A novel hybridization capture method for direct whole genome sequencing of clinical specimens to inform Legionnaires' disease investigations.

The unprecedented precision and resolution of whole genome sequencing (WGS) can provide definitive identification of infectious agents for epidemiological outbreak tracking. WGS approaches, however, are frequently impeded by low pathogen DNA recovery from available primary specimens or unculturable samples. A cost-effective hybrid capture assay for Legionella pneumophila WGS analysis directly on primary specimens was developed. DNA from a diverse range of sputum and autopsy specimens PCR-positive for L. pneumophila serogroup 1 (LPSG1) was enriched with this method, and WGS was performed. All tested specimens were determined to be enriched for Legionella reads (up to 209,000-fold), significantly improving the discriminatory power to compare relatedness when no clinical isolate was available. We found the WGS data from some enriched specimens to differ by less than five single-nucleotide polymorphisms (SNPs) when compared to the WGS data of a matched culture isolate. This testing and analysis retrospectively provided previously unconfirmed links to environmental sources for clinical specimens of sputum and autopsy lung tissue. The latter provided the additional information needed to identify the source of these culture-negative cases associated with the South Bronx 2015 Legionnaires' disease (LD) investigation in New York City. This new method provides a proof of concept for future direct clinical specimen hybrid capture enrichment combined with WGS and bioinformatic analysis during outbreak investigations.IMPORTANCELegionnaires' disease (LD) is a severe and potentially fatal type of pneumonia primarily caused by inhalation of Legionella-contaminated aerosols from man-made water or cooling systems. LD remains extremely underdiagnosed as it is an uncommon form of pneumonia and relies on clinicians including it in the differential and requesting specialized testing. Additionally, it is challenging to obtain clinical lower respiratory specimens from cases with LD, and when available, culture requires specialized media and growth conditions, which are not available in all microbiology laboratories. In the current study, a method for Legionella pneumophila using hybrid capture by RNA baiting was developed, which allowed us to generate sufficient genome resolution from L. pneumophila serogroup 1 PCR-positive clinical specimens. This new approach offers an additional tool for surveillance of future LD outbreaks where isolation of Legionella is not possible and may help solve previously unanswered questions from past LD investigations.

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.

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.

Hazenella coriacea gen. nov., sp. nov., isolated from clinical specimens.

A Gram-staining-positive, endospore-forming rod was isolated independently from clinical specimens in New York State, USA, once in 2009 and twice in 2011. The three isolates had identical 16S rRNA gene sequences and, based on their 16S rRNA gene sequence, are most closely related to the type strains of Laceyella sediminis and L. sacchari (94.6 % similarity). The partial 23S rRNA gene sequences of the three strains were also 100 % identical. Maximum-likelihood phylogenetic analysis suggests that the new isolates belong to the family Thermoactinomycetaceae. Additional biochemical and phenotypic characteristics of the strains support the family designation and suggest that the three isolates represent a single species. In each of the strains, the predominant menaquinone is MK-7, the diagnostic diamino acid is meso-diaminopimelic acid and the major cellular fatty acids are iso-C15 : 0, anteiso-C15 : 0 and iso-C13 : 0. The polar lipids are phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, four unknown phospholipids, four unknown aminophospholipids and an unknown lipid. It is proposed that the novel isolates represent a single novel species within a new genus, for which the name Hazenella coriacea gen. nov., sp. nov. is proposed. The type strain of Hazenella coriacea is strain 23436(T) ( = DSM 45707(T) = LMG 27204(T)).

Sporosarcina newyorkensis sp. nov. from clinical specimens and raw cow's milk.

Twelve independent isolates of a gram-positive, endospore-forming rod were recovered from clinical specimens in New York State, USA, and from raw milk in Flanders, Belgium. The 16S rRNA gene sequences for all isolates were identical. The closest species with a validly published name, based on 16S rRNA gene sequence, is Sporosarcina koreensis (97.13 % similarity). DNA-DNA hybridization studies demonstrate that the new isolates belong to a species distinct from their nearest phylogenetic neighbours. The partial sequences of the 23S rRNA gene for the novel strains and their nearest neighbours also provide support for the novel species designation. Maximum-likelihood phylogenetic analysis of the 16S rRNA gene sequences confirmed that the new isolates are in the genus Sporosarcina. The predominant menaquinone is MK-7, the peptidoglycan has the type A4α L-Lys-Gly-D-Glu, and the polar lipids consist of diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. The predominant fatty acids are iso-C(14 : 0), iso-C(15 : 0) and anteiso-C(15 : 0). In addition, biochemical and morphological analyses support designation of the twelve isolates as representatives of a single new species within the genus Sporosarcina, for which the name Sporosarcina newyorkensis sp. nov. (type strain 6062(T)  = DSM 23544(T)  = CCUG 59649(T)  = LMG 26022(T)) is proposed.

