scholarly journals Low nutrient levels reduce the fitness cost of MexCD-OprJ efflux pump overexpression in ciprofloxacin-resistant Pseudomonas aeruginosa

2018 ◽  
Author(s):  
Wenfang Lin ◽  
Kun Wan ◽  
Jie Zeng ◽  
Jingjing Li ◽  
Xi Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Antibiotic Resistance ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Fitness Cost ◽  
Resistant Bacteria ◽  
Nutrient Levels ◽  
High Nutrient ◽  
Key Factor ◽  
Nutrient Conditions

AbstractThe long-term persistence of antibiotic resistance in the environment is a public health concern. Expression of an efflux pump, an important mechanism of resistance to antibiotics, is usually associated with a fitness cost in bacteria. In this study, we aimed to determine why antibiotic resistance conferred by overexpression of an efflux pump persists in environments such as drinking and source water in which antibiotic selective pressure may be very low or even absent. Competition experiments between wild-type Pseudomonas aeruginosa and ciprofloxacin-resistant mutants revealed that the fitness cost of ciprofloxacin resistance (strains cip_1, cip_2, and cip_3) significantly decreased (P < 0.05) under low-nutrient (0.5 mg/l total organic carbon (TOC)) relative to high-nutrient (500 mg/l TOC) conditions. Mechanisms underlying this fitness cost were analyzed. MexD gene expression in resistant bacteria (cip_3 strain) was significantly lower (P < 0.05) in low-nutrient conditions, with 10 mg/l TOC (8.01 ± 0.82-fold), than in high-nutrient conditions, with 500 mg/l TOC (48.89 ± 4.16-fold). Moreover, rpoS gene expression in resistant bacteria (1.36 ± 0.13-fold) was significantly lower (P < 0.05) than that in the wild-type strain (2.78 ± 0.29-fold) under low-nutrient conditions (10 mg/l TOC), suggesting a growth advantage. Furthermore, the difference in metabolic activity between the two competing strains was significantly smaller (P < 0.05) in low-nutrient conditions (5 and 0.5 mg/l TOC). These results suggest that nutrient levels are a key factor in determining the persistence and spread of antibiotic resistance conferred by efflux pumps in the natural environment with trace amounts or no antibiotics.ImportanceThe widespread of antibiotic resistance has led to an increasing concern about the environmental and public health risks. Mechanisms associated with antibiotic resistance including efflux pumps often increase bacterial fitness cost. Our study showed that the fitness cost of ciprofloxacin resistance conferred by overexpression of MexCD-OprJ efflux pump significantly decreased under low-nutrient relative to high-nutrient conditions. The significance of our research is to reveal that nutrient levels are key factor in determining the persistence of antibiotic resistance conferred by efflux pumps under conditions with trace amounts or no antibiotics, which can be mediated by some mechanisms including MexD gene expression, SOURs differences, and rpoS gene regulation.

scholarly journals Clinically Relevant Chromosomally Encoded Multidrug Resistance Efflux Pumps in Bacteria

2006 ◽  
Vol 19 (2) ◽  
pp. 382-402 ◽  
Author(s):  
Laura J. V. Piddock
Keyword(s):  
Antibiotic Resistance ◽  
Drug Transport ◽  
Efflux Pump ◽  
Regulatory Gene ◽  
Structural Data ◽  
Efflux Pumps ◽  
Resistant Bacteria ◽  
Multiple Drug ◽  
Transporter Gene ◽  
Resistance Nodulation Division

SUMMARY Efflux pump genes and proteins are present in both antibiotic-susceptible and antibiotic-resistant bacteria. Pumps may be specific for one substrate or may transport a range of structurally dissimilar compounds (including antibiotics of multiple classes); such pumps can be associated with multiple drug (antibiotic) resistance (MDR). However, the clinical relevance of efflux-mediated resistance is species, drug, and infection dependent. This review focuses on chromosomally encoded pumps in bacteria that cause infections in humans. Recent structural data provide valuable insights into the mechanisms of drug transport. MDR efflux pumps contribute to antibiotic resistance in bacteria in several ways: (i) inherent resistance to an entire class of agents, (ii) inherent resistance to specific agents, and (iii) resistance conferred by overexpression of an efflux pump. Enhanced efflux can be mediated by mutations in (i) the local repressor gene, (ii) a global regulatory gene, (iii) the promoter region of the transporter gene, or (iv) insertion elements upstream of the transporter gene. Some data suggest that resistance nodulation division systems are important in pathogenicity and/or survival in a particular ecological niche. Inhibitors of various efflux pump systems have been described; typically these are plant alkaloids, but as yet no product has been marketed.

