Comparison of the Etest and agar dilution for in vitro antimicrobial susceptibility testing of Campylobacter

Abstract

The performance of the Etest and agar dilution for in vitro antimicrobial susceptibility of Campylobacter spp. was evaluated using a quality control strain Campylobactor jejuni ATCC 33560, and 81 C. jejuni and 54 Campylobacter coli isolates recovered from retail raw meats. Seven antimicrobial agents: chloramphenicol, ciprofloxacin, doxycycline, erythromycin, gentamicin, nalidixic acid and tetracycline, were tested using the two methods, whereas azithromycin was tested using the Etest only. The correlation between the Etest and agar dilution MICs varied greatly depending on the antimicrobial agents tested. The overall agreement of MICs (±1 log₂ dilution) between the two methods was 61.9%, ranging from 21.4% for nalidixic acid to 92.6% for gentamicin. MICs obtained using the Etest were generally lower than those by agar dilution regardless of the species of organism tested. MIC₅₀ and/or MIC₉₀ values were at least one dilution lower for the Etest than for agar dilution when testing chloramphenicol, ciprofloxacin, doxycycline, erythromycin and nalidixic acid. Based on the agar dilution MICs, the resistant rate of the 135 Campylobacter isolates was highest for tetracycline (82.2%), followed by doxycycline (78.5%), nalidixic acid (21.5%), ciprofloxacin (20.7%) and erythromycin (17.0%). None of the isolates demonstrated resistance to chloramphenicol or gentamicin. The study indicated that the Etest results were not in complete agreement with the agar dilution test. Although the Etest has been proven to be a satisfactory testing method, its use for Campylobacter susceptibility testing requires further standardization. The study also showed that C. jejuni and C. coli isolates resistant to antimicrobials used for treating campylobacteriosis were common in retail raw meats.

Received 7 February 2002; returned 3 May 2002; revised 13 June 2002; accepted 27 June 2002

Introduction

Campylobacter species, primarily Campylobacter jejuni and Campylobacter coli, are recognized as a major cause of human gastroenteritis worldwide, with an estimated 2.4 million cases each year in the United States.1 Campylobacteriosis is considered a zoonotic disease, with raw or undercooked poultry being an important source of Campylobacter infection.2 Contaminated milk, water, pork, beef, lamb and seafood also contribute to human infections.3

Campylobacter infection is usually a mild to moderate self-limiting diarrhoeal disease. However, severe and prolonged cases of enteritis, bacteraemia, septic arthritis and other extra-intestinal infections have also been reported.4C. jejuni has been identified as the predominant cause of antecedent infection in Guillain–Barré syndrome (GBS) and Miller Fisher syndrome, two frequent forms of acute inflammatory polyneuropathy.5 When antimicrobial agents are recommended for the treatment of patients with severe, prolonged or relapsing campylobacteriosis, erythromycin or a fluoroquinolone such as ciprofloxacin is the drug of choice. Tetracycline, doxycycline and chloramphenicol are sometimes listed as alternative drugs for treatment. Serious systemic infections may also be treated with an aminoglycoside such as gentamicin.2^,4 However, an increasing number of Campylobacter strains resistant to several of these drugs have been isolated from clinical samples in many European countries,6^–11 Canada12 and the United States.13^,14 Erythromycin resistance has been documented much more frequently in C. coli than C. jejuni.9^,15^,16

There is a growing concern that veterinary use of antimicrobials in food animals can select for resistant Campylobacter spp., which may subsequently be transmitted to humans through the food chain.13^,17^–19 However, only a few reports6^,13^,16^,20^,21 have examined the current trend of antimicrobial susceptibility in Campylobacter isolates from healthy animals or retail meats. Surveillance data at the retail level in the US are especially lacking.

Several testing methods, including disc diffusion, broth microdilution, agar dilution and the Etest (AB Biodisk, Sweden), have been used to determine the in vitro susceptibilities of Campylobacter to antimicrobial agents.22^–25 However, until recently none of these methods has been standardized on either a national or international scale. Thus, it is difficult to compare results from various studies due to a large number of testing variables as well as to the different interpretive criteria used. The latter is especially problematic since there are no NCCLS-approved MIC interpretive criteria for Campylobacter. Recently, the NCCLS subcommittee on Veterinary Antimicrobial Susceptibility Testing approved the agar dilution test as a standard susceptibility testing method for Campylobacter, and C. jejuni ATCC 33560 as the quality control (QC) organism. The aims of this study were to evaluate the validity and accuracy of the Etest as a method for performing in vitro antimicrobial susceptibility testing of Campylobacter using agar dilution as a reference method, and to determine antimicrobial susceptibility profiles of C. jejuni and C. coli isolated from retail meats.

