International Microbiology (2018) 21:23–33

https://doi.org/10.1007/s10123-018-0002-5

ORIGINAL ARTICLE

Comparison of selected disinfectants efficiency against Listeria

monocytogenes biofilm formed on various surfaces
Krzysztof Skowron 1 & Karolina Hulisz 2 & Grzegorz Gryń 3 & Halina Olszewska 4 & Natalia Wiktorczyk 1 &
Zbigniew Paluszak 2

Received: 12 February 2018 / Revised: 10 March 2018 / Accepted: 13 March 2018 / Published online: 11 April 2018
# The Author(s) 2018

Abstract
Listeria monocytogenes is a main etiological factor of listeriosis, spread mainly by food products. In recent years, an increasing
number of patients with listeriosis and an augmentation in L. monocytogenes antibiotic resistance, e.g. to penicillin and ampi-
cillin, has been reported. The aim of the study was to characterise the L. monocytogenes strains isolated from fish-processed food
products. Species identification, based on the multiplex-PCR reaction, was performed, and the genetic similarity of the isolates
was analysed with the RAPD technique. The strains, in the form of planktonic cells and a biofilm, were subjected to drug-
susceptibility analysis, and the effect of disinfectants on the bacillus cells was evaluated. All of the analysed strains were of the
Listeria monocytogenes species. Three genetically distant strains were detected, i.e. Lm I, Lm II and Lm III. Approximately
66.6% penicillin-resistant and 66.6% cotrimoxazole-resistant strains were found. No erythromycin-resistant strain was detected.
The Lm II strain was simultaneously resistant to four antibiotics, i.e. penicillin, ampicillin, meropenem and cotrimoxazole. The
strongest biofilm was formed on aluminium foil and the weakest on rubber. The tested disinfectant antibiofilm effectiveness was
related to the type of surface. The most effective agent was paracetic acid and hydrogen peroxide (elimination rate 5.10–6.62 log
CFU × cm−2 and 5.70–7.39 log CFU × cm−2 after 1- and 5-min exposure, respectively) and the least—sodium hydroxide
(elimination rate 0.52–1.20 log CFU × cm−2 and 0.98–1.81 log CFU × cm−2 after 1- and 5-min exposure, respectively).
Further studies on a greater number of L. monocytogenes strains are recommended.

Keywords Listeria monocytogenes . Biofilm . Disinfectants . Fish . Antibiotic resistance

Introduction Wiedmann 2007). It has the ability to grow in a wide range

of pH and temperature conditions, as well as a reduced sensi-
Listeria monocytogenes is a Gram-positive, non-capsular, fac- tivity to vacuum conditions and UV radiation (Fontana et al.
ultative anaerobic, rod-shaped bacterium (Sauders and 2015; Khan et al. 2013). L. monocytogenes is widely distrib-
uted in the natural environment, e.g. in the soil, sewage, sur-
face waters and decomposing plant matter (Abdala 2013;
* Krzysztof Skowron Chambel et al. 2007). Food products, i.e. raw meat, fish,
skowron238@wp.pl vegetables, fruits and dairy products, are a popular source
of those pathogenic bacilli (Sauders and Wiedmann 2007).
1
Department of Microbiology, Nicolaus Copernicus University in Secondary food contamination results from the bacteria
Toruń, Collegium Medicum of L. Rydygier in Bydgoszcz, 9 M. ability to form a biofilm on the surface of equipment used
Skłodowskiej-Curie St, 85-094 Bydgoszcz, Poland in food processing plants (Colagiorgi et al. 2016;
2
Department of Microbiology and Food Technology, UTP University Sokunrotank et al. 2013). Within the biofilm produced,
of Science and Technology in Bydgoszcz, 6 Bernardyńska St, the microorganisms metabolic and physiological processes
85-029 Bydgoszcz, Poland
are regulated by auto-inducing quorum sensing (QS) sig-
3
Plant Breeding and Acclimatization Institute – National Research nalling molecules (Garmyn et al. 2009). In the biofilm
Institute, Al, PowstańcówWlkp. 10, 85-090 Bydgoszcz, Poland
structure, the bacilli are characterised by an increased
4
Department of Animal Hygiene and Environmental Microbiology, antibiotic- and disinfectant-resistance (Sokunrotank
UTP University of Science and Technology in Bydgoszcz, 28
Mazowiecka St, 85-084 Bydgoszcz, Poland
et al. 2013). This results from the biofilm specific
24 Int Microbiol (2018) 21:23–33

