The effects of antibiotic use on the dynamics of the microbiome and resistome in pigs.

Antibiotics are widely used in pig farming across the world which has led to concerns about the potential impact on human health through the selection of antibiotic resistant pathogenic bacteria. This worry has resulted in the development of a production scheme known as pigs Raised Without Antibiotics (RWA), in which pigs are produced in commercial farms, but are ear-tagged as RWA until slaughter unless they receive treatment, thus allowing the farmer to sell the pigs either as premium priced RWA or as conventional meat. Development of antibiotic resistance in pig farming has been studied in national surveys of antibiotic usage and resistance, as well as in experimental studies of groups of pigs, but not in individual pigs followed longitudinally in a commercial pig farm. In this study, a cohort of RWA designated pigs were sampled at 10 time points from birth until slaughter along with pen-mates treated with antibiotics at the same farm. From these samples, the microbiome, determined using 16S sequencing, and the resistome, as determined using qPCR for 82 resistance genes, was investigated, allowing us to examine the difference between RWA pigs and antibiotic treated pigs. We furthermore included 176 additional pigs from six different RWA farms which were sampled at the slaughterhouse as an endpoint to substantiate the cohort as well as for evaluation of intra-farm variability. The results showed a clear effect of age in both the microbiome and resistome composition from early life up until slaughter. As a function of antibiotic treatment, however, we observed a small but significant divergence between treated and untreated animals in their microbiome composition immediately following treatment, which disappeared before 8 weeks of age. The effect on the resistome was evident and an effect of treatment could still be detected at week 8. In animals sampled at the slaughterhouse, we observed no difference in the microbiome or the resistome as a result of treatment status but did see a strong effect of farm origin. Network analysis of co-occurrence of microbiome and resistome data suggested that some resistance genes may be transferred through mobile genetic elements, so we used Hi-C metagenomics on a subset of samples to investigate this. We conclude that antibiotic treatment has a differential effect on the microbiome vs. the resistome and that although resistance gene load is increased by antibiotic treatment load, this effect disappears before slaughter. More studies are needed to elucidate the optimal way to rear pigs without antibiotics.

Mixed effect modeling of tetracycline resistance levels in Danish slaughter pigs.

Mathematical and statistical modeling can be a very useful tool in understanding and fighting antimicrobial resistance (AMR). Here we present investigations of mixed effect models of varying complexity in order to identify and address possible management factors affecting the tetracycline AMR levels in Danish pig farms. Besides antimicrobial exposure during pigs life cycle, the type of production seems to also have an influence. The results concludes that not only fully integrated farms (CHR integrated) but also farms in a production network with a single ownership (CVR integrated) might have a preventive effect on levels of tetracycline AMR compared to more complex trading patterns.

A dynamic model for spread of livestock-associated methicillin-resistant Staphylococcus aureus on a pig farm, incorporating bacterial load and human exposure through air.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) is widely distributed in the pig population in many countries, where its presence is undesirable, because as an opportunistic human pathogen, it poses a threat to human health. At present, there is a lack of knowledge regarding successful methods for eradication of LA-MRSA on a pig farm, which does not involve emptying the farm and culling all pigs. Some studies have reported an association between levels of LA-MRSA in the barn air and LA-MRSA carriage among humans entering or working in the pig barns. Therefore, interventions that are able to reduce the amount of LA-MRSA carried by the pigs and/or the concentration of LA-MRSA in the barn air, might be highly relevant if aiming for reducing the spread of LA-MRSA into the general human population. In the present study, an existing agent-based simulation model for spread of LA-MRSA within a pig herd was extended to also include LA-MRSA load and spread through air. This makes it possible to use the model for studying the air exposure to LA-MRSA for humans entering the pig barns. The model was used for simulating various types of interventions in contaminated herds. At present quantitative data for nasal carriage of LA-MRSA in pigs are sparse, and many knowledge gaps regarding spread of LA-MRSA remain. Thus, our goal of building this model was not to provide exact values for risk reduction, but to avail a model that can be used for studying the effect of various types of interventions mechanistically, once more relevant data become available. Collection of more data on the influence of load is crucial for getting a better understanding of which possible interventions strategies, that might still have some potential in countries, where LA-MRSA has already spread to the majority of the pig population.

Within-farm prevalence and environmental distribution of livestock-associated methicillin-resistant Staphylococcus aureus in farmed mink (Neovison vison).

