Objective To establish and compare the pulsed-field gel electrophoresis （PFGE）, multiple-locus variable number tandem repeat analysis （MLVA） and automated ribotyping for subtyping of Citrobacter strains. Methods P...Objective To establish and compare the pulsed-field gel electrophoresis （PFGE）, multiple-locus variable number tandem repeat analysis （MLVA） and automated ribotyping for subtyping of Citrobacter strains. Methods PFGE protocol was optimized in terms of plug preparation procedure, restriction enzymes and configuration of electrophoretic parameters. MLVA method was evaluated by finding variable number tandem repeats in two genomes of Citrobacter strains. The ribotyping was performed by using the automated RiboPrinter system. Results We optimized the plug preparation procedure, focused on the cell suspension concentration （turbidity of 2.5 to 3.5）, SDS addition （no SDS needed） and lysis time （1 h）, and selected the appropriate restriction enzyme （Xbal） and the electrophoretic parameters （1.0 s-20.0 s for 19 h） of PFGE. There was nearly no discriminatory power of MLVA between Citrobacter strains. For 51 Citrobacter strains, automated ribotyping gave a D-value of 0.9945, while PFGE gave a D-value of 0.9969. Both PFGE and automated ribotyping clustered strains from the same sources （with the same species from the same place at the same time identified as the same source） and divided strains from different sources （from different years, places and hosts） into different subtypes. Conclusion PFGE protocol established in this paper and automated ribotyping are suitable for application in Citrobacter subtyping.展开更多
In order to develop a rapid and reliable method for B. cereus genotyping, factors influencing PFGE results, including preparation of bacterial cells embedded in agarose, lysis of embedded cells, enzymatic digestion of...In order to develop a rapid and reliable method for B. cereus genotyping, factors influencing PFGE results, including preparation of bacterial cells embedded in agarose, lysis of embedded cells, enzymatic digestion of intact genomic DNA, and electrophoresis parameters allowing for reproducible and meaningful DNA fragment separation, were controlled. Optimal cellular growth （Luria-Bertani agar plates for 12-18 h） and lysis conditions （4 h incubation with 500 μg/mL lysozyme） produced sharp bands on the gel.展开更多
Background The rise of the production of CTX-M class extended spectrum β-lactamases （ESBLs） has been well documented in traveling countries but no data are found for Macao, an international travel city. The objecti...Background The rise of the production of CTX-M class extended spectrum β-lactamases （ESBLs） has been well documented in traveling countries but no data are found for Macao, an international travel city. The objectives of this study were to identify the antimicrobial resistance pattern, and determine the prevalence, genotype and clonal relationship of ESBLs in 209 clinical Escherichia coli strains from Macao, China. Methods Antimicrobial susceptibility test was performed to determine the resistance patterns of the isolates using the disk diffusion method with 17 antimicrobial agents. Phenotypic detection was screened and confirmed according to the Clinical and Laboratory Standards Institute. Genotypic characterization was detected by isoelectric focusing analysis, polymerase chain reaction and sequencing. The clonal relationship between the different ESBL isolates was studied by pulsed-field gel electrophoresis （PFGE）. Results Imipenem and meropenem exhibited 100% susceptible among 209 strains. Overall, 82.3%, 67.3%, 52.9%, 51.2% and 51.0% of the isolates displayed resistance to ampicillin, tetracylcline, ciprofloxacin, sulfamethoxazole trimethoprin and gentamycin. The prevalence rate of ESBLs was 30.1%. Antibiotic resistances were found to be significantly higher among the ESBL producing group compared to non-ESBL producing group. We detected CTX-M-14 to be the major genotypic characterization of ESBLs （76.2%）. Two strains showed indistinguishable patterns by PFGE. Conclusions The prevalence of antimicrobial resistance is alarming high in Macao. Antimicrobial resistance is significantly higher among the ESBL producing group. This study documented CTX-M-14 as the predominant ESBL type. Although indistinguishable pattern was found between two strains, it was too small to decide whether any of the investigated strains was epidemic. Our findings may be also pertinent for other geographic areas undergoing similar travel characteristics to understand the corresponding effects on bacterial populations.展开更多
