Evaluating the Expression of Efflux Pumps in Pseudomonas aeruginosa in Exposure to Sodium Dodecyl Sulfate, Didecyldimethylammonium Chloride, and Octenidine Dihydrochloride.

Emerging resistance of Gram-negative bacteria, including Pseudomonas aeruginosa, to commonly used detergents and disinfectant is encountering us with hazard. Inappropriate use of disinfectants has forced bacteria to gain resistance. The ability of bacteria to extrude substrates from the cellular interior to the external environment has enabled them to persist in exposure to toxic compounds, which is due to existence of transport proteins. Efflux pumps, in Gram-negative bacteria, are proteins responsible for exporting molecules outside of the cell, by crossing the two membranes. In this study, 40 P. aeruginosa strains from hospitals, clinics, and burn center laundries and 40 P. aeruginosa strains from urban laundries were collected. This study evaluated the minimum inhibitory concentration (MIC) level of sodium dodecyl sulfate (SDS), didecyldimethylammonium chloride (DDAC), and octenidine dihydrochloride (Od) in P. aeruginosa strains. The real-time PCR was carried out to evaluate the expression of MexAB-OprM, MexCD-OprJ, and MexXY-OprM efflux system. The obtained results indicated a higher MIC level for SDS, DDAC, and Od in medical laundries. The sub-MIC level of DDAC and Od increased the expression level of MexAB-OprM, MexCD-OprJ, and MexXY-OprM in P. aeruginosa strains, suggesting that efflux pumps contribute to disinfectant resistance in P. aeruginosa.

Major facilitator superfamily efflux pumps in human pathogens: Role in multidrug resistance and beyond.

The major facilitator superfamily (MFS) of proteins constitutes a large group of related solute transporters found across all known living taxa of organisms. The transporters of the MFS contain an extremely diverse array of substrates, including ions, molecules of intermediary metabolism, and structurally different antimicrobial agents. First discovered over 30 years ago, the MFS represents an important collection of integral membrane transporters. Bacterial microorganisms expressing multidrug efflux pumps belonging to the MFS are considered serious pathogens, accounting for alarming morbidity and mortality numbers annually. This review article considers recent advances in the structure-function relationships, the transport mechanism, and modulation of MFS multidrug efflux pumps within the context of drug resistance mechanisms of bacterial pathogens of public health concerns.

Molecular Mechanisms of Bacterial Resistance to Antimicrobial Peptides in the Modern Era: An Updated Review.

Antimicrobial resistance (AMR) poses a serious global health concern, resulting in a significant number of deaths annually due to infections that are resistant to treatment. Amidst this crisis, antimicrobial peptides (AMPs) have emerged as promising alternatives to conventional antibiotics (ATBs). These cationic peptides, naturally produced by all kingdoms of life, play a crucial role in the innate immune system of multicellular organisms and in bacterial interspecies competition by exhibiting broad-spectrum activity against bacteria, fungi, viruses, and parasites. AMPs target bacterial pathogens through multiple mechanisms, most importantly by disrupting their membranes, leading to cell lysis. However, bacterial resistance to host AMPs has emerged due to a slow co-evolutionary process between microorganisms and their hosts. Alarmingly, the development of resistance to last-resort AMPs in the treatment of MDR infections, such as colistin, is attributed to the misuse of this peptide and the high rate of horizontal genetic transfer of the corresponding resistance genes. AMP-resistant bacteria employ diverse mechanisms, including but not limited to proteolytic degradation, extracellular trapping and inactivation, active efflux, as well as complex modifications in bacterial cell wall and membrane structures. This review comprehensively examines all constitutive and inducible molecular resistance mechanisms to AMPs supported by experimental evidence described to date in bacterial pathogens. We also explore the specificity of these mechanisms toward structurally diverse AMPs to broaden and enhance their potential in developing and applying them as therapeutics for MDR bacteria. Additionally, we provide insights into the significance of AMP resistance within the context of host-pathogen interactions.

