RESUMO
Diphtheria is caused by a toxigenic bacterium Corynebacterium diphtheria which is being an emerging pathogen in India. Since diphtheria morbidity and mortality continues to be high in the country, the present study aimed to study the molecular epidemiology of C. diphtheriae strains from India. A total of 441 diphtheria suspected specimens collected as part of the surveillance programme between 2015 and 2020 were studied. All the isolates were confirmed as C. diphtheriae with standard biochemical tests, ELEK's test, and real-time PCR. Antimicrobial susceptibility testing for the subset of isolates showed intermediate susceptibility to penicillin and complete susceptible to erythromycin and cefotaxime. Isolates were characterized using multi locus sequence typing method. MLST analysis for the 216 C. diphtheriae isolates revealed major diversity among the sequence types. A total of 34 STs were assigned with 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 also seen. The identified STs were grouped into 8 different clonal complexes (CC). The majority belongs to CC5 followed by CC466, CC574 and CC209, however a single non-toxigenic strain belongs to CC42. This epidemiological analysis revealed the emergence of novel STs and the clones with better dissemination properties. This study has also provided information on the circulating strains of C. diphtheriae among the different regions of India. The molecular data generated through surveillance system can be utilized for further actions in concern.
Assuntos
Antibacterianos/farmacologia , Corynebacterium diphtheriae/classificação , Corynebacterium diphtheriae/efeitos dos fármacos , Monitoramento Epidemiológico , Cefotaxima/farmacologia , Corynebacterium diphtheriae/genética , Corynebacterium diphtheriae/isolamento & purificação , Difteria/epidemiologia , Eritromicina/farmacologia , Humanos , Índia/epidemiologia , Testes de Sensibilidade Microbiana , Epidemiologia Molecular , Tipagem de Sequências Multilocus , Penicilinas/farmacologiaRESUMO
BACKGROUND: Sporadic cases of infection with non-toxigenic Corynebacterium diphtheriae (C. diphtheriae) isolates have been reported in regions covered by the Diphtheria-Tetanus-Pertussis vaccine, but no information describing the whole genome of non-toxigenic strains collected in China is available. Therefore, in this work, the complete genome of a non-toxigenic strain of C. diphtheriae from a hospital located in southeastern China was performed. RESULTS: This non-toxigenic isolate belonged to the belfanti biotype and possessed a unique ST (assigned as ST799 in pubMLST). ErmX was present in the genome sequence and this isolate owned the resistance to erythromycin and clindamycin. Genes coding for virulence factors involved in adherence, iron-uptake and regulation of diphtheria toxin were also found. Two genes were involved in the interaction between pathogen and host. The phylogenetic analysis revealed that this newly isolated strain was similar to the strain NCTC10838, CMCNS703 and CHUV2995. CONCLUSION: Non-toxigenic C. diphtheriae strain contained virulence factors, thus it is able to cause an infectious disease, aspect that could be clarified by performing the whole genome sequencing analysis.
Assuntos
Corynebacterium diphtheriae/classificação , Corynebacterium diphtheriae/genética , Difteria/microbiologia , China , Corynebacterium diphtheriae/patogenicidade , Hospitais , Humanos , Pessoa de Meia-Idade , Filogenia , Fatores de Virulência/genéticaRESUMO
Between July 2018 and May 2019, Corynebacterium diphtheriae was isolated from eight patients with non-respiratory infections, seven of whom experienced homelessness and had stayed at shelters in King County, WA, USA. All isolates were microbiologically identified as nontoxigenic C. diphtheriae biovar mitis. Whole-genome sequencing confirmed that all case isolates were genetically related, associated with sequence type 445 and differing by fewer than 24 single-nucleotide polymorphisms (SNPs). Compared to publicly available C. diphtheriae genomic data, these WA isolates formed a discrete cluster with SNP variation consistent with previously reported outbreaks. Virulence-related gene content variation within the highly related WA cluster isolates was also observed. These results indicated that genome characterization can readily support epidemiology of nontoxigenic C. diphtheriae.
