Phenylalanine-arginine Β-naphthylamide Research Articles

Investigation on the mechanisms of carbapenem resistance among the non-carbapenemase-producing carbapenem-resistant Klebsiella pneumoniae.

In Malaysia, an increase in non-carbapenemase-producing carbapenem-resistant Klebsiella pneumoniae (NC-CRKP) has been observed over the years. Previously, four NC-CRKP with increased susceptibility to ciprofloxacin in the presence of phenylalanine-arginine β-naphthylamide (PAβN) were identified. However, no contribution of the PAβN-inhibited efflux pump to carbapenem resistance was observed. All four NC-CRKP harboured non-carbapenemase β-lactamase, with two also exhibiting porin loss. In this study, we further investigated the genomic features and resistance mechanisms of these four isolates. All four NC-CRKP were subjected to whole-genome sequencing, followed by comparative genomic and phylogenetic analyses. Multi-locus sequence typing (MLST) analysis divided the four NC-CRKP into different sequence types: ST392, ST45, ST14, and ST5947. Neither major nor rare carbapenemase genes were detected. Given the presence of non-carbapenemase β-lactamase in all isolates, we further investigated the potential mechanisms of resistance by identifying related chromosomal mutations. Deletion mutation was detected in the cation efflux system protein CusF. Insertion mutation was identified in the nickel/cobalt efflux protein RcnA. Missense mutation of ompK36 porin was detected in two isolates, while the loss of ompK36 porin was observed in another two isolates. This study revealed that NC-CRKP may confer carbapenem resistance through a combination of non-carbapenemase β-lactamase and potential chromosomal mutations including missense mutation or loss of ompK36 porin and/or a frameshift missense mutation in efflux pump systems, such as cation efflux system protein CusF and nickel/cobalt efflux protein RcnA. Our findings highlighted the significance of implementing whole-genome sequencing into clinical practice to promote the surveillance of carbapenem resistance mechanisms among NC-CRKP.

Resistance to aztreonam-avibactam among clinical isolates of Escherichia coli is primarily mediated by altered penicillin-binding protein 3 and impermeability

This study was conducted to investigate decreased susceptibility (minimum inhibitory concentrations [MICs] 0.25–4 mg/L) and resistance (MICs > 4 mg/L) to aztreonam-avibactam (ATM-AVI). Contemporary non-replicate clinical isolates of carbapenemase-producing Escherichia coli (CP-EC) (n=90) and ESBL-producing E. coli (EP-EC) (n=12) were used. CP-EC belonged to 25 distinct sequence types (STs) and all EP-EC belonged to ST405. All strains were isolated from 2019 to 2022 at the Karolinska University Laboratory, Stockholm, Sweden. ATM-AVI MICs were determined using broth microdilution. The EUCAST epidemiological cut-off value of 0.125 mg/L was used to define the wild type (WT). Whole-genome sequences (Illumina) were analysed for detecting resistance determinants among WT vs. non-WT isolates. Among 102 isolates, 40 (39%) and 62 (61%) were WT and non-WT, respectively. Among non-WT isolates, resistance was noted for 20 and decreased susceptibility for 42. Resistance was observed among 14/47 New Delhi metallo-β-lactamase (NDM)-producers, 5/43 OXA-48 group producers, and 1/12 EP-EC. Decreased susceptibility was observed among 29/47 NDM, 13/43 OXA-48 group, and 3/12 EP-EC. Resistant isolates predominantly belonged to ST405, followed by STs 410, 361, 167, 617, and 1284. Penicillin-binding protein 3 (PBP3) inserts (YRIK/YRIN) were observed in 20/20 and CMY-42 in 5/20 resistant isolates. Several mutations in the ftsI (encoding PBP3) and regulatory genes of outer membrane proteins (OmpC and OmpF) and efflux pumps (AcrAB-TolC) were detected. A ≥ 2-fold reduction in MICs was observed among 20/20 vs. 7/20 isolates tested in the presence of the membrane permeabiliser, polymyxin B nanopeptide (PMBN) and efflux inhibitor, phenylalanine arginine β-naphthylamide (PAβN), respectively. In conclusion, resistance to ATM-AVI is a result of interplay of various determinants, including target alterations, deactivating enzymes, and decreased permeability.

Molecular basis of the persistence of chloramphenicol resistance among Escherichia coli and Salmonella spp. from pigs, pork and humans in Thailand.

