Aquaculture Extension Manual No. 37
Laboratory Manual of Standardized Methods for Antimicrobial
Sensitivity Tests for Bacteria Isolated from Aquatic Animals and Environment
Lila Ruangpan and Eleonor A. Tendencia
Southeast Asian Fisheries Development Center Aquaculture Department
Government of Japan Trust Fund
December 2004
Aquaculture Extension Manual No. 37
Laboratory Manual of Standardized Methods for Antimicrobial Sensitivity Tests for
Bacteria Isolated from Aquaculture Animals and Environment
December 2004
ISBN 971-8511-74-1
Coryright 2004
Southeast Asian Fisheries Development Center, Aquaculture Department
Tigbauan 5021, Iloilo, Philippines
All Rights Reserved
No part of this publication may be reproduced or transmitted in any form or by any
means, electronic or mechanical, including photocopy, recording, or any information
storage and retrieval system, without the permission in writing from the publisher.
Citation is as follows:
Ruangpan, L. and E.A. Tendencia. 2004. Laboratory manual of standardized methods
for antimicrobial sensitivity tests for bacteria isolated from aquaculture. Southeast
Asian Fisheries Development Center, Aquaculture Department, Iloilo, Philippines.
Published by:
Southeast Asian Fisheries Development Center
Aquaculture Department
Tigbauan 5021, Iloilo, Philippines
For inquiries and comments:
Fish Health Section
Southeast Asian Fisheries Development Center
Aquaculture Department
Tigbauan 5021, Iloilo, Philippines
Fax: (63 33) 335 1008
Tel : (63 33) 336 2965
E-mail: aqdchief@aqd.seafdec.org.ph, training@aqd.seafdec.org.ph
AQD website: http://www.seafdec.org.ph/
Worldwide trade in aquaculture products demands strict adherence to standards for good quality and safe seafood for the consumers. Thus, information on good
aquaculture practices, specifically those that have direct influence on marketable
products, should be disseminated. One of the most needed methods concerns health
maintenance and disease control. While fish products are generally considered wholesome and healthy, farming practices during production may affect their quality.
The use and misuse of antibiotics in aquaculture have led to rejection of exported
products. Thus, farmers and all sectors involved in the production and the marketing
process need to know about the hazards of employing practices that may lead to product contamination.
Realizing the importance of this issue and in response to growing need to evaluate
the extent of antibiotic usage in aquaculture, the Government of Japan has, since the
year 2000, funded the Southeast Asian Fisheries Development Center (SEAFDEC) through the Trust Fund for the Regional Fish Disease Project entitled
"Development
of Fish Disease Inspection Methodologies for Artificially-Bred Seeds." Under this
regional project, various research studies were conducted, and two authors (Dr. Lila Ruangpan, Chanthaburi Coastal Fisheries Research and Development Center, Thailand and Ms. Eleonor A. Tendencia, SEAFDEC Aquaculture Department,
Philippines) of this manual were among the study leaders. The manual is one of the
important outputs of a collection of studies related to antibiotic usage in order to
come up with guidelines for its prudent usage. It offers a complete guide for testing
bacterial susceptibility and resistance through the use of simple techniques for diskagar
diffusion tests, and a guide to do a more thorough study to test therapeutic levels
using microbial inhibitory concentration.
Kazuya NAGASAWA, Ph.D.
Fish Disease Expert and Leader of the Regional Fish Disease Project
Southeast Asian Fisheries Development Center, Aquaculture Department
Tigbauan 5021, Iloilo, Philippines
December 2004
Foreword
III
Table of Contents
V
Acknowledgments
VII
Introduction
1
Chapter 1. Bacterial Isolation, Identification and Storage
3
Principle
4
Isolation
5
Storage
7
Identification
8
References
9
Appendix 1.1. Important bacterial characteristics taken into
consideration when coding or labeling isolated bacteria
10
Appendix 1.2. Scheme for the identification of bacterial genera that
are important in aquaculture
11
Chapter 2. Disk Diffusion Method
13
Principle
14
Media
14
Inoculum
17
Antimicrobial disk
20
Control plate
21
Incubation
21
Reading and measurement of zones of inhibition
22
Interpretation of results
24
Rejection criteria
24
References
26
Appendix 2.1. Acceptable inhibitory zone diameter (mm) limit of control
strains recommended for use in the disk diffusion antimicrobial
sensitivity testing of bacteria isolated from animals
27
Appendix 2.2. Zone diameter interpretative standard for veterinary pathogens
28
Chapter 3. Minimal Inhibitory Concentration (MIC) Test and
Determination of Antimicrobial Resistant Bacteria
31
Principle
32
Media
32
Inoculum
37
Control Plate
41
Incubation
41
Reading the MIC values
44
Determination of Antimicrobial Resistant Bacteria
44
Inhibitory concentration at 50% and 90%
44
Determination of antimicrobial resistant bacteria
44
References
45
Appendix 3.1. List of culture apparatus, culture media, solvents, control strains,
and antimicrobial agents needed for the minimal inhibitory
concentration (MIC) test
47
Appendix 3.2. Preparation of antimicrobial agent stock solution
49
Appendix 3.3. List of solvents and diluents needed for the preparation of
stock solutions of antimicrobial agents
50
Appendix 3.4. Preparation of dilutions of antimicrobial agents for use in the agar
dilution method of minimal inhibitoryconcentration (MIC) test
51
Appendix 3.5. Interpretative categories and correlative minimal inhibitory
concentration (MICs) for food-borne pathogens
52
Appendix 3.6. Determination of inhibitory concentration at
50% (IC50) and 90 % (IC90) values
53
Appendix 3.7. Table of assigned values to evaluate the breakpoint, which
correlates to the minimal inhibitory concentration (MIC)
54
Appendix 3.8. Notes on laboratory practice
The Government of Japan Trust Fund through SEAFDEC Aquaculture Department
provided financial support for the research project and publication of this manual. We
would like to express our sincere thanks to Dr. Yasuo Inui and Dr. Kazuya
Nagasawa, the former and current Fish Disease Experts and Leaders of the Regional Fish Disease
Project, for their encouragement and critical suggestions; and the staff of the Fish
Health Section, SEAFDEC Aquaculture Department, for their kind cooperation, especially to Dr. Celia R. Lavilla-Pitogo for carefully reading the manuscript and Dr.