Materials and methods

Campylobacter strains and culture conditions

C. jejuni ATCC 33560 was used as the QC organism for both agar dilution and the Etest. Eighty-one C. jejuni (75 from 32 chickens, four from a beef sample and two from a pork sample) and 54 C. coli isolates (39 from 14 chickens, seven from three turkeys and eight from three pork samples) were selected from our culture collection of a retail meat study in the Greater Washington DC area during the summer and autumn of 1999. Campylobacter isolates were stored at –80°C in brain–heart infusion broth (Becton Dickinson Microbiology System, Cockeysville, MD, USA) containing 50% glycerol. Before conducting agar dilution or the Etest, isolates were streaked from the –80°C stock onto 5% horse blood agar (Lampire Biological Laboratories Inc., Pipersville, PA, USA) and allowed to grow for 40–48 h under a microaerobic environment (5% O₂, 10% CO₂ and 85% N₂).

Antimicrobial agents

For agar dilution, the following antimicrobial agents were tested: chloramphenicol, ciprofloxacin, doxycycline, erythromycin, gentamicin, nalidixic acid and tetracycline. Ciprofloxacin was obtained from Pentex, Miles Inc., Kankakee, IL, USA, and the remaining antimicrobials were procured from Sigma, St Louis, MO, USA. Concentrations of antimicrobial agents for agar dilution testing ranged from 0.06 to 32.0 mg/L, except for ciprofloxacin (range 0.008–4.0 mg/L), and for chloramphenicol and nalidixic acid (range 0.25 to 128.0 mg/L) (Table 1). Etest strips were purchased from AB Biodisk (Piscataway, NJ, USA). Azithromycin was also tested using the Etest method; however, it was not available for agar dilution testing. The concentration gradient of each antimicrobial agent on the Etest strips was 0.016–256.0 mg/L, with the exception of ciprofloxacin, for which the gradient was 0.002–32.0 mg/L.

Antimicrobial susceptibility testing of Campylobacter

For both Etest and agar dilution testing, suspensions of Campylobacter were prepared from grown organisms on blood agar plates into 3 mL of Mueller–Hinton II broth (Difco Laboratories) and adjusted to a turbidity of a 0.5 McFarland standard. Colony counts were performed regularly to validate the Campylobacter inoculum density and to ensure inoculum compatibility between the two test methods.

Agar dilution test

Mueller–Hinton agar plates, supplemented with 5% defibrinated sheep blood (Cleveland Scientific, Cleveland, OH, USA) and the appropriate concentrations of antimicrobial agent, were prepared and inoculated with Campylobacter cell suspensions, as recommended by the NCCLS26 using a Cathra replicator (Oxoid Inc., Ogdensburg, NY, USA) with 1 mm pins. The plates were incubated at 37°C in the same microaerobic atmosphere for 48 h. MICs of antimicrobials were recorded as the lowest concentration of antimicrobial agent that completely inhibited Campylobacter growth on the agar plates.

Etest

Etest strips were used in accordance with the manufacturer’s instructions. They were removed from –30°C storage and brought to room temperature prior to use. Mueller–Hinton agar plates (150 mm in diameter) supplemented with 5% defibrinated sheep blood (Hardy Diagnostics, Santa Marie, CA, USA) were inoculated by swabbing evenly in three directions with a 0.5 McFarland standard of the test organism. Four Etest strips were applied to the surface of the plate in an equidistance radial manner, with the lowest concentration toward the centre. Plates were incubated under the same condition as for agar dilution. MICs were read directly from the test strip at the point where the zone of inhibition intersected the MIC scale on the strip.

Data analysis

MIC agreement between the two methods was defined as the same MIC ± 1 log₂ dilution.27 Off-scale MIC results obtained from both methods were not included in the agreement calculation. The following MIC breakpoints defined by the NCCLS for the Enterobacteriaceae were employed, with the exception of azithromycin and erythromycin, for which the breakpoints were those used by the National Antimicrobial Resistance Monitoring System (NARMS28): azithromycin 2 mg/L, chloramphenicol 32 mg/L, ciprofloxacin 4 mg/L, doxycycline 16 mg/L, erythromycin 8 mg/L, gentamicin 16 mg/L, nalidixic acid 32 mg/L and tetracycline 16 mg/L. The χ² test and Fisher’s exact two-tailed test (SAS for Windows Version 8; SAS Institute Inc., Cary, NC, USA) were performed to compare the resistance rates between C. jejuni and C. coli isolates.

