Efflux Pump Inhibitors Research Articles

TolC-AcrA complex formation monitored by time dependent single-channel electrophysiology

Characterizing protein-protein interaction on a single molecular level is a challenge, experimentally as well as interpretation of the data. For example, Gram-negative bacteria contain protein complexes spanning the outer and inner cell wall devoted to efflux effectively cell toxic substances. Recent seminal work revealed the high-resolution structure of such a tripartic composition TolC-AcrA-AcrB suggesting to design inhibitors preventing efflux of antibiotics. To show that electrophysiology can provide supporting information here, we reconstitute single TolC homotrimer into a planar lipid membrane, apply a transmembrane voltage and follow the assembly of AcrA to TolC using the modulation of the ion current through TolC channel during binding. In particular, the presence of AcrA in solution increases the average ionic current through TolC and, moreover, reduces the ion-current fluctuations caused by flickering of TolC. Here, we show that statistical properties of ion-current fluctuations (the power spectral density) provide a complementary measure of the interaction of the TolC-AcrA complex in presence of putative efflux pump inhibitors. Both characteristics, the average ion current across TolC and the current noise, taken into consideration together, point to a stiffening of the tip of TolC which might reduce the formation of the complex.

Anti-staphylococcal and antibiotic-potentiating activities of botanicals from nine Cameroonian food plants towards multidrug-resistant phenotypes

Background: Staphylococcus aureus is a commensal and pathogenic bacterium responsible for both community and nosocomial infections, superficial or deep, and benign or lethal. Staphylococcus aureus is a commensal and pathogenic bacterium responsible for both community and nosocomial infections, superficial or deep, and benign or lethal. Because of its infectious potential and its ability to develop resistance to many antibiotics, staphylococcal infections remain the target of reinforced clinical surveillance. To contribute to the fight against resistant staphylococcal infections, the in vitro assessment of the anti-staphylococcal activity of methanol extracts (or botanicals) of nine food plants from Cameroon, Persea americana, Psidium guajava, Syzygium jambos, Vernonia amygdalina, Citrus sinensis, passiflora edulis, Carica papaya, Aframomum letestuanum, and Garcinia kola), as well as the effects of the association of some of these botanicals with antibiotics against resistant and multidrug-resistant staphylococci. Methods: The plant secondary metabolites were extracted by maceration in methanol; the microdilution method using the rapid para-Iodonitrotetrazolium chloride (INT) colorimetric method was applied to evaluate the antibacterial activities of the botanicals as well as the effects of combining these extracts with antibiotics. Results: The botanicals had a minimum inhibitory concentration (MIC) range of 64-2048 µg/mL on the 17 staphylococcal strains and isolates tested. Extracts from Aframomum letestuanum seeds and Psidium guajava leaves and bark had the broadest activity spectra, inhibiting the growth of 95% and 85% of the studied bacteria, respectively. In the presence of an efflux pump inhibitor, reserpine, methanol extracts from Syzygium jambos leaves, Psidium guajava bark and epicarp, and Afromomum letestuanum epicarp showed a considerable increase in their activity. Botanicals from the leaves of Syzygium jambos improved the activities of tetracycline, ceftriaxone, chloramphenicol, and ampicillin against more than 80% of the tested bacteria. Conclusion: The investigated pants, mostly Psidium guajava, Syzygium jambos, and Aframomum letestuanum could be used in the treatment of staphylococcal infections with multidrug-resistant phenotypes.

