MEM INST OSWALDO CRUZ, RIO DE JANEIRO, 95(2) Mar/Apr 2000
PAGES: 247-249 DOI: Short communication
Antimicrobial Susceptibility of Campylobacter jejuni subsp. jejuni Assessed by E-test and Double Dilution Agar Method in Southern Chile

H Fernández +, M Mansilla, V González*

Instituto de Microbiología Clínica, Universidad Austral de Chile, Casilla Postal 567, Valdivia, Chile
*Escuela de Medicina, Universidad de Concepción, Concepción, Chile

Abstract

The susceptibility patterns of 108 Campylobacter jejuni subsp. jejuni clinical strains, to six antimicrobial agents was determined by using the E-test and the double dilution agar methods. Using both metods, no strain was found to be resistant to ciprofloxacin, erythromycin and gentamicin, but two (1.8%) were resistant to tetracycline and all to aztreonam. Seven (6.5%) strains were resistant to ampicillin by the E-test and five (4.6%) by the double dilution agar method and by both meyhods. No great discrepancies were observed between both methods.

Campylobacter jejuni subsp. jejuni is one of the most frequent causes of infectious diarrhea in both, developed and developing countries (Fernández 1992, Allos & Blaser 1995). Although C. jejuni subsp. jejuni diarrhea is a self-limited disease, in some clinical instances it is necessary the prescription of antibiotics, being erythromycin the drug of choice (Allos & Blaser 1995). For several years however, higher levels of resistance to this drug, as well as to others, such as tetracycline and fluoroquinolones, have been reported in several countries (Taylor & Courvalin 1988).

The information on bacteriological, biological, pathogenical, clinical and epidemiological aspects of C. jejunisubsp. jejuni generated in South America is vast and valuable (Fernández 1992). However, the susceptibility patterns of this bacterium have not been well defined in this geographical region.

The aim of this study was to determine the susceptibility patterns of C. jejuni subsp. jejuni clinical strains, to six antimicrobial agents, in Southern Chile by using two quantitative methods.

A total of 108 C. jejuni subsp. jejuni strains submitted in this study, had been isolated and identified from pediatric diarrheic stools specimens using standard procedures between March 1996 and December 1997. Strains were stored at -35ºC in freezing medium until analyzed. Staphylococcus aureus ATCC 25923 andEscherichia coli and E. coli ATCC 25922 were used as control strains.

The antimicrobial susceptibility patterns of the strains under study were determined by means of the E-test and the double dilution agar method (Baker et al. 1991).

To perform the E-test, several colonies of each strain, obtained from a fresh culture in blood agar plate, were suspended in 5 ml of Mueller-Hinton broth to achieve a turbidity equal to the 0.5 Mac Farland standard. The suspensions were inoculated with sterile swabs onto 150 mm diameter Mueller-Hinton 5% sheep blood agar plates and after the agar surfaces were allowed to dry, six E-test strips were applied on each plate. Plates were incubated at 37ºC for 48 h under microaerobic conditions and inhibitory concentrations were read at the point where the elliptical zone of inhibition intersected the E-test strip.

For the agar dilution method, Mueller-Hinton agar plates containing serial twofold dilutions of each antimicrobial agent from 0.125 to 128 µg/ ml were prepared. Inocula of the strains were prepared diluting 1:10 each bacterial suspension made in Mueller-Hinton broth with a turbidity equal to the 0.5 Mac Farland standard. Plates were inoculated with a multipoint replicator delivering spots of 4 µl of the diluted bacterial suspensions, starting from the lowest to the highest concentration of each antimicrobial drug. At the beginning of each series a Mueller-Hinton agar plate without antibiotics was inoculated as a viability control of the strains under study. The inoculated plates were incubated under the same conditions described above. For this method, minimal inhibitory concentrations (MICs) were defined as the lowest antibiotic concentrations yielding no growth.

The antimicrobial agents tested in this study were erythromycin, tetracycline, gentamicin, ciprofloxacin, ampicillin and aztreonam. The susceptibility criteria were those defined by the National Committee for Clinical Laboratory Standards (National Comimttee for Clinical Laboratory Standards 1995).

Table shows the MICs of the six antimicrobial agents tested. Using both metods, no strain was found to be resistant to ciprofloxacin, erythromycin and gentamicin, but all were resistant to aztreonam showing MICs ³256 µg/ml. The MICs for ampicillin ranged from 0.064-48 µg/ml and from £0.125-32 µg/ml with the E-test and the double dilution agar method respectively. Seven (6.5%) strains were resistant to ampicillin by the E-test method, whereas five (4.6%) presented this characteristic with the double dilution agar method. We believe that these results are not in great discrepancy with both methods since they only differ in one step dilution, being MIC90the same for E-test and agar dilution method (8 µg/ml). Similar observations were reported previously by Baker
et al. (1991) for other bacteria and by Baker (1992) for C. jejuni.

The highest MIC found for tetracycline was 32 µg/ml and the same two strains (1.8%) were resistant to tetracycline by both methods. With the exception of two strains that appeared to be resistent to ampicillin with the E-test and susceptible with the double dilution agar method, no other major discrepancies were observed between both methods. The MICs obtained by both methods were very similar.

