伊朗肉类及肉制品中放线菌种的筛选、分子鉴定、种群多样性及抗生素敏感性模式

Screening, molecular identification, population diversity, and antibiotic susceptibility pattern of Actinomycetes species isolated from meat and meat products of slaughterhouses, restaurants, and meat stores of a developing country, Iran

作者信息Tahereh Motallebirad, Omid Mardanshah, Mehdi Safarabadi, Kazem Ghaffari, Mohammad Ali Orouji, Behnam Abedi, Davood Azadi
PMID37520382
发布时间2023-07-13
DOI10.3389/fmicb.2023.1134368

实验完整度

研究包含样本采集、形态生化鉴定、分子鉴定和药敏试验,但缺乏体内或体外功能验证,实验层级单一。

主要模型

60份肉类及肉制品样本 21株放线菌分离株

重点核对

样本类型及数量:包括碎肉、羊肉、牛肉、鸡肉、汉堡、香肠等共60份 采样地点:Markazi省屠宰场、肉店和餐馆 分离培养条件:样本经3%十二烷基硫酸钠和1% NaOH去污染处理,接种于血琼脂、Sauton培养基、LJ培养基,在不同温度和CO2条件下培养 鉴定方法:常规生化试验结合hsp65和16S rRNA基因PCR扩增及序列分析 药敏试验:采用肉汤微量稀释法,测定10种抗生素的MIC,并参照CLSI 2021标准判读

摘要

Introduction: Actinomycetes can colonize surfaces of tools and equipment and can be transferred to meat and meat products during manufacture, processing, handling, and storage. Moreover, washing the meat does not eliminate the microorganisms; it only spreads them. As a result, these opportunistic pathogens can enter the human body and cause various infections. Therefore, the aim of the current study was to screen, identify, and determine the antibiotic susceptibility of Actinomycetes species from meat and meat products in the Markazi province of Iran. Methods: A total of 60 meat and meat product samples, including minced meat, mutton, beef, chicken, hamburgers, and sausages, were collected from slaughterhouses, butchers, and restaurants in the Markazi province of Iran. The samples were analyzed using standard microbiological protocols for the isolation and characterization of Actinomycetes. PCR amplification of hsp65 and 16SrRNA genes and sequence analysis of 16SrRNA were used for genus and species identification. The minimum inhibitory concentrations (MICs) of antimicrobial agents were determined by the broth microdilution method and interpreted according to the CLSI guidelines. Results: A total of 21 (35%) Actinomycetes isolates from 5 genera and 12 species were isolated from 60 samples. The most prevalent Actinomycetes were from the genus Mycobacterium, with six (28.6%) isolates (M. avium complex, M. terrae, M. smegmatis, and M. novocastrense), followed by the genus Rhodococcus with five (23.8%) isolates (R. equi and R. erythropolis), the genus Actinomyces with four (19.1%) isolates (A. ruminicola and A. viscosus), the genus Nocardia with four (19.1%) isolates (N. asiatica, N. seriolae, and N. niigatensis), and the genus Streptomyces with two (9.5%) isolates (S. albus). Chicken and sausage samples had the highest and lowest levels of contamination, with six and one isolates. Respectively, the results of drug susceptibility testing (DST) showed that all isolates were susceptible to Ofloxacin, Amikacin, Ciprofloxacin, and Levofloxacin, whereas all of them were resistant to Doxycycline and Rifampicin. Discussion: The findings suggest that meat and meat products play an important role as a reservoir for the transmission of Actinomycetes to humans, thus causing life-threatening foodborne diseases such as gastrointestinal and cutaneous disorders. Therefore, it is essential to incorporate basic hygiene measures into the cycle of meat production to ensure food safety.

实验结论

提炼研究问题、关键发现与证据,快速把握文章的核心贡献。

研究问题
肉类及肉制品中放线菌的污染情况、种群多样性及其抗生素敏感性如何?
核心机制
放线菌可通过肉类及肉制品作为载体传播给人类,引起胃肠道和皮肤等食源性疾病。
主要证据
从60份样本中分离出21株放线菌,经16S rRNA基因序列分析鉴定为5属12种,包括分枝杆菌属、红球菌属、放线菌属、诺卡菌属和链霉菌属。药敏试验显示所有分离株对氧氟沙星、阿米卡星、环丙沙星和左氧氟沙星敏感,对多西环素和利福平耐药。
研究意义
研究表明肉类及肉制品可能作为放线菌传播给人的重要储库,强调在肉类生产循环中纳入基本卫生措施以确保食品安全。

研究路径

按研究推进顺序梳理实验设计、验证步骤与关键观察。

1

样本采集与处理

获取肉类及肉制品样本以检测放线菌污染情况

从伊朗Markazi省的屠宰场、肉店和餐馆共采集60份样本,包括羊肉、牛肉、鸡肉、碎肉、香肠、热狗和汉堡。样本在24小时内送至实验室,并测量温度pH。

2

去污染与培养

减少杂菌并促进放线菌的生长以进行后续分离

取2-4克样本在均质袋中均质,用3%十二烷基硫酸钠和1% NaOH去污处理10分钟,离心后取上清和沉淀接种于血琼脂、Sauton培养基和LJ培养基,在20°C、30°C、37°C、10% CO2环境下培养2个月。

3

表型鉴定

初步鉴定放线菌属并区分不同种

使用常规微生物学方法包括抗酸染色、革兰染色等,并进行了CAMP试验、吡嗪酰胺酶、脲酶等生化试验。

4

分子鉴定

精确鉴定到属和种水平

提取DNA,通过PCR扩增16S rRNA和hsp65基因,并进行序列分析。利用属特异性PCR鉴定属,测序确定种。

5

药敏试验

测定分离株对各类抗生素的敏感性

使用肉汤微量稀释法,根据CLSI 2021指南进行。测试了10种抗生素,记录MIC值。

研究方法

按研究目的归类文中使用的方法,便于定位所需技术。

产品清单

实验环节名称品牌货号
十二烷基硫酸钠--151-21-3
血琼脂----
Sauton培养基----
罗氏培养基----
头孢西丁--35607-66-0
阿米卡星--37517-28-5
多西环素--564-25-0
环丙沙星--85721-33-1
利福平--13292-46-1
卡那霉素--59-01-8
异烟肼--54-85-3
链霉素--57-92-1
左氧氟沙星--100986-85-4
亚胺培南--64221-86-9

关键环节

汇总复现实验时建议重点确认的条件及原文阅读提示。

环节核对要点
样本采集与处理
样本类型、数量、采样地点及运输条件(24小时内)
阅读提示:Methods: 研究设计、采样和去污染部分
去污染与培养
去污染试剂浓度(3% SDS, 1% NaOH)及处理时间(10分钟),离心条件,培养基种类(血琼脂、Sauton、LJ),培养温度(20、30、37°C)和CO2浓度(10%),培养时间(2个月)
阅读提示:Methods: 研究设计、采样和去污染部分
表型鉴定
使用的生化试验项目(CAMP、脲酶、过氧化氢酶等)及其结果判读标准
阅读提示:Methods: 微生物学鉴定部分及表1
分子鉴定
PCR引物、扩增条件(596 bp、829 bp、620 bp片段),测序方法及数据分析使用的软件(jPhydit)
阅读提示:Methods: 分子鉴定部分及表1
药敏试验
抗生素种类、浓度范围(0.06-512 mg/L),微量肉汤稀释法具体步骤,培养时间(4天),接种菌量(0.5麦氏浊度),判读标准(CLSI 2021)
阅读提示:Methods: 药敏试验部分及表2