ABSTRACT
Acinetobacter baumannii is a critical opportunistic pathogen associated with nosocomial infections. The high rates of antibiotic-resistance acquisition make most antibiotics ineffective. Thus, new medical countermeasures are urgently needed. Outer membrane proteins (OMPs) are prime candidates for developing novel drug targets and antibacterial strategies. However, there are substantial gaps in our knowledge of A. baumannii OMPs. This study reports the impact of OmpA-like protein on bacterial physiology and virulence in A. baumannii strain AB5075. We found that PsaB (ABUW_0505) negatively correlates to stress tolerance, while ArfA (ABUW_2730) significantly affects bacterial stiffness, cell shape, and cell envelope thickness. Furthermore, we expand our knowledge on YiaD (ABUW_3045), demonstrating structural and virulence roles of this porin, in addition to meropenem resistance. This study provides solid foundations for understanding how uncharacterized OMPs contribute to A. baumannii's physiological and pathological processes, aiding the development of innovative therapeutic strategies against A. baumannii infections.
Subject(s)
Acinetobacter Infections , Acinetobacter baumannii , Anti-Bacterial Agents , Bacterial Outer Membrane Proteins , Porins , Acinetobacter baumannii/pathogenicity , Acinetobacter baumannii/drug effects , Acinetobacter baumannii/physiology , Bacterial Outer Membrane Proteins/metabolism , Bacterial Outer Membrane Proteins/genetics , Virulence , Anti-Bacterial Agents/pharmacology , Porins/metabolism , Porins/genetics , Acinetobacter Infections/microbiology , Acinetobacter Infections/drug therapy , Animals , Drug Resistance, Bacterial , Mice , Microbial Sensitivity Tests , FemaleABSTRACT
Non-carbapenemase-producing carbapenem-resistant Enterobacterales (non-CP CRE) may be associated with a grave outcome. The common underlying mechanism is beta-lactamases and mutations in outer membrane porins. We report a case of a deep-seated infection caused by Klebsiella pneumoniae ST395 not amenable to source control, involving recurrent bloodstream infection, resulting in in vivo selection of carbapenem resistance under therapy. Three consecutive K. pneumoniae blood isolates were studied using short- and long-read sequencing. The genomes were subject to resistome and virulome, phylogenetic, and plasmid analyses. ompK36 porins were analyzed at the nucleotide and amino acid levels. Genomes were compared to 297 public ST395 K. pneumoniae genomes using cgMLST, resistome, and porin analyses and the EuSCAPE project. Relevant ompK36 and micF sequences were extracted and analyzed as above. The three sequential K. pneumoniae blood isolates belonged to the same clone. Subsequent CR isolates revealed a new large deletion of the ompK36 gene also involving the upstream region (deletion of micF). Comparison with public ST395 genomes revealed the study isolates belonged to clade B, representing a separate clone. N-terminal large ompK36 truncations were uncommon in both public data sets. In vivo selection of non-CP CRE K. pneumoniae could have substantial clinical implications. Such selection should be scrutinized through repeated cultures and frequent susceptibility testing during antimicrobial treatment, especially in the context of persistent or recurrent bloodstream infections and when adequate source control cannot be achieved. The occurrence of an unusually large deletion involving the ompK36 locus and upstream micF should be further studied.
Subject(s)
Anti-Bacterial Agents , Bacterial Proteins , Carbapenems , Klebsiella Infections , Klebsiella pneumoniae , Microbial Sensitivity Tests , Porins , Klebsiella pneumoniae/genetics , Klebsiella pneumoniae/drug effects , Klebsiella pneumoniae/isolation & purification , Humans , Klebsiella Infections/drug therapy , Klebsiella Infections/microbiology , Carbapenems/pharmacology , Carbapenems/therapeutic use , Porins/genetics , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Bacterial Proteins/genetics , Male , Bacteremia/microbiology , Bacteremia/drug therapy , Phylogeny , Genome, Bacterial/genetics , Plasmids/genetics , beta-Lactamases/geneticsABSTRACT
Meningococcal (Neisseria meningitidis) serogroup B (MenB) strain antigens are diverse and a limited number of strains can be evaluated using the human serum bactericidal antibody (hSBA) assay. The genetic Meningococcal Antigen Typing System (gMATS) was developed to predict the likelihood of coverage for large numbers of isolates by the 4CMenB vaccine, which includes antigens Neisseria adhesin A (NadA), Neisserial Heparin-Binding Antigen (NHBA), factor H-binding protein (fHbp), and Porin A (PorA). In this study, we characterized by whole-genome analyses 284 invasive MenB isolates collected from 2010 to 2014 by the Argentinian National Laboratories Network (52-61 isolates per year). Strain coverage was estimated by gMATS on all isolates and by hSBA assay on 74 randomly selected isolates, representative of the whole panel. The four most common clonal complexes (CCs), accounting for 81.3% of isolates, were CC-865 (75 isolates, 26.4%), CC-32 (59, 20.8%), CC-35 (59, 20.8%), and CC-41/44 (38, 13.4%). Vaccine antigen genotyping showed diversity. The most prevalent variants/peptides were fHbp variant 2, NHBA peptides 24, 21, and 2, and PorA variable region 2 profiles 16-36 and 14. The nadA gene was present in 66 (23.2%) isolates. Estimated strain coverage by hSBA assay showed 78.4% of isolates were killed by pooled adolescent sera, and 51.4% and 64.9% (based on two different thresholds) were killed by pooled infant sera. Estimated coverage by gMATS (61.3%; prediction interval: 55.5%, 66.7%) was consistent with the infant hSBA assay results. Continued genomic surveillance is needed to evaluate the persistence of major MenB CCs in Argentina.