structure, which is stabilised by the so-called 201707 norm (Polish Norm PN-EN-ISO 11290–1:2017:07
extracellurar polimeric substances (EPS) that form the 2017). The samples were incubated in half-Fraser broth (24 h,
biofilm matrix (Sokunrotank et al. 2013). EPS is an impor- 37 °C). Next, 0.1 ml of the suspension was transferred to the
tant element that protects cells from drying, phagocytosis and Fraser broth (9.9 ml, incubation: 48 h, 37 °C). A surface culture
the penetration of antimicrobial agents into the inner layer of was initiated on the agar substrate for Listeria, according to
the biofilm (Abdala 2013; Kołzwan 2011). The increasing Ottaviani and Agosti (ALOA) (MERCK), from the potentially
resistance of microorganisms enforces the use of various positive samples, i.e. for which a black discolouration of the
methods of their elimination. Disinfection is one of them. It substrate was reported (incubation: 24 h, 37 °C). The incubation
is performed by using physical or chemical methods. The was prolonged for another day, if there was no growth after the
procedure allows to reduce the number of bacilli to a certain first 24 h. Plates with green-blue colonies, surrounded by an
level. The most commonly applied chemical disinfectants are opaque zone, were used in the further analysis.
the following: (1) halogens and their derivatives: hypochlo-
rite, chloramine T, chlorine dioxide; (2) iodophors; (3) DNA isolation
peroxygen compounds: hydrogen peroxide, peracetic acid;
(4) ozone; (5) alcohols: ethyl, propyl, isopropyl; (6) formalde- Total genomic DNA was isolated from samples using a
hyde and glutaric aldehyde; (7) surfactants; (8) quaternary Genomic Mini AXE Bacteria Spin column kit (A&A
ammonium compounds; (9) nitrogen compounds: polyam- Biotechnology, Poland), according to the manufacturer
ides; (10) organic and inorganic acids and their derivatives: procedure.
acetic acid; (11) heavy metal compounds. Depending on the
concentration, disinfectants may act bacteriostatic or Isolates identification (multiplex-PCR)
bactericidally. Disinfectants may cause the following: (1) de-
struction of the cell wall; (2) damage to the cytoplasm; (3) The PCR reaction was utilised to identify the isolates (Bubert
oxidation of bacteria cell membrane, proteins, double bonds, et al. 1999). Two primer pairs were applied: L1/L2 and LM1/
enzymes, RNA and DNA, as well as sulphhydryl groups to LM2 (Table 1) (Abdala 2013; Leclerq et al. 2010). Each 25 μL
disulphide bridges (hypochlorite and peroxyacids); (4) in- reaction volume contained 1 × PCR bufor (Promega); 25 mM
hibition of active transport across the cell membrane (qua- MgCl2 (Promega); 10 mMdNTP Solution Mix (Promega);
ternary ammonium compounds); (5) protein coagulation 10 μM of each primer pair (Oligo.pl); 1 U Taq DNA polymer-
and/or inhibition of their synthesis (aldehydes); (6) ase (Promega); 2 μL template DNA and sterile, double-
blocking the active enzyme centers (Baranowska et al. distilled water to volume. Amplification was performed as
2014). follows: one cycle of 2 min at 94 °C for initial DNA denatur-
The aim of the study was to characterise L. monocytogenes ation; 30 cycles of 30 s at 94 °C for denaturation, 30 s at 50 °C
strains isolated from a fish processing plant. Species identification for annealing and 1 min at 72 °C for DNA extension. The last
(via multiplex-PCR) and genetic similarity analysis (RAPD-PCR) cycle was followed by a final extension step of 5 min at 72 °C.
of isolates were performed. The drug-susceptibility and the effect The amplified DNA fragments were separated on 1.5% (w/
of disinfectants on the bacteria in both planktonic and biofilm v) agarose gel, in a TBE buffer, and detected by staining with
forms were evaluated. Midori Green (NIPPON Genetics EUROPE gmbH).
Molecular weights of the fragments were estimated using a
100–1000 bp DNA molecular marker (A&A Biotechnology,
Material and methods Poland). Listeria monocytogenes ATTC 7644 was used as the
reference strain.
Materiał
Genetic similarity evaluation (RAPD-PCR)
The research involved 20 samples of raw material, semi-final and
final product obtained from the fish processing plant (sampling in The isolates’ genetic similarity evaluation was performed with
accordance with the PN-ISO 18593 norm) (Polish Norm PN- the RAPD technique (Random Amplification of Polymorphic
ISO-18593 2005). DNA) (Park et al. 2012). The reaction was performed using
the OPA-11 primer with the 5’–CAATCGCCGT–3′ sequence
Methods (Ozbey et al. 2006). Each 25 μL reaction volume contained
1 × PCR bufor with 2 mM MgCl2 (Promega); 200 μM dNTP
Detection of L. monocytogenes in food samples Solution Mix (Promega); 1 μM single OPA-11 primer
(Oligo.pl); 1.25 U Taq DNA polymerase (Promega); 3 μL
Isolation of L. monocytogenes bacilli from the samples was template DNA and water to volume. Amplification was per-
performed in accordance with the PN-EN ISO 11290-1: formed as follows: one cycle of 1 min at 94 °C for initial DNA
Int Microbiol (2018) 21:23–33 25

Table 1 Primer sequence

(Abdala 2013; Leclerq et al. Primer Primer sequence (5′ → 3′) Target gene Amplicon size [bp]
2010)
L1 CAG CAG CCG CGG TAA TAC rrs 938
L2 CTC CAT AAA GGT GAC CCT
LM1 CCT AAG ACG CCA ATC GAA hlyA 700
LM2 AAG CAC TTG CAA CTG CTC

