Adopted by the Task Force on 19 November 2007

Transcription

1 Report of the Task Force on Zoonoses Data Collection including a proposal for technical specifications for a baseline survey on the prevalence of Methicillin Resistant Staphylococcus aureus (MRSA) in breeding pigs 1 Adopted by the Task Force on 19 November For citation purposes: Report of the Task Force on Zoonoses Data Collection on a proposal for technical specifications for a baseline survey on the prevalence of Methicillin Resistant Staphylococcus aureus (MRSA) in breeding pigs, The EFSA Journal (2007) 129, European Food Safety Authority,

2 Summary Methicillin Resistant Staphylococcus aureus (MRSA) is known for its impact on public health. Recently, the MRSA clonal complex ST398 has been reported to occur in pigs and several other animal species. Several Member States are undertaking studies to understand better the impact of these findings. The Task Force on Zoonoses Data Collection was asked by the European Food Safety Authority (EFSA) to provide recommendations on the necessity to survey the occurrence of MRSA in animals. The Task Force recommends that in addition to national studies it is important to undertake a common survey at European Community level, in particular to understand and quantify the spread of MRSA in some farm animals throughout the EU as well as the relevance of different strains to understand whether there is a clonal spread. The Task Force proposes to combine such a survey to the forth coming Salmonella baseline survey in breeding pigs. The survey will focus on breeding pigs, but it is proposed to sample for MRSA also the premises of fattening pigs, if present on the breeding pig holdings. The inclusion of fattening pigs will be useful to examine the transfer of MRSA within the holding, but it will not provide a representative estimate of the prevalence of MRSA in fattening pigs in the country. This report describes the technical specifications for the MRSA survey including the sampling techniques and analytical methods. European Food Safety Authority,

3 Contents Summary...2 A. Background...4 B. Rationale for the choices made in the proposal...6 C. Proposed technical specifications for a baseline survey on the prevalence of MRSA in breeding pigs Definitions Objectives Primary objective Secondary objectives Sampling frame Sample collection in the herds Type and detail of sample Sample information Transport of samples Laboratory analytical methods Laboratories Receipt of samples Sample analysis Preparation of the specimen Selective enrichment and isolation of MRSA Identification and verification Sub-typing Antimicrobial susceptibility testing Storage of isolates Reporting from the Member States Overall description on the implementation of the survey Complete data on each holding sampled and corresponding tests results...12 Task Force on Zoonoses Data Collection members...13 Acknowledgements...13 References...13 Annex...14 European Food Safety Authority,

