The Expression of Efflux Pump Genes in Methicillin-Resistant Staphylococcus aureus (MRSA) Strains Isolated from Blood Cultures in Iran

2020 ◽  
Vol 15 (5) ◽  
Author(s):  
Arezoo Bostanmaneshrad ◽  
Jamileh Norouzi ◽  
Gita Eslami ◽  
Ali Hashemi
Keyword(s):  
Staphylococcus Aureus ◽  
Efflux Pump ◽  
Resistance Mechanism ◽  
Expression Level ◽  
Resistance Pattern ◽  
Dilution Method ◽  
University Hospitals ◽  
Efflux Pump Inhibitor ◽  
Disk Diffusion Test ◽  
Methicillin Resistant

Background: Efflux pump is a significant resistance mechanism in Staphylococcus aureus. A total of 100 patients with bacteremia from Shahid Beheshti University Hospitals of Tehran in Iran were tested for the expression of efflux pump genes, contributing to S. aureus antimicrobial resistance. Objectives: this study was conducted to identify resistance pattern, and to evaluate the inhibitory effect of efflux pump, MIC of ciprofloxacin, and expression levels of norA, norB, and norC efflux pump genes in the presence of an efflux pump inhibitor against MDR S. aureus. Methods: A total of 100 MRSA isolates were investigated in different hospitals of Shahid Beheshti University of Medical Sciences from April 2017-2018. Owing to new consensus guidelines from the Clinical and Laboratory Standards Institute (CLSI), both the Kirby-Bauer disk diffusion test and micro-dilution method were used to evaluate antimicrobial susceptibility. Efflux pump activity using carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was identified as a chemical efflux pump inhibitor. E-test was used to determine Vancomycin-resistant antibiotic. Broth micro-dilution method for S. aureus isolates resistant to ciprofloxacin has been developed for minimum inhibitory concentration (MIC) of ciprofloxacin and CCCP and their composition. Quantitative Real-time Polymerase Chain Reaction (qRT-PCR) was used to investigate the expression level of norA, norB, and norC efflux pump genes. Results: A total of 38 of 45 MRSA isolates (84.4%) showed resistance to ciprofloxacin. Moreover, 100% of isolates had the norA and norB genes. Further, 95% of S. aureus isolates had the norC gene. According to this study, ciprofloxacin MIC has decreased by CCCP compared to ciprofloxacin. There was an increase in the expression level of norA, norB, and norC efflux pump genes in methicillin-resistant and ciprofloxacin-resistant S. aureus strains based on RT- PCR. In this study, four different spA types were obtained as the most prevalent type of spA by t037and t790 (23.3%) and t030 (14.1%) and t044 (12.2%). Conclusions: This study indicates that the prevalence of ciprofloxacin-resistant S. aureus strains has a rising trend among MRSA clinical isolates. The ability of S. aureus isolates to be converted into drug-resistant strains using efflux pump mechanism has become a widespread concern.

scholarly journals Efflux-Related Resistance to Norfloxacin, Dyes, and Biocides in Bloodstream Isolates of Staphylococcus aureus

2007 ◽  
Vol 51 (9) ◽  
pp. 3235-3239 ◽  
Author(s):  
Carmen E. DeMarco ◽  
Laurel A. Cushing ◽  
Emmanuel Frempong-Manso ◽  
Susan M. Seo ◽  
Tinevimbo A. A. Jaravaza ◽  
...  
Keyword(s):  
Gene Expression ◽  
Staphylococcus Aureus ◽  
Efflux Pump ◽  
Resistance Mechanism ◽  
Efflux Pump Inhibitor ◽  
Qrt Pcr ◽  
Reverse Transcription Pcr ◽  
Environmental Surfaces ◽  
Genes Encoding ◽  
High Level

