Vol. 39, Septamber, 2014.

1/39 PHENOTYPIC DETECTION OF VARIOUS Β-LACTAMASES AMONG MULTIDRUG RESISTANT ISOLATES OF ESCHERICHIA COLI AND KLEBSEILLA PNEUMONIAE ISOLATED FROM URINARY TRACT INFECTIONS IN SOME EGYPTIAN HOSPITALS

Abdel Rahman K. Raslan1, Reham Wasfi1, Moselhy S. Mansy2, Mohamed S. Ashour1,2

Microbiology & Immunology Department, Faculty of Pharmacy, October University for Modern Sciences and Arts, Giza, Egypt1,

Microbiology & Immunology Department, Faculty of Pharmacy - Al-Azhar University, Cairo, Egypt2.

In the present study a total of 205 isolates were recovered from186 different cases from urinary tract infections ward in some Egyptian hospitals (Dar El-Fouad Hospital, EL-Demerdash Hospital, El-Sheikh Zayed Hospital and Kasr Al-Ainy Hospital) from September 2011 to October 2012. The most predominant bacteria isolated from urine samples was Escherichia coli 49.76% (102/205), followed by Klebseilla pneumonia represented 14.63% (35/205).The growing number and rapid increase in antibiotic resistance among E. coli, Klebsiella spp. has prompted us to investigate the resistance mechanisms among these isolates. It was found that the rates of potential ESâL production among isolates of E. coli and Klebsiella spp. were 46.08% (47/102) and 60% (21/35) respectively. While, the rates of potential ESβLs and AmpC β-lactamase production among E. coli and Klebsiella spp., were 22.55% (23/102) and 25.71% (9/35), respectively. Our study revealed that the rates of carbapenemases production among E. coli and Klebsiella spp. were 0.98% (1/102) and 8.57% (3/35), respectively.

2/39 COMPARATIVE STUDIES ON DIFFERENT MOLECULAR METHODS FOR EPIDEMIOLOGICAL TYPING OF MULTI-DRUG-RESISTANCE KLEBSIELLAPNEUMONIAE ISOLATED FROM MANSOURA HOSPITALS

Ramadan Hassan, Rasha Barwa, Mona I. Shaaban and Lamiaa Adel*

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

*Student Hospital, Mansoura University, Egypt.

The present study aims to evaluate two molecular methods for epidemiological typing of multi drug resistant Klebsiella pneumoniae isolated from Mansoura Hospitals. In this study, a total of 300 clinical isolates were collected from different patients distributed among Mansoura Hospitals, Dakahlia governorate, Egypt. Ninety six isolates were identified as K. pneumoniae using standard biological methods. Most isolates were obtained from urinary tract infection (70%). The susceptibility to eleven antimicrobials was determined and analyzed among isolates. The antimicrobial susceptibility test showed that Forty five isolates (47%) were multi drug resistance (MDR). Plasmids isolation from all MDR isolates revealed that 80% of these isolates harbored plasmids. Thirteen plasmid patterns were observed upon single digestion with EcoR1. However, the absence of plasmids from 20% of isolates decreases the type ability power of this technique. Hence, Random Amplified Polymorphic DNA (RAPD) analysis was applied as another molecular typing method using two individual primers, AP3 and OPA13. OPA13 showed eight distinct patterns (RA1-RA8) representing 37 isolates. Primer AP3 showed more discriminatory patterns (R1-R18) and can be used individually for better interpretation in typing a large number of isolates. In conclusion, RAPD typing is efficient and cost-effective while maintaining reproducible results for analyzing large numbers of clinical isolates and useful to understand the distribution of K. pneumoniae during investigation of outbreaks.

3/39 CYTOMEGALOVIRUS INFECTIONS AMONG LOW BIRTH WEIGHT INFANTS IN A MAJOR HOSPITAL IN CAIRO, EGYPT, (ELGALAE).

Hebatallah M. Elwa, Aymen S. Yassin*, Hoda B. Hussein**, Magdy A. Amin* and Nagwa A. Meguid

Department of Research on Children with Special Needs, National Research Center, Dokki, Giza, Egypt.

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

**ElGalaa Teaching Hospital, Cairo, 11698, Egypt.

Human Cytomegalovirus (HCMV) is one of the leading causes of congenital infections, which can lead to severe foetal anomalies or even foetal loss. In order to determine the incidence of congenital HCMV infection in low birth weight neonates and the most prevalent genotype, cord blood samples were collected from a 102 full-term low birth weight neonates at the time of delivery as well as blood samples from their mothers during a four month period. Another 102 blood samples were also included in a normal weight control group. Samples were subjected to DNA extraction, PCR amplification and genotyping of the glycoprotein gB gene. Five cord blood samples were found to be HCMV positive (2.45%); four of which were low birth weight (3.92%) while, only one sample from the control group was HCMV positive (0.98%). Real time PCR was used for quantitative analysis of the virus among the positive samples. The Genotyping of the positive five cases revealed that four cases were gB1 and only one was gB2. The genotypes gB3 and gB4 were absent. Low birth weight is a major risk factor for congenital HCMV infection. The incidence of infection in Egypt is among the highest infection rates found in developing countries. The most prevalent genotype in Egypt is gB1.

