Identification and Analysis of the Klebsiella pneumoniae Species Complex in Laboratory Rats and Mice

doi:10.12300/j.issn.1674-5817.2025.174

Abstract

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

CLC Number:

Q95-33

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.

Figures/Tables 6

References 25

[1]	国家市场监督管理总局, 国家标准化管理委员会. 实验动物微生物、寄生虫学等级及监测: [S]. 北京: 中国标准出版社, 2022.
	State Administration for Market Regulation, National Standardization Administration. Laboratory animal—Microbiological and parasitical standards and monitoring: [S]. Beijing: Standard Press of China, 2022.
[2]	WYRES K L, LAM M M C, HOLT K E. Population genomics of Klebsiella pneumoniae [J]. Nat Rev Microbiol, 2020, 18(6): 344-359. DOI: 10.1038/s41579-019-0315-1 .
[3]	BARRIOS-CAMACHO H, SILVA-SÁNCHEZ J, CERCAS-AYALA E, et al. PCR system for the correct differentiation of the main bacterial species of the Klebsiella pneumoniae complex[J]. Arch Microbiol, 2021, 204(1): 73. DOI: 10.1007/s00203-021-02668-x .
[4]	VERBURG I, HERNÁNDEZ LEAL L, WAAR K, et al. Klebsiella pneumoniae species complex: From wastewater to the environment[J]. One Health, 2024, 19: 100880. DOI: 10.1016/j.onehlt.2024.100880 .
[5]	HOLT K E, WERTHEIM H, ZADOKS R N, et al. Genomic analysis of diversity, population structure, virulence, and antimicrobial resistance in Klebsiella pneumoniae, an urgent threat to public health[J]. Proc Natl Acad Sci USA, 2015, 112(27): E3574-E3581. DOI: 10.1073/pnas.1501049112 .
[6]	朱昱蓉, 刘荣华. 肺炎克雷伯菌、类肺炎克雷伯菌和变栖克雷伯菌的研究进展[J]. 中国感染控制杂志, 2023, 22(8): 983-989. DOI: 10.12138/j.issn.1671-9638.20232720 .
	ZHU Y R, LIU R H. Advance in Klebsiella pneumoniae, Klebsiella quasipneumoniae and Klebsiella variicola [J]. Chin J Infect Control, 2023, 22(8): 983-989. DOI: 10.12138/j.issn.1671-9638.20232720 .
[7]	PERALTA-MACOTELA P, FLORES-MONZÓN B, LIRA DE LEÓN K I, et al. Complete genome sequences of Klebsiella pneumoniae, Klebsiella quasipneumoniae, and Klebsiella variicola clinical isolates from an epidemiology study[J]. Microbiol Resour Announc, 2025, 14(3): e01060-e01024. DOI: 10.1128/mra.01060-24 .
[8]	WATANABE N, WATARI T, OTSUKA Y, et al. Clinical characteristics and antimicrobial susceptibility of Klebsiella pneumoniae, Klebsiella variicola and Klebsiella quasipneumoniae isolated from human urine in Japan[J]. J Med Microbiol, 2022, 71(6): 001546. DOI: 10.1099/jmm. 0.001546 .
[9]	RODRÍGUEZ-SANTIAGO J, ALVARADO-DELGADO A, RODRÍGUEZ-MEDINA N, et al. Colistin-resistant Klebsiella pneumoniae species complex: The scenario in Mexico[J]. J Glob Antimicrob Resist, 2025, 43: 203-212. DOI: 10.1016/j.jgar.2025.04.024 .
[10]	国家质量监督检验检疫总局. 实验动物肺炎克雷伯杆菌检测方法: [S]. 北京: 中国标准出版社, 2002.
	General Administration of Quality Supervision, Inspection and Quarantine. Laboratory animal—Method for examination ofKlebsiella pneumonia: [S]. Beijing: Standard Press of China, 2022.
[11]	LI S R, WANG X N, LU Y L, et al. Co-infections of Klebsiella pneumoniae and Elizabethkingia miricola in black-spotted frogs (Pelophylax nigromaculatus)[J]. Microb Pathog, 2023, 180: 106150. DOI: 10.1016/j.micpath.2023.106150 .
[12]	于灵芝, 谢建芸, 冯丽萍, 等. 金黄色葡萄球菌荧光定量PCR检测方法的建立及其在大鼠、小鼠粪便检测中的应用[J]. 实验动物与比较医学, 2023, 43(5): 566-573. DOI: 10.12300/j.issn.1674-5817.2023.022 .
	YU L Z, XIE J Y, FENG L P, et al. Establishment of fluorescence qPCR method for detection of Staphylococcus aureus and its application in feces detection of rats and mice[J]. Lab Anim Comp Med, 2023, 43(5): 566-573. DOI: 10.12300/j.issn.1674-5817.2023.022 .
