Parmi les desserts à base d’ovoproduits, l’île flottante est reconnue comme particulièrement sensible d’un point de vue microbiologique car sa commercialisation souffre de la survenue intempestive d’altérations que les industriels souhaitent maîtriser. Les travaux réalisés au cours de cette thèse avaient pour objectif de mieux appréhender ces phénomènes afin de mieux les contrôler. Nous avons montré que l’altération de l’île flottante concernait principalement la crème anglaise et qu’elle s’accompagnait d’un développement bactérien conséquent, d’une fréquente diminution du pH et de modifications sensorielles liées à l’aspect et à l’odeur. Les principales bactéries détectées ont été identifiées comme appartenant au groupe Bacillus cereus et aux genres Staphylococcus et Enterococcus. Le blanc d’œuf pasteurisé, utilisé pour la fabrication des œufs en neige, s’est avéré être une source de contamination possible.Cependant, l’implication de bactéries installées sous forme de biofilms dans l’environnement de production ou véhiculées par d’autres ingrédients a aussi été fortement envisagée. La ré-inoculation, en culture pure, d’une collection bactérienne représentative dans de la crème anglaise stérile a montré que différents types de modifications sensorielles et physico-chimiques s’exprimaient d’un genre bactérien à l’autre et qu’ils étaient notamment corrélés à la capacité des bactéries à consommer les sucres et les protéines de la crème anglaise et à produire des métabolites dont des composés volatils odorants. Ces résultats à l’appui, différents tests ont pu être proposés, p / Among the chilled egg products-based desserts, the French dessert “île flottante” is recognized as particularly sensitive from a microbiological point of view, because marketing is suffering from untimely spoilage occurrence that manufacturers wish to control. The work done in this thesis aimed to better understand these phenomena in order to better control them. We have shown that the dessert spoilage mainly concerned the custard cream and it was characterized by high bacterial count, frequent pH decreasing and sensory changes of appearance and smell. The main bacteria detected were identified as belonging to the Bacillus cereus group and Staphylococcus and Enterococcus genera. The possible involvement of bacteria from the pasteurized egg white, used for the egg white foaming, in the dessert spoilage issue was established.However, the involvement of bacteria from biofilms installed in the production environment or provided by other ingredients was also strongly suspected. The spoilage potential assessment of pure culture of a representative bacterial collection in sterile custard cream has shown that different types of sensory and physicochemical changes were expressed according to bacterial genus and that these changes were particularly correlated with the ability of bacteria to consume sugars and proteins of custard and to produce various volatile compounds with specific odorous. With these results, various tests have been proposed for a better control of the white egg batches orientation according to their microbiological quality and so to guarantee their safety with

Bacillus cereus sensu stricto (ss) est un pathogène alimentaire majeur représentant la 2e cause de toxiinfectionalimentaire en France en 2012. Cette espèce fait partie du groupe Bacillus cereus sensu lato (sl)constitué d’espèces ubiquitaires génétiquement très proches et incluant d’autres pathogènes comme B.anthracis, B. thuringensis et B. cytotoxicus. Les souches de B. cereus sl sont d’autre part réparties en septgroupes phylogénétiques présentant des gammes de température de croissance variées et caractérisés partrois thermotypes principaux: thermotolérants, mésophiles, psychrotolérants. L’adaptation au froid dessouches B. cereus ss est un mécanisme clé car il conditionne sa capacité à se développer dans les alimentsréfrigéré pour atteindre des doses qui peuvent être dangereuse pour les consommateurs. Le but de cetteétude a été d’étudier les mécanismes moléculaires impliqués dans l’adaptation au froid de la diversité desouches représentant B. cereus sl.Nous avons mis en évidence que les gènes codant pour le système à deux composants CasK/R sontsurexprimés à basse température. CasK/R s’est révélé être un système générique d’adaptation de B. cereussl au froid, car son rôle a été mis en évidence lors de l’étude de quatre souches de thermotypes différents etleurs mutants isogéniques ΔcasK/R respectifs. Une étude transcriptomique réalisée sur une souche ATCC14579 et son mutant ΔcasK/R a révélé que seize des gènes différentiellement exprimés en début de phaseexponentielle et en phase stationnaire, à basse température, codent pour des protéines impliquées dans lemétabolisme des acides gras. Nous avons mis en évidence le rôle de CasK /R dans la modification de lacomposition en acides gras membranaires via une augmentation de la proportion en acides gras insaturéslors de la croissance de B. cereus au froid. Par ailleurs, le gène codant pour la désaturase DesA,principalement responsable des insaturations des acides gras à basse température est régulée positivementpar CasK/R au froid.Nous avons également démontré que les gènes casK/R sont organisés en opéron avec un gène codant pourun régulateur RpiR-like. De manière originale, cet opéron est négativement régulé par CasK/R à bassetempérature en phase stationnaire. Le promoteur individuel du rpiR est réprimé à basse température maisaussi à température optimale de croissance, ce qui suggère un rôle de CasK/R, même à températureoptimale / Bacillus cereus sensu stricto (ss) is a major foodborne pathogen representing the second cause of foodpoisoning in France in 2012. This species belongs to Bacillus cereus sensu lato (sl) consisting of ubiquitousspecies genetically close-related and including other pathogens such as B. anthracis, B. thuringiensis and B.cytotoxicus. The strains of B. cereus sl are divided into seven phylogenetic groups with various growthtemperature ranges and characterized by three main thermotypes: thermotolerant, mesophilic,psychrotolerant. The