Complement Susceptibility in Relation to Genome Sequence of Recent Klebsiella pneumoniae Isolates from Thai Hospitals. - Related Documents




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481801.0000Complement Susceptibility in Relation to Genome Sequence of Recent Klebsiella pneumoniae Isolates from Thai Hospitals. The capacity to resist the bactericidal action of complement (C') is a strong but poorly understood virulence trait in Klebsiella spp. Killing requires activation of one or more C' pathways, assembly of C5b-9 membrane attack complexes (MACs) on the surface of the outer membrane (OM), and penetration of MACs into the target bilayer. We interrogated whole-genome sequences of 164 Klebsiella isolates from three tertiary hospitals in Thailand for genes encoding surface-located macromolecules considered to play a role in determination of C' resistance. Most isolates (154/164) were identified as Klebsiella pneumoniae, and the collection conformed to previously established population structures and antibiotic resistance patterns. The distribution of sequence types (STs) and capsular (K) types were also typical of global populations. The majority (64%) of isolates were resistant to C', and the remainder were either rapidly or slowly killed. All isolates carried genes encoding capsular polysaccharides (K antigens), which have been strongly linked to C' resistance. In contrast to previous reports, there were no differences in the amount of capsule produced by C'-resistant isolates compared to C'-susceptible isolates, nor was there any correlation between serum reactivity and the presence of hypermucoviscous capsules. Similarly, there were no correlations between the presence of genes specifying lipopolysaccharide O-side chains or major OM proteins. Some virulence factors were found more frequently in C'-resistant isolates but were considered to reflect clonal ST expansion. Thus, no single gene accounts for the C' resistance of the isolates sequenced in this study.IMPORTANCE Multidrug-resistant Klebsiella pneumoniae is responsible for an increasing proportion of nosocomial infections, and emerging hypervirulent K. pneumoniae clones now cause severe community-acquired infections in otherwise healthy individuals. These bacteria are adept at circumventing immune defenses, and most survive and grow in serum; their capacity to avoid C'-mediated destruction is correlated with their invasive potential. Killing of Gram-negative bacteria occurs following activation of the C' cascades and stable deposition of C5b-9 MACs onto the OM. For Klebsiella, studies with mutants and conjugants have invoked capsules, lipopolysaccharide O-side chains, and OM proteins as determinants of C' resistance, although the precise roles of the macromolecules are unclear. In this study, we sequenced 164 Klebsiella isolates with different C' susceptibilities to identify genes involved in resistance. We conclude that no single OM constituent can account for resistance, which is likely to depend on biophysical properties of the target bilayer.201830404929
626610.9997Bacterial gene loss as a mechanism for gain of antimicrobial resistance. Acquisition of exogenous DNA by pathogenic bacteria represents the basis for much of the acquired antimicrobial resistance in pathogenic bacteria. A more extreme mechanism to avoid the effect of an antibiotic is to delete the drug target, although this would be predicted to be rare since drug targets are often essential genes. Here, we review and discuss the description of a novel mechanism of resistance to the cephalosporin drug ceftazidime caused by loss of a penicillin-binding protein (PBP) in a Gram-negative bacillus (Burkholderia pseudomallei). This organism causes melioidosis across south-east Asia and northern Australia, and is usually treated with two or more weeks of ceftazidime followed by oral antibiotics for three to six months. Comparison of clinical isolates from six patients with melioidosis found initial ceftazidime-susceptible isolates and subsequent ceftazidime-resistant variants. The latter failed to grow on commonly used culture media, rendering these isolates difficult to detect in the diagnostic laboratory. Genomic analysis using pulsed-field gel electrophoresis and array based genomic hybridisation revealed a large-scale genomic deletion comprising 49 genes in the ceftazidime-resistant strains. Mutational analysis of wild-type B. pseudomallei demonstrated that ceftazidime resistance was due to deletion of a gene encoding a PBP 3 present within the region of genomic loss. This provides one explanation for ceftazidime treatment failure, and may be a frequent but undetected event in patients with melioidosis.201223022568
505920.9997Site-selective modifications by lipid A phosphoethanolamine transferases linked to colistin resistance and bacterial fitness. Genes encoding lipid A modifying phosphoethanolamine transferases (PETs) are genetically diverse and can confer resistance to colistin and antimicrobial peptides. To better understand the functional diversity of PETs, we characterized three canonical mobile colistin resistance (mcr) alleles (mcr-1, -3, -9), one intrinsic pet (eptA), and two mcr-like genes (petB, petC) in Escherichia coli. Using an isogenic expression system, we show that mcr-1 and mcr-3 confer similar phenotypes of decreased colistin susceptibility with low fitness costs. mcr-9, which is phylogenetically closely related to mcr-3, and eptA only provide fitness advantages in the presence of sub-inhibitory concentrations of colistin and significantly reduce fitness in media without colistin. PET-B and PET-C were phenotypically distinct from bonafide PETs; neither impacted colistin susceptibility nor caused considerable fitness cost. Strikingly, we found for the first time that different PETs selectively