Efflux Pump Genes Research Articles

Mutations in the efflux regulator gene oqxR provide a simple genetic switch for antimicrobial resistance in Klebsiella pneumoniae.

Klebsiella pneumoniae is a pathogen of major concern in the global rise of antimicrobial resistance and has been implicated as a reservoir for the transfer of resistance genes between species. The upregulation of efflux pumps is a particularly concerning mechanism of resistance acquisition as, in many instances, a single point mutation can simultaneously provide resistance to a range of antimicrobials and biocides. The current study investigated mutations in oqxR, which encodes a negative regulator of the RND-family efflux pump genes, oqxAB, natively found in the chromosome of K. pneumoniae. Resistant mutants in four K. pneumoniae strains (KP6870155, NTUH-K2044, SGH10, and ATCC43816) were selected from single exposures to 30 µg/mL chloramphenicol and 12 mutants were selected for whole genome sequencing to identify mutations associated with resistance. Resistant mutants generated by single exposures to chloramphenicol, tetracycline, or ciprofloxacin at ≥4 X MIC were replica plated onto all three antibiotics to observe simultaneous cross-resistance to all compounds, indicative of a multidrug resistance phenotype. A variety of novel mutations, including single point mutations, deletions, and insertions, were found to disrupt oqxR leading to significant and simultaneous increases in resistance to chloramphenicol, tetracycline, and ciprofloxacin. The oqxAB-oqxR locus has been mobilized and dispersed on plasmids in many Enterobacteriaceae species and the diversity of these loci was examined to evaluate the evolutionary pressures acting on these genes. Comparison of the promoter regions of oqxR in plasmid-borne copies of the oqxR-oqxAB operon indicated that some constructs may produce truncated versions of the oqxR transcript, which may impact on oqxAB regulation and expression. In some instances, co-carriage of chromosomal and plasmid encoded oqxAB-oqxR was found in K. pneumoniae, implying that there is selective pressure to maintain and expand the efflux pump. Given that OqxR is a repressor of oqxAB, any mutation affecting its expression or function can lead to multidrug resistance. This is in contrast to antibiotic target site mutations that must occur in limited sequence space to be effective and not impact the fitness of the cell. Therefore, oqxR may act as a simple genetic switch to facilitate resistance via OqxAB mediated efflux.

Sublethal Sodium Hypochlorite Exposure: Impact on Resistance-Nodulation-Cell Division Efflux Pump Overexpression and Cross-Resistance to Imipenem.

Sodium hypochlorite (NaOCl) is widely used in public healthcare facilities; this exposure can result in the development of bacterial tolerance to disinfectants, which has known links to antibiotic cross-resistance. However, the mechanism through which cross-resistance to antibiotics and disinfectants develops remains ambiguous. Therefore, this study aimed to examine the phenotypic and transcriptomic changes caused by disinfectant exposure in Gram-negative bacteria and determine the cause of cross-resistance to antibiotics. The results demonstrated that the misuse of disinfectants plays an important role in the emergence of disinfectant resistance and in the increase in antibiotic resistance. Antibiotic resistance may occur from the exposure of Gram-negative bacteria to subminimal inhibitory concentrations (MICs) of NaOCl. Ten passages of Gram-negative bacteria in increasingly higher subMICs of the NaOCl disinfectant were sufficient to increase the MIC to >2500 µg/mL NaOCl, particularly in K. pneumoniae and P. aeruginosa. To determine the development of cross-resistance to antibiotics due to NaOCl exposure, the MICs for each antibiotic before and after the exposure of each strain to sublethal concentrations of NaOCl were compared. After overnight incubation with a sublethal concentration of NaOCl, a statistically significant increase in MIC was only observed for imipenem (p < 0.01). An investigation of the mechanism of cross-resistance by means of transcriptome analysis revealed that 1250 µg/mL of NaOCl-adapted K. pneumoniae and P. aeruginosa strains increased resistance to imipenem due to the increased expression of resistance-nodulation-cell division (RND) efflux pumps, such as AcrAB-TolC and MexAB/XY-OprM. Therefore, we suggest that exposure to NaOCl can influence the expression of RND efflux pump genes, contributing to imipenem cross-resistance.

