دو ماهنامه میکروب شناسی پزشکی ایران
سال چهارم شماره 4 (زمستان 1389)

Nonfermenting Gram-Negative Bacteria (NFGNB) are opportunistic pathogens responsible for many nosocomial infections. The most prevalent NFGNB species are Pseudomonas aeruginosa, Acinetobacter baumannii, Stenotrophomonas maltophilia. These bacteria expressed high level of resistance to antibiotics usually through the most general mechanism of resistance by production of Extended-Spectrum β-lactamases (ESBLs). The present study was conducted to determine the prevalence rate of NFGNB, their antibiotic resistances and ESBLs production during 2007-2008 in Kerman. A total of 110 NFGNB isolates from hospitalized patients and outpatients (Afzali Poor, Bahonar, and Shafa hospitals) were collected during 2007-2008 in Kerman. The isolates were identified by biochemical methods as 93 isolates (84.5%) of Pseudomonas aeruginosa, 11 isolates (10%) of Stenotrophomonas maltophilia and 6 isolates (5.5%) of Acinetobacter baumannii. The minimum inhibitory concentrations (MICs) of the isolates to 11 common antibiotics were determined using agar dilution method. These isolates are capable of producing β-lactamase, and they were determined using nitrocefin disks. The ESBL producing isolates were detected by DCDT method (double disk test & combined disk test). P. aeruginosa was the most common isolate among all isolated NFGNB. Resistance to cefotaxime, ceftizoxime, nalidixic acid, amoxicillin, cephalexin, for all the isolates was very high (74-100%), whereas the lowest rate of resistance was observed against imipenem, ceftazidime and ciprofloxacin with the rate of 0.9%, 13.6% and 24.5% respectively. β-lactamase production was positive for 50 (45.5%) of the isolate and 47.2% of the isolate were ESBL producers. Antibiotic resistance, β-lactamase and ESBL production were more common in Acinetobacter baumannii isolates. NFGNB, especially Acinetobacter baumannii, showed high antibiotic resistance to common drugs. Ceftazidime and imipenem are still useful antibiotics and they can be used for the treatment of infections caused by these bacteria.

Listeria monocytogenes is a gram-positive, facultatively intracellular, non spore forming bacillus and enters a human body after ingestion of contaminated foods such as milk products, meats and vegetables. Epidemiological studies were shown that this bacterium is the cause of abortion and fetal abnormalities in a human. Although the immunological aspect of listeriosis was much studied experimentally during pregnancy, it needs to be studied from the viewpoints of bacteriology and embryology as well. In this study, haploid mice, the sensitive model to listeriosis was used to evaluate abortion and fetal abnormalities caused by experimental listeria infection. Unlethal dose of different serotypes (1/2a, 1/2b,4ab, 4a, 4c, 4d) of L. monocytogenes was determined in mice. Seventy inbred BALB/c gestating pregnant mice were divided in two groups as a control and a test group. In control group, mice were injected intraperitoneally with normal saline and in test group; mice were injected with 200µl of 1/2 logCFU/ ml L. monocytogenes. On day zero of pregnancy to 30, a few numbers of mice were randomly selected to remove their uterus after sedation. The position and number of placenta were checked and counted. They were compared with the position and number of fetuses in each uterus to determine the number of abortions. Remaining pregnant mice were delivered by cesarean section on day 24 of pregnancy to determine contaminated organs, size of organs, abortion numbers, embryo weights, embryo abnormalities during the first hour of delivery. Groups comparison was made by Post Hoc. Injection of different serotypes of L.monocytogenes in equal concentrations showed no death in pregnant mice in both test and control groups. There was a significant difference (P≤ 0.05) in abortion rates between test and control groups that were injected with suspension of L.monocytogenes strain 4b. There were also no significant differences in weight and height of fetuses in both groups. No fetal abnormalities were observed in heads, number of fingers and size of the organs in the embryos of all mice groups. No significant differences in weights and heights in the embryos of pregnant mice were observed. In both test and control groups the abortion rates were found to be significantly different.

