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1.
Mol Genet Genomics ; 295(4): 891-909, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32189066

RESUMO

Wolbachia is an obligate intracellular Gram-negative alpha-proteobacterium that has diverse effects on reproduction of arthropod hosts, including cytoplasmic incompatibility, male killing, feminization, and parthenogenesis. Some of these effects have important potential for control of insect pests, including mosquitoes that vector pathogens of humans. In mosquitoes, and in most other arthropods, elimination of Wolbachia by antibiotic treatment has no effect on host survival and reverses the Wolbachia-associated phenotype. Elimination of Wolbachia strain wFol, which enables parthenogenetic reproduction of the Collembolan, Folsomia candida, would result in population extinction. However, F. candida adults remain viable and resume reproduction when antibiotics are removed, suggesting that wFol survives antibiotic treatment in a quiescent persister state similar to that induced by chromosomally encoded toxin-antitoxin (TA) modules in free-living bacteria. Computational approaches were used to document the presence of antitoxin genes upstream of Wolbachia RelE/ParE, Fic, and AbiEii toxin genes. Moreover, this analysis revealed that Wolbachia RatA toxin is encoded by a single copy gene associated with an ssrS noncoding RNA gene. Documentation of potentially functional TA modules expands our understanding of the metabolic capabilities of Wolbachia, and provides an explanation for variable and sometimes contradictory results of antibiotic treatments. The presence of chromosomal TA modules in Wolbachia genomes suggests that wFol, and potentially other strains of Wolbachia, can enter a quiescent persister state.


Assuntos
Partenogênese/genética , Reprodução/genética , Sistemas Toxina-Antitoxina/genética , Wolbachia/genética , Animais , Cromossomos Bacterianos/genética , Culicidae/microbiologia , DNA Topoisomerase IV/genética , Genoma Bacteriano/genética , Humanos , Masculino , Controle de Pragas , Simbiose/genética , Wolbachia/patogenicidade
2.
Ann Lab Med ; 40(1): 27-32, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31432636

RESUMO

BACKGROUND: Mutations in the quinolone resistance-determining regions (QRDRs) of Acinetobacter baumannii DNA gyrase (gyrA) and topoisomerase IV (parC) are linked to fluoroquinolone (FQ) resistance. We developed a mismatched PCR-restriction fragment length polymorphism (RFLP) assay to detect mutations in the gyrA and parC QRDRs associated with FQ resistance in A. baumannii. METHODS: Based on the conserved sequences of A. baumannii gyrA and parC, two primer sets were designed for mismatched PCR-RFLP to detect mutations in gyrA (codons 83 and 87) and parC (codons 80 and 84) by introducing an artificial restriction enzyme cleavage site into the PCR products. This assay was evaluated using 58 A. baumannii strains and 37 other Acinetobacter strains that have been identified by RNA polymerase ß-subunit gene sequence analysis. RESULTS: PCR amplification of gyrA and parC was successful for all A. baumannii strains. In 11 FQ -susceptible strains, the gyrA and parC PCR products were digested by the selected restriction enzymes at the site containing gyrA (codons 83 and 87) and parC (codons 80 and 84). PCR products from 47 FQ-resistant strains containing mutations in gyrA and parC were not digested by the restriction enzymes at the site containing the mutation. As for the non-baumannii Acinetobacter strains, although amplification products for gyrA were obtained for 28 strains, no parC amplification product was obtained for any strain. CONCLUSIONS: This assay specifically amplified gyrA and parC from A. baumannii and detected A. baumannii gyrA and parC mutations with FQ resistance.


Assuntos
Acinetobacter baumannii/genética , DNA Girase/genética , DNA Topoisomerase IV/genética , Farmacorresistência Bacteriana/genética , Reação em Cadeia da Polimerase/métodos , Acinetobacter baumannii/efeitos dos fármacos , Antibacterianos/farmacologia , Pareamento Incorreto de Bases , DNA Bacteriano/química , DNA Bacteriano/metabolismo , Fluoroquinolonas/farmacologia , Testes de Sensibilidade Microbiana , Mutação , Polimorfismo de Fragmento de Restrição , Análise de Sequência de DNA
3.
BMC Infect Dis ; 19(1): 898, 2019 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-31660876

RESUMO

BACKGROUND: Salmonella infection poses significant public health threat globally, especially in resource-limited countries. Emergence and spread of antibiotic resistant strains to fluoroquinolones have led to treatment failures and increased mortality in Salmonella infection. However, there is dearth of information regarding mechanisms of resistance to fluoroquinolones in Ghana. This study therefore sought to identify chromosomal mutations and plasmid-mediated resistance as possible mechanisms of fluoroquinolone resistance from clinical isolates in Ghana. METHODS: This was a retrospective study of archived isolates biobanked at Kumasi Centre for Collaborative Research in Tropical Medicine, Ghana. Isolates were obtained from blood, stool and oropharynx samples at two hospitals, between May, 2016 and January, 2018. Salmonella identification was done using standard microbiological protocols and antibiotic susceptibility testing performed by Kirby-Bauer disc diffusion method. Isolates with intermediate susceptibility and/or resistance to nalidixic acid and/or ciprofloxacin were selected and examined for chromosomal mutations by Sanger sequencing and plasmid-mediated resistance by PCR. RESULTS: Of 133 biobanked isolates cultured, 68 (51.1%) and 16 (12%) were identified as Salmonella Typhi and non-typhoidal Salmonella (NTS), respectively. Sequence analysis of gyrA gene revealed the presence of 5 different nonsynonymous mutations, with the most frequent mutation (Ile203Ser) occurring in 12 out of 13 isolates tested. Gyrase B (gyrB) gene had 1 nonsynonymous mutation in 3 out of 13 isolates, substituting phenylalanine with leucine at codon 601 (Phe601Leu). No mutation was observed in parC and parE genes. Two NTS isolates were found to harbour qnrS plasmid-mediated resistant gene of molecular size 550 bp with high ciprofloxacin MIC of 0.5 µg/ml. CONCLUSION: This study reports for the first time in Ghana plasmid-mediated fluoroquinolone resistant gene qnrS in Salmonella clinical isolates. Nonsynonymous mutations of gyrA and gyrB genes likely to confer Salmonella reduced susceptibility to ciprofloxacin were also reported.


