# Cleaning and disinfection of biofilms composed of Listeria monocytogenes and background microbiota from meat processing surfaces

Annette Fagerlund, Trond Møretrø, Even Heir, Romain Briandet, Solveig Langsrud

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Annette Fagerlund, Trond Møretrø, Even Heir, Romain Briandet, Solveig Langsrud. Cleaning and disinfection of biofilms composed of Listeria monocytogenes and background microbiota from meat processing surfaces. Applied and Environmental Microbiology, 2017, 83 (17), pp.AEM.01046-17. 10.1128/AEM.01046-17. hal-01607143

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1 Cleaning and disinfection of biofilms

2 composed of *Listeria monocytogenes* and

3 background microbiota from meat

4 processing surfaces

5 Annette Fagerlundª#, Trond Møretrøª, Even Heirª, Romain Briandetᵇ, Solveig Langsrudª

6 Nofima, Norwegian Institute of Food, Fisheries and Aquaculture Research, Ås, Norwayª; Micalis

7 Institute, INRA, AgroParisTech, Université Paris-Saclay, Jouy-en-Josas, Franceᵇ

8 Running Head: Cleaning and disinfection of biofilms

9 Keywords: *Listeria monocytogenes*, conveyor belt, biofilm, cleaning, disinfection

10 #Address correspondence to Annette Fagerlund, annette.fagerlund@nofima.no
11 ABSTRACT

12 Surfaces of food processing premises are exposed to regular cleaning and disinfection (C&D)
13 regimes, using biocides that are highly effective against bacteria growing as planktonic cells.

14 However, bacteria growing in surface associated communities (biofilms) are typically more
15 tolerant towards C&D than their individual free cells counterparts, and survival of pathogens

16 such as *Listeria monocytogenes* may be affected by interspecies interactions within biofilms. In
17 this study, *Pseudomonas* and *Acinetobacter* were the most frequently isolated genera surviving

18 on conveyor belts subjected to C&D in meat processing plants. In the laboratory, *Pseudomonas*,
19 *Acinetobacter* and *L. monocytogenes* dominated the community both in suspensions and in

20 biofilms formed on conveyor belts, when cultures were inoculated with eleven-genera cocktails
21 of representative bacterial strains from the identified background flora. When biofilms were
22 exposed to daily C&D cycles, mimicking treatments used in food industry, the levels of

23 *Acinetobacter* and *Pseudomonas mandelii* diminished, and biofilms were instead dominated by
24 *Pseudomonas putida* (65-76%), *Pseudomonas fluorescens* (11-15%) and *L. monocytogenes* (3-

25 11%). The dominance of certain species after daily C&D correlated with high planktonic growth
26 rates at 12°C and tolerance to C&D. In single-species biofilms, *L. monocytogenes* developed

27 higher tolerance to C&D over time, both for the peracetic acid and quaternary ammonium
28 disinfectant, indicating that a broad-spectrum mechanism was involved. Survival after C&D

29 appeared to be a common property of *L. monocytogenes* strains, as both persistent and
30 sporadic subtypes showed equal survival in complex biofilms. Biofilms established preferentially
31 in surface irregularities of conveyor belts, potentially constituting harborage sites for persistent
32 contamination.

33 **IMPORTANCE**

34 In food industry, efficient production hygiene is a key measure to avoid accumulation of
35 spoilage bacteria and eliminate pathogens. Persistence of bacteria is however a withstanding
36 problem in food processing environments. This study demonstrated that environmental
37 bacteria can survive foam cleaning and disinfection (C&D) at user concentrations in the
38 industrial environment. The phenomenon was replicated in laboratory experiments. Important
39 characteristics of persisting bacteria were high growth rate at low temperature, tolerance to the
40 cleaning agent and ability to form biofilm. This study also supports other recent research
41 suggesting that strain-to-strain variation cannot explain why certain subtypes of *Listeria*
42 *monocytogenes* persist in food processing environments while others are found only
43 sporadically. The present investigation highlights the failure of regular C&D and a need for
44 research on improved agents efficiently detaching the biofilm matrix.

45 **INTRODUCTION**

46 Food production premises are regularly subjected to cleaning and disinfection (C&D) regimes
47 designed to reduce bacterial load and eliminate pathogens. Peracetic acid (PAA) and quaternary
48 ammonium compounds (QAC) such as benzalkonium chloride are widely used as disinfectants in
49 the food industry and in healthcare facilities. Disinfectants are agents that have multiple targets
50 in the cell, and typically kill bacteria by disruption of the bacterial membrane (1). The use of
51 chemical disinfectants in food processing environments is usually based on their efficacy in tests

52 performed with planktonic bacteria (2). However, in natural and industrial environments,

53 bacteria often grow as biofilms, which are complex and structured microbial communities

54 encased in a self-produced protective extracellular matrix composed of polysaccharides,

55 proteins and/or extracellular DNA. The formation of biofilms is important for microbial survival

56 in the food industry, and cells in biofilms typically exhibit increased tolerance towards

57 antimicrobial agents compared with their planktonic counterparts (3, 4). Possible mechanisms

58 contributing to the low efficacy of conventional biocides on biofilms include diffusion-reaction

59 limitation associated with the biofilm matrix, slow growth and development of persister cell

60 subpopulations (4).

61 The microbiota found in food processing plant surfaces after C&D is commonly reported to be

62 diverse and include foodborne pathogens and food spoilage bacteria. Predominant genera in

63 meat processing plants after C&D include *Pseudomonas*, *Acinetobacter*, *Staphylococcus* and

64 *Serratia* (5-7). One of the pathogens regularly encountered in such environments is *Listeria*

65 monocytogenes, which causes the life-threatening disease listeriosis. This bacterium poses a

66 significant food safety challenge given its wide distribution in nature and its ability to grow at

67 refrigeration temperatures and to survive and persist on equipment in food processing

68 environments. Contamination of food products with *L. monocytogenes* mainly occurs in the

69 food production environment, and is a concern especially with regard to ready-to-eat (RTE)

70 products such as cold meat cuts. Transfer of *L. monocytogenes* from food contact surfaces such
71 as conveyor belts onto processed food products have been documented, and in some cases

72 shown to result in outbreaks of listeriosis (8, 9).

73 Certain strains of *L. monocytogenes* can establish in the production environment and persist for

74 months or even years, especially in humid areas and areas where C&D is difficult. Persistent

75 strains of *L. monocytogenes* often belong to certain molecular subtypes, while other subtypes

76 are found only sporadically (10-14). Several studies have investigated whether phenotypic traits

77 such as the ability to form biofilms and survive biocide action may be responsible for the

78 prolonged persistence of certain strains on food processing plant surfaces (15-18). Individual

79 strains of *L. monocytogenes* have been shown to vary in their ability to form biofilms (19, 20)

80 and differ in their tolerance towards disinfectants (21, 22). However, no single genetic

81 determinant or individual trait responsible for *L. monocytogenes* persistence has been

82 identified, and it is now generally thought that the perceived persistence of certain subtypes of

83 *L. monocytogenes* is due to a complex combination of factors (13, 14).

84 The resident background microflora is recognized to play an important role with respect to

85 protecting and sheltering pathogenic strains within food processing environments. Weak biofilm

86 formers can for instance improve their survival by joining a multispecies biofilm (23-25).

87 Additionally, it appears that biofilms composed of multiple genera are generally less susceptible

88 to biocide action than their single-species counterparts (4, 23, 26, 27). For example, under most

89 conditions, dual species biofilms of *L. monocytogenes* and *Lactobacillus plantarum* were more

90 tolerant to benzalkonium chloride and PAA than were the corresponding single species biofilms

91 (28). Nevertheless, specific bacterial interactions, which include competition, coaggregation and

5
92 metabolic cross-feeding, may have variable effects on the survival of individual biofilm

93 community members (23). Growth of *L. monocytogenes* in dual-species biofilms with

94 representative strains from food production environments has for instance resulted in both

95 enhanced and reduced cell numbers of *L. monocytogenes* (29). It is, however, not clear to what

96 extent these effects vary between strains or subtypes of *L. monocytogenes*, or how different *L.*

97 *monocytogenes* strains survive in more complex multigenera biofilms subject to conditions

98 similar to those found in food industry.

99 The purpose of this study was to examine biofilm formation and survival of strains belonging to
100 bacterial genera commonly isolated from conveyor belts in meat processing environments,

101 under conditions simulating those encountered in these environments. This included an
102 assessment of the efficacy of C&D under relevant conditions, and an examination of how the
103 background microbiota may affect growth and survival of persistent and sporadic L.

104 *monocytogenes* subtypes in biofilms exposed to C&D. Initially, the microbiota surviving C&D of
105 conveyor belts in meat processing plants was identified. An experimental biofilm model system

106 was then set up using conditions realistic for food industry, including growth on coupons cut
107 from conveyor belt material and exposure to daily cycles of C&D. Biofilms composed of L.

108 *monocytogenes* strains were compared with complex multigenera biofilms inoculated with both

109 *L. monocytogenes* and selected strains dominating the bacterial flora identified in meat

110 processing environments. The development of the biofilm microbiota was investigated using

111 viability counting, amplicon sequencing and imaging techniques.
112 RESULTS

113 **Identification of microbiota on conveyor belts in meat processing plants.** Sampling of
114 nine conveyor belts after sanitation in two meat processing plants resulted in identification of a
115 total of 121 isolates from a total of 22 genera (Table 1). Eight genera were common for both
116 plants, but overall, the microbiota after sanitation differed between plants and between single
117 conveyor belts. For two of the six conveyor belts sampled in Plant A, the bacterial numbers were
118 very low and four or less isolates were collected (conveyors 4 and 5). For conveyors with higher
119 bacterial numbers, *Pseudomonas* was most frequently isolated and dominated alone in one
120 sample, together with *Psychrobacter* in another, and with *Acinetobacter* on a third conveyor
121 belt. For one conveyor belt, which was associated with a permanent *L. monocytogenes*
122 (MF5377) reoccurrence, a diverse microbiota was found in which *Microbacterium* dominated
123 together with *Epilithonimonas*. In Plant B, *Sphingomonas* dominated together with *Rhodococcus*
124 on one conveyor and with *Acinetobacter* on another. Only five isolates were collected from the
125 third conveyor belt. A total of 16 isolates were selected for the present study, representing the
126 most dominant bacteria (Table 2).

