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Objective@#To evaluate the in vitro activity of ceftazidime-avibactam (CAZ-AVI) alone or in combination with colistin (COL) against clinically isolated extensively drug-resistant Pseudomonas aeruginosa (XDR-PA).@*Methods@#Minimum inhibitory concentration (MIC) of 16 clinical XDR-PA isolates was determined by broth dilution method and chessboard design when CAZ-AVI and COL were used alone or in combination, then the combined inhibitory concentration index (FICI) was calculated. Class A [Klebsiella pneumoniae carbapenemase β-lactamase (blaKPC), Guiana extended-spectrum β-lactamase (blaGES)], Class B [imipenemase β-lactamase (blaIMP), Verona-Integronmetallo β-lactamase (blaVIM), New Delhi metallo β-lactamase (blaNDM), German imipenemase β-lactamase (blaGIM), Sao Paulo metallo -β- lactamase (blaSPM)], Class C [AmpC β-lactamase (blaAmpC)], Class D [oxacillinase β-lactamase (blaOXA)] β- lactamase-related resistance genes were detected by polymerase chain reaction. Drug-resistant mutation frequencies of each strain were determined on a drug-containing plate. The time kill curves of three XDR-PA were plotted by colony counting method. A biofilm model was established in vitro, and the synergistic effect of CAZ-AVI and COL on biofilm inhibition was detected by methythiazolyl tetrazolium assay (MTT).@*Results@#The MICs of 16 XDR-PA for CAZ-AVI ranged from 1 mg/L to 128 mg/L, and three of the isolates showed resistance (MIC > 8 mg/L). The FICI range of CAZ-AVI combined with COL was 0.312-1.000. Four isolates were synergistic, while the other 12 isolates were additive. Three isolates resistant to CAZ-AVI contained Class B resistance genes such as blaIMP and blaVIM, while 13 susceptible isolates carried resistance genes belonging to Class A, C or D. The logarithm values of mutation frequencies of drug resistance in CAZ-AVI group, COL group and combination group were -4.81±0.88, -7.06±0.69 and -9.70 (-9.78, -9.53), respectively. There were significant differences among the three groups (H = 33.601, P < 0.001), and between every two groups (adjusted P < 0.05). In time kill curves, the phytoplankton load of three XDR-PA decreased more than 6 log CFU/L when these two drugs were used together, and number of PA1819 planktonic bacteria decreased more than 5.1 log CFU/L compared with monotherapy group. Viable quantity in biofilm (A490) of normal saline group, CAZ-AVI group, COL group and CAZ-AVI-COL group were 0.665±0.068, 0.540±0.072, 0.494±0.642 and 0.317±0.080, respectively. There was significant difference between the other two groups (all P < 0.001), except for that between CAZ-AVI group and COL group (P = 0.109).@*Conclusions@#CAZ-AVI combined with COL can effectively improve the bactericidal effect of each drug alone on XDR-PA. The regimen can also reduce the production of drug-resistant bacteria and inhibit the formation of biofilm. Therefore, it is a potential treatment for XDR-PA infection.
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Objective To evaluate the in vitro activity of ceftazidime-avibactam (CAZ-AVI) alone or in combination with colistin (COL) against clinically isolated extensively drug-resistant Pseudomonas aeruginosa (XDR-PA). Methods Minimum inhibitory concentration (MIC) of 16 clinical XDR-PA isolates was determined by broth dilution method and chessboard design when CAZ-AVI and COL were used alone or in combination, then the combined inhibitory concentration index (FICI) was calculated. Class A [Klebsiella pneumoniae carbapenemase β-lactamase (blaKPC), Guiana extended-spectrum β-lactamase (blaGES)], Class B [imipenemase β-lactamase (blaIMP), Verona-Integronmetallo β-lactamase (blaVIM), New Delhi metallo β-lactamase (blaNDM), German imipenemase β-lactamase (blaGIM), Sao Paulo metallo -β- lactamase (blaSPM)], Class C [AmpC β-lactamase (blaAmpC)], Class D [oxacillinase β-lactamase (blaOXA)] β- lactamase-related resistance genes were detected by polymerase chain reaction. Drug-resistant mutation frequencies of each strain were determined on a drug-containing plate. The time kill curves of three XDR-PA were plotted by colony counting method. A biofilm model was established in vitro, and the synergistic effect of CAZ-AVI and COL on biofilm inhibition was detected by methythiazolyl tetrazolium assay (MTT). Results The MICs of 16 XDR-PA for CAZ-AVI ranged from 1 mg/L to 128 mg/L, and three of the isolates showed resistance (MIC > 