Genomic analysis of three cheese-borne pseudomonas lactis with biofilm and spoilage-associated behavior

Psychrotrophic pseudomonads cause spoilage of cold fresh cheeses and their shelf-life reduction. Three cheese-borne Pseudomonas sp., ITEM 17295, ITEM 17298, and ITEM 17299 strains, previously isolated from mozzarella cheese, revealed distinctive spoilage traits based on molecular determinants requiring further investigations. Genomic indexes (ANI, isDDH), MLST-based phylogeny of four housekeeping genes (16S rRNA, gyrB, rpoB and rpoD) and genome-based phylogeny reclassified them as Pseudomonas lactis. Each strain showed distinctive phenotypic traits at 15 and 30◦C: ITEM 17298 was the highest biofilm producer at both temperatures, whilst ITEM 17295 and ITEM 17299 showed the strongest proteolytic activity at 30◦C. A wider pattern of pigments was found for ITEM 17298, while ITEM 17295 colonies were not pigmented. Although the high genomic similarity, some relevant molecular differences supported this phenotypic diversity: ITEM 17295, producing low biofilm amount, missed the pel operon involved in EPS synthesis and the biofilm-related Toxin-Antitoxin systems (mqsR/mqsA, chpB/chpS); pvdS, required for the pyoverdine synthesis, was a truncated gene in ITEM 17295, harboring, instead, a second aprA involved in milk proteolysis. This work provided new insight into the food spoiler microbiota by identifying these mozzarella cheese spoilers as P. lactis; molecular targets to be exploited in the development of novel preservative strategies were also revealed

Biofilm and Pathogenesis-Related Proteins in the Foodborne P. fluorescens ITEM 17298 With Distinctive Phenotypes During Cold Storage

In food chain, Pseudomonas spp. cause spoilage by reducing shelf life of fresh products, especially during cold storage, with a high economic burden for industries. However, recent studies have shed new light on health risks occurring when they colonize immunocompromised patient tissues. Likewise to P. aeruginosa, they exhibit antibiotic resistance and biofilm formation, responsible for their spread and persistence in the environment. Biofilm formation might be induced by environmental stresses, such as temperature fluctuations causing physiological and metabolic changes exacerbating food spoilage (by protease and pigment synthesis), and the production of adhesion molecules, chemotactic or underestimated virulence factors. In order to provide a new insight into phenotypic biodiversity of Pseudomonas spoilers isolated from cold stored cheese, in this work 19 Pseudomonas spp. were investigated for biofilm, pigments, exopolysaccharide production and motility at low temperature. Only nine strains showed these phenotypic traits and the blue pigmenting cheese strain P. fluorescens ITEM 17298 was the most distinctive. In addition, this strain decreased the survival probability of infected Galleria mellonella larvae, showing, for the first time, a pathogenic potential. Genomic and proteomic analyses performed on the ITEM 17298 planktonic cells treated or not with lactoferrin derived antibiofilm peptides allowed to reveal specific biofilm related-pathways as well as proteins involved in pathogenesis. Indeed, several genes were found related to signaling system by cGMP-dependent protein kinases, cellulose, rhamnolipid and alginate synthesis, antibiotic resistance, adhesion and virulence factors. The proteome of the untreated ITEM 17298, growing at low temperature, showed that most of the proteins associated with biofilm regulation, pigmentation motility, antibiotic resistance and pathogenecity were repressed, or decreased their levels in comparison to that of the untreated cultures. Thus, the results of this work shed light on the complex pathways network allowing psychrotrophic pseudomonads to adapt themselves to food-refrigerated conditions and enhance their spoilage. In addition, the discovery of virulence factors and antibiotic resistance determinants raises some questions about the need to deeper investigate these underestimated bacteria in order to increase awareness and provide input to update legislation on their detection limits in foods

Development of a real-time loop-mediated isothermal amplification method for monitoring Pseudomonas lurida in raw milk throughout the year of pasture

IntroductionThe psychrophilic bacterium Pseudomonas lurida (P. lurida) and its thermostable alkaline proteases can seriously damage raw milk quality.MethodsIn this study, specific primers were designed for P. lurida’s gyrB and aprX genes, and a real-time loop-mediated isothermal amplification (RealAmp) rapid detection method was developed for the early monitoring of P. lurida and its proteases in raw milk. A phylogenetic tree of the gyrB and aprX genes of P. lurida was constructed to analyze the homology of the design sequence of the RealAmp primer. The DNA of 2 strains of P. lurida and 44 strains of non-P. lurida were detected via RealAmp to analyze the specificity of the primer.ResultsIt was found that aprX-positive proteases were produced by P. lurida-positive strains only when Pseudomonas fluorescens was negative. The dissociation temperatures of gyrB and aprX in the RealAmp-amplified products were approximately 85.0°C and 90.0°C, respectively. Moreover, DNA was detected through a 10-fold dilution of P. lurida in a pure bacterial solution and artificially contaminated skimmed milk. The limit of detection of P. lurida DNA copy number in the pure bacterial solution was 8.6 copies/μL and that in the 10% skimmed milk was 5.5 copies/μL. Further, 144 raw milk samples throughout the year from three farms in Hebei province were analyzed using RealAmp. The highest detection rate of P. lurida was 56% in the first and third quarters, and that of proteases was 36% in the second quarter. The detection rates of P. lurida and its proteases were the highest in samples collected from pasture 2 (52 and 46%, respectively), and the ability of P. lurida to produce proteases reached 88%.DiscussionIn conclusion, RealAmp established an early and rapid method for the detection of P. lurida and its proteases in raw milk samples, allowing the identification and control of contamination sources in a timely manner to ensure the quality of milk and dairy products