4/39 PRODUCTION OF DNA POLYMERASE BY RECOMBINANT PET-17B/PFU-POL AND ITS OPTIMIZATION FOR PCR

Mona I. Shaaban

Microbiology Department, Faculty of Pharmacy, Mansoura University, Egypt

Polymerase chain reaction (PCR) is one of the most widely used techniques in molecular biology and biotechnology. Thermostable DNA polymerase is the key enzyme that catalyzes the amplification of DNA templates during PCR cycles. Although this enzyme has been produced worldwide, there is no reported cloning or production of polymerases in Egypt. In the current work, plasmid coding Pfu polymerase enzyme (pET-17b/Pfu-Pol) was transformed into E. coli Top10. The plasmid coding Pfu- polymerase was confirmed by restriction analysis using HindIII (5.5Kbp) and by double digestion with HindIII/XhoI (3.6 and 1.95Kbp). Furthermore, the confirmed plasmid was transformed into E. coli BL21 (DE3) pLysS cells for protein expression. Enzyme production was induced with 0.5mM IPTG and precipitated with ammonium sulfate. The SDS-PAGE analysis of the enzyme showed single protein band at 86KDa. Different conditions were assessed for optimization of the prepared enzyme including Mg ion concentration, the amount of used enzyme and the extension temperature. Taq enzyme showed more PCR potential activity using 4mM MgCl2 with 0.5µl of the prepared enzyme in the final reaction mixture (25µl). Also, the enzyme was active when PCR was conducted at extension temperature 72º C. Moreover, the produced Pfu-polymerase showed similar enzyme activity compared to the commercially available polymerases.

5/39 Characterization of Staphylococcus aureus nasal colonization rates among healthcare workers, clinical students and community in the eastern region, Al-Ahsa, Saudi Arabia

Taghrid S. El-Mahdy and Riham M. Shawky

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

Carriers of Staphylococcus aureus have an important role in its dissemination. The colonization rates of S. aureus in anterior nose nares from 210 healthy volunteers (70 from the non-hospital adult personnel in the community, 68 from clinical students and 72 from healthcare workers “HCWs” in 6 hospitals) in the eastern region, Al-Ahsa, Saudi Arabia were determined. The percentage of nasal carriers of S. aureus in the community was 37%, whereas it was 26% in both clinical students and HCWs groups. Methicillin-resistant S. aureus (MRSA) colonization was 9% and it was only isolated from clinical students and HCWs. Antimicrobial susceptibility testing showed that 90.5% of the 63 strains isolated from all volunteers were ampicillin resistant. Three multi-resistant strains to three antibiotics or more were identified; two from HCWs (one of them is MRSA), one from clinical students and none from the community group. No resistance was detected to linezolid and vancomycin in all isolates. Coagulase negative staphylococci carriage was 87% in all our volunteers and was highest in the HCWs (93%). In conclusion, the study shows the need for a periodic screening of both the community and hospital personnel to adopt strategies for treating S. aureus carriers; specifically MRSA carriers; hence overcome acquiring S. aureus infections.

6/39 PCR DETECTION OF INDICATOR GENES IN METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS (MRSA) ISOLATED FROM THREE SAUDI HOSPITALS

Wael A. Elnaggar, Ishraga E. A-Elbasit, Hussam H. Arafat* and Ahmed Elsherbini**

Department of Health Sciences, Faculty of Pharmacy, Northern Border University, Rafha, KSA.

*Department of Biology, Faculty of Science and Arts, Northern Border University, Rafha, KSAandDepartment of Botany,faculty of Science, Minia University, Minia, Egypt.

**Department of Medical Emergency Services, HealthSciencesCollege, Umm Al-QuraUniversity (Al-Qunfudah) KSA.

The aim of the present study is to examine the recovered strains of methicillin-resistant Staphylococcus aureus (MRSA) phenotypically by conventional identification. Genotypicalexamination was made also by polymerase chain reaction (PCR) to detect the genes; lukF encoding Panton-Valentine Leukocidin(PVL) and arcA an indicator of the arginine catabolic mobile element (ACME).Twinty-eight strains of Staphylococcus aureus, collected in 2013 from three Saudi central hospitals, were characterized by streaking on Mannitol salt agar plates and biochemically identified as Staphylococcus aureus. Resistance towards eight antimicrobial agents revealed that most of the tested strains of Staphylococcus aureus showed resistance to the tested antimicrobials in the following order; Oxacillin 100%, Tetracycline 71%, Cefoxitin 71%, Erythromycin 71%, Ciprofloxacin 71%, Imipenem 68%, Amikacin 60% and Vancomycin 7 %. All the tested strains produced the gene arcA, while 80% showed the presence of lukF.

