Food and Drug Administration, Office of Research, Center for Veterinary Medicine, Laurel, Maryland 20708
JMI Laboratories, North Liberty, Iowa 52317
Quality control ranges were developed for broth microdilution testing of Campylobacter jejuni ATCC 33560 against 14 antimicrobials. Cation-adjusted Mueller-Hinton broth containing 2.5% laked horse blood was the preferred medium, with incubation in a microaerobic atmosphere of 10% CO2, 5% O2, and 85% N2 at 36°C for 48 h or 42°C for 24 h.
Members of the genus Campylobacter are well-known as leading causes of bacterial gastroenteritis in humans (2). While intestinal campylobacteriosis typically resolves without antimicrobial intervention, treatment is warranted to manage severe or invasive illness. The treatment of choice traditionally has been a macrolide or fluoroquinolone (1). Historically, Campylobacter spp. have been susceptible to these and several other agents; however, there is evidence of increasing resistance, most notably to the fluoroquinolones (1, 5, 5a, 7).
Presently, the Clinical and Laboratory Standards Institute (CLSI; formerly the National Committee for Clinical Laboratory Standards) recognizes only agar dilution as a standardized method for the in vitro antimicrobial susceptibility testing of Campylobacter spp. (4). This method was developed with quality control (QC) ranges for five antimicrobials—ciprofloxacin, doxycycline, gentamicin, erythromycin, and meropenem—using C. jejuni ATCC 33560 as the QC organism (6). Agar dilution is labor-intensive and requires specialized equipment and training. It is cost-prohibitive when a small number of isolates is being tested and is therefore difficult to implement in most laboratory settings. The main purpose of this study was to identify testing conditions that would yield reproducible results with a broth microdilution format. Because broth microdilution is a familiar method that can easily be used to test one or more isolates and because it is amenable to semi-automation, it has clear technical advantages. In addition, we sought to establish QC ranges for a larger number of antimicrobial agents to include those of value to clinical laboratories and to researchers and for the monitoring efforts of national public health surveillance systems.
The data were generated in a multilaboratory study following the guidelines described in National Committee for Clinical Laboratory Standards document M23-A5 (8). The QC organism, C. jejuni ATCC 33560, was stored at –70°C in Brucella broth with 20% glycerol and recovered from freezer stocks by overnight incubation in a microaerobic atmosphere (10% CO2, 5% O2, 85% N2) on tryptic soy agar with 5% defibrinated sheep blood. Testing was performed with commercially prepared frozen panels (Trek Diagnostics, Cleveland, Ohio) containing serial twofold antimicrobial drug dilutions in three different lots of cation-adjusted MH broth supplemented with 2.5% laked horse blood. Testing was done with a humid microaerobic atmosphere (as described above), generated by using either gas-regulated incubators or sealed plastic boxes containing gas-generating sachets (Remel, Lenexa, KS; Oxoid, Ogdensburg, NY). In each laboratory, the QC organism was tested in parallel at 36°C for 48 h and 42°C for 24 h over 10 consecutive days.
Inocula were prepared from overnight growth on blood agar plates by suspension in sterile distilled water, saline, or MH broth to obtain a turbidity equivalent to that of a 0.5 McFarland standard. The suspension was added to inoculum trays, and the wells of the microtiter panels were seeded using a 96-pin inoculator, which produced a concentration of approximately 5.0 x 105 CFU/ml in each well. Inoculated microtiter panels were sealed with perforated gas-permeable covers (Trek Diagnostics) and stacked no more than four high to ensure uniform temperature and atmosphere throughout the incubation period. During the study, laboratories performed colony counts to ensure proper inoculum densities. The mean colony counts ranged from 1.0 x 104 to 2.7 x 106 CFU/ml.
