Journal of Antimicrobial Chemotherapy Advance Access published September 26, 2013

J Antimicrob Chemother doi:10.1093/jac/dkt366

High genetic diversity of methicillin-susceptible Staphylococcus aureus (MSSA) from humans and animals on livestock farms and presence of SCCmec remnant DNA in MSSA CC398 Stien Vandendriessche1,2*, Wannes Vanderhaeghen2,3, Jesper Larsen4, Ricardo de Mendonc¸a1, Marie Hallin5, Patrick Butaye2,3, Katleen Hermans2, Freddy Haesebrouck2 and Olivier Denis1 1

*Corresponding author. Tel: 003225556971; Fax: 00325553110; E-mail: [email protected]

Received 10 June 2013; returned 23 July 2013; revised 12 August 2013; accepted 20 August 2013 Objectives: To investigate the genetic diversity of methicillin-susceptible Staphylococcus aureus (MSSA) carriage isolates from animals and humans on pig, veal, dairy, beef and broiler farms. Methods: S. aureus isolates were genotyped using spa typing and multilocus sequence typing (MLST). Antimicrobial susceptibility phenotypes and genotypes were determined. The presence of staphylococcal cassette chromosome mec (SCCmec)-associated DNA was characterized by PCR and sequencing among isolates of clonal complex (CC) 398. Results: Overall, 41 MSSA isolates in humans and 141 in animals were found, originating from all farm types. These MSSA were mainly assigned to CC398, CC1, CC5, CC9, CC30, CC97, CC133 and CC705/151. MSSA CC398 showed resistance to tetracycline, trimethoprim, macrolides and/or lincosamides, aminoglycosides, ciprofloxacin, spectinomycin and quinupristin/dalfopristin, whereas non-CC398 MSSA showed considerably less resistance. Three porcine MSSA CC398-t011 isolates harboured remnant DNA of a composite SCCmec V(5C2&5)c element that lacked the mec gene complex. This resulted from an MRSA-to-MSSA conversion due to recombination between the ccrC genes flanking the mec gene complex. The SCC remnant still contained an intact J1 region harbouring czrC and tet(K), encoding zinc and tetracycline resistance, respectively, thereby illustrating the capacity of S. aureus CC398 to adapt to different antibiotic selection pressures in the farming environment. Processes such as mec gene complex deletion probably contribute to the enormous diversity of SCC(mec) elements observed in staphylococci. Conclusions: MSSA CC398 precursors from which MRSA CC398 might (re)emerge were present on pig, veal and broiler farms, all of which are livestock sectors commonly known to be affected by MRSA CC398. The multiresistance phenotype of S. aureus CC398 appears to be independent of methicillin resistance. Keywords: molecular epidemiology, antimicrobial resistance, food animals, heavy metal resistance, carriage

Introduction Staphylococcus aureus is an opportunistic pathogen that can cause serious infections in humans and animals.1,2 Molecular epidemiological studies have shown that S. aureus strains circulating in different environments, such as hospitals, the community or animal farms, may be distinct from each other and this appears to be especially true for methicillin-resistant S. aureus (MRSA).2 – 4 At the beginning of the 2000s, a specific MRSA lineage assigned to clonal

complex (CC) 398 by multilocus sequence typing (MLST) emerged in livestock and persons in professional contact with livestock.4 – 6 Since its initial description, the livestock-associated (LA)-MRSA CC398 clone has appeared in different animal species worldwide.4 Although other MRSA clones have been detected in animals,1,7 the CC398 lineage seems to predominate in most food animal populations from Europe and North America. In contrast to the large number of reports on MRSA CC398,4,5,8 – 10 only limited attention has been given to the ecology of methicillin-susceptible S. aureus

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National Reference Centre for S. aureus, Department of Microbiology, Hoˆpital Erasme, Universite´ Libre de Bruxelles, Brussels, Belgium; 2 Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium; 3 Department of General Bacteriology, Veterinary and Agrochemical Research Centre, Brussels, Belgium; 4Department of Microbiological Surveillance and Research, Statens Serum Institut, Copenhagen, Denmark; 5Centre de Diagnostic Mole´culaire iris-Lab, Hoˆpital Saint-Pierre, Universite´ Libre de Bruxelles, Brussels, Belgium

Vandendriessche et al.

