Characteristics and diversity of mutations in regulatory genes of resistance-nodulation-cell division efflux pumps in association with drug-resistant clinical isolates of Acinetobacter baumannii

Abstract Background: This study aimed to characterize the regulation and expression of three putative resistance-nodulation-cell division (RND)-type efflux systems and their contribution to multidrug efflux in clinical isolates of Acinetobacter baumannii.Methods: Antimicrobial susceptibility testing (AST) of 95 A. baumannii isolates was determined by Kirby-Bauer disk diffusion for 18 antibiotics and minimum inhibitory concentration (MIC) of colistin was determined by broth microdilution method. Moreover, MIC of five classes of antibiotics was assessed using E-test strips in the presence and absence of phenylalanine-arginine beta-naphthylamide (PAβN). Regulatory genes of RND efflux pumps (AdeRS, AdeL, AdeN and BaeSR) were subjected to sequencing. The relative expression of adeB. adeG and adeJ genes was determined by quantitative real-time PCR (RT-PCR).Results: Overall, majority of isolates (93%) were extensively drug-resistant (XDR). In the phenotypic assay, efflux pump activity was observed in 40% of isolates against multiple antibiotics mainly tigecycline, but not to imipenem. Several amino acid substitutions were detected in the regulatory genes; except in AdeN. Of note, G186V in AdeS were found to be associated with overexpression of their relative efflux pumps. No insertion sequences (ISs) were detected.Conclusions: Our findings outline the role of RND efflux pumps in resistance of A. baumannii against multiple antibiotics particularly tigecycline, and point out importance of a variety of single mutations in the corresponding regulatory systems. Even though it has been concluded that multidrug resistance occurs as a result of a complex sets of different resistant mechanisms.

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Characteristics and diversity of mutations in regulatory genes of resistance-nodulation-cell division efflux pumps in association with drug-resistant clinical isolates of Acinetobacter baumannii

10.21203/rs.3.rs-46743/v4 ◽

2020 ◽

Author(s):

Bahare Salehi ◽

Zohreh Ghalavand ◽

Abbas Yadegar ◽

Gita Eslami

Keyword(s):

Cell Division ◽

Acinetobacter Baumannii ◽

Efflux Pump ◽

Efflux Pumps ◽

Regulatory Genes ◽

Clinical Isolates ◽

Drug Resistant ◽

Broth Microdilution Method ◽

Rnd Efflux Pumps ◽

Multiple Antibiotics

Abstract Background: This study aimed to characterize the regulation and expression of three putative resistance-nodulation-cell division (RND)-type efflux systems and their contribution to multidrug efflux in clinical isolates of Acinetobacter baumannii. Methods: Antimicrobial susceptibility testing (AST) of 95 A. baumannii isolates was determined by Kirby-Bauer disk diffusion for 18 antibiotics and minimum inhibitory concentration (MIC) of colistin was determined by broth microdilution method. Moreover, MIC of five classes of antibiotics was assessed using E-test strips in the presence and absence of phenylalanine-arginine beta-naphthylamide (PAβN). Regulatory genes of RND efflux pumps (AdeRS, AdeL, AdeN and BaeSR) were subjected to sequencing. The relative expression of adeB. adeG and adeJ genes was determined by quantitative real-time PCR (RT-PCR).Results: Overall, majority of isolates (93%) were extensively drug-resistant (XDR). In the phenotypic assay, efflux pump activity was observed in 40% of isolates against multiple antibiotics mainly tigecycline, but not to imipenem. Several amino acid substitutions were detected in the regulatory genes; except in AdeN. Of note, G186V in AdeS were found to be associated with overexpression of their relative efflux pumps. No insertion sequences (ISs) were detected. Conclusions: Our findings outline the role of RND efflux pumps in resistance of A. baumannii against multiple antibiotics particularly tigecycline, and point out importance of a variety of single mutations in the corresponding regulatory systems. Even though it has been concluded that multidrug resistance occurs as a result of a complex sets of different resistant mechanisms.

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Characteristics and diversity of mutations in regulatory genes of resistance-nodulation-cell division efflux pumps in association with drug-resistant clinical isolates of Acinetobacter baumannii

10.21203/rs.3.rs-46743/v1 ◽

2020 ◽

Author(s):

Bahare Salehi ◽

Zohreh Ghalavand ◽

Abbas Yadegar ◽

Gita Eslami

Keyword(s):

