Novel mutation in efflux pump Rv1258c (Tap) gene in drug resistant clinical isolates of Mycobacterium tuberculosis in Iran.

INTRODUCTION: Tuberculosis (TB) remains a serious public health problem worldwide. Drug-resistant TB is considered a major and growing global threat. Despite the great variety of described mutations in Mycobacterium tuberculosis (MTB) resistance genes, the mechanisms of drug resistance are still controversial. Recently, a report on the role of efflux pump genes in drug resistance added to this complexity. Therefore, a thorough understanding of efflux pump genes in drug-resistant TB clinical isolates is needed. METHODOLOGY: We performed molecular analysis of the efflux pump gene (Rv1258c) in 33 drug-resistant and 20 drug-sensitive clinical MTB isolates by sequencing the amplicons' targets in both the forward and reverse directions. RESULTS: A novel mutation of the Rv1258c gene was identified at G442A (Ala148Thr) in rifampicin mono-resistant clinical strain, as compared to the H37Rv reference strain. In addition, a cytosine nucleotide insertion was found between the positions 580 and 581 (denominated Tap580) in two drug-sensitive strains at identical gene positions. CONCLUSIONS: These results indicated the possibility of mutation in the efflux pump genes and the important role of Tap efflux pump genes in drug-resistant MTB isolates. However, further research is required to determine the direct association of these mutations with resistant MTB.

Improvement of tomato yield and quality using slow release NPK fertilizers prepared by carnauba wax emulsion, starch-based latex and hydrogel nanocomposite combination.

The modern agriculture is working on introducing new generation of fertilizers that apt to slow down the nutrients release to be more in synchrony with plant's need throughout growth season, enhance fertilizer performance, and decrease nutrient losses into the environment. The aim of this research was to develop an advanced NPK slow-release fertilizer (SRF) and investigate its effect on yield, nutritional and morphological responses of tomato plant (Lycopersicon esculentum Mill.) as a model crop. To this goal, three water-based bio-polymeric formulations including starch-g-poly (acrylic acid-co-acrylamide) nanocomposite hydrogel, starch-g-poly(styrene-co-butylacrylate) latex, and carnauba wax emulsion were synthesized and used for production of NPK-SRF samples. Different samples of coated fertilizers (urea, potassium sulfate, and superphosphate granules) were prepared using different ratios of latex and wax emulsion, and for phosphorus and potash (R-treatment). Moreover, some of coated fertilizers (15 and 30 wt.%) was replaced with nanocomposite hydrogel containing fertilizers, named D and H treatments, respectively. The effect of SRF samples were compared with commercial fertilizers (NPK treatment) and a commercial SRF (T treatment), on the growth of tomato in the greenhouse, at two different levels (100 and 60). The efficiency of all the synthesized formulations were higher than NPK and T treatments, and among them, H100 significantly improved the morphological and physiological characteristics of tomato. For instance, amount of residual elements (nitrogen, phosphorus and potassium) as well as micro elements of calcium, iron and zinc in tomato cultivation bed and accordingly the uptake of these elements in the roots, aerial parts and fruits were increased in the R, H, and D treatments. The highest yield (1671.54 g), highest agricultural agronomy efficiency of fertilizer, and the highest dry matter percentage (9.52%) were obtained in H100. The highest amount of lycopene, antioxidant capacity and vitamin C was also observed in H100. Nitrate accumulation in tomato fruit in the synthesized SRF samples were decreased significantly compared to NPK100, and the lowest amount was observed in H100, which was 55.24% less than NPK100. Accordingly, it is suggested that combination of natural-based nanocomposite hydrogels along with coating latexes and wax emulsions can be a successful method to synthesize efficient NPK-SRF formulations for improvement of crop growth and quality.

Molecular Detection of blaOXA -type Carbapenemase Genes and Antimicrobial Resistance Patterns among Clinical Isolates of Acinetobacter baumannii.

