Vol. 36, September, 2013

1/36 BACTERIURIA AND ANTIBIOTICS RESISTANCE IN ILEAL NEOBLADDERS: PREVALENCE AND CLINICAL SIGNIFICANCE

Ramadan Hassan, Hassan Abol-Enein, Esam Elsawy, Mohamed M. El-Sokary, Hany Kenawy and Wafaa. Ezz El-Arab

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

Urology and Nephrology Center, Mansoura University, Egypt.

The incorporation of intestinal segments in the urinary tract favors bacterial growth of the skin flora, anaerobic bacteria, and uropathogenic strains.Bacteriuria is common in all kinds of reconstruction. Bacteria identified in urine culture were mainly intestinal species. Drug resistance of pathogens is a serious medical problem, because of very fast arise and spread of mutant strains that are insusceptible to medical treatment. Microorganisms use varied mechanisms to acquire drug resistance, for example horizontal gene transfer may be sufficiently common to drive the dissemination of antibiotic resistance alone. Frequent horizontal gene transfer would broadly distribute resistance genes, resulting in many resistance gene combinations. The present study aims to determine the presence of bacteriuria and if it symptomatic or asymptomatic also to show extend of antibiotics resistance in patients with ileal neobladder. Overall 600 urine samples from the 200 patients underwent ileal neobladder (between December 1991 and May 2010), collected from Urology and Nephrology Center, Mansoura University (UNC). The bacterial cultures for these patients were screened randomly at the first time and screened for 2nd and 3rd time after taking suitable antibiotics if they were positive growth. To show the role of horizontal gene transfer in trimethoprim-sulphamethoxazol resistant E. coli strain, first we examined the presence of type I and type II integron, in a total of 45 strains of E. coli, there were 20 (44.5%) strains contain type I integron, 9 (20%) strains contain type II integron and 10 (22.2%) strains contain both type, and 6 (13.3%) strains were negative for both type of Integrons. Second by detection of sul gene in the same E. coli isolates. There were 43 strains capture Sul1 by 95%, (16) strains capture Sul 2by 35%, 14 strains contain both Sul1&Sul2 by 31% and only one strain carry Sul3 by 2.2%. According to association to type I integron, it was found that 27 (62. 8%) strains caring Sul1was associated with type I integron and there was no connection between Sul2& Sul3 with type I integron. Then a sequencing to 7 samples contain type I& type II were done to show dfrs genes in both types, in this study there were dfr A1, dfr5 and dfr7.

2/36 INCIDENCE OF FUNGAL INFECTIONS IN CASES OF HAEMATOLOGICAL MALIGNANCIES

Wageih S. El Naghy, Hanan S. Abdel Khalek, Mona O. Ramadanand Atef Taha*

Department of Medical Microbiology and Immunology, Faculty of Medicine, Tanta University &*Internal Medicine Department, Faculty of Medicine, Tanta University.

Haematologic malignancies come from bone marrow and lymphatic cells. They comprise a group of cancers that include the acute, chronic leukaemias, Hodgkin's disease (HL), non-Hodgkin's lymphomas (NHL) and multiple myeloma. Approximately 20 – 50% of patients of haematological malignancies have evidence of invasive fungal infections at autopsy. The most common species were Aspergillus, Candida, Penicillium and Mucor genera. In this study, 100 patients of haematological malignancies were selected from Haematology Unit, Internal Medicine Department, Faculty of Medicine, Tanta University and Tanta Cancer Institute. The patients who received cytotoxic drugs or radiotherapy were diagnosed according to clinical and laboratory investigations. The samples collected included nasal swabs, sputum and skin lesions swabs.Laboratory examination of the samples were done through direct microscopic examination. Each sample was cultured on Sabaraud's dextrose agar containing chloramphenicol (0.05 gm / L) incubated at room temperature and examined daily, over at least 2 weeks, for the appearance of growth. Identification of the suspected culture was done using the following scheme: 1) Gross colonial appearance: Texture, appearance and color of the colony. 2) Direct microscopical examination of the suspected intact culture from suspected colonies. The present study found that 94% of total number chosen for this study gave fungal growth. Also it was found that most of cases had neutropaenia (73%). They were different in severity; mild (20%) with ANC (absolute neutrophilic count) (1500 – 1000), moderate (33%) with ANC (1000-500) and severe neutropenia (20%) with ANC < 500. The present study revealed that the most common isolated fungi were Penicillium genus (39.4%) followed by Candida genus (38.3%) then Aspergillus genus (17%) and lastly Mucor genus (5.3%).As regards to the type of Aspergillus spp. isolated, it was found that Aspergillus fumigatus was (37.5%) and Aspergillus niger was (62.5%). Penicillium genus was the most frequently encountered (39.4%), Candida genus comes next (38.3%), followed by Aspergillus genus (17%) and Mucor genus (5.3%).

