Efflux Pump Activity Research Articles

Valproate modulates the activity of multidrug resistance efflux pumps, as a chemoresistance factor in gastric cancer cells.

Drug resistance is one of the most critical problems in gastric cancer therapy. This study was performed to investigate the valproic acid effects on the proliferation of sensitive and resistant cell lines of human gastric cancer, and to explore the mechanism of the agent on multi drug resistance and apoptosis genes. The cytotoxicity effect of valproic acid on the EPG85.257 and EPG85.257RDB cells was assessed by the MTT assay, and the IC50 concentration was evaluated. Apoptosis, genotoxicity, and drug resistance pump activity were evaluated using comet assay, Real-time PCR, and flow cytometry, respectively. Cell proliferation was assayed using a scratch test. Dose-dependent toxicity was recorded after treatment of cells with valproic acid. Valproic acid represented a significant growth inhibition on EPG85.257 cells with IC50 values of 5.84 µM and 4.78 µM after 48 h and 72 h treatment, respectively. In contrast, the drug-resistant counterpart represented 8.7 µM and 7.02 µM IC50 values after the same treatment time. Valproic acid induced PTEN, Bcl2, P53, Bax, P21, and caspase3 expression in EPG85.257 cells, whereas p21, p53, PTEN, and ABCB1 were overexpressed in EPG5.257RDB. Valproic acid hindered cell migration in both cell lines (P < 0.01). Valproate genotoxicity was significantly higher in the parent cells than in their resistant EPG85.257RDB counterparts. Valproate led to a 62% reduction in the daunorubicin efflux of the MDR1 pump activity. Valproate can affect drug resistance in gastric cancer via a unique mechanism independent of MDR1 expression.

Genotypic and phenotypic characterization of determinants that mediate antimicrobial resistance in Escherichia coli strains of clinical origin in South-Western Nigeria.

Multidrug resistant bacterial pathogens employ different mechanisms in evading the action of antibiotics. Multidrug resistance is wide spread among strains of Escherichia coli implicated in several infections including urinary tract infections, gastrointestinal infections, meningitis and bacteraemia. This study investigates the antibiotic resistance profile, efflux pump activity and biofilm formation ability of E. coli strains isolated from clinical samples. A total of 32E. coli strains isolated from clinical samples were characterized and subjected to antibiotic susceptibility testing using standard methods. Isolates were screened phenotypically for biofilm formation and efflux pump activity. While molecular detection of genes encoding curli fimbriae and efflux pump activity was done by PCR. All 32 (100%) E. coli isolates were resistant to ceftazidime, cefuroxime, cefixime, amoxicillin-clavulanate, ofloxacin and ciprofloxacin. While 30 (93.8%) were resistant to gentamicin, 27 (84.4%) were resistant to cefepime and the least resistance of 15.6% was to imipenem. Efflux pump encoding gene tolC was detected in 13(40.6%) of the isolates, while 1(3.1%) harboured acrA gene. acrB gene was not detected in any of the isolates. Seven (21.9%) of the isolates were strong biofilm formers, while 5 (15.6%) and 20 (62.5%) were moderate and weak biofilm formers respectively. csgA gene was detected in all E. coli isolates. High antibiotic resistance of E. coli strains observed in this study is of public health significance. . It is therefore important to scale up efforts in regular monitoring of antibiotic resistance in both community and hospital settings.

Synergistic Antibacterial Activity of Curcuma domestica Val. Extract with Tetracycline Against Multidrug-resistant Acinetobacter baumannii

Infections caused by multidrug-resistant (MDR) bacteria are on the rise globally. MDR is facilitated by overexpression of efflux pump and permeability changes of membrane. Acinetobacter baumannii is a pathogenic germ that causes a major problem in infection, also, there has been an increase in the incidence of resistance to various antibiotics. The present study highlights the synergistic of ethanolic extract of Curcuma domestica (EECD) rhizome with tetracycline against multidrug-resistant A. baumannii (MDR-Ab). Assessment of Minimum Inhibitory Concentration (MIC) was determined by microdilution using 96-well plates. The synergistic effect of EECD and tetracycline was determined by checkerboard method. The effect of EECD and tetracycline combination was investigated by bacteriolytic activity and inhibition of efflux pump by Ethidium Bromide (EtBr) accumulation assay. EECD presented the MIC value 250 µg/mL against MDR-Ab. Fractional Inhibitory Concentration Index (FICI) value of EECD and tetracycline combination was 0.4, which showed their synergistic effect. Additionally, the combination of EECD and tetracycline could inhibit the efflux pump in MDR-Ab. This combination can also compromise cell integrity by altering membrane permeability thus lysing the bacteria cells. According to these results, EECD and tetracycline combination has synergistic effects at some sites of action, and thus could be used as a breakthrough to overcome infection problems due to MDR-Ab.&#x0D; &#x0D; Keywords: Acinetobacter baumannii, antibacterial, Curcuma domestica, multidrug-resistant, tetracycline

