Working and living in close contact with domestic animals facilitates bidirectional interspecies transmission of microbiota. Concerns about the importance of animals as sources of antibiotic resistant pathogens have been heightened by the unveiling of healthy livestock as reservoirs of methicillin-resistant Staphylococcus aureus (MRSA) in many parts of the world [1–7]. While most research has focused on the ST398 lineage of livestock associated MRSA that predominates in Europe, several genotypes of S. aureus are adapted to livestock, and their relative prevalence varies geographically and among livestock species [2, 8–10].

In developed countries, approximately 20 to 30% of healthy people harbor S. aureus in the nasal cavity, and nasal colonization is associated with elevated risk of clinical infections [11, 12]. The most recent (2003–2004) national data for the USA estimated 28.6% and 1.5% of the population to harbor S. aureus and MRSA, respectively, in their nasal cavities [13]. S. aureus colonization is heterogeneously distributed across subsets of the population classified as ‘persistent’ (or permanent), ‘intermittent’ or ‘non’ carriers, although the criteria for defining persistent carriage vary [14, 15]. Bacterial, host, microbiome, and other environmental factors may influence the likelihood and duration of nasal colonization of humans with S. aureus [16–18], but detailed longitudinal studies of the dynamics of nasal carriage and bacterial genetic diversity are necessary to better understand this phenomenon [14]. Because some lineages of S. aureus are known to be host-adapted to particular avian or mammalian species [19, 20], and subtle genomic changes can alter host tropism [21], regular exposure of people to S. aureus of animal origin further complicates the poorly understood biology of nasal staphylococcal colonization.

S. aureus is considered part of the normal bacterial flora of pigs [22], and people working with live pigs are at elevated risk of being culture positive for S. aureus and MRSA. Notably, the predominant genotypes detected in humans with animal contact are typically those present in the animal populations with which they have contact [1, 23–29]. Because S. aureus are among the most numerous bacteria in bioaerosols of swine barns [30, 31], discriminating between transient contamination of superficial anatomical sites of people (e.g., upper airways or skin) and sustained colonization is problematic, particularly for workers with regular animal contact. Transient contamination may be the most common outcome in people after short term exposure to MRSA positive swine herds [28, 32, 33]. To date there have only been 2 substantial longitudinal studies of MRSA and S. aureus colonization in occupationally exposed swine workers, both in the Netherlands. A study of 110 farm workers sampled 6 times over a year reported that 38% were ‘persistent nasal carriers’ of MRSA, but the possibility of repeated exposure and recontamination could not be eliminated [34]. A study of 137 swine veterinarians sampled 5 times over a 2 year period classified 13% of subjects to be persistently colonized with MRSA based on consistent molecular typing of isolates [24]. The rather different estimates (38% vs 13%) of persistent carriage reported in these 2 studiesmay be an artifact of the different sampling protocols and/or experimental subjects (farmers vs. veterinarians).

Fundamental questions remain about the capacity for S. aureus lineages disseminated from animals to colonize and cause disease in humans. Veterinarians are likely more informative subjects than farmers for elucidating long term colonization patterns following interspecies exposure as they typically are exposed to multiple herds rather than a single animal population. The goal of this study was to analyze long term patterns of S. aureus (including MRSA) colonization in an intensively sampled cohort of US swine veterinarians.

The specific aims of the study were to describe the frequency and duration of positive S. aureus and MRSA nasal cultures in a cohort of veterinarians having regular contact with varied populations of commercial swine in the USA, and to characterize the genotypes of the isolates detected. The intensity of sampling (monthly for 18 months) was designed to enable more detailed understanding of S. aureus colonization patterns than in previous studies of swine workers.

