International Journal of Food Microbiology 407 (2023) 110389

Contents lists available at ScienceDirect

International Journal of Food Microbiology

journal homepage: www.elsevier.com/locate/ijfoodmicro

Assessment of biofilm, enzyme production and antibiotic susceptibility of

bacteria from milk pre- and post-pasteurization pipelines in Algeria
Nassima Didouh a, b, Medjahdi Khadidja a, b, c, Carla Campos d, Benedita Sampaio-Maia e, f,
Moussa Boudjemaa Boumediene a, b, Ricardo Araujo e, *
a
Université Abou Bekr Belkaid Tlemcen, Algeria
b
Laboratoire de Microbiologie Appliqué à l'Agroalimentaire au Biomédical et à l'Environnement, 13000 Tlemcen, Algeria
c
Université Hassiba Benbouali Chlef, Algeria
d
Instituto Português de Oncologia (IPO) do Porto Francisco Gentil, Porto, Portugal
e
Nephrology & Infectious Diseases R&D Group, INEB - Instituto de Engenharia Biomédica, i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto,
Porto, Portugal
f
Faculdade de Medicina Dentária, Universidade do Porto, Porto, Portugal

A R T I C L E I N F O A B S T R A C T

Keywords: Bacterial biofilm is a major concern of dairy industry due to its association with milk contamination and its
Biofilm derived products. Algerian pasteurized milk shelf-life does not exceed one day, which may reflect the high level
Pasteurization of contamination of this product and presence of extracellular enzymes such as lipases and proteases. This work
Milk
aimed to investigate the microbial biodiversity in milk-processing surfaces of a dairy plant in Algeria. Therefore,
MALDI-TOF
Enzyme production
stainless steel cylinders were placed in piping system of the dairy system before and after pasteurization of the
Antimicrobial susceptibility milk, being removed after 7 days, for biofilm maturation and microorganism isolation and identification by mass
Acinetobacter spectrometry.
Bacillus Fifty-nine Gram-positive isolates were identified, namely Bacillus altitudinis, Bacillus cereus, Bacillus pumilus,
Enterobacter Bacillus subtilis, Bacillus weithenstephanensis, Enterococcus casseliflavus, Enterococcus faecium, and Staphylococcus
Enterococcus epidermidis. In addition, twenty-four Gram-negative isolates were identified, namely Acinetobacter schindleri
Enterobacter cloacae, Enterobacter xiangfangensis, Leclercia adecarboxylata, and Raoultella ornithinolytica. Bacterial
isolates showed ability for production of extracellular enzymes, being 49 % capable of both proteolytic and
lipolytic activities. Milk isolates were tested for the ability to form biofilms on stainless steel. The cell numbers
recovered on plate count agar plates from stainless steel biofilms ranged from 3.52 to 6.92 log10 CFU/cm2, being
the maximum number detected for Enterococcus casseliflavus. Bacterial isolates showed intermediate and/or
resistant profiles to multiple antibiotics. Resistance to amoxicillin, cefoxitin and/or erythromycin was commonly
found among the bacterial isolates.

1. Introduction
The most widely consumed milk in the world is ultra-high temper­
ature (UHT) milk because of its long shelf life followed by pasteurized
milk. While pasteurized milk shelf-life was reported to be fourteen days
in the United States (Martin et al., 2018), Algerian pasteurized milk
shelf-life does not exceed one day, which may reflect the high level of
contamination of this product and presence of extracellular enzymes
such as lipases and proteases (Hales, 2018). The control of these spoilage
enzymes and their activity, milk can be engineered for extended shelf
life (Glantz et al., 2020). Algerian pasteurized milk is consumed by large
parts of the population, including less than 2 year-old infants, and this
fact may represent a health risk for consumers. To our knowledge, no
episodes of food poisoning have been reported after the consumption of
pasteurized milk in Algeria. The main reason for this being the fact the
product is usually boiled by consumers before consumption (Malek
et al., 2013). These heat treatments that food generally undergoes are
the cause of various chemical changes that affect their nutritional value
(Amine and Bourais, 2013).
The main source of contamination of dairy products is often

* Corresponding author at: Nephrology & Infectious Diseases R&D Group, i3S - Instituto de Investigação e Inovação em Saúde, R. Alfredo Allen 208, 4200-135
Porto, Portugal.
E-mail address: ricjparaujo@yahoo.com (R. Araujo).

