MICROBIAL DRUG RESISTANCE Volume 10, Number 3, 2004 © Mary Ann Liebert, Inc.
Antimicrobial Susceptibility Patterns of Arcobacter butzleri and Arcobacter cryaerophilus Strains Isolated from Humans and Broilers KURT HOUF,1 LUC A. DEVRIESE,2 FREDDY HAESEBROUCK,2 OLIVIER VANDENBERG,3 JEAN-PAUL BUTZLER,3 JAN VAN HOOF,1 and PETER VANDAMME4
ABSTRACT The MICs of five antimicrobial agents were determined by the agar dilution method for 98 Arcobacter butzleri and 28 Arcobacter cryaerophilus strains from humans, and poultry. With gentamicin, a MIC of 16 mg/ml was recorded for one A. butzleri strain isolated from poultry, whereas for the other strains MICs ranged from 0.25 to 4 mg/ml. With ciprofloxacin, a bimodal distribution of susceptibility levels was seen for human A. butzleri isolates (0.015–0.03 versus 0.12–0.25), whereas MICs for 65 of the 68 A. butzleri poultry strains ranged from 0.12 to 0.5 mg/ml and three strains from three different broilers were resistant with a MIC of 16 mg/ml. One A. cryaerophilus strain from poultry was resistant to erythromycin at a MIC of 128 mg/ml, whereas MICs for the other Arcobacter strains ranged from 2 to 32 mg/ml. No difference in susceptibility or resistance among the human and poultry strains tested was observed with doxycycline and nalidixic acid. The presence of acquired resistance to erythromycin and ciprofloxacin among poultry isolates is a matter of concern, because the two antimicrobials are generally prescribed as first-line drugs for the treatment of infections with Campylobacteraceae in humans.
INTRODUCTION
T
HE GENUS ARCOBACTER was described in 1991 as a second genus within the family Campylobacteraceae to encompass bacteria formerly known as aerotolerant campylobacters.22,23 Three species, Arcobacter butzleri, Arcobacter cryaerophilus, and Arcobacter skirrowii, are associated with reproductive disorders,5,19 mastitis,14 and gastric ulcers21 in farm animals, but have also been isolated from healthy livestock.25 Although A. skirrowii has recently been isolated from a patient with enteritis,26 A. butzleri and A. cryaerophilus are incriminated predominantly with enteritis and bacteremia in humans,3,16 but the nature of their pathogenicity is still unclear. The fourth species, Arcobacter nitrofigilis, is a nitrogen-fixing bacterium associated with the roots of Spartina alterniflora, a salt marsh plant.17 No association with animals or humans is known. Poultry products are commonly contaminated with A. butzleri and A. cryaerophilus on the surfaces.8 Contamination lev-
els as high as several thousands cells per gram of skin or meat have been reported.8 Studies involving biotyping2,12 and serotyping13 have shown that the same types of these organisms are similarly distributed among human and poultry isolates. Although the exact contribution of contaminated poultry products to human infection remains to be determined, the handling of raw poultry, cross-contamination, and the consumption of undercooked poultry products are probable routes of transmission. Diarrhea caused by members of the Campylobacteraceae, and Arcobacter in particular, is presumed to be a self-limiting disease, although severity or duration of symptoms may necessitate antibacterial therapy.20 When an antibiotic is recommended for treatment, the most commonly prescribed drugs are erythromycin or a fluoroquinolone such as ciprofloxacin.15 Tetracycline, doxycycline, and gentamicin are sometimes listed as alternative drugs for treatment. Because arcobacters may be transferred from poultry car-
1Department of Veterinary Public Health, and 2Department of Pathology, Bacteriology and Poultry Diseases, Faculty of Veterinary Medicine, Ghent University, 9820 Merelbeke, Belgium. 3Center for Enteric Campylobacter, Saint-Peter, Queen Fabiola and Brugmann University Hospitals, 1000 Brussels, Belgium. 4Laboratory of Microbiology, Faculty of Sciences, Ghent University, K.L. 9000 Ghent, Belgium.
243
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HOUF ET AL.
casses and meat to humans, the possible development of antimicrobial resistance in Arcobacter species due to the use of antimicrobial agents during raising of the birds is another matter of concern. The present study was conducted to determine current susceptibility of A. butzleri and A. cryaerophilus strains isolated from broiler carcasses and clinical infections in humans in Belgium to five antimicrobials, including those that are recommended therapeutically.
