Maroua Gdoura-Ben

Antoine Amor

Clarisse Culot

Mousa Techer

Mohd Alreshidi

Sophie Adnan

Florence Jan

Noël Baron

Mejdi Grosset

Radhouane Snoussi

Gdoura

Maroua Gdou-Ben

Amor

Antoine Culot
email: antoine.culot@mixscience.eu

Clarisse Tech
email: techerclarisse@yahoo.fr

Mousa Alreshidi

Mohd Adnan
email: drmohdadnan@gmail.com

Sophie Jan
email: sophie.jan@agrocampus-ouest.fr

Florence Baron
email: florence.baron@agrocampus-ouest.fr

Noël Grosset
email: noel.grosset@agrocampus-ouest.fr

Mejdi Snoussi

Radhouane Gdoura
email: gdoura.radhouane@gmail.com

Michel Gautier
email: michel.gautier@agrocampus-ouest.fr

Isolation, Partial Characterization and Application of Bacteriophages in Eradicating Biofilm Formation by Bacillus cereus on Stainless Steel Surfaces in Food Processing Facilities

Keywords: B. cereus group, biofilm, stainless steel, bacteriophage, sewage, characterization, eradication, inhibition

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

B. cereus group bacteria are widely distributed in the environment. Several species from the B. cereus group are associated with food poisoning cases by producing two different kinds of toxins: an emetic one and a diarrheal one [START_REF] Granum | Bacillus cereus and its toxins[END_REF]. B. cereus can be found in biofilm form in the food industry. Biofilms consist of complex communities of surface attached bacteria, surrounded by a polymeric extracellular material that typically consists of extracellular polysaccharides, proteins and sometimes eDNA [START_REF] Branda | Biofilms: The matrix revisited[END_REF]. The extracellular matrix of a biofilm protects the involved bacteria from environmental damage, antimicrobial agents, and host immune defenses [START_REF] Flemming | The biofilm matrix[END_REF]. Therefore, biofilms are extremely difficult to remove, exhibiting resistance to cleaning and disinfecting processes, as is the case of B. cereus biofilms, which exhibit resistance to the action of quaternary ammonium Pathogens 2022, 11, 872 2 of 17 compound and sodium hypochlorite [START_REF] Peng | Inactivation and removal of Bacillus cereus by sanitizer and detergent[END_REF]. The resistance of B. cereus to chemical treatments is due to the bacterium's ability to form spores and produce toxins that, in turn, can be harmful to humans and the food industry [START_REF] Marchand | Biofilm formation in milk production and processing environments; influence on milk quality and safety[END_REF][START_REF] Rajkovic | Risk Profile of the Bacillus cereus Group Implicated in Food Poisoning[END_REF]. Thus, the negative impact of biofilm formation on food safety and economic losses in food processing [START_REF] Tauveron | Variability among Bacillus cereus strains in spore surface properties and influence on their ability to contaminate food surface equipment[END_REF][START_REF] Van Houdt | Biofilm formation and the food industry, a focus on the bacterial outer surface[END_REF] have enhanced the development of different new approaches in order to control and/or eradicate biofilm formation, such as the use of enzymes, bacteriophages, microbial interactions, and metabolic molecules [START_REF] Srey | Biofilm formation in food industries: A food safety concern[END_REF]. Recently, new research on the use of bacteriophages as potential agents for biofilm eradication has increased. Indeed, several reports on the interactions between phages and biofilms have been published for a wide range of bacteria, such as: Escherichia coli [START_REF] Corbin | Bacteriophage T4 multiplication in a glucose-limited Escherichia coli biofilm[END_REF], Enterococcus faecalis [START_REF] Teng | Further Characterization of the epa Gene Cluster and Epa Polysaccharides of Enterococcus faecalis[END_REF], Listeria monocytogenes [START_REF] Hibma | Infection and removal of L-forms of Listeria monocytogenes with bred bacteriophage[END_REF][START_REF] Soni | Removal of Listeria monocytogenes Biofilms with Bacteriophage P100[END_REF],

Pseudomonas fluorescens [START_REF] Sillankorva | Pseudomonas fluorescens biofilms subjected to phage phiIBB-PF7A[END_REF], Staphylococcus aureus [START_REF] Resch | Phage release from biofilm and planktonic Staphylococcus aureus cells[END_REF], Salmonella [START_REF] Garcia | Bacteriophage use to control Salmonella biofilm on surfaces present in chicken slaughterhouses[END_REF][START_REF] Gong | Application of bacteriophages to reduce Salmonella attachment and biofilms on hard surfaces[END_REF][START_REF] Karaca | Effects of P22 bacteriophage on Salmonella enterica subsp. enterica serovar Typhimurium DMC4 strain biofilm formation and eradication[END_REF][START_REF] Sadekuzzaman | Application of bacteriophages for the inactivation of Salmonella spp. in biofilms[END_REF], and Streptococcus mutans [START_REF] Dalmasso | Isolation of a Novel Phage with Activity against Streptococcus mutans Biofilms[END_REF].

Bacteriophages, more commonly known as phages, are obligate parasites of bacteria. They are ubiquitous in nature, and they have been isolated from different environments including soil, water, and several food products such as meat, dairy, and vegetable products [START_REF] Atterbury | Isolation and characterization of Campylobacter bacteriophages from retail poultry[END_REF][START_REF] Binetti | Thermal and chemical inactivation of indigenous Streptococcus thermophilus bacteriophages isolated from Argentinian dairy plants[END_REF][START_REF] Aprea | Isolation and Morphological Characterization of New Bacteriophages Active against Campylobacter jejuni[END_REF][START_REF] Hsu | Enteric bacteriophages as potential fecal indicators in ground beef and poultry meat[END_REF][START_REF] Surekhamol | Isolation and characterization of broad spectrum bacteriophages lytic to Vibrio harveyi from shrimp farms of Kerala, India[END_REF][START_REF] Whitman | Isolation of psychrophilic bacteriophage-host systems from refrigerated food products[END_REF]. Several bacteriophage infecting B. cereus group bacteria have been isolated from soils, muds, food wastes and fermented foods [START_REF] Lee | Characterization and complete genome sequence of a virulent bacteriophage B4 infecting food-borne pathogenic Bacillus cereus[END_REF][START_REF] Oh | Isolation and Characterization of Bacillus cereus Bacteriophages from Foods and Soil[END_REF][START_REF] Shin | Prevalence of Bacillus cereus bacteriophages in fermented foods and characterization of phage JBP901[END_REF][START_REF] Shin | Characterization and genome analysis of the Bacillus cereus-infecting bacteriophages BPS10C and BPS13[END_REF]. Although there have been, as cited above, several studies published investigating the effect of phages against biofilms, no study has proposed phages as alternative biocontrol agents against B. cereus biofilm. Therefore, the aim of the present study was (i) to isolate bacteriophages against B. cereus group strains from different sewage pool samples (P1-P4); (ii) to characterize bacteriophages by the analysis of their host range, morphology, protein patterns and restriction enzyme digestion profiles; (iii) to determine the potential of isolated bacteriophages for the prevention and eradication B. cereus biofilm on stainless steel coupons.

Results

Isolation and Host Spectrum Evaluation of Bacteriophages

In total, 62 bacteriophages infecting the B. cereus group were isolated by enrichment from four different sewage pools (P1-P4). As mentioned in Table 1, the enrichment of the four pools (P1, P2, P3 and P4) allowed amplification of 16, 15, 19 and 12 bacteriophages infecting the B. cereus group, respectively. The purified bacteriophages were screened on the basis of host range analysis using the 174 B. cereus group strains in the collection. Plaques formed by phages on appropriate host bacteria were classified as clear, moderately clear and turbid (data not shown). Based on the quality of plaques and the lytic activity spectrum, seven phages (Ø1BC478; Ø1BC3990; Ø2BC4663; Ø3BC4663; Ø4BC4663(1); Ø4BC4663 [START_REF] Branda | Biofilms: The matrix revisited[END_REF] and Ø1BC4663) were selected as having the broadest lytic spectrum (Table S1). Plaque morphology on agar plates of isolated phages is illustrated in Figure 1. The isolated phages showed a lytic efficiency on tested B. cereus strains that ranged from 8.6% to 13.2% (Table 2). Selected bacteriophages were examined by transmission electron microscopy (Figure 2). On the basis of their morphological features, all phages were considered to be part of the Siphoviridae family in Caudovirales [START_REF] Ackermann | Bacteriophage observations and evolution[END_REF], because they exhibited a typical icosahedral head and long, flexible and non-contractile tail structure in TEM micrographs. The dimensions and features of the phages are given in the Table 3. Within our phage samples, there were light and dark phage heads. Dark heads demosntrated loss of their nucleic acid [START_REF] Aprea | Isolation and Morphological Characterization of New Bacteriophages Active against Campylobacter jejuni[END_REF][START_REF] Lee | Isolation and characterization of phages infecting Bacillus cereus[END_REF].

Bacteriophage Characterization

Morphological Observation

Selected bacteriophages were examined by transmission electron microscopy (Figure 2). On the basis of their morphological features, all phages were considered to be part of the Siphoviridae family in Caudovirales [START_REF] Ackermann | Bacteriophage observations and evolution[END_REF], because they exhibited a typical icosahedral head and long, flexible and non-contractile tail structure in TEM micrographs. The dimensions and features of the phages are given in the Table 3. Within our phage samples, there were light and dark phage heads. Dark heads demosntrated loss of their nucleic acid [START_REF] Aprea | Isolation and Morphological Characterization of New Bacteriophages Active against Campylobacter jejuni[END_REF][START_REF] Lee | Isolation and characterization of phages infecting Bacillus cereus[END_REF]. SDS-PAGE was used to determine the structural protein content of each phage (Figure 3). A similar profile was observed for phages Ø2BC4663 and Ø4BC4663(2), with four predominant protein bands corresponding to approximately 220, 180, 39 and 28 kDa. Phages Ø1BC3990, Ø3BC4663, Ø4BC4663(1) and Ø1BC4663 were found to produce two major protein bands corresponding, respectively, to 39, 32 kDa (Ø1BC3990); 39, 28 kDa (Ø3BC4663); 39, 31 kDa (Ø4BC4663(1)); and 42, 31 kDa (Ø1BC4663). Phage Ø1BC478 contained four major protein bands at molecular masses of approximately 120, 50, 39 and 28 kDa. 3). A similar profile was observed for phages Ø2BC4663 and Ø4BC4663(2), with four predominant protein bands corresponding to approximately 220, 180, 39 and 28 kDa. Phages Ø1BC3990, Ø3BC4663, Ø4BC4663(1) and Ø1BC4663 were found to produce two major protein bands corresponding, respectively, to 39, 32 kDa (Ø1BC3990); 39, 28 kDa (Ø3BC4663); 39, 31 kDa (Ø4BC4663(1)); and 42, 31 kDa (Ø1BC4663). Phage Ø1BC478 contained four major protein bands at molecular masses of approximately 120, 50, 39 and 28 kDa.