Assuntos
Infecções por Corynebacterium/diagnóstico , Corynebacterium diphtheriae/classificação , Polimorfismo de Nucleotídeo Único , Sequenciamento Completo do Genoma/métodos , Adulto , Idoso , Corynebacterium diphtheriae/genética , Corynebacterium diphtheriae/isolamento & purificação , Surtos de Doenças , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Pessoas Mal Alojadas , Humanos , Masculino , Pessoa de Meia-Idade , Filogenia , Fatores de Virulência/genética , WashingtonRESUMO
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.
Assuntos
Corynebacterium diphtheriae/efeitos dos fármacos , Corynebacterium diphtheriae/genética , Farmacorresistência Bacteriana/genética , Genes Bacterianos/genética , Metagenômica , Antibacterianos/farmacologia , Corynebacterium diphtheriae/classificação , Corynebacterium diphtheriae/isolamento & purificação , DNA Bacteriano/genética , Difteria/microbiologia , Toxina Diftérica/genética , Estudo de Associação Genômica Ampla , Genômica , Humanos , Macrolídeos/farmacologia , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus , Filogenia , Estudos ProspectivosRESUMO
A group of six clinical isolates previously identified as Corynebacterium diphtheriae biovar Belfanti, isolated from human cutaneous or peritoneum infections and from one dog, were characterized by genomic sequencing, biochemical analysis and MALDI-TOF mass spectrometry. The six isolates were negative for the diphtheria toxin gene. Phylogenetic analyses showed that the six isolates (including FRC0190T) are clearly demarcated from C. diphtheriae, Corynebacterium belfantii, Corynebacterium ulcerans and Corynebacterium pseudotuberculosis. The average nucleotide identity of FRC0190T with C. diphtheriae NCTC11397T was 92.6%, and was 91.8% with C. belfantii FRC0043T. C. diphtheriae subsp. lausannense strain CHUV2995T appeared to be a later heterotypic synonym of C. belfantii (ANI, 99.3%). Phenotyping data revealed an atypical negative or heterogeneous intermediate maltose fermentation reaction for the six isolates. MALDI-TOF mass spectrometry differentiated the new group from the other Corynebacterium taxa by the presence of specific spectral peaks. rpoB sequences showed identity to atypical, maltose-negative C. diphtheriae biovar Belfanti isolates previously described from two cats in the USA. We propose the name Corynebacterium rouxii sp. nov. for the novel group, with FRC0190T (= CIP 111752T = DSM 110354T) as type strain.
Assuntos
Corynebacterium diphtheriae/classificação , Corynebacterium/classificação , Técnicas de Tipagem Bacteriana , Corynebacterium/química , Corynebacterium/genética , Infecções por Corynebacterium/microbiologia , Corynebacterium diphtheriae/química , Corynebacterium diphtheriae/genética , Humanos , Filogenia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Sequenciamento Completo do GenomaRESUMO
In some parts of the world, Corynebacterium diphtheriae has reemerged as a pathogen, especially as a cause of infections among impoverished and marginalized populations. We performed whole-genome sequencing (WGS) on all cutaneous C. diphtheriae isolates (n = 56) from Vancouver's inner-city population over a 3-year time period (2015 to 2018). All isolates with complete genome assembly were toxin negative, contained a common set of 22 virulence factors, and shared a highly conserved accessory genome. One of our isolates harbored a novel plasmid conferring macrolide and lincosamide resistance. Fifty-two out of 56 isolates were multilocus sequence type 76, and single nucleotide variants (SNV) and core-genome multilocus sequence typing (cgMLST) analysis demonstrated tight clustering of our isolates relative to all publicly available C. diphtheriae genomes. All sequence type 76 (ST76) study isolates were within a median of 22 SNVs and 13 cgMLST alleles of each other, while NCBI genomes were within a median of 17,436 SNVs and 1,552 cgMLST alleles of each other (both P < 2.2 × 10-16). A single strain of C. diphtheriae appears to be causing cutaneous infections in the low-income population of Vancouver. Further research is needed to elucidate transmission networks in our study population and standardize C. diphtheriae epidemiological typing when whole genomes are sequenced.
