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Open AccessArticle

Arthrobacter woluwensis Bacteremia: A Clinical and Genomic Report

1
Department of Infectious Diseases, Taichung Veterans General Hospital, Taichung 40705, Taiwan
2
Division of Infectious Disease, Department of Internal Medicine, Tungs’ Taichung Metroharbor Hospital, Taichung 433402, Taiwan
3
Division of Infectious Diseases, Department of Internal Medicine, E-Da Hospital, Kaohsiung 840, Taiwan
4
School of Medicine, College of Medicine, I-Shou University, Kaohsiung 840, Taiwan
5
Department of Computer Science and Information Engineering, National Chung Cheng University, Chiayi 62102, Taiwan
6
Division of Clinical Toxicology, Department of Emergency Medicine, Taichung Veterans General Hospital, Taichung 40705, Taiwan
7
School of Medicine, National Defense Medical Center, Taipei 11490, Taiwan
8
Rong Hsing Research Center for Translational Medicine, National Chung Hsing University, Taichung 402, Taiwan
9
Ph.D. Program in Translational Medicine, National Chung Hsing University, Taichung 402, Taiwan
*
Authors to whom correspondence should be addressed.
Shu-Yuan Li and Chin-Chuan Kao contributed equally to this work.
Academic Editor: Carmelo Biondo
Pathogens 2021, 10(4), 443; https://doi.org/10.3390/pathogens10040443
Received: 15 January 2021 / Revised: 28 March 2021 / Accepted: 5 April 2021 / Published: 8 April 2021
(This article belongs to the Special Issue Advances in Human Pathogens Infections)

Abstract

Arthrobacter woluwensis is a Gram-positive, aerobic Actinobacteria that is widely distributed in the environment worldwide. Little is known about A. woluwensis infection and it is commonly mis-identified by culturing with commercial kits. To date, only six cases of bacteremia caused by A. woluwensis have been reported in the literature. Herein, we report a case of Arthrobacter woluwensis bacteremia in an immunocompromised host. In this case report, the results of antimicrobial susceptibility testing showed that this clinical isolate of A. woluwensis is sensitive to vancomycin, teicoplanin, but resistant to penicillin, cephalosporin and ciprofloxacin. Additionally, whole genome sequencing analysis identified common subunits of the urease system.
Keywords: Arthrobacter woluwensis; bacteremia; ureC Arthrobacter woluwensis; bacteremia; ureC

1. Introduction

Arthrobacter woluwensis is a Gram-positive, aerobic Actinobacteria that is widely distributed in the environment, mainly in soil, and it sometimes represents the majority of single bacterial groups in aerobic plate counts of soil specimens [1]. Arthrobacter strains have relatively low pathogenic potential but they can be pathogenic to immunocompromised hosts. Arthrobacter spp. was first considered as a Corynebacterium in 1896 when the species Corynebacterium was first proposed, but it was later assessed to be a distinct species [2]. Considering its rarity and similarity to other corynebacteriae, it is commonly mis-identified in culturing by commercial kits, thus, proper identification often requires assistance from molecular biological methods, such as chemotaxonomic methods or ribosomal RNA/DNA sequencing. To the best of our knowledge, only six cases of bacteremia related to Arthrobacter woluwensis have been reported to date [1,3,4,5,6,7].
Here, we present a case of a newly diagnosed gastric cancer patient who suffered from Arthrobacter woluwensis bacteremia during their hospital admission.

1.1. Specimen Collection and Antibiotic Susceptibility Testing

A 93-year-old man received total gastrectomy, D1 lymph node dissection, and Roux-en-Y anastomosis due to adenocarcinoma of the stomach and developed a postoperative wound infection. Blood culture from peripheral blood yielded unidentified Gram-positive bacillus. The strain was designated as QTS. Antimicrobial susceptibility testing of the isolate was performed with VITEK2 (bioMérieux) and the strain was susceptible to penicillin, vancomycin, trimethoprim/sulfamethoxazole (TMP/SMX), and resistant to ciprofloxacin, clindamycin, and gentamicin. E test MIC values of penicillin, vancomycin, amikacin and daptomycin were 0.75 µg/mL, 1.0 µg/mL, 2 µg/mL and 3 µg/mL.

