Association of Salmonella mutants with germ-free rats: characterization of the reverse mutational response to 2-nitrofluorene.

Salmonella typhimurium strain TA1538 described by Ames, in association with otherwise germ-free rats, colonizes the gastrointestinal tract. The revertants enumerated in the feces of each of these rats varies in a coordinated manner in relation to the day on which the measurement is made. In response to the oral ingestion of a single dose of 2-nitrofluorene, the concentration of revertants in the feces increases and then returns essentially to control values within 6 days. When these rats are challenged repeatedly with a similar oral dose of 2-nitrofluorene the revertent concentration in the feces remains elevated for a progressively longer time. A change in phenotype of strain TA1538 during prolonged association rather than a change in the biology of the rat seems to explain this phenomenon. Firstly, germ-free rats exposed repeatedly to 2-nitrofluorene and then associated with strain TA1538 do not have the prolonged response characteristic of multiple challenges with 2-nitrofluorene. Secondly, strain TA1538 reisolated after several weeks of association with the otherwise germ-free rat shows other alterations such as decreased sensitivity to 2-nitrofluorene in the pour plate assay and a decreased sensitivity to crystal violet. In spite of limitations imposed by these alterations in strain TA1538, it is possible to demonstrate a dose-response relationship between the amount of 2-nitrofluorene ingested and the concentration of revertants in the feces of exposed rats.     

Estrogenic activity of an environmental pollutant, 2-nitrofluorene,after metabolic activation by rat liver microsomes

In this study, the metabolic activation of 2-nitrofluorene (NF) to estrogenic compounds was examined. NF was negative in estrogen reporter assays using estrogen-responsive yeast and human breast cancer cell line MCF-7. However, the compound exhibited estrogenic activity after incubation with liver microsomes of 3-methylcholanthrene-treated rats in the presence of NADPH. Minor estrogenic activity was observed when liver microsomes of untreated or phenobarbital-treated rats were used instead of those from 3-methylcholanthrene-treated rats. When the compound was incubated with the liver microsomes of 3-methylcholanthrene-treated rats in the presence of NADPH, 7-hydroxy-2-nitrofluorene (7-OH-NF) was formed as a major metabolite. However, little of the metabolite was formed by liver microsomes of untreated or phenobarbital-treated rats. Rat recombinant cytochrome P450 1A1 exhibited a significant oxidase activity toward NF, affording 7-OH-NF. Liver microsomes of phenobarbital-treated rats also enhanced oxidase activity toward NF. In this case, 9-hydroxy-2-nitrofluorene was formed. 7-OH-NF exhibited a significant estrogenic activity, while the activity of 9-hydroxy-2-nitrofluorene was much lower. These results suggest that the estrogenic activity of NF was due to formation of the 7-hydroxylated metabolite by liver microsomes.     

环丙沙星药物使用频度与细菌耐药性的相关性分析

目的回顾性分析我院喹诺酮类抗菌药物环丙沙星的使用频率和与临床分离主要细菌耐药性的相关性,为临床合理使用抗菌药物提供依据。方法回顾性分析中山大学附属第三医院2013年1月至2015年6月各季度环丙沙星使用频率与临床分离的5种主要细菌的耐药率,并对二者的相关性进行分析。结果 2013年1月至2015年6月各季度环丙沙星的使用DDDs值呈下降的趋势,随着环丙沙星使用频度的降低,大肠埃希菌和肺炎克雷伯杆菌对其耐药率呈正相关,差异有统计学意义(r=0.68,P0.05;r=0.68,P0.05)。鲍曼不动杆菌对环丙沙星的耐药率呈负相关,差异有统计学意义(r=-0.66,P0.05),铜绿假单胞菌和金黄色葡萄球菌其耐药率与药物使用频度无关(r=0.07,P0.05;r=0.01,P0.05)。结论细菌的耐药性和环丙沙星的使用频度具有一定相关性,应注意合理使用抗菌药物,减少多重耐药菌株的产生。     

Sublethal ciprofloxacin treatment leads to resistance via antioxidant systems in Proteus mirabilis

