Whole Genome Sequencing Methods Research Articles

Genomic analysis of two Acinetobacter baumannii strains belonging to two different sequence types (ST172 and ST25)

ObjectivesAcinetobacter baumannii is an opportunistic nosocomial pathogen that is the main focus of attention in clinical settings owing to its intrinsic ability to persist in the hospital environment and its capacity to acquire determinants of resistance and virulence. Here we present the genomic sequencing, molecular characterisation and genomic comparison of two A. baumannii strains belonging to two different sequence types (STs), one sporadic and one widely distributed in our region. MethodsWhole-genome sequencing (WGS) of Ab42 and Ab376 was performed using Illumina MiSeq-I and the genomes were assembled with SPAdes. ARG-ANNOT, CARD-RGI, ISfinder, PHAST, PlasmidFinder, plasmidSPAdes and IslandViewer were used to analyse both genomes. ResultsGenome analysis revealed that Ab42 belongs to ST172, an uncommon ST, whilst Ab376 belongs to ST25, a widely distributed ST. Molecular characterisation showed the presence of two antibiotic resistance genes in Ab42 and nine in Ab376. No insertion sequences were detected in Ab42, however 22 were detected in Ab376. Moreover, two prophages were found in Ab42 and three in Ab376. In addition, a CRISPR-cas type I-Fb and two plasmids, one of which harboured an AbGRI1-like island, were found in Ab376. ConclusionsWe present WGS analysis of twoA. baumannii strains belonging to two different STs. These findings allowed us to characterise a previously undescribed ST (ST172) and provide new insights to the widely studied ST25.

Porcine reproductive and respiratory syndrome virus whole-genome sequencing efficacy with field clinical samples using a poly(A)-tail viral genome purification method.

The genomic surveillance of porcine reproductive and respiratory syndrome virus (PRRSV) is based on sequencing of the ORF5 gene of the virus, which covers only 4% of the entire viral genome. It is expected that PRRSV whole-genome sequencing (WGS) will improve PRRSV genomic data and allow better understanding of clinical discrepancies observed in the field when using ORF5 sequencing. Our main objective was to implement an efficient method for WGS of PRRSV from clinical samples. The viral genome was purified using a poly(A)-tail viral genome purification method and sequenced using Illumina technology. We tested 149 PRRSV-positive samples: 80 sera, 33 lungs, 33 pools of tissues, 2 oral fluids, and 1 processing fluid (i.e., castration liquid). Overall, WGS of 67.1% of PRRSV-positive cases was successful. The viral load, in particular for tissues, had a major impact on the PRRSV WGS success rate. Serum was the most efficient type of sample to conduct PRRSV WGS poly(A)-tail assays, with a success rate of 76.3%, and this result can be explained by improved sequencing reads dispersion matching throughout the entire viral genome. WGS was unsuccessful for all pools of tissue and lung samples with Cq values > 26.5, whereas it could still be successful with sera at Cq ≤ 34.1. Evaluation of results of highly qualified personnel confirmed that laboratory skills could affect PRRSV WGS efficiency. Oral fluid samples seem very promising and merit further investigation because, with only 2 samples of low viral load (Cq = 28.8, 32.8), PRRSV WGS was successful.

Coronavirus Disease 2019 (COVID-19) Re-infection by a Phylogenetically Distinct Severe Acute Respiratory Syndrome Coronavirus 2 Strain Confirmed by Whole Genome Sequencing

BackgroundWaning immunity occurs in patients who have recovered from COVID-19. However, it remains unclear whether true re-infection occurs.MethodsWhole genome sequencing was performed directly on respiratory specimens collected during two episodes of COVID-19 in a patient. Comparative genome analysis was conducted to differentiate re-infection from persistent viral shedding. Laboratory results, including RT-PCR Ct values and serum SARS-CoV-2 IgG, were analyzed.ResultsThe second episode of asymptomatic infection occurred 142 days after the first symptomatic episode in an apparently immunocompetent patient. During the second episode, there was serological evidence of elevated C-reactive protein and SARS-CoV-2 IgG seroconversion. Viral genomes from first and second episodes belong to different clades/lineages. Compared to viral genomes in GISAID, the first virus genome has a stop codon at position 64 of orf8 leading to a truncation of 58 amino acids, and was phylogenetically closely related to strains collected in March/April 2020, while the second virus genome was closely related to strains collected in July/August 2020. Another 23 nucleotide and 13 amino acid differences located in 9 different proteins, including positions of B and T cell epitopes, were found between viruses from the first and second episodes.ConclusionsEpidemiological, clinical, serological and genomic analyses confirmed that the patient had re-infection instead of persistent viral shedding from first infection. Our results suggest SARS-CoV-2 may continue to circulate among the human populations despite herd immunity due to natural infection or vaccination. Further studies of patients with re-infection will shed light on protective correlates important for vaccine design.

