实验大鼠和小鼠中肺炎克雷伯菌复合体的鉴定与分析

doi:10.12300/j.issn.1674-5817.2025.174

摘要/Abstract

摘要：

目的分析复旦大学实验动物中心入驻大鼠和小鼠中肺炎克雷伯菌分离株的类群，同时优化肺炎克雷伯菌的鉴定方法，并进行毒力基因和药物敏感性检测，为实验动物设施微生物的检测策略和防控措施提供实践依据。方法本研究收集了2023年9月至2025年9月从实验动物供应商购入的实验大鼠、小鼠中采集的回盲肠内容物，从中分离并冻存了11株肺炎克雷伯菌，对其进行β-内酰胺酶基因多重PCR检测，以进行分群。在此基础上，挑取不同类群的代表性菌株进行生化鉴定、质谱检测、16S rRNA和tyrB基因测序、毒力基因检测和药物敏感性检测。结果 β-内酰胺酶基因的多重PCR检测结果显示，11株菌中4株为肺炎克雷伯菌（Klebsiella pneumoniae，Kp1），4株为类肺炎克雷伯菌（Klebsiella quasipneumoniae，Kp2），3株为变栖克雷伯菌（Klebsiella variicola，Kp3）。Kp1～Kp3无法通过生化鉴定、质谱检测和16S rRNA基因序列鉴别，而β-内酰胺酶基因多重PCR和tyrB基因序列可以实现三者的同源性分群。Kp1～Kp3在荚膜基因（荚膜相关基因G、镁转运系统ATP结合蛋白A）和铁载体基因（肺炎克雷伯菌铁摄取系统基因）的检出率上有差异，且对不同药物的敏感性也有较大差异。结论从商品化生产的实验大鼠和小鼠中分离到的肺炎克雷伯菌类群与人类临床样本中的菌株具有相似性，且同样包括肺炎克雷伯菌复合体中的多个类群；本研究优化了Kp1～Kp3类群的鉴定方法，并对Kp1～Kp3进行了部分毒力基因和耐药性的初步检测，可针对实验动物设施内出现的不同类群感染提供精准治疗参考。

关键词: 肺炎克雷伯菌复合体, β-内酰胺酶基因, 多重PCR, 实验大鼠和小鼠

Abstract:

Objective To analyze the phylogroups of Klebsiella pneumoniae isolates from laboratory rats and mice housed at the Laboratory Animal Center Fudan University, optimize the identification method for Klebsiella pneumoniae, and perform virulence genes detection and antimicrobial susceptibility testing, thereby providing a practical basis for microbial detection strategies and prevention and control measures in laboratory animal facilities. Methods Between September 2023 and September 2025, ileocecal contents were collected from laboratory rats and mice purchased from laboratory animal suppliers, from which 11 strains of Klebsiella pneumoniae were isolated and cryopreserved. These strains were tested for β-lactamase genes using multiplex PCR for phylogrouping. Furthermore, representative strains from different phylogroups were selected for biochemical tests, mass spectrometry analysis, 16S rRNA gene and tyrB gene sequencing, as well as virulence genes detection and antimicrobial susceptibility testing. Results Multiplex PCR detection of β-lactamase genes revealed that among 11 strains, 4 were identified as Klebsiella pneumoniae (Kp1), 4 as Klebsiella quasipneumoniae (Kp2), and 3 as Klebsiella variicola (Kp3). Kp1-Kp3 could not be differentiated using biochemical tests, mass spectrometry analysis, or 16S rRNA gene sequencing, whereas multiplex PCR of β-lactamase genes and tyrB gene sequencing enabled phylogrouping of all three. There were differences in the detection rates of capsular genes (capsule associated gene G and magnesium transport system ATP-binding protein A) and siderophore genes (Klebsiella pneumoniae iron uptake system genes) among Kp1-Kp3; susceptibility to different antibiotics also varied considerably. Conclusion Klebsiella pneumoniae phylogroups isolated from commercially produced laboratory rats and mice are similar to those in human clinical samples and likewise include multiple phylogroups of the species complex. In this study, the identification methods of Kp1-Kp3 were optimized,furthermore selected virulence genes and antimicrobial resistance in Kp1-Kp3 were preliminarily investigated, which could provide precise reference for the treatment of infections caused by different phylogroups occurring in laboratory animal facilities.