(MSSA) CC398 on livestock farms.11 Yet this knowledge might contribute greatly to the understanding of the emergence and spread of MRSA CC398 among livestock, as it may reveal the existence of a reservoir from which MRSA CC398 strains can emerge. MRSA emerges by site-specific integration of staphylococcal cassette chromosome mec (SCCmec) into the chromosomal orfX locus of an MSSA strain.12 SCCmec is composed of an mec gene complex that contains the mecA or mecC gene, a ccr gene complex that encodes one or more cassette chromosome recombinases necessary for excision/integration of the whole cassette and three non-essential joining regions J1, J2 and J3.13,14 To date, 11 SCCmec types and numerous subtypes have been described in MRSA.13,15,16 Studies have revealed enormous variation in SCCmec types and subtypes,12,13 including composite6,13 and mosaic6,10,17 SCCmec elements that result from complex recombination and rearrangement processes. Such processes might be especially important in LA-MRSA, considering that 3 of the 11 SCCmec types described so far have been discovered in MRSA from animals15,16 and that LA-MRSA strains frequently carry composite or non-typeable SCCmec elements.4 – 6,8,10,16 Additionally, it has been shown repeatedly that SCCmec is unstable in certain MRSA strains or under specific conditions.6,18 – 20 SCCmec deletion can be complete,21 although some have proposed that partial deletion occurs even more frequently,20 thereby generating SCCmec DNA fragments that remain in the S. aureus chromosome.6,19,22 The aim of the present study was to investigate the genetic background of MSSA strains isolated from humans and animals on pig, veal calf, dairy cattle, beef cattle and broiler farms to gain insight into the genetic diversity of livestock-associated S. aureus. A second aim was to look for SCCmec remnant DNA in MSSA CC398 and to compare it with SCCmec of MRSA CC398 isolated from the same animal.

Materials and methods Study population and strain collection Farms with five types of production animals—pigs (n¼10), veal calves (n ¼20), dairy cattle (n¼10), beef cattle (n ¼10) and broilers (n ¼20)— were selected as previously described.23 On every farm, samples were taken from humans and from a fixed number of animals as previously described.23 Overall, samples were collected from 149 farmers and family members, and from 200 pigs, 200 veal calves, 100 beef cows, 100 dairy cows and 800 broilers. This study was approved by the Medical Ethical Commission of the ULB-Erasme Hospital in Brussels (reference P2009/065); all human participants signed an informed consent form.

Isolation, identification and molecular typing of S. aureus Nasal swabs were placed into Amies transport medium (Copan, Italy) and processed individually within 8 h, except for the broiler swabs, for which eight samples were pooled (five pools/farm). Isolation of S. aureus, DNA extraction and identification were performed as previously described.23 Only the S. aureus isolates negative for mecA and mecC were included in this study and will henceforth be referred to as MSSA. Molecular typing and assessment of genetic relatedness were performed as previously described,23 by spa typing and Based Upon Repeat Pattern (BURP) clustering for all isolates and by MLST for a subset of strains. MLST was performed for each spa type that was detected in more than one isolate (regardless of the host). For these spa types (n ¼23 types), we aimed to perform MLST for one MSSA per host type

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(i.e. human, pig, veal calf, dairy cow, beef cow or broiler). Additionally, MLST was performed for one strain with a spa type that occurred only once (t937), but for which CC398 was suspected.

Antimicrobial susceptibility testing and determination of resistance genes Susceptibility to 16 antimicrobials was determined as previously described for all human-derived MSSA (n¼41), all animal-derived MSSA for which, based on the spa type, CC398 was suspected (n¼22) and one animal-derived MSSA per non-CC398 spa type per farm (n¼53).23 For the above-mentioned strains, the presence of resistance genes for tetracycline [tet(M), tet(K) and tet(L)], for macrolides and/or lincosamides [erm(A), erm(B), erm(C), erm(T), msr(A) and lnu(A)], for aminoglycosides [aac(6 ′ )-aph(2 ′′ ), aph(3 ′ ) and ant(4,4 ′′ )] and for trimethoprim (dfrK) was determined by PCR as previously described.23

