Comparative molecular analysis of Staphylococcus aureus from intensive livestock farming with emphasis on LA-MRSA of poultry origin

Since a few years Staphylococcus aureus, especially livestock-associated methicillin-resistant S. aureus (LA-MRSA), has been the focus of many studies, but only limited information on LA-MRSA from poultry origin was available at the beginning of this Ph.D. project. The aims of this study were (i) to comprehensively characterize livestock-associated S. aureus of poultry origin, (ii) to answer the question if MRSA is a food-borne pathogen, (iii) to provide an overview on antimicrobial resistance genes in staphylococci, and (iv) to identify novel antimicrobial resistance genes in LA-MRSA isolates. In the first part of the Ph.D. project, three different test populations were investigated. The first test population included 131 isolates from clinically ill turkeys and chickens (CHAPTER 3). Among the turkey isolates (n=80), the clonal complex (CC) 398 predominated and 21.2 % were LA-MRSA. The majority of chicken isolates (n=51) belonged to CC5 and only 9.8 % were LA-MRSA, however, all were identified as LA-MRSA CC398. In the other two studies 37 LA-MRSA CC398 isolates from four different broiler farms (broilers n=11, broiler houses n=15, farm residences n=5, humans living and/or working on the farms n=6) (CHAPTER 4) and 46 LA-MRSA CC398 and CC9 isolates from broilers at four different slaughterhouses (n=28) and workers at the respective poultry slaughterhouses (n=18) (CHAPTER 6) were investigated in order to explore transmission between different reservoirs. Molecular analysis of all LA-MRSA isolates from the three test populations showed two SCCmec types (IV and V), seven spa types (mainly t011) and twelve dru types (mainly dt11a or dt10q). Susceptibility testing, DNA microarray analysis and/or gene specific PCR assays, respectively, revealed highly variable resistance phenotypes (n=41) and genotypes (n=40). All LA-MRSA isolates (except three LA-MRSA CC9 isolates) were considered as multi-resistant due to their resistance to three or more classes of antimicrobial agents. These results showed that the current dissemination of LA-MRSA also included chickens and turkeys. The LA-MRSA isolates from the same farm or flock at slaughter exhibited a high homogeneity indicating transmission between poultry at farm or flock level. In contrast, the heterogeneity of the LA-MRSA isolates from the abattoir workers might reflect their occupational contact with animals from numerous poultry flocks. In addition, a rare observation was made in a LA-MRSA CC398-t3015 from a broiler farm (CHAPTER 5). This isolate exhibited two different erm(C)-carrying plasmids, pSWS371 and pSWS372. The larger plasmid harbored two more genes, a cop-6 gene and a novel pre/mob gene. Besides the additional genes, both plasmids differed in their rep genes, which assigned them to different incompatibility groups and explains why they can stably coexist in the same bacterial cell. In the second part of this Ph.D. project, the available tools to track the spread of MRSA were considered and the current knowledge about these organisms from food producing animals and food with respect to the role of MRSA to act as a food-borne pathogen were summarized (CHAPTER 7). Based on the published literature it was concluded that only the presence of MRSA in/on food intended for human consumption does not equate to MRSA being classified as a food-borne pathogen. The consumer-related aspects, e.g. age or health status, and also the adherence to kitchen hygiene play an equally important role. In the second review (CHAPTER 8), the latest information on resistance genes so far detected in staphylococci from healthy as well as diseased livestock, wild animals and pets were described. It became obvious that more than 40 different resistance genes have been identified in staphylococci from animals, which confer resistance to virtually all classes of antimicrobial agents approved for use in animals. These resistance genes can be located on mobile genetic elements, which play a major role as carriers of antimicrobial resistance genes and facilitate the exchange of resistance genes with staphylococci of human origin but also with other Gram-positive bacteria. In the third part of the Ph.D. project, the novel lincosamide/ pleuromutilin/streptogramin A resistance gene lsa(E) was identified in an enterococcal multi-resistance gene cluster in MRSA ST398 and MSSA ST9 isolates of human origin from Spain (CHAPTER 9). The lsa(E) gene was the first lsa gene in staphylococci which confers this resistance phenotype and confirms that there is a gene flux between E. faecalis and S. aureus. The lsa(E) gene was also identified in 16 porcine MRSA ST9-t899-IVa isolates from China (CHAPTER 10). In one of these isolates, lsa(E) was located in a slightly different multi-resistance gene cluster on a plasmid. Consequently, the entire plasmid was sequenced to gain insight into the structure and organization of this plasmid. The multi-resistance plasmid pV7037 proved to be 40,971 bp in size and carried, besides the previously determined multi-resistance gene cluster, a functionally active tet(L) gene, a complete cadDX operon and also a functionally deleted variant of the β-lactamase transposon Tn552 (CHAPTER 12). In addition, a comprehensively analysis of the two different types of multi-resistance gene clusters identified the novel spectinomycin resistance gene spw (CHAPTER 11). In a further study, the novel resistance genes lsa(E) and spw were also detected in eleven MRSA CC9 isolates from chicken (n=8) and pig carcasses (n=2) and from a butcher (n=1) from six different wet markets and a supermarket in Hong Kong, as well as in a single MRSA CC398 isolate of a nasally colonized human patient from a German hospital (CHAPTER 13). Ten overlapping PCR assays, which covered the entire cluster, and multi-resistance gene cluster-carrying contigs, which were obtained by whole genome sequencing, showed that all 12 MRSA isolates carried a large part of the pV7037-associated multi-resistance gene cluster. This part included the resistance genes aadE, spw, lsa(E) and lnu(B). These and previous observations suggest that a enterococcal multi-resistance gene cluster with two novel resistance genes has been acquired by MRSA/MSSA CC9 and CC398 of various origins several times at different occasions in Europe and Asia. Moreover, the novel spectinomycin resistance gene spd, recently identified on plasmid pDJ91S, was found in 30 LA-MRSA CC398 isolates from broilers and humans on farms (n=21) or at the slaughterhouses (n=4) in The Netherlands, from turkey meat/-products (n=4) in Germany and Austria, from a diseased pig (n=1) in Germany and also in a single LA-MSSA ST433 isolate from a diseased pig in Germany (CHAPTER 14). Electrotransformation and sequencing identified the spd gene on a novel plasmid, pSWS2889, which is 30 bp smaller and carried a different rep gene in comparison to plasmid pDJ91S. Restriction analysis showed that 22 isolates carried plasmid pSWS2889, whereas plasmid pDJ91S was present in the remaining nine isolates. Furthermore, the analysis of the genetic basis of elevated enrofloxacin MIC´s of ≥ 1 mg/L among 21 MRSA and 15 MSSA isolates of CC 398, CC9, CC5 or sequence type 2269 from pigs, cattle, poultry or food of poultry origin revealed different mutations in the quinolone resistance determining regions of the genes gyrA, gyrB, grlA and grlB as well as in the promoter region of norA (CHAPTER 15). In addition to the previously reported GrlA substitutions, all CC398 isolates, regardless of their origin and their methicillin resistance status, exhibited a Glu422Asp substitution in GrlB and a specific set of norA regulator mutations. The detection of four novel mutations (Ser80Leu and Glu84Asp in GrlA, Glu88Asp and Ser84Ala in GyrA) additionally illustrates the variety and the potential for mutational resistance to emerge.