modify different phosphates of lipid A; MCR-1, MCR-3, and PET-C selectively modify the 4'-phosphate, whereas MCR-9 and EptA modify the 1-phosphate. However, 4'-phosphate modifications facilitated by MCR-1 and -3 are associated with lowered colistin susceptibility and low toxicity. Our results suggest that PETs have a wide phenotypic diversity and that increased colistin resistance is associated with specific lipid A modification patterns that have been largely unexplored thus far. IMPORTANCE: Rising levels of resistance to increasing numbers of antimicrobials have led to the revival of last resort antibiotic colistin. Unfortunately, resistance to colistin is also spreading in the form of mcr genes, making it essential to (i) improve the identification of resistant bacteria to allow clinicians to prescribe effective drug regimens and (ii) develop new combination therapies effective at targeting resistant bacteria. Our results demonstrate that PETs, including MCR variants, are site-selective in Escherichia coli and that site-selectivity correlates with the level of susceptibility and fitness costs conferred by certain PETs. Site selectivity associated with a given PET may not only help predict colistin resistance phenotypes but may also provide an avenue to (i) improve drug regimens and (ii) develop new combination therapies to better combat colistin-resistant bacteria.202439611852
438630.9997Large-scale screening of a targeted Enterococcus faecalis mutant library identifies envelope fitness factors. Spread of antibiotic resistance among bacteria responsible for nosocomial and community-acquired infections urges for novel therapeutic or prophylactic targets and for innovative pathogen-specific antibacterial compounds. Major challenges are posed by opportunistic pathogens belonging to the low GC% gram-positive bacteria. Among those, Enterococcus faecalis is a leading cause of hospital-acquired infections associated with life-threatening issues and increased hospital costs. To better understand the molecular properties of enterococci that may be required for virulence, and that may explain the emergence of these bacteria in nosocomial infections, we performed the first large-scale functional analysis of E. faecalis V583, the first vancomycin-resistant isolate from a human bloodstream infection. E. faecalis V583 is within the high-risk clonal complex 2 group, which comprises mostly isolates derived from hospital infections worldwide. We conducted broad-range screenings of candidate genes likely involved in host adaptation (e.g., colonization and/or virulence). For this purpose, a library was constructed of targeted insertion mutations in 177 genes encoding putative surface or stress-response factors. Individual mutants were subsequently tested for their i) resistance to oxidative stress, ii) antibiotic resistance, iii) resistance to opsonophagocytosis, iv) adherence to the human colon carcinoma Caco-2 epithelial cells and v) virulence in a surrogate insect model. Our results identified a number of factors that are involved in the interaction between enterococci and their host environments. Their predicted functions highlight the importance of cell envelope glycopolymers in E. faecalis host adaptation. This study provides a valuable genetic database for understanding the steps leading E. faecalis to opportunistic virulence.201122194979
482140.9997Enterobacter hormaechei replaces virulence with carbapenem resistance via porin loss. Pathogenic Enterobacter species are of increasing clinical concern due to the multidrug-resistant nature of these bacteria, including resistance to carbapenem antibiotics. Our understanding of Enterobacter virulence is limited, hindering the development of new prophylactics and therapeutics targeting infections caused by Enterobacter species. In this study, we assessed the virulence of contemporary clinical Enterobacter hormaechei isolates in a mouse model of intraperitoneal infection and used comparative genomics to identify genes promoting virulence. Through mutagenesis and complementation studies, we found two porin-encoding genes, ompC and ompD, to be required for E. hormaechei virulence. These porins imported clinically relevant carbapenems into the bacteria, and thus loss of OmpC and OmpD desensitized E. hormaechei to the antibiotics. Our genomic analyses suggest porin-related genes are frequently mutated in E. hormaechei, perhaps due to the selective pressure of antibiotic therapy during infection. Despite the importance of OmpC and OmpD during infection of immunocompetent hosts, we found the two porins to be dispensable for virulence in a neutropenic mouse model. Moreover, porin loss provided a fitness advantage during carbapenem treatment in an ex vivo human whole blood model of bacteremia. Our data provide experimental evidence of pathogenic Enterobacter species gaining antibiotic resistance via loss of porins and argue antibiotic therapy during infection of immunocompromised patients is a conducive environment for the selection of porin mutations enhancing the multidrug-resistant profile of these pathogens.202539977318