Characterization of MSSA CC398 strains For strains identified as MSSA CC398 (n¼28), the MICs of cefoxitin and oxacillin were determined by Etest according to the manufacturer’s (bioMe´rieux, France) instructions. An in-house PCR assay was developed, amplifying a 167 bp region from the 3′ end of orfX to the intergenic region downstream of orfX in S. aureus CC398 lacking SCCmec. Primers and PCR conditions are shown in Table S1 (available as Supplementary data at JAC Online). For three porcine MSSACC398 isolates that failed to yield a 167 bp amplicon, the presence of SCCmec remnant DNA was investigated using two complementary approaches. First, the presence of a ccr gene complex and an mec gene complex was determined.24 Second, the presence of eight fragments (A –H) that are distributed over the entire SCCmec V(5C2&5)c element, was investigated using a PCR assay and primers that have been described elsewhere.16 In the latter assay, the LA-MRSA CC398 strain JCSC694416 was used as a positive control for the PCR amplifications. PFGE using Cfr9I was performed on the three porcine MSSA CC398 isolates that failed to yield a 167 bp amplicon using a method that has been described elsewhere.25 Six porcine LA-MRSA CC398 isolates from the same farm were included for comparison. Of these LA-MRSACC398 isolates, five harboured an SCCmec V(5C2&5)c element.23 The Cfr9I genomic digest of S. aureus NCTC 8325 was used as a molecular size marker.

Genome resequencing and assembly of MSSA CC398 harbouring SCCmec remnant DNA and of MRSA CC398, isolated from the same animal Two isolates carried by the same animal were selected for paired-end sequencing on an Illumina HiSeq 2000 Analyzer (Illumina Inc., San Diego, CA, USA): MSSA P211 harbouring SCCmec remnant DNA and MRSA P12623 carrying SCCmec V(5C2&5)c. Both isolates have the same spa type and, except for methicillin resistance, the same resistance phenotype and genotype. To extract the genomic DNA, isolates were grown for 16 –18 h at 378C in 5 mL of brain heart infusion broth. A 3 mL aliquot was centrifuged for 10 min at 8000 rpm, the supernatant was discarded and the pellet was resuspended with 100 mL of lysis buffer (40 mM Tris– HCl, pH 8.0, 4 mM EDTA and 2.4% Triton X-100). The 100 mL suspension was incubated for 60 min at 378C in the presence of 50 mL of lysozyme (40 mg/mL) and 50 mL of lysostaphin (1 mg/mL). The DNA was then extracted by using the DNeasy Tissue Kit (Qiagen) according to the manufacturer’s instructions. The integrity and concentration of the extracted DNA was estimated by agarose gel electrophoresis and spectrophotometric measurement (Qbit, Invitrogen), respectively. Library preparation and genome sequencing were performed according to the manufacturer’s standard protocols. Briefly, following enzymatic processing (end repair, phosphorylation, A-tailing and adapter ligation), DNA fragments of 500 bp were purified by gel electrophoresis and enriched

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SCCmec remnant DNA in MSSA CC398

by PCR. The paired-end library was sequenced to a depth of 100× or more. After cleaning the data, the short reads were assembled into scaffolds using SOAPdenovo (version 1.05); the order of the scaffolds was determined by alignment to the S0385 genome (GenBank accession number AM990992) using SOAPaligner (version 2.21). The nucleotide sequences of SCCmec V(5C2&5)c in MRSA P126 and SCCmec remnant DNA in MSSA P211 were further extracted and annotated from the preliminary draft chromosomal sequence of both strains. To map SCCmec (remnant) DNA, we blasted every scaffold of MRSA P126 and MSSA P211 against SCCmec V(5C2&5)c as described by Li et al.16 (GenBank accession number AB505629). Positive scaffolds were aligned against AB505629 and gaps were closed by PCR followed by Sanger sequencing. The recombination site present in SCC of MSSA P211 was confirmed by PCR followed by Sanger sequencing. Primers and PCR conditions are displayed in Table S1.

Results Frequency of occurrence of MSSA isolates on livestock farms MSSA was detected in 41 (28%) of 149 human samples. Sixteen percent of persons on pig farms carried MSSA, as did 9% of humans on veal calf farms, 45% on dairy farms, 38% on beef farms and 42% on broiler farms. Overall, 141 MSSA isolates were collected from animals. MSSA carriage in animals was 27.5% for pigs, 7% for veal calves, 25% for dairy cattle and 18% for beef cattle. MSSA was detected in 29 pools (out of 100) from eight broiler farms. Table 1 summarizes the results of MSSA carriage in humans and animals according to the farm type. In a study performed in parallel to the present work, on the same farms and with the same animals, 40 and 303 MRSA were isolated from humans and animals, respectively.23

