Vibrio parahaemolyticus is a food-borne pathogen commonly associated with the consumption of raw or undercooked seafood resulting in primary infections of the human gastrointestinal tract. It is estimated to cause about 4500 illnesses each year in the United States. However, infection from this food-borne pathogen can be avoided if this organism is detected in the implicated food, prior to consumption. Current standard methods of detecting this organism are dependent on the culturability of the bacteria. Detection based on an organism’s culturability may be problematic as V. parahaemolyticus has been known to exist in a viable but nonculturable (VBNC) state. Bacteria in the VBNC state are characterized by low levels of metabolic activity and the inability to be cultured by standard laboratory practices. When bacteria enter the VBNC state, their gene expression profile may be different than the culturable counterpart. We were interested in comparing the expression of two virulence-associated genes between VBNC and culturable cells of V. parahaemolyticus. V. parahaemolyticus RIMD2210633 was incubated at 4°C in modified Morita mineral salt solution supplemented with 0.5% NaCl (MMS) or trypticase soy broth supplemented with 2% NaCl (TSBS), which represented nutrient poor and rich conditions, respectively. The number of VBNC and culturable cells were determined by standard plate count and fluorescence microscopy. The expression levels of virulence-associated genes tdh2 and escU, were measured relative to the housekeeping gene, pvsA, by qRT-PCR. Nutrient availability and temperatures exerted variable effects on the virulence gene expression. It is possible that VBNC V. parahaemolyticus cells may retain their pathogenicity potential.

Conversion of <i>Campylobacter upsaliensis</i> to the nonculturable but viable coccoid form was characterized. Chloramphenicol did not prevent the conversion. Severe decreases in isocitrate dehydrogenase activity and oxygen uptake and extensive degradation of ribosomal RNA suggest that the coccoid form is a degenerative form rather than part of a life cycle. The autolysins of spiral and coccoid forms of <i>C. upsaliensis</i> were also studied. Autolytic activity in the soluble and sediment fractions of sonicates of the spiral and the coccoid form of <i>C. upsaliensis</i> could not be demonstrated by native (nondenaturing) PAGE. Autolysins were detected, however, by using denaturing SDS-PAGE gels containing either purified <i>E. coli </i> peptidoglycan or whole cells of <i>Micrococcus luteus</i> as the turbid substrate, with subsequent renaturation by treatment with Triton X-100 buffer. In renaturing gels that contained <i>E. coli</i> peptidoglycan, 14 autolytic bands were detected ranging from 200 kDa to 12 kDa. In similar gels containing whole cells of <i>M. luteus</i> , only a single band appeared having a molecular weight of 34 kDa. This band corresponded to one of the bands present in the gels containing <i>E. coli </i> peptidoglycan. This common autolysin was isolated by adsorbing it from <i>C. upsaliensis</i> lysates onto <i>M. luteus</i> cells and then subjecting these cells to renaturing SDS-PAGE in gels containing <i>E. coli</i> peptidoglycan. The 34 kDa autolysin differed from a single 51 kDa autolysin unique to the <i>M. luteus cells</i>. The 34 kDa autolysin was isolated from an SDS-PAGE gel and was pure when tested by isoelectric focusing. The N-terminal amino acid sequence analysis showed the first 15 amino acids of the 34 kDa autolysin to have 67% identity with a part of antigenic protein PEB4 of <i>Campylobacter jejuni</i>. The purified autolysin was used to immunize rabbits and the antibodies produced precipitated autolytic activity from cell lysates. The specificity of the antibodies was shown by Western blotting: only a single specific band occurred, with a molecular weight of 34 kDa, and thus it seems unlikely that the 34 kDa autolysin was derived from any of the other autolysins that were detected. / Ph. D.

Following active growth, the aquatic gram-negative rod Prolinoborus fasciculus (Aquaspirillum fasciculus) exhibited a mass conversion from its culturable rod form to a nonculturable coccoid form. Chloramphenicol did not prevent the conversion. Attempts to obtain variants that would not convert to the coccoid form were unsuccessful. Although the coccoid form fluoresced with acridine orange, agarose gel electrophoresis revealed extensive ribosomal RNA degradation. Poly-Ã -hydroxybutyrate, abundant in the vegetative rods, was not detectable in the coccoid cells. The results suggest that the coccoid form of A. fasciculus is a degenerative form rather than part of a life cycle. / Master of Science

