Multidrug Resistance Efflux Pumps Research Articles

Genomic Insights Into a Strong Biofilm‐Forming Enterococcus faecalis MTR_EFS01 Strain Isolated From a Shrimp in Bangladesh

ABSTRACTEnterococcus faecalis is known for its ability to form strong biofilms and its role as an opportunistic pathogen. In this study, we screened and characterized a multidrug‐resistant (MDR) and strong biofilm‐forming E. faecalis isolate obtained from a shrimp sample to determine its genetic diversity, molecular epidemiology, and underlying factors associated with antimicrobial resistance genes (ARGs) and virulence factor genes (VFGs). The E. faecalis MTR_EFS01 strain was isolated using culturing, staining, and biochemical tests and MALDI‐TOF methods. The MDR profile of the strain was determined through the disk diffusion test. The complete genomic sequence of E. faecalis MTR_EFS01 was obtained using the Illumina NextSeq2000 platform. The de novo assembly of the E. faecalis MTR_EFS01 genome revealed a total length of 2,862,301 bp with 80.0 × coverage. This genome comprised 38 contigs, a G + C content of 37.4%, and identified two CRISPR arrays, seven prophages, and 55 RNA genes. The E. faecalis MTR_EFS01 strain was classified as ST862 with a high pathogenicity index of 0.896. The strain harbored eight ARGs conferring resistance to tetracycline, erythromycin, trimethoprim, and MDR efflux pumps. Furthermore, 27 VFGs were identified in this strain, linked to antiphagocytosis, adherence, biofilm formation, enzymes, and immune invasion. Metabolic functional analysis revealed that our strain had 243 subsystems, with the most abundant genes associated with carbohydrate metabolism, amino acids and derivatives, and protein metabolism. The findings in this study underscore the importance of continuous monitoring, research, and collaborative efforts to address the growing threat of MDR and biofilm‐forming pathogens in diverse settings.

Natural Inhibitors of Salmonella MDR Efflux Pumps AcrAB and AcrD: An Integrated In Silico, Molecular, and In Vitro Investigation.

Multidrug-resistant (MDR) Salmonella remains a significant global health threat. This study aimed to explore the potential of essential oil components as novel inhibitors of the Salmonella MDR efflux pumps AcrAB and AcrD. Salmonella isolates were characterized for serotype, antibiotic resistance, and efflux pump activity. Essential oil components were screened for inhibitory effects using phenotypic and genotypic methods. In silico docking and molecular dynamics simulations were conducted to investigate binding interactions and stability. Salmonella Typhimurium was the predominant serotype with high MDR rates. Efflux pump activity was prevalent. Cumin and cinnamon oils demonstrated promising inhibitory effects on these pumps. Molecular docking simulations revealed strong binding affinities of analyzed compounds to the AcrAB and AcrD binding pocket. The 2-methyl-1-(p-tolyl)propan-2-ol exhibited higher stability within the AcrAB binding pocket compared to (1S,3R,5R)-1-isopropyl-4-methylenebicyclo[3.1.0]hexan-3-ol within the AcrD binding pocket. Treatment with these oils significantly downregulated efflux pump genes (robA, acrB, mdtB, acrF, acrD, soxS, mdsB, marA). The novel approach of combining in silico and molecular dynamics simulations with precise gene expression analysis provides a valuable framework for future studies aimed at combating MDR Salmonella efflux pumps.

Genomic Insights of Multidrug-Resistant Enterococcus faecalis and Acinetobacter baumannii Isolated from a Sepsis Patient with Pauci-Immune Crescentic Glomerulonephritis, India.