B. cereus ss cold adaptation is a key mechanism because it determines B. cereusability to grow in refrigerated foods and achieve doses that can be dangerous to consumers. The aim of thisstudy was to study the molecular mechanisms involved in the cold adaptation of strains representing B.cereus sl diversity.We demonstrated that the genes encoding the two component system CasK/R are overexpressed at lowtemperature. CasK/R was found to be a generic mechanism for B. cereus sl cold adaptation as its role washighlighted in the study of four strains with different thermotypes and their respective isogenic mutantsΔcasK/R. A transcriptomic study on a B. cereus ATCC 14579 strain and its ΔcasK/R mutant strain revealedthat sixteen of the genes differentially expressed in both early log phase and stationary phase at lowtemperature encode proteins involved in the fatty acids metabolism. We showed the role of CasK/R in themodification of the membrane fatty acid composition via an increase of the proportion of unsaturated fattyacids during growth of B. cereus at low temperature. Furthermore, the gene encoding the desaturase DesA,mainly responsible of the fatty acids unsaturation at a low temperature is upregulated by CasK/R at lowtemperature.We also demonstrated that casK/R genes were organized in operon with a gene encoding a RpiR-likeregulator. Interstingly,, this operon is negatively regulated by CasK/R at low temperature in the stationaryphase. The individual rpiR promoter is repressed by CasK/R at low temperature but also optimal growthtemperature, suggesting also a role for CasK/R at optimal temperature

As bactérias patogênicas são os principais agentes que contaminam alimentos e podem prejudicar a saúde humana. Para tentar combater e controlar a contaminação de alimentos investigam-se novos compostos que apresentam atividade antimicrobiana. O ramnolipídeo (RL) é um biossurfatante (BS) produzido por Pseudomonas spp. que apresenta elevada biodegradabilidade e, baixa toxicidade além de potencial antimicrobiano. O objetivo desse trabalho foi estudar a atividade antimicrobiana do RL frente às bactérias patogênicas Gram positivas, Bacillus cereus (ATCC 33018), Listeria monocytogenes (ATCC 19112), Staphylococcus aureus (ATCC 8095) e Gram negativas, Escherichia coli (EHEC) (ATCC 43895) e Salmonella enterica (ATCC 13076) além de contribuir na elucidação do mecanismo de ação destes compostos. Os testes de susceptibilidade ao RL foram realizados a partir da determinação da concentração inibitória mínima (CIM) e concentração bactericida mínima (CBM) utilizando a técnica de micro-diluição. O efeito do pH sobre a atividade antimicrobiana foi avaliado na faixa de pH 5 a 9. Para avaliação do mecanismo de ação foram realizados ensaios de permeabilidade celular, espectroscopia de infravermelho e hidrofobicidade celular. O RL apresentou atividade antimicrobiana para as bactérias B. cereus em CIM 19,5 &#956g/mL e CBM 39,1 &#956g/mL, e para L. monocytogenes CIM 156,2 &#956g/mL e CBM 312,5 &#956g/mL. Para B. cereus apresentou efeito bactericida a partir de 30 minutos na CBM, e para L. monocytogenes em 8 horas de incubação com o RL na CBM. As bactérias Gram negativas E. coli e S. enterica mostraram-se resistentes ao RL. O pH influenciou a ação antimicrobiana do RL sendo mais efetivo em pH mais ácidos. O tratamento com RL promoveu redução da hidrofobicidade da superfície celular das bactérias sensíveis. Os espectros infravermelhos evidenciaram alterações na composição química da membrana/parede celular principalmente para bactérias Gram positivas. A permeabilidade da membrana celular aumentou de acordo com o aumento da concentração de RL. A atividade antimicrobiana do RL foi evidenciada para as bactérias Gram positivas sendo mais sensíveis B. cereus e L. monocytogenes. Os resultados obtidos neste trabalho sugerem que o RL promove alterações na permeabilidade e composição química da membrana celular bacteriana sendo um agente potencial para controle de bactérias Gram positivas de importância alimentar. / Pathogenic bacteria are main agents that contaminate food and are harmful to human health. The search for new compounds to combat and control food pathogens is of increasing interest. Rhamnolipid (RL) is a biosurfactant (BS) typically produced by Pseudomonas spp., showing high biodegradability, low toxicity and antimicrobial activity. This study aimed to evaluate the antimicrobial activity of RL against the food pathogenic Gram positive bacteria Bacillus cereus (ATCC 33018), Listeria monocytogenes (ATCC 19112), Staphylococcus aureus (ATCC 8095) and Gram negative, Escherichia coli (EHEC) (ATCC 43895) and Salmonella enterica (ATCC 13076) and also contribute to the elucidation of RL mechanism of action. Susceptibility tests were performed by determination of the minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) using the broth microdilution method. The effect of pH on antimicrobial action was also investigated ranging from 5 to 9. Mechanism of action was studied using membrane permeability, infrared spectroscopy and cell hydrophobicity assays. The MIC value for B. cereus was 19.5 &#956g/mL and MBC was 39.1 &#956g/mL. L. monocytogenes was inhibited at concentration 156.2 &#956g/mL showing MBC of 312.5 &#956g/mL. B. cereus presented bactericidal effect after 30 minutes and for L. monocytogenes after 8 hours. The Gram-negative E. coli and S. enterica were resistant to RL. The pH influence antimicrobial activity of the RL showing decreasing MIC values at acidic conditions. Cell hydrophobicity was reduced by RL for the sensitive bacteria. Infrared spectroscopy showed that RL induced changes in chemical composition of cell membrane/ wall especially for the Gram positive bacteria. Cell permeability also increases as RL concentration increases. Antimicrobial activity of RL was evidenced for Gram positive bacteria and the most sensitive were B. cereus and L. monocytogenes. The results of this study suggest that rhamnolipid biosurfactant promotes changes in the permeability and membrane chemical composition showing potential to control foodborne Gram positive bacteria.

