Decontamination of Spores on Model Stainless-Steel Surface by Using Foams Based on Alkyl Polyglucosides.

In the food industry, the surfaces of processing equipment are considered to be major factors in the risk of food contamination. The cleaning process of solid surfaces is essential, but it requires a significant amount of water and chemicals. Herein, we report the use of foam flows based on alkyl polyglucosides (APGs) to remove spores of Bacillus subtilis on stainless-steel surfaces as the model-contaminated surface. Sodium dodecyl sulfate (SDS) was also studied as an anionic surfactant. Foams were characterized during flows by measuring the foam stability and the bubble size. The efficiency of spores' removal was assessed by enumerations. We showed that foams based on APGs could remove efficiently the spores from the surfaces, but slightly less than foams based on SDS due to an effect of SDS itself on spores removal. The destabilization of the foams at the end of the process and the recovery of surfactant solutions were also evaluated by using filtration. Following a life cycle assessment (LCA) approach, we evaluated the impact of the foam flow on the global environmental footprint of the process. We showed significant environmental impact benefits with a reduction in water and energy consumption for foam cleaning. APGs are a good choice as surfactants as they decrease further the environmental impacts.

Treatment with disinfectants may induce an increase in viable but non culturable populations of Listeria monocytogenes in biofilms formed in smoked salmon processing environments.

The objectives of this study were 1) to evaluate the impact of two industrial disinfectants on the viability of Listeria monocytogenes populations in biofilm and 2) to investigate the viability state of L. monocytogenes cells present on contact surfaces in the smoked salmon processing environment. In the first step, we cultured mono species and mixed species biofilms containing L. monocytogenes on stainless steel or polyvinyl chloride (PVC) at 8 °C for 48h. The biofilms were then exposed to quaternary ammonium- and hydrogen peroxide-based disinfectants. Residual total populations of L. monocytogenes were measured by qPCR, and viable culturable (VC) cell populations were quantified using standard microbiological culture-based techniques and by a quantitative PCR (qPCR) assay coupled with a propidium monoazide treatment. Decreases in VC populations and the appearance of viable but non culturable (VBNC) populations were observed in response to treatment with the disinfectants. An 8 month sampling campaign in 4 smoked salmon processing plants was also carried out to detect L. monocytogenes in environmental samples. VBNC cells were detected mainly after the cleaning and disinfection operations. This study showed that industrial disinfectants did not inactivate all L. monocytogenes cells on inert surfaces. The presence of VBNC populations of L. monocytogenes in the smoked salmon processing environment is a public health concern.

Hydrophobicity of abiotic surfaces governs droplets deposition and evaporation patterns.

Surface contamination with droplets containing bacteria is of concern in the food industry and other environments where hygiene control is essential. Deposition patterns after the drying of contaminated droplets is affected by numerous parameters. The present study evaluated the rate of evaporation and the shape of deposition patterns after the drying of water droplets on a panel of materials with different surface properties (topography, hydrophobicity). The influence of the particle properties (in this study 1 µm-microspheres and two bacterial spores) was also investigated. Polystyrene microspheres were hydrophobic, while Bacillus spores were hydrophilic or hydrophobic, and surrounded by different surface features. In contrast to material topography, hydrophobicity was shown to deeply affect droplet evaporation, with the formation of small, thick deposits with microspheres or hydrophilic spores. Among the particle properties, the spore morphology (size and round/ovoid shape) did not clearly affect the deposition pattern. Conversely, hydrophobic spores aggregated to form clusters, which quickly settled on the materials and either failed to migrate, or only migrated to a slight extent on the surface, resulting in a steady distribution of spores or spore clusters over the whole contaminated area. Adherent bacteria or spores are known to be highly resistant to many stressful environmental conditions. In view of all the quite different patterns obtained following drying of spore-containing droplets, it seems likely that some of these would entail enhanced resistance to hygienic processes.

Glycosylation of BclA Glycoprotein from Bacillus cereus and Bacillus anthracis Exosporium Is Domain-specific.

