effects on the survival of enterococcus faecium in dishwater

Foodservice Research International

15

(2004) 118–128.

All Rights Reserved.

118 ©

Copyright 2004, Blackwell Publishing

EFFECTS ON THE SURVIVAL OF

ENTEROCOCCUS FAECIUM

IN DISHWATER

Erik JOHANSSON

1

, EVA STÅHL WERNERSSON

1,3

and HÅKAN HÅKANSON

2

1

Environmental LaboratoryGS Development AB Jägershillgatan 15 SE-213 75 Malmö

Sweden

2

Department of BiotechnologyUniversity of Lund

Box 124, SE-221 00 Lund Sweden

Accepted for Publication April 29, 2005

ABSTRACT

Microorganisms have been frequently found in dishwaters despite hightemperatures and pH levels. Therefore, this study investigates the dishwaterin dishwashing processes to determine the survival rate of a heat-resistant testorganism, Enterococcus faecium. The experiments are conducted in a 2-Lbeaker, and the temperature and the pH are varied in the spans typical forhand dishwashing and mechanical dishwashers (45–65C and pH 7–9). Trypticsoy is added to the beaker to study the influence of nutrition (which normallyexists in dishwater). A three-log reduction of E. faecium after 50 min at 65Cis noted and compared with 300 min at 55C, at pH 7. For 45C, no reductionof

E. faecium

is observed. The findings emphasize the importance of main-taining a high temperature in dishwaters during dishwashing processes.

INTRODUCTION

There is a potential risk of the spread of foodborne diseases because ofcross-contamination in the kitchen, where the food is prepared and stored.This is evident for both professional as well as domestic kitchens. Even though

Blackwell Science, LtdOxford, UKFRIFoodservice Research International1524-8275Copyright 2005 by Food & Nutrition Press, Inc., Trumbull, Connecticut.2005153/4118128Original Article

SURVIVAL OF ENTEROCOCCUS FAECIUM

IN DISHWATERE. JOHANSSON, E.S. WERNERSSON and H. HÅKANSON

3

Corresponding author. TEL:

+

46-40-671-50-00; FAX:

+

46-40-21-58-20; EMAIL: [email protected]

SURVIVAL OF


IN DISHWATER 119

work is performed to improve kitchen hygiene, there is no evidence that thespread of infection is reduced over time (Cliver 2002; Nestle 2003). The mostimportant factor for such spread is the personal hygiene of the workers in thekitchen (Huddle

et al.

2002). Other important factors are the storage temper-atures of the food and cross-contamination, which can occur between thecontaminated kitchen equipment and food (Barrie 1996; Chen

et al.

2001;Michaels

et al.

2002). Even in dishwashers, there is a risk of cross-contamination from the dishwater to the dishware (Ståhl Wernersson

et al.

2004a).Today, most dishware cleaning is performed by hand and at a tempera-

ture of about 45C (Mattick

et al.

2003). However, in professional kitchens,dishwashers are more frequently used to clean dishware. When dishwashersare used, the dishwater temperature is higher (55–65C). In dishwashers forpots and pans, the temperature is normally 65C during the cleaning process.Another difference between hand dishwashing and dishwashers is the use ofdifferent detergents. In most dishwashing done by hand, the detergent has amild disinfecting effect and a pH between 7 and 8. In dishwashers, detergentswith a high pH (11–14) are normally used, which give the dishwater pHsolutions of 8–11. At the end of the dishwashing cycle, the dishwashingmachines have a rinsing period of about 15–30 s, when fresh hot water issprayed over the dishware to rinse off the detergent and raise surface temper-atures to obtain hygienically clean dishware. Rinsing during hand dishwash-ing is more unpredictable and uses tap water that has a lower temperature(

ª

55C) than that of dishwashers (

ª

85C). In a dishwasher, exposure time ofdishware to dishwater is in the order of 1–10 min, while for hand dishwash-ing, it is 10–120 min, including soaking. In dishwashers, dishwater is nor-mally reused for about 20–30 wash cycles, but is diluted by about 10% withthe fresh, hot rinsing water in each cycle. For hand dishwashing, the dish-water is used both for the soaking and cleaning of dishware. The frequencyof the changing of dishwater depends strongly on the person performing thecleaning.

It has been shown in several investigations that bacteria can survive indishwater from both hand and machine dishwashing, even with high pH and/or temperature (Mosupye and von Holy 2000; Ståhl Wernersson

et al.

2003).The log reduction in the numbers of

Enterococcus faecium

remaining ondishware has been tested for two different dishwashing processes, using theDeutsches Institute für Normung (DIN) formula (Ståhl Wernersson

et al.

