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574800.9835Nosocomial Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus: Sensitivity to Chlorhexidine-Based Biocides and Prevalence of Efflux Pump Genes. The widespread use of disinfectants and antiseptics has led to the emergence of nosocomial pathogens that are less sensitive to these agents, which in combination with multidrug resistance (MDR) can pose a significant epidemiologic risk. We investigated the susceptibility of nosocomial Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus to a 0.05% chlorhexidine (CHX) solution and a biocidal S7 composite solution based on CHX (0.07%) and benzalkonium chloride (BAC, 0.055%). The prevalence of efflux pump genes associated with biocide resistance and their relationship to antibiotic resistance was also determined. Both biocides were more effective against Gram-positive S. aureus than Gram-negative bacteria. The most resistant strains were P. aeruginosa strains, which were mainly killed by 0.0016% CHX and by 0.0000084% (CHX)/0.0000066% (BAC) S7. The S7 bactericidal effect was observed on P. aeruginosa and S. aureus after 10 min, while the bactericidal effect of CHX was only observed after 30 min. qacEΔ1 and qacE efflux pump genes were prevalent among E. coli and K. pneumoniae, while mexB was more often detected in P. aeruginosa. norA, norB, mepA, mdeA, and sepA were prevalent in S. aureus. The observed prevalence of efflux pump genes highlights the potential problem whereby the sensitivity of bacteria to biocides could decline rapidly in the future.202539796210
233710.9833Klebsiella pneumoniae susceptibility to biocides and its association with cepA, qacΔE and qacE efflux pump genes and antibiotic resistance. BACKGROUND: Although antiseptics are some of the most widely used antibacterials in hospitals, there is very little information on reduced susceptibility to these biocides and its relationship with resistance to antibiotics. AIM: To determine the relationship between reduced susceptibility to biocides and the carriage of antiseptic resistance genes, cepA, qacΔE and qacE, as well as identifying the role of efflux pumps in conferring reduced susceptibility. METHODS: Susceptibility was assessed for five biocides: chlorhexidine, benzalkonium chloride, Trigene, MediHex-4, Mediscrub; and for 11 antibiotics against 64 isolates of Klebsiella pneumoniae. Susceptibility to all compounds was tested by the agar double dilution method (DDM) and the effect of efflux pumps on biocides determined by repeating the susceptibility studies in the presence of the efflux pump inhibitor carbonyl cyanide m-chlorophenyl hydrazone (CCCP). The presence of the cepA, qacΔE and qacE genes was identified by polymerase chain reaction. FINDINGS: The bacteria were not widely antibiotic resistant though a few showed reduced susceptibility to cefoxitin, chloramphenicol and rifampicin and later-generation cephalosporins but not to carbapenems. Biocide susceptibility, tested by DDM, showed that 50, 49 and 53 strains had reduced susceptibility to chlorhexidine, Trigene and benzalkonium chloride, respectively. The antiseptic resistance genes cepA, qacΔE and qacE were found in 56, 34 and one isolates respectively and their effects as efflux pumps were determined by CCCP (10 mg/L), which decreased the minimum inhibitory concentrations (MICs) of chlorhexidine and Medihex-4 by 2-128-fold but had no impact on the MICs of benzalkonium chloride, Trigene and Mediscrub. CONCLUSION: There was a close link between carriage of efflux pump genes, cepA, qacΔE and qacE genes and reduced biocide susceptibility, but not antibiotic resistance, in K. pneumoniae clinical isolates.201222498639