127 Three *Pseudomonas* sp. and two *Acinetobacter* sp. were subjected to whole genome sequencing
128 and phylogenetic analysis to further determine their taxonomic status. This analysis showed
129 that strain MF6396 belonged to the *Pseudomonas putida* group, and that strains MF6394 and
130 MF4836 belonged to the *Pseudomonas fluorescens* and *Pseudomonas mandelii* subgroups,
131 respectively, within the *P. fluorescens* complex (Fig. S1 in the Supplemental Material). Thus all
132 three strains belong to the *P. fluorescens* lineage. For simplicity, these strains are referred to as
133 *P. putida* MF6396, *P. fluorescens* MF6394 and *P. mandelii* MF4836 in the remainder of this text.

134 Both *Acinetobacter* strains included in the experiments (MF4640 and MF4642) were determined

135 to belong to the species *A. johnsonii* using *in silico* multilocus sequence typing (MLST) (Fig. S2 in

136 the Supplemental Material).

137 *Pseudomonas* and *Acinetobacter* dominated in laboratory multigenera biofilms. A

138 biofilm model system was set up to examine biofilm formation and survival under conditions

139 simulating food production environments. Biofilms were grown on conveyor belt coupons

140 placed vertically in 24-well plates with BHI broth at 12°C, which is a temperature typically found

141 in Norwegian meat processing facilities. In addition to the 16 strains from the background

142 microbiota found on conveyors in meat processing plants (described above), seven *L.*

143 *monocytogenes* strains belonging to different phylogenetic clusters were selected for inclusion

144 in biofilm experiments (according to selection criteria in Materials and Methods section). Four

145 belonged to MLST sequence types (STs) responsible for persistent contaminations in Norwegian

146 food processing plants, while three strains belonged to STs which were only sporadically

147 encountered in Norwegian food industry (Table 2) (22). Coupons were inoculated with a

148 suspension of either the 16 background microbiota strains plus the seven *L. monocytogenes*

149 strains (referred to as multigenera biofilms), or with only the seven *L. monocytogenes* strains (*L.*

150 *monocytogenes* biofilms). The biofilms were allowed to develop for four days and subsequently

151 subjected to C&D on Days 4 to 7, using a chlorinated alkaline cleaning agent (Alkalifoam) and

152 disinfection with either a QAC- or PAA-based disinfectant, at user concentrations recommended

153 by the manufacturers. Wells containing multigenera biofilms usually contained a floating pellicle
154 that was attached to the coupon at the air-liquid interface. Visible biofilm deposits were
155 generally observed in this zone of the coupons after C&D.

156 The development of the microbiota in the multigenera biofilms was investigated using 16S rRNA
157 amplicon sequencing. The results showed that after four days of biofilm growth, one of the A.
158 *johnsonii* strains (MF4640) dominated the biofilm, while after seven days of growth, the *P.*
159 *putida* strain (MF6396) had taken over as the dominant strain. The proportion of *L.*

160 *monocytogenes* in the multigenera biofilm was higher on Day 7 than on Day 4 (Fig. 1A). To
161 investigate whether the shift in microbiota from Day 4 to Day 7 was only due to establishment
162 of a more mature biofilm, or also affected by the C&D cycles, new experiments were conducted
163 in which only the three dominating *Pseudomonas* spp. strains and *A. johnsonii* strain MF4640
164 were included. Here, coupons that were rinsed daily with H₂O were included in addition to
165 coupons treated with C&D agents. The results presented in Fig. 1B show that the bacterial strain
166 composition identified on coupons subjected to C&D in these two additional experiments were
167 similar to those obtained in the first three experiments in which biofilms were inoculated with
168 all 16 background microbiota strains (Fig. 1A and Fig. S3 in the Supplemental Material).

169 However, in the absence of C&D, no significant shift in the microbiota composition was
170 observed from Day 4 to Day 7, and the *A. johnsonii* strain dominated, followed by *P. putida*. This
171 indicated that daily exposure to C&D selected for *P. putida*, *P. fluorescens* and *L. monocytogenes*
172 and almost eliminated the *P. mandelii* and *A. johnsonii* strains.

173 **No selection between different L. monocytogenes strains was observed in biofilms.** To
174 determine whether the different *L. monocytogenes* strains had different fitness during growth
175 in biofilms subjected to C&D, strain identification of single colonies collected after Day 7 of
176 biofilm growth were performed by sequencing of the *dapE* MLST allele (Fig. 2A and Fig. S4 in the
177 Supplemental Material). The frequencies of each strain across all tested samples ranged from
178 5% for MF5378, to 28% for MF5360. The four strains belonging to persistent subtypes had an
179 overall frequency of 51% across all samples, indicating that these strains did not have a greater
180 ability to survive in biofilms exposed to C&D than strains belonging to sporadic subtypes. No
181 evidence for selection between different *L. monocytogenes* strains was observed, neither in the
182 multigenera biofilms where *L. monocytogenes* was grown in the presence of 16 background
183 flora strains, nor in biofilms containing *L. monocytogenes* only.

184 **Strains dominating in the multigenera biofilm showed high growth rates in planktonic culture.** The relative amounts of each bacterial strain in planktonic cultures inoculated with the same bacteria as were used in the multigenera biofilm experiments is shown in Fig. 1C. As in the biofilm experiments, the bacterial composition developed towards *A. johnsonii*, *Pseudomonas* spp. and *L. monocytogenes*. In contrast to during growth in biofilm conditions, however, both *A. johnsonii* strains (MF4640 and MF4642) seemed to compete equally well under planktonic culture conditions. Similarly, the three *Pseudomonas* spp. strains were in approximately equal proportions in the planktonic cultures, while in the biofilms, there was significantly more of *P. putida* MF6396 than of *P. fluorescens* MF6394 and especially *P. mandelii* MF4836.

193 All seven *L. monocytogenes* strains were retained in approximately equal amounts when grown
194 together in planktonic culture, both when they were grown alone and when they were grown
195 together with the 16 background microbiota strains (Fig. 2B and Fig. S4 in the Supplemental

196 Material). The proportion of each strain present in the cultures containing only *L.*

197 *monocytogenes*, determined using *dapE* amplicon sequencing, ranged from an average 9%

198 (MF5376/ST7) to 21% (MF5377/ST8) after 72 h of growth. When the seven *L. monocytogenes*

199 strains were grown together with the 16 background microbiota strains, the proportion of each

200 *L. monocytogenes* strain after 72 h ranged from 12% (MF5376/ST7 and MF5634/ST121) to 18%

201 (MF5377/ST8). These results indicated that during planktonic growth at 12°C, none of the seven

202 *L. monocytogenes* strains appeared to have a growth advantage allowing them to outcompete

203 any of the other strains.

204 When the individual strains were grown in separate wells in a Bioscreen C instrument (Fig. 3 and

205 Table S1 in the Supplemental Material), the largest maximal growth rates during the exponential

206 phase of growth was attained by *P. fluorescens* MF6394, followed by *P. mandelii* MF4836, the

207 two *A. johnsonii* strains and then *P. putida* MF6396. The cultures containing *Pseudomonas*

208 strains ultimately reached higher values of OD₆₀₀ than the *Acinetobacter* cultures. Other strains

209 with high growth rates were the seven *L. monocytogenes* strains (which all had similar growth

210 curves) and *Epilithonimonas* strain MF6392, followed by the *Psychrobacter* and *Microbacterium*

211 strains (MF4641 and MF4634). It thus seems like the strains showing rapid planktonic growth at

212 12°C in BHI culture medium are highly competitive in the biofilms grown on conveyor belt

213 coupons.
214 The *Pseudomonas* genomes contained different sets of known biofilm-associated genes.

215 The difference in competitiveness between the three *Pseudomonas* strains and between

216 the two *A. johnsonii* strains in planktonic culture compared to growth in the conveyor belt

217 biofilm model (Fig. 1) could possibly be due to differences in the ability to form biofilms.

218 Therefore, the *Pseudomonas* spp. and *A. johnsonii* genomes were screened for known biofilm-associated genes using BLAST analysis. All three *Pseudomonas* strains contained the *alg* operon

219 required for alginate synthesis, and homologs to the *lapABCD* and *lapG* genes required for

220 expression of the large surface protein LapA on the cell surface. The genes responsible for Pel,

221 Psl, and cellulose synthesis were however each only present in one of the three strains: *P.*

222 *fluorescens* MF6394 contained a *psl* operon, *P. mandelii* MF4836 contained a *pel* operon, while

223 a homolog to the *wss* operon required for cellulose synthesis was present in *P. putida* MF6396.

224 It thus appears that all three strains harbor genetic factors enabling biofilm formation (Table S2

225 in the Supplemental Material). With respect to the two *A. johnsonii* strains, not much is known

226 about biofilm formation in non-*baumanii* *Acinetobacter* strains, and no homologs to genes

227 shown to be involved in biofilm formation in *Acinetobacter baumanii* were identified in the

228 genomes of the two *A. johnsonii* strains employed in the current study. The two strains did

229 however have different genome sizes, as the genome of *A. johnsonii* MF4640 was 13% larger

230 than that of the 3.36 Mbp large genome of strain MF4642. A large portion of the additional

231 genetic material in MF4640 appears to constitute plasmids and other mobile genetic elements.

232

233 The sanitation regime was inefficient at killing bacteria in conveyor belt biofilms. To

234 assess sanitation efficacy in the biofilm model system, the total number of colony-forming units
235 (CFUs) in biofilms growing on conveyor belt coupons was determined both before and after coupons were subjected to C&D.

