Zhou Jian, Mao Sha, Liu Ying, Gu Tao, Zhou Jingzhu, Chen Fengming, Hu Yong, Li Shijun
School of Public Health, The Key Laboratory of Environmental Pollution Monitoring and Disease Control, Ministry of Education, Guizhou Medical University, Guiyang, 550025, China.
Key Laboratory of Microbio and Infectious Disease Prevention and Control in Guizhou Province, Guizhou Center for Disease Control and Prevention, Guiyang, 550004, China.
BMC Microbiol. 2025 Apr 12;25(1):211. doi: 10.1186/s12866-025-03941-5.
Niviventer, a rodent species widely distributed in Asian forests, serves as a significant reservoir for pathogens. Bordetella pseudohinzii(B. pseudohinzii), a recently identified Bordetella species with unclear pathogenic potential, poses challenges in species identification and understanding of its pathogenicity, its biological traits and antibiotic resistance are not well understood.
B. pseudohinzii(strains 21F10, 22F12, and 27F25) were isolated from lung tissue of wild niviventer rodents in Guizhou, China. Initial identification was performed using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) and 16 S rRNA gene sequencing. A phylogenetic tree based on the 16 S rRNA gene sequences was constructed using the neighbor-joining method implemented in MEGA 11. Whole-genome sequencing (WGS) was conducted on all three strains, and strain 21F10 underwent hybrid assembly of second- and third-generation sequencing to achieve high-quality sequences. Average Nucleotide Identity (ANI) and digital DNA-DNA hybridization (dDDH) were used as gold standards for strain identification, with thresholds set at 95% and 70%, respectively. Gene annotation was performed using nine databases, including KEGG, VFDB, CARD, PHI, COG, and NR. Antimicrobial susceptibility testing was carried out using the drug-sensitive plate method.
Initial MALDI-TOF MS identification misclassified the strains as B. avium and B. hinzii. However, PCR amplification of the 16 S rRNA gene (primers 27 F and 1492R) revealed that the strains were identified as B. hinzii (identity > 99%). Further analysis of the 16 S rRNA gene sequences obtained from WGS showed identities greater than 99% with both B. pseudohinzii and B. hinzii. Phylogenetic analysis of the 16 S rRNA gene sequences showed that the strains were closely related to B. hinzii, followed by B. pseudohinzii. Ultimately, the ANI values of all three strains with B. pseudohinzii were greater than 95%, and dDDH values exceeded 70%, confirming the strains as B. pseudohinzii. Strain 21F10 exhibited notable findings in terms of virulence factors and antibiotic resistance genes. Antimicrobial susceptibility testing revealed significant resistance to several cephalosporins (cefoxitin, cefuroxime, cefotaxime, cefazolin, and ceftiofur). The 16 S rRNA and WGS of strain 21F10 have been deposited in GenBank and Genome Sequence Archive (GSA)under accession numbers PQ881859 and CRA022358, respectively.
The first isolation of B. pseudohinzii from the lung tissue of wild niviventer was reported, and the limitations of traditional methods for identifying B. pseudohinzii were demonstrated. We highlight the superiority of WGS for accurate species identification. The findings reveal a complex pathogenic profile and notable antibiotic resistance, providing important insights for the future prevention and treatment of B. pseudohinzii infections in humans, as well as underscoring the need for monitoring B. pseudohinzii in rodent populations.
针毛鼠是一种广泛分布于亚洲森林的啮齿动物，是病原体的重要宿主。伪欣茨氏博德特氏菌（B. pseudohinzii）是最近发现的一种博德特氏菌，其致病潜力尚不清楚，在物种鉴定以及对其致病性的理解方面存在挑战，其生物学特性和抗生素耐药性也未得到充分了解。
从中国贵州野生针毛鼠的肺组织中分离出伪欣茨氏博德特氏菌（菌株21F10、22F12和27F25）。最初使用基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF MS）和16S rRNA基因测序进行鉴定。使用MEGA 11中实现的邻接法，基于16S rRNA基因序列构建系统发育树。对所有三个菌株进行全基因组测序（WGS），菌株21F10进行了第二代和第三代测序的混合组装以获得高质量序列。平均核苷酸同一性（ANI）和数字DNA-DNA杂交（dDDH）用作菌株鉴定的金标准，阈值分别设定为95%和70%。使用包括KEGG、VFDB、CARD、PHI、COG和NR在内的九个数据库进行基因注释。使用药敏平板法进行药敏试验。
最初的MALDI-TOF MS鉴定将这些菌株错误分类为鸟博德特氏菌和欣茨氏博德特氏菌。然而，16S rRNA基因（引物27F和1492R）的PCR扩增显示，这些菌株被鉴定为欣茨氏博德特氏菌（同一性>99%）。对从WGS获得的16S rRNA基因序列的进一步分析表明，与伪欣茨氏博德特氏菌和欣茨氏博德特氏菌的同一性均大于99%。16S rRNA基因序列的系统发育分析表明，这些菌株与欣茨氏博德特氏菌密切相关，其次是伪欣茨氏博德特氏菌。最终，所有三个菌株与伪欣茨氏博德特氏菌的ANI值均大于95%，dDDH值超过70%，证实这些菌株为伪欣茨氏博德特氏菌。菌株21F10在毒力因子和抗生素耐药基因方面表现出显著特征。药敏试验显示对几种头孢菌素（头孢西丁、头孢呋辛、头孢噻肟、头孢唑林和头孢噻呋）有明显耐药性。菌株21F10的16S rRNA和WGS已分别以登录号PQ881859和CRA022358保藏于GenBank和基因组序列存档库（GSA）。
报道了首次从野生针毛鼠的肺组织中分离出伪欣茨氏博德特氏菌，并证明了传统的伪欣茨氏博德特氏菌鉴定方法的局限性。我们强调了WGS在准确物种鉴定方面的优越性。研究结果揭示了复杂的致病特征和显著的抗生素耐药性，为未来人类伪欣茨氏博德特氏菌感染的预防和治疗提供了重要见解，同时也强调了在啮齿动物种群中监测伪欣茨氏博德特氏菌的必要性。
