Alterations in the gut microbiota and their metabolites in human intestinal epithelial cells of patients with colorectal cancer.

BACKGROUND: The gut microbiota has become one of the main risk factors for the formation and development of colorectal cancer (CRC). CRC intensification may be due to the microbial pathogens' colonization and their released metabolites. Here, we analyzed Bacteroidetes and Clostridia bacteria in CRC patients and studied bacterial metabolome in cancerous tissues compared to their adjacent normal tissues. METHODS AND RESULTS: The population of selected bacteria in biopsy specimens of 30 patients with CRC was studied by RT-qPCR. The mutagenicity and cytotoxicity effects of microbiota metabolites were evaluated by Ames test and MTT Assay, respectively. Moreover, gene expression in carcinogenic pathways was studied by RT-qPCR, and genes with different expressions in tumor and non-tumor tissues were diagnosed. Based on microbiota analysis, the relative abundance of Clostridia and C. difficile was significantly higher in CRC tissue, whereas C. perfringens showed higher relative abundance in normal tissue. AIMES test confirmed the proliferation and mutagenicity effects of the bacterial metabolites in CRC patients. Significant upregulation of C-Myc, GRB2, IL-8, EGFR, PI3K, and AKT and downregulation of ATM were observed in CRC samples compared to the control. CONCLUSIONS: The influence of bacterial metabolites on inflammation and altered expression of genes in the cell signaling pathways was observed. The findings confirm the role gut microbiota composition and bacterial metabolites as key players in CRC onset and development.

The characteristic and potential therapeutic effect of isolated multidrug-resistant Acinetobacter baumannii lytic phage.

BACKGROUND: Widespread misuse of antibiotics caused bacterial resistance increasingly become a serious threat. Bacteriophage therapy promises alternative treatment strategies for combatting drug-resistant bacterial infections. In this study, we isolated and characterized a novel, potent lytic bacteriophage against multi-drug resistant (MDR) Acinetobacter baumannii and described the lytic capability and endolysin activity of the phage to evaluate the potential in phage therapy. METHODS: A novel phage, pIsf-AB02, was isolated from hospital sewage. The morphological analysis, its host range, growth characteristics, stability under various conditions, genomic restriction pattern were systematically investigated. The protein pattern of the phage was analyzed, and the endolysin activity of the phage was determined under the non-denaturing condition on SDS-PAGE. The optimal lytic titer of phage was assessed by co-culture of the phage with clinical MDR A. baumannii isolates. Finally, HeLa cells were used to examine the safety of the phage. RESULTS: The morphological analysis revealed that the pIsf-AB02 phage displays morphology resembling the Myoviridae family. It can quickly destroy 56.3% (27/48) of clinical MDR A. baumannii isolates. This virulent phage could decrease the bacterial host cells (from 108 CFU/ml to 103 CFU/ml) in 30 min. The optimum stability of the phage was observed at 37 °C. pH 7 is the most suitable condition to maintain phage stability. The 15 kDa protein encoded by pIsf-AB02 was detected to have endolysin activity. pIsf-AB02 did not show cytotoxicity to HeLa cells, and it can save HeLa cells from A. baumannii infection. CONCLUSION: In this study, we isolated a novel lytic MDR A. baumannii bacteriophage, pIsf-AB02. This phage showed suitable stability at different temperatures and pHs, and demonstrated potent in vitro endolysin activity. pIsf-AB02 may be a good candidate as a therapeutic agent to control nosocomial infections caused by MDR A. baumannii.

Prevalence, population structure, distribution of serotypes, pilus islands and resistance genes among erythromycin-resistant colonizing and invasive Streptococcus agalactiae isolates recovered from pregnant and non-pregnant women in Isfahan, Iran.

