Inhibitor Phenylalanine-arginine Β-naphthylamide Research Articles

Genome-wide analysis of genes involved in efflux function and regulation within Escherichia coli and Salmonella enterica serovar Typhimurium.

The incidence of multidrug-resistant bacteria is increasing globally, with efflux pumps being a fundamental platform limiting drug access and synergizing with other mechanisms of resistance. Increased expression of efflux pumps is a key feature of most cells that are resistant to multiple antibiotics. Whilst expression of efflux genes can confer benefits, production of complex efflux systems is energetically costly and the expression of efflux is highly regulated, with cells balancing benefits against costs. This study used TraDIS-Xpress, a genome-wide transposon mutagenesis technology, to identify genes in Escherichia coli and Salmonella Typhimurium involved in drug efflux and its regulation. We exposed mutant libraries to the canonical efflux substrate acriflavine in the presence and absence of the efflux inhibitor phenylalanine-arginine β-naphthylamide. Comparisons between conditions identified efflux-specific and drug-specific responses. Known efflux-associated genes were easily identified, including acrAB, tolC, marRA, ramRA and soxRS, confirming the specificity of the response. Further genes encoding cell envelope maintenance enzymes and products involved with stringent response activation, DNA housekeeping, respiration and glutathione biosynthesis were also identified as affecting efflux activity in both species. This demonstrates the deep relationship between efflux regulation and other cellular regulatory networks. We identified a conserved set of pathways crucial for efflux activity in these experimental conditions, which expands the list of genes known to impact on efflux efficacy. Responses in both species were similar and we propose that these common results represent a core set of genes likely to be relevant to efflux control across the Enterobacteriaceae.

N1-Benzofused Modification of Fluoroquinolones Reduces Activity Against Gram-Negative Bacteria.

The fluoroquinolone class of antibiotics has a well-established structure–activity relationship (SAR) and a long history in the clinic, but the effect of electron-rich benzofused substituents at the N1 position remains poorly explored. Because groups at this position are part of the topoisomerase–DNA binding complex and form a hydrophobic interaction with the major groove of DNA, it was hypothesized that an electron-rich benzofused N1 substituent could enhance this interaction. Molecular modeling techniques were employed to evaluate the binding of certain N1-modified fluoroquinolones to DNA gyrase targets from both Staphylococcus aureus and Klebsiella pneumoniae species compared with ciprofloxacin and norfloxacin. Seven N1-modified fluoroquinolones were subsequently synthesized and tested against a panel of Gram-negative pathogens to determine minimum inhibitory concentration (MIC) values. Gram-negative outer membrane penetration was investigated using the membrane permeabilizer polymyxin B nonapeptide and compound efflux via resistance–nodulation–division-family efflux transporters was evaluated using the known efflux pump inhibitor phenylalanine–arginine β-naphthylamide. Additionally, the target inhibitory activity of representative compound 6e was determined in a cell-free environment. A correlation between N1 substituent hydrophobicity and activity was observed across the MIC panel, with compound activity decreasing with increased hydrophobicity. Those compounds with highest hydrophobicity were inactive because of poor solubility profiles whereas compounds with intermediate hydrophobicity were inactive because of impaired outer membrane penetration, and reduced inhibition of topoisomerase targets, the latter in contrast to modeling predictions. This study adds new information to the fluoroquinolone SAR and suggests limited utility of large hydrophobic substituents at the N1 position of fluoroquinolones.

MexAB-OprM Efflux Pump of Pseudomonas aeruginosa Offers Resistance to Carvacrol: A Herbal Antimicrobial Agent.

Carvacrol is a herbal antimicrobial agent with in vitro activity against several bacterial pathogens. However, multidrug resistant strains of Pseudomonas aeruginosa are resistant to herbal antimicrobial compounds including carvacrol. Resistance of P. aeruginosa to carvacrol is not well studied. This study was aimed to identify the gene(s) associated with carvacrol resistance, thus to understand its mechanisms in P. aeruginosa. A herbal drug resistant strain was isolated from a hospital environment. Carvacrol sensitive mutant was generated using transposon mutagenesis. The inactivated gene in the mutant was identified as mexA, which is part of the mexAB-oprM operon. Inactivation of the mexA gene resulted in a >31-fold reduction in MIC of carvacrol, whereas a >80-fold reduction was observed in the presence of drug efflux inhibitor phenylalanine-arginine β-naphthylamide (PAβN). The parental herbal-resistant strain was completely killed within 3 h of incubation in the presence of carvacrol and PAβN. The mexA inactivation did not affect the resistance to other herbal compounds used. The results demonstrate that resistance to carvacrol in P. aeruginosa is mediated by the MexAB-OprM efflux pump.

