Genogrouping of type II-A CRISPR array in Streptococcus dysgalactiae subsp. equisimilis from humans and companion animals compared to multilocus sequence and emm typing.

We evaluated the feasibility of type II-A clustered regularly interspaced short palindromic repeats (CRISPR) array-based genogrouping using Streptococcus dysgalactiae subsp. Equisimilis isolates from 32 humans and 8 companion animals and compared Simpson's diversity index of this genogrouping to those of multilocus sequence typing (MLST) and emm genotyping. CRISPRCasFinder detected a type II-A CRISPR array with the same repeat sequences in three whole-genome sequences. Subsequently, optimized polymerase chain reaction-based II-A CRISPR array amplification was performed to sequence the region around the leader and terminal repeat sequences. We conducted spacer genogrouping by evaluating the spacer sequence similarities. A phylogenetic dendrogram was constructed, and spacer content and polymorphisms were illustrated. Simpson's diversity indices were calculated for the CRISPR array genogrouping, MLST, and emm genotyping. We analyzed the association between the spacer genogroup with sequence type (ST)/emm genotype for each isolate. Of the 40 isolates, 39 with the II-A CRISPR array were amplified, sequenced, and assigned to 13 genogroups (A-M). The Simpson's diversity indices for the three typing were 0.874, 0.914, and 0.924, respectively. We found genetic lineages between genogroup M and ST127/stG245.0 and between genogroup I and ST29/stG485.0. These observations suggest the feasibility of II-A CRISPR array genogrouping and the genetic relationship between spacer genogroups and STs/emm genotypes in the isolates.

Biofilm production ability and associated characteristics of Streptococcus agalactiae isolates from companion animals and humans.

OBJECTIVE: We evaluated biofilm production ability (BPA) of Streptococcus agalactiae isolates from companion animals/humans and clarified the relationship between BPA populations and other microbiological features. METHODS: Companion animal-/human-origin isolates were collected with host information. We measured BPA using crystal violet staining, via virulence-associated gene profiling (hylB-pavA-pilB-spb1-srtC1-brpA), capsular genotyping, multilocus sequence typing, and antimicrobial resistance (AMR) phenotyping/genotyping. Significant difference in BPA of isolates from different hosts was assessed. We analyzed the association between BPA populations and the virulence genotypes, capsular genotypes, sequence types/clonal complexes, and AMR phenotypes/genotypes. Inhibitory effect of berberine on BPA was evaluated. RESULTS: Five, twenty-six, and twenty-six isolates belonged to strong, moderate, and weak biofilm producers, whereas seventeen showed no biofilm production. We defined strong, moderate, or weak biofilm producers as the producer group (n = 57) to conduct a comparative analysis between the producer and non-producer populations. There was a significant correlation between the producer population and vaginal specimen. We found significant associations between the producer group and presence (57.9%) of pilB and between the non-producer population and presence (70.6%) of spb1. There was no association between the producer group and capsular genotypes, sequence types/clonal complexes, and AMR phenotypes/genotypes (except for a significant correlation between the producer group and AMR to minocycline). We confirmed inhibitory effect of berberine at sub-minimum inhibitory concentrations (MICs) against the type strain on BPA. CONCLUSION: Our observations suggest that S. agalactiae harboring pilB is more capable of producing biofilms, with berberine inhibitory effect at sub-MICs on BPA.

Intracellular invasion ability of Streptococcus agalactiae among non-invasive isolates from human adults and companion animals in Japan.

OBJECTIVE: This study evaluated the cell invasion ability (CIA) of Streptococcus agalactiae isolates from humans and companion animals and clarified the relationship between CIA populations and their microbiological features. METHODS: Human-origin and companion animal-origin isolates were collected along with host information. We measured CIA using human-lineage colon cancer epithelium (Caco-2) and keratinocyte (HaCaT) cell lines, via virulence-associated gene profiling (bca-rib-bac-lmb-cylE-hylB-pavA-pilB-spb1-srtC1-brpA), capsular genotyping, multilocus sequence typing, and antimicrobial resistance (AMR) phenotyping/genotyping. Significant differences in data regarding CIA into epithelium and keratinocytes and those of isolates from different hosts were assessed. We analyzed the association of CIA populations with the virulence genotypes, capsular genotypes, sequence types/clonal complexes, and AMR phenotypes/genotypes. RESULTS: A comparative analysis was performed between human (n = 15) and canine (n = 17) non-invasive isolates. There was a difference in CIA data between Caco-2 and HaCaT cells using human and animal isolates. For percent invasion ability into Caco-2 cells, we designated values ≥ 0.1 as high-frequency CIA and values < 0.1 as low-frequency CIA. Fourteen isolates harbored high-frequency and 18 isolates harbored low-frequency strains. There was no association between the high-frequency population and the virulence genotypes, capsular genotypes, sequence types/clonal complexes, and AMR phenotypes/genotypes. CONCLUSION: This is the first report assessing the invasion ability of S. agalactiae into HaCaT and Caco-2 cells. Our observations suggest that S. agalactiae is more capable of entering Caco-2 rather than HaCaT.