Anti-Klebsiella and antibiotic-potentiation activities of the methanol extracts of seven Cameroonian dietary plants against multidrug-resistant phenotypes over-expressing AcrAB-TolC efflux pumps

Background: Bacterial infections continue to wreak havoc around the world with high death rates. African medicinal plants and the phytoconstituents showed high efficiency in impeding the growth of the resistance phenotypes of bacteria The present work was designed to determine the antibacterial activity of seven Cameroonian dietary plants against clinical MDR isolated of Klebsiella sp. These plants included Persea americana Miller (Lauraceae), Psidium guajava Linn. (Myrtaceae), Mangifera indica Linn. (Anacardiaceae), Citrus sinensis Linn. (Rutaceae), Passiflora edulis Sims (Passifloraceae), Garcinia kola Heckel (Guttiferae), and Artocarpus heterophylus Lam. (Moraceae). Methods: Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) determinations on the used bacterial strains alone, in the presence of an efflux pump inhibitor, phenylalanine arginine beta naphthylamide (PAβN), and in combination with antibiotics, were performed by the microbroth dilution method using a rapid colorimetric para-Iodonitrotetrazolium chloride (INT) assay. Results: The tested botanicals had different extend of antibacterial activities, with MIC ranges of between 256 μg/mL and 2048 μg/mL. Botanicals from Persea americana, Psidium guajava, Mangifera indica, Artocarpus heterophyllus, and Garcinia kola bark had detected MIC values on all 15 tested Klebsiella strains. PAβN potentiated the activity of the botanicals on all tested bacteria, with the increase of activity ranging from 4 to more than 128-fold. The most significant increase of 4 to more than 128-fold was observed with botanicals from leaves and bark of Psidium guajava and Mangifera indica. The botanicals from the leaves of Mangifera indica potentiated the activity of eight out of ten tested antibiotics (Ceftriaxone, Chloramphenicol, Levofloxacin, Ampicillin, Tetracycline, Imipenem, Doxycycline, and Levofloxacin) against 100% of the tested bacteria. Conclusion: In the present study, it was demonstrated that botanicals from Persea Americana, Psidium guajava, Mangifera indica, Artocarpus heterophyllus, and Garcinia kola had the highest spectrum of activity, and can be used to combat the resistance of Klebsiella species

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.

Repurposing Antidepressants and Phenothiazine Antipsychotics as Efflux Pump Inhibitors in Cancer and Infectious Diseases.

Multidrug resistance (MDR) is a major obstacle in the therapy of infectious diseases and cancer. One of the major mechanisms of MDR is the overexpression of efflux pumps (EPs) that are responsible for extruding antimicrobial and anticancer agents. EPs have additional roles of detoxification that may aid the development of bacterial infection and the progression of cancer. Therefore, targeting EPs may be an attractive strategy to treat bacterial infections and cancer. The development and discovery of a new drug require a long timeline and may come with high development costs. A potential alternative to reduce the time and costs of drug development is to repurpose already existing drugs. Antidepressants and antipsychotic agents are widely used in clinical practice in the treatment of psychiatric disorders and some somatic diseases. Antidepressants and antipsychotics have demonstrated various beneficial activities that may be utilized in the treatment of infections and cancer. This review aims to provide a brief overview of antibacterial and anticancer effects of selective serotonin reuptake inhibitors (SSRIs), tricyclic antidepressants (TCAs) and phenothiazine antipsychotics, while focusing on EPs. However, it should be noted that the antimicrobial activity of a traditionally non-antibiotic drug may have clinical implications regarding dysbiosis and bacterial MDR.

Myoinositol and methyl stearate increases rifampicin susceptibility among drug-resistant Mycobacterium tuberculosis expressing Rv1819c.

