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1.
J Agric Food Chem ; 70(51): 16106-16116, 2022 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-36524955

RESUMO

The valorization of poultry byproducts, like feathers (processed to feather meal), in animal feed could contribute to the presence of veterinary drugs, including antibiotics. An animal study was carried out to study the fate of sulfadiazine, trimethoprim, and oxytetracycline in feathers, plasma, and droppings of broiler chickens. Cage and floor housing, different from current farm practices, were studied. Samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). A longer presence of antibiotics was observed in feathers compared to plasma, with sulfadiazine being present the most. The internal presence (via blood) and the external presence (via droppings) of antibiotics in/on feathers were shown. Analysis of Escherichia coli populations, from droppings and feathers, highlighted that resistant bacteria could be transferred from droppings to feathers in floor-housed animals. The overall results suggest that feathers are a potential reservoir of antimicrobial residues and could contribute to the selection of antibiotic-resistant bacteria in the environment, animals, and humans.


Assuntos
Antibacterianos , Oxitetraciclina , Humanos , Animais , Antibacterianos/análise , Oxitetraciclina/análise , Galinhas , Plumas/química , Sulfadiazina/farmacologia , Sulfadiazina/análise , Trimetoprima/farmacologia , Trimetoprima/análise , Cromatografia Líquida , Espectrometria de Massas em Tandem/métodos
2.
Microbiologyopen ; 11(5): e1316, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36314749

RESUMO

Antibiotic resistance is becoming increasingly prevalent amongst bacterial pathogens and there is an urgent need to develop new types of antibiotics with novel modes of action. One promising strategy is to develop resistance-breaker compounds, which inhibit resistance mechanisms and thus resensitize bacteria to existing antibiotics. In the current study, we identify bacterial DNA double-strand break repair as a promising target for the development of resistance-breaking co-therapies. We examined genetic variants of Escherichia coli that combined antibiotic-resistance determinants with DNA repair defects. We observed that defects in the double-strand break repair pathway led to significant resensitization toward five bactericidal antibiotics representing different functional classes. Effects ranged from partial to full resensitization. For ciprofloxacin and nitrofurantoin, sensitization manifested as a reduction in the minimum inhibitory concentration. For kanamycin and trimethoprim, sensitivity manifested through increased rates of killing at high antibiotic concentrations. For ampicillin, repair defects dramatically reduced antibiotic tolerance. Ciprofloxacin, nitrofurantoin, and trimethoprim induce the promutagenic SOS response. Disruption of double-strand break repair strongly dampened the induction of SOS by these antibiotics. Our findings suggest that if break-repair inhibitors can be developed they could resensitize antibiotic-resistant bacteria to multiple classes of existing antibiotics and may suppress the development of de novo antibiotic-resistance mutations.


Assuntos
Infecções por Escherichia coli , Proteínas de Escherichia coli , Humanos , Escherichia coli/metabolismo , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Nitrofurantoína/metabolismo , Nitrofurantoína/farmacologia , Reparo do DNA , Ciprofloxacina/farmacologia , Testes de Sensibilidade Microbiana , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Trimetoprima/metabolismo , Trimetoprima/farmacologia
3.
Huan Jing Ke Xue ; 43(10): 4536-4544, 2022 Oct 08.
Artigo em Chinês | MEDLINE | ID: mdl-36224139

RESUMO

The co-exposure of antibiotics has important effects on antibiotic resistance genes (ARGs) and microbial community aggregation in wastewater treatment plants (WWTPs). However, it is unclear whether differences in historical antibiotic exposure stress can determine responses of microbes and ARGs to combined antibiotics. By selecting a high concentration (30 mg·L-1) of sulfamethoxazole (SMX) and trimethoprim (TMP) as historical exposure stress conditions, the effects of SMX and TMP-combined pollution on ARGs, bacterial communities, and their interactions were explored in short-term experiments. Based on high-throughput quantitative PCR, a total of 13 ARGs were detected, and the absolute abundance was 2.21-5.42 copies·µL-1 (logarithm, DNA, the same below). Among them, sul2, ermB, mefA, and tetM-01 were the main subtypes in the samples, and the absolute abundance was between 2.95 and 5.40 copies·µL-1. The combined contamination of SMX and TMP could cause the enrichment of ARGs and mobile genetic elements (MGEs); however, their effects on each subtype were different, and the historical legacy effect of SMX was higher than that of TMP. Under the different exposure histories, the co-occurrence and co-exclusion patterns existed between ARGs. Moreover, MGEs (especially intI-1) were significantly correlated with sulfonamides (sul1 and sul2), tetracyclines[tet(32)], and macrolide-lincosamide-streptogramin (MLSB) resistance genes (ermB). Based on the full-scale classification of microorganisms, it was found that the microbial community structure of various groups responded differently to combined pollution, and the conditionally abundant taxa (CAT) were obviously enriched. Thauera, Pseudoxanthomonas, and Paracoccus were the dominant resistant bacterial genera. Furthermore, a total of 31 potential hosts of ARGs were identified with network analysis, which were dominated with conditionally rare taxa (CRT). Particularly, Candidatus_Alysiosphaera and Fusibacter were positively correlated with most of the ARGs, being the common protentional hosts. Importantly, some rare genera (RT, Variibacter, Aeromonas, Cloacibacterium, etc.) were potential hosts of transposon IS613, which played an important role in the proliferation and spread of ARGs. In conclusion, this study revealed the legacy effects of historical antibiotic stress on ARGs and their hosts, which could provide new ideas and theoretical basis for reducing ARGs pollution in WWTPs.


