RESUMO
OBJECTIVES: To investigate the potential relationship between quinolone resistance and biofilm production in a collection of Salmonella enterica clinical isolates and in S. enterica serovar Typhimurium serial mutants with increasing resistance to ciprofloxacin. METHODS: Nalidixic acid susceptibility and biofilm formation were assessed in a collection of 122 S. enterica clinical isolates. An in vitro quinolone-resistant mutant, 59-64, was obtained from a biofilm-producing and quinolone-susceptible clinical isolate, 59-wt, in a multistep selection process after increasing ciprofloxacin concentrations. The quinolone resistance mechanisms [target gene and multidrug resistance (MDR) regulatory mutations, MICs of several antibiotics, cell envelope protein analysis, real-time PCR and ciprofloxacin accumulation] were characterized for mutant strains. In addition, analysis of fitness, biofilm formation, rdar morphotype and expression of biofilm-related genes by real-time PCR were also determined. RESULTS: Nalidixic acid-susceptible S. enterica strains were more prevalent in producing biofilm than the resistant counterparts. Strain 59-64 acquired five target gene mutations and showed an MDR phenotype. AcrAB and acrF overexpression were ruled out, whereas TolC did show increased expression in 59-64, which, in addition, accumulated less ciprofloxacin. Consistently, increased ramA expression was seen in 59-64 and attributed to a mutation within its promoter. Reduced biofilm production related to diminished csgB expression as well as reduced fitness was seen for 59-64, which was unable to form the rdar morphotype. CONCLUSIONS: Quinolone resistance acquisition may be associated with decreased production of biofilm due to lower csgB expression. Efflux, biofilm production and fitness seem to be interrelated.
Assuntos
Antibacterianos/farmacologia , Biofilmes/crescimento & desenvolvimento , Farmacorresistência Bacteriana , Quinolonas/farmacologia , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/fisiologia , Proteínas de Bactérias/análise , Ciprofloxacina/farmacologia , Perfilação da Expressão Gênica , Humanos , Testes de Sensibilidade Microbiana , Mutação , Ácido Nalidíxico/farmacologia , Reação em Cadeia da Polimerase em Tempo Real , Infecções por Salmonella/microbiologia , Salmonella typhimurium/isolamento & purificação , Seleção Genética , Inoculações SeriadasRESUMO
The aim of this study was to compare different fluoroquinolones against Campylobacter spp., analysing the molecular mechanisms of resistance. Moxifloxacin exhibited the greatest activity of the quinolones tested, being active against isolates carrying a single mutation in the gyrA gene. High resistance levels to moxifloxacin were related to the presence of a double gyrA mutation.
Assuntos
Antibacterianos/farmacologia , Compostos Aza/farmacologia , Campylobacter/efeitos dos fármacos , DNA Girase/genética , Farmacorresistência Bacteriana/genética , Mutação , Quinolinas/farmacologia , Substituição de Aminoácidos/genética , Campylobacter/genética , DNA Girase/efeitos dos fármacos , Fezes/microbiologia , Fluoroquinolonas , Humanos , Testes de Sensibilidade Microbiana , MoxifloxacinaRESUMO
A total of 33 Stenotrophomonas maltophilia clinical isolates were tested for their susceptibility to clinafloxacin in comparison with ciprofloxacin, levofloxacin, moxifloxacin, nalidixic acid, norfloxacin, sparfloxacin and trovafloxacin. The MIC(50) and MIC(90) were as follows: ciprofloxacin 4 and 64 microg/mL; clinafoxacin 0.5 and 4 microg/mL; levofloxacin 2 and 32 microg/mL; moxifloxacin 1 and 8 microg/mL; nalidixic acid 8 and 128 microg/mL; norfloxacin 64 and 256 microg/mL; sparfloxacin 1 and 16 microg/mL; and trovafloxacin 1 and 8 microg/mL. Clinafloxacin was the most active quinolone, with only a 15.1% of strains showing resistance. When the MICs were determined in the presence of 25 microg/ml of reserpine, the MIC(90) of trovafloxacin and moxifloxacin did not change, whereas decreased 2-fold for clinafloxacin, levofloxacin, sparfloxacin and nalidixic acid, and 4- and 8-fold for ciprofloxacin and norfloxacin respectively. No clinafloxacin-resistant strains were observed when the MIC was performed in the presence of reserpine. Therefore, clinafloxacin shows the better "in vitro"activity against these 33 strains of S.maltophilia.