Application of Whole Genome Sequencing and Pan-Family Multi-Locus Sequence Analysis to Characterize Relationships Within the Family Brucellaceae.

The bacterial family Brucellaceae is currently composed of seven genera, including species of the genus Brucella, a number of which are significant veterinary and zoonotic pathogens. The bacteriological identification of pathogenic Brucella spp. may be hindered by their close phenotypic similarity to other members of the Brucellaceae, particularly of the genus Ochrobactrum. Additionally, a number of novel atypical Brucella taxa have recently been identified, which exhibit greater genetic diversity than observed within the previously described species, and which share genomic features with organisms outside of the genus. Furthermore, previous work has indicated that the genus Ochrobactrum is polyphyletic, raising further questions regarding the relationship between the genus Brucella and wider Brucellaceae. We have applied whole genome sequencing (WGS) and pan-family multi-locus sequence analysis (MLSA) approaches to a comprehensive panel of Brucellaceae type strains, in order to characterize relationships within the family. Phylogenies based on WGS core genome alignments were able to resolve phylogenetic relationships of 31 non-Brucella spp. type strains from within the family, alongside type strains of twelve Brucella species. A phylogeny based on concatenated pan-family MLSA data was largely consistent with WGS based analyses. Notably, recently described atypical Brucella isolates were consistently placed in a single clade with existing species, clearly distinct from all members of the genus Ochrobactrum and wider family. Both WGS and MLSA methods closely grouped Brucella spp. with a sub-set of Ochrobactrum species. However, results also confirmed that the genus Ochrobactrum is polyphyletic, with seven species forming a separate grouping. The pan-family MLSA scheme was subsequently applied to a panel of 50 field strains of the family Brucellaceae, isolated from a wide variety of sources. This analysis confirmed the utility of the pan-Brucellaceae MLSA scheme in placing field isolates in relation to recognized type strains. However, a significant number of these isolates did not cluster with currently identified type strains, suggesting the existence of additional taxonomic diversity within some members of the Brucellaceae. The WGS and pan-family MLSA approaches applied here provide valuable tools for resolving the identity and phylogenetic relationships of isolates from an expanding bacterial family containing a number of important pathogens.

Hospital MRSA outbreaks: Multiplex PCR-reverse line blot binary typing as a screening method for WGS, and the role of the environment in transmission.

Whole-genome sequencing (WGS) can provide useful information on methicillin-resistant Staphylococcus aureus (MRSA) transmission in hospitals. However, it is expensive and laborious, especially for environmental screening programs which generate large numbers of isolates. Multiplex PCR-reverse line blot binary typing (mPCR-RLB BT) is a rapid, high throughput, inexpensive typing method which could be useful to screen isolates for WGS. This study assessed the strategy of screening isolates with mPCR-RLB BT to reduce the need for WGS; and to assess the role of the environment in transmission. MRSA transmission in a Burns Unit and its related Intensive Care Unit was studied. mPCR-RLB BT was performed on 238 isolates; this, combined with epidemiological data, was used to choose 97 isolates for WGS. Relationships between isolates by WGS demonstrated several outbreaks. There was a significant contribution of environmental isolates to transmission, and several problem areas were identified. There was a substantial cost saving from screening isolates with mPCR-RLB BT. The use of an inexpensive, rapid screening method for MRSA typing is useful to reduce expenditure and time spent on hospital infection control programs, and reductions are likely to be even more considerable in a non-outbreak setting. Environmental screening and WGS are useful to determine exact sources of transmission to focus mitigation strategies.

First isolation and characterization of Brucella suis from yak.