Restriction Enzyme Digestion Pattern Analysis

The extracted isolated bacteriophages DNAs were digested with five restriction endonucleases (BamHI, EcoRI, HindIII, Hin6I and TaqI). Neither of the extracted phages DNAs were susceptible to BamHI and HindIII, whereas the EcoRI, Hin6I and TaqI enzymes resulted in very similar restriction profiles for selected phages (Figure 4). These

Restriction Enzyme Digestion Pattern Analysis

The extracted isolated bacteriophages DNAs were digested with five restriction endonucleases (BamHI, EcoRI, HindIII, Hin6I and TaqI). Neither of the extracted phages DNAs were susceptible to BamHI and HindIII, whereas the EcoRI, Hin6I and TaqI enzymes resulted in very similar restriction profiles for selected phages (Figure 4). These results indicate that the selected phages have similar genome sizes and possibly are highly related. Selected phages have estimated genome sizes of around 37 kb, as calculated by adding together the fragment sizes obtained with the three enzymes above.

Bacteriophage Action on B. cereus Biofilms

Effectiveness of Phage Treatment in Reducing Pre-formed Biofilms

The eradication activity of each phage on pre-formed biofilms of 24, 48 and 72 h of the corresponding B. cereus host strain was evaluated by stainless steel application. Phage post-treatment of different preformed biofilms was performed using appropriate phage dilutions (10 5 -10 8 PFU/mL), followed by six hours of incubation at 30 °C. The phage posttreatment effects on preformed biofilms are presented as percentage reduction of the biofilm in Figure 5.

All phage titrations (10 5 -10 8 PFU/mL) were found to have removal efficiency against 24 h-old biofilm. In fact, bacteriophage post-treatments of each tested bacteriophage with titers of 10 8 , 10 7 , 10 6 and 10 5 PFU/mL reduced the corresponding pre-formed 24 h-old biofilm by 28.0 to 59.4%, 22.5 to 49.1%, 18.4% to 42.1% and 6.1 to 32.2%, respectively. The highest biomass removal activity of 59.4% was observed with Ø4BC4663(2) post-treatment at a titer of 10 8 PFU/mL against 24 h-old biofilm (Figure 5F). Results showed that only higher phage titrations were found to be effective on already formed 48h-old biofilm (≥10 6 PFU/ mL). In the 48 h-old biofilms, the highest removal efficiency of approximatively 37% was observed with Ø1BC3990 phage post-treatment at a titer of 10 7 and 10 8 PFU/mL (Figure 5B), whereas no eradication activity against 48 h-old biofilm was found with Ø2BC4663 (Figure 5C), Ø4BC4663(1) (Figure 5E) and Ø4BC4663(2) (Figure 5F) post-treatment at a titer of 10 5 PFU/mL. Removal efficiency of phage post-treatment was dramatically decreased for the 72 h-old biofilm samples. No eradication against 72 h-old biofilms was detected for all phages titrations of both phage Ø1BC3990 (Figure 5B) and Ø2BC4663 (Figure 5C). However, the Ø1BC4663 post-treatment with titration of 10 8 PFU/mL was found to be the most effective against 72 h-old biofilms, resulting in biomass removal of 25.6% (Figure 5G).

Bacteriophage Action on B. cereus Biofilms 2.3.1. Effectiveness of Phage Treatment in Reducing Pre-formed Biofilms

The eradication activity of each phage on pre-formed biofilms of 24, 48 and 72 h of the corresponding B. cereus host strain was evaluated by stainless steel application. Phage post-treatment of different preformed biofilms was performed using appropriate phage dilutions (10 5 -10 8 PFU/mL), followed by six hours of incubation at 30 • C. The phage post-treatment effects on preformed biofilms are presented as percentage reduction of the biofilm in Figure 5.

All phage titrations (10 5 -10 8 PFU/mL) were found to have removal efficiency against 24 h-old biofilm. In fact, bacteriophage post-treatments of each tested bacteriophage with titers of 10 8 , 10 7 , 10 6 and 10 5 PFU/mL reduced the corresponding pre-formed 24 h-old biofilm by 28.0 to 59.4%, 22.5 to 49.1%, 18.4% to 42.1% and 6.1 to 32.2%, respectively. The highest biomass removal activity of 59.4% was observed with Ø4BC4663(2) posttreatment at a titer of 10 8 PFU/mL against 24 h-old biofilm (Figure 5F). Results showed that only higher phage titrations were found to be effective on already formed 48 h-old biofilm (≥10 6 PFU/mL). In the 48 h-old biofilms, the highest removal efficiency of approximatively 37% was observed with Ø1BC3990 phage post-treatment at a titer of 10 7 and 10 8 PFU/mL (Figure 5B), whereas no eradication activity against 48 h-old biofilm was found with Ø2BC4663 (Figure 5C), Ø4BC4663(1) (Figure 5E) and Ø4BC4663(2) (Figure 5F) posttreatment at a titer of 10 5 PFU/mL. Removal efficiency of phage post-treatment was dramatically decreased for the 72 h-old biofilm samples. No eradication against 72 h-old biofilms was detected for all phages titrations of both phage Ø1BC3990 (Figure 5B) and Ø2BC4663 (Figure 5C). However, the Ø1BC4663 post-treatment with titration of 10 8 PFU/mL was found to be the most effective against 72 h-old biofilms, resulting in biomass removal of 25.6% (Figure 5G).

Effectiveness of Phage Treatment in Inhibiting Biofilms

The results of bacteriophage treatment of each isolated phage with different titers (10 5 , 10 6 , 10 7 and 10 8 PFU/mL) in inhibiting biofilm formation at different incubation periods (24, 48 and 72 h), are shown in Figure 6. All tested phage titrations prevented biofilm formation at the end of the 24 h and 48 h incubation periods, by 65.3 to 97.1% and 48.2 to 91.9%, respectively. Due to the long incubation period (72 h), the inhibition capability of all tested bacteriophages was found to be less effective at the end of the 72h incubation

Effectiveness of Phage Treatment in Inhibiting Biofilms

The results of bacteriophage treatment of each isolated phage with different titers (10 5 , 10 6 , 10 7 and 10 8 PFU/mL) in inhibiting biofilm formation at different incubation periods (24, 48 and 72 h), are shown in Figure 6. All tested phage titrations prevented biofilm formation at the end of the 24 h and 48 h incubation periods, by 65.3 to 97.1% and 48.2 to 91.9%, respectively. Due to the long incubation period (72 h), the inhibition capability of all tested bacteriophages was found to be less effective at the end of the 72h incubation period. The log reduction percentage ranged from 16.8 to 28.4%, 37.9 to 58.3%, 66.7 to 76.8% and 79.1 to 87.1% for 10 5 , 10 6 , 10 7 and 10 8 PFU/mL, respectively. The highest inhibition, of 97.1%, was observed with Ø4BC4663(1) treatment at a titer of 10 8 PFU/mL against the host strain biofilm formation after 24 h (Figure 6E). For phage treatment against biofilm formation after 48h of incubation, the highest inhibition, of 91.1%, was observed with phage Ø3BC4663 treatment at a titer of 10 8 PFU/mL (Figure 6D). The lowest inhibition efficiency was detected for Ø1BC478 treatment (10 5 -10 8 PFU/mL) against biofilm formation after different incubation periods (24, 48 and 72 h) (Figure 6A).

Pathogens 2022, 11, x FOR PEER REVIEW 8 of 17 period. The log reduction percentage ranged from 16.8 to 28.4%, 37.9 to 58.3%, 66.7 to 76.8% and 79.1 to 87.1% for 10 5 , 10 6 , 10 7 and 10 8 PFU/mL, respectively. The highest inhibition, of 97.1%, was observed with Ø4BC4663(1) treatment at a titer of 10 8 PFU/mL against the host strain biofilm formation after 24 h (Figure 6E). For phage treatment against biofilm formation after 48h of incubation, the highest inhibition, of 91.1%, was observed with phage Ø3BC4663 treatment at a titer of 10 8 PFU/mL (Figure 6D). The lowest inhibition efficiency was detected for Ø1BC478 treatment (10 5 -10 8 PFU/mL) against biofilm formation after different incubation periods (24, 48 and 72 h) (Figure 6A).

Discussion

In this study, we first studied the prevalence of B. cereus phages in sewage pool samples. A specific enrichment phase using a mix of 174 B. cereus strains was developed to allow the amplification of phages present in very minute numbers in a sample and, thus, to enhance the probability of isolating a wider range of phages quickly and at low cost. Previous studies [START_REF] Aprea | Isolation and Morphological Characterization of New Bacteriophages Active against Campylobacter jejuni[END_REF][START_REF] Surekhamol | Isolation and characterization of broad spectrum bacteriophages lytic to Vibrio harveyi from shrimp farms of Kerala, India[END_REF] have used such an experimental protocol to isolate a variety of phages infecting Campylobacter jejuni and Vibrio harveyi strains. Among the 62 isolated phages, seven phages were chosen for further characterization, due to their host spectrum of activity and plaques quality (Table S1). The lytic efficiency of the selected phages on tested B. cereus strains varied from 8.6 to 13.2% (Table 2). Although the majority of bacteriophage studies have concentrated on the characterization of high percentage lysis bacteriophages and their application as agents for biocontrol in the food industry, our study aims to test the possibility of using low percentage lysis bacteriophages in biofilm treatment. Indeed, and as reported in previous work [START_REF] Liu | Characterization of the narrowspectrum bacteriophage LSE7621 towards Salmonella Enteritidis and its biocontrol potential on lettuce and tofu[END_REF], low percentage lysis bacteriophages can more effectively lyse a small number of host strains. Therefore, a broadening of the host spectrum of a bacteriophage is often counterbalanced by a limitation of the virulence of that bacteriophage towards its own host. [START_REF] Ford | Frequency and fitness consequences of bacteriophage Φ6 host range mutations[END_REF][START_REF] Keen | Tradeoffs in bacteriophage life histories[END_REF]. The application of bacteriophages that act only on a specific host can simultaneously reduce the likelihood of developing resistance at the community level and have a relatively low impact on the microbial flora [START_REF] De Jonge | Molecular and evolutionary determinants of bacteriophage host range[END_REF]. Furthermore, these bacteriophages can be combined to be exploited as novel biocontrol agents in a cocktail form [START_REF] Ciacci | Characterization of vB_Kpn_F48, a newly discovered lytic bacteriophage for Klebsiella pneumoniae of sequence type 101[END_REF][START_REF] Wang | Characterization and genome analysis of novel phage vB_EfaP_IME195 infecting Enterococcus faecalis[END_REF] or explored for specific detection.