Detection of efflux pump CmeABC in enrofloxacin resistant Campylobacter spp. strains isolated from broiler chickens (Gallus gallus domesticus) in the state of Rio de Janeiro, Brazil

ABSTRACT: Fowls are the main reservoirs of the highly important food-originating pathogen called Campylobacter spp. and broilers’ meat and byproducts are the main vehicles of this microorganism. Increasing of Campylobacter spp. resistant strains to fluorquinolones, an antimicrobial class often employed in poultry farming and in human medicine has become a great concern to poultry breeders. In fact, several studies evaluated increasing bacterial resistance against these antimicrobial agents. The role of CmeABC efflux system has been underscored among the resistance mechanisms in Campylobacter spp. to fluorquinolones. This study investigated the occurrence of CmeABC efflux pump in 81 and 78 enrofloxacin resistant strains of Campylobacter jejuni and C. coli respectively, isolated from broilers collected from six abattoirs situated at São José do Vale do Rio Preto/RJ poultry center and from two commercial abattoirs situated at Metropolitan Region of Rio de Janeiro, from 2013 to 2016. The resistance to enrofloxacin was assessed by agar dilution to determine minimum inhibitory concentration (MIC). The CmeABC efflux system was investigated through the detection of genes genes cmeA, cmeB and cmeC by PCR. The activity of CmeABC efflux pump was investigated in 20 strains by using the efflux pump inhibitor Phenylalanine-Arginine β-Naphthylamide (PAβN). The three genes cmeA, cmeB and cmeC were detected in 94.3% of the strains (C. jejuni = 80 and C. coli = 70), whereas the system was absent or incomplete in 5.7% of strains (C. jejuni = 1 and C. coli = 8). MIC varied between 0.5μg/ml and 64μg/ml, and 88.7% of strains were enrofloxacin resistant and 11.3% featuring intermediate resistance. The inhibition of the efflux pump by PAβN reduced the MIC to enrofloxacin up to eight times in fifteen strains (75%). These results indicate that this system is frequent and active in Campylobacter spp. Resistant strains in the presence of enrofloxacin.

New N-phenylpyrrolamide DNA gyrase B inhibitors: Optimization of efficacy and antibacterial activity

The ATP binding site located on the subunit B of DNA gyrase is an attractive target for the development of new antibacterial agents. In recent decades, several small-molecule inhibitor classes have been discovered but none has so far reached the market. We present here the discovery of a promising new series of N-phenylpyrrolamides with low nanomolar IC50 values against DNA gyrase, and submicromolar IC50 values against topoisomerase IV from Escherichia coli and Staphylococcus aureus. The most potent compound in the series has an IC50 value of 13 nM against E. coli gyrase. Minimum inhibitory concentrations (MICs) against Gram-positive bacteria are in the low micromolar range. The oxadiazolone derivative 11a, with an IC50 value of 85 nM against E. coli DNA gyrase displays the most potent antibacterial activity, with MIC values of 1.56 μM against Enterococcus faecalis, and 3.13 μM against wild type S. aureus, methicillin-resistant S. aureus (MRSA) and vancomycin-resistant Enterococcus (VRE). The activity against wild type E. coli in the presence of efflux pump inhibitor phenylalanine-arginine β-naphthylamide (PAβN) is 4.6 μM.

The efflux pump inhibitor phenylalanine-arginine β-naphthylamide (PAβN) increases resistance to carbapenems in Chilean clinical isolates of KPC-producing Klebsiella pneumoniae.

KPC-producing strains present a wide range of carbapenem minimum inhibitory concentrations (MICs). This variation may be due to differential expression of blaKPC and porin genes, efflux pump activity and the production of extended-spectrum β-lactamases and/or AmpC β-lactamases. The aim of this study was to determine the role of efflux pumps inhibited by phenylalanine-arginine β-naphthylamide (PAβN) in resistance to carbapenems in Chilean clinical isolates of blaKPC-harbouring Klebsiella pneumoniae. MICs were determined by the agar dilution method for imipenem, meropenem, ertapenem and ciprofloxacin in the presence and absence of PAβN (25mg/L) in 17 carbapenem-resistant KPC-producing K. pneumoniae strains. Outer protein membrane (OMP) profiles were determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Expression levels of the ompK35 and ompK36 genes were also determined by real-time quantitative reverse transcription PCR (qRT-PCR). No contribution of PAβN-inhibited efflux pumps to carbapenem resistance was found, unlike ciprofloxacin resistance. However, a ≥4-fold increase in the MIC of at least one carbapenem was observed in 13 isolates in the presence of PAβN. Additionally, decreased gene expression of ompK35 and ompK36 in the presence of PAβN was detected, however no obvious differences in porin band intensity were observed by SDS-PAGE. The presence of PAβN resulted in an increase in carbapenem MICs unrelated to efflux pump inhibition, and a decrease in the expression of ompK35 and ompK36 genes without an obvious difference in OMP profiles observed by SDS-PAGE. Therefore, additional factors are responsible for the increase in carbapenem MIC in the presence of PAβN.