8 mg/L). The FICI range of CAZ-AVI combined with COL was 0.312-1.000. Four isolates were synergistic, while the other 12 isolates were additive. Three isolates resistant to CAZ-AVI contained Class B resistance genes such as blaIMP and blaVIM, while 13 susceptible isolates carried resistance genes belonging to Class A, C or D. The logarithm values of mutation frequencies of drug resistance in CAZ-AVI group, COL group and combination group were -4.81±0.88, -7.06±0.69 and -9.70 (-9.78, -9.53), respectively. There were significant differences among the three groups (H = 33.601, P < 0.001), and between every two groups (adjusted P < 0.05). In time kill curves, the phytoplankton load of three XDR-PA decreased more than 6 log CFU/L when these two drugs were used together, and number of PA1819 planktonic bacteria decreased more than 5.1 log CFU/L compared with monotherapy group. Viable quantity in biofilm (A490) of normal saline group, CAZ-AVI group, COL group and CAZ-AVI-COL group were 0.665±0.068, 0.540±0.072, 0.494±0.642 and 0.317±0.080, respectively. There was significant difference between the other two groups (all P < 0.001), except for that between CAZ-AVI group and COL group (P = 0.109). Conclusions CAZ-AVI combined with COL can effectively improve the bactericidal effect of each drug alone on XDR-PA. The regimen can also reduce the production of drug-resistant bacteria and inhibit the formation of biofilm. Therefore, it is a potential treatment for XDR-PA infection.
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Objective To evaluate the in vitro activity of ceftazidime-avibactam (CAZ-AVI) alone or in combination with colistin (COL) against clinically isolated extensively drug-resistant Pseudomonas aeruginosa (XDR-PA). Methods Minimum inhibitory concentration (MIC) of 16 clinical XDR-PA isolates was determined by broth dilution method and chessboard design when CAZ-AVI and COL were used alone or in combination, then the combined inhibitory concentration index (FICI) was calculated. Class A [Klebsiella pneumoniae carbapenemase β-lactamase (blaKPC), Guiana extended-spectrum β-lactamase (blaGES)], Class B [imipenemase β-lactamase (blaIMP), Verona-Integronmetallo β-lactamase (blaVIM), New Delhi metallo β-lactamase (blaNDM), German imipenemase β-lactamase (blaGIM), Sao Paulo metallo -β- lactamase (blaSPM)], Class C [AmpC β-lactamase (blaAmpC)], Class D [oxacillinase β-lactamase (blaOXA)] β- lactamase-related resistance genes were detected by polymerase chain reaction. Drug-resistant mutation frequencies of each strain were determined on a drug-containing plate. The time kill curves of three XDR-PA were plotted by colony counting method. A biofilm model was established in vitro, and the synergistic effect of CAZ-AVI and COL on biofilm inhibition was detected by methythiazolyl tetrazolium assay (MTT). Results The MICs of 16 XDR-PA for CAZ-AVI ranged from 1 mg/L to 128 mg/L, and three of the isolates showed resistance (MIC > 8 mg/L). The FICI range of CAZ-AVI combined with COL was 0.312-1.000. Four isolates were synergistic, while the other 12 isolates were additive. Three isolates resistant to CAZ-AVI contained Class B resistance genes such as blaIMP and blaVIM, while 13 susceptible isolates carried resistance genes belonging to Class A, C or D. The logarithm values of mutation frequencies of drug resistance in CAZ-AVI group, COL group and combination group were -4.81±0.88, -7.06±0.69 and -9.70 (-9.78, -9.53), respectively. There were significant differences among the three groups (H = 33.601, P < 0.001), and between every two groups (adjusted P < 0.05). In time kill curves, the phytoplankton load of three XDR-PA decreased more than 6 log CFU/L when these two drugs were used together, and number of PA1819 planktonic bacteria decreased more than 5.1 log CFU/L compared with monotherapy group. Viable quantity in biofilm (A490) of normal saline group, CAZ-AVI group, COL group and CAZ-AVI-COL group were 0.665±0.068, 0.540±0.072, 0.494±0.642 and 0.317±0.080, respectively. There was significant difference between the other two groups (all P < 0.001), except for that between CAZ-AVI group and COL group (P = 0.109). Conclusions CAZ-AVI combined with COL can effectively improve the bactericidal effect of each drug alone on XDR-PA. The regimen can also reduce the production of drug-resistant bacteria and inhibit the formation of biofilm. Therefore, it is a potential treatment for XDR-PA infection.