Assuntos
Anti-Infecciosos/farmacologia , Fluoroquinolonas , Reserpina/farmacologia , Stenotrophomonas maltophilia/efeitos dos fármacos , Farmacorresistência Bacteriana , Infecções por Bactérias Gram-Negativas/microbiologia , Humanos , Testes de Sensibilidade Microbiana/métodos , Testes de Sensibilidade Microbiana/tendênciasRESUMO
The MIC of gemifloxacin and five other quinolones was tested against 31 clinical isolates of Neisseria gonorrhoeae; strains were analyzed for the presence of mutations in both the gyrA and parC genes. Only seven strains were resistant to nalidixic acid due to a mutation in the gyrA gene but not in the parC gene, with six and two considered intermediate to ciprofloxacin and levofloxacin, respectively. The activity of gemifloxacin was similar to that of trovafloxacin and moxifloxacin, but was more active than nalidixic acid, ciprofloxacin or levofloxacin against the gyrA mutant strains. Gemifloxacin is a valid therapeutic alternative to treat infections with N. gonorrhoeae, retaining its activity against strains already presenting a mutation in gyrA.
Assuntos
Anti-Infecciosos/farmacologia , Compostos Aza , DNA Girase/genética , Fluoroquinolonas , Genes Bacterianos , Naftiridinas/farmacologia , Neisseria gonorrhoeae/efeitos dos fármacos , Neisseria gonorrhoeae/genética , Quinolinas , Ciprofloxacina/farmacologia , DNA Topoisomerase IV/genética , Farmacorresistência Bacteriana , Gemifloxacina , Gonorreia/tratamento farmacológico , Gonorreia/microbiologia , Humanos , Técnicas In Vitro , Levofloxacino , Testes de Sensibilidade Microbiana , Moxifloxacina , Mutação , Ácido Nalidíxico/farmacologia , Neisseria gonorrhoeae/enzimologia , Neisseria gonorrhoeae/isolamento & purificação , Ofloxacino/farmacologiaRESUMO
BACKGROUND: Nalidixic acid resistance among Salmonella Typhimurium clinical isolates has steadily increased, whereas the level of ciprofloxacin resistance remains low. The main objective of this study was to characterize the fluoroquinolone resistance mechanisms acquired in a S. Typhimurium mutant selected with ciprofloxacin from a susceptible isolate and to investigate its invasion ability. METHODOLOGY/PRINCIPAL FINDINGS: Three different amino acid substitutions were detected in the quinolone target proteins of the resistant mutant (MIC of ciprofloxacin, 64 microg/ml): D87G and G81C in GyrA, and a novel mutation, E470K, in ParE. A protein analysis revealed an increased expression of AcrAB/TolC and decreased expression of OmpC. Sequencing of the marRAB, soxRS, ramR and acrR operons did not show any mutation and neither did their expression levels in a microarray analysis. A decreased percentage of invasion ability was detected when compared with the susceptible clinical isolate in a gentamicin protection assay. The microarray results revealed a decreased expression of genes which play a role during the invasion process, such as hilA, invF and the flhDC operon. Of note was the impaired growth detected in the resistant strain. A strain with a reverted phenotype (mainly concerning the resistance phenotype) was obtained from the resistant mutant. CONCLUSIONS/SIGNIFICANCE: In conclusion, a possible link between fluoroquinolone resistance and decreased cell invasion ability may exist explaining the low prevalence of fluoroquinolone-resistant S. Typhimurium clinical isolates. The impaired growth may appear as a consequence of fluoroquinolone resistance acquisition and down-regulate the expression of the invasion genes.
