Tox Gene Research Articles

Spatiotemporal persistence of multiple, diverse clades and toxins of Corynebacterium diphtheriae

Diphtheria is a respiratory disease caused by the bacterium Corynebacterium diphtheriae. Although the development of a toxin-based vaccine in the 1930s has allowed a high level of control over the disease, cases have increased in recent years. Here, we describe the genomic variation of 502 C. diphtheriae isolates across 16 countries and territories over 122 years. We generate a core gene phylogeny and determine the presence of antimicrobial resistance genes and variation within the tox gene of 291 tox+ isolates. Numerous, highly diverse clusters of C. diphtheriae are observed across the phylogeny, each containing isolates from multiple countries, regions and time of isolation. The number of antimicrobial resistance genes, as well as the breadth of antibiotic resistance, is substantially greater in the last decade than ever before. We identified and analysed 18 tox gene variants, with mutations estimated to be of medium to high structural impact.

Prevalence of the Genes Associated with Biofilm and Toxins Synthesis amongst the Pseudomonas aeruginosa Clinical Strains.

Pseudomonas aeruginosa is one of the most commonly isolated bacteria from clinical specimens, with an increasing isolation frequency in nosocomial outbreaks. The hypothesis tested was whether carbapenem-resistant P. aeruginosa strains display an altered carriage of the virulence factor genes, depending on the type of carbapenem resistance. The aim of the study was to investigate, by PCR, the frequency of 10 chosen virulence factors genes (phzM, phzS, exoT, exoY, exoU, toxA, exoS, algD, pilA and pilB) and the genotype distribution in 107 non-duplicated carbapenem-resistant P. aeruginosa isolates. P. aeruginosa genes involved in phenazine dyes and exoenzyme T synthesis were noted with the highest frequency (100%). Fimbriae-encoding genes were detected with the lowest incidence: 15.9% and 4.7% for pilin A and B, respectively. The differences observed between the exoS gene prevalence amongst the carbapenemase-positive and the carbapenemase-negative strains and the pilA gene prevalence amongst the strains of different origins were statistically significant. Virulence genes’ prevalence and the genotype distribution vary amongst P. aeruginosa strains resistant to carbapenems, especially in terms of their carbapenemase synthesis ability and the strain origin.

Molecular diagnostic of toxigenic Corynebacterium diphtheriae strain by DNA sensor potentially suitable for electrochemical point-of-care diagnostic

The presented study is focused on the development of electrochemical genosensor for detection of tox gene fragment of toxigenic Corynebacterium diphtheriae strain. Together with our previous studies it fulfils the whole procedure for fast and accurate diagnostic of diphtheria at its early stage of infection with the use of electrochemical methods. The developed DNA sensor potentially can be used in more sophisticated portable device. After the electrochemical stem-loop probe structure optimization the conditions for real asymmetric PCR (aPCR) product detection were selected. As was shown it was crucial to optimize the magnesium and organic solvent concentrations in detection buffer. Under optimal conditions it was possible to selectively detect as low as 20.8 nM of complementary stand in 5 min or 0.5 nM in 30 min with sensitivity of 12.81 and 0.24 1⋅μM−1 respectively. The unspecific biosensor response was elucidated with the use of new electrode blocking agent, diethyldithiocarbamate. Its application in electrochemical genosensors lead to significant higher current values and the biosensor response even in conditions with magnesium ion depletion. The developed biosensor selectivity was examined using samples containing genetic material originated from a number of non-target bacterial species which potentially can be present in the human upper respiratory tract.

Distinct Tumor-Resident Memory HBV-Specific T Cell Responses Correlate with Relapse-Free Survival in Patients with HBV-Associated Hepatocellular Carcinoma

In the context of hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC), despite a range of possibilities, the antigen specificities of tumor-infiltrating T cells and their relevance to control of is largely unknown. Using highly multiplexed peptide-MHC tetramer staining of unexpanded cells from blood, liver and tumor tissues from 46 HCC patients, we detected 91 different CD8 T cell populations specific for epitopes derived from HBV, tumor-associated and neoantigens (NeoAg), as well as disease-unrelated antigens. Parallel high-dimensional analysis delineated distinct tissue-resident memory T cells (TRM) populations among other highly diverse lymphocytes profiles observed in these compartments. Intratumoral and intrahepatic HBV-specific T cells were particularly enriched for three TRM phenotypes and the majority expressed relatively low levels of PD-1 receptor and TOX gene, inconsistent with reported terminal exhausted T cells (TEX) despite being clonally expanded within tumors. High frequencies of terminal TEX in these tumors was uncommon, whereas patients who had detectable and less exhausted tumor-infiltrating HBV- or NeoAg-specific CD8 TRM had superior long-term relapse-free survival. Thus, non-terminally-exhausted tumor-resident HBV-specific CD8 TRM show hallmarks of active involvement and effective antitumor response, implying that these cells could be harnessed for therapeutic purposes.