BACKGROUND: The information on antibiotic resistance and molecular features of Group B Streptococcus (GBS) are essential for epidemiological purposes as well as vaccine development. Therefore, we aimed to assess the antimicrobial resistance profiles and molecular characteristics of GBS isolates in Isfahan, Iran. A total number of 72 colonizing and invasive GBS were collected from pregnant and non-pregnant women. The GBS isolates were analyzed for resistance profiles, capsular genotyping, and detection of PI-1, PI-2a, PI-2b, hvgA, ermB, ermTR, lnuB and, mefA genes. Besides, erythromycin-resistant strains were subjected to multilocus sequence typing (MLST). RESULTS: The prevalence of colonizing and invasive GBS were 11 and 0.05%, respectively. The frequency of capsular serotypes was as follows: III (26.3%), Ia (20.83%), Ib and V (each 15.2%), IV (9.7%), II (8.3%), VII (2.7%), and VI (1.3%). Overall frequencies of PIs were as follows: PI-1, 37.5%, PI-1 + PI-2a, 30.5%, PI-1 + PI-2b, 29.1% and PI-2b, 2.7%. Two maternal colonizing GBS (2.6%) were hvgA positive and were belonged to ST-17/CPS-III/PI-1 + PI-2b lineage. Among 30(41.6%) erythromycin resistant GBS, 21 isolates (70%) harbored ermB gene, followed by ermTR (23.3%) and mefA (10%). One clindamycin-resistant isolate harbored the lnuB gene. MLST analysis revealed the following five clonal complexes (CCs) and nine STs: (CC-19/ST-335, ST-19, and ST-197), (CC-12/ST-43, ST-12), (CC-23/ST-163, ST-23), (CC-17/ST-17) and (CC-4/ST-16). CONCLUSION: The study shows an alarmingly high prevalence of erythromycin-resistant GBS in Iran. In addition, we report dissemination of ST-335/CPS-III clone associated with tetracycline and erythromycin resistance in our region. The distribution of capsular and pilus genotypes varies between invasive and colonizing GBS that could be helpful for vaccine development.

Cellular miR-101-1 Reduces Efficiently the Replication of HSV-1 in HeLa Cells.

INTRODUCTION: Herpes simplex viruses (HSVs) are widely distributed in the human population. HSV type 1 (HSV-1) is responsible for a spectrum of diseases, ranging from gingivostomatitis to keratoconjunctivitis, and encephalitis. The HSVs establish latent infections in nerve cells, and recurrences are common. Their frequent reactivation in elderly and immunosuppressed patients causes serious health complications. OBJECTIVES: Due to the growing resistance to its main drug, acyclovir, alternative treatments with different mechanisms of action are required. MicroRNAs regulate host and viral gene expression posttranscriptionally. Previous studies reported that mir-101-2 expression has widely participated in the regulation of HSV-1 replication. In this study, we investigate the effect of hsa-miR-101-1 in the replication of HSV-1. METHODS: We found that transfection of miR-101-1 into HeLa cells could reduce effectively HSV-1 replication using plaque assay and real-time PCR methods. RESULTS: We showed that overexpression of miR-10-1 produced less viral progeny and manifested a weaker cytopathic effect, without affecting cell viability. DISCUSSION/CONCLUSION: This result can give us new insights into the control of HSV-1 infections.

Efficient process development for high-level production, purification, formulation, and characterization of recombinant mecasermin in Escherichia coli.

Overproduction of recombinant mecasermin was achieved by investigation of effect of three factors, temperature, inducer amount, and culture media, at three levels according to the Taguchi statistical design in Escherichia coli in a bench-scale bioreactor. In optimal conditions (induction temperature 28 °C, terrific broth with glucose (TB+G) medium, with 0.1 mM IPTG as inducer) 0.84 g/L mecasermin with expression levels of 38% of total protein and 4.13 g/L final dry cell biomass was produced, that is one of the highest values of recombinant protein has been reported in the batch system. The cell disruption was done by lysozyme pretreatment with sonication to the efficient purification of mecasermin. The isolated and washed inclusion bodies were solubilized in Gdn-HCl at pH 5.4 and folded with glutathione and purified with gel filtration. The purified rhIGF-1 (mecasermin) was formulated with arginine. Mecasermin protein remained t stable at 4 °C for up to 2 years. The quantitative and qualitative control indicated that mecasermin is expressed correctly (without the initial methionine by mass spectrometry), pure (without endotoxin and other protein impurities), correct folding (FTIR, RF-HPLC), monomer form (SEC-HPLC), and active (bioactivity test). Also, the purification results revealed that expression at low temperature results in the efficient purification of the overproduced mecasermin with high quantity and quality.

Phylogenetic analysis of human bocavirus in children with acute respiratory infections in Iran.

