水性抗生素耐药细菌的分离和鉴定及其抗生素耐药基因的分子表征
Summary
在这里，我们提出了一个详细的方案，用于从水中分离和鉴定抗生素耐药细菌及其抗生素抗性基因（ARG）的分子特征。使用基于培养物和非基于培养物（宏基因组分析）技术提供了有关印度孟买淡水中存在的细菌多样性和不同ARG的总库的完整信息。
Abstract
抗生素耐药性（AR）通过与淡水水体相关的微生物群的发展和传播是一个主要的全球健康问题。在本研究中，使用传统的基于培养的技术和高通量非培养物的宏基因组方法收集和分析淡水样品的总细菌多样性和AR基因（ARG）。本文提出了一种系统方案，用于从淡水样品中计数总细菌和抗生素耐药菌，并测定可培养分离株中的表型和基因型耐药性。此外，我们报告了使用从淡水样品中提取的总宏基因组DNA的全宏基因组分析来鉴定整体细菌多样性，包括不可培养的细菌，以及鉴定水体中不同ARG（抗性组）的总库。按照这些详细的方案，我们观察到抗生素耐药细菌负荷在9.6×10 5-1.2×109 CFU/mL范围内。大多数分离株对多种测试的抗生素耐药，包括头孢噻肟、氨苄西林、左氧氟沙星、氯霉素、头孢曲松、庆大霉素、新霉素、甲氧苄啶和环丙沙星，多种抗生素耐药性（MAR）指数为≥0.2，表明分离株的耐药性水平很高。16S rRNA测序鉴定了潜在的人类病原体，如肺炎克雷伯菌，以及机会性细菌，如Comamonas spp.、Micrococcus spp.、Arthrobacter spp.和Aeromonas spp.。 分离株的分子表征表明存在各种ARG，例如blaTEM，blaCTX-M（β-内酰胺），aadA，aac（6’）-Ib（氨基糖苷类）和dfr1（甲氧苄啶），这也得到了全宏基因组DNA分析的证实。在宏基因组DNA中还检测到编码抗生素外排泵的其他ARGs-mtrA，macB，mdtA，acrD，β-内酰胺酶-SMB-1，VIM-20，ccrA，ampC，blaZ，氯霉素乙酰转移酶基因catB10和利福平耐药基因rphB-的高患病率。在本研究中讨论的方案的帮助下，我们确认了具有不同AR表型和基因型性状的水性MAR细菌的存在。因此，全宏基因组DNA分析可以用作传统基于培养的技术的补充技术，以确定水体的整体AR状态。
Introduction
抗菌素耐药性（AMR）已被确定为最紧迫的全球问题之一。抗微生物药物耐药性的快速演变及其在世界范围内的传播是人类健康和全球经济面临的最大威胁之一，就与之相关的健康成本而言1.抗生素的过度使用和滥用导致AR增加。COVID-19大流行突出了这一点，在此期间，在许多情况下，由于受影响患者的AMR，相关继发感染的治疗受到巨大损害2。除了人类直接使用/滥用抗生素外，在农业和畜牧业中过度使用和滥用抗生素以及不适当地排放到环境（包括水体）中也是一个主要问题3。细菌中新的耐药性状和多重耐药性的兴起迫切需要更好地了解导致AR发展及其传播的因素。多种抗生素耐药细菌，通常在质粒等移动遗传元件上携带多个AR基因（ARG），可以将这些耐药基因转移到非耐药微生物，包括潜在的人类病原体，从而导致超级细菌的出现，即使是最后的抗生素也无法治愈4。这些多重抗生素耐药细菌，如果存在于水生态系统中，可以通过食用受污染的水基食物（如鱼、螃蟹和软体动物）直接进入人体肠道。先前的研究表明，AR细菌在天然水系统中的传播也可以到达其他供水，包括饮用水，从而可以进入人类食物链5，6，7。
本研究的目的是使用基于培养物和非基于培养物（全宏基因组分析）技术的组合来提供全面的方案，以获得有关印度孟买水体中存在的细菌多样性和不同ARG的总库的完整信息。传统上，基于培养的技术已被用于研究水体中的细菌多样性。由于可培养微生物仅占任何生态位中总微生物群的一小部分，为了更好地了解细菌多样性的整体状况以及任何样品中普遍存在的各种耐药性状，必须同时使用各种基于培养和独立于培养的技术。一种强大而可靠的非培养技术是全宏基因组DNA分析。这种高通量方法已成功用于细菌多样性的各种研究或各种ARG的功能注释8，9。该技术使用宏基因组（样品中的总遗传物质）作为各种分析的起始材料，因此与培养无关。本研究中的方案可用于全宏基因组DNA分析，以获取有关水样中总细菌多样性和各种ARG（抗性组）的信息。
Protocol
1. 样品采集和处理 样品采集在无菌样品容器中收集适当体积的水样，确保填充不超过容器的 3/4。 收集后尽快将样品在无菌条件下运输到实验室，并立即进行处理。 来样加工通过无菌平纹细布无菌过滤水样，以去除任何颗粒物质。 对过滤水进行适当的连续稀释以进行进一步分析。 2. 估计细菌…
Representative Results
细菌总载量和抗生素耐药 （AR） 细菌计数通过在改良培养基R2A琼脂上散布10-4至10-6倍稀释的水样品来计数总细菌负荷。为了计数AR细菌计数，将10-3至10-6倍稀释液散布在含有抗生素的培养基板上（图3）。总细菌计数和AR细菌计数计算为CFU/mL，所有电镀实验一式两份。按照上述方案，本研究表明细菌总数为3.0×109CFU / mL。?…
Discussion
样本收集和处理起着重要作用，可能会影响研究结果和解释。因此，为了排除样品的变化，重要的是在所研究的淡水体的多个位置进行采样。在处理此类样品时保持适当的无菌环境条件可以防止污染。此外，为了防止细菌组成的变化可能影响提取核酸的质量和数量，运输条件应保持在4°C，从样品收集点到后续处理的时间最短。一些研究强调，如果不仔细进行，样品收集和处理之间的过渡期可能会…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作得到了孟买大学科技部-大学研究和科学卓越促进（DST-PURSE）计划的财政资助的部分支持。Devika Ghadigaonkar在该计划下担任项目研究员。科学和技术部科学与工程研究委员会（DST-SERB）项目编号：CRG/2018/003624高级研究员Harshali Shinde提供的技术支持得到了认可。
Materials
| 100 bp DNA ladder | Himedia | MBT049-50LN | For estimation of size of the amplicons |
| 2x PCR Taq mastermix | HiMedia | MBT061-50R | For making PCR reaction mixture |
| 37 °C Incubator | GS-192, Gayatri Scientific | NA | For incubation of bacteria |
| 6x Gel Loading Buffer | HiMedia | ML015-1ML | Loading and Tracking dye which helps to weigh down the DNA sample and track the progress of electrophoresis |
| Agarose powder | Himedia | MB229-50G | For resolving amplicons during Agarose Gel Electrophoresis (AGE) |
