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1.
Anticancer Res ; 40(9): 5025-5033, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32878790

RESUMO

BACKGROUND/AIM: This study aimed to investigate the effect of a new 7-(4-(N-substituted carbamoylmethyl) piperazin-1-yl) ciprofloxacin-derivative on the proliferation and migration abilities of HeLa cells. MATERIALS AND METHODS: Cell viability and morphological alterations were examined. Changes in migration were detected using wound healing and colony formation assays. Flow cytometry and western blotting were used to investigate the molecular mechanisms underlying this ciprofloxacin-derivative's action in HeLa cells. RESULTS: The examined ciprofloxacin-derivative reduced viability of HeLa cells in a concentration-dependent manner and altered cellular morphology, indicating cell death. Furthermore, it significantly inhibited wound closure, even in a non-cytotoxic concentration, and reduced HeLa cell colony formation. In addition, apoptosis was increased probably through significant up-regulation of Bax protein expression and the generation of active cleaved caspase-3 protein. CONCLUSION: Our new derivative inhibits proliferation and induces apoptosis of HeLa cells. Furthermore, it suppressed the migration and colony formation abilities of HeLa cells. Therefore, it represents an attractive agent for drug development against cervical cancer based on its anti-metastatic effect.


Assuntos
Antineoplásicos/farmacologia , Ciprofloxacino/análogos & derivados , Ciprofloxacino/farmacologia , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ciprofloxacino/química , Relação Dose-Resposta a Droga , Células HeLa , Humanos , Estrutura Molecular , Ensaio Tumoral de Célula-Tronco
2.
PLoS Biol ; 18(8): e3000805, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32810152

RESUMO

Antibiotics are losing efficacy due to the rapid evolution and spread of resistance. Treatments targeting bacterial virulence factors have been considered as alternatives because they target virulence instead of pathogen viability, and should therefore exert weaker selection for resistance than conventional antibiotics. However, antivirulence treatments rarely clear infections, which compromises their clinical applications. Here, we explore the potential of combining antivirulence drugs with antibiotics against the opportunistic human pathogen Pseudomonas aeruginosa. We combined two antivirulence compounds (gallium, a siderophore quencher, and furanone C-30, a quorum sensing [QS] inhibitor) together with four clinically relevant antibiotics (ciprofloxacin, colistin, meropenem, tobramycin) in 9×9 drug concentration matrices. We found that drug-interaction patterns were concentration dependent, with promising levels of synergies occurring at intermediate drug concentrations for certain drug pairs. We then tested whether antivirulence compounds are potent adjuvants, especially when treating antibiotic resistant (AtbR) clones. We found that the addition of antivirulence compounds to antibiotics could restore growth inhibition for most AtbR clones, and even abrogate or reverse selection for resistance in five drug combination cases. Molecular analyses suggest that selection against resistant clones occurs when resistance mechanisms involve restoration of protein synthesis, but not when efflux pumps are up-regulated. Altogether, our work provides a first systematic analysis of antivirulence-antibiotic combinatorial treatments and suggests that such combinations have the potential to be both effective in treating infections and in limiting the spread of antibiotic resistance.


Assuntos
Antibacterianos/farmacologia , Ciprofloxacino/farmacologia , Colistina/farmacologia , Furanos/farmacologia , Gálio/farmacologia , Meropeném/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Tobramicina/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Combinação de Medicamentos , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Testes de Sensibilidade Microbiana , Biossíntese de Proteínas/efeitos dos fármacos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/metabolismo , Percepção de Quorum/efeitos dos fármacos , Virulência
3.
Nat Commun ; 11(1): 3970, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32769975

RESUMO

The rise of antibiotic resistance in many bacterial pathogens has been driven by the spread of a few successful strains, suggesting that some bacteria are genetically pre-disposed to evolving resistance. Here, we test this hypothesis by challenging a diverse set of 222 isolates of Staphylococcus aureus with the antibiotic ciprofloxacin in a large-scale evolution experiment. We find that a single efflux pump, norA, causes widespread variation in evolvability across isolates. Elevated norA expression potentiates evolution by increasing the fitness benefit provided by DNA topoisomerase mutations under ciprofloxacin treatment. Amplification of norA provides a further mechanism of rapid evolution in isolates from the CC398 lineage. Crucially, chemical inhibition of NorA effectively prevents the evolution of resistance in all isolates. Our study shows that pre-existing genetic diversity plays a key role in shaping resistance evolution, and it may be possible to predict which strains are likely to evolve resistance and to optimize inhibitor use to prevent this outcome.


