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Comparison of the disinfectionefficacy of chlorine-based productsfor inactivation of viral indicators andpathogenic bacteria in produce washwaterCristobal Chaidez a , Maria Moreno a , Werner Rubio a , MiguelAngulo a & Benigno Valdez ba Department of Food Safety , Centro de Investigacion enAlimentacion y Desarrollo , Culiacan, 80129, Mexicob Instituto Tecnologico de Estudios Superiores de Monterrey ,MexicoPublished online: 21 Jul 2010.

To cite this article: Cristobal Chaidez , Maria Moreno , Werner Rubio , Miguel Angulo & BenignoValdez (2003) Comparison of the disinfection efficacy of chlorine-based products for inactivationof viral indicators and pathogenic bacteria in produce wash water, International Journal ofEnvironmental Health Research, 13:3, 295-302, DOI: 10.1080/0960312031000122442

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Comparison of the disinfection efficacy ofchlorine-based products for inactivation of viralindicators and pathogenic bacteria in producewash water

CRISTOBAL CHAIDEZ1, MARIA MORENO1, WERNER RUBIO1, MIGUEL ANGULO1

and BENIGNO VALDEZ2

1Department of Food Safety, Centro de Investigacion en Alimentacion y Desarrollo, Culiacan, Mexico, 80129, 2Instituto

Tecnologico de Estudios Superiores de Monterrey, Mexico

Outbreaks of pathogenic bacteria infections associated with the consumption of fresh produce has

occurred with increased frequency in recent years. This study was undertaken to determine the efficacy of

three commonly used disinfectants in packing-houses of Culiacan, Mexico (sodium hypochlorite [NaOCl],

trichlor-s-triazinetrione [TST] and thrichlormelamine [TCM]) for inactivation of viral indicators and

pathogenic bacteria inoculated onto produce wash water. Each microbial challenge consisted of 2 L of

water containing approximately 8 log10 bacterial CFU ml71, and 8 log10 viral PFU ml71 treated with 100

and 300 mg l71 of total chlorine with modified turbidity. Water samples were taken after 2 min of contact

with chlorine-based products and assayed for the particular microorganisms. TST and NaOCl were found

to effectively reduce for bacterial pathogens and viral indicators 8 log10 and 7 log10, respectively (a=0.05).

The highest inactivation rate was observed when the turbidity was low and the disinfectant was applied at

300 mg l71. TCM did not show effective results when compared with the TST and NaOCl (P5 0.05).

These findings suggest that turbidity created by the organic and inorganic material present in the water

tanks carried by the fresh produce may affect the efficacy of the chlorine-based products.

Keywords: Disinfection; chlorine-based products; E. coli; S. typhimurium; MS2.

Introduction

The increasing demand for fresh fruits and vegetables along with market globalization is a real

possibility for an increase of foodborne illness as a result of, contamination of foods with

pathogenic micro-organisms. Raw fresh produce can become contaminated with microbial

pathogens while growing in the field, during harvesting, processing, and distribution (Beuchat

1996; Gross and Wang 1999). The mechanisms by which the pathogens reach the fresh produce

are not fully understood; however, one hypothesis states that the fruits and vegetables become

contaminated with water used at the dump tanks (Bartz and Showalter 1980). Chlorination is

widely practiced as a disinfection process for microbial control in water used to wash fruits and

vegetables at packing-houses. When properly applied, chlorine-based products are efficient.

Correspondence: Cristobal Chaidez, Centro de Investigacion en Alimentacion y Desarrollo,

Carretera a Eldorado Km. 5.5, Culiacan, Sinaloa, Mexico, 80129. Tel/Fax: 01152 (667) 760-5536;

E-mail: chaqui@ciad.edu.mx

International Journal of

Environmental Health Research 13(3), 295 – 302 (September 2003)

ISSN 0960-3123 printed/ISSN 1369-1619 online/03/030295-08 # 2003 Taylor & Francis Ltd

DOI: 10.1080/0960312031000122442

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However several drawbacks have been identified, including the protection exerted by the

organic and inorganic matter to chlorine disinfection (Karch and Loftis 1998). Available

research has shown that increased resistance to disinfection may result from the attachment of

micro-organisms to various surfaces, including particles, algae, and carbon fines (le Chevallier

et al. 1988). Ridgway and Olson (1982), have shown that the majority of viable bacteria in

chlorinated drinking water are attached to particles. Presumably, micro-organisms entrapped in

particles are shielded from disinfectants. Therefore, the ineffectiveness of chlorine and other

disinfectants may depend on whether or not the target organisms are readily accessible

(Solomon et al. 2002).

