RESUMO
In many eukaryotes, Argonaute proteins, guided by short RNA sequences, defend cells against transposons and viruses. In the eubacterium Thermus thermophilus, the DNA-guided Argonaute TtAgo defends against transformation by DNA plasmids. Here, we report that TtAgo also participates in DNA replication. In vivo, TtAgo binds 15- to 18-nt DNA guides derived from the chromosomal region where replication terminates and associates with proteins known to act in DNA replication. When gyrase, the sole T. thermophilus type II topoisomerase, is inhibited, TtAgo allows the bacterium to finish replicating its circular genome. In contrast, loss of gyrase and TtAgo activity slows growth and produces long sausage-like filaments in which the individual bacteria are linked by DNA. Finally, wild-type T. thermophilus outcompetes an otherwise isogenic strain lacking TtAgo. We propose that the primary role of TtAgo is to help T. thermophilus disentangle the catenated circular chromosomes generated by DNA replication.
Assuntos
Proteínas Argonautas/metabolismo , Proteínas de Bactérias/metabolismo , DNA Girase/metabolismo , Replicação do DNA/genética , DNA/metabolismo , Thermus thermophilus/metabolismo , Proteínas Argonautas/genética , Proteínas de Bactérias/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Cromossomos/metabolismo , Ciprofloxacina/farmacologia , DNA/genética , Replicação do DNA/efeitos dos fármacos , Endonucleases/metabolismo , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Proteínas Recombinantes , Recombinação Genética/efeitos dos fármacos , Recombinação Genética/genética , Imagem Individual de Molécula , Espectrometria de Massas em Tandem , Thermus thermophilus/genética , Thermus thermophilus/crescimento & desenvolvimento , Thermus thermophilus/ultraestrutura , Inibidores da Topoisomerase II/farmacologiaRESUMO
Antibiotic resistance is a global health threat and often results from new mutations. Antibiotics can induce mutations via mechanisms activated by stress responses, which both reveal environmental cues of mutagenesis and are weak links in mutagenesis networks. Network inhibition could slow the evolution of resistance during antibiotic therapies. Despite its pivotal importance, few identities and fewer functions of stress responses in mutagenesis are clear. Here, we identify the Escherichia coli stringent starvation response in fluoroquinolone-antibiotic ciprofloxacin-induced mutagenesis. Binding of response-activator ppGpp to RNA polymerase (RNAP) at two sites leads to an antibiotic-induced mutable gambler-cell subpopulation. Each activates a stress response required for mutagenic DNA-break repair: surprisingly, ppGpp-site-1-RNAP triggers the DNA-damage response, and ppGpp-site-2-RNAP induces σS-response activity. We propose that RNAP regulates DNA-damage processing in transcribed regions. The data demonstrate a critical node in ciprofloxacin-induced mutagenesis, imply RNAP-regulation of DNA-break repair, and identify promising targets for resistance-resisting drugs.
Assuntos
Proteínas de Escherichia coli , Proteínas de Escherichia coli/metabolismo , Guanosina Tetrafosfato/metabolismo , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Ciprofloxacina/farmacologia , DNA/metabolismo , RNA/metabolismo , Regulação Bacteriana da Expressão GênicaRESUMO
In recent years, new evidence has shown that the SOS response plays an important role in the response to antimicrobials, with involvement in the generation of clinical resistance. Here we evaluate the impact of heterogeneous expression of the SOS response in clinical isolates of Escherichia coli on response to the fluoroquinolone, ciprofloxacin. In silico analysis of whole genome sequencing data showed remarkable sequence conservation of the SOS response regulators, RecA and LexA. Despite the genetic homogeneity, our results revealed a marked differential heterogeneity in SOS response activation, both at population and single-cell level, among clinical isolates of E. coli in the presence of subinhibitory concentrations of ciprofloxacin. Four main stages of SOS response activation were identified and correlated with cell filamentation. Interestingly, there was a correlation between clinical isolates with higher expression of the SOS response and further progression to resistance. This heterogeneity in response to DNA damage repair (mediated by the SOS response) and induced by antimicrobial agents could be a new factor with implications for bacterial evolution and survival contributing to the generation of antimicrobial resistance.
