Phylogenetic diversity and mutational analysis of New Delhi Metallo-β-lactamase (NDM) producing E. coli strains from pediatric patients in Pakistan

The evolution of NDM genes (bla_NDM) in E. coli is accounted for expansive multidrug resistance (MDR), causing severe infections and morbidities in the pediatric population. This study aimed to analyze the phylogeny and mutations in NDM variants of E. coli recovered from the pediatric population. Carbapenem-resistant clinical strains of E. coli were identified using microbiological phenotypic techniques. PCR technique used to amplify the bla_NDM genes, identified on agarose gel, and analyzed by DNA sequencing. The amino acid substitutions were examined for mutations after aligning with wild types. Mutational and phylogenetic analysis was performed using Lasergene, NCBI blastn, Clustal Omega, and MEGA software, whereas PHYRE2 software was used for the protein structure predictions. PCR amplification of the bla_NDM genes detected 113 clinical strains of E. coli with the contribution of bla_NDM-1 (46%), bla_NDM-4 (3.5%), and bla_NDM-5 (50%) variants. DNA sequencing of bla_NDM variants showed homology to the previously described bla_NDM-1, bla_NDM-4, and bla_NDM-5 genes available at GenBank and NCBI database. In addition, the mutational analysis revealed in frame substitutions of Pro60Ala and Pro59Ala in bla_NDM-4 and bla_NDM-5, respectively. The bla_NDM-1 was ortholog with related sequences of E. coli available at GenBank. The phylogenetic analysis indicated that the NDM gene variants resemble other microbes reported globally with some new mutational sites.     

Phylogenetic diversity and mutational analysis of New Delhi Metallo-β-lactamase (NDM) producing E. coli strains from pediatric patients in Pakistan

The evolution of NDM genes (bla_NDM) in E. coli is accounted for expansive multidrug resistance (MDR), causing severe infections and morbidities in the pediatric population. This study aimed to analyze the phylogeny and mutations in NDM variants of E. coli recovered from the pediatric population. Carbapenem-resistant clinical strains of E. coli were identified using microbiological phenotypic techniques. PCR technique used to amplify the bla_NDM genes, identified on agarose gel, and analyzed by DNA sequencing. The amino acid substitutions were examined for mutations after aligning with wild types. Mutational and phylogenetic analysis was performed using Lasergene, NCBI blastn, Clustal Omega, and MEGA software, whereas PHYRE2 software was used for the protein structure predictions. PCR amplification of the bla_NDM genes detected 113 clinical strains of E. coli with the contribution of bla_NDM-1 (46%), bla_NDM-4 (3.5%), and bla_NDM-5 (50%) variants. DNA sequencing of bla_NDM variants showed homology to the previously described bla_NDM-1, bla_NDM-4, and bla_NDM-5 genes available at GenBank and NCBI database. In addition, the mutational analysis revealed in frame substitutions of Pro60Ala and Pro59Ala in bla_NDM-4 and bla_NDM-5, respectively. The bla_NDM-1 was ortholog with related sequences of E. coli available at GenBank. The phylogenetic analysis indicated that the NDM gene variants resemble other microbes reported globally with some new mutational sites.     

鲍曼不动杆菌耐药性监测与碳青霉烯酶基因型研究

目的：对鲍曼不动杆菌耐药性及碳青霉烯酶基因型进行研究,以指导临床合理应用抗生素。方法：收集青岛市海慈医疗集团2009年6月至2010年6月从临床分离的鲍曼不动杆菌60株,用琼脂稀释法测定最低抑菌浓度（M IC）,改良Hodge试验检测碳青霉烯酶,用PCR法检测OXA-23,OXA-24,OXA-58基因,并对PCR产物进行测序。结果：①鲍曼不动杆菌检出率前两位是ICU病房和呼吸科病房,分别占32.3%和27.4%,多重耐药鲍曼不杆菌阳性率最高的是ICU,为70.6%（12/17）,其次为呼吸科病房,为35.0%（7/20）,哌拉西林、哌拉西林/他唑巴坦、头孢曲松、头孢他啶、头孢吡肟、亚胺培南、美罗培南、庆大霉索、阿米卡星、环丙沙星、左氧氟沙星、加替沙星、头孢哌酮/舒巴坦、氨曲南耐药率分别为92.3%、55.4%、88.6%、86.3%、80.3%、30.0%、35.0%、76.6%、79.6%、75.1%、87.1%、48.3%、42.0%和79.6%.②在21株耐碳青霉烯类鲍曼不动杆菌,有14株碳青霉烯酶表型阳性,检出率为66.7%,有18株PCR扩增出OXA-23基因,检出率85.7%,全部菌株blaOXA-24及blaOXA-58PCR扩增均为阴性,PCR产物测序表明与鲍曼不动杆菌（AY795964.1）blaOXA-23基因序列100%同源。结论：鲍曼不动杆菌多重耐药性严重;表型和基因型检测证实本院临床分离鲍曼不动杆菌对碳青霉烯类耐药机制主要是产OXA-23型酶。     