MATERIALS AND METHODS Bacterial isolates In the present study, 38 arcobacters isolated from human feces were included. Nine A. butzleri strains were obtained from the BCCM/LMG Bacteria collection, Ghent University (Ghent, Belgium). The other 29 arcobacters were isolated from stool samples of patients with acute or persistent diarrhea. They were admitted to the Brugmann, Queen Fabiola, and Saint-Peter University Hospitals in Brussels over a 6-year period (November, 1995, to August, 2001).24 These human isolates were obtained by the procedure developed by de Boer et al. which comprises cefoperazone (32 mg/L), piperacillin (75 mg/L), trimethoprim (20 mg/L), and cycloheximide (100 mg/L) as selective substances,2 and identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of whole-cell proteins.23 The isolates were lyophilized and stored until inclusion in the present susceptibility test. From February, 2001, to October, 2002, 52 fresh broiler carcasses were collected in 52 different Belgian retail stores. From each carcass, samples of 10 grams of neck and breast skin were homogenized with 10 ml of peptone water in a stomacher blender for 1 min at the normal speed. One hundred microliters of each skin homogenate was inoculated by the spiral plating method onto an Arcobacter-selective agar plate containing 28 grams of Arcobacter broth (Oxoid Ltd., Basingstoke, Hampshire, England) per liter, 12 grams of Agar Technical no. 3 (Oxoid) per liter, and the previously developed selective supplement, comprising amphotericin B (10 mg/L), cefoperazone (16 mg/L), 5-fluorouracil (100 mg/L), novobiocin (32 mg/L), and trimethoprim (64 mg/L).8 Plates were incubated for 48 hr at 28°C under microaerobic conditions by evacuating 80% of the normal atmosphere and introducing a gas mixture of 8% CO2, 8% H2, and 84% N2 into the jar. After incubation, a maximum of five randomly selected colonies per agar plate were picked and preserved at 280°C until further examination.
Identification and characterization All isolates were subcultured on nonselective blood agar plates consisting of Mueller–Hinton agar (CM 337-Oxoid) supplemented with 10% horse blood (E&O Laboratories, Bonnybridge, UK). Following incubation for 48 hr at 28°C in a microaerobic atmosphere, the bacterial growth was harvested and suspended in 1 ml of sterile distilled water. The suspensions were centrifuged for 5 min at 13,000 rpm to pellet the cells and the supernatants were discarded. The cell pellets were resuspended in 500 ml of sterile distilled water and boiled for 10 min. After a quick spin, 2 ml of each supernatant was used as
a DNA template and analyzed by multiplex-PCR (m-PCR) for species-level identification.10 For strain typing, 1 ml of each supernatant was used in ERIC-PCR analysis as previously described.9 For convenience, the Pearson product moment correlation coefficient is expressed as a percentage similarity. Arcobacter isolates grouped by cluster analysis of the ERIC patterns at a similarity level of 88% or more were subsequently further characterized by RAPD-PCR to confirm their clonal relatedness.9 Only one strain of each genotype was selected for further susceptibility testing.
Susceptibility testing The MICs were determined by the agar dilution method. The following antibacterial preparations were tested: ciprofloxacin (Bayer, Brussels, Belgium), doxycycline (Sigma, St. Louis, MO), erythromycin (Sigma), gentamicin (Kela Laboratory, Sint-Niklaas, Belgium), and nalidixic acid (Sigma). The antimicrobial agents were dissolved in appropriate solvents to make stock solutions containing 1,000 mg/ml and further diluted in sterile distilled water according to the methods recommended by the National Committee for Clinical Laboratory Standards (NCCLS M100-S7).18 A control plate without antibiotic was inoculated at the end of the procedure. Arcobacter strains were grown in brain heart infusion broth (Oxoid) and incubated at 28°C under microaerobic conditions. Inocula were prepared by diluting the overnight cultures in buffered saline to a density of 0.5 on the McFarland turbidity scale and diluted 40-fold before inoculating. MIC tests were carried out on Mueller–Hinton II agar (Becton Dickinson, Cockeysville, MD) containing doubling dilutions of the antibacterial agents. Plates were seeded with approximately 1 3 105 colony-forming units (CFU) with a Steers inoculum replicator (MAST, London, UK) and incubated for 24 hr at 28°C under microaerobic conditions. An antibiotic-free agar plate and the Staphylococcus aureus ATCC 29213 and Escherichia coli ATCC 25922 strains used for susceptibility tests were included in the agar dilution test as controls. The MIC was recorded as the lowest concentration that nearly completely inhibited growth, thus disregarding faint hazes of growth or single colonies.