Assuntos
Corynebacterium diphtheriae/classificação , Genoma Bacteriano , Filogenia , Pobreza/estatística & dados numéricos , Sequenciamento Completo do Genoma , Técnicas de Tipagem Bacteriana , Canadá/epidemiologia , Cidades/epidemiologia , Corynebacterium diphtheriae/isolamento & purificação , Corynebacterium diphtheriae/patogenicidade , Difteria/epidemiologia , Difteria/transmissão , Humanos , Tipagem de Sequências Multilocus , Pele/microbiologia , Fatores de VirulênciaRESUMO
Infections caused by Corynebacterium diphtheriae remain endemic in many countries. Since the implementation of the DTP (Diphtheria-Tetanus-Pertussis) vaccination program in 1977, only sporadic diphtheria cases have been reported in Thailand. In 2012, a diphtheria outbreak occurred in rural Thailand and 38 cases were reported, with the majority being adults (mean 22.1â¯years, range 5-72â¯years). The current study determined the genetic diversity of C. diphtheriae isolated from 83 individuals associated with either sporadic (nâ¯=â¯34) from 1994, 1996, 1997, 1998, 1999, 2000, 2012, and 2018, or 2012 outbreak (nâ¯=â¯49) diphtheria occurrences in Thailand. Antimicrobial susceptibility testing was performed on 41/83 isolates using broth microdilution. All sporadic (nâ¯=â¯27) and epidemic (nâ¯=â¯14) C. diphtheriae isolates (41/41; 100%) were susceptible to erythromycin (≤0.5⯵g/ml), clindamycin (≤0.5⯵g/ml), gentamicin (≤ 4⯵g/ml), ciprofloxacin (≤1⯵g/ml), and vancomycin (2⯵g/ml), except tetracycline with a resistance rate of 34.1% (14/41 isolates). All isolates were intermediately resistant to penicillin (MIC range, 0.25-2⯵g/ml). Multilocus sequence typing (MLST) revealed 17 sequence types (STs) among 83C. diphtheriae isolates. For the 2012 outbreak isolates, the predominant ST was ST243 (nâ¯=â¯34/49; 69.4%), followed by ST245 (nâ¯=â¯5/49; 10.2%) and ST244 (nâ¯=â¯4/49; 8.1%), whereas the main STs among the sporadic isolates were ST248 (nâ¯=â¯15/34; 44.1%), followed by ST209 (nâ¯=â¯7/34; 20.6%) and ST258 (nâ¯=â¯3/34; 8.8%). The ST243 outbreak strain was a single-locus variant of sporadic ST258. Phylogenetic analysis using concatenated sequences of 7 MLST genes from 17 STs revealed that ST243, ST248, and ST258 were located in the same cluster and ST243 appeared to have evolved from ST258, an endemic strain. This study highlights the importance of epidemiological surveillance together with characterization of C. diphtheriae strains to help inform the future control and prevention of diphtheria.
Assuntos
Antibacterianos/farmacologia , Infecções por Corynebacterium/epidemiologia , Infecções por Corynebacterium/microbiologia , Corynebacterium diphtheriae/efeitos dos fármacos , Corynebacterium diphtheriae/genética , Surtos de Doenças , Infecções por Corynebacterium/história , Corynebacterium diphtheriae/classificação , História do Século XXI , Humanos , Testes de Sensibilidade Microbiana , Epidemiologia Molecular , Tipagem de Sequências Multilocus , Filogenia , Vigilância em Saúde Pública , Tailândia/epidemiologiaRESUMO
Introduction. Diphtheria is caused by toxigenic strains of Corynebacterium diphtheriae, Corynebacterium ulcerans and Corynebacterium pseudotuberculosis. For diagnostic purposes, species identification and detection of toxigenic strains (diphtheria toxin (tox)-positive strains) is typically performed using end-point PCR. A faster quadruplex real-time PCR (qPCR) was recently developed (De Zoysa et al. J . Med . Microbiol. 2016;65(12):1521-1527).Aims. We aimed to improve the quadruplex method by adding a 16S rRNA gene target as an internal processing control, providing confirmation of the presence of bacterial DNA in the assays, thus avoiding the possibility of false-negative reporting.Methodology. Universal 16S rRNA gene primers and a probe were defined. The novel method was tested using 36 bacterial isolates and 17 clinical samples. Experimental robustness to temperature and reagent concentration variations was assessed.Results. The method allows detection of the tox gene and distinguishing C. diphtheriae (including the newly described species Corynebacterium belfantii) from C. ulcerans and C. pseudotuberculosis. Complete diagnostic specificity, sensitivity and experimental robustness were demonstrated. The lower limit of detection for C. diphtheriae, C. ulcerans and tox targets was 1.86 genome copies per 5 µl reaction volume. The method was successfully used on two distinct qPCR technologies (LightCycler 480, Roche Diagnostics and Rotor-Gene Q, Qiagen) and in two laboratories (Institut Pasteur, Paris, France and Public Health England - National Infection Service, London, UK).Conclusion. This work describes validation of the improved qPCR quadruplex method and supports its implementation for the biological diagnosis of diphtheria.
