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1.
Nucleic Acids Res ; 48(4): 2035-2049, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-31950157

RESUMO

Negative supercoiling by DNA gyrase is essential for maintaining chromosomal compaction, transcriptional programming, and genetic integrity in bacteria. Questions remain as to how gyrases from different species have evolved profound differences in their kinetics, efficiency, and extent of negative supercoiling. To explore this issue, we analyzed homology-directed mutations in the C-terminal, DNA-wrapping domain of the GyrA subunit of Escherichia coli gyrase (the 'CTD'). The addition or removal of select, conserved basic residues markedly impacts both nucleotide-dependent DNA wrapping and supercoiling by the enzyme. Weakening CTD-DNA interactions slows supercoiling, impairs DNA-dependent ATP hydrolysis, and limits the extent of DNA supercoiling, while simultaneously enhancing decatenation and supercoil relaxation. Conversely, strengthening DNA wrapping does not result in a more extensively supercoiled DNA product, but partially uncouples ATP turnover from strand passage, manifesting in futile cycling. Our findings indicate that the catalytic cycle of E. coli gyrase operates at high thermodynamic efficiency, and that the stability of DNA wrapping by the CTD provides one limit to DNA supercoil introduction, beyond which strand passage competes with ATP-dependent supercoil relaxation. These results highlight a means by which gyrase can evolve distinct homeostatic supercoiling setpoints in a species-specific manner.


Assuntos
Trifosfato de Adenosina/metabolismo , DNA Girase/genética , DNA Bacteriano/genética , DNA Super-Helicoidal/química , Trifosfato de Adenosina/química , Catálise , Cromossomos Bacterianos/genética , DNA Girase/química , DNA Bacteriano/química , DNA Super-Helicoidal/genética , Escherichia coli/enzimologia , Modelos Moleculares , Mutação/genética , Conformação de Ácido Nucleico , Ligação Proteica , Domínios Proteicos/genética
2.
Int J Infect Dis ; 92: 241-246, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31978580

RESUMO

OBJECTIVES: To compare the prevalence of levofloxacin (LFX) resistance and the population structure of Mycobacterium tuberculosis (MTB) with different mutations conferring LFX resistance between 2005 and 2015. METHODS: A total 542 MTB isolates were randomly selected from pulmonary tuberculosis (TB) patients in 2005 and 2015 and analyzed regarding minimum inhibitory concentrations (MICs) and quinolone resistance-determining regions (QRDR). RESULTS: One hundred and eleven of the 542 MTB isolates analyzed (20.5%) were resistant to LFX. There were 42 and 69 LFX-resistant isolates from 2005 and 2015, respectively, and MIC high-level LFX resistance was significantly higher in 2015 (40.6%, 28/69) than in 2005 (16.7%, 7/42) (p = 0.02). There were 87 (78.4%) mutations of these 111 LFX-resistant isolates. In addition, a significant difference in proportion was observed in the isolates with mutations in codon 90 of the gyrA gene between 2005 and 2015 (11.9% in 2005 versus 29.0% in 2015, p = 0.04). CONCLUSIONS: There was an alarming increase in prevalence of LFX-resistant TB in China between 2005 and 2015. This dynamic change is mostly attributed to the increase in high-level LFX resistance. Moreover, a significant difference was noted in the proportion of LFX-resistant isolates harboring specific mutations within the gyrA gene between 2005 and 2015.


Assuntos
Farmacorresistência Bacteriana , Levofloxacino/uso terapêutico , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Tuberculose Pulmonar/tratamento farmacológico , Adulto , China/epidemiologia , DNA Girase/genética , Feminino , Fluoroquinolonas/farmacologia , Fluoroquinolonas/uso terapêutico , Humanos , Levofloxacino/farmacologia , Masculino , Testes de Sensibilidade Microbiana , Mutação , Mycobacterium tuberculosis/isolamento & purificação , Prevalência , Tuberculose Pulmonar/epidemiologia , Tuberculose Pulmonar/microbiologia
3.
Int J Syst Evol Microbiol ; 70(1): 406-438, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31617837

