Iranian Journal of Microbiology
Volume:5 Issue: 2, Jun 2013

Recently, nosocomial infections have been discussed as a critical issue among intubated patients leading to significant morbidity and mortality. Hence, the pattern of microbiological colonization and antibiotic resistance are much valuable in this regard. We aimed to investigate the pattern of microorganism colonization and antibiotic resistance in patients with endotracheal tube or tracheostomy to propose a proper empirical antibiotic therapy in this setting. This cross sectional study was conducted among 880 patients admitted in Imam Khomeini hospital between 2008 and 2011 who were subsequently intubated or underwent tracheostomy due to insufficient self ventilation. Samples for microbiological cultures were obtained after extubation and then sent to the central laboratory for further assessment. Antibiograms and microbiological cultures were obtained for each sample. Of 880 patients enrolled in this study, 531 (60.3%) were male and 349 (39.7%) were female. Nineteen different organisms were isolated including Acinetobacter (213, 24.2%), Pseudomonas aeruginosa (147, 16.7%), Staphylococcus aureus (106, 12%), Proteus mirabilis (90, 10.2%), and other organisms (324, 36.8%). Antibiotic resistance was mainly seen in Acinetobacter (ciprofloxacin, ceftazidim, cefepim, and penicillin), S. aureus (imipenem) and Klebsiella (pipracillin-tazobactam and ampicillin-sulbactam). This study presents the most common microorganisms colonizing tracheal tube of hospitalized patients and their pattern of antibiotic resistance. Acinetobacter was the most common microorganism isolated from endotracheal tube. Hence, it may be possible to initiate the empiric antibiotic treatment before the results of culture are ready. Ciprofloxacin was also the most prevalent antibiotic revealing resistant pattern. Moreover, most of the microorganisms were sensitive to imipenem and pipracillin-tazobactam.

The emergence of extended-spectrum β-lactamase (ESBL)-producing Shigella spp. is of increasing clinical concern specially in children worldwide. The aim of this study was to investigate the occurrence of extended-spectrum β-lactamase producing Shigella spp. in Tehran, IranThe study includedall Shigella isolates recovered from pediatric patients aged less than 12 years admitted to a major pediatric hospital in Tehran, Iran, from 2008 to 2010. Bacterial identification, antimicrobial susceptibility testing, extended spectrum β-lactamases (ESBLs) screening and confirmatory testswere performed according to the standard guidelines. Conjugal transfer experiments and plasmid analysis were also carried out. Polymerase chain reaction and sequencing were used to identify the genetic determinants responsible for ESBL production. Fourout of 55Shigella isolates,includingthreeS.sonnei and one S. flexneri, showed an ESBL-positive phenotype.Plasmid transfer of the ESBL phenotype was successful for the S. flexneri isolate only. By PCR and sequencing, one S. sonneiisolatetested positive for the CMY-59 gene, while the other two S. sonneiand the S. flexneriisolatestested positive for the blaTEM-1 and blaCTX-M-15 genes. We found the prevalence of ESBL producing Shigella isolates was higher than detection rates observed in many other countries. Our finding raise concerns about the dissemination of ESBL among the strains of endemic S. sonnei throughout the country, because this species is now the most frequently isolated Shigella species in Iran and shigellosis by such strains in the community can pose a significant threat to patients and presents a challenge for disease management.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 has been recognized as a major food borne pathogen responsible for frequent hemorrhagic colitis and hemolytic uremic syndrome in humans. Cattle are important reservoirs of E. coli O157:H7, in which the organism colonizes the intestinal tract and is shed in the feces. Vaccination of cattle has significant potential as a pre-harvest intervention strategy for E. coli O157:H7. The aim of this study was to evaluate active and passive immunization against E. coli O157:H7 using a recombinant protein. The recombinant FepA protein induced by IPTG was purified by nickel affinity chromatography. Antibody titre was determined by ELISA in FepA immunized rabbits sera. Sera collected from vaccinated animals were used for bacterial challenge in passive immunization studies. The results demonstrate that passive immunization with serum raised against FepA protects rabbits from subsequent infection. Significant recognition by the antibody of ferric enterobactin binding protein may lead to its application in the restriction of Enterobacteriaceae propagation.

