http://www.plosone.org/article/info:doi/10.1371/journal.pone.

0004258

ST398 - FDA
Background
Recent research has demonstrated that many swine and swine farmers in the Netherlands and Canada are colonized with MRSA. However, no studies to date have investigated carriage of MRSA among swine and swine farmers in the United States (U.S.).

Methods
We sampled the nares of 299 swine and 20 workers from two different production systems in Iowa and Illinois, comprising approximately 87,000 live animals. MRSA isolates were typed by pulsed field gel electrophoresis (PFGE) using SmaI and EagI restriction enzymes, and by multi locus sequence typing (MLST). PCR was used to determine SCCmec type and presence of the pvl gene.

Results
In this pilot study, overall MRSA prevalence in swine was 49% (147/299) and 45% (9/20) in workers. The prevalence of MRSA carriage among production system A's swine varied by age, ranging from 36% (11/30) in adult swine to 100% (60/60) of animals aged 9 and 12 weeks. The prevalence among production system A's workers was 64% (9/14). MRSA was not isolated from production system B's swine or workers. Isolates examined were not typeable by PFGE when SmaI was used, but digestion with EagI revealed that the isolates were clonal and were not related to common human types in Iowa (USA100, USA300, and USA400). MLST documented that the isolates were ST398.

Conclusions
These results show that colonization of swine by MRSA was very common on one swine production system in the midwestern U.S., suggesting that agricultural animals could become an important reservoir for this bacterium. MRSA strain ST398 was the only strain documented on this

farm. Further studies are examining carriage rates on additional farms. Citation: Smith TC, Male MJ, Harper AL, Kroeger JS, Tinkler GP, et al. (2009) Methicillin-Resistant Staphylococcus aureus (MRSA) Strain ST398 Is Present in Midwestern U.S. Swine and Swine Workers. PLoS ONE 4(1): e4258. doi:10.1371/journal.pone.0004258 Editor: Ulrich Dobrindt, University of Wrzburg, Germany

Received: October 9, 2008; Accepted: December 19, 2008; Published: January 23, 2009 Copyright: 2009 Smith et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was funded with departmental startup funds (TCS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction
Staphylococcus aureus is one of the most common and devastating human pathogens [1]. Though approximately a third of the population is colonized with S. aureus [2], [3], colonization by strains of S. aureus that are resistant to methicillin (methicillin-resistant S. aureus, MRSA) is less common. A recent publication estimated that 1.5% of the United States (U.S.) population (~4.1 million persons) is colonized with MRSA [4]. Klevens et al. recently showed that deaths from MRSA infections in the U.S. have eclipsed those from many other infectious diseases, including HIV/AIDS. On the basis of data from several major metropolitan areas in the U.S., these investigators estimated that MRSA caused 94,000 infections and over 18,000 deaths in the U.S. in 2005 [5]. Moreover, MRSA has been found in a variety of animals, including horses [6], [7], cattle [8], dogs, cats [9], and swine [10], [11], [12]. Voss et al. reported that the prevalence of MRSA among pig farmers was >760 times

higher than that among patients admitted to Dutch hospitals [13]. Multi locus sequence typing (MLST) suggested that these MRSA isolates belonged to sequence type 398 (ST398), and had been transmitted from pigs to pig farmers, among pig farmers and their family members, and from the colonized son of a swine veterinarian to a hospital nurse. A subsequent study found that 4.6% of veterinarians and veterinary students were colonized with MRSA compared with a population-based estimate of 1% [14]. Additional studies in swine have shown that isolates obtained from swine and their human caretakers are frequently indistinguishable, suggesting transmission between the two animal species [11], [12]. Indeed, investigations in the Netherlands demonstrated that ST398 now accounts for 20% of all MRSA detected in that country, documenting the importance of considering livestock and other animals when examining the epidemiology of MRSA [15]. However, despite research examining swineassociated MRSA in the Netherlands and Canada [10], [12], currently the prevalence of MRSA in swine or their caretakers is unknown in the U.S. In a rural state such as Iowa, which produces 25% of the swine raised in the U.S., transmission of MRSA on swine farms or in veterinary facilities could complicate efforts to reduce MRSA transmission statewide and beyond. Therefore, we conducted a pilot culture survey to examine the prevalence of MRSA in swine and swine workers in two swine farming production systems in Iowa and Illinois.

