Vol. 23, May, 2009

1/23 PECTINASE PRODUCTION BY SOME FUNGI AND ITS APPLICATION

Adel A. EL-Mehallawy, Bothaina M. Youssef*, Hisham M. Swailam*

and Nermin M. Gomaa*

Microbiology Department, Faculty of Science, Ain-Shams University.

* National Center for Radiation Research and Technology (NCRRT), Atomic Energy Aurthority (AEA). Egypt.

The present investigation was carried out in order to study the optimum nutritional and environmental parameters for fungal pectinase production and its application. Sixty five fungal isolates were isolated from 8 pectin-rich wastes. Out of the 65 fungal isolates, 12 isolates showed pectinolytic activity. They belonged to the genera Aspergillus, Rhizopus, Penicillium, Trichoderma and Fusarium. Two of the 12 isolates were the powerful and they were identified as Aspergillus niger and Trichoderma viride. It was found that A. niger gave the highest enzyme production at the 3rd day of incubation, while T. viride gave the highest enzyme production at the 4th day of incubation. Citrus pectin (1%) was found to be the best carbon source for enzyme production by both A. niger and T. viride. The optimum pH value for enzyme production by A. niger was 5.0, while it was 5.5 in case of T. viride. Ammonium sulphate was found to be the best nitrogen source for enzyme production by A. niger, while in case of T. viride, it was ammonium oxalate. Both the two fungal genera used gave high enzyme production when grown under static than under shaking conditions. As an application of the excreted enzyme, it was found that A. niger pectinase enzyme showed excellent clarification of apple juice, while T. viride showed a limited clarification of apple juice preparation.

 

2/23 IDENTIFICATION AND CHARACTERIZATION OF A NOVEL HCV-NS4 ANTIGEN IN SERA OF HEPATITIS C INFECTED PATIENTS

Fathy A. Mansour and Sameh B. Abou-Zahra

Departments of Botany (Microbiology), Faculty of Science,

Mansoura University, Mansoura, Egypt.

The identification of native HCV antigens may be very useful in the early diagnosis and treatment of HCV infections. The present investigation aimed at the utilization of the western blot and ELISA techniques to identify the native HCV-NS4 antigen in sera of HCV infected patients in Internal Medicine University Hospital. The results herein reported demonstrated that the purified HCV antigen appeared as a polypeptide band at 21-kDa when analyzed by silver stained SDS-PAGE. A highly significant correlation (r = 0.797, p < 0.0001) has been observed between serum concentrations of the HCV-NS4 antigen and HCV-RNA. The present study clearly established the fact that the NS4 antigenemia based on ELISA test can serve as a useful addition to HCV diagnostic methods, especially under field condition.

 


3/23 CHARACTERIZATION OF ALLERGENS OF POLLEN GRAINS OF THE DELONIX REGIA (POINCIANA)

Khalil A. El Halfway, El Desouki E. Fouda and Mahmoud A. Abd El Naby

Industrial Biotechnology Depatment, Genetic Engeneering & Biotechnology Institute Al-Monfeia University

Allergy & Immunology Center Al-Azhar University

Allergic disorders are one of the most common disorders in the world and various studies have demonstrated a steady and significant increase in world wide prevalence in the last two decades .This study aims at pointing out the allergenicity of Delonix regia pollen grains and seeds in Egypt and characterization of its allergens.Pollen grains have been found to be very important in causing human Immunoglobulin (E) mediated allergic disorders e.g.: asthma or rhinitis.The study of these allergens in area of interest for allergy diagnosis and therapy. Quite a number of plants producing allergenically potent pollen grains have been reported all over the world. Delonix regia (Royal Poinciana) are plants which predominant grew in Egypt during summer in may places of the country, So we will study their pollen grains as a possible causes of aero allergenic diseases in Egypt. So it has to be characterized and major allergens should be identified. The familial syndrome of atopy (allergic asthma, seasonal rhinitis, and/or eczemaurticaria) accompanied by increased levels of total serum Immunoglobulin (E).The present work aimed to identify major allergens in Delonix regia pollen grains.

 

4/23 OPTIMIZATION OF BIOSURFACTANT PRODUCTION FROM PSEUDOMONAS AERUGINOSA THROUGH FACTORIAL DESIGNS

Mervet A. Kassem, Norhan Fanaki, Hameda Abou-Shleib, Fatma Dabbous and

Yasser R. Abdel-Fattah*

Department of Microbiology, Faculty of Pharmacy, Alexandria University, Egypt.

* Bioprocess Development Dept., Genetic Engineering and Biotechnology Research Institute, Mubarak City for Scientific Research.

In a screen program, a total of 44 microbial isolates were collected from different clinical and environmental sources and thereafter only those capable of biosurfactant production were subjected to microscopic and biochemical identification. Qualitative screening for biosurfactant production by the tested microbial isolates was carried out using emulsification index, oil spreading technique and surface tension measurement using drop number methods. Among the tested 44 isolates, P103 was selected as a potent biosurfactant producer. Optimization of biosurfactant-production was carried out by applying factorial designs including Plackett-Burman and Box-Behnken designs, and their results were statistically analyzed and subjected to model verification. From the 12 variables examined, medium initial pH and levels of yeast extract and glycerol were the most significant factors affecting biosurfactant production and consequently were further tested through the Box-Behnken design to find out optimum levels. According to the solver function of the Microsoft Excel tools, it was found that 0.12% yeast extract, 0.38% glycerol and pH of 7.27 would be optimum with a predicted surface tension of 18.25 mN/m. Monitoring of cell growth and biosurfactant production in both shake flask and fermentor for 3 days revealed that the microbial growth level in the shake flask is higher than that of the fermentor and also the surface tension decrease in the shake flask is lower than that in the fermentor.