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CRISPR/Cas9 system, consisting of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins, is a prokaryotic immune system that confers resistance to foreign genetic elements such as those present within plasmids and phages. A simple version of the CRISPR/Cas system, type Ⅱ CRISPR, has been modified to edit genomes. By delivering the Cas9 nuclease together with a synthetic guide RNA (sgRNA) into cells, genome can be edited at desired loci site. CRISPR/Cas genome editing techniques have been widely implemented in various species and research areas. In this review, we summarize the several applications of CRISPR/Cas9 in the field of drug discovery and development, which include target gene screening and editing, drug target screening and validation, generation of animal models and treatment of genetic disease, etc. The defects and improvements of CRISPR/Cas9 technology is discussed as well.
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Objective To observe whether the classic drug-resistant mutations can be induced in various concentrations of adefovir (ADV)-treated HepG2.2.15 cells persistently and explore the mechanism for emergence of drug resistance.Methods HepG2.2.15 cells were cultured continually in 12-well plates with medium containing 0,0.01,0.1,1.0μmol/L concentration of ADV,and passaged every 3 days up to the 110th generations.The intracellular and supernatant HBV DNA was extracted every 10 generations.Intracellular HBV cccDNA was amplified by plasmid-safe ATP dependent DNase (PSAD) digestion in combination with rolling circle amplification and gap-spanning semi-nested PCR assay.And the RT region of supernatant HBV DNA was amplified by one-tube nested PCR assay.Then the classic drug-resistant mutations of the RT region of intracellular cccDNA and supernatant HBV DNA were analyzed using direct PCR sequencing combined with clonal sequencing (more than 20 clones/sample).Results HBV DNA stably replicated in ADV-untreated cells (control group).The intracellular total DNA and cccDNA levels,supernatant HBV DNA level decreased continuously with the prolonged ADV culture duration in 0.01 μmol/L and 0.1μmol/L ADV group.Drug resistant mutations were not detected up to the 110th generation in 0.01 μmol/L ADV group;while rtA181V+N236T mutations were detected at the110th generation in 0.1μmol/L ADV group.The 1.0μmol/L ADV group was ceased of culture at the 15th generation due to inhibited cell growth.Conclusion HBV cccDNA exists in HepG2.2.15 cells,and the classical drug-resistant mutations of rtA181V+N236T could be induced by proper concentration of ADV.
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Objective@#To investigate the genetic characteristics of Lamivudine-resistant mutation patterns and HBV S gene mutants in patients with chronic hepatitis disease of different disease progression.@*Methods@#Blood samples of LAM-resistant patients with chronic hepatitis disease were collected. HBV RT gene nucleotide sequences were obtained, and then differences in drug-resistant mutation patterns, drug susceptibility and HBV S gene mutants characteristics between the two groups were analyzed.@*Results@#Forty-seven chronic hepatitis B (CHB) patients and 16 HBV-related liver cirrhosis (LC)/HBV-related hepatocellular carcinoma (HCC) patients were included in this study. M204I single point mutation and L180M+ M204I/V were the most common pattern during patients with chronic hepatitis disease (35/63, 55.56%). The numbers of resistant to three nucleos(t)ide analogues in LC/HCC group was higher than CHB group’s (62.50% vs 34.04%, P=0.046). In HBV S gene, more immune associated HBsAg-escape mutations were detected in LC/HCC group than that in CHB group (62.50% vs 31.91%, P=0.031). I126T/V and G145A (for LCC/HCC group, 60%), I126S/T and S117T (for CHB group, 46.67%) were showed as the most common form for HBsAg escape mutations in the two groups. The two groups both detected RT mutations concomitantly with stop codon mutations in S gene (rtA181T/sW172* and rtM204I/sW196*).@*Conclusions@#Different characteristics in Lamivudine-resistant mutations and associated HBV S gene mutants were found in patients with chronic hepatitis disease of different disease progression, and LC/HCC patients exhibit more multi-drug resistant variants and immune associated HBsAg-escape mutants than CHB patients.
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Objective:To compare the correlation of four drug-resistance interpretation system including HIVDB, ANRS, REGA and HIV GRADE for testing HIV-1 genotype drug resistance. Methods:Trendy subtypes and restructuring model of HIV-1 including B’,CRF01_AE and CRF07_BC were selected,each genotype was 200 series,600 samples were analyzed by four drug?resistance interpretation system, the results were divided into three levels including resistance (R), possible drug?resistance (I) and susceptible (S). Results:Four drug?resistance interpretation system had a high correlation in genotypic drug?resistance consequence (rs>0.57, P<0.01), CRF07_BC had the highest correlation, followed by subtype B, CRF01_AE was slightly lower.Conclusion:Four drug?resistance interpretation system has a high correlation in analyzing antiviral drug resistance for major epidemic of HIV in China.