Assuntos
Antibacterianos , Ciprofloxacina , Proteínas de Escherichia coli , Escherichia coli , Testes de Sensibilidade Microbiana , Recombinases Rec A , Resposta SOS em Genética , Resposta SOS em Genética/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Ciprofloxacina/farmacologia , Humanos , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Antibacterianos/farmacologia , Recombinases Rec A/genética , Recombinases Rec A/metabolismo , Farmacorresistência Bacteriana/genética , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Dano ao DNA/efeitos dos fármacos , Sequenciamento Completo do Genoma , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/tratamento farmacológico , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Adaptação Fisiológica , Reparo do DNA/efeitos dos fármacos , Proteínas de Ligação a DNARESUMO
Collateral sensitivity is an evolutionary trade-off whereby acquisition of the adaptive phenotype of resistance to an antibiotic leads to the nonadaptive increased susceptibility to another. The feasibility of harnessing such a trade-off to design evolutionary-based approaches for treating bacterial infections has been studied using model strains. However, clinical application of collateral sensitivity requires its conservation among strains presenting different mutational backgrounds. Particularly relevant is studying collateral sensitivity robustness of already-antibiotic-resistant mutants when challenged with a new antimicrobial, a common situation in clinics that has hardly been addressed. We submitted a set of diverse Pseudomonas aeruginosa antibiotic-resistant mutants to short-term evolution in the presence of different antimicrobials. Ciprofloxacin selects different clinically relevant resistance mutations in the preexisting resistant mutants, which gave rise to the same, robust, collateral sensitivity to aztreonam and tobramycin. We then experimentally determined that alternation of ciprofloxacin with aztreonam is more efficient than ciprofloxacintobramycin alternation in driving the extinction of the analyzed antibiotic-resistant mutants. Also, we show that the combinations ciprofloxacinaztreonam or ciprofloxacintobramycin are the most effective strategies for eliminating the tested P. aeruginosa antibiotic-resistant mutants. These findings support that the identification of conserved collateral sensitivity patterns may guide the design of evolution-based strategies to treat bacterial infections, including those due to antibiotic-resistant mutants. Besides, this is an example of phenotypic convergence in the absence of parallel evolution that, beyond the antibiotic-resistance field, could facilitate the understanding of evolution processes, where the selective forces giving rise to new, not clearly adaptive phenotypes remain unclear.
Assuntos
Antibacterianos , Ciprofloxacina , Sensibilidade Colateral a Medicamentos , Farmacorresistência Bacteriana , Infecções por Pseudomonas , Pseudomonas aeruginosa , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Ciprofloxacina/farmacologia , Ciprofloxacina/uso terapêutico , Sensibilidade Colateral a Medicamentos/genética , Farmacorresistência Bacteriana/genética , Humanos , Testes de Sensibilidade Microbiana , Mutação , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/genéticaRESUMO
Ciprofloxacin-resistant Salmonella Typhimurium (S. Typhimurium) causes a significant health burden worldwide. A wealth of studies has been published on the contributions of different mechanisms to ciprofloxacin resistance in Salmonella spp. But we still lack a deep understanding of the physiological responses and genetic changes that underlie ciprofloxacin exposure. This study aims to know how phenotypic and genotypic characteristics are impacted by ciprofloxacin exposure, from ciprofloxacin-susceptible to ciprofloxacin-resistant strains in vitro. Here, we investigated the multistep evolution of resistance in replicate populations of S. Typhimurium during 24 days of continuously increasing ciprofloxacin exposure and assessed how ciprofloxacin impacts physiology and genetics. Numerous studies have demonstrated that RamA is a global transcriptional regulator that prominently perturbs the transcriptional landscape of S. Typhimurium, resulting in a ciprofloxacin-resistant phenotype appearing first; the quinolone resistance-determining region mutation site can only be detected later. Comparing the microbial physiological changes and RNA sequencing (RNA-Seq) results of ancestral and selectable mutant strains, the selectable mutant strains had some fitness costs, such as decreased virulence, an increase of biofilm-forming ability, a change of "collateral" sensitivity to other drugs, and inability to utilize galactitol. Importantly, in the ciprofloxacin induced, RamA directly binds and activates the gatR gene responsible for the utilization of galactitol, but RamA deletion strains could not activate gatR. The elevated levels of RamA, which inhibit the galactitol metabolic pathway through the activation of gatR, can lead to a reduction in the growth rate, adhesion, and colonization resistance of S. Typhimurium. This finding is supported by studies conducted in M9 medium as well as in vivo infection models. IMPORTANCE: Treatment of antibiotic resistance can significantly benefit from a deeper understanding of the interactions between drugs and genetics. The physiological responses and genetic mechanisms in antibiotic-exposed bacteria are not well understood. Traditional resistance studies, often retrospective, fail to capture the entire resistance development process and typically exhibit unpredictable dynamics. To explore how clinical isolates of S. Typhimurium respond to ciprofloxacin, we analyzed their adaptive responses. We found that S. Typhimurium RamA-mediated regulation disrupts microbial metabolism under ciprofloxacin exposure, affecting genes in the galactitol metabolic pathways. This disruption facilitates adaptive responses to drug therapy and enhances the efficiency of intracellular survival. A more comprehensive and integrated understanding of these physiological and genetic changes is crucial for improving treatment outcomes.