The most common clinical manifestations of invasive meningococcal disease include meningitis and septicemia, which can be deadly, and many survivors suffer long-term serious after-effects. Most cases of invasive meningococcal disease are caused by six meningococcal serogroups (types), including serogroup B. Although vaccines are available against meningococcal serogroup B infection, these vaccines target antigens that are highly diverse. Consequently, the effectiveness of vaccination may vary from country to country because the meningococcal serogroup B strains circulating in particular regions carry different forms of the target vaccine antigens. This means it is important to test serogroup B strains isolated from specific populations to estimate the percentage of strains that a vaccine is likely to be effective against (known as 'vaccine strain coverage'). The genetic Meningococcal Antigen Typing System (gMATS) was developed to predict strain coverage by the four-component meningococcal serogroup B vaccine, 4CMenB, against large numbers of serogroup B strains. In this study, we analyzed 284 invasive meningococcal serogroup B isolates collected between 2010 and 2014 in Argentina. Genetic analyses showed that the vaccine antigens of the isolates were diverse and some genetic characteristics had not been found in isolates from other countries. However, vaccine strain coverage estimated by gMATS was consistent with that reported in other parts of the world and with strain coverage results obtained for a subset via another method, the human serum bactericidal antibody (hSBA) assay. These results highlight the need for continued monitoring of circulating bacterial strains to assess the estimated strain coverage of meningococcal serogroup B vaccines.
Subject(s)
Antigens, Bacterial , Meningococcal Infections , Meningococcal Vaccines , Neisseria meningitidis, Serogroup B , Humans , Argentina/epidemiology , Meningococcal Vaccines/immunology , Meningococcal Vaccines/administration & dosage , Meningococcal Infections/microbiology , Meningococcal Infections/prevention & control , Meningococcal Infections/epidemiology , Infant , Adolescent , Child , Antigens, Bacterial/genetics , Antigens, Bacterial/immunology , Child, Preschool , Young Adult , Neisseria meningitidis, Serogroup B/genetics , Neisseria meningitidis, Serogroup B/isolation & purification , Neisseria meningitidis, Serogroup B/immunology , Adult , Female , Male , Whole Genome Sequencing , Bacterial Proteins/genetics , Bacterial Proteins/immunology , Genotype , Adhesins, Bacterial/genetics , Adhesins, Bacterial/immunology , Middle Aged , Porins/genetics , Porins/immunology , Serum Bactericidal Antibody Assay , Aged , Neisseria meningitidis/genetics , Neisseria meningitidis/immunology , Neisseria meningitidis/isolation & purification , Neisseria meningitidis/classificationABSTRACT
The cell envelope fortifies bacterial cells against antibiotics and other insults. Species in the Mycobacteriales order have a complex envelope that includes an outer layer of mycolic acids called the mycomembrane (MM) and a cell wall composed of peptidoglycan and arabinogalactan. This envelope architecture is unique among bacteria and contributes significantly to the virulence of pathogenic Mycobacteriales like Mycobacterium tuberculosis. Characterization of pathways that govern envelope biogenesis in these organisms is therefore critical in understanding their biology and for identifying new antibiotic targets. To better understand MM biogenesis, we developed a cell sorting-based screen for mutants defective in the surface exposure of a porin normally embedded in the MM of the model organism Corynebacterium glutamicum. The results revealed a requirement for the conserved σD envelope stress response in porin export and identified MarP as the site-1 protease, respectively, that activate the response by cleaving the membrane-embedded anti-sigma factor. A reporter system revealed that the σD pathway responds to defects in mycolic acid and arabinogalactan biosynthesis, suggesting that the stress response has the unusual property of being induced by activating signals that arise from defects in the assembly of two distinct envelope layers. Our results thus provide new insights into how C. glutamicum and related bacteria monitor envelope integrity and suggest a potential role for members of the σD regulon in protein export to the MM.
Subject(s)
Cell Membrane , Cell Wall , Corynebacterium glutamicum , Mycolic Acids , Sigma Factor , Cell Wall/metabolism , Cell Wall/genetics , Corynebacterium glutamicum/genetics , Corynebacterium glutamicum/metabolism , Mycolic Acids/metabolism , Sigma Factor/metabolism , Sigma Factor/genetics , Cell Membrane/metabolism , Stress, Physiological , Porins/metabolism , Porins/genetics , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Galactans/metabolism , Gene Expression Regulation, Bacterial , Peptidoglycan/metabolismABSTRACT
Nanopores are increasingly powerful tools for single molecule sensing, in particular, for sequencing DNA, RNA and peptides. This success has spurred efforts to sequence non-canonical nucleic acid bases and amino acids. While canonical DNA and RNA bases have pKas far from neutral, certain non-canonical bases, natural RNA modifications, and amino acids are known to have pKas near neutral pHs at which nanopore sequencing is typically performed. Previous reports have suggested that the nanopore signal may be sensitive to the protonation state of an individual moiety. We sequenced ion currents with the MspA nanopore using a single stranded DNA containing a single non-canonical DNA base (Z) at various pH conditions. The Z-base has a near-neutral pKa â¼ 7.8. We find that the measured ion current is remarkably sensitive to the protonation state of the Z-base. We demonstrate how nanopores can be used to localize and determine the pKa of individual moieties along a polymer. More broadly, these experiments provide a path to mapping different protonation sites along polymers and give insight in how to optimize sequencing of polymers that contain moieties with near-neutral pKas.
