Diversity Among Methicillin-Resistant Non-Staphylococcus

Veterinary Microbiology 158 (2012) 123–128

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Veterinary Microbiology

jo urnal homepage: www.elsevier.com/locate/vetmic

Species and staphylococcal cassette chromosome mec (SCCmec) diversity

among methicillin-resistant non-Staphylococcus aureus staphylococci isolated from pigs

a,b, a,b,c a,b a

Wannes Vanderhaeghen *, Stien Vandendriessche , Florence Crombe´ , Marc Dispas ,

c b b a,b

Olivier Denis , Katleen Hermans , Freddy Haesebrouck , Patrick Butaye

Department of General Bacteriology, Veterinary and Agrochemical Research Centre, Groeselenbergstraat 99, B-1180 Ukkel, Belgium

Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium

Laboratoire de Re´fe´rence MRSA-Staphylocoques, Department of Microbiology, Hoˆpital Erasme, Universite´ Libre de Bruxelles, 1070 Anderlecht, Belgium

A R T I C L E I N F O A B S T R A C T

While methicillin-resistant Staphylococcus aureus (MRSA) ST398 is known to be

Article history:

Received 6 September 2011 widespread in pig farms, few studies have investigated the species diversity and SCCmec

Received in revised form 16 January 2012 types of methicillin-resistant non-S. aureus staphylococci (MRNAS) residing in the nose of

Accepted 19 January 2012 pigs. We examined nasal swab samples of 200 pigs originating from 10 Belgian pig farms

previously found positive for MRSA ST398. Suspected staphylococcal isolates were

Keywords: subjected to a 16S rRNA-mecA-nuc PCR. Conﬁrmed MRNAS were genotypically identiﬁed

Coagulase-negative staphylococci

to the species level and investigated with a SCCmec typing PCR. MRNAS (n = 72) were

Methicillin resistance

detected on all 10 farms and were carried by 29.5% of the pigs. Seven MRNAS species were

Pigs

found: Staphylococcus epidermidis (38.9%), Staphylococcus sciuri (18.1%), Staphylococcus

Reservoir

SCCmec pasteuri (18.1%), Staphylococcus rostri (12.5%), Staphylococcus warneri (8.3%), Staphylo-

coccus haemolyticus (2.7%) and Staphylococcus hominis (1.4%). SCCmec cassettes were of

Methicillin-resistant Staphylococcus epider-

midis type IVa (29.2%), type IVc (25%), type III (22.2%), type V (5.6%) or could not be assigned to

any of the known types (NT types) (18.1%). Five distinct NT types were found. The

predominance of methicillin-resistant S. epidermidis (MRSE) in our samples is remarkable,

as MRSE is mainly associated with humans. The ﬁnding of three different SCCmec elements

(IVa, V, NT type 1) in MRNAS that also prevail or predominate in MRSA ST398 shows that

MRNAS might be an important SCCmec reservoir for MRSA in pigs. Yet, the occurrence of

multiple other SCCmec types illustrates that further studies are required to understand the

presence and spread of SCCmec in methicillin-resistant staphylococci from animals.

1. Introduction some mec (SCCmec). Based on structural composition, 11

different types (I–XI) and numerous subtypes of SCCmec

Methicillin-resistant Staphylococcus aureus (MRSA) have been recognized in MRSA so far (website of the

originates from methicillin-susceptible S. aureus (MSSA) International Working Group on the Classiﬁcation of

by acquisition of mecA, a gene carried within a mobile Staphylococcal Cassette Chromosome Elements, IWG-

genetic element called staphylococcal cassette chromo- SCC; www.sccmec.org). In non-S. aureus staphylococci

(NAS), mecA/SCCmec has been demonstrated to prevail as

well (Ibrahem et al., 2009). In human clinical samples, the

* Corresponding author at: Department of General Bacteriology, presence of methicillin-resistant NAS (MRNAS) has been

Veterinary and Agrochemical Research Centre, Groeselenbergstraat 99,

well-studied, and the MRNAS species most frequently

B-1180 Ukkel, Belgium. Tel.: +32 2 379 04 35; fax: +32 2 379 06 70.

encountered are methicillin-resistant Staphylococcus epi-

E-mail addresses: [email protected],

dermidis (MRSE) and methicillin-resistant Staphylococcus

[email protected] (W. Vanderhaeghen).