Assuntos
Infecções por Corynebacterium/diagnóstico , Corynebacterium/isolamento & purificação , Difteria/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real/métodos , Corynebacterium/classificação , Corynebacterium/genética , Infecções por Corynebacterium/microbiologia , Corynebacterium diphtheriae/classificação , Corynebacterium diphtheriae/genética , Corynebacterium diphtheriae/isolamento & purificação , DNA Bacteriano/genética , Difteria/microbiologia , Toxina Diftérica/genética , Humanos , RNA Ribossômico 16S/genéticaRESUMO
BACKGROUND: Diphtheria toxin (DT) is produced by toxigenic strains of the human pathogen Corynebacterium diphtheriae as well as zoonotic C. ulcerans and C. pseudotuberculosis. Toxigenic strains may cause severe respiratory diphtheria, myocarditis, neurological damage or cutaneous diphtheria. The DT encoding tox gene is located in a mobile genomic region and tox variability between C. diphtheriae and C. ulcerans has been postulated based on sequences of a few isolates. In contrast, species-specific sequence analysis of the diphtheria toxin repressor gene (dtxR), occurring both in toxigenic and non-toxigenic Corynebacterium species, has not been done yet. We used whole genome sequencing data from 91 toxigenic and 46 non-toxigenic isolates of different pathogenic Corynebacterium species of animal or human origin to elucidate differences in extracted DT, DtxR and tox-surrounding genetic elements by a phylogenetic analysis in a large sample set. RESULTS: Sequences of both DT and DtxR, extracted from whole genome sequencing data, could be classified in four distinct, nearly species-specific clades, corresponding to C. diphtheriae, C. pseudotuberculosis, C. ulcerans and atypical C. ulcerans from a non-toxigenic toxin gene-bearing wildlife cluster. Average amino acid similarities were above 99% for DT and DtxR within the four groups, but lower between them. For DT, subgroups below species level could be identified, correlating with different tox-comprising mobile genetic elements. In most C. diphtheriae, tox genes were located within known prophages. In contrast, in C. ulcerans diverse tox-including mobile elements could be identified: either prophages differing from C. diphtheriae prophages or an alternative pathogenicity island (PAI) described previously. One isolate showed a different, shorter tox-comprising putative PAI. Beyond the tox-overlapping elements, most isolates harbored a variety of additional prophages. CONCLUSION: Our NGS data from 137 isolates indicate the existence of different genetic backgrounds of DT-mediated pathogenicity in different Corynebacterium species and evolution of once acquired pathogenicity features with the strains. Different groups of pathogenicity-related elements within C. ulcerans imply that tox transmission pathways between isolates may differ in the zoonotic species and contribute to their emerging pathogenic potential.