<p>In&nbsp; this&nbsp; study,&nbsp; tolerance&nbsp; of&nbsp; soil&nbsp; bacteria&nbsp; to&nbsp; hexavalent&nbsp; chromium&nbsp; (Cr(VI))&nbsp; was&nbsp; investigated.&nbsp; First,&nbsp; influence&nbsp; of&nbsp; high chromium levels of anthropogenic and geogenic origin on the&nbsp; soil cultivable&nbsp; bacterial community was examined. Next, a number&nbsp; of&nbsp; bacterial&nbsp; strains&nbsp; with&nbsp; high&nbsp; Cr(VI)&nbsp; tolerance&nbsp; were&nbsp; isolated&nbsp; from&nbsp; diverse&nbsp;&nbsp; environmental&nbsp; samples&nbsp; such&nbsp; as&nbsp; soil, sediment, water and waste material.&nbsp; Strains were&nbsp; identified&nbsp; and&nbsp; tested for&nbsp; the&nbsp; level of&nbsp; Cr(VI) tolerance&nbsp; and&nbsp; the&nbsp; ability to<br />reduce toxic Cr(VI) to more innocuous Cr(III). Selected&nbsp; <em>Bacillus cereus</em>&nbsp; group strains&nbsp; were further characterized&nbsp; -&nbsp; their morphological&nbsp; and&nbsp; biochemical&nbsp; characteristics,&nbsp; 16S&nbsp; rRNA&nbsp; and&nbsp; pycA&nbsp; gene&nbsp; sequences,&nbsp; biofilm&nbsp; formation&nbsp; potential&nbsp; and resistance to other heavy metals were determined. Also, more detailed study of their tolerance level and&nbsp; Cr(VI) reduction was&nbsp; conducted.&nbsp; Strain&nbsp; with&nbsp; the highest&nbsp; resistance&nbsp; together&nbsp; with the&nbsp; control&nbsp; chromate&nbsp; sensitive&nbsp; strain&nbsp; were&nbsp; analyzed&nbsp; by STEM EDS for their cellular and endospore Cr content under different conditions. Results indicate Cr(VI) tolerant bacteria are&nbsp; present&nbsp; both&nbsp; in&nbsp; low&nbsp; and&nbsp; high&nbsp; Cr&nbsp; environments.&nbsp; Majority&nbsp; of&nbsp; isolates&nbsp; belonged&nbsp; to&nbsp; the&nbsp;<em> B.&nbsp; cereus&nbsp;</em> group&nbsp; indicating&nbsp; its overall high tolerance to&nbsp; Cr(VI). Certain strains exhibited high&nbsp; tolerance and reduction&nbsp; ability,&nbsp; indicating their possible<br />usefulness&nbsp; in practical&nbsp; bioremediation&nbsp; application.&nbsp; STEM&nbsp; EDS&nbsp; analysis&nbsp; of&nbsp; Cr(VI)-sensitive&nbsp;<em> B.&nbsp; subtilis&nbsp;</em> PY79&nbsp; strain&nbsp; and Cr(VI)-resistant&nbsp; <em>B. cereus&nbsp;</em> group strain&nbsp; NCr1a revealed&nbsp; significant differences in their response to Cr(VI)&nbsp; and in&nbsp; their&nbsp; Cr cellular and endospore content.</p> / <p>U ovom radu ispitana je tolerantnost&nbsp; zemlji&scaron;nih&nbsp; bakterija na &scaron;estovalentni hrom (Cr(VI)). Prvo, ispitan je uticaj visokog nivoa&nbsp; hroma&nbsp; antropogenog&nbsp; i&nbsp; geogenog&nbsp; porekla&nbsp; na&nbsp; kultivabilnu&nbsp; bakterijsku&nbsp; zajednicu&nbsp; zemlji&scaron;ta.&nbsp; Dalje,&nbsp; izolovani&nbsp; su bakterijski sojevi sa visokom tolerancijom na Cr(VI) iz različitih sredinskih uzoraka &nbsp; kao &scaron;to su zemlji&scaron;te, sediment, voda i otpadni materijal. Sojevi su identifikovani i određen je nivo njihove Cr(VI) tolerancije i sposobnost redukcije toksičnog Cr(VI)&nbsp; u&nbsp; manje&nbsp; toksični&nbsp; Cr(III).&nbsp; Odabrani&nbsp; sojevi&nbsp; <em>Bacillus&nbsp; cereus&nbsp;</em> grupe&nbsp; su&nbsp; dalje&nbsp; karakterisani&nbsp; &ndash;&nbsp; određene&nbsp; su&nbsp; njihove morfolo&scaron;ke i biohemijske karakteristike, 16S rDNK i&nbsp; pycA&nbsp; sekvence, potencijal formiranja biofilma i otpornost na druge te&scaron;ke&nbsp; metale.&nbsp; Takođe,&nbsp; sprovedeno&nbsp; je&nbsp; detaljnije&nbsp; ispitivanje&nbsp; njihove&nbsp; tolerancije&nbsp; i&nbsp; redukcije&nbsp; Cr(VI).&nbsp; Soj&nbsp; sa&nbsp; najvi&scaron;om otporno&scaron;ću&nbsp; je&nbsp; uporedo&nbsp; sa&nbsp; kontrolnim&nbsp; osetljivim&nbsp; sojem&nbsp; analiziran&nbsp; pomoću&nbsp; STEM&nbsp; EDS&nbsp; na&nbsp; sadržaj&nbsp; hroma&nbsp; u&nbsp; ćelijama&nbsp; I endosporama u različitim uslovima. Rezultati ukazuju da su bakterije tolerantne na Cr(VI) prisutne i u sredinama sa niskim i&nbsp; sa&nbsp; visokim&nbsp; koncentracijama&nbsp; hroma.&nbsp; Većina&nbsp; izolata&nbsp; pripadala&nbsp; je&nbsp; B.&nbsp; cereus&nbsp; grupi&nbsp; &scaron;to&nbsp; ukazuje&nbsp; na njenu&nbsp; uop&scaron;teno&nbsp; visoku otpornost na Cr(VI). Pojedini sojevi su pokazali visoku otpornost i sposobnost&nbsp; redukcije Cr(VI), &scaron;to ukazuje na mogućnost njihove praktične primene u bioremedijaciji. STEM EDS analiza osetljivog<em> B. subtilis</em> PY79 soja i Cr(VI)- rezistentnog soja <em>B.&nbsp; cereus</em>&nbsp; grupe&nbsp; NCr1a&nbsp; otkrila&nbsp; je&nbsp; značajne&nbsp; razlike&nbsp; u&nbsp; njihovom&nbsp; odgovoru na&nbsp; Cr(VI)&nbsp; i&nbsp; sadržaju&nbsp; Cr&nbsp; u njihovim&nbsp; ćelijama&nbsp; i endosporama.</p>