Plant Growth-Promoting Activity of Pseudomonas aeruginosa FG106 and Its Ability to Act as a Biocontrol Agent against Potato, Tomato and Taro Pathogens

Simple Summary Microbial bio-stimulants are attracting increasing attention in agricultural research. In particular, plant growth-promoting rhizobacteria (PGPR) have great potential to improve crops' productivity and tolerance of biotic and abiotic stresses. It is anticipated that PGPR could eventually replace synthetic fungicides in agriculture. This research evaluated Pseudomonas aeruginosa strain FG106-which was isolated from tomato plants- as a potential biocontrol agent against several plant pathogens. This strain displayed multiple plant growth-promoting attributes and high in vitro and in vivo inhibition of growth and pathogenicity of tested phytopathogens. It is thus a multifunctional PGPR with potential applications as a biocontrol agent to control fungal and bacterial pathogens. P. aeruginosa strain FG106 was isolated from the rhizosphere of tomato plants and identified through morphological analysis, 16S rRNA gene sequencing, and whole-genome sequencing. In vitro and in vivo experiments demonstrated that this strain could control several pathogens on tomato, potato, taro, and strawberry. Volatile and non-volatile metabolites produced by the strain are known to adversely affect the tested pathogens. FG106 showed clear antagonism against Alternaria alternata, Botrytis cinerea, Clavibacter michiganensis subsp. michiganensis, Phytophthora colocasiae, P. infestans, Rhizoctonia solani, and Xanthomonas euvesicatoria pv. perforans. FG106 produced proteases and lipases while also inducing high phosphate solubilization, producing siderophores, ammonia, indole acetic acid (IAA), and hydrogen cyanide (HCN) and forming biofilms that promote plant growth and facilitate biocontrol. Genome mining approaches showed that this strain harbors genes related to biocontrol and growth promotion. These results suggest that this bacterial strain provides good protection against pathogens of several agriculturally important plants via direct and indirect modes of action and could thus be a valuable bio-control agent

Investigating biofilm adhesion and cohesion forces in respect to cleaning applications