https://doi.org/10.1016/j.ijfoodmicro.2023.110389
Received 17 May 2023; Received in revised form 1 September 2023; Accepted 2 September 2023
Available online 9 September 2023
0168-1605/© 2023 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
N. Didouh et al.
associated to the formation of biofilms on the surfaces of milk transport
pipes, milking containers, and accessories in the dairy industries (Srey
et al., 2013). In order to form biofilms, both viable and non-viable
bacteria adhere reversibly and irreversibly to surfaces and produce
extracellular polymeric substances for its protection (Donlan and Cos­
terton, 2002). Hence, the bacteria present in milk can generally form
biofilms in the canalization of dairy industry (Marchand et al., 2012)
Therefore, the quality and the safety of milk and its products can be
influenced negatively by established bacterial biofilms when new milk
passes through the pipelines (Austin and Bergeron, 1995). Such
contamination events are frequently related to obstinacy on poorly
maintained equipment of food industry, representing a hygiene issue
(Abdallah et al., 2014; Sosnowski et al., 2016) and an economic problem
(Bremer et al., 2006; Lee et al., 2016). The inhibition of biofilm for­
mation on stainless steel surfaces with a peptide-based coating can be
safely used in the dairy industry because it does not have an effect on the
technological properties of dairy products (Friedlander et al., 2019). The
over-use of antibiotics, either to prevent or supervise the diseases
associated to the dairy food by lacking other controlling measures, may
support the emergence of antibiotic resistant bacteria (Collignon and
McEwen, 2019). The transmission of antibiotic resistance genes (ARGs)
between humans and animals is mainly due to the indiscriminate use of
antibiotics in animal husbandry, especially in poultry, exerts a signifi­
cant impact on the accumulation of the resistoma in humans (Hu et al.,
2014, 2016). Then resistant bacteria results in a huge hazard for public
health (Holmes and Zadoks, 2011).
Human gut microbiome can be enriched by resistance genes associ­
ated to the consumption of certain food products, such as milk (Van den
Braak et al., 1998). According to Liu et al. (2020), a dramatic enrich­
ment of the bacterial populations present in raw marked milk and in­
crease in the abundance and frequency of ARG when the milk is stored at
room temperature. Metagenomic sequencing showed that raw milk
contained more ARGs than pasteurized milk, and a conjugation assay
documented active transmission of blaCMY-2, a ceftazidime resistance
gene present in raw milk-borne E. coli, across multiple bacterial species.
This work aimed to investigate the microbial biodiversity in milk-
processing surfaces of a dairy plant in Algeria. To follow the process
of biofilm formation, we built experiments with stainless steel cylinders
into the piping system of the dairy system for 7 days. Tracking biofilm
formation on stainless steel cylinders helped to identify bacterial species
that may also trigger regular biofilm formation on the equipment
commonly used in the dairy environment. Samples were collected before
and after pasteurization cycles in both cold and hot seasons. The isolated
microorganisms were identified by matrix-assisted laser desorption/
ionization time-of-flight (MALDI-TOF), the biofilm-forming capabilities
assessed by measuring their ability to attach onto polystyrene and
stainless steel and the susceptibility to multiple antibiotics evaluated.
2. Material and methods
2.1. Sample collection and handling
The samples were taken at the dairy plant located at Ain Defla,
(North of Algeria), during the period from January to July 2017. Twelve
samples were taken from pre-pasteurization and post-pasteurization raw
cow milk pipelines. AISI 304 L Stainless steel cylinders (7.5 cm length ×
6.5 cm diameter) (supplied by the factory) were cleaned by immersion
in ethanol/acetone (v/v) for at least 1 h. The cylinders are then rinsed
with sterile distilled water then submerged in NaOH solution (2 %) for 5
min at 70 ◦ C, followed by a second rinse with sterile distilled water, then