583750.9996The secondary resistome of multidrug-resistant Klebsiella pneumoniae. Klebsiella pneumoniae causes severe lung and bloodstream infections that are difficult to treat due to multidrug resistance. We hypothesized that antimicrobial resistance can be reversed by targeting chromosomal non-essential genes that are not responsible for acquired resistance but essential for resistant bacteria under therapeutic concentrations of antimicrobials. Conditional essentiality of individual genes to antimicrobial resistance was evaluated in an epidemic multidrug-resistant clone of K. pneumoniae (ST258). We constructed a high-density transposon mutant library of >430,000 unique Tn5 insertions and measured mutant depletion upon exposure to three clinically relevant antimicrobials (colistin, imipenem or ciprofloxacin) by Transposon Directed Insertion-site Sequencing (TraDIS). Using this high-throughput approach, we defined three sets of chromosomal non-essential genes essential for growth during exposure to colistin (n = 35), imipenem (n = 1) or ciprofloxacin (n = 1) in addition to known resistance determinants, collectively termed the "secondary resistome". As proof of principle, we demonstrated that inactivation of a non-essential gene not previously found linked to colistin resistance (dedA) restored colistin susceptibility by reducing the minimum inhibitory concentration from 8 to 0.5 μg/ml, 4-fold below the susceptibility breakpoint (S ≤ 2 μg/ml). This finding suggests that the secondary resistome is a potential target for developing antimicrobial "helper" drugs that restore the efficacy of existing antimicrobials.201728198411
992160.9996Identification of Multiple Low-Level Resistance Determinants and Coselection of Motility Impairment upon Sub-MIC Ceftriaxone Exposure in Escherichia coli. Resistance to third-generation cephalosporins among Gram-negative bacteria is a rapidly growing public health threat. Among the most commonly used third-generation cephalosporins is ceftriaxone. Bacterial exposure to sublethal or sub-MIC antibiotic concentrations occurs widely, from environmental residues to intermittently at the site of infection. Quality of ceftriaxone is also a concern, especially in low- and middle-income countries, with medicines having inappropriate active pharmaceutical ingredient (API) content or concentration. While focus has been largely on extended-spectrum β-lactamases and high-level resistance, there are limited data on specific chromosomal mutations and other pathways that contribute to ceftriaxone resistance under these conditions. In this work, Escherichia coli cells were exposed to a broad range of sub-MICs of ceftriaxone and mutants were analyzed using whole-genome sequencing. Low-level ceftriaxone resistance emerged after as low as 10% MIC exposure, with the frequency of resistance development increasing with concentration. Genomic analyses of mutants revealed multiple genetic bases. Mutations were enriched in genes associated with porins (envZ, ompF, ompC, and ompR), efflux regulation (marR), and the outer membrane and metabolism (galU and pgm), but none were associated with the ampC β-lactamase. We also observed selection of mgrB mutations. Notably, pleiotropic effects on motility and cell surface were selected for in multiple independent genes, which may have important consequences. Swift low-level resistance development after exposure to low ceftriaxone concentrations may result in reservoirs of bacteria with relevant mutations for survival and increased resistance. Thus, initiatives for broader surveillance of low-level antibiotic resistance and genomic resistance determinants should be pursued when resources are available. IMPORTANCE Ceftriaxone is a widely consumed antibiotic used to treat bacterial infections. Bacteria, however, are increasingly becoming resistant to ceftriaxone. Most work has focused on known mechanisms associated with high-level ceftriaxone resistance. However, bacteria are extensively exposed to low antibiotic concentrations, and there are limited data on the evolution of ceftriaxone resistance under these conditions. In this work, we observed that bacteria quickly developed low-level resistance due to both novel and previously described mutations in multiple different genes upon exposure to low ceftriaxone concentrations. Additionally, exposure also led to changes in motility and the cell surface, which can impact other processes associated with resistance and infection. Notably, low-level-resistant bacteria would be missed in the clinic, which uses set breakpoints. While they may require increased resources, this work supports continued initiatives for broader surveillance of low-level antibiotic resistance or their resistance determinants, which can serve as predictors of higher risk for clinical resistance.202134787446
482470.9996Chemogenomic Screen for Imipenem Resistance in Gram-Negative Bacteria. Carbapenem-resistant Gram-negative bacteria are considered a major threat to global health. Imipenem (IMP) is used as a last line of treatment against these pathogens, but its efficacy is diminished by the emergence of resistance. We applied a whole-genome screen in Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa isolates that were submitted to chemical mutagenesis, selected for IMP resistance, and characterized by next-generation sequencing. A comparative analysis of IMP-resistant clones showed that most of the highly mutated genes shared by the three species encoded proteins involved in transcription or signal transduction. Of these, the rpoD gene was one of the most prevalent and an E. coli strain disrupted for rpoD displayed a 4-fold increase in resistance to IMP. E. coli and K. pneumoniae also specifically shared several mutated genes, most involved in membrane/cell envelope biogenesis, and the contribution in IMP susceptibility was experimentally proven for amidases, transferases, and transglycosidases. P. aeruginosa differed from the two Enterobacteriaceae isolates with two different resistance mechanisms, with one involving mutations in the oprD porin or, alternatively, in two-component systems. Our chemogenomic screen performed with the three species has highlighted shared and species-specific responses to IMP.IMPORTANCE Gram-negative carbapenem-resistant bacteria are a major threat to global health. The use of genome-wide screening approaches to probe for genes or mutations enabling resistance can lead to identification of molecular markers for diagnostics applications. We describe an approach called Mut-Seq that couples chemical mutagenesis and next-generation sequencing for studying resistance to imipenem in the Gram-negative bacteria Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa The use of this approach highlighted shared and species-specific responses, and the role in resistance of a number of genes involved in membrane biogenesis, transcription, and signal transduction was functionally validated. Interestingly, some of the genes identified were previously considered promising therapeutic targets. Our genome-wide screen has the potential to be extended outside drug resistance studies and expanded to other organisms.201931744905