The spores of the Bacillus cereus group (B. cereus, Bacillus anthracis, and Bacillus thuringiensis) are surrounded by a paracrystalline flexible yet resistant layer called exosporium that plays a major role in spore adhesion and virulence. The major constituent of its hairlike surface, the trimerized glycoprotein BclA, is attached to the basal layer through an N-terminal domain. It is then followed by a repetitive collagen-like neck bearing a globular head (C-terminal domain) that promotes glycoprotein trimerization. The collagen-like region of B. anthracis is known to be densely substituted by unusual O-glycans that may be used for developing species-specific diagnostics of B. anthracis spores and thus targeted therapeutic interventions. In the present study, we have explored the species and domain specificity of BclA glycosylation within the B. cereus group. First, we have established that the collagen-like regions of both B. anthracis and B. cereus are similarly substituted by short O-glycans that bear the species-specific deoxyhexose residues anthrose and the newly observed cereose, respectively. Second we have discovered that the C-terminal globular domains of BclA from both species are substituted by polysaccharide-like O-linked glycans whose structures are also species-specific. The presence of large carbohydrate polymers covering the surface of Bacillus spores may have a profound impact on the way that spores regulate their interactions with biotic and abiotic surfaces and represents potential new diagnostic targets.

Inverse Saffman-Taylor Experiments with Particles Lead to Capillarity Driven Fingering Instabilities.

Using air to displace a viscous fluid contained in a Hele-Shaw cell can create a fingering pattern at the interface between the fluids if the capillary number exceeds a critical value. This Saffman-Taylor instability is revisited for the inverse case of a viscous fluid displacing air when partially wettable hydrophilic particles are lying on the walls. Though the inverse case is otherwise stable, the presence of the particles results in a fingering instability at low capillary number. This capillary-driven instability is driven by the integration of particles into the interface which results from the minimization of the interfacial energy. Both axisymmetric and rectangular geometries are considered in order to quantify this phenomenon.

Presence and function of a thick mucous layer rich in polysaccharides around Bacillus subtilis spores.

This study was designed to establish the presence and function of the mucous layer surrounding spores of Bacillus subtilis. First, an external layer of variable thickness and regularity was often observed on B. subtilis spores. Further analyses were performed on B. subtilis 98/7 spores surrounded by a thick layer. The mechanical removal of the layer did not affect their resistance to heat or their ability to germinate but rendered the spore less hydrophilic, more adherent to stainless steel, and more resistant to cleaning. This layer was mainly composed of 6-deoxyhexoses, ie rhamnose, 3-O-methyl-rhamnose and quinovose, but also of glucosamine and muramic lactam, known also to be a part of the bacterial peptidoglycan. The specific hydrolysis of the peptidoglycan using lysozyme altered the structure of the required mucous layer and affected the physico-chemical properties of the spores. Such an outermost mucous layer has also been seen on spores of B. licheniformis and B. clausii isolated from food environments.

Sporulation conditions influence the surface and adhesion properties of Bacillus subtilis spores.

Spore-forming bacteria of the Bacillus subtilis group are responsible for recurrent contamination of processing lines in the food industry which can lead to food spoilage. The persistence of B. subtilis would be due to the high resistance of spores to extreme environmental condition and their propensity to contaminate surfaces. While it is well known that sporulation conditions modulate spore resistance properties, little is known about their effect on surface and adhesion properties. Here, we studied the impact of 13 sporulation conditions on the surface and adhesion properties of B. subtilis 168 spores. We showed that Ca2+ or Mg2+ depletion, lower oxygen availability, acidic pH as well as oxidative stresses during sporulation lead to the release of more hydrophobic and adherent spores. The consequences of these sporulation conditions on crust composition in carbohydrates and proteins were also evaluated. The crust glycans of spores produced in a sporulation medium depleted in Ca2+ or Mg2+ or oxygen-limited conditions were impaired and contained lower amounts of rhamnose and legionaminic acid. In addition, we showed that lower oxygen availability or addition of hydrogen peroxide during sporulation decreases the relative amount of two crust proteins (CgeA and CotY) and the changes observed in these conditions could be due to transcriptional repression of genes involved in crust synthesis in late stationary phase. The fact that sporulation conditions affect the ease with which spores can contaminate surfaces could explain the frequent and recurrent presence of B. subtilis spores in food processing lines.

Role played by exosporium glycoproteins in the surface properties of Bacillus cereus spores and in their adhesion to stainless steel.

