Commission Decision of 20 December 2007 concerning a financial contribution from the Community towards a survey on the prevalence of Salmonella spp. and Methicillin-resistant Staphylococcus aureus in herds of breeding pigs to be carried out in the Member States (notified under document number C(2007) 6579) (2008/55/EC)

Part C: Sample collection and analysis for the MRSA survey

1. Type and detail of sample

1.1. Sample collection

Five dust samples shall be gathered using five dry sterile swabs of about 500 cm² each from five of the 10 pens selected for sampling under part A. These five pens shall be chosen in a way that breeding pigs in different production stages are included. For each pen dorsal surfaces of pen partition walls shall be swabbed. In case there is not enough dust present, then ventilator ducts etc. shall be sampled in addition. After use, the soiled swab shall be placed in a sterile plastic bag.

The creation of aerosol in the building shall be avoided during sampling.

1.2. Sample information

Each sample and its sample form shall be labelled with a unique number which shall be used from sampling to testing. The competent authority shall arrange for the issue and use of a unique numbering system.

1.3. Transport of samples

Samples shall be kept at constant temperature between + 2 °C and 25 °C (room temperature) and free of external contamination during storage and transportation. The samples shall be sent to the laboratory as quickly as possible and reach the laboratory no later than 10 days after sampling.

2. Laboratory analytical methods

2.1. Laboratories

Analysis and subtyping of MRSA shall take place in experienced laboratories. This shall preferably be the National Reference Laboratories (NRL’s) for 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 authority shall decide to designate other experienced laboratories or a NRL in another Member State to perform the analyses. These laboratories shall have proven experience of using the required methods and have an accreditation system in place according to ISO 17025. An up-to-date list of authorised laboratories can be consulted on the website of the Community Reference Laboratory for antimicrobial resistance (CRL-AR) in Copenhagen, Denmark.

2.2. Receipt of samples

Samples arriving 10 days after sampling shall be discarded unless bacteriological examination can be started within 13 days. 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.

2.3. Sample analysis

2.3.1. Selective enrichment

In the laboratory the five dust swabs shall be pooled in a 100 ml of Mueller-Hinton broth supplemented with 6,5 % NaCl and incubated at 37 °C for 16-20 h. One millilitre of this shall then be inoculated into 9 ml Tryptone Soy Broth + 3,5 mg/l cefoxitin and 75 mg aztreonam and shall be incubated for a further 16-20 h at 37 °C. One loop-full of this shall then be spread onto a chromogenic agar selective for MRSA and incubated for 24-48 h at 37 °C. The specific agar recommended by the CRL-AR shall be used. This agar is described in the CRL-AR website.

Based on colony morphology and colour, up to five colonies indicative for being MRSA shall be subcultivated on blood agar. Presumptive Staphylococcus aureus (S. aureus) shall at this stage either be stored under appropriate conditions (– 80 °C) for later identification and characterisation or processed immediately.

2.3.2. Identification of MRSA

Presumptive S. aureus shall be identified as S. aureus and MRSA by PCR. The identification shall be performed using a multiplex PCR with simultaneous identification of the mecA-gene or two different PCR shall be performed. To limit the amount of work only one of the five presumptive S. aureus isolates shall initially be identified. If this isolate is identified as MRSA, it shall be stored. No further testing of the remaining four isolates is required if the first isolate is identified as MRSA and they can be discarded. If the first isolate is not identified as MRSA, the next of the initial five isolates shall be tested. This process shall continue until one MRSA has been identified or all five isolates have been tested. Alternatively, identification by PCR as a first step can be done on a pool of the five presumptive colonies from a sample. In case of a positive PCR, the analysis shall be repeated on individual colonies to identify a positive colony.

For quality assurance, 16 presumptive S. aureus isolates which were not identified as MRSA, as well as 16 MRSA strains, sampled over the whole year 2008 shall be sent to the CRL-AR. A proportion of these isolates shall be sent to the CRL-AR on a quarterly basis. In case less than 16 isolates were verified as MRSA all these isolates shall be sent.

2.3.3. Subtyping for possible link to human isolates

Positive MRSA’s shall be tested for Staphylococcus type A (Spa-typing). The typing shall be performed at the NRL or under its supervision, or isolates shall be forwarded to the CRL-AR, which shall then perform the typing.

On a subset of representative isolates (about 2 % of the number of pooled samples) MLST-typing shall be performed by the NRL or the CRL-AR.

2.3.4. Antimicrobial susceptibility testing

Antimicrobial susceptibility testing is optional. If carried out, MRSA isolates shall be tested for antimicrobial susceptibility 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. Reporting on antimicrobial susceptibility shall be carried out in accordance with Article 9(1) of Directive 2003/99/EC.

2.4. Storage of isolates

The 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 five years. This is in order to allow, for instance, later testing for antimicrobial susceptibility or other types of characterisation. Also isolates sent to the CRL-AR shall be stored for a minimum of five years. Isolates shall be stored under conditions not allowing changes in properties (– 80 °C). If the laboratory in charge do not have the available storage capability, isolates shall be forwarded to the CRL-AR, which shall store these isolates.

3. Reporting from the laboratories

All analytical results shall be sent on a confidential basis only from the laboratory to the competent authority of the Member States where the dust samples were collected.