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.

Antibacterial Potential of Symmetrical Twin-Drug 3,6-Diaminoxanthones.

Global health faces a significant issue with the rise of infectious diseases caused by bacteria, fungi, viruses, and parasites. The increasing number of multi-drug resistant microbial pathogens severely threatens public health worldwide. Antibiotic-resistant pathogenic bacteria, in particular, present a significant challenge. Therefore, there is an urgent need to identify new potential antimicrobial targets and discover new chemical entities that can potentially reverse bacterial resistance. The main goal of this research work was to create and develop a library of 3,6-disubstituted xanthones based on twin drugs and molecular extension approaches to inhibit the activity of efflux pumps. The process involved synthesizing 3,6-diaminoxanthones through the reaction of 9-oxo-9H-xanthene-3,6-diyl bis(trifluoromethanesulfonate) with various primary and secondary amines. The resulting 3,6-disubstituted xanthone derivatives were then tested for their in vitro antimicrobial properties against a range of pathogenic strains and their efficacy in inhibiting the activity of efflux pumps, biofilm formation, and quorum-sensing. Several compounds have exhibited effective antibacterial properties against the Gram-positive bacterial species tested. Xanthone 16, in particular, has demonstrated exceptional efficacy with a remarkable MIC of 11 µM (4 µg/mL) against reference strains Staphylococcus aureus ATCC 25923 and Enterococcus faecalis ATCC 29212, and 25 µM (9 µg/mL) against methicillin-resistant S. aureus 272123. Furthermore, some derivatives have shown potential as antibiofilm agents in a crystal violet assay. The ethidium bromide accumulation assay pinpointed certain compounds inhibiting bacterial efflux pumps. The cytotoxic effect of the most promising compounds was examined in mouse fibroblast cell line NIH/3T3, and two monoamine substituted xanthone derivatives with a hydroxyl substituent did not exhibit any cytotoxicity. Overall, the nature of the substituent was critical in determining the antimicrobial spectra of aminated xanthones.

Escherichia coli resistance mechanism AcrAB-TolC efflux pump interactions with commonly used antibiotics: a molecular dynamics study

While antibiotic resistance poses a threat from both Gram-positive bacteria (GPB) and Gram-negative bacteria (GNB), GNB pose a more imminent public health hazard globally. GNB are a threat to growing antibiotic resistance because of the complex makeup of the membrane. The AcrAB-TolC efflux pump is a known resistance mechanism of Escherichia coli (E. coli) cells. This study utilized molecular dynamics modeling to visualize some of the changes occurring at a molecular level when airborne bacteria are exposed to stress and antibiotics. This study was conducted to build upon previous experimental research showing that there is an increase in antibiotic resistance and efflux pump activity when exposed to aerosolization. AcrB and AcrAB-TolC proteins were simulated under standard and increased pressure to compare the effect of aerosolization on the binding to the three different antibiotics (puromycin (PUY), ampicillin (AMP) and sulfamethoxazole-trimethoprim (SXT)) to the AcrB binding site. Analysis such as root-mean-square deviation of atomic positions and root-mean-square fluctuation, the opening of TolC, and the significant molecular mechanics with generalized Born and surface area solvation (MM-GBSA) scores associated with specific ligands were recorded. Resistance in experimental data indicated a relationship between the docking scores and some ligand–protein interactions. Results showed that there was more flexibility in the proteins within simulations conducted under standard pressure for the AcrB protein and the full tripartite complex AcrAB-TolC, showing that increased pressure causes more rigidity. MM-GBSA scores, used to calculate the free energy of ligand–protein binding, did not show a significant change, but interestingly, the strongest MM-GBSA scores were for ligands that moved to another binding pocket and did not result in resistance or opening of the efflux pump. However, the ligand moved from the binding site and did not cause the opening of TolC to increase significantly, whereas PUY and AMP were bound to the binding site for the duration of all simulations. AMP ligands under increased pressure showed the largest change in opening of the TolC efflux pump and aligns with experimental data showing E. coli cells had the most resistance to AMP after aerosolization. These results, in addition to other real-time changes such as OM proteins and mutations of targets within the cell, could be used to delineate and mitigate antibiotic resistance mechanisms.

