Open Access Original Research Article

Listeria spp. and Enterobacteriaceae Group in Sandwiches of Meat and Meat Products

Rashed A. Zaghloul, Moustafa A. El-Shenawy, Naseem A. Neweigy, Hamed E. Abou-Aly, Raouf K. El-dairouty, Wagih I. El-Kholy, Mohamed T. Fouad, J. M. Soriano, J. Mañes, Lydia Micó

Microbiology Research Journal International, Page 360-368
DOI: 10.9734/BMRJ/2014/7417

Aims: To evaluate the Egyptian street-vended sandwiches of meat and meat products for the presence of Listeria monocytogenes and Enterobacteriaceae group. To evaluate the microbiological quality of street-vended sandwiches of meat and meat products sold in Cairo-Egypt.
Place and Duration of Study: Department of Food Microbiology, National Research Center, Cairo, at during the period of January 2011 to September 2012.
Methology: Seventy sandwiches of meat and meat products including ten samples each of burger, hawawshi, kofta, liver, luncheon, sausages and shawerma sandwiches were randomly collected from the street-vendors and food shops in Great-Cairo governorate. Samples were investigated for their loads of Enterobacteriaceae counts as well as the presence of L. monocytogenes. Enterobacteriaceae counts was done using the conventional International method (FDA), however isolation and identification of L. monocytogenes was carried out using three different microbiological examination methods including classic selective conventional media, chromogenic media, as well as rapid identification method “food-system kits”. 168 isolates of Listeria spp were identified following the biochemical identification tests (Bergey’s Manual) and confirmed using Hi Listeria identification kits and latex test kits.
Results: Enterobacteriaceae group were detected in 51% of the examined samples with an average counts of 37x102 cfu/g. Listeria spp. were detected in range between 24% and 36%, depended on the method used, with numbers ranged from 16x102 to 23x102 cfu/g. All samples that were contaminated with Listeria spp. harboured L. monocytogenes. Listeria spp. was not detected in all the examined Hawawshi sandwiches with an exception of one positive sample detected using the chromogenic media. The obtained results indicated that 37 out 68 (54%) isolates, picked up from classic selective conventional media, and 62 out of 100 (62%) isolates from chromogenic medium were confirmed as L. monocytogenes indicating that chromogenic medium may be the superior for isolation of the pathogen from ready-to-eat sandwiches of meat and products.
Conclusion: The obtained results indicated that these foods presented a source of infection to the consumer. Measures to control the quality of the raw material, environmental and hygienic conditions during preparation and serving should be taken. The chromogenic media was the most efficient for the isolation of the pathogen during this course of study.

Open Access Original Research Article

Isolation, Identification and Optimization of Culture Conditions of Bacillus sp. Strain PM1 for Alkalo-thermostable Amylase Production

Kinshuk Sharma, Sarita Bhutty, S. M. Paul Khurana, Urvashi Kumar Kohli

Microbiology Research Journal International, Page 369-380
DOI: 10.9734/BMRJ/2014/6873

Aims: To isolate and optimize the culture conditions for thermo stable and alkaline amylase production from bacteria.
Study Design: Optimization of different physiological and nutritional parameters for amylase production and kinetic studies of amylase.
Place and Duration of Study: Soil Samples: Herbal garden of Amity University Haryana, Gurgaon (Manesar), India, between April 2012 and September 2012.
Methodology: Amylolytic isolates were selected by flooding the nutrient agar plates containing 2% starch with Lugol solution. Isolates were selected on the basis of higher ratio of clear zone to colony size and grown in nutrient broth containing 2% starch. The level of amylase was detected in the culture filtrate. The selected isolate showing maximum amylase production was identified on the basis of 16S rDNA amplification.
Results: An Alkalo-thermostable amylase producing bacterial isolate from soil was identified as Bacillus sp. strain PM1 on the basis of 16S rRNA. It yielded 3.5 U/ml of amylase in medium containing (%) starch 2.0, beef extract 0.5, NaCl 0.5 at 50ºC, pH 7.0 at 180 rpm after 72 h. The optimum pH and temperature for amylase activity was 8.0 and 50°C, respectively. The enzyme exhibited 67% activity after 60 minute incubation at 50ºC. At pH 8.0, the enzyme retained 78% activity after 4 h.
Conclusion: The properties of the isolated enzyme are adequate for its use in starch processing and baking industry.