This study aimed to investigate the potential mechanisms associated with the persistence of chloramphenicol (CHP) resistance in Escherichia coli and Salmonella enterica isolated from pigs, pork, and humans in Thailand. The CHP-resistant E. coli (n = 106) and Salmonella (n = 57) isolates were tested for their CHP susceptibility in the presence and absence of phenylalanine arginine β-naphthylamide (PAβN). The potential co-selection of CHP resistance was investigated through conjugation experiments. Whole genome sequencing (WGS) was performed to analyze the E. coli (E329, E333, and E290) and Salmonella (SA448, SA461, and SA515) isolates with high CHP MIC (32-256 μg/mL) and predominant plasmid replicon types. The presence of PAβN significantly reduced the CHP MICs (≥4-fold) in most E. coli (67.9%) and Salmonella (64.9%). Ampicillin, tetracycline, and streptomycin co-selected for CHP-resistant Salmonella and E. coli-transconjugants carrying cmlA. IncF plasmids were mostly detected in cmlA carrying Salmonella (IncFIIAs) and E. coli (IncFIB and IncF) transconjugants. The WGS analysis revealed that class1 integrons with cmlA1 gene cassette flanked by IS26 and TnAs1 were located on IncX1 plasmid, IncFIA(HI1)/HI1B plasmids and IncFII/FIB plasmids. IncFIA(HI1)/HI1B/Q1in SA448 contained catA flanked by IS1B and TnAs3. In conclusion, cross resistance through proton motive force-dependent mechanisms and co-selection by other antimicrobial agents involved the persistence of CHP-resistance in E. coli in this collection. Dissemination of CHP-resistance genes was potentially facilitated by mobilization via mobile genetic elements.

Detection of qacΔE Efflux Pump Gene among the Clinical Isolates of Escherichia coli and its Correlation with Resistance to Disinfectants.

Disinfectants and antiseptics inhibit the dissemination of pathogenic organisms in hospitals but often cause disinfectant-resistant microorganisms, an important factor for nosocomial infection. This study aimed to evaluate the correlation between qacΔE efflux pump gene and its resistance to disinfectants among Escherichia coli clinical isolates. A total of 97 E. coli isolates were isolated from patients with urinary tract infections. The minimum inhibition concentration (MIC) value of chlorhexidine and benzalkonium chloride was determined using broth microdilution method. Effect of efflux pumps was assessed by MIC test in the presence of phenylalanine-arginine β-naphthylamide (PAβN), and then the qacΔE efflux pump gene was detected using polymerase chain reaction (PCR). Of the isolates, 85.6% and 61.9% were resistant to chlorhexidine and benzalkonium chloride, respectively. Following the treatment of isolates with the efflux pump's inhibitor, PAβN, the MIC value of chlorhexidine and benzalkonium chloride decreased in 75.2% and 57.7% of the isolates, respectively. A significant correlation was found between PAβN treatment and the change in the resistant strains to susceptible strains (p = 0.021). The qacΔE gene was detected in 84.5% (n = 82) of the isolates, and the presence of the gene amongst disinfectant-resistant strains was also significant (p < 0.001). It is suggested to conduct other studies on other efflux pumps, as well as to periodically monitor the resistance to disinfectants. Substances inhibiting efflux pumps and neutral compounds are effective in the reduction of resistance to disinfectants. New disinfectants and drugs should be designed.

Effect of Berberine as efflux pump inhibitor in multidrug-resistant Klebsiella pneumoniae isolated from urinary tract infections

The urinary tract infections with K. pneumoniae have increased over many years. The emergence of antibiotic resistance among these bacteria presents a challenging problem for the clinician regarding the management and treatment of infections. The multidrug resistance in K. pneumoniae is due to several mechanisms, one of which is the role of efflux pumps. The current study investigated the role of Efflux Pump Inhibitors Phenylalanine-Arginine β-Naphthylamide (PAβN) and Berberine as antibacterial agents with multidrug-resistant K. pneumonia isolates from urinary tract infections. The collection of study samples took place between December 2021 and completed at the end of April 2022; it included 260 urine samples collected from outpatients and inpatients suffering from urinary tract infections during this period, from both genders with ages ranging from 15 to 72 years in five hospitals in Baghdad. The results of selective media, biochemical tests, and the VITEK2 system identified 76 isolates (65.5%) as K. pneumoniae from all collected bacterial cultures. The results of the antimicrobial susceptibility test using the disc diffusion method for the isolates under study showed that K. pneumoniae clinical isolates were moderately resistant to most antibiotics tested. Most K. pneumoniae isolates were highly resistant to Amoxicillin (96.1%) and Trimethoprim (80.3%). Also, there was apparent resistance to Gentamicin and Amikacin, while the lowest percentage of resistance was for Meropenem (55.1%) and Ciprofloxacin (53.9%). The susceptibility of the strains to Ciprofloxacin was highly increased in the presence of the efflux pump inhibitor (PAβN). The PAβN reduced the minimum inhibitory concentrations (MICs) by 4- to 64-fold. The results of minimum inhibitory concentrations (MICs) of Berberine against ten K. pneumoniae isolates with multidrug resistance revealed that the range of MICs of Berberine was (3.9-500 µg/ml) and it was obvious that there is a significant effect of Berberine on the growth of K. pneumoniae at deficient concentrations. This study concluded that using Berberine as an efflux pump inhibitor and antimicrobial agent may become a new generation of urgently needed antimicrobials that can overcome bacterial resistance mechanisms. Keywords: UTI infections, MDR, Berberine, Klebsiella pneumoniae

