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SOURCES OF MICROBIAL CONTAMINANTS IN BIOSAFETY ... · computer software. • Means separated using...
Transcript of SOURCES OF MICROBIAL CONTAMINANTS IN BIOSAFETY ... · computer software. • Means separated using...
PRESENTER: DENNIS NYACHAE MOSE
KENYATTA UNIVERSITY
18/8/2016
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SOURCES OF MICROBIAL CONTAMINANTS IN
BIOSAFETY LABORATORIES IN KENYA
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.
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• 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.
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Objectives
To determine sources of microbial contaminations
in biosafety laboratories.
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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
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
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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
Colony on
NA
H2S gas Motility Gram
stain Catalase Starch
hydrolysis Citrate
utilization Indole Lactose Oxidase Isolate identity
White,
smooth,
creamy and
round
- - + Coccus
in clusters + - - - - - Staphylococcus
aureus
Green , glossy
pigmented,
thin
- - - Bacillus + - + - - + Pseudomonas
aeruginosa
White, moist,
glistening
growth
- + + Cocci + - - + AG - Escherichia coli
White glossy
membranous - - + Bacillus + + - - - - Bacillus subtilis
Clear, small,
round,
irregular
- - - Bacillus + - + - + - Enterobacter sp
Grayish,
granular,
limited growth
- - + Bacillus + - - - - - Corynebacteria sp
Translucent-
creamy,
mucoid, round
- - - Bacillus + - + - AG - Klebsiella sp
BIOCHEMICAL IDENTIFICATION OF ISOLATES
+ Positive; - Negative; AG Acid Gas
SITE MICROBES IDENTIFIED ON EACH SITE
Laboratory walls
Staphylococcus aureus, E. coli, Rhizopium sp, Fusarium sp, Bacillus subtillis, Aspergillus sp, Enterobacter aerogenes, Pseudomonas aeruginosa and Cladosporium sp.
Tables
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 MICROBES IDENTIFIED ON EACH SITE
Preparation rooms
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’
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.
A B C
E
Y M N
Y M Y M
D
Microscopic and Gram’s characteristics of identified
bacteria
Bacterial species Shape Arrangements Gram reaction
Motility
Escherichia coli Straight rods, cocobacilliary
Singles/ pairs G-ve Non-motile
Pseudomonas aeruginosa
Straight and slightly curved rods
Singles G-ve Motile
Shigella sp. Short rods Singles, clustered
G-ve Non-
motile
Salmonella sp. Straight rods Paired G-ve Motile
Bacillus pumilus Rods Singles, pairs G +ve Motile
Bacillus subtillis Rods Singles, pairs G+ve Motile
Staphylococcus aureus Cocci Singles, pairs and
irregular clusters G+ve Non-
motile
Gel electrophoresis of DNA
5000-
12000-
2000 -
M 1 2 3 4 5 6
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).
1650 - 1000 -
850 -
650 - 500 -
400 -
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).
2000 -
3000 -
M 1 2 3 4 5 6 7 8
1000 -
800 -
600 - 500 -
M 1 2 3 4 5 6 7 8
2000 - 5000 -
12000 -
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).
1650 -
1000-
850
- 650-
500-
400-
200-
DISCUSSION Sources of contaminants
Water used in systems
During collection of specimen raw materials
Improper cleaning of procedures
Improper techniques in hood or lab bench
Mobile phones, bags, pens, notebooks and shoes,
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• 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.
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• 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
THANK YOU