Prevalence and Characterization of CRISPR Locus 2.1 Spacers in Escherichia coli Isolates Obtained from Feces of Animals and Humans.

The clustered regularly interspaced short palindromic repeat (CRISPR) has been studied as an immune system in prokaryotes for the survival of bacteriophages. The CRISPR system in prokaryotes records the invasion of bacteriophages or other genetic materials in CRISPR loci. Accordingly, CRISPR loci can reveal a history of infection records of bacteriophages and other genetic materials. Therefore, identification of the CRISPR array may help trace the events that bacteria have undergone. In this study, we characterized and identified the spacers of the CRISPR loci in Escherichia coli isolates obtained from the feces of animals and humans. Most CRISPR spacers were found to stem from phages. Although we did not find any patterns in CRISPR spacers according to sources, our results showed that phage-derived spacers mainly originated from the families Inoviridae, Myoviridae, Podoviridae, and Siphoviridae and the order Caudovirales, whereas plasmid-derived CRISPR spacers were mainly from the Enterobacteriaceae family. In addition, it is worth noting that the isolates from each animal and human source harbored source-specific spacers. Considering that some of these taxa are likely found in the gut of mammalian animals, CRISPR spacers identified in these E. coli isolates were likely derived from the bacteriophageome and microbiome in closed gut environments. Although the bacteriophageome database limits the characterization of CRISPR arrays, the present study showed that some spacers were specifically found in both animal and human sources. Thus, this finding may suggest the possible use of E. coli CRISPR spacers as a microbial source tracking tool. IMPORTANCE We characterized spacers of CRISPR locus 2.1 in E. coli isolates obtained from the feces of various sources. Phage-derived CRISPR spacers are mainly acquired from the order Caudovirales, and plasmid-derived CRISPR spacers are mostly from the Enterobacteriaceae family. This is thought to reflect the microbiome and phageome of the gut environment of the sources. Hence, spacers may help track the encounter of bacterial cells with bacterial cells, viruses, or other genetic materials. Interestingly, source-specific spacers are also observed. The identification of source-specific spacers is thought to help develop the methodology of microbial source tracking and understanding the interactions between viruses and bacteria. However, very few spacers have been uncovered to track where they originate. The accumulation of genome sequences can help identify the hosts of spacers and can be applied for microbial source tracking.

Resistome Study in Aquatic Environments.

Since the first discovery of antibiotics, introduction of new antibiotics has been coupled with the occurrence of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs). Rapid dissemination of ARB and ARGs in the aquatic environments has become a global concern. ARB and ARGs have been already disseminated in the aquatic environments via various routes. Main hosts of most of ARGs were found to belong to Gammaproteobacteria class, including clinically important potential pathogens. Transmission of ARGs also occurs by horizontal gene transfer (HGT) mechanisms between bacterial strains in the aquatic environments, resulting in ubiquity of ARGs. Thus, a few of ARGs and MGEs (e.g., strA, sul1, int1) have been suggested as indicators for global comparability of contamination level in the aquatic environments. With ARB and ARGs contamination, the occurrence of critical pathogens has been globally issued due to their widespread in the aquatic environments. Thus, active surveillance systems have been launched worldwide. In this review, we described advancement of methodologies for ARGs detection, and occurrence of ARB and ARGs and their dissemination in the aquatic environments. Even though numerous studies have been conducted for ARB and ARGs, there is still no clear strategy to tackle antibiotic resistance (AR) in the aquatic environments. At least, for consistent surveillance, a strict framework should be established for further research in the aquatic environments.

Dynamics of Genotypic and Phenotypic Antibiotic Resistance in a Conventional Wastewater Treatment Plant in 2 Years.

Wastewater treatment plants (WWTPs) are considered a sink and a source of antibiotic resistance. In this study, we applied both culture-dependent and SmartChip-based culture-independent approaches for the investigation of antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) at Jungnang (JN), located in the Han River, Seoul, South Korea, for 2 years, i.e., 2017 and 2018. The JN WWTP reduced the diversity and abundance of ARB and ARGs but was not sufficient for removing them all. Interestingly, through the treatment process in the JN WWTP, the composition of diverse multidrug-resistant (MDR) bacteria was concentrated mainly into some genera of the Gammaproteobacteria class (Citrobacter, Escherichia-Shigella, and Stenotrophomonas), which could be key carriages to spread ARGs into the environments. In addition, SmartChip analyses showed that the relative abundance and the number of ARGs were significantly decreased from the influents to the effluents in both 2017 and 2018. SmartChip analyses for 2 years also allowed to notify the core ARGs in the influents and the effluents with the presence of clinically relevant core ARGs, such as vanC, bla OXA , and bla NDM , which persisted in the treatment process. Considering diverse bacterial mechanisms for exchanging and transferring ARGs, the occurrence of MDR bacteria and core ARGs could be a source for the blooming of the antibiotic resistome in the WWTP and nearby environments.