Results

Quality control strain C.jejuni ATCC 33560 was used to evaluate and compare the Etest with the NCCLS recommended agar dilution test. MICs from four agar dilution experiments were all within the QC range for six of the seven antimicrobial agents tested, with the exception of chloramphenicol for which no QC range was established for Campylobacter (Table 1). Etest MIC results were also within QC ranges for tetracycline, gentamicin, erythromycin and ciprofloxacin. However, the QC ranges of MIC generated via the Etest were at times lower by two dilutions for doxycycline and nalidixic acid (Table 1). Overall, MICs obtained from the Etest for C. jejuni ATCC 33560 were always one to several dilutions lower than those obtained from the agar dilution.

A similar observation was also made when the 135 Campylobacter meat isolates were tested (Table 2). For each antimicrobial agent, Etest MICs were always one to two dilutions lower than those obtained via agar dilution at the susceptible end of the MIC ranges. On the other hand, the Etest tended to yield much higher resistant MICs than those measured by agar dilution at the resistant end of the MIC ranges. Table 2 also shows the MIC₅₀ and MIC₉₀ values of the eight antimicrobials tested. There were often two- to four-fold differences in these values between the two methods. However, the MIC₅₀ and MIC₉₀ values of gentamicin and tetracycline correlated well.

Agreement of the MICs between agar dilution and the Etest among the Campylobacter isolates is summarized in Table 3. The overall agreement of MICs (±1 log₂ dilution) between the two methods was 61.9%, ranging from 21.4% with nalidixic acid to 92.6% with gentamicin. Ciprofloxacin MIC agreement between the two methods was 85.2%, followed by erythromycin (65.6%), doxycycline (59.0%), tetracycline (57.7%) and chloramphenicol (51.9%). Large MIC discrepancies (> four dilutions) between the Etest and agar dilution methods were observed for several antimicrobial agents tested, including nalidixic acid, ciprofloxacin, doxycycline and tetracycline.

Since there were no recommended antimicrobial susceptibility interpretive criteria for Campylobacter, resistant isolates were identified based on the NCCLS interpretive criteria for Enterobacteriaceae, with the exception of azithromycin and erythromycin, for which the breakpoints were those used by the NARMS. Based on MICs from agar dilution, Campylobacter isolates (n = 135) demonstrated the greatest resistance to tetracycline (82.2%), followed by doxycycline (78.5%), nalidixic acid (21.5%), ciprofloxacin (20.7%) and erythromycin (17.0%) (Figure 1). The resistance rate to azithromycin was 4.4% according to the Etest. None of the Campylobacter isolates demonstrated resistance to chloramphenicol or gentamicin. C. coli isolates exhibited a significantly higher resistance rate (P < 0.05) than C. jejuni to erythromycin (40.7% versus 1.2%), ciprofloxacin (33.3% versus 12.3%), nalidixic acid (31.5% versus 14.8%) and azithromycin (9.3% versus 1.2%), whereas the resistance rates to tetracycline and doxycycline were slightly higher among C. jejuni isolates with no statistical significance (Figure 1).

Multidrug resistance was also observed among the Campylobacter isolates. Sixty-one (45%) isolates were resistant to two antimicrobials, 21 (15.6%) to four, and 20 (14.8%) to three antimicrobials. Two C. coli isolates, one recovered from a chicken sample and one from a turkey sample, were resistant to five antimicrobials, including ciprofloxacin, doxycycline, erythromycin, nalidixic acid and tetracycline. However, 12 Campylobacter isolates (8.9%) were susceptible to all eight antimicrobial agents tested.

Discussion

Certain fastidious bacteria, including Brucella, Helicobacter and Campylobacter, present difficulties in antimicrobial susceptibility testing due to both unique growth requirements and test conditions.29 The Etest, an agar-based stable concentration gradient method for MIC determination, has been used to determine the susceptibility profiles of Campylobacter isolates. The methodology has proven to be a relatively accurate method to test antimicrobial susceptibilities of fastidious organisms, including Helicobacter pylori and C. jejuni.22^,25^,30^–32 Previous studies22^–25 compared disc diffusion, broth microdilution, agar dilution and the Etest in the validity and accuracy of determining Campylobacter susceptibilities to antimicrobial agents. The overall conclusion from these reports was that the Etest was a preferable method for susceptibility testing of Campylobacter,24 and that Etest and agar dilution had an overall agreement of 82.9% for C. jejuni.22 Hayward et al.32 previously reported a high correlation (r = 0.88) between the Etest and agar dilution results, but the study did not include nalidixic acid.