Materials and Methods Description of farms and swine sampled

Production system A (PSA) is a conventional commercial confinement operation consisting of a 5200 head breed-to-wean sow farm with multiple age-segregated nurseries, finishing, and wean-to-finish sites scattered throughout northern Illinois and eastern Iowa. Collectively, approximately 60,000 swine are present at any one time. Sows in this herd originated from both Canada (Manitoba, Ontario and Quebec) and the U.S. (Minnesota and Illinois). The crossbred sows are from a major swine genetic supplier. The sow herd is relatively young, having been repopulated in 2006. Samples (n = 210) were taken from swine housed at 7 geographically distinct farms within this closed system. The nares of adult sows and swine ages 9, 12, 15, 18, 21, and 24 weeks were swabbed (30 per age group). Animals were not co-mingled prior to sampling. One sample was eliminated because it became contaminated. Nine weeks after

the first visit, 20 additional cultures were obtained from swine that were initially 12 and 15 weeks old (10 from each age group). Sixteen weeks after the first visit, 20 samples were obtained from randomly selected dampiglet pairs. Production system B (PSB) is also a relatively young herd sow herd comprising approximately 2600 sows at the single sow farm location and 27,000 total animals housed at multiple, age-segregated nursery, finisher and wean-to-finish sites throughout eastern Iowa. The sow farm was populated in 2006 with crossbred females originating solely in the United States (Michigan). The breeding stock females in this herd are also from a major swine genetic supplier, but different than those of PSA. Thirty samples were taken from swine in each of 3 age groups: adult sows, and pigs at 11 and 20 weeks of age (n = 90). Animals were not co-mingled prior to sampling.

Human participants
Human caretakers (n = 20) provided nasal and oropharyngeal swabs. Employees filled out a questionnaire providing demographic data, potential risk factors for MRSA infection, information about contact with swine, and use of personal protective equipment. The institutional review board (IRB) and the institutional animal care and use committee (IACUC) approved the protocols. All human participants gave written informed consent prior to enrollment.

Sample collection and bacterial isolation

One naris from each animal and both nares from caretakers were sampled with sterile swabs. Cultures were done as described previously [12]. Briefly, samples were collected using sterile swabs and inserted into Stuart's medium at 4C for transportation. Samples were inoculated into 2 mL enrichment broth containing 10 g tryptone/L, 75 g NaCl/L, 10 g mannitol/L and 2.5 g yeast extract/L. After 24 h incubation at 35C, a loopful of broth was inoculated onto selective MRSA agar plates (BBL CHROMagar MRSA, Becton, Dickinson and Company). These plates were incubated 2448 hours at 35C and examined for MRSA. Isolates were confirmed to be S. aureus by examining their appearance on Gram stain, and by doing the catalase test, the tube coagulase test and the S. aureus latex agglutination assay (Pastorex Staph-plus, Bio-Rad). Methicillin resistance was confirmed by testing for the presence of penicillin binding protein 2 (PBP2) (MRSA latex agglutination test, Oxoid Ltd., Hants, UK).

MRSA isolates were stored at 80C.

Molecular testing
All human isolates and 15 isolates from swine (representing all age groups) were selected for molecular typing. Pulsed field gel electrophoresis (PFGE) was performed as previously described [16]. Isolates that were non-typeable after SmaI digestion were examined after digestion with EagI. Isolates from this study were compared with the type strains for USA100, USA300, and USA400 [17]. For SCCmec typing and pvl PCR, genomic DNA was extracted using the Wizard Genomic DNA preparation kit (Promega). The multiplex SCCmec PCR included ten primer sets: CIF2 F2/R2, mecI P2/P3, RIF5 F10/R13, dcs F2/R1, mecA P4/P7, kdp F1/R1, SCCmec III J1 F/J1 R, ccrB2 F2/R2, SCCmec V J1 F/J1 R, and ccrC F2/R2 [18]. The presence of pvl was determined by an additional PCR [19]. Multi locus sequence typing (MLST) was performed on a subset of isolates which were identical by PFGE and analyzed as previously described [20]. All molecular procedures employed known positive and negative controls.

Antimicrobial susceptibility testing

All human MRSA isolates and the 15 swine isolates evaluated by molecular typing were tested for antimicrobial susceptibility by the broth dilution method described by the Clinical and Laboratory Standards Institute [21]. Isolates were tested for susceptibility to penicillin, oxacillin, tetracycline, erythromycin, clindamycin, trimethoprim-sulfamethoxazole, quinupristin/dalfopristin, gentamicin, levofloxacin, moxifloxacin, linezolid, daptomycin, vancomycin, and rifampin.

Survey/data analysis
Questionnaire and laboratory data were linked by a unique specimen number. Initially, potential risk factor associations were assessed with Fisher's exact test. Bivariate and multivariate modeling of risk factors were performed by exact logistic regression. A trend in prevalence of MRSA in swine by age group was tested with the Cochran-Armitage trend test. A significance level of 0.05 was used in the analyses. Analyses were performed using SAS software version 9.1 (SAS Institute Inc., Cary, NC).

