Mycobacterium abscessus (Mab), a rapidly growing mycobacterial species with intrinsic and acquired resistance to multiple antibiotics, is an emerging public health concern. The rise in clinical cases of treatment-refractory infections of M. abscessus has propelled its research toward novel therapeutic approaches. The number of publications entitled “ Mycobacterium abscessus” has increased by ~300% over the last decade, of which the majority of studies exploring the fundamental biology and pathogenesis of Mab have used the reference strain ATCC19977. However, whole-genome sequence analyses, combined with transposon-seq based functional genomics, reveal an open pan-genome with significant variations in the essential genes across ATCC19977 and clinical isolates. These new discoveries demand a careful selection of strains and growth conditions in experimental design. In this minireview, we discuss these challenges and propose a framework for future M. abscessus studies in silico , including a new web-based resource for pangenome analysis, in vitro, and in animal models.

Mycobacterium abscessus infections are frequently drug resistant and can cause severe disease, particularly among vulnerable populations in healthcare settings. In June 2024, a cluster of M. abscessus infection cases was reported at a hospital in Maryland, USA. An investigation involving hospital infection prevention staff, the Maryland Department of Health, the Centers for Disease Control and Prevention, and the Wadsworth Laboratory of the New York State Department of Health included initiation of mitigation efforts such as tap water use restrictions, point-of-use water filters, hospitalwide water flushing, and staff education regarding sink safety. Whole-genome sequencing of patient and environmental isolates, coupled with epidemiologic data, revealed a strong association (0-24 single-nucleotide polymorphism differences) between environmental and patient samples, indicating that infections were likely a result of patient exposure to M. abscessus. Ensuring initiation of mitigation initiatives likely prevented additional patient infections. Although effective in this investigation, not all mitigation strategies are equal or sustainable.

Isoniazid (INH) is a critical antibiotic used worldwide for the treatment and prophylaxis of tuberculosis. Drug resistance (DR) to INH is the single most common type of DR, mediated by multiple genes/loci, including katG, inhA, mabA, mabA-inhA, and the oxyR-ahpC intergenic region. Over the course of 6 years, we performed a two-phase study of 3,696 Mycobacterium tuberculosis complex (MTBC) strains, aiming to determine the molecular basis of INH resistance and assess whole-genome sequencing (WGS) for predicting resistance. In phase 1, we performed a side-by-side study, including 1,767 strains with paired phenotypic drug susceptibility testing (DST) and genotypic DST. We found WGS capable of accurately predicting INH resistance with a sensitivity of 90.3% and a specificity of 99.8%. The negative predictive value of WGS for INH susceptibility was 98.8%. Based on these findings, we developed a molecular testing algorithm where phenotypic DST (pDST) was reduced and applied this new testing algorithm in phase 2 to 1,929 MTBC strains, resulting in streamlined testing, reduced cost, and decreased turnaround time (TAT). The prevalence of INH resistance among MTBC strains in New York was found to be 10.2%. Of the 3,696 isolates tested, 337 were predicted INH-resistant by WGS. Of 41 additional strains exhibiting phenotypic INH resistance, 38 were found to have mutations in genes known to be associated with INH resistance. This study demonstrates the utility of WGS as a molecular tool for predicting INH DR and shows that the vast majority of INH resistance in MTBC has a molecular basis in known resistance loci. IMPORTANCE:Isoniazid (INH) is one of the two most critical antibiotics used as part of standard treatment of tuberculosis and is also used as preventative therapy for contacts of tuberculosis patients, despite having a higher rate of drug resistance than all other antibiotics used in standard therapy. Furthermore, isoniazid resistance typically precedes rifampin resistance in the development of multidrug-resistant TB. As such, the reliable detection of INH resistance is crucial for case management and to limit the acquisition of additional drug resistance. The present study describes a whole-genome sequencing approach to predicting INH resistance from clinical isolates and models how this technology can be used within a reduced phenotypic drug susceptibility testing algorithm to limit duplicate testing, saving resources and time while maintaining the sensitivity of resistance detection.