they are submerged in HNO3 (1 %) for 5 min at 70 ◦ C, followed by a final
rinse with sterile distilled water. Finally, the cylinders are covered with
aluminium and sterilized them at 120 ◦ C for 1 h as suggested by Peng
et al. (2001).
Stainless-steel cylinders were inserted at the pre- and post-
pasteurization pipe for 7 days in order to have a mature biofilm. The
2
International Journal of Food Microbiology 407 (2023) 110389
cylinders were then recovered in sterile glass jars containing 300 mL of a
dilution solution: tryptone-salt-water (TSE; 1 g of tryptone and 8.5 g of
NaCl per L of distilled water; Sigma Aldrich Chimie S.a.r.l, St. Quentin
Fallavier Cedex, France) with 2 % Tween 80 to ensure maximum re­
covery of germs adhered to the surfaces of the cylinders. The samples
were stored at 4 ◦ C then transported to the laboratory to be analysed.
Jars of TSE (with 2 % Tween 80) containing the cylinders were put in an
ultrasonic bath for 2.5 min, then stirred for a minute by vortex (this
operation was repeated) to detach the microorganisms and constitute
the stock solution, which were later used for the preparation of other
decimal dilutions (Faille et al., 2013).
2.2. Phenotypic characterization of bacterial isolates
Serial dilutions were spread on the plate count agar (peptone of
casein 5 g, yeast extract 2.5 g, glucose 1 g, agar 15 g per L of distilled
water; Laboratorios Conda S.A., Madrid, Spain). Plates were incubated
at 30 ◦ C for 72 h. Three to five colonies with different colour, texture and
size were chosen from each sample/plate according to their morphology
on plate agar. Bacterial isolates were examined for cell morphology and
Gram staining.
2.3. MALDI-TOF identification
The identification of bacterial isolates was performed by MALDI-TOF
mass spectrometry (MS) processed on the Bruker MALDI Biotyper®
system according to the manufacturer's instructions (Bruker, MA, USA).
A bacterial suspension of 1 μL of a saturated solution was used and
allowed to air-drying at room temperature. Isolates were only consid­
ered to be identified if they obtained an identification score value equal
to or higher than 2.0, corresponding to probable identification to species
level (Bizzini and Greub, 2010).
2.4. Production of proteases and lipases by bacterial isolates
The isolates were plated on agar plates containing the appropriate
substrates. Production of proteolytic enzymes was determined on plate
count agar containing 10 % skim milk powder (Vanderzant and Splitt­
stoesser, 1992). After incubation at 30 ◦ C for 72 h, plates were flooded
with 1 N HCl to observe clearance zones. Lipase production was assessed
on plate count agar containing 1 % of Tween 80. After incubation at
30 ◦ C for up to 5 days, plates were observed for the presence of a halo
around the bacterial growth was observed. The assay was done in
triplicate.
2.5. Ability to polystyrene biofilm formation
Biofilm-forming ability was measured in 96-well polystyrene
microplates. A standardized culture of each isolate in tryptone soya
broth (TSB) (casein peptone 17 g/L, dipotassium hydrogen phosphate
2.5 g/L, glucose 2.5 g/L, sodium chloride5 g/L, soya peptone 3 g/L;
Merck KGaA, Darmstadt, Germany) was added with 0.2 % of glucose to
the microplate and the test was run 5 times. Negative control wells
contained TSB with glucose were used. Samples were dispensed into
wells (200 μL for each) and the microplates incubated at 37 ◦ C for 24 h.
The planktonic cells were removed from each well, and the plates
were rinsed three times with deionised water. Excess moisture was
removed by tapping the microplates on sterile napkins, and the plates
were dried for 15 min. Then, biofilm formation was quantified by crystal
violet assay, as reported by Sabaeifard et al. (2014) with some modifi­
cation. The wells were stained with 200 μL of 0.1 % crystal violet
(Sigma-Aldrich) solution at room temperature for 30 min, and then the
microplates were washed with deionised water to remove the stain.
After drying, crystal violet was solubilised in 250 μL of 30 % acetic acid
glacial.
The absorbance (optical density, OD) at 590 nm was measured. Five
N. Didouh et al. International Journal of Food Microbiology 407 (2023) 110389

