Molecular Characterisation of Corynebacterium diphtheriae Isolates of Faucial Diphtheria Cases from Assam: A Cross-sectional Study
Correspondence Address :
Dr. Gargi Choudhury,
Associate Professor, Department of Microbiology, Assam Medical College and Hospital,
Dibrugarh, Assam, India.
E-mail: gargicb@gmail.com
Introduction: Diphtheria caused by toxigenic Corynebacterium diphtheriae is an acute respiratory infection characterised by pseudomembrane formation in the throat. Although diphtheria is often reported sporadically across India, molecular characterisation of C.diphtheriae strains could play a crucial role in epidemiological investigations.
Aim: The present study aimed at molecular characterisation of Corynebacterium diphtheriae isolates of faucial diphtheria cases from Assam, North-East India utilising Multi-Locus Sequence Typing (MLST).
Materials and Methods: In this cross-sectional study, three isolates were obtained from three different cluster of cases that occurred across two districts of Assam, India for a period from November 2019 to June 2020. MLST of these three C.diphtheriae isolates were performed in Multidisciplinary Research Unit of Assam Medical College and Hospital by using the 7-gene MLST alleles of C.diphtheriae to determine the Sequence Types (ST).
Results: The MLST of the three isolates of this study revealed two distinct STs, ST-588 and ST-576 with multi-clonal lineages with ST-466, reported in C.diphtheriae strains of North Kerala and ST-301 related strains of Bangladesh respectively.
Conclusion: In spite of frequent reports of diphtheria cases from Assam, molecular typing of the C.diphtheriae strains found in North-Eastern region of India has not yet been performed. This is one of the initial investigations from this region of India to use MLST for typing C.diphtheriae strains. Therefore, studies related to molecular characterisation of C.diphtheriae strains found throughout India could immensely help in determining the clonality and diversity existing among these strains.
Clonal distribution, Diphtheria, Epidemiological investigation, Multi-locus sequence typing, Phylogenetic tree
Diphtheria caused by Corynebacterium diphtheriae is an acute infectious disease ranging from mild infection to acute airway obstruction, toxaemia and sudden death (1). The disease can have a fatality rate as high as 30.8% as reported in a previous study from Assam (2). This disease is not only prevalent in Assam, a North-Eastern state of India, but also in other parts of India (3). However, due to widespread immunisation coverage in children, a shift in burden of diphtheria cases has been observed in adolescents and adults (4). However, cases are still reported in children of marginalised population (5).
Faucial diphtheria is characterised by the formation of a pseudo-membrane on one or both tonsils that extends to the tonsillar pillars, uvula, soft palate, oropharynx, and nasopharynx. Further extension of the pseudo-membrane into the larynx or trachea or dislodgement of a piece of it may even lead to fatal airway obstruction (6).
The causative agent, Corynebacterium diphtheriae, isolated from diphtheria cases needs to be monitored and characterised to know the prevailing ST, to be able to detect any evolution in the bacteria and contain any possible spread of Corynebacterium diphtheria strains. Various reports on diphtheria cases from Assam have been published (2),(7),(8),(9). However, studies on molecular typing of C.diphtheriae using techniques like RFLP, MLST, etc., have not been done from the North-East part of India. Hence, this study was done to utilise the 7-gene MLST alleles of C.diphtheriae to determine the ST and illustrate the epidemiology of the strains prevalent in this endemic region.
A laboratory based cross-sectional study was undertaken in the Multi-disciplinary Research Unit of Assam Medical College & Hospital, Dibrugarh during the period from November 2019 to June 2020.This study was approved by the Institutional Ethics Committee of Assam Medical College and Hospital, Dibrugarh (IEC No. AMC/EC/185; Reg.no. ECR/636/Inst/AS/2014).
Inclusion criteria: Corynebacterium diphtheriae isolated from index cases of diphtheria outbreaks.
Exclusion criteria: Corynebacterium diphtheriae isolated from cases other than the index cases of diphtheria outbreaks.
