Vol. 32, May, 2012.

1/32 LISTERIA MONOCYTOGENES ENHANCES AND IS KILLED BY NEUTROPHILS EXTRACELLULAR TRAPS

Walid K. A. Mohamed

Department of Microbiology, Faculty of Pharmacy, Al-Azhar University,

Assiut; Egypt

Neutropohils together with macrophages and natural killer (NK) cells play a mandatory role in the early phase of Listeria infection. They encounter and kill Listeria intracellularly upon phagocytosis when their antimicrobial granuoles fused with the phagosome. However, a little is known if neutrophils can kill Listeria extracellularly. In this study, the phagocytic ability of neutrophils was blocked and they have been examined how they react when coming in contact with Listeria monocytogenes. It is revealed that Listeria activates the release of neutrophil extracellular traps (NETs) which in turn ensnare and inhibit survival of Listeria.

 

 

2/32 EVALUATION OF SOME MICROBIAL BIOCONTROL AGENTS AND COMPOST AGAINST ROOT-KNOT NEMATODE

(MELOIDOGYNE JAVANICA)

Neveen M. Galal; *Ebtsam, M. Morsy and *Osama N. Massoud

Plant Pathol. Res. Inst., Agricultural Research Center (ARC)

*Soils, Water and Environment Res. Inst., Agricultural Research Center (ARC)

Different biocontrol agents via; Streptomyces antibioticus, Bacillus subtilis, Arbuscular mycorrhizal fungi, Trichoderma harzianum and Pasteuria penetrans were tested against root-knot nematode Meloidogyne javanica in vitro and in vivo on tomato plants. Results of in vitro experiment showed that all the tested microorganisms decreased significantly percentage of nematode hatching compared to the control. The greatest inhibition of egg hatching was with B. subtilis, where it gave 100%, 99%, 95%, 98% at 24, 48, 72 and 96 hours, respectively. In greenhouse experiment, results showed that the number of galls, egg masses, females/root system and number of juveniles/250 g soil were significantly reduced as a result of biocontrol agent treatments where the highest reduction percentages per root system obtained with the treatment B. subtilis it gave 81% (in galls), 88% (in egg masses), 83% (in females) and 71% (in eggs/egg masses), whereas the highest reduction in juveniles recorded with mixture treatment (93%). Applications of biocontrol agents particularly AMF, S. antibioticus and mixed treatments led to a marked increase in P. penetrans infected females / root system. Mixture of bioagents contributed in increasing percentage of AM colonization in plant roots, mean number of AMF spores in soil and NPK percentages in plants. This promoted the plants to grow in a good healthy state.

 

 

3/32 IDENTIFICATION OF LESIONAL CD4+CD25+ REGULATORY T CELLS IN SYSTEMIC LUPUS ERYTHEMATOSUS.

Manal Darwish, Makram Attalah andSherin M. Hosney*

Department of Microbiology and Immunology, *Internal Medicine and Rheumatology, Faculty of Medicine, Ain Shams University

Natural CD4+CD25+ regulatory T cells (T regs) show a potent immunosuppressive function and contribute to immunologic self tolerance by suppressing potentially auto-reactive T cells. Depletion of these cells leads to the induction of severe autoimmune diseases in animal models. In systemic lupus erythematosus (SLE), conflicting data on the role of CD4+CD25+ T regs in the disease have been presented in previous literature. Decreased numbers of peripheral blood T regs have been reported by most studies on SLE patients with active disease, but non-impaired or even increased CD4+CD25+ T regs numbers have also been described. In addition, both deficient and normal suppressive capacity of isolated T regs has been observed in SLE. The aim of this study was to characterize and quantify CD4+CD25+ T regs in the peripheral blood of SLE patients and to correlate it with disease activity and severity hoping that the manipulation of these T regulatory cells could have important implication in the management of this life threatening autoimmune disease. The present study was conducted on 37 subjects (25 SLE patients and 12 controls). All patients fulfilled at least four of the revised SLE criteria of the American College of Rheumatology. The patients group was divided according to SLE disease activity index into 2 groups: group 1 categorized as quiescent lupus patients with SLEDAI= 2, and group 2 as active lupus with SLEDAI ≥4. All subjects were subjected to full medical history and full clinical examination. Flowcytometry was used to assess the quantity of CD3+/CD4+/CD25+ T cells. The results showed that patients with SLE had statistically significant lower levels of CD4CD25 T cells compared with normal controls. Quiescent SLE Patients who were compliant to glucocorticoids treatment represented in clinical improvement and ESR declination are directly correlated to high level of CD4CD25 T cells in blood, and on the contrary was the non compliant patients. Therefore we concluded that SLE patients have impaired Treg production or maintenance, a trait strongly associated with SLE disease