Genotype distribution of MSSA isolates Detailed results of the genotype distribution are displayed in Table 2. Overall, 44 different spa types were found, of which 21 were represented by a single isolate. The majority of MSSA isolates grouped into five spa CCs corresponding to five MLST CCs: spa CC002-CC5 (n¼ 34), spa CC337-CC9 (n¼28), spa CC034-CC398 (n¼ 27), spa CC021-CC30 (n¼ 15) and spa CC267-CC97 (n¼ 12). The remaining isolates

comprised 16 singletons (represented by 36 isolates), while six spa types (represented by 30 isolates) were excluded from BURP clustering. Two singletons were assigned to CC97 and CC398, respectively. One excluded spa type belonged to CC9. Other singletons and excluded spa types belonged to different CCs, including CC1 (n¼ 12), CC133 (n¼ 13) and CC705/151 (n¼ 14). Some genetic backgrounds were limited to a single production type, such as CC5 on broiler farms and CC9 on pig farms, while others were detected on farms of different production types: CC97 on veal calf and beef farms, CC133 and CC705/151 on beef and dairy cattle farms and CC398 on pig, veal, broiler and dairy farms. The single MSSA CC398 isolate collected from a dairy cow, which had spa type t937, was assigned to sequence type (ST) 291, a double-locus variant of ST398. On 11 (16%) farms, MSSA isolates with the same spa type were detected in both humans and animals from that farm: MSSA t127 and t337 each on one pig farm, t011 on a veal calf farm, t034 on a broiler farm, t3478 on four broiler farms, t190 and t1247 each on one dairy cattle farm and t1403 on a beef cattle farm.

Antimicrobial susceptibility and resistance determinants All strains were susceptible to chloramphenicol, fusidic acid, linezolid, mupirocin, rifampicin and co-trimoxazole. The percentage of isolates resistant to the other antimicrobials and the associated resistance genes are presented in Table 3.

Characterization of MSSA CC398 strains All 28 CC398 strains were susceptible to cefoxitin (MIC range 2 –4 mg/L) and oxacillin (MIC range 0.125–0.75 mg/L). A total of 25 MSSA CC398 isolates carried an intact orfX gene, whereas 3 porcine MSSA CC398 isolates from a single farm yielded a negative result, carried ccrC, lacked the mec gene complex and contained fragments A–H of SCCmec V(5C2&5)c, which are located in the J1 and J3 regions flanking the ccr gene complexes (Figure 1). The three porcine MSSA CC398 with SCCmec remnant DNA and the porcine LA-MRSA CC398 isolates obtained from the same farm/animals were highly homogeneous in terms of spa types (t011), resistance patterns and resistance gene profiles (data not shown). However, Cfr9I-PFGE demonstrated a three-band difference between the three porcine MSSA CC398 with SCCmec

Table 1. Occurrence of MSSA in humans and animals on pig, veal, dairy, beef and broiler farms Humans

Animals

Farm type

No. of farms

no. of humans sampled

no. of MSSA carriers (%)

no. of farms with MSSA carriers

no. of animals sampled

no. of MSSA carriers (%)

no. of farms with MSSA carriers

Pig Veal Dairy Beef Broiler

10 20 10 10 20

25 45 22 21 36

4 (16) 4 (9) 10 (45) 8 (38) 15 (42)

3 3 9 6 10

200 200 100 100 800

55 (27.5) 14 (7) 25 (25) 18 (18) 29 poolsa

8 7 8 8 8

Total

70

149

41 (28)

31

NA

NA

NA

NA, not applicable. Of 100 pools analysed.

a

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Vandendriessche et al.

Table 2. Genotype distribution of MSSA strains isolated from humans and animals on pig, veal, dairy, beef and broiler farms MLST

spa

CC

ST

CC

type

spa repeat succession

No. of strains

5

5 5 8 7 1 81 9 9 9 9 97 97 97 352 352 352 389 30 433 433 433 SLV45 45 133 133 398 398 398 291 1380 522 504 ND ND ND ND ND ND ND ND ND ND ND ND ND

002 002 Si 7 Si 2 Si 4 Si 4 337 337 337 Ex 5 Si 12 267 267 267 267 267 Si 6 021 021 021 021 Si 1 Si 3 Ex 4 Ex 6 034 034 034 Si 11 Ex 1 Si 15 Ex 2 021 021 Si 5 Si 8 Si 9 002 Si 10 002 Ex 3 021 Si 13 Si 14 Si 16

t002 t3478 t190 t091 t127 t127 t337 t2315 t1430 t1939 t1247 t1236 t2421 t267 t2802 t5727 t164 t021 t318 t1298 t3427 t015 t095 t1403 t8862 t011 t034 t571 t937 t528 t3576 t529 t012 t122 t136 t213 t286 t311 t493 t539 t777 t822 t1313 t1402 t3906

