The rate and importance of Clostridium difficile in colorectal cancer patients.

AIM: The aim of this study was to analyze the Clostridium difficile and their toxins in cancerous tissues in comparison to their adjacent healthy tissues in patients with colorectal cancer (CRC) in Iran. BACKGROUND: Intestinal infection or colonization by microbial pathogens and their released metabolites may have a role in the exacerbation of CRC. METHODS: A total of 60 biopsy samples from 30 cancerous and 30 adjacent healthy tissues were collected from patients with CRC. Biopsies were homogenized and cultured in cycloserine cefoxitin fructose agar-agar medium to investigate the presence of C. difficile. DNA was extracted, PCR was performed on pure colonies for bacteria detection, and toxin genes were evaluated in each bacterium positive cases. Real-time PCR was performed on extracted DNA for quantitative comparison of Clostridium difficile in healthy and tumor tissues in CRC patients. RESULTS: Clostridium difficile was isolated from 18 of the cancerous tissue (60%) and 6 of their healthy adjacent tissue (20%) in the culture medium, but toxin genes were positive just in one sample in both groups. Real-time PCR showed the colonization in all samples. CONCLUSION: This study showed a higher prevalence of Clostridium difficile in cancerous lesions in comparison to healthy tissues. We suggest that the investigation of the rate of CD of colorectal cancer patients before surgery is critical for patients. Further studies with more samples size to study the importance of this bacterium and its toxins in the investigation of colorectal cancer patients survey is recommended.

The effect of intestinal microbiota metabolites on HT29 cell line using MTT method in patients with colorectal cancer.

AIM: The aim of this study was to evaluate the effect of intestinal microbiota metabolites in colorectal cancer patients on HT29 cell line using MTT assay. BACKGROUND: Colorectal cancer is one of the most common malignant tumors. Human guts harbor abundant microbes that adjust many aspects of the host physiology. Increasing studies suggest that gut microbiota play a significant role in the incidence and expansion of CRC, as a result of virulence factors, bacterial metabolites, or inflammatory pathways. METHODS: In this cross-sectional study, 60 biopsy samples including 30 cancerous and 30 adjacent healthy tissues were collected from patients with CRC during 2017. Biopsy samples were first cultured on Thioglycollate broth medium for 24hr after which the microbiota metabolites were filtered and stored at -20 C° for further evaluation. HT29 cells were treated by microbiota metabolites at different times (3, 6, 12, 18h) and its viability was assessed by MTT assay. RESULTS: The cells treated with microbiota metabolites showed increased viability and proliferation in time-dependent analysis by MTT assay, but there was not significant differences between the two groups. CONCLUSION: It seems that microbial metabolites are able to induce proliferation and increase cell viability and thus induce colorectal cancer.

Role of gut microbiota in the pathogenesis of colorectal cancer; a review article.

Colorectal cancer (CRC) is one of the most frequently diagnosed cancers worldwide. Lifestyle is identified as one of the most important risk factors for CRC, especially in sporadic colorectal cancer. The natural composition of the gut microbiota changes rapidly during the first decade of life. Maintaining homeostasis in the gut is essential as structural and metabolic functions of the commensal microbiota inhibit gut colonization of pathogens. Dysbiosis, imbalance in function or structure of gut microbiota, has been associated with a variety of diseases, such as colorectal cancer. The aim of this review was to investigate the possible links between the dysbiosis in gut microbiota and colorectal cancer, and the potential role of anaerobic gut microbiota in the pathogenesis of colorectal cancer. Based on this review, various studies have shown that some of the gut microbiota such as anaerobic bacteria significantly increased in CRC patients, but we suggest more investigations are required to assess the importance of these bacteria and their metabolites in the pathogenesis of CRC are required.

The study of H. pylori putative candidate factors for single- and multi-component vaccine development.