1.2. Genome Sequencing, Assembly, Annotation, and Phylogenetic Analysis

One colony of trypticase soy agar with 5% sheep blood was transferred into 5 mL of Müller–Hinton medium and incubated overnight at 37 °C. DNA isolation was done according to the manufacturer’s specifications with the QIAGEN Genomic-tip 100/G100 kit and the Genomic DNA Buffer (Gentra Bio, Paisley, UK), measuring the DNA concentration with a Qubit 2.0 fluorometer (Life Technologies, Carlsbad, US). The QTS whole genome was sequenced by Oxford Nanopore GridION using R9.4 flow cell at 122x coverage. The raw signals were basecalled by Guppy 3.4 into long reads. The adaptors that remained in the long reads were trimmed by Porechop. These clean reads were assembled by Flye v. 2.6 into a circular chromosome of 3.68 Mbp. The sequencing errors left on the genome were polished by four runs of Racon, one run of Medaka, and finally, one run of Homopolish. The resulting genome nearly reached 100% CheckM completeness. The protein-coding genes in the QTS genome were annotated via the NCBI Prokaryotic Genome Annotation Pipeline (PGAP). Virulence factors in the genome were identified by DIAMOND alignment against the virulence factor database (VFDB). Whole-genome average nucleotide identity (ANI) was computed by OrthoANI. The phylogeny of QTS and other related genomes was reconstructed by MEGA X. The circular comparative genome map was plotted by Circos.

2. Result

The information regarding gene sequencing and assembly is summarized in Table 1. The genomic contents of the QTS genome are illustrated in Figure 1. The phylogenetic tree based on the 16S rRNA sequence and ANI was constructed to show the phylogenetic position of A. woluwensis QTS (Figure 2). A. woluwensis QTS is closely related to other A. woluwensis strains in terms of nucleotide sequences, sharing an ANI > 96%. As shown in Figure 3, the alignment revealed an obvious syntenic relationship between strains QTS and DSM 10495. Candidate virulence genes are presented in the Supplementary Materials, Table S1, including urease operon. Common subunits of the urease system were identified, including ureA, ureC, ureD, ureE, ureF and ureG [8].

3. Discussion

Pubmed was used to search the literature published before March 2021 with the keywords: Arthrobacter woluwensis, Arthrobacter, and bacteremia. The characteristics of patients, diagnosis, treatment and outcome are summarized in Table 2.
Four of six patients in the literature review demonstrated features of poor immunity, including AIDS, terminal colon cancer, extreme old age and multiple myeloma under chemotherapy treatment. In 3 of 5 patients, a central venous catheter was in place when bacteremia occurred. Case 3 and 6 were intravenous drug users and vulnerable to bloodstream infection.
The antimicrobial susceptibility is summarized in Table 3. Most isolates showed sensitivity to vancomycin, teicoplanin, tetracycline and showed resistance to penicillin, cephalosporin, gentamicin and ciprofloxacin.
In the literature review, patients with immunocompromised status and central venous catheter insertion were vulnerable to A. woluwensis bacteremia. Arthrobacter strains have been isolated from aqueous/vitreous fluid [9], placenta [10], inguinal lymph node [11], implantable cardioverter defibrillator cultures [12], wound swab [13], urine, cervix, vaginal swab, neck abscess [14], and peritoneal dialysis fluid [15]. A. cumminisii and A. oxydans were the most common species found in human clinical specimens [14]. Despite being found in various organs in the human body, they rarely cause infections, indicating the low pathogenicity of Arthrobacter spp.
In the present study, commercial kits failed to identify Arthrobacter spp. Further phenotypic features and genotyping methods may help with more precise identification. Due to the lack of precision of the identification methodologies, the true incidence of infection caused by Arthrobacter spp. may be underestimated.
The patients mentioned in the literature were treated successfully with ampicillin, vancomycin, linezolid, and teicoplanin. However, MIC results were variable and antibiotic resistance to β-lactam and ciprofloxacin was found. Relatively high vancomycin MIC was observed in some isolates. Empirical therapy for these cases can be challenging before MIC results are available. In most of the patients, a central venous catheter was in place when A. woluwensis bacteremia occurred. Multidimensional central-line bundle care and catheter removal may be an essential measure in the prevention and treatment of A. woluwensis-related infection. It was reported that central line-associated bloodstream infection was significantly reduced by 12.2% after care bundle implementation in intensive care units in Taiwan [16].
Urea hydrolysis has been observed in Arthrobacter woluwensis isolates in previous reports [1,3,4,6] and in some other Arthrobacter spp. [1,17]. Previous studies have reported that bacterial urease and associated proteins were encoded by ureA, ureB, ureC, ureD, ureE, ureF and ureG, as shown in Figure 4 [8]. UreC gene was found in the isolate of this report. Friedrich et al. reported ureC gene was more frequent among Shiga toxin-producing E. coli (STEC). The clinical STEC isolates contained ureC but seldom expressed urease activity [18]. Urease is an important factor for Helicobacter pylori colonization in human gastric mucosa [19]. Hence, ureC gene could be used for H. pylori detection and was a potential target for vaccine production and therapeutic antibody [20,21,22]. The association between urease genes and the virulence of Arthrobacter spp. is still unclear.

