Conservation of resistance nodulation cell division efflux pump mediated antibiotic resistance in Burkholderia cepacia complex and Burkholderia pseudomallei complex species

Burkholderia cepacia complex (Bcc) and Burkholderia pseudomallei complex (Bpc) species include pathogens that are typically multidrug resistant. Dominant intrinsic and acquired multidrug resistance mechanisms are efflux mediated by pumps of the resistance nodulation cell division (RND) family. From comparative bioinformatic and, in many instances, functional studies we infer that RND pump-based resistance mechanisms are conserved in Burkholderia . We propose to use these findings as a foundation for adoption of a uniform RND efflux pump nomenclature.

Comparison of the A. baumannii reference strains ATCC 17978 and ATCC 19606 in antimicrobial resistance mediated by the AdeABC efflux pump

The Acinetobacter baumannii RND efflux pump AdeABC is regulated by the 2-component regulator AdeRS. In this study, we compared regulation and expression of AdeABC of the reference strains ATCC 17978 and ATCC 19606. A clearly stronger efflux activity was demonstrated for ATCC 19606. An amino acid substitution at residue 172 of adeS was identified as potential cause for differential expression of the pump. Therefore, we recommend caution with exclusively using single reference strains for research.

Characterization of TMexCD3-TOprJ3, an RND-type efflux system conferring resistance to tigecycline in Proteus mirabilis, and its associated Integrative Conjugative Element

The emergence and transmission of novel antimicrobial resistance genes pose a great threat to public health globally. Recently, the plasmid-encoding RND efflux pump TMexCD1-TOprJ1 in Klebsiella pneumoniae was reported to reduce the sensitivity of multiple antimicrobials. Herein, we identified a pandrug-resistant Proteus mirabilis isolate, which harbored the novel tmexCD3-toprJ3 gene cluster located on SXT/R391 ICE. This study expands current knowledge in transfer mechanism of tmexCD1-toprJ1-like gene clusters among P. mirabilis and warrant further genomic epidemiology investigations.

Resistance-Nodulation-Division Efflux Pump, LexABC, Contributes to Self-Resistance of the Phenazine Di-N-Oxide Natural Product Myxin in Lysobacter antibioticus

Antibiotic-producing microorganisms have developed several self-resistance mechanisms to protect them from autotoxicity. Transporters belonging to the resistance- nodulation-division (RND) superfamily commonly confer multidrug resistance in Gram-negative bacteria. Phenazines are heterocyclic, nitrogen-containing and redox-active compounds that exhibit diverse activities. We previously identified six phenazines from Lysobacter antibioticus OH13, a soil bacterium emerging as a potential biocontrol agent. Among these phenazines, myxin, a di-N-oxide phenazine, exhibited potent activity against a variety of microorganisms. In this study, we identified a novel RND efflux pump gene cluster, designated lexABC, which is located far away in the genome from the myxin biosynthesis gene cluster. We found a putative LysR-type transcriptional regulator encoding gene lexR, which was adjacent to lexABC. Deletion of lexABC or lexR gene resulted in significant increasing susceptibility of strains to myxin and loss of myxin production. The results demonstrated that LexABC pump conferred resistance against myxin. The myxin produced at lower concentrations in these mutants was derivatized by deoxidation and O-methylation. Furthermore, we found that the abolishment of myxin with deletion of LaPhzB, which is an essential gene in myxin biosynthesis, resulted in significant downregulation of the lexABC. However, exogenous supplementation with myxin to LaPhzB mutant could efficiently induce the expression of lexABC genes. Moreover, lexR mutation also led to decreased expression of lexABC, which indicates that LexR potentially positively modulated the expression of lexABC. Our findings reveal a resistance mechanism against myxin of L. antibioticus, which coordinates regulatory pathways to protect itself from autotoxicity.

Involvement of MexS and MexEF-OprN in Resistance to Toxic Ion Chelators in Pseudomonas putida KT2440

Bacteria must be able to cope with harsh environments to survive. In Gram-negative bacteria like Pseudomonas species, resistance-nodulation-division (RND) transporters contribute to this task by pumping toxic compounds out of cells. Previously, we found that the RND system TtgABC of Pseudomonas putida KT2440 confers resistance to toxic metal chelators of the bipyridyl group. Here, we report that the incubation of a ttgB mutant in medium containing 2,2’-bipyridyl generated revertant strains able to grow in the presence of this compound. This trait was related to alterations in the pp_2827 locus (homolog of mexS in Pseudomonas aeruginosa). The deletion and complementation of pp_2827 confirmed the importance of the locus for the revertant phenotype. Furthermore, alteration in the pp_2827 locus stimulated expression of the mexEF-oprN operon encoding an RND efflux pump. Deletion and complementation of mexF confirmed that the latter system can compensate the growth defect of the ttgB mutant in the presence of 2,2’-bipyridyl. To our knowledge, this is the first report on a role of pp_2827 (mexS) in the regulation of mexEF-oprN in P. putida KT2440. The results expand the information about the significance of MexEF-OprN in the stress response of P. putida KT2440 and the mechanisms for coping with bipyridyl toxicity.