Assuntos
Antibacterianos , Ciprofloxacina , Farmacorresistência Bacteriana , Regulação Bacteriana da Expressão Gênica , Salmonella typhimurium , Ciprofloxacina/farmacologia , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Virulência , Farmacorresistência Bacteriana/genética , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Animais , Camundongos , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Testes de Sensibilidade Microbiana , MutaçãoRESUMO
BACKGROUND: Antimicrobial resistance (AMR) remains a significant global health threat particularly impacting low- and middle-income countries (LMICs). These regions often grapple with limited healthcare resources and access to advanced diagnostic tools. Consequently, there is a pressing need for innovative approaches that can enhance AMR surveillance and management. Machine learning (ML) though underutilized in these settings, presents a promising avenue. This study leverages ML models trained on whole-genome sequencing data from England, where such data is more readily available, to predict AMR in E. coli, targeting key antibiotics such as ciprofloxacin, ampicillin, and cefotaxime. A crucial part of our work involved the validation of these models using an independent dataset from Africa, specifically from Uganda, Nigeria, and Tanzania, to ascertain their applicability and effectiveness in LMICs. RESULTS: Model performance varied across antibiotics. The Support Vector Machine excelled in predicting ciprofloxacin resistance (87% accuracy, F1 Score: 0.57), Light Gradient Boosting Machine for cefotaxime (92% accuracy, F1 Score: 0.42), and Gradient Boosting for ampicillin (58% accuracy, F1 Score: 0.66). In validation with data from Africa, Logistic Regression showed high accuracy for ampicillin (94%, F1 Score: 0.97), while Random Forest and Light Gradient Boosting Machine were effective for ciprofloxacin (50% accuracy, F1 Score: 0.56) and cefotaxime (45% accuracy, F1 Score:0.54), respectively. Key mutations associated with AMR were identified for these antibiotics. CONCLUSION: As the threat of AMR continues to rise, the successful application of these models, particularly on genomic datasets from LMICs, signals a promising avenue for improving AMR prediction to support large AMR surveillance programs. This work thus not only expands our current understanding of the genetic underpinnings of AMR but also provides a robust methodological framework that can guide future research and applications in the fight against AMR.
Assuntos
Antibacterianos , Escherichia coli , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Escherichia coli/genética , Farmacorresistência Bacteriana/genética , Ciprofloxacina/farmacologia , Ciprofloxacina/uso terapêutico , Ampicilina , Cefotaxima , Aprendizado de Máquina , NigériaRESUMO
Rapid point-of-care tests that diagnose gonococcal infections and identify susceptibility to antibiotics enable individualized treatment. This could improve patient outcomes and slow the emergence and spread of antibiotic resistance. However, little is known about the long-term impact of such diagnostics on the burden of gonorrhea and the effective life span of antibiotics. We used a mathematical model of gonorrhea transmission among men who have sex with men in the United States to project the annual rate of reported gonorrhea cases and the effective life span of ceftriaxone, the recommended antibiotic for first-line treatment of gonorrhea, as well as 2 previously recommended antibiotics, ciprofloxacin and tetracycline, when a rapid drug susceptibility test that estimates susceptibility to ciprofloxacin and tetracycline is available. The use of a rapid drug susceptibility test with ≥50% sensitivity and ≥95% specificity, defined in terms of correct ascertainment of drug susceptibility and nonsusceptibility status, could increase the combined effective life span of ciprofloxacin, tetracycline, and ceftriaxone by at least 2 years over 25 years of simulation. If test specificity is imperfect, however, the increase in the effective life span of antibiotics is accompanied by an increase in the rate of reported gonorrhea cases even under perfect sensitivity.
Assuntos
Gonorreia , Minorias Sexuais e de Gênero , Masculino , Humanos , Estados Unidos/epidemiologia , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Gonorreia/tratamento farmacológico , Gonorreia/epidemiologia , Ceftriaxona/uso terapêutico , Ceftriaxona/farmacologia , Homossexualidade Masculina , Longevidade , Neisseria gonorrhoeae , Testes de Sensibilidade Microbiana , Ciprofloxacina/farmacologia , Ciprofloxacina/uso terapêutico , Tetraciclina/farmacologia , Tetraciclina/uso terapêutico , Farmacorresistência BacterianaRESUMO
Quorum sensing is a type of cell-cell communication that modulates various biological activities of bacteria. Previous studies indicate that quorum sensing contributes to the evolution of bacterial resistance to antibiotics, but the underlying mechanisms are not fully understood. In this study, we grew Pseudomonas aeruginosa in the presence of sub-lethal concentrations of ciprofloxacin, resulting in a large increase in ciprofloxacin minimal inhibitory concentration. We discovered that quorum sensing-mediated phenazine biosynthesis was significantly enhanced in the resistant isolates, where the quinolone circuit was the predominant contributor to this phenomenon. We found that production of pyocyanin changed carbon flux and showed that the effect can be partially inhibited by the addition of pyruvate to cultures. This study illustrates the role of quorum sensing-mediated phenotypic resistance and suggests a strategy for its prevention.
Assuntos
Antibacterianos , Ciprofloxacina , Farmacorresistência Bacteriana , Testes de Sensibilidade Microbiana , Fenazinas , Pseudomonas aeruginosa , Piocianina , Percepção de Quorum , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/genética , Ciprofloxacina/farmacologia , Percepção de Quorum/efeitos dos fármacos , Fenazinas/farmacologia , Fenazinas/metabolismo , Antibacterianos/farmacologia , Piocianina/biossíntese , Farmacorresistência Bacteriana/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Quinolonas/farmacologiaRESUMO
Understanding the evolution of antibiotic resistance is important for combating drug-resistant bacteria. In this work, we investigated the adaptive response of Pseudomonas aeruginosa to ciprofloxacin. Ciprofloxacin-susceptible P. aeruginosa ATCC 9027, CIP-E1 (P. aeruginosa ATCC 9027 exposed to ciprofloxacin for 14 days) and CIP-E2 (CIP-E1 cultured in antibiotic-free broth for 10 days) were compared. Phenotypic responses including cell morphology, antibiotic susceptibility, and production of pyoverdine, pyocyanin and rhamnolipid were assessed. Proteomic responses were evaluated using comparative iTRAQ labelling LC-MS/MS to identify differentially expressed proteins (DEPs). Expression of associated genes coding for notable DEPs and their related regulatory genes were checked using quantitative reverse transcriptase PCR. CIP-E1 displayed a heterogeneous morphology, featuring both filamentous cells and cells with reduced length and width. By contrast, although filaments were not present, CIP-E2 still exhibited size reduction. Considering the MIC values, ciprofloxacin-exposed strains developed resistance to fluoroquinolone antibiotics but maintained susceptibility to other antibiotic classes, except for carbapenems. Pyoverdine and pyocyanin production showed insignificant decreases, whereas there was a significant decrease in rhamnolipid production. A total of 1039 proteins were identified, of which approximately 25â% were DEPs. In general, there were more downregulated proteins than upregulated proteins. Noted changes included decreased OprD and PilP, and increased MexEF-OprN, MvaT and Vfr, as well as proteins of ribosome machinery and metabolism clusters. Gene expression analysis confirmed the proteomic data and indicated the downregulation of rpoB and rpoS. In summary, the response to CIP involved approximately a quarter of the proteome, primarily associated with ribosome machinery and metabolic processes. Potential targets for bacterial interference encompassed outer membrane proteins and global regulators, such as MvaT.