637120.9832Bioactive compounds from the African medicinal plant Cleistochlamys kirkii as resistance modifiers in bacteria. Cleistochlamys kirkii (Benth) Oliv. (Annonaceae) is a medicinal plant traditionally used in Mozambique to treat infectious diseases. The aim of this study was to find resistance modifiers in C. kirkii for Gram-positive and Gram-negative model bacterial strains. One of the most important resistance mechanisms in bacteria is the efflux pump-related multidrug resistance. Therefore, polycarpol (1), three C-benzylated flavanones (2-4), and acetylmelodorinol (5) were evaluated for their multidrug resistance-reverting activity on methicillin-susceptible and methicillin-resistant Staphylococcus aureus and Escherichia coli AG100 and AG100 A strains overexpressing and lacking the AcrAB-TolC efflux pump system. The combined effects of antibiotics and compounds (2 and 4) were also assessed by using the checkerboard microdilution method in both S. aureus strains. The relative gene expression of the efflux pump genes was determined by real-time reverse transcriptase quantitative polymerase chain reaction. The inhibition of quorum sensing was also investigated. The combined effect of the antibiotics and compound 2 or 4 on the methicillin-sensitive S. aureus resulted in synergism. The most active compounds 2 and 4 increased the expression of the efflux pump genes. These results suggested that C. kirkii constituents could be effective adjuvants in the antibiotic treatment of infections.201829464798
574930.9830Antibiotic resistance as an indicator of bacterial chlorhexidine susceptibility. The antibiotic and chlorhexidine (CHX) susceptibility of 70 distinct clinical isolates: Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Staphylococcus aureus (not MRSA), Streptococcus pyogenes and Enterococcus faecalis (10 of each) were tested using minimal bactericidal (MBC) and/or minimal inhibitory (MIC) concentrations. Non-fermentative bacteria tolerated CHX at high concentrations; Gram-positive cocci, especially S. pyogenes, were the most susceptible. We found a good correlation between CHX and antibiotic susceptibility in both MIC and MBC among Gram-negative bacteria, and mainly in MBC among Gram-positive bacteria. Resistance to ciprofloxacin, imipenem, cefotaxime, ceftazidime, gentamicin and aztreonam appeared to indicate increased CHX resistance among Gram-negative bacteria. This finding gives clinicians the ability to predict CHX susceptibility according to routine antibiotic resistance testing.200212090797
637240.9828Sensitizing multi drug resistant Staphylococcus aureus isolated from surgical site infections to antimicrobials by efflux pump inhibitors. BACKGROUND: Staphylococcus aureus is a common hospital acquired infections pathogen. Multidrug-resistant Methicillin-resistant Staphylococcus aureus represents a major problem in Egyptian hospitals. The over-expression of efflux pumps is a main cause of multidrug resistance. The discovery of efflux pump inhibitors may help fight multidrug resistance by sensitizing bacteria to antibiotics. This study aimed to investigate the role of efflux pumps in multidrug resistance. METHODS: Twenty multidrug resistant S. aureus isolates were selected. Efflux pumps were screened by ethidium bromide agar cartwheel method and polymerase chain reaction. The efflux pump inhibition by seven agents was tested by ethidium bromide agar cartwheel method and the effect on sensitivity to selected antimicrobials was investigated by broth microdilution method. RESULTS: Seventy percent of isolates showed strong efflux activity, while 30% showed intermediate activity. The efflux genes mdeA, norB, norC, norA and sepA were found to play the major role in efflux, while genes mepA, smr and qacA/B had a minor role. Verapamil and metformin showed significant efflux inhibition and increased the sensitivity to tested antimicrobials, while vildagliptin, atorvastatin, domperidone, mebeverine and nifuroxazide showed no effect. CONCLUSION: Efflux pumps are involved in multidrug resistance in Staphylococcus aureus. Efflux pump inhibitors could increase the sensitivity to antimicrobials.202034394224
