Marijke Verhegghe

Evidence of possible methicillin-resistant Staphylococcus aureus ST398 spread between pigs and other animals and people residing on the same farm

Methicillin-resistant Staphylococcus aureus (MRSA) has emerged in a wide variety of animal species. However, little is known about the transmission routes of MRSA ST398 between different animal species, the barn environment and people residing on the same farm. In this study, two pig farms, two poultry-pig and two dairy-pig farms were investigated with respect to the presence of MRSA. On each farm, samples were collected from all animal species present, the barn environment, the farmer, household members and the herd veterinarians. Besides the MRSA prevalence, the obtained spa-, SCCmec-type and antimicrobial susceptibility profiles were also compared. Multilocus sequence typing (MLST) showed that MRSA ST398 was found in all animal species, in humans present on the farms and also in the pig barn environment. The presence of MRSA with the same spa-, SCCmec-type and antibiotic profile in the different animal species in direct or indirect contact with pigs suggests MRSA transfer. Furthermore, different pig age categories were investigated, with weaned piglets having the highest MRSA prevalence (86.3%). The herd-level prevalence was highly correlated (r=0.86, p=0.03) between sows and pre-weaned piglets. The results also indicate that companion animals, rats, mice and farmers could play an important role in the dissemination of MRSA, emphasizing the importance of internal biosecurity. However, external biosecurity is equally important because other spa-, SCCmec-types or antimicrobial resistances can be introduced through purchase of gilts. In this study we demonstrated that MRSA likely spreads between animal species, humans and the pig barn environment, which is why it is important to accurately implement control practices, in which not only pigs should be targeted, but also all other animal species present on farms.

Methicillin‐Resistant Staphylococcus aureus (MRSA) ST398 in Pig Farms and Multispecies Farms

During the last few years, methicillin‐resistant Staphylococcus aureus (MRSA) ST398 has been isolated frequently from livestock, especially from pigs and to a lesser extent from cattle and poultry. To gain insight into the distribution of this bacterium in pig farms versus multispecies farms, 30 Belgian farms (10 pig, 10 pig/poultry and 10 pig/cattle farms) were screened for the presence of MRSA. On each farm, 10 nasal swabs were taken from pigs. When present, cattle (n = 10) were sampled in the nares and poultry (n = 10) in the nares, earlobes and cloaca. A selection of the obtained isolates were further characterized using multilocus sequence typing (MLST), spa typing, SCCmec typing, pulsed field gel electrophoresis (PFGE), multiple‐locus variable‐number tandem repeat analysis (MLVA) and antimicrobial susceptibility testing. On 26 of 30 farms, MRSA was isolated from pigs. Furthermore, MRSA was also isolated from poultry and cattle on one pig/poultry and five pig/cattle farms, respectively. All tested MRSA isolates belonged to ST398. Eight spa types (t011, t034, t567, t571, t1451, t2974, t3423 and t5943) were detected, among which t011 predominated. SCCmec cassettes type IVa and V were present in 20% and 72% of the isolates, respectively. When combining the results of the two remaining typing methods, PFGE and MLVA, eighteen genotypes were obtained of which one genotype predominated (56% of the positive farms). All MRSA isolates were resistant to tetracycline. Resistance to trimethoprim, aminoglycosides, macrolides, lincosamides, fluoroquinolones and chloramphenicol was also observed. In conclusion, there was no effect of the farm type on the MRSA status of the pigs. A statistically significant difference was observed when comparing the pig/poultry or the pig/cattle MRSA status on the multispecies farms. Additionally, a wide variety of MRSA ST398 strains was found within certain farms when combining different typing methods.

Screening of poultry-pig farms for methicillin-resistant Staphylococcus aureus: sampling methodology and within herd prevalence in broiler flocks and pigs

Many reports described the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in different livestock animals from one-species farms. However, in no published reports the prevalence on mixed poultry-pig farms was mentioned, nor the possible relation in MRSA colonization between those two species on one farm, and the possible role of the farmer in the dissemination of MRSA between those two species. Furthermore, no data is available on the optimal sampling site to detect MRSA in broilers. Therefore this study aimed to determine the most suitable sample location in broiler chickens for MRSA and the within flock prevalence of MRSA in various broiler flocks and compared this with the MRSA prevalence in pigs, the colonization of the farmer and the contamination in the barn environment in three mixed poultry-pig farms. MRSA was most frequently isolated from the cloaca and nose shell and to a lesser extent from the skin beneath the wing and the pharynx. The relative sensitivity of the different anatomical sites was, 44.4% for the cloaca, 33.3% for the nose shell, 16.7% for the skin beneath the wing and 5.6% for the pharynx. Based upon these relative sensitivities combining cloaca and nose shell would increase the chance of MRSA detection. A rather low within flock prevalence of MRSA varying between 0% and 28% was detected in broilers, whereas in pigs on the same farms the within herd prevalence varied between 82% and 92%. No MRSA contamination in the direct barn environment of the broilers was found, this in contrast to the environment of the pigs, indicating a relationship between MRSA prevalence and contamination in the environment. Two farmers were continuously colonized, while the third one was only once. In conclusion, a major difference was seen in MRSA occurrence between broilers and pigs from the same farm. This may suggest that broilers are naturally less susceptible to MRSA ST398 colonization than pigs. Conversely, short production time in broilers, vacancy of the barn environment during one week and the higher frequency of disinfection might also explain the lower prevalence in broilers. The farmer may play an important role in the dissemination of MRSA from pigs to poultry, especially in mixed farms where pigs are highly colonized and may act as a reservoir for MRSA ST398 carriage in humans.

