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1.
Int J Infect Dis ; 106: 295-299, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33864922

RESUMO

OBJECTIVE: WFQ-228 is a novel developed fluoroquinolone (FQ) displaying potent antimicrobial activity against various clinical isolates of pathogens, including FQ-resistant isolates. The aim was to comparatively analyze in vitro susceptibilities of WFQ-228, levofloxacin (LFX), and moxifloxacin (MFX) against Mycobacterium tuberculosis (MTB) isolates, especially with gyrA mutations. METHODS: We selected a panel of 75 MTB isolates, consisting of 25 FQ-susceptible and 50 FQ-resistant isolates determined by conventional drug susceptibility testing. The minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of FQs to MTB isolates were assessed. RESULTS: MFX exhibited the most potent activity against FQ-susceptible MTB, demonstrating a MIC50 of 0.031 mg/L, which was lower than that of LFX and WFQ-228. Against FQ-resistant MTB isolates, the MIC50 of WFQ-228 was higher than that of MFX but lower than that of LFX. For WFQ-228, there was a significant overlap existing in the MIC distributions between the probable susceptible (PS) and probable resistant (PR) groups. Six out of 50 PR isolates were classified as susceptible based on a proposed critical concentration (CC) of 0.5 mg/L, yielding a poor sensitivity of 88.0%. These discordant isolates had GyrA substitution in Ala90Val, Ser91Pro, and Asp94Tyr. Additionally, MFX exhibited bactericidal activity against MTB isolates without gyrA mutations, which was significantly higher than that of isolates with gyrA mutations. CONCLUSION: WFQ-228 is more efficacious than LFX in isolates with specific mutations conferring low-level FQ resistance. The bactericidal effect is noted more frequently in FQ-susceptible isolates than FQ-resistant isolates for MFX.


Assuntos
Antibacterianos/farmacologia , Fluoroquinolonas/farmacologia , Levofloxacino/farmacologia , Moxifloxacina/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , DNA Girase/genética , Farmacorresistência Bacteriana/genética , Humanos , Testes de Sensibilidade Microbiana , Mutação/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação
2.
Arch Biochem Biophys ; 701: 108786, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33548211

RESUMO

DNA Gyrase is a type II topoisomerase that utilizes the energy of ATP hydrolysis for introducing negative supercoils in DNA. The protein comprises two subunits GyrA and GyrB that form a GyrA2GyrB2 heterotetramer. GyrB subunit contains the N-terminal domain (GBNTD) for ATPase activity and the C-terminal domain (GBCTD) for interaction with GyrA and DNA. Earlier structural studies have revealed three different conformational states for GBNTD during ATP hydrolysis defined as open, semi-open, and closed. Here we report, the three-dimensional structure of a new transient closed conformation of GBNTD from Salmonella Typhi (StGBNTD) at 1.94 Å resolution. Based on the structural analysis of this transient closed conformation, we propose the role of protein in the mechanism of ATP hydrolysis. We further explored the effect of pH on ATPase activity and structural stability of the GBNTD using CD and fluorescence spectroscopy at varying pH environment. Kinetic parameters obtained from the ATPase assay were correlated with its secondary and tertiary structure at their respective pH environment. The protein possessed maximum ATPase activity and structural stability at optimum pH 8. At acidic pH, a remarkable decrease in both enzymatic activity and structural stability was observed whereas at alkaline pH there was no significant change. The structural analysis of StGBNTD reveals the role of polar interactions in stabilizing the overall dimeric conformation of the protein.


Assuntos
Adenosina Trifosfatases/química , DNA Girase/química , Salmonella typhi/enzimologia , Adenosina Trifosfatases/genética , Cristalografia por Raios X , DNA Girase/genética , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Cinética , Domínios Proteicos , Salmonella typhi/genética
3.
BMC Infect Dis ; 20(1): 950, 2020 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-33308173