 

 

4/32 QUANTIFICATION OF VIRAL LOAD OF HEPATITIS B AND HEPATITIS C VIRUSES IN PLASMA: COMPARING CONVENTIONAL PCR, REAL-TIME PCR AND ISOTHERMAL NASBA

Mohamed S. Badr, Ola I. Ahmed٭, Amany S. Awad٭, Mohamed S. Salama٭٭

Molecular Biology Department, Medical Research Center, Ain Shams University

٭Medical Microbiology & Immunology Department, Faculty of Medicine,

Ain Shams University

٭٭Molecular Biology Department, Faculty of Science, Ain Shams University

Detection and monitoring of microbial load of variety of microbial targets could contribute immensely to research efforts aimed at understanding the pathogenicity of organisms and progression of microbial diseases. In the present study we aimed to find a specific and sensitive quantification method for detection and quantification of nucleic acids of HBV and HCV. The present study was carried out on 40 patients recruited from Ain Shams University Hospital; 20 patients diagnosed clinically and laboratory as having chronic hepatitis B and 20 with chronic hepatitis C. We compared two methods of nucleic acid extraction; manual and automated methods and different techniques of quantification of nucleic acids (End point polymerase chain reaction (PCR), real-time PCR and nucleic acid sequence-based amplification (NASBA) technique). Results showed that the yield of nucleic acid extracted by the automated technique was higher than that extracted by the manual method (p<0.001). However, the difference in the purity of extracted nucleic acid was not statistically significant between the 2 methods of extraction. The automated technique is time saving, less tedious, with less risk of contamination, however it is more expensive. By comparing the three methods of nucleic acid amplification and quantification regarding dynamic range, median and mean rank of HBV and HCV loads, results showed a statistically highly significant difference among the three methods (P<0.001). Thus, it could be concluded that automated extraction systems are recommended to be used as a routine diagnostic tool. Both the real-time NASBA and real-time PCR are reliable methods for quantification of targets, with low risks of contamination and broad dynamic range. NASBA technique has another advantage being isothermal in nature thus; do not require thermal cycling apparatus.

 

 

5/32 BASELINE-SURVEILLANCE OF DEVICE ASSOCIATED INFECTIONS (DAIS) IN A SURGICAL-MEDICAL INTENSIVE CARE UNIT AT ZAGAZIG UNIVERSITY HOSPITAL, EGYPT

Abeer El Nakera, Takwa E. Meawed* and Mona Aboserea**

Ansethiology & Intensive Care, * Microbiology & Medical Immunology and **Public Health & Preventive Medicine, Faculty of Medicine, Zagazig University.

There are no available statistical data about device-associated infections (DAIs) rate in Zagazig University Hospitals’ intensive care units. As we cannot control what we could not measure, we aimed to detect microbial and antimicrobial profile of device associated infections (DAIs), determine the severity of illness and mortality in infected patients and identify the main obstacles to implement infection control program. A prospective surveillance study was carried out in one-year duration for patients with device associated infection in a mixed surgical-medical intensive care unit (ICU). Adult patients admitted to the studied ICU for more than 48 hours and had devices inserted, were observed for criteria of DAI. Data collected were patients' scoring according to Acute Physiology and Chronic Health Evaluation (APACHE II), Sequential Organ Failure Assessment (SOFA) and injury severity scores, identification of DAIs’ microbial and antimicrobial profile, patients’ ICU/hospital stay and mortality. A questionnaire format distributed to ICU health care providers about infection control program implementation. Out of 912 patients admitted to the studied ICU and managed with invasive devices, 358 patients acquired DAI. Overall DAI rate was 39%. Ventilator-associated pneumonia (VAP) was the most frequent infection with an incidence of 64.5 / 1000 ventilator days. Methecillin-Resistant Staph Aureus (MRSA) was the most frequent organism (28%). High DAIs-related mortality was detected especially among CR-BSI's patients. There was a significant relation between mortality and Klebsiella infection. The main obstacle to implement infection control program was inadequate resources (39%).Implementation of strict infection control guidelines, planning for budget rearrangement and health care provider training courses are highly recommended to guard against DAIs in the ICU. A Second setting study is needed to assess the degree and effectiveness of interventions after at least two years.