Open Access Original Research Article

Adhesion, Autoaggregation and Hydrophobicity of Six Lactobacillus Strains

Anwar A. Abdulla, Thikra A. Abed, A. M. Saeed

Microbiology Research Journal International, Page 381-391
DOI: 10.9734/BMRJ/2014/6462

Aims: The aim of this study was to assess probiotic attributes such as adhesion, auto aggregation, hydrophobicity and antibacterial activity of Lactobacillus strains from dairy products.
Methodology: In this study, the autoaggregation, coaggregation, hydrophobicity and adhering abilities and antimicrobial activities of six Lactobacillus strains belonging to different species were assessed. Hydrophobicity was determined by bacterial adherence to hydrocarbons, xylene, n-hexadecane and chloroform.
Results: The percentage of hydrophobicity of the strains range from 29.5% to 77.4% as measured by the described test. The autoaggregation among Lactobacillus strains range from 15.8% to 63.1%, while coaggregation range from 18.6% to 55.1%. Adhesion of the tested strains to buccal epithelial cells range from 8.0% to 50%. The tested Lactobacillus strains demonstrated variable inhibitory activity against pathogenic bacteria.
Conclusion: Our findings indicated that one Lactobacillus strain expressed broad antibacterial activities against a group of bacterial pathogens and along 2 other strains exhibited ability to adhere to epithelial cells as shown by aggregation, coaggregation and hydrophobicity, indicating that such isolates can be good candidates for probiotic use.

Open Access Original Research Article

Lipolytic Activity and Molecular Identification of Pseudomonas aeruginosa and Lysinibacillus sphaericus Isolated from Domestic Oil Rich Wastewater

A. T. Odeyemi, B. I. Aderiye, E. I. Adeyeye, E. Donbraye, T. Faleye

Microbiology Research Journal International, Page 392-404
DOI: 10.9734/BMRJ/2014/6587

Microbial lipases have been heightened in bioremediation and various industries. Place and Duration of Study: Department of Microbiology, Ekiti State University, Ado-Ekiti, Ekiti State, Nigeria, between September 2010 and August 2011. To identify the lipolytic enzyme producing microbial strains in domestic oil rich wastewater, the 16S rRNA gene was amplified and sequenced. The sequences were used to identify the strains by comparing with related sequences in database using BLAST analysis. The enzyme activity was quantified by HPLC analysis. All the lipolytic bacteria showed appreciable growth rates in the wastewater (between 0.67 and 1.67 mg/day) within 5 days. The most effective lipolytic bacteria isolates in the oil-rich wastewater were two species of the genus Pseudomonas and one of Bacillus. Comparing the weights on the first day to the twelfth day values when lipolytic organisms were grown in palm oil, some appreciable increases in weight difference were recorded in some isolates: 28.3%, 7.84%, 4.44% and 6.98% for Pseudomonas, Staphylococcus, Bacillus and Klebsiella, respectively. The weight increase of each of the microbial cells in palm oil culture was usually lesser than what was obtained in the oil-rich wastewater culture. Two isolates showed high similar sequence (99%) to that of Pseudomonas aeruginosa and Lysinibacillus sphaericus, respectively. From palm oil, Lysinibacillus sp. produced various forms of fatty acids in the medium, including myristic acid (2.61%), palmitic acid (6.22%), stearic acid (5.18%) and arachidic (3.66%). These strains are versatile in utilizing the limited nutrient and had the ability to grow appreciably in the toxic condition (soap solution), suggesting that they may serve as candidates in treating dietary oil-rich wastewater.

