Overexpression Of Efflux Pumps Research Articles

7-Year (2015-21) longitudinal surveillance of lefamulin in vitro activity against bacterial pathogens collected worldwide from patients with respiratory tract infections including pneumonia and characterization of resistance mechanisms.

Lefamulin (Xenleta™), a pleuromutilin antibiotic, was approved for the oral and IV treatment of community-acquired bacterial pneumonia (CABP) in adults in 2019/2020. This study evaluated the in vitro activity of lefamulin and comparators against 19 584 unique bacterial isolates collected from patients with community-acquired respiratory tract infections and hospitalized patients with pneumonia within the global SENTRY Antimicrobial Surveillance Program during 2015-21. Isolates were susceptibility tested by the CLSI broth microdilution method, and resistance mechanisms were investigated in isolates with elevated lefamulin MICs. Lefamulin exhibited potent antibacterial activity against the most common and typical CABP pathogens tested, including Streptococcus pneumoniae [MIC50/90, 0.06/0.25 mg/L; 99.9% susceptible (S)], Staphylococcus aureus (MIC50/90, 0.06/0.12 mg/L; 99.6% S), Haemophilus influenzae (MIC50/90, 0.5/2 mg/L; 99.1% S) and Moraxella catarrhalis (MIC50/90, 0.06/0.12 mg/L; 100.0% S). Potent activity was also observed against the less common pneumonia pathogens: β-haemolytic (MIC50/90 of 0.03/0.06 mg/L) and viridans group Streptococcus spp. (MIC50/90 of 0.06/0.25 mg/L) and Haemophilus parainfluenzae (MIC50/90 of 1/4 mg/L). Lefamulin's activity was not adversely affected by resistance to macrolides, penicillin, tetracyclines, fluoroquinolones and other resistance phenotypes. Non-susceptibility/resistance to lefamulin was rare and primarily determined by ribosomal protection through vga(A) variants in S. aureus, overexpression of AcrAB-TolC efflux pump in H. influenzae or modifications in L3, L4 and 23SrRNA in Streptococcus spp. Based on the coverage of the most important CABP pathogens and lacking cross-resistance, lefamulin may represent a valuable empirical treatment option for ambulatory and hospitalized patients with CABP, particularly in settings with high prevalence of resistance.

Prenylated isoflavonoids from Fabaceae against the NorA efflux pump in Staphylococcus aureus

Overexpression of NorA efflux pumps plays a pivotal role in the multidrug-resistance mechanism in S. aureus. Here, we investigated the activities of prenylated isoflavonoids, present in the legume plant family (Fabaceae), as natural efflux pump inhibitors (EPIs) in fluoroquinolone-resistant S. aureus. We found that four prenylated isoflavonoids, namely neobavaisoflavone, glabrene, glyceollin I, and glyceollin III, showed efflux pump inhibition in the norA overexpressing S. aureus. At sub-inhibitory concentrations, neobavaisoflavone (6.25 µg/mL, 19 µM) and glabrene (12.5 µg/mL, 39 µM), showed up to 6 times more Eth accumulation in norA overexpressing S. aureus than in the control. In addition, these two compounds boosted the MIC of fluoroquinolones up to eightfold. No fluoroquinolone potentiation was observed with these isoflavonoids in the norA knockout strain, indicating NorA as the main target of these potential EPIs. In comparison to the reported NorA EPI reserpine, neobavaisoflavone showed similar potentiation of fluoroquinolone activity at 10 µM, higher Eth accumulation, and less cytotoxicity. Neobavaisoflavone and glabrene did not exhibit membrane permeabilization effects or cytotoxicity on Caco-2 cells. In conclusion, our findings suggest that the prenylated isoflavonoids neobavaisoflavone and glabrene are promising phytochemicals that could be developed as antimicrobials and resistance-modifying agents to treat fluoroquinolone-resistant S. aureus strains.