 

 

6/32 COMPARATIVE STUDIES OF DIFFERENT EFFLUX PUMP INHIBITORS (EPIs) EFFECT ON PSEUDOMONAS AERUGINOSA RESISTANCE TO FLUOROQUINOLONES

Ehsan Abdel Saboor, Salwa S. Seif Eldin, Amany M. Nafei and Marwa Awad*

Department of Microbiology & Immunology, Faculty of Medicine, Assiut University

* Drug Research Center, Assiut University

Pseudomonas aeruginosa (Ps.aeruginosa) represents a phenomenon of antibiotic resistance, and demonstrates practically all known mechanisms of bacterial resistance. Active efflux is an important mechanism of resistance in Ps.aeruginosa. It contributes to the development of multiple resistances to all strategic antipseudomonal antibiotics. More than five hundred urine samples were collected from patients in Assiut University Hospital. Ps.aeruginosa was identified by conventional methods. The antibiotic susceptibity testing of isolates showed that 68% of isolates were resistant to ciprofloxacin and 62% were resistant to levofloxacin. In this study we compare effect of three efflux pump inhibitors (Reserpine, Pantoprazole and Carbonyl cyanide m-chlorophenylhydrazone (CCCP)) on the activity of ciprofloxacin and levofloxacin. By measuring ability of these agents to potentiate effect of ciprofloxacin and levofloxacin against resistant Ps.aeruginosa isolates. The results showed that reserpine was able to potentiate effect of ciprofloxacin in 50% of isolates, and in 5.5% for levofloxacin. Pantoprazole results were 33.3% for ciprofloxacin, 16.7% for levofloxacin. Finally CCCP potentiate effect of both ciprofloxacin and levofloxacin in 11.1% of isolates.

7/32 ROLE OF BACTERIAL, FUNGAL AND ATYPICAL PATHOGENS INFECTIONS IN ACUTE EXACERBATIONS OF CHRONIC OBSTRUCTIVE PULMONARY DISEASE

Alia H. Ahmed, Ola I. Ahmed, Eman R. Ali* and Narges M. Elaish

Medical Microbiology & Immunology Department,*Chest Department, Faculty of Medicine, Ain Shams University.

Chronic obstructive pulmonary disease (COPD) affects large numbers of patients and is associated with significant morbidity, disability, and mortality. COPD is complicated by frequent and recurrent acute exacerbations (AEs), which are associated with enormous health care expenditures and high morbidity. The Aim of the present study is to define the role of bacterial, fungal and the atypical pathogens Chlamydia pneumoniae and Mycoplasma pneumoniae infections as risk factors causing AEs of COPD in order to introduce proper antimicrobial agents as an effective therapy. The present study was conducted on 40 patients divided into two groups: the first group included 25 patients suffering from AE of COPD and the second group included 15 patients with stable COPD as a control group. Semi quantitative method for counting potential pathogens in sputum samples was done and the presence of potential pathogen in counts more than 106/ml of sputum is considered significant microbial growth. This was followed by isolation and identification of the potential pathogens by conventional methods while C. pneumoniae and M. pneumoniae were detected using multiplex real time Polymerase Chain Reaction (PCR). Results showed that 16 (64%) of group I patients had significant microbial growth while none of group II had significant microbial growth. The difference between the two groups was statistically highly significant (P<0.001). Strep. pneumoniae was the most commonly isolated organism. It was isolated from 5 (20%) patients with AE of COPD followed by Staph. aureus and Candida albicans. Each of them was isolated from 4 (16%) patients. Using multiplex real time PCR, C. pneumoniae was detected in 5 patients (20%) of group I versus 2 patients (14.29%) of group II while M. pneumoniae was detected in 5 patients (20 %) of group I versus one patient (7.7%)of group II and the difference between the two groups was statistically insignificant (p>0.05). Thus, it could be concluded that bacterial and fungal infections play an important role in pathogenesis of AE in COPD patients. However, the role of C. pneumoniae and M. pneumoniae in the development of AE is not yet clear and need more studies to clarify it.

