FMM/RAS/298: Strengthening capacities, policies and national action plans on prudent and responsible use of antimicrobials in fisheries Workshop 2 in cooperation with Malaysia Department of Fisheries and INFOFISH 7-9 August 2017, Kuala Lumpur, Malaysia

Design of antimicrobial susceptibility testing programmes relevant to aquaculture and aquacultural products Peter Smith [email protected] “An expert is someone who knows some of the worst mistakes that can be made in his subject, and how to avoid them.” Werner Heisenburg SELECTING A STANDARD TESTING PROTOCOL FOR YOUR SPECIES OF INTEREST

Medium & incubation temperature All members of species should give sufficient growth

Availability of QC ranges Protocol should have QC ranges for antimicrobials of interest

Availability of interpretive criteria(epidemiological cut-off values) These are protocol and species specific. Few are available, They are essential but easy to set (NRI and ECOFFinder). Should not influence selection of any testing protocol GROUP 1 NON FASTIDIOUS G-VE SPECIES

Aeromonas caviae Aeromonas veronii Edwardsiella tarda Aeromonas hydrophila Acinetobacter spp Edwardsiella anguillarum Aeromonas jandaei Citrobacter spp Pseudomonas anguilliseptica Aeromonas salmonicida Edwardsiella piscicida Pseudomonas fluorescens Aeromonas sobria Edwardsiella ictaluri Yersinia ruckeri

Medium All can be tested with unmodified Mueller-Hinton

Temperature All (except Aeromonas salmonicida) can be tested at 35°C, 28°C & 22°C

THERE ARE STANDARD PROTOCOLS FOR ALL GROUP 1 SPECIES QC acceptable ranges using M-H media

MIC Disc Temperature (°C) 35 28 & 22 35 28 & 22 Agents used in aquaculture Amoxicillin/ampicillin Chloramphenicol Doxycycline Enrofloxacin Erythromycin Florfenicol Flumequine Nitrofurantoin Ormetoprim-sulfadimethoxine Oxolinic acid Oxytetracycline Trimethoprim Trimethoprim-sulfamethoxazole Sulphamethoxazole Agents used in humans Various 92 0 89 0 GROUP 2 NON-FASTIDIOUS G-VE FACULTATIVE AND OBLIGATE HALOPHILES

Mesophiles Vibrio anguillarum Vibrio harveyi Vibrio parahaemolyticus Vibrio vulnificus Photobacterium damselae

Psychrophile Aliivibrio salmonicida

DO ANY REQUIRE ADDITION OF 1% NaCl TO MUELLER- HINTON? QC acceptable ranges using M-H media + 1% NaCl

MIC Disc Temperature (°C) 35 28 & 22 35 28 & 22 Agents used in aquaculture Amoxicillin/ampicillin Chloramphenicol Doxycycline Enrofloxacin Erythromycin Florfenicol Flumequine Nitrofurantoin Ormetoprim-sulfadimethoxine Oxolinic acid Oxytetracycline Trimethoprim Trimethoprim-sulfamethoxazole Sulphamethoxazole Agents used in humans Various 0 0 0 0 QC acceptable ranges using M-H media

MIC Disc Temperature (°C) 35 28 & 22 35 28 & 22 Agents used in aquaculture Amoxicillin/ampicillin Chloramphenicol Doxycycline Enrofloxacin Erythromycin Florfenicol Flumequine Nitrofurantoin Ormetoprim-sulfadimethoxine Oxolinic acid Oxytetracycline Trimethoprim Trimethoprim-sulfamethoxazole Sulphamethoxazole Agents used in humans Various 92 0 89 0 GROUP 2A NON-FASTIDIOUS G-VE FACULTATIVE HALOPHILES

Medium There is evidence that all the mesophilic halophiles listed have been tested with unmodified Mueller-Hinton

Temperature There is evidence that the mesophilic halophiles can be tested at 28°C & 22°C. Most (except V. anguillarum?) can be tested at 35°C GROUP 2B NON-FASTIDIOUS G-VE OBLIGATE HALOPHILES

If any halophilic species are encountered that cannot be tested on un-modified Mueller-Hinton it is recommended that 1% NaCl should be added.

THERE ARE NO QC ACCEPTABLE RANGES FOR MUELLER-HINTON MEDIA WITH 1% NaCl GROUP 3 FLAVOBACTERIA AND RELATED SPECIES

Flavobacterium branchiophilum Flavobacterium psychrophilum Flavobacterium columnare Tenacibaculum maritimum

Media Standard protocols for MIC testing of the Flavobacteria spp. specify the use of dilute Mueller-Hinton broth

Temperature F. columnare is tested at 28°C F. psychrophilum and F. branchiophilum at 18°C

THERE ARE NO STANDARD PROTOCOLS FOR T. maritimum OR FOR DISC DIFFUSION TESTING OF ANY OF THESE 4 SPECIES MIC QC acceptable ranges using dilute M-H media

28°C 18 °C Reference strain E. coli A. sal E. coli A. sal Agents used in aquaculture Amoxicillin/ampicillin Chloramphenicol Doxycycline Enrofloxacin Erythromycin Florfenicol Flumequine Nitrofurantoin Ormetoprim-sulfadimethoxine Oxolinic acid Oxytetracycline Trimethoprim Trimethoprim-sulfamethoxazole Sulphamethoxazole Agents used in humans Various 0 0 0 0 GROUP 4 G+VE COCCI

Aerococcus viridans Streptococcus iniae Lactococcus garvieae Streptococcus phocae Streptococcus agalactiae Weissella spp. Streptococcus dysgalactiae

DO THESE SPECIES REQUIRE ADDITION OF BLOOD PRODUCTS? GROUP 4 G+VE COCCI

Medium The current recommendation is the M-H media should be supplemented

with blood products and incubation should be in 5% CO2

There is evidence that the G+ve cocci listed can be tested with unmodified Mueller-Hinton incubated in air.

