Ultraviolet (UV) Radiation Inactivation of Cronobacter sakazakii in

Dry Infant Formula Observed Using Fourier Transform Infrared

Spectroscopy and Electron Microscopy

By

Qian Liu

A thesis submitted in partial fulfillment of

The requirements for the degree of

Master of Science in Food Science

WASHINGTON STATE UNIVERSITY

School of Food Science

May 2011

ii

To the Faculty of Washington State University:

The members of the Committee appointed to examine the thesis of QIAN LIU find it

satisfactory and recommend that it be accepted.

Barry G. Swanson, Ph.D., Chair

Dong Hyun Kang, Ph.D.

Barbara Rasco, Ph.D.

iii

Acknowledgements

First, I would like to express my gratitude to Dr. Dong-Hyun Kang for his support and

encouragement during my study in Washington State University. He gave me an opportunity

to work in food science and led me to scientific research. Besides the guidance on my

research work, he enabled me to obtain many possibilities in my life. I am a very lucky

person to have him as my first advisor. His philosophy and enthusiasm on life and work

enlightens me to establish a career in science.

I am also very grateful to Dr. Barry Swanson and Dr. Barbara Rasco at Washington

State University who care about me as their own child. Without great support and

encouragement from them, this work could not have been completed. I would also express

my thanks to Dr. Gulhan Unlu who helped me in both course and research work, and Dr.

Shyam Sablani who allowed me to use the radiometer. I also want to thank Dr. Valerie Lynch-

Holm at Franceschi Microscopy and Imaging Center, who did not only provide valuable

information in electron microscopy works but also share with me her research experience to

encourage me to go through the depressed part in my life.

I would like thank staff members in our department, Jodi Anderson who took care of me

as a mama bear and helped me get used to life in America. I also thank Barbara Smith and

Carolee Armfield, who helped me with financial materials. A special thank goes to Frank

Younce for providing help on usage of equipment in the Pilot Plant.

I would like to express my appreciation to my lab members who consider me as a family

member: Peter Gray, A Reum Han and Tahir Zahoor. They contributed their patience and

hard work to teach me important lab skills in the food safety area. This work could not have

iv

been done without their help and support. I also thank my dear friends in Pullman, Beata

Vixie and her family, Yuhui Chao, Peichi Yang, Donglei Luan and his wife. They made my

life in Pullman easy and happy.

Last, I would express my great gratitude to my family and my boyfriend Xiaonan Lu.

No matter how far away they are, they are always ready to support me and help me to fulfill

my dreams. I cannot live in this world without them.

v

Ultraviolet (UV) Radiation Inactivation of Cronobacter sakazakii in

Dry Infant Formula Observed Using Fourier Transform Infrared

Spectroscopy and Electron Microscopy

Abstract

By Qian Liu, M.S. Washington State University

May, 2011

Chair: Barry Swanson

Cronobacter sakazakii is an opportunistic pathogen associated with dry infant formula

which poses a high risk to low birth weight neonates. In the current study, inactivation of C.

sakazakii in dry infant formula by ultraviolet (UV) radiation alone and in combination with

heat treatment at temperatures of 55, 60 and 65oC were applied. UV radiation with doses in a

range of 12.1 to 72.8 kJ/m2 at room temperature had a significant effect on the inactivation of

C. sakazakii in dry infant formula (p

vi

TABLE OF CONTENTS

ACKNOWLEDGEMENTS…………………………………………………………………..iii

ABSTRACT…………………………………………………………………………………...v

LIST O TABLES…………………………………………………………………………….viii

LIST OF FIGURES……………………………………………………………………………x

1 INTRODUCTION .................................................................................................................. 1

1.1 Infant formula processing ................................................................................................. 1

1.2 Biochemical characteristics and taxonomy of C. sakazaki ............................................... 2

1.3 Environment and food sources.......................................................................................... 4

1.4 Growth requirements of C. sakazakii................................................................................ 5

1.5 Biofilm formation ............................................................................................................. 6

1.6 Resistance of C. sakazakii to environmental stress ........................................................ 10

1.6.1 Heat resistance ....................................................................................................... 10

1.6.2 Osmotic and desiccation resistance ....................................................................... 11

1.6.3 Antibiotics resistance ............................................................................................. 13

1.7 Virulence factors ............................................................................................................. 14

1.8 Detection methods .......................................................................................................... 16

1.9 UV radiation treatments .................................................................................................. 20

1.10 Fourier Transform Infrared Spectroscopy .................................................................... 23

2 MATERIAL AND METHODS ............................................................................................. 26

2.1 Bacterial strains, culture methods and preparation of stock cultures .............................. 26

vii

2.2 Sample preparation and inoculation ................................................................................ 26

2.3 Ultraviolet radiation treatment ........................................................................................ 27

2.4 Combined UV radiation and hot water treatment ........................................................... 27

2.5 Bacterial enumeration. .................................................................................................... 28

2.6 Membrane filtration and FT-IR spectroscopy ................................................................. 28

2.7 Electron microscopy analysis ......................................................................................... 29

2.8 Data preprocessing and chemometrics ............................................................................ 30

2.9 Statistical analysis ........................................................................................................... 31

3 RESULTS AND DISCUSSION ............................................................................................ 32

3.1 C.sakazakii inactivation effect of UV radiation .............................................................. 32

3.2 Combined treatments of heat and UV radiation ............................................................. 34

3.3 FT-IR spectroscopy decoding and cluster analysis. ........................................................ 36

3.4 Electron microscopy analysis. ........................................................................................ 38

4 CONCLUSION ..................................................................................................................... 39

References ................................................................................................................................ 40

viii

LIST OF TABLES

Table 1: Biochemical traits suitable for differentiation between C. sakazakii and E. cloacae 56

Table 2. DNA-DNA relatedness of Coronobacter strains ....................................................... 57

Table 3. Food and environmental sources of C. sakazakii ....................................................... 58

Table 4. D values of C. sakazakii strains in Artificial media and reconstituted infant formula

.............................................................................................................................................. 59

Table 5. z values of C. sakazakii strains in different media ..................................................... 65

Table 6. UV radiation treatment time and dose. ...................................................................... 67

Table 7. D values of C.sakazakii inoculated in infant formula for UV radiation in combination

with hot water treatments and hot water treatments only ..................................................... 68

ix

LIST OF FIGURES

Figure 1. FDA standard protocol for the isolation of C. sakazakii from infant formula ......... 69

Figure 2. Microbial survival curve of C. sakazakii inoculated in infant formula and treated

with UV radiation (254 nm) for 5 min, 10 min, 15min, 20 min, 25 min and 30 min. ......... 70

Figure 3. Population reduction of C. sakazakii inoculated in infant formula after UV radiation

exposure with a sample layer of 0.032 cm, 0.16 cm and 0.32 cm ........................................ 71

Figure 4(a). Microbial survival population of C. sakazakii inoculated in infant formula after

UV radiation 20 min and 55oC hot water treatment and 55oC heat treatment only ............. 72

Figure 4(b). Microbial survival population of C.sakazakii inoculated in infant formula after

UV radiation 20 min and 60oC hot water treatment and 60oC hot water treatment. ............ 73

Fig.4(c). Microbial survival population of C.sakazakii inoculated in infant formula after UV

radiation 20 min and 65oC hot water treatment and 65oC hot water treatment only ............ 74

Figure. 5. Raw infrared spectra of non-fat milk powder and non-fat milk powder inoculated

with C.sakazakii. .................................................................................................................. 75

Figure 6 (a). FT-IR spectroscopy: second derivative of spectra of UV-treated C. sakazakii and

intact C. sakazakii in the fingerprint region (1800 – 900 cm-1) ........................................... 76

Figure 6 (b). FT-IR spectroscopy: second derivative of spectra of UV-treated C.sakazakii and

intact C.sakazakii between 3400 – 2800 cm-1 ...................................................................... 77

Figure 7. Principal component analysis of C. sakazakii treated with UV and control. The

cluster analysis model was validated by new treatments ..................................................... 78

Figure 8(a). Scanning electron micrograph of C. sakazakii in infant formula at 29,978×. ..... 79

x

Figure 8(b). Scanning electron micrograph of C. sakazakii in infant formula treated by UV

radiation for 20 min at 30,017×. ........................................................................................... 80

Figure 8(c). Scanning electron micrograph of infant formula at 29,987×. .............................. 81

Figure 9(a). Transmission electron micrograph of thin section of C. sakazakii and infant

formula mixture. ................................................................................................................... 82

Figure 9(b). Transmission electron micrograph of thin section of UV radiation of 20 min

treated C. sakazakii and infant formula mixture. ................................................................. 83

Figure 9(c). Transmission electron micrograph of thin section of infant formula.. ................. 84

1

1 Introduction

1.1 Infant formula processing

Infant formula is an important breast milk substitute formulated to resemble

nutrient composition of human breast milk. Bovine’s milk is a primary protein source for

infant formula production, although soy protein is also used. To simulate breast milk, fat is

removed from milk. Whey protein, carbohydrates, fat, minerals and vitamins are then added

back. In the process of infant formula manufacture, skim milk is pasteurized and evaporated

followed by addition and blending of dry ingredients. Addition of these ingredient leaves the

possibility for microbial contamination, thus a heat treatment is required either before or after

evaporation and is a critical control point in the production process. Some heat sensitive

nutrients must be added after drying to avoid quality change and nutrition retention.