ABSTRACT Efflux is an important resistance mechanism in Staphylococcus aureus, but its frequency in patients with bacteremia is unknown. Nonreplicate bloodstream isolates were collected over an 8-month period, and MICs of four common efflux pump substrates, with and without the broad-spectrum efflux pump inhibitor reserpine, were determined (n = 232). A reserpine-associated fourfold decrease in MIC was considered indicative of efflux. Strains exhibiting efflux of at least two of the four substrates were identified (“effluxing strains” [n = 114]). For these strains, MICs with or without reserpine for an array of typical substrates and the expression of mepA, mdeA, norA, norB, norC, and qacA/B were determined using quantitative real-time reverse transcription-PCR (qRT-PCR). A fourfold or greater increase in gene expression was considered significant. The most commonly effluxed substrates were ethidium bromide and chlorhexidine (100 and 96% of effluxing strains, respectively). qRT-PCR identified strains overexpressing mepA (5 [4.4%]), mdeA (13 [11.4%]), norA (26 [22.8%]), norB (29 [25.4%]), and norC (19 [16.7%]); 23 strains overexpressed two or more genes. Mutations probably associated with increased gene expression included a MepR-inactivating substitution and norA promoter region insertions or deletions. Mutations possibly associated with increased expression of the other analyzed genes were also observed. Effluxing strains comprised 49% of all strains studied (114/232 strains), with nearly half of these overexpressing genes encoding MepA, MdeA, and/or NorABC (54/114 strains). Reduced susceptibility to biocides may contribute to persistence on environmental surfaces, and efflux of drugs such as fluoroquinolones may predispose strains to high-level target-based resistance.

scholarly journals The Prevalence and the Resistance Profile of Methicillin-Resistant Staphylococcus aureus Colonized in the Nostrils of Cancer Patients in Namazi Teaching Hospital, Shiraz, Iran, 2011-12

2015 ◽  
Vol 4 (1) ◽  
pp. 21-25
Author(s):  
Mohammad Reza Kandehkar Ghahraman ◽  
Razyeh Hassanzadeh ◽  
Mohammad Motamedifar ◽  
Abdolsamad Ashrafzadeh ◽  
Zahra Hashemizadeh
Keyword(s):  
Staphylococcus Aureus ◽  
Cancer Patients ◽  
Antibiotic Susceptibility ◽  
Resistance Pattern ◽  
Susceptibility Pattern ◽  
In Vitro Susceptibility ◽  
Disk Diffusion Test ◽  
Vast Number ◽  
Methicillin Resistant

Background: Methicillin-resistant (MRSA) Staphylococcus aureus (S. aureus) are responsible for a vast number of nosocomial infections especially in immunocompromised subjects such as cancer patients. The presence of comorbidities including malignancies has been associated, with S. aureus bacteremia mortality. Thus, the detection of MRSA in this patients and the antibiotic susceptibility pattern of the isolates eases the selection of first-line medications and the prevention from further complications in cancer patients. The aim of this study was to determine the prevalence of MRSA infection and antibiotic susceptibility patterns of the isolates in pre and post-chemotherapy course in cancer patients.Materials and Methods: From May 2011 to July 2012, 200 nostril samples of cancerous patients were obtained and cultured on blood agar plates. After isolation and confirmation of S. aureus, antibiotic resistance pattern of the isolates was determined pre-chemotherapy and after the chemotherapy against vancomycin, tigecycline, linezolid, chloramphenicol, and oxacillin using disk diffusion test following CLSI guidelines. Chi-square test was used for data analysis.Results: Among a total number of 200 various cancer patients (64.5% females), 42 (21%) cases were positive for S. aureus and 7 (3.5 %) were MRSA carriers. Mean ages of MSSA and MRSA infected patients were 50.97±15.94 and 53.57±18.28 years old, respectively. In vitro susceptibility pattern of the MRSA and MSSA isolates to the 4 tested agents did not differed significantly after the chemotherapy in contrast with pre-chemotherapy state.Conclusions: This study showed that chemotherapy does not change the susceptibility pattern of MRSA species to antibiotics of choice in cancer patients. However, the importance of controlling methicillin resistant staphylococcal infections in critical cases, specifically cancer cases, necessitates the early detection, further investigations on more effective medications.

scholarly journals Overexpression of the adeB Efflux Pump Gene in Tigecycline-Resistant Acinetobacter baumannii Clinical Isolates and Its Inhibition by (+)Usnic Acid as an Adjuvant