5/23 EFFICIENCY OF THE ORGANIC SOLVENTS EXTRACTS OF SOME MEDICINAL AND AROMATIC PLANTS FOR THE CONTROL OF SQUASH POWDERY MILDEW

Ameena Moran Al Surhanee

Girl College of Education-Al Jouf University-KSA

Experiments were conducted under laboratory, greenhouse and field conditions to investigate the possible utilization of plant extracts to control Sphaerotheca fuliginea, the causal pathogen of squash powdery mildew. Six air dried plant materials, i.e. anise seeds, blue gum leaves, chamomile flowers, cumin seeds, marjoram herb and thyme herb, as well as three organic solvents (acetone, methanol and petroleum ether) were used for extraction. Methanolic extracts (ME) of blue gum and thyme gave significant decrease in spore germination of S. fuliginea. Treating detached leaves with the organic solvents extracts (OSE) as preventive and curative treatments proved that the first was more effective than the second in controlling the disease. Spraying with (ME) of blue gum and thyme decreased powdery mildew disease incidence and increased total phenols. Also, spraying squash in greenhouse and field with the (OSE) as preventive and curative treatments gave sufficient control to the disease in most cases. However, the efficiency of the methanolic extract of thyme herb in controlling the disease as curative treatment was always comparable to that of the fungicide Afugan.

 

 

6/23 BETA-LACTAMASE GENE IN HEAVY METALS AND ANTIMICROBIAL RESISTANT BACTERIAL ISOLATES FROM FRESH WATER

Mervat F. El Sherif

Department of Biological and Geological Sciences, Faculty of Education, Alexandria University, Egypt.

The aim of the present study was to determine the level of antibiotic resistance patterns and distribution of heavy metal resistance of bacterial isolates from fresh water and to determine if there is a relationship between antibiotic and heavy metal resistance. We undertook studies in the River Nile branch (Rossetta branch). The concentration of heavy metal in the studied water samples ranged from 0.0002 to 0.01 mg /L The resistance of selected 8 bacterial isolates to 10 different antibiotics, and to 5 heavy metals, was investigated by agar diffusion and agar dilution methods, respectively. Tetracycline, ampcillin and amoxcillin were found to be the antibiotics that the majority of isolates can resist, then followed by cephalexin, novamycin, gentamycin, kanamycin, chloramphinicol, cepharidine, streptomycin. Multiple antibiotic resistant (MAR) was expressed by 100% with 25% of them were resistant to five or more antibiotics and reached as high as 100% for one species. Most isolates showed tolerance to different concentrations of heavy metals and while Streptococcus sp. showed high resistant against studied heavy metals. Molecular sizes of the detected plasmids ranged from 23.45 kbp and 55.8 kbp in E. coli. Heavy metals and antimicrobial resistant strains that showed resistance to amoxicillin were tested for the presence of TEM-family of beta-lactamase genes using a novel primer pair. The sensitivity of PCR detection of TEM-class gene with the boiling method was determined by making 10- fold dilution of bacterial suspension. Antimicrobial drug susceptibility testing demonstrated that multi-drug resistance was common in most isolates. The 858 bp specific amplification of PCR product was produced after testing of eight potentially isolated bacterial pathogens. This study permitted an evaluation of the frequencies of multidrug resistance with special emphasis on amoxicillin resistance of microorganisms from fresh water in Egypt. The results show that River Nile has a significant proportion of antibiotic and heavy metal resistant bacteria, and these bacteria constitute a potential risk for public health.

 

7/23 OTOMYCOSIS IN QALYOBIA GOVERNORATE:

A CLINICOMYCOLOGICAL STUDY.

Gamal A. Amer and Kassem M. Kassem*

Departments of Microbiology & Immunology and Otorhinolaryngology*, Faculty of Medicine, Benha University, Egypt

Otomycosis is a fungal infection of external auditory canal. We have tried to clarify the different predisposing factors and etiological agents for otomycosis in Qalyobia governorate. Sixty six patients with confirmed otomycosis were included in the study. Their age ranged from 7 to 59 years (mean ± SD = 30.97 + 13.43y). They were 37 (56.06%) females and 29 (43.94%) males. The study also included 20 age and sex matched healthy control. For all, swabs were taken from the lesion in the external auditory canal by ENT specialist. Smears stained with KOH and Gram stains and culture on Sabouraud's dextrose agar were done for each patient. The culture growth was identified according to the standard methods. The results showed that otomycosis is common in adults than in children (31.82% in the age group between 20 and 30 years, but 7.58% in children less than 10 years of age) a difference which is statistically significant (P < 0.01). The disease is more common in females (56.06%) compared to (43.94%) in males. The disease is usually unilateral and right ear is affected more than left ear (48.49% to right ear, compared to 42.42% to left ear). The most common predisposing factors are self cleaning (45.45% of cases), local use of antibiotic eardrops (25.76% of cases) and immune deficiency (diabetes – malignancy) in 18.18% of cases. The most common fungi isolated from otomycosis in our study were Aspergillus niger (45.45% of cases) followed by Candida albicans (27.27%) of cases. Conclusion: Otomycosis is not uncommon disease. It usually affects adult females, usually unilateral, right ear is more affected than left ear. The most common predisposing factors are self cleaning of the ear and local use of antibiotic otic drops. The most common isolated fungi are Aspergillus niger and Candida albicans.