absorbance values obtained for each strain were used to calculate mean Table 1
values (and standard deviation), by subtracting the value of the negative Distribution of isolates obtained pre- and post-pasteurization on stainless steel
control from the mean value for each milk sample. Then, the strains pipes used in the dairy industry. The Chi-square (Chi2) test of independence was
were classified as: 1) non-biofilm producers when OD at 590 nm was used to analyse the hypothesis of similarity between the bacterial populations
lower than 0.1, 2) weak-biofilm producers when OD at 590 nm was pre- and post-pasteurization. The Chi2 value observed was 0.442, confirming
this similarity.
between 0.1 and 1.0, 3) moderate-biofilm producers when OD at 590 nm
was between 1.1 and 3.0, and 4) strong-biofilm producers when OD at Isolate identify by MALDI-TOF No. of isolates Total
590 nm was higher than 3.0 (Rossi et al., 2016). Pre- Post-
pasteurization pasteurization
2.6. Ability to stainless steel biofilm formation Bacillus altitudinis 1 0 1
Bacillus cereus 21 12 33
Stainless steel coupons (15 × 45 mm, type AISI 304 L) were used as Bacillus pumilus 1 1 2
Bacillus subtilis 6 2 8
test surfaces. These were cleaned as described above. The cleaned
Bacillus weithenstephanensis 1 0 1
stainless steel was placed horizontally in a tube containing 5 ml of Enterococcus casseliflavus 0 2 2
ultrahigh-temperature-treated skim milk for 2 h at room temperature to Enterococcus faecium 5 6 11
get the conditioning film. The coupons were then removed and rinsed Staphylococcus epidermidis 1 0 1
three times with distilled water. Thereafter, the coupons were individ­ Acinetobacter schindleri 2 0 2
Enterobacter cloacae 11 8 19
ually dipped into tubes containing brain-heart infusion broth (Lio­ Enterobacter xiangfangensis 1 0 1
filchem Srl, Roseto degli Abruzzi, Italy) spiked with 108 CFU/ml of the Leclercia adecarboxylata 0 1 1
tested strain. Each coupon was then incubated for 24 h at 30 ◦ C (optimal Raoultella ornithinolytica 0 1 1
growth temperature for Bacillus spp. and Enterobacter spp.) or 37 ◦ C
(optimal growth temperature for Acinetobacter spp., Enterococcus spp.,
and Acinetobacter schindleri were exclusively isolated in pre-
Leclercia spp., Raoultella spp. and Staphylococcus spp.) before being
pasteurization pipes, while rare isolates of Leclercia adecarboxylata,
removed with sterile forceps. Non-adherent cells were removed by
Raoultella ornithinolytica and Staphylococcus hominis were exclusively
rinsing the coupon three times in PBS buffer. The coupons were then re-
isolated in post-pasteurization pipes (Fig. 1).
immersed in 5 mL of PBS buffer (Kruszewski et al., 2013) and the
adherent cells were detached from the coupon by sonication in an ul­
trasonic bath for 2.5 min and then vortexed for 1 min (this process was 3.2. Production of proteases and lipases by bacterial isolates
repeated) to detach the microorganisms and form the stock solution that
was later used to prepare other decimal dilutions (Faille et al., 2013). Forty-one isolates belonging to the genera Bacillus, Enterococcus,
Dilutions were plated on PCA and incubated for 48 h. The tests were run Staphylococcus, Enterobacter, and Acinetobacter were both proteolytic
in triplicate. and lipolytic. Then, 2 isolates showed exclusively production of pro­
teases and 27 isolates produced exclusively lipases. The isolates of
2.7. Antibiotic susceptibility Leclercia adecarboxylata and Raoultella ornithinolytica showed neither
proteolytic nor lipolytic activities, in addition to 11 other isolates.
Antibiotic susceptibility was evaluated using the Kirby–Bauer disk-
diffusion method for the following antibiotics: amoxicillin (AMX, 25 3.3. Biofilm-forming ability of bacterial strains
μg), cefoxitin (FOX, 30 μg), ciprofloxacin (CIP, 5 μg), clindamycin (CD,
2 μg), chloramphenicol (C, 30 μg), erythromycin (E, 30 μg), gentamycin The polystyrene biofilm-forming capability of individual isolates,
(GEN, 10 μg), sulfamethoxazole/trimethoprim (SXT, 1.25/23.75 μg) and classified on the basis of OD at 590 nm, is shown in Table 2. The majority
tetracycline (TET, 30 μg). Assays were conducted as recommended by of isolates capable to produce biofilms were classified as weak biofilm
Clinical & Laboratory Standards Institute (CLSI) (CLSI, 2017). As a producers (n = 16), while 6 isolates were classified as moderate pro­
quality control, Escherichia coli strain ATCC 25922 was included in all ducers. Only 3 isolates were characterized as strong biofilm producers,
assays. Bacterial susceptibility profiles were classified using the CLSI being identified as Bacillus cereus, Enterococcus faecium and Acinetobacter
and European Committee on Antimicrobial Susceptibility Testing schindleri. The remaining collection of the isolates (70 %; 58 out of 83
(EUCAST) recommendations (CLSI, 2015; EUCAST, 2022). isolates) showed no ability for biofilm formation.
Regarding to stainless steel biofilm formation, the cell numbers
2.8. Data and statistical analysis recovered on plate count agar plates from stainless steel biofilms ranged
from 3.52 to 6.92 log10 CFU/cm2 (Fig. 2), being the maximum number
All data analysis was carried out using IBM SPSS Statistics for Win­ detected for Enterococcus casseliflavus. No association could be found
dows, Version 23.0 (IBM Corp, NY, US). All biofilm experiments were between the ability to form polystyrene or stainless steel biofilms. The
done in triplicate. Chi-square independence test was used to analyse isolates classified as moderate or high polystyrene biofilm producers
hypotheses regarding the categorical variables. t-Test for independent were not the isolates with ability to form larger stainless steel biofilms.
samples was used for the continuous variables, using Levene's test for
equal variances. A significance level of 0.05 was considered. 3.4. Antibiotic susceptibility

