Rapid selection of quinolone resistance in Campylobacter jejuni but not in Escherichia coli in individually housed broilers

Michiel van Boven1,*, Kees T. Veldman2, Mart C. M. de Jong1 and Dik J. Mevius2

1 Animal Sciences Group, Wageningen University and Research Centre, P.O. Box 65, 8200 AB Lelystad; 2 Central Institute for Animal Disease Control, CIDC-Lelystad, P.O. Box 2004, 8203 AA Lelystad, The Netherlands

Received 11 May 2003; returned 27 May 2003; revised 3 July 2003; accepted 4 July 2003


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Objective: To determine the within-host population dynamics of Campylobacter jejuni and Escherichia coli in chickens during and after treatment with fluoroquinolones.

Materials and methods: Total and resistant faecal counts were determined from cloacal swabs during and after treatment with enrofloxacin. Chickens were housed individually to avoid confounding as a result of interaction between animals, and to be able to focus solely on the within-host dynamics. To determine the molecular basis of resistance, a number of isolates were checked for mutations in gyrA.

Results: Treatment with enrofloxacin at doses routinely prescribed (50 ppm) rapidly reduced the faecal counts of E. coli below the detection limit and did not induce resistance. In C. jejuni, on the other hand, treatment with enrofloxacin quickly selected for high frequencies of fluoroquinolone-resistant strains. In all phenotypically resistant isolates, resistance was traced to mutations in the gyrA gene.

Conclusions: (1) A licensed dosage (50 ppm) of enrofloxacin in drinking water of chickens is effective (i.e. markedly reduced faecal counts) and is safe on a short time scale in E. coli (i.e. did not rapidly select for resistance), but is neither safe nor effective in C. jejuni. (2) The rapid emergence of resistance to quinolones in C. jejuni does not necessarily result from horizontal transmission of resistant strains among chickens, but could solely be the result of de novo selection of resistance in individual chickens.

Keywords: enrofloxacin, antimicrobial resistance, in vivo selection, chickens


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Campylobacter jejuni is one of the main causes of morbidity caused by food-borne gastrointestinal infections in man. It is widely believed that the emergence and increase in frequency of fluoroquinolone-resistant strains of C. jejuni in humans resulted, at least in part, from the use of fluoroquinolones in the poultry industry.14 The same may be true, perhaps to a lesser extent, for the emergence of fluoroquinolone-resistant Escherichia coli in humans.5

Several studies have investigated the consequences of fluoroquinolone use in poultry.14 All these studies focused on a single bacterial species, and studied either the molecular mechanisms of resistance or the patterns of resistance observed at the population level. Here we took an alternative approach that aimed to unravel the effects of fluoroquinolone use on the within-host dynamics of two important bacterial species. By simultaneously studying the dynamics of two bacterial species in individual chickens, we investigated whether selection of resistance was a rare event or whether it was easily induced, and whether the within-host dynamics of susceptible and resistant E. coli and C. jejuni were largely congruent or fundamentally different.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Experimental design

Sixteen 1-day-old Ross 308 broiler chickens were placed in one isolator for 21 days. Throughout, the chickens had free access to drinking water and standard pelleted broiler feed without antibiotics. The chickens were administered lyophilized intestinal microflora of SPF chickens through the drinking water in the prescribed dosage on day 1 (Aviguard; Bayer AG, Leverkusen, Germany).

On days 1, 5 and 8, the faeces of the animals were checked for the presence of Campylobacter spp. and E. coli and were negative for both.

On day 8, a fluoroquinolone-susceptible C. jejuni (C356) strain [enrofloxacin MIC 0.06 mg/L, determined with Etest strips (AB-Biodisk, Solna Sweden)], isolated from chickens and generously supplied by Dr Jacobs-Reitsma (Animal Sciences Group, Edelhertweg 15, 82 19 PH Lelystad, The Netherlands) was added to the drinking water at a concentration of approximately 103 cfu/mL.

On day 10, a mixture of three fluoroquinolone-susceptible E. coli strains (CIDC collection: CEESA 1.1, 1.2 and 1.4, enrofloxacin MIC <= 0.015 mg/L, determined according to NCCLS document M31-A2, concentration 106 cfu/mL) isolated in 1999 from faeces of normal broilers was added to the drinking water.

Faecal samples were taken on days 12, 15 and 19 from the cloaca using charcoal swabs (Greiner Bio-One B. V., Alphen a/d Rijn, The Netherlands, art. no. 425011) to determine whether C. jejuni and E. coli had colonized the gut. On day 21, eight chickens were selected from the group of 16 (four male and four female). Each chicken was individually housed in a separate isolator. From day 21 through day 30, 50 ppm enrofloxacin (Baytril 10% oral solution, Reg-NL 2929, batch no. ZW8304; Bayer AG, Leverkusen, Germany) was added daily to the drinking water of six chickens in accordance with the instructions on the package insert. Two chickens received no antibiotics and acted as control animals. In each isolator, the water was refreshed daily at 24 h intervals.