denaturation; six cycles of 2 min at 30 °C for annealing and sodium thiosulphate (7.84 g l−1) and Tween 80 (30.0 g l−1)
1 min at 72 °C for DNA extension; 35 cycles of 15 s at 94 °C (Krzywicka et al. 1993) and incubated for 2 min at room tem-
for initial DNA denaturation; 40 s at 37 °C for annealing and perature. Next, 3 μl of the neutralised solution, for each concen-
35 s at 72 °C for DNA extension. The last cycle was followed tration tested, was taken by a multi-channel pipette and cultured
by a final extension step of 10 min at 72 °C. on the Columbia Agar with 5.0% sheep blood. The growth on a
The amplified DNA fragments were separated on 2.0% (w/v) solid medium was evaluated after 24- and 96-h incubation
agarose gel, in a TBE buffer, and detected by staining with (37 °C). The disinfectant concentration at which there was no
Midori Green. To evaluate the genetic similarity, a phylogenetic bacteria development was considered as the MBC for a given
dendrogram was plotted in the CLIQS 1D Pro software dilution series.
(TotalLab). The clustering analysis was performed using the
UPGMA hierarchical grouping technique (Unweighted Pair Biofilm formation by L. monocytogenes strains on various
Group Method of Aritmetic Means). Measures of genetic unifor- surfaces and the effect of disinfectants on the bacilli
mity among recovered individuals were determined using the in the biofilm
dice dissimilarity coefficient.
The surfaces tested included sterile elements made of rubber,
Drug-susceptibility analysis stainless steel, polypropylene and aluminium foil (size: 10 mm ×
20 mm). Suspensions of the tested L. monocytogenes strains and
The antibiotic susceptibility of the strains tested was determined the ATTC 7644 L. monocytogenes reference strain, with a 0.5
using the disk-diffusion method. In the study, the strains’ suscep- optical density on the MacFarland scale, were prepared in test
tibility to penicillin (1 IU), ampicillin (2 μg), meropenem tubes containing 4 ml of sterile brain-heart (BHI) broth (Beton-
(10 μg), erythromycin (15 μg) and cotrimoxazole (1.25– Dickinson). The surfaces tested were immersed in the suspension
23.75 μg) was evaluated. The prepared antibiograms were incu- and transferred to a fresh sterile BHI broth every 24 h. After 72 h,
bated at 35 °C for 20 h. After the incubation period, growth the surfaces were rinsed twice with buffered saline (0.9% PBS;
inhibition zones around the antibiotic discs were measured. The Avantor). These surfaces were used in evaluation of biofilm for-
results were analysed in accordance with the EUCAST ver. 7.0 mation by examined strains and assesement of antilisterial effec-
recommendations. tiveness of tested disinfectants.
For the determination of ability to biofilm formation by tested
Determination of the minimum bactericidal concentration strains, the surface with biofilm was placed in a tube containing
of disinfectants 3 ml of PBS. Then sonication was performed using the sonicator
Ultrasonic DU-4 (Nickel-Electro Ltd.). After sonication, serial
To determine the value of the disinfectant minimum bactericidal tenfold dilutions of the obtained suspension were made and in-
concentration (MBC), against the tested L. monocytogenes oculated on the Columbia Agar medium with 5.0% sheep blood
strains and the L. monocytogenes ATTC 7644 reference strain, (Becton Dickinson), 100 μl on each. Twenty-four-hour incuba-
dilution series were prepared in hard water of composition tion was made at 37 °C in the aerobic atmosphere, and the ob-
compliant with the PN-EN-1276 norm (Krzywicka et al. tained result was presented as logarithm of the number of colony
1993). Disinfectant concentrations of 0.001, 0.01, 0.05, 0.1, forming units (CFU) per 1 cm2 of the surface tested.
0.5, 1.0, 2.0% and 0.1, 0.5, 1.0% were prepared for 1- and 5- The influence of four disinfectants, which contained the fol-
min exposure, respectively. lowing: peracetic acid and hydrogen peroxide, quaternary am-
A 20 μl sample of the tested strain suspension (0.5 on the monium compounds, sodium hydroxide or sodium hypochlorite
MacFarland scale) was placed in the well of a titration plate, and as active compounds, was analysed. The surfaces tested, with a
180 μl of the disinfectant, at the described above concentrations, biofilm, were placed in disinfectant solutions prepared according
was added. The disinfectant action was terminated after 1 and to the PN-EN-1276 norm (Polish Norm PN-EN-1276 2000).
5 min. For this purpose, 20 μl of the sample tested was trans- The experiment included a 0.5% disinfectant concentration for
ferred to 180 μl neutralising solution, i.e. nutrient broth both exposure times. After a given time, the surfaces were im-
(1000 ml), lecithin (3.0 g l−1), histidine 1 (1.0 g l−1), anhydrous mersed in the neutralising solution and then sonicated for 10 min
26 Int Microbiol (2018) 21:23–33

(30 kHz, 150 W). A series of tenfold dilutions was prepared in five L. monocytogenes isolates used in the study, three genetically
physiological saline, and a surface culture was initiated on the different strains could be found. It was shown that two strains
Columbia Agar with 5.0% sheep blood (bioMerieux); incuba- included genetically identical isolates (Lm II + Lm V and Lm
tion at 37 °C for 24–48 h. The procedure was repeated three III + Lm IV). Two of the most genetically similar strains were
times with each strain tested L. monocytogenes strains treated Lm I and Lm II. The level of their genetic similarity reached
with a solution in which the disinfectant was replaced with hard approx. 5.0%. The lowest level of genetic similarity, approx.
water, in equivalent volume, and was used as the control. The 1.0%, was found between Lm I and Lm III strains.
recovered colonies were counted and expressed as the loga-
rithm of the number of colony-forming units (CFU) per 1 cm2 Drug-susceptibility analysis
of the surface tested. Logarithmic declines in the number of
L. monocytogenes bacilli after disinfectant action, relative to Penicillin- and cotrimoxazole-resitance were the most often
the control, were calculated. reported among the strains tested. Resistance to penicillin
Additionally, the cell viability in the biofilm was evaluated (66.6% of strains tested) and cotrimoxazole (66.6%) was
microscopically, without and after the disinfectant treatment, found in Lm I and Lm II strains. Resistance to ampicillin
using the LIVE/DEAD BacLight Bacterial Viability Kit (33.3%) and meropenem (33.3%) was found in Lm II strain.
(ThermoFisher). There was no erythromycin-resistance reported among the
studied population (Table 3). Three susceptibility profiles
Statistical analysis could be distinguished. The Lm II strain (profile II) was si-
multaneously resistant to four antibiotics (penicillin, ampicil-
The normality of data distribution was checked, based on the lin, meropenem and cortimoxazole). The Lm III strain was
Shapiro-Wilk test, for the calculated logarithmic declines in the sensitive to all antibiotics tested (profile III) (Table 3).
number of L. monocytogenes bacilli and the means for all strains
tested were calculated. The results were statistically analysed Determination of the minimum bactericidal
with the two-way analysis of variance (ANOVA), and the com- concentration of disinfectants
parisons of means were made with Tukey post hoc test (p ≤ 0.05)
using Statistica 12.0 PL tools (StatSoft). The surface and disin- The disinfectant concentration at which no growth of bacteria
fectant types were considered as the independent variables, while was observed after further culturing on the disinfectant-free
the logarithmic decline of bacteria number as a dependent substrate was considered as the MBC for a given series of
variable. dilutions. It was found that peracetic acid and hydrogen per-
oxide were the most effective in inhibiting the bacteria strains
growth after 24-h incubation. The peracetic acid and hydrogen
Results peroxide MBC value, during 60-s contact with the disinfec-
tant, was 0.001% with the L. monocytogenes ATTC 7644
Isolates identification (multiplex-PCR) reference and Lm III strains and 0.01% for Lm I and Lm II
strains. The MBC value increased (0.01%) with the Lm III
The PCR reaction was performed on five isolates (Table 2). strain after 96-h incubation. It was reported that the MBC
All isolates were confirmed to be L. monocytogenes, presence value for 5-min exposure to peracetic acid and hydrogen per-
of the hlyA and rrs gene. oxide, after 24- and 96-h incubation, reached 0.1% for all
strains tested. As for the 60-s exposure, the bacilli growth
Genetic similarity valuation (RAPD-PCR) inhibition was observed at 0.01% sodium hypochlorite con-
centration with L. monocytogenes ATCC 7644, Lm I and Lm
The phylogenetic dendrogram of the bacilli tested (Fig. 1) indi- III strains, and at 0.05% for the Lm II strain. An increase in the
cates the presence of two major phylogenetic lines. Among the MBC value (0.05%) was observed with the Lm I strain after