Novel diverse sequences of the Streptococcus canis M-like protein (SCM) gene and their prevalence in diseased companion animals: Association of their alleles with sequence types.

OBJECTIVE: We aimed to determine novel alleles and their prevalence in Streptococcus canis M-like protein (SCM) and to elucidate association of their alleles with sequence types (STs)/clonal complexes (CCs) and antimicrobial resistance (AMR) phenotypes/genotypes. METHODS: We amplified and sequenced scm, by using primers reported by Pinho recently, for 40 isolates in 2015 and 2017, in which the sequences could not be determined with conventional primers. Isolates, for which SCM alleles, STs, and AMR phenotypes/genotypes were previously determined, were included as controls. A phylogenetic tree of SCM amino acid sequences was constructed. Alleles, based on the tree positions with their prevalence, as well as STs/CCs and AMR phenotypes/genotypes were characterized. RESULTS: Although one isolate possessed SCM allele type 1, 39 isolates had novel allele types 10-15, based on cluster analysis. The 11 and 12 allele types were firstly found in this study. We designated novel allele types as group II and non-novel allele types as group I. Prevalence of group II alleles was 29.9% and 16.2% in 2015 and 2017. Prevalent group II types were allele 10 (10.3%), allele 11 (2.7%), and allele 15 (3.3%) through both periods. There was a significant difference in distribution of STs/CCs between groups I/II SCM populations. We found significant differences in distribution of macrolide/lincosamide AMR genotype (7.7% vs. 26.8%) and AMR rates of fluoroquinolone (0% vs. 12.5%) between the two populations. CONCLUSION: Our study presents group II scm sequences and their prevalence among diseased companion animals in Japan, with association of their alleles with STs.

Biofilm Production Ability and Other Microbiological Features of Streptococcus canis.

This study assessed the biofilm production ability (BPA) and other microbiological features of Streptococcus canis strains. Forty strains of companion-animal origin, including the host information, from 2015 and 2017 were randomly selected, and three strains of blood-origin from two humans and one dog were included. We measured BPA using crystal violet staining, along with S. canis M-like protein (SCM) allele typing, sequence type (ST) determination, antimicrobial resistance (AMR) phenotyping/genotyping, and virulence-associated gene profiling (gbp, ap1, fp1, and brp). BPA measurements revealed 35 strains with BPA and 48 strains without BPA. There was an association between the producer and the isolation year (2017). Moreover, we observed an association between the non-producer and SCM allele 1 and ST9, and between the producer and SCM allele 10 and ST21. Furthermore, we observed a correlation between the producer and the presence of AMR genotypes. Specifically, there was an association between the producer and ap1 detection, and between non-producer and gbp detection. Our results suggest a correlation between biofilm producers and other microbiological features (i.e. isolation year, SCM allele type 10, ST21, presence of AMR genotypes, and ap1 detection).

Comparing Genomic Characteristics of Streptococcus pyogenes Associated with Invasiveness over a 20-year Period in Korea.

