Raising the stakes: Loss of efflux-pump regulation decreases meropenem susceptibility in Burkholderia pseudomallei

AbstractBurkholderia pseudomallei, the causative agent of the high-mortality disease melioidosis, is a Gram-negative bacterium that is naturally resistant to many antibiotics. There is no vaccine for melioidosis, and effective eradication is reliant on biphasic and prolonged antibiotic administration. The carbapenem drug, meropenem, is the current gold-standard option for treating severe melioidosis. Intrinsic B. pseudomallei resistance towards meropenem has not yet been documented; however, resistance could conceivably develop over the course of infection, leading to prolonged sepsis and treatment failure. Here, we document 11 melioidosis cases in which B. pseudomallei isolates developed decreased susceptibility towards meropenem during treatment, including two cases not treated with this antibiotic. Meropenem minimum inhibitory concentrations increased over time from 0.5-0.75 to 3-8 μg/mL. Using comparative genomics, we identified multiple mutations affecting multidrug resistance-nodulation-division (RND) efflux pump regulators, leading to over-expression of their corresponding pumps. The most commonly affected pump was AmrAB-OprA, although alterations in the local regulators of BpeEF-OprC or BpeAB-OprB were observed in three cases. This study confirms the role of RND efflux pumps in decreased meropenem susceptibility in B. pseudomallei. Further, we document two concerning examples of severe melioidosis where the reduced treatment efficacy of meropenem was associated with a fatal outcome.Significance StatementThe bacterium Burkholderia pseudomallei, which causes the often-fatal tropical disease melioidosis, is difficult to eradicate. Due to high levels of intrinsic antibiotic resistance, only a handful of antibiotics are effective against this pathogen. One of these, meropenem, is commonly used in the treatment of melioidosis patients who are unresponsive to other treatments or are critically ill. Here, we describe 11 melioidosis cases whereby patients exhibited prolonged or repeated infections that were associated with the development of decreased meropenem susceptibility. We identified the molecular basis for this decreased susceptibility in latter B. pseudomallei isolates obtained from these patients, and functionally confirmed the mechanism conferring this phenotype. Our findings have important ramifications for the diagnosis, treatment and management of life-threatening melioidosis cases.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00920-21 ◽

2021 ◽

Author(s):

Nawarat Somprasong ◽

Jinhee Yi ◽

Carina M. Hall ◽

Jessica R. Webb ◽

Jason W. Sahl ◽

...

Keyword(s):

Cell Division ◽

Burkholderia Cepacia ◽

Burkholderia Pseudomallei ◽

Efflux Pump ◽

Multidrug Resistant ◽

Resistance Mechanisms ◽

Burkholderia Cepacia Complex ◽

Complex Species ◽

Functional Studies ◽

Rnd Efflux Pump

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.

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

Microorganisms ◽

10.3390/microorganisms8111782 ◽

2020 ◽

Vol 8 (11) ◽

pp. 1782

Author(s):

Tania Henriquez ◽

Tom Baldow ◽

Yat Kei Lo ◽

Dina Weydert ◽

Andreas Brachmann ◽

...

Keyword(s):

Pseudomonas Putida ◽

Efflux Pump ◽

Toxic Metal ◽

Growth Defect ◽

Metal Chelators ◽

Pseudomonas Putida Kt2440 ◽

Pseudomonas Species ◽

Resistance Nodulation Division ◽

Rnd Efflux Pump

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.

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Characterization of a Novel Pyranopyridine Inhibitor of the AcrAB Efflux Pump of Escherichia coli

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01866-13 ◽

2013 ◽

Vol 58 (2) ◽

pp. 722-733 ◽

Cited By ~ 91

Author(s):

Timothy J. Opperman ◽

Steven M. Kwasny ◽

Hong-Suk Kim ◽

Son T. Nguyen ◽

Chad Houseweart ◽

...