Investigation of Genetic Diversity of Pasteurella multocida Isolated from Diseased Poultry in Korea

Pasteurella multocida causes fowl cholera which is an economically important disease in poultry industries around the world. In this study, we analyzed the capsular genotype, lipopolysaccharide (LPS) genotype, virulence-associated genes (VAGs) patterns, antimicrobial resistance and genetic diversity in a total of 9 P. multocida isolates from poultry with fowl cholera between 2014 and 2019 in Korea. When combining the capsular types with the LPS genotypes, two isolates of the 9 isolates were A:L3, and the others were non-typeable (NT): L3. Of the 23 VAGs, all the isolates harbored ptfA, fimA, hsf-1, hsf-2, pfhA, exbB, exbD, tonB, hgbA, hgbB, fur, sodA, sodC, pmHAS, ompA, ompH, oma87, plpB, psl, and nanH, whereas toxA gene was not detected in any of the 9 isolates. In addition, among the 11 antimicrobials, most of the isolates except for one isolate resistant to florfenicol, exhibited susceptibility to all the antimicrobials. Multi-locus sequence typing (MLST) analysis revealed 5 different sequence types (ST): ST8, ST351, ST352, ST353, and ST368. The ST351, ST352, ST353, and ST368 were identified for the first time in this study, and ST352 and ST353 isolates were largely prevalent nationwide. These STs isolates should be monitored continuously because in some cases, ST352 and ST353 isolates demonstrated high mortality rates. Although only limited numbers of isolates have been analyzed, our findings provide overall characteristics and epidemiological information of the P. multocida strains recently prevalent in Korea.

A novel PCR method for simultaneously detecting Acute hepatopancreatic Necrosis Disease (AHPND) and mutant-AHPND in shrimp

Acute Hepatopancreatic Necrosis Disease (AHPND) is a globally serious threat, especially for Vietnam shrimp culture. This disease is caused by toxin-carrying bacteria Vibrio parahaemolyticus which plasmid encodes binary toxins ToxA and ToxB. The disease has a mortality rate by up to 100% after 3–5 days post infection and fast spread among shrimp culturing regions. In 2017, a mutant of this disease named mutant-AHPND was recorded with a 1053 bp transposon inserted into toxA gene, toxB gene was not affected but the mortality rate was still up to 50% and the disease still spread quickly. There is no PCR test which can simultaneously detect both the normal and mutant plasmids of this disease commercially available. In this study, we developed a PCR test with single primer pairs which can specifically and simultaneously detect both types of the disease in shrimp. The PCR technique is established with a sensitivity of 2.5 pg per 20 μl reaction for plasmid solution and is four-times higher than that of the AP3 PCR kit which is commercially used in detection of AHPND disease in shrimp. Whereas the specificity of the method analyzed using two Vp-AHPND strains, one mutant Vp-AHPND strain, thirteen non-AHPND Vibrio species and White spot syndrome virus (WSSV) revealed that the method does not give unspecific reaction with non-AHPND bacteria and WSSV. The established PCR reaction is the core step in developing a PCR kit that helps detect both types of disease-causing plasmid in shrimp. With optimized parameters, this PCR kit can also be a cost-effective and rapid tool which can be used by shrimp cultivators and organizations in disease detection for epidemic monitoring.

Molecular Typing and Virulence Analysis of Pseudomonas Aerugınosa Isolated From Burn Infections Recovered From Duhok and Erbil Hospitals/Iraq.