This study investigates new aspects of the possible role of antioxidant defenses in the mechanisms of resistance to ciprofloxacin in Proteus mirabilis. Four ciprofloxacin-resistant variants (CRVs), selected in vitro by repeated cultures in a sub-minimum inhibitory concentration (MIC) concentration of ciprofloxacin, attained different levels of antibiotic resistance and high Ferric reducing antioxidant power, with 10(-6) frequencies. However, no mutations occurred in positions 83 or 87 of gyrA, 464 or 466 of gyrB, or 78, 80 or 84 of parC, suggesting that resistance took place without these typical mutations in DNA gyrase or topoisomerase IV. Assays with ciprofloxacin and the pump inhibitor carbonyl cyanide m-chlorophenylhydrazone showed that in addition to the antioxidant mechanisms, the influx/efflux mechanism also contributed to the increase in the resistance to ciprofloxacin in one CRV. Moreover, lipid oxidation to malondialdehyde and protein oxidation to carbonyls and advanced oxidation protein products were higher in sensitive than in the resistant strains, as a new factor involved in the mechanisms of resistance in P. mirabilis. The oxidative stress cross-resistance to telluride in CRVs enhanced the role of the antioxidants in the ciprofloxacin resistance of P. mirabilis, which was reinforced during the assays of reduction of susceptibility to ciprofloxacin by glutathione and ascorbic acid.     

Contribution of the multidrug efflux pump LfrA to innate mycobacterial drug resistance

Multidrug resistance (MDR) in bacteria has been associated with efflux pumps that export structurally unrelated compounds and decrease cytoplasmic drug accumulation. To investigate MDR in mycobacteria, we studied the Mycobacterium smegmatis mutant mc(2)11, which is resistant to doxorubicin, tetracycline, rhodamine, ethidium bromide and the hydrophilic fluoroquinolones. A genomic library constructed from this mutant was used to select clones conferring resistance to doxorubicin. Surprisingly, the clone selected encodes the efflux pump LfrA, which has been reported to confer resistance to hydrophilic fluoroquinolones, ethidium bromide, rhodamine, and acriflavine. To define the contribution of LfrA to the innate mycobacterial drug resistance and to the MDR phenotype in mc(2)11, the lfrA gene was disrupted in both the mc(2)11 mutant and the mc(2)155 wild-type parent. LfrA disruption of the wild-type strain decreased resistance to ethidium bromide and acriflavine, and increased accumulation of ethidium bromide. However, disruption of lfrA gene results only in a 2-fold decrease in minimal inhibitory concentrations (MICs) for ciprofloxacin, doxorubicin, rhodamine, and accumulation of [(14)C]ciprofloxacin was unchanged. LfrA disruption of the MDR strain mc(2)11 produced a similar phenotype. Thus, LfrA contributes significantly to the intrinsic MICs of M. smegmatis for ethidium bromide and acriflavine, but not for ciprofloxacin, doxorubicin or rhodamine.     

Mutations in the gyrA and parC genes in ciprofloxacin-resistant clinical isolates of Acinetobacter baumannii in Korea

Mutation with Ser-83-->Leu in gyrA gene was associated with the principal mutation for ciprofloxacin resistance in clinical isolates of Acinetobacter baumannii. Double mutation, Ser-83-->Leu in gyrA gene and Ser-80-->Leu in parC gene, was the most frequently detected among ciprofloxacin-resistant isolates. A novel mutation with Ser-80-->Trp in parC gene, in addition to mutation in gyrA gene, was associated with a high-level ciprofloxacin resistance. These results suggested that the presence of an additional mutation in the parC gene contributed to a higher-level of ciprofloxacin resistance than a single mutation in the gyrA gene (geometric mean MICs of ciprofloxacin, 44.1 versus 16 microg/ml, P < 0.05).     

The role of RamA on the development of ciprofloxacin resistance in Salmonella enterica serovar Typhimurium

Active efflux pump is a primary fluoroquinolone resistant mechanism of clinical isolates of Salmonella enterica serovar Typhimurium. RamA is an essential element in producing multidrug resistant (MDR) S. enterica serovar Typhimurium. The aim of the present study was to elucidate the roles of RamA on the development of ciprofloxacin, the first choice for the treatment of salmonellosis, resistance in S. enterica serovar Typhimurium. Spontaneous mutants were selected via several passages of S. enterica serovar Typhimurium CVCC541 susceptible strain (ST) on M-H agar with increasing concentrations of ciprofloxacin (CIP). Accumulation of ciprofloxacin was tested by the modified fluorometric method. The expression levels of MDR efflux pumps were determined by real time RT-PCR. In ST and its spontaneous mutants, the ramA gene was inactivated by insertion of the kan gene and compensated on a recombinant plasmid pGEXΦ(gst-ramA). The mutant prevention concentration (MPC) and mutant frequencies of ciprofloxacin against ST and a spontaneous mutant in the presence, absence and overexpression of RamA were tested. Four spontaneous mutants (SI1-SI4) were obtained. The SI1 (CIP MICs, 0.1 mg/L) without any target site mutation in its quinolone resistant determining regions (QRDRs) and SI3 (CIP MICs, 16 mg/L) harboring the Ser83→Phe mutation in its QRDR of GyrA strains exhibited reduced susceptibility and resistance to multidrugs, respectively. In SI1, RamA was the main factor that controlled the susceptibility to ciprofloxacin by activating MdtK as well as increasing the expression level of acrAB. In SI3, RamA played predominant role in ciprofloxacin resistance via increasing the expression level of acrAB. Likewise, the deficiency of RamA decreased the MPCs and mutant frequencies of ST and SI2 to ciprofloxacin. In conclusion, the expression of RamA promoted the development of ciprofloxacin resistant mutants of S. enterica serovar Typhimurium. The inhibition of RamA could decrease the appearance of the ciprofloxacin resistant mutants.     

