Iranian Journal of Microbiology
Volume:2 Issue: 3, Sep 2010

Pseudomonas aeruginosa is one of the most important causative agents of nosocomial infections especially in ICU and burn units. P. aeruginosa infections are normally difficult to eradicate due to acquired resistance to many antibiotics. Recent appearance of carbapenem resistant P. aeruginosa isolates is considered a major healthcare problem. The present study was conducted to detect class 1 integron and antibiotic susceptibility profiles of imipenem-sensitive and resistant clinical isolates of P. aeruginosa. Antibiotic susceptibility profiles and minimum inhibitory concentration against imipenem was studied in 160 clinical isolates of P. aeruginosa by disk agar diffusion method and Etest, respectively. Detection of class 1 integron was performed by the PCR method. Demographic and microbiological data were compared between imipenem susceptible and non-susceptible isolates by the SPSS software. PCR results showed that 90 (56.3%) of P. aeruginosa isolates carried class 1 integron. Antibiotic susceptibility results revealed that 93 (58.1%) were susceptible and 67 (41.9%) were non-susceptible to imipenem. Comparison of antibiotic susceptibility patterns showed high level of drug resistance among imipenem non-susceptible isolates. We found that MDR phenotype, presence of class 1 integron and hospitalization in ICU and burn units were significantly associated with imipenem non-susceptible isolates. The high frequency of imipenem resistance was seen among our P. aeruginosa isolates. Since carbapenems are considered as the last drugs used for treatment of P. aeruginosa infections, it is crucial to screen imipenem non-susceptible isolates in infection control and optimal therapy.

Enterotoxigenic Escherichia coli (ETEC) is one of the most common agents of diarrhea among other bacterial agents. Designing and producing vaccine against these bacteria is one of the major purposes of World Health Organization (WHO). Due to presence of diverse clones of ETEC strains in the world, the use of global vaccines for ETEC infection is controversial. B subunit of heat labile toxin (LTB) was introduced as a vaccine candidate molecule by several investigators. The expression of LTB gene isolated from a local bacterial strain and investigation of its immunological property was the objective of this study. LTB gene was isolated from a local isolated ETEC, cloned and expressed using pET28a expression vector. For LTB gene expression, the three main expression parameters (IPTG concentration, time and temperature of induction) were investigated. The recombinant protein was purified (> 95%) with Ni-NTA column using 6XHis-tag and used as an antigen in ELISA test. The immunological analyses showed production of high titer of specific antibody in immunized mice. Anti LTB Antibody could bind to whole toxin and neutralize the toxin through inhibition of its binding to the Ganglioside M1 receptor. The recombinant LTB protein is a highly immunogenic molecule. Considering the LTB role in ETEC pathogenesis, it can be taken into account as one of the most important components of vaccines against local ETEC.

Dairy cattle have been implicated as principal reservoir of Verotoxin-Producing Escherichia coli (VTEC), with undercooked ground beef and raw milk being the major vehicles of food borne outbreaks. VTEC has been implicated as an etiological agent of individual cases and outbreaks in developed countries. This study was designed to determine the prevalence of VETEC in diarrheic dairy calves up to 20 days of age in Najaf, Iraq. 326 fecal samples from diarrheic calves were collected for isolation of Escherichia coli O157:H7 and non-O157 VTEC isolates. Non-sorbitol fermentation, enterohemolysin phenotype, and slide agglutination with antisera were used for screening and detection of these serotypes. Nineteen (5.8%) non-sorbitol fermenting and 3 (0.9%) enterohemolysin-producing E. coli were obtained. Only 9 were agglutinated with available antisera and none of them belonged to the O157:H7 serotype. Three were found to be verotoxin positive on Vero cell monolayers. These included serotype O111 (2 isolates) and serotype O128 (1 isolate). All three VTEC isolates were resistant to ampicillin and streptomycin. Two exhibited adherence phenotype on HEp-2 cells. E. coli O157:H7 serotype is not prevalent in diarrheic dairy calves, and VTEC is not a frequent cause of diarrhea in calves in Najaf/ Iraq.

Staphylococcus aureusis a one of THE most frequent causes of food poisoning (FP) in dairy products. The main etiologic agents of FP are staphylococcal enterotoxins (SE). There are different types of SE; types A (SEA) and B (SEB) are the most clinically important enterotoxins. Traditional dairy products are still produced in small batches and sold by some vendors without a permit from the Ministry of Health. This study focuses on the molecular and serological detection of enterotoxigenic Staphylococcus aureus SEA and SEB genes and its products, respectively from samples of such traditional products. 100 samples from dairy products were produced under sterile conditions via traditional methods and were transported to the laboratory. The samples were cultured and identified by routine bacteriological methods. The isolated bacteria were evaluated by PCR tests for detection of the genes encoding SEA and SEB. Subsequently, the ability of these strains to produce enterotoxin was examined by Sac's culture method and was confirmed by Sigel Radial Immounodiffussion (SRID). The results indicated that 32% of the dairy products were contaminated by S. aureus (cream 18%, cheese 10%, milk 4%). The PCR results showed that 15.6% of the S. aureus isolates possessed the SEA gene, 9.3% had the SEB gene, and 6.2% possessed both genes. The evaluation of enterotoxin production indicated that 80% of SEA and 33% of SEB genes were expressed. Enterotoxins SEA and SEB are heat stable and consequently; heating has no effect on dairy products contaminated by entertoxins. Subsequently, gastritis may occur within several hours after consumption. Our findings suggest that PCR is a rapid, sensitive, specific, and inexpensive method for detecting SE and can replace the traditional assays.

