Attachment of and Biofilm Formation by Enterobacter sakazakii on Stainless Steel and Enteral Feeding Tubes

ABSTRACT Enterobacter sakazakii has been reported to form biofilms, but environmental conditions affecting attachment to and biofilm formation on abiotic surfaces have not been described. We did a study to determine the effects of temperature and nutrient availability on attachment and biofilm formation by E. sakazakii on stainless steel and enteral feeding tubes. Five strains grown to stationary phase in tryptic soy broth (TSB), infant formula broth (IFB), or lettuce juice broth (LJB) at 12 and 25ï¿½C were examined for the extent to which they attach to these materials. Higher populations attached at 25ï¿½C than at 12ï¿½C. Stainless steel coupons and enteral feeding tubes were immersed for 24 h at 4ï¿½C in phosphate-buffered saline suspensions (7 log CFU/ml) to facilitate the attachment of 5.33 to 5.51 and 5.03 to 5.12 log CFU/cm2, respectively, before they were immersed in TSB, IFB, or LJB, followed by incubation at 12 or 25ï¿½C for up to 10 days. Biofilms were not produced at 12ï¿½C. The number of cells of test strains increased by 1.42 to 1.67 log CFU/cm2 and 1.16 to 1.31 log CFU/cm2 in biofilms formed on stainless steel and feeding tubes, respectively, immersed in IFB at 25ï¿½C; biofilms were not formed on TSB and LJB at 25ï¿½C, indicating that nutrient availability plays a major role in processes leading to biofilm formation on the surfaces of these inert materials. These observations emphasize the importance of temperature control in reconstituted infant formula preparation and storage areas in preventing attachment and biofilm formation by E. sakazakii.

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Attachment and Biofilm Formation by Escherichia coli O157:H7 on Stainless Steel as Influenced by Exopolysaccharide Production, Nutrient Availability, and Temperature

Journal of Food Protection ◽

10.4315/0362-028x-67.10.2123 ◽

2004 ◽

Vol 67 (10) ◽

pp. 2123-2131 ◽

Cited By ~ 62

Author(s):

JEE-HOON RYU ◽

HOIKYUNG KIM ◽

LARRY R. BEUCHAT

Keyword(s):

Escherichia Coli ◽

Stainless Steel ◽

Nutrient Availability ◽

Biofilm Formation ◽

Escherichia Coli O157 ◽

Strain Atcc ◽

Wild Type ◽

E Coli ◽

Nutrient Environment ◽

Phosphate Buffered Saline

The influence of exopolysaccharide (EPS) production, nutrient availability, and temperature on attachment and biofilm formation by Escherichia coli O157:H7 strains ATCC 43895 (wild type) and 43895-EPS (extensive EPS-producing mutant) on stainless steel coupons (SSCs) was investigated. Cells grown on heated lettuce juice agar and modified tryptic soy agar were suspended in phosphate-buffered saline (PBS). SSCs were immersed in the cell suspension (109 CFU/ml) at 4°C for 24 h. Biofilm formation by cells attached to SSCs as affected by immersing in 10% tryptic soy broth (TSB), lettuce juice broth (LJB), and minimal salts broth (MSB) at 12 and 22°C was studied. A significantly lower number of strain 43895-EPS cells, compared to strain ATCC 43895 cells, attached to SSCs during a 24-h incubation (4°C) period in PBS suspension. Neither strain formed a biofilm on SSCs subsequently immersed in 10% TSB or LJB, but both strains formed biofilms in MSB. Populations of attached cells and planktonic cells of strain ATCC 43895 gradually decreased during incubation for 6 days in LJB at 22°C, but populations of strain 43895-EPS remained constant for 6 days at 22°C, indicating that the EPS-producing mutant, compared to the wild-type strain, has a higher tolerance to the low-nutrient environment presented by LJB. It is concluded that EPS production by E. coli O157:H7 inhibits attachment to SSCs and that reduced nutrient availability enhances biofilm formation. Biofilms formed under conditions favorable for EPS production may protect E. coli O157:H7 against sanitizers used to decontaminate lettuce and produce processing environments. Studies are under way to test this hypothesis.

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Biofilm Formation and Low pH Viability of Cronobacter spp. (Enterobacter sakazakii) Isolated from Powdered Infant Formula and Infant Foods in Korea

Korean Journal for Food Science of Animal Resources ◽

10.5851/kosfa.2009.29.6.702 ◽

2009 ◽

Vol 29 (6) ◽

pp. 702-708 ◽

Cited By ~ 7

Author(s):

Sun-Ae Kim ◽

Yu-Mi Lee ◽

Se-Wook Oh ◽

Hyo-Sun Gwak ◽

In-Gyun Hwang ◽

...

