Pairwise Single Nucleotide Polymorphism Research Articles

Mycobacterium immunogenum acquisition from hospital tap water: a genomic and epidemiologic analysis.

We identified 23 cases of Mycobacterium immunogenum respiratory acquisition linked to a colonized plumbing system at a new hospital addition. We conducted a genomic and epidemiologic investigation to assess for clonal acquisition of M. immunogenum from hospital water sources and improve understanding of genetic distances between M. immunogenum isolates. We performed whole-genome sequencing on 28 M. immunogenum isolates obtained from August 2013 to July 2021 from patients and water sources on four intensive care and intermediate units at an academic hospital. Study hospital isolates were recovered from 23 patients who experienced de novo respiratory isolation of M. immunogenum and from biofilms obtained from five tap water outlets. We also analyzed 10 M. immunogenum genomes from previously sequenced clinical (n = 7) and environmental (n = 3) external control isolates. The 38-isolate cohort clustered into three clades with pairwise single-nucleotide polymorphism (SNP) distances ranging from 0 to 106,697 SNPs. We identified two clusters of study hospital isolates in Clade 1 and one cluster in Clade 2 for which clinical and environmental isolates differed by fewer than 10 SNPs and had less than 0.5% accessory genome variation. A less restrictive combined threshold of 40 SNPs and 5% accessory genes reliably captured additional isolates that met clinical criteria for hospital acquisition, but 12 (4%) of 310 epidemiologically unrelated isolate pairs also met this threshold. Core and accessory genome analyses confirmed respiratory acquisition of multiple clones of M. immunogenum from hospital water sources to patients. When combined with epidemiologic investigation, genomic thresholds accurately distinguished hospital acquisition.

Increase in invasive group A streptococcal infections in Milan, Italy: a genomic and clinical characterization.

Group A Streptococcus (GAS) causes multiple clinical manifestations, including invasive (iGAS) or even life-threatening (severe-iGAS) infections. After the drop in cases during COVID-19 pandemic, in 2022 a sharp increase of GAS was reported globally. GAS strains collected in 09/2022-03/2023 in two university hospitals in Milan, Italy were retrospectively analyzed. Clinical/epidemiological data were combined with whole-genome sequencing to: (i) define resistome/virulome, (ii) identify putative transmission chains, (iii) explore associations between emm-types and clinical severity. Twenty-eight isolates were available, 19/28 (67.9%) from adults and 9/28 (32.1%) from pediatric population. The criteria for iGAS were met by 19/28 cases (67.9%), of which 11/19 (39.3%) met the further criteria for severe-iGAS. Pediatric cases were mainly non-invasive infections (8/9, 88.9%), adult cases were iGAS and severe-iGAS in 18/19 (94.7%) and 10/19 (52.6%), respectively. Thirteen emm-types were detected, the most prevalent being emm1 and emm12 (6/28 strains each, 21.4%). Single nucleotide polymorphism (SNP) analysis of emm1.0 and emm12.0 strains revealed pairwise SNP distance always >10, inconsistent with unique transmission chains. Emm12.0-type, found to almost exclusively carry virulence factors speH and speI, was mainly detected in children and in no-iGAS infections (55.6 vs. 5.3%, p = 0.007 and 66.7 vs. 0.0%, p < 0.001, respectively), while emm1.0-type was mainly detected in severe-iGAS (0.0 vs. 45.5%, p = 0.045). This study showed that multiple emm-types contributed to a 2022/2023 GAS infection increase in two hospitals in Milan, with no evidence of direct transmission chains. Specific emm-types could be associated with disease severity or invasiveness. Overall, these results support the integration of classical epidemiological studies with genomic investigation to appropriately manage severe infections and improve surveillance.

Insidious transmission of Mycobacterium tuberculosis in Ordos, China: a molecular epidemiology study.

