Genetic Variations in MexAB-OprM Efflux Pump Regulators and Their Association with Antibiotic Resistance and Sequence type in Clinical and Epidemiologically High-risk Clones of Pseudomonas Aeruginosa.

Abstract Background: Pseudomonas aeruginosa is a major opportunistic pathogen involved in healthcare-associated infections with high mortality rates. This bacterium exhibits elevated resistance to a wide range of antibiotics, resulting in part from the overexpression of efflux pumps, among which MexAB-OprM stands out as constitutive. Antibiotic resistance in clinical isolates is associated with mutations in the mexR, nalC, and nalD repressors that modulate the expression of this efflux pump. This study identifies point mutations in the mexR, nalC, and nalD genes and investigates their associations with antibiotic resistance and sequence type in clinical and epidemiologically high-risk clones of P. aeruginosa. Results: A total of 91 P. aeruginosa strains isolated at a pediatric hospital in Mexico (2007–2015) were classified according to their resistance to antibiotics. The strains were typed by multilocus sequencing of 7 genes. The MexAB-OprM efflux pump phenotype was determined using the minimal inhibitory concentration for the reporter antibiotic carbenicillin in the presence/absence of the efflux pump inhibitor Phe-Arg-β-naphthylamine. Sequencing of the mexR, nalC, and nalD genes to identify mutations was performed. Genetic relationship among the strains was evaluated by a phylogenetic inference analysis using maximum likelihood to construct a phylogenetic network. The relationship between variables was determined by a principal component analysis. STs revealed six main complexes. Mutations in the mexR, nalC, and nalD genes revealed 27 different haplotypes. Pan-drug and extensive drug resistant profiles were associated with specific STs with haplotypes 1 (ST1725, endemic clone), 8, 12 (ST233, epidemiologically high-risk clone), and 5 [related to dead when compared to ST1725 and ST233 (RRR 23.34; p=0.009 and RRR 32.01; p=0.025)], however the resistance in these strains was not mainly attributed to the MexAB-OprM phenotype. Strains with the same haplotype and resistant profile showed different pump behavior.Conclusions: A significant relationship between ST and resistant profiles was observed; on one hand, the mexR-nalC-nalD haplotypes were not related to the MexAB-OprM efflux pump phenotypic behavior. On the other hand, the relationship between mexR-nalC-nalD haplotypes and phylogenetically related ST, suggest mutations in these repressors are highly maintained within these STs.

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Efflux Pump-Driven Antibiotic and Biocide Cross-Resistance in Pseudomonas aeruginosa Isolated from Different Ecological Niches: A Case Study in the Development of Multidrug Resistance in Environmental Hotspots

Microorganisms ◽

10.3390/microorganisms8111647 ◽

2020 ◽

Vol 8 (11) ◽

pp. 1647 ◽

Cited By ~ 2

Author(s):

Anteneh Amsalu ◽

Sylvia A. Sapula ◽

Miguel De Barros Lopes ◽

Bradley J. Hart ◽

Anh H. Nguyen ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Multidrug Resistance ◽

Antimicrobial Resistance ◽

Molecular Mechanisms ◽

Efflux Pump ◽

Principal Component ◽

Cross Resistance ◽

Clinical Samples ◽

Ecological Niches ◽

Efflux Pump Inhibitor

Pseudomonas aeruginosa is an opportunistic pathogen displaying high intrinsic antimicrobial resistance and the ability to thrive in different ecological environments. In this study, the ability of P. aeruginosa to develop simultaneous resistance to multiple antibiotics and disinfectants in different natural niches were investigated using strains collected from clinical samples, veterinary samples, and wastewater. The correlation between biocide and antimicrobial resistance was determined by employing principal component analysis. Molecular mechanisms linking biocide and antimicrobial resistance were interrogated by determining gene expression using RT-qPCR and identifying a potential genetic determinant for co- and cross-resistance using whole-genome sequencing. A subpopulation of P. aeruginosa isolates belonging to three sequence types was resistant against the common preservative benzalkonium chloride and showed cross-resistance to fluoroquinolones, cephalosporins, aminoglycosides, and multidrug resistance. Of these, the epidemiological high-risk ST235 clone was the most abundant. The overexpression of the MexAB-OprM drug efflux pump resulting from amino acid mutations in regulators MexR, NalC, or NalD was the major contributing factor for cross-resistance that could be reversed by an efflux pump inhibitor. This is the first comparison of antibiotic-biocide cross-resistance in samples isolated from different ecological niches and serves as a confirmation of laboratory-based studies on biocide adapted isolates. The isolates from wastewater had a higher incidence of multidrug resistance and biocide-antibiotic cross-resistance than those from clinical and veterinary settings.

