Exploring the hemicellulolytic properties and safety of Bacillus paralicheniformis as stepping stone in the use of new fibrolytic beneficial microbes.

Bacillus strains from the Moroccan Coordinated Collections of Microorganisms (CCMM) were characterised and tested for fibrolytic function and safety properties that would be beneficial for maintaining intestinal homeostasis, and recommend beneficial microbes in the field of health promotion research. Forty strains were investigated for their fibrolytic activities towards complex purified polysaccharides and natural fibres representative of dietary fibres (DFs) entering the colon for digestion. We demonstrated hemicellulolytic activities for nine strains of Bacillus aerius, re-identified as Bacillus paralicheniformis and Bacillus licheniformis, using xylan, xyloglucan or lichenan as purified polysaccharides, and orange, apple and carrot natural fibres, with strain- and substrate-dependent production of glycoside hydrolases (GHs). Our combined methods, based on enzymatic assays, secretome, and genome analyses, highlighted the hemicellulolytic activities of B. paralicheniformis and the secretion of specific glycoside hydrolases, in particular xylanases, compared to B. licheniformis. Genomic features of these strains revealed a complete set of GH genes dedicated to the degradation of various polysaccharides from DFs, including cellulose, hemicellulose and pectin, which may confer on the strains the ability to digest a variety of DFs. Preliminary experiments on the safety and immunomodulatory properties of B. paralicheniformis fibrolytic strains were evaluated in light of applications as beneficial microbes' candidates for health improvement. B. paralicheniformis CCMM B969 was therefore proposed as a new fibrolytic beneficial microbe candidate.

Sperm-fluid-cell interplays in the bovine oviduct: glycosaminoglycans modulate sperm binding to the isthmic reservoir.

When entering the oviduct for fertilisation, spermatozoa come into contact with the oviduct fluid (OF) and can bind to luminal epithelial cells in the isthmus to form a sperm reservoir. The objective of this study was to examine how the OF modulates sperm adhesion to the oviduct reservoir using an in vitro model of oviduct epithelial spheroids (OES). Bovine oviducts from a local slaughterhouse were used to collect OF and isthmic fragments for the in vitro incubation of OES. Compared to a non-capacitating control medium, the pre-ovulatory OF significantly decreased by 80-90% the density of spermatozoa bound to OES without affecting sperm motility, membrane integrity, or sperm-cilia interactions. This effect on sperm binding was reproduced with (1) OF from different cycle stages and anatomical regions of the oviduct; (2) OF fractions of more than 3 kDa; (3) modified OF in which proteins were denatured or digested and (4) heparan sulphate but not hyaluronic acid, two glycosaminoglycans present in the OF. In conclusion, the OF significantly decreased the number of spermatozoa that bind to oviduct epithelial cells without affecting sperm motility and this effect was due to macromolecules, including heparan sulphate.

Neonatal necrotizing enterocolitis: Clostridium butyricum and Clostridium neonatale fermentation metabolism and enteropathogenicity.

Bacterial colonization in the gut plays a pivotal role in neonatal necrotizing enterocolitis (NEC) development, but the relationship between bacteria and NEC remains unclear. In this study, we aimed to elucidate whether bacterial butyrate end-fermentation metabolites participate in the development of NEC lesions and confirm the enteropathogenicity of Clostridium butyricum and Clostridium neonatale in NEC. First, we produced C.butyricum and C.neonatale strains impaired in butyrate production by genetically inactivating the hbd gene encoding ß-hydroxybutyryl-CoA dehydrogenase that produces end-fermentation metabolites. Second, we evaluated the enteropathogenicty of the hbd-knockout strains in a gnotobiotic quail model of NEC. The analyses showed that animals harboring these strains had significantly fewer and less intense intestinal lesions than those harboring the respective wild-type strains. In the absence of specific biological markers of NEC, the data provide original and new mechanistic insights into the disease pathophysiology, a necessary step for developing potential novel therapies.

Three Distinct Proteases Are Responsible for Overall Cell Surface Proteolysis in Streptococcus thermophilus.