2004b). However, these food residues were less adhesive to the dishwater thanexpected and the

E. faecium

were moved into the dishwater, which then isreused as wash water for the next washing cycle. In this article, the survivalof

E. faecium

has been studied at different temperatures and pH in order todetermine the period of time that there is a risk of cross-contamination

120 E. JOHANSSON, E.S. WERNERSSON and H. HÅKANSON

between contaminated dishwater and dishware. Nutrition was used in thesolutions, given that soiled dishwater always contains food residues that canbe used as nutrition for the microorganisms. The microorganism

E. faecium

was chosen because of its resistance to high temperatures and is common ininternational standards regarding dishwashing (DIN 10510, 2001; DIN 10512,2001) and in the literature. A comparison between hand dishwashing anddishwashers is performed and discussed.

MATERIALS AND METHODS

Bacterial Strain and Media

The test microorganism used was

E. faecium

(CIP 106742, DSM 2146,American Type Culture Collection 6057), a bacterium that can cause food-borne diseases (Cowan

et al.

1995; Krüger and Zschaler 1996). It is knownto be fairly thermo-stable (Bradley and Fraise 1996) and its thermal resistancehas previously been determined at 70C for 10 min (Ståhl Wernersson

et al.

2004a).

E. faecium

has been utilized for microbiological testing of dishwash-ing in different types of dishwashers (Francis and Newsom 1987; Ebner

et al.

2000) and it is used for the evaluation of the cleaning results of dishwashersaccording to DIN standards (DIN 10510, 2001; DIN 10512, 2001).

A freeze-dried culture (Culture Collection, University of Göteborg,Sweden) of

E. faecium

was mixed with 1 mL of a physiological saline solution(0.9% NaCl) and transferred to a flask with 100 mL of tryptic soy broth (casein–peptone soymeal–peptone broth, MERCK 1-05459, Darmstadt, Germany).With the flask being shaken, the bacteria were grown for 24 h at 30C. Theprocedure was repeated twice more, using 1 mL of the previous suspension.After the final 24 h, the culture was harvested by centrifugation (BB3V, JOUANS.A., St. Herblain, France) at 2500

¥

g for 5 min. The cells were centrifugedand washed with three separate 10-mL volumes of physiological saline solu-tion. The last pellet was resuspended in 10 mL of the said solution, resultingin a working cell suspension of 9

¥

10

9

cfu/mL. To determine the cell density,the cell suspension was serially diluted to a final dilution of 10

2

, and 0.05 mLof all the dilutions were spread on kanamycin aesculin azide agar (OxoidCM0591, Baisingstoke, Hampshire, England) including a kanamycin sulfatesupplement (Oxoid SR92). The plates were inoculated at 36C for 2 days.

In the experiments, the survival of the microorganism was analyzed bythe spread plate technique with dilutions on tryptic soy agar (casein–peptonesoymeal–peptone agar, MERCK 1-05458). An aliquot of 0.05 mL fromthe samples was spread on the plates that were incubated at 30C for 5 days.

E. faecium

was verified through inspection of colony morphology andGram-staining.

SURVIVAL OF


IN DISHWATER 121

Bacterial Survival in Dishwashing Solutions

To mimic the conditions in dishwater that can be found in dishwashersor during dishwashing by hand, pH, temperature and nutrient supply werecontrolled. Sterilized water (2 L) with 3 or 25% tryptic soy broth was contin-uously agitated with a magnetic stirrer (700 rpm) in a glass beaker.

For the major part of the experiments, 2 mL/L (0.2%) of a detergent (usedfor potwashing machines) was added to the beaker (Gigant 1997), accordingto the suppliers’ recommendations. Gigant has a pH of 14 and it consists of5–10% NaOH, 20–40% nitrilotriacetic acid trisodium salt (a complexingagent) and 1–5% polycarboxylate and water. In the experiment representinghand dishwashing, an estimation of normal dosage resulted in 0.5 mL/L(0.05%) of a washing up liquid (Dizzy 2002), which is in accordance with theliterature (Charnock 2003). Dizzy has a pH of 7.5 and consists of severalcomponents: sodium lauryl tri(oxyethyl) sulfate, sodium lauryl sulfate, decylglycosides, alkyl dimethylamine oxide (C

13

–C

15

), cocoamidopropyl betaine,color and perfume.