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<p><b>OBJECTIVE</b>To investigate distinctive features in drug-resistant mutations (DRMs) and interpretations for reverse transcriptase inhibitors (RTIs) between proviral DNA and paired viral RNA in HIV-1-infected patients.</p><p><b>METHODS</b>Forty-three HIV-1-infected individuals receiving first-line antiretroviral therapy were recruited to participate in a multicenter AIDS Cohort Study in Anhui and Henan Provinces in China in 2004. Drug resistance genotyping was performed by bulk sequencing and deep sequencing on the plasma and whole blood of 77 samples, respectively. Drug-resistance interpretation was compared between viral RNA and paired proviral DNA.</p><p><b>RESULTS</b>Compared with bulk sequencing, deep sequencing could detect more DRMs and samples with DRMs in both viral RNA and proviral DNA. The mutations M184I and M230I were more prevalent in proviral DNA than in viral RNA (Fisher's exact test, P<0.05). Considering 'majority resistant variants', 15 samples (19.48%) showed differences in drug resistance interpretation between viral RNA and proviral DNA, and 5 of these samples with different DRMs between proviral DNA and paired viral RNA showed a higher level of drug resistance to the first-line drugs. Considering 'minority resistant variants', 22 samples (28.57%) were associated with a higher level of drug resistance to the tested RTIs for proviral DNA when compared with paired viral RNA.</p><p><b>CONCLUSION</b>Compared with viral RNA, the distinctive information of DRMs and drug resistance interpretations for proviral DNA could be obtained by deep sequencing, which could provide more detailed and precise information for drug resistance monitoring and the rational design of optimal antiretroviral therapy regimens.</p>
Subject(s)
Adult , Female , Humans , Male , Middle Aged , Antiviral Agents , Pharmacology , China , DNA, Viral , Genetics , Metabolism , Drug Resistance, Viral , Genetics , HIV Infections , Drug Therapy , HIV-1 , Genetics , Metabolism , High-Throughput Nucleotide Sequencing , Mutation , Proviruses , Genetics , Metabolism , RNA, Viral , Genetics , Metabolism , RNA-Directed DNA PolymeraseABSTRACT
This study aimed to evaluate emerging trends of drug resistance to nucleoside reverse transcriptase inhibitors (NRTIs) and nonnucleoside reverse transcriptase inhibitors (NNRTIs) among 290 former blood donor HIV-1 infected patients in Hubei,China,from 2004 to 2006,all of whom had received anti-HIV-1 therapy.The presence of NRTI- and NNRTI-associated mutations were established by sequencing; genotypic and predicted phenotypic drug resistance were evaluated using HIVdb Program version 5.0.1 (http://hivdb.stanford.edu/pages/algs/HIVdb.html).Genotypic drug resistance analysis showed significant increases in percentages of patients carrying HIV-1 strains with M41L,T215Y/F,D67N,K103N,G190A/S,Y181C/F or L210W mutations.Of the variants' predicted phenotypic drug resistance,highly significant increases were detected in percentages of patients carrying HIV-1 with high resistance to zidovudine (AZT) or stavudine (D4T) in NRTIs,and to delavirdine (DLV),efavirenz (EFV) or nevirapine (NVP) in NNRTIs; intermediate resistance to abacavir (ABC),AZT,D4/T,didanosine (DDI) or tenofovir disoproxil fumarate (TDF) in NRTIs,and to etravirine (ETR) in NNRTIs; and low and potential low resistance to lamivudine (3TC),ABC,emtricitabine (FTC) or TDF in NRTIs,and to ETR in NNRTIs.
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Generally absolute majority of wild-type microbial strains do not produce bioactive metabolites, resulting in large numbers of so-called 'useless strains' stocked or destroyed. These strains, however, would become a great source of bioactive meabolites if their secondary metabolism could be altered to produce diverse metabolites. We have therefore undertaken a research work on exploiting microbial new strain resources for drug screening by altering secondary metabolism of the 'useless strains' to discover bioactive metabolites. A considerable progress with expectant advantage desired has been made in the studies on marine-derived actinomycetic and fungal strains. This paper summarizes our research results including several new developments in brief.
