Logo
homeHome
toggle_offCompany ProfilegroupsBoards & Staffsworkspace_premiumMemberships & PartnersschoolAcademy of Journalismperson_addCareerslinkBlog(EN)emailContact Us
list_altOur JournalslightbulbInstruction for AuthorsbookmarkBrieflands PolicieshandshakePublish My Researchplay_circleJournal Management Systemcredit_cardArticle Processing Chargeszoom_inOpen Peer ReviewimagePublishing Services
menu_bookPublish My Book

Jundishapur Journal of Microbiology

Ahvaz Jundishapur University of Medical Sciences

homeHome
play_arrowCurrent IssuelistAll Issuesformat_indent_increaseIn Presscheck_circleAccepted ManuscriptssearchSearch
lightbulbInstructions
infoJournal InformationgroupsEditors & BoardsshareIndexing and Listing Sourcesshow_chartJournal Metricszoom_inOpen Peer Review (OPR)balancePublication Ethics and Malpractice Statementassignment_indReviewer and AE Registration FormemailSupportphoneContact Us
format_list_bulletedAPC
Authors GuideSubmit Manuscript

Outlines

https://doi.org/10.5812/jjm-121534

Polymorphisms of dtxR Gene of Corynebacterium diphtheriae Isolated from Diphtheria Outbreak in Indonesia

Author(s):
Fitriana FitrianaFitriana Fitriana1, Novi AmaliaNovi Amalia2, Yudi HartoyoYudi Hartoyo2, Sundari NursofiahSundari Nursofiah2, Nelly PuspandariNelly PuspandariNelly Puspandari ORCID2, Khariri KhaririKhariri Khariri1, Kambang SariadjiKambang Sariadji2, Fauzul MunaFauzul Muna2, Yuni RukminiatiYuni RukminiatiYuni Rukminiati ORCID2, Aulia RizkiAulia Rizki2, Dwi FebriyanaDwi FebriyanaDwi Febriyana ORCID2, Tati FebriantiTati FebriantiTati Febrianti ORCID2, Ida SusantiIda Susanti2, Ratih Dian SaraswatiRatih Dian SaraswatiRatih Dian Saraswati ORCID2, Sunarno SunarnoSunarno SunarnoSunarno Sunarno ORCID1,*
1National Research and Innovation Agency, Jakarta, Indonesia
2Center for Health Resilience and Resource Policy, Health Policy Agency, Jakarta, Indonesia
Jundishapur Journal of Microbiology:Vol. 15, issue 4; e121534
Published online:Jun 19, 2022
Article type:Research Article
Received:Dec 02, 2021
Accepted:Jun 01, 2022
How to Cite:Fitriana F, Amalia N, Hartoyo Y, Nursofiah S, Puspandari N, et al. Polymorphisms of dtxR Gene of Corynebacterium diphtheriae Isolated from Diphtheria Outbreak in Indonesia. Jundishapur J Microbiol. 2022;15(4):e121534. doi: https://doi.org/10.5812/jjm-121534

Abstract

Background:

In Corynebacterium diphtheriae, the dtxR gene plays a role in regulating diphtheria toxin synthesis. The dtxR gene is often used as a marker for identifying C. diphtheriae by the polymerase chain reaction (PCR) method because it is present in all strains of this bacterium. Mutations in the dtxR gene can cause the over-synthesis of diphtheria toxin and reduce PCR assays' sensitivity.

Objectives:

This study aimed to describe the polymorphisms in the dtxR gene of C. diphtheriae isolated from a diphtheria outbreak in Indonesia.

Methods:

Forty-eight isolates of C. diphtheriae were obtained from clinical samples (throat/nasopharyngeal swabs) of diphtheria cases and close contacts. The isolates were revived on a blood agar plate (BAP), bacterial colonies were harvested, and deoxyribonucleic acid (DNA) was extracted. The DNA sequencing was carried out using a Whole-genome Sequencing (WGS) approach. The data were converted and analyzed with U-gene software. The dtxR gene analysis was performed with C. diphtheriae PW8 as references.

Results:

There were 59-point mutation locations in 48 isolates examined. None of these single nucleotide polymorphisms (SNPs) coded for amino acid changes. Based on the mutation pattern, seven clades/groups of the dtxR gene of 48 C. diphtheriae isolates were examined.

