AcrAB Multidrug Efflux Pump Is Associated with Reduced Levels of Susceptibility to Tigecycline (GAR-936) in Proteus mirabilis

Abstract

Tigecycline has good broad-spectrum activity against many gram-positive and gram-negative pathogens with the notable exception of the Proteeae. A study was performed to identify the mechanism responsible for the reduced susceptibility to tigecycline in Proteus mirabilis. Two independent transposon insertion mutants of P. mirabilis that had 16-fold-increased susceptibility to tigecycline were mapped to the acrB gene homolog of the Escherichia coli AcrRAB efflux system. Wild-type levels of decreased susceptibility to tigecycline were restored to the insertion mutants by complementation with a clone containing a PCR-derived fragment from the parental wild-type acrRAB efflux gene cluster. The AcrAB transport system appears to be associated with the intrinsic reduced susceptibility to tigecycline in P. mirabilis.The growing threat of acquired resistance in the Enterobacteriaceae (6,10,20) indicates the crucial need for new antibiotics for continued effective treatment of bacterial infection. Tigecycline, the 9-t-butylglycylamido derivative of minocycline, is a new antimicrobial agent belonging to a novel class of tetracyclines, the glycylcyclines (24). It has good activity against gram-positive pathogens, including penicillin-resistant Streptococcus pneumoniae, vancomycin-resistant enterococci, and methicillin-resistant Staphylococcus aureus (2,5,18). Tigecycline has good activity against most gram-negative pathogens, including Klebsiella pneumoniae and Escherichia coli (4,18). Tigecycline also has good activity against organisms with a resistance determinant from the major facilitator family, including E. coli with tet(A), tet (B) , tet(C), tet(D), and tet(M); S. aureus with tet(K) and tet(M); and Enterococcus faecalis with tet(M) (18).Proteus mirabilis is a notable exception to the activity of tigecycline, which routinely shows MICs of 4 g/ml for the organism in tests.It is important to identify current and emerging resistance mechanisms. Identification and mechanistic studies of bacterial resistance mechanisms can help to further reduce health care costs due to bacterial infection. Therefore, a study was performed to identify the mechanism responsible for the reduced susceptibility of P. mirabilis to tigecycline (M. A. Visalli, E. Murphy, S. J. Projan, and P. A. Bradford, Abstr. 41st Intersci. Conf. Antimicrob. Agents Chemother., abstr. C1-2019, 2001). MATERIALS AND METHODSBacterial strains and growth conditions. The bacterial strains, plasmids, and transposons used in this study are listed in Table 1. A typical clinical isolate of P. mirablis, G151, was selected from a tigecycline phase II clinical trial. The E. coli strains used included Top10 (Invitrogen, Carlsbad, Calif.), Transformax EC100D pir-116 (Epicentre, Madison, Wis.), DM1 (Gibco Life Technologies, Rockville, Md.), and two laboratory strains, one wild type and the other an acrAB deletion mutant (17). The strains were grown in Luria-Bertani (LB) broth or agar in the presence of the following selective antibiotics when required: 50 g of kanamycin/ml, 50...

Effects of Efflux Transporter Genes on Susceptibility of Escherichia coli to Tigecycline (GAR-936)

“…Bcr expression did not result in any change in the tigecycline MIC. Recently, it was reported that the AcrB homologue is involved in the tigecycline resistance of P. mirabilis (28), which is in good agreement with the result obtained here. The MICs for the strains expressing AcrAB and AcrEF were restored to the MIC for strain TG1, which has an intact AcrB pump, but did not reach the level of clinical resistance.…”

“…Tigecycline is a broadspectrum glycylcycline derivative (2) and is efficacious against highly resistant bacteria (1), including methicillin-resistant Staphylococcus aureus and penicillin-resistant Streptococcus pneumoniae. However, it is less active against clinically problematic opportunistic pathogens, such as Pseudomonas aeruginosa (6) and Proteus mirabilis (3,28).…”

The Challenge of Efflux-Mediated Antibiotic Resistance in Gram-Negative Bacteria

“…A nearly 30-fold overexpression of adeB was observed for tigecycline-nonsusceptible isolates (533). Thus, although tigecycline displays activity against isolates possessing tetracycline-specific resistance mechanisms (ribosomal protection and efflux) (539), broad-substrate-specificity RND pumps play a key role in the emergence of tigecycline resistance in Acinetobacter spp., similar to observations for many other Gram-negative bacteria (16,138,268,310,312,324,393,540). (Additionally, non-pump-mediated tigecycline insusceptibility occurred due to a deletion mutation in the trm gene that encodes S-adenosyl-L-methionine-dependent methyltransferase [541], showing the complexity of tigecycline resistance mechanisms.)…”

“…The substrate range of AcrB from P. mirabilis includes, as expected, a number of antibiotics, dyes, and detergents (310,311). A similar range was found in a tigecycline-resistant clinical isolate of M. morganii overexpressing AcrAB, whose inactivation has led to hypersusceptibility to multiple agents, including a 130-fold reduction of the tigecycline MIC value (312).…”

Clinically Relevant Chromosomally Encoded Multidrug Resistance Efflux Pumps in Bacteria

“…The modest in vitro activity of the macrolides for Haemophilus influenzae has also been attributed to efflux via AcrAB in this species (159,197). In addition, the new glycycline, tigecycline, has been shown to be less active against P. aeruginosa and Proteus mirabilis due to the MexXY-OprM and AcrAB-TolC efflux pumps, respectively (37,225). As there are now increasing numbers of MDR gramnegative bacteria, the focus on drug development will inevitably turn to extending the spectrum of activity of these agents, with efforts to identify agents that are not substrates of efflux pumps.…”