Assuntos
Ciprofloxacina , Infecções por Pseudomonas , Humanos , Ciprofloxacina/farmacologia , Pseudomonas aeruginosa/genética , Cromatografia Líquida , Proteômica , Piocianina , Espectrometria de Massas em Tandem , Antibacterianos/farmacologiaRESUMO
Introduction. Bacterial keratitis, particularly caused by Pseudomonas aeruginosa, is challenging to treat because of multi-drug tolerance, often associated with the formation of biofilms. Antibiotics in development are typically evaluated against planktonic bacteria in a culture medium, which may not accurately represent the complexity of infections in vivo.Hypothesis/Gap Statement. Developing a reliable, economic ex vivo keratitis model that replicates some complexity of tissue infections could facilitate a deeper understanding of antibiotic efficacy, thus aiding in the optimization of treatment strategies for bacterial keratitis.Methodology. Here we investigated the efficacy of three commonly used antibiotics (gentamicin, ciprofloxacin and meropenem) against Pseudomonas aeruginosa cytotoxic strain PA14 and invasive strain PA01 using an ex vivo porcine keratitis model.Results. Both strains of P. aeruginosa were susceptible to the MIC of the three tested antibiotics. However, significantly higher concentrations were necessary to inhibit bacterial growth in the minimum biofilm eradication concentration (MBEC) assay, with both strains tolerating concentrations greater than 512 mg l-1 of meropenem. When MIC and higher concentrations than MBEC (1024 mg l-1) of antibiotics were applied, ciprofloxacin exhibited the highest potency against both P. aeruginosa strains, followed by meropenem, while gentamicin showed the least potency. Despite this, none of the antibiotic concentrations used effectively cleared the infection, even after 18 h of continuous exposure.Conclusions. Further exploration of antibiotic concentrations and aligning dosing with clinical studies to validate the model is needed. Nonetheless, our ex vivo porcine keratitis model could be a valuable tool for assessing antibiotic efficacy.
Assuntos
Antibacterianos , Biofilmes , Ciprofloxacina , Modelos Animais de Doenças , Ceratite , Testes de Sensibilidade Microbiana , Infecções por Pseudomonas , Pseudomonas aeruginosa , Animais , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/crescimento & desenvolvimento , Antibacterianos/farmacologia , Suínos , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/microbiologia , Biofilmes/efeitos dos fármacos , Ceratite/microbiologia , Ceratite/tratamento farmacológico , Ciprofloxacina/farmacologia , Gentamicinas/farmacologia , Meropeném/farmacologiaRESUMO
Supplementary nucleic acid amplification testing for Neisseria gonorrhoeae (NG) is widely used to circumvent specificity problems associated with extragenital sites. Here, we compared different supplementary approaches for confirming NG-positive samples from the cobas 4800 CT/NG (c4800) and cobas 6800 CT/NG (c6800) assays using the ResistancePlusGC (RP-GC) assay, which in addition to detecting NG, also predicts ciprofloxacin susceptibility via NG gyrA characterization. Two different nucleic acid extraction techniques were investigated for RP-GC detection; extracts from c4800 (c4800-RP-GC) and MagNA Pure 96 (MP96-RP-GC). NG-positive (n = 300) and -negative (n = 150) samples in cobas PCR media from routine c4800 testing were retrospectively retested with c4800, c6800, c4800-RP-GC, and MP96-RP-GC. Selected samples were also tested with Xpert CT/NG (Xpert) for discrepant analysis. The gyrA status was compared to ETEST ciprofloxacin susceptibility or non-susceptibility for recovered isolates (n = 63). Extragenital confirmatory rates were higher for MP96-RP-GC (131/140; 93.6%) compared to c4800-RP-GC (126/146; 86.3%), albeit not significantly (P = 0.6677). Of 9 samples testing positive by c6800 and negative by MP96-RP-GC, 7/9 (77.8%) were also negative by Xpert. By contrast, the number of samples returning a valid gyrA status was significantly (P = 0.0003) higher for MP96-RP-GC (270/293; 92.2%) compared to c4800-RP-GC (245/298; 82.2%). The overall MP96-RP-GC gyrA status correlated 98.4% (61/62) with the reported ciprofloxacin sensitive (35/36; 97.2%) or non-susceptible (26/26; 100%) phenotype. Improved RP-GC confirmatory rates and reported gyrA status were observed using MP96 nucleic acids compared to c4800 extracts. The data further highlight the ongoing need for NG supplemental testing for oropharyngeal samples.