The correlation between the Etest and agar dilution method, as compared in this study, varied greatly depending on the antimicrobial agent tested. For ciprofloxacin and erythromycin, the drugs of choice for treating campylobacteriosis, the results of the two methods correlated relatively well. However, MICs for nalidixic acid varied greatly between the two testing methods, suggesting that the Etest should probably not be used for susceptibility testing of Campylobacter to this agent. Another study22 also concluded that the Etest should not be used to test C. jejuni against clindamycin. The correlation in our study, however, was not affected by the species of the organism tested, i.e. C. jejuni versus C. coli (data not shown).

In previous reports comparing the Etest with agar dilution, the Etest tended to give lower MIC readings than agar dilution for susceptible organisms.22^,24 Our study showed the same trend when testing both the QC organism and the 135 Campylobacter meat isolates. One of the reasons why the Etest may result in lower MICs for Campylobacter of certain antimicrobial agents is that it has not been standardized and validated for Campylobacter as has agar dilution. Perhaps if the Etest Campylobacter method was subjected to the same validation process required by the NCCLS for the agar dilution method, a better correlation of the two methods could be established. Further multicentre laboratory evaluations of the Etest may be warranted to establish clearly the usefulness of this technique for Campylobacter susceptibility testing.

Technically speaking, the Etest is a simple method for conducting susceptibility testing, and does not require a special or dedicated instrument. It is especially flexible when testing a few isolates against several antimicrobial agents. However, reading of Etest plates can be subjective and variable. A technical problem that arose during this study was poor growth of some Campylobacter isolates on Etest plates, causing difficulty in interpreting the Etest results. However, re-testing some of the isolates using the Etest did give satisfactory results. Agar dilution, on the other hand, is a reference method for susceptibility testing. It is relatively labour-intensive compared with the Etest. The disadvantage, however, diminishes when a large number of isolates are to be tested. The amount of labour involved can be reduced further by use of 1 mm pins instead of 3 mm pins for inoculation because it avoids an additional step of 1:10 dilution of the 0.5 McFarland inoculum and takes less time for the inoculated plates to dry. Our preliminary study showed that the size of the pins had no effect on MICs using 35 randomly selected Campylobacter isolates.

When performing in vitro antimicrobial susceptibility tests, it is important that standardized testing methods are used in order to demonstrate intra- and inter-laboratory reproducibility of results. The agar dilution method described in the present study was performed in accordance with the NCCLS standard M31-A2. However, although the NCCLS has approved the agar dilution test as a standardized in vitro antimicrobial susceptibility testing method for Campylobacter, it has not established interpretive standards of susceptible, intermediate and resistant breakpoints. The NCCLS interpretive criteria for the Enterobacteriaceae were used to interpret data for Campylobacter in this study.

Our study suggested that chloramphenicol and gentamicin might be more effective against Campylobacter than ciprofloxacin or erythromycin. C. coli, in particular, displayed significantly higher resistance rates to erythromycin, ciprofloxacin, azithromycin and nalidixic acid. However, erythromycin is still relatively effective for treating campylobacteriosis since C. jejuni accounts for ∼95% of human Campylobacter infections.33 Multidrug resistance has been observed in many Campylobacter isolates tested. Several investigators34^,35 have reported that the incidence of human C. jejuni and C. coli infection has increased considerably in many parts of the world for the last decade, as has the number of quinolone-resistant and, to a lesser extent, macrolide-resistant Campylobacter strains associated with human illness. Since C. jejuni and C. coli demonstrate different susceptibility profiles, it is important to differentiate Campylobacter at the species level, and to provide antimicrobial susceptibility data for each species, in order to monitor better the trend in antimicrobial resistance among Campylobacter isolates and to ensure effective treatment of Campylobacter infections.

In summary, the data generated in this study demonstrated that MICs obtained by the Etest were not in complete agreement with MICs generated by agar dilution. The correlation of MICs depended on the antimicrobial agents in question but not on the species of the organisms tested, although the susceptibility profiles of C. jejuni and C. coli could be very different. Although the Etest has proven to be a satisfactory testing method, its use for Campylobacter susceptibility testing requires further standardization for certain antimicrobial agents. In addition, this study showed that C. jejuni and C. coli with elevated MICs, especially of those agents used for treating campylobacteriosis, were commonly present in retail raw meats. Therefore, surveillance programmes on antimicrobial-resistant food-borne pathogens in retail meat products are needed.

Acknowledgements

This study was supported in part by grants from the Maryland Agricultural Experimental Station and the University of Maryland Joint Institute for Food Safety and Applied Nutrition.

References

Author notes

1Department of Nutrition and Food Science, University of Maryland, College Park, MD 20742; 2Division of Animal and Food Microbiology/Office of Research, Center for Veterinary Medicine, Food & Drug Administration, Laurel, MD 20708, USA