A novel mechanism of action of ketoconazole: inhibition of the NorA efflux pump system and biofilm formation in multidrug-resistant Staphylococcus aureus

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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Higher biofilm formation in multidrug-resistant clinical isolates of Staphylococcus aureus

International Journal of Antimicrobial Agents ◽

10.1016/j.ijantimicag.2008.02.009 ◽

2008 ◽

Vol 32 (1) ◽

pp. 68-72 ◽

Cited By ~ 59

Author(s):

An Sung Kwon ◽

Gwang Chul Park ◽

So Yeon Ryu ◽

Dong Hoon Lim ◽

Dong Yoon Lim ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Biofilm Formation ◽

Multidrug Resistant ◽

Clinical Isolates

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Clinically Approved Drugs Inhibit the Staphylococcus aureus Multidrug NorA Efflux Pump and Reduce Biofilm Formation

Frontiers in Microbiology ◽

10.3389/fmicb.2019.02762 ◽

2019 ◽

Vol 10 ◽

Cited By ~ 3

Author(s):

Saskia Zimmermann ◽

Mareike Klinger-Strobel ◽

Jürgen A. Bohnert ◽

Sindy Wendler ◽

Jürgen Rödel ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Biofilm Formation ◽

Efflux Pump ◽

Approved Drugs

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Prevalence of multidrug-resistant Staphylococcus aureus isolates with strong biofilm formation ability among animal-based food in Shanghai

Food Control ◽

10.1016/j.foodcont.2020.107106 ◽

2020 ◽

Vol 112 ◽

pp. 107106 ◽

Cited By ~ 2

Author(s):

Chujun Ou ◽

Daiqi Shang ◽

Jingxian Yang ◽

Bo Chen ◽

Jiang Chang ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Biofilm Formation ◽

Multidrug Resistant

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Investigation on the effect of known potent S. aureus NorA efflux pump inhibitors on the staphylococcal biofilm formation

RSC Advances ◽

10.1039/c7ra03859c ◽

2017 ◽

Vol 7 (59) ◽

pp. 37007-37014 ◽

Cited By ~ 7

Author(s):

Stefano Sabatini ◽

Miranda Piccioni ◽

Tommaso Felicetti ◽

Stefania De Marco ◽

Giuseppe Manfroni ◽

...

Keyword(s):

Biofilm Formation ◽

Efflux Pump ◽

Multidrug Resistant ◽

Efflux Pump Inhibitors

The emergence of multidrug resistant microorganisms has triggered the impending need of developing effective antibacterial strategies.

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Ethidium Bromide MIC Screening for Enhanced Efflux Pump Gene Expression or Efflux Activity in Staphylococcus aureus

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01058-10 ◽

2010 ◽

Vol 54 (12) ◽

pp. 5070-5073 ◽

Cited By ~ 49

Author(s):

Diixa Patel ◽

Christos Kosmidis ◽

Susan M. Seo ◽

Glenn W. Kaatz

Keyword(s):

Staphylococcus Aureus ◽

Ethidium Bromide ◽

Efflux Pump ◽

Large Strain ◽

Efflux Pumps ◽

Multidrug Resistant ◽

Reverse Transcription Pcr ◽

Biocide Resistance ◽

Straightforward Method ◽

Short Period

ABSTRACT Multidrug resistance efflux pumps contribute to antimicrobial and biocide resistance in Staphylococcus aureus. The detection of strains capable of efflux is time-consuming and labor-intensive using currently available techniques. A simple and inexpensive method to identify such strains is needed. Ethidium bromide is a substrate for all but one of the characterized S. aureus multidrug-resistant (MDR) efflux pumps (NorC), leading us to examine the utility of simple broth microtiter MIC determinations using this compound in identifying efflux-proficient strains. Quantitative reverse transcription-PCR identified the increased expression of one or more MDR efflux pump genes in 151/309 clinical strains (49%). Ethidium bromide MIC testing was insensitive (48%) but specific (92%) in identifying strains with gene overexpression, but it was highly sensitive (95%) and specific (99%) in identifying strains capable of ethidium efflux. The increased expression of norA with or without other genes was most commonly associated with efflux, and in the majority of cases that efflux was inhibited by reserpine. Ethidium bromide MIC testing is a simple and straightforward method to identify effluxing strains and can provide accurate predictions of efflux prevalence in large strain sets in a short period of time.