RESUMO

BACKGROUND: Antimicrobial resistance in M. genitalium is a growing clinical problem. We investigated the mutations associated with macrolide and fluoroquinolone resistance, two commonly used medical regimens for treatment in China. Our aim is to analyze the prevalence and diversity of mutations among M. genitalium-positive clinical specimens in Guangzhou, south China. METHODS: A total of 154 stored M. genitalium positive specimens from men and women attending a STI clinic were tested for macrolide and fluoroquinolone mutations. M. genitalium was detected via TaqMan MGB real-time PCR. Mutations associated with macrolide resistance were detected using primers targeting region V of the 23S rRNA gene. Fluoroquinolone resistant mutations were screened via primers targeting topoisomerase IV (parC) and DNA gyrase (gyrA). RESULTS: 98.7% (152/154), 95.5% (147/154) and 90.3% (139/154) of M. genitalium positive samples produced sufficient amplicon for detecting resistance mutations in 23S rRNA, gyrA and parC genes, respectively. 66.4% (101/152), 0.7% (1/147) and 77.7% (108/139) samples manifested mutations in 23S rRNA, gyrA and parC genes, respectively. A2072G (59/101, 58.4%) and S83I (79/108, 73.1%) were highly predominating in 23S rRNA and parC genes, respectively. Two samples had amino acid substitutions in gyrA (M95I and A96T, respectively). Two samples had two amino acid substitutions in parC (S83I + D87Y). 48.6% (67/138) of samples harbored both macrolide and fluoroquinolone resistance-associated mutations. The most common combination of mutations was A2072G (23S rRNA) and S83I (parC) (40/67, 59.7%). One sample had three amino acid changes in 23S rRNA, gyrA and parC genes (A2072G + A96T + S83I). CONCLUSIONS: The high antimicrobial resistance rate of M. genitalium in Guangzhou is a very worrying problem and suggests that antimicrobial resistance testing and the development of new antibiotic regimens are crucially needed.


Assuntos
Antibacterianos/uso terapêutico , Farmacorresistência Bacteriana Múltipla/genética , Fluoroquinolonas/uso terapêutico , Macrolídeos/uso terapêutico , Mutação , Infecções por Mycoplasma/tratamento farmacológico , Mycoplasma genitalium/genética , Doenças Bacterianas Sexualmente Transmissíveis/tratamento farmacológico , China/epidemiologia , DNA Girase/genética , DNA Topoisomerase IV/genética , DNA Bacteriano/genética , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Feminino , Humanos , Masculino , Infecções por Mycoplasma/epidemiologia , Infecções por Mycoplasma/microbiologia , Mycoplasma genitalium/isolamento & purificação , Prevalência , RNA Ribossômico 23S/genética , Reação em Cadeia da Polimerase em Tempo Real , Estudos Retrospectivos , Doenças Bacterianas Sexualmente Transmissíveis/epidemiologia , Doenças Bacterianas Sexualmente Transmissíveis/microbiologia
4.
BMC Infect Dis ; 20(1): 866, 2020 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-33213390

RESUMO

BACKGROUND: Mycolicibacterium fortuitum is a species of the rapidly growing mycobacteria that can cause pulmonary infection. It is susceptible to multiple antibiotics both in vitro and in clinical practice, so that any combination of susceptible drugs is effective. However, we encountered a case of infection due to fluoroquinolone-resistant M. fortuitum. In this study, we report the case and describe the mechanism of resistance. CASE PRESENTATION: A 65-year-old man with a history of total gastrectomy and immunosuppressant treatment for rheumatoid arthritis developed a recurrence of pulmonary infection caused by M. fortuitum. He was treated with clarithromycin and levofloxacin as a first-line treatment, based on the favorable susceptibility at that time. After recurrence, a high minimum inhibitory concentration to fluoroquinolones was detected. DNA sequencing of the pathogen showed the substitution of serine for tryptophan at residue 83 in the gyrA gene. He was successfully treated with a combination of other antibiotics. CONCLUSION: This is the first report on the treatment of fluoroquinolone-resistant M. fortuitum and investigation of the mechanism of resistance. We suggest that the susceptibility test remains effective for determining the next line of treatment after a pathogen has acquired resistance, and resistance to fluoroquinolones in M. fortuitum can be attributed to a single change of amino acid.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana/genética , Fluoroquinolonas/farmacologia , Pneumopatias/diagnóstico , Infecções por Mycobacterium não Tuberculosas/diagnóstico , Mycobacterium fortuitum/efeitos dos fármacos , Idoso , Substituição de Aminoácidos , DNA Girase/química , DNA Girase/genética , DNA Girase/metabolismo , Humanos , Pneumopatias/microbiologia , Masculino , Testes de Sensibilidade Microbiana , Infecções por Mycobacterium não Tuberculosas/microbiologia , Mycobacterium fortuitum/genética , Mycobacterium fortuitum/isolamento & purificação , Recidiva , Análise de Sequência de DNA
5.
BMC Infect Dis ; 20(1): 518, 2020 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-32677920