TEM analysis showed that all isolated B. cereus bacteriophages are members of the Siphoviridae familiy. This finding is consistent with previous studies, which identify the morphology of B. cereus bacteriophage isolates as part of the Siphoviridae family [START_REF] Oh | Isolation and Characterization of Bacillus cereus Bacteriophages from Foods and Soil[END_REF][START_REF] Geng | Identification and genomic comparison of temperate bacteriophages derived from emetic Bacillus cereus[END_REF]. In contrast, other studies have showed that bacteriophages infecting the B. cereus group belonged to the Myoviridae family [START_REF] Lee | Characterization and complete genome sequence of a virulent bacteriophage B4 infecting food-borne pathogenic Bacillus cereus[END_REF][START_REF] Shin | Prevalence of Bacillus cereus bacteriophages in fermented foods and characterization of phage JBP901[END_REF][START_REF] Lee | Isolation and characterization of phages infecting Bacillus cereus[END_REF][START_REF] Peng | Characterization of a novel phage infecting the pathogenic multidrug-resistant Bacillus cereus and functional analysis of its endolysin[END_REF] as well as to the Tectiviridae family [START_REF] Gillis | Prevalence, genetic diversity, and host range of tectiviruses among members of the Bacillus cereus group[END_REF].

The structural proteins of the seven phages isolated were analyzed by using SDS-PAGE. The major structural proteins and several minor proteins were identified in the isolated phages. In order to evaluate the protein molecular weights in the samples, the migration of each band was checked against the migration patterns of standard proteins with known molecular weight. The B. cereus phages Ø2BC4663 and Ø4BC4663(2) showed a similar banding pattern. However, the protein profile varied among the remaining phages. Hence, the structural proteins are unique to each phage and depend on their morphotype, even though the phage is part of the same family. Similar results have been reported previously using Escherichia coli [START_REF] Hantke | Major outer membrane protein of Escherichia coli k12 serve as receptors for the phage T2 (protein 1a) and 434 (protein 1b)[END_REF] and Salmonella bacteriophages [START_REF] Capparelli | Bacteriophage-Resistant Salmonella rissen: An In Vitro Mitigated Inflammatory Response[END_REF]. The difference in protein profiles could explain phage host and plaque morphology diversity. Therefore, structural protein analysis was suggested as a method of determining phage morphotype, characterization and differentiation [START_REF] Ngangbam | Molecular characterization of Salmonella bacteriophage isolated from natural environment and its potential role in phage therapy[END_REF].

According to the results of this study, all of the selected phages have the same restriction pattern, with an estimated genome size of around 37 kb. These results may suggest that the seven selected phages are quite similar in genome size and may also have similar DNA sequences. The size of the phages' genomes is comparable to that published in previous studies [START_REF] Klumpp | Complete Nucleotide Sequence and Molecular Characterization of Bacillus Phage TP21 and its Relatedness to Other Phages with the Same Name[END_REF][START_REF] Yuan | Genomic analysis of a phage and prophage from a Bacillus thuringiensis strain[END_REF]. However, phages specific to the B. cereus bacteria have been described as having a genome size of more than 130 kb [START_REF] Lee | Isolation and characterization of phages infecting Bacillus cereus[END_REF]47,[START_REF] Stewart | The genome of Bacillus subtilis bacteriophage SPO1[END_REF].

Comparative analysis of restriction fragment profiles showed that the genomes of the isolated phages were very similar. These results are consistent with those obtained in previous studies, which reported that several phages specific to the B. cereus group exhibited high genetic congruence [START_REF] Yuan | Genomic analysis of a phage and prophage from a Bacillus thuringiensis strain[END_REF][START_REF] Fouts | Sequencing Bacillus anthracis typing phages gammaand cherry reveals a common ancestry[END_REF][START_REF] Minakhin | Genome sequence and gene expression of Bacillus anthracis bacteriophage Fah[END_REF][START_REF] Schuch | Detailed genomic analysis of the Wbeta and gamma phages infecting Bacillus anthracis: Implications for evolution of environmental fitness and antibiotic resistance[END_REF][START_REF] Moumen | Sequence analysis of inducible prophage phIS3501 integrated into the haemolysin IIgene of Bacillus thuringiensis var israelensis ATCC35646[END_REF]. The results of these studies suggest that bacteriophages with similar genomes are likely to be found in a variety of environments. Although the genomes of these phages are generally very similar, several genes show a great diversity, and very variable regions in the genomes have been observed, such as the genes coding for proteins having functions in lysis of the host and in DNA replication [START_REF] Yuan | Genomic analysis of a phage and prophage from a Bacillus thuringiensis strain[END_REF]. Sequencing the genome of the phages isolated in this work could allow a better comparison.

The present study evaluated whether isolated bacteriophages could prevent or eradicate B. cereus biofilm formation on a stainless steel surface, commonly found in the food industry. It was found that phage treatment at all titers (10 5 -10 8 PFU/mL) was more effective in reducing biofilm formation (ranging from 0.1 to 1.5 log 10 ) than eradicating already formed biofilms (from 0 to 0.5 log 10 ). The low eradication percentage reported for 48 h-and 72 h-old biofilms could be attributed to the complex structure of preformed biofilms, that can pose a serious obstacle for phage penetration in multiple ways, such as the diffusion barrier imposed by the biofilm matrix and the physiological and metabolic state of biofilm cells [START_REF] Gong | Application of bacteriophages to reduce Salmonella attachment and biofilms on hard surfaces[END_REF].

B. cereus biofilms are composed mainly of vegetative cells, but during the maturation and aging process, B. cereus has the ability to form spores among established biofilms. Another study showed high levels (over 50%) of sporulation in 48 h submerged biofilms of different strains of the B. cereus group on stainless steel slides [START_REF] Faille | Sporulation of Bacillus spp. within biofilms: A potential source of contamination in food processing environments[END_REF]. Sporulation of B. cereus in biofilms has been evidenced in several studies, although further studies are needed to determine whether biofilms play a role in sporulation and the direct link between biofilm formation and B. cereus sporulation.

The production and accumulation of enterotoxins/emetic toxins in the biofilm matrix may function as an autoinducer of quorum sensing and, in particular, may contribute to biofilm development and play an anti-competitive or defensive role in protecting an inhabited niche by preventing invasion by other species [START_REF] Huang | Bacillus cereus spores and toxins-The potential role of biofilms[END_REF].

During the initial step of attachment in the biofilm formation process, bacterial cells may detach from the surface and return to a planktonic phase to colonize a new surface and, thus, bacteriophage treatment has high efficiency against these planktonic bacteria, as demonstrated in previous studies [START_REF] Heringa | Use of a mixture of bacteriophages for biological control of Salmonella enterica strains in compost[END_REF][START_REF] Kinley | Prevalence and Biological Control of Salmonella Contamination in Rendering Plant Environments and the Finished Rendered Meals[END_REF]. Therefore, the application of bacteriophages during the bacterial attachment step is more effective than on mature biofilms. Similar findings were reported during the application of bacteriophage P22 to reduce attachment and biofilms of Salmonella typhimurium on a stainless steel surface [START_REF] Karaca | Effects of P22 bacteriophage on Salmonella enterica subsp. enterica serovar Typhimurium DMC4 strain biofilm formation and eradication[END_REF]. The researchers found that treatment with phage P22 was significantly more effective in inhibiting biofilm formation (from 0.08 to 1.2 log 10 ) than in eradicating pre-formed mature biofilm, after treatment (from 0 to 0.5 log 10 ). Likewise, a study on the application of bacteriophages to reduce the attachment and biofilms of E. coli O157:H7 on a stainless steel surface [START_REF] Sharma | Inactivation of Escherichia coli O157:H7 in biofilm on stainless steel by treatment with an alkaline cleaner and a bacteriophage[END_REF] showed a reduction of 1.2 log 10 CFU/coupon after bacteriophage treatment of bacterial attachment; however, the same bacteriophage treatment was not able to significantly reduce pre-formed biofilms under the same conditions.

Our results show that the duration of phage treatment, as well as the age of the biofilm, influences the efficiency of inhibition of biofilm formation and eradication of mature biofilms of B. cereus. This is in agreement with the results of other studies [START_REF] Corbin | Bacteriophage T4 multiplication in a glucose-limited Escherichia coli biofilm[END_REF][START_REF] Sharma | Inactivation of Escherichia coli O157:H7 in biofilm on stainless steel by treatment with an alkaline cleaner and a bacteriophage[END_REF] that have revealed significant reductions in biofilms (ranging from 1 to 6 log10), dependent upon biofilm constituents, biofilm age, phage selection, and treatment duration.

As mentioned above, for mature pre-formed biofilm, the rate of biomass reduction was dependent on the biofilm matrix, that would render phage penetration difficult, rather than on the physiological status of the cells. Indeed, bacteriophages preferably infect planktonic cells in the exponential phase [START_REF] Jamal | Characterization of Siphoviridae phage Z and studying its efficacy against multidrug-resistant Klebsiella pneumoniae planktonic cells and biofilm[END_REF], such as the newly divided biofilm-surface bacteria and, thus, may not present sufficient efficiency against persistent cells with reduced metabolic activity, enclosed in biofilms, resulting in reducing bacteriophage treatment effectiveness [START_REF] Abedon | Bacteriophage exploitation of bacterial biofilms: Phage preference for less mature targets?[END_REF][START_REF] Gutiérrez | Bacteriophages as weapons against bacterial biofilms in the food industry[END_REF].

