Crystal violet binding assay for assessment of biofilm formation by isolates of Listeria monocytogenes and Listeria spp from a typical tropical abattoir on wood, steel and glass surfaces.

Abstract

Microorganisms attach to surfaces and develop biofilms which are a concern in food and environmental safety. This study, quantified biofilms in 20 isolates viz: Listeria monocytogenes (14) and Listeria spp (6) from meat tables in Ibadan municipal abattoir, Nigeria on wood, glass and steel surfaces using crystal-violet binding assay. Listeria isolates formed biofilms on all three surfaces. At 24hours Listeria monocytogenes(SLM) formed more biofilms on wood(0.22±0.01) and glass(0.09±0.01) surfaces than Listeria spp(SLS) which was wood(0.17±0.02) and glass (0.06±0.01). But the reverse was the case on steel where Listeria spp (SLS) formed more biofilms (0.11±0.01) than Listeria monocytogenes (SLM) (0.10±0.00). A significant difference (P<0.01) exists between isolates for wood and glass at 24hours and (P<0.05) for wood at 72hours. Significant differences (P<0.05) occured in biofilms produced between 24hours and 72hours. It can be concluded that selection of suitable surfaces could reduce the ability of bacteria forming biofilms on meat contact-surfaces. This finding is very relevant in developing countries where wood is still in use as a major meat contact-surface in abattoirs.

Full text

Global Veterinaria 6 (1): 06-10, 2011
ISSN 1992-6197
© IDOSI Publications, 2011
Corresponding Author: Victoria O. Adetunji, Department of Veterinary, Public Health and Preventive Medicine,
University of Ibadan, Ibadan, Nigeria, E-mail: vadetunji@gmail.com & vo.adetunji@mail.ui.edu.ng.
6
Crystal Violet Binding Assay for Assessment of Biofilm Formation
by Listeria monocytogenes and Listeria spp on Wood, Steel and Glass Surfaces
Victoria O. Adetunji and Tajudeen O. Isola
Department of Veterinary, Public Health and Preventive Medicine, University of Ibadan, Ibadan, Nigeria
Abstract: Micro-organisms attach to surfaces and develop biofilms which are a concern in food and
environmental safety. This study, quantified biofilms in 20 isolates viz: Listeria monocytogenes (14) and
Listeria spp (6) from meat tables in Ibadan municipal abattoir, Nigeria on wood, glass and steel surfaces using
crystal-violet binding assay. Listeria isolates formed biofilms on all three surfaces. At 24hours Listeria
monocytogenes(SLM) formed more biofilms on wood(0.22±0.01) and glass(0.09±0.01) surfaces than Listeria
spp(SLS) which was wood(0.17±0.02) and glass (0.06±0.01). But the reverse was the case on steel where Listeria
spp (SLS) formed more biofilms (0.11±0.01) than Listeria monocytogenes (SLM) (0.10±0.00). A significant
difference (P<0.01) exists between isolates for wood and glass at 24hours and (P<0.05) for wood at 72hours.
Significant differences (P<0.05) occured in biofilms produced between 24hours and 72hours. It can be
concluded that selection of suitable surfaces could reduce the ability of bacteria forming biofilms on meat
contact-surfaces. This finding is very relevant in developing countries where wood is still in use as a major meat
contact-surface in abattoirs.
Key words: Biofilm
Listeria spp Abattoir Meat Contact-surfaces
INTRODUCTION Various methods have been used for biofilm
Nearly 99% of micro-organisms living on the earth assay as described by Stepanovic and others [4, 22]. In
live in microbial communities known as biofilms [1]. this assay, the dye bound to adherent cells is
Biofilms are formed by adhesion of bacterial cells to resolubilized and measured in optical density (OD).
surfaces through an exopolymeric matrix. This matrix is Listeria spp are gram positive bacilli and are
important in formation and structure of the biofilm and typified by Listeria monocytogenes, the causative
also on the protection of the bacterial cells as it prevents agent of listeriosis. Clinical infections are primarily by
antimicrobials and xenobiotics from gaining access to Listeria monocytogenes although Listeria ivanovii is
the cells inside the biofilm [2]. Biofilm formation can also pathogenic being particularly associated with
compromise the cleanliness of food contact surfaces and abortion in ruminants. The remaining species are
environmental surfaces by spreading detached organisms recognized as non pathogenic. Listeria monocytogenes is
to food and other areas of processing plants [3]. Biofilms quite hardy and resists dexterous effects of freezing,
formed in food processing environments are important drying and heat remarkably well for a bacterium that does
as they have the potential to act as a chronic source of not form spore [7].
microbial contamination that eventually leads to food Listeria’s tolerance has attributed to it being
spoilage and transmission of diseases [1, 4]. Biofilms lead isolated from various meat samples, with chicken
to serious hygienic problems and hence, the important being the best host for growth of the pathogen [8].
aspect of controlling biofilms represents one of the Listeriosis is contracted through the consumption of
most persistent challenges within food and industrial contaminated foods [9]. Listeria monocytogenes is
environments where microbial communities are found in a variety of food products such as soft
problematic [5]. Biofilm forming bacteria persist and cheeses, dairy products raw foods, ready to eat
produce after sanitation thus making biofilms a potential products and equipment surfaces [10, 11]. Listeria
threat to food safety [6]. monocytogenes is a food borne pathogen that
assessment; one of these is the crystal violet binding