In infant formula industry, efforts to reduce contamination with C. sakazakii focus on

environmental monitoring, hygiene practice and end-product testing for the organisms

(Farber, 2003). Control measures are necessary on receipt of raw materials, such as milk.

Development of validated detection methods improves detection efficiency of the organism in

the raw materials.

Heat treatment is a traditional method to reduce C. sakazakii contamination in the

industry. C. sakazakii exhibited greater heat and desiccation resistance compared to other

bacteria in Enterobacter family. Thus, C. sakazakii can survive for long periods in spray dried

infant formula. Because infant formula is not a sterile product, there is potential risk of

contamination in packaging, storage and distribution. For example, bacteria can be introduced

into spray dried products by addition of non-heat-treated ingredients. Raw bovine milk has to

2

undergo short time high temperature sterilization to eliminate all viable microorganisms. Raw

ingredients which cannot undergo heat treatment must be stored in a sterilized room

separated from ingredients pasteurized. Heat treatment either before or after drying will

improve control of microbial contamination. The dry area of manufacturing facilities must be

hygienic to avoid undesirable entry of contaminants. The low moisture area is also a critical

control point for post pasteurization contamination. The packaging area must also be

separated from the processing and storage area to avoid exposure of finished products to

contamination. The blenders, utensils and equipments used in processing should be

disinfected and sanitary to avoid biofilm growth. Alternative sanitizing methods such as

radiation should be studied and applied to infant formula industry to reduce the

contamination, particularly the formation of biofilms on food contact surfaces that may be a

source of contamination.

When preparing and handling reconstituted infant formula, professionals should follow

preparation and hygiene recommendations provided by public health officials and

organizations such as American Dietetic Association (Farber, 2003) and instruct parents and

infant caregivers accordingly. Particularly, controlling the temperature of hot water used to

reconstitute infant formula and the “holding time” (amount of time a formula is at room

temperature in the feeding bag or bottle and accompanying lines during enteral tube feeding)

of reconstituted infant formula are good strategies to prevent C. sakazakii contamination of

milk (Farmer, 2003; Chen et al., 2009).

1.2 Biochemical characteristics and taxonomy of C. sakazakii

C. sakazakii is motile peritrichous, Gram negative rod (Farmer, 1980). C. sakazakii was

3

first characterized as yellow pigment producing Enterobacter cloacae in the 8th edition of

Bergey’s Manual of Determinative Bacteriology (Sakazaki, 1974). To classify this organism

into Enterobactericeae is based upon DNA-DNA hybridization, biochemical characterization,

pigment production, and antibiotic susceptibility. By DNA-DNA hybridization, the type

strain of E. sakazakii was 83 to 89% related to other strains of E. sakazakii but only 31 to 49%

related to strains of E. clocae. This result provided the fundamental information that C.

sakazakii is a new species rather than a phenotypically distinct subgroup within the existing E.

cloacae species (Farmer et al, 1980). In the same study, Farmer et al. (1980) provided details

on biochemical traits and antibiotic susceptibility of C. sakazakii (Table 1). Unlike E.

cloacae, C. sakazakii produces yellow-pigmented colonies on trypticase soy agar, brain heart

infusion (BHI) agar and blood agar during incubation of 48 to 72 h. The yellow pigments are

more pronounced after incubation at 25 than at 36oC, and the intensity of the pigmentation

varies from strain to strain (Lehner, 2004). E. sakazakii cannot ferment D-sorbitol and

exhibits delayed extracellular DNase activity. In enzyme activity studies, Muytjens et al.

(1984) reported that α-glucosidase activity can be used to differentiate C. sakazakii from

other Enterobacter species. Detection of α-glucosidase activity enables the development of

selective and differential media for C. sakazakii, such as Oh and Kang media (Oh and Kang,

2004), Leuschner, Baird, Donald and Cox media (Leuschner, 2004), and Druggan-Forsythe-

Iversen media (Iversen et al., 2004b). The absence of lecithinase and production of Tween

esterase are also distinctive characteristics of C. sakazakii. (Farmer, 1980).

Iverson et al. (2008) reclassified the isolates described as C. sakazakii and proposed a

novel genus. The formal description of these organisms is completed by using Biotype 100

4

and Biolog Phenotype MicroArray data (Iversen et al., 2008). Based on further DNA-DNA

hybridization experiments, phenotypically different strains within the formal species C.

sakazakii were reclassified as Cronobacter sakazakii gen. nov., comb. nov. Cronobacter

malonaticus sp. nov., Cronobacter turicensis sp. nov.; Cronobacter muytjensii sp. nov.

Cronobacter dublinensis sp. nov.; Cronobacter dublinensis sub sp. Dublinensis subsp. nov.;

Cronobacter dublinensis subsp. Lausannensis subsp. nov. and Cronobacter dublinensis subsp.

Lactaridi subsp. Nov. (Iverson et al, 2008). The DNA-DNA relatedness values for species

presented below in Table 2.

1.3 Environment and food sources

The natural habitat or reservoir of C. sakazakii is unknown. The organism is not found in

surface water but is found in mud, grind, rotting wood, bird dung, rodents, domestic animals,

cattle, and raw bovine’s milk (Muytjens and Kollee, 1990). A possible environmental

reservoir of C. sakazakii was the gut of insects such as the Mexican fruit fly Anastrepha

ludens and the stable fly Stomoxys calcitrans (Kuzina et al., 2001; Hamilton et al., 2003.).

Flies serve as vehicles in C. sakazakii transmission because they are fed on blood of warm-

blooded animals and are found wherever cattle, pigs and horses are kept (Iversen and

Forthyse, 2004).

Kandha et al. (2004) investigated the presence of C. sakazakii in food manufacturing

factories and households by adaptive cultivation methods, biochemical characterization and

ribotyping. The environmental samples from factories were obtained by scraping or sweeping

surfaces in the production-line environment or by sampling vacuum-cleaner bags. Samples

from households were taken from vacuum-cleaner bags. Kandhai (2004) found 8 out of 9

5

environmental samples from factories and 5 out of 16 household samples contained C.

sakazakii. The organism was isolated from a range of foods including cheese, fermented

bread, tofu, sour tea, cured meats, minced beef and sausage meat. C. sakazakii may occur in a

large range of food ingredients due to its wide distribution in soil, however the detail is not

reported. The efficiency and accuracy of isolation and detection methods involved in these

studies are unclear so estimations of the prevalence of C. sakazakii may not be precise.

Members of the family Enterobacteriaceae were cultured from 52.5% of 141 milk

substitute infant formulas which were obtained in 35 countries (Muytjens et al., 1988b). C.

sakazakii was one of the most frequently isolated species (Muytjens et al., 1988b). Postupa

and Aldova (1984) isolated six C. sakazakii strains from powdered milk. Both Simmons et al.

(1989) in the United States and Bierling et al. (1989) in Iceland also isolated C. sakazakii

from dried infant formula.

1. 4 Growth requirements of C. sakazakii

C. sakazakii grows on media such as MacConkey, eosin methylene blue and violet red

bile agar used to isolate enteric organism Two morphologically different colony types are

observed when pure culture of C. sakazakii are streaked on agar plate: scallop-edged rubbery

colonies and smooth colonies (Farmer et al., 1980). C. sakazakii can grow to ca 109 CFU/ml

overnight from 104 at 37 or 44oC in trypticase soy broth (Iversen et al., 2004a). C. sakazakii

can grow at a temperature range of 6-47oC with an optimum of 37-43oC depending on the

medium. The doubling time at 37oC varied from 14 to 29 min in whitley impedance broth,

brain heart infusion medium and trpticase soy broth medium. In infant formula milk, the

6

doubling times at 6 and 21oC are 13.7 and 1.7 h (Iversen et al., 2004a).

Survival and growth of C. sakazakii in dry infant formula powder was investigated in the

last decade. C. sakazakii is reported to survive for at least two years in powdered infant

formula at a aw of 0.14 (Edelson-Mammel et al., 2005; Barron and Forsythe, 2007).

