Determining antimicrobial MICs against aquaculture pathogens using Sensititre® plates

ASSOCIATE PROFESSOR AQUATIC ANIMAL HEALTH
Thad Cochran National Warmwater
Aquaculture Center
Department of Pathobiology and Population Medicine
College of Veterinary Medicine
Mississippi State University
By Patricia S. Gaunt, DVM, PhD, DABVT


SUMMARY

  • In US aquaculture, where bacterial infections are a leading cause of death, monitoring bacterial pathogen susceptibility to antibiotics is important because there are only three commercially available antimicrobials for food-fish use.

  • When conducted according to standardized methods, the minimal inhibitory concentration (MIC) assay can reliably assess the in vitro susceptibility of bacteria to an antibiotic.

  • The MIC value, coupled with pharmacokinetic and field efficacy data, can be used to predict the therapeutic plasma antibiotic concentrations needed to obtain optimal efficacy.

Introduction

Bacterial pathogens are a leading cause of mortality in aquacultured fish.1 However, only three antimicrobials - florfenicol (Aquaflor®), oxytetracycline (Terramycin®) and sulfadimethoxine/ ormetoprim (Romet 30®) - are commercially available for treating bacterial infections in the US food-fish industry. Consequently, it is important to monitor the susceptibility of bacterial pathogens to these antimicrobials.

Clinical breakpoints (interpretive criteria) define whether bacteria are susceptible, intermediately susceptible or resistant to an antibiotic, and their values are derived from extensive in vitro, pharmacokinetic and field trial studies.2,3 While clinical breakpoints are in place for many bacterial pathogens of terrestrial animals, the only aquaculture pathogen with established clinical breakpoints is Aeromonas salmonicida.4,5 Data to generate clinical breakpoints for fish pathogens should be established in a standardized fashion so they can be consistently reproduced in any laboratory.

Bacteria isolated from fish require different laboratory testing conditions compared to those for terrestrial animals because there are physiological differences, such as the temperature variation between “cold-blooded” fish (poikilotherms) and “warm-blooded” terrestrial animals (homeotherms).4 Diagnostic laboratories that routinely test aquatic bacteria have established their own laboratory-specific clinical breakpoints based on two parameters: specific in vitro assays and the clinical field response to antimicrobial use.

In an effort to establish formal breakpoints for aquatic bacteria, the Clinical and Laboratory Standards Institute (CLSI) has published guidelines for the methodology of minimal inhibitory concentration (MIC) assays.2 The MIC technique is a quantitative antimicrobial susceptibility assay.2,3 MIC assays can be conducted using either large volumes (≥1 mL) of inoculum (broth-macrodilution assay) or small volumes (50-100 μl) of inoculum (broth-microdilution assay). Most laboratories prefer the broth-microdilution assay because it requires smaller inocula volumes, is less labor intensive and is contained within a single tray or plate.

Although CLSI provides the procedures for in-house preparation of broth microdilution plates with serial dilutions of antimicrobial standards, microdilution plates are commercially available with lyophilized antimicrobials. For illustration purposes, the following discussion will focus on the commercially prepared microdilution plates (Sensititre® Plate, Trek Diagnostic Systems Ltd., Cleveland, OH) containing the antibiotic florfenicol (Figure 1). Florfenicol (FFC), the active ingredient in Aquaflor, was recently approved in the US for control of mortality associated with Edwardsiella ictaluri in catfish and Flavobacterium psychrophilum and Aeromonas salmonicida in freshwater-reared salmonids.6,7,8 In addition, Aquaflor®-CA1 (florfenicol) is conditionally approved for control of mortality in catfish due to columnaris disease associated with Flavobacterium columnare.9

Determining FFC MICs with the broth-microdilution assay

Prior to in vitro susceptibility testing, the bacteria must be purified and identified using routine morphological, biochemical and/or polymerase chain reaction assays. Sensititre microdilution MIC assays utilize sterile 96-well microdilution plates arranged in rows and columns. The last column of wells contains 0 μg/mL FFC and serves as a positive control to assess the viability of organisms (Figure 2). Each of the following columns contains FFC in a series of two-fold dilutions that begin with 0.125 μg/mL FFC; the concentration then doubles in each subsequent column to a maximum FFC concentration of 128 μg/mL. Following is the procedure for setting up the MIC assay according to CLSI guidelines.2

Microdilution plate for broth dilution susceptibility testing of bacteria

Figure 1

Microdilution plate for broth dilution susceptibility testing of bacteria
Florfenicol (FFC) dilutions double in each vertical column of wells with the exception of the last column, which contained no FFC (see arrow).