Assuntos
Antibacterianos , Esgotos , Antibacterianos/análise , Antibacterianos/farmacologia , Bactérias , Resistência Microbiana a Medicamentos/genética , Genes Bacterianos , Lincosamidas/análise , Lincosamidas/farmacologia , Macrolídeos/farmacologia , Esgotos/microbiologia , Estreptograminas/farmacologia , Sulfametoxazol/farmacologia , Tetraciclinas/análise , Tetraciclinas/farmacologia , Trimetoprima/análise , Trimetoprima/farmacologia , /microbiologia
4.
Ann Med ; 54(1): 2500-2510, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36120867

RESUMO

Objective: To determine the minimum inhibitory concentration (MIC) distribution of antibacterial drugs and the susceptibility of non-tuberculous mycobacterial (NTM) isolates to provide a reference basis for the clinical selection of an effective starting regimen.Methods: The common clinical isolates of NTM in the respiratory tract, which met the standards of the American Thoracic Society for NTM lung disease, were collected. The MICs of 81 isolates were determined using the microbroth dilution method (Thermo Fisher Scientific, USA), as recommended by the Clinical and Laboratory Standards Institute, USA.Results: Included were 43 Mycobacterium avium complex (MAC) strains, 24 M. abscessus complex (MAB) strains, and 14 M. kansasii strains. The sensitivity rates of MAC to clarithromycin and amikacin were 81.4% and 79.1%, respectively, while the sensitivity rates to linezolid and moxifloxacin were only 20.9% and 9.3%; the MIC of rifabutin was the lowest (MIC50% was just 2 µg/mL). After incubation for 3-5 days, the sensitivity rate of MAB to clarithromycin was 87.5%; this decreased to 50% after 14 days' incubation. Most of them were susceptible to amikacin (91.6%), and most were resistant to moxifloxacin (95.8%), ciprofloxacin (95.8%), imipenem (95.8%), amoxicillin/clavulanate (95.8%), tobramycin (79.1%), doxycycline (95.8%) and trimethoprim/sulfamethoxazole (95.8%). intermediate (83.3%) and resistant (16.7%) to cefoxitin. The susceptibility to linezolid was only 33.3%. The sensitivity and resistance breakpoints of tigecycline were set to ≤0.5 and ≥8 µg/mL, respectively, and the sensitivity and resistance rates were 50% and 0%, respectively. M. kansasii was susceptible to clarithromycin, amikacin, linezolid, moxifloxacin, rifampicin and rifabutin (100%).Discussion: In Wenzhou, clarithromycin, amikacin and rifabutin have good antibacterial activity against MAC, while linezolid and moxifloxacin have high resistance. Amikacin and tigecycline have strong antibacterial activity against MAB, while most other antibacterial drugs are resistant to varying degrees. Most antibacterial drugs are susceptible to M. kansasii and have good antibacterial activity.Conclusion: The identification of NTM species and the detection of their MICs have certain guiding values for the treatment of NTM lung disease.Key MessageThe three most common respiratory non-tuberculous mycobacterial (NTM) isolates with clinical significance in the Wenzhou area were tested for drug susceptibility. The broth microdilution method was used to determine the minimum inhibitory concentration distribution of antibacterial drugs and the susceptibility of NTM isolates to provide a reference basis for the clinical selection of an effective starting regimen.


Assuntos
Pneumopatias , Infecções por Mycobacterium não Tuberculosas , Amicacina/farmacologia , Amicacina/uso terapêutico , Amoxicilina , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Cefoxitina/farmacologia , Cefoxitina/uso terapêutico , Ciprofloxacina/farmacologia , Ciprofloxacina/uso terapêutico , Claritromicina/farmacologia , Ácido Clavulânico/farmacologia , Ácido Clavulânico/uso terapêutico , Doxiciclina/farmacologia , Doxiciclina/uso terapêutico , Humanos , Imipenem/farmacologia , Imipenem/uso terapêutico , Linezolida/farmacologia , Linezolida/uso terapêutico , Testes de Sensibilidade Microbiana , Moxifloxacina/farmacologia , Moxifloxacina/uso terapêutico , Infecções por Mycobacterium não Tuberculosas/tratamento farmacológico , Infecções por Mycobacterium não Tuberculosas/microbiologia , Micobactérias não Tuberculosas , Sistema Respiratório , Rifabutina/farmacologia , Rifabutina/uso terapêutico , Rifampina/farmacologia , Rifampina/uso terapêutico , Sulfametoxazol/farmacologia , Sulfametoxazol/uso terapêutico , Tigeciclina/farmacologia , Tigeciclina/uso terapêutico , Tobramicina/farmacologia , Tobramicina/uso terapêutico , Trimetoprima/farmacologia , Trimetoprima/uso terapêutico
5.
J Clin Invest ; 132(18)2022 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-36106638