For controlling biofilm formation, bacteriophage treatment is not effective enough when it is extended. Indeed, biofilm formation is concluded at the end of the incubation period, due to the decrease in phage concentration [START_REF] Karaca | Effects of P22 bacteriophage on Salmonella enterica subsp. enterica serovar Typhimurium DMC4 strain biofilm formation and eradication[END_REF] or the appearance of resistance mechanisms of bacteria against phages, which negatively affects phage treatment efficacy [START_REF] Karaca | Effects of P22 bacteriophage on Salmonella enterica subsp. enterica serovar Typhimurium DMC4 strain biofilm formation and eradication[END_REF][START_REF] Sadekuzzaman | Application of bacteriophages for the inactivation of Salmonella spp. in biofilms[END_REF][START_REF] Hudson | Bacteriophages as biocontrol agents in food[END_REF]. As reported in previous studies [START_REF] Donlan | Preventing biofilms of clinically relevant organisms using bacteriophage[END_REF][START_REF] Labrie | Bacteriophage resistance mechanisms[END_REF], phage resistance within biofilms in laboratory conditions could be due to mutations in bacterial receptors or the loss of phage receptors. However, phages can overcome bacterial resistance by adapting to new receptors [START_REF] Samson | Revenge of the phages: Defeating bacterial defences[END_REF]. Moreover, during bacteriophage-mediated lysis, bacterial extracellular DNA can release and accumulate to a sufficient concentration, which would explain the increase in biofilm levels at this time [START_REF] Gödeke | Phage-induced lysis enhances biofilm formation in Shewanella oneidensis MR-1[END_REF].

Materials and Methods

Bacterial Strains and Culture Conditions

The bacterial strains used in this study for phage induction and host spectrum consisted of a collection of 174 B. cereus group strains, previously isolated from a total of 687 Tunisian food samples (cereals; spices; cooked food; canned products; seafood products; dairy products; fresh-cut vegetables; raw and cooked poultry meats) [START_REF] Gdoura-Ben Amor | Isolation, Identification, Prevalence, and Genetic Diversity of Bacillus cereus Group Bacteria from Different Foodstuffs in Tunisia[END_REF]. Brain heart infusion medium (BHI) (Fisher Bioblock, Illkirch, France), supplemented with yeast extract (YE) (Fisher Bioblock), was used to grow the B. cereus strains used in this study. All BHI-YE plates or broth cultures were incubated for 24 h at 30 • C.

Isolation of B. cereus-Specific Phages and Host Spectrum Evaluation 4.2.1. Sampling

Bacteriophages were isolated from sewage, before and after treatment, collected from a wastewater treatment plant (Rennes, France) and from a total of 50 samples containing animal wastewater [START_REF] Aprea | Isolation and Morphological Characterization of New Bacteriophages Active against Campylobacter jejuni[END_REF], slurry [START_REF] Tauveron | Variability among Bacillus cereus strains in spore surface properties and influence on their ability to contaminate food surface equipment[END_REF], fecal waste (11) sewage ( 5) and soil samples (4) collected from an experimental farm specializing in feed production for pigs, poultry and rabbits (Paris, France). For the absolute presence of bacteriophages, all samples were divided into 4 groups (P1-P4). The first group (P1) was made up of 25 samples taken from the experimental farm. The pooled sample of the first group combined 15 animal wastewater samples (settling ponds [START_REF] Tauveron | Variability among Bacillus cereus strains in spore surface properties and influence on their ability to contaminate food surface equipment[END_REF], pig effluent (5) and poultry effluent samples (3)), 6 samples of fecal waste and 4 samples of soil, altogether. The second group (P2) contained the remaining samples (25) taken from the experimental farm. Seven slurry, 5 fecal waste, 5 settling pond, 5 sewage and 3 rabbit effluent samples made up the combined sample of P2. For the third (P3) and fourth (P4) groups, each contained a mixture of sewage effluent samples, taken from the wastewater treatment plant before and after treatment, respectively.

Enrichment Phase

The collection of 174 B. cereus group strains was used to multiply bacteriophages in each pool sample (P1-P4) as follows: after incubation of each B. cereus group strain in BHI-YE broth for 5 h at 30 • C, 500 µL of each culture was mixed and added to 500 mL of BHI-YE broth to form the host culture broth. After 3 h of contact, 10 mL of each pooled sample (P1-P4) were added separately to the host culture broth. After incubation at 30 • C for 24 h, the culture supernatant was harvested by centrifugation at 7000 rpm for 20 min and sterilized by filter-sterilization (0.22 µm in pore size) (Starstedt, Nümbrecht, Germany). The filtrates obtained by this process were stored at 4 • C.

Phage Isolation, Propagating and Purifying

The bacteriophages infecting the B. cereus group were isolated using a spot assay technique [START_REF] Aprea | Isolation and Morphological Characterization of New Bacteriophages Active against Campylobacter jejuni[END_REF], with slight modifications. Briefly, 5 mL of BHI-YE soft agar (0.8% agar) inoculated with 100 µL of each B. cereus strain culture were overlaid on a pre-solidified BHI-YE agar (1.5% agar) plate. Once the overlay was dry, 10 µL of enriched suspension of each pooled sample (P1-P4) was spotted on the plate. The plates were left for 30 min until the spots were dry and then incubated overnight at 30 • C. In the case of phage detection (forming of lysis zones), plaques were individually picked up from the agar, dispensed into a sterile falcon tube containing 3 mL of BHI-YE broth and left at room temperature overnight to allow the phages to be diffused from the agar. Then, samples were mixed gently, and the liquid was filtered with a 0.22 µm filter. Subsequently, a phage titer was determined following a soft agar overlay method [START_REF] Shin | Prevalence of Bacillus cereus bacteriophages in fermented foods and characterization of phage JBP901[END_REF][START_REF] Lee | Isolation and characterization of phages infecting Bacillus cereus[END_REF]67,[START_REF] Wommack | Methods for the isolation of viruses from environmental samples[END_REF]. Briefly, the filtrate of the propagated plaque was diluted serially 10 times in TS buffer (8.5 g NaCl and 1 g tryptone per liter), mixed with 200 µL of each cultured B. cereus host strain and left for 15 min at room temperature to allow phages to adhere on the bacterial cells. Once the incubation was completed, the culture was added to 3 mL of soft agar (BHI-YE broth with 0.8% agar) and poured onto pre-solidified BHI-YE agar (1.5% agar). The plate was then incubated at 30 • C overnight. After this, plates were then observed for plaque formation, and this was expressed as plaque forming units/mL (PFU/mL) of supernatant. A sample was considered positive for phages when plaques were noted on the bacterial lawn of the plates. This procedure was repeated at least three times until the morphologies of the plaques became consistent. Phage-free cultures (containing only bacterial host) and host-free cultures (containing only phage) were used as controls.

Host Range Assay

A total of 62 phages infecting the B. cereus group were isolated. Host range analysis was performed to separate the broad-spectrum lytic phages from the 62 generated phage lysates. The host range of the 62 phages was tested by a spotting technique [START_REF] Lee | Isolation and characterization of phages infecting Bacillus cereus[END_REF] on 174 B. cereus group strains, conducted by spotting 10 µL of purified phage lysate onto host bacterial lawns prepared on a pre-solidified BHI-YE agar (1.5% agar) plate. The plates were left for 30 min until the spots were dry and then incubated overnight at 30 • C for lysis zone formation. As a control, each bacterial strain was also infected using sterile phage buffer. Lytic effectiveness of each phage lysate on the host bacteria was confirmed by plaque formation. Plaques were classified into the following three categories: clear, moderately clear, and turbid (data not shown). Seven B. cereus phages were selected for having produced clear plaques against a maximum number of bacterial strains (Table S1) and were subjected to further characterization steps.

Bacteriophage Characterization 4.3.1. High-Titer Phage Stock Preparation

The phage concentration step was performed according to a modified method described in previous studies [START_REF] Lee | Isolation and characterization of phages infecting Bacillus cereus[END_REF]67]. The filtrate of purified phages was diluted serially by ten-fold in TS buffer to achieve a concentration allowing confluent lysis of the host in a soft agar overlay plate. The dilution producing the most confluent lysis was chosen. Each phage stock was then plated by using the overlay method to achieve confluent lysis on 20 plates for each sample. After an overnight incubation, 5 mL of BHI-YE broth was added to each of these 20 plates. The suspension was recovered and left at 4 • C for 3 to 4 h, to allow the phages to diffuse from the overlay. The suspension was then centrifuged at 9000 rpm for 20 min, and this was sufficient to separate the overlay agar. The supernatant was harvested and filtered (0.22 µm), followed by ultracentrifugation at 35,000 rpm (Sorvall Discovery 90SE ultracentrifuge; T-865 rotor) for 2 h at 20 • C. The supernatant was then gently discarded, and the pellet was resuspended in 300 µL of TE buffer (10 mM Tris-HCL, 1 mM EDTA; pH = 8) that was added to each tube. The titer of the phage stock was then counted by the overlay method, using the appropriate host strain.

Morphology Observation by Transmission Electron Microscopy

Using a transmission electron microscope (TEM), phage morphologies were examined by a single negative staining method. The target titer of the stocks was >10 10 PFU/mL to allow visualization by electron microscopy. Thirty microliters of phage stock was spotted onto 200-mesh copper grids with carbon-coat formvar films (Electron Microscopy Sciences, Hatfield, PA, USA) and left for 1 min. The grids were then stained with 2% aqueous uranyl acetate for 1 min. Excess liquid was blotted with filter paper and the grid was air dried. Bacteriophage morphology was examined by electron microscopy in a JOEL JEM-1400 transmission electron microscope.

Structural Protein Analysis

As described by [START_REF] Geng | Identification and genomic comparison of temperate bacteriophages derived from emetic Bacillus cereus[END_REF] [START_REF] Geng | Identification and genomic comparison of temperate bacteriophages derived from emetic Bacillus cereus[END_REF], phage stock (10 9 PFU/mL) was diluted 1:1 in a dissociation buffer containing 95% (v/v) of 2× Laemmli Sample Buffer (Bio-Rad, Marnes-la-Coquette France) and 5% (v/v) of ß-mercaptoethanol (Sigma-Aldrich, Saint Quentin, Fallavier, France) and then boiled for 10 min to denature the viral proteins. The phage proteins were separated by SDS-PAGE in precast 4-20% SDS-polyacrylamide gel (Mini-PROTEAN TGX TM ) (Bio-Rad). Electrophoresis was carried out at 200 V for approximatively 30 to 40 min. Gels were stained with Bio-Safe Coomassie blue G250 (Bio-Rad) for 1 to 2 h. Protein mass determination was performed using Precision Plus Protein TM unstained standards (Bio-Rad).