Acinetobacter baumannii is a bacterium found in most places, especially in clinics and hospitals, and an important agent of nosocomial infections. The presence of class D enzymes such as OXA-type carbapenemases in A. baumannii is proven to have a key function in resistance to carbapenem. The aim of the current study is to determine the blaOXA -type carbapenemase genes and antimicrobial resistance among clinically isolated samples of A. baumannii. We assessed 100 clinically isolated specimens of A. baumannii from patients in intensive care units of educational hospitals of Hamadan, West of Iran. The A. baumannii isolates' susceptibility to antibiotics was performed employing disk diffusion method. Multiplex polymerase chain reaction was used to identify the bla OXA-24-like , bla OXA-23-like , bla OXA-58-like , and bla OXA-51-like genes. The bla OXA-23-like , bla OXA-24-like , and bla OXA-58-like genes' prevalence were found to be 84, 58, and 3%, respectively. The highest coexistence of the genes was for bla OXA-51/23 (84%) followed by bla OXA-51/24-like (58%). The bla OXA-51/23- like pattern of genes is a sort of dominant gene in resistance in A. baumannii from Hamadan hospitals. The highest resistance to piperacillin (83%) and ciprofloxacin (81%) has been observed in positive isolates of bla OXA-23-like . The A. baumannii isolates with bla OXA-58-like genes did not show much resistance to antibiotics. Based on the results of the phylogenetic tree analysis, all isolates have shown a high degree of similarity. This study showed the high frequency of OXA -type carbapenemase genes among A. baumannii isolates from Hamadan hospitals, Iran. Thus, applying an appropriate strategy to limit the spreading of these strains and also performing new treatment regimens are necessary.

The Potential Effects of Fungicides on Association of Rhizophagus irregularis with Maize and Wheat

Abstract The effect of different fungicides on mycorrhizal fungi should be investigated in different plants and environmental conditions. Thus, the purpose of this study was to appraise the effect of simultaneous fungicides application (including benomyl, rovral TS, mancozeb, and tilt) on the efficiency of Rhizophagus irregularis in cultivations of maize and wheat. This study was conducted in two separate experiments in the laboratory and greenhouse. The results of the laboratory stage showed that the use of all four fungicides significantly reduced the spore number compared to the conditions of non-use of the fungicide, although only rovral TS and mancozeb led to a significant reduction in root colonization percentage of R. irregularis. In the greenhouse, the benomyl significantly increased root dry weight in maize although tilt significantly reduced root colonization of maize with R. irregularis. The tilt and rovral TS had a positive effect and benomyl had a negative effect on wheat growth traits, but the root colonization of wheat with R. irregularis was not affected by fungicides. Generally, benomyl (2 g L-1) in maize and tilt (2 mL L-1) in wheat and rovral TS in both plants could be recommended with the combined application of R. irregularis inoculants. Therefore, depending on the type of fungicide and the host plant, the effect of the fungicide on colonization and association of mycorrhiza varies.

Designing a chimeric subunit vaccine for influenza virus, based on HA2, M2e and CTxB: a bioinformatics study.

BACKGROUND: Type A influenza viruses are contagious and even life-threatening if left untreated. So far, no broadly protective vaccine is available due to rapid antigenic changes and emergence of new subtypes of influenza virus. In this study, we exploited bioinformatics tools in order to design a subunit chimeric vaccine from the antigenic and highly conserved regions of HA and M2 proteins of H7N9 subtype of influenza virus. We used mucosal adjuvant candidates, including CTxB, STxB, ASP-1, and LTB to stimulate mucosal immunity and analyzed the combination of HA2, M2e, and the adjuvant. Furthermore, to improve the antigen function and to maintain their three-dimensional structure, 12 different linkers including six rigid linkers and six flexible linkers were used. The 3D structure model was generated using a combination of homology and ab initio modeling methods and the molecular dynamics of the model were analyzed, either. RESULTS: Analysis of different adjuvants showed that using CtxB as an adjuvant, results in higher overall vaccine stability and higher half-life among four adjuvant candidates. Fusion of antigens and the CTxB in the form of M2e-linker-CTxB-linker-HA2 has the most stability and half life compared to other combination forms. Furthermore, the KPKPKP rigid linker showed the best result for this candidate vaccine among 12 analyzed linkers. The changes in the vaccine 3D structure made by linker insertion found to be negligible, however, although small, the linker insertion between the antigens causes the structure to change slightly. Eventually, using predictive tools such as Ellipro, NetMHCpan I and II, CD4episcore, CTLpred, BepiPred and other epitope analyzing tools, we analyzed the conformational and linear epitopes of the vaccine. The solubility, proteasome cleavage sites, peptidase and potential chemical cutters, codon optimization, post translational modification were also carried out on the final vaccine. CONCLUSIONS: It is concluded that M2e-Linker-CTxB-Linker-HA2 combination of chimeric vaccine retains its 3D structure and antigenicity when KPKPKP used as linker and CTxB used as adjuvant.