The lactic acid bacterium Streptococcus thermophilus was believed to display only two distinct proteases at the cell surface, namely, the cell envelope protease PrtS and the housekeeping protease HtrA. Using peptidomics, we demonstrate here the existence of an additional active cell surface protease, which shares significant homology with the SepM protease of Streptococcus mutans. Although all three proteases-PrtS, HtrA, and SepM-are involved in the turnover of surface proteins, they demonstrate distinct substrate specificities. In particular, SepM cleaves proteins involved in cell wall metabolism and cell elongation, and its inactivation has consequences for cell morphology. When all three proteases are inactivated, the residual cell-surface proteolysis of S. thermophilus is approximately 5% of that of the wild-type strain. IMPORTANCE Streptococcus thermophilus is a lactic acid bacterium used widely as a starter in the dairy industry. Due to its "generally recognized as safe" status and its weak cell surface proteolytic activity, it is also considered a potential bacterial vector for heterologous protein production. Our identification of a new cell surface protease made it possible to construct a mutant strain with a 95% reduction in surface proteolysis, which could be useful in numerous biotechnological applications.

Do Primocolonizing Bacteria Enable Bacteroides thetaiotaomicron Intestinal Colonization Independently of the Capacity To Consume Oxygen?

Aerobic bacteria are frequent primocolonizers of the human naive intestine. Their generally accepted role is to eliminate oxygen, which would allow colonization by anaerobes that subsequently dominate bacterial gut populations. In this hypothesis-based study, we revisited this dogma experimentally in a germfree mouse model as a mimic of the germfree newborn. We varied conditions leading to the establishment of the dominant intestinal anaerobe Bacteroides thetaiotaomicron Two variables were introduced: Bacteroides inoculum size and preestablishment by bacteria capable or not of consuming oxygen. High Bacteroides inoculum size enabled its primocolonization. At low inocula, we show that bacterial preestablishment was decisive for subsequent Bacteroides colonization. However, even non-oxygen-respiring bacteria, a hemAEscherichia coli mutant and the intestinal obligate anaerobe Clostridium scindens, facilitated Bacteroides establishment. These findings, which are supported by recent reports, revise the long-held assumption that oxygen scavenging is the main role for aerobic primocolonizing bacteria. Instead, we suggest that better survival of aerobic bacteria ex vivo during vectorization between hosts could be a reason for their frequent primocolonization.

Highly sensitive detection of Campylobacter spp. In chicken meat using a silica nanoparticle enhanced dot blot DNA biosensor.

Paper-based DNA biosensors are powerful tools in point-of-care diagnostics since they are affordable, portable, user-friendly, rapid and robust. However, their sensitivity is not always as high as required to enable DNA quantification. To improve the response of standard dot blots, we have applied a new enhancement strategy that increases the sensitivity of assays based on the use of biotinylated silica-nanoparticles (biotin-Si-NPs). After immobilization of a genomic Campylobacter DNA onto a paper membrane, and addition of a biotinylated-DNA detection probe, hybridization was evidenced using streptavidin-conjugated to horseradish peroxidase (HRP) in the presence of luminol and H2O2. Replacement of the single biotin by the biotin-Si-NPs boosted on average a 30 fold chemiluminescent read-out of the biosensor. Characterization of biotin-Si-NPs onto a paper with immobilized DNA was done using a scanning electron microscope. A limit of detection of 3 pg/µL of DNA, similar to the available qPCR kits, is achieved, but it is cheaper, easier and avoids inhibition of DNA polymerase by molecules from the food matrices. We demonstrated that the new dot blot coupled to biotin-Si-NPs successfully detected Campylobacter from naturally contaminated chicken meat, without needing a PCR step. Hence, such an enhanced dot blot paves the path to the development of a portable and multiplex paper based platform for point-of-care screening of chicken carcasses for Campylobacter.

Freeze-dried fecal samples are biologically active after long-lasting storage and suited to fecal microbiota transplantation in a preclinical murine model of Clostridioides difficile infection.