Global Veterinaria, 6 (1): 06-10, 2011
7
account for less than 1% of food borne illness but is from the broth culture for biofilm quantification using the
responsible for 28% of deaths caused by food borne crystal violet binding assay described by Stepanovic et
diseases [12]. al. [4]. Each set of chips was washed 3 times with 5ml of
Several studies have been conducted to determine sterile distilled water. The remaining adhered bacteria were
the ability of Listeria monocytogenes to adhere and form fixed with 2.5ml of methanol per chip. Each chip was
biofilms on food contact surfaces. All of which stained with crystal violet (Fishers scientific, USA) for 15
determined that L. monocytogenes attach to food industry minutes and then the excess stain washed off under
surfaces such as plastics, glass, stainless steel and rubber running tap water. After the chip was air dried, the dye
surfaces [13-20]. However, it has been noted that there are bound to the adherent cells was re- solubilized with 2.5ml
differences in both the extent and the rate of adsorption. of 33% glacial acetic acid (Fishers scientific, USA) for
Luden et al [21] demonstrated that the most prevalent each chip. The re-solubilized liquid for each chip was
strain of L. monocytogenes (strain 1/ c) found in food poured into a cuvette. The absorbance (optical density)
2
processing plants has good adhesion ability and required of each re-solubilized liquid was measured against the
only a short contact time for attachment. optical density of blank reading without inoculation
This work presents an assessment of biofilms formed (control) at wavelength of 620nm for the Listeria
by isolates of Listeria monocytogenes and Listeria spp strains using a spectrophotometer (Springfield, UK). The
from a tropical abattoir on wood, glass and steel surfaces absorbance of negative control was subtracted from the
using the crystal violet binding assay. absorbance values to determine the actual values [23].
MATERIALS AND METHODS Statistical Analysis: All results were presented in
Preparation of Listeria Isolates Culture for Attachment analysis of variance (ANOVA) was used to determine
and Biofilm: Listeria monocytogenes isolates (SLM 14)
and Listeria spp (SLS 6) were isolated from meat tables in
Ibadan municipal abattoir, Nigeria. Modified oxford agar
base (MOX) for Listeria isolation was supplemented with
antibiotic supplements (acriflavine, nalidixic acid and
cycloheximide) (Becton, Dickinson and company) was
used for Listeria isolation. Further identification of the
isolates was based on procedures described by Barrow
and Feltham [22]. One colony of each Listeria isolates
was cultured on nutrient agar slope (Fluka 7014, Germany)
and was transferred into each of the 20 sterile glass jars
with lids containing 150ml of nutrient broth (Fluka 7014,
Germany). One glass jar not inoculated with Listeria
isolate served as control in this study. The inoculated
broth culture was incubated at 37°C for 24hours. Two
hundred and seventy-six (276) chips (90 each (4cm x 2cm
x 1cm) for glass (Easy way medical, England), steel (type
304, #4, Ajaokuta steel co., Nigeria) and wood (softwood,
Oak) were used. The chips were washed with detergent
(Unilever, Lagos, Nigeria), rinsed with sterile distilled
water and air-dried before being placed into hot air oven
(Elektro-Helios, Sweden) at 75°C for 30mins. After 24hours
incubation of the inoculated broth culture, four of steel,
glass and wood chips were aseptically put into each of
the glass jars making a total of 12 chips per glass jar. All
the glass jars were incubated at ambient temperature of
(26-28°C for 24 hours and 72 hours. At the end of each
)
incubation period, a set of chips were aseptically removed
mean±standard error of mean (Mean±SEM). A one-way
significant differences between means for each surface
and strains. Student t- test was used to assess significant
differences between strains. All data were analyzed using
SPSS 15 [24], Chicago and IL., USA. Statistical
significance was evaluated at P<0.05 and P<0.01. Charts
were plotted using Microsoft Excel [25].
RESULTS
The mean absorbance values at 24hours showed
that Listeria monocytogenes (SLM) (Figure 1) formed
more biofilms on wood (0.22±0.01) and glass (0.09±0.01)
surfaces than Listeria spp (SLS) which was wood
(0.17±0.02) and glass (0.06±0.01). But the reverse
was the case on steel where Listeria spp (SLS) formed
more biofilms (0.11±0.01) than Listeria monocytogenes
(SLM) (0.10±0.00) respectively (Table 1). The mean
absorbance value at 72hours showed that Listeria
monocytogenes (SLM) formed more biofilms on wood
(0.35±0.01) and glass (0.12±0.01) surfaces than Listeria
spp (SLS) which was wood (0.28±0.01) and glass
(0.11±0.01) respectively (Table 1). The mean absorbance
values (nm) of biofilm adherence to surfaces were
observed at 24 and 72hours on wood more significantly
(P<0.05) than on glass and steel surfaces for the isolates
except Listeria spp at 24hours where there were
significant differences (P<0.05) in biofilm formation on the
three surfaces (Table 1).