Keyword(s):

Infant Formula ◽

Biofilm Formation ◽

Low Ph ◽

Powdered Infant Formula ◽

Enterobacter Sakazakii ◽

Infant Foods

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Incidence, Survival, and Growth of Enterobacter sakazakii in Infant Formula

Journal of Food Protection ◽

10.4315/0362-028x-60.3.226 ◽

1997 ◽

Vol 60 (3) ◽

pp. 226-230 ◽

Cited By ~ 137

Author(s):

MARIA NAZAROWEC-WHITE ◽

JEFFREY M. FARBER

Keyword(s):

Infant Formula ◽

Severe Form ◽

Neonatal Meningitis ◽

Enterobacter Sakazakii ◽

Potential Growth ◽

Lack Of Information ◽

Generation Times ◽

Sporadic Cases ◽

Survival And Growth ◽

And Storage

Enterobacter sakazakii has been implicated in a severe form of neonatal meningitis. Although studies have failed to identify an environmental source for the organism, dried infant formula has been implicated in outbreaks and sporadic cases of E. sakazakii meningitis. The high mortality rate (50 to 75%), the severity of the infection in infants, and the lack of information on the incidence, survival, and growth of E. sakazakii in foods led to this study. Experiments were undertaken to determine the incidence of E. sakazakii in dried infant formula, the temperature range for growth, and the growth characteristics of E. sakazakii in reconstituted dried infant formula. Strains of E. sakazakii were isolated from dried infant formula available on the Canadian retail market. The prevalence varied from 0 to 12% in samples from five different companies. For both clinical and food isolates, minimum growth temperatures of 5.5 to 8.0°C were observed by using a temperature-gradient incubator. The potential growth of E. sakazakii was followed by using a mixture of food and clinical isolates in three different formulas incubated at 4, 10, and 23°C. Average generation times were 40 min at 23°C and 4.98 h at 10°C. E. sakazakii strains did not grow at 4°C and began to die off during storage at this temperature. The results of this study stress the importance of using aseptic methods and proper temperature control in the preparation, use, and storage of dried infant formula.

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Effect of trans-Cinnamaldehyde on Inhibition and Inactivation of Cronobacter sakazakii Biofilm on Abiotic Surfaces

Journal of Food Protection ◽

10.4315/0362-028x.jfp-10-296 ◽

2011 ◽

Vol 74 (2) ◽

pp. 200-208 ◽

Cited By ~ 48

Author(s):

MARY ANNE ROSHNI AMALARADJOU ◽

KUMAR VENKITANARAYANAN

Keyword(s):

Quantitative Pcr ◽

Infant Formula ◽

Reverse Transcription ◽

Biofilm Formation ◽

Feeding Tube ◽

Cronobacter Sakazakii ◽

Cinnamon Oil ◽

Abiotic Surfaces ◽

Expression Of Genes ◽

Reverse Transcription Quantitative Pcr

The efficacy of trans-cinnamaldehyde (TC), an ingredient in cinnamon oil, for inhibiting biofilm synthesis (560 and 750 μM TC) and inactivating mature biofilms (23 and 38 mM TC) of Cronobacter sakazakii was investigated at 24 and 12°C in the presence and absence of reconstituted infant formula on polystyrene plates, stainless steel coupons, feeding bottle coupons, and enteral feeding tube coupons. Additionally, TC's effect on the expression of genes critical for biofilm formation in C. sakazakii was determined by reverse transcription quantitative PCR. TC inhibited and inactivated C. sakazakii biofilms on all matrices tested at both temperatures. C. sakazakii was reduced by >4.0 and 3.0 log CFU/ml after 96 h of exposure to 38 mM and 750 μM TC, respectively. Reverse transcription quantitative PCR results revealed that TC significantly (P ≤ 0.05) down-regulated biofilm-associated genes in C. sakazakii. TC could potentially be used to control C. sakazakii biofilms on infant formula feeding equipment and preparatory areas.