Conclusions:

At least seven types of DNA sequences and more than 50 SNPs of the dtxR gene were identified in 48 C. diphtheriae isolates from a diphtheria outbreak in Indonesia. Although all of them are silent mutations, they must be considered in the design of PCR examination in diphtheria laboratories.

Keywords
Corynebacterium diphtheriae
DNA
Indonesia
Mutation

1. Background

Indonesia has experienced diphtheria outbreaks over the past few years. Based on the World Health Organization (WHO) data, Indonesia is almost always among the top five countries with the most diphtheria cases in the world (1). Meanwhile, data from the Indonesian Ministry of Health show that diphtheria cases are almost found in all provinces. In 2018, diphtheria cases reported from 28 out of 34 provinces in Indonesia, ranging from one to 385 cases. The case fatality rate (CFR) of diphtheria in Indonesia in 2018 was 2.09% nationally, less than globally (2). Several analyses have linked high diphtheria cases in Indonesia to low immunization coverage (3, 4).
Diphtheria is mainly caused by Corynebacterium diphtheriae, while C. ulcerans and C. pseudotuberculosis cause a small number of cases. The main virulence factor in diphtheria-causing bacteria is the diphtheria toxin. Corynebacterium diphtheriae contains the dtxR gene that plays a role in regulating diphtheria toxin synthesis (5, 6). The dtxR gene is often used as a marker for identifying C. diphtheriae by the polymerase chain reaction (PCR) method because it is present in all strains of this bacterium (7, 8). Mutations in the dtxR gene can cause over-synthesis of diph-theria toxin and reduce the sensitivity of PCR assays.

2. Objectives

This study describes the polymorphisms in the dtxR gene of C. diphtheriae isolated from a diphtheria outbreak in Indonesia.

3. Methods

3.1. Samples

Forty-eight isolates of C. diphtheriae were used as samples in this study in 2017. They were stored by the National Institute of Health Research and Development. The bacterial isolates were isolated from clinical samples (throat/nasopharyngeal swabs) of diphtheria cases and close contacts in several provinces of Indonesia (Table 1). The isolates were revived on a blood agar plate (BAP) and incubated at 37°C for 24 hours. The bacterial colonies were harvested and put into 500 µL aquadest for deoxyribonucleic acid (DNA) extraction. Table 1 shows that most of the samples came from Jakarta and Banten, accounting for about 70.8% of the total sample. In contrast, the least samples came from Aceh, Central Java, East Java, and West Kalimantan, with one isolate each. Some provinces of Indonesia have no representative isolates. Several regions, especially Eastern Indonesia, referred their diphtheria samples to BBLK Surabaya for the examination so that they were not included in the list of isolates examined.
Table 1.Sample Distribution by Province and Year of Isolation
ProvinceYear of IsolationQuantityPercent
1. Jakarta2015, 2016, 20171531.3
2. Banten2010, 2014, 2015, 2016, 20171939.6
3. West Java2016, 2017612.5
4. Central Java201712.1
5. East Java201512.1
6. Aceh201712.1
7. West Kalimantan201312.1
8. Central Kalimantan2012, 201648.3
Total2010, 2012, 2013, 2014, 2015, 2016, 201748100

3.2. DNA Extraction

The DNA extraction was performed using a commercial QiaAmp kit (Qiagen) following the manufacturer's protocol. The extracted DNA was stored in 50 µL molecular-grade water. The DNA quality and quantity were checked using Qubit and gel electrophoresis to meet the MiSeq machine (Illumina) protocol (8-10).

3.3. DNA Sequencing

The DNA sequencing was carried out using a Whole-genome Sequencing (WGS) approach by a MiSeq machine (Illumina). The data were converted and analyzed with U-gene software. The dtxR gene analysis was performed with C. diphtheriae PW8 as references (8-10).

