Vol. 35, January, 2013.

1/35 HEPATITIS C VIRUS GENOTYPING BY INNO-LIPA HCV II AND DIRECT SEQUENCING OF THE 5'UTR

Ola Abd El- Kader, Dalia E. Metwally, Abeer A. Ghazal,Ghada F. E-Helaly, Manal M. Mahmoud* and Marwa A. Hassan**

Microbiology Department, Medical Research Institute University of Alexandria.

*Internal medicineDepartment, Medical Research Institute University of Alexandria.

** Department of Microbiology, Faculty of Sciences, University of Alexandria

The determination of Hepatitis C virus (HCV) genotypes is known to be one of the main independent factors that influence the outcome of antiviral therapy, and is an important factor in the pre-treatment evaluation. The aim of this study was to compare the FDA(food and drug administration) approved polymerase chain reaction (PCR) based reverse hybridization INNO-LiPA test with direct sequencing of the 5`UTR for HCV genotyping and to evaluate the role of genotypes in relation to clinical, biochemical, histopathological and virological data. A total of 20 HCV RNA positive samples obtained from chronic hepatitis C patients were examined for their genotypes/subtypes by the Versant HCV genotype assay INNO-LIPA; genotype (1a) was diagnosed i n one case, sub-genotyping was possible only in 1(5.2%) out of the 19 HCV genotype 4, thirteen (65%) out of the 19 HCV genotype 4 could not be further sub-genotyped. Eleven (73.3%) out of the 15 cases tested by sequencing the 5`UTR region have been successfully genotyped and sub-genotyped. Nine (60%) out of them previously diagnosed by INNO-LiPA as genotype 4 or (4a, 4c, 4d) were found to be 4a, 1 (6.6%) was sub-genotyped as 4f and the other (6.6%) was 4o. Four (26.7%) isolates belonging to genotype 4 could not be further sub-genotyped. In the present study, 12 (66.6%) out of the 18 HCV4 genotyped isolates showed no or mild fibrosis and 8(44.4%) had a low metavir grading grade (A1). As   the majority (60%) of our 15 isolates subjected to sequencing were sub-typed as HCV 4a, no relation between HCV-4 subtypes and the degree of liver fibrosis could be deduced from this study. Sequencing of the 5`UTR is a reliable test for HCV genotyping for the detection of major types and subtypes detection, while INNO-LiPA is a good test at the genotype level but unreliable for subtyping especially in the Egyptian population.

 

2/35 EMERGENCE OF EXTENDED SPECTRUM Β-LACTAMASES IN CLINICAL ISOLATES OF PSEUDOMONAS AERUGINOSA IN ISMAILIA GOVERNORATE, EGYPT

Hany Rashad Hashem, Nora Fahmy Mahmoud, Alaa Mahmoud Shawky* and Salah Mohamed Abdallah

Department of Microbiology and Immunology, Faculty of Pharmacy,

Suez Canal University.

*Department of Microbiology and Immunology, Faculty of Pharmacy,

Cairo University.

Pseudomonas aeruginosa (P. aeruginosa) is an important pathogen commonly implicated in serious nosocomial infections, a growing number of β-lactamases have been reported worldwide in P. aeruginosa isolates. The aim of this study was to delineate the antibiotic susceptibility patterns of clinical isolates of P. aeruginosa. A total of 48 isolates of P. aeruginosa were collected from laboratories of Suez Canal University Educational Hospital and Community Clinical Laboratory in Ismailia Governorate, Egypt. Susceptibility to 12 antimicrobial agents was performed by disk diffusion method. ESBLS-phenotypic confirmatory detection was carried out by two methods, disk combination method and using double-disk synergy test (DDST).The sensitivity patterns of P. aeruginosa isolates demonstrated showed a very high resistance rate that reach up to100% toward ampicillin, cefoxitin, cefuroxime, and cefazoline. While it reached to 89.6, 75, 64.6 and 47.9% towards cefotaxime, cefotriaxone, ceftazidime, and cefepime respectively. However, isolates were sensitive to imipenem, aztreonam, ciprofloxacin, and gentamicin in a rate of 77.1, 75, 68.8 and 66.7% respectively. The analyzed samples results for the production of ESBLs phenotypically from p. aeruginosa were 70.8%.