3/36 INHIBITION OF QUORUM SENSINGAND RELATED VIRULENCE FACTORSBY SUB-LETHAL CONCENTRATIONS OF AMINOGLYCOSIDESIN PSEUDOMONAS AERUGINOSA PAO1

Somaia A. El-Mowafy, Khaled H. Abd El Galil, El-Sayed E. Habib, Mona I. Shaaban.

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

Antipathogenic therapy is a new approach for controlling bacterial infection through bacterial attenuation without induction of microbial resistance. This could be achieved by inhibition of cell-cell communication through targeting quorum-sensing (QS), and inhibition of autoinducer dependent virulent gene expression. This study examined the effect of sub-inhibitory concentrations of aminoglycosides (Ak and Gm) on quorum sensing, virulence factors and biofilms of Pseudomonas aeruginosa PAO1. The QS inhibitory effects of aminoglycosides were assessed by measuring the changes in the quorum-sensing signal molecules N-acyl homoserine lactones, and other virulence factors such as elastase, protease, hemolysin, pyocyanin, biofilms formation and bacterial motility. Treated P. aeruginosa with sub-inhibitory concentrations of aminogylcosides showed significant reduction (P<0.01 and P<0.05) in levels of N-acyl homoserine lactones, and subsequent decrease in the quorum sensing controlled virulence factors such as elastase, protease, hemolysin. Biofilm formation was also attenuated yet, in the presence of lower concentrations of aminoglycosides up to 1/20 or 1/50 of MIC. Furthermore, In the presence of non-lethal concentrations of aminoglycosides, bacterial motility was significantly attenuated.In conclusion, aminoglycosides showed potential QS inhibitory activity at sub-MIC concentrations that could be effective in controlling quorum sensing and related virulence factors in P. aeruginosa.


4/36 PREVALENCE OF H. PYLORI ICE A1 AND ICE A2 GENE ALLELES AMONG PATIENTS WITH DIFFERENT GASTRIC LESIONS

Nalgaa F. Abd El Haliem, Zeinab N. Said, Sumaya H. El-Shazly*, Safia M. Mustafa*, Nagwa M. Abd El Rahman**, Khaled S. El bassel*** and Mona M. Metawea***

Microbiology & Immunology, Tropical Medicine*, Pathology** Departments,         Faculty of Medicine (for Girls), Al-Azhar University, Cairo, Egypt

Gastroenterology, Hepatology and Endoscopy***

Educational Hospital and Institute Organization.

Helicobacter pylori (H. pylori) are important human pathogens that cause severe gastrointestinal diseases. Several virulence factors of H. pylori play crucial role in the pathogenesis of the infections.The role of H. pylori different gene alleles on the clinical outcome is an area of extensive studies. The aim of this work is to investigate the prevalence of H. pylori ice A1 and ice A2 gene alleles in patients with different gastric lesions and to evaluate any relation existed between these genotypes and the clinical outcome. A prospective analysis of ninety eight patients who underwent endoscopy was included in this study. Patients were divided into three groups according to the endoscopic and histopathological findings, 37 patients with gastritis, 30 patients had gastric ulcer and 31 patients with gastric mass. H. pyloriDNA was isolated from antral biopsies and the presence of ice A1 and ice A2 genotypes were determined by polymerase chain reaction (Multiplex-PCR) technique. Results: H. pylori were detected by histopathology in 76.5 % (75/98) while H. pyloriDNA detection by PCR was 68.4% (67/98).The ice A1 and ice A2 genotypes were detected in 43.3% (29/67) and 53.7% (36/67), respectively among H. pyloriDNA positive patients. Peptic ulcer patients recorded significant difference of ice A2 detection 64% (16/25) than the other groups (P < 0.05). Cases with H. pylori positive ice A2 are at 3.5 more risk to get ulcer than those with negative result (odds ratios (OR) = 3.56, with (95% CI), P < 0.05). There was significant relation of ice A2 and grading ofmononuclear cell (MNC) infiltration in all patients, while, it was only between ice A1 and gastric carcinoma. Conclusion: These finding reinforces the importance of the interplay between the virulence of H. pylori genotypes ice A and host susceptibility in the development of more severe illness as peptic ulcer disease (PUD) and gastric carcinoma. Large scaled studies are needed for better evaluation of H. pylori ice A gene and disease relationship.