Phylogenetic diversity and spatiotemporal dynamics of bacterial and microeukaryotic plankton communities in Gwangyang Bay of the Korean Peninsula.

Nutrient dynamics function globally, flowing from rivers to the ocean (estuarine-coastal zone), and are vulnerable to climate change. Microbial habitats can be affected by marine nutrient dynamics and may provide a clue to predict microbial responses to environmental heterogeneity in estuarine-coastal zones. We surveyed surface seawater in Gwangyang Bay, a semi-enclosed estuary in Korea, from 2016 to 2018 using a metabarcoding approach with prokaryotic 16S and eukaryotic 18S rRNA genes. Bacterial and microeukaryotic communities in these waters showed distinct local communities in response to environmental heterogeneity and community transition at spatiotemporal scales in the estuarine-coastal zone. The relative abundance of prokaryotic and eukaryotic operational taxonomic units suggested a microbial trophic interaction in the Gwangyang Bay waters. We found that the community assembly process in prokaryotic communities was primarily influenced by biological interaction (immigration-emigration), whereas that in eukaryotic communities was more affected by environmental stress (habitat specificity) rather than by biotic factors. Our findings in the Gwangyang Bay waters may provide information on underlying (biotic or abiotic) factors of the assembly process in microbial communities in the estuarine-coastal zone.

Higher abundance of core antimicrobial resistant genes in effluent from wastewater treatment plants.

Wastewater treatment plants (WWTPs) receive sewage water from a variety of sources, including livestock farms, hospitals, industries, and households, that contain antimicrobial resistant bacteria (ARB) and antimicrobial resistant genes (ARGs). Current treatment technologies are unable to completely remove ARB and ARGs, which are eventually released into the aquatic environment. This study focused on the core resistome of urban WWTPs that are persistent through wastewater treatment processes. We adopted the Hiseq-based metagenomic sequencing approach to identify the core resistome, their genetic context, and pathogenic potential of core ARGs in the influent (IN) and effluent (EF) samples of 12 urban WWTPs in South Korea. In this study, the abundance of ARGs ranged from 0.32 to 3.5 copies of ARGs per copy of the 16S rRNA gene, where the IN samples were relatively higher than the EF samples, especially for the macrolide-lincosamide-streptogramin (MLS)- and tetracycline- resistant genes. On the other hand, there were 43 core ARGs sharing up to 90% of the total, among which the relative abundance of sul1, APH(3'')-lb, and RbpA was higher in EF than in IN (p < 0.05). Moreover, tetracycline and sulfonamide-related core ARGs in both EF and IN were significantly more abundant on plasmids than on chromosomes (p < 0.05). We also found that the majority of core ARGs were carried by opportunistic pathogens such as Acinetobacter baumannii, Enterobacter cloacae, and Pseudomonas aeruginosa in both IN and EF. In addition, phages were the only mobile elements whose abundance correlated with that of core ARGs in EF, suggesting that transduction may play a major role in disseminating ARGs in the receiving water environment of the urban WWTP. The persistent release of core ARGs with pathogenic potential into environmental water is of immediate concern. The mobility of ARGs and ARBs in the environment is a major public health concern. These results should be taken into consideration when developing policy to mitigate environmental dissemination of ARG by WWTPs.

Emergence of High Level Carbapenem and Extensively Drug Resistant Escherichia coli ST746 Producing NDM-5 in Influent of Wastewater Treatment Plant, Seoul, South Korea.