Clostridia that produce neurotoxins are highly relevant organisms to public health. While cases of botulism [caused by C. botulinum and other organisms that produce botulinum neurotoxin (BoNT)] are rare, the severity of this disease necessitates robust epidemiologic surveillance to promptly identify and mitigate outbreaks. Next generation sequencing (NGS) can provide additional support to these investigations through single nucleotide polymorphism (SNP)-based analysis, phylogenetic reconstruction, toxin subtyping, and structural analysis. Until recently, testing for this disease was restricted to traditional culture or molecular methods such as polymerase chain reaction (PCR) to detect bont genes, while mouse bioassay and endopeptidase-mass spectrometry (Endopep-MS) methods confirmed the presence of enzymatically active toxin. The New York State Department of Health (NYSDOH) Wadsworth Center Biodefense Laboratory performed a retrospective whole genome sequence (WGS) analysis of approximately 240 Clostridium spp. isolates from the past 40 years to supplement traditional test results and further characterize these organisms. Genomic analyses identified seven BoNT serotypes/serotype combinations, including A4(B5), A5(B2’), and B5F2 that were uncharacteristic of samples typically received. Additionally, SNP-based analysis and de novo genome assemblies retrospectively validated several epidemiology links or differentiated samples previously tested with only traditional methods. Our work highlights the clinical utility of supplementing conventional data with NGS to further characterize BoNT-producing organisms and underscores the importance of incorporating WGS into laboratory workflows to support epidemiologic investigations. However, several obstacles still exist which may prevent implementation. These include the expertise needed to execute bioinformatic analyses and interpret the resulting data, a lack of standardized bioinformatic workflows, and difficulty in determining SNP-based thresholds to identify linked samples without incorporation of additional data and analyses. Supplementing or replacing short-read sequencing with long-read sequencing (LRS) and the use of metagenomic or capture-based enrichment for analysis of primary specimens could increase the leverage obtained from WGS in epidemiological investigations.

ABSTRACT 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. IMPORTANCE Legionnaires’ 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.

Mycobacterium tuberculosis complex (MTBC) infections are treated with combinations of antibiotics; however, these regimens are not as efficacious against multidrug and extensively drug resistant MTBC. Phenotypic (growth-based) drug susceptibility testing on slow growing bacteria like MTBC requires many weeks to months to complete, whereas sequencing-based approaches can predict drug resistance (DR) with reduced turnaround time. We sought to develop a multiplexed, targeted next generation sequencing (tNGS) assay that can predict DR and can be performed directly on clinical respiratory specimens. A multiplex PCR was designed to amplify a group of thirteen full-length genes and promoter regions with mutations known to be involved in resistance to first- and second-line MTBC drugs. Long-read amplicon libraries were sequenced with Oxford Nanopore Technologies platforms and high-confidence resistance mutations were identified in real-time using an in-house developed bioinformatics pipeline. Sensitivity, specificity, reproducibility, and accuracy of the tNGS assay was assessed as part of a clinical validation study. In total, tNGS was performed on 72 primary specimens and 55 MTBC-positive cultures and results were compared to clinical whole genome sequencing (WGS) performed on paired patient cultures. Complete or partial susceptibility profiles were generated from 82% of smear positive primary specimens and the resistance mutations identified by tNGS were 100% concordant with WGS. In addition to performing tNGS on primary clinical samples, this assay can be used to sequence MTBC cultures mixed with other mycobacterial species that would not yield WGS results. The assay can be effectively implemented in a clinical/diagnostic laboratory with a two to three day turnaround time and, even if batched weekly, tNGS results are available on average 15 days earlier than culture-derived WGS results. This study demonstrates that tNGS can reliably predict MTBC drug resistance directly from clinical specimens or cultures and provide critical information in a timely manner for the appropriate treatment of patients with DR tuberculosis.

