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BACKGROUND: Corynebacterium diphtheriae complex was formed by the species C. diphtheriae, Corynebacterium ulcerans and Corynebacterium pseudotuberculosis in the recent past. In addition to C. diphtheriae, C. ulcerans and C. pseudotuberculosis species can carry the tox gene, which encodes diphtheria toxin. Currently, three new species have been included in the complex: Corynebacterium rouxii, Corynebacterium silvaticum, and Corynebacterium belfantii. C. rouxii is derived from the ancient Belfanti biovar of C. diptheriae. We provide the complete genome sequences of two non-toxigenic strains C. rouxii isolated from a cat with a purulent infection in Brazil. The taxonomic status and sequence type, as well as the presence of resistance and virulence genes, and CRISPR-Cas system were additionally defined. RESULTS: The genomes showed an average size of 2.4 Mb and 53.2% GC content, similar to the type strain of the species deposited in Genbank/NCBI. Strains were identified as C. rouxii by the rMLST database, with 95% identity. ANI and DDH in silico were consistent with values above the proposed cut-off points for species limit, corroborating the identification of the strains as C. rouxii. MLST analyses revealed a new ST, which differs from ST-537 only by the fusA allele. No horizontal transfer resistance gene was predicted in both genomes and no mutation was detected in the constitutive genes gyrA and rpoB. Some mutations were found in the seven penicillin-binding proteins (PBPs) detected. The tox gene was not found, but its regulatory gene dtxR was present. Among the predicted virulence genes are those involved in iron uptake and adherence, in addition to the DIP0733 protein involved in epithelial cell adhesion and invasion. The CRISPR-Cas type I-E system was detected in both genomes, with 16 spacer sequences each. Of them, half are unknown according to the databases used, indicating that there is an unexplored reservoir of corynebacteriophages and plasmids. CONCLUSIONS: This is the first genomic study of C. rouxii reported in Brazil. Here we performed taxonomic analysis and the prediction of virulence factors. The genomic analyses performed in this study may help to understand the potential pathogenesis of non-toxigenic C. rouxii strains.

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A patient with a renal transplant after an autologous stem cell transplant for multiple myeloma developed non-toxigenic Vibrio cholerae diarrhea after travel to Mexico. This is a rare cause of diarrhea in transplant recipients, and the patient had not had pre-travel counseling. This case reflects the lack of referral of transplant recipients for travel infectious disease review before overseas travel and the role of the live attenuated cholera vaccine.

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Vibrio parahaemolyticus non-toxigenic strains are responsible for about 10% of acute gastroenteritis associated with this species, suggesting they harbor unique virulence factors. Zonula occludens toxin (Zot), firstly described in Vibrio cholerae, is a secreted toxin that increases intestinal permeability. Recently, we identified Zot-encoding genes in the genomes of highly cytotoxic Chilean V. parahaemolyticus strains, including the non-toxigenic clinical strain PMC53.7. To gain insights into a possible role of Zot in V. parahaemolyticus, we analyzed whether it could be responsible for cytotoxicity. However, we observed a barely positive correlation between Caco-2 cell membrane damage and Zot mRNA expression during PMC53.7 infection and non-cytotoxicity induction in response to purified PMC53.7-Zot. Unusually, we observed a particular actin disturbance on cells infected with PMC53.7. Based on this observation, we decided to compare the sequence of PMC53.7-Zot with Zot of human pathogenic species such as V. cholerae, Campylobacter concisus, Neisseria meningitidis, and other V. parahaemolyticus strains, using computational tools. The PMC53.7-Zot was compared with other toxins and identified as an endotoxin with conserved motifs in the N-terminus and a variable C-terminal region and without FCIGRL peptide. Notably, the C-terminal diversity among Zots meant that not all of them could be identified as toxins. Structurally, PMC53.7-Zot was modeled as a transmembrane protein. Our results suggested that it has partial 3D structure similarity with V. cholerae-Zot. Probably, the PMC53.7-Zot would affect the actin cytoskeletal, but, in the absence of FCIGRL, the mechanisms of actions must be elucidated.