Global Veterinaria, 6 (1): 06-10, 2011
8
Fig. 1: Showing differences in biofilms produced by harveyi. Cunliffe et al. [33]; Sinde and Carballo [19];
SLM-Listeria monocytogenes and SLS-Listeria Donlan [34] also reported that Listeria monocytogenes
spp on the 3 surfaces at 24 and 72hours adhere in higher numbers to more hydrophobic materials.
Table 1: Comparison of Listeria biofilms on wood, steel and glass surfaces
at 24 and 72 hours incubation period
SURFACES
---------------------------------------------------------------
Strains Wood (mean Steel(mean Glass (mean
(Codes) Periods absorbance (nm) absorbance (nm) absorbance (nm)
SLM 24hrs 0.22±0.01 0.10±0.00 0.09±0.01
ab b
SLS 24hrs 0.17±0.02 0.11±0.01 0.06±0.01
ab c
SLM 72hrs 0.35±0.02 0.13±0.01 0.12±0.01
ab b
SLS 72hrs 0.28±0.03 0.13±0.01 0.11±0.00
ab b
* *Means with the same superscript are not significantly different at
0.05 levels
DISCUSSION
In this study, biofilm formation by Listeria
monocytogenes and Listeria spp revealed that these
bacteria possess a high capacity for biofilm formation on
the 3 surfaces used (wood, glass and steel) but with
differences in the extent of adhesion. Previous studies
have confirmed the biofilm formation by strains of
Listeria monocytogenes and Listeria spp on these
surfaces [13, 14, 19, 20, 26]. The higher level of biofilm
production by isolates of Listeria monocytogenes when
compared to Listeria spp on wood and glass at 24hours
has been explained in other studies done by Hood and
Zottola [13]; Gulsun et al. [27] where they reported that
the level of biofilm production of isolates is a virulence
characteristic of such isolates. In the current work, the
mean absorbance values (nm) of biofilms produced by
Listeria showed that wood retained the greatest biofilms
followed by steel and glass. This result is in agreement
with the report of Sinde and Carballo, [19]; Donlan [28]
who reported that glass and stainless steel are hydrophilic
materials while wood and plastic are hydrophobic
materials. Hydrophobic materials are reported as surfaces
that provide a greater bacterial adherence [29]. Fletcher
and Loeb [30] noted that large numbers of bacteria
attached to hydrophobic surfaces with little or no surface
charge and moderate numbers attached to hydrophobic
metals with positive charge or neutral charge and very few
attached to hydrophilic negatively charged substrates.
The importance of hydrophobicity in bacteria attachment
has also been highlighted by other investigators [31].
Variation in biofilm density depending on surface was
also reported by Karunasagar et al. [32] with Vibrio
Donlan [34] also reported that adhesion is the first step in
the complex process of biofilm formation.
The finding of significant differences (P < 0.05) in
biofilm formation between 24 and 72 hours incubation
periods for all the Listeria strains (Figure 1) in this study
has also been demonstrated by Moltz and Martin; [35];
Adetunji [36]; Adetunji and Adegoke [37] on stainless
steel chips and quantitative recovery of purified biofilm
and cellulose from culture respectively. In these studies
increase in biofilms formation with extension of incubation
period was reported.
Use of wood as a food contact surface has reduced
in the developed countries because it is a porous and
absorbent material where organic matter along with
bacteria can become entrapped; cross-contamination is a
main concern [38] but wood is still in use in the
developing countries for food processing because it's
readily available and cheap. The preferred surface for food
processing is glass. Glass is sometimes used for food
contact surfaces because of its smooth and corrosion-
resistant surface [39]. On the other hand, stainless steel
resists impact damage better than glass but is vulnerable
to corrosion, while rubber surfaces are prone to
deterioration and may develop surface cracks where
bacteria can accumulate [40]. Equally important in meat
contact surface is the cleanability of the surface once
bacteria forming biofilm have attached. The use of wood
tables for meat display should be discouraged in the
developing countries based on the findings in this study.
CONCLUSION
It is concluded that Listeria monocytogenes and
Listeria spp isolates from meat tables in the abattoir
under study are capable of forming biofilms on wood,

Global Veterinaria, 6 (1): 06-10, 2011
9
steel and glass surfaces but with some variations. 12. Pan, Y.F., J.R. Breidt and S. Kathariono, 2006.
The food processing units in the developing countries
should therefore consider the application of findings in
this study with a view to preventing biofilms in food
processing environment. Further studies will be necessary
to understand whether the ability to form biofilms may
influence the survival of strains in meat contact surfaces.
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