Decoding host cell interaction- and fluconazole-induced metabolic alterations and drug resistance in Candida auris.

Candida auris is an emerging drug-resistant pathogen associated with high mortality rates. This study aimed to explore the metabolic alterations and associated pathogenesis and drug resistance in fluconazole-treated Candida auris-host cell interaction. Compared with controls, secreted metabolites from fluconazole-treated C. auris and fluconazole-treated C. auris-host cell co-culture demonstrated notable anti-Candida activity. Fluconazole caused significant reductions in C. auris cell numbers and aggregated phenotype. Metabolites produced by C. auris with potential fungal colonization, invasion, and host immune evasion effects were identified. Metabolites known to enhance biofilm formation produced during C. auris-host cell interaction were inhibited by fluconazole. Fluconazole enhanced the production of metabolites with biofilm inhibition activity, including behenyl alcohol and decanoic acid. Metabolites with potential Candida growth inhibition activity such as 2-palmitoyl glycerol, 1-tetradecanol, and 1-nonadecene were activated by fluconazole. Different patterns of proinflammatory cytokine expression presented due to fluconazole concentration and host cell type (fibroblasts versus macrophages). This highlights the immune response's complexity, emphasizing the necessity for additional research to comprehend cell-type-specific responses to antifungal therapies. Both host cell interaction and fluconazole treatment increased the expression of CDR1 and ERG11 genes, both associated with drug resistance. This study provides insights into pathogenesis in C. auris due to host cell interaction and fluconazole treatment. Understanding these interactions is crucial for enhancing fluconazole sensitivity and effectively combating C. auris.

Response of Fusarium oxysporum soil isolate to amphotericin B and fluconazole at the proteomic level.

Fusarium oxysporum is a cross-kingdom pathogen that infects humans, animals, and plants. The primary concern regarding this genus revolves around its resistance profile to multiple classes of antifungals, particularly azoles. However, the resistance mechanism employed by Fusarium spp. is not fully understood, thus necessitating further studies to enhance our understanding and to guide future research towards identifying new drug targets. Here, we employed an untargeted proteomic approach to assess the differentially expressed proteins in a soil isolate of Fusarium oxysporum URM7401 cultivated in the presence of amphotericin B and fluconazole. In response to antifungals, URM7401 activated diverse interconnected pathways, such as proteins involved in oxidative stress response, proteolysis, and lipid metabolism. Efflux proteins, antioxidative enzymes and M35 metallopeptidase were highly expressed under amphotericin B exposure. Antioxidant proteins acting on toxic lipids, along with proteins involved in lipid metabolism, were expressed during fluconazole exposure. In summary, this work describes the protein profile of a resistant Fusarium oxysporum soil isolate exposed to medical antifungals, paving the way for further targeted research and discovering new drug targets.

Inside-out, antimicrobial resistance mediated by efflux pumps in clinical strains of Acinetobacter baumannii isolated from burn wound infections.

