فصلنامه زیست شناسی میکروارگانیسم ها
پیاپی 12 (زمستان 1393)

One of the main applications of chitosan is for heavy metals removal from waste waters. Industrially, chitosan is produced through deacetylation of chitin present in shellfish waste. Another source of chitosan is the cell wall of zygomycetes fungi with several advantages over shellfish wastes. Materials and method s: Fungal chitosan purified from biomass of Mucor indicus and shrimp chitosan were applied and compared for removal of copper ions from aqueous solution. The effects of pH (3 to 5.5), copper ion concentration (5 to 52 mg l-1), the amount of chitosan (200 to 3000 mg l-1), adsorption time, temperature, and presence of other metal ions on the biosorption of Cu2+ were investigated. Maximum adsorption capacities for fungal and shrimp chitosans were 58.5 and 60.7 mg g-1, respectively. T he rate of copper adsorption by the fungal chitosan was significantly higher than that by the shrimp chitosan. Among p seudo-first order, pseudo-second order, intra-particle diffusion, and Elovich models, Ho’s pseudo-second order model was the best model for fitting the kinetic data. The adsorption capacity increased for both types of chitosans by increasing the solution pH. However, temperature and presence of other ions did not show significant effects on the biosorption capacity of copper. The isotherm data were very well described by Langmuir, Freundlich, and Redlich-Peterson models. Discussion and Both fungal and shrimp chitosans can effectively be used for removal of copper ions from aqueous solutions. Adsorption process for fungal chitosan is fast, while the process is slower for the shrimp chitosan. Therefore, from the kinetics point of view, the fungal chitosan is preferable compared with the shrimp chitosan.

Actinomycetes are widely distributed in natural and man-made environments and have capability for degradation of organic matter. They are also well known as a rich source of antibiotics and bioactive molecules and are of considerable importance in industry. In this study, a rare actinomycete was isolated and subjected to polyphasic identification. Identification of this rare actinomycetes was carried on according to polyphasic taxonomic approach which has been outlined by International Committee on Systematics of Prokaryotes (ICSP). The cell wall of strain LO5 contained meso-diaminopimelic acid as diaminoacid and galactose, mannose and rhamnose as diagnostic sugars. The phospholipids consisted of diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. Phylogenetic analysis based on nearly complete 16S rRNA gene sequence comparison revealed affiliation to the family Pseudonocardiaceae and its similarity to the most closely related neighbor the Lechevalieria fradiae CGMCC 4.3506T was 98.6%. The level of DNA–DNA relatedness between the novel strain and Lechevalieria fradiae CGMCC 4.3506T was only 75 %. So according to the recommendations of a threshold value of 70% DNA-DNA similarity, LO5 belongs to the species Lechevalieria fradiae CGMCC 4.3506T. On the basis of genomic and phenotypic properties, the subspecies Lechevalieria fradiae subsp. Iranica IBRC-M 10378 is proposed.Discussion and A polyphasic approach based on phenotypic, chemotaxonomic and phylogenetic investigations has been proposed for categorizing of a rare actinomycete in subspecies level. The techniques used to obtain the data required for determination of the taxonomic status of this isolate are based on minimal standards that have been established by some of the taxonomic subcommittees of the International Committee on Systematics of Prokaryotes (ICSP) for specific groups of organisms.

Infections caused by Pseudomonas aeruginosa biofilm are the major causes of death in patients with cystic fibrosis (CF). Some studies revealed that biofilms are resistant to several antibiotics because of their impermeable structures. In order to re-sensitize bacteria to different antibiotics, biofilm formation should be inhibited. In this research, evaluation of antibiofilm activity of n-butanolic Cyclamen coum extract as a medici­nal plant from Myrsinaceae family, in combination with ciprofloxacin was carried out. The biofilm formation ability by P. aeruginosa PAO1 and one clinically isolated P. aeruginosa (PA214) was confirmed by microtiter plate method. Extraction of the tubers of Cyclamen coum was done by fractionation method. The antibiofilm and antibacterial properties of n-butanolic C. coum extract (which includes saponin compounds) alone and in combination with ciprofloxacin by using microdilution and crystal violet methods were examined. The cytotoxicity effect of the n-butanolic extract on HT-29 cells was assayed by MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl-tetrazolium bromide) test. The biofilm formation ability by P. aeruginosa strains was quantitatively confirmed. Saponin content of the n-butanolic C.coum extract was 156 µg/mL. The extract revealed antibacterial activity against the growth of planktonic P. aeruginosa strains. The combination of n-butanolic C.coum extract and ciprofloxacin significantly inhibited P.aeruginosa biofilm formation (ΣFBIC = 0.5). The n-butanolic C.coum extract showed insignificant cytotoxic effect against HT-29 human cancer cell line after 48 hours and 72 hours incubation. Discussion and It can be concluded that n-butanolic C.coum extract in combination with ciprofloxacin significantly revealed antibiofilm activity against P. aeruginosa biofilm however, further clinical investigations are required.