RESUMO

BACKGROUNDSeveral molecular imaging strategies can identify bacterial infections in humans. PET affords the potential for sensitive infection detection deep within the body. Among PET-based approaches, antibiotic-based radiotracers, which often target key bacterial-specific enzymes, have considerable promise. One question for antibiotic radiotracers is whether antimicrobial resistance (AMR) reduces specific accumulation within bacteria, diminishing the predictive value of the diagnostic test.METHODSUsing a PET radiotracer based on the antibiotic trimethoprim (TMP), [11C]-TMP, we performed in vitro uptake studies in susceptible and drug-resistant bacterial strains and whole-genome sequencing (WGS) in selected strains to identify TMP resistance mechanisms. Next, we queried the NCBI database of annotated bacterial genomes for WT and resistant dihydrofolate reductase (DHFR) genes. Finally, we initiated a first-in-human protocol of [11C]-TMP in patients infected with both TMP-sensitive and TMP-resistant organisms to demonstrate the clinical feasibility of the tool.RESULTSWe observed robust [11C]-TMP uptake in our panel of TMP-sensitive and -resistant bacteria, noting relatively variable and decreased uptake in a few strains of P. aeruginosa and E. coli. WGS showed that the vast majority of clinically relevant bacteria harbor a WT copy of DHFR, targetable by [11C]-TMP, and that despite the AMR, these strains should be "imageable." Clinical imaging of patients with [11C]-TMP demonstrated focal radiotracer uptake in areas of infectious lesions.CONCLUSIONThis work highlights an approach to imaging bacterial infection in patients, which could affect our understanding of bacterial pathogenesis as well as our ability to better diagnose infections and monitor response to therapy.TRIAL REGISTRATIONClinicalTrials.gov NCT03424525.FUNDINGInstitute for Translational Medicine and Therapeutics, Burroughs Wellcome Fund, NIH Office of the Director Early Independence Award (DP5-OD26386), and University of Pennsylvania NIH T32 Radiology Research Training Grant (5T32EB004311-12).


Assuntos
Infecções Bacterianas , Trimetoprima , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Bactérias , Infecções Bacterianas/diagnóstico por imagem , Infecções Bacterianas/tratamento farmacológico , Radioisótopos de Carbono , Escherichia coli , Humanos , Trimetoprima/farmacologia , Trimetoprima/uso terapêutico
6.
ACS Chem Biol ; 17(10): 2877-2889, 2022 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-36122928

RESUMO

Destabilizing domains (DDs) are an attractive strategy allowing for positive post-transcriptional small molecule-regulatable control of a fusion protein's abundance. However, in many instances, the currently available DDs suffer from higher-than-desirable basal levels of the fusion protein. Accordingly, we redesigned the E. coli dihydrofolate reductase (ecDHFR) DD by introducing a library of ∼1200 random ecDHFR mutants fused to YFP into CHO cells. Following successive rounds of fluorescence-activated cell sorting, we identified six new ecDHFR DD clones with significantly enhanced proteasomal turnover in the absence of a stabilizing ligand, trimethoprim (TMP). One of these clones, designated as "C12", contained four unique missense mutations (W74R/T113S/E120D/Q146L) and demonstrated a significant 2.9-fold reduction in basal levels compared to the conventional ecDHFR DD (i.e., R12Y/G67S/Y100I). This domain was similarly responsive to TMP with respect to dose response and maximal stabilization, indicating an overall enhanced dynamic range. Interestingly, both computational and wet-lab experiments identified the W74R and T113S mutations of C12 as the main contributors toward its basal destabilization. However, the combination of all the C12 mutations was required to maintain both its enhanced degradation and TMP stabilization. We further demonstrate the utility of C12 by fusing it to IκBα and Nrf2, two stress-responsive proteins that have previously been challenging to regulate. In both instances, C12 significantly enhanced the basal turnover of these proteins and improved the dynamic range of regulation post stabilizer addition. These advantageous features of the C12 ecDHFR DD variant highlight its potential for replacing the conventional N-terminal ecDHFR DD and improving the use of DDs overall, not only as a chemical biology tool but for gene therapy avenues as well.


Assuntos
Escherichia coli , Tetra-Hidrofolato Desidrogenase , Animais , Cricetinae , Tetra-Hidrofolato Desidrogenase/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Inibidor de NF-kappaB alfa , Fator 2 Relacionado a NF-E2 , Ligantes , Cricetulus , Trimetoprima/farmacologia
7.
Mol Syst Biol ; 18(9): e10490, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-36124745

RESUMO

Dose-response relationships are a general concept for quantitatively describing biological systems across multiple scales, from the molecular to the whole-cell level. A clinically relevant example is the bacterial growth response to antibiotics, which is routinely characterized by dose-response curves. The shape of the dose-response curve varies drastically between antibiotics and plays a key role in treatment, drug interactions, and resistance evolution. However, the mechanisms shaping the dose-response curve remain largely unclear. Here, we show in Escherichia coli that the distinctively shallow dose-response curve of the antibiotic trimethoprim is caused by a negative growth-mediated feedback loop: Trimethoprim slows growth, which in turn weakens the effect of this antibiotic. At the molecular level, this feedback is caused by the upregulation of the drug target dihydrofolate reductase (FolA/DHFR). We show that this upregulation is not a specific response to trimethoprim but follows a universal trend line that depends primarily on the growth rate, irrespective of its cause. Rewiring the feedback loop alters the dose-response curve in a predictable manner, which we corroborate using a mathematical model of cellular resource allocation and growth. Our results indicate that growth-mediated feedback loops may shape drug responses more generally and could be exploited to design evolutionary traps that enable selection against drug resistance.


Assuntos
Antibacterianos , Tetra-Hidrofolato Desidrogenase , Antibacterianos/farmacologia , Escherichia coli/genética , Retroalimentação , Tetra-Hidrofolato Desidrogenase/genética , Tetra-Hidrofolato Desidrogenase/farmacologia , Trimetoprima/farmacologia
8.
Microbiol Spectr ; 10(5): e0125722, 2022 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-36047803