5/36 DETECTION OF Klebsiella Pneumoniae Carbapenemases AMONG Gram Negative Clinical Isolates

Marwa Shabban and Safaa Abdel-Rahman

Medical Microbiology and Immunology Department, Faculty of Medicine, Ain Sham University

Carbapenem-resistant Gram negative bacteria have recently evolved as a significant problem in the health care. One of the mechanisms of resistance to carbapenems is related to their ability to produce Klebsiellapneumoniae Carbapenemase (KPC) enzymes. This study aimed to evaluate carbapenemresistance in Gram negative clinical isolates with particular emphasis on KPC detection as the probable mechanism ofresistance among these isolates. Out of 120 different clinical samples 66 Gram negative bacilli were isolated, identified, and subjected to antimicrobial susceptibility testing by disk diffusion testagainst three carbapenem antibiotics (Imipenem, Ertapenem, and Meropenem). Isolates resistant to any of the tested carbapenems were further screened for carbapenemase by the modified Hodge test (MHT). KPC productionwas tested for by phenylboronic acid - combined disk (PBA-CD) test and detection of blaKPCgene by polymerase chain reaction (PCR).Totally, out of the 66 Gram negative isolates, 23 (34.8%) showed resistance to carbapenems. The carbapenem resistant Gram negative bacilli were; Acinetobacterbaumanni (5/11; 45.5%), Pseudomonas aeruginosa (8/21; 38.1%), and Enterobacteriacae (10/34; 29.4%). Resistance to carbapenems amongEnterobacteriacae was detected in 7 isolates of Klebsiellapneumoniae (20.6%) and 3 isolates of Proteus mirabilis (8.8%). Among thecarbapenem resistant isolates the blaKPC gene was detected in 6 isolates (26.1%); all were Klebsiellapneumoniae (6/7; 85.7%). The MHT detected 4 out of 6 blaKPC producing isolates, meanwhile, the PBA-CD test detected all blaKPC producing isolates. The blaKPC gene was not detected in carbapenem resistant isolates of either Pseudomonas aeruginosaor Acinetobacterbaumanni. The agreement between blaKPC PCR and thePBA-CD test and MHT was 82.6% and 73.9% respectively. In conclusion, KPC production is an important mechanism of carbapenem resistance among Enterobacteriacae. Rapid identification of KPC would be a key to limit the spread of such resistant strains. PBA-CD test can be a simple rapid screening test for detecting KPC mediated carbapenem resistance.

6/36 DETECTION OF BIOFILM GENES AMONG CLINICAL ISOLATESOF PSEUDOMONAS AERUGINOSA RECOVERED FROMSOME EGYPTIAN HOSPITALS.

Khaled Abd El Galil, M.S. AbdelGhani*, Sebak M.A.* and El-Naggar W.

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

*Microbiology and Immunology Department, Faculty of Pharmacy, Beni Suef University, Egypt.