Assuntos
Gonorreia , Ácidos Nucleicos , Humanos , Neisseria gonorrhoeae/genética , Ciprofloxacina/farmacologia , Estudos Retrospectivos , Sensibilidade e Especificidade , Tomografia Computadorizada por Raios X , Gonorreia/diagnósticoRESUMO
Campylobacter fetus is known to cause human disease, particularly in elderly and immunocompromised hosts. There are limited published data for antimicrobial susceptibility patterns with this organism, and no interpretive criteria are available. We reviewed antimicrobial susceptibilities of C. fetus isolates tested at a tertiary care center and reference laboratory over an 11-year period. C. fetus isolates from patients treated at Mayo Clinic and those sent as referrals for identification and susceptibility were included. Antimicrobial susceptibility testing was performed using agar dilution for ciprofloxacin, doxycycline, erythromycin, gentamicin, meropenem, and tetracycline. Geographic distribution, culture source, organism minimal inhibitory concentration (MIC) distributions, and MIC50 and MIC90 were examined. Excluding duplicates, 105 unique isolates were identified from 110 positive cultures. Blood cultures represented the most common source, followed by body fluids, skin and soft tissue, and central nervous system. Gentamicin and meropenem had favorable MIC50 and MIC90 of 1 µg/mL. Ciprofloxacin demonstrated an MIC50 of 1 µg/mL; however, the MIC90 was >2 µg/mL. Erythromycin demonstrated MIC50 and MIC90 of 2 µg/mL. Tetracycline and doxycycline were tested on a limited number of isolates and showed a wide range of MICs. Gentamicin and meropenem demonstrated favorable MICs in C. fetus isolates. These may represent therapeutic options for consideration in serious C. fetus infections, pending susceptibility results. Ciprofloxacin, which showed variable results, may be more appropriate for use only after susceptibility testing. C. fetus interpretive criteria are needed to aid clinicians in selection of both empiric and definitive therapies. IMPORTANCE: Our findings contribute to the scant literature on Campylobacter fetus antimicrobial susceptibility test results. We used a reference test method of agar dilution and provide MICs for a large number of organisms and antimicrobial agents.
Assuntos
Anti-Infecciosos , Campylobacter , Humanos , Idoso , Campylobacter fetus , Doxiciclina/farmacologia , Meropeném , Ágar , Antibacterianos/farmacologia , Ciprofloxacina/farmacologia , Eritromicina/farmacologia , Tetraciclina , Gentamicinas/farmacologia , Testes de Sensibilidade MicrobianaRESUMO
BACKGROUND: Risk factors for ciprofloxacin or MDR in primary care urine specimens are not well defined. OBJECTIVES: We created a primary care-specific antibiogram for Escherichia coli isolates from cases with complicated and uncomplicated urinary tract infection (UTI) and evaluated risk factors for ciprofloxacin, trimethoprim/sulfamethoxazole and MDR among Enterobacterales. METHODS: We conducted a cross-sectional study to determine resistance and risk factors by collecting urine cultures from all patients (≥18 years) presenting with provider-suspected UTI at two primary care, safety-net clinics in Houston, TX, USA between November 2018 and March 2020. RESULTS: Among 1262 cultures, 308 cultures grew 339 uropathogens. Patients with Enterobacterales (nâ=â199) were mostly female (93.5%) with a mean age of 48.5 years. E. coli was the predominant uropathogen isolated (nâ=â187/339; 55%) and had elevated trimethoprim/sulfamethoxazole (43.6%) and ciprofloxacin (29.5%) resistance, low nitrofurantoin (1.8%) resistance, and no fosfomycin resistance. Among E. coli, 10.6% were ESBL positive and 24.9% had MDR. Birth outside the U.S.A., prior (2 year) trimethoprim/sulfamethoxazole resistance, and diabetes mellitus were associated with trimethoprim/sulfamethoxazole resistance. Prior (60 day) fluoroquinolone use, prior ciprofloxacin resistance and both diabetes mellitus and hypertension were strongly associated with ciprofloxacin resistance. Prior fluoroquinolone use and a history of resistance to any studied antibiotic were associated with MDR, while pregnancy was protective. CONCLUSIONS: We found elevated resistance to UTI-relevant antimicrobials and novel factors associated with resistance; these data can be incorporated into clinical decision tools to improve organism and drug concordance.