26-23-17-34-17-20-17-12-17-16 26-23-17-34-17-20-17-12-17-16-12-17-16 11-17-34-24-34-22-25 07-23-21-17-34-12-23-02-12-23 07-23-21-16-34-33-13 07-23-21-16-34-33-13 07-16-23-23-02-12-23-02-34 07-16-23-23-12-23-02-34 07-16-23-02-12-23-02-34 07-23-02-34 07-23-34-34-33-34 26-23-12-21-17-34-34-34-33-34 26-34-34-33-34 07-23-12-21-17-34-34-34-33-34 07-23-21-17-34-34-34-33-34 07-23-12-21-21-17-34-34-34-33-34 07-06-17-21-34-34-22-34 15-12-16-02-16-02-25-17-24 15-12-16-16-02-16-02-25-17-24 15-16-02-16-02-25-17-24 15-16-16-02-16-02-25-17-24 08-16-02-16-34-13-17-34-16-34 08-16-02-16-34-34 03-23-24 03 08-16-02-25-34-24-25 08-16-02-25-02-25-34-24-25 08-16-02-25-02-25-34-25 08-16-34-24-34-34-17-17 04 04-31-17-25-17 04-34 15-12-16-02-16-02-25-17-24-24 08-16-02-16-02-25-17-24-24 04-44-33-31-12-16-34-16-12-25-22-22-34 07-23-12-21-24-33-22-17 07-23-13-34-16-34-33-13 26-23-17-34-20-17-12-17-16 04-34-17-66-32-17-23-24 26-23-17-34-17-12-17-16 26-23-17 15-12-16-02-16-02-25-16-17-24-24 14-12-23-02-34-34 09-02-16-34-34-13-17-34-16-34 04-44-33-31-12-16-34

3 29 2 3 12

V (1), BC (1) B (6) DC (1) B (2) P (3), V (1)

H (3) H (5), B (24) H (1), DC (1) H (3) H (2), P (9), V (1)

17 1 10 1 3 2 1 4 3 2 3 2 6 1 2 2 1 12 1 13 11 3 1 1 2 14 2 1 1 1 1 1 1 1 1 1 1 1 1

P (6) P (1) P (2) P (1) DC (1) V (1) V (1) BC (1) BC (1) BC (1) BC (1) P (1) P (1) P (1) P (1) DC (1), B (1) V (1) DC (5), BC (3) DC (1) P (1), V (4) P (1), B (3) P (1) DC (1) DC (1) BC (1) DC (5), BC (1) BC (1) BC (1) DC (1) P (1) B (1) DC (1) DC (1) DC (1) BC (1) BC (1) BC (1) DC (1) V (1)

H (1), P (16) P (1) P (10) P (1) H (2), DC (1) V (2) V (1) BC (4) BC (3) BC (2) BC (3) P (2) P (6) P (1) P (2) H (2) V (1) H (1), BC (3), DC (8) DC (1) H (1), P (3), V (9) H (5), P (1), B (5) P (3) DC (1) DC (1) BC (2) BC (1), DC (13) H (2) H (1) H (1) H (1) H (1) H (1) H (1) H (1) H (1) H (1) H (1) H (1) H (1)

8 7 1 9

97

20 30

45 133 398

479 522 705 — — — — — — — — — — — — —

Type of farm (no. of farms)

Host (no. of strains)

ND, not determined; Si, singleton; Ex, excluded; H, human; P, pig; V, veal; DC, dairy cattle; BC, beef cattle; B, broiler. Total number of strains ¼182.

remnant DNA and porcine LA-MRSA CC398 isolates from the same farm/animals (Figure S1, available as Supplementary data at JAC Online). Two bands of 90 and 140 kb present in LA-MRSA CC398

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were replaced by one band of 220 kb in MSSA CC398. These differences can be explained by a partial deletion of the SCCmec V(5C2&5)c element, which carries two Cfr9I restriction sites in

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SCCmec remnant DNA in MSSA CC398