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Effectiveness of Disinfectants in Killing Enterobacter sakazakii in Suspension, Dried on the Surface of Stainless Steel, and in a Biofilm

Applied and Environmental Microbiology ◽

10.1128/aem.01766-06 ◽

2006 ◽

Vol 73 (4) ◽

pp. 1256-1265 ◽

Cited By ~ 73

Author(s):

Hoikyung Kim ◽

Jee-Hoon Ryu ◽

Larry R. Beuchat

Keyword(s):

Stainless Steel ◽

Infant Formula ◽

Exposure Time ◽

Day Care ◽

Food Service ◽

Organic Matrix ◽

Enterobacter Sakazakii ◽

Planktonic Cells ◽

Day Care Centers ◽

Organic Matrices

ABSTRACT The effectiveness of 13 disinfectants used in hospitals, day-care centers, and food service kitchens in killing Enterobacter sakazakii in suspension, dried on the surface of stainless steel, and in biofilm was determined. E. sakazakii exhibited various levels of resistance to the disinfectants, depending on the composition of the disinfectants, amount and type of organic matrix surrounding cells, and exposure time. Populations of planktonic cells suspended in water (7.22 to 7.40 log CFU/ml) decreased to undetectable levels (<0.30 log CFU/ml) within 1 to 5 min upon treatment with disinfectants, while numbers of cells in reconstituted infant formula were reduced by only 0.02 to 3.69 log CFU/ml after the treatment for 10 min. The presence of infant formula also enhanced the resistance to the disinfectants of cells dried on the surface of stainless steel. The resistance of cells to disinfectants in 6-day-old and 12-day-old biofilms on the surface of stainless steel was not significantly different. The overall order of efficacy of disinfectants in killing E. sakazakii was planktonic cells > cells inoculated and dried on stainless steel > cells in biofilms on stainless steel. Findings show that disinfectants routinely used in hospital, day-care, and food service kitchen settings are ineffective in killing some cells of E. sakazakii embedded in organic matrices.

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Biofilm formation on enteral feeding tubes by Cronobacter sakazakii, Salmonella serovars and other Enterobacteriaceae

International Journal of Food Microbiology ◽

10.1016/j.ijfoodmicro.2009.08.007 ◽

2009 ◽

Vol 136 (2) ◽

pp. 227-231 ◽

Cited By ~ 56

Author(s):

E. Hurrell ◽

E. Kucerova ◽

M. Loughlin ◽

J. Caubilla-Barron ◽

S.J. Forsythe

Keyword(s):

Enteral Feeding ◽

Biofilm Formation ◽

Cronobacter Sakazakii ◽

Feeding Tubes ◽

Salmonella Serovars

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Fate of Enterobacter sakazakii Attached to or in Biofilms on Stainless Steel Upon Exposure to Various Temperatures or Relative Humidities

Journal of Food Protection ◽

10.4315/0362-028x-71.5.940 ◽

2008 ◽

Vol 71 (5) ◽

pp. 940-945 ◽

Cited By ~ 47

Author(s):

HOIKYUNG KIM ◽

JIHYUN BANG ◽

LARRY R. BEUCHAT ◽

JEE-HOON RYU

Keyword(s):

Stainless Steel ◽

Relative Humidity ◽

Infant Formula ◽

Storage Temperature ◽

Enterobacter Sakazakii ◽

Steel Surfaces

Survival of Enterobacter sakazakii dried on the surface of stainless steel and exposed to 43% relative humidity, as affected by temperature, was studied. Populations of E. sakazakii (7.4 to 8.6 log CFU per coupon) on coupons dried for 2 h at 22°C decreased significantly (P ≤ 0.05) at 4, 25, and 37°C within 10, 3, and 1 day(s), respectively, but the pathogen remained viable for up to 60 days. At a given storage temperature and time, reductions were significantly greater when cells had been suspended in water rather than in infant formula before drying. Formation of biofilm by E. sakazakii on stainless steel immersed in M9 medium, which contains minimal concentrations of nutrients, and infant formula at 25°C and subsequent survival of cells at 25°C as affected by exposure to 23, 43, 68, 85, and 100% relative humidity were investigated. Some of the cells in these biofilms survived under all test relative humidities for up to 42 days. The overall order of survival as affected by relative humidity was 100 > 23 = 43 = 68 > 85% relative humidity, regardless of the medium in which the biofilm was formed. Reduction in viability of cells was significantly greater in biofilm that had formed in M9 medium than in biofilm formed in infant formula. Results indicate that infant formula provides protection for attached cells, as well as cells in biofilm, against lethality on exposure to desiccation. These results are useful when predicting the survival characteristics of E. sakazakii on stainless steel surfaces in processing and preparation kitchen environments.

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