Methods for Antimicrobial Susceptibility Testing

1 Methods for Antimicrobial Susceptibility Testing Dr. Yuting Deng Pearl River Fisheries Research Institute (PRFRI), Chinese Academy of Fishery Sciences (CAFS), China OUTLINE. General Susceptibility considerations Standardized Susceptibility Testing protocols Interpretation of test results Quality control in AST. General Susceptibility considerations Choice of Methods for AST. Antimicrobial s u s c e p t i b i l i t y Te s t i n g Broth Agar E-test Disk dilution dilution diffusion Macrodilution Microdilution The tests have to be performed in accordance with internationally accepted procedures such as those published by the Clinical and Laboratory Standards Institute (CLSI) or the European Committee on Antimicrobial Susceptibility Testing (EUCAST).

2 U Disk Diffusion Method Discription Diffusion of an Antimicrobial agent from a disk or tablet containing a specified concentration of the agent into a solid culture medium (normally M ller Hinton agar, MHA) that has been inoculated with a defined concentration of a pure culture. -- photo by Deng YT. Advantages Disadvantages Easy to perform Precision deceases as incubation is Low cost lowered Reproducible if standardized Misleading results can occur when Does not require expensive equipment certain agents are tested against Possibility to semi-automate specific organisms u Broth Dilution Method Discription A technique in which a suspension of a pure culture of a bacterium of a defined concentration is tested against serial twofold dilutions of an Antimicrobial Microdilution agent in a liquid medium (normally cation adjusted M ller Hinton broth, CAMHB).

3 -- photo by Deng YT. Advantages Disadvantages Reference test method More reproducible than agar disk Requires more advanced capacities diffusion More costly Commercial preparations are available, Less flexible than agar dilution or disk which with a harmonized test protocol diffusion in adjusting to the changing supports comparability of results needs of the surveillance/monitoring among laboratories programme Possibility to automate u Agar Dilution Method Discription A technique in which a suspension of a pure culture of a bacterium of a defined concentration is tested against serial twofold dilutions of an Antimicrobial agent in a solid medium --Mani I et al., Acta Microbiologica Hellenica, 2018. (M ller Hinton agar, MHA). Advantages Disadvantages Can test multiple bacteria (except bacteria that swarm) Labor-intensive Can potentially improve the Require substantial economic and identification of MIC endpoints and technical resources extend the Antimicrobial concentration Short shelf-life of prepared plates (1-3.)

4 Range weeks). Possibility to semi-automate The endpoints are not always easy to Recommended for fastidious organisms read such as anaerobes u E-test Discription E-test consists of a predefined gradient of antibiotic concentrations on a plastic strip and is used to determine the Minimum Inhibitory Concentration (MIC) of antibiotics. --Tenover FC, Encyclopedia of Microbiology (Third Edition), 2009. Advantages Disadvantages Recommended for fastidious, slow- growing or nutritionally deficient micro-organisms High cost Easy to perform Can read the MIC endpoint Recommendations : For purposes of surveillance and in the interest of comparability of the data provided by each country in the region, standardized-quantitative Methods providing MIC are recommended ( dilution method).

5 AST should be conducted using standardized dilution Methods (either agar- dilution or broth dilution Methods ) as described by the CLSI or EUCAST. Each MIC determination must include quality control bacterial strains as recommended by CLSI to monitor performance and reproducibility of the test system. Harmonized panel of antimicrobials for monitoring The common test panel of Antimicrobial agents in the monitoring programme should be concise but provide valuable information about the possible resistance to a much broader group of agents. Additional Antimicrobial agents can be included, depending on country needs and preferences. u Consideration for assigning antimicrobials agents If the surveillance is mainly concerned with protecting public health, agents of relevance in therapy of humans should be prioritized.