4 A. Background Methicillin Resistant Staphylococcus aureus (MRSA) is known for its impact on public health. MRSA is typically a health-care related problem (hospital-acquired HA-MRSA) although increasingly also community-acquired strains (CA-MRSA) are reported. Recently, the MRSA clonal complex ST398 seems to have found a reservoir in animals and it has been reported to occur in pigs, cattle, horses, poultry and dogs. This MRSA clone has been shown to be capable of infecting humans and therefore this clone of MRSA is a zoonosis, with direct public health impact. For certain professional groups (e.g. pig holders and their families, veterinarians) it should be considered an occupational health risk. Triggered by the Dutch findings on the presence of MRSA in pigs and other food producing animals and the spread to pig holders, increasing number of studies from other countries demonstrate the presence of MRSA in several animal species, including pet, wild and farm animals. This includes a report on a prevalence survey of MRSA in Belgium in pigs and in pig farmers and other human population. MRSA has been found in both breeding and finishing pig herds in the Netherlands. MRSA have been detected in cattle, chickens, horses, pigs, dogs, rabbits, birds and cats. Studies from different countries have varying designs, which makes it difficult to infer the magnitude and public health importance of MRSA found in animals. Although current knowledge suggests that the majority of human MRSA is not acquired from animal contact, the Task Force believes that it is necessary to have monitoring in place to evaluate the epidemic situation and the trend of MRSA prevalence in production animals in EU. This will help to assess the risk of MRSA derived from animals to colonize and possibly infect humans. Indeed, it may be urgent to start such monitoring at the EU-level. Some Member States may be only at the start of an epidemic and may be in a position to prevent (further) spread and stay ahead of the curve. A first step would be to conduct a survey to understand the spread of MRSA at present. It seems cost-effective to invest appropriate resources since the cost of prevention is lower than cost of infection. In addition, there are expectations from the public since MRSA attracts media attention and questions are raised on the risk for consumers and the actions to be taken by EU and national authorities. At present there are many open questions that need answers in order to mount an appropriate response: a. How wide-spread is MRSA in food animals? (prevalence within Member States (MS), and which MS are affected to what extent?) b. On which types of pig farms are they prevalent? (finishers, breeders, etc.) c. Which types occur in animals? Is it mainly the ST398 clone? d. What is the occurrence of ST398 clone in humans in different MS? e. What are risk factors for occurrence at MRSA (selective pressure, husbandry factors, colonized breeding herds and animal movements) and how could spread be limited? f. What is the origin of the problem? Is it a new problem? (earliest reports are from 2003) g. What is impact on human health in relation to MSSA (Methicillin Susceptible S. aureus) HA-MRSA and/or CA-MRSA? To address these questions some MS are undertaking studies in order to understand the significance of MRSA strains found in animals compared to the occurrence of MRSA strains in European Food Safety Authority,

5 humans. In addition to these national approaches it is important to undertake a common survey at European Community level, in particular to address the first questions: i.e. to understand and quantify the spread of MRSA strains in some farm animals throughout the EU as well as the relevance of different strains to understand whether there is a clonal spread and to understand the significance of this to the overall occurrence of MRSA strains in humans. Recently there is good experience in studying the prevalence of different zoonotic agents at Community level through base-line surveys and it seems cost-effective to build on the base-line study approach. The upcoming Salmonella baseline survey in breeding pigs, starting on 1 January 2008, is an excellent opportunity to do this in a harmonized and cost-effective way where MS authorities are visiting a representative sample of pig holdings and can take few additional dust samples for MRSA detection in each selected holding without much extra cost. The Task Force on Zoonoses Data Collection was asked by EFSA to provide among others recommendations on the necessity to survey the occurrence of MRSA in certain animal species and indicate possible approaches. The Task Force hereby presents a protocol to survey MRSA in breeding pigs building on the upcoming baseline survey on Salmonella in breeding pigs. This protocol has been developed together with the Community Reference Laboratory for antimicrobial resistance (CRL-AR) in collaboration with researchers in The Netherlands and the Working Group on developing harmonized schemes for monitoring antimicrobial resistance in zoonotic agents. The Task Force is grateful for CRL-AR offer to provide assistance in testing or to provide assistance in finding laboratories capable of performing such testing. In developing this protocol the aim was to build as much as possible on the Salmonella baseline survey adding as little as possible additional burden on the MS. Therefore reference is made to the report of the Task Force with technical specifications for a baseline survey on the prevalence of Salmonella in breeding pigs 1 and Commission Decision 2 on Salmonella breeding pigs baseline survey for definitions used and sampling design. It is obvious that this survey will not provide answers to all of the many questions we have today around MRSA in animals and it should rather be considered a first EU-wide prevalence survey of MRSA in breeding pigs, possibly providing preliminary data related to MRSA in slaughter pigs. It must be acknowledged that the data on fattening pigs that is collected from breeding holdings cannot be extrapolated to fattening pig holdings in general. However, it is an excellent opportunity to understand the spread of MRSA and which Sequence Types (clones) occur in pigs. MRSA has also been detected in poultry and cattle herds. Thus, it would be advisable in the future to survey the occurrence also in these animal species if there were indications that they are a significant hazard to humans. Without a more thorough knowledge of the types of MRSA clones in humans, it may not be possible to assess the significance of any findings in animals. 1 Report of the Task Force on Zoonoses Data Collection on a proposal for technical specifications for a baseline survey on the prevalence of Salmonella in breeding pigs. The EFSA Journal (2007) 99, Commission Decision 2007/636/EC of 28 September 2007 concerning a financial contribution from the Community towards a survey on the prevalence of Salmonella spp. in herds of breeding pigs to be carried out in the Member States (notified under document number C(2007) 4434). OJ L 257, , p European Food Safety Authority,