Keyword(s):

Escherichia Coli ◽

Efflux Pump ◽

Efflux Pumps ◽

Gram Negative ◽

Content Type ◽

E Coli ◽

Resistance Nodulation Division ◽

Rnd Efflux Pumps ◽

Acrab Efflux Pump

ABSTRACTMembers of the resistance-nodulation-division (RND) family of efflux pumps, such as AcrAB-TolC ofEscherichia coli, play major roles in multidrug resistance (MDR) in Gram-negative bacteria. A strategy for combating MDR is to develop efflux pump inhibitors (EPIs) for use in combination with an antibacterial agent. Here, we describe MBX2319, a novel pyranopyridine EPI with potent activity against RND efflux pumps of theEnterobacteriaceae. MBX2319 decreased the MICs of ciprofloxacin (CIP), levofloxacin, and piperacillin versusE. coliAB1157 by 2-, 4-, and 8-fold, respectively, but did not exhibit antibacterial activity alone and was not active against AcrAB-TolC-deficient strains. MBX2319 (3.13 μM) in combination with 0.016 μg/ml CIP (minimally bactericidal) decreased the viability (CFU/ml) ofE. coliAB1157 by 10,000-fold after 4 h of exposure, in comparison with 0.016 μg/ml CIP alone. In contrast, phenyl-arginine-β-naphthylamide (PAβN), a known EPI, did not increase the bactericidal activity of 0.016 μg/ml CIP at concentrations as high as 100 μM. MBX2319 increased intracellular accumulation of the fluorescent dye Hoechst 33342 in wild-type but not AcrAB-TolC-deficient strains and did not perturb the transmembrane proton gradient. MBX2319 was broadly active againstEnterobacteriaceaespecies andPseudomonas aeruginosa. MBX2319 is a potent EPI with possible utility as an adjunctive therapeutic agent for the treatment of infections caused by Gram-negative pathogens.

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Loss of Methyltransferase Function and Increased Efflux Activity Leads to Doxycycline Resistance in Burkholderia pseudomallei

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00268-17 ◽

2017 ◽

Vol 61 (6) ◽

Cited By ~ 12

Author(s):

Jessica R. Webb ◽

Erin P. Price ◽

Bart J. Currie ◽

Derek S. Sarovich

Keyword(s):

Burkholderia Pseudomallei ◽

Molecular Mechanisms ◽

Efflux Pump ◽

Intrinsic Resistance ◽

Resistance Development ◽

Content Type ◽

Treatment Regimens ◽

Resistance Nodulation Division ◽

Lengthy Treatment ◽

Prolonged Antibiotic Therapy

ABSTRACT The soil-dwelling bacterium Burkholderia pseudomallei is the causative agent of the potentially fatal disease melioidosis. The lack of a vaccine toward B. pseudomallei means that melioidosis treatment relies on prolonged antibiotic therapy, which can last up to 6 months in duration or longer. Due to intrinsic resistance, few antibiotics are effective against B. pseudomallei. The lengthy treatment regimen required increases the likelihood of resistance development, with subsequent potentially fatal relapse. Doxycycline (DOX) has historically played an important role in the eradication phase of melioidosis treatment. Both primary and acquired DOX resistances have been documented in B. pseudomallei; however, the molecular mechanisms underpinning DOX resistance have remained elusive. Here, we identify and functionally characterize the molecular mechanisms conferring acquired DOX resistance in an isogenic B. pseudomallei pair. Two synergistic mechanisms were identified. The first mutation occurred in a putative S-adenosyl-l-methionine-dependent methyltransferase (encoded by BPSL3085), which we propose leads to altered ribosomal methylation, thereby decreasing DOX binding efficiency. The second mutation altered the function of the efflux pump repressor gene, amrR, resulting in increased expression of the resistance-nodulation-division efflux pump, AmrAB-OprA. Our findings highlight the diverse mechanisms by which B. pseudomallei can become resistant to antibiotics used in melioidosis therapy and the need for resistance monitoring during treatment regimens, especially in patients with prolonged or recrudesced positive cultures for B. pseudomallei.