scholarly journals Dynamic Boolean modelling reveals the influence of energy supply on bacterial efflux pump expression

2021 ◽  
Author(s):  
Ryan Kerr ◽  
Sara Jabbari ◽  
Jessica M A Blair ◽  
Iain Johnston
Keyword(s):  
Gene Expression ◽  
Antimicrobial Resistance ◽  
Energy Supply ◽  
Regulatory Networks ◽  
Efflux Pump ◽  
Bacterial Species ◽  
Efflux Pumps ◽  
Resistant Bacteria ◽  
Energy Availability ◽  
Bacterial Populations

Antimicrobial resistance (AMR) is a global health issue. One key factor contributing to AMR is the ability of bacteria to export drugs through efflux pumps, which relies on the ATP-dependent expression and interaction of several controlling genes. Recent studies have shown significant cell-to-cell ATP variability exists within clonal bacterial populations, but the contribution of intrinsic cell-to-cell ATP heterogeneity is generally overlooked in understanding efflux pumps. Here, we consider how ATP variability influences gene regulatory networks controlling expression of efflux pump genes in two bacterial species. We develop and apply a generalisable Boolean modelling framework, developed to incorporate the dependence of gene expression dynamics on available cellular energy supply. Theoretical results show differences in energy availability can cause pronounced downstream heterogeneity in efflux gene expression. Cells with higher energy availability have a superior response to stressors. Further, in the absence of stress, model bacteria develop heterogeneous pulses of efflux pump gene expression which contribute to a sustained sub-population of cells with increased efflux expression activity, potentially conferring a continuous pool of intrinsically resistant bacteria. This modelling approach thus reveals an important source of heterogeneity in cell responses to antimicrobials and sheds light on potentially targetable aspects of efflux pump-related antimicrobial resistance.

scholarly journals Expression ofPseudomonas aeruginosaAntibiotic Resistance Genes Varies Greatly during Infections in Cystic Fibrosis Patients

2018 ◽  
Vol 62 (11) ◽  
Author(s):  
Lois W. Martin ◽  
Cynthia L. Robson ◽  
Annabelle M. Watts ◽  
Andrew R. Gray ◽  
Claire E. Wainwright ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cystic Fibrosis ◽  
Antibiotic Resistance ◽  
Efflux Pump ◽  
Antibiotic Resistance Gene ◽  
Resistance Mechanisms ◽  
Laboratory Culture ◽  
Bacterial Cells ◽  
Content Type ◽  
Key Factor

ABSTRACTThe lungs of individuals with cystic fibrosis (CF) become chronically infected withPseudomonas aeruginosathat is difficult to eradicate by antibiotic treatment. Two keyP. aeruginosaantibiotic resistance mechanisms are the AmpC β-lactamase that degrades β-lactam antibiotics and MexXYOprM, a three-protein efflux pump that expels aminoglycoside antibiotics from the bacterial cells. Levels of antibiotic resistance gene expression are likely to be a key factor in antibiotic resistance but have not been determined during infection. The aims of this research were to investigate the expression of theampCandmexXgenes during infection in patients with CF and in bacteria isolated from the same patients and grown under laboratory conditions.P. aeruginosaisolates from 36 CF patients were grown in laboratory culture and gene expression measured by reverse transcription-quantitative PCR (RT-qPCR). The expression ofampCvaried over 20,000-fold and that ofmexXover 2,000-fold between isolates. The median expression levels of both genes were increased by the presence of subinhibitory concentrations of antibiotics. To measureP. aeruginosagene expression during infection, we carried out RT-qPCR using RNA extracted from fresh sputum samples obtained from 31 patients. The expression ofampCvaried over 4,000-fold, whilemexXexpression varied over 100-fold, between patients. Despite these wide variations, median levels of expression ofampCin bacteria in sputum were similar to those in laboratory-grown bacteria. The expression ofmexXwas higher in sputum than in laboratory-grown bacteria. Overall, our data demonstrate that genes that contribute to antibiotic resistance can be highly expressed in patients, but there is extensive isolate-to-isolate and patient-to-patient variation.