Efflux Pump Inhibitor Phenylalanine-Arginine Β-Naphthylamide Effect on the Minimum Inhibitory Concentration of Imipenem in Acinetobacter baumannii Strains Isolated From Hospitalized Patients in Shahid Motahari Burn Hospital, Tehran, Iran.

Background:Acinetobacter baumannii has emerged as a highly troublesome pathogen and a leading cause of mortality and morbidity among hospitalized burn patients.Objectives:The aims of this study were to determine the frequency of the AdeABC genes and the role of the efflux pump (s) in the imipenem resistance of A. baumannii strains isolated from burn patients.Materials and Methods:This study was conducted on 60 A. baumannii isolates collected from 240 wound samples of burn patients admitted to the Burn Unit of Shahid Motahari Burn hospital, Tehran, Iran. Antibiotic susceptibility tests were performed using the Kirby-Bauer disc diffusion and broth microdilution according to the clinical and laboratory standards institute (CLSI) guidelines. The activity of the efflux pump was evaluated using the efflux pump inhibitor, the phenylalanine-arginine Β-naphthylamide (PAΒN). The AdeABC genes were detected by polymerase chain reaction (PCR) and sequencing.Results:In this study, 100% of the isolates were resistant to cefotaxime, ceftazidime, ceftriaxone, ciprofloxacin, cefepime, piperacillin, meropenem, co-trimoxazole, and piperacillin/tazobactam; 56 (94%) to gentamicin; 50 (81%) to amikacin; 58 (97%) to imipenem; and 45 (76%) to tetracycline. Additionally,all the isolates were susceptible to colistin. The susceptibility of the strains to imipenem was highly increased in the presence of the efflux pump inhibitor such that for 58 (96.6%) of the isolates, the PAΒN reduced the minimum inhibitory concentrations (MIC) by 4- to 64-fold. The adeA and adeB genes were detected in 60 (100%) of the isolates, and the adeC gene was present in 51 (85%).Conclusions:The efflux pump may play a role in antibiotic resistance in A. baumannii isolates. The ability of A. baumannii isolates to acquire drug resistance by the efflux pump mechanism is a concern. Thus, new strategies are required in order to eliminate the efflux transport activity from resistant A. baumannii isolates causing nosocomial infections.

Efflux Pump Inhibitor Phenylalanine-Arginine Β-Naphthylamide Effect on the Minimum Inhibitory Concentration of Imipenem in Acinetobacter baumannii Strains Isolated From Hospitalized Patients in Shahid Motahari Burn Hospital, Tehran, Iran

Background:: Acinetobacter baumannii has emerged as a highly troublesome pathogen and a leading cause of mortality and morbidity among hospitalized burn patients. Objectives:: The aims of this study were to determine the frequency of the AdeABC genes and the role of the efflux pump (s) in the imipenem resistance of A. baumannii strains isolated from burn patients. Materials and Methods:: This study was conducted on 60 A. baumannii isolates collected from 240 wound samples of burn patients admitted to the Burn Unit of Shahid Motahari Burn hospital, Tehran, Iran. Antibiotic susceptibility tests were performed using the Kirby-Bauer disc diffusion and broth microdilution according to the clinical and laboratory standards institute (CLSI) guidelines. The activity of the efflux pump was evaluated using the efflux pump inhibitor, the phenylalanine-arginine Β-naphthylamide (PAΒN). The AdeABC genes were detected by polymerase chain reaction (PCR) and sequencing. Results:: In this study, 100% of the isolates were resistant to cefotaxime, ceftazidime, ceftriaxone, ciprofloxacin, cefepime, piperacillin, meropenem, co-trimoxazole, and piperacillin/tazobactam; 56 (94%) to gentamicin; 50 (81%) to amikacin; 58 (97%) to imipenem; and 45 (76%) to tetracycline. Additionally,all the isolates were susceptible to colistin. The susceptibility of the strains to imipenem was highly increased in the presence of the efflux pump inhibitor such that for 58 (96.6%) of the isolates, the PAΒN reduced the minimum inhibitory concentrations (MIC) by 4- to 64-fold. The adeA and adeB genes were detected in 60 (100%) of the isolates, and the adeC gene was present in 51 (85%). Conclusions:: The efflux pump may play a role in antibiotic resistance in A. baumannii isolates. The ability of A. baumannii isolates to acquire drug resistance by the efflux pump mechanism is a concern. Thus, new strategies are required in order to eliminate the efflux transport activity from resistant A. baumannii isolates causing nosocomial infections.