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Growth-related changes in intracellular spermidine and its effect on efflux pump expression and quorum sensing in Burkholderia pseudomallei

Microbiology ◽

10.1099/mic.0.032888-0 ◽

2010 ◽

Vol 156 (4) ◽

pp. 1144-1154 ◽

Cited By ~ 13

Author(s):

Ying Ying Chan ◽

Kim Lee Chua

Keyword(s):

Quorum Sensing ◽

Cell Density ◽

Burkholderia Pseudomallei ◽

Efflux Pump ◽

Regulatory Region ◽

Resistance Nodulation Division ◽

Dose Dependent ◽

Exogenous Spermidine

The Burkholderia pseudomallei BpeAB-OprB resistance-nodulation-division (RND) family pump effluxes aminoglycoside and macrolide antibiotics as well as acylhomoserine lactones (AHLs) involved in quorum sensing. Expression of bpeA–lacZ was cell density-dependent and was inducible in the presence of these compounds. Intracellular levels of spermidine and N-acetylspermidine increased with cell density in wild-type B. pseudomallei KHW, but were always lower in the bpeAB pump mutant at all growth phases. The significance of changes in intracellular spermidine on efflux pump expression was demonstrated by the disruption of the binding of the BpeR repressor protein to the bpeABoprB regulatory region in vitro in the presence of increasing spermidine concentrations. This was supported by dose-dependent activation of bpeA–lacZ transcription in vivo in the presence of exogenous spermidine and N-acetylspermidine, thus implicating the involvement of the BpeAB-OprB pump in spermidine homeostasis in B. pseudomallei. Consequently, inhibition of intracellular spermidine synthesis reduced the efflux of AHLs by BpeAB-OprB. Other potential therapeutic applications of spermidine synthase inhibitors include the reduction of swimming motility and biofilm formation by B. pseudomallei.

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Ever-Adapting RND Efflux Pumps in Gram-Negative Multidrug-Resistant Pathogens: A Race against Time

Antibiotics ◽

10.3390/antibiotics10070774 ◽

2021 ◽

Vol 10 (7) ◽

pp. 774

Author(s):

Martijn Zwama ◽

Kunihiko Nishino

Keyword(s):

Legionella Pneumophila ◽

Bacterial Infections ◽

Efflux Pump ◽

Efflux Pumps ◽

Multidrug Resistant ◽

Bacterial Strains ◽

Gram Negative ◽

Over Expression ◽

Wide Range ◽

Resistance Nodulation Division

The rise in multidrug resistance (MDR) is one of the greatest threats to human health worldwide. MDR in bacterial pathogens is a major challenge in healthcare, as bacterial infections are becoming untreatable by commercially available antibiotics. One of the main causes of MDR is the over-expression of intrinsic and acquired multidrug efflux pumps, belonging to the resistance-nodulation-division (RND) superfamily, which can efflux a wide range of structurally different antibiotics. Besides over-expression, however, recent amino acid substitutions within the pumps themselves—causing an increased drug efflux efficiency—are causing additional worry. In this review, we take a closer look at clinically, environmentally and laboratory-evolved Gram-negative bacterial strains and their decreased drug sensitivity as a result of mutations directly in the RND-type pumps themselves (from Escherichia coli, Salmonella enterica, Neisseria gonorrhoeae, Pseudomonas aeruginosa, Acinetobacter baumannii and Legionella pneumophila). We also focus on the evolution of the efflux pumps by comparing hundreds of efflux pumps to determine where conservation is concentrated and where differences in amino acids can shed light on the broad and even broadening drug recognition. Knowledge of conservation, as well as of novel gain-of-function efflux pump mutations, is essential for the development of novel antibiotics and efflux pump inhibitors.

