Salmonella entericaserovar Enteritidis is a significant cause of gastrointestinal illness in the United States; however, current molecular subtyping methods lack resolution for this highly clonal serovar. Advances in next-generation sequencing technologies have made it possible to examine whole-genome sequencing (WGS) as a potential molecular subtyping tool for outbreak detection and source trace back. Here, we conducted a retrospective analysis ofS. Enteritidis isolates from seven epidemiologically confirmed foodborne outbreaks and sporadic isolates (not epidemiologically linked) to determine the utility of WGS to identify outbreaks. A collection of 55 epidemiologically characterized clinical and environmentalS. Enteritidis isolates were sequenced. Single nucleotide polymorphism (SNP)-based cluster analysis of theS. Enteritidis genomes revealed well supported clades, with less than four-SNP pairwise diversity, that were concordant with epidemiologically defined outbreaks. Sporadic isolates were an average of 42.5 SNPs distant from the outbreak clusters. Isolates collected from the same patient over several weeks differed by only two SNPs. Our findings show that WGS provided greater resolution between outbreak, sporadic, and suspect isolates than the current gold standard subtyping method, pulsed-field gel electrophoresis (PFGE). Furthermore, results could be obtained in a time frame suitable for surveillance activities, supporting the use of WGS as an outbreak detection and characterization method forS. Enteritidis.
We describe a Salmonella Poona outbreak involving 31 infant cases in France. Following outbreak detection on 18 January 2019, consumption of rice-based infant formula manufactured at a facility in Spain was identified as the probable cause, leading to a recall on 24 January. Whole genome sequencing analysis linked present outbreak isolates to a 2010–11 S. Poona outbreak in Spain associated with formula manufactured in the same facility, indicating a persistent source of contamination.
AbstractWhole-genome sequencing is likely to become increasingly used by local clinical microbiology laboratories, where sequencing volume is low compared with national reference laboratories. Here, we describe a universal protocol for simultaneous DNA extraction and sequencing of numerous different bacterial species, allowing mixed species sequence runs to meet variable laboratory demand. We assembled test panels representing 20 clinically relevant bacterial species. The DNA extraction process used the QIAamp mini DNA kit, to which different combinations of reagents were added. Thereafter, a common protocol was used for library preparation and sequencing. The addition of lysostaphin, lysozyme or buffer ATL (a tissue lysis buffer) alone did not produce sufficient DNA for library preparation across the species tested. By contrast, lysozyme plus lysostaphin produced sufficient DNA across all 20 species. DNA from 15 of 20 species could be extracted from a 24-h culture plate, while the remainder required 48–72 h. The process demonstrated 100% reproducibility. Sequencing of the resulting DNA was used to recapitulate previous findings for species, outbreak detection, antimicrobial resistance gene detection and capsular type. This single protocol for simultaneous processing and sequencing of multiple bacterial species supports low volume and rapid turnaround time by local clinical microbiology laboratories.
Background: Hospital-acquired infections pose a significant threat to patient health. Laboratories are starting to consider whole-genome sequencing (WGS) as a molecular method for outbreak detection and epidemiological surveillance. The objective of this study was to assess the use of the iSeq100 platform (Illumina, San Diego, CA) for accurate sequencing and WGS-based outbreak detection using the bioMérieux EPISEQ CS, a novel cloud-based software for sequence assembly and data analysis. Methods: In total, 25 isolates, including 19 MRSA isolates and 6 ATCC strains were evaluated in this study: A. baumannii ATCC 19606, B. cepacia ATCC 25416, E. faecalis ATCC 29212, E. coli ATCC 25922, P. aeruginosa ATCC 27853 and S. aureus ATCC 25923. DNA extraction of all isolates was performed on the QIAcube (Qiagen, Hilden, Germany) using the DNEasy Ultra Clean Microbial kit extraction protocol. DNA libraries were prepared for WGS using the Nextera DNA Flex Library Prep Kit (Illumina) and sequenced at 2×150-bp on the iSeq100 according to the manufacturer’s instructions. The 19 MRSA isolates were previously characterized by the DiversiLab system (bioMérieux, France). Upon