Background: Almost all foals develop transient diarrhoea within the first weeks of life. Studies indicated different viral, bacterial, and parasitic causes, such as rotavirus, Clostridium perfringens, Escherichia coli, and Cryptosporidium are discussed. But little is known about the development of intestinal microflora in foals. The present study investigated whether the supplementation with Bacillus cereus var. toyoi would modify the developing intestinal microflora and consequently reduce diarrhoea in foals. From birth, the foals were randomly assigned to three treatment groups: placebo (10 mL isotonic NaCl, n = 8), low dosage (LD; 5 × 108 cfu B. cereus var. toyoi, n = 7) and high dosage (HD; 2 × 109 cfu B. cereus var. toyoi, n = 10). Treatment groups were supplemented orally once a day for 58 days. Faeces scoring and sampling were performed within the first 24 h after birth and on day 9, 16, 23, 30, 44, 58 of the foal’s life and also on the first day of diarrhoea. Culture-plate methods were used to analyse the bacterial microflora. Results: Eighty-eight per cent of the foals developed diarrhoea (placebo 7/8, LD 5/7, HD 10/10) during the first 58 days of life. Bacillus cereus var. toyoi supplementation had no effect on bacterial microflora. Clostridium perfringens and enterobacteria were equally prevalent in foals with diarrhoea and those who were not afflicted. Conclusions: We conclude that the supplementation of B. cereus var. toyoi had no effect on the occurrence of diarrhoea and health status in the foals.

Diarrhoe ist eines der häufigsten Probleme beim equinen Neonaten. Nahezu alle Fohlen entwickeln Durchfall innerhalb der ersten Lebenswochen. Unterschiedliche virale, bakterielle und parasitäre Ursachen werden diskutiert. In diesen Zeitraum fällt ebenfalls die erste Rosse der Stute, sodass der Durchfall um den 5. - 15. Lebenstag (LT) bei den Fohlen als „Fohlenrossedurchfall“ bezeichnet wird. Es wird vermutet, dass die Entwicklung der intestinalen Mikroflora und die Reifung der Darmschleimhaut im Wesentlichen für das Durchfallgeschehen verantwortlich sind. Bisher ist jedoch wenig bekannt über die Entwicklung der intestinalen Mikroflora bei Fohlen. Einige Probiotika sind als Darmflorastabilisatoren bei Tieren zugelassen. Studien belegten positive Effekte von Toyocerin® (B. cereus var. toyoi) auf die Darmgesundheit bei anderen Tierarten wie z.B. Kälbern, Ferkeln, Broilern, Puten und Mastkaninchen. Die vorliegende Arbeit sollte klären, ob die Supplementierung von B. cereus var. toyoi zu einer Stabilisierung der sich entwickelnden intestinalen Mikroflora und damit zu einer Verringerung der Durchfälle bei Fohlen führt. Die Untersuchung wurde an 25 Mutterstuten eines Vollblutgestüts und ihren Fohlen durchgeführt. Alle Fohlen wurden von Februar bis Mai 2011 geboren. Von Geburt an wurden die Fohlen randomisiert in drei Behandlungsgruppen eingeteilt: Placebo-Gruppe (10 ml isotone Kochsalzlösung, n=8), 50 mg Toyocerin-Gruppe (5 x 108 KbE B. cereus var. toyoi gelöst in 10 ml isotoner Kochsalzlösung, n=7) und 200 mg Toyocerin-Gruppe (2 x 109 KbE B. cereus var. toyoi gelöst in 10 ml isotoner Kochsalzlösung, n=10). Die Placebo- und Behandlungsgruppen wurden einmal täglich vom 1. – 58. LT supplementiert. Herz- und Atemfrequenz, Körperinnentemperatur und die Körpermasseentwicklung wurden nach einem standardisierten Protokoll erhoben. Kotproben konnten mit Hilfe von Kotsammelbeuteln oder durch rektale Entnahme innerhalb von 24 Stunden nach der Geburt sowie an LT 9, 16, 23, 30, 44, 58 und am ersten Durchfalltag gewonnen werden. Blutproben wurden aus der Vena jugularis externa am 1., 9., 16., 30., 58. LT sowie am ersten Durchfalltag entnommen. Die bakteriologische Untersuchung erfolgte mit Hilfe des Kulturverfahrens. Die Bestimmung der Gesamt-IgG-Werte wurde mit einem kompetitiven ELISA, die Bestimmung der spezifischen Antikörper IgG-anti-LPS von E. coli J5 und IgG-anti-PLC-von-C. perfringens-1a mit einem indirekten ELISA durchgeführt. 88 % der Fohlen entwickelten Durchfall (Placebo 7/8, 50 mg Toyocerin 5/7, 200 mg Toyocerin 10/10) mit einer hohen Inzidenz zwischen dem 8. und 16. LT. Das Allgemeinbefinden und die Bewegungs- und Sauglust blieben dabei unbeeinflusst. Zum Zeitpunkt des ersten Östrus der Stute zeigten 59 % der Fohlen Diarrhoe. Unter den 41 %, die keinen Durchfall zum Zeitpunkt der Fohlenrosse hatten, waren Fohlen, die nie Durchfall vom 1. – 58. LT zeigten, aber auch welche die Diarrhoe entwickelten, als die Mutterstute sich nicht in Rosse befand. Ein Zusammenhang zwischen der Fohlenrosse der Stute und Durchfall bei ihrem Fohlen konnte nicht hergestellt werden. Es zeigte sich eine Tendenz, dass hohe Spiegel der Gesamt-IgG (>20 mg/ml) und IgG-anti-LPS von E. coli J5 (>120 RE/ml) nach der Kolostrumaufnahme im Zusammenhang mit einer geringeren Anzahl von Durchfalltagen innerhalb der ersten zwei Lebensmonate standen. C. perfringens und Enterobakterien waren gleichermaßen nachweisbar bei Fohlen mit Durchfall als auch bei unauffälligen Fohlen. Aus der Supplementierung von B. cereus var. toyoi ergab sich kein Effekt auf die Kotflora der Fohlen, außer auf die Gesamtkeimzahlen (GKZ) der aeroben Bakterien. Bei den Aerobiern im Fohlenkot konnte ein signifikanter Behandlungseffekt (p=0,012) festgestellt werden. Im ersten Milchkot der Fohlen waren GKZ von 4,5 x 104 KbE/g (200 mg Toyocerin-Gruppe) bis 5,0 x 105 KbE/g (50 mg Toyocerin-Gruppe) bei den aeroben Bakterien und GKZ von 2,4 x 105 KbE/g (200 mg Toyocerin-Gruppe) bis 4,7 x 106 KbE/g (50 mg Toyocerin-Gruppe) median bei den Anaerobiern nachweisbar. Danach stieg der Gehalt der aeroben und anaeroben Bakterien weiter bis zum 3. LT und stagnierte bis zum 16. LT. Während dieser Stagnationsphase trat bei 92 % der Fohlen (23/25) eine Veränderung der Kotkonsistenz bis hin zu Durchfällen auf. Vom 16. bis zum 58. LT sanken die Gehalte moderat bei den Aerobiern median am 58. LT auf 2,7 x 105 KbE/g (Placebo-Gruppe) bis 2,2 