 

3/35 NOSOCOMIAL DEVICE RELATED INFECTIONS INSIDE INTENSIVE CARE UNITS AND NHIBITORY EFFECT OF RESERPINE ON CLINICAL ISOLATES OF CIPROFLOXACIN RESISTANT ACINETOBACTER BAUMANNII

Moslhey S. Mansy, Hamido M. Hefni, *Wael M. Tawakol and

*Mohamed F. El-Badawy

Department Microbiology and Immunology, Faculty of Pharmacy, Al-Azhar University, Cairo, Egypt.

*Department Microbiology and Immunology, Faculty of Pharmacy, Misr University for Science and Technology, 6th October, Egypt.

Patients are admitted to intensive care unit (ICU) due to some medical problems which require medical intervention using medical devices which contribute to the incidence of nosocomial infection. Antimicrobial resistance is a serious problem inside ICUs, among the mechanisms of resistance to antimicrobial agents is the efflux pump system. The goal of this research is to investigate the prevalence of nosocomial device related infections (DRIs) and to study the effect of reserpine as efflux pump inhibitor (EPI) among ciprofloxacin resistant Acinetobacter baumannii that were meropenem resistant. In this study 141 nosocomially infected cases that were device and undevice related examined for occurrence of DRIs. These cases revealed 157 bacterial isolates from different sites as sputum, endotracheal tube (ETT) , central venous catheter (CVC), central venous pressure (CVP), nasogastric tube (NGT), cerebrospinal fluid (CSF),nasal, urine, pus, bedsore, blood, surgical drain and surgical wound . Susceptibility to ciprofloxacin and meropenem for Acinetobacter baumannii isolates was done by Kirby Bauer disc diffusion method. Also minimum inhibitory concentration (MIC) for ciprofloxacin was determined in presence and absence of 25mg/L reserpine by microtitre broth dilution method. The rate of DRI was 47.77% and the rate of undeviced related infection was 53.23%. Microbiological identification revealed that 31.20% of isolates were Gram positive and 68.80% were Gram negative. Presence of reserpine with ciprofloxacin decreased the MIC four folds and one fold in 56% and 20% of meropenem resistant Acinetobacter baumannii (MRAB) isolates respectively, on the other hand the MIC of (24%) of MRAB isolates were not affected by the presence of reserpine. The study demonstrated that invasive medical intervention is an important contributory factor for the incidence of nosocomial infection as well as reserpine is an EPI for ciprofloxacin resistant Acinetobacter baumannii.

 


4/35 CHARACTERIZATION OF BETA LACTAMASE IN PSEUDOMONAS AERUGINOSA

Abd El Gawad Hashem, Mona Wasef*, Yasser Al Mohamady and Doaa Mostafa Al-Eraky

Department of Microbiology and Immunology, Faculty of Pharmacy, Cairo University

*Department of Clinical and Chemical Pathology, Faculty of Medicine, Cairo University