Botanical from the bark of Zizyphus jujuba Mill. (Rhamnaceae) had weak anti-Klebsiella activity, but strongly potentiated the effects of antibiotics against multidrug-resistant phenotypes

Background: Klebsiella pneumoniae is medically the most important species of this genus. Klebsiella oxytoca also cause infections in human but to a much lesser degree than K. pneumoniae. In this work, the antibacterial potential of the methanol extract from the bark of Zizyphus jujuba (ZJB) was evaluated against the multidrug-resistant (MDR) clinical isolates of Klebsiella pneumoniae and Klebsiella oxytoca overexpressing AcrAB-TolC efflux pumps. Methods: The broth microdilution method combined with the rapid para-iodonitrotetrazolium chloride (INT) colorimetric technique was used to determine the minimal inhibitory concentration (MIC) and the minimal bactericidal concentration (MBC) of ZJB alone, in the presence of an efflux pump inhibitor (EPI) phenylalanine-arginine β-naphthylamide (PAβN), or in the presence of antibiotics. The phytochemical screening of ZJB was evaluated using standard methods. Results: ZJB displayed weak antibacterial activities with MIC values above 625 μg/mL in all the 14 tested Klebsiella species. In the presence of PAβN, the activity of ZJB increased by 4- to more than 128-fold on all the tested bacteria. At MIC/2 and MIC/4, ZJB potentiated the activity of doxycycline (DOX), levofloxacin (LEV), imipenem (IMI), ciprofloxacin (CIP), ceftriaxone (CRO), and tetracycline (TET) against at least 80% of the MDR bacterial strains tested. ZJB contains alkaloids, flavonoids, triterpenes, saponins, phenols, and anthocyanins. Conclusion: This study has demonstrated that ZJB could be used as an antibacterial agent if it is combined with an efflux pump inhibitor or with antibiotics against MDR bacteria over-expressing active efflux pumps. Keywords: Antibacterial activity; antibiotics; efflux pumps; Klebsiella; multidrug resistance; Zizyphus jujuba

Antibacterial potential and modes of action of methanol extracts of flowers and leaves of Vernonia glabra (Steetz) Vatke (Asteraceae) against multidrug-resistant Gram-negative bacteria overexpressing efflux pumps

Background: Bacterial infections caused by multidrug-resistant (MDR) Gram-negative bacteria remain a public health problem and have contributed to a reduction in the range of antibiotics available for antibiotic therapy. The search for new antibacterial substances is becoming increasingly important, and plants represent an important reservoir of therapeutic molecules. In the present study, the antibacterial activity and modes of action of Vernonia glabra against Gram-negative and multi-resistant bacteria were evaluated. Methods: The antibacterial activity of Vernonia glabra extracts was assessed using the broth microdilution method, and the effects of the flower extract on bacterial growth kinetics and on the H+-proton pump/ATPase of Providencia stuartii ATCC29916 were carried out using standard experimental protocols; qualitative reference methods were used to identify the secondary metabolites present in the extracts. Results: Phytochemical screening of Vernonia glabra flower and leaf extracts revealed the presence of alkaloids, phenols, flavonoids, tannins, triterpenes, saponins and anthocyanins. The flower and leaf extracts showed antibacterial activity of 100% and 93.33% respectively against the bacteria tested. With minimum inhibitory concentrations (MICs) ranging from 32 µg/mL to 2048 µg/mL, the flower extract showed excellent activity against Escherichia coli AG100 and Providencia stuartii ATCC29916 with a MIC of 32 µg/mL, while the leaf extract showed good activity against Klebsiella pneumoniae ATCC11296 and Providencia stuartii PS2636 with a MIC of 256 µg/mL. The flower extract inhibited the growth of Providencia stuartii ATCC22916 at the exponential phase and inhibited its H+ proton pumps/ATPase. In the presence of the efflux pump inhibitor, phenylalanine-arginine β-naphthylamide (PAβN), the activity of the leaf extract was enhanced on 90.90% of the bacteria tested. With activity enhancement factors ranging from 2 to 128, both extracts potentiated the activity of antibiotics (imipenem, ampicillin, levofloxacin, tetracycline, vancomycin, ceftriaxone, ciprofloxacin, and doxycycline) against at least 70% of the bacteria tested. Conclusion: The results obtained in the present work show that Vernonia glabra is a source of antibacterial molecules that can be used against MDR Gram-negative bacteria. Keywords: Antibacterial; Asteraceae; efflux pumps; modes of action; multidrug resistance; Vernonia glabra.