Fecal microbiota transplantation is now recommended for treating recurrent forms of Clostridioides difficile infection. Recent studies have reported protocols using capsules of either frozen or freeze-dried stool allowing oral administration in in- and out-patient settings. However, a central question remains the viability, engraftment, and efficacy of the microbiome over time during storage life. This study shows that both the freeze-drying and freezing procedures for fecal samples allowed preserving viability, short-chain fatty acids concentration, and anti-Clostridioides difficile properties of microbiota without significant alteration after storage for 12 months. Fecal transplantation with freeze-dried microbiota allowed engraftment of microbiota leading to clearance of Clostridioides difficile infection in a preclinical murine model with a survival rate of 70% versus 53-60% in mice treated with frozen inocula, and 20% in the untreated group. Moreover, the freeze-dried powder can be used to fill oral hard capsules using a very low amount (0.5%) of glidant excipient, allowing oral formulation. Altogether, this study showed that freeze-dried inocula can be used for the treatment of Clostridioides difficile infection with long-lasting stability of the fecal microbiota. This formulation facilitates biobanking and allows the use of hard capsules, an essential step to simplify patient access to treatment.

Identification of New Factors Modulating Adhesion Abilities of the Pioneer Commensal Bacterium Streptococcus salivarius.

Biofilm formation is crucial for bacterial community development and host colonization by Streptococcus salivarius, a pioneer colonizer and commensal bacterium of the human gastrointestinal tract. This ability to form biofilms depends on bacterial adhesion to host surfaces, and on the intercellular aggregation contributing to biofilm cohesiveness. Many S. salivarius isolates auto-aggregate, an adhesion process mediated by cell surface proteins. To gain an insight into the genetic factors of S. salivarius that dictate host adhesion and biofilm formation, we developed a screening method, based on the differential sedimentation of bacteria in semi-liquid conditions according to their auto-aggregation capacity, which allowed us to identify twelve mutations affecting this auto-aggregation phenotype. Mutations targeted genes encoding (i) extracellular components, including the CshA surface-exposed protein, the extracellular BglB glucan-binding protein, the GtfE, GtfG and GtfH glycosyltransferases and enzymes responsible for synthesis of cell wall polysaccharides (CwpB, CwpK), (ii) proteins responsible for the extracellular localization of proteins, such as structural components of the accessory SecA2Y2 system (Asp1, Asp2, SecA2) and the SrtA sortase, and (iii) the LiaR transcriptional response regulator. These mutations also influenced biofilm architecture, revealing that similar cell-to-cell interactions govern assembly of auto-aggregates and biofilm formation. We found that BglB, CshA, GtfH and LiaR were specifically associated with bacterial auto-aggregation, whereas Asp1, Asp2, CwpB, CwpK, GtfE, GtfG, SecA2 and SrtA also contributed to adhesion to host cells and host-derived components, or to interactions with the human pathogen Fusobacterium nucleatum. Our study demonstrates that our screening method could also be used to identify genes implicated in the bacterial interactions of pathogens or probiotics, for which aggregation is either a virulence trait or an advantageous feature, respectively.

Impact of Cell Surface Molecules on Conjugative Transfer of the Integrative and Conjugative Element ICESt3 of Streptococcus thermophilus.

Integrative conjugative elements (ICEs) are chromosomal elements that are widely distributed in bacterial genomes, hence contributing to genome plasticity, adaptation, and evolution of bacteria. Conjugation requires a contact between both the donor and the recipient cells and thus likely depends on the composition of the cell surface envelope. In this work, we investigated the impact of different cell surface molecules, including cell surface proteins, wall teichoic acids, lipoteichoic acids, and exopolysaccharides, on the transfer and acquisition of ICESt3 from Streptococcus thermophilus The transfer of ICESt3 from wild-type (WT) donor cells to mutated recipient cells increased 5- to 400-fold when recipient cells were affected in lipoproteins, teichoic acids, or exopolysaccharides compared to when the recipient cells were WT. These mutants displayed an increased biofilm-forming ability compared to the WT, suggesting better cell interactions that could contribute to the increase of ICESt3 acquisition. Microscopic observations of S. thermophilus cell surface mutants showed different phenotypes (aggregation in particular) that can also have an impact on conjugation. In contrast, the same mutations did not have the same impact when the donor cells, instead of recipient cells, were mutated. In that case, the transfer frequency of ICESt3 decreased compared to that with the WT. The same observation was made when both donor and recipient cells were mutated. The dominant effect of mutations in donor cells suggests that modifications of the cell envelope could impair the establishment or activity of the conjugation machinery required for DNA transport.IMPORTANCE ICEs contribute to horizontal gene transfer of adaptive traits (for example, virulence, antibiotic resistance, or biofilm formation) and play a considerable role in bacterial genome evolution, thus underlining the need of a better understanding of their conjugative mechanism of transfer. While most studies focus on the different functions encoded by ICEs, little is known about the effect of host factors on their conjugative transfer. Using ICESt3 of S. thermophilus as a model, we demonstrated the impact of lipoproteins, teichoic acids, and exopolysaccharides on ICE transfer and acquisition. This opens up new avenues to control gene transfer mediated by ICEs.