 

8/23 MORPHOLOGICAL AND MOLECULAR CHARACTERIZATION OF SOME EGYPTIAN TRICHODERMA ISOLATES

Ilham M. El-Refai, Susan M. W. Assawah, Saida M. Amer and Mohamed S. Draz

Botany Department, Faculty of Science, Tanta University, Tanta, Egypt.

In the present study, thirteen isolates of Trichoderma belonging to three taxonomic sections (Trichoderma, Pachybasium and longibarachiatum) and under seven species were collected from Egyptian soil identified. The conventional morphological approach was used for characterization and discrimination of different Trichoderma isolates. The growth pattern of the investigated isolates showed significant variations in the nature of mycelial growth, sporulation and pigmentation with the tested isolates and growth media. The present results generally emphasized the variability and plasticity of the available morphological characters and ensure the importance of using molecular characterization to aid in precise identification and taxonomy of Trichoderma isolate. The data of sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed 27 bands with molecular weight ranging from 18 to 116 KDa. All bands were polymorphic indicating a high genetic variation within and among the investigated Trichoderma isolates. The constructed dendrogram based on SDS-PAGE indicates that the isolates from the same Trichoderma sections were distributed among different clusters. Genomic DNA isolated from the thirteen isolates of Trichoderma was analyzed by RAPD-PCR using five primers. A total number of 131 DNA fragments were detected. All the amplified fragments were polymorphic. The generated cluster based on SDS-PAGE and RAPD-PCR combination analysis showed more reliability.

 

 

9/23 IMPROVEMENT OF SORGHUM BICOLOR L. GROWTH AND YIELD IN RESPONSE TO AZOTOBACTER CHROOCOCCUM, COMPOST WATER EXTRACTS AND ARBASCULAR MYCORRHIZA FUNGI: DIFFERENT APPLICATION METHODS

Ebtsam M. Morsy, Nadia H. El-Batanony* and Osama N. Massoud

Soil, Water and Environment Research Institute, Agricultural Research Center, Giza, Egypt.

*Environmental Studies and Research Institute (ESRI), Sadat City, Menoufiya University, Egypt.

Two field experiments were conducted on a sandy clay loam soil, in a private farm at Kafr Dawood, Menoufiya, Egypt during two successive summer seasons of 2006-2007 to assess the response of Sorghum bicolor L. to inoculation with Azotobacter chroococcum and/or AM mycorrhia fungi, applied by two different inoculation methods in soil amended by compost water extracts (CWE). The growth, microbiolgical, physiological and yield parameters of sorghum plants were determined. The results proved that, shoot and root dry weights of sorghum, total bacterial population dynamic in sorghum rhizospheric zoon were positively affected when a combination of A. chroococcum and/or AM mycorrhia fungi and CWE. Furthermore, the combined inoculations significantly increased nitrogenase activity by 3.5 times, dehydrogenase activity by two times over control (full NPK) especially the treatment CWE + AMSA + A.C. AM mycorrhizal infections % in sorghum roots as well as AM mycorrhizal spore numbers in soil after harvest was increased in the treatment CWE + AMSA + A.C (The infection was 100% and 379 spore/g soil). On the other hand the total NPK contents of sorghum shoots were significantly increased with combined treatments. Moreover, the two treatments CWE +AMSA + A.C and CWE +AMGC + A.C showed almost 1.2 times increase in grain yield(g/plant) over control (full NPK). There was insignificant increase in sorghum grain yield per Feddan (ardab/fed), the two treatments CWE +AMSA +A.C and CWE +AMGC+ A.C gave almost 28.0 ard/fed. In addition, there was also a significant increase in grains nitrogen contents and total carbohydrates. In spite of the slight superiority of the treatment CWE +AMSA + A.C over the treatment CWE +AMGC + A.C in all parameter tested, we recommended the use of grain coating inoculation method because this method is more easier, has an economic impact in saving more quantities of inocula that used to expand more areas and also shipping costs. In addition, we can also recommended that the use of CWE in agriculture combined with free-living nitrogen fixers and AM mycorrhiza inoculants is considered to be more efficient than using it as single inoculant.

10/23 PRODUCTION, PURIFICATION AND PARTIAL CHARACTERIZATION OF KERATINASE FROM STREPTOMYCES THERMOVULGARIS FHM 8

Fathy A. Mansour, Hend A. Hamedo* and Majdah M.Y. Al-Tuwaijri**

Botany Department, Faculty of Science, Mansoura University;

*Biology Department, Faculty of   Education, at El Arish, Suez Canal University

**Botany Department, Faculty of Applied Science (Girls Branch), Umm Al Qura University, Saudi Arabia

A number of 28 isolates of actinomycetes were isolated from compost and fertile soil samples collected from Meet-Khamees village at Mansoura district. These isolates were screened for their capabilities of degrading chicken feather. Keratinase producing abilities. A highly active thermophilic strain, identified as Streptomyces thermovulgaris FHM8 proved by far to possess the highest keratin-degrading potential as compared to the other isolates tested. The optimum conditions for growth and keratinase production byS .thermovulgaris were elucidated. The results showed that the optimum temperature for keratinase production was 55ºC and the optimum pH was 8.5. Starch appeared to surpass other carbon sources examined in enhancing the keratinolytic activity of the bacterium. SDS-PAGE revealed that the keratinase enzyme produced by S. thermovulgaris was obtained as a monomeric enzyme protein with molecular weight about 45 KDa.