Acinetobacter baumannii belongs to the ESKAPE group. It is classified as a critical priority group by the World Health Organization and a global concern on account of its capacity to acquire and develop resistance mechanisms to multiple antibiotics. Data from the United States indicates 500 deaths annually. Resistance mechanisms of this bacterium include enzymatic pathways such as ß-lactamases, carbapenemases, and aminoglycoside-modifying enzymes, decreased permeability, and overexpression of efflux pumps. A. baumannii has been demonstrated to possess efflux pumps, which are classified as members of the MATE family, RND and MFS superfamilies, and SMR transporters. The aim of our work was to assess the distribution of efflux pumps and their regulatory gene expression in clinical strains of A. baumannii isolated from burned patients. METHODS: From the Clinical Microbiology Laboratory at the Instituto Nacional de Rehabilitación Luis Guillermo Ibarra Ibarra collection in Mexico, 199 strains were selected. Antibiotics susceptibilities were performed by broth microdilutions to determine minimal inhibitory concentrations. Phenotypic assays with efflux pump inhibitors were conducted using carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and phenylalanine-arginine ß-naphthylamide (PAßN) in conjunction with amikacin, ceftazidime, imipenem, meropenem and levofloxacin. A search was conducted for structural genes that are linked to efflux pumps, and the relative expression of the adeR, adeS, and adeL genes was analyzed. RESULTS: Among a total of 199 strains, 186 exhibited multidrug resistance (MDR). Fluoroquinolones demonstrated the highest resistance rates, while minocycline and amikacin displayed comparatively reduced resistance rates (1.5 and 28.1, respectively). The efflux activity of fluorquinolones exhibited the highest phenotypic detection (from 85 to 100%), while IMP demonstrated the lowest activity of 27% with PAßN and 43.3% with CCCP. Overexpression was observed in adeS and adeL, with adeR exhibiting overexpression. Concluding that clinical strains of A. baumannii from our institution exhibited efflux pumps as one of the resistance mechanisms.

Methyl gallate attenuates virulence and decreases antibiotic resistance in extensively drug-resistant Pseudomonas aeruginosa.

Pseudomonas aeruginosa infections have become a serious threat to public health due to the increasing emergence of extensively antibiotic-resistant strains and high mortality rates. Therefore, the search for new therapeutic alternatives has become crucial. In this study, the antivirulence and antibacterial activity of methyl gallate was evaluated against six clinical isolates of extensively antibiotic-resistant P. aeruginosa. Methyl gallate exhibited minimal inhibitory concentrations of 256 to 384 µg/mL; moreover, the use of subinhibitory concentrations of the compound inhibited biofilm formation, swimming, swarming, proteolytic activity, and pyocyanin production. Methyl gallate plus antipseudomonal antibiotics showed a synergistic effect by reduced the MICs of ceftazidime, gentamicin and meropenem. Furthermore, the potential therapeutic effect of methyl gallate was demonstrated in an infection model. This study evidenced the antivirulence and antimicrobial activity of methyl gallate as a therapeutic alternative against P. aeruginosa.

Targeting bacterial biofilm-related genes with nanoparticle-based strategies.

Persistent infection caused by biofilm is an urgent in medicine that should be tackled by new alternative strategies. Low efficiency of classical treatments and antibiotic resistance are the main concerns of the persistent infection due to biofilm formation which increases the risk of morbidity and mortality. The gene expression patterns in biofilm cells differed from those in planktonic cells. One of the promising approaches against biofilms is nanoparticle (NP)-based therapy in which NPs with multiple mechanisms hinder the resistance of bacterial cells in planktonic or biofilm forms. For instance, NPs such as silver (Ag), zinc oxide (ZnO), titanium dioxide (TiO2), copper oxide (Cu), and iron oxide (Fe3O4) through the different strategies interfere with gene expression of bacteria associated with biofilm. The NPs can penetrate into the biofilm structure and affect the expression of efflux pump, quorum-sensing, and adhesion-related genes, which lead to inhibit the biofilm formation or development. Therefore, understanding and targeting of the genes and molecular basis of bacterial biofilm by NPs point to therapeutic targets that make possible control of biofilm infections. In parallel, the possible impact of NPs on the environment and their cytotoxicity should be avoided through controlled exposure and safety assessments. This study focuses on the biofilm-related genes that are potential targets for the inhibition of bacterial biofilms with highly effective NPs, especially metal or metal oxide NPs.

Mechanistic Insights into Cellular and Molecular Targets of Zinc Oxide Quantum Dots (ZnO QDs) in Fungal Pathogen, Candida albicans: One Drug Multi-Targeted Therapeutic Approach.

