Figure 4. Rela%ve abundance of selected bacterial genera in samples of several concentra%ons on surfaces recovered with different swabs. (†: Each result was composed by the mean of 4 samples; Heatmap realized with Prism 7) 95.66 39.74 5.93 0.18 0.00 14.41 1.69 0.19 0.21 0.14 94.83 49.74 10.19 0.08 0.00 72.11 34.34 4.60 0.08 0.05 0.02 0.45 1.44 0.80 0.62 4.58 0.95 4.19 0.41 0.03 0.00 0.52 0.74 0.84 2.38 5.19 17.33 0.71 0.56 0.80 2.40 0.09 0.23 0.43 0.00 0.00 0.00 0.11 1.00 0.10 0.00 0.00 0.26 0.15 0.01 0.00 0.00 0.15 0.08 0.00 0.00 0.00 0.01 0.36 5.20 7.97 8.21 0.00 0.11 0.16 0.19 0.08 0.00 0.23 4.84 9.11 7.16 1.26 0.16 2.36 3.58 2.85 0.00 3.25 0.87 10.85 24.56 22.74 0.00 0.37 1.74 1.24 0.65 0.00 0.97 12.77 24.83 16.00 13.23 1.51 10.79 20.56 11.56 0.00 28.14 0.54 9.02 11.15 14.49 62.27 0.74 0.74 0.67 0.79 0.85 0.68 7.20 13.74 23.75 25.38 0.52 6.03 8.24 12.90 61.37 0.34 0.00 0.01 0.00 0.00 0.00 19.21 21.60 16.42 25.70 32.56 0.00 0.04 0.17 0.10 0.00 0.00 0.03 0.00 0.05 0.00 0.01 0.05 0.67 1.01 1.09 4.18 0.05 0.43 0.13 0.16 0.00 0.08 0.39 0.49 1.64 0.00 4.04 0.35 1.61 1.76 6.18 0.05 Bacillus Esch eric hia-Sh igel la Salm onel la Broc hoth rix Carn obac teriu m Eliza beth king ia Micr obac teriu m Staph yloc occu s Cotton pad A Cotton pad B Cotton pad C Cotton pad D MRD cotton pad Sponge A Sponge B Sponge C Sponge D MRD Sponge Gauze pad A Gauze pad B Gauze pad C Gauze pad D MRD gauze pad Sponge-Stick A Sponge-Stick B Sponge-Stick C Sponge-Stick D MRD Sponge-Stick Mean Matrix (Chicken)
0 20 40 60 80
Comparison between swabbing devices in order to analyze the microbial flora
found on surfaces of community kitchens by classical microbiology and 16S
rRNA amplicon sequencing
Krings S.*
1, Crèvecoeur S.
1, Rodriguez C.
1, Fall P. A.
2, Taminiau B.
1, Daube G.
11University of Liege, Faculty of Veterinary Medicine, Department of Food Science & FARAH, Liège, Belgium 2Genalyse Partner SA, Herstal, Belgium *skrings@ulg.ac.be The work in community kitchens involves several manual steps bearing the risk of transmiVng pathogenic microorganisms. This fact jus%fies accurate control of surfaces to prevent foodborne illnesses. In this study, the efficiency of different swabbing devices was tested in terms of recovery of microorganisms by culture methods and culture-independent 16S rRNA amplicon sequencing. Solu%on of 2-%mes diluted chicken (+/- 102 CFU/mL) 1 mL spread with L-spreader LeT to dry for 10 min Sterile polypropylene cuUng board (PP, rough) Sterile stainless steel (SS, smooth) 1mL of each of those 3 bacteria (in droplets): • Bacillus cereus ATCC 13061 (Bc) • Escherichia coli (Ec) • Salmonella enterica ssp. enterica serovar EnteridiYs (Sa) At 3 concentra%ons:
A: 104 CFU/mL of Bc, 104 CFU/mL of Ec, 104 CFU/mL of Sa
B: 103 CFU/mL of Bc, 103 CFU/mL of Ec, 103 CFU/mL of Sa
C: 102 CFU/mL of Bc, 102 CFU/mL of Ec, 102 CFU/mL of Sa
D: Only chicken LeT to dry for 10 min 1) Culture method: • PCA Bio-Rad 356-4475 • MYP Oxoid CM0929 + SR0047+ SR0099 • Rapid’E.coli 2 Bio-Rad 355-5299 • Rapid’Salmonella Bio-Rad 356-3961
Co`on pads Colruyt Gauze pads Mercurochrome Sponges (with Neutralizing Buffer) 3M™ HS10NB2G Sponge-S%cks 3M™ SSL100
4 sterile swabbing devices: 2) V1-V3 16S rRNA amplicon sequencing: • DNA extrac%on Qiagen Blood & Tissue kit, Cat.No/ID: 69506 • Illumina MiSeq Analysis by mothur AS BS CS AR BR CR 0 1 2 3 4 5 log (CFU/mL) Bacillus cereus Initial concentration Cotton pad recovered Sponge recovered Gauze pad recovered Sponge-Stick recovered ** *** **** **** **** *** * * ** **** **** *** ***** ** AS BS CS AR BR CR 0 1 2 3 4 5 log (CFU/mL) Escherichia coli Initial concentration Cotton pad recovered Sponge recovered Gauze pad recovered Sponge-Stick recovered ** **** ** **** ** ** **** **** **** *** AS BS CS AR BR CR 0 1 2 3 4 5 log (CFU/mL) Salmonella enterica Initial concentration Cotton pad recovered
Sponge recovered Gauze pad recovered Sponge-Stick recovered *** ** **** ** ** *** **** *** * **** *** *** ** The perfect swab for kitchen analyses should recover the highest number of viable bacteria with a high populaYon diversity from the surfaces. Classical culture method showed best recovery numbers for the 3 inoculated bacteria with Sponge-S%cks (no significant difference between inocula%on dosis and recovered number of bacteria, p > 0.1234). The 16S rRNA amplicon sequencing allows to conclude that sponge samples were loaded with Microbacterium genus (from Neutralizing buffer). Furthermore, a high relaYve abundance of Bacillus genus was found in co`on pad, gauze pad and Sponge-S%ck samples. Salmonella genus was detected in only low proporYons, whereas Escherichia genus are problema%c as their DNA can be contaminants of reagents used during library crea%on. However, differences in recovery or
enumera%on with each method must be considered, as they can induce es%ma%on bias on the ini%al concentra%on or recovered CFU/mL. Finally, low amount of DNA in controls lead to the emergence of free DNA contaminants like Elizabethkingia popula%on, which can be considered as a bias. Further studies will be performed to assess the recovery of bacteria aZer longer drying Ymes in order to approach real kitchen condi%ons and make the final swab decision. Figures 1, 2 and 3. Comparison between inocula%on dosis and recovery numbers of Bacillus cereus, Escherichia coli and Salmonella enterica, respec%vely, by the use of different swabs and different concentra%ons. (AS/AR: concentra%on A (104 CFU/mL) on the smooth/rough surface; BS/BR: concentra%on B (103 CFU/mL) on the smooth/rough surface; CS/CR: concentra%on C (102 CFU/mL) on the smooth/rough surface; CFU/mL: colony-forming unit/milliliter; * = 0.0332; ** = 0.0021; *** = 0.0002; **** < 0.0001; †: Each sample was realized in duplicate; 2-way ANOVA and Tukey’s mul\ple comparisons performed with Prism 7) AS BS CS AR BR CR 0 1 2 3 4 5 log (CFU/mL) Bacillus cereus Inoculation dosis
Cotton pad recovered Sponge recovered Gauze pad recovered Sponge-Stick recovered