Two veterinarians withdrew from the study due to emigration or altered work circumstances, leaving a cohort of 66 swine veterinarians who provided monthly nasal swabs. One veterinarian stopped working with swine after 7 months, but completed the sampling protocol and was retained in the study. Compliance with swab submission and survey completion was over 99% for both nasal swab submission (1179/1188) and survey submission (1177/1188). The median interval between sample collection and sample processing was 4 days (IQ range: 3–5 days; range: 1-35 days) and was not associated with the likelihood of culture positivity (p = 0.99).

Overall, S. aureus was detected in 63.7% (757/1188) of monthly nasal swab samples (yielding 1356 S. aureus isolates characterized) and MRSA in 9.5% (113/1188) samples (yielding 213 MRSA isolates characterized). The monthly apparent prevalence of S. aureus ranged from 58% to 82%, while apparent prevalence of MRSA ranged from 6 to 15%, and there was no indication of seasonal or longer term trends in prevalence over the course of the study (Fig. 1). MRSA was detected at least once during the study in 18 (27%) of subjects.

At the individual level, the proportion of positive sampling events ranged from 0% (one veterinarian) to 100% (18 veterinarians) and was clearly bimodally distributed (Fig. 2). In univariate analyses, the likelihood of a culture positive result was negatively associated with the interval between the last contact with pigs and collection of the sample (P = 0.001), and positively associated with the hours of pig contact per week (P = 0.02), but not associated with the number of farms visited (P = 0.09) in the previous week. Multivariable analysis to adjust for repeated observations on the same veterinarians showed the odds or a positive culture were reduced by 33% for samples collected one day or longer after pig contact (OR = 0.67; 95% CI: 0.46–0.96) compared with same day collection. Three subjects reported having a staphylococcal infection during the study (2 MSSA, 1 MRSA), all of which were described as localized and did not lead to hospitalization or time off work. This corresponds with an incidence of 2.5 cases per 1000 person months.

Of the 66 subjects, 31 (47%) were classified as intermittent carriers and 34 (52%) were classified as persistent carriers of S. aureus. Based on consistent detection of a specific spa type at all culture positive samplings, 14 (21%) veterinarians were classified as true persistent carriers (TPC). The TPC group included the veterinarian who ceased working with pigs after 7 months but remained positive with ST398/t034 (methicillin susceptible) for the remaining 10 months. The majority (60%) of MRSA isolations were from 4 PC subjects who were positive for MRSA (all ST398) on at least 15 occasions. One of the PC MRSA subjects, who consistently harbored ST398/t034 MRSA, worked exclusively in one production system where this spa type occurs at high prevalence, and which was used as a MRSA positive control farm in the related swine study [8].

Whole genome sequencing

Comparison of the isolates from 9PC subjects (3 each of sequence types ST398, ST9 and ST5) generally indicated close genetic similarity between the first and last isolates collected. All paired isolates within subjects had 0.01 and 0.2 nucleotide differences per site within ST5 and ST398 respectively (Fig. 3). Pairs of isolates of the same genotype (ST9-t337) showed greater similarity within subjects than between subjects (Fig. 3b).

Overall, the S. aureus isolates were distributed among 27 spa types within 8 MLST sequence types (Table 1). Three sequence types (ST398, ST5, ST9) constituted over 94% of the S. aureus isolates. Over the course of the study, ST398 isolates were detected at least once in 63 (83%) veterinarians; ST5 isolates in 43 (65%) veterinarians, ST9 in 29 (56%) veterinarians; and other MLST types in 8 (12%) veterinarians. The 3 predominant MLST types (ST398, ST5, ST9) were all isolated at least once from 23 (35%) subjects. Within each of these 3 MLST types, a single spa type (t034, t002, and t337, respectively) constituted approximately 70% of isolates. Two of these sequence types, ST398 (80.8%) and ST5 (14.1%), accounted for almost 95% of all MRSA isolates, with remainder being ST8/t008, a common human MRSA variant considered unlikely to be of swine origin.