515680.9996Pseudomonas aeruginosa strains isolated from animal with high virulence genes content and highly sensitive to antimicrobials. OBJECTIVES: P. aeruginosa is one of the most metabolically versatile bacteria having the ability to survive in multiple environments through its accessory genome. An important hallmark of P. aeruginosa is the high level of antibiotic resistance, which often makes eradication difficult and sometimes impossible. Evolutionary forces have led to this bacterium to develop high antimicrobial resistance with a variety of elements contributing to both intrinsic and acquired resistance. The objectives were to genetically and phenotypically characterizer P. aeruginosa strains isolated from companion animals of different species. METHODS: We characterized a collection of 39 P. aeruginosa strains isolated from infected animals. The genetic characterization was in relation to chromosomal profile by PFGE; content of virulence gene; presence of genomic islands (GIs); genes of the cytotoxins exported by T3SS: exoU, exoS, exoT and exoY; and type IV pili allele. The phenotypic characterization was based on patterns of susceptibility to different antimicrobials. RESULTS: Each strain had a PFGE profile, a high virulence genes content, and a large accessory genome. However, most of the strains presented high sensitivity to almost all antimicrobials tested, showing no acquired resistance (no β-lactamases). The exception to this lack of resistance was seen with penicillin. CONCLUSIONS: P. aeruginosa could be a naturally sensitive bacterium to standard antimicrobials but could rapidly develop intrinsic and acquired resistance when the bacterium is exposed to pressure exerted by antibiotics, as observed in hospital settings.202438452900
628090.9996Genomic variation in Pseudomonas aeruginosa clinical respiratory isolates with de novo resistance to a bacteriophage cocktail. Pseudomonas aeruginosa is an opportunistic pathogen that can cause sinus infections and pneumonia in cystic fibrosis (CF) patients. Bacteriophage therapy is being investigated as a treatment for antibiotic-resistant P. aeruginosa infections. Although virulent bacteriophages have shown promise in treating P. aeruginosa infections, the development of bacteriophage-insensitive mutants (BIMs) in the presence of bacteriophages has been described. The aim of this study was to examine the genetic changes associated with the BIM phenotype. Biofilms of three genetically distinct P. aeruginosa strains, including PAO1 (ATCC 15692), and two clinical respiratory isolates (one CF and one non-CF) were grown for 7 days and treated with either a cocktail of four bacteriophages or a vehicle control for 7 consecutive days. BIMs isolated from the biofilms were detected by streak assays, and resistance to the phage cocktail was confirmed using spot test assays. Comparison of whole genome sequencing between the recovered BIMs and their respective vehicle control-treated phage-sensitive isolates revealed structural variants in two strains, and several small variants in all three strains. These variations involved a TonB-dependent outer membrane receptor in one strain, and mutations in lipopolysaccharide synthesis genes in two strains. Prophage deletion and induction were also noted in two strains, as well as mutations in several genes associated with virulence factors. Mutations in genes involved in susceptibility to conventional antibiotics were also identified in BIMs, with both decreased and increased antibiotic sensitivity to various antibiotics being observed. These findings may have implications for future applications of lytic phage therapy.IMPORTANCELytic bacteriophages are viruses that infect and kill bacteria and can be used to treat difficult-to-treat bacterial infections, including biofilm-associated infections and multidrug-resistant bacteria. Pseudomonas aeruginosa is a bacterium that can cause life-threatening infections. Lytic bacteriophage therapy has been trialed in the treatment of P. aeruginosa infections; however, sometimes bacteria develop resistance to the bacteriophages. This study sheds light on the genetic mechanisms of such resistance, and how this might be harnessed to restore the sensitivity of multidrug-resistant P. aeruginosa to conventional antibiotics.202540162801