 

11/23 SYNERGETIC EFFECT OF PLANT-GROWTH PROMOTING RHIZOBACTERIA AND ARBUSCULAR MYCORRHIZA FUNGI ON ONION (ALLIUM CEPA) GROWTH AND ITS BULBS QUALITY AFTER STORAGE

Nadia H. El-Batanony

Environmental Studies and Research Institute (ESRI), Sadat City, Menoufiya University, Egypt

Two pot experiments were carried out with a sandy-loam soil during two successive winter seasons (2006-2007) to evaluate the effect of different plant-growth promoting rhizobacteria Bacillus circulans, mixture of Azospirillum lipoferum + Azotobacter chrococcoum + Bacillus polymyxa, Rhizobium sp. and Arbuscular mycorrhiza fungi, alone or in combination of all on onion growth parameters and onion bulbs quality. The growth, microbiolgical, physiological and yield parameters of onion plants were evaluated as well as the quality of onion bulbs after storage. It was noticed that, most of the vegetative growth parameters and total bacterial population dynamic in onion rhizospheric zoon were positively affected by inoculating onion plants with the different treatments as compared to the control (C-without). The treatment number 5 gave the highest significant values of all the tested growth parameters in comparison with the two controls {C-without and control with full dose of NPK (FNPK)}. The lowest value of the % of onion shoot water content being 29.42 as well as onion bulb water content being 40.97 was recorded in the treatment number 5 at the growth 3. The highly increased for bulb dry weight and bulb diameter values were recorded with treatment number 5 being (42.3 g and 8.0 cm, respectively) at growth interval 4. The highest speed of growth over all growth period (RGR4-1) was given in treatment number 5, followed by treatments No. 3, 1, 2 and 4 as compared to the two controls. Furthermore, the highest value of nitrogenase activity as well as % of AM mycorrhiza infection of onion roots were 41.98 µmole C2H4/h/g RDW and 95% for treatment number 5 at the growth interval 3. The total NPK% contents of onion dry shoots compared with control FNPK at the different growth intervals showed significant increase in the most of the treatments. The highest increases were obtained with treatment 5 (4.0 % N, 1.97% P and 2.91 % K).At the growth interval 3 the increase in organic carbon % of onion bulbs was significant for all the treatments compared to the controls (FNPK and C-without). Treatment No.5 gave the highest mean of total carbohydrate value (29.23 mg/g DW) followed by the treatment No.3 (28.77 mg/g DW), then treatment No.1 (24.9 mg/g DW). The highly protective effect against storage caused diseases was given by the treatments No. 2, No. 3 and No. 5; they gave 100% healthy onion bulbs in comparison with the two controls. It could be concluded that, there is important synergism observed on onion plant growth when the inoculants used contain a mixture of organisms. Thus, in order to have future beneficial inoculants for field grown crops, one approach should consider performing inoculation assays containing a mixture of soil organisms instead of a single strain.

12/23 IMPACT OF SALINITY STRESS ON THE FREE AMINO ACID POOLS OF SOME SUGARBEET PATHOGENS

Aisha M.H. Al-Rajhi

Collage of Training Teachers, Riyadh, Saudi Arabia

This investigation was designed to compare the constitution of intracellular free amino acid pools of the sugarbeet root rot fungi Fusarium solani Mart. and Sclerotium rolfsii Sacc. (Indigenous to saline soils), with those of the wilt fungi Fusarium oxysporum f. sp. lycopersici and F. oxysporum f. sp. vasinfectum (indigenous to non-saline agricultural soils) under the impact of salinity stress induced by different NaCl concentrations. The results obtained showed that tolerance of NaCl salinity by the fungi varied according to the species and the developmental stage of the fungus. F. solani and S. rolfsii appeared to be more tolerant of the salinity stresses applied than the two formae speciales of F. oxysporum. The results also indicated that in F. solani and S. rolfsii, both synthesis of amino acids as well as their incorporation into proteins are stimulated under salinity stress, whereas in the less tolerant form species of F. oxysporum, whether synthesis of amino acids was stimulated (f. sp. lycopersici) or inhibited (f. sp. vasinfectum), protein synthesis was depressed in either case. Considerable variations in the types and quantities of free amino acids produced by the fungi were detected under salinity stress. Tryptophan, proline, cystine and cysteine were not detected in the two form species of F. oxysporum, but were detected in concentrations that were stimulated with increasing salt concentrations in F. solani and S. rolfsii. The accumulation under salinity stress of alanine and proline in F. solani, and of proline and histidine in S. rolfsii might suggest that these amino acids have a significant role in osmoregulation by these fungi.

13/23 PNEUMOCOCCAL BACTEREMIA: ANTIMICROBIAL-DRUGS USE ANDRESISTANCE OF STREPTOCOCCUS PNEUMONIAE TO BETA-LACTAMANTIBIOTICS

Ayman Khater, Manal Mokhtar*, Mohamed Osman Helme**

and Enas Abdel –Magid***

Chest Department, *Microbiology and Immunology Department, Faculty of Medicine, Ain Shams University, Cairo, Egypt.

