Genomic Analysis of Reduced Susceptibility to Tigecycline in Enterococcus faecium

Abstract

c Tigecycline (TIG) is approved for use for the treatment of complicated intra-abdominal infections, skin and skin structure infections, as well as pneumonia. Acquired resistance or reduced susceptibility to TIG has been observed in Gram-negative rods, has seldom been reported in Gram-positive organisms, and has not yet been reported in Enterococcus faecium. Using the serial passage method, in vitro mutant AusTig and in vitro mutants HMtig1 and HMtig2 with decreased TIG susceptibility (MICs, 0.25 g/ml) were obtained from strains E. faecium Aus0004 and HM1070 (MICs, 0.03 g/ml), respectively. In addition, two vancomycin-resistant E. faecium clinical isolates (EF16 and EF22) with reduced susceptibility to TIG (MICs, 0.5 and 0.25 g/ml, respectively) were studied. Compared to the wild-type strains, the in vitro mutants also showed an increase in the MICs of other tetracyclines. An efflux mechanism did not seem to be involved in the reduced TIG susceptibility, since the presence of efflux pump inhibitors (reserpine or pantoprazole) did not affect the MICs of TIG. Whole-genome sequencing of AusTig was carried out, and genomic comparison with the Aus0004 genome was performed. Four modifications leading to an amino acid substitution were found. These mutations affected the rpsJ gene (efau004_00094, coding for the S10 protein of the 30S ribosomal subunit), efau004_01228 (encoding a cation transporter), efau004_01636 (coding for a hypothetical protein), and efau004_02455 (encoding the L-lactate oxidase). The four other strains exhibiting reduced TIG susceptibility were screened for the candidate mutations. This analysis revealed that three of them showed an amino acid substitution in the same region of the RpsJ protein.In this study, we characterized for the first time genetic determinants linked to reduced TIG susceptibility in enterococci.

The Ribosomal S10 Protein Is a General Target for Decreased Tigecycline Susceptibility

“…While the data presented in Fig. 1 and those reported by Cattoir et al and Villa et al show a correlation between mutations in rpsJ and reduced TGC susceptibility, there has been no direct confirmation that mutations in rpsJ alone are able to significantly decrease susceptibility (8,9). To confirm the causal link between adaptive mutations in rpsJ to reduced TGC susceptibility, we used whole-genome sequencing to isolate a strain of E. faecalis S613 that contained only a mutation in rpsJ (10) Using comparative whole-genome sequencing between S613 and clones isolated from an adapted bioreactor population, a strain with a single mutation in rpsJ was identified, referred to here as S613(S10 R53Q-⌬54-57ATHK ).…”

“…Particularly, in the Gram-positive species, D60 of S10 was frequently mutated to a tyrosine. In this study, the S10 D60Y allele was independently isolated from 10 different populations, and the same allele was also identified in a previous study, where E. faecium was also adapted in vitro to TGC (8) (Fig. 1).…”

“…We made several unsuccessful attempts to perform allelic replacement of resistance-associated rpsJ alleles into the susceptible ancestral strains of E. coli BW25113 and A. baumannii AB210. In addition, Cattoir et al reported that attempts to perform allelic replacement at this locus in E. faecium Aus0004 also failed (8). This could be due to the essential role of S10 in translation and transcription; S10 is both a component of the 30S ribosomal subunit and an important transcription factor involved in lambda N-mediated antitermination (20).…”

“…The emergence of these alleles in a diverse range of species strongly suggests that mutation of rpsJ is a general strategy to achieve decreased susceptibility to TGC. Previous studies identified mutations affecting the loop of S10 in E. faecium and K. pneumoniae strains that developed nonsusceptibility to TGC in patients undergoing TGC therapy, which highlights the relevance of these mutations to the clinical setting (8,9). Our observation that the growth rates of E. faecalis S613(S10 R53Q-⌬54-57ATHK ) are comparable to those of the original susceptible strain in the absence of antibiotic suggests that there is little fitness cost incurred by this adaptive mutation in the organism and that this would be a stable and persistent allele within the population once established.…”

Differential Penicillin-Binding Protein 5 (PBP5) Levels in the Enterococcus faecium Clades with Different Levels of Ampicillin Resistance

“…When we analyzed the region upstream of pbp5 in the draft genomes of TX82 A1 and 1.230.933 A1 , we found that the sequences in this area contain a series of gaps; however, alignments designed to close these gaps (the primers used are listed in Table S2), along with PCR and sequencing results, indicated a genetic environment similar to that in the subclade A1 strain Aus0004 (Table 2 and Fig. 2D), which is also highly resistant (MIC ϭ 256 g/ml, as described previously [24] and confirmed here). The region between psr and pbp5 in Aus0004 includes three different open reading frames (ORFs), ISEfm1, gnat, and hisJ, predicted to encode a transposase, an acetyltransferase, and a histidinol phosphate phosphatase, respectively (Fig.…”