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Foodborne Salmonella enterica serovars: Characterization of quinolone resistance mechanism and expression analysis of multi-drug efflux pump

식품유래 살모넬라 엔테리카 혈청형: 퀴놀론 내성기전 특성과 다제내성 유출펌프 발현 분석

  • Koo-Youn Kim (김구연, 대학원 생명공학과 식품공학전공)

초록/요약

In this study, antimicrobial resistance in a total of 67 Salmonella enterica serovars (Salmonella enterica serovar Typhimurium, S. Enteritidis, S. Haardt, and S. Rissen) isolated from various foods (ready-to eat food, raw chicken meat, raw beef, and fruit-vegetables) in Korea was characterized using antimicrobial susceptibility testing and PCR to determine resistance phenotype and genotype, respectively. Among these strains, quinolone and multi-drug resistant isolates were selected to characterize the quinolone resistance mechanism using PCR and sequence analysis of plasmid-mediated quinolone resistance (PMQR) genes and quinolone resistance determining region (QRDR), and to analyze the expression of multi-drug efflux pump by using quantitative real-time PCR (qRT-PCR), respectively. The epidemiological relationship among the resistant strains was determined by pulsed-field gel electrophoresis (PFGE). Antimicrobial susceptibility testing was performed by disk diffusion method recommended by Clinical and Laboratory Standards Institute (CLSI). Resistance patterns were shown to be diverse to the following antibiotics: ampicillin (14.9%), streptomycin (13.4%), gentamicin (1.5%), chloramphenicol (6.0%), nalidixic acid (23.9%), and tetracycline (10.4%). Resistance genotype was determined by PCR and sequence analysis for each target genes. Ampicillin (blaTEM-1), streptomycin (strA, strB), chloramphenicol (cat), and tetracycline (tetA) resistant strains had each plasmid-mediated resistance determinants and also showed high level of minimal inhibitory concentrations (MICs) for each antimicrobial. Transferability of these determinants was characterized by conjugation assay and determining the MICs for corresponding antimicrobials. MICs for each antimicrobial in transconjugants were shown to be increased by 32 to 128 fold of recipient’s. Plasmid DNAs of each resistant isolates harboring resistance determinants were extracted to analyze the molecular size and possessive characters. Each resistant isolates carried more than one of the various sizes (above 1 kb) of plasmid DNA. Sixteen Salmonella strains were isolated from ready-to-eat (RTE) food, kimbab, and raw chicken meat and found to have high minimal inhibitory concentrations (MICs) against nalidixic acid (512 ~ 4,096 μg/mL). Among them, 4 S. Haardt isolates showed multi-drug resistance (MDR) patterns with reduced susceptibility (generally recognized as 0.125 ~ 1 µg/mL of ciprofloxacin MICs) to 5 fluoroquinolones (ciprofloxacin, ofloxacin, norfloxacin, levofloxacin, and moxifloxacin). The mechanisms of quinolone resistance in the nalidixic acid resistant strains were characterized by PCR and sequence analysis to investigate the PMQR genes and amino acid changes in QRDR, and inhibition testing for efflux pump overexpression. No PMQR gene was detected in any of the nalidixic acid resistant isolates. However, several mutations associated with quinolone resistance were found in the QRDR: single mutations in gyrA (Asp87Tyr, Asp87Gly, and Asp87Asn) and double mutations in gyrA (Ser83Thr) and parC (Thr57Ser), but not a single mutation found in parC (Thr57Ser). MICs for nalidixic acid were reduced by 2 to 32 fold by the efflux pump inhibitor, phe-arg-β-naphthylamide (PAβN). Pulsed-field gel electrophoresis (PFGE) was carried out to confirm the epidemiological relationship between the nalidixic acid resistant strains. The PFGE patterns of XbaI-digested genomic DNA could be classified into 5 groups, based on having greater than 80% genetic similarity. The results showed that the resistant strain isolated from each sample in this study can spread from one source to another by cross-contamination. qRT-PCR was used to analysis the molecular expression levels of efflux pump transporters (acrB, acrF) and transcriptional regulatory genes (marA, ramA, robA, and soxS) in wild type Salmonella enterica serovar Haardt isolates and in in vitro-selected fluoroquinolone resistant mutants. Efflux pump inhibition tests and detection of additional mutations in the quinolone resistance determining region (QRDR) were also performed. In two mutant strains (ciprofloxacin MICs = 4 and 8 µg/mL, respectively), overexpression of acrB (1.81 and 3.82 fold increase over wild type) and ramA (1.94 and 4.24 fold) was shown to be the main factor involved in increasing resistance level of fluoroquinolones and other classes of antimicrobial, while not the other genes. Also, an additional QRDR mutation (Asp87Asn in gyrA) was found in a mutant with a ciprofloxacin MIC of 8 µg/mL. In conclusion, though relative lower level of resistance than previous reports of veterinary and clinical fields was shown in this study, resistance to specific antimicrobials such as ampicillin, streptomycin, and nalidixic acid revealed that use of zoonotic antimicrobials in veterinary fields might be concerned with acquisition of resistance in foodborne Salmonella. Plasmid-mediated resistance genes inducing resistance to extended-spectrum antimicrobials were not detected in this study. However, considering the frequently reported worldwide studies about the determinants in foodborne isolates, continuous monitoring of these determinants is necessary with national or international level of surveillance program. And the clonal spread of the nalidixic acid resistant isolates, which is confirmed in this study, may be the main source of spread of the resistance in food chain. To control these pathogens, the following points have to be performed: Banning or accurate restricting the use of certain drugs, hygiene in animal originated food producing, improvement of individual hygiene.

