ABSTRACTSince its discovery in the early 2000s, methicillin-resistantStaphylococcus aureus(MRSA) clonal complex 398 (CC398) has become a rapidly emerging cause of human infections, most often associated with livestock exposure. We applied whole-genome sequence typing to characterize a diverse collection of CC398 isolates (n= 89), including MRSA and methicillin-susceptibleS. aureus(MSSA) from animals and humans spanning 19 countries and four continents. We identified 4,238 single nucleotide polymorphisms (SNPs) among the 89 core genomes. Minimal homoplasy (consistency index = 0.9591) was detected among parsimony-informative SNPs, allowing for the generation of a highly accurate phylogenetic reconstruction of the CC398 clonal lineage. Phylogenetic analyses revealed that MSSA from humans formed the most ancestral clades. The most derived lineages were composed predominantly of livestock-associated MRSA possessing three different staphylococcal cassette chromosomemecelement (SCCmec) types (IV, V, and VII-like) including nine subtypes. The human-associated isolates from the basal clades carried phages encoding human innate immune modulators that were largely missing among the livestock-associated isolates. Our results strongly suggest that livestock-associated MRSA CC398 originated in humans as MSSA. The lineage appears to have undergone a rapid radiation in conjunction with the jump from humans to livestock, where it subsequently acquired tetracycline and methicillin resistance. Further analyses are required to estimate the number of independent genetic events leading to the methicillin-resistant sublineages, but the diversity of SCCmecsubtypes is suggestive of strong and diverse antimicrobial selection associated with food animal production.IMPORTANCEModern food animal production is characterized by densely concentrated animals and routine antibiotic use, which may facilitate the emergence of novel antibiotic-resistant zoonotic pathogens. Our findings strongly support the idea that livestock-associated MRSA CC398 originated as MSSA in humans. The jump of CC398 from humans to livestock was accompanied by the loss of phage-carried human virulence genes, which likely attenuated its zoonotic potential, but it was also accompanied by the acquisition of tetracycline and methicillin resistance. Our findings exemplify a bidirectional zoonotic exchange and underscore the potential public health risks of widespread antibiotic use in food animal production.

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Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of healthcare-associated (HA), community-associated (CA) and livestock-associated (LA) infections. Recently, the discovery of human and bovine MRSA isolates carrying a new mecA gene homologue, mecA(LGA251) (now designated mecC), has caused concern because they are not detected by conventional, confirmatory tests for MRSA. Very little is known about their frequency, epidemiology and possible transmission between livestock and humans. In this study, the epidemiology of the mecC isolates in Denmark was investigated by screening the national collections of MRSA cases (from 1988 onwards) and S. aureus bacteraemia cases (from 1958 onwards). Isolates carrying mecC were only recovered infrequently before 2003 (n = 2) but now seem to be increasing, with 110 cases in 2003-2011. Clinical data on mecC-carrying MRSA demonstrated that mecC-MRSA were primarily community-acquired (CA-MRSA) and affected persons typically living in rural areas, being older than other CA-MRSA patients. Among 22 cases in Region Zealand, four reported contact with cattle and sheep. Two of these persons lived on farms with livestock positive for mecC-carrying MRSA, sharing spa type (t843), MLVA (MT429) and PFGE pattern with the human isolates. These observations indicate that mecC-carrying MRSA can be exchanged between humans and ruminants.

SCCmec in MRSA is acknowledged to be of importance not only because it contains the mecA or mecC gene but also for staphylococcal adaptation to different environments, e.g., in hospitals, the community, and livestock. Typing of SCCmec by PCR techniques has, because of its heterogeneity, been challenging, and whole-genome sequencing has only partially solved this since no good bioinformatic tools have been available. In this article, we describe the development of a new bioinformatic tool, SCCmecFinder, that includes most of the needs for infection control professionals and researchers regarding the interpretation of SCCmec elements. The software detects all of the SCCmec elements accepted by the International Working Group on the Classification of Staphylococcal Cassette Chromosome Elements, and users will be prompted if diverging and potential new elements are uploaded. Furthermore, SCCmecFinder will be curated and updated as new elements are found and it is easy to use and freely accessible.

Several methicillin-resistant Staphylococcus aureus (MRSA) lineages that carry a novel mecA homologue (mecC) have recently been described in livestock and humans. In Denmark, two independent human cases of mecC-MRSA infection have been linked to a livestock reservoir. We investigated the molecular epidemiology of the associated MRSA isolates using whole genome sequencing (WGS). Single nucleotide polymorphisms (SNP) were defined and compared to a reference genome to place the isolates into a phylogenetic context. Phylogenetic analysis revealed two distinct farm-specific clusters comprising isolates from the human case and their own livestock, whereas human and animal isolates from the same farm only differed by a small number of SNPs, which supports the likelihood of zoonotic transmission. Further analyses identified a number of genes and mutations that may be associated with host interaction and virulence. This study demonstrates that mecC-MRSA ST130 isolates are capable of transmission between animals and humans, and underscores the potential of WGS in epidemiological investigations and source tracking of bacterial infections.

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.Page 1 not detected in any of the samples. BRSV was detected in diseased calves in two herds but not in 35 the clinically healthy animals. Among the diseased calves in these two herds a significant increase 36 in haptoglobin and serum amyloid A levels was observed compared to the healthy calves. The 37 results indicate, that haptoglobin might be the best choice for detecting disease under field 38 conditions. For H. somni and M. haemolytica, a higher percentage of the samples were found 39 positive by PCR than by cultivation, whereas the opposite result was found for P. multocida. 40Detection of P. multocida by PCR or cultivation was found to be significantly associated with the 41 disease status of the calves. For H. somni a similar association with disease status was only 42 observed for cultivation and not for PCR. 43 44

Livestock constitutes a potential reservoir of meticillin-resistant Staphylococcus aureus isolates belonging to a recently derived lineage within clonal complex 398 (MRSA CC398-IIa). Since its discovery in the early 2000s, this lineage has become a major cause of human disease in Europe, posing a serious public health challenge in countries with intensive livestock production. To retrace the history of human colonisation and infection with MRSA CC398-IIa in Denmark, we conducted a nationwide, retrospective study of MRSA isolates collected from 1999 to 2011. Among 7,429 MRSA isolates screened, we identified 416 MRSA CC398-IIa isolates. Of these, 148 were from people with infections, including 51 from patients reporting no livestock exposure. The first cases of MRSA CC398-IIa infection in Denmark occurred in 2004. Subsequently, the incidence of MRSA CC398-IIa infection showed a linear annual increase of 66% from 2004 to 2011 (from 0.09 to 1.1 per 100,000 person-years). There were clear temporal and spatial relationships between MRSA CC398-IIa-infected patients with and without livestock exposure. These findings suggest substantial dissemination of MRSA CC398-IIa from livestock or livestock workers into the Danish community and underscore the need for strategies to control its spread both on and off the farm.