Results

MRSA prevalence in swine

Nasal swabs were taken from 209 swine representing 7 different age groups at PSA. The overall prevalence of MRSA was 70% (147/209). Figure 1 illustrates the significant decreasing trend in prevalence found with the increase in age group (Cochran-Armitage trend test, p-value <0.01). Swine 15 weeks or younger had higher odds of MRSA colonization (OR: 2.17, 95% confidence interval = 1.6 to infinity) when compared to adult swine.

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Figure 1. Prevalence of MRSA in swine from production system A by age aroup. doi:10.1371/journal.pone.0004258.g001 At a follow-up visit 9 weeks later, 20 of 20 samples obtained from the youngest animals were positive. Twelve of 20 (60%) samples obtained 16 weeks after the first visit from 10 randomly selected piglet-dam pairs were positive. Results were concordant for 4 pairs (in 1 pair both dam and piglet had negative nares cultures; in 3 pairs both animals had positive nares cultures) and discordant for 6 pairs (in 2 pairs the dam's nares culture grew MRSA and the piglet's culture did not; in 4 pairs the piglet's nares culture grew MRSA and the dam's culture did not). Swine from a second production system (PSB) were also tested. Because the prevalence of MRSA carriage was high among younger pigs at PSA, we sampled fewer age groups at PSB. We collected 90 samples from 3 age groups (11 weeks, 20 weeks, and adult). We did not detect MRSA in

any of these swabs.

MRSA prevalence in humans and risk factors for MRSA carriage

Persons working in these swine facilities were invited to participate in the study. PSA employed 18 staff who had contact with swine at the sow farm; 14 (77%) volunteered to provide swabs and respond to a questionnaire. PSB employed 7 staff in contact with swine at the sow farm; 6 (86%) participated in our study. Overall, 9/20 (49%) carried MRSA, all of whom were employed at PSA (9/14 persons sampled at PSA, 64% prevalence). Seven persons were colonized in the nares only and 2 were colonized in both the nares and throat. As all swine and human samples obtained at PSB were negative for MRSA, only PSA was included in the risk factor analyses. Age, gender, use of tobacco products, underlying medical conditions, respiratory illness in the prior 12 months, use of antimicrobial agents in the prior 3 months, exposure to healthcare facilities (including long-term-care facilities), a history of skin and soft tissue infections or of having MRSA in the prior 12 months, duration of employment, the number of swine contacted per day, eating pork products, and exposure to raw pork were not associated with nasal carriage of MRSA (see Table 1). All14 PSA subjects work with breeding swine. Persons who do not obtain blood or other specimens from swine (separate analyses) were at higher risk of carrying MRSA than staff that did do these chores.

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Table 1. Characteristics of Production System A swine workers and MRSA prevalence. doi:10.1371/journal.pone.0004258.t001

Molecular typing
As previously described [22], the isolates from swine and caretakers were not typeable when the DNA was digested with SmaI (Fig. 2a) but were typeable when EagI was used (Fig. 2b). All isolates from swine and from swine workers were closely related by the Tenover criteria [23] and they were distinct from common human strains (USA100, USA300, and USA400, not shown).

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Figure 2. A: PFGE of MRSA isolates from swine and swine workers; DNA digested with SmaI. B: PFGE of MRSA isolates from swine and swine workers; DNA digested with EagI. Lane 1: molecular weight ladder. Lanes 2, 12, 25: NCTC 8325 (control strain). Lanes 311: isolates from swine workers. Lanes 1324: isolates from swine. doi:10.1371/journal.pone.0004258.g002 All isolates were SCCmec type V and pvl-negative (data not shown). MLST analysis of a subset of isolates confirmed that these isolates were ST398.

Antibiotic resistance
All isolates from swine and from humans were resistant to penicillin, oxacillin and tetracycline, and all were susceptible to trimethoprim-

sulfamethoxazole, gentamicin, levofloxacin, moxifloxacin, linezolid, daptomycin, vancomycin, and rifampin. Three of 15 (20%) swine isolates were resistant to erythromycin, 2 (13%) were resistant to quinupristindalfopristin, and 13 (87%) were resistant to clindamycin. None of the 9 human isolates tested were resistant to erythromycin or quinupristindalfopristin, but 1 (11%) was resistant to clindamycin. The unusual pattern of erythromycin susceptibility and clindamycin resistance among 11 of the isolates was confirmed with repeat testing.