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

Frontiers in Microbiology ◽

10.3389/fmicb.2021.618513 ◽

2021 ◽

Vol 12 ◽

Author(s):

Yangyang Zhao ◽

Jiayu Liu ◽

Tianping Jiang ◽

Rongxian Hou ◽

Gaoge Xu ◽

...

Keyword(s):

Gene Cluster ◽

Efflux Pump ◽

Essential Gene ◽

Resistance Mechanism ◽

Resistance Mechanisms ◽

Biosynthesis Gene Cluster ◽

Regulatory Pathways ◽

Redox Active ◽

Resistance Nodulation Division ◽

Rnd Efflux Pump

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.

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Emergence of a Plasmid-Encoded Resistance-Nodulation-Division Efflux Pump Conferring Resistance to Multiple Drugs, Including Tigecycline, in Klebsiella pneumoniae

mBio ◽

10.1128/mbio.02930-19 ◽

2020 ◽

Vol 11 (2) ◽

Cited By ~ 9

Author(s):

Luchao Lv ◽

Miao Wan ◽

Chengzhen Wang ◽

Xun Gao ◽

Qiwen Yang ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Gene Cluster ◽

Efflux Pump ◽

Gene Clusters ◽

Site Specific ◽

Content Type ◽

E Coli ◽

Carbapenem Resistant ◽

Resistance Nodulation Division ◽

Rnd Efflux Pump

ABSTRACT Transporters belonging to the chromosomally encoded resistance-nodulation-division (RND) superfamily mediate multidrug resistance in Gram-negative bacteria. However, the cotransfer of large gene clusters encoding RND-type pumps from the chromosome to a plasmid appears infrequent, and no plasmid-mediated RND efflux pump gene cluster has yet been found to confer resistance to tigecycline. Here, we identified a novel RND efflux pump gene cluster, designated tmexCD1-toprJ1, on plasmids from five pandrug-resistant Klebsiella pneumoniae isolates of animal origin. TMexCD1-TOprJ1 increased (by 4- to 32-fold) the MICs of tetracyclines (including tigecycline and eravacycline), quinolones, cephalosporins, and aminoglycosides for K. pneumoniae, Escherichia coli, and Salmonella. TMexCD1-TOprJ1 is closely related (64.5% to 77.8% amino acid identity) to the MexCD-OprJ efflux pump encoded on the chromosome of Pseudomonas aeruginosa. In an IncFIA plasmid, pHNAH8I, the tmexCD1-toprJ1 gene cluster lies adjacent to two genes encoding site-specific integrases, which may have been responsible for its acquisition. Expression of TMexCD1-TOprJ1 in E. coli resulted in increased tigecycline efflux and in K. pneumoniae negated the efficacy of tigecycline in an in vivo infection model. Expression of TMexCD1-TOprJ1 reduced the growth of E. coli and Salmonella but not K. pneumoniae. tmexCD1-toprJ1-positive Enterobacteriaceae isolates were rare in humans (0.08%) but more common in chicken fecal (14.3%) and retail meat (3.4%) samples. Plasmid-borne tmexCD1-toprJ1-like gene clusters were identified in sequences in GenBank from Enterobacteriaceae and Pseudomonas strains from multiple continents. The possibility of further global dissemination of the tmexCD1-toprJ1 gene cluster and its analogues in Enterobacteriaceae via plasmids may be an important consideration for public health planning. IMPORTANCE In an era of increasing concerns about antimicrobial resistance, tigecycline is likely to have a critically important role in the treatment of carbapenem-resistant Enterobacteriaceae, the most problematic pathogens in human clinical settings—especially carbapenem-resistant K. pneumoniae. Here, we identified a new plasmid-borne RND-type tigecycline resistance determinant, TMexCD1-TOprJ1, which is widespread among K. pneumoniae isolates from food animals. tmexCD1-toprJ1 appears to have originated from the chromosome of a Pseudomonas species and may have been transferred onto plasmids by adjacent site-specific integrases. Although tmexCD1-toprJ1 still appears to be rare in human clinical isolates, considering the transferability of the tmexCD1-toprJ1 gene cluster and the broad substrate spectrum of TMexCD1-TOprJ1, further dissemination of this mobile tigecycline resistance determinant is possible. Therefore, from a “One Health” perspective, measures are urgently needed to monitor and control its further spread. The current low prevalence in human clinical isolates provides a precious time window to design and implement measures to tackle this.