Subject(s)
DNA, Single-Stranded , Nanopores , Hydrogen-Ion Concentration , DNA, Single-Stranded/chemistry , DNA/chemistry , Protons , Porins/chemistry , Porins/genetics , Sequence Analysis, DNA/methodsABSTRACT
Antimicrobial peptides (AMPs) are becoming next-generation alternative antibacterial agents because of the rapid increase in resistance in bacteria against existing antibiotics, which can also be attributed to the formation of resilient biofilms. However, their widespread use is limited because of their poor absorption, higher dosage requirements, and delayed onset of the bioactivity to elicit a desired response. Here we developed a short AMP that specifically targeted Fusobacterium nucleatum. We conjugated 23R to a statherin-derived peptide (SDP) through rational design; this conjugate binds to FomA, a major porin protein of F. nucleatum. The SDP-tagged 23R exhibited rapid and highly specific bactericidal efficacy against F. nucleatum. Further, IC50 values were in the nanomolar range, and they were 100-fold lower than those obtained with unconjugated 23R. In a human gut microbiota model, 0.1 nM SDP-23R achieved 99% clearance of F. nucleatum ATCC 25586 without markedly altering resident microbiota. Here we demonstrated that binding-peptide-coupled AMPs show increased killing efficacy and specificity for the target pathogen without affecting the resident microbiota.
Subject(s)
Anti-Bacterial Agents , Antimicrobial Peptides , Fusobacterium nucleatum , Fusobacterium nucleatum/drug effects , Antimicrobial Peptides/pharmacology , Antimicrobial Peptides/chemistry , Humans , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/chemistry , Microbial Sensitivity Tests , Gastrointestinal Microbiome/drug effects , Biofilms/drug effects , Porins/metabolism , Porins/genetics , Porins/chemistry , Bacterial Outer Membrane Proteins/metabolism , Bacterial Outer Membrane Proteins/geneticsABSTRACT
BACKGROUND: Pseudomonas aeruginosa is a common cause of nosocomial infections. However, the emergence of multidrug-resistant strains has complicated the treatment of P. aeruginosa infections. While polymyxins have been the mainstay for treatment, there is a global increase in resistance to these antibiotics. Therefore, our study aimed to determine the prevalence and molecular details of colistin resistance in P. aeruginosa clinical isolates collected between June 2019 and May 2023, as well as the genetic linkage of colistin-resistant P. aeruginosa isolates. RESULTS: The resistance rate to colistin was 9% (n = 18) among P. aeruginosa isolates. All 18 colistin-resistant isolates were biofilm producers and carried genes associated with biofilm formation. Furthermore, the presence of genes encoding efflux pumps, TCSs, and outer membrane porin was observed in all colistin-resistant P. aeruginosa strains, while the mcr-1 gene was not detected. Amino acid substitutions were identified only in the PmrB protein of multidrug- and colistin-resistant strains. The expression levels of mexA, mexC, mexE, mexY, phoP, and pmrA genes in the 18 colistin-resistant P. aeruginosa strains were as follows: 88.8%, 94.4%, 11.1%, 83.3%, 83.3%, and 38.8%, respectively. Additionally, down-regulation of the oprD gene was observed in 44.4% of colistin-resistant P. aeruginosa strains. CONCLUSION: This study reports the emergence of colistin resistance with various mechanisms among P. aeruginosa strains in Ardabil hospitals. We recommend avoiding unnecessary use of colistin to prevent potential future increases in colistin resistance.
Subject(s)
Anti-Bacterial Agents , Bacterial Proteins , Colistin , Microbial Sensitivity Tests , Pseudomonas Infections , Pseudomonas aeruginosa , Transcription Factors , Colistin/pharmacology , Pseudomonas aeruginosa/genetics , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/isolation & purification , Anti-Bacterial Agents/pharmacology , Humans , Bacterial Proteins/genetics , Pseudomonas Infections/microbiology , Pseudomonas Infections/epidemiology , Prevalence , Drug Resistance, Multiple, Bacterial/genetics , Biofilms/drug effects , Biofilms/growth & development , Hospitals , Drug Resistance, Bacterial/genetics , Cross Infection/microbiology , Cross Infection/epidemiology , Membrane Transport Proteins/genetics , Porins/geneticsABSTRACT
This study investigated resistance evolution mechanisms of conjugated plasmids and bacterial hosts under different concentrations of antibiotic pressure. Ancestral strain ECNX52 was constructed by introducing the blaNDM-5-carrying IncX3 plasmid into E. coli C600, and was subjected to laboratory evolution under different concentrations of meropenem pressure. Minimal inhibitory concentrations and conjugation frequency were determined. Fitness of these strains was assessed. Whole genome sequencing and transcriptional changes were performed. Ancestral host or plasmids were recombined with evolved hosts or plasmids to verify plasmid or host factors in resistance evolution. Role of the repA mutation on plasmid copy number was determined. Two out of the four clones (EM2N1 and EM2N3) exhibited four-fold increase in MIC when exposed to a continuous pressure of 2 µg/mL MEM (1/32 MIC), by down regulating expression of outer membrane protein ompF. Besides, all four clones displayed four-fold increase in MIC and higher conjugation frequency when subjected to a continuous pressure of 4 µg/mL MEM (1/16 MIC), attributing to increasing plasmid copy number generated by repA D140Y (GATâTAT) mutation. Bacterial hosts and conjugative plasmids can undergo resistance evolution under certain concentrations of antimicrobial pressure by reducing the expression of outer membrane proteins or increasing plasmid copy numbers.