Discussion
This study is the first to document MRSA in U.S. swine and swine workers, and to our knowledge, the first to report the presence of ST398 (also reported as non-typeable MRSA, NT-MRSA) [15] in the U.S. Like previous studies in Canada, Denmark, and the Netherlands [11], [12], [24], ST398 was found in both animals and humans, suggesting transmission between the two. The prevalence of MRSA colonization among swine and swine workers was high at one farm system that we examined in the Midwestern U.S., suggesting that agricultural animals could become an important reservoir for this bacterium. Strain ST398 was the only MRSA identified among the swine and swine workers. This strain has been the predominant strain among swine in the Netherlands and Canada. However, Khanna et al. in Canada recently found both ST398 and ST5/USA100 colonizing the nares of swine and swine workers [12]. This difference may indicate that the epidemiology of MRSA on Canadian swine farms is different than on the affected farm system in Iowa and Illinois. On the other hand, the difference may have resulted from differing sampling methodologies. Khanna et al. sampled a small number of humans and swine on 20 farms whereas we took a larger number of samples from a smaller number of farms in two corporate systems. Furthermore, because we did not type all isolates in this pilot study, additional strain types may be present that we did not detect. The rate of MRSA colonization in both humans and swine on the farms in one of the corporate systems in our study was high, suggesting that once MRSA is introduced, it may spread broadly among both swine and their caretakers. Other investigators have postulated that this spread may be facilitated by use of tetracycline in swine farming [10]. The ST398 isolates identified in our study were resistant to tetracycline, and thus, could have been selected by antimicrobial pressure on this farm. However, both production systems that we sampled employ similar protocols for prophylactic and therapeutic use of antimicrobial agents, including

tetracycline. Therefore, our data do not allow us to speculate on the relationship between antimicrobial use and MRSA carriage. In addition to tetracycline resistance, we found an unusual macrolide-lincosamide resistance phenotype among a subset of isolates (erythromycin susceptible, clindamycin resistant), one which is not explained by the commonly-recognized mechanisms of macrolide-lincosamide resistance [25]. At present, we do not know why one farm system had a high prevalence rate of MRSA among its swine and its swine handlers. The two production systems did have several differences. First, they raised different breeds of swine. Second, PSA was an older, more established operation that had approximately twice the number of animals as PSB. Additionally, a portion of the sows at PSA were imported from Canada, while those from PSB originated in Michigan. Canada is the most important exporter of live hogs to the U.S. [26]. Thus, it is possible that ST398 may have been brought into the U.S. via live swine or pork products. However, this study was not designed to identify the source of the MRSA and additional research should further examine this question. In addition, our survey did not help us understand why a high proportion of PSA staff carried MRSA. Most of the potential risk factors examined were not statistically different between the carriers and the non-carriers. We cannot explain the observation that staff who do not obtain blood and other samples from the animals were more likely to be carriers than were staff who obtained such samples. Additional studies in larger populations will be needed to identify risk factors and to assess whether this association is real. Investigators in other countries have documented that ST398 causes infections in humans [11], [13], [15] and Wulf et al. have recently described a hospital-based outbreak in the Netherlands [27]. Iowa ranks first in the nation in swine production, with over 19 million hogs at any time point distributed over more than 10,000 farms [28], [29]. Therefore, one would expect that Iowa would be a good state in which to assess the prevalence of infections caused by ST398 among humans. None of the swine workers in this small study reported prior MRSA infections. In addition, we have not identified this strain among the hundreds of human MRSA isolates examined in several ongoing studies of MRSA (including invasive infections) in Iowa [30], [31]. Our study had several limitations. We demonstrated that MRSA can remain in a population of swine for up to 6 months. However, we did not

re-test the same animals over time. Thus, we cannot comment about duration of carriage in particular animals and we could not determine whether the lower rate of colonization in older animals observed at PSA was a true difference related to biological mechanisms or an incidental finding. The latter observation contrasts with prior research that found no significant difference in the rates of MRSA carriage by age group [12]. In addition, we did not evaluate whether the environment was contaminated and could have been a source of transmission for swine or for humans, or whether transmission occurred through direct contact with a colonized animal or human. Moreover, we studied only 9 farms in 2 production systems. Thus, our results may not be generalizable to other swine farms in Iowa and Illinois or to other areas of the U.S. In summary, we report the first isolation of MRSA from swine and swine workers in the U.S. Although the extent of this problem in the U.S. is currently unknown, our findings may have important implications for the epidemiology of MRSA disease. For example, Van Loo et al. identified MRSA in meat products in the Netherlands [32], suggesting that persons who handle raw pork products might be at risk for acquiring MRSA. Future studies should assess the risk of MRSA disease among swine workers and their contacts, survey retail meat products for MRSA contamination, study larger populations of swine and humans to define the epidemiology of MRSA within swine operations, and assess MRSA carriage rates in other livestock.

Acknowledgments
We thank the production systems that allowed us to study their swine and employees. Portions of this research were presented at the 2008 International Conference for Emerging Infectious Diseases and the 2008 American Society for Microbiology General Meeting (abstract Y-036). The authors report no conflicts of interest. Research was approved by the University of Iowa Institutional Review Board (#200802740) and University Animal Care and Use Committee (#0804064).

