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Population genomics and antimicrobial resistance in Corynebacterium diphtheriae.

ABSTRACT: BACKGROUND:Corynebacterium diphtheriae, the agent of diphtheria, is a genetically diverse bacterial species. Although antimicrobial resistance has emerged against several drugs including first-line penicillin, the genomic determinants and population dynamics of resistance are largely unknown for this neglected human pathogen. METHODS:Here, we analyzed the associations of antimicrobial susceptibility phenotypes, diphtheria toxin production, and genomic features in C. diphtheriae. We used 247 strains collected over several decades in multiple world regions, including the 163 clinical isolates collected prospectively from 2008 to 2017 in France mainland and overseas territories. RESULTS:Phylogenetic analysis revealed multiple deep-branching sublineages, grouped into a Mitis lineage strongly associated with diphtheria toxin production and a largely toxin gene-negative Gravis lineage with few toxin-producing isolates including the 1990s ex-Soviet Union outbreak strain. The distribution of susceptibility phenotypes allowed proposing ecological cutoffs for most of the 19 agents tested, thereby defining acquired antimicrobial resistance. Penicillin resistance was found in 17.2% of prospective isolates. Seventeen (10.4%) prospective isolates were multidrug-resistant (??3 antimicrobial categories), including four isolates resistant to penicillin and macrolides. Homologous recombination was frequent (r/m?=?5), and horizontal gene transfer contributed to the emergence of antimicrobial resistance in multiple sublineages. Genome-wide association mapping uncovered genetic factors of resistance, including an accessory penicillin-binding protein (PBP2m) located in diverse genomic contexts. Gene pbp2m is widespread in other Corynebacterium species, and its expression in C. glutamicum demonstrated its effect against several beta-lactams. A novel 73-kb C. diphtheriae multiresistance plasmid was discovered. CONCLUSIONS:This work uncovers the dynamics of antimicrobial resistance in C. diphtheriae in the context of phylogenetic structure, biovar, and diphtheria toxin production and provides a blueprint to analyze re-emerging diphtheria.

SUBMITTER: Hennart M

PROVIDER: S-EPMC7694903 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Population genomics and antimicrobial resistance in Corynebacterium diphtheriae.

Hennart Melanie M Panunzi Leonardo G LG Rodrigues Carla C Gaday Quentin Q Baines Sarah L SL Barros-Pinkelnig Marina M Carmi-Leroy Annick A Dazas Melody M Wehenkel Anne Marie AM Didelot Xavier X Toubiana Julie J Badell Edgar E Brisse Sylvain S

Genome medicine 20201127 1

<h4>Background</h4>Corynebacterium diphtheriae, the agent of diphtheria, is a genetically diverse bacterial species. Although antimicrobial resistance has emerged against several drugs including first-line penicillin, the genomic determinants and population dynamics of resistance are largely unknown for this neglected human pathogen.<h4>Methods</h4>Here, we analyzed the associations of antimicrobial susceptibility phenotypes, diphtheria toxin production, and genomic features in C. diphtheriae. W ...[more]

PMID: 33246485

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Project description:Corynebacterium diphtheriae is the etiological agent of diphtheria, a disease caused by the presence of the diphtheria toxin. However, an increasing number of records report non-toxigenic C. diphtheriae infections. Here, a C. diphtheriae strain was recovered from a patient with a past history of bronchiectasis who developed a severe tracheo-bronchitis with multiple whitish lesions of the distal trachea and the mainstem bronchi. Whole-genome sequencing (WGS), performed in parallel with PCR targeting the toxin gene and the Elek test, provided clinically relevant results in a short turnaround time, showing that the isolate was non-toxigenic. A comparative genomic analysis of the new strain (CHUV2995) with 56 other publicly available genomes of C. diphtheriae revealed that the strains CHUV2995, CCUG 5865 and CMCNS703 share a lower average nucleotide identity (ANI) (95.24 to 95.39%) with the C. diphtheriae NCTC 11397T reference genome than all other C. diphtheriae genomes (>98.15%). Core genome phylogeny confirmed the presence of two monophyletic clades. Based on these findings, we propose here two new C. diphtheriae subspecies to replace the lineage denomination used in previous multilocus sequence typing studies: C. diphtheriae subsp. lausannense subsp. nov. (instead of lineage-2), regrouping strains CHUV2995, CCUG 5865, and CMCNS703, and C. diphtheriae subsp. diphtheriae subsp. nov, regrouping all other C. diphtheriae in the dataset (instead of lineage-1). Interestingly, members of subspecies lausannense displayed a larger genome size than subspecies diphtheriae and were enriched in COG categories related to transport and metabolism of lipids (I) and inorganic ion (P). Conversely, they lacked all genes involved in the synthesis of pili (SpaA-type, SpaD-type and SpaH-type), molybdenum cofactor and of the nitrate reductase. Finally, the CHUV2995 genome is particularly enriched in mobility genes and harbors several prophages. The genome encodes a type II-C CRISPR-Cas locus with 2 spacers that lacks csn2 or cas4, which could hamper the acquisition of new spacers and render strain CHUV2995 more susceptible to bacteriophage infections and gene acquisition through various mechanisms of horizontal gene transfer.