237 After the initial four days of biofilm development, the cell densities in multigenera biofilms reached about $1\times10^8$ CFUs per coupon (3 cm$^2$ surface area). Coupons were then subjected to daily cycles of C&D for three days, and sampled again on Day 7 after allowing 24 h of regrowth after the last C&D cycle. Control coupons were rinsed with sterile deionized water ($H_2O$) every day. There was no significant difference in cell densities on coupons with multigenera biofilms sampled prior to C&D on Day 4 and Day 7, regardless of whether coupons had been treated with QAC, PAA, or rinsed in $H_2O$ ($P>0.05$; Fig. 4, grey bars). Thus neither the QAC- nor PAA-based C&D regimes altered the total amount of biofilm on conveyor belt material present 24 h after C&D treatment.

246 However, while the total number of CFUs on each coupon in the multigenera biofilm was similar in all tested samples, the amount of *L. monocytogenes* in the biofilm increased about tenfold from Day 4 to Day 7 (Fig. 4, yellow bars). The fraction of *L. monocytogenes* in the multigenera biofilms increased from 2.3% (SE±1.1%) on Day 4 to 9% (SE±2%), 18% (SE±4%) and 32% (SE±7%) in the $H_2O$-rinsed, QAC-treated, and PAA-treated biofilms, respectively, harvested on Day 7. In the biofilms where *L. monocytogenes* were grown alone, however, there was no statistically significant difference in *L. monocytogenes* counts per coupon between Days 4 and 7 ($P>0.05$; Fig. 4, green bars), with around $2\times10^7$ CFUs per coupon on both days and across the different treatments.
255 When the total numbers of CFUs per coupon before and after C&D were compared, between
256 0.6 and 0.9 log₁₀ reductions in total CFUs were observed on Day 4 and Day 7, respectively, for
257 coupons harboring multigenera biofilms (Fig. 5, grey bars). The difference in log₁₀ reductions
258 between treatments or day of sampling was not statistically significant (*P*>0.05).

259 The log₁₀ reduction for the *L. monocytogenes* component of the multigenera biofilm was
260 significantly lower than the log₁₀ reduction in total CFUs per coupon when the Day 4 coupons
261 were treated with PAA disinfection (0.6 vs. 0.9 log₁₀ reduction; *P*=0.04). For the other
262 treatments (QAC-treatment on Days 4 and 7, and PAA-treatment on Day 7), there was no
263 difference in survival of the flora strains and the *L. monocytogenes* strains in the multigenera
264 biofilm upon C&D (*P*>0.05; Fig. 5, compare grey and yellow bars). This indicates that the
265 proportion of *L. monocytogenes* cells in the biofilm was relatively stable during a cycle of C&D.

266 For the *L. monocytogenes* biofilms, on Day 4, the reduction in CFUs per coupon upon sanitation
267 treatment was about the same as for the multispecies biofilms. On Day 7, however, there was
268 almost no reduction in bacterial numbers upon C&D, with average reductions in cell numbers of
269 only 0.13 and 0.26 log₁₀ CFUs per coupon upon QAC- and PAA-treatment of the biofilms,
270 respectively (Fig. 5, green bars).

271 Overall, these experiments indicated that biofilms on conveyor belt materials were not
272 eliminated when exposed to a C&D regime relevant for the food industry. Little or no
273 development of tolerance to C&D agents was observed for the multigenera biofilms during the
274 course of the experiment. The *L. monocytogenes* biofilms, however, did develop increased
275 tolerance over time, as no significant reductions in CFU was observed during the C&D process

276 after the coupons had been exposed to three daily cycles of cleaning followed by either

277 disinfection with PAA or a QAC.

278 All strains were susceptible to the sanitation agents in suspension tests. To examine

279 whether any of the strains included in the multigenera biofilms had a specific tolerance towards

280 the employed C&D agents that could explain survival, bactericidal suspension tests were

281 performed on each strain, using both QAC and PAA disinfection agents as well as the Alkalifoam

282 cleaning agent. For all strains, the bacterial reductions were over 4 log₁₀ units after exposure to

283 recommended user concentrations of the QAC and PAA disinfectants for 5 min at 12°C (Table S3

284 in the Supplemental Material). Most strains also showed the same level of tolerance to the

285 cleaning agent alone. The exceptions were the two *Corynebacterium* sp. strains and the

286 *Micrococcus* sp. strain, which showed only between 10 and 100-fold reduction in CFUs upon

287 treatment with the cleaning agent, and the two *Kocuria* sp. strains, *P. putida* strain MF6396, and

288 the *Psychrobacter* sp. strain, which showed 3 log₁₀ to 4 log₁₀ reductions in CFU per ml upon

289 treatment with the cleaning agent. These results indicate that all strains were susceptible to the

290 C&D treatment when grown in suspension.

291 CLSM analysis showed that biofilms predominantly settle on the underside of the

292 conveyor belt material. Confocal laser scanning microscopy (CLSM) was employed to examine

293 the spatial organization of biofilms formed on the conveyor belt coupons. The three-

294 dimensional image reconstructions shown in Fig. 6 and Fig. 7 were obtained by scans of several
295 pre-defined location patterns on each coupon (see Fig. 6D), and were selected from 174

296 acquired confocal Z-stack scans (see Table S4 in the Supplemental Material). In the majority of
297 captured scans, relatively few sparse cells – attached singly or as small clusters – were observed

298 on the coupon surface. However, a significant number of images showed the presence of large
299 heterogeneous three-dimensional biofilms. These were also observed on some of the coupons

300 examined immediately after cleaning and disinfection with QAC or PAA. In the multigenera
301 biofilms, GFP expressing *L. monocytogenes* cells were often absent despite observations of
302 significant numbers of background flora cells. When present, *L. monocytogenes* were spatially
303 organized as single cells mixed in between the cells of the background flora strains. In some of
304 the images, the biofilm also appeared to have a slightly layered structure, with *L.*

305 *monocytogenes* cells found closer to the bottom layer of the biofilms (Fig. 7K). No separate *L.*
306 *monocytogenes* monospecies microcolonies were observed on the coupons in which
307 multigenera biofilms were grown.

308 The top face of the conveyor belt is coated with PVC, and is a matt antistatic surface (Fig. 6D).

309 The underside of the conveyor belt is an urethane-impregnated woven polyester fabric. The
310 photomicrograph in Fig. 7B, taken of the underside of a coupon, shows the linen weave pattern

311 with single smooth warp threads and weft threads composed of bundles of smaller fibers. The
312 difference between the flat top face and the heterogeneous topography of the rear face of the
313 conveyor belt coupons can be seen in the overview images obtained by stitching together

314 multiple CLSM scans – acquired across the length of the coupon from top to bottom – shown in
315 Fig. 6E and Fig. 7D. Notably, for the rear side of the coupons, scans were only obtained for the
316 most elevated parts of the fabric, since the microscope was not able to focus in the areas

317 constituting the «valleys» in the fabric surface. Most striking were images acquired for biofilms

318 formed on weft threads composed of bundles of smaller fibers on the rear side of the conveyor

319 belt coupons, as shown in Fig. 7E and F. Both the background flora and *L. monocytogenes* cells

320 are predominantly found in the gap between these fibers. In the multigenera biofilms,
321 mushroom shaped biofilm structures could be observed to protrude upwards from the cleft
322 harboring bacterial cells (Fig. 7G).

323 Quantitative analysis of the biovolume of GFP expressing *L. monocytogenes* cells in the biofilms
324 was performed by analysis of the green channel of the acquired CLSM image stacks (Table 3 and
325 Fig. S5 in the Supplemental Material). The calculated biovolume of *L. monocytogenes* cells was
326 higher prior to C&D than after treatment with QAC or PAA. Also, the results suggest that the
327 total *L. monocytogenes* biovolume was higher in biofilms harvested on Day 7 compared to
328 biofilms harvested on Day 4, both in multigenera biofilms and in *L. monocytogenes* single
329 species biofilms. Finally, the analysis strongly indicates that significantly more *L. monocytogenes*
330 cells were attached to the woven-structured underside of the conveyor belt than on the PVC-
331 coated top surface. The strongest effect was seen for *L. monocytogenes* biofilms rinsed in H₂O
332 daily from Days 4 to 7 and harvested on Day 7, in which 14 (SE±11) µm³ and 2841 (SE±1439)
333 µm³ *L. monocytogenes* cells were found on the top and bottom faces of the conveyor belt
334 coupons, respectively.

335 In summary, the microscopy showed that *L. monocytogenes* cells were spatially intermixed with
336 background flora species in the multigenera biofilms. Furthermore, bacteria appeared to be
337 predominantly situated in the gaps between filament fibers on the underside of the conveyor

338 belts.

