High-Throughput Detection of Bacterial Community and Its Drug-Resistance Profiling From Local Reclaimed Wastewater Plants

Abstract

Treated wastewater from reclaimed facilities (WWTP) has become a reusable source for a variety of applications, such as agricultural irrigation. However, it is also a potential reservoir of clinically-relevant multidrug resistant (MDR) pathogens, including ESKAPE (Enterococcus faecium and Streptococcus surrogates, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species along with the emerging nosocomial Escherichia strains). This study was performed to decipher the bacterial community structure through Illumina high throughput 16S rRNA gene sequencing, and to determine the resistance profile using the Sensititre antimicrobial susceptibility test (AST) conforming to clinical lab standards (NCCLS). Out of 1747 bacterial strains detected from wastewater influent and effluent, Pseudomonas was the most predominant genus related to ESKAPE in influent, with sequence reads corresponding to 21.356%, followed by Streptococcus (6.445%), Acinetobacter (0.968%), Enterococcus (0.063%), Klebsiella (0.038%), Escherichia (0.028%) and Staphylococcus (0.004%). Despite the different treatment methods used, the effluent still revealed the presence of some Pseudomonas strains (0.066%), and a wide range of gram-positive cocci, including Staphylococcus (0.194%), Streptococcus (0.63%) and Enterococcus (0.037%), in addition to gram-negative Acinetobacter (0.736%), Klebsiella (0.1%), and Escherichia sub-species (0.811%). The AST results indicated that the strains Escherichia along with Klebsiella and Acinetobacter, isolated from the effluent, displayed resistance to 11 antibiotics, while Pseudomonas was resistant to 7 antibiotics, and Streptococcus along with Staphylococcus were resistant to 9 antibiotics. Results herein, proved the existence of some nosocomial MDR pathogens, known for ESKAPE, with potential drug resistance transfer to the non-pathogen microbes, requiring targeted remediation.

Reducing the Risk of Transmission of Critical Antimicrobial Resistance Determinants From Contaminated Pork Products to Humans in South-East Asia

“…Most recently, molecular high throughput (HPT) and nextgeneration sequencing (metagenomic analysis, transcriptome, and resistome) are among the techniques that facilitate studies of the microbial community and their diverse genomes and gene expression without a requirement for culture. Moreover, these techniques are very valuable since they can compute numerous target genes without knowledge of the bacteria or genes in the samples, are not limited by specific primer sets, and are even faster to perform than the conventional molecular methods such as PCR and qPCR (Rizzo et al, 2013;Limayem et al, 2019;Lira et al, 2020). They also provide a deeper insight into antibiotic resistance conditions related to both vertical and horizontal gene transfer occurrences (Von Wintersdorff et al, 2016).…”

Towards risk assessment for antibiotic resistant pathogens in recycled water: a systematic review and summary of research needs

“…In two studies, shortread sequences were assembled to generate contigs, which were annotated to identify ARGs (Garner et al, 2018;Chopyk et al, 2020). Limayem et al (2019) employed a unique approach in which multidrug-resistant bacteria were isolated via tryptic soy agar paired with the Vizion Sensititre method for resistance determination, then short-read sequencing (Illumina MiSeq) was utilized to identify putative ESKAPE pathogens (i.e., E. coli, S. aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterococcus faecium). The data presented to summarize the findings of these methods varied for each study.…”

“…The data presented to summarize the findings of these methods varied for each study. Data were presented in terms of number of contigs containing an ARG (Chopyk et al, 2020), log gene copies normalized to 16S rRNA genes (Garner et al, 2018(Garner et al, , 2019Zhang et al, 2020), and percent of isolates resistant (Limayem et al, 2019). Authors typically noted the most abundant resistance classes and ARGs, which are summarized in Table 3 along with other key parameters reported.…”

“…Unlike qPCR, metagenomic sequencing provides the ability to detect thousands of ARGs simultaneously without needing to design specific primers for each target ARG (Ishii, 2020). Of the compiled studies, six utilized metagenomic sequencing to characterize the resistome (Table 3) (Garner et al, 2018(Garner et al, , 2019Stamps et al, 2018;Limayem et al, 2019;Chopyk et al, 2020;Zhang et al, 2020). The compiled studies utilized a variety of metagenomic approaches to characterize ARGs.…”

Effect of Chlorination on Microbiological Quality of Effluent of a Full-Scale Wastewater Treatment Plant

“…Despite the fact that the effectiveness of common disinfection methods has been examined in various research works using microbial indicators [ 17 , 20 , 22 , 23 ], a few studies have been performed through the use of culture-independent techniques to access microbiological effluent quality [ 3 , 24 ]. The aim of the present study was to evaluate the effectiveness of chlorination as a disinfection method against bacterial pathogens and key physiological groups of bacteria detected in the effluent of a full-scale wastewater treatment plant.…”