Three glycosylated serine-rich repeat proteins play a pivotal role in adhesion and colonization of the pioneer commensal bacterium, Streptococcus salivarius.

Bacterial adhesion is a critical step for colonization of the host. The pioneer colonizer and commensal bacterium of the human gastrointestinal tract, Streptococcus salivarius, has strong adhesive properties but the molecular determinants of this adhesion remain uncharacterized. Serine-rich repeat (SRR) glycoproteins are a family of adhesins that fulfil an important role in adhesion. In general, Gram-positive bacterial genomes have a unique SRR glycoprotein-encoding gene. We demonstrate that S. salivarius expresses three large and glycosylated surface-exposed proteins - SrpA, SrpB and SrpC - that show characteristics of SRR glycoproteins and are secreted through the accessory SecA2/Y2 system. Two glycosyltransferases - GtfE/F - encoded outside of the secA2/Y2 locus, unusually, perform the first step of the sequential glycosylation process, which is crucial for SRR activity. We show that SrpB and SrpC play complementary adhesive roles involved in several steps of the colonization process: auto-aggregation, biofilm formation and adhesion to a variety of host epithelial cells and components. We also show that at least one of the S. salivarius SRR glycoproteins is important for colonization in mice. SrpA, SrpB and SrpC are the main factors underlying the multifaceted adhesion of S. salivarius and, therefore, play a major role in host colonization.

Forced- and Self-Rotation of Magnetic Nanorods Assembly at the Cell Membrane: A Biomagnetic Torsion Pendulum.

In order to provide insight into how anisotropic nano-objects interact with living cell membranes, and possibly self-assemble, magnetic nanorods with an average size of around 100 nm × 1 µm are designed by assembling iron oxide nanocubes within a polymeric matrix under a magnetic field. The nano-bio interface at the cell membrane under the influence of a rotating magnetic field is then explored. A complex structuration of the nanorods intertwined with the membranes is observed. Unexpectedly, after a magnetic rotating stimulation, the resulting macrorods are able to rotate freely for multiple rotations, revealing the creation of a biomagnetic torsion pendulum.

Impact of temperature on Marinobacter hydrocarbonoclasticus SP17 morphology and biofilm structure during growth on alkanes.

Alkanes are widespread pollutants found in soil, freshwater and marine environments. Marinobacter hydrocarbonoclasticus (Mh) strain SP17 is a marine bacterium able to use many hydrophobic organic compounds, including alkanes, through the production of biofilms that allow their poor solubility to be overcome. This study pointed out that temperature is an environmental factor that strongly affects the biofilm formation and morphology of Mh on the model alkanes, hexadecane and paraffin. We showed that Mh biofilm formation and accumulation of intracytoplasmic inclusions are higher on solid alkanes (hexadecane at 10 °C and paraffin at 10 °C and 30 °C) than on liquid alkane (hexadecane at 30 °C) or soluble substrate (lactate at both temperatures). We also found that Mh produces more extracellular polymeric substances at 30 °C than at 10 °C on alkanes and none on lactate. We observed that bacterial length is significantly higher at 10 °C than at 30 °C on lactate and hexadecane. On paraffin, at 30 °C, the cell morphology is markedly altered by large rounded or irregularly shaped cytoplasmic inclusions. Altogether, the results showed that Mh is able to adapt and use alkanes as a carbon source, even at low temperature.

