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BRIEF COMMUNICATION
Year : 2019  |  Volume : 37  |  Issue : 3  |  Page : 423-425
 

Insights to the diphtheria toxin encoding prophages amongst clinical isolates of Corynebacterium diphtheriae from India


1 Department of Clinical Microbiology, Christian Medical College, Vellore, India
2 Department of Paediatrics, Christian Medical College, Vellore, India
3 World Health Organization Country Office, New Delhi, India
4 King George's Medical University, Lucknow, Uttar Pradesh, India
5 State Public Health Laboratory, Thiruvananthapuram, India
6 Maharishi Valmiki Infectious Diseases Hospital, New Delhi, India
7 Choithram Hospital and Research Centre, Indore, Madhya Pradesh, India

Date of Submission12-Dec-2019
Date of Decision12-Dec-2019
Date of Acceptance22-Dec-2019
Date of Web Publication29-Jan-2020

Correspondence Address:
Dr. Shalini Anandan
Department of Clinical Microbiology, Christian Medical College, Vellore - 632 004, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmm.IJMM_19_469

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 ~ Abstract 


Diphtheria is a dreadful disease caused by Corynebacterium diphtheriae. Lysogenised bacteriophages carrying toxin gene in C. diphtheriae can make the strain toxigenic. However, such phage disseminates the toxin genes to other strains when it undergoes lytic phase. As little is known about the phage diversity in C. diphtheriae in India, the present study was undertaken to investigate the prophages integrated into the genome of 29 clinical isolates of C. diphtheriae using whole-genome shotgun sequencing. Amongst these isolates, 27 were toxigenic, while 2 were non-toxigenic strains. Of the 27 toxigenic strains, all harbored known phages carrying toxin gene and two other phages with unknown function. However, the two non-toxin strains did not harbour any of the phages in the genome. It is imperative to devise prevention strategies that hinder the dissemination of toxin by prophages, as it may increase the complications of diphtheria post-immunisation.


Keywords: Bacteriophage, Corynebacterium diphtheriae, non-toxigenic, toxin, whole-genome sequencing


How to cite this article:
Muthuirulandi Sethuvel DP, Subramanian N, Pragasam AK, Inbanathan FY, Gupta P, Johnson J, Sharma NC, Hemvani N, Veeraraghavan B, Anandan S, Sangal L. Insights to the diphtheria toxin encoding prophages amongst clinical isolates of Corynebacterium diphtheriae from India. Indian J Med Microbiol 2019;37:423-5

How to cite this URL:
Muthuirulandi Sethuvel DP, Subramanian N, Pragasam AK, Inbanathan FY, Gupta P, Johnson J, Sharma NC, Hemvani N, Veeraraghavan B, Anandan S, Sangal L. Insights to the diphtheria toxin encoding prophages amongst clinical isolates of Corynebacterium diphtheriae from India. Indian J Med Microbiol [serial online] 2019 [cited 2020 Apr 7];37:423-5. Available from: http://www.ijmm.org/text.asp?2019/37/3/423/277074





 ~ Introduction Top


Diphtheria is a dreadful disease caused by Corynebacterium diphtheriae. World's 80% of Diphtheria cases are seen in India.[1] Diphtheria is a vaccine-preventable bacterial infection that kills many despite immunisation. There are several cases reported across India in the recent times.[2],[3],[4],[5],[6] The toxigenic phenotype of C. diphtheriae is generally determined by temperate corynephages whose genomes carry tox gene that encodes diphtheria toxin. The pathogen is known to be lysogenised with bacteriophage (corynephage beta, omega and gamma) harbouring the toxin gene, theoretically when undergoes lytic phase can disseminate the toxin gene.[7] The present study investigates the prophages integrated into the genome of clinical isolates of C. diphtheriae using whole-genome shotgun sequencing.


 ~ Methodology Top


During the years 2015 and 2017, a total of 343 diphtheria suspected cases were included in this study. The specimens were screened for the causative agent C. diphtheriae by culture and real-time polymerase chain reaction as described previously.[8] The isolates were biochemically confirmed for C. diphtheriae. The toxigenicity testing was performed by Elek's test according to the previously reported method.[9] Further, whole-genome shotgun sequencing was done using Ion torrent PGM (Life Technologies, CA, USA) with 400 bp library fragmentation protocol as described by the manufacturer. The raw reads were assembled using SPAdes 5.0.0.0 embedded in Ion torrent Server. The genome sequence was annotated using the NCBI Prokaryotic Genome Annotation Pipeline (http://www.ncbi.nlm.nih.gov/genomes/static/Pipeline.html). The annotated genomes were screened for prophages through PHAge Search Tool Enhanced Release (PHASTER) analysis.[10],[11]


 ~ Results and Discussion Top


Amongst the 343 specimens screened, 149 (43%) were positive for C. diphtheriae with 9% being toxin negative (13/149). Amongst this, a total of 29 isolates of C. diphtheriae were randomly chosen. About 27 (93%) isolates harboured toxin genes and were positive for Elek's test, whereas 2 (7%) isolates were negative by both methods. PHASTER analysis of the genome sequences revealed diverse phages that are integrated into the genome of the study isolates.