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Screening of Natural Products and Derivatives for the Identification of RND Efflux Pump Inhibitors

Combinatorial Chemistry & High Throughput Screening ◽

10.2174/1386207319666160720101502 ◽

2016 ◽

Vol 19 (9) ◽

pp. 705-713 ◽

Cited By ~ 9

Author(s):

Debarati Choudhury ◽

Anupam Talukdar ◽

Pankaj Chetia ◽

Amitabha Bhattacharjee ◽

Manabendra Choudhury

Keyword(s):

Natural Products ◽

Efflux Pump ◽

Rnd Efflux Pump ◽

Efflux Pump Inhibitors

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A Simple Method for Assessment of MDR Bacteria for Over-Expressed Efflux Pumps

The Open Microbiology Journal ◽

10.2174/1874285801307010072 ◽

2013 ◽

Vol 7 (1) ◽

pp. 72-82 ◽

Cited By ~ 39

Author(s):

Marta Martins ◽

Matthew P McCusker ◽

Miguel Viveiros ◽

Isabel Couto ◽

Séamus Fanning ◽

...

Keyword(s):

Ethidium Bromide ◽

Efflux Pump ◽

Efflux Pumps ◽

Outer Cell ◽

Gram Negative Bacteria ◽

Drug Resistant ◽

Topoisomerase Iv ◽

Simple Method ◽

Gram Negative ◽

Over Expression

It is known that bacteria showing a multi-drug resistance phenotype use several mechanisms to overcome the action of antibiotics. As a result, this phenotype can be a result of several mechanisms or a combination of thereof. The main mechanisms of antibiotic resistance are: mutations in target genes (such as DNA gyrase and topoisomerase IV); over-expression of efflux pumps; changes in the cell envelope; down regulation of membrane porins, and modified lipopolysaccharide component of the outer cell membrane (in the case of Gram-negative bacteria). In addition, adaptation to the environment, such as quorum sensing and biofilm formation can also contribute to bacterial persistence. Due to the rapid emergence and spread of bacterial isolates showing resistance to several classes of antibiotics, methods that can rapidly and efficiently identify isolates whose resistance is due to active efflux have been developed. However, there is still a need for faster and more accurate methodologies. Conventional methods that evaluate bacterial efflux pump activity in liquid systems are available. However, these methods usually use common efflux pump substrates, such as ethidium bromide or radioactive antibiotics and therefore, require specialized instrumentation, which is not available in all laboratories. In this review, we will report the results obtained with the Ethidium Bromide-agar Cartwheel method. This is an easy, instrument-free, agar based method that has been modified to afford the simultaneous evaluation of as many as twelve bacterial strains. Due to its simplicity it can be applied to large collections of bacteria to rapidly screen for multi-drug resistant isolates that show an over-expression of their efflux systems. The principle of the method is simple and relies on the ability of the bacteria to expel a fluorescent molecule that is substrate for most efflux pumps, ethidium bromide. In this approach, the higher the concentration of ethidium bromide required to produce fluorescence of the bacterial mass, the greater the efflux capacity of the bacterial cells. We have tested and applied this method to a large number of Gram-positive and Gram-negative bacteria to detect efflux activity among these multi-drug resistant isolates. The presumptive efflux activity detected by the Ethidium Bromide-agar Cartwheel method was subsequently confirmed by the determination of the minimum inhibitory concentration for several antibiotics in the presence and absence of known efflux pump inhibitors.

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