In this study, we conducted this population-based study to evaluate the genetic diversity and clustering rate of Mycobacterium tuberculosis (MTB) strains using the whole-genome sequencing (WGS), to better understand its transmission in Ordos. All patients with culture-positive TB notified in Ordos from January 2021 to December 2022 were recruited. WGS was performed to analyze single-nucleotide polymorphism (SNP) and to identify genotypic drug susceptibilities of MTB isolates. Overall, a total of 186 patients were included in the present study, of whom 35 (18.8%) had no symptoms suggestive of active TB. Lineage 2 was the predominant MTB sublineage, accounting for 186 of isolates tested. When the pairwise SNP difference ≤ 12 was used as the cutoff for WGS-based clusters, we identified 17 genotypic clusters, and 38 isolates belonged to these 17 clusters, resulting in a clustering rate of 20.4%. The Beijing genotype was an independent factor associating with genomic-clustering (adjusted OR 4.219, 95% CI 0.962-18.502). The overall sensitivity on WGS-based resistance prediction was 85.7% for rifampicin, 73.1% for isoniazid, 60.0% for Ethambutol, 72.7% for streptomycin, and 72.7% for fluoroquinolones. To conclude, the present study demonstrates the extensive recent transmission of Beijing genotype strains in the community of Ordos. The failure to provide a comprehensive pattern of transmission indicated the missed diagnosis of active TB within the community. A substantial proportion of subclinical TB cases are recognized in the bacteria-positive cases, emphasizing that we must interrupt transmission by finding people with active TB before they infect others.

Transmission dynamics variability of lineage 2 Mycobacterium tuberculosis strains in Kobe, Japan, determined using population-based whole-genome sequencing analysis.

Currently, tuberculosis (TB) in Japan is highly prevalent among elderly patients who were born during a time when TB was highly prevalent. Mycobacterium tuberculosis (Mtb) lineage 2 (L2) is the predominant strain in the country. Moreover, the proportion of foreign-born patients with TB has been increasing. This epidemiological situation in Japan motivated us to explore the heterogeneity in transmission dynamics among the sublineages of Mtb L2 within this aging population. For this purpose, we conducted a population-based whole genome sequencing analysis of 550 Mtb strains in Kobe, Japan, and employed pairwise single nucleotide polymorphism (SNP) distance clustering and terminal branch length (TBL) distribution analysis to assess Mtb transmission. The genomic clustering rate with a threshold of ≤5 SNPs was significantly lower in elderly patients aged 70years or higher than in non-elderly patients. The elderly patient group showed significantly longer TBL than the non-elderly group. These results supported the notion that reactivation of distant infection is a major driving force for the high incidence of TB in elderly individuals. The age group distribution and frequency of lineages/sublineages were found to significantly differ between foreign-born and Japan-born patients. The increased proportion of foreign-born patients might have resulted in more strain diversity in Japan. The L2.2.A sublineage demonstrated a significant association with elderly patients and exhibited lower transmission rates, which indicate to be prone to reactivate from long-term latency. In contrast, L2.2.Modern, showed a strong association with younger and foreign-born patients. This sublineage showed a high genomic cluster rate, suggesting its high transmissibility. The other three major sublineages, namely L2.2.AA2, L2.2.AA3.1, and L2.2.AA3.2, exhibited a consistent increase in cluster rates across varying SNP thresholds, indicating their relatively recent emergence as endemic sublineages in Japan. In conclusion, this study highlights distinct differences in the transmission dynamics of L2 sublineages within an aging society.

Genome-wide case-only analysis of gene-gene interactions with known Parkinson’s disease risk variants reveals link between LRRK2 and SYT10

The effects of one genetic factor upon Parkinson’s disease (PD) risk may be modified by other genetic factors. Such gene-gene interaction (G×G) could explain some of the ‘missing heritability’ of PD and the reduced penetrance of known PD risk variants. Using the largest single nucleotide polymorphism (SNP) genotype data set currently available for PD (18,688 patients), provided by the International Parkinson’s Disease Genomics Consortium, we studied G×G with a case-only (CO) design. To this end, we paired each of 90 SNPs previously reported to be associated with PD with one of 7.8 million quality-controlled SNPs from a genome-wide panel. Support of any putative G×G interactions found was sought by the analysis of independent genotype-phenotype and experimental data. A total of 116 significant pairwise SNP genotype associations were identified in PD cases, pointing towards G×G. The most prominent associations involved a region on chromosome 12q containing SNP rs76904798, which is a non-coding variant of the LRRK2 gene. It yielded the lowest interaction p-value overall with SNP rs1007709 in the promoter region of the SYT10 gene (interaction OR = 1.80, 95% CI: 1.65–1.95, p = 2.7 × 10−43). SNPs around SYT10 were also associated with the age-at-onset of PD in an independent cohort of carriers of LRRK2 mutation p.G2019S. Moreover, SYT10 gene expression during neuronal development was found to differ between cells from affected and non-affected p.G2019S carriers. G×G interaction on PD risk, involving the LRRK2 and SYT10 gene regions, is biologically plausible owing to the known link between PD and LRRK2, its involvement in neural plasticity, and the contribution of SYT10 to the exocytosis of secretory vesicles in neurons.