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초록/요약

본 연구에서는 한국 내 다양한 유형의 식품 (즉석섭취식품, 육계, 소고기, 과채류) 에서 분리된 총 67주의 Salmonella enterica serovars의 항생제 내성 특성을 연구하기 위해 감수성검사와 중합효소연쇄반응 (PCR)을 이용하여 항생제 내성 표현형과 유전형을 결정하였다. 이들 중 퀴놀론계 항생제 내성 균주와 다제내성 균주를 대상으로 각각, 플라스미드 매개 퀴놀론 내성 유전자 (plasmid-mediated quinolone resistance gene, PMQR)와 퀴놀론 내성 결정 부위 (quinolone resistance determining region, QRDR)에 대해 실시한 PCR과 염기서열 분석법 (sequence analysis)을 이용해 퀴놀론계 항생제 내성기전 특성을 알아보고, 정량적 실시간 중합효소연쇄반응 (quantitative real-time PCR, qRT-PCR)을 이용한 다제내성 유출펌프 (efflux pump)의 발현량을 분석하였다. 내성 균주간의 역학적 연관성은 pulsed-field gel electrophoresis (PFGE) 로 알아보았다. 항생제 감수성 검사는 CLSI에서 권장하는 방법에 따라 디스크 확산법으로 실시하였다. 내성유형은 다음 항생제에 대해 다양하게 나타났다: Ampicillin (14.9%), streptomycin (13.4%), gentamicin (1.5%), chloramphenicol (6.0%), nalidixic acid (23.9%), and tetracycline (10.4%). 내성 유전형은 각 대상 유전자에 대해 PCR과 sequence analysis로 결정하였다. Ampicillin (blaTEM-1), streptomycin (strA, strB), chloramphenicol (cat), tetracycline (tetA) 내성주들은 각각의 plasmid 매개 내성유전자를 지니고 있었고 각 항생제에 대해 높은 수준의 최소저해농도 (minimal inhibitory concentrations, MICs)를 지니고 있었다. 내성유전자의 전달성은 접합실험과 관련 항생제의 MIC를 측정하여 확인하였다. 피전달접합균주 (transconjugant)의 MIC는 수여자 (recipient)에 비해 32~128배가 증가한 것으로 나타났다. 내성 유전자를 보유한 각 내성주의 plasmid DNA를 추출해 각각의 molecular size와 보유 특성을 분석하였다. 각 내성주들은 하나 이상의 다양한 크기 (1 kb 이상) 를 지닌 plasmid를 보유하고 있었다. 즉석섭취식품인 김밥과 계육에서 분리된 16주의 nalidixic acid 내성주는 nalidixic acid에 대한 높은 수준의 MIC (512~4,096 μg/mL)를 지니고 있었다. 이 중 4주의 S. Haardt는 다제내성 패턴과 함께 5가지 플루오로퀴놀론계 항생제 (ciprofloxacin, ofloxacin, norfloxacin, levofloxacin, moxifloxacin)에 대해 저하된 감수성 (일반적으로 0.125~1 µg/mL의 ciprofloxacin MIC로 인식됨)을 가지고 있었다. Nalidixic acid 내성주의 퀴놀론계 항생제 내성기전은 PCR과 sequence analysis를 이용한 PMQR의 검출과 QRDR에서의 아미노산 변화, efflux pump 과발현 저해 시험을 이용하여 알아보았다. PMQR은 모든 nalidixic acid 내성 균주에서 검출되지 않았다. 하지만 QRDR에서의 몇몇의 mutation (gyrA에서 single mutation [Asp87Tyr, Asp87Gly, Asp87Asn], gyrA[Ser83Thr]와 parC[Thr57Ser]에서 double mutation)이 퀴놀론 내성에 관여하는 것을 알 수 있었고. parC에서의 single mutation (Thr57Ser)은 퀴놀론 내성을 유도하지 않았다. Efflux pump inhibitor인 phe-arg-β-naphthylamide (PAβN)에 의해 nalidixic acid의 MIC가 2~32배로 감소하는 것을 관찰할 수 있었다. PFGE를 통해 nalidixic acid 내성주간의 역학적 관계를 알아본 결과, XbaI 제한효소로 절단한 genomic DNA의 PFGE 패턴을 80%의 유전적 유사성에 근거하여 5개의 그룹으로 나눌 수 있었다. 본 연구에서 각 sample에서 분리된 내성주들은 한 유래에서 다른 유래로 교차오염이 되어 확산되었다는 것을 알 수 있었다. qRT-PCR을 이용하여 플루오로퀴놀론계에 대해 저하된 감수성을 지닌 다제내성 S. Haardt를 대상으로 wild type isolate와 in vitro-selected fluoroquinolone resistant mutant의 of efflux pump (acrB, acrF)와 transcriptional regulatory genes (marA, ramA, robA, soxS)의 발현량을 측정하였다. Efflux pump 저해 시험과 추가적인 QRDR mutation의 여부 역시 수행하였다. 타 유전자들과 달리, mutant에서 acrB (wild의 1.81, 3.82배)와 ramA (1.94, 4.24배)의 과발현은 fluoroquinolone계 항생재와 다른 계열의 항생제 내성의 수준을 증가시키는데 관련이 있다는 것을 알 수 있었다. 또한, ciprofloxacin MIC가 8 µg/mL인 mutant에서 추가적인 QRDR mutation (gyrA의 Asp87Asn)도 관찰할 수 있었다. 결론적으로, 본 연구에서 축산과 임상에 비해 낮은 수준의 내성을 보였지만 인수공통으로 사용되는 특정 항생제에 대한 내성이 눈에 띄게 나타나는 것은 동물유래 식품의 생산에서의 항생제 사용이 식품유래 병원성균의 항생제 내성획득에 연관이 될 수 있다는 것을 확인하였다. 또한, 광범위 항생제에 대한 내성을 유도하는 plasmid 매개 유전자는 검출되지 않았지만 식품유래 균주들의 유전자 보유에 대한 최근 연구의 잦은 보고를 고려해볼 때, 국가적, 국제적 단위의 프로그램에 지속적 모니터링이 필요할 것으로 여겨진다. 그리고 내성균주의 클론의 확산은 food chain에서 퀴놀론 내성 확산의 주요한 경로인 것을 알 수 있었으며 이러한 균주들의 관리를 위해, 특정 항생제의 규제 및 제한의 정확한 명시, 축산물 생산에서의 위생, 개인위생의 향상등이 이루어져야 할 것이다.

