A Nordic standard procedure for detection and enumeration of thermotolerant Campylobacter in foods

Transcription

1 A Nordic standard procedure for detection and enumeration of thermotolerant Campylobacter in foods Results of a collaborative study on NMKL* 119, rev. *Nordic Committee on Food Analysis

2 Aim To develop a (Nordic) standard procedure for quantification of thermotolerant Campylobacter in food To validate this method through a collaborative study To have the method approved by the Nordic Committee on Food Analysis Hanne Rosenquist 2

3 Background year 2000 The standard methods (NMKL, ISO) for detecting thermotolerant Campylobacter in food were qualitative (Result: Detected/Not detected) Quantification was needed for Quantitative Microbiological Risk Assessment and increasing research on interventions (aiming at reducing numbers rather than eliminating the organism) Hanne Rosenquist 3

4 NMKL 119, rev. includes 3 procedures Qualitative detection When detected or not detected is an acceptable result Principle: Selective enrichment of a specified amount of sample material followed by plating on a selective media. Semi-quantitative detection When an estimate of the level of Campylobacter contamination is needed When a low number of Campylobacter is expected, or When the level of accompanying flora is relatively high Principle: Selective enrichment of a serial dilution followed by plating on selective media. Quantitative determination When an estimate of the actual number of Campylobacter in the sample is needed Principle: Direct plating on a selective media from a serial dilutions of a sample followed by enumeration of typical colonies. Hanne Rosenquist 4

5 NMKL 119, rev. includes 3 procedures Qualitative detection When detected or not detected is an acceptable result Principle: Selective enrichment of a specified amount of sample material followed by plating on a selective media. Semi-quantitative detection When an estimate of the level of Campylobacter contamination is needed When a low number of Campylobacter is expected, or When the level of accompanying flora is relatively high Principle: Selective enrichment of a serial dilution followed by plating on selective media. Quantitative determination When an estimate of the actual number of Campylobacter in the sample is needed Principle: Direct plating on a selective media from a serial dilutions of a sample followed by enumeration of typical colonies. Hanne Rosenquist 5

6 NMKL 119, rev. includes 3 procedures Qualitative detection When detected or not detected is an acceptable result Principle: Selective enrichment of a specified amount of sample material followed by plating on a selective media. Semi-quantitative detection When an estimate of the level of Campylobacter contamination is needed When a low number of Campylobacter is expected, or When the level of accompanying flora is relatively high Principle: Selective enrichment of a serial dilution followed by plating on selective media. Quantitative determination When an estimate of the number of Campylobacter in the sample is needed Principle: Direct plating on a selective media from a serial dilutions of a sample followed by enumeration of typical colonies. Hanne Rosenquist 6

7 NMKL 119, rev.: Flowsheet Qualitative detection Semi-quantitative detection Quantative determination Test sample 25 g in 225 ml Bolton broth Test sample 15 g stomachated in 120 ml complete Bolton broth. Serial dilutions in Bolton Broth Test sample 10 g stomachated in 90 ml BPW. Serial dilutions With preincubation: Incubation at 37.0 ± 1.0 C for 2-4 hours and then the selective supplement is added. Incubation is continued at 41.5 ± 1.0 C for 48 ± 4 hours in microaerobic atmosphere 10 μl surface spread onto mccda or AHB (+TCC) Incubation at 41.5 ± 1.0 C for 48 ± 4 hours in microaerobic atmosphere Without preincubation: Incubation at 41.5 ± 1.0 C for 48 ± 4 hours in microaerobic atmosphere Incubation at 41.5 ± 1.0 C for 48 ± 4 hours in microaerobic atmosphere 10 μl surface spread onto mccda or AHB (+TCC) from dilutions. Incubation at 41.5 ± 1.0 C for 48 ± 4 hours in microaerobic atmosphere Reading results and confirmation by phase contrast microscopy 100 μl surface spread onto mccda and AHB (+TCC) from dilutions. Incubation at 41.5 ± 1.0 C for 48 ± 4 hours in microaerobic atmosphere Hanne Rosenquist 7

8 NMKL 119, rev.: Further identification Biochemical tests Oxidase test Catalase test Hydrolysis of hippurate Hydrolysis of indoxylacatate Commercially available identification systems PCR Hanne Rosenquist 8

9 Plating media: AHB or mccda Abeyta-Hunt-Bark (AHB) + TTC Heart infusion agar 40 g Yeast extract 2 g Sodium metabisulphite 0.25 g Ferrosulphate, FeSO 4, 7H 2 O 0.25 g Sodium pyruvate 0.25 g Cefoperazone 32 mg Rifampicin 10 mg Amphotericin B 2 mg Triphenyltetrazoliumchloride 1% 10 ml Dist. water 950 ml ph 7.2 ± 0.2 at 25 C Modified Charcoal Cephoperazone Desoxycholate Agar (mccda) Agar 12 g Meat extract 10 g peptone 10 g Sodium chloride 5 g Bacteriological charcoal 4 g Casein hydrolysate 3 g Sodium desoxycholate 1 g Ferrous sulphate, FeSO 4, 7H 2 O 0.25 g Sodium pyruvate 0.25 g Cephoperazone 32 mg Amphotericin B Dist. Water 10 mg 1000 ml ph 7.4 ± 0.2 at 25 C Hanne Rosenquist 9