Human bocavirus (HBoV) was first characterized in nasopharyngeal aspirates from young children with acute respiratory infections. It is prevalent among children with acute wheezing. This study was carried out in order to analyze the infection frequency and coinfection rates of HBoV with respiratory syncytial virus (RSV) and to perform phylogenetic analysis of HBoV in samples of children with acute respiratory infection in Isfahan, Iran. During the time period 2016-2017, altogether 75 respiratory samples from children hospitalized with acute respiratory infection were collected. The samples were first screened for RSV by direct immunofluorescence method and then subjected to detect HBoV DNA by PCR. Genotyping of HBoV-positive samples was conducted by direct sequencing of PCR products using NP and VP1/VP2 genes. Out of 75 respiratory samples, 20 (26.7%) and 10 (13.3%) were positive for RSV and HBoV, respectively. The coinfection rate was 40% (p = 0.048). Considering the seasonal distribution, winter has the highest extent outbreak (p = 0.036). Sequence analysis of positive samples exhibits that all of the isolated HBoV were related to genotype 1 (HBoV-1) with minimal sequence variations. Increasing frequency of HBoV suggests that the virus is related to acute respiratory infection in children. A single genetic lineage of HBoV1 seems to be the major genotype in Iran.

Frequency Determination of Carbapenem-Resistant Klebsiella Pneumoniae (CRKP) Isolated from hospitals in Isfahan of Iran and Evaluation of Synergistic Effect of Colistin and Meropenem on them.

Overuse and misuse of Carbapenems among Klebsiella pneumoniae isolates have caused Carbapenem-Resistant Klebsiella Pneumonia (CRKP) during recent years. Colistin is one of the last available options, and there are increasing concerns about the dosage and resistance to this agent in long-term monotherapies. This study was designed to identification of carbapenemase producing isolates of K. pneumoniae via phenotypic and genotypic methods as well as evaluation of colistin-meropenem combination therapy potential. This study was carried out in Isfahan, of Iran on 100 samples from Alzahra and Khorshid hospitals in 2017. The Modified Hodge Test (MHT) was used to investigate the carbapenemase presence. The minimum inhibitory concentration (MIC) and the Fractional Inhibitory Concentration (FIC) were determined using broth macrodilution and checkerboard assays (respectively) for both meropenem and colistin. The bla-KPC gene was studied by polymerase chain reaction (PCR).The highest and the lowest rate of resistance were observed for piperacillin (84%) and ertapenem (50%) respectively. 68 isolates by MHT were CRKP, but None of them were positive for bla-KPC gene. 21 isolates from CRKP cases were high resistant to used antimicrobial agents in the study that both MIC and FIC results showed significant synergy for this antibiotics in checkerboard test (p-value < 0.05). 21 resistant isolates from CRKP cases showed statistically significant synergy potential for meropenem and colistin. The meropenem-colistin combination therapy can be applied as a suitable antibiotic synergy but it requires further investigation in clinical assay. Regarding to our findings, Probably other mechanisms of resistance to Carbapenems ,except bla-kpc genes are involved.

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.

The Bioinformatics Report of Mutation Outcome on NADPH Flavin Oxidoreductase Protein Sequence in Clinical Isolates of H. pylori.

frxA gene has been implicated in the metronidazole nitro reduction by H. pylori. Alternatively, frxA is expected to contribute to the protection of urease and to the in vivo survival of H. pylori. The aim of present study is to report the mutation effects on the frxA protein sequence in clinical isolates of H. pylori in our community. Metronidazole resistance was proven in 27 of 48 isolates. glmM and frxA genes were used for molecular confirmation of H. pylori isolates. The primer set for detection of whole sequence of frxA gene for the effect of mutation on protein sequence was used. DNA and protein sequence evaluation and analysis were done by blast, Clustal Omega, and T COFFEE programs. Then, FrxA protein sequences from six metronidazole-resistant clinical isolates were analyzed by web-based bioinformatics tools. The result of six metronidazole-resistant clinical isolates in comparison with strain 26695 showed ten missense mutations. The result with the STRING program revealed that no change was seen after alterations in these sequences. According to consensus data involving four methods, residue substitutions at 40, 13, and 141 increase the stability of protein sequence after mutation, while other alterations decrease. Residue substitutions at 40, 43, 141, 138, 169, and 179 are deleterious, while, V7I, Q10R, V34I, and V96I alterations are neutral. As FrxA contribute to survival of bacterium and in regard to the effect of mutations on protein function, it might affect the survival and bacterium phenotype and it need to be studied more. Also, none of the stability prediction tool is perfect; iStable is the best predictor method among all methods.