Open Access Original Research Article

Phenotypic and Genotypic Characteristics of Rhizobia Isolated from Meknes-tafilalet Soils and Study of Their Ability to Nodulate Bituminaria bituminosa

Btissam Ben Messaoud, Imane Aboumerieme, Laila Nassiri, Elmostafa El Fahime, Jamal Ibijbijen

Microbiology Research Journal International, Page 405-417
DOI: 10.9734/BMRJ/2014/6172

Aims: The objectives were to isolate and characterize phenotypically and genotypically the rhizobial strains from the soils belonging to the Meknes-Tafilalet region in order to select strains that are able to nodulate Bituminaria bituminosa.
Study Design: An experimental study.
Place and Duration of Study: Department of biology (Soil & Environment Microbiology Unit) Faculty of Sciences, Moulay Ismail University and Technical Support Unit for Scientific Research, CNRST in Rabat; between January and August 2010.
Methodology: Samples from 23 different sites belonging to the Meknes-Tafilalet region were collected in order to select rhizobial strains that are able to nodulate Bituminaria bituminosa. The morphological, cultural and phenotypic parameters of isolated strains were studied. The phenotypic characteristics include colony morphology, growth speed, tolerances to temperature, salt and pH. To assess the genotypic diversity among the isolates, molecular characteristics based on 16S rDNA gene sequencing were performed.
Results: The majority of the isolated strains showed fast-growing capacity (75%). Most strains tolerate neutral to alkaline pH, however some strains (18%) showed weak growth capacity at pH 4. All isolates were tolerant to high salt stress ([NaCl] = 3%). The genotypic characterization based on16S rDNA gene sequencing of the twelve strains showed a high diversity between the isolates.
Conclusion: Taken together, our results highlight the important biodiversity of the isolated rhizobial strains and open opportunities for the development of new bio-fertilizer.

Open Access Original Research Article

Salmonella Enteritidis Flagella may Enhance Attachment and Invasion of Hen Ovarian Granulosa Cells and Induce Protective Immune Response in Egg-laying Hens

Anwar Ali Abdulla, Mokhtar Arshad, A. Mahdi Saeed

Microbiology Research Journal International, Page 418-427
DOI: 10.9734/BMRJ/2014/7425

Aims: To study the effect of flagellin on bacterial attachment and invasion of avian ovary cells in vitro by comparing the attachment and invasion of wild-type S. Enteritidis with non-motile mutants. To assess the immunogenic properties of extracted flagellin against Salmonella Enteritidis experimental infection in laying hens.
Methodology: Non-flagellated mutants for wild-type S. Enteritidis (phage type 8, 13A and 28) were produced by using a strain of S. Enteritidis, SA4502, which carried an fliC::Tn 10 to transfer fliC::Tn 10 insertion into the wild type strains using phage 22 (P22)-mediated transduction with selection for antibiotic resistance encoded within the mutant alleles. Granulosa cells were harvested from Single Comb White Leghorn hens between 18-45 weeks of age. Flagellin was purified from the studied bacterial cultures of Salmonella Enteritidis following reported methods. Laying hens were immunized with the flagellin with adjuvant
Results: Non-motile mutants of S. Enteritidis phage wild types were analyzed to confirm the elimination of H1 flagellin synthesis. Wild-type and fliC mutant strains were assessed for their ability to adhere to hen's ovarian granulosa cells. The adherence of the mutant strain was reduced nearly ten-fold compared with that of the wild-type phage 8. Similarly, light microscopic observation of fixed cover slips from wild-type phage types and its mutant strain revealed fewer numbers of the bacterial mutants adhered to the cultured granulosa cell monolayer. Light microscopy revealed similar findings for mutant phage types 28 and 13 A when compared to the wild-type control. There was five folds rise in the egg yolk antibody during the 2-3 weeks post-immunization. No rise was detected in the egg yolk samples from the control hens injected with the placebo mixture without flagellin.
Conclusion: It was concluded that Flagellin has an important role in the attachment and invasion of Salmonella Enteritidis to avian ovary cells and that it can be used as immunogenic components to induce a protective immune response in vaccinated hens against challenge infection with the wild type strains.