Nano-carriers effects on the viability and efficiency of Pseudomonas strains as phosphate solubilizing bacteria.

In order to develop nanotechnology application in the agricultural systems achieving more sustainability in the environment, we have used different nano-carriers for phosphate solubilizing bacteria. The viability and efficacy of two bacterial species; Pseudomonas putida (PP20) and Pseudomonas kilonensis (PK11) in solubilizing phosphate sources (i.e., tricalcium phosphate and hydroxyapatite) with different nano-carriers including nanoclay, natural char micro-particles (NCMPs), nanoclay + alginate, NCMPs + alginate, and natural char nano-particles (NCNPs)+alginate were investigated. Clay, talc powder, and natural char (NC) were included for comparison. The synthesized NCNPs and NCMPs were characterized using FTIR, SEM and Boehm titration analyses. The results confirmed that the chemical oxidation of pristine char made many oxygenated functional groups on the surface of tiny and spherical NCNPs (14.8 nm) which caused their effective incorporation in the matrix of alginate beads. Results of phosphate solubilizing study showed that P. kilonensis was the superior species for viability and stability of its performance on solubilizing phosphorus. The six months evaluation showed that NCNPs + alginate and nanoclay + alginate carriers at both temperatures (4 °C and 28 °C), were the proficient carriers for preserving both bacteria. The results of solubilizing phosphorus sources revealed that both bacteria solubilized tricalcium phosphate more than hydroxyapatite and PK11 showed more privilege in this regard. In addition, the solubilizing index determined after storage for 6 months at 4 °C was higher for all the carriers. Analysis of variance for phosphatase activity revealed that embedding both bacteria in nanoclay + alginate carrier guaranteed the highest phosphatase activity, even though differences between this carrier and NCNPs + alginate and NCMPs + alginate were not significant for the PK11.

Sequence-based detection of first-line and second-line drugs resistance-associated mutations in Mycobacterium tuberculosis isolates in Isfahan, Iran.

Tuberculosis is an infectious disease, which requires special medical attention due to the emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains. The present study aimed to assess drug resistance to first-line anti-mycobacterial drugs, including rifampin (RIF), isoniazid (INH), and ethambutol (EMB), as well as second-line drugs, including ofloxacin (OFX), kanamycin (KAN), amikacin (AMK), and capreomycin (CAP). The following eight loci were investigated to evaluate drug resistance: rpoB, katG, inhA, and embB, associated with resistance to RIF, INH, and EMB and gyrA, rrs, eis, and tlyA, associated with resistance to OFX, AMK, KAN, and CAP. A total of 482 patients with tuberculosis, who were referred to Molla Haadi Sabzevari Healthcare Center (Isfahan, Iran) during 2014-2017, were studied. Of 482 patients with tuberculosis, 32 (6.63%) Mycobacterium tuberculosis isolates were resistant to the first-line anti-mycobacterial drugs. Overall, 23 (71.8%), 13 (40.6%), and 3 (9.3%) isolates were resistant to INH, RIF, and EMB, respectively. Also, 13 (100%), 6 (46.1%), and 1 (7.6%) out of 13 MDR/RIF-resistant isolates were resistant to CAP and KAN, AMK, and OFX, respectively. Among the eight loci, non-synonymous substitutions were observed in rpoB (n = 7), katG (n = 10), inhA (n = 7), gyrA (n = 13), and rrs (n = 3), whereas synonymous substitutions were seen in tlyA and gyrA. On the other hand, no mutation was detected in embB or eis. Based on the present results, mutations in the eis promoter region and embB locus may not be involved in resistance to KAN and EMB in our study population. Also, the gyrA Asp94Asn mutation may be an indicator of resistance to OFX. We did not detect any XDR isolates, whereas MDR and pre-XDR isolates were found, which can be alarming.

Prevalence of intestinal parasites in Isfahan city, central Iran, 2014.