The alarming increase in multidrug resistance, which includes Bedaquiline and Delamanid, stumbles success in Tuberculosis treatment outcome. Mycobacterium tuberculosis gains resistance to rifampicin, which is one of the less toxic and potent anti-TB drugs, through genetic mutations predominantly besides efflux pump mediated drug resistance. In recent decades, scientific interventions are being carried out to overcome this hurdle using novel approaches to save this drug by combining it with other drugs/molecules or by use of high dose rifampicin. This study reports five small molecules namely Ellagic acid, Methyl Stearate, Myoinositol, Rutin, and Shikimic acid that exhibit synergistic inhibitory activity with rifampicin against resistant TB isolates. In-silico examinations revealed possible blocking of Rv1819c-an ABC transporter efflux pump that was known to confer resistance in M. tuberculosis to rifampicin. The synergistic anti-TB activity was assessed using a drug combination checkerboard assay. Efflux pump inhibition activity of ellagic acid, myoinositol, and methyl stearate was observed through ethidium bromide accumulation assay in the drug-resistant M. tuberculosis clinical strains and recombinant Mycobacterium smegmatis expressing Rv1819c in coherence with the significant reduction in the minimum inhibitory concentration of rifampicin. Cytotoxicity of the active efflux inhibitors was tested using in silico and ex vivo methods. Myoinositol and methyl stearate were completely non-toxic to the hematological and epithelial cells of different organs under ex vivo conditions. Based on these findings, these molecules can be considered for adjunct TB therapy; however, their impact on other drugs of anti-TB regimen needs to be tested.

Biofilm inhibition based on controlling the transmembrane transport and extracellular accumulation of quorum sensing signals

The regulation of bacterial quorum sensing (QS) has been used to inhibit biofouling in wastewater treatment plants and the formation of biofilms. In contrast to traditional QS regulation strategies, this study aimed to obstruct the transmembrane transport process of QS signals to decrease their extracellular accumulation. Three phytochemicals, astragaloside IV, eugenol, and baicalin were selected, their effects on biofilm formation by Pseudomonas aeruginosa PA14 were studied, and the mechanisms determined. The inhibition efficiency of biofilm formation by 50 mg/L astragaloside IV, 1 mg/L eugenol, and 1 mg/L baicalin were 37%, 26%, and 26%, respectively. Confocal laser scanning microscopy and analysis of extracellular polymeric substances indicated that the three inhibitors affected the structure and composition of the biofilms. Furthermore, bacterial motility and a variety of QS-related virulence factors were suppressed by the inhibitor treatment due to changes in bacterial QS. Notably, the three inhibitors all decreased the extracellular concentration of the QS signaling molecule 3-oxo-C12-homoseine lactone by affecting the function of efflux pump MexAB-OprM. This indirectly interfered with the bacterial QS system and thus inhibited biofilm formation. In conclusion, this study revealed the inhibitory effects and inhibition mechanism of three phytochemicals on efflux pump and QS of P. aeruginosa and realized the inhibition on biofilm formation. We update the efflux pump inhibitor library and provide a new way for biofilm contamination control.

Plants Derived Efflux Pump Inhibitors: An approach against Multidrug-Resistant Gram-negative bacteria Klebsiella pneumoniae

The AcrAB/TolC efflux pump-mediated drug resistance in gram-negative bacteria might be controlled by efflux pump inhibitors (EPIs). This study was planned to discover a compound from plants that can be used as an EPI. Forty isolates of K. pneumoniae and 33 different plants were collected. The methanolic extracts of parts of plants were prepared. Chloramphenicol, tetracycline, and Ciprofloxacin were used for the sensitivity assay. Plant extracts were tested for EPI activity using Berberine and Et

Antibiotic Resistance of Hemophilus influenzae Isolated from Children in Southwest China.