**Pediatrics Department, Faculty of Medicine, Cairo University,

***Clinical Pharmacist, Cairo University.

Objective: To assess the clinical impact of emerging drug resistance for S. pneumoniae to clarify antibiotic recommendations for community-acquired pneumonia. Methods: We performed a study of one hundred – five hospitalized patients with blood cultures positive for Streptococcus pneumoniae (S. pneumoniae) were enrolledin Dr. Soliman Fakeeh Hospitals inJeddah, Kingdom of Saudi Arabia, during the period of December 2004 through May 2007.  Results: Sixteen (16.16 %) of S. pneumoniae isolates had invitro intermediate susceptibility to penicillin (minimum inhibitory concentration MIC, 0.12-1 µg/mL), and eight (8.08%) of isolates were resistant (MIC ≥ 2 µg/mL). Age, severity of illness, and underlying disease with immunosuppression were significantly associated with mortality. Penicillin resistance was not a risk factor for mortality. The impact of concordant antibiotic therapy (Concordant therapy was definedas receipt for thefirst two days afterthe blood was obtainedfor culture of asingle antibiotic that hadinvitro activity, sensitive, i.e.,neither intermediate nor resistantinvitro against theS. pneumoniae isolated), versus discordant antibiotic therapy (Discordant therapywas defined as receiptfor the first twodays after the bloodsample was obtained forculture of a singleantibiotic that was inactive, intermediate or resistant, invitro against theS. pneumoniae isolated) on mortality was assessed at fourteen days. Discordant therapy with Penicillins, Cefotaxime, and Ceftriaxone (but not Cefuroxime) did not result in a higher mortality rate. Similarly, time required for abatement of a fever and frequency of suppurative complications were not associated with concordance of ß-lactam antibiotic therapy. ß-Lactam antibiotics should still be useful for treatment of pneumococcal infections that do not involve cerebrospinal fluid, regardless of in vitro susceptibility, as determined by currentClinical and Laboratory Standards Institute (CLSI) breakpoints. We evaluated risk factors for emergence of drug resistance, performed in vitro susceptibility testing for blood isolates, and, most importantly, recorded data on the antibiotics administered, including timing of antibiotic therapy in relation to the onset of symptoms, dose, duration of therapy, and route of administration. Conclusion: This study confirms that ß-lactamagents, including penicillin, arestill effective for thetreatment of penicillin-non susceptible pneumococcal pneumonia. It may be indicatedthat penicillin should bemore widely used.

 

14/23 RAPID DETECTION OF LEGIONELLA PNEUMOPHILA IN RESPIRATORY SPECIMENS BY POLYMERASE CHAIN REACTION

Manal Mokhtar, Magdi Khalil*,Ayman Khater* and Mohamed Osman Helme**

Microbiology and Immunology, *Chest and **Pediatrics Departments, Faculty of Medicine, Ain Shams University and **Cairo University

Objective: Legionella species are a common cause of community-acquired respiratory tract infections and an occasional cause of nosocomialpneumonia. Significantmortality rates among the elderly and patients with severe underlyingdisease may occur as a result of infection with this pathogen.Microbial cultures are available to diagnosis of Legionella but they require a relative longer time to yield definitive results. The present work compared Polymerase Chain Reaction (PCR)to culture with respiratory specimens from patients suspected ofhaving Legionella pneumophila infection. Methods: From December 2005 through December 2007 one hundred respiratory samples from patients suspected of having pneumoniacaused by Legionella were submitted to Dr. Soliman Fakeeh Hospitals,Jeddah, Kingdom Saudi Arabian, Microbiology Laboratory. The number and type of specimens submitted were seventy two sputum, eight bronchial washing, fifteen bronchoalveolar lavage fluid (BAL) and five pleural fluid specimens. All the samples were subjected to both culture and PCR with gel detection. Results: We found that Legionella was detected in 24 out of 100 specimens (24%) by PCR and 18 out of 100 specimens (18%) were positive for Legionella by microbial culture. All the culture positive specimens were also positive by PCR and six of eighty two culture-negative sampleswere PCR positive for Legionella pneumophila. Considering culture as a gold stander and reference method, the sensitivity and specificity of PCR were 100% and 93%, respectively. There was association of infection with age, malignancy and immunosuppressive therapy. Conclusion, Legionella pneumophila is an important cause of pneumonia in immunosuppressed patients (those who have malignancy or receiving immunosuppressive therapy). PCR assay demonstrates high sensitivity and high specificity and can be used as a screening, rapid and specific test for routine detection of L. pneumophila directly from respiratory specimens.

 

15/23 ANTIBACTERIAL ACTIVITY OF COMMERCIALLY MOUTHWASH PREPARATIONS AGAINST CARIOGENIC STREPTOCOCCUS MUTANS

Ghada H. Shaker

Department of Microbiology, Faculty of Pharmacy, Zagazig University, Egypt.

Twenty seven clinical isolates of Streptococci among 45 samples isolated from different volunteers of both sexes were examined for production of crude glucosyltransferase enzyme (GTF). The Streptococcus mutans (S. mutans) isolate no.13 among 27 clinical isolates of Streptococci gave the highest crude enzymatic activity recording a value of 1164 unit/ml. The minimum inhibitory concentrations (MICs) for the five selected mouthwashes against isolate no.13 were determined by broth dilution method in Todd Hewitt medium with or without 5% serum. When the medium was supplemented with serum, higher MIC values were observed. The short- term antibacterial effect of the selected mouthwashes on salivary and plaque Streptococcus mutans was tested in 25 volunteers. Preparation No.2 which contains cetrimide in combination with chlorhexidine gluconate was the most effective mouthwash preparation, while preparationNo. 4 which contains povidone iodine was the least effective in reducing S. mutans levels of oral microorganisms giving 95% and 67% reduction in viable counts respectively after 180 min. as compared to control (counts obtained after mouth rinsing with sterile water).

