鳞状皮屑裸小鼠木糖葡萄球菌的分离鉴定

doi:10.12300/j.issn.1674-5817.2024.166

实验动物与比较医学 ›› 2025, Vol. 45 ›› Issue (3): 368-375.DOI: 10.12300/j.issn.1674-5817.2024.166

鳞状皮屑裸小鼠木糖葡萄球菌的分离鉴定

孔志豪¹, 魏晓锋¹()(), 于灵芝¹()(), 冯丽萍¹, 朱琦¹, 施国君², 王晨²

^1.上海实验动物研究中心, 上海 201203
^2.英崴沃(上海)生物科技有限公司, 上海 201203

收稿日期:2024-11-12 修回日期:2025-02-05 出版日期:2025-07-07 发布日期:2025-06-25
通讯作者: 魏晓锋（1980—），男，硕士，副研究员，主要从事实验动物质量控制研究。E-mail： wei.xf@outlook.com。 ORCID： 0009-0009-5089-8342；
于灵芝（1980—），女，博士，助理研究员，主要从事实验动物病原体核酸检测方法研究。E-mail： yulingzhi@slarc.org.cn。 ORCID：
作者简介:孔志豪（2001—），男，大专，主要从事实验动物质量控制研究。E-mail：kongzhihao@slarc.org.cn
基金资助:
上海实验动物研究中心科技创新计划新星项目“木糖葡萄球菌快速分子检测方法的建立及应用”(2024NS05)

Isolation and Identification of Staphylococcus xylosus in Nude Mice with Squamous Skin Scurfs

KONG Zhihao¹, WEI Xiaofeng¹()(), YU Lingzhi¹()(), FENG Liping¹, ZHU Qi¹, SHI Guojun², WANG Chen²

^1.Shanghai Laboratory Animal Research Center, Shanghai 201203, China
^2.INVIVO (Shanghai) Biotechnology Co. , Ltd. , Shanghai 201203, China

Received:2024-11-12 Revised:2025-02-05 Published:2025-06-25 Online:2025-07-07
Contact: WEI Xiaofeng (ORCID: 0009-0009-5089-8342), E-mail: wei.xf@outlook.com;
YU Lingzhi (ORCID: 0009-0004-1835-3493), E-mail: yulingzhi@slarc.org.cn

摘要/Abstract

摘要：

目的拟从鳞状皮屑裸小鼠的皮肤上分离病原菌，并进行病原鉴定、溯源分析和致病性研究，以期为鳞状皮屑裸小鼠的病原体诊断提供新的思路。方法对1只患鳞状皮屑皮肤病的裸小鼠的皮肤进行拭子采样，通过核酸检测、细菌分离培养、生化鉴定、16S rDNA 基因扩增测序、全基因组测序构建系统进化树等方法鉴定菌株。然后取15只 BALB/c 裸小鼠，随机分为涂擦生理盐水的对照组、涂擦1.8×10⁸ CFU/mL分离病菌液的高浓度组和涂擦1.8×10⁷ CFU/mL分离病菌液的低浓度组，通过动物感染试验及HE染色观察皮肤组织病理学变化，进行该菌株的致病性分析。结果送检裸小鼠的皮肤拭子样本中牛棒状杆菌核酸阴性，排除了牛棒状杆菌的感染。进一步分离培养的病原菌经高盐甘露醇琼脂平板和血琼脂平板培养以及革兰染色提示为革兰阳性葡萄球菌。16S rDNA 测序和全自动微生物鉴定系统鉴定结果显示，该菌株为木糖葡萄球菌。全基因组测序后的系统进化树分析显示，该菌株与韩国叶菜分离株（GenBank GCA_00207825.1）的亲缘关系最近。动物感染试验显示，高浓度和低浓度分离菌液分别感染17 d和20 d时裸小鼠头颈部和背部开始出现鳞状皮屑，之后逐渐扩散至其他部位；而且两组的皮屑症状均表现为一过性，分别持续7 d和3 d皮屑消失；高浓度组和低浓度组的感染率均为33.33%。与对照组相比，感染后的裸小鼠皮肤组织病理学观察未发现明显异常，提示该菌株对裸小鼠的致病力存在明显的个体差异。结论从患鳞状皮屑裸小鼠皮肤上分离鉴定出1株木糖葡萄球菌菌株。该菌株是一种机会性感染病原体，临床表现为一过性的鳞状皮屑症状，组织病理学并未发生明显改变，并且裸小鼠对该菌株的敏感性存在个体差异。本研究结果为免疫缺陷小鼠或基因敲除小鼠的病原体诊断提供了数据支撑。