Assuntos
Corynebacterium diphtheriae/patogenicidade , Toxina Diftérica/genética , Difteria/transmissão , Filogenia , Fatores de Virulência/genética , Animais , Corynebacterium diphtheriae/classificação , DNA Bacteriano/genética , Ilhas Genômicas , Humanos , Especificidade da Espécie , Virulência , Sequenciamento Completo do GenomaRESUMO
BACKGROUND: Corynebacterium diphtheriae is the main etiological agent of diphtheria, a global disease causing life-threatening infections, particularly in infants and children. Vaccination with diphtheria toxoid protects against infection with potent toxin producing strains. However a growing number of apparently non-toxigenic but potentially invasive C. diphtheriae strains are identified in countries with low prevalence of diphtheria, raising key questions about genomic structures and population dynamics of the species. This study examined genomic diversity among 48 C. diphtheriae isolates collected in Australia over a 12-year period using whole genome sequencing. Phylogeny was determined using SNP-based mapping and genome wide analysis. RESULTS: C. diphtheriae sequence type (ST) 32, a non-toxigenic clone with evidence of enhanced virulence that has been also circulating in Europe, appears to be endemic in Australia. Isolates from temporospatially related patients displayed the same ST and similarity in their core genomes. The genome-wide analysis highlighted a role of pilins, adhesion factors and iron utilization in infections caused by non-toxigenic strains. CONCLUSIONS: The genomic diversity of toxigenic and non-toxigenic strains of C. diphtheriae in Australia suggests multiple sources of infection and colonisation. Genomic surveillance of co-circulating toxigenic and non-toxigenic C. diphtheriae offer new insights into the evolution and virulence of pathogenic clones and can inform targeted public health actions and policy. The genomes presented in this investigation will contribute to the global surveillance of C. diphtheriae both for the monitoring of antibiotic resistance genes and virulent strains such as those belonging to ST32.
Assuntos
Corynebacterium diphtheriae/genética , Genoma Bacteriano , Pulmão/microbiologia , Pele/microbiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Austrália , Corynebacterium diphtheriae/classificação , Corynebacterium diphtheriae/isolamento & purificação , DNA Bacteriano/química , DNA Bacteriano/isolamento & purificação , DNA Bacteriano/metabolismo , Feminino , Estudo de Associação Genômica Ampla , Humanos , Masculino , Pessoa de Meia-Idade , Tipagem de Sequências Multilocus , Filogenia , Polimorfismo de Nucleotídeo Único , Sequenciamento Completo do Genoma , Adulto JovemRESUMO
Clinical isolates belonging to Corynebacterium diphtheriae biovar Belfanti were characterized by genomic sequencing and biochemical and chemotaxonomic analyses. Phylogenetic analyses indicated that biovar Belfanti represents a branch that is clearly demarcated from C. diphtheriae strains of biovars Mitis and Gravis. The average nucleotide identity of isolates of biovar Belfanti with C. diphtheriae type strain NCTC 11397T (biovar Gravis) was 94.85â%. The inability to reduce nitrate differentiated biovar Belfanti from other strains of C. diphtheriae. On the basis of these results, we propose the name Corynebacterium belfantii sp. nov. for the group of strains previously considered as C. diphtheriaebiovar Belfanti. The type strain of C. belfantii is FRC0043T (=CIP 111412T=DSM 105776T). Strains of C. belfantii were isolated mostly from human respiratory samples.
Assuntos
Corynebacterium diphtheriae/classificação , Filogenia , Sistema Respiratório/microbiologia , Técnicas de Tipagem Bacteriana , Composição de Bases , Corynebacterium diphtheriae/genética , Corynebacterium diphtheriae/isolamento & purificação , DNA Bacteriano/genética , Ácidos Graxos/química , França , Genes Bacterianos , Humanos , Fosfolipídeos/química , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Vitamina K 2/químicaRESUMO
OBJECTIVE: There is a lack of study in Corynebacterium diphtheriae isolates in Malaysia. The alarming surge of cases in year 2016 lead us to evaluate the local clinical C. diphtheriae strains in Malaysia. We conducted single nucleotide polymorphism phylogenetic analysis on the core and pan-genome as well as toxin and diphtheria toxin repressor (DtxR) genes of Malaysian C. diphtheriae isolates from the year 1986-2016. RESULTS: The comparison between core and pan-genomic comparison showed variation in the distribution of C. diphtheriae. The local isolates portrayed a heterogenous trait and a close relationship between Malaysia's and Belarus's, Africa's and India's strains were observed. A toxigenic C. diphtheriae clone was noted to be circulating in the Malaysian population for nearly 30 years and from our study, the non-toxigenic and toxigenic C. diphtheriae strains can be differentiated significantly into two large clusters, A and B respectively. Analysis against vaccine strain, PW8 portrayed that the amino acid composition of toxin and DtxR in Malaysia's local strains are well-conserved and there was no functional defect noted. Hence, the change in efficacy of the currently used toxoid vaccine is unlikely to occur.