Restriction Digest Profile Analysis of B. cereus Bacteriophages

Genomic DNA from a high titer of phages, prepared as described above (Section 4.3.1), was extracted using the phenol/chloroform method according to [START_REF] Sambrook | Molecular Cloning: A Laboratory Manual[END_REF] [START_REF] Sambrook | Molecular Cloning: A Laboratory Manual[END_REF]. The phage suspension (1 mL) was treated with DNase (invitrogen, Thermo Fisher Scientific, Vilnius, Lithuania) for 30 min, followed by the addition of 40 µL of proteinase K (Qiagen, Hilden, Germany) and incubation at 56 • C for 30 min. Then, 100 µL of 10% SDS was added, and the solution was incubated at 65 • C for 30 min. To remove proteins from the phage suspension, the mixture was treated with equal volumes of phenol/chloroform/isoamyl alcohol (25:24:1, v/v) twice. Subsequently, phage DNA was precipitated by adding 1/10 volume of aqueous 3 mol sodium acetate (pH 4.8) and 2.5 volumes of 100% ethanol and washed with 70% ethanol, and finally dissolved in 40-50 µL of milliQ water (Sigma-Aldrich). The quality of the extracted DNA was evaluated by using a NanoDrop ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, NC, USA), as measured by the absorbance ratio at 260 and 280 nm. The quality of the extracted DNA was also examined by 1% agarose-gel electrophoresis at 80 V for about 90 min. The genomic DNA was used for a restriction enzyme treatment with BamHI, EcoRI, HindIII, Hin6I and TaqI in an appropriate buffer, according to the manufacturer's instructions. The digested products were then analyzed by electrophoresis at 80 V for approximately 2 h on a 1% agarose gel in 1× TAE buffer (Promega, Madison, WI, USA) (Promega, Madison, WI, USA), and stained with ethidium bromide using a 10 kb SmartLadder (Eurogentec, Seraing, Belgium) as a molecular weight marker.

Assessment of Bacteriophage Action on B. cereus Biofilms 4.4.1. Preparation of Stainless Steel Coupons

As stainless steel is frequently used in food processing areas, 304 L stainless steel coupons measuring 2 mm thick and 15 mm in diameter (Bretagne Laser, Guer, France), were used in this study. The coupons were prepared for treatment assays as previously described by [START_REF] Jan | Biodiversity of psychrotrophic bacteria of the Bacillus cereus group collected on farm and in egg product industry[END_REF] [START_REF] Jan | Biodiversity of psychrotrophic bacteria of the Bacillus cereus group collected on farm and in egg product industry[END_REF]. After immersion in 2% (v:v) RBS 35 (Chemical Products R. BORGHGRAEF, Brussels, Belgium) in distilled water, the coupons were rinsed 5 times (for 5 min per rinse) with distilled water at 50 • C and 5 times with distilled water at room temperature. The coupons were then autoclaved for 20 min at 120 • C and transferred to the bottom of the wells of a 24-well microplate, using one coupon per well.

Reducing Pre-Formed Biofilm Experiment (Phage Post-Treatment)

An aliquot of 300 µL of each bacterial host used for propagation (strain 478, strain 3990 and strain 4663) [START_REF] Gdoura-Ben Amor | Isolation, Identification, Prevalence, and Genetic Diversity of Bacillus cereus Group Bacteria from Different Foodstuffs in Tunisia[END_REF] was taken from exponentially growing cultures (10 8 CFU/mL) and deposited in each well. The plates were incubated statically for 24, 48 and 72 h at 30 • C. Following the incubation period, the cell suspensions were removed, and the coupons held in the wells were rinsed with 9 g/L NaCl solution in order to discard non-adherent cells. To assess the effectiveness of phage treatment on the reduction of preformed biofilm of the B. cereus group, the methodology described in previous studies [START_REF] Garcia | Bacteriophage use to control Salmonella biofilm on surfaces present in chicken slaughterhouses[END_REF][START_REF] Karaca | Effects of P22 bacteriophage on Salmonella enterica subsp. enterica serovar Typhimurium DMC4 strain biofilm formation and eradication[END_REF] was followed with some adjustments. In brief, already formed biofilm samples on stainless steel coupons were treated with 300 µL of the appropriate phage dilutions (10 5 -10 8 PFU/mL) and incubated for 6h at 30 • C. Tests were performed twice in triplicate. One triplicate included, and the other excluded, the addition of bacteriophages, with each sample being used as its own positive control. After the bacteriophage treatment, each well was rinsed with 9 g/L NaCl solution 3 times and allowed to air-dry. Then, each coupon was moved to a new tube filled with 5 mL of 0.02% aqueous Tween80 solution (Thermo Fisher Scientific) and five sterile glass beads (3 mm of diameter). The coupons were then vortexed for 1 min at the highest intensity. To separate residual phages, the recovered suspension was centrifuged for 10 min at 7000 rpm at 25 • C. Cell pellets were resuspended in 1 mL of TS buffer and enumeration of bacteria was carried out by a plate counting micro-method [START_REF] Baron | Rapid and cost-effective method for microorganism enumeration based on miniaturization of the conventional plate-counting technique[END_REF]. The CFU (colony forming unit) count per square centimeter was converted to a logarithmic value. Results are presented as the effectiveness of bacteriophage treatment on reducing pre-formed biofilm of the B. cereus group. The percentage of biofilm eradication was determined using the following formula [START_REF] Karaca | Effects of P22 bacteriophage on Salmonella enterica subsp. enterica serovar Typhimurium DMC4 strain biofilm formation and eradication[END_REF]: [PK = (1 -10 -LR ) × 100%] where PK is the percentage killing and LR is the log reduction [log 10 (untreated viable cell density) -log 10 (treated viable cell density)].

Inhibiting Biofilm Formation Experiment (Phage Treatment)

The protocol evaluation of the inhibition potential of bacteriophages on B. cereus biofilm formation was adapted and optimized (with modifications) from previous work [START_REF] Karaca | Effects of P22 bacteriophage on Salmonella enterica subsp. enterica serovar Typhimurium DMC4 strain biofilm formation and eradication[END_REF]. Briefly, each B. cereus host strain was cultured with appropriate phage suspensions adjusted to 10 4 -10 8 PFU/mL in a test medium. The plates with stainless steel coupons were prepared as described in Section 4.4.1. An aliquot of 300 µL of each bacterial host (10 8 CFU/mL) and 300 µL of the appropriate phage suspension, adjusted to 10 5 -10 8 PFU/mL in TE buffer, were deposited in each well. The control well was inoculated only with host bacterial suspension. Tests were done in triplicate. The plates were incubated for 24, 48 and 72 h at 30 • C. Experimental data were obtained as described in Section 4.4.2. Results are presented as the effectiveness of bacteriophage treatment on the inhibition of B. cereus biofilm formation. The logarithmic reduction of biofilm cells was also calculated by the same formula given above.

Conclusions

The isolation and characterization of phages specific to the B. cereus group represents the first accomplishment of this study. Following characterization, the seven selected phages were found to be different in terms of identity. The second accomplishment of this work was to evaluate the effectiveness of phages to treat B. cereus attachment and biofilms on stainless steel surfaces. We found that phage treatment had the potential to inhibit up to 97.1% of biofilm formation and to reduce up to 59.4% of pre-formed biofilms. Our findings suggest that the use of bacteriophages in food processing facilities may reduce the possibility that finished products will be re-contaminated with B. cereus. Nevertheless, to evaluate the efficacity on real environment biofilms, some further experiments are needed, such as optimizing the bacteriophage cocktail to remove mixed B. cereus biofilm that occurs with other microflora on food contact surfaces. Moreover, the application of phage treatment as a highly potent approach to inactivate B. cereus attachment and biofilms in the food industry should be verified in a broader range of temperature, humidity and pH conditions.

Supplementary Materials:

The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/pathogens11080872/s1, Table S1. Host Range: Host specificity of each of the seven phage isolates in the B. cereus strains collection (n = 174). The sensitivity of different strains of the B. cereus group to different isolated phages was tested by plaquing; "+" (colored in yellow): sensitive to phages (formation of clear plaques); "-" (no color): resistant to phages (no formation of plaques).

Figure 1 .

 1 Figure 1. (A-G) Plaque morphology on agar plates of isolated phages. (A) Ø1BC478, (B) Ø1BC3990, (C) Ø2BC4663, (D) Ø3BC4663; (E) Ø4BC4663(1); (F) Ø4BC4663(2); and (G) Ø1BC4663.

Figure 1 .

 1 Figure 1. (A-G) Plaque morphology on agar plates of isolated phages. (A) Ø1BC478, (B) Ø1BC3990, (C) Ø2BC4663, (D) Ø3BC4663; (E) Ø4BC4663(1); (F) Ø4BC4663(2); and (G) Ø1BC4663.

Figure 2 .

 2 Figure 2. (A-G) Transmission electron micrographs of seven B. cereus-specific bacteriophages isolated in this study. (A) Ø1BC478; (B) Ø1BC3990; (C) Ø2BC4663; (D) Ø3BC4663; (E) Ø4BC4663(1); (F) Ø4BC4663(2); and (G) Ø1BC4663. The virions were negatively stained with uranyl acetate and visualized at a magnification of 100,000× (B,D,F), 120,000× (C,G) and 150,000× (A,E). The scale bar indicates 100 nm.

Figure 2 .

 2 Figure 2. (A-G) Transmission electron micrographs of seven B. cereusspecific bacteriophages isolated in this study. (A) Ø1BC478; (B) Ø1BC3990; (C) Ø2BC4663; (D) Ø3BC4663; (E) Ø4BC4663(1); (F) Ø4BC4663(2); and (G) Ø1BC4663. The virions were negatively stained with uranyl acetate and visualized at a magnification of 100,000× (B,D,F), 120,000× (C,G) and 150,000× (A,E). The scale bar indicates 100 nm.

