Association of tcdA+/tcdB+ Clostridium difficile Genotype with Emergence of Multidrug-Resistant Strains Conferring Metronidazole Resistant Phenotype

Background:

Reduced susceptibility of Clostridium difficile to antibiotics is problematic in clinical settings. There is new evidence indicating the cotransfer of toxin-encoding genes and conjugative transposons encoding resistance to antibiotics among different C. difficile strains. To analyze this association, in the current study, we evaluated the frequency of toxigenic C. difficile among the strains with different multidrug-resistant (MDR) profiles in Iran.

Methods:

Antimicrobial susceptibility patterns and minimal inhibitory concentrations (MIC) of the isolates were determined against metronidazole, imipenem, ceftazidime, amikacin, and ciprofloxacin by agar dilution method. The association of the resistance profiles and toxigenicity of the strains were studied by PCR targeting tcdA and tcdB genes.

Results:

Among 86 characterized strains, the highest and lowest resistance rates were related to ciprofloxacin (97%) and metronidazole (5%), respectively. The frequency of resistance to other antibiotics was as follow: imipenem (48%), ceftazidime (76%), and amikacin (76.5%). Among the resistant strains, double drug resistance and MDR phenotypes were detected in the frequencies of 10.4% and 66.2%, respectively. All of the metronidazole-resistant strains belonged to tcdA ⁺/tcdB ⁺ genotype with triple or quintuple drug resistance phenotypes. MIC₅₀ and MIC₉₀ for this antibiotic was equally ≤ 8 μg/ml.

Conclusion:

These results proposed the association of tcdA ⁺/tcdB ⁺ genotype of C. difficile and the emergence of resistance strains to broad-spectrum antibiotics and metronidazole. Key Words: Multidrug resistance, Clostridium difficile, Metronidazole     

Morphological evidences for onion-induced growth inhibition of Trichophyton rubrum and Trichophyton mentagrophytes

The antifungal activity of onion (Allium cepa L.) on two important dermatophytes, Trichophyton rubrum and Trichophyton mentagrophytes, with special reference to morphological aspects was studied. Growth of both fungi was found to be strongly inhibited by aqueous onion extract (AOE) as a dose-dependent manner. The extract showed fungicidal effect for both fungi at concentrations >3.12% (v/v). The fungus T. mentagrophytes was more affected by the onion as compared to T. rubrum at all concentrations used. Morphological effects of onion exposure were examined in correlation with fungal growth. Corresponding to the growth inhibition, light and electron microscopy observations revealed morphological anomalies in hyphal compartments. The results demonstrated that AOE targets the cell membrane of the fungi as breaking down of both inner and outer membranes with consequent extrution of materials into the surrounding medium. Cytoplasmic membranes and other membranous structures of organelles, such as nuclei and mitochondria, were also disrupted. In correlation to the fungal growth, morphological alterations occurred to a less content for T. rubrum compared with T. mentagrophytes. The hyphae of T. rubrum were found to be mainly affected by converting to resistant forms, i.e., chlamidospores as a consequence of phenotype switching response to AOE. Plasmolysis accompanied by an almost complete depletion and disorganization of cytoplasmic structures were found to be the final event which led to cell death. Ultrastructural evidences obtained from this study strongly support that morphological changes of T. rubrum and T. mentagrophytes caused by AOE are associated with its fungistatic and fungicidal activities. With respect to the morphological results and the preliminary data on fungal biochemistry, a mechanism of action by interacting of AOE with thiol (-SH) groups present in essential compartments of the fungal cells was postulated.     

Efficacy of essential oils combination on performance,ileal bacterial counts,intestinal histology and immunocompetence of laying hens fed alternative lipid sources

