PLOS ONE

RESEARCH ARTICLE

Divergent evolution of Corynebacterium

diphtheriae in India: An update from National
Diphtheria Surveillance network
Naveen Kumar Devanga Ragupathi ID1,2☯, Dhiviya Prabaa Muthuirulandi Sethuvel1☯,
Dhivya Murugan1, Ranjini Ranjan1, Vikas Gautam ID3, Prashanth Gupta ID4,
Jaichand Johnson5, Naresh Chand Sharma ID6, Ankur Mutreja7,8, Pradeep Haldar ID9,
Arun Kumar ID10, Pankaj Bhatnagar ID10, Lucky Sangal10, Balaji Veeraraghavan ID1*
1 Department of Clinical Microbiology, Christian Medical College, Vellore, India, 2 Department of Chemical
a1111111111
and Biological Engineering, The University of Sheffield, Sheffield, United Kingdom, 3 Department of Medical
a1111111111 Microbiology, Postgraduate Institute of Medical Education & Research, Chandigarh, India, 4 King George’s
a1111111111 Medical University, Lucknow, Uttar Pradesh, India, 5 State Public Health Laboratory, Thiruvananthapuram,
a1111111111 India, 6 Maharishi Valmiki Infectious Diseases Hospital, New Delhi, India, 7 Department of Medicine,
a1111111111 Addenbrookes Hospital, University of Cambridge, Cambridge, United Kingdom, 8 Translational Health
Science and Technology Institute (THSTI), Delhi-NCR, India, 9 Ministry of Health and Family Welfare,
Government of India, New Delhi, India, 10 World Health Organization, New Delhi, India

☯ These authors contributed equally to this work.

* vbalaji@cmcvellore.ac.in
OPEN ACCESS

Citation: Devanga Ragupathi NK, Muthuirulandi

Sethuvel DP, Murugan D, Ranjan R, Gautam V,
Gupta P, et al. (2021) Divergent evolution of
Abstract
Corynebacterium diphtheriae in India: An update
Diphtheria is caused by a toxigenic bacterium Corynebacterium diphtheria which is being an
from National Diphtheria Surveillance network.
PLoS ONE 16(12): e0261435. https://doi.org/ emerging pathogen in India. Since diphtheria morbidity and mortality continues to be high in
10.1371/journal.pone.0261435 the country, the present study aimed to study the molecular epidemiology of C. diphtheriae
Editor: Yung-Fu Chang, Cornell University, UNITED strains from India. A total of 441 diphtheria suspected specimens collected as part of the
STATES surveillance programme between 2015 and 2020 were studied. All the isolates were con-
Received: September 7, 2021 firmed as C. diphtheriae with standard biochemical tests, ELEK’s test, and real-time PCR.
Antimicrobial susceptibility testing for the subset of isolates showed intermediate suscepti-
Accepted: December 1, 2021
bility to penicillin and complete susceptible to erythromycin and cefotaxime. Isolates were
Published: December 15, 2021
characterized using multi locus sequence typing method. MLST analysis for the 216 C.
Peer Review History: PLOS recognizes the diphtheriae isolates revealed major diversity among the sequence types. A total of 34 STs
benefits of transparency in the peer review
were assigned with majority of the isolates belonged to ST466 (30%). The second most
process; therefore, we enable the publication of
all of the content of peer review and author common ST identified was ST405 that was present in 14% of the isolates. The international
responses alongside final, published articles. The clone ST50 was also seen. The identified STs were grouped into 8 different clonal com-
editorial history of this article is available here: plexes (CC). The majority belongs to CC5 followed by CC466, CC574 and CC209, however
https://doi.org/10.1371/journal.pone.0261435
a single non-toxigenic strain belongs to CC42. This epidemiological analysis revealed the
Copyright: © 2021 Devanga Ragupathi et al. This is emergence of novel STs and the clones with better dissemination properties. This study has
an open access article distributed under the terms
also provided information on the circulating strains of C. diphtheriae among the different
of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and regions of India. The molecular data generated through surveillance system can be utilized
reproduction in any medium, provided the original for further actions in concern.
author and source are credited.

Data Availability Statement: All relevant data are

within the manuscript and its supporting
information files.