Propionibacterium freudenreichii est une bactérie très utilisée par l’industrie laitière. Elle appartient aux Actinomycètes connus pour leur survie pendant de longues périodes, dans des conditions environnementales défavorables. Pour mieux comprendre ce phénomène, la caractérisation phénotypique de 8 souches de P. freudenreichii a été réalisée sur 11 jours dans un milieu en carence nutritionnelle. Le taux de survie bactérienne a été mesuré par densité optique, par énumération et évaluation de la viabilité cellulaire. En outre, l’absence de lyse cellulaire a été évaluée par PCR quantitative. La croissance de P. freudenreichii a été décrite en phases exponentielle, stationnaire, stationnaire tardive et survie à long terme.Dans nos conditions expérimentales pendant la période de survie à long terme, les bactéries sont restées viables. La caractérisation phénotypique a montré que P. freudenreichii CIRM-BIA138 était la plus résistante à la carence nutritionnelle et entrait dans un état viable mais non-cultivable. Cette souche a été utilisée pour une étude fonctionnelle par RNA-Seq ainsi que pour des analyses biochimiques sur les surnageants de culture, en phases exponentielle et stationnaire. L’association de ces données transcriptomiques et métabolomiques a permis de déduire les stratégies impliquées dans la survie de cette bactérie. La préparation à l’état de dormance, la diminution du métabolisme et l’utilisation de sources alternatives d’énergie semblent impliquées dans l’adaptation et la persistence de P. freudenreichii CIRM-BIA138 en carence nutritionnelle durant de long / Propionibacterium freudenreichii is a dairy bacterium belonging to the Actinobacteria group, which is known to survive for long periods in harsh environmental conditions. In order to investigate the long-term survival phenomenon in P. freudenreichii, 8 strains were phenotypically characterized for a period of 11 days in nutrient shortage condition. Bacterial survival rate was assessed by optical density, CFU counting and live-dead cellular viability. In addition, the absence of cell lysis was evaluated by quantitative PCR. P. freudenreichii growth phases were classified as exponential, stationary, late stationary and long-term survival. Moreover, it was observed that bacterial viability was maintained during long-term survival.Phenotypical characterization indicated that P. freudenreichii CIRM-BIA138 was more resistant to nutrient shortage being able to enter into a viable but nonculturable dormant state. In addition, functional studies of this strain were conducted by RNA-Seq on cultures sampled in exponential and stationary growth phases. Concomitantly, several biochemical analyses were carried out on the culture supernatant. An integrative approach of metabolomic and transcriptomic data allowed us to infer strategies associated with the survival of this bacterium, such as preparation for the dormant state, slow down of metabolic activity and utilization of alternative sources of energy, which altogether might allow P. freudenreichii CIRM-BIA 138 to adapt and persist through nutrient shortage for long periods.

Abstract The plant can be considered a superorganism that consists of the plant per se and numerous populations of pro- and eukaryotic microorganisms. The interactions between the plant and endophytic microorganisms colonizing plant internal tissues are typically commensalistic or mutualistic. However, information on the role of endophytes in plant defense is limited because pathways are only partly known and systemic responses are typically not seen. The aim of this thesis was to study the priming capacities of endophytic Methylobacterium sp. IMBG290 on potato (Solanum tuberosum L.). Priming of plants by non-pathogenic bacteria allows the host to save energy and to reduce time needed for development of defense reaction during a pathogen attack. Priming phenomenon was demonstrated for Methylobacterium sp. IMBG290 as an activation of salicylic acid and jasmonate/ethylene-dependent defense pathways after challenge inoculation with the pathogen. Moderate activation of plant antioxidant system may also contribute to resistance induction by the strain. The viable but nonculturable state is presumably a survival strategy observed for the majority of bacterial endophytes. Pathogen attack or environmental changes can activate these quiescent forms. Thus Methylobacterium+ sp. IMBG290 became cultivable upon plant inoculation by nonpathogenic bacteria. I observed that the composition of the endophyte community changed in response to Methylobacterium sp. IMBG290 inoculation in shoot tissues and correlated with potato disease resistance and growth promotion. Therefore, the activation of endophytic bacterial populations as a putative mechanism of plant disease resistance was proposed. Endophytes have a high agricultural potential. Growth- and resistance-promoting capacities of Methylobacterium sp. IMBG290 on potato were highly variable depending on the cultivar, pathogen, inoculum density, and environmental conditions. Context-dependent efficacy requires more attention when designing complex microbial inoculants capable influencing positively plant growth, resistance, and nutritional properties. / Tiivistelmä Kasvia voidaan pitää superorganismina, joka koostuu kasvista itsestään ja lukuisista pro-ja eukaryottisista mikrobipopulaatioista. Kasvin ja sen sisäosia asuttavien endofyyttisten mikro-organismien väliset vuorovaikutukset ovat yleensä kommensalistisia tai mutualistisia. Endofyyttien rooli kasvin puolustuksessa on kuitenkin huonosti tunnettu, koska reitit tiedetään vain osittain eikä järjestelmällisiä vasteita usein havaita. Tämän väitöskirjatyön tavoitteena oli tutkia endofyyttisen Methylobacterium sp. IMBG290-kannan kykyä vahvistaa perunan (Solanum tuberosum L.) puolustusta. Tautia aiheuttamattomat bakteerit kykenevät vahvistamaan kasvien puolustusta, mikä auttaa isäntäkasvia säästämään energiaa ja nopeuttamaan puolustusreaktiota patogeenihyökkäyksen aikana. Methylobacterium sp. IMBG290-kannan vahvistuskyvyn osoitettiin perustuvan salisyylihappo- ja jasmonaatti/etyleeni-riippuvaisten puolustusreittien aktivoimiseen patogeeni-istutuksen jälkeen. Antioksidanttijärjestelmän lievä aktivoituminen voi myös vaikuttaa kannan aiheuttamaan vastustuskyvyn lisääntymiseen. Suurimmalle osalle bakteeriendofyyteistä ‘elävä mutta viljelemätön’-olotila on luultavasti selviytymisstrategia. Patogeenihyökkäys tai muutokset ympäristössä voivat aktivoida tällaiset hiljaiset olomuodot. Methylobacterium sp. IMBG290-kanta muuttui viljeltävissä olevaan muotoon kun kasviin istutettiin tautia aiheuttamaton bakteeri. Selvitin, että endofyytti-yhteisön koostumus muuttuu vasteena Methylobacterium sp. IMBG290-kannan istuttamiseen kasvin verson solukoissa, korreloiden lisääntyneen perunan vastustuskyvyn ja kasvun kanssa. Siksi endofyyttisten bakteeripopulaatioiden aktivoitumista esitettiin uutena kasvin puolustusmekanismina. Endofyyteillä on suuret mahdollisuudet maataloudessa. Methylobacterium sp. IMBG290-kannan kasvua ja vastustuskykyä lisäävät ominaisuudet perunalla vaihtelivat lajikkeen, patogeenin, lisätyn bakteeriympin ja ympäristöolosuhteiden mukaan. Suunniteltaessa monimutkaisia bakteeriymppejä kasvien kasvin, vastustuskyvyn ja ravintosisällön lisäämiseksi, täytyy tällainen tilanteesta riippuva tehokkuus ottaa enemmän huomioon.