Pseudomonas aeruginosa is one of the most common pathogens causing infections in hospitals, and shows increasing resistance to β-lactam antibiotics by producing different classes of beta-lactamases. It is also predictable to find a single isolate that expresses multiple β-lactamase enzymes, further complicating the treatment options. Thus, in this study, we aimed to determine the coexistence of different beta-lactamase enzymes in clinical isolates of P. aeruginosa from some Egyptian hospitals. A total of 108 clinical isolates of Pseudomonas species from 3 hospitals in Egypt were identified and tested for the presence of different beta-lactamase enzymes: extended spectrum beta lactamase (ESBL) and metallo β-lactamases (MBL) in the period from Jan 2010 to July 2010. In vitro susceptibility pattern of beta lactam antibiotics was done by the Kirby Bauer disc diffusion method. All the 3rd generation cephalosporin-resistant and imipenem-resistant isolates were go for screening and confirmatory test for ESBL and MBL respectively then positive isolates for phenotype tests were screened for OXA gene and VIM and IMP genes respectively through the PCR method using the bioinformatics tools. A total of 60 (56%) isolates were confirmed to be Pseudomonas areuginosa. Out of the sixty isolate a total of 5 (8%) and 10 (17%) isolates were positive ESBL and MBL respectively, and one isolate confirmed to have both blaOXA and blaVIM genes. A total of 10 (17%) isolates were resistant to all beta lactam antibiotics Piperacillin/tazobactum and ceplalosporin/sulbactam showed different sensitivity with 78% and 68% respectively. The most effective antibiotic was Imipenem where 83% of isolates showed sensitivity to this antibiotic. This study reveals the high prevalence of P. aeruginosa producing beta-lactamase enzymes of different mechanisms in Egyptian hospitals which afford serious therapeutic challenge. Thus proper antibiotic policy and measures to restrict the indiscriminate use of cephalosporins and carbapenems should be taken to minimize the emergence of this multiple beta -lactamase producing pathogen.

 

 

5/35 EPIDEMIOLOGICAL AND MICROBIOLOGICAL STUDY OF INFECTIOUS   BLOODY DIARRHEA IN CHILDREN: MOLECULAR IDENTIFICATION OF DIARRHEAGENIC ESCHERICHIA COLI

Mohamed H. Bahbah, Mohsen H. Hussein*, Azza Z. Labeb**, Fathy F. Ghaly***

Pediatrics Department, Faculty of Medicine, Menoufiya University

*Department Pediatrics National Liver Institute, Menoufiya University

**Microbiology and Immunology, Faculty of Medicine, Menoufiya University

***Pediatrics Department, Fever Hospital

In the developing world acute infectious   bloody diarrhea is an important cause of morbidity and mortality in children less than 5 years .The aim of this study was to demonstrate bacterial pathogens causing bloody diarrhea, with molecular differentiation of diarrheagenic Escherichia coli among children and to highlight antimicrobial resistance patterns of bacterial isolates. This study was carried out at Menouf Fever Hospital, Menufia Governorate, Egypt. In total, our work included 150 infants and children, [80 male and 70 female], aged from 6 months to 15 years old. Fifty patients, with bloody diarrhea and 100 patients with acute watery diarrhea. General stool examination and stool culture were done for all patients. Bacterial strains had been presumptively identified by culturing on selective media according to the standard microbiological methods, and their antimicrobial susceptibility patterns were detected. In bloody diarrhea group, diarrheagenic E. coli (DEC) were further examined for genes coding for virulence factors using a multiplex PCR method. In this study, the prevalence of bloody diarrhea was 33.3%, both groups of bloody and watery diarrhea were more common in rural area (60% and 86%), in females (52 % and 56%). Growth of bacteria was detected in 40 (80%) stool samples of bloody diarrhea group and 48 (48%) samples of watery diarrhea. In bloody diarrhea E. coli was the most common pathogen isolated in 15 children (30%), followed by 10 (20%) Shigella spp. and 10 (20%) Campylobacter spp. and only 5 Salmonella isolates were obtained. In watery diarrhea group E. coli (40 %) was the commonest bacteria isolated. Enteropathogenic E. coli were the most prevalent, accounted for 18 % of all cases of bloody diarrhea. A total of 5 isolates were stx gene positive and proved to be enterohaemorrhagicE. coli, they represented 10% of cases of bloody diarrhea. Only one case of enteroinvasiveE. coli proved with ipaH gene was detected in our bloody diarrhea isolates. All bacterial isolates of bloody diarrhea group were 100% sensitive to ciprofloxacin, ceftriaxone and ceftazidime. High resistant of our bacterial isolates were observed towards ampicillin (100%), co-trimoxazole (95%) and chloramphenicol (72.5%). In conclusions, E. coli was the most prevalent isolate in bloody diarrhea and was more common in children under 5 years. Constant antibiotic surveillance is warranted as bacteria were highly resistant to many antimicrobial agents. Shigella spp. was multi-resistant and reached alarming resistance rate.