目的以脉冲场凝胶电泳(pulsed field gel electrophoresis,PFGE)为金标准,评价基质辅助激光解吸电离飞行时间质谱(matrix-assisted laser desorption/ionization time-of-flight mass spectrometry,MALDI-TOF MS)在多重耐药铜绿假单胞菌...目的以脉冲场凝胶电泳(pulsed field gel electrophoresis,PFGE)为金标准,评价基质辅助激光解吸电离飞行时间质谱(matrix-assisted laser desorption/ionization time-of-flight mass spectrometry,MALDI-TOF MS)在多重耐药铜绿假单胞菌(multi-drug resistant Pseudomonas aeruginosa,MDR-PA)同源性分析中应用的可行性。方法收集陆军军医大学第三附属医院2017年1-12月重症监护病房住院患者分离的20株MDR-PA非重复菌株,利用VITEK MS SARAMIS软件对MDR-PA蛋白图谱进行聚类分析,同时对其进行PFGE同源性分析,并将两种方法所得的分型结果进行对比分析。结果 MALDITOF MS同源性分析结果与PFGE同源性分析结果比较显示,20株MDR-PA通过MALDI-TOF MS聚类分析将其分为三大簇(Ⅰ型13株,Ⅱ型6株,Ⅲ型1株),而依据PFGE分型结果则将其分为6种亚型(A型2株,B型3株,C型4株,D型3株,E型7株,F型1株),其中有C型和E型中具有相同基因型菌株间,其MALDI-TOF MS亲缘性关系也较近,而其余4种亚型菌株间亲缘性比较,发现PFGE分型结果与MALDI-TOF MS聚类分析结果不完全相符。结论与PFGE同源性分析相比,MALDI-TOF MS同源性分析结果一致性不稳定,具有一定局限性,尚不能取代PFGE同源性分析。展开更多
目的以肠道定植碳青霉烯类耐药肠杆菌科细菌(carbapenem resistance Enterobacteriaceae,CRE)与同一患者后期感染细菌的关系为出发点,同源性检测和耐药基因筛查为中心,从重症医学科(intensive care unit,ICU)患者肠道CRE的定植情况、CR...目的以肠道定植碳青霉烯类耐药肠杆菌科细菌(carbapenem resistance Enterobacteriaceae,CRE)与同一患者后期感染细菌的关系为出发点,同源性检测和耐药基因筛查为中心,从重症医学科(intensive care unit,ICU)患者肠道CRE的定植情况、CRE肠道定植与后期感染的关系层次上,进行CRE防治的应用基础研究,从而为临床及时有效的抗感染治疗提供一定指导。方法收集2018—2019年来自ICU病房及由其他科室转入ICU的共11位患者的临床资料,分别分离同一患者肠道定植CRE菌株和后期其他感染部位的菌株,对所有菌株进行药物敏感性试验和耐药基因携带情况检测,采用多位点序列分析(MLST)、脉冲场凝胶电泳(PAGE)试验的方法对定植CRE菌株和后期其他感染部位的菌株进行同源性分析。结果95位ICU患者中有19位患者肠道CRE筛查阳性,定植率为20.00%。其中发生后期其他部位感染的患者11位,目标菌株耐碳青霉烯酶基因检测结果显示22株菌株中有21株检出耐药基因,占95.45%(21/22)。其中19株检出KPC-2耐药基因,阳性率为86.36%(19/22);2株检出NDM-1耐药基因,阳性率为9.09%(2/22)。其他碳青霉烯酶基因检测均为阴性。22株目标菌的MLST分型共分为3个型,主要为ST11型,11位患者中除了1位患者的后期感染菌株与定植菌株差异明显外,其余患者的肠道定植菌株与后期感染菌株均为相同的ST型;PAGE检出22株菌的分型可分为A群和B群,共7个型别,其中7位患者的肠道定植菌株与后期感染菌株之间条带位置与数目相同,视为同一克隆型,3位患者的肠道定植菌株与后期感染菌株条带有2~3个差异,同源性极高,视为高度相关菌株;1位患者的肠道定植菌株与后期感染菌株的条带异超过7条,视为不相关菌株。结论ICU患者定植率高,应加强入院CRE定植筛查;ICU患者部位检出的定植及感染CRE菌株MIC值高,为高耐药性菌株;ICU患者发生CRE感染的菌株与自身定植的菌株有极高的同源性。展开更多
基金supported by the project (grant 2005CB522904 and 2005CB522905) from the Ministry of Scientific Technologythe project (grant 2008ZX10004-001, 2008ZX10004-008, and 2009ZX10004-101) from the Ministry of Scientific Technology and the Ministry of Health of the People’s Republic of China
文摘Objective To establish and compare the pulsed-field gel electrophoresis （PFGE）, multiple-locus variable number tandem repeat analysis （MLVA） and automated ribotyping for subtyping of Citrobacter strains. Methods PFGE protocol was optimized in terms of plug preparation procedure, restriction enzymes and configuration of electrophoretic parameters. MLVA method was evaluated by finding variable number tandem repeats in two genomes of Citrobacter strains. The ribotyping was performed by using the automated RiboPrinter system. Results We optimized the plug preparation procedure, focused on the cell suspension concentration （turbidity of 2.5 to 3.5）, SDS addition （no SDS needed） and lysis time （1 h）, and selected the appropriate restriction enzyme （Xbal） and the electrophoretic parameters （1.0 s-20.0 s for 19 h） of PFGE. There was nearly no discriminatory power of MLVA between Citrobacter strains. For 51 Citrobacter strains, automated ribotyping gave a D-value of 0.9945, while PFGE gave a D-value of 0.9969. Both PFGE and automated ribotyping clustered strains from the same sources （with the same species from the same place at the same time identified as the same source） and divided strains from different sources （from different years, places and hosts） into different subtypes. Conclusion PFGE protocol established in this paper and automated ribotyping are suitable for application in Citrobacter subtyping.