Population reduction of C. sakazakii at aw of 0.43-0.40 is greater than reduction in powders at

aw of 0.25-0.30 at 4oC for 6 months. Decreases in populations were greater in dry infant

formula stored at 30oC than at 21 or 4 oC. Survival of C. sakazakii does not demonstrate

significant differences (p

7

Leberkuhne and Wagner, 1986). The proposed mechanism of enhanced bacteria resistance to

environmental stress in biofilms is extracellular polymeric substances produced in biofilm

formation provide a protective barrier against environmental stresses (Kim et al., 2007). C.

sakazakii cells at or near the surface of biofilm also act as protective barriers for the cells

deeply in the biofilm. Although cells deep in the biofilm matrix cannot access oxygen and

nutrients in the environment, the cells undergo starvation leading to increased resistance to

environmental stress. Biofilms formed by Listeria monocytogenes on stainless steel and

Teflon coupons adapt to sanitizing agents while cells removed from biofilms individually are

not resistant to sanitizing agents. The development of resistance to environmental stress may

be attributed to extracellular substances instead of layering the cells in biofilms (Pan et al.,

2006). Lehner et al. (2005) observed calcofluor stained fibrils in biofilm matix formed by C.

sakazakii in Luria-Bertani broth and brain heart infusion broth suggesting the presence of

cellulose as an extracellular compound in this type of biofilm. Extracellular polysaccharides

produced by 24 C. sakazakii isolates analyzed with high performance liquid chromatography

revealed the presence of glucose, galactose, fucose and glucuronic acid (Lehner et al., 2005).

Dancer et al. (2009) determined that a polysaccharide capsule may not be a necessary

determinant of biofilm density by transmission electron microscopy. Hartmann et al. (2010)

conducted research to investigate the genetic basis of biofilm formation by Cronobacter spp.

on polystyrene surfaces by screening a library of random transponson mutant strain ES 5 for

decreased biofilm formation. Genes associated with flagellar structure and flow cell biofilm

architecture rather than genes associated with cellulose biosynthesis contributed to the

attachment of Cronobacter to polystyrene surfaces.

8

Cell-to-cell signaling, also known as quorum sensing, is demonstrated to play a role in

biofilm formation by foodborne pathogens (Annous et al., 2009). In Gram negative bacteria,

the homologues for Vibrio fischeri LuxI-LuxR regulatory genes are the key factor of quorum

sensing system. The auto-inducer acylated homoserine lactones (AHLs) are synthesized by

the LuxI homologues in the cell and secreted to the external environment. When the AHLs

reach a threshold concentration, AHLs enter bacteria cells and bind to the LuxR homologues

to activate or repress target gene transcription. Thus, the quorum sensing system can regulate

biofilm formation. In Gram positive bacteria, the auto-inducer is secreted external to the cells

similarly, but the auto-inducer regulates target gene transcription by binding to the receptors

on the cell surface instead of reentering the cells (Miller and Bassler, 2001). Quorum sensing

systems of a large number of bacteria also depend on molecules other than autoinducer-1 (AI-

1). For example, autoinducer-2 (AI-2) is a byproduct of the activated methyl cycle catalyzed

by LuxS enzyme (Smith et al., 2004; Vendeville et al., 2005). Certain food pathogens such as

Escherichia, Shigella, Salmonella, Yersinia and other Gram negative bacteria possess the

auto-inducer-3/epinephrine/norepinephrine (AI-3/epi/norepi) signaling system (Walters and

Sperandio, 2006). Salmonella, Escherichia, Shigella, and Klebsiella that do not produce

AHL detect AHLs produced by other bacteria (Michael et al., 2001). In C. sakazakii strains,

the presence of AHLs is determined by thin-layer chromatography. Lehner et al. (2005)

analyzed ethyl acetate extracts of cell supernatants for 56 selected C. sakazakii strains and

observed production of AHLs by C. sakazakii.

Attachment and biofilm formation on the abiotic surface of C. sakazakii is influenced by

temperature, nutrient availability and humidity. In one study, stainless steel coupons and

9

enteral feeding tubes were immersed in phosphate-buffer saline cell suspension of five strains

of C. sakazakii grown to stationary phase (7 log CFU/ml) to gain the attachment of 5.33 to

5.51 and 5.03 to 5.12 log CFU/cm2 at 4oC. Then coupons and tubes attached by C. sakazakii

were immersed into tryptic soy broth, infant formula and lettuce juice broth followed by

incubation for 10 days at 12 or 25oC. Biofilms were not observed at 12oC, and the cells

increased 1.42 to 1.67 and 1.16 to 1.31 log CFU/cm2 on stainless steel coupons and enteral

feeding tubes in infant formula at 25oC (Kim et al., 2006). Biofilms were formed on abiotic

surfaces immersed in infant formula, but not on abiotic surfaces immersed in typtic soy broth

and lettuce juice suggesting that nutrient availability play a role in biofilm formation (Kim et

al., 2006). Dancer et al. (2009) concluded that nitrogen source is a more important

determinant than carbohydrate source in biofilm formation of C. sakazakii in milk compared

with carbohydrates (Dancer et al., 2009). Quantification of biofilm formation on plastic

surfaces by 72 seletected C. sakazakii strains in brain heart infusion broth, nutrient broth,

tryptic soy broth, 1/20 tryptic soy broth and reconstituted infant formula revealed that

reconstituted infant formula supported biofilm formation more effectively than artificial

media (Oh et al., 2007). Survival of C. sakazakii cells in biofilms on stainless steel coupons

immersed in M9 medium and reconstituted infant formula were investigated when exposed to

23, 43, 68, 85 and 100% relative humidity. The overall order of survival as affected by

humidity was 100 > 23 = 43 = 68 > 85% relative humidity regardless of matrices (Kim et al.,

2008). Temperature control of infant feeding preparation and storage conditions choice is

important for preventing C. sakazakii biofilm formation.

10

1.6 Resistance of C. sakazakii to environmental stress

1.6.1 Heat resistance

Preliminary studies of D values of C. sakazakii strains were conducted in artificial

medium and reconstituted infant formula from 50 to 70oC. D55 of selected strains in

reconstituted infant formula varied from 1.51 to 14.21 min according to Al-Holy et al. (2009).

D55 range selected C. sakazakii strains was 3.27 min to 17.07 min in phosphate buffer pH 7.0

reported by Dancer et al. (2009). Considerable variations in the heat resistance of the tested

C.sakazakii strains were observed. Physiological properties of the tested strains are an

important factor that influences heat resistance. C. sakazakii ATCC 29544 exhibited a D55

value of 14.83 min while the D55 value of C. sakazakii 55 is 1.51 min in reconstituted infant

formula. Edelson-Mammel and Farmer (2004) concluded that C. sakazakii may have a set of

genetic determinants for heat resistance based on two distinct phenotypes presented in D

value profile. Williams (2005) reported a protein only expressed in heat resistant strains and

identified as homologous to a hypothetical protein found in the heat resistant bacteria,

Methylobacillus flagellatus KT by a top-down proteomics approach. Asakura (2007) studied

the genetic heat resistance of C. sakazakii, and concluded that infB gene which encodes for a

translation initiation factor is expressed in a higher amount in heat resistant strains than in

heat sensitive strains.

Iversen et al. (2004a), Dancer et al. (2009) and Al-Holy et al. (2008) reported D60 values of

C. sakazakii strains. Iversen et al. (2004a) compared heat resistance of type and capsulated

strains in typtone soy broth and reconstituted infant formula. The medium used for bacteria

culture influences the heat resistance of selected strains. D60 value of type strain of C.

11

sakazakii was 0.9 min in tryptone soy broth and 1.1 min in reconstituted infant formula

(Iversen et al., 2004a). Moreover, C. sakazakii 55 demonstrated a D55 of 1.51 min in

reconstituted infant formula (Al-Holy et al., 2008) and 3.27 min in phosphate buffer with pH

7.0 (Dancer et al., 2009). D60 values ranged from 0.17 min to 2.71 min in both reconstituted

infant formula and liquid medium except C. sakazakii 607 with a D60 value of 264.4 min in

reconstituted infant formula (Edelson-Mammel and Buchanan, 2004; Al-Holy et al., 2008;

Dancer et al., 2009). D65 value of C. sakazakii 607 was 35.2 min in reconstituted infant

formula reported by Edelson-Mammel and Buchanan (2004) (Table 4).

Overall z value for selected food and clinical strains of C. sakazakii calculated by

Nazarowec-White and Farmer (1997) was 5.82oC based on D values at 52, 54, 56, 58 and

60oC in reconstituted infant formula. z value for C. sakazakii 1387-2 was 3.1oC calculated

with D values of 53, 54, 56, 58oC in phosphate buffer pH 7.0. The most heat resistant C.

sakazakii 607 strain reviewed exhibited a z value of 5.6oC in reconstituted infant formula

based on D values of 56, 58, 60, 65, 70oC (Edelson-Mammel and Buchanan, 2004). Iversen et

al. (2004a) presented different z values of C. sakazakii NCTC 11467 and capsulated strain C.

sakazakii 823 at 5.8oC and 5.7oC in typtone soy broth with D values of 54, 56, 58, 60, 62 oC

(Iversen et al., 2004a). Al-Holy et al. (2009) reported z values of tested C. sakazakii strains in

reconstituted infant formula varied from 3.76oC to 10.11oC based on D values of 55, 60, 63oC.

The selected C. sakazakii strains exhibited a range of z values from 6.26oC to 10.86oC in

phosphate buffer pH 7.0 based on D values of 50, 55, 60oC (Dancer et al., 2009) (Table 5).