6 B. Rationale for the choices made in the proposal While preparing the technical specifications for the baseline survey of MRSA in breeding pigs, some decisions and choices regarding the scope and design of the specifications were made. These decisions and their rationales are described below: MRSA have been detected in breeding pig herds. Breeding pigs are at the top of the production pyramid and even a low MRSA prevalence in these herds may contribute to a significant part of the MRSA infections among fattening pigs. Breeding herds may disseminate the infections to a large number of other herds because most of the breeding herds have trade contacts with several fattening pig herds. A recent publication from The Netherlands indicate that trade of animals is a key factor in the spread of MRSA between herds. There are differences in the numbers of breeding as opposed to fattening pigs traded between and within EU Member States. The current MRSA situation is considered unlikely to be static, so it is important to gather information from both breeding and production herds of pigs. Particularly if some potential control measures might depend on having some breeding and production herds free from colonization. It is well established for other bacterial pathogens, such as Salmonella, that breeding pigs may play an important role in the maintenance and transmission of the infection either to the slaughter generation (fattening herds), or act as a source of infection to the breeding pigs whose progeny will become the slaughter generation (nucleus and multiplier herds, production herds). It is proposed that primarily both breeding and production herds of breeding pigs are sampled. If there are fattening pigs present on the holding one additional set of samples should be taken from them. Studies in The Netherlands and Belgium have indicated that MRSA is more prevalent among fattening pigs. However, the situation in The Netherlands and Belgium might not hold true for all other Member States. This report limits itself to farm animals and does not consider food-borne transmission. Further recommendations regarding farm animals species other that breeding pigs may be issued later. Currently EFSA is working on a Scientific Opinion on food-borne antimicrobial resistance as a biological hazard considering also S. aureus resistance. The samples to be collected will be dust samples. The optimum sampling site for detecting MRSA is currently not known, although data from the Netherlands and Belgium indicate that 60 nasal swabs from pigs to be taken per holding is an optimum. In a recent, yet unpublished Dutch study in pigs the sensitivity of taking 5 dust samples (Sodibox swabs) was found only slightly lower than that of 60 nasal swabs. Thus dust samples yield high isolation rates and they are much less expensive in terms of time, of sampling and human resources. Since it seems not feasible to collect up to 60 additional nostril samples per visited holding in the proposed Salmonella survey in breeding pigs it is proposed to collect combined dust samples instead. For outdoor holdings dust samples can best be taken from indoor facilities on these holdings, if present. Thus, the purpose of this survey is to estimate the prevalence of holdings with breeding pigs and if present of fattening pigs colonized with MRSA that may present a risk to humans or a risk to other pigs (either breeding herds lower down the production pyramid or directly to fattening pigs). European Food Safety Authority,

7 It is suggested that the pigs are sampled at the holding level. It is important to emphasise that the survey is not intended to estimate the prevalence of MRSA colonization within each holding but rather to estimate the prevalence of colonized holdings. Current knowledge indicates that MRSA on pig farms in some Member States is an occupational risk for people working or living on those farms. Data on the effect of exposure of slaughterhouse workers are incomplete. Both breeding animals and fattening pigs do enter the food chain at the end of their productive lives, although so far no indications of spread through the food chain have been identified. However, cross contamination may take place during transport and stay at the slaughterhouse. Hence, sampling at the slaughterhouse would not provide a good estimate for the number of colonized herds. The proposed survey approach is likely to provide an estimate of the minimal prevalence of holdings with breeding and, if available, fattening pigs in these holdings colonized with MRSA. This is because it is not sure that not finding a MRSA equals to absence of MRSA in that holding. Assuming that a positive dust sample equates to the presence of MRSA in that pig holding it is more accurate to say that the study detects the prevalence of holdings on which MRSA has been detected in the environment (dust). Besides information on the prevalence of MRSA in EU Member States in pigs, this study will result in information on the clonal spread of MRSA isolates found. Appropriate typing techniques will demonstrate the genetic relations of strains isolated and the antimicrobial resistance pattern. European Food Safety Authority,