Brucella spp., facultative intracellular pathogens that can persistently colonize animal host cells and cause zoonosis, affect public health and safety. A Brucella strain was isolated from yak in Qinghai Province. To detect whether this isolate could cause an outbreak of brucellosis and to reveal its genetic characteristics, several typing and whole-genome sequencing methods were applied to identify its species and genetic characteristics. Phylogenetic analysis based on MLVA and whole-genome sequencing revealed the genetic characteristics of the isolated strain. The results showed that the isolated strain is a B. suis biovar 1 smooth strain, and this isolate was named B. suis QH05. The results of comparative genomics and MLVA showed that B. suis QH05 is not a vaccine strain. Comparison with other B. suis strains isolated from humans and animals indicated that B. suis QH05 may be linked to specific animal and human sources. In conclusion, B. suis QH05 does not belong to the Brucella epidemic species in China, and as the first isolation of B. suis from yak, this strain expands the host range of B. suis.

Comparison of commercially available whole-genome sequencing kits for variant detection in circulating cell-free DNA

Circulating cell-free DNA (ccfDNA) has great potential for non-invasive diagnosis, prognosis and monitoring treatment of disease. However, a sensitive and specific whole-genome sequencing (WGS) method is required to identify novel genetic variations (i.e., SNVs, CNVs and INDELS) on ccfDNA that can be used as clinical biomarkers. In this article, five WGS methods were compared: ThruPLEX Plasma-seq, QIAseq cfDNA All-in-One, NEXTFLEX Cell Free DNA-seq, Accel-NGS 2 S PCR FREE DNA and Accel-NGS 2 S PLUS DNA. The Accel PCR-free kit did not produce enough material for sequencing. The other kits had significant common number of SNVs, INDELs and CNVs and showed similar results for SNVs and CNVs. The detection of variants and genomic signatures depends more upon the type of plasma sample rather than the WGS method used. Accel detected several variants not observed by the other kits. ThruPLEX seemed to identify more low-abundant SNVs and SNV signatures were similar to signatures observed with the QIAseq kit. Accel and NEXTFLEX had similar CNV and SNV signatures. These results demonstrate the importance of establishing a standardized workflow for identifying non-invasive candidate biomarkers. Moreover, the combination of variants discovered in ccfDNA using WGS has the potential to identify enrichment pathways, while the analysis of signatures could identify new subgroups of patients.

The spread of chloramphenicol-resistant Neisseria meningitidis in Southeast Asia

ObjectivesInvasive disease caused by Neisseria meningitidis is a significant health concern globally, but our knowledge of the prevailing serogroups, antimicrobial susceptibility patterns, and genetics of N. meningitidis in Southeast Asia is limited. Chloramphenicol resistance in N. meningitidis has rarely been reported, but was first described in isolates from Vietnam in 1998. We aimed to characterise eight chloramphenicol resistant meningococcal isolates collected between 2007 and 2018 from diagnostic microbiology laboratories in Cambodia, Thailand and the Lao People's Democratic Republic (Laos). MethodsWhole-genome sequencing was used to generate genome sequences from 18 meningococcal isolates including the eight chloramphenicol resistant isolates. We identified antimicrobial resistance genes present in these strains, and examined the phylogenetic relationships between strains. ResultsThe eight resistant strains all contain the same chloramphenicol resistance gene first described in 1998, and are closely related to each other. Strains resistant to penicillin, tetracycline, and ciprofloxacin were also observed, including a chloramphenicol-resistant strain which has acquired penicillin and ciprofloxacin resistance. ConclusionsThis study suggests that chloramphenicol-resistant N. meningitidis is more widespread than previously thought, and that the previously-identified resistant lineage is now found in multiple countries in Southeast Asia.