Antibacterial potential and modes of action of the methanol extracts of Elephantopus mollis Kunth (Asteraceae) against multidrug-resistant Gram-negative bacteria overexpressing efflux pumps

Background: Bacterial drug resistance still constitutes a major clinical issue. In the present study, the in vitro antibacterial potential, and modes of action of Elephantopus mollis were investigated. Methods: The antibacterial activity of methanol extracts of the various parts of E. mollis, their association with an efflux pump inhibitor, phenylalanine-arginine β-naphthylamide (PAβN) and the potentiating effect of several standard antibiotics were determined using the broth microdilution method. The effects of E. mollis leaf extract on H+ proton pump/ATPase function and bacterial growth kinetics were determined using standard methods. Phytochemical screening of the extracts was carried out using standard qualitative methods. Results: The crude extract (botanicals) from E. mollis leaf and flower had antibacterial activity with a 100% inhibition spectrum against bacterial strains and isolates with MICs ranging from 16 to 256 µg/mL and 64 to 1024 µg/mL respectively. Botanicals from the leaf extract showed excellent activity with a MIC of 16 µg/mL against K. pneumoniae KP55, a MIC of 32 µg/mL against K. pneumoniae (K2), and P. stuartti (NEA16). Botanicals from the leaf extract inhibited the exponential growth phase and H+-proton pump/ATPases of K. pneumoniae ATCC11296. In the presence of PAβN, the activity of E. mollis extracts was improved on 90% (leaves and flowers) and 63% (roots) of multidrug-resistant (MDR) bacterial strains and isolates. The various extracts of E. mollis potentiated the activities of the antibiotics: doxycycline, levofloxacin, vancomycin, imipenem, ceftriaxone, and ciprofloxacin against at least 70% of bacterial strains and isolates, with factors of increase in activity ranging from 2 to 128. Extracts from all parts of E. mollis contained alkaloids, flavonoids, tannins, and phenols. Conclusion: The results show that E. mollis is a source of antibacterial phytomedicine that can be used to treat bacterial infections caused by Gram-negative bacteria expressing MDR phenotypes. Keywords: Antibiotics; Asteraceae; bacteria; efflux pumps; Elephantopus mollis; multidrug resistance.

A moderately active crude methanol extract from the whole plant of Kalanchoe crenata (Andrews) Haw. (Crassulaceae) strongly potentiated the effect of antibiotics against multidrug-resistant Gram-negative bacteria

Background: Resistant bacteria develop a high level of resistance to multiple drugs, limiting treatment options and increasing morbidity and mortality. This work was planned to evaluate the antibacterial potential of the methanol extract from the whole plant of Kalanchoe crenata (KCW) against multidrug-resistant (MDR) Gram-negative bacteria. Methods: The minimal inhibitory concentrations (MIC) and the minimal bactericidal concentrations (MBC) of KCW alone, in the presence of an efflux pump inhibitor (EPI) phenylalanine-arginine β-naphthylamide (PAβN), or in the presence of antibiotics were assessed using the broth microdilution method combined with the rapid para-iodonitrotetrazolium chloride (INT) colorimetric technique. Results: KCW displayed weak antibacterial activities with MIC values ranging from 128 to 1024 μg/mL against 10 of the 15 tested bacterial strains. Moderate antibacterial activities with MIC values ranging from 128-625 μg/mL were recorded against some strains belonging to Klebsiella pneumoniae, Escherichia coli, and Providencia stuartii. PAβN does not significantly enhance the activity KCW. At MIC/2, KCW potentiated the activity of doxycycline (DOX), levofloxacin (LEV), imipenem (IMI), ciprofloxacin (CIP), ceftriaxone (CRO), and tetracycline (TET) against at least 80% of the MDR bacterial strains tested. Conclusion: The present study demonstrated that KCW is a moderately active antibacterial agent, but a good efflux pump inhibitor that could potentiate the activity of antibiotics against MDR bacteria over-expressing active efflux pumps. Keywords: Antibacterial; antibiotic-potentiation; Crassulaceae; efflux pumps; Kalanchoe crenata; multidrug resistance.