First report of a carbapenem-resistant Aeromonas veronii environmental isolate in the United States co-harboring two carbapenemase genes

ObjectivesThe spread of carbapenem-resistant bacteria (CRB), and especially carbapenemase-producing CRB, is a global public health threat. Among them, Aeromonas species are of increasing concern because these emerging opportunistic pathogens are widespread in the environment and have increasingly been found to be resistant to carbapenems. The aim of this study was to investigate the genome and carbapenem-resistance determinants of Aeromonas veronii SS-M2–3, a highly carbapenem-resistant, carbapenemase-producing, river isolate from California (U.S.). MethodsWe first used disk diffusion assays to characterize the susceptibility profile to carbapenems and other antibiotics of A. veronii SS-M2–3. We next used whole-genome sequencing using the Illumina platform and bioinformatics analysis to characterize the resistome of this isolate and identify its carbapenemase genes. ResultsA. veronii SS-M2–3 was resistant to all carbapenems tested and amoxicillin-clavulanic acid, whereas it was sensitive to cefotaxime and all non-β-lactam antibiotics tested. Whole genome sequencing of this isolate revealed a complex resistome that included multidrug efflux pump genes and three chromosomal β-lactamase genes. These three genes encoded for highly conserved variants (82% to 97% amino acid identity) of the ChpA3 subclass B2 metallo-carbapenemase, OXA-12 class D carbapenemase and the FOX-2 class C β-lactamase. This is the first report of an environmental A. veronni isolate from the U.S. co-harbouring two carbapenemase genes. ConclusionsThese findings reveal that natural aquatic environments in the U.S. represent an underappreciated reservoir of carbapenem-resistant Aeromonas veronii isolates that can carry multiple carbapenemase genes.

Unravelling the complex mechanisms of multidrug resistance in bovine mastitis pathogens: Insights into antimicrobial resistance genes, biofilm dynamics, and efflux systems

Mastitis remains a paramount economic threat to dairy livestock, with antibiotic resistance severely compromising treatment efficacy. This study provides an in-depth investigation into the multidrug resistance (MDR) mechanisms in bacterial isolates from bovine mastitis, emphasizing the roles of antimicrobial resistance genes (ARGs), biofilm formation, and active efflux systems. A total of 162 Staphylococci, eight Escherichia coli, and seven Klebsiella spp. isolates were obtained from 215 milk samples of clinical and subclinical mastitis cases. Antibiotic susceptibility testing identified Twenty Staphylococci (12.35 %), six E. coli (75 %) and seven Klebsiella (100 %) identified as MDR displaying significant resistance to β-lactams and tetracyclines The Multiple Antibiotic Resistance (MAR) index of these isolates ranged from 0.375 to 1.0, highlighting extensive resistance. Notably, 29 of the 33 MDR isolates produced biofilms on Congo red agar, while all exhibited biofilm formation in the Microtitre Plate assay. Critical ARGs (blaZ, blaTEM, blaCTX-M, tetM, tetA, tetB, tetC, strA/B, aadA) and efflux pump genes (acrB, acrE, acrF, emrB, norB) regulating active efflux were identified. This pioneering study elucidates the synergistic contribution of ARGs, biofilm production, and efflux pump activity to MDR in bovine mastitis pathogens. To our knowledge, this comprehensive study is the first of its kind, offering novel insights into the complex resistance mechanisms. The findings underscore the imperative need for advanced antibiotic stewardship and strategic interventions in dairy farming to curb the rise of antibiotic-resistant infections, thereby protecting both animal and public health.

A first study of meat-borne enterococci from butcher shops: prevalence, virulence characteristics, antibiotic resistance and clonal relationship.

IntroductionEnterococcus, which used to be thought of as a harmless commensal living in the digestive tract, has now become a highly resistant and highly contagious pathogen that makes nosocomial infections much more common. This study examined enterococci species and their antibiotic resistance phenotypes and genotypes and virulence gene content in Turkish ground beef samples. Methodology A total of 100 ground beef samples were analyzed between May 2020 and May 2021. The isolated strains were identified via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and confirmed using polymerase chain reaction (PCR) after which they were divided into several species using PCR and tested for antibiotic resistance against 19 antimicrobial agents using the disc diffusion method. The genetic similarity analysis, pulsed-field gel electrophoresis (PFGE) was performed. Results A total of 93 isolates in ground beef were identified, comprised of E. faecalis 72.04%; E. hirae- 11.82%; E. casseliflavous- 7.52%; E. faecium- 5.3%; E. gallinarium- 3.23%. The virulence genes observed in Enterococcus species were distributed as follows: gelE 88.1%, ace 53.7%, efaA 40.8%, asaI 19.3%, esp 6.4%, and cylA 1.07%. A high antibiotic resistance was recorded for tetracycline (43.01%), followed by ampicilin (17.2%), and chloramphenicol (13.9%). 17.2% of Enterococcus isolates were multidrug-resistant. The study determined the high prevalence of antibiotic resistance genes, specifically for tet(L) 10 (10.7%), aac(6')Ie-aph(2")-la 3 (3.2%), and ermB 3 (3.2%). The presence of efflux pump genes were identified in 74.1% of Enterococcus isolates. Genetic characterization of 67 E. faecalis isolates by PFGE revealed 41 pulsed-field gel electrophoresis (PFGE) patterns that were grouped into 15 clusters, which presented more than one strain with 100% similarity. Conclusion Isolates obtained from several areas and butchers had comparable patterns of PFGE, suggesting that the presence of circulating E. faecalis poses a potential public health concern in diverse districts. To mitigate the health hazards associated with the contamination of enterococci from raw to cooked meats, it is necessary to enhance the disinfection of butcheries, promote excellent hand hygiene among butchers, and implement appropriate meat storage and handling methods to prevent bacterial development.