Lyme disease is the most common vector-borne disease in North America and Europe. The clinical manifestations of Lyme disease vary based on the genospecies of the infecting Borrelia burgdorferi spirochete, but the microbial genetic elements underlying these associations are not known. Here, we report the whole genome sequence (WGS) and analysis of 299 B. burgdorferi (Bb) isolates derived from patients in the Eastern and Midwestern US and Central Europe. We develop a WGS-based classification of Bb isolates, confirm and extend the findings of previous single- and multi-locus typing systems, define the plasmid profiles of human-infectious Bb isolates, annotate the core and strain-variable surface lipoproteome, and identify loci associated with disseminated infection. A core genome consisting of ~900 open reading frames and a core set of plasmids consisting of lp17, lp25, lp36, lp28-3, lp28-4, lp54, and cp26 are found in nearly all isolates. Strain-variable (accessory) plasmids and genes correlate strongly with phylogeny. Using genetic association study methods, we identify an accessory genome signature associated with dissemination in humans and define the individual plasmids and genes that make up this signature. Strains within the RST1/WGS A subgroup, particularly a subset marked by the OspC type A genotype, have increased rates of dissemination in humans. OspC type A strains possess a unique set of strongly linked genetic elements including the presence of lp56 and lp28-1 plasmids and a cluster of genes that may contribute to their enhanced virulence compared to other genotypes. These features of OspC type A strains reflect a broader paradigm across Bb isolates, in which near-clonal genotypes are defined by strain-specific clusters of linked genetic elements, particularly those encoding surface-exposed lipoproteins. These clusters of genes are maintained by strain-specific patterns of plasmid occupancy and are associated with the probability of invasive infection.

Genome-scale analyses have revealed many transcription factor binding sites within, rather than upstream of, genes, raising questions as to the function of these binding sites. Here, we use complementary approaches to map the regulon of the Escherichia coli transcription factor PhoB, a response regulator that controls transcription of genes involved in phosphate homeostasis. Strikingly, the majority of PhoB binding sites are located within genes, but these intragenic sites are not associated with detectable transcription regulation and are not evolutionarily conserved. Many intragenic PhoB sites are located in regions bound by H-NS, likely due to shared sequence preferences of PhoB and H-NS. However, these PhoB binding sites are not associated with transcription regulation even in the absence of H-NS. We propose that for many transcription factors, including PhoB, binding sites not associated with promoter sequences are transcriptionally inert and hence are tolerated as genomic "noise."IMPORTANCE Recent studies have revealed large numbers of transcription factor binding sites within the genes of bacteria. The function, if any, of the vast majority of these binding sites has not been investigated. Here, we map the binding of the transcription factor PhoB across the Escherichia coli genome, revealing that the majority of PhoB binding sites are within genes. We show that PhoB binding sites within genes are not associated with regulation of the overlapping genes. Indeed, our data suggest that bacteria tolerate the presence of large numbers of nonregulatory, intragenic binding sites for transcription factors and that these binding sites are not under selective pressure.

Whole-genome sequencing (WGS) can predict drug resistance and antimicrobial susceptibility in Mycobacterium tuberculosis complex (MTBC) and has shown promise in partially replacing culture-based phenotypic drug susceptibility testing (pDST). We performed a two-year side by side study comparing the prediction of drug resistance and antimicrobial susceptibility by WGS molecular DST (mDST) to pDST to determine resistance at the critical concentration by Mycobacterial Growth Indicator Tube (MGIT) and agar proportion testing. Negative predictive values of WGS results were consistently high for the first-line drugs: rifampin (99.9%), isoniazid (99.0%), pyrazinamide (98.5%), and ethambutol (99.8%); the rates of resistance to these drugs, among strains in our population, are 2.9%, 10.4%, 46.3%, and 2.3%, respectively. WGS results were available an average 8 days earlier than first-line MGIT pDST. Based on these findings, we implemented a new testing algorithm with an updated WGS workflow in which strains predicted pan-susceptible were no longer tested by pDST. This algorithm was applied to 1177 isolates between October 2018 and September 2020, eliminating pDST for 66.6% of samples and reducing pDST for an additional 22.0%. This algorithm change resulted in faster turnaround times and decreased cost while maintaining comprehensive antimicrobial susceptibility profiles of all culture-positive MTBC cases in New York.

