MexAB-OprM and MexXY-OprM efflux pumps overexpression; additional mechanism for carbapenems resistance among nosocomial Pseudomonas aeruginosa isolates

2020 ◽  
Vol EJMM29 (4) ◽  
pp. 17-25
Author(s):  
Asmaa M. Elbrolosy ◽  
Amira H. Elkhayat ◽  
Dina M. Hassan ◽  
Eman H. Salem
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Target Genes ◽  
Efflux Pump ◽  
Early Recognition ◽  
Efflux Pumps ◽  
Diffusion Method ◽  
University Hospitals ◽  
Pcr Assay ◽  
Efflux Pump Inhibitor ◽  
Disk Diffusion Method

Background: Multidrug-resistant pathogens have been on the rise during the last few years. Pseudomonas aeruginosa is commonly encountered in nosocomial infections with remarkable ability to develop antimicrobial resistance of which carbapenems are of great concern. Objectives: To explore the role of MexAB-OprM and MexXY-OprM efflux pumps overexpression as carbapenems resistance mechanisms among nosocomial P. aeruginosa isolates at both Menoufia and Kasr Al Ainy University Hospitals by phenotypic and molecular characterization methods. Methodology: A total of 120 P. aeruginosa isolates were collected from patients with hospital-acquired infections and subjected to antibiotic susceptibility testing by the Kirby-Bauer disk diffusion method. Carbapenems-resistant isolates were selected and investigated phenotypically for the contribution of MexAB-OprM and MexXY-OprM efflux pumps by both disk synergy and MIC reduction assays with cyanide-m-chlorophenyl hydrazone (CCCP) as an efflux pump inhibitor. Real time PCR assay verified the existence of mexA and mexX genes as regulators of MexAB-OprM and MexXY-OprM overexpression. Laboratory results were correlated with data regarding patients' clinical findings as well as risk factors. Results: Out of 120 P. aeruginosa isolates, 88 (73.3%) isolates were carbapenems-resistant of which 100% were MDR isolates. The highest resistance rate was for piperacillin and piperacillin/tazobactam (100% for each) and the lowest rate was seen against colistin (7.5%).The RT-PCR assay revealed that, 54/88 (61.3%) P. aeruginosa isolates harbored the target genes: 21 isolates (38.9%) were positive for mexA alone, 12 isolates (22.2%) were positive for mexX alone and 21 isolates (38.9%) showed co-existence of the two genes. In relation to PCR results, the sensitivity, specificity and accuracy of CCCP disk synergy test respectively were 46%, 94% and 64.8% while, those for MIC method were 88.9%, 55.9% and 76.1% respectively. Conclusion: Carbapenems resistance mediated by the overexpression of efflux pumps has also now emerged. Early recognition of this resistance mechanism to allow the use of alternative b-lactams is imperative.

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

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.

scholarly journals Role of efflux pump inhibitor in decreasing antibiotic cross-resistance of Pseudomonas aeruginosa in a burn hospital in Iran

2016 ◽  
Vol 10 (06) ◽  
pp. 600-604 ◽  
Author(s):  
Mahshid Talebi-Taher ◽  
َAli Majidpour ◽  
Abbas Gholami ◽  
Samira Rasouli-Kouhi ◽  
Maryam Adabi
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Disk Diffusion ◽  
Diffusion Method ◽  
Cross Resistance ◽  
Beta Lactams ◽  
Efflux Pump Inhibitor ◽  
Efflux Pump Inhibitors

Introduction: Multidrug resistance in Pseudomonas aeruginosa may be due to efflux pump overexpression. This study phenotypically examined the role of efflux pump inhibitors in decreasing antibiotic cross-resistance between beta-lactams, fluoroquinolones, and aminoglycosides in P. aeruginosa isolates from burn patients in Iran. Methodology: A total of 91 phenotypically and genotypically confirmed P. aeruginosa samples were studied. Multidrug cross-resistance was determined using the disk diffusion method and minimum inhibitory concentration (MIC) test. The contribution of efflux pumps was determined by investigating MIC reduction assay to markers of beta-lactams, fluoroquinolones, and aminoglycosides in the absence and presence of an efflux pump inhibitor. All the isolates were also tested by polymerase chain reaction for the presence of mexA, mexC, and mexE efflux genes. Results: Of the isolates, 81 (89%) and 83 (91.2%) were multidrug resistant according to the disk diffusion and MIC method, respectively. Cross-resistance was observed in 67 (73.6%) and 68 (74.7%) of isolates according to the disk diffusion and MIC method, respectively. In the presence of the efflux pump inhibitor, twofold or higher MIC reduction to imipenem, cefepime, ciprofloxacin, and gentamicin was observed in 59, 65, 55, and 60 isolates, respectively. Except for two isolates that were negative for mexC, all isolates were positive for mexA, mexC, and mexE genes simultaneously. Conclusion: Efflux pumps could cause different levels of resistance based on their expression in clinical isolates. Early detection of different efflux pumps in P. aeruginosa could allow the use of other antibiotics and efflux pump inhibitors in combination with antibiotic therapy.