Hemophilus influenzae (Hi) is one of the major pediatric bacterial pneumonia pathogens that heavily threatens children's lives and global health. With widespread usage as first-line treatment, the prevalence of β-lactam-resistant strains is increasing sharply. In order to treat Hi more effectively, a systematic study on the antibiotic resistance profiles, β-lactamase-negative ampicillin-resistant (BLNAR) strains isolation rate, and potential BLNAR resistance mechanism in our region is needed. This study analyzed antimicrobial susceptibility of Hi, and clinical data of Hi-infected patients retrospectively. BLNAR and β-lactamase-positive ampicillin-clavulanate resistant strains (BLPACR) were confirmed by the Kirby-Bauer method and β-lactamase test. ftsI gene in BLNAR was sequenced to find out whether resistance was induced by penicillin-binding protein mutation. Ampicillin susceptibility test with or without efflux pump inhibitors were done to assess efflux pump contribution in BLNAR. RT-PCR was performed to evaluate the efflux pump genes' transcription levels. A total of 2,561 Hi strains were isolated in our hospital from January 2016 to December 2019. Male to female ratio was 1.52:1. Median age was 10 months. Infant (< 3 years old) infection accounted for 83.72%. Hi resistance rates to sulfamethoxazole-trimethoprim, ampicillin, cefathiamidine, cefaclor, cefuroxime, cephalothin, amoxicillin-clavulanate, tetracycline, chloramphenicol, ofloxacin, cefotaxime, and rifampin were 84.28%, 78.01%, 49.80%, 41.98%, 36.58%, 33.64%, 4.55%, 4.1%, 3.37%, 1.77%, 0.99%, and 0.12%, respectively, while 1.33% were BLNAR. BLNARs were classified into four groups by mutation patterns in ftsI gene and most strains were divided to Group Ⅲ/Ⅲ-like. EmrB, ydeA and norM transcription levels in some ampicillin-resistant strains were higher than their sensitive counterparts. Ampicillin is not sufficiently effective as a first-line Hi infection treatment. However, ampicillin-clavulanate and cefotaxime may be a better choice. Efflux pumps, emrB, ydeA and norM play roles in the high resistance to ampicillin.

Efflux pump inhibitor combined with ofloxacin decreases MRSA biofilm formation by regulating the gene expression of NorA and quorum sensing.

Carbonyl cyanide p-nitrophenylhydrazone (2e) displayed a lone or synergistic efficacy against MRSA (RSC Adv., 2020, 10, 17854). In this work, the synergistic mechanism of 2e with ofloxacin was studied. MRSA2858 had potential for biofilm formation, and the value of MBEC of 2e alone was 0.78-1.56 μM, while that of 2e + ofloxacin was 0.39-0.78 μM. 2e combined with ofloxacin showed a synergistic anti-biofilm effect against MRSA. Efflux pump inhibitor 2e can better bind to NorA protein. After MRSA2858 was treated with 2e of 1/2MIC (0.78 μM) and ofloxacin of 1/8MIC (0.097 μM), the transcript levels of efflux genes (norA) and quorum-sensing (QS) regulatory genes (agrA, sarA, icaA, hla) were substantially down-regulated, and alpha-hemolysin (Hla) was inhibited by 99.15%. 2e combined with ofloxacin was more effective than 2e alone in reducing bacterial load in vivo. All in all, efflux pump inhibitor 2e enhanced the bactericidal activities of antibiotics through regulating the gene expression of NorA and QS system.

Evaluation of efflux pump inhibitory activity of some plant extracts and using them as adjuvants to potentiate the inhibitory activity of some antibiotics against Staphylococcus aureus

Antibiotic-resistant pathogens became a real global threat to human and animal health. This needs to concentrate the efforts to minimize and control these organisms. Efflux pumps are considered one of the important strategies used by bacteria to exclude harmful materials from the cell. Inhibition of these pumps can be an active strategy against multidrug resistance pathogens. There are two sources of efflux pump inhibitors that can be used, chemical and natural inhibitors. The chemical origin efflux pump inhibitors have many toxic side effects while the natural origin is characterized by a wide margin of safety for the host cell. In this study, the ability of some plant extracts like (propolis show rosemary, clove, capsaicin, and cumin) to potentiate the inhibitory activity of some antibiotics such as (ciprofloxacin, erythromycin, gentamycin, tetracycline, and ampicillin) against Staphylococcus aureus pathogen were tested. Efflux pump inhibitory activity of the selected plant extracts was tested using an ethidium bromide (EtBr) accumulation assay. The results have shown that Propolis has a significant synergistic effect in combination with ciprofloxacin, erythromycin, and gentamycin. While it has no effect with tetracycline or ampicillin. Also, no synergic effect was noticed in a combination of the minimum inhibitory concentration for the selected plant extracts (rosemary, clove, capsaicin, and cumin) with any of the tested antibiotics. Interestingly, according to the results of the EtBr accumulation assay, Propolis has potent inhibitory activity against the S. aureus (MRS usa300) pump system. This study suggests that Propolis might act as a resistance breaker that is able to restore the activity of ciprofloxacin, erythromycin, and gentamycin against S. aureus strains, in case of the efflux-mediated antimicrobial resistance mechanisms.