In this study, no strain of C. jejuni subsp. jejuni was found to be resistant to erythromycin, the antibiotic of choice to treat Campylobacter enteritis, and to ciprofloxacin and gentamicin, antimicrobial agents indicated to treat acute diarrhea and extraintestinal infections due to Campylobacter respectively (Allos & Blaser 1995), by either methods. In a previous study, using C. jejuni subsp. jejuni strains isolated from hens, we did not find resistance to gentamicin and erythromycin (Tejero et al. 1996). On the other hand, using the disk difussion method employed routinely in our laboratory as a screening method, we have isolated through various years only three erythromycin-resistant strains (unpublished data). With these results, we can speculate that erythromicyn resistance is not a common phenomenon observed in C. jejuni subsp. jejuni strains isolated in Southern Chile. However, we can not rule out that it could be an emerging problem in the future, as it occurred in other countries (Taylor & Courvalin 1988). With regard to ciprofloxacin, we have previously isolated C. jejunisubsp. jejuni resistant strains from domestic animals recognized as reservoir and as source of infection for human beings. These strains showed concomitant resistance to other quinolones (Fernández et al. 1996). This suggests that in the future we could isolate, from animals and from human beings, C. jejuni subsp. jejunistrains showing resistance to ciprofloxacin and other quinolones. Resitance to quinolones in Campylobacterhas been increasingly reported since the past decade (Gootz & Martin 1991, Gaudreau & Gilbert 1998).

All our strains were highly resistant to aztreonam. This was reported previously in C. jejuni strains by Goossens et al. (1985) and by our group (Tejero et al. 1996) in C. jejuni subsp. jejuni and C. coli strains isolated from hens. Bearing in mind the high resistance to aztreonam observed in C. jejuni subsp. jejuni and the high activity of this antimicrobial drug against several enteropathogens, we have successfuly tested a new selective antimicrobial mixture that includes aztreonam, to isolate C. jejuni subsp. jejuni and C. coli from different sources (manusc. in prep.).

We have previously found a low percentage (2.3%) of tetracycline resistance in strains isolated from hens (Tejero et al. 1996), and in the present study only two (1.8%) out of the 108 clinical strains tested were resistant to this antibiotic. So, based on the evidence mentioned above, we can conclude that in this geographical region tetracycline is not yet a problem as it is in other countries (Taylor & Courvalin 1988, Gaudreau & Gilbert 1998, Li et al. 1998).

Using the E- test we found seven strains (6.5%) resistant to ampicillin. Five of them (4.6%) were also resistant by the double diluition agar method. Since we have not isolated ampicillin-resistant strains (Tejero et al. 1996) before, these are the first documented data about ampicillin resistance in C. jejuni subsp. jejuni in our region. Probably this could be an emerging problem that could require the establishment of a laboratory surveillance system to assess its real magnitude.

In agreement with the reports of Baker et al. (1991) and Funke et al. (1993), no great discrepancies were observed between both methods. This, and the fact that the E-test is technically simple and needs no special equipment, it could be become a reliable method for susceptibility testing in C. jejuni subsp. jejuni.

 

REFERENCES

Allos BM, Blaser MJ 1995. Campylobacter jejuni and the expanding spectrum of related infections. Clin Infect Dis20: 1092-1101.

Baker CN 1992. The E-test and Campylobacter jejuniDiagn Microbiol Infect Dis 15: 469-472.

Baker CN, Stocker SA, Culver DH, Thornsberry C 1991. Comparison of the E test to agar dilution, broth microdilution, and agar diffusion susceptibility testing techniques by using a special challenge set of bacteria. J Clin Microbiol 29: 533-538.

Fernández H 1992. Thermotolerant Campylobacter species associated with human diarrhea in Latin America.Ciênc Cult 44: 39-43.

Fernández H, Martin R, Thibaut J 1996. Campylobacter jejuni and Campylobacter coli resistant to fluoroquinolones in domestic animals. Arch Med Vet 28: 151-154.

Funke G, Lüthy-Hottenstein J, Altwegg M 1993. The E test for antyimicrobial susceptibility testing of clinicalCampylobacter jejuni and Campylobacter coli isolates. Med Microbiol Lett 2: 168-175.

Gaudreau C, Gilbert H 1998. Antimicrobial resistance of clinical strains of Campylobacter jejuni subsp. jejuniisolated from 1985 to 1997 in Quebec, Canada. Antimcrob Agents Chemother 42: 2106-2108.

Goossens H, Mol De P, Coigneau H, Levy J, Grados O, Ghysels G, Innocent H, Butzler JP 1985. Comparative activities of aztreonam, ciprofloxacin, norfloxacin, ofloxacin, HR 810 (a new cephalosporin), RV 28965 (a new macrolide) and other agents against enteropathogens. Antimicrob Agents Chemother 27: 288-292.

Gootz TD, Martin BA 1991. Characterization of high-level quinolone resistance in Campylobacter jejuni.Antimicrob Agents Chemother 35: 840-845.

Li Ch-Ch, Chiu Ch-H, Wu J-L, Huang Y-C, Lin T-Y 1998. Antimicrobial susceptibilities of Camp-ylobacter jejuniand coli by using E-test in Taiwan. Scand J Infect Dis 30: 39-42.

National Comimttee for Clinical Laboratory Standards 1995. Performance standards for antimicrobial susceptibilities testing. M7-A3. Villanova, PA.

Taylor DE, Courvalin P 1988. Mechanisms of antibiotic resistance in Campylobacter species. Antimicrob Agents Chemother 32: 1107-1112.

Tejero A, Salazar R, Fernández H 1996. Campylobacter jejuni in two groups of hens and study of antimicrobial susceptibility. Arch Med Vet 28: 155-157.

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