1.6.2 Osmotic and desiccation resistance

Dried infant formula powder has a water activity about 0.2. To survive under this

12

extreme condition, C. sakazakii possesses osmotic and desiccation resistant mechanisms.

Breeuwer et al. (2003) reported that C. sakazakii strains inoculated in BHI with 40% sorbitol

(aw = 0.934) decreased about 1 log after 2 months, making the most resistant strain to osmotic

stress compared to the nine microorganisms within Enterobactericeae tested as part of this

study. At a sorbitol concentration of 75% (aW = 0.811), C. sakazakii strains 1387-2 and 1360

were detectable after four weeks. The addition of trehalose, proline or glycine betaine to

sorbitol stressed C. sakazakii did not improve survival. In contrast, C. sakazakii cannot grow

in M9 medium with 1 mol/L NaCl, but addition of glycine betaine to medium enhance

growth (Breeuwer et al., 2003).

In the study of desiccation resistance, the C. sakazakii cell populations in dry infant

formula at day 46 exhibited a 1.0-1.5 log10 cfu/g reduction in stationary phase in dry infant

formula with a aw of 0.14 at 20oC. Similarly, Edelson-Mammel et al. (2005) reported a 2.4

log10 cfu/g reduction was observed at the initial 5 months and an additional 1 log10 cfu/g

reduction during the subsequent 19 months. Barron and Forsythe (2007) also reported that

some capsulated C. sakazakii strains were recoverable after 2.5 year storage in dry infant

formula powder while other selected strains belonged to Enterobacteriaceae were not

recovered after 6, 15 and 24 months.

Research to explain the osmoadapation of bacteria suggests organisms can protect

themselves from a NaCl concentration stress environment by pumping out sodium and

accumulating potassium through a proton gradient that impart enzymes with more negative

charged amino acid residues than those in normal environment. To survive in an anhydrous

environment, a compatible solute such as trehalose is accumulated by the microorganism to

13

stabilize dry cell membranes by interaction with –OH groups on phospholipid membrane.

Moreover, trehalose also stabilizes dry proteins by interaction with –OH groups on polar

residues in dry protein. The hydrogen bonds formed between trehalose and phosphate or

amino acid residues provide a water shell for these structure compounds (Crowe, 1992).

C. sakazakii can survive in desiccation and high osmotic pressure environments by

stabilizing macromolecules.

1.6.3 Antibiotics resistance

Muytjens et al. (1988a) determined the activities of 29 antimicrobial agents against C.

sakazakii and compared antibiotic resistance with seven other Enterobacter species. C.

sakazakii was resistant to 27 out of 29 antibiotics, except for cephalothin and

sulfamethoxazole. The minimum inhibitory concentration (MIC) for C. sakazakii was at least

twofold higher than the MIC for E. cloacae. C. sakazakii also exhibited exclusive resistant to

ampicillin in the tested strains with MIC of 8 µg/ml and MIC for cephalothin in a

concentration range from 256 to 512 µg/ml (Muytjens, 1988a). Farmer et al. (1980) reported

that C. sakazakii is susceptible to gentamicin, kanamycin, chloramphenicol and ampicillin;

More than 87% were susceptible to nalidixic acid, streptomycin, tetracycline and

carbenicillin. All selected 24 strains of C. sakazakii were resistant to penicillin (Farmer,

1980). Nazarowec-White and Farber (1999) reported that 13 out of 24 strains were

susceptible to ampicillin, cefotaxime, chloramphenicol, gentamicin, kanamycin, polymyxin B,

trimethoprimsulphamethoxazole, tetracycline and streptomycin, but resistant to

sulphisoxazole and cephalotin. One of the food strains was resistant to sulphisoxazole,

cephalothin, chloramphenicol and ampicillin. Kuzina et al. (2003) reported that C. sakazakii

14

isolated from M fruit flies is resistant to ampicillin, cephalothin, erythromycin, novobiocin,

and penicillin. Lai (2001) reported that C. sakazakii clinical isolates examined exhibited

uniform resistance to ampicillin, cefazolin and extended spectrum penicillins and variable

resistance to the 3rd generation cephalosporines and the quinolones. Compared with the

results previously reported, the C.sakazakii isolates from M fruit flies were susceptible to

ampicillin, tetracycline, chloramphenicol, gentamycin and the 3rd generation cephalosporines.

All selected isolates were susceptible to aminoglycosides and trimethoprim-sulfamethoxazole.

Denisson and Morris (2002) reported that an isolate from an infected vascular graft and thigh

wound was resistant to ampicillin, gentamicin and cefotaxime. The prevalence of antibiotic

resistance among isolates of C. sakazakii may relate to the increasing trend of antibiotic

resistance among Enterobacter species. The intensive units of antibiotics applied to patients

impart selective pressure on the isolates in infection sites and result in increasing antibiotic

resistance among those isolates. Moreover, transmission of antibiotic resistance is considered

through a transpron among antibiotic resistant isolates and antibiotic susceptible isolates

(Lehner, 2004).

1. 7 Virulence factors

C. sakazakii causes life threatening bacterial infections in low birth weight, premature and

immunosuppressed infants. There were at least 111 cases and 26 reported deaths resulting

from C. sakazakii infection worldwide reported 1958. Infant formula contaminated by C.

sakazakii was associated with at least five infections in the US and Europe from 1958 to 2010

(Anonymous, 2010).

15

Potential virulence factors are required to evaluate the pathogenicity of C. sakazakii to

humans. Successful colonization, establishment and ultimately production of disease are

properties necessary for a pathogenic microorganism to cause a disease epidemically. To date

only a few studies were conducted to examine the mechanisms of pathogenicity for C.

sakazakii.

Pagotto et al. (2003) used 18 clinical and foodborne isolates of C. sakazakii and the

suckling mice assay to evaluate enterotoxin production. The tested strains were lethal to

suckling mice at a dose of 108 CFU. In in vitro assays, CHO, Vero and Y-1 cells exhibited

lysis and loss of cell morphology in the presence of C. sakazakii strain LA filtrates, which

may contain enterotoxin produced by the bacteria. Four out of eighteen C. sakazakii strains

were positive for enterotoxin production. The adherence of 50 C. sakazakii strains to

epithelial cell lines HEp-2, Caco-2 and brain microvascular endothelial cell line (HBMEC)

were examined by Mange et al. (2008). Diffusion and formation of localized clusters of

C.sakazakii on the cell surfaces were observed for three cell lines. C. sakazakii infection is

linked with outbreaks of neonatal meningitis and necrotizing enterocolitis (Anonymous,

2010). The mortality rates of meningitis and necrotizing enterocolitis caused by C. sakazakii

infection are 10-55% and 40-80%, respectively. (Iversen and Forsythe, 2003).

Necrotizing enterocolitis (NEC) resulting from consumption of powdered infant formula

is common in neonates and characterized by intestinal necrosis and pneumatosis intestinalis.

In an NEC outbreak in 1998, a total of 11 C. sakazakii strains were isolated from stomach

aspirate, anal swabs, and blood (Van Acker, 2001).

The first reported case of meningitis due to “yellow pigmented E. clocae” occurred in

16

England in 1961 (Iversen and Forsythe, 2003). Like most microorganisms, in order to cause

meningitis, C. sakazakii must adhere to and colonize intestine surfaces, translocate into the

blood stream, escape from host immune system, cross the blood/brain barrier, and survive in

the cerebral spinal fluid. Most reported outbreaks of meningitis due to C. sakazakii are

resulting from hospital nurseries and neonatal intensive care units (Iversen and Forsythe,

2003). Muytjens et al. (1983) reported eight cases of neonatal meningitis occurred in the

Netherlands during the last six years with 75% fatality. It is probable that C. sakazakii

exhibits developmental dependence on access to the central nervous system (Iversen and

Forsythe, 2003).

1.8 Detection methods

The FDA recommended detection protocol is a presumptive, most probable number

(MPN) assay based on three tubes of enrichment broth to allow the detection of small

population of C. sakazakii present in reconstituted infant formula. The approved method

requires five days to complete. Figure 1 illustrates a summary of the method. This method

was first used by Mutjyens (1988a). Mutjyens (1988a) reconstituted infant formula powder

with buffered peptone water rather than distilled water, and subcultured growth in sheep

blood agar and eosin-methylene blue agar rather than trypticase soy agar before the API

biochemical tests. The FDA recommended method involves three main elements:

preenrichment, enrichment and selection. In the preenrichment step, a total of 333 g infant

formula powder is reconstituted with distilled water overnight at 36oC, followed by

enrichment in Enterobacteriaceae Enrichment (EE) broth for overnight at 36oC. Point one ml

17

or loopful culture is spread on violet red bile glucose (VRBG) agar and incubated at 36oC

overnight to select presumptive Enterobacteriaceae. The presumptive positive clonines are

subcultured on trypticase soy agar (TSA) and incubated for 42 to 72 h at 25oC to encourage

the yellow pigment production. The yellow pigmented colonies are inoculated into the API

20E system and incubated at 36oC for 18 – 24 h to subject to the biochemical identification

(U.S. FDA, 2002; Figure 1). API 20E system can determine determine the metabolic

capabilities, the genus, and species of enteric bacteria in the family Enterobacteraceae by

reactions in miniaturized test couples with different agents The detection limit of this method

is < 1 cell in 25 g (how can it be less than 1 cell? )infant formula powder (Mansfield, 2000).

