Nine strains resistant to five fluoroquinolones （Ciprofloxacin, Ofloxacin, Enrofloxacin, Danofloxacin, Sarafloxacin） were isolated from clinical samples and extracted the chromosomal DNA of these strains. Designed p...Nine strains resistant to five fluoroquinolones （Ciprofloxacin, Ofloxacin, Enrofloxacin, Danofloxacin, Sarafloxacin） were isolated from clinical samples and extracted the chromosomal DNA of these strains. Designed primers to amplify the Quinolone-resistance-determining region （QRDR） of gyrA and par（？,, then the PCR products were sequenced and analyzed. In comparision with NCTC5776, a single mutation was found at base 371 in gyrA of strain 38 which changed from C to T, and a single mutation was found at base 350 in gyrA of strain 60 which changed from A to C. No mutation was found in gyrA of the rest The mutation of strain 38 led to an amino acid substitution of Arg99Cys and the mutation of 60 led to an amino acid substitution of Met 92 Leu. No mutation was found in parC QRDR of all the isolates. These results indicats that the DNA gyrase will be the primary target to salmonella of fluoroquinolone.展开更多
By means of mathematical modeling methods, we analyzed the relationship between the resistance to fluoroquinolones and GyrA mutation of Salmonella from animal isolates. We found that considering the influence of the r...By means of mathematical modeling methods, we analyzed the relationship between the resistance to fluoroquinolones and GyrA mutation of Salmonella from animal isolates. We found that considering the influence of the resistance to ciprofloxacin hydrochloride, enrofloxacin, ofloxacin, pefloacin mesylate and norfloxacin nicotinate of the five types of fluoroquinolones to GyrA mutation of animal Salmonella, the resistance of pefloacin mesylate had the most significant effect, while the resistance of ciprofloxacin hydrochloride and enrofloxacin were the least significant factors. Nearly half of the Salmonella supports such a rule that the MIC of norfloxacin nicotinate reaching 64 or 128 might lead to the mutation Ser83→Phe, MIC exceeding 512 might lead to the mutation Ser83→Gly; 60% of the sample supported that the MIC of enrofloxacin reaching 32 or 64 might lead to the mutation Asp87 →Asn. 80% of them agreed to the fact that the MIC of neither ciprofloxacin hydrochloride under 64 nor pefloacin mesylate below 512 might result in the gene mutation in 119 site. All Salmonella isolates supported the conclusion that the mutation Alal19→Val took place, if and only if the MIC of norfloxacin nicotinate exceeded 512.展开更多
本研究对重庆荣昌采后发病姜块进行病原菌分离鉴定,分离到一株新的生姜致病菌。通过细菌形态、生理生化、16S r DNA遗传分析鉴定为沙门氏菌(Salmonella enterica)。将沙门氏菌回接姜苗、姜块,发现姜苗叶片失绿,褶皱;姜块表面水渍并有分...本研究对重庆荣昌采后发病姜块进行病原菌分离鉴定,分离到一株新的生姜致病菌。通过细菌形态、生理生化、16S r DNA遗传分析鉴定为沙门氏菌(Salmonella enterica)。将沙门氏菌回接姜苗、姜块,发现姜苗叶片失绿,褶皱;姜块表面水渍并有分泌物;动态污染研究表明:沙门氏菌能在姜块中迅速繁殖,显著提高姜块超氧化物歧化酶(superoxide dismutase,SOD)、过氧化物酶(peroxidase,POD)、过氧化氢酶(catalase,CAT)的活性。该研究表明,沙门氏菌能以生姜为寄主存活。展开更多
Nine strains resistant to five fluoroquinolones (Ciprofloxacin, Ofloxacin, Enrofloxacin, Danofloxacin, Sarafloxacin) were isolated from clinical samples and extracted the chromosomal DNA of these strains. Designed pri...Nine strains resistant to five fluoroquinolones (Ciprofloxacin, Ofloxacin, Enrofloxacin, Danofloxacin, Sarafloxacin) were isolated from clinical samples and extracted the chromosomal DNA of these strains. Designed primers to amplify the Quinolone-resistance-determining region(QRDR) of gyrB gene, then the PCR products were cloned and the sequence was analyzed. In comparison with the standarded strain NCTC5776, no mutation was found in the QRDR of gyrB gene of all resistant strains. The result indicated that the QRDR of gyrB has little relationship with fluoroquinolone resistance to salmonella.展开更多
Salmonella gallinarum has shown multiple drug resistance(MDR),especially high level fluoroquinolone(FQ)resistance in recent years.To determine whether the active efflux system was responsible for high-level FQ resista...Salmonella gallinarum has shown multiple drug resistance(MDR),especially high level fluoroquinolone(FQ)resistance in recent years.To determine whether the active efflux system was responsible for high-level FQ resistance,this research studied AcrAB efflux pump in Salmonella gallinarum on molecular level.The resistant strains were induced by standard strain C79-13 with ciprofloxacin in vitro.With carbonylcyanide-p-chlorophenyl hydrazone(CCCP)as an energy inhibitor,efflux inhibition test initially showed the potential impact of efflux pump on drug resistance.Sequence analysis of acrA gene indicated that gene mutation of AcrAB efflux pump was not definitely associated with MDR and drug resistance level of Salmonella gallinarum.Detected by competitive RT-PCR,the mRNA expression of acrA and acrB genes in the resistant strains significantly increased(p<0.01)compared with that of the control strain C79-13.The mRNA expression level of acrB gene(increased from 1.6-to 2.9-folds)was consistent with that of acrA gene(increased from 1.6-to 2.8-folds),which increased with the drug resistance level.However,gene mutation of acrA gene showed no correlation with its mRNA expression level,indicating that gene mutation did not affect the expression of AcrAB pump itself.The results suggested that the overexpression rather than the gene mutation of AcrAB efflux pump was an important factor causing the high level drug resistance of Salmonella gallinarum.展开更多