Acinetobacter baumannii and Enterococcus faecalis are opportunistic bacteria frequently associated with hospital-acquired infections. A. baumannii nosocomial infections in intensive care units are a worldwide problem, with high mortality rates. It may also develop rapidly multidrug resistance (MDR), extensive drug resistance (XDR), and even pan-drug resistance (PDR). Colistin resistance which is an example of pan-drug resistance, is highly alarming as it's used as a last-line antibiotic. Microbes capable of crossing epithelial barriers such as E. faecalis have developed novel strategies to counter antimicrobial agents and cause bacteremia in immunocompromised patients. However, the coinfection of these bacteria in the same patient is unusual. Here, we report a genomic investigation of the extensively drug-resistant E. faecalis and A. baumannii isolated from the blood sample of a patient diagnosed with pauci-immune crescentic glomerulonephritis (PICGN). Identification of cultures isolated from blood sample was carried out using whole-genome sequencing and resistome profiles were mapped. Whole genome sequencing revealed that E. faecalis SVJ-EF01 had a genome size of2,935,226bp and GC content of 37.4%, whereas A. baumannii SVJ-AC01 had a genome size of 3,730,857bp and GC content of 39%. Draft genomes were functionally annotated demonstrating that the organism harbors multiple virulence factors and antimicrobial-resistant mechanisms including MDR efflux pumps. A. baumannii genome possessed a CRISPR-Cas system which might contribute to antimicrobial resistance. This highlights the significance of polymicrobial nature in ESKAPE pathogenesis research. This genomic investigation helps to gain insights into the virulence, resistance profile, and functional potential of these pathogens.

Guidelines in Designing a Universal Primer Mixture to Probe and Quantify Antibiotic-Resistant Genes Using the Polymerase Chain Reaction (PCR).

Multidrug resistance efflux pumps (MDREPs) in biofilm communities have become an increasingly expensive problem in clinical settings. Polymerase chain reaction (PCR)-based detection can be used to diagnose and characterize these genes, but this requires effective primer design to minimize false positives and negatives in test conclusions. A universal primer approach has previously been used to detect conserved core genes but not for accessory genes such as MDREPs. This study describes a guideline for the design of primers used in the detection of MDREP genes and an optimization approach for creating primers by using multiple sequence alignments to target conserved regions in silico, progressing from in silico to in vitro to generate working primers. Using thisapproach, this paper was able to generate primers to target sugE, a small multidrug resistance (SMR) protein found in microbial species. Primers were tested positively against synthetic DNA sequences but were inconsistent with DNA extracted from the organism of interest. Primer design informs the shortfalls of this detection technique and the difficulty in characterizing such genomic elements.

A Comparative Metagenomic Analysis of Specified Microorganisms in Groundwater for Non-Sterilized Pharmaceutical Products

In pharmaceutical manufacturing, ensuring product safety involves the detection and identification of microorganisms with human pathogenic potential, including Burkholderia cepacia complex (BCC), Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica, Staphylococcus aureus, Clostridium sporogenes, Candida albicans, and Mycoplasma spp., some of which may be missed or not identified by traditional culture-dependent methods. In this study, we employed a metagenomic approach to detect these taxa, avoiding the limitations of conventional cultivation methods. We assessed the groundwater microbiome’s taxonomic and functional features from samples collected at two locations in the spring and summer. All datasets comprised 436–557 genera with Proteobacteria, Bacteroidota, Firmicutes, Actinobacteria, and Cyanobacteria accounting for > 95% of microbial DNA sequences. The aforementioned species constituted less than 18.3% of relative abundance. Escherichia and Salmonella were mainly detected in Hot Springs, relative to Jefferson, while Clostridium and Pseudomonas were mainly found in Jefferson relative to Hot Springs. Multidrug resistance efflux pumps and BlaR1 family regulatory sensor-transducer disambiguation dominated in Hot Springs and in Jefferson. These initial results provide insight into the detection of specified microorganisms and could constitute a framework for the establishment of comprehensive metagenomic analysis for the microbiological evaluation of pharmaceutical-grade water and other non-sterile pharmaceutical products, ensuring public safety.

Repurposing harmaline as a novel approach to reverse tmexCD1-toprJ1-mediated tigecycline resistance against klebsiella pneumoniae infections

BackgroundA novel plasmid-mediated resistance–nodulation–division (RND) efflux pump gene cluster tmexCD1-toprJ1 in Klebsiella pneumoniae tremendously threatens the use of convenient therapeutic options in the post-antibiotic era, including the “last-resort” antibiotic tigecycline.ResultsIn this work, the natural alkaloid harmaline was found to potentiate tigecycline efficacy (4- to 32-fold) against tmexCD1-toprJ1-positive K. pneumoniae, which also thwarted the evolution of tigecycline resistance. Galleria mellonella and mouse infection models in vivo further revealed that harmaline is a promising candidate to reverse tigecycline resistance. Inspiringly, harmaline works synergistically with tigecycline by undermining tmexCD1-toprJ1-mediated multidrug resistance efflux pump function via interactions with TMexCD1-TOprJ1 active residues and dissipation of the proton motive force (PMF), and triggers a vicious cycle of disrupting cell membrane integrity and metabolic homeostasis imbalance.ConclusionThese results reveal the potential of harmaline as a novel tigecycline adjuvant to combat hypervirulent K. pneumoniae infections.