 


16/23 STUDY ON SOME POTENTIAL VIRULENCE FACTORS OF LOCALLY ISOLATED CLINICAL ISOLATES OF CANDIDA ALBICANS

Ghada H. Shaker, Fathy M. Serry, Hassan A. Abdel-Salam, and Amira El-Ganiny

Department of Microbiology and Immunology, Faculty of Pharmacy, University of Zagazig, Egypt.

Twenty clinical and commensal isolates of Candida albicans were recovered from different sources. Candida albicans was identified by biochemical activities, dimorphic morphology, germ tube formation and was confirmed by API20C Aux system. To study the virulence factors, the clinical and commensal isolates of C. albicans were tested for the production of phospholipase, and lipase enzymes, and for their adherence to epithelial cells in vitro. Total cell protein from yeast and hyphal forms of selected isolates were analyzed by polyacrylamide gel electrophoresis. Vaginal isolates demonstrated significantly higher adherence capacity (9.53 ± 1.83) compared with oral (6.69 ± 0.77) and commensal isolates (3.39 ± 0.41), that could be correlated with virulence. All the tested isolates were phospholipase positive with statistically non significant (p > 0.05) differences. Testing for lipase activity using tributyrin method revealed that oral isolates showed statistically significant (p < 0.05) wider zones of lipolysis (12.88 ± 1.38 mm) compared with vaginal (11.77 ± 0.96 mm) and commensal isolates (9.9 ± 1.08 mm) that could correlate with isolate type and virulence. Assay of specific lipase activity using olive oil emulsion in liquid media showed that vaginal isolates had higher activity (2.38± 0.93 U/ml) compared with oral (2±0.85 U/ml) and commensal isolates (1.9± 0.274 U/ml) but the differences were statistically non significant. Protein analysis by SDS-PAGE revealed the absence of protein bands of sizes about 120, 115, 70, and 40 KDa from the yeast forms compared with the hyphal forms. For the yeast forms, vaginal isolate showed two extra protein bands of about 112 and 100 KDa compared with oral and commensal isolates. Vaginal and commensal isolates showed protein bands of about 45 and 35 KDa that were absent in oral isolates. Oral and vaginal isolates showed protein band of about 30 KDa that did not appear with commensal isolates and the latter showed extra protein band of about 26 KDa. For the hyphal forms, vaginal and commensal isolates had a protein band of about 32 KDa that was absent in oral isolate, while oral and vaginal isolates showed a protein band of about 28 KDa that was missing in commensal strains.

 

17/23 REMOVAL OF CADMIUM IONS BY LIVING AND DEAD BIOMASS OF PSEUDOMONAS AERUGINOSA

Wesam A. Hassanein, Nadia M. Awny, Eman Y. Tohamy and Seham M. Meselhy

Botany Department, Faculty of Science, Zagazig University, Egypt

Pseudomonas aeruginosa, strain was examined for its resistance to increasing concentrations of three heavy metal ions, namely Cd+2, Zn+2 and Cu+2. The minimum inhibitory concentrations were 110.38, 81.69 and 72.28 mg/l for Cd+2, Zn+2 and Cu+2 respectively. Increasing concentrations of heavy metal ions decreased the bacterial growth and metal accumulation for tested bacteria. In this work the useful method for possibility of using metal resistant bacteria for removing Cd+2 from polluted effluent is the primary aim. Due to well known pathogenisty of Ps. aeruginosa, it was decided to use it as dead and dried biomass. Cd+2 biosorption was found to be affected by different factors. The maximum biosorption was achieved at the initial Cd+2 concentration; 85.85 mg/l, contact time; 60 min, pH; 5, temperatures; 25oC, amount of the dried biomass; 1g/l and shaking speeds ; 70rpm/min. The EDAX analysis of dead biomass indicated higher uptake of Cd+2 if compared with other metals associated with dried biomass. The weight percentage of Cd+2 biosorption was 78.33%.On other hand, the cadmium removal was reversible processes. The % of Cd+2 released was increased by repeating the washing cycle and third stage of desorption with 15 mM HNO3 or 10 mM EDTA was sufficient to reduce Cd+2 biosorped to 2.59 and 1.4 % respectively. Also, the dead biomass of Ps. aeruginosa was used in Cd+2 removal from seven different polluted industrial waste-waters collected from different sites in Sharkie province. Dry biomass of Ps. aeruginosa proved to be a good candidate for Cd+2 removal from polluted industrial waters.

 

18/23 MOLECULAR IDENTIFICATION OF ANTIBIOTICS RESISTANT PSEUDOMONAS AERUGINOSA WT

Wesam, A. Hassanein

Botany Department, Faculty of Science, Zagazig University, Egypt.

