Livestock-associated Staphylococcus aureus on Polish pig farms

Background Livestock-associated Staphylococcus aureus (LA-SA) draws increasing attention due to its particular ability to colonize farm animals and be transmitted to people, which in turn leads to its spread in the environment. The aim of the study was to determine the dissemination of LA-SA on pig farms selected throughout Poland, characterize the population structure of identified S. aureus, and assess the prevalence of LA-SA carriage amongst farmers and veterinarians being in contact with pigs. Methods and findings The study was conducted on 123 pig farms (89 farrow-to-finish and 34 nucleus herds), located in 15 out of 16 provinces of Poland. Human and pig nasal swabs, as well as dust samples were analyzed. S. aureus was detected on 79 (64.2%) farms from 14 provinces. Amongst these farms LA-SA-positive farms dominated (71/79, 89.9%, 95% CI [81.0%, 95.5%]). The prevalence of LA-MRSA-positive farms was lower than LA-MSSA-positive (36.6% of LA-SA-positive farms, 95% CI [25.5%, 48.9%] vs. 74.6%, 95% CI [62.9%, 84.2%]). In total, 190 S. aureus isolates were identified: 72 (38%) MRSA and 118 (62%) methicillin-susceptible S. aureus (MSSA), of which 174 (92%) isolates were classified to three livestock-associated lineages: CC398 (73%), CC9 (13%), and CC30/ST433 (6%). All CC398 isolates belonged to the animal clade. Four LA-MRSA clones were detected: ST433-IVa(2B) clone (n = 8, 11%), described to the best of our knowledge for the first time, and three ST398 clones (n = 64, 89%) with the most prevalent being ST398-V(5C2&5)c, followed by ST398-V(5C2), and ST398-IVa(2B). Nasal carriage of LA-SA by pig farmers was estimated at 13.2% (38/283), CC398 carriage at 12.7% (36/283) and ST398-MRSA carriage at 3.2% (9/283), whereas by veterinarians at 21.1% (8/38), 18.4% (7/38) and 10.5% (4/38), respectively. Conclusions The prevalence of LA-MRSA-positive pig farms in Poland has increased considerably since 2008, when the first MRSA EU baseline survey was conducted in Europe. On Polish pig farms CC398 of the animal clade predominates, this being also reflected in the prevalence of CC398 nasal carriage in farmers and veterinarians. However, finding a new ST433-IVa(2B) clone provides evidence for the continuing evolution of LA-MRSA and argues for further monitoring of S. aureus in farm animals.

to contribute in the study. As a consequence, in the period from August 2010 to November 2012 a cross-sectional study was conducted on 123 pig farms (89 farrow-to-finish and 34 nucleus herds), located in 15 out of 16 provinces of Poland. The number of farms selected was proportional to the headage of pig per 100 ha in a particular province. The study did not include the Lower Silesia Province, where pig production is amongst the lowest in the country. The size of the farrow-to-finish herds ranged from ten to 2000 sows (median 80) and of the nucleus herds from nine to 1000 (median 54). Sixty-four (52%) farms had their own pig production, while 59 (48%) purchased gilts and/or boars from other sources. Thirty-nine (32%) farms obeyed the rules of good management applying the All-In-All-Out (AIAO) procedure and 57 (46%) farms did not. For 27 (22%) of farms no data were obtained. In the AIAO procedure the animals are kept together in groups. The groups are closely matched by age, weight, production stage and condition. The animals are moved into and out of facilities in groups. The facilities are cleaned and thoroughly disinfected between groups of animals. Most of the farms weaned their piglets between 21-49 days of life, with an average of 28 days.

Sampling procedure
There were three sources of material: human and pig nasal swabs and environmental dust samples. From each farm, both pig nasal swabs and dust samples were collected during one-off screening (one visit at the farm) in three different sectors: farrowing unit (sows), nursery and fattening unit. Five nasal swabs from five healthy pigs and five environmental dust samples were collected from each sector. Dust samples were taken mainly from pens and ventilator ducts. Whenever possible, five pigs from each age group were selected using a convenience sampling scheme that avoided sampling more than one pig in co-mingled groups. If all three age groups were not present on the farm, pigs from the available groups were sampled.
Nasal swabs were collected at the same time, from consenting owners of the farms, individual workers taking care of animals (hereinafter all referred to as"pig farmers") and from veterinarians supervising pig farms. The vets working on several farms were screened only once. The number of nasal swabs collected from farm personnel depended on the number of workers employed at each farm and individual agreement to be sampled given by every person.
One swab per volunteer on each farm was taken from both nares personally after swabbing training. In total, 321 human nasal swabs (283 from pig farmers and 38 from vets), 1845 pig nasal swabs and 1845 dust samples were collected. All samples were stored at 4˚C and passed on directly to the laboratory. Nasal cotton swabs were transported in Amies medium (Medlab Products, Raszyn, Poland).
At the time of the project application and realization ethical approval was not required for taking nasal swabs according to local and national regulations (the project was reviewed and accepted by the Ministry of Science and Higher Education, Poland and additional approval was not recquired). All human participants signed an informed consent form. Collected data were anonymized before analysis at the National Veterinary Research Institute, Puławy.