The antimicrobial agents and the twofold dilution ranges tested for each drug were as follows: azithromycin, 0.015 to 16 μg/ml; chloramphenicol, 0.03 to 64 μg/ml; ciprofloxacin, 0.004 to 8 μg/ml; clarithromycin, 0.015 to 16 μg/ml; clindamycin, 0.008 to 8 μg/ml; doxycycline, 0.008 to 32 μg/ml; erythromycin, 0.015 to 16 μg/ml; florfenicol, 0.03 to 64 μg/ml; gentamicin, 0.015 to 16 μg/ml; levofloxacin, 0.004 to 8 μg/ml; meropenem, 0.001 to 8 μg/ml; nalidixic acid, 0.12 to 256 μg/ml; telithromycin, 0.03 to 64 μg/ml; and tetracycline, 0.06 to 32 μg/ml.
Table 1 shows the frequency distributions of C. jejuni ATCC 33560 for the 14 antimicrobial agents for both testing conditions. Slightly different MIC ranges were generated depending upon the incubation time and temperature used for testing. These differences were, however, within a single twofold dilution increment. Reproducibility at either temperature was not affected by the use of different lots of medium (data not shown). The proposed QC concentration ranges included either a 3 or a 4 log2 dilution range for all agents. These MIC QC limits encompassed 95% of the observed values under both incubation conditions, except for erythromycin at 36°C and meropenem. For erythromycin, 94% of the values were within the proposed three-dilution QC ranges for all 10 laboratories; achievement of 95% would have required extension to an unacceptable five-dilution range. When the meropenem data for all 10 laboratories was included, the agreement was only 80%, with one laboratory being consistently one dilution below the QC range and another being consistently one dilution above the QC range; there was a 97% agreement when the data were adjusted for eight labs. Based on the data provided, the CLSI Subcommittees on Antimicrobial Susceptibility Testing and Veterinary Antimicrobial Susceptibility Testing approved the QC ranges indicated in boldface type in Table 1. This method and the QC limits will be published in upcoming CLSI documents.
In the course of developing the method, it was discovered that many isolates failed to grow at either 43°C or 35°C. Thus, it is recommended that stable temperatures of 36 to 37°C and 42°C be maintained. It was also observed that sealable plastic bags, such as those commonly used in primary isolation, were not adequate for maintaining a stable atmosphere and should not be used for susceptibility testing. In addition, one commercially available microaerobic atmosphere-generating system did not yield consistent results. Thus, it is important to adhere to the CLSI recommendation of repeated preliminary testing of the QC organism before the implementation of a new testing method.
The antimicrobial agents tested in this study were chosen based on those considered important for treating campylobacteriosis and for the surveillance needs of the National Antimicrobial Resistance Monitoring System (http://www.fda.gov/cvm/narms_pg.html#Data) for monitoring trends in susceptibility among agents used in human and veterinary medicine. The availability of a standardized broth microdilution susceptibility testing method for Campylobacter will facilitate routine clinical testing and help ensure the accuracy and comparability of data from researchers and public health monitoring systems.
Contributing members of the Campylobacter Susceptibility Testing Working Group include the following: Timothy Barrett and Kevin Joyce, CDC, Atlanta, GA; Steve Brown and Maria Traczewski, CMI, Wilsonville, OR; Jennifer Streit, Paul Rhomberg, and Jeff Kirby, JMI Laboratories, North Liberty, IA; Cindy Knapp, Scott Killian, and Nikki Holiday, Trek Diagnostics, Cleveland, OH; Jianghong Meng, Beilei Ge, and Yifan Zhang, University of Maryland, College Park, MD; Irving Nachamkin and Mei Yu, University of Pennsylvania, Philadelphia, PA; Barth Reller and Rachel Addison, Duke University Medical Center, Durham, NC; Robert Rennie, LeAnn Turnbill, and Cheryl Brosnikoff, University of Alberta Hospital, Edmonton, Alberta, Canada; Gary Stein and Sharon Schooley, Michigan State University, East Lansing, MI; and Qijing Zhang and Sonia Pereira, Iowa State University, Ames, IA.
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