Elsevier

Food Microbiology

Volume 82, September 2019, Pages 177-193
Food Microbiology

Proteomic analysis of the food spoiler Pseudomonas fluorescens ITEM 17298 reveals the antibiofilm activity of the pepsin-digested bovine lactoferrin

https://doi.org/10.1016/j.fm.2019.02.003Get rights and content

Highlights

  • Biofilm biomass by P. fluorescens increased at 15 °C compared to 30 °C.

  • Bovine lactoferrin hydrolysate reduced biofilm regardeless growth temperature.

  • Bovine lactoferrin hydrolysate affected swarming, twiching, swimming motility.

  • Comparative proteomic analysis revelead the affected pathways.

Abstract

Pseudomonas fluorescens is implicated in food spoilage especially under cold storage. Due to its ability to form biofilm P. fluorescens resists to common disinfection strategies increasing its persistance especially across fresh food chain. Biofilm formation is promoted by several environmental stimuli, but gene expression and protein changes involved in this lifestyle are poorly investigated in this species.

In this work a comparative proteomic analysis was performed to investigate metabolic pathways of underlying biofilm formation of the blue cheese pigmenting P. fluorescens ITEM 17298 after incubation at 15 and 30 °C; the same methodology was also applied to reveal the effects of the bovine lactoferrin hydrolysate (HLF) used as antibiofilm agent.

At 15 °C biofilm biomass and motility increased, putatively sustained by the induction of regulators (PleD, AlgB, CsrA/RsmA) involved in these phenotypic traits. In addition, for the first time, TycC and GbrS, correlated to indigoidine synthesis (blue pigment), were detected and identified. An increase of virulence factors amounts (leukotoxin and PROKKA_04561) were instead found at 30 °C. HLF caused a significant reduction in biofilm biomass; indeed, at 15 °C HLF repressed PleD, TycC and GbrS and induced the negative regulators of alginate biosynthesis; at both temperatures induced the cyclic-di-GMP-binding biofilm dispersal mediator (PROKKA_02061).

In conclusion, in this work protein determinats of biofilm formation were revelead in ITEM 17298 under the low temperature; the synthesis of these latter were inhibited by HLF confirming its possible exploitation as antibiofilm agent for biotechnological applications in cold stored foods.

Introduction

Pseudomonas fluorescens are widespread psychrotrophic Gram-negative bacteria implicated in food spoilage, especially under cold storage, causing the reduction of shelf-life and loss of foodstuffs (Baruzzi et al., 2012; Caldera et al., 2016). Pseudomonas spp. contaminations in food chain are mostly derived from water and pipe surfaces where these bacteria grow as biofilms (Srey et al., 2013). During biofilm formation, the transition from planktonic (free living) cells to the attached aggregated form is triggered by de-novo expression of transcriptional regulators and key genes responsible for surface-cell and intracellular interactions, metabolic pathways, virulence and resistance mechanisms (Waite et al., 2005). Moreover, the formation of a biofilm is considered a strategy to counteract microbial competition (Oliveira et al., 2015).

Biofilm formation can be influenced and promoted by different factors, such as nutrients, kind of surfaces, stress response (Monds and O'Toole, 2009). Recently, a positive correlation between low temperatures and biofilm production by foodborne P. fluorescens was found by Rossi et al. (2018) reporting that the number of biofilm-forming strains at 15 °C was higher than that at 30 °C. Likewise, Chierici et al. (2016) and Caputo et al. (2015) reported that low temperatures (4 and 15 °C) induced pigment production for this bacterial species. In P. aeruginosa the role of pigments in biofilm formation (Mavrodi et al., 2013; Park et al., 2014), as well as other genes and factors involved in the transition to aggregated cells and biofilm maintenance has been studied for a long time. By contrast, to the best of our knowledge, no metabolic pathways have been deeply investigated to explain P. fluorescens responses to environmental conditions. After all, the human risks correlated with the spread of this species had been underestimated. It is only recently that some studies identified P. fluorescens in clinical environment (Dickson et al., 2014; Nishimura et al., 2017) and correlated them to human diseases (Madi et al., 2010; Nishimura et al., 2017). In addition to this, P. fluorescens harbors an enormous pool of antibiotic and biocide resistance genes that can be transmitted to human and animals via horizontal gene transfer through contaminated foods (Donnarumma et al., 2010; Naghmouchi et al., 2012). It is clear that these results highlighted the urgent need for further researches to better characterize and counteract the spread of this microorganism.