Phenotypic characteristics of environmental Pseudomonas aeruginosa: an in vitro study on epidemiological aspects

Pseudomonas aeruginosa is one of the most commonly isolated species among non-fermenting Gram-negative bacteria, both from clinical samples and from environmental sources. The survival of the species in harsh conditions is ensured by the production of a protective biofilm; assessment of biofilm-forming capacity aids future pathogen eradication strategies. The aim of our present study was to assess the relationship between antibiotic resistance, biofilm-forming capacity and other phenotypic virulence factors in environmental P. aeruginosa isolates. One hundred and fourteen (n = 114) isolates were included in the study, which were obtained from various geographical regions and environmental origins. Antimicrobial susceptibility testing was carried out using standard protocols. Biofilm-forming capacity and pyocyanin pigment production were tested using microtiter plate-based methods. Swarming, swimming and twitching motility, and siderophore-production were assessed using agar-plate based methodologies. Resistance in environmental isolates were highest for levofloxacin/ciprofloxacin 49.12% (n = 56), ceftazidime 42.98% (n = 49) and cefepime 35.96% (n = 41), while lowest for colistin 0% (n = 0); overexpression of RND-type efflux pumps was seen in 33.33% (n = 33) of isolates. 21.93% (n = 25) met the criteria to be classified as multidrug resistant (MDR). 17.54% (n = 20) of isolates were weak/non-biofilm producers, while (25.45%, n = 29) and (57.01%, n = 65) were moderate and strong biofilm producers, respectively. No significant differences were noted in biofilm-formation (OD570 values non-MDR [mean ± SD]: 0.396 ± 0.138 vs. MDR: 0.348 ± 0.181; p > 0.05) or pyocyanin pigment production (OD686 values non-MDR: 0.403 ± 0.169 vs. MDR: 0.484 ± 0.125; p > 0.05) between MDR and non-MDR environmental P. aeruginosa. Highest motility values were observed for swarming motility, followed by swimming and twitching motility; no relevant differences (p > 0.05) in motility were noted in the context of MDR status or biofilm-formation in the tested isolates. P. aeruginosa is an opportunistic pathogen with high medical importance, being a causative agent of recalcitrant infections, which are becoming difficult to treat with the onset of MDR. Further studies are warranted to assess biofilm-forming capacity, and to provide insights into the mechanisms underlying biofilm-formation both in isolates of clinical and environmental origins.

Co-targeting HSP90 alpha and CDK7 overcomes resistance against HSP90 inhibitors in BCR-ABL1+ leukemia cells

HSP90 has emerged as an appealing anti-cancer target. However, HSP90 inhibitors (HSP90i) are characterized by limited clinical utility, primarily due to the resistance acquisition via heat shock response (HSR) induction. Understanding the roles of abundantly expressed cytosolic HSP90 isoforms (α and β) in sustaining malignant cells’ growth and the mechanisms of resistance to HSP90i is crucial for exploiting their clinical potential. Utilizing multi-omics approaches, we identified that ablation of the HSP90β isoform induces the overexpression of HSP90α and extracellular-secreted HSP90α (eHSP90α). Notably, we found that the absence of HSP90α causes downregulation of PTPRC (or CD45) expression and restricts in vivo growth of BCR-ABL1+ leukemia cells. Subsequently, chronic long-term exposure to the clinically advanced HSP90i PU-H71 (Zelavespib) led to copy number gain and mutation (p.S164F) of the HSP90AA1 gene, and HSP90α overexpression. In contrast, acquired resistance toward other tested HSP90i (Tanespimycin and Coumermycin A1) was attained by MDR1 efflux pump overexpression. Remarkably, combined CDK7 and HSP90 inhibition display synergistic activity against therapy-resistant BCR-ABL1+ patient leukemia cells via blocking pro-survival HSR and HSP90α overexpression, providing a novel strategy to avoid the emergence of resistance against treatment with HSP90i alone.

Molecular Characterization of Hetero-Pathogenic and Diarrheagenic Escherichia coli Pathotypes in Diarrheic Children under Five Years and Exposure Environment in Ogun State, South-West Nigeria.