In this study, 225 isolates of Pseudomonas aeruginosa were recovered from burn wounds in major hospitals in Duhok and Erbil, Iraq, between April 2015 and September 2015. A total of 136 of these isolates were from men, comprising 60.4% of the total, whereas 89 (39.6%) were recovered from women. One hundred of these isolates were selected (50 from each province of Erbil and Duhok) and subjected to 16 different antibiotics using the disc diffusion method. The isolates showed a high level of resistance to most of the tested antibiotics, with 90% of the isolates being multidrug resistant. Imipenem was considered as the most effective antibiotic against these isolates with a resistant rate of 47%. The genome of all of these isolates were successfully amplified and produced a single band for the 16S rDNA locus with a molecular weight of about 956 base pairs, which was used to confirm, at the molecular level, that all these isolates were indeed P. aeruginosa. The results of the detection of five virulence-related genes including opr1, toxA, exoS, lasB, and nan1 revealed that 10 of these isolates, accounting for 10%, lacked any of the tested virulence markers. The opr1 gene, as a marker for the presence of a pathogenicity island, was the most dominant marker among all the virulence markers and was detected in 90 isolates (90%), followed by the toxA and exoS genes, which were both observed in 86 (86%) isolates, whereas the lasB gene was found in 82 (82%) isolates and the nan1 gene in 35 (35%) of the isolates, respectively.

Virulence genotyping of drug resistant Pseudomonas aeruginosa clinical isolates in Egypt using multiplex PCR

Pseudomonas aeruginosa is a serious Gram- negative pathogen capable of causing several nosocomial and community acquired infections. It produces many toxins such as: exotoxin A, pyocyanin, elastase and type III secretion system proteins. P. aeruginosa is genotyped according to the produced type III secretion system protein into invasive and cytotoxic genotypes. This study aimed to determine the prevalent virulence genotypes as well as the prevalence of five virulence genes using multiplex PCR. The antimicrobial resistance of the collected 134 P. aeruginosa isolates was detected using the disc diffusion method. The five exotoxin genes were detected by conventional multiplex PCR. Seventy percent of isolates were multidrug resistant. The phz, lasB, toxA, exoS and exoU genes were detected in 93%, 90%, 88%, 57% and 43% of isolates, respectively. Fifty two percent of isolates represented the invasive genotype and 38.8% represented the cytotoxic genotype. Clinical P. aeruginosa strains exhibit high antimicrobial resistance. The invasive and cytotoxic genotypes are associated with different types of clinical specimens and both genotypes can co-exist in the same isolates. Therefore, following up the antibiotic resistance of P. aeruginosa and its virulence factors and produced toxins is recommended to judge the bacterial virulence in outbreaks and epidemiologic studies.

Prevalence, Virulence, Antimicrobial Resistance, and Molecular Characterization of Pseudomonas aeruginosa Isolates From Drinking Water in China.

Pseudomonas aeruginosa is an important opportunistic pathogen and remains a major threat to the microbial safety of drinking water. There is a lack of comprehensive data on P. aeruginosa contamination in drinking water in China. Therefore, this study aimed to determine the prevalence, genetic diversity, virulence genes, and antimicrobial resistance of P. aeruginosa isolated from mineral water and spring water in China. From January 2013 to January 2014, 314 drinking water samples were collected from 23 cities in China. Of the collected samples, 77 (24.5%) were contaminated with P. aeruginosa, and these comprised 34 raw water (30.4%), 39 activated carbon-filtered water (30.6%), and four final water product (3.9%). A total of 132 P. aeruginosa isolates were obtained, and all of them showed the presence of virulence genes, with the detection rates of ExoU, ExoS, phzM, toxA, and lasB genes being 7.6, 86.3, 95.5, 89.4, and 100%, respectively. All isolates were sensitive to the 14 antibiotics (ciprofloxacin, levofloxacin, ofloxacin, norfloxacin, gentamicin, tobramycin, amikacin, polymyxin B, imipenem, meropenem, aztreonam, ceftazidime, cefepime, and piperacillin/tazobactam) tested. The 132 isolates were categorized into 42 sequence types according to multilocus sequence typing, and ST235 accounted for 8.3% (11) of the total isolates. Thus, this study provides comprehensive data on the prevalence and characteristics of P. aeruginosa in drinking water in China and can aid in developing preventive measures against contamination during the drinking water treatment process.

Serotyping, Genotyping and Virulence Genes Characterization of Pasteurella multocida and Mannheimia haemolytica Isolates Recovered from Pneumonic Cattle Calves in North Upper Egypt.