x 106 KbE/g (50 mg Toyocerin-Gruppe) und bei den Anaerobiern median am 58. LT auf 3,8 x 105 KbE/g (Placebo-Gruppe) bis 2,9 x 106 KbE/g (200 mg Toyocerin-Gruppe). Bis zum 58. LT näherte sich der Medianwert der aeroben und anaeroben Bakterien im Kot der Placebo-Gruppe dem Wert der Mutterstuten (gemessen am ersten Tag nach der Geburt) an. Innerhalb der ersten Lebenstage war eine hohe aerobe sowie anaerobe Keimzahl im Kot der Fohlen nachzuweisen, die sich oberhalb der Keimzahlen befand, die im Kot der Mutterstuten zum Zeitpunkt der Geburt gemessen wurde. Im Rahmen der Entwicklung und Etablierung der bakteriellen intestinalen Mikroflora wurde das Fohlenrossedurchfallgeschehen bei den Fohlen beobachtet. B. cereus var. toyoi hatte dabei keinen Einfluss auf die Anzahl der Fohlen mit Durchfall und den Gesundheitsstatus der Fohlen. / Diarrhoea is probably one of the most common problems in equine neonates. Almost all foals develop transient diarrhoea within the first weeks of life. Different viral, bacterial and parasitic causes are discussed. Between the 5th and the 15th day of the foal’s life, when their dam’s first post partum (p.p.) oestrus is expected, diarrhoea in foals is observed quite often. That is why it’s called “foal heat diarrhoea”. In literature establishment of intestinal microflora and maturation of the intestinal mucosa is responsible for the occurrence of diarrhoea in this period of life. But little is known about the development of the intestinal microflora in foals. Many probiotics are authorised as gut flora stabilisers in animal nutrition. Some studies proved positive effects of Bacillus (B.) cereus var. toyoi (Toyocerin®) on intestinal health in other species e.g. calves, piglets, broiler chicken, poultry and growing rabbits. The present study deals with the question if a supplementation of B. cereus var. toyoi lead to a stabilisation of the developing intestinal microflora and therefore to a reduction of diarrhoea in foals. A total of 25 mares and foals of a thoroughbred stud were included into the study. Foals were born between February and May 2011. From birth, the foals were randomly assigned to three treatment groups: placebo group (10 ml isotonic saline solution, n=8), 50 mg Toyocerin group (5 x 108 cfu B. cereus var. toyoi solved in 10 ml isotonic saline solution, n=7) and 200 mg Toyocerin group (2 x 109 cfu B. cereus var. toyoi solved in 10 ml isotonic saline solution, n=10). Placebo- and treatment groups were orally supplemented once a day starting on the 1st through to the 58th day of life. Determination of heart and respiratory rate, body temperature, body weight was realised according to a standardised protocol. Within the first day of life, on day 9, 16, 23, 30, 44, 58 and on the first day of diarrhoea faecal samples has been taken from the rectum or by the use of a collection bag. Blood samples were taken via jugular venipuncture on day 1, 9, 16, 30, 58 and on the first day of diarrhoea. Culture-depend methods were used to analyse the bacterial microflora. Serum IgG was analysed by a competitive ELISA, IgG-anti-LPS from E. coli J5 and IgG-anti-PLC-from-C. perfringens-1a by an indirect ELISA. 88 % of the foals developed diarrhoea (placebo 7/8, 50 mg Toyocerin 5/7, 200 mg Toyocerin 10/10) with a high incidence between the 8th and the 16th day of the foal’s life. Meanwhile, foals remained bright and alert and continued to nurse. At the time point of the first p.p. oestrus in the mares, 59 % of their foals showed signs of diarrhoea. Within the remaining 41 % there are foals that had no diarrhoea but there are also foals which had diarrhoea when the mare had not been in heat. Neonatal diarrhoea in foals is not linked to p.p. oestrus in their mares. There was a tendency, that high serum-IgG (> 20 mg/ml) and IgG-anti-LPS from E. coli J5 (> 120 RE/ml) after colostrum uptake were associated with lower diarrhoea severity in the first 58 days of the foal’s life. C. perfringens and enterobacteria can be found equally in foals with diarrhoea and in foals which are not afflicted. B. cereus var. toyoi supplementation had no effect on faecal bacteria in foals, except on aerobic bacteria (p=0,012). In the first milk faeces aerobic bacteria were detected in median from 4,5 x 104 cfu/g (200 mg Toyocerin-group) to 5,0 x 105 cfu/g (50 mg Toyocerin-group) and anaerobic bacteria were detected in median from 2,4 x 105 cfu/g (200 mg Toyocerin-group) to 4,7 x 106 cfu/g (50 mg Toyocerin-group). Afterwards the counts increased towards the 3rd day of life and stayed on a high level till the 16th day of life. During this stagnation in 92 % of the foals a change in faecal consistency and diarrhoea was observed. Afterwards, from the 16th though to the 58th day of life, the bacteria counts in the faeces moderately decreased in median for the aerobic bacteria on the 58th day of life down to 3,8 x 105 cfu/g (placebo-group) till 2,9 x 106 cfu/g (200 mg Toyocerin-group). On the 58th day of life the counts of aerobic and anaerobic bacteria in the faeces of the placebo-group approached the counts in the faeces of the mare (measured at the time point of birth). In the first days of foals’ life detection of aerobic and anaerobic bacteria in the faeces were high, and above the level of the bacteria counts in the faeces of the mare at the time point of birth. Foal heat diarrhoea is observed as a part of the development and establishment of bacterial intestinal microflora. B. cereus var. toyoi had no effect on the percentage of foals with diarrhoea and health status in the foals at that point.

