. Chemother. 64, 640–662. Nimmo, G.R., Coombs, G.W., 2008. Community-associated methicillinresistant Staphylococcus aureus (MRSA) in Australia. Int. J. Antimicrob. Agents 31, 401–410. Pan, A., Battisti, A., Zoncada, A., Bernieri, F., Boldini, M., Franco, A., Giorgi, M., Iurescia, M., Lorenzotti, S., MArtinotti, M., Monaci, M., Pantosti, A., 2009. Community-acquired methicillin-resistant Staphylococcus aureus ST398 infection, Italy. Emerg. Infect. Dis. 15, 845–847. ˜ lvarez-Fernandez, M., Constenla, L., A ˜ lvarez, P., Perez, S., 2010. Potel, C., A First human isolates of methicillin-resistant Staphylococcus aureus sequence type 398 in Spain. Eur. J. Clin. Microbiol. Infect. Dis. 29, 351–352. Price, L.B., Stegger, M., Hasman, H., Aziz, M., Larsen, J., Andersen, P.S., Pearson, T., Waters, A.E., Foster, J., Schupp, J., Gillec, J., Driebe, E., Liu, C.M., Laurent, F., Springer, B., Zdovc, I., Battisti, A., Franco, A., Zmudzki, J., Schwarz, S., Butaye, P., Jouy, E., Pomba, C., Porreo, M.C., Smith, T.C., Robinson, D.A., Weese, J.S., Arriola, C.O., Yu, F., Ruimy, R., Keim, P., Skov, R., Aarestrup, F.M., 2012. Staphylococcus aureus CC398: host adaptation and emergence of methicillin resistance in livestock. mBio 3, e00305–e00311. Schijffelen, M.J., Boel, C.H., van Strijp, J.A., Fluit, A.C., 2010. Whole genome analysis of a livestock-associated methicillin-resistant Staphylococcus aureus ST398 isolate from a case of human endocarditis. BMC Genomics 11, 376. Schwarz, S., Silley, P., Simjee, S., Woodford, N., van Duijkeren, E., Johnson, A.P., Gaastra, W., 2010. Assessing the antimicrobial susceptibility of bacteria obtained from animals. Vet. Microbiol. 141, 1–4. Shittu, A., Nu¨bel, U., Udo, E., Lin, J., Gaogakwe, S., 2009. Characterisation of methicillin-resistant Staphylococcus aureus isolates from hospitals in KwaZulu-Natal province, Republic of South Africa. J. Med. Microibiol. 58, 1219–1226. Soavi, L., Stellini, R., Signorini, L., Antonini, B., Pedroni, P., Zanetti, L., Milanesi, B., Pantosti, A., Matteelli, A., Pan, A., Carosi, G., 2010. Methicillin-resistant Staphylococcus aureus ST398, Italy. Emerg. Infect. Dis. 16, 346–348. Van Duijkeren, E., Ikawaty, R., Broekhuizen-Stins, M.J., Jansen, M.D., Spalburg, E.C., de Neeling, A.J., Allaart, J.G., van Nes, A., Wagenaar, J.A., Fluit, A.C., 2008. Transmission of methicillin-resistant Staphylococcus aureus strains between different kinds of pig farms. Vet. Microbiol. 126, 383–389. Van Loo, I., Huijsdens, X., Tiermersma, E., de Neeling, A., van de SandeBruinsma, N., Beaujean, D., Voss, A., Klytmans, J., 2007. Emergence of methisillin-resistant Staphylococcus aureus of animal origin in humans. Emerg. Infect. Dis. 13, 1834–1839. Vanderhaeghen, W., Cerpentier, T., Adriaensen, C., Vicca, J., Hermans, K., Butaye, P., 2010. Methicillin-resistant Staphylococcus aureus (MRSA) ST398 associated with clinical and subclinical mastitis in Belgian cows. Vet. Microbiol. 144, 166–171. Vestergaard, M., Cavaco, L.M., Sirichote, P., Unahalekhaka, A., Dangsakul, W., Svendsen, C.A., Aarestrup, F.M., Hendriksen, R.S., 2012. SCCmec type IX element in methicillin resistant Staphylococcus aureus spa type t337 (CC9) isolated from pigs and pork in Thailand. Front. Microbiol. 3, 103. Wagenaar, J.A., Yue, H., Pritchard, J., Broekhuizen-Stins, M., Huijsdens, X., Mevius, D.J., Bosch, T., van Duijkeren, E., 2009. Unexpected sequence types in livestock associated methicillin-resistant Staphylococcus aureus (MRSA): MRSA ST9 and a single locus variant of ST9 in pig farming in China. Vet. Microbiol. 139, 405–409. Walther, B., Monecke, S., Ruscher, C., Friedrich, A.W., Ehricht, R., Slickers, P., Soba, A., Wleklinski, C.G., Wieler, L.H., Lubke-Becker, A., 2009. Comparative molecular analysis substantiates a zoonotic potential of equine methicillin-resistant Staphylococcus aureus (MRSA). J. Clin. Microbiol. 47, 704–710. Wulf, M., van Nes, A., Eikelenboom-Boskamp, A., de Vries, J., Melchers, W., Klaassen, C., Voss, A., 2006. Methicillin-resistant Staphylococcus aureus in veterinary doctors and students, The Netherlands. Emerg. Infect. Dis. 12, 1939–1941.