RESUMO

The genus Bacillus, harbouring 293 species/subspecies, constitutes a phylogenetically incoherent group. In the absence of reliable means for grouping known Bacillus species into distinct clades, restricting the placement of new species into this genus has proven difficult. To clarify the evolutionary relationships among Bacillus species, 352 available genome sequences from the family Bacillaceae were used to perform comprehensive phylogenomic and comparative genomic analyses. Four phylogenetic trees were reconstructed based on multiple datasets of proteins including 1172 core Bacillaceae proteins, 87 proteins conserved within the phylum Firmicutes, GyrA-GyrB-RpoB-RpoC proteins, and UvrD-PolA proteins. All trees exhibited nearly identical branching of Bacillus species and consistently displayed six novel monophyletic clades encompassing 5-23 Bacillus species (denoted as the Simplex, Firmus, Jeotgali, Niacini, Fastidiosus and Alcalophilus clades), interspersed with other Bacillaceae species. Species from these clades also generally grouped together in 16S rRNA gene trees. In parallel, our comparative genomic analyses of Bacillus species led to the identification of 36 molecular markers comprising conserved signature indels in protein sequences that are specifically shared by the species from these six observed clades, thus reliably demarcating these clades based on multiple molecular synapomorphies. Based on the strong evidence from multiple lines of investigations supporting the existence of these six distinct 'Bacillus' clades, we propose the transfer of species from these clades into six novel Bacillaceae genera viz. Peribacillus gen. nov., Cytobacillus gen. nov., Mesobacillus gen. nov., Neobacillus gen. nov., Metabacillus gen. nov. and Alkalihalobacillus gen. nov. These results represent an important step towards clarifying the phylogeny/taxonomy of the genus Bacillus.


Assuntos
Bacillus/classificação , Genômica , Filogenia , Sequência de Aminoácidos , Técnicas de Tipagem Bacteriana , Sequência de Bases , DNA Girase/genética , DNA Bacteriano/genética , Genes Bacterianos , Mutação INDEL , RNA Ribossômico 16S/genética , Análise de Sequência de DNA
4.
Int J Food Microbiol ; 312: 108374, 2020 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-31669765

RESUMO

Salmonella enterica outbreaks in sprouts originate from contaminated seeds; conventional prevention technologies have been reported from many research institutes. In this study, we applied a biological control approach to inhibit S. enterica growth using the seed-dwelling non-antagonistic bacteria. We isolated non-antibacterial seed-dwelling bacteria from vegetable sprouts. A total of 206 bacteria exhibiting non-antibacterial activity against S. enterica were subjected to alfalfa sprout development tests. Eight isolates exhibiting no deleterious effect on the growth of alfalfa sprouts were tested for S. enterica growth inhibition on alfalfa seeds and sprouts, and an isolate EUS78 was finally selected for further investigation. Based on 16S rRNA, gyrB, and rpoB gene sequence analyses, strain EUS78 was identified as Erwinia persicina. In population competition, the S. enterica population increased by >3 log CFU/g after 6 days of alfalfa sprout growth, whereas S. enterica growth was significantly inhibited by treatment with EUS78 (P < .05). This effect of S. enterica growth inhibition by EUS78 was sustained until the end of the alfalfa sprout harvest. Overall, bacterial strain EUS78 significantly reduced S. enterica growth on alfalfa sprouts in a manner consistent with competitive exclusion. These findings led us to monitor EUS78 behavior on seeds during early sprout development using fluorescence and scanning electron microscopy. Strain EUS78 initially colonized alfalfa sprout seed coat edges, cotyledons, and finally root surfaces during early sprout germination. As alfalfa sprouts grew, EUS78 bacterial cells established colonies on newly emerged plant tissues such as root tips. The results of this study suggest that strain EUS78 has potential as a biological control agent to inhibit S. enterica contamination in the sprout food industry.


Assuntos
Antibiose/fisiologia , Agentes de Controle Biológico , Erwinia/fisiologia , Medicago sativa/microbiologia , Salmonella enterica/crescimento & desenvolvimento , Sementes/microbiologia , DNA Girase/genética , RNA Polimerases Dirigidas por DNA/genética , Erwinia/genética , Microbiologia de Alimentos , Indústria de Processamento de Alimentos , Germinação/fisiologia , Medicago sativa/química , RNA Ribossômico 16S/genética , Verduras/microbiologia
5.
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
6.
Vet Microbiol ; 239: 108459, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31767067