In this protocol, VRBG and TSA are not specific for C. sakazakii, time consuming and is not

sensitive.

Muytjens et al. (1984) first demonstrated C. sakazakii isolates produce α-glucosidase

while other isolates of E. cloacae, E. aerogenes and E. agglomerans do not produce α-

glucosidase by performing α-glucosidase test. The special α-glucosidase activity indicates

that the detection of α-glucosidase can be used as a biomarker of rapid detection and

differentiation of C. sakazakii from other Enterobacter species. Several selective media are

formulated based on α-glucosidase production. Iversen, Druggan and Forsythe developed a

chromogenic medium (Druggan-Forsythe-Iversen, DFI) for the selective detection of C.

sakazakii. DFI medium detects α-glucosidase activity by using 5-bromo-4 chloro-3-indolyl-α,

D-glucopyranoside (XαGlc). C.sakazakii hydrolyzes XαGlc to an indigo pigment, producing

blue-green colonies on this medium (Iversen et al., 2004b). Compared to FDA recommended

methods, the DFI medium requires three days to complete detection instead of five days

18

(Iversen et al., 2004b). DFI medium exhibits improved accuracy because it is specific for C.

sakazakii and differentiates C. sakazakii from other Enterobacter species. Restaino et al.

(2005) used two chronogenic substrates, 5-bromo-4-chloro-3-indoxyl-β-D-cellobioside and

5-bromo-4-chloro-3-indoxyl- α-D-glucopyranoside as indicators of α-glucosidase activity.

Pure cultures of C. sakazakii strains yield blue-black raised colonies, and other enteric

organisms or Pseudomonas aeruginosa yield white, yellow, green or clear colonies with or

without clear halos. The medium may give false positive readings for Shigella sonnei or

Pantoea strain (Restaino, 2005). This new detection method exhibited 100% sensitivity and

96.6% specificity for 240 samples (Restaino et al., 2005).

Oh and Kang (2004) developed a fluorogenic medium using 4-methylumbelliferyl-α-D

glucoside as a marker to exhibit α-glucosidase activity. 4-methylumbelliferyl-α-D glucoside

is digested by α-glucosidase into a free 4-methylumbelliferyl moiety and is fluororecent when

exposed to UV radiation with a wavelength of 365 nm.

A cationic-magnetic-bead capture technique is used to detect C. sakazakii by

combination charged paramagnetic beads and chromogenic medium. The positively charged

magnetic beads interact with negatively charged lipopolysaccharide on surface of Gram

negative bacteria. Capture by ionic interaction collects C. sakazakii cells to achieve a

detectable concentration. This method needs 6-h enrichment at 42oC in buffered peptone

water, a 30 min cationic bead capture and selective culture on DFI agar. This method can

detect 1 to 5 CFU cells in 500 g infant formula powder within 24 h (Mullane, 2006 a&b).

Detection by culturing methods is labor intensive, time consuming and can have a low

sensitivity. Molecular based methods are developing to overcome the limitations of

19

phenotypic detection. Molecular based methods include chromosomal DNA restriction

analysis, plasmid typing, ribotyping, pulsed field gel electrophoresis (PFGE) and random

amplification of polymorphic DNA (RAPD).

Malorny developed a real time PCR targeting 16 rRNA gene for specific detection of C.

sakazakii. This real time PCR assay is applied to diagnostic detection of C. sakazakii in

infant formula with a 100% detection probability when the C. sakazakii cell concentration is

103 CFU/ml (Malorny, 2005). Seo and Brackett (2005) developed a real time PCR targeted

specific sequence within the macromolecular synthesis (MMS) operon. The detection limit of

this newly developed assay is 100 CFU/ml in pure culture and in reconstituted infant formula.

The assay can detect C. sakazakii with highly specificity without any enrichment steps (Seo

and Brackett, 2005).

Bacterial typing systems are based on the principle that clonally related isolates share

characteristics by which they can be differentiated from unrelated isolates. Nazarowec-White

and Farber (1999) clustered C. sakazakii infant formula and clinical isolates by using

phenotypic (biotype and antibiograms) and genotypic (ribotyping, RAPD and PFGE)

methods. Clark et al. (1990) used a combination of typing methods (plasmid analysis,

antibiograms, chromosomal restriction endonuclease analysis, ribotying and multilocus

enzyme electrophoresis) to evaluate the isolates from patients and infant formulas and

determine their relatedness. These methods trace the isolates from patients back to the source

infant formula by comparing the typing patterns of the isolates.

Molecular based methods are valuable analytical tools to identify and trace the source of

contamination of infant formula. These methods are required to be standardized and verified

20

to apply in epidemiological research of C. sakazakii infection.

1.9 UV radiation treatments

Ultraviolet radiation is well developed for water treatment, air disinfection and surface

decontamination. The US Food and Drug Administration (FDA) approved UV light as an

alternative treatment to thermal pasteurization of fresh juice products (US FDA, 2000). The

performance criterion defined by FDA for fruit and vegetable juice processing is a five log 10

reduction in the number of the target pathogen of concern (US FDA, 2000).

UV radiation processing involves the use of radiation from the UV region of the

electromagnetic spectrum for purposes of disinfection (US FDA, 2009). The wavelength

range from 100 to 400 nm is classified as UV radiation. UV radiation is subdivided into

vacuum UV (100 to 200 nm), UVA (315 to 400 nm), UVB (280-315 nm) and UVC (200 to

280 nm) (Srikanth, 1998). The vacuum UV has high energy and produces reactive radicals,

but most radiation with wavelengths in this range is blocked by ozone. UVA is the largest

contributor of UV in sunlight and responsible for skin tanning. UVB penetrates the skin and

leads to skin cancer. UVC effectively inactivates bacteria and viruses by damaging DNA

within defined germicidal range. Inactivation of microorganism by UV radiation is achieved

through absorption of UV radiation by DNA molecules and the dimerization of thymine bases.

A limitation of UV radiation to food processing is its poor transmissivity within a food

product. The geometric configuration of reactors, the power, wavelength and arrangement of

UV sources, and the geometric and chemical properties of products influence the effective

inactivation of microorganisms. UV radiation is used with other processing technologies,

21

such as chemical disinfectants (Ha, 2010), oxidizing catalysts (Kim, 2009) and powerful

oxidizing agents (Hadjok, 2008) that may exhibit synergistic effects for inactivating

microbial pathogens.

Ha et al. (2010) reported that combined treatments of UV radiation and ethanol

inactivate Bacillus cereus, C. sakazakii, Staphlococcus aureus, Escherichia coli and

Salmonella enteric Typhimurium in bovine albumin solutions. Hadjok et al. (2009)

demonstrated that treating fresh lettuce with combined aqueous hydrogen peroxide and UV

radiation inactivates Salmonella, E.coli O157:H7, Pectobacterium carotovora and

Pseudomonas fluorescens on the surfaces and within fresh produce without quality

deterioration (Hadjok, 2008). Jung (2008) applied combined UV radiation and ozone

treatments to inactivate Bacillus subtilis spores and obtain a synergistic effect, because ozone

absorbs UV radiation and produces radicals, which exhibit a powerful oxidizing effect. Kim

et al. (2009) investigated the efficacy of titanium dioxide – UV photocatalytic disinfection

treatment on the shelf life of iceberg lettuce. Titanium dioxide produces hydroxyl radicals by

photocatalytically reacting with UV radiation, providing a germicidal effect. The TiO2-UV

system inactivates more pathogens than UV treatments or NaOCl treatments alone and

inhibits the growth of survivors at 4 and 25 oC (Kim, 2009).

UV radiation is well established in liquid food processing. Parts of water receive a UV

radiation exposure of at least 400 J/m2 at 254 nm to reduce human pathogen by at least four

logs (Bernhardt, 1994). UV radiation sensitivity of microorganisms is a key factor affecting

efficacy of UV treatment on microorganisms in liquid foods. Microorganism sensitivity is

variable due to cell wall structure and composition, proteins and nucleic acid structures.

22

Generally, bacterial spores are the most resistant forms. Gram positive bacteria are more

resistant than Gram negative bacteria (Koutchma, 2009). Physico-chemical parameters are

also considered in UV reactor design to increase bacteria inactivation efficacy including

fluid dynamic parameters, transmissivity and absorptive properties.

Chemical components in a liquid system reduce antimicrobial activity of UV

treatments. For example, dissolved organic solutes and compounds such as fructose,

sucrose, glucose and malic acid, attenuate bacteriacidal effects by absorbing UV radiation

(Fan and Geveke, 2007). Suspended solids scatter, absorb and block UV radiation as well

as protect bacteria by providing particulate surfaces for aggregation (Christenen and

Linden, 2001). Thus, the homogeneity of the flow pattern should be controlled to achieve

uniform inactivation. Product composition, solids content, color, starches and other

chemical properties of foods are also major factors that influence bacterial inactivation.