Assuntos
Antibacterianos/efeitos adversos , Antibacterianos/uso terapêutico , Farmacorresistência Bacteriana/genética , Fluoroquinolonas/efeitos adversos , Fluoroquinolonas/uso terapêutico , Genes Bacterianos/genética , Plasmídeos/metabolismo , Infecções por Salmonella/tratamento farmacológico , Salmonella enterica/genética , Adolescente , Pré-Escolar , Ciprofloxacino/efeitos adversos , Ciprofloxacino/uso terapêutico , DNA Girase/genética , DNA Topoisomerase IV/genética , Testes de Sensibilidade a Antimicrobianos por Disco-Difusão , Feminino , Gana , Humanos , Masculino , Mutação , Estudos Retrospectivos , Salmonella enterica/isolamento & purificação , Adulto Jovem
4.
Nat Commun ; 10(1): 4828, 2019 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-31645551

RESUMO

Shigella sonnei increasingly dominates the international epidemiological landscape of shigellosis. Treatment options for S. sonnei are dwindling due to resistance to several key antimicrobials, including the fluoroquinolones. Here we analyse nearly 400 S. sonnei whole genome sequences from both endemic and non-endemic regions to delineate the evolutionary history of the recently emergent fluoroquinolone-resistant S. sonnei. We reaffirm that extant resistant organisms belong to a single clonal expansion event. Our results indicate that sequential accumulation of defining mutations (gyrA-S83L, parC-S80I, and gyrA-D87G) led to the emergence of the fluoroquinolone-resistant S. sonnei population around 2007 in South Asia. This clone was then transmitted globally, resulting in establishments in Southeast Asia and Europe. Mutation analysis suggests that the clone became dominant through enhanced adaptation to oxidative stress. Experimental evolution reveals that under fluoroquinolone exposure in vitro, resistant S. sonnei develops further intolerance to the antimicrobial while the susceptible counterpart fails to attain complete resistance.


Assuntos
Farmacorresistência Bacteriana/genética , Disenteria Bacilar/microbiologia , Fluoroquinolonas , Genoma Bacteriano/genética , Shigella sonnei/genética , Antibacterianos/uso terapêutico , Ásia Sudeste/epidemiologia , Ásia Ocidental/epidemiologia , Teorema de Bayes , Ciprofloxacino/uso terapêutico , DNA Girase/genética , DNA Topoisomerase IV/genética , Evolução Molecular Direcionada , Disenteria Bacilar/tratamento farmacológico , Disenteria Bacilar/epidemiologia , Europa (Continente)/epidemiologia , Evolução Molecular , Humanos , Epidemiologia Molecular , Mutação , Filogenia , Polimorfismo de Nucleotídeo Único , Shigella sonnei/fisiologia
5.
J Microbiol ; 57(12): 1056-1064, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31555989

RESUMO

We employed a stepwise selection model for investigating the dynamics of antibiotic-resistant variants in Escherichia coli K-12 treated with increasing concentrations of ciprofloxacin (CIP). Firstly, we used Sanger sequencing to screen the variations in the fluoquinolone target genes, then, employed Illumina NGS sequencing for amplicons targeted regions with variations. The results demonstrated that variations G81C in gyrA and K276N and K277L in parC are standing resistance variations (SRVs), while S83A and S83L in gyrA and G78C in parC were emerging resistance variations (ERVs). The variants containing SRVs and/or ERVs were selected successively based on their sensitivities to CIP. Variant strain 1, containing substitution G81C in gyrA, was immediately selected following ciprofloxacin exposure, with obvious increases in the parC SRV, and parC and gyrA ERV allele frequencies. Variant strain 2, containing the SRVs, then dominated the population following a 20× increase in ciprofloxacin concentration, with other associated allele frequencies also elevated. Variant strains 3 and 4, containing ERVs in gyrA and parC, respectively, were then selected at 40× and 160× antibiotic concentrations. Two variants, strains 5 and 6, generated in the selection procedure, were lost because of higher fitness costs or a lower level of resistance compared with variants 3 and 4. For the second induction, all variations/indels were already present as SRVs and selected out step by step at different passages. Whatever the first induction or second induction, our results confirmed the soft selective sweep hypothesis and provided critical information for guiding clinical treatment of pathogens containing SRVs.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana/fisiologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/fisiologia , Fluoroquinolonas/farmacologia , Estresse Fisiológico/fisiologia , Ciprofloxacino/farmacologia , DNA Girase/genética , DNA Topoisomerase IV/genética , DNA Bacteriano/genética , Farmacorresistência Bacteriana/genética , Escherichia coli/genética , Escherichia coli K12/efeitos dos fármacos , Escherichia coli K12/genética , Genes Bacterianos/genética , Ensaios de Triagem em Larga Escala , Testes de Sensibilidade Microbiana , Mutação , Sequenciamento Completo do Genoma
6.
Med Hypotheses ; 131: 109305, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31443754

RESUMO

Infections due to resistant bacteria are the life-threatening and leading cause of mortality worldwide. The current therapy for bacterial infections includes treatment with various drugs and antibiotics. The misuse and over usage of these antibiotics leads to bacterial resistance. There are several mechanisms by which bacteria exhibit resistance to some antibiotics. These include drug inactivation or modification, elimination of antibiotics through efflux pumps, drug target alteration, and modification of metabolic pathway. However, it is difficult to treat infections caused by resistant bacteria by conventional existing therapy. In the present study binding affinities of some glitazones against ParE and MurE bacterial enzymes are investigated by in silico methods. As evident by extra-precision docking and binding free energy calculation (MM-GBSA) results, rivoglitazone exhibited higher binding affinity against both ParE and MurE enzymes compared to all other selected compounds. Further molecular dynamic (MD) simulations were performed to validate the stability of rivoglitazone/4MOT and rivoglitazone/4C13 complexes and to get insight into the binding mode of inhibitor. Thus, we hypothesize that structural modifications of the rivoglitazone scaffold can be useful for the development of an effective antibacterial agent.