Rationally designed multitargeted drugs, known as network therapeutics/multimodal drugs, have emerged as versatile therapeutic solutions to combat drug-resistant microbes. Here, we report novel mechanistic insights into cellular and molecular targets of ZnO quantum dots (QDs) against Candida albicans, a representative of fungal pathogens. Stable, monodispersed 4-6 nm ZnO QDs were synthesized using a wet chemical route, which exhibited dose-dependent inhibition on the growth dynamics of Candida. Treatment with 200 µg/mL ZnO QDs revealed an aberrant morphology and a disrupted cellular ultrastructure in electron microscopy and led to a 23% reduction in ergosterol content and a 53% increase in intracellular reactive oxygen species. Significant increase in steady-state fluorescence polarization and fluorescence lifetime decay of membrane probe 1,6-diphenyl-1,3,5-hexatriene (DPH) in treated cells, respectively, implied reduction in membrane fluidity and enhanced microviscosity. The observed reduction in passive diffusion of fluorescent Rhodamine 6G across the membrane validated the intricate relationship between ergosterol, membrane fluidity, and microviscosity. An inverse relationship existing between ergosterol biosynthetic genes, ERG11 and ERG3 in treated cells, related well with displayed higher susceptibilities. Furthermore, treated cells exhibited impaired functionality and downregulation of ABC drug efflux pumps. Multiple cellular targets of ZnO QDs in Candida were validated by in silico molecular docking. Thus, targeting ERG11, ERG3, and ABC drug efflux pumps might emerge as a versatile, nano-ZnO-based strategy in fungal therapeutics to address the challenges of drug resistance.

Central role of the ramAR locus in the multidrug resistance in ESBL-Enterobacterales.

The aim of this study was to evaluate the proportion of resistance to a temocillin, tigecycline, ciprofloxacin, and chloramphenicol phenotype called t2c2 that resulted from mutations within the ramAR locus among extended-spectrum ß-lactamases-Enterobacterales (ESBL-E) isolated in three intensive care units for 3 years in a French university hospital. Two parallel approaches were performed on all 443 ESBL-E included: (i) the minimal inhibitory concentrations of temocillin, tigecycline, ciprofloxacin, and chloramphenicol were determined and (ii) the genomes obtained from the Illumina sequencing platform were analyzed to determine multilocus sequence types, resistomes, and diversity of several tetR-associated genes including ramAR operon. Among the 443 ESBL-E strains included, isolates of Escherichia coli (n = 194), Klebsiella pneumoniae (n = 122), and Enterobacter cloacae complex (Ecc) (n = 127) were found. Thirty-one ESBL-E strains (7%), 16 K. pneumoniae (13.1%), and 15 Ecc (11.8%) presented the t2c2 phenotype in addition to their ESBL profile, whereas no E. coli presented these resistances. The t2c2 phenotype was invariably reversible by the addition of Phe-Arg-ß-naphthylamide, indicating a role of resistance-nodulation-division pumps in these observations. Mutations associated with the t2c2 phenotype were restricted to RamR, the ramAR intergenic region (IR), and AcrR. Mutations in RamR consisted of C- or N-terminal deletions and amino acid substitutions inside its DNA-binding domain or within key sites of protein-substrate interactions. The ramAR IR showed nucleotide substitutions involved in the RamR DNA-binding domain. This diversity of sequences suggested that RamR and the ramAR IR represent major genetic events for bacterial antimicrobial resistance.IMPORTANCEMorbimortality caused by infectious diseases is very high among patients hospitalized in intensive care units (ICUs). A part of these outcomes can be explained by antibiotic resistance, which delays the appropriate therapy. The transferable antibiotic resistance gene is a well-known mechanism to explain the high rate of multidrug resistance (MDR) bacteria in ICUs. This study describes the prevalence of chromosomal mutations, which led to additional antibiotic resistance among MDR bacteria. More than 12% of Klebsiella pneumoniae and Enterobacter cloacae complex strains presented mutations within the ramAR locus associated with a dysregulation of an efflux pump called AcrAB-TolC and a porin: OmpF. These dysregulations led to an increase in antibiotic output notably tigecycline, ciprofloxacin, and chloramphenicol associated with a decrease of input for beta-lactam, especially temocillin. Mutations within transcriptional regulators such as ramAR locus played a major role in antibiotic resistance dissemination and need to be further explored.

