Methicillin-Resistant Staphylococcus aureus in Poultry

Methicillin-resistant Staphylococcus aureus (MRSA) has been detected in several species and animal-derived products. To determine whether MRSA is present in poultry, we sampled 50 laying hens and 75 broiler chickens. MRSA was found in some broiler chickens but no laying hens. In all samples, spa type t1456 was found.

S taphylococcus aureus is a well-known pathogen of humans and animals. Methicillin resistance in this bacterial species represents a threat to human health. Originally, methicillin-resistant S. aureus (MRSA) was a nosocomial pathogen, but in the 1990s, MRSA spread into communities worldwide.
Recently, pigs were shown to be a major reservoir for MRSA multilocus sequence type 398 (ST398). Because this sequence type has also been isolated from other animal species, it is referred to as livestock-associated MRSA (1). It has also shown potential for zoonotic transmission (2). Among ST398 isolates, a variation in spa types has been found (3). MRSA has been isolated from raw chicken meat or carcasses in Korea (4,5) and Japan (6); however, these strains were human-associated and not the livestock-associated strains. Thus, the possibility of human contamination of poultry carcasses by slaughterhouse employees cannot be ruled out. We investigated whether livestock-associated MRSA is present in commercial broiler chickens and laying hens.

The Study
In 2007, from randomly selected farms in Belgium, we sampled 5 laying hens from each of 10 farms and 5 broiler chickens from each of 14 farms. One broiler farm was sampled twice (4 months apart, from different fl ocks in the same house, leaving 1 production round unsampled). Samples were taken from the cloaca and nasal cavity of these 50 laying hens and 75 broiler chickens.
Samples were fi rst incubated in a brain-heart infusion broth supplemented with nalidixic acid and colistin, each at a concentration of 10 μg/mL. After overnight incubation at 37°C, 1 μL of this broth was streaked onto an MRSA Chro-mID plate (bioMérieux, Marcy l'Etoile, France) and incubated for 24-48 h at 37°C. To differentiate phenotypes of S. aureus, we purifi ed colonies that showed typical growth on MRSA Chrom-ID plates by transferring them to modifi ed Baird-Parker agar (7) and Columbia blood agar (Oxoid, Hampshire, UK) for DNAse and catalase testing.
The phenotypically identifi ed MRSA strains were then confi rmed by 16S rRNA-mecA-nuc triplex PCR as previously described (8). For all strains, the spa type was determined, and for 3 strains, multilocus sequence typing (MLST) was performed as described (9). Relatedness with other spa types from porcine origin (10) was determined by using Ridom SpaServer software version 1.3 (Ridom GmbH; Würzburg, Germany; www.ridom.de/spa-server).
Disk susceptibility of the strains was tested by using the Kirby-Bauer disk-diffusion method. Clinical Laboratory Standards Institute guidelines (M31-A3) were followed for inoculum standardization. After plates were incubated for 18 h, inhibition zones were measured in millimeters and interpreted according to Neo-Sensitabs manufacturer's instructions (http://rosco.dk). S. aureus ATCC 25923 was included for internal quality control.
MRSA was not isolated from any laying hen samples. This fi nding may indicate that MRSA is absent or present only in low numbers in laying hens, possibly because of the limited use of antimicrobial drugs in these animals. Use of certain antimicrobial drugs in human hospitals has been shown to be a risk factor for acquiring MRSA infection, especially when the chosen treatment is inappropriate or insuffi cient (11). Antimicrobial-drug use may also be a risk factor for MRSA colonization of animals. The antimicrobial drugs used in the fl ocks included in this study were tylosin, amoxicillin, trimethoprim-sulfamethoxazole, lincomycin, tetracycline, and colistin.
MRSA was isolated from 8 broiler chickens from 2 of the 14 farms sampled. Low prevalence in poultry has also been found by Kitai et al. (6) and Lee (5), although they sampled chicken carcasses from slaughterhouses and did not fi nd any livestock-associated strains. Given our relatively small sample size, our data did not permit us to estimate the within-and between-fl ock prevalence.
In the MRSA-positive fl ocks, the number of positive samples varied between 1/5 (20%) and 5/5 (100%). From the 1 MRSA-positive farm that was sampled twice, MRSA was isolated on both occasions. This fi nding indicates that MRSA may persist on a farm and colonize future fl ocks. MRSA was found in nearly equal numbers from the nares samples and the cloaca samples. Of the 8 MRSA-positive animals (16 samples), MRSA was found in all samples except for 1 cloacal swab, for a total of 15 MRSA isolations.
Molecular typing showed that the strains all belonged to spa type t1456 of the livestock-associated ST398, which is typically not typeable by pulsed-fi eld gel electrophoresis. To our knowledge, this spa type has not been found in other animal species (11,12). Its relatedness to other spa types isolated from pigs is shown in the Table. A shortening of variable number tandem repeat composition seems to be present in spa type t1456. Whether t1456 is a clone typically associated with poultry, or specifi cally broiler chickens, and whether it is spreading internationally needs further investigation.

Conclusions
We confi rmed the presence of MRSA in broiler chickens, but we were unable to fi nd it in laying hens. All isolates belonged to 1 spa type, t1456, and thus differed from the other strains belonging to ST398 isolated from other animal species in Belgium and abroad. Whether this spa type is typically associated with poultry still needs to be confi rmed. More detailed data are also needed to gain further insight in the true within-and between-fl ock prevalence of MRSA in poultry and its evolution over time.  Pig  t011  008 -16 -02 -25 -----34 -24 -25  Pig  t034  008 -16 -02 -25 -02 -25 -34 -24 -25  Pig  t108  008 -16 -02 -25 -------24 -25  Pig  t567