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

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.

Determination of imipenem efflux-mediated resistance in Acinetobacter spp., using an efflux pump inhibitor

2019 ◽  
Author(s):  
Ghazale Amiri ◽  
Maryam Abbasi Shaye ◽  
Masoumeh Bahreini ◽  
Asghar Mafinezhad ◽  
Kiarash Ghazvini ◽  
...  
Keyword(s):  
Efflux Pump ◽  
Efflux Pumps ◽  
Diffusion Method ◽  
Efflux Pump Inhibitor ◽  
Acinetobacter Species ◽  
Fold Reduction ◽  
Carbonyl Cyanide ◽  
Acinetobacter Spp

Background and Objectives: In recent years, reports of Acinetobacter strains resistant to all known antibiotics have caused a great concern in medical communities. Overexpression of efflux pumps is one of the major causes of resistance in bacteria. The aim of this study was to investigate the role of efflux pumps in conferring resistance to imipenem in clinically important Acinetobacter spp; Acinetobacter baumannii and Acinetobacter lwoffii. Materials and Methods: A total number of 46 clinical Acinetobacter isolates, including 33 A. baumannii and 13 A. lwoffii isolates, previously collected from Shahid Kamyab and Ghaem hospitals of Mashhad, Iran were used in this study. Imipenem susceptibility testing was carried out by the disc diffusion method. Imipenem minimum inhibitory concentration (MIC) for resistant Acinetobacter isolates were determined both in the presence and absence of the efflux pumps inhibitor, carbonyl cyanide 3-chlorophenylhydrazone (CCCP). Results: Resistance to imipenem was observed in 38 isolates including 30 A. baumannii and 8 A. lwoffii isolates. Experiments in the presence of CCCP showed a 2 to 16384 fold reduction in imipenem MICs in 14 A. baumannii and 2 A. lwoffii isolates. Conclusion: The results obtained showed high levels of resistance to imipenem and contribution of efflux pumps in conferring resistance in both Acinetobacter species in this study. Moreover, imipenem efflux mediated resistance highlights the importance of this mechanism not only in A. baumannii but also in non-baumannii Acinetobacter Spp. which have been neglected in antibiotic resistance studies.

scholarly journals Detection of OqxAB and QepA Efflux Pumps and Their Association with Antibiotic Resistance in Klebsiella pneumoniae Isolated From Urinary Tract Infection

2020 ◽  
Vol 7 (4) ◽  
Author(s):  
Manijeh Dehnamaki ◽  
Maryam Ghane ◽  
Laleh Babaeekhou
Keyword(s):  
Urinary Tract Infection ◽  
Antibiotic Resistance ◽  
Urinary Tract ◽  
Tract Infection ◽  
Klebsiella Pneumoniae ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Diffusion Method ◽  
Beta Lactam ◽  
Disk Diffusion Method

Background: The emergence and spread of drug resistance among Klebsiella pneumoniae clinical isolates have limited the treatment options for these bacteria. Efflux pumps are considered as one of the key mechanisms of antibiotic resistance in K. pneumoniae isolates. Objectives: The present study aimed to detect oqxA, oqxB, and qepA efflux genes in K. pneumoniae isolated from urinary tract infection (UTI) and survey their association with antibiotic resistance. Methods: In total, 100 K. pneumoniae isolates were obtained from urine samples, and an antimicrobial susceptibility test was conducted using the disk diffusion method according to the Clinical and Laboratory Standards Institute (CLSI) instructions. Polymerase chain reaction (PCR) was done for the detection of efflux pump genes including, oqxA, oqxB, and qepA, and their association was statistically analyzed with resistance to antibiotics. Results: The highest rate of resistance was obtained against trimethoprim-sulfamethoxazole (72%), amikacin (70%), levofloxacin (68%), gentamicin (56%), ceftazidime (56%), and ceftriaxone (51%), and the lowest resistance was against imipenem (10%). Thirty one percent of isolates were multidrug resistant (MDR). Molecular distribution test showed that 57% and 56% of isolates carried the oqxA and oqxB genes, respectively. Also, the frequency of qepA genes was 21%. The presence of oqxA/oqxB and qepA efflux genes were significantly associated with fluoroquinolone and beta-lactam resistance phenotypes (P < 0.05). Conclusions: The high frequency of efflux genes showed that this resistance mechanism is the main way, along with other resistance mechanisms in K. pneumoniae isolates. It is necessary to adopt appropriate treatment to reduce the incidence of resistance.