339 DISCUSSION

340 The current study aimed to decipher growth and survival of *L. monocytogenes* on conveyor belts

341 in food industry using conditions relatively realistic for those found in meat production

342 environments. This included growing strains of *L. monocytogenes* in multigenera biofilms with

343 strains from the background microbiota isolated in these environments. Initial investigation of

344 the microbiota on conveyor belts after C&D in two RTE meat-processing plants resulted in

345 isolation of a relatively small number of bacteria, but nevertheless, a high diversity was found

346 between and also within samples (Table 1). A relatively diverse microbiota was therefore used

347 in the initial biofilm experiments (Table 2). Similar to what has been found in other studies,

348 *Pseudomonas* was relatively common after C&D (5-7, 30-32). *Enterobacteriaceae* has also been

349 reported to be common in meat processing environments (5-7, 30, 31, 33, 34), but was absent

350 in our study. Instead, microbiota of conveyor belts were dominated by bacteria less frequently

351 reported in previous studies, such as *Acinetobacter*, *Microbacterium*, *Sphingomonas* and

352 *Epilithonimonas* (Table 1). The composition of the microbiota is dependent on a number of

353 factors such as the sanitation regime, the temperature and the humidity. Biofilm formation

354 reflecting all these varying conditions would not be possible in *in vitro* laboratory studies. In this

355 study, we chose to simulate conditions with high humidity and nutrient content at a

356 temperature relevant for meat processing environments (12°C), and apply C&D cycles similar to

357 those found in food industry.
The composition of the biofilms formed on conveyor belt coupons under these conditions was largely stable – with a dominance of *Pseudomonas* and *Acinetobacter* strains – regardless of whether four or 16 background strains were used as inocula, and regardless of whether coupons had been treated with QAC, PAA, or rinsed in H₂O (Fig. 1A and B). Stable coexistence of *Acinetobacter* and *Pseudomonas* strains in biofilms has been reported previously (35, 36). In the current study, the composition of the biofilms shifted from an *Acinetobacter*-dominated biofilm in the Day 4 samples to a *P. putida*-dominated biofilm in the Day 7 samples subjected to daily C&D (Fig. 1A and B). This transition was not seen in biofilms instead subjected to daily rinse in H₂O on Days 4 to 7 (Fig. 1B; column labelled H₂O). Furthermore, in suspension, *P. putida* MF6396 had a higher tolerance towards the lethal effect of the chloralkali cleaning agent than *A. johnsonii* and the other included *Pseudomonas* strains. This suggests that the dominance of the *P. putida* strain in biofilms subjected to daily C&D could be a consequence of the C&D treatments and tolerance of the *P. putida* strain towards the cleaning agent. However, the relative levels of *Acinetobacter* decreased over time also in the planktonic competition experiments performed in the current study (Fig. 1C) and in multigenera biofilm experiments performed in a previous study, in which the effect of C&D was not assessed (37). Potentially, interspecies interactions such as competition for limiting nutrient sources may also have contributed to the observed transition in microbial composition between the Day 4 and Day 7 biofilms.

Specific bacteria may show enhanced survival in biofilms challenged by biocides by means of interspecies interactions such as coaggregation and metabolic cross-feeding (27). Interactions
379 with other bacteria in biofilms may potentially explain the persistence of pathogens such as *L.*

380 *monocytogenes* in food production environments. In the current study, the proportion of *L.*

381 *monocytogenes* in the multigenera biofilms increased during the course of the experiment,

382 concomitant with the shift towards a *P. putida*-dominated biofilm. This is consistent with *L.*

383 *monocytogenes* specifically interacting with the *P. putida* strain. Interestingly, this specific strain

384 (MF6396) was isolated from a conveyor belt which was persistently contaminated with *L.*

385 *monocytogenes*, and from which the persistent ST8 strain *L. monocytogenes* MF5377 was

386 isolated (see Table 1) (38). It is therefore likely that MF6396 and MF5377 may have originated

387 from the same microhabitat in the meat production plant. Examination of biofilms using CLSM

388 in the current study showed that cells of *L. monocytogenes* were found intermixed with

389 background flora cells, with no spatially segregated *L. monocytogenes* microcolonies observed

390 within the multigenera biofilms (Fig. 6 and Fig. 7). Such spatial distribution patterns in

391 multispecies biofilms are indicative of interspecies coaggregation and cooperation (39, 40),

392 further suggesting that *L. monocytogenes* cells may directly interact with one or more of the

393 other species found in the biofilm. Previous studies have shown that co-culture of *L.*

394 *monocytogenes* and resident apathogenic bacteria from food production environments have

395 resulted in both positive and negative effects on the biomass of *L. monocytogenes* (29, 41).

396 Potential specific interactions between the individual strains examined in the current study are

397 subject to further examination in our laboratory.

398 The observation that certain subtypes of *L. monocytogenes* are more likely than others to

399 persist in food processing environments has prompted several investigators to examine whether
400 genetic determinants or various phenotypic traits could be associated with this ability. One of
401 the aims of the current study was to examine whether this perceived persistence may be linked
402 to strain-specific differences in the ability of *L. monocytogenes* to interact with the resident
403 microflora in biofilms. Few studies have addressed this point specifically, although in a recent
404 study, Overney et al. (42) found that two reference strains of *L. monocytogenes* (EGD-e and
405 LO28) did not differ in survival rate when they were grown in dual culture biofilms with a *P*.

406 *fluorescens* strain, when biofilms were subject to daily cycles of C&D and desiccation. A similar
407 result was obtained in the current study, where seven *L. monocytogenes* strains – four of which
408 belonged to subtypes linked to persistent contaminations in food production facilities – were
409 shown to be equally capable of growth and survival in biofilms exposed to C&D (Fig. 2). This
410 result was obtained both with monospecies and multigenera biofilms, and is consistent with the
411 growing consensus that individual genetic traits linked to specific subtypes do not account for
412 the existence of persistent subtypes of *L. monocytogenes* (13, 14).

413 It is widely acknowledged that the efficacy of C&D agents is lower for biofilms than for bacteria
414 growing in planktonic culture (3, 4, 27). A high level of tolerance to C&D was also observed for
415 biofilms in the current study, with less than 1 log₁₀ reductions in total CFUs per coupon obtained
416 across treatments, when the C&D agents were applied at the concentrations recommended by
417 the manufacturers (Fig. 5). A similar level of efficacy of C&D agents applied at recommended
418 user concentrations was seen in a study by Pan et al. (43), where *L. monocytogenes* biofilms –
419 grown on stainless steel or Teflon coupons and subjected to daily cycles of sanitation followed
420 by starvation and incubation in dilute culture medium – were followed over a period of three
421 weeks. In their study, treatments of biofilms with minimum recommended user concentrations

422 of peroxide, QAC or chloride disinfection resulted in less than 0.3 log₁₀ CFU cm⁻² after the first

423 week of their simulated food processing regimen. However, not all studies find the efficacy of

424 C&D agents against biofilms to be this low – in some studies the disinfection agents have to be

425 diluted below recommended user concentrations in order to maintain enough cells above the

426 detection threshold after disinfection of biofilm coupons (42). Also, previous observations of

427 PAA being more effective against *L. monocytogenes* biofilms than QAC (44) was not supported

428 by the results obtained in the current study. In any case, the explanation for the low efficacy of

429 C&D seen in the current study cannot be attributed to the greater tolerance towards biocides

430 commonly observed for multispecies biofilms compared with their single species counterparts

431 (4, 23, 26), because the opposite was actually observed: *L. monocytogenes* biofilms were shown

432 to become more tolerant to daily C&D than the multigenera biofilms (Fig. 5). Since no significant

433 difference in survival of bacteria was observed between treatments with QAC or PAA

434 disinfectants, the low efficacy of C&D was furthermore not likely to be a result of specific

435 resistance mechanisms such as the presence of efflux pumps conferring resistance towards

436 chemical agents. This is supported by the observation that no selection between different *L.*

437 *monocytogenes* strains was seen despite two of the strains possessing the *qacH* gene encoding

438 an efflux pump conferring increased tolerance to low concentrations of QAC compounds (Table

439 2) (45).

440 The explanation for the low efficacy of the C&D treatment could instead, at least partly, be

441 ascribed to features of the coupon material on which biofilms were grown. Within food
442 processing plants, conveyor belts have been shown to be favorable to contaminations with *L.*

443 *monocytogenes* that are difficult to remove (9, 46). Furthermore, cracks or scratches in the
444 surfaces of materials used in food industry been shown to support development of *L.*

445 *monocytogenes* biofilms deeply rooted in microscopic sutures and ridges (47). The underside of
446 the conveyor belt used as the surface for biofilm growth in the current study had a woven
447 surface with filament fiber threads. When coupons were viewed using CLSM, bacteria could be
448 seen to shelter in the clefts between these fibers (Fig. 7), and quantitative biovolume analysis
449 furthermore suggested that significantly more *L. monocytogenes* cells were attached to the
450 underside of the conveyor belt than on the smooth top coating (Table 3). Bacteria could also be
451 expected to find harborage sites on the cut edges of the conveyor belt coupons, which –
452 although likely to be sealed to prevent penetration of soiling and bacteria when conveyors are
453 initially installed in food production plants – could be said to model situations where worn or
454 frayed conveyors are employed in a production facility.

455 The observed increase in tolerance to C&D by *L. monocytogenes* biofilms over time both for the
456 QAC and PAA disinfectants (Fig. 5) concurs with results obtained in the study by Pan et al. (43),
457 in which *L. monocytogenes* appeared to develop similar levels of biofilm-specific resistance to
458 disinfection with peroxide, QAC and chloride during the course of the experiment. This indicates
459 that a broad-spectrum mechanism, probably related to the biofilm mode of growth, was
460 responsible for the increased tolerance seen in both studies. This increase may potentially also
461 be linked to attributes of the coupon surface on which biofilms were grown. When biofilms
462 were examined using CLSM in the current study, larger *L. monocytogenes* biofilm aggregates
463 were always seen confined to the clefts and surface structures on the underside of the conveyor

464 belt material, while the multigenera biofilms were regularly observed to protrude outwards

465 from the crevices in which they were rooted. Conceivably, spatial growth patterns and/or a

466 relatively modest growth rate could account for *L. monocytogenes* biofilms not extending

467 beyond the shelter of the crevices during the 24 h separating two cycles of C&D, thereby

468 resulting in the observed lower reduction in *L. monocytogenes* numbers upon C&D on Day 7

469 compared to on Day 4 (Fig. 5).

470 In summary, the results from the present study showed that *L. monocytogenes* can grow and

471 survive in multigenera biofilms formed from bacteria belonging to the background microbiota

472 isolated in meat industry environments, even after several rounds of C&D. Furthermore, the

473 results suggest that regular C&D agents used in food industry fail at removing biofilms from

474 heterogeneous surfaces harboring cracks or crevices. Although the underside of a conveyor belt

475 is not intended to be in direct contact with food, it may confer harborage sites from which

476 bacteria can shelter and cross-contaminate food-contact surfaces during processing. Further

477 research into more efficient methods for removal of biofilms and a greater focus on hygienic

478 design of food processing equipment is warranted.