Open Access Original Research Article

Optimization of Process Parameters for Improved Production of Bioactive Metabolites by Streptomyces tritolerans DAS 165T

Usha Kiranmayi Mangamuri, Vijayalakshmi Muvva, Sudhakar Poda, Dayanand Agasar

Microbiology Research Journal International, Page 428-442
DOI: 10.9734/BMRJ/2014/7086

Aims: To optimize the process parameters for enhanced production of bioactive metabolites by Streptomyces tritolerans DAS 165T.
Place and Duration of Study: Department of Botany and Microbiology, April 2012 to August 2012.
Methodology: Agar well diffusion assay was employed to study the effect of environmental parameters such as incubation period, pH, temperature and salt concentration and influence of various nutrients such as carbon and nitrogen sources and minerals on the bioactive metabolite production by Streptomyces tritolerans DAS 165T.
Results: The production of antimicrobial metabolite was high when the strain was cultured for six days at 35ºC in medium (pH 7.5) with sucrose at the concentration of 2% (carbon source), soya peptone at the concentration of 1% (nitrogen source) and sodium chloride at the concentration of 5%.
Conclusion: This is the first report on the optimization of bioactive metabolite production by Streptomyces tritolerans DAS 165T. As the strain exhibited potent antimicrobial activity, it may be explored for biotechnological purposes.

Open Access Original Research Article

Rapid Detection of Extended Spectrum Β-lactamase (ESBL) Producing Strain of Escherichia coli in Urinary Tract Infections Patients in Benha University Hospital, Egypt

Enas Sh. Khater, Hammouda W. Sherif

Microbiology Research Journal International, Page 443-453
DOI: 10.9734/BMRJ/2014/7376

Background: An increase in extended spectrum β-lactamase (ESBL)-producing Escherichia coli (E. coli) has been observed.
Aims: Of this study was done to detect the prevelance of ESBL, AmpC producing and ESBL and AmpC co-producing strains of Escherichia coli (E. coli) in urinary tract infections patients in Benha University Hospital and to evaluate the performance of CHROMagar™ ESBL media for rapid screening of ESBL producing E. coli.
Place and Duration of Study: This is a Six-months Cross sectional study conducted in Urology and Microbiology & Immunology departments, Benha University, Egypt.
Methodology: All patients under study were subjected to: Full history taking and clinical examination. Bacteriological study included; urine sample collection from each patient and subjected to urine analysis, urine culture on cysteine lactose electrolyte deficient agar (CLED) agar, CHROMagar™ ESBL media and MacConkey agar supplemented with 2 mg/liter ceftazidime (MCKC). ESBL detection in E. coli isolated on CLED agar by phenotypic screening by clinical and laboratory standards institute (CLSI) method then phenotypic confirmation by E. test. The presence AmpC beta-lactamase ESBL was detected by AmpC disc test and detection of AmpC beta-lactamase and ESBL co-producers by cefepime and Cefepime + Clavulanate E test.
Results: In this study out of 45 E. coli strains 24 (53.3%) ESBL producers were detected by E. test (golden method for confirmation of ESBL according to CLSI) and 21(46.7%) strains were non ESBL producers. There was no significant difference between ESBL isolation from community acquired and health care associated UTI patients; out of the 24 isolated ESBL producing E.coli strains 9 (37.5%) were detected in community acquired UTI patients while 15 (62.5%) were detected in health care associated UTI patients. The sensitivity of both MCKC and CHROMagar™ ESBL media were 100%(95%CL: 85.6% to 100%).While specificity were 87.5% (95%CL:67.6% to 97.2%) and 80.8% (95%CL: 60.6% to 93.4%) respectively. In our study out of 45 isolated E. coli strains 14 (31.1%) were AmpC producers by AmpC test, 4 (8.9%) were AmpC and ESBL co-producers by cefepime/ cefepime clavulanic E.test.
Conclusion: It is important to know the prevalence of ESBL, AmpC producing and ESBL&AmpC co-producing organisms so that judicious use of antibiotics could be done and increase awareness about the need for routine detection of AmpC and ESBL in clinical isolates. CHROMagar™ ESBL media detect ESBL producers from clinical specimen and give rapid presumptive identification by means of colony colour after 24h with good sensitivity and specificity.

