Impact of erythromycin resistance on the virulence properties and fitness of Campylobacter jejuni

Abstract

Epidemiological studies of macrolide resistance in Campylobacter jejuni demonstrated that infections with macrolide-resistant C. jejuni could be associated with an increased risk of adverse events, development of invasive illness or death compared to macrolide-susceptible isolates. In this study, an in vitro induction experiment was conducted using susceptible C. jejuni strain and erythromycin as a selecting agent to obtain Ery-resistant mutant with 23S rRNA gene mutation (A2074C). Changes in the virulence characteristics and fitness between the susceptible parent strain and Ery-resistant mutant were examined. Ery-resistant mutant demonstrated slightly more resistance to bile in the bile tolerance assay compared to the susceptible strain but with no statistical significant difference. However Ery-resistant mutant apparently demonstrated reduced adhesion and invasion characteristics to intestinal epithelial cells, murine macrophage and short time intracellular survivability within macrophage compared to the susceptible strain. Co-inoculation of the two strains in the mice resulted in low colonization level of the resistant strain compared to the susceptible strain. Competition experiments resulted in mutant that grew significantly slower than the susceptible parent strain and the mutation imposed a fitness cost in Ery-resistant mutant. Taken together these findings demonstrated the increment of the virulence characteristics of Ery-susceptible strain rather than Ery-resistant strain. The adverse events previously observed in the epidemiological studies for macrolide-resistant strains infection, we suggested this maybe attributed to the resistivity of the resistant strains to the treatment and consequently prolonged the symptoms and compromised the disease in patients.

Introduction

Campylobacter, especially Campylobacter jejuni, comes up on top of the principal bacterial agents that currently considered as the leading cause of human bacterial gastroenteritis [1]. Human gastroenteritis caused by Campylobacter infection largely attributed to the consumption of contaminated or undercooked food and is mostly often a self-limiting diarrheal illness where patients do not usually require antimicrobial treatment. Nevertheless severe and prolonged gastroenteritis required antimicrobial treatment. In such cases macrolides and quinolones are the drugs of choice [2]. In severe cases the most notable post-infection complication of campylobacteriosis is the development of Guillain-Barré syndrome, acute ascending bilateral paralysis [3]. Macrolides inhibit bacterial growth by binding to ribosomes and interfering with the bacterial protein synthesis. They bind to the 50S ribosomal subunit, causing blockage of transpeptidation and/or translocation [4].

Macrolide resistance in C. jejuni occurs mainly through chromosomal target gene mutation, particularly 23S rRNA gene point mutations (A2075G, A2074C, A2074G) [5]. Furthermore an efflux pump system (CmeABC) plays a significant role in Campylobacter multidrug resistance and extrusion of antibiotics, bile and dyes [6]. Recently modifications in ribosomal proteins L4 and L22 were reported to act synergistically with CmeABC to confer macrolide resistance in Campylobacter [7].

Resistance to clinically important antimicrobial agents, particularly macrolides and fluoroquinolones, is increasing among Campylobacter isolates but few studies have explored the human health consequences of such resistance. Despite wealthy information about the adverse events associated with antimicrobial drug resistance in Salmonella infections [8], [9], [10], limited information exists on the clinical consequences of resistance in Campylobacter infection. Data supporting an increase in virulence of drug-resistant isolates of C. jejuni are starting to emerge and epidemiological studies have examined the clinical impact of antibiotic resistance in Campylobacter infections. For instance, investigations from the United States, Thailand and Denmark have provided information that infections with macrolide-resistant Campylobacter isolates could be associated with increased risk of adverse events, development of invasive illness or death compared to infections with macrolide-susceptible isolates [4], [11], [12], [13]. Moreover some epidemiological reports demonstrated that quinolone resistance in C. jejuni is also associated with adverse events, longer duration of diarrhea, and development of invasive illness [14], [15], [16], [17]. However all reports were dependent upon epidemiological studies results. Furthermore no scientific evidence that clearly illustrated if macrolide resistance in C. jejuni could be associated with increased virulence of the resistant strains over the susceptible strains.

Indeed, in case of mutations leading to acquisition of antibiotic resistance, some evidences demonstrated the possibility of fitness reduction, which may also reduce bacterial virulence [18], [19]. The 23S rRNA gene mutations (A2074C/A2075G) were shown to impose a fitness cost in macrolide-resistant mutants of the chicken isolate h1 and human isolate C. jejuni 81–176 [20], [21]. Although significant progress has been made in elucidating the fitness of macrolide resistance in Campylobacter, examination of the fitness of macrolide-resistant mutants especially those derived from the same parent strain is still essential due to strain diversity, different selection environment and/or different type of mutation may have different effect on the bacterial fitness.

The present study was therefore undertaken to examine whether acquisition of macrolide resistance through 23S rRNA might modulate the virulence of C. jejuni resistant mutant. This was assessed through the ability of macrolide-susceptible and macrolide-resistant C. jejuni to tolerate the deleterious effect of bile salts, since bile tolerance is prerequisite for successful colonization. Furthermore we examined their ability to adhere and invade the intestinal epithelial cells and murine macrophage, as well as their ability to survive in murine macrophage. Moreover, animal colonization model between the two strains was studied. Furthermore we elucidated the relationship between C. jejuni virulence and fitness due to macrolide resistance.