Emerging cystic fibrosis pathogens: incidence and antimicrobial resistance

We examined the frequency of isolation and the antimicrobial resistance of Burkholderia cepacia complex, Stenotrophomonas maltophilia and Achromobacter xylosoxidans in cystic fibrosis patients from 2000 to 2004. Strains susceptibility to tobramycin, piperacillin/tazobactam, imipenem, gentamicin, ciprofloxacin and ceftazidime was determined by disc diffusion assay. B. cepacia complex showed a very high resistance also to ciprofloxacin reaching 100% in 2004. S. maltophilia and A. xvylosoxidans showed high rates of antimicrobial resistance both aminoglycoside and ciprofloxacin. It is very important to monitor the percentage of isolation of these species over time to verify strains resistance to antibiotics and also to test new combinations of antimicrobial agents.     

Analysis of the Membrane Proteome of Ciprofloxacin-Resistant Macrophages by Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC)

Overexpression of multidrug transporters is a well-established mechanism of resistance to chemotherapy, but other changes may be co-selected upon exposure to drugs that contribute to resistance. Using a model of J774 macrophages made resistant to the fluoroquinolone antibiotic ciprofloxacin and comparing it with the wild-type parent cell line, we performed a quantitative proteomic analysis using the stable isotope labeling with amino acids in cell culture technology coupled with liquid chromatography electrospray ionization Fourier transform tandem mass spectrometry (LC-ESI-FT-MS/MS) on 2 samples enriched in membrane proteins (fractions F1 and F2 collected from discontinuous sucrose gradient). Nine hundred proteins were identified with at least 3 unique peptides in these 2 pooled fractions among which 61 (F1) and 69 (F2) showed a significantly modified abundance among the 2 cell lines. The multidrug resistance associated protein Abcc4, known as the ciprofloxacin efflux transporter in these cells, was the most upregulated, together with Dnajc3, a protein encoded by a gene located downstream of Abcc4. The other modulated proteins are involved in transport functions, cell adhesion and cytoskeleton organization, immune response, signal transduction, and metabolism. This indicates that the antibiotic ciprofloxacin is able to trigger a pleiotropic adaptative response in macrophages that includes the overexpression of its efflux transporter.     

环丙沙星耐药的铜绿假单胞菌药敏情况分析

目的了解对环丙沙星耐药的铜绿假单胞菌的药敏情况。方法细菌的鉴定及药敏试验,均采用法国生物梅里埃公司的VITEK2微生物鉴定与药敏系统,选择经VITEK2测定耐环丙沙星的铜绿假单胞菌(耐药株)68株及对环丙沙星敏感的铜绿假单胞菌(敏感株)67株,统计分析两者对亚胺培南、阿米卡星、庆大霉素、头孢他啶、哌拉西林和哌拉西林他唑巴坦等几种临床上常用抗生素的药敏情况。结果耐药株对以上6种抗生素的敏感率分别为亚胺培南706%,阿米卡星206%,庆大霉素191%,头孢他啶279%,哌拉西林632%,哌拉西林他唑巴坦721%。而敏感株对6种抗生素的敏感率则分别为925%、328%、687%、657%、881%与896%,明显高于耐药株,经χ2检验,除阿米卡星005相似文献   

Antimutagenic effects of mushrooms

A heat-resistant factor in ethanol extracts of the fungus Craterellus cornucopioides completely inhibited the mutagenicity of aflatoxin B1, benzo[a]pyrene, the acridine half mustard ICR-191 and 2-nitrofluorene in a forward-mutation system using Salmonella typhimurium TM677 (screening for 8-azaguanine resistance). There was no inhibitory effect on the mutagenic activity of 4-nitroquinoline-N-oxide, methyl methanesulfonate or N-methyl-N'-nitro-N-nitrosoguanidine. Experiments performed to elucidate the mechanism of the antimutagenic effect showed that neither an alteration of cell viability nor an interference with the excision-repair and the inducible SOS-repair system was involved. The conceivable mechanisms for the antimutagenicity of the ethanol extract include direct chemical interaction with the mutagen and/or inhibition of the activation process in the case of the promutagens. The antimutagenic activity of Craterellus cornucopioides is not unique among mushroom species. The ethanol extracts of 6 other mushrooms showed a similar antimutagenic activity.     