Assuntos
Antibacterianos/farmacologia , Infecções Bacterianas/tratamento farmacológico , DNA Topoisomerase IV/antagonistas & inibidores , Peptídeo Sintases/antagonistas & inibidores , Tiazolidinedionas/farmacologia , Tiazolidinas/farmacologia , Antibacterianos/química , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , DNA Topoisomerase IV/química , Resistência Microbiana a Medicamentos , Humanos , Ligação de Hidrogênio , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Peptídeo Sintases/química , Relação Estrutura-Atividade , Tiazolidinedionas/química , Tiazolidinas/química
7.
Proc Natl Acad Sci U S A ; 116(29): 14740-14748, 2019 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-31262826

RESUMO

Global growth in antibiotic resistance is a major social problem. A high level of resistance to fluoroquinolones requires the concurrent presence of at least 3 mutations in the target proteins-2 in DNA gyrase (GyrA) and 1 in topoisomerase IV (ParC), which occur in a stepwise manner. In the Escherichia coli chromosome, the gyrA and parC loci are positioned about 1 Mb away from each other. Here we show that the 3 fluoroquinolone resistance mutations are tightly associated genetically in naturally occurring strains. In the latest pandemic uropathogenic and multidrug-resistant E. coli clonal group ST1193, the mutant variants of gyrA and parC were acquired not by a typical gradual, stepwise evolution but all at once. This happened as part of 11 simultaneous homologous recombination events involving 2 phylogenetically distant strains of E. coli, from an uropathogenic clonal complex ST14 and fluoroquinolone-resistant ST10. The gene exchanges swapped regions between 0.5 and 139 Kb in length (183 Kb total) spread along 976 Kb of chromosomal DNA around and between gyrA and parC loci. As a result, all 3 fluoroquinolone resistance mutations in GyrA and ParC have simultaneously appeared in ST1193. Based on molecular clock estimates, this potentially happened as recently as <12 y ago. Thus, naturally occurring homologous recombination events between 2 strains can involve numerous chromosomal gene locations simultaneously, resulting in the transfer of distant but tightly associated genetic mutations and emergence of a both highly pathogenic and antibiotic-resistant strain with a rapid global spread capability.


Assuntos
Farmacorresistência Bacteriana Múltipla/genética , Fluoroquinolonas/farmacologia , Loci Gênicos , Recombinação Homóloga , Escherichia coli Uropatogênica/genética , Cromossomos Bacterianos/genética , DNA Girase/genética , DNA Topoisomerase IV/genética , Proteínas de Escherichia coli/genética , Fluoroquinolonas/uso terapêutico , Transferência Genética Horizontal , Humanos , Testes de Sensibilidade Microbiana , Mutação , Pandemias , Infecções Urinárias/tratamento farmacológico , Infecções Urinárias/epidemiologia , Infecções Urinárias/microbiologia , Escherichia coli Uropatogênica/patogenicidade
8.
Eur J Med Chem ; 179: 576-590, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31279292

RESUMO

A series of novel fluoroquinolone-Safirinium dye hybrids was synthesized by means of tandem Mannich-electrophilic amination reactions from profluorophoric isoxazolones and antibiotics bearing a secondary amino group at position 7 of the quinoline ring. The obtained fluorescent spiro fused conjugates incorporating quaternary nitrogen atoms were characterized by 1H NMR, IR, MS, and elemental analysis. All the synthetic analogues (3a-h and 4a-h) were evaluated for their in vitro antimicrobial, bactericidal, and antibiofilm activities against a panel of Gram positive and Gram-negative pathogenic bacteria. The most active Safirinium Q derivatives of lomefloxacin (4d) and ciprofloxacin (4e) exhibited molar-based antibacterial activities comparable to the unmodified drugs and displayed considerable inhibitory potencies in E. coli DNA gyrase supercoiling assays with IC50 values in the low micromolar range. Zwiterionic hybrids were noticeably less lipophilic than the parent quinolones in micellar electrokinetic chromatography (MECK) experiments. The tests performed in the presence of phenylalanine-arginine ß-naphthylamide (PAßN) or carbonyl cyanide m-chlorophenylhydrazone (CCCP) revealed that the conjugates are to some extent subject to bacterial efflux and cellular accumulation, respectively. Moreover, the hybrids did not exhibit notable cytotoxicity towards the HEK 293 control cell line and demonstrated low propensity for resistance development, as exemplified for compounds 3g and 4b. Finally, molecular docking experiments revealed that the synthesized compounds were able to bind in the fluoroquinolone-binding mode at S. aureus DNA gyrase and S. pneumoniae topoisomerase IV active sites.


Assuntos
Antibacterianos/farmacologia , Inibidores Enzimáticos/farmacologia , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Compostos de Amônio Quaternário/farmacologia , Quinolonas/farmacologia , Antibacterianos/síntese química , Antibacterianos/química , DNA Girase/metabolismo , DNA Topoisomerase IV/antagonistas & inibidores , DNA Topoisomerase IV/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Bactérias Gram-Negativas/metabolismo , Bactérias Gram-Positivas/metabolismo , Células HEK293 , Humanos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Compostos de Amônio Quaternário/síntese química , Compostos de Amônio Quaternário/química , Quinolonas/química , Relação Estrutura-Atividade
9.
Eur J Med Chem ; 179: 166-181, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31254919