Helicobacter pylori has grown to colonize inside the stomach of nearly half of the world's population, turning into the most prevalent infections in the universe. Medical care failures noticeably confirm the need for a vaccine to hinder or deal with H. pylori. This review is planned to discuss the most known factors as a vaccine candidate, including single (AhpC, BG, CagA, KatA, Fla, Hsp, HWC, Lpp, LPS, NAP, OMP, OMV, SOD, Tpx, Urease, VacA) and multi-component vaccines. Many promising results in the field of single and multivalent vaccine can be seen, but there is no satisfactory outcome and neither a prophylactic nor a therapeutic vaccine to treat or eradicate the infection in human has been acquired. Hence, selecting suitable antigen is an important factor as an appropriate adjuvant. Taken all together, the development of efficient anti-H. pylori vaccines relies on the fully understanding of the interactions between H. pylori and its host immune system. Therefore, more work should be done on epitope mapping, analysis of molecular structure, and determination of the antigen determinant region as well due to design a vaccine, preferably a multi-component vaccine to elicit specific CD4 T-cell responses that are required for H. pylori vaccine efficacy.

Genetic Lineages of Mycobacterium tuberculosis Isolates in Isfahan, Iran.

In this study, we aimed to identify the genetic lineages of Mycobacterium tuberculosis isolates in Isfahan via the mycobacterial interspersed repetitive-unit-variable number tandem repeat typing method based on 15 loci. Forty-nine M. tuberculosis isolates were collected between 2013 and 2015 from Tuberculosis patients in Mollahadi Sabzevari Tuberculosis Center in Isfahan. All isolates were typed by 15-locus MIRU-VNTR typing. The highest percentage of isolates, 44.89 % (22/49), belonged to the Euro-American lineage, while the frequencies of the East-African-Indian, East-Asian, and Indo-Oceanic lineages were 28.57 % (14/49), 24.4 % (12/49), and 2.04 % (1/49), respectively. Among the 22 isolates of the Euro-American lineage, those belonging to the NEW-1 sub-lineage were most prevalent (24.4 %). Approximately, the same proportion of isolates belonging to the Delhi/CAS, Beijing, and NEW-1 sub-lineages were identified in Iranian and Afghan immigrant patients. The Delhi/CAS and Beijing sub-lineage isolates were prevalent among patients who had been previously treated for TB. Results showed that all of the 49 MIRU-VNTR patterns were unique and the clustering rate of the 15-locus MIRU-VNTR was 0.0 (minimum recent transmission). The results of this study show that the lineages of M. tuberculosis isolates in Isfahan are similar to those reported in the Eastern Mediterranean region (indicative of the epidemiological relationship between the countries in the region). The low clustering rate in our results reveals that transmission of tuberculosis in Isfahan is, in most cases, a reactivation of previous tuberculosis infection and the role of recently transmitted disease is minor.

Phylogenetic Analysis of Prevalent Tuberculosis and Non-Tuberculosis Mycobacteria in Isfahan, Iran, Based on a 360 bp Sequence of the rpoB Gene.

BACKGROUND: Taxonomic and phylogenetic studies of Mycobacterium species have been based around the 16sRNA gene for many years. However, due to the high strain similarity between species in the Mycobacterium genus (94.3% - 100%), defining a valid phylogenetic tree is difficult; consequently, its use in estimating the boundaries between species is limited. The sequence of the rpoB gene makes it an appropriate gene for phylogenetic analysis, especially in bacteria with limited variation. OBJECTIVES: In the present study, a 360bp sequence of rpoB was used for precise classification of Mycobacterium strains isolated in Isfahan, Iran. MATERIALS AND METHODS: From February to October 2013, 57 clinical and environmental isolates were collected, subcultured, and identified by phenotypic methods. After DNA extraction, a 360bp fragment was PCR-amplified and sequenced. The phylogenetic tree was constructed based on consensus sequence data, using MEGA5 software. RESULTS: Slow and fast-growing groups of the Mycobacterium strains were clearly differentiated based on the constructed tree of 56 common Mycobacterium isolates. Each species with a unique title in the tree was identified; in total, 13 nods with a bootstrap value of over 50% were supported. Among the slow-growing group was Mycobacterium kansasii, with M. tuberculosis in a cluster with a bootstrap value of 98% and M. gordonae in another cluster with a bootstrap value of 90%. In the fast-growing group, one cluster with a bootstrap value of 89% was defined, including all fast-growing members present in this study. CONCLUSIONS: The results suggest that only the application of the rpoB gene sequence is sufficient for taxonomic categorization and definition of a new Mycobacterium species, due to its high resolution power and proper variation in its sequence (85% - 100%); the resulting tree has high validity.