Key words: Klebsiella pneumoniae species complex, β-lactamase genes, Multiplex PCR, Laboratory rats and mice

中图分类号:

Q95-33

强苏静,李冬婷,徐瑾华,等. 实验大鼠和小鼠中肺炎克雷伯菌复合体的鉴定与分析[J]. 实验动物与比较医学, 2026, 46(4): 564-572. DOI: 10.12300/j.issn.1674-5817.2025.174.

QIANG Sujing,LI Dongting,XU Jinhua,et al. Identification and Analysis of the Klebsiella pneumoniae Species Complex in Laboratory Rats and Mice[J]. Laboratory Animal and Comparative Medicine, 2026, 46(4): 564-572. DOI: 10.12300/j.issn.1674-5817.2025.174.

图/表 6

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基因 Gene	引物序列(5'→3') Primer sequences (5'→3')	片段大小/bp Fragment length/bp	退火温度/℃ Annealing temperature/℃
rmpA	F：ACTGGGCTACCTCTGCTTCA； R：CTTGCATGAGCCATCTTTCA	535	55
wcaG	F：GGTTGGKTCAGCAATCGTA； R：ACTATTCCGCCAACTTTTGC	169	58
magA	F：GGTGCTCTTTACATCATTGC； R：GCAATGGCCATTTGCGTTAG	1 283	53
wabG	F：CGGACTGGCAGATCCATATC； R：ACCATCGGCCATTTGATAGA	683	58
uge	F：GATCATCCGGTCTCCCTGTA； R：TCTTCACGCCTTCCTTCACT	535	55
fimH	F：TGCTGCTGGGCTGGTCGATG； R：GGGAGGGTGACGGTGACATC	534	55
Aer	F：GCATAGGCGGATACGAACAT； R：CACAGGGCAATTGCTTACCT	556	55
ybtA	F：ATGACGGAGTCACCGCAAAC； R：TTACATCACGCGTTTAAAGG	960	56
kfu	F：GAAGTGACGCTGTTTCTGGC； R：TTTCGTGTGGCCAGTGACTC	797	55
ureA	F：GCTGACTTAAGAGAACGTTATG； R：GATCATGGCGCTACCTCA	337	55

基因 Gene	引物序列(5'→3') Primer sequences (5'→3')	片段大小/bp Fragment length/bp	退火温度/℃ Annealing temperature/℃
rmpA	F：ACTGGGCTACCTCTGCTTCA； R：CTTGCATGAGCCATCTTTCA	535	55
wcaG	F：GGTTGGKTCAGCAATCGTA； R：ACTATTCCGCCAACTTTTGC	169	58
magA	F：GGTGCTCTTTACATCATTGC； R：GCAATGGCCATTTGCGTTAG	1 283	53
wabG	F：CGGACTGGCAGATCCATATC； R：ACCATCGGCCATTTGATAGA	683	58
uge	F：GATCATCCGGTCTCCCTGTA； R：TCTTCACGCCTTCCTTCACT	535	55
fimH	F：TGCTGCTGGGCTGGTCGATG； R：GGGAGGGTGACGGTGACATC	534	55
Aer	F：GCATAGGCGGATACGAACAT； R：CACAGGGCAATTGCTTACCT	556	55
ybtA	F：ATGACGGAGTCACCGCAAAC； R：TTACATCACGCGTTTAAAGG	960	56
kfu	F：GAAGTGACGCTGTTTCTGGC； R：TTTCGTGTGGCCAGTGACTC	797	55
ureA	F：GCTGACTTAAGAGAACGTTATG； R：GATCATGGCGCTACCTCA	337	55