Diarrheagenic Escherichia coli (DEC) is one of the most common etiological agents of moderate-to-severe diarrhea in Low- and Middle-Income Countries (LMICs). Therefore, determining the source(s) of DEC in index cases and exposure environment is important for developing a prevention strategy. The current study aims to investigate the prevalence of DEC among children under 5 years and their exposure environment in Ogun State, Nigeria. Samples from 228 diarrheic children and their exposure environment were collected and screened for E. coli. Bio-chemically compatible distinct colonies were molecularly characterized using a 7-virulence-gene multiplex PCR with virulence factors (VFs) indicative of four pathotypes of E. coli: enterotoxigenic (ETEC), verotoxigenic (VTEC), enteropathogenic (EPEC), and enteroinvasive (EIEC). Representative pathotypes were subjected to antimicrobial susceptibility and over-expressed efflux pump assays. One or more VFs typical of specific pathotypes were detected in 25.9% (59/228) diarrhea cases consisting of ETEC (21.5%) and EPEC (0.4%), while hetero-pathogenic pathotypes were found in 4.0% of cases. Of the food sources, 27.9% (101/362) were positive for DEC, of which ETEC accounted for 21.0%, VTEC 1.9%, EPEC 0.6%, EIEC 0.6%, and hetero-pathogenic pathotypes were 3.9%. Furthermore, ETEC was the only pathotype detected in the wastewater (4/183). Interestingly, the consumption of street-vended foods was the most significant (p = 0.04) risk factor for DEC infection in the study area. A total of 73.3% of selected DEC pathotypes showed resistance to antimicrobials, while 27.5% demonstrated over-expression of efflux pump activity. The high prevalence of ETEC across all sources and the occurrence of hetero-pathogenic DEC in diarrheic children and food sources emphasizes the importance of establishing a better strategy for the control and prevention of diarrhea among children in low- and medium-income households.

Escherichia coli from Human Wounds: Analysis of Resistance to β-Lactams and Expression of RND Efflux Pumps.

Resistance of pathogenic strains of Escherichia coli to β-lactams, particularly to ampicillin, is on the rise and it is attributed to intrinsic and acquired mechanisms. One important factor contributing to resistance, together with primarily resistance mechanisms, is a mutation and/or an over-expression of the intrinsic efflux pumps in the resistance-nodulation-division (RND) superfamily. Among these efflux pumps, AcrA, AcrB, TolC, and AcrD play an important role in antimicrobial co-resistance, including resistance to β-lactams. Twelve E. coli isolates obtained from patients' wounds and the control strain of E. coli ATCC 25922 were analyzed. The phenotypic resistance of these isolates to selected β-lactams was assessed by determination of the minimal inhibitory concentration. Additionally, the prevalence of β-lactamase genes (blaTEM, blaCTX-M, blaSHV, and blaAmpC) was screened by PCR. Real-time qPCR was used to determine the expression of the selected efflux pumps acrA, acrB, tolC, and acrD and the repressor acrR after the exposure of E. coli to ampicillin. Phenotypic resistance to β-lactams was detected in seven isolates, mainly to ampicillin and piperacillin. This was corroborated by the presence of at least one acquired bla gene in each of these isolates. Although E. coli strains varied in the expression of RND-family efflux pumps after the ampicillin exposure, their gene expression indicated that these pumps did not play a major role in the phenotypic resistance to ampicillin. Each E. coli isolate displayed unique characteristics, differing in minimum inhibitory concentration (MIC) values, prevalence of acquired blaTEM and blaCTX-M genes, and expression of the RND-family pumps. This together demonstrates that these clinical isolates employed distinct intrinsic or acquired resistance pathways for their defense against ampicillin. The prevalence and spread of ampicillin resistant E. coli has to be monitored and the search for ampicillin alternatives is needed.