Molecular characterization of extended-spectrum beta-lactamases and carbapenemases producing Enterobacteriaceae isolated from North Eastern region of India

Objectives: This study is aimed to investigate the prevalence of genes encoding extended-spectrum β-lactamases (ESBLs) and carbapenemases production among Enterobacteriaceae isolated from North East India. Materials and Methods: A total of 210 non-duplicate multi-drug resistant Enterobacteriaceae (MDRE) strains were included in this investigation. The isolates were resistant to third-generation cephalosporins, aminoglycosides, and fluoroquinolones. First, the strains were subjected to phenotypic assays to determine ESBLs and carbapenemases production; then, multiplex polymerase chain reaction (mPCR) assays were done to detect ESBLs and carbapenemases genes. In addition, efflux pump activity was determined by phenylalanine-arginine b-naphthylamide assay. Statistical Analysis: The frequency of ESBLs and carbapenemase genes among MDRE strains was shown as percentages. The data analysis was done using Microsoft Excel computer software. Results: Among 210 MDRE clinical isolates, ESBLs production was observed in 72.86% (153) isolates. During mPCR assay, gene encoding ESBLs were detected in 55.24% (116) MDRE strains beta-lactamase Temoniera (blaTEM) (26.67%, 56), beta-lactamase Cefotaxime-Munich (blaCTX-M) (19.52%, 41), and beta-lactamase sulfhydryl reagent variable (blaSHV) (9.05%, 19)]. In addition, 55 (26.2%) and 53 (25.26%) strains were found to be meropenem and imipenem resistant, respectively. Carbapenemase nordmann-poirel (Carba-NP) test for carbapenemases activity was found to be positive in 18.58% (39) MDRE strains. The genes encoding carbapenemases production was observed in 18.58% (39) MDRE [beta-lactamase New Delhi metallo-β-lactamases-1(blaNDM-1) (8.10%, 17), beta-lactamase oxacillinase-48 (blaOXA-48) (2.86%, 6), beta-lactamase Verona imipenemase (blaVIM) (1.43%, 3), and blaOXA-48 and blaVIM (6.19%, 13)]. Efflux pump activity was observed in 5 (2.3%) of Carbapenem-resistant Enterobacteriaceae isolates. Conclusions: For the first time in this region, we have detected the presence of blaOXA-48 and blaVIM in a single MDRE isolate as high as 6.1%. Therefore, clinicians need to detect the ESBLs and carbapenemases producing Enterobacteriaceae on priority in hospital settings for therapeutic options as well as stringent infection control strategies to be adopted as precautions.

Nonlethal Molecular Nanomachines Potentiate Antibiotic Activity Against Gram-Negative Bacteria by Increasing Cell Permeability and Attenuating Efflux.

Antibiotic resistance is a pressing public health threat. Despite rising resistance, antibiotic development, especially for Gram-negative bacteria, has stagnated. As the traditional antibiotic research and development pipeline struggles to address this growing concern, alternative solutions become imperative. Synthetic molecular nanomachines (MNMs) are molecular structures that rotate unidirectionally in a controlled manner in response to a stimulus, such as light, resulting in a mechanical action that can propel molecules to drill into cell membranes, causing rapid cell death. Due to their broad destructive capabilities, clinical translation of MNMs remains challenging. Hence, here, we explore the ability of nonlethal visible-light-activated MNMs to potentiate conventional antibiotics against Gram-negative bacteria. Nonlethal MNMs enhanced the antibacterial activity of various classes of conventional antibiotics against Gram-negative bacteria, including those typically effective only against Gram-positive strains, reducing the antibiotic concentration required for bactericidal action. Our study also revealed that MNMs bind to the negatively charged phospholipids of the bacterial inner membrane, leading to permeabilization of the cell envelope and impairment of efflux pump activity following light activation of MNMs. The combined effects of MNMs on membrane permeability and efflux pumps resulted in increased antibiotic accumulation inside the cell, reversing antibiotic resistance and attenuating its development. These results identify nonlethal MNMs as pleiotropic antibiotic enhancers or adjuvants. The combination of MNMs with traditional antibiotics is a promising strategy against multidrug-resistant Gram-negative infections. This approach can reduce the amount of antibiotics needed and slow down antibiotic resistance development, thereby preserving the effectiveness of our current antibiotics.