Table 2 Specification of
L. monocytogenes-positive Isolate symbol Sample specification Collection date
samples
Lm I Semi-finished product; after smoking 22 August 2016
Lm II Semi-finished product; after smoking 22 August 2016
Lm III Semi-finished product; product ripening, 06 September 2016
warehouse prior to the confectionery hall
Lm IV Semi-finished product; before cutting, confection hall 06 September 2016
Lm V Product; slices on a tray before packaging, confection hall 06 September 2016
Int Microbiol (2018) 21:23–33 27

Fig. 1 Genetic similarity dendrogram of the tested isolates

96-h incubation. A 0.1% MBC value, after 5-min exposure to Biofilm formation by L. monocytogenes strains
sodium hypochlorite, was reported for all strains after 24- and on various surfaces and the effect of disinfectants
96-h incubation. As for quaternary ammonium compounds, on the bacilli in the biofilm
the bacteria growth inhibition was reported at 0.01% concen-
tration with the Lm II strain and at 0.05% with the remaining All tested strains formed biofilm on examined sur-
strains, for both 24- and 96-h incubation. On the other hand, faces. Regardless of the strain, the highest number of
the MBC value after 5-min exposure to the disinfectant was L. monocytogenes was isolated from the biofilm on the alu-
0.1% for all strains tested and both incubation periods. It was minium foil and the smallest from the biofilm on the rubber
found that sodium hydroxide was less effectively inhibiting (Table 5). Among the tested strains, the strongest biofilm was
bacilli growth in the biofilm formation. It was reported that the formed by the strain Lm I, for which the number of bacilli
MBC value, after a 60-s exposure to the abovementioned dis- isolated from the biofilm ranged from 6.90 log CFU × cm−2
infectant, was 1.0% with the L. monocytogenes ATTC on rubber to 7.99 log CFU × cm −2 on aluminium foil
7644 reference and Lm III strains or 2.0% for the Lm I (Table 5). In turn, the weakest biofilm was formed by the Lm
and Lm II strains. As for 96-h incubation, the MBC III strain, for which the number of re-isolated bacteria ranged
value increased to 2.0% with the Lm I strain. Growth from 5.94 log CFU × cm−2 from biofilms on rubber to 7.74 log
inhibition after 5-min exposure to sodium hydroxide CFU × cm−2 from biofilms on aluminium foil (Table 5).
was found at 0.5% concentration for L. monocytogenes The obtained results showed differences in the antilisterial
ATTC 7644 reference and Lm III strains and at 1.0% for Lm I effect of disinfectants tested, depending on the active substance
and Lm II strains (Table 4). contained, surface type and exposure duration (Figs. 2 and 3).

Table 3 Drug-susceptibility
evaluation and drug-susceptibility Number (percentage) of strains Profile name Drug-susceptibility profile Number of strains
profiles of the tested resistant to antibiotics
L. monocytogenes strains
P—2 (66.6%) I R: P, SXT 1 (33.3%)—Lm I
AM—1 (33.3%) S: AM, MEM, E
MEM—1 (33.3%) II R: P, AM, MEM, SXT 1 (33.3%)—Lm II
E—0 (0.0%) S: E
SXT—2 (66.6%) III R: --- 1 (33.3%)—Lm III
S: P, AM, MEM, E, SXT

P penicillin, AM ampicillin, MEM meropenem, E erythtomycin, SXT trimetophrim/sulfamethoxazole, R resis-

tance, S susceptible
28 Int Microbiol (2018) 21:23–33

Table 4 Disinfectants effect on the growth of planktonic bacilli; C control, S susceptible strain, R resistant strain

Exposure duration 60 s 5 min

Incubation duration After 24 h incubation After 96 h incubation After 24 h incubation After 96 h incubation C

Concentration [%] 0.001 0.01 0.05 0.1 0.5 1.0 2.0 0.001 0.01 0.05 0.1 0.5 1.0 2.0 0.1 0.5 1.0 0.1 0.5 1.0

Strain Agent type Sodium hydroxide

ATCC 7644 R R R R R S S R R R R R S S R S S R S S R
Lm I R R R R R R S R R R R R R S R R S R R S R
Lm II R R R R R R S R R R R R R S R R S R R S R
Lm III R R R R R S S R R R R R R S R S S R S S R
Strain Agent type Sodium hypochlorite
ATCC 7644 R S S S S S S R S S S S S S S S S S S S R
Lm I R S S S S S S R S S S S S S S S S S S S R
Lm II R R S S S S S R R S S S S S S S S S S S R
Lm III R S S S S S S R S S S S S S S S S S S S R
Strain Agent type Quaternary ammonium compounds
ATCC 7644 R R S S S S S R R S S S S S S S S S S S R
Lm I R R S S S S S R R S S S S S S S S S S S R
Lm II R S S S S S S R S S S S S S S S S S S S R
Lm III R R S S S S S R R S S S S S S S S S S S R
Strain Agent type Peracetic acid and hydrogen peroxide
ATCC 7644 S S S S S S S S S S S S S S S S S S S S R
Lm I R S S S S S S R S S S S S S S S S S S S R
Lm II R S S S S S S R S S S S S S S S S S S S R
Lm III S S S S S S S R S S S S S S S S S S S S R