7/39 BIOLOGICAL ACTIVITIES OF SOME XYLOOLIGOSACCHARIDES FROM LIGNOCELLULOSIC WASTES

Mayada H. Aly, Salwa A. Megahed*, Mohamed A. Ramadan* and

Mohamed M. Hussein**

Department of Crime Investigation Research, National Center for Social and Criminological Research, Cairo, Egypt

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

**Department of Natural and Microbial Products, National Research Center, Dokki, Cairo, Egypt

Xylooligosaccharides (XOS's) exhibited considerable biological activities and can be incorporated into many food products and in pharmaceutical and drug industry. XOS's were produced from xylose-containing polysaccharides (XPS's) obtained from natural, xylan-rich, agro-industrial wastes, i.e., corncobs and sugarcane bagasse. The yields of XPS's were 32.10% and 27.20% from corncobs and sugarcane bagasse, respectively. The preparation of XOS's was doneby partial hydrolysis of XPS's obtained from corncobs and sugarcane bagasse,either chemically by oxalic acid (0.25N) or by fungal attack by Aspergillus niger and by Trichoderma reesei. The partial hydrolysis of XPS's; resulted in production of (8) mixtures comprising total of (18) individual oligosaccharides. This was followed by evaluation of their biological activities, including their prebiotic, anticoagulant and fibrinolytic actions. The results showed that the tested XOS-mixtures enhanced the growth of the probiotic bacteria. This indicates that the studied XOS's can be considered as promising prebiotics. Additionally, the tested XOS's showed very low anticoagulant effect, and for fibrinolytic activity ranged between, moderate to very low activity.

8/39 MICROBIOLOGICAL STUDIES ON BACTERIAL ISOLATES FROM PENICILLINS FILLING CLEANROOM

Moustafa Ahmed El-Nakeeb, Amal Mohamed Khalil and Ahmed Fathi Gasser

Faculty of Pharmacy, Alexandria University, Egypt

*Pharco Pharmaceuticals, Egypt

Aseptic processing is a critical method for the preparation of thermolabile sterile parenteral drug products. Sterile β-lactam antibiotics are extremely deactivated by heat, so the method of choice for their processing is through aseptic filling. The consequences of contamination on aseptically-filled products are harmful to the patient and having financial risk to the manufacturer. The aim of this work is isolation of bacterial contaminants from Classes A and B of aseptic penicillins filling area in Pharco Pharmaceuticals Egypt, studying the ability of these contaminants to produce penicillinase and studying the sensitivity of these contaminants to biocides. In addition, possible interaction between the commonly used biocides was checked. Thirty-one bacterial isolates were collected from cleanroom class A and seventy isolates from class B. Isolates were identified using macroscopic, microscopic examination, and the identity was confirmed by biochemical methods. Staphylococcus sp. were the predominant microorganism isolated, which include S. epidermidis (50%) and S. aureus (5%), followed by Micrococcus sp. (27%) then Corynebacterium sp. (10%). Bacillus sp. and P. aeroginosa were in the same proportion (4%). Isolates showed a heterogeneous susceptibility toward β-lactams when applying Kirby-Bauer disk diffusion technique. Accordingly, isolates were introduced into the aseptic area through different sources. Fifty-two isolates were found to be penicillinase producers. The effectiveness of biocides was tested according to modified suspension method and surface contact method. Isolates had different sensitivity toward the tested biocides. Based on the suspension method, at five minutes contact time, 1% Minncare was effective against 23 isolates out of 27 tested. While 1% Kleencare DS607 was effective against 21 isolates out of 27 tested and 70%isopropyl alcohol was effective against 23 isolates out of 27 tested. Two hard surfaces were selected for surface contact test, aluminum foil and powder free sterile surgical gloves. Results were similar to a great extent when biocides were applied to either aluminum surface or rubber surface, except an intangible difference in few cases. The concomitant use of at least two biocides within the cleanroom is necessary. Interactions between the selected biocides were also examined using macrodilution checkerboard method. No interaction between hydrogen peroxide and DS 607 was observed. The effectiveness of isopropyl alcohol was enhanced when DS607 present as 6.25 ppm in isopropyl alcohol. The activity of hydrogen peroxide and isopropyl alcohol was reduced when mixed to each other.