RESUMO

Antimicrobial-resistant Salmonella enterica poses a significant public health concern worldwide. However, the dissemination of Salmonella enterica among food animals in eastern China has not been fully addressed. Here, we demonstrated the antimicrobial resistance (AMR) patterns and the whole-genome characterization of 105 S. enterica isolates from 1,480 fecal samples and anal swabs collected from 22 different farms (chickens, ducks, and pigs) and two live animal markets located in Zhejiang and Fujian Provinces in eastern China in 2019. The prevalence of isolates in duck farms (19.17%, 23/120) was statistically significantly higher (P < 0.001) than that in chicken farms (6.61%, 37/523) and pig farms (3.50%, 7/200). Among these isolates, 75.26% (79/105) were multidrug resistant, with the highest rates of resistance to tetracycline (76.20%) and ampicillin (67.62%) and the lowest resistance rate to meropenem (0.00%). The serotypes were consistent with sequence types and were closely related to the sampling animal species and sites. S. enterica serotype Kentucky (20.95%, 22/105) was the most frequent serotype and harbored more AMR patterns and genes than others. Furthermore, IncFII(S) and IncHI2 were the most prevalent replicons. A total of 44 acquired AMR genes were found. Among those genes, aac(6')-Iaa, blaTEM-1B, floR, dfrA14, fosA7, mph(A), qnrS1, sul1, tet(A), and ARR-3 were the dominant AMR genes mediating the AMR toward aminoglycosides, ß-lactams, phenicol, trimethoprim, fosfomycin, macrolide, quinolone, sulfonamides, tetracycline, and rifampin, respectively. The consistency of acquired AMR genes with AMR phenotypes for ampicillin, ceftiofur, ceftazidime, meropenem, sulfamethoxazole-trimethoprim, and tetracycline was >90%. Together, our study highlights the application of whole-genome sequencing to assess veterinary public health threats. IMPORTANCE Public health is a significant concern in China, and the foodborne pathogen Salmonella, which is spread via the animal-borne food chain, plays an important role in the overall disease burden in China annually. The development of advanced sequencing technologies has introduced a new way of understanding emerging pathogens. However, the routine surveillance application of this method in China remains in its infancy. Here, we applied a pool of all isolates from the prevalence data in Zhejiang and Fujian for whole-genome sequencing and combined these data with the cutting-edge bioinformatic analysis pipeline for one-step determination of the complete genetic makeup for all 105 genomes. The illustrated method could provide a cost-effective approach, without labor-intensive laboratory characterization, for predicting serotypes, genotypes, plasmid types, antimicrobial resistance genes, and virulence genes, and thus would provide essential knowledge for emerging pathogens. Our findings and perspectives are essential for delivering updated knowledge on foodborne pathogens in an understudied region in China.


Assuntos
Anti-Infecciosos , Fosfomicina , Quinolonas , Salmonella enterica , Animais , Suínos , Salmonella enterica/genética , Galinhas , Patos , Farmacorresistência Bacteriana Múltipla/genética , Fazendas , Meropeném/farmacologia , Fosfomicina/farmacologia , Ceftazidima/farmacologia , Rifampina/farmacologia , Antibacterianos/farmacologia , Anti-Infecciosos/farmacologia , Quinolonas/farmacologia , Genômica , Ampicilina/farmacologia , Aminoglicosídeos/farmacologia , Macrolídeos/farmacologia , Trimetoprima/farmacologia , Sulfonamidas/farmacologia , Sulfametoxazol/farmacologia , Tetraciclinas/farmacologia , Testes de Sensibilidade Microbiana
9.
Cell Chem Biol ; 29(9): 1446-1464.e10, 2022 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-35835118

RESUMO

Chemogenetic methods enabling the rapid translocation of specific proteins to the plasma membrane (PM) in a single protein-single ligand manner are useful tools in cell biology. We recently developed a technique, in which proteins fused to an Escherichia coli dihydrofolate reductase (eDHFR) variant carrying N-terminal hexalysine residues are recruited from the cytoplasm to the PM using the synthetic myristoyl-d-Cys-tethered trimethoprim (mDcTMP) ligand. However, this system achieved PM-specific translocation only when the eDHFR tag was fused to the N terminus of proteins, thereby limiting its application. In this report, we engineered a universal PM-targeting tag for mDcTMP-induced protein translocation by grafting the hexalysine motif into an intra-loop region of eDHFR. We demonstrate the broad applicability of the new loop-engineered eDHFR tag and mDcTMP pair for conditional PM recruitment and activation of various tag-fused signaling proteins with different fusion configurations and for reversibly and repeatedly controlling protein localization to generate synthetic signal oscillations.


Assuntos
Tetra-Hidrofolato Desidrogenase , Trimetoprima , Membrana Celular/metabolismo , Escherichia coli/metabolismo , Ligantes , Proteínas , Transdução de Sinais , Tetra-Hidrofolato Desidrogenase/metabolismo , Trimetoprima/farmacologia
10.
Pak J Pharm Sci ; 35(2): 487-492, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-35642404

RESUMO

Escherichia coli is the most studied among those bacteria causing urinary tract infections. This study was aimed to find out antibacterial activity and minimum inhibitory concentration of selected antibacterial agents against E. coli isolates of hospitalized UTI patients. The specimens were inoculated on Eosin Methylene Blue medium. E. coli isolates were identified via colonial morphology, biochemical testing and API-20 kit. The susceptibility pattern of antibacterial agents was determined applying disc diffusion method (Kirby-Bauer) and dilution tube method. Among all, 38.82% (n=158/407) specimens were positive for E. coli, while the rest showed either no growth or exhibited colonies other than E. coli. while observing the susceptibility pattern, Imipenem was found the most effective (73.42%) antibacterial agent, followed by nitrofurantoin (52.53%), cefpirome (44.94%) and tazobactam/ piperacillin (44.94%), whereas the E. coli isolates were highly resistant to sulfamethoxazole/trimethoprim (71.52%), followed by Amoxicillin-clavulanic acid (67.72%), nalidixic acid (66.46%) and Tobramycin (62.03%), when tested by disc diffusion method. The isolates were susceptible to cefpirome (39.87%) and tobramycin (39.87%) and resistant to sulfamethoxazole/trimethoprim (75.32%), followed by levofloxacin (61.39%), when tested by tube dilution method. The study concluded high degree of resistance against Sulfamethoxazole/trimethoprim, in contrast, cephalosporin and Imipenem exhibited good potency which can be recommended for UTI.