Whole-genome sequencing of carbapenem-resistant Enterobacterales isolates and evaluation of hospital-acquired infections

Background: Multidrug-resistant organisms (MDROs) are increasingly implicated in nosocomial outbreaks worldwide. We evaluated whole-genome sequencing (WGS) as an adjunctive epidemiological tool to identify infection clusters and MDRO transmission in the healthcare setting. Methods: Clinical isolates of carbapenem-resistant Enterobacterales (CRE) from July 1, 2021, to June 30, 2022, underwent Illumina WGS. Assembled genomes were taxonomically classified with GTDB-Tk software and were typed using multilocus sequence typing (MLST). Average nucleotide identity (ANI) was calculated between genomes. Numbers of differences among core single-nucleotide polymorphisms (SNPs) were calculated for pairs within taxonomic groups, and the data were evaluated in the context of patient dates and locations of care obtained from the electronic medical record. Results: In total, 39 CRE isolates underwent WGS (Fig. 1). Klebsiella pneumoniae represented the largest number of isolates (n = 18). Using MLST, 2 distinct groups of K. pneumoniae were identified (ST307 and ST258) with 5 and 4 isolates, respectively (Fig. 2). Within ST307, SNP differences ranged between 8 and 115. 3 isolates (CRE8, CRE10, and CRE12) were collected within 4 weeks of each other and had ≤26 pairwise SNP differences. Notably, CRE8 and CRE10 were located on the same unit at the same time and used the same MRI scanner on the same day. CRE35 had &gt;95 SNP differences and was admitted 8 months prior to others in ST307 but had surgery in the same OR as CRE8. Within ST258, pairwise comparison of samples revealed 139–588 SNP differences. CRE21, CRE31, and CRE33 had SNP differences of ≤150. These patients were in the same hospital room (CRE33 and CRE21) and unit (CRE31 and CRE33), but they did not overlap temporally. CRE37 had &gt;580 SNP differences, with no overlap in hospitalization dates or locations with other patients. Conclusions: Two closely related K. pneumoniae isolate populations were identified using WGS. Strong temporal and spatial commonalities were identified among isolates with few SNP differences. Isolate pairs with intermediate SNP differences shared spatial commonalities, suggesting possible indirect transmission between patients. No common exposures were identified for pairs with large numbers of SNP differences. WGS is an evolving tool to detect outbreak clonal populations of MDRO not identified through traditional epidemiologic techniques. WGS can provide insight into transmission patterns and the role of environmental contamination in propagating these nosocomial infections. More studies are needed to define the role and clinical significance of isolates with intermediate SNP differences in transmission of these pathogens between hosts and the healthcare environment.Disclosures: None

Worldwide transmission of ST11-KL64 carbapenem-resistant Klebsiella pneumoniae: an analysis of publicly available genomes.

ST11-KL64 is an internationally distributed lineage of carbapenem-resistant Klebsiella pneumoniae and is the most common type in China. The international and interprovincial (in China) transmission of ST11-KL64 CRKP remains to be elucidated. We used both static clusters defined based on a fixed cutoff of ≤21 pairwise single-nucleotide polymorphisms and dynamic groups defined by modeling the likelihood to be linked by a transmission threshold to investigate the transmission of ST11-KL64 strains based on genome sequences mining. We analyzed all publicly available genomes (n = 730) of ST11-KL64 strains, almost all of which had known carbapenemase genes with KPC-2 being dominant. We identified 4 clusters of international transmission and 14 clusters of interprovincial transmission across China of ST11-KL64 strains. We found that dynamic grouping could provide further resolution for determining clonal relatedness in addition to the widely adopted static clustering and therefore increases the confidence for inferring transmission.IMPORTANCECarbapenem-resistant Klebsiella pneumoniae (CRKP) is a serious challenge for clinical management and is prone to spread in and between healthcare settings. ST11-KL64 is the dominant CRKP type in China with a worldwide distribution. Here, we used two different methods, the widely used clustering based on a fixed single-nucleotide polymorphism (SNP) cutoff and the recently developed grouping by modeling transmission likelihood, to mine all 730 publicly available ST11-KL64 genomes. We identified international transmission of several strains and interprovincial transmission in China of a few, which warrants further investigations to uncover the mechanisms for their spread. We found that static clustering based on ≤21 fixed SNPs is sensitive to detect transmission and dynamic grouping has higher resolutions to provide complementary information. We suggest the use of the two methods in combination for analyzing transmission of bacterial strains. Our findings highlight the need of coordinated actions at both international and interprovincial levels for tackling multi-drug resistant organisms.

Clonal Dissemination of Antifungal-Resistant Candida haemulonii, China.

