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[从临床样本中分离的棒状杆菌属细菌生物膜形成、群体感应抑制活性及耐药性研究]

[Investigation of Biofilm Formation, Anti-Quorum Sensing Activity and Antimicrobial Resistance in Corynebacterium Species Isolated from Clinical Samples].

作者信息

Keskin Banu Hümeyra, Şahin İdris, Kahraman Gözde, Duran Pelin Kamuran, Dülger Görkem, Durmuş Mehmet Akif, Ceylan Ayşe Nur, Çalışkan Emel, Öksüz Şükrü

机构信息

Zonguldak Gynecology and Obstetrics Hospital, Medical Microbiology Laboratory, Zonguldak, Türkiye.

Düzce University Faculty of Medicine, Department of Medical Microbiology, Düzce, Türkiye.

出版信息

Mikrobiyol Bul. 2024 Jul;58(3):239-258. doi: 10.5578/mb.20249704.

Abstract

An increasing number of different clinical infections caused by Corynebacteria have been reported in the last decade. The aim of this study was to evaluate the antibiotic resistance rates, biofilm formation capacities and to investigate the ''anti-quorum-sensing (anti-QS)'' activities of corynebacteria, which were divided into three groups according to the type of growth in culture (pure, with another pathogenic bacterium and polymicrobial growth). In total 240 Corynebacterium spp. isolates from different clinical specimens sent to the medical microbiology laboratories of Düzce University Faculty of Medicine Hospital and Başakşehir Çam and Sakura City Hospital between June 2021 and June 2022 were classified into three groups: pure, isolated with another pathogen and polymicrobial, according to their growth patterns in culture. Bacteria were identified by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) Biotyper (Bruker, Germany) at an external centre. Antibiotic susceptibilities were determined by disc diffusion method and for vancomycin broth microdilution method was used. Results were interpreted according to EUCAST recommendations. The biofilm-forming properties of the isolates were determined quantitatively. Bioactive components of 17 isolates with strong biofilm formation were extracted and anti-QS activity was determined by agar diffusion method using Chromobacterium violaceum ATCC 12472 strain and then violacein pigment production was measured quantitatively. Of the 240 Corynebacterium spp. isolates, 138 (58%) were pure, 52 (22%) were isolated with another pathogen and 50 (20%) were part of a polymicrobial infection. Of the isolates, 140 were identified as C.striatum, 34 as C.amycolatum and 24 as Corynebacterium afermentans. When the antibiotic resistance rates of the Corynebacterium isolates were analysed according to the groups, the resistance rates to rifampicin and tetracycline antibiotics were found to be statistically significantly lower in the polymicrobial group than in the other groups. The resistance rates to penicillin, clindamycin, ciprofloxacin, moxifloxacin, rifampicin, tetracycline and linezolid were 96.7%, 88.3%, 86.3%, 73.8%, 62.5%, 59.2% and 0.8%, respectively. While all isolates were susceptible to vancomycin, linezolid resistance was detected in two C.afermentans isolates. When the biofilm formation ability was analysed, it was observed that 87 (36.3%) isolates formed biofilm. The biofilm formation rate of the isolates in the polymicrobial growth group was lower than the other two groups. The anti-QS activity of 17 isolates with strong biofilm formation was investigated and none of the Corynebacterium extracts tested were found to have anti-QS activity (inhibition of violacein pigment production without inhibiting bacterial growth) in the QS study with C.violaceum, whereas five isolate extracts had antibacterial activity (inhibition of bacterial growth). Four of the bacterial extracts with antimicrobial activity belonged to C.amycolatum and one to C.afermentans. In conclusion, when both antibiotic resistance rates and biofilm formation rates were analysed, the corynebacteria growing in culture with another pathogen showed similar characteristics to the corynebacteria growing as a pure culture. Therefore, it was thought that corynebacteria growing with another pathogen should not be ignored. In addition, the antimicrobial effects of some corynebacterial extracts suggested that more QS studies should be carried out with microbiota bacteria.

摘要

在过去十年中,由棒状杆菌引起的不同临床感染报告数量不断增加。本研究旨在评估棒状杆菌的抗生素耐药率、生物膜形成能力,并研究其“群体感应抑制(抗QS)”活性。根据培养中的生长类型(纯培养、与另一种病原菌混合培养和多菌混合生长),棒状杆菌被分为三组。2021年6月至2022年6月期间,从杜兹大学医学院医院和巴沙克谢希尔·卡姆和樱花市医院医学微生物实验室送检的不同临床标本中分离出的240株棒状杆菌属菌株,根据其在培养中的生长模式分为三组:纯培养、与另一种病原体一起分离和多菌混合。细菌由外部中心的基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)Biotyper(德国布鲁克公司)鉴定。采用纸片扩散法测定抗生素敏感性,万古霉素采用肉汤微量稀释法测定。结果根据欧盟CAST推荐标准进行解释。对分离株的生物膜形成特性进行定量测定。提取17株具有强生物膜形成能力的分离株的生物活性成分,采用紫色杆菌ATCC 12472菌株通过琼脂扩散法测定抗QS活性,然后定量测定紫色菌素色素产量。在240株棒状杆菌属分离株中,138株(58%)为纯培养,52株(22%)与另一种病原体一起分离,50株(20%)是多菌混合感染的一部分。在分离株中,140株被鉴定为纹带棒状杆菌,34株为无枝菌酸棒状杆菌,24株为发酵棒状杆菌。当根据组分析棒状杆菌分离株的抗生素耐药率时,发现多菌混合组对利福平和四环素类抗生素的耐药率在统计学上显著低于其他组。对青霉素、克林霉素、环丙沙星、莫西沙星、利福平、四环素和利奈唑胺的耐药率分别为96.7%、88.3%、86.3%、73.8%、62.5%、59.2%和0.8%。虽然所有分离株对万古霉素敏感,但在两株发酵棒状杆菌分离株中检测到利奈唑胺耐药。当分析生物膜形成能力时,观察到87株(36.3%)分离株形成生物膜。多菌混合生长组分离株的生物膜形成率低于其他两组。研究了17株具有强生物膜形成能力的分离株的抗QS活性,在与紫色杆菌的QS研究中,未发现所测试的棒状杆菌提取物具有抗QS活性(抑制紫色菌素色素产生而不抑制细菌生长),而五种分离株提取物具有抗菌活性(抑制细菌生长)。四种具有抗菌活性的细菌提取物属于无枝菌酸棒状杆菌,一种属于发酵棒状杆菌。总之,当分析抗生素耐药率和生物膜形成率时,与另一种病原体一起在培养中生长的棒状杆菌表现出与纯培养生长的棒状杆菌相似的特征。因此,认为与另一种病原体一起生长的棒状杆菌不应被忽视。此外,一些棒状杆菌提取物的抗菌作用表明,应使用微生物群细菌进行更多的QS研究。

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