6/35 BIODEGRADATION OF POLYETHYLENE BY THERMOPHILIC BACTERIA BACILLUS LICHENIFORMIS AND STREPTOMYCES SPP.

Hassan E. Abd Elsalam, Hoda H. Abo-Ghalia*, Azhar A. Hussain*,

Fatma A. El-nakeib**, Mohamed A. Abu-Saied*** and Amany G. Ibrahim*.

Soil and Water Technol. Department., Arid Land Cultivation Research Institute, City for Scientific Research and Technology Applications (CSAT), New Borg El-Arab, Alex., Egypt.

*Botany Department., Women's College of Arts, Science & Education, Ain Shams Univ., Cairo, Egypt.

**Environ. Biotechnol. Department., Genetic Eng. Biotechnol. Research Institute, CSAT, Alex., Egypt.

***Polymer Materials Res. Department. Adv. Techn. & New Materials Res. Institute, CSAT, Alex., Egypt.

Plastic and polyethylene waste accumulating in the environment are posing an ever increasing ecological threat. The plastic sheets or bags do not allow water and air to go into earth, which causesinfertility of soil, preventing degradation of other normal substances and danger to animal life. One of the most problematic plastics in this regard is polyethylene (PE). Isolation, screening and identification of the most efficient bacteria (Streptomyces spp. and Bacillus licheniformis) degrading linear low density polyethylene were obtained in the previous study. However, the factors that affect the degradation rate of PE were studied in this work. The selected isolates that can degrade polyethylene were found to degrade PE at pH 7-7.5. However, the optimum temperature was at 50ºC and optimum agitation was at 150 rpm. The degree of polyethylene degradation was increased when Streptomyces spp. and Bacillus licheniformis were combined together. The activity of the bacterial biofilm could be measured by hydrolysis of fluoroscein diacetate (FDA). It was found that Bacillus licheniformis colonized PE surface and formed a massive biofilm on it, thus the biofilm formation increased the biodegradability of the isolated Bacillus licheniformis than Streptomyces spp.

7/35 THE VITAL ROLE OF CYANOBACTERIA, AZOLLA AND YEAST APPLICATION AS BIOFERTILIZER IN IMPROVING ALLIUM CEPA L CROP

Azza A.M. Abd El-All, Ismail Yasso* and Hassan Ismail

Soils Water and Environ. Research Institute, Agric. Res .Center.

*Noubaria Research Station, Crops Research Institute, Agric. Res. Center.

Onion plays a vital role in the human life because of its pharmaceutical and nutritional properties. Several attempts were performed to enhance the quality and quantity of onion crop. In this study, one third of the recommended nitrogen fertilizer was substituted by the microbiota application. Results indicate that the use of biofertilizers (cyanobacteria and azolla either single or in combination with yeast extract) can compensate the reduction of onion nitrogenous requirements by 30 % without any significant changes in quality and quantity of onion crop. The yield reached in azolla extract with yeast extract plus 60 kg N/fed (T6) treatment to 95.05 % of control which took 90 kg N/fed (recommended dose of nitrogen) in the first season while in the second season, it reached to 106.34 % increase for the same treatment in proportional to control. The same treatment (T6) gave high valuesin the average of weight bulb where the results were 96.03% of control   and 108.89 % increase than control that wasfertilized by the recommended dose of nitrogen at the first and second season, respectively. An important role in decreasing the time of maturity and the percentage of boltersof onion crop was ascertained to biofertilizers. Cyanobacteria extractwith azolla extract plus 60 kg N/fed treatment (T4) was the best in decreasing the time of maturity that diminished from 152.67 to 151.67 day in the first season and decreased from 155.5 to 154.75 day in the second season. The percentage of bolters also decreased from 2.67 to 1.67 % for the mixture of biological agents plus 60 kg N/fed (T7) in the first season while at second season, all biological treatments were equaled to control in the percentage of bolters except the treatment of azolla plus 60 kg N/fed (T2) which increased to 1.67% while the control was 1.33%   Generally, almost results in the second season were better than that of the first season.