Evaluation of real-time nanopore sequencing for Salmonella serotype prediction

The use of whole genome sequencing (WGS) data generated by short-read sequencing technologies such as the Illumina sequencing platforms has been shown to provide reliable results for Salmonella serotype prediction. Emerging long-read sequencing platforms developed by Oxford Nanopore Technologies (ONT) provide an alternative WGS method to meet the needs of industry for rapid and accurate Salmonella confirmation and serotype classification. Advantages of the ONT sequencing platforms include portability, real-time base-calling and long-read sequencing. To explore whether WGS data generated by an ONT sequencing platform could accurately predict Salmonella serotypes, 38 Salmonella strains representing 34 serotypes were sequenced using R9.4 flow cells on an ONT sequencer for up to 2 h. The downstream bioinformatics analysis was performed using pipelines with different assemblers including Canu, Wdbtg2 combined with Racon, or Miniasm combined with Racon. In silico serotype prediction programs were carried out using both SeqSero2 (raw reads and genome assemblies) and SISTR (genome assemblies). The WGS data of the same strains were also obtained from Illumina Hiseq (200 x depth of coverage per genome) as a benchmark of accurate serotype prediction. Predictions using WGS data generated after 30 min, 45 min, 1 h, and 2 h of ONT sequencing time all matched the prediction results from Illumina WGS data. This study demonstrated the comparable accuracy of WGS-based serotype prediction between ONT and Illumina sequencing platforms. This study also sets a start point for future validation of ONT WGS as a rapid Salmonella confirmation and serotype classification tool for the food industry.

Whole genome bisulfite sequencing of human spermatozoa reveals differentially methylated patterns from type 2 diabetic patients.

Aims/IntroductionThe incidence of type 2 diabetes mellitus is increasing worldwide, and it might partly cause metabolic disorder and type 2 diabetes mellitus susceptibility in patients’ offspring through epigenetic modification. However, the underlying mechanisms remain largely unclear. Recent studies have shown a potential link between deoxyribonucleic acid methylation in paternal sperm and susceptibility to type 2 diabetes mellitus in offspring, so this article focuses on whether the whole‐genome methylation profiles of spermatozoa in type 2 diabetes mellitus patients have changed.Materials and MethodsWe investigated the genome‐wide deoxyribonucleic acid methylation profiles in spermatozoa by comparing eight individuals with type 2 diabetes mellitus and nine non‐diabetic controls using whole‐genome bisulfite sequencing method.ResultsFirst, we found that the proportion of methylated cytosine in the whole genome of the type 2 diabetes mellitus group was slightly lower than that of the control group. Interestingly, the proportion of methylated cytosines in the CG context decreased, and the proportion of methylated cytosines in the CHG context (H = A, T or C) increased in the type 2 diabetes mellitus group, but the proportion of methylated cytosines in the CHH context (H = A, T or C) barely changed. The methylated cytosines in the CG context were mainly distributed at the high methylated level, whereas methylated cytosines in the CHG context and methylated cytosines in the CHH context were mainly distributed at the low and middle methylated level in both groups. Second, functional enrichment analysis showed that differentially methylated genes played a significant role in nervous system development and cell metabolism. Finally, we identified 10 top type 2 diabetes mellitus‐related differentially methylated genes, including IRS1, PRKCE, FTO, PPARGC1A, KCNQ1, ATP10A, GHR, CREB1, PRKAR1A and HNF1B.ConclusionsOur study provides the first evidence for deoxyribonucleic acid methylation reprogramming in spermatozoa of type 2 diabetes mellitus patients, and provides a new basis for explaining the complex mechanism of type 2 diabetes mellitus susceptibility in offspring.

Blood-based detection of early-stage colorectal cancer using multiomics and machine learning.

66 Background: Despite population screening efforts, screening rates for colorectal cancer (CRC) remain suboptimal. A non-invasive, blood-based screening test with high sensitivity and specificity in early-stage disease should improve adherence and ultimately reduce mortality; however, tests based only on tumor-derived biomarkers have limited sensitivity. Here we used a multiomic, machine learning platform to discover, refine, and combine tumor- and immune-derived signals to develop a blood test for the detection of early-stage CRC. Methods: Samples from 591 participants enrolled in a prospective study including average-risk screening and case-control cohorts (NCT03688906) were included in this analysis (CRC: n = 43; colonoscopy-confirmed CRC-negative controls: n = 548). Participants with CRC were 60% male with a mean age of 63, and controls were 55% male with a mean age of 60. Stage distribution was 54% early (I/II) and 34% late (III/IV) with 11% unknown. Plasma was analyzed by whole-genome sequencing, bisulfite sequencing, and protein quantification methods. Computational methods were used to assess and infer the performance of individual and combined assays. Results: For colorectal adenocarcinoma, which represents ~95% of all CRCs, our multiomic test achieved a mean sensitivity of 92% in early stage (n = 17) and 84% in late stage (n = 11) at a specificity of 90%. Across all CRC pathological subtypes, our test achieved a mean sensitivity of 80% in early stage (n = 19) and 83% in late stage (n = 12) at a specificity of 90%; the test detected the single squamous cell carcinoma but missed both neuroendocrine tumors. Individual assays achieved a mean sensitivity of 50% in early stage and 66% in late stage at a specificity of 90%. Conclusions: In a prospective cohort, we demonstrated high sensitivity and specificity for early-stage adenocarcinoma by combining tumor- and immune-derived signals from cfDNA, epigenetic, and protein biomarkers. While most CRCs are adenocarcinomas, detection of all pathological subtypes is required to maximize sensitivity in a screening population. Further analysis of molecular and pathological subtypes, as well as the entire ~3000 patient cohort, is underway. Clinical trial information: NCT03688906.

