2017–2019年中国南方五省规模化养猪场副猪格拉菌血清型、耐药性及β-内酰胺类耐药基因bla-TEM分子特征分析

Characterization of serotypes,antibiotic resistance,and bla-TEM gene of Glaesserella parasuis in large-scale pig farms in 5 southern provinces of China during 2017–2019

【目的】旨在分析当前规模化养殖场副猪格拉菌(Glaesserellaparasuis)优势血清型、耐药特性、耐药基因与分子特征。【方法】对源自规模化养猪场21株副猪格拉菌临床分离株,采用PCR鉴定血清型;利用K-B纸片扩散法鉴定其对25种抗生素的耐药表型;采用PCR检测bla-、bla-和bla-等7种耐药基因,并采用Chi-square test和Fisher exact test分析耐药表型和耐药基因型的相关性;耐药基因目的条带测序,并应用CLC Sequence Viewer软件分析β-内酰胺类耐药基因(bla-)编码蛋白氨基酸关键位点差异与耐药性的关系。【结果】21株副猪格拉菌临床分离株的优势血清型为4和12型;对β-内酰胺类药物苯唑西林的耐药性较强,耐药菌占比达61.9%(13/21);多重耐药菌株占比高达90.5%(19/21);β-内酰胺类耐药基因bla-携带率较高(52.4%,11/21),且bla-与β-内酰胺类药物青霉素G、苯唑西林和头孢拉定的耐药性显著相关,部分bla-编码氨基酸存在可能与副猪格拉菌耐药能力有关的差异位点。【结论】本研究表明,规模化养猪场的副猪格拉菌多重耐药情况仍很严重,并明确了被调查区域β-内酰胺类药物耐药率高的主要原因是携带耐药基因bla-,为加强对规模化养猪场副猪格拉菌耐药性监测提供理论依据。

[Objective]To analyze the serotypes,antibiotic resistance,and resistance genes of 21 isolates of Glaesserella parasuis in large-scale pig farms.[Methods]Serotypes were identified by PCR and the resistance to 25 antibiotics with the K-B disk diffusion method.Seven resistance genes,including bla-,bla-,and bla-,were detected by PCR.Then chi-square test and Fisher’s exact test were used to analyze the association between resistance phenotypes and genotypes.Theβ-lactam resistance gene bla-was sequenced and the sequences of amino acids encoded by bla-from different isolates were compared.Finally,the relationship between the differential sites of the amino acids and antibiotic resistance was analyzed by CLC Sequence Viewer.[Results]The isolates were dominated by serotypes 4 and 12.They showed high rate(61.9%,13/21)of resistance toβ-lactam antibiotic oxacillin and 90.5%(19/21)of the isolates demonstrated multi-antibiotic resistance.bla-was identified in 52.4%(11/21)of the isolates,which was significantly correlated with the resistance phenotype of penicillin G,oxacillin,and cefradine.There were mutations of the amino acids encoded by bla-among isolates,which may be related to the antibiotic resistance of the isolates.[Conclusion]Multi-antibiotic resistance of G.parasuis isolates is prevalent in large-scale pig farms,and the high rate ofβ-lactam resistance is attributed to the inborn resistance gene bla-.The conclusion lays a theoretical basis for surveillance of antibiotic resistance of G.parasuis in large-scale pig farms.