Microbial Detection of Enterobacter sakazakii: Food and Clinical

Donald H. BurrDonald H. Burr

CFSAN/FDACFSAN/FDA

ObjectiveObjective

• Provide a summary of the methods for Provide a summary of the methods for isolating and quantifying levels of isolating and quantifying levels of E. E. sakazakiisakazakii in food and clinical samples. in food and clinical samples.

• Not allNot all of the material included in your of the material included in your White Paper will be discussed. White Paper will be discussed.

Initial Isolation Reports of Initial Isolation Reports of E. sakazakiiE. sakazakii

• 1980 - Farmer et. al.1980 - Farmer et. al.

• 1983 - Muytjens et. al.1983 - Muytjens et. al.

• 1984 – Postupa and Aldova1984 – Postupa and Aldova

• **All non-quantitativeAll non-quantitative

1980 - Farmer et. al.1980 - Farmer et. al.

• In designating In designating E. sakazakiiE. sakazakii as a new as a new species a can of dried milk listed as the species a can of dried milk listed as the source of one isolate. source of one isolate.

• No isolation details were provided. No isolation details were provided.

1983 - Muytjens et. al.1983 - Muytjens et. al.

• Studied 8 cases of neonatal meningitis associated Studied 8 cases of neonatal meningitis associated with with E. sakazakiiE. sakazakii..

• Isolated Isolated E. sakazakiiE. sakazakii several times from several times from prepared prepared formulaformula but never from either the powdered but never from either the powdered formula itself or the water used in preparing the formula itself or the water used in preparing the formula.formula.

• No information was reported on the quantity of No information was reported on the quantity of powdered formula analyzed.powdered formula analyzed. However,However,

– On 3/4/03, Dr. Muytjens informed FDA that the On 3/4/03, Dr. Muytjens informed FDA that the quantity analyzed was 10 g. quantity analyzed was 10 g.

1984 – Postupa and Aldova1984 – Postupa and Aldova

• Described 4 strains of Described 4 strains of E. sakazakiiE. sakazakii from from powdered milk and 2 strains from powdered milk and 2 strains from powdered milk infant formula. powdered milk infant formula.

• Isolated on deoxycholate-citrate agar Isolated on deoxycholate-citrate agar incubated at 37incubated at 37ooC for 48 hrs. C for 48 hrs.

• No details on the quantity analyzed. No details on the quantity analyzed.

Quantitative Methods Quantitative Methods DevelopmentDevelopment

• 1988 - Muytjens and co-workers:1988 - Muytjens and co-workers:“European Method”“European Method”

• 1997 – Nazarowec-White and Farber: 1997 – Nazarowec-White and Farber: “Canadian Method”“Canadian Method”

• 2002 - FDA Method2002 - FDA Method• Minor modifications only in the latter Minor modifications only in the latter

two methods; Sample size and sensitivity two methods; Sample size and sensitivity remains the same.remains the same.

European MethodEuropean Method• First described by Muytjens et. al. in 1988.First described by Muytjens et. al. in 1988.• In referring back to their 1983 paper they In referring back to their 1983 paper they

commented that “although it was not cultured commented that “although it was not cultured from the formula powder itself, this might have from the formula powder itself, this might have been due to an unequal distribution in the powder been due to an unequal distribution in the powder or its presence at such a low concentration that it or its presence at such a low concentration that it escaped detection by escaped detection by conventional methodsconventional methods”. (i.e., ”. (i.e., a 10 g sample).a 10 g sample).

• Therefore, they decided to culture large quantities Therefore, they decided to culture large quantities of powdered substitutes for breast milk for the of powdered substitutes for breast milk for the presence of all presence of all EnterobacteriaceaeEnterobacteriaceae including including E. E. sakazakiisakazakii. .