Table 3. Antimicrobial susceptibility profile and distribution of resistance genes in MSSA CC398 versus non-CC398 MSSA from humans and animals (n ¼116) CC398a pig farms

totalb Resistance phenotype, genetic determinant

H

Non-CC398

A

veal farms

broiler farms

H

A

H

A

H

A

totalb Ac

H

Total no. of MSSA isolates

6

21

0

7

1

9

5

5

35

53

Tetracycline tet(M) tet(K) tet(L)

6 (100%) 6 1 1

21 (100%) 21 11 8

/ / / /

7 7 6 0

1 1 0 1

9 9 0 8

5 5 1 0

5 5 5 0

6 (17%) 0 4 0

8 (15%) 4 6 1

Trimethoprim dfrK

3 (50%) 1

18 (86%) 13

/ /

7 7

1 1

6 6

2 0

5 0

0 (0%) 0

3 (6%) 1

Macrolides and/or lincosamidesd erm(A) erm(B) erm(C) erm(T) lnu(A)

5 (83%) 3 1 2 0 0

18 (86%) 1 5 10 8 5

/ / / / / /

4 1 0 3 0 0

1 0 0 1 0 0

9 0 0 7 8 0

4 3 1 1 0 0

5 0 5 0 0 5

1 (3%) 0 0 1 0 0

4 (8%) 1 1 2 0 0

Aminoglycosides aac(6 ′ )-aph(2 ′′ ) ant(4,4 ′′ ) aph(3 ′ )

1 (17%) 1 0 0

9 (43%) 9 4 0

/ / / /

0 0 0 0

1 1 0 0

9 9 4 0

0 0 0 0

0 0 0 0

2 (6%) 0 2 0

3 (6%) 3 2 0

Ciprofloxacin

0 (0%)

4 (19%)

/

3

0

1

0

0

0 (0%)

2 (4%)

Spectinomycin

4 (67%)

6 (29%)

/

1

0

1

4

4

0 (0%)

1 (2%)

Quinupristin/dalfopristin

2 (33%)

4 (19%)

/

0

0

0

2

4

0 (0%)

2 (4%)

H, human; A, animal. Results are displayed for human (n ¼6) and animal (n¼21) isolates; one multi-susceptible MSSA CC398 from a dairy cow is not included. b The percentage of resistant isolates is displayed in brackets. c Two MSSA ST97 from veal calves were resistant to macrolides and/or lincosamides (n ¼2) due to erm(B)+erm(C), aminoglycosides (n¼2) due to ant(4,4 ′′ ), tetracycline (n¼2) due to tet(M)+tet(K) +tet(L), trimethoprim (n ¼1) due to dfrK and spectinomycin (n¼1). d The resistance gene msr(A) was not detected. a

close proximity to each other, thereby eliminating the two restriction sites located in SCCmec V(5C2&5)c (Figure 1).

Nucleotide sequence of SCCmec remnant DNA in MSSA P211 and of SCCmec V(5C2&5)c in MRSA P126 The draft chromosomal sequence of MSSA P211 and MRSA P126 consisted of 38 and 36 scaffolds, respectively. For both isolates, four scaffolds aligned completely or partially with SCCmec V(5C2&5)c (AB505629); together these four scaffolds completely covered the SCC element present in MSSA P211 and MRSA P126 (Figure 1). The SCCmec V(5C2&5)c element in MRSA P126 (47 214 bp; GenBank accession number KF593809) differed from the SCCmec remnant DNA in MSSA P211 (30 604 bp; GenBank accession number KF593810) only by the absence of a 16.6 kb region, encoding all genes located between ccrC1 allele 8 and ccrC1 allele 2 (Figure 1). The alignment of the nucleotide sequences

of the ccrC genes of MRSA P126 and MSSA P211 showed that the ccrC gene detected in MSSA P211 resulted from a recombination within a 25 bp region in the 3′ end of ccrC1 allele 8 and the 5′ start of ccrC1 allele 2. The recombined ccrC gene in MSSA P211 consists of: 1357 bp originating from ccrC1 allele 8; a 25 bp recombination site present in both ccrC1 allele 8 and ccrC1 allele 2; and a 298 bp region originating from ccrC1 allele2.