4728100.9996Antibiotic Resistance Profile, Outer Membrane Proteins, Virulence Factors and Genome Sequence Analysis Reveal Clinical Isolates of Enterobacter Are Potential Pathogens Compared to Environmental Isolates. Outer membrane proteins (OMPs) of gram-negative bacteria play an important role in mediating antibacterial resistance, bacterial virulence and thus affect pathogenic ability of the bacteria. Over the years, prevalence of environmental antibiotic resistant organisms, their transmission to clinics and ability to transfer resistance genes, have been studied extensively. Nevertheless, how successful environmental bacteria can be in establishing as pathogenic bacteria under clinical setting, is less addressed. In the present study, we utilized an integrated approach of investigating the antibiotic resistance profile, presence of outer membrane proteins and virulence factors to understand extent of threat posed due to multidrug resistant environmental Enterobacter isolates. Also, we investigated clinical Enterobacter isolates and compared the results thereof. Results of the study showed that multidrug resistant environmental Enterobacter isolates lacked OmpC, lacked cell invasion abilities and exhibited low reactive oxygen species (ROS) production in neutrophils. In contrast, clinical isolates possessed OmpF, exhibited high invasive and adhesive property and produced higher amounts of ROS in neutrophils. These attributes indicated limited pathogenic potential of environmental Enterobacter isolates. Informations obtained from whole genome sequence of two representative bacterial isolates from environment (DL4.3) and clinical sources (EspIMS6) corroborated well with the observed results. Findings of the present study are significant as it highlights limited fitness of multidrug resistant environmental Enterobacter isolates.202032154188
6248110.9996Characterization of a stable, metronidazole-resistant Clostridium difficile clinical isolate. BACKGROUND: Clostridium difficile are gram-positive, spore forming anaerobic bacteria that are the leading cause of healthcare-associated diarrhea, usually associated with antibiotic usage. Metronidazole is currently the first-line treatment for mild to moderate C. difficile diarrhea however recurrence occurs at rates of 15-35%. There are few reports of C. difficile metronidazole resistance in the literature, and when observed, the phenotype has been transient and lost after storage or exposure of the bacteria to freeze/thaw cycles. Owing to the unstable nature of the resistance phenotype in the laboratory, clinical significance and understanding of the resistance mechanisms is lacking. METHODOLOGY/PRINCIPAL FINDINGS: Genotypic and phenotypic characterization was performed on a metronidazole resistant clinical isolate of C. difficile. Whole-genome sequencing was used to identify potential genetic contributions to the phenotypic variation observed with molecular and bacteriological techniques. Phenotypic observations of the metronidazole resistant strain revealed aberrant growth in broth and elongated cell morphology relative to a metronidazole-susceptible, wild type NAP1 strain. Comparative genomic analysis revealed single nucleotide polymorphism (SNP) level variation within genes affecting core metabolic pathways such as electron transport, iron utilization and energy production. CONCLUSIONS/SIGNIFICANCE: This is the first characterization of stable, metronidazole resistance in a C. difficile isolate. The study provides an in-depth genomic and phenotypic analysis of this strain and provides a foundation for future studies to elucidate mechanisms conferring metronidazole resistance in C. difficile that have not been previously described.201323349739
6267120.9996Beta-lactamase dependent and independent evolutionary paths to high-level ampicillin resistance. The incidence of beta-lactam resistance among clinical isolates is a major health concern. A key method to study the emergence of antibiotic resistance is adaptive laboratory evolution. However, in the case of the beta-lactam ampicillin, bacteria evolved in laboratory settings do not recapitulate clinical-like resistance levels, hindering efforts to identify major evolutionary paths and their dependency on genetic background. Here, we used the Microbial Evolution and Growth Arena (MEGA) plate to select ampicillin-resistant Escherichia coli mutants with varying degrees of resistance. Whole-genome sequencing of resistant isolates revealed that ampicillin resistance was acquired via a combination of single-point mutations and amplification of the gene encoding beta-lactamase AmpC. However, blocking AmpC-mediated resistance revealed latent adaptive pathways: strains deleted for ampC were able to adapt through combinations of changes in genes involved in multidrug resistance encoding efflux pumps, transcriptional regulators, and porins. Our results reveal that combinations of distinct genetic mutations, accessible at large population sizes, can drive high-level resistance to ampicillin even independently of beta-lactamases.202438918379
5058130.9996Widespread Fosfomycin Resistance in Gram-Negative Bacteria Attributable to the Chromosomal fosA Gene. Fosfomycin is a decades-old antibiotic which is being revisited because of its perceived activity against many extensively drug-resistant Gram-negative pathogens. FosA proteins are Mn(2+) and K(+)-dependent glutathione S-transferases which confer fosfomycin resistance in Gram-negative bacteria by conjugation of glutathione to the antibiotic. Plasmid-borne fosA variants have been reported in fosfomycin-resistant Escherichia coli strains. However, the prevalence and distribution of fosA in other Gram-negative bacteria are not known. We systematically surveyed the presence of fosA in Gram-negative bacteria in over 18,000 published genomes from 18 Gram-negative species and investigated their contribution to fosfomycin resistance. We show that FosA homologues are present in the majority of genomes in some species (e.g., Klebsiella spp., Enterobacter spp., Serratia marcescens, and Pseudomonas aeruginosa), whereas they are largely absent in others (e.g., E. coli, Acinetobacter baumannii, and Burkholderia cepacia). FosA proteins in different bacterial pathogens are highly divergent, but key amino acid residues in the active site are conserved. Chromosomal fosA genes conferred high-level fosfomycin resistance when expressed in E. coli, and deletion of chromosomal fosA in S. marcescens eliminated fosfomycin resistance. Our results indicate that FosA is encoded by clinically relevant Gram-negative species and contributes to intrinsic fosfomycin resistance.IMPORTANCE