C control, S susceptible strain, R resistant strain

Italicised data—MBC value

After 1 min exposure, peracetic acid and hydrogen perox- 0.52 log CFU × cm−2 on aluminium foil to 1.20 log CFU ×
ide caused the greatest decreases in the number of cells isolat- cm−2 on polypropylene; however, significant differences were
ed from the biofilm, i.e. from 5.10 log CFU × cm−2 (rubber) to observed only with peracetic acid and hydrogen peroxide-
6.63 log CFU × cm−2 (stainless steel). The recorded logarith- based disinfectants on all surfaces, quaternary ammonium
mic decrease in the bacteria number was significantly higher compounds on stainless steel and with all tested disinfectants
in comparison to all other compounds, regardless of the sur- on aluminium foil (Fig. 2).
face type, only with exception of aluminium foil (Fig. 2). On For sodium hydroxide, the greatest decrease in the
the other hand, the lowest reduction in the L. monocytogenes L. monocytogenes bacilli number isolated from the biofilm
bacilli number, recovered from the biofilm after 1-min disin- (1.20 log CFU × cm−2), after 1-min contact, was found on
fection, was recorded after applying sodium hydroxide on the polypropylene surface. In turn, for quaternary ammonium
each surface. The recorded decrease values reached from compounds and for sodium hypochlorite, the greatest reduc-
tions of L. monocytogenes in biofilm (5.10 and 3.84 log
Table 5 Number of L. monocytogenes isolated from biofilm on
CFU × cm −2, respectively) were obtained on aluminium
different surfaces foil (Fig. 2). For paraacetic acid and hydrogen perox-
ide, the highest effectiveness against L. monocytogenes
Surface Number of bacteria [log CFU × cm−2]
(6.63 log CFU × cm−2) was noticed on stainless steel
Strain Rubber Polypropylene Stainless steel Aluminium foil (Fig. 2).
Lm I 6.90 ± 1.26* 7.41 ± 1.93 7.10 ± 0.95 7.99 ± 0.64 The lowest efficiency of majority of tested disinfectants
Lm II 6.51 ± 1.12 7.90 ± 1.40 6.90 ± 1.08 7.82 ± 1.21
(without sodium hydroxide) was demonstrated on the rubber
Lm III 5.94 ± 1.66 7.19 ± 2.36 6.20 ± 1.87 7.74 ± 1.05
surface (Fig. 2). Significant differences in the effectiveness of
particular disinfectants, depending on the surface type, were
*Standard deviation found for the quaternary ammonium-based compounds,
Int Microbiol (2018) 21:23–33 29

Fig. 2 Bacilli number decrease (average for all tested strains) in the significantly, K—average for all strains initial number of
biofilm on the tested surfaces after 60-s exposure to various disinfectants L. monocytogenes (prior disinfection) in biofilm on particular
(a, b, c, ...—values marked with different letters differ statistically surfaces)

acting on the biofilm on rubber vs. stainless steel and alumin- For sodium hydroxide, the greatest decrease in the number
ium foil and on polypropylene vs. aluminium foil, as well as of L. monocytogenes isolated from the biofilm after 5-min
for sodium hypochlorite acting on the biofilm on aluminium contact with the disinfectant (1.81 log CFU × cm−2), similarly
foil vs. rubber and stainless steel (Fig. 2). as for 1-min exposure, was found on the polypropylene. In
Similarly to the 1-min contact, after 5-min exposure to the turn, for quaternary ammonium compounds and and sodium
disinfectant, the highest decline in the cell number, i.e. 5.7– hypochlorite, the best antibiofilm activity (6.02 and 4.63 log
7.39 log CFU × cm−2, was reported for peracetic acid and CFU × cm−2, respectively) was stated on aluminium foil, just
hydrogen peroxide, regardless of the surface type. A signifi- like in case of 1-min exposure (Fig. 3). For peracetic acid and
cantly greater logarithmic decrease in the bacteria number was hydrogen peroxide, the greatest decrease in the number of
recorded on each surface in comparison to other agents, ex- L. monocytogenes isolated from the biofilm (7.39 log
cept the quatery ammoinium compounds acting against bio- CFU × cm−2) was found on polypropylene.
film on stainless steel and aluminium foil (Fig. 3). On the other After 5-min, the lowest efficiency of all agents tested
hand, the lowest reduction in the number of bacilli isolated against the biofilm of L. monocytogenes was demonstrated
from the biofilm, after 5-min exposure to the disinfec- on the rubber surface (Fig. 3). Significant differences in the
tant, was demonstrated for the sodium hydroxide-based effectiveness of individual disinfectants, in regard to the sur-
agent, for each tested surface. The recorded decrease face type, were found only for the quaternary ammonium
values reached from 0.98 log CFU × cm−2 (aluminium compunds and sodium hypochlorite, acting on the biofilm
foil) to 1.81 log CFU × cm−2 (polypropylene). Significant dif- on aluminium foil vs. biofilm on rubber and polypropylene
ferences in the decrease values were reported for sodium (Fig. 3). Moreover, the significant differences in the
hydroxide vs. other tested disinfectants acting against antibiofilm effectiveness were noticed for sodium hydroxide
biofilm on stainless steel and aluminium foil and vs. acting on aluminium foil vs. on polypropylene (Fig. 3).
peracetic acid and hydrogen peroxide on rubber and The microscopic observations confirmed the results regard-
polypropylene (Fig. 3). ing the microbiocidal effectiveness of disinfectants obtained
30 Int Microbiol (2018) 21:23–33