8/32 SLC11A1 GENE AND VDR GENE POLYMORPHISMS IN ASSOCIATION WITH SUSCEPTIBILITY TO PULMONARY TUBERCULOSIS

Reham A. Khalifa, Marwa S. Fathi, Hala M. Salem* and Nehad M. Osman*

Medical Microbiology and Immunology,   *Chest department, Faculty of Medicine, Ain Shams University

       Tuberculosis (TB) is a global public health problem, resulting in 1.6 million deaths worldwide in 2005. About 10% of all people infected with Mycobacterium tuberculosis (Mtb) develop active pulmonary disease, suggesting differences in susceptibility to progression from infection to disease. Polymorphism in Solute Carrier Family 11 member 1 (SLC11A1) (formerly NRAMP1) gene and vitamin D receptor (VDR) gene has been implicated in host susceptibility to TB. The aim of the present study was to study SLC11A1 gene and VDR gene polymorphisms among primary and reactivational pulmonary TB patients in comparison with healthy controls and their correlation with susceptibility to Mtb infection. Thirty well defined pulmonary TB patients and 30 unrelated healthy controls were enrolled. Genetic polymorphisms of VDR gene (TaqI and FokI) and SLC11A1 gene (INT4, D543N and 3_UTR) were analyzed using PCR and RFLP. QuantiFERON-TB Gold In-Tube (QFT-GIT) test was performed for healthy controls to detect latent tuberculosis (LTB) infection. Results showed a highly significant difference between cases and controls regarding homozygote genotypes of D543N, 3_UTR and FokI polymorphisms where homozygote wild genotype represented 86.7%, 83.3% and 70% among controls in comparison to 30%, 26.7% and 26.7% among cases, and homozygote mutant genotype represented 43.3%, 53.3% and 60% among cases in comparison to 6.7% , 10% and 6.7% among controls respectively. Also, a highly significant difference was found between quantiferonpositive(QT+ve) and quantiferon negative (QT-ve) individuals as regards FokI polymorphism of VDR gene. In conclusion: Polymorphisms in the VDR and SLC11A1 genes were associated with susceptibility to TB. QFT-GIT was found to be a reliable test that can be used for periodic screening of HCW in healthcare facilities caring for TB patients, especially those with genotypes associated with high susceptibility to TB.

9/32 GENOTYPIC DETECTION FOR FLOUROQUINOLONE RESISTANCE AMONG MULTIDRUG RESISTANT PSEUDOMONAS AEURGINOSA ISOLATES

Salma M. Mansour, Nabila A.Elsheikh*, Wafaa El-Tayeb** and Magdy A. Amin***

Microbiology & Immunology Department, Faculty of Pharmacy, German University in Cairo; *Microbiology & Immunology Department, Faculty of Medicine, Al- Azhar University; **Microbiology Deptartement, Faculty of Pharmacy, Misr International University and ***Microbiology & Immunology Department, Faculty of Pharmacy, Cairo University.

In this study, the resistance of Pseudomonas aeruginosa to fluoroquinolones due to mutation DNA gyrase was investigated. And since this was not the only mean by which Pseudomonas aeruginosa acquires resistance, multidrug efflux pump overproduction and their effect on raising the MICs of both ciprofloxacin and levofloxacin were studied. Two efflux pump inhibitors (EPIs) including omperazole (100ug/ml) and reserpine (20ug/ml) were included in the present study. Meanwhile we screened for mutation in the two DNA gyrase subunits (gyrA and gyrB). Using single strand conformation polymorphism (SSCP). Our results revealed that mutation in DNA gyrase and efflux pump overproduction were shown to be the major contributors to the resistance of our isolates. On the other hand, some isolates showed neither mutation in DNA gyrase nor any change in the MICs of flouroquinolones upon the addition of EPIs.

