Prevalence and characterization of Campylobacter jejuni from chicken meat sold in French retail outlets

Highlights

• 76% of the French chicken meat was contaminated by Campylobacter.

• The C. jejuni population was genetically diverse and the ST-21 complex predominated.

• More than 25% of the isolates were potentially neuropathic with LOS class A or B.

• Isolates were susceptible to erythromycin, but more than 30% were ciprofloxacin-resistant.

• ST-21 complex isolates had mostly LOS class C and a high proportion was ciprofloxacin-resistant.

Abstract

Campylobacter was detected in 76% of broiler meat products collected in retail outlets during a monitoring plan carried out in France throughout 2009. Campylobacter jejuni was the most prevalent species (64.7% of products being contaminated). The 175 C. jejuni isolates collected were characterized. MLST typing results confirmed substantial genetic diversity as the 175 C. jejuni isolates generated 76 sequence types (STs). The ST-21, ST-45 and ST-464 complexes predominated accounting for 43% of all isolates.

A class-specific PCR to screen the sialylated lipooligosaccharide (LOS) locus classes A, B and C showed that 50.3% of the C. jejuni isolates harbored sialylated LOS.

The antimicrobial resistance profiles established using a subset of 97 isolates showed that resistance to tetracycline was the most common (53.6%), followed with ciprofloxacin and nalidixic acid (32.9%, and 32.0% respectively). All the tested isolates were susceptible to erythromycin, chloramphenicol and gentamicin.

Clear associations were demonstrated between certain clonal complexes and LOS locus classes and between certain clonal complexes and antimicrobial resistance.

This work paints a representative picture of C. jejuni isolated from poultry products circulating in France, providing data on STs, LOS locus classes and antibiotic resistance profiles in isolates recovered from products directly available to the consumer.

Introduction

Campylobacter jejuni is the major cause of bacterial gastroenteritis in developed countries. Over 214,000 human cases were reported in 2012 in the European Union (EFSA and ECDC, 2014). Broiler chickens are generally recognized as the major source of contamination and the handling, preparation and consumption of broiler meat may account for 20 to 30% of human campylobacteriosis cases (EFSA, 2010b).

Campylobacteriosis is usually a self-limiting disease lasting only a few days. Treatment with antimicrobials is not usually required except in severe cases or immunocompromised patients. Macrolides, such as erythromycin are now generally considered to be the optimal drug for treating campylobacteriosis, with fluoroquinolones as an alternative therapy (Allos, 2001). However, the prevalence of antimicrobial resistant Campylobacter strains in humans is increasing especially resistance to fluoroquinolones and to a lesser extent, macrolides (Cody et al., 2010, EFSA (European Food Safety Authority) and ECDC (European Center for Disease Prevention and Control), 2011). Multiple resistance patterns to several classes of antibiotics are also emerging. Trends in microbial resistance have shown a clear link between use of antibiotics in the poultry industry and resistant isolates of Campylobacter in humans (Moore et al., 2006). These resistant zoonotic bacteria are of special concern since they might compromise the effective treatment of infections in humans.

C. jejuni is also a recognized antecedent cause of post-infectious neuropathies such as Guillain–Barré syndrome (GBS) or Miller–Fischer syndrome (MFS) (Nachamkin et al., 1998). Among the numerous factors involved in the development of such autoimmune diseases, molecular mimicry between the lipooligosaccharide (LOS) structures present on the cell surface of C. jejuni and gangliosides in peripheral nerves play a crucial role (Ang et al., 2004, Ang et al., 2002, Godschalk et al., 2004).

Nineteen distinct classes of LOS biosynthesis loci have been described based on major genetic differences, gene content and organization (Parker et al., 2008). Five of these classes (A, B, C, M and R) harbor sialyltransferase genes involved in incorporating sialic acids into the LOS (Gilbert et al., 2002, Parker et al., 2008, Parker et al., 2005). Sialylation of the LOS core has been associated with ganglioside mimicry and it has been shown that in GBS or MFS related to Campylobacter strains, the majority of strains express the LOS gene cluster A or B (Godschalk et al., 2004, Islam et al., 2009, Koga et al., 2006).

Moreover, C. jejuni expressing sialylated LOS (classes A, B and C) appear to be more invasive for intestinal cells in vitro than those expressing non-sialylated LOS (classes D and E) (Habib et al., 2009a, Louwen et al., 2008). Sialylation of the LOS was also associated with increased severity of enteric disease by Mortensen et al. (2009) but these results were not recently supported by Ellström et al. (2013).

Molecular typing is an important tool for elucidating the diversity and transmission routes of Campylobacter isolates contaminating the food chain. Various molecular typing methods are currently available for studying the population structure of Campylobacter (Wassenaar and Newell, 2000). These genotypic methods have shown that Campylobacter has marked genomic diversity and a slightly clonal population structure, in which the different lineages and the relatedness between isolates cannot be easily determined, particularly within the framework of long-term epidemiological studies. Based on a concept similar to multilocus enzyme electrophoresis, multilocus sequence typing (MLST) exploits the relative conservation in sequence of certain genes in which variations are more likely to be selectively neutral because of their housekeeping functions (Maiden et al., 1998). This approach was successfully developed to study Campylobacter populations (Dingle et al., 2001) and is now recognized as the gold standard typing method for this bacterial genus (Sheppard et al., 2009).

In France, little is known about the prevalence of pathogenic Campylobacter in chicken and their molecular characteristics. No population-based surveys have been conducted to investigate the molecular epidemiology of C. jejuni in chicken meat at points close to human consumption. In this study, a survey was carried out to investigate the prevalence of Campylobacter in the chicken meat supply chain. C. jejuni isolates were further characterized as in France, more than 80% of campylobacteriosis are due to this species. Thus MLST was used to determine the diversity and clonal relationships among the 175 C. jejuni isolates collected. In addition, we correlated the LOS classes assigned by PCR and antimicrobial resistance of the isolates with the genotypes assigned by MLST.