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Antibiotic resistance patterns of efflux pumps MexAB-Opr M in pathogenic Pseudomonas aeruginosa isolates

10.21203/rs.3.rs-38864/v1 ◽

2020 ◽

Author(s):

Javad rasouli ◽

Behnam hashemi ◽

Hamed Afkhami ◽

Mansoor Khaledi ◽

Reza valadan ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Antibiotic Resistance ◽

Efflux Pump ◽

Efflux Pumps ◽

Drug Susceptibility ◽

Diffusion Method ◽

Efflux Pump Inhibitor ◽

Disk Diffusion Method ◽

Burn Victims ◽

Resistance Patterns

Abstract Objectives Pseudomonas aeruginosa is one of the most important causes of Hospital infection especially in burn victims. The current study aimed to determine antibiotic resistance of the efflux Pumps MexAB-Opr M. In the present study, 115 samples of urine, blood, sputum, and ICU were collected from the reconstructive section of the patients. The drug susceptibility patterns were determined by disk diffusion method. Phenotypic activity of the efflux pump from the E-test was evaluated, in the presence and without the presence of efflux pump inhibitor. The MexAB gene was analyzed by PCR reaction. Results The resistant isolated was shown to be Ciprofloxacin 33.91%, Nurfloxacin 38.26%, Gentamicin 71.7%, Nalidixic acid 95.95%, Ceftazidim 38.46%, Emipenem 24.34%, Meropenem 26.36%, and Cefotaxim 40.86%. The highest and lowest resistance rates were Co-trimoxazole and Piperacilin, respectively. The findings of PCR reaction among 115 P. aeruginosa isolates indicated that 62.62% was MexAB gene. The results of MIC with E-test revealed that the role of efflux pumps in antibiotic resistance was 19 isolated. Due to the importance of antibiotic resistance to investigate other efflux pumps, comparison of efflux pump involvement in antibiotic resistance, and relationship between efflux pumps MexAB-Opr M are highly required and suggested.

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The Impact of an Efflux Pump Inhibitor on the Activity of Free and Liposomal Antibiotics against Pseudomonas aeruginosa

Pharmaceutics ◽

10.3390/pharmaceutics13040577 ◽

2021 ◽

Vol 13 (4) ◽

pp. 577

Author(s):

Douweh Leyla Gbian ◽

Abdelwahab Omri

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Efflux Pump ◽

Antimicrobial Activities ◽

Dilution Method ◽

Efflux Pump Inhibitor ◽

Signal Production ◽

Lung Infections ◽

The Impact ◽

Microbroth Dilution

The eradication of Pseudomonas aeruginosa in cystic fibrosis patients has become continuously difficult due to its increased resistance to treatments. This study assessed the efficacy of free and liposomal gentamicin and erythromycin, combined with Phenylalanine arginine beta-naphthylamide (PABN), a broad-spectrum efflux pump inhibitor, against P. aeruginosa isolates. Liposomes were prepared and characterized for their sizes and encapsulation efficiencies. The antimicrobial activities of formulations were determined by the microbroth dilution method. Their activity on P. aeruginosa biofilms was assessed, and the effect of sub-inhibitory concentrations on bacterial virulence factors, quorum sensing (QS) signals and bacterial motility was also evaluated. The average diameters of liposomes were 562.67 ± 33.74 nm for gentamicin and 3086.35 ± 553.95 nm for erythromycin, with encapsulation efficiencies of 13.89 ± 1.54% and 51.58 ± 2.84%, respectively. Liposomes and PABN combinations potentiated antibiotics by reducing minimum inhibitory and bactericidal concentrations by 4–32 fold overall. The formulations significantly inhibited biofilm formation and differentially attenuated virulence factor production as well as motility. Unexpectedly, QS signal production was not affected by treatments. Taken together, the results indicate that PABN shows potential as an adjuvant of liposomal macrolides and aminoglycosides in the management of lung infections in cystic fibrosis patients.