Sequence type	Spa type	S. aureus (n = 1356)	MRSA (n = 213)
ST398	t034	462 (34.1)	116 (54.5)
	t571	71 (5.2)	0
	t011	63 (4.6)	12 (5.6)
	t337a	20 (1.5)	0
	t3446a	19 (1.4)	0
	t1250	8 (0.6)	0
	t2330	6 (0.4)	44 (20.7)
	t2876	24 (1.8)	0
	t7160	13 (1.0)	0
	t1255	3 (0.2)	0
		650 (51.8)	172 (80.8)
ST5	t002	238 (17.6)	27 (12.7)
	t045	69 (5.1)	0
	t062	19 (1.4)	0
	t242	0	3 (1.4)
	t570	12 (0.9)	0
	t856	3 (0.2)	0
		341 (25.1)	30 (14.1)
ST9	t337a	178 (13.1)	0
	t2498	47 (3.5)	0
	t10494	11 (0.8)	0
	t3446a	9 (0.7)	0
	t1334	3 (0.2)	0
	t1430	2 (0.1)	0
		289 (18.4)	0
ST8	t008	0	11 (5.1)
	t2196	18 (1.3)	0
ST30	t338	4 (0.3)	0
	t363	1 (0.1)	0
ST72	t126	30 (2.2)	0
ST278	t330	22 (1.6)	0
ST2007	t8314	1 (0.1)	0

^aTwo sequence types were identified within t337 and t3446 isolates

The detection patterns of specific spa types over time varied enormously from highly consistent presence of individual spa types in TPC subjects, to very inconsistent patterns with multiple spa types detected in individual veterinarians over time (Fig. 4a, b). More than one spa type was detected in 71 (6%) of the monthly nasal swab samples. It is also notable, that in some of the PC and IC subjects a single spa type was detected over multiple consecutive months, but thereafter other spa types were detected.

In the quantitative comparison of PC and IC veterinarians among 41 AASV members sampled in 2014, PC veterinarians (18 of 22; 82%) were more likely to be nasal culture positive than IC (4 of 19, 21%). Culture positive swabs from PC subjects harbored approximately 2 logs more S. aureus per swab than positive swabs from IC subjects (P = 0.05). Spa typing of isolates from this non-selective procedure yielded the same spa types found at other months for each TPC subject, and the same spa type detected in the most recent positive culture for 2 PC subjects.

The high prevalence of S. aureus recovered from nasal cultures reflects the increased exposure to S. aureus occurring in livestock environments [4, 26, 39]. Although some influence of methodological differences cannot be eliminated, the overall S. aureus prevalence (64%) in swine veterinarians is approximately double that estimated in studies of the overall US population [13] and in other developed countries [11, 12]. However, it is very similar to a 72% prevalence in a study of Dutch swine veterinarians [24]. The prevalence of MRSA (9.5%) was also higher than reported in the US population (1.5%), but was substantially lower than in the Dutch swine veterinarians (44%), which likely reflects the lower prevalence of MRSA in the US swine industry relative to the Netherlands [4, 8, 40].

A substantial majority (>84%) of S. aureus isolates in this study were deemed likely to be of swine origin based on several criteria. A parallel study of 38 US swine farms, sampled by a subset of the veterinarians in the current study, found that three MLST sequence types (ST9, ST398, ST5) constituted over 99% of swine isolates, with spa types t337, t034, and t002 predominating [8]. The same sequence types and spa types were similarly predominant among the veterinarians, generally lacked the IEC genes, and were predominantly (85%) tetracycline resistant [41]. The absence of the IEC genes, together with tetracycline resistance, has been used previously to differentiate isolates of human and animal origin [42, 43] and our data suggest that S. aureus acquired from swine may largely displace S. aureus of human origin in the nasal flora of swine veterinarians. However, given that more than one spa type was often detected in individual samples, and only 2 isolates per sample were categorized, it is possible that carriage of relatively low numbers of human S. aureus by veterinarians went undetected [44], and that animal contact added to, rather than displaced human S. aureus. Regardless, the data suggest that animal exposure alters the composition of the nasal S. aureus populations of swine workers. Any resultant impact on risk of clinical infection will depend on the relative persistence, transmissibility, and pathogenicity of S. aureus of swine origin compared with human adapted variants.