Cell Division ◽

Acinetobacter Baumannii ◽

Efflux Pump ◽

Efflux Pumps ◽

Regulatory Genes ◽

Clinical Isolates ◽

Drug Resistant ◽

Broth Microdilution Method ◽

Rnd Efflux Pumps ◽

Multiple Antibiotics

Abstract Background This study aimed to characterize the regulation and expression of three putative resistance-nodulation-cell division (RND)-type efflux systems and their contribution to multidrug efflux in clinical isolates of Acinetobacter baumannii. Methods Antimicrobial susceptibility testing (AST) of 95 A. baumannii isolates was determined by Kirby-Bauer disk diffusion for 18 antibiotics and minimum inhibitory concentration (MIC) of colistin was determined by broth microdilution method. Moreover, MIC of five classes of antibiotics was assessed using E-test strips in the presence and absence of phenylalanine-arginine beta-naphthylamide (PAβN). Regulatory genes of RND efflux pumps (AdeRS, AdeL, AdeN and BaeSR) were subjected to sequencing. The relative expression of adeB. adeG and adeJ genes was determined by quantitative real-time PCR (RT-PCR). Results Overall, majority of isolates (93%) were extensively drug-resistant (XDR). In the phenotypic assay, efflux pump activity was observed in 40% of isolates against multiple antibiotics mainly tigecycline, but not to imipenem. Several amino acid substitutions were detected in the regulatory genes; except in AdeN. Of note, A136V in AdeR and G186V in AdeS were found to be associated with overexpression of their relative efflux pumps. No insertion sequences (ISs) were detected. Conclusions Our findings outline the role of RND efflux pumps in resistance of A. baumannii against multiple antibiotics particularly tigecycline, and point out importance of a variety of single mutations in the corresponding regulatory systems. Even though it has been concluded that multidrug resistance occurs as a result of a complex sets of different resistant mechanisms.

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Characteristics and diversity of mutations in regulatory genes of resistance-nodulation-cell division efflux pumps in association with drug-resistant clinical isolates of Acinetobacter baumannii

10.21203/rs.3.rs-46743/v2 ◽

2020 ◽

Author(s):

Bahare Salehi ◽

Zohreh Ghalavand ◽

Abbas Yadegar ◽

Gita Eslami

Keyword(s):

Cell Division ◽

Acinetobacter Baumannii ◽

Efflux Pump ◽

Efflux Pumps ◽

Regulatory Genes ◽

Clinical Isolates ◽

Drug Resistant ◽

Broth Microdilution Method ◽

Rnd Efflux Pumps ◽

Multiple Antibiotics

Abstract Background: This study aimed to characterize the regulation and expression of three putative resistance-nodulation-cell division (RND)-type efflux systems and their contribution to multidrug efflux in clinical isolates of Acinetobacter baumannii. Methods: Antimicrobial susceptibility testing (AST) of 95 A. baumannii isolates was determined by Kirby-Bauer disk diffusion for 18 antibiotics and minimum inhibitory concentration (MIC) of colistin was determined by broth microdilution method. Moreover, MIC of five classes of antibiotics was assessed using E-test strips in the presence and absence of phenylalanine-arginine beta-naphthylamide (PAβN). Regulatory genes of RND efflux pumps (AdeRS, AdeL, AdeN and BaeSR) were subjected to sequencing. The relative expression of adeB. adeG and adeJ genes was determined by quantitative real-time PCR (RT-PCR).Results: Overall, majority of isolates (93%) were extensively drug-resistant (XDR). In the phenotypic assay, efflux pump activity was observed in 40% of isolates against multiple antibiotics mainly tigecycline, but not to imipenem. Several amino acid substitutions were detected in the regulatory genes; except in AdeN. Of note, A136V in AdeR and G186V in AdeS were found to be associated with overexpression of their relative efflux pumps. No insertion sequences (ISs) were detected. Conclusions: Our findings outline the role of RND efflux pumps in resistance of A. baumannii against multiple antibiotics particularly tigecycline, and point out importance of a variety of single mutations in the corresponding regulatory systems. Even though it has been concluded that multidrug resistance occurs as a result of a complex sets of different resistant mechanisms

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Characteristics and diversity of mutations in regulatory genes of resistance-nodulation-cell division efflux pumps in association with drug-resistant clinical isolates of Acinetobacter baumannii

Antimicrobial Resistance and Infection Control ◽

10.1186/s13756-021-00924-9 ◽

2021 ◽

Vol 10 (1) ◽

Author(s):

Bahare Salehi ◽

Zohreh Ghalavand ◽

Abbas Yadegar ◽

Gita Eslami

Keyword(s):