Pasteurella (P.) multocida and Mannheimia (M.) haemolytica are the most two common pathogenic bacterial agents causing pneumonia in calves. Both bacteria are associated with significant economic losses in the cattle industry due to high morbidity and mortality rates, especially in the case of severe infections. The objectives of the present study were to perform serotyping and genotyping, as well as characterization of the virulence-associated genes in 48 bacterial isolates; 33 P. multocida and 15 M. haemolytica. All strains were isolated from pneumonic cattle calves showing respiratory manifestations such as fever, nasal discharges, and rapid breathing in North Upper Egypt governorates (Beni-Suef and El-Fayoum). PCR was applied as a confirmatory test using a specific universal gene, kmt1, and rpt2 for P. multocida and M. haemolytica, respectively. The results show that 29 (87.9%) P. multocida and 15 (100%) M. haemolytica isolates were positive for the corresponding universal gene. The results of serotyping indicate that 86.2% of P. multocida isolates belonged to serotype B:2, while 13.8% were untyped. Meanwhile, 60% and 40% of M. haemolytica isolates belonged to serotype 2 and serotype 1, respectively. Investigation of virulence-associated genes showed that all the tested P. multocida isolates harbored nanB, omp87, and toxA genes. Four M. haemolytica isolates harbored both gcp and lktC genes and of these, three isolates harbored the ssa gene. Sequencing of toxA gene of P. multocida and lktC gene of M. haemolytica in the current strains indicated a great homology with strains uploaded in gene banks from different hosts and localities worldwide.

Emerging MDR-Pseudomonas aeruginosa in fish commonly harbor oprL and toxA virulence genes and blaTEM, blaCTX-M, and tetA antibiotic-resistance genes

This study aimed to investigate the prevalence, antibiogram of Pseudomonasaeruginosa (P.aeruginosa), and the distribution of virulence genes (oprL,exoS, phzM, and toxA) and the antibiotic-resistance genes (blaTEM, tetA, and blaCTX-M). A total of 285 fish (165 Oreochromisniloticus and 120 Clariasgariepinus) were collected randomly from private fish farms in Ismailia Governorate, Egypt. The collected specimens were examined bacteriologically. P. aeruginosa was isolated from 90 examined fish (31.57%), and the liver was the most prominent infected organ. The antibiogram of the isolated strains was determined using a disc diffusion method, where the tested strains exhibited multi-drug resistance (MDR) to amoxicillin, cefotaxime, tetracycline, and gentamicin. The PCR results revealed that all the examined strains harbored (oprL and toxA) virulence genes, while only 22.2% were positive for the phzM gene. On the contrary, none of the tested strains were positive for the exoS gene. Concerning the distribution of the antibiotic resistance genes, the examined strains harbored blaTEM, blaCTX-M, and tetA genes with a total prevalence of 83.3%, 77.7%, and 75.6%, respectively. Experimentally infected fish with P.aeruginosa displayed high mortalities in direct proportion to the encoded virulence genes and showed similar signs of septicemia found in the naturally infected one. In conclusion, P.aeruginosa is a major pathogen of O.niloticus and C.gariepinus.oprL and toxA genes are the most predominant virulence genes associated with P.aeruginosa infection. The blaCTX-M,blaTEM, and tetA genes are the main antibiotic-resistance genes that induce resistance patterns to cefotaxime, amoxicillin, and tetracycline, highlighting MDR P.aeruginosa strains of potential public health concern.

Diagnosis, genetic variations, virulence, and toxicity of AHPND-positive Vibrio parahaemolyticus in Penaeus monodon

