18/8/2016 SOURCES OF MICROBIAL CONTAMINANTS IN BIOSAFETY LABORATORIES IN KENYA PRESENTER: DENNIS NYACHAE MOSE KENYATTA UNIVERSITY 1
INTRODUCTION Contamination occurs through avoidable procedural errors Modern laboratories are busy environments with personnel sharing equipment across overlapping work stations that may be near high-traffic areas and busy instruments Can be influenced by factors for example temperature, humidity, nutrient media used in the labs as well as storage conditions of the media. 24-Aug-16 2
Microorganism (filamentous fungi, yeasts, bacteria viruses and viroids) and micro-arthropods (mites and thrips) have been identified as contaminants. Some of the most basic laboratory procedures are the most important, including using proper aseptic technique, wearing clean lab coats and washing hands in order to reduce the risk of introducing microorganisms into mammalian cell cultures. 24-Aug-16 3
Objectives To determine sources of microbial contaminations in biosafety laboratories. 24-Aug-16 4
Significance of the study assist the personnel to be careful when performing standard manipulations of microbiological specimens. Hence this will help in reducing the costs associated with the application of the technology in biosafety laboratories 24-Aug-16 5
METHOD Media preparation Sample collection from lab sites using nutrient broth Isolation of the microbial contaminants to 0.1 % NA and PDA Incubation at 25 C for 72 h for fungal growth PDA and 0.1 % NA 37 C for 24 h for bacterial growth Identification of fungus and bacteria isolates using biochemical methods (gram stain and microscopy) and morphological Characteristics 24-Aug-16 6
Continued Isolation of persistent bacterial strains Molecular identification of persistent bacteria isolates DNA isolation PCR amplification (27F 5 -AGAGTTTGATCMTGGCTCAG-3 and (R1525 5 - AAGGAGGTGWTCCARCC -3 ) Restriction Fragment Length Polymorphism (RFLP) analysis
Data analysis Percentage data on incidences of contamination transformed using square root method. Data on bacterial contamination analyzed using ANOVA with statistical GENESTAT version 6 computer software. Means separated using Tukeys Honest Significance Difference at 5 % level.
RESULTS Isolation and identification of contaminants in biosafety laboratories Thirteen bacterial and fungal isolates obtained and identified from different laboratory places. All sites tested contained both bacterial and fungi No site was negative for both bacteria and fungi
BIOCHEMICAL IDENTIFICATION OF ISOLATES Colony on NA H 2 S gas Motility Gram stain Catalase Starch hydrolysis Citrate utilization Indole Lactose Oxidase Isolate identity White, smooth, creamy and round Green, glossy pigmented, thin White, moist, glistening growth - - + Coccus in clusters + - - - - - Staphylococcus aureus - - - Bacillus + - + - - + Pseudomonas aeruginosa - + + Cocci + - - + AG - Escherichia coli White glossy membranous - - + Bacillus + + - - - - Bacillus subtilis Clear, small, round, irregular Grayish, granular, limited growth - - - Bacillus + - + - + - Enterobacter sp - - + Bacillus + - - - - - Corynebacteria sp Translucentcreamy, mucoid, round - - - Bacillus + - + - AG - Klebsiella sp + Positive; - Negative; AG Acid Gas
SITE MICROBES IDENTIFIED ON EACH SITE Laboratory walls Tables Staphylococcus aureus, E. coli, Rhizopium sp, Fusarium sp, Bacillus subtillis, Aspergillus sp, Enterobacter aerogenes, Pseudomonas aeruginosa and Cladosporium sp. Staphylococcus aureus, E. coli, Salmonella sp, Shigella sp, Pseudomonas aeruginosa, Bacillus pimillis and Cladosporium sp. Dust coats and gloves Staphylococcus aureus, E. coli, Enterobacter aerogenes, Salmonella sp, Shigella sp and Cladosporium sp. Biosafety cabinets Salmonella sp, Aspergillus sp, E. coli, Rhizopus sp, Penicillium sp and Cladosporium sp. Door knobs Staphylococcus aureus, E. coli, Aspergillus sp, Fusarium sp and Cladosporium sp.