479 MATERIALS AND METHODS

480 Isolation of bacteria from conveyor belts in meat processing plants. Two plants

481 processing RTE meats were visited. Samples from a total of nine conveyors – six from Plant A

482 and three from Plant B – were taken after C&D, before start of production. The daily sanitation
483 included a chloralkali agent for cleaning followed by disinfections using QAC in Plant A and PAA
484 in Plant B. In addition, the conveyors in Plant B were disinfected with 70% ethanol several times
485 during the production day, between processing of different products and before breaks. An area
486 of approximately 900 cm² was sampled with neutralizing sampling cloths (Sodibox, Nevez,
487 France). The cloths were stored at 4°C and analyzed within 36 h. Ten ml peptone water (1 g l⁻¹
488 peptone [Oxoid], 0.85% NaCl, pH 7.2) was added to the plastic bag containing the cloth, and
489 after 30 seconds treatment in a Stomacher, 1 ml samples were plated to blood agar directly and
490 after dilution in order to obtain single well separated colonies for identification. The agar plates
491 were incubated at 20°C for 5 days. Up to 20 colonies were picked at random, restreaked for
492 purification and subjected to 16S rRNA sequencing (V3-V4 region) for identification using the
493 universal 16S rRNA primers tcctacgggaggcagcagt and ggactaccagggtatctaatcctgtt (48), as
494 previously described (37). The taxonomy of each strain was assigned by using the SeqMatch tool
495 of the Ribosomal Database Project (RDP), with database v.11.5 (https://rdp.cme.msu.edu).

496 **Selection criteria for background microbiota strains included in biofilm experiments.** A
497 total of 16 strains isolated from conveyor belts in meat processing Plants A and B were selected
498 for inclusion in multigenera biofilm experiments (Table 2). Of these, 14 isolates represented the
499 nine most frequently found genera after C&D. All these genera were among the dominating
500 (>20% of the colonies) in at least one sample. More than one isolate were chosen from genera
501 with diversity in the 16S rRNA amplicon sequences. In addition, *Kocuria* isolates were included
502 because *Kocuria* spp. has been reported to promote biofilm formation of *L. monocytogenes* in
503 an earlier study (29). All strains included in the experiments had unique 16S rRNA amplicon

504 sequences, enabling their differentiation by 16S rRNA sequencing.

505 **Selection criteria for L. monocytogenes** strains. Seven *L. monocytogenes* strains from three

506 different meat processing facilities [Plant A and B and a third plant; Plant C; corresponding to

507 plants M2, M4, and M1, respectively, from Møretrø et al. (22)] were selected for inclusion in

508 experiments in the current study (Table 2). These strains had been collected as part of two

509 research projects where nine Norwegian food processing plants were sampled, resulting in

510 isolation of a total of 680 *L. monocytogenes* strains subsequently typed using multiple locus

511 variable number tandem-repeats analysis (MLVA) (22). Of the seven strains used in the current

512 study, four were from MLVA profiles that were identified as persistent in the said projects using

513 the statistical approach described in Malley et al. (49) (results to be published separately), and

514 which were detected after C&D in more than one of the nine sampled Norwegian facilities. The

515 four included persistent strains were furthermore selected from individual strains isolated after

516 C&D at sampling points in which the same MLVA genotype had been found on several

517 occasions. Three *L. monocytogenes* strains were selected from so-called sporadic MLVA profiles.

518 These strains were selected based on the criteria that they should be isolated during

519 production, and that their MLVA profiles were not commonly found after C&D. All seven

520 selected strains had different *dapE* alleles, enabling their differentiation by sequencing the *dapE*

521 MLST allele (50). Alleles and sequence types for MLST were compared with those available in

522 the Institute Pasteur's *L. monocytogenes* MLST database

523 (http://bigsdb.web.pasteur.fr/listeria/listeria.html).
524 **Whole genome sequencing.** DNA isolation, whole genome sequencing and *de novo* genome assembly was performed essentially as previously described (38), with 300 bp paired-end sequencing on a MiSeq instrument (Illumina), except that genome assembly was performed with v3.10.0 of SPAdes (51) and inclusion of six k-mer sizes (21,33,55,77,99,127). Contigs with size <500 bp and with coverage <35 were removed from the assemblies. The sequences were annotated using the NCBI Prokaryotic Genomes Automatic Annotation Pipeline (PGAAP) server (http://www.ncbi.nlm.nih.gov/genome/annotation_prok/).

531 **Sequence search for known biofilm genes.** The *Pseudomonas* and *Acinetobacter* genome assemblies from the current study were analyzed for the presence of genes known to be involved in biofilm formation in these genera (52-54) using BLAST+ v2.2.30 (55). The following genes were used as queries in the analysis: *pslA-R* (PA2231-PA2246), *pelA-B* (PA3064-PA3058), the *alg* operon (PA3540-3551), and *cdrA* (PA4625) from *P. aeruginosa* PAO1 (Accession AE004091), genes *wssA-J* from *P. fluorescens* SBW25 (Accession AY074776), genes *lapA-G* (PP018-PP0164), *lapF* (PP0806), the *bcs* operon (PP2629-PP2638), *peaA-I* (PP3133-P3141), and the *peb* locus (PP1795-PP1788) from *P. putida* K4220 (Accession AE015451), the *csuA-E* genes encoding the pilus usher-chaperone assembly system from *A. baumannii* 19606 (Accession AY241696), *pgaA-D* (A1S_2160-2 and A1S_3792) from *A. baumannii* ATCC 17978 (Accession CP000521), and the gene encoding Bap from *A. baumannii* 307-0294 (Accession EU117203). Genome comparisons were performed using Mauve (56).
543 **Phylogenetic analysis.** The sequences of single genes or whole genomes from reference

544 strains used in phylogenetic analyses were downloaded from Genbank, and their accession

545 numbers are listed in Table S5 in the Supplemental Material. *Acinetobacter* strains were typed

546 *in silico* using the MLST scheme described by Diancourt et al. (57), while *Pseudomonas* strains

547 were analyzed using the MLSA scheme described by Mulet et al. (58). The concatenated

548 sequences of the seven MLST alleles (for *Acinetobacter*) or the four MLSA alleles (for

549 *Pseudomonas*) were aligned using CLCMain Workbench 7 (CLCbio). Phylogenetic trees were

550 then inferred from the alignments in MEGA7 (59) using the Neighbor-Joining method. The

551 evolutionary distances were computed using the Jukes-Cantor method and bootstrap

552 confidence values were generated using 1000 replicates.

553 **C&D agents.** C&D agents used in the current study were selected to represent products with

554 concentrations of active ingredients typical of industrial formulations. The industrial chlorinated

555 alkaline cleaning agent ISS Alkalifoam 27 (Ecolab, Norway), referred to as «Alkalifoam»

556 throughout the text, was used at a 1% concentration, which is the minimum recommended user

557 concentration indicated by the manufacturer. At this concentration the solution contains

558 minimum 0.02% NaOH and 0.03% sodium hypochlorite. Two industrial disinfection agents were

559 used. One was Aco Hygiene Des QA (Aco Kjemi, Norway), which is a formulation based on

560 quaternary ammonium compounds, referred to as «QAC» throughout the text. The second was

561 Diverfoam active (Lilleborg, Norway), which is based on peracetic acid, and referred to as «PAA»

562 throughout the text. Both are used at the indicated minimum user concentrations, which was

563 1% for QAC and 1.5% for PAA. At these concentrations, the QAC solution contains minimum
564 0.05% benzalkonium chloride while the PAA solution contains minimum 0.02% peracetic acid,

565 0.05% acetic acid, and 0.15% hydrogen peroxide.

566 Growth conditions in planktonic culture. Bacteria were grown in brain heart infusion (BHI)

567 broth (Oxoid) throughout all experiments. Overnight cultures and precultures were grown in 5

568 ml volumes in culture tubes and 50 ml Nunc-tubes, respectively, with shaking at 30°C, except for

569 *Sphingomonas* sp. MF4632, which was grown at 20°C. All biofilm and growth experiments were

570 carried out at 12°C. For plating, RAPID'L.mono (RLM) agar (Bio-Rad) and BHI agar (Oxoid) plates

571 were used.

572 For generation of growth curves for single strains, overnight cultures were diluted to

573 approximately 10⁵ CFU ml⁻¹ and inoculated in volumes of 250 µl in 100-well polystyrene

574 microwell plates (Oy Growth Curves Ab Ltd). The plates were incubated for 7 days at 12°C in a

575 Bioscreen C instrument (MTX Lab Systems Inc), with continuous shaking and recording of

576 OD₆₀₀nm every hour. Blank wells contained BHI broth only, and values for blanks were subtracted

577 from sample values to obtain actual absorbance measurements. Triplicate wells were used for

578 each sample and each strain was tested three or four times.

579 For the planktonic competition experiment, overnight cultures were mixed in roughly equal CFU

580 numbers in an inoculum diluted to a final total concentration of 10⁵ CFU ml⁻¹. Fifty ml culture

581 volumes were incubated in 500 ml baffled Erlenmeyer bottles at 12°C with shaking at 200 rpm.

582 Every 24 h, samples were withdrawn and plated to determine CFU count, and cells were
583 pelleted by centrifugation and stored at -20°C for use in amplicon sequencing analysis (see below).

585 **Construction of GFP-labelled *L. monocytogenes*.** *L. monocytogenes* strains were transformed with plasmid pNF8, from which the green fluorescent protein (GFP) is constitutively expressed (60). The pNF8 plasmid was a kind gift from Hanne Ingmer at the University of Copenhagen. Transformation was performed using the procedure described by Monk et al. (61).
586
587
588
589 Erythromycin at a concentration of 10 µg ml⁻¹ was used for selection of pNF8. The identity of all strains after transformation was confirmed by PCR amplification and sequencing the *dapE* MLST allele (50) using primers *ggtttcccagtcacgacgttgtacgactaatgggcatgaagaacaag* and *ttgtgagcggataacaatttcatcgaaactatgggcatcttttacc* for PCR (overhangs underlined) and primers *gttttcccagtcacgacgttgta* and *ttgtgagcggataacaatttc* for sequencing.

