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Using gBlocks™ HiFi Gene Fragments to explore novel pathways of antibiotic resistance

Dulanto Chiang A, Patil PP, Beka L, et al. Hypermutator strains of Pseudomonas aeruginosa reveal novel pathways of resistance to combinations of cephalosporin antibiotics and beta-lactamase inhibitors. PLoS Biol. 2022;20(11):e3001878.

Chiang et al. used whole genome sequencing (WGS) and transcriptional profiling to identify transcriptional and mutational changes underlying antibacterial resistance in mismatch repair (MMR) deficient P. aeruginosa isolates. The authors focused on two Resistance-Nodulation-Division (RND) efflux pumps—the MexVW efflux pump and the mexB gene of the MexAB-OprM efflux pump—to characterize their roles in mediating ceftazidime/avibactam (CZA) resistance in hypermutated P. aeruginosa isolates. They used IDT’s gBlocks HiFi Gene Fragments to generate mutant alleles of the MexVW efflux pump and the mexB gene of the MexAB-OprM efflux pump to further characterize its role in the rapid development of CZA resistance.

Pseudomonas aeruginosa is a pathogen known to cause serious infections. Multidrug resistant (MDR) P. aeruginosa strains have been shown to contain hypermutations in DNA repair pathways. Hypermutation is the result of mutations in the ATPase domain of MutS mismatch repair (MMR) protein [1]. MMR gene deficiency leads to a rapid development of CZA resistance in P. aeruginosa [2]. CZA is an antimicrobial combination of an anti-pseudomonal cephalosporin and a novel beta-lactamase inhibitor which is potent for inhibiting P. aeruginosa PDC (AmpC) cephalosporinase [3]. An important caveat is that CZA-resistant P. aeruginosa strains have been shown to have an overexpression of the Resistance-Nodulation-Division (RND) MexAB-OprM efflux pump due to point mutations in cephaloporinase [1].

Whole-genome sequencing (WGS) and transcriptional profiling of the MMR-deficient strains were performed to identify mutational and transcriptional changes that underlie CZA resistance. The results revealed that the MMR-deficient strains can also lead to a rapid development of resistance without the modification of well-established resistance genes. Chiang et al. identified that an early inactivation of the MexB protein was due to a mutation in the mexB gene of the MexAB-OprM efflux pump. The MexAB-OprM efflux pump is part of the RND class of efflux pumps which mediates ceftazidime/avibactam (CZA) resistance in P. aeruginosa [4]. The RND family of efflux pumps are widely distributed in Gram-negative bacteria. These transporters have the essential functions for transporting antibiotics across cytoplasmic membrane [5]. It has been identified that this early inactivation of the mexB gene of the MexAB-OprM efflux pump is a driving factor for alternative resistance mechanisms in P. aeruginosa.

Chiang et al. used gBlocks HiFi Gene Fragments to further investigate how the mexB gene of the MexAB-OprM efflux pump and the MexVW efflux pump play a role in antibiotic resistance. They genetically engineered MMR-deficient strains containing mutations in the mexB gene and the MexVW RND efflux pump. Three strains were generated—one strain for each mutation and a double mutant strain—to study how these two mutations in the MexVW RND efflux pump resulted in an increase of antibiotic resistance in P. aeruginosa

Learn more about how IDT gene fragments can be used for antibiotic resistance research.

Additional resources


2.Khil PP, Dulanto Chiang A, Ho J, et al. Dynamic Emergence of Mismatch Repair Deficiency Facilitates Rapid Evolution of Ceftazidime-Avibactam Resistance in Pseudomonas aeruginosa Acute Infection. mBio. 2019;10(5):e01822-19.

3. Zhanel GG, Lawson CD, Adam H, et al. Ceftazidime-avibactam: a novel cephalosporin/β-lactamase inhibitor combination. Drugs. 2013;73(2):159-177. 

4. Middlemiss JK, Poole K. Differential impact of MexB mutations on substrate selectivity of the MexAB-OprM multidrug efflux pump of Pseudomonas aeruginosaJ Bacteriol. 2004;186(5):1258-1269.

5. Nikaido H, Takatsuka Y. Mechanisms of RND multidrug efflux pumps. Biochim Biophys Acta. 2009;1794(5):769-781.



Published Jul 3, 2023
Revised/updated May 17, 2023