Sewage sludge composting: quality assessment for agricultural application.

In order to use sewage sludge (SS) composts in agriculture, it is extremely important to estimate the quality of compost products. The aim of this study was to investigate the quality of composted SS as a fertilizer and soil amendment especially in semi-arid areas. To determine the quality and agronomic value of the SS compost products, analyses on pH, electrical conductivity, organic matter content, C/N ratio, phytotoxicity, microbial load, and heavy metal content of composted anaerobically digested SS, with different proportions (1:1, 1:2, and 1:3 v/v) of green and dry plant waste, as bulking agents, were performed. The 1:2 and 1:3 mixtures of SS and green/dry plant waste were the most beneficial for composting, with final composts attaining high organic matter degradation and exhibiting low amounts of heavy metals, a relatively high germination index, and significant reduction of pathogens, suggesting the agricultural relevance of composted SS and green/dry plant waste at 1:2 and 1:3 (v/v) proportions. pH and electrical conductivity were also within the permissible limits. With respect to international standards, it appears that composted SS and green/dry plant waste at 1:2 and 1:3 proportions pose no threat to soil or plant quality if used in agriculture or land restoration.

Antibiotic profile classification of Proteus mirabilis using machine learning: An investigation into multidimensional radiomics features.

Antimicrobial resistance (AMR) presents a significant threat to global healthcare. Proteus mirabilis causes catheter-associated urinary tract infections (CAUTIs) and exhibits increased antibiotic resistance. Traditional diagnostics still rely on culture-based approaches, which remain time-consuming. Here, we study the use of machine learning (ML) to classify bacterial resistance profiles using straightforward microscopic imaging of P. mirabilis for resistance classification integrated with radiomics feature analysis and ML models. From 150 P. mirabilis strains isolated from catheters of patients diagnosed with a CAUTI, 30 % displayed multidrug resistance using the standardized disk diffusion method, and 60 % showed strong biofilm activity in microtiter plate assays. As a more rapid alternative, we introduce wavelet-based and regular microscopy imaging with feature extraction/selection, following image preprocessing steps (image denoising, normalization, and mask creation). These features enable training and testing different ML models with 5-fold cross-validation for P. mirabilis resistance classification. From these models, the Random Forest (RF) algorithm exhibited the highest performance with ACC = 0.95, specificity = 0.97, sensitivity = 0.88, and AUC = 0.98 among the other ML algorithms considered in this study for P. mirabilis resistance classification. This successful application of wavelet-based feature Radiomics analysis with RF model represents a crucial step towards a precise, rapid, and cost-effective method to distinguish antibiotic resistant P. mirabilis strains.

Assessment of cagE and babA mRNA expression during morphological conversion of Helicobacter pylori from spiral to coccoid.

Helicobacter pylori (H. Pylori) is an actively dividing spiral bacterium that changes to coccoid morphology under stressful environments. The infectivity of the coccoids is still controversial. The aim of this study was to determine the viability and expression of two important virulence genes (babA and cagE), in antibiotic-induced coccoid forms. Three strains of H. pylori, the standard 26695 and two clinical isolates (p1, p2) were converted to coccoid form by amoxicillin. Coccoids were identified according to Gram-staining and microscopic morphology. The viability of the cells was analyzed by flow cytometry. The expression of cagE and babA in coccoid forms were evaluated and compared to the spirals by quantitative PCR assay. The coccoid forms were developed after 72 h exposure of H. pylori to ½ MIC of amoxicillin, and the conversion form was completed (100 %) at 144 h in all of three isolates. Flow cytometry analyses showed that the majority of the induced coccoids (90-99.9 %) were viable. Expression of cagE and babA was seen in coccoids; however, in lower rate (cagE, ~3-fold and babA, ~10-fold) than these in spiral forms. Coccoid forms of two clinical isolates significantly expressed higher rate of cagE and babA than standard 26695 strain (P = 0.01). These results suggest that the induced coccoid form of H. pylori is not a passive entity but can actively infect the human by expression of the virulence genes for long time in stomach and probably play a role in chronic and severe disease.