Biofilm formation in Pseudomonas aeruginosa (P. aeruginosa) is controlled by about 1% of its chromosomal genes; from which four genes were selected for prospective work. The aim of this study was to determine the biofilm formation in P. aeruginosa clinical isolates and to evaluate the role of the selected genes in biofilm formation. A total of fifty isolates were recovered from different clinical samples isolated fromsome Egyptian hospitals by isolation on cetrimideagar media and then biochemically identified as P. aeruginosa. The antibiogram of the planktonic cells of all isolates was determined and showed that amikacin was the most potent antibiotic against all isolates. Quantification of biofilm formation of isolates was done by the microtiter plate method using crystal violet (CV) assay. According to the optical density (OD) readings, isolates were classified into the following categories: strong, moderate or weak biofilm producers. Screening for some selected biofilm genes as RhlI, PilA, PilT and PelB genes in some isolates using PCR, revealed the presence of these genes in both strong and weak biofilm producer isolates. These final results suggest the importance of these genes in biofilm formation and suggest the presence of other factors which may contribute in determining the degree of biofilm formation in P. aeruginosa.

7/36 ANTIMICROBIAL ACTIVITY OF POMEGRANATE EXTRACTS (PUNICA GRANATUM.) AGAINST FOOD-BORN BACTERIA

Maha Hewedy, Afaf Amin*, Camilia Michel** and Soha Zalat*

Botany Department, university college of Women for Arts, Science and Education, Ain Shams University,

*Food Safety Department, National Nutrition Institute, Cairo,

**Pharmacognosy Department, Faculty of Pharmacy, Cairo University.

All over the world, it is said that the use of plants bypeople for thetreatment of many diseases is an old tradition. Despitethe remarkable progress in synthetic organic chemistry ofthe twentieth century, more than 25% of prescribed medicinesin industrialized countries are derived either directly or indirectly from plants.However, plants used in traditional medicine are still understudied, particularly in clinical microbiology. The aim of the present study was to investigate the antimicrobial activity of polyphenolics of different parts of pomegranate (Punica granatum.), ethanol 70% extracts. Fractions obtained from the pomegranate ethanol 70% extracts (petroleum ether, butanol and ethyl acetate) were screened for their inhibitory effects on ten bacterial strains (Escherichia coli, Bacillus cereus, Staphylococcus aureus and seven different strains of Salmonella ) using the agar diffusion method. It was shown that ethanol 70% extract and its fractions of inner and outer pomegranate inhibited all the ten strains. Inner and outer pomegranate peel were better extracts than other parts for using as natural extracts had antibacterial activity. The minimum inhibitory concentration (MIC) of the inner and outer pomegranate peel extract ranged from 0.625 to 5 and 0.312 to 10 mg/ml respectively while the minimum bactericidal concentration (MBC) was between 2.5 to 5 and 2.5 to >10 mg/ml respectively.

8/36 STUDIES ON ENTEROTOXINS OF ESCHERICHIA COLI STRAINS ISOLATED FROM VARIOUS SOURCES

Ramadan H. Ibrahim, Eman Abdelmegeed and Dhafer Alwayli.

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

Enterotoxigenic Escherichia coli (ETEC) colonize the intestine and adhere to epithelium results in diarrhea. ETEC strains which produce enterotoxins are a significant cause of morbidity and mortality in the developing world. ETEC produce one or both of two enterotoxins; the heat stable (ST) and heat labile (LT) toxins encoded by plasmid-borne ST and LT genes respectively. In this study, a total of 100 E. coli isolates were collected from various clinical sources from some Hospitals of Mansoura, Egypt, and tested for resistance to eleven antimicrobial agents. Resistance was high for ampicillin (92%), amoxicillin (91%), cefotaxime (53%), cefoperazone (52%), ciprofloxacin (61%) and levofloxacin (52%). However, resistance was low for cefoxitin (4%) followed by tobramycin (8%) and gentamicin (13%). The isolated strains of E. coli were screened for heat-stable enterotoxin encoded genes (STp and estA2) and heat-labile enterotoxin encoded genes (LTA and eltB) as well as astA gene, which encodes enteroaggregative E. coli heat-stable enterotoxin 1 (EAST1) using PCR. We found that 83% of E. coli strains were positive, heat stable enterotoxin genes were found in 53% of total E. coli strains tested; 57 strains (57%) were positive for heat labile enterotoxin genes with eltB was more predominant and astA gene was distributed among 55%. Finally, typing some isolated E. coli which contain enterotoxins by investigation their plasmid profile and restriction fragment length polymorphism (RFLP).