RESULTS The arcobacters isolated from the stool samples were identified as A. butzleri (n 5 21) or A. cryaerophilus (n 5 8). Characterization of all 38 human isolates by ERIC-PCR resulted in 38 different genotypes, with 78% as the highest similarity level noticed (data not shown). Arcobacters were isolated from 34 of the 52 broiler carcasses examined, and a total of 147 colonies was subcultured for further analysis. They were identified in the m-PCR assay as A. butzleri (n 5 119) and A. cryaerophilus (n 5 28). Characterization of these isolates resulted in 68 A. butzleri and 20 A. cryaerophilus genotypes. The results of the MIC testing of the 98 A. butzleri and 28 A. cryaerophilus strains from human (n 5 38) and poultry (n 5 88) origin are shown in Tables 1 and 2. In general, comparison of the range of the MICs failed to reveal significant differences among the two Arcobacter species, as the value for each dif-
245
ARCOBACTER SUSCEPTIBILITY TO ANTIMICROBIALS TABLE 1.
DISTRIBUTION
OF
MICS
FOR
30 A.
BUTZLERI AND
8 A.
CRYAEROPHILUS
STRAINS ISOLATED
FROM
HUMANS
Number of strains with MIC (mg/ml) of Antimicrobial agents Ciprofloxacin
Species A. A. A. A. A. A. A. A. A. A.
Doxycycline Erythromycin Gentamicin Nalidixic acid
butzleri cryaerophilus butzleri cryaerophilus butzleri cryaerophilus butzleri cryaerophilus butzleri cryaerophilus
0.015
0.03
10
6
0.06
DISCUSSION In the present study, MIC concentrations for Arcobacter species were determined by the agar dilution method. Specific standardized procedures for susceptibility testing of Campylobacteraceae and resistance breakpoints have not been estab-
DISTRIBUTION
OF
MICS
0.25
6 7
8 1 1
0.5
1
2
4
8
16
1 1
3 1
15 6 3 1 14 1
5
4
1
3 7 15
18
2 1
8 6
1 6 1
1
fered by no more than two dilutions. Source related differences in susceptibility were only detected within A. butzleri for ciprofloxacin: a bimodal distribution of susceptibility levels was seen with human A. butzleri strains (0.015–0.03 versus 0.12–0.25) (Table 1). MICs of 65 poultry strains ranged from 0.12 to 0.5 mg/ml, and a frank resistance to this antibiotic was seen in three strains isolated from three different broilers that had a ciprofloxacin MIC of 16 mg/ml. All three strains have a MIC for nalidixic acid of 64 mg/ml, which was not clearly different from the results obtained with the other strains (Table 2). One of these strains was also less susceptible to gentamicin at a MIC of 16 mg/ml. One A. cryaerophilus strain isolated from a broiler carcass was found resistant to erythromycin at a MIC of 128 mg/ml, whereas MICs for the other Arcobacter strains ranged from 2 to 32 mg/ml. The MICs of doxycycline and nalidixic acid for A. butzleri of both human and poultry origin fell within the range of seven dilutions.
TABLE 2.