Biofilms are structural communities of bacterial cells enshrined in a self produced polymeric matrix. The studies on biofilm formation of Pasteurella multocida have become imperative since it is a respiratory pathogen and its biofilm mode could possibly be one of its virulence factors for survival inside a host. The present study describes a biofilm assay for P. multocida on inert hydrophilic material called bentonite clay. The potential of the organism to form in vitro biofilm was assessed by growing the organism under nutrient restriction along with the inert substrate bentonite clay, which will provide a surface for attachment. For quantification of biofilm, plate count by the spread plate method was employed. Capsule production of the attached bacteria was demonstrated by light microscopic examination following Maneval staining and capsular polysaccharide estimation was done using standard procedures. Results and The biofilm formation peaked on the third day of incubation (1.54 ×106 cfu/g of bentonite clay) while the planktonic cells were found to be at a maximum on day one post inoculation (8.10 ×108 cfu/ml of the broth). Maneval staining of late logarithmic phase biofilm cultures revealed large aggregates of bacterial cells, bacteria appearing as chains or as a meshwork. The capsular polysaccharide estimation of biofilm cells revealed a 3.25 times increase over the planktonic bacteria. The biofilm cells cultured on solid media also produced some exclusive colony morphotypes.

The Escherichia coli strains are greatly important in nosocomial and community acquired infections. The aim of this study was to determine the transmission of bacterial infections using genetic analysis. Two hundred and thirty Escherichia coli strains, isolated from different clinical samples, were characterized by enterobacterial repetitive intergenic consensus (ERIC)–PCR technique. The results and the similarity between the strains were determined on the basis of Jaccard similarity coefficient in the SAHN program of the NTSYS-pc software.The ERIC–PCR profiles allowed typing of the 230 isolates into 205 ERIC-types which were grouped into twenty main clusters (C01–C20).The first group makes up 4.34% of the total isolates. Out of the 230 isolates, 34.2% belonged to D phylogenic group which were associated with extra-intestinal samples. Our results showed high diversity in E. coli isolates indicating low rate of hospital infection in our university hospitals. The majority of the isolates belonged to the D phylogenic group.

Childhood infectious diseases are one of the most known environmental pathogenic causesof childhood asthma. The high prevalence of both Helicobacter pylori infection and asthma in our country prompted us to assess anyprobable association between them in childhood. This cross-sectionalstudy recruited 196 children aged 6 to 12 years old comprising 98 asthmatic (case group) and 98 healthy (control group) individuals. Urea breath test was performed for all of the children and H. pyloriinfection was compared between the two groups according to the urea breath test results.Urea breath test was positive in 18 asthmatic (18.36) and 23 (23.36) healthy subjects but was not significantly different between the case and controls(p=0.380).Furtheranalysis in the asthmatic group revealed association ofH. pyloriinfection withage (p<0.001) and duration of asthma (p=0.010). However, no significant correlation was found between sex, severity of asthma, controledasthma or abnormal pulmonary function testswith H. pyloriinfection (p= 0.804, 0.512, 0.854 and 0.292, respectively).Given the results of the study, H. pylori infection was not significantly differentbetween asthmatic and healthy children.In asthmatic patients, there wasnosignificant association between H.pyloriinfection andsex,severity of disease, control status of disease andnormal or abnormal pulmonary function tests.H. Pylori infection had a significant association withincreasing age and duration of asthma.

Entomopathogenic nematodes, belonging to the family heterorhabditis and steinernematidae, are reported to be symbiotically associated with specific bacteria and the secondary metabolites produced by these bacteria possess antimicrobial activity. In this study, bacteria were isolated from nematodes belonging to the family rhabditidae, and the antimicrobial activity was tested against four bacteria viz. Bacillus subtilis MTCC 2756, Staphylococcus aureus MTCC 902, Escherichia coli MTCC 2622, and Pseudomonas aeruginosa MTCC 2642 and five fungi viz. Aspergillus flavus MTCC 183, Candida albicans MTCC 277, Fusarium oxysporum MTCC 284, Rhizoctonia solani MTCC 4634 and Penicillium expansum MTCC 2006. The isolated bacteria was cultured in nutrient broth (NB), Luria broth (LB) and Tryptic soya broth (TSB) at 25, 30 and 35 ºC. Cell free culture filtrate was prepared by centrifugation and was separated into organic and aqueous fractions. Organic fraction was concentrated and tested for antimicrobial activity. The culture filtrate of the bacteria isolated from the entomopathogenic Rhabditis sp. was found to possess antimicrobial activity against the four bacteria and five fungi tested. The bacterium grew well in TSB, LB and NB media though in TSB yield and activity were higher. Antimicrobial activity was higher at 30ºC as compared with 25 or 35ºC. HPLC analysis indicated major differences in peak areas and retention times at different temperatures. Increased number of peaks with higher peak areas was obtained at 30ºC. The study suggests that the bacteria could produce more bioactive molecules effective against medically and agriculturally important bacteria and fungi depending on culture media and temperature. Modified media could yield different types of molecules effective against diseases/disorders of plant, animals and humans.