Phosphorus is one of the most essential macroelements for bacterial cells. Since phosphate (PO4-3) limitation is frequently encountered in soils, bacteria developed some mechanisms in response to this sever condition. Phosphate transporter (PstS) and proteins involved in quorum sensing (QS) signaling pathway are affected by mediating PhoB, response regulator, following phosphate starvation. QS system of Sinorhizobium meliloti composed of at least three genes of sinI (autoinducer synthase), sinR and expR (autoinducer activated receptor) which involved in its free living and symbiotic functions. The optical density (OD600) of different S. meliloti transformed strains carrying pLK004 (a pstS promoter-egfp fusion), pLK64 (a sinI promoter-egfp fusion), pLK65 (a sinR promoter-egfp fusion), pLK66 (an expR promoter-egfp fusion) and control (promoterless-egfp fusion) plasmids were read under different phosphate concentrations of 0.1 (phosphate deficiency), 0.5 and 2 mM (sufficient phosphate) at several time points of 16, 24 and 40h. The promoter activity of different genes of pstS, sinI, sinR and expR were measured as emitted fluorescence per bacterial cell density (OD600) under different phosphate concentrations. By reducing phosphate concentration in the medium, the growth rate of transformed bacteria decreased, especially at 40h. The promoter activity of pstS, sinI and sinR, but not expR, genes was activated following phosphate starvation. Discussion and S. meliloti can upregulate PstS to partly compensate phosphate deficiency in the environment. The gene of sinR is also activated in a PhoB dependent manner as phosphate starvation is encountered. SinR is the activator of sinI, so the upregulation of QS pathway under phosphate deficiency may be facilitate free living and symbiotic bacterial functions.

Candida albicans has been isolated in up to 65% of healthy individuals without signs of clinical disease. Mouthwashes can be used for many preventative and therapeutic purposes. This study evaluates the effectiveness of Iranian and foreign mouthwashes against C.albicans as a common fungal flora in the mouth. In this research, standard strain of C.albicans, ATCC 10231 is used. The suspension is provided by a fresh culture of C.albicans (24 hours) and the OD is read in 530 nm. The C. albicans suspension was cultured on Sabouraud dextrose agar plate. In the next step, two wells created on SDA, filling with mouthwashes (100 µl). After incubation at 37ºC for 24 hours, the inhibition zone was measured. Minimum inhibitory concentration (MIC) and Minimum fungicidal concentration (MFC) of mouthwashes were determined. The data are analyzed by SPSS software, independent T-tests and one sided variance analysis (ANOVA-one way). Findings of our study showed in which agar diffusion, MIC and MFC tests, there are no significant differences between the antifungal effect of the Iranian and foreign mouthwashes on C.albicans (P value>0.05). Discussion and This study shows that both Iranian and foreign mouthwashes have a good effect against C.albicans, as a common fungal flora in the mouth.

This study was to determine the prevalence of verotoxigenic E.coli and their virulence factors isolated from coldwater fishes (Rainbow trout) using multiplex PCR. A total of 100 samples of feces and skins were collected at the final step of production in the production pools. Bacteriological and biochemical examination were done for isolation and confirmation that the isolates belonged to the E.coli species. Multiplex PCR were used to identifying the Stx1, Stx2, eae and hly genes. The results showed that none of the isolates was E.coli O157: H7, but other non- O157 verotoxigenic strains were isolated. Totally 14% of the skin isolates and 4% of fecal isolates contain Stx1, Stx2, eae genes. Discussion and The results of this study revealed that the Rainbow trout meat may play a role as a vehicle to transmit the verotoxigenic E.coli to human. Water may contaminate with feces of animals, birds, contaminated fish foods and workers. Controlling the contaminating ones is recommended.

Biomass and biochemical composition content (particular fatty acid profile, protein, Pigment) play an important role in commercial scale production of microorganisms. Nitrogen concentration is the most effective factor in biochemical composition and growth rate changes. Materials and method s: The unicellular fresh water microalgae, Desmodesmus cuneatus (Scenedesmaceae, Chlorophyta), was grown on BM medium under various concentration of nitrogen at 25°C and pH 7.0in a period of 12 days. Daily cell counting was done using Neubauer haemocytometer to determine the growth rate, cell density and exponential time. The grown cells were harvested in the stationary phase to determine fatty acid and protein content. The maximum growth rate (0.35 ± 0.09 day−1) and cell density (36.00 ± 1.01 ×105 cell.ml-1) was achieved in 2.5 mM of nitrogen concentration. The maximum level of PUFA, was 25.31 % of the total fatty acids under N-sufficient conditions (5 mM nitrogen) compared to 17.01 % under N-free. The maximum precentage of protein content (36.81 %) was found at 5 mM nitrogen content and minimum (11.14 %) was seen at nitrogen free medium. Discussion and The results showed that control of nutrients plays an important role in microalgae culture. Any increase or decrease, depending on the type of nutrient, causes metabolic changes as well as changes in physiology and the nutritional value of microalgae. The nitrogen concentration of 2.5 mM may be more beneficial than other concentrations, as cell number is sustained in exponential phase longer.