Author Contributions
Conceived and designed the experiments: TCS MJM LAH DJD. Performed the experiments: TCS MJM ALH JSK GPT EDMK. Analyzed the data: TCS AC. Contributed reagents/materials/analysis tools: TCS DJD. Wrote the paper: TCS MJM LAH DJD. Critically revised the manuscript: TCS MJM JSK GPT EDMK LAH DJD.

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36.

http://www.ncbi.nlm.nih.gov/pubmed/22375071

Identification of a highly transmissible animalindependent Staphylococcus aureus ST398 clone with distinct genomic and cell adhesion properties.
Uhlemann AC, Porcella SF, Trivedi S, Sullivan SB, Hafer C, Kennedy AD, Barbian KD, McCarthy AJ, Street C, Hirschberg DL, Lipkin WI, Lindsay JA, DeLeo FR, Lowy FD. Source

Department of Medicine, Division of Infectious Diseases, College of Physicians and Surgeons, Columbia University, New York, New York, USA. au2110@columbia.edu

Abstract
A methicillin-resistant Staphylococcus aureus (MRSA) clone known as ST398 has emerged as a major cause of acute infections in individuals who have close contact with livestock. More recently, the emergence of an animal-independent ST398 methicillin-sensitive S. aureus (MSSA) clone has been documented in several countries. However, the limited surveillance of MSSA has precluded an accurate assessment of the global spread of ST398 and its clinical relevance. Here we provide evidence that ST398 is a frequent source of MSSA infections in northern Manhattan and is readily transmitted between individuals in households. This contrasts with the limited transmissibility of livestock-associated ST398 (LA-ST398) MRSA strains between humans. Our whole-genome sequence analysis revealed that the chromosome of the human-associated ST398 MSSA clone is smaller than that of the LA-ST398 MRSA reference strain S0385, due mainly to fewer mobile genetic elements (MGEs). In contrast, human ST398 MSSA isolates harbored the prophage 3 and the human-specific immune evasion cluster (IEC) genes chp and scn. While most of the core genome was conserved between the human ST398 MSSA clone and S0385, these strains differed substantially in their repertoire and composition of intact adhesion genes. These genetic changes were associated with significantly enhanced adhesion of human ST398 MSSA isolates to human skin keratinocytes and keratin. We propose that the human ST398 MSSA clone can spread independent of animal contact using an optimized repertoire of MGEs and adhesion molecules adapted to transmission among humans. IMPORTANCE: Staphylococcus aureus strains have generally been considered to be species specific. However, cross-species transfers of S. aureus clones, such as ST398 methicillin-resistant S. aureus (MRSA), from swine to humans have been reported. Recently, we observed the emergence of ST398 methicillin-susceptible S. aureus (MSSA) as a colonizing strain of humans in northern Manhattan. Here we report that ST398 is a frequent cause of MSSA infections in this urban setting. The ST398 MSSA clone was readily transmitted within households, independent of animal contact. We discovered that human ST398 MSSA genomes were smaller than that of the LA-ST398 strain S0385 due to fewer mobile genetic elements. Human and LA-ST398 strains also differed in their composition of adhesion genes and their ability to bind to human skin keratinocytes, providing a potential mechanism of S. aureus host adaptation. Our findings illustrate the importance of implementing molecular surveillance of MSSA

given the evidence for the rapid and clinically undetected spread of ST398 MSSA.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3294494/

Colonization and Transmission of MethicillinResistant Staphylococcus aureus ST398 in Nursery Piglets

Florence Cromb, a,b Wannes Vanderhaeghen,a,b Jeroen Dewulf,c Katleen Hermans,b Freddy Haesebrouck,b and Patrick Butayea,b
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ABSTRACT
A transmission experiment was performed to evaluate the spread of methicillin-resistant Staphylococcus aureus (MRSA) ST398 in nursery piglets. Reproduction ratios (R0) in three experimental groups were found to vary between 3.92 and 52.54, indicating that after introduction, MRSA ST398 will spread easily among weaned piglets, with a tendency to become established.
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TEXT
Methicillin-resistant Staphylococcus aureus (MRSA) ST398 has been reported worldwide to prevail in livestock, on retail meat, and in people in close contact with affected animals (2, 7, 9, 12, 18, 19, 21). To gain a better understanding, a number of recent studies have focused on the colonization and transmission dynamics of MRSA ST398 in pigs, in the field as well as under experimental conditions (1, 4, 16, 17, 24). However, so far, quantitative data on the progression of MRSA colonization in a group of susceptible animals are quite sparse. Furthermore,