| Ampicillin antibiotic disc | HiMedia | SD002 | For performing AST |
| Autoclave | Equitron | NA | Required for sterilization of media, glass plates, test tubes, etc |
| Bioanalyzer 2100 | Agilent Technologies | NA | To check the quality and quantity of the amplified library |
| Bisafety B2 Cabinet | IMSET | IMSET BSC-Class II Type B2 | Used for microbiological work like bacterial culturing, AST etc. |
| Cefotaxime antibiotic disc | HiMedia | SD295E-5VL | For performing AST |
| Cefotaxime antibiotic powder | HiMedia | TC352-5G | For preparation of antibiotic stock solution required during isolation of antibiotic resistant bacteria |
| Ceftriaxone antibiotic disc | HiMedia | SD065 | For performing AST |
| Centrifuge Minispin | Eppendorf | Minispin Plus-5453 | Used to pellet the debris during crude DNA preparation |
| Chloramphenicol antibiotic disc | HiMedia | SD006-5x50DS | For performing AST |
| Ciprofloxacin antibiotic disc | HiMedia | SD060-5x50DS | For performing AST |
| Ciprofloxacin antibiotic powder | HiMedia | TC447-5G | For preparation of antibiotic stock solution required during isolation of antibiotic resistant bacteria |
| Colorimeter | Quest | NA | For checking the OD of culture suspensions |
| Comprehensive Antibiotic Resistance Database (CARD) database | functional annotation of ARGs; https://card.mcmaster.ca/ | ||
| Cooling Shaker Incubator | BTL41 Allied Scientific | NA | For incubation of media plates for culturing bacteria |
| Deep Freezer (-40 °C) | Haier | DW40L, Haier Biomedicals | For storage of glycerol stocks |
| DNA Library Prep Kit | NEB Next Ultra DNA Library Prep Kit for Illumina | NA | Paired-end sequencing library preparation |
| EDTA | HiMedia | GRM1195-100G | For preparation of Gel running buffer for Agarose Gel Electrophoresis (AGE) |
| Electrophoresis Apparatus | TechResource | 15 cm gel casting tray | For making the agarose gel and carrying out electrophoresis |
| Electrophoresis Power pack with electrodes | Genei | NA | For running the AGE |
| Erythromycin antibiotic disc | HiMedia | SD222-5VL | For performing AST |
| Erythromycin antibiotic powder | HiMedia | CMS528-1G | For preparation of antibiotic stock solution required during isolation of antibiotic resistant bacteria |
| Erythromycin antibiotic powder | HiMedia | TC024-5G | For preparation of antibiotic stock solution required during isolation of antibiotic resistant bacteria |
| Escherichia coli ATCC 25922 | HiMedia | 0335X-1 | Used as a control while performing AST |
| Ethidium Bromide | HiMedia | MB071-1G | Intercalating agent and visualizaion of DNA after electrophoresis under Gel Documentation System |
| Fluorometer | Qubit 2.0 | NA | For determining concentration of extracted metagenomic DNA |
| Gel Documentation System | BioRad | Used for visualizing PCR amplicons after electrophoresis | |
| Gentamicin antibiotic disc | HiMedia | SD170-5x50DS | For performing AST |
| Glacial Acetic Acid | HiMedia | AS119-500ML | For preparation of Gel running buffer for Agarose Gel Electrophoresis (AGE) |