Efflux pump gene single-nucleotide variants associated with resistance in Mycobacterium tuberculosis isolates with discrepant drug genotypes.

INTRODUCTION: Single-nucleotide variants (SNVs) in Mycobacterium tuberculosis (M. tuberculosis) genomes can predict multidrug resistance (MDR) but not all phenotype-genotype correlations can be explained. We investigated SNVs in efflux pumps (EPs) in the context of M. tuberculosis drug resistance. METHODS: We analysed 2221 M. tuberculosis genomes from 1432 susceptible and 200 MDR, 172 pre-extensively drug resistant (XDR) and 417 XDR isolates. Analysis of 47 EP genes was conducted using MTB-VCF, an in-house bioinformatics pipeline. SNVs were categorized according to their SIFT/Polyphen scores. Resistance genotypes were also called using the TB-Profiler tool. RESULTS: Genome comparisons between susceptible and drug resistant (DR) isolates identified 418 unique SNVs in EP of which; 53.5% were in MDR, 68.9% in pre-XDR and 61.3% in XDR isolates. Twenty EPs had unique SNVs with a high SIFT/PolyPhen score, comprising 38 unique SNVs. Sixteen SNVs across 12 EP genes were significantly associated with drug resistance and enriched in pre-XDR and XDR strains. These comprised 12 previously reported SNVs (in Rv0191, Rv0507, Rv0676, Rv1217, Rv1218, Rv1273, Rv1458, Rv1819, and Rv2688) and 4 novel SNVs (in Rv1877 and Rv2333). We investigated their presence in genomes of 52 MDR isolates with phenotype-genotype discrepancies to rifampicin (RIF), isoniazid (INH), or fluoroquinolones. SNVs associated with RIF and INH (Rv1217_1218, Rv1819, Rv0450, Rv1458, Rv3827, Rv0507, Rv0676, Rv1273, and Rv2333), and with fluoroquinolone (Rv2688) resistance were present in these discrepant strains. CONCLUSIONS: Considering SNVs in EPs as part of M. tuberculosis genome-based resistance interpretation may add value, especially in evaluation of XDR resistance in strains with phenotype-genotype discrepancies.

Role of bacterial multidrug efflux pumps during infection.

Multidrug efflux pumps are protein complexes located in the cell envelope that enable bacteria to expel, not only antibiotics, but also a wide array of molecules relevant for infection. Hence, they are important players in microbial pathogenesis. On the one hand, efflux pumps can extrude exogenous compounds, including host-produced antimicrobial molecules. Through this extrusion, pathogens can resist antimicrobial agents and evade host defenses. On the other hand, efflux pumps also have a role in the extrusion of endogenous compounds, such as bacterial intercommunication signaling molecules, virulence factors or metabolites. Therefore, efflux pumps are involved in the modulation of bacterial behavior and virulence, as well as in the maintenance of the bacterial homeostasis under different stresses found within the host. This review delves into the multifaceted roles that efflux pumps have, shedding light on their impact on bacterial virulence and their contribution to bacterial infection. These observations suggest that strategies targeting bacterial efflux pumps could both reinvigorate the efficacy of existing antibiotics and modulate the bacterial pathogenicity to the host. Thus, a comprehensive understanding of bacterial efflux pumps can be pivotal for the development of new effective strategies for the management of infectious diseases.

Decoding antimicrobial resistance: unraveling molecular mechanisms and targeted strategies.