RESUMO

BACKGROUND: Campylobacter jejuni is a leading cause of bacterial diarrhea worldwide, and increasing rates of fluoroquinolone (FQ) resistance in C. jejuni are a major public health concern. The rapid detection and tracking of FQ resistance are critical needs in developing countries, as these antimicrobials are widely used against C. jejuni infections. Detection of point mutations at T86I in the gyrA gene by real-time polymerase chain reaction (RT-PCR) is a rapid detection tool that may improve FQ resistance tracking. METHODS: C. jejuni isolates obtained from children with diarrhea in Peru were tested by RT-PCR to detect point mutations at T86I in gyrA. Further confirmation was performed by sequencing of the gyrA gene. RESULTS: We detected point mutations at T86I in the gyrA gene in 100% (141/141) of C. jejuni clinical isolates that were previously confirmed as ciprofloxacin-resistant by E-test. No mutations were detected at T86I in gyrA in any ciprofloxacin-sensitive isolates. CONCLUSIONS: Detection of T86I mutations in C. jejuni is a rapid, sensitive, and specific method to identify fluoroquinolone resistance in Peru. This detection approach could be broadly employed in epidemiologic surveillance, therefore reducing time and cost in regions with limited resources.


Assuntos
Infecções por Campylobacter/diagnóstico , Campylobacter jejuni/genética , DNA Girase/genética , Farmacorresistência Bacteriana/genética , Fluoroquinolonas/uso terapêutico , Mutação Puntual , Reação em Cadeia da Polimerase em Tempo Real/métodos , Substituição de Aminoácidos , Infecções por Campylobacter/tratamento farmacológico , Infecções por Campylobacter/microbiologia , Campylobacter jejuni/isolamento & purificação , Criança , Ciprofloxacina/uso terapêutico , Análise Mutacional de DNA/métodos , Diarreia/diagnóstico , Diarreia/tratamento farmacológico , Diarreia/microbiologia , Humanos , Isoleucina/genética , Testes de Sensibilidade Microbiana , Peru , Treonina/genética
6.
Sci Rep ; 10(1): 7817, 2020 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-32385379

RESUMO

The essentiality of DNA Gyrase in basic cellular processes in bacterial pathogens makes it an ideal drug target. Though the Gyrase has a conserved mechanism of action, the complete DNA wrapping and binding process is still unknown. In this study, we have identified six arginine residues R556, R612, R667, R716, R766, and R817 in the DNA GyraseA - C-terminal domain from Salmonella enterica serovar Typhi (StGyrA-CTD) to be essential for DNA wrapping and sliding by a sequence and structure analysis. Through site-directed mutagenesis and EMSA studies, we observed that the substitution of R667 (blade 3) and R716 (blade 4) in StGyrA-CTD led to loss of DNA binding. Whereas, upon mutation of residue R612 (blade2), R766 (blade5) and R817 (blade6) along with supporting residue R712 (blade 4) a decrease in binding affinity was seen. Our results indicate that R667 and R716 act as a pivot point in DNA wrapping and sliding during gyrase catalytic activity. In this study, we propose that the DNA wrapping mechanism commences with DNA binding at blade3 and blade4 followed by other blades to facilitate the DNA sliding during supercoiling activity. This study provides a better understanding of the DNA binding and wrapping mechanism of GyrA-CTD in DNA Gyrase.


Assuntos
Arginina/genética , DNA Girase/genética , Conformação Proteica em Folha beta/genética , Salmonella typhi/genética , Sequência de Aminoácidos/genética , DNA Girase/ultraestrutura , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação/genética , Ligação Proteica/genética , Domínios Proteicos/genética , Salmonella typhi/enzimologia , Salmonella typhi/patogenicidade
7.
Sci Rep ; 10(1): 8497, 2020 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-32444702

