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      Genomic Analysis of Enterococcus spp. Isolated From a Wastewater Treatment Plant and Its Associated Waters in Umgungundlovu District, South Africa


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          We investigated the antibiotic resistome, mobilome, virulome, and phylogenomic lineages of Enterococcus spp. obtained from a wastewater treatment plant and its associated waters using whole-genome sequencing (WGS) and bioinformatics tools. The whole genomes of Enterococcus isolates including Enterococcus faecalis ( n = 4), Enterococcus faecium ( n = 5), Enterococcus hirae ( n = 2), and Enterococcus durans ( n = 1) with similar resistance patterns from different sampling sites and time points were sequenced on an Illumina MiSeq machine. Multilocus sequence typing (MLST) analysis revealed two E. faecalis isolates that had a common sequence type ST179; the rest had unique sequence types ST841, and ST300. The E. faecium genomes belonged to 3 sequence types, ST94 ( n = 2), ST361 ( n = 2), and ST1096 ( n = 1). Detected resistance genes included those encoding tetracycline [ tet(S), tet(M), and tet(L)], and macrolides [ msr(C), msr(D), erm(B), and mef(A)] resistance. Antibiotic resistance genes were associated with insertion sequences (IS6, ISL3, and IS982), and transposons (Tn3 and Tn6000). The tet(M) resistance gene was consistently found associated with a conjugative transposon protein (TcpC). A total of 20 different virulence genes were identified in E. faecalis and E. faecium including those encoding for sex pheromones ( cCF10, cOB1, cad, and came), adhesion ( ace, SrtA, ebpA, ebpC, and efaAfs), and cell invasion ( hylA and hylB). Several virulence genes were associated with the insertion sequence IS256. No virulence genes were detected in E. hirae and E. durans. Phylogenetic analysis revealed that all Enterococcus spp. isolates were more closely related to animal and environmental isolates than clinical isolates. Enterococcus spp. with a diverse range of resistance and virulence genes as well as associated mobile genetic elements (MGEs) exist in the wastewater environment in South Africa.

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          The Sequence Alignment/Map format and SAMtools

          Summary: The Sequence Alignment/Map (SAM) format is a generic alignment format for storing read alignments against reference sequences, supporting short and long reads (up to 128 Mbp) produced by different sequencing platforms. It is flexible in style, compact in size, efficient in random access and is the format in which alignments from the 1000 Genomes Project are released. SAMtools implements various utilities for post-processing alignments in the SAM format, such as indexing, variant caller and alignment viewer, and thus provides universal tools for processing read alignments. Availability: http://samtools.sourceforge.net Contact: rd@sanger.ac.uk
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            FastTree 2 – Approximately Maximum-Likelihood Trees for Large Alignments

            Background We recently described FastTree, a tool for inferring phylogenies for alignments with up to hundreds of thousands of sequences. Here, we describe improvements to FastTree that improve its accuracy without sacrificing scalability. Methodology/Principal Findings Where FastTree 1 used nearest-neighbor interchanges (NNIs) and the minimum-evolution criterion to improve the tree, FastTree 2 adds minimum-evolution subtree-pruning-regrafting (SPRs) and maximum-likelihood NNIs. FastTree 2 uses heuristics to restrict the search for better trees and estimates a rate of evolution for each site (the “CAT” approximation). Nevertheless, for both simulated and genuine alignments, FastTree 2 is slightly more accurate than a standard implementation of maximum-likelihood NNIs (PhyML 3 with default settings). Although FastTree 2 is not quite as accurate as methods that use maximum-likelihood SPRs, most of the splits that disagree are poorly supported, and for large alignments, FastTree 2 is 100–1,000 times faster. FastTree 2 inferred a topology and likelihood-based local support values for 237,882 distinct 16S ribosomal RNAs on a desktop computer in 22 hours and 5.8 gigabytes of memory. Conclusions/Significance FastTree 2 allows the inference of maximum-likelihood phylogenies for huge alignments. FastTree 2 is freely available at http://www.microbesonline.org/fasttree.
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              ISfinder: the reference centre for bacterial insertion sequences

              ISfinder () is a dedicated database for bacterial insertion sequences (ISs). It has superseded the Stanford reference center. One of its functions is to assign IS names and to provide a focal point for a coherent nomenclature. It is also the repository for ISs. Each new IS is indexed together with information such as its DNA sequence and open reading frames or potential coding sequences, the sequence of the ends of the element and target sites, its origin and distribution together with a bibliography where available. Another objective is to continuously monitor ISs to provide updated comprehensive groupings or families and to provide some insight into their phylogenies. The site also contains extensive background information on ISs and transposons in general. Online tools are gradually being added. At present an online Blast facility against the entire bank is available. But additional features will include alignment capability, PsiBLAST and HMM profiles. ISfinder also includes a section on bacterial genomes and is involved in annotating the IS content of these genomes. Finally, this database is currently recommended by several microbiology journals for registration of new IS elements before their publication.

                Author and article information

                Front Microbiol
                Front Microbiol
                Front. Microbiol.
                Frontiers in Microbiology
                Frontiers Media S.A.
                14 June 2021
                : 12
                : 648454
                [1] 1Antimicrobial Research Unit, College of Health Sciences, University of Kwazulu-Natal , Durban, South Africa
                [2] 2Department of Applied Biology and Biochemistry, National University of Science and Technology , Bulawayo, Zimbabwe
                [3] 3Sequencing Core Facility, National Institute for Communicable Diseases, National Health Laboratory Service , Johannesburg, South Africa
                Author notes

                Edited by: Jorge Andres Olivares Pacheco, Pontificia Universidad Católica de Valparaíso, Chile

                Reviewed by: Ana P. Tedim, Institute of Health Sciences Studies of Castilla y León (IECSCYL), Spain; Ana Paula Guedes Frazzon, Federal University of Rio Grande do Sul, Brazil

                This article was submitted to Antimicrobials, Resistance and Chemotherapy, a section of the journal Frontiers in Microbiology

                Copyright © 2021 Mbanga, Amoako, Abia, Allam, Ismail and Essack.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                : 31 December 2020
                : 19 May 2021
                Page count
                Figures: 4, Tables: 5, Equations: 0, References: 44, Pages: 13, Words: 0
                Original Research

                Microbiology & Virology
                enterococcus spp.,whole-genome sequencing,wastewater treatment plant,antibiotic resistance,south africa


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