| Summary: | 碩士 === 國立嘉義大學 === 微生物免疫與生物藥學系研究所 === 100 === Background: Escherichia coli is a diverse genetic bacteria as normal flora of intestine and opportunistic pathogens that cause gastroenteritis, bacteremia, bladder infection, meningitis or pus. Recently, multidrug resistant E. coli emerges. Soil microorganisms plays key roles in maintaining a healthy soil, including nitrogen fixation, phosphate solubilization, enhanced plant growth promotion, and organic matter degradation, but some plant pathogenic bacteria are able to resist the bactericidal components in environment. Loss of the antimicrobial against these pathogens would be a public health problem.
Objectives: (1) To characterize the antimicrobial resistance and its related genes, and genes for virulence factors of clinical E. coli. (2) To analyze the microflora distribution and the antimicrobial resistance in soil microorganisms isolated from National Chiayi University.
Methods: (1) E. coli isolates were collected from Chiayi Chang Gung Memorial Hospital (CGMH, n=131), Tainan Hospital (TNH, n=40) and National Chiayi University Veterinary Teaching Hospital (NCYUVH, n=57). Susceptibility to beta-lactam antimicrobials, and nalidixic acid as well as ciprofloxacin of quinolone antimicrobials were examined for each isolate. Genes for beta-lactamase, qnr and virulence factors, plasmid profiles and genetic relatedness of isolates were investigated by multiplex PCR, Kado-Liu method, Southern blotting, transformation and PFGE, respectively. The sequences flanking the qnr genes were determined by inverse PCR and sequencing. The mutations in quinolone resistance-determining region (QRDR) of gyrA and parC of quinolone resistance isolates were determined. (2) All bacterial isolates were obtained from soil samples from National Chiayi University. Phenotyping and genotyping of isolates were determined by physiological, biochemical method and 16S rDNA-RFLP. Susceptibility to beta-lactam, aminoglycoside, quinolone, chloramphenicol, tetracycline and sulfonamide antimicrobials was examined for each isolate.
Results: (1) Kado-Liu method identified that 91% of isolates harbored plasmid and the prevalence of major plasmid sizes differed among three counties. The prevalence of antimicrobial resistance was 71% for ampicillin, 30% for 3rd generation cephalosporins, 20% for extended-spectrum beta-lactamase (ESBL) isolates, and 12% for carbapenem non-susceptible (CNS) isolates, respectively. 3rd generation cephalosporins resistant E. coli isolates were more in TNH (78%) than those CGMH (23%) and NCYUVH (19%). Twenty-seven CNS isolates were not associated with carbapenemase. 79% of isolates contained class A or C bla genes and six related genes identified by multiplex PCR were blaTEM, blaCMY, blaSHV, blaCTX-M-14, blaCTX-M-3 and blaDHA with the prevalence of 61%, 44%, 9%, 7%, 6% and 1%, respectively. The blaTEM was the most prevalent gene in CGMH and NCYUVH, and blaCMY was the most prevalent gene in TNH. The qnr genes were identified in isolates collected from Tainan Hospital, Taichung Veterans General Hospital Chiayi Branch and National Chiayi University Veterinary Teaching Hospital, with the prevalence of 17.5% (7/40), 3.7% (5/135) and 8.8% (5/57), respectively. Multiplex PCR identified qnrA, qnrB (qnr2- and qnrB4-like) and qnrS genes. The qnr-positive isolates consisted of at least one of the following genes: blaTEM, blaCTX-M-14, blaDHA, blaCMY-2 and blaSHV. Examination of transformant revealed that plasmid with the qnr genes were associated with different bla genes and transposon, for example, sulI and qnrB2 coexisted on about 90-kb plasmid, the qnrB4 with blaDHA-1 on about 90-kb plasmid, and the qnrS7 with blaSHV-120 for ESBL on a large than 90-kb plasmid, and a novel qnrA inserted with partial IS26. PFGE pattern differed among the qnr-positive isolates. Susceptibility test revealed that 65% (11/17) of the qnr-positive isolates were resistance to three fluoroquinolones and Qnr only conferred reduced susceptibility to ciprofloxacin. Sequencing of QRDR of the fluoroquinolone resistant isolates confirmed mutations at two codons Ser83Leu and Asp87Asn (or Asp87Tyr) in the GyrA and at one codon Ser80Ile in the ParC. The prevalence of virulence genes differed among these hospitals, and fimH, kpsMTII, sat, iroN, iutA, iucD, fyuA and usp were important factor to infect human than cow. (2) Actinomycetales bacterium, Streptomyces polychromogenes, Bacillus licheniformis, Bacillus cereus, Bacillus pumilus, Azospirillum zeae, Cupriavidus taiwanensis, Chitinophaga terrae, Pseudomonas pseudoalcaligenes and Pseudomonas alcaligenes were identified The prevalence of antimicrobial resistance was 80% for ampicillin, 60% for 3rd generation cephalosporins, and 7% for carbapenems. 16% isolates ESBL produced. The prevalence of antimicrobial resistance in Gram-positive soil bacteria was higher than that of Gram-negative soil bacteria except for chloramphenicol, and Chitinophaga terrae is resistant to all antimicrobials except for sulfamethoxazole/trimethoprim.
Conclusion: (1) Plasmid profiles, antimicrobial resistance and genes for resistance as well as virulence factors of E. coli isolates differed among counties and sources. (2) All antimicrobial resistance was obtained in soil bacterial isolates in southern Taiwan.
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