Antibiotic Resistance Patterns and Genetic Diversity in Clinical Isolates of Pseudomonas aeruginosa Isolated From Patients of a Referral Hospital, Isfahan, Iran.

BACKGROUND: Pseudomonas aeruginosa is a well-known opportunistic pathogen, which affects hospitalized patients in different wards due to its natural resistance to drugs. OBJECTIVES: The purpose of the current study was to determine the antibiotic susceptibility profiles and genetic relatedness in P. aeruginosa isolated from patients admitted to a referral hospital in Isfahan, Iran. MATERIALS AND METHODS: Out of 150 analyzed samples, 54 P. aeruginosa isolates were recovered and were subjected to antibiotic resistance patterns and genetic diversity determination by Kirby-Bauer's disk diffusion method and RAPD-PCR, respectively. RESULTS: The highest percentage of resistance was observed against ceftazidime and imipenem with 30 (55.6%) isolates; meanwhile all isolates were sensitive to polymyxin B. Twenty-eight (51.8%) isolates revealed resistance to all applied antibiotics. RAPD-PCR (Random Amplified Polymorphic DNA- Polymerase Chain Reaction) results showed 54 unique genotypes, which were divided into 39 clusters. CONCLUSIONS: Although different source of P. aeruginosa may involve in patient colonization, genetically related strains were isolated from different wards and or the same ward of the hospital. Our results pointed to the restriction of currently used antibiotics in studied hospital. We hope that our results cast light on the control and transmission of the infection in the investigated hospital.

Clonal Relatedness among Imipenem-Resistant Pseudomonas aeruginosa Isolated from ICU-Hospitalized Patients.

Imipenem-resistant Pseudomonas aeruginosa (P. aeruginosa) has become an increasingly important problem in healthcare settings worldwide. The aim of the present study was to evaluate clonal spread among imipenem-resistant P. aeruginosa isolated from ICU-hospitalized patients. Totally, 150 wound specimens were analyzed. Antibiotic resistance profiles and clonal diversity were evaluated using Kirby-Bauer's disk diffusion method and Random Amplified Polymorphic DNA- (RAPD-) PCR, respectively. The isolates showed a high frequency of antibiotic resistance against meropenem, and imipenem (100%) followed by ciprofloxacin, and ceftazidime (90%); meanwhile resistance to polymyxin B was not observed. Eighteen (40%) of P. aeruginosa isolates were MBL-positive via ethylenediaminetetraacetic acid (EDTA) combined disk test. Our findings showed high genetic diversity, with 37 different RAPD types detected. RAPD typing results showed cross-acquisition of P. aeruginosa in investigated hospital, suggesting failure in infection control practices. Incidence of MBL-positive isolates is high and should be regarded as a threat to hospitalized patients.

Morphological and Bactericidal Effects of Amikacin, Meropenem and Imipenem on Pseudomonas aeruginosa.

BACKGROUND: Pseudomonas aeruginosa might be converted to coccoid bacteria under antibiotic stress. Bacterial conversion would increase resistance to antibiotics due to changes in cell wall crosslink or decreased metabolic activity. Morphology of P. aeruginosa under stress conditions (presence of antibiotics) can be changed to elongated bacilli, U shape and finally coccoid bacteria. Results of several researches showed that coccoid bacteria are one of the most important aspects of drug resistance. It would be the major reason for treatment failure. OBJECTIVES: The aim of this study was to determine in vitro morphological and bactericidal effects of amikacin, meropenem and imipenem on P. aeruginosa isolated from clinical specimens. MATERIALS AND METHODS: Eight P. aeruginosa isolates obtained from clinical samples of burned patients and standard strain ATCC 27853 were used in this study. Isolates were identified by biochemical tests and confirmed by PCR method using ITS specific primer. Minimum inhibitory concentrations (MICs) of three antibiotics were determined by E-test method. Bacteria were exposed to antibiotics at different concentrations. Bacterial morphology in different days was examined by specific microscope and viability of isolates was examined by flow cytometry. RESULTS: All used antibiotics at sub MIC concentration had capability to induce coccoid bacteria. The highest rate of induced coccoid bacteria was 98.2% after 8 days, with contribution of imipenem and meropenem at 2 µg/mL concentration. Amikacin at 4 µg/mL concentration induced lower rate of coccoid bacteria (55.05%). Amikacin had a strong bactericidal effect on coccoid bacteria at 8 µg/mL concentration. Imipenem and meropenem showed very weak bactericidal effect on coccoid bacteria. CONCLUSIONS: Induction of coccoid form of P. aeruginosa may be one of the important reasons for antibiotic treatment failure; therefore, prescribed dose of antibiotics should be carefully managed to prevent increasing antibiotic resistance and coccoid bacteria induction.