Cell Division ◽

Acinetobacter Baumannii ◽

Efflux Pump ◽

Efflux Pumps ◽

Regulatory Genes ◽

Clinical Isolates ◽

Drug Resistant ◽

Rnd Efflux Pumps ◽

Multiple Antibiotics

Abstract Background This study was aimed to characterize the genetic diversity and expression of three putative resistance-nodulation-cell division (RND)-type efflux systems and their contribution to multidrug efflux in clinical isolates of Acinetobacter baumannii. Methods Antimicrobial susceptibility testing of 95 A. baumannii isolates was determined by Kirby-Bauer disk diffusion for 18 antibiotics and minimum inhibitory concentration (MIC) of colistin was determined by the broth microdilution method. Moreover, the MIC of five classes of antibiotics was assessed using E-test strips in the presence and absence of phenylalanine-arginine beta-naphthylamide (PAβN). Regulatory genes of the RND efflux pumps (adeRS, adeL, adeN and baeSR) were subjected to sequencing. The relative expression of adeB, adeG and adeJ genes was determined by quantitative real-time PCR (qRT-PCR). Results Overall, the majority of isolates (94%) were extensively drug-resistant (XDR). In the phenotypic assay, efflux pump activity was observed in 40% of the isolates against multiple antibiotics mainly tigecycline. However, we found no efflux activity against imipenem. Several amino acid substitutions were detected in the products of regulatory genes; except in AdeN. Of note, G186V mutation in AdeS was found to be associated with overexpression of its efflux pump. No insertion sequences were detected. Conclusions Our findings outlined the role of RND efflux pumps in resistance of A. baumannii to multiple antibiotics particularly tigecycline, and pointed out the importance of a variety of single mutations in the corresponding regulatory systems. Further studies are required to decipher the precise role of RND efflux pumps in multidrug-resistant clinical isolates of A. baumannii.

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Characteristics and Diversity of Mutations in Regulatory Genes of Resistance-nodulation-cell Division Efflux Pumps in Association With Drug-resistant Clinical Isolates of Acinetobacter Baumannii

10.21203/rs.3.rs-46743/v5 ◽

2021 ◽

Author(s):

Bahare Salehi ◽

Zohreh Ghalavand ◽

Abbas Yadegar ◽

Gita Eslami

Keyword(s):

Cell Division ◽

Acinetobacter Baumannii ◽

Efflux Pump ◽

Efflux Pumps ◽

Regulatory Genes ◽

Clinical Isolates ◽

Drug Resistant ◽

Rnd Efflux Pumps ◽

Multiple Antibiotics

Abstract Background: This study was aimed to characterize the genetic diversity and expression of three putative resistance-nodulation-cell division (RND)-type efflux systems and their contribution to multidrug efflux in clinical isolates of Acinetobacter baumannii.Methods: Antimicrobial susceptibility testing of 95 A. baumannii isolates was determined by Kirby-Bauer disk diffusion for 18 antibiotics and minimum inhibitory concentration (MIC) of colistin was determined by the broth microdilution method. Moreover, the MIC of five classes of antibiotics was assessed using E-test strips in the presence and absence of phenylalanine-arginine beta-naphthylamide (PAβN). Regulatory genes of the RND efflux pumps (adeRS, adeL, adeN and baeSR) were subjected to sequencing. The relative expression of adeB, adeG and adeJ genes was determined by quantitative real-time PCR (RT-PCR).Results: Overall, the majority of isolates (93%) were extensively drug-resistant (XDR). In the phenotypic assay, efflux pump activity was observed in 40% of the isolates against multiple antibiotics mainly tigecycline. However, we found no efflux activity against imipenem. Several amino acid substitutions were detected in the products of regulatory genes; except in AdeN. Of note, G186V mutation in AdeS was found to be associated with overexpression of its efflux pump. No insertion sequences were detected. Conclusions: Our findings outlined the role of RND efflux pumps in resistance of A. baumannii to multiple antibiotics particularly tigecycline, and pointed out the importance of a variety of single mutations in the corresponding regulatory systems. Further studies are required to decipher the precise role of RND efflux pumps in multidrug-resistant clinical isolates of A. baumannii.

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Diversity of mutations in regulatory genes of resistance-nodulation-cell division efflux pumps in association with tigecycline resistance in Acinetobacter baumannii

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dky083 ◽

2018 ◽

Vol 73 (6) ◽

pp. 1501-1508 ◽

Cited By ~ 16

Author(s):

Stefanie Gerson ◽

Jennifer Nowak ◽

Esther Zander ◽

Julia Ertel ◽

Yurong Wen ◽

...