594 **Biofilm experiments with C&D.** Precultures of each strain were inoculated from glycerol stocks, prepared from exponential phase cultures and maintained at -80°C, grown separately to logarithmic phase, and mixed in roughly equal CFU numbers in an inoculum diluted to a final total concentration of ~10⁶ CFU ml⁻¹. The bacterial suspensions were inoculated in 24-well plates containing coupons of food grade PVC conveyor belt material (Forbo-Siegling Transilon; E 8/2 U0/V5 MT white FDA) cut to 1.0 cm × 1.5 cm, autoclaved, and placed vertically in each well.
595
596
597
598
599 One ml inoculum was added to each well so that wells were half-filled with culture broth, resulting in the air/liquid interface crossing the length of the coupon (see Fig. 6D). The plates 600
602 were incubated at 12°C with gentle orbital shaking, and the culture medium was refreshed on
603 Day 3.

604 Control coupons not subjected to C&D were harvested after four days of biofilm development.

605 Sets of coupons subjected to C&D (see below) on Day 4 were either harvested after treatment
606 or – for coupons to be harvested on Day 7 – placed in a new 24-well tray containing 1 ml BHI in
607 each well and incubated as before for 24 h. The cycles of C&D followed by incubation in BHI was
608 repeated on Days 5 and 6. On Day 7, sets of coupons treated with either QAC or PAA on Days 4
609 to 6 were harvested prior to and after C&D treatment. Coupons sampled prior to C&D (both on
610 Days 4 and 7) were rinsed three times in ~10ml H₂O (in 15 ml Falcon tubes) to remove non-
611 adherent bacteria before harvest. Control coupons subjected to rinsing in H₂O instead of
612 treatment with C&D agents on Days 4 to 7 were included in selected experiments.

613 Treatment with C&D agents was performed as follows: C&D agents were applied as foam (as
614 intended by the manufacturers), produced in foam pump bottles (Sunvita, Norway). Each
615 coupon was rinsed three times in ~10 ml H₂O (in 15 ml Falcon tubes) and placed vertically in
616 wells of a clean 24-well tray. The wells were filled with 1% Alkalifoam, coupons were incubated
617 5 minutes, rinsed as before in H₂O, and placed in a second clean 24-well plate. The wells were
618 then filled with 1% QAC or 1.5% PAA, coupons were incubated 5 minutes, and finally rinsed as
619 before in H₂O. The average weight of foam applied to each well was ~350 mg.

620 Cells attached to coupons were harvested as follows: Each coupon was transferred to a glass

621 tube containing 4.5 ml peptone water and 2 g glass beads of diameter ~2 mm (Assistant, No.
622 1401/2). Tubes were then vortexed for 30 seconds and sonicated for 10 minutes (Branson 3510
623 ultrasonic cleaner) to dislodge attached cells and disperse cell aggregates. After withdrawing
624 45.5 µl or 500 µl for plating dilutions on agar plates (to determine total and L. monocytogenes
625 CFU count per coupon), the remaining cells were pelleted by centrifugation (16000×g for 5 min)
626 and stored at -20°C. The identity of single L. monocytogenes colonies from dilutions plated after
627 harvesting coupons subjected to sanitation on Day 7 was determined by PCR amplification and
628 sequencing the dapE MLST allele (50) as described above.

629 Biofilms analyzed using CLSM were grown and subjected to rinsing or C&D as described above,
630 with the following exceptions: For L. monocytogenes, the strains labelled with GFP were used,
631 and overnight cultures for these were grown in the presence of 10 µg ml⁻¹ erythromycin. The
632 biofilm inoculum was prepared from overnight cultures diluted to an OD₆₀₀nm of 0.01. These
633 were mixed so that the inoculum contained 12.5% v/v of each of the four background
634 microbiota strains Acinetobacter MF4640 and Pseudomonas strains MF4836, MF6394 and
635 MF6396, and 50% of a mixture of equal amounts of the seven L. monocytogenes strains (Table
636 2). Biofilms were grown under static conditions. Rinsing of coupons in H₂O before and after
637 treatment with C&D agents was performed three times in 2.5 ml volumes of H₂O in 24-well
638 plates. After coupons were either subjected to C&D or rinsed in H₂O (to remove non-adherent
639 bacteria from control coupons), coupons were left in BHI until imaging the same day.

640 DNA isolation and amplicon sequencing. For purification of genomic DNA for amplicon
641 sequencing analysis, cells were lysed using Lysing Matrix B and a FastPrep-24 instrument (both
642 MP Biomedicals) and DNA isolated using either the PowerSoil-htp 96 Well Soil DNA Isolation Kit
643 (MoBio) (biofilm experiments #1 to #3) or the DNeasy Blood and Tissue Kit (Qiagen) (biofilm experiments #4 and #5 and planktonic competition experiments). Libraries for amplicon sequencing to analyze microbial composition were prepared following the 16S Metagenomic Sequencing Library Preparation protocol from Illumina (62). Briefly, amplicon PCR was performed with primers targeting either the V3-V4 region of the 16S rRNA gene or the *dapE* gene of *L. monocytogenes*, followed by an index PCR performed using the Nextera XT index kit (Illumina). The primers used to amplify the 16S rRNA gene were
650 *tcgtcggcagcgtcagatgtgtataagagacagcctacgggnggcwgcag* and
651 *gtctcgtgggctcggagatgtgtataagagacaggactachvgggtatctaatcc*, and those used to amplify *dapE*
652 were *tcgtcggcagcgtcagatgtgtataagagacagcgactaatgggcatgaagaacaag* and
653 *gtctcgtgggctcggagatgtgtataagagacagcatcgaaactatgggcatttttacc* (overhangs underlined). PCR products were purified using the AMPure XP system (Agencourt) after each PCR and after pooling. Purified indexed PCR products and the pooled sample were quantified using the Quant-iT Picogreen dsDNA kit (Invitrogen). The library was spiked with 10% PhiX control and sequenced using MiSeq v3 reagents using paired 300 bp reads on a MiSeq instrument (Illumina).

658 **Metagenomic Analysis using Qiime software.** Demultiplexed raw reads from the MiSeq run were processed with the Qiime software package (Quantitative Insights Into Microbial Ecology) v1.9.1 (63): After paired end reads were joined, they were quality filtered on q20. Then, samples amplified with *dapE* primers were assigned to their respective *dapE* allele using a closed reference OTU picking protocol against a custom reference file containing the *dapE* allele sequences of the seven *L. monocytogenes* strains (*dapE*-alleles numbers 4, 6, 7, 8, 9, 18, 20, and
664 21 as listed at the Institute Pasteur's *L. monocytogenes* MLST database at
665 http://bigsdb.web.pasteur.fr/listeria/listeria.html). The OTU picking script was run with default
666 parameters except that the sequence similarity threshold was set to 1. For samples amplified
667 with 16S rRNA primers, samples were analyzed using an open reference OTU picking protocol, in
668 which reads were first matched against a custom reference file containing the 16S rRNA allele
669 sequences of *L. monocytogenes* plus the 16 background flora strains included in the
670 experiments. The 16S rRNA reference file is included as Table S6 in the Supplemental Material.

671 **Bactericidal suspension test.** Overnight cultures were diluted to approximately 10⁸ CFU ml⁻¹
672 in peptone water and 1 ml of the diluted culture was added directly to 9 ml of H₂O (control) or
673 user concentrations of Alkalifoam (1%), QAC (1%) or PAA (1.5%) resulting in a final cell
674 concentration of approximately 10⁷ CFU ml⁻¹. After 5 min, 0.5 ml of the solution was transferred
675 to Dey Engley (D/E) neutralizing broth (Difco) and dilutions were plated on BHI agar plates. The
676 tests were performed with all solutions at 12°C. The experiment was performed three to four
677 times for each strain.

678 **Confocal laser scanning microscopy (CLSM).** Surface-associated bacteria on conveyor belt
679 coupons were stained with the cell-permeant Syto 61 red fluorescent nucleic acid strain (Life
680 Technologies), diluted to 5 mM in DMSO and used at a 1:2000 dilution. *L. monocytogenes* were
681 pinpointed in the complex biofilm through specific emission of their green GFP expression.
682 Images were acquired using a Leica SP8 confocal laser scanning microscope (Leica
683 Microsystems) at the MIMA2 microscopy platform (www6.jouy.inra.fr/mima2). Images were
684 obtained using a HC PL APO 63× long distance water objective with a numerical aperture of

685 NA=1.2. The GFP green emitted fluorescence signal from *L. monocytogenes* cells was collected

686 on a hybrid detector in the range 500-550 nm after excitation at 488 nm with an Argon laser set

687 at 20% of its maximal intensity. The red fluorescence emitted by the bacteria labeled with Syto

688 61 was collected on a photomultiplier in the range 645-675 nm after excitation with a 633 nm

689 HeNe laser. In order to contrast the surface topography, reflected signal from the 633 nm HeNe

690 laser was collected. Samples were scanned at 600 Hz every micron with 246×246 µm images in

691 order to acquire multi-color 3D stacks.

692 Representative CLSM images from each coupon were acquired by scanning Z-stacks on different

693 locations of the sample with a fixed pattern on the coupon surface. In most cases, three or five

694 stacks were taken from both the top face and underside of the conveyor belt coupon, at even

695 intervals following a line from the bottom to the top of the coupon, as marked with crosses in

696 the photograph of the coupon in Fig. 6D. The number of obtained Z-stack CLSM scans for the

697 different samples and treatments is summarized in Table S4 in the Supplemental Material. On a

698 few coupons, a larger view of the coupon was obtained thanks to a mosaic 3D meta-image,

699 obtained using a motorized stage that automatically moves the sample between scans and tiles

700 the adjacent fields. All CLSM stacks were processed using IMARIS (Bitplane) to projection images

701 of the biofilms. Quantitative analysis of each Z-stack to quantify the green biovolume

702 corresponding to *L. monocytogenes* subpopulation of the biofilm was performed using the ICY

703 image analysis software (64) using a homemade script previously described, with some

704 adaptations (65). The biomass software algorithm did not reliably score low cell numbers
705 correctly, therefore the biovolume of scans where ≤5 cells were observed in the green channel
706 of the image projection was set to 0, when the scored biovolume was above a threshold of 800
707 µm³. The biovolume of the red channel was not extracted since red fluorescent background
708 interfered with the Syto 61 specific signal.