10 Collaborative study: Overview 14 laboratories (S, N, I, DK, SF, B, NL) 3 matrices (raw minced chicken meat, fresh cut lettuce, pasteurised full milk) 3 methods (qualitative, semi-quantitative, quantitative) 2 strains (C. jejuni SLV-542 and C. coli SLV-271) 3 concentrations (low, medium, high plus blanks) Hanne Rosenquist 10

11 Collaborative study on NMKL 119, rev. Overview of methods, matrices, strains and levels Method Detection limit (cfu/g, ml) NMKL 119, rev. Qualitative detection 0.04 NMKL 119, rev. Semi-quantitative detection 0.1 NMKL 119, rev. Quantitative detection 100 Strain Level* Expected mean concentration** (cfu/g, ml) C. jejuni Low 1.0 x 10 0 High 4.4 x 10 1 C. coli Low 1.0 x 10 0 High 3.9 x 10 1 C. jejuni Low 1.0 x 10 0 Medium 4.4 x 10 1 High 7.9 x 10 2 C. coli Low 1.0 x 10 0 Medium 3.9 x 10 1 High 8.1 x 10 2 C. jejuni Low 7.9 x 10 2 Medium 4.0 x 10 3 High 7.7 x 10 3 C. coli Low 8.1 x 10 2 Medium 8.7 x 10 3 High 7.4 x 10 4 Matrix inoculated chicken milk lettuce chicken milk chicken milk Hanne Rosenquist 11

12 Results: Specificity Qualitative detection Semi-quantitative detection Quantitative determination Number of blank samples tested Specificity Hanne Rosenquist 12

13 Results: Sensitivities qualitative detection Qualitative detection Sensitivity (%) 100,0 90,0 80,0 70,0 60,0 50,0 40,0 30,0 20,0 10,0 0,0 Chicken meat Milk Fresh cut lettuce with preincubation Fresh cut lettuce without preincubation Total Low level (ca. 1 cfu/g) High level (ca. 40 cfu/g) Hanne Rosenquist 13

14 Results: Sensitivities semi-quantitative detection Semi-quantitative detection Sensitivity (%) 100,0 90,0 80,0 70,0 60,0 50,0 40,0 30,0 20,0 10,0 0,0 Chicken meat Milk Total Low level (ca. 1 cfu/g) Medium level (ca. 40 cfu/g) High level (ca. 800 cfu/g) Hanne Rosenquist 14

15 Results: Semi-quantitative detection Sensitivity distribution within intervals Expected mean concentration Matrix No. of positives / number of samples analysed Percentage of the results within the following intervals of thermotolerant Campylobacter (cfu/g) < >10000 Low Chicken 17/ ( 1 cfu/g) Milk 16/ Medium Chicken 23/ ( 40 cfu/g) Milk 23/ High Chicken 25/ ( 800 cfu/g) Milk 24/ Hanne Rosenquist 15

16 Results: Sensitivities quantitative determination Quantitative detection Sensitivity (%) 100,0 90,0 80,0 70,0 60,0 50,0 40,0 30,0 20,0 10,0 0,0 Chicken meat Milk Total Chicken meat Milk Total AHB mccda Low level (ca. 800 cfu/g) Medium level (ca cfu/g for C.j.; ca cfu/g for C.c.) High level (ca cfu/g for C.j. and ca cfu/g for C.c.) Hanne Rosenquist 16

17 Results: quantitative determination - low and medium level Matrix Organism Level Selective agar N Median (log) Std. Dev Chicken C. jejuni Low AHB C. coli Low AHB C. jejuni Low mccda C. coli Low mccda C. jejuni Medium AHB C. coli Medium AHB C. jejuni Medium mccda C. coli Medium mccda Milk C. jejuni Low AHB C. coli Low AHB C. jejuni Low mccda C. coli Low mccda C. jejuni Medium AHB C. coli Medium AHB C. jejuni Medium mccda C. coli Medium mccda Hanne Rosenquist 17

18 Results: quantitative determination - high level Matrix Organism Level Selective agar N Median (log) Mk SDk s r r(log) RSD r S R R(log) RSD R Chicken C. jejuni High AHB C. coli High AHB C. jejuni High mccda C. coli High mccda Milk C. jejuni High AHB C. coli High AHB C. jejuni High mccda C. coli High mccda Chicken in total Milk in total AHB in total mccda in total TOTAL Repeatability (within laboratories) Reproducibility (between laboratories) Hanne Rosenquist 18

19 Conclusions A method including qualitative, semi-quantitative and quantitative detection of thermotolerant Campylobacter in food has been developed The specificity of the methods were satisfactory The sensitivity of the methods were OK The repeatability (within labs) of the quantitative analysis was satisfactory The reproducibility (between labs) of the quantitative analysis was quite high (not satisfactory?) reflects the difficulties in handling this organism Hanne Rosenquist 19

20 Thanks to The staff at the participating laboratories Niels Ladefoged Nielsen, Danish Food Administration Christina Normark, Livsmedelsverket Anja Bengtsson & Tina Bech Hansen, DFVF Winnie Grebell, Tina Birk, Bodil Linghartz, Kate Vibefeldt, Bodil Madsen & Louise Boysen, DFVF Hanne Rosenquist 20