scholarly journals Burkholderia collagen-like protein 8, Bucl8, is a unique outer membrane component of a tetrapartite efflux pump in Burkholderia pseudomallei and Burkholderia mallei

2020 ◽  
Author(s):  
Megan E Grund ◽  
Soo J Choi ◽  
Dudley H McNitt ◽  
Mariette Barbier ◽  
Gangqing Hu ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Burkholderia Pseudomallei ◽  
Triple Helix ◽  
Efflux Pump ◽  
Fusaric Acid ◽  
Efflux Pumps ◽  
Resistant Bacteria ◽  
Burkholderia Mallei ◽  
Membrane Component ◽  
Collagen Triple Helix

AbstractBacterial efflux pumps are an important pathogenicity trait because they extrude a variety of xenobiotics. Our laboratory previously identified in silico Burkholderia collagen-like protein 8 (Bucl8) in the Tier one select agents Burkholderia pseudomallei and Burkholderia mallei. We hypothesize that Bucl8, which contains two predicted tandem outer membrane efflux pump domains, is a component of a putative efflux pump. Unique to Bucl8, as compared to other outer membrane proteins, is the presence of an extended extracellular region containing a collagen-like (CL) domain and a non-collagenous C-terminus (Ct). Molecular modeling and circular dichroism spectroscopy with a recombinant protein, corresponding to this extracellular CL-Ct portion of Bucl8, demonstrated that it adopts a collagen triple helix, whereas functional assays screening for Bucl8 ligands identified binding to fibrinogen. Bioinformatic analysis of the bucl8 gene locus revealed it resembles a classical efflux-pump operon. The bucl8 gene is co-localized with downstream fusCDE genes encoding fusaric acid (FA) resistance, and with an upstream gene, designated as fusR, encoding a LysR-type transcriptional regulator. Using RT-qPCR, we defined the boundaries and transcriptional organization of the fusR-bucl8-fusCDE operon. We found exogenous FA induced bucl8 transcription over 80-fold in B. pseudomallei, while deletion of the entire bucl8 locus decreased the MIC of FA 4-fold in its isogenic mutant. We furthermore showed that the Bucl8 pump expressed in the heterologous Escherichia coli host confers FA resistance. On the contrary, the Bucl8 pump did not confer resistance to a panel of clinically-relevant antimicrobials in Burkholderia and E. coli. We finally demonstrated that deletion of the bucl8-locus drastically affects the growth of the mutant in L-broth. We determined that Bucl8 is a component of a novel tetrapartite efflux pump, which confers FA resistance, fibrinogen binding, and optimal growth.Author SummaryBurkholderia pseudomallei and Burkholderia mallei are highly infectious and multidrug resistant bacteria that are classified by the National Institute of Allergy and Infectious Diseases as Tier one select agents partly due to the intrinsic multidrug resistance associated with expression of the efflux pumps. To date, only few efflux pumps predicted in Burkholderia spp. have been studied in detail. In the current study we introduce Bucl8, an outer membrane component of an unreported putative efflux pump with a unique extended extracellular portion that forms a collagen triple helix and binds fibrinogen. We demonstrate Bucl8’s role in fusaric acid resistance by defining its operon via bioinformatic and transcriptional analyses, as well as by employing loss-of-function and gain-of-function genetic approaches. Our studies also implicate the Bucl8-associated pump in metabolic and physiologic homeostasis. Understanding how Bucl8 efflux pump contributes to Burkholderia pathology will foster development of pump inhibitors targeting transport mechanism or identifying potential surface-exposed vaccine targets.