Production of IFN-γ and IL-4 Against Intact Catalase and Constructed Catalase Epitopes of Helicobacter pylori From T-Cells.

BACKGROUND: Helicobacter pylori infection is highly prevalent in the developing countries. It causes gastritis, peptic ulcer disease, and gastrocarcinoma. Treatment with drugs and antibiotics is problematic due to the following reasons: cost, resistance to antibiotics, prolonged treatment and using multiple drugs. Catalase is highly conserved among the Helicobacter species and is important to the survival of the organism. It is expressed in high amounts and is exposed to the surface of this bacterium; therefore it represents a suitable candidate vaccine antigen. OBJECTIVES: A suitable approach in H. pylori vaccinology is the administration of epitope based vaccines. Therefore the responses of T-cells (IFN-γ and IL-4 production) against the catalase of H. pylori were determined. Then the quality of the immune responses against intact catalase and three epitopes of catalase were compared. MATERIALS AND METHODS: In this study, a composition of three epitopes of the H. pylori catalase was selected based on Propred software. The effect of catalase epitopes on T-cells were assayed and immune responses identified. RESULTS: The results of IFN-γ, IL-4 production against antigens, epitopes, and recombinant catalase by T-cells were compared for better understanding of epitope efficiency. CONCLUSIONS: The current research demonstrated that epitope sequence stimulates cellular immune responses effectively. In addition, increased safety and potency as well as a reduction in time and cost were advantages of this method. Authors are going to use this sequence as a suitable vaccine candidate for further research on animal models and humans in future.

Morphological and Bactericidal Effects of Different Antibiotics on Helicobacter pylori.

BACKGROUND: Helicobacter pylori (H. pylori) is a spiral Gram negative bacteria that can transform to the coccoid form in adverse conditions. OBJECTIVES: The aim of this study was to determine the in vitro morphological and bactericidal effects of metronidazole, amoxicillin and clarithromycin on H. pylori. MATERIALS AND METHODS: The standard strain 26695 of H. pylori was cultured on Brucella agar (BA) and the minimum inhibitory concentrations (MICs) of three antibiotics were determined by E-test method. The bacteria were exposed to antibiotics at 1/2 MIC, MIC and 2X MIC concentrations in Brucella broth (BB). Induced coccoid forms were confirmed by Gram staining and light microscopy. The viability of cells as well as the susceptibility of viable coccoids to antibiotics were examined using the flow cytometry method. RESULTS: All of the three antibiotics at sub-MIC induced coccoid forms. The highest rates of coccoids (> 90%) were induced at 0.008 µg/mL concentration (1/2 MIC) of amoxicillin, 72 hours postexposure. Metronidazole and clarithromycin with 1/2 MIC (0.5 and 0.125 µg/mL respectively) induced lower rates of coccoid forms (60% and 40% respectively). Potent bactericidal effects on coccoids were observed with Metronidazole at 2X MIC and clarithromycin at MIC (0.25 µg/mL) (80 - 90%). Amoxicillin with MIC and 2X MIC had no bactericidal effect on coccoid forms. CONCLUSIONS: Despite the good in vitro bactericidal effect of amoxicillin on spiral forms of H. pylori, this antibiotic has little effect on induced coccoids that may develop after the inappropriate in vivo antibacterial treatment. Hence, for successful therapy, it is essential not only to eradicate the spiral forms, but to eliminate the viable coccoids.

Detection of Helicobacter pylori in city water, dental units' water, and bottled mineral water in Isfahan, Iran.