4. Results

There were 59-point mutation locations in 48 isolates (Table 2). None of these SNPs coded for amino acid changes or silent mutations. Based on the mutation pattern, there were seven clades/groups of the dtxR gene in 48 C. diphtheriae isolates from a diphtheria outbreak in Indo-nesia. Table 2 and Figure 1 show that some examined isolates demonstrated similarities to the reference sequence (C. diphtheria PW8). Type 7 was very prominent because there was the larg-est sequence difference compared to other isolates. Figure 1 shows the grouping of C. diphtheriae isolated from several parts of Indonesia based on the mutation pattern.
Table 2.Position of Point Mutations in the dtxR Gene of 48 Corynebacterium diphtheriae Iso-lates
Base PositionsPW8 aType 1Type 2Type 3Type 4Type 5Type 6Type 7
39T......C
54G...A...
66A.....T.
72C......T
75T......C
93T......C
126C..TT...
180C......T
195A......G
198G......A
204C......T
207C......T
210T......G
225T....C.C
234A......G
246T......A
270T......C
273C.T..T..
309C......A
318C......T
321T......C
339A......G
357A......G
358T......C
372T......C
373C......A
393A......G
402T......G
405C......T
414C......A
423C......T
429T......C
456T......C
459T......C
462C......T
471C......A
474C.....T.
493A......G
504T.....A.
507C.....TT
516T.....CC
521T......C
534G......A
537T......C
540A......G
552T......C
558C.....TT
564T.....A.
567T......C
579C.....T.
585T......C
600C......G
603C......T
613A......C
639C....ATG
640C.....AA
652G......A
654T.....C.
663T......C

a Reference

Phylogenetic description based on the mutation pattern of the <i>dtxR</i> gene
Figure 1.

Phylogenetic description based on the mutation pattern of the dtxR gene

5. Discussion

A previous study on the variation of the dtxR gene was conducted by Nakao et al. on isolates from diphtheria outbreaks in Russia and its surroundings using PCR-SSCP (single-strand conformation polymorphisms) (11). The identification results showed 12 types of the dtxR gene. Furthermore, De Zoysa et al. did the same study on a sample from the United Kingdom. The results showed at least four variants of the dtxR gene from 26 isolates examined (12). In Indonesia, a previous study was reported by Mulyastuti et al. with four isolates from the Java and Kalimantan islands. The results showed three variants of the dtxR gene with three mutation locations (13). Furthermore, Sunarno et al. reported 10 partial sequences (162 bases) of the dtxR gene in isolates from the Java and Kalimantan islands. The results showed at least two variants with three mutation locations (14).
The samples examined in this study were only 48 isolates that were not proportional by province (Table 1). It is one of the limitations of this study. However, this is the first study to show a lot of sequence variations (seven types) and DNA mutations (59 SNPs) of the dtxR gene of C. diphtheriae isolated from a diphtheria outbreak in Indonesia (Table 2). This study showed that mutations occurred in approximately 9% of 683 bases in the dtxR gene. Most mutations were found in one isolate (53 S18), which had a similar sequence to the C. diphtheriae strain Dong-yang (CP074413.1) reported from China in 2021, strain CHUV2995 (LT990688.1) reported from Switzerland in 2018, and strain CMCNS703 (CP038789.1) reported from India in 2019 (NCBI Blast: Nucleotide Sequence (nih.gov)). The mutations do not cause changes in amino acids, so it is predicted that they will not affect the function of the protein formed.
Knowledge of dtxR mutations is essential to predict the accuracy of PCR assays for identifying diphtheria-causing bacteria. Several previous studies used the dtxR gene as a marker for diphtheria-causing bacteria. Initially, Pimenta et al. developed a PCR assay to detect C. diphtheriae with the dtxR gene as a marker. The results showed that the dtxR gene could be used to identify toxigenic and non-toxigenic C. diphtheriae (7). Furthermore, De Zoysa et al., Pimenta et al., and Torres et al. also developed a PCR targeting the dtxR gene as a marker of C. diphtheriae (15-17). The dtxR gene is also known as a marker for C. ulcerans and C. pseudotuberculosis.
The dtxR gene is predicted better than other genes. Sunarno et al. showed that the dtxR gene was more conserved than the pld gene and differentiated between species more than the 16s rRNA gene. Therefore, the dtxR gene was used as a target for PCR examination to simultaneously identify three bacterial species causing diphtheria (8, 18). In addition, the dtxR gene functions as a regulator of diphtheria toxin synthesis. Mutations in certain regions have been shown to cause uncontrolled diphtheria toxin synthesis. In this case, the dtxR gene function is closely related to the availability of Fe in the environment where bacteria grow (19, 20). A literature search showed that the mutations found in this study did not affect the effectiveness of the established PCR examination because they were not located at the attachment site of the PCR primer or its probe. How-ever, this is a warning in the PCR examination for diphtheria.