The primary goal of this study was to understand the persistence of S. aureus of swine origin in occupational groups that are in close contact with pigs. Several previous studies of livestock workers have examined this question, with varying outcomes [3–5, 26, 29, 32, 39]. Most studies focused on MRSA alone, and the frequency and duration of sampling has varied widely. A common obstacle to inference has been the inability to differentiate repeated contamination of the nasal mucosa from true persistent colonization [45], particularly for farmers who are repeatedly exposed to the same herd (and S. aureus populations) [39]. We specifically studied veterinarians because they generally visit multiple farms and therefore should be exposed to more diverse S. aureus populations.

Longitudinal studies of nasal carriage of S. aureus in humans typically classify subjects as persistent carriers, intermittent carriers, and non-carriers [14, 46]. It is believed that a subset (usually of the order of 20%) of healthy people are persistently colonized with S. aureus [15, 47], and this is associated with higher risk of S. aureus clinical infections [48, 49]. Both host genetic factors and microbial factors may play a role in determining duration of carriage [16], and it was recently reported that presence of S. lugdunensis in the nose may suppress S. aureus populations [18]. Currently, there is no accepted consensus for defining persistent carriers, and categorization of individuals will be influenced by study design (particularly the frequency and duration of sampling), and the cut-off (carrier index) used to define carriage status [46]. We employed a post-hoc epidemiological approach to establish a cut-off (carriage index >0.8) to define persistent carriage. The same criterion has been employed in previous studies [14, 46]. The bimodal distribution of the frequency of culture positive samples among veterinarians (Fig. 2) suggests that even in environments with high exposure to S. aureus of animal origin, individual host characteristics are likely important determinants of the persistence of colonization. This inference is further supported by the substantially higher numbers of S. aureus recovered from nasal swabs from PC compared with IC veterinarians, which is consistent with quantitative studies performed in humans both with [39] and without [50] known livestock association.

The proportion of persistent carriers (52%, 34/66) was considerably higher, and the proportion of non-carriers (<2%, 1/66) considerably lower, than reported in people without livestock contact [14, 46, 51]. This may reflect unusually frequent exposure to S. aureus that can occur in intensive swine facilities [52]. Very similar rates of apparent persistent carriage of S. aureus were also observed in recent longitudinal studies of swine farmers (52%, [39]) and swine veterinarians (47%, [24]) in the Netherlands, although the sampling protocols and criteria for defining persistence in those studies differed.