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PLOS ONE Divergent evolution of Corynebacterium diphtheriae

Funding: The study was funded by World Health 1. Introduction

Organization, Country Office, New Delhi, India
(Ref. No: 2014/488695). Diphtheria is a vaccine preventable disease caused by the human pathogen toxigenic strains of
Corynebacterium diphtheriae, which causes severe infections like myocarditis and peripheral
Competing interests: The authors declare that no
competing interest exist.
neuropathy. Humans are the only known reservoir of C. diphtheriae [1]. Whereas the other
two toxin producing Corynebacterium species such as Corynebacterium ulcerans and Coryne-
bacterium pseudotuberculosis are of zoonotic origin. Non-toxigenic strains of C. diphtheriae
can also cause disease with less severity. Diphtheria, if not detected early and treated promptly
can lead to significant mortality and morbidity [2]. According to WHO, India reported the
highest number of diphtheria cases with more than half of the overall global diphtheria cases
between 1980 and 2019 [3].
Recently, effective vaccination with diphtheria toxoid has reduced the mortality and mor-
bidity of diphtheria infection globally. However, fully vaccinated individuals also get affected
for unknown reasons including waning immunity, personal health, nutritional status, and
infection due to non-toxigenic or variant strains [1]. In 2014, the Government of India
launched “Mission Indradhanush” to strengthen the immunization programme and to rapidly
achieve full immunization coverage of all children and pregnant women against seven vaccine
preventable diseases including diphtheria. In 2015, WHO India also initiated a national sur-
veillance programme on vaccine preventable diseases. In which, Christian Medical College,
Vellore, acts as a National reference laboratory (NRL) for diphtheria and pertussis
surveillance.
Outbreaks associated with displaced population, socioeconomic conditions of the people
and vaccination rates emphasize the continued threat posed by diphtheria. Sporadic outbreaks
of diphtheria have continued to be reported from different parts of India among both vacci-
nated and non-vaccinated individuals [2, 4, 5]. Information on the clonality of C. diphtheriae
strains is not well established in India. Previous MLST studies have shown the dominance of
certain clones in a specific geographical area [1]. Preliminary investigations in India have
reported novel sequence types in most places including the outbreak reported in Northern
Kerala in 2016 [6, 7]. This investigation shows that the genome of C. diphtheriae is constantly
evolving and necessitates the monitoring of the spread and evolutional changes in endemic
region like India. Owing to the continuous diphtheria outbreak in the country, the present
study investigated the molecular epidemiology of geographically diverse collection of C.
diphtheriae strains from India.

2. Results
2.1. Culture, AST and real-time PCR
A total of 441 diphtheria suspected specimens [isolates (n = 204); throat swabs (n = 218); other
samples (n = 19)] were received and screened for diphtheria by both culture and real-time
PCR. These included samples collected at study participating centres. All these samples were
confirmed at the NRL for diphtheria at CMC, Vellore. Detailed breakdown of positives by cul-
ture and PCR were given in S1 Table. Out of 218 swabs received, 32 were culture positive and
43 were PCR positive for C. diphtheriae. One isolate was identified to be toxin (toxA) negative
by PCR. Of the 204 isolates received from participating centres, 180 were confirmed as C.
diphtheriae by PCR with 8.3% (n = 15) toxA negative isolates. Among the 19 other samples, 10
were found to be positive, of which 7 were toxin negative. Antimicrobial susceptibility testing
for the subset of 33 isolates (CMC) showed intermediate susceptibility to penicillin. Whereas
all tested isolates were susceptible to erythromycin and cefotaxime according to CLSI-M45
guidelines.

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PLOS ONE Divergent evolution of Corynebacterium diphtheriae

Fig 1. Distribution of sequence types over the study period (2015–2020).