5.1. Conclusions

At least seven types of DNA sequences and more than 50 Single Nucleotide Polymorphisms (SNPs) of the dtxR gene were identified in 48 C. diphtheriae isolates from a diphtheria outbreak in Indonesia. Although all of them are silent mutations, they must be considered in the design of the PCR examination in diphtheria laboratories.

Acknowledgments

Footnotes

References

  • 1.
    World Health Organization. Diphteria Reported Cases. World Health Organization; 2020. Available from: https://apps.who.int/immunization_monitoring/globalsummary/timeseries/tsincidencediphtheria.html.
  • 2.
    Kurniawan R, Hardhana B, Siswanti T. Indonesia Health Profile Year 2018. Jakarta, Indonesia: Kementerian Kesehatan Republik Indonesia; 2019.
  • 3.
    Karyanti MR, Nelwan EJ, Assyidiqie IZ, Satari HI, Hadinegoro SR. Diphtheria epidemiology in Indonesia during 2010-2017. Acta Med Indones. 2019;51(3):205-13. [PubMed ID: 31699943].
  • 4.
    Tosepu R, Gunawan J, Effendy DS, Ahmad OAI, Farzan A. The outbreak of diphtheria in Indonesia. Pan Afr Med J. 2018;31:249. [PubMed ID: 31448005]. [PubMed Central ID: PMC6691314]. https://doi.org/10.11604/pamj.2018.31.249.16629.
  • 5.
    Sharma NC, Efstratiou A, Mokrousov I, Mutreja A, Das B, Ramamurthy T. Diphtheria. Nat Rev Dis Primers. 2019;5(1):81. [PubMed ID: 31804499]. https://doi.org/10.1038/s41572-019-0131-y.
  • 6.
    Tao X, Schiering N, Zeng HY, Ringe D, Murphy JR. Iron, DtxR, and the regulation of diphtheria toxin expression. Mol Microbiol. 1994;14(2):191-7. [PubMed ID: 7830565]. https://doi.org/10.1111/j.1365-2958.1994.tb01280.x.
  • 7.
    Pimenta FP, Hirata R, Rosa ACP, Milagres LG, Mattos-Guaraldi AL. A multiplex PCR assay for simultaneous detection of Corynebacterium diphtheriae and differentiation between non-toxigenic and toxigenic isolates. J Med Microbiol. 2008;57(Pt 11):1438-9. [PubMed ID: 18927428]. https://doi.org/10.1099/jmm.0.2008/000414-0.
  • 8.
    Muna F, Sariadji K, Rukminiati Y, Febriyana D; Sunarno; Khariri, et al. New approach for the identification of potentially toxigenic Corynebacterium sp. using a multiplex PCR assay. J Microbiol Methods. 2021;184:106198. [PubMed ID: 33713727]. https://doi.org/10.1016/j.mimet.2021.106198.
  • 9.
    Sunarno S, Rukminiati Y, Saraswati RD. ST534: the new sequence type of Corynebacterium diphtheriae causing diphteria in Jakarta and surrounding areas, Indonesia. Turk J Med Sci. 2020;50(1):267-70. https://doi.org/10.3906/sag-1909-4.
  • 10.
    Nelwan EJ, Puspandari N, Paramita RI, Erlina L, Renesteen E, Fadilah F. Whole genome sequencing data of Escherichia coli isolated from bloodstream infection patients in Cipto Mangunkusumo National Hospital, Jakarta, Indonesia. Data Brief. 2020;30:105631. [PubMed ID: 32395590]. [PubMed Central ID: PMC7210415]. https://doi.org/10.1016/j.dib.2020.105631.
  • 11.
    Nakao H, Pruckler JM, Mazurova IK, Narvskaia OV, Glushkevich T, Marijevski VF, et al. Heterogeneity of diphtheria toxin gene, tox, and its regulatory element, dtxR, in Corynebacterium diphtheriae strains causing epidemic diphtheria in Russia and Ukraine. J Clin Microbiol. 1996;34(7):1711-6. [PubMed ID: 8784575]. [PubMed Central ID: PMC229100]. https://doi.org/10.1128/JCM.34.7.1711-1716.1996.
  • 12.
    De Zoysa A, Efstratiou A, Hawkey PM. Molecular characterization of diphtheria toxin repressor (dtxR) genes present in nontoxigenic Corynebacterium diphtheriae strains isolated in the United Kingdom. J Clin Microbiol. 2005;43(1):223-8. [PubMed ID: 15634975]. [PubMed Central ID: PMC540142]. https://doi.org/10.1128/JCM.43.1.223-228.2005.