Microdilution plate for broth dilution susceptibility testing of bacteria

Figure 2

Sensititre Plate inoculated with Edwardsiella ictaluri to determine the florfenicol (FFC) minimal inhibitory concentration (MIC)

Row A (see arrow) contained uninoculated broth (negative control). Note the clear appearance in the wells, indicating no bacterial growth.

Rows B-H of the last column contained no FFC. Without the presence of the antibiotic, there was bacterial growth appearing as a cloudy dense button in the well bottom (positive control).

The remaining wells contained FFC in dilutions beginning at 0.125 μg/mL and doubling in each column, from left to right, to a final concentration of 128 μg/mL.

Row B was inoculated with E. coli ATCC 25922 to serve as the quality control. The first five wells in Row B, which contained FFC concentrations ranging from 0.125 μg/mL, showed growth of the bacterium. The MIC for FFC against E. coli ATCC 25922 was 4 μg/mL.

Rows C-H were inoculated with E. ictaluri. The first two wells in each row showed growth of the bacterium. The MIC for FFC against the E. ictaluri isolate was 0.5 μg/mL.

1
Three to five (3 to 5) bacterial colonies are removed from a 24- to 48-hour non-selective agar plate and are inoculated directly into broth or sterile saline. To ensure uniformity in methodology, cation-adjusted Mueller Hinton broth (CAMHB) is the broth of choice for culturing most aquatic pathogens. Some fastidious organisms require supplemented media, and information on these special requirements is provided by CLSI M49-A.2

2
The density of testing inocula is standardized to a 0.5 McFarland standard. This is equivalent to a cell density of ~1-2 x 108 cfu/mL, which should be verified periodically using plate counts.

3
The standardized inocula suspensions are diluted in CAMHB so that each well on the plate contains ~5 x 105 cfu/mL. A known inoculum volume per well is used to make this calculation. For example, if 100 μL of medium and an inoculum volume of 5 μL are used, the 1 x 108 dilution suspension must be diluted to yield 107 cfu/mL. This will yield a final test concentration of bacteria in the well of ~5 x 104 cfu/well (~5 x 105 cfu/mL).

Each test organism is inoculated in a separate row. The test inoculum suspension should be subcultured on agar plates to check for purity.

Included in each tray should be a column of wells containing no antimicrobial agent (positive control wells) to assess the viability of organisms.

4
One row of wells per plate is inoculated with a quality-control organism (Figure 2). Escherichia coli ATCC 25922 or Aeromonas salmonicida subspsalmonicida ATCC 33658 is recommended by CLSI as the control organism for most aquatic pathogens tested between 22° C to 28° C (71.6° F to 82.4° F). For organisms tested at higher temperatures or other special requirements, the reader is referred to CLSI M-49 for additional qualitycontrol strain recommendations.

5
One row of wells per plate is filled with un-inoculated broth to serve as a negative control.

6
To prevent drying during incubation, each plate is sealed with a tight-fitting plastic cover before incubation. The plates are stacked ≤ four plates high in the incubator.

7
The microdilution trays are incubated at 22° C to 28° C (71.6° F to 82.4° F) } 2° C for either 24 to 28 hours or 44 to 48 hours for bacteria such as E. ictaluri, F. columnare and A. salmonicida. Extended incubation of inoculated plates is discouraged because antimicrobial deterioration could result in falsely elevated MICs. For assaying FFC's MIC against F. psychrophilum, which requires a lower incubation temperature, the reader is referred to CLSI M49-A.2

8
Bacterial growth appears as a dense button in the well bottom. The well with the lowest concentration of FFC exhibiting no observable growth is the MIC against the test organism. For the results to be valid, a button of bacteria ≥2 mm in diameter must grow in the positive control well.

Discussion

When conducted according to standardized methods, the MIC assay will reliably assess the in vitro susceptibility of bacteria to an antibiotic.2,3,4 The culture of the same organism by different personnel and laboratories using different media, antibiotic strengths, incubation temperatures and durations has produced highly variable results,4 which will confound the usefulness of in vitro testing.

In an effort to establish reliable clinical breakpoints for aquatic bacteria, the CLSI has approved guidelines for determining MIC assays for nonfastidious bacteria such as E. ictaluriand A. salmonicida.2 Standards are provided for media preparation, inoculation densities, incubation temperature, quality-control organisms and interpretation of results to ensure these procedures are rigorous and reproducible between laboratories. However, guidelines for certain groups of bacteria, e.g., gliding bacteria such as F. columnare and F. psychrophilum, are only provisional.

