EFFLUX-PUMP EXPRESSION IN MULTIDRUG-RESISTANT BACTERIA: CASE IN A UNIVERSITY TEACHING HOSPITAL OF CAMEROON (original) (raw)
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Detection of efflux pump activity among clinical isolates of Staphylococcus and Micrococcus species
Tropical Journal of Pharmaceutical Research
Purpose: To detect efflux pump activity (EPA) and screening a suspected efflux pump inhibitor (EPI) [1-(3-(trifluoromethyl)benzyl]-piperazine (TFMBP)], which could help in reducing multi-drug resistance (MDR). Methods: Eighteen isolates, viz, 14 S. aureus, 2 S. lentus, 1 S. xylosus and 1 Micrococcus species from various hospital infections of admitted patients were screened for antibiotics susceptibility to 11 classes of antibiotics including oxacillin and β-lactamase production. Efflux pump activity (EPA) was determined by minimum inhibitory concentration (MIC) technique in the presence and absence of TFMBP, the isolates were also screened for MDR genes. Results: All the isolates were resistant to ampicillin (10 µg) and penicillin (10 µg), but sensitive to bacitracin (10 µg). Majority of the isolates were MDR 12/18 (66.7 %), 10 (55.6 %) were inducible βlactamase producers and 3 (16.7 %) were intrinsic β-lactamase producers. Seven (38.9 %) were resistant to oxacillin and also produced carbapenemase enzyme. Eight (66.7 %) of the 12 MDR isolates gave evidence of EPA with TFMBP. However, no MDR genes were detected. Conclusion: Staphylococcus and Micrococcus species exhibit EPA in antibiotic resistance while a suitable EPI such as TFMBP when combined with specific antibiotics could help combat this menace.
Antibiotic resistance and efflux pumps
2019
The main purpose of this manuscript is to review the resistance against antibiotics and efflux pumps, one of the mechanisms important in resistance against antibiotics. As a definition, the resistance against antibiotics is accepted as the capability of a microorganism to resist the activity of antimicrobials, which were successfully used to kill the microorganism once. Antibiotic resistance is characterized by several antibiotic susceptibility tests. The wide consumption of antibiotics; the over prescription of antimicrobial drugs by medical doctors; unnecessary, incorrect and inadequate self-medication by the patient and use of several antimicrobial agents either to support a healthy growth or therapeutic purposes in animals consumed as food triggered severe antibiotic resistance. Therefore, the resistance against antimicrobials became a considerable, wide-spread issue in all around the world and the studies have been initiated to overcome the resistance against antibiotics. There...
Observing the presence of efflux pump activities in some clinically isolated bacterial strains
International Journal of Biology and Chemistry, 2022
Chemotherapeutic agents are substances that cure infectious diseases for many years. However, problems arose due to drug resistance that developed over time and it was observed that the drugs failed to stance that adversely st stance mechast the cell. Thus, the microorganism becomes resistant to the drug used. If this resistance is inhibited, the drug may work. A variety of chemical or natural inhibitors are available for inhibition. However, since they cause toxicity problems, their clinical use is not currently available. Studies in this area are ongoing. There are st st stant clinically isolated strains using EtBr dye. The strains that are Acinetobacter baumannii, Candida albicans, Candida glabrata, Candida tropicalis, Klebsiella pneumoniae, Providencia rustigianii, Serratia odorifera, , Staphylococcus aureus and Streptococcus pneumoniae microorganisms. Cartwheel
Egyptian Journal of Medical Microbiology
Background: Antimicrobial resistance is one of the most serious public health threats of the twenty-first century, Uropathogenic Escherichia coli (UPEC) are one of the main bacteria causing urinary tract infections (UTIs). The rate of UPEC with high resistance towards antibiotics has increased dramatically in recent years. Objectives: This study aimed to assess the antibiotic resistance pattern of UPEC and to detect the relationship of antibiotic resistance with the presence of efflux pump genes (AcrA-AcrB-TolC). Methodology: This study included 50 UPEC strains, Identification of E.coli by Gram stain, culture and biochemical reactions was done, Antibiotic susceptibility for isolated E.coli strains by vitek system and detection of AcrA-AcrB-TolC genes by conventional PCR among isolated strains were also performed. Results: the prevalence of MDR was 70%,UPEC isolates showed high level of resistance to : ampicillin(94%), nalidixic acid (84%), ticacillin (82%), ciprofloxacin (76%) and trimethoprim/sulfamethoxazole (76%), low level of resistance of UPEC to:gentamicin (34%), amoxicillin/clavulinic acid (28%), ceftazidime (21%), cefoxitin (16%), piperacillin/ tazobactam (8%), tobramycin(2%) and ertapenem (2%) but no resistance to amikacin , imipenem and nitrofurantoin. 50%, 66% and 68% of isolates had genes acrA, acrB and tolC respectively. there was a significant correlation between tol C gene and MDR phenotype. Conclusion: the rate of MDR UPEC is rising, efflux pumps play an important role in mediating antibiotic efflux and increase the rate of antibiotic rasistance. The frequency of tol C gene was significantly higher in MDR than non MDR, while the acr A B level showed non significant variation among MDR and non MDR.