Assuntos
Infecções por Escherichia coli , Infecções Urinárias , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Escherichia coli , Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/microbiologia , Humanos , Imipenem/farmacologia , Sulfametoxazol/farmacologia , Tobramicina/farmacologia , Trimetoprima/farmacologia , Infecções Urinárias/tratamento farmacológico , Infecções Urinárias/microbiologia
11.
Microb Drug Resist ; 28(5): 545-550, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35512733

RESUMO

Burkholderia cepacia complex (Bcc) in airways of patients with cystic fibrosis (CF) is associated with an increased morbidity and mortality. A huge range of intrinsic antimicrobial resistances challenges the treatment of Bcc infections. The aim was to assess the susceptibility of Bcc to ceftazidime/avibactam and standard drugs for the treatment for CF patients and to determine the respective genomic determinants of resistance. Bcc isolates (n = 64) from a prospective multicenter study of CF airway pathogens (2004-2020, Germany) were subjected to broth microdilution and minimal inhibitory concentrations were interpreted with European Committee on Antimicrobial Susceptibility Testing and Clinical & Laboratory Standards Institute breakpoints. A synergism between aztreonam and avibactam was tested using ceftazidime/avibactam disks with or without aztreonam. Plasmids and chromosomes of all isolates were screened for antimicrobial resistance genes. The highest susceptibility rate was detected for trimethoprim/sulfamethoxazole (83%), followed by ceftazidime/avibactam (78%), ceftazidime (53%), levofloxacin (39%) and meropenem (27%). The median inhibition zone diameters of ceftazidime-avibactam and ceftazidime/avibactam plus aztreonam were equal. This was in line with the absence of known class B metallo-ß-lactamases in any of the isolates. The majority of isolates carried blapenA (98%) and blaampC (86%). Trimethoprim/sulfamethoxazole and ceftazidime/avibactam showed high susceptibility rates. Aztreonam in combination with ceftazidime/avibactam had no synergistic effect in our Bcc isolates.


Assuntos
Complexo Burkholderia cepacia , Fibrose Cística , Antibacterianos/farmacologia , Compostos Azabicíclicos/farmacologia , Compostos Azabicíclicos/uso terapêutico , Aztreonam/farmacologia , Aztreonam/uso terapêutico , Ceftazidima/farmacologia , Ceftazidima/uso terapêutico , Fibrose Cística/tratamento farmacológico , Combinação de Medicamentos , Humanos , Testes de Sensibilidade Microbiana , Estudos Prospectivos , Sulfametoxazol/farmacologia , Trimetoprima/farmacologia
12.
BMC Microbiol ; 22(1): 144, 2022 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-35610571

RESUMO

BACKGROUND: Although urinary tract infections (UTIs) are extremely common, isolation of causative uropathogens is not always routinely performed, with antibiotics frequently prescribed empirically. This study determined the susceptibility of urinary isolates from two Health and Social Care Trusts (HSCTs) in Northern Ireland to a range of antibiotics commonly used in the treatment of UTIs. Furthermore, we determined if detection of trimethoprim resistance genes (dfrA) could be used as a potential biomarker for rapid detection of phenotypic trimethoprim resistance in urinary pathogens and from urine without culture. METHODS: Susceptibility of E. coli and Klebsiella spp. isolates (n = 124) to trimethoprim, amoxicillin, ceftazidime, ciprofloxacin, co-amoxiclav and nitrofurantoin in addition to susceptibility of Proteus mirabilis (n = 61) and Staphylococcus saprophyticus (n = 17) to trimethoprim was determined by ETEST® and interpreted according to EUCAST breakpoints. PCR was used to detect dfrA genes in bacterial isolates (n = 202) and urine samples(n = 94). RESULTS: Resistance to trimethoprim was observed in 37/124 (29.8%) E. coli and Klebsiella spp. isolates with an MIC90 > 32 mg/L. DfrA genes were detected in 29/37 (78.4%) trimethoprim-resistant isolates. Detection of dfrA was highly sensitive (93.6%) and specific (91.4%) in predicting phenotypic trimethoprim resistance among E. coli and Klebsiella spp. isolates. The dfrA genes analysed were detected using a culture-independent PCR method in 16/94 (17%) urine samples. Phenotypic trimethoprim resistance was apparent in isolates cultured from 15/16 (94%) dfrA-positive urine samples. There was a significant association (P < 0.0001) between the presence of dfrA and trimethoprim resistance in urine samples containing Gram-negative bacteria (Sensitivity = 75%; Specificity = 96.9%; PPV = 93.8%; NPV = 86.1%). CONCLUSIONS: This study demonstrates that molecular detection of dfrA genes is a good indicator of trimethoprim resistance without the need for culture and susceptibility testing.