RESUMO

This work did a new exploration towards aminothiazolquinolone oximes as potentially multi-targeting antimicrobial agents. A class of novel hybrids of quinolone, aminothiazole, piperazine and oxime fragments were designed for the first time, conveniently synthesized as well as characterized by 1H NMR, 13C NMR and HRMS spectra. Biological activity showed that some of the synthesized compounds exhibited good antimicrobial activities in comparison with the reference drugs. Especially, O-methyl oxime derivative 10b displayed excellent inhibitory efficacy against MRSA and S. aureus 25923 with MIC values of 0.009 and 0.017 mM, respectively. Further studies indicated that the highly active compound 10b showed low toxicity toward BEAS-2B and A549 cell lines and no obvious propensity to trigger the development of bacterial resistance. Quantum chemical studies have also been conducted and rationally explained the structural features essential for activity. The preliminarily mechanism exploration revealed that compound 10b could not only exert efficient membrane permeability by interfering with the integrity of cells, bind with topoisomerase IV-DNA complex through hydrogen bonds and π-π stacking, but also form a steady biosupramolecular complex by intercalating into DNA to exert the efficient antibacterial activity. The supramolecular interaction between compound 10b and human serum albumin (HSA) was a static quenching, and the binding process was spontaneous, where hydrogen bonds and van der Waals force played vital roles in the supramolecular transportation of the active compound 10b by HSA.


Assuntos
Antibacterianos/farmacologia , Desenho de Fármacos , Oximas/farmacologia , Quinolonas/farmacologia , Antibacterianos/síntese química , Antibacterianos/química , DNA/efeitos dos fármacos , DNA Topoisomerase IV/antagonistas & inibidores , DNA Topoisomerase IV/metabolismo , Relação Dose-Resposta a Droga , Humanos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Oximas/síntese química , Oximas/química , Teoria Quântica , Quinolonas/síntese química , Quinolonas/química , Albumina Sérica Humana/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Relação Estrutura-Atividade
10.
J Med Microbiol ; 68(8): 1227-1232, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31215858

RESUMO

PURPOSE: Haemophilus influenzae strains with low susceptibility to quinolones have recently emerged in the paediatric field in Japan. These strains are judged as 'susceptible' in routine susceptibility tests, although they may survive after quinolone treatment. Therefore, we aimed to construct a simple and cost-effective identification method for low-susceptibility strains using disc diffusion assays. METHODOLOGY: A total of 33 H. influenzae clinical isolates and a control strain were used. For the disc diffusion assay, levofloxacin, norfloxacin, nalidixic acid and pipemidic acid were employed. Correlations between the inhibition zone diameter and amino acid substitutions were evaluated. RESULTS: All of the tested strains formed clear inhibition zones on both levofloxacin and norfloxacin discs. By contrast, none of the low-susceptibility strains showed inhibition zones against nalidixic acid, while the low-susceptibility strains with amino acid substitutions in both GyrA and ParC did not show inhibition zones against pipemidic acid discs, indicating that low-susceptibility strains can be detected with high sensitivity and specificity by the presence or absence of inhibition zones for earlier quinolones. CONCLUSION: A disc diffusion test combining results from nalidixic acid and pipemidic acid can detect low-susceptibility strains harbouring amino acid substitutions without the need for genetic analysis. This test can help reduce inappropriate and unnecessary fluoroquinolone use.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Infecções por Haemophilus/microbiologia , Haemophilus influenzae/efeitos dos fármacos , Quinolonas/farmacologia , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Criança , DNA Girase/genética , DNA Topoisomerase IV/genética , Testes de Sensibilidade a Antimicrobianos por Disco-Difusão , Farmacorresistência Bacteriana/genética , Haemophilus influenzae/genética , Haemophilus influenzae/isolamento & purificação , Humanos , Japão , Ácido Nalidíxico/farmacologia , Ácido Pipemídico/farmacologia , Sensibilidade e Especificidade
11.
mBio ; 10(3)2019 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-31186328

RESUMO

The emergence of fluoroquinolone resistance in nosocomial pathogens has restricted the clinical efficacy of this antibiotic class. In Acinetobacter baumannii, the majority of clinical isolates now show high-level resistance due to mutations in gyrA (DNA gyrase) and parC (topoisomerase IV [topo IV]). To investigate the molecular basis for fluoroquinolone resistance, an exhaustive mutation analysis was performed in both drug-sensitive and -resistant strains to identify loci that alter ciprofloxacin sensitivity. To this end, parallel fitness tests of over 60,000 unique insertion mutations were performed in strains with various alleles in genes encoding the drug targets. The spectra of mutations that altered drug sensitivity were found to be similar in the drug-sensitive and gyrA parC double-mutant backgrounds, having resistance alleles in both genes. In contrast, the introduction of a single gyrA resistance allele, resulting in preferential poisoning of topo IV by ciprofloxacin, led to extreme alterations in the insertion mutation fitness landscape. The distinguishing feature of preferential topo IV poisoning was enhanced induction of DNA synthesis in the region of two endogenous prophages, with DNA synthesis associated with excision and circularization of the phages. Induction of the selective DNA synthesis in the gyrA background was also linked to heightened prophage gene transcription and enhanced activation of the mutagenic SOS response relative to that observed in either the wild-type (WT) or gyrA parC double mutant. Therefore, the accumulation of mutations that result in the stepwise evolution of high ciprofloxacin resistance is tightly connected to modulation of the SOS response and endogenous prophage DNA synthesis.IMPORTANCE Fluoroquinolones have been extremely successful antibiotics due to their ability to target multiple bacterial enzymes critical to DNA replication, the topoisomerases DNA gyrase and topo IV. Unfortunately, mutations lowering drug affinity for both enzymes are now widespread, rendering these drugs ineffective for many pathogens. To undermine this form of resistance, we examined how bacteria with target alterations differentially cope with fluoroquinolone exposures. We studied this problem in the nosocomial pathogen A. baumannii, which causes drug-resistant life-threatening infections. Employing genome-wide approaches, we uncovered numerous pathways that could be exploited to raise fluoroquinolone sensitivity independently of target alteration. Remarkably, fluoroquinolone targeting of topo IV in specific mutants caused dramatic hyperinduction of prophage replication and enhanced the mutagenic DNA damage response, but these responses were muted in strains with DNA gyrase as the primary target. This work demonstrates that resistance evolution via target modification can profoundly modulate the antibiotic stress response, revealing potential resistance-associated liabilities.