关键词: 裸小鼠, 鳞状皮屑, 木糖葡萄球菌, 系统进化树

Abstract:

Objective To isolate pathogenic bacteria from the skin of a nude mouse exhibiting squamous skin scurfs, and perform bacterial identification, traceability analysis, and pathogenicity studies to provide a new approach for the diagnosis of pathogens in nude mice with squamous skin scurfs. Methods Skin swab samples were collected from a nude mouse exhibiting squamous skin scurfs for nucleic acid testing, bacterial isolation and culture, biochemical identification, 16S rDNA gene amplification and sequencing, and whole genome sequencing to construct a phylogenetic tree. Fifteen BALB/c nude mice were randomized into a saline-treated control group, a high-concentration group treated with 1.8×10? CFU/mL of the isolated bacterial suspension, and a low-concentration group treated with 1.8×10? CFU/mL of the isolated bacterial suspension. Pathogenicity was assessed by animal infection experiments and observation of histopathological changes in skin tissue using HE staining. Results The nucleic acid test for Corynebacterium bovis was negative, excluding infection by this organism. The pathogen isolated on mannitol salt agar and blood agar, combined with Gram staining, suggested a Gram-positive Staphylococcus species. The isolated strain was identified by 16S rDNA sequencing and a fully automated microbial identification system as Staphylococcus xylosus. Phylogenetic tree analysis based on whole genome sequencing showed that the strain was most closely related to an isolate from leafy vegetables in South Korea (GenBank GCA_00207825.1). In the high-concentration group, squamous skin scurfs appeared on the head, neck, and back of nude mice on the 17th day post-infection, while in the low concentration group, similar symptoms appeared on the 20th day post-infection and gradually spread to other areas. The scaling symptoms were transient, lasting for 7 days in the high-concentration group and 3 days in the low-concentration group, after which the skin returned to normal. The infection rate was 33.33% in both the high- and low-concentration groups. No significant pathological changes were observed in the skin tissues of infected mice compared to the control group, indicating marked individual differences in the pathogenicity of the strain in nude mice. Conclusion A strain of Staphylococcus xylosus was isolated from the skin of a nude mouse exhibiting squamous skin scurfs. The strain is an opportunistic pathogen that causes transient squamous skin scurfs without significant histopathological changes, and there are individual differences in the sensitivity of nude mice to this strain. These findings can provide valuable data for pathogen identification in immunodeficient or gene knockout mice.

Key words: Nude mice, Squamous skin scurfs, Staphylococcus xylosus, Phylogenetic tree

中图分类号:

Q95-33

孔志豪,魏晓锋,于灵芝,等. 鳞状皮屑裸小鼠木糖葡萄球菌的分离鉴定[J]. 实验动物与比较医学, 2025, 45(3): 368-375. DOI: 10.12300/j.issn.1674-5817.2024.166.

KONG Zhihao,WEI Xiaofeng,YU Lingzhi,et al. Isolation and Identification of Staphylococcus xylosus in Nude Mice with Squamous Skin Scurfs[J]. Laboratory Animal and Comparative Medicine, 2025, 45(3): 368-375. DOI: 10.12300/j.issn.1674-5817.2024.166.

图/表 6

图 1 实时荧光定量PCR检测显示牛棒状杆菌阴性注：红色曲线代表阳性对照，黑色曲线代表阴性对照，绿色曲线代表样本。蓝色直线代表阈值线。

Figure 1 Detection of Corynebacterium bovis was negative by real-time fluorescence PCRNote： Red curve shows the positive control. Black curve shows the negative control. Green curve shows the sample. Blue line shows the threshold line.

图2 分离菌株在高盐甘露醇琼脂平板（A）和血琼脂平板（B）上的菌落形态图以及革兰染色镜检图（C）

Figure 2 Colony morphology on mannitol salt agar plate （A） and blood agar plate （B） and Gram staining microscopic examination （C） of the isolated strain

表 1 分离菌株的生化鉴定结果显示为木糖葡萄球菌

Table 1 Biochemical identification results indicating that the isolated strain was Staphylococcus xylosus

