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

Isolation, Identification and genomic pattern of Mycobacterium tuberculosis from tuberculin positive cattle from Qom province with RFLP metod by PGRS prob

Introduction:Mycobacterium bovis is the principal cause of bovine tuberculosis in bovids. In the present work, isolation, identification and genotyping of Mycobacterium tuberculosis complex (MTbC) isolates collected from slaughtered reactor (tuberculine-positive) cattle in Qum province using RFLP-PGRS are addressed. Materials and methods: From January 2012 to February 2013, major head and trunk lymph nodes from a total of 30 carcasses originating from reactor cattle were collected at Qum abbatoir. These were all cultured on gycerinated and pyruvated Lowenstein-Jensen slopes and incubation at 37°C 8-12 weeks. Slopes bearing acid-fast bacterial growth were subjected PCR-IS6110 in searching for MTbC isolates. The identified MTbC complex isoltes were strain typed by RFLP-PGRS. Results: Totally, PCR-IS6110 experiment detected MTbC in 21 specimens. Genotyping these by RFLP-PGRS resulted characterization of 3 different genotypes. Discussion and conclusion: Isolation of MTbC bacteria from tuberculinated cattle of Qum province raises public health concern due to the likely transmission of infection to human subjects. Identification of three RFLP-PGRS types between the 21 collected isolates from Qum is a reflection of co-existence of different MTbC strains and genetic diversity in the region, the extent of this diversity however, remains to be assessed by further epidemiological works. Taking the current level of genetic diversity between MTbC bacteria in Qum into account, re-consideration of the existing test-and-slaughter scheme seems necessary

Molecular Identification of Extended-Spectrum β-lactamase and Integron Genes in Klebsiella pneumonia

Introduction: Infections caused by Gram negative bacteria, producing extended-spectrum β-lactamase, including Klebsiella pneumoniae are increasing all over the world with high morbidity and mortality. The aim of the present study was determined antimicrobial profile susceptibility and the prevalence of antibiotic resistance genes by multiplex PCR. Methods: In the present study, we obtained one-hundred isolates of K. pneumoniae from different clinical samples. The antibiotic susceptibility testing was done in thirteen antibiotic and, therefore, M-PCRs were conducted using the DNA amplification for detection of ESBLs (blaTEM, blaCTX-M, blaSHV) and int (I, II, III) genes. Results: The results of resistance to amoxicillin/clavulanate, ciprofloxacin, amikacin, trimethoprim-sulfamethoxazole, cefotaxime, ampicillin, aztreonam, imipenem, gentamicin, ceftazidime, Cefepime, ceftriaxone and levofloxacin were obtained 37%, 37%, 93%, 84%, 52%, 87%, 59%, 8%, 24%, 67%, 52%, 43% and 26%, respectively. The frequency of the extended-spectrum β-lactamase K. pneumoniae was obtained 37%. The prevalence of resistance genes of ESBLs in the M-PCR method showed that the blaTEM, blaCTX and blaSHV were 38%, 24% and 19%, respectively, however, only 8 (8%) out of 100 isolates were found to have positive outcomes for the existence of class 1 integrons and there were no detected class 2 or class 3 integrons. Conclusions: Our results recommend the likely co-carriage of some ESBLs genes and antibiotic resistance integrons on the same plasmids harboring the MDR genes. Keywords: fKlebsiella pneumonia, integrons, drug resistance. | PubMe

Frequency of exoY, exoS, exoT and exoU genes among Pseudomonas aeruginosa Isolated from patients in Tehran hospitals by Multiplex PCR

Background and Aim: Pseudomonas aeruginosa is a gram-negative pathogen that causes a variety of serious infections predominantly in immunocompromised patients. To promote severe illness, P. aeruginosa uses a type III secretion system to inject toxic effector proteins into the cytoplasm of eukaryotic cells. Four effector proteins have been described in P. aeruginosa: ExoU, ExoS, ExoT, and ExoY. The aim of the study was to determine the prevalence of the type III secretion system toxins-encoding genes among P. aeruginosa isolates collected from different clinical specimens such as urine, wound, blood and respiratory secretions from patients. Materials and Methods: 55 Pseudomonas aeruginosa isolates were identified from hospitalized patients in Tehran during 2015 – 2016, using conventional microbiological tests. The susceptibility of isolates to antibiotics were assessed using disk diffusion test. After DNA extraction, Multiplex PCR was performed on the P. aeruginosa isolates to detect the secretion toxins-encoding genes. Results: High resistance rates were seen for cefipime (89%), ceftazidime (85.45%), aztreonam (83.63%), tobramycin (78.18%) and gentamicin (60%). The prevalence of the genes among all isolates was as follows; exoT (76.32%), exoS (30.90%), exoY (14.54%) and exoU (67.27%). exoU was more prevalent among MDR than in non-MDR strains (81.3% versus16.6%). exoU+ isolates were more likely to be fluoroquinolone-resistant than exoS+ isolates (32% versus 17%). Conclusions: Type III secretion system toxins-encoding genes found in isolated P.aeruginosa, in which exoT, exoU and exoS gene detected in most isolates while exoY gene was detected in minaroty of the isolates