various methods have been applied in previous experimental studies, all bearing certain limitations. For example, in a recent study from Denmark, the MRSA status of the 2-week-old animals was investigated only once a week by nasal sampling, hampering a detailed analysis of the transmission dynamics (17). In another recent study from the Netherlands, Broens et al. (4) found that nasal inoculation of piglets with MRSA did not result in stable colonization. For that reason, the researchers had to resort to an oral inoculation model which led to the development of lethal necrotizing pneumonia in 80% of their inoculated animals. In this paper, we report on the successful use of a combined nasal-skin inoculation model for the colonization of weaned piglets without the use of antimicrobials. Furthermore, we quantified the spread of MRSA ST398 among nursery piglets by estimating the basic reproduction ratio (R0). By additionally investigating environmental contamination and the presence of methicillin-susceptible Staphylococcus aureus (MSSA) on the piglets, we aimed at gaining more insight into the transmission and colonization dynamics of MRSA ST398. Thirty-one 3-week-old cross-bred piglets (Landrace by Pitrain, Malves-St. Marie, Belgium) from four different sows were obtained from a MRSA-free herd. The MRSA-negative status of all piglets was confirmed upon arrival at the experimental site and once more 4 days before inoculation, as described below. The animals were equally distributed over three experimental groups (13) of eight piglets and one negative control group of seven piglets. After 1 week of acclimatization, at 28 days of age, two piglets (seeder piglets) from each experimental group were randomly selected for inoculation with a suspension of MRSA strain C26 (ST398, spa type t011, SCCmec type V). To achieve this suspension, overnight brain heart infusion (BHI) broth (BioRad, France) cultures were diluted 1:250, grown to exponential phase, and centrifuged (2,167 g, room temperature, 10 min). After being washed twice with 1 phosphate-buffered saline (PBS), pH 7.4 (Invitrogen, Belgium), the bacterial pellet was resuspended in PBS, pH 7.4, and adjusted to an optical density at

600 nm (OD600) of 0.5, corresponding to a concentration of ~3 108 CFU/ml. Inoculation was done using a sterile 1-ml syringe (Becton Dickinson, Belgium) by administering 1 ml (~3 108 CFU) of the MRSA suspension in both nares and 0.5 ml (~1.5 108 CFU) on two spots of the skin behind the ears, for a total of ~9 108 CFU. To ensure that the correct amounts of CFU were applied, the concentration of the inoculum was checked afterwards by a serial dilution on 5% sheep blood agar plates (Bio-Rad, Belgium). Similar to the seeders, five piglets of the negative-control group were inoculated with sterile PBS at pH 7.4. The inoculation day was set as day 0 postinoculation (0 DPI). Two DPI, the seeders were returned to their pens. Transmission was monitored during a period of 6 weeks, corresponding to the nursery period mostly applied under field conditions in Western Europe (15). Swab samples were collected separately from both anterior nares, the skin behind the ears, and the perineum, using a moistened sterile swab. Environmental contamination was examined once a week by streaking 10 cm2 of the wall (piglet height) and the feeder (floor height) with a sterile moistened sponge. After euthanasia (43 DPI), samples were taken from the throat. This study was approved by the Ethical Committee for Animal Experiments of the Veterinary and Agrochemical Research center (VAR), Brussels, Belgium. All samples were processed within 24 h. Swab samples were inoculated in 3 ml of Mueller-Hinton broth (MH) (Oxoid, Germany) supplemented with 6.5% NaCl, while sponges were inoculated in 50 ml of MH supplemented with 6.5% NaCl. Both swabs and sponges were incubated aerobically for 24 h at 37C. Then 1 l of each broth was inoculated on ChromID MRSA plates (bioMrieux, France) and incubated aerobically at 37C for 48 h. Swabs taken 4, 2 (nostril samples only), 14, 22, 34, and 43 DPI were also investigated for the presence of MSSA, using ChromID S. aureus plates (bioMrieux, France). Suspected MRSA and MSSA colonies were purified on Columbia agar plates with 5% sheep blood (Bio-Rad, Belgium). From purified isolates, DNA

was extracted as previously described (20), and both MRSA and MSSA were identified by multiplex PCR using the method of Maes et al. (14). To certify the isolates' characteristics, one MRSA strain originating from one randomly selected animal of each group per sampling occasion was characterized by spa typing (10). From each MSSA-positive piglet on 4 and 43 DPI, one strain was likewise characterized. Additionally, MRSA and/or MSSA strains isolated from the environmental samples on 4 and 42 DPI were characterized by spa typing (10). Multilocus sequence typing (MLST) was effectuated on one MSSA isolate of each spa type (8). Quantification of transmission of MRSA was done using a stochastic infection model as described by Velthuis et al. (22). Briefly, transmission among piglets was assumed to be in accordance with the susceptible-infectious-susceptible (SIS) model. In this model, it is assumed that animals can carry MRSA intermittently. An animal was classified as infectious when MRSA was detected on at least one of the three sampling sites and as susceptible when MRSA was not detected. Given that the population (N) is composed of susceptible animals (S) and infectious animals (I), two events can occur: an infection event, (S, I) (S 1, I + 1), and a recovery event without immunity, (S + 1, I 1). The rate at which an infection event occurs depends on the density of S animals, the number of I animals, and the transmission parameter (i.e., the number of cases affected by I animals during every period out of the number of S animals at the start of the period). The rate at which a recovery event occurs depends on the number of I animals and the recovery parameter (i.e., the number of recovered animals per unit of time) (22, 23). From these expression results, the average number of secondary cases caused by one infectious individual in a totally susceptible population, termed the reproduction ratio (R0), equals / (23). Consequently, R0 can be assessed after estimating these transmission parameters. This was done separately for the three experimental groups.