Helicobacter pylori is the causative agent of peptic ulcer disease and a co-factor in development of gastric malignancies. LPS are among toxic substances produced by H. pylori exhibiting low endotoxic activity compared to typical bacterial LPS. The aim of this study was to investigate bioactivity of LPS produced by different serotypes of Helicobacter pylori compared to Escherichia coli and Brucella abortus LPS. Bacterial LPS was extracted by the hot phenol-water method. Biological activities of LPS were determined via the limulus lysate assay, pyrogenic assay, and blood pressure and PBMC induction test in rabbits. Biological activity of O2 serotype LPS of H. pylori was less than the biological activity of other H. pylori serotypes. Our data supported the hypothesis that the unique bacterial LPS of the O2 serotype must be included in the formulation of a multivalent H. pylori vaccine.

This paper describes chemical composition and antimicrobial activity of Cymbopogon nardus citronella essential oil against Edwardsiella spp. (n = 21), Vibrio spp. (n = 6), Aeromonas spp. (n = 2), Escherichia coli (n = 2), Salmonella spp. (n = 2), Flavobacterium spp. (n = 1), Pseudomonas spp. (n = 1) and Streptococcus spp. (n = 1) isolated from internal organs of aquatic animals. Due to the ban of antibiotics for aquaculture use, this study was carried out to evaluate the potential of citronella essential oil as alternative to commercial antibiotic use against systemic bacteria in cultured aquatic animals. The essential oil of C. nardus was prepared by using the steam distillation method and the chemical composition of the essential oil was analyzed by gas chromatography–mass spectroscopy (GC–MS). Minimum inhibitory concentration (MIC) of the essential oil tested against bacterial isolates from various aquatic animals and ATCC type strains were determined using two-fold broth micro dilution method with kanamycin and eugenol as positive controls. A total of 22 chemical compounds were detected in C. nardus essential oil with 6-octenal, 3, 7-dimethyl- or citronellal representing the major compounds (29.6%). The MIC values of the citronella oil ranged from 0.244 µg/ml to 0.977 µg/ml when tested against the bacterial isolates. The results of the present study revealed the potential of C. nardus essential oil as alternative to commercial antibiotics for aquaculture use.

Sodium dodecyl sulfate (SDS) is one of the main surfactant components in detergents and cosmetics, used in high amounts as a detergent in products such as shampoos, car wash soap and toothpaste. Therefore, its bioremediation by suitable microorganisms is important. Alkylsulfatase is an enzyme that hydrolyses sulfate -ester bonds to give inorganic sulfate and alcohol. The purpose of this study was to isolate SDS–degrading bacteria from Tehran city car wash wastewater, study bacterial alkylsulfatase enzyme activity and identify the alkylsulfatase enzyme coding gene. Screening of SDS-degrading bacteria was carried out on basal salt medium containing SDS as the sole source of carbon. Amount of SDS degraded was assayed by methylene blue active substance (MBAS).Results and Identification of the sdsA gene was carried by PCR and subsequent sequencing of the 16S rDNA gene and biochemical tests identified Pseudomonas aeruginosa. This bacterium is able to degrade 84% of SDS after four days incubation. Bacteria isolated from car wash wastewater were shown to carry the sdsA gene (670bp) and the alkylsulfatase enzyme specific activity expressed from this gene was determined to be 24.3 unit/mg. The results presented in this research indicate that Pseudomonas aeruginosa is a suitable candidate for SDS biodegradation.

