食品动物源hvKP与cKP菌株毒力基因、耐药性和整合子−基因盒特征比较分析

董含倩; 林子清; 李旭婷; 杨惟一; 林居纯

doi:10.7671/j.issn.1001-411X.202506009

食品动物源hvKP与cKP菌株毒力基因、耐药性和整合子−基因盒特征比较分析

Profiles of virulence genes, antimicrobial resistance and integron-cassettes in hvKP versus cKP strains from food animals

摘要

摘要:
目的比较四川省食品动物源高毒力肺炎克雷伯菌(Hypervirulent Klebsiella pneumoniae，hvKP)与经典肺炎克雷伯菌(Classical Klebsiella pneumoniae，cKP)的毒力基因、耐药性及整合子−耐药基因盒系统差异，揭示hvKP独特的致病性与耐药性机制，为兽医临床hvKP感染提供新的防控和治疗策略。
方法采用拉丝试验和毒力基因PCR法对139株肺炎克雷伯菌(Klebsiella pneumoniae，KP)进行hvKP、cKP分类鉴定；分别采用微量肉汤稀释法和PCR测序法检测菌株的药物敏感性和整合子−基因盒分布特征；采用多位点序列分型(Multilocus sequence typing，MLST)对hvKP菌株进行分子分型。
结果 139株KP菌株中共检出16株hvKP(高黏液型，均携带rmpA-iucA-iutA)和123株cKP。相比于cKP，hvKP菌株中rmpA、iucA、aero、iroB、ybtA的携带率更高(P≤0.01)，且多毒力谱更复杂。所有KP菌株对抗菌药物呈不同水平的耐药性，多重耐药谱广泛，hvKP对卡那霉素、庆大霉素、多西环素及恩诺沙星的耐药率显著低于cKP(P≤0.05或P≤0.01)。本研究KP菌株中，IntI 1检出率较高(hvKP：100.00%，cKP：99.19%)，只有1株hvKP携带基因盒，为arr3-dfrA27，31株cKP携带基因盒，包含9种不同阵列，以dhfrI-aadA2和dfrA12-orf-aadA2为主。在3株cKP中检出IntI 2，但未携带任何基因盒。分子分型显示，hvKP的主要流行型为ST25(43.75%)和ST60(31.25%)，且这2种ST型菌株均表现出多重耐药性与复杂毒力谱并存的特征。
结论四川省食品动物源KP菌株耐药性严重，整合子介导的耐药机制增加了耐药性传播的风险。尤其需要警惕hvKP，该类菌株携带复杂毒力因子并表现出严重耐药性，给兽医临床治疗带来严峻挑战。因此，在兽医临床实践中，快速准确地鉴别高毒力菌株、持续监测其耐药性，并据此制定最优治疗方案，尤为重要。

Abstract:
Objective To characterize the differences in virulence genes, antimicrobial resistance profiles, and integron-resistance gene cassette systems between hypervirulent Klebsiella pneumoniae (hvKP) and classical Klebsiella pneumoniae (cKP) isolates from food-producing animals in Sichuan Province, elucidate the unique pathogenicity and antimicrobial resistance mechanism of hvKP, and provide a foundation for new strategies for the prevention and treatment of hvKP infection in veterinary clinical practice.
Method A total of 139 K. pneumoniae (KP) isolates were classified as hvKP or cKP using the string test and PCR detection of virulence genes. The broth microdilution method and PCR sequencing were used to detect the antimicrobial susceptibility and integron-cassette distribution patterns in these isolates, respectively. Multilocus sequence typing (MLST) was conducted for hvKP isolates.
Result Among the 139 KP isolates, 16 were identified as hvKP, which were high viscosity and carried rmpA-iucA-iutA, the other 123 were cKP. Compared to cKP, the hvKP isolates demonstrated a significantly higher prevalence of rmpA, iucA, aero, iroB, and ybtA (P≤0.01) and possessed more complex virulence profiles. All KP isolates exhibited varying levels of antimicrobial resistance and widespread multidrug resistance profile. hvKP demonstrated significantly lower resistance rates to kanamycin, gentamicin, doxycycline, and enrofloxacin compared to cKP (P≤0.05 or P≤0.01). The IntI 1 was detected in nearly all isolates (100.00% in hvKP and 99.19% in cKP). Among these, only one hvKP isolate carried cassette, which was arr3-dfrA27. In contrast, 31 cKP isolates harbored cassettes, including nine distinct arrays, with dhfrI-aadA2 and dfrA12-orf-aadA2 being the main ones. The IntI 2 gene was rare, found in only three cKP isolates, all of which contained empty cassettes. MLST analysis identified the predominant sequence types (STs) among hvKP were ST25 (43.75%) and ST60 (31.25%). Notably, both of these STs isolates displayed concurrent multidrug resistance and complex virulence profiles.
Conclusion Antimicrobial resistance in KP isolates from food animals in Sichuan Province is significant. The integron-mediated resistance mechanisms heightens the risk of antimicrobial resistance dissemination. Of particular concern is hvKP, which combines complex virulence factors with serious antimicrobial resistance, complicating veterinary clinical treatment. Therefore, the rapid and accurate identification of hypervirulent strains, continuous monitoring of antimicrobial resistance, and the development of optimal treatments are urgent priorities in veterinary practice.

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