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Biocontrol of Antimicrobial Resistant Salmonella enterica Serovars by Using Bacteriophage

  • 강현월 (대학원 생명공학과 식품공학전공)

초록/요약

Emergence of foodborne pathogens with resistant phenotypes against various drugs is considered as one of the leading public health concern because it can cause a therapeutic failure. In this study, a total of 98 Salmonella enterica strains were isolated from chicken meats obtained from Korean traditional markets. CTX-M-15 extended-spectrum β-lactamase (ESBL)-producing Salmonella isolates recovered from chicken meats (16 strains) were characterized to determine resistance phenotype and genotype. Two serotypes of cefotaxime resistant Salmonella isolates (Enteritidis and Virchow) harbored the identical sequence of blaCTX-M-15. Ciprofloxacin susceptible isolate (Sal10-FC-KU12) recovered from non-frozen chicken meat was artificially exposed to ciprofloxacin containing nutrient agar media. Induced ciprofloxacin resistant strains (SalML, SalMM, SalMH) were obtained. Minimal inhibition concentrations (MICs) of four strains against 12 antibiotics (ciprofloxacin, ofloxacin, levofloxacin, norfloxacin, nalidixic acid, ampicillin, streptomycin, cephalothin, cefoxitin, cefotaxim, chloramphenicol, tetracycline) were measured in the presence and absence of efflux pump inhibitor. Expression level of efflux pump related genes (acrB, acrF, marA, ramA, robA and soxS) were determined using real-time RT-PCR (RT-qPCR). Derived mutants showed elevated MICs against various antibiotics. Comparison between efflux pump inhibitor containing antibiotics shows the elevation of MICs against 11 antibiotics is caused by the action of efflux pump. RT-qPCR showed increased expression level of marA, ramA, and acrB compare to the family strain. This result of suggests that continuous usage of ciprofloxacin in various field, especially against food producing animals, might induce the emergence of Salmonella strains shows resistant phenotype against not only fluoroquinolone but also several antimicrobials in other classes. Because of the antimicrobial-resistant Salmonella strains are easily detected in foods and food processing environments, bacteriophages have recently surfaced as an alternative biocontrol tool. Virulent bacteriophage (wksl3) that could specifically infect Salmonella Enteritidis, S. Typhimurium and several additional serovars is isolated in this study. TEM revealed that phage wksl3 belonged to the family Siphoviridae. Complete genome sequence analysis and bioinformatics analysis revealed that phage wksl3 DNA is composed of 42,766 bp with 64 open reading frames. Since wksl3 does not encode any phage lysogeny factors, toxins, pathogen-related genes, or food-borne allergens, phage wksl3 may be considered a virulent phage with no side effects. Strong stable lytic activity, significant reduction in the number of S.Enteritidis after application on food, and lack of clinical symptoms of this phage suggests that wksl3 may be a useful agent for the protection of foods against S.Enteritidis contamination in foods.

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TABLE OF CONTENTS
ABSTRACT i
TABLE OF CONTENTS iii
CHAPTER 1 Accelerating spread of CTX-M-15 Extended-Spectrum β-Lactamase Among Nontyphoid Salmonella Isolated from Chicken Meat 1
Abstract 2
1.1 Introduction 3
1.2 Materials and Methods 5
1.2.1 Cefotaxime resistant Salmonella isolates 5
1.2.2 Antimicrobial susceptibility test 5
1.2.3 Phenotypic and genotypic characterization of ESBL 6
1.2.4 rep-PCR DNA fingerprinting and analysis 6
1.2.5 Inc/replicon typing 7
1.3 Results and Discussion 8

References 14

CHAPTER 2 Increase of multidrug efflux pump expression in fluoroquinolone-resistant Salmonella mutants induced by ciprofloxacin selective pressure 17
Abstract 18
2.1 Introduction 19
2.2 Materials and Methods 21
2.2.1 Bacterial strains, in vitro selection of mutants, and culture conditions 21
2.2.2 Antimicrobial susceptibility test 21
2.2.3 Efflux pump inhibition test 22
2.2.4 Expression analysis of transporter genes and transcriptional regulator genes 22
2.2.5 Sequence analysis of QRDR region 23

2.3 Results 25
2.3.1 Resistance phenotypes and QRDR mutations of Salmonella isolate and corresponding mutants 25
2.3.2 Effects of efflux pump inhibitor on mutants 27
2.3.3 Expression analysis of acrB, acrF and transcriptional regulator genes in wild-type strain and mutants 28

2.4 Discussion 30
References 35

CHAPTER 3 A new biocontrol agent, wksl3, for Salmonella Enteritidis and Typhimurium in Foods: Characterization, Application, Sequence Analysis, and Oral Acute Toxicity Study 43
Abstract 44
3.1 Introduction 46
3.2 Materials and Methods 49
3.2.1 Bacterial strains and culture conditions 49
3.2.2 Isolation of Salmonella phage 49
3.2.3 Determination of the phage host spectrum 52
3.2.4 Transmission electron microscopy (TEM) 53
3.2.5 One-step growth curve 53
3.2.6 Nucleotide extraction, sequencing, and genomic analysis 53
3.2.7 Oral toxicity studies in mice 55
3.2.8 Application of phages to control Salmonella on chicken skin 57
3.2.9 Statistical analysis 58
3.2.10 Nucleotide sequence accession number 58

3.3 Results 59
3.3.1 Bacteriophage wksl3 isolation and host spectrum determination 59
3.3.2 Phage wksl3 microscopy 59
3.3.3 One-step growth curve 60
3.3.4 DNA sequence analysis 60
3.3.5 Metabolism-related operons 68
3.3.6 Structure- and cell wall lysis-related operons 70
3.3.7 Acute oral toxicity study at mice 72
3.3.8 Effectiveness of wksl3 for the control of Salmonella on chicken skin 73
3.4 Discussion 76
References 84
ABSTRACT IN KOREAN 98
ACKNOWLEDGEMENTS 100

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