Abstract 2072: Novel lead compounds demonstrating anti-biofilm properties in Staphylococcus aureus may act as Nor-A efflux pump inhibitors

Abstract 2072: Novel lead compounds demonstrating anti-biofilm properties in Staphylococcus aureus may act as Nor-A efflux pump inhibitors

Discovery of a novel AcrAB-TolC pump inhibitor using the multistep virtual screening, synthesis and biological evaluation of asymmetric imidazole-4,5-dicarboxamide derivatives

By virtual screening, we identify the potential of asymmetric imidazole-4,5-dicarboxamide derivatives as E. coli-AcrB efflux pump inhibitors. The compounds are synthesized and their inhibitory activity is evaluated by a biological assay.

Analysis of Efflux Pump System and Other Drug Resistance Related Gene Mutations in Tigecycline-Resistant Acinetobacter baumannii.

The isolation of tigecycline-resistant Acinetobacter baumannii in recent years has brought great difficulties to clinical prevention and treatment. To explore the effect of efflux pump system and other resistance related gene mutations on tigecycline resistance in Acinetobacter baumannii. Fluorescence quantitative PCR was used to detect the expression levels of major efflux pump genes (adeB, adeJ, and adeG) in extensive drug-resistant Acinetobacter baumannii. The minimum inhibitory concentration (MIC) of tigecycline was detected by the broth microdilution testing and efflux pump inhibition experiment to assess the role of efflux pump in tigecycline resistance of Acinetobacter baumannii. Efflux pump regulatory genes (adeR and adeS) and tigecycline resistance related genes (rpsJ, trm, and plsC) were amplified by PCR and sequenced. By sequence alignment, tigecycline sensitive and tigecycline-insensitive Acinetobacter baumannii were compared with standard strains to analyze the presence of mutations in these genes. The relative expression of adeB in the tigecycline-insensitive Acinetobacter baumannii was significantly higher than that in the tigecycline sensitive Acinetobacter baumannii (114.70 (89.53-157.43) vs 86.12 (27.23-129.34), P = 0.025). When efflux pump inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was added, the percentage of tigecycline-insensitive Acinetobacter baumannii with tigecycline MIC decreased was significantly higher than that of tigecycline-sensitive Acinetobacter baumannii (10/13 (76.9%) vs 26/59 (44.1%)), P = 0.032); the relative expression of adeB in the MIC decreased group was significantly higher than that in the MIC unchanged group (110.29 (63.62-147.15) vs 50.06 (26.10-122.59), P = 0.02); The relative expression levels of efflux pumps adeG and adeJ did not increase significantly, and there was no significant difference between these groups. One adeR point mutation (Gly232Ala) and eight adeS point mutations (Ala97Thr, Leu105Phe, Leu172Pro, Arg195Gln, Gln203Leu, Tyr303Phe, Lys315Asn, Gly319Ser) were newly detected. Consistent mutations in trm and plsC genes were detected in both tigecycline-insensitive and tigecycline-sensitive Acinetobacter baumannii, but no mutation in rpsJ gene was detected in them. Tigecycline-insensitive Acinetobacter baumannii efflux pump adeABC overexpression was an important mechanism for tigecycline resistance, and the mutations of efflux pump regulator genes (adeR and adeS) are responsible for adeABC overexpression. The effect of trm, plsC, and rpsJ gene mutations on the development of tigecycline resistance in Acinetobacter baumannii remains controversial.