8 C. Proposed technical specifications for a baseline survey on the prevalence of MRSA in breeding pigs 1. Definitions Since this protocol is building on the baseline survey on the prevalence of Salmonella in breeding pigs the definitions given in the Task Force report published in the EFSA Journal (2007) 99, 1-28 apply. 2. Objectives 2.1. Primary objective The main objective of this survey is to obtain an estimate of the minimal prevalence of holdings with breeding pigs where MRSA has been detected in dust samples, indicating the presence of MRSA in pigs on the holding. This prevalence will be evaluated separately in breeding holdings, as well as in production holdings in the Community and in the MSs. The data from the breeding and production holdings will give information on the presence of MRSA in breeding pigs and the clonal diversity of MRSA will be assessed by spa-typing followed by MLST typing of selected isolates Secondary objectives The survey also provides the opportunity to collect data on a restricted set of holding-level and sample-level variables that may be associated with MRSA prevalence. However, sample size calculations have not been predicated on this secondary aim. Also premises of fattening pigs present on the production holdings that are visited are investigated. The data from the fattening pigs on production holdings may provide further information to assess the presence of MRSA in fattening pigs and the clonal diversity of MRSA. However, it should be acknowledged that the data on fattening pigs are collected from breeding holdings are can not be extrapolated to fattening pig holdings in general. Lastly, MRSA strains will be available for antibiotic susceptibility testing to give valuable information on the prevalence of resistance to different antimicrobials. 3. Sampling frame The assumptions, in terms of a priori prevalence (50%), desired confidence level (95%) as well as accuracy (7.5%), used to calculate the primary sample size, i.e. the number of breeding holdings and the number of production holdings to be included in each Member State for the Salmonella survey, can also be made for the MRSA survey. Therefore the sample of holdings to be investigated in the Salmonella baseline survey in breeding pigs can also be used for the MRSA survey. More details about the sampling frame are available in Annex 1 of the Commission Decision 2007/636/EC. European Food Safety Authority,

9 The secondary sample size is one composite sample for selected holdings without fattening pigs, whereas on holdings with fattening pigs it is two composite samples. 4. Sample collection in the herds 4.1. Type and detail of sample Five dust samples are gathered using 5 sterile dry swabs (approximately the size of 18 cm x 32 cm) from 5 of the 10 pens selected for sampling in the frame of the Salmonella baseline survey. These 5 pens should be chosen in a way that pigs in different production stages are included. For each pen dorsal surfaces of pen partition walls are swabbed covering approximately a surface area of 50x50 cm 2. In case there is not enough dust present or the surface area is less than 50x50 cm 2, then other surfaces adjacent to the pen, such as ventilator ducts etc., should be sampled in addition. After use, the soiled swab is placed in a sterile plastic bag. If a holding with breeding pigs also holds fattening pigs, 5 additional samples of dust are taken by means of 5 sterile dry swabs from 5 different pens of the fattening pigs from premises on the holding containing animals at the size between kg. Where all animals present on site of this weight are housed in one building, the 5 pens should be chosen from different parts of this building. Where fattening pigs are housed in multiple buildings on the holding the samples should be taken from distinct buildings. The creation of aerosol building should be avoided during sampling. More detailed guidance on sampling is provided in the Annex. For outdoor holdings dust samples can be taken from indoor facilities of these holdings, if present Sample information Each sample and its sample form should be labelled with a unique number which should be used from sampling to testing. The competent authority must arrange for the issue and use of a unique numbering system Transport of samples Samples should be kept at constant temperature between +2 C and +25 C (room temperature) and free of external contamination during storage and transportation. Studies have shown that MRSA survive for a long time (up to 1 year) in dust. It is, however; recommended that the samples should be sent to the laboratory as quickly as possible. In any case the sample should reach the laboratory no later than 10 days after sampling. 5. Laboratory analytical methods 5.1. Laboratories Analysis, subtyping and susceptibility testing of MRSA shall take place in experienced laboratory(ies). This should preferably be the National Reference Laboratories (NRL s) for European Food Safety Authority,