European MethodEuropean Method

• In triplicate mix 100, 10 and 1 gram samples with In triplicate mix 100, 10 and 1 gram samples with 900, 90 and 9 ml, respectively, of buffered 900, 90 and 9 ml, respectively, of buffered peptone water at 45peptone water at 45ooC until completely dissolved. C until completely dissolved. Incubate overnight at 36Incubate overnight at 36ooC.C.

• Inoculate 10 ml from each flask into 90 ml of Inoculate 10 ml from each flask into 90 ml of EnterobacteriaceaeEnterobacteriaceae enrichment (EE) broth. enrichment (EE) broth. Incubate overnight at 36Incubate overnight at 36ooC. C.

• In duplicate, inoculate 1 ml from each enrichment In duplicate, inoculate 1 ml from each enrichment broth into 20 ml of fluid Violet-Red-Bile Glucose broth into 20 ml of fluid Violet-Red-Bile Glucose (VRBG) agar. Incubate overnight at 36(VRBG) agar. Incubate overnight at 36ooC.C.

European MethodEuropean Method• Suspect colonies subcultured to sheep Suspect colonies subcultured to sheep

blood and eosin-methylene blue agars.blood and eosin-methylene blue agars.• Identify strains with API-20E system. Identify strains with API-20E system. • Additional testing for Additional testing for E. sakazakiiE. sakazakii

includedincluded production of yellow colonies production of yellow colonies on nutrient agar after 48 hr at 25on nutrient agar after 48 hr at 25ooC, C, production of extracellular DNase and a production of extracellular DNase and a positive alpha-glucosidase reaction. positive alpha-glucosidase reaction.

API 20E-System

                                                                                                                  

                 The API 20E system has become popular for rapid identification of members of the Enterobacteriaceae and other Gram-negative bacteria. The plastic strips consist of

20 small wells containing dehydrated media components (top row). The bacterium to be tested is suspended in sterile saline and added to each well, then the strip is

incubated for 16-24 hours and the colour reactions are noted as either positive or negative. The test results can be entered into a computer programme to identify the bacterium. Four strips inoculated with four different bacteria are shown in the Figure.

In each case the spectrum of results was different.

Representative API Strip for Representative API Strip for E. sakazakiiE. sakazakii

Representative API Strip Results Representative API Strip Results for for E. sakazakiiE. sakazakii

European MethodEuropean Method

• Levels of Levels of E. sakazakiiE. sakazakii in the sample in the sample determined by the most probable number determined by the most probable number (MPN)(MPN) procedure. procedure.

MPN ProcedureMPN Procedure• Statistical method assuming that the bacteria Statistical method assuming that the bacteria

are separate and the conditions of incubation are separate and the conditions of incubation such that every inoculum that contains even one such that every inoculum that contains even one viable organism will produce detectable growth.viable organism will produce detectable growth.

• Based on the number of positive samples from Based on the number of positive samples from each of the series of triplicate cultures of the each of the series of triplicate cultures of the three inoculation levels (100g,10g,1g). three inoculation levels (100g,10g,1g).

• MPN Table provides MPN and 95% confidence MPN Table provides MPN and 95% confidence interval.interval.

Table 1. For 3 tubes each at 0.1, 0.01, and 0.001 g inocula, the MPNs per gram and 95 percent confidence intervals.

Pos. tubes Conf. lim. Pos. tubes Conf. lim.