One of the most important resistance determinants in Enterobacteriaceae are extended spectrum β-lactamases (ESBLs). During the last decade, CTX-M types ESBLs have increased considerably and become the most common ESBLs worldwide which are the major causes of the urinary tract infections (UTIs). The aim of this study was to determine the antibiotic resistance patterns and the frequency of the CTX-M β-lactamases among multi-drug resistant (MDR) Escherichia coli (E. coli) isolates from northern Iranian patients with UTI. Thirty three E. coli isolates from urine samples were applied in this study. Double disk synergy test (DDST) was applied for identification of ESBL phenotypes in E. coli isolates. The ESBL related genes, CTX-M group (1, 2, 8 and 9), were amplified by polymerase chain reaction (PCR). All E. coli isolates showed sensitivity to piperacillin and 55% of the isolates were resistant to 3rd and 4th cephalosporins. The presence of the blaCTX-M gene in 88% of the ESBL producing isolates was approved based on molecular method. CTX-M (1, 2, 8 and 9) containing E. coli isolates showed resistance to more antibiotics than non-CTX-M isolates. CTX-M-1 was the most prevalent CTX-M determinant in ESBL producing E. coli isolates. Discussion and Based on the results of the present study, the preferred antibiotic against CTX-M type ESBL-producing E. coli strains in north of Iran, Rasht, should be piperacillin. Although, CTX-M type ESBLs prevalence was nearly low in the studied MDR E. coli isolates, but controlling these low prevalence isolates is important.

In the present study, the inhibitory effect of nisin-producing Lactococcus lactis during co-culture and pure standard nisin were assessed against selected foodborne pathogenes in growth medium and Iranian UF Feta cheese. In comparison L lactis, not only proves flavor but also plays a better role in microbial quality of Iranian UF Feta cheese as a model of fermented dairy products. L. lactis subsp. lactis as nisin producer strain, Listeria monocytogenes, Escherichia coli and Staphylococcus aureus as pathogenic strains were inoculated in Ultra-Filtered Feta cheese. Growth curve of bacterial strains were studied by colony count method in growth medium and UF Feta cheese separately and during co-culture with L. lactis. Nisin production was determined by agar diffusion assay method against susceptible test strain and confirmed by RP-HPLC analysis method. Counts of L. monocytogenes decreased in cheese sample containing L. lactis and standard nisin, to 103 CFU/g after 7 days and it reached to undetectable level within 2 weeks. S. aureus counts remained at its initial number, 105 CFU/g, after 7 days then decreased to 104 CFU/g on day 14 and it was not detectable on day 28. E. coli numbers increased in both treatments after 7 days and then decreased to 104 CFU/g after 28 days. Despite the increasing number of E. coli in growth medium containing nisin, due to the synergistic effect of nisin and other metabolites produced by Lactococcus lactis and starter cultures, the number of E. coli decreased with slow rate. Discussion and The results showed, L. monocytogenes was inhibited by L. lactis before entering the logarithmic phase during co-culture. S. aureus was also inhibited during co-culture, but it showed less sensitivity in comparison with L. monocytogenes. However, the number of E. coli remained steady in co-culture with L. lactis. Also, we found that, in all cheese samples, E. coli decreased slowly after 28 days which may be due to the synergistic inhibitory effects of nisin and other metabolites produced by L. lactis and starter culture strains. These conditions are compatible to UF Feta cheese making processes. The usage of L. lactis is more effective in terms of pathogenic inhibitory in comparison with free nisin. Using L. lactis as an adjunct starter culture can assist microbial quality improvement and prevent important pathogens, which may survive during food processing, because of the production of beneficial metabolites.

Environmental contaminations due to petrochemical plastic usage have forced researchers to search new biological methods for biodegradable polymer production. The aim of this study was to find native PHA producing bacteria from Abadan oil refinery in order to be used in biodegradable polymer production studies. For this purpose soil samples were harvested from oil sludge contaminated soil of Abadan refinery. After primary enrichment, screening of PHA producing bacteria was done by PHA- Detection agar and was confirmed by Sudan black and Nile Blue A staining methods. These isolates were identified based on phenotypic methods and sequencing of 16s rRNA. Polymer extraction was performed and optimized using different concentrations of HClO and SDS. As a result of this study 26 different bacterial isolates were obtained from which 17 isolates were PHA producer with different potentiality. Based on the polymer accumulation 4 isolates were selected for further studies. The efficiency of PHA production in these isolates was 75.53±5.08, 82±19.05, 81.06±6.92 and 79.86±11.84%. Based on sequence analysis in NCBI database, these isolates were identified as Bacillus cereus. Discussion and With respect to the results of this study it can be suggested that oil contaminated soils due to high C/N and C/P ratios and also different carbohydrate contents are suitable candidates for PHA producer bacteria isolation. So the native strains in such habitats with high carbon content can be optimized for industrial polymer production.