Bacterial isolates
Material from five pig nasal swabs, collected in each farm sector, were pooled into one sample, placed in a 100 ml Mueller-Hinton Broth (BD BBL, France) supplemented with 6.5% NaCl and incubated at 37˚C for 16-20 h. The same was done with dust swabs. In total, 738 cultures were set up (369 from pigs and 369 from dust samples). One milliliter of each bacterial culture was inoculated into 9 ml Trypticase Soy Broth (BD BBL, France) supplemented with aztreonam (75 mg/l) and incubated for a further 16-20 h at 37˚C. One loop-full of each positive culture, as well as nasal swabs from humans, were spread at the same time onto Chapman agar (bioMérieux, France) and chromogenic agar selective for S. aureus species (SAID, bioMérieux, France) and incubated for 24-48 h at 37˚C. Grown colonies were subcultured on 5% sheep blood agar (BD BBL, France). Bacteria were stored at -80˚C for further identification.
Species identification was based on phenotypic criteria including colony morphology, and both CF and coagulase tests. Additionally Microgen TM Staph (Microgen Bioproducts Limited, Camberley, Surrey, UK) rapid confirmatory latex agglutination test for S. aureus was used.
All S. aureus isolates were tested for methicillin resistance by using the cefoxitin-disc diffusion method. The analysis was performed according to procedure and breakpoints proposed by EUCAST recommendations [21] with S. aureus ATCC29213 as the reference strain. Isolates were also screened by PCR for the presence of the mecA and the mecC gene as described earlier [22,23].

Total DNA preparation
Total DNA of the S. aureus isolates was purified using Genomic Mini DNA kit (A&A Biotechnology, Gdynia, Poland) according to the manufacturer's instructions.

Genetic background determination
Initially, S. aureus isolates were screened by PCR for identification of CC398 [24] and in all accessory gene regulator (agr) allotypes were determined [25]. Amongst CC398 isolates discrimination between human and animal clade was performed [26].
spa typing was performed [27] and spa-types were determined by using the Ridom Staph-Type software v.2.1.1 (Ridom GmbH) [28]. The BURP algorithm was used to assign spa-types into spa-clonal complexes (spa-CCs) with defined default parameters "exclude spa-types shorter than 5 repeats" and "cluster spa-types into the same group if cost distances are less than 4" [29].
Multilocus Sequence Typing (MLST) was performed on selected isolates, 36 MRSA and 62 MSSA [30]. At least one isolate per each spa-type detected in each source of material was analyzed. The sequence type (ST) was also determined for three non-spa-typeable isolates. Allele numbers and STs were assigned through the S. aureus MLST database (http://saureus.mlst. net).
Clonal complexes, CCs, were determined using the eBURSTv3 algorithm (http://saureus. mlst.net/eburst) and restricted to STs that share five or more alleles of seven loci examined with founder or subfounders, within each predicted clonal group [31]. The analysis was performed on April 29 th , 2016.

SCCmec typing
SCCmec types and subtypes were determined [32,33], and those of type V were analyzed by additional strategies [34,35]. The presence of the czrC gene encoding cadmium and zinc resistance was detected [36]. The SCCmec elements were classified according to guidelines proposed by the International Working Group on the Classification of SCC elements [37]. Direct repeat unit (dru) VNTR regions associated with SCCmec elements were determined [38] and dru-types were assigned through dru-typing database (http://dru-typing.org). ACME and toxin genes profiling. Detection of the arginine catabolic mobile element (ACME) was performed in duplex PCR with primers AIPS.29 and AIPS.28 (locus arc) and AIPS.45 and AIPS.46 (locus opp3) [39].

Detection of prophage of integrase type 3 (φSaint3) and IEC types classification.
The presence of φSaint3 was determined by detection of the prophage integrase type 3 (int3) gene [44]. The composition of sak, chp, and scn genes from the human-specific Immune Evasion Cluster (IEC), carried on φSaint3, was determined by PCR and IEC types were classified [45].