Fig. 3 Bacilli number decrease (average for all tested strains) in differ statistically significantly, K—average for all strains initial
the biofilm on the tested surfaces after 5-min exposure to various number of L. monocytogenes (prior disinfection) in biofilm on
disinfectants (a, b, c, ...—values marked with different letters particular surfaces)

in classical culturing methods. Exemplary changes in the per- to penicillin (n = 7/43, 16.3%) and ampicillin (n = 9/43,
centage of live and dead cells in the selected biofilm layer of 20.9%). Opposite results were obtained by Gelbíčová and
L. monocytogenes produced on the rubber surface are shown Karpiškova (2012), who did not report the presence of a
in Fig. 4. penicillin-resistant strain among the isolates tested.
Research conducted by Korsak et al. (2012) in the Polish
food processing environment showed that all isolates tested
Discussion were ampicillin-sensitive. On the other hand, Majczyna and
Białasiewicz (2006) found that no strain tested was resis-
In recent years, the number of patients with listeriosis has been tant to cotrimaxazole. Approximately 33.3% of the tested
increasing. Fresh and smoked fish are considered to be one of population strains were resistant to meropenem. In a study
the main sources of pathogenic L. monocytogenes (Ertas and by Ruiz-Bolivar et al. (2011), it was reported that 44.0% of
Seker 2005; Fallah et al. 2013). The increasing resistance of strains were meropenem-resistant. In the present study, no
bacillia to antibiotics, e.g. penicillin and ampicillin, is a seri- strains with erythromycin resistance were found. Similar
ous problem. The present study showed that 66.6% of strains results were recorded by Korsak et al. (2012), who did
were penicillin- or catotrimoxazole-resistant. It was also found not detect L. monocytogenes strains resistant to erythromy-
that 33.3% of strains were resistant to ampicillin. cin. Doménech et al. (2015) showed that only two of the 69
Abdollahzadeh et al. (2016) evaluated the susceptibility of samples tested were erythromycin resistant, and they
L. monocytogenes strains isolated from seafood to eight anti- originated from smoked salmon. Research conducted by
microbials, including penicillin, ampicillin and trimeroxazole. Jamali and Thong (2014) confirmed the presence of
They found a high-resistance level of the strains tested to erythromycin-resistant strains (6.3%).
penicillin (57.0%) and ampicillin (100.0%) (Abdollahzadeh Due to its ability to form biofilm on surfaces of various
et al. 2016). Jamali et al. (Jamali and Thong 2014) reported porosity, e.g. stainless steel, rubber, polypropylene and glass,
that strains isolated from open fish markets were resistant L. monocytogenes poses a serious threat to the food industry
Int Microbiol (2018) 21:23–33 31

ceramic tiles, polypropylene and glass (microscopic examina-

tion). Also in the present study, it was shown that the strains
formed a biofilm on the tested surfaces: rubber, polypropylene,
stainless steel and aluminium foil after 24 h. It was shown that
the number of living cells adhering to the surface of stainless
steel was lower in comparison to the number of cells inhabiting
polypropylene or rubber. The strongest biofilm was formed on
aluminium foil surface. Poimeniodou et al. (2016) found that
the surface type significantly influenced biofilm formation by
L. monocytogenes. They reported that the average population
size of biofilm cells on polystyrene (5.6 log CFU × cm−2) was
greater than on stainless steel (4.7 log CFU × cm−2). Yun H.
et al. (2010) showed that the number of reisolated
L. monocytogenes after inoculation was the highest from alu-
minium foil, what confirmed our results.
Effective disinfection is an important aspect in food process-
ing plants. The increasing resistance of microorganisms to
commonly used agents, e.g. based on sodium hypochlorite or
sodium hydroxide, is a serious problem. The present study
evaluated the effectiveness of four disinfectants against bacilii
cells in the biofilm formation. It was found that peracetic acid
and hydrogen peroxide, as well as sodium hypochlorite, were
the agents, which most strongly inhibited the growth of those
organisms. It was reported that the MBC value for the Lm III
strain increased (0.01%) after 96-h incubation, 60-s exposure to
peracetic acid and sodium hydroxide. The observed increase in
the MBC value after 96-h incubation may be due to the pres-
ence of damaged cells or cells with a reduced metabolism that
needed time to regenerate and multiply. It was shown that the
greatest logarithmic decline in the colony count, after the
application of peracetic acid and hydrogen peroxide, was
recorded on the polypropylene surface. Beltrome et al. (2015)
reported that treatments with peracetic acid and sodium hypo-
chlorite were effective in eliminating L. monocytogenes from
the polyethylene cutting board used in a food processing plant.
Lee et al. (2016), on the other hand, observed the highest anti-
microbial activity of 0.5% peracetic acid against
L. monocytogenes and S. aureus biofilm isolated from stainless
steel surface in dairy plants. They also found that peracetic acid
was ineffective against the cells adjacent to the polystyrene
surface (Lee et al. 2016). In the other hand, Cabeca et al.
(2012) reported a very high sensitivity of L. monocotogenes
strains colonising a stainless steel surface to low concentrations
of peracetic acid. In the present study, it was shown that sodium
Fig. 4 Percentage of viable (white) and dead (grey) cells in the selected hypochlorite effectively eliminated bacilli in the biofilm forma-
L. monocytogenes (Lm I strain) biofilm layer on rubber surface after
treatment with disinfectants; a control (without disinfectant), b peracetic
tion from the surfaces tested, especially from stainless steel. An
acid, c quaternary ammonium compounds, d sodium hypochlorite, e so- opposite result was described by Krysinski et al. (1999), who
dium hydroxide reported the lowest antimicrobial activity of this compound.
Also, Chen et al. (2015) found that peracetic acid and sodium
hypochlorite were ineffective against the studied microorgan-
(Borucki et al. 2003). Doijad and Sukhadeo (2015) showed that isms (L. monocytogenes, S. Typhimurium, E. coli) in the bio-
after 24 h, the tested bacilli strains formed strong biofilms on film formation on the stainless steel surface. They found that
surfaces used in the food industry, such as stainless steel, dodecyl sulfate sodium salt (SDS) was the only agent which
32 Int Microbiol (2018) 21:23–33