Assuntos
Antibacterianos , Infecções Urinárias , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Farmacorresistência Bacteriana , Escherichia coli , Humanos , Testes de Sensibilidade Microbiana , Trimetoprima/farmacologia , Resistência a Trimetoprima/genética , Infecções Urinárias/microbiologia
13.
Transl Vis Sci Technol ; 11(5): 26, 2022 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-35612831

RESUMO

Purpose: Pseudomonas aeruginosa is a leading cause of corneal infections. Recently, we discovered an antimicrobial drug combination, polymyxin B/trimethoprim (PT) + rifampin, that displayed impressive efficacy toward P. aeruginosa in both in vitro and in vivo studies. As such, this combination was further evaluated as a potential keratitis therapeutic through testing the combination's efficacy against a diverse set of P. aeruginosa clinical isolates. Methods: Minimum inhibitory concentrations (MICs) of moxifloxacin, levofloxacin, erythromycin, tobramycin, PT, polymyxin B (alone), trimethoprim (alone), and rifampin were determined for 154 ocular clinical P. aeruginosa isolates, 90% of which were derived from corneal scrapings. Additionally, the efficacy of PT + rifampin was evaluated utilizing fractional inhibitory concentration (FIC) testing. Results: While 100% of isolates were resistant to erythromycin (average MIC 224 ± 110 µg·mL-1) and trimethoprim (alone) (206 ± 67.3 µg·mL-1), antibiotic resistance was generally found to be low: moxifloxacin (2% of isolates resistant; average MIC 1.08 ± 1.61 µg·mL-1), levofloxacin (3.9%; 1.02 ± 2.96 µg·mL-1), tobramycin (1%; 0.319 ± 1.31 µg·mL-1), polymyxin B (0%; 0.539 ± 0.206 µg·mL-1), PT (0%; 0.416 ± 0.135 µg·mL-1), and rifampin (0%; 23.4 ± 6.86 µg·mL-1). Additionally, FIC testing revealed that PT + rifampin eradicated 100% of isolates demonstrating additive or synergistic activity in 95% of isolates (average FIC index 0.701 ± 0.132). Conclusions: The drug combination of PT + rifampin was effective against a large panel of clinically relevant P. aeruginosa strains and, as such, may represent a promising therapeutic for P. aeruginosa keratitis. Translational Relevance: This work furthers the preclinical development of a novel antibiotic combination for the treatment of corneal infections (bacterial keratitis).


Assuntos
Ceratite , Infecções por Pseudomonas , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Bacitracina/farmacologia , Bacitracina/uso terapêutico , Combinação de Medicamentos , Eritromicina/farmacologia , Eritromicina/uso terapêutico , Framicetina/farmacologia , Framicetina/uso terapêutico , Humanos , Ceratite/tratamento farmacológico , Ceratite/microbiologia , Levofloxacino/farmacologia , Levofloxacino/uso terapêutico , Moxifloxacina/farmacologia , Moxifloxacina/uso terapêutico , Polimixina B/farmacologia , Polimixina B/uso terapêutico , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa , Rifampina/farmacologia , Rifampina/uso terapêutico , Tobramicina/farmacologia , Tobramicina/uso terapêutico , Trimetoprima/farmacologia , Trimetoprima/uso terapêutico
14.
Clin Pharmacol Ther ; 112(3): 687-698, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35527512

RESUMO

Endogenous biomarkers for transporter-mediated drug-drug interaction (DDI) predictions represent a promising approach to facilitate and improve conventional DDI investigations in clinical studies. This approach requires high sensitivity and specificity of biomarkers for the targets of interest (e.g., transport proteins), as well as rigorous characterization of their kinetics, which can be accomplished utilizing physiologically-based pharmacokinetic (PBPK) modeling. Therefore, the objective of this study was to develop PBPK models of the endogenous organic cation transporter (OCT)2 and multidrug and toxin extrusion protein (MATE)1 substrates creatinine and N1 -methylnicotinamide (NMN). Additionally, this study aimed to predict kinetic changes of the biomarkers during administration of the OCT2 and MATE1 perpetrator drugs trimethoprim, pyrimethamine, and cimetidine. Whole-body PBPK models of creatinine and NMN were developed utilizing studies investigating creatinine or NMN exogenous administration and endogenous synthesis. The newly developed models accurately describe and predict observed plasma concentration-time profiles and urinary excretion of both biomarkers. Subsequently, models were coupled to the previously built and evaluated perpetrator models of trimethoprim, pyrimethamine, and cimetidine for interaction predictions. Increased creatinine plasma concentrations and decreased urinary excretion during the drug-biomarker interactions with trimethoprim, pyrimethamine, and cimetidine were well-described. An additional inhibition of NMN synthesis by trimethoprim and pyrimethamine was hypothesized, improving NMN plasma and urine interaction predictions. To summarize, whole-body PBPK models of creatinine and NMN were built and evaluated to better assess creatinine and NMN kinetics while uncovering knowledge gaps for future research. The models can support investigations of renal transporter-mediated DDIs during drug development.


Assuntos
Cimetidina , Pirimetamina , Biomarcadores , Cimetidina/farmacocinética , Creatinina , Interações Medicamentosas , Humanos , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Preparações Farmacêuticas , Pirimetamina/farmacologia , Trimetoprima/farmacologia
15.
Anal Bioanal Chem ; 414(9): 3121-3135, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35141763

RESUMO

Antibiotics are some of the most widely used drugs. Their release in the environment is of great concern since their consumption is a major factor for antibiotic resistance, one of the most important threats to human health. Their occurrence and fate in agricultural systems have been extensively investigated in recent years. Yet whilst their biotic and abiotic degradation pathways have been thoroughly researched, their biotransformation pathways in plants are less understood, such as in case of trimethoprim. Although trimethoprim has been reported in the environment, its fate in higher plants still remains unknown. A bench-scale experiment was performed and 30 trimethoprim metabolites were identified in lettuce (Lactuca sativa L.), of which 5 belong to phase I and 25 to phase II. Data mining yielded a list of 1018 ions as possible metabolite candidates, which was filtered to a final list of 87 candidates. Molecular structures were assigned for 19 compounds, including 14 TMP metabolites reported for the first time. Alongside well-known biotransformation pathways in plants, additional novel pathways were suggested, namely, conjugation with sesquiterpene lactones, and abscisic acid as a part of phase II of plant metabolism. The results obtained offer insight into the variety of phase II conjugates and may serve as a guideline for studying the metabolization of other chemicals that share a similar molecular structure or functional groups with trimethoprim. Finally, the toxicity and potential contribution of the identified metabolites to the selective pressure on antibiotic resistance genes and bacterial communities via residual antimicrobial activity were evaluated.