Contribution of genetic factors towards cefotaxime and ciprofloxacin resistance development among Extended spectrum beta-lactamase producing-Quinolone resistant pathogenic Enterobacteriaceae

β-lactams and quinolones are widely utilised to treat pathogenic Enterobacterial isolates worldwide. Due to improper use of these antibiotics, both ESBL producing and quinolone resistant (ESBL-QR) pathogenic bacteria have emerged. Nature of contribution of beta-lactamase (bla)/quinolone resistant (QR) genes, efflux pumps (AcrAB-TolC) over-expression and outer membrane proteins (OMPs) /porin loss/reduction and their combinations towards development of this phenotype were explored in this study. Kirby–Bauer disc diffusion method was used for phenotypic characterization of these bacteria and minimum inhibitory concentration of cefotaxime and ciprofloxacin was determined by broth micro dilution assay. Presence of bla, QR, gyrA/B genes was examined by PCR; acrB upregulation by real-time quantitative PCR and porin loss/reduction by SDS-PAGE. Based on antibiogram, phenotypic categorization of 715 non-duplicate clinical isolates was: ESBL+QR+ (n = 265), ESBL+QR- (n = 6), ESBL-QR+ (n = 346) and ESBL-QR-(n = 11). Increased OmpF/K35 and OmpC/K36 reduction, acrB up-regulation, prevalence of bla, QR genes and gyrA/B mutation was observed among the groups in following order: ESBL+QR+> ESBL-QR+> ESBL+QR-> ESBL-QR-. Presence of bla gene alone or combined porin loss and efflux pump upregulation or their combination contributed most for development of a highest level of cefotaxime resistance of ESBL+QR+ isolates. Similarly, combined presence of QR genes, porin loss/reduction, efflux pump upregulation and gyrA/B mutation contributed towards highest ciprofloxacin resistance development of these isolates.

Effect of piperine on the inhibitory potential of MexAB-OprM efflux pump and imipenem resistance in carbapenem-resistant Pseudomonas aeruginosa

The escalating prevalence of carbapenem-resistant Pseudomonas aeruginosa (CRPA) poses a significant threat to global public health through the spread of its ‘high-risk’ clones. Immediate and decisive research into antimicrobial agents against CRPA is crucial for the development of effective measures and interventions. Overexpression of the MexAB-OprM efflux pump is one of the major mechanisms of CRPA. Since the active efflux of antibacterial agents plays a significant role in mediating drug resistance in CRPA, the inhibition of efflux pumps has become a promising strategy to restore antibacterial potency. Piperine (PIP) has been proven to be a promising efflux pump inhibitor in some bacteria. However, there are no studies on whether PIP can act as a potential efflux pump inhibitor in CRPA. The present study aimed to identify the antibacterial activity of PIP against CRPA and to evaluate the effect on the MexAB-OprM efflux pump. Molecular docking was used to analyze the possible interaction of PIP with the proteins of the MexAB-OprM efflux pump in CRPA. The effect of PIP on the expression of the MexAB-OprM efflux pump was investigated by real-time quantitative PCR (qPCR) and ethidium bromide accumulation efflux assay. The effect of PIP on CRPA imipenem (IPM) resistance was investigated by the checkerboard dilution method. The results demonstrated that PIP exhibited the lowest binding affinity of −9.1 kcal towards efflux pump proteins. A synergistic effect between PIP and IPM on CRPA was observed. More importantly, PIP effectively hindered the efflux of ethidium bromide and IPM by up-regulating MexR gene expression while down-regulating MexA, MexB, and OprM gene expressions. In conclusion, PIP could enhance the antibacterial activity of IPM by inhibiting the MexAB-OprM efflux pump. Our work proved that PIP had the potential to be an efflux pump inhibitor of CRPA.

Detection of Multidrug-Resistant RND Efflux Pumps and Regulatory Proteins in Antibiotic-Resistant P. aeruginosa Recovered from Hospital Wastewater Effluent in the Eastern Cape Province of South Africa