Subject(s)
Anti-Bacterial Agents , Escherichia coli Proteins , Escherichia coli , Microbial Sensitivity Tests , Plasmids , Porins , Escherichia coli/genetics , Escherichia coli/drug effects , Plasmids/genetics , Anti-Bacterial Agents/pharmacology , Porins/genetics , Porins/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Carbapenems/pharmacology , Meropenem/pharmacology , Mutation , Evolution, Molecular , Conjugation, Genetic , Carbapenem-Resistant Enterobacteriaceae/genetics , Carbapenem-Resistant Enterobacteriaceae/drug effects , Whole Genome Sequencing , Gene Dosage , beta-Lactamases/geneticsABSTRACT
Pseudomonas aeruginosa is a notorious multidrug-resistant pathogen that poses a serious and growing threat to the worldwide public health. The expression of resistance determinants is exquisitely modulated by the abundant regulatory proteins and the intricate signal sensing and transduction systems in this pathogen. Downregulation of antibiotic influx porin proteins and upregulation of antibiotic efflux pump systems owing to mutational changes in their regulators or the presence of distinct inducing molecular signals represent two of the most efficient mechanisms that restrict intracellular antibiotic accumulation and enable P. aeruginosa to resist multiple antibiotics. Treatment of P. aeruginosa infections is extremely challenging due to the highly inducible mechanism of antibiotic resistance. This review comprehensively summarizes the regulatory networks of the major porin proteins (OprD and OprH) and efflux pumps (MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY) that play critical roles in antibiotic influx and efflux in P. aeruginosa. It also discusses promising therapeutic approaches using safe and efficient adjuvants to enhance the efficacy of conventional antibiotics to combat multidrug-resistant P. aeruginosa by controlling the expression levels of porins and efflux pumps. This review not only highlights the complexity of the regulatory network that induces antibiotic resistance in P. aeruginosa but also provides important therapeutic implications in targeting the inducible mechanism of resistance.
Subject(s)
Anti-Bacterial Agents , Gene Expression Regulation, Bacterial , Membrane Transport Proteins , Pseudomonas aeruginosa , Pseudomonas aeruginosa/genetics , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/metabolism , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/metabolism , Humans , Membrane Transport Proteins/metabolism , Membrane Transport Proteins/genetics , Pseudomonas Infections/drug therapy , Pseudomonas Infections/microbiology , Drug Resistance, Multiple, Bacterial/genetics , Porins/metabolism , Porins/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Biological TransportABSTRACT
Pseudomonas aeruginosa is a significant pathogen responsible for severe multisite infections with high morbidity and mortality rates. This study analyzed carbapenem-resistant Pseudomonas aeruginosa (CRPA) at a tertiary hospital in Shandong, China, using whole-genome sequencing (WGS). The objective was to explore the mechanisms and molecular characteristics of carbapenem resistance. A retrospective analysis of 91 isolates from January 2022 to March 2023 was performed, which included strain identification and antimicrobial susceptibility testing. WGS was utilized to determine the genome sequences of these CRPA strains, and the species were precisely identified using average nucleotide identification (ANI), with further analysis on multilocus sequence typing and strain relatedness. Some strains were found to carry the ampD and oprD genes, while only a few harbored carbapenemase genes or related genes. Notably, all strains possessed the mexA, mexE, and mexX genes. The major lineage identified was ST244, followed by ST235. The study revealed a diverse array of carbapenem resistance mechanisms among hospital isolates, differing from previous studies in mainland China. It highlighted that carbapenem resistance is not due to a single mechanism but rather a combination of enzyme-mediated resistance, AmpC overexpression, OprD dysfunction, and efflux pump overexpression. This research provides valuable insights into the evolutionary mechanisms and molecular features of CRPA resistance in this region, aiding in the national prevention and control of CRPA, and offering references for targeting and developing new drugs.