4. Conclusions

Here, we report a case of Arthrobacter woluwensis bacteremia in an immunocompromised host treated successfully with ampicillin and catheter removal in Taiwan. Whole genome sequencing identified common subunits of the urease system.

Supplementary Materials

The following are available online at https://www.mdpi.com/article/10.3390/pathogens10040443/s1, Table S1: Candidate virulence genes.

Author Contributions

Conceptualization, P.-Y.L.; Data curation, S.-Y.L., C.-H.L. and Y.-T.H.; Formal analysis, Y.-T.H. and P.-Y.L.; Funding acquisition, C.-C.K. and Y.-C.M.; Investigation, C.-H.L., Y.-C.M. and P.-Y.L.; Methodology, C.-H.L. and Y.-C.M.; Project administration, Y.-P.J.; Resources, C.-C.K.; Software, Y.-P.J. and Y.-C.M.; Validation, Y.-P.J.; Visualization, Y.-P.J.; Writing—original draft, S.-Y.L., Y.-C.H., Y.-T.H. and P.-Y.L.; Writing—review & editing, C.-C.K. and Y.-T.H. All authors have read and agreed to the published version of the manuscript.

Funding

Y.-T.H. was supported in part by the Ministry of Science and Technology (109-2221-E-194-038-MY3). P.-Y.L. was supported in part by the by the Ministry of Science and Technology (109-2314-B-075A-009) and Taichung Veterans General Hospital (TCVGH-1103901C).

Institutional Review Board Statement

The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board of Taichung Veterans General Hospital (CE210007A).

Informed Consent Statement

Informed consent was obtained from all subjects involved in the study.

Data Availability Statement

This Whole Genome Shotgun project has been deposited at GenBank under the accession CP049819.

Acknowledgments

We would like to thank Pan Lee for the support in Tokyo.

Conflicts of Interest

The authors declare no conflict of interest.