Antibiotics ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 1037
Author(s):  
Nagaraju Bankan ◽  
Fathimunnisa Koka ◽  
Rajagopalan Vijayaraghavan ◽  
Sreekanth Reddy Basireddy ◽  
Selvaraj Jayaraman
Keyword(s):  
Acinetobacter Baumannii ◽  
Efflux Pump ◽  
Resistance Mechanism ◽  
Usnic Acid ◽  
Pcr Analysis ◽  
Dilution Method ◽  
High Morbidity ◽  
Efflux Pump Inhibitor ◽  
Acinetobacter Species ◽  
Pump Activity

Acinetobacter species are among the most life-threatening Gram-negative bacilli, causing hospital-acquired infections, and they are associated with high morbidity and mortality. They show multidrug resistance that acts via various mechanisms. In Acinetobacter baumannii, efflux pump-mediated resistance to many antimicrobial compounds, including tigecycline, has been widely reported. Natural compounds have been used for their various pharmacological properties, including anti-efflux pump activity. The present study aimed to evaluate the efflux pump-mediated resistance mechanism of Acinetobacter baumannii and the effect of (+)Usnic acid as an efflux pump inhibitor with tigecycline. For detecting the efflux pump activity of tigecycline-resistant Acinetobacter baumannii isolates, microbroth dilution method and real-time quantitative reverse transcription–polymerase chain reaction was used. (+)Usnic acid was added to tigecycline and tested by the checkerboard method to evaluate its efficacy as an efflux pump inhibitor. qRT-PCR analysis was carried out to show the downregulation of the efflux pump in the isolates. Out of 42 tigecycline-resistant Acinetobacter baumannii isolates, 19 showed efflux pump activity. All 19 strains expressed the adeB gene. (+)Usnic acid as an adjuvant showed better efficacy in lowering the minimum inhibitory concentration compared with the conventional efflux pump inhibitor, carbonyl cyanide phenylhydrazone.

Prevalence of vanA Gene among Methicillin Resistant S. aureus Strains Isolated from Burn Wound Infections in Menoufia University Hospitals

2020 ◽  
Vol 29 (3) ◽  
pp. 97-104
Author(s):  
Amira H. Elkhyat ◽  
Amal F. Makled ◽  
Ahmed M. Albeltagy ◽  
Tarek F. Keshk ◽  
Amal M. Dawoud
Keyword(s):  
Staphylococcus Aureus ◽  
Vancomycin Resistance ◽  
Control Measures ◽  
Diffusion Method ◽  
Clinical Samples ◽  
Dilution Method ◽  
University Hospitals ◽  
Burn Wound ◽  
Methicillin Resistant ◽  
Vana Gene

Background: Staphylococcus aureus is a leading cause of burn wound infection. Vancomycin resistance among methicillin-resistant Staphylococcus aureus (MRSA) is becoming a worldwide growing threat. Objectives: to detect the prevalence of MRSA in burn patients and its antibiotic susceptibility patterns. In addition, the resistance patterns of MRSA to vancomycin and the prevalence of vanA gene among MRSA isolates were investigated. Methodology: A total 250 clinical samples were obtained from patients admitted to Burn Unit in Menoufia University Hospitals. Identification and antimicrobial susceptibility testing of S. aureus isolates were performed. Cefoxitin disk diffusion method was used to identify MRSA strains. Vancomycin resistance was determined by agar dilution method. Detection of mecA and vanA genes by multiplex PCR was done. Results: Staphylococcus aureus represented 43.3% of all isolates. By cefoxitin disc diffusion method, 94% (79/84) of isolated S. aureus were MRSA that showed a high resistance to most antimicrobials used with rates ranged from 40.5 % to 100%. Phenotypically among MRSA isolates, vancomycin sensitive S. aureus (VSSA), vancomycin-intermediate S. aureus (VISA) and vancomycin-resistant S. aureus (VRSA) were 59.5%, 15.2%, 25.3% respectively. Among MRSA isolates, 17 (21.5%) isolates had vanA gene by PCR (16 isolates were VRSA and one isolate was VSSA).Conclusion: This study is considered as an alarm demonstrating that implementation of proper infection control measures is mandatory to control spread of such resistant strains in our hospital.

scholarly journals Mutation-Based Antibiotic Resistance Mechanism in Methicillin-Resistant Staphylococcus aureus Clinical Isolates