BACKGROUND: Few studies have investigated the invasiveness of Streptococcus pyogenes based on whole-genome sequencing (WGS). Using WGS, we determined the genomic features associated with invasiveness of S. pyogenes strains in Korea. METHODS: Forty-five S. pyogenes strains from 1997, 2006, and 2017, including common emm types, were selected from the repository at Gyeongsang National University Hospital in Korea. In addition, 48 S. pyogenes strains were randomly selected depending on their invasiveness between 1997 and 2017 to evaluate the genetic evolution and the associations between invasiveness and genetic profiles. Using WGS datasets, we conducted virulence-associated DNA sequence determination, emm genotyping, multi-locus sequence typing (MLST), and superantigen gene profiling. RESULTS: In total, 87 strains were included in this study. There were no significant differences in the genomic features throughout the study periods. Four genes, csn1, ispE, nisK, and citC, were detected only in invasive strains. There was a significant association between invasiveness and emm cluster type A-C3, including, emm1.0, emm1.18, emm1.3, and emm1.76 (P<0.05). The predominant emm1 lineage belonged to ST28. There were no associations between invasiveness and superantigen gene profiles. CONCLUSIONS: This is the first study using WGS datasets of S. pyogenes strains collected between 1997 and 2017 in Korea. Streptococcal invasiveness is associated with the presence of csn1, ispE, nisK, and citC. The emm1 lineage and ST28 clone are explicitly associated with invasiveness, whereas genomic features remained stable over the 20-year period.

Comparative Genomic Features of Streptococcus canis Based on Pan-Genome Orthologous Group Analysis According to Sequence Type.

Using bacterial pan-genomes obtained through whole genome sequencing (WGS), coding DNA sequences (CDSs) can be clustered into pan-genome orthologous groups (POGs). We aimed to investigate comparative genomic features of Streptococcus canis based on POG analysis and to determine CDSs specific to prevalent sequence type (ST) 9. Twenty WGS datasets from S. canis strains, including invasive and non-invasive specimens, were retrieved from the National Center for Biotechnology Information Assembly database. Based on the WGS data, we performed comparative genome hybridization (CGH), pan- and core-genome prediction, Venn diagram testing with five ST9 strains, and phylogenetic analysis with ST determination. We compared the CDSs of seven ST9 and 13 non-ST9 strains. We observed genomic diversity based on CGH and Venn diagram analyses. The predicted pan- and core-genomes contained 4,772 and 1,403 genes, respectively. We found five clades consisting of different STs (ST1, ST44/2, ST13/14, ST21/15/41, and ST9) based on the phylogenetic tree. There were differences in four pathways (DNA restriction-modification system, DNA-mediated transposition, extracellular region, and response to oxidative stress) regulated by CDSs specific to ST9. Our findings describe genomic diversity in CGH and Venn diagram testing, pan- and core-genomes, five clades of genomes consisting of different STs, and unique CDS features associated with ST9.

Decisive diagnostic clue for infectious abdominal aortic aneurysm caused by Arthrobacter russicus in a diabetic elderly woman with renal dysfunction: A case report and literature review.

Infectious aortic aneurysm (IAA) can be a rare but potentially fatal sequela of infectious inflammatory disease of the aortic wall with a high incidence of rupture. The definitive diagnosis is based on vascular imaging of the aneurysm using contrast-enhanced computed tomography (CE-CT) and identification of the causative microorganism from positive blood cultures (BCs). However, IAA remains extremely difficult to diagnose and treat in patients with prior antimicrobial treatment or with renal dysfunction. Here we describe a case of an 85-year-old woman with IAA caused by Arthrobacter russicus presenting with abdominal pain and fever that was initially diagnosed as a presumptive urinary tract infection and treated with empiric antimicrobial therapy. However, persistent abdominal pain with increased serological inflammation necessitated further evaluation. Unenhanced multimodality imaging considering the renal dysfunction revealed infectious aortitis of the infrarenal abdominal aorta, together with the initial culture results, leading to the tentative diagnosis of Klebsiella pneumoniae aortitis. Thereafter, serial monitoring with unenhanced magnetic resonance angiography (MRA) using thin-slab maximum intensity projection (TS-MIP) revealed acute aortic expansion strongly suggestive of a pseudoaneurysm that was successfully treated with early surgical repair under adequate infection control. Despite negative Gram staining and tissue culture results for the excised aortic wall, a definitive diagnosis of IAA secondary to A. russicus rather than K. pneumoniae was finally made by confirming the histologic findings consistent with IAA and the identification of A. russicus 16S rRNA on the resected aortic wall. The patient also developed a vascular graft infection during the postoperative course that required long-term systemic antimicrobial therapy. This case highlights the value of unenhanced MRA in the early detection of IAA in patients with renal dysfunction and the importance of a molecular diagnosis for identifying the causative microorganism in cases of culture- or tissue-negative IAA.