MrkD Gene as a Regulator of Biofilm Formation with Correlation to Antibiotic Resistance among Clinical Klebsiella pneumoniae Isolates from Menoufia University Hospitals

2020 ◽  
Vol 29 (3) ◽  
pp. 137-144
Author(s):  
Asmaa M. Elbrolosy ◽  
Naira A. Eissa ◽  
Nahed A. Al-Rajhy ◽  
Esraa El-Sayed A. El-Mahdy ◽  
Rasha G. Mostafa
Keyword(s):  
Antibiotic Resistance ◽  
Klebsiella Pneumoniae ◽  
Biofilm Formation ◽  
Diffusion Method ◽  
Clinical Samples ◽  
Resistance Pattern ◽  
University Hospitals ◽  
Pcr Assay ◽  
Disk Diffusion Method ◽  
Microbiological Methods

Background: Klebsiella pneumoniae (K. pneumoniae) is a common pathogen involved in a diverse array of life-threatening infections. Increasing frequent acquisition of antibiotic resistance by K. pneumoniae has given rise to multidrug-resistant pathogen mostly at the hospital level. Objectives: To assess the prevalence and antibiotic resistance pattern of the clinical K. pneumoniae isolates at Menoufia University Hospitals (MUHs) as well as to explore the role of mrkD gene as a regulator of biofilm formation. Methodology: A total of 340 different clinical samples were obtained from 270 patients who were admitted to MUHs and those from Outpatient clinics during the period from April 2018 to September 2019. 84 K. pneumoniae isolates were identified by the standard microbiological methods and vitek-2 system. The antimicrobial resistance pattern was determined by disk diffusion method. The biofilm-forming ability of all K. pneumoniae isolates was demonstrated phenotypically by the modified Congo red agar method (MCRA) and PCR assay verified the presence of mrkD gene as a genetic determinant of biofilm formation. Results: Klebsiella spp. represented 34.7% of the collected isolates and the predominant spp. was K. pneumoniae (91.3%). The highest resistance rates were for ceftriaxone (69%) followed by aztreonam (67.9%), 66.7% for each of piperacillin and ceftazidime, while the least resistance rate was for fosfomycin (8.3%). Biofilm production was detected among 83.3% of the isolates by MCRA method. A highly significant statistical difference was noted between biofilm- and non- biofilm - producing K. pneumoniae isolates regarding resistance to cefepieme and amikacin (P <0.001) and similarly regarding resistance to aztreonam, imipenem, meropenem, ertapenem and tobramycin (P<0.05). Conventional PCR assay showed that, 92% of the isolates harbored mrkD gene with a highly significant association with biofilm formation. Conclusion: The increasing prevalence and remarkable ability to acquire antibiotic resistance among K. pneumoniae isolates together with biofilm formation should alert even more regarding the hazard of this pathogen in hospital settings.

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

Pharmaceutics ◽  
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.

scholarly journals 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

2018 ◽  
Vol 63 (2) ◽  
pp. e01718-18 ◽  
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.

scholarly journals Presence of different beta-lactamase classes among clinical isolates of Pseudomonas aeruginosa expressing AmpC beta-lactamase enzyme

2010 ◽  
Vol 4 (04) ◽  
pp. 239-242 ◽  
Author(s):  
Supriya Upadhyay ◽  
Malay Ranjan Sen ◽  
Amitabha Bhattacharjee
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Treatment Options ◽  
Clinical Isolates ◽  
Diffusion Method ◽  
Beta Lactamase ◽  
Beta Lactam ◽  
Disk Diffusion Method ◽  
Extended Spectrum Beta Lactamase ◽  
Extended Spectrum ◽  
Enzyme Detection

Introduction: Infections caused by Pseudomonas aeruginosa are difficult to treat as the majority of isolates exhibit varying degrees of beta-lactamase mediated resistance to most of the beta-lactam antibiotics. It is also not unusual to find a single isolate that expresses multiple β-lactamase enzymes, further complicating the treatment options. Thus the present study was designed to investigate the coexistence of different beta-lactamase enzymes in clinical isolates of P. aeruginosa. Methodology: A total of 202 clinical isolates of P. aeruginosa were tested for the presence of AmpC beta-lactamase, extended spectrum beta-lactamase (ESBL) and metallo beta-lactamase (MBL) enzyme. Detection of AmpC beta-lactamase was performed by disk antagonism test and a modified three-dimensional method, whereas detection of ESBL was done by the combined disk diffusion method per Clinical and Laboratory Standards Institute (CLSI) guidelines and MBL were detected by the Imipenem EDTA disk potentiation test. Results: A total of 120 (59.4%) isolates were confirmed to be positive for AmpC beta-lactamase. Among them, 14 strains (7%) were inducible AmpC producers. Co-production of AmpC along with extended spectrum beta-lactamase and metallo beta-lactamase was reported in 3.3% and 46.6% isolates respectively. Conclusion: The study emphasizes the high prevalence of multidrug resistant P. aeruginosa producing beta-lactamase enzymes of diverse mechanisms. Thus proper antibiotic policy and measures to restrict the indiscriminative use of cephalosporins and carbapenems should be taken to minimize the emergence of this multiple beta-lactamase producing pathogens.

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