Discussion The present study is unique in that it is the first to systematically sample animals and humans across different livestock production sectors in order to investigate the genetic diversity of MSSA healthy carriage isolates. The presented data provide an insight into the MSSA distribution at farm level and, due to the use of a uniform protocol for the different farm types, they allow comparison of the genetic backgrounds of MSSA from animals and humans. In

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Figure 1. (a) Schematic representation of the intact SCCmec V(5C2&5)c element in LA-MRSA CC398 P126 and the corresponding SCC remnant DNA in LA-MSSA CC398 P211 isolated from the same animal. Bars with the letters A– H indicate the fragments A– H, which are distributed over the entire JCSC6944 SCCmec V(5C2&5)c element.16 Red arrowheads (DR1, DR2 and DR3) indicate the locations of integration site sequences for the SCC. The ccr gene complexes are shaded in green, the mec gene complex is shaded in orange, insertion sequences (IS) are indicated in yellow, the czrC gene is indicated in dark blue and tet(K), which is carried on plasmid pT181, is indicated in light blue. Genomic Cfr9I restriction sites, determined using Webcutter 2.0, of SCCmec V(5C2&5)c (GenBank accession number AB565029) and of LA-MRSA S0385 (GenBank accession number AM990992) are indicated by red arrows. (b) Schematic representation of the recombined ccrC gene detected in MSSA P211. (c) Comparison of nucleotide sequences of ccrC genes of MRSA P126 [ccrC1 allele 8 (black, bold) and ccrC1 allele 2 (red)] and of MSSA P211 (recombined ccrC). The 25 bp recombination site is boxed and divergent nucleotides in the depicted ccrC genes are underlined. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

general, the distribution of MSSA clones per production type was in agreement with results of previous surveys, which have shown the presence of MSSA CC1, CC9, CC30 and CC398 in pigs,11 CC97, CC133 and CC705/151 in bovines26 and CC5 in broilers.11 Despite the rather small study population, MSSA displaying identical spa types and with identical or highly similar resistance profiles were recovered from animals and humans on a considerable proportion of farms. In the majority of cases, the MSSA isolates had a genetic background that is thought to be specific for the animal species raised on that farm,3,11,27 indicating that these cases could represent transmission events from animals to farmers. PFGE analysis would, however, be required to confirm the clonal relationship of human and animal-associated MSSA from individual farms.

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Remarkably, as previously described for human carriers, MRSA seemed to add to the burden of S. aureus rather than replacing it. Indeed, combined results from the present survey and a parallel survey23 showed high carriage rates for MSSA and MRSA in a single study population. As previously suggested for MRSA CC398,1 MSSA CC398 showed only restricted host specificity as they were detected in humans and animals on all farm types except for beef cattle farms. This contrasts with a previous report from Denmark that found MSSA CC398 in pigs, but not in broilers or bovines.11 The present study detected MSSA CC398 in all livestock sectors for which MRSA CC398 has previously been reported.4 Additionally, the observed spa type distribution of MSSA CC398 mirrored the most frequently