9/39 EMERGENCE OF KLEBSIELLA PNEUMONIAE CLINICAL ISOLATES HARBORING KLEBSIELLA PNEUMONIAE CARBAPENEMASE AND METALLO-Â-LACTAMASE IN TWO HOSPITALS IN EGYPT

Abeer K. Abdulall*, Hadir A. El-Mahallawy**, Samia G. Abdo*** and Naiema K. Aly****

*Department of Microbiology and Immunology – Faculty of Pharmacy (Girls) - Al-Azhar University

**Department of Clinical pathology, National Cancer Institute, Cairo University

***Department of Clinical pathology, Faculty of medicine, Ain-Shams University

****Department of Clinical pathology, Faculty of medicine, Al-Azhar University

Klebsiella pneumoniae carbapenemases (KPCs) as well as metallo- β-lactamases (MBL) producing Enterobacteriaceae are associated with severe and often fatal infections in severely ill patients. This study was carried out to investigate and confirm the emergence of carbapenemase producing Enterobacteriaceae in Egyptian Hospitals. Particularly the molecular class A KPC and the molecular class B MBL namely the imipenem resistant phenotype carbapenemase (IMP) and verona integron-encoded metallo-beta-lactamase (VIM) producers. To pursue this aim, 134 non-duplicate clinical isolates belonging to Enterobacteriaceae were collected from three Hospitals in Cairo Egypt. The antimicrobial resistance patterns of all isolates included in the study were following the screening criteria of carbapenemase producing bacteria. Detection of carbapenemase producing isolates was carried out by the modified Hodge test (MHT). Phenotypic detection of KPCs and MBLs was by inhibitors combined disc tests, in MHT positive isolates, and was confirmed by detection of bla KPC, bla IMP and bla VIM. Concomitant existence of: KPCs and IMP was confirmed in clinical isolates of K. pneumoniae from inpatients hospitalized in the National Cancer Institute (NCI) Hospital and Al-Demerdash University Hospital (DUH). bla KPC and bla VIM genes were detected together in two isolates from inpatients in the NCI. bla KPC genewas detected alone in three isolates from inpatients in the NCI and two isolates from inpatients in the DUH. bla IMP gene was detected alone in two isolates from inpatients in the DUH and one from NCI Hospital. To our knowledge this was the first report on emergence of KPC-VIM and IMP–KPC producing K. pneumoniae isolates in Egyptian hospitals. Weconcluded that, emergence of KPC and MBL-producing Enterobacteriaceae should be investigated in other hospitals located in other geographic locations in Egypt to assess the magnitude of the problem. Also, infection control practices and antibiotic policies should be strengthened to avoid the blowout of these microbial bums in our hospitals.

10/39 THE PROMOTIVE EFFECT OF N2 FIXERS, BACILLUS CIRCULANS AND SACCHAROMYCES CEREVISIAE ON THE VIABILITY OF NATIVE ARBUSCULAR MYCORRHIZAL FUNGI AND THE IMPACT ON THE PRODUCTIVITY OF ALFALFA (MEDICAGO SATIVA L.)

Osama N. Massoud, Ebtsam M. Morsy and Mounira M. Bishara

Soils, Water and Environment Research Institute, Agricultural Research Center,

Giza, Egypt.

This study was carried out to investigate the effects of inoculation with symbiotic (Rhizobium meloti), Asymbiotic (Azospirillium lipoferum), K- solubilizers (B. circulans) and Saccharomyces cerevisiae (as plant growth promoters) on the viability of native mycorrhizal spores (endogenous) and the increase of its propagula and the impact on growth and productivity of Alfa-Alfa .This study was carried out in field experiment during winter season (2011/2012) at an experimental farm of Giza Research Station, Agriculture Research Center (ARC), Giza, Egypt. These strains were capable of producing hormone like substances and exo- polysaccharides in variable quantities besides the ability of nitrogen fixation by R. meloti and A. lipoferum in their specific media. The obtained results revealed that the populations of Azospirillum spp., B. circulans and S. cerevisiae in the rhizospheric area were higher with the treatment mixture compared to control. The inoculation with these microorganisms led to the increase of native mycorrhizal colonization (%) in rhizospheric area and abundance of mycorrhizal spores in soil in addition the number of nodules in Alfalfa roots. The mixture treatments was still the superior one where it gave the highest enzymes activity represented in Dehydrogenase (µg TPF/g dry soil/day), nitrogenase (µmole C2H4/g rhizosphere/h) and acid and alkaline phosphatase (µg/g dry soil). This treatments also indicated the highest values of carbohydrates crude protein and NPK (%) therefore, this treatment recorded the highest dry highest dry weight (ton/fed) as yield parameter during the first and second cuts, respectively. Its values are: 1.35 and 2.55 (ton/fed), respectively, more than the control and other treatments. It was concluded that the inoculation with these beneficial microorganisms enhanced and promoted the other native microorganisms to exist and colonize the rhizospheric area of plants and hence the increase of soil fertility and plant productivity.

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