L’apport de fer est essentiel pour la plupart des organismes vivants, incluant la majorité des bactéries pathogènes. Cependant, le fer libre est toxique : il est lié à des protéines de stockage et de transport (e.g. ferritine, hémoprotéines…) et voit son homéostasie finement régulée. Afin d’extraire le fer de ces protéines, les bactéries utilisent divers systèmes tels que des protéines de surface ou encore des sidérophores. Bacillus cereus est une bactérie Gram-positive sporulante, pathogène opportuniste chez l’homme, 2ème cause en France de toxi-infection alimentaire collective. Chez B. cereus, la protéine de surface IlsA et le sidérophore bacillibactine (BB) sont impliqués dans l’acquisition du fer de la ferritine exogène et elles sont importantes pour l’infection de l’insecte modèle Galleria mellonella. Mes travaux présentaient deux parties : tout d’abord, l’étude de l’import du complexe BB-Fe3+ dans la cellule par FeuA, protéine de liaison de ce complexe à la surface de la bactérie, souligne le rôle central du couple BB-FeuA. La délétion des gènes codants pour ces deux molécules limite l’acquisition par B. cereus du fer de la ferritine, de l’hème, de l’hémoglobine et du fer inorganique in vitro. En revanche, elle présente un phénotype de virulence in vivo comparable à la souche de référence dans le cas d’injection intra-hémocœlique de larves de G. mellonella. Ce résultat surprenant suggère un probable rétrocontrôle sur l’expression de facteurs de virulence lorsque B. cereus ne produit ni BB ni FeuA, et se trouve par conséquent fortement carencé en fer. Le second volet de mes travaux s’intéresse à l’expression des gènes liés à l’homéostasie du fer in vivo, au cours de l’infection de l’intestin de larves de G. mellonella axéniques. Nous avons choisi une approche de type microgénomique, en prélevant les échantillons par microdissection laser, sur de façon à prélever de petits échantillons dans une zone définie, puis en analysant l’expression de quelques gènes ciblés par RT-qPCR et ddPCR à 3h et 16h post ingestion. Nos résultats montrent que : i) la colonisation intestinale de G. mellonella est impactée lorsque B. cereus est dépourvu du couple BB-FeuA ; ii) ilsA est exprimé lors de l’infection intestinale ; iii) les gènes ciblés impliqués dans l’homéostasie du fer sont activés dès le début de l’infection, suggérant un rôle dans l’adaptation et la pathogénicité ; iv) une faible modulation de l’expression est observée entre les deux temps. Ces travaux ouvrent de nouvelles connaissances fondamentales sur l’homéostasie du fer et des perspectives quant à l’utilisation de nouvelles techniques pour l’étude in situ des interactions hôte-pathogène. / Iron acquisition is essential for most living organisms, including many pathogenic bacteria. However, free iron is toxic: it is bound into storage or transport proteins (e.g. ferritin, hemoproteins…) and iron homeostasis is tightly regulated. To scavenge iron from these sources, bacteria possess several systems to acquire the bound iron, by surface proteins or siderophores. Bacillus cereus is a sporeforming Gram-positive bacterium, opportunistic human pathogen, 2nd cause of food-borne disease in France. It has been demonstrated that the B. cereus surface protein IlsA and the siderophore bacillibactin (BB) are involved in iron acquisition from ferritin and that these two molecules are important for infection of the insect model G. mellonella. My thesis project focused on two parts: first the study of the BB-Fe3+ complex import into the cell by the siderophore binding protein FeuA highlights the central role of both BB and FeuA. The deletion of the genes encoding for these two molecules limits iron acquisition by B. cereus from ferritin, heme, hemoglobin and inorganic iron in vitro. On the other hand, the virulence phenotype during intra-haemocelic infection of G. mellonella is similar to the Wild-type strain. These results suggest a possible feedback on the expression of virulence factor genes when B. cereus is unable to synthetize both BB and FeuA, and therefore are under high stress. The second part of my work focused on the expression of genes involved in iron homeostasis in vivo, during gut infection of germ-free larvae of G. mellonella. We chose to perform a microgenomic approach, using laser-capture microdissection to get small samples in targeted areas, and then analysing the expression of chosen genes by RT-qPCR and ddPCR at two time points post ingestion The results show that : i) the colonisation of G. mellonella gut is impacted when B. cereus is deprived of both BB and FeuA ; ii) ilsA is expressed during gut infection ; iii) iron homeostasis is involved in adaptation and pathogenicity from the early step of infection of the insect gut ; iv) only weak gene expression modulation occured between the two timepoints This work gives new fundamental knowledge about B. cereus iron homeostasis, and highlights the use of new techniques regarding the in situ study of host-pathogen interactions.