Edgar C. Amar for serving as the over-all coordinator in the preparation of drafts.
Thanks also to Dr. Hisatsugu Wakabayashi, Emeritus Professor of Fish Pathology Laboratory at the University of Tokyo, for reviewing the manuscript.
Bacteria are ubiquitous in the environment and may cause disease and mortality
in cultured shrimp and fish both in the hatchery and grow-out ponds. Accordingly,
antimicrobial agents are widely used by farmers especially in the intensive culture
system. Misuse of drugs and non-compliance of treatment regimens among users can
cause treatment to be less effective and prolong the duration of disease. Furthermore,
they may increase the prevalence of transferable resistance genes to the bacteria in
the environment and in food fish that may indirectly affect human health due to limited therapeutic options.
Since several antimicrobial agents are widely used in various areas of shrimp and fish culture, testing for the susceptibility and resistance of bacteria to different agents need to be conducted. The data gathered from the tests are essential in the selection of the most effective antimicrobial agents against pathogenic bacteria. Furthermore, the values obtained in the tests are useful in determining the dosage regimen of each antimicrobial. Different methods have been employed in laboratories to determine the susceptibility of microorganisms to antimicrobial agents. Two main methods for the in vitro determination of the susceptibility of microorganisms against antimicrobials are the disk agar diffusion test, wherein antibiotic-impregnated disks are used with an agar medium, and the dilution techniques, wherein the test microorganism is exposed to increasing concentration of an antibiotic either in broth or agar.
The disk agar diffusion method is the most widely used laboratory technique for antimicrobial susceptibility test. This method is simple, requires a short duration, and easy to perform. It also provides an accurate determination of the susceptibility of an organism to a measured amount of test antibiotics. However, only a few antimicrobials have veterinary-specific interpretative criteria; therefore, human interpretative criteria are used for the majority of the chemotherapeutants. Human interpretative criteria could, however, vary from veterinary-specific data due to strain differences in the bacterial species.
The dilution technique, using broth and agar media, is used to measure
quantitatively the in vitro activity of antimicrobial agents in terms of susceptibility
and resistance by bacteria (Thornsberry et al., 1977; Thornsberry and McDougal, 1983). The minimal inhibitory concentration (MIC) using agar dilution is one of the
appropriate in vitro tests widely used among several laboratories (McDonald et al.,
1995). The results obtained by MIC dilution test can give an indication as to the
concentration of antimicrobial agent needed to inhibit or eradicate the infectious
organism. Basically, a series of plates is prepared with an agar medium to which
various concentrations of the antimicrobial agents are added. The plates are then
inoculated with a suitable standardized suspension of the test bacteria. After the
plates are incubated overnight under a certain temperature, examination of the test
results is performed, MIC values are read, and drug resistance of the bacterial strains
is determined.
In both techniques, it is important that control strains be included to check the quality of batches of media and to monitor the performance of the assay protocol. The selection of the control strain should be based on the kind of bacteria being investigated in any set of experiments. The National Committee for Clinical Laboratory Standards (NCCLS) recommended the use of the following organisms for the quality control of antimicrobial susceptibility test: Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus faecalis, Actinobacillus pleuropneumoniae, Haemophilus somnus, Campylobacter jejuni, Streptococcus pneumoniae, and Klebsiella pneumoniae.
The series of procedures, materials preparation, as well as the interpretative criteria to obtain the final results in antimicrobial sensitivity testing are influenced greatly by the method used, which must be standardized and carefully controlled. The standard methods found in this manual are written for the students, teachers, laboratory technicians, researchers, and scientists who work in this field.
REFERENCES
McDonald, C.L., W.E. Maher and R.J. Fass. 1995. Revised interpretation of oxacillin
MICs for Staphylococcus epidermidis based on MecA detection. Antimicrob.
Agents Chemother., 39: 982-984.
Thornsberry, C., T.L. Gaven and E.H. Gerlach. 1977. New developments in
antimicrobial agent susceptibility testing. Cumitech, 6: 1-13.
Thornsberry, C. and L.K. McDougal. 1983. Successful use of broth microdilution
in susceptibility tests for methicillin-resistant (heteroresistant) stapthylococci.
Clin. Microbiol., 18: 1084-1091.