There is a critical need to identify alternate approaches to treat infections caused by extensively drug-resistant (XDR) Gram-negative bacteria. Fosfomycin is an old antibiotic which is routinely used for the treatment of urinary tract infections, although there is substantial interest in expanding its use to systemic infections caused by XDR Gram-negative bacteria. In this study, we show that fosA genes, which encode dimeric Mn(2+)- and K(+)-dependent glutathione S-transferase, are widely distributed in the genomes of Gram-negative bacteria-particularly those belonging to the family Enterobacteriaceae-and confer fosfomycin resistance. This finding suggests that chromosomally located fosA genes represent a vast reservoir of fosfomycin resistance determinants that may be transferred to E. coli Furthermore, they suggest that inhibition of FosA activity may provide a viable strategy to potentiate the activity of fosfomycin against XDR Gram-negative bacteria.201728851843
5060140.9996Nonclonal Emergence of Colistin Resistance Associated with Mutations in the BasRS Two-Component System in Escherichia coli Bloodstream Isolates. Infections by multidrug-resistant Gram-negative bacteria are increasingly common, prompting the renewed interest in the use of colistin. Colistin specifically targets Gram-negative bacteria by interacting with the anionic lipid A moieties of lipopolysaccharides, leading to membrane destabilization and cell death. Here, we aimed to uncover the mechanisms of colistin resistance in nine colistin-resistant Escherichia coli strains and one Escherichia albertii strain. These were the only colistin-resistant strains of 1,140 bloodstream Escherichia isolates collected in a tertiary hospital over a 10-year period (2006 to 2015). Core-genome phylogenetic analysis showed that each patient was colonized by a unique strain, suggesting that colistin resistance was acquired independently in each strain. All colistin-resistant strains had lipid A that was modified with phosphoethanolamine. In addition, two E. coli strains had hepta-acylated lipid A species, containing an additional palmitate compared to the canonical hexa-acylated E. coli lipid A. One E. coli strain carried the mobile colistin resistance (mcr) gene mcr-1.1 on an IncX4-type plasmid. Through construction of chromosomal transgene integration mutants, we experimentally determined that mutations in basRS, encoding a two-component signal transduction system, contributed to colistin resistance in four strains. We confirmed these observations by reversing the mutations in basRS to the sequences found in reference strains, resulting in loss of colistin resistance. While the mcr genes have become a widely studied mechanism of colistin resistance in E. coli, sequence variation in basRS is another, potentially more prevalent but relatively underexplored, cause of colistin resistance in this important nosocomial pathogen.IMPORTANCE Multidrug resistance among Gram-negative bacteria has led to the use of colistin as a last-resort drug. The cationic colistin kills Gram-negative bacteria through electrostatic interaction with the anionic lipid A moiety of lipopolysaccharides. Due to increased use in clinical and agricultural settings, colistin resistance has recently started to emerge. In this study, we used a combination of whole-genome sequence analysis and experimental validation to characterize the mechanisms through which Escherichia coli strains from bloodstream infections can develop colistin resistance. We found no evidence of direct transfer of colistin-resistant isolates between patients. The lipid A of all isolates was modified by the addition of phosphoethanolamine. In four isolates, colistin resistance was experimentally verified to be caused by mutations in the basRS genes, encoding a two-component regulatory system. Our data show that chromosomal mutations are an important cause of colistin resistance among clinical E. coli isolates.202032161146
9897150.9996The fitness connection of antibiotic resistance. More than three decades ago multidrug-resistant (MDR) clones of the pathogens: Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Clostridioides difficile, Enterococcus faecium, Pseudomonas aeruginosa and Acinetobacter baumannii have started to disseminate across wide geographical areas. A characteristic feature of all these MDR lineages is the carriage of some mutations in the quinolone resistance-determining regions (QRDRs) of DNA gyrase and topoisomerase IV which besides conferring resistance to fluoroquinolones are associated with a fitness benefit. Several lines of evidence strongly suggest that extra fitness conferred by these mutations facilitated the dissemination of the international MDR lineages. MDR pathogens require extra energy to cover the fitness cost conferred by the excess antibiotic resistance gene cargo. However, extra energy generated by upgraded metabolic activity was demonstrated to increase the uptake of antibiotics enhancing susceptibility. Accordingly, MDR bacteria need additional positive fitness schemes which, similarly to the QRDR advantage, will not compromise resistance. Some of these, not clone-specific effects are large genomes, the carriage of low-cost plasmids, the transfer of plasmid genes to the chromosome, the application of weak promoters in integrons and various techniques for the economic control of the activity of the integrase enzyme including a highly sophisticated system in A. baumannii. These impacts - among others - will confer a fitness advantage promoting the spread of MDR pathogens. However, even the potential of extra fitness generated by the combined effect of various schemes is not without limit and virulence-related genes or less relevant antibiotic resistance gene cargoes will often be sacrificed to permit the acquisition of high-priority resistance determinants. Accordingly major MDR clone strains are usually less virulent than susceptible isolates. In summary, a fitness approach to the research of antibiotic resistance is very useful since the fitness status of MDR bacteria seem to profoundly impact the capacity to disseminate in the healthcare setting.202540276228