 

9/36 PHENOTYPIC AND GENOTYPIC DETECTION OF Β-LACTAMASE PRODUCED BY CLINICAL STAPHYLOCOCCUS AUREUS ISOLATES

Sameh M. AbdelGhani, Khaled H. Abd El Galil*, Mostafa N. Taha**and

Wael A. El-Naggar*

Microbiology Department, Faculty of Pharmacy, Beni Suef University, Egypt.

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

** Microbiology Department, Faculty of Pharmacy, Nahda University, Egypt.

The aim of the present study is to compare between phenotypic and genotypic testing of β-Lactamase production by Staphylococcus aureus (S. aureus). A total of fifty isolates of S.aureus were recovered from different clinical sources by isolation on Mannitol salt agar media and biochemically identified as S. aureus. The antimicrobial susceptibility pattern was estimated and the tested clinical isolates were phenotypically screened for β-Lactamase production. They were also genotypically examined by PCR for the presence of genes for β-Lactamase, blaZ and mecA genes. Among fifty isolates of S. aureus, the antibiotic susceptibility testing showed that vancomycin was the most active antibiotic against all isolates (100%) followed by cefoperazone/sulbactam (86%) then, amikacin (78%), while all isolates were resistant to penicillin (100%). The phenotypic detection using nitrocefin test revealed that 43 isolates (86%) testedpositive for β-Lactamase production where 7 isolates (14%) were negative β-Lactamase production. For genotypic detection, it was found that 75% of the selected isolates were positive for blaZ gene, 70 % of the selected samples were positive for mecA gene. The present study shows that genotypic characterization was more prominent compared to the phenotypic detection of the β-lactamase production. These findings confirm the importance of these genes in β-Lactamase production and suggest the presence of other factors which may contribute to β-Lactamase production in S. aureus.

 

10/36 CHARACTERIZATION OF ERYTHROMYCIN, CLINDAMYCIN AND TETRACYCLINE RESISTANCE RATES IN COAGULASE-NEGATIVE STAPHYLOCOCCI ISOLATED FROM THE FACES OF ACNE PATIENTS AND CONTROLS IN THE EASTERN REGION, SAUDI ARABIA

Taghrid S. El-Mahdy and Mohamed Emara

Dept. of Microbiology and Immunology, Faculty of Pharmacy, Helwan University

Use of long courses of antibiotics is common for treatment of acne. During treatment course, selective pressure is exerted on commensal skin flora, including coagulase-negative staphylococci (CNS). These bacteria are prevalent opportunistic pathogens and may act as reservoirs of resistance genes for pathogenic strains. The study aimed to investigate the prevalence of resistance to most commonly antibiotics used for acne treatment; erythromycin, clindamycin and tetracycline; in CNS isolated from faces of acne patients and controls in Al-Ahsa, Saudi Arabia. A total of 15 acne patients and 15 controls were participated in the study and were asked to fill questionnaire about their treatment histories and skin care measures. Dermatologists in King Fahd hospital-Hofuf were requested to fill questionnaire regarding their acne treatment management in their clinics. Facial swabs from all participants were cultured on plates containing the examined antibiotics and CNS were isolated, scored and identified. Prevalence degree of clindamycin and erythromycin resistant CNS is more common than tetracycline resistance. Acne patients currently treating with antibiotics were no more likely to carry resistant CNS than patients using other or no medication. All clindamycin resistant strains were also resistant to erythromycin. In our study, 34 of 60 (57 %) of erythromycin resistant CNS exhibited constitutive Macrolide, Lincosamide and Streptogramin B (MLSB)phenotype, whereas, 23 % and 20 % of those isolates showed inducible MLSB and MSB phenotypes, respectively. In conclusion, the study reveals the high prevalence of clindamycin and erythromycin resistant CNS in this region in both controls and patients groups. This finding is worrisome and short antibiotic courses are recommended for treatment of acne to overcome the spread of resistance genes from CNS. The study also recommends adoption of testing antibiotic susceptibility for bacterial flora isolated from acne patients unresponsive to therapy.