O Bacillus cereus é um patógeno amplamente distribuído na natureza, relacionado a intoxicações alimentares e causador da coagulação doce do leite. A intoxicação pode ocorrer após a ingestão de alimentos nos quais a bactéria produziu a toxina. Em laticínios, o B. cereus é problemático por sua habilidade de formar esporos resistentes ao calor e sobreviver aos tratamentos térmicos, como a pasteurização e UHT. As espécies de Bacillus são freqüentemente isoladas de leite pasteurizado e UHT e evidências indicam que os biofilmes formados nas superfícies dos equipamentos de processamento são uma das principais fontes de contaminação. No biofilme, as células ou esporos aderidos, têm aumentada sua resistência e podem resistir ao processo de sanificação, se tornando focos de disseminação de bactérias no processo. A matriz polimérica do biofilme reage e neutraliza os agentes sanificantes, expondo as bactérias em seu interior a doses subletais, podendo levar a aquisição de resistência. Neste estudo, biofilmes de células vegetativas B. cereus foram desenvolvidos na superfície de aço inoxidável, tipo 304, com filme de condicionamento de leite. A adesão média das células foi de 1,4 x 105UFC/cm2. Também foram desenvolvidos biofilmes de esporos, com adesão média em aço inoxidável de 1,4 x 104UFC/cm2. Foi avaliada a resistência das células vegetativas e esporos de B. cereus em biofilmes, ao processo simulado de higienização por clean in place (CIP) realizado em laticínios. Após a limpeza a adesão das células vegetativas em aço inoxidável foi reduzida a aproximadamente 1CFU/cm2. Após a sanificação, os resultados permaneceram inalterados. A contaminação remanescente foi relacionada com a maior resistência das células nos biofilmes formados. Após a limpeza e sanificação a adesão média dos esporos foi de 8,9 x 101 UFC/cm2 e 3,3 x 101 UFC/cm2, respectivamente. Tendo em vista este resultado, foi observado que esporos de B. cereus são mais difíceis de remover de superfícies de aço inoxidável do que células vegetativas, com procedimentos CIP. Finalmente, foi realizada a exposição de células vegetativas de B. cereus a três concentrações subletais (3,9; 1,8 e 0,9µg/mL) de cloreto de alquil amidopropil dimetil benzil amônio para avaliação da adaptação pelo método da concentração inibitória mínima (CIM). A aquisição de resistência foi avaliada pela comparação do valor inicial de CIM (7,4µg/mL), com os valores finais, após 28 dias de exposição. Após o período de adaptação, o valor de CIM encontrado foi o dobro do original (14,9µg/mL) indicando aquisição de resistência ao sanificante pela cepa. Foi constatado ainda que o cultivo da cepa na menor concentração subletal apresentou resultados mais coerentes, sugerindo que exposição a doses subletais mais brandas resulta em uma maior resposta adaptativa. / Bacillus cereus is a pathogen widely distributed in the environment and a serious problem in the dairy industry because of its ability to form heat-resistant spores that can survive pasteurization and UHT treatments. This bacteria is responsible for foodborne diseases in man due to production of toxins that may cause gastroenteritis, diarrhoea and vomiting. In addition, B. cereus is responsible for spoilage of pasteurized milk and cream referred to as \"sweet curdling\". Bacillus strains are frequently isolated from pasteurized and UHT treated milk and evidence indicates that biofilm formed on processing equipment surfaces are major sources of milk contamination. In biofilms, adherent cells and spores acquire increased resistance and persist to the sanitization process, becoming sources of bacteria dissemination in food processing. The extracellular polymeric matrix neutralizes the sanitizing agents and exposing the bacteria in biofilms to sublethal concentrations can lead to an increased resistance. In this study, biofilms of B. cereus vegetative cells were developed in a 304 stainless steel (SS) surface with a milk conditioning film. The mean adhesion of cells on SS surface was 1.4 x 105CFU/cm2. Biofilms of B. cereus spores were also developed, and the mean adhesion found was 1.4 x 104CFU/cm2. The resistance of B. cereus vegetative cells and spores in biofilms to a simulated clean-in-place (CIP) procedure used in dairy industry was evaluated. After cleaning procedure, the population of vegetative cells in the SS surface was reduced to approximated 1 CFU/cm2. After sanitizing procedure, the cell count remained unaltered. The remaining contamination was related with the increased resistance of cells in the developed biofilms. After cleaning and sanitizing procedures, the mean of spore adhesion found was 8.9 x 101 CFU/cm2 and 3.3 x 101 CFU/cm2, respectively. These results show that B. cereus spores are more difficult to remove from SS surfaces than vegetative cells using CIP procedures. Finally, B. cereus vegetative cells were exposed to three different sublethal concentrations (3.9; 1.8 and 0.9µg/mL) of alkyl amidopropyl dimethyl benzyl ammoniun cloride, for evaluation of adaptation, using the Minimum Inhibitory Concentration (MIC) test. The acquired resistance was evaluated by comparing the initial MIC value (7.4µg/mL) with final MICs, after 28 days of exposure. After the adaptation period, with exception of an experiment where the MIC was threefold the original value (29.8µg/mL), in the other experiments the MICs found was double (14.9µg/mL) of the original MIC, indicating acquisition of resistance by the B. cereus strain. It was observed that the growth of cells in the mildest sublethal concentration (0.9µg/mL) resulted in more consistent effects, suggesting that the exposure to mildest sublethal concentrations results in higher adaptive responses.