Overexpression of Enterococcus faecalis elr operon protects from phagocytosis.

BACKGROUND: Mechanisms underlying the transition from commensalism to virulence in Enterococcus faecalis are not fully understood. We previously identified the enterococcal leucine-rich protein A (ElrA) as a virulence factor of E. faecalis. The elrA gene is part of an operon that comprises four other ORFs encoding putative surface proteins of unknown function. RESULTS: In this work, we compared the susceptibility to phagocytosis of three E. faecalis strains, including a wild-type (WT), a ΔelrA strain, and a strain overexpressing the whole elr operon in order to understand the role of this operon in E. faecalis virulence. While both WT and ΔelrA strains were efficiently phagocytized by RAW 264.7 mouse macrophages, the elr operon-overexpressing strain showed a decreased capability to be internalized by the phagocytic cells. Consistently, the strain overexpressing elr operon was less adherent to macrophages than the WT strain, suggesting that overexpression of the elr operon could confer E. faecalis with additional anti-adhesion properties. In addition, increased virulence of the elr operon-overexpressing strain was shown in a mouse peritonitis model. CONCLUSIONS: Altogether, our results indicate that overexpression of the elr operon facilitates the E. faecalis escape from host immune defenses.

Plant-derived compounds as natural antimicrobials to control paper mill biofilms.

Biofilms can cause severe problems in industrial paper mills, particularly of economic and technological types (clogging of filters, sheet breaks or holes in the paper, machine breakdowns, etc.). We present here some promising results on the use of essential oil compounds to control these biofilms. Biofilms were grown on stainless-steel coupons with a microbial white water consortium sampled from an industrial paper mill. Five essential oil compounds were screened initially in the laboratory in terms of their antimicrobial activity against planktonic cells and biofilms. The three most active compounds were selected and then tested in different combinations. The combination finally selected was tested at the pilot scale to confirm its efficiency under realistic conditions. All the compounds tested were as active against biofilms as they were against planktonic cells. The most active compounds were thymol, carvacrol, and eugenol, and the most efficient combination was thymol-carvacrol. At a pilot scale, with six injections a day, 10 mM carvacrol alone prevented biocontamination for at least 10 days, and a 1 mM thymol-carvacrol combination enabled a 67 % reduction in biofilm dry matter after 11 days. The use of green antimicrobials could constitute a very promising alternative or supplement to the treatments currently applied to limit biofilm formation in the environment of paper mill machines.

Enterococcus faecalis prophage dynamics and contributions to pathogenic traits.

Polylysogeny is frequently considered to be the result of an adaptive evolutionary process in which prophages confer fitness and/or virulence factors, thus making them important for evolution of both bacterial populations and infectious diseases. The Enterococcus faecalis V583 isolate belongs to the high-risk clonal complex 2 that is particularly well adapted to the hospital environment. Its genome carries 7 prophage-like elements (V583-pp1 to -pp7), one of which is ubiquitous in the species. In this study, we investigated the activity of the V583 prophages and their contribution to E. faecalis biological traits. We systematically analyzed the ability of each prophage to excise from the bacterial chromosome, to replicate and to package its DNA. We also created a set of E. faecalis isogenic strains that lack from one to all six non-ubiquitous prophages by mimicking natural excision. Our work reveals that prophages of E. faecalis V583 excise from the bacterial chromosome in the presence of a fluoroquinolone, and are able to produce active phage progeny. Intricate interactions between V583 prophages were also unveiled: i) pp7, coined EfCIV583 for E. faecalis chromosomal island of V583, hijacks capsids from helper phage 1, leading to the formation of distinct virions, and ii) pp1, pp3 and pp5 inhibit excision of pp4 and pp6. The hijacking exerted by EfCIV583 on helper phage 1 capsids is the first example of molecular piracy in Gram positive bacteria other than staphylococci. Furthermore, prophages encoding platelet-binding-like proteins were found to be involved in adhesion to human platelets, considered as a first step towards the development of infective endocarditis. Our findings reveal not only a role of E. faecalis V583 prophages in pathogenicity, but also provide an explanation for the correlation between antibiotic usage and E. faecalis success as a nosocomial pathogen, as fluoriquinolone may provoke release of prophages and promote gene dissemination among isolates.