233850.9825Characterization of disinfectant susceptibility profiles among clinical isolates of Acinetobacter baumannii in Ardabil, Iran. Antimicrobial disinfectants have been extensively used to control hospital-acquired infections worldwide. Prolonged exposure to bacteria could promote resistance to antimicrobial disinfectants. This study evaluated the antimicrobial activity of four commonly used disinfectants; triclosan, chlorhexidine digluconate, benzalkonium chloride, and formaldehyde against Acinetobacter baumannii clinical isolates. This study also determined the prevalence and association of efflux pumps encoding genes qacE, qacED1, emrA, and aceI with tolerance to disinfectants. A total of 100 A. baumannii isolates were included in the current study. The antimicrobial disinfectants' minimum inhibitory concentration (MIC) was determined using an agar dilution method. Genes involved in resistance to disinfectants were investigated by PCR method. The benzalkonium chloride MICs ranged between 32 and 128 μg mL-1, chlorhexidine digluconate 8-64 μg mL-1, triclosan 1-32 μg mL-1, and formaldehyde 128 μg mL-1. Overall, the highest MIC90 value was identified for formaldehyde (128 μg mL-1), followed by benzalkonium chloride and chlorhexidine digluconate (64 μg mL-1, each one) and triclosan (4 μg mL-1). In the present study, the qacE, qacED1, emrA, and aceI genes were found in 91%, 55%, 100%, and 88% of isolates, respectively. The qacG gene was not identified in our A. baumannii isolates. The qacED1 gene was associated with higher MICs for all disinfectants tested (P < 0.05), while the qacE and aceI genes were associated with higher MICs for benzalkonium chloride and chlorhexidine. This study indicated that triclosan is the most effective disinfectant against A. baumannii isolates.202338063878
245960.9821In vitro antimicrobial activity and resistance mechanisms of cefiderocol against clinical carbapenem-resistant gram-negative bacteria. BACKGROUND: The rise of carbapenem-resistant gram-negative bacteria (CRGNB) necessitates new therapeutic options such as cefiderocol. OBJECTIVE: To evaluate the in vitro efficacy of cefiderocol against clinical CRGNB and investigate associated resistance mechanisms. METHODS: A total of 370 CRGNB isolates were analyzed. Minimum inhibitory concentration (MIC) values were determined, and whole genome sequencing, efflux pump inhibition assays, and RT-qPCR were conducted to assess resistance-related mutations, gene loss, and expression changes. RESULTS: Cefiderocol demonstrated potent in vitro activity, with high susceptibility rates in C. freundii (100%), K. pneumoniae (93.3%), and E. hormaechei (92.2%), and notable activity against P. aeruginosa (80.0%) and Escherichia coli (76.8%). Efflux pump inhibition by Carbonyl Cyanide m-Chlorophenyl Hydrazone (CCCP) significantly reduced MICs in resistant strains. Key resistance mechanisms included β-lactamase gene variants (bla (OXA-66), bla (OXA-23), bla (SHV-12)), mutations in envZ, cirA, nuoC, ampC, and loss or altered expression of iron transporter genes (piuA, pirA, fepA). CONCLUSION: Cefiderocol is highly effective against CRGNB; however, resistance may arise through diverse mechanisms, including efflux pump activity. Continued surveillance of emerging resistance is essential to guide its optimal clinical use.202541113641
233970.9818Phenotypic ESBL and non-phenotypic ESBL isolates of Klebsiella pneumoniae exhibit differing responses to induced antimicrobials resistance and subsequent antibiotic cross-resistance. AIM: To investigate the effect of adapting Klebsiella pneumoniae clinical isolates harboring ESBL genes to cetrimide (CT) in terms of subsequent cross-resistance to other biocides and antibiotics, and to investigate changes to virulence markers, such as biofilm formation and efflux activity. The changes between phenotypic extended spectrum β-lactamases (pESBL) expressing isolates and non-phenotypic ESBL (npESBL) isolates was compared. METHODS AND RESULTS: Kl. pneumoniae isolates (14 pESBL and 17 npESBL) were adapted to increasing concentrations of CT, until 4 × MIC was reached. The MIC of the adapted isolates was tested against chloroxylenol (CX) and chlorhexidine. Disk diffusion techniques were used to determine the susceptibility of the isolates to different antibiotics. Biofilm formation was assessed for the isolates using the crystal violet method and efflux pump activity was studied using the ethidium bromide assay. After CT adaptation, 100% of npESBL isolates and 85.7% of pESBL isolates showed increase in CT MIC after CT adaptation. While 41.2% of npESBL and 57.1% of the pESBL isolates showed