Unveiling the potential of recently FDA-approved drugs as quorum sensing inhibitors against P. Aeruginosa using high-performance computational techniques

Through cell-to-cell communication, activation of efflux pumps, formation of biofilms, and other mechanisms, pseudomonas aeruginosa’s quorum sensing systems (QSS), notably the lasl/las-r system, contribute a vital role in the development of anti-microbial resistance (AMR). Identifying potential drugs against these targets could have significant implications for combating pseudomonal infections. The current study aims to identify promising recently FDA-approved drugs against lasl/las-r proteins. The ligands were selected from the FDA-approved drug lists of the last 5 years. Out of 202, 78 drugs were checked for interaction with lasl/las-r protein and 4 drugs revealed top binding conformations characterized by favorable energetic profiles within the active site of the las-r protein which were further assigned for 250-ns molecular dynamics (MD) simulation. The MD analysis confirmed the dynamical stability of brexanolone and oteseconazole with las-r protein. The root mean square deviation (RMSD), radius of gyration (Rg) and solvent-accessible surface area (SASA) analysis have indicated less deviation, more compactness of protein and less exposure of protein ligand complex to its surroundings as compared to the reference ligand-protein complex. The hydroxyl group in the oteseconazole whereas hydroxyl and ketone group in the brexanolone were responsible for hydrogen bonds with the active site residue of las r ptotein as indicated by ligand-protein contacts diagram. The binding energies per residue analysis revealed TYR-47 as the most contributing amino acid residue for interaction with oteseconazole and brexanolone. The identified drugs may be potential repurposing candidates against pseudomonal infections through inhibition of las-r protein. Communicated by Ramaswamy H. Sarma

Tryptophan transport gene inactivation promotes the development of antibiotic resistance in Escherichia coli.

Indole serves as a signaling molecule that could regulate different bacterial physiological processes, including antibiotic resistance through biofilm formation and drug efflux pump activity. In Escherichia coli, indole is produced through the tryptophan pathway, which involves three permeases (Mtr, AroP, and TnaB) that can transport the amino acid tryptophan. Although these permeases play distinct roles in the secretion of indole biosynthesis, their impact on multidrug resistance mediated by indole remaines unclear. This study was designed to investigate the connection between the tryptophan transport system and antibiotic resistance by constructing seven gene deletion mutants from E. coli MG1655 (wild type). Our result showed that deletion of the aroP or tnaB gene led to increased antibiotic resistance as evaluated by MICs for different antibiotics. Efflux activity test results revealed that the increased antibiotic resistance was related with the AcrAB-Tolc drug efflux pump in the mutants. The transcriptome analysis further demonstrated that decreased susceptibility to kanamycin and ampicillin in E. coli was accompanied by reduced accumulation of reactive oxygen species and decreased motility. These findings highlight the substantial influence of the tryptophan transport system on antibiotic resistance in E. coli, which is crucial for developing strategies against antibiotic resistance in bacterial infections.

Transcriptome reveals the role of the htpG gene in mediating antibiotic resistance through cell envelope modulation in Vibrio mimicus SCCF01.

HtpG, a bacterial homolog of the eukaryotic 90 kDa heat-shock protein (Hsp90), represents the simplest member of the heat shock protein family. While the significance of Hsp90 in fungal and cancer drug resistance has been confirmed, the role of HtpG in bacterial antibiotic resistance remains largely unexplored. This research aims to investigate the impact of the htpG gene on antibiotic resistance in Vibrio mimicus. Through the creation of htpG gene deletion and complementation strains, we have uncovered the essential role of htpG in regulating the structural integrity of the bacterial cell envelope. Our transcriptomics analysis demonstrates that the deletion of htpG increases the sensitivity of V. mimicus to antimicrobial peptides, primarily due to upregulated lipopolysaccharide synthesis, reduced glycerophospholipid content, and weakened efflux pumps activity. Conversely, reduced sensitivity to β-lactam antibiotics in the ΔhtpG strain results from decreased peptidoglycan synthesis and dysregulated peptidoglycan recycling and regulation. Further exploration of specific pathway components is essential for a comprehensive understanding of htpG-mediated resistance mechanisms, aiding in the development of antimicrobial agents. To our knowledge, this is the first effort to explore the relationship between htpG and drug resistance in bacteria.