Increasing antimicrobial susceptibility of MDR Salmonella with the efflux pump inhibitor 1-(1-Naphthylmethyl)-piperazine

Salmonella is a widespread foodborne pathogen that can exhibit multidrug resistance (MDR; resistance to ≥3 antimicrobial classes). Therefore, the development of new preventative measures against MDR Salmonella is highly important. Bacterial antibiotic resistance is commonly mediated by efflux pumps. In this study, two compounds that block efflux pump activity, 1-(1-Naphthylmethyl)-Piperazine (NMP) and Phenylalanine-arginine β-naphthylamide (PaβN), were tested with the antibiotic tetracycline to determine if a synergistic reduction in resistance could be achieved in tetracycline-resistant Salmonella. The efflux pump inhibitors (EPIs) reduced Salmonella resistance to tetracycline by 16 to 32-fold in several tetracycline resistant isolates. For example, the tetracycline minimum inhibitory concentration (MIC) for MDR Salmonella enterica serovar I 4,[5],12:i:- USDA15WA-1 (SX 238) was 256 μg/mL. However, in the presence of NMP (250 μg/mL), the MIC dropped to 8 μg/mL which is below the Clinical Laboratory Standards Institute (CLSI) breakpoint for tetracycline resistance in Salmonella (≥16 μg/mL). Confocal and transmission electron microscopy revealed NMP-mediated damage to Salmonella membranes at a higher concentration (1000 μg/mL), implying that the EPI disrupts membrane morphology which can lead to cell death; however, this effect was dependent on NMP concentration, as NMP blocked efflux activity with less of a membrane-disrupting effect at a lower concentration (250 μg/mL). These findings suggest that the use of EPIs can reduce the MIC of tetracycline and restore the effectiveness of the antibiotic against tetracycline-resistant Salmonella.

Boosting the Antibacterial Activity of Azithromycin on Multidrug-Resistant Escherichia coli by Efflux Pump Inhibition Coupled with Outer Membrane Permeabilization Induced by Phenylalanine-Arginine β-Naphthylamide.

The global spread of multidrug-resistant (MDR) bacteria increases the demand for the discovery of new antibiotics and adjuvants. Phenylalanine-arginine β-naphthylamide (PAβN) is an inhibitor of efflux pumps in Gram-negative bacteria, such as the AcrAB-TolC complex in Escherichia coli. We aimed to explore the synergistic effect and mechanism of action of PAβN combined with azithromycin (AZT) on a group of MDR E. coli strains. Antibiotic susceptibility was tested for 56 strains, which were screened for macrolide resistance genes. Then, 29 strains were tested for synergy using the checkerboard assay. PAβN significantly enhanced AZT activity in a dose-dependent manner in strains expressing the mphA gene and encoding macrolide phosphotransferase, but not in strains carrying the ermB gene and encoding macrolide methylase. Early bacterial killing (6 h) was observed in a colistin-resistant strain with the mcr-1 gene, leading to lipid remodeling, which caused outer membrane (OM) permeability defects. Clear OM damage was revealed by transmission electron microscopy in bacteria exposed to high doses of PAβN. Increased OM permeability was also proven by fluorometric assays, confirming the action of PAβN on OM. PAβN maintained its activity as an efflux pump inhibitor at low doses without permeabilizing OM. A non-significant increase in acrA, acrB, and tolC expression in response to prolonged exposure to PAβN was noted in cells treated with PAβN alone or with AZT, as a reflection of bacterial attempts to counteract pump inhibition. Thus, PAβN was found to be effective in potentiating the antibacterial activity of AZT on E. coli through dose-dependent action. This warrants further investigations of its effect combined with other antibiotics on multiple Gram-negative bacterial species. Synergetic combinations will help in the battle against MDR pathogens, adding new tools to the arsenal of existing medications.

Scalable and Chromatography-Free Synthesis of Efflux Pump Inhibitor Phenylalanine Arginine β-Naphthylamide for Its Validation in Wild-Type Bacterial Strains.

Phenylalanine arginine β-naphthylamine, or PAβN, is a C-terminus capped dipeptide discovered in 1999 as an RND-type efflux pump inhibitor (EPI). Since then, PAβN has become a standard tool compound in EPI research and development. Despite this, PAβN lacks a detailed or efficient synthesis, and standard parameters for its use in wild-type bacterial strains are inconsistent or non-existent. Therefore, a scalable and chromatography-free synthesis of PAβN was developed using streamlined traditional solution-phase peptide coupling chemistry. With this procedure, gram scale quantities of PAβN were synthesized alongside analogues and stereoisomers to build a focused library to evaluate simple structure activity relationships. While most analogues were less active than the broadly utilized L,L-PAβN itself, we identified that its enantiomer, D,D-PAβN, also provided 8- to 16-fold potentiation of the antibiotic levofloxacin at 40 to 50 μg/mL concentrations of EPI in various wild-type Pseudomonas aeruginosa strains. Additionally, D,D-PAβN was shown to be significantly more hydrolytically stable than L,L-PAβN, indicating that it may be a useful, and now readily synthesized, tool compound facilitating future EPI research.