3. Results The antimicrobial susceptibilities of the isolates to the tested anti­

3.1. Bacterial isolates
Eighty-three isolates were obtained based on the colony morphology
and phenotypic characterization from 12 samples taken at pre- and post-
pasteurization of raw cow milk pipelines procedures. Most microbial
species were found in both sampling points (before and after pasteuri­
zation), as seen in Table 1. Rare isolates of Bacillus weithenstephanensis,
Bacillus altitudinis, Staphylococcus epidermidis, Enterobacter xiangfangensis
biotics, namely ciprofloxacin, clindamycin, erythromycin, amoxicillin,
cefoxitin, chloramphenicol, gentamicin, tetracycline, and trimetho­
prim/sulfamethoxazole, is shown in Table 3. The susceptibility profiles
were very distinct among the isolates and resistance to all antibiotics
was observed within the tested collection, except for chloramphenicol.
No isolates were resistant to all the tested antibiotics but some isolates
were resistant to multiple antibiotics. A set of 5 isolates of E. cloacae was
resistant to 4 antibiotics, namely amoxicillin, cefoxitin, tetracycline, and
trimethoprim/sulfamethoxazole, while 2 other E. cloacae isolates were

3
N. Didouh et al. International Journal of Food Microbiology 407 (2023) 110389

Fig. 1. Venn diagram representing the bacterial species collected pre- and post-pasteurization in both cold and hot seasons.

resistant to amoxicillin, cefoxitin, and trimethoprim/sulfamethoxazole.

Table 2
Then there were 17 isolates resistant to two antibiotics from different
Biofilm-forming ability onto a polystyrene surface by bacterial isolates collected
families. Inversely, a set of 21 isolates was not resistant to any of the
from stainless steel pipes from a milk-processing dairy plant.
tested antibiotics but intermediate profiles were observed in all these
Species Negative Weak Moderate Strong isolates to some antibiotics.
Bacillus altitudinis (n = 1) 1 – – – Among the resistance profiles, the most commonly found resistances
Bacillus cereus (n = 33) 25 3 4 1 in this collection of isolates were to amoxicillin and cefoxitin among
Bacillus pumilus (n = 2) 2
E. cloacae isolates and erythromycin among B. cereus isolates, but some
– – –
Bacillus subtilis (n = 8) 8 – – –
Bacillus weithenstephanensis (n = 1) 1 – – – isolates of E. faecium were also resistant to amoxicillin. The rarest
Enterococcus casseliflavus (n = 2) 2 – – – resistance was observed for chloramphenicol (no resistant isolates were
Enterococcus faecium (n = 11) 7 2 1 1 found), gentamicin (2 isolates; S. epidermidis and E. cloacae) and clin­
Staphylococcus epidermidis (n = 1) 1 – – – damycin (4 isolates; Bacillus cereus and Bacillus pumilus).
Acinetobacter schindleri (n = 2) 1 – – 1
Enterobacter cloacae (n = 19) 8 10 1 –
Enterobacter xiangfangensis (n = 1) 0 1 – – 4. Discussion
Leclercia adecarboxylata (n = 1) 1 – – –
Raoultella ornithinolytica (n = 1) 1 – – – One of the major problems in the food industry is dealing with
bacterial biofilm. Bacteria in biofilm can tolerate adverse environmental
conditions, such as nutrient deficiency or treatment with antimicrobial
substances (Brooks and Flint, 2008; Jahid and Ha, 2014). It is the case of

4
N. Didouh et al. International Journal of Food Microbiology 407 (2023) 110389

Fig. 2. Ability of bacteria isolated from milk (n = 83) to form biofilms on stainless steel. Isolates with similar ability to form biofilm on stainless steel were grouped.
Error bars indicate standard deviation.