Assuntos
Farmacorresistência Bacteriana , Fluoroquinolonas/farmacologia , Mutação , Salmonella typhimurium/genética , Antibacterianos/farmacologia , Ciprofloxacina/farmacologia , Regulação Bacteriana da Expressão Gênica , Células HeLa , Humanos , Modelos Genéticos , Ácido Nalidíxico/farmacologia , Invasividade Neoplásica , Fenótipo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição GênicaRESUMO
INTRODUCTION: The objective of this investigation was to identify the mechanism of decreased susceptibility to gentamicin in a Salmonella clinical isolate, leading to the detection of a aminoglycoside acetyltransferase gene found in a class 1 integron. METHODS: A multidrug-resistant Salmonella strain was recovered from feces of a traveler to Egypt. The antimicrobial susceptibility test to 12 antimicrobial agents was performed with the Kirby-Bauer method. The presence of class 1 integron was determined by PCR. The amplified product was recovered and sequenced in order to establish the genes carried. In addition, susceptibility to gentamicin C1a, gentamicin C1, sisomicin, neomycin, dibekacin, kanamycin, tobramycin, amikacin, netilmicin, apramycin, dactimicin, spectinomycin, streptomycin, lividomycin and butirosin, was established. The Champion pET101 Directional TOPO Expression Kit was used to clone and express the aac(3)-I gene. RESULTS: The isolate was identified as Salmonella enterica serovar Haifa, showing resistance to nalidixic acid, tetracycline and decreased susceptibility to gentamicin. One integron with a size circa 1,500 bp, encoding an aac(3)-Id plus aadA7 genes was observed. The analysis of the susceptibility to different aminoglycosides in the E. coli TOP10F' transformed with the vector carrying aac(3)-Id gene showed resistance to gentamicin C1a, gentamicin C1, and dactimicin, in accordance with the presence of this enzyme but, was susceptible to sisomicin. The homology of the amino acid and nucleotide sequences with the AAC(3)-Id enzyme was of 100%. CONCLUSION: The presence of the AAC(3)-Id enzyme was described for the first time in a S. Haifa.
Assuntos
Acetiltransferases/isolamento & purificação , Diarreia/microbiologia , Salmonella enterica/classificação , Salmonella enterica/enzimologia , Viagem , Humanos , Salmonella enterica/isolamento & purificaçãoRESUMO
Molecular analysis of 63 Escherichia coli urine isolates showed that pyelonephritis (n=23) and prostatitis (n=17) isolates exhibited more virulence factors (VFs) among the 35 sought than did cystitis isolates (n=23). Several nontraditional VFs--including bmaE (M fimbriae), gafD (G fimbriae), fyuA (yersiniabactin receptor), ireA and iroN (novel siderophore receptors), cvaC (colicin [microcin] V), traT (serum-resistance associated), ibeA (invasion of brain endothelium), ompT (outer membrane protease T), and malX (pathogenicity island marker)--either differentiated significantly between syndromes (despite small numbers of isolates and possible multiple-comparison artifacts) or were broadly prevalent. Thus, interventions that target conserved uro-VFs may be possible, despite the likely existence of syndrome-specific pathogenetic mechanisms and/or host defense systems.
Assuntos
Cistite/microbiologia , Infecções por Escherichia coli/microbiologia , Escherichia coli/genética , Prostatite/microbiologia , Pielonefrite/microbiologia , Fatores de Virulência/genética , Adesinas Bacterianas/genética , Adulto , Idoso , Proteínas da Membrana Bacteriana Externa/genética , Bacteriocinas/genética , Escherichia coli/classificação , Escherichia coli/isolamento & purificação , Escherichia coli/patogenicidade , Proteínas de Escherichia coli/genética , Feminino , Proteínas de Fímbrias/genética , Fímbrias Bacterianas/genética , Ilhas Genômicas/genética , Humanos , Lectinas/genética , Masculino , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Peptídeo Hidrolases , Fenóis/análise , Porinas/genética , Receptores de Superfície Celular/genética , Espanha , Tiazóis/análise , Urina/microbiologia , Fatores de Virulência/análiseRESUMO
The prevalence of 31 virulence factors was analyzed among nalidixic acid-susceptible and -resistant Escherichia coli strains from phylogenetic group B2. Hemolysin, cytotoxic necrotizing factor 1, and S and F1C fimbriae genes were less prevalent among nalidixic acid-resistant E. coli strains. Quinolone resistance may be associated with a decrease in the presence of some virulence factors.
Assuntos
Anti-Infecciosos/farmacologia , Farmacorresistência Bacteriana , Escherichia coli/efeitos dos fármacos , Quinolonas/farmacologia , Doenças Urológicas/microbiologia , Fatores de Virulência/genética , Escherichia coli/classificação , Escherichia coli/genética , Escherichia coli/patogenicidade , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Feminino , Humanos , Masculino , Testes de Sensibilidade Microbiana , Ácido Nalidíxico/farmacologia , Filogenia , Virulência , Fatores de Virulência/metabolismoRESUMO
The distribution of beta-lactamases in a group of 20 epidemiologically well defined Acinetobacter baumannii clinical isolates and the in vitro activity of Syn 2190, a novel beta-lactamase AmpC inhibitor, were determined. Twenty-five per cent of the strains carried and expressed a TEM-type beta-lactamase, whereas 35% had an OXA-type beta-lactamase. In nine out of 11 (82%) ceftazidime-resistant and four out of 13 (30.7%) cefepime-resistant strains, the MIC of these beta-lactam antibiotics decreased when determined in the presence of Syn 2190. Thus, our results suggest that in a high percentage of A. baumannii clinical isolates the increased production of AmpC, in combination or not with other resistance mechanisms, contributes to the resistance pattern in A. baumannii to beta-lactams.