Prevalence and Genetic Diversity of Livestock-Associated Methicillin-Resistant Staphylococcus aureus on Belgian Pork.

Since the first description of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA), a high prevalence was observed in pigs. At present, questions remain about the transmission of LA-MRSA to the general human population through pork. The objectives of the present study were to determine the prevalence of LA-MRSA in Belgian pork and to determine the role of the pork production chain and butcheries in transmission of LA-MRSA to the human population. Pig meat samples (chops, bacon, minced pork, ribs, forelimbs, and ears; n = 137) originating from four butcheries (A through D) were spread plated on ChromID MRSA plates both before and after overnight enrichment culture. Suspect colonies were confirmed using a MRSA-specific triplex PCR assay and a CC398-specific PCR assay. The isolates (n = 147) were further characterized by SCCmec typing, multiple-locus variable number tandem repeat analysis, and antimicrobial susceptibility testing, a selection of isolates were subjected to pulsed-field gel electrophoresis and spa typing. Direct plating revealed a MRSA prevalence of 8%. After enrichment, MRSA was isolated from 98 (72%) of 137 samples of which the majority were from rib, ear, and forelimb. The majority (97%) of obtained isolates belonged to CC398, the main LA-MRSA type. A high level of genetic diversity was noted among the isolates from one butchery. Thirty antimicrobial susceptibility profiles were found; 13 and 9% of the isolates had Cip-Tet-Tri and Gen-Kan-Tet-Tob-Tri profiles, respectively. These results indicate the importance of enrichment for MRSA detection of pork. The observed genetic diversity of the isolates indicated that the pork production chain can be considered a source of multiple MRSA types that could be transmitted to the human population through cross-contaminated meat.

Reduction of Mycobacterium avium ssp. paratuberculosis in colostrum: Development and validation of 2 methods, one based on curdling and one based on centrifugation

The aim of this study was to develop and validate 2 protocols (for use on-farm and at a central location) for the reduction of Mycobacterium avium ssp. paratuberculosis (MAP) in colostrum while preserving beneficial immunoglobulins (IgG). The on-farm protocol was based on curdling of the colostrum, where the IgG remain in the whey and the MAP bacteria are trapped in the curd. First, the colostrum was diluted with water (2 volumes colostrum to 1 volume water) and 2% rennet was added. After incubation (1 h at 32°C), the curd was cut and incubated again, after which whey and curd were separated using a cheesecloth. The curd was removed and milk powder was added to the whey. Approximately 1 log reduction in MAP counts was achieved. A reduction in total proteins and IgG was observed due to initial dilution of the colostrum. After curd formation, more than 95% of the immunoglobulins remained in the whey fraction. The semi-industrial protocol was based on centrifugation, which causes MAP to precipitate, while the IgG remain in the supernatant. This protocol was first developed in the laboratory. The colostrum was diluted with skimmed colostrum (2 volumes colostrum to 1 volume skimmed colostrum), then skimmed and centrifuged (at 15,600 × g for 30 min at room temperature). We observed on average 1.5 log reduction in the MAP counts and a limited reduction in proteins and IgG in the supernatant. To obtain a semi-industrial protocol, dairy pilot appliances were evaluated and the following changes were applied to the protocol: after 2:1 dilution as above, the colostrum was skimmed and subsequently clarified, after which the cream was heat treated and added to the supernatant. To investigate the effect of the colostrum treatment on the nutritional value and palatability of the colostrum and the IgG transfer, an animal experiment was conducted with 24 calves. Six received the dams colostrum, 6 were given untreated purchased colostrum (control), and 2 groups of 6 calves received colostrum treated according to both of the above-mentioned methods. No significant differences were found between the test groups and the dams colostrum group in terms of animal health, IgG uptake in the blood serum, milk, or forage uptake. Two protocols to reduce MAP in colostrum (for use on-farm or at a central location) were developed. Both methods preserve the vital IgG.

Preliminary evaluation of good sampling locations on a pig carcass for livestock-associated MRSA isolation

BackgroundThe presence of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) in livestock animals, especially in pigs, gave rise to concerns of pork being a possible MRSA source to the human population. Monitoring the flow-through of LA-MRSA throughout the meat production chain could be useful. Here, the optimal sampling location for LA-MRSA isolation on pig carcasses was determined.FindingsIn one slaughterhouse, 40 cooled carcass halves from one LA-MRSA-positive herd were sampled on six carcass sites (ham, belly, back, forelimb, sternum and abdominal cavity). The obtained MRSA isolates were characterized using Pulsed Field Gel Electrophoresis. Without enrichment of the samples, no MRSA was isolated from the carcasses. After enrichment, MRSA was isolated from 19 out of 40 (47.5%) carcasses. The forelimb appeared to be the most contaminated part of the carcass (17/19 carcasses). Three pulsotypes were detected and the predominant pulsotype was also the herd pulsotype that was determined in our previous study.ConclusionsThe present study demonstrated that the forelimb is a good sampling location for LA-MRSA. For good determination of LA-MRSA on carcasses, enrichment is needed. Only LA-MRSA was isolated. Moreover, the farm strain was isolated from the carcasses, which indicates that transmission from the primary production throughout the slaughterhouse occurred. The results suggest that good hygiene practices in slaughterhouses are important to reduce the transmission of LA-MRSA to the human population.