Assuntos
Proteínas de Bactérias/metabolismo , Resistência Microbiana a Medicamentos , Evolução Molecular , Staphylococcus aureus/genética , Staphylococcus aureus/isolamento & purificação , Ciprofloxacino/farmacologia , Resistência Microbiana a Medicamentos/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Genoma Bacteriano , Mutação/genética , Filogenia , Staphylococcus aureus/efeitos dos fármacos , Transcriptoma/efeitos dos fármacos , Transcriptoma/genética
4.
PLoS One ; 15(7): e0235892, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32663201

RESUMO

Today the development of antibiotic resistance, especially in the treatment of bacterial infections associated with biofilms, has led to increasing the importance of antimicrobial peptides (AMPs). In this work, antimicrobial and synergistic activity of three truncated HNP-1 analogs (2Abz14S29, 2Abz23S29, and HNP1ΔC18A) with ß-lactam (amoxicillin and cefixime) and fluoroquinolones (ciprofloxacin and norfloxacin) antibiotics against multidrug-resistant (MDR) uropathogenic E. coli clinical isolates were evaluated. The anti-biofilm potential of peptides at different stages was also investigated. All peptides exhibited additive activity just with ß-lactam antibiotics in a checkerboard synergy assay. Inhibition and eradication of MDR uropathogenic E. coli biofilm were shown by all test peptides at different concentrations. Thus, truncated HNP-1 analogs (2Abz14S29, 2Abz23S29, and HNP1ΔC18A) may have the potential for the treatment of urinary tract infections (UTIs) caused by biofilm-forming MDR uropathogenic E. coli.


Assuntos
Antibacterianos/farmacologia , Biofilmes , Escherichia coli Uropatogênica/efeitos dos fármacos , alfa-Defensinas/farmacologia , Amoxicilina/farmacologia , Antibacterianos/toxicidade , Cefixima/farmacologia , Ciprofloxacino/farmacologia , Farmacorresistência Bacteriana , Sinergismo Farmacológico , Norfloxacino/farmacologia , alfa-Defensinas/toxicidade
5.
BMC Infect Dis ; 20(1): 514, 2020 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-32677988

RESUMO

BACKGROUND: Worldwide, an increase in antimicrobial resistance (AMR) of Neisseria gonorrhoeae has been observed. Until now, no protocol for an external quality assessment (EQA) has been available for Germany. The German gonococcal resistance network (GORENET) performed an EQA of primary laboratories in Germany in order to assess quality of antibiotic susceptibility testing, to gain information about laboratory procedures and to assess the impact of these procedures on test results. METHODS: Laboratories assessed drug susceptibility to cefixime, ceftriaxone, azithromycin, penicillin and ciprofloxacin for five N. gonorrhoeae strains, using their standard laboratory protocols. Minimal inhibitory concentrations (MICs) were compared to World Health Organisation (WHO) consensus results (or, if not available, reference laboratory results), while deviation by +/- one doubling dilution was accepted. Data on laboratory procedures were collected via a standardised questionnaire. Generalized linear models and conditional inference trees (CTREE) were used to assess relationships between laboratory procedures and testing outcomes. RESULTS: Twenty-one primary laboratories participated in the EQA in June 2018. 96% of ciprofloxacin MICs were reported within accepted deviations, as well as 88% for cefixime, 85% for ceftriaxone, 79% for penicillin and 70% for azithromycin. The use of interpretation standards and general laboratory procedures like agar base, incubation settings or the use of control strains strongly differed between laboratories. In statistical analysis, incubation time of cultures < 24 h was associated with correct measurements. Additionally, a 5% CO2 concentration was associated with correct results regarding azithromycin compared to 3%. CTREE analysis showed that incubation time, humidity and CO2 concentration had the greatest influence on the average deviation from consensus results. CONCLUSIONS: In conclusion, we report the development of a protocol for N. gonorrhoeae antimicrobial susceptibility testing in Germany. While testing results were in accordance with the expected consensus results in 70-96%, depending on the antibiotic agent, laboratory methodology was heterogeneous and may significantly affect the testing quality. We therefore recommend the development of a standard operating procedure (SOP) for N. gonorrhoeae susceptibility testing in Germany.


Assuntos
Antibacterianos/uso terapêutico , Farmacorresistência Bacteriana/efeitos dos fármacos , Gonorreia/tratamento farmacológico , Laboratórios/normas , Ensaio de Proficiência Laboratorial , Neisseria gonorrhoeae/efeitos dos fármacos , Antibacterianos/farmacologia , Azitromicina/farmacologia , Azitromicina/uso terapêutico , Cefixima/farmacologia , Cefixima/uso terapêutico , Ceftriaxona/farmacologia , Ceftriaxona/uso terapêutico , Ciprofloxacino/farmacologia , Ciprofloxacino/uso terapêutico , Alemanha , Gonorreia/microbiologia , Humanos , Ensaio de Proficiência Laboratorial/métodos , Testes de Sensibilidade Microbiana , Penicilinas/farmacologia , Penicilinas/uso terapêutico , Controle de Qualidade , Padrões de Referência , Inquéritos e Questionários
6.
MMWR Morb Mortal Wkly Rep ; 69(24): 735-739, 2020 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-32555137