Subject(s)
Anti-Bacterial Agents , Bacterial Proteins , Carbapenems , Microbial Sensitivity Tests , Multilocus Sequence Typing , Pseudomonas Infections , Pseudomonas aeruginosa , Whole Genome Sequencing , beta-Lactamases , Pseudomonas aeruginosa/genetics , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/classification , Pseudomonas aeruginosa/isolation & purification , China , Carbapenems/pharmacology , Humans , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/genetics , Pseudomonas Infections/microbiology , Retrospective Studies , beta-Lactamases/genetics , Porins/genetics , Genome, Bacterial/genetics , Membrane Transport Proteins/genetics , Tertiary Care Centers , Bacterial Outer Membrane Proteins/geneticsABSTRACT
The EnvZ-OmpR two-component system of Escherichia coli regulates the expression of the ompF and ompC porin genes in response to medium osmolarity. However, certain mutations in envZ confer pleiotropy by affecting the expression of genes of the iron and maltose regulons not normally controlled by EnvZ-OmpR. In this study, we obtained two novel envZ and ompR pleiotropic alleles, envZT15P and ompRL19Q, among revertants of a mutant with heightened envelope stress and an outer membrane (OM) permeability defect. Unlike envZ, pleiotropic mutations in ompR have not been described previously. The mutant alleles reduced the expression of several outer membrane proteins (OMPs), overcame the temperature-sensitive growth defect of a protease-deficient (ΔdegP) strain, and lowered envelope stress and OM permeability defects in a background lacking the BamB protein of an essential ß-barrel assembly machinery complex. Biochemical analysis showed OmpRL19Q, like wild-type OmpR, is readily phosphorylated by EnvZ, but the EnvZ-dependent dephosphorylation of OmpRL19Q~P was drastically impaired compared to wild-type OmpR. This defect would lead to a prolonged half-life for OmpRL19Q~P, an outcome remarkably similar to what we had previously described for EnvZR397L, resulting in pleiotropy. By employing null alleles of the OMP genes, it was determined that the three pleiotropic alleles lowered envelope stress by reducing OmpF and LamB levels. The absence of LamB was principally responsible for lowering the OM permeability defect, as assessed by the reduced sensitivity of a ΔbamB mutant to vancomycin and rifampin. Possible mechanisms by which novel EnvZ and OmpR mutants influence EnvZ-OmpR interactions and activities are discussed.IMPORTANCEMaintenance of the outer membrane (OM) integrity is critical for the survival of Gram-negative bacteria. Several envelope homeostasis systems are activated when OM integrity is perturbed. Through the isolation and characterization of novel pleiotropic ompR/envZ alleles, this study highlights the involvement of the EnvZ-OmpR two-component system in lowering envelope stress and the OM permeability defect caused by the loss of proteins that are involved in OM biogenesis, envelope homeostasis, and structural integrity.
Subject(s)
Anti-Bacterial Agents , Bacterial Outer Membrane Proteins , Escherichia coli Proteins , Escherichia coli , Gene Expression Regulation, Bacterial , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli/drug effects , Bacterial Outer Membrane Proteins/genetics , Bacterial Outer Membrane Proteins/metabolism , Anti-Bacterial Agents/pharmacology , Alleles , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Porins/genetics , Porins/metabolism , Mutation , Stress, Physiological , Phosphorylation , Multienzyme Complexes , Trans-ActivatorsABSTRACT
BACKGROUND: Escherichia coli (E. coli) is a multidrug resistant opportunistic pathogen that can cause secondary bacterial infections in patients with COVID-19. This study aimed to determine the antimicrobial resistance profile of E. coli as a secondary bacterial infection in patients with COVID-19 and to assess the prevalence and characterization of genes related to efflux pumps and porin. METHODS: A total of 50 nonduplicate E. coli isolates were collected as secondary bacterial infections in COVID-19 patients. The isolates were cultured from sputum samples. Confirmation and antibiotic susceptibility testing were conducted by Vitek 2. PCR was used to assess the prevalence of the efflux pump and porin-related genes in the isolates. The phenotypic and genotypic evolution of antibiotic resistance genes related to the efflux pump was evaluated. RESULTS: The E. coli isolates demonstrated high resistance to ampicillin (100%), cefixime (62%), cefepime (62%), amoxicillin-clavulanic acid (60%), cefuroxime (60%), and ceftriaxone (58%). The susceptibility of E. coli to ertapenem was greatest (92%), followed by imipenem (88%), meropenem (86%), tigecycline (80%), and levofloxacin (76%). Regarding efflux pump gene combinations, there was a significant association between the acrA gene and increased resistance to levofloxacin, between the acrB gene and decreased resistance to meropenem and increased resistance to levofloxacin, and between the ompF and ompC genes and increased resistance to gentamicin. CONCLUSIONS: The antibiotics ertapenem, imipenem, meropenem, tigecycline, and levofloxacin were effective against E. coli in patients with COVID-19. Genes encoding efflux pumps and porins, such as acrA, acrB, and outer membrane porins, were highly distributed among all the isolates. Efflux pump inhibitors could be alternative antibiotics for restoring tetracycline activity in E. coli isolates.