8/35 ENTEROCOCCUS FAECALIS IN ENDODONTIC INFECTIONS: INCIDENCE, ANTIBIOTIC SUSCEPTIBILITY AND DETECTION OF VIRULENCE GENES WITH EVALUATION OF BIOFILM REMOVAL TECHNIQUES

Reham A. Khalifa, Slsabyl M. Ibrahim*, Hossam Y. Rashid*and Ghada M. Eid*

Medical Microbiology and Immunology Department, Faculty of Medicine,

Ain Shams University,  

*Endodontic Department, Faculty of Dentistry, Cairo University

Enterococcus faecalis (E. faecalis), is one of the most frequently isolated species amongst all the bacteria associated with endodontic infections. The virulence factors of E. faecalis that may be related to endodontic infection include the phenotypic markers gelatinase, haemolysin, and biofilm formation. Sodium hypochlorite and chlorhexidine are antimicrobial agents frequently used in the treatment of endodontic and periodontal infections. The aim of this work was to determine incidence, antibiotic susceptibility and virulence factors of E.faecalis isolated from necrotic pulp infections with detection of Gel E & Cyl A virulence genes by real time polymerase chain reaction (PCR) and to evaluate two different biofilm removal techniques. Paper point samples were collected from infected canals of 30 patients for determining incidence of E. faecaliswith detection of virulence factors; gelatinase, haemolysin and biofilm formation, and virulence determinants; Gel E and Cyl A genes. One of the isolated virulent E. faecalis strains was then used forex vivobiofilm formation in root canals that were treated by twotechniques; the plastic syringe and the EndoActivator, to be irrigated by saline, chlorhexidine or sodium hypochlorite with EDTA. Pre- and post-irrigation bacteriological count and scanning electron microscopy (SEM) examination was done to evaluate the used irrigates and irrigation techniques. E. faecalis was isolated from 80% of cases. Enterococcal isolates exhibited antimicrobial resistance to erythromycin (62.5%), chloramphenicol (33.3%) and doxycycline (12.5%). Seventeen of the isolates were positive for Gel E gene, which was expressed in 64.7% of Gel E gene positive strains, 13 isolates were positive for Cyl A gene, which was expressed in 61.5% of Cyl A gene positive strains and 8 isolates were positive for both Gel E gene and Cyl A gene. Sodium hypochlorite and chlorhexidine were both effective ingelatinase and haemolysin positive E. faecalis biofilm removal with no statistically significant difference between the plastic syringe or EndoActivator Sonic system in biofilm removal. E. faecalis was isolated from the majority necrotic pulp infections. It showed resistance to certain antibiotics andwas multi-virulent, with production of gelatinase, hemolysin and biofilm formation. Sodium hypochlorite and chlorhexidine were both effective in removing E. faecalis biofilm using plastic syringe or EndoActivator.


9/35 PREVALENCE OF EXTENDED SPECTRUM BETA LACTAMASES IN URINARY ISOLATES OF ESCHERICHIA COLI AND KLEBSIELLA PNEUMONIAE

Hazem Abdel-Wahab Ahmed, Mona Abdel-Monem Ismail, Mohammed Sayed Mohammed, Wedad Mahmoud Abdel-Rahim