CONT SITE Preparation rooms MICROBES IDENTIFIED ON EACH SITE Staphylococcus aureus, Klebsiella pneumonia, E. coli, Enterobacter aerogenes, Salmonella sp, Shigella sp, Pseudomonas aeruginosa, Bacillus pimillis and Cladosporium sp. Incubating room Salmonella sp, Aspergillus sp, E. coli, Rhizopus sp, Penicillium sp and Cladosporium sp. Laboratory indoor air E. coli, Penicillium sp, Rhizopus sp and Cladosporium sp. Floor Staphylococcus aureus, E. coli, Enterobacter aerogenes, Salmonella sp, Shigella sp, Pseudomonas aeruginosa, Bacillus pimillis and Cladosporium sp.
Cont Y M N A B C Y M Y M D A, Staphylococcus aureus on mannitol agar isolated from KUPTL floor; B, Corynebacteria on nutrient agar isolated from KARI floor; Biochemical tests. C, TSI test; D, Methyl red test for microbes, E, Simmons test; Y, are positive; M, are the controls. E
Microscopic and Gram s characteristics of identified bacteria Bacterial species Shape Arrangements Gram reaction Motility Escherichia coli Pseudomonas aeruginosa Straight rods, cocobacilliary Straight and slightly curved rods Singles G-ve Motile Shigella sp. Short rods Singles, clustered G-ve Salmonella sp. Straight rods Paired G-ve Motile Bacillus pumilus Rods Singles, pairs G +ve Motile Singles/ pairs G-ve Nonmotile Nonmotile Bacillus subtillis Rods Singles, pairs G+ve Motile Staphylococcus aureus Cocci Singles, pairs and G+ve Nonmotile irregular clusters
Gel electrophoresis of DNA M 1 2 3 4 5 6 12000-5000- 2000-1650 - 1000-850 - 650-500 - 400 - Gel electrophoresis of bacterial genomic DNA. Lanes 1, Bacillus sp; Lane 2, Shigella sp; Lanes 3, Pseudomonas sp; Lane 4, Corynebacteria sp and lanes 5-6, Staphylococcus sp; M, the standard 1 kb plus marker(promega).
M 1 2 3 4 5 6 7 8 3000-2000 - 1000-800 - 600-500 - Electrophoresis in 1 % agarose gel of PCR products. Lanes 1-2, Shigella sp; Lane 3-4, B. subtilis; Lane 5-6, P. aeruginosa; Lane 7-8, S. aureus. Lane M, 100 bp DNA marker (Sigma).
12000-5000 - 2000-1650 - 1000-850 - 650-500- 400-200- M 1 2 3 4 5 6 7 8 Hae III digestion patterns of PCR products from standard bacteria and bacterial isolates from biosafety level II laboratory. Lanes 1-2, S. aureus (Lane 1, biosafety sample and Lane 2 standard sample NCO7447); Lanes 3-4, Shigella (Lane 3, biosafety sample and Lane 4 standard sample ATCC 25922); Lane 5-6, P. aeruginosa (Lane 5, standard sample NC12924 and Lane 6 biosafety sample); Lanes 7-8, B. subtilis (Lane 7, standard samples NCO8241 and Lane 8, biosafety sample); Lane M 1 kb plus marker (Promega).
Water used in systems DISCUSSION Sources of contaminants During collection of specimen raw materials Improper cleaning of procedures Improper techniques in hood or lab bench Mobile phones, bags, pens, notebooks and shoes, 24-Aug-16 18
E.coli bacterium was frequently isolated in biosafety laboratories. Associated with infections such as diarrhea. Pseudomonas sp reportedly associated with wet surfaces of air- conditioning systems, cooling coils, drain pans and sump pumps PCR followed by RFLP can be used to identify the above bacteria was rapid and effective.
Conclusion contaminants were still found in biosafety cabinets even after disinfection. Bacterial and fungal contamination remains a continuing threat in biosafety laboratories, but techniques for reducing contamination are available. It was noted that certain specific microorganisms like Salmonella, Staphylococcus, Aspergillus sp and Cladosporium were still found on gloves and biosafety cabinets.
RECOMMENDATION All personnel must use dust coats which should be cleaned daily and must wear laboratory canvas once in the labs. Labs to have QAOs for SOPs Keep the cabinet fully closed when not in use There is need to increase the concentration of disinfectants or change to others due to persistence.
Lab cleaning should include any high flat surfaces, such as the tops of refrigerators, freezers and incubators, which can collect dust and other potential contaminants There is need for laboratories to periodically use open plates and swabs to know the levels of contamination in each lab to determine their disinfection procedures 24-Aug-16 22
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