Assuntos
Alface , Trimetoprima , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Biotransformação , Humanos , Alface/química , Compostos Fitoquímicos/metabolismo , Trimetoprima/metabolismo , Trimetoprima/farmacologia
16.
PLoS Comput Biol ; 18(2): e1009855, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-35143481

RESUMO

Antimicrobial resistance presents a significant health care crisis. The mutation F98Y in Staphylococcus aureus dihydrofolate reductase (SaDHFR) confers resistance to the clinically important antifolate trimethoprim (TMP). Propargyl-linked antifolates (PLAs), next generation DHFR inhibitors, are much more resilient than TMP against this F98Y variant, yet this F98Y substitution still reduces efficacy of these agents. Surprisingly, differences in the enantiomeric configuration at the stereogenic center of PLAs influence the isomeric state of the NADPH cofactor. To understand the molecular basis of F98Y-mediated resistance and how PLAs' inhibition drives NADPH isomeric states, we used protein design algorithms in the osprey protein design software suite to analyze a comprehensive suite of structural, biophysical, biochemical, and computational data. Here, we present a model showing how F98Y SaDHFR exploits a different anomeric configuration of NADPH to evade certain PLAs' inhibition, while other PLAs remain unaffected by this resistance mechanism.


Assuntos
Antagonistas do Ácido Fólico , Infecções Estafilocócicas , Farmacorresistência Bacteriana/genética , Antagonistas do Ácido Fólico/química , Antagonistas do Ácido Fólico/farmacologia , Humanos , NADP/metabolismo , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Tetra-Hidrofolato Desidrogenase/química , Tetra-Hidrofolato Desidrogenase/genética , Tetra-Hidrofolato Desidrogenase/metabolismo , Trimetoprima/química , Trimetoprima/metabolismo , Trimetoprima/farmacologia
17.
Ir J Med Sci ; 191(1): 279-282, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33599916

RESUMO

BACKGROUND: Urinary tract infection (UTI) is one of the common infections in childhood. Prompt diagnosis and treatment reduces the risk of complications. The choice of antibiotic to treat UTI varies from region to region. Rational use and appropriately chosen antibiotic reduces the emergence of resistant uropathogens. OBJECTIVE: We investigated the resistance pattern of uropathogens for commonly used antibiotics to treat UTI locally. METHODS: Data was collected between 2009 and 2019 on all infants and children under 16 years of age with culture proven UTI. Results were compared with previously published figures between 2002 and 2008. RESULTS: A total of 1002 samples were analysed (91/year). Male to female ratio was 1:4.6. About 94% of the samples grew E. coli. As before, high resistance rates were recorded to Amoxicillin and Trimethoprim (Z = -0.325: P = 0.7452; not significant). Overall, average resistance has decreased for Nitrofurantoin from 10% between 2002 and 2008 to 5.84% between 2009 and 2019 (Z = 3.002: P = 0.0027). On the other hand, Cefalexin resistance has increased from 7.4 to 14.56% between the two study periods (Z = -4.2: P = < 0.0002). CONCLUSION: Despite rising resistance rates, we recommend that Cefalexin should cautiously remain the antibiotic of choice for empirically treating uncomplicated urinary tract infections in secondary care pending urine culture. Nitrofurantoin should be reserved for treating non-coliform/atypical UTIs or multi-drug resistant UTIs. There is an ongoing need for clinicians in all geographic regions to continue to monitor antibiotic resistance rates every few years.


Assuntos
Infecções Urinárias , Sistema Urinário , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Criança , Farmacorresistência Bacteriana , Escherichia coli , Feminino , Humanos , Lactente , Masculino , Testes de Sensibilidade Microbiana , Trimetoprima/farmacologia , Trimetoprima/uso terapêutico , Infecções Urinárias/tratamento farmacológico , Infecções Urinárias/epidemiologia
18.
J Bacteriol ; 204(1): e0037021, 2022 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-34633866

RESUMO

Cells that cannot synthesize one of the DNA precursors, dTTP, due to thyA mutation or metabolic poisoning, undergo thymineless death (TLD), a chromosome-based phenomenon of unclear mechanisms. In Escherichia coli, thymineless death is caused either by denying thyA mutants thymidine supplementation or by treating wild-type cells with trimethoprim. Two recent reports promised a potential breakthrough in TLD understanding, suggesting significant oxidative damage during thymine starvation. Oxidative damage in vivo comes from Fenton's reaction when hydrogen peroxide meets ferrous iron to produce hydroxyl radical. Therefore, TLD could kill via irreparable double-strand breaks behind replication forks when starvation-caused single-strand DNA gaps are attacked by hydroxyl radicals. We tested the proposed Fenton-TLD connection in both thyA mutants denied thymidine, as well as in trimethoprim-treated wild-type (WT) cells, under the following three conditions: (i) intracellular iron chelation, (ii) mutational inactivation of hydrogen peroxide (HP) scavenging, and (iii) acute treatment with sublethal HP concentrations. We found that TLD kinetics are affected by neither iron chelation nor HP stabilization in cultures, indicating no induction of oxidative damage during thymine starvation. Moreover, acute exogenous HP treatments completely block TLD, apparently by blocking cell division, which may be a novel TLD prerequisite. Separately, the acute trimethoprim sensitivity of the rffC and recBCD mutants demonstrates how bactericidal power of this antibiotic could be amplified by inhibiting the corresponding enzymes. IMPORTANCE Mysterious thymineless death strikes cells that are starved for thymine and therefore replicating their chromosomal DNA without dTTP. After 67 years of experiments testing various obvious and not so obvious explanations, thymineless death is still without a mechanism. Recently, oxidative damage via in vivo Fenton's reaction was proposed as a critical contributor to the irreparable chromosome damage during thymine starvation. We have tested this idea by either blocking in vivo Fenton's reaction (expecting no thymineless death) or by amplifying oxidative damage (expecting hyperthymineless death). Instead, we found that blocking Fenton's reaction has no influence on thymineless death, while amplifying oxidative damage prevents thymineless death altogether. Thus, oxidative damage does not contribute to thymineless death, while the latter remains enigmatic.