a cross-resistance with chlorhexidine. CT adaptation resulted in a significant decrease in the susceptibility of npESBL isolates to aztreonam and cefotaxime compared to pESBL isolates, which could be linked to the increase in efflux activity of npESBL compared to pESBL. Biofilm formation was significantly increased after CT adaptation regardless of the type of isolate. CONCLUSIONS: The extensive use of biocides in the environment can induce cross-resistance to other biocides and antibiotics, and can increase the ability of bacteria to form biofilms. The response of bacteria to biocide adaptation differs between pESBL and npESBL isolates, although the effect is strain specific.202336724268
229880.9817Burden of biocide resistance among multidrug-resistant bacteria isolated from various clinical specimens in a tertiary care hospital. BACKGROUND: Most studies on biocide resistance and its genetic determinants arise from environmental or food-borne microbial isolates and only a few from clinically relevant isolates. OBJECTIVES: This study determines the proportion of biocide resistance against five commonly used biocides and detects biocide resistance genes among MDR bacterial isolates using PCR. METHODS: Consecutive MDR isolates (n ​= ​180) were included (30 each of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Staphylococcus aureus, and Enterococcus species) from clinical specimens of various inpatient units at JIPMER. The isolates were challenged at 0.5,1 and 2 Macfarland (McF) inoculum with discrete dilutions of disinfectants. The minimum bactericidal concentrations (MBCs) for 70% Ethanol, 1.5% Cresol, 2% Glutaraldehyde, 1% Cetrimide, and 1% Chlorhexidine were determined for the isolates using ATCC reference strains as controls. PCR was performed targeting qac A/B, G; smr; and nfx B genes. RESULTS: For all biocides, MDR isolates had MBCs less than the maximum MBCs of ATCC strains. For MDR K. pneumoniae, A. baumannii, and P. aeruginosa, the highest MBCs of chlorhexidine and cetrimide were ≥75 and ​≥ ​150 ​μg/ml respectively at 0.5 McF inoculum; whereas these organisms grew at higher inoculum (2McF) even at commercially recommended biocidal concentration (1%) corresponding to 750 and 1500 ​μg/ml of chlorhexidine and cetrimide respectively. Meanwhile, the highest MBCs of MDR E. coli were 75 and 150 ​μg/ml for chlorhexidine and cetrimide respectively. Interestingly, the Gram-positive cocci survived the action of up to 35% ethanol. The nfxB and qacG genes were detected in 87% and 6.67% of MDR P. aeruginosa isolates respectively with no biocide resistance genes detected among the other organisms. CONCLUSIONS: Biocide dilutions challenged with higher inoculum indicated a narrow margin of effectiveness for certain biocides. Although a significant proportion of clinical MDR isolates of P. aeruginosa harbored biocide resistance genes, this finding had no phenotypic correlation.202337769586
247890.9817Study on the resistance mechanism via outer membrane protein OprD2 and metal β-lactamase expression in the cell wall of Pseudomonas aeruginosa. The aim of the present study was to evaluate the imipenem-resistant mechanism via the outer membrane protein (OMP) OprD2 and metal β-lactamase expression in the cell wall of Pseudomonas aeruginosa. The Pseudomonas aeruginosa was clinically separated and validated by VITEK-2 full-automatic bacteria analyzer. Drug resistance, sensitive antibiotics and minimum inhibitory concentration (MIC) were tested using the drug sensitivity analysis system. The phenotype positive strains of MBL genes were screened using the Kirby-Bauer diffusion method by adding metal ion-chelating agent EDTA on the imipenem susceptibility paper. IMP-1, VIM-1 and SPM metaloenzyme genes were tested by polymerase chain reaction (PCR)-telomeric repeat amplification protocol (TRAP). The OMP OprD2 genes were tested by PCR-TRAP, and the protein expression was tested using western blot analysis. The location of OMP OprD2 was confirmed using the sodium salicylate inhibition test. The results showed that 80 portions (40%) of MBL-positive strains were screened out of 200 specimens. Imipenem-resistant Pseudomonas