6275160.9996Resistance to fosfomycin: Mechanisms, Frequency and Clinical Consequences. Fosfomycin has been used for the treatment of infections due to susceptible and multidrug-resistant (MDR) bacteria. It inhibits bacterial cell wall synthesis through a unique mechanism of action at a step prior to that inhibited by β-lactams. Fosfomycin enters the bacterium through membrane channels/transporters and inhibits MurA, which initiates peptidoglycan (PG) biosynthesis of the bacterial cell wall. Several bacteria display inherent resistance to fosfomycin mainly through MurA mutations. Acquired resistance involves, in order of decreasing frequency, modifications of membrane transporters that prevent fosfomycin from entering the bacterial cell, acquisition of plasmid-encoded genes that inactivate fosfomycin, and MurA mutations. Fosfomycin resistance develops readily in vitro but less so in vivo. Mutation frequency is higher among Pseudomonas aeruginosa and Klebsiella spp. compared with Escherichia coli and is associated with fosfomycin concentration. Mutations in cAMP regulators, fosfomycin transporters and MurA seem to be associated with higher biological cost in Enterobacteriaceae but not in Pseudomonas spp. The contribution of fosfomycin inactivating enzymes in emergence and spread of fosfomycin resistance currently seems low-to-moderate, but their presence in transferable plasmids may potentially provide the best means for the spread of fosfomycin resistance in the future. Their co-existence with genes conferring resistance to other antibiotic classes may increase the emergence of MDR strains. Although susceptibility rates vary, rates seem to increase in settings with higher fosfomycin use and among multidrug-resistant pathogens.201930268576
4819170.9996Gram-Negative Bacteria. Gram-negative bacteria (GNB) are among the world's most significant public health problems due to their high resistance to antibiotics. These microorganisms have significant clinical importance in hospitals because they put patients in the intensive care unit (ICU) at high risk and lead to high morbidity and mortality. Two large groups, Enterobacteriaceae and the non-fermenters, are responsible for most clinical isolates; nevertheless, other clinically concerning gram-negative organisms exist, including but not limited to Neisseria, Haemophilus spp., Helicobacter pylori, and Chlamydia trachomatis. Enterobacteriaceae   Enterobacteriaceae are a heterogeneous group widely dispersed in nature. They account for about 80% of gram-negative isolates with a myriad of disease-causing general/species in humans, including urinary tract infections, pneumonia, diarrhea, meningitis, sepsis, endotoxic shock, and many others. The general/species that frequently affect humans are Escherichia, Proteus, Enterobacter, Klebsiella, Citrobacter, Yersinia, Shigella, and Salmonella, among others. Laboratory characterization is an essential component when it comes to microorganisms; therefore, it is imperative to expose characteristics of Enterobacteriaceae, which are bacilli, non-sporulated, have variable motility, grow in the presence and absence of oxygen, ferment organisms of glucose, are cytochrome oxidase negative, and can reduce nitrate to nitrite. Non-Fermenters The non-fermenter, gram-negative bacilli (BNF) have a lower frequency of isolation when compared to Enterobacteriaceae; however, they are a relevant group since they cause severe, fatal infections, especially in the hospital environment. They also cause opportunistic diseases in ICU patients who undergo invasive procedures. The main BNF microorganisms that cause human disease are Pseudomonas aeruginosa, Acinetobacter baumannii, Burkholderia cepacia, Burkholderia pseudomallei, Stenotrophomonas., Alcaligenes, and Moraxella. These stand out for being aerobic and non-sporulated; they are incapable of fermenting sugars, using them through the oxidative route. The critical issue regarding BNF, when it comes to the antimicrobial sensitivity profile, is undoubtedly their intrinsic resistance since they produce a variety of genes with multiple mechanisms capable of mitigating the microbicidal action. Thus, it stands out in P. aeruginosa, cephalosporinase of type AmpC, and efflux systems that confer resistance to b-lactams. The most frequent are MexAB-OprM; and loss of OprD (which gives impermeability to the bacterial cell due to the loss of porin.) Acinetobacter baumannii naturally produces AmpC cephalosporinase and oxacillinase (OXA), leaving it spontaneously immune to many drugs. The genetic ingenuity of this microorganism goes further, and it combines high impermeability with genetic plasticity, combining with the resistance of mechanisms such as extended-spectrum b-lactamases (ESBL). The Stenotrophomonas exhibit a pattern of intrinsic multi-resistance, especially in patients who have had contact with carbapenems. Thus, Stenotrophomonas present several efflux pumps and produce two carbapenemases – L1 (resistance to all carbapenems) and L2 (cephalosporinase).  These mechanisms, associated or separate, restrict the treatment options to an alarming level. Sulfamethoxazole-trimethoprim remains the mainstay of treatment.  