Assuntos
Antibacterianos/farmacologia , Escherichia coli/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Timina/farmacologia , Trimetoprima/farmacologia , Replicação do DNA , DNA Bacteriano , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Peróxido de Hidrogênio , Ferro/metabolismo , Viabilidade Microbiana , Timina/metabolismo
19.
Poult Sci ; 101(1): 101538, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34788713

RESUMO

One hundred and twenty chicken samples from feces (n = 80), the carcass surface at slaughter at 2 meat chicken farms (n = 20), and retail chicken meat from 5 markets (n = 20) collected during 2018 and 2019 were examined for the prevalence of plasmid-mediated quinolone resistance (PMQR) in Escherichia coli. We detected qnrS-positive E. coli in a total of 74 samples from feces (n = 59), the carcass surface (n = 7), and retail meat (n = 8). These 74 qnrS-positive isolates were tested for antimicrobial susceptibility to determine the minimum inhibitory concentrations (MICs) of certain antimicrobials and genetically characterized. Ampicillin-resistance accounted for 71 of the 74 isolates (96%), followed by resistance to oxytetracycline (57/74; 77%), enrofloxacin (ERFX) (56/74; 76%), sulfisoxazole (SUL) (56/74; 76%), trimethoprim (TMP) (49/74; 66%), and dihydrostreptomycin (48/74; 65%). All farm-borne SUL- and TMP-resistant isolates except one were obtained from samples from farm A where a combination of sulfadiazine and TMP was administered to the chickens. Concentrations of ERFX at which 50 and 90% of isolates were inhibited were 2 µg/mL and 32 µg/mL, respectively. Diverse pulsed-field gel electrophoresis (PFGE) patterns of XbaI-digested genomic DNA were observed in the qnrS-positive isolates from fecal samples. Several isolates from feces and the carcass surface had identical XbaI-digested PFGE patterns. S1-nuclease PFGE and Southern blot analysis demonstrated that 7 of 11 dfrA13-positive fecal isolates carried both the qnrS and dfrA13 genes on the same plasmid, and 2 of 3 dfrA1-positive isolates similarly carried both qnrS and dfrA1 on the same plasmid, although the PFGE patterns of XbaI-digested genomic DNA of the isolates were different. These results suggest that the qnrS gene is prevalent in chicken farms via horizontal transfer of plasmids and may partly be co-selected under the use of TMP.


Assuntos
Galinhas , Proteínas de Escherichia coli , Animais , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Fazendas , Plasmídeos/genética , Prevalência , Tailândia , Trimetoprima/farmacologia
20.
Br J Pharmacol ; 179(11): 2659-2677, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-34855201

RESUMO

BACKGROUND AND PURPOSE: The zinc finger transcription factor Snail is aberrantly activated in many human cancers and strongly associated with poor prognosis. As a transcription factor, Snail has been traditionally considered an 'undruggable' target. Here, we identified a potent small-molecule inhibitor of Snail, namely trimethoprim, and investigated its potential antitumour effects and the underlying mechanisms. EXPERIMENTAL APPROACH: The inhibitory action of trimethoprim on Snail protein and the related molecular mechanisms were revealed by molecular docking, biolayer interferometry, immunoblotting, immunoprecipitation, qRT-PCR, pull-down and cycloheximide pulse-chase assays. The anti-proliferative and anti-metastatic effects of trimethoprim via targeting Snail were tested in multiple cell-based assays and animal models. KEY RESULTS: This study identified trimethoprim, an antimicrobial drug, as a potent antitumour agent via targeting Snail. Molecular modelling analysis predicted that trimethoprim directly binds to the arginine-174 pocket of Snail protein. We further discovered that trimethoprim strongly interrupts the interaction of Snail with CREB-binding protein (CBP)/p300, which consequently suppresses Snail acetylation and promotes Snail degradation through the ubiquitin-proteasome pathway. Furthermore, trimethoprim sufficiently inhibited the proliferation, epithelial-mesenchymal transition (EMT) and migration of cancer cells in vitro via specifically targeting Snail. More importantly, trimethoprim effectively reduced Snail-driven tumour growth and metastasis to vital organs such as lung, bone and liver. CONCLUSIONS AND IMPLICATIONS: These findings indicate, for the first time, that trimethoprim suppresses tumour growth and metastasis via targeting Snail. This study provides insights for a better understanding of the anticancer effects of trimethoprim and offers a potential anticancer therapeutic agent for clinical treatment.


Assuntos
Fatores de Transcrição , Trimetoprima , Animais , Antibacterianos/farmacologia , Linhagem Celular Tumoral , Movimento Celular , Simulação de Acoplamento Molecular , Metástase Neoplásica , Fatores de Transcrição da Família Snail/metabolismo , Fatores de Transcrição/metabolismo , Trimetoprima/farmacologia
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