The disinfection conditions notably affect the efficacy of the operation. Many packing-houses

that employ chlorine as a primary disinfectant are unable to achieve desired microbial

inactivation levels. In addition, pathogens of concern to the agriculture industry, such as E. coli

0157:H7, S. typhimurium and enteric viruses (i.e. Hepatitis A virus [HAV]), are able to evade the

chlorine concentrations typically applied for water treatment (McDonell and Russell 1999;

Beuchat 1998). Moreover, the micro-organisms differ in sensitivity to disinfectants depending

on the type of cell wall, membrane composition, age and cycle of growth, biofilm production,

and clump formation (McDonell and Russell 1999). A disinfectant is considered to be an

effective microbicidal when it is capable of achieving a 6-log, 4-log, and 3-log reduction of

bacteria, virus, and protozoan, respectively (Geldreich 1996). The official method has stated a

reduction level of 5 log10 of selected surrogates for an effective sanitizer (AOAC 1988).

Several researchers have investigated the effectiveness of chemicals in killing pathogenic

bacteria. Taormina and Beuchat (1999), studied the efficacy of chemical treatments in

eliminating 2.0 to 3.0 log10 E. coli 0157:H7 per g of alfalfa seeds. Significant (=0.05)

reductions in populations were observed after treatment with 500 or 1000 mg l71 of chlorine

{Ca(OCl)}. S. montevideo was isolated from fresh tomatoes and conclusive evidence showed

that raw tomatoes were the source of contamination (Asplund and Esko 1991). The tomatoes

were traced to a packer where tomatoes were dumped into a chlorinated warm water bath

containing 40 – 60 mg l71 of chlorine for a short period of time before being packed. It is

unclear how S. montevideo implicated in the outbreak could survive on tomatoes following

chlorine treatment at the packinghouse (Zhuang and Beuchat 1995). However, the possible low

concentration of free chlorine, the short contact time, or the presence of organic matter at the

dump tanks carried by the tomatoes could have protected the bacteria from the effect of

chlorine-based product (Wei et al. 1995).

Pathogenic strains of E. coli, mainly serotype 0157:H7, had been recognized as a food-borne

disease agent. Most outbreaks have been linked to prepared food, being the most frequently

vehicle of infection (Doyle 1991). However, lettuce, cantaloupe, cabbage, unpasteurized apple

juice and alfalfa sprouts have also been implicated in some outbreaks (Ackers et al. 1996; Besser

et al. 1993; Beuchat 1996; del Rosario and Beuchat 1994; Golden et al. 1992; US Department of

Healthy and Human Services 1997). Hepatitis A virus (HAV), has also been implicated in a

variety of food-borne outbreaks of HAV (Bidawid et al. 2000). Outbreaks of enteric viruses

have occurred as a result of consumption of fruits improperly disinfected.

Current techniques to concentrate enteric viruses from water are cumbersome, expensive and

time-consuming. Microbial indicators of these pathogens have been used to evaluate the

efficacy of disinfectants (Chauret et al. 2001). Bacteriophages have been proposed as a viral

indicator because their handling is simple and inexpensive and does not require specialized

personnel or sophisticated facilities (Leclec et al. 2000). Most disinfection studies have been

conducted with bacteriophages as models for human enteric viruses (Abad et al. 1994).

296 Chaidez et al.

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The purpose of the present study was to evaluate the effectiveness of chlorine based-products

applied to dump water tanks at the packinghouses in the inactivation/reduction of viral

indicators and pathogenic bacteria.

Materials and methods

Preparation of the inocula and procedures for chlorine challenges

Escherichia coli and Salmonella typhimurium bacterial assays Two pure cultures of

Escherichia coli (ATCC 15597) and Salmonella typhimurium (ATCC 19585) provided by Dr

Charles P. Gerba, Universisty of Arizona, were selected and subjected to chlorination. Bacteria

were selected on the basis of their relevance to the fresh produce contamination. Bacterial

cultures were grown in trypticase soy agar for 24 h at 378C in a shaker and finally harvested by

centrifugation at 13,0806 g for 10 min. The cellular pellets were washed three times in a

0.01 M phosphate buffer saline (PBS) solution. This washing procedure was repeated three

times in order to minimize the concentration of non-cell associated constituents’ solution that

could react with chlorine. The target numbers was quantified and the final concentration of

viable bacteria in suspension after incubation was 109 CFU per ml. The final wash was diluted

10-fold to achieve a final concentration of approximately 107 CFU ml71.