Compared to water, a food matrix is a complex system and many factors must be

manipulated to obtain ideal bacteria inactivation similar to bacterial inactivation in water.

Some components in food sensitive to UV radiation must be considered to minimize

quality changes. Vitamin A, carotenes, cyanobalamin (vitamin B12), vitamin D, folic acid,

vitamin K, riboflavin (vitamin B2), tocopherols (vitamin E), tryptophan, unsaturated fatty

acids and phospholipids are “light sensitive” (Spikes, 1981). These chemicals in foods easily

absorb photons and promote reactions to break or form bonds. Milk and milk products are

highly light sensitive. UV radiation applied in milk industry may result in off-flavors and

nutrients loss.

23

1.10 Fourier Transform Infrared Spectroscopy

The infrared light originates from laser source is absorbed by analyzing samples. The

functional groups in macromolecules change vibrational stretching structures and can be

observed in the mid-infrared (4,000 to 400 cm-1) spectral regions. Thus, the chemical

composition of bacterial membranes is derived from FT-IR spectrum.

Application of vibrational spectroscopy for rapid determination of food contamination is

increasing in recent years due to short and non-destructive sample preparation steps and

quick analysis times. Several types of vibrational spectroscopy are used to detect microbial

contamination in food, including mid-infrared (mid-IR, 4000-650 cm-1) spectroscopy and

Fourier transform infrared (FT-IR, 4000 – 400 cm-1) spectroscopy. Compared to classical

microbiology methods, PCR-based molecular methods and antigen-antibody immunology

reaction based methods, FT-IR can detect and potentially quantify bacteria at populations of

105 CFU/g powder in food powders within 5 min instead of days. Furthermore, spectroscopic

methods are being explored to study inactivation mechanism by identifying changes in

composition of the cell membranes and DNA.

These spectroscopic techniques are employed to study bacterial injury after inactivation

treatments such as sonication (Lin et al., 2004), heat (Al-Qadiri et al., 2008) or antibiotics

(Neugebauer et al., 2007). Lin et al. (2004) employed FT-IR (4000-600 cm-1) to discriminate

intact and sonication-injured Listeria monocytogenes ATCC 19114 by segregation of intact

and injured strains through principal component analysis. Al-Qadiri et al. (2008) detected

sublethally heat-injured microorganisms Salmonella enterica Typhimurium and Listeria

monocytogenes by FT-IR spectroscopy (4000-600 cm-1). The analysis of spectra also revealed

different cell injury mechanisms for Gram positive and Gram negative bacteria. FI-IR and

24

Raman spectroscopy is applied to elucidate the action mode of drugs in new drug

development. The cell composition changes of Gram positive bacteria Staphylococcus

epidermidis occurring in the present of moxifloxacin drugs were investigated by vibrational

spectroscopy (Neugebauer, 2007).

Although FT-IR spectroscopy is an attractive method to differentiate biochemical

composition before and after disinfection, many factors must be controlled to increase

sensitivity. Cell recovery methods and filtration techniques are key factors to minimize

detection limits (Lu and Rasco, 2010). A filtration membrane that can remove spectral

interference produced by media residue and obtain a uniform layer of bacteria cells is ideal.

Application 500 μl of the bacterial suspension onto an aluminum oxide membrane filter of

0.2 μm pore size and 25 mm outer diameter is widely used filtration technique (Lu and Rasco,

2010).

Davis isolated Samonella enteric serovars from chicken breast by filtration and

immunomagnetic separation and collected the FT-IR spectra (Davis, 2010). The physiological

state of the microorganisms and culture medium also contribute variability in the FT-IR

spectra (Lu and Rasco, 2010). Use of microbes in the stationary phase is generally

recommended for FT-IR spectroscopy because spectral features will be more consistent due

to the stable physiological state (Lu and Rasco, 2010, al-Qadiri et al. 2007). Van der Mei et

al.(2004) and Filip et al. (2008) reported that the culture age, composition of the media and

quantity of nutrients can influence spectral features.

In conclusion, dry infant formula contaminated by C. sakazakii is of great concern to the

public. It is extremely important to explore novel methods to inactivate C. sakazakii in dry

25

infant formula. In this study, the efficacy of UV radiation for inactivating C. sakazakii in dry

infant formula was evaluated. The efficacy of combined treatment was evaluated by

reconstituting dry infant formula exposed to UV radiation in hot water at 55, 60 and 65oC.

The changes in cell membranes and morphology of the bacterial pathogen were investigated

by FT-IR and electron microscopy.

26

2 Materials and Methods

2.1 Bacterial strains, culture methods and preparation of stock cultures

Three strains of C. sakazakii (American Type Culture Collection [ATCC] 51329, 29544

and 12868) were obtained from the School of Food Science culture collection at Washington

State University (Pullman, WA, USA). All bacteria strains were stored at -80oC in tryptic soy

broth (TSB, Difco Laboratories, Detroit, MI) with 15% glycerol. Frozen cultures of the three

strains were streaked onto tryptic soy agar (TSA, Difco Laboratories, Detroit, MI) and

incubated 24h at 37oC. The grown cultures were stored at 4oC for routine use. Cultures of

each strain were combined together to construct a culture cocktail and harvested by

centrifugation at 4,000 rpm for 20 min at room temperature (centra CL2, 4675×g,West

Chester, PA). Cultures were washed twice with buffered peptone water (Difco). The pellets

were resuspended in buffered peptone water, corresponding to ~108 to 109 CFU/ml.

2.2 Dry infant formula sample preparation and inoculation

Dry infant formula powder (Enfamil, Mead Johnson & Company, Evansville, IN) was

purchased from a local grocery store. Five grams of dry infant formula powder was placed

into a Petri dish (150×15 mm, Becton Dickinson & Co., Franklin Lakes, NJ) and spread into

a layer of ca 0.032 mm. One hundred microliters of the cocktail was inoculated to dry infant

formula powder by depositing droplets in five locations with a micropipette. The dry infant

formula was dried in a laminar flow biosafety hood with the fan running for 30 min. After

drying, clumps of inoculated infant formula powder were crushed with a sterile spatula and

thoroughly shaken to produce a homogeneous dispersal of inoculums throughout the dry

infant formula.

27

2.3 Ultraviolet radiation treatment

The UV light chamber for treatment of dry infant formula was custom built at the School

of Food Science, Washington State University (Pullman, WA). The chamber contains two

45.4 cm-long UV lamps 10 cm apart (Sun Ray Technologies, Inc. Killington, VT). The lamps

are suspended across the chamber with a distance of 15 cm from the base of the chamber. UV

intensity at 253.7 nm was determined by a UV radiometer (Steril-Aire, Burbank, CA) at the

surface of the Petri dish. The radiometer sensor was placed under the UV lamp with the lamp

on for 30 min to obtain a consistent reading. The interior of chamber is lined with a highly

reflective metal foil (Sun Ray Technologies) to increase UV intensity and to minimize any

shadowing effect on irregular surface shaped dry infant formula samples. Plates of inoculated

dry infant formula were individually subjected to UV treatment. The intensity was kept

constant and selected exposure times were applied to allow respective doses (Table 6). The

Petri dish was shaken at selected time intervals to expose dry infant formula granules to UV

radiation more evenly. To analyze the influence of layer thickness on UV radiation

inactivation efficacy, 5, 15 and 25 g of inoculated dry infant formula were placed onto Petri

dishes to obtain layer thickness of 0.032, 0.16 and 0.32 cm.

2.4 Combined UV radiation and hot water treatment.

Five grams of inoculated and air dried infant formula was placed into a Petri dish and

spread into a thin layer. After exposure to UV radiation for 20 min at room temperature, UV

radiation exposed infant formula was reconstituted in 10 ml sterile distilled water at room

temperature in a stomacher bag and homogenized for 2 min with a Seward stomacher

(Stomacher 80, Seward, London, UK). Hot water treatment was conducted in a water bath

28

(VWR Scientific, West Chester, PA) for selective time intervals at 55oC, 60oC and 65oC. The

temperature of the dry infant formula samples was monitored by a type omega thermocouple

(OMEGA Engineering Inc., Stamford, CT) and temperature controlled within ±0.5oC. The

treatment time was controlled by a stopwatch (Thermo Fisher Scientific Inc., Waltham, MA).

The reconstituted infant formula was removed from the water bath and immersed in ice

immediately after each treatment to stop heating.

2.5 Bacterial enumeration.

UV treated dry infant formula powder was rehydrated as described previously. One

milliliter sample aliquots of reconstituted infant formula were serially diluted tenfold in 9 ml

of sterile 0.2 % peptone water (Difco) and 0.1 ml of samples were spread-plated onto Petri

plates of OK Medium (OK; Acumedia, Lansing, MI), a medium for selective recovery and

enumeration of C. sakazakii. The bacteria were enumerated on to TSA to recover the injured

cells for the combined treatments of UV radiation and hot water. Agar plates were incubated

at 37°C for 24 h and colonies were enumerated. Colonies fluorescing when illuminated with

365 nm UV light were counted.