Lyme disease is the most common vector-borne disease in North America and Europe. The clinical manifestations of Lyme disease vary based on the genospecies of the infecting Borrelia burgdorferi spirochete, but the microbial genetic elements underlying these associations are not known. Here, we report the whole genome sequence (WGS) and analysis of 299 patient-derived B. burgdorferi sensu stricto ( Bbss ) isolates from patients in the Eastern and Midwestern US and Central Europe. We develop a WGS-based classification of Bbss isolates, confirm and extend the findings of previous single- and multi-locus typing systems, define the plasmid profiles of human-infectious Bbss isolates, annotate the core and strain-variable surface lipoproteome, and identify loci associated with disseminated infection. A core genome consisting of ∼800 open reading frames and a core set of plasmids consisting of lp17, lp25, lp36, lp28-3, lp28-4, lp54, and cp26 are found in nearly all isolates. Strain-variable (accessory) plasmids and genes correlate strongly with phylogeny. Using genetic association study methods, we identify an accessory genome signature associated with dissemination and define the individual plasmids and genes that make up this signature. Strains within the RST1/WGS A subgroup, particularly a subset marked by the OspC type A genotype, are associated with increased rates of dissemination. OspC type A strains possess a unique constellation of strongly linked genetic changes including the presence of lp56 and lp28-1 plasmids and a cluster of genes that may contribute to their enhanced virulence compared to other genotypes. The patterns of OspC type A strains typify a broader paradigm across Bbss isolates, in which genetic structure is defined by correlated groups of strain-variable genes located predominantly on plasmids, particularly for expression of surface-exposed lipoproteins. These clusters of genes are inherited in blocks through strain-specific patterns of plasmid occupancy and are associated with the probability of invasive infection.

We report the unusual genotypic characterization of a bacterium isolated from a clinical sample of a patient who grew up in Bangladesh and lives in the United States. Using whole-genome sequencing, we identified the bacterium as a member of the Mycobacterium tuberculosis complex (MTBC). Phylogenetic placement of this strain suggests a new MTBC genotype. Even though it had the same spoligotype as M. caprae strains, single-nucleotide polymorphism–based phylogenetic analysis placed the isolate as a sister lineage distinct from M. caprae, most closely related to 5 previously sequenced genomes isolated from primates and elephants in Asia. We propose a new animal-associated lineage, La4, within MTBC.

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.

Nontuberculous mycobacteria (NTM) are environmental bacteria commonly found in soil and water in almost every part of the world. While usually non-pathogenic, they can cause acute respiratory and cutaneous infections under certain circumstances or in patients with underlying medical conditions. Contrary to members of the Mycobacterium tuberculosis complex, documented human-to-human transmissions of NTM have been rarely reported and most cases result from direct environmental exposure. Here we describe the identification of a new NTM species isolated from a hand laceration of a New York State patient after a fall. This new NTM forms rough, orange pigmented colonies and is naturally resistant to doxycycline and tobramycin. Whole genome analysis reveal no close relatives present in public databases, and our findings are in accordance with the recognition of a new taxonomic species of NTM. We propose the name Mycobacterium salfingeri sp. nov. for this new NTM representative. The type strain is 20-157661T (DSM = 113368T, BCCM = ITM 501207T).

Additional file 3: Table S3. Functional categories of DEGs.

