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

Abstract

e Tigecycline is a translational inhibitor with efficacy against a wide range of pathogens. Using experimental evolution, we adapted Acinetobacter baumannii, Enterococcus faecium, Escherichia coli, and Staphylococcus aureus to growth in elevated tigecycline concentrations. At the end of adaptation, 35 out of 47 replicate populations had clones with a mutation in rpsJ, the gene that encodes the ribosomal S10 protein. To validate the role of mutations in rpsJ in conferring tigecycline resistance, we showed that mutation of rpsJ alone in Enterococcus faecalis was sufficient to increase the tigecycline MIC to the clinical breakpoint of 0.5 g/ml. Importantly, we also report the first identification of rpsJ mutations associated with decreased tigecycline susceptibility in A. baumannii, E. coli, and S. aureus. The identified S10 mutations across both Gram-positive and -negative species cluster in the vertex of an extended loop that is located near the tigecycline-binding pocket within the 16S rRNA. These data indicate that S10 is a general target of tigecycline adaptation and a relevant marker for detecting reduced susceptibility in both Gram-positive and -negative pathogens. Tigecycline is an FDA-approved antibiotic used to treat complicated skin and skin structure infections (cSSSI), complicated intra-abdominal infections (cIAI), and community-associated bacterial pneumonia (CABP). Increasingly, tigecycline and other drugs of last resort, such as vancomycin and daptomycin, are used "off label" to combat infections arising from multidrug-resistant pathogens (1). As infections with multidrug-resistant pathogens increase, they pose a major threat to public health, with at least two million infections and 23,000 deaths annually in the United States (2). With increased usage, tigecycline resistance will inevitably develop. By understanding how drug resistance emerges, we can be more proactive in identifying the spread of alleles associated with nonsusceptibility or even move toward a preemptive strategy to block the emergence of resistance through new classes of "antievolution" drugs that could work with current antibiotics.The antibiotic tigecycline (TGC) has good in vitro activity against a broad spectrum of multidrug-resistant pathogens and is often used as part of a combination regimen in severe infections caused by multidrug-resistant isolates (3). TGC is a semisynthetic drug belonging to the tetracycline-derived glycylcycline family. Like tetracycline, TGC inhibits the elongation step of translation by binding reversibly to the 16S rRNA and blocking the entry of tRNAs into the A site. The therapeutic success of TGC is due, in part, to its high affinity for the ribosome, which is about 20-fold higher than that of tetracycline (4). TGC has a bulky t-butylglycylamido modification at the 9 position that has been proposed to limit the activity of common tetracycline-resistance mechanisms, such as ribosomal protection (tetM, tetO, etc.) and efflux pumps (tetK, tetA, etc.) (3).Despite the promising in vitro activity of TGC...

Contrasting approaches to genome-wide association studies impact the detection of resistance mechanisms in Staphylococcus aureus

“…5A). Consistent with previous observations (Beabout et al 2015), the most common site for significant associations was K57 (Fig. 5B).…”

“…The mutations significantly associated with resistance (rpsJ-K57MQR, Y58D and D60Y) have been observed in S. aureus or E. faecium in a previous experimental evolution study which selected for resistance to tigecycline. Allelic replacement to verify these mutations have failed to produce a viable mutant (Beabout et al 2015;Cattoir et al 2015). Studies in Neisseria gonorrhoeae have also described a V57M intermediate resistance mutation in the corresponding site (Hu et al 2005).…”

Vancomycin-Resistant Enterococci

“…116 Analysis of resistant strains found that the changes in S10 are clustered in an extended loop of the protein that lies in close proximity to the tigecycline binding site of the 16S rRNA, possibly interfering with access to or binding of its target. 117 Even modest increases in the tigecycline MIC are problematic in serious infections such as bacteremia or endocarditis because of the low achievable serum concentration of this antibiotic.…”

Mutations at the Ribosomal S10 Gene in Clinical Strains of Staphylococcus aureus with Reduced Susceptibility to Tigecycline

“…T igecycline (TGC) resistance in Staphylococcus aureus is a rare phenomenon that has been associated with overexpression of a multidrug and toxic compound extrusion (MATE) family efflux pump (mepA) in passaged mutants and clinical strains (1,2). Recently, specific mutations on the tip of the extended loop of the ribosomal S10 protein (rpsJ) in close proximity to the 16SrRNA TGC-binding site have also been associated to TGC-resistance in passaged mutants (3). But, as far as we know, there are no studies reporting clinical S. aureus isolates with mutations at rpsJ.…”