Bacterial efflux pumps excrete SYTO™ dyes and lead to false-negative staining results.

Multidrug efflux pumps excrete a range of small molecules from bacterial cells. In this study, we show that bacterial efflux pumps have affinity for a range of SYTO™ dyes that are commonly used to label bacteria. Efflux pump activity will there lead to false negative results from bacterial staining and SYTO™ dyes should be used with caution on live samples.

Naturally derived efflux pump inhibitor among tetracycline-resistant Acinetobacter baumannii isolates

Abstract Background: The antimicrobial resistance of Acinetobacter baumannii is of major concern. It is one of the commonly distributed nosocomial pathogens, and its strains are frequently reported to demonstrate resistance to the most routinely prescribed antibiotics in varying degrees. Objective: This study aimed to detect the inhibitory effect of chamomile extract against the efflux pump of A. baumannii. Materials and Methods: Clinical samples (125) were collected from different sites (urine, sputum, wound, and burn swabs), followed by A. baumannii isolation and identification, and detection of tetracycline susceptibility test by disk-diffusion. Later on bacterial efflux pump activity was detected by ethidium bromide cartwheel assay and water extract of chamomile was used as an inhibitor of efflux pump A. baumannii. Results: The percentage of A. baumannii isolation 23/125 (18.4%) that distributed as 25% isolates were obtained from sputum, 17.9% isolates from burns, whereas 16% and 12.5% isolates from urine and wounds respectively. It showed various resistances against different members of the three generations of tetracyclines, with the highest resistance against minocycline 47.8% followed by resistance to tetracycline and doxycycline as 43.5% and 39%, respectively. Efflux pump was detected in all cases, water extract of chamomile showed a significant inhibitory effect especially at 80 mg/mL concentration. Conclusion: Efflux pump is an important mechanism for tetracycline resistance among A. baumannii isolates, and the watery extract of chamomile can be considered a prompt inhibitor for this pump.

Prevalence of Bacteriuria, and Characterization of Their Antimicrobial Sensitivity Pattern and Efflux Pumps Activity in Diabetic and Non-diabetic Uti Patients

Rationale: Urinary tract infections (UTI) occur more frequently in patients with type 2 diabetes. In developing countries, impairments in the defense system, metabolic disorder during diabetes, and incomplete bladder emptying all contribute to the pathogenesis of UTI in diabetic patients. This comparative study is undertaken to reveal the prevalence of uropathogens and their antimicrobial resistance patterns, and to assess hematological biomarker in diabetic and non-diabetic UTI patients. Methods: The prevalence of uropathogens were tested in 40 diabetic and 40 non-diabetic UTI patients. The antimicrobial susceptibility pattern of bacterial isolates were assessed by the Kirby-Buer disc-diffusion method. Moreover, the bacterial load, Gram-staining, biochemical and serological assays were performed using appropriate methods. Results: The findings showed that diabetic patients are more likely to UTI compared to non-diabetic patients. In terms of uropathogenic prevalence, 61% Escherichia coli, 34% Klebsiella pneumoniae, 2% Staphylococcus aureus, 2% Pseudomonas aureginosa and 1% others. The antimicrobial resistance profile demonstrated that most of the bacterial isolates were resistant to amoxicillin, ampicillin, erythromycin and methicillin but susceptible to azithromycin, chloramphenicol and ciprofloxacin. Moreover, the drug efflux pumps activity and bacterial burden were significantly high among antimicrobial resistant bacterial isolates. Besides, the C-reactive protein, Erythrocyte sedimentation rate, glycosylated hemoglobin A1C, neutrophils and lymphocytes were significantly high in diabetic UTI patients. Conclusion: This study found E. coli was the most frequent bacteria isolates, and this observation is consistent others. Diabetic UTI patients are more vulnerable with high bacterial burden and systemic inflammation. Bioresearch Commu. 10(1): 1413-1419, 2024 (January)

Type I Photosensitizer Targeting Glycans: Overcoming Biofilm Resistance by Inhibiting the Two-Component System, Quorum Sensing, and Multidrug Efflux.

