2016; 36(4): 367-370
Ann Lab Med 2021; 41(3): 323-327
Published online May 1, 2021 https://doi.org/10.3343/alm.2021.41.3.323
Copyright © Korean Society for Laboratory Medicine.
A Novel Species of the Genus Arsenicicoccus Isolated From Human Blood Using Whole-Genome Sequencing
1Department of Laboratory Medicine, Daejeon Eulji Medical Center, Eulji University, Daejeon, Korea; 2Department of Laboratory Medicine, Chung-Ang University College of Medicine, Seoul, Korea; 3Department of Urology, Chung-Ang University College of Medicine, Seoul, Korea; 4Department of Integrative Biology and Physiology, University of California, Los Angeles, USA
Correspondence to: Mi-Kyung Lee, M.D., Ph.D.
Department of Laboratory Medicine, Chung-Ang University College of Medicine, 102 Heukseok-ro, Dongjak-gu, Seoul 06973, Korea.
Whole-genome sequencing (WGS) is an easily accessible and valuable tool in clinical microbiology, which can be used for identifying novel and rare species. We isolated gram-positive cocci from the blood of a pediatric patient, which could not be phenotypically identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) (BioM?rieux, Marcy-l’?toile, France). We could not identify the isolate to the species level using 16S ribosomal RNA (rRNA) sequencing. WGS was performed using the Illumina MiSeq platform (Illumina, San Diego, CA, USA); however, the subsequent genomic sequence database search using the TrueBac ID-Genome system (ChunLab, Inc., Seoul, Korea) did not yield any hits with an average nucleotide identity value >95.0%, which is the cut-off for species-level identification. Phylogenetic analysis suggested that the isolate belonged to a new Arsenicicoccus species, forming a subcluster with Arsenicicoccus bolidensis. Our data demonstrate that WGS allows a more accurate annotation of microbial genomes than other clinical microbiology tools, such as MALDI-TOF MS and 16S rRNA sequencing. This is the first report of the isolation of a novel Arsenicicoccus species from a clinical sample.
Keywords: Novel Arsenicicoccus species, 16S rRNA sequencing, Whole-genome sequencing
We investigated a case of bloodstream infection caused by gram-positive cocci that were not accurately identified by routine testing. The isolate was identified as belonging to a novel
A 17-month-old boy presenting with fever, nonprojectile vomiting, cyclic abdominal pain, and decreased urination was admitted to the emergency department (ED) of Chung-Ang University Hospital, Seoul, Korea, in May 2019. At admission, the patient’s temperature was 37.6°C, and blood analysis indicated a white blood cell count of 10.46 × 109/L, with a 70.0% of neutrophils. The C-reactive protein serum concentration was normal (< 0.1 mg/L), and the patient was discharged from the ED after 5-6 hours following clinical improvement after hydration therapy. However, the patient’s gastrointestinal symptoms aggravated soon thereafter; he presented with persistent vomiting and new symptoms, including diarrhea. In the evening, the patient was readmitted to the ED and diagnosed as having gastroenteritis. Blood analysis showed no significant changes compared to previous ones. Microbial analyses of stool specimens, including PCR analysis for detecting viral pathogens (rotavirus, norovirus, enteric adenovirus, and astrovirus) and a stool culture for detecting bacterial pathogens (
An aerobic blood culture was performed using a pediatric blood culture bottle and the BacTAlert 3D blood culture system (BioMérieux, Inc., Durham, NC, USA); positive signals were detected (BacT/Alert PF Plus; BioMérieux, Inc., Marcy-l’Étoile, France) after approximately 48 hours of incubation at 35.0°C. Subculture on blood and chocolate agar plates were also prepared. After 24 hours, colonies on both media showed a slow growth rate, with a dry, pale yellow, pinpoint morphology (Fig. 1). These isolates were gram-positive clusters of cocci and presented delayed catalaseand coagulase-positive results. The Vitek 2 system (BioMérieux, Marcy-l’Étoile, France) identified the isolate as
Figure 1. Subculturing of the isolate on blood (A, B, C) and chocolate (D, E, F) agar plates. G. Gram staining of the isolate.
Sequencing of the 16S rRNA gene in conjunction with a GenBank Basic Local Alignment Search Tool (BLAST) search identified the pathogen as
For a more accurate identification of the isolate, WGS using the Illumina MiSeq platform (Illumina, San Diego, CA, USA) and data analysis using the TrueBac ID-Genome system (www.truebacid.com; ChunLab, Inc., Seoul, Korea) were performed . The genome size of the pathogen obtained by WGS was 3,423,857 bp. We further determined a GC content of 71.98%, with an average sequencing depth of 281.8 ×, a total of 62 contigs, and an N50 value of 160,716 bp. The TrueBac ID database search showed that
A phylogenetic tree analysis using the neighbor-joining method with 16S rRNA gene sequences revealed that the newly isolated species (MKL-02) formed a subcluster with
Figure 2. Phylogenetic analysis based on (A) 16S rRNA sequences using the neighbor-joining method or (B) whole-genome sequencing combined with unweighted pair group and arithmetic mean clustering methods as well as average nucleotide identity values. The isolated pathogen (MKL-02) is underlined. Abbreviation: rRNA, ribosomal RNA.
Antimicrobial susceptibility was tested using the Vitek 2 system, AST-ST01. As the Vitek 2 system identified the isolate as
As no susceptibility criteria (MIC or disk diffusion values) are available for
Our contradictory results can be explained by the “90-60 rule.” While 90% of the susceptibility results are expected to predict success, only 60% of the resistance results are expected to do the same .
In clinical microbiology, automated phenotypical identification systems and matrix-assisted laser desorption/ionization time-offlight mass spectrometry (MALDI-TOF MS) have been widely adopted for routine bacterial identification. However, discrepancies among methods are frequently encountered, even if the identification score is high enough to identify organisms at the species level. This problem may originate from overlapping biochemical or proteomic profiles among different strains, the limited number of databases/libraries in the analyzer, or both. Likewise, 16S rRNA gene sequence similarities above cut-off levels (98.7–99.0%) do not guarantee the correct identification of certain strains, as nearly identical 16S rRNA gene sequences have been reported in different species [6, 9]. These species identification difficulties encountered using molecular or phenotypic typing can occur more frequently for strains rarely found in clinical specimens, as in the present case. In such cases, genomebased identification can be useful. Unlike 16S rRNA gene sequencing, WGS provides clear-cut criteria for bacterial classification [10, 11]. The ANI value is most widely used for analyzing genomic sequence similarity and is thus regarded as a possible next-generation gold standard for species delineation. It represents the mean of the similarity values between homologous genomic regions shared by two genes. It is now generally accepted that ANI values of 95–96% equate to a DNA-DNA hybridization value of 70%, which is the accepted standard for bacterial species identification .
In conclusion, we identified a clinical isolate to be a grampositive coccus belonging to a novel
Research conception & design: Lee MK. Data acquisition: Jeong JH, Kweon OJ, Kim HR, Kim TH, and Lee MK. Data analysis and interpretation: Jeong JH, Kweon OJ, and Lee MK. Figures: Ha SM. Drafting of the manuscript: Jeong JH. Critical revision of the manuscript: Lee MK. Approval of the final manuscript: all authors.
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