Candida haemulonii, a relative of C. auris, frequently shows antifungal resistance and is transmissible. However, molecular tools for genotyping and investigating outbreaks are not yet established. We performed genome-based population analysis on 94 C. haemulonii strains, including 58 isolates from China and 36 other published strains. Phylogenetic analysis revealed that C. haemulonii can be divided into 4 clades. Clade 1 comprised strains from China and other global strains; clades 2-4 contained only isolates from China, were more recently evolved, and showed higher antifungal resistance. Four regional epidemic clusters (A, B, C, and D) were identified in China, each comprising ≥5 cases (largest intracluster pairwise single-nucleotide polymorphism differences <50 bp). Cluster A was identified in 2 hospitals located in the same city, suggesting potential intracity transmissions. Cluster D was resistant to 3 classes of antifungals. The emergence of more resistant phylogenetic clades and regional dissemination of antifungal-resistant C. haemulonii warrants further monitoring.

Genome-wide comparisons reveal broad variations in intraspecific SNP frequencies among species in Agaricomycetes, Basidiomycota

Background:Genome sequence analyses can provide crucial information for understanding population history, speciation, and taxonomy. In Class Agaricomycetes where most mushroom-forming fungi belong, most species so far have been defined based on morphological, ecological, and/or molecular features. At present, there is little information on how species defined based on such features reflect their genome sequence diversity. In this study, we investigated genome-wide single nucleotide polymorphism (SNP) frequencies between strains within species to understand the patterns of variation.Methods:A total of 112 species in 72 genera of Agaricomycetes contained the nuclear and/or mitochondrial genome sequences from at least two strains each in public databases. Together, we obtained 398 and 106 available nuclear and mitochondrial genomes respectively from these taxa. Pairwise strain comparisons of the nuclear and mitochondrial genomes within individual species were conducted to obtain their SNP frequencies.Results:The SNP frequencies between nuclear genomes within individual species ranged 0–7.69% while for the mitochondrial genome, the pairwise strain SNP frequencies ranged 0–4.41%. The Spearman’s non-parametric rank correlation test showed a weak but statistically significant positive correlation between the paired nuclear and mitochondrial genome SNP frequencies. Overall, we observed a significantly higher SNP frequency in the nuclear genome than in the mitochondrial genomes between strains within most species. Interestingly, across the broad Basidiomycetes, the ratios of mitochondrial genome SNPs and nuclear genome SNPs between pairs of strains within each species were almost all lower than 1, with a mean of 0.24.Conclusions:Our analyses revealed broad variations among species in their intraspecific SNP frequencies in both the nuclear and mitochondrial genomes. However, there was broad consensus among the examined species in their mitochondrial to nuclear genome SNP ratios, suggesting that such a ratio could potentially serve as an indicator for genome sequence-based species identification.

1374. Utilizing Genomic Epidemiology to Explore SARS CoV-2 Transmission Patterns and Support Outbreak Investigations in Long Term Care Facilities, Washington State, April-October 2021

Abstract Background In Washington State, COVID-19 cases in long-term care facilities (LTCF) have accounted for less than 3% of all cases, yet 30% of all COVID-19 deaths. Understanding transmission patterns and outbreak epidemiology informs outbreak management. From April to October 2021, two large LTCFs experienced COVID-19 outbreaks. Whole genome sequencing and phylogenetic analysis were leveraged to explore transmission patterns and complement outbreak epidemiology. Methods Epidemiologic data was exported from the Washington Disease Reporting System. Sequences, retrieved from GISAID, were aligned to the Wuhan-1 reference genome using Nextalign version 1.11.0. Pairwise single nucleotide polymorphism (SNP) distance matrices were calculated using SNP-Dists version 0.8.2. Phylogenetic trees for each outbreak were generated using IQ-Tree multicore version 2.2.0-beta COVID-edition using the GTR+F+G4 nucleotide substitution model with 1000 bootstrap replicates. MicrobeTrace was used to visualize the phylogeny, SNP heatmap, and identify clusters among sequences. Results Weekly, LTCF A tested 162 residents and 800 staff, and LTCF B tested 60 residents and 144 staff. Of all cases in LTCF A (n= 119), 23% (n =27) were residents and 77% (n = 92) were staff, compared to 78% (n =28) residents and 22% (n =7) staff among total LTCF B cases (n=36). In LTCF A, 34% (n=40) of the cases had high-quality sequences available. Seven clusters of two or more genetically related sequences and thirteen genetically unrelated sequences were identified. Five of the clusters involved resident and staff cases, linked by unit. Two clusters and remaining unrelated sequences were among staff. In LTCF B, 40% (n=14) of the cases had high-quality sequences available. One cluster of genetically related sequences was identified, all from residents of two floors. The SNP differences between sequences from LTCF A ranged from 0 to 70, whereas SNP differences between LTCF B sequences ranged from 0 to 6. Conclusion Phylogenetic analysis of the two outbreaks confirms differences in disease transmission patterns. Multiple independent introductions of SARS-CoV-2 were identified in LTCF A, compared to a single introduction in LTCF B. Genomic epidemiology is a valuable resource for outbreak investigation and management. Disclosures All Authors: No reported disclosures.