RESUMO

For the last decades, forensic microbiology became an emerging complementary tool in criminalistics. Although the insect-microbe interactions regarding pathogen transmission were extensively studied, only scarce information is available on bacterial transfer from necrophagous insects to host tissues. Our data provides the first report on the occurrence of Wohlfahrtiimonas chitiniclastica and Ignatzschineria indica in Lucilia illustris Meigen, 1826 (Diptera: Calliphoridae), and the quantitative dynamics of the two bacterial species along the insect life-stages and transfer to beef and pork host tissues using qPCR gyrase b specific primers. The content of both bacterial species increased along the insect life stages. W. chitiniclastica was detected in all developmental stages independent of the feeding substrate. I. indica was measurable with 102 gene copies ng-1 DNA threshold starting from the third instar larvae when feeding on beef, and from the egg stage with a 102× higher representation when using the pork substrate. The transfer of bacterial species to both tissues occurred after 3 colonization days except for I. indica that was visible in beef liver only during day 5. Considering the utilization of pork tissues as human analogues, these quantitative microbial dynamics data provides first insect-specific bacterial candidates as potential colonization biomarkers in forensic investigations.


Assuntos
Proteínas de Bactérias/genética , Biomarcadores/análise , DNA Girase/genética , DNA Bacteriano/análise , Dípteros/microbiologia , Gammaproteobacteria/isolamento & purificação , Animais , Bovinos , DNA Bacteriano/genética , Dípteros/crescimento & desenvolvimento , Dípteros/metabolismo , Ciências Forenses , Gammaproteobacteria/genética , Gammaproteobacteria/metabolismo
8.
Vet Microbiol ; 242: 108566, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32122581

RESUMO

Antimicrobial resistance reported in bacteria of animal origin is considered a major challenge to veterinary public health. In this study, the genotypic and phenotypic characterisation of twelve Escherichia coli isolates of bovine origin is reported. Twelve bacterial isolates of animal origin were selected from a previous study based on their multidrug resistant (MDR) profile. Efflux pump activity was measured using ethidium bromide (EtBr) and the biofilm forming ability of the individual strains was assessed using a number of phenotypic assays. All isolates were resistant to tetracyclines and a number of isolates expressed resistance to fluoroquinolones which was also confirmed in silico by the presence of these resistance markers. Amino acid substitutions in the quinolone resistance-determining regions were identified in all isolates and the presence of several siderophores were also noted. Whole genomesequence (WGS) data showed different STs that were not associated with epidemic STs or virulent clonal complexes. Seven isolates formed biofilms in minimal media with some isolates showing better adaptation at 25 °C while others at 37 °C. The capacity to efflux EtBr was found to be high in 4 isolates and impaired in 4 others. The pathogenicity of three selected isolates was assessed in zebrafish embryo infection models, revealing isolates CFS0355 and CFS0356 as highly pathogenic. These results highlight the application of NGS technologies combined with phenotypic assays in providing a better understanding of E. coli of bovine origin and their adaptation to this niche environment.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Infecções por Escherichia coli/veterinária , Proteínas de Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Animais , Biofilmes/crescimento & desenvolvimento , Bovinos/microbiologia , Simulação por Computador , DNA Girase/genética , Embrião não Mamífero , Infecções por Escherichia coli/virologia , Testes de Sensibilidade Microbiana , Virulência , Peixe-Zebra/virologia
9.
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
10.
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
11.
J Appl Microbiol ; 128(6): 1624-1633, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31951091

RESUMO

AIMS: To study the association between number and positions of mutations with MICs of fluoroquinolone non-susceptible Haemophilus influenzae. METHODS AND RESULTS: More than 40% of 48 H. influenzae isolated from nursing home residents were not susceptible to fluoroquinolone. Amino acid changes in the quinolone resistance determining regions, and correlation with MICs and inhibition zone diameters were analysed. All isolates with reduced susceptibility to fluoroquinolones (MIC ≥0·125 µg ml-1 ) had at least one mutation in gyrA at position 84 and were resistant to nalidixic acid. Compared to isolates with reduced susceptibility, resistant isolates were associated with mutations in gyrA at positions 88 and 134, and in parC at position 88 (P < 0·001). Inhibition zone diameter for nalidixic acid disk ≥23 mm may detect susceptible isolates. CONCLUSIONS: Reduced susceptibility to fluoroquinolones was associated with mutations at position 84 in gyrA. A further increase in fluoroquinolone MIC was associated with mutations in gyrA at positions 88 and 134, and parC at position 88. SIGNIFICANCE AND IMPACT OF THE STUDY: Due to limited resistant H. influenzae strains, prior studies on association between positions of mutations and fluoroquinolone MICs were inconclusive. The comparison of mutations between isolates with susceptibility, reduced susceptibility and high resistance supported the importance of the present study.