https://doi.org/10.1371/journal.pone.0261435.g001

2.2. MLST analysis

MLST analysis for the 216 C. diphtheriae isolates representing the Indian population revealed
major diversity among the sequence types. A total of 34 STs were assigned to the 216 C. diph-
theria isolates. The distribution of STs over the study period was shown in Fig 1. The identified
STs were grouped into 8 different clonal complexes (CC). The majority belongs to CC5 fol-
lowed by CC466, CC574 and CC209, however a single non-toxigenic strain belongs to CC42.
Majority of the isolates belonged to ST466 (30%). The second most common ST identified
was ST405 that was present in 14% of the isolates. The international clone ST50 was seen in
11% of the study isolates. Further ST377 and ST469 were identified in 9.2% and 7.4% of the
isolates, respectively.
2.2.1. Region specific clones. ST301 was identified only in Lucknow and was not seen in
any other state. The non-toxigenic clone was mainly seen in the Kerala isolates. The region-
wide distribution of observed C. diphtheriae STs is given in S2 Table. Other STs observed were
ST295, ST308 including novel STs such as ST408, ST409, ST422, ST443, ST446, ST468, ST470,
ST540, ST541, ST542, ST548, ST566, ST567, ST568, ST569, ST57, ST573, ST574, ST575,
ST576, ST587, ST588, ST590, ST591, ST592 and ST599.
Most of the novel STs were identified in isolates from Kerala. All the novel STs identified in
this study were SLV/DLV/TLV of the existing STs with a minimum of one SNP to a maximum
of 26 SNPs except for two STs (ST466 and ST566), which stand separate (Table 1). The non-
toxigenic isolates were observed to have the following sequence types ST542, ST409, ST295,

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PLOS ONE Divergent evolution of Corynebacterium diphtheriae

Table 1. SNP analysis of sequence types identified among the study isolates.
Sequence type atpA dnaE dnaK fusA leuA odhA rpoB SNPs Clonal complex
ST3 3 3 3 2 3 3 3 Known ST CC209
ST295 2 1 20 19 3 3 14 Known ST CC509
ST50 2 2 4 1 3 3 2 Known ST CC5
ST377 2 43 87 4 59 3 6 Known ST CC540
ST301 2 10 3 1 7 3 2 Known ST CC574
ST308 4 12 4 1 18 3 13 Known ST CC5
�
ST405 4 12 4 1 18 3 2 SLV of ST308 by 3 SNPs CC5
�
ST408 8 2 3 1 18 3 13 TLV of ST308 by atpA—4 dnaE—5 dnaK—11 SNPs
�
ST409 6 7 21 12 9 12 11 SLV of ST163 by 1 SNP CC42
ST422 2 2 38 58 3 3 22 TLV of ST50 by dnaK—17 fusA—6 rpoB—3 SNPs
ST443 3 1 20 19 37 3 35 TLV of ST295 by atpA—2 leuA—4 rpoB—1 SNPs
ST446 4 2 4 1 3 3 2 SLV of ST50 by 4 SNPs CC5
�
ST466 2 3 3 1 2 3 13 None CC466
�
ST468 3 2 4 19 59 3 13 DLV of ST136 by fusA—7 leuA—4 CC5
�
ST469 4 10 3 1 7 3 13 DLV of ST301 by atpA—4 rpoB—3 SNPs
�
ST470 3 2 99 1 20 3 13 TLV of ST408 by atpA—2 dnaK—7 leuA—6 SNPs
�
ST540 2 43 87 4 2 3 6 SLV of ST377 by 8 SNPs CC377
�
ST541 2 3 3 1 59 3 13 SLV of ST466 by 8 SNPs CC466
�
ST542 3 1 4 19 5 3 9 TLV of ST468 by dnaE—1 leuA—3 rpoB—3 SNPs
ST548 3 3 4 2 3 3 5 SLV of ST209 by 4 SNPs CC209
ST566 19 9 4 19 30 26 22 None
ST567 23 3 3 1 2 3 13 SLV of ST466 by 1 SNP CC466
ST568 3 1 49 19 3 3 6 TLV of ST542 by dnaK—10 leuA—1 rpoB—3 SNPs
ST569 3 2 4 19 3 3 13 SLV of ST468 by 4 SNPs CC5
ST570 3 3 3 2 41 3 3 SLV of ST3 by 2 SNPs CC209
ST573 2 3 76 1 25 3 13 DLV of ST466 by dnaK—5 leuA—3 SNPs CC466
ST574 2 10 3 1 3 3 2 SLV of ST301 by 6 SNPs CC574
ST575 3 1 4 58 5 3 22 DLV of ST542 by fusA -1 rpoB—5 SNPs
ST576 2 10 3 64 7 3 2 SLV of ST301 by 1 SNP CC574
ST587 2 3 97 1 2 3 35 DLV of ST466 dnaK—8 rpoB—1 SNPs CC466
ST588 2 3 3 1 2 16 13 SLV of ST466 by odhA—4 SNP CC466
ST590 2 10 3 1 3 57 2 DLV of ST301 by leuA—6 odhA—1 SNPs CC574
ST591 2 3 38 1 3 3 22 DLV of ST422 by dnaE—1 fusA—6 SNPs
ST592 2 12 87 4 59 3 2 DLV of ST377 by dnaE—4 rpoB—1 SNPs
ST599 2 12 4 1 18 3 2 SLV of ST405 by atpA—4 SNP CC5
https://doi.org/10.1371/journal.pone.0261435.t001