RESUMO

Helicobacter suis is a fastidious, Gram negative bacterium that colonizes the stomach of pigs and non-human primates. It has also been associated with gastric disease in humans. A combined agar and broth dilution method was used to analyze the activity of 15 antimicrobial agents against 20 and 15 H. suis isolates obtained from pigs and macaques, respectively. After 48 h microaerobic incubation, minimal inhibitory concentrations (MICs) were determined by software-assisted calculation of bacterial growth as determined by quantitative real-time PCR. A monomodal distribution of MICs was seen for ß-lactam antibiotics, macrolides, gentamicin, neomycin, doxycycline, metronidazole, and rifampicin. Presence of a bimodal distribution of MICs indicated that 2 porcine isolates did not belong to the wild type population (WTP) for fluoroquinolones. This was also the case for 1 porcine isolate for tetracycline, 1 porcine and 2 primate isolates for lincomycin, and 1 primate isolate for spectinomycin. Single nucleotide polymorphisms (SNPs) were present in the gyrA gene of the isolates not belonging to the WTP for fluoroquinolones and in ribosomal protein encoding genes of the isolates not belonging to the WTP for tetracycline and spectinomycin. MICs of ampicillin, tetracycline and doxycycline were higher for porcine H. suis isolates compared to primate isolates and in these porcine isolates SNPs were detected in genes encoding penicillin binding and ribosomal proteins. This study indicates that acquired resistance occasionally occurs in H. suis isolates and that zoonotically important porcine isolates may be intrinsically less susceptible to ß-lactam antibiotics and tetracyclines than primate isolates.


Assuntos
Antibacterianos/farmacologia , Infecções por Helicobacter/microbiologia , Helicobacter heilmannii/efeitos dos fármacos , Doenças dos Macacos/microbiologia , Doenças dos Suínos/microbiologia , Animais , DNA Girase/genética , Farmacorresistência Bacteriana , Helicobacter heilmannii/isolamento & purificação , Macaca/microbiologia , Testes de Sensibilidade Microbiana , Polimorfismo de Nucleotídeo Único , Suínos/microbiologia
7.
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
8.
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
9.
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
10.
Future Microbiol ; 14: 1123-1132, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31512520

RESUMO

Aim: Multidrug-resistant Staphylococcus aureus isolates have become a serious concern in clinical microbiology. Antisense strategy, which specifically targets essential genes, could be helpful. Materials & methods: S. aureus cultures were treated with peptide conjugate-peptide nucleic acid (PPNA) specific for the gyrA gene. In addition, antimicrobial synergy with ciprofloxacin was tested. Results: The results indicated anti-gyrA-PPNA dramatically inhibited the growth of S. aureus isolates in Mueller Hinton Broth with complete elimination of bacteria observed on cell cultures. Specifically, PPNA reduced the gyrA transcripts up to 50%. With antisense interference, growth inhibition was augmented through combination with ciprofloxacin. Conclusion: This study suggested that anti-gyrA-PPNAs could be introduced as a novel candidate for developing antisense antibiotic to treat all S. aureus infections.


Assuntos
Antibacterianos/farmacologia , DNA Girase/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Ácidos Nucleicos Peptídicos/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento , Ciprofloxacino/farmacologia , DNA Girase/genética , Sinergismo Farmacológico , Testes de Sensibilidade Microbiana , Staphylococcus aureus/genética
11.
Chemosphere ; 237: 124421, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31382196

RESUMO

Antibiotics in the effluents of municipal wastewater treatment plants (WWTP) may create selective pressures to induce antibiotic resistance in bacteria downstream. This study evaluates ciprofloxacin (CIP) removal by a freshwater alga, Scenedesmus dimorphus, to assess the efficacy of algae-based tertiary treatment in reducing effluent-induced CIP resistance. Results show significant CIP removal in light-exposed samples without algae and experimental algae (EA) samples: 53% and 93%, respectively, over 144 h. A residual antibiotic potency assay reveals that untreated CIP is significantly more growth-inhibiting to a model bacterium (Escherichia coli) than the algae-treated and light-exposed samples during short exposures (6 h). Adaptive laboratory evolution (ALE), again using E. coli, reveals that treated samples exhibit reduced capacity to elicit CIP resistance during sustained exposures compared to untreated CIP. Finally, observed CIP resistance in the CIP-exposed ALE lineages is corroborated via genotype characterization, which reveals the presence of resistance-associated mutations in gyrase subunit A (gyrA) that are not present in ALE lineages exposed to algae treated or light-exposed samples. As such, algae-mediated tertiary treatment could be effective in suppressing CIP resistance in bacterial communities downstream from WWTP. In addition, ALE is useful for assessing the potential of wastewater-relevant samples to elicit antibiotic resistance downstream.