Effectiveness of Plant Beneficial Microbes: Overview of the Methodological Approaches for the Assessment of Root Colonization and Persistence.

Issues concerning the use of harmful chemical fertilizers and pesticides that have large negative impacts on environmental and human health have generated increasing interest in the use of beneficial microorganisms for the development of sustainable agri-food systems. A successful microbial inoculant has to colonize the root system, establish a positive interaction and persist in the environment in competition with native microorganisms living in the soil through rhizocompetence traits. Currently, several approaches based on culture-dependent, microscopic and molecular methods have been developed to follow bioinoculants in the soil and plant surface over time. Although culture-dependent methods are commonly used to estimate the persistence of bioinoculants, it is difficult to differentiate inoculated organisms from native populations based on morphological characteristics. Therefore, these methods should be used complementary to culture-independent approaches. Microscopy-based techniques (bright-field, electron and fluorescence microscopy) allow to obtain a picture of microbial colonization outside and inside plant tissues also at high resolution, but it is not possible to always distinguish living cells from dead cells by direct observation as well as distinguish bioinoculants from indigenous microbial populations living in soils. In addition, the development of metagenomic techniques, including the use of DNA probes, PCR-based methods, next-generation sequencing, whole-genome sequencing and pangenome methods, provides a complementary approach useful to understand plant–soil–microbe interactions. However, to ensure good results in microbiological analysis, the first fundamental prerequisite is correct soil sampling and sample preparation for the different methodological approaches that will be assayed. Here, we provide an overview of the advantages and limitations of the currently used methods and new methodological approaches that could be developed to assess the presence, plant colonization and soil persistence of bioinoculants in the rhizosphere. We further discuss the possibility of integrating multidisciplinary approaches to examine the variations in microbial communities after inoculation and to track the inoculated microbial strains.

Methylome Variation Predicts Exemestane Resistance in Advanced ER+ Breast Cancer.

Background:More than 30% of estrogen receptor-positive breast cancers are resistant to primary hormone therapy, and about 40% that initially respond to hormone therapy eventually acquire resistance. Although the mechanisms of hormone therapy resistance remain unclear, aberrant DNA methylation has been implicated in oncogenesis and drug resistance.Purpose:We investigated the relationship between methylome variations in circulating tumor DNA and exemestane resistance, to track hormone therapy efficacy.Methods:We prospectively recruited 16 patients who were receiving first-line therapy in our center. All patients received exemestane-based hormone therapy after enrollment. We collected blood samples at baseline, first follow-up (after 2 therapeutic cycles) and at detection of disease progression. Disease that progressed within 6 months under exemestane treatment was considered exemestane resistance but was considered relatively exemestane-sensitive otherwise. We obtained circulating tumor DNA-derived methylomes using the whole-genome bisulfide sequencing method. Methylation calling was done by BISMARK software; differentially methylated regions for exemestane resistance were calculated afterward.Results:Median follow-up for the 16 patients was 19.0 months. We found 7 exemestane resistance-related differentially methylated regions, located in different chromosomes, with both significantly different methylation density and methylation ratio. Baseline methylation density and methylation ratio of chromosome 6 [32400000-32599999] were both high in exemestane resistance. High baseline methylation ratios of chromosome 3 [67800000-67999999] (P = .013), chromosome 3 [140200000-140399999] (P = .037), and chromosome 12 [101200000-101399999] (P = .026) could also predict exemestane resistance. During exemestane treatment, synchronized changes in methylation density and methylation ratio in chromosome 6 [32400000-32599999] could accurately stratify patients in terms of progression-free survival (P = .000033). Cutoff values of methylation density and methylation ratio for chromosome 6 [149600000-149799999] were 0.066 and 0.076, respectively.Conclusion:Methylation change in chromosome 6 [149600000-149799999] is an ideal predictor of exemestane resistance with great clinical potential.