2.6 Membrane filtration and FT-IR spectroscopy.

Non fat milk powder was used as a surrogate for dry infant formula for UVC treatments to

eliminate the background noise raised by large fat molecules. Two one hundredths of a gram

of non fat milk was dissolved in 50 ml of sterile saline water (0.85% w/v) and filtered

through an aluminum oxide membrane filter (0.2 μm pore size, 25 mm OD) (Anodisc,

Whatman Inc., Clifton, NJ) using a Whatman vacuum glass membrane filter holder

29

(Whatman Catalog No. 1960-032) to harvest bacterial cells. The anodisc filters were removed

from the filtration apparatus and air-dried under laminar flow at room temperature (ca. 22°C)

for 10 min to allow a homogeneous film of bacterial cells to form.

FT-IR spectra were collected using a Nicolet 380 FT-IR spectrometer (Thermo Electron

Inc., San Jose, USA). The aluminum oxide membrane filter coated with a uniform and thin

layer of bacterial cells was placed in direct contact with the diamond crystal cell (30,000 –

200 cm-1) of attenuated total reflectance (ATR) detector. Infrared spectra were recorded from

4002 to 399 cm-1 at a resolution of 8 cm-1. Each spectrum was acquired by adding together 32

interferograms. Five spectra were acquired for intact and UV treated C. sakazakii at different

locations on the aluminum oxide filter for a total of 15 spectra for each group of cells.

Triplicate experiments (N=3) were conducted and spectra from the first two experimental

runs were used to establish chemometric models while the spectra from the third experiment

were used for model validation.

2.7 Electron microscopy analysis.

Scanning electron microscopy (SEM) was performed to examine morphological changes

of C. sakazakii cells before and after UV irradiation treatment of dry infant formula powder.

The dry infant formula sample was reconstituted in sterile water at room temperature and C.

sakazakii cells were obtained by centrifuge. First, C. sakazakii cells were fixed with 2%

glutaraldehyde, 2% paraformaldehyde in 0.1 M phosphate buffer overnight. The fixed cells

were rinsed with double distilled water, followed by freeze drying in a Virtis Lyophilizer (The

Virtis Co., Inc., Gardiner, NY, USA). The freeze dried cells were mounted onto SEM stubs

and sputter-coating with gold. The coated cells were observed under a FEI Quanta 200F Field

30

Emission scanning electron microscope (Field Emission Instruments, Hillsboro, OR, USA)

using an accelerating voltage of 30 kV.

Transmission electron microscopy (TEM) was employed to study the influence of UV

radiation on the inner structures of C. sakazakii cells. Uninoculated infant formula powder

was designated as a control. C. sakazakii cells in dry infant formula untreated and treated

with UV radiation were placed into the primary fixative overnight at 4°C. The fixed cells

were rinsed three times with 0.1M phosphate buffer (10 min each) and post-fixed by 2%

osmium tetroxide for 2 h at room temperature. The post fixed cells were quickly rinsed twice

with 0.1 M of buffer (10 min each), followed by dehydration with sequential ethanol

solutions (30%, 50%, 70%, 95%, and 100%), and 100% propylene oxide twice (10 min each).

The bacteria cells were infiltrated by propylene oxide: Spurr’s (1:1) overnight and 100%

Spurr’s twice (overnight each). The samples were embedded in Spurr’s resin. The

composition of Spurr’s is vinyl ten gram of cyclohexene dioxide, six gram of diglycidyl ether

of polypropylene glycol, twenty six gram of nonenyl succinic anhydride, and four tenth gram

of dimethylaminoethanol (Spurr, 1969). The cells were observed in Philips electron

microscope (Field Emission Instruments, Hillsboro, OR, USA) operating at 200 kV.

2.8 Data preprocessing and chemometrics.

Infrared spectra were first pre-processed by EZ OMNIC 7.1 a (Thermo Electron Inc.).

Relevant background was subtracted from each raw spectrum. Automatic baseline correction

was employed to flatten the baseline, following by a smooth of five (Gaussian function of

9.643 cm-1). The pre-processed spectra were read by Matlab 2010a (Math Works Inc., Natick,

31

MA, USA) with .xls format by Excel (Microsoft Inc., Redmond, WA, USA). The

reproducibility of vibrational spectra from four independent experiments (N=3) was

investigated by calculating Dy1y2 according to equations (1) and (2) (Wang et al., 2010). In the

equations, y1i and y2i are signal intensities of two selected spectra while 1y and 2y are mean

values of signal intensities of two selected spectra; n represents the data points in the selected

wavenumber region. The Dy1y2 ranges from 0 to 2000. The smaller values define good

reproducibility of spectra. Zero means the two spectra are identical; One thousand means that

the two spectra are totally unrelated. Two thousand means the two spectra are negatively

related.

ry1y2=∑∑

∑

==

=

−−

−

n

ii

n

ii

n

iii

ynyyny

yynyy

1

22

22

1

21

21

12121

(1)

Dy1y2 =（1- ry1y2）1000 (2)

Second derivative transforms (with a gap value of 10 cm-1) were performed for spectral

processing in Matlab. Principal component analysis (PCA) was employed . PCA is an

unsupervised chemometric tool to reduce the dimensionality of multivariate data while

preserving most of the variances and provides a two or three dimensional cluster of results for

group segregation (Lu et al., 2010).

2.9 Statistical analysis.

All experiments were repeated three times with duplicate samples. Data were analyzed by

one-way analysis of variance (ANOVA, P≤0.05) followed by T-test using Matlab.

32

3 Results and Discussion

3.1 C. sakazakii inactivation effect of UV radiation

UV radiation for 25 min of dry infant formula was the most effective treatment with a

1.38 log10 CFU reduction per gram. A curve of C.sakazakii survival population versus UV

radiation treatment time was plotted (Figure 2). An S-shaped survival curve was observed

with initial rapid inactivation in the first five minutes followed by a period of rapid

inactivation at 25-30 min. The initial rapid inactivation resulted from the UV radiation

exposure and the second rapid inactivation may result from the raised temperature in the

chamber after 20 min. After 20 min of UV radiation treatment, the temperature in UV

chamber was about 37oC. Complete inactivation was not be achieved because the UV

radiation did not completely penetrate the dry infant formula.

The germicidal effect of UV radiation is influenced by food matrices and the

physiological state of bacteria. Although UV radiation is well established for sanitation of air,

water, liquid food pasteurization and surface decontamination, UV radiation of powdered

food was investigated. U.S. FDA recommendations state that to achieve a four-log microbial

inactivation, the UV radiation exposure must be at least 400 J/m2 for all parts of the product

(U.S.FDA, 2009). In this experiment, a 25 min UV radiation treatment led to a radiation dose

of ca 60.7 kJ/m2 (Table 6). The radiation dose required for liquid food listed by FDA is too

small for food powders. Compared to water, powdered food exhibits a range of optical and

physical properties and diverse chemical composition that influences UV transmittance, dose

delivery, momentum transfer and consequently microbial inactivation (Koutchma, 2009). The

transmittance of UV radiation in infant formula is much smaller compared to transmittance in

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liquid food. Infant formula particles absorb and scatter UV radiation due to chemical

composition, optical properties and shape. Moreover, the large clumps formed during

inoculation can provide a site for the aggregation of bacteria in the core of the clump

surrounded by infant formula particles (Koutchma, 2009). The clumps protect C. sakazakii

cells from UV radiation. In infant formula processing, UV radiation is preferred as a post

pasteurization process to avoid C. sakazakii contamination resulting from poor environment

hygiene (Johler et al., 2010)

Sensitivity of microorganisms to UV radiation also influences the efficacy of bacteria

inactivation. Cell wall structure, thickness and composition, absorbent compounds and

nucleic acid structure are important to bacterial sensitivity exposed to UV radiation

(Koutchma, 2009). C. sakazakii in the stationary phase produce carotenoids known to

stabilize cellular membranes, influence cellular membrane fluidity, scavenge reactive oxygen

species, and play a role in the survival of C. sakazakii in stressful environments (Gruszecki &

Strzalka, 2005). Johler et al. (2005) identified genes involved in pigment expression in C.

sakazakii strain ES 5 and reported mutants that cannot produce yellow pigmentation were

more sensitive to UVB radiation than wild type C. sakazakii. Lehner et al. (2006) applied

bacterial artificial chromosome approach and heterologous expression of the yellow pigment

in Escherichia coli to elucidate the molecular structure of the genes responsible for pigment

production in C. sakazakii ES 5. Lehner et al. (2006) ascertained the carotenogenic nature of

the pigment by in situ visible microspectroscopy and resonance Raman microspectroscopy.

Besides, trehalose, which improves desiccation tolerance of C. sakazakii may also contribute

to UV radiation. Trehalose acts as a counterbalance to extracellular osmotic pressure,

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stabilizing phospholipid membranes and proteins by replacing the shell of water surrounding

the membranes and proteins preventing irreversible damage of the cells (Mullane, 2006a).