Bacillus cereus spores are surrounded by a loose-fitting layer called the exosporium, whose distal part is mainly formed from glycoproteins. The role played by the exosporium glycoproteins of B. cereus ATCC 14579 (BclA and ExsH) was investigated by considering hydrophobicity and charge, as well as the properties of spore adhesion to stainless steel. The absence of BclA increased both the isoelectric point (IEP) and hydrophobicity of whole spores while simultaneously reducing the interaction between spores and stainless steel. However, neither the hydrophobicity nor the charge associated with BclA could explain the differences in the adhesion properties. Conversely, ExsH, another exosporium glycoprotein, did not play a significant role in spore surface properties. The monosaccharide analysis of B. cereus ATCC 14579 showed different glycosylation patterns on ExsH and BclA. Moreover, two specific glycosyl residues, namely, 2-O-methyl-rhamnose (2-Me-Rha) and 2,4-O-methyl-rhamnose (2,4-Me-Rha), were attached to BclA, in addition to the glycosyl residues already reported in B. anthracis.

Domains of BclA, the major surface glycoprotein of the B. cereus exosporium: glycosylation patterns and role in spore surface properties.

The role of the BclA domains of B. cereus ATCC 14579 was investigated in order to understand the phenomena involved in the interfacial processes occurring between spores and inert surfaces. This was done by (i) creating deletions in the collagen-like region (CLR) and the C-terminal domain (CTD) of BclA, (ii) building BclA proteins with various lengths in the CLR and (iii) modifying the hydrophobic upper surface in the CTD. First, it was demonstrated that the CLR was substituted by three residues already reported in the CLR of B. anthracis, viz. rhamnose, 3-O-methyl-rhamnose, and GalNH(2) residues, while the CTD was also substituted by two additional glycosyl residues, viz. 2-O-methyl-rhamnose and 2,4-O-methyl-rhamnose. Regarding the properties of the spores, both CLR and CTD contributed to the adhesion of the spores, which was estimated by measuring the resistance to detachment of spores adhered to stainless steel plates). CLR and CTD also impacted the hydrophobic character and isoelectric point of the spores. It was then shown that the resistance to detachment of the spores was not affected by the physicochemical properties, but by the CLR length and the presence of hydrophobic amino acids on the CTD.

Influence of the design of fresh-cut food washing tanks on the growth kinetics of Pseudomonas fluorescens biofilms.

Mitigation of cross-contamination of fresh-cut food products at the washing step was studied by investigating how the vat design would affect the biofilm contamination surfaces. Hygienic design features such as no horizontal surfaces and only open angles exceeding 100° were proposed. The flow organization (velocity streamlines, wall shear stresses, and dynamics of the flow) was identified by means of computational fluid dynamics (CFD) calculation. Pseudomonas fluorescens PF1 biofilm growth kinetics were then mapped. The change in some geometrical features induced a better flow organization reducing "dead zones". This significantly changed the biofilm growth kinetics, delaying the detection of biofilms from 20 hr to 24 hr. Critical areas such as welds, corners, and interfaces appeared far less prone to strong bacterial development. This would mean milder or less chemicals required at the washing step and faster and easier cleaning.

Selective adhesion of Bacillus cereus spores on heterogeneously wetted silicon nanowires.

The article reports on the selective adhesion of Bacillus cereus spores on patterned and heterogeneously wetted superhydrophobic silicon nanowires surfaces. Superhydrophilic patterns on superhydrophobic silicon nanowire (SiNW) surfaces were prepared by a standard optical lithography technique. Exposure of the patterned surface to a suspension of B. cereus spores in water led to their specific adsorption in superhydrophobic areas. Comparable results were obtained on a patterned hydrophobic/hydrophilic flat silicon (Si) surface even though a higher concentration of spores was observed on the hydrophobic areas, as compared to the superhydrophobic regions of the SiNW substrate. The surfaces were characterized using scanning electron microscopy (SEM), fluorescence spectroscopy, and contact angle measurements.

Using enzymes to remove biofilms of bacterial isolates sampled in the food-industry.