ST443, ST422, ST566, and ST568, whereas the STs identified in both toxigenic and non-toxi-
genic isolates were ST408, ST466, ST591 and ST575, respectively. The MLST sequences
obtained, and its distribution among the study isolates are given in the S2 Table. The phyloge-
netic tree showed the relatedness of the isolates based on the concatenated MLST gene
sequences (Fig 2). Individual isolate details on toxigenicity, ST, region and year of isolation
were given in S3 Table. The possible routes of transmission of STs were mapped according to
their pattern of first occurrence and their sample collection date (Fig 3).

3. Discussion
Diphtheria remains endemic in many parts of the world and continues to be a major public
health problem in India. There have been numerous reports of diphtheria from several parts of

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PLOS ONE Divergent evolution of Corynebacterium diphtheriae

Fig 2. Maximum likelihood phylogeny of MLST alleles from 216 C. diphtheriae isolates. The clades are highlighted based on the common ST types identified in the
study. STs associated with non-toxigenic strains are highlighted in yellow. The inner ring represents the toxigenicity results of the isolates followed by date of collection.
The outer ring represents the region of isolation.
https://doi.org/10.1371/journal.pone.0261435.g002

India for the past 10 years [8]. The occurrence of novel sequence types and their circulation in
different geographical regions has recently become a serious concern. To the best of our
knowledge, this is the first extensive study of the epidemiology of C. diphtheriae from India.
Amongst the total C. diphtheriae positive cases in this study, most of them were from Tamil
Nadu, Kerala and Uttar Pradesh (Lucknow) which indicates the burden of diphtheria in the
respective localities. Epidemiological surveillance provides significant understanding of the
clonal population of C. diphtheriae particularly in endemic region like India. The presented
MLST analysis of the C. diphtheriae isolates revealed great diversity among the isolates over
the years 2015–2020. The MLST analysis also includes isolates from the Kerala outbreak
reported in 2016 [6].

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Fig 3. Illustration of the probable routes of transmission of STs identified in this study. The coloured circles represent the first occurrence of the ST and
the dotted arrow with colour indicates the possible transmission routes according to the year of isolation. The red dotted lines highlight the hotspot for novel
sequence types. Map outline was created using mapchart.net. Republished from mapchart.net under a CC BY license, with permission from MapChart,
original copyright 2021.
https://doi.org/10.1371/journal.pone.0261435.g003

The predominant sequence type in this study was ST466 (28.6%), a novel sequence type
which was first observed in the Kerala outbreak in 2016. Later in 2017, the ST was consistently
seen in Kerala and was also found in Lucknow and Chandigarh. While in 2018, it was found in
significant numbers in Lucknow, Uttar Pradesh and Madhya Pradesh which shows the ability
of this clone to disseminate rapidly. The second most common sequence type was ST405
(14.5%). This is an SLV of ST308 and was first observed in the year 2015 in Andhra Pradesh.
This clone further spread to Kerala in 2016. While in 2017, it was also seen in Tamil Nadu and
Karnataka. However, in 2018, ST405 was found endemic in Karnataka. Notably, all the identi-
fied novel sequence types evolved from established known sequence types by locus variations.
ST377 (9.2%) which was identified among isolates from Tamil Nadu in 2015, began to be
seen in Kerala after 2016. Whereas ST469 (7.8%) was first identified in Kerala in 2016 and
from 2017 it was also seen in Tamil Nadu.