Ureaplasma urealyticum is an opportunistic bacteria which is able to cause some human diseases, such as: urethritis, abortion, respiratory dysfunction syndrome in infants and infertility. Culture of clinical specimen is the main diagnosis method of U. urealyticum which is not normally done in clinical laboratories due to unreasonable costs. One of the important reasons for difficulty and costly of U. urealyticum culture is presence of normal horse serum in its media as a source of cholesterol. In this study, it was tried to reduce the rate of expenses and difficulty of U. urealyticum cultivation with proposing an inexpensive and suitable complement instead of horse serum. In this research dry milk was examined as an alternative complement.

Vaginal specimen sampling and cultivation on PPLO broth and PPLO agar was used for isolation U. urealyticum. Different concentrations of dry milk (20, 10, 5, 2/5, 1/25%) was added into PPLO Broth medium before autoclaving. PCR was used for confirmation of presence of U. urealyticum in vaginal specimen (and also confirmation of growth of U. urealyticum on media with dry milk) as well as attention to colony shape and using of MnCl2.

This study showed that U. urealyticum can grow on a media with dry milk as a cholesterol source complement. Media containing less concentration of dry milk showed more to provide U. urealyticum growth. The maximum growth was found in a medium with 1.25% of dry milk while a medium with 20% dry milk did not show any growth of U. urealyticum.

U. urealyticum can grow on media that horse serum is replaced by dry milk as a cholestrol source.

High mutation rate and immunosuppressive effect of some key proteins of hepatitis C virus (HCV) encouraged researchers to find conserved and small immunogenic units to induce immune responses. Thus epitope-based vaccines were introduced to find out any potential immunogens. However, the relatively weak immune responses against these vaccines require a creation of additional measures. This study aimed to construct plasmids carrying CTL epitope of hepatitis C virus using immunoinformatics modeling method and assessment of their preliminary immunogenicity. One H-2Dd and two HLA-A*0201-restricted CD8+ T-cell epitopes from E2, E1 and core regions of HCV were selected and immunoinformatically analyzed for optimum sequentiality. The multiepitope sequence was constructed by SOEing PCR and cloned in pcDNA3.1+ vector. PADRE, endoplasmic reticulum signal sequence (ERss) and hepatitis B surface antigen (HBsAg) immuno-enhancer sequences were fused to the 5′ end of multiepitope constructs. In vitro, expression of each plasmid was analyzed by RT-PCR, dot-blot, Western-blot and immunofluorescence techniques. Preliminary in vivo immunogenicity of the construct was assessed by delayed-type hypersensitivity (DTH) response in BALB/c mice. In vitro, analyses confirmed the expression of plasmids. Preliminary, in vivo assessments indicated the processing and presentation of the H-2Dd epitope in BALB/c mice. Moreover, although the immunostimulation effects of ERss and PADRE were shown, fusion of HBsAg did not enhance the DTH response. This study shows the value of immunoinformatics prediction of hepatitis C virus epitopes as a multiepitope vaccine candidate and DTH assay for preliminary analysis using BALB/c mice. Data obtained, provide enough support for further evaluation of the designed constructs in HLA-A2 transgenic mice.

In the present decade, the term ‘scientific production’ was considered as an important topic. The aim of this study was to investigate current international situation of Iranian researchers in parasitology field. This scientometric study was performed using bibliographic records from the ISI databases during a period of 1980-2009 related to Iranian parasitology researches. Out of 72,229 papers written by Iranian scientists during 1980-2009, a total of 392 articles (0.54 %) were in the domain of parasitology. Some of these articles were collaborative works and some were non-collaborative. Iranian parasitologist had many collaborative works with their counterparts in United Kingdom (U.K). Mohebali as one of the Iranian parasitologist, with 26 articles was the most productive scientist and Tehran university of medical sciences with 114 records (29.08%) was the most productive institution in the field of parasitology. Our results indicate that the trend of scientific researches and papers on the base of parasitology works were increased significantly since 2008.