Open Access Original Research Article

Determination of Vancomycin and Methicillin Resistance in Clinical Isolates of Staphylococcus aureus in Iranian Hospitals

Abbas Farhadian, Qorban Behzadian Nejad, Shahin Najar peerayeh, Mohammad Rahbar, Farzam Vaziri

Microbiology Research Journal International, Page 454-461
DOI: 10.9734/BMRJ/2014/4836

Aims: The aim of this study was to determine the prevalence of methicillin resistant S. aureus (MRSA), vancomycin resistant or vancomycin intermediate resistant S. aurues (aureus) (VRSA/VISA) among clinical isolates.
Study Design: S.aureus isolates used in this study were randomly collected from in-patient and outpatient of several hospitals of 7 cities in Iran (Tehran, Shiraz, Zahedan, Tabriz, Sannandaj, Sari, and Ahvaz) during 2006-2008.
Methodology: Antibiotic susceptibility of 250 strains of Staphylococcus aureus isolated from Iranian hospitals were determined by disk diffusion method. Minimum inhibitory concentration (MICs) were determined for oxacillin and vancomycin by E-test. PCRs were used by specific primers (PCR used specific primers) for detection of mecA, vanA, vanB genes.
Results: The percentage of resistance by disk diffusion method was as below: methicillin 46%, vancomycin 0%, penicillin 86%, erythromycin 42%, ciprofloxacin 29%, gentamicin 39% and clindamycin 33%. E-test MIC method showed that 43% isolates were resistant to methicillin and 4% isolates were VISA (≤ 8µg/ml). The prevalence of resistance genes in the clinical isolates were: mecA 44%, vanA 0%, vanB 0%.
Conclusion: This study revealed that clinical isolates have rather high resistance to methicillin, erythromycin, gentamicin, penicillin and clindamycin We did not observe resistance to vancomycin. In order to avoid a possible outbreak involving VISA), vancomycin should be used carefully as a drug for treatment of S. aureus infections.

Open Access Original Research Article

Prevalence and Antibiotic Resistance Profile of Salmonella Isolates from Commercial Poultry and Poultry Farm-handlers in Jos, Plateau State, Nigeria

G. O. A. Agada, I. O. Abdullahi, M. Aminu, M. Odugbo, S. C. Chollom, P. R. Kumbish, A. E. J. Okwori

Microbiology Research Journal International, Page 462-479
DOI: 10.9734/BMRJ/2014/5872

Aim: This study was designed to investigate the prevalence and antibiotic resistance profile of Salmonella serovars from poultry and poultry farm-handlers.
Study Design: Investigative
Place and Duration of Study: Samples were analyzed at the Central Diagnostic Laboratory, National Veterinary Research Institute Vom and Department of Microbiology, Ahmadu Bello University, Zaria. This work was carried out between August 2012 and April 2013.
Methodology: Samples were pre-enriched in buffered peptone water followed by selective enrichment using Selenite Faeces Broth and Rappaport-Vassilidis Broth. Isolation and identification was made by inoculating the selectively enriched sample on to Salmonella-Shigella agar, Xylose Lysine Deoxycholate agar and Brilliant Green agar followed by confirmation of presumptive colonies using different biochemical tests and analytical profile index 20 E. Polyvalent (O) and (H) Salmonella antisera were used for serotyping the Salmonella isolates. The CLSI, 2010 method was used for antimicrobial susceptibility testing
Results: A prevalence rate of 10.9% was observed from the 450 samples. Serovars of Salmonella detected were S. Gallinarum 57.2%, S. Typhimurium 8.2%, S. Typhi 20.4%, S. Pullorum 6.1%, S. Enteritidis 6.1% and S. Paratyphi A 2.0%. Statistically, significant difference (p<0.05) was observed between isolates and occurrence at different sample sites. The isolates were 100% resistant to oxacillin, 96.0% to ampicillin, 93.9% tylosin, 83.7 5 ceftazidime and 63.3% oxytetracycline. Five of the isolates were 100% resistant to more than five different antibiotics. There was statistical significant difference (p<0.01) in antimicrobial resistance patterns exhibited by the serovars. However, the isolates showed sensitivity to gentamycin 100%, gendox 83.7%, ciprofloxacin 81.6% and amoxicillin-clavulanic acid 57.1%.
Conclusion: The study revealed emergence of multiple-drug resistant Salmonella serovars from poultry and poultry farm handlers. We therefore suggest further epidemiological studies.