Bacteriophage The MS-2 phage (ATCC 15597-B1) provided by The University of Arizona,

prepared from infected lawns of the bacterial host (Escherichia coli ATCC 15597). The MS-2

phage was grown, propagated and titered using standard procedures (APHA 1998). Serial

dilutions (1:10) of the samples were made in 0.01 M PBS and added to test tubes containing

3 ml of molten top agar /tryptic soy broth with 1% bacto agar (Difco, Detroit, MI, USA)

containing 0.1 ml of a 4 – 6 h culture of the E. coli host strain. The suspension was gently

vortexed and poured onto solid tryptic soy agar (Difco, Detroit, MI, USA) plates. The plates

were incubated for 18 – 24 h at 378C and enumerated with the aid of a Quebec colony counter.

The final concentration of 108 PFU ml71 was achieved for the bacteriophage challenges.

Chemical treatments

The physical/chemical conditions of the water monitored in this study are listed in Table 1. The

table expresses the turbidity, pH, total chlorine and temperature of both ‘average’ and ‘worst-

case’ water. The ‘average’ water obtained from the municipal distribution system was

inoculated with E. coli, S. typhimurium or MS2 phage at concentrations of 108 CFU ml71 and

Table 1. Chemical/physical quality parameters of test water

Average Worst-casea

Turbidity (NTU) 2 200PH 7.0 7.0Total chlorine (mg l71) 100/300b 100/300b

Temperature (8C) 25 25

aThe characteristics for the worst-case water as specified by the USEPA Guide Standard (1987);bmg l71 of total chlorine as Sodium hypochlorite; Trichlormelamine; Trichlor-S-triazinetrione

297Microbial disinfection

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109 PFU ml71 for bacterial and viral challenges, respectively. The ‘worst-case’ water was spiked

with high turbidity [200 NTU with Arizona Fine Test Dust (General Motors, Flint, MI, USA)],

also containing the same level of bacterial or viral challenge. It should be noted that the ‘worst-

case’ condition was taken from the US EPA Guide Standard for Testing Microbiological Water

Purifiers (USEPA 1987) adjusted to the needs of the present study.

‘Average’ and ‘worst-case’ water treated in separate sterile 2-litre beaker: 100 and

300 mg l71 of chlorine-based products [as NaOCl (sodium hypochlorite), TST (trichlor-s-

triazinetrione), and TCM (trichloromelamine)]. Concentrations of total chlorine was

determined with the spectrophotometer 2010 DPD colorimetric method (Hach Co., Ames,

IA, USA) as described on the Standard Methods for the Examination of Water and

Wastewater APHA (1998).

Microbiological analysis

Immediately after a contact time of 2 min, each microorganism was assayed by the appropriate

method. Samples of control and treatment solutions from water inoculated with E. coli and S.

typhimurium were spread plated on Hecktoen and M-Endo agar less (Difco), respectively.

Samples from water inoculated with MS2 phage were assayed by the agar overlay method

(Abbaszadegan 1997).

Statistical analysis

Three replicates for each set of experimental parameters were conducted. Data were subjected

to the MINITAB (Version 12 1998) system for the analysis of variance as described by

Montgomery (1991).

Results

The results shown in Table 2 correspond to reductions of E. coli after treatments with chlorine-

based compounds in average and worst-case water conditions. The results shown on Table 2 are

numbers of E. coli reduced after the treatments with chlorine based-compounds to water under

‘average’ and ‘worst-case’ water condition of turbidity. Differences in inactivation/reductions of

Table 2. Reduction of Escherichia coli in the test watera

Reduction (log10 CFU ml71)NaOClb TCMc TSTd

Test water (NTU) 100 300 100 300 100 300

2e 6 8 1 5 8 8200f 5 6 1 3 6 6

aTwo minutes contact time;bmg l71 of chlorine as Sodium hypochlorite;cTrichlormelamine;dTrichlor-S-triazinetrione;eAverage;fWorst case water with contact time of 2 min

298 Chaidez et al.

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E. coli numbers of E. coli reduced after 2 min treatments were minimal with NaOCl and TST.

Lower reductions were achieved by the TCM treatment. Treatment of inoculated water with

100 and 300 mg l71 of NaOCl and TST for 2 min resulted in a reduction that ranged between 8

and 5 log10 for the ‘average’ and ‘worst-case’ water. No significant differences among

treatments were observed (P5 0.05), whereas treatment with TCM at concentrations of 100

and 300 mg l71 reduced the population to 1 and 3 log10, respectively.

