Elsevier

Food Control

Volume 30, Issue 1, March 2013, Pages 222-226
Food Control

A study of the molecular basis of quinolone and macrolide resistance in a selection of Campylobacter isolates from intensive poultry flocks

https://doi.org/10.1016/j.foodcont.2012.06.044Get rights and content

Abstract

The aim of this study was to investigate the molecular basis for observed high-level quinolone and macrolide resistance in poultry Campylobacter isolates. Seventeen Campylobacter isolates displaying high-level resistance to nalidixic acid, ciprofloxacin and/or erythromycin were investigated. Minimum inhibitory concentrations were initially determined using both the broth microdilution and E-test methods. The contribution of target gene mutations and active efflux to the observed resistances were then investigated using PCR and sequencing methods. High-level resistance to nalidixic acid was attributed to amino acid substitutions Thr-86-Ile and Asn-203-Ser in GyrA in some but not all isolates. Contrary to previous reports, the Thr-86-Ile substitution did not confer universal resistance to all quinolones. Strains displaying a high level of resistance to erythromycin carried the 23S rRNA transition mutation A2075G and/or carried mutations in the L4 and/or L22 ribosomal-encoding proteins. Interestingly and in contrast to previous studies, not all of the isolates carrying substitutions within the β-hairpin region of the L22 ribosomal protein displayed erythromycin resistance. With the exception of a single isolate, efflux did not contribute to either quinolone or macrolide resistance.

This study further expands our understanding of the molecular basis of quinolone and macrolide resistance in Campylobacter spp. and suggests that other factors, remaining to be elucidated, may also contribute to the resistant phenotypes observed.

Highlights

► Thr-86-Ile and Asn-203-Ser GyrA substitutions conferred nalidixic acid resistance. ► The Thr-86-Ile substitution did not confer universal resistance to all quinolones. ► Erythromycin resistance was due to 23S rRNA (A2075G) and/or L4/L22 mutations. ► Not all L22 β-hairpin mutations confer erythromycin resistance. ► In general, efflux did not contribute to antibiotic resistance.

Introduction

Campylobacteriosis is the most common cause of acute bacterial gastroenteritis in developed countries (Threfall, Ward, Frost, & Willshaw, 2000). While the majority of cases are self-limiting and do not require therapeutic intervention, severe cases are normally treated with erythromycin or ciprofloxacin (Engberg, Aarestrup, Taylor, Gerner-Smidt, & Nachamkin, 2001). However, over the last decade antibiotic resistance has been widely reported in Campylobacter giving rise to serious public health concerns (Soonthornchaikul et al., 2006). While antimicrobial resistance to quinolones and macrolides has been attributed, at least in part, to their use in poultry production (Humphrey et al., 2005, Luber et al., 2003), control of the emergence and dissemination of antimicrobial resistance in Campylobacter requires a fundamental understanding of the molecular basis of the observed resistant phenotypes.

Multiple mechanisms for antibiotic resistance have been reported for Campylobacter (Taylor & Tracz, 2005) including modification (mutation) of target genes and active efflux pump systems. In gram-negative bacteria DNA gyrase is the primary target of quinolones. Resistance to this class of antibiotics is usually associated with amino acid substitutions in the gyrA-encoding subunit of the DNA gyrase (Dionisi et al., 2004, Griggs et al., 2005) within the DNA-binding domain in a region termed the quinolone resistance determining region (QRDR). In the absence of a secondary target for quinolones in Campylobacter (Payot et al., 2006), the Thr-86-Ile amino acid substitution in the QRDR is sufficient to confer a resistant phenotype in Campylobacter jejuni and Campylobacter coli (Cooper et al., 2002, Luo et al., 2003, Payot et al., 2002, Piddock et al., 2003). Other modifications of the gyrA-encoding subunit have also been associated with quinolone resistance including Asp-203-Ser (Lucey et al., 2002, Luo et al., 2003, Piddock et al., 2003).

Macrolide drugs bind to bacterial ribosomes causing dissociation of the peptidyl-tRNA thereby interfering with protein synthesis and preventing bacterial growth. Two mechanisms of macrolide resistance have been described in Campylobacter including: [a] modification of the antibiotic target and [b] removal from the bacterial cell by efflux (Taylor & Tracz, 2005). The former is the most common mechanism and usually occurs by mutation. The large (50S) bacterial ribosomal subunit contains 23S rRNA that is the primary target of macrolides. Modification by mutation reduces macrolide binding thereby conferring a resistant phenotype. Point mutations at positions 2074 and/or 2075 have been associated with high levels of erythromycin resistance (Corcoran et al., 2006, Payot et al., 2004, Taylor and Tracz, 2005, Vacher et al., 2003).

