PRESENTER: DENNIS NYACHAE MOSE

KENYATTA UNIVERSITY

18/8/2016

1

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