RESUMO

Meningococcal disease is a sudden-onset, life-threatening illness caused by the bacterium Neisseria meningitidis. Prompt empiric antibiotic treatment can reduce morbidity and mortality among patients, and antibiotic prophylaxis can prevent secondary disease in close contacts. Historically, N. meningitidis isolates in the United States have largely been susceptible to the antibiotics recommended for treatment and prophylaxis, including penicillin and ciprofloxacin. This report describes detection of penicillin-resistant and ciprofloxacin-resistant N. meningitidis serogroup Y (NmY) isolates in the United States. NmY isolates containing a blaROB-1 ß-lactamase enzyme gene conferring resistance to penicillins (1) were recovered from 33 cases reported during 2013-2020. Isolates from 11 of these cases, reported during 2019-2020, harbored a ciprofloxacin resistance-associated mutation in a chromosomal gene (gyrA). Cases were reported from 12 geographically disparate states; a majority of cases (22 of 33, 67%) occurred in Hispanic persons. These cases represent a substantial increase in penicillin-resistant and ciprofloxacin-resistant meningococci in the United States since 2013. Ceftriaxone and cefotaxime, the recommended first-line agents for empiric bacterial meningitis treatment, can continue to be used for treatment, but health care providers should ascertain susceptibility of meningococcal isolates to penicillin before switching to penicillin or ampicillin. Ongoing monitoring for antimicrobial resistance among meningococcal isolates and prophylaxis failures will be important to inform treatment and prophylaxis recommendations.


Assuntos
Ciprofloxacino/farmacologia , Resistência Microbiana a Medicamentos , Neisseria meningitidis/isolamento & purificação , beta-Lactamases/biossíntese , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Neisseria meningitidis/efeitos dos fármacos , Neisseria meningitidis/genética , Sorogrupo , Estados Unidos , Adulto Jovem
7.
Int J Nanomedicine ; 15: 3393-3404, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32523339

RESUMO

Introduction: The efficacy of several antimicrobial agents has been hindered due to the increasing frequency of multidrug-resistant (MDR) Pseudomonas aeruginosa strains. So, the need for new antibacterial drugs or drug combinations is urgent. Recently, desirable antibacterial effects were reported for many metals nanoparticles such as TiO2 nanoparticles (TDNs). Purpose: This study aims to investigate the prevalence of MDR P. aeruginosa and assess the efficiency of TDN in the treatment of MDR P. aeruginosa-associated infections. Materials and Methods: The synthesis of TDN by the sol-gel method was carried out. Particle size measurements and morphology were done using dynamic light scattering (DLS) and high-resolution transmission electron microscopy (HR-TEM). To investigate the physical and chemical changes of drugs due to the combination, the tested drugs, both alone and in combination with TDN, were subjected to differential scanning calorimetry (DSC), infrared (IR) spectroscopy, and X-ray diffraction studies. Antimicrobial susceptibility was detected by agar disc-diffusion assay. The minimum inhibitory concentration (MIC) of TDN and the tested antibiotics were assessed by the agar dilution method. Checkerboard analysis was performed to determine the combined effect of TDN and the tested antibiotics against 25 MDR P. aeruginosa strains. Results: TDNs were prepared with an average particle size of 64.77 ± 0.14 nm with an accepted polydispersity index (PDI) value of 0.274 ± 0.004. TEM showed that the particles were shaped into irregular spheres. Twenty-five P. aeruginosa isolates that were absolutely resistant to cefepime (100%), highly resistant to ceftriaxone (96%), amikacin (80%), and ciprofloxacin (76%) were selected. Superior antibacterial activity of TDN was observed against the selected 25 MDR P. aeruginosa isolates. The combination of TDN and cefepime were found to show synergistic activity against all tested isolates followed by ceftriaxone (96%), amikacin (88%), and ciprofloxacin (80%). Conclusion: Using TDN in combination with antibiotics can help in the treatment of MDR P. aeruginosa-associated infections. So, preparation of topical pharmaceutical dosage forms containing a combination of these antibiotics and TDN can be useful against MDR P. aeruginosa.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Nanopartículas/química , Pseudomonas aeruginosa/efeitos dos fármacos , Titânio/farmacologia , Antibacterianos/uso terapêutico , Calorimetria , Ciprofloxacino/farmacologia , Ciprofloxacino/uso terapêutico , Humanos , Testes de Sensibilidade Microbiana , Nanopartículas/ultraestrutura , Tamanho da Partícula , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/isolamento & purificação , Espectroscopia de Infravermelho com Transformada de Fourier , Difração de Raios X
8.
Anticancer Res ; 40(5): 2739-2749, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32366419

RESUMO

BACKGROUND/AIM: Ciprofloxacin has been used as an antibiotic in the clinic for decades. Recently, ciprofloxacin and its derivatives have shown promising anti-proliferative and cytotoxic activities against several malignant cells. The aim of this study was to investigate the effect of a new derivative of ciprofloxacin on colorectal cancer (HCT116) and non-small lung carcinoma (A549) cells. MATERIALS AND METHODS: Cell viability was detected by the MTT assay. Flow cytometry was used to examine the cell cycle and apoptosis. Expression of bax, bcl2, p53 and p21 was investigated by qRT-PCR and western blotting. RESULTS: Ciprofloxacin-derivative had an anti-proliferative effect on both cell lines in a concentration-dependent manner and caused cell cycle arrest at the G2/M phase and apoptosis. p53 and Bax proteins were overexpressed, while p21 and bcl2 gene expression was decreased after treatment with the ciprofloxacin derivative. CONCLUSION: This new ciprofloxacin derivative can be potentially used for the treatment of colorectal cancer and non-small lung carcinoma.


