Sattar Fazal, Hu Xiao, Saxena Anugrah, Mou Kathy, Shen Huigang, Ali Hazrat, Ghauri Muhammad Afzal, Sarwar Yasra, Ali Aamir, Li Ganwu
National Institute for Biotechnology and Genetic Engineering College, Pakistan Institute of Engineering and Applied Sciences (NIBGE-C, PIEAS), Faisalabad 38000, Punjab, Pakistan.
Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA 50011, USA.
Antibiotics (Basel). 2024 Oct 4;13(10):937. doi: 10.3390/antibiotics13100937.
Wastewater is a major source of Antibiotic-Resistant Bacteria (ARB) and a hotspot for the exchange of Antibiotic-Resistant Genes (ARGs). The occurrence of Carbapenem-Resistant Bacteria (CRB) in wastewater samples is a major public health concern. This study aimed to analyze Antibiotic resistance in bacteria from wastewater sources in Pakistan. We analyzed 32 bacterial isolates, including 18 , 4 , and 10 other bacterial isolates using phenotypic antibiotic susceptibility assay and whole-genome sequencing. This study identified the ARGs, plasmid replicons, and integron genes cassettes in the sequenced isolates. One representative isolate was further sequenced using Illumina and Oxford nanopore sequencing technologies. Our findings revealed high resistance to clinically important antibiotics: 91% of isolates were resistant to cefotaxime, 75% to ciprofloxacin, and 62.5% to imipenem, while 31% showed non-susceptibility to gentamicin. All isolates were resistant to cephalosporins, with 72% also resistant to carbapenems. Sequence analysis showed a diverse resistome, including carbapenamases (blaNDM-5, blaOXA-181), ESBLs (blaCTX-M-15, blaTEM), and AmpC-type β-lactamases (blaCMY). Key point mutations noticed in the isolates were pmrB_Y358N (colistin) and ftsI_N337NYRIN, ftsI_I336IKYRI (carbapenem). The isolates had 11 different STs, with ST410 predominating (28%). Notably, the phylogroup A isolate 45EC1, (ST10886) is reported for the first time from wastewater, carrying blaNDM-5, blaCMY-16, and pmrB_Y358N with class 1 integron gene cassette of 12-2- on a plasmid-borne contig. Other carbapenamase, blaNDM-1 and blaOXA-72, were detected in 22EB1 and 51AC1, respectively. The integrons with the gene cassettes encoding antibiotic resistance, and the transport and bacterial mobilization protein, were identified in the sequenced isolates. Ten plasmid replicons were identified, with IncFIB prevalent in 53% of isolates. Combined Illumina and Oxford nanopore sequencing revealed blaNDM-5 on an IncFIA/IncFIC plasmid and is identical to those reported in the USA, Myanmar, and Tanzania. These findings highlight the environmental prevalence of high-risk and WHO-priority pathogens with clinically important ARGs, underscoring the need for a One Health approach to mitigate ARB isolates.
废水是抗生素耐药菌（ARB）的主要来源，也是抗生素耐药基因（ARG）交换的热点。废水样本中碳青霉烯耐药菌（CRB）的出现是一个重大的公共卫生问题。本研究旨在分析巴基斯坦废水源细菌中的抗生素耐药性。我们使用表型抗生素敏感性试验和全基因组测序分析了32株细菌分离株，包括18株、4株和10株其他细菌分离株。本研究确定了测序分离株中的ARG、质粒复制子和整合子基因盒。使用Illumina和牛津纳米孔测序技术对一株代表性分离株进行了进一步测序。我们的研究结果显示，这些分离株对临床上重要的抗生素具有高度耐药性：91%的分离株对头孢噻肟耐药，75%对环丙沙星耐药，62.5%对亚胺培南耐药，而31%对庆大霉素不敏感。所有分离株均对头孢菌素耐药，72%也对碳青霉烯类耐药。序列分析显示了一个多样的耐药组，包括碳青霉烯酶（blaNDM-5、blaOXA-181）、超广谱β-内酰胺酶（blaCTX-M-15、blaTEM）和AmpC型β-内酰胺酶（blaCMY）。在分离株中发现的关键突变是pmrB_Y358N（黏菌素）和ftsI_N337NYRIN、ftsI_I336IKYRI（碳青霉烯类）。这些分离株有11种不同的序列类型，其中ST410占主导（28%）。值得注意的是，系统发育A群分离株45EC1（ST10886）首次从废水中报道，在一个质粒携带的重叠群上携带blaNDM-5、blaCMY-16和pmrB_Y358N以及12-2-的1类整合子基因盒。其他碳青霉烯酶blaNDM-1和blaOXA-72分别在22EB1和51AC1中检测到。在测序分离株中鉴定出了带有编码抗生素耐药性、转运和细菌移动蛋白的基因盒的整合子。鉴定出10种质粒复制子，其中IncFIB在53%的分离株中普遍存在。Illumina和牛津纳米孔测序相结合显示，在一个IncFIA/IncFIC质粒上存在blaNDM-5，与美国、缅甸和坦桑尼亚报道的相同。这些发现突出了携带临床上重要ARG的高风险和世界卫生组织重点病原体在环境中的流行情况，强调了采取一体化健康方法来减轻ARB分离株的必要性。