Study Procedure
Three C.diphtheriae isolates were isolated during the period from 2017 to 2019 from index cases of diphtheria outbreaks which occurred in Jorhat and Dibrugarh districts of Assam were subjected to MLST. Among these isolates, two isolates (AMC1 and AMC2) were isolated in the Department of Microbiology, Assam Medical College and Hospital, Dibrugarh and one isolate (JMC1) isolated in Department of Microbiology, Jorhat Medical College and Hospital, Jorhat. Further molecular characterisation of these isolates was performed in the Multi-disciplinary Research Unit of Assam Medical College and Hospital. These isolates were confirmed as C.diphtheriae by standard biochemical methods and PCR amplification of rpoB and toxA gene of C.diphtheriae.
Bacterial DNA extraction: Bacterial DNA was extracted using QIAamp DNA mini kit (Qiagen Inc., USA) following manufacturer’s instructions and the extracted DNA was quantified using the μDrop plate in Multiskan GO (Thermo Scientific, USA).
PCR identification of C.diphtheriae isolates: The PCR targets of rpoB gene of C.diphtheriae, C. ulcerans, and toxA gene fragment of C.diphtheriae were used for identification of the C.diphtheriae isolates. The PCR amplification was carried out in a 25 μL reaction volume consisting of 12.5 μL of 2X Bioline MyTaq HS Mix (Meridian Biosciences, USA) and 500 nM of each forward and reverse primers. The PCR conditions consisted of an initial denaturation of 95°C for 1 minute followed by 35 cycles of denaturation at 95°C for 15 seconds, annealing at 55°C for 15 seconds, and extension at 72°C for 10 seconds. The PCR amplicons were visualised for respective PCR bands on a 2% agarose gel stained with ethidium bromide (7).
Multi-Locus Sequence Typing (MLST): MLST for C.diphtheriae isolates were performed by PCR amplification and sequencing of the seven housekeeping genes (atpA, dnaE, dnaK, fusA, leuA, odhA and rpoB) as per the protocol described by Bolt F et al., (10). Purification of the amplified PCR products was performed by using ExoSAP-IT PCR product clean-up reagent (ThermoFisher Scientific, USA) by following manufacturer’s instructions. Cycle sequencing was performed by using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, USA) and sequencing was performed using Genetic Analyser 3500 (Applied Biosystems, USA). Consensus FASTA sequences were generated in BioEdit sequence alignment editor (Version 7.2.5) by multiple sequence alignment of both forward and reverse reads (11).
The population snapshot analysis of the 1357 isolates available till date in the C.diphtheriae MLST database was performed by utilising goeBURST algorithm in Phyloviz (Version 2.0) (12). The phylogenetic tree was constructed using the concatenated allele sequences in MEGA (Version X) using Neighbour-Joining method and Jukes Cantor algorithm. The test of phylogeny was done with 1000 bootstrap replicates (13). The allele sequences of the isolates of this study were submitted to Corynebacterium diphtheriae and C.ulcerans MLST sequence database (http://pubmlst.org/cdiphtheriae) and STs were assigned to each of the three isolates of this study. The isolates AMC1, AMC2, and JMC1 have been submitted to Corynebacterium diphtheriae and C. ulcerans MLST isolate database with isolate ids 851, 852, and 853 respectively (14).
Statistical Analysis
As this study involves only three isolates of C. diphtheriae, statistical methods could not be applied.
Population snapshot and phylogenetic analysis: Total three Corynebacterium diphtheriae isolates from each index case of three different diphtheria outbreaks were studied for MLST. The allele sequences of these isolates of this study were submitted to the Corynebacterium diphtheriae MLST sequence database (https://bigsdb.pasteur.fr/diphtheria/) and STs were assigned to each of the three isolates of this study. The isolates AMC1, AMC2, and JMC1 have been submitted to Corynebacterium diphtheriae MLST isolate database with isolate ids 2113, 2114, and 2115, respectively.
The population snapshot analysis of the 856 isolates available till date in the Corynebacterium diphtheriae MLST database revealed that the isolates AMC1 and AMC2 bearing ST-588 was assigned to the ST-466 Clonal Complex (CC) and is a Single Locus Variant (SLV) of ST-466 (Table/Fig 1). The JMC1 isolate with ST-576 was assigned to the ST-574 CC and is a SLV of ST301 (Table/Fig 1). The phylogenetic tree constructed using the concatenated allele sequences of seven housekeeping genes of the three C.diphtheriae isolates of this study along with reference strains (NCTC 05011, HCO1, HC04, C7betatox+, NCTC 13129, and INCA402) obtained from different parts of the world (Table/Fig 2). The isolates AMC1, AMC2, and JMC1 of this study were found to be in close relation with the NCTC 05011 strain with ST-143 originating form United Kingdom. None of the three isolates of this study were related to toxigenic reference strains of C7 beta tox (ST-26) and NCTC 13129 (ST-8).