Stubborn biofilm infections pose serious threats to human health due to the persistence, recurrence, and dramatically magnified antibiotic resistance. Photodynamic therapy has emerged as a promising approach to combat biofilm. Nevertheless, how to inhibit the bacterial signal transduction system and the efflux pump to conquer biofilm recurrence and resistance remains a challenging and unaddressed issue. Herein, a boric acid-functionalized lipophilic cationic type I photosensitizer, ACR-DMP, is developed, which efficiently generates •OH to overcome the hypoxic microenvironment and photodynamically eradicates methicillin-resistant Staphylococcus aureus (MRSA) and biofilms. Furthermore, it not only alters membrane potential homeostasis and osmotic pressure balance due to its strong binding ability with plasma membrane but also inhibits quorum sensing and the two-component system, reduces virulence factors, and regulates the activity of the drug efflux pump attributed to the glycan-targeting ability, helping to prevent biofilm recurrence and conquer biofilm resistance. In vivo, ACR-DMP successfully obliterates MRSA biofilms attached to implanted medical catheters, alleviates inflammation, and promotes vascularization, thereby combating infections and accelerating wound healing. This work not only provides an efficient strategy to combat stubborn biofilm infections and bacterial multidrug resistance but also offers systematic guidance for the rational design of next-generation advanced antimicrobial materials.

Assessing the Effect of Oxytetracycline on the Selection of Resistant Escherichia coli in Treated and Untreated Broiler Chickens.

Oxytetracycline (OTC) is administered in the poultry industry for the treatment of digestive and respiratory diseases. The use of OTC may contribute to the selection of resistant bacteria in the gastrointestinal tract of birds or in the environment. To determine the effect of OTC on the selection of resistant Escherichia coli strains post-treatment, bacteria were isolated from droppings and litter sampled from untreated and treated birds. Bacterial susceptibility to tetracyclines was determined by the Kirby-Bauer test. A total of 187 resistant isolates were analyzed for the presence of tet(A), (B), (C), (D), (E), and (M) genes by PCR. Fifty-four strains were analyzed by PFGE for subtyping. The proportion of tetracycline-resistant E. coli strains isolated was 42.88%. The susceptibility of the strains was treatment-dependent. A high clonal diversity was observed, with the tet(A) gene being the most prevalent, followed by tet(C). Even at therapeutic doses, there is selection pressure on resistant E. coli strains. The most prevalent resistance genes were tet(A) and tet(C), which could suggest that one of the main mechanisms of resistance of E. coli to tetracyclines is through active efflux pumps.

Isolation and identification of antimicrobial susceptibility, biofilm formation, efflux pump activity, and virulence determinants in multi-drug resistant Pseudomonas aeruginosa isolated from freshwater fishes.

The present study was undertaken to evaluate the prevalence, underlying resistance mechanism, and virulence involved in Pseudomonas aeruginosa (n = 35) isolated from freshwater fishes in Andhra Pradesh, India. Antibiogram studies revealed that 68.5, 62.8, 37.1, 11.4, 8.5, 57.1, 54.2, and 48.5% of isolates had resistance to oxytetracycline, co-trimoxazole, doxycycline, enrofloxacin, ciprofloxacin, cefotaxime, ceftazidime, and ampicillin, respectively. The resistant isolates harboured the tetA (85.7%), tetD (71.4%), tetM (91.4%), sul1 (80%), blaCTX-M (57.1%), blaTEM (42.8%), and blaSHV (48.5%) genes. In total, 50% of the isolates were altered as multi-drug resistant, and the multiple antibiotic resistance index was calculated as 0.4. Furthermore, 37.3, 48.5, and 14.2% of isolates were categorized as strong, moderate, and weak biofilm formers, possessing pslA (91.5%) and pslD (88.6%) biofilm encoding genes. In total, 82.8% of the isolates exhibited efflux pump activity and harboured the mexA (74.2%), mexB (77.1%), and oprM (37.1%) genes. Virulent genes oprL, toxA, exoS, and phzM were detected in 68.5, 68.5, 100, and 17.1% of isolates, respectively. The data suggested that P. aeruginosa harbours multiple resistance mechanisms and virulence factors that may contribute to antibiotic resistance and pathogenicity, and their distribution in fish culture facilities highlights the public health hazards of the food chain.