Assuntos
Anti-Infecciosos/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Fluoroquinolonas/farmacologia , Haemophilus influenzae/efeitos dos fármacos , DNA Girase/genética , DNA Topoisomerase IV/genética , Infecções por Haemophilus/microbiologia , Haemophilus influenzae/genética , Haemophilus influenzae/isolamento & purificação , Humanos , Testes de Sensibilidade Microbiana , Mutação , Casas de Saúde , Taiwan
12.
Int J Food Microbiol ; 317: 108461, 2020 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-31794931

RESUMO

Vibrio parahaemolyticus is the leading cause of foodborne bacterial poisoning in China. The aim of this research is to conduct a study on the prevalence, virulence, and antimicrobial resistance of V. parahaemolyticus from different types of food samples in 12 different cities of China. Since fluoroquinolones are the major choice of treatment for V. parahaemolyticus infections, the genetic basis for fluoroquinolone resistance in V. parahaemolyticus were also investigated. V. parahaemolyticus was detected in 163 of the 784 food samples collected from 12 different cities in China, resulting in a prevalence of 20.79%. The prevalence of V. parahaemolyticus in ready-to-eat (RTE) food (4.96%) was much lower than those of shrimp (32.62%) and fish (22.00%). Virulence gene screening showed that 44 (27.00%) V. parahaemolyticus strains carried at least one virulence gene. Four isolates from shrimp and three isolates from fish contained both the virulence genes tdh and trh. In addition, the trh was firstly detected in one isolate collected from RTE food. All isolates exhibited relatively high resistance rates to ampicillin (82.21%), gentamicin (19.63%), and tetracycline (14.11%), while <10% of strains were resistant to ciprofloxacin (4.91%), levofloxacin (4.91%), and tetracycline (4.29%). Eight fluoroquinolone-resistant V. parahaemolyticus were selected to determine the molecular basis for fluoroquinolone resistance. These eight isolates belonged to three different types according to enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR). A Ser83Ile substitution in GyrA was deteted in seven fluoroquinolone-resistant strains, except V209 which harbored a Ser83Phe substitution in GyrA. Moreover, A Ser85Leu substitution in ParC was found in five isolates (V52, V53, V61, V163, and V209). Plasmid-mediated quinolone resistance (PMQR) genes were detected in all eight fluoroquinolone-resistant V. parahaemolyticus strains. This is the first report of Ser83Phe substitution in GyrA, qnrD and qnrS1 in V. parahaemolyticus. The information generated in this study will provide valuable information for risk assessment of V. parahaemolyticus infections and future control of antibiotic-resistant V. parahaemolyticus species in China.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana/genética , Fluoroquinolonas/farmacologia , Doenças Transmitidas por Alimentos/epidemiologia , Vibrio parahaemolyticus/efeitos dos fármacos , Vibrio parahaemolyticus/genética , Substituição de Aminoácidos/genética , Ampicilina/farmacologia , Animais , China/epidemiologia , DNA Girase/genética , Fast Foods/microbiologia , Doenças Transmitidas por Alimentos/microbiologia , Gentamicinas/farmacologia , Humanos , Levofloxacino/farmacologia , Plasmídeos/genética , Prevalência , Alimentos Marinhos/microbiologia , Tetraciclina/farmacologia , Vibrio parahaemolyticus/isolamento & purificação , Vibrio parahaemolyticus/patogenicidade , Virulência/genética
14.
Artigo em Inglês | MEDLINE | ID: mdl-31740556

