The X-ray structure of L-threonine dehydrogenase from the common hospital pathogen Clostridium difficile

In many prokaryotes, the first step of threonine metabolism is catalysed by the enzyme threonine dehydrogenase (TDH), which uses NAD+ to oxidize its substrate to 2-amino-3-ketobutyrate. The absence of a functional TDH gene in humans suggests that inhibitors of this enzyme may have therapeutic potential against pathogens which are reliant on this enzyme. Here, TDH from Clostridium difficile has been cloned and overexpressed, and the X-ray structure of the apoenzyme form has been determined at 2.6 Å resolution.

Identification of Pathogenic Variations in Seafood Vibrio parahaemolyticus Isolates by Comparing Genome Sequences

In this study, to investigate the pathogenic characteristics of the isolates, the pathogenic gene ( tdh gene) was identified, and adherence and cytotoxicity assays were performed. Furthermore, genome sequences of the seafood isolates were analyzed using the Illumina Hi-seq 2500 platform. The isolated strains were then mapped by comparing the genomes to the reference genome, and variations in the nucleotide sequences and amino acids were identified with the CLC Genomics Workbench program. As a result, the tdh gene was identified in four isolates of V. parahaemolyticus , three (SMFM201809-CPC7-3, SMFM201809-CF8-2, and SMFM201809-CF8-3) of which showed high cytotoxicity and differences in cell adhesion. Thus, they were selected to identify virulence factors and genomic variations. All three isolates had the same virulence factors, such as adherence, secretion systems, and toxin. Additionally, this study identified amino acid variants in the regions of type IV pilus, T3SS1, T3SS1 secreted effectors, and thermolabile hemolysin. These results indicate that variations in amino acids found in regions related to adherence and cytotoxicity led to differences in adhesion efficiency and cytotoxicity. Therefore, the isolates may cause more serious foodborne illness. .

Antibiotic resistance pattern and its correlation to the presence of tdh gene and CRISPR-Cas system in Vibrio parahaemolyticus strains isolated from seafood

Vibrio parahaemolyticus is a pathogen native to the aquatic environment. In this study, 46 environmental V. parahaemolyticus isolates were subjected to a correlational analysis to find the association between their antimicrobial susceptibility pattern, prevalence of CRISPR-Cas system and thermostable direct hemolysin (tdh) gene. Antibiotic resistance profiling against eleven antibiotics revealed the isolates to be multidrug resistant. Isolates exhibited highest resistance to vancomycin (97.8%) followed by ampicillin (91.3%), cefotaxime (69.6%), ceftazidime/clavulanic acid (54.4%), ceftazidime (45.7%) and gentamicin (39.1%). CRISPR loci and tdh gene were detected in 47.83 and 58.7% of strains respectively. No significant correlation was observed between antibiotic resistance to presence of CRISPR, except in the case of gentamicin wherein, a negative correlation was seen (r=-0.272, p<0.10). Similarly, tdh did not correlate to antibiotic resistance. Seventeen strains in this study harboured the CRISPR loci as well as tdh gene, the association of which was found to be statistically significant.

Virulence and Antibiotic Resistance of Vibrio parahaemolyticus Isolates from Seafood from Three Developing Countries and of Worldwide Environmental, Seafood, and Clinical Isolates from 2000 to 2017

ABSTRACT Vibrio parahaemolyticus is a leading cause of seafood-associated illness. This study investigated the prevalence, virulence, and antibiotic resistance of V. parahaemolyticus in three low- and middle-income countries. Freshly caught fish samples (n = 330) imported to Jordan from Yemen, India, and Egypt were tested. The overall prevalence of V. parahaemolyticus was 15% (95% confidence interval: 11 to 19%). Three isolates (6%) were positive for the thermostable direct hemolysin–related (trh) gene, and all isolates was negative for the thermostable direct hemolysin (tdh) gene. All isolates were resistant to colistin sulfate, neomycin, and kanamycin, and 51 and 43% of isolates were resistant to tetracycline and ampicillin, respectively. Only 4% of the isolates were resistant to cefotaxime and chloramphenicol, and no isolates were resistant to sulfamethoxazole-trimethoprim, streptomycin, gentamicin, ciprofloxacin, and nalidixic acid. All isolates were resistant to two classes of antibiotics, and 86% were multidrug resistant (resistant to at least one drug in three or more classes of antibiotics). A literature review of clinical, seafood, and environmental V. parahaemolyticus isolates worldwide revealed high rates of gentamicin and ampicillin resistance, emerging resistance to third-generation cephalosporins, and limited resistance to sulfamethoxazole-trimethoprim, ciprofloxacin, nalidixic acid, and chloramphenicol. Thus, last-resort antibiotics could be ineffective for treating V. parahaemolyticus infections. Several global reports also documented illness outbreaks in humans caused by trh- and tdh-negative V. parahaemolyticus strains. More research is needed to determine whether the presence of these genes is sufficient to classify the strains as virulent.