Objective To establish a loop-mediated isothermal amplification( LAMP) method for detecting diarrhea pathogens( Shigella and Salmonella) in rhesus monkeys and evaluate the application of the LAMP method for detecting ...Objective To establish a loop-mediated isothermal amplification( LAMP) method for detecting diarrhea pathogens( Shigella and Salmonella) in rhesus monkeys and evaluate the application of the LAMP method for detecting bacterial diseases in nonhuman primate laboratory animals. Materials and Methods A total of 205 fecal samples of rhesus monkeys were detected in this LAMP assay. The specificity and sensitivity of LAMP for Shigella and Salmonella were analyzed,and real-time polymerase chain reaction( REAL-TIME PCR) assay was employed as control. Results The LAMP method established here needed only 45 min to complete the reaction at 63℃. Its detection limit was 10 pg / μL and with a high specificity. The positive rate of Shigella and Salmonella was 1. 5% and 6. 3%,respectively. Conclusions Here we have established a fast and simple Shigella and Salmonella LAMP detection method that has strong specificity and high sensitivity and is suitable for rapid detection of bacterial disease in macaques. The development of this rapid detection kit is underway,and it will be helpful to the diarrhea detection.展开更多
Salmonella enterica serovar Choleraesuis strain C500 is a live attenuated vaccine that has been widely used in Chi-na for over 50 years to prevent piglet paratyphoid.However,as C500 is obtained by chemical methods,the...Salmonella enterica serovar Choleraesuis strain C500 is a live attenuated vaccine that has been widely used in Chi-na for over 50 years to prevent piglet paratyphoid.However,as C500 is obtained by chemical methods,the genetic background of this strain remained unclear.In this study,we compared the genomic differences between the virulent reference strain C78-2 and C500 by suppression subtractive hybridization combined with the mirror orientation selection method(MOS-SSH).Six genes(asr,ydgF,ydgD,ydgE,rpoS,and ptsG)were lost in C500 strain.Using-real-time PCR analysis,we demonstrated that the genes regulated by rpoS,a vital transcriptional regulator playing an important role in Salmonella infection,were downregulated in C500.Additionally,the virulence of the rpoS mutant strain C78-2ArpoS was 100000 times lower than the parental strain in BALB/c mice.So loss of rpoS gene is the major factor leading to the attenuation of C500 strain.展开更多
文摘Nine strains resistant to five fluoroquinolones （Ciprofloxacin, Ofloxacin, Enrofloxacin, Danofloxacin, Sarafloxacin） were isolated from clinical samples and extracted the chromosomal DNA of these strains. Designed primers to amplify the Quinolone-resistance-determining region （QRDR） of gyrA and par（？,, then the PCR products were sequenced and analyzed. In comparision with NCTC5776, a single mutation was found at base 371 in gyrA of strain 38 which changed from C to T, and a single mutation was found at base 350 in gyrA of strain 60 which changed from A to C. No mutation was found in gyrA of the rest The mutation of strain 38 led to an amino acid substitution of Arg99Cys and the mutation of 60 led to an amino acid substitution of Met 92 Leu. No mutation was found in parC QRDR of all the isolates. These results indicats that the DNA gyrase will be the primary target to salmonella of fluoroquinolone.
基金Supported by the National Natural Science Foundation of China (11171244)
文摘By means of mathematical modeling methods, we analyzed the relationship between the resistance to fluoroquinolones and GyrA mutation of Salmonella from animal isolates. We found that considering the influence of the resistance to ciprofloxacin hydrochloride, enrofloxacin, ofloxacin, pefloacin mesylate and norfloxacin nicotinate of the five types of fluoroquinolones to GyrA mutation of animal Salmonella, the resistance of pefloacin mesylate had the most significant effect, while the resistance of ciprofloxacin hydrochloride and enrofloxacin were the least significant factors. Nearly half of the Salmonella supports such a rule that the MIC of norfloxacin nicotinate reaching 64 or 128 might lead to the mutation Ser83→Phe, MIC exceeding 512 might lead to the mutation Ser83→Gly; 60% of the sample supported that the MIC of enrofloxacin reaching 32 or 64 might lead to the mutation Asp87 →Asn. 80% of them agreed to the fact that the MIC of neither ciprofloxacin hydrochloride under 64 nor pefloacin mesylate below 512 might result in the gene mutation in 119 site. All Salmonella isolates supported the conclusion that the mutation Alal19→Val took place, if and only if the MIC of norfloxacin nicotinate exceeded 512.