Intracellular Invasion Ability and Associated Microbiological Characteristics of Streptococcus canis in Isolates from Japan.

This study evaluated the cell invasion ability (CIA) of Streptococcus canis isolates, and clarified the relationship between high-frequency CIA and its microbiological features. Of the companion animal-origin isolates (n = 117) that were obtained in 2017, 40 isolates were randomly selected with the host information, with two human blood-origin isolates included. CIA was measured using human colon carcinoma epithelium and the hemolytic activity (HA) using sheep blood, along with S. canis M-like protein (SCM) allele typing, sequence type (ST) determination, and antimicrobial resistance (AMR) phenotyping/genotyping. CIA measurements revealed that 19 and 24 isolates had high- and low-frequencies, respectively. HA assessment revealed that 24 and 19 isolates were categorized as high- and low- level, respectively. No difference was observed in the high-/low-level HA between the high- /low-frequency CIA populations. A significant difference was found in the high-/low-frequency CIA between the SCM group I/II populations. Additionally, a significantly higher CIA was found in the SCM allele type 10/type 11 than in the others. A significant association was observed between high-frequency CIA and the ST21/ST41 populations. No difference was found in the high-/low-frequency CIA between the presence and absence of the AMR phenotype/genotype. These observations suggest a relationship between high-frequency CIA and its microbiological characteristics (SCM allele type 10/type 11 or ST21/ST41).

Non-Necrotizing Soft Tissue Infection and Streptococcal Toxic Shock Syndrome Caused by a Novel Streptococcus pyogenes Subtype (emm76.10).

We report a case of non-necrotizing soft tissue infection and streptococcal toxic shock syndrome (STSS) caused by a novel emm subtype (emm76.10) of group A Streptococcus pyogenes (GAS). A 54-year-old Japanese woman suffering from fever, fatigue, and lower abdominal pain along with erythema for 3 days was admitted to our hospital. Additionally, she presented with hypotension and multiple organ failure. Exploratory incision was performed due to the presence of STSS and for an examination of the necrotizing soft tissue infection from her lower abdomen to the left thigh. Tissue cultures from the exudates and fascia yielded positive results for GAS growth, although blood cultures returned as negative. After 15 days of antimicrobial therapy, she recovered fully without any complications. Genotyping of the isolate indicated a novel emm subtype (emm76.10), with 5 amino acid substitutions in the emm76.0 subtype sequence and the full-length sequence of 780 bp. This isolate was resistant to tetracyclines, macrolides-lincosamides, and fluoroquinolones, owing to the presence of antibiotic resistance determinants, tet(M) and erm(B), and point mutations, Ser79Phe/Ser81Phe, in the quinolone resistance-determining regions of parC/gyrA. In conclusion, our observations suggest the importance of early-stage exploratory incision and drainage from the infected region for the isolation and characterization of causative bacteria to facilitate selection of appropriate antibiotics for treatment.

Novel Quinolone Nonsusceptible Streptococcus canis Strains with Point Mutations in Quinolone Resistance-Determining Regions and Their Related Factors.

This study investigated quinolone nonsusceptible Streptococcus canis with point mutations in quinolone resistance-determining regions (QRDRs). After selecting targets from 185 isolates, we tested antimicrobial susceptibility using levofloxacin, ciprofloxacin, norfloxacin, and moxifloxacin. We also determined the amino acid sequences of QRDRs in gyrA/gyrB/parC/parE genes and their point mutations. Finally, we performed S. canis-derived M-like protein (SCM) allele typing, multilocus sequence typing, and antimicrobial resistance genotyping. Correlations between nonsusceptible strains and their related factors were examined. We found 13 (7.0%) nonsusceptible isolates consisting of two classes, high-level minimum inhibitory concentrations (MICs) (n = 7, 3.8%) and low-level MICs (n = 6, 3.2%). Mutations Ser81Phe/Ser81Tyr/Glu85Lys in gyrA, Ser67Phe/Ser67Tyr/Asp71Tyr in parC, Asp438Asn in parE, and Gly408Asp in gyrB were observed in these nonsusceptible strains. Common mutations included Ser81 and Ser67/Asp71; additionally, we found one strain each with Glu85, Asp438, and Gly408 mutations. There was a significant correlation between nonsusceptible isolates and the presence of SCM allele type 2, sequence type 46, tetracyclineresistance genes, and macrolide/lincosamide-resistance genes. These results could be used in future, by veterinarians while treating companion animals with clinical symptoms of streptococcal infections.