10/32 PHENOTYPIC AND GENOTYPIC CHARACTERIZATION OF SOME VIRULENCE FACTORS IN PSEUDOMONAS AERUGINOSA STRAINS ISOLATED FROM DIFFERENT CLINICAL SOURCES IN MANSOURA UNIVERSITY HOSPITALS.

Dina Eid, Wael El-Naggar, Rasha Barwa and Mohammed A. El-Sokkary.

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

The present study aims to determine the extent of production of some virulence factors and antimicrobial resistance of P. aeruginosa isolated from different clinical sources in Mansoura University Hospitals. In addition, this research was done to characterize five bacterial genes that encode virulence determinants on both chromosomal and plasmid DNA using PCR technique. Ninety eight isolates of P. aeruginosa from diverse clinical sources were employed..Isolates were mainly recovered from endotracheal tube parts, urine, burns and wounds. In this study, the susceptibility to 10 antimicrobials was determined and analyzed among isolates. The antimicrobial susceptibility test showed that amikacin and imipenem were the most active antibiotics against tested isolates. All isolates were resistant to amoxicillin- clavulanic acid, cefadroxil, cefotaxime and cefepime, they manifested different degrees of sensitivity to the rest antimicrobials. The phenotypic detection of virulence factors revealed that pyocyanin was produced by all isolates, but higher amounts were produced by urine isolates. High percent of haemolysis was manifested by 77% of isolates, where urine and burn isolates exhibited higher hemolysis percent. Regarding total protease and staphylolytic activity, 82% of isolates were positive protease producers where higher protease and staphylolysis activity were noticed among blood and endotracheal isolates. Most of isolates (92.8%) were positive lipase producers and both wound and endotracheal isolates were shown to have higher production levels. Strong biofilm formation was observed for 18% of isolates and the highest level of biofilm production was noticed for endotracheal isolates as twelve out of twenty six gave OD ˃0.2. For genotypic detection, it was observed that among the fifty tested isolates, 100% were positive for toxA, aprA and lasB genes. A high percentage constituting of 74% and 94% were positive for exoS and exoU genes respectively. Plasmid extraction showed that 93% of tested isolates contained plasmids and the tested virulence genes were harbored by most of them (toxA, lasB and exoU genes were carried by 92% of tested isolates while aprA and exoS genes were carried by 84.6% and 61.5% respectively). The present study confirmed previous observations that the virulence of P. aeruginosa is multifactorial and resistance to antimicrobials was not generally associated with the level of production of the pathogenicity factors. Plasmid studies confirmed their crucial role in the horizontal transfer of virulence.

11/32 IMPROVEMENT OF HYGROMYCIN A PRODUCTION BY A MUTANT STRAIN OF STREPTOMYCES HYGROSCOPICUS NRRL2338 USING SHAKE FLASK

Rasha Barwa, Khaled Abd El Galil, El- Sayed E. Habib

Department of Microbiology, Faculty of Pharmacy, Mansoura University,

Mansoura, Egypt.

Hygromycin A, is a fermentation derived natural product. It was first isolated as antibacterial agent from Streptomyces hygroscopicus to inhibit the ribosomal peptidyl transferase activity. The aim of this study is to enhance the production of hygromycin A through optimization of the production media and condition then isolating true mutant which carry beneficial mutations to be used for the production of hygromycin A. Streptomyces hygroscopicus biomass was tested for the production of hygromycin A using different media and different production conditions. Ultraviolet mutants were selected and tested for the production of hygromycin A. Maximum productivity of Streptomyces hygroscopicus (5000 μg/ ml) was obtained in the six day upon using Isp2 +0.75% galactose. Greater production of hygromycin A was obtained when using 16% inoculum size rather than 10% and 20% inoculum size. Regarding temperature, Greater production of hygromycin A was obtained upon incubation at 30°C rather than 28° C and 32°C. In the strain improvement study, the genetic modification of Streptomyces hygroscopicus NRRL 2388 was carried out by UV irradiation method. The result showed that mutant 3 has the highest hygromycin A productivity (6294 μg/ ml).