Intestinal parasites are important enteric pathogens. Poverty, low quality of food and water supply and poor sanitation systems are the important factors associated with intestinal parasitic infections. These kinds of infections can be a good index for hygienic and sanitation status of the society. This study aimed to determine the prevalence of intestinal parasitic infections among humans referred to Dr. Sharifi Clinical Laboratory, Isfahan, Iran, 2014. In this cross sectional study, 652 fecal samples (286 males and 366 females) from humans who had stool examination test from January to August 2014 were chosen. Microscopic examination for parasitic infections has been carried out using wet mount method. Indistinguishable samples underwent trichrome staining method for accurate identification of protozoa. Intestinal parasitic infections were observed in 68 (10.42 %) out of 652 studied humans. Forty eight Blastocystis hominis (7.36 %), thirteen Endolimax nana (1.99 %), nine Giardia lamblia (1.38 %), five Entamoeba coli (0.76 %), four Chilomastix mesnili (0.61 %) and two Iodamoeba butschlii (0.15 %) were the observed protozoa in the studied population. B. hominis, E. nana and C. mesnili were found to be significantly more prevalent in people with loose stool specimen. Considering the helminthic infections, only one case (0.15 %) that was excreted Taenia saginata proglottids has been documented among 652 studied humans. Based on the findings of the present study intestinal parasitic infections in Isfahan city has been dramatically decreased over the past years and shows a good hygienic and sanitation status of the city.

Parasites and immunotherapy: with or against?

Immunotherapy is a sort of therapy in which antibody or antigen administrates to the patient in order to treat or reduce the severity of complications of disease. This kind of treatment practiced in a wide variety of diseases including infectious diseases, autoimmune disorders, cancers and allergy. Successful and unsuccessful immunotherapeutic strategies have been practiced in variety of parasitic infections. On the other hand parasites or parasite antigens have also been considered for immunotherapy against other diseases such as cancer, asthma and multiple sclerosis. In this paper immunotherapy against common parasitic infections, and also immunotherapy of cancer, asthma and multiple sclerosis with parasites or parasite antigens have been reviewed.

Prevalence of ESBL and MBL encoding genes in Acinetobacter baumannii strains isolated from patients of intensive care units (ICU).

The aim of this study was to investigate the prevalence of ESBL and MBL encoding genes among A. baumannii isolates. In this cross sectional study, 100 A. baumannii strains were isolated from ICU wards of 3 educational hospitals of Hamadan City, Iran in 2011. Phenotypic identification of the production of ESBLs and MBLs has been carried out by using E-test and DDST methods, respectively. PCR technique was used for amplification of the ESBL and MBL encoding genes, namely: CTX-M, SHV, TEM, OXA-51, VIM-Family, IMP-Family, SPM-1, SIM-1, and GIM-1. Eighty seven (87%), 95 (95%), 98 (98%) and 95 (95%) out of 100 A. baumannii isolates were resistant to imipenem, meropenem, ceftazidime and cefotaxime, respectively. Also, 99% and 7% of the isolates were MBLs and ESBLs produced phenotypically. Thirty (30%), 20 (20%) and 58 (58%) out of 100 A. baumannii isolates have been confirmed to harbor the bla VIM-family, TEM and SHV genes, respectively. Our results show no significant relationship between the detected gens with production of MBLs and ESBLs in spite of high prevalence of MBL encoding and drug resistant A. baumannii. Probably some other genes rather than what we studied are involved in phenotypic production of MBLs and ESBLs and subsequent drug resistance in Hamadan area, Iran.

Comparison of fecal antigen detection using enzyme linked immunosorbent assay with the auramine phenol staining method for diagnosis of human cryptosporidiosis.

BACKGROUND: Fecal antigen detection using the enzyme linked immunosorbent assay (ELISA) and oocyst detection using auramine phenol (AP) staining methods, are told to be more sensitive compared to other conventional methods, for diagnosis of cryptosporidiosis. OBJECTIVES: The aim of the present study was to evaluate the antigen-detection capacity in the stool specimens using ELISA and oocyst detection by AP staining methods, for the diagnosis of human cryptosporidiosis. MATERIALS AND METHODS: A total of 228 fecal samples were collected from residents of rural areas of Hamadan, West of Iran. Each fecal sample was divided into two parts, one kept frozen at -20˚C for Ag-capture ELISA and the other in 10% formalin for the AP staining method. Cryptosporidium Ag-detection ELISA procedure was performed according to the manual of the manufacturer. The preserved samples concentrated using the formalin-ether concentration technique were stained with AP and then investigated under florescent microscopy. RESULTS: Eight (3.5%) and three (1.3%) out of 228 fecal samples were positive for Cryptosporidium infection by ELISA and AP staining methods, respectively. Cryptosporidium Ag-detection using ELISA showed an increased frequency of the infection, compared to the AP staining method (P = 0.062). CONCLUSIONS: For epidemiological studies and diagnostic purposes of the Cryptosporidium infection, especially in asymptomatic individuals, Ag-detection ELISA is an easy to perform and accurate method, compared to other conventional microscopic methods.