effectively eliminated the bacilli. The authors reported that all Open Access This article is distributed under the terms of the Creative
Commons Attribution 4.0 International License (http://
three pathogens studied could synthesise catalase, which pro- creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
tects the embedded cells, preventing the full penetration of distribution, and reproduction in any medium, provided you give appro-
hydrogen peroxide into the biofilm (Chen et al. 2015). In the priate credit to the original author(s) and the source, provide a link to the
present research, it was found that the effectiveness of quater- Creative Commons license, and indicate if changes were made.
nary chemical compounds was surface type-dependent. As for
quaternary ammonium compounds, the highest logarithmic de-
cline in the colony count, i.e. 5.01 log CFU × cm−2, 5-min
exposure, was recorded for the stainless steel surface, while References
the lowest, 3.14 log CFU × cm−2, 5-min exposure, on the rub-
ber surface. Poimenidou et al. (2016) found that quaternary Abdala S (2013) Mechanisms of biofilm formation by Listeria
monocytogenes. Doctoral Thesis, University of Leicester, pp 1–256
ammonium compounds were more effective against the biofilm Abdollahzadeh E, Ojagh SM, Hosseini H, Ghaemi EA, Irajan G, Heidarlo
formed on polystyrene as compared to the one formed on stain- MN (2016) Antimicrobial resistance of Listeria monocytogenes iso-
less steel. In contrast, Ortiz et al. (2016) reported the resistance lated from seafood and humans in Iran. Microb Pathog 100:1–5
of bacilli to quaternary ammonium compounds caused by Baranowska M, Chojnowski W, Nowak H (2014) Disinfection in
diaryplants. Nauki Inżynieryjskie i Technologie 4(15):9–22
the long-term use of those particular disinfectants. In
Beltrame CA, Martelo EB, Mesquitaetet R’z A et al (2015) Adhesion of
the present study, it was found that sodium hydroxide Listeria monocytogenes to cutting board surfaces and removal by
was the least efficient in eliminating L. monocytoges different sanitizers. J Consum Protection Food Saf 10:41–47
strains. Similar results were reported by Chen et al. (2015), Borucki KM, Peppin JD, White D, Loge F, Call DR (2003) Variation in
who claim that sodium hydroxide was ineffective in eliminat- biofilm formation among strains of Listeria monocytogenes. Appl
Environ Microbiol 69(12):7336–7342
ing the biofilm formed by L. monocytogenes, E. coli or Bubert A, Hein I, Rauch M, Lehner A, Yoon B, Goebel W, Wagner M
S. typhimurium. Common disinfectants, based on sodium hy- (1999) Detection and differentiation of Listeria spp. by a single
droxide or hypochlorite, are becoming less and less effec- reaction based on multiplex PCR. Appl Environ Microbiol 65:
tive, due to the increasing tollerance of bacilli. The use of 4688–4692
Cabeca TK, Pizolitto AC, Pizolitto EL (2012) Activity of disinfectants
optimised concentrations of various disinfectants, carefully against foodborne pathogens in suspension and adhered to stainless
selected depending on the surface type, seems to be a rec- steel surfaces. Braz J Microbiol 43(3):1112–1119
ommended solution. Also, the improvement of existing Chambel L, Sol M, Fernandes I, Barbosa M, Zilhão I, Barata B, Jordan S,
bacteria screening and elimination strategies, or the devel- Perni S, Shama G, Adrião A (2007) Occurrence and persistence of
Listeria spp. in the environment of ewe and cow's milk cheese dair-
opment of new ones, should be considered (Krysinski et al.
ies in Portugal unveiled by an integrated analysis of identification,
1999). typing and spatial-temporal mapping along production cycle. Int J
The increasing number of listeriosis cases and resistance Food Microbiol 116:52–63
of bacilli to antimicrobials and conventional disinfectants, Chen D, Zhao T, Doyle MP (2015) Control of pathogens in biofilms on
including those based on sodium hydroxide, is an important the surface of stainless steel bylevulinic acid plus sodium dodecyl
sulfate. Int J Food Microbiol 207:1–7
public health problem. Major sources of bacilli are food Colagiorgi A, Pierluigi Di C, Zanardi E, Ghidini S, Ianieri A (2016) A
products that are re-contaminated in food industry plants, look inside the Listeria monocytogenes biofilms extracellular ma-
e.g. at the raw product processing stage. Disinfectants com- trix. MDPI. Journal 4(22):1–12
monly used in processing plants eliminate planktonic bio- Doijad S, Sukhadeo B. Barbuddhe, Sandeep Gargeetet al. (2015)
Biofilm-forming abilities of Listeria monocytogenes serotypes iso-
film forms, but are not effective with the mature biofilm lated from different sources. PLOS One. https://doi.org/10.1371/
structure. The results of the present study provide prelimi- journal.pone.013704
nary information on fish contamination with potentially vir- Doménech E, Jimenez-Belenguer A, Amoros JA, Ferrus MA, Escriche I
ulent L. monocytogenes strains. Nevertheless, further studies (2015) Prevalence and antimicrobial resistance of Listeria
monocytogenes and Salmonella strains isolated in ready-to-eat foods
on a greater number of strains isolated from fish processing
in Eastern Spain. Food Control 47:120–125
plants are recommended. Ertas HB, Seker E (2005) Isolation of Listeria monocytogenes from fish
intenstines and RAPD analysis. Turk J Vet Anim Sci 29:1007–1011
Funding information This research was in part financially supported Fallah S, Aziz A, Saei-Dehkordi S, Mahzounieh M (2013) Occurence and
by the Nicolaus Copernicus University with funds from the mainte- antibiotic resistance profiles of Listeria monocytogenes isolated
nance of the research potential of the Department of Microbiology from sea food products and market and market and processing en-
DS-UPB no. 782. vironments in Iran. Food Control 34:630–636
Fontana C, Coconelli Pier S, Vignalo G, Saavedra L (2015) Occurence of
Compliance with ethical standards antylisterial structural bacteriocins gens in meat borne lacticacid
bacteria. Food Control 47:53–59
Garmyn D, Gal L, Lemaitre JP, Hartman A, Pivetau P (2009)
This article does not contain any studies with human participants or an- Communication and autoinduction in the species Listeria
imals performed by any of the authors. monocytogenes. A central role for the agr system. Communicative
and Integr Biol 2(4):371–374
Int Microbiol (2018) 21:23–33 33