Acute hepatopancreatic necrosis disease (AHPND) is an emerging shrimp (Penaeus monodon) disease caused by Vibrio parahaemolyticus (VP) since 2013 in Bangladesh. The aim of this work was to evaluate a PCR and RT-PCR techniques as rapid methods for detecting V. parahaemolyticus AHPND-positive P. monodon using genetic markers. Healthy and diseased shrimp (P. monodon) samples were collected from three monitoring stations. The samples were enriched in TCBS plates and DNA extraction from the cultured bacteria. DNA quantifications, PCR amplification, RT-PCR, and gene sequencing were done for the detection of V. parahaemolyticus AHPND-positive P. monodon. The sequence of PCR amplicons showed 100% identity and significant alignment with V. parahaemolyticus. The primers used provided high specificity for V. parahaemolyticus in PCR detection compared with another Vibrio species. In the PCR, amplification resulted positive amplicons, whereas, non-AHPND isolates showed no amplicons. Neighbor-joining methods indicated that all genes evolved from a common ancestor and clades have different traits with very low genetic distance and low variability. The pairwise alignment scores of atpA, tox, blaCARB, 16S rRNA, and pirA genes were 100.0, 98.90, 98.89, 95.53, and 41.42, respectively. The RT-qPCR exposed variable expression levels for all genes in the AHPND-positive strain. Homology analysis and distance matrix exhibited all genes to have the lowest similarity and most divergence, offering the highest specificity. In this study, the expression and variability of target genes confirmed the presence of V. parahaemolyticus in all sampling sites. The results suggested that PCR amplification, RT-qPCR, and gene sequencing can be used for the rapid detection of V. parahaemolyticus in AHPND-positive P. monodon that may lead to subsequent prevention and treatment research in the future for managing this disease.

PacBio genome sequencing reveals new insights into the genomic organisation of the multi-copy ToxB gene of the wheat fungal pathogen Pyrenophora tritici-repentis

BackgroundNecrotrophic effector proteins secreted by fungal pathogens are important virulence factors that mediate the development of disease in wheat. Pyrenophora tritici-repentis (Ptr), the causal agent of wheat tan spot, has a race structure dependent on the combination of effectors. In Ptr, ToxA and ToxB are known proteinaceous effectors responsible for necrosis and chlorosis respectively. While Ptr ToxA is encoded by the single gene ToxA, ToxB has multiple loci in the Ptr genome, which is postulated to be directly related to the level of ToxB production and leaf chlorosis. Although previous analysis has indicated that the majority of the ToxB loci lie on a single chromosome, the exact number and chromosomal locations for all the ToxB loci have not been fully identified.ResultsIn this study, we have sequenced the genome of a race 5 ToxB-producing isolate (DW5), using PacBio long read technology, and found that ToxB duplications are nested in the complex subtelomeric chromosomal regions. A total of ten identical ToxB gene copies were identified and based on flanking sequence identity, nine loci appeared associated with chromosome 11 and a single copy with chromosome 5. Chromosome 11 multiple ToxB gene loci were separated by large sequence regions between 31 and 66 kb within larger segmental duplications in an alternating pattern related to loci strand, and flanked by transposable elements.ConclusionThis work provides for the first time the full accompaniment of ToxB loci and surrounding regions, and identifies the organization and distribution of ten ToxB loci to subtelomeric regions. To our knowledge, this is the first report of an interwoven strand-related duplication pattern event. This study further highlights the importance of resolving the highly complex distal chromosomal regions, that remain difficult to assemble, and can harbour important effectors and virulence factors.

Expression pattern, regulation, and clinical significance of TOX in breast cancer.

Thymocyte selection-associated high mobility group box protein (TOX) is a transcription factor implicated in the regulation of T cell exhaustion during chronic infection and cancer. While TOX is being targeted for cancer immunotherapy, limited information is available about its significance in breast cancer and other solid tumors. We performed a comprehensive analysis of TOX gene expression, its epigenetic regulation, protein localization, relation to tumor infiltrating immune cell composition, and prognostic significance in breast cancer using publicly available datasets. Our results suggest an inverse correlation between TOX expression and DNA methylation in tumor cells. However, its expression is elevated in tumor infiltrating immune cells (TIICs), which may compensates for the total TOX levels in the tumor as a whole. Furthermore, higher TOX levels in tumors are associated with T cell exhaustion signatures along with presence of active inflammatory response, including elevated levels of T cell effector cytokines. Survival analysis also confirmed that higher expression of TOX is associated with better prognosis in breast cancer. Therefore, expression of TOX may serve as a novel prognostic marker for this malignancy.

TOX correlates with prognosis, immune infiltration, and T cells exhaustion in lung adenocarcinoma.