High prevalence of multidrug resistance and metallo-beta-lactamase (MßL) producing Acinetobacter baumannii isolated from patients in ICU wards, Hamadan, Iran.

BACKGROUND: Acinetobacter baumannii is gram- negative opportunistic coccobacilli, the most important agent in nosocomial infections with high mortality rate. Multidrug resistance in strains isolated from nosocomial infections, making it difficult to treat and sometimes impossible. The aim of the present study was to investigate antibiotic resistance in A. baumannii isolates from Iranian patients in Hamadan, west of Iran. METHODS: In this cross sectional study 100 A. baumannii isolated from trachea, blood, urine, sputum and wound samples of patients bedridden in Intensive care unit (ICU) wards of three educational hospitals during June 2011 to October 2012 was included. Isolates confirmed at species level using biochemical tests and tracing blaOXA-51 gene using Polymerase chain reaction (PCR) and preserved frozen at -70 °C until examination. Their susceptibility to 17 antibiotics was performed using Kirby-Bauer disc diffusion method. Determination of minimum inhibitory concentration and Metallo-beta-lactamase production was carried out using E-test method. RESULTS: Resistance rate of isolates were 94%, 85%, 84%, 97%, 95% and 98% against meropenem, imipenem, amikacin, ciprofloxacin, piperacillin/tazobactam and cefotaxime, respectively. No resistant isolate was observed against tigecycline and also no sensitive isolate seen against aztreonam and cefotaxime. Results of E-test illustrated that 99% of all isolates were Metallo-beta-lactamase (MßL) producing, which were resistance to imipenem; also 85% of them were resistance to meropenem. MIC50 and MIC90 of the isolates were ≥256 and ≥32 mg/ml for imipenem and meropenem, respectively. CONCLUSIONS: The antibiotic resistance against most of the antibiotics, especially carbapenems is very high in Hamadan region. In addition colistin sulfate and tigecycline were most effective antibiotics and to be used in A. baumannii infections.

Seroprevalence of Toxoplasma gondii infection and related risk factors in Tabriz City, Iran, 2008.

BACKGROUND: Toxoplasma gondii is a worldwide obligate intercellular parasite. Felids are its definitive host and warm-blooded animals including humans are its intermediate host. The aim of this seroepidemiological study was to investigate the frequency of human infection using ELISA method and related risk factors in Tabriz City, northwestern Iran. METHODS: In this cross sectional study, 171 blood samples were collected randomly from clients referred to Alinasab Hospital diagnostic laboratory, Tabriz, Iran in 2008. Simultaneously data about risk factors such as having soil related jobs (for example: building construction workers and farmers), cat contact, eating raw vegetables at restaurants, the method of washing vegetables, eating undercooked game meat and the quantity of red meat consumption (undercooked) were collected by questionnaires. Anti-Toxoplasma IgG titers were determined in samples using ELISA method. Data were analyzed by Chi-square, One Way ANOVA and t-test using SPSS v.16 Software. RESULTS: Sixty (35.1%) out of 171 serum samples were anti-Toxoplasma IgG positive. There was statistically significant difference about seropositivity between soil related jobs and others (P=0.007, OR=2.43; 95% CI: 1.27, 4.66). In addition there was significant relationship between seropositivity and eating vegetables at restaurants (P=0.039, OR=1.94; 95% CI: 1.02, 1.68). No significant statistical differences were observed about seropositivity considering the other mentioned risk factors. CONCLUSION: The prevalence rate of Toxoplasma chronic infection was relatively high in the studied people. However, having soil related jobs and eating vegetables at restaurants increases the probability of acquiring the infection.