Assuntos
Diabetes Mellitus , Gammaproteobacteria , Gravidez , Humanos , Feminino , Pessoa de Meia-Idade , Masculino , Ciprofloxacina/farmacologia , Estudos Transversais , Escherichia coli , Combinação Trimetoprima e Sulfametoxazol/farmacologia , Fatores de Risco , Fluoroquinolonas , Testes de Sensibilidade Microbiana , Resistência a Múltiplos Medicamentos , Atenção Primária à SaúdeRESUMO
INTRODUCTION: Antimicrobial resistance in Neisseria gonorrhoeae compromises gonorrhoea treatment and rapid antimicrobial susceptibility testing (AST) would be valuable. We have developed a rapid and accurate flow cytometry method (FCM) for AST of gonococci. METHODS: The 2016 WHO gonococcal reference strains, and WHO Q, R and S (nâ=â17) were tested against seven clinically relevant antibiotics (ceftriaxone, cefixime, azithromycin, spectinomycin, ciprofloxacin, tetracycline and gentamicin). After 4.5 h incubation of inoculated broth, the fluorescent dye Syto™ 9 was added, followed by FCM analysis. After gating, the relative remaining population of gonococci, compared with unexposed growth control samples, was plotted against antimicrobial concentration, followed by non-linear curve regression analysis. Furthermore, the response at one single concentration/tested antibiotic was evaluated with the intention to use as a screening test for detection of resistant gonococci. RESULTS: A dose-dependent response was seen in susceptible isolates for all tested antimicrobials. There was a clear separation between susceptible/WT and resistant/non-WT isolates for ceftriaxone, cefixime, spectinomycin, ciprofloxacin and tetracycline. In contrast, for azithromycin, only high-level-resistant isolates were distinguished, while resistant isolates with MICs of 4 mg/L were indistinguishable from WT (MICâ≤â1 mg/L) isolates. For gentamicin, all tested 17 isolates were WT and FCM analysis resulted in uniform dose-response curves. Using a single antibiotic concentration and a 50% remaining cell population cut-off, the overall sensitivity and specificity for resistance detection were 93% and 99%, respectively. CONCLUSIONS: By providing results in <5 h for gonococcal isolates, FCM-based AST can become a rapid screening method for antimicrobial resistance or antimicrobial susceptibility in gonococci.
Assuntos
Anti-Infecciosos , Gonorreia , Humanos , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Neisseria gonorrhoeae , Azitromicina/farmacologia , Ceftriaxona/farmacologia , Espectinomicina/farmacologia , Cefixima/farmacologia , Citometria de Fluxo , Farmacorresistência Bacteriana , Gonorreia/epidemiologia , Anti-Infecciosos/farmacologia , Ciprofloxacina/farmacologia , Tetraciclina/farmacologia , Testes de Sensibilidade Microbiana , Gentamicinas/farmacologiaRESUMO
BACKGROUND: Enterococcus faecium is a Gram-positive bacterium, naturally present in the human intestinal microbiota, but is also an opportunistic pathogen responsible for healthcare-associated infections. Persisters are individuals of a subpopulation able to survive by arrest of growth coping with conditions that are lethal for the rest of the population. These persistent cells can grow again when the stress disappears from their environment and can cause relapses. RESULTS: In this study, we highlighted that ciprofloxacin (10-fold the MIC) led to the formation of persister cells of E. faecium. The kill curve was typically biphasic with an initial drop of survival (more than 2 orders of magnitude reduction) followed by a constant bacterial count. Growth curves and antimicrobial susceptibility tests of these persisters were similar to those of the original cells. In addition, by genomic analyses, we confirmed that the persisters were genotypically identical to the wild type. Comparative proteomic analysis revealed that 56 proteins have significantly different abundances in persisters compared to cells harvested before the addition of stressing agent. Most of them were related to energetic metabolisms, some polypeptides were involved in transcription regulation, and seven were stress proteins like CspA, PrsA, ClpX and particularly enzymes linked to the oxidative stress response. CONCLUSIONS: This work provided evidences that the pathogen E. faecium was able to enter a state of persister that may have an impact in chronic infections and relapses. Moreover, putative key effectors of this phenotypical behavior were identified by proteomic approach.
Assuntos
Enterococcus faecium , Humanos , Enterococcus faecium/genética , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Proteômica , Ciprofloxacina/farmacologia , Recidiva , Testes de Sensibilidade MicrobianaRESUMO
BACKGROUND: The emergence of antimicrobial resistance in bacterial pathogens is a growing concern worldwide due to its impact on the treatment of bacterial infections. The "Trojan Horse" strategy has been proposed as a potential solution to overcome drug resistance caused by permeability issues. OBJECTIVE: The objective of our research was to investigate the bactericidal activity and mechanism of action of the "Trojan Horse" strategy using enterobactin conjugated with Ciprofloxacin and Fosfomycin against the antibiotic-resistant Escherichia coli strain OQ866153. METHODOLOGY: Enterobactin, a mixed ligand of E. coli OQ866153, was conjugated with Ciprofloxacin and Fosfomycin individually to aid active absorption via specific enterobactin binding proteins (FepABCDG). The effectiveness of the conjugates was assessed by measuring their bactericidal activity against E. coli OQ866153, as well as their ability to inhibit DNA gyrase enzyme and biofilm formation. RESULTS: The Fe+3-enterobactin-Ciprofloxacin conjugate effectively inhibited the DNA gyrase enzyme (Docking score = -8.597 kcal/mol) and resulted in a lower concentration (25 µg/ml) required to eliminate supercoiled DNA plasmids compared to the parent drug (35 µg/ml; Docking score = -6.264 kcal/mol). The Fe+3-Enterobactin-Fosfomycin conjugate showed a higher inhibition percentage (100%) of biofilm formation compared to Fosfomycin (21.58%) at a concentration of 2 mg/ml, with docking scores of -5.481 and -3.756 kcal/mol against UDP-N acetylglucosamine 1-carboxyvinyltransferase MurA. CONCLUSION: The findings of this study suggest that the "Trojan Horse" strategy using enterobactin conjugated with Ciprofloxacin and Fosfomycin can effectively overcome permeability issues caused by efflux proteins and enhance the bactericidal activity of these drugs against antibiotic-resistant strains of E. coli.