aeruginosa (IRPA) and MIC values were significantly higher than quality control bacteria and control bacteria (P<0.05). A total of 35 cases with IMP-1 positive, 20 with VIM-1 positive, 16 with SPM positive, 5 with 2 positive genes and 4 with 3 positive genes were screened among MBL positive strains. A total of 150 portions (75%) of OprD2 deficiencies were screened from 200 specimens. The standard strains and sensitive strains showed OprD2 protein bands at 45 kDa while no OprD2 protein bands appeared in OprD2 deficiency strains. It was in accordance with gene detection. In conclusion, OMP OprD2 deficiency and MBL phenotype positivity may be important mechanisms of IRPA.201627882088
5375100.9816Mechanism of Eravacycline Resistance in Clinical Enterococcus faecalis Isolates From China. Opportunistic infections caused by multidrug-resistant Enterococcus faecalis strains are a significant clinical challenge. Eravacycline (Erava) is a synthetic fluorocycline structurally similar to tigecycline (Tige) that exhibits robust antimicrobial activity against Gram-positive bacteria. This study investigated the in vitro antimicrobial activity and heteroresistance risk of Eravacycline (Erava) in clinical E. faecalis isolates from China along with the mechanism of Erava resistance. A total of 276 non-duplicate E. faecalis isolates were retrospectively collected from a tertiary care hospital in China. Heteroresistance to Erava and the influence of tetracycline (Tet) resistance genes on Erava susceptibility were examined. To clarify the molecular basis for Erava resistance, E. faecalis variants exhibiting Erava-induced resistance were selected under Erava pressure. The relative transcript levels of six candidate genes linked to Erava susceptibility were determined by quantitative reverse-transcription PCR, and their role in Erava resistance and heteroresistance was evaluated by in vitro overexpression experiments. We found that Erava minimum inhibitory concentrations (MICs) against clinical E. faecalis isolates ranged from ≤0.015 to 0.25 mg/l even in strains harboring Tet resistance genes. The detection frequency of Erava heteroresistance in isolates with MICs ≤ 0.06, 0.125, and 0.25 mg/l were 0.43% (1/231), 7.5% (3/40), and 0 (0/5), respectively. No mutations were detected in the 30S ribosomal subunit gene in Erava heteroresistance-derived clones, although mutations in this subunit conferred cross resistance to Tige in Erava-induced resistant E. faecalis. Overexpressing RS00630 (encoding a bone morphogenetic protein family ATP-binding cassette transporter substrate-binding protein) in E. faecalis increased the frequency of Erava and Tige heteroresistance, whereas RS12140, RS06145, and RS06880 overexpression conferred heteroresistance to Tige only. These results indicate that Erava has potent in vitro antimicrobial activity against clinical E. faecalis isolates from China and that Erava heteroresistance can be induced by RS00630 overexpression.202032523563
5380110.9814In Vitro Screening of a 1280 FDA-Approved Drugs Library against Multidrug-Resistant and Extensively Drug-Resistant Bacteria. Alternative strategies against multidrug-resistant (MDR) bacterial infections are suggested to clinicians, such as drug repurposing, which uses rapidly available and marketed drugs. We gathered a collection of MDR bacteria from our hospital and performed a phenotypic high-throughput screening with a 1280 FDA-approved drug library. We used two Gram positive (Enterococcus faecium P5014 and Staphylococcus aureus P1943) and six Gram negative (Acinetobacter baumannii P1887, Klebsiella pneumoniae P9495, Pseudomonas aeruginosa P6540, Burkholderia multivorans P6539, Pandoraea nosoerga P8103, and Escherichia coli DSM105182 as the reference and control strain). The selected MDR strain panel carried resistance genes or displayed phenotypic resistance to last-line therapies such as carbapenems, vancomycin, or colistin. A total of 107 compounds from nine therapeutic classes inhibited >90% of the growth of the selected Gram negative and Gram positive bacteria at a drug concentration set at 10 µmol/L, and 7.5% were anticancer drugs. The common hit was the antiseptic chlorhexidine. The activity of niclosamide, carmofur, and auranofin was found against the selected methicillin-resistant S. aureus. Zidovudine was effective against colistin-resistant E. coli and carbapenem-resistant K. pneumoniae. Trifluridine, an antiviral, was effective against E. faecium. Deferoxamine mesylate inhibited the growth of XDR P. nosoerga. Drug repurposing by an in vitro screening of a drug library is a promising approach to identify effective drugs for specific bacteria.202235326755