0.10 0.01 0.001

MPN/g

Low High 0.10 0.01 0.001

MPN/g

Low High

0 0 0 <3.0 -- 9.5 2 2 0 21 4.5 42

0 0 1 3.0 0.15 9.6 2 2 1 28 8.7 94

0 1 0 3.0 0.15 11 2 2 2 35 8.7 94

0 1 1 6.1 1.2 18 2 3 0 29 8.7 94

0 2 0 6.2 1.2 18 2 3 1 36 8.7 94

0 3 0 9.4 3.6 38 3 0 0 23 4.6 94

1 0 0 3.6 0.17 18 3 0 1 38 8.7 110

1 0 1 7.2 1.3 18 3 0 2 64 17 180

1 0 2 11 3.6 38 3 1 0 43 9 180

1 1 0 7.4 1.3 20 3 1 1 75 17 200

1 1 1 11 3.6 38 3 1 2 120 37 420

1 2 0 11 3.6 42 3 1 3 160 40 420

1 2 1 15 4.5 42 3 2 0 93 18 420

1 3 0 16 4.5 42 3 2 1 150 37 420

2 0 0 9.2 1.4 38 3 2 2 210 40 430

2 0 1 14 3.6 42 3 2 3 290 90 1,000

2 0 2 20 4.5 42 3 3 0 240 42 1,000

2 1 0 15 3.7 42 3 3 1 460 90 2,000

2 1 1 20 4.5 42 3 3 2 1100 180 4,100

2 1 2 27 8.7 94 3 3 3 >1100 420

E. sakazakii MPN/100g = (MPN/g from Table 1) divided by 1000 to adjust for the larger sample sizes then multiplied by 100 to obtain per 100g.

European Survey Results European Survey Results

• From 35 countries, 141 powdered formula From 35 countries, 141 powdered formula samples were analyzed. samples were analyzed.

• E. sakazakiiE. sakazakii was isolated from 20 samples was isolated from 20 samples collected from 13 of the countries. collected from 13 of the countries.

• The levels of The levels of E. sakazakiiE. sakazakii recovered ranged recovered ranged from 0.36 to 66 Colony Forming Units from 0.36 to 66 Colony Forming Units (CFU)/100 g. (CFU)/100 g.

• The lowest level of detection reported in The lowest level of detection reported in this method was 0.36 CFU/100 g.this method was 0.36 CFU/100 g.

Table 1. For 3 tubes each at 0.1, 0.01, and 0.001 g inocula, the MPNs per gram and 95 percent confidence intervals.

Pos. tubes Conf. lim. Pos. tubes Conf. lim.

0.10 0.01 0.001

MPN/g

Low High 0.10 0.01 0.001

MPN/g

Low High

0 0 0 <3.0 -- 9.5 2 2 0 21 4.5 42

0 0 1 3.0 0.15 9.6 2 2 1 28 8.7 94

0 1 0 3.0 0.15 11 2 2 2 35 8.7 94

0 1 1 6.1 1.2 18 2 3 0 29 8.7 94

0 2 0 6.2 1.2 18 2 3 1 36 8.7 94

0 3 0 9.4 3.6 38 3 0 0 23 4.6 94

1 0 0 3.6 0.17 18 3 0 1 38 8.7 110

1 0 1 7.2 1.3 18 3 0 2 64 17 180

1 0 2 11 3.6 38 3 1 0 43 9 180

1 1 0 7.4 1.3 20 3 1 1 75 17 200

1 1 1 11 3.6 38 3 1 2 120 37 420

1 2 0 11 3.6 42 3 1 3 160 40 420

1 2 1 15 4.5 42 3 2 0 93 18 420

1 3 0 16 4.5 42 3 2 1 150 37 420

2 0 0 9.2 1.4 38 3 2 2 210 40 430

2 0 1 14 3.6 42 3 2 3 290 90 1,000

2 0 2 20 4.5 42 3 3 0 240 42 1,000

2 1 0 15 3.7 42 3 3 1 460 90 2,000

2 1 1 20 4.5 42 3 3 2 1100 180 4,100

2 1 2 27 8.7 94 3 3 3 >1100 420

0.36 /100g detection limit vs 0.30/100g lowest possible limit

Canadian MethodCanadian Method 1997 – Nazarowec-White and Farber1997 – Nazarowec-White and Farber

• Minor modification of Dr. Muytjens’ method:Minor modification of Dr. Muytjens’ method:– Dried infant formula suspended in sterile water.Dried infant formula suspended in sterile water.– Suspect colonies from VRBG plates subcultured to Suspect colonies from VRBG plates subcultured to

TSB-YE agar.TSB-YE agar.

