Suchanta Nutchaba, Ullah Naeem, Santanirand Pitak, Am-In Nutthee, Chaichanawongsaroj Nuntaree
Center of Excellence for Innovative Diagnosis of Antimicrobial Resistance, Department of Transfusion Medicine and Clinical Microbiology, Faculty of Allied Health Sciences, Chulalongkorn University, Pathumwan, Bangkok 10330, Thailand.
Program of Molecular Sciences in Medical Microbiology and Immunology, Department of Transfusion Medicine and Clinical Microbiology, Faculty of Allied Health Sciences, Chulalongkorn University, Pathumwan, Bangkok 10330, Thailand.
Animals (Basel). 2024 Sep 10;14(18):2630. doi: 10.3390/ani14182630.
The commensal in the gut of pigs is a major reservoir of antimicrobial resistance and can result in possible transmission to humans through the food chain. Direct detection of from fecal samples is challenging and can be used as a bioindicator of antimicrobial resistance. This study aimed to compare the antimicrobial susceptibility profiles in commensal from antibiotic- and nonantibiotic-using pig farms and developed the direct detection of ESBL genes in pig fecal samples using nested PCR (nPCR) and multiplex PCR (mPCR) techniques. All direct genotypic results were validated with the results of PCR sequencing of isolated colonies. The ESBL-producing were found in 98.6% (145 isolates) and 96.6% (144 isolates) of antibiotic-using and nonantibiotic-using farms, respectively, predominantly CTX-M-55. The nPCR decreased the limit of detection (LOD) from sPCR about 100 times, and the lower LODs of 10, 10, and 1 CFU/mL were reached after incubating samples in an enrichment medium for 2, 4, and 8 h, respectively. The mPCR, sPCR, and nPCR techniques showed sensitivities of 30.15%, 69.85%, and 91.91%, respectively, compared to PCR sequencing. The stability and recycling of ESBL genes were independent of antibiotic usage in commensal originating in pig farms.
猪肠道中的共生菌是抗生素耐药性的主要储存库，可能通过食物链传播给人类。直接从粪便样本中检测共生菌具有挑战性，可将其用作抗生素耐药性的生物指标。本研究旨在比较来自使用抗生素和未使用抗生素猪场的共生菌的药敏谱，并开发利用巢式PCR（nPCR）和多重PCR（mPCR）技术直接检测猪粪便样本中ESBL基因的方法。所有直接基因型结果均通过分离菌落的PCR测序结果进行验证。分别在98.6%（145株分离菌）和96.6%（144株分离菌）的使用抗生素和未使用抗生素的猪场中发现了产ESBL的共生菌，主要为CTX-M-55。nPCR将检测限（LOD）比单重PCR降低了约100倍，分别在富集培养基中孵育样本2、4和8小时后，达到了10、10和1 CFU/mL的较低检测限。与PCR测序相比，mPCR、单重PCR和nPCR技术的灵敏度分别为30.15%、69.85%和91.91%。ESBL基因的稳定性和循环与源自猪场的共生菌中的抗生素使用无关。