After UV radiation treatments of 20 min with UV dose of 48.6 kJ/m2, the reduction of viable

microorganism on OK medium was 0.69, 0.25 and 0.07 log 10 CFU/g infant formula with an

infant formula layer of 0.032, 0.16 and 0.32 cm, respectively (Figure 3). Layer thickness is

an important factor influencing UV radiation inactivation efficacy. The thincker the layer was,

the more bacteria survived through UV radiation treatment. Oteiza et al. (2005) explained the

effect of apple juice thickness on efficacy of UV radiation as a bactericide with the Lambert-

Beer Law:

P= P0 exp(-abc).

When UV radiation power of P0 penetrates a liquid, the food matrix absorbs and scatters UV

radiation so the transmitted radiation power is reduced to P. The transmittance of a solution is

the fraction of incident radiation transmitted by the solution, T=P/ P0 (Oteiza, 2005). In this

equation, a is the specific absorptivity (l /mol/ cm), b is the distance the radiation travels

through the sample (cm), c is the particle concentration of the solution (mol/l) (Oteiza, 2005).

Due to the exponential nature of the Lambert-Beer law, the transmittance power is reduced

rapidly with an increase in the travel distance of UV radiation. As a result of UV radiation

power attenuation, the bactericidal effect of UV radiation is reduced dramatically as the

clumping of the infant formula increased.

3.2 Combined treatments of UV radiation and heat inactivation C. sakazakii in reconstituted infant formula

Inactivation effect of C. sakazakii in dry infant formula using UV radiation and

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reconstituted in 55oC, 60oC or 65oC sterilized hot water was evaluated. Table 7 presents D

values of C.sakazakii contaminating infant formula subjected to hot water treatment only and

C. sakazakii contaminating infant formula subjected to UV radiation and hot water treatment.

D values for C. sakazakii culture cocktail contaminating infant formula reconstituted with 55,

60 or 65oC water were 3.99±1.99, 0.87±0.11, 1.10±0.55 min, respectively. D values obtained

in this experiment were smaller than D values in previous studies. Al-Holy et al. (2009)

reported D55 values for C. sakazakii ATCC 12868 and ATCC 29544 in reconstituted infant

formula were 14.21±3.58 and 14.83±3.50 min. D60 values for C. sakazakii ATCC 12868 and

ATCC 29544 in reconstituted infant formula were 0.53±0.08 and 2.71±0.32 min in

reconstituted infant formula (Al-Holy et al., 2009). Inoculating C. sakazakii cocktail culture

into dry infant formula and air drying may result in death and injury of desiccation sensitive

cells (Edelson-Mammel, 2005).

D values for C.sakazakii in reconstituted infant formula after UV radiation treatment are

1.88±1.05, 1.00±0.06 and 0.89±0.10 at 55, 60 and 65oC. Compared to D55 and D65 values for

C. sakazakii in reconstituted infant formula subjected to hot water treatments only, D55 and

D65 values decreased (Figure 4 a c).

Reconstituting dry infant formula with 70-90oC water can contribute a four to six log

reduction of C. sakazakii (Chen et al., 2009). The highest water temperature tested was less

than 70oC and was not achieved ideal C. sakazakii inactivation effect before cooling down.

UV radiation treatment before reconstitution may be a method to increase the hot water

treatment effect.

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3.3 FT-IR spectroscopy decoding and cluster analysis.

Due to interference of fat in dry infant formula, non-fat milk powder was used for

spectral analysis. The fat content of dry infant formula is 24.9% w/w and 0 in non fat milk

powder. Traditional microbiological experiment was performed to validate that there was no

significant difference (P < 0.05) of bacterial inactivation by UV radiation treatment between

infant formula powder and non-fat milk powder. Figure 5 presents the raw FT-IR spectral

features of non-fat milk powder and non-fat milk powder inoculated with C. sakazakii.

Distinct differences on raw spectra were observed and provided prerequisite for subtraction

control spectra form sample spectra (Lu and Rasco, 2010). The intra-group variation of

spectral features is significantly (p < 0.05) smaller than the inter-group variation of spectral

features. D values were calculated for each group and ranged between 36.93 ± 15.13 and

76.32 ± 31.4, which demonstrates good reproducibility of spectral features of each sample.

The differences of raw FT-IR spectra for UV radiation treated and non-treated C.

sakazakii contaminating dry infant formula were not visually discernible. Second derivative

transformation of raw spectra was applied to magnify small variability (Figure 6 a, b). The

peak at 970 cm-1 is assigned to the symmetric stretching mode of dianionic phosphate

monoesters in cellular nucleic acids (Argov et al., 2004). The peak at 1080 cm-1 is assigned to

symmetric PO2- of nucleic acids (Wood et al., 1998). The peak at 1240 cm-1 is assigned to

P=O stretching (asymmetric) of > PO2- phosphodiesters from nucleic acids (Naumann, 2001).

Spectral bands at 1080 and 1240 cm-1 reflect the functional groups information of DNA. The

peak at 1317 cm-1 is assigned to amide III components of proteins (Yang et al., 2005). The

peak at 1515 cm-1 is assigned to amide II (Lu et al., 2010a). The peak at 1637 cm-1 is assigned

to amide I of β-pleated sheet structures (Naumann, 2001). The peak at 1655 cm-1 is assigned

37

to amide I of α-helical structures (Lu and Rasco, 2010). The amide bands provide information

about α-helix, β sheet and random coil conformations in proteins. The peak at 1400 cm-1 is

assigned to C=O symmetric stretching of COO- in proteins (Naumann, 2001). The peak at

1455 cm-1 is assigned to symmetric bending modes of methyl groups in skeletal proteins

(Fung et al., 1996). The peak at 1740 cm-1 is assigned to >C=O stretching of esters (Naumann,

2001). Those bands at 1317, 1400, 1455, 1515, 1637, 1655 and 1740 cm-1 are related to

protein secondary structure (Figure 6a). The peak at 2850 cm-1 is assigned to C-H

symmetric stretching of >CH2 in fatty acids (Naumann, 2001). The peak at 2918 cm-1 is

assigned to C-H asymmetric stretching of >CH2 in fatty acids (Lu and Rasco, 2010). These

peaks at 2850 and 2918 cm-1 are related to fatty acids in bacterial cell membranes (Figure

6b). Spectral variations indicated that the function of protein, lipid and DNA are closely

associated with bacterial survival under treatment of UV readiation.

Principal component analysis (PCA) was employed to segregate control C. sakazakii

extracts from UV treated extracts (Figure 7). Cells from the control treatment were tightly

clustered, but variations were observed among UV treatments indicating differences in the

degree of cell injury.

Infrared spectroscopy was useful to monitor variability in cell membrane composition

that is associated with bacterial injury and survival. Lin et al. (2004) discriminated intact L.

monocytogenes cells from sonication-injured cells by ATR spectroscopy using PCA

segregation clusters, noting that injury was attributed to macromolecular shearing and

subsequent redistribution of cell wall components along with possible denaturation of

intracellular proteins. Lu et al. (2010b) employed FT-IR coupled with loading plot analysis

38

and PCA segregation to sort bacterial injury populations after cold and freeze treatments and

demonstrated that pathogens produce oligosaccharides and potentially other components in

response to stress. Alvarez-Ordóñez and Prieto (2010) used FT-IR and studied the

ultrastructure changes of Salmonella enterica cells under acid, alkaline, heat and oxidative

stressed conditions. A wide variety of cellular compounds involved in bacterial resistance to

unfavorable conditions (Al-Holy et al., 2006; Al-Qadiri et al., 2008).

3.4 Electron microscopy analysis.

No detectable changes of cell surface were clearly delineated in scanning electron

micrographs of UV radiation treated C. sakazakii (Figure 8). The lack of clearly delineated

differences on cell surfaces was indicated little UV damage following UV radiation treatment.

Cells were not clearly delineated in transmission electron micrographs based on treatment

(Figure 9). The lack of no clearly delineated cells may be attributed to decomposition of cells

from UV radiation. Casein particles were visible in C. sakazakii micrographs.

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4 Conclusions

In this study, UV radiation dose in a range of 12.1 to 72.8 kJ/m2 inactivated C.

sakazakii in dry infant formula. UV radiation of 60.7 kJ/m2 resulted in a 1.38 log10 CFU/g

reduction at room temperature. UV dose required by FDA to inactivate bacteria in liquid food

is too low for desiccation food powder. The UV radiation in combination with 55oC and 65oC

hot water treatment decreases D values of C. sakazakii in reconstituted infant formula. FT-IR

analysis suggested UV radiation results in structural changes in protein, DNA and lipids of

cell injury. Electron microscopy is not an effective method to investigate the cell injury

resulting from UV radiation. A high energy radiation is considered as a pasteurization method

to inactivate C. sakazakii in dry infant formula (Lee et al., 2006). Photocatalyst can be added

into food to increase UV radiation inactivation effect on foodborne pathogen (Benabbou et al.,

2007). UV penetration material should be developed to increase UV transmittivity.

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