维吾尔族与汉族新生儿感染产超广谱β-内酰胺酶与头孢菌素酶病原菌的耐药性及基因型分析

Analysis of antibiotic resistance and genotypes on extended spectrum β-lactamase and AmpC β-lactamase producing strains isolated from Uygur and Han newborns

目的 了解乌鲁木齐地区维吾尔族与汉族新生儿中肺炎克雷伯菌和大肠埃希菌产超广谱β-内酰胺酶（ESBLs）与头孢菌素酶（AmpC酶）的耐药性及基因型分布.方法 采用纸片扩散（K-B）法测定299株病原菌对22种抗菌药的耐药率;利用多重PCR技术检测产ESBLs或AmpC酶菌株耐药基因,并用DNA序列分析确认基因亚型;采用数字表法随机抽取产ESBLs的TEM、SHV、CTX-M-1、CTX-M-9组阳性菌株及产AmpC酶的DHA、CIT阳性菌株共148株进行测序.耐药率采用世界耐药监测网Whonet 5.4软件进行统计,数据对比应用医学PEMS 3.1统计软件采用卡方检验进行统计学处理,P＜0.05认为差异有统计学意义.结果 在耐药率方面,维吾尔族与汉族新生儿中产ESBLs肺炎克雷伯菌对复方新诺明[80.0%（40/50）和56.0%（28/50）,x2=6.6176,P=0.0101]、产ESBLs大肠埃希菌对头孢哌酮＋舒巴坦[54.2%（32/59）和94.0%（47/50）,x2=21.4512,P=0.0000]、产AmpC酶肺炎克雷伯菌对头孢哌酮＋舒巴坦[100.0%（20/20）和72.2%（26/36）,x2=6.7633,P=0.0093]和对阿米卡星[65.0%（13/20）和25.0%（9/36）,x2=8.6246,P=0.0033]耐药率差异有统计学意义.基因测序结果显示,ESBLs基因型除SHV在大肠埃希菌中维吾尔族新生儿只检出3.4%（2/59）而汉族新生儿未检出外,其他TEM、CTX-M-1、CTX-M-9组基因检出率均在38.0%（19/50）以上;其中TEM、CTX-M-1组基因分别为单一的TEM-1型和CTX-M-15型,SHV、CTX-M-9组阳性基因分别检出SHV-1、2、11、12、27、61、99和CTX-M-9、14、24、27、65等不同型别基因.肺炎克雷伯菌和大肠埃希菌中检出2种或2种以上ESBLs基因型的菌株占56.7%（42/74）～90.0%（63/70）.携带AmpC酶基因的2种菌在2个民族新生儿中基因型都只集中在DHA-1和CMY-44型,其他型未检出.2个民族新生儿间只有产TEM型的ESBLs大肠埃希菌检出率为维吾尔族新生儿高于汉族新生儿[71.2%（42/59）和50.0%（25/50）,x2=5.1291,P=0.0235],其余各种耐药基因型检出率差异无统计学意义（x2＜3.7780,P＞0.05）.结论 2个民族新生儿间部分菌株耐药率和耐药基因型分布差异有明显统计学意义,而且肺炎克雷伯菌和大肠埃希菌携带多种耐药基因并对β-内酰胺类抗生素呈高耐药性.

Objective This study aimed to investigate drug resistance and genotypes of the extended spectrum β-lactamase （ESBLs） and AmpC β-lactamase-producing Escherichia coli and Klebsiella pneumoniae isolated from Uygur and Han newborns in Urumqi. Methods Disk diffusion test （Kirby-Bauer） was used for detecting drug resistance of 299 strains to twenty two kinds of antibiotics. Resistance genes of the ESBLs and AmpC β-lactamase-producing strains were amplified by multiplex PCR and subtypes were confirmed by DNA sequence analysis. Total 148 strains were selected with random number table and sequenced,which included TEM-, SHV-, CTX-M-1-, or CTX-M-9- positive ESBLs-producing strains and DHA-,or CIT- positive AmpC β-lactamase-producing strains. Antibiotic resistant rates were analyzed by Whonet 5.4 and statistic analysis was performed by chi-square （ x2 ） test with PEMS 3. 1. Results The antibiotic resistant rates between Uygur and Han newborns significantly differ in ESBLs-producing Klebsiella pneumoniae to Suffamethoxazolum-Trimethoprimum （80. 0% （40/50） and 56. 0% （28/50） ,x2 = 6. 6176,P = 0. 0101 ） ,in ESBLs-producing Escherichia coli to Sulbactam and Cefopcrazone （54. 2% （32/59） and 94. 0% （47/S0）, x2 = 21.4512, P = 0. 0000 ）, and in AmpC β-lactamase-producing Klebsiella pneumoniae to Sulbactam and Cefopcrazone （ 100. 0% （20/20） and 72. 2% （26/36） ,x2 =6. 7633 ,P =0. 0093） and to Amikacin （65.0% （13/20） and 25.0% （9/36）, X2 = 8.6246, P= 0.0033）. Although SHV gene of ESBLs-producing Escherichia coli was detected from Uygur newborns at only 3.4% （2/59） and not detectable from Han newborns, TEM, CTX-M-1, and CTX-M-9 group genes were all detected over 38.0%（19/50）. Among the detected strains,the subtypes of TEM and CTX-M-1 were mainly TEM-1 and CTX-M-15,respectively; whereas the subtypes of SHV and CTX-M-9 included SHV-1,2,11,12,27 ,61,99 and CTX-M-9,14,24,27,65, respectively. The strains of Escherichia coli and Klebsiella pneumoniae carrying two or more kinds of ESBLs genotypes were 56. 7% （42/74） -90. 0% （63/70）. Two species carrying the AmpC gene in two kinds of newborns were only grouped in the subtypes of DHA-1 and CMY-44 ,and other subtypes were not detected at all. Moreover,TEM-positive ESBLs-producing Escherichia coli were detected from Uygur newborns at the higher rate than that from Han newborns （71.2% （42/59） and 50. 0% （25/50）, x2 =5. 1291 ,P= 0. 0235 ）, while there was no difference in other genotypes detected between two kinds of newborns （ x2 〈 3. 7780, P 〉 0. 05 ）. Conclusion There were significant differences in antibiotic resistance and genotype distribution of Klebsiella pneumoniae and Escherichia coli between two nationality newborns, and these two bacteria detected in this study carried multi-resistance genes and showed high resistant to β-lactamase antibiotics.