Statistical analysis
Exact (permutation) chi-square tests were used to detect differences between frequencies of S. aureus types in farm/sample categories, and interval estimates for proportions and odds ratios were found by exact methods using SAS 9.4 (SAS Institute, 2014) [46].
Genetic diversity, on the grounds of spa-types, was estimated using Simpson's index of diversity (SID) with 95% confidence intervals [47,48].

Definitions
Farms were defined as S. aureus-positive, when at least one S. aureus isolate was identified in a farm sample from pig farmer, pig or environmental dust. Equivalent criteria were applied for defining of LA-SA-positive farms. Isolates were classified as LA-SA when they belonged to livestock-associated CCs (LA-CCs): CC398, CC9 or CC30/ST433.
MRSA clones were defined based on ST and SCCmec type/subtype association [30].
No differences were demonstrated in distribution of LA-SA-positive farms throughout the country but, due to modest sample size their existence cannot be excluded (wide confidence intervals for odds ratios, not shown). Regarding LA-SA-negative farms, which encompassed 35.8% (44/123) of all farms studied, we did not notice any differences concerning geographic location, type of farm, size of herd or application of AIAO procedure, compared with LA-SApositive ones.

Characteristics of S. aureus isolates from LA-CCs
On 10 farms ST398 isolates were identified in both pig farmers and non-human reservoirs. The isolates from the same farm shared their characteristics (spa-type, SCCmec-type and drutype in case of MRSA or spa-type in case of MSSA).
CC9, the second largest CC, was detected on 13 (10.6%) farms from seven provinces and embraced only MSSA isolates. Seven spa-types were identified, with the predominant types: t1430 (36.0%) and, observed exclusively in pigs, t337 (28.0%). All spa-types but one were grouped into one spa-CC 1334.
CC30/ST433 genetic lineage was distinguished within CC30, the third numerous CC in this study. MRSA and MSSA isolates of CC30/ST433, were detected on two and three farms, respectively, each from a different province. Isolates were assigned to three spa-types clustered into one spa-CC 021, with the most prevalent being t318. All MRSA isolates belonged to a new clone ST433-IVa(2B).

Discussion
The epidemiology of S. aureus in Western and Central Europe has changed significantly in the past decade due to the emergence of genetic lineages adapted to livestock animals, especially to pigs. Here we present the results of the first structured survey conducted on pig farms in Poland, which partially fill the gap concerning the prevalence and molecular characteristics of livestock-associated S. aureus, both MRSA and MSSA, in the central and eastern EU countries.
In 2008, when the first MRSA EU baseline survey on dust samples from pig holdings was conducted in Europe, Poland was classified amongst countries with the lowest occurrence of MRSA-positive farms [10]. Since that time, as our study shows, the prevalence of LA-MRSApositive farms has increased considerably, from 2.1% to 20.6% in breeding (nucleus) holdings (95% CI for Odds Ratio [2.6, 77]), and from 3.4% to 13.5% in production (farrow-to-finish) holdings (95% CI for OR [1.48, 14.9]). However, it still remains lower than those observed in