JAC

SCCmec remnant DNA in MSSA CC398

detected spa types for MRSA CC398: t011 and t034. These results suggest that on most farm types MSSA CC398 precursors, from which MRSA CC398 could locally emerge, are present. Another similarity between MRSA and MSSA CC398 was the almost uniform tetracycline resistance, due to tet(M) alone or combined with tet(K) and/or tet(L). Moreover, although isolated from the same study population, MSSA CC398 were generally more resistant than non-CC398 MSSA, and these ‘additional’ resistances and resistance genes were frequently the same as those observed in MRSA CC398.23 This indicates that the multiresistance phenotype of CC398 is associated with lineage rather than with methicillin resistance. Indeed, it has been shown before that the CC398 lineage lacks the type I restriction-modification (RM) system present on genomic island b (y Sb),10 making it probably more prone to acquisition of foreign DNA by horizontal gene transfer as compared with other S. aureus lineages. Furthermore, the MSSA CC398 isolates mainly originated from pig, veal calf and broiler farms, all of which are livestock production sectors characterized by intensive animal farming and high antimicrobial pressure,28 – 30 whereas non-CC398 MSSA were frequently detected on beef and dairy cattle farms also. Higher antimicrobial use on pig, veal and broiler farms as compared with beef and dairy cattle farms28 – 30 probably contributes to the higher resistance rates observed for MSSA CC398 than for non-CC398 MSSA. Partial excision of SCCmec has been frequently described for S. aureus,6,18,19 but so far only in human isolates. In the present study, we report on three porcine MSSA CC398-t011 strains harbouring SCCmec remnant DNA, resulting from an MRSA-to-MSSA conversion due to recombination between the ccrC genes that flank the mec gene complex. This hypothesis resulted from the fact that the three MSSA CC398 strains did not amplify the orfX region, but did amplify fragments A–H located in J1 and J3 regions flanking the ccr gene complexes. Additionally, the MSSA isolates had spa types and resistance phenotypes and genotypes identical to those of the MRSA isolates obtained from the same animals. PFGE analysis showed the replacement of two bands of 90 and 140 kb in MRSA CC398 by one band of 220 kb in MSSA CC398, consistent with the deletion of the region between the two ccrC gene complexes. Sequencing confirmed that the 30.6 kb SCCmec remnant in MSSA P211 was identical to SCCmec V(5C2&5)c in MRSA P126, except that it lacked a 16.6 kb region between the ccrC genes encompassing the mec gene complex. The fact that three MSSA with SCCmec DNA were detected on a single farm can be explained by independent SCCmec excision events or by partial SCCmec deletion followed by clonal spread. Most cases of (partial) SCCmec excision for which the deletion mechanism has been investigated concerned complete excision mediated by ccr genes12,18 or partial excision mediated by IS431 elements flanking the mec complex.18,22 In contrast, partial excision due to homologous recombination has been reported only once in S. aureus, in the composite SCCmec V(5C2&5)b element.6 Possibly, composite SCCmec elements carrying two ccrC genes are more prone to diversification by recombination. Several studies that investigated the SCCmec type diversity in methicillinresistant Staphylococcus haemolyticus (MRSH) from humans found that this species frequently carries SCCmec V, and in a considerable number of strains the mec complex was flanked by ccrC-carrying units.31 At the same time, investigation of the SCCmec region in methicillin-susceptible S. haemolyticus (MSSH) showed that half of them carried the ccrC gene while they lacked

mecA. As in the present study, Bouchami et al.32 suggested that MRSH-to-MSSH conversion had occurred, and homologous recombination was proposed as the deletion mechanism. As SCCmec formation is believed to occur by the acquisition of a mec gene complex by an SCC element,16 the presence of a completely functional non-mec SCC in MSSA CC398 provides ideal conditions for the subsequent emergence of new SCCmec types or subtypes. It can be hypothesized that partial SCCmec excision in animal MSSA CC398 strains represents a way for S. aureus to retain important resistance genes whilst eliminating the mec complex. Indeed, the czrC and tet(K) genes that remained present in the intact J1 region of the non-mec SCC confer resistance to cadmium and zinc and to tetracycline, respectively. It has been proposed that the use of zinc in animal farming might have contributed to the selection of MRSA within the CC398 lineage, as the zinc resistance gene czrC is located within J1 of SCCmec V(5C2&5)c.16,33 In this respect, the presence of a non-mec SCC element that carries the czrC gene might confer an advantage on MSSA CC398 in specific circumstances, namely when no antibiotic pressure due to b-lactam use is exerted. This is consistent with the fact that the piglets from which the three MSSA strains with SCC remnant DNA were isolated did not receive antibiotics from the moment of their birth until the day of sampling (data not shown). Further study is, however, required to investigate this hypothesis. Whether the presence of tet(K) confers an advantage on the MSSA CC398 isolates remains to be elucidated. Indeed, tet(K) confers low-level tetracycline resistance,34 but the overwhelming majority of CC398 isolates are already tetracycline resistant due to tet(M).5,8,9,23 Furthermore, previous research has shown that the Tet(K) transporter protein also catalyses efflux of tetracycline – divalent metal ion complexes,34 including Cu2+ and Zn2+, which are frequently used as feed additives in animal farming.33 In conclusion, MSSA CC398 precursors from which MRSA CC398 could emerge locally were frequently observed in the livestock production sectors most affected by MRSA CC398. A non-mec SCC element harbouring czrC and tet(K), generated by partial excision due to homologous recombination, was detected. These kinds of genetic event probably contribute to the formation of new SCCmec types as well as to the diversification of SCCmec elements in the CC398 lineage.

Acknowledgements We thank Raf de Ryck for performing PFGE and Professor Marc Struelens for critical review of the manuscript prior to submission.

Funding This work was supported by a research grant of the Belgian Federal Public Service of Public Health, Food Chain Safety and Environment (to S. V.), Contract RF6189 MRSA, by a grant from Ghent University (to S. V.), Contract number 30242860 and by the National Reference Center for MRSA— Staphylococci.

Transparency declarations None to declare.

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Supplementary data Table S1 and Figure S1 are available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/).

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