Subject(s)
COVID-19 , Coinfection , Escherichia coli Infections , Humans , Escherichia coli , Ertapenem/pharmacology , Levofloxacin/pharmacology , Meropenem/pharmacology , Tigecycline/pharmacology , Anti-Bacterial Agents/pharmacology , Escherichia coli Infections/microbiology , Imipenem/pharmacology , Porins/genetics , Porins/pharmacology , Microbial Sensitivity TestsABSTRACT
AIMS: Acinetobacter baumannii is a nosocomial pathogen known to be multidrug-resistant (MDR), especially to drugs of the carbapenem class. Several factors contribute to resistance, including efflux pumps, ß-lactamases, alteration of target sites, and permeability defects. In addition, outer membrane proteins (OMPs), like porins are involved in the passage of antibiotics, and their alteration could lead to resistance development. This study aimed to explore the possible involvement of porins and OMPs in developing carbapenem resistance due to differential expression. METHODS AND RESULTS: The antibiotic-susceptible and MDR isolates of A. baumannii were first studied for differences in their transcriptional levels of OMP expression and OMP profiles. The antibiotic-susceptible isolates were further treated with imipenem, and it was found that the omp genes were differentially expressed. Six of the nine genes studied were upregulated at 1 h of exposure to imipenem. Their expression gradually decreased with time, further confirmed by their OMP profile and two-dimensional gel electrophoresis. CONCLUSIONS: This study could identify OMPs that were differentially expressed on exposure to imipenem. Hence, this study provides insights into the role of specific OMPs in antibiotic resistance in A. baumannii.
Subject(s)
Acinetobacter baumannii , Anti-Bacterial Agents , Bacterial Outer Membrane Proteins , Imipenem , Microbial Sensitivity Tests , Acinetobacter baumannii/drug effects , Acinetobacter baumannii/genetics , Acinetobacter baumannii/metabolism , Imipenem/pharmacology , Bacterial Outer Membrane Proteins/genetics , Bacterial Outer Membrane Proteins/metabolism , Anti-Bacterial Agents/pharmacology , Gene Expression Regulation, Bacterial/drug effects , Drug Resistance, Multiple, Bacterial/genetics , Acinetobacter Infections/microbiology , Humans , Porins/genetics , Porins/metabolismABSTRACT
Antimicrobial resistance transmission from farm animals to humans is a critical health concern and hence a detailed molecular surveillance is essential for tracking the spread and consequent evolution of antimicrobial resistance. In this study, a pan-drug resistant Klebsiella pneumoniae MS1 strain was isolated from a healthy broiler farm and studied. From the results of the study, MS1 was found to be is resistant to 18 tested antibiotics and has a high-risk to be pathogenic to humans with a probability of 0.80. The whole genome sequencing data of MS1 was used to predict the presence of antimicrobial resistance genes and pathogenicity. The genome analysis has revealed MS1 to have 34 AMR genes. Out of these, the AMR gene OmpK37 codes for an important protein involved in cell permeability and hence in antibiotic resistance. Further analysis was carried out by using an in-genome analysis method to understand the evolution of OmpK37 and the underlying reason for the emergence of resistance. From the detailed analysis, the current study could demonstrate for the first time the evolution of OmpK37 from OmpC. Though structurally OmpK37 was very similar to other porins present in MS1, it was found to have higher mutability as a distinguishing feature which makes it an important protein in monitoring the evolving resistances in microorganisms.
Subject(s)
Chickens , Feces , Klebsiella pneumoniae , Animals , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/genetics , Chickens/microbiology , Drug Resistance, Bacterial/genetics , Drug Resistance, Multiple, Bacterial/genetics , Feces/microbiology , Genome, Bacterial , Klebsiella pneumoniae/genetics , Klebsiella pneumoniae/drug effects , Porins/genetics , Whole Genome SequencingABSTRACT
The outer membrane (OM) in gram-negative bacteria contains proteins that regulate the passive or active uptake of small molecules for growth and cell function, as well as mediate the emergence of antibiotic resistance. This study aims to explore the potential mechanisms for restoring bacteria to azithromycin susceptibility based on transcriptome analysis of bacterial membrane-related genes. Transcriptome sequencing was performed by treating multidrug-resistant Escherichia coli T28R with azithromycin or in combination with colistin and confirmed by reverse transcription-quantitative PCR (RT-qPCR). Azithromycin enzyme-linked immunosorbent assay (ELISA) test, ompC gene overexpression, and molecular docking were utilized to conduct the confirmatory research of the potential mechanisms. We found that colistin combined with azithromycin led to 48 differentially expressed genes, compared to azithromycin alone, such as downregulation of tolA, eptB, lpxP, and opgE and upregulation of ompC gene. Interestingly, the addition of colistin to azithromycin differentially downregulated the mph(A) gene mediating azithromycin resistance, facilitating the intracellular accumulation of azithromycin. Also, overexpression of the ompC elevated azithromycin susceptibility, and colistin contributed to further suppression of the Mph(A) activity in the presence of azithromycin. These findings suggested that colistin firstly enhanced the permeability of bacterial OM, causing intracellular drug accumulation, and then had a repressive effect on the Mph(A) activity along with azithromycin. Our study provides a novel perspective that the improvement of azithromycin susceptibility is related not only to the downregulation of the mph(A) gene and conformational remodeling of the Mph(A) protein but also the upregulation of the membrane porin gene ompC.IMPORTANCEUsually, active efflux via efflux pumps is an important mechanism of antimicrobial resistance, such as the AcrAB-TolC complex and MdtEF. Also, bacterial porins exhibited a substantial fraction of the total number of outer membrane proteins in Enterobacteriaceae, which are involved in mediating the development of the resistance. We found that the upregulation or overexpression of the ompC gene contributed to the enhancement of resistant bacteria to azithromycin susceptibility, probably due to the augment of drug uptakes caused and the opportunity of Mph(A) function suppressed by azithromycin with colistin. Under the combination of colistin and azithromycin treatment, OmpC exhibited an increased selectivity for cationic molecules and played a key role in the restoral of the antibiotic susceptibility. Investigations on the regulation of porin expression that mediated drug resistance would be important in clinical isolates treated with antibiotics.