Assuntos
Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/genética , Antibacterianos/farmacologia , Ciprofloxacino/farmacologia , Farmacorresistência Bacteriana/genética , Prófagos/fisiologia , Resposta SOS em Genética , Acinetobacter baumannii/virologia , Alelos , Dano ao DNA , DNA Girase/genética , DNA Topoisomerase IV/genética , Perfilação da Expressão Gênica , Testes de Sensibilidade Microbiana , Mutação , Fenótipo , Prófagos/genética , Replicação Viral , Sequenciamento Completo do Genoma
12.
Pol J Microbiol ; 68(1): 59-69, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31050254

RESUMO

The widespread of infections caused by methicillin-resistant Staphylococcus aureus (MRSA), has necessitated the search for alternative therapies; introduction of new agents being a suggestion. This study compares the in vitro and in vivo activities of zabofloxacin, a novel fluoroquinolone, with moxifloxacin, levofloxacin and ciprofloxacin against clinical isolates of MRSA from patients hospitalized in the Alexandria Main University hospital; a tertiary hospital in Alexandria, Egypt, where zabofloxacin has not been yet introduced. The strains tested showed the highest percentage of susceptibility to zabofloxacin (61.2%) among the tested fluoroquinolones with the most effective MIC50 and MIC90 (0.25 and 2 µg/ml, respectively). Time-kill curve analysis revealed a rapid bactericidal activity of zabofloxacin after 6 h of incubation with a quinolone-resistant isolate and complete killing when tested against a quinolone-sensitive isolate with inhibition of regrowth in both cases. PCR amplification and sequencing of QRDRs in selected strains revealed the following amino acid substitutions: Ser-84→Leu in GyrA, Ser-80→Phe in GrlA and Pro-451→Ser in GrlB. The in vivo studies demonstrated that zabofloxacin possessed the most potent protective effect against systemic infection in mice (ED50: 29.05 mg/kg) with lowest count in the dissected lungs (3.66 log10 CFU/ml). The histopathological examination of lung specimens of mice treated with zabofloxacin displayed least congestion, inflammation, oedema and necrosis with clear alveolar spaces and normal vessels. In conclusion, zabofloxacin was proved to possess high in vitro and in vivo efficacy encompassing its comparators and could be considered as a possible candidate for the treatment of infections caused by MRSA. To our knowledge, this is the first study evaluating the in vitro and in vivo activity of zabofloxacin against Egyptian MRSA clinical isolates.The widespread of infections caused by methicillin-resistant Staphylococcus aureus (MRSA), has necessitated the search for alternative therapies; introduction of new agents being a suggestion. This study compares the in vitro and in vivo activities of zabofloxacin, a novel fluoroquinolone, with moxifloxacin, levofloxacin and ciprofloxacin against clinical isolates of MRSA from patients hospitalized in the Alexandria Main University hospital; a tertiary hospital in Alexandria, Egypt, where zabofloxacin has not been yet introduced. The strains tested showed the highest percentage of susceptibility to zabofloxacin (61.2%) among the tested fluoroquinolones with the most effective MIC50 and MIC90 (0.25 and 2 µg/ml, respectively). Time-kill curve analysis revealed a rapid bactericidal activity of zabofloxacin after 6 h of incubation with a quinolone-resistant isolate and complete killing when tested against a quinolone-sensitive isolate with inhibition of regrowth in both cases. PCR amplification and sequencing of QRDRs in selected strains revealed the following amino acid substitutions: Ser-84→Leu in GyrA, Ser-80→Phe in GrlA and Pro-451→Ser in GrlB. The in vivo studies demonstrated that zabofloxacin possessed the most potent protective effect against systemic infection in mice (ED50: 29.05 mg/kg) with lowest count in the dissected lungs (3.66 log10 CFU/ml). The histopathological examination of lung specimens of mice treated with zabofloxacin displayed least congestion, inflammation, oedema and necrosis with clear alveolar spaces and normal vessels. In conclusion, zabofloxacin was proved to possess high in vitro and in vivo efficacy encompassing its comparators and could be considered as a possible candidate for the treatment of infections caused by MRSA. To our knowledge, this is the first study evaluating the in vitro and in vivo activity of zabofloxacin against Egyptian MRSA clinical isolates.


Assuntos
Antibacterianos/farmacologia , Fluoroquinolonas/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Animais , Carga Bacteriana/efeitos dos fármacos , Ciprofloxacino/farmacologia , DNA Girase/efeitos dos fármacos , DNA Girase/genética , DNA Topoisomerase IV/efeitos dos fármacos , DNA Topoisomerase IV/genética , Egito , Hospitais Universitários , Humanos , Levofloxacino/farmacologia , Pulmão/microbiologia , Pulmão/patologia , Masculino , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Camundongos , Testes de Sensibilidade Microbiana , Moxifloxacina/farmacologia , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia
13.
J Med Microbiol ; 68(6): 903-909, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31090535

