Which mechanisms of azithromycin resistance are selected when efflux pumps are inhibited?

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Abstract

The aim of this study was to develop in vitro azithromycin (AZM)-resistant mutants of Escherichia coli and Shigella spp. in the presence of Phe-Arg β-naphthylamide (PAβN) and to observe which AZM resistance mechanisms other than efflux pumps were inhibited by PAβN emerge. The frequency of mutation ranged between <6.32 × 10−10 and 5.22 × 10−7 for E. coli and between <5.32 × 10−10 and 1.69 × 10−7 for Shigella spp. The E. coli mutants showed an increase in the AZM minimum inhibitory concentration (MIC) up to 128-fold, whilst the Shigella spp. mutants presented increases in MIC levels of up to 8-fold. In one mutant, the insertion of nucleotides encoding the amino acid sequence IMPRAS was found in the rplV gene. Increases in OmpW expression were observed in all E. coli mutants compared with their respective parental isolates. The combination of antibiotics and efflux pump inhibitors appears to be a good option to reduce the frequency of mutation in clinical isolates.

Introduction

Diarrhoeagenic Escherichia coli and Shigella spp. rank among the main aetiological causes of diarrhoea [1]. The clinical manifestations of shigellosis vary from simple watery diarrhoea to full-blown dysentery that frequently requires antibiotic treatment [2], whilst E. coli usually produces a self-limiting illness. However, in the case of E. coli infections, use of antimicrobial agents such as ampicillin, trimethoprim/sulfamethoxazole and ciprofloxacin may sometimes be required owing to the duration or severity of symptoms [3], [4]. Levels of antimicrobial resistance to the aforementioned agents have progressively increased worldwide in recent years [3], [4], making it necessary to search for alternatives.

In Peru, as in other South American areas, infantile diarrhoea has particular relevance in peri-urban and rural areas [5]. Previous studies have shown high levels of antibiotic resistance in diarrhoeagenic pathogens in the country [4] as well as its extension to commensal micro-organisms [6]. In this regard, antibiotic resistance is a severe health problem worldwide that can lead to inefficacy of antimicrobial agents and therapeutic failure [7]. Thus, the possible development of antimicrobial resistance should be closely monitored. Furthermore, a better understanding of the mechanisms that underlie this resistance is necessary to find therapeutic alternatives.

Azithromycin (AZM) is an antimicrobial agent belonging to the macrolide family that has mainly been used to treat infections caused by Gram-positive micro-organisms [8]. However, it has been shown that this antimicrobial agent possesses good activity against various Gram-negative micro-organisms [9]. In fact, AZM currently plays a relevant role in the treatment of Campylobacter spp., and its use has been recommended in the treatment of other diarrhoeagenic bacteria such as E. coli and Shigella spp. [10]. Unfortunately, despite the absence of an established breakpoint, recent reports have described the presence of AZM resistance both in E. coli and Shigella spp. [4], [11].

To date, different molecular mechanisms involved in the development of resistance to AZM have been described. The most important are probably those encoded on mobile elements, such as ere(A), ere(B), mef(A), mef(B) and others [11]. However, other mechanisms, such as punctual mutations in the rplD, rplV and 23S rRNA genes, have also been described [12], [13]. In addition, the potential of macrolides to act as a substrate of some chromosomal efflux pumps has been reported [14]. In a previous study with in vitro AZM-resistant mutants, we saw that the efflux pumps inhibitable by Phe-Arg β-naphthylamide (PAβN) were the principal mechanism of acquired resistance [15]. Thus, the aim of this study was to obtain in vitro AZM-resistant E. coli and Shigella spp. mutants when inhibiting the action of efflux pumps with PAβN, analysing the frequency of mutation, stability and cross-resistance with other agents and establishing which mechanisms of AZM resistance are selected. Furthermore, the potential of combining an efflux pump inhibitor with AZM to minimise the emergence of AZM resistance was evaluated.

Section snippets

Bacterial strains

Four clinical isolates of E. coli and three Shigella spp. (one Shigella flexneri, one Shigella boydii and one Shigella sonnei) were isolated from faecal samples collected from children with diarrhoea under 5 years of age in Lima (Peru), identified by conventional microbiological and biochemical methods and assessed by amplification of the uidA and ipaH genes [16], [17]. In the case of Shigella spp., identification to species level was performed by serotyping (Probac, São Paulo, Brazil). These

Frequency of mutation

All selected mutant strains showed the same REP-PCR pattern as their parental isolate (data not shown), being considered as reliably derived AZM-resistant mutants.

The frequency of mutation ranged between <6.32 × 10−10 and 5.22 × 10−7 for E. coli and between <5.32 × 10−10 and 1.69 × 10−7 for Shigella isolates (Table 1). The E. coli mutants showed a 2- to 128-fold increase in the MIC of AZM with respect to the parental isolates, with one strain (Ec 2.7) reaching a MIC of 256 mg/L (Table 2). Meanwhile, the

Discussion

The ability of an antimicrobial agent to select resistance is a relevant factor that affects its utility and may diminish its useful life. Along this line, several studies have been designed to analyse and characterise clinical isolates and in vitro mutants of different micro-organisms [15], [21]. In the present work, the ability of AZM to select resistant mutants of Shigella spp. and E. coli in the presence of an inhibitor of some efflux pumps, and which mechanisms responsible for this

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