Aim: The present study aims to estimate the phenotypic and genotypic characteristics of efflux pump systems in Enterobacteriacae clinical isolates. Methods: A total of 60 isolates of Escherichia coli and Klebsiella pneumoniae were studied for antibacterial susceptibility pattern, Effect of efflux pump inhibitors (reserpine, carbonyl cyanide m-chloropheylhydorazone [CCCP] and omperazole) on the MIC of antibacterial agents & ethidium bromide (EtBr) efflux assay, preparing outer membrane proteins, polymerase chain reaction for the detection of efflux genes. Sequencing of some efflux genes, constructing the phylogenetic tree and determining the copy number of the resistance genes in both chromosomal and plasmid DNA. Results: Nearly all the clinical isolates were multiple resistant as they were resistant to most antimicrobial classes used in this study. CCCP was found to have the highest effect on decreasing the MIC of different antibiotics comparable with reserpine and omperazole. It was found that all tested E. coli and K. pneumoniae isolates extrude EtBr resulting in decrease in fluorescence over the time of assay. In E. coli isolate No.25 and K. pneumoniae No. 12, only the control cells extrude EtBr resulting in significant loss in fluorescence. In presence of each of reserpine, CCCP and omperazole, an insignificant loss in fluorescence was observed, reflecting blockage of EtBr by these compounds at different levels. All E. coli isolates produced high amount of outer membrane proteins with apparent molecular mass of 37 & 39 and 50 KDa. These proteins may be designated as OmpC, OmpF and TOlC respectively. Regarding K. pneumonia, all the isolates produced high amount of outer membrane proteins with apparent molecular mass of 37 & 50 KDa and only six isolates produced an outer membrane protein of apparent molecular mass of 39 KDa these proteins may be designated as ompK35 & TOlC and ompK 36 respectively. AcrA and AcrB and TOlC genes was detected and amplified in most of the tested isolates of E. coli and K. pneumoniae genomic and plasmid DNA. AcrA amplicons were visualized at 500 bp for E. coli and 200 bp for K. pneumoniae, AcrB amplicon was detected at 500 bp and TOlC amplicon was detected at 700 bp for both E. coli and K. pneumonia isolates. Moving to the sequencing results, the phylogeny was used to determine the relatedness between different isolates with the observation that different genes like AcrA or AcrB were very closely related to each other on chromosomal DNA and that was the same observation for their gene sequence on plasmid DNA that differs from the chromosomal sequence. It was observed that the chromosomal copy number of AcrA and AcrB genes of the E. coli and K. pneumoniae isolates was nearly the same and after the acquisition of the plasmid, the copy number of these genes was significantly increased.
A Simple Method for Assessment of MDR Bacteria for Over-Expressed Efflux Pumps
2013
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.