Assuntos
Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Ciprofloxacino/farmacologia , Células A549 , Anexina A5/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HCT116 , Humanos , Concentração Inibidora 50 , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo
9.
PLoS One ; 15(4): e0230423, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32236115

RESUMO

Campylobacter jejuni is one of the most prevalent causes of bacterial gastroenteritis worldwide, and it is largely associated with consumption of contaminated poultry. Current Campylobacter control measures at the poultry production level remain insufficient, and hence there is the need for alternative control strategies. We evaluated the potential of the monoterpene (-)-α-pinene for control of C. jejuni in poultry. The antibacterial and resistance-modulatory activities of (-)-α-pinene were also determined against 57 C. jejuni strains. In addition, the anti-quorum-sensing activity of (-)-α-pinene against C. jejuni NCTC 11168 was determined for three subinhibitory concentrations (125, 62.5, 31.25 mg/L) over three incubation times using an autoinducer-2 bioassay based on Vibrio harveyi BB170 bioluminescence measurements. The effects of a subinhibitory concentration of (-)-α-pinene (250 mg/L) on survival of C. jejuni, and in combination with enrofloxacin on fluoroquinolone resistance development in C. jejuni, were determined in a broiler chicken model, by addition of (-)-α-pinene to the broiler water supply. The reduction of C. jejuni numbers by (-)-α-pinene was further determined in broiler chickens that were colonized with either fluoroquinolone-susceptible or -resistant strains, by direct gavage treatment. We observed weak in vitro antimicrobial activity for (-)-α-pinene alone (MIC >500 mg/L), but strong potentiating effects on antibiotics erythromycin and ciprofloxacin against different Campylobacter strains (>512 fold change). After 24 h of treatment of C. jejuni with (-)-α-pinene, its quorum-sensing signaling was reduced by >80% compared to the untreated control. When given in the drinking water, (-)-α-pinene did not show any significant inhibitory effects on the level of C. jejuni in the colonized chickens, and did not reduce fluoroquinolone resistance development in combination with enrofloxacin. Conversely, when (-)-α-pinene was administered by direct gavage, it significantly reduced the number of fluoroquinolone susceptible C. jejuni in the colonized broiler chickens. These results demonstrate that (-)-α-pinene modulates quorum-sensing in Campylobacter, potentiates antibiotics against different Campylobacter strains, and reduces Campylobacter colonization in broiler chickens.


Assuntos
Antibacterianos/farmacologia , Monoterpenos Bicíclicos/farmacologia , Campylobacter jejuni/efeitos dos fármacos , Percepção de Quorum/efeitos dos fármacos , Animais , Antibacterianos/uso terapêutico , Monoterpenos Bicíclicos/uso terapêutico , Infecções por Campylobacter/patologia , Infecções por Campylobacter/prevenção & controle , Campylobacter jejuni/fisiologia , Ceco/microbiologia , Galinhas , Ciprofloxacino/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Eritromicina/farmacologia , Fluoroquinolonas/farmacologia , Fluoroquinolonas/uso terapêutico , Testes de Sensibilidade Microbiana , Doenças das Aves Domésticas/patologia , Doenças das Aves Domésticas/prevenção & controle
11.
BMC Complement Med Ther ; 20(1): 92, 2020 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-32192484

RESUMO

BACKGROUND: Acinetobacter baumannii is an opportunistic pathogen that causes nosocomial infections especially in patients in intensive care units (ICUs). Accordingly, the aim of our study was to detection of adeABC efllux pump encoding genes and antimicrobial effect of the essential oil of Mentha longifolia and Menthol on the minimum inhibitory concentration (MIC) of imipenem and ciprofloxacin in clinical isolates of A. baumannii. METHODS: A total of 75 clinical isolates of A. baumannii were collected. The presence of efflux pump genes was detected by polymerase chain reaction (PCR). The minimum inhibitory concentration (MIC) of the essential oil of Mentha longifolia and Menthol and their combined effect with antibiotics were measured by microbroth dilution method and fractional inhibitory concentration (FIC) index. RESULTS: The frequency of adeA, adeB, and adeC genes in clinical isolates of A. baumannii were 86.7, 90.7, and 92%, respectively. When the essential oil of Mentha longifolia was combined with ciprofloxacin and imipenem, MICs decreased 4- and 8-fold, respectively. In the combination of menthol with imipenem, the resistance to imipenem was reduced from 0- to 16-fold in 90% (63/70) of the isolates. CONCLUSION: The presence of efflux pump genes in more than 90% of A. baumannii isolates indicates its potential role in inducing imipenem- and ciprofloxacin-resistance in this bacterium. Menthol has an antimicrobial effect as an active ingredient in Mentha longifolia. In the future, the combination of medicinal plants with antibiotics can be used as a complement in treating diseases caused by drug-resistant bacteria such as A. baumannii infections.