Pilus biogenesis in Lactococcus lactis: molecular characterization and role in aggregation and biofilm formation.

The genome of Lactococcus lactis strain IL1403 harbors a putative pilus biogenesis cluster consisting of a sortase C gene flanked by 3 LPxTG protein encoding genes (yhgD, yhgE, and yhhB), called here pil. However, pili were not detected under standard growth conditions. Over-expression of the pil operon resulted in production and display of pili on the surface of lactococci. Functional analysis of the pilus biogenesis machinery indicated that the pilus shaft is formed by oligomers of the YhgE pilin, that the pilus cap is formed by the YhgD pilin and that YhhB is the basal pilin allowing the tethering of the pilus fibers to the cell wall. Oligomerization of pilin subunits was catalyzed by sortase C while anchoring of pili to the cell wall was mediated by sortase A. Piliated L. lactis cells exhibited an auto-aggregation phenotype in liquid cultures, which was attributed to the polymerization of major pilin, YhgE. The piliated lactococci formed thicker, more aerial biofilms compared to those produced by non-piliated bacteria. This phenotype was attributed to oligomers of YhgE. This study provides the first dissection of the pilus biogenesis machinery in a non-pathogenic Gram-positive bacterium. Analysis of natural lactococci isolates from clinical and vegetal environments showed pili production under standard growth conditions. The identification of functional pili in lactococci suggests that the changes they promote in aggregation and biofilm formation may be important for the natural lifestyle as well as for applications in which these bacteria are used.

Biofilms of a Bacillus subtilis hospital isolate protect Staphylococcus aureus from biocide action.

The development of a biofilm constitutes a survival strategy by providing bacteria a protective environment safe from stresses such as microbicide action and can thus lead to important health-care problems. In this study, biofilm resistance of a Bacillus subtilis strain (called hereafter ND(medical)) recently isolated from endoscope washer-disinfectors to peracetic acid was investigated and its ability to protect the pathogen Staphylococcus aureus in mixed biofilms was evaluated. Biocide action within Bacillus subtilis biofilms was visualised in real time using a non-invasive 4D confocal imaging method. The resistance of single species and mixed biofilms to peracetic acid was quantified using standard plate counting methods and their architecture was explored using confocal imaging and electronic microscopy. The results showed that the ND(medical) strain demonstrates the ability to make very large amount of biofilm together with hyper-resistance to the concentration of PAA used in many formulations (3500 ppm). Evidences strongly suggest that the enhanced resistance of the ND(medical) strain was related to the specific three-dimensional structure of the biofilm and the large amount of the extracellular matrix produced which can hinder the penetration of peracetic acid. When grown in mixed biofilm with Staphylococcus aureus, the ND(medical) strain demonstrated the ability to protect the pathogen from PAA action, thus enabling its persistence in the environment. This work points out the ability of bacteria to adapt to an extremely hostile environment, and the necessity of considering multi-organism ecosystems instead of single species model to decipher the mechanisms of biofilm resistance to antimicrobials agents.

Characteristics of microbial biofilm on wooden vats ('gerles') in PDO Salers cheese.