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.

Colistin Resistance among Enterobacterales Isolates: Underlying Mechanisms and Alternative Treatment Options

Global dissemination of multidrug-resistant (MDR) and extensively drug-resistant (XDR) Gram-negative bacteria (GNB) such as carbapenemase-producing Enterobacterales has resulted in reviving colistin as a final therapeutic alternative. Colistin resistance foretold a catastrophe. We aimed to detect the rates of carbapenems and colistin resistance among hospital-acquired Enterobacterales species, verify the underlying mechanisms and provide antibiogram for colistin-resistant isolates. The collected Enterobacterales isolates were tested for their antimicrobial susceptibility by the disk diffusion method and agar dilution was utilized for both imipenem and colistin. The production of ESβLs and carbapenemases was phenotypically assessed by the combined disk (CDT) and modified carbapenem inactivation (mCIM) tests, respectively. Possible attributes for colistin resistance were explored by detection of both plasmid- and efflux pump-mediated mechanisms. By multiplex PCR assay, carbapenem resistance (blaNDM-1 &amp; blaOXA-48) and mobilized colistin-resistant-1 (mcr-1) genes were identified. A total of 160 Enterobacterales isolates were obtained of which 68.8% were MDR, 25% were XDR and 6.3% were pandrug-resistant (PDR) isolates with no statistically significant difference among Enterobacterales species (P&gt; 0.05). Carbapenems resistance was detected in 41.3% (66/160) while colistin resistance was detected in 22% (36/160) of isolates. Proteus mirabilis expressed the highest rate of colistin resistance (100%; 16/16), followed by Enterobacter aerogenes (23.1%; 6/26), E. coli (13%; 6/46) and K.pneumoniae (11.1%; 8/72). One hundred percent (36/36) of colistin-resistant isolates proved efflux pump activity for colistin. However; only 2% (2/100) of tested Enterobacterales carried mcr-1 gene through molecular analysis. Colistin-resistant isolates exhibited variable susceptibility to the tested antimicrobial agents of which fosfomycin was the highest (94.1%). Efflux pump activity played a major role for colistin resistance among Enterobacterales species and fosfomycin could be a promising therapeutic option.

A Review of the Resistance Mechanisms for β-Lactams, Macrolides and Fluoroquinolones among Streptococcus pneumoniae.

Streptococcus pneumoniae (S. pneumoniae) is a bacterial species often associated with the occurrence of community-acquired pneumonia (CAP). CAP refers to a specific kind of pneumonia that occurs in individuals who acquire the infection outside of a healthcare setting. It represents the leading cause of both death and morbidity on a global scale. Moreover, the declaration of S. pneumoniae as one of the 12 leading pathogens was made by the World Health Organization (WHO) in 2017. Antibiotics like β-lactams, macrolides, and fluoroquinolones are the primary classes of antimicrobial medicines used for the treatment of S. pneumoniae infections. Nevertheless, the efficacy of these antibiotics is diminishing as a result of the establishment of resistance in S. pneumoniae against these antimicrobial agents. In 2019, the WHO declared that antibiotic resistance was among the top 10 hazards to worldwide health. It is believed that penicillin-binding protein genetic alteration causes β-lactam antibiotic resistance. Ribosomal target site alterations and active efflux pumps cause macrolide resistance. Numerous factors, including the accumulation of mutations, enhanced efflux mechanisms, and plasmid gene acquisition, cause fluoroquinolone resistance. Furthermore, despite the advancements in pneumococcal vaccinations and artificial intelligence (AI), it is not feasible for individuals to rely on them indefinitely. The ongoing development of AI for combating antimicrobial resistance necessitates more research and development efforts. A few strategies can be performed to curb this resistance issue, including providing educational initiatives and guidelines, conducting surveillance, and establishing new antibiotics targeting another part of the bacteria. Hence, understanding the resistance mechanism of S. pneumoniae may aid researchers in developing a more efficacious antibiotic in future endeavors.