Large-scale bacterial genomic and metagenomic analysis reveals Pseudomonas aeruginosa as potential ancestral source of tigecycline resistance gene cluster tmexCD-toprJ

BackgroundThe global dissemination of the multidrug resistance efflux pump gene cluster tmexCD-toprJ has greatly weakened the effects of multiple antibiotics, including tigecycline. However, the potential origin and transmission mechanisms of the gene cluster remain unclear. MethodsHere, we concluded a comprehensive bioinformatics analysis on integrated 73,498 bacterial genomes, including Pseudomonas spp., Klebsiella spp., Aeromonas spp., Proteus spp., and Citrobacter spp., along with 1,152 long-read metagenomic datasets to trace the origin and propagation of tmexCD-toprJ. ResultsOur results demonstrated that tmexCD-toprJ was predominantly found in Pseudomonas aeruginosa sourced from human hosts in Asian countries and North American countries. Phylogenetic and genomic feature analyses showed that tmexCD-toprJ was likely evolved from mexCD-oprJ of some special clones of P. aeruginosa. Furthermore, metagenomic analysis confirmed that P. aeruginosa is the only potential ancestral bacterium for tmexCD-toprJ. A putative mobile genetic structure harboring tmexCD-toprJ, int-int-hp-hp-tnfxB-tmexCD-toprJ, was the predominant genetic context of tmexCD-toprJ across various bacterial genera, suggesting that the two integrase genes play a pivotal role in the horizontal transmission of tmexCD-toprJ. ConclusionsBased on these findings, it is almost certain that the tmexCD-toprJ gene cluster was derived from P. aeruginosa and further spread to other bacteria.

In silico Exploration of Essential Oil Constituents in Combating Multidrug-Resistant Staphylococcus aureus Infected Wounds

This research explores the multifaceted pharmacological actions of essential oils and its constituents, derived as secondary metabolites from aromatic plants, with a particular focus on their potent wound healing and antibacterial activities, elucidating their significance in therapeutic approach towards infected wounds. An in silico screening was carried out to identify the interaction between the bioactive essential oil contituents (EOC) such as cinnamaldehyde, citral, geraniol, linalool, and p-cymene, docked against various target proteins associated with antibiotic resistance and wound healing, including mec A (PDB ID- 4DK1), nor A (PDB ID- 7LO8), TGF- β1 (PDB ID- 1PY5), TGF- β2 (PDB ID- 1M9Z), VEGF (PDB ID-3QTK), GSK-3β (PDB ID-1Q5K) and MMP-9 (PDB ID-5UE4). The docking was done with AutoDock V 4.0 using five EOCs against seven receptors and the binding energy was gaged. The binding energy of EOCs were observed to be ranging from -5.3 kcal/mol to -2.55 kcal/mol. Notably, all the screened EOCs exhibited favourable binding affinity with GSK-3β, indicating their potential role in the inflammatory phase of wound healing. Additionally, towards antibiotic resistance, all EOC displayed adequate binding affinity with norA, suggesting their potential in modulating multidrug resistant efflux pumps. Compliance with Lipinski's rule, positions these EOC as promising candidates for drug development, particularly in the context of wound healing and antibiotic resistance. This study holds the promise of contributing novel insights to the field of wound care and combating antibiotic resistance, paving the way for innovative approaches in addressing the challenges posed by multi-drug resistant Staphylococcus aureus (MDRSA) infected wounds.

Extending the Potency and Lifespan of Antibiotics: Inhibitors of Gram-Negative Bacterial Efflux Pumps.