Journal of Infection in Developing Countries, 2023
Introduction: Enterococcus faecium is a major cause of community and hospital-acquired infections. Due to limited options for infection with fluoroquinolones-resistant Enterococci, novel therapeutics are urgently needed. Efflux pumps are contributed to fluoroquinolones resistance phenotype in this bacterium and novel inhibitors that target these efflux pumps could be effective in patients. In this research, the possible synergistic effect of an efflux pump inhibitor (EPI), thioridazine, with ciprofloxacin was investigated against clinical isolates of E. faecium. Methodology: A total of 88 isolates of E. faecium from clinical specimens were studied from August 2017 to September 2018. Conventional phenotypic and molecular methods characterized all the isolates. Standard susceptibility tests and molecular assays determined the antibiotic resistance profiles and the frequency of efflux pump genes. Minimum inhibitory concentrations (MICs) to ciprofloxacin (CIP) in the presence and absence of thioridazine were measured by the micro-broth dilution method. Results: The highest antibiotic resistance rate among E. faecium isolates was related to ciprofloxacin (96.8%), levofloxacin (94.3%), and imipenem (90.9%), respectively. The highest frequency of efflux pump determinants was related to efmA (60, 68%), followed by emeA (48, 54.5%), and efrA and/or efrB genes (45, 51%). The efflux pump inhibitor showed ≥ 2-fold decrease in the MIC value of ciprofloxacin in 48.2% of the isolates. Conclusions: Efflux pump inhibitor genes efrAB, efmA, and emeA are common among the E. faecium clinical isolates. Our results supported the administration of thioridazine, as an efflux pump inhibitor, in fluoroquinolone-resistant E. faecium infections due to its synergistic effect with CIP.
International Journal of Microbiology
Antibiotic resistance mechanisms in Enterobacteriaceae are causative agents of global health problems. Bacterial infections due to multidrug resistance (MDR) may be mediated by the overexpression of efflux pumps. In this study, we investigated the prevalence of oqxA and oqxB genes as two encoding agents of efflux pumps and the determination of antibiotic resistance rate in clinical isolates of Enterobacteriaceae. In this study, 100 Enterobacteriaceae isolates collected from different clinical specimens of infectious patients, such as wounds, urine, blood, discharge, and abscesses except stool, were examined. Identification of the isolates was performed using standard biochemical tests such as TSI, citrate, urea, lysine, SIM, MR-VP, and gas production. The antimicrobial susceptibility test was carried out by the Kirby–Bauer disk diffusion method according to CLSI guidelines, and finally, the oqxA and oqxB genes were detected by the PCR method. Among 100 Enterobacteriaceae isolates, E...
The Egyptian Journal of Medical Microbiology
Background: The efflux pumps are one of the main mechanisms of the antibiotic resistance in Escherichia coli .The efflux pump inhibitors (chlorpromazine and omeprazole) were tested for their effect on the antibiotic resistance by inhibiting efflux pump activity. Objective: The present study aims to estimate the effect of some efflux pump inhibitors on the antibiotic resistance of some Escherichia coli isolates. Methodology: A total of 100 isolates of Escherichia coli were studied for antibacterial susceptibility pattern by disk diffusion method with and without efflux pump inhibitors chlorpromazine (25 µg) and omeprazole (100 µg), determination of the MIC of amikacin and gentamicin on 60 E.coli resistant isolates, the effect of the efflux pump inhibitors on the MIC of amikacin and gentamicin and PCR amplification of the efflux pump genes AcrD and MdfA genes. Results: The difference between all tested antibiotics in the change of resistance to totally sensitive E.coli isolates after addition of CPZ and OMP by disk diffustion method were statistically highly significant (p value <0.001), in which the highest percentage value were reported for aminoglycoside antibiotics (amikacin and gentamicin). The highest reduction in the MIC of amikacin and gentamicin was observed with chlorpromazine than omeprazole (p<0.05) .The proportion of isolates with greater than twofold reductions in MIC in the presence of CPZ were 69.2% and 50.9% for amikacin and gentamicin respectively (p>0.05) while in the presence of OMP were 46.2% and 30.9% for amikacin and gentamicin respectively, (p>0.05). PCR detection of efflux pump genes detected a high level of AcrD gene detection than MdfA gene (p value <0.05).The percentage of AcrD detection in amikacin and gentamicin resistant isolates were 77% and 87.3% respectively, while for MdfA gene detection in amikacin and gentamicin resistant isolates were 59% and 71% respectively. Conclusion: Antibacterial efflux pumps are involved in establishment of resistance among the tested isolates. Chlorpromazine and Omperazole were capable of inhibiting the efflux pump with higher activity on aminoglycoside antibiotics. Chlorpromazine was more effective than Omeprazole as EPI. PCR results showed that AcrD and MdfA efflux pump genes contributed to the resistance of the tested aminoglycosides.