A Comprehensive Genomic Analysis of the Emergent Klebsiella pneumoniae ST16 Lineage: Virulence, Antimicrobial Resistance and a Comparison with the Clinically Relevant ST11 Strain.

Klebsiella pneumoniae is considered an opportunistic pathogen frequently involved with healthcare-associated infections. The genome of K. pneumoniae is versatile, harbors diverse virulence factors and easily acquires and exchanges resistance plasmids, facilitating the emergence of new threatening clones. In the last years, ST16 has been described as an emergent, clinically relevant strain, increasingly associated with outbreaks, and carrying virulence factors (such as ICEKp, iuc, rmpADC/2) and a diversity of resistance genes. However, a far-reaching phylogenetic study of ST16, including geographically, clinically and temporally distributed isolates is not available. In this work, we analyzed all publicly available ST16 K. pneumoniae genomes in terms of virulence factors, including capsular lipopolysaccharide and polysaccharide diversity, plasmids and antimicrobial resistance genes. A core genome SNP analysis shows that less than 1% of studied sites were variant sites, with a median pairwise single nucleotide polymorphism difference of 87 SNPs. The number and diversity of antimicrobial resistance genes, but not of virulence-related genes, increased consistently in ST16 strains during the studied period. A genomic comparison between ST16 and the high-risk clone ST11 K. pneumoniae, showed great similarities in their capacity to acquire resistance and virulence markers, differing mostly in the great diversity of capsular lipopolysaccharide and polysaccharide types in ST11, in comparison with ST16. While virulence and antimicrobial resistance scores indicated that ST11 might still constitute a more difficult-to-manage strain, results presented here demonstrate the great potential of the ST16 clone becoming critical in public health.

Assessing the effect of interaction between gut microbiome and inflammatory bowel disease on the risks of depression

BackgroundGut microbiome and inflammatory bowel disease (IBD) are implicated in the development of depression, but the effect of their interactions on the risk of depression remains unclear. We aim to analyze the effect of interactions between gut microbiome and IBD on the risk of depression, and explore candidate genes involving the interactions. MethodsUsing the individual genotype and depression traits data from the UK Biobank, we calculated the polygenetic risk scores (PRS) of 114 gut microbiome, ulcerative colitis (UC), Crohn's disease (CD), and total IBD (CD + UC) respectively. The effects of interactions between gut microbiome and IBD on depression were assessed through a linear regression model. Moreover, for observed significant interactions between gut microbiome PRS and IBD PRS, PLINK software was used to test pair-wise single nucleotide polymorphisms (SNPs) interaction of corresponding gut microbiome PRS and IBD PRS on depression. ResultsWe found 64 candidate interactions between gut microbiome and IBD on four phenotypes of depression, such as F_Lachnospiraceae (RNT) × (CD + UC) for patient health questionnaire-9 (PHQ-9) score (P = 1.48 × 10−3), F_Veillonellaceae (HB) × UC for self-reported depression (P = 2.83 × 10−3) and P_Firmicutes (RNT) × CD for age at first episode of depression (P = 8.50 × 10−3). We observed interactions of gut-microbiome-associated SNPs × IBD-associated SNPs, such as G_Alloprevotella (HB)-associated rs147650986 (GPM6A) × IBD-associated rs114471990 (QRICH1) (P = 2.26 × 10−4). ConclusionOur results support the effects of interactions between gut microbiome and IBD on depression risk, and reported several novel candidate genes for depression.

One clone to rule them all: Culture-independent genomics of Chlamydia psittaci from equine and avian hosts in Australia.