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Biofilm Formation in Methicillin-Resistant Staphylococcus aureus Isolated in Cystic Fibrosis Patients Is Strain-Dependent and Differentially Influenced by Antibiotics

Frontiers in Microbiology ◽

10.3389/fmicb.2021.750489 ◽

2021 ◽

Vol 12 ◽

Author(s):

Agathe Boudet ◽

Pauline Sorlin ◽

Cassandra Pouget ◽

Raphaël Chiron ◽

Jean-Philippe Lavigne ◽

...

Keyword(s):

Cystic Fibrosis ◽

Staphylococcus Aureus ◽

Biofilm Formation ◽

Multidrug Resistant ◽

Inhibitory Effect ◽

Ring Test ◽

Methicillin Resistant ◽

Lung Dysfunction ◽

Chronic Colonization ◽

Strain Dependent

Cystic fibrosis (CF) is a genetic disease with lung abnormalities making patients particularly predisposed to pulmonary infections. Staphylococcus aureus is the most frequently identified pathogen, and multidrug-resistant strains (MRSA, methicillin-resistant S. aureus) have been associated with more severe lung dysfunction leading to eradication recommendations. Diverse bacterial traits and adaptive skills, including biofilm formation, may, however, make antimicrobial therapy challenging. In this context, we compared the ability of a collection of genotyped MRSA isolates from CF patients to form biofilm with and without antibiotics (ceftaroline, ceftobiprole, linezolid, trimethoprim, and rifampicin). Our study used standardized approaches not previously applied to CF MRSA, the BioFilm Ring test® (BRT®), the Antibiofilmogram®, and the BioFlux™ 200 system which were adapted for use with the artificial sputum medium (ASM) mimicking conditions more relevant to the CF lung. We included 63 strains of 10 multilocus sequence types (STs) isolated from 35 CF patients, 16 of whom had chronic colonization. The BRT® showed that 27% of the strains isolated in 37% of the patients were strong biofilm producers. The Antibiofilmogram® performed on these strains showed that broad-spectrum cephalosporins had the lowest minimum biofilm inhibitory concentrations (bMIC) on a majority of strains. A focus on four chronically colonized patients with inclusion of successively isolated strains showed that ceftaroline, ceftobiprole, and/or linezolid bMICs may remain below the resistance thresholds over time. Studying the dynamics of biofilm formation by strains isolated 3years apart in one of these patients using BioFlux™ 200 showed that inhibition of biofilm formation was observed for up to 36h of exposure to bMIC and ceftaroline and ceftobiprole had a significantly greater effect than linezolid. This study has brought new insights into the behavior of CF MRSA which has been little studied for its ability to form biofilm. Biofilm formation is a common characteristic of prevalent MRSA clones in CF. Early biofilm formation was strain-dependent, even within a sample, and not only observed during chronic colonization. Ceftaroline and ceftobiprole showed a remarkable activity with a long-lasting inhibitory effect on biofilm formation and a conserved activity on certain strains adapted to the CF lung environment after years of colonization.

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Trp-Containing Antibacterial Peptides Impair Quorum Sensing and Biofilm Development in Multidrug-Resistant Pseudomonas aeruginosa and Exhibit Synergistic Effects With Antibiotics

Frontiers in Microbiology ◽

10.3389/fmicb.2021.611009 ◽

2021 ◽

Vol 12 ◽

Author(s):

Dejing Shang ◽

Xue Han ◽

Wanying Du ◽

Zhiru Kou ◽

Fengquan Jiang

Keyword(s):