The purpose of this study was to characterize microbial biofilms from 'gerles' (wooden vats for making PDO Salers cheese) and identify their role in milk inoculation and in preventing pathogen development. Gerles from ten farms producing PDO Salers cheese were subjected to microbial analysis during at least 4 periods spread over two years. They were distinguished by their levels of Lactobacillus (between 4.50 and 6.01 log CFU/cm(2)), Gram negative bacteria (between 1.45 and 4.56 log CFU/cm(2)), yeasts (between 2.91 and 5.57 log CFU/cm(2)), and moulds (between 1.72 and 4.52 log CFU/cm(2)). They were then classed into 4 groups according their microbial characteristics. These 4 groups were characterized by different milk inoculations (with either sour whey or starter culture, daily or not), and different washing procedures (with water or whey from cheese making). The farm gerles were not contaminated by Salmonella, Listeria monocytogenes or Staphylococcus aureus. Only one slight, punctual contamination was found on one gerle among the ten studied. Even when the milk was deliberately contaminated with L. monocytogenes and S. aureus in the 40 L experimental gerles, these pathogens were found neither on the gerle surfaces nor in the cheeses. Using 40 L experimental gerles it was shown that the microbial biofilms on the gerle surfaces formed in less than one week and then remained stable. They were mainly composed of a great diversity of lactic acid bacteria (Leuconostoc pseudomesenteroides, Lactococcus lactis, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus hilgardii,…), Gram positive catalase positive bacteria (Curtobacterium flaccumfaciens, Curtobacterium oceanosedimentum Citrococcus spp., Brachybacterium rhamnosum, Kocuria rhizophila, Arthrobacter spp.…) and yeast (Kluyveromyces lactis, Kluyveromyces marxianus). In less than 1 min, even in a 500 L farm gerle, the gerle's microbial biofilm can inoculate pasteurized milk with micro-organisms at levels superior to those in raw milk.

The effect of in vitro growth conditions on the resistance of Acanthamoeba cysts.

Despite increasing concerns of direct pathogenicity and/or their role as hosts for other microorganisms there are currently no standard methods for the inactivation of amoebae that belong to the genus Acanthamoeba. Methods used to grow amoebae and produce cysts for these tests may be important as they can dramatically modify cyst susceptibility. We compared resistance of cysts produced from trophozoites grown in peptone-yeast extract-glucose broth or by feeding on HEp-2 cells and then encysted in Neff's medium. We observed that trophozoites grown using HEp-2 cells as a nutrient source produce cysts that are significantly more resistant to SDS and to most biocides tested, including heat. Increased resistance is likely due to a higher proportion of mature cysts presenting thicker cell walls as demonstrated using transmission electron microscopy. This was confirmed by calcofluor white staining demonstrating higher cellulose content in cysts produced from trophozoites grown using HEp-2 cells as a feeding source. These results demonstrate that not only methods used to produce cysts from trophozoites are critical, but that methods used to grow trophozoites before encystment should also be chosen carefully. This should be taken into account for the development of protocols to evaluate biocides and antimicrobials against amoebal cysts.

The canine oocyte: uncommon features of in vivo and in vitro maturation.

The biology of the canine oocyte is unusual compared with that of other mammalian females. The present paper reviews both in vivo and in vitro specificities of canine oocytes. Final follicular growth in the bitch is characterised by an early appearance of LH binding sites in the granulosa, a high proportion of polyovular follicles and a preovulatory luteinisation, starting at the time of the LH surge. Through follicular fluid, preovulatory oocytes are thus exposed to high levels of progesterone, as high as 1000-fold plasma concentrations. The composition of the follicular fluid is affected by the size of the female. The more specific aspect of oocyte biology in the bitch is ovulation: oocytes are expelled immature, at the Prophase I stage. Ovulatory follicles are 6-8 mm in diameter, releasing oocytes from 110 µm, with dark cytoplasm. Resumption of meiosis occurs from 48 h postovulation, MII stages appearing 48-54 h after ovulation. The mechanisms controlling such a late meiotic resumption are still unknown. Granulosa cells seem to play a central role as in other mammalian species, but not with cAMP as the principal mediator. The importance of a transient reactivation of oocyte transcription a few hours before meiotic resumption is to be explored. These specific features may contribute to the low efficiency of IVM. Only 10-20% oocytes reach the metaphase stage and suffer from a poor cytoplasmic maturation. Moreover, in vitro culture of canine oocytes is associated with a high proportion of degeneration. To date, IVM of the oocytes is the main limiting factor for the development of assisted reproductive techniques in the canine. A better knowledge of the basic physiology of folliculogenesis and the molecular mechanisms controlling oocyte meiosis resumption in this species may allow us to overcome this obstacle.