Table 3
Susceptibility of bacterial isolates to multiple antibiotics (CIP – ciprofloxacin; CD – clindamycin; E – erythromycin; AMX – amoxicillin; FOX – cefoxitin; C – chlor­
amphenicol; GEN – gentamicin; TET – tetracycline; SXT - trimethoprim/sulfamethoxazole); nd – not done; R – resistant; I - intermediate; S – susceptible.
n CIP CD E AMX FOX C GEN TET SXT

Bacillus altitudinis 1 R S R nd nd nd nd nd nd
Bacillus cereus 33 I 4%R/96% 82%R/ nd nd nd nd nd nd
S 18%S
Bacillus pumilus 2 I R R nd nd nd nd nd nd
Bacillus subtilis 8 I S 13%R/ nd nd nd nd nd nd
87%S
Bacillus 1 I S S nd nd nd nd nd nd
weihenstephanensis
Enterococcus casseliflavus 2 50%R/50%S nd 50%R/ 50%R/50%S nd S nd S nd
50%I
Enterococcus faecium 11 44%R/44%I/ nd I 30%R/70%S nd 10%I/ nd 80%I/20% nd
12%S 90%S S
Staphylococcus 1 I S I nd R S R S S
epidermidis
Acinetobacter schindleri 2 I nd nd S R nd I S R
Enterobacter cloacae 19 S nd nd 88%R/6%I/ 94%R/4% S 6%R/35%I/ 29%R/ 41%R/
6%S S 59%S 71%S 59%S
Enterobacter 1 S nd nd R R S I S S
xiangfangensis
Leclercia adecarboxylata 1 S nd nd S S S S S S
Raoultella ornithinolytica 1 S nd nd I I S S S R
Total 83