Pseudomonas Aeruginosa ◽

Antibacterial Activity ◽

Quorum Sensing ◽

Virulence Factors ◽

Biofilm Formation ◽

Efflux Pump ◽

Synergistic Effects ◽

Multidrug Resistant ◽

Biofilm Development ◽

Antibiotic Efflux

Pseudomonas aeruginosa uses quorum sensing (QS) to control virulence, biofilm formation and antibiotic efflux pump expression. The development of effective small molecules targeting the QS system and biofilm formation represents a novel attractive strategy. In this present study, the effects of a series of Trp-containing peptides on the QS-regulated virulence and biofilm development of multidrug-resistant P. aeruginosa, as well as their synergistic antibacterial activity with three classes of traditional chemical antibiotics were investigated. The results showed that Trp-containing peptides at low concentrations reduced the production of QS-regulated virulence factors by downregulating the gene expression of both the las and rhl systems in the strain MRPA0108. Biofilm formation was inhibited in a concentration-dependent manner, which was associated with extracellular polysaccharide production inhibition by downregulating pelA, algD, and pslA transcription. These changes correlated with alterations in the extracellular production of pseudomonal virulence factors and swarming motility. In addition, the combination of Trp-containing peptides at low concentration with the antibiotics ceftazidime and piperacillin provided synergistic effects. Notably, L11W and L12W showed the highest synergy with ceftazidime and piperacillin. A mechanistic study demonstrated that the Trp-containing peptides, especially L12W, significantly decreased β-lactamase activity and expression of efflux pump genes OprM, MexX, and MexA, resulting in a reduction in antibiotic efflux from MRPA0108 cells and thus increasing the antibacterial activity of these antibiotics against MRPA0108.

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EVALUATION OF EFFLUX PUMP ACTIVITY AND BIOFILM FORMATION IN MULTIDRUG RESISTANT KLEBSIELLA PNEUMONIAE ISOLATES IN TANTA, EGYPT

International Research Journal of Pharmacy ◽

10.7897/2230-8407.1203123 ◽

2021 ◽

Vol 12 (3) ◽

pp. 1-5

Author(s):

Tarek El-Said El-Banna ◽

Fatma Ibrahim Sonbol ◽

Heba M El-Dawy ◽

Lamiaa A Al-Madboly

Keyword(s):

Drug Resistance ◽

Klebsiella Pneumoniae ◽

Biofilm Formation ◽

Efflux Pump ◽

Treatment Options ◽

Multidrug Resistant ◽

High Morbidity ◽

Biochemical Tests ◽

Antibiotic Resistance Profile ◽

Pump Activity

Nosocomial and community acquired infections that caused by multidrug-resistant (MDR) Klebsiella pneumoniae isolates are widespread recently resulting in high morbidity and mortality due to limited number of treatment options with effective antibiotics. The aim of this study is to evaluate the antibiotic resistance profile, biofilm formation and efflux pump activity of MDR K. pneumoniae isolates collected from different hospitals in Tanta, Egypt. A total of 70 K. pneumoniae isolates characterized by standard biochemical tests and confirmed by MALDI-TOF/MS were screened for antibiotic susceptibility, efflux pump activity and biofilm formation. Isolates displayed high resistance to penicillins, cephalosporins, trimethoprim-sulfamethoxazole and the majority of tested fluoro/-quinolones and decreased resistance to imipenem, amikacin, chloramphenicol, tigecycline and colistin. Out of 70 K. pneumoniae isolates, 2 isolates exhibited Pan Drug-Resistance (PDR) profile while 57 (81.4%) and 11 (15.7%) exhibited MDR and Extensively drug-resistance (XDR) profiles, respectively. Sixty-four (91.4%) isolates exhibited efflux pump activity while all tested isolates had the ability to form biofilm with varied degrees as 40 (57.1%), 26 (37.1%), and 4 (5.7%) isolates were strong, moderate and weak biofilm producers, respectively. Also, a strong relation between efflux pump activity and biofilm formation per isolate was detected. In conclusion, Multidrug resistance, biofilm formation and efflux pump capabilities in K. pneumoniae have serious public health implications in the management and control of infections caused by this bacterium. Therefore, a multifaceted approach and precise planning are recommended in controlling these infections

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