P. aeruginosa (P. aeruginosa) is a problematic hospital agent that is a global challenge due to the ineffectiveness of some conventional antimicrobial therapies. Multidrug-resistant (MDR) P. aeruginosa has distinct action modes, including beta-lactamase production, porin gene repression, and efflux pump overexpression. This current research work focuses on efflux pumps (MexAB-OprM, MexCD-OprJ, MexXY-OprN) and their regulatory proteins (NfxB, MexR, MexZ, NalC, NalD) in MDR P. aeruginosa isolated from hospital wastewater effluent. The sequence analysis of the main transporter MexB was also performed. Following antibiotic resistance profiling and polymerase chain reaction (PCR) amplification of the efflux pump genes, the association of the efflux pump proteins with antibiotic resistance was investigated and analysed statistically. Fifty-seven (57) multidrug-resistant isolates were obtained from 81 PCR-confirmed P. aeruginosa isolates. Of the MDR P. aeruginosa isolates, the following rates were recorded: MexA (96.5%), MexB (100%), OprM (96.5%), MexC (100%), MexD (74.1%), OprJ (63.7%), MexX (89.6%), and OprN (51.7%). Additionally, the regulatory proteins had the following rates: NfxB (31.6%), NalC (15.8%), NalD (12.2%), MexZ (3.5%), and MexR (3.5%). The efflux pumps and regulatory proteins are strongly associated with antibiotic resistance, implying that P. aeruginosa antibiotic resistance is heavily influenced by these efflux pumps and regulatory genes. The MexB DNA sequences had numerous substitutions and poor alignment with divergent regions, and hence they have a possible role in increased antibiotic resistance. The absence of regulatory genes in most MDR P. aeruginosa isolates in the current research may have permitted transcription of the efflux pump operons, thus also increasing the antibiotic resistance burden.

Efficacy of Plant Extracts against Carbapenem Resistant Acinetobacter baumannii: A Notorious Pathogen in the Intensive Care Unit

Carbapenem was originally regarded as a cornerstone therapeutic for Gram-negative bacterial infections that could be fatal, but these pathogens are rapidly acquiring resistance to it by various methods such as forming biofilms, mutations in porin channels, overexpression of efflux pumps, altering or modifying antibiotics, and by horizontal gene transfer of resistance biomarkers. Among Gram-negative pathogens, the World Health Organization has prioritized Acinetobacter baumannii as a “pathogen of concern.” A. baumannii strains have a variety of antibiotics resistance pathways, making them resistant to the majority of antibiotics. Antibiotics are used indiscriminately and excessively, causing extensive natural selection pressure and allowing just the most versatile variants of A. baumannii to flourish. Evolutionary changes are unavoidable, but the widespread utilization of antimicrobials in communities, hospitals, and agriculture industries is certainly contributing to the outbreak. Antibiotic resistance has resulted in a number of adverse outcomes such as inadequacy of antimicrobials, persistent infections, and physical ailments that cause the patients to lose momentum, longer stay in the hospital, increased mortality, and morbidity, as well as increased cost that can overburden the patients and even the hospitals. Therefore, optimizing efficient alternate natural antibacterial compounds is a critical aspect of addressing the problem of antibiotic resistance. Various studies (in vitro) done by scientists and researchers have illustrated the efficacy of plant extracts and their essential oils against A. baumannii and other pathogens. The most urgent need is to do research in vivo and more screening of natural products or plant parts, which might lead to the discovery of novel therapeutically efficient antibacterial agents against resistant bacterial infections.

Acinetobacter baumannii IC2 and IC5 Isolates with Co-Existing blaOXA-143-like and blaOXA-72 and Exhibiting Strong Biofilm Formation in a Mexican Hospital.

Acinetobacter baumannii is an opportunistic pathogen responsible for healthcare-associated infections (HAIs) and outbreaks. Antimicrobial resistance mechanisms and virulence factors allow it to survive and spread in the hospital environment. However, the molecular mechanisms of these traits and their association with international clones are frequently unknown in low- and middle-income countries. Here, we analyze the phenotype and genotype of seventy-six HAIs and outbreak-causing A. baumannii isolates from a Mexican hospital over ten years, with special attention to the carbapenem resistome and biofilm formation. The isolates belonged to the global international clone (IC) 2 and the Latin America endemic IC5 and were predominantly extensively drug-resistant (XDR). Oxacillinases were identified as a common source of carbapenem resistance. We noted the presence of the blaOXA-143-like family (not previously described in Mexico), the blaOXA-72 and the blaOXA-398 found in both ICs. A low prevalence of efflux pump overexpression activity associated with carbapenem resistance was observed. Finally, strong biofilm formation was found, and significant biofilm-related genes were identified, including bfmRS, csuA/BABCDE, pgaABCD and ompA. This study provides a comprehensive profile of the carbapenem resistome of A. baumannii isolates belonging to the same pulse type, along with their significant biofilm formation capacity. Furthermore, it contributes to a better understanding of their role in the recurrence of infection and the endemicity of these isolates in a Mexican hospital.