Metal tolerance in enterococci isolated from seabirds in Abrolhos Archipelago, Brazil: Evaluating their role as bioindicators of marine pollution

Microbiota exposed to pollution provide insights into host physiology and ecosystem disruption. This study evaluated Enterococcus spp. tolerant to arsenic (As), copper (Cu), and mercury (Hg) from red-billed tropicbirds (Phaethon aethereus) and brown boobies (Sula leucogaster), which previously showed these metals in their blood and feathers, and their potential use as bioindicators of metal contamination. Enterococcus casseliflavus (47.9 %), E. faecalis (34.1 %), E. hirae (11.7 %), and E. faecium (5.3 %) were identified. Both seabird species had a high incidence of As-tolerant bacteria (84.0 %), with 40.4 % of these strains containing As efflux system genes (arsA_I and arsA_II). Cu efflux pump gene (tcrB) was detected in 30.9 % of strains, while Hg reductase genes (mer) were not found. As- and Cu-tolerance in enterococci observed in this study underlines their potential as bioindicators in metal-polluted marine environments. Further research may elucidate the role of these metal-tolerant enterococci in seabird gut and their adaptability to polluted environments.

Green synthesized silver nanoparticles from Ocimum sanctum: A potent inhibitor of biofilm forming ability and efflux pumps in bacteria causing bovine mastitis

Therapeutic management of mastitis faces significant challenges due to multidrug resistance. In the present study, multi-drug-resistant (MDR) Staphylococcus spp, Klebsiella pneumoniae, and Escherichia coli were isolated from bovine clinical mastitis cases and the phenotypic and genotypic multidrug resistance profiling was carried out. Silver nanoparticles (AgNPs) were biosynthesized using Ocimum sanctum leaf extracts and characterized via UV Vis absorption, Fourier Transform Infrared Spectroscopy, X-ray diffraction studies, Energy dispersive spectroscopy and Electron Microscopy. The determined minimum inhibitory concentration and minimum bactericidal concentration of the AgNPs against the recovered MDR isolates were 62.5 μg/ml and 125 μg/ml respectively. At a concentration of 50 μg/ml, the AgNPs demonstrated biofilm inhibitory activities of 80.35 % for MDR E. coli, 71.29 % for S. aureus and 60.18 % for MDR K. pneumoniae. Post-treatment observations revealed notable differences in biofilm formation across bacterial isolates. Furthermore, AgNP treatment led to significant downregulation of expression of the efflux pump genes acrB, acrE, acrF, and emrB in Gram-negative isolates and norB in Staphylococci isolates. This research underscores the potential for the development of an eco-friendly antimicrobial alternative in the form of green synthesized silver nanoparticles to combat drug resistance offering potential antibiofilm and efflux pump inhibitory activities.

Downregulation of Klebsiella pneumoniae RND efflux pump genes following indole signal produced by Escherichia coli

