FMM/RAS/298: Strengthening capacities, policies and national action plans on prudent and responsible use of antimicrobials in fisheries Laboratory determination of the susceptibility to antibiotics of bacteria isolated from aquatic animals Peter Smith peter.smith@nuigalway.ie Aquatic AMR Workshop 1: 10-11 April 2017, Mangalore, India
In-vitro susceptibility testing Laboratory methods for measuring in vitro susceptibility (MIC and disc diffusion) were developed over 50 years ago They are very easy to perform So is banging a drum (ask any three-year old) Playing the drums well is much more difficult (ask the parents of any three-year old)
Using in-vitro susceptibility testing to detect resistance A two step process 1. Obtain a measure of in-vitro susceptibility (disc diffusion or MIC) 2. Interpret the meaning of that measure (resistant or sensitive)
In-vitro susceptibility testing protocols Susceptibility tests are not robust The results you get are totally dependent on the protocol you use Media Temperature Inoculation Time
PROBLEM 1 When different laboratories use different susceptibility test protocols They generate different data
Published % distributions of Flavobacterium psychrophilum oxytetracycline MIC using various non-standardised protocols MIC Lab A Lab B Lab C Lab D Lab E 0.03 8 4 8 0.06 11 5 7 3 0.12 12 21 7 0.25 43 5 6 0.5 12 6 5 1 2 3 15 10 5 2 17 8 20 4 2 22 14 15 8 2 24 10 33 16 2 5 15 32 15 3 64 40 <64 14 39
The need for standard test protocols We need to compare susceptibility data 1. Generated in the same laboratory at different times and by different workers 2. Generated in different laboratories and different countries We need internationally agreed standardised protocols for susceptibility testing
The need for standard susceptibility test protocols for aquaculture In 2000 there were many testing protocols being used 24 scientists from 17 countries met Many of the protocols being used were valid and they did not try to determine which was best They decided to adopt a single protocol on which they could all agree
Standard test protocols for aquaculture CLSI guidelines Disc Diffusion VET03-A replaces M42-A MIC VET04-A2 replaces M49-A Quality control and meaning VET03/VET04-S2
CLSI quality control For each susceptibility test protocol the acceptable range of results for reference strains are set Every day they test isolates laboratories MUST also test reference strains The results are valid ONLY IF the results with the reference strains are within the acceptable range
Quality control for test protocol using Mueller - Hinton at 22 c ± 2 for 44 48h Acceptable ranges for Aeromonas salmonicida subsp. salmonicida ATCC 33658 AMP ENRO FFN FLU OTC OXA O.002 0.004 0.008 0.015 0.03 0.06 0.12 0.25 0.5 1 2
Distributions of F. Psychrophilum oxytetracycline MIC Red data obtained using CLSI standard protocols Black data obtained using non-standard protocols MIC Lab 1 Lab 2 Lab 3 Lab 4 Lab A Lab B Lab C Lab D Lab E 0.015 1 14 0.03 24 40 9 18 8 4 8 0.06 25 5 32 58 11 5 7 3 0.12 4 12 21 7 0.25 1 5 43 5 6 0.5 4 9 1 12 6 5 1 13 9 2 3 15 10 5 2 20 16 5 17 8 20 4 11 3 23 12 2 22 14 15 8 2 24 10 33 16 2 5 15 32 15 3 64 40 Off-scale 25 10 14 39
Take home message 1 The internationally agreed and standardised CLSI test protocols should always be adopted When they are available
What do susceptibility test data mean? Can they tell us if a strain is sensitive or resistant? How do we define resistance? Can we set cut-off values for susceptibility data?
What do we mean by resistance? Clinical resistance Detected using clinical breakpoints The bacterium is clinically resistant if, because of its reduced susceptibility, an antibiotic therapy cannot reduce the morbidity or mortality resulting from its infections. Epidemiological (microbial) resistance Detected using epidemiological cut off values The bacterium is less susceptible than other members of its species. Genetic resistance Detected using molecular techniques The bacterium possesses genetic sequences that encode a mechanism that reduces susceptibility.