Gelbíčová T, Karpiškova R (2012) Outdoor environment as a source of Turkey and RAPD analysis of Listeria monocytogenes isolates. Ir
Listeria monocytogenes in food chain. Czech J Food Sci 30(1):83–88 Vet J 59:342–344
Jamali H, Thong KL (2014) Genotypic characterization and antimicrobial Park S, Jung J, Set C et al (2012) Molecular characterization of Listeria
resistance of Listeria monocytogenes from ready-to-eats foods. monocytogenes based on the PFGE and RAPD in Korea. Adv
Food Control 44:1–6 Microbiol 2:605–616
Khan JA, Rathore RS, Khan S, Ahmad I (2013) In vitro detection of Poimenidou SV, Chrysadaakou M, Tzakoniati A, Bikouli VC, Nychas G-
pathogenic Listeria monocytogenes from food sources by J, Skandamis PN (2016) Variability of Listeria monocytogenes
convencional, molecular and cell culture method. Braz J Microbiol strains in biofilm formation on stainless steel and polystyrene mate-
44(3):751–758 rials and resistance to peracetic acid and quaternary ammonium
Kołzwan B (2011) Analysis of biofilms—their formation and function- compounds. Int J Food Microbiol 237:164–171
ing. Ochrona Środowiska 33(4):3–14 Polish Norm PN-EN-1276 (2000) Chemiczne środki dezynfekcyjne i
Korsak D, Borek A, Daniluk S, Grabowska A, Pappelbaum K (2012) antyseptyczne- Ilościowa zawiesinowa metoda określania działania
Antimicrobial susceptibilities of Listeria monocytogenes strains iso- bakteriobójczego chemicznych środków dezynfekcyjnych i
lated from food and food processing environment in Poland. Int J antyseptycznych stosowanych w sektorze żywnościowym,
Food Microbiol 158:203–208 warunkach przemysłowych i domowych oraz zakładach
Krysinski EP, Brown LJ, Marchisello TJ (1999) Effect of cleaners and użyteczności publicznej – Metoda badania i wymagania (faza 2, etap
sanitizeres on L. monocytogenes attached to product contact sur- 1). Polski Komitet Normalizacyjny
faces. J Food Prot 55(4):246–251 Polish Norm PN-EN-ISO 11290–1:2017:07 (2017) Mikrobiologia
Krzywicka H, Janowska J, Tadeusiak B, Zarzycka E (1993) Metoda łańcucha żywnościowego – Horyzontalna metoda wykrywania i
określania stężeń użytkowych preparatów dezynfekcyjnych. oznaczania liczby Listeria monocytogenes i innych Listeria spp. -
Metoda nośnikowa. Wydawnictwo Metodyczne Państwowego Część 1: Metoda wykrywania. Polski Komitet Normalizacyjny
Zakładu Higieny, Warszawa
Polish Norm PN-ISO-18593 (2005) Mikrobiologia żywności i pasz-
Leclerq A, Clermont D, Bizet C, Grimont PAD, Le Fleche-Mateos A,
Horyzontalne metody pobierania próbek z powierzchni z użyciem
Roche SM, Buchriser C, Cadet-Daniel V, La Monnier A, Lecuit M,
płytek kontaktowych i wymazów. Polski Komitet Normalizacyjny
Allerberger F (2010) Listeria rocourtiae sp. nov. Int J Syst Evol
Microbiol 60:2210–2214 Ruiz-Bolivar Z, Neuque-Rico MC, Poutou-Pinales RA et al (2011)
Lee SHI, Cappato LP, Corassin CH, Cruz AG, Oliviera CAF (2016) Effect of Antimicrobial susceptibility of Listeria monocytogenes food isolates
paracetic acid on biofilm formes by Staphylococcus aureus and Listeria from different cities in Columbia. Foodborne Pathog Dis 8(8):913–919
monocytogenes isolated from dairy products. J Dairy Sci 99:1–7 Sauders DB, Wiedmann M (2007) Ecology of Listeria species and
Majczyna D, Białasiewicz D (2006) Characteristic of Listeria spp. bacte- L. monocytogenes in natural environment. In: Ryser ET, Marth EH
ria isolated from food products. Med Dośw Mikrobiol 58:119–126 (eds) Listeria, listeriosis, and food safety. Taylar and Francis CRC
Ortiz S, López-Alonso V, Rodríguez P, Martínez-Suáreza JV (2016) The Pres, BocaRaton, pp 21–45
connection between persistent, disinfectant-resistant Listeria Sokunrotank S, Iqbal KJ, Sang-Do H (2013) Biofilm formation in food
monocytogenes strains from two geographically separate Iberian industries. A food safety concern. Food Control 31:572–585
pork processing plants: evidence from comparative genome analy- Yun H, Kim B, Jung S, Kruk Z, Kim D, Choe W, Jo C (2010) Inactivation
sis. Appl Environ Microbiol 82(1):308–312 of Listeria monocytogenes inoculated on disposable plastic tray,
Ozbey G, Hassan Ertas HB, FilizKok F (2006) Prevalence of Listerias aluminum foil, and paper cup by atmospheric pressure plasma.
pecies in camel sausages from retail markers in Aydin province in Food Control 21:1182–1186