Diphtheria owing to its high mortality rate was a much-feared disease before the introduction of a mass immunisation programme in India. However, there have been resurgences of this infectious disease in many parts of the country despite the high immunisation coverage. Outbreaks and sporadic cases of diphtheria have been reported occasionally from Assam (Table/Fig 3) (2),(7),(8),(9),(15). In one of the previous studies from Assam, 33 cases were reported and 10 cases were confirmed as C.diphtheriae by culture (7). MLST plays a crucial role in epidemiological investigation of pathogens. In 2016, C.diphtheriae strains with ST-466 were isolated during a national program surveillance in North Kerala. These ST-466 strains were found to be toxigenic as confirmed by Modified Elek’s Test (3). The isolates AMC1 and AMC2 of this study with ST-588 is a SLV of ST-446 and were found to be positive for the toxA gene as confirmed by PCR. During December 2017, an outbreak among the Forcibly Displaced Myanmar Nationals (FDMN) in Bangladesh occurred, in response to which Centers for Disease Control and Prevention (CDC) provided technical aid to study and characterise the C.diphtheriae isolates. This reported outbreak included three novel STs, two of which were also closely related to ST-301 (16). The ST-576 was assigned JMC1 isolate of this study is a SLV of ST301. The Indian state of Assam shares an international border of 263 kilometres with Bangladesh which may explain this clonality existing among the C.diphtheriae isolates of Assam and Bangladesh.
The MLST database of C.diphtheriae includes a total 1357 isolate data submitted till date from various parts of the world (14). However, only thirteen isolate data including the three isolates reported in this study have been submitted to the database from India. Though sporadic cases have been reported frequently all over the country and a nationwide surveillance programme is still going on under Integrated Disease Surveillance Programme (IDSP), molecular typing of the isolates has not been performed in most of the cases (17).
Limitation(s)
This study only included three C.diphtheriae isolates from index cases from three different diphtheria outbreaks. However, a greater number of isolates are required to determine the prevalent STs in this region. Moreover, Elek’s Gel precipitation test to determine the toxigenicity of these isolates was not performed in this study.
Two distinct STs, ST-588 and ST-576 with multi-clonal lineages with ST-466, reported in C.diphtheriae strains of North Kerala and ST-301 related strains of Bangladesh respectively, were revealed. In spite of frequent reports of diphtheria cases from Assam, molecular typing of the C.diphtheriae strains found in this region has not yet been performed. This is one of the initial investigations from the North-Eastern region of India to use MLST for typing C.diphtheriae strains found in this region. More studies from entire India on ST of C.diphtheriae strains could help immensely on establishing the prevalent STs; clonality and diversity existing among the C.diphtheriae strains found in India. The MLST on a large number of isolates from various regions of North East India could be a future perspective. Also, the determination of the toxigenic potential of all outbreak isolates of the regions could be another scope for future research. Moreover, expanded diphtheria surveillance including other anatomical sites (cutaneous, mucosal) etc., could be placed for early management of the cases.
The authors sincerely acknowledge the Indian Council of Medical Research (ICMR) for providing the platform for this study in the Multidisciplinary Research Unit (MRU-ICMR), Assam Medical College, Dibrugarh. Authors are also thankful to the technical staff of MRU-ICMR.
DOI: 10.7860/JCDR/2022/52934.16379
Date of Submission: Oct 26, 2021
Date of Peer Review: Dec 30, 2021
Date of Acceptance: Feb 22, 2022
Date of Publishing: May 01, 2022
AUTHOR DECLARATION:
• Financial or Other Competing Interests: No
• Was Ethics Committee Approval obtained for this study? Yes
• Was informed consent obtained from the subjects involved in the study? NA
• For any images presented appropriate consent has been obtained from the subjects. NA
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