Fatty acids and their derivatives were found in many plant and animal products. The aim of this study was to investigate the inhibitory effects of fatty acids (lauric acid, capric acid, palmitoleic acid and oleic acid) and its monoglycerids (monolaurin, monocaprin, monopalmitolein and monolein) derivatives on E.coli O157:H7. Antibacterial efficiency of the agents was determined according to the broth dilution test in BHI broth. Among with bacterial inoculum, appropriate quantities of each agent were added to BHI broth to give final concentrations of 0.16, 0.31, 0.63, 1.25 and 2.5 Mm.Then samples were incubated at 37°C. Control samples consisted of inoculated broth without fatty acids and their monoglycerids. Data were analyzed by ANOVA test. The results showed that capric acid and its monoglycerid in comparison with other fatty acids showed significantly higher inhibitory effect on E. coli (P<0.05). The antibacterial effects of capric acid were marked at 0.63 Mm and higher dilution. Fatty acids and their monoglycerids, especially capric acid and its monoglicerid have antibacterial effects on strain O157: H7. The effectiveness is directly correlated with capric acid concentration.

In addition to the use of chemicals in food, pharmaceutical, cosmetic and hygiene industries, use of herbal and natural compounds have also increased. It seems natural ingredients compared with the chemical components show less side effects, such as mutagenicity and carcinogenesis; however, their use can also be associated with side effects and toxicity. Some significant medicinal effects of saffron have been proven, which reveals the necessity to investigate the toxicity of the plant. This study aimed to evaluates the cytotoxic effects of saffron on cell lines Vero, HeLa, and Hep2 using MTT assay method. Firstly the stigma of saffron was extracted by methanol: water 80:20 using maceration method. Cytotoxic effects of different dilutions of saffron extract on Vero normal cell lines and HeLa and Hep2 cancerous cell lines was investigated using MTT assay method in a 96 well microplate. Reading optical density (OD) was done by microplate reader apparatus and IC50 (Inhibition Concentration 50%) was calculated for each cell line. Aqueous - methanolic saffron extract on cell lines Vero, HeLa, and Hep2, showed IC50 equal 162, 653 and 208 μg/ml respectively. Saffron has a toxic effect on normal Vero cell line. Therefore, must be cautious to not to use it in food and as herbal medicine in high concentrations. Also the extract of this plant revealed toxic effects on two cancerous cell lines, Hepa2 and Hela. However, its toxicity, especially on HeLa cancerous cell line was lower than the Vero normal cell line.

Survey on bacterial contamination is one of the most important ways for hygienic evaluation of meats at different points of processing and marketing. In the last decade application of ozone as an antiseptic agent was proposed in foods (meats, vegetables, fruits, etc.) processing. The aim of this study was to evaluate the effects of ozone gas for reduction of bacterial contamination of poultry meats at an industrial slaughterhouse in Tehran.

In this study, 210 whole chicken carcasses in 7 groups (one control group and 6 test groups) randomly were selected and exposed to ozone gas with various concentration of 4, 6, and 8 ppm, and exposure time of 4 and 10 minutes. Standard methods were used for total bacterial count and detection of Salmonella in the samples. Data were analyzed using descriptive statistical indices and ANOVAs test.

The results showed that the mean of total number of bacteria in the control group was 1.02×105 cfu/g while in the test groups with two exposure times of 4 and 10 minutes consisting of groups; with the gas concentration of 4 ppm were 9.14×104 and 1.76×104, with the gas concentration of 6 ppm were 3.51×104 and 1.15×103 and with the gas concentration of 8 ppm were 5.32×102 and 1.06×102 cfu/g, respectively. There was no Salmonella bacterium in the samples of the test groups of 8 ppm gas concentration with both exposure times of 4 and 10 minutes.

The results of this study are indicated that application of ozone as an antiseptic agent on poultry carcasses at slaughterhouse has a proper positive effect on microbial load reduction of the meats.