Evaluation of Antibiotic Resistance and Detection of papC and papG genes in Escherichia coli Strains Isolated from Patients with Urinary Tract Infection

Abstract Background and Objectives: Escherichia coli is the most common etiologic factor of urinary tract infection, which its most important virulence factor is P fimbriae. Uropathogenic E. coli expresses various types of adhesins, such as pili adhesins (pyelonephritis-associated pili, Pap) that mediates binding to the surface of epithelial cells of the urinary tract. This study aims to identify papC and papG genes and to evaluate antibiotic resistance level in the isolated E. coli samples. Methods: In this descriptive cross-sectional study, 50 samples were collected from patients with urinary tract infection and after isolation of bacteria and DNA extraction, antibiotic susceptibility tests was performed by disc diffusion method using related antibiotics. Presence of papG and papC genes (class I, II, and III) was assessed by multiplex PCR method. Statistical data were analyzed using descriptive t-test. Results: The isolated E. coli samples were susceptible to amikacin (100%) and cefepime (72%) and resistant to ampicillin (100%) and nitrofurantoin (94%). Eighteen samples (32.7%) had papG gene, of which 17 (30.9%) samples had papGII gene and 1 sample (1.8%) had papGIII gene; papGI gene was not detected in any of the samples. Conclusion: The results of the present study showed that papC and papGI genes are the most common Pap fimbriae adhesion-encoding genes in E. coli isolated from urinary tract infection. The difference between the results of this study with those of other studies is due to geographic diversity. Keywords: Escherichia coli; Adhesion pap, Disk diffusion antimicrobial tests; Multiplex polymerase chain reaction

In-silico design, expression, and purification of novel chimeric Escherichia coli O157:H7 OmpA fused to LTB protein in Escherichia coli.

E. coli O157:H7, one of the major EHEC serotypes, is capable of developing bloody diarrhea, hemorrhagic colitis (HC), and fatal hemolytic uremic syndrome (HUS) and is accompanied by high annual economic loss worldwide. Due to the increased risk of HC and HUS development following antibiotic therapy, the prevention of infections caused by this pathogen is considered to be one of the most effective ways of avoiding the consequences of this infection. The main aim of the present study was to design, express, and purify a novel chimeric protein to develope human vaccine candidate against E. coli O157:H7 containing loop 2-4 of E. coli O157:H7, outer membrane protein A (OmpA), and B subunit of E. coli heat labile enterotoxin (LTB) which are connected by a flexible peptide linker. Several online databases and bioinformatics software were utilized to choose the peptide linker among 537 analyzed linkers, design the chimeric protein, and optimize the codon of the relative gene encoding this protein. Subsequently, the recombinant gene encoding OmpA-LTB was synthesized and cloned into pET-24a (+) expression vector and transferred to E. coli BL21(DE3) cells. The expression of OmpA-LTB chimeric protein was then carried out by induction of cultured E. coli Bl21 (DE3) cells with 1mM isopropyl-β-D-thiogalactopyranoside (IPTG). The purification of OmpA-LTB was then performed by nickel affinity chromatography. Expression and purification were analyzed by sodium dodecyl sulphate poly acrylamide gel electrophoresis. Moreover, the identity of the expressed protein was analyzed by western blotting. SDS-PAGE and western immunoblotting confirmed the successful expression of a 27 KDa recombinant protein after 24 hours at 37°C post-IPTG induction. OmpA-LTB was then successfully purified, using nickel affinity chromatography under denaturing conditions. The yield of purification was 12 mg per liter of culture media. Ultimately, we constructed the successful design and efficient expression and purification of OmpA-LTB divalent under the above-mentioned conditions

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