[13]	JEONG E S, LEE K S, HEO S H, et al. Rapid identification of Klebsiella pneumoniae, Corynebacterium kutscheri, and Streptococcus pneumoniae using triplex polymerase chain reaction in rodents[J]. Exp Anim, 2013, 62(1): 35-40. DOI: 10.1538/expanim.62.35 .
[14]	DENISSEN J, HAVENGA B, REYNEKE B, et al. Comparing antibiotic resistance and virulence profiles of Enterococcus faecium, Klebsiella pneumoniae, and Pseudomonas aeruginosa from environmental and clinical settings[J]. Heliyon, 2024, 10(9): e30215. DOI: 10.1016/j.heliyon.2024.e30215 .
[15]	LEE C R, LEE J H, PARK K S, et al. Antimicrobial resistance of hypervirulent Klebsiella pneumoniae: epidemiology, hypervirulence-associated determinants, and resistance mechanisms[J]. Front Cell Infect Microbiol, 2017, 7: 483. DOI: 10.3389/fcimb.2017.00483 .
[16]	KOCSIS B. Hypervirulent Klebsiella pneumoniae: An update on epidemiology, detection and antibiotic resistance[J]. Acta Microbiol Immunol Hung. 2023, 70(4): 278-287. DOI: 10.1556/030.2023.02186 .
[17]	RUSSO T A, OLSON R, FANG C T, et al. Identification of biomarkers for differentiation of hypervirulent Klebsiella pneumoniae from classical K. pneumoniae [J]. J Clin Microbiol, 2018, 56(9): e00776-18. DOI: 10.1128/JCM.00776-18 .
[18]	FONSECA E L, VEIGA RAMOS N DA, ANDRADE B G N, et al. A one-step multiplex PCR to identify Klebsiella pneumoniae, Klebsiella variicola, and Klebsiella quasipneumoniae in the clinical routine[J]. Diagn Microbiol Infect Dis, 2017, 87(4): 315-317. DOI: 10.1016/j.diagmicrobio.2017.01.005 .
[19]	沈兰凤, 胡仁静, 万林, 等. 变栖克雷伯菌全基因组测序及毒力特征分析[J]. 职业与健康, 2024, 40(6): 732-738. DOI: 10.13329/j.cnki.zyyjk.2024.0139 .
	SHEN L F, HU R J, WAN L, et al. Whole-genome sequencing and virulence characteristics of Klebsiella variicola [J]. Occup Health, 2024, 40(6): 732-738. DOI: 10.13329/j.cnki.zyyjk.2024.0139 .
[20]	MORGADO S, FONSECA E, VICENTE A C. Genomics of Klebsiella pneumoniae species complex reveals the circulation of high-risk multidrug-resistant pandemic clones in human, animal, and environmental sources[J]. Microorganisms, 2022, 10(11): 2281. DOI: 10.3390/microorganisms10112281 .
[21]	BRISSE S, VAN HIMBERGEN T, KUSTERS K, et al. Development of a rapid identification method for Klebsiella pneumoniae phylogenetic groups and analysis of 420 clinical isolates[J]. Clin Microbiol Infect, 2004, 10(10): 942-945. DOI: 10.1111/j.1469-0691.2004.00973.x .
[22]	VOELLMY I K, LANG C, GASSER M, et al. Antibiotic resistance surveillance of Klebsiella pneumoniae complex is affected by refined MALDI-TOF identification, Swiss data, 2017 to 2022[J]. Euro Surveill, 2022, 27(45): 2200104. DOI: 10.2807/1560-7917.ES.2022.27.45.2200104 .
[23]	RODRIGUES C, PASSET V, RAKOTONDRASOA A, et al. Identification of Klebsiella pneumoniae, Klebsiella quasipneumoniae, Klebsiella variicola and related phylogroups by MALDI-TOF mass spectrometry[J]. Front Microbiol, 2018, 9: 3000. DOI: 10.3389/fmicb.2018.03000 .
[24]	STACKEBRANDT E, GOEBEL B M. Taxonomic note: a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology[J]. Int J Syst Evol Microbiol, 1994, 44(4): 846-849. DOI: 10.1099/00207713-44-4-846 .
[25]	KOT B, PIECHOTA M, SZWEDA P, et al. Virulence analysis and antibiotic resistance of Klebsiella pneumoniae isolates from hospitalised patients in Poland[J]. Sci Rep, 2023, 13: 4448. DOI: 10.1038/s41598-023-31086-w .

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	+	+	+