709 **Accession numbers.** This Whole Genome Shotgun projects have been deposited at
710 DDBJ/ENA/GenBank under the accession numbers MVOJ00000000, MVOK00000000,
711 MVOL00000000, MVOM00000000, and MVON00000000. The versions described in this paper
712 are versions XXXX01000000. The raw reads are available from the National Center for
713 Biotechnology Information (NCBI) Sequence Read Archive (SRA) under accession numbers
714 SRR5273317, SRR5273318, SRR5273319, SRR5273320, and SRR5273321.

715 **SUPPLEMENTAL MATERIAL**

716 Supplemental material for this article may be found in the file:

717 «Supplemental Material», PDF file, 1.2 MB

718 **ACKNOWLEDGEMENTS**

719 We are grateful to Bjørn C. T. Schirmer for isolation of microbiota strains from meat processing
720 plants, and to Merete Rusås Jensen, Anette Wold Åsli, Tove Maugesten, Signe M. Drømtorp,
721 and Julien Deschamps for excellent technical assistance. This work was supported by Norwegian
722 Research Funding for Agriculture and Food Industry (grant no. 234355). Collection of strains
723 used in this work was funded by the Norwegian Research Funding for Agriculture and Food

724 Industry (grant no. 207765) and The Norwegian Seafood Research Fund (grant no. FHF-900521).
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925 TABLES

926 Table 1: Microbiota found on conveyor belts in meat processing plants

<table><thead><tr><th rowspan="3"></th><th colspan="6">Number of colonies of each genus<br/>isolated from each conveyor belt</th><th rowspan="3">Total<br/>number of<br/>colonies of<br/>each genus:</th></tr><tr><th colspan="5">Plant A</th><th colspan="3">Plant B</th></tr><tr><th>1</th><th>2</th><th>3</th><th>4</th><th>5</th><th>6<sup>b</sup></th><th>1</th><th>2</th><th>3</th></tr></thead><tbody><tr><td>Pseudomonas</td><td>7<sup>a</sup></td><td>5</td><td>24<sup>a</sup></td><td></td><td></td><td>1<sup>a</sup></td><td></td><td></td><td></td></tr><tr><td>Acinetobacter</td><td>1</td><td>6</td><td></td><td></td><td></td><td></td><td>6<sup>a</sup></td><td></td><td></td></tr><tr><td>Microbacterium</td><td></td><td></td><td></td><td></td><td></td><td>10<sup>a</sup></td><td>1</td><td></td><td></td></tr><tr><td>Sphingomonas</td><td></td><td></td><td></td><td></td><td></td><td>1</td><td>10<sup>a</sup></td><td></td><td></td></tr><tr><td>Epilithonimonas</td><td></td><td></td><td></td><td></td><td></td><td>8<sup>a</sup></td><td></td><td></td><td></td></tr><tr><td>Micrococcus</td><td></td><td></td><td></td><td>4</td><td></td><td>1<sup>a</sup></td><td></td><td>1</td><td></td></tr><tr><td>Psychrobacter</td><td>4</td><td></td><td></td><td></td><td></td><td></td><td>2<sup>a</sup></td><td></td><td></td></tr><tr><td>Rhodococcus</td><td></td><td></td><td></td><td></td><td></td><td>1</td><td>4<sup>a</sup></td><td></td><td></td></tr><tr><td>Corynebacterium</td><td></td><td></td><td></td><td></td><td></td><td></td><td>2<sup>a</sup></td><td>2</td><td></td></tr><tr><td>Brevundomonas</td><td></td><td></td><td></td><td></td><td></td><td>1</td><td>2</td><td>1</td><td></td></tr><tr><td>Vagococcus</td><td>3</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>3</td></tr><tr><td>Erysipelothrix</td><td>2</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>2</td></tr><tr><td>Kocuria</td><td></td><td></td><td></td><td></td><td>1<sup>a</sup></td><td></td><td>1<sup>a</sup></td><td></td><td>2</td></tr><tr><td>Chryseobacterium</td><td></td><td></td><td></td><td></td><td></td><td></td><td>1</td><td></td><td>1</td></tr><tr><td>Exigubacterium</td><td></td><td></td><td></td><td>1</td><td></td><td></td><td></td><td></td><td>1</td></tr><tr><td>Leucobacter</td><td></td><td></td><td></td><td></td><td></td><td></td><td>1</td><td></td><td>1</td></tr><tr><td>Lysinibacillus</td><td>1</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>1</td></tr><tr><td>Moraxella</td><td></td><td></td><td></td><td>1</td><td></td><td></td><td></td><td></td><td>1</td></tr><tr><td>Paenibacillus</td><td></td><td>1</td><td></td><td></td><td></td><td></td><td></td><td></td><td>1</td></tr><tr><td>Rhothia</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>1</td><td>1</td></tr><tr><td>Roseomonas</td><td></td><td></td><td></td><td></td><td>1</td><td></td><td></td><td></td><td>1</td></tr><tr><td>Variovorax</td><td></td><td></td><td></td><td></td><td></td><td></td><td>1</td><td></td><td>1</td></tr><tr><td>Total number of<br/>colonies isolated from<br/>each conveyor belt:</td><td>18</td><td>12</td><td>24</td><td>4</td><td>2</td><td>25</td><td>20</td><td>11</td><td>5</td><td>SUM: 121</td></tr></tbody></table>

927

48

<footnote><sup>a</sup>Sample with isolates used in biofilm experiments in the current study.</footnote>

<footnote><sup>b</sup>L. monocytogenes was isolated from this conveyor belt on several other occasions (MF5377).</footnote>
<table><caption>928 Table 2: Bacterial strains used in biofilm experiments</caption><thead><tr><th colspan="3">Background microbiota from conveyor belts</th></tr><tr><th>Strain</th><th>Plant<sup>a</sup></th><th>Bacterial species or genera</th></tr></thead><tbody><tr><td>MF4640</td><td>B</td><td>Acinetobacter johnsonii</td></tr><tr><td>MF4642</td><td>B</td><td>Acinetobacter johnsonii</td></tr><tr><td>MF4643</td><td>B</td><td>Corynebacterium sputi</td></tr><tr><td>MF4645</td><td>B</td><td>Corynebacterium sp.</td></tr><tr><td>MF6392</td><td>A</td><td>Epilithonimonas sp.</td></tr><tr><td>MF4644</td><td>B</td><td>Kocuria sp.</td></tr><tr><td>MF6395</td><td>A</td><td>Kocuria rhizophila</td></tr><tr><td>MF4634</td><td>B</td><td>Microbacterium sp.</td></tr><tr><td>MF6393</td><td>A</td><td>Micrococcus sp.</td></tr><tr><td>MF6396</td><td>A</td><td>Pseudomonas sp. (P. putida group)</td></tr><tr><td>MF6394</td><td>A</td><td>Pseudomonas sp. (P. fluorescens subgroup)</td></tr><tr><td>MF4836</td><td>A</td><td>Pseudomonas sp. (P. mandelii subgroup)</td></tr><tr><td>MF4641</td><td>B</td><td>Psychrobacter sp.</td></tr><tr><td>MF4633</td><td>B</td><td>Rhodococcus erythropolis</td></tr><tr><td>MF4637</td><td>B</td><td>Rhodococcus fascians</td></tr><tr><td>MF4632</td><td>B</td><td>Sphingomonas sp.</td></tr></tbody></table><table><thead><tr><th colspan="3">L. monocytogenes strains from meat processing environments<sup>b</sup></th></tr><tr><th>Strain</th><th>Plant<sup>a</sup></th><th>MLST sequence type, MLVA profile<sup>c</sup></th></tr></thead><tbody><tr><td>MF4536</td><td>C</td><td>ST9, MLVA 6-11-15-18-6 (persistent, qacH positive)</td></tr><tr><td>MF5376</td><td>A</td><td>ST7, MLVA 7-7-10-10-6 (persistent)</td></tr><tr><td>MF5634</td><td>B</td><td>ST121, MLVA 6-7-14-10-6 (persistent, qacH positive)</td></tr><tr><td>MF5377</td><td>A</td><td>ST8, MLVA 6-9-18-16-6 (persistent)</td></tr><tr><td>MF4565</td><td>C</td><td>ST18, MLVA 8-8-17-21-6 (sporadic)</td></tr><tr><td>MF5630</td><td>C</td><td>ST19, MLVA 6-9-18-10-6 (sporadic)</td></tr><tr><td>MF5378</td><td>A</td><td>ST394, MLVA 6-9-19-10-6 (sporadic)</td></tr></tbody></table>

<footnote><sup>a</sup> Plants A, B, and C correspond to plants M2, M4, and M1, respectively, described in Møretrø et al. (22).</footnote>

<footnote><sup>b</sup> L. monocytogenes strains were isolated in Møretrø et al. (22).</footnote>