124 W. Vanderhaeghen et al. / Veterinary Microbiology 158 (2012) 123–128

haemolyticus (Petinaki et al., 2001; Ibrahem et al., 2009). In colony of each different suspected morphology was selected

veterinary medicine, MRNAS have mostly been studied as and puriﬁed on Columbia agar with 5% sheep blood (Bio-

pathogens in ruminant mastitis (Rajala-Schultz et al., Rad, Belgium). Isolates were grown for 48–72 h at 37 8C

2009). Only a few studies also investigated carriage of followed by another 24–48 h at room temperature, to be

MRNAS in livestock (Bagcigil et al., 2007; Zhang et al., able to fully judge their morphotype (Kloos and Bannerman,

2009; Huber et al., 2011) and companion animals (van 1994). Afterwards, for each sample, the puriﬁed isolates

Duijkeren et al., 2004; Bagcigil et al., 2007). However, little from the four different agar plates were compared. Isolates

is yet known on the carriage of MRNAS in pigs or on the showing identical morphotypes were judged as being

MRNAS species involved. identical to each other (Kloos and Bannerman, 1994) and

It has been suggested that MRNAS function as a mecA/ only one such isolate was further included in the study. If

SCCmec reservoir associated with the formation of MRSA identical morphotypes were found on (one of) the agar

from MSSA (Barbier et al., 2010; Bloemendaal et al., 2010). plates with cefoxitin (ChromID MRSA, blood agar + cefox-

Although this has mainly been studied in human medicine, itin) and (one of) the plates without cefoxitin (ChromID S.

it could also be relevant for animals. Indeed, in the past aureus, colistin-aztreonam agar), an isolate originating from

years, the emergence of a speciﬁc MRSA lineage, sequence (one of) the cefoxitin-containing agar plates was selected.

type (ST) 398, was reported in domestic animals (Voss Hereafter, an ‘isolate’ refers to a pure culture showing a

et al., 2005; Van den Eede et al., 2009; Vanderhaeghen colony morphotype unique for a given sample. Isolates

et al., 2010b). While MRSA ST398 strains have been shown were kept at À80 8C in 50% glycerol until further use.

to carry predominantly SCCmec cassettes of type IV(a) and

V (Vanderhaeghen et al., 2010b), little is known on the 2.3. Detection of mecA and identiﬁcation of MRNAS

types of SCCmec elements that are present in MRNAS from

animals. From all isolates, DNA was extracted as previously

The objectives of this study were to assess the carriage described (Vanderhaeghen et al., 2010a). Then, all isolates

and species distribution of MRNAS on pig farms previously were examined with a 16S rRNA-mecA-nuc triplex PCR

found positive for MRSA ST398 and to investigate the (Maes et al., 2002). MRNAS isolates, recognized as showing

SCCmec elements that they possess. ampliﬁcation of 16S rRNA and mecA, were further

identiﬁed to the species level using tRNA intergenic spacer

PCR combined with capillary gel electrophoresis (Baele

2. Materials and methods

et al., 2000). It was taken into consideration that certain

staphylococcal species, such as S. simulans and Staphylo-

2.1. Sampling

coccus sciuri, give poor or no ampliﬁcation of 16S rRNA in

Ten Belgian pig farms were visited between August and the triplex PCR (Maes et al., 2002); therefore, also isolates

December 2009. Farms were selected based on the that were only mecA positive in the triplex PCR but had a

presence of MRSA ST398 positive pigs as determined in clear staphylococcal morphology were included in a tRNA

a 2007 survey (Crombe´ et al., 2011). Four farms harboured identiﬁcation assay.

only fattening pigs, two farms were breeding farms, with In case tRNA intergenic spacer PCR was not sufﬁcient to

sows and piglets, and four farms were farrow-to-ﬁnish identify an isolate, rpoB sequencing was performed, with

farms, with sows, piglets and fattening pigs. On each farm, the primers and conditions reported by Drancourt and

a convenience sample of ten pigs of each represented age Raoult (2002). An isolate was identiﬁed when there was

group was sampled, resulting in a total of 200 pigs (60 98% or more sequence similarity with GenBank sequences

sows, 60 piglets and 80 fattening pigs). All pigs were of one species.

healthy at the moment of sampling.