Assuntos
Corynebacterium diphtheriae/classificação , Corynebacterium diphtheriae/genética , Toxoide Diftérico , Difteria/microbiologia , Difteria/prevenção & controle , Genoma Bacteriano/genética , Filogenia , Toxoide Diftérico/farmacologia , Humanos , MalásiaRESUMO
From 2016 through the middle of 2017, the German Consiliary Laboratory on Diphtheria noted an increase in nontoxigenic Corynebacterium diphtheriae isolates submitted from cities in northern Germany. Many patients for whom epidemiologic data were available were homeless, alcohol or drug abusers, or both. After performing routine diagnostics and multilocus sequence typing (MLST), we analyzed isolates of sequence type (ST) 8 and previously submitted isolates by whole-genome sequencing. Results were analyzed for phylogenetic relationship by core genome MLST (cg-MLST) and whole-genome single-nucleotide polymorphism profiles. Next-generation sequencing-based cg-MLST revealed several outbreak clusters caused by ST8; the geographic focus was in the metropolitan areas of Hamburg and Berlin. To achieve enhanced analytical depth, we used additional cg-MLST target genes and genome-wide single-nucleotide polymorphisms. We identified patient characteristics and detected transmission events, providing evidence that nontoxigenic C. diphtheriae infection is a potential public health threat in industrialized countries.
Assuntos
Infecções por Corynebacterium/epidemiologia , Infecções por Corynebacterium/microbiologia , Corynebacterium diphtheriae/classificação , Corynebacterium diphtheriae/genética , Infecções por Corynebacterium/história , Surtos de Doenças , Feminino , Genoma Bacteriano , Alemanha/epidemiologia , História do Século XXI , Humanos , Masculino , Tipagem de Sequências Multilocus , Polimorfismo de Nucleotídeo Único , Vigilância em Saúde Pública , Sequenciamento Completo do GenomaAssuntos
Corynebacterium diphtheriae/isolamento & purificação , Difteria/microbiologia , Animais , Técnicas de Tipagem Bacteriana , Chile , Corynebacterium diphtheriae/classificação , Difteria/tratamento farmacológico , Difteria/patologia , Eritromicina/uso terapêutico , Penicilina G/administração & dosagemRESUMO
BACKGROUND: Corynebacterium diphtheriae is a re-emerging pathogen in Europe causing invasive infections in vaccinated persons and classical diphtheria in unvaccinated persons. In the presented study we analysed genetic changes in C. diphtheriae isolates collected in Poland from the period before the introduction of the mass anti-diphtheria vaccination to the present time when over 98% of the population is vaccinated. METHODS: A total of 62 C. diphtheriae isolates collected in the 1950s-1960s, 1990s and 2000-2016 in Poland were investigated. Examined properties of the isolates included toxigenic status, presence of tox gene, biotype, MLST type (ST) and type of infection. RESULTS: A total of 12 sequence types (STs) were identified among the analysed C. diphtheriae isolates. The highest variability of STs was observed among isolates from diphtheria and asymptomatic carriers collected in the XX century. Over 95% of isolates collected from invasive and wound infections in 2004-2016 belonged to ST8. Isolates from the XX century represented all four biotypes: mitis, gravis, intermedius and belfanti, but the belfanti biotype appeared only after the epidemic in the 1990s. All except three isolates from the XXI century represented the biotype gravis. CONCLUSIONS: During a diphtheria epidemic period, non-epidemic clones of C. diphtheriae might also disseminate and persist in a particular area after the epidemic. An increase of the anti-diphtheria antibody level in the population causes not only the elimination of toxigenic strains from the population but may also influence the reduction of diversity of C. diphtheriae isolates. MLST types do not reflect the virulence of isolates. Each ST can be represented by various virulent variants representing various pathogenic capacities, for example toxigenic non-invasive, nontoxigenic invasive and nontoxigenic non-invasive.