Efflux is a natural process found in all prokaryotic and eukaryotic cells that removes a diverse range of substrates from inside to outside. Many antibiotics are substrates of bacterial efflux pumps, and modifications to the structure or overexpression of efflux pumps are an important resistance mechanism utilized by many multidrug-resistant bacteria. Therefore, chemical inhibition of bacterial efflux to revitalize existing antibiotics has been considered a promising approach for antimicrobial chemotherapy over two decades, and various strategies have been employed. In this review, we provide an overview of bacterial multidrug resistance (MDR) efflux pumps, of which the resistance nodulation division (RND) efflux pumps are considered the most clinically relevant in Gram-negative bacteria, and describe over 50 efflux inhibitors that target such systems. Although numerous efflux inhibitors have been identified to date, none have progressed into clinical use because of formulation, toxicity, and pharmacokinetic issues or a narrow spectrum of inhibition. For these reasons, the development of efflux inhibitors has been considered a difficult and complex area of research, and few active preclinical studies on efflux inhibitors are in progress. However, recently developed tools, including but not limited to computational tools including molecular docking models, offer hope that further research on efflux inhibitors can be a platform for research and development of new bacterial efflux inhibitors.

3-O-Substituted Quercetin: an Antibiotic-Potentiating Agent against Multidrug-Resistant Gram-Negative Enterobacteriaceae through Simultaneous Inhibition of Efflux Pump and Broad-Spectrum Carbapenemases.

The discovery of safe and efficient inhibitors against efflux pumps as well as metallo-β-lactamases (MBL) is one of the main challenges in the development of multidrug-resistant (MDR) reversal agents which can be utilized in the treatment of carbapenem-resistant Gram-negative bacteria. In this study, we have identified that introduction of an ethylene-linked sterically demanding group at the 3-OH position of the previously reported MDR reversal agent di-F-Q endows the resulting compounds with hereto unknown multitarget inhibitory activity against both efflux pumps and broad-spectrum β-lactamases including difficult-to-inhibit MBLs. A molecular docking study of the multitarget inhibitors against efflux pump, as well as various classes of β-lactamases, revealed that the 3-O-alkyl substituents occupy the novel binding sites in efflux pumps as well as carbapenemases. Not surprisingly, the multitarget inhibitors rescued the antibiotic activity of a carbapenem antibiotic, meropenem (MEM), in NDM-1 (New Delhi Metallo-β-lactamase-1)-producing carbapenem-resistant Enterobacteriaceae (CRE), and they reduced MICs of MEM more than four-fold (synergistic effect) in 8-9 out of 14 clinical strains. The antibiotic-potentiating activity of the multitarget inhibitors was also demonstrated in CRE-infected mouse model. Taken together, these results suggest that combining inhibitory activity against two critical targets in MDR Gram-negative bacteria, efflux pumps, and β-lactamases, in one molecule is possible, and the multitarget inhibitors may provide new avenues for the discovery of safe and efficient MDR reversal agents.

Crucifer Lesion-Associated Xanthomonas Strains Show Multi-Resistance to Heavy Metals and Antibiotics.

Copper resistance in phytopathogens is a major challenge to crop production globally and is known to be driven by excessive use of copper-based pesticides. However, recent studies have shown co-selection of multiple heavy metal and antibiotic resistance genes in bacteria exposed to heavy metal and xenobiotics, which may impact the epidemiology of plant, animal, and human diseases. In this study, multi-resistance to heavy metals and antibiotics were evaluated in local Xanthomonas campestris pv. campestris (Xcc) and co-isolated Xanthomonas melonis (Xmel) strains from infected crucifer plants in Trinidad. Resistance to cobalt, cadmium, zinc, copper, and arsenic (V) was observed in both Xanthomonas species up to 25 mM. Heavy metal resistance (HMR) genes were found on a small plasmid-derived locus with ~ 90% similarity to a Stenotrophomonas spp. chromosomal locus and a X. perforans pLH3.1 plasmid. The co-occurrence of mobile elements in these regions implies their organization on a composite transposon-like structure. HMR genes in Xcc strains showed the lowest similarity to references, and the cus and ars operons appear to be unique among Xanthomonads. Overall, the similarity of HMR genes to Stenotrophomonas sp. chromosomal genomes suggest their origin in this genus or a related organism and subsequent spread through lateral gene transfer events. Further resistome characterization revealed the presence of small multidrug resistance (SMR), multidrug resistance (MDR) efflux pumps, and bla (Xcc) genes for broad biocide resistance in both species. Concurrently, resistance to antibiotics (streptomycin, kanamycin, tetracycline, chloramphenicol, and ampicillin) up to 1000 µg/mL was confirmed.