呼吸科住院患者耐碳青霉烯鲍曼不动杆菌抗菌药物敏感性及其分子特征

目的调查深圳市人民医院呼吸科住院患者耐碳青霉烯鲍曼不动杆菌（CRAB）的抗菌药物敏感性和耐药分子机制,以及克隆流行情况。方法收集2010年深圳市人民医院呼吸科住院患者临床分离CRAB29株,琼脂稀释法测定亚胺培南等15种抗菌药对CRAB的最低抑菌浓度（MIC）,PCR和DNA测序分析CRAB碳青霉烯酶基因型,脉冲场凝胶电泳（PFGE）分析菌株同源性。结果多粘菌素B对29株CRAB抗菌活性最强,敏感性100％,MIC50／MIC90为1／1μg/mL,其次米诺环素,敏感性96．6％,MIC50／MIC90为4／4μg/mL,替加环素中介率高达96．6％,MIC50／MIC90为4／4μg/mL。96．6％（28／29）CRAB携带ISAba1—blaOXA-23-like,对亚胺培南和美罗培南高度耐药,亚胺培南和美罗培南的MIC集中分布在32—64μg／mL;1株携带ISAba1—blaOXA-51-like CRAB,对亚胺培南和美罗培南中度耐药,亚胺培南和美罗培南的MIC分别为4μg/mL和8μg/mL。29株CRAB未发现blaOXA-24-like、blaOXA-143-like、金属酶基因及KPC酶基因。29株CRAB经PFGE分型共分2型,以A型28株（96．6％）为主要流行克隆,均携带ISAba1-blaOXA-23-like。结论2010年我院呼吸科临床分离CRAB主要携带ISAba1—blaOXA-23-like基因,并以克隆播散流行。     

鲍曼不动杆菌碳青霉烯酶基因分布与耐药相关性研究

目的 研究鲍曼不动杆菌碳青霉烯酶基因的分布与其耐药的相关性。方法 收集2015年1月至2017年3月瑞安市两家市级医院的100株耐碳青霉烯类抗生素鲍曼不动杆菌,应用PCR技术检测碳青霉烯酶的相关基因,应用ERIC-PCR法对筛选结果阳性菌株进行DNA同源性分析。结果 100株耐碳青霉烯类抗生素鲍曼不动杆菌中,有50株携带OXA-51基因,26株携带OXA-23基因,同时携带OXA-51和OXA-23基因的有10株,未检出其他碳青霉烯酶基因。对筛选出的58株鲍曼不动杆菌进行同源性分析,得出的DNA指纹条带数为7条,其大小为200~2000 bp。根据片段数目和大小,分为A、B、C、D、E共5种基因型,分别有38、32、21、5、4个克隆株。结论 本地区鲍曼不动杆菌对碳青霉烯类药物耐药的机制主要为携带OXA-23基因,克隆传播是主要的传播途径。     

鲍曼不动杆菌耐药性监测与碳青霉烯酶基因型研究

目的:对鲍曼不动杆菌耐药性及碳青霉烯酶基因型进行研究,以指导临床合理应用抗生素。方法:收集青岛市海慈医疗集团2009年6月至2010年6月从临床分离的鲍曼不动杆菌60株,用琼脂稀释法测定最低抑菌浓度(M IC),改良Hodge试验检测碳青霉烯酶,用PCR法检测OXA-23,OXA-24,OXA-58基因,并对PCR产物进行测序。结果:①鲍曼不动杆菌检出率前两位是ICU病房和呼吸科病房,分别占32.3%和27.4%,多重耐药鲍曼不杆菌阳性率最高的是ICU,为70.6%(12/17),其次为呼吸科病房,为35.0%(7/20),哌拉西林、哌拉西林/他唑巴坦、头孢曲松、头孢他啶、头孢吡肟、亚胺培南、美罗培南、庆大霉索、阿米卡星、环丙沙星、左氧氟沙星、加替沙星、头孢哌酮/舒巴坦、氨曲南耐药率分别为92.3%、55.4%、88.6%、86.3%、80.3%、30.0%、35.0%、76.6%、79.6%、75.1%、87.1%、48.3%、42.0%和79.6%.②在21株耐碳青霉烯类鲍曼不动杆菌,有14株碳青霉烯酶表型阳性,检出率为66.7%,有18株PCR扩增出OXA-23基因,检出率85.7%,全部菌株blaOXA-24及blaOXA-58PCR扩增均为阴性,PCR产物测序表明与鲍曼不动杆菌(AY795964.1)blaOXA-23基因序列100%同源。结论:鲍曼不动杆菌多重耐药性严重;表型和基因型检测证实本院临床分离鲍曼不动杆菌对碳青霉烯类耐药机制主要是产OXA-23型酶。