RESUMO

Quinolone resistance is increasing in Neisseria meningitidis, with its prevalence in China being high (>70%), but its origin remains unknown. The aim of this study was to investigate the donors of mutation-harboring gyrA alleles in N. meningitidis A total of 198 N. meningitidis isolates and 293 commensal Neisseria isolates were collected between 2005 and 2018 in Shanghai, China. The MICs of ciprofloxacin were determined using the agar dilution method. The resistance-associated genes gyrA and parC were sequenced for all isolates, while a few isolates were sequenced on the Illumina platform. The prevalences of quinolone resistance in the N. meningitidis and commensal Neisseria isolates were 67.7% (134/198) and 99.3% (291/293), respectively. All 134 quinolone-resistant N. meningitidis isolates possessed mutations in T91 (n = 123) and/or D95 (n = 12) of GyrA, with 7 isolates also harboring ParC mutations and exhibiting higher MICs. Phylogenetic analysis of the gyrA sequence identified six clusters. Among the 71 mutation-harboring gyrA alleles found in 221 N. meningitidis isolates and genomes (n = 221), 12 alleles (n = 103, 46.6%) were included in the N. meningitidis cluster, while 20 alleles (n = 56) were included in the N. lactamica cluster, 27 alleles (n = 49) were included in the N. cinerea cluster, and 9 alleles (n = 10) were included in the N. subflava cluster. Genomic analyses identified the exact N. lactamica donors of seven mutation-harboring gyrA alleles (gyrA92, gyrA97, gyrA98, gyrA114, gyrA116, gyrA151, and gyrA230) and the N. subflava donor isolate of gyrA171, with the sizes of the recombinant fragments ranging from 634 to 7,499 bp. Transformation of gyrA fragments from these donor strains into a meningococcal isolate increased its ciprofloxacin MIC from 0.004 µg/ml to 0.125 or 0.19 µg/ml and to 0.5 µg/ml with further transformation of an additional ParC mutation. Over half of the quinolone-resistant N. meningitidis isolates acquired resistance by horizontal gene transfer from three commensal Neisseria species. Quinolone resistance in N. meningitidis increases in a stepwise manner.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana/genética , Neisseria meningitidis/efeitos dos fármacos , Neisseria meningitidis/genética , Neisseria/efeitos dos fármacos , Quinolonas/farmacologia , China/epidemiologia , Ciprofloxacina/farmacologia , DNA Girase/genética , DNA Topoisomerase IV/genética , Transferência Genética Horizontal , Humanos , Testes de Sensibilidade Microbiana , Mutação/genética , Neisseria/genética , Filogenia , Prevalência , Transformação Bacteriana/genética
15.
Antonie Van Leeuwenhoek ; 113(4): 589-592, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31736013

RESUMO

The resistance to fluoroquinolones in corynebacteria is due to mutations occurring in the quinolone-resistance-determining region (QRDR) of the gyrA gene encoding the enzyme gyrase A subunit. In recent years we can observe an increasing number of infections caused by multidrug-resistant Corynebacterium striatum, Corynebacterium jeikeium and Corynebacterium urealyticum, including wide range of disorders, such as invasive infections. In this study 14 Corynebacterium spp. isolated from intravenous sites were sequenced and new combinations of mutations in the QRDR of the gyrA gene were found in C. jeikeium and C. urealyticum. Nowadays, no study comparing mutations in this region and the susceptibility to fluoroquinolones in C. jeikeium and C. urealyticum has been described. All the isolates that showed double mutation (position 87 and 91) in the QRDR gyrA gene had high MIC to the fluoroquinolones tested.


Assuntos
Antibacterianos/farmacologia , Corynebacterium/efeitos dos fármacos , Corynebacterium/genética , DNA Girase/genética , Farmacorresistência Bacteriana Múltipla/genética , Bacteriemia/sangue , Bacteriemia/microbiologia , Infecções Relacionadas a Cateter/microbiologia , Infecções por Corynebacterium/microbiologia , DNA Girase/metabolismo , Humanos , Injeções Intravenosas , Mutação
16.
G3 (Bethesda) ; 10(1): 79-88, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31744901

RESUMO

In light of the rising prevalence of antimicrobial resistance (AMR) and the slow pace of new antimicrobial development, there has been increasing interest in the development of adjuvants that improve or restore the effectiveness of existing drugs. Here, we use a novel small RNA (sRNA) screening approach to identify genes whose knockdown increases ciprofloxacin (CIP) sensitivity in a resistant strain of Escherichia coli 5000 sRNA constructs were initially screened on a gyrA S83L background, ultimately leading to 30 validated genes whose disruption reduces CIP resistance. This set includes genes involved in DNA replication, repair, recombination, efflux, and other regulatory systems. Our findings increase understanding of the functional interactions of DNA Gyrase, and may aid in the development of new therapeutic approaches for combating AMR.