The results shown on Table 3 correspond to reductions of S. typhimurium after the

treatments with chlorine for the same water conditions specified above. Again, the NaOCl and

TST treatment were more effective than the TCM. Reductions of 8 log for populations of S.

typhimurium were achieved after NaOCl and TST treatment under both water conditions. TCM

achieved 6 log10 at 300 mg l71 in both ‘average’ and ‘worst-case’ water conditions.

Results of experiments using the MS2 phage are presented in Table 4. Reductions in viral

populations were ranged between 4 and 8 log10 in ‘average’ case water when using NaOCl or

Table 3. Reduction of Salmonella typhimurium in the test watera

Reduction (log10 CFU ml71)NaOClb TCMc TSTd

Test water (NTU) 100 300 100 300 100 300

2e 8 8 3 6 8 8200f 6 8 2 6 7 8

aTwo minutes contact time;bmg l71 of chlorine as Sodium hypochlorite;cTrichlormelamine;dTrichlor-S-triazinetrione;eAverage;fWorst case water.

Table 4. Reduction of MS-2 in the test watera

Reduction (log10 CFU ml71)NaOClb TCMc TSTd

Test water (NTU) 100 300 100 300 100 300

2e 4 8 1 5 5 8200f 5 0.1 3 5 0.1 2 5 0.1 4

aTwo minutes contact time;bmg l71 of chlorine as Sodium hypochlorite;cTrichlormelamine;dTrichlor-S-triazinetrione;eAverage;fWorst case water contact time of 2 min.

299Microbial disinfection

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TST, whereas treatment with TCM only achieved a 5 log10 reduction at 300 mg l71. On the

contrary, a significant decrease on viral inactivation occurred in ‘worst-case’ water. The highest

reduction achieved was 4 log10 at 300 mg l71 of TST.

Discussion

For the bacterial reduction/inactivation testing, the chemical disinfectants were challenged with

each test bacterium individually during separate test periods. The chemical disinfectants were

found to remove Escherichia coli and Salmonella thyphimurium by greater than 6 log10 at the

initial test point. It is known that under conditions of high water quality, waterborne vegetative

bacteria are highly susceptible to relatively low doses of chlorine.

Washing with chlorinated tap water is intended to reduce microbial contamination on raw

fruits and vegetables coming from the field. Although washing produce in chlorinated water

may have some effectiveness in removing soil and other debris, it should not be relied upon to

completely remove micro-organisms. Several factors control the disinfection of water such as;

type of disinfectant, type of micro-organisms, disinfection concentration and contact time,

physical and chemical interference with disinfection (Beuchat et al. 2001). Chemical compounds

that interfere with disinfection are inorganic and organic nitrogenous compounds, iron,

manganese, and hydrogen sulphide. Turbidity in water is composed of inorganic (e.g., silt, clay,

iron oxides) and organic matter as well as microbial cells (Silverman et al. 1983; le Chevallier et

al. 1988).

Factors such as the amount of organic matter surrounding the target organisms are likely to

influence the adhesion characteristics of cells and the lethal effect of sanitizers. Results of this

study do show, however, that the amount of organic material present in the wash water

influences the efficacy of disinfectants. Results also show that similar and significant reductions

in populations of E. coli and S. typhimurium occur in water used to wash fruits and vegetables

at the packinghouses using 100 or 300 mg l71 of NaOCL or TST in both average and worst-

case water conditions. On the contrary, significant reduction of MS2 phage was only achieved

using 300 mg l71 of NaOCl or TST in ‘average-case’ water, whereas in the ‘worst-case’ water

challenge neither NaOCl nor TST nor TCM were effective.

We recognize that the levels of microbial organisms used in this study are far greater than

what may be found on water dump tanks; however, numbers of viral and bacterial organisms

were used that could be readily detected by the assays used in the present study. Under natural

conditions, even a low level of contamination could present a significant human health risk,

since the infective dose of viral particles (HAV) or bacterial cells (E. coli 0157:H7) are less

than 10 PFU and 1000 cells, respectively (Schiff et al. 1984; Zhao et al. 1994).

The microbial disinfection efficacy of NaOCl and TST evaluated in this study was found to

exceed the requirements as set forth in the official definition (AOAC 1998) of sanitizing for food

product surfaces, which indicates a reduction level of 5 log10 of selected surrogates under

average case water conditions.

Acknowledgements

This work was supported by a Sistema de Investigaciones del Mar de Cortes (SIMAC) grant

(990106021).

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