10 Staphylococcus aureus and/or antimicrobial resistance in the Member States. In case the NRL does not have the capacity or experience to perform the analyses or if it is not the laboratory that performs detection routinely, the competent authorities may decide to designate other experienced laboratories or a NRL in another Member State to perform the analyses. These laboratories should have proven experience of using the required methods and have an accreditation system in place according to EN-ISO An up-to-date list of authorised laboratories can be consulted on the website of the Community Reference Laboratory for antimicrobial resistance (CRL-AR) Receipt of samples Samples arriving 10 days after sampling shall be discarded. At the laboratory, samples shall be kept at a constant temperature between +2 C and +25 C until bacteriological examination, which shall be carried out within 13 days after sampling Sample analysis Preparation of the specimen In the laboratory the 5 swabs from pens of breeding pigs are pooled to one composite sample, and the 5 swab samples from fattening pig premises are in a similar way pooled to one sample Selective enrichment and isolation of MRSA In carriage the number of MRSA bacteria can be very low and other bacterial species might exist in far greater numbers. Using an enrichment broth increases the sensitivity and it is therefore necessary and recommended. The 5 dust swabs are pooled in 100 ml of Mueller-Hinton broth with 6.5% NaCl and incubated aerobically at 37 o C for h. One ml of this is then inoculated into 9 ml Tryptone Soy Broth containing 3.5 mg/l cefoxitin and 75 mg/l aztreonam and incubated for a further 16-20h at 37 C. One loop-full of this is then spread onto a chromogenic agar selective for MRSA and incubated for 24h-48h at 37 C. The specific agar recommended by the CRL-AR in Copenhagen, Denmark, shall be used. This agar is described in CRL-AR web site ( Based on colony morphology and colour, up to five colonies indicative for being MRSA are subcultivated on blood agar. Presumptive S. aureus can at this stage either be stored under appropriate conditions (-80 C) for later identification and characterization or processed immediately Identification and verification Presumptive S. aureus should be identified by PCR. CRL-AR will publish a list of suitable PCR procedures on their website ( The identification can be performed using a multiplex PCR with simultaneous identification of the meca-gene or two different PCR can be performed. To limit the amount of work only one of the five presumptive S. aureus should initially be identified and if positive as S. aureus colonies verified as MRSA. If this isolate is positive and verified as MRSA this isolate should be stored at -80 C. No further testing of the remaining four isolates are required and they can be discarded. If the first isolate is not confirmed as S. aureus or as MRSA, the next of the initial five isolates should be tested. This process should continue until one MRSA has been identified or all five isolates tested. Alternatively, verification by PCR as a European Food Safety Authority,