Assuntos
Antibacterianos/farmacologia , Ciprofloxacina/farmacologia , Farmacorresistência Bacteriana/genética , Genes Bacterianos , DNA Girase/genética , DNA Girase/metabolismo , Escherichia coli , Interferência de RNA , Genética Reversa
17.
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
18.
J Infect Dis ; 221(5): 804-811, 2020 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-31573602

RESUMO

BACKGROUND: Tick-borne relapsing fever (TBRF) is a neglected zoonotic bacterial disease known to occur on 5 continents. We report a laboratory-acquired case of TBRF caused by Borrelia caucasica, which is endemic in Ukraine and transmitted by Ornithodoros verrucosus ticks. METHODS: We isolated spirochetes and characterized them by partially sequencing the 16s ribosomal ribonucleic acid (rrs), flagellin (flaB), and deoxyribonucleic acid gyrase (gyrB) genes and conducting a phylogenetic analysis. RESULTS: These analyses revealed a close relationship of Ukrainian spirochetes with the Asian TBRF species, Borrelia persica. The taxonomic and nomenclature problems related to insufficient knowledge on the spirochetes and their vectors in the region are discussed. CONCLUSIONS: Although these findings enhance our understanding of species identities for TBRF Borrelia in Eurasia, further work is required to address the neglected status of TBRF in this part of the world. Public health practitioners should consider TBRF and include the disease into differential diagnosis of febrile illnesses with unknown etiology.


Assuntos
Borrelia/genética , Ornithodoros/microbiologia , Febre Recorrente/diagnóstico , Febre Recorrente/epidemiologia , Spirochaetales/genética , Animais , Borrelia/isolamento & purificação , DNA Girase/genética , DNA Bacteriano/genética , Flagelina/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Camundongos , Ornithodoros/genética , Filogenia , RNA Ribossômico 16S/genética , Febre Recorrente/microbiologia , Febre Recorrente/transmissão , Análise de Sequência de DNA , Spirochaetales/isolamento & purificação , Ucrânia/epidemiologia
19.
Int J Med Microbiol ; 310(1): 151359, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31585716

RESUMO

Allicin (diallylthiosulfinate) is a potent antimicrobial substance, produced by garlic tissues upon wounding as a defence against pathogens and pests. Allicin is a reactive sulfur species (RSS) that oxidizes accessible cysteines in glutathione and proteins. We used a differential isotopic labelling method (OxICAT) to identify allicin targets in the bacterial proteome. We compared the proteomes of allicin-susceptible Pseudomonas fluorescens Pf0-1 and allicin-tolerant PfAR-1 after a sublethal allicin exposure. Before exposure to allicin, proteins were in a predominantly reduced state, with approximately 77% of proteins showing less than 20% cysteine oxidation. Protein oxidation increased after exposure to allicin, and only 50% of proteins from allicin-susceptible Pf0-1, but 65% from allicin-tolerant PfAR-1, remained less than 20% oxidised. DNA gyrase was identified as an allicin target. Cys433 in DNA gyrase subunit A (GyrA) was approximately 6% oxidized in untreated bacteria. After allicin treatment the degree of Cys433 oxidation increased to 55% in susceptible Pf0-1 but only to 10% in tolerant PfAR-1. Allicin inhibited E. coli DNA gyrase activity in vitro in the same concentration range as nalidixic acid. Purified PfAR-1 DNA gyrase was inhibited to greater extent by allicin in vitro than the Pf0-1 enzyme. Substituting PfAR-1 GyrA into Pf0-1 rendered the exchange mutants more susceptible to allicin than the Pf0-1 wild type. Taken together, these results suggest that GyrA was protected from oxidation in vivo in the allicin-tolerant PfAR-1 background, rather than the PfAR-1 GyrA subunit being intrinsically less susceptible to oxidation by allicin than the Pf0-1 GyrA subunit. DNA gyrase is a target for medicinally important antibiotics; thus, allicin and its analogues may have potential to be developed as gyrase inhibitors, either alone or in conjunction with other therapeutics.


Assuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , DNA Girase/metabolismo , Alho/química , Ácidos Sulfínicos/farmacologia , Inibidores da Topoisomerase II/farmacologia , Bactérias/enzimologia , Cisteína/metabolismo , DNA Girase/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Oxirredução , Proteoma , Pseudomonas fluorescens/efeitos dos fármacos
20.
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
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