Discovery of novel dihydronaphthalene—imidazole ligands as potential inhibitors of Staphylococcus aureus multidrug resistant NorA efflux pump: A combination of experimental and in silico molecular docking studies

Overexpression of the efflux pump is a predominant mechanism by which bacteria show antimicrobial resistance (AMR) and leads to the global emergence of multidrug resistance (MDR). In this work, the inhibitory potential of library of dihydronapthyl scaffold-based imidazole derivatives having structural resemblances with some known efflux pump inhibitors (EPI) were designed, synthesized and evaluated against efflux pump inhibitor against overexpressing bacterial strains to study the synergistic effect of compounds and antibiotics. Out of 15 compounds, four compounds (Dz-1, Dz-3, Dz-7, and Dz-8) were found to be highly active. DZ-3 modulated the MIC of ciprofloxacin, erythromycin, and tetracycline by 128-fold each against 1199B, XU212 and RN4220 strains of S. aureus respectively. DZ-3 also potentiated tetracycline by 64-fold in E. coli AG100 strain. DZ-7 modulated the MIC of both tetracycline and erythromycin 128-fold each in S. aureus XU212 and S. aureus RN4220 strains. DZ-1 and DZ-8 showed the moderate reduction in MIC of tetracycline in E. coli AG100 only by 16-fold and 8-fold, respectively. DZ-3 was found to be the potential inhibitor of NorA as determined by ethidium bromide efflux inhibition and accumulation studies employing NorA overexpressing strain SA-1199B. DZ-3 displayed EPI activity at non-cytotoxic concentration to human cells and did not possess any antibacterial activity. Furthermore, molecular docking studies of DZ-3 was carried out in order to understand the possible binding sites of DZ-3 with the active site of the protein. These studies indicate that dihydronaphthalene scaffolds could serve as valuable cores for the development of promising EPIs.

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.

Whole-genome sequencing and analysis of Chryseobacterium arthrosphaerae from Rana nigromaculata

Chryseobacterium arthrosphaerae strain FS91703 was isolated from Rana nigromaculata in our previous study. To investigate the genomic characteristics, pathogenicity-related genes, antimicrobial resistance, and phylogenetic relationship of this strain, PacBio RS II and Illumina HiSeq 2000 platforms were used for the whole genome sequencing. The genome size of strain FS91703 was 5,435,691 bp and GC content was 37.78%. A total of 4,951 coding genes were predicted; 99 potential virulence factors homologs were identified. Analysis of antibiotic resistance genes revealed that strain FS91703 harbored 10 antibiotic resistance genes in 6 categories and 2 multidrug-resistant efflux pump genes, including adeG and farA. Strain FS91703 was sensitive to β-lactam combination drugs, cephem, monobactam and carbapenems, intermediately resistant to phenicol, and resistant to penicillin, aminoglycosides, tetracycline, fluoroquinolones, and folate pathway inhibitors. Phylogenetic analysis revealed that strain FS91703 and C. arthrosphaerae CC-VM-7T were on the same branch of the phylogenetic tree based on 16 S rRNA; the ANI value between them was 96.99%; and the DDH values were 80.2, 72.2 and 81.6% by three default calculation formulae. These results suggested that strain FS91703 was a species of C. arthrosphaerae. Pan-genome analysis showed FS91703 had 566 unique genes compared with 13 other C. arthrosphaerae strains, and had a distant phylogenetic relationship with the other C. arthrosphaerae strains of the same branch in phylogenetic tree based on orthologous genes. The results of this study suggest that strain FS91703 is a multidrug-resistant and highly virulent bacterium, that differs from other C. arthrosphaerae strains at the genomic level. The knowledge about the genomic characteristics and antimicrobial resistance of strain FS91703 provides valuable insights into this rare species, as well as guidance for the treatment of the disease caused by FS91703 in Rana nigromaculata.

Metagenomic analysis reveals impact of acyl homoserine endolipid-like signaling molecules on the aqueous sediment resistome under florfenicol stress

Quorum sensing potentially helps microorganisms adapt to antibiotic stress encountered in the environment. This experiment investigated the effect of acyl homoserine endolipid-like signaling molecules on microbial antibiotic resistance gene structures in aqueous sediments under florfenicol stress. Additional acyl homoserine endolipid-like signaling molecules (AHLs) alter the structure of multidrug resistance genes in florfenicol-stressed sediments, particularly the multidrug resistance efflux pump gene family. Prophages and integrative and conjugative elements (ICEs) determined the resistance genes structure, and pathways related to mobile genetic elements (MGEs) transfer may play an essential role in this process. The practical application of AHLs to regulate quorum sensing systems may alter bacterial stress responses to environmental florfenicol residues, thereby reducing the development of antibiotic resistance in the environment.