Antimicrobial resistance poses a significant global health threat, necessitating innovative approaches for combatting it. This review explores various mechanisms of antimicrobial resistance observed in various strains of bacteria. We examine various strategies, including antimicrobial peptides (AMPs), novel antimicrobial materials, drug delivery systems, vaccines, antibody therapies, and non-traditional antibiotic treatments. Through a comprehensive literature review, the efficacy and challenges of these strategies are evaluated. Findings reveal the potential of AMPs in combating resistance due to their unique mechanisms and lower propensity for resistance development. Additionally, novel drug delivery systems, such as nanoparticles, show promise in enhancing antibiotic efficacy and overcoming resistance mechanisms. Vaccines and antibody therapies offer preventive measures, although challenges exist in their development. Non-traditional antibiotic treatments, including CRISPR-Cas systems, present alternative approaches to combat resistance. Overall, this review underscores the importance of multifaceted strategies and coordinated global efforts to address antimicrobial resistance effectively.

AdeIJK Pump-Specific Inhibitors Effective against Multidrug Resistant Acinetobacter baumannii.

Multidrug-resistant Acinetobacter baumannii is a serious threat pathogen rapidly spreading in clinics and causing a range of complicated human infections. The major contributor to A. baumannii antibiotic resistance is the overproduction of AdeIJK and AdeABC multidrug efflux pumps of the resistance-nodulation-division (RND) superfamily of proteins. The dominant role of efflux in antibiotic resistance and the relatively high permeability of the A. baumannii outer membrane to amphiphilic compounds make this pathogen a promising target for the discovery of clinically relevant efflux pump inhibitors. In this study, we identified 4,6-diaminoquoniline analogs with inhibitory activities against A. baumannii AdeIJK efflux pump and followed up on these compounds with a focused synthetic program to improve the target specificity and to reduce cytotoxicity. We identified several candidates that potentiate antibacterial activities of antibiotics erythromycin, tetracycline, and novobiocin not only in the laboratory antibiotic susceptible strain A. baumannii ATCC17978 but also in multidrug-resistant clinical isolates AB5075 and AYE. The best analogs potentiated the activities of antibiotics in low micromolar concentrations, did not have antibacterial activities on their own, inhibited AdeIJK-mediated efflux of its fluorescent substrate ethidium ion, and had low cytotoxicity in A549 human lung epithelial cells.

Whole-genome sequencing, multilocus sequence typing, and resistance mechanism of the carbapenem-resistant Pseudomonas aeruginosa in China.

Pseudomonas aeruginosa is a significant pathogen responsible for severe multisite infections with high morbidity and mortality rates. This study analyzed carbapenem-resistant Pseudomonas aeruginosa (CRPA) at a tertiary hospital in Shandong, China, using whole-genome sequencing (WGS). The objective was to explore the mechanisms and molecular characteristics of carbapenem resistance. A retrospective analysis of 91 isolates from January 2022 to March 2023 was performed, which included strain identification and antimicrobial susceptibility testing. WGS was utilized to determine the genome sequences of these CRPA strains, and the species were precisely identified using average nucleotide identification (ANI), with further analysis on multilocus sequence typing and strain relatedness. Some strains were found to carry the ampD and oprD genes, while only a few harbored carbapenemase genes or related genes. Notably, all strains possessed the mexA, mexE, and mexX genes. The major lineage identified was ST244, followed by ST235. The study revealed a diverse array of carbapenem resistance mechanisms among hospital isolates, differing from previous studies in mainland China. It highlighted that carbapenem resistance is not due to a single mechanism but rather a combination of enzyme-mediated resistance, AmpC overexpression, OprD dysfunction, and efflux pump overexpression. This research provides valuable insights into the evolutionary mechanisms and molecular features of CRPA resistance in this region, aiding in the national prevention and control of CRPA, and offering references for targeting and developing new drugs.

Molecular mechanisms of tigecycline-resistance among Enterobacterales.