Chlamydia psittaci is an avian pathogen with zoonotic potential. In Australia, C. psittaci has been well reported as a cause of reproductive loss in mares which subsequently have been the source of infection and illness in some in-contact humans. To date, molecular typing studies describe the predominant and clonal C. psittaci sequence type (ST)24 strains in horse, psittacine, and human infections. We sought to assess the clonality between ST24 strains and the emergence of equine ST24 with a comprehensive genomics approach. We used culture-independent probe-based and metagenomic whole-genome sequencing to investigate 13 C . psittaci genomes from horses, psittacines, and a pigeon from Australia. Published genomes of 36 C . psittaci strains were also used to contextualise our Australian dataset and investigate lineage diversity. We utilised a single-nucleotide polymorphism (SNP) based clustering and multi-locus sequence typing (MLST) approach. C. psittaci has four major phylogenetic groups (PG1-4) based on core-genome SNP-based phylogeny. PG1 contained clonal global and Australian equine, psittacine, and human ST24 genomes, with a median pairwise SNP distance of 68 SNPs. PG2, PG3, and PG4 had greater genomic diversity, including diverse STs collected from birds, livestock, human, and horse hosts from Europe and North America and a racing pigeon from Australia. We show that the clustering of C. psittaci by MLST was congruent with SNP-based phylogeny. The monophyletic ST24 clade has four major sub-lineages. The genomes of 17 Australian human, equine, and psittacine strains collected between 2008 and 2021 formed the predominant ST24 sub-lineage 1 (emerged circa 1979). Despite a temporal distribution of 13 years, the genomes within sub-lineage 1 had a median pairwise SNP distance of 32 SNPs, suggesting a recent population expansion or potential cross-host transmission. However, two C. psittaci genomes collected in 2015 from Victorian parrots clustered into distinct ST24 sub-lineage 4 (emerged circa 1965) with ovine strain C19/98 from Germany. This work describes a comprehensive phylogenomic characterisation of ST24 and identifies a timeline of potential bird-to-equine spillover events.

Clonal Dissemination of Multidrug-Resistant and Hypervirulent Klebsiella pneumoniae Clonal Complex in a Chinese Hospital.

The emergence of high antimicrobial-resistant and hypervirulent Klebsiella pneumoniae (hvKp) clones in clinics has become a cause of concern in recent years. Despite the global spread of the clonal complex (CC) 258, hvKp of other non-CC258 subgroups also emerged. Here, by performing a retrospective study from July 2019 to August 2020 in a Chinese hospital, we obtained 25 K. pneumoniae isolates belonging to CC15. By antimicrobial susceptibility testing and whole genome sequencing and analysis, we obtained the resistant phenotypes and genotypes of these isolates. Twenty-one isolates (84%) were carbapenem-resistant, and eighteen were blaKPC-2 positive. In addition, ten isolates were identified as putative hvKp and seven were carbapenem-resistant hvKp. Nine isolates carried the pLVPK-like virulence plasmid, which contains the fragment including rmpA2, peg-589, iutA, and iucABCD. Another isolate carried iucA. Phylogenetic analysis revealed that the isolates belonged to four lineages, and the putative hvKp isolates were identified in three of these. Two independent sublineages of putative hvKp were caused by the acquisition of pLVPK-like virulence plasmid. Based on comparative genomic analysis, the number of pairwise single nucleotide polymorphisms amongst the four sublineages, Lineage 1a, 1b, 2a, and 2b, were 1–43, 2–13, 129–279, and 3–4, respectively, indicating clonal transmission of Lineage 1a, 1b, and 2b. These results indicate that multiple lineages of CC15 carbapenem-resistant hvKp have emerged in the hospital and caused nosocomial transmission, and that the spreading of virulence plasmids among classic K. pneumoniae subtypes might become more common and happen more easily. These findings highlight the importance of surveillance of local epidemics of non-CC258 subgroups in hospitals.

Molecular epidemiology characteristics and detecting transmission of carbapenemase-producing enterobacterales in southwestern China

BackgroundTo investigate the genotype and clinical characteristics of carbapenem-resistant Enterobacteriaceae (CRE) strains in southwest China and provide information on the treatment stopping the spread of the infection. MethodsThe clinical information of CRE isolates was collected from 19 hospitals in 12 cities across Sichuan Province, China, between June 2018 and April 2019. The isolates were detected by DNA sequencing of genes encoding carbapenem enzymes and multilocus sequence types (MLSTs). ResultsA total of 166 nonrepetitive CRE isolates were isolated during the study period from sputum, blood, urine, and other samples. Klebsiella pneumoniae carbapenemase (KPC) was dominant in Klebsiella pneumoniae (53.9%), followed by New Delhi metallo-β-lactamase (NDM) (42.1%). A total of 43 STs were detected. The most common ST of K. pneumoniae was ST11, and that of Escherichia coli was ST410. Pairwise single nucleotide polymorphism (SNP) distances and the likelihood of local transmission by epidemiology were plotted for each species. About 65% of these pairs had ≤ 20 pairwise SNPs. ConclusionA large number of CRE strains carried carbapenemase. Although NDM-ST12 K. pneumoniae should not be disregarded, KPC-ST11is the predominant strain. Thus, the possibility of transmission between E. coli and K. pneumoniae could not be ignored.