The aim of this study was to analyze the cleaning efficiency of polysaccharidases and proteolytic enzymes against biofilms of bacterial species found in food industry processing lines and to study enzyme effects on the composition of extracellular polymeric substances (EPS) and biofilm removal in a Clean-in-Place (CIP) procedure. The screening of 7 proteases and polysaccharidases for removal of biofilms of 16 bacterial species was first evaluated using a microtiter plate assay. The alkaline pH buffer removed more biofilm biomass as well as affecting a larger range of bacterial species. The two serine proteases and alpha-amylase were the most efficient enzymes. Proteolytic enzymes promoted biofilm removal of a larger range of bacterial species than polysaccharidases. Using three isolates derived from two bacterial species widely found in food processing lines (Pseudomonas fluorescens and the Bacillus cereus group), biofilms were developed on stainless steel slides and enzymatic solutions were used to remove the biofilms using CIP procedure. Serine proteases were more efficient in removing cells of Bacillus biofilms than polysaccharidases. However, polysaccharidases were more efficient in removing P. fluorescens biofilms than serine proteases. Solubilization of enzymes with a buffer containing surfactants, and dispersing and chelating agents enhanced the efficiency of polysaccharidases and proteases respectively in removing biofilms of Bacillus and P. fluorescens. A combination of enzymes targeting several components of EPS, surfactants, dispersing and chelating agents would be an efficient alternative to chemical cleaning agents.

Comparison of the performance of the biofilm sampling methods (swab, sponge, contact agar) in the recovery of Listeria monocytogenes populations considering the seafood environment conditions.

AIMS: The aim of this study was to evaluate the performance of sampling methods [contact plates, sponges, and swabs] in the recovery of biofilm Listeria monocytogenes populations considering the seafood environment conditions (nature of conditioning, of materials and bacterial species). METHODS AND RESULTS: Different materials (stainless steel, polyvinyl chloride, polyurethane) were conditioned with two fish filtrates, the ready-to-eat the most consumed in Europe (smoked salmon, cod). After, we added the suspension of Listeria monocytogenes, alone or with Pseudomonas fluorescens or Carnobacterium strains, and incubated for 48 h at 8 °C. Then, the 48 h-biofilms were sampled with different methods (contact plates, sponges, and swabs). The cultivable bacterial populations were enumerated on agar, while the L. monocytogenes total and viable populations were quantified by qPCR and propidium monoazide-qPCR (PMA-qPCR), respectively. The amount of L. monocytogenes in biofilms was affected only by the nature of the conditioning with lowest adherent bacteria with cod versus with smoked salmon conditioning. Considering the amount of total population, the swab displayed the lowest values versus the sponges and the contact plates. An explanation was that the observations of the swab by microscopy showed the bacteria trapped within it. The recovery of cultivable bacterial populations was not significantly different with the three sampling methods. On the contrary, we showed that the VBNC populations were only detached by two of three methods (contact plates, sponges) while for the dead populations, those were contact plates and swabs. CONCLUSIONS: The nature of the conditioning influenced the amount of the bacteria in biofilms. And the performance of the recovery of the bacterial populations (dead, VBNC, cultivable) was dependent on the methods used. SIGNIFICANCE AND IMPACT OF THE STUDY: This study showed that the seafood environmental conditions influenced the biofilm formation and the assessment of the efficiency of cleaning and disinfectant operations could be significantly affected by the used sampling methods.

The sps Genes Encode an Original Legionaminic Acid Pathway Required for Crust Assembly in Bacillus subtilis.

The crust is the outermost spore layer of most Bacillus strains devoid of an exosporium. This outermost layer, composed of both proteins and carbohydrates, plays a major role in the adhesion and spreading of spores into the environment. Recent studies have identified several crust proteins and have provided insights about their organization at the spore surface. However, although carbohydrates are known to participate in adhesion, little is known about their composition, structure, and localization. In this study, we showed that the spore surface of Bacillus subtilis is covered with legionaminic acid (Leg), a nine-carbon backbone nonulosonic acid known to decorate the flagellin of the human pathogens Helicobacter pylori and Campylobacter jejuni We demonstrated that the spsC, spsD, spsE, spsG, and spsM genes of Bacillus subtilis are required for Leg biosynthesis during sporulation, while the spsF gene is required for Leg transfer from the mother cell to the surface of the forespore. We also characterized the activity of SpsM and highlighted an original Leg biosynthesis pathway in B. subtilis Finally, we demonstrated that Leg is required for the assembly of the crust around the spores, and we showed that in the absence of Leg, spores were more adherent to stainless steel probably because of their reduced hydrophilicity and charge.IMPORTANCEBacillus species are a major economic and food safety concern of the food industry because of their food spoilage-causing capability and persistence. Their persistence is mainly due to their ability to form highly resistant spores adhering to the surfaces of industrial equipment. Spores of the Bacillus subtilis group are surrounded by the crust, a superficial layer which plays a key role in their adhesion properties. However, knowledge of the composition and structure of this layer remains incomplete. Here, for the first time, we identified a nonulosonic acid (Leg) at the surfaces of bacterial spores (B. subtilis). We uncovered a novel Leg biosynthesis pathway, and we demonstrated that Leg is required for proper crust assembly. This work contributes to the description of the structure and composition of Bacillus spores which has been under way for decades, and it provides keys to understanding the importance of carbohydrates in Bacillus adhesion and persistence in the food industry.

