Owaid Hekmat A, Al-Ouqaili Mushtak T S
Department of Biology, College of Science, University of Anbar, Ramadi, Iraq.
Department of Microbiology, College of Medicine, University of Anbar, Anbar Governorate, Ramadi, Iraq.
Heliyon. 2025 Jan 6;11(1):e41670. doi: 10.1016/j.heliyon.2025.e41670. eCollection 2025 Jan 15.
The presence of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system in the superbug presents a unique opportunity to precisely target and edit bacterial genomes to modify their drug resistance. The objective was to detect the prevalence of CRISPR in extensively and pan-drug-resistant and to determine the utility of whole-genome sequencing (WGS) for the analysis of the entire genome for such strains. The antimicrobial susceptibilities of one hundred isolates were assessed using the antibiotic susceptibility test (AST) card of the VITEK system. The presence of the CRISPR/Cas system was determined via specific primers using conventional polymerase chain reaction (PCR). Further, WGS was conducted using a DNA nanoball sequencing platform via BGI-Tech for the isolates of interest. Out of 54 resistant isolates 33 (33.0 %) were metallo-β-lactamase producers. Cas1, Cas3, CRISPR1, and CRISPR2 were positive in 6.0 % of isolates, while incomplete CRISPR1-Cas systems alone were found in 15.0 %. Also, CRISPR2-type was found intact in 26 % of isolates. The prevalence of resistance to antimicrobials in isolates was significantly greater in the CRISPR/Cas-negative group compared to the CRISPR/Cas-positive. Significant relationships for variables were examined using Fisher's exact tests using Chi-squared and a P-value of <0.05 as a statistical threshold. Further, on examination of CRs as a collective entity, encompassing both extensive drug resistance (XDR) and pan-drug resistance (PDR), it becomes evident that the vast majority of these strains (n = 29; 87.8 %) lacked CRISPR/Cas systems. In phylogenic analysis, PDR- revealed a very close evolutionary relationship with those originating from Kazakhstan, while XDR was globally unique. Further, the entire genome showed the presence of unique virulence and resistant pseudomonal genes. The CRISPR/Cas system and drug resistance are antagonistic to one another. XDR and PDR represent a potential threat to public health and contribute to the seriousness of associated illnesses by leading to resistant infections. Further, WGS for the two strains revealed resistance to multiple antibiotics. It is important to examine specific antimicrobial resistance (AMR) pathways, which suggests that a significant number of resistant genes in these isolates indicate a loss of CRISPR genes in the two strains. Furthermore, the WGS approach can lead to a better understanding of the genomic mechanism of pseudomonal resistance to antibiotics.
超级细菌中存在的成簇规律间隔短回文重复序列（CRISPR）/Cas系统为精确靶向和编辑细菌基因组以改变其耐药性提供了独特的机会。目的是检测广泛耐药和泛耐药细菌中CRISPR的流行情况，并确定全基因组测序（WGS）在分析此类菌株全基因组方面的效用。使用VITEK系统的抗生素敏感性测试（AST）卡评估了100株分离菌的抗菌药敏性。通过常规聚合酶链反应（PCR）使用特异性引物确定CRISPR/Cas系统的存在。此外，通过BGI-Tech的DNA纳米球测序平台对感兴趣的分离菌进行WGS。在54株耐药分离菌中，33株（33.0%）是金属β-内酰胺酶产生菌。Cas1、Cas3、CRISPR1和CRISPR2在6.0%的分离菌中呈阳性，而仅不完全的CRISPR1-Cas系统在15.0%的分离菌中被发现。此外，CRISPR2型在26%的分离菌中完整存在。与CRISPR/Cas阳性组相比，CRISPR/Cas阴性组分离菌中对抗菌药物的耐药率显著更高。使用卡方检验的Fisher精确检验和P值<0.05作为统计阈值来检验变量之间的显著关系。此外，在将广泛耐药（XDR）和泛耐药（PDR）的CR视为一个整体实体进行检查时，很明显这些菌株中的绝大多数（n = 29；87.8%）缺乏CRISPR/Cas系统。在系统发育分析中，PDR-显示出与源自哈萨克斯坦的菌株有非常密切的进化关系，而XDR在全球范围内是独特的。此外，全基因组显示存在独特的毒力和耐药假单胞菌基因。CRISPR/Cas系统和耐药性彼此拮抗。XDR和PDR对公共卫生构成潜在威胁，并通过导致耐药感染增加相关疾病的严重性。此外，对这两种菌株的WGS显示它们对多种抗生素耐药。研究特定的抗菌耐药（AMR）途径很重要，这表明这些分离菌中的大量耐药基因表明这两种菌株中CRISPR基因的缺失。此外，WGS方法可以更好地理解假单胞菌对抗生素耐药的基因组机制。