The associations observed between culture positivity and time since last pig contact and hours of pig contact in the prior week are consistent with previous studies indicating that occupational exposures to animals often result in transient contamination [28, 32, 53]. Collection of swabs on the same day following pig contact substantially increases the odds of a positive culture, therefore sampling schedules will influence estimates of prevalence. The variability of spa types detected over time in the IC group (Fig. 4b) also attests to multiple exposures of swine veterinarians to diverse S. aureus populations. Although early research of persistent carriage in humans suggested that people were colonized over the long term by a single S. aureus variant, more recent studies indicate that S. aureus variants are often replaced over time [14, 46, 51]. Ritchie (2015) sampled 122 healthy young adults weekly over 13 weeks and described 3 patterns of persistent carriage, being continuous carriage of a single spa type; an abrupt change from one spa type to another; and periods of co-carriage with two spa types. Although ‘strain turnover’ was observed in both intermittent and persistent carriers in that study, the majority (63%) of PC subjects were colonized by a single spa type. We made similar observations in the veterinary cohort where all 3 patterns of PC were observed, and a substantial proportion (41%; 14 of 34 PC) of the PC veterinarians were classified as ‘true persistent colonization’ due to the repeated presence of a single spa type over 18 months. This slightly lower proportion (41% vs. 63%) of TPC could be an artifact of the longer sampling period (18 months vs. 13 weeks) providing more opportunity for strain turnover, or the additional exposure to S. aureus variants that occurs in the livestock environment. Our observations with WGS provided further evidence that true persistent colonization with animal origin of S. aureus can occur in some subjects. The paired isolates from each persistent carrier (18 months apart) had shorter genomic distances than seen between isolates of same spa type but from different individuals. A similar inference was reached in a previous study using whole genome mapping of isolates from 16 Dutch veterinarians [54]. Although it is not possible to eliminate the possibility of repeated reacquisition of the same variant from pigs by veterinarians, this is considered unlikely over an extended period unless they were exposed to homogeneous swine populations harboring few S. aureus variants. However, repeated reacquisition from pigs can be eliminated for one subject in our study who remained culture positive for ST398-t034 MSSA for 9 months after leaving swine practice. Current evidence suggests that a substantial proportion of swine veterinarians become colonized with S. aureus of swine origin for at least 18 months to 2 years [24], and can harbor substantial numbers of these organisms. Furthermore, although several studies indicate that ST398 MRSA are less transmissible among people than MRSA of human origin, transmission from veterinarians to their families, who also may become persistently infected, has been clearly demonstrated [54, 55]. Also, persistent colonization of humans with swine S. aureus isolates may be a mechanism for transmission among herds. This is arguably inconsequential given that S. aureus is part of the normal commensal flora of swine, but could be problematic for efforts to prevent MRSA transmission among herds [56].

The fact that some swine workers harbor substantial numbers of S. aureus with genotypes consistent with swine origin has implications regarding occupational health. However, the human health consequences of livestock associated S. aureus are not well defined, and available information is largely limited to ST398 MRSA. In the USA, to date S. aureus of animal origin appear to have had negligible impact on human health. The reported incidence of S. aureus infections in this study (2.5 per 1000 person months, or 3% per veterinary-year) is similar to that reported in a study of Iowa residents (2.7 per 1000 person months). Furthermore, spa types linked to livestock represented only 1% of S. aureus isolates, and 0.24% of MRSA isolates, from human clinical infections in the pig dense state of Iowa [57]. In contrast, a substantial study in Denmark showed increased likelihood of infection with specific livestock associated MRSA, but not increased MRSA infection risk overall, in people living in pig dense areas [58]. Transmission from swine workers into the broader community is a plausible explanation. However, despite the substantial exposure to S. aureus of swine origin that occurs in intensive production environments, there are remarkably few reports of medically significant infections occurring in swine workers [45, 59]. To date, we are unaware of any studies demonstrating increased risk of clinical S. aureus infections in livestock workers. A recent prospective study of swine farm workers in Holland found that carriage of ST398 MRSA was not associated with elevated risk of infections, healthcare contact, or measures of reduced quality of life [60]. Somewhat surprisingly, the small numbers of fatal human cases of ST398 infection have occurred in people without known livestock contact, but who were generally medically compromised [61]. Even though there are a small number of fatal human cases of ST398 infections, it is important to better understand the interchange of S. aureus between humans and animals and the implications for the transfer of resistance elements.

Swine veterinarians are continually exposed to S. aureus of swine origin when working with pigs. This longitudinal study confirmed that the outcomes of this occupational exposure range from short term contamination of the nasal cavities (reflected in higher prevalence of detection in samples collected soon after animal exposure) to apparent long term colonization (at least 18 months) in some individuals in whom the same spa type was detected repeatedly throughout the study. Exposure to pigs did not lead to prolonged colonization in most subjects, and the higher numbers of S. aureus in PC subjects suggests that unknown host factors may determine the likelihood of prolonged colonization by S. aureus of livestock origin. Although exposure to S. aureus and persistent colonization of swine veterinarians was common, the few reports of clinical S. aureus infections reported were of minimal medical significance.