Molecular Characterization and Phylodynamics of Livestock-associated Methicillin-Resistant Staphylococcus aureus

Autor: Min-Tao Wan, 萬民濤
Rok vydání: 2014
Druh dokumentu: 學位論文 ; thesis
Popis: 102
Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) has emerged as a potential zoonotic pathogen since it was first identified in Europe in 2003. The overall objective of this dissertation was to investigate the molecular characteristics and phylodynamics of Taiwan LA-MRSA isolated from finishing pig and livestock environments. The specific aims were: (1) to compare the dynamics of MRSA-related resistance genes and the genotypic and phenotypic characteristics of MRSA isolates in municipal and swine wastewater; (2) to investigate and compare the virulence factors, toxic gene profiles, and phylogenetic relationships between Taiwan LA-MRSA ST9 and human clinical ST9; (3) to compare the presence of adhesion factors in LA-MRSA populations isolated directly from pigs and from swine slaughterhouse wastewater, and to compare their abilities to produce biofilms and (4) to investigate the effects of the LA-MRSA Taiwan clone on the population dynamics. For aim (1), the concentrations of resistance genes (mecA, intI1, and qacEΔ1) were quantified using real-time quantitative polymerase chain reaction (real-time qPCR) in 72 samples taken from a swine slaughterhouse (the second largest swine auction market for pigs ready to slaughter in Taiwan) and 24 samples from the largest secondary municipal wastewater treatment plant in Taiwan. One hundred and thirteen MRSA isolates (86 from municipal wastewater and 27 from swine slaughterhouse wastewater) were recovered and were characterized by antimicrobial susceptibility testing, minimum inhibitory concentrations (MICs), and staphylococcal cassette chromosome mec (SCCmec) typing. For aim (2), a total of 152 MRSA ST9 isolates, including 147 LA-MRSA isolates and 5 human clinical isolates, were screened for the presence of the accessory gene regulator (agr) and 20 enterotoxin genes (se), the exfoliatoxin gene, and toxic shock syndrome toxin-1 (TSST-1) gene. The evolutionary history of staphylocoagulase (SC) in Taiwan MRSA ST9 was reconstructed using phylogenetic methods. For aim (3), this study investigated the dynamics of biofilm formation gene, 10 microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) - MRSA infection and colonization, and biofilm expression in MRSA from 147 asymptomatic nasal swine isolates and 86 swine slaughterhouse wastewater isolates. Finally, 140 MRSA (135 piggery and 5 human clinical) isolates which collected from 3 populations of western Taiwan, central eastern Taiwan, and Penghu Island were used to reconstruct the population structure by analysis of the mec-associated direct repeat unit (dru). The ancestral population dynamics of the dru type were estimated by constructing a minimum spanning network (MSN). Past population dynamics of LA-MRSA isolates were estimated using a Bayesian skyline plot (BSP) model. The results revealed that resistance genes are highly abundant throughout the year in swine slaughterhouse wastewater (6.1–10.8 log10 copies/mL). More than 60% of the MRSA isolates from swine slaughterhouse wastewater carried the antiseptic resistance gene (qacE△1) with multidrug resistance (MDR) and high MICs. The municipal MRSA isolates harbored SCCmec II, III, IV, and VII, whereas those isolates from the swine slaughterhouse harbored only SCCmec V and IX. Most Taiwan LA-MRSA ST9 harbored agr-II with a combination of the enterotoxin gene cluster (egc)-2 and the tst gene. Further, Taiwan LA-MRSA ST9 and human clinical MRSA ST9 belonged to a novel SC XIc subtype with similar genetic toxin profiles. Taiwan LA-MRSA isolates from swine and swine slaughterhouse wastewater carried many MSCRAMMs. Biofilm production was higher in piggery nasal swab samples than in swine-related wastewater samples. Finally, population genetic analyses identified 28 dru types (24 novel types) and 15 dru-Clonal Complexes (CCs) in Taiwan LA-MRSA ST9 subpopulation. MSN and analysis of the genetic structure of sub-populations revealed that dru types in Taiwan LA-MRSA ST9 had at least of two origins. BSP analysis indicated that MRSA ST9 is a stable population with a long evolutionary history in Taiwan. In conclusions, Taiwan LA-MRSA and its resistance genes are widely distributed in swine populations and swine-related environments. These Taiwan LA-MRSA isolates carry several virulence factors and are able to form biofilm- pose potential threat to public health. Phylogenetic and population genetic analyses indicate a large and stable population of LA-MRSA clone and may have evolved unique regional subpopulations in Taiwan. Further epidemiological and evolutionary studies of these novel LA-MRSA are needed in order to assess future public health implications.
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