A dry cotton swab was used to collect a sample from 2.4. Cefoxitin disk diffusion testing

both nostrils of each animal. Samples were stored in Stuart

transporter medium (Meus s.r.l., Italy) for transport to the The correspondence between presence of mecA and

laboratory, and laboratory processing of the samples was phenotypic cefoxitin resistance was investigated for all

commenced three to six hours after sampling. MRNAS isolates with the disk diffusion test, using 30 mg

cefoxitin disks (Rosco Diagnostics, Denmark) and follow-

2.2. Bacterial isolates ing CLSI recommendations M31-A3 (Clinical and Labora-

tory Standards Institute, 2008). S. aureus strains ATCC

Each sample was grown overnight at 37 8C in 7.5% NaCl 25923 and ATCC 43300 were used as internal quality

Brain Heart Infusion (BHI) broth (Becton Dickinson, US) controls. The strength of the correlation was assessed by

and then subcultured on four different agars: ChromID estimating the occurrence of resistance to cefoxitin in the

MRSA agar (BioMe´rieux, France), Columbia Agar (Oxoid, mecA-positive population, using logistic regression analy-

Germany) supplemented with 5% sheep blood (BioMe´r- sis with signiﬁcance level of 5%.

ieux, France) and 3.5 mg/l cefoxitin (Sigma-Aldrich, US),

ChromID S. aureus agar (BioMe´rieux, France), and Colum- 2.5. Determination of oxacillin and cefoxitin minimum

bia colistin-aztreonam agar with 5% sheep blood (Oxoid, inhibitory concentration (MIC)

France). Plates were incubated at 37 8C. After 24–36 h, each

of the four plates was examined for colonies showing a For those MRNAS isolates that had discordant results

staphylococcal morphology; per plate, one representative for mecA PCR and cefoxitin resistance in the disk diffusion

W. Vanderhaeghen et al. / Veterinary Microbiology 158 (2012) 123–128 125

Table 1

Distribution of methicillin-resistant non-S. aureus (MRNAS) species (n isolates) and their SCCmec types on the 10 farms (F1–10).

MRNAS species F1 F2 F3 F4 F5 F6 F7 F8 F9 F10

S. epidermidis 1 6 1 8 – 12 – – – –

(n = 28) NT type 3 IVa V IVa, IVc IVa, IVc, V

S. haemolyticus – – – – – 1 – – – 1

(n = 2) V V

S. hominis – – – – – – 1 – – –

(n = 1) NT type 5

S. pasteuri – – – 1 3 1 8 – – –

(n = 13) IVc IVc IVc IVc

S. rostri – – – – – 6 3 – – –

(n = 9) III III, IVa, NT type 2

S. sciuri – 2 2 2 1 2 1 1 2 –

(n = 13) NT type 4 III III NT type 4 III IIIA III III, NT type 2

S. warneri – – – – – – 6 – – –

(n = 6) NT type 1

NT types: SCCmec cassettes that could not be assigned to any of the known types; the details of these cassettes are described in Table 2.

test, oxacillin and cefoxitin MICs were determined using S. sciuri (n = 13; 18.1%), Staphylococcus rostri (n = 9; 12.5%),

Etest according to the manufacturer’s instructions (Bio- Staphylococcus warneri (n = 6; 8.3%), S. haemolyticus (n = 2;

Me´rieux, France). S. aureus strains ATCC 25923 and ATCC 2.8%) and Staphylococcus hominis (n = 1; 1.4%).