Subject(s)
Escherichia coli Proteins , Escherichia coli , Azithromycin/pharmacology , Colistin/pharmacology , Up-Regulation , Molecular Docking Simulation , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/metabolism , Porins/genetics , Porins/metabolism , Microbial Sensitivity Tests , Escherichia coli Proteins/metabolismABSTRACT
Carbapenem-resistant Enterobacterales (CRE), specifically those resistant to only ertapenem among carbapenems (ETP-mono-resistant), are increasingly reported, while the optimal therapy options remain uncertain. To investigate the prevalence and characteristics of ETP-mono-resistant CRE, CRE strains were systematically collected from 102 hospitals across China between 2018 and 2021. A 1:1 randomized matching study was conducted with ETP-mono-resistant strains to meropenem- and/or imipenem-resistant (MEM/IPM-resistant) strains. Antimicrobial susceptibility testing, whole-genome sequencing, carbapenem-hydrolysing activity and the expression of carbapenemase genes were determined. In total, 18.8% of CRE strains were ETP-mono-resistant, with relatively low ertapenem MIC values. ETP-mono-resistant strains exhibited enhanced susceptibility to ß-lactams, ß-lactam/ß-lactamase inhibitor combinations, levofloxacin, fosfomycin, amikacin and polymyxin than MEM/IPM-resistant strains (P < 0.05). Phylogenetic analysis revealed high genetic diversity among ETP-mono-resistant strains. Extended-spectrum ß-lactamases (ESBLs) and/or AmpC, as well as porin mutations, were identified as potential major mechanisms mediating ETP-mono-resistance, while the presence of carbapenemases was found to be the key factor distinguishing the carbapenem-resistant phenotypes between the two groups (P < 0.001). Compared with the MEM/IPM-resistant group, limited carbapenemase-producing CRE (CP-CRE) strains in the ETP-mono-resistant group showed a significantly lower prevalence of ESBLs and porin mutations, along with reduced expression of carbapenemase. Remarkably, spot assays combined with modified carbapenem inactivation method indicated that ETP-mono-resistant CP-CRE isolates grew at meropenem concentrations eightfold above their corresponding MIC values, accompanied by rapidly enhanced carbapenem-hydrolysing ability. These findings illustrate that ETP-mono-resistant CRE strains are relatively prevalent and that caution should be exercised when using meropenem alone for treatment. The detection of carbapenemase should be prioritized.
Subject(s)
Anti-Bacterial Agents , Carbapenems , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , beta-Lactamases/metabolism , Carbapenems/pharmacology , Ertapenem/pharmacology , Meropenem , Microbial Sensitivity Tests , Phylogeny , Porins/genetics , Prevalence , ChinaABSTRACT
Background: Carbapenem resistance in Pseudomonas aeruginosa is a serious global health problem. We investigated the clonal distribution and its association with the carbapenem resistance mechanisms of carbapenem-non-susceptible P. aeruginosa isolates from three Korean hospitals. Methods: A total of 155 carbapenem-non-susceptible P. aeruginosa isolates collected between 2011 and 2019 were analyzed for sequence types (STs), antimicrobial susceptibility, and carbapenem resistance mechanisms, including carbapenemase production, the presence of resistance genes, OprD mutations, and the hyperproduction of AmpC ß-lactamase. Results: Sixty STs were identified in carbapenem-non-susceptible P. aeruginosa isolates. Two high-risk clones, ST235 (N=41) and ST111 (N=20), were predominant; however, sporadic STs were more prevalent than high-risk clones. The resistance rate to amikacin was the lowest (49.7%), whereas that to piperacillin was the highest (92.3%). Of the 155 carbapenem-non-susceptible isolates, 43 (27.7%) produced carbapenemases. Three metallo-ß-lactamase (MBL) genes, blaIMP-6 (N=38), blaVIM-2 (N=3), and blaNDM-1 (N=2), were detected. blaIMP-6 was detected in clonal complex 235 isolates. Two ST773 isolates carried blaNDM-1 and rmtB. Frameshift mutations in oprD were identified in all isolates tested, regardless of the presence of MBL genes. Hyperproduction of AmpC was detected in MBL gene-negative isolates. Conclusions: Frameshift mutations in oprD combined with MBL production or hyperproduction of AmpC are responsible for carbapenem resistance in P. aeruginosa. Further attention is required to curb the emergence and spread of new carbapenem-resistant P. aeruginosa clones.
