Resistance mechanism and homology of ampicillin-resistant Haemophilus influenzae in children

doi:10.3969/j.issn.1673-8640.2020.06.001

Abstract

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

CLC Number:

R378.4

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.

Figures/Tables 8

References 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