Figure 3 .

 3 Figure 3. SDS-PAGE analysis of phage structural proteins. Lane 1; 5; 8 and 10: Precision Plus Protein TM unstained standards (Biorads). Lane 2; 3; 4; 6; 7; 9 and 11: Ø1BC478; Ø1BC3990; Ø2BC4663; Ø3BC4663; Ø4BC4663(1); Ø4BC4663(2) and Ø1BC4663; respectively.

Figure 3 .

 3 Figure 3. SDS-PAGE analysis of phage structural proteins. Lane 1; 5; 8 and 10: Precision Plus Protein TM unstained standards (Biorads). Lane 2; 3; 4; 6; 7; 9 and 11: Ø1BC478; Ø1BC3990; Ø2BC4663; Ø3BC4663; Ø4BC4663(1); Ø4BC4663(2) and Ø1BC4663; respectively.

Pathogens 2022 ,

 2022 11, x FOR PEER REVIEW 6 of 17results indicate that the selected phages have similar genome sizes and possibly are highly related. Selected phages have estimated genome sizes of around 37 kb, as calculated by adding together the fragment sizes obtained with the three enzymes above.

Figure 4 .

 4 Figure 4. Restriction enzyme digestion pattern analysis of isolated bacteriophages DNAs. Lane 2 to Lane 8: TaqI digestion of Ø1BC478; Ø1BC3990; Ø2BC4663; Ø3BC4663; Ø4BC4663(1); Ø4BC4663(2) and Ø1BC4663, respectively. Lane 10 to Lane 16: Hin6I digestion of Ø1BC478; Ø1BC3990; Ø2BC4663; Ø3BC4663; Ø4BC4663(1), Ø4BC4663(2) and Ø1BC4663, respectively. Lane 18 to Lane 24: EcoRI digestion of Ø1BC478; Ø1BC3990; Ø2BC4663; Ø3BC4663; Ø4BC4663(1); Ø4BC4663(2) and Ø1BC4663, respectively. Lanes 1, 9, 17 and 25: Molecular marker (SmartLadder 10 kb).

Figure 4 .

 4 Figure 4. Restriction enzyme digestion pattern analysis of isolated bacteriophages DNAs. Lane 2 to Lane 8: TaqI digestion of Ø1BC478; Ø1BC3990; Ø2BC4663; Ø3BC4663; Ø4BC4663(1); Ø4BC4663(2) and Ø1BC4663, respectively. Lane 10 to Lane 16: Hin6I digestion of Ø1BC478; Ø1BC3990; Ø2BC4663; Ø3BC4663; Ø4BC4663(1), Ø4BC4663(2) and Ø1BC4663, respectively. Lane 18 to Lane 24: EcoRI digestion of Ø1BC478; Ø1BC3990; Ø2BC4663; Ø3BC4663; Ø4BC4663(1); Ø4BC4663(2) and Ø1BC4663, respectively. Lanes 1, 9, 17 and 25: Molecular marker (SmartLadder 10 kb).

Figure 5 .

 5 Figure 5. (A-G) Effect of phage post-treatments at different titrations (10 5 -10 8 PFU/mL) on preformed biofilms of 24, 48 and 72 h and the reduction percentages of biofilm in a stainless steel application (bars represent standard deviation). (A) Ø1BC478; (B) Ø1BC3990; (C) Ø2BC4663; (D) Ø3BC4663; (E) Ø4BC4663(1); (F) Ø4BC4663(2); and (G) Ø1BC4663.

Figure 5 .

 5 Figure 5. (A-G) Effect of phage post-treatments at different titrations (10 5 -10 8 PFU/mL) on preformed biofilms of 24, 48 and 72 h and the reduction percentages of biofilm in a stainless steel application (bars represent standard deviation). (A) Ø1BC478; (B) Ø1BC3990; (C) Ø2BC4663; (D) Ø3BC4663; (E) Ø4BC4663(1); (F) Ø4BC4663(2); and (G) Ø1BC4663.

Figure 6 .

 6 Figure 6. (A-G): Effect of phage treatments at different titrations (10 5 -10 8 PFU/mL) on biofilm formation during incubation periods of 24, 48 and 72h and the inhibition percentages of biofilm formation in a stainless steel application (bars represent standard deviation). (A) Ø1BC478; (B) Ø1BC3990; (C) Ø2BC4663; (D) Ø3BC4663; (E) Ø4BC4663(1); (F) Ø4BC4663(2); and (G) Ø1BC4663.

Figure 6 .

 6 Figure 6. (A-G): Effect of phage treatments at different titrations (10 5 -10 8 PFU/mL) on biofilm formation during incubation periods of 24, 48 and 72 h and the inhibition percentages of biofilm formation in a stainless steel application (bars represent standard deviation). (A) Ø1BC478; (B) Ø1BC3990; (C) Ø2BC4663; (D) Ø3BC4663; (E) Ø4BC4663(1); (F) Ø4BC4663(2); and (G) Ø1BC4663.

Author

  Contributions: Conceptualization, M.G.-B.A. and M.A. (Mousa AlReshidi); methodology, M.G.-B.A., A.C., C.T. and N.G.; validation, M.A. (Mohd Adnan), S.J., C.T. and M.G.; formal analysis, M.G.-B.A., M.S. and A.C.; writing-original draft preparation, M.G.-B.A., M.A. (Mousa AlReshidi), M.A. (Mohd Adnan) and M.S.; writing-review and editing, S.J., C.T., R.G. and M.G.; supervision, S.J., F.B., R.G. and M.G. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding.

Table 1 .

 1 The details of isolation of B. cereus phages.

	Pooled Sample	Number of Isolated Phages	Isolated Phages
			Ø1BC13; Ø1BC14; Ø1BC18; Ø1BC27; Ø1BC28; Ø1BC396;
	P1	16	Ø1BC478; Ø1BC499; Ø1BC2893; Ø1BC2976; Ø1BC3296;
			Ø1BC3940; Ø1BC3990; Ø1BC4663; Ø1BC4785; and Ø1BC4851
			Ø2BC12; Ø2BC13; Ø2BC14; Ø2BC27; Ø2BC28; Ø2BC396;
	P2	15	Ø2BC2893; Ø2BC2976; Ø2BC3296; Ø2BC3990; Ø2BC4663;
			Ø2BC4785; Ø2BC4851; Ø2BC4855; and Ø2BC5659
			Ø3BC13; Ø3BC14; Ø3BC18; Ø3BC24; Ø3BC27; Ø3BC396;
	P3	19	Ø3BC478; Ø3BC2893; Ø3BC3296; Ø3BC3940; Ø3BC3990; Ø3BC4160; Ø3BC4165; Ø3BC4663; Ø3BC4785; Ø3BC4851;
			Ø3BC4855; Ø3BC5659; and Ø3BCE10
			Ø4BC13; Ø4BC24; Ø4BC27; Ø4BC396; Ø4BC478; Ø4BC2893;
	P4	12	Ø4BC3990; Ø4BC4663(1); Ø4BC4663(2); Ø4BC4785; Ø4BC4851;
			and Ø4BCE10

Table 2 .

 2 Percentage infectivity of each isolated bacteriophage, using a spot test method against 174 B. cereus strains.

	Isolated Phages	Percentage Lysis (%)
	Ø1BC3990	13.2
	Ø1BC478	13.2
	Ø3BC4663	12.6
	Ø2BC4663	10.3
	Ø4BC4663(2)	10.3
	Ø4BC4663(1)	9.7
	Ø1BC4663	8.6

2.2. Bacteriophage Characterization 2.2.1. Morphological Observation

Table 3 .

 3 Morphological specificity of B. cereus phages as demonstrated by transmission electron microscopy.

			Total Length	Head Diameter
	Phages	Characteristics	(±Standard Deviation)	(±Standard Deviation)	Family
			(nm)	(nm)
	Ø1BC478		330.5 ± 90.2	75.0 ± 31.8
	Ø1BC3990		411.4 ± 86.9	125.5 ± 20.8
	Ø2BC4663 Ø3BC4663 Ø4BC4663(1)	icosahedral head, non-contarctile tail	340.0 ± 93.8 355.6 ± 59.6 200.3 ± 74.9	113.3 ± 38.9 86.4 ± 27.6 87.5 ± 40.3	Siphoviridae
	Ø4BC4663(2)		337.7 ± 60.8	112.0 ± 29.9
	Ø1BC4663		314.2 ± 87.9	97.61 ± 18.9
		2.2.2. Protein Pattern Analysis

Table 3 .

 3 Morphological specificity of B. cereus phages as demonstrated by transmission electron microscopy.

	Phages

Characteristics Total Length (±Standard Devia- tion) (nm) Head Diameter (±Standard Devia- tion) (nm)

					Family
	Ø1BC478		330.5 ± 90.2	75.0 ± 31.8
	Ø1BC3990		411.4 ± 86.9	125.5 ± 20.8
	Ø2BC4663	icosahedral	340.0 ± 93.8	113.3 ± 38.9
	Ø3BC4663	head, non-con-	355.6 ± 59.6	86.4 ± 27.6	Siphoviridae
	Ø4BC4663(1)	tarctile tail	200.3 ± 74.9	87.5 ± 40.3
	Ø4BC4663(2)		337.7 ± 60.8	112.0 ± 29.9
	Ø1BC4663		314.2 ± 87.9	97.61 ± 18.9
	2.2.2. Protein Pattern Analysis
	SDS-PAGE was used to determine the structural protein content of each phage (Fig-
	ure

Data Availability Statement: Not applicable.

Conflicts of Interest:

The authors declare no conflict of interest.