We estimated the transmission parameter using a generalized linear model (GLM) with a complementary log-log link function and log I/N as the offset variable, as described by Velthuis et al. (22). The duration of the infectious period (1/) was defined as the average number of days at which each animal was classified as infectious during the experiment. As depicted in Fig. 1, highly efficient transmission of MRSA strain C26 from seeders to contact animals was observed. In all three experimental groups, all contact animals were MRSA positive on the first sampling occasion (4 DPI), 2 days after the introduction of the seeders with the contact animals. In contrast, all animals of the negative control group remained MRSA negative during the whole experiment. The corresponding R0 values were all significantly higher than 1 (Table 1). In general, an R0 score above 1 means that a primary case generates more than one secondary case and that the agent spreads in the population (6). Consequently, when our results are extrapolated to a field situation, a single introduction of a MRSA ST398positive piglet in a susceptible population of weaned piglets is likely to cause an infection of multiple animals. In other words, our results prove that MRSA ST398, or at least the strain we used (spa type t011, SCCmec type V), has high spreading potential in weaned piglets. Recently, two other studies have investigated the transmission potential of MRSA ST398 in piglets (4, 17). Even though different methodologies were used in these studies, efficient transmission and persistence of MRSA ST398 among the animals were also described, illustrating the robustness of this observation.

Fig 1 MRSA detection status of the piglets during the time of the experiment (expressed in days postinoculation [DPI]). (A) Distribution of

MRSA-positive (MRSA+) seeder animals (n = 2/group). (B) Distribution of MRSA-positive contact animals (n = 6/group) and ...

Table 1 Estimation of transmission parameters by the generalized linear model methoda An important issue to consider is the dose required for successful transmission of MRSA ST398 to susceptible animals. In this study, the dose used to inoculate the seeders was relatively high (~9 108 CFU), which possibly resulted in a higher level of infectiousness of the seeders, i.e., a higher number of bacteria transmitted than the number of bacteria transmitted under field conditions (23). Still, preliminary data showed that such a high dose was necessary to achieve colonization (unpublished data). In this study, the amount of bacteria present on the animals was not quantified, rendering it unclear whether the contact animals were carrying doses similar to those of the seeders or whether the animals were only transiently contaminated. However, it must be noted that semiquantitative approaches have already shown a high variability in MRSA counts in naturally colonized animals (17). In addition, one must consider that quantification techniques will always be hampered by the sampling procedure as well as by the sensitivity of the isolation method. At this time, well-defined criteria to describe true colonization with S. aureus in animals are absent. Numerous studies have reported MRSA ST398 in pigs or other animals and in their caretakers, but without indicating whether all the positive individuals were truly colonized. It appears that a MRSA-positive animal is not the sole source for contamination of susceptible pen mates (11). Our study suggests that the environment may also play a role in the spread of MRSA ST398. Indeed, after introduction of the seeders, the

environmental samples from all three experimental groups were shown to harbor the inoculated strain (Table 2). This means that besides the seeders (and their secondary cases) the environment can start to function as a source of MRSA supply. Hence, our study suggests that a pen housing MRSA-negative piglets will, upon introduction of a MRSA-positive animal, become a dynamic system of interacting reservoirs, regardless of whether the entities of this system are truly colonized or not. Even if certain entities in this system, such as an individual animal, lose the MRSA ST398 strain for a while, other sources will be present to resupply it. In this respect, it is not hard to imagine that the farmer or other contact animals, such as dogs or rats, might act as vectors to spread MRSA ST398 through different parts of the farm, expanding the dynamic system from a single pen to the entire farm.