Chronic industrial perturbation and seasonal change induces shift in the bacterial community from gammaproteobacteria to betaproteobacteria having catabolic potential for aromatic compounds at Amlakhadi canal.

Escalating proportions of industrially contaminated sites are one of the major catastrophes faced at the present time due to the industrial revolution. The difficulties associated with culturing the microbes, has been circumvent by the direct use of metagenomic analysis of various complex niches. In this study, a metagenomic approach using next generation sequencing technologies was applied to exemplify the taxonomic abundance and metabolic potential of the microbial community residing in Amlakhadi canal, Ankleshwar at two different seasons. All the metagenomes revealed a predominance of Proteobacteria phylum. However, difference was observed within class level where Gammaproteobacteria was relatively high in polluted metagenome in Summer while in Monsoon the abundance shifted to Betaproteobacteria. Similarly, significant statistical differences were obtained while comparing the genera amongst contaminated sites where Serratia, Achromobacter, Stenotrophomonas and Pseudomonas were abundant in summer season and the dominance changed to Thiobacillus, Thauera, Acidovorax, Nitrosomonas, Sulfuricurvum, Novosphingobium, Hyphomonas and Geobacter in monsoon. Further upon functional characterization, the microbiomes revealed the diverse survival mechanisms, in response to the prevailing ecological conditions (such as degradation of aromatic compounds, heavy metal resistance, oxidative stress responses and multidrug resistance efflux pumps, etc.). The results have important implications in understanding and predicting the impacts of human-induced activities on microbial communities inhabiting natural niche and their responses in coping with the fluctuating pollution load.

Phenotypic and genomic insights into the pathogenicity and antimicrobial resistance of an Enterobacter roggenkampii strain isolated from diseased silver arowana (Osteoglossum bicirrhosum).

Enterobacter roggenkampii is an opportunistic pathogen that causes infections in a wide range of hosts. A bacterial strain named EOBSR_19 was isolated from diseased silver arowana, Osteoglossum bicirrhosum. This bacterium was identified as E. roggenkampii based on the phenotypic characteristics and sequence analysis of the16S rDNA and gyrB genes. Average nucleotide identity and phylogenetic analysis based on the whole genome sequence further confirmed the bacterial taxonomy of EOBSR_19. Artificial experimental infection indicated that EOBSR_19 was pathogenic to fish. Antimicrobial susceptibility test showed it was multi-drug resistant. The EOBSR_19 was found to be resistant to 18 antibiotics belonging to quinolones, macrolides, sulfonamides, aminoglycosides, and β-lactams classes. The whole genome sequencing analysis showed that EOBSR_19 carried 730 virulence genes that were annotated for different functional modules, such as adhesion and invasion, secretion system, siderophore transport system and bacterial toxin. Among them, the virulence genes related to adhesion and invasion were the most abundant. In addition, drug resistance genes involved in multiple mechanisms of antimicrobial resistance were identified in its genomics, including multidrug resistance efflux pumps, antibiotic inactivating enzymes, and antibiotic binding site mutations. Its genomic analysis via whole-genome sequencing provided insights into the pathogenicity and antimicrobial resistance.

Antibiotic Resistance Characterization and Molecular Characteristics of Enterococcus Species Isolated from Combination Probiotic Preparations in China.

Enterococci can act as reservoirs for antibiotic-resistant genes that are potentially at risk of being transferred to other bacteria that inhabit in the gastrointestinal tract. The aim of this study was to determine the phenotypic and molecular characteristics of antibiotic-resistant enterococci isolated from probiotic preparations. In total, we isolated 15 suspected Enterococcus species from 5 compound probiotics, which were identified by 16S rDNA as 12 Enterococcus faecium and 3 Enterococcus faecalis. Determination of antimicrobial susceptibility by the microdilution broth method showed widespread resistance to sulfamethoxazole (100%), norfloxacin (99.3%), azithromycin (99.3%), gentamicin (86.7%), and chloramphenicol (20%). Whole genome sequencing of five resistant strains revealed that all had circular DNA chromosomes and that E. faecium J-1-A to J-4-A contained a plasmid, while E. faecalis J-5-A did not. The results of the resistance gene analysis revealed that each strain contained approximately 30 resistance genes, with the antibiotic resistance genes and the multidrug resistance efflux pump genes mdtG, lmrC, and lmrD detected in all strains. The chloramphenicol resistance genes ykkC and ykkD were first identified in E. faecalis. And there were 21, 19, 21, 21, and 29 virulence factors involved in strains, respectively. Further analysis of the gene islands (GIs) revealed that each strain contained more than 10 GIs. The above results confirm the existence of hidden dangers in the safety of probiotics and remind us to carefully select probiotic preparations containing enterococcal strains to avoid the potential spread of resistance and pathogenicity.