孔号 Well	实验 Test	Kp1	Kp2	Kp3	孔号 Well	实验 Test	Kp1	Kp2	Kp3
2	丙氨酸-苯丙氨酸-脯氨酸芳胺酶	-	-	-	33	蔗糖	+	+	+
3	侧金盏花醇	+	+	-	34	D-塔格糖	-	-	-
4	吡咯烷基芳胺酶	+	+	+	35	D-海藻糖	+	+	+
5	L-阿拉伯醇	-	-	-	36	柠檬酸盐	+	+	+
7	D-纤维二糖	+	+	+	37	丙二酸盐利用	+	+	+
9	β-半乳糖苷酶	+	+	+	39	5-酮-葡萄糖苷	-	+	+
10	H₂S产生	-	-	-	40	L-乳酸产碱	+	+	+
11	β-N-乙酰氨基葡萄糖苷酶	+	+	-	41	α-葡萄糖	+	+	+
12	谷氨酰芳胺酶pNA	-	-	-	42	琥珀酸盐产碱	+	+	+
13	D-葡萄糖	+	+	+	43	N-乙酰-β-半乳糖苷酶	-	-	-
14	γ-谷氨酰转移酶	+	+	+	44	α-半乳糖苷酶	+	+	+
15	发酵/葡萄糖	+	+	+	45	磷酸酶	+	+	+
17	β-葡萄糖苷酶	+	+	+	46	氨基乙酸芳胺酶	+	+	+
18	D-麦芽糖	+	+	+	47	鸟氨酸脱羧酶	-	-	-
19	D-甘露醇	+	+	+	48	赖氨酸脱羧酶	+	+	+
20	D-甘露糖	+	+	+	53	组氨酸同化	-	+	-
21	β-木糖苷酶	+	+	+	56	COURMARATE	-	-	+
22	β-丙氨酸芳胺酶pNA	-	-	-	57	β-葡萄糖苷酸酶	-	-	-
23	L-脯氨酸芳胺酶	+	+	-	58	0/129耐受	+	+	+
26	脂酶	-	-	-	59	谷氨酸-甘氨酸-精氨酸芳胺酶	-	-	-
27	古老糖	+	+	+	61	L-苹果酸盐同化	-	+	-
29	酪氨酸芳胺酶	+	+	-	62	ELLMAN	-	-	-
31	尿素酶	+	+	+	63	L-乳酸盐同化	-	-	-
32	D-山梨醇	+	+	+

孔号 Well	实验 Test	Kp1	Kp2	Kp3	孔号 Well	实验 Test	Kp1	Kp2	Kp3
2	丙氨酸-苯丙氨酸-脯氨酸芳胺酶	-	-	-	33	蔗糖	+	+	+
3	侧金盏花醇	+	+	-	34	D-塔格糖	-	-	-
4	吡咯烷基芳胺酶	+	+	+	35	D-海藻糖	+	+	+
5	L-阿拉伯醇	-	-	-	36	柠檬酸盐	+	+	+
7	D-纤维二糖	+	+	+	37	丙二酸盐利用	+	+	+
9	β-半乳糖苷酶	+	+	+	39	5-酮-葡萄糖苷	-	+	+
10	H₂S产生	-	-	-	40	L-乳酸产碱	+	+	+
11	β-N-乙酰氨基葡萄糖苷酶	+	+	-	41	α-葡萄糖	+	+	+
12	谷氨酰芳胺酶pNA	-	-	-	42	琥珀酸盐产碱	+	+	+
13	D-葡萄糖	+	+	+	43	N-乙酰-β-半乳糖苷酶	-	-	-
14	γ-谷氨酰转移酶	+	+	+	44	α-半乳糖苷酶	+	+	+
15	发酵/葡萄糖	+	+	+	45	磷酸酶	+	+	+
17	β-葡萄糖苷酶	+	+	+	46	氨基乙酸芳胺酶	+	+	+
18	D-麦芽糖	+	+	+	47	鸟氨酸脱羧酶	-	-	-
19	D-甘露醇	+	+	+	48	赖氨酸脱羧酶	+	+	+
20	D-甘露糖	+	+	+	53	组氨酸同化	-	+	-
21	β-木糖苷酶	+	+	+	56	COURMARATE	-	-	+
22	β-丙氨酸芳胺酶pNA	-	-	-	57	β-葡萄糖苷酸酶	-	-	-
23	L-脯氨酸芳胺酶	+	+	-	58	0/129耐受	+	+	+
26	脂酶	-	-	-	59	谷氨酸-甘氨酸-精氨酸芳胺酶	-	-	-
27	古老糖	+	+	+	61	L-苹果酸盐同化	-	+	-
29	酪氨酸芳胺酶	+	+	-	62	ELLMAN	-	-	-
31	尿素酶	+	+	+	63	L-乳酸盐同化	-	-	-
32	D-山梨醇	+	+	+

基因类别 Gene category	基因 Gene	Kp1	Kp2	Kp3
荚膜Capsule	rmpA	-	-	-
	wcaG	-	+	-
	magA	-	+	-
脂多糖Lipopolysaccharide	wabG	+	+	+
脂多糖Lipopolysaccharide	uge	+	+	+
菌毛Pili	fimH	+	+	+
铁载体Siderophore	Aer	-	-	-
	ybtA	-	-	-
	kfu	+	-	+
尿素酶Urease	ureA	+	+	+