6 If the purpose of the surveillance is to facilitate prudent Antimicrobial use in aquaculture or to study the epidemiology of AMR in aquaculture, those Antimicrobial agents most frequently used in therapy of aquatic animals should be prioritized. Clinical efficacy Resistance prevalence Minimizing emergence of resistance Drug cost Applicable regulatory and accreditation requirements considerations for good clinical practice u Antimicrobial agent class used in global aquaculture CLASS Antimicrobial agent CLASS Antimicrobial agent Doxycycline, Tetracycline, Beta-Lactams Ampicilin, Amoxicillin Tetracyclines Oxytetracycline Neomycin, Gentamicin, Aminoglycosides Quinolones Flumequine, Enrofloxacin Kanamycin Sulfamonomethoxine Folate Thiamphenicol Sulfanilamide/TMP.

7 Phenicols pathway Florfenicol Sulfamethazine/TMP. Antagonists Sulfamethoxazole/TMP. Macrolides Erythromycin Rifampin Nitrofurans Nitrofurantoin Flumequine, Oxolinic acid u Selection Guidelines In general, routine tests should include only one representative of each group of related drugs (class) with activity against a nearly identical spectrum of organisms and for which interpretive results would be nearly always the same. Tetracycline is tested as the class representative for chlortetracycline, doxycycline, minocycline, and oxytetracycline. Trimethoprim-sulfamethoxazole (SXT) can be tested to represent the potentiated sulfonamides, including trimethoprim-sulfadiazine and ormetoprim-sulfamethoxine. u Harmonized panel of antimicrobials for tests performed at <35.

8 With respect to surveillance programs to facilitate prudent Antimicrobial use in aquaculture or to study the epidemiology of AMR, the first task that must be addressed is the establishment of protocol specific epidemiological cut-off values (ECVs )for tests performed at temperatures <35 . The first step to establish new ECVs is to decide which agents to test. It is urged that at present all studies aimed at setting ECVs must include these agents but that they may additionally include other agents of more local interest. Availability of QC data for suggested panel of Antimicrobial agents, disc diffusion method --CLSI VET 04. Aeromonas salmonicida subsp. Escherichia coli (ATCC. Antimicrobial class Antimicrobial agent Disc contents salmonicida (ATCC 33658)Disc 25922)Disc (MHA)a (MHA)A.

9 Aminoglycosides Gentamicin 10 g . Trimethoprim- Anti-folates . sulfamethoxazole g -lactams Ampicillin 10 g . Macrolides Erythromycin 15 g . Phenicols Florfenicol 30 g . Enrofloxacin 5 g - . Quinolones Oxolinic acid 2 g . Tetracyclines Oxytetracycline 30 g . Availability of QC data for suggested panel of Antimicrobial agents, broth microdilution method --CLSI VET 04. Aeromonas salmonicida subsp. Escherichia coli (ATCC 25922)MIC. Antimicrobial class Antimicrobial agent salmonicida (ATCC 33658)MIC. (CAMHB)a (CAMHB)a Aminoglycosides Gentamicin . Trimethoprim- Anti-folates . sulfamethoxazole -lactams Ampicillin . Macrolides Erythromycin - . Phenicols Florfenicol . Enrofloxacin - . Quinolones Oxolinic acid . Tetracyclines Oxytetracycline . u Intrinsic resistance Some bacterial species are innately resistant to specific Antimicrobial agents or Antimicrobial agent classes.

10 The vast majority of mesophilic Aeromonas spp. and many Vibrio spp. (but not all) are innately resistant to some beta-lactam agents (ampicillin, amoxycillin and 1st generation cephalosporins). Standardized Susceptibility Testing protocols Protocol of disk diffusion method Disk diffusion AST Process 1 2 3 4. Reagent and Inoculum Inocuating the Applying disk Material preparation preparation test plates 5 6 7. Incubating the plates Reading plates Record the results 1. Reagents and materials preparation u Preparing Plates Mueller-Hinton agar (MHA) is considered the best medium for routine AST of nonfastidius bacteria because: acceptible batch-to-batch reproducibility for AST. low in inhibitors that affect certain drugs suseptibility test results support satisfactory growth of most nonfastidious pathogens 1) Prepare from commercially available dehydrated MHA according to the manufacturer's instructions.