In addition to assessing bacterial susceptibility, the MIC value is often used to predict therapeutic plasma antibiotic concentrations needed in patients to obtain optimal efficacy.10,11 The therapeutic plasma concentration of an antibiotic should exceed its MIC value against a particular pathogen for the interdose interval or by a concentration factor depending on whether the antibiotic acts via a timedependent or concentration-dependent mechanism, respectively.12

However, there are limitations when using this calculation to predict therapeutic outcomes because marked dissimilarities occur between in vitro and in vivo conditions.3,4 Internal and external factors influence the antimicrobial plasma concentration calculated from in vitro methods and the concentration of antibiotic at the site of infection in a piscine patient. These factors include: drug administration route and pharmacokinetics, species and disease state of fish, water temperature and salinity, virulence of the pathogen and its susceptibility to antimicrobial treatment, as well as the presence of multiple pathogens.3,4,10

Before predicting the therapeutic outcome with an antibiotic administered to diseased fish, data from both in vitro and in vivo factors should be assessed. In vitro antimicrobial data obtained by standardized MIC methodology should be correlated with both piscine pharmacokinetic and field efficacy data, allowing universal clinical breakpoints to be established for aquatic bacterial pathogens. This will enable diagnosticians to reliably monitor antimicrobial susceptibility between laboratories and choose the appropriate antimicrobial therapy when needed.


Regarding the use of Aquaflor in the US:
CAUTION: Federal law restricts medicated feed containing this veterinary feed directive (VFD) drug to use by or on the order of a licensed veterinarian.

References

1 Plumb J. Epizootiology of Fish Diseases. Health Maintenance and Principal Microbial Diseases of Cultured Fishes. Iowa State University, Ames, Iowa, USA, 1999:24-28.

2 Clinical and Laboratory Standards Institute (CLSI). Methods for Broth Dilution Susceptibility Testing of Bacteria Isolated From Aquatic Animals; Approved Guideline. CLSI document M49-A (ISBN 1-56238-612-3). Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898, USA. 2006.

3 Forbes BA, Sahm DF, Weissfeld AS. Laboratory Methods for Detection of Antibacterial Resistance. Bailey & Scott's Diagnostic Microbiology. St. Louis, Missouri: Mosby, 2002:229-250.

4 Miller R, Reimschuessel R. Epidemiologic cutoff values for antimicrobial agents against Aeromonas salmonicida isolates determined by frequency distributions of minimal inhibitory concentration and diameter of zone of inhibition data. American Journal of Veterinary Research 2006;67:1837-1843.

5 Clinical and Laboratory Standards Institute (CLSI). Performance Standards for Antimicrobial Susceptibility Testing of Bacteria Isolated From Aquatic Animals: First Informational Supplement. CLSI document M42/M49-S1 (ISBN 1-56238- 727-8). Clinical and Laboratory Standards Institute, 940 West Valley Road, Site 1400, Wayne, Pennsylvania 19087-1898 USA, 2010.

6 United States Food and Drug Administration, Center for Veterinary Medicine. FDA Approves New Antimicrobial for Catfish. October 25, 2005. Available at: http://www.fda.gov/AnimalVeterinary/ NewsEvents/CVMUpdates/ucm048397.htm. Accessed September 03, 2009.

7 United States Food and Drug Administration. FDA Approval of New Antimicrobial for Salmonids. March 22, 2007. Available at: http://www.fda.gov/AnimalVeterinary/NewsEvents/CVMUpd ates/ucm045829.htm. Accessed August 12, 2009.

8 United States Food and Drug Administration. FDA Approves New Antimicrobial for Salmonids for Treatment of Furunculosis. November 6,2007. Available at:http://www.fda.gov/AnimalVeterinary/NewsEvents/CVMU pdates/ucm045818.htm. Accessed August 12, 2009.

9 United States Food and Drug Administration. First FDA Conditionally Approved New Animal Drug for Columnaris Disease in Catfish. April 18, 2007. Available at: http://www.fda.gov/AnimalVeterinary/NewsEvents/CVMUpd ates/ucm045571.htm. Accessed August 12, 2009.

10 Samuelsen OB, Bergh Ø, Arne E. Pharmacokinetics of florfenicol in cod Gadus morhua and in vitro antibacterial activity against Vibrio anguillarum. Diseases of Aquatic Organisms 2003;56:127-133.

11 Stamm JM. In vitro resistance by fish pathogens to aquacultural antibacterials, including the quinolones difloxacin (A-56619) and sarafloxacin (A-56620). Journal of Aquatic Animal Health 1989;1:135-141.

12 AliAbadi FS and Lees P. Antibiotic treatment for animals: effect on bacterial population and dosage regimen optimization. International Journal of Antimicrobial Agents 2000;14:307-313.


Terramycin® is a registered trademark of Phibro Animal Health Corporation. Romet® is a registered trademark of PHARMAQ. Sensititre® is a registered trademark of Trek Diagnostic Systems.




 

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