In this regard, several strategies preventing biofilm formation have been investigated and also identified from diverse natural sources, such as plant-derived compounds (Hentzer et al., 2003; Caputo et al., 2018). In this context, the application of natural cationic peptides was reported as a promising antibiofilm strategy against different species (Rajput and Kumar, 2018); however, biophysical properties required for antibiofilm activity and its mechanism are not fully known.

In our previous works, we investigated the antimicrobial efficacy of bovine lactoferrin-derived peptides (BLFPs) in counteracting the growth of foodborne pseudomonads (Quintieri et al., 2012, 2013a); the antimicrobial efficacy of these peptides was demonstrated in vitro, in cold stored foods and on functionalized coatings (Baruzzi et al., 2015; Quintieri et al., 2013b, 2015); BLFPs were also able to block the blue discoloration of Mozzarella cheese contaminated by the pigmenting P. fluorescens ITEM 17298 (Caputo et al., 2015). Studies by other authors showed that peptides derived from human lactoferrin significantly inhibited these phenotypic traits also in other microorganisms (Morici et al., 2016; Xu et al., 2010; Sánchez-Gómez et al., 2015); however, no results revealed how these compounds act.

Therefore, in this work we firstly investigated the ability of the pigmenting P. fluorescens ITEM 17298 to form biofilm under two temperatures (15 °C and 30 °C); then, we present a comparative proteomic analysis of P. fluorescens ITEM 17298 planktonic cells, grown under the assayed temperatures in order to reveal metabolic pathways and physiological changes that characterize strain adaptation to these conditions. In addition to this, the same methodology was applied on the planktonic cells treated with bovine lactoferrin hydrolysate (HLF) acting as “antibiofilm agent” at its sub-lethal concentration. The results of this study reveal some protein targets and metabolic pathways involved in the expression of biofilm phenotype at the assayed temperatures and affected by peptide treatment.

Section snippets

Bacterial strain, growth conditions and genome analysis

The foodborne Pseudomonas fluorescens ITEM 17298 (previously named as 84095) from the ISPA-CNR microbial collection (http://server.ispa.cnr.it/ITEM/Collection/; Fanelli et al., 2017) was freshly streaked onto Luria Bertani agar (LB broth: 10.0 g of tryptone, 5.0 g of yeast extract, 10.0 g of NaCl per liter added with 16 g/L of technical agar, Sigma-Aldrich, Milan, Italy) and grown overnight at 30 °C. After incubation a single colony was inoculated into LB broth (5 mL) and incubated overnight

Genomic features of P. fluorescens ITEM 17298

The draft genome sequencing resulted in 18 MB of 125 bp paired-end reads and indicated a genomic size of 6,318,747 bp with a GC content of 59%. The evaluation of the raw data quality performed by FastQC software indicated that more that 95% of reads per sample showed an average quality score higher than 30. Reads were assembled into 247 contigs >200 bp (Fanelli et al., 2017). Analysis of protein domains categorized 48 of the predicted proteins as involved in antibiotic and cationic

Discussion

P. fluorescens exhibits a broad temperature adaptability affecting its spoilage activity mainly in cold stored foods. This behavior causes an evident competitive microbial advantage that is also favoured by biofilm formation and the ability to tackle to environmental changes. In this context, the mechanisms underlying physiological and spoilage traits of this microorganisms have been poorly studied. To this purpose, we firstly investigated strain phenotypic traits (biofilm biomass produced and

Conclusion

For the first time the proteome profile of a blue pigmenting and biofilm forming P. fluorescens was presented in this work. Proteomic results were consistent with microbiological ones favoring at the low temperature both the highest biofilm biomass and an increase of different protein determinants related with biofilm formation, cell motility, and adhesion. Conversely, at 30 °C some virulence factors such as leukotoxin were detected, highlighting the need to further investigate this strain.

Author contributions

LQ, KR designed research; FF, VCL performed genomic analysis; LQ, DZ performed proteomic analysis; DB and CH performed mass spectrometry analyses; LQ and LC performed and analyzed microbiological data; LQ and DZ analyzed proteomic data; LQ and DZ wrote the paper; LQ, DZ, AFL, LC, FF and KR revised the manuscript.

The authors are thankful to Dr Lucia Decastelli (Istituto Zooprofilattico Sperimentale del Piemonte, Liguria 62 e Valle d'Aosta, Turin, Italy) for having supplied the strain used in

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