The aim of the present study was to identify the animal prevalence and environmental reservoir of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) in mink farms. LA-MRSA on mink constitutes a human health hazard to farmers and farm workers, who handle the animals and are at risk of bites and scratches from colonized sites. The primary route of LA-MRSA colonization of mink is suspected to be by ingestion of contaminated pig by-products. We performed a cross-sectional study with repeated measurements during May-July 2017. A total of 644 mink carcasses (542 mink kits and 102 breeding animals) from five Danish farms were sampled. From each carcass, pharynx was swabbed and the right forepaw dissected. In addition, environmental samples covering feed, air, glove, cages (top and between) and nest boxes were collected on the farms. MRSA was selectively cultured from each sample and suspect colonies were assessed using matrix-assisted laser desorption ionisation (MALDI-TOF) for species confirmation. Further, from each farm, three isolates from mink and one isolate per positive environmental site were sent for whole genome sequencing. We isolated LA-MRSA from mink in four out of the five farms, but LA-MRSA bacterium was detected on all farms. On farms with LA-MRSA positive animals, the overall apparent animal prevalence ranged from 20% [13;29] CI95% to 29% [22;38]CI95%. LA-MRSA was isolated from kits before weaning, most likely due to a contaminated environment or transfer from the dam. Further, the apparent prevalence of LA-MRSA in kits tended to increase during the first months of age. On farms where LA-MRSA was isolated from mink, LA-MRSA was also isolated from the environment. LA-MRSA was isolated from all environmental sites tested (i.e. glove, on top of and between the cages and in the nest boxes), apart from air. The negative air samples contrast with the high concentrations of LA-MRSA in air found in the pig production. Hence, the risk of human exposure to LA-MRSA on mink-farms tends to be associated mainly with direct contact with contaminated environmental sites and the handling of colonized mink. All sequenced isolates were confirmed as LA-MRSA CC398 and genetically similar to clones previously isolated from the Danish pig production, supporting the hypothesis of LA-MRSA being transmitted by contaminated pig by-products.

A detailed investigation of the porcine skin and nose microbiome using universal and Staphylococcus specific primers.

MRSA is an increasing problem in humans as well as livestock. The bacterial co-colonization of the skin in MRSA carriers has been poorly investigated and moreover, there have been no methods for high resolution investigations of the Staphylococcus genus apart from tediously culturing or doing multiple PCRs. On 120 samples from pig ear, skin and nose, we generated amplicons from the V1-V2 region of the 16S rRNA gene to gather an overview of the genus-level microbiome, along with using MRSA specific plates to count MRSA. In parallel with this, amplicons of the tuf gene were generated, targeting only a region of the tuf gene found only in the Staphylococcus genus. Using these methods, we determined a core microbiota across the healthy pig and determined the Staphylococcus genus to be dominated by S. equorum. Moreover, we found Streptococcus to be inversely associated with Staphylococcus and MRSA, suggesting a role for this genus in combating MRSA. In this work, we have thoroughly investigated the skin and nose microbiome of the pig and developed a high throughput method for profiling the Staphylococcus genus which we believe will be useful for further investigations.

Livestock-associated methicillin-resistant Staphylococcus aureus is widespread in farmed mink (Neovison vison).

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) clonal complex (CC) 398 is widespread in the Danish pig production with around 90% of herds being positive. Since 2009, cases of CC398 LA-MRSA infections in Danish mink farmers have been observed. The objective of the study was to examine the presence of LA-MRSA in farmed mink. The investigation comprised three different sample types 1) clinical samples from carcasses submitted to the laboratory for diagnostic examination, 2) paws and pharyngeal swabs from healthy animals collected at pelting, and 3) feed samples from mink feed producers. In clinical samples, LA-MRSA was found in 34% of submissions and was most prevalent in samples from paws (33%) and pharynx (17%), followed by nasal and intestinal samples (each 13%), whereas it was never detected in perineal samples. LA-MRSA was found in healthy animals on 40% of the investigated farms, including paw samples (29%) and pharyngeal samples (16%). Twenty out of the 108 feed samples from feed producers were positive for LA-MRSA. The dominant spa-types were t034 and t011 associated to CC398, corresponding to the dominant spa-types detected in the Danish pig production, from which slaughter offal is used for mink feed. The spa-types, the high prevalence of LA-MRSA on paws and in pharynx, and its detection in feed samples, suggest feed as a possible source of LA-MRSA in mink.