BK virus excretion in acquired immunocompromised children: A comparison between kidney transplant recipients and steroid resistant nephrotic syndrome.

BACKGROUND: BK virus (BKV) is ubiquitous in human beings. Virus reactivation may occur in immunocompromised settings. The aim of this study was to compare BKV excretion in acquired immunocompromised children (kidney transplant recipients and steroid resistant nephrotic syndrome) with normal population. MATERIALS AND METHODS: One hundred and thirty one participants less than 20 years were recruited in the case-control study from June 2009 to December 2010. The participants consisted of 40 patients with steroid resistant nephrotic syndrome (subgroup 1), 39 kidney transplant recipients (subgroup 2) and 52 normal populations as control group. The first morning urine samples were analyzed in duplicate by conventional polymerase chain reaction (PCR) method for BKV. RESULTS: Nine participants out of 131 had positive results for BKV. Three patients in subgroup 1 (7.5%), two patients in subgroup 2 (5.1%) and six people (11.5%) in the control group had positive PCR results for urinary BKV. No significant difference was noted among groups, P = 0.53. The mean of glomerolar filtration rates in participants with positive and negative results for BKV were 125.5 ± 30.8 ml/min/m(2) and 132.2 ± 42.5 ml/min/m(2) respectively, P = 0.8. CONCLUSION: Acquired immunocompromised conditions did not increase the chance of urine BKV excretion in our study.

Resistance pattern of Helicobacter pylori strains to clarithromycin, metronidazole, and amoxicillin in Isfahan, Iran.

BACKGROUND: Helicobacter pylori (H. pylori) resistance to antibiotics has become a global problem and is an important factor in determining the outcome of treatment of infected patients. The purpose of this study was to determine the H. pylori resistance to clarithromycin, metronidazole, and amoxicillin in gastrointestinal disorders patients. MATERIALS AND METHODS: In this study, a total of 260 gastric antrum biopsy specimens were collected from patients with gastrointestinal disorders who referred to Endoscopy Section of the Isfahan Hospitals. The E-test and Modified Disk Diffusion Method (MDDM) were used to verify the prevalence of antibiotic resistance in 78 H. pylori isolates to the clarithromycin, metronidazole, and amoxicillin. RESULTS: H. pylori resistance to clarithromycin, metronidazole, and amoxicillin were 15.3, 55.1, and 6.4%, respectively. In this study, we had one multidrug resistance (MDR) isolates from patient with gastritis and peptic ulcer disease. CONCLUSION: Information on antibiotic susceptibility profile plays an important role in empiric antibiotic treatment and management of refractive cases. According to the results obtained in this study, H. pylori resistance to clarithromycin and metronidazole was relatively high. MDR strains are emerging and will have an effect on the combination therapy.

Enhanced taxane production and secretion from Taxus baccata cell culture by adding dimethylsulfoxide.

The ability of an aprotic solvent, dimethylsulfoxide (DMSO), to induce taxane synthesis and release from cell suspension culture of Taxus baccata was examined. The results showed that applying DMSO in optimal conditions not only led to enhancement in taxane excretion from the cells but also led to improvement in taxol synthesis. Maximum yields for taxol [3.34 mg/g dry cell weight (DCW)] were achieved by adding 5% DMSO to the culture at the late-exponential phase of cell growth (day 14) and culturing for 21 days, which was 2.26-fold of that for the control (1.48 mg/g DCW). However, adding 5% DMSO did not affect the yield of 10-deacetyl baccatin III and baccatin III. This condition also resulted in maximum extracellular taxane (4.86 mg/L); this value was 2.82-fold higher than that in the control (1.72 mg/L). We demonstrated that the late-exponential phase of cell growth could be the best time to add elicitor for maximizing the yield of taxol. Adding DMSO at earlier times (days 1 and 7) or in other concentrations (1% and 3%) had negative effects on taxane synthesis. Overall results suggest that DMSO has good potential to enhance synthesis and release taxol from cell suspension culture of T. baccata L.

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.

Optimization of the basal medium for improving production and secretion of taxanes from suspension cell culture of Taxus baccata L.