0.12
FOR
68 A.
32
64
128
256
6
12 1
2
5
lished. Consequently, a number of different testing methods, such as broth microdilution,4 disc diffusion,1,7 and the E-test,27 have been used for Arcobacter susceptibility testing in clinical, veterinary, and food microbiology. Furthermore, due to the fastidious nature and the microaerobic growth requirements of those microorganisms, the quality-control limits given for nonfastidious organisms in aerobic atmosphere are not adequate.6 It is known that an increased level of carbon dioxide does decrease the effect of certain antimicrobials such as macrolides and fluoroquinolones. This will certainly occur in the microaerobic atmosphere required for the growth of Campylobacteraceae, and should be taken into account when interpreting susceptibility patterns.6 Recent comparison of the broth microdilution, the E-test, and the agar dilution method showed overall comparable results for Campylobacter susceptibility testing when performed under the same microaerobic conditions and incubation temperature. Especially the MICs obtained by the three methods of ciprofloxacin and erythromycin were in accordance with each other.15 Although the comparison of the results in different publications on Arcobacter susceptibilities is hindered by the lack of standardized protocols, the MICs obtained in previous studies using broth or agar dilution are, with some exceptions, comparable to those obtained in the present study.4,11 In 1992, Kiehlbauch et al. applied the broth microdilution technique un-
BUTZLERI AND
20 A.
CRYAEROPHILUS
STRAINS ISOLATED
FROM
POULTRY
Number of strains with MIC (mg/ml) of Antimicrobial agents Ciprofloxacin Doxycycline Erythromycin Gentamicin Nalidixic acid
Species A. A. A. A. A. A. A. A. A. A.
butzleri cryaerophilus butzleri cryaerophilus butzleri cryaerophilus butzleri cryaerophilus butzleri cryaerophilus
0.015
0.03
0.06
0.12
0.25
0.5
2
49 18
14
2
1
2 4
7 9
14 3
38 7
6
1
2
4
8
16
32
64
128
3 22 6 4 10 12 4 1
27 1 13 9 3 1
8
1
16
32
3
1
1 20 15
26 3
10 1
11
256
246
HOUF ET AL.
der aerobic atmosphere for susceptibility testing of the same panel of antimicrobials for A. butzleri and A. cryaerophilus isolates.11 The MICs for ciprofloxacin, erythromycin, doxycycline, and nalidixic acid differ by no more than one dilution from those obtained in the present study. However, with gentamicin, MICs for A. butzleri and A. cryaerophilus ranged from #0.12 to 0.5 mg/ml, whereas in the present study, the MICs ranged from 0.5 to 4 and 0.25 to 2 mg/ml, respectively, and one poultry A. butzleri strain with a MIC of 16 mg/ml (Tables 1 and 2). It is unknown whether the higher carbon dioxide concentration may decrease the activity of aminoglycosides as well. In the recent susceptibility study by Fera et al.,4 even higher MIC ranges for both ciprofloxacin and gentamicin than those in the present study were obtained, although the same incubation atmosphere and temperature were applied. Whether the different origin of the strains in the studies, water versus human stools and poultry carcasses, is the cause of those MIC shifts, is not clear. In contrast to thermophilic Campylobacter species, of which some strains demonstrate resistance to quinolones and cross-resistance between nalidixic acid and quinolones, most of the Arcobacter strains tested were susceptible to both antimicrobial agents. Remarkable, however, is the lowered susceptibility and even resistance to ciprofloxacin of A. butzleri strains isolated from poultry (Table 2). The latter finding is also demonstrated by the concentrations required to inhibit growth of 50% of the strains (MIC50); the MIC50 for A. butzleri isolated from poultry is 0.12 whereas the MIC50 for human strains is 0.03. The use of fluoroquinolones for treatment of poultry may be the basis for this decreased susceptibility. Because poultry products in particular are incriminated for the transmission of arcobacters to humans, the presence of antimicrobial-resistant Arcobacter species in fresh poultry products can have public health implications. Our findings indicated that some strains from retail poultry were resistant and that multidrug resistance occurs. Especially the resistance to erythromycin and the decreased susceptibility to ciprofloxacin may have human health implications, as the two antimicrobials are generally prescribed as first-line drugs for the treatment of infections with Campylobacteraceae.
4.
5.
6.
7.
8.
9.
10.
11.
12. 13. 14.
15.
ACKNOWLEDGMENTS 16.
We thank the BCCM/LMG Bacteria collection, Ghent University (Ghent, Belgium) for providing the Arcobacter collection strains used in this study. The skilled technical assistance provided by Arlette Van de Kerckhove, Bieke Verbeke and Linda Vlaes was greatly appreciated.
17.
18.
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Address reprint requests to: Dr. Kurt Houf Department of Veterinary Public Health Ghent University Salisburylaan 133 B-9820 Merelbeke, Belgium E-mail:
[email protected]