Assuntos
Acinetobacter baumannii/efeitos dos fármacos , Antibacterianos/farmacologia , Proteínas de Membrana Transportadoras/genética , Mentha/química , Mentol/farmacologia , Óleos Voláteis/farmacologia , Proteínas de Bactérias/genética , Ciprofloxacino/farmacologia , Quimioterapia Combinada , Imipenem/farmacologia , Testes de Sensibilidade Microbiana
12.
Microbiol Res ; 236: 126466, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32193126

RESUMO

Since the advent of biologics in human welfare various bio-molecules have been explored. Different bacterial exopolysaccharides have proved their worth in many industrial and commercial applications. In this perspective, while exploring a surfactant exopolysaccharide of Ochrobactrum pseudintermedium C1, it is strikingly observed that it possesses a potent antibacterial property which encourages its bio-medical applications. Following isolation and purification of the said exopolysaccharide, its structural configuration and functional attributes are studied by several analytical procedures involving FTIR, 13C- NMR, CHN-analysis, estimation of zeta potential, XRD-study and digital tensiometry. When treated with pathological samples in vitro, it distinctly elicits its antibacterial property by exhibiting a characteristic zone of inhibition. Combined with a standard antibiotic (like ciprofloxacin), it enhances the action of antibiotic also. Mechanism of its antibacterial action is evaluated by crystal violet entrapment assay with UV-vis spectrophotometry, bacterial cell viability assay by trypan blue staining and SEM study. Results show that its basic surfactant property, anionic character, crystalline nature and scaffolding architecture are supposed to facilitate its antibacterial property which is manifested by its capability of disrupting bacterial cell envelope causing eventual cell death. In the current global scenario, an increasing threat of antibiotic resistance is prevailing due to their indiscriminate use. If used as an adjuvant with a judicious dose of antibiotic, this bio-molecule might play a significant role in bio-medicine to combat such threat.


Assuntos
Antibacterianos/biossíntese , Antibacterianos/farmacologia , Ochrobactrum , Polissacarídeos Bacterianos/biossíntese , Tensoativos/metabolismo , Antibacterianos/metabolismo , Quimioterapia Adjuvante , Ciprofloxacino/farmacologia , Escherichia coli/efeitos dos fármacos , Humanos , Técnicas In Vitro , Testes de Sensibilidade Microbiana , Ochrobactrum/metabolismo , Polissacarídeos Bacterianos/química , Polissacarídeos Bacterianos/metabolismo , Staphylococcus aureus/efeitos dos fármacos
13.
Microb Genom ; 6(2)2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32048983

RESUMO

Antimicrobial resistance (AMR) poses a threat to public health. Clinical microbiology laboratories typically rely on culturing bacteria for antimicrobial-susceptibility testing (AST). As the implementation costs and technical barriers fall, whole-genome sequencing (WGS) has emerged as a 'one-stop' test for epidemiological and predictive AST results. Few published comparisons exist for the myriad analytical pipelines used for predicting AMR. To address this, we performed an inter-laboratory study providing sets of participating researchers with identical short-read WGS data from clinical isolates, allowing us to assess the reproducibility of the bioinformatic prediction of AMR between participants, and identify problem cases and factors that lead to discordant results. We produced ten WGS datasets of varying quality from cultured carbapenem-resistant organisms obtained from clinical samples sequenced on either an Illumina NextSeq or HiSeq instrument. Nine participating teams ('participants') were provided these sequence data without any other contextual information. Each participant used their choice of pipeline to determine the species, the presence of resistance-associated genes, and to predict susceptibility or resistance to amikacin, gentamicin, ciprofloxacin and cefotaxime. We found participants predicted different numbers of AMR-associated genes and different gene variants from the same clinical samples. The quality of the sequence data, choice of bioinformatic pipeline and interpretation of the results all contributed to discordance between participants. Although much of the inaccurate gene variant annotation did not affect genotypic resistance predictions, we observed low specificity when compared to phenotypic AST results, but this improved in samples with higher read depths. Had the results been used to predict AST and guide treatment, a different antibiotic would have been recommended for each isolate by at least one participant. These challenges, at the final analytical stage of using WGS to predict AMR, suggest the need for refinements when using this technology in clinical settings. Comprehensive public resistance sequence databases, full recommendations on sequence data quality and standardization in the comparisons between genotype and resistance phenotypes will all play a fundamental role in the successful implementation of AST prediction using WGS in clinical microbiology laboratories.


Assuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Bactérias/genética , Farmacorresistência Bacteriana , Genoma Bacteriano , Bactérias/classificação , Bactérias/isolamento & purificação , Carbapenêmicos/farmacologia , Ciprofloxacino/farmacologia , Biologia Computacional , Humanos , Testes de Sensibilidade Microbiana
14.
PLoS One ; 15(2): e0228509, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32074119

RESUMO

In this study, a drug discovery programme that sought to identify novel dual bacterial topoisomerase II inhibitors (NBTIs) led to the selection of six optimized compounds. In enzymatic assays, the molecules showed equivalent dual-targeting activity against the DNA gyrase and topoisomerase IV enzymes of Staphylococcus aureus and Escherichia coli. Consistently, the compounds demonstrated potent activity in susceptibility tests against various Gram-positive and Gram-negative reference species, including ciprofloxacin-resistant strains. The activity of the compounds against clinical multidrug-resistant isolates of S. aureus, Clostridium difficile, Acinetobacter baumannii, Neisseria gonorrhoeae, E. coli and vancomycin-resistant Enterococcus spp. was also confirmed. Two compounds (1 and 2) were tested in time-kill and post-antibiotic effect (PAE) assays. Compound 1 was bactericidal against all tested reference strains and showed higher activity than ciprofloxacin, and compound 2 showed a prolonged PAE, even against the ciprofloxacin-resistant S. aureus BAA-1720 strain. Spontaneous development of resistance to both compounds was selected for in S. aureus at frequencies comparable to those obtained for quinolones and other NBTIs. S. aureus BAA-1720 mutants resistant to compounds 1 and 2 had single point mutations in gyrA or gyrB outside of the quinolone resistance-determining region (QRDR), confirming the distinct site of action of these NBTIs compared to that of quinolones. Overall, the very good antibacterial activity of the compounds and their optimizable in vitro safety and physicochemical profile may have relevant implications for the development of new broad-spectrum antibiotics.


Assuntos
Antibacterianos/farmacologia , DNA Girase/efeitos dos fármacos , DNA Topoisomerases Tipo II/efeitos dos fármacos , Inibidores da Topoisomerase II/farmacologia , Animais , Células CHO , Ciprofloxacino/farmacologia , Cricetulus , DNA Topoisomerases Tipo II/metabolismo , DNA Bacteriano/efeitos dos fármacos , Farmacorresistência Bacteriana/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Células Hep G2 , Humanos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Staphylococcus aureus/efeitos dos fármacos , Testes de Toxicidade
15.
PLoS One ; 15(2): e0228591, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32023304

RESUMO

Biofilms are currently considered as a predominant lifestyle of many bacteria in nature. While they promote survival of microbes, biofilms also potentially increase the threats to animal and public health in case of pathogenic species. They not only facilitate bacteria transmission and persistence, but also promote spreading of antibiotic resistance leading to chronic infections. In the case of Francisella tularensis, the causative agent of tularemia, biofilms have remained largely enigmatic. Here, applying live and static confocal microscopy, we report growth and ultrastructural organization of the biofilms formed in vitro by these microorganisms over the early transition from coccobacillary into coccoid shape during biofilm assembly. Using selective dispersing agents, we provided evidence for extracellular DNA (eDNA) being a major and conserved structural component of mature biofilms formed by both F. subsp. novicida and a human clinical isolate of F. philomiragia. We also observed a higher physical robustness of F. novicida biofilm as compared to F. philomiragia one, a feature likely promoted by specific polysaccharides. Further, F. novicida biofilms resisted significantly better to ciprofloxacin than their planktonic counterparts. Importantly, when grown in biofilms, both Francisella species survived longer in cold water as compared to free-living bacteria, a trait possibly associated with a gain in fitness in the natural aquatic environment. Overall, this study provides information on survival of Francisella when embedded with biofilms that should improve both the future management of biofilm-related infections and the design of effective strategies to tackle down the problematic issue of bacteria persistence in aquatic ecosystems.


Assuntos
Biofilmes , Farmacorresistência Bacteriana , Francisella/fisiologia , Água Doce/microbiologia , Adaptação Fisiológica , Antibacterianos/farmacologia , Ciprofloxacino/farmacologia , Sequência Conservada , DNA Bacteriano/química , Francisella/efeitos dos fármacos , Francisella/genética , Francisella/patogenicidade , Infecções por Bactérias Gram-Negativas/microbiologia , Humanos
16.
Pathog Dis ; 78(1)2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-32083661

