儿童耐氨苄西林流感嗜血杆菌耐药机制及同源性研究

doi:10.3969/j.issn.1673-8640.2020.06.001

摘要/Abstract

摘要：

目的探讨儿童耐氨苄西林流感嗜血杆菌（Hi）的耐药机制及同源性。方法收集Hi临床分离株117株。采用微量肉汤稀释法测定菌株耐药情况;采用硝酸噻吩试纸法测定氨苄西林耐药菌株β-内酰胺酶;采用聚合酶链反应（PCR）扩增产β-内酰胺酶氨苄西林耐药（BLPAR）菌TEM-1和ROB-1基因;采用PCR扩增耐药菌株ftsI基因,并对ftsI基因进行测序,通过与Hi RdKW20标准菌株的ftsI基因进行Blast比对确定其氨基酸置换形式,明确ftsI基因突变介导的不产β-内酰胺酶氨苄西林耐药（BLNAR）菌耐药机制;采用多重PCR测定Hi同源性,明确其分子流行病特点。结果 117株Hi中氨苄西林耐药率为56.4%（66/117）,β-内酰胺酶检出率为46.2%（54/117）,产酶基因均为TEM-1基因。BLNAR菌检出率为10.3%（12/117）,ftsI基因一代测序BLPAR菌、BLNAR菌突变率分别为68.5%（37/54）、75.0%（9/12）,ftsI基因型Ⅲ型分别为73.0%（27/37）、77.8%（7/9）,BLPAR菌与BLNAR菌发生Asn526Lys置换合并Ser385Thr置换分别占51.4%（19/37）、77.8%（7/9）;BLPAR菌多重PCR分型分为a、b、c、d和e型,BLNAR菌多重PCR分型分为a、e型,2种菌均具有克隆传播趋势,a型分别占63.0%（34/54）、75%（9/12）。结论耐氨苄西林Hi的耐药机制以产TEM-1型β-内酰胺酶为主,不产酶菌株的耐药机制主要是由KTG基序附近发生Asn526Lys置换合并SSN基序附近发生Ser385Thr置换介导ftsI基因突变所致。BLPAR菌与BLNAR菌 ftsI基因型均以Ⅲ型为主,且均具有克隆传播趋势。

关键词: 流感嗜血杆菌, 氨苄西林, 耐药机制, 同源性

Abstract:

Objective To study the resistance mechanism and homology of ampicillin-resistant Haemophilus influenzae（Hi） in children. Methods Totally,117 isolates of Hi were isolated. Antimicrobial susceptibility test was performed by microbroth dilution method. Beta-lactamase was determined by nitrocefin disk test. Beta-lactamase-positive ampicillin-resistance（BLPAR） genes,including TEM-1 and ROB-1,were amplified by polymerase chain reaction（PCR）,and ftsI gene was sequenced. Blast was compared with that of Hi RdKW20 to clarify the resistance mechanism of beta-lactamase-negative ampicillin-resistance（BLNAR）. The homology of Hi was determined by multiplex PCR,and the molecular epidemiological characteristics of Hi were clarified. Results Among the 117 isolates,the ampicillin resistance rate was 56.4%（66/117）,the beta-lactamase positive rate was 46.2%（54/117）,and TEM-1 gene was determined in all beta-lactamase-positive isolates. The determination rate of BLNAR was 10.3%（12/117）. The mutation rates of ftsI in BLPAR and BLNAR were 68.5%（37/54） and 75.0%（9/12）,and the genotype Ⅲ was the main type [73.0%（27/37） and 77.8%（7/9）]. The 51.4%（19/37） and 77.8%（7/9） of Asn526Lys replacement combined with Ser385Thr replacement occurred between BLPAR and BLNAR. The multiplex PCR typing of BLPAR was classified into 5 types,including a,b,c,d and e. The multiplex PCR typing of BLNAR was classified into 2 types,a and e,and these 2 types had clone-transmitted trends,and the proportions of type a in the 2 kinds of isolates were 63.0%（34/54） and 75%（9/12）,respectively. Conclusions The main resistance mechanism of beta-lactamase-positive Hi is to produce TEM-1. The main resistance mechanism of non-beta-lactamase Hi is caused by Asn526Lys replacement near KTG motif combined with Ser385Thr replacement near SSN motif. The ftsI genotype of BLPAR and BLNAR is mainly type Ⅲ. Both BLPAR and BLNAR have the trend of clone-transmission.