2021 ◽  
Vol 14 (5) ◽  
pp. 420
Author(s):  
Tanveer Ali ◽  
Abdul Basit ◽  
Asad Mustafa Karim ◽  
Jung-Hun Lee ◽  
Jeong-Ho Jeon ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Antibiotic Resistance ◽  
Active Site ◽  
Methicillin Resistant Staphylococcus Aureus ◽  
Meca Gene ◽  
Resistance Mechanism ◽  
Ligand Docking ◽  
Clinical Samples ◽  
Allosteric Site ◽  
Methicillin Resistant

β-Lactam antibiotics target penicillin-binding proteins and inhibit the synthesis of peptidoglycan, a crucial step in cell wall biosynthesis. Staphylococcus aureus acquires resistance against β-lactam antibiotics by producing a penicillin-binding protein 2a (PBP2a), encoded by the mecA gene. PBP2a participates in peptidoglycan biosynthesis and exhibits a poor affinity towards β-lactam antibiotics. The current study was performed to determine the diversity and the role of missense mutations of PBP2a in the antibiotic resistance mechanism. The methicillin-resistant Staphylococcus aureus (MRSA) isolates from clinical samples were identified using phenotypic and genotypic techniques. The highest frequency (60%, 18 out of 30) of MRSA was observed in wound specimens. Sequence variation analysis of the mecA gene showed four amino acid substitutions (i.e., E239K, E239R, G246E, and E447K). The E239R mutation was found to be novel. The protein-ligand docking results showed that the E239R mutation in the allosteric site of PBP2a induces conformational changes in the active site and, thus, hinders its interaction with cefoxitin. Therefore, the present report indicates that mutation in the allosteric site of PBP2a provides a more closed active site conformation than wide-type PBP2a and then causes the high-level resistance to cefoxitin.

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 Staphylococcus aureus Mutants Isolated via Exposure to Nonfluorinated Quinolones: Detection of Known and Unique Mutations

2001 ◽  
Vol 45 (12) ◽  
pp. 3422-3426 ◽  
Author(s):  
Siddhartha Roychoudhury ◽  
Tracy L. Twinem ◽  
Kelly M. Makin ◽  
Mark A. Nienaber ◽  
Chuiying Li ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Sequence Analysis ◽  
Efflux Pump ◽  
Serial Passage ◽  
Efflux Pump Inhibitor ◽  
In Vitro Development ◽  
Development Of Resistance ◽  
High Level ◽  
Vitro Development

ABSTRACT The in vitro development of resistance to the new nonfluorinated quinolones (NFQs; PGE 9262932, PGE 4175997, and PGE 9509924) was investigated in Staphylococcus aureus. At concentrations two times the MIC, step 1 mutants were isolated more frequently with ciprofloxacin and trovafloxacin (9.1 × 10−8 and 5.7 × 10−9, respectively) than with the NFQs, gatifloxacin, or clinafloxacin (<5.7 × 10−10). Step 2 and step 3 mutants were selected via exposure of a step 1 mutant (selected with trovafloxacin) to four times the MICs of trovafloxacin and PGE 9262932. The step 1 mutant contained the known Ser80-Phe mutation in GrlA, and the step 2 and step 3 mutants contained the known Ser80-Phe and Ser84-Leu mutations in GrlA and GyrA, respectively. Compared to ciprofloxacin, the NFQs were 8-fold more potent against the parent and 16- to 128-fold more potent against the step 3 mutants. Mutants with high-level NFQ resistance (MIC, 32 μg/ml) were isolated by the spiral plater-based serial passage technique. DNA sequence analysis of three such mutants revealed the following mutations: (i) Ser84-Leu in GyrA and Glu84-Lys and His103-Tyr in GrlA; (ii) Ser-84Leu in GyrA, Ser52-Arg in GrlA, and Glu472-Val in GrlB; and (iii) Ser84-Leu in GyrA, Glu477-Val in GyrB, and Glu84-Lys and His103-Tyr in GrlA. Addition of the efflux pump inhibitor reserpine (10 μg/ml) resulted in 4- to 16-fold increases in the potencies of the NFQs against these mutants, whereas it resulted in 2-fold increases in the potencies of the NFQs against the parent.

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