 

 

12/32 TRANSFER OF ANTIBIOTIC RESISTANCE GENES FROM LACTIC ACID BACTERIA TO SOME PATHOGENIC BACTERIA

Gamal Fadl M. Gad, Ahmed M. Abdel-Hamid* and Zeinab Shawky H. Farag

Department Microbiology, Faculty of Pharmacy, Minia University

*Department of Botany and Microbiology, Faculty of Science, Minia University

The ability of lactic acid bacteria (LAB) strains, isolated from pharmaceutical and dairy products, to transfer tetracycline and erythromycin resistance genes encoded by tet(M) and erm(B) to some pathogenic strains was examined through conjugation. The pathogenic strains were isolated and identified from clinical samples of patients suffering from food poisoning and urinary tract infections. Strains that were sensitive to tetracycline and/or erythromycin and resistant to one or more of the other antibiotics were selected as recipients. The LAB strains were first tested for their antibacterial activity against the selected pathogenic strains. LAB strains had antibacterial activities were excluded from mating experiment. In vitro transfer experiments were carried out using plate and broth mating techniques. tet(M) or erm(B) genes were transferred from donor LAB to Enterococcus and Listeria spp. Transfer frequency was lower in case of broth mating when compared with plate mating.Transconjugants were confirmed by using antibiotic agar dilution test to determine MICs and by detecting the corresponding marker genes through PCR.

13/32 DETECTION OF EXTENDED-SPECTRUM-Β-LACTAMASE-PRODUCING   ENTEROBACTERIACEAESTRAINS ISOLATED FROM INTENSIVE CARE UNITS' PATIENTS SUFFERING FROM HOSPITAL ASSOCIATED URINARY TRACT INFECTIONS

Hend A. Sharaf, Randa S. Abd El-latif and Tarek M. Sherif*

MedicalMicrobiology & Immunologyand *Surgury Departments,

Faculty of Medicine, Zagazig University

Extended-spectrum- beta - lactamases (ESBLs) are a large, rapidly evolving group of enzymes that confer resistance to oxyimino cephalosporins and monobactams and are inhibited by clavulanate. Rapid reliable detection of ESBL production is a prerequisite for successful infection management and for monitoring resistance trends and implementation of intervention strategies. This work aimed to identify ESBL producing Enterobacteriaceae isolates among patients in Intensive Care Units (ICUs) suffering from hospital associated urinary tract infections (UTIs) and study some characters of their plasmids. This study was conducted from April 2010 to April 2011, at Medical Microbiology and Immunology Department, Zagazig University. Urine samples were collected from 220 patients admitted to ICUs of Internal Medicine Department of Zagazig University Hospitals. Patients were suspected to have hospital associated UTIs. Enterobacteriaceae isolated from positive cases of UTIs were identified using API 20E. ESBLs production was tested for Enterobacteriaceae isolates using double disk diffusion test. Plasmids from all isolates were purified and their characters were compared between ESBL producing and non producing isolates. Enterobacteriaceae was isolated from 192 (87.27%) out of 220 urine samples collected. Escherichia coli (E. coli) was detected in 145 (65.9%) samples and found to be the leading cause of UTIs among patients of ICUs. Sixty one (31.77%) out of 192 Enterobacteriaceae isolates were proven positive for the production of ESBL. Klebsiella pneumonia (K. pneumonia) isolates showed the highest percentage of ESBL production (70%). The number of ESBL producing strains containing plasmids were higher than those of ESBL non producing strains (50-100 and 33.33- 45.04 % respectively). Big plasmids (more than 23.13 kbp) were found only in ESBL producing strains. The increasing prevalence of ESBL-producing Enterobacteriaceae is becoming a major threat necessitating construction and implementation of new infection control policies and procedures to overcome this enormously growing problem.