Evaluation of the antibacterial activity of hecogenin acetate and its inhibitory potential of NorA and MepA efflux pumps from Staphylococcus aureus.

Antimicrobial drugs are of great importance in the control of bacterial infections. Its indiscriminate use contributes to the consolidation of bacterial resistance. Its applicability is due to its secondary metabolites, such as saponins, which are compounds with relevant antibacterial action. Hecogenin acetate is a saponin present in plants of the agave genus with analgesic, antioxidant, antinociceptive, cardioactive, anticancer, antifungal and antimicrobial activity. The present work aimed to identify the antibacterial activity of hecogenin acetate against strains of E. coli, P. aeruginosa and S. aureus and to investigate the NorA and MepA efflux pump inhibitory activity of S. aureus strains. The Minimum Inhibitory Concentration was evaluated by broth microdilution. The Antibiotic Activity Modifier effect and the assessment of efflux pump inhibition were evaluated by microdilution with sub-inhibitory concentrations. Hecogenin acetate showed minimal inhibitory concentration without significant relevance. In the evaluation of the potentiating activity of the antibiotic action, a greater antagonistic behavior is noticed. In the analyzes performed with the efflux pump, it was noticed that the hecogenin acetate does not interfere in the efflux pump mechanism of the analyzed bacteria.

The fungal expel of 5-fluorocytosine derived fluoropyrimidines mitigates its antifungal activity and generates a cytotoxic environment.

Invasive aspergillosis remains one of the most devastating fungal diseases and is predominantly linked to infections caused by the opportunistic human mold pathogen Aspergillus fumigatus. Major treatment regimens for the disease comprise the administration of antifungals belonging to the azole, polyene and echinocandin drug class. The prodrug 5-fluorocytosine (5FC), which is the only representative of a fourth class, the nucleobase analogs, shows unsatisfactory in vitro activities and is barely used for the treatment of aspergillosis. The main route of 5FC activation in A. fumigatus comprises its deamination into 5-fluorouracil (5FU) by FcyA, which is followed by Uprt-mediated 5FU phosphoribosylation into 5-fluorouridine monophosphate (5FUMP). In this study, we characterized and examined the role of a metabolic bypass that generates this nucleotide via 5-fluorouridine (5FUR) through uridine phosphorylase and uridine kinase activities. Resistance profiling of mutants lacking distinct pyrimidine salvage activities suggested a minor contribution of the alternative route in 5FUMP formation. We further analyzed the contribution of drug efflux in 5FC tolerance and found that A. fumigatus cells exposed to 5FC reduce intracellular fluoropyrimidine levels through their export into the environment. This release, which was particularly high in mutants lacking Uprt, generates a toxic environment for cytosine deaminase lacking mutants as well as mammalian cells. Employing the broad-spectrum fungal efflux pump inhibitor clorgyline, we demonstrate synergistic properties of this compound in combination with 5FC, 5FU as well as 5FUR.

Xanthene Derivatives Targeting Bacterial Efflux Pumps, Quorum-Sensing, and Biofilm Formation

The rise of multidrug resistance (MDR) bacteria in nosocomial and health-care institutions is widespread and is currently recognized as a major medical challenge. Mechanisms of bacterial resistance, namely, quorum sensing (QS), biofilm formation, and efflux pumps, have been identified as critical biological processes in MDR bacteria. Following previous reports on the activity of phenothiazines against mechanisms of bacterial resistance, in this work we focus on the synthesis of xanthene derivatives aiming to discover phenothiazine bioisosteres with improved activity. Four compounds were obtained from the conjugation of xanthydrol with sulfonamides and aniline and were fully characterized. Their antibacterial activity was assessed considering their minimum inhibitory concentration (MIC) against Gram-positive and Gram-negative strains, efflux pump inhibition, influence on biofilm formation and quorum-sensing (QS) inhibition. It was observed that the MIC of all the tested compounds was above 64 µg/mL The four 9-xanthenyl derivatives obtained, particularly the xanthene sulfonamide derivatives 3b and 3c, showed promising results on QS inhibition with a reduction of pigment production of 48 and 41 mm, and on biofilm formation with a reduction of 78 and 79%, respectively.