Comparative analysis of multiplexed PCR and short- and long-read whole genome sequencing to investigate a large Klebsiella pneumoniae outbreak in New York State.

In 2017, the New York State Department of Health investigated a large Klebsiella pneumoniae outbreak in a health care facility. A retrospective analysis was conducted to compare the use of multiple molecular typing methods for characterizing the outbreak. Forty-four isolates were characterized using the rapid real-time PCR OpGen Acuitas® AMR Gene Panel. Additionally, short-read whole genome sequencing (WGS) analysis was used to identify antimicrobial resistance (AMR) genes and assess isolate relatedness. Long-read Oxford Nanopore MinION WGS was used to characterize the plasmid content of a subset of isolates. All methods showed overall concordance, identifying four clusters, with a few discrepancies in the clustering of individual isolates. Though short- and long-read WGS results provided a more nuanced understanding of the molecular epidemiology of this outbreak, this study highlights the utility of the Acuitas® PCR-based approach, which can more easily be performed by health care facilities, for rapid clustering of patient isolates.

Neisseria wadsworthii sp. nov. and Neisseria shayeganii sp. nov., isolated from clinical specimens.

An analysis of 16S rRNA gene sequences from archived clinical reference specimens has identified two novel Neisseria species. For each species, two strains from independent sources were identified. Amongst species with validly published names, the closest species to the newly identified organisms were Neisseria canis, N. dentiae, N. zoodegmatis, N. animaloris and N. weaveri. DNA-DNA hybridization studies demonstrated that the newly identified isolates represent species that are distinct from these nearest neighbours. Analysis of partial 23S rRNA gene sequences for the newly identified strains and their nearest neighbours provided additional support for the species designation. Bayesian analysis of 16S rRNA gene sequences suggested that the newly identified isolates belong to distinct but related species of the genus Neisseria, and are members of a clade that includes N. dentiae, N. bacilliformis and N. canis. The predominant cellular fatty acids [16 : 0, summed feature 3 (16 : 1ω7c and/or iso-15 : 0 2-OH) and 18 : 1ω7c], as well as biochemical and morphological analyses further support the designation of Neisseria wadsworthii sp. nov. (type strain 9715(T) =DSM 22247(T) =CIP 109934(T)) and Neisseria shayeganii sp. nov. (type strain 871(T) =DSM 22246(T) =CIP 109933(T)).

Design and implementation of a protocol for the detection of Legionella in clinical and environmental samples.

Our laboratory has developed a novel real-time polymerase chain reaction (PCR) assay for the detection of Legionella pneumophila and differentiation from other Legionella spp. in clinical and environmental samples. The 23S rRNA gene was used as a target to detect all Legionella spp., and the mip gene was targeted for the specific detection of L. pneumophila in this multiplex Taqman real-time PCR assay. The 23S rRNA gene is a novel target for Legionella testing; it detects all species and serogroups of Legionella without the contamination issues that accompany the use of the 16S rRNA gene as a target. This assay provides an analytical sensitivity of <1 colony-forming unit and a specificity of 100%. Because culture is important and provides a means for molecular typing via pulsed-field gel electrophoresis (PFGE), we developed a testing algorithm that includes both the new real-time PCR assay and culture for clinical and environmental samples and applied this algorithm during a period of 3 years. Of the 64 clinical samples received by our laboratory for Legionella testing during this period, PCR was found to be an essential diagnostic tool because only 13.3% (2/15) clinical samples that were determined to be L. pneumophila were detected by culture during this period. Of the 276 environmental samples received for Legionella testing during this period, 140 were found to be positive for L. pneumophila. Of these 140 samples, 69.3% were detected by both PCR and culture methods, 29.3% were positive by PCR alone, and 1.4% were positive by culture methods alone. We feel these results indicate that our algorithm, including both PCR and culture, should be used for environmental samples. Among both the clinical and environmental Legionella samples identified by PCR, a subset was not suitable for culture because of issues of lengthy transport, antimicrobial treatment, or bacterial overgrowth. Samples like these are commonly submitted to our laboratory, so the use of our testing algorithm combining these methods is critical. We conclude that molecular and culture methods must be used in combination to provide the best and most comprehensive approach to laboratory detection and investigation of legionellosis.

Complete Genome Sequences of Bordetella pertussis Isolates with Novel Pertactin-Deficient Deletions.

Clinical isolates of the respiratory pathogen Bordetella pertussis in the United States have become predominantly deficient for the acellular vaccine immunogen pertactin through various independent mutations. Here, we report the complete genome sequences for four B. pertussis isolates that harbor novel deletions responsible for pertactin deficiency.