Contribution of efflux and mutations in fluoroquinolone susceptibility in MDR enterobacterial isolates: a quantitative and molecular study.

The emergence of MDR strains is a public health problem in the management of associated infections. Several resistance mechanisms are present, and antibiotic efflux is often found at the same time as enzyme resistance and/or target mutations. However, in the laboratory routinely, only the latter two are identified and the prevalence of antibiotic expulsion is underestimated, causing a misinterpretation of the bacterial resistance phenotype. The development of a diagnostic system to quantify the efflux routinely would thus improve the management of patients. A quantitative technique based on detection of clinically used fluoroquinolones was investigated in Enterobacteriaceae clinical strains with a high or basal efflux activity. The detail of efflux involvement was studied from MIC determination and antibiotic accumulation inside bacteria. WGS was carried out on selected strains to determine the genetic background associated with efflux expression. Only 1 Klebsiella pneumoniae isolate exhibited a lack of efflux whereas 13 isolates had a basal efflux and 8 presented efflux pump overexpression. The antibiotic accumulation evidenced the efficacy of the efflux mechanism in strains, and the contribution of dynamic expulsion versus target mutations in fluoroquinolone susceptibility. We confirmed that phenylalanine arginine β-naphthylamide is not a reliable marker of efflux due to the affinity of the AcrB efflux pump for different substrates. We have developed an accumulation test that can be used efficiently on clinical isolates collected by the biological laboratory. The experimental conditions and protocols ensure a robust assay that with improvements in practice, expertise and equipment could be transferred to the hospital laboratory to diagnose the contribution of efflux in Gram-negative bacteria.

Multifactorial resistance mechanisms associated with resistance to ceftazidime-avibactam in clinical Pseudomonas aeruginosa isolates from Switzerland.

Increasing reports of multidrug resistance (MDR) in clinical Pseudomonas aeruginosa have led to a necessity for new antimicrobials. Ceftazidime-avibactam (CZA) is indicated for use against MDR P. aeruginosa across a broad range of infection types and particularly those that are carbapenem resistant. This study sought to determine the molecular mechanisms of CZA and imipenem (IPM)-resistance in clinical P. aeruginosa isolates obtained from Swiss hospitals. Clinical P. aeruginosa isolates were obtained from inpatients in three hospitals in Switzerland. Susceptibility was determined by either antibiotic disc testing or broth microdilution according to EUCAST methodology. AmpC activity was determined using cloxacillin and efflux activity was determined using phenylalanine-arginine β-naphthylamide, in agar plates. Whole Genome Sequencing was performed on 18 clinical isolates. Sequence types (STs) and resistance genes were ascertained using the Centre for Genomic Epidemiology platform. Genes of interest were extracted from sequenced isolates and compared to reference strain P. aeruginosa PAO1. Sixteen different STs were identified amongst the 18 isolates in this study indicating a high degree of genomic diversity. No carbapenemases were detected but one isolate did harbor the ESBL bla PER-1. Eight isolates were CZA-resistant with MICs ranging from 16-64 mg/L, and the remaining ten isolates had either low/wildtype MICs (n=6; 1-2 mg/L) or elevated, but still susceptible, MICs (n=4; 4-8 mg/L). Ten isolates were IPM-resistant, seven of which had mutations resulting in truncations of OprD, and the remaining nine IPM-susceptible isolates had intact oprD genes. Within CZA-R isolates, and those with reduced susceptibility, mutations resulting in ampC derepression, OprD loss, mexAB overexpression and ESBL (bla PER-1) carriage were observed in various combinations and one harbored a truncation of the PBP4 dacB gene. Within the six isolates with wildtype-resistance levels, five had no mutations that would affect any antimicrobial resistance (AMR) genes of interest when compared to PAO1. This preliminary study highlights that CZA-resistance in P. aeruginosa is multifactorial and could be caused by the interplay between different resistance mechanisms including ESBL carriage, increased efflux, loss of permeability and derepression of its intrinsic ampC.