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.

Prenylated phenolics from Morus alba against MRSA infections as a strategy for wound healing.

Antimicrobial resistance is a public health threat and the increasing number of multidrug-resistant bacteria is a major concern worldwide. Common antibiotics are becoming ineffective for skin infections and wounds, making the search for new therapeutic options increasingly urgent. The present study aimed to investigate the antibacterial potential of prenylated phenolics in wound healing. Phenolic compounds isolated from the root bark of Morus alba L. were investigated for their antistaphylococcal potential both alone and in combination with commonly used antibiotics. The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) were determined by microdilution and agar method. Synergy was investigated using the checkerboard titration technique. Membrane-disrupting activity and efflux pump inhibition were evaluated to describe the potentiating effect. Prenylated phenolics inhibited bacterial growth of methicillin-resistant Staphylococcus aureus (MRSA) at lower concentrations (MIC 2-8μg/ml) than commonly used antibiotics. The combination of active phenolics with kanamycin, oxacillin, and ciprofloxacin resulted in a decrease in the MIC of the antimicrobial agent. Kuwanon C, E, T, morusin, and albafuran C showed synergy (FICi 0.375-0.5) with oxacillin and/or kanamycin. Prenylated phenolics disrupted membrane permeability statistically significantly (from 28 ± 16.48% up to 73 ± 2.83%), and membrane disruption contributes to the complex antibacterial activity against MRSA. In addition, kuwanon C could be considered an efflux pump inhibitor. Despite the antibacterial effect on MRSA and the multiple biological activities, the prenylated phenolics at microbially significant concentrations have a minor effect on human keratinocyte (HaCaT) viability. In conclusion, prenylated phenolics in combination with commonly used antibiotics are promising candidates for the treatment of MRSA infections and wound healing, although further studies are needed.

Effect of Carbonyl Cyanide M-chlorophenylhydrazone on Ciprofloxacin Resistance and Biofilm Formation in Hospital-acquired Uropathogenic Escherichia coli and Klebsiella pneumoniae

Antibiotic resistant and biofilm forming uropathogenic Enterobacteriaceae are rising. This study was conducted to evaluate the efflux pump and plasmid mediated efflux genes in ciprofloxacin (CIP) resistant hospital acquired uropathogenic Escherichia coli and Klebsiella pneumoniae. Also, to assess the anti-biofilm action of carbonyl cyanide m-chlorophenylhydrazone (CCCP). Uropathogenic E. coli and K. pneumoniae isolates were collected from Mansoura University Hospitals in Mansoura, Egypt. The effect of Sub- minimum inhibitory concentration (MIC) of CCCP on CIP MIC was evaluated and the MIC decrease factor (MDF) was calculated. The presence of oqxAB and qepA genes was detected by PCR. The effect CCCP on biofilm was detected in strong biofilm formers. 56 and 47 CIP-resistant uropathogenic E. coli and K. pneumoniae isolates respectively were detected. Significant MDF by CCCP was observed in 55.3% of these isolates. The qepA gene was only present in E. coli. However, oqxAB genes were found only in K. pneumoniae. Biofilm formation was detected in 58.9% and 72.3% of CIP-resistant E. coli and K. pneumoniae isolates, respectively. Biofilm formation was significantly decreased by CCCP. According to these findings, CIP resistance and plasmid-mediated efflux pumps in uropathogenic E. coli and K. pneumoniae are of rising concern. Efflux pump inhibitor CCCP represents a possible option to decrease the biofilm formation in these resistant urinary pathogens.