43300 were used as quality control strains. The distribution of the MRNAS species over the different

farms is presented in Table 1. As shown there, methicillin-

2.6. Testing for PBP2a production resistant S. sciuri was the most widespread species, being

detected on eight farms. Four other MRNAS species were

In addition to MIC determination, MRNAS isolates that detected on multiple farms: MRSE, present in ﬁve farms,

had discordant results for mecA PCR and cefoxitin methicillin-resistant S. pasteuri, found in four farms, and

resistance in the disk diffusion test were also tested for methicillin-resistant S. haemolyticus and methicillin-resis-

the production of PBP2a, the protein encoded by mecA, tant S. rostri, both detected in two farms. Methicillin-

using the Clearview Exact PBP2a test assay (Alere, resistant S. hominis and methicillin-resistant S. warneri were

Belgium). S. aureus strains ATCC 25923 and ATCC 43300 detected in samples from only one farm.

were used as quality control strains. Some pigs carried more than one MRNAS: 11 pigs carried

two MRNAS and one pig carried three MRNAS. In all but one

2.7. Typing of SCCmec elements samples, the different MRNAS were also different MRNAS

species. In the one exception, two MRSE were detected,

All MRNAS were included in the PCR method described which were, however, found to carrydifferent SCCmec types.

by Kondo et al. (2007) for typing of the SCCmec element.

The ﬁrst three multiplex PCRs (M-PCRs) described in this 3.2. Cefoxitin disk diffusion testing

method were used, respectively, for typing of the mec-

complex (M-PCR 1), typing of the ccr-complex (M-PCR 2) A correlation of 86.1% (95% CI: 76.1–92.4%) was observed

and type IV subtyping (M-PCR 3) (Kondo et al., 2007). In M- between cefoxitin disk diffusion testing and presence of

PCR 2, an internal control for mecA is included. The mecA (p < 0.05). Only the nine isolates of S. rostri that carried

following control strains were used: NCTC 10442 for type I mecA appeared to be susceptible to cefoxitin, with zone

SCCmec; N315 for type II; 85/2082 for type III; 8/6-3P (JCSC diameters between 26 mm and 32 mm.

1978) for type IVb; MR108 for type IVc; JCSC 4469 for type

IVd; and two ﬁeld isolates previously characterized in the 3.3. Cefoxitin and oxacillin MIC determination and PBP2a

Belgian Reference Laboratory for Staphylococci-MRSA as testing

being type IVa and V.

The nine S. rostri isolates with discordant mecA PCR –

cefoxitin disk diffusion test results tested positive for PBP2a

3. Results

production, proving the presence and expression of mecA.

However, Etest showed cefoxitin MICs to be 1 mg/ml and

3.1. Detection and identiﬁcation of MRNAS

oxacillin MICs to be 0.125 mg/ml (7 isolates) or 0.25 mg/ml (2

Presence of mecA was set as the gold standard to isolates), which is susceptible according to CLSI clinical

consider a NAS isolate as methicillin-resistant (a methi- breakpoints (Clinical and Laboratory Standards Institute,

cillin-resistant NAS, MRNAS). In total, the mecA gene was 2008).

detected in 72 isolates from 59 of the 200 samples (29.5%)

and from all ten farms (100%). Among these 72 MRNAS, 3.4. Typing of SCCmec elements

seven species were identiﬁed (Table 1). In descending

order of number of isolates these were S. epidermidis The SCCmec typing results of the 72 MRNAS are shown

(n = 28; 38.9%), Staphylococcus pasteuri (n = 13; 18.1%), in Table 2. Ten different types of SCCmec cassettes were

126 W. Vanderhaeghen et al. / Veterinary Microbiology 158 (2012) 123–128

Table 2

Characteristics of SCCmec elements found in 72 methicillin-resistant non-Staphylococcus aureus staphylococci (MRNAS) isolated from pigs.

a,b a c

ccr-Complex mec-Complex SCCmec type n Isolates Species (n isolates)

A3/B3 A III 15 S. rostri (7), S. sciuri (8)

A3/B3 + C A IIIA (III + SCCHg) 1 S. sciuri (1)

A2/B2 B IVa 21 S. epidermidis (20), S. rostri (1)

A2/B2 B IVc 18 S. epidermidis (5), S. pasteuri (13)