Biofilm formation and cell surface properties among pathogenic and nonpathogenic strains of the Bacillus cereus group.

Biofilm formation by 102 Bacillus cereus and B. thuringiensis strains was determined. Strains isolated from soil or involved in digestive tract infections were efficient biofilm formers, whereas strains isolated from other diseases were poor biofilm formers. Cell surface hydrophobicity, the presence of an S layer, and adhesion to epithelial cells were also examined.

Effect of different Bacillus subtilis lipopeptides on surface hydrophobicity and adhesion of Bacillus cereus 98/4 spores to stainless steel and Teflon.

Various lipopeptides produced by Bacillus subtilis were examined for their ability to modify the surface hydrophobicity of two substrata, stainless steel (SS) and Teflon. These modifications were evaluated by water contact angle measurements. The effects depended on the lipopeptide, its concentration, and the tested substratum. Treatment of SS with different concentrations of surfactin S1 showed an increase of the hydrophobicity between 1 and 100 mg l(-1). On the same substratum, fengycin increased hydrophobicity up to its critical micelle concentration (6.25 mg l(-1)). With higher concentrations of fengycin, hydrophobicity decreased. Surfactin, mycosubtilin, and iturin A decreased hydrophobicity on Teflon. The different effects of these three families of lipopeptides were related to their structural differences. A good correlation was shown between hydrophobicity modifications of surfaces and the attachment of B. cereus 98/4 spores. Enhancement in the hydrophobicity of the surfaces increased the number of adhering spores.

Evaluation of the hydrophobic properties of latex microspheres and Bacillus spores. Influence of the particle size on the data obtained by the MATH method (microbial adhesion to hydrocarbons).

The current experimental study investigates the influence of latex microsphere particles' size on the assessment of their hydrophilic/hydrophobic character, using the method known as "Microbial Adhesion to Hydrocarbons" (MATH). Since bacteria surfaces often change according to the environment in which they find themselves, most of the experiments here were carried out using the calibrated latex microspheres Polybeads® and Yellow-green Fluoresbrite® (Polyscience) microspheres with diameters between 0.2 µm and 4.5 µm. All the beads had a density of ˜1.05 g/cm3. The first set of experiments was performed to adapt the procedure for measurements of water contact angles to microsphere lawns. It was found that all the microspheres tested were hydrophobic, when using a water contact angle of around 110-118°. However, wide differences were observed using the MATH method. The smaller microspheres (0.2 µm, 0.5 µm +/- 0.75 µm) exhibited a poor affinity to hexadecane, even after long contact times, suggesting a hydrophilic character. In contrast, larger microspheres quickly adhered to hexadecane, which is consistent with the values obtained for the water contact angles observed. These results suggest that, at least where hydrophobic particles are concerned, the MATH method is not suitable for the assessment of the hydrophobic character of particles with diameters of less than 1.0 µm. We lastly investigated whether the data obtained for Bacillus spores could also be affected by spore size. The hydrophobicity of spores of eight Bacillus strains was analysed by both MATH and contact angle. Some discrepancies were observed between both methods but could not be related their size (length or width).

Comparative evaluation of DNA extraction methods for amplification by qPCR of superficial vs intracellular DNA from Bacillus spores.