| Glycerol | HiMedia | GRM1027-500ML | For making glycerol stocks |
| Imipenem antibiotic disc | HiMedia | SD073 | For performing AST |
| Kaiju Database | NA | NA | For taxonomical classification of reads; https://kaiju.binf.ku.dk/ |
| Kanamycin antibiotic disc | HiMedia | SD017-5x50DS | For performing AST |
| Kanamycin antibiotic powder | HiMedia | MB105-5G | For preparation of antibiotic stock solution required during isolation of antibiotic resistant bacteria |
| Levofloxacin antibiotic disc | HiMedia | SD216-5VL | For performing AST |
| Luria Bertani broth | Himedia | M1245-500G | For enrichment of cultures |
| McFarland Standards | Himedia | R092-1No | To compare density of culture suspension |
| Molecular Biology water | HiMedia | TCL018-500ML | For making PCR reaction mixture |
| Mueller-Hinton Agar (MHA) | HiMedia | M173-500G | For performing Antibiotc Susceptibility Testing (AST) |
| Neomycin antibiotic disc | HiMedia | SD731-5x50DS | For performing AST |
| PCR Gradient Thermal Cycler | Eppendorf | Mastercycler Nexus Gradient 230V/50-60 Hz | Used for performing PCR for amplification of 16S rRNA region and various Antibiotic Resistance genes |
| Primers | Xcelris | NA | For PCR amplication |
| R2A Agar, Modified | HiMedia | M1743 | For preparation of media plates for isolation of total and antibiotic resistant (AR) bacterial load |
| Scaffold generation | CLC Genomics Workbench 6.0 | NA | For generation of scaffolds |
| Sequencer | Illumina platform (2 x 150 bp chemistry) | NA | Sequencing of amplified library |
| Sodium Chloride | HiMedia | TC046-500G | For preparation of 0.85% saline for serially diluting the water sample |
| Soil DNA isolation Kit | Xcelgen | NA | For extraction of whole metagenomic DNA from the filtered water sample |
| Staphylococcus aureus subsp. aureus ATCC 29213 | HiMedia | 0365P | Used as a control while performing AST |
| Taxonomical Classification | Kaiju ioinformatics tool | NA | For classification of reads into different taxonomic groups from phylum to genus level |
| The Comprehensive Antibiotic Resistance Database (CARD) | NA | NA | For functional annotation of ARGs |
| Tigecycline antibiotic disc | HiMedia | SD278 | For performing AST |
| Trimethoprim antibiotic disc | HiMedia | SD039-5x50DS | For performing AST |
| Tris base | HiMedia | TC072-500G | For preparation of Gel running buffer for Agarose Gel Electrophoresis (AGE) |
| Vancomycin antibiotic powder | HiMedia | CMS217 | For preparation of antibiotic stock solution required during isolation of antibiotic resistant bacteria |
| Weighing Balance | Mettler Toledo | ME204 Mettler Toledo | Used for weighing media powders, reagent powders etc. |
| NA – Not Applicable |
References
- Prestinaci, F., Pezzotti, P., Pantosti, A. Antimicrobial resistance: A global multifaceted phenomenon. Pathogens and Global Health. 109 (7), 309-318 (2015).
- Knight, G., et al. Antimicrobial resistance and COVID-19: Intersections and implications. Elife. 10, 64139 (2021).