RESUMO

PURPOSE: To prevent severe invasive pneumococcal infection, pneumococcal conjugate vaccines (PCVs) were introduced in Japan in 2010, and in 2013 a pneumococcal 13-valent conjugate vaccine (PCV13) was included in the routine vaccination schedule for infants. In this study, we analysed the antimicrobial susceptibilities and capsular types of pneumococci isolated from non-invasive patient sites from 2007 to 2016 to assess the impact of the introduction of PCV13. METHODOLOGY: A total of 618 pneumococcal isolates collected at a teaching hospital from 2007 to 2016 were used. These isolates were characterized by capsular typing, multilocus sequence typing and antimicrobial susceptibility testing. RESULTS: Capsular typing indicated that, after the introduction of the PCV, the proportion of PCV13 serotypes decreased (P<0.01), while non-PCV13 serotypes became diverse. In particular, increases in 22 F, 15A and 23A were noted among non-PCV13 serotypes. Regarding antimicrobial susceptibility, the non-susceptibility rate to penicillin of pneumococci that showed higher minimum inhibitory concentrations (MICs) than the susceptibility breakpoint decreased, and pneumococci tended to become susceptible. However, all type 23A pneumococci and 77.8  % of type 15A pneumococci showed the reverse trend, with low susceptibility to penicillin. Furthermore, all 15A and 23A isolates had macrolide resistance genes. CONCLUSION: These data suggest that PCVs can prevent infections caused by PCV serotypes. However, since non-PCV13-type pneumococci, in particular 15A and 23A, which have acquired multidrug resistance, have already emerged over time, the development of a novel vaccine targeting a broader spectrum of pneumococci is warranted.


Assuntos
Infecções Pneumocócicas/prevenção & controle , Vacinas Pneumocócicas/imunologia , Streptococcus pneumoniae/imunologia , Antibacterianos/farmacologia , Cápsulas Bacterianas/imunologia , Técnicas de Tipagem Bacteriana , Portador Sadio , DNA Girase/genética , DNA Topoisomerase IV/genética , Hospitais de Ensino , Humanos , Japão/epidemiologia , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus , Penicilinas/farmacologia , Infecções Pneumocócicas/epidemiologia , Infecções Pneumocócicas/microbiologia , Sorogrupo , Sorotipagem , Streptococcus pneumoniae/genética , Vacinas Conjugadas/imunologia
14.
J Prev Med Hyg ; 60(1): E25-E30, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31041407

RESUMO

Background: Fluoroquinolone resistant Escherichia coli isolates have become an important challenge in healthcare settings in Iran. In this study, we have determined Fluoroquinolone resistant E. coli isolates (from both outpatients and inpatients) and evaluated mutations of gyrA and parC within the quinolone resistance-determining regions (QRDR) of these clinical isolates. Materials and methods: Clinical isolates were recovered from the urine sample of patients with urinary tract infections admitted at Alzahra hospital, Iran, between September and February 2013. We assessed antimicrobial susceptibility of all isolates and determined mutations in QRDR of gyrA and parC genes from 13 fluoroquinolone-resistant isolates by DNA sequencing. Results: A total of 135 E. coli strains were obtained from 135 patients (91 outpatients and 44 inpatients). The resistance rate of fluoroquinolones (Ciprofloxacin, Norfloxacin and Ofloxacin) among our strains was 45.2%. Two E. coli isolates were shown just a single mutation, but other isolates possessed 2-5 mutations in gyrA and parC genes. Mutations in the QRDR regions of gyrA were at positions Ser83 and Asp87 and parC at positions Ser80, Glu84, Gly78. Conclusions: Ciprofloxacin is the most common antimicrobial agent used for treating urinary tract infections (UTIs) in healthcare settings in Iran. Accumulation of different substitutions in the QRDR regions of gyrA and parC confers high-level resistance of fluoroquinolones in clinical isolates.


Assuntos
Antibacterianos , Infecções por Escherichia coli/epidemiologia , Fluoroquinolonas , Infecções Urinárias/epidemiologia , Adulto , Assistência Ambulatorial , Infecções Relacionadas a Cateter/epidemiologia , Infecções Relacionadas a Cateter/microbiologia , Criança , DNA Girase/genética , DNA Topoisomerase IV/genética , Farmacorresistência Bacteriana/genética , Escherichia coli/genética , Escherichia coli/fisiologia , Infecções por Escherichia coli/microbiologia , Feminino , Hospitalização , Humanos , Recém-Nascido , Irã (Geográfico)/epidemiologia , Masculino , Epidemiologia Molecular , Complicações Pós-Operatórias/epidemiologia , Complicações Pós-Operatórias/microbiologia , Prevalência , Procedimentos Cirúrgicos Operatórios , Cateterismo Urinário , Infecções Urinárias/microbiologia
15.
BMC Microbiol ; 19(1): 76, 2019 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-30961546

RESUMO

BACKGROUND: Phenotypic fluoroquinolone resistance was first reported in Western Kenya in 2009 and later in Coastal Kenya and Nairobi. Until recently gonococcal fluoroquinolone resistance mechanisms in Kenya had not been elucidated. The aim of this paper is to analyze mutations in both gyrA and parC responsible for elevated fluoroquinolone Minimum Inhibitory Concentrations (MICs) in Neisseria gonorrhoeae (GC) isolated from heterosexual individuals from different locations in Kenya between 2013 and 2017. METHODS: Antimicrobial Susceptibility Tests were done on 84 GC in an ongoing Sexually Transmitted Infections (STI) surveillance program. Of the 84 isolates, 22 resistant to two or more classes of antimicrobials were chosen for analysis. Antimicrobial susceptibility tests were done using E-test (BioMerieux) and the results were interpreted with reference to European Committee on Antimicrobial Susceptibility Testing (EUCAST) standards. The isolates were sub-cultured, and whole genomes were sequenced using Illumina platform. Reads were assembled de novo using Velvet, and mutations in the GC Quinolone Resistant Determining Regions identified using Bioedit sequence alignment editor. Single Nucleotide Polymorphism based phylogeny was inferred using RaxML. RESULTS: Double GyrA amino acid substitutions; S91F and D95G/D95A were identified in 20 isolates. Of these 20 isolates, 14 had an additional E91G ParC substitution and significantly higher ciprofloxacin MICs (p = 0.0044*). On the contrary, norfloxacin MICs of isolates expressing both GyrA and ParC QRDR amino acid changes were not significantly high (p = 0.82) compared to MICs of isolates expressing GyrA substitutions alone. No single GyrA substitution was found in the analyzed isolates, and no isolate contained a ParC substitution without the simultaneous presence of double GyrA substitutions. Maximum likelihood tree clustered the 22 isolates into 6 distinct clades. CONCLUSION: Simultaneous presence of amino acid substitutions in ParC and GyrA has been reported to increase gonococcal fluoroquinolone resistance from different regions in the world. Our findings indicate that GyrA S91F, D95G/D95A and ParC E91G amino acid substitutions mediate high fluoroquinolone resistance in the analyzed Kenyan GC.