validation of the iSeq100 platform, a new outbreak analysis was performed using WGS analysis using EPISEQ CS. ATCC sequences were compared to assembled reference genomes from the NCBI GenBank to assess the accuracy of the iSeq100 platform. The FASTQ files were aligned via BowTie2 version 2.2.6 software, using default parameters, and FreeBayes version 188.8.131.52-0 was used to call homozygous single-nucleotide polymorphisms (SNPs) with a minimum coverage of 5 and an allele frequency of 0.87 using default parameters. ATCC sequences were analyzed using ResFinder version 3.2 and were compared in silico to the reference genome. Results: EPISEQ CS classified 8 MRSA isolates as unrelated and grouped 11 isolates into 2 separate clusters: cluster A (5 isolates) and cluster B (6 isolates) with similarity scores of ≥99.63% and ≥99.50%, respectively. This finding contrasted with the previous characterization by DiversiLab, which identified 3 clusters of 2, 8, and 11 isolates, respectively. The EPISEQ CS resistome data detected the mecA gene in 18 of 19 MRSA isolates. Comparative analysis of the ATCCsequences to the reference genomes showed 99.9986% concordance of SNPs and 100.00% concordance between the resistance genes present. Conclusions: The iSeq100 platform accurately sequenced the bacterial isolates and could be an affordable alternative in conjunction with EPISEQ CS for epidemiological surveillance analysis and infection prevention.Funding: NoneDisclosures: None
ABSTRACTAlthoughSalmonella entericacan produce life-threatening colitis in horses, certain serotypes are more commonly associated with clinical disease. Our aim was to evaluate the proportional morbidity attributed to different serotypes, as well as the phenotypic and genotypic antimicrobial resistance (AMR) ofSalmonellaisolates from patients at an equine referral hospital in the southern United States. A total of 255Salmonellaisolates was obtained from clinical samples of patients admitted to the hospital between 2007 and 2015. Phenotypic resistance to 14 antibiotics surveilled by the U.S. National Antimicrobial Resistance Monitoring System was determined using a commercially available panel. Whole-genome sequencing was used to identify serotypes and genotypic AMR. The most common serotypes wereSalmonella entericaserotype Newport (18%),Salmonella entericaserotype Anatum (15.2%), andSalmonella entericaserotype Braenderup (11.8%). Most (n= 219) of the isolates were pansusceptible, while 25 were multidrug resistant (≥3 antimicrobial classes). Genes encoding beta-lactam resistance, such asblaCMY-2,blaSHV-12,blaCTX-M-27, andblaTEM-1B, were detected. TheqnrB2 andaac(6′)-Ib-crgenes were present in isolates with reduced susceptibility to ciprofloxacin. Genes encoding resistance to gentamicin (aph(3′)-Ia,aac(6′)-IIc), streptomycin (strA andstrB), sulfonamides (sul1), trimethoprim (dfrA), phenicols (catA), tetracyclines [tet(A) andtet(E)], and macrolides [ere(A)] were also identified. The main predicted incompatibility plasmid type was I1 (10%). Core genome-based analyses revealed phylogenetic associations between isolates of common serotypes. The presence of AMRSalmonellain equine patients increases the risk of unsuccessful treatment and causes concern for potential zoonotic transmission to attending veterinary personnel, animal caretakers, and horse owners. Understanding the epidemiology ofSalmonellain horses admitted to referral hospitals is important for the prevention, control, and treatment of salmonellosis.IMPORTANCEIn horses, salmonellosis is a leading cause of life-threatening colitis. At veterinary teaching hospitals, nosocomial outbreaks can increase the risk of zoonotic transmission, lead to restrictions on admissions, impact hospital reputation, and interrupt educational activities. The antimicrobials most often used in horses are included in the 5th revision of the World Health Organization's list of critically important antimicrobials for human medicine. Recent studies have demonstrated a trend of increasing bacterial resistance to drugs commonly used to treatSalmonellainfections. In this study, we identify temporal trends in the distribution ofSalmonellaserotypes and their mechanisms of antimicrobial resistance; furthermore, we are able to determine the likely origin of several temporal clusters of infection by using whole-genome sequencing. These data can be used to focus strategies to better contain the dissemination and enhance the mitigation ofSalmonellainfections and to provide evidence-based policies and guidelines to steward antimicrobial use in veterinary medicine.