This study was carried out to assess the effects of a blend of herbal essential oils (namely Thymus vulgaris, Mentha piperita, Rosmarinus offisinalis and Anethum graveolens) and different dietary lipid sources on the performance, ileal bacterial enumeration, intestinal histological alterations and immune responses in laying hens. For this purpose, a total of 150 laying hens were randomly allocated to six experimental treatments with five replicates of five birds each. Dietary treatments consisted of three levels of a mixture of essential oils (0, 100 and 200 mg/kg) and two sources of lipid (soybean oil and fish oil containing different ratios of n‐6 to n‐3 fatty acids) fed to the animals during an 80‐days feeding trial. Findings indicated that dietary administration of fish oil not only increased significantly the spleen relative weight (p < 0.01) and the antibody titre against Newcastle virus (p < 0.05) but also led to reductions in liver relative weight (p < 0.05) and feed conversion ratio (p < 0.05). Moreover, the mixture of herbal essential oils brought about declines in hepatic relative weight, heterophile to lymphocyte ratio (p < 0.05) and intestinal pathogen populations (p < 0.01). Mention must also be made of the improvements it made in spleen weight (p < 0.01), antibody titres against SRBC (p < 0.01) and Newcastle virus (p < 0.05), villus height to crypt depth ratio (p < 0.01), goblet cell numbers (p < 0.05), lamina propria lymphatic follicle diameters (p < 0.01) and feed conversion ratio (p = 0.06). It may be claimed that the enhancements observed in the performance of laying hens fed fish oil and 200 mg/kg of the blend of essential oils could have potentially been associated with improved intestinal health indices as well as cellular and humoral immune responses.     

Hydrocortisone Reduces Toll-Like Receptor 4 Expression on Peripheral CD14+ Monocytes in Patients Undergoing Percutanoues Coronary Intervention

Bacground: Evidence from several lines of investigations suggests that Toll-like receptor 4 (TLR4) is involved in atherosclerosis as a bridge between innate and acquired immunity. Percutaneous coronary intervention (PCI) can trigger inflammation through activation of human TLR4 (hTLR4) on monocytes. Hydrocortisone as an anti-inflammatory and immuno-suppressant agent has multiple mechanisms of action. In this study, we aimed at assessing the effects of hydrocortisone on monocyte expression and activity of hTLR4 in patients underwent PCI. Methods: Blood samples were taken from a total of 71 patients with chronic stable angina who were scheduled for a PCI, before the intervention. Thirty patients received 100 mg hydrocortisone prior to the procedure. Control group was composed of 41 patients underwent PCI without receiving hydrocortisone. Blood collection was repeated 2 and 4 h after PCI. The expression of hTLR4 on the surface of CD14⁺ monocytes and the serum levels of TNF-α and IL-1β were measured using flowcytometry and Sandwich ELISA. Results: Compared with controls, hydrocortisone significantly reduced monocyte expression of hTLR4 in test group (P<0.01). In addition, it had a significant effect on reduction of serum concentrations of TNF-α and IL-1β in test group in a time-dependent manner (P<0.01). Conclusion: In this study, hydrocortisone was able to reduce the hTLR4/CD14 positive monocytes and its related pro-inflammatory cytokines, thus it can decrease inflammatory responses following PCI. Key Words: Toll-like receptor 4, Cytokines, Hydrocortisone     

Association between p53?Codon 72 (Arg72Pro) Polymorphism and Primary Open-Angle Glaucoma in Iranian Patients

Background: Glaucomatous neuropathy is a type of cell death due to apoptosis. The p53 gene is one of the regulatory genes of apoptosis. Recently, the association between the p53 gene encoding for proline at codon 72 and primary open-angle glaucoma (POAG) has been studied in some ethnic groups. This study is the first association analysis of POAG and p53 codon 72 polymorphism in Iranian patients. Methods: A cohort of 65 unrelated patients with POAG (age range from 12-62 years, mean ± SD of 40.16 ± 17.51 years) and 65 unrelated control subjects (without glaucoma, age range of 14-63 years, mean ± SD of 35.64 ± 13.61 years) were selected. In Iranian POAG patients and normal healthy controls, the p53 codon 72 polymorphism in exon 4 was amplified using polymerase chain reaction. The amplified DNA fragments were digested with the BstUI restriction enzyme, and the digestion patterns were used to identify the alleles for the polymorphic site. Results: Comparisons revealed significant differences in allele and genotype frequencies of Pro72Arg between POAG patients and control group. A higher risk of POAG was associated with allele Pro (OR = 2.1, 95% CI = 1.2–3.4) and genotype Pro/Pro (OR = 3.9, 95% CI = 0.13-12.7). Conclusion: The p53 Pro72 allele was more frequent in Iranian POAG patients than in the control group (P<0.05). The present findings show that the individuals with the Pro/Pro genotype may be more likely to develop POAG. However, additional studies are necessary to confirm this association. Key Words: Primary open-angle glaucoma (POAG), Glaucoma, p53, Codon 72, Iran     

A bioinformatic insight into the genetic diversity within pomegranate cultivars: from nuclear to chloroplast genes

Genetic Resources and Crop Evolution - The pomegranate (Punica granatum L.) is an ancient fruit plant which has been consumed for its nutritional value and medicinal properties by mankind. Several...     