C C2 V 4 S. epidermidis (2), S. haemolyticus (2)

A2/B2 + C B NT type 1 6 S. warneri (6)

A2/B2 mecA NT type 2 2 S. rostri (1), S. sciuri (1)

C mecA NT type 3 1 S. epidermidis (1)

– A NT type 4 3 S. sciuri (3)

A2/B2 + A4/B4 + C + UK B NT type 5 1 S. hominis (1)

The mecA gene is present in the mec-complex; nonetheless, the internal mecA-control used in the typing method is included in the multiplex PCR (M-

PCR) for ccr-complex determination (Kondo et al., 2007). In the ﬁrst (left) column, ‘–’ then means that no other bands than the mecA-control were ampliﬁed.

In the second column ‘mecA’ means that no bands were ampliﬁed in the M-PCR for mec-complex determination but that the mecA-control was ampliﬁed in

the M-PCR for ccr-complex determination.

UK (unknown): ampliﬁcation of a fragment not corresponding to any expected band length.

NT: non-typeable cassette; the categorization into ‘type 1, 2. . .’ was done based on the moment of detection.

detected. Cassettes of type IV predominated, with 21 predominant MRNAS species, mainly because of the high

cassettes of type IVa (29.2%) and 18 of type IVc (25%). Also detection rate of MRSE on three farms (Table 1). This

16 cassettes of type III (22.2%) and 4 of type V (5.6%) were ﬁnding was unexpected: MRSE is the most frequently

found. The remaining 13 cassettes (18.1%) could not be detected MRNAS species in humans, both in hospitals

assigned to any of the known types (Table 2). Six of them (Kloos and Bannerman, 1994) and in the community

showed a pattern suggestive of being an as yet unassigned (Jamaluddin et al., 2008), while it is infrequently found in

variant of type IV cassettes (designated ‘NT type 1’ in Table animals (Huber et al., 2011; Tulinski et al., 2012).

2). Other cassettes (NT types 2, 3 and 4) had characteristics Moreover, our MRSE isolates were mostly carrying SCCmec

indicative of either being incomplete cassettes, either elements of type IVa, which is also observed in human

having modiﬁed primer attachment sequences, either MRSE (Wisplinghoff et al., 2003; Barbier et al., 2010). This

possessing mec- or ccr-complexes that were not included suggests that the MRSE isolates detected in our pig

in the primer sets used. The cassette found in the samples may have a human origin. A possible link between

methicillin-resistant S. hominis strain (NT type 5) showed our animal MRSE strains and human strains will be the

ampliﬁcation of four different ccr-sequences (Table 2). subject of further research.

The highest diversity of SCCmec cassettes was found for Another surprising result was the ﬁnding of methicillin-

S. sciuri and S. epidermidis, in which four different types resistant S. rostri, a species only recently described as a

were detected. Yet, cassettes of type III predominated in S. major part of the nasal bacterial ﬂora of pigs in Switzerland

sciuri while in S. epidermidis, the most prevalent cassette (Riesen and Perreten, 2010). In a subsequent Swiss study

was by far type IVa (Table 2). Two species harboured that investigated antimicrobial resistance in a population

cassettes of only one type: all S. pasteuri harboured of S. rostri, none of the isolates phenotypically showed

cassettes of type IVc, while all S. warneri harboured the oxacillin resistance (Stegmann and Perreten, 2010). The

aforementioned cassettes NT type 1. Eight of the nine presence of mecA was, however, not tested in that study.

isolates of S. rostri were found to carry complete cassettes Although our S. rostri isolates carried mecA and produced

of well-known types, III or IVa; the remaining isolate PBP2a, they appeared phenotypically susceptible accord-

carried a non-typeable cassette (NT type 2) with a ccr- ing to CLSI guidelines. To elucidate how to interpret results

complex of type 2 but no ampliﬁcation of mec-complex of phenotypical methicillin resistance-testing in S. rostri, a

sequences except mecA (Table 2). larger study including a signiﬁcant amount of mecA-

As shown in Table 1, on three farms similar SCCmec positive and mecA-negative S. rostri isolates will be

types were detected in different MRNAS species. necessary.