BackgroundMore than a century has passed since it was discovered that many bacteria produce indole, but research into the actual biological roles of this molecule is just now beginning. The influence of indole on bacterial virulence was extensively investigated in indole-producing bacteria like Escherichia coli. To gain a deeper comprehension of its functional role, this study investigated how indole at concentrations of 0.5-1.0 mM found in the supernatant of Escherichia coli stationary phase culture was able to alter the virulence of non-indole-producing bacteria, such as Pseudomonas aeruginosa, Proteus mirabilis, and Klebsiella pneumoniae, which are naturally exposed to indole in mixed infections with Escherichia coli.ResultsBiofilm formation, antimicrobial susceptibility, and efflux pump activity were the three phenotypic tests that were assessed. Indole was found to influence antibiotic susceptibly of Pseudomonas aeruginosa, Proteus mirabilis and Klebsiella pneumoniae to ciprofloxacin, imipenem, ceftriaxone, ceftazidime, and amikacin through significant reduction in MIC with fold change ranged from 4 to 16. Biofilm production was partially abrogated in both 32/45 Pseudomonas aeruginosa and all eight Proteus mirabilis, while induced biofilm production was observed in 30/40 Klebsiella pneumoniae. Moreover, acrAB and oqxAB, which encode four genes responsible for resistance-nodulation-division multidrug efflux pumps in five isolates of Klebsiella pneumoniae were investigated genotypically using quantitative real-time (qRT)-PCR. This revealed that all four genes exhibited reduced expression indicated by 2^−ΔΔCT < 1 in indole-treated isolates compared to control group.ConclusionThe outcomes of qRT-PCR investigation of efflux pump expression have established a novel clear correlation of the molecular mechanism that lies beneath the influence of indole on bacterial antibiotic tolerance. This research provides novel perspectives on the various mechanisms and diverse biological functions of indole signaling and how it impacts the pathogenicity of non-indole-producing bacteria.

Proposition of a phagosensor with a unique Teseptimavirus SAL_R1S on a carbon nanotube platform for efficient detection of typhoid pathogen

The detection of the typhoid pathogen, Salmonella enterica serotype Typhi (S. Typhi), holds massive clinical, public health, and epidemiological significance around the globe. Conventional diagnosis relies on bacterial isolation having a set of challenges when it comes to accurate detection, therapeutic intervention and disease management. Substantial reviews and reports exist on the advantages of bacteriophage-based biosensors (phagosensors) concerning Salmonella. However, phagosensor for Salmonella Typhi point of care detection at a lower limit of detection (LOD) has yet to be reported. This study is the earliest endeavor to develop a multi-wall carbon nanotubes (MWCNTs) based electrochemical phagosensor utilizing a unique bacteriophage SAL_R1S as a biomolecular recognition element, selectively binding S. Typhi DMS_A1 at LOD of 1 CFU/ml. S. Typhi DMS_A1, retrieved from patient's blood, consists of 10 pathogenicity islands and a wide range of efflux pump genes in its whole genome, which has not yet been documented for Salmonella. Subsequent screening for its specific bacteriophage from a sewage sample pinpointed the phage SAL_R1S of class Caudoviricetes, family Autographiviridae and genus Teseptimavirus. The whole genome- and tail-fiber protein- based alignment was close to Salmonella phage Vi06 covering 88.8 % and 90 % similarity, respectively. SAL_R1S exclusively binds S. Typhi in a specific manner and also possess excellent genetic feature as a candidate for developing a highly sensitive electrochemical phagosensor. Therefore, it was covalently immobilized onto a modified SPE/MWCNT/PANI-based electrode surface, allowing charge-directed, oriented immobilization which then confirmed through scanning electron microscopy. The electrode surface was featured via field emission scanning electron microscopy, electrochemical impedance spectroscopy, and cyclic voltammetry. The pathogen detection process of the phagosensor is quick (∼ 20 min). It has exceptional selectivity for typhoid pathogens from blood, wastewater or within mixed populations, indicating the application of this proposed phagosensor in clinical settings as a rapid, alternative to available conventional detection techniques, and low-cost surveillance tool.

Effect of Mentha longifolia essential oil on oqxA efflux pump gene expression and biofilm formation in ciprofloxacin-resistant Klebsiella pneumoniae strains.

Today, medicinal plants and their derivatives are considered to reduce the prevalence of antibiotic resistance. The aim of this study was to investigate the effect of Mentha longifolia essential oil on oqxA efflux pump gene expression and biofilm formation in ciprofloxacin-resistant Klebsiella pneumoniae strains. A total of 50 clinical strains of K. pneumoniae resistant to ciprofloxacin were studied. The minimum inhibitory concentration (MIC) of M. longifolia essential oil and its synergistic effect with ciprofloxacin were determined using the microbroth dilution method and the fractional inhibitory concentration (FIC) method. Minimum biofilm inhibition concentration (MBIC) of M. longifolia essential oil was detected. The effect of essential oils on the expression level of the oqxA gene was detected by Real-time PCR. M. longifolia essential oil showed inhibitory activity against ciprofloxacin-resistant strains of K. pneumoniae. When M. longifolia essential oil was combined with ciprofloxacin, the MIC was reduced 2-4 times. In 28% of the strains, M. longifolia with ciprofloxacin showed a synergistic effect. M. longifolia essential oil reduces the strength of biofilm formation and alters the biofilm phenotype. A significant decrease in oqxA gene expression was observed in all isolates after treatment with M. longifolia essential oil. Based on the results of this study, it was observed that supplementing M. longifolia essential oil can help reduce ciprofloxacin resistance and inhibit biofilm formation in fluoroquinolone-resistant K. pneumoniae strains.