RESUMO

Current study aims to characterize the influence of sub-minimum inhibitory concentration (sub-MIC) of ciprofloxacin on Salmonella intracellular survival and host virulence. Herein, Salmonella resistance patterns to various antibiotics were in agreement with those reported in previous studies. Moreover, intracellular survival of both ciprofloxacin-sensitive and -resistant Salmonella was markedly reduced upon treatment with sub-MIC of ciprofloxacin as determined by gentamicin protection assay. These findings were further confirmed using immunostaining indicating an inhibitory effect of sub-MIC of ciprofloxacin on Salmonella intracellular survival. RT-qPCR revealed that expression of genes encoding Salmonella type three secretion system (TTSS) decreased upon bacterial exposure to sub-MIC of ciprofloxacin. Furthermore, bacterial exposure to sub-MIC of ciprofloxacin significantly reduced expression of both sifA and sifB, which are important for Salmonella filaments formation within the host. Treatment of Salmonella with sub-MIC of ciprofloxacin reduced bacterial capacity to kill mice infection models. A lower mortality rate was observed in mice injected with Salmonella treated with sub-MIC of ciprofloxacin as compared with mice inoculated with untreated bacteria. Collectively, current findings indicate that, in addition to its bactericidal potential, sub-MIC of ciprofloxacin could inhibit Salmonella intracellular survival, virulence genes expression as well as host pathogenesis, providing another mechanism for ciprofloxacin in limiting Salmonella host infection.


Assuntos
Proteínas de Bactérias/genética , Ciprofloxacino/farmacologia , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Proteínas de Membrana/genética , Infecções por Salmonella/microbiologia , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/fisiologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Humanos , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos , Testes de Sensibilidade Microbiana , Mutação , Infecções por Salmonella/imunologia , Infecções por Salmonella/metabolismo , Virulência/genética
17.
PLoS One ; 15(1): e0214833, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31899775

RESUMO

It is a common view that an organism's microbiota has a profound influence on host fitness; however, supporting evidence is lacking in many organisms. We manipulated the gut microbiome of Daphnia magna by chronic exposure to different concentrations of the antibiotic Ciprofloxacin (0.01-1 mg L-1), and evaluated whether this affected the animals fitness and antioxidant capacity. In line with our expectations, antibiotic exposure altered the microbiome in a concentration-dependent manner. However, contrary to these expectations, the reduced diversity of gut bacteria was not associated with any fitness detriment. Moreover, the growth-related parameters correlated negatively with microbial diversity; and, in the daphnids exposed to the lowest Ciprofloxacin concentrations, the antioxidant capacity, growth, and fecundity were even higher than in control animals. These findings suggest that Ciprofloxacin exerts direct stimulatory effects on growth and reproduction in the host, while microbiome- mediated effects are of lesser importance. Thus, although microbiome profiling of Daphnia may be a sensitive tool to identify early effects of antibiotic exposure, disentangling direct and microbiome-mediated effects on the host fitness is not straightforward.


Assuntos
Daphnia/microbiologia , Microbioma Gastrointestinal/efeitos dos fármacos , Aptidão Genética/genética , Reprodução/efeitos dos fármacos , Animais , Antibacterianos/farmacologia , Antioxidantes/metabolismo , Ciprofloxacino/farmacologia , Daphnia/efeitos dos fármacos , Daphnia/genética , Microbioma Gastrointestinal/genética
18.
Nat Microbiol ; 5(2): 256-264, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31959970

RESUMO

Despite the sporadic detection of fluoroquinolone-resistant Shigella in Asia in the early 2000s and the subsequent global spread of ciprofloxacin-resistant (cipR) Shigella sonnei from 2010, fluoroquinolones remain the recommended therapy for shigellosis1-7. The potential for cipR S. sonnei to develop resistance to alternative second-line drugs may further limit future treatment options8. Here, we aim to understand the evolution of novel antimicrobial resistant (AMR) S. sonnei variants after introduction into Vietnam. We found that cipR S. sonnei displaced the resident ciprofloxacin-susceptible (cipS) lineage while rapidly acquiring additional resistance to multiple alternative antimicrobial classes. We identified several independent acquisitions of extensively drug-resistant/multidrug-resistant-inducing plasmids, probably facilitated by horizontal transfer from commensals in the human gut. By characterizing commensal Escherichia coli from Shigella-infected and healthy children, we identified an extensive array of AMR genes and plasmids, including an identical multidrug-resistant plasmid isolated from both S. sonnei and E. coli in the gut of a single child. We additionally found that antimicrobial usage may impact plasmid transfer between commensal E. coli and S. sonnei. These results suggest that, in a setting with high antimicrobial use and a high prevalence of AMR commensals, cipR S. sonnei may be propelled towards pan-resistance by adherence to outdated international treatment guidelines.