6359120.9813Drug resistance of oral bacteria to new antibacterial dental monomer dimethylaminohexadecyl methacrylate. Only two reports exist on drug-resistance of quaternary ammonium monomers against oral bacteria; both studies tested planktonic bacteria for 10 passages, and neither study tested biofilms or resins. The objectives of this study were to investigate the drug-resistance of Streptococcus mutans, Streptococcus sanguinis and Streptococcus gordonii against dimethylaminohexadecyl methacrylate (DMAHDM), and to evaluate biofilms on resins with repeated exposures for 20 passages for the first time. DMAHDM, dimethylaminododecyl methacrylate (DMADDM) and chlorhexidine (CHX) were tested with planktonic bacteria. Biofilms were grown on a resin containing 3% DMAHDM. Minimum-inhibitory concentrations were measured. To detect drug-resistance, the survived bacteria from the previous passage were used as inoculum for the next passage for repeated exposures. S. gordonii developed drug-resistance against DMADDM and CHX, but not against DMAHDM. Biofilm colony-forming units (CFU) on DMAHDM-resin was reduced by 3-4 log; there was no difference from passages 1 to 20 (p > 0.1). No drug-resistance to DMAHDM was detected for all three bacterial species. In conclusion, this study showed that DMAHDM induced no drug-resistance, and DMAHDM-resin reduced biofilm CFU by 3-4 log, with no significant change from 1 to 20 passages. DMAHDM with potent antibacterial activities and no drug-resistance is promising for dental applications.201829615732
2299130.9812Determining the resistance of carbapenem-resistant Klebsiella pneumoniae to common disinfectants and elucidating the underlying resistance mechanisms. INTRODUCTION: Carbapenem-resistant Klebsiella pneumoniae (CRKP) infection is a serious problem in hospitals worldwide, posing a particular risk to immunocompromised patients. Elimination strategies may prevent these drug-resistant bacteria from spreading within hospital environments. Here, the susceptibility of patient-derived CRKP strains to common chemical disinfectants and possible correlations between the presence of drug-resistance genes and increased resistance to disinfectants were investigated. METHODS: The minimum inhibitory (MIC) and the minimum bactericidal concentrations (MBC) of common chemical disinfectants against each CRKP strain were determined using agar dilution; K. pneumoniae ATCC700603 served as a standard. The presence of the drug-resistance genes qacΔE, qacA, acrA and qacE was determined using PCR. RESULTS: A total of 27 clinically isolated CRKP strains collected in our hospital from 2011 to 2013 exhibited sensitivity to the following common chemical disinfectants in decreasing order of sensitivity: 75% ethyl alcohol > 2% glutaraldehyde > "84" disinfectant > 0.2% benzalkonium bromide > 2% iodine tincture > 1% iodophor > 0.1% chlorhexidine acetate. Of the 27 strains, 59, 41, 19 and 15% contained qacΔE, qacA, acrA and qacE resistance genes; 15% carried acrA, qacΔE and qacA, and 26% carried both qacA and qacΔE. Comparative analysis indicated that drug-resistance genes were correlated with higher MIC values. CONCLUSION: These pan-resistant pathogenic CRKP strains contained various drug-resistance genes and exhibited relatively high resistance to ethyl alcohol, chlorhexidine acetate and iodophor. Monitoring the drug-resistance rates of CRKP strains displaying disinfectant resistance may facilitate appropriate and effective sterilisation and thus preventing the spread of these pan-resistant strains.201526184804