The global emergence of antimicrobial resistance to multiple antibiotics has recently become a significant concern. Gram-negative bacteria, known for their ability to acquire mobile genetic elements such as plasmids, represent one of the most hazardous microorganisms. This phenomenon poses a serious threat to public health. Notably, the significance of tigecycline, a member of the antibiotic group glycylcyclines and derivative of tetracyclines has increased. Tigecycline is one of the last-resort antimicrobial drugs used to treat complicated infections caused by multidrug-resistant (MDR) bacteria, extensively drug-resistant (XDR) bacteria or even pan-drug-resistant (PDR) bacteria. The primary mechanisms of tigecycline resistance include efflux pumps' overexpression, tet genes and outer membrane porins. Efflux pumps are crucial in conferring multi-drug resistance by expelling antibiotics (such as tigecycline by direct expelling) and decreasing their concentration to sub-toxic levels. This review discusses the problem of tigecycline resistance, and provides important information for understanding the existing molecular mechanisms of tigecycline resistance in Enterobacterales. The emergence and spread of pathogens resistant to last-resort therapeutic options stands as a major global healthcare concern, especially when microorganisms are already resistant to carbapenems and/or colistin.

Anti-bacterial and anti-biofilm activities of arachidonic acid against the cariogenic bacterium Streptococcus mutans.

Streptococcus mutans is a Gram-positive, facultative anaerobic bacterium, which causes dental caries after forming biofilms on the tooth surface while producing organic acids that demineralize enamel and dentin. We observed that the polyunsaturated arachidonic acid (AA) (ω-6; 20:4) had an anti-bacterial activity against S. mutans, which prompted us to investigate its mechanism of action. The minimum inhibitory concentration (MIC) of AA on S. mutans was 25 µg/ml in the presence of 5% CO2, while it was reduced to 6.25-12.5 µg/ml in the absence of CO2 supplementation. The anti-bacterial action was due to a combination of bactericidal and bacteriostatic effects. The minimum biofilm inhibitory concentration (MBIC) was the same as the MIC, suggesting that part of the anti-biofilm effect was due to the anti-bacterial activity. Gene expression studies showed decreased expression of biofilm-related genes, suggesting that AA also has a specific anti-biofilm effect. Flow cytometric analyses using potentiometric DiOC2(3) dye, fluorescent efflux pump substrates, and live/dead SYTO 9/propidium iodide staining showed that AA leads to immediate membrane hyperpolarization, altered membrane transport and efflux pump activities, and increased membrane permeability with subsequent membrane perforation. High-resolution scanning electron microscopy (HR-SEM) showed remnants of burst bacteria. Furthermore, flow cytometric analysis using the redox probe 2',7'-dichlorofluorescein diacetate (DCFHDA) showed that AA acts as an antioxidant in a dose-dependent manner. α-Tocopherol, an antioxidant that terminates the radical chain, counteracted the anti-bacterial activity of AA, suggesting that oxidation of AA in bacteria leads to the production of cytotoxic radicals that contribute to bacterial growth arrest and death. Importantly, AA was not toxic to normal Vero epithelial cells even at 100 µg/ml, and it did not cause hemolysis of erythrocytes. In conclusion, our study shows that AA is a potentially safe drug that can be used to reduce the bacterial burden of cariogenic S. mutans.

Characterization of genes related to the efflux pump and porin in multidrug-resistant Escherichia coli strains isolated from patients with COVID-19 after secondary infection.