14/32 THE IMPACT OF SOME BIOCONTROL AGENTS AGAINST SOME PATHOGENIC FUNGI OF FABA BEAN

Nadia F. Emam, Mohamed Zakaria, Esaad H. Bedawi* and Mona M. Ali*

Agriculture Microbiology Department, Fac. Agric., Cairo Univ., Giza, Egypt *Agriculture Microbiology Department, Soil Water and Environment, Agric., Research Center, Giza, Egypt   

A pot experiment was conducted to determine the efficiency of faba bean seed inoculated with Rhizobium leguminasarum, AM fungi and / or compost as biocontrol agents for damping off root-rot diseases of faba bean plants. Twenty isolates of Rhizobium spp were isolated from nodules of faba bean plants grow under naturally infested soil with pathogenic fungi. The isolate R12 was identified as Rhizobium leguminasarum as it was obligate symbiosis on faba bean and exhibited high activity of nitrogenase enzyme. Eleven isolates of filamentous fungi were isolated and classified as F. oxysporium, F. Solani, F. moniliform and Rhizoctonia solani. The isolate No.12 of R. leguminasarum (R12) inhibited 99% of the isolated fungi. Among five different types of composts, compost (C2) recorded the highest inhibition value against all tested pathogenic fungi. Seeds treatment with Rhizobium leguminasarum (R12), AM Fungi, compost (C2) in individual reduced the damping off faba bean plants compared with untreated control. The combined treatment with the tested Rhizobium, AM fungi and compost was the most effective treatment as indicated by increasing plant height and shoot dry weight up to 72 % and 78 %, respectively. Regarding the root length and dry weight, the values scored significant increases of (55% and 76 %) respectively compared with control treatment. Plants supplemented with Rhizobium + AM Fungi and compost influence positively on nodule number, dry weight as well as pod number and weight of 100 seed.

ِِِِ15/32 EVALUATION OF THE ANTIMICROBIAL ACTIVITIES OF SOME PLANT ESSENTIAL OILS

Ahmed Abdelaziz, Tarek El-Banna, Amal Abo Kamar and *Marwa Azzam

Microbiology Department, Faculty of Pharmacy, Tanta University,Egypt and

*Ministry of Health,Egypt.

Twenty essential oils were screened to determine there antimicrobial activities against 3 reference strains (Staphylococcus aureus NCTC 6571, Escherichia coli ATCC 10418, and Candida albicans ATCC 10231) using the cup-plate diffusion method. Chamomile, cinnamon, cumin, and wild basil oils showed the highest antibacterial activity followed by clove, parsley, and rose red oils. Camphor tree, garlic, ginger, lemon, rosemary, spearmint, tea tree, thyme and turmeric oils showed moderate to low antibacterial activity. In contrast, marjoram, nutmeg, radish, and sage oils showed no antibacterial activity. On the other hand, cinnamon, rose red, and wild basil oils showed the highest anticandidal activity followed by clove, cumin, and lemon oils. Camphor tree, garlic, ginger, spearmint, and thyme oils showed moderate to low anticandidal activity whereas, chamomile, marjoram, nutmeg, parsley, radish, rosemary, sage, tea tree, and turmeric oils showed no anticandidal activity. On the basis of promising activity the most active essential oils were chosen for further study. The Minimum Inhibitory Concentrations (MIC) of chamomile, cinnamon, cumin, parsley, rose red, and wild basil oils were determined against 83 bacterial isolates (13 Staph aureus, 15 Pseudomonas aeruginosa, 15 Klebsiella pneumoniae, 15 Escherichia coli, and 25 Proteus mirabilis) in addition to S. aureus NCTC 6571 and E. coli ATCC 10418 reference strains. Also MICs of cinnamon, clove, cumin, lemon, rose red, and wild basil oils were determined against 6 C. albicans isolates and C. albicans ATCC 10231. MIC50, MIC90, and geometric mean were calculated. Rose red, wild basil, cumin, and cinnamon oils were the most active antibacterials. S. aureus was the most susceptible bacteria while Pr. mirabilis was the least susceptible. On the other hand wild basil oil and lemon oil exhibited the highest and the least active against C. albicans, respectively.

16/32 SYNERGISTIC EFFECTS OF SOME PLANT ESSENTIAL OILS COMBINATIONS WITH ANTIBIOTICS ON MULTIPLY RESISTANT BACTERIAL PATHOGENS

Tarek El-Banna, Ahmed Abdelaziz, Amal Abo Kamar and *Marwa Azzam

Microbiology Department, Faculty of Pharmacy, Tanta University,Egypt and

*Ministry of Health,Egypt.