Biofilms are complex microbial ecosystems formed by one or more bacteria species immersed in an extracellular matrix of different compositions depending on the environment and the colonizing species. While bacteria are beneficial for several technological bioprocesses, they could be catastrophic for our everyday lives as humans. In schools, healthcare facilities, food processing lines, our homes, we try to keep every surface clean and sterilized from our invisible “friends” in order to prevent ourselves from infectious diseases. Since biofilms are living communities, not traceable with the human eye, reliable methods are needed for the investigation of their attachment, growth and removal on surfaces, three different phenomena all regulated by forces. The goal of this PhD was to understand how the different material surfaces affect the above phenomena. In the first study the initial biofilm growth as well as the removal of Pseudomonas fluorescens and Pseudomonas putida biofilms from different material surfaces was investigated. After 30 minutes of growth at 25 °C for P. fluorescens and at 30oC for P. putida it was found that P. fluorescens showed higher percentage of surface coverage comparing to P. putida on all surfaces. In terms of different materials, the percentage of the area covered by bacteria was significantly lower on plastic surfaces (PET, PTFE and polypropylene) than on more hydrophilic surfaces like glass, hydroxyapatite and stainless steel. The biofilm residual contamination was investigated using a parallel-plate flow chamber, developed for this thesis, where three different cleaning conditions were tested on stainless steel, polycarbonate and plasma-treated polycarbonate surfaces: 1) Water rinsing under shear stress conditions, 2) NaOH cleaning in static conditions and 3) NaOH cleaning under shear stress conditions. It was found that the procedure that combined NaOH and shear stress was more effective for all material surfaces. In terms of surfaces, it was seen that stainless steel was cleaned more efficiently compared to plastic surfaces. In terms of biofilm residual contamination, a more distinct biofilm removal was observed for P. putida than for P. fluorescens. In the second study the removal of real mixed-microbial biofilm from common artificial surfaces was investigated using commercial enzymatic detergents and disinfectants used in the food industry. A mixed-microbial sample was sourced from a meat packaging line and biofilm was grown under high shear conditions on stainless steel and PET surfaces and the synergistic effect of enzymes in biofilm cleaning was studied. The cleaning effectiveness was evaluated in response to different formulations containing non-foaming commercial surfactants among with amylase, protease and lipase at neutral pH. The microscopic observation of changes in biofilm structure using SEM and confocal analyses indicated that enzymes were very effective in biofilm removal, especially on stainless steel surfaces. It was observed that the combination of enzymes was more efficient than formulations based in a single enzyme regardless of surfaces. The treatment with formulation combining amylase, protease and lipase, effectively decreased the total biofilm mass, the bacteria viability and the polysaccharide content in the biofilm formulations containing non-foaming commercial surfactants among with amylase, protease and lipase at neutral pH. The microscopic observation of changes in biofilm structure using SEM and confocal analyses indicated that enzymes were very effective in biofilm removal, especially on stainless steel surfaces. It was observed that the combination of enzymes was more efficient than formulations based in a single enzyme regardless of surfaces. The treatment with formulation combining amylase, protease and lipase, effectively decreased the total biofilm mass, the bacteria viability and the polysaccharide content in the biofilm. The last chapter of this PhD was focused on the biofilm EPS and the role that the forces between EPS and the surrounding interphases play in biofilm cleaning. Regardless of the bacteria species, EPS is generally comprised of soluble, gel-forming polysaccharides, proteins and eDNA, as well as insoluble components such as amyloids, cellulose, fimbriae and pili. Thus, a polysaccharide and specifically alginic acid was chosen as an EPS-related material to be studied. Moreover, surface modification can play a significant role in the prevention of biofilm attachment and growth and consequently the achievement of more effective cleaning. For this reason, the goal of that study was to measure directly the adhesion and cohesion forces, developed between an EPS-related material and surfaces while in air or under simulations of cleaning conditions like water and different pH solutions. Several polymers were studied as material for polycarbonate surface modification under two different pH conditions (3 and 11) and the adhesion and cohesion forces of alginic acid were measured under air, water, NaOH and HCl solutions. Overall, it was seen that during acidic conditions the cohesive strength of alginic acid increases, while in water and in NaOH solution it decreases. Nonetheless, the adhesive strength showed decline during all cleaning conditions which depended highly on the surface. Furthermore, the polymer surface modification of the polycarbonate surfaces had a significant impact on the adhesive strength of the alginic acid in all cases. Of great interest were two polymers, Lupasol and Poly-(2-ethyl-2-oxazoline), as they caused the most important reduction in the adhesive strength of the alginic acid. Since the results from all studies have been very interesting a future recommendation would be to expand the experiments through the combination of the relevant conditions. Thus, the technique used to measure adhesion and cohesion forces on alginic acid could be adjusted at the micron-mm scale to measure model P. fluorescens and P. putida biofilms under the optimal enzymatic conditions

Facultative bacterial symbionts from European Orius species: Evidence for an ancestral symbiotic association

Pest control in agriculture employs diverse strategies, among which the use of predatory insects has steadily increased. The use of several species within the genus Orius in pest control is widely spread, particularly in Mediterranean Europe. The use of predatory insects in pest control in agriculture has spread worldwide and increased significantly, especially in the use of various Orius species. Currently, most studies about Orius species have been focused on the diet manipulation or selective breeding methods to reduce the rearing costs and improve the efficiency, only a few studies were associated to their Wolbachia symbionts. The characterisation and contribution of microbial symbionts to Orius sp. fitness, behaviour, and potential impact on human health has been neglected. Therefore, there is a lack of knowledge regarding Orius’ symbionts such as their taxonomic characterisation, the functions of the symbionts and potential influences on human health. This project was focused on the first comparative genomics report of genome sequences level description of the predominant culturable facultative bacterial symbionts associated with the analyses of draft genomes of facultative symbionts using Next Generation Sequencing (NGS) technique related to five Orius species (Orius laevigatus, Orius niger, Orius pallidicornis, Orius majusculus and Orius albidipennis) and collected from various European countries (Greece, Italy, and Spain). Initially, coxl (COI) based taxonomic classification of the Orius species used was performed, followed by the isolation of culturable bacteria from live insects. The whole genome sequences of the bacterial isolates were generated and assembled into draft genomes using NGS. The isolates of two predominant bacteria belong to Serratia and Leucobacter genera, the third predominant bacteria are most likely to be a new genus within the Erwiniaceae. Orius sp. Serratia isolates genomes are more similar to Serratia sp. SCBI. Pan-genome analysis of Serratia sp. Orius isolates evidenced an open pan-genome, and 279 accessory genes were related to the insect symbiosis trait. Additionally, pan-genome analyses of the Serratia sp. isolates offered clues linking Type VI secretion system effector–immunity proteins from the Tai4 sub-family to the symbiotic lifestyle. These symbionts were found to colonise all the insect specimens tested, which evidenced an ancestral symbiotic association between these bacteria and the genus Orius. Additionally, plasmid sequence analyses suggest sequence exchanges between Serratia sp. Orius isolates and pathogenic Serratia species, which may have implications for food safety and human health