High level carbapenem and extensively drug resistant (XDR) Escherichia coli strain N7, which produces a variant of New Delhi metallo-ß-lactamase (NDM-5), was isolated from the influent of the Jungnang wastewater treatment plant located on Han River, Seoul, South Korea. Phenotypic and genotypic resistances to carbapenem were tested using agar and broth dilution methods, and polymerase chain reaction. Whole-genome sequencing was performed to characterize the genetic structure of strain N7. E. coli strain N7, which harbors the bla NDM-5 gene, showed high level of carbapenem resistance at concentrations of doripenem (512 mg/L) and meropenem (256 mg/L), and XDR to 15 antibiotics. Based on the genomic sequence analysis, two plasmids, a hybrid IncHI2/N-type and an IncX3 type, were present. The former contains a cluster (bla NDM-5-ble MBL -trpF-dsbD) bracketed by multi-insertional sequences, IS3000, ISAba125, IS5, and IS26. The latter carries the following resistance genes: bla CTX-14, aac(3)-IV, aadA1, aadA2, aph(3')-Ia, aph(4)-Ia, sul1, sul2, sul3, dfrA12, fosA3, oqxA, oqxB, mph(A), and floR, and cmlA1. The chromosome, contig3, and contig5 also carry bla CTX-64 and mdf(A), tet(A), and erm(B), tet(M) and aadA22, respectively. Strain N7 also harbors virulence factors such as fimH, flu, ecpABCDE, sfmA, hlyE, and gadA. This study demonstrates the emergence of high level carbapenem resistant XDR E. coli strain N7 containing bla NDM-5 in aquatic environment, Seoul, South Korea. Due to the presence of mobile genetic elements, this strain could horizontally transfer resistance genes, including bla NDM-5 to environmental bacteria. Thus, it is necessary to conduct continuous surveillance for carbapenem resistance in various aquatic environments.

Metagenomic exploration of antibiotic resistome in treated wastewater effluents and their receiving water.

Environmental dissemination of antimicrobial resistance is a global health problem. Antimicrobial-resistant bacteria and antibiotic-resistant genes (ARGs) are constantly released into the environment through effluents (EFs) from wastewater treatment plants (WWTPs). Thus, requiring a better understanding of the selection and fate of ARGs in wastewater treatment processes. Therefore, we investigated the impacts of urban WWTP EFs on receiving water in the context of their resistomes and mobilomes. We used a HiSeq-based short read metagenomic approach to address the dynamics and diversity of ARGs in WWTP EF as well as the upstream (UP) and downstream (DN) river waters, followed by an investigation of plasmid-mediated ARGs. The abundance of ARGs at each site varied from 7.2 × 10-2 to 7.4 × 10-1 ARG copies per 16S rRNA gene copy, and EF samples showed the highest abundance, followed by DN and UP water samples. ARG diversity ranged from 121 to 686 types per site, and EF had the most diverse ARGs. Commonly identified ARGs in the EF and DN samples were clinically important and were absent in UP samples. The abundance of ARGs, mobile genetic elements (MGEs), and plasmid contigs found only in EF and DN were positively correlated with each other, indicating the importance of mobilomes in the dissemination of ARGs in the environment. Moreover, the proportions of plasmid-mediated ARGs was highest in the EF samples, followed by the DN and UP samples. These findings suggest that WWTP EF may act as a driving factor shaping the resistomes and mobilomes of receiving waters. In particular, a higher abundance of plasmid-mediated ARGs in WWTP EF suggests higher transmissibility in the DN environment.

Metagenomic analysis reveals the prevalence and persistence of antibiotic- and heavy metal-resistance genes in wastewater treatment plant.

The increased antibiotic resistance among microorganisms has resulted into growing interest for investigating the wastewater treatment plants (WWTPs) as they are reported to be the major source in the dissemination of antibiotic resistance genes (ARGs) and heavy metal resistance genes (HMRGs) in the environment. In this study, we investigated the prevalence and persistence of ARGs and HMRGs as well as bacterial diversity and mobile genetic elements (MGEs) in influent and effluent at the WWTP in Gwangju, South Korea, using high-throughput sequencing based metagenomic approach. A good number of broad-spectrum of resistance genes (both ARG and HMRG) were prevalent and likely persistent, although large portion of them were successfully removed at the wastewater treatment process. The relative abundance of ARGs and MGEs was higher in effluent as compared to that of influent. Our results suggest that the resistance genes with high abundance and bacteria harbouring ARGs and MGEs are likely to persist more through the treatment process. On analyzing the microbial community, the phylum Proteobacteria, especially potentially pathogenic species belonging to the genus Acinetobacter, dominated in WWTP. Overall, our study demonstrates that many ARGs and HMRGs may persist the treatment processes in WWTPs and their association to MGEs may contribute to the dissemination of resistance genes among microorganisms in the environment.