Microbiology and Immunology Department, Faculty of Medicine, Minia University

Extended spectrum beta-lactamases (ESBLs) are defined as β-lactamases capable of hydrolyzing oxyiminocephalosporins and are inhibited by β-lactamase inhibitors and due to mutations in the TEM-1, TEM-2, or SHV-1 genes, which are commonly found in the Enterobacteriaceae family. The aim of this work was to find out prevalence of extended spectrum beta-lactamases (ESBLs) producers in urinary isolates of E.coli and K. pneumoniae among patients with urinary tract infection in Minia University Hospital, identification of the molecular types of extended spectrum beta-lactamases (ESBL) produced by E. coli and K. pneumoniae. Also, know the antimicrobial susceptibility pattern of all isolates and ESBL producers. A total number of 230 mid - steam urine samples that were collected from patients that were clinically suspected to have UTI, the samples were collected from in- patient and out- patient Departments of Minia University Hospital, the samples were send the microbiology laboratory for identification of ESBL producers of E.coli and K. pneumoniae. The study found ESBLS production was prevalent in E. coli and K. pneumoniae urine isolates, as well as, high prevalence of TEM and SHV producing isolates as TEM producing isolates was 57%, SHV was 20%, 9% both and 14% was non gene producers.Our study concluded that ESBLS production was prevalent in E. coli and K. pneumoniae urine isolates, as well as, high prevalence of TEM and SHV producing isolates. In addition, resistance of these organisms common antimicrobial agents used for treatment of UTI. Strict antibiotic policy and effective infection control procedures implementation are the key partners in the control of such infections caused by ESBLs producing organisms.

10/35 ANTIFUNGAL SUSCEPTIBILITY TESTING OF CANDIDA SPECIES ISOLATED BY TWO CHROMOGENIC MEDIA FROM DIABETIC PATIENTS WITH DETECTION OF OVEREXPRESSION OF CDR1, CDR2 AND MDR1 IN CANDIDA ALBICANS.

Reham A. Khalifa, Marwa S. Fathi, Mohamed E. Elserafy* and Wael M. Saudi**

Medical Microbiology and Immunology Department,*General Surgery Department, Faculty of Medicine, Ain Shams University and**Dermatology & Venereology Department, Faculty of Medicine, Misr University for Science & Technology(MUST)

Candida albicans (C. albicans) and Non-albicans Candida (NAC) infections represent an overlooked cause of life threatening infections among high risk patients as diabetic patients. NAC are more prone to exhibit antifungal resistance, thus species level of identification of Candida species and proper determination of antifungal susceptibility patterns is vital for administration of proper antifungal therapy to patients with suspected yeast infections. The aim of the present study was to evaluate the two different chromogenic media used for isolation and identification of different Candida species isolated from diabetic patients with assessment of their antifungal susceptibility and detection of overexpression of antifungal resistance determinants CDR1, CDR2 and MDR1 among C. albicans isolates. The study was conducted on 80 diabetic patients (48 males and 32 females) with age range 18 - 65 years old (46±14.7), who were admitted to surgery department and surgicalintensive care unit (ICU) of Ain Shams University Hospitals (ASUHs) during the period from November 2009 to April 2011. They were suffering from diabetic foot infections, surgical site infections (SSI), catheter associated urinary tract infections (CAUTI), localized abscesses and ventilator associated pneumonia (VAP). Samples were cultured on two chromogenic media; Brilliance Candida agar (BCA) and Candiselect 4 (CS4), to evaluate their ability to isolate and identify different Candida species. Antifungal susceptibility was performed using disc diffusion, MIC test strip and Fungitest panel. Real time RT-PCR was done to evaluate overexpression of CDR1, CDR2 and MDR1 among C. albicans isolates. NAC was found to be a major cause of Candida infections among diabetic patients with different risk factors and they showed high incidence of antifungal resistance. The use of BCA provided a rapid and accurate mean for species level identification of most of the isolated Candida species. Fungitest panel for antifungal susceptibility testing showed acceptable agreement with MIC test strips for some antifungal drugs asamphotericin B (Amp B), yet it showed less agreement for fluorocytosine (5FC) and it didn't allow assessment of minimal inhibition concentration (MIC) of resistant isolates. Overexpression of CDR1, CDR2 and MDR1 was observed among fluconazole (FL) resistant C. albicans isolates which emphasize their role in azole resistance. Further studies are needed to clarify the effect of different risk factors on the expression of genes implicated in antifungal resistance.      

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