Prevalence of Major Methicillin-Resistant Staphylococcus aureus Clones in Korea Between 2001 and 2008
2016; 36(6): 536-541
Ann Lab Med 2021; 41(5): 489-492
Published online September 1, 2021 https://doi.org/10.3343/alm.2021.41.5.489
Copyright © Korean Society for Laboratory Medicine.
Jun Ho Jeon, Ph.D.1* , Chi-Hwan Choi, Ph.D.2* , Jeong Hyun Kim, M.D.3 , Junghee Hyun, M.S.4 , Eun-Sun Choi, Ph.D.1 , Sang-Yoon Choi, Ph.D.1 , Yong-Woo Shin, Ph.D.1 , Seong Wook Pyo, M.S.1 , Dae-Won Kim, Ph.D.2 , Byung Hak Kang, Ph.D.1 , Young Joon Park, M.D.5 , and Gi-eun Rhie, Ph.D.1
1Division of High-risk Pathogens, 2Division of Zoonotic and Vector Borne Disease Research, 3Division of Zoonotic and Vector Borne Disease Control, 4Division of Emerging Infectious Disease Response, 5Director for Epidemiological Investigation Analysis, Korea Disease Control and Prevention Agency, Cheongju, Korea
Correspondence to: Gi-eun Rhie, Ph.D.
Division of High-risk Pathogens, Korea Disease Control and Prevention Agency, 187 Osongsaengmyeong 2-ro, Cheongju 28159, Korea
* These authors contributed equally to this study.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Botulism is a neuroparalytic disease caused by a neurotoxin produced by Clostridium botulinum. This study aimed to genetically characterize C. botulinum strain isolated from the first case of infant botulism in Korea reported on June 17, 2019. We isolated C. botulinum strain CB-27 from a stool sample of the patient and analyzed the toxin types and toxin gene cluster compositions of the strain using a mouse bioassay, real-time PCR, and genome sequencing. Toxin gene cluster analysis showed that strain CB-27 possesses a C. botulinum neurotoxin type A harboring an unexpressed B gene. Although the nucleotide and amino acid sequences of toxin genes as well as the toxin gene cluster arrangements in strain CB-27 were identical to those of the known strain CDC_69094, the total nucleotide sequences of the toxin gene clusters of CB-27 differed from those of CDC_69094 by 0.47%, indicating genetic diversity of toxin gene clusters of CB-27 among other previously reported C. botulinum strains. To our knowledge, this is the first description of a C. botulinum strain with two separate toxin gene clusters in Korea.
Keywords: Clostridium botulinum, Toxin type, Toxin gene cluster, Genetic diversity, Neurotoxin
Botulism is a neuroparalytic disease caused by the seven immunologically distinct toxins (A to G) produced by
On June 7, 2019, a 4-month-old Korean baby with suspected infant botulism was admitted to Ajou University Hospital, Suwon, Korea, and on June 13, clinical samples, including serum and stool samples, were submitted to the Korea Centers for Disease Control and Prevention (KCDC) to identify
A small amount of the stool sample was inoculated into cooked-meat medium (Difco, Franklin Lakes, USA) and cultured under anaerobic conditions at 37°C for three days. The culture was mixed 1:1 with 100% ethanol and incubated at 37°C for 1 hour. Serial culture dilutions were plated on egg yolk agar under anaerobic conditions to isolate
The culture filtrate of strain CB-27 was diluted 1:10,000 in phosphate-buffered saline containing 0.2% gelatin and then analyzed using a mouse bioassay. The mice were injected with 0.05 international units of botulinum antitoxins A and B (National Institute for Biological Standards and Control, Potters Bar, UK). Botulinum antitoxin A and a mixture of antitoxins A and B showed neutralizing ability against the CB-27 culture filtrate (Table 1) in accordance with the mouse bioassay results obtained using stool samples.
To confirm the discrepancy between the mouse bioassay and real-time PCR analysis results, genomic DNA was extracted from the CB-27 strain and subjected to whole genome sequencing using a PacBio RS II (Pacific Biosciences, Menlo Park, CA, USA; https://www.pacb.com) and an Ion S5 (Thermo Fisher Scientific, Waltham, MA USA; https://www.thermofisher.com) sequencer. The toxin gene cluster sequences of CB-27 were assembled de novo using the Hierarchical Genome Assembly Process version 3 (Pacific Biosciences). High-quality Ion S5 reads were used to correct potential sequencing errors in the PacBio long reads in Proovread version 2.14 (https://github.com/BioInf-Wuerzburg/proovread) . The cluster sequence of CB-27 (67,538 bp) has been deposited at GenBank under accession number MT199282. Genes were annotated using rapid prokaryotic genome annotation (Prokka version 1.14.5, https://github.com/tseemann/prokka) . Gene annotation showed that the cluster sequence of CB-27 harbored a
To phylogenetically characterize the toxin gene clusters of CB-27, we downloaded sequence data of 240 strains representing different toxin types and subtypes from Pathosystems Resource Integration Center (PATRIC) (https://www.patricbrc.org) . All-against-all pairwise sequence comparisons were conducted using Basic Local Alignment Search Tool (https://blast.ncbi.nlm.nih.gov/) to determine the closest relatives of CB-27. Multiple sequence alignments were conducted using MAFFT version 7.453 (https://mafft.cbrc.jp/alignment/software/), with a maximum of 1,000 iterations. We constructed maximum-likelihood phylogenetic trees using RAxML-NG version 0.9.0 (https://github.com/amkozlov/raxml-ng) with TVM+F+G4, the best nucleotide substitution model recommended by ModelFinder [15, 16]. The
We examined genomic arrangements of the toxin gene clusters of CB-27 using the multiple genome alignment tool MAUVE version 2.4.0 (http://darlinglab.org/mauve/mauve.html) . Fig. 2 shows a schematic diagram of the toxin A and B gene cluster structures of CB-27 and other representative strains (subtypes A1, A2, A3, and A(B)). The toxin gene cluster structures of CB-27 were identical to those of CDC_69094. Both CB-27 and CDC_69094 possess not only
In conclusion, we characterized the toxin gene clusters of
Conceptualization: Rhie G. Data curation: Jeon JH, Choi CH, Kim JH, Hyun J, Choi ES, Choi SY, Shin YW, Pyo SW, Kim DW, Kang BH, Park YJ. Writing-review and editing: Rhie G, Jeon JH. Final approval of the manuscript: all authors.
The authors have declared no conflicts of interest.
This work was supported by the KDCA (4840-302-210-13).