Assuntos
Antibacterianos , Fosfomicina , Antibacterianos/química , Fosfomicina/farmacologia , Ciprofloxacina/farmacologia , Escherichia coli , Enterobactina/química , Enterobactina/metabolismo , Enterobactina/farmacologia , DNA Girase , Testes de Sensibilidade MicrobianaRESUMO
BACKGROUND: The emergence of fluoroquinolone resistance in clinical isolates of Klebsiella pneumoniae is a growing concern. To investigate the mechanisms behind this resistance, we studied a total of 215 K. pneumoniae isolates from hospitals in Bushehr province, Iran, collected between 2017 and 2019. Antimicrobial susceptibility test for fluoroquinolones was determined. The presence of plasmid mediated quinolone resistance (PMQR) and mutations in quinolone resistance-determining region (QRDR) of gyrA and parC genes in ciprofloxacin-resistant K. pneumoniae isolates were identified by PCR and sequencing. RESULTS: Out of 215 K. pneumoniae isolates, 40 were resistant to ciprofloxacin as determined by E-test method. PCR analysis revealed that among these ciprofloxacin-resistant isolates, 13 (32.5%), 7 (17.5%), 40 (100%), and 25 (62.5%) isolates harbored qnrB, qnrS, oqxA and aac(6')-Ib-cr genes, respectively. Mutation analysis of gyrA and parC genes showed that 35 (87.5%) and 34 (85%) of the ciprofloxacin-resistant isolates had mutations in these genes, respectively. The most frequent mutations were observed in codon 83 of gyrA and codon 80 of parC gene. Single gyrA substitution, Ser83â Ile and Asp87âGly, and double substitutions, Ser83âPhe plus Asp87âAla, Ser83âTyr plus Asp87âAla, Ser83âIle plus Asp87âTyr, Ser83âPhe plus Asp87âAsn and Ser83âIle plus Asp87âGly were detected. In addition, Ser80âIle and Glu84âLys single substitution were found in parC gene. CONCLUSIONS: Our results indicated that 90% of isolates have at least one mutation in QRDR of gyrA orparC genes, thus the frequency of mutations was very significant and alarming in our region.
Assuntos
Antibacterianos , DNA Girase , DNA Topoisomerase IV , Farmacorresistência Bacteriana , Infecções por Klebsiella , Klebsiella pneumoniae , Testes de Sensibilidade Microbiana , Mutação , Plasmídeos , Quinolonas , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/isolamento & purificação , DNA Girase/genética , Plasmídeos/genética , DNA Topoisomerase IV/genética , Humanos , Antibacterianos/farmacologia , Infecções por Klebsiella/microbiologia , Infecções por Klebsiella/epidemiologia , Farmacorresistência Bacteriana/genética , Quinolonas/farmacologia , Ciprofloxacina/farmacologia , Irã (Geográfico) , Proteínas de Bactérias/genética , Prevalência , Fluoroquinolonas/farmacologiaRESUMO
BACKGROUND: The study aims to investigate the effect of combining silver nanoparticles (AGNPs) with different antibiotics on multi-drug resistant (MDR) and extensively drug resistant (XDR) isolates of Pseudomonas aeruginosa (P. aeruginosa) and to investigate the mechanism of action of AGNPs. METHODS: AGNPs were prepared by reduction of silver nitrate using trisodium citrate and were characterized by transmission electron microscope (TEM) in addition to an assessment of cytotoxicity. Clinical isolates of P. aeruginosa were collected, and antimicrobial susceptibility was conducted. Multiple Antibiotic Resistance (MAR) index was calculated, and bacteria were categorized as MDR or XDR. Minimum inhibitory concentration (MIC) of gentamicin, ciprofloxacin, ceftazidime, and AGNPs were determined. The mechanism of action of AGNPs was researched by evaluating their effect on biofilm formation, swarming motility, protease, gelatinase, and pyocyanin production. Real-time PCR was performed to investigate the effect on the expression of genes encoding various virulence factors. RESULTS: TEM revealed the spherical shape of AGNPs with an average particle size of 10.84 ± 4.64 nm. AGNPS were safe, as indicated by IC50 (42.5 µg /ml). The greatest incidence of resistance was shown against ciprofloxacin which accounted for 43% of the bacterial isolates. Heterogonous resistance patterns were shown in 63 isolates out of the tested 107. The MAR indices ranged from 0.077 to 0.84. Out of 63 P. aeruginosa isolates, 12 and 13 were MDR and XDR, respectively. The MIC values of AGNPs ranged from 2.65 to 21.25 µg /ml. Combination of AGNPs with antibiotics reduced their MIC by 5-9, 2-9, and 3-10Fold in the case of gentamicin, ceftazidime, and ciprofloxacin, respectively, with synergism being evident. AGNPs produced significant inhibition of biofilm formation and decreased swarming motility, protease, gelatinase and pyocyanin production. PCR confirmed the finding, as shown by decreased expression of genes encoding various virulence factors. CONCLUSION: AGNPs augment gentamicin, ceftazidime, and ciprofloxacin against MDR and XDR Pseudomonas isolates. The efficacy of AGNPs can be attributed to their effect on the virulence factors of P. aeruginosa. The combination of AGNPs with antibiotics is a promising strategy to attack resistant isolates of P. aeruginosa.