5
N. Didouh et al.
standard cleaning and sanitation procedures, which are not effective to
remove biofilms (Bremer et al., 2006). In the dairy industry, the raw
milk contains a large number of microorganisms able to adhere to sur­
faces and form biofilm. Even if the primary biofilm formers are neither
putrefactive organisms nor pathogens, established biofilms can become
an attractive habitat for other harmful organisms (Weiler et al., 2013).
These organisms will be in contact with pasteurized products and pose a
risk to human health and product quality (Latorre et al., 2010; Sharma
and Anand, 2002). This can also occur as a result of post-pasteurization
contamination leading to a reduction in the shelf life of the product due
to microbial spoilage, which can cause enormous economic and indus­
trial damages (Alles et al., 2018; González-Rivas et al., 2018; Reich et al.,
2017).
In this study, pipe-adhered bacteria from a milk-processing dairy
were identified from two different time-points, before and after
pasteurization. To the best of our knowledge, this is the first time such
sampling strategy is used in order to isolate bacteria adhered to the
pipelines of the dairy industry. A diverse microbiota consisting of both
gram-positive and gram-negative bacteria belonging to thirteen species
was isolated from the two time-points. This strongly suggests that the
pasteurization process does not kill all bacteria but a large fraction. The
microbial species identified in this work have previously been reported
to attach to stainless steel tubing surfaces widely used in the food and
pharmaceutical industries due to their high corrosion resistance and
excellent mechanical properties (Cherif-Antar et al., 2016; Weber et al.,
2019; Zaffora et al., 2021). The ability to adhere to stainless steel of
various bacterial pathogens is due to their flagella, curli, pili, and
membrane adhesins, but also extrapolymeric substances and surface
proteins used by food processing industry and produce biofilms. The
supporting environmental conditions (temperature, pressure, fluctua­
tions in climatic conditions), stainless steel properties (surface energy,
hydrophobicity, surface roughness, topography) and type of food raw
materials, pre-processing and processing conditions play an important
role in enhancing bacterial adhesion and favourable conditions for
biofilm formation (Jindal et al., 2016; Dula et al., 2021).
The persistence of B. cereus, even after pasteurization, is due to heat
resistance of endospores (Sinnelä et al., 2021). Main mechanism of
spoilage is endospore subsequent germination and bacterial growth with
the production of enzymes in pasteurized milk (Gopal et al., 2015). The
pasteurization process, which involves subjecting milk to moderate heat
treatment, is inefficient against bacterial spores. The most widespread
sporulated bacteria belong to Bacillus and related genera (Malek, 2019).
The increasing recovery of these species continues even after the
cleaning in place (CIP) process, which is responsible for cleaning the
interior surface of pipelines, vessels, filters, process equipment and
associated things without dismantling the equipment (Didouh et al.,
2015, 2022). We can explain the presence of some species only in post
pasteurization by development of their ability to adhere to stainless steel
surface, which is under control of several genes and needs activation
through exposure to adverse environment such as pasteurization (Non­
aka et al., 2006; Rockabrand et al., 1998; Yeh et al., 1997). The ability to
adhere to stainless steel surface can also be associated to bacterial
thermotolerance, an inherent feature often expressed after exposure
either to sub-boiling temperature (Spinks et al., 2006), high pressure
conditions (Hauben et al., 1997) or chlorination (Folsom and Frank,
2000).
In this study it was observed a dominance of cultivable Bacillus iso­
lates (53 %). This microbiota may come from the dairy farm; it over­
comes processing barriers and ultimately may lead to a decrease in the
quality of the final product used in several dairy products, such as liquid
milk or cheese. Bacteria are ubiquitous in the natural environment,
especially around dairy farms, and are introduced into raw milk mainly
during milking procedures (Martin et al., 2019). Some studies have
investigated the role of agricultural practices and sources in the transfer
of bacteria from the environmental niche to raw milk, with litter, forage
and parlour practices reported to play an important role (Magnusson
6
International Journal of Food Microbiology 407 (2023) 110389
et al., 2007; Martin et al., 2019; Murphy et al., 2019; Masiello et al.,
2014; Vissers et al., 2006). A combination of both biofilm formation
potential and heat resistance may further exacerbate the problem of
bacterial persistence (Bojer et al., 2011). Therefore, some strategies have
been employed to remove, prevent, or delay the formation of Bacillus
biofilms in the dairy industry, but with limited success. A lack of un­
derstanding of the structure and behaviour of Bacillus biofilm under
conditions relevant to dairy environments may be partly responsible for
this situation (Shemesh and Ostrov, 2020). Therefore, it is crucial to
control the different enzymes present in raw milk, their activities and
their impact on dairy product quality, shelf life and functionality (Glantz
et al., 2020). In 1989, Wessels et al. (1989) found that some isolates of
Enterobacter and Klebsiella were proteolytic at 7 ◦ C, while others of
Enterobacter, Klebsiella and Serratia showed lipolytic activity at 30 ◦ C.
These enzymes affect shelf life and lead to taste, odour and product
defects, which in turn lead to increase waste of milk (Glantz et al., 2020).
Enterococci, particularly E. faecalis and E. faecium, are representative
indicators of hygiene (Kim et al., 2020) and their resistance to
pasteurization conditions (Sanz Perez et al., 1982) supports our results,