References

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Figure 1. A graphical circular map of Arthrobacter woluwensis QTS. Graphical depiction from outside to the center: DNA coordinate, protein-coding genes on forward strand and reverse strand (colored by COG categories), tRNA gene, rRNA gene, GC content and GC skew.
Figure 1. A graphical circular map of Arthrobacter woluwensis QTS. Graphical depiction from outside to the center: DNA coordinate, protein-coding genes on forward strand and reverse strand (colored by COG categories), tRNA gene, rRNA gene, GC content and GC skew.
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Figure 2. Phylogenetic analysis using 16S rRNA and ANI. (a) Phylogeny reconstruction using 16S rRNA of QTS and seven closely-related species under the Arthrobacter genus. Shewanella algae MARS14 is used as an outgroup. The bootstrap values and branch length are shown on the nodes and edge, respectively. (b) Phylogenetic cluster using pairwise ANI between the eight Arthrobacter species and the outgroup genomes. The heat map contains pairwise ANI of any two genomes. QTS shares >96% ANI with four A woluwensis genomes.
Figure 2. Phylogenetic analysis using 16S rRNA and ANI. (a) Phylogeny reconstruction using 16S rRNA of QTS and seven closely-related species under the Arthrobacter genus. Shewanella algae MARS14 is used as an outgroup. The bootstrap values and branch length are shown on the nodes and edge, respectively. (b) Phylogenetic cluster using pairwise ANI between the eight Arthrobacter species and the outgroup genomes. The heat map contains pairwise ANI of any two genomes. QTS shares >96% ANI with four A woluwensis genomes.
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Figure 3. Circos plot of genomes of A.woluwensis QTS and A.woluwensis DSM 10495.
Figure 3. Circos plot of genomes of A.woluwensis QTS and A.woluwensis DSM 10495.
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Figure 4. Arthrobacter woluwensis QTS and several Arthrobacter spp. contained a similar gene structure associated with urea hydrolysis.
Figure 4. Arthrobacter woluwensis QTS and several Arthrobacter spp. contained a similar gene structure associated with urea hydrolysis.
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Table 1. Sequencing and assembly.
Table 1. Sequencing and assembly.
StrainsCoverageGenome SizeGenesSequencing TechnologyAssembly MethodCountryHostIsolation SourceDNAAccession Number
QTS122.0x36809713369Oxford Nanopore GridIONFlye v. 2.6TaiwanHomo speciesBloodCircularCP049819
Table 2. Reported cases of Arthrobacter woluwensis bacteremia.
Table 2. Reported cases of Arthrobacter woluwensis bacteremia.
Case No.Age/SexUnderlying ConditionDiagnosisSpecimenTreatmentRisk FactorOutcome
1 [1]33FHIV infection, stage C-3BacteremiaBlood2 weeks ampicillinPort-A catheterSurvival
2 [6]56MMetastatic colon cancerBacteremiaBloodVancomycin, catheter removalSubclavian catheterDied 1
3 [3]39MIVDU, TBMitral valve endocarditisBlood6 weeks teicoplaninIVDUSurvival
4 [4]91FIschemic strokeBacteremiaBlood10 days linezolidHospital acquired infectionSurvival
5 [5]76FMultiple myeloma, HTN, DMBacteremiaBlood19 days teicoplanin, catheter removalChemoportSurvival
6 [7]52Hepatitis CMitral and aortic valve endocarditisIntraoperative samples and bloodOperation 2, Teicoplanin, TMP/SMX, linezolid 3IVDUSurvival
Present case93MCAD, HTN, prostate cancer, newly diagnosised gastric cancerBacteremiaBlood2 weeks ampicillinCVC, post-OP woundSurvival
1 Died due to underlying malignancy. 2 Mitral valve replacement with a biological prosthetic valve and an aortic vegetectomy. 3 Teicoplanin + TMP/SMX after operation, switch to linezolid and TMP/SMX before discharge, total 4 weeks course of treatment following operation. HIV: human immunodeficiency virus; IVDU: Intravenous drug user; TB: tuberculosis; HTN: hypertension; DM: diabetes mellitus; TMP/SMX: Trimethoprim/sulfamethoxazole; CAD: coronary artery disease; CVC: central venous catheter.
Table 3. Antimicrobial susceptibility profile of Arthrobacter woluwensis in reported cases.
Table 3. Antimicrobial susceptibility profile of Arthrobacter woluwensis in reported cases.
CaseAMCAMCROCXMCECIPCLIERYGMIPMPERIFTECTETVADAPLIN
1 [1]R #RRRRRRRRRR(4 *)RSSS(2)
2 [6] R(4) S(1.5)
3 [3] RRRRRI I R(4)SSSS(2)
4 [4] SSS SIS S S
5 [5] IR I(1) S(2)
Present case RR R I(0.75) S(1)R
AMC, amoxicillin-clavulanic acid; AM, ampicillin; CRO, ceftriaxone; CXM, cefuroxime; CE, cefalothin; CIP, ciprofloxacin; CLI, clindamycin; ERY, erythromycin; GM, gentamicin; IPM, imipenem; PE, penicillin G; RIF, rifampin; TEC, teicoplanin; TET, tetracycline; VA, vancomycin; DAP, daptomycin; LIN, linezolid. # According to CLSI breakpoints. * (): MIC result.
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