Antibacterial potential and modes of action of botanicals from Momordica foetida (Cucurbitaceae) against multidrug-resistant bacteria expressing active efflux pumps

Background: Momordica foetida (cucurbitaceae) known as a bitter cucumber in Cameroon is used as a medicinal plant. This plant is widely used in the treatment of microbial infections, diabetes, hypertension, stomach, and intestinal irritation. The leaves are also used to treat malaria, earache, and toothache. This study aimed at investigating the antibacterial activity of the methanol extract of M. foetida seeds against multidrug-resistant bacteria expressing active efflux pumps and to characterize the mode of action of this extract. Methods: The antibacterial activity of the crude extract of M. foetida in the presence and absence of an efflux pump inhibitor, phenylalanine-arginine β-naphthylamide (PAβN), was evaluated by the serial micro-dilution method. The determination of the effect of this sample on bacterial H+-ATPase proton pumps was performed by a standard method using a pH meter and the study of the effect of the same sample on cell growth kinetics, bacterial membrane, and biofilms was performed by a spectrophotometric method. Results: The phytochemical composition of the crude extract of M. foetida, evaluated using standard qualitative methods, showed a rather selective distribution of secondary metabolites (presence of polyphenols, flavonoids, alkaloids, triterpenes, sterols, and saponins). The crude extract of this plant virtually inhibited the growth of 90.90% (20/22) of the bacteria studied with minimal inhibitory concentrations (MIC) ranging from 64 to 1024 µg/mL. The lowest MIC of 64 µg/mL was observed against Escherichia coli (AG102, MC4100, W3110), Klebsiella pneumoniae (KP55 and ATCC 11296), Providencia stuartii (NEA16, ATCC 29916 and PS2636), Pseudomonas aeruginosa (PA124) and Staphylococcus aureus (MRSA9) strains. In the presence of PAβN, the activity of the extract was increased against all strains and isolates used. The crude extract caused a prolongation of the lag phase and a general decrease in the growth of Escherichia coli AG102 bacteria due to the blocking of the H+-ATPase proton pumps of this bacterium, resulting in a decrease in the acidity of the medium. This sample destroyed the bacterial membrane and showed significant activity on the biofilms of this strain tested. Conclusion: The results obtained in the present work provide important and significant data that could allow the probable use of Momordica foetida in the control of bacterial infections.

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.

Phenylalanine-arginine β-naphthylamide could enhance neomycin-sensitivity on Riemerella anatipestifer in vitro and in vivo.

Riemerella anatipestifer is an important duck pathogen responsible for septicemia and infectious serositis, which has caused great economic losses to the duck industry. Phenylalanine-arginine β-naphthylamide (PAβN) is an efflux pump inhibitor, which mainly reduces the efflux effect by competing with antibiotics for efflux pump channels. Here, we found that R. anatipestifer strain GD2019 showed resistances to gentamicin, amikacin, kanamycin, and neomycin. Notably, PAβN could significantly reduce the Minimal inhibitory concentrations (MICs) of neomycin on the GD2019 strain. Moreover, PAβN combined with neomycin significantly decreased bacterial loads, relieved pathological injury and increase survival rate (p < 0.05) for the ducks lethally challenged by the GD2019 strain. Therefore, our results suggested, in vitro and in vivo, PAβN could reduce neomycin-resistant of R. anatipestifer. Importantly, finding of this study provide a new approach for treating antibiotic-resistant R. anatipestifer infection.

Antibacterial activities of thirteen naturally occuring compounds from two Kenyan medicinal plants: Zanthoxylum paracanthum (Mildbr). Kokwaro (Rutaceae) and Dracaena usambarensis Engl. (Asparagaceae) against MDR phenotypes

• Root extract of Zanthoxylum paracanthum with good antibacterial activities. • Efflux pump inhibitor improved antimicrobial activities of Z. paracanthum extract. • All compounds exhibited activities on at least 2/13 bacteria tested. • Nitrogen-containing compounds showed the best activities against bacteria tested. • The phenolic amide, 1 was bactericidal against the standard and MDR isolates. The antibacterial potency of thirteen compounds reported from two distinct genera, Zanthoxylum (Rutaceae) and Dracaena (Asparagaceae) against a panel of sensitive and multi-drug resistant Gram-positive and Gram-negative bacteria is described. The minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) determinations on the tested bacteria were established using modified rapid iodonitrotetrazolium chloride (INT) colorimetric assay. The root extract (50% MeOH/CH 2 Cl 2 ) of Zanthoxylum paracanthum was also combined with an efflux pump inhibitor, phenylalanine arginine β-naphthylamide (PAβN) (at 30 μg/mL), to evaluate the role of efflux pumps in the susceptibility of bacteria. Structure-Activity-Relationship (SAR) of related congeners is also described. The results of the MICs ranged from 4 (against Staphylococcus aureus ) to 16 μg/mL (towards Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae, Providencia stuartii, Pseudomonas aeruginosa ) for Z. paracanthum extract and from 16 (against S. aureus MRSA3) to 1024 μg/mL (towards S. aureus ATCC25923 and MRSA3) for the compounds. Addition of phenylalanine arginine β-naphthylamide (PAβN) in the root extract of Z. paracanthum significantly increased the antibacterial potency of the extract against the tested bacteria, with the highest MICs values being observed at 1 μg/mL (against S. aureus and E. coli strains). The root extract of Z. paracanthum and most of the compounds exhibited an MIC/MBC ≤ 4 except for compound 1 , indicating their bactericidal status. Overall, this study provides baseline information on the antibacterial profiles of the studied samples against both Gram-positive and Gram-negative pathogenic bacteria. Structure modification of the moderately active compounds originating from Z. paracanthum and Dracaena usambarensis should be attempted in future studies towards the improvement of their antibacterial potencies.