E. faecium

was added to the beaker, resulting in initial bacterial numbersof 50,000 cfu/mL. The solution (25 mL) was transferred aseptically – at 0, 2,5, 10, 20, 40, 70, 100, 150 and 250 min – after the addition of the microor-ganisms to ice-cold, sterile glass bottles. The samples were neutralized to pH 7with sterile 0.1 M HCl and instantly returned to icy water. They were thenspread in duplicate on tryptic soy agar.

To simulate the conditions with hand dishwashing parameters, a modeltest was designed. The volume of water was increased to 18 L and a 3% trypticsoy broth concentration was chosen. Gigant was added (0.2%) and the pH wasadjusted to pH 8 with 0.1 M HCl. Then 0.1 mL of the cell suspension(9

¥

10

9

cfu/mL

E. faecium

) was added to the tank, and samples were trans-ferred aseptically from the tank at the same time intervals as for the smallbeaker experiments (which were treated likewise). The initial temperature was57C, which corresponds to the upper limit of hot tap water, and the tempera-ture was allowed to decrease, as it would do in a sink, to 42C.

The conductivity was measured, as this is the most common parameterfor monitoring the dosage of detergent in dishwashers. A CD641T conductiv-ity cell and a CDM210 conductivity meter (Radiometer, Copenhagen,Denmark) were used. The cell was calibrated to the International Union ofPure and Applied Chemistry (IUPAC) standard 0.1D KCl (12.85 mS/cm). APHM210 pH meter with a pHC2015-8 electrode was used to measure the pH(Radiometer, Copenhagen, Denmark). The electrode was calibrated usingIUPAC standards for pH 7 and pH 12.45. Chemical oxygen demand (COD) –a parameter that reflects the nutrition contents of the solutions – was measuredusing a Spectroquant NOVA 60 spectrophotometer and a COD Cell Test


(Spectroquant MERCK). The temperature was sampled in the solutions, usinga calibrated sensor, Pt100.

RESULTS

In Table 1, the conditions for the various experiments are listed togetherwith the results of temperature, pH, conductivity and COD measurements.Figure 1a shows the reduction of bacterial numbers as a function of time atdifferent temperatures, at pH 7. Measured values below the detection level(

<

1 cfu/mL) are marked with symbols at the sampling time. In Fig. 1b, thepH dependency is shown at 65C as a function of time. The influence of thebroth concentration, 3 and 25%, at 45C and pH 7 is depicted in Fig. 2a. Acomparison of the bacterial survival using hand detergent and machine deter-gent is shown in Fig. 2b. The pH was adjusted to pH 7 for both detergentsand the temperature was 45C. In Fig. 3, the bacterial survival for the modeltest is shown as a function of time.

DISCUSSION

Temperature Influence on

E. faecium

Survival

Bacterial survival is strongly affected by the temperature, as can be seenin Fig. 1a. The reduction of

E. faecium

at pH 7 after 50 min shows thattemperatures around 65C decrease bacterial survival by three log units. Toachieve the same reduction for 55C, a time of 300 min is needed. At 45C thereis no reduction in the population of

E. faecium

.

TABLE 1.PARAMETERS FOR THE EXPERIMENTS AND MEASURED TEMPERATURE, pH,

CONDUCTIVITY AND CHEMICAL OXYGEN DEMAND (COD) IN THE SOLUTIONS

Experimentnumber

Volume(L)

Detergenttype

Broth(%)

Temperature(C)

pH–

Conductivity(mS/cm)

COD(mg/L)

1 2 Gigant 25 65 9 5.1 86002 2 Gigant 25 65 8 5.0 74003 2 Gigant 25 65 7 4.8 83004 2 Gigant 25 55 7 4.8 83005 2 Gigant 25 45 7 4.8 83006 2 Gigant 3 45 7 1.7 12007 2 Dizzy 25 45 7 4.0 96008 18 Gigant 3 57–42 8 1.7 1200

SURVIVAL OF


IN DISHWATER 123

FIG. 1. SURVIVAL OF


AS A FUNCTION OF TIME (a) FOR DIFFERENT TEMPERATURES (

�

, 45C;

�

, 55C;

�

, 65C) IN SOLUTIONS CONTAINING BROTH (25%) AND DETERGENT AT pH 7; AND (b) FOR DIFFERENT pH (

�

, pH 7;

�

, pH 8;

�

, pH 9) IN SOLUTIONS CONTAINING BROTH (25%) AND DETERGENT AT 65CThe detection limit is marked with a solid line.