After the birds were housed separately, faecal samples were taken daily from day 21 through day 41, and every 2 to 3 days from day 43 through day 55. Cloacal swabs and faecal samples were directly transported to the laboratory and weighed. Each faecal sample was 1:10 diluted in Buffered Peptone Water.

The number of enrofloxacin-resistant C. jejuni mutants and the total number of C. jejuni were determined by standard bacterial counting techniques on Campylobacter Blood Free Selective agar (CCDA; Oxoid CM0739B + selective supplement SR155) with and without 4 mg/L enrofloxacin. To check the activity of the enrofloxacin in CCDA, each batch was inoculated with enrofloxacin-susceptible C. jejuni C356 and enrofloxacin-resistant C. jejuni C2912; MIC 8 mg/L. This was repeated for E. coli on MacConkey agar plates (Oxoid CM115) with and without 1 mg/L enrofloxacin. To check the activity of enrofloxacin in the MacConkey agar, each batch was inoculated with enrofloxacin-susceptible E. coli ATCC 25922 (MIC 0.03 mg/L) and enrofloxacin-resistant E. coli (CIDC collection; CEESA 1.13; MIC 8 mg/L). Throughout the experiment, the enrofloxacin-susceptible C. jejuni C356 and E. coli ATCC 25922 strains failed to grow on the respective agar plates with enrofloxacin. The resistant strains showed normal growth on these plates.

MICs of enrofloxacin were determined by agar dilution according to NCCLS document M31-A2 for 19 E. coli isolates (six isolated before treatment on day 19, seven isolated on the last treatment day (day 30), and six isolated on day 55) obtained from MacConkey agar without enrofloxacin. E. coli ATCC 25922 was used as a quality control strain.

MICs of enrofloxacin were also determined for 29 C. jejuni isolates, including eight isolated before treatment, five isolated on the third day of treatment (day 23), eight isolated on the last day of treatment (day 30), and eight isolated on day 55. The MIC determinations were carried out on CCDA according to NCCLS document M31-A2, except that plates were incubated micro-aerobically at 37°C for 48 h. C. jejuni C356 and C. jejuni C2912 were used as quality control strains.

The study was controlled and approved by the local ethics committee of the Animal Sciences Group, Lelystad, The Netherlands.

DNA isolation and nucleotide sequence analysis

A selection of 20 C. jejuni isolates was screened for mutations in the gyrase A gene (gyrA). The selection consisted of four susceptible isolates from faecal samples of two control broiler chickens, six susceptible isolates collected before the start of the treatment, and 10 resistant isolates that were collected after treatment of the broilers. The gyrA sequences of the challenge strain C. jejuni C356 and reference strain C. jejuni NCTC 11168 were also verified.

Chromosomal DNA was purified using the Puregene DNA isolation kit (Genta Systems, Minneapolis, USA). Primers gyrA PmF (5'-TGGTTTAAAGCCTGTTCATAG-3') and gyrA PmR (5'-AGTTGCCTTGTCCTGTAATA-3') were used to amplify a 209-bp product containing the quinolone resistance-determining region of gyrA.6 PCR reactions were carried out using high fidelity Tgo polymerase (Roche Molecular Biochemicals, Mannheim, Germany). Subsequently, the correct PCR product was purified using the High Pure PCR Product Purification Kit (Roche Molecular Biochemicals, Mannheim, Germany) and sequenced using the PmF and PmR primers.


    Results and discussion
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Escherichia coli

In the control animals, the bacterial counts increased within 10 days from below the detection limit (200 cfu/g) to approximately 108 cfu/g (Figure 1). From then on, the bacterial counts remained high (107 cfu/g in animal ‘a’ and 2.3 x 106 cfu/g in animal ‘b’) in both control animals. However, variation in the bacterial counts was considerable, ranging from 105 cfu/g to almost 109 cfu/g.



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Figure 1. Selection of enrofloxacin resistance in E. coli. Open circles denote the total faecal count, whereas filled circles represent the faecal count of resistant E. coli. Panels a and b show the time course of the faecal counts of two control animals. Panels c–h give the faecal counts of six animals treated with enrofloxacin from day 21 through day 30 (shaded area).

 
In animals ‘c’ to ‘h’, treatment with enrofloxacin resulted in a rapid reduction in the faecal counts of E. coli to levels below the detection limit. However, the counts started to increase again less than a week after cessation of treatment, and were back at the pre-treatment level at the end of the experimental period in five of six treated animals. Animal ‘c’ remained negative for E. coli on all but 2 days after cessation of the antimicrobial treatment. No selection of resistance was observed in E. coli during and after treatment. In fact, enrofloxacin MICs ranged from <=0.03 to 0.06 mg/L throughout the experiment (n = 19).

Campylobacter jejuni

In the control animals ‘a’ and ‘b’, high faecal counts were observed from day 12 onwards (106–107 cfu/g) (Figure 2). However, in both animals the faecal counts tended to decrease over time (–0.063 log10 step per day, P < 0.001). No resistant isolates were found in the control animals. Enrofloxacin MICs of the strains isolated from the control animals varied from 0.06 to 0.125 mg/L (n = 8).