Identification of novel drug targets to counteract efflux pump mediated multidrug resistance in Acinetobacter baumannii

The emergence of multidrug-resistant (MDR) Acinetobacter baumannii poses an escalating threat to the healthcare system worldwide. A significant factor contributing to increasing resistance is the overexpression of chromosomally encoded efflux pumps, which expel antibiotics from bacterial cells, thereby rendering treatments less effective. The efflux pumps not only mediate resistance to antibiotics through drug efflux but also work synergistically with other resistance mechanisms, thereby doubling the resistance. Despite their crucial role in antibiotic resistance, understanding of the structure, function, mechanisms of action, and regulation of efflux pumps remains limited, which is necessary for devising effective strategies to restore drug susceptibility and to combat MDR isolates. In this context, the present study evaluated the prevalence of efflux pump overexpression in clinical A. baumannii isolates using phenotypic and genotypic methods and identified potential therapeutic targets employing a network-based approach. A total of 172 A. baumannii isolates were collected and subjected to antibiotic susceptibility tests using the Kirby-Bauer disk diffusion method. All the isolates were found to be MDR, with 94.76 % showing resistance to carbapenems. Efflux pump overexpression was detected in 54.65 % of isolates using the Ethidium-Bromide Agar Cartwheel method, and efflux pump inhibitory activity was observed in 68.71 % of isolates using cyanide m-chlorophenylhydrazone (CCCP). A total of thirteen efflux pump genes were detected in the tested isolates using diagnostic PCR, which were considered for interaction network analysis using STRING. Clustering analysis of the merged network identified two highly interconnected clusters, each comprising functional partners crucial for efflux pump function and regulation. Key hub genes, including AdeB, AdeJ, AdeK, AdeC, macB, tolC, AIL80285.1, AdeR, and AdeS, were identified as primary targets due to their significant influence on the network. Additionally, 24 clustered genes were pinpointed as potential drug targets for developing novel therapeutics to combat the formidable challenge of efflux pump-mediated MDR in A. baumannii.

Identification and characterisation of two functional antibiotic MATE efflux pumps in the archaeon Halorubrum amylolyticum

Multidrug efflux pumps have been found to play a crucial role in drug resistance in bacteria and eukaryotes. In this study, we investigated the presence of functional multidrug and toxic compound extrusion (MATE) efflux pumps, inferred from whole genome sequencing, in the halophilic archaeon Halorubrum amylolyticum CSM52 using Hoechst 33342 dye accumulation and antimicrobial sensitivity tests in the presence and absence of efflux pump inhibitors (EPIs). The whole genome sequence of H. amylolyticum CSM52 contained two putative MATE-type efflux pump genes, which may contribute to the inherent resistance to conventional antimicrobial agents reported in archaea. Antimicrobial susceptibility of the wild-type H. amylolyticum CSM52 testing revealed a lack of sensitivity to a wide range of antimicrobials, including glycopeptides, aminoglycosides, macrolides, fluoroquinolones, tetracycline, and chloramphenicol. However, the presence of EPIs, such as thioridazine, fluoxetine, and chlorpromazine, significantly increased the susceptibility of H. amylolyticum CSM52 to a number of these antimicrobials, indicating the potential involvement of efflux pumps in the observed resistance. A molecular modelling study with EPIs and substrate antimicrobials provided important insights into the molecular interactions with the putative transporter. It suggests that the occupancy of the transporter channel by EPIs has the potential to impact the efflux of antimicrobials. Phylogenetic analysis of the amino acid sequences of both MATE pumps showed low similarity with bacterial representatives, suggesting the presence of novel and distinct MATE efflux pumps in archaea. Our findings provide the first experimental evidence of active antibiotic efflux mechanisms in archaea and their potential roles in antimicrobial resistance, broadening our understanding of mechanisms of archaeal antimicrobial resistance, an overlooked aspect of AMR research.