Keyword(s):

Cell Division ◽

Acinetobacter Baumannii ◽

Efflux Pumps ◽

Regulatory Genes

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In-silico interaction studies on the efflux pumps suggest polymyxins to be better substrate for RND efflux pumps in multi-drug resistant strains of Acinetobacter baumannii

10.7287/peerj.preprints.3091 ◽

2017 ◽

Author(s):

Privita Verma ◽

Vishvanath Tiwari

Keyword(s):

Free Energy ◽

Multidrug Resistance ◽

In Silico ◽

Efflux Pump ◽

Binding Free Energy ◽

Efflux Pumps ◽

Drug Resistant ◽

Rnd Efflux Pumps ◽

Resistant Strains ◽

Drug Resistant Strains

Bacterial efflux pumps have emerged as antibiotic resistance determinants and confers multidrug resistance to a broad range of antimicrobials as well as non-antibiotic substances by potentially extruding them out. These are also associated with the virulence and biofilm formation. Thus, development of inhibitors against these effective antibacterial targets can be a highly beneficial approach in order to reverse the multidrug resistance (MDR) in the clinical relevant strains. A keen study on the efflux of foregoing or commonly used antibiotics will contribute in finding substrates that could bind efficiently with these efflux pumps. In the present study, we have explored efflux pumps belonging to Resistance-Nodulation-Division (RND) family as they are found prevalent in gram negative pathogens such as A. baumannii. The two RND efflux pumps under consideration are AdeABC and AcrAB-TolC. For comparative analysis, a non-RND family efflux pump i.e. NorM is selected here. We employed a series of computational techniques ranging from molecular docking to binding free energy estimation and molecular dynamics simulations to determine the binding affinity for different classess of drugs namely aminoglycosides, polymyxins, β-lactams, tetracyclines, glycylcyclines, quinolones and metronidazole against the efflux pumps under study. Our results revealed that class polymyxins has the highest binding affinity with the RND efflux pumps i.e. AcrAB-TolC and AdeABC as well as non-RND efflux pump (NorM). The experimental data was found to much extent in accordance with the binding free energy scores obtained from our in silico analysis. To recapitulate the outcome, polymyxins can possibly be the most efficient substrate for both RND and non-RND efflux pumps, thus, can be used as a reference drug for designing potent efflux pump inhibitors against multi-drug resistant strains.

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Amotosalen is a bacterial multidrug efflux pump substrate potentially affecting its pathogen inactivation activity

10.1101/2021.03.15.435562 ◽

2021 ◽

Author(s):

Alex B. Green ◽

Katelyn E. Zulauf ◽

Katherine A. Truelson ◽

Lucius Chiaraviglio ◽

Meng Cui ◽

...

Keyword(s):

Acinetobacter Baumannii ◽

Efflux Pump ◽

Efflux Pumps ◽

Multidrug Resistant ◽

Pathogen Inactivation ◽

Multidrug Efflux ◽

Multidrug Efflux Pump ◽

Minimal Inhibitory Concentrations ◽

E Coli ◽

Rnd Efflux Pumps

AbstractPathogen inactivation is a strategy to improve the safety of transfusion products. The Cerus Intercept technology makes use of a psoralen compound called amotosalen in combination with UVA light to inactivate bacteria, viruses and protozoa. Psoralens have structural similarity to bacterial multidrug-efflux pump substrates. As these efflux pumps are often overexpressed in multidrug-resistant pathogens and with recent reported outbreaks of transfusion-associated sepsis with Acinetobacter, we tested whether contemporary drug-resistant pathogens might show resistance to amotosalen and other psoralens based on multidrug efflux mechanisms through microbiological, biophysical and molecular modeling analysis. The main efflux systems in Enterobacterales and Acinetobacter baumannii, tripartite RND (resistance-nodulation-cell division) systems which span the inner and outer membranes of Gram-negative pathogens and expel antibiotics from the bacterial cytoplasm into the extracellular space, were specifically examined. We found that amotosalen was an efflux substrate for the TolC-dependent RND efflux pumps in E. coli and the AdeABC efflux pump from Acinetobacter baumannii, and that minimal inhibitory concentrations for contemporary bacterial isolates in vitro approached and exceeded the concentration of amotosalen used in the approved platelet and plasma inactivation procedures. These findings suggest that otherwise safe and effective inactivation methods should be further studied to exclude possible gaps in their ability to inactivate contemporary, multidrug-resistant bacterial pathogens.ImportancePathogen inactivation is a strategy to enhance the safety of transfused blood products. We identify the compound, amotosalen, widely used for pathogen inactivation, as a bacterial multidrug efflux substrate. Specifically, experiments suggest that amotosalen is pumped out of bacteria by the major TolC-dependent RND efflux pumps in E. coli and the AdeABC efflux pump in Acinetobacter baumannii. Such efflux pumps are often overexpressed in multidrug-resistant pathogens. Importantly, the minimal inhibitory concentrations for contemporary multidrug-resistant Enterobacterales, Acinetobacter baumannii, Pseudomonas aeruginosa, Burkholderia spp., and Stenotrophomonas maltophilia isolates approached or exceeded the amotosalen concentration used in approved platelet and plasma inactivation procedures, potentially as a result of efflux pump activity. Although there are important differences in methodology between our experiments and blood product pathogen inactivation, these findings suggest that otherwise safe and effective inactivation methods should be further studied to exclude possible gaps in their ability to inactivate contemporary, multidrug-resistant bacterial pathogens.

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