Divergent evolution of Corynebacterium diphtheriae in India: An update from National Diphtheria Surveillance network

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.

Monitoring of cyanobacteria using derivative spectrophotometry and improvement of the method detection limit by changing pathlength

Effective monitoring tools and methods are needed for the early detection and management of cyanobacteria in water bodies to minimize their harmful impacts on the environment and public health. This research investigated changing the cuvette pathlength (10-, 50-, and 100-mm) to improve the detection of cyanobacteria using UV-Vis spectrophotometry with subsequent application of derivative spectrophotometry and Savitzky-Golay (S-G) transformation. A non-toxigenic strain of blue-green cyanobacteria, Microcystis aeruginosa (CPCC 632), and a green algae strain for comparison, Chlorella vulgaris (CPCC 90), were studied in a wide range of concentrations (955,000–1,855 cells/mL). In each concentration range, method detection limits were established with absorbance measurements and S-G first derivative of absorbance using 10-, 50-, and 100-mm cuvette pathlengths. Increasing the cuvette pathlength from 10 to 100 mm resulted in a 15-fold improvement in sensitivity with absorbance and a 13-fold improvement with S-G first derivative of absorbance for M. aeruginosa. Overall, adoption of 100 mm pathlength and application of S-G derivative spectra improved the method detection limit for M. aeruginosa from 337,398 cells/mL to 4,916 cells/mL, which is below the WHO guideline for low probability of adverse health effects (<20,000 cells/mL). Similarly, the detection limit for C. vulgaris was improved from 650,414 cells/mL to 11,661 cells/mL. The results also showed that spectrophotometry could differentiate M. aeruginosa from C. vulgaris based on the variations in their pigment absorbance peaks.

Whole genome sequence of a non-toxigenic Corynebacterium diphtheriae strain from a hospital in southeastern China

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.

Clinical case of combined intestinal infection caused by enteroaggregative Escherichia coli and toxigenic strain Clostridium difficile in a child with cystic fibrosis

The goal is to study the peculiarities of the clinical picture and the distant outcome of intestinal infection due to enteroaggregative escherichiosis and the toxigenic strain of C. difficile in a child with cystic fibrosis.Material and methods. To verify the etiology of intestinal infection, the following studies were conducted. — fecal PCR “OKI-screen” tests to detect viral and bacterial pathogens, fecal bacteriopsy for pathogenic and opportunistic microbes; determination of C. difficile A and B toxins in feces by enzyme-linked fluorescence analysis.Results. The combined intestinal infection caused by en-teroaggregative escherichiosis and a toxigenic strain of C. difficile, in a child with pulmonary-intestinal form of cystic fibrosis was characterized by a wavy course, a pronounced intoxication syndrome, excicosis, hemorrhagic enterocolitis, signs of systemic and local inflammation, metabolic disorders. There were no recurrences of C. difficile-infection in catamnese.Conclusion. It is necessary to continue research on the course of intestinal infections caused by bacterial associations of pathogens.

Comparison of Host Cytokine Response in Piglets Infected With Toxigenic and Non-toxigenic Staphylococcus hyicus

Staphylococcus hyicus is the most common causative agent of exudative epidermitis (EE) in piglets. Staphylococcus hyicus can be grouped into toxigenic and non-toxigenic strains based on its ability to cause EE in pigs. However, the inflammatory response of piglets infected with toxigenic and non-toxigenic S. hyicus has not been elucidated. In this study, we evaluated the serum cytokine profile in piglets inoculated with toxigenic and non-toxigenic S. hyicus strains and recorded the clinical signs in piglets. Fifteen piglets were divided into three groups (n = 5) and inoculated with a toxigenic strain (ZC-4), a non-toxigenic strain (CF-1), and PBS (control), respectively. The changes in serum levels of cytokines (interleukin [IL]-1β, IL-4, IL-6, IL-8, IL-10, IL-12, granulocyte-macrophage colony-stimulating factor, interferon-γ, transforming growth factor-β1, and tumor necrosis factor-α) were evaluated using a cytokine array at 6, 24, 48, and 72 h post inoculation. The results showed that piglets infected with the toxigenic strain exhibited more severe clinical signs and higher mortality than those infected with the non-toxigenic strain. The serum levels of pro-inflammatory cytokine IL-1β were significantly increased in toxigenic-and non-toxigenic-strain-infected piglets compared to those in the control group (p < 0.05), while the anti-inflammatory cytokine IL-10 was significantly up-regulated only in toxigenic group than in control group (p < 0.05). These results indicated that piglets infected with toxigenic and non-toxigenic S. hyicus showed differential infection status and inflammatory responses. Both toxigenic- and non-toxigenic- S. hyicus infection could induce a pro-inflammatory reaction in piglets. In addition, the toxigenic strain induced a strong anti-inflammatory response in piglets as indicated by the increased serum level of IL-10, which may be associated with the severe clinical signs and increased mortality and may be the key cytokine response responsible for pathogenic mechanisms of S. hyicus.

PEPTIDE IMUNOFAN AND MOXIFLOXACIN COMBINED EFFECTS ON TOXIGENIC STRAIN CLOSTRIDIUM DIFFICILE IN VITRO

Moxifloxacin and imunofan peptide concentrations - dependent Clostridium difficile growth rate was analyzed in vitro. In the course of our study, it was revealed imunofan peptide at concentrations 0.05, 0.25 μg/ml has antimicrobial characteristics against toxigenic C. difficile strain. At the same time, with the peptide and the antibiotic combined interaction, we observed moxifloxacin concentration 0-1.5 MIC stimulates C. difficile growth, regardless of the imunofan concentration. Concentrations of maximum growth inhibition for C. difficile were also established with the combined effects peptide imunofan and antibiotic moxifloxacin - 1.5 μg/ml and 2.5 MIC, respectively.

Naphtho-Gamma-Pyrones Produced by Aspergillus tubingensis G131: New Source of Natural Nontoxic Antioxidants

Seven naphtho-gamma-pyrones (NγPs), including asperpyrone E, aurasperone A, dianhydroaurasperone C, fonsecin, fonsecinone A, fonsecin B, and ustilaginoidin A, were isolated from Aspergillus tubingensis G131, a non-toxigenic strain. The radical scavenging activity of these NγPs was evaluated using ABTS assay. The Trolox equivalent antioxidant capacity on the seven isolated NγPs ranged from 2.4 to 14.6 μmol L−1. The toxicity and ability of the NγPs to prevent H2O2-mediated cell death were evaluated using normal/not cancerous cells (CHO cells). This cell-based assay showed that NγPs: (1) Are not toxic or weakly toxic towards cells and (2) are able to protect cells from oxidant injuries with an IC50 on H2O2-mediated cell death ranging from 2.25 to 1800 μmol mL−1. Our data show that A. tubingensis G131 strain is able to produce various NγPs possessing strong antioxidant activities and low toxicities, making this strain a good candidate for antioxidant applications in food and cosmetic industries.