Small RNA-regulated expression of efflux pump affects tigecycline resistance and heteroresistance in clinical isolates of Klebsiella pneumoniae

Tigecycline and the newly Food and Drug Administration-approved tetracyclines, including eravacycline and omadacycline, are regarded as last-resort treatments for multidrug-resistant Enterobacterales. However, tigecycline resistance in Klebsiella pneumoniae has increased, especially the underlying mechanism of heteroresistance is unclear. This study aimed to elucidate the mechanisms underlying tigecycline resistance and heteroresistance in clinical K. pneumoniae isolates. A total of 153 clinical K. pneumoniae isolates were collected, and identified 15 tigecycline-resistant and three tigecycline-heteroresistant isolates using broth microdilution and population analysis profile methods, respectively. Total RNAs from K. pneumoniae ATCC13883 and the laboratory-induced tigecycline-resistant strain were extracted and sequenced on an Illumina platform. Differentially expressed genes and regulatory small RNAs (sRNAs) were analyzed and validated in clinical isolates of K. pneumoniae using quantitative real-time PCR. RNA sequencing results showed that mdtABC efflux pump genes were significantly upregulated in the tigecycline-resistant strains. Overexpression of mdtABC was observed in a clinical K. pneumoniae isolate, which increased tigecycline minimum inhibitory concentrations (MICs) and was involved in tigecycline heteroresistance. Sequencing analysis of sRNA demonstrated that candidate sRNA-120 directly interacted with the mdtABC operon and was downregulated in tigecycline-resistant strains. We generated an sRNA-120 deletion mutation strain and a complemented strain of K. pneumoniae. The sRNA-120 deletion strain displayed increased mRNA levels of mdtA, mdtB, and mdtC and an increase in MICs of tigecycline. The complemented strain of sRNA-120 restored the mRNA levels of these genes and the susceptibility to tigecycline. RNA antisense purification and parallel reaction monitoring mass spectrometry were performed to verify the interactions between sRNA-120 and mdtABC. Collectively, our study highlights that the post-transcriptional repression of mdtABC through sRNA-120 may provide an additional layer of efflux pump gene expression control, which is important for resistance and heteroresistance in clinical K. pneumoniae isolates.

Ocimum basilicum and Lagenaria siceraria Loaded Lignin Nanoparticles as Versatile Antioxidant, Immune Modulatory, Anti-Efflux, and Antimicrobial Agents for Combating Multidrug-Resistant Bacteria and Fungi.

Lignin nanoparticles emerged as a promising alternative for drug delivery systems owing to their biodegradability and bioactive properties. This study investigated the antimicrobial activity of the ethanolic extract of Ocimum basilicum-loaded lignin nanoparticles (OB-LNPs) and Lagenaria siceraria seed oil-loaded lignin nanoparticles (LS-LNPs) to find a solution for antimicrobial resistance. OB-LNPs and LS-LNPs were tested for their antimicrobial potential against Escherichia coli, Enterococcus faecalis, Klebsiella pneumoniae, Staphylococcus aureus, Salmonella enterica, Trichophyton mentagrophytes, Trichophyton rubrum, and Microsporum canis. OB-LNPs and LS-LNPs were further tested for their anti-efflux activity against ciprofloxacin-resistant Salmonella enterica strains and for treating Salmonella infection in a rat model. We also investigated the antifungal efficacy of OB-LNPs and LS-LNPs for treating T. rubrum infection in a guinea pig model. Both OB-LNPs and LS-LNPs showed strong antimicrobial potential against S. Typhimurium and T. rubrum infections. LS-LNPs showed antibacterial activity against Salmonella enterica species with a MIC range of 0.5-4 µg/mL and antifungal activity against T. rubrum with a MIC range of 0.125-1 µg/mL. OB-LNPs showed antibacterial activity against Salmonella enterica species with a MIC range of 0.5-2 µg/mL and antifungal activity against T. rubrum with a MIC range of 0.25-2 µg/mL. OB-LNPs and LS-LNPs downregulated the expression of ramA and acrB efflux pump genes (fold change values ranged from 0.2989 to 0.5434; 0.4601 to 0.4730 for ramA and 0.3842-0.6199; 0.5035-0.8351 for acrB). Oral administration of OB-LNPs and LS-LNPs in combination with ciprofloxacin had a significant effect on all blood parameters, as well as on liver and kidney function parameters. Oxidative stress mediators, total antioxidant capacity, and malondialdehyde were abolished by oral administration of OB-LNPs and LS-LNPs (0.5 mL/rat once daily for 5 days). Interferon-γ and tumor necrosis factor-α were also reduced in comparison with the positive control group and the ciprofloxacin-treated group. Histopathological examination of the liver and intestine of OB-LNPs and LS-LNPs-treated rats revealed an elevation in Salmonella clearance. Treatment of T. rubrum-infected guinea pigs with OB-LNPs and LS-LNPs topically in combination with itraconazole resulted in a reduction in lesion scores, microscopy, and culture results. In conclusion, OB-LNPs and LS-LNPs possess immunomodulatory and antioxidant potential and can be used as naturally derived nanoparticles for drug delivery and treatment of Salmonellosis and dermatophytosis infections.