Project description:An increasing number of isolations of Corynebacterium diphtheriae has been observed in recent years in the archipelago of New Caledonia. We aimed to analyze the clinical and microbiological features of samples with C. diphtheriae. All C. diphtheriae isolates identified in New Caledonia from May 2015 to May 2019 were included. For each case, a retrospective consultation of the patient files was conducted. Antimicrobial susceptibility phenotypes, tox gene and diphtheria toxin expression, biovar, and the genomic sequence were determined. Core genome multilocus sequence typing (cgMLST), 7-gene MLST, and search of genes of interest were performed from genomic assemblies. Fifty-eight isolates were included, with a median age of patients of 28 years (range: 9 days to 78 years). Cutaneous origin accounted for 51 of 58 (87.9%) isolates, and C. diphtheriae was associated with Staphylococcus aureus and/or Streptococcus pyogenes in three-quarters of cases. Half of cases came either from the main city Noumea (24%, 14/58) or from the sparsely populated island of Lifou (26%, 15/58). Six tox-positive isolates were identified, associated with recent travel to Vanuatu; 5 of these cases were linked and cgMLST confirmed recent transmission. Two cases of endocarditis in young female patients with a history of rheumatic fever involved tox-negative isolates. The 58 isolates were mostly susceptible to commonly used antibiotics. In particular, no isolate was resistant to the first-line molecules amoxicillin or erythromycin. Resistance to tetracycline was found in a genomic cluster of 17 (29%) isolates, 16 of which carried the tetO gene. There were 13 cgMLST sublineages, most of which were also observed in the neighboring country Australia. Cutaneous infections may harbor nontoxigenic C. diphtheriae isolates, which circulate largely silently in nonspecific wounds. The possible introduction of tox-positive strains from a neighboring island illustrates that diphtheria surveillance should be maintained in New Caledonia, and that immunization in neighboring islands must be improved. Genomic sequencing uncovers how genotypes circulate locally and across neighboring countries. IMPORTANCE The analysis of C. diphtheriae from the tropical archipelago of New Caledonia revealed a high genetic diversity with sublineages that may be linked to Polynesia, Australia, or metropolitan France. Genomic typing allowed confirming or excluding suspected transmission events among cases and contacts. A highly prevalent tetracycline-resistant sublineage harboring the tetO gene was uncovered. Toxigenic isolates were observed from patients returning from Vanuatu, showing the importance of improving vaccination coverage in settings where it is insufficient. This study also illustrates the importance for diphtheria surveillance of the inclusion of isolates from cutaneous sources in addition to respiratory cases, in order to provide a more complete epidemiological picture of the diversity and transmission of C. diphtheriae.

Project description:We assessed changes in gene expression in response to manganese availability for the human pathogen Corynebacterium diphtheriae. Total RNA was harvested from wild-type C. diphtheriae strain 1737 and an isogenic ΔmntR (dip0619) grown in semi-defined metal-limited media (mPGT) without or with 5 µM manganese chloride supplementation. Three biological replicates were prepared and five genes were assessed by real-time PCR to validate the array results: dip0124, dip0169, dip0615, dip1923, and dip2261. (Abstract) Corynebacterium diphtheriae is the causative agent of a severe respiratory disease in humans. The bacterial systems required for infection are poorly understood, but the acquisition of metals such as manganese (Mn) are likely critical for host colonization. MntR is a Mn-dependent transcriptional regulator in C. diphtheriae and was previously shown to repress the expression of the mntABCD genes, which encode an ABC metal transporter. However, other targets of Mn and MntR regulation in C. diphtheriae have not been identified. In this study, we use comparisons between the gene expression profiles of wild-type C. diphtheriae strain 1737 grown without or with Mn supplementation and comparisons of gene expression between wild-type and an mntR mutant to characterize the C. diphtheriae Mn and MntR regulon. MntR was observed to both repress and induce the expression of various target genes in a Mn-dependent manner. Genes induced by MntR include the Mn-superoxide dismutase, sodA, and the putative ABC transporter locus, iutABCD. DNA binding studies showed that MntR interacts at the promoter regions for several genes identified in the expression study, and a 17-bp consensus MntR DNA binding site was identified. We found that an mntR mutant displayed increased sensitivity to Mn and Cd that could be alleviated by the additional deletion of the mntA-D transport locus, providing evidence that the MntABCD transporter functions as a Mn uptake system in C. diphtheriae. The findings in this study further our understanding of metal uptake systems and global metal regulatory networks in this important human pathogen.

Dataset Information

Population genomics and antimicrobial resistance in Corynebacterium diphtheriae.

Publications

Population genomics and antimicrobial resistance in Corynebacterium diphtheriae.

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