Mourad Chemek

Samaher Chebil

Ben Sarra

Dalila Amor

Eric Khlaifia

Ayoub Haj Faulques

Kamel Said

Alimi

• Sarra

Ben Amor

Dalila Khlaifia

Eric Faulques

Ayoub Haj Said

Kamel Alimi
email: kamel.alimi@fsm.rnu.tn

Chemical insertion of anthracene moiety into the backbone of a newly synthesized oligophenylene (OMPA): effect on the photo-physical properties

Keywords: 4-(methoxyphenyl)acetonitrile, Anthracene, Optical properties, FT-IR, Time-resolved photoluminescence spectroscopy

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Introduction

The report of the electroluminescence (EL) of poly para-phenylenevinylene (PPV) in 1990 by Friend et al., led to major eff ts to improve the luminescence properties of conjugated polymers as luminescent active layers [1][2][3]. Indeed, an augmentation in EL intensity and an increase in the effi of the prepared electroluminescent devices based on conjugated-non-conjugated sequences were observed [3,4]. Recently in our laboratory, an improvement of the luminescence properties in solution and solid state (thin fi was observed when the non-con-jugated poly(9-vinylcarbazole) (PVK) was added in the chemical synthesis of poly(3-alkylthiophenes) [5,6]. The preparation of organic light-emitting diodes with blue emission is of great interest. Indeed, phenylene based polymer such as poly-p-phenylene (PPP) or oligophenylenes are an interesting class of blue emit-ting materials [7]. Grem et all reported a blue light emitting EL device using PPP [8,9]. However, the low EL quantum effi found for that device limits its exploitation and fabrication [8,9]. Indeed, as usual for many π-conjugated polymers, poor luminescence properties are obtained in solid state-e.g., thin fi due to the high aromatic π-π interaction and an inevitable fast migration of the photo-generated species [7,10,11]. Consequently, a quenching of redshifted PL is observed in solid state, with low quantum effi of the luminescence and a short fl escence lifetime [10,11]. In order to enhance the blue emission of any poly-p-phenylene derivative it is necessary to combine the organic mate-rial in hybrid organic/inorganic heterostructures [10][11][12]. Insertion of an inor-ganic material in the prepared hybrids reduces the π-π stacking interaction and improves the blue emission [10][11][12]. On the other hand, anthracene derivatives are an important class of fl ophores that has been widely used as co-monomers for designing and synthesizing a new class of blue light emitters [13,14]. Actu-ally, anthracene reduces in these compounds the aromatic π-π interactions in solid state and improves the luminescence properties [15][16][17].

In our previous work, we have synthesized new oligophenylene derivatives by electro-oxidative oligomerization of the 4-(methoxyphenyl acetonitrile) monomer [18]. The obtained oligomer is oligo-[4-(methoxyphenyl)acetonitrile]n (n = 1-5) denoted (OMPA). The chemical structure, as well as the optical properties of the obtained oligomer were also elucidated previously. The possible mechanism of the electro-oxidative oligomerization was supported theoretically by density functional theory (DFT) calculations [19]. Theoretical and experimental investi-gations show that the average length chain is of about 5-MPA units [18,19].

In order to improve the photo-physical properties of the synthesized OMPA oli-gomer, the chemical structure was modified by Knoevenagel condensation of OMPA with 9-anthtraldehyde. The chemical modification procedure is fully described below and the photo-physical properties of the obtained oligomer denoted OMPA-ANTH were studied with optical absorption, steady-state, and time-resolved photo-luminescence spectroscopies. Although the grafting of the anthracene group into the backbone of OMPA is partial, a significant change of the photo-physical properties was observed which is a consequence of modifications in the electronic structure.

Experimental details

4-Methoxyphenylacetonitrile, 4-methoxybezaldehyde, 9-anthraldhyde and tetrabuty-lammonium tetrafluoroborate (Bu) 4 N(BF 4 ) were purchased from ACROS ORGAN-ICS. Chloroform and dichloromethane were purchased from PROLABO. Diethyl ether, ethanol, petroleum ether, and acetonitrile were from PANREAC. The synthe-sis of OMPA oligomer was described elsewhere in our previous paper [19]. The pos-sible mechanism for the electro-oligomerization and the obtained chemical structure were supported by different experimental analyses as well as theoretical calculations [19,20].

Infrared spectra were obtained with a Bruker vector 22 Fourier transform spec-trophotometer. Samples were prepared in pellets of KBr mixed with the organic compound under study. Optical absorption measurements were carried out at room temperature (RT) by using a Varian Cary 5G spectrophotometer. Continuous-wave photoluminescence (PL) spectra were collected at RT from the front face geometry of the samples with a Jobin-Yvon Fluorolog 3 spectrometer equipped with a CCD camera using a xenon lamp (450 W) as an excitation source. Time-resolved photolu-minescence experiments (TRPL) were carried out at RT with a regeneratively ampli-fied femtosecond Ti: Sapphire laser system (Spectra-Physics Hurricane X) generat-ing 100 fs pulses at 800 nm with a repetitive rate of 1 kHz and an average power of 1 W. The laser line is frequency-tripled to obtain an excitation line ƛ exc = 266 nm. The Emission are temporally resolved with a high dynamic range Hamamatsu C7700 streak camera coupled to an imaging spectrograph with a temporal resolution less than 20 ps and processed using the HPDTA Hamamatsu software.

Optical measurements of the samples (OMPA and OMFA) were investigated in dilute chloroform solution and as thin films. For optical measurements, a diluted chloroform solution of OMPA and OMPA-ANTH (C =1.5 × 10 -5 M) was prepared. Thin films were obtained by simple deposition of a prepared chloroform solution (C =1.5 × 10 -3 M) by drop-coasting onto silica.

The voltammetric study was performed with a Voltalab10 apparatus from a Radi-ometer driven by the Volta Master software. The working electrode was a 2 mm diameter platinum disk; the reference electrode was an SCE (Tacussel SR110) and the counter electrode was a platinum wire. The preparative electrolyses were carried out in a two-compartment cell, under nitrogen, at a controlled potential versus an SCE (Tacussel C12). The potentiostat and the current integrator were Tacussel PRT 1-100 and IG 5, respectively. The working electrode and the counter electrode were a 4 cm 2 and a 0.25 cm 2 platinum gauze, respectively. The separation between cell compartments was realized by with four glass frits.

Results and discussion

1-Chemical modification of MPA/OMPA by 9-anthraldhyde using the Knoevenagel condensation way

The unmodified oligomer oligo [4-Methoxyphenylacetonitrile] (OMPA) was electrochemically prepared by the electrolysis of (4-methoxyphenyl) acetonitrile at a con-trolled potential of 1.8 V as described in our previous work [18]. First, we describe the modification of the chemical structure of the 4methoxyphenylacetonitrile (MPA) monomers by Knoevenagel condensation of MPA with the 9-anthraldehyde: a 50 mL reaction vessel was equipped with a magnetic stirrer, reflux condenser, gas inlet, and a rubber septum. It was charged with 1 mmol of MPA dissolved in 20 mL of anhydrous ethanol. To this mixture was added drop-wise, with stirring, 5 mL of a solution of sodium ethanolate (2 M) and then 1mmoL of 9-anthtraldehyde. The reaction was controlled by thinlayer chromatography. When the reaction was over, the mixture was cooled in an ice bath, then the modified monomer MPA-ANTH pre-cipitated as a yellow powder and it was removed by filtration and washed with dis-tilled water and then by ethanol. To purify this product, it was taken in chloroform and precipitated in petroleum ether. The yield of the reaction is 67%. The obtained MPA-ANTH exhibits good solubility in commonly used organic solvents such as chloroform (CHCl 3 ), dichloromethane (CH 2 Cl 2 ), and THF. The chemical structure of the modified and unmodified monomers (MPA/MPA-ANTH) was checked first by 1 H and 13 C NMR spectroscopies in CDCl 3 given in the supplementary material. of a solution of sodium ethanolate (2 M) and then 1.2 mL (10 -3 M) of anthraldehyde. The mixture was refl ed and stirred under argon atmosphere for 24 h, the mixture was cooled in an ice bath, then the obtained sample was precipi-tated and washed with distilled water and then by ethanol and fi ed.

To purify the sample, OMPA-ANTH was taken in chloroform and precipitated in petro-leum ether. The obtained oligomer exhibits good solubility in commonly used solvents (CHCl 3 , CH 2 Cl 2 , THF…). 1 H and 13 C spectroscopies (supplementary material) confi m the modifi and the chemical structure according to the presented Schemes 2 And 3.

2-Photo-physical properties of the OMPA-ANTH 2-1-Infrared analysis of the OMPA-ANTH

Normalized infrared spectra of the modified and unmodified OMPA oligomers are presented in Fig. 1, and the corresponding depicted vibrational modes are sum-marized in Table 1. The infrared spectrum of the unmodified OMPA prepared oli-gomer shows vibrations at around 665, 757, 927, 1026, 1180, 1413, 1461, 1504, 1608, 1664 cm -1 , resulting in the vibrations of the characteristic chemical group of OMPA oligomer [18]. Elsewhere, by analyzing the normalized infrared spectrum of the anthracene modified OMPA oligomer (OMPA-ANTH) we note firstly the pres-ence of the main vibrations of unmodified OMPA, however their positions and their intensities are slightly modified (see Fig. 1 and Table 1). Besides, we detect the dis-appearance of a few main vibrations of pure OMPA oligomer at 1082, 1372, and 1115 cm -1 and the appearance of new vibrations at 619, 773, 885, and 1624 cm -1 , which are attributed to the main vibrations of anthracene chemical group [20,21]. These observations confirm the formation of OMPA-ANTH chains.

2-2-Electrochemical measurements

Cyclic voltammetry measurements were performed using a three-electrode cell setup with 0.1 M tetrabutylammonium tetrafluoroborate (Bu) 4 N(BF 4 ) as a supporting electrolyte in degassed acetonitrile. Figure 2 illustrate the cyclic voltam-mograms of OMPA-ANTH oligomer. The E HOMO and the E LUMO were calculated according to the empirical method [22,23], where: E HOMO (eV) = -(E onset oxi + 4,4) and E LUMO (eV) = -(E onset red + 4,4) with the onset potentials measured vs. an ECS electrode. E onset oxi and E onset red were estimated from the obtained cyclic voltammo-grams (E onset oxi = 1.08 eV, E onset red = -0.79 eV). Therefore, E HOMO and E LUMO were estimated to be respectively -5.48 eV and -3.61 eV. Consequently, the energy gap E g = E HOMO -E LUMO is estimated to be 1.87 eV. Note that going from unmodified OMPA to anthracene modified OMPA leads to an observed increase in E HOMO and E LUMO , from -5.83 eV and -3.5 eV, respectively, for pure OMPA [18] to -5.48 and -3.61, respectively, for the anthracene modified oligomer. Consequently, the energy gap Eg = E HOMO -E LUMO decreases from 2.33 eV for the unmodified oli-gomer to 1.87 eV for the modified one.