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Target (MexB)- and Efflux-Based Mechanisms Decreasing the Effectiveness of the Efflux Pump Inhibitor D13-9001 in Pseudomonas aeruginosa PAO1: Uncovering a New Role for MexMN-OprM in Efflux of β-Lactams and a Novel Regulatory Circuit (MmnRS) Controlling MexMN Expression

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01718-18 ◽

2018 ◽

Vol 63 (2) ◽

pp. e01718-18 ◽

Cited By ~ 1

Author(s):

Srijan Ranjitkar ◽

Adriana K. Jones ◽

Mina Mostafavi ◽

Zachary Zwirko ◽

Oleg Iartchouk ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Efflux Pump ◽

Response Regulator ◽

Heavy Metal Resistance ◽

Metal Resistance ◽

Rna Seq ◽

Efflux Pump Inhibitor ◽

Content Type ◽

Regulatory Circuit ◽

Outer Membrane Channel

ABSTRACT Efflux pumps contribute to antibiotic resistance in Gram-negative pathogens. Correspondingly, efflux pump inhibitors (EPIs) may reverse this resistance. D13-9001 specifically inhibits MexAB-OprM in Pseudomonas aeruginosa. Mutants with decreased susceptibility to MexAB-OprM inhibition by D13-9001 were identified, and these fell into two categories: those with alterations in the target MexB (F628L and ΔV177) and those with an alteration in a putative sensor kinase of unknown function, PA1438 (L172P). The alterations in MexB were consistent with reported structural studies of the D13-9001 interaction with MexB. The PA1438L172P alteration mediated a >150-fold upregulation of MexMN pump gene expression and a >50-fold upregulation of PA1438 and the neighboring response regulator gene, PA1437. We propose that these be renamed mmnR and mmnS for MexMN regulator and MexMN sensor, respectively. MexMN was shown to partner with the outer membrane channel protein OprM and to pump several β-lactams, monobactams, and tazobactam. Upregulated MexMN functionally replaced MexAB-OprM to efflux these compounds but was insusceptible to inhibition by D13-9001. MmnSL172P also mediated a decrease in susceptibility to imipenem and biapenem that was independent of MexMN-OprM. Expression of oprD, encoding the uptake channel for these compounds, was downregulated, suggesting that this channel is also part of the MmnSR regulon. Transcriptome sequencing (RNA-seq) of cells encoding MmnSL172P revealed, among other things, an interrelationship between the regulation of mexMN and genes involved in heavy metal resistance.

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Complete Genome Sequence of Pseudomonas aeruginosa K34-7, a Carbapenem-Resistant Isolate of the High-Risk Sequence Type 233

Microbiology Resource Announcements ◽

10.1128/mra.00886-18 ◽

2018 ◽

Vol 7 (4) ◽

Cited By ~ 2

Author(s):

George Taiaroa ◽

Ørjan Samuelsen ◽

Tom Kristensen ◽

Ole Andreas Løchen Økstad ◽

Adam Heikal

Keyword(s):

Pseudomonas Aeruginosa ◽

High Risk ◽

Genome Sequence ◽

Complete Genome Sequence ◽

Complete Genome ◽

Sequence Type ◽

Resistant Isolate ◽

Content Type ◽

Carbapenem Resistant ◽

New Antibiotics

Carbapenem-resistant Pseudomonas aeruginosa is defined as a “critical” priority pathogen for the development of new antibiotics. Here we report the complete genome sequence of an extensively drug-resistant, Verona integron-encoded metallo-β-lactamase-expressing isolate belonging to the high-risk sequence type 233.

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Curcumin as Efflux Pump Inhibitor Agent for Enhancement Treatment Against Multidrug Resistant Pseudomonas aeruginosa Isolates

Iraqi Journal of Science ◽

10.24996/ijs.2020.61.1.6 ◽

2020 ◽

pp. 59-67

Author(s):

Sulaiman D. Sulaiman ◽

Ghusoon A. Abdulhasan

Keyword(s):

Pseudomonas Aeruginosa ◽

Inhibitory Concentration ◽

Efflux Pump ◽

Multidrug Resistant ◽

Diffusion Method ◽

Disc Diffusion Method ◽

Efflux Pump Inhibitor ◽

Before And After ◽

Chromogenic Agar ◽

Susceptibility Patterns

Pseudomonas aeruginosa is considered as a developing opportunistic nosocomial pathogen and is well-known for its multidrug resistance that can be efficiently treated by a combination of antibiotics andefflux pump inhibitors (EPI). Therefore, the purpose of this study was to investigate the effect of curcumin as an EPI for the enhancement of the effectiveness of antibiotics against multidrug resistant (MDR) isolates ofP. aeruginosa. Susceptibility patterns of suspected bacteria was determined using the disc diffusion method andresistant bacteria were identified using chromogenic agar and 16S rDNA. The effectsof curcuminon the enhancement of antibiotics’s activity was evaluated usingthe broth microdilution method.The susceptibility patterns for 50 (67.6%) suspectedP. aeruginosaisolates showed that 36 (72%) of these isolateswere resistant to one of the used antibiotics,whereasonly 21 (42%) were MDR. The highest percentage of resistance was observedtoceftazidime (66%) followed by ciprofloxacin and levofloxacin (40%). Only 35 isolates were specified by chromogenic agar and 16S rDNAas P. aeruginosa.The minimal inhibitory concentration (MIC) of 35 isolates for ciprofloxacin resistant was between 4 and128 µg/ml while for ceftazidime was between 64and 512 µg/ml. After the addition of 50 μg/ml curcumin with ciprofloxacin, there wasa significant increase in the sensitivity (p≤ 0.01) of 13 MDR P.aeroginosa isolates whereas no differences in the sensitivity to ceftazidime were recorded before and after addition ofcurcumin. In conclusion, the results of this study show that curcumin can decrease the MIC value of ciprofloxacin in MDR isolates of P. aeruginosaand can be used as a native compound to enhance the treatment of resistant isolates with ciprofloxacin.