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목차

ABBREVIATIONS 1

PURPOSE OF THIS STUDY 3

CHAPTER 1. Diversity of antimicrobial resistance characteristics in foodborne Salmonella enterica serovars, isolated from various foods 4
1. Introduction 5
2. Materials and Methods 9
2.1 Salmonella strains 9
2.2 Serotyping 9
2.3 Antimicrobial susceptibility testing 9
2.4 Detection of antimicrobial resistance determinants 10
2.5 Conjugation assay 11
2.6 Plasmid DNA extraction 12
3. Results 13
3.1 Distribution of serotype 13
3.2 Resistance phenotype 13
3.3 Resistance genotype and the corresponding MICs 14
3.4 Characteristics of transconjugant 15
3.5 Characteristics of plasmid DNA 16
4. Discussion 17
5. References 20
6. Figures and Tables 24

CHAPTER 2. Characterization of quinolone resistance mechanism in foodborne Salmonella enterica serovars with high level nalidixic acid resistance 31
1. Introduction 32
2. Materials and Methods 35
2.1 Quinolone resistant strains and determination of quinolone MICs 35
2.2 Detection of PMQR genes and other resistance genes 35
2.3 Detection of chromosomal mutations in the QRDR 35
2.4 Efflux pump inhibition test by using phe-arg-β-naphthylamide (PAβN) 36
2.5 Pulsed-field gel electrophoresis (PFGE) 36
3. Results 38
3.1 Selection of nalidixic acid resistant isolates and MICs for quinolone and fluoroquinolones 38
3.2 PMQR genes 38
3.3 Amino acid changes in the QRDR 38
3.4 Effects of efflux pump inhibition by PAβN 39
3.5 Epidemiological relationship between the tested strains 39
4. Discussion 41
5. References 47
6. Figures and Tables 54

CHAPTER 3. Expression of the multi-drug efflux pumps AcrB, AcrF, and their regulatory genes in multi-drug resistant Salmonella enterica serovar Haardt with reduced fluoroquinolone susceptibility 57
1. Introduction 58
2. Materials and Methods 62
2.1 Bacterial strains 62
2.2 In vitro selection of fluoroquinolone resistant mutants 62
2.3 Sequence analysis of QRDR mutations 63
2.4 Determining MICs 63
2.5 Total RNA extraction and first strand cDNA synthesis 63
2.6 Expression analysis of acrB, acrF and the regulatory genes, marA, ramA, robA, and soxS by qRT-PCR 64
3. Results 66
3.1 Resistance phenotype of wild strains and fluoroquinolone resistant mutants 66
3.2 QRDR mutations 66
3.3 Efflux inhibition effect 67
3.4 Expression level of efflux pump transporter and the regulatory genes 67
4. Discussion 68
5. References 71
6. Figures and Tables 78

ACKNOWLEDGEMENTS 82

ABSTRACT IN KOREAN 83

SPECIAL THANKS TO 87

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