Methicillin resistant Staphylococcus aureus (MRSA) is a major nosocomial pathogen causing significant morbidity and mortality. The aim of this study was to evaluate in vitro activities of different antibiotics against methicillin resistant Staphylococcus aureus. The study was conducted over a period of one year (January 2009 - December 2009) in the Department of Microbiology, Army Medical College, the National University of Sciences and Technology, Pakistan. One hundred and thirty-nine Methicillin resistant Staphylococcus aureus isolated from the clinical specimens at Rawalpindi Military Hospital were subjected to in vitro susceptibility against various antimicrobials using Kirby Bauer disc diffusion technique. All the isolated MRSA organisms were uniformly susceptible to vancomycin, linezolid and tigecycline. Other drugs which were found to be effective were chloramphenicol, and rifampacin. Most of the MRSA were isolated from pus samples.. Vancomycin, tigecycline and linezolid were effective against methicillin resistant strains of S.aureus. This study suggests that chloramphenicol and rifampacin also have good in vitro efficacy for methicillin resistant S. aureus infections. Oral dosing option for linezolid, chloramphenicol and rifampacin can allow earlier discharge of hospitalized patients and thus reduce health care expenses as well as help reduce the chances of vancomycin resistant strains emergence.

Although members of the Lactobacillus casei group are known to survive under acidic conditions, the underlying mechanisms of growth at acidic condition and the impact of low pH on the relative level of protein expression at the cell surface remain poorly studied. After confirming the taxonomy of L. casei strain GCRL 12 which was originally isolated from cheese and confirmed by 16S rRNA sequence analysis, the impact of acidic pH on growth rate was determined. Late log-phase cells cultured at pH 4.0 showed obvious changes in Gram staining properties while transmission electron microscopy analysis revealed evidence of structural distortions of the cell surface relative to the controls cultured at pH 6.5. When comparing cytosolic or whole cell preparations on SDS-PAGE, few changes in protein profiles were observed under the two growth conditions. However, analysis of surface protein extracted by 5M LiCl demonstrated changes in the proportions of proteins present in the molecular weight range of 10 to 80 kDa, with some proteins more dominant at pH 6.5 and other at pH 4. These data suggest that surface proteins of this strain are associated with growth and survival at low pH. The function of these proteins is subject to further investigation.

Bacillus subtilis HBsu is a 10 kD heat-stable protein shown to be involved in binding to DNA and is encoded by the hbs gene. Large-scale production for biochemical analysis is achieved through cloning and expression of the recombinant protein. This gene was amplified from B. subtilis ATCC 6633 using PCR and cloned into pET28a (+) expression vector. The construct was used to transform Escherichia coli BL21 (DE3). The expression of the protein was induced by the addition of 1mM IPTG. To confirm the expression of the cloned gene, SDS-PAGE was carried out and production of an approximately 11 KD recombinant tagged protein was confirmed for the cloned hbs gene.Results and The identity of the recombinant HBsu was verified and characterized by SDS-PAGE which can then be utilized for further applications.

In the present study, isolation of Streptomyces associated with marine sponges and its bioactive potential against fish and shellfish pathogens were assessed. The Streptomyces sp. were isolated from the marine sponges namely Callyspongia diffusa, Mycale mytilorum, Tedania anhelans and Dysidea fragilis collected from Vizhinjam port, situated in the South-West coast of India. The Streptomyces associated with marine sponges were isolated using specific ISP media. The isolates of Streptomyces were characterized for their colony characteristics, morphological properties, physiological and biochemical properties and were tentatively identified. The strains were cultivated on a lab scale level as shake-flask cultures and the crude extracts of the bioactive compounds obtained with ethyl acetate were screened biologically and chemically. By biological screening, the extracts were analyzed for their activity against fish and shellfish pathogens namely Aeromonas hydrophila, Serratia sp. and Vibrio spp, using the disk and agar-well diffusion bioassay method, while by chemical screening the crude culture extracts were analyzed by TLC and UV-Vis spectrophotometer. Ninety-four isolates were found to be associated with marine sponges, among them only seven strains showed antagonism against fish and shellfish pathogens. Analysis of morphological, physiological and biochemical characteristics suggested that these strains belonged to the genus Streptomyces. The initial screening of the isolates by spot inoculation method exhibited antibacterial activity against Aeromonas hydrophila. In-vitro screening of the submerge culture extracts showed positive inhibition against the fish and shellfish pathogens namely Aeromonas hydrophila, Serratia sp. and Vibrio spp. The screening of bioactive compounds confirmed the production of polyene substances by UV spectrum, which resulted in absorbance peaks ranging from 225 to 245 nm and TLC analysis yielded Rf values ranging from 0.40 to 0.78. The results suggest that the seven Streptomyces strains isolated from marine sponges produce potential antibacterial compounds against fish and shellfish pathogens.

We report a catalase-negative Staphylococcus aureus isolated from a 56-year-old male diabetic patient with foot ulcer who attended our surgery ward. Species identification was confirmed by Gram staining, standard biochemical tests and PCR amplification of the nuc and fem genes. Antibiotic susceptibility showed that the strain was sensitive to imepenem, chloramphenicol, amoxicillin, vancomycin and resistant to oxacillin, penicillin, ceftriaxone, erythromycin, clindamycin, and amikacin. Clinicians and microbiologists must be encouraged to identify and report these atypical strains and the infections associated with them in order to establish their role in pathogenesis