RamA, a transcriptional regulator conferring florfenicol resistance in Leclercia adecarboxylata R25

Due to the inappropriate use of florfenicol in agricultural practice, florfenicol resistance has become increasingly serious. In this work, we studied the novel florfenicol resistance mechanism of an animal-derived Leclercia adecarboxylata strain R25 with high-level florfenicol resistance. A random genomic DNA library was constructed to screen the novel florfenicol resistance gene. Gene cloning, gene knockout, and complementation combined with the minimum inhibitory concentration (MIC) detection were conducted to determine the function of the resistance-related gene. Sequencing and bioinformatics methods were applied to analyze the structure of the resistance gene-related sequences. Finally, we obtained a regulatory gene of an RND (resistance-nodulation-cell division) system, ramA, that confers resistance to florfenicol and other antibiotics. The ramA-deleted variant (LA-R25ΔramA) decreased the level of resistance against florfenicol and several other antibiotics, while a ramA-complemented strain (pUCP24-prom-ramA/LA-R25ΔramA) restored the drug resistance. The whole-genome sequencing revealed that there were five RND efflux pump genes (mdtABC, acrAB, acrD, acrEF, and acrAB-like) encoded over the chromosome, and ramA located upstream of the acrAB-like genes. The results of this work suggest that ramA confers resistance to florfenicol and other structurally unrelated antibiotics, presumably by regulating the RND efflux pump genes in L. adecarboxylata R25.

Development and Characterization of Acidic-pH Tolerant Mutants of Zymomonas mobilis through Adaptation and Next Generation Sequencing based Genome Resequencing and RNA-Seq

Background: Acid pretreatment is a common strategy used to break down the hemicellulose component of the lignocellulosic biomass to release pentoses, and a subsequent enzymatic hydrolysis step is usually applied to release hexoses from the cellulose. The hydrolysate after pretreatment and enzymatic hydrolysis containing both hexoses and pentoses can then be used as substrates for biochemical production. However, the acid-pretreated liquor can also be directly used as the substrate for microbial fermentation, which has an acidic pH and contains inhibitory compounds generated during pretreatment. Although the natural ethanologenic bacterium Zymomonas mobilis can grow in a broad range of pH 3.5~7.5, cell growth and ethanol fermentation are still affected under acidic-pH conditions below pH 4.0. Results: In this study, adaptive laboratory evolution (ALE) strategy was applied to adapt Z. mobilis under acidic-pH conditions. Two mutant strains named 3.6M and 3.5M with enhanced acidic-pH tolerance were selected and confirmed, of which 3.5M grew better than ZM4 but worse than 3.6M in acidic-pH conditions that is served as a reference strain between 3.6M and ZM4 to help unravel the acidic-pH tolerance mechanism. Mutant strains 3.5M and 3.6M exhibited 50~130% enhancement on growth rate, 4~9 h reduction on fermentation time to consume glucose, and 20~63% improvement on ethanol productivity than wild-type ZM4 at pH 3.8. Next-generation sequencing (NGS)-based whole genome resequencing (WGR) and RNA-Seq technologies were applied to unravel the acidic-pH tolerance mechanism of mutant strains. WGR result indicated that compared to wild-type ZM4, 3.5M and 3.6M have seven and five single nucleotide polymorphisms (SNPs) respectively, among which four are shared in common. Additionally, RNA-Seq result showed that the upregulation of genes involved in glycolysis and the downregulation of flagellar and mobility related genes would help generate and redistribute cellular energy to resist acidic pH while keeping normal biological processes in Z. mobilis. Moreover, genes involved in RND efflux pump, ATP-binding cassette (ABC) transporter, proton consumption, and alkaline metabolite production were significantly upregulated in mutants under the acidic-pH condition compared with ZM4, which could help maintain the pH homeostasis in mutant strains for acidic-pH resistance. Furthermore, our results demonstrated that in mutant 3.6M, genes encoding F1F0 ATPase to pump excess protons out of cells were upregulated under pH 3.8 compared to pH 6.2. This difference might help mutant 3.6M manage acidic conditions better than ZM4 and 3.5M. A few gene targets were then selected for genetics study to explore their role on acidic-pH tolerance, and our results demonstrated that the expression of two operons in the shuttle plasmids, ZMO0956-ZMO0958 encoding cytochrome bc1 complex and ZMO1428-ZMO1432 encoding RND efflux pump, could help Z. mobilis tolerate acidic-pH conditions. Conclusion: An acidic-pH tolerant mutant 3.6M obtained through this study can be used for commercial bioethanol production under acidic fermentation conditions. In addition, the molecular mechanism of acidic-pH tolerance of Z. mobilis was further proposed, which can facilitate future research on rational design of synthetic microorganisms with enhanced tolerance against acidic-pH conditions. Moreover, the strategy developed in this study combining approaches of ALE, genome resequencing, RNA-Seq, and classical genetics study for mutant evolution and characterization can be applied in other industrial microorganisms.