2-3 Optical absorption observations

The ultraviolet-visible absorption spectra of the anthracene modified oligomer (OMPA-ANTH) are investigated in a diluted solution state, using chloroform (CHCl 3 ) as a solvent and in solid state (thin film). The normalized optical absorption spectra of the OMPA-ANTH oligomer are depicted together with that of unmodified OMPA oligomer in Fig. 3. Both obtained spectra of OMPA-ANTH exhibited a fine vibronic structure typically obtained for anthracene and anthracene derivatives [25,26], oppositely to the optical absorption spectrum of pure OMPA oligomer (Fig. 3) [18]. The optical absorption spectrum of OMPA-ANTH in solution and solid state displays a narrow intense transition at 260 nm, with three weak ones at 356, 370, and a last one at 390 nm, corresponding to the π-π* transition. Noting that, going from solution to solid state, no red-shift detected on the obtained optical transition. As mentioned above unmodified OMPA absorbs in solution in the UV part at 240, 270, and 340 nm [18]. A red-shift of the optical transition and in particular the π-π* transition from 340 to 390 nm under the chemical insertion of the anthracene group is observed. Using the well-known formula E g = 1240/λ onset where λ onset is the onset of the absorption wavelength [26], the optical bandgap E g is estimated. The onset of absorption of modified OMPA is at 490 nm in solution. Going from OMPA to OMPA-ANTH a red-shift of λ onset from 400 nm [18] to 490 nm is obtained in solu-tion, indicating consequently a decrease in the optical bandgap from 3.1 eV for unmodified OMPA [18] to 2.53 eV for OMPA-ANTH in solution state. Going from OMPA-ANTH solution to solid state a red-shift of the onset of the absorption wave-length is observed from 490 to 514 nm, implying an optical band gap at around 2.4 eV in solid state.

2-4-Steady-state photoluminescence observations

The excited state of the as-prepared samples is, first, investigated by steadystate photoluminescence measurement (PL) in a diluted chloroform solution state. The normalized photoluminescence spectra of OMPA and OMPA-ANTH, under UV excitations at λ 1exc = 260, λ 2exc = 350 and λ 3exc = 390 nm, are presented in Fig. 4. Under λ 1exc = 260 nm excitation, the unmodified OMPA oligomer presents an intense and large emission with a vibronic profile showing two maximum emissions at 380 and 490 nm. For λ 2exc = 350 and λ 3exc = 390 nm excitations, broad PL spectra were obtained in solution, with maximum emission λ emi2 and λ emi3 at 470 and 490 nm, respectively. Upon insertion of anthracene, the emission properties of the oligomers were greatly modified and all obtained spectra of OMPA-ANTH exhibited vibronic structures typical of anthracene and anthracene derivatives [24,25] for different excitation wavelengths.

2-5-Transient photoluminescence observations

In this part, time-resolved photoluminescence spectroscopy (TRPL) has been used to better understand the possible photo-physical mechanisms -such as inter and intra-exciton formation and migration of the photo-generated speciesinto the modified and unmodified oligomers. a semi-logarithmic scale in the range 0-5 ns. Figure 5b presents TRPL decays of thin films of these oligomers on a semi-logarithmic scale in the range 0-1 ns. A single monoexponential fit cannot reproduce the experimental decays especially in solid state. This suggests the presence of multiple exciton decay mechanism as it could be expected in solid state film. All obtained decays are simulated using two coupled exponential convoluted with a Gaussian laser pulse according to the model presented by Faulques et al. [START_REF] Faulques | Spectroscopy of Emerging Materials[END_REF]28]. In this model, the two coupled exponential decays convoluted with the contribution of apparatus function given by the Gaussian temporal dependence G (t) of the laser pulse, according t o t h e f o l l o w i n g r a t e e q u a t i o n s

1 = 2
In these equations n1 and n2 are the populations of the excited states levels 1 and 2, respectively and k 1 and k 2 are the inverse of the lifetimes τ 1 and τ 2 . In this simple model [START_REF] Faulques | Spectroscopy of Emerging Materials[END_REF], the populations of levels 1 and 2 are coupled in order to account indirectly for a migration process from the short to the long segments [START_REF] Faulques | Spectroscopy of Emerging Materials[END_REF]28]. Photogenerated charges populating the higher energy level 1 migrate toward defects and fast relax on the lower energy state 2 with lifetime τ 1 . At longer time, the photogenerated charges on level 2 are less mobile and consequently survive longer with a slower time constant τ 2 . Furthermore, n 1 and n 2 represent the total populations of photogenerated charges in the energy states 1 and 2. These populations include photogenerated charges recombining radiatively and nonradiatively.

The decaying population is defined as n = A 1 n 1 + A 2 n 2 , where A 1 and A 2 are proportional to the PL intensity from levels 1 and 2, respectively. The average intensityweighted lifetime τ mean is defined according to the following formula:

A 1 r 2 + A 2 r 2  1 mean A r 1 2 + A 2 r
For clarity, the weight corresponding to the relative population of photogenerated charges contributing to each of the decay times (A i , i ) is calculated by the following formula:

P i (%) = A i r i ΣA i r i
All deduced parameters from the simulation of decays of OMPA and OMPA-ANTH solution and thin film are presented in Table 2. For both oligomers prepared in solution, TRPL decays tend to have a single exponential behavior with a slower decay time. When going to the condensed prepared films, TRPL spectra of both samples present a faster decay with a biexponential character. These results are essentially due to different photo-generated species under laser wavelength excitation going from solutions to thin films. Indeed, previous studies [6,29,30] show that the strong macromolecular interactions in the condensed state favor the migration of the photo-generated species from short chains to longer ones leading to photoluminescence quenching [6,28,29]. The effect of the chemical grafting of anthracene units into the backbone of the prepared OMPA oligomer on the photo-physical properties is observed in the solution or condensed state. Indeed, TRPL decays of OMPA modified by anthracene prepared in solution or thin films are slower compared to those of the unmodified OMPA oligomer in both states. Thus, this result shows that solid-state interactions favor a fast migration of the photo-generated species through a fast decay channel with a lower average lifetime τ mean . Actually, it has been shown that an increase of τ 2 and τ mean is a signature of the increase of the effective conjugation length on the chemical structure 29]. Indeed, the chemical insertion of the anthracene group into the backbone of the OMPA oligomer induces an increase of τ 2 and τ mean lifetimes (see Table 2) in the diluted and condensed state. One can conclude first that the conjugation in the oli-gomers increases, which is confirmed by the augmentation of the optical bandgap and the increase in the onset and the maximum of absorption. Second, the increase in the PL lifetime is a sign that the inter-chain interaction in OMPA-ANTH is less important than that in the OMPA oligomer.

2-6-Thermal investigations

Thermal properties of the modified and unmodified oligomers were investigated by thermogravimetric analysis (TGA) under nitrogen atmosphere. Figure 6 shows the TGA curve of pure OMPA and OMPA-ANTH oligomer measured for the tempera-ture range varying from 0 to 700 °C. Modified and unmodified oligomers remain stable until around 300 °C. In the studied range of temperature, we can distinguish four thermal stability regions. A slow and negligible weight loss is observed until 130 °C for both oligomers (at around 5% at 130 °C). These behaviors are most prob-ably due to the departure of residual solvents used in the synthesis step and also Fig. 6 TGA curve of OMPA and OMPA-ANTH to unreacted used substance under chemical reactions. Between 130 °C and 300 °C, both oligomers remain stable with a weak weight loss of about 15% at 300 °C. Elsewhere, until 300 °C pure OMPA decomposes slowly compared to modified OMPA (OMPA-ANTH). This feature is due to the degradation of the added anthracene group in this range of temperature. Anthracene and anthracene-based polymer remain stable until 300 °C and undergo a fast degradation between around 300 °C and 480 °C [30]. In the last range (T > 480 °C), a fast decomposition is observed for both oligomers, which is slower for the OMPA-ANTH.

Conclusion

In this work, we have successfully modified the chemical structure of the synthe-sized OMPA oligomer using the chemical Knoevenagel condensation route. The grafting of the anthracene functional group induces a decrease in the optical and electronic bandgap. Added to that a substantial change is observed in the emission properties. Finally, we showed that the post-synthetic chemical modification of our oligophenylene is a versatile molecular engineering tool enabling the design of new materials with modulated functionalities and photophysical properties.

1 HScheme 2

 12 Scheme 1 Chemical structure of the obtained OMPA oligomer

Fig. 1 1 Fig. 2

 112 Fig. 1 Experimental FT-IR spectra of OMPA and OMPA-ANTH

Fig. 3

 3 Fig. 3 Normalized optical absorption spectra of unmodified OMPA in solution, and OMPA-ANTH in chloroform solution and thin film

Fig. 4

 4 Fig. 4 Steady-state photoluminescence spectra of OMPA and OMPA-ANTH for different excitation wavelength (λ 1exc = 260 nm, λ 2exc = 350 nm and λ 3exc = 390 nm) in chloroform solution

Fig. 5 (

 5 Fig. 5 (A): Decay time of OMPA and OMPA-ANTH in chloroform solution with 0-5 ns window. (B) Decay time of OMPA and OMPA-ANTH in condensed state (thin film) with 0-1 ns window

Table 2

 2 Decay times (τ 1 , τ 2 ,

	(A 1 , A 2 ) and percentage τ mean ), pre-exponential factor	Samples	(P 1% ) τ 1 (ns) (A 1 )	(P 2% ) τ 2 (ns) (A 2 )	τ mean (ns)
	contributions P 1 , P 2 to emission of levels 1 and 2 of OMPA and OMPA-ANTH oligomers in	(Solution) OMPA	(8.7) 0.4	(4.2) 2.32	1.82
	solution and solid states		(31)	(68)
		OMPA (Thin film)	0.034 (15.17)	0.23 (1.67)	0.11
		OMPA-ANTH (Solution)	(55) (6) 0.34	(45) (3.6) 3.22	3
		OMPA-ANTH	(6)	(94)
		(Thin film)	0.041 (3.56)	0.37 (31)	0.2
			(50)	(49)

Acknowledgements

The authors are grateful to Dr Jany Wéry-Venturini and to Dr Florian Massuyeau for their help on optical measurements at IMN Nantes.

Supplementary Information

The online version contains supplementary material available at https://doi. org/10.1007/s11164-021-04481-z.