Genomic Profiling of Multidrug Efflux Pumps and Heavy Metal Proteins in Multidrug-resistant Campylobacter fetus Isolated from Sheath Wash Samples of Bulls in South Africa

A substantial evolution of resistance mechanisms among zoonotic bacteria has resulted from anthropogenic factors related to the application of antibiotics in human and veterinary medicine, particularly in contemporary agriculture. This issue associated with the presence of heavy metal-laced protein in zoonotic bacteria should be taken seriously with regard to the health of animals and the general people. To address this issue, the present study employed whole genome sequencing to identify the antimicrobial resistance patterns of Campylobacter fetus subsp. fetus (Cff) and Campylobacter fetus subsp. venerealis (Cfv), resistance and virulence genes, as well as heavy metal protein. Based on culture method biochemical testing and PCR amplification using particular primer pairs (MG3F-MG4R and VenSF-VenSR), bacteria were isolated and identified as C. fetus subsp. fetus and C.fetus subsp. venerealis. Subsequently, antimicrobial disc diffusion tests and whole genome sequencing were performed. Isolated bacteria were resistant to tetracycline at 65%, amoxicillin, and doxycycline at 60%. The resistance was also observed against neomycin at (55%), streptomycin (60%), and gentamycin (55%). Through comprehensive genome sequencing analysis and PCR, multiple efflux pumps linked to multidrug resistance were identified, including the broad-specificity multidrug efflux pump (YkkD), along with CmeA, CmeB, CmeC, and gryA.The genome sequence also revealed genes associated with the production of Cytotoxin (Cdt A, B, and C), adhesion and colonization (VirB10 and VirB9), and invasion (CiaB). In addition, different genomic features in heavy metal resistance included Cobalt-zinc-cadmium resistance protein (CzcD), Tellurite resistance protein (TehA), and arsenic efflux pump protein. The findings of the current study revealed that the emergence of bacterial multidrug resistance is increasingly associated with the substantial and growing contribution of Multidrug resistance efflux pumps, as evident in Cff and Cfv. Therefore, it is crucial to tighten the control of Cff and Cfv in livestock production to prevent the transfer of genes resistant to humans through the food chain.

Hydrophobic moment drives penetration of bacterial membranes by transmembrane peptides

With antimicrobial resistance (AMR) remaining a persistent and growing threat to human health worldwide, membrane-active peptides are gaining traction as an alternative strategy to overcome the issue. Membrane-embedded multi-drug resistant (MDR) efflux pumps are a prime target for membrane-active peptides, as they are a well-established contributor to clinically relevant AMR infections. Here, we describe a series of transmembrane peptides (TMs) to target the oligomerization motif of the AcrB component of the AcrAB-TolC MDR efflux pump from Escherichia coli. These peptides contain an N-terminal acetyl-A-(Sar)3 (sarcosine; N-methylglycine) tag and a C-terminal lysine tag-a design strategy our lab has utilized to improve the solubility and specificity of targeting for TMs previously. While these peptides have proven useful in preventing AcrB-mediated substrate efflux, the mechanisms by which these peptides associate with and penetrate the bacterial membrane remained unknown. In this study, we have shown peptide hydrophobic moment (μH)-the measure of concentrated hydrophobicity on one face of a lipopathic α-helix-drives bacterial membrane permeabilization and depolarization, likely through lateral-phase separation of negatively-charged POPG lipids and the disruption of lipid packing. Our results show peptide μH is an important consideration when designing membrane-active peptides and may be the determining factor in whether a TM will function in a permeabilizing or non-permeabilizing manner when embedded in the bacterial membrane.