 

11/36 RESPONSE OF POTATO CROP INOCULATED WITH SILICATE BACTERIA AND CYANOBACTERIA UNDER TWO LEVELS OF POTASSIUM SULPHATE AND FELDSPAE.

Hager I. Tolba, El-Sayeda H. M. El-Badawy and Hanaa A. Abo-Kora

Soils, Water and Environ. Res. Inst., Agric., Res. Center, Giza, Egypt

A field experiment was carried out at El-Nubariea Research Station, El-Bohera Governorate, Egypt, during winter season (2011/2012) to study impact of Bacillus circulans (silicate bacteria) and cyanobacteria under two sources and levels of potassium fertilizers on plant growth parameters, and crop component (minerals content, total carbohydrates % and soluble sugar content %) and the soil biological activity (dehydrogenase activity, nitrogenase activity and total bacteria). Results indicated that microbial inoculation caused significant increase in shoots dry weight after 60 and 90 days of planting compared with non inoculation treatments. Feldspar increased the plant growth and yield more than potassium sulphate. Moreover a slight increase in nitrogen content of plants after cyanobacteria inoculation was observed. Furthermore, phosphorus and potassium percentage were slightly increased after inoculation with B. circulans. comparedwith non inoculation treatments. Results revealed that, high total carbohydrates (%) were estimated with (full dose of feldspar and B. circulans, however, the highest soluble sugar content was determined with (full dose of feldspar and cyanobacteria). Data also revealed that dehydrogenase and nitrogenase activities were significantly increased after microbial inoculation and also feldspar caused significant increase more than potassium sulphate in combination with microbial inoculation. Moreover, B. circulans caused significant increase more than cyanobacteria. The total microbial count showed the same trend. So, the use of feldspar (K-rock) in combination with biofertilizers may be agronomically more useful and environmentally more feasible than chemical potassium ferlizer.

 


12/36 AN ENVIRONMENTAL CHITOSANASE-PRODUCING ISOLATE FROM ALEXANDRIA GOVERNORATE, EGYPT: ITS MOLECULAR CHARACTERIZATION, BIOFILM FORMATION AND ENZYME PURIFICATION AND CHARACTERIZATION

Mervat Kassem, Nabil El-Toukhy*, Rana Magdy, Nourhan Fanakiand Hamida Abou-Shleib

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

*Pharmaceutical Bioproduct Research Department, Genetic Engineering and Biotechnology Research Institute, City for Scientific Research and Technology Applications, New Borg El-Arab, Alexandria, Egypt.

Five different biopolymer-degrading microorganisms were isolated among thirty samples collected from various locations in Alexandria Governorate, Egypt. Only the isolate coded R3 was found to have a chitosan-degrading ability. The identity of isolate R3 was reached by molecular identification rather than by biochemical characterization. After the molecular identification and constructing Maximum Likelihood phylogenetic tree using GenBank database, the isolate R3 was identified as Pseudomonas geniculata strain that close matches to Pseudomonas geniculata strain XJUHX-10. In order to determine optimal conditions for the growth of the strain, and hence for its biodegradation activity, the effect of different factors, such as temperature, metal ions concentration and culture media on the growth pattern were studied. Optimum growth was observed at temperature 37°C, The effect of different culture media on the formation of biofilm by this isolate using the surface viable count was studied as well and the results were further confirmed by scanning electron microscopy. The isolate R3 showed high number of biofilm viable cells in the gelatin containing medium and the lowest number in the chitosan-containing medium. Chitosanase enzyme was purified using combination of chromatographic techniques and resulted in a purified enzyme with only 17% activity compared with the initial activity. Analysis of the enzyme for molecular mass using SDS-PAGE revealed one band of 28 kDa. The optimum pH was 6 with highest stability at 4-6, while the optimum temperature was 60°C with thermal instability at high temperatures. The chitosanase activity was increased up to 1.5% substrate concentration and the kinetic parameters Vmax and Km were 400 U/mg and 0.600 mg/ml, respectively. Using different divalent cations decreased the chitosanase activity at all concentrations used.

 

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