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Rapid nanopore-based DNA sequencing protocol of antibiotic-resistant bacteria for use in surveillance and outbreak investigation.

ABSTRACT: Outbreak investigations are essential to control and prevent the dissemination of pathogens. This study developed and validated a complete analysis protocol for faster and more accurate surveillance and outbreak investigations of antibiotic-resistant microbes based on Oxford Nanopore Technologies (ONT) DNA whole-genome sequencing. The protocol was developed using 42 methicillin-resistant Staphylococcus aureus (MRSA) isolates identified from former well-characterized outbreaks. The validation of the protocol was performed using Illumina technology (MiSeq, Illumina). Additionally, a real-time outbreak investigation of six clinical S. aureus isolates was conducted to test the ONT-based protocol. The suggested protocol includes: (1) a 20 h sequencing run; (2) identification of the sequence type (ST); (3) de novo genome assembly; (4) polishing of the draft genomes; and (5) phylogenetic analysis based on SNPs. After the sequencing run, it was possible to identify the ST in 2 h (20 min per isolate). Assemblies were achieved after 4 h (40 min per isolate) while the polishing was carried out in 7 min per isolate (42 min in total). The phylogenetic analysis took 0.6 h to confirm an outbreak. Overall, the developed protocol was able to at least discard an outbreak in 27 h (mean) after the bacterial identification and less than 33 h to confirm it. All these estimated times were calculated considering the average time for six MRSA isolates per sequencing run. During the real-time S. aureus outbreak investigation, the protocol was able to identify two outbreaks in less than 31 h. The suggested protocol enables identification of outbreaks in early stages using a portable and low-cost device along with a streamlined downstream analysis, therefore having the potential to be incorporated in routine surveillance analysis workflows. In addition, further analysis may include identification of virulence and antibiotic resistance genes for improved pathogen characterization.

SUBMITTER: Ferreira FA

PROVIDER: S-EPMC8208686 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Outbreaks of methicillin-resistant Staphylococcus aureus (MRSA) are well described in the neonatal intensive care unit (NICU) setting. Genomics has revolutionized the investigation of such outbreaks; however, to date, this has largely been completed retrospectively and has typically relied on short-read platforms. In 2022, our laboratory established a prospective genomic surveillance system using Oxford Nanopore Technologies sequencing for rapid outbreak detection. Herein, using this system, we describe the detection and control of an outbreak of sequence-type (ST)97 MRSA in our NICU. The outbreak was identified 13 days after the first MRSA-positive culture and at a point where there were only two known cases. Ward screening rapidly defined the extent of the outbreak, with six other infants found to be colonized. There was minimal transmission once the outbreak had been detected and appropriate infection control measures had been instituted; only two further ST97 cases were detected, along with three unrelated non-ST97 MRSA cases. To contextualize the outbreak, core-genome single-nucleotide variants were identified for phylogenetic analysis after de novo assembly of nanopore data. Comparisons with global (n=45) and national surveillance (n=35) ST97 genomes revealed the stepwise evolution of methicillin resistance within this ST97 subset. A distinct cluster comprising nine of the ten ST97-IVa genomes from the NICU was identified, with strains from 2020 to 2022 national surveillance serving as outgroups to this cluster. One ST97-IVa genome presumed to be part of the outbreak formed an outgroup and was retrospectively excluded. A second phylogeny was created using Illumina sequencing, which considerably reduced the branch lengths of the NICU isolates on the phylogenetic tree. However, the overall tree topology and conclusions were unchanged, with the exception of the NICU outbreak cluster, where differences in branch lengths were observed. This analysis demonstrated the ability of a nanopore-only prospective genomic surveillance system to rapidly identify and contextualize an outbreak of MRSA in a NICU.

Project description:Whole genome sequencing (WGS) has become the new gold standard for bacterial outbreak investigation, due to the high resolution available for typing. While sequencing is currently predominantly performed on Illumina devices, the preceding library preparation can be performed using various protocols. Enzymatic fragmentation library preparation protocols are fast, have minimal hands-on time, and work with small quantities of DNA. The aim of our study was to compare three library preparation protocols for molecular typing: Nextera XT (Illumina); Nextera Flex (Illumina); and QIAseq FX (Qiagen). We selected 12 ATCC strains from human Gram-positive and Gram-negative pathogens with %G+C-content ranging from 27% (Fusobacterium nucleatum) to 73% (Micrococcus luteus), each having a high quality complete genome assembly available, to allow in-depth analysis of the resulting Illumina sequence data quality. Additionally, we selected isolates from previously analyzed cases of vancomycin-resistant Enterococcus faecium (VRE) (n = 7) and a local outbreak of Klebsiella aerogenes (n = 5). The number of protocol steps and time required were compared, in order to test the suitability for routine laboratory work. Data analyses were performed with standard tools commonly used in outbreak situations: Ridom SeqSphere+ for cgMLST; CLC genomics workbench for SNP analysis; and open source programs. Nextera Flex and QIAseq FX were found to be less sensitive than Nextera XT to variable %G+C-content, resulting in an almost uniform distribution of read-depth. Therefore, low coverage regions are reduced to a minimum resulting in a more complete representation of the genome. Thus, with these two protocols, more alleles were detected in the cgMLST analysis, producing a higher resolution of closely related isolates. Furthermore, they result in a more complete representation of accessory genes. In particular, the high data quality and relative simplicity of the workflow of Nextera Flex stood out in this comparison. This thorough comparison within an ISO/IEC 17025 accredited environment will be of interest to those aiming to optimize their clinical microbiological genome sequencing.

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Rapid nanopore-based DNA sequencing protocol of antibiotic-resistant bacteria for use in surveillance and outbreak investigation.

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