Cytotoxic activity, virulence plasmid profiles and comparative proteomic analysis of Campylobacter jejuni strains

Human disease due to food-borne pathogens remains a medical problem worldwide. Campylobacter jejuni is the most common cause of bacterial food-borne diarrhoeal disease, but the mechanisms by which it causes disease remain unclear. This bacterium is also the most common antecedent to the peripheral neuropathies Guillain-Barre syndrome (GBS) and Miller-Fisher syndrome (MFS). The main aim of this project was to compare cytotoxicity of isolates of C. jejuni towards mammalian cells in culture, to purify any cytotoxic activity and to compare strains by plasmid and proteomic analysis. Using the API Campy identification kit, 37 of the 39 strains were identified as C. jejuni jejuni serotype 1 or 2 with identification (ID) scores > 98.3%; and two strains were identified as C. jejuni jejuni 1 with ID scores of 88.3%. GOT (Gamma Glutamyl Transferase) test was the most important test to differentiate between C. jejuni jejuni 1 and 2 which showed negative and positive results, respectively. The cytotoxic activity against Vero and Caco-2 cells of some of the typed strains of C. jejuni was tested. Cytotoxic activity was clearly demonstrated using Vero and Caco-2 cells and these activities peaked after incubation of the cell culture with sample for 16h. Comparison of wild-type strains 81-176 and 11168 with mutants of these strains lacking cytolethal distending toxin (CDT) or putative haemolysin or phospholipase toxins indicated that the toxicity observed was not due to any of these proteins, as cytotoxicity was unaffected by their absence. Some isolates (e.g. C0L12) showed low cytotoxicity while others, especially C. jejuni 81-176, consistently produced high cytotoxin activity which was heat-labile and lethal to tissue culture cells. Cytotoxicity comparisons of inner and outer membrane preparations with a cytoplasmic fraction of C. jejuni strain 81-176 and C0L12 indicated that greatest activity was found in the inner membrane fraction. Proteins in the membrane preparation could not be easily fractionated and cell-free extracts of strains with low (COL12) and high (81-176) cytotoxic effects were chosen for further purification and characterisation studies. Although some differences were detected after DEAE- Sepharose fractionation and two-dimensional electrophoretic (2-DE) separation of proteins, no individual proteins could be clearly related to cytotoxicity. However, proteomic analysis did reveal some interesting properties of the C. jejuni strains. Of the proteins characterised by mass spectrometry after 2-DE, oxidoreductases were the most frequently identified in the virulent strain 81-176, but not in the type strain 11168. There was no apparent correlation between toxin production and clinical symptoms in patients from whom the strains were isolated. The study indicated that not all C. jejuni strains tested produced cytotoxin (s), but that cytotoxin producers seemed to be predominantly human strains. The presence of virB11 and tetO genes carried by plasmids was examined by PCR in the 39 C. jejuni isolates together with strains 81-176 and NCTC 11168. Detection rates for the virB11 and tetO genes in the clinical isolates were 28.2% and 12.82%, respectively

Streptococcus pneumoniae quorum sensing and biofilm formation are affected by Thymus daenensis, Satureja hortensis, and Origanum vulgare essential oils

The aim of this study was to investigate the effect of Thymus daenensis L., Satureja hortensis L., and Origanum vulgare L. essential oils (EOs) on the planktonic growth, biofilm formation, quorum sensing (QS), and competence system (CS) of Streptococcus pneumoniae. The anti-biofilm activity of EOs was determined by Microtiter-Plate Test (MtP) and scanning electron microscope (SEM). The QS and CS inhibitory activities were determined on the pre-grown biofilm by gene expression analysis using quantitative real-time RT-PCR. Using gas chromatography–mass spectrometry analysis, the major components of the tested EOs were detected. The MtP and SEM detected a significant inhibitory effect of the three EOs on biofilm formation at sub-minimum inhibitory concentrations (MICs). The most anti-biofilm activity was seen for T. daenensis. LuxS and pfs genes (genes involved in QS) downregulated the following treatment with MIC/2 of Thymus and Satureja EOs. Thymol, carvacrol, p-cymene, pulegone, and 1,8-cineole were the major components of the tested EOs. The used EOs seem to be good candidates for preventing biofilm formation and subsequent colonization of S. pneumoniae. This study introduced T. daenensis and S. hortensis as new anti-biofilm and QS inhibitor agents with a natural origin

Expression of virulence factor genes in co-infections with Trueperella pyogenes isolates and other bacterial pathogens; an in vivo study

Trueperella pyogenes is an opportunistic bacterial pathogen causing several infectious diseases, including metritis, mastitis and abscesses in domestic animals such as dairy cattle. Several virulence proteins are released by T. pyogenes strains contributing to the pathogenic and causing disease potential of this pathogen. So far, many aspects of T. pyogenes pathogenesis are unknown. In this study, expression levels of plo, fimA, nanH and cbpA genes encoding pyolysin, fimbriae, neuraminidase and collagen-binding protein, respectively in T. pyogenes isolated from totally 15 metritis, mastitis and cutaneous abscesses convenience samples in response to co-culture with other pathogens including E. coli, St. dysgalactiae, S. aureus, F. necrophorum and L. plantarum strains in mice study model have been investigated. We found that expression levels of plo, fimA, nanH and cbpA genes in T. pyogenes isolates in response to co-culture with F. necrophorum and E. coli were significantly increased; however, no significant changes was seen in the level of expression of these genes in the isolates in response to co-culture with St. dysgalactiae and S. aureus. Notably, expression of all virulence factor genes was suppressed in T. pyogenes in response to co-culture with L. plantarum. We observed that L. plantarum might be used to prevent infectious diseases caused by T. pyogenes