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Interestingly, a region-specific sequence type was identified in this study. ST301 which was
restricted to Lucknow was not found in any other region in India. ST301 was first identified in
2017 and continue to persist in Lucknow. A SLV of ST301 was identified and assigned the
novel type as ST576. This shows region specific emergence of novel clones due to SNPs in the
existing clones.
Results from this study reveal that C. diphtheriae strains are highly diverse. Till date differ-
ent STs have reported from different countries [9–11]. Such global outbreak associated clones
include, ST8, ST12, ST52, and ST66 from United Kingdom [9]. Similarly, ST378 and ST395
seems to be common in South Africa [11]. Whereas, in India ST405, ST466 and ST377 are
common as observed in this study. Moreover, the international clone, ST50 was identified in
2016 in Tamil Nadu and Andhra Pradesh. In 2017 and 2018 it was common in Karnataka
which shows the inter-state spread of the ST50 clone. Most of the STs identified in India are
diverse but the reason for this and the source of these clones is not well established. Socio-geo-
graphical approach is required to understand and identify the ancestral lineage of these clones.
It has been postulated that non-toxigenic strains occur more frequently in individuals who
have been previously immunized [12]. In this study, though the immunization status was not
known, 14% of strains were negative for toxin by PCR. Further, phylogenetic analysis revealed
that majority of the non-toxigenic strains were clustered in two different clades associated
with ST408 and ST542. These were mostly seen in isolates from Kerala. Non-toxigenic ST408
and ST542 were found to have evolved from the existing sequence types of toxigenic ST301
and ST377 respectively. This scenario could be due to the loss of genes that affect the expres-
sion of tox gene during the evolution.
MLST is a useful tool for evolutionary studies and for tracking the dissemination of clones.
However, there is a limitation in discriminating strains within the same ST due to continuing
change in their accessory genomes. Previous studies revealed that approximately one-third of
the C. diphtheriae genome encodes accessory genes that vary widely between strains. The
strains within individual STs differed by the presence or absence of up to 290 genes, which are
mostly present on the genomic islands [13]. This study clearly indicates the evolving genome
diversity of C. diphtheriae strains in India.
Clonal variation among the C. diphtheriae strains was observed in the study population.
The epidemiological analysis revealed the emergence of novel STs and the clones with better
dissemination properties. This indicates a rapid evolution of C. diphtheriae in India. In the
present study, ST466 was found to be the predominant sequence type with better adaptive and
dissemination capabilities. ST301 was identified as a region-specific toxigenic clone, from
which the non-toxigenic ST408 has evolved. This study has now revealed the pattern of circu-
lating STs of C. diphtheriae among the different regions of India.

4. Materials and methods

Samples collected as part of the National Diphtheria Surveillance Programme, India were
included in this study. Specimens (throat, ear and nasal swabs, and pieces of throat membrane)
were collected from diphtheria suspected patients attending CMC, Vellore. Isolates were also
received from participating centres, various district hospitals and government health centres.
All the specimens were subjected to culture identification and were confirmed by real-time
PCR.
The collected samples were representative of the Indian population. The centre from South
India includes, Christian Medical College, Vellore; Coimbatore Medical College, Coimbatore;
Madurai Medical College, Madurai; Kauvery Hospital, Trichy; Govt. Villupuram Medical Col-
lege, Villupuram; Kanchi Kamakoti Hospital and Southern Railway Hospital, Chennai; State

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PLOS ONE Divergent evolution of Corynebacterium diphtheriae

Public Health, Trivandrum, Kerala; St. John’s Hospital, RIMS, Raichur and S. Nijalingappa
Medical College, Karnataka. The samples representing the North Indian population were col-
lected from the following centres: PGIMER, Chandigarh; KGMC, Lucknow; Bharathi Vidya
Peeth, Sangli, Maharashtra; and LTM Medical College, Mumbai.

4.1. Bacterial culture and characterisation

The swabs were processed, and initial identification was done by staining and culture on blood
agar (10% sheep blood) and serum tellurite agar. C. diphtheriae colonies were further con-
firmed on tinsdale agar (cystinase test) (DIFCO, USA) as black colonies with brown halo. The
species identification was done with standard biochemical testing based on the utilization of
sugars such as glucose, dextrose, sucrose, maltose followed by nitrate and urease tests (Fisher
Scientific, Massachusetts, USA).

4.2. Antimicrobial susceptibility testing

Minimum inhibitory concentration (MIC) was determined for the subset of the isolates using
E-test against antibiotics such as penicillin, cefotaxime, and erythromycin. The results were
interpreted according to CLSI-M45 guidelines [14].