Environmental contamination of the pens used in the transmission trialOne might question what causes the animals to be MRSA negative on certain occasions. First of all, the MRSA population on a specific animal might have simply diminished and ultimately vanished because the animal was not truly colonized. However, investigation of the postmortem samples showed that MRSA was present in the throats of two contact animals of group 1 and all contact animals of group 3, as well as in five out of the six seeders (data not shown), suggesting that at least these animals were stably colonized. Hence, other possibilities also need to be considered. For example, the MRSA strain might still have been present but in a quantity below the detection threshold of the sampling and/or isolation method. Another factor that might have played a role is the presence of other bacteria in the collected samples. This will have differed for

different animals and on different occasions. It has been reported that some bacteria might inhibit in vitro growth of MRSA, resulting in false-negative results (3). Of particular interest in this respect is the indigenous microflora of the individual animals, which could exert an antagonistic effect on stable colonization by MRSA (4, 5, 13, 16, 17). Dall'Antonia et al. (5) illustrated that in humans, MSSA competes with MRSA for colonization of the anterior nares. Interestingly, in our study MSSA strains (spa type t3446 and occasionally spa type t337, MLST ST9) were also isolated, and more frequently, from the animals belonging to the groups where the MRSA status fluctuation was more pronounced (groups 1 and 2) (data not shown). The competitive effect between MSSA (ST9 or other STs) and MRSA is an interesting issue to consider in further studies in order to determine the true impact of interference on colonization status and its usefulness in MRSA eradication programs. In conclusion, this study demonstrates that a combined nasalskin inoculation model results in colonization of weaned piglets with MRSA ST398. In all three groups, the R0 values were shown to be significantly above 1, indicating that MRSA ST398 tends to become established. In this respect, groups of pigs or whole farms might function as dynamic systems with various interacting entities that maintain a MRSA-positive status. Still, further research is needed to gain more insight into the different carrier states of pigs and to investigate the usefulness of interference strains as a tool for eradicating MRSA carriage. We believe that, using our model, additional transmission studies should be performed in order to evaluate the efficacy of various treatments (e.g., medication) and to determine the influence of control strategies on the spread of MRSA ST398.
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ACKNOWLEDGMENTS
Financial support was received from Institute for the Promotion of Innovation by Science and Technology in Flanders (IWT) project 070596.

We thank Willem Van Campe and his team for excellent technical assistance.
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FOOTNOTES
Published ahead of print 22 December 2011

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REFERENCES
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[PubMed] 9. Graveland H, Wagenaar JA, Bergs K, Heesterbeek H, Heederik D. 2011. Persistence of livestock associated MRSA CC398 in humans is dependent on intensity of animal contact. PLoS One 6:e16830. [PMC free article] [PubMed] 10. Harmsen D, et al. 2003. Typing of methicillin-resistant Staphylococcus aureus in a university hospital setting by using novel software for spa repeat determination and database management. J. Clin. Microbiol. 41:54425448. [PMC free article] [PubMed] 11. Hsieh JM, Chen RS, Tsai TY, Pan TM, Chou CC. 2008. Phylogenetic analysis of livestock oxacillin-resistant Staphylococcus aureus. Vet. Microbiol. 126:234242. [PubMed] 12. Lewis HC, et al. 2008. Pigs as source of methicillin-resistant Staphylococcus aureus CC398 infections in humans, Denmark. Emerg. Infect. Dis. 14:13831389. [PMC free article] [PubMed] 13. Lina G, et al. 2003. Bacterial competition for human nasal cavity colonization: role of staphylococcal agr alleles. Appl. Environ. Microbiol. 69:1823. [PMC free article] [PubMed] 14. Maes N, Magdalena J, Rottiers S, De Gheldre Y, Struelens MJ. 2002. Evaluation of a triplex PCR assay to discriminate Staphylococcus aureus from coagulase-negative staphylococci and determine methicillin resistance from blood cultures. J. Clin. Microbiol. 40:15141517. [PMC free article] [PubMed] 15. Meyns T, et al. 2004. Quantification of the spread of Mycoplasma hyopneumoniae in nursery pigs using transmission experiments. Prev. Vet. Med. 66:265275. [PubMed] 16. Moodley A, Latronico F, Guardabassi L. 2011. Experimental colonization of pigs with methicillin-resistant Staphylococcus aureus (MRSA): insights into the colonization and transmission of livestock-associated MRSA. Epidemiol. Infect. 139:15941600. [PubMed] 17. Moodley A, Nielsen SS, Guardabassi L. 2011. Effects of tetracycline and zinc on selection of methicillin-resistant Staphylococcus aureus (MRSA) sequence type 398 in pigs. Vet. Microbiol. doi:10.1016/j.vetmic.2011.05.025. 18. van de Giessen AW, et al. 2009. Occurrence of methicillinresistant Staphylococcus aureus in rats living on pig farms. Prev.

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7625 CAS Dropper Pipet Sufficient for 50 tests at 20 ppm 1 drop = 1.25 ppm HEDP 1 drop = 1.4 ppm PBTC Chromazurol S method may be used for Dequest, Bayhibit, Belcor 575 and Belsperse 161 phosphonates. Indicator changes from yellow to pink at the pH ideal for the reaction, then thorium nitrate is added until the solution turns purple.
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