Seroprevalence of Neospora caninum in slaughtered native cattle in Kurdistan province, Iran

Neospora caninum is a worldwide distributed pathogen which causes abortion in cattle leading to economic loss in the cattle industry. The aim of this study was to determine the seroprevalence of N. caninum antibodies in the native cattle slaughtered in various areas of Kurdistan province (western Iran) from September 2010 to September 2011. Serum samples from 368 cattle slaughtered in seven slaughterhouses in this region were taken for detection of anti-N. caninum antibodies using commercial N. caninum ELISA kit. Antibodies to N. caninum were found in 29 samples (7.80%). The present study was the first report of Neospora infection in this region and indicated that native cattle of Kurdistan province were exposed to this parasite

Detection of Methicillin-Resistance Gene in Staphylococcus aureus Isolated from Traditional White Cheese in Iran

Background & Aims of the Study: Methicillin-resistant staphylococcus aureus (MRSA) is considered as a major pathogen in public health concern. The objectives of this study were to firstly determine antibiotic sensitivity among Staphylococcus aureus isolated from traditional Iranian white cheese during 2015 from Hamedan province of Iran; and secondly to estimate the presence of methicillin-resistant S. aureus. Materials &Methods: This cross-sectional study was done by collecting 120 Iranian white cheeses (traditional and industrial) which were available in different markets; and tested for the presence of S. aureus by culture methods. The obtained isolates were subjected to disc diffusion antimicrobial susceptibility tests followed by PCR detection of the mecA gene. Results: Out of 120 examined cheese samples, 19 samples (31.67%) were contaminated with S. aureus. The highest rate of antibiotic resistance was observed for penicillin, as all of the 19 isolates (100%) were found to be resistant to this antibiotic using disk diffusion method. Three out of 19 S. aureus isolates (15.7%) were phenotypically resistant to methicillin (disk diffusion), while 4 (21.05%) of them were genotypically confirmed as MRSA strains. Furthermore, none of the isolates were found resistant to vancomycin. Conclusion: The results of the study confirm the presence of methicillin resistant strains of S. aureus in Iranian white cheese. It should be considered to constitute a potential health risk for consumers, suggesting usage of more stringent hygiene measures

Investigation of antimicrobial susceptibility and virulence factor genes in Trueperella pyogenes isolated from clinical mastitis cases of dairy cows

Trueperella pyogenes is an opportunistic pathogen causing important diseases including mastitis and metritis in domestic animals such as dairy cows leading to prominent economic losses in food production industry. The aim of this study was to investigate bacterial species, antimicrobial susceptibility, and presence of virulence factor genes and genotyping of T. pyogenes isolates associated with summer mastitis cases from 22 different farms around Tehran, Iran. Fifty-five percent of dairy cows with clinical mastitis symptoms was infected by T. pyogenesis indicated that this pathogen is the most important contributor to clinical mastitis in dairy cows in the present study. A significant correlation was illustrated between presence of virulence factor genes of isolated pathogen, biochemical patterns, and the utter infected types. Multidrug resistance susceptibility observed between isolates indicated the important need for prudent use of antimicrobials in treatment of mastitis caused by T. pyogenes and increased concerning of consumer health associated with recent problems of antimicrobial resistance. The categorization of isolates was implemented into seven different clonal related types by COX-PCR at 80% of similarity cutoff with significance relationship to clonal types, CAMP test result and sampling time and biochemical profile. Regarding to the results obtained at the present study, T. pyogenes can be considered as an important typically cause of purulent and acute form of clinical bovine mastitis and loss of dairy productivity. Further studies with more sample size and high-throughput omic methods in various sampling time and areas are suggested for study of this pathogen precisely. KEYWORDS antimicrobial susceptibility, dairy cow, Trueperella pyogenes, virulence factor gen

Complete genome sequence of Trueperella pyogenes strain Arash114, isolated from the uterus of a water buffalo (Bubalus bubalis) in Iran

Objective: Trueperella pyogenes has been considered a major causative agent of metritis, abortion, and death in a broad range of domestic and wild animals, including cattle, swine, sheep, goats, camels, buffalo, deer, antelopes, reptiles, and birds. Data description: Here, we report the complete chromosome sequence of Trueperella pyogenes strain Arash114, isolated from the uterus of a water buffalo (Bubalus bubalis) died due to the infection caused by this pathogen. The genome assembly comprised 2,338,282 bp, with a 59.5% GC content. Annotation of the genome showed 46 tRNA genes, 6 rRNA, 1 CRISPR and 2059 coding sequences. Also, several genes coding for antimicrobial resistance such as tetW and virulence factor including plo, nanH, nanP, cbp and 4 fimbrial proteins were found. This study will advance our knowledge regarding the metabolism, virulence factors, antibiotic resistance and evolution of Arash114 strain and serve as an appropriate template for future researches. Keywords: Complete genome sequencing; Trueperella pyogenes; Uterus infection; Water buffalo