Detection of Acinetobacter pittii ST220 co-producing NDM-1 and OXA-820 carbapenemases from a hospital sink in a non-endemic country of NDM

ObjectivesNDM-1 is by far one of the most commonly prevalent carbapenemases in Enterobacteriaceae and Acinetobacter baumannii. This study presented an Acinetobacter pittii (A. pittii) isolate co-harboring blaNDM-1 and blaOXA-820 from a university hospital sink, where New Delhi metallo-β-lactamase (NDM) producers have not been found in either patients or their environments. MethodsWhole-genome sequencing was performed on the HiSeq 4000 platform, and the reads were de novo assembled using the A5-miSeq Assembly pipeline. Annotation of the resulting scaffolds were performed by using the DDBJ Fast Annotation and Submission Tool (DFAST). The blaNDM-1-carrying plasmid was determined. ResultsThe A. pittii ST220 strain SU1805 detected from a sink strainer in the treatment room was resistant to imipenem and meropenem. Antimicrobial resistance genes blaNDM-1, blaOXA-820, blaADC-43, and aphA6 were found in this strain. The blaNDM-1 was found to be located downstream of an ISAba125 element on a plasmid pSU1805NDM with a size of 41,022 bp, and GC content of 38.3% harbouring 48 protein-coding genes. The aphA6 gene was also located upstream of the ISAba125 on the same plasmid. The A. pittii intrinsic blaOXA-213-like gene blaOXA-820 was located between fxsA and yncA genes in the chromosome. The strain also harboured biofilm-associated genes such as ompA, the csu operon and their regulating genes bfmRS. ConclusionThis study described the first isolation of NDM-1-producing A. pittii in Japan, and highlighted the importance of proper implementation of measures against AMR for sink drainage systems, since NDM producers may have already been hidden in such environments in a non-endemic country of NDM.

Phylogenetic surveillance of travel-related Zika virus infections through whole-genome sequencing methods

In 2018, the World Health Organization identified the Zika virus (ZIKV) as a pathogen that should be prioritized for public health research due to its epidemic potential. In this study, whole-genome sequencing (WGS) of travel-acquired ZIKV infections was used to examine the limitations of phylogenetic analysis. WGS and phylogenetic analysis were performed to investigate geographic clustering of samples from five Canadians with travel-acquired ZIKV infections and to assess the limitations of phylogenetic analysis of ZIKV sequences using a phylogenetic cluster approach. Genomic variability of ZIKV samples was assessed and for context, compared with hepatitis C virus (HCV) samples. Phylogenetic analysis confirmed the suspected region of ZIKV infection for one of five samples and one sample failed to cluster with sequences from its suspected country of infection. Travel-acquired ZIKV samples depicted low genomic variability relative to HCV samples. A floating patristic distance threshold classified all pre-2000 ZIKV sequences into separate clusters, while only Cambodian, Peruvian, Malaysian, and South Korean sequences were similarly classifiable. While phylogenetic analysis of ZIKV data can identify the broad geographical region of ZIKV infection, ZIKV’s low genomic variability is likely to limit precise interpretations of phylogenetic analysis of the origins of travel-related cases.

Characterization of a community-acquired methicillin-resistant sequence type 338 Staphylococcus aureus strain containing a staphylococcal cassette chromosome mec type VT