11 first step could be done on a pool of the five presumptive colonies from a sample. In case of a positive PCR, the analysis will be repeated on individual colonies to identify a positive colony. For quality assurance, 16 isolates of non-confirmed MRSA strains as well as 16 MRSA strains, sampled over the whole year 2008 are sent to the CRL-AR. A proportion of these isolates should be sent to the CRL-AR on a quarterly basis. In case less than 16 isolates were identified as MRSA all these isolates should be sent Sub-typing Positive MRSA s should be spa-tested 1 for possible link to humans ( The typing can be performed at the isolating laboratory or isolates can be forwarded to the CRL-AR, which will then perform the typing. On a subset of representative isolates MLST-typing will be performed by the NRL or the CRL- AR. Laboratories will be asked to forward selected isolates to the CRL-AR for this. SCCmec typing and other characterization of selected isolates might also be performed by the CRL-AR or NRL on request of the Commission Antimicrobial susceptibility testing Antimicrobial susceptibility testing according to internationally accepted guidelines is strongly recommended. No guidelines for antimicrobial susceptibility testing of S. aureus have been published by EFSA. However, for the purpose of this study it is proposed that MRSA isolates are susceptibility tested using micro-dilution at least to the following antimicrobial agents: ciprofloxacin, erythromycin, fusidic acid, gentamicin, linezolide, mupirocin, sulphametoxazole, trimethoprim, tetracycline, chloramphenicol, vancomycin and quinupristin/dalfopristin Storage of isolates The MRSA isolates shall be stored at the National Reference Laboratories (NRL s) using the method for NRL culture collection ensuring viability and no changes in properties of the strains for a minimum of 5 years. This is in order to allow, for instance, later testing for antimicrobial susceptibility or other types of characterisation if requested by the Commission. Also isolates sent to the CRL-AR will be stored for a minimum of 5 years. Isolates shall be stored under conditions not allowing changes in their properties (-80 C). If the laboratory in charge do not have the available storage capability, isolates could be forwarded to the CRL-AR, which will store these isolates. 6. Reporting from the Member States 6.1. Overall description on the implementation of the survey A report in text format shall include at least: (a) Number of dust samples from pens of breeding pigs obtained and analysed: 1 Spa-typing is a sequence based typing for Staphylococcus aureus, where the isolate is assigned a type based on its sequence of the gene encoding Staphylococcual protein A (spa); a gene specific for S. aureus. European Food Safety Authority,

12 (i) From breeding holding as defined in Part 2(1) of Annex I to Decision 2007/636/EC. (ii) From production holdings as defined in Part 2(1) of Annex I to Decision 2007/636/EC (iii) Number of dust samples from pens of slaughter pigs obtained and analysed from production holdings. (b) (c) Overall results: Prevalence of MRSA in breeding pigs of breeding holdings and of breeding pigs and fattening pigs on production holdings, based on detection and confirmation by PCR. List of laboratories responsible in the baseline survey for MRSA i) Isolation ii) PCR (species identification, meca, SCCmec) iii) Spa-typing iv) MLST-typing v) Antimicrobial susceptibility testing 6.2. Complete data on each holding sampled and corresponding tests results: The Member States shall submit the results of the investigations electronically in the form of raw data using a data dictionary and data collection forms provided by the Commission. A common data dictionary and data collection form shall be used as the one used in accordance with Decision 2007/636/EC. The Member States shall arrange that each sample can be linked to the information collected in accordance with the provisions in Part 5(2) of Annex I to Decision 2007/636/EC. The following information shall be collected in Member States for each sample sent to the laboratory: (a) Code of the sample Sample from breeding or fattening unit (b) Code/Name of the laboratory involved in the detection (c) Date of sample collection (d) Date laboratory analysis begun (e) Result of detection (positive/negative) (f) Code/Name of the laboratory involved in the PCR (g) Result of PCR (meca, species identification, SCCmec) (h) Code/Name of the laboratory involved in the spa-typing (g) Result of spa-typing. (h) Code/Name of the laboratory involved in the MLST-typing (if not CRL) (i) Result of MLST-typing (j) Code/Name of the laboratory involved in the antimicrobial susceptibility testing (if not CRL) (k) Antimicrobial susceptibility test results European Food Safety Authority,