Background Transmission of pathogens by vector mosquitoes is intrinsically linked with mosquito’s reproductive strategy because anautogenous mosquitoes require vertebrate blood to develop a batch of eggs. Each cycle of egg maturation is tightly linked with the intake of a fresh blood meal for most species. Mosquitoes that acquire pathogens during the first blood feeding can transmit the pathogens to susceptible hosts during subsequent blood feeding and also vertically to the next generation via infected eggs. Large-scale gene-expression changes occur following each blood meal in various tissues, including ovaries. Here we analyzed mosquito ovary transcriptome following a blood meal at three different time points to investigate blood-meal induced changes in gene expression in mosquito ovaries. Results We collected ovaries from Aedes aegypti that received a sugar meal or a blood meal on days 3, 10 and 20 post blood meal for transcriptome analysis. Over 4000 genes responded differentially following ingestion of a blood meal on day 3, and 660 and 780 genes on days 10 and 20, respectively. Proteins encoded by differentially expressed genes (DEGs) on day 3 include odorant binding proteins (OBPs), defense-specific proteins, and cytochrome P450 detoxification enzymes. In addition, we identified 580 long non-coding RNAs that are differentially expressed at three time points. Gene ontology analysis indicated that genes involved in peptidase activity, oxidoreductase activity, extracellular space, and hydrolase activity, among others were enriched on day 3. Although most of the DEGs returned to the nonsignificant level compared to the sugar-fed mosquito ovaries following oviposition on days 10 and 20, there remained differences in the gene expression pattern in sugar-fed and blood-fed mosquitoes. Conclusions Enrichment of OBPs following blood meal ingestion suggests that these genes may have other functions besides being part of the olfactory system. The enrichment of immune-specific genes and cytochrome P450 genes indicates that ovaries become well prepared to protect their germ line from any pathogens that may accompany the blood meal or from environmental contamination during oviposition, and to deal with the detrimental effects of toxic metabolites.

Additional file 2: Table S2. Significant DEGs between BF and SF samples at three different time points.

Since 1978, the New York State Department of Health's public health laboratory, Wadsworth Center (WC), in collaboration with epidemiology and environmental partners, has been committed to providing comprehensive public health testing for Legionella in New York. Statewide, clinical case counts have been increasing over time, with the highest numbers identified in 2017 and 2018 (1,022 and 1,426, respectively). Over the course of more than 40 years, the WC Legionella testing program has continuously implemented improved testing methods. The methods utilized have transitioned from solely culture-based methods for organism recovery to development of a suite of reference testing services, including identification and characterization by PCR and pulsed-field gel electrophoresis (PFGE). In the last decade, whole-genome sequencing (WGS) has further refined the ability to link outbreak strains between clinical specimens and environmental samples. Here, we review Legionnaires' disease outbreak investigations during this time period, including comprehensive testing of both clinical and environmental samples. Between 1978 and 2017, 60 outbreaks involving clinical and environmental isolates with matching PFGE patterns were detected in 49 facilities from the 157 investigations at 146 facilities. However, 97 investigations were not solved due to the lack of clinical or environmental isolates or PFGE matches. We found 69% of patient specimens from New York State (NYS) were outbreak associated, a much higher rate than observed in other published reports. The consistent application of new cutting-edge technologies and environmental regulations has resulted in successful investigations resulting in remediation efforts. IMPORTANCE Legionella, the causative agent of Legionnaires' disease (LD), can cause severe respiratory illness. In 2018, there were nearly 10,000 cases of LD reported in the United States (https://www.cdc.gov/legionella/fastfacts.html; https://wonder.cdc.gov/nndss/static/2018/annual/2018-table2h.html), with actual incidence believed to be much higher. About 10% of patients with LD will die, and as high as 90% of patients diagnosed will be hospitalized. As Legionella is spread predominantly through engineered building water systems, identifying sources of outbreaks by assessing environmental sources is key to preventing further cases LD.

Carbapenemase-producingEnterobacteriaceaeare a major threat to global public health.Klebsiella pneumoniaecarbapenemase (KPC) is the most commonly identified carbapenemase in the United States and is frequently found on mobile genetic elements including plasmids, which can be horizontally transmitted between bacteria of the same or different species. Here we describe the results of an epidemiological investigation of KPC-producing bacteria at two healthcare facilities. Using a combination of short-read and long-read whole-genome sequencing, we identified an identical 44 kilobase plasmid carrying thebla(KPC-2)gene in four bacterial isolates belonging to three different species (Citrobacter freundii,Klebsiella pneumoniae, andEscherichia coli). The isolates in this investigation were collected from patients who were epidemiologically linked in a region in which KPC was uncommon, suggesting that the antibiotic resistance plasmid was transmitted between these bacterial species. This investigation highlights the importance of long-read sequencing in investigating the relatedness of bacterial plasmids, and in elucidating potential plasmid-mediated outbreaks caused by antibiotic resistant bacteria.