Genomic insights into blaNDM-carrying carbapenem-resistant Klebsiella pneumoniae clinical isolates from a university hospital in Thailand

The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates is a serious threat to global health. Here, we elucidate the genetic features of blaNDM-carrying CRKP clinical isolates from a university hospital in Thailand. The entire genomes of 19 CRKP isolates were extracted and then sequenced using the MGISEQ200 platform. Using various bioinformatics tools, we analyzed the antimicrobial resistance (AMR), virulence factors, gene transfer, bacterial defense mechanisms, and genomic diversity of the CRKP isolates. The sequence type (ST) 16 was found in most of the isolates, along with carriages of the blaNDM-1, blaOXA-232, and blaCTX-M-15 genes. The IncFIB(pQil), Col440II, and ColKP3 plasmids were identified with high frequency. The CRKP isolates harbored genes encoding for virulence factors such as adherence, biofilm formation, immune evasion, and iron uptake. The CRISPR-Cas region in the CRKP9 isolate consisted of 28 distinct spacer sequences. The genomes of the CRKP isolates presented restriction-modification (R-M) sites (M.Kpn34618Dcm and M.Kpn928I) and integrated bacteriophage genomes (Klebsiella phage ST16-OXA48phi5.4 and Enterobacteria phage mEp390). Bottromycin and sactipeptides were also identified. The isolates could be separated into three clades according to STs and pairwise single nucleotide polymorphism (SNP) distance. Pairwise average nucleotide identity (ANI) values revealed intra-species. These findings support the importance of whole-genome sequencing (WGS) to the rapid and accurate genomic analysis of clinical isolates of CRKP.

SAM-TB: a whole genome sequencing data analysis website for detection of Mycobacterium tuberculosis drug resistance and transmission.

Whole genome sequencing (WGS) can provide insight into drug-resistance, transmission chains and the identification of outbreaks, but data analysis remains an obstacle to its routine clinical use. Although several drug-resistance prediction tools have appeared, until now no website integrates drug-resistance prediction with strain genetic relationships and species identification of nontuberculous mycobacteria (NTM). We have established a free, function-rich, user-friendly online platform for MTB WGS data analysis (SAM-TB, http://samtb.szmbzx.com) that integrates drug-resistance prediction for 17 antituberculosis drugs, detection of variants, analysis of genetic relationships and NTM species identification. The accuracy of SAM-TB in predicting drug-resistance was assessed using 3177 sequenced clinical isolates with results of phenotypic drug-susceptibility tests (pDST). Compared to pDST, the sensitivity of SAM-TB for detecting multidrug-resistant tuberculosis was 93.9% [95% confidence interval (CI) 92.6–95.1%] with specificity of 96.2% (95% CI 95.2–97.1%). SAM-TB also analyzes the genetic relationships between multiple strains by reconstructing phylogenetic trees and calculating pairwise single nucleotide polymorphism (SNP) distances to identify genomic clusters. The incorporated mlstverse software identifies NTM species with an accuracy of 98.2% and Kraken2 software can detect mixed MTB and NTM samples. SAM-TB also has the capacity to share both sequence data and analysis between users. SAM-TB is a multifunctional integrated website that uses WGS raw data to accurately predict antituberculosis drug-resistance profiles, analyze genetic relationships between multiple strains and identify NTM species and mixed samples containing both NTM and MTB. SAM-TB is a useful tool for guiding both treatment and epidemiological investigation.

Mutation of Mycobacterium tuberculosis and Implications for Using Whole-Genome Sequencing for Investigating Recent Tuberculosis Transmission.

Tuberculosis (TB) control programs use whole-genome sequencing (WGS) of Mycobacterium tuberculosis (Mtb) for detecting and investigating TB case clusters. Existence of few genomic differences between Mtb isolates might indicate TB cases are the result of recent transmission. However, the variable and sometimes long duration of latent infection, combined with uncertainty in the Mtb mutation rate during latency, can complicate interpretation of WGS results. To estimate the association between infection duration and single nucleotide polymorphism (SNP) accumulation in the Mtb genome, we first analyzed pairwise SNP differences among TB cases from Los Angeles County, California, with strong epidemiologic links. We found that SNP distance alone was insufficient for concluding that cases are linked through recent transmission. Second, we describe a well-characterized cluster of TB cases in California to illustrate the role of genomic data in conclusions regarding recent transmission. Longer presumed latent periods were inconsistently associated with larger SNP differences. Our analyses suggest that WGS alone cannot be used to definitively determine that a case is attributable to recent transmission. Methods for integrating clinical, epidemiologic, and genomic data can guide conclusions regarding the likelihood of recent transmission, providing local public health practitioners with better tools for monitoring and investigating TB transmission.