Assuntos
Antibacterianos/farmacologia , Ciprofloxacino/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Scenedesmus/metabolismo , Eliminação de Resíduos Líquidos/métodos , Antibacterianos/isolamento & purificação , Antibacterianos/metabolismo , Ciprofloxacino/isolamento & purificação , Ciprofloxacino/metabolismo , DNA Girase/genética , Farmacorresistência Bacteriana/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Genótipo , Testes de Sensibilidade Microbiana , Mutação , Águas Residuárias/química , Poluentes Químicos da Água/isolamento & purificação , Poluentes Químicos da Água/metabolismo , Poluentes Químicos da Água/farmacologia
12.
Biosci Biotechnol Biochem ; 83(12): 2249-2256, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31382821

RESUMO

The inhibitory effect of WQ-3810 on DNA gyrase was assayed to evaluate the potential of WQ-3810 as a candidate drug for the treatment of quinolone resistant Salmonella Typhymurium infection. The inhibitory effect of WQ-3810, ciprofloxacin and nalidixic acid was compared by accessing the drug concentration that halves the enzyme activity (IC50) of purified S. Typhimurium wildtype and mutant DNA gyrase with amino acid substitution at position 83 or/and 87 in subunit A (GyrA) causing quinolone resistance. As a result, WQ-3810 reduced the enzyme activity of both wildtype and mutant DNA gyrase at a lower concentration than ciprofloxacin and nalidixic acid. Remarkably, WQ-3810 showed a higher inhibitory effect on DNA gyrase with amino acid substitutions at position 87 than with that at position 83 in GyrA. This study revealed that WQ-3810 could be an effective therapeutic agent, especially against quinolone resistant Salmonella enterica having amino acid substitution at position 87.


Assuntos
Antibacterianos/farmacologia , Azetidinas/farmacologia , DNA Girase/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Fluoroquinolonas/farmacologia , Quinolonas/farmacologia , Salmonella typhimurium/efeitos dos fármacos , DNA Girase/genética , DNA Girase/metabolismo , Farmacorresistência Bacteriana , Concentração Inibidora 50 , Testes de Sensibilidade Microbiana , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Salmonella typhimurium/enzimologia
13.
Diagn Microbiol Infect Dis ; 95(3): 114865, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31405631

RESUMO

We present a case of subcutaneous infection caused by Bordetella hinzii in a healthy male. The isolate was successfully identified by gyrB gene sequencing. B. hinzii cannot be distinctively identified using 16S rRNA gene sequencing or by biochemical methods. The number of cases infected with B. hinzii might be underestimated owing to the difficulty in accurate identification, which can be achieved by gyrB gene sequencing to gain knowledge about the species.


Assuntos
Abscesso/microbiologia , Infecções por Bordetella/diagnóstico , Bordetella/fisiologia , Abscesso/diagnóstico , Abscesso/tratamento farmacológico , Abscesso/patologia , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Proteínas de Bactérias/genética , Bordetella/genética , Infecções por Bordetella/tratamento farmacológico , Infecções por Bordetella/microbiologia , Infecções por Bordetella/patologia , DNA Girase/genética , DNA Bacteriano/genética , Humanos , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Pele/microbiologia , Resultado do Tratamento
14.
Molecules ; 24(15)2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31375003

RESUMO

Antibiotic resistance is one of the main public health concerns of this century. This resistance is also associated with oxidative stress, which could contribute to the selection of resistant bacterial strains. Bearing this in mind, and considering that flavonoid compounds are well known for displaying both activities, we investigated a series of hydroxy-3-arylcoumarins with structural features of flavonoids for their antibacterial activity against different bacterial strains. Active compounds showed selectivity against the studied Gram-positive bacteria compared to Gram-negative bacteria. 5,7-Dihydroxy-3-phenylcoumarin (compound 8) displayed the best antibacterial activity against Staphylococcus aureus and Bacillus cereus with minimum inhibitory concentrations (MICs) of 11 g/mL, followed by Staphylococcus aureus (MRSA strain) and Listeria monocytogenes with MICs of 22 and 44 g/mL, respectively. Moreover, molecular docking studies performed on the most active compounds against Staphylococcus aureus tyrosyl-tRNA synthetase and topoisomerase II DNA gyrase revealed the potential binding mode of the ligands to the site of the appropriate targets. Preliminary structure-activity relationship studies showed that the antibacterial activity can be modulated by the presence of the 3-phenyl ring and by the position of the hydroxyl groups at the coumarin scaffold.


