2021—2024年安徽省马鞍山市市售肉与肉制品中产气荚膜梭菌污染特征及病原学特征分析

Contamination characteristics and pathogenic characteristics of Clostridium perfringens in commercial meat and meat products in Ma’anshan City, Anhui Province from 2021 to 2024

中文关键词:  产气荚膜梭菌  肉与肉制品  污染特征  毒力基因  耐药基因组学  多位点序列分型  核心基因组系统发育

英文关键词:Clostridium perfringens  meat and meat products  contamination characteristics  toxin genes  drug resistance genomics  multilocus sequence typing  core genome phylogeny

中文摘要:

      目的 了解马鞍山市市售肉与肉制品中产气荚膜梭菌的污染现状、毒力基因携带模式、耐药表型特征及基因组进化规律。方法 2021—2024年, 在马鞍山市3区3县及网购渠道采集300份肉与肉制品, 通过分离培养和聚合酶链式反应(polymerase chain reaction, PCR)技术鉴定产气荚膜梭菌; 采用多重荧光PCR检测7种毒力基因, 琼脂稀释法测定12种抗生素的药敏谱, 全基因组测序(whole-genome sequencing, WGS)结合生物信息学分析进行多位点序列分型(multilocus sequence typing, MLST)、耐药基因预测及核心基因组系统发育树构建。结果 300份样品中产气荚膜梭菌总阳性检出率为18.67% (56/300), 不同年份、样品类别、采样区域及场所的检出率差异具有统计学意义(P<0.05), 其中生禽肉(45.16%)和农贸市场样品(30.82%)污染最为严重。7种毒力基因(cpa、cpb、iap、etx、cpe、netB、cbp2)检测显示, cpa基因阳性率100.00%, cpe和cbp2阳性率分别为7.14%和48.21%, 菌株以A型为主(92.86%), 同时检出E型(1.79%)和F型(5.36%)。药敏试验表明, 菌株对四环素(64.29%)和克林霉素(32.14%)耐药率较高, 26.79%的菌株存在多重耐药现象; 头孢哌酮、美罗培南和氯霉素敏感性达100.00%。全基因组分析显示, 56株菌包含50种序列型别, 其中26种为新发现型别; 共鉴定出9种耐药基因, mprF、tetA(P)和tetB(P)携带率居前3位(85.71%、73.21%、69.64%); 核心基因组系统发育树揭示不同年份、区域的菌株存在同源性, 提示跨区域传播风险。结论 马鞍山市市售肉与肉制品产气荚膜梭菌污染较严重, 毒力基因携带率高, 多重耐药与遗传多样性突出, 存在跨区域传播与食物中毒风险, 需加强生禽肉与农贸市场环节监管。

英文摘要:

      Objective To investigate the contamination status, toxin genes carriage patterns, antimicrobial resistance phenotypes and genomic evolution patterns of Clostridium perfringens in commercial meat and meat products in Ma’anshan City. Methods A total of 300 meat and meat product samples were collected from 3 districts, 3 counties and online shopping channels in Ma’anshan City from 2021 to 2024. Clostridium perfringens was identified by isolation culture and polymerase chain reaction (PCR). The 7 kinds of toxin genes were detected by multiplex fluorescence PCR. The antimicrobial susceptibility profiles of 12 kinds of antibiotics were determined by agar dilution method. Whole-genome sequencing (WGS) combined with bioinformatics analysis was used to perform multilocus sequence typing (MLST), antimicrobial resistance gene prediction and core-genome phylogenetic tree construction. Results The total positive detection rate of Clostridium perfringens in 300 samples was 18.67% (56/300). There were statistically significant differences in the detection rates among different years, sample types, sampling regions and sites (all P<0.05), among which raw poultry meat (45.16%) and farmers’ market samples (30.82%) were the most heavily contaminated. The detection of 7 kinds of toxin genes (cpa, cpb, iap, etx, cpe, netB, cbp2) showed that the positive rate of cpa gene was 100.00%, and the positive rates of cpe and cbp2 were 7.14% and 48.21%, respectively. The strains were dominated by type A (92.86%), and type E (1.79%) and type F (5.36%) were also detected. Antimicrobial susceptibility testing showed that the strains had high resistance rates to tetracycline (64.29%) and clindamycin (32.14%) and 26.79% of the strains exhibited multidrug resistance; the susceptible rates to cefoperazone, meropenem and chloramphenicol were 100.00%. Whole-genome analysis revealed that the 56 strains contained 50 kinds of MLST, including 26 kinds of newly discovered types. A total of 9 kinds of antimicrobial resistance genes were identified and the carriage rates of mprF, tetA(P), and tetB(P) ranked the top 3 (85.71%, 73.21% and 69.64%, respectively). The core-genome phylogenetic tree indicated genetic homology among strains from different years and regions, suggesting a risk of cross-regional transmission. Conclusion The contamination of Clostridium perfringens in commercial meat and meat products in Ma’anshan City is relatively severe, with high toxin gene carriage rates, prominent multidrug resistance and genetic diversity. There are risks of cross-regional transmission and food poisoning, so it is necessary to strengthen the supervision of raw poultry meat and farmers’ market links.