Key words: Haemophilus influenzae, Ampicillin, Resistance mechanism, Homology

中图分类号:

R378.4

王姜琳, 孙杰, 杨慧健, 王冰洁, 潘芬, 张泓. 儿童耐氨苄西林流感嗜血杆菌耐药机制及同源性研究[J]. 检验医学, 2020, 35(6): 513-518.

WANG Jianglin, SUN Jie, YANG Huijian, WANG Bingjie, PAN Fen, ZHANG Hong. Resistance mechanism and homology of ampicillin-resistant Haemophilus influenzae in children[J]. Laboratory Medicine, 2020, 35(6): 513-518.

图/表 8

参考文献 17

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基因名称	引物序列（5'~3'）	退火温度/℃	扩增片段/bp
TEM-1	F：TGGGTGCACGAGTGGGTTAC R：TTATCCGCCTCCATCCAGTC	56	526
ROB-1	F：CGTTTATGTATGGGATACAGAA R：GGGTTACGTTATCGCCTAATT	52	338
ftsI	F：TTGCACATATCTCCGATGAG R：AGCTGCTTCAGCATCTTGC	52	1 050

基因名称	引物序列（5'~3'）	退火温度/℃	扩增片段/bp
TEM-1	F：TGGGTGCACGAGTGGGTTAC R：TTATCCGCCTCCATCCAGTC	56	526
ROB-1	F：CGTTTATGTATGGGATACAGAA R：GGGTTACGTTATCGCCTAATT	52	338
ftsI	F：TTGCACATATCTCCGATGAG R：AGCTGCTTCAGCATCTTGC	52	1 050

基因名称	引物序列（5'~3'）	退火温度/℃	扩增片段/bp
Hinf-1	AAGCAAAACAAAAAGTTGAGGATAC	45	317
Hinf-7	AAATTAAACCTATTATTTTGTCGCT	45
Hamp-1	CAGGTGGCGTTTATATTGATTCTAG	45	518
Hamp-3	AGCGGCTCTATCTTGTAATGACACA	45

基因名称	引物序列（5'~3'）	退火温度/℃	扩增片段/bp
Hinf-1	AAGCAAAACAAAAAGTTGAGGATAC	45	317
Hinf-7	AAATTAAACCTATTATTTTGTCGCT	45
Hamp-1	CAGGTGGCGTTTATATTGATTCTAG	45	518
Hamp-3	AGCGGCTCTATCTTGTAATGACACA	45

抗菌药物	折点			耐药株数	耐药率/%	敏感率/%
抗菌药物	敏感/（μg/mL）	中介/（μg/mL）	耐药/（μg/mL）	耐药株数	耐药率/%	敏感率/%
氨苄西林	≤1	2	≥4	66	56.4	27.8
氨苄西林-舒巴坦	≤2/1		≥4/2	39	33.3	66.7
头孢呋辛	≤4	8	≥16	16	13.7	84.4
头孢曲松	≤2					93.2
头孢吡肟	≤2					90.6
克拉霉素	≤8	16	≥32	38	32.5	53.0
氯霉素	≤2	4	≥8	4	3.4	96.6
左氧氟沙星	≤2					94.9
美罗培南	≤0.5					93.2
复方磺胺甲噁唑	≤0.5/9.5	1/19~2/38	≥2/76	94	80.3	14.4