Role of the multi-drug efflux systems on the baseline susceptibility to ceftazidime/avibactam and ceftolozane/tazobactam in clinical isolates of non-carbapenemase-producing carbapenem-resistant Pseudomonas aeruginosa.

Carbapenem-resistant Pseudomonas aeruginosa (CRPA) is one of the pathogens that urgently needs new drugs and new alternatives for its control. The primary strategy to combat this bacterium is combining treatments of beta-lactam with a beta-lactamase inhibitor. The most used combinations against P. aeruginosa are ceftazidime/avibactam (CZA) and ceftolozane/tazobactam (C/T). Although mechanisms leading to CZA and C/T resistance have already been described, among which are the resistance-nodulation-division (RND) efflux pumps, the role that these extrusion systems may play in CZA, and C/T baseline susceptibility of clinical isolates remains unknown. For this purpose, 161 isolates of non-carbapenemase-producing (Non-CP) CRPA were selected, and susceptibility tests to CZA and C/T were performed in the presence and absence of the RND efflux pumps inhibitor, Phenylalanine-arginine β-naphthylamide (PAβN). In the absence of PAβN, C/T showed markedly higher activity against Non-CP-CRPA isolates than observed for CZA. These results were even more evident in isolates classified as extremely-drug resistant (XDR) or with difficult-to-treat resistance (DTR), where CZA decreased its activity up to 55.2% and 20.0%, respectively, whereas C/T did it up to 82.8% (XDR), and 73.3% (DTR). The presence of PAβN showed an increase in both CZA (37.6%) and C/T (44.6%) activity, and 25.5% of Non-CP-CRPA isolates increased their susceptibility to these two combined antibiotics. However, statistical analysis showed that only the C/T susceptibility of Non-CP-CRPA isolates was significantly increased. Although the contribution of RND activity to CZA and C/T baseline susceptibility was generally low (two-fold decrease of minimal inhibitory concentrations [MIC]), a more evident contribution was observed in a non-minor proportion of the Non-CP-CRPA isolates affected by PAβN [CZA: 25.4% (15/59); C/T: 30% (21/70)]. These isolates presented significantly higher MIC values for C/T. Therefore, we conclude that RND efflux pumps are participating in the phenomenon of baseline susceptibility to CZA and, even more, to C/T. However, the genomic diversity of clinical isolates is so great that deeper analyzes are necessary to determine which elements are directly involved in this phenomenon.

Cellular modes of action of the methanol extract from the aerial parts of Psychotria sycophylla (K.Schum.) Petit (Rubiaceae) against multidrug-resistant bacteria

Background: Infectious diseases caused by multidrug-resistant bacteria (MDR) remain a global health issue. The population uses several plants from the Cameroonian pharmacopeia to treat bacterial infections. The methanol extract from the aerial parts of Psychotria sycophylla (K.Schum.) Petit (Rubiaceae) (PSA) previously showed antibacterial activities against MDR bacteria. In the present study, the antibacterial activities of PSA were determined in the presence of an efflux pump inhibitor (EPI), phenylalanine-arginine β-naphthylamide (PAβN). The effects of PSA on the bacterial membrane and its ability to inhibit the bacterial biofilm were also assessed. Methods: The evaluation of antibacterial activity of PSA in the presence of PAβN was evaluated using the microdilution method. The modification of the bacterial membrane permeability as well as the inhibition of biofilm were evaluated using spectrophotometric methods. Results: When tested with PAβN, the inhibitory effects of PSA increased vis à vis all the tested bacteria. PSA also destroyed the bacterial membrane and showed significant inhibition of biofilms of Escherichia coli AG102 strain. Conclusion: The reported data provide complementary information on the antibacterial effect of Psychotria sycophylla and might be helpful in formulating a phytomedicine to combat infections due to multidrug-resistant bacteria. Keywords: Bacteria; modes of action; multidrug resistance; Psychotria sycophylla; Rubiaceae.