Leicester School Allied Health Sciences, Faculty of Health & Life Sciences, De Montfort University, Leicester LE1 9BH, UK.
Int J Environ Res Public Health. 2022 Apr 14;19(8):4788. doi: 10.3390/ijerph19084788.
In light of the limited therapeutic options with Carbapenem-Resistant Enterobacterales (CRE) infections, understanding the bacterial risk factors, such as biofilm formation and related gene expression of CRE, is vital. This study investigates the biofilm formation and biofilm-related gene expression of two enteric Enterobacterales with major CR determinants IMP and NDM-1, which were seen in high prevalence in most common bacterial infections over the past few years. To our knowledge, this is the first study that demonstrated the relationship between biofilm formation and the related gene expression, to understand the potential molecular mechanisms during the biofilm formation in CRE. Biofilms were quantified by tissue culture plate assay at the stages of the biofilm development: initial attachment (6 h), microcolony formation (12 h), maturation (24 h), and dispersion (48 h). In a dispersion, event bacteria detach without any mechanical means and colonise another area. To investigate the influence of different growth conditions on biofilm formation, biofilms were quantified under different growth conditions. In parallel, quantitative real-time PCR (qPCR) assessed the biofilm-related gene expression of a cluster of genes, including biofilm maturation, quorum sensing, stress survival, and antibiotic resistance. Structural changes during biofilm development were assessed via confocal laser scanning microscopy (CLSM). We observed that the biofilm formation of CRE is correlated with the biofilm development stages, with maximum biofilm observed at 24 h at the maturation stage. Our data also showed that biofilm growth, under the condition tested, is the major factor influencing the variability of biofilm gene expression quantification assays. qPCR analyses have demonstrated that the expression of biofilm-related genes is highly correlated with phenotypic biofilm development, and these findings can be further expanded to understand the variation in regulation of such genes in these significant CRE pathogens. Our study demonstrated that both CRE strains, IMP and NDM-1, are high biofilm formers, and genes involved in biofilm development are upregulated during biofilm growth. The characteristic of the increased biofilm formation with the upregulation of antibiotic-resistant and biofilm-related genes indicates the successful pathogenic role of biofilms of these selected CRE and is attributed to their multi-drug resistance ability and successful dissemination of CRE in common bacterial infections.
鉴于耐碳青霉烯肠杆菌科（CRE）感染的治疗选择有限，了解细菌的风险因素，如 CRE 的生物膜形成和相关基因表达，至关重要。本研究调查了两种肠杆菌属 Enterobacterales 的生物膜形成和生物膜相关基因表达，这两种 Enterobacterales 具有主要的 CR 决定因素 IMP 和 NDM-1，它们在过去几年中最常见的细菌感染中普遍存在。据我们所知，这是第一项研究表明生物膜形成与相关基因表达之间的关系，以了解 CRE 中生物膜形成过程中的潜在分子机制。生物膜的形成通过组织培养板测定在生物膜发展的各个阶段进行量化：初始附着（6 小时）、微菌落形成（12 小时）、成熟（24 小时）和分散（48 小时）。在分散中，细菌无需任何机械手段即可脱落并定植到另一个区域。为了研究不同生长条件对生物膜形成的影响，在不同的生长条件下对生物膜进行了量化。同时，通过定量实时 PCR（qPCR）评估了包括生物膜成熟、群体感应、应激存活和抗生素耐药性在内的基因簇的生物膜相关基因表达。通过共聚焦激光扫描显微镜（CLSM）评估生物膜形成过程中的结构变化。我们观察到，CRE 的生物膜形成与生物膜发展阶段相关，在成熟阶段 24 小时达到最大生物膜。我们的数据还表明，在测试条件下，生物膜生长是影响生物膜基因表达定量测定变异性的主要因素。qPCR 分析表明，生物膜相关基因的表达与表型生物膜发育高度相关，这些发现可以进一步扩展，以了解这些重要的 CRE 病原体中这些基因调节的变化。我们的研究表明，两种 CRE 菌株 IMP 和 NDM-1 都是高生物膜形成者，并且在生物膜生长过程中，参与生物膜发育的基因上调。生物膜形成增加与抗生素耐药性和生物膜相关基因上调的特征表明，这些选定的 CRE 生物膜的成功致病作用归因于它们的多药耐药能力和 CRE 在常见细菌感染中的成功传播。