Assuntos
Antibacterianos/farmacologia , DNA Girase/genética , DNA Topoisomerase IV/genética , Fluoroquinolonas/farmacologia , Neisseria gonorrhoeae/efeitos dos fármacos , Neisseria gonorrhoeae/genética , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Monitoramento Epidemiológico , Feminino , Gonorreia/microbiologia , Humanos , Quênia , Masculino , Testes de Sensibilidade Microbiana , Mutação , Estudos Retrospectivos
16.
Nucleic Acids Res ; 47(10): 5231-5242, 2019 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-30957856

RESUMO

DNA topoisomerases play essential roles in chromosome organization and replication. Most bacteria possess multiple topoisomerases which have specialized functions in the control of DNA supercoiling or in DNA catenation/decatenation during recombination and chromosome segregation. DNA topoisomerase I is required for the relaxation of negatively supercoiled DNA behind the transcribing RNA polymerase. Conflicting results have been reported on the essentiality of the topA gene encoding topoisomerase I in the model bacterium Bacillus subtilis. In this work, we have studied the requirement for topoisomerase I in B. subtilis. All stable topA mutants carried different chromosomal amplifications of the genomic region encompassing the parEC operon encoding topoisomerase IV. Using a fluorescent amplification reporter system we observed that each individual topA mutant had acquired such an amplification. Eventually, the amplifications were replaced by a point mutation in the parEC promoter region which resulted in a fivefold increase of parEC expression. In this strain both type I topoisomerases, encoded by topA and topB, were dispensable. Our results demonstrate that topoisomerase IV at increased expression is necessary and sufficient to take over the function of type 1A topoisomerases.


Assuntos
Bacillus subtilis/enzimologia , Bacillus subtilis/genética , DNA Topoisomerase IV/metabolismo , DNA Topoisomerases Tipo I/metabolismo , Proteínas de Bactérias/metabolismo , Cromossomos Bacterianos , Replicação do DNA , DNA Bacteriano/genética , DNA Super-Helicoidal/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Genoma Bacteriano , Mutação , Fenótipo , Mutação Puntual , Regiões Promotoras Genéticas
17.
PLoS One ; 14(3): e0214304, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30913237

RESUMO

Escherichia coli is a normal inhabitant of the intestinal microbiota of chickens, a small proportion of which may be avian pathogenic E. coli (APEC) or potential extraintestinal pathogenic E. coli (ExPEC), capable of causing disease in humans. These E. coli may also be resistant to antimicrobials of critical importance in human or veterinary health. This study aims to 1) determine the prevalence of antimicrobial resistance (AMR) and resistance genes, multidrug resistance (MDR), chromosomal mechanisms of quinolone-resistance and virulence profiles of E. coli isolated from healthy chicken farms in the region of Dakar, Senegal, 2) investigate the spread of third-generation cephalosporins (3GC) resistance in E. coli isolated from healthy chicken farms with respect to virulence and resistance genes, serogroups, Pulsed-Field Gel Electrophoresis (PFGE), phylogenetic groups, plasmid types and transferability and 3) determine whether nonsusceptibility against 3GC on farms could be linked to risk factors. More than 68% of isolates from environmental faecal and drinking water samples, carcasses and carcass washes collected on 32 healthy chicken farms were multidrug resistant (MDR), resistance to antimicrobials critical in human health (3GC or ciprofloxacin) being found in all types of samples. Ciprofloxacin resistance was due to mutations in the gyrA and parC genes, 95% of tested farms harboring isolates carrying three mutations, in gyrA (Ser83Ile and Asp87Asn) and parC (Ser80Ile). Nine of the 32 farms (28.1%) demonstrated the presence of one or more 3GC-nonsusceptible indicator isolates but none of the potential risk factors were significantly associated with this presence on farms. Following ceftriaxone enrichment, presumptive extended-spectrum beta-lactamase/AmpC-beta-lactamase (ESBL/AmpC)-producer isolates were found in 17 of the 32 farms. 3GC resistance was mediated by blaCMY-2 or blaCTX-M genes, blaCTX-M being of genotypes blaCTX-M-1, blaCTX-M-8 and for the first time in chickens in Senegal, the genotype blaCTX-M-15. Clonally related ESBL/AmpC-producer isolates were found on different farms. In addition, blaCTX-M genes were identified on replicon plasmids I1 and K/B and blaCMY-2 on K/B, I1 and B/O. These plasmids were found in isolates of different clusters. In addition, 18 isolates, some of which were ESBL/AmpC-producers, were defined as potential human ExPEC. In conclusion, E. coli isolates potentially pathogenic for humans and demonstrating MDR, with resistance expressed against antimicrobials of critical importance in human health were found in healthy chickens in Senegal. Our results suggest that both clonal spreading and horizontal gene transfer play a role in the spread of 3GC-resistance and that chickens in Senegal could be a reservoir for AMR and ExPEC for humans. These results highlight the importance of raising awareness about compliance with biosecurity measures and prudent use of antimicrobials.