Biotransformation of 1-nitropyrene and 2-nitrofluorene to novel metabolites, the corresponding formylamino compounds, in animal bodies

The present study provides the first evidence that nitropolycyclic aromatic hydrocarbons such as 1-nitropyrene and 2-nitrofluorene are metabolized to the corresponding formylamino compounds in animal bodies. The study shows that the formation of such novel metabolites, 1-formylaminopyrene and 2-formylaminofluorene, is due to N-formylation of the nitroreduction products, 1-aminopyrene and 2-aminofluorene, by liver formamidase in the presence of N-formyl-L-kynurenine.     

Antimicrobial-resistant Campylobacter species from retail raw meats

The antimicrobial susceptibilities of 378 Campylobacter isolates were determined. Resistance to tetracycline was the most common (82%), followed by resistance to doxycycline (77%), erythromycin (54%), nalidixic acid (41%), and ciprofloxacin (35%). Campylobacter coli displayed significantly higher rates of resistance to ciprofloxacin and erythromycin than Campylobacter jejuni, and Campylobacter isolates from turkey meat showed a greater resistance than those from chicken meat.     

The effect of phenolic and polyphenolic compounds on the development of drug resistance

The effect of two phenolic compounds vanillin (4-hydroxy-3-methoxybenzaldehyde) and lignin on the development of drug/antibiotic resistance in Salmonella typhimurium was studied. Using the modified Ames test we have shown that vanillin alone has negligible effect on spontaneous mutability to ciprofloxacin and gentamicin resistance. At the tested concentrations vanillin reduces the toxicity of 4-nitroquinoline-N-oxide (4NQO) and reduces the ability of this compound to induce mutations leading to ciprofloxacin but not to gentamicin resistance. Lignin at higher concentrations increases mutagenicity to ciprofloxacin resistance and possess considerable inhibition effect on the spontaneous and 4NQO induced mutability to gentamicin resistance.     

Changes in Acholeplasma laidlawii plasma membrane functions during development of resistance to ciprofloxacin and tetracycline]

The role of transport activity of Acholeplasma laidlawii plasmatic membrane in the development of resistance to ciprofloxacin and tetracycline was investigated. It was shown that one of the important steps of resistance development in acholeplasms is a complex of adaptation metabolic reactions providing limited antibiotic accumulation by the cells. In the case of ciprofloxacin resistance metabolism changes concerning transport systems took place before mutations in target genes. Development of tetracycline resistance of mollecutes after incubation in the medium with enhancing antibiotic concentrations and not connected with the presence of tet(M) determinant was demonstrated for the first time.     

Resistance to ciprofloxacin by enhancement of antioxidant defenses in biofilm and planktonic Proteus mirabilis

Antibiotic resistance and antioxidant defense were induced by ciprofloxacin in planktonic Proteus mirabilis and compared with the natural antibiotic resistance of biofilm. Resistant variants (1X and 1Y) were obtained from cultures of the sensitive wild type “wt” strain 1 in the presence of the antibiotic. Planktonic strain 1 exhibited oxidative stress with increases in the reactive oxygen species (ROS) and consumption of NO in the presence of ciprofloxacin, whereas 1X and 1Y suffered non-significant rises in ROS generation, but produced and consumed more NO than sensitive strain 1. The two resistant variants were more resistant to telluride than wt and showed increased levels of intracellular superoxide dismutase (SOD) and glutathione (GSH). However, ciprofloxacin did not stimulate oxidative stress in biofilm. The production of ROS and NO with or without ciprofloxacin was less significant in biofilms than in an equivalent number of planktonic bacteria; sensitive and resistant strains did not present differences. On the other hand, SOD and GSH were more elevated in the biofilm than in planktonic bacteria. In summary, these results indicate that ciprofloxacin can induce resistance by the enhancement of antioxidant defense in planktonic bacteria, similar to the natural resistance occurring in biofilm. This feature may be added to the factors that regulate the susceptibility to this antibiotic.     

Influence of recA mutations on gyrA dependent quinolone resistance.