- Ventola, C. L. The antibiotic resistance crisis: Part 1: Causes and threats. Pharmacy and Therapeutics. 40 (4), 277-283 (2015).
- Naik, O. A., Shashidhar, R., Rath, D., Bandekar, J. R., Rath, A. Metagenomic analysis of total microbial diversity and antibiotic resistance of culturable microorganisms in raw chicken meat and mung sprouts (Phaseolus aureus) sold in retail markets of Mumbai. India. Current Science. 113 (1), 71-79 (2017).
- Naik, O. A., Shashidhar, R., Rath, D., Bandekar, J., Rath, A. Characterization of multiple antibiotic resistance of culturable microorganisms and metagenomic analysis of total microbial diversity of marine fish sold in retail shops in Mumbai, India. Environmental Science and Pollution Research. 25 (7), 6228-6239 (2018).
- Czekalski, N., GascónDíez, E., Bürgmann, H. Wastewater as a point source of antibiotic-resistance genes in the sediment of a freshwater lake. The ISME Journal. 8 (7), 1381-1390 (2014).
- Kraemer, S., Ramachandran, A., Perron, G. Antibiotic pollution in the environment: From microbial ecology to public policy. Microorganisms. 7 (6), 180 (2019).
- Edmonds-Wilson, S., Nurinova, N., Zapka, C., Fierer, N., Wilson, M. Review of human hand microbiome research. Journal of Dermatological Science. 80 (1), 3-12 (2015).
- de Abreu, V., Perdigão, J., Almeida, S. Metagenomic approaches to analyze antimicrobial resistance: An overview. Frontiers in Genetics. 11, 575592 (2021).
- Carlson, S., et al. Detection of multiresistant Salmonella typhimurium DT104 using multiplex and fluorogenic PCR. Molecular and Cellular Probes. 13 (3), 213-222 (1999).
- Breakpoint tables for interpretation of MICs and zone diameters, Version 12.0. European Committee on Antimicrobial Susceptibility Testing Available from: https://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/Breakpoint_tables/v_12.0_Breakpoint_Tables.pdf (2022)
- Bharti, R., Grimm, D. Current challenges and best-practice protocols for microbiome analysis. Briefings in Bioinformatics. 22 (1), 178-193 (2019).
- Choo, J., Leong, L., Rogers, G. Sample storage conditions significantly influence faecal microbiome profiles. Scientific Reports. 5, 16350 (2015).
- Clinical and Laboratory Standards Institute. . Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically, 11th edition. , (2015).
- Bayot, M., Bragg, B. Antimicrobial Susceptibility Testing. StatPearls. , (2021).
- Joseph, A. A., Odimayo, M. S., Olokoba, L. B., Olokoba, A. B., Popoola, G. O. Multiple antibiotic resistance index of Escherichia coli isolates in a tertiary hospital in South-West Nigeria. Medical Journal of Zambia. 44 (4), 225-232 (2017).
- Lorenz, T. Polymerase chain reaction: Basic protocol plus troubleshooting and optimization strategies. Journal of Visualized Experiments. (63), e3998 (2012).
- Rolin, J. Food and human gut as reservoirs of transferable antibiotic resistance encoding genes. Frontiers in Microbiology. 4, 173 (2013).
- Racewicz, P., et al. Prevalence and characterisation of antimicrobial resistance genes and class 1 and 2 integrons in multiresistant Escherichia coli isolated from poultry production. Scientific Reports. 12, 6062 (2022).
- Gebreyes, W., Thakur, S. Multidrug-resistant Salmonella enterica serovar Muenchen from pigs and humans and potential interserovar transfer of antimicrobial resistance. Antimicrobial Agents and Chemotherapy. 49 (2), 503-511 (2005).
- Li, L., et al. Prevalence and characteristics of extended-spectrum β-lactamase and plasmid-mediated fluoroquinolone resistance genes in Escherichia coli isolated from chickens in Anhui Province, China. PLoS One. 9 (8), 104356 (2014).
- Akers, K., et al. Aminoglycoside resistance and susceptibility testing errors in Acinetobacter baumannii-calcoaceticus complex. Journal Of Clinical Microbiology. 48 (4), 1132-1138 (2010).
- Ciesielczuk, H. . Extra-intestinal pathogenic Escherichia coli in the UK: The importance in bacteraemia versus urinary tract infection, colonisation of widespread clones and specific virulence factors. , (2015).
Citer Cet Article
Ghadigaonkar, D., Rath, A. Isolation and Identification of Waterborne Antibiotic-Resistant Bacteria and Molecular Characterization of their Antibiotic Resistance Genes. J. Vis. Exp. (193), e63934, doi:10.3791/63934 (2023).