Activity of the efflux pump inhibitor phenylalanine-arginine β-naphthylamide against the AdeFGH pump of Acinetobacter baumannii

AdeFGH is a multidrug resistance efflux pump of Acinetobacter baumannii belonging to the resistance–nodulation–cell division (RND) family. Analysis of expression of this pump established it as the most prevalent RND pump in clinical isolates of A. baumannii from Canadian hospitals. In this study, the activity of the efflux pump inhibitor (EPI) phenylalanine-arginine β-naphthylamide (PAβN) against the RND efflux pump AdeFGH of A. baumannii was analysed. The adeFGH operon was cloned under the control of the tac promoter and was introduced in single copy into a surrogate Pseudomonas aeruginosa strain that is devoid of its six native efflux pumps. Isopropyl-β-d-thiogalactopyranoside (IPTG) was used to induce expression of the pump and the sensitivity of strains expressing the adeFGH operon to PAβN was analysed in the surrogate strain and in clinical isolates of A. baumannii by measuring the minimum inhibitory concentrations of trimethoprim, chloramphenicol and clindamycin (substrates of AdeFGH) in the presence and absence of PAβN. PAβN was found to inhibit the ability of the AdeFGH pump to efflux all three antibiotics tested in the surrogate P. aeruginosa strain as well as in the A. baumannii strains. However, in A. baumannii strains the inhibition was more profound for clindamycin than for trimethoprim and chloramphenicol. In addition, these data also support the importance of a strain devoid of efflux pumps for screening of EPIs, since the inhibition of AdeFGH by PAβN was found to be more effective in the surrogate P. aeruginosa strain than in the clinical isolates of A. baumannii.

Chemical modification of capuramycins to enhance antibacterial activity

To extend capuramycin spectrum of activity beyond mycobacteria and improve intracellular drug activity. Three capuramycin analogues (SQ997, SQ922 and SQ641) were conjugated with different natural and unnatural amino acids or decanoic acid (DEC) through an ester bond at one or more available hydroxyl groups. In vitro activity of the modified compounds was determined against Mycobacterium spp. and representative Gram-positive and Gram-negative bacteria. Intracellular activity was evaluated in J774A.1 mouse macrophages infected with Mycobacterium tuberculosis (H37Rv). Acylation of SQ997 and SQ641 with amino undecanoic acid (AUA) improved in vitro activity against most of the bacteria tested. Conjugation of SQ922 with DEC, but not AUA, improved its activity against Gram-positive bacteria. In the presence of efflux pump inhibitor phenylalanine arginine β-naphthyl amide, MICs of SQ997-AUA, SQ641-AUA and SQ922-DEC compounds improved even further against drug-susceptible and drug-resistant Staphylococcus aureus. In Gram-negative bacteria, EDTA-mediated permeabilization caused 4- to 16-fold enhancement of the activity of AUA-conjugated SQ997, SQ922 and SQ641. Conjugation of all three capuramycin analogues with AUA improved intracellular killing of H37Rv in murine macrophages. Conjugation of capuramycin analogues with AUA or DEC enhanced in vitro activity, extended the spectrum of activity in Gram-positive bacteria and increased intracellular activity against H37Rv.

Mechanisms of quinolone resistance in Aeromonas species isolated from humans, water and eels

Mechanisms of resistance were determined in 33 quinolone-resistant isolates of the species Aeromonas hydrophila, Aeromonas caviae, Aeromonas media, Aeromonas salmonicida, Aeromonas popoffii and Aeromonas veronii, recovered from humans, freshwater and eels. The quinolone resistance-determining regions (QRDRs) of gyrA and parC genes were sequenced in these resistant strains, as well as in 8 quinolone-sensitive Aeromonas used as controls. All quinolone-resistant Aeromonas carried point mutations in the gyrA QRDR at codon 83, respectively giving rise to substitutions Ser 83 → Ile (32 strains) or Ser 83 → Val (1 strain). Almost half of these isolates (48%) carried additional point mutations in the gyrA QRDR at codon 92 and/or in the parC QRDR at codon 80 corresponding to substitutions Leu 92 → Met and Ser 80 → Ile. In all cases, MICs of quinolones were determined in the presence and absence of the efflux pump inhibitor phenylalanine-arginine β-naphthylamide (PAβN). Addition of PAβN had no effect on the levels of resistance observed in these isolates. In conclusion, the mechanism of quinolone resistance in the Aeromonas isolates studied was related to mutations in QRDR regions of gyrA and parC genes, with little obvious involvement of pumps inhibited by PAβN.