Antibiotic Resistance These organisms have a range of mechanisms to prevent the action of many antimicrobials used in clinical medicine. Some of the mechanisms of resistance include efflux pumps, alteration of the drug binding site and membrane permeability, degradation enzymes, and the conformational change of the drug culminating in its inactivation. GNB have two membranes, an external and an internal. The external membrane expresses a potent immune response inducer, lipopolysaccharide (LPS), which is composed of three units: a hydrophilic polysaccharide, O antigen, and a hydrophobic domain known as lipid A. Lipid A are responsible for the higher endotoxic activity of these bacteria. However, the LPS is heterogeneous in the various bacterial groups, and some bacteria manifest this antigen weakly due to genetic changes and are not recognized by Toll-like receptors. In contrast, there are BGN groups that can trigger such a response in large proportions. Thus, LPS can trigger the innate immune response through Toll-like receptors 4 (TLR4), which occurs in many immune cells such as monocytes, macrophages, dendritic cells, and neutrophils. The resulting activation of the innate immune response mediated by LPS together with TLR4 receptors culminates in an exacerbated response with the production of cytokines, chemokines, and interferons and their suppression. Enterobacteriaceae diffuse their plasmids by conjugation, which gives rise to resistance to almost all existing antibiotics. The family of enzymes carbapenemase – KPC, NDM-1, IMP, VIM, OXA-48 – is undoubtedly one of the most significant health challenges of the century, given the potential for dissemination between species and mortality rates due to infections caused by bacteria with such plasmids. Colistin, one of the few antibiotics that still treat multiresistant infections, already has a mobile resistance gene, mcr-1, and Enterobacteriaceae has a crucial role in the spread of this gene, with worldwide reports. Moreover, a further concern is that they usually associate these genes with other resistance genes (CTX-M, NDM, IMP), producing resistance to cephalosporins and carbapenems, enhancing the deleterious effects caused by these microorganisms.202530855801
9914180.9996Identification of host genetic factors modulating β-lactam resistance in Escherichia coli harbouring plasmid-borne β-lactamase through transposon-sequencing. Since β-lactam antibiotics are widely used, emergence of bacteria with resistance to them poses a significant threat to society. In particular, acquisition of genes encoding β-lactamase, an enzyme that degrades β-lactam antibiotics, has been a major contributing factor in the emergence of bacteria that are resistant to β-lactam antibiotics. However, relatively few genetic targets for killing these resistant bacteria have been identified to date. Here, we used a systematic approach called transposon-sequencing (Tn-Seq), to screen the Escherichia coli genome for host genetic factors that, when mutated, affect resistance to ampicillin, one of the β-lactam antibiotics, in a strain carrying a plasmid that encodes β-lactamase. This approach enabled not just the isolation of genes previously known to affect β-lactam resistance, but the additional loci skp, gshA, phoPQ and ypfN. Individual mutations in these genes modestly but consistently affected antibiotic resistance. We have identified that these genes are not only implicated in β-lactam resistance by itself but also play a crucial role in conditions associated with the expression of β-lactamase. GshA and phoPQ appear to contribute to β-lactam resistance by regulating membrane integrity. Notably, the overexpression of the uncharacterized membrane-associated protein, ypfN, has been shown to significantly enhance β-lactam resistance. We applied the genes identified from the screening into Salmonella Typhimurium and Pseudomonas aeruginosa strains, both critical human pathogens with antibiotic resistance, and observed their significant impact on β-lactam resistance. Therefore, these genes can potentially be utilized as therapeutic targets to control the survival of β-lactamase-producing bacteria.202540231449
4913190.9996Multiple Plasmids Contribute to Antibiotic Resistance and Macrophage Survival In Vitro in CMY2-Bearing Salmonella enterica. Multiple drug resistance (MDR) in bacteria represents a notable problem but if carried on plasmid their spread could become a significant threat to public health. Plasmids in members of the Enterobacteriaceae family and in particular Salmonella and Escherichia coli strains have been implicated in the spread of antibiotic resistance genes. However, the mechanisms involved in the transfer of plasmid-borne resistance genes are not fully understood. Here, we analyzed the ability of Salmonella enterica clinical isolates to transfer plasmid-borne MDR to E. coli. We also determined whether possession of an Inc A/C plasmid by a S. enterica isolate would confer increased fitness compared to an isolate not carrying the plasmid. Sixteen human and animal isolates of S. enterica were screened using a three-panel multiplex PCR assay, and simplex PCR for the blaCMY-2 gene. Using these data we selected a suitable strain as a plasmid donor for the construction of a new Salmonella strain with an Inc A/C plasmid. This allowed us to compare isogenic strains with and without the Inc A/C plasmid in multiple growth, fitness, and invasion assays. The results showed that possession of Inc A/C plasmid confers significant fitness advantage when tested in J774 macrophages as opposed to HEp-2 cells where no significant difference was found. In addition, stress assays performed in vitro showed that the possession of this large plasmid by Salmonella strains tested here does not appear to incur a significant fitness cost. Gaining a better understanding of molecular mechanisms of plasmid transfer between pathogenic bacteria will allow us to characterize the role of MDR in pathogenicity of bacteria and to identify methods to reduce the frequency of dissemination of multiple antibiotic resistance genes.201627070176