We examined, in Escherichia coli, the influence of recA mutant alleles on the level of quinolone resistance promoted by mutations in the gyrA gene. We found that the recA142 mutation, abolishing all the activities of RecA protein, greatly reduced the level of resistance to the quinolone ciprofloxacin, whereas the recA430 allele affecting the SOS inducing ability of RecA, reduced ciprofloxacin resistance to a lesser extent. The recA142 mutation did not cause enhancement of ciprofloxacin induced DNA breakage in gyrA mutants, indicating that the stabilization of DNA-gyrase complexes by the quinolone is not influenced by a RecA mutant protein. We suggest that RecA protein plays a role in the repair of quinolone damage, principally through a recombinational mechanism and, to a lesser degree, through the induction of the SOS response.     

The SOS response increases bacterial fitness,but not evolvability,under a sublethal dose of antibiotic

Exposure to antibiotics induces the expression of mutagenic bacterial stress–response pathways, but the evolutionary benefits of these responses remain unclear. One possibility is that stress–response pathways provide a short-term advantage by protecting bacteria against the toxic effects of antibiotics. Second, it is possible that stress-induced mutagenesis provides a long-term advantage by accelerating the evolution of resistance. Here, we directly measure the contribution of the Pseudomonas aeruginosa SOS pathway to bacterial fitness and evolvability in the presence of sublethal doses of ciprofloxacin. Using short-term competition experiments, we demonstrate that the SOS pathway increases competitive fitness in the presence of ciprofloxacin. Continued exposure to ciprofloxacin results in the rapid evolution of increased fitness and antibiotic resistance, but we find no evidence that SOS-induced mutagenesis accelerates the rate of adaptation to ciprofloxacin during a 200 generation selection experiment. Intriguingly, we find that the expression of the SOS pathway decreases during adaptation to ciprofloxacin, and this helps to explain why this pathway does not increase long-term evolvability. Furthermore, we argue that the SOS pathway fails to accelerate adaptation to ciprofloxacin because the modest increase in the mutation rate associated with SOS mutagenesis is offset by a decrease in the effective strength of selection for increased resistance at a population level. Our findings suggest that the primary evolutionary benefit of the SOS response is to increase bacterial competitive ability, and that stress-induced mutagenesis is an unwanted side effect, and not a selected attribute, of this pathway.     

Ciprofloxacin is an inhibitor of the Mcm2-7 replicative helicase

Most currently available small molecule inhibitors of DNA replication lack enzymatic specificity, resulting in deleterious side effects during use in cancer chemotherapy and limited experimental usefulness as mechanistic tools to study DNA replication. Towards development of targeted replication inhibitors, we have focused on Mcm2-7 (minichromosome maintenance protein 2–7), a highly conserved helicase and key regulatory component of eukaryotic DNA replication. Unexpectedly we found that the fluoroquinolone antibiotic ciprofloxacin preferentially inhibits Mcm2-7. Ciprofloxacin blocks the DNA helicase activity of Mcm2-7 at concentrations that have little effect on other tested helicases and prevents the proliferation of both yeast and human cells at concentrations similar to those that inhibit DNA unwinding. Moreover, a previously characterized mcm mutant (mcm4chaos3) exhibits increased ciprofloxacin resistance. To identify more potent Mcm2-7 inhibitors, we screened molecules that are structurally related to ciprofloxacin and identified several that compromise the Mcm2-7 helicase activity at lower concentrations. Our results indicate that ciprofloxacin targets Mcm2-7 in vitro, and support the feasibility of developing specific quinolone-based inhibitors of Mcm2-7 for therapeutic and experimental applications.     

Antibiotic resistant N. gonorrhoeae in Trinidad and Tobago.

We tested the susceptibility patterns of 128 N. gonorrhoeae isolates to six antimicrobials; penicillin, tetracycline, spectinomycin, ceftriaxone, ciprofloxacin and azithromycin, and examined whether certain demographic or behavioral factors related to antibiotic use increased the likelihood of infection by a resistant strain. There was a low rate of resistance to penicillin; penicillinase-producing and chromosomal-mediated penicillin resistant gonorrhea were estimated to be 0.8%. A much higher proportion of isolates were resistant to tetracycline (up to 15%). All isolates were sensitive to spectinomycin, ciprofloxacin and ceftriaxone. However, less than 2% of isolates displayed intermediate resistance to both ciprofloxacin and ceftriaxone, and 9% exhibited intermediate resistance to spectinomycin. Patients who had obtained medication before attending the clinic and had taken all of the medication were more likely (p = 0.03) to be infected with a resistant strain of gonococcus. Also, patients who were asked by a clinic doctor to return for a test of cure during an earlier clinic visit, but who did not return were more likely to be infected with a resistant organism (p = 0.006) compared to those who returned at the doctor's request. These findings have important implications for antibiotic use and educational programs in Trinidad and Tobago.