Thermophilic bacteria are less studied but important group of microorganisms due to their ability to produce industrial enzymes. In this study, thermophilic bacteria were isolated from hot spring of Tarabalo, India. A bacterium that could tolerate high temperatures was characterized by morphology, biochemistry and sequencing of its 16S rRNA gene. The isolate was screened for protease and amylase activity. Phylogenetic affiliations and G+C content of the isolate was studied. The bacterium with the ability to tolerate high temperatures was identified as Bacillus sp. both by morphology, biochemistry and sequencing of its 16S rRNA gene. BLAST search analysis of the sequence showed maximum identity with Bacillus amyloliquefaciens (99% similarity). Strain exhibited considerable protease activity. Phylogenetic analysis of the isolate revealed close affiliation with thermophilic Bacillus species. The G+C content was found to be 54.7%. The study confirmed that the isolated Bacillus sp. to be a true thermophile and could be a source of thermostable protease which can be exploited for pharmaceutical and industrials applications.

Virosomes are Virus Like Particles (VLP) assembled in-vitro. Influenza virosomes maintain the cell binding and membrane fusion activity of the wild type virus but are devoid of viral genetic material or internal proteins. Influenza virosomes mimic the natural antigen presentation route of the influenza virus. Virosomes were prepared by membrane solubilization and reconstitution. Briefly, the Madine-Darby Canine kidney (MDCK) cell line was cultivated on microcarrier beads inoculated with influenza virus strain A/X-47 (H3N2). The culture medium was harvested and clarified. Subsequently, virus was concentrated and purified by ultrafiltration and ultracentrifugation. The purified viral membrane was dissolved by adding 375 μl of 200 mM 1, 2-dicaproyl-sn-glycero-3-phosphocholine (DCPC) in HEPES-buffered saline (HBS). Nucleocapsid was removed by ultracentrifugation. The supernatant consisting of phospholipids and glycoproteins of the influenza virus was reconstituted by removal of DCPC using overnight dialysis against Hank''s Buffered Saline (HBS) solution at 4°C. After dialysis, crude virosome preparation was layered over a discontinuous sucrose gradient in order to separate non-incorporated material from the reconstituted virus membranes. The virosome harvested from the boundary of the two sucrose layers successfully was identified by the Hemagglutination assay and western blotting. Use of a dialyzable short-chain phospholipid (DCPC) is an efficient procedure for solubilization and reconstitution of influenza virus virosomes and has not caused structural changes in a major envelope glycoprotein (hemagglutinin protein) on the surface of virosome.

Hepatitis E virus (HEV) is a major public health concern in developing countries. HEV transmission occurs primarily by the fecal-oral route. It has also been reported that blood donors are potentially able to cause transfusion-associated hepatitis E in endemic areas. This study aimed to determine the seroprevalence of HEV infection among volunteer blood donors in Central province of Iran in 2012. A total of 530 consecutive blood donor samples collected from Blood Transfusion Organization, Central Province of Iran. All samples were tested for the presence of IgG Hepatitis E antibody (anti-HEV) using enzyme-linked immunosorbent assay (ELISA). From 530 blood donors, 91.9% were male and 8.1% were female. Overall, anti-HEV was found in 76 of 530 samples (14.3%). There was no significant difference in HEV seropositivity between the subjects regarding gender and area of residence (urban vs. rural). Anti-HEV was distributed among all age groups. Although people aged 31-50 years had the highest prevalence, but there was no statistical difference between the age groups. This study shows a relatively high prevalence of anti-HEV in the blood donors of Central province of Iran. More investigations are needed to assess the potential benefit of adding HEV screening of blood products to the current blood donor selection criteria.

Current chemotherapies of cutaneous leihmaniasis have faced to some problems and limitations; development of new anti-leishmanicidal drugs from different sources like herbal plants, are crucially important. The objective of the present study was evaluation of in vitro activity of Alkanna frigida extracts in comparison with glucantime against Leishmania major. L. major promastigotes were exposed to different concentrations of the A. frigida extracts, processed by ethyl acetate, ethanol, hexane and chloroform. The inhibitory effect, as the IC50, were calculated after 24, 48 and 72 hours by linear regression analysis values of the concentrations employed. The significant inhibition was observed after 24 and 48 hours with different concentrations of compounds (p<0.05 in all tests). All extracts had potent activity against proliferation of the promastigotes, comparing to the untreated negative control. It could compete with the glucantime efficacy in some concentrations. Ethyl acetate and ethanol extractions showed potent IC50 value, 106 µg/ml and 86 µg/ml, respectively. Hexane and chloroform extractions had poor efficacy after 24 hours; however, the efficacy increased after 48 and 72 hours. The results indicated that the A. frigida has appropriate inhibitory effects on the growth of L. major promastigotes in vitro and can be of herbal targets for further investigation in vivo.