Implementation of a high-throughput whole genome sequencing approach with the goal of maximizing efficiency and cost effectiveness to improve public health.

This manuscript describes the development of a streamlined, cost-effective laboratory workflow to meet the demands of increased whole genome sequence (WGS) capacity while achieving mandated quality metrics. From 2020 to 2021, the Wadsworth Center Bacteriology Laboratory (WCBL) used a streamlined workflow to sequence 5,743 genomes that contributed sequence data to nine different projects. The combined use of the QIAcube HT, Illumina DNA Prep using quarter volume reactions, and the NextSeq allowed the WCBL to process all samples that required WGS while also achieving a median turn-around time of 7 days (range 4 to 10 days) and meeting minimum sequence quality requirements. Public Health Laboratories should consider implementing these methods to aid in meeting testing requirements within budgetary restrictions. IMPORTANCE: Public Health Laboratories that implement whole genome sequencing (WGS) technologies may struggle to find the balance between sample volume and cost effectiveness. We present a method that allows for sequencing of a variety of bacterial isolates in a cost-effective manner. This report provides specific strategies to implement high-volume WGS, including an innovative, low-cost solution utilizing a novel quarter volume sequencing library preparation. The methods described support the use of high-throughput DNA extraction and WGS within budgetary constraints, strengthening public health responses to outbreaks and disease surveillance.

Streptococcus sciuri sp. nov., Staphylococcus marylandisciuri sp. nov. and Staphylococcus americanisciuri sp. nov., isolated from faeces of eastern grey squirrel (Sciurus carolinensis).

One novel Streptococcus strain (SQ9-PEAT) and two novel Staphylococcus strains (SQ8-PEAT and GRT3T) were isolated from faeces of a wild eastern grey squirrel. The strains were non-spore-forming, non-motile Gram-positive cocci, facultative anaerobes. The genomes for these strains were sequenced. The 16S rRNA gene and core-genome-based phylogenetic analyses showed that strain SQ9-PEAT was closely related to Streptococcus hyointestinalis, strain SQ8-PEAT to Staphylococcus pettenkoferi and Staphylococcus argensis, and strain GRT3T to Staphylococcus rostri, Staphylococcus muscae and Staphylococcus microti. Average nucleotide identity and pairwise digital DNA-DNA hybridization values calculated for these novel strains compared to type strain genomes of phylogenetically related species within the genera Streptococcus and Staphylococcus clearly revealed that strain SQ9-PEAT represents a novel species of the genus Streptococcus and strains SQ8-PEAT and GRT3T represent two novel species of the genus Staphylococcus. Phenotypical features of these novel type strains differed from the features of the type strains of other phylogenetically related species. MALDI-TOF mass spectrometry supported identification of these novel species. Based on these data, we propose one novel species of the genus Streptococcus, for which the name Streptococcus sciuri sp. nov. with the type strain SQ9-PEAT (=DSM 114656T=CCUG 76426T=NCTC 14727T) is proposed, and two novel species of the genus Staphylococcus, for which the names Staphylococcus marylandisciuri sp. nov. with the type strain SQ8-PEAT (=DSM 114685T=CCUG 76423T=NCTC 14723T) and Staphylococcus americanisciuri sp. nov. with the type strain GRT3T (=DSM 114696T=CCUG 76427T=NCTC 14722T) are proposed. The genome G+C contents are 38.29, 36.49 and 37.26 mol% and complete draft genome sizes are 1 692 266, 2 371 088 and 2 237 001 bp for strains SQ9-PEAT, SQ8-PEAT and GRT3T, respectively.

Neisseria montereyensis sp. nov., Isolated from Oropharynx of California Sea Lion (Zalophus californianus): Genomic, Phylogenetic, and Phenotypic Study.

A novel Neisseria strain, designated CSL10203-ORH2T, was isolated from the oropharynx of a wild California sea lion (Zalophus californianus) that was admitted to The Marine Mammal Center in California, USA. The strain was originally cultured from an oropharyngeal swab on BD Phenylethyl Alcohol (PEA) agar with 5% sheep blood under aerobic conditions. Phylogenetic analyses based on 16S rRNA, rplF, and rpoB gene sequences and the core genome sequences indicated that the strain was most closely related to only N. zalophi CSL 7565T. The average nucleotide identity and digital DNA-DNA hybridization values between strain CSL10203-ORH2T and the closely related species N. zalophi CSL 7565T were 89.84 and 39.70%, respectively, which were significantly lower than the accepted species-defined thresholds for describing novel prokaryotic species at the genomic level. Both type strains were phenotypically similar but can be easily and unambiguously distinguished between each other by the analysis of their housekeeping genes, e.g., rpoB, gyrB, or argF. The major fatty acids in both type strains were C12:0, C16:0, C16:1-c9, and C18:1-c11. Based on the genomic, phenotypic, and phylogenetic properties, the novel strain represents a novel species of the genus Neisseria, for which the name Neisseria montereyensis sp. nov. with the type strain CSL10203-ORH2T (= DSM 114706T = CCUG 76428T = NCTC 14721T) is proposed. The genome G + C content is 45.84% and the complete draft genome size is 2,310,535 bp.

The Use of a Shared Services Model for Mycobacteriology Testing: Lessons Learned.

OBJECTIVES: Public health laboratories (PHLs) provide essential services in the diagnosis and surveillance of diseases of public health concern, such as tuberculosis. Maintaining access to high-quality laboratory testing is critical to continued disease detection and decline of tuberculosis cases in the United States. We investigated the practical experience of sharing tuberculosis testing services between PHLs through the Shared Services Project. METHODS: The Shared Services Project was a 9-month-long project funded through the Association of Public Health Laboratories and the Centers for Disease Control and Prevention during 2012-2013 as a one-time funding opportunity to consortiums of PHLs that proposed collaborative approaches to sharing tuberculosis laboratory services. Submitting PHLs maintained testing while simultaneously sending specimens to reference laboratories to compare turnaround times. RESULTS: During the 9-month project period, 107 Mycobacterium tuberculosis complex submissions for growth-based drug susceptibility testing and molecular detection of drug resistance testing occurred among the 3 consortiums. The median transit time for all submissions was 1.0 day. Overall, median drug susceptibility testing turnaround time (date of receipt in submitting laboratory to result) for parallel testing performed in house by submitting laboratories was 31.0 days; it was 43.0 days for reference laboratories. The median turnaround time for molecular detection of drug resistance results was 1.0 day (mean = 2.8; range, 0-14) from specimen receipt at the reference laboratories. CONCLUSIONS: The shared services model holds promise for specialized tuberculosis testing. Sharing of services requires a balance among quality, timeliness, efficiency, communication, and fiscal costs.

Rapid prediction of inducible clarithromycin resistance in Mycobacterium abscessus.

We have developed a single tube TaqMan(®) real-time PCR assay that differentiates the full-length and truncated erm(41) gene to predict inducible resistance to clarithromycin in Mycobacterium abscessus. A study of 87 clinical isolates found this assay to be 90.8% concordant to conventional drug susceptibility testing results for the prediction of inducible clarithromycin drug resistance.