Complete Genome Sequences of Four Streptococcus canis Strains Isolated from Dogs in South Korea.

This study reports the complete genome sequences of four strains of Streptococcus canis isolated from dogs in South Korea. Their genomes ranged from 2.157 to 2.265 Mbp, with a G+C content of 39.6% to 39.7%. The sequence types, antimicrobial resistance genes, and S. canis M-like protein alleles were characterized.

Draft Genome Sequence of Blood-Origin Streptococcus canis Strain FU149, Isolated from a Dog with Necrotizing Soft Tissue Infection.

The draft genome sequence of the blood-origin Streptococcus canis strain FU149, isolated from a dog with a necrotizing soft tissue infection in Japan, is reported. The genome size was 2.108 Mbp, with a G+C content of 39.5%. Sequences unmapped to the reference genome sequence of NCTC 12191T (GenBank accession number LR134293) were characterized.

Draft Genome Sequences of Seven Streptococcus canis Strains Isolated from Diseased Companion Animals in Japan.

The draft genomes of seven Streptococcus canis isolates from diseased dogs and cats in Japan are reported here. The genome sizes ranged from 1.901 Mbp to 2.203 Mbp, with G+C contents of 39.5% to 39.8%. The sequence types, antimicrobial resistance genotypes, and S. canis M-like protein alleles were all characterized.

Comparison of Streptococcus agalactiae Isolates from Humans and Companion Animals Reveals Genotypic and Phenotypic Differences.

This study assessed whether Streptococcus agalactiae isolates from companion animals differed from those of human origin. Beta-hemolytic S. agalactiae was collected from a veterinary laboratory center and a university hospital. Strains were identified using 16S rRNA amplicon sequencing and amplification of the species-specific dltS gene. We conducted virulence gene profiling, capsular genotyping, determination of clonal complex (CC), and antimicrobial resistance (AMR) phenotyping or genotyping. The 20 non-invasive isolates obtained from animals and 15 non-invasive isolates from adult humans were comparatively analyzed in this study. We found significant differences in the virulence gene profiles of bca-rib-lmb-cylE (40.0% vs. 93.3%) and the possession of bac (30.0% vs. 0%) between animal-origin and human-origin non-invasive strains. We observed a significant difference in the distribution of CC1 between the two non-invasive populations. There were significant differences in the prevalence of tetracycline resistance genotypes (60.0% vs. 20.0%) and absence of AMR genotypes (30.0% vs. 80.0%), and AMR rates of tetracycline (35.0% vs. 0%) and fluoroquinolone (20.0% vs. 66.7%) between the two non-invasive populations. These observations suggest that there were different features, in terms of virulence gene profile, CC, and AMR genotype/phenotype in the non-invasive isolates of animal origin compared to those of human origin.

Species Identification of ß-Hemolytic Streptococci from Diseased Companion Animals and Their Antimicrobial Resistance Data in Japan (2017).

This study aimed to identify the species and assess the antimicrobial resistance (AMR) of ß-hemolytic streptococci isolated from companion animals in Japan. Strains were isolated from clinical specimens of 131 companion animals that exhibited symptoms in April-May 2017. We identified strains by 16S rRNA sequencing and assessed their antimicrobial susceptibility using the broth microdilution method. AMR genes erm(A)-erm(B)-mef(A) and tet(M)-tet(O)-tet(K)-tet(L)-tet(S) in all isolates were amplified by PCR. 16S rRNA sequencing identified ß-hemolytic streptococcal species as Streptococcus canis (n = 117, 89.3%), S. agalactiae (n = 7), S. dysgalactiae subsp. equisimilis (n = 5), S. dysgalactiae subsp. dysgalactiae (n = 1), and S. equi subsp. zooepidemicus (n = 1). Overall AMR rates were 39.7% for minocycline, 19.8% for erythromycin, and 17.6% for clindamycin, with a minimum inhibitory concentration (MIC90) of > 4, > 2, and > 1 µg/mL, respectively. AMR genotyping showed the presence of single or mixed types: erm(B)-mef(A) and tet(M)-tet(O)-tet(L)-tet(S). There was a significant relationship between tetracycline-resistance genotypes and open pus/skin-derived specimens. These observations identify some unique features of ß-hemolytic streptococcal isolates from companion animals in Japan, such as the dominant isolation of S. canis and resistance to tetracycline, macrolide, and lincosamide antibiotics, in terms of species identification and AMR properties.