This study was designed to assess the efficiency of eight extraction methods regarding their ability to release superficial (exogenous) and intracellular (endogenous) DNA from B. cereus spores for subsequent analysis by quantitative PCR (qPCR). B. cereus spore suspensions were subjected to both commercial DNA extraction kits and mechanical DNA extraction methods. The spores were observed by transmission electron microscopy to evaluate any damage caused during extraction. The efficiency of both extraction and purification were assessed using a qPCR assay targeting the bclA gene. Most of the extraction methods assessed, except the passage through the French press or the use of the QIAamp DNA Blood Mini kit without 95°C treatment, allowed the amplification of significant amounts of DNA. By using propidium monoazide, which is a photoreactive DNA-binding dye, the presence of non-negligible amounts of amplifiable DNA at the spore surface was highlighted. A further set of extraction assays was then performed on spores previously treated with PMA. The results of this study show that both superficial and intracellular spore DNA can be released by extraction methods to a greater or lesser extent and then further amplified by qPCR. The Precellys extraction allowed the detection of both intracellular and superficial DNA, the DNeasy Blood & Tissue kit the specific detection of intracellular DNA, while the Instagene kit detected only superficial DNA. Of the methods tested in this study, the Precellys extraction was the most efficient in terms of further DNA detection. SIGNIFICANCE AND IMPACT OF THE STUDY: In order to verify the presence or absence of B. cereus spores in food or on surfaces in the food environment, the use of an efficient extraction method is required, followed by a qPCR analysis on the DNA released. Conversely, in order to quantify the population of Bacillus spores, any superficial DNA must be blocked, e.g. with PMA, prior to intracellular DNA extraction and further amplification.

The absence of N-acetylglucosamine in wall teichoic acids of Listeria monocytogenes modifies biofilm architecture and tolerance to rinsing and cleaning procedures.

The wall teichoic acid (WTA) is the major carbohydrate found within the extracellular matrix of the Listeria monocytogenes biofilm. We first addressed the frequency of spontaneous mutations in two genes (lmo2549 and lmo2550) responsible for the GlcNAcylation in 93 serotype 1/2a strains that were mainly isolated from seafood industries. We studied the impact of mutations in lmo2549 or lmo2550 genes on biofilm formation by using one mutant carrying a natural mutation inactivating the lmo2550 gene (DSS 1130 BFA2 strain) and two EGD-e mutants that lack respective genes by in-frame deletion of lmo2549 or lmo2550 using splicing-by-overlap-extension PCR, followed by allelic exchange mutagenesis. The lmo2550 gene mutation, occurring in around 50% isolates, caused a decrease in bacterial adhesion to stainless steel compared to wild-type EGD-e strain during the adhesion step. On the other hand, bacterial population weren't significantly different after 24h-biofilm formation. The biofilm architecture was different between the wild-type strain and the two mutants inactivated for lmo2549 or lmo2550 genes respectively with the presence of bacterial micro-colonies for mutants which were not observed in the wild-type EGD-e strain biofilm. These differences might account for the stronger hydrophilic surface exhibited by the mutant cells. Upon a water flow or to a cleaning procedure at a shear stress of 0.16 Pa, the mutant biofilms showed the higher detachment rate compared to wild-type strain. Meanwhile, an increase in the amount of residual viable but non-culturable population on stainless steel was recorded in two mutants. Our data suggests that the GlcNAc residue of WTA played a role in adhesion and biofilm formation of Listeria monocyctogenes.

Effect of incubation duration, growth temperature, and abiotic surface type on cell surface properties, adhesion and pathogenicity of biofilm-detached Staphylococcus aureus cells.

The goal of this study was to investigate the effect of growth conditions such as the temperature (20, 30 and 37 °C), incubation duration (24 and 48 h) and surface type (stainless steel and polycarbonate) on the cell surface physicochemical properties and adhesion to abiotic surfaces of biofilm-detached and planktonic Staphylococcus aureus cells. This study tested also the hypothesis that S. aureus planktonic cells exhibit distinct pathogenic properties compared with their sessile counterparts. The results showed that the changes of the growth conditions promoted changes in the zeta potential, hydrophobicity, electron donor/acceptor character of the studied cell populations. Biofilm-detached cells showed a greater adhesion to stainless steel and polycarbonate compared with planktonic cells. Compared with planktonic cells, sessile ones showed higher cytotoxic effect against HeLa cells, DNase activity, and siderophore levels. The higher cytotoxic effect and production of DNase and siderophore increased with the increase of temperature and duration of incubations. Based on the obtained data, the S. aureus biofilm-detached cells were found to be distinct in many physiological properties compared with their planktonic counterparts.