Herd-level prevalence of Mycobacterium avium subspecies paratuberculosis by bulk-tank milk PCR in Fars province (southern Iran) dairy herds

A cross-sectional study was conducted from March to August 2006 in dairy herds in Fars province, southern Iran to determine the herd-level prevalence of Mycobacterium avium subspecies paratuberculosis (MAP) infection. Bulk-tank milk samples were collected from 110 dairy herds in the 3 districts (Shiraz, Marvdasht and Sepidan) of the province. Among study populations, 12 herds (11%, 95%CI: 5-17%) were positive for MAP infection based on IS900 nested PCR. The prevalence of positive milk samples in the three districts of Fars province was different ranging from 8.6% to 23% which was not statistically significant (P=0.19). It is recommended to conduct further epidemiologic studies to determine cow-level prevalence and risk factors for infection, and to evaluate the economic consequences of the MAP infection in the region.     

Isolation of Streptococci from milk samples of normal, acute and subclinical mastitis cows and determination of their antibiotic susceptibility patterns

Streptococci are frequently isolated from bovine mastitis in dairy cows with only limited information available on the antimicrobial susceptibility of these organisms. A total of 42 Streptococci isolated from 148 milk samples of normal, sub acute and acute bovine mastitis cases. Overall, 35% of the strains tested were Streptococcus dysgalactiae, Streptococcus agalactiae 26%, Streptococcus uberis 18 and 4% were Enterococcus sp. Differences between the number of isolations in acute and sub acute groups were statistically significant, (p<0.5). The antimicrobial susceptibility for these organisms was determined for the following antimicrobial agents: cephalexine, penicillin, clindamycin, cloxaciline, gentamicin, streptomycin, amoxicillin, tetracycline, kanamycin, oxytetracycline, ampicillin, chloramphenicol and erythromycin. S. agalactiae, S. dysgalactiae, S. uberis and Enterococci demonstrated high level of resistance against streptomycin, penicillin and cloxaciline. Low level of sensitivity to other tested antimicrobials was demonstrated.     

Molecular genotyping and epidemiology of Mycobacterium bovis strains obtained from cattle in Iran

Restriction fragment length polymorphism (RFLP) genotyping was employed to analyze the population genetics of Mycobacterium bovis in Iran. One hundred and twenty-three isolates collected from slaughtered tuberculosis-suspect cattle and one clinically asymptomatic buffalo were subjected to RFLP analysis with probes of the polymorphic GC-rich sequence (PGRS) and the direct repeat sequence (DR) using DNA digested with PvuII and AluI. All these methods detected a large homogeneous population in which only a few isolates had variant genotypes. Only AluI-based RFLPs of both the PGRS and DR sequences were able to clearly differentiate between BCG and field strains of M. bovis. As in previous reports, these findings seem to reflect a recent dispersal of one or a few strains in Iran following the substantial expansion of Holstein-Friesian cattle over the last few decades.     

Growth and physiological responses of Quercus brantii seedlings inoculated with Biscogniauxia mediterranea and Obolarina persica under drought stress

Charcoal canker of oak is a common disease in the western regions of Iran, which has widely spread due to climate change and increasing drought in recent years. Biscogniauxia mediterranea and Obolarina persica are known as two agents of oak charcoal canker in Zagros forests causing the dieback of oak trees. The effects of charcoal disease agent pathogens on the growth and physiological response of two‐year‐old seedlings of Quercus brantii were evaluated under drought stress in greenhouse over a period of nine months. Survival was 21.7% lower in seedlings inoculated with B. mediterranea subjected to drought stress compared with control treatment. The length of lesions on stems was 68% more under charcoal pathogens in drought stressed compared with the length of lesions caused by charcoal pathogens only. On the other hand, stem lesion length caused by B. mediterranea was 25% greater than those caused by O. persica. Some morphophysiological characteristics were affected by charcoal pathogens alone and/or in combination with drought. Diameter, height and biomass decreased the most in seedlings inoculated with charcoal pathogens under drought stress. Pathogenic agents alone and in combination with drought stress reduced photosynthetic rates, stomatal conductance, transpiration, the maximum Rubisco activity (V_cmax), maximum photochemical efficiency of photosystem II (F_v/F_m) and xylem water potential. In general, drought increased the aggressiveness of fungus and intensification of their destructive effects in Quercus brantii. Moreover, there was no significant difference between the effect of B. mediterranea and O. persica on the morphophysiological parameters studied.