The MRNAS isolates detected in our study carried a

4. Discussion large diversity of SCCmec elements. Yet, similar to what

was observed in MRNAS from Dutch pigs (Tulinski et al.,

Only a few studies have been performed on the 2012), known SCCmec types constituted the majority of the

occurrence of MRNAS in pigs. Here, we found 29.5% of elements. SCCmec types IVa and V, which predominate in

200 pigs to carry at least one MRNAS. Similarly, in MRSA ST398 (Van den Eede et al., 2009; Vanderhaeghen

Switzerland 36.3% of 716 pigs from 241 farms were et al., 2010b; Crombe´ et al., 2011), also formed a

MRNAS carriers (Huber et al., 2011), whereas no MRNAS considerable part of the SCCmec types detected in our

could be detected in pig samples obtained from three MRNAS. Moreover, the cassette NT type 1 we detected has

Danish pig farms (Bagcigil et al., 2007). recently been reported to occur in MRSA ST398 isolated

The MRNAS species diversity we observed was quite from Italian pigs (Battisti et al., 2010).

different from the diversity recently described in Dutch Interestingly, simultaneously performed research on

pigs (Tulinski et al., 2012). In our study, MRSE was the the presence of MRSA in our farms (Vandendriessche

W. Vanderhaeghen et al. / Veterinary Microbiology 158 (2012) 123–128 127

et al., 2011) showed that similar SCCmec types were 5. Conclusions

present in MRSA ST398 and MRNAS on ﬁve farms. On these

farms, a total of thirteen pigs were found concurrently MRNAS were detected on all ten pig farms and were

carrying MRNAS and MRSA with the same SCCmec type, carried by almost one third of the sampled pigs. Among

including 11 type IVa cassettes, carried by S. epidermidis seven different MRNAS species, MRSE predominated,

(n = 10) and S. rostri (n = 1) and two type V cassettes, which is remarkable due to the general association of

carried by S. epidermidis (n = 1) and S. haemolyticus (n = 1). MRSE with humans. Also methicillin-resistant S. rostri was

This strongly suggests that SCCmec elements are shared found, a species only recently described as an important

between MRNAS and MRSA in pigs, a conclusion that was constituent of the nasal ﬂora of Swiss pigs. Our results

inferred from the results of the recent Dutch study as well indicate that MRNAS and particularly MRSE could act as an

(Tulinski et al., 2012). The direction of transfer is hard to important SCCmec-reservoir for MRSA ST398. Yet, the

assess and could very well work in both directions. Yet, considerable presence of SCCmec types not associated with

MRSE has previously been described as SCCmec donor of a MRSA from pigs should be further studied, to elucidate the

type IVa cassette to S. aureus (Bloemendaal et al., 2010). To mechanisms explaining the presence and spread of

further investigate the similarity between the SCCmec SCCmec cassettes in animal staphylococci.

elements in our MRNAS and those in MRSA ST398 strains,

techniques with a higher discriminative power than the Acknowledgements

current classic PCR typing techniques will be necessary.

A considerable number of the SCCmec elements were

The authors thank Katrien Geurts for excellent technical

type IVc and type III. In the literature, type IVc has not yet

assistance and Claire Nonhoff for aid in testing the S. rostri

been reported to occur in MRSA ST398, though it must be

isolates for phenotypical methicillin resistance. We also

noted that several studies reporting on MRSA ST398 did

thank Drs. Lotte De Ridder, Drs. Ste´phanie Ne´meghaire and

not perform type IV subtyping (Vanderhaeghen et al.,

Drs. Leon Oosterik for their critical review of the manu-

2010b). Also in non-ST398 MRSA in pigs, type IVc is

script. This research was funded by the Belgian Federal

infrequently detected (Overesch et al., 2011). In contrast,

Public Service of Health, Food Chain Safety and Environ-

type IVc is often found in various human community-

ment, Project No. RF-6189 MRSA.

acquired MRSA clones (Orendi et al., 2010; Higashiyama

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