Assuntos
Corynebacterium diphtheriae/classificação , Corynebacterium diphtheriae/isolamento & purificação , Difteria/epidemiologia , Técnicas de Tipagem Bacteriana , Corynebacterium diphtheriae/genética , Corynebacterium diphtheriae/patogenicidade , Difteria/microbiologia , Surtos de Doenças , Humanos , Tipagem de Sequências Multilocus , Polônia/epidemiologiaRESUMO
Corynebacterium diphtheriae is a globally important Gram-positive aerobic Actinobacterium capable of causing the toxin-mediated disease, diphtheria. Diphtheria was a major cause of childhood mortality prior to the introduction of the toxoid vaccine, yet it is capable of rapid resurgence following the breakdown of healthcare provision, vaccination or displacement of people. The mechanism and treatment of toxin-mediated disease is well understood, however there are key gaps in our knowledge on the basic biology of C. diphtheriae particularly relating to host colonisation, the nature of asymptomatic carriage, population genomics and host adaptation.
Assuntos
Corynebacterium diphtheriae , Difteria/epidemiologia , Difteria/microbiologia , Surtos de Doenças/prevenção & controle , Antibacterianos/uso terapêutico , Corynebacterium diphtheriae/classificação , Corynebacterium diphtheriae/patogenicidade , Corynebacterium diphtheriae/fisiologia , Difteria/tratamento farmacológico , Difteria/prevenção & controle , Antitoxina Diftérica/uso terapêutico , Toxina Diftérica/biossíntese , Toxina Diftérica/intoxicação , Toxoide Diftérico/administração & dosagem , Toxoide Diftérico/efeitos adversos , Surtos de Doenças/estatística & dados numéricos , Genoma Bacteriano , Humanos , Filogenia , Vacinação/normasRESUMO
In 2015, a cluster of respiratory diphtheria cases was reported from KwaZulu-Natal Province in South Africa. By using whole-genome analysis, we characterized 21 Corynebacterium diphtheriae isolates collected from 20 patients and contacts during the outbreak (1 patient was infected with 2 variants of C. diphtheriae). In addition, we included 1 cutaneous isolate, 2 endocarditis isolates, and 2 archived clinical isolates (ca. 1980) for comparison. Two novel lineages were identified, namely, toxigenic sequence type (ST) ST-378 (n = 17) and nontoxigenic ST-395 (n = 3). One archived isolate and the cutaneous isolate were ST-395, suggesting ongoing circulation of this lineage for >30 years. The absence of preexisting molecular sequence data limits drawing conclusions pertaining to the origin of these strains; however, these findings provide baseline genotypic data for future cases and outbreaks. Neither ST has been reported in any other country; this ST appears to be endemic only in South Africa.
Assuntos
Corynebacterium diphtheriae/classificação , Corynebacterium diphtheriae/genética , Difteria/epidemiologia , Difteria/microbiologia , Surtos de Doenças , Adolescente , Adulto , Sistemas CRISPR-Cas , Criança , Pré-Escolar , Corynebacterium diphtheriae/isolamento & purificação , Difteria/história , Feminino , Genoma Viral , História do Século XXI , Humanos , Lactente , Masculino , Tipagem de Sequências Multilocus , Filogenia , Sistema de Registros , África do Sul/epidemiologia , Sequenciamento Completo do Genoma , Adulto JovemRESUMO
In this study, we report the comparative genomics and phylogenetic analysis of Corynebacterium diphtheriae strain B-D-16-78 that was isolated from a clinical specimen in 2016. The complete genome of C. diphtheriae strain B-D-16-78 was sequenced using PacBio Single Molecule, Real-Time sequencing technology and consists of a 2,474,151-bp circular chromosome with an average GC content of 53.56%. The core genome of C. diphtheriae was also deduced from a total of 74 strains with complete or draft genome sequences and the core genome-based phylogenetic analysis revealed close genetic relationship among strains that shared the same MLST allelic profile. In the context of CRISPR-Cas system, which confers adaptive immunity against re-invading DNA, 73 out of 86 spacer sequences were found to be unique to Malaysian strains which harboured only type-II-C and/or type-I-E-a systems. A total of 48 tox genes which code for the diphtheria toxin were retrieved from the 74 genomes and with the exception of one truncated gene, only nucleotide substitutions were detected when compared to the tox gene sequence of PW8. More than half were synonymous substitution and only two were nonsynonymous substitutions whereby H24Y was predicted to have a damaging effect on the protein function whilst T262V was predicted to be tolerated. Both toxigenic and non-toxigenic toxin-gene bearing strains have been isolated in Malaysia but the repeated isolation of toxigenic strains with the same MLST profile suggests the possibility of some of these strains may be circulating in the population. Hence, efforts to increase herd immunity should be continued and supported by an effective monitoring and surveillance system to track, manage and control outbreak of cases.