1397. Modern Lineages of Mycobacterium tuberculosis Were Recently Introduced in Western India and Demonstrate Increased Transmissibility

Background Mycobacterium tuberculosis (Mtb) transmissibility may vary between lineages (or variants) and this may contribute to the slow decline of tuberculosis incidence. The objective of our study was to compare transmissibility across four major lineages (L1-4) of Mtb in Pune, India.MethodsWe performed whole-genome sequencing (WGS) of Mtb isolated from sputum culture of adult patients with pulmonary TB. We performed genotypic susceptibility testing for both first- and second-line drugs using a previously validated random forest predictor. We identified single nucleotide polymorphisms and generated a multiple sequence alignment excluding drug resistance conferring mutations to avoid skewing the phylogeny due to convergent evolution in these regions. We used Bayesian molecular dating to generate phylogenies and compared tree characteristics using a two-sample Kolmogorov-Smirnov (KS) test.ResultsOf the 642 isolates from distinct study participants that underwent WGS, 612 met quality criteria. The median age of participants was 31 years (range 18-74), the majority were male (64.7%) and sputum smear-positive (83.3%), and 6.7% had co-infection with HIV (Table 1). There was no significant difference in baseline characteristics between lineages. The majority of isolates belonged to L3 (44.6%). The majority (61.1%) of multidrug-resistant (MDR, resistant to isoniazid and rifampin) isolates belonged to L2. In phylogenetic analysis, we found evidence of higher transmissibility of L2 as indicated by shorter branch lengths (i.e., less time had elapsed between transmission and sampling) and more genetic similarity (smaller pairwise single nucleotide polymorphism [SNP] distances) among L2 isolates as compared to other lineages (Figure 1). Branching times for L2 and L4 were smaller than L1 and L3 indicating recent introduction into the region (p < 0.001 [KS test]). Figure 1: Lineage-wise distribution of A) phylogenetic tree branch lengths (log) and B) pairwise single nucleotide polymorphism (SNP) distance, using 612 tuberculosis isolates from Pune, India. P values calculated using two-sample Kolmogorov-Smirnov test. Table 1: Demographic characteristics of study participants included in the study, by lineage.ConclusionModern Mtb lineages (L2 and L4) were relatively recently introduced in western India, as compared to older lineages (L1 and L3), with the more drug-resistant L2 showing higher transmissibility. These findings highlight the need for early detection and treatment initiation to interrupt transmission with important implications for antimicrobial stewardship and heightened surveillance of TB resistance rates.Disclosures All Authors: No reported disclosures

The pattern of linkage disequilibrium in Dual-Purpose Belgian Blue cattle.

Quantifying the level of linkage disequilibrium (LD), non-random association of alleles at two or more loci, is important to determine the number of markers needed for genomic selection. The aims of this study were to evaluate the extent of LD in Dual-Purpose Belgian Blue (DPBB) and to compare the level of LD in DPBB with that of Walloon Holstein. Data of 28,427 single nucleotide polymorphisms (SNP), located on 29 Bos taurus autosomes(BTA), of 639 DPBB and 398 Holstein bulls were used. The level of LD between pairwise SNPs separated by up to 10Mb was evaluated, separately for each breed, using the squared correlation of the alleles at two loci. The analysis of molecular variance showed that the percentage of variation within populations (85.48%) was higher than between populations (14.52%). However, permutation tests showed a significant genetic differentiation between the two studied populations (p<.01). The average LD found between adjacent SNP pairs in DPBB (0.16 (SD=0.22)) was generally lower than in Holstein (0.23 (SD=0.27)). The proportion of SNPs in useful LD (r2 >0.30) within a genomic distance of ≤0.10Mb between SNPs was 18.58% and 28.23% in DPBB and Holstein bulls, respectively. In both breeds, the effective population size decreased over generations; however, the decline was greater in DPBB than that in Holstein. Based on results, it can be concluded that at least 68,000 SNPs are needed for implementing genomic selection in DPBB cattle with enough accuracy.