Assuntos
Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/enzimologia , Antibacterianos/farmacologia , Proteínas de Bactérias , Monobactamas/farmacologia , Inibidores de beta-Lactamases , Infecções por Acinetobacter/tratamento farmacológico , Infecções por Acinetobacter/epidemiologia , Infecções por Acinetobacter/microbiologia , Acinetobacter baumannii/isolamento & purificação , Inibidores Enzimáticos/farmacologia , Humanos , Testes de Sensibilidade Microbiana/estatística & dados numéricos , beta-Lactamases/metabolismoRESUMO
The evolution of antimicrobial resistance in Salmonella isolates causing traveler's diarrhea (TD) and their mechanisms of resistance to several antimicrobial agents were analyzed. From 1995 to 2002, a total of 62 Salmonella strains were isolated from stools of patients with TD. The antimicrobial susceptibility to 12 antibiotics was determined, and the molecular mechanisms of resistance to several of them were detected as well. The highest levels of resistance were found against tetracycline and ampicillin (21 and 19%, respectively), followed by resistance to nalidixic acid (16%), which was mainly detected from 2000 onward. Molecular mechanisms of resistance were analyzed in 16 isolates. In these isolates, which were resistant to ampicillin, two genes encoding beta-lactamases were detected: oxa-1 (one isolate) and tem-like (seven isolates [in one strain concomitantly with a carb-2]). Resistance to tetracycline was mainly related to tetA (five cases) and to tetB and tetG (one case each). Resistance to chloramphenicol was related to the presence of the floR and cmlA genes and to chloramphenicol acetyltransferase activity in one case each. Different genes encoding dihydrofolate-reductases (dfrA1, dfrA12, dfrA14, and dfrA17) were detected in trimethoprim-resistant isolates. Resistance to nalidixic acid was related to the presence of mutations in the amino acid codons 83 or 87 of the gyrA gene. Further surveillance of the Salmonella spp. causing TD is needed to detect trends in their resistance to antimicrobial agents, as we have shown in our study with nalidixic acid. Moreover, such studies will lead to better treatment and strategies to prevent and limit their spread.
Assuntos
Antibacterianos/farmacologia , Diarreia/tratamento farmacológico , Diarreia/microbiologia , Infecções por Salmonella/microbiologia , Salmonella/efeitos dos fármacos , Viagem , Tipagem de Bacteriófagos , DNA Girase/genética , Primers do DNA , DNA Topoisomerase IV/genética , DNA Bacteriano/genética , Farmacorresistência Bacteriana , Testes de Sensibilidade Microbiana , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Salmonella/genética , Infecções por Salmonella/tratamento farmacológico , SorotipagemRESUMO
The in vitro activity of clinafloxacin was studied in comparison with ciprofloxacin, levofloxacin, moxifloxacin, nalidixic acid, sparfloxacin and trovafloxacin against Acinetobacter baumannii clinical isolates. Clinafloxacin showed a MIC(90) of 4 mg/L, whereas the remaining quinolones showed a MIC(90) equal to or higher than 16 mg/L. MIC(50) determination in the presence of reserpine resulted in a two-fold decrease, except for trovafloxacin, which decreased four-fold, and for moxifloxacin and nalidixic acid, which did not change. The effect of reserpine was most pronounced among strains with a low level of resistance to quinolones. The MIC of clinafloxacin for strains with no mutation in either gyrA or parC genes ranged from 0.008 to 0.25 mg/L. In strains with a single mutation at amino acid codon Ser83 of the gyrA gene, the MIC of clinafloxacin ranged from 0.12 to 1 mg/L, whereas strains with a double mutation, one in the gyrA gene and another in the parC gene, showed a range of MIC of clinafloxacin from 1 to 8 mg/L. Therefore, clinafloxacin shows good activity against strains carrying a single mutation in the gyrA gene, and hence a second mutation is required for the microorganism to express resistance.