scholarly journals Expression of Pseudomonas aeruginosa Multidrug Efflux Pumps MexA-MexB-OprM and MexC-MexD-OprJ in a Multidrug-Sensitive Escherichia coli Strain

1998 ◽  
Vol 42 (1) ◽  
pp. 65-71 ◽  
Author(s):  
Ramakrishnan Srikumar ◽  
Tatiana Kon ◽  
Naomasa Gotoh ◽  
Keith Poole
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Antibiotic Resistance ◽  
Crystal Violet ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Multidrug Efflux ◽  
Efflux System ◽  
E Coli ◽  
Multidrug Efflux Pumps

ABSTRACT The mexCD-oprJ and mexAB-oprM operons encode components of two distinct multidrug efflux pumps inPseudomonas aeruginosa. To assess the contribution of individual components to antibiotic resistance and substrate specificity, these operons and their component genes were cloned and expressed in Escherichia coli. Western immunoblotting confirmed expression of the P. aeruginosa efflux pump components in E. coli strains expressing and deficient in the endogenous multidrug efflux system (AcrAB), although only the ΔacrAB strain, KZM120, demonstrated increased resistance to antibiotics in the presence of the P. aeruginosa efflux genes. E. coli KZM120 expressing MexAB-OprM showed increased resistance to quinolones, chloramphenicol, erythromycin, azithromycin, sodium dodecyl sulfate (SDS), crystal violet, novobiocin, and, significantly, several β-lactams, which is reminiscent of the operation of this pump in P. aeruginosa. This confirmed previous suggestions that MexAB-OprM provides a direct contribution to β-lactam resistance via the efflux of this group of antibiotics. An increase in antibiotic resistance, however, was not observed when MexAB or OprM alone was expressed in KZM120. Thus, despite the fact that β-lactams act within the periplasm, OprM alone is insufficient to provide resistance to these agents. E. coli KZM120 expressing MexCD-OprJ also showed increased resistance to quinolones, chloramphenicol, macrolides, SDS, and crystal violet, though not to most β-lactams or novobiocin, again somewhat reminiscent of the antibiotic resistance profile of MexCD-OprJ-expressing strains ofP. aeruginosa. Surprisingly, E. coli KZM120 expressing MexCD alone also showed an increase in resistance to these agents, while an OprJ-expressing KZM120 failed to demonstrate any increase in antibiotic resistance. MexCD-mediated resistance, however, was absent in a tolC mutant of KZM120, indicating that MexCD functions in KZM120 in conjunction with TolC, the previously identified outer membrane component of the AcrAB-TolC efflux system. These data confirm that a tripartite efflux pump is necessary for the efflux of all substrate antibiotics and that the P. aeruginosa multidrug efflux pumps are functional and retain their substrate specificity in E. coli.

scholarly journals Persistence of Transferable Extended-Spectrum-β-Lactamase Resistance in the Absence of Antibiotic Pressure

2012 ◽  
Vol 56 (9) ◽  
pp. 4703-4706 ◽  
Author(s):  
Jennifer L. Cottell ◽  
Mark A. Webber ◽  
Laura J. V. Piddock
Keyword(s):  
Antibiotic Resistance ◽  
Resistance Gene ◽  
Resistance Genes ◽  
Antibiotic Resistance Genes ◽  
Fitness Cost ◽  
Resistant Bacteria ◽  
Bacterial Host ◽  
Antibiotic Resistant ◽  
M 14 ◽  
Host Strains