Macrolide binding may also be inhibited by mutations in the ribosomal proteins L4 and L22. However, not all mutations confer erythromycin resistance. The A103V substitution in the L22 protein has been identified in high-level erythromycin-resistant C. jejuni and C. coli but K15I, E111A, T114A in L22 and V121A and V196A in L4 are located outside the important target region and have been found in susceptible Campylobacter strains (Corcoran et al., 2006).

Other factors may also contribute to the resistance phenotype. Efflux of drug(s) was first proposed in 1995 as a mechanism that conferred a multi-drug resistance (MDR) phenotype to Campylobacter. In 2002, the chromosomally encoded multi-drug resistance-nodulation-cell division (RND) efflux system, CmeABC efflux pump, was described in C. jejuni (Lin et al., 2002, Luo et al., 2003) and in C. coli (Corcoran, Quinn, Cotter, & Fanning, 2005). This efflux pump is known to promote both intrinsic (Lin et al., 2002, Pumbwe and Piddock, 2000) and acquired resistance (Ge et al., 2005, Luo et al., 2003, Payot et al., 2002) to a range of antimicrobial agents including quinolones and macrolides in Campylobacter species. The structure of this pump includes an outer membrane protein (CmeC), an inner membrane efflux transporter of the RND superfamily (CmeB) and a periplasmic fusion protein (CmeA) (Payot et al., 2004). This pump, which is normally present in wild-type Campylobacter strains, is also capable of extruding a wide range of substances including antimicrobial agents, detergents and bile salts (Pumbwe & Piddock, 2000).

The aim of this study was therefore to investigate the role of both target gene mutations and efflux pump activity in both fluoroquinolone and macrolide resistance in Campylobacter isolates from intensively reared poultry thus adding to the body of knowledge that already exists in this area that is providing the scientific basis for the design of next generation antibiotics and the development of strategies to control antibiotic resistance in Campylobacter.

Section snippets

Bacterial isolates and growth conditions

Fifteen poultry Campylobacter isolates, previously shown to have quinolone and/or macrolide resistance phenotypes, were obtained from the Teagasc Campylobacter collection at the Teagasc Food Research Centre. These had been isolated from several different flocks within 12 months of this study (Ashtown). Two reference strains NCTC 11168 (C. jejuni, human isolate) and NCTC 11366 (C. coli, porcine isolate) were also included.

Broth microdilution

Broth microdilution was performed as described by Luber et al. (2003) with

Results

C. jejuni, isolates T204, T208, T216, T227, T233, T234, T242, T244, T253, T256 and T273 and C. coli T26 exhibited high-level resistance (>256 μg/ml) to nalidixic acid. C. jejuni strains T216, T227, T242, T253, T256, T273 and T277 and C. coli T14 exhibited high-level resistance (>32 μg/ml) to ciprofloxacin. C. jejuni T208 and C. coli T26 exhibited high-level resistance (>256 μg/ml) to erythromycin. All other isolates displayed low-intermediate resistance to these antibiotics (Table 2).

The

Discussion

The prevalence of Campylobacter carriage in European poultry flocks has been increasing year on year with an average reported carcass incidence of 75.8% (Anonymous, 2010). The emergence of resistance to therapeutically important antimicrobials in bacterial pathogens is a serious threat to public health. Campylobacter spp. are becoming increasingly resistant to quinolones and erythromycin which are the drugs of choice for the treatment of complicated cases of campylobacteriosis (Aarestrup &

Conclusions

This study concluded that; the Thr-86-Ile or Asn-203-Ser amino acid substitution in the GyrA-encoding subunit of DNA gyrase is the primary contributor to the high-level quinolone resistance in some but not all resistant isolates and further investigation of alternative mechanisms would be required; the Thr-86-Ile substitution does not confer universal resistance to all quinolone antibiotics; the 23S rRNA point mutation A2075G and/or the L4/L22 mutations were responsible for the high-level

Acknowledgements

This work was funded by safefood, the Food Safety Promotion Board, Project number 04-RESR-04.

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