Assuntos
Antibacterianos , Biofilmes , Farmacorresistência Bacteriana Múltipla , Nanopartículas Metálicas , Testes de Sensibilidade Microbiana , Infecções por Pseudomonas , Pseudomonas aeruginosa , Prata , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/fisiologia , Biofilmes/efeitos dos fármacos , Prata/farmacologia , Prata/química , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Nanopartículas Metálicas/química , Antibacterianos/farmacologia , Humanos , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/tratamento farmacológico , Ciprofloxacina/farmacologia , Fatores de Virulência/genética , Gentamicinas/farmacologia , Microscopia Eletrônica de Transmissão , Ceftazidima/farmacologiaRESUMO
Recents reports have shown increases in the abuse of anti-malaria, antibiotic and analgesic drugs. This study evaluated the effects of co-administration of artemether-lumefantrine (AL), ciprofloxacin (CPX) and diclofenac (DFC) on inflammatory and immunological status of female Wistar rats. Ninety-six female Wistar rats were assigned into eight groups of twelve animals each. Group A was control, groups B, C, D, E, F, G and H were administered AL, CPX, DFC, AL + CPX, AL + DFC, CPX + DFC and AL + CPX + DFC respectively. Dosages of administered drugs were 178 mg/kg b/w of AL, 185 mg/kg b/w of CPX and 9 mg/kg b/w of DFC. Animals were sacrificed after 6 and 12 weeks of oral administration. Blood was obtained through cardiac puncture. The liver was harvested and processed for immunohistochemical analysis. Differential leukocyte count and neutrophil adhesion test was conducted on whole blood. Immunological response was assessed by the serum levels of C-reactive protein (CRP), interleukin-1ß (Il-1ß), interleukin-6 (Il-6), monocyte chemoattractant protein-1 (MCP-1), vascular cell adhesion molecule-1 (VCAM-1), myeloperoxidase, and total immunoglobulin G. Data were analyzed with Graph pad prism 5, using one way analysis of variance at 5 % level of significance. Total leukocyte, lymphocyte and basophils count increased (p<0.05) in B, C, E, F, G and H, while neutrophil count decreased (p<0.05) in D, E, G and H at 6 weeks. Neutrophil adhesion decreased (p<0.05) in B, E, F, G and H at 6 weeks. There was no significant difference (p>0.05) in the expression of Il-6, MCP-1 and VCAM-1 across the groups. Il-1ß decreased in H, while CRP increased in H at 6 weeks and 12 weeks. MPO activity decreased (p<0.05) in B, C, D, E, G and H at 6 weeks, but increased (p<0.05) in D and G at 12 weeks. Immunohistochemical analysis indicated increase (p<0.05) in tumour necrosis factor-α in liver tissues of B, C, D, E, F and G, while nuclear factor erythroid 2-related factor 2 increased (p<0.05) in C, D, E, F and G, but decreased (p<0.05) in H at 12 weeks. The co-administration of AL, CPX and DFC induced inflammatory responses with attendant immunological dysfunctions and liver damage.
Assuntos
Antimaláricos , Ratos , Animais , Feminino , Ratos Wistar , Diclofenaco/farmacologia , Combinação Arteméter e Lumefantrina , Ciprofloxacina/farmacologia , Interleucina-6 , Molécula 1 de Adesão de Célula Vascular , Artemeter , Fator de Necrose Tumoral alfaRESUMO
Antibiotic resistance is a critical topic worldwide with important consequences for public health. So considering the rising issue of antibiotic-resistance in bacteria, we explored the impact of nitrogen and phosphorus eutrophication on drug resistance mechanisms in Enterococcus faecalis, especially ciprofloxacin, oxytetracycline, and ampicillin. For this purpose we examined the antibiotic-resistance genes and biofilm formation of Enterococcus faecalis under different concentration of nitrogen and phosphorus along with mentioned antibiotics. Mesocosms were designed to evaluate the impact of influence of eutrophication on the underlying mechanism of drugn resistence in Enterococcus faecalis. For this purpose, we explored the potential relation to biofilm formation, adhesion ability, and the expression levels of the regulatory gene fsrA and the downstream gene gelEI. Our results demonstrated that the isolates of all treatments displayed high biofilm forming potential, and fsrA and gelE genes expression. Additionally, the experimental group demonstrated substantially elevated Enterococcus faecalis gelE expression. Crystal violet staining was applied to observe biofilm formation during bacterial development phase and found higher biofilm formation. In conclusion, our data suggest that E. faecalis resistance to ciprofloxacin, oxytetracycline, and ampicillin is related to biofilm development. Also, the high level of resistance in Enterococcus faecalis is linked to the expression of the fsrA and gelE genes. Understanding these pathways is vital in tackling the rising problem of bacterial resistance and its potential effect on human health.