ABSTRACTThe treatment of infections caused by antibiotic-resistant bacteria is one of the great challenges faced by clinicians in the 21st century. Antibiotic resistance genes are often transferred between bacteria by mobile genetic vectors called plasmids. It is commonly believed that removal of antibiotic pressure will reduce the numbers of antibiotic-resistant bacteria due to the perception that carriage of resistance imposes a fitness cost on the bacterium. This study investigated the ability of the plasmid pCT, a globally distributed plasmid that carries an extended-spectrum-β-lactamase (ESBL) resistance gene (blaCTX-M-14), to persist and disseminate in the absence of antibiotic pressure. We investigated key attributes in plasmid success, including conjugation frequencies, bacterial-host growth rates, ability to cause infection, and impact on the fitness of host strains. We also determined the contribution of theblaCTX-M-14gene itself to the biology of the plasmid and host bacterium. Carriage of pCT was found to impose no detectable fitness cost on various bacterial hosts. An absence of antibiotic pressure and inactivation of the antibiotic resistance gene also had no effect on plasmid persistence, conjugation frequency, or bacterial-host biology. In conclusion, plasmids such as pCT have evolved to impose little impact on host strains. Therefore, the persistence of antibiotic resistance genes and their vectors is to be expected in the absence of antibiotic selective pressure regardless of antibiotic stewardship. Other means to reduce plasmid stability are needed to prevent the persistence of these vectors and the antibiotic resistance genes they carry.

scholarly journals Two resistance nodulation division-family efflux pumps inChromobacteriumspecies and their role in antibiotic resistance and tolerance

2019 ◽  
Author(s):  
Saida Benomar ◽  
Kara C Evans ◽  
Robert L Unckless ◽  
Josephine R Chandler
Keyword(s):  
Antibiotic Resistance ◽  
Efflux Pump ◽  
Bacterial Species ◽  
Efflux Pumps ◽  
Resistance Mechanisms ◽  
Antibiotic Resistant ◽  
Burkholderia Thailandensis ◽  
New Information ◽  
Resistance Nodulation Division ◽  
Culture Growth

ABSTRACTVery little is known of the antibiotic resistance mechanisms of members of theChromobacteriumgenus. In previous studies ofChromobacterium subtsugae(formerlyC. violaceum) strain CV017, we identified a resistance nodulation division (RND)-family efflux pump (CdeAB-OprM). Here, we show thecdeAB-oprMgenes are widely distributed in members of theChromobacteriumgenus. We use antimicrobial susceptibility testing with a CV017cdeAB-oprMmutant to show the products of these genes confers resistance to a variety of antibiotics including ciprofloxacin, a clinically important antibiotic. We also identified a related RND-family pump,cseAB-oprN, in the genome of CV017 and otherC. subtsugaespecies, that is not present in other members of theChromobacteriumgenus. We demonstrate that CdeAB-OprM and CseAB-OprN are both transcriptionally induced in CV017 cells treated with sub-lethal antibiotic concentrations and they are important for induction of tolerance to different antibiotics. While CdeAB-OprM has a broad antibiotic specificity, the CseAB-OprN system is highly specific for a ribosome-targeting antibiotic produced by the saprophytic bacteriumBurkholderia thailandensis,bactobolin. Finally, we use a previously developedB. thailandensis-C. subtsugaeCV017 co-culture model to demonstrate that adding sub-lethal bactobolin at the beginning of co-culture growth increases the ability of CV017 to compete withB. thailandensisin a manner that is dependent on the CseAB-OprN system. Our results provide new information on the antibiotic resistance mechanisms ofChromobacteriumspecies and highlight the importance of efflux pumps during competition with other bacterial species.IMPORTANCEThis study describes two closely related efflux pumps in members of theChromobacteriumgenus, which includes opportunistic but often-fatal pathogens and species with highly versatile metabolic capabilities. Efflux pumps remove antibiotics from the cell and are important for antibiotic resistance. One of these pumps is broadly distributed in theChromobacteriumgenus and increases resistance to clinically relevant antibiotics. The other efflux pump is present only inChromobacterium subtsugaeand is highly specific for bactobolin, an antibiotic produced by the soil saprophyteBurkholderia thailandensis. We demonstrate these pumps can be activated to increase resistance by their antibiotic substrates, and that this activation is important forC. subtsugaeto survive in a laboratory competition experiment withB. thailandensis.These results have implications for managing antibiotic-resistantChromobacteriuminfections, bioengineering ofChromobacteriumspecies, and for understanding the evolution of efflux pumps.

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