4.3. Real-time PCR detection

DNA was extracted using the AQIAamp DNA blood mini kit (QIAGEN, Hilden, Germany).
The target genes include rpoB specific for C. diphtheria and C. ulcerans, toxA. RNaseP was
used as an internal control. Primers, probes and cyclic conditions used in this study were as
previously described [15]. Briefly, the reaction set-up includes 95˚C for 10 mins; followed by
45 cycles of 95˚C for 15 secs and 60˚C for 30 secs. Ct cut off values for positivity were �34 for
C. diphtheriae, �31 for C. ulcerans, and �35 for toxA.

4.4. MLST typing

C. diphtheriae isolates were subjected to multi-locus sequence typing (MLST) according to the
C. diphtheriae PubMLST protocol. Briefly, the DNA fragments on each strand were sequenced
with the sequencing primers by using an ABI PRISM BigDye Terminator Cycle Sequencing kit
(Applied Biosystems, CA, USA) and an ABI 3500 Genetic Analyser (Applied Biosystems,
USA). The Allele profiles, ST and clonal complexes (CC) were assigned by using the PubMLST
database (https://pubmlst.org/organisms/corynebacterium-diphtheriae) [9]. Alleles and ST
that had not been previously described were submitted to the database and were assigned new
allele numbers and STs. A clonal complex was defined as a cluster of related STs linked as sin-
gle-, double- or triple- locus variants to another ST in the group. MLST alleles of C. diphtheriae
isolates were used to build a phylogeny based on the maximum-likelihood method using
MEGA (v7.0.26) [16]. Metadata were added to the phylogenetic tree using iTOL (v4.3) online
software [17]. Further, predicted routes of transmission of strains studied here was mapped
using mapchart.net.

4.5. goeBURST analysis

goeBURST analysis was performed to identify the related genotypes in the population, and to
identify the founding genotype (ST) of each group. goeBURST analysis was performed using
the PHYLOViZ 2.0 tool [18].

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Supporting information
S1 Table. Laboratory confirmation of C. diphtheriae cases at NRL for diphtheria, CMC,
Vellore.
(DOCX)
S2 Table. Distribution of sequence types among different states in India.
(DOCX)
S3 Table. Metadata including isolate ID, toxigenicity, STs, region and year of isolation for
C. diphtheriae isolates depicted in the MLST phylogeny Fig 2.
(XLSX)

Acknowledgments
The authors gratefully acknowledge the Institutional Review Board of the Christian Medical
College, Vellore (83-i/11/13) for approving the study and providing lab space and facilities.

Author Contributions
Conceptualization: Naveen Kumar Devanga Ragupathi, Dhiviya Prabaa Muthuirulandi
Sethuvel, Lucky Sangal, Balaji Veeraraghavan.
Data curation: Dhiviya Prabaa Muthuirulandi Sethuvel.
Formal analysis: Naveen Kumar Devanga Ragupathi, Vikas Gautam, Prashanth Gupta, Jaic-
hand Johnson, Naresh Chand Sharma, Ankur Mutreja, Arun Kumar, Pankaj Bhatnagar,
Balaji Veeraraghavan.
Funding acquisition: Pradeep Haldar, Arun Kumar, Pankaj Bhatnagar, Lucky Sangal.
Investigation: Naveen Kumar Devanga Ragupathi, Vikas Gautam, Prashanth Gupta, Jaichand
Johnson, Naresh Chand Sharma.
Methodology: Dhivya Murugan, Ranjini Ranjan.
Resources: Vikas Gautam, Prashanth Gupta, Jaichand Johnson, Naresh Chand Sharma, Pra-
deep Haldar, Arun Kumar, Pankaj Bhatnagar, Lucky Sangal.
Software: Naveen Kumar Devanga Ragupathi.
Supervision: Naveen Kumar Devanga Ragupathi, Balaji Veeraraghavan.
Validation: Naveen Kumar Devanga Ragupathi, Pradeep Haldar, Pankaj Bhatnagar, Lucky
Sangal, Balaji Veeraraghavan.
Writing – original draft: Naveen Kumar Devanga Ragupathi, Dhiviya Prabaa Muthuirulandi
Sethuvel, Lucky Sangal, Balaji Veeraraghavan.
Writing – review & editing: Ankur Mutreja, Pradeep Haldar, Arun Kumar, Pankaj Bhatnagar,
Lucky Sangal, Balaji Veeraraghavan.

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