Assuntos
Farmacorresistência Bacteriana Múltipla/genética , Escherichia coli/genética , Fluoroquinolonas/farmacologia , Fatores R/genética , Shigella sonnei/efeitos dos fármacos , Shigella sonnei/genética , Criança , Ciprofloxacino/farmacologia , Sistema Digestório/microbiologia , Reservatórios de Doenças/microbiologia , Disenteria Bacilar/tratamento farmacológico , Disenteria Bacilar/epidemiologia , Disenteria Bacilar/microbiologia , Epidemias , Escherichia coli/isolamento & purificação , Genes Bacterianos , Humanos , Filogenia , Shigella sonnei/classificação , Simbiose/genética , Vietnã/epidemiologia
19.
J Med Microbiol ; 69(3): 372-378, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31961790

RESUMO

Introduction. The emergence of novel strains of Vibrio cholerae O1 El Tor biotype has gained attention due to causing several epidemics around the world. Variant strains have evolved as a result of the acquisition of genes that confer extended virulence and pathogenicity.Aim. This study aimed to determine the presence of the most recently emerging Haitian-like genetic traits among the isolates from Jawaharlal Institute of Postgraduate Medical Education and Research, Pondicherry, Southern India. We also wanted to detect the prevalence of the sulfamethoxazole and trimethoprim (SXT) element, which is an integrating conjugative element (ICE) and the antimicrobial resistance genes present in our isolates.Methodology. Identification of Haitian-specific alleles was done by mismatched amplification mutation assay PCR (MAMA-PCR). The presence of SXT elements was carried out by PCR by detecting int, eex, att-prfC and setR genes. Detection of antibiotic resistance determinant, sul(1,2,3); dfr(A1,18,5) for trimethoprim resistance, tet(A,B,C,D,E,Y,G,M), tet34 for tetracycline resistance and erm(A,B,C), mph(A,B), ere(A,B), msr(A,D) for azithromycin resistance were targeted by PCR. The MIC of tetracycline, ciprofloxacin and azithromycin was determined by the E-test method.Results. Of the 95 isolates, 60 % of the isolates were found to carry Haitian-specific alleles of ctxB, tcpA and rtxA gene, 100 % of the isolates were found to carry SXT elements. All the isolates harboured the four conserved genes of the SXT element, except one which had only eex, att-prfC, setR genes. About 99 % harboured sul2 and dfrA1 genes. No tet and macrolide genes were detected. We observed a progressive increase in the MIC of azithromycin ranging from 0.75 µg ml-1 to 2 µg ml-1.Conclusion. None of the isolates were the prototype El Tor biotype. All the isolates were a Haitian variant. The presence of SXT elements across all our isolates and their creeping MIC of azithromycin is a matter of concern. Further testing for other genetic determinants of resistance will be carried out in our future studies.


Assuntos
Antibacterianos/farmacologia , Azitromicina/farmacologia , Cólera/epidemiologia , Farmacorresistência Bacteriana/genética , Vibrio cholerae O1/genética , Alelos , Cólera/microbiologia , Ciprofloxacino/farmacologia , Fezes/microbiologia , Transferência Genética Horizontal , Genótipo , Haiti , Humanos , Índia/epidemiologia , Testes de Sensibilidade Microbiana , Mutação , Fenótipo , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Tetraciclina/farmacologia , Vibrio cholerae O1/isolamento & purificação
20.
Nat Microbiol ; 5(4): 630-641, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31959968

RESUMO

Intestinal microbiotas contain beneficial microorganisms that protect against pathogen colonization; treatment with antibiotics disrupts the microbiota and compromises colonization resistance. Here, we determine the impact of exchanging microorganisms between hosts on resilience to the colonization of invaders after antibiotic-induced dysbiosis. We assess the functional consequences of dysbiosis using a mouse model of colonization resistance against Escherichia coli. Antibiotics caused stochastic loss of members of the microbiota, but the microbiotas of co-housed mice remained more similar to each other compared with the microbiotas among singly housed animals. Strikingly, co-housed mice maintained colonization resistance after treatment with antibiotics, whereas most singly housed mice were susceptible to E. coli. The ability to retain or share the commensal Klebsiella michiganensis, a member of the Enterobacteriaceae family, was sufficient for colonization resistance after treatment with antibiotics. K. michiganensis generally outcompeted E. coli in vitro, but in vivo administration of galactitol-a nutrient that supports the growth of only E. coli-to bi-colonized gnotobiotic mice abolished the colonization-resistance capacity of K. michiganensis against E. coli, supporting the idea that nutrient competition is the primary interaction mechanism. K. michiganensis also hampered colonization of the pathogen Salmonella, prolonging host survival. Our results address functional consequences of the stochastic effects of microbiota perturbations, whereby microbial transmission through host interactions can facilitate reacquisition of beneficial commensals, minimizing the negative impact of antibiotics.


Assuntos
Disbiose/microbiologia , Microbioma Gastrointestinal/fisiologia , Klebsiella/fisiologia , Interações Microbianas , Simbiose/fisiologia , Animais , Antibacterianos/farmacologia , Bacteroidetes/classificação , Bacteroidetes/isolamento & purificação , Ciprofloxacino/farmacologia , Contagem de Colônia Microbiana , Disbiose/induzido quimicamente , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/patogenicidade , Firmicutes/classificação , Firmicutes/isolamento & purificação , Vida Livre de Germes , Klebsiella/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/crescimento & desenvolvimento , Salmonella typhimurium/patogenicidade , Estreptomicina/farmacologia , Verrucomicrobia/classificação , Verrucomicrobia/isolamento & purificação
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