Les nouvelles pratiques utilisées pour l’élaboration du vin amènent à une recrudescence des altérations microbiennes. C’est pourquoi, de nouvelles méthodes doivent être développées afin de quantifier ces microorganismes de façon précise, rapide et avec de faibles coûts. Les principales altérations du vin sont dues aux bactéries acétiques (BA) (A. aceti, A. pasteurianus, G. oxydans et Ga. liquefaciens) et à Brettanomyces bruxellensis. Par l’action d’enzymes, les 1ères transforment l’éthanol en acide acétique alors que B. bruxellensis transforme les acides hydroxycinnamiques en éthyles phénols (EP) (molécules odorantes désagréables). La cytométrie en flux couplée à la technique d’hybridation in situ en fluorescence a tout d’abord été étudiée. Aucun résultat reproductible n’a été développé pour les BA en vin rouge alors que pour B. bruxellensis, le protocole existant a été amélioré avec une quantification possible en 18 h. La PCR en temps réel a également été utilisées afin de quantifier ces microorganismes. Un protocole a été développé pour la quantification des BA en vin rouge (103 cellules/mL) avec l’utilisation d’un témoin interne microbiologique permettant de valider le rendement de l’extraction de l’ADN. Pour B. bruxellensis, trois kits commerciaux ont été analysés lors d’une étude interlaboratoires. Les quantifications se sont révélées significativement différentes des énumérations sur boite de Pétri avec une quantification des cellules mortes. De plus, il a été étudié et validé l’effet population de B. bruxellensis sur l’efficacité du SO2. Il ressort également de ces expérimentations que les cellules en état viable mais non cultivable ne produisent pas d’EP. / New practices used to elaborate wine lead to an increase of wine spoilage due to microorganisms. That is why, new technics have to be developed to quantify these microorganisms accurately, quickly and with low costs. The main wine spoilages are due to acetic acid bacteria (AAB) (A. aceti, A. pasteurianus, G. oxydans and Ga. liquefaciens) and Brettanomyces bruxellensis development. AAB transforms ethanol to acetic acid while B. bruxellensis transforms hydroxycinnamic acids to ethyl phenols (EP) (unpleasant odor molecules). In order to detect these wine spoilage microrganisms, flow cytometry coupled to fluorescent in situ hybridization has been assessed. No reproducible results have been developed for AAB in red wine while for B. bruxellensis, the existing protocol has been improved with a possible quantification after 18 h compared to 48-72 h in the previous protocol. The real-time PCR was also used to quantify these microorganisms. A protocol has been developed for the AAB quantification in red wine (103 cells/mL) with the use of a microbiological internal control to validate the DNA yield after extraction. For B. bruxellensis, three commercial kits were analyzed in an interlaboratory study. Quantifications were significantly different to the enumerations by Petri dish, with dead cell quantifications. Moreover, we demonstrated that the effectiveness of sulfite is dependent of the B. bruxellensis population. It also appears from these experiments that cells in viable but not culturable state do not produce EP.