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Figure 2. Selection of enrofloxacin resistance in C. jejuni. Open circles denote the total faecal count, whereas filled circles represent the faecal count of resistant C. jejuni. Panels a and b show the time course of the faecal counts of two control animals. Panels c–h give the faecal counts of six animals treated with enrofloxacin from day 21 through day 30 (shaded area).

 
Within 2 days after the start of the treatment, fluoroquinolone-resistant strains of C. jejuni arose in four of the six treated animals. Enrofloxacin MICs of resistant strains isolated after treatment varied from 1 to 8 mg/L (n = 17). During treatment, the faecal counts were restored to a level comparable with that of the control animals.

Two of the treated animals showed a different pattern. In one animal, the antimicrobial treatment reduced the faecal count below the detection limit for the whole experimental period (panel ‘f’), whereas in the other animal, the antimicrobial treatment resulted in a temporary reduction of the faecal count below the detection limit (panel ‘d’). Four days after the treatment was stopped, the faecal counts started to increase again in the latter animal. Interestingly, the population consisted largely or exclusively of resistant C. jejuni.

Mutations in gyrA are the most common cause of resistance to quinolones.7,8 To verify whether mutations in gyrA had occurred in our enrofloxacin-resistant isolates, we determined the nucleotide sequence of gyrA of a number of isolates. All tested isolates including the reference strain NCTC 11168 and the challenge strain C356 showed one nucleotide difference compared with the published sequence, at codon 86 (ACT -> ACA).6 In 10 enrofloxacin susceptible C. jejuni strains, no other mutations were found. In eight enrofloxacin-resistant strains isolated from animals ‘c’, ‘e’, ‘g’ and ‘h’ with MIC values varying from 4 to 8 mg/L, a substitution of Thr-86 to Ile had occurred (ACA -> ATA). Two resistant strains with enrofloxacin MIC values of 1 and 2 mg/L showed a mutation of Asp-90 to Asn (GAT -> AAT). The latter two isolates were obtained from animal ‘d’ in Figure 2 after the treatment had stopped.

Our results revealed that enrofloxacin treatment at doses routinely prescribed (50 ppm) gives a marked, albeit temporary, reduction in the faecal counts of E. coli and probably does not select for resistance. In C. jejuni, on the other hand, fluoroquinolone treatment quickly selects for very high levels of resistance.9 Hence, if we had focused solely on E. coli we could have reached the false conclusion that treatment with fluoroquinolones is effective as well as safe from the point of view of the development of resistance. Our findings further show that the rapid emergence of resistance in Campylobacter spp. at the population level is not necessarily the result of transmission of resistant strains among chickens but could conceivably be due solely to de novo selection of resistance in individual chickens.


    Acknowledgements
 
We thank Tineke Bergsma for indispensable technical assistance and Elly Katsma for critical reading of the manuscript.


    Footnotes
 
* Corresponding author. Tel: +31-320-238525; Fax: +31-320-238961; E-mail: michiel.vanboven{at}wur.nl Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Endtz, H. P., Ruijs, G. J., van Klingeren, B. et al. (1991). Quinolone resistance in Campylobacter isolated from man and poultry following the introduction of fluoroquinolones in veterinary medicine. Journal of Antimicrobial Chemotherapy 27, 199–208.[Abstract]

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3 . Pearson, A. D., Greenwood, M. H., Donaldson, J. et al. (2000). Continuous source outbreak of campylobacteriosis traced to chicken. Journal of Food Protection 63, 309–14.[ISI][Medline]

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5 . Garau, J., Xercavins, M., Rodriguez-Carbaleira, M. et al. (1999). Emergence and dissemination of quinolone-resistant Escherichia coli in the community. Antimicrobial Agents and Chemotherapy 43, 2736–41.[Abstract/Free Full Text]

6 . Wang, Y., Huang, W. M. & Taylor, D. E. (1993). Cloning and nucleotide sequence of the Campylobacter jejuni gyrA gene and characterization of quinolone resistance mutations. Antimicrobial Agents and Chemotherapy 37, 457–63.[Abstract]

7 . Gibreel, A., Sjogren, E., Kaijser, B. et al. (1998). Rapid emergence of high-level resistance to quinolones in Campylobacter jejuni associated with mutational changes in gyrA and parC. Antimicrobial Agents and Chemotherapy 44, 3276–8.

8 . Walsh, C. (2000). Molecular mechanisms that confer antibacterial drug resistance. Nature 406, 775–81.[CrossRef][ISI][Medline]

9 . McDermott, P. F., Bodeis, S. M., English, L. L. et al. (2002). Ciprofloxacin resistance in Campylobacter jejuni evolves rapidly in chickens treated with fluoroquinolones. Journal of Infectious Diseases 185, 837–40.[CrossRef][ISI][Medline]