Short Communication
Deletions in a ribosomal protein-coding gene are associated with tigecycline resistance in Enterococcus faecium

https://doi.org/10.1016/j.ijantimicag.2015.07.009Get rights and content

Highlights

  • Whole-genome sequencing permits de novo detection of antibiotic resistance in Enterococcus faecium.

  • Reduced susceptibility to tigecycline is associated with substitutions in rpsJ.

  • Resistance to tigecycline is associated with deletions in rpsJ.

  • Protein modelling indicates that these deletions distort the tigecycline-binding site.

Abstract

Enterococcus faecium is an emerging nosocomial pathogen associated with antibiotic therapy in the hospital environment. Whole-genome sequences were determined for three pairs of related, consecutively collected E. faecium clinical isolates to determine putative mechanisms of resistance to tigecycline. The first isolates (1S, 2S and 3S) in each of the three pairs were sensitive to tigecycline [minimum inhibitory concentration (MIC) of 0.125 mg/L]. Following tigecycline therapy, the second isolate in each pair demonstrated increased resistance to tigecycline. Two isolates (1R and 2R) were resistant (MIC of 8 mg/L) and one isolate (3I) demonstrated reduced susceptibility (MIC of 0.5 mg/L). Mutations distinguishing each pair of sensitive and resistant isolates were determined through alignment to a reference genome and variant detection. In addition, a de novo assembly of each isolate genome was constructed to confirm mutations. A total of 16 mutations in eleven coding sequences were determined. Mutations in the rpsJ gene, which encodes a structural protein forming part of the 30S ribosomal subunit, were detected in each of the pairs. Mutations were in regions proximal to the predicted tigecycline-binding site. Predicted amino acid substitutions were detected in 1R and 3I. The resistant strains were additionally associated with deletions of 15 nucleotides (2R) and 3 nucleotides (1R). This study confirms that amino acid substitutions in rpsJ contribute towards reduced susceptibility to tigecycline and suggests that deletions may be required for tigecycline resistance in E. faecium.

Introduction

Enterococcus faecium is a nosocomial pathogen associated with antibiotic therapy in the hospital environment that is increasing in prevalence [1], [2]. Compared with Enterococcus faecalis, which remains the most common Enterococcus species in clinical infections, E. faecium is intrinsically resistant to a greater number of antibiotics and can efficiently acquire additional antibiotic resistance mechanisms necessitating the use of alternative antibiotics for therapy. Vancomycin resistance is a common and pressing clinical problem in Enterococcus. This results from transposon-mediated insertion of genes coding for vancomycin-inactivating enzymes. Tigecycline, a derivative of minocycline, has bacteriostatic activity through binding to the 30S subunit of the bacterial ribosome and inhibiting protein synthesis [2], [3]. It provides broad-spectrum activity against multiresistant pathogens such as meticillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), and Enterobacteriaceae with extended-spectrum β-lactamases (ESBLs) or carbapenemases. Tigecycline resistance has been reported [3], mainly in Gram-negative pathogens, e.g. Acinetobacter baumannii and Klebsiella pneumoniae [4], [5], although rarely also in Gram-positive pathogens such as E. faecalis and MRSA [6], [7]. Tigecycline resistance is most commonly associated with overexpression of efflux pumps, e.g. MarA-mediated overexpression of AcrAB in Escherichia coli [3], [8].

Whole-genome sequencing (WGS) is a powerful technique that can be used to pinpoint mutations that arise in clinical isolates which have been exposed to antibiotics during treatment. This technique has allowed detailed analysis of pairs of consecutively isolated tigecycline-susceptible and -resistant isolates of Gram-negative pathogens to be undertaken [5], [9]. In this study, WGS was used to investigate the nature of potential mechanism(s) associated with tigecycline resistance and reduced susceptibility in three VRE clinical isolates.

Section snippets

Bacterial isolates

Two pairs of isolates (1S and 1R, and 3S and 3I) were obtained from patients in a hospital in the West Midlands, UK. Both patients were previous liver transplant recipients and had been diagnosed with hepatic artery thrombosis and intrahepatic collections. A tigecycline-resistant VRE (1R) was isolated from the first patient following a total of 38 days of treatment with tigecycline. The VRE isolate with reduced susceptibility to tigecycline (3I) was isolated following two courses of

Results and discussion

The antibiotic susceptibility profiles revealed increased resistance to tigecycline in each of the three post-therapy isolates (Table 1). Each of the pairs showed different mutations in rpsJ, which encodes the S10 structural protein in the small 30S ribosomal subunit.

Comparison of genomes for pair 1 showed a codon deletion (ATC) and a non-synonymous transition (T>C) mutation. Pair 2 had a deletion of five codons (ATCCGTGCGACTCAT). Pair 3 had two non-synonymous transversion (C>A and A>G)

Acknowledgments

The authors wish to thank Daniele Meunier for supplying the isolates and Kate Martin for PFGE comparisons [both at the Antimicrobial Resistance and Healthcare Associated Infections (AMRHAI) Reference Unit, Public Health England, London, UK], and Gabby Philips (Department of Microbiology, Ninewells Hospital, Dundee, UK) for her helpful input into the Scottish isolates.
Funding: This article presents independent research funded by the National Institute for Health Research Surgical Reconstruction

References (15)

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