  • 13.
    Mulyastuti Y, Rahayu SI, Sunarno S, Wasito EB. Short Communication: Investigation of Diphtheria in Indonesia: dtxR and tox genes analysis of Corynebacterium diphtheriae collected from outbreaks. Biodiversitas. 2017;18(2):773-9. https://doi.org/10.13057/biodiv/d180247.
  • 14.
    Sunarno S, Mulyastuti Y, Puspandari N, Sariadji K. DNA sequence analysis of dtxR gene (partial) of Corynebacterium diphtheriae causing diphtheria in Jawa and Kalimantan Islands, Indonesia. Indones Biomed J. 2017;9(2). https://doi.org/10.18585/inabj.v9i2.268.
  • 15.
    De Zoysa A, Efstratiou A, Mann G, Harrison TG, Fry NK. Development, validation and implementation of a quadruplex real-time PCR assay for identification of potentially toxigenic corynebacteria. J Med Microbiol. 2016;65(12):1521-7. [PubMed ID: 27902437]. https://doi.org/10.1099/jmm.0.000382.
  • 16.
    Pimenta FP, Matias GA, Pereira GA, Camello TC, Alves GB, Rosa AC, et al. A PCR for dtxR gene: application to diagnosis of non-toxigenic and toxigenic Corynebacterium diphtheriae. Mol Cell Probes. 2008;22(3):189-92. [PubMed ID: 18378421]. https://doi.org/10.1016/j.mcp.2008.01.001.
  • 17.
    Torres Lde F, Ribeiro D, Hirata RJ, Pacheco LG, Souza MC, dos Santos LS, et al. Multiplex polymerase chain reaction to identify and determine the toxigenicity of Corynebacterium spp with zoonotic potential and an overview of human and animal infections. Mem Inst Oswaldo Cruz. 2013;108(3):272-9. [PubMed ID: 23778659]. [PubMed Central ID: PMC4005569]. https://doi.org/10.1590/S0074-02762013000300003.
  • 18.
    Sunarno S, Fitri N, Puspandari N, Sariadji K. The dtxR Gene: A New Alternative Marker to Identify Corynebacterium ulcerans and Corynebacterium pseudotuberculosis by PCR Assay. Indones Biomed J. 2018;10(3):236-42. https://doi.org/10.18585/inabj.v10i3.403.
  • 19.
    Qian Y, Lee JH, Holmes RK. Identification of a DtxR-regulated operon that is essential for siderophore-dependent iron uptake in Corynebacterium diphtheriae. J Bacteriol. 2002;184(17):4846-56. [PubMed ID: 12169610]. [PubMed Central ID: PMC135300]. https://doi.org/10.1128/JB.184.17.4846-4856.2002.
  • 20.
    Yellaboina S, Ranjan S, Chakhaiyar P, Hasnain SE, Ranjan A. Prediction of DtxR regulon: identification of binding sites and operons controlled by Diphtheria toxin repressor in Corynebacterium diphtheriae. BMC Microbiol. 2004;4:38. [PubMed ID: 15447793]. [PubMed Central ID: PMC524172]. https://doi.org/10.1186/1471-2180-4-38.
Import into EndNoteImport into BibTex
Indexed in
logoWeb of Sciences Core Collections
logoScopus
Crossmark
Crossmark
Checking
Share on
Comments
Number of Comments:0
Cited by
Metrics
Get Permission (article level)

Purchasing Reprints

  • Copyright Clearance Center (CCC) handles bulk orders for article reprints for Brieflands. To place an order for reprints, please click here (   https://www.copyright.com/landing/reprintsinquiryform/ ). Clicking this link will bring you to a CCC request form where you can provide the details of your order. Once complete, please click the ‘Submit Request’ button and CCC’s Reprints Services team will generate a quote for your review.
Search Relations

Author(s):

Related Articles

Related Article in PubMed

Related Article in Google Scholar

Create Citation Alert via Google Reader