Assuntos
Antibacterianos/farmacologia , Cefalosporinas/farmacologia , Galinhas/microbiologia , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Escherichia coli/isolamento & purificação , Animais , Proteínas de Bactérias/genética , DNA Girase/genética , DNA Topoisomerase IV/genética , Farmacorresistência Bacteriana Múltipla/genética , Escherichia coli/metabolismo , Escherichia coli/patogenicidade , Fazendas , Testes de Sensibilidade Microbiana , Mutação , Doenças das Aves Domésticas/diagnóstico , Doenças das Aves Domésticas/epidemiologia , Doenças das Aves Domésticas/microbiologia , Prevalência , Fatores de Risco , Senegal/epidemiologia , Virulência/genética , beta-Lactamases/genética , beta-Lactamases/metabolismo
18.
Comput Biol Chem ; 79: 177-184, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30836319

RESUMO

With the aim of reducing the adverse effects of fluoroquinolones in the environment, a complete design and screening system for the low biological enrichment and high photodegradabilities of 29 fluoroquinolones was established through a three-dimensional quantitative structure-activity relationship (3D-QSAR) model and molecular docking. The interaction mechanisms of the fluoroquinolones with Gram-negative bacteria (DNA gyrase in Escherichia coli) and Gram-positive bacteria (Topoisomerase IV in Staphylococcus aureus) were also evaluated. Consequently, the 3D-QSAR model showed that the 3- and 18-positions of the fluoroquinolones strongly affected their biological enrichment, and that the introduction of electropositive or hydrophobic groups at these positions reduced the logarithm of the octanol-water partition coefficient. Using nadifloxacin as a template, 23 derivatives with lower biological enrichment than nadifloxacin (decreased by 30.12%-94.18%) were designed. Meanwhile, the photodegradabilities of 15 derivatives were increased compared with nadifloxacin. Finally, the further screening by molecular docking of nadifloxacin and the above 15 derivatives with DNA gyrase and Topoisomerase IV showed that 13 of the derivatives had lower biological enrichment (decreased by 0.30%-16.76%) than nadifloxacin in the bacteria.


Assuntos
DNA Girase/química , DNA Topoisomerase IV/química , Fluoroquinolonas/química , Simulação de Acoplamento Molecular , Relação Quantitativa Estrutura-Atividade , DNA Girase/metabolismo , DNA Topoisomerase IV/metabolismo , Escherichia coli/enzimologia , Fluoroquinolonas/metabolismo , Estrutura Molecular , Staphylococcus aureus/enzimologia
19.
Molecules ; 24(5)2019 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-30862066

RESUMO

Twenty-five new derivatives of 8-hydroxycycloberberine (1) were synthesized and evaluated for their activities against Gram-positive bacteria, taking 1 as the lead. Part of them displayed satisfactory antibacterial activities against methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA), as well as vancomycin-intermediate Staphylococcus aureus (VISA). Especially, compound 15a displayed an excellent anti-MRSA activity with MICs (minimum inhibitory concentrations) of 0.25⁻0.5 µg/mL, better than that of 1. It also displayed high stability in liver microsomes and whole blood, and the LD50 value of over 65.6 mg·kg-1 in mice via intravenous route, suggesting a good druglike feature. The mode of action showed that 15a could effectively suppress topo IV-mediated decatenation activity at the concentration of 7.5 µg/mL, through binding a different active pocket of bacterial topo IV from quinolones. Taken together, the derivatives of 1 constituted a promising kind of anti-MRSA agents with a unique chemical scaffold and a specific biological mechanism, and compound 15a has been chosen for the next investigation.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Berberina/química , Berberina/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Berberina/análogos & derivados , DNA Topoisomerase IV/antagonistas & inibidores , DNA Topoisomerase IV/química , Relação Dose-Resposta a Droga , Estabilidade de Medicamentos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Relação Estrutura-Atividade
20.
PLoS One ; 14(3): e0212936, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30856202

RESUMO

Plasmid-mediated quinolone resistance (PMQR) is frequent among Escherichia coli from various food products and animals in several countries. The objective of this study was to characterize quinolone resistant E. coli (QREC) from Norwegian turkey meat regarding resistance profiles, genetic mechanisms for quinolone resistance, genetic relatedness, and to investigate whether PMQR genes were present. In total, 78 QREC were isolated by a selective method from 156 samples throughout 2013. Isolates were subjected to susceptibility testing, characterization of resistance mechanisms, serotyping, phylotyping and multi-locus variable-tandem repeat analysis (MLVA). All 78 isolates were resistant to ciprofloxacin, while 77 were also resistant to nalidixic acid. The nalidixic acid sensitive isolate had a resistance profile indicating the presence of a PMQR gene. Both PCR and whole genome sequencing confirmed the presence of a 47 304 kb IncX1 plasmid containing the qnrS1 gene. The mechanism conferring quinolone resistance in the remaining isolates was mediated by mutations in the quinolone resistance-determining region of the chromosomal gyrA gene and for most of the isolates also in the parC gene. Molecular typing by MLVA showed a high degree of genetic diversity, although four clusters dominated. Two clusters contained strains belonging to phylogroup D/serogroup O176, the third contained isolates of phylogroup B1/serogroup O19, whereas the fourth contained isolates of phylogroup B1/non-typeable serogroup. Isolates within the latter cluster had MLVA profiles identical to QREC isolated from day-old imported turkey parent animals investigated in a preliminary study at the Norwegian Veterinary Institute. This finding suggests that QREC obtained from turkey may have been introduced via import of breeding animals to Norway. This is the first time the qnrS1 gene is described from E. coli isolated from Norwegian turkey meat. Compared to available qnrS1 carrying plasmids in Genbank, the current IncX1 plasmid showed high degree of similarity to other IncX1 plasmids containing qnrS1 isolated from both Shigella flexneri and E. coli found in different geographical areas and sources. To conclude, this study showed that mutations in gyrA and parC are the main mechanism conferring quinolone resistance in E. coli isolated from Norwegian turkey meat, and that PMQR has not been widely dispersed throughout the E. coli population in Norwegian turkey.


Assuntos
Farmacorresistência Bacteriana/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Produtos Avícolas/microbiologia , Animais , Antibacterianos/farmacologia , DNA Girase/genética , DNA Topoisomerase IV/genética , Escherichia coli/isolamento & purificação , Fluoroquinolonas/farmacologia , Testes de Sensibilidade Microbiana , Mutação , Noruega , Plasmídeos/genética , Perus/microbiologia
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