BackgroundThymocyte selection‐associated high mobility group box (TOX) plays a crucial role on the development of innate immunity and tumor microenvironment. This study aims to explore the prognostic potential of TOX and comprehensively analyze the correlations between TOX, immune infiltration, and T cells function in diverse cancers particularly lung adenocarcinoma (LUAD).MethodsTIMER was used to analyze TOX expression in different cancers. Potential prognostic value of TOX was evaluated by the PrognoScan, Kaplan‐Meier Plotter, and GEPIA2. The relationships between TOX, immune infiltration, and related gene marker sets were analyzed by TIMER and GEPIA2. Single‐cell RNA‐seq for T cells in LUAD was analyzed to further investigate the correlations between TOX expression and different T cells populations.ResultsTOX downregulates in most of the cancer types and correlates with poor prognosis in LUAD. TOX shows significant impacts on survival of LUAD with early stage, ever‐smoking, or low‐TMB status. Increased TOX expression positively correlates with high immune infiltration levels in most of the immune cells and functional T cells including exhausted T cells. Moreover, multiple key genes of exhausted T cells comprising PD‐1, TIM‐3, TIGHT, and CXCL13 have remarkable interaction with TOX. Specifically, TOX is observed with high enrichment in exhausted CD4+ and CD8+ T cells populations in single‐cell RNA‐seq analysis for LUAD.ConclusionTOX is a prognosis‐related biomarker for multiple cancer types especially LUAD. Increased TOX expression significantly increase immune infiltration levels in most of the immune cells comprising CD8+ T cells, CD4+ T cells, mast cells, and functional T cells. Moreover, we verified that TOX highly correlates with exhausted T cells and is probable a critical regulator promoted T cells exhaustion in LUAD. Detection of TOX expression could help to predict prognosis and regulating TOX expression in exhausted T cells may offer a novel strategy in maximizing immunotherapy efficacy for LUAD.

SnToxA, SnTox1, and SnTox3 originated in Parastagonospora nodorum in the Fertile Crescent

AbstractThe centre of origin of the globally distributed wheat pathogen Parastagonospora nodorum has remained uncertain because only a small number of isolates from the Fertile Crescent were included in earlier population genetic and phylogeographic studies. We isolated and genetically analysed 193 P. nodorum strains from three naturally infected wheat fields distributed across Iran using 11 neutral microsatellite loci. Compared to previous studies that included populations from North America, Europe, Africa, Australia, and China, the populations from Iran had the highest genetic diversity globally and also exhibited greater population structure over smaller spatial scales, patterns typically associated with the centre of origin of a species. Genes encoding the necrotrophic effectors SnToxA, SnTox1, and SnTox3 were found at a high frequency in the Iranian population. By sequencing 96 randomly chosen Iranian strains, we detected new alleles for all three effector genes. Analysis of allele diversity showed that all three effector genes had higher diversity in Iran than in any population included in previous studies, with Iran acting as a hub for the effector diversity that was found in other global populations. Taken together, these findings support the hypothesis that P. nodorum originated either within or nearby the Fertile Crescent with a genome that already encoded all three necrotrophic effectors during its emergence as a pathogen on wheat. Our findings also suggest that P. nodorum was the original source of the ToxA genes discovered in the wheat pathogens Phaeosphaeria avenaria f. sp. tritici 1, Pyrenophora tritici‐repentis, and Bipolaris sorokiniana.

Expression of the ToxA and PtrPF2 genes of the phytopathogenic fungus Pyrenophora tritici-repentis at the beginning of the infection process

Background. Pyrenophora tritici-repentis causing a tan spot of wheat produces host-specific toxins.
 Materials and methods. Two P. tritici-repentis isolates with different ability to cause necrosis on the leaves of wheat cultivar Glenlea (nec+ and nec) and with different expression level of ToxA and PtrPf2 (factor transcription gene) in vitro were used for analysis. ToxA gene expression in P. tritici-repentis isolates in planta was characterized using quantitative PCR.
 Results. The expression of the ToxA gene in P. tritici-repentis ToxA+ isolates significantly increased when infected the wheat leaves compared to ToxA expression results obtained in vitro. The levels of ToxA expression in both isolates differed significantly after 24, 48 and 96 h after inoculation, however, the dynamics of the trait change over time were similar. However, the highest ToxA expression in the virulent (nec+) isolate in contrast with the avirulent (nec) isolate was observed at a point of 48 h. Whereas the expression of regulating transcription factor PtrPf2 in planta differed imperceptibly from expression in vitro throughout the observation period.
 Conclusion. Obviously, the role of the fungal transcription factor in regulating the effector gene expression weakens in planta, and other mechanisms regulating the expression of pathogen genes at the biotrophic stage of the disease develop.