Inhibition of β-lactamase function by de novo designed peptide.

Antimicrobial resistance is a great public health concern that is now described as a "silent pandemic". The global burden of antimicrobial resistance requires new antibacterial treatments, especially for the most challenging multidrug-resistant bacteria. There are various mechanisms by which bacteria develop antimicrobial resistance including expression of β-lactamase enzymes, overexpression of efflux pumps, reduced cell permeability through downregulation of porins required for β-lactam entry, or modifications in penicillin-binding proteins. Inactivation of the β-lactam antibiotics by β-lactamase enzymes is the most common mechanism of bacterial resistance to these agents. Although several effective small-molecule inhibitors of β-lactamases such as clavulanic acid and avibactam are clinically available, they act only on selected class A, C, and some class D enzymes. Currently, none of the clinically approved inhibitors can effectively inhibit Class B metallo-β-lactamases. Additionally, there is increased resistance to these inhibitors reported in several bacteria. The objective of this study is to use the Resonant Recognition Model (RRM), as a novel strategy to inhibit/modulate specific antimicrobial resistance targets. The RRM is a bio-physical approach that analyzes the distribution of energies of free electrons and posits that there is a significant correlation between the spectra of this energy distribution and related protein biological activity. In this study, we have used the RRM concept to evaluate the structure-function properties of a group of 22 β-lactamase proteins and designed 30-mer peptides with the desired RRM spectral periodicities (frequencies) to function as β-lactamase inhibitors. In contrast to the controls, our results indicate 100% inhibition of the class A β-lactamases from Escherichia coli and Enterobacter cloacae. Taken together, the RRM model can likely be utilized as a promising approach to design β-lactamase inhibitors for any specific class. This may open a new direction to combat antimicrobial resistance.

Uncovering the Resistance Mechanisms in Extended-Drug-Resistant Pseudomonas aeruginosa Clinical Isolates: Insights from Gene Expression and Phenotypic Tests.

(1) Background: The purpose of the study was to describe the activity of mex efflux pumps in Multidrug-Resistant (MDR) clinical isolates of Pseudomonas aeruginosa and to compare the carbapenem-resistance identification tests with PCR; (2) Methods: Sixty MDR P. aeruginosa were analyzed for detection of carbapenemase by disk diffusion inhibitory method, carbapenem inactivation method and Modified Hodge Test. Endpoint PCR was used to detect 7 carbapenemase genes (blaKPC, blaOXA48-like, blaNDM, blaGES-2, blaSPM, blaIMP, blaVIM) and mcr-1 for colistin resistance. The expression of mexA, mexB, mexC, mexE and mexX genes corresponding to the four main efflux pumps was also evaluated; (3) Results: From the tested strains, 71.66% presented at least one carbapenemase gene, with blaGES-2 as the most occurring gene (63.3%). Compared with the PCR, the accuracy of phenotypic tests did not exceed 25% for P. aeruginosa. The efflux pump genes were present in all strains except one. In 85% of the isolates, an overactivity of mexA, mexB and mostly mexC was detected. Previous treatment with ceftriaxone increased the activity of mexC by more than 160 times; (4) Conclusions: In our MDR P. aeruginosa clinical isolates, the carbapenem resistance is not accurately detected by phenotypic tests, due to the overexpression of mex efflux pumps and in a lesser amount, due to carbapenemase production.