The Minimum Inhibitory Concentrations (MICs) of six essential oils including: chamomile, cinnamon, cumin, parsley, rose red, and wild basil oils against 53 bacterial isolates (13 Staph aureus, 15 Escherichia coli, and 25 Proteus mirabilis) were determined. The possible synergistic effects when these oils were combined with the antibiotics (cefotaxime, tetracycline, and erythromycin) against antibiotic-resistant isolates were also evaluated. S. aureus was more susceptible to essential oils than E. coli and Pr. mirabilis. Rose red, wild basil, and cinnamon oils showed the highest inhibitory effect. Rose red oil had interesting activity against Pr. mirabilis (40% of isolates presented MIC values ≤8 µg/ml). On the other hand, parsley oil was the least active. Essential oils had a significant synergistic effect on tested antibiotics and the most of the studied combinations (95.6%), were synergistic. Low concentrations (1/4 × MIC) of essential oils produced significant restoration of susceptibility to antibiotics by as much as 4-fold, 8-fold, and 16-fold in the majority of tested combinations (23.1%, 27.5% and 20.9%, respectively). Interestingly, 8 of tested combinations had 64-fold and one combination had 128-fold decrease in the MIC of the antibiotic, respectively demonstrating dramatically significant enhancement of the killing activity of antibiotics exerted by essential oils. Moreover, essential oils changed the resistance pattern of bacterial isolates in 83.5% of tested combinations from resistant to sensitive against antibiotics. The synergy between essential oils and antibiotics was observed to be bacterial, essential oil as well as antibiotic specific.

17/32 IMMUNOFLUORESCENCE FOLLOWED BY TISSUE CULTURE FOR DIAGNOSIS OF INFLUENZA A AND B VIRUSES AND DETECTION OF PANDEMIC (H1N1) 2009 SUBTYPE

Maha Kamal Gohar and Rehab Rabie

Medical Microbiology and Immunology, Faculty of Medicine, Zagazig University

Influenza virus (family Orthomyxoviridae), is a common infection of the respiratory tract. There are three influenza A virus subtypes circulating in humans: H3N2, seasonal H1N1, and 2009 H1N1 pandemic strain. This study was conducted for diagnosis of influenza virus infection as a cause of influenza like illness (ILI) and to compare Immunofluoresence (IF) as a rapid technique for diagnosis of influenza virus infection in nasopharyngeal swabs versus virus culture as a gold standard for its diagnosis. Detection of Pandemic (H1N1) 2009 was performed by one step RT- PCR. This study was conducted in Medical Microbiology and Immunology department, Faculty of Medicine, Zagazig University, during the period from September 2010 to August 2011. The study included 75 patients (46 males and 29 females) with age range (4 days - 65 years), median=19.Nasopharyngeal swabs were subjected to IF detection of the virus directly on the sample followed by tissue culture on MDCK cells and IF staining on tissue culture. Then detection of influenza A Pandemic (H1N1) 2009 subtype by one step RT- PCR. Age group (< 2 years) was the most susceptible age group to influenza like illness and there was male predominance (61.3%). Indirect immunofluoresence test performed directly on the nasopharyngeal samples revealed 23 positive cases out of 75 (30.7%). Virus culture revealed that 26 out of 75 (34.7%) cases were confirmed as influenza viruses. Influenza A virus represented 21 cases and influenza B virus 5 cases. In comparing IF versus the virus isolation as the gold standard, its sensitivity =76.9 %, specificity = 93.9 % and accuracy = 88 %. As regard IF before and after isolation technique, it was statistically non significant. None of the vaccinated individuals was positive for influenza indicating the value of vaccination for protection against influenza. Fever and dry cough were the best predictors for influenza virus infection. Six out of 21 tissue culture positive for influenza type A were Pandemic (H1N1) 2009 subtype. Two of them had GIT manifestation and three had febrile seizures indicating the need for further studies to detect influenza A (H1N1) 2009 shedding in stool and viral load in patients with febrile seizures.  

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