The analysis of 16S ribosomal RNA gene sequence is a good tool for the detection of Pseudomonas aeruginosa in clinical specimens. Pseudomonas aeruginosa Wt was confirmed for the identification to the species level based on alignment of 16S rRNA gene sequence available in Genbank. DNA product at the predicted size 1kb was obtained. BLAST search indicated the homology between deduced sequence of Wt strain and P. aeruginosa D12A06 16S rRNA gene (accession number FJ655799.1). The antibiotic susceptibility of tested Wt strain against different antibiotics was carried using disc diffusion method. The inhibition zones were recorded only for 5 antibiotics. Resistance of Wt to tested antibiotics was arranged in the following order tetracycline (TE), nalidixic acid (NA) > norfloxacin (NOR) > chloramphilinical (C) > ciprofloxacin (CIP). Antibiotic susceptibility of Wt to selected antibiotics and the diameter of inhibition zones were affected by different cultural conditions as incubation period, incubation temperatures, pH values, carbon and nitrogen sources and types of agar media. Using agar dilution method MICs of TE and CIP were 100 and 80 µg/ml respectively for tested P. aeruginosa Wt. On the other hand plasmid profile analysis indicated that resistance of Wt strain to CIP may be plasmid DNA carrier while it may be chromosomal DNA for TE.

19/23 DEVELOPMENT OF AFLATOXIN BINDING ASSAY USING PROBIOTIC BACTERIA

Amal S. Hathout and Soher E. Aly

Toxicology & Food Contaminants Department, National Research Center,

Cairo, Egypt

Aflatoxins are a group of mycotoxins that are produced by the aflatoxigenic fungi Aspergillus parasiticus. Aflatoxins contaminate many foods and animal feeds, and thus cause serious chronic disease outbreaks such as cancer and even death. Due to the significant commercial interest in using probiotic bacteria as a natural food preservative to enhance food safety and stability, the ability of Lactobacillus casei and L. reuteri to bind aflatoxins B1, B2, G1 and G2 at different levels of incubation period, temperature, and pH was evaluated. L. casei was more effective and bound 51.10%, 94.85%, 99.99% and 95.67% of aflatoxins B1, B2, G1 and G2 respectively after 30 minutes of incubation at 37ºC and pH 6.6. On the other hand maximum aflatoxins bound to L. reuteri were 98.60%, 94.32% and 99.70% for aflatoxins B2, G1 and G2 respectively after 24 hours at 37ºC and pH 3.3. On studying the stability of bacteria / aflatoxin complex by washing for 5 times, variable amounts of aflatoxins were released from the lactobacilli / aflatoxin complex, especially aflatoxin B1. The aflatoxins bound to L. reuteri were more stable and lower amounts of aflatoxins were released on washing than those bound to L. casei.

 

20/23 THREE YEARS STUDY TO EVALUATE THE EFFECT OF EXPOSURE OF HEALTHY HOSPITAL STAFF WORKERS TO LEGIONELLA PNEUMOPHILA CONTAMINATED HOSPITAL WATER SYSTEMS.

Wafaa El-tayab*, Sahar Radwan, Amany El-Sharif and Fatma Gomaa

Microbiology and Immunology Departments, Faculty of Pharmacy (Girls), Al-Azhar University & *Misr International University, Cairo, Egypt

The study was based on immunological response to Legionella pneumophila exposure.  Legionella IgG/IgA/IgM ELISA test system was used to detect antibodies in sera obtained from healthy hospital staff workers. The results obtained were compared with results obtained with blood donors' sera. This study was carried out to assess the risk of exposure of healthy hospital staff members to Legionella pneumophila contaminated water system. Our study revealed that long exposure of healthy people to Legionella spp. induces production of antibodies. Increased awareness to hazards of Legionella spp. & recognition of the methods of evaluating Legionella pneumophila in hospital water is highly recommended.

 

21/23 RAPID DETECTION OF METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS (MRSA) USING MRSASELECT MEDIUM AND POLYMERASE CHAIN REACTION (PCR) OF MECA GENE IN SCREENING SWABS

Salwa S. Afifi, Tayseer Ramadan* and Magda H. Mahran**

Microbiology & Immunology Dept., Faculty of Pharmacy and Medicine*,**(for Girls), Al Azhar University* and Research of Ophthalmology**

Accurate and rapid identification of Methicillin resistant Staphylococcus aureus (MRSA) in clinical specimens is essential for time decision on isolation procedures and effective antimicrobial chemotherapy. This study evaluated the sensitivities and specificities of MRSA detection by two methods; MRSASelect medium and PCR amplification of mecA gene, without the time-consuming step of identifying a bacterial isolate by using Phonotypical methods which are routinely used to detect Methicillin resistance in Staphylococci. The study was carried out on one hundred and sixteen patients presented with infected ulcers, infected wounds, and burn ulcers. 14/116 samples were positive by MRSASelect after 18 -48 hours. mecA gene was amplified by PCR in 11 samples. The 3 negative swabs were the ‘false positive by MRSASelect. PCR and MRSASelect had sensitivity and specificity of 100% and 100% & 97.14% respectively. The mean PCR turnaround time was 14.5 h. In conclusion, MRSA select agar detects a majority of cases of MRSA swab patients in 18- 24 h and is easy to use. Compared to MRSA agar, the mec A Gene PCR assay was more specific, provided significantly faster turnaround times, and resulted in more-prompt isolation of MRSA in clinical specimens.