13 Task Force on Zoonoses Data Collection members José Ignacio Arraz Recio, Andrea Ammon, Harry Bailie, Marta Bedriova, Melanie Picherot, Birgitte Borck, Karen Camilleri, Georgi Chobanov, Adriana Costache, Kris De Smet, Matthias Hartung, Birgitte Helwigh, Merete Hofshagen, Vaidotas Kiudulas, Elina Lahti, Peter Much, Lisa O Connor, Rob A.A. Van Oosterom, Jacek Osek, José Luis Paramio Lucas, Manca Pavšič, Christodoulos Pipis, Saara Raulo, Antonia Ricci, Tatjana Ribakova, Valentina Rizzi, Petr Šatrán, Joseph Schon, Jelena Sõgel, Petra Szabados, Patricia Tavares Santos, Kilian Unger, Luc Vanholme and Dimitris Vourvidis. Acknowledgements The Task Force on Zoonoses Data Collection wishes to acknowledge the contribution of the Working Group that prepared this report: Frank Aarestrup, Antonio Battisti, Bjorn Bengtsson, Segundo Piriz Duran, Hanne-Dorthe Emborg, Dik Johan Mevius, Getraud Regula, Pascal Sanders, Andreas Schroeter, Christopher Teale, Dariusz Wasyl, Pierre-Alexandre Beloeil, Kris De Smet, Pia Mäkelä and Stef Bronzwaer. References Poulsen AB, Skov R, Pallesen LV. Detection of methicillin resistance in coagulase-negative staphylococci and in staphylococci directly from simulated blood cultures using the EVIGENE MRSA Detection Kit. J. Antimicrob. Chemother. 2003; 51: Friedrich AW, Witte W, Harmsen D, de Lencastre H, Hryniewicz W, Scheres J, Westh H; SeqNet.org participants. SeqNet.org: a European laboratory network for sequence-based typing of microbial pathogens. Euro Surveill :E Strommenger B, Kettlitz C, Weniger T, Harmsen D, Friedrich AW, Witte W. Assignment of Staphylococcus isolates to groups by spa typing, SmaI macrorestriction analysis, and multilocus sequence typing. J. Clin. Microbiol : European Food Safety Authority,

14 Annex Details on the dust-sampling procedure in the holdings Five dust samples are taken from each breeding pig holding. The samples must be collected wearing disposable gloves. It is best to prepare the sampling material beforehand [disposable gloves, sterile dry swabs (e.g. Sodibox or similar swabs)]. The swab is a sterile square piece of cotton cloth (18 cm 32 cm) that is used to wipe dust. One dry swab is used to sample each pen selected. This sampling should be compatible with the within-holding swab-sampling for Salmonella (see Annex 2 of the technical specifications for a baseline survey on the prevalence of Salmonella in breeding pigs, The EFSA Journal (2007) 99, 1-28). Together 5 of the 10 pens selected for sampling in the frame of the Salmonella baseline survey are chosen for the MRSA sampling. It should be ensured that these 5 pens contain breeders in different physiological situation (service area, lactation phase, pregnancy phase). In farrow-to-finish farm of the production level, 5 additional dust samples are collected. The dust samples must be gathered from 5 pens containing fattening pigs weighting between kg. If those fattening pigs are bred in more than one building, the 5 pens sampled should be taken from the different buildings. The sampling procedure is as follows: 1. At a sample pen side put on a pair of disposable gloves and a mask. 2. Open a swab bag (e.g. Sodibox) and unfold the swab. 3. Wipe dust from the top of pen partitions of the selected pen. Approximately an area corresponding 50x50 cm 2 should be wiped. If dust is very sparse or the area to be covered would be less than 50x50 cm 2, then collect dust from other surfaces adjacent to the pen wherever dust is available. 4. After wiping, place the swab with dust in the sterile plastic bag and close the bag. 5. Proceed to next sample pen and follow instructions 2-4. In case the pens of fattening pigs are also samples, the disposable gloves and mask should be changed before proceeding to that sampling. 6. Ensure that the farm identifier, the sample number and the sample type (breeding or fattening pigs) is clearly marked on the sample container or fully cross-referenced on a sample record sheet. European Food Safety Authority,