Assuntos
Antibacterianos/química , Infecções Bacterianas/tratamento farmacológico , Cumarínicos/química , Relação Estrutura-Atividade , Antibacterianos/farmacologia , Bacillus cereus/efeitos dos fármacos , Bacillus cereus/patogenicidade , Infecções Bacterianas/microbiologia , Cumarínicos/farmacologia , DNA Girase/química , DNA Girase/genética , Humanos , Listeria monocytogenes/efeitos dos fármacos , Listeria monocytogenes/patogenicidade , Simulação de Acoplamento Molecular , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/patogenicidade
15.
Helicobacter ; 24(5): e12628, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31282059

RESUMO

BACKGROUND: Fluoroquinolones hinder bacterial DNA replication by inhibiting DNA gyrase. However, mutations, in the QRDR segment of its A subunit (GyrA), cause antibiotic resistance. Here, the interactions of levofloxacin (LVX), gemifloxacin (GXN), and moxifloxacin (MXN) with Helicobacter pylori GyrA, in LVX-resistant vs -sensitive strains, were studied. METHODS: Levoflixacin-sensitive (n = 4) and -resistant (n = 9) H pylori strains, randomly selected from another antibiotic susceptibility study, underwent PCR amplification of gyrA gene, spanning the QRDR segment. The amplified gene fragments were sequenced and aligned. The homology model of H pylori GyrA was built based on that of Escherichia coli, and energy minimization was done. The interaction patterns of LVX, GXN, and MXN with GyrA were analyzed via molecular docking studies. RESULTS: Sequence alignment of the 13 studied strains, created 5 categories of strains: (A) wild type-like (H pylori ATCC26695), (B) N87K-only, (C) D91N-only, (D) N87K + V94L, and (E) D91N + A97V mutations. The minimum inhibitory concentrations (MIC) for LVX-sensitive (category A) and -resistant (categories B-E) strains were <1 mg/L and ≥32 mg/L, respectively. The binding mode of GyrA in category A with LVX identified G35/N87/Y90/D91/V94/G114/S115/I116/D117/G118/D119, as key residues, some residing outside the QRDR segment. Category B strains lost only one interaction (G35), which led to elevated binding free energy (∆G) and full LVX resistance. Categories C-E lost more contacts, with higher ∆G and again full LVX resistance. GXN bound to GyrA of categories A and B via a different set of key residues, while MXN retained the lost contact (G35) in LVX-resistant, category B strains. CONCLUSION: Using molecular docking tools, we identified the key residues responsible for interaction of GyrA with LVX, GXN, and MXN. In the presence of N87K-only mutation, the loss of one of these contacts (ie, G35) led to full LVX resistance. Yet, GXN and MXN overcame this mutation, by retaining all key contacts with GyrA.


Assuntos
Antibacterianos/farmacologia , DNA Girase/metabolismo , Farmacorresistência Bacteriana , Gemifloxacina/farmacologia , Helicobacter pylori/efeitos dos fármacos , Levofloxacino/farmacologia , Moxifloxacina/farmacologia , Antibacterianos/metabolismo , DNA Girase/química , DNA Girase/genética , Gemifloxacina/metabolismo , Helicobacter pylori/enzimologia , Humanos , Levofloxacino/metabolismo , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Moxifloxacina/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação , Reação em Cadeia da Polimerase , Ligação Proteica , Análise de Sequência de DNA
16.
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 , 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
17.
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
18.
Mol Biol Rep ; 46(4): 4049-4061, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31093874

RESUMO

Rapid and species-specific detection, and virulence evaluation of opportunistic pathogens such as Pseudomonas aeruginosa, are issues that increasingly has attracted the attention of public health authorities. A set of primers and hydrolysis probe was designed based on one of the P. aeruginosa housekeeping genes, gyrB, and its specificity and sensitivity was evaluated by TaqMan qPCR methods. The end point PCR and SYBR Green qPCR were used as control methods. Furthermore, multiplex RT-qPCRs were developed for gyrB as reference and four virulence genes, including lasB, lasR, rhlR and toxA. Totally, 40 environmental samples, two clinical isolates from CF patients, two standard strains of P. aeruginosa, and 15 non-target reference strains were used to test the sensitivity and specificity of qPCR assays. In silico and in vitro cross-species testing confirmed the high specificity and low cross-species amplification of the designed gyrB418F/gyrB490R/gyrB444P. The sensitivity of both TaqMan and SYBR Green qPCRs was 100% for all target P. aeruginosa, and the detected count of bacteria was below ten genomic equivalents. The lowest M value obtained from gene-stability measurement was 0.19 that confirmed the suitability of gyrB as the reference gene for RT-qPCR. The developed qPCRs have enough detection power for identification of P. aeruginosa in environmental samples including clean and recreational water, treated and untreated sewage and soil. The short amplicon length of our designed primers and probes, alongside with a low M value, make it as a proper methodology for RT-qPCR in virulence genes expression assessment.


Assuntos
DNA Girase/genética , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/isolamento & purificação , Proteínas de Bactérias/genética , Primers do DNA/genética , DNA Bacteriano/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Especificidade da Espécie , Virulência , Fatores de Virulência/genética
19.
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
20.
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
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