Emergence of a clinical Klebsiella pneumoniae harboring an acrAB-tolC in chromosome and carrying the two repetitive tandem core structures for bla KPC-2 and bla CTX-M-65 in a plasmid.

The emergence of clinical Klebsiella pneumoniae strains harboring acrAB-tolC genes in the chromosome, along with the presence of two repetitive tandem core structures for bla KPC-2 and bla CTX-M-65 genes on a plasmid, has presented a significant clinical challenge. In order to study the detailed genetic features of K. pneumoniae strain SC35, both the bacterial chromosome and plasmids were sequenced using Illumina and nanopore platforms. Furthermore, bioinformatics methods were employed to analyze the mobile genetic elements associated with antibiotic resistance genes. K. pneumoniae strain SC35 was found to possess a class A beta-lactamase and demonstrated resistance to all tested antibiotics. This resistance was attributed to the presence of efflux pump genes, specifically acrAB-tolC, on the SC35 chromosome. Additionally, the SC35 plasmid p1 carried the two repetitive tandem core structures for bla KPC-2 and bla CTX-M-65, as well as bla TEM-1 with rmtB, which shared overlapping structures with mobile genetic elements as In413, Tn3, and TnAs3. Through plasmid transfer assays, it was determined that the SC35 plasmid p1 could be successfully transferred with an average conjugation frequency of 6.85 × 10-4. The structure of the SC35 plasmid p1 appears to have evolved in correlation with other plasmids such as pKPC2_130119, pDD01754-2, and F4_plasmid pA. The infectious strain SC35 exhibits no susceptibility to tested antibioticst, thus effective measures should be taken to prevent the spread and epidemic of this strain.

Antimicrobial resistance, biofilm formation, and molecular detection of efflux pump and biofilm genes among Klebsiella pneumoniae clinical isolates from Northern Jordan

The current study aimed to investigate the antimicrobial susceptibility profiles, biofilm production capabilities, and the prevalence of efflux pump and biofilm-associated genes among Klebsiella pneumoniae clinical isolates. One hundred sixty-seven K. pneumoniae isolates were collected from microbiology laboratories in Northern Jordan hospitals. Antimicrobial susceptibility was tested using the Kirby-Bauer method. The double-disk synergy test was used to detect the extended-spectrum beta-lactamase (ESBL) phenotype. PCR was used to detect the frequency of acrAB,tolC, and mdtk efflux pump genes and fimH-1,mrkA, and mrkD biofilm-associated genes among the isolates. The highest nonsusceptibility was observed against azithromycin (87.4 %) and nitrofurantoin (85.0 %). Among the isolates, 75.4 % and 92.2 % were multidrug resistant and produced biofilms, respectively. Efflux pump genes acrAB, tolC, and mdtK were found in 96.4 %, 95.2 %, and 90.4 % of the isolates, respectively. Biofilm-associated genes mrkD, mrkA, and fimH-1 were found in 92.2 %, 89.2 %, and 88.6 % of the isolates, respectively. The presence of the mrkA was significantly associated with biofilm formation. Overall, high percentages of multi-drug resistance, efflux pump, and biofilm-associated genes were observed among the isolates. Subsequent studies are recommended to monitor changes in the prevalence of resistance phenotypes and genotypes of isolates.