Occurrence of Vibrio vulnificus and Toxigenic Vibrio parahaemolyticus on Sea Catfishes from Galveston Bay, Texas

Dorsal and pectoral fin spines from two species of sea catfishes (Bagre marinus and Ariopsis felis) landed at 54 sites in Galveston Bay, Texas, and its subbays from June to October 2005 were screened with traditional cultivation-based assays and quantitative PCR assays for Vibrio vulnificus and Vibrio parahaemolyticus. V. vulnificus was present on 51.2% of fish (n = 247), with an average of 403 ±337 SD cells g−1. V. parahaemolyticus was present on 94.2% (n = 247); 12.8% tested positive for the virulence-conferring tdh gene, having an average 2,039 ± 2,171 SD cells g−1. The increasing trend in seafood consumption of “trash fishes” from lower trophic levels, such as sea catfishes, warrants evaluation of their life histories for association with pathogens of concern for human consumption.

Characterization of Vibrio Parahaemolyticus isolated from oysters and mussels in São Paulo, Brazil

Vibrio parahaemolyticus is a marine bacterium, responsible for gastroenteritis in humans. Most of the clinical isolates produce thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) encoded by tdh and trh genes respectively. In this study, twenty-three V. parahaemolyticus, previously isolated from oysters and mussels were analyzed by PCR using specific primers for the 16S rRNA and virulence genes (tdh, trh and tlh) and for resistance to different classes of antibiotics and PFGE. Nineteen isolates were confirmed by PCR as V. parahaemolyticus. The tlh gene was present in 100% of isolates, the tdh gene was identified in two (10.5%) isolates, whereas the gene trh was not detected. Each isolate was resistant to at least one of the nine antimicrobials tested. Additionally, all isolates possessed the blaTEM-116 gene. The presence of this gene in V. parahaemolyticus indicates the possibility of spreading this gene in the environment. Atypical strains of V. parahaemolyticus were also detected in this study.

Rapid and Specific Detection of the Thermostable Direct Hemolysin Gene in Vibrio parahaemolyticus by Loop-Mediated Isothermal Amplification

Several investigators have reported that thermostable direct hemolysin (TDH) and TDH-related hemolysin are important virulence factors of Vibrio parahaemolyticus, but it has been difficult to detect these factors rapidly in seafood and other environmental samples. A novel nucleic acid amplification method, termed the loop-mediated isothermal amplification (LAMP), which amplifies DNA with high specificity and rapidity under isothermal conditions, was applied. In this study, we designed tdh gene-specific LAMP primers for detection of TDH-producing V. parahaemolyticus. The specificity of this assay was evaluated with 32 strains of TDH-producing V. parahaemolyticus, one strain of TDH-producing Grimontia hollisae, 10 strains of TDH-nonproducing V. parahaemolyticus, and 94 strains of TDH-nonproducing bacteria, and the sensitivity was high enough to detect one cell per test. Moreover, to investigate the detection of TDH-producing V. parahaemolyticus in oysters, the LAMP assay was performed with enrichment culture in alkaline peptone water of oyster samples inoculated with TDH-producing V. parahaemolyticus and TDH-nonproducing V. parahaemolyticus and V. alginolyticus after enrichment in alkaline peptone water. These results suggest that the LAMP assay targeting tdh gene has high sensitivity and specificity and is useful to detect TDH-producing V. parahaemolyticus in oyster after enrichment.