Assuntos
Corynebacterium diphtheriae/classificação , Corynebacterium diphtheriae/genética , Difteria/microbiologia , Genoma Bacteriano , Genômica , Filogenia , Sistemas CRISPR-Cas , Biologia Computacional/métodos , Corynebacterium diphtheriae/isolamento & purificação , Difteria/epidemiologia , Toxina Diftérica/genética , Genômica/métodos , Humanos , Malásia/epidemiologia , Anotação de Sequência Molecular , Sequenciamento Completo do GenomaRESUMO
Corynebacterium diphtheriae is typically recognized as a colonizer of the upper respiratory tract (respiratory diphtheria) and the skin (cutaneous diphtheria). However, different strains of Corynebacteriumdiphtheriae can also cause invasive infections. In this study, the characterization of a non-toxigenic Corynebacteriumdiphtheriae strain (designated BR-INCA5015) isolated from osteomyelitis in the frontal bone of a patient with adenoid cystic carcinoma was performed. Pathogenic properties of the strain BR-INCA5015 were tested in a Caenorhabditis elegans survival assay showing strong colonization and killing by this strain. Survival rates of 3.8±2.7 %, 33.6±7.3 % and 0 % were observed for strains ATCC 27010T, ATCC 27012 and BR-INCA5015, respectively, at day 7. BR-INCA5015 was able to colonize epithelial cells, showing elevated capacity to adhere to and survive within HeLa cells compared to other Corynebacteriumdiphtheriae isolates. Intracellular survival in macrophages (THP-1 and RAW 264.7) was significantly higher compared to control strains ATCC 27010T (non-toxigenic) and ATCC 27012 (toxigenic). Furthermore, the ability of BR-INCA5015 to induce osteomyelitis was confirmed by in vivo assay using Swiss Webster mice.
Assuntos
Corynebacterium diphtheriae/isolamento & purificação , Corynebacterium diphtheriae/patogenicidade , Osteomielite/microbiologia , Adulto , Animais , Caenorhabditis elegans , Corynebacterium diphtheriae/classificação , Corynebacterium diphtheriae/genética , Células Epiteliais/microbiologia , Feminino , Humanos , Macrófagos/microbiologia , Masculino , Camundongos , Células RAW 264.7 , VirulênciaRESUMO
INTRODUCTION: Corynebacterium diphtheriae can cause various infections such as diphtheria, wound infections, septic arthritis, bacteraemia and endocarditis. Different virulence properties of the isolates might be related to different virulence factors expressed by the isolates. The objective of this study was to explore whether whole cell protein profiling might be useful in prediction of pathogenic properties of C. diphtheriae isolates. METHODS: C. disphtheriae isolates collected from diphtheria, invasive and local infections and from asymptomatic carriers in Poland, France, New Caledonia and Canada in 1950-2014 were investigated using whole cell protein profile analysis. RESULTS: All the examined isolates were divided into two clades: A and B with similarity about 47%, but clade B was represented by only one isolate. The clade A was divided in two subclades A.I NS .II with similarity 53,2% and then into four groups: A.Ia, A.Ib, A.Ic and A.Id. The comparative analysis did not distinguish clearly toxigenic and nontoxigenic isolates as well as invasive and noninvasive isolates. CONCLUSIONS: Whole cell protein profile analysis of C. diphtheria exhibits good concordance with other genotyping methods but this method is not able to distinguish clearly invasive from non-invasive isolates.