FIG. 2. SURVIVAL OF


AS A FUNCTION OF TIME (a) FOR DIFFERENT BROTH CONCENTRATIONS (

�

, 3%;

�

, 25%) IN SOLUTIONS CONTAINING DETERGENT AT 45C AND pH 7; AND (b) FOR DIFFERENT DETERGENTS (

�

, DISHWASHER DETERGENT;

�

, HAND DISHWASHING DETERGENT) IN SOLUTIONS CONTAINING BROTH (25%) AT 45C AND pH 7

The detection limit is marked with a solid line.

SURVIVAL OF


IN DISHWATER 125

Influence of pH on

E. faecium

Survival

In Fig. 1b, the pH dependency of

E. faecium

is shown in a solutioncontaining 25% broth and 0.2% dishwasher detergent, at 65C. This tempera-ture is common for dishwashers. In most dishwashers, the dishwater is reusedand only diluted by a small amount of fresh hot water at the end of thedishwashing cycle. Even with a detergent with a strong alkaline content, thedishwater pH will decrease as the dishwater becomes soiled because ofthe increasing organic load. This justifies the selected pH range from 7 to 9.As a summary based on Fig. 1, it is obvious that a temperature raised over acertain limit reduces the microbiological activity more than an increasing pH,which is in accordance with the literature (Jay 1996; Prescot

et al.

1999).

Effects of Nutrients in the Solutions

As dishwater is reused in dishwashers, the organic content increases.Once a threshold organic load is reached, the short-term influence of the brothconcentration on the bacterial survival is low (Fig. 2a). The organic load ofdishwashers has been reported to be up to a COD level of 6000 mg/L (Harpel

et al.

1994). With the selected broth concentrations this level is covered (seeTable 1). In most experiments, 25% broth was used in order to be sure that

FIG. 3. SURVIVAL OF


AS A FUNCTION OF TIME FOR THE MODEL TEST (

�

)

AND THE TEMPERATURE THROUGHOUT THE EXPERIMENT (

�

)The detection limit is marked with a solid line.


the nutrition content is not limiting bacterial growth. The conductivity wasfound to be in the range of 1.7–5.5 mS/cm that is comparable with data fromdishwashers in professional kitchens (Ståhl Wernersson

et al.

2003).

Effects of Type of Detergent

The compositions of hand dishwashing and dishwasher detergents aredifferent. Dishwasher detergents utilize pH and complexing agents to improvethe cleaning process. The hand dishwashing detergents utilize surface activeagents, and a pH that is acceptable for bare hands. A comparison of

E.faecium

’s survival in solutions, with the two detergents used in this study, isshown in Fig. 2b. It can be seen that the dishwasher detergent has very littleinfluence on the numbers of bacteria over time. For the hand dishwashingdetergent a decrease of the numbers of

E. faecium

occurs shortly after expo-sure, similar to that of a raised temperature (Fig. 1a, 55C). However, thereduction rate decreases with time and after 250 min a high number of bacteriawas measured. This is in accordance with Charnock (2003), who measuredbacterial activity as a function of time with a different hand dishwashingdetergent. At a similar concentration and even with antibacterial handdishwashing liquid, the reduction of bacterial activity is very slow(Kusumaningrum

et al.

2002).

Scale Up and Model Test of Hand Dishwashing

Conditions for hand dishwashing were simulated in the 18-L experimentwith a broth concentration corresponding to an organic load of COD 1200 mg/L. The temperature was then allowed to decrease over time to 42C. In Fig. 3,the cfu/mL of

E. faecium

versus time is shown. The 18-L curve shows a verysmall reduction of

E. faecium

, even after 250 min, which is similar to that ofthe experiment at 45C and pH 7, Fig. 1a.

If the temperature is maintained at 55C, there will be a significantdecrease of

E. faecium

, but after

>

120 min (Fig. 1a). This explains the lowreduction of the microorganisms in the 18-L experiment, because the temper-ature after 2 h has decreased to 47C and, therefore, the reduction rate iscomparable to the beaker experiments at 45C.

CONCLUSION

The most important parameter to reduce the number of

E. faecium

indishwater is to maintain its temperature at a high level, preferably around 65C.The bacteria’s survival is less dependent on the pH, the organic load of the

SURVIVAL OF


IN DISHWATER 127

dishwater (the broth concentration) and the type of detergent. For hand dish-washing, the exposure time of the dishware to the dishwater is 10–120 min ata temperature of about 45C. For dishwashers, the exposure time is 1–10 minat 55–65C. However, the extended exposure time in hand dishwashing doesnot compensate for the reduction of bacterial counts at the lower temperature.Therefore, the risk of cross-contamination in the kitchen is higher for handdishwashing than for dishwashers.

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