Analysis of the Type II-A CRISPR-Cas System in Streptococcus canis Isolated from Diseased Companion Animals and One Human Patient in Japan.

We determined the whole-genome sequence (WGS) of Streptococcus canis strain TA4 harboring the M-like protein gene (scm); the strain was isolated from a human patient presenting with bacteremia. The potential of type II-A clustered regularly interspaced short palindromic repeats (CRISPR) array-based typing was evaluated, and the genetic relation was elucidated between spacer genogroups and scm prevalence and/or polymorphisms among the isolates from 19 diseased companion animals and the human patient. CRISPRFinder and CRISPRCasFinder detected the type II-A locus with the same repeat sequences in strain TA4 and another WGS of S. canis strain, isolated from a cow with mastitis. An optimized PCR-based amplification method was used to sequence the region covering the locus around the leader and terminal repeat sequences. Among the 20 isolates sequenced, 16 strains (including TA4) were identified with the CRISPR array. We conducted comparative analysis of the homologous spacer sequences and performed grouping based on the successive common ancestral spacer types. These 16 isolates were assigned to five genogroups (A to E) with scm being absent in genogroup A. We found a relationship between genogroups C and E and allele type 1 of the deduced M-like protein. These preliminary findings suggest the feasibility of CRISPR array-based typing and a genetic relation between the spacer genogroups and scm prevalence and/or polymorphisms in the isolates.

Draft Genome Sequence of Streptococcus canis Clinical Strain OT1, Isolated from a Dog Owner with Invasive Infection without a Dog Bite in Japan.

Streptococcus canis is a ß-hemolytic bacterium that can cause invasive infections in animals and humans. Here, we report a draft genome sequence of S. canis strain OT1, isolated from a female dog owner with bacteremia without a dog bite. The draft genome comprises 2,030,366 bp in 48 contigs.

Prevalence and diversity of M-like protein (SCM) gene in Streptococcus canis isolates from diseased companion animals in Japan: Implication of SCM allele.

Streptococcus canis (Sc)-origin M-like protein (SCM) binds to plasminogen and immunoglobulin G and facilitates anti-phagocytic properties. We aimed to determine the prevalence and diversity of the scm gene in Sc isolates from diseased companion animals in Japan and to propose potential SCM alleles of amino acid (AA) sequences. We collected ß-hemolytic streptococci from diseased animals with host information nationwide in 2015 and 2017. After Sc identification and scm gene amplification and sequencing, the gene's prevalence and relationship between its presence and host information were determined. Furthermore, phylogenetic trees of AA sequences were constructed, and classification and distribution of SCM alleles based on variations of AA sequences were conducted. The scm detection rates were 70.6% (n = 48, 2015) and 82.9% (n = 97, 2017). There was a relationship between scm presence and Tokyo in 2015 and 2017. We found an association between scm detection and dogs in 2017 alone. Major sequence sizes were 1311 bp, 1308 bp, and 1305 bp. Using the phylogenetic trees of AA sequences, we confirmed shared positions of five identical sequence patterns in both periods. Nine SCM alleles were determined with six signal-peptide types. Most prevalent alleles were type 1, type 2, and type 4 in both periods. Our observations suggest prevalence and diversity of scm in animal-origin Sc isolates in Japan.

Draft Genome Sequence of Streptococcus canis Clinical Strain TA4, Harboring the M-Like Protein Gene and Isolated in Japan from a Patient with Bacteremia.

Streptococcus canis is an animal-origin ß-hemolytic bacterium that can cause severe infections in animals and occasionally infects humans. Here, we report a draft genome sequence of an S. canis strain harboring the M-like protein gene. This strain was isolated from a patient with bacteremia (reported by Taniyama et al. [D. Taniyama, Y. Abe, T. Sakai, T. Kikuchi, and T. Takahashi, IDCases 7:48-52, 2017, https://doi.org/10.1016/j.idcr.2017.01.002]). The draft genome comprises 2,129,080 bp in 60 contigs.