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Clostridioides difficile is a Gram positive, sporulated, rod-shape, anaerobic pathogen responsible for nosocomial diarrhea and colitis, mainly in antibiotic treated patients. C. difficile produce two toxins responsible for disease, toxin A (TcdA) and toxin B (TcdB), although not all strains produce them. Non-toxigenic C. difficile (NTCD) strains are able to colonize the intestinal mucosa and are often isolated from asymptomatic individuals. NTCD are poorly studied, their evolutionary history has not been elucidated, and their relationship with illness remains controversial. The aim of this work was to analyze the phenotype of NTCD strains isolated from clinical cases in hospitals of México, and whether NTCD strains present characteristics that differentiate them from the toxigenic strains. Seventy-four C. difficile strains isolated from patients were tested for cytotoxicity and 14 were identified as NTCD strains. We analyzed phenotypical characteristics that are important for the biology of C. difficile like colony morphology, antibiotic resistance, motility, sporulation, and adherence. Strains were also genotyped to determine the presence of genes coding for TcdA, TcdB and binary toxin and ribotyped for 027 type. When compared with toxigenic strains, NTCD strains presented an enlarged branched colony morphology, higher resistance to metronidazole, and increased sporulation efficiency. This phenotype has been reported associated with mutations that regulates phenotypic characteristics like swimming, sporulation or adhesion. Our results show that phenotype of NTCD strains is heterogeneous but still present characteristics that differentiate them from toxigenic strains.

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Vibrio parahaemolyticus is the leading cause of seafood-borne gastroenteritis worldwide. As reported in other countries, after the rise and fall of the pandemic strain in Chile, other post-pandemic strains have been associated with clinical cases, including strains lacking the major toxins TDH and TRH. Since the presence or absence of tdh and trh genes has been used for diagnostic purposes and as a proxy of the virulence of V. parahaemolyticus isolates, the understanding of virulence in V. parahaemolyticus strains lacking toxins is essential to detect these strains present in water and marine products to avoid possible food-borne infection. In this study, we characterized the genome of four environmental and two clinical non-toxigenic strains (tdh-, trh-, and T3SS2-). Using whole-genome sequencing, phylogenetic, and comparative genome analysis, we identified the core and pan-genome of V. parahaemolyticus of strains of southern Chile. The phylogenetic tree based on the core genome showed low genetic diversity but the analysis of the pan-genome revealed that all strains harbored genomic islands carrying diverse virulence and fitness factors or prophage-like elements that encode toxins like Zot and RTX. Interestingly, the three strains carrying Zot-like toxin have a different sequence, although the alignment showed some conserved areas with the zot sequence found in V. cholerae. In addition, we identified an unexpected diversity in the genetic architecture of the T3SS1 gene cluster and the presence of the T3SS2 gene cluster in a non-pandemic environmental strain. Our study sheds light on the diversity of V. parahaemolyticus strains from the southern Pacific which increases our current knowledge regarding the global diversity of this organism.

RESUMO

Vibrio parahaemolyticus is a leading cause of raw seafood-associated bacterial gastroenteritis in the world. Its pathogenesis is likely to be multifactorial, although the most characteristic virulence-associated factors are the toxins TDH and TRH, in addition to the Type-III Secretion System-2, which codes for diverse effectors involved in cytotoxicity and enterotoxicity. However, diarrhea cases produced by clinical strains lacking all of these main virulence factors (non-toxigenic strains) have been reported in many countries and they can represent up to 9-10% of the clinical isolations. So far, although there have been significant advances in the description of the virulence factors of V. parahaemolyticus, the ability of non-toxigenic strains to cause illness is still not completely understood. To elucidate this question it is necessary to have adequate infection models. The susceptibility of G. mellonella to the infection with non-toxigenic strains seems to be the response to identifying new virulence factors and consequently providing new insights into mechanisms of the virulence of non-toxigenic strains. This new model means an invaluable contribution to public health, since the understanding of virulence in strains lacking the traditional major toxins is essential to detect these strains present in waters and marine products and avoid possible food-borne infection.

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