A análise microbiológica atua como ferramenta fundamental para a obtenção de dados sobre a qualidade, sanidade, higiene e segurança na produção de alimentos; desta forma, tem sido adotada na indústria alimentícia para o controle de qualidade. Por sua composição completa e balanceada, o leite é um substrato ideal para o desenvolvimento de diversos grupos de microrganismos. Com o objetivo de pesquisar bactérias da espécie Bacillus cereus em amostras de leite fluido, bem como a capacidade de germinação de esporos e a multiplicação dessa bactéria após processo de fervura, com manutenção das amostras à temperatura ambiente e à temperatura de refrigeração por períodos de 1, 2, 4, 6, 8, 10 e 12 horas, foram analisadas 75 amostras de leite, conforme as metodologias recomendadas por Silva et al. (2007). Destas, 46 amostras (61,3%) mostraram-se com algum grau de contaminação pela bactéria antes de serem submetidas à fervura. Por sua vez, as amostras mantidas à temperatura ambiente após a fervura, tiveram suas contagens bacterianas, principalmente a partir da 8a hora, superiores à contagem inicial, inclusive atingindo níveis capazes de desencadear uma toxinfecção alimentar, demonstrando a ocorrência da germinação dos esporos e a multiplicação das células vegetativas. Por outro lado, alíquotas dessas mesmas amostras mantidas sob refrigeração (7ºC) não atingiram populações preocupantes, enfatizando, desse modo, a importância da necessidade da refrigeração do leite após a fervura. / The microbiological analysis acts as basic tool for the attainment of data on the quality, health, hygiene and security in the food production, in such a way, she has been adopted in the nourishing industry for the quality control. For its complete and balanced composition, milk is an ideal substratum for the development of diverse groups of microorganisms. With the objective to search cereus bacteria of the Bacillus species in fluid milk samples, as well as the capacity of germination of spores and the multiplication of this bacterium after boil process, with maintenance of the samples to the ambient temperature and the temperature of refrigeration for periods of 1, 2, 4, 6, 8, 10 and 12 hours, 75 milk samples had been analyzed, as the methodologies recommended for Silva et al. (2007). Of these, 46 samples (61.3%) had revealed with some degree of contamination for the bacterium before being submitted to the boil. In turn, the samples kept to the ambient temperature after the boil, had its bacterial countings, mainly from 8a hour, superiors to the initial counting, also reaching levels capable to unchain an alimentary toxinfection, demonstrating to the occurrence of the germination of the spores and the multiplication of the vegetative cells. On the other hand, aliquot of these same samples kept under refrigeration (7ºC) had not reached preoccupying populations, emphasizing, in this manner, the importance of the necessity of the refrigeration of milk after the boil.

Bacillus cereus est un pathogène opportuniste à l'origine de deux types de toxi-infections alimentaires classées en syndrome émétique ou diarrhéique. Le syndrome diarrhéique résulte de la production d'entérotoxines (Hbl, Nhe et CytK) au niveau de l'intestin grêle de l'hôte, caractérisé par une atmosphère anaérobie et un faible potentiel d'oxydo-réduction (POR). La capacité de B. cereus à se développer et à produire des entérotoxines dans ces conditions est sous le contrôle de deux systèmes qui agissent, en partie, indépendamment du régulateur pléiotrope connu, PlcR (Phospholipase C Regulator). Il s'agit du système à deux composants ResDE et de la protéine Fnr (Fumarate Nitrate Reductase). Le but de cette étude a été de caractériser d'un point de vue fonctionnel l'implication du régulateur Fnr et du système ResDE dans la toxinogenèse de B. cereus. Les résultats ont montré que la régulation de la transcription de hbl et nhe était sous le contrôle direct et indirect de Fnr et de ResD. En aérobiose, la fixation de Fnr (forme Apo) sur les régions promotrices des gènes de structure des entérotoxines (pnhe et phbl) et des gènes de régulation (presDE, pfnr et pplcR) dépend des conditions redox. L'affinité de ResD pour pnhe, phbl, presDE, pfnr et pplcR dépend des séquences de ces régions promotrices et son affinité pour les régions promotrices presDE et pfnr dépend de son état de phosphorylation. ResD et ApoFnr sont capables de se fixer simultanément sur les régions promotrices étudiées et sont également capables d'interagir physiquement en l'absence d'ADN. Nous avons proposé un modèle de régulation de la toxinogenèse dans lequel ResDE et Fnr pourraient agir en synergie. Enfin des expériences de double hybride ont permis de mettre en évidence que la protéine PlcR pourrait interagir in vivo avec les régulateurs ResD et Fnr. La régulation de la toxinogenèse impliquerait donc la formation d'un complexe multi-moléculaire