<footnote><sup>c</sup> Listed MLVA profiles correspond to variable number tandem repeat (VNTR) loci LMV6-LMV1-LMV2-LMV7-LMV9 described in Lindstedt et al. (66). Persistent or sporadic MLVA profiles are determined using the criteria described in the Materials and Methods section.</footnote>
<table><caption>930 Table 3: Biovolume of *L. monocytogenes* on conveyor belt couponsª</caption><thead><tr><th rowspan="2">Front of coupon</th><th rowspan="2"></th><th colspan="2">Before C&D</th><th colspan="2">After C&D</th></tr><tr><th>H<sub>2</sub>O</th><th>QAC</th><th>QAC</th><th>PAA</th></tr></thead><tbody><tr><td rowspan="2">Multigenera biofilms</td><td>Day 4</td><td>149 (±32)</td><td>NA</td><td>39 (±23)</td><td>0.06 (±0.03)</td></tr><tr><td>Day 7</td><td>35 (±22)</td><td>1.3 (±1.2)</td><td>0.67 (±0.61)</td><td>NT</td></tr><tr><td rowspan="2">L. monocytogenes biofilms</td><td>Day 4</td><td>3.4 (±1.1)</td><td>NA</td><td>0.18 (±0.11)</td><td>0.1 (±0.08)</td></tr><tr><td>Day 7</td><td>14 (±11)</td><td>NT</td><td>NT</td><td>NT</td></tr><tr><th rowspan="2">Back of coupon</th><th rowspan="2"></th><th colspan="2">Before C&D</th><th colspan="2">After C&D</th></tr><tr><th>H<sub>2</sub>O</th><th>QAC</th><th>QAC</th><th>PAA</th></tr><tr><td rowspan="2">Multigenera biofilms</td><td>Day 4</td><td>331 (±141)</td><td>NA</td><td>11 (±5)</td><td>0 (±0)</td></tr><tr><td>Day 7</td><td>62 (±14)</td><td>68 (±66)</td><td>0.1 (±0.06)</td><td>NT</td></tr><tr><td rowspan="2">L. monocytogenes biofilms</td><td>Day 4</td><td>172 (±73)</td><td>NA</td><td>0.61 (±0.61)</td><td>NT</td></tr><tr><td>Day 7</td><td>2841 (±1439)</td><td>11 (±3,6)</td><td>6.3 (±3.9)</td><td>NT</td></tr></tbody></table>

NA: not applicable

NT: not tested

931

50

<footnote>ª Values are obtained from CLSM Z-scans using the ICY image analysis software (64) and are given as averages of the total volume (µm³) per scan +/- the standard error of the mean for each tested combination. The number of coupons tested for each condition is given in Table S4 in the Supplemental Material.</footnote>
932 FIGURE LEGENDS

933 **FIG 1** Development of the microbiota in multigenera biofilms on conveyor belt coupons (A, B)
934 and planktonic cultures (C) at 12°C. Inocula were composed of *L. monocytogenes* plus either 16
935 (A, C) or four (B) background microbiota strains. The frequencies of different bacterial strains
936 were determined by 16S rRNA amplicon sequencing. (A, B) Biofilms were allowed to develop for
937 four days before being subjected to daily cleaning with Alkalifoam and treatment with either a
938 QAC or PAA disinfection agent or (B) a daily rinse in H2O. Coupons were harvested either before
939 or after C&D on the day of harvest, as indicated. (C) Development of microbiota in planktonic
cultures, grown with shaking in Erlenmeyer flasks for a total of 72 h. Presented results are
940 averages from three (A, C) or two (B) independent experiments. Results for individual
941 experiments are shown in Fig. S3 in the Supplemental Material.
942

943 **FIG 2** Competition between *L. monocytogenes* strains during biofilm (A) and planktonic growth
944 (B) in BHI at 12°C. Inoculated suspensions were composed of equal amounts of seven different
945 *L. monocytogenes* strains, grown either together with 16 background microbiota strains
946 (multigenera biofilm/culture) or alone (*L. monocytogenes* biofilm/culture). (A) Frequencies of *L.
947 monocytogenes* strains surviving in biofilms on conveyor belt coupons. Biofilms were allowed to
develop for four days and then subjected to daily cleaning with Alkalifoam and disinfection with
948 either a QAC- or PAA-based disinfectant on four consecutive days before harvest. The identity of
949 single colonies of *L. monocytogenes* was determined by Sanger sequencing of the *dapE* allele.
950 Results are pooled from three replicate experiments in which ten single *L. monocytogenes*
951 colonies were identified from each of the four conditions analyzed; 120 colonies in total. (B)
952
953 Time-course experiment showing the relative abundance of *L. monocytogenes* strains in
954 planktonic cultures, grown with shaking in Erlenmeyer flasks for a total of 72 h. The frequencies
955 of different *L. monocytogenes* strains was determined by *dapE* amplicon sequencing. Presented
956 results are averages from three independent experiments. Results for individual experiments
957 are shown in Fig. S4 in the Supplemental Material.

958 **FIG 3** Growth of the 16 background microbiota strains and the seven *L. monocytogenes* strains
959 in BHI medium at 12°C. The experiment was performed in a Bioscreen C instrument, with
960 measurement of the absorbance at 600nm once every hour. Results shown are averages of
961 triplicate wells in one representative experiment out of three experiments performed. Strains
962 included in the biofilm experiments with four background microbiota strains (Fig. 1B) are
963 represented by solid colored lines. Dashed lines represent selected strains that also show good
964 growth properties. Grey lines represent the remaining eight strains.

965 **FIG 4** Total bacterial numbers in biofilms on conveyor belt coupons prior to C&D. Biofilms were
966 allowed to develop undisturbed until Day 4 before being subjected to rinsing in H₂O or to
967 cleaning with Alkalifoam and disinfection with either a QAC- or a PAA-based disinfection agent
968 on three consecutive days. The microbiota on coupons harvested on Day 7 had been allowed to
969 regrow for 24 h after the last disinfection step. The *L. monocytogenes* count in multigenera
970 biofilms was determined by plating on selective agar. Mean values of five replicates are shown,
971 except for the sample labelled H₂O, where the mean of two replicates is shown. Error bars show
972 standard error of the mean. Asterisks represent differences in *L. monocytogenes* CFU per
973 coupon relative to in multigenera biofilms harvested on Day 4 (*, P = 0.08; **, P < 0.05; two-tailed paired Student t tests).

975 **FIG 5** Tolerance of biofilms to C&D regimes relevant in the food industry. The log₁₀ reduction
976 upon C&D for multigenera biofilms and biofilms inoculated with seven *L. monocytogenes* strains
977 grown on conveyor belt coupons is shown. Biofilms were allowed to develop undisturbed until
978 Day 4 before being subjected to cleaning with Alkalifoam and disinfection with either a QAC- or
979 a PAA-based disinfection agent on four consecutive days. Calculated reductions are relative to
980 control coupons rinsed in H₂O only. The *L. monocytogenes* count in multigenera biofilms was
981 determined by plating on selective agar. Mean values of five experiments and standard error of
982 the mean are shown. Asterisks represent comparison of samples using the two-tailed paired
983 Student t tests (*, P = 0.06; **, P < 0.05).

984 **FIG 6** Biofilms on the top coating of conveyor belt material examined using CLSM. Green
985 represents GFP-expressing *L. monocytogenes* cells, while *Acinetobacter* and *Pseudomonas*
986 background flora strains are shown in red. CLSM images shown are Easy3D shadow projection
987 reconstructions obtained from the confocal Z-stack series using the IMARIS software. Coupons
988 with biofilm were imaged either on Day 4 (B) or on Day 7 (A, C, E) after initiation of biofilm
989 growth, and harvested after rinsing in H₂O (C, E) or after C&D with QAC (A, B). Panel (D) shows a
990 photograph of the front side of a conveyor belt coupon. The arrow indicates the approximate
991 location of the air-liquid interface during biofilm development, and crosses show the
992 approximate locations on each coupon where the CLMS image acquisitions were acquired
993 (when only three images were acquired, the locations indicated by the large crosses were used).
994 Panel (E) shows a mosaic 3D meta-image, obtained using a motorized stage that

995 automatically moves the sample between scans and tiles the adjacent fields. The box drawn

996 with dashed line in (D) approximately corresponds to the area of the coupon covered by the

997 CSLM image in (E).

998 **FIG 7** Biofilms formed on the underside of the conveyor belt examined using CLSM.

999 *Pseudomonas* and *Acinetobacter* background flora strains (shown in red) and GFP-expressing *L.*

1000 *monocytogenes* strains (in green) were used as inoculum, except in panel (F), where biofilms

1001 were inoculated with *L. monocytogenes* strains only. (A, B) Photographs showing the underside

1002 of a clean conveyor belt coupon. The image in panel (B), photographed using an USB

1003 microscope lens, shows the weave pattern of the fabric with the smooth warp thread indicated

1004 by an arrow. Panels (C) to (H) show biofilms formed on the weft thread of the woven fabric, on

1005 coupons rinsed in H₂O on Days 4 to 7 (C, D, F), on a coupon rinsed in H₂O on Day 7 after

1006 treatment with QAC on Days 4 to 6 (E), and on coupons harvested on Day 4 for after treatment

1007 with PAA (G) or QAC (H). In panels (C, E, F), the smaller fibers constituting the weft thread is

1008 represented by the reflection signal (in grey). (C, D) Mosaic 3D meta-image, shown in section

1009 view mode (C) and Easy3D blend representation (D), with warp threads indicated by arrows. The

1010 boxes drawn with dashed lines in (A) and (B) show the size and location of the area of a coupon

1011 depicted by the CSLM images shown in panels (D) and (C), respectively. Panels (I) to (K) are

1012 different representations of the same scan, showing biofilm harvested on Day 4 after rinsing in

1013 H₂O, formed on the warp thread of the woven fabric. Images are shown as three-dimensional
1014 (E, F, K) and Easy 3D (G, H, I, J) IMARIS representations. The scale is the same in the Easy3D

1015 blend images in panels (G) to (J). The red channel is not shown in panels (F) and (J).
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Comment citer ce document :
Fagerlund, A., Møretrø, Heir, Briandet, R., Langsrud, S. (2017). Cleaning and disinfection of biofilms composed of Listeria monocytogenes and background microbiota from meat processing surfaces . Applied and Environmental Microbiology. DOI: 10.1128/AEM.01046-17
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