Frequency of ToxA and ExoS genes in Pseudomonas aeruginosa isolates obtained from different clinical specimens

The present study carried out in Azady teaching general hospital in Kirkuk city during the period from August 2017 to May 2018, to determine the frequency of ToxA gene and ExoS gene in P. aeruginosa isolates. Pseudomonas aeruginosa (100) isolates had been obtained from (1260) different clinical specimens, High prevalence of P. aeruginosa found in isolates obtained from burn swabs. Regarding to genders and ages, P. aeruginosa presented in females (58%) more than males (42%), regarding to age groups, P. aeruginosa presented more frequently (26%) in age group (20-30) followed by children (1-10) years that formed (22%). Molecular detection of virulence genes had been done by using conventional PCR technique, regarding to prevalence of ToxA and ExoS genes, ToxA gene presented (96%), ExoS gene (92%). Regarding to the distribution of genes in clinical specimens, ToxA gene present (100%) in all clinical specimens except in urine samples (90.9%) and low percentage found in sputum (50%). ExoS gene present (100%) in all isolates, while ExoS gene less frequently presented in ear swab isolates ( 83.3%) than other specimens, followed by urine specimens (81.8%), in bronchial washes isolates ExoS gene was absent. This study aims to determine the frequency of ToxA gene and ExoS gene in P. aeruginosa isolates that obtained from different clinical specimens.

Phylogenomic characterisation of a novel corynebacterial species pathogenic to animals

The genus Corynebacterium includes species of biotechnological, medical and veterinary importance. An atypical C. ulcerans strain, W25, was recently isolated from a case of necrotizing lymphadenitis in a wild boar. In this study, we have analysed the genome sequence of this strain and compared the phenotypic and virulence properties with other corynebacterial pathogens. Phylogenomic analyses revealed that strain W25 belongs to a novel species along with PO100/5 and KL1196. The latter strains were isolated from a pig and a roe deer, respectively; hence, this species appears to be associated to animals. The isolate W25 is likely a non-toxigenic tox gene bearing strain and may have compromised abilities to adhere to pharyngeal and laryngeal epithelial cells due to potential loss of the gene functions in spaBC and spaDEF pilus gene clusters. A number of corynebacterial virulence genes are present including pld encoding phospholipase D. Therefore, this strain may be able to cause severe invasive infections in animals and zoonotic infections in humans.

Investigation of biotype, genotype and virulence associated genes inPasteurella multocida capsular type A strains from the respiratory tract of cattle and their relationship with disease cases

Pasteurella (P.) multocida, which causes respiratory disease in cattle, can also be found commensally in the upper respiratory tract. Therefore, it is epidemiologically important to determine the possible difference between pathogenic and commensal strains for the diagnosis of the disease. In this study, phenotypic and genotypic characteristics and virulence associated genes of P. multocida capsular type A strains isolated from the respiratory tract of sick and healthy cattle were investigated. Also, the relationship between the disease status of animals and the characteristics of the isolates were evaluated. Total of 51 P. multocida capsular type A strains were biotyped and genotyped by using commercial bacteria identification kits and PCR-fingerprint, respectively. Four major biotypes generated from xylose and sucrose test results were detected in 39 (76.47%) isolates. Eleven different biotypes were detected in the remaining 12 isolates (23.53%), which were separated from the 4 major biotypes by mannitol, indole, sorbitol, nitrate, VP, lactose, arabinose, salicin, and rhamnose test. Four genotypes were detected in P. multocida isolates and most of them (90.2%) had genotype I profile. Virulence associated genes, such as hgbA, exbB, exbD, tbpA, ptfA, hsf-2, tadD, nanB, nanH, pmHAS, sodA, sodC, and oma87 were detected in all isolates whereas hgbB, toxA, and hsf-1 genes were not found by PCR. PfhA gene were found in 49 isolates (96.07%) but, ompH gene could be detected only in 10 (18.86%) isolates. The findings of the study indicated that P. multocida capsular type A strains generated as xylose positive/sucrose negative (biotype II) and genotype II may be associated with respiratory diseases of cattle. However, ompH gene was observed to be associated with sucrose negative isolates, but not with disease cases. As a result, it was concluded that xylose positive/sucrose negative biotype and genotype II determined in P. multocida capsular type A strains isolated from respiratory diseases of cattle, might be important epidemiological criteria in the differentiation of commensal and pathogenic isolates.