• API-20E confirmation. No additional API-20E confirmation. No additional biochemicals.biochemicals.

• Levels determined by MPN.Levels determined by MPN.• 0.36/100g sensitivity. 0.36/100g sensitivity.

Canadian Survey ResultsCanadian Survey Results

• E. sakazakiiE. sakazakii was isolated from 8 of 120 was isolated from 8 of 120 cans, representing 5 manufacturers, at a cans, representing 5 manufacturers, at a level of 0.36 CFU/100 g.level of 0.36 CFU/100 g.

FDA Method - 2002FDA Method - 2002

• Minor modifications of the Canadian method: Minor modifications of the Canadian method: – Direct spreading or streaking of the overnight EE broth rather Direct spreading or streaking of the overnight EE broth rather

than VRBG pour plates.than VRBG pour plates.– Five presumptive colonies subcultured to Trypticase Soy Agar Five presumptive colonies subcultured to Trypticase Soy Agar

and incubated at 25and incubated at 25ooC for 48-72 hours. C for 48-72 hours. – Only yellow pigmented colonies from the TSA plates are Only yellow pigmented colonies from the TSA plates are

confirmed using the API 20E system.confirmed using the API 20E system.

• No additional biochemical testing is recommended. No additional biochemical testing is recommended.

• Level of detection by MPN is 0.36/100g.Level of detection by MPN is 0.36/100g. • Can detect levels of Can detect levels of E. sakazakiiE. sakazakii much lower than the much lower than the

recommended Food and Agriculture Organization recommended Food and Agriculture Organization (FAO) level of 3.0 CFU/g of powdered infant formula. (FAO) level of 3.0 CFU/g of powdered infant formula.

Left, Unmixed; Right, Mixed. (Photograph courtesy of Sharon Edelson Mammel)

VRBG agar: Typical colonies will appear as purple colonies surrounded by a purple halo of precipitated bile acids.

TSA agar: Typical colonies will appear as yellow-pigmented colonies after 48-72 hr incubation at 25°C

Clinical IsolationClinical Isolation

• E. sakazakiiE. sakazakii is isolated from clinical samples is isolated from clinical samples using standard methods for the isolation of using standard methods for the isolation of EnterobacteriaceaeEnterobacteriaceae. .

• No special media has been developed for No special media has been developed for E. E. sakazakiisakazakii..

• Grows well on blood agar, MacConkey, eosin Grows well on blood agar, MacConkey, eosin methylene blue, deoxycholate agar and tergitol methylene blue, deoxycholate agar and tergitol 7 agars.7 agars.

• Has been confirmed with either the API-20 or Has been confirmed with either the API-20 or the Enterotube II systemthe Enterotube II system..

ConclusionsConclusions

• Procedures for isolating Procedures for isolating E. sakazakiiE. sakazakii from from powdered formula and clinical samples follow powdered formula and clinical samples follow the standard microbiological methods for the the standard microbiological methods for the isolation of other members of the family isolation of other members of the family Enterobacteriaceae.Enterobacteriaceae.

• Normally, sterile clinical samples pose no Normally, sterile clinical samples pose no major problems for isolating major problems for isolating E. sakazakiiE. sakazakii..

Conclusions - continuedConclusions - continued

• Because of the very low levels of Because of the very low levels of E. sakazakiiE. sakazakii in in powdered formula samples and its non-random powdered formula samples and its non-random distribution in the powder, larger quantities distribution in the powder, larger quantities and sub-samples should be cultured for and sub-samples should be cultured for isolation. isolation.

• In both clinical and food microbiology In both clinical and food microbiology laboratories, appropriate incubation times and laboratories, appropriate incubation times and temperatures should be applied in diagnostic temperatures should be applied in diagnostic tests for precise identification of tests for precise identification of E. sakazakiiE. sakazakii..