Bacteriophage Gordon GMA4 (NC030939) was found to be an intact prophage and seen lysogenised among 35% (n = 9) of the isolates, encodes the toxin gene within the attachment sites. Around 31% (n = 8) of the isolates were lysogenised with bacteriophage Gordon Gsput1 (NC030932), encoding the toxin within its genome. Due to the integrity of these phage genomes, the lytic properties are believed to exist which increases the vulnerability of the phage to undergo lytic cycle and disseminate the toxin gene to other non-toxigenic C. diphtheria.[12] Incomplete prophage elements of Bacteriophages Gordon CaptainKirk2 (NC031072) and Gordon BritBrat (NC030942) were seen among 5 (19%) toxigenic C. diphtheriae [Table 1]. These phages did not code for the toxin gene but were adjacent to the toxin gene and few hypothetical proteins on the bacterial genome. This indicates the possibility of novel bacteriophages yet to be characterised that might encode the toxin genes within its genome.
Table 1: Description of the toxin encoding prophages and other prophages seen in the study isolates of Corynebacterium diphtheriae and control strains of Corynebacterium diphtheriae by PHAge search tool enhanced release analysis

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The prophages seen in the toxigenic strains had high sequence similarities with the previously reported corynephages of C. diphtheriae NCTC 13129, C. diphtheriae C7 (beta) and C. diphtheriae PW8 [Figure 1]. The remaining 2 (7.7%) toxigenic isolates did not harbour any prophage elements to be associated to the toxin gene but had transposase encoding gene, of which the function remains uncertain. The non-toxigenic strains did not have any of the toxin encoding phage elements, but had incomplete phage elements (<16 kb) and had no correlation with the prophages mentioned amongst the toxigenic strains. The absence of intact prophages in the non-toxigenic strains makes it highly susceptible for phage infection, consecutively explains the theoretical toxin production by lytic phages in non-toxigenic strains of C. diphtheriae. Besides, carriage of non-toxigenic strains in healthy individuals, as part of the normal upper respiratory tract flora can act as a potential reservoir that can undergo phage conversion and dissemination.[13]
Figure 1: Comparison of prophages in the reference strains and study isolates of Corynebacterium diphtheria

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 ~ Conclusion Top


This is the first report from India on the diverse toxin encoding prophages of C. diphtheriae. It is imperative to devise prevention strategies that hinder the dissemination of toxin by prophages, as it increases the complications of diphtheria post-immunisation. Genomic evidence of the absence of intact phages provides a possible explanation for clinical diphtheria caused by non-toxigenic strains of C. diphtheria e, creating a need for treatment strategies that hinder the replication of bacteriophages and thereby eliminates the possible acquisition of toxin gene in the future.

Financial support and sponsorship

This work was funded by the World Health Organisation Country Office, New Delhi, India.

Conflicts of interest

There are no conflicts of interest.



 
 ~ References Top

1.
Dandinarasaiah M, Vikram BK, Krishnamurthy N, Chetan AC, Jain A. Diphtheria Re-emergence: Problems faced by developing countries. Indian J Otolaryngol Head Neck Surg 2013;65:314-8.  Back to cited text no. 1
    
2.
Bhagat S, Grover SS, Gupta N, Roy RD, Khare S. Persistence of Corynebacterium diphtheriae in Delhi & National Capital Region (NCR). Indian J Med Res 2015;142:459-61.  Back to cited text no. 2
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3.
Jain A, Samdani S, Meena V, Sharma MP. Diphtheria: It is still prevalent!!! Int J Pediatr Otorhinolaryngol 2016;86:68-71.  Back to cited text no. 3
    
4.
Patil N, Gawade N, Gaidhane A, Syed ZQ. Investigating diphtheria outbreak: A qualitative study in rural area. Int J Med Sci Public Health 2014;3:1.  Back to cited text no. 4
    
5.
Iyer V, Azhar GS, Choudhury N, Dhruwey VS, Dacombe R, Upadhyay A. Infectious disease burden in Gujarat (2005-2011): Comparison of selected infectious disease rates with India. Emerg Health Threats J 2014;7:22838.  Back to cited text no. 5
    
6.
Sangal L, Joshi S, Anandan S, Balaji V, Johnson J, Satapathy A, et al. Resurgence of diphtheria in North Kerala, India, 2016: Laboratory supported case-based surveillance outcomes. Front Public Health 2017;5:218.  Back to cited text no. 6
    
7.
Holmes RK. Biology and molecular epidemiology of diphtheria toxin and the Tox gene. J Infect Dis 2000;181 Suppl 1:S156-67.  Back to cited text no. 7
    
8.
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;65:1521-7.  Back to cited text no. 8
    
9.
Engler KH, Glushkevich T, Mazurova IK, George RC, Efstratiou A. A modified Elek test for detection of toxigenic corynebacteria in the diagnostic laboratory. J Clin Microbiol 1997;35:495-8.  Back to cited text no. 9
    
10.
Arndt D, Grant JR, Marcu A, Sajed T, Pon A, Liang Y, et al. PHASTER: A better, faster version of the PHAST phage search tool. Nucleic Acids Res 2016;44:W16-21.  Back to cited text no. 10
    
11.
Zhou Y, Liang Y, Lynch KH, Dennis JJ, Wishart DS. PHAST: A fast phage search tool. Nucleic Acids Res 2011;39:W347-52.  Back to cited text no. 11
    
12.
Abedon ST, Lejeune JT. Why bacteriophage encode exotoxins and other virulence factors. Evol Bioinform Online 2007;1:97-110.  Back to cited text no. 12
    
13.
Sangal V, Hoskisson PA. Evolution, epidemiology and diversity of Corynebacterium diphtheriae: New perspectives on an old foe. Infect Genet Evol 2016;43:364-70.  Back to cited text no. 13
    


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