ObjectivesTo determine the molecular characteristics of a sequence type 338 community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) strain and the relationship among MRSA strains from various lineages and areas. MethodsWhole-genome sequencing, genomic comparison, antimicrobial susceptibility testing, and hemolysis analysis were performed to identify the resistance determinants and virulence factors of strain ZY05 and the relationships among CC59 clones. ResultsMRSA strain ZY05 was resistant to tetracycline, erythromycin, and clindamycin, and the resistance genes erm(B) and tet(K) were detected in the genome. ZY05 harbors the genomic islands νSaα, νSaβ, νSaγ, and ΦSa2, the pathogenicity island νSa1, and virulence factors such as Panton–Valentine leukocidin, phenol-soluble modulins, alpha-hemolysin, enterotoxin B, enterotoxin K, and enterotoxin Q, which are the same as those present in ST59 strains. In addition, the virulence potential of ST338 did not differ from that of ST59. This strain contains the staphylococcal cassette chromosome mec (SCCmec) type VT, a distinct SCCmec type previously reported from Taiwan. The results of core genome multilocus sequence typing (cgMLST) analysis showed that the gene distances between ST59 and ST338 were close among CC59 isolates, while strains from Taiwan were identical to isolates from the Chinese mainland with respect to these two sequence types. ConclusionsThe ST338 strain ZY05, which has a close genetic relationship to ST59 strains, is multidrug-resistant and highly virulent. Strains of two identical lineages, ST59 and ST338, from Taiwan and the Chinese mainland may have the same genetic background.

2602. Genetic Basis of Staphylococcus aureus Virulence

BackgroundAlthough multiple different virulence factors have been identified for Staphylococcus aureus, there is limited information on genetic variation present between different strains of S. aureus in the clinical setting. To better define whether differing virulence factors could contribute to differing clinical manifestations of S. aureus infections we undertook a comparison of the frequency of virulence and antibiotic resistance genes present in S. aureus isolates from different clinical sites.MethodsWhole-genome sequencing was performed on a convenience sample of S. aureus isolates from clinical or surveillance cultures obtained at an academic medical center over a 27-month period. Genomic assemblies were generated and annotated to define protein-coding regions. The prevalence of 28 genes previously defined as being associated with S. aureus virulence or antimicrobial resistance, including MSCRAMM genes, was then analyzed in relation to nine specific culture sources including only a single isolate from each culture source per patient using a likelihood ratio χ 2 analysis.ResultsThere were 1286 S. aureus isolates with draft assemblies and annotations, and there was a statistically significant (P < 0.01) difference in gene frequencies between culture sources for 18 genes that included 13 of 19 virulence factors, 4 of 7 antibiotic resistance genes and 1 of 2 MSCRAMM genes. The most notable variation was seen for the presence of the sec, sep, entB, lukS, lufK, fosB, mecA, and ermA genes (all with P < 0.0001). There were also significant variations in overall gene frequency patterns between isolates from wound, blood, and respiratory isolates (P < 0.0001), as well as significant differences in the frequency of cna and hlY genes between surveillance and clinical isolates (P < 0.0001).ConclusionThis study demonstrates a difference in the prevalence of virulence and antibiotic resistance genes in S. aureus isolates based on the culture source. As the culture location can be considered a surrogate for different types of infections (such as bacteremia, pneumonia, urinary tract infections) these differences in gene frequency may contribute to variation in the clinical manifestations of infections by differing S. aureus strains.Disclosures All authors: No reported disclosures.

Multi-omics study in monozygotic twins confirm the contribution of de novo mutation to psoriasis

BackgroundGenome-wide association studies have identified over 120 risk loci for psoriasis. However, most of the variations are located in non-coding region with high frequency and small effect size. Pathogenetic variants are rarely reported except HLA-C*0602 with the odds ratio being approximately 4.0 in Chinese population. Although rare variations still account for a small proportion of phenotypic variances in complex diseases, their effect on phenotypes is large. Recently, more and more studies focus on the low-frequency functional variants and have achieved a certain amount of success. MethodWhole genome sequencing and sanger sequencing was performed on 8 MZ twin pairs discordant for psoriasis to scan and verified the de novo mutations (DNMs). Additionally, 665 individuals with about 20 years' medical history versus 2054 healthy controls and two published large population studies which had about 8 years’ medical history (including 10,727 cases versus 10,582 controls) were applied to validate the enrichment of rare damaging mutations in two DNMs genes. Besides, to verify the pathogenicity of candidate DNM in C3, RNA-sequencing for CD4+, CD8+ T cells of twins and lesion, non-lesion skin of psoriasis patients were carried out. Meanwhile, the enzyme-linked immunosorbent assay kit was used to detect the level of C3, C3b in the supernatant of peripheral blood. ResultA total of 27 DNMs between co-twins were identified. We found six of eight twins carry HLA-C∗0602 allele which have large effects on psoriasis. And it is interesting that a missense mutation in SPRED1 and a splice region mutation in C3 are found in the psoriasis individuals in the other two MZ twin pairs without carrying HLA-C*0602 allele. In the replication stage, we found 2 loss-of-function (LOF) variants of C3 only in 665 cases with about 20 years' medical history and gene-wise analysis in 665 cases and 2054 controls showed that the rare missense mutations in C3 were enriched in cases (OR = 1.91, P = 0.0028). We further scanned the LOF mutations of C3 in two published studies (about 8 years’ medical history), and found one LOF mutation in the case without carrying HLA-C*0602. In the individual with DNM in C3, RNA sequencing showed the expression level of C3 in skin was significant higher than healthy samples in public database (TPM fold change = 1.40, P = 0.000181) and ELISA showed protein C3 in peripheral blood was higher (~2.2-fold difference) than the other samples of twins without DNM in C3. ConclusionTo the best of our knowledge, this is the first report that DNM in C3 is the likely pathological mutations, and it provided a better understanding of the genetic etiology of psoriasis and additional treatments for this disease.