6190140.9810Identifying Escherichia coli genes involved in intrinsic multidrug resistance. Multidrug resistance is a major cause of clinical failure in treating bacterial infections. Increasing evidence suggests that bacteria can resist multiple antibiotics through intrinsic mechanisms that rely on gene products such as efflux pumps that expel antibiotics and special membrane proteins that block the penetration of drug molecules. In this study, Escherichia coli was used as a model system to explore the genetic basis of intrinsic multidrug resistance. A random mutant library was constructed in E. coli EC100 using transposon mutagenesis. The library was screened by growth measurement to identify the mutants with enhanced or reduced resistance to chloramphenicol (Cm). Out of the 4,000 mutants screened, six mutants were found to be more sensitive to Cm and seven were more resistant compared to the wild-type EC100. Mutations in 12 out of the 13 mutants were identified by inverse polymerase chain reaction. Mutants of the genes rob, garP, bipA, insK, and yhhX were more sensitive to Cm compared to the wild-type EC100, while the mutation of rhaB, yejM, dsdX, nagA, yccE, atpF, or htrB led to higher resistance. Overexpression of rob was found to increase the resistance of E. coli biofilms to tobramycin (Tob) by 2.7-fold, while overexpression of nagA, rhaB, and yccE significantly enhanced the susceptibility of biofilms by 2.2-, 2.5-, and 2.1-fold respectively.200818807027
5755150.9810Effects of Efflux Pump Inhibitors on Colistin Resistance in Multidrug-Resistant Gram-Negative Bacteria. We tested the effects of various putative efflux pump inhibitors on colistin resistance in multidrug-resistant Gram-negative bacteria. Addition of 10 mg/liter cyanide 3-chlorophenylhydrazone (CCCP) to the test medium could significantly decrease the MICs of colistin-resistant strains. Time-kill assays showed CCCP could reverse colistin resistance and inhibit the regrowth of the resistant subpopulation, especially in Acinetobacter baumannii and Stenotrophomonas maltophilia These results suggest colistin resistance in Gram-negative bacteria can be suppressed and reversed by CCCP.201626953203
2287160.9810Expression of norA, norB and norC efflux pump genes mediating fluoroquinolones resistance in MRSA isolates. INTRODUCTION: Although fluoroquinolones are used to treat methicillin-resistant Staphylococcus aureus (MRSA)-induced infections, acquisition of antibiotic resistance by bacteria has impaired their clinical relevance. We aimed to evaluate the frequency of norA, norB, and norC efflux pump genes-mediating fluoroquinolones resistance and measure their expression levels in MRSA isolates. METHODOLOGY: 126 S. aureus isolates were collected from different clinical samples of adult hospitalized patients and identified by conventional microbiological methods. MRSA was diagnosed by cefoxitin disc diffusion method and minimum inhibitory concentration (MIC) of ciprofloxacin by broth microdilution method. The expression levels of efflux pump genes were measured by quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: 80 (63.5%) MRSA isolates were identified and showed high level of resistance to erythromycin (80%), gentamicin (75%), clindamycin (65%) and ciprofloxacin (60 %). norA, norB and norC were detected in 75%, 35% and 55% of the MRSA isolates respectively. norC was the most commonly overexpressed gene measured by qRT-PCR, occurring in 40% of MRSA isolates, followed by norA (35%) and norB (30%). The expression of these genes was significantly higher in ciprofloxacin-resistant than quantitative real-time PCR ciprofloxacin-sensitive MRSA isolates. CONCLUSIONS: This study showed high prevalence and overexpression of efflux pump genes among MRSA isolates which indicates the significant role of these genes in the development of multidrug resistance against antibiotics including fluoroquinolones.202438635612