BACKGROUND: Escherichia coli (E. coli) is a multidrug resistant opportunistic pathogen that can cause secondary bacterial infections in patients with COVID-19. This study aimed to determine the antimicrobial resistance profile of E. coli as a secondary bacterial infection in patients with COVID-19 and to assess the prevalence and characterization of genes related to efflux pumps and porin. METHODS: A total of 50 nonduplicate E. coli isolates were collected as secondary bacterial infections in COVID-19 patients. The isolates were cultured from sputum samples. Confirmation and antibiotic susceptibility testing were conducted by Vitek 2. PCR was used to assess the prevalence of the efflux pump and porin-related genes in the isolates. The phenotypic and genotypic evolution of antibiotic resistance genes related to the efflux pump was evaluated. RESULTS: The E. coli isolates demonstrated high resistance to ampicillin (100%), cefixime (62%), cefepime (62%), amoxicillin-clavulanic acid (60%), cefuroxime (60%), and ceftriaxone (58%). The susceptibility of E. coli to ertapenem was greatest (92%), followed by imipenem (88%), meropenem (86%), tigecycline (80%), and levofloxacin (76%). Regarding efflux pump gene combinations, there was a significant association between the acrA gene and increased resistance to levofloxacin, between the acrB gene and decreased resistance to meropenem and increased resistance to levofloxacin, and between the ompF and ompC genes and increased resistance to gentamicin. CONCLUSIONS: The antibiotics ertapenem, imipenem, meropenem, tigecycline, and levofloxacin were effective against E. coli in patients with COVID-19. Genes encoding efflux pumps and porins, such as acrA, acrB, and outer membrane porins, were highly distributed among all the isolates. Efflux pump inhibitors could be alternative antibiotics for restoring tetracycline activity in E. coli isolates.

Differential development of antibiotic resistance and virulence between Acinetobacter species.

The two species that account for most cases of Acinetobacter-associated bacteremia in the United Kingdom are Acinetobacter lwoffii, often a commensal but also an emerging pathogen, and Acinetobacter baumannii, a well-known antibiotic-resistant species. While these species both cause similar types of human infection and occupy the same niche, A. lwoffii (unlike A. baumannii) has thus far remained susceptible to antibiotics. Comparatively little is known about the biology of A. lwoffii, and this is the largest study on it conducted to date, providing valuable insights into its behaviour and potential threat to human health. This study aimed to explain the antibiotic susceptibility, virulence, and fundamental biological differences between these two species. The relative susceptibility of A. lwoffii was explained as it encoded fewer antibiotic resistance and efflux pump genes than A. baumannii (9 and 30, respectively). While both species had markers of horizontal gene transfer, A. lwoffii encoded more DNA defense systems and harbored a far more restricted range of plasmids. Furthermore, A. lwoffii displayed a reduced ability to select for antibiotic resistance mutations, form biofilm, and infect both in vivo and in in vitro models of infection. This study suggests that the emerging pathogen A. lwoffii has remained susceptible to antibiotics because mechanisms exist to make it highly selective about the DNA it acquires, and we hypothesize that the fact that it only harbors a single RND system restricts the ability to select for resistance mutations. This provides valuable insights into how development of resistance can be constrained in Gram-negative bacteria. IMPORTANCE: Acinetobacter lwoffii is often a harmless commensal but is also an emerging pathogen and is the most common cause of Acinetobacter-derived bloodstream infections in England and Wales. In contrast to the well-studied and often highly drug-resistant A. baumannii, A. lwoffii has remained susceptible to antibiotics. This study explains why this organism has not evolved resistance to antibiotics. These new insights are important to understand why and how some species develop antibiotic resistance, while others do not, and could inform future novel treatment strategies.

Repurposing approved drugs as potential efflux pump inhibitors in multidrug-resistant Pseudomonas aeruginosa.

Aim: To evaluate the action of promethazine, fluoxetine and carbonyl cyanide 3-chlorophenylhydrazone as efflux pump inhibitors (EPIs) against multidrug-resistant Pseudomonas aeruginosa. Methods: The effect of the compounds was evaluated in planktonic cells and bacterial biofilms. Accumulation tests were performed with ethidium bromide to prove their action as EPIs. Then, they were associated with antimicrobials. Results: Effect on planktonic cells and biofilms was found. Assays with ethidium bromide indicate their action as EPIs. Significant reductions in the metabolic activity of biofilms were observed after the association with the antimicrobials, especially for meropenem. Conclusion: It is possible to prove the action of these compounds as EPIs for P. aeruginosa and demonstrate the relevance of efflux pumps in antimicrobial resistance.

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