Temperature There is evidence that the G+ve cocci can be tested at 28°C & 22°C. Most (except S. phocae ) can be tested at 35°C QC acceptable ranges at 35°C with and without blood products

MIC Disc

Additions none Blood CO2 none Blood CO2 Reference strain E. coli S. E. coli S. pneumoniae pneumoniae Agents used in aquaculture Amoxicillin/ampicillin Chloramphenicol Doxycycline Enrofloxacin Erythromycin Florfenicol Flumequine Nitrofurantoin Ormetoprim-sulfadimethoxine Oxolinic acid Oxytetracycline Trimethoprim Trimethoprim-sulfamethoxazole Sulphamethoxazole Agents used in humans Various 92 62 89 67 QC acceptable ranges at 28°C with and without blood products

MIC Disc

Additions none Blood CO2 none Blood CO2 Reference strain E. coli/A.sal S. E. coli S. pneumoniae pneumoniae Agents used in aquaculture Amoxicillin/ampicillin Chloramphenicol Doxycycline Enrofloxacin Erythromycin Florfenicol Flumequine Nitrofurantoin Ormetoprim-sulfadimethoxine Oxolinic acid Oxytetracycline Trimethoprim Trimethoprim-sulfamethoxazole Sulphamethoxazole Agents used in humans Various 0 0 0 0 GROUP 5 MYCOBACTERIA AND RELATED SPECIES

Mycobacterium fortuitum M. marinum Nocardia asteroides N. crassostreae N. seriolae

PROTOCOLS FOR TESTING THESE SPCIES ARE AVALABLE IN CLSI GUIDELINE M42-A2 GROUP 6 MISCELLANEOUS SPECIES

Clostridium botulinum Francisella noatunensis Hepatobacter penaei Piscirickettsia salmonis Renibacterium salmoninarum

THERE ARE NO STANDARD PROTOCOLS FOR THESE SPECIES SELECTING A STANDARD TESTING PROTOCOL FOR YOUR SPECIES OF INTEREST

Summary

CLSI standard susceptibility test protocols with QC ranges for species or groups

Number examined 42 MIC protocols available 35 Disc protocols available 28 SELECTING A STANDARD TESTING PROTOCOL FOR YOUR SPECIES OF INTEREST

Summary

Species for which no standard protocols have been set Aliivibrio salmonicida Clostridium botulinum Francisella noatunensis Hepatobacter penaei Piscirickettsia salmonis Renibacterium salmoninarum Tenacibaculum maritimum

Antimicrobial agents listed as used in aquaculture for which no QC ranges have been set Josamycin, Neomycin, Thiamphenicol, Sulfamethoxazole

Antimcrobial agents which can be ‘paired’ Amoxycillin / Ampicillin Oxytetracycline / Doxycycline Oxolinic acid / Flumequine Ormetoprim-sulfadimethoxine / Trimethoprim-sulfamethoxazole STUDY DESIGN To establish the susceptibility to the antimicrobial agents used as therapeutic agents in aquaculture of the bacterial species that are the primary targets of that use.

Species Those that are the most frequent targets of antibiotic therapy in aquaculture

Agents Those that are most frequently used in aquaculture

Strain collection Diagnostic investigations of infected animals or sampling of the immediate environment of infected populations To investigate the public heath implications of the presence of bacteria with reduced antimicrobial susceptibility in aquaculture products

Species Those that are the most frequent causes of infections in humans requiring antimicrobial therapy mediated by the consumption of or contact with aquacultural products

Agents Those that are the most frequently used in humans as therapeutic treatments of the food-borne or contact-transmitted infections associated with aquacultural products

Strain collection From aquaculture product at ‘point-of-sale’ To investigate the public heath implications of antimicrobial agent use in aquaculture mediated through aquaculture products

Species Those that are the most frequent causes of infections in humans mediated by the consumption of or contact with aquacultural products

Agents Those that are the most frequently used in humans as therapeutic treatments of the food-borne or contact-transmitted infections associated with aquacultural products

Strain collection Strains must be collected both from the water entering the farm and the aquaculture product leaving the farm To investigate the impact of antimicrobial agent use in aquaculture on the susceptibility to antimicrobial agents of the microflora in the local environment of aquaculture enterprises

Studies of this type involve the detection of gene sequences and do not involve the isolation or phenotypic study of bacteria “Science is built of facts just as a house is built of bricks, but a pile of facts is no more a science than a pile of bricks is a house.” Henri Poincaré

It is easy to generate data

It is only when we use appropriate methods in well designed studies that we learn what they mean GOOD LUCK IN ALL YOUR WORK