文摘本研究对重庆荣昌采后发病姜块进行病原菌分离鉴定,分离到一株新的生姜致病菌。通过细菌形态、生理生化、16S r DNA遗传分析鉴定为沙门氏菌(Salmonella enterica)。将沙门氏菌回接姜苗、姜块,发现姜苗叶片失绿,褶皱;姜块表面水渍并有分泌物;动态污染研究表明:沙门氏菌能在姜块中迅速繁殖,显著提高姜块超氧化物歧化酶(superoxide dismutase,SOD)、过氧化物酶(peroxidase,POD)、过氧化氢酶(catalase,CAT)的活性。该研究表明,沙门氏菌能以生姜为寄主存活。
文摘Nine strains resistant to five fluoroquinolones (Ciprofloxacin, Ofloxacin, Enrofloxacin, Danofloxacin, Sarafloxacin) were isolated from clinical samples and extracted the chromosomal DNA of these strains. Designed primers to amplify the Quinolone-resistance-determining region(QRDR) of gyrB gene, then the PCR products were cloned and the sequence was analyzed. In comparison with the standarded strain NCTC5776, no mutation was found in the QRDR of gyrB gene of all resistant strains. The result indicated that the QRDR of gyrB has little relationship with fluoroquinolone resistance to salmonella.
基金Supported by the Application Technology Research and Development Project in Heilongjiang Province of China(PC13S03)the Foundation of Heilongjiang Province Educational Committee of China(11541030)。
文摘Salmonella gallinarum has shown multiple drug resistance(MDR),especially high level fluoroquinolone(FQ)resistance in recent years.To determine whether the active efflux system was responsible for high-level FQ resistance,this research studied AcrAB efflux pump in Salmonella gallinarum on molecular level.The resistant strains were induced by standard strain C79-13 with ciprofloxacin in vitro.With carbonylcyanide-p-chlorophenyl hydrazone(CCCP)as an energy inhibitor,efflux inhibition test initially showed the potential impact of efflux pump on drug resistance.Sequence analysis of acrA gene indicated that gene mutation of AcrAB efflux pump was not definitely associated with MDR and drug resistance level of Salmonella gallinarum.Detected by competitive RT-PCR,the mRNA expression of acrA and acrB genes in the resistant strains significantly increased(p<0.01)compared with that of the control strain C79-13.The mRNA expression level of acrB gene(increased from 1.6-to 2.9-folds)was consistent with that of acrA gene(increased from 1.6-to 2.8-folds),which increased with the drug resistance level.However,gene mutation of acrA gene showed no correlation with its mRNA expression level,indicating that gene mutation did not affect the expression of AcrAB pump itself.The results suggested that the overexpression rather than the gene mutation of AcrAB efflux pump was an important factor causing the high level drug resistance of Salmonella gallinarum.
文摘Objective To establish a loop-mediated isothermal amplification( LAMP) method for detecting diarrhea pathogens( Shigella and Salmonella) in rhesus monkeys and evaluate the application of the LAMP method for detecting bacterial diseases in nonhuman primate laboratory animals. Materials and Methods A total of 205 fecal samples of rhesus monkeys were detected in this LAMP assay. The specificity and sensitivity of LAMP for Shigella and Salmonella were analyzed,and real-time polymerase chain reaction( REAL-TIME PCR) assay was employed as control. Results The LAMP method established here needed only 45 min to complete the reaction at 63℃. Its detection limit was 10 pg / μL and with a high specificity. The positive rate of Shigella and Salmonella was 1. 5% and 6. 3%,respectively. Conclusions Here we have established a fast and simple Shigella and Salmonella LAMP detection method that has strong specificity and high sensitivity and is suitable for rapid detection of bacterial disease in macaques. The development of this rapid detection kit is underway,and it will be helpful to the diarrhea detection.
文摘Salmonella enterica serovar Choleraesuis strain C500 is a live attenuated vaccine that has been widely used in Chi-na for over 50 years to prevent piglet paratyphoid.However,as C500 is obtained by chemical methods,the genetic background of this strain remained unclear.In this study,we compared the genomic differences between the virulent reference strain C78-2 and C500 by suppression subtractive hybridization combined with the mirror orientation selection method(MOS-SSH).Six genes(asr,ydgF,ydgD,ydgE,rpoS,and ptsG)were lost in C500 strain.Using-real-time PCR analysis,we demonstrated that the genes regulated by rpoS,a vital transcriptional regulator playing an important role in Salmonella infection,were downregulated in C500.Additionally,the virulence of the rpoS mutant strain C78-2ArpoS was 100000 times lower than the parental strain in BALB/c mice.So loss of rpoS gene is the major factor leading to the attenuation of C500 strain.