Detecting clinical resistance in aquatic animal medicine A bacterium should be considered as clinically resistant if, as a result of its reduced susceptibility to an antimicrobial agent, it would be able to continue to contribute to morbidity and mortality in a population during and after a particular administration of that agent to that population Clinical resistance depends on The susceptibility of the bacterium The properties and environment of the administration The nature and history of the treated population
Detecting clinical resistance in aquatic animal medicine In aquaculture Populations treated Diversity of aquatic animal species Diversity of environments (temperature, salinity) Poor standardisation of dosing Serious lack of data It will a long time before we can set clinical breakpoints of general value for aquaculture
Epidemiological cut-off values Epidemiological cut-off values depend on the in-vitro phenotypes of isolates and are protocol and species-specific They are not affected by the conditions of any therapeutic use and can be universally valid The OIE Aquatic Animal Health Code recommends that epidemiological cut-off values should be used to interpret the susceptibility data generated in monitoring and surveillance programmes
Epidemiological cut off values Wild type WT Susceptibility within the range established for fully susceptible members of the species Non wild type NWT Susceptibility outside the range established for fully susceptible members of the species The terms resistant and sensitive should not be used for categories established by epidemiological cut-off values
Establishing the range of zone sizes for fully susceptible members of a species The zone sizes for fully susceptible (WT) members of a bacterial species are normally distributed. The mean minus 2.5 standard deviations of their distribution represents the lower limit zones for 97.7% of the fully susceptible WT isolates This lower limit represent the epidemiological cut-off value
Percentage Zones for tetracycline against S. pyogenes WT distribution calculated by NRI Mean 28.0 mm Standard deviation 2.1 mm Epidemiological cut-off value 22 mm 20 18 16 14 12 10 8 6 4 2 0 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 Zone size (mm) WT NWT Normal distribution
Epidemiological cut-off values for MIC data Percentage For MIC data the log 2 transformed data for WT isolates are normally distributed Epidemiological cut-off value 4μg/ml 40 35 30 E. coli/tetracycline WT NWT 25 20 15 10 5 0 NRI 0.125 0.25 0.5 1 2 4 8 16 32 64 128 256 MIC
Statistically based objective methods for setting epidemiological cut-off values Disc diffusion data Normalised Resistance Interpretation (NRI) MIC data Normalised Resistance Interpretation (NRI) ECOFFinder Excel spreadsheets for performing these analyses automatically are available on-line NRI @ http://www.bioscand.se/nri/ ECOFFinder @ clsi.org/standards/micro/ecoffinder/
Take home message 2 The meaning of susceptibility test data should be established by applying internationally agreed interpretive criteria When they are available
Quantity of data needed to set epidemiological cut-off values Individual laboratories Can generate local values for application to their own data Data sets with a minimum of 30 WT observations are needed International authorities Can generate universal cut-off values Data sets from at least 3 laboratories with a minimum of 100 WT observations Terminology Cut-off values set locally are termed CO WT Cut-off values set by CLSI are termed ECVs Cut-off values set by EUCAST are termed ECOFFs
Application of ECVs in the clinical context ECVs have no inherent clinical relevance Possible laboratory comments If WT Laboratory susceptibility tests have detected full susceptibility There are no reason why therapy should not be initiated If NWT Laboratory susceptibility tests have detected reduced susceptibility. The isolate may contain resistance mechanisms. Initiation, or continuation with therapy would be imprudent and is likely to result in clinical failure
Take home message 3 Understanding each other s work is essential If we are to address our common problem of Antibiotic Resistance
Thank you for your attention
Distributions of F. Psychrophilum oxytetracycline MIC Red data obtained using CLSI standard protocols Black data obtained using non-standard protocols MIC Lab 1 Lab 2 Lab 3 Lab 4 Lab A Lab B Lab C Lab D Lab E 0.015 1 14 0.03 24 40 9 18 8 4 8 0.06 25 5 32 58 11 5 7 3 0.12 4 12 21 7 0.25 1 5 43 5 6 0.5 4 9 1 12 6 5 1 13 9 2 3 15 10 5 2 20 16 5 17 8 20 4 11 3 23 12 2 22 14 15 8 2 24 10 33 16 2 5 15 32 15 3 64 40 Off-scale 25 10 14 39
Detecting clinical resistance in human medicine Clinical breakpoints can be used to determine whether an isolate has such reduced susceptibility that antibiotic therapy would fail In human medicine clinical breakpoints have been set for antibiotics against many bacterial species Very large amounts of data (PK/PD and clinical outcomes) are required In human medicine we deal with treatments of individual patients at a single temperature with well standardised doses.