Molecular identification of Pseudomonas aeruginosa and antibiotic resistance testing

Pseudomonas aeruginosa is a Gram-negative bacillus considered an opportunistic pathogen, usually associated with nosocomial infections in immunocompromised patients. Among the infections caused, the most frequent are urinary tract infections, wound infections, pneumonia, and bacteremia. It has intrinsic and acquired resistance to antibiotics, due to the low permeability of its outer membrane, the overexpression of efflux pumps, and the production of enzymes that degrade the antibiotic. In this study, 61 samples of P. aeruginosa collected from the General Hospital “Dr. Aquiles Calles Ramírez" from the city of Tepic, which were phenotypically identified using the Vitek2 automated system. Subsequently, they were molecularly identified with the polymerase chain reaction technique, targeting the gyrB gene that codes for the B subunit of DNA gyrase. In 60 of the samples, amplification of the gyrB gene was obtained and only one, no amplification was observed. The results showed that infections caused by P. aeruginosa are more frequent in male patients, in age range of 41-60 years. Urine culture being the origin of the sample that most predominated. The sensitivity-resistance analysis to antibiotics showed that most of the strains are resistant to β-lactams, except for meropenem, which presented a high sensitivity in most of the strains, as well as in the case of ciprofloxacin, amikacin, and gentamicin.

Portrait of a killer: Uncovering resistance mechanisms and global spread of Acinetobacter baumannii.

Antibiotic resistance is a growing global concern in the field of medicine as it renders bacterial infections difficult to treat and often more severe. Acinetobacter baumannii is a gram-negative bacterial pathogen causing a wide range of infections, including pneumonia, sepsis, urinary tract infections, and wound infections. A. baumannii has emerged as a significant healthcare-associated pathogen due to its high level of antibiotic resistance. The global spread of antibiotic-resistant strains of A. baumannii has resulted in limited treatment options, leading to increased morbidity and mortality rates, especially in vulnerable populations such as the elderly and immunocompromised individuals, as well as longer hospital stays and higher healthcare costs. Further complicating the situation, multi- and pan-drug-resistant strains of A. baumannii are becoming increasingly common, and these deadly strains are resistant to all or almost all available antibiotics. A. baumannii employs various clever strategies to develop antibiotic resistance, including horizontal transfer of resistance genes, overexpression of inherent efflux pumps that remove drugs from the cell, intrinsic mutations, combined with natural selection under antibiotic selective pressure leading to emergence of successful resistance clones. The typical multidrug resistance phenotype of A. baumannii is, therefore, an orchestrated collimation of all these mechanisms combined with the worldwide spread of "global clones," rendering infections caused by this pathogen challenging to control and treat. To address the escalating problem of antibiotic resistance in A. baumannii, there is a need for increased surveillance, strict infection control measures, and the development of new treatment strategies, requiring a concerted effort by healthcare professionals, researchers, and policymakers.

Trimeric Tobramycin/Nebramine Synergizes β-Lactam Antibiotics against Pseudomonas aeruginosa.

β-Lactam antibiotics remain one of the most effective therapeutics to treat infections caused by Gram-negative bacteria (GNB). However, since ancient times, bacteria have developed multiple resistance mechanisms toward this class of antibiotics including overexpression of β-lactamases, suppression of porins, outer membrane impermeability, overexpression of efflux pumps, and target modifications. To cope with these challenges and to extend the lifetime of existing β-lactam antibiotics, β-lactamase inhibitors are combined with β-lactam antibiotics to prevent antibiotic inactivation by β-lactamases. The combination therapy of an outer membrane permeabilizer with β-lactam antibiotics is an alternative approach to overcoming bacterial resistance of β-lactams in GNB. This approach is of particular interest for pathogens with highly impermeable outer membranes like Pseudomonas aeruginosa. Previous studies have shown that outer membrane permeabilizers can be designed by linking tobramycin and nebramine units together in the form of dimers or chimeras. In this study, we developed trimeric tobramycin and nebramine-based outer membrane permeabilizers presented on a central 1,3,5-triazine framework. The resultant trimers are capable of potentiating outer membrane-impermeable antibiotics but also β-lactams and β-lactam/β-lactamase inhibitor combinations against resistant P. aeruginosa isolates. Furthermore, the microbiological susceptibility breakpoints of ceftazidime, aztreonam, and imipenem were reached by a triple combination consisting of an outer-membrane permeabilizer/β-lactam/β-lactamase inhibitor in β-lactam-resistant P. aeruginosa isolates. Overall, our results indicate that trimeric tobramycins/nebramines can rescue clinically approved β-lactams and β-lactam/β-lactamase inhibitor combinations from resistance.