Helicobacter pylori infection in human is one of the most common infections worldwide. However, the origin and transmission of this bacterium has not been clearly explained. One of the suggested theories is transmission via water. This study was conducted to determine the prevalence rate of H. pylori in tap water, dental units' water, and bottled mineral water in Iran. In the present study, totally 200 water samples were collected in Isfahan province and tested for H. pylori by cultural method and polymerase chain reaction (PCR) by the detection of the ureC (glmM) gene. Using cultural method totally 5 cultures were positive. Two out of 50 tap water samples (4%), 2 out of 35 dental units' water (5.8%) samples, and 1 out of 40 (2.5%) from water cooler in public places were found to be contaminated with H. pylori. H. pylori ureC gene was detected in 14 (7%) of water samples including 5 tap water (10%), 4 dental units' water (11.4%), 1 refrigerated water with filtration, and 4 (10%) water cooler in public places samples. This may be due to the coccoid form of bacteria which is detected by PCR method.

Rapid and accurate identification of Mycobacterium tuberculosis complex and common non-tuberculous mycobacteria by multiplex real-time PCR targeting different housekeeping genes.

Rapid and accurate identification of mycobacteria isolates from primary culture is important due to timely and appropriate antibiotic therapy. Conventional methods for identification of Mycobacterium species based on biochemical tests needs several weeks and may remain inconclusive. In this study, a novel multiplex real-time PCR was developed for rapid identification of Mycobacterium genus, Mycobacterium tuberculosis complex (MTC) and the most common non-tuberculosis mycobacteria species including M. abscessus, M. fortuitum, M. avium complex, M. kansasii, and the M. gordonae in three reaction tubes but under same PCR condition. Genetic targets for primer designing included the 16S rDNA gene, the dnaJ gene, the gyrB gene and internal transcribed spacer (ITS). Multiplex real-time PCR was setup with reference Mycobacterium strains and was subsequently tested with 66 clinical isolates. Results of multiplex real-time PCR were analyzed with melting curves and melting temperature (T (m)) of Mycobacterium genus, MTC, and each of non-tuberculosis Mycobacterium species were determined. Multiplex real-time PCR results were compared with amplification and sequencing of 16S-23S rDNA ITS for identification of Mycobacterium species. Sensitivity and specificity of designed primers were each 100 % for MTC, M. abscessus, M. fortuitum, M. avium complex, M. kansasii, and M. gordonae. Sensitivity and specificity of designed primer for genus Mycobacterium was 96 and 100 %, respectively. According to the obtained results, we conclude that this multiplex real-time PCR with melting curve analysis and these novel primers can be used for rapid and accurate identification of genus Mycobacterium, MTC, and the most common non-tuberculosis Mycobacterium species.

Two Dimensional Structural Analysis and Expression of a New Staphylococcus aureus Adhesin Based Fusion Protein.

OBJECTIVES: Staphylococcus aureus is a foremost source of numerous nosocomial and community acquired infections. Antibiotic therapy for vancomycin resistant S. aureus (VRSA) can not promise the eradication of infections. Since adhesion is the major route of infections, adhesin based vaccine could suppress S. aureus infections. Fibronectin binding protein A (FnBPA) and clumping factor A (ClfA) are major responsible adhesions involved in S. aureus infections, so they could be candidate vaccine molecules against an extensive range of infections. This project intended to express a new fusion protein construct and analysis of biological activity regarding binding activity. MATERIALS AND METHODS: pfnbA- ClfA construct was transformed to Escherichia coli BL21 (DE3). Transformant E. coli were grown in LB broth and induced with IPTG and cellular extracts were separated on SDS-PAGE. RT-PCR was performed to verify expression. Binding activity of fusion protein was studied using human gingival fibroblast (HGF) cell line. D1-D3 protein from unpublished study was used as control. RESULTS: The expected fusion protein fragment showed by SDS-PAGE. RT-PCR verified the existence of mRNA relating to expressed fusion protein. Binding activity of S. aureus decreased after treatment of HGF cells with fusion protein. CONCLUSION: In total, binding activity of fusion protein was approximately two fold lesser than D1-D3 protein. It is supposed that the fusion protein could not be attached to its ligand easily and would be more accessible to antigen presenting cells and consequently protective antibodies will be produced. This project is pending for in vivo infection study in animal model.