Prosthetic joint infections present diverse and unique microbial communities using combined whole-genome shotgun sequencing and culturing methods.

Introduction. Prosthetic joint infections (PJIs) are challenging to treat therapeutically because the infectious agents often are resistant to antibiotics and capable of abundant growth in surface-attached biofilms. Though infection rates are low, ca. 1-2 %, the overall increase in the sheer number of joint replacement surgeries results in an increase in patients at risk.Aims. This study investigates the consensus of microbial species comprising PJI ecology, which is currently lacking.Methodology. In this study, PJI populations from seven patients were analysed using combined culturing and whole-genome shotgun sequencing (WGSS) to establish population profiles and compare WGSS and culture methods for detection and identification of the PJI microbiome.Results. WGSS detected strains when culture did not, notably dormant, culture-resistant and rare microbes. The CosmosID algorithm was used to predict micro-organisms present in the PJI and discriminate contaminants. However, culturing indicated the presence of microbes falling below the WGSS algorithm threshold. In these instances, microbes cultured are believed to be minor species. The two strategies were combined to build a population profile.Conclusions. Variability between and among PJIs showed that most infections were distinct and unique. Comparative analysis of populations revealed PJIs to form clusters that were related to, but separate from, vaginal, skin and gut microbiomes. Fungi and protists were detected by WGSS, but the role of fungi is just beginning to be understood and for protists it is unknown. These micro-organisms and their novel and strain-specific microbial interactions remain to be determined in current clinical tests.

Genomic surveillance of invasive meningococcal disease in the Czech Republic, 2015-2017.

IntroductionThe study presents the results of the genomic surveillance of invasive meningococcal disease (IMD) in the Czech Republic for the period of 2015–2017.Material and methodsThe study set includes all available IMD isolates recovered in the Czech Republic and referred to the National Reference Laboratory for Meningococcal Infections in 2015–2017, a total of 89 Neissseria meningitidis isolates—from 2015 (n = 20), 2016 (n = 27), and from 2017 (n = 42). All isolates were studied by whole genome sequencing (WGS).ResultsSerogroup B (MenB) was the most common, followed by serogroups C, W, and Y. Altogether 17 clonal complexes were identified, the most common of which was hypervirulent complex cc11, followed by complexes cc32, cc41/44, cc269, and cc865. Over the three study years, hypervirulent cc11 (MenC) showed an upward trend. The WGS method showed two clearly differentiated clusters of N. meningitidis C: P1.5,2:F3-3:ST-11 (cc11). The first cluster is represented by nine isolates, all of which are from 2017. The second cluster consisted of five isolates from 2016 and eight isolates from 2017. Their genetic discordance is illustrated by the changing nadA allele and subsequently by the variance in BAST type. Clonal complex cc269 (MenB) also increased over the time frame. WGS identified the presence of MenB vaccine antigen genes in all B and non-B isolates of N. meningitidis. Altogether 49 different Bexsero antigen sequence types (BAST) were identified and 10 combinations of these have not been previously described in the PubMLST database.ConclusionsThe genomic surveillance of IMD in the Czech Republic provides data needed to update immunisation guidelines for this disease. WGS showed a higher discrimination power and provided more accurate data on molecular characteristics and genetic relationships among invasive N. meningitidis isolates.