 

22/23 PREVALENCE OF VANCOMYCIN RESISTANT ENTEROCOCCI IN A GOVERNMENTAL HOSPITAL IN CAIRO-EGYPT

Wafaa N. El-Tayeb, Magda A. Gamil**, Mohamed S.E. Ashour*

and Maha A. Ismail***

Microbiology Department, Faculty of Pharmacy, Misr International University, *University of Modern Sciences and Arts, **Faculty of Medicine (Girls), Al-Azhar University, ***Mansheit El Bakry Hospital

Two hundred specimens were collected from hospitalized patients suspected of having vancomycin resistant enterococci (VRE). Each specimen was subjected to bacteriological study including microscopical examination of Gram stained smears, culture on Bile esculin azide agar medium and identification of isolates by conventional biochemical reactions and API 20 strep. Drug susceptibility testing was performed 30 ug vancomycin disc diffusion method and E-test Strep to determine minimum inhibitory concentration (MIC).This study showed that the overall positive VRE cases from two hundred cases were twenty five cases representing 12.5%. A total of 86 enterococcal isolates were isolated representing 43%, only twenty five (29.1%) isolates were vancomycin resistant. Biochemical reactions revealed that the most isolated VRE species were as follows: 9 (36%) E. avium, 6 (24%) E. faecium, 5 (20%) E. casseliflavus, 2 (8%) E. Faecalis, 1(4%) E. mundtii and 1 (4%) E. pseudoavium and 1 (4%) unidentified. According to E-test results the MIC for the 25 VRE isolates ranged were from 24 to 64 μg/ml. Fifteen of VRE isolates of MIC 32 μg/ml (Van B),one isolate has MIC 64 μg/ml, (Van B), seven isolates have MIC 24 μg/ml (Van B), two isolates have MIC 48 μg/ml, (Van B).

23/23 Production of yoghurt and bio-Fermented Milk from Camel’s milk

El-Sayed M. Abd El-Wahed

Department of food science, Faculty of agriculture, Zagazig University, Egypt.

Camel’s and cow’s milk was used for production of yoghurt and probiotic-fermented milk. Preliminary experiments showed that the storage period at (7±1°C) of the fermented camel’s milk extended to about 10 days without any defects in its appearance. After 12 days of storage, slight wheying off were observed. Comparison between effect of mixed camel’s and cow’s milk on chemical properties of traditional yoghurt and bio-fermented milk. It was clear that the treatment (50%) camel’s milk treated with yoghurt culture had the highest values of TS% than that of all treatments, while treatment (50%) camel’s milk treated with yoghurt culture and probiotic culture had the highest values of Fat/DM, Ash, N/DM, NPN/DM, acidity % and TVFA. The statistical analysis indicated that there were significant differences (p<0.05) between all treatments in TS, Fat/DM, Ash, N/DM, NPN/DM, Acidity % and TVFA. The microbiological analysis of yoghurt and bio-fermented milk during storage at (7±1°C) was clear that the enterobacterial group, lipolytic and proteolytic bacteria were not detected until the end of storage. While yeast and moulds were not detected until the end of storage.

24/23 INDUCTION OF STARTERS CULTURES AND THEIR

APPLI CATION IN SOFT CHEESE PROCESSING

Mohamed M. El-Abbassy, Mostafa M. Ashour, Mohamed M. Omar

and El-Sayed M. Abd El-Sattar

Food Science Department, Faculty of Agriculture, Zagazig University, Zagazig, Egypt

Starter culture was induced by the use of gradual increasing of concentration of streptomycin until achieving induced starters. The same repeated with gentamycin. The induced starters were used in milk with ratio of 2%. First treatment was made from skim buffaloe's spiked with streptomycin and YC-11, the second treatment was made from skim milk spiked with streptomycin (100 µg / ml) and induced YC-11, the third treatment was made from acidified (adjust the pH of milk to 6 using lactic acid) spiked skim milk with streptomycin (100 µg / ml) and YC-11 and the fourth treatment was made from acidified spiked skim milk with streptomycin (100 µg / ml) and induced YC-11. The four treatments were repeated with gentamycin (10µg / ml).the control treatment was carried out with (skim milk free from antibiotics + YC-11). The cheese samples were subjected to chemical, microbiological, and sensory evaluation on the following intervals the fresh samples, and after 7 and 14 days. the fourth treatment was higher than the other three treatments but less than the control So that, the induced starters and direct acidification were selected to be conducted in milk containing antibiotic residues.

25/23 INDUCTION OF STARTER CULTURES AND THEIR APPLI CATION IN FERMENTED MILK PROCESSING

Mostafa M. Ashour, Mohamed M. Omar, Mohamed M. El-Abbassy

and El-Sayed M. Abd El-Sattar

Food Science Department, Faculty of Agriculture, Zagazig University, Zagazig, Egypt

In this study the yoghurt starter culture (YC-11) was subjected to induction by using gradual concentrations of the two antibiotics: streptomycin and gentamycin. The induction was repeated until obtaining an induced starters. Induced starters were used in yoghurt manufacture by using cow’s milk. Four treatments were carried out in addition to the control treatment when it was manufactured from cow’s milk free from antibiotic and YC-11. The treatments were as follow the first treatment was made from spiked antibiotic milk and YC-11, the second treatment was made from spiked antibiotic milk and induced YC-11, the third treatment was made from spiked antibiotic acidified milk(adjust the pH of milk to 6 using lactic acid) and YC-11 and the fourth treatment was made from spiked antibiotic acidified milk and induced YC-11). These experiments were repeated for each antibiotic separately. The chemical analysis, microbiological, and sensory evaluation were carried out on the fresh samples, after 3 and 7 days throughout the storage period at 7 ± 2°C. The results indicated that it is possible to induce the YC-11 starter to the two understudy, antibiotics successfully and can be used it in yoghurt manufacture. The results showed that the fourth treatment was equal to control in relation to sensory evaluation, microbiology and chemical analysis. So that, the induced starters and direct acidification were selected to be conducted in milk containing antibiotic residues.

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