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Objective To obtain antibiotic-resistant mutants producing metabolites with antitumor activity from wild-type actinomycete strains without antitumor activity. Methods An actinomycete strain L35-1 was used as an initial strain for obtaining antibiotic-resistant mutants, which is a marine-derived wild-type strain without antitumor activity with an inhibition rate of 2.8% at the 1000 μg/ml of high sample concentration on K562 cells. The antibiotic-resistant mutants both from auto-mutagenesis and chemical mutagen-induced mutagenesis were selected by single colony isolation on antibiotic-containing plates according to the method for obtaining drug-resistant mutants in ribosome engineering. The antitumor activity was assayed by the MTT method using K562 cells for the mutants with aqueous acetone extracts of the whole broth of their fermentation.Results A total of 114 neomycin-resistant (ner) and 68 streptomycin-resistant (str) mutants, all from auto-mutagenesis, was obtained on drug-containing plates. Among them, the 7 ner and 3 str mutants appeared to be bioactive with an inhibition rate above 20% at the 100 μg/ml sample concentration on K562 cells. On the other hand, 41 str and 32 ner mutants from DES-induced mutagenesis and 46 ner mutants from NTG-induced mutagenesis were obtained by mutagen-induced mutation coupled with the single colony isolation on antibiotic-containing plates, among which, one str mutant from DES-induced mutagenesis and one ner mutant from NTG-induced mutagenesis were bioactive with an inhibition rate over 20% at the 100 μg/ml sample concentration on K562 cells. Conclusions The present result has revealed that the wild-type actinomycete strains without bioactivity might become a great source initial strains to obtain bioactive mutants by drug-resistant mutation technique.
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Objective To isolate stable passage primary HIV-1 drug resistant strains and observe replication dynamics of the drug resistant isolates and evolvement tendency of the drug resistant mutations in vitro.Methods Peripheral blood mononuclear cells(PBMCs)from 15 AIDS patients receiving highly active antiretroviral therapy(HAART)were collected,and the primary HIV-1 stains were separated utilizing co-cultivated with PBMCs from normal people.HIV-1 pol genes from those strains were obtained by RT-PCR and sequenced.The drug resistant mutations were analyzed in the Stanford HIV Drug Resistance Database.Results Eight strong positive strains were isolated from 15 AIDS patients with viral loads higher than 1000 copies/ml,and two of them were drug resistant.Drug resistant mutations of the two strains were respectively K103N/K238T and M184V/K103N/Y181C/H221Y which show high-level resistance to NVP and 3TC/NVP,respectively.K103N,M184V,Y181C and H221Y exist stably in the environment without drug pressure,however,RT K238T reverted to K238.Conclusion Two drug resistant strains were successfully isolated in vitro without drug pressure.Strains with K103N shows superior fitness and can exist steadily.Strains with M184V and K103N/Y181C/H221Y can also replicate stably in vitro without drug pressure.NNRTI mutation K238T reproduces astatically,which suggests that RT 238 codon might revert gradually to wild genotype.
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BACKGROUND: Despite significant progress in vaccine and therapeutic regimen, hepatitis B virus (HBV) infection remains one of the major diseases. Long-term use of lamivudine can induce the emergence of drug resistance. TRUGENE(TM) HBV Genotyping (Visible Genetics Inc., Ga, USA) is an assay that reports the viral genotype and mutations likely to confer resistance to antiviral therapy. In this study, we analyzed HBV genotype and mutations and correlated them with the histologic grade and stage of the liver disease to provide the useful information about the therapy of chronic liver disease. METHODS: HBV DNA was isolated from 86 patients with HBV-associated chronic liver diseases and analyzed by TRUGENE(TM) HBV Genotyping. Histologic grade and stage were correlated with RESULTS: HBV genotypes of 86 patients were all C (100%). Mutations associated with lamivudine resistance were detected in 10 patients (11.6%) and M204I (YIDD) mutant was the most common. Unknown mutation such as L180F was also detected. Statistical analysis showed that the number of coding changes at HBsAg region was significantly correlated with the lobular activity (P=0.01). CONCLUSIONS: All patients were genotype C and lamivudine resistant mutations were detected in 11.6%. L180F mutation, not known previously, was detected in one case. Number of coding changes at HBsAg region was significantly correlated with the lobular activity. It was considered that follow-up studies about the clinical significance of coding changes in HBsAg are needed, and that a further study such as in vitro transfection is necessary to confirm the possibility of a novel mutation of L180F.