as well as their ability to produce biofilms (El-Zamkan and Mohamed,
2021; Necidová et al., 2009). Gomes et al. (2008) stated that some
foodborne isolates of E. faecalis and E. faecium had the ability to form
weak, moderate, or strong biofilms, while some isolates did not form any
biofilms at all. Similar results were also found in this study. There was a
high abundance of non-biofilm producers in the milk samples collected
here, but in fact such isolates can attach to the biofilms of other bacteria
thereby forming mixed species biofilms (Lourenço et al., 2011). In
addition, Enterobacter cloacae, Enterobacter xiangfangensis, Leclercia
adecarboxylata and Raoultella ornithinolytica were found in the Algerian
dairy pipelines. These Enterobacteriaceae are usually indicators of poor
sanitation (Amreen and Viswanath, 2023). According to De Siqueira
et al. (2021), the presence of multiple pathogenic bacterial species in
devices indicates errors in the cleaning process. To ensure the hygiene
quality of the end products, the reassessment of hygiene practices and
quality control in dairies for processed products is necessary. Innovative
and promising approaches to combat bacteria and their biofilms are
being developed, namely methods based on natural ingredients and the
use of nanoparticles (Mevo et al., 2021).
In this study we found differences between the ability of bacterial
isolates to adhere to two surfaces, polystyrene and stainless steel.
Polystyrene is hydrophobic while stainless steel is relatively hydrophilic,
therefore this fact can justify the differential ability of bacterial isolates
to be attached to these surfaces (Djordjevic et al., 2002). Stainless steel is
used for all equipment in contact with milk samples, consequently
bacterial adhesion to steel surfaces is of greater concern to the dairy
industry. It has been shown that the surface properties of stainless steel
change after treatment with skimmed milk by forming a protein layer on
the steel, which can affect bacterial adhesion (Barnes et al., 1999;
Hamadi et al., 2014). The average biofilm production of multiple iso­
lates of S. aureus in TSB was lower compared to their average biofilm
production in skim milk, suggesting that the risk of such biofilm for­
mation on stainless steel may increase significantly (Fabres-Klein et al.,
2015).
The application of antibiotics may also prevent bacterial growth but
the emergence of resistant bacteria is a major concern, reporting the
World Health Organization (WHO) in 2023 that 1.27 million deaths can
be attributable to bacterial antimicrobial resistance per year (Balkhy
and Balachandran, 2023). Makovec and Ruegg (2003) reported an in­
crease over the years of bacterial isolates resistant to erythromycin in
dairy cows' milk, namely Staphylococcus aureus, Escherichia coli, Entero­
bacter spp., Enterococcus spp., and Pasteurella spp. In 2012, Daka et al.
(2012) reported multiple isolates of S. aureus resistant to penicillin,
ampicillin, amoxicillin-clavulanic acid, ciprofloxacin, erythromycin,
ceftriaxone, trimethoprime-sulfamethoxazole, oxacillin and vancomy­
cin from cow's raw milk. Another study performed in Iran by Hassani
et al. (2022) highlighted the high prevalence of antimicrobial resistance
N. Didouh et al.
of pathogenic bacteria isolated from bovine milk to amoxicillin, peni­
cillin and cephalexin. In Algeria, a few studies have investigated the
antimicrobial resistance of bacterial strains isolated from cattle or milk
samples. Barour et al. (2019) reported a wide variety of resistance in
E. coli isolated from cattle in eastern Algeria, being only 32 % of the
isolates susceptible to the tested ten antibiotics. The National Network
for the Surveillance of the Resistance of Bacteria to Antibiotics in Algeria
is still recording significant levels of resistance to chloramphenicol and
furans in multiple bacterial isolates (Rahal et al., 2021). In this study,
some antibiotic resistant isolates were also reported namely to amoxi­
cillin, cefoxitin and erythromycin. Drug resistance may result from
inappropriate use of antibiotics in veterinary being a major concern
among healthcare researchers. Several reports show that a relationship
could be established between infections by antimicrobial resistant
pathogens in humans and antimicrobial use and drug resistance in food-
producing animals (Bennani et al., 2020; Choffnes et al., 2012; Pokharel
et al., 2020), however the extension of the problem is still unknown. Use
of antimicrobials drives the emergence of resistance, hence the extensive
over and misuse in livestock is of concern (Magnusson et al., 2021).
5. Conclusion
The standard pasteurization protocol applied in dairy industry in
Algeria is insufficient to eliminate Gram-positive bacteria (e.g. Bacillus,
Enteroccocus) and Gram-negative bacteria (e.g. Enterobacter). Therefore,
it is necessary to implement additional approaches to reduce contami­
nation in dairy farms and, subsequently, in raw cow milk or to imple­
ment temperature-time pasteurization parameters in accordance with
the microbial loads present in raw milk. Such strategies may be able to
help reducing the presence of bacteria and prevent the formation of
biofilm which can shelter pathogens, being some of these organisms
resistant to multiple antibiotics.
Funding
R.A. was supported by Individual Call to Scientific Employment
Stimulus—Second Edition (grant number CEECIND/01070/2018).
CRediT authorship contribution statement
Conceptualization - ND, MK, MBB, RA; Data curation - ND, RA;
Formal analysis - ND, RA; Resources - CC, BSM; Writing original draft -
ND; Writing review & editing - all authors.
Declaration of competing interest
All authors declare no conflict of interest.
Data availability
Data will be made available on request.
Acknowledgments
Thanks go to the Algerian manufactory “Giplait” Ain Defla for their
enormous support and kind encouragement.
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