Botanicals from Aframomum letestuanum Gagnep. (Zingiberaceae) can overcome the multidrug resistance of Klebsiella species overexpressing AcrAB-TolC efflux pumps

Background: Bacteria belonging to the genus Klebsiella have developed resistance mechanisms to clinically used antibiotics, leading to their loss of efficacy. In the present study, the in vitro antibacterial activity of methanol extracts from Aframomum letestuanum, as well as their modes of action against a panel of 14 bacterial species belonging to the genus Klebsiella including multidrug-resistant (MDR) phenotypes overexpressing efflux pumps were evaluated. Methods: The broth microdilution method was used to assess the antibacterial activities of plant extracts (botanicals), while standard experimental protocols were used to study modes of action. Harborne's qualitative reference methods were used to identify the major groups of secondary metabolites present in the botanicals. Results: Phytochemical screening revealed that botanicals contain alkaloids, terpenoids, phenols, flavonoids, tannins, saponins, and anthocyanins. Botanicals from A. letestuanum seed pulp and seed inhibited the growth of all the bacteria tested, with MICs ranging from 16 to 512 μg/mL (pulp) and 64 to 2048 μg/mL (seed). The pulp extract had excellent activity, with MIC values of 16 μg/mL against K2, 32 μg/mL against Kp 80, and Kp 126, and 64 μg/mL against Kp55, Kp63, and ATCC11296. The seed extract displayed a MIC of 64 μg/mL against Kp58 and Kp2. A. letestuanum seed pulp extract inhibited bacterial growth in the exponential phase and induced inhibition of ATPase-H+-dependent proton pumps in K. pneumoniae ATCC11296. Conclusion: Botanicals from A. letestuanum are potential phytomedicines that deserve further investigation to develop novel drugs to overcome the multidrug resistance of Klebsiella species. Keywords: Aframomum letestuanum; antibacterial; antibiotics; efflux pumps; multidrug resistance; Klebsiella species.

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.

Combination Therapy with Ciprofloxacin and Pentamidine against Multidrug-Resistant Pseudomonas aeruginosa: Assessment of In Vitro and In Vivo Efficacy and the Role of Resistance-Nodulation-Division (RND) Efflux Pumps.

The aim of this work was to (i) evaluate the efficacy of a combination treatment of pentamidine with ciprofloxacin against Galleria mellonella larvae infected with an MDR strain of P. aeruginosa and (ii) determine if pentamidine acts as an efflux-pump inhibitor. Resistant clinical isolates, mutant strains overexpressing one of three RND efflux pumps (MexAB-OprM, MexCD-OprJ, and MexEF-OprN), and a strain with the same three pumps deleted were used. MIC assays confirmed that the clinical isolates and the mutants overexpressing efflux pumps were resistant to ciprofloxacin and pentamidine. The deletion of the three efflux pumps induced sensitivity to both compounds. Exposure to pentamidine and ciprofloxacin in combination resulted in the synergistic inhibition of all resistant strains in vitro, but no synergy was observed versus the efflux-pump deletion strain. The treatment of infected G. mellonella larvae with the combination of pentamidine and ciprofloxacin resulted in enhanced efficacy compared with the monotherapies and significantly reduced the number of proliferating bacteria. Our measurement of efflux activity from cells revealed that pentamidine had a specific inhibitory effect on the MexCD-OprJ and MexEF-OprN efflux pumps. However, the efflux activity and membrane permeability assays revealed that pentamidine also disrupted the membrane of all cells. In conclusion, pentamidine does possess some efflux-pump inhibitory activity, in addition to a more general disruptive effect on membrane integrity that accounts for its ability to potentiate ciprofloxacin activity. Notably, the enhanced efficacy of combination therapy with pentamidine and ciprofloxacin versus MDR P. aeruginosa strains in vivo merits further investigation into its potential to treat infections via this pathogen in patients.