生化项目 Biochemical items	鉴定结果 Results
D-苦杏仁甙 D-amygdalin	-
磷脂酰肌醇磷脂酶C Phosphatidylinositol phospholipase C	-
D-木糖 D-xylose	+
精氨酸双水解酶1 Arginine dihydrolase 1	+
β-半乳糖苷酶 β-galactosidase	+
α-葡糖苷酶 α-glucosidase	+
丙氨酸-苯丙氨酸-脯氨酸芳胺酶 Ala-Phe-Pro-arylamidase	-
环糊精 Cyclodextrin	-
L-天冬氨酸芳胺酶 L-aspartate arylamidase	-
β-半乳糖吡喃糖苷酶 β-galactopyranosidase	-
α-甘露糖苷酶 α-mannosidase	-
磷酸酶 Phosphatase	+
亮氨酸芳胺酶 Leucine arylamidase	-
L-脯氨酸芳胺酶 L-proline arylamidase	-
β-葡萄糖醛酸酶 β-glucuronidase	+
α-半乳糖苷酶 α-galactosidase	-
L-吡咯烷酮芳胺酶 L-pyrrolidonyl-arylamidase	+
β-葡萄糖醛酸酶 β-glucuronidase	+
丙氨酸芳胺酶 Alanine arylamidase	-
酪氨酸芳胺酶 Tyrosine arylamidase	-
D-山梨醇 D-sorbitol	-
尿素酶 Urease	+
多黏菌素B Polymyxin B	-
D-半乳糖 D-galactose	-
D-核糖 D-ribose	+
L-乳酸盐碱化 L-lactate alkalinization	+
乳糖 Lactose	+
N-乙酰-D-氨基葡萄糖 N-acetyl-D-glucosamine	+
D-麦芽糖 D-maltose	+
杆菌肽耐药 Bacitracin resistance	+
新生霉素耐药 Novobiocin resistance	+
6.5%NaCl中生长 Growth in 6.5% NaCl	+
D-甘露醇 D-mannitol	+
D-甘露糖 D-mannose	+
甲基-β-D-吡喃葡萄糖苷 Methyl-β-D-glucopyranoside	+
支链淀粉 Pullulan	-
D-棉子糖 D-raffinose	-
O/129耐药 O/129 resistance	+
水杨素 Salicin	-
蔗糖 Saccharose/Sucrose	+
D-海藻糖 D-trehalose	+
精氨酸双水解酶2 Arginine dihydrolase 2	-
奥普托欣耐药 Optochin resistance	+

图3 分离菌株基于单拷贝基因集的系统进化树分析注：1代表分离菌株 slarc01。Bp 代表自展值百分比。

Figure 3 Phylogenetic tree analysis based on single-copy orthologue of the isolated strainNote： “1” represents the isolated strain slarc01. “Bp” represents the bootstrap percentage.

图4 不同组别裸小鼠感染分离菌液后出现皮屑的临床表现注：A，颈背部皮肤涂擦生理盐水的对照组；B，颈背部皮肤涂擦1.8×107 CFU/mL分离培养菌液的低浓度组；C，颈背部皮肤涂擦1.8×108 CFU/mL分离培养菌液的高浓度组腹部症状；D，颈背部皮肤涂擦1.8×108 CFU/mL分离培养菌液的高浓度组背部症状。

Figure 4 Clinical manifestations of scaling in different groups of nude mice infected with isolated bacterial suspensionNote： A， Control group of applying physiological saline solution to the skin on the neck and back； B， Low concentration group of 1.8×107 CFU/mL isolated bacterial solution applied to the skin on the neck and back； C， Abdominal symptoms in the high concentration group of isolated bacterial solution with a concentration of 1.8×108 CFU/mL applied to the skin on the neck and back； D， Back symptoms in the high concentration group treated with 1.8×108 CFU/mL isolated bacterial solution applied to the skin on the neck and back.

图5 分离菌感染后鳞状皮屑裸小鼠的皮肤组织病理学变化（HE染色，×100）注：A，对照组；B，低浓度组；C，高浓度组。黑色箭头指示真皮毛囊，红色箭头指示皮脂腺（比例尺大小为200 μm）。

Figure 5 Pathological changes in skin tissues of nude mice with squamous skin scurfs after infection with isolated bacteria （HE staining， ×100）Note： A， Control group； B， Low concentration group； C， High concentration group. Black arrow indicates dermal hair follicle； red arrow indicates sebaceous gland （Scale bar： 200 μm）.

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鳞状皮屑裸小鼠木糖葡萄球菌的分离鉴定

Isolation and Identification of Staphylococcus xylosus in Nude Mice with Squamous Skin Scurfs

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