2283170.9809Association of qacE and qacEDelta1 with multiple resistance to antibiotics and antiseptics in clinical isolates of Gram-negative bacteria. Clinical isolates of Enterobacter cloacae, Citrobacter freundii, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia were tested for resistance to antibiotics and to the antiseptics benzalkonium chloride and cetyltrimethylammonium bromide. Furthermore, they were examined for the presence of the resistance genes qacE and qacEDelta1. qacEDelta1 was detected by PCR in 10% of all (n=103) and in 81% of multiply antibiotic-resistant strains (n=15). qacE was found in only one of 37 P. aeruginosa strains. The minimum inhibitory concentrations of benzalkonium chloride, cetyltrimethylammonium bromide, and ethidium bromide were not significantly different for qacEDelta1/qacE-positive or -negative strains. Our data indicate that multiply antibiotic-resistant Gram-negative bacteria are not necessarily more resistant to quaternary ammonium compounds than antibiotic-sensitive strains even though qacE or qacEDelta1 is present.200010650208
2285180.9809Efflux genes and active efflux activity detection in Malaysian clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA). Efflux-mediated resistance has been recognized as an important contributor of antibiotic resistance in bacteria, especially in methicillin-resistant Staphylococcus aureus (MRSA) isolates. This study was carried out to detect and analyze efflux genes (norA and mdeA) and active efflux activity in a collection of Malaysian MRSA and methicillin-sensitive S. aureus (MSSA) clinical isolates. Nineteen isolates including three ATCC S. aureus reference strains were subjected to PCR detection and DNA sequence analysis for norA and mdeA and active efflux detection using modified minimum inhibitory concentration (MIC) assay. From the 19 isolates, 18 isolates harboured the mdeA gene while 16 isolates contained norA gene. DNA sequence analysis reveals 98-100% correlation between the PCR product and the published DNA sequences in GenBank. In addition, 16 isolates exhibited active efflux activity using the ethidium bromide (EtBr)-reserpine combination MIC assay. To our knowledge, this is the first report on the detection of efflux genes and active efflux activity amongst Malaysian clinical isolates of MRSA/MSSA. Detection of active efflux activity may explain the previous report on efflux-mediated drug resistance profile amongst the local clinical isolates.200818720500
2276190.9807Role of gyrase A/B double mutations along with Qnr genes in development of higher ciprofloxacin resistance among pathogenic Escherichia coli and Klebsiella pneumoniae. Contribution of quinolone resistant (QR) genes, efflux pumps (AcrB) over-expression and outer membrane proteins (OMPs) loss/reduction, gyrA/B mutation towards development of quinolone resistance of pathogenic E.coli and Klebsiella sp was explored. Phenotypic characterization of 715 bacteria, isolated from Indian patients during 2011-2017 was performed by Kirby-Bauer disc diffusion assay. Minimum inhibitory concentration of ciprofloxacin was determined by broth microdilution assay. Presence of QR, gyrA/B genes was examined by PCR; acrB upregulation by quantitative PCR and porin profile by SDS-PAGE. Catalytic pockets of modelled proteins were characterized and their interaction with ciprofloxacin was analyzed using AutoDock. Isolates were phenotypically categorized into QR1-QR4 groups according to their resistance against single-four quinolones. Percent prevalence of QR-genes among isolates increased gradually with resistance against increasing number of quinolone antibiotics. Gradual increase in % partial reduction/complete loss of porins was observed from QR1 to QR4 groups with highest fold of Omp reduction. Similar trend was also observed in % prevalanace of upregulated acrB genes among these phenotypic groups with highest fold of upregulation observed among QR2 group. Isolates with GyrA-Ser83Leu + Asp87Asn and GyrB-Asn440Thr + Ser463Ala mutants harbouring Qnr genes mostly demonstrated highest MICs. This is also evident from greater hydrolytic efficiency (ΔG◦ value) of double mutants than their wild types. Dislocation of drug binding site among mutated-GyrA might explain their lower affinity towards quinolones -thus lowering their drug susceptibility. These findings underscore GyrA/B double mutants' role in higher QR among pathogenic E.coli and Klebsiella species, which might guide future antimicrobial therapy.202540784534