Subject(s)
Anti-Bacterial Agents , Bacterial Proteins , Carbapenems , Hospitals , Microbial Sensitivity Tests , Pseudomonas Infections , Pseudomonas aeruginosa , beta-Lactamases , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/genetics , Pseudomonas aeruginosa/isolation & purification , Carbapenems/pharmacology , Republic of Korea , beta-Lactamases/genetics , beta-Lactamases/metabolism , Humans , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Pseudomonas Infections/microbiology , Porins/genetics , Porins/metabolism , MutationABSTRACT
Bacteriocins, which have narrow-spectrum activity and limited adverse effects, are promising alternatives to antibiotics. In this study, we identified klebicin E (KlebE), a small bacteriocin derived from Klebsiella pneumoniae. KlebE exhibited strong efficacy against multidrug-resistant K. pneumoniae isolates and conferred a significant growth advantage to the producing strain during intraspecies competition. A giant unilamellar vesicle leakage assay demonstrated the unique membrane permeabilization effect of KlebE, suggesting that it is a pore-forming toxin. In addition to a C-terminal toxic domain, KlebE also has a disordered N-terminal domain and a globular central domain. Pulldown assays and soft agar overlay experiments revealed the essential role of the outer membrane porin OmpC and the Ton system in KlebE recognition and cytotoxicity. Strong binding between KlebE and both OmpC and TonB was observed. The TonB-box, a crucial component of the toxin-TonB interaction, was identified as the 7-amino acid sequence (E3ETLTVV9) located in the N-terminal region. Further studies showed that a region near the bottom of the central domain of KlebE plays a primary role in recognizing OmpC, with eight residues surrounding this region identified as essential for KlebE toxicity. Finally, based on the discrepancies in OmpC sequences between the KlebE-resistant and sensitive strains, it was found that the 91st residue of OmpC, an aspartic acid residue, is a key determinant of KlebE toxicity. The identification and characterization of this toxin will facilitate the development of bacteriocin-based therapies targeting multidrug-resistant K. pneumoniae infections.
Subject(s)
Bacteriocins , Klebsiella pneumoniae , Anti-Bacterial Agents/metabolism , Anti-Bacterial Agents/pharmacology , Bacteriocins/genetics , Bacteriocins/metabolism , Bacteriocins/pharmacology , Bacteriocins/toxicity , Klebsiella pneumoniae/genetics , Klebsiella pneumoniae/metabolism , Porins/genetics , Porins/metabolism , Cell Membrane Permeability/drug effects , Cell Membrane Permeability/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Protein Domains , Drug Resistance, Multiple, Bacterial/drug effectsABSTRACT
Outer membrane proteins perform essential functions in uptake and secretion processes in bacteria. MspA is an octameric channel protein in the outer membrane of Mycobacterium smegmatis and is structurally distinct from any other known outer membrane protein. MspA is the founding member of a family with more than 3000 homologs and is one of the most widely used proteins in nanotechnological applications due to its advantageous pore structure and extraordinary stability. While a conserved C-terminal signal sequence is essential for folding and protein assembly in the outer membrane of Gram-negative bacteria, the molecular determinants of these processes are unknown for MspA. In this study, we show that mutation and deletion of methionine 183 in the highly conserved C-terminus of MspA and mutation of the conserved tryptophan 40 lead to a complete loss of protein in heat extracts of M. smegmatis. Swapping these residues partially restores the heat stability of MspA indicating that methionine 183 and tryptophan 40 form a conserved sulfur-π electron interaction, which stabilizes the MspA monomer. Flow cytometry showed that all MspA mutants are surface-accessible demonstrating that oligomerization and membrane integration in M. smegmatis are not affected. Thus, the conserved C-terminus of MspA is essential for its thermal stability, but it is not required for protein assembly in its native membrane, indicating that this process is mediated by a mechanism distinct from that in Gram-negative bacteria. These findings will benefit the rational design of MspA-like pores to tailor their properties in current and future applications.
Subject(s)
Mycobacterium , Tryptophan , Tryptophan/metabolism , Porins/chemistry , Porins/genetics , Porins/metabolism , Mycobacterium smegmatis/genetics , Mycobacterium smegmatis/metabolism , Methionine/metabolismABSTRACT
OBJECTIVES: K. pneumoniae is a common cause of severe hospital-acquired infections. In the present study, we have characterised the whole-genome of two K. pneumoniae ST437 belonging to the clonal complex CC258. METHODS: The whole-genome sequencing was performed by MiSeq Illumina, with a 2 × 300bp paired-end run. ResFinder 4.4.2 was used to detect acquired antimicrobial resistance genes (ARGs) and chromosomal mutations. Mobile genetic elements (plasmids and ISs) were identified by MobileElementFinder v1.0.3. The genome was also assigned to ST using MLST 2.0.9. Virulence factors were detected using the Virulence Factor Database (VFDB). RESULTS: K. pneumoniae KPNAQ_1/23 and KPNAQ_2/23 strains, isolated from urine samples of hospitalised patients, showed resistance to most antibiotics, including ceftazidime-avibactam, ceftolozane-tazobactam, and meropenem-vaborbactam combinations. Both strains were susceptible only to cefiderocol. Multiple mechanisms of resistance were identified. Resistance to ß-lactams was due to the presence of NDM-5, OXA-232, CTX-M-15, SHV-182 ß-lactamases, and OmpK36 and OmpK37 porin mutations. Resistance to fluoroquinolones was mediated by chromosomal mutations in acrR, oqxAB efflux pumps, and the bifunctional gene aac(6')-Ib-cr. CONCLUSION: The presence of different virulence genes makes these KPNAQ_1/23 and KPNAQ_2/23 high-risk clones.