CC (n)
agr ST spa-type SCCmec-type dru-type SEs genes Humans a n = 13(4) Pigs n = 31 Dust n = 28 sea, seh, sek, seq 2008 on pig farms in Belgium, Germany, or Spain [10]. One valid explanation of the observed higher percentage of LA-MRSA-positive farms may be the increase in imports of piglets, over the past years to Poland, from countries like Denmark, Germany and the Netherlands [49], where the rates of LA-MRSA colonized pigs are high [14,50]. The lower prevalence of LA-MRSA-positive than LA-MSSA-positive farms, observed in our study, may arise from the fact that one-fourth of LA-SA-positive farms have been classified based on S. aureus isolates detected exclusively in samples from pig farmers, where the occurrence of LA-MSSA is higher than LA-MRSA.
The nasal carriage of ST398-MRSA by farmers exposed to pigs in this study was low (3.2%) compared with rates in similar populations from other countries, ranging from about 20% in Canada and USA [5,7] to over 80% in Belgium, Denmark and Germany [12,51]. Concurrently, CC398-MRSA carriage amongst Polish pig farmers was three times lower than CC398-MSSA. The reason for this remains unclear, however, it was earlier observed in Germany that the frequency of transfer events of CC398-MSSA amongst non-exposed to pigs people living on pig farms was twice as high as for CC398-MRSA [12]. Our results may support these findings and/or may suggest that within the CC398 lineage MSSA strains are better adopted to humans than MRSA. However, since our findings are contrary to results described by Denis et al., who showed considerably higher nasal carriage of ST398-MRSA than ST398-MSSA in pig farmers from Belgium [52], only further studies could clarify this issue.
Regarding the prevalence of ST398-MRSA amongst Polish veterinarians (10.5%), it seems to be on a moderate level, comparable with that reported in Belgian livestock veterinarians (7.5%), but lower than observed in veterinarians exposed to pigs in Germany, estimated at 45% [12,53].
Three CCs, CC398, CC9, and CC30/ST433, have been identified as the most frequent, detected on over half of the pig farms studied. All these CCs are presently recognized as the main pig-associated ones worldwide. In Western and Central Europe the predominant CC398 comprise both MRSA and MSSA, while the other two CCs are generally represented by MSSA strains [54][55][56].
LA-SA isolates analyzed in the current study shared features typical for pig-associated strains, like the absence of toxin genes such as exfoliative toxins, toxic-shock syndrome 1 toxin as well as Panton-Valentine leukocidin genes [57,58]. With the one exception, CC398-MSSA-t1928 from human, they carried neither φSaint3 prophage nor the IEC genes, which is also characteristic for isolates adapted to animals [59][60][61]. All CC398 isolates analyzed belonged to the animal clade and the majority of them represented three classical, in Western and Central Europe, pig-borne spa-types, t011, t034 and t108 [14,[62][63][64][65][66]. However, the distribution of major spa-types varied between MRSA and MSSA. While in the MRSA population two predominant spa-types, t011 and t034, occurred at similar levels, amongst MSSA exclusively t034 dominated. This observation is in contrast to other reports, where the spa-type distribution of CC398-MSSA mirrored the most frequently detected spa-types for CC398-MRSA, like t011 and t034 in Belgium [56] or t034 in Denmark [54,67].
Even though almost 90% of LA-MRSA isolates characterized in this study were assigned to CC398, the remaining MRSA, from CC30/ST433 genetic background should deserve particular attention. These MRSA isolates belonged to clone ST433-t318-IVa, that to the best of our knowledge is described here for the first time and may constitute a new LA-MRSA clone of pig origin. The isolates of the emerged clone were identified in non-human samples on two pig farms located in two, non-bordering provinces; one in the northern-eastern (Podlaskie) and second in the southern (Małopolskie) part of Poland. There were no relationships between the two farms and both purchased gilts and/or boars from other sources. Whereas CC30/ST433 is a well established LA-MSSA genetic lineage in Europe for more than 20 years [3,54], MRSA of this lineage had not been observed till 2009. The first and the only one, until now, clone ST433-MRSA, characterized by spa-type t1333 and SCCmecV with czrC gene, conferring cadmium and zinc resistance, was isolated from pigs in Denmark [68], where the ST433-t1333 genotype had been earlier identified as the second most common MSSA genotype amongst pigs [54]. During our study only one ST433-MSSA isolate of spa-type t318 was found, however, this spa-type was the most frequent amongst MSSA from pig nasal swabs collected at a slaughterhouse in South-Western Poland [69]. We do not have evidence that the new clone emerged in Poland, nevertheless we cannot exclude that it arose de novo by the acquisition of the SCCmec IVa element by S. aureus of the genetic background disseminated in our country.
Identification of a new MRSA-ST433-IVa clone provides further proof for the spread of SCCmec elements in pig-associated S. aureus genetic lineage. Additional characterization of CC30/ST433 strains, both MRSA and MSSA, from different geographical regions may be crucial for better understanding the evolution of this lineage and its impact on global epidemiology.
We are aware of some of the limitations of this work. First, the study was extended in time for over two years and some of the farms could have changed their status from MRSA-positive to MRSA-negative and vice versa during the study period. Second, the prevalence of S. aureus isolates from pigs and environmental dust could be underestimated due to the sampling method and the pooling procedure used in the study. Nevertheless, the study allowed to establish a reference point for epidemiological investigations to be held on livestock animals, especially pigs, in the future.
In conclusion, S. aureus from pig farms in Poland belong to well established in Western and Central Europe LA-SA genetic lineages, CC398, CC9 and CC30/ST433. CC398 of the animal clade predominates on Polish pig farms, which is also reflected in the prevalence of CC398 nasal carriage in both farmers and veterinarians, having professional contacts with pigs. However, finding a new CC30/ST433-IVa(2B) clone and increasing the rate of CC398-MRSA during the last five years provides evidence for the continuing evolution and expansion of LA-MRSA in Poland. Further monitoring of S. aureus in farm animals is strongly required due to possible impact on public health.