Efflux pumps encoding genes (adeA and adeS) in relation to antibiotic resistance pattern in Acinetobacter baumannii strains isolated from Benha university hospital

Background Acinetobacter baumannii become a growing problem in hospitals as a predominant multidrug-resistant (MDR). This resistance capacity is generated by various mechanisms including efflux pumps that can direct antibiotics outwards and prevent antibiotics from affecting the bacteria. Methods Clinical samples were cultured on blood agar and MacConkey agar medium, identified by Gram stain and biochemical reactions, and then identified to the species level by Vitek2 automated system. The adeA and adeS genes among isolated strains were detected by conventional PCR. This study included 50 A. baumannii strains to assess the antibiotic resistance pattern of A. baumannii. Results A. baumannii strains were fully resistant to Piperacillin (100%) and lowest resistance to Imipenem (54%) was observed. Out of 50 isolates, 41 (82%) of A. baumanii isolates had adeA gene and 32 (64%) had adeS gene. The clinical strains that had adeA gene and adeS gene showed significantly higher resistance to Ciprofloxacin (92.7%) and (100%), respectively. Additionally, borderline significant differences were reported regarding Gentamycin (P = 0.065), Ceftazidime (P = 0.08), and Meropenem (P = 0.08), with the resistance being higher in the adeA positive group. Conclusion Efflux pumps genes (adeA, adeS) played a key role in antibiotic resistance of A. baumanii.

Characterization of ST15-KL112 Klebsiella pneumoniae Co-Harboring Bla oxa-232 and rmtF in China.

This study aimed to investigate the emergence and characteristics of carbapenem-resistant Klebsiella pneumoniae (CRKP) strains that demonstrate resistance to multiple antibiotics, including aminoglycosides and tigecycline, in a Chinese hospital. A group of ten CRKP strains were collected from the nine patients in a Chinese hospital. Antimicrobial Susceptibility Testing (AST) and phenotypic inhibition assays precisely assess bacterial antibiotic resistance. Real-time quantitative PCR (RT-qPCR) was used to analyze the mRNA levels of efflux pump genes (acrA/acrB and oqxA/oqxB) and the regulatory gene (ramA). The core-genome tree and PFGE patterns were analyzed to assess the clonal and horizontal transfer expansion of the strains. Whole-genome sequencing was performed on a clinical isolate of K. pneumoniae named Kpn20 to identify key resistance genes and antimicrobial resistance islands (ARI). The CRKP strains showed high resistance to carbapenems, aminoglycosides (CLSI, 2024), and tigecycline (EUCAST, 2024). The mRNA expression levels of efflux pump genes and regulatory genes were detected by RT-qPCR. All 10 isolates had significant differences compared to the control group of ATCC13883. The core-genome tree and PFGE patterns revealed five clusters, indicating clonal and horizontal transfer expansion. Three key resistance genes (blaoxa-232, blaCTX-M-15 , and rmtF) were observed in the K. pneumoniae clinical isolate Kpn20. Mobile antibiotic resistance islands were identified containing bla CTX-M-15 and rmtF, with multiple insertion sequences and transposons present. The coexistence of bla oxa-232 and rmtF in a high-risk K. pneumoniae strain was reported. Conjugation assay was utilized to investigate the transferability of bla oxa-232-encoding plasmids horizontally. The study highlights the emergence of ST15-KL112 high-risk CRKP strains with multidrug resistance, including to aminoglycosides and tigecycline. The presence of mobile ARI and clonal and horizontal transfer expansion of strains indicate the threat of transmission of these strains. Future research is needed to assess the prevalence of such isolates and develop effective control measures.

Acr AB and Oxq AB Efflux Pump Genes Among Resistant Klebsiella Pneumoniae Isolated from Zagazig University Hospitals

Acr AB and Oxq AB Efflux Pump Genes Among Resistant Klebsiella Pneumoniae Isolated from Zagazig University Hospitals

Mechanisms of Helicobacter pylori Resistance to Antibiotics

Background: Helicobacter pylori is the most frequent human bacterial infection worldwide; it is prevalent in approximately half of the world's population. H.pylori poses a major health risk because it possesses distinct virulence factors and highly resistant to antibiotics. Multidrug resistance (MDR) and single-drug resistance are two distinct resistance profiles. Chromosome mutations influence antibiotic activity via target-mediated pathways. Inadequate drug absorption, efflux pumps activity, biofilm development, and cocci formation represent supplementary biological mechanisms by which H.pylori acquires drug resistance. The overexpression of efflux pumps genes in mutant isolates is readily detectable in post-therapy courses. Objective: Characterization of the biological and molecular mechanisms contributing to the development of antibiotic resistance in H.pylori. Conclusion: An alarming rate of drug-resistant H.pylori is on the rise. Primary and acquired resistance to clarithromycin and metronidazole has increased globally. Bacterial factors including biofilms, efflux pumps, and molecular mechanism are in association with H.pylori resistance. It is important to continue to monitor the resistance profiles of H.pylori isolates. Keywords: Helicobacter pylori, MDR, Efflux pump, Biofilm, antibiotic resistance.