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Association of Efflux Pump and OMPs with Antibiotic Susceptibility Among ESBL-Producing Klebsiella Pneumoniae Clinical Strains in Malaysia

10.21203/rs.3.rs-375267/v1 ◽

2021 ◽

Author(s):

Golnaz Mobasseri ◽

Thong Kwai Lin ◽

Cindy Shuan Ju Teh

Keyword(s):

Antibiotic Resistance ◽

Klebsiella Pneumoniae ◽

Antibiotic Susceptibility ◽

Efflux Pump ◽

Outer Membrane Proteins ◽

Multidrug Resistant ◽

Efflux Pump Inhibitor ◽

Pump System ◽

Extended Spectrum Beta Lactamases ◽

Amoxicillin Clavulanate

Abstract Multidrug-resistant (MDR) Klebsiella pneumoniae (K. pneumoniae) poses a serious public health threat. K. pneumoniae strains that produce extended-spectrum beta-lactamases (ESBL) are becoming increasingly reported in nosocomial and community-acquired infections. Besides resistance genes, integrons, and plasmids, altered membrane permeability caused by porin loss and energy-dependent efflux have also contributed to antibiotic resistance in K. pneumoniae. The objective of this study was to determine the correlation between the reduction of antibiotic susceptibility and overexpression of efflux pump as well as the lack of outer membrane proteins (OMPs) among clinical ESBLs resistant K. pneumoniae. The expression levels of ramA, acrA, ompK35 and ompK36 in 12 MDR K. pneumoniae strains with varying MICs levels were analyzed using quantitative real time-Polymerase Chain Reaction (qRT-PCR). The role of efflux pump on antibiotic resistance was also studied by using minimum inhibitory concentration (MICs) method with//without efflux pump inhibitor. The result indicated that the strains with highest resistance to cefotaxime showed the lowest level of ompK35 and ompK36 genes expression while the strains with lowest MIC level of resistance to cefotaxime showed the highest level of expression of acrA and ramA. Our finding also revealed the effect of efflux pump inhibitor phenyl-arginine-b-naphthylamide (PAβN) on the MIC levels of ceftazidime, amoxicillin-clavulanate and cefotaxime which were significantly reduced around 1–7 folds MIC levels. These results suggest that Efflux pump system and deficiently of OMPs contributing role in antibiotic susceptibility which should be taken seriously to prevent the treatment failure due to antimicrobial resistance.

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P089 Synergistic activities of an efflux pump inhibitor and antibiotics against Pseudomonas aeruginosa isolates from cystic fibrosis patients

Journal of Cystic Fibrosis ◽

10.1016/s1569-1993(18)30385-0 ◽

2018 ◽

Vol 17 ◽

pp. S84

Author(s):

D.L. Gbian ◽

A. Omri

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Efflux Pump ◽

Efflux Pump Inhibitor

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Ultraviolet irradiation sensitizes Pseudomonas aeruginosa PAO1 to multiple antibiotics

Environmental Science Water Research & Technology ◽

10.1039/c8ew00293b ◽

2018 ◽

Vol 4 (12) ◽

pp. 2051-2057 ◽

Cited By ~ 1

Author(s):

Fuzheng Zhao ◽

Qing Hu ◽

Hongqiang Ren ◽

Xu-Xiang Zhang

Keyword(s):

Pseudomonas Aeruginosa ◽

Antibiotic Resistance ◽

Uv Irradiation ◽

Resistance Genes ◽

Ultraviolet Irradiation ◽

Efflux Pump ◽

Antibiotic Resistance Genes ◽

Regulatory System ◽

Pseudomonas Aeruginosa Pao1 ◽

Multiple Antibiotics

UV irradiation disturbs the regulatory system of efflux pump proteins to sensitize P. aeruginosa to multiple antibiotics. The increasing susceptibility to rifampicin and vancomycin might be caused by UV-mediated mutations in antibiotic resistance genes.

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