BACKGROUND AND PURPOSE OF THE STUDY: Taxol is one of the most effective anticancer drugs that isolated from Taxus sp. due to the slow growth of Taxus trees and low concentration of Taxol in the tissues, the biotechnological approaches especially plant cell culture have been considered to produce Taxol in commercial scale. METHODS: We investigated the effects of basal medium type used in culture media on production of Taxol and other taxane compounds from cell suspension culture of T. baccata L. Briefly, five commonly basal media including Gamborg, Murashige and Skoog, Woody Plant, Schenk and Hildebrandt, and Driver and Kuniyuki medium were used for preparing separate suspension culture media. The intra- and extra-cellular yields of taxanes were analyzed by using HPLC after 21 days period of culturing. RESULTS: The yields of taxanes were significantly different for the cultures prepared by different basal media. Moreover, the effects of basal medium on the yield of products differed for varius taxane compounds. Maximum yields of Baccatin III (10.03 mgl-1) and 10-deacetyl baccatin III (4.2 mgl-1) were achieved from the DKW basal media, but the yield of Taxol was maximum (16.58 mgl-1) in the WPM basal media. Furthermore, the secretion of taxanes from the cells into medium was also considerably affected by the type of basal medium. The maximum extra-cellular yield of Taxol (7.81 mgl-1), Baccatin III (5.0 mgl-1), and 10-deacetyl baccatin III (1.45 mgl-1) were also obtained by using DKW basal medium that were significantly higher than those obtained from other culture media.

Pseudomonas aeruginosa infections in patients, hospital means, and personnel's specimens.

BACKGROUND: Pseudomonas aeruginosa is a common cause of nosocomial infections. It exhibits innate resistance to a wide range of antibiotics. This study was performed to determine clonal characteristic of P. aeruginosa isolated from clinical specimens, hospital means, and hospital personnel by PCR- ribotyping patterns. METHODS: A total of 104 P. aeruginosa were isolated from clinical and environmental samples (59 clinical, 45 hospital means and hospital personnel). P. aeruginosa was identified by standard bacteriological methods, mucoid colony morphotypes, and antibiotic resistance rate. The genomes of isolates were extracted and all considered species were confirmed by 16S rDNA- based PCR assay. Then all isolates were genotyped by the 16S - 23SrDNA and Hinf1 restriction enzyme technique. RESULTS: Antibacterial sensitivity pattern of isolates showed clinical and environmental specimens were approximately identical (high antibiotic resistance to Ceftazidime and low antibiotic resistance to Amikacin). Colony morphotypes of specimens revealed that mucoid type of clinical isolates were more than that of environmental isolates. Among clinical and environmental strains P1; (570 bp) was the most prevalence pattern. CONCLUSIONS: Antibiotic resistance, phenotypic characterization, and PCR- ribotyping pattern showed there is clonal relatedness between clinical and environmental isolates and environment could be a main reservoir for P. aeruginosa infections in hospital.

Development of PCR-based method for detection of Enterobacteriaceae in septicemia.

OBJECTIVE: Sepsis is a systemic inflammatory response associated with high mortality rates in the clinical setting. A multiplex endpoint polymerase chain reaction (PCR) based assay for rapid detection of enterobacteriaceae involved in septicemia, which included Internal Control (IC) and 16S rDNA, is presented here. To develop a panel of primers for DNA fragments of 16S rDNA, enterobacteriaceae, IC, and evaluate analytical sensitivity and specificity of the test. MATERIALS AND METHODS: Primers for amplification of enterobacteriaceae, IC, and16S rDNA were designed, and then PCR was performed. Minimal analytical sensitivity was determined by cloning and colony PCR, and specificity was tested on the basis of their respective standard strains. This study is a cross-sectional Model. RESULTS: Our results showed the rpoB gene as the most promising target for detection of enterobacteriaceae by PCR amplification. Specificity and sensitivity of endpoint PCR were 100%, 100%, and 100%, and 10, 1, and 100 copies/reaction for enterobacteriaceae, IC, and 16S rDNA, respectively. CONCLUSION: The molecular panel presented offers the advantage of an easy, reliable, and cost-effective system when compared to other molecular detection methods. However, further evaluation is needed. Our assay holds promising for more rapid pathogens related in clinical sepsis.