基金supported by the National Special Fund for the Development of Major Research Equipment and Instruments(2012YQ09019706)from the Ministry of Science and Technologythe Priority Project on Infectious Disease Control and Prevention(2012ZX10004215 and 2008ZX10004-008)from the National Health and Family Planning Commission of the People's Republic of China
文摘In order to develop a rapid and reliable method for B. cereus genotyping, factors influencing PFGE results, including preparation of bacterial cells embedded in agarose, lysis of embedded cells, enzymatic digestion of intact genomic DNA, and electrophoresis parameters allowing for reproducible and meaningful DNA fragment separation, were controlled. Optimal cellular growth （Luria-Bertani agar plates for 12-18 h） and lysis conditions （4 h incubation with 500 μg/mL lysozyme） produced sharp bands on the gel.
文摘Background The rise of the production of CTX-M class extended spectrum β-lactamases （ESBLs） has been well documented in traveling countries but no data are found for Macao, an international travel city. The objectives of this study were to identify the antimicrobial resistance pattern, and determine the prevalence, genotype and clonal relationship of ESBLs in 209 clinical Escherichia coli strains from Macao, China. Methods Antimicrobial susceptibility test was performed to determine the resistance patterns of the isolates using the disk diffusion method with 17 antimicrobial agents. Phenotypic detection was screened and confirmed according to the Clinical and Laboratory Standards Institute. Genotypic characterization was detected by isoelectric focusing analysis, polymerase chain reaction and sequencing. The clonal relationship between the different ESBL isolates was studied by pulsed-field gel electrophoresis （PFGE）. Results Imipenem and meropenem exhibited 100% susceptible among 209 strains. Overall, 82.3%, 67.3%, 52.9%, 51.2% and 51.0% of the isolates displayed resistance to ampicillin, tetracylcline, ciprofloxacin, sulfamethoxazole trimethoprin and gentamycin. The prevalence rate of ESBLs was 30.1%. Antibiotic resistances were found to be significantly higher among the ESBL producing group compared to non-ESBL producing group. We detected CTX-M-14 to be the major genotypic characterization of ESBLs （76.2%）. Two strains showed indistinguishable patterns by PFGE. Conclusions The prevalence of antimicrobial resistance is alarming high in Macao. Antimicrobial resistance is significantly higher among the ESBL producing group. This study documented CTX-M-14 as the predominant ESBL type. Although indistinguishable pattern was found between two strains, it was too small to decide whether any of the investigated strains was epidemic. Our findings may be also pertinent for other geographic areas undergoing similar travel characteristics to understand the corresponding effects on bacterial populations.
文摘目的以肠道定植碳青霉烯类耐药肠杆菌科细菌(carbapenem resistance Enterobacteriaceae,CRE)与同一患者后期感染细菌的关系为出发点,同源性检测和耐药基因筛查为中心,从重症医学科(intensive care unit,ICU)患者肠道CRE的定植情况、CRE肠道定植与后期感染的关系层次上,进行CRE防治的应用基础研究,从而为临床及时有效的抗感染治疗提供一定指导。方法收集2018—2019年来自ICU病房及由其他科室转入ICU的共11位患者的临床资料,分别分离同一患者肠道定植CRE菌株和后期其他感染部位的菌株,对所有菌株进行药物敏感性试验和耐药基因携带情况检测,采用多位点序列分析(MLST)、脉冲场凝胶电泳(PAGE)试验的方法对定植CRE菌株和后期其他感染部位的菌株进行同源性分析。结果95位ICU患者中有19位患者肠道CRE筛查阳性,定植率为20.00%。其中发生后期其他部位感染的患者11位,目标菌株耐碳青霉烯酶基因检测结果显示22株菌株中有21株检出耐药基因,占95.45%(21/22)。其中19株检出KPC-2耐药基因,阳性率为86.36%(19/22);2株检出NDM-1耐药基因,阳性率为9.09%(2/22)。其他碳青霉烯酶基因检测均为阴性。22株目标菌的MLST分型共分为3个型,主要为ST11型,11位患者中除了1位患者的后期感染菌株与定植菌株差异明显外,其余患者的肠道定植菌株与后期感染菌株均为相同的ST型;PAGE检出22株菌的分型可分为A群和B群,共7个型别,其中7位患者的肠道定植菌株与后期感染菌株之间条带位置与数目相同,视为同一克隆型,3位患者的肠道定植菌株与后期感染菌株条带有2~3个差异,同源性极高,视为高度相关菌株;1位患者的肠道定植菌株与后期感染菌株的条带异超过7条,视为不相关菌株。结论ICU患者定植率高,应加强入院CRE定植筛查;ICU患者部位检出的定植及感染CRE菌株MIC值高,为高耐药性菌株;ICU患者发生CRE感染的菌株与自身定植的菌株有极高的同源性。
