No description available.

No description available.

Enterococcus faecium è un componente fondamentale del microbiota di diversi alimenti fermentati quali formaggi e salumi e viene spesso isolato in alto numero in alimenti pronti al consumo. É inoltre largamente utilizzato come probiotico sia per l’uomo che per gli animali. Allo stesso tempo, però, questa specie batterica rappresenta una delle cause principali di infezioni nosocomiali quali endocarditi ed infezioni al tratto urinario. Studi recenti hanno dimostato che la specie E. faecium è costituita da due sub-popolazioni principali: la prima è denominate hospital associated (HA) clade “A” ed include la maggior parte dei ceppi responsabili di infezioni umane; la seconda è chiamata community associated (CA) clade “B”, e contiene principalmente ceppi commensali dell’uomo. Analisi più approfondite hanno rivelato un ulteriore suddivisione all’interno del clade A, nel sub-clade A1 (che raggruppa la maggioranza dei ceppi clinici) e nel sub-clade A2, associato agli animali e più sporadicamente ad infezioni umane. Nel 2012, EFSA ha redatto una linea guida per la valutazione della sicurezza di E. faecium usato come probiotico per gli animali, concludendo che i cepi appartenenti all’hospital-associated clade non devono essere utilizzati in nutrizione animale. Comunque, la distinzione tra le due sub-popolazioni è stata fatta utilizzando dati ottenuti prevalentemente da isolati umani e animali e solo un numero limitato di ceppi isolati dagli alimenti è stato considerato. Obiettivo di questa tesi di dottorato è stato quello di valutare la sicurezza di E. faecium negli alimenti fermentati, considerando ceppi isolati da formaggi artigianali e prodotti carnei e utilizzando sia tecniche di genomica che analisi fisiologiche. Nessuno dei ceppi alimentari studiati è risultato parte del clade A1, ma un ceppo isolato da un salame stagionato pronto al consumo ha rivelato diversi tratti tipici dei ceppi A1, tra cui particolari IS, transposase e geni di resistenza agli antibiotici. Questi risultati, così come altri dati, sottolineano la necessità di approfondire le conoscenze circa il ruolo dei ceppi di E. faecium isolati da alimenti come fattore di rischio per la salute umana. / Enterococcus faecium is commonly found in high numbers in ready to eat foods, being a member of the bacterial communities of a variety of fermented foods, including cheese and sausages, and is widely used as human and animal probiotic. However, this bacterial species is a leading cause of nosocomial infection, mainly endocarditis and urinary tract infections. Recent studies have demonstrated that E. faecium species consists of two very distinct clades: the hospital associated (HA) clade “A”, which includes most of the strains responsible for human infections, and the community associated (CA) clade “B”, that contains primarily human commensal isolates. Deeper analysis revealed a further split within clade A into sub-clade A1 (which groups the vast majority of clinical isolates), and sub-clade A2, associated with animals and sporadic human infections. In 2012, the European Food Safety Authority has issued a guideline for the safety assessment of E. faecium used as animal probiotics, concluding the strains belonging to the hospital-associated clade should not be used in animal nutrition. However, the differentiation of the two clades has been performed using data mainly deriving from human and animal isolates, and only a limited number of strains from the food chain were considered. Aim of this doctoral thesis was to assess the safety of E. faecium in fermented food, considering strains isolated from artisanal cheese and meat products, and using both whole genome-based techniques and physiological studies. None of the food isolates studied in this work belong to the epidemic clade A1, however a strain isolated from a ready to eat salami revealed several A1-specific traits, such as specific IS, transposases and antibiotic resistance genes. These results, as well as other data, underline the emergency of deeper understanding the role of E. faecium isolated from fermented foods as risk factor for human health.

Inledning: Antibiotikaresistens är ett av de största hoten mot global folkhälsa. Meticillinresistent Staphylococcus aureus, tidigare främst associerat med sjukhusvård, överförs nu mellan individer i samhället (samhällsförvärvad meticillinresistent staphylococcus aureus). Att identifiera riskfaktorer är centralt för att kunna bedriva effektivt preventivt arbete mot smittöverföring. Syfte: Syftet med uppsatsen var att identifiera och beskriva riskfaktorer associerade med förekomst av samhällsförvärvad meticillinresistent staphylococcus aureus. Metod: Litteraturstudie baserad på 20 internationella artiklar. Huvudfynden i artiklarna kategoriserades i teman. Resultat: Riskfaktorer på samhälls-, hushålls- och individnivå kunde identifieras, bland annat rörande klimat, tidigare antibiotikaanvändning och samsjuklighet.  Diskussion: Ett fåtal enkelt påverkbara riskfaktorer kunde identifieras. Av de identifierade riskfaktorerna är troligen inte alla generaliserbara till en svensk kontext. I flera tidigare studier framhålls att samhällsförvärvad meticillinresistent staphylococcus aureus främst drabbar individer som sedan tidigare är friska, vilket fynden i föreliggande uppsats delvis motsäger då samsjuklighet i exempelvis HIV, diabetes och fetma identifierades som riskfaktorer associerade med samhällsförvärvad meticillinresistent staphylococcus aureus. / Introduction: Antibiotic resistance a threat to global public health. Methicillin resistant staphylococcus aureus, previously primarily associated with hospital care, is now being transmitted in the community (community acquired methicillin resistant staphylococcus aureus). Identifying risk factors is central to enable effective preventive efforts against transmission of community acquired methicillin resistant staphylococcus aureus.   Aim: The aim of this essay was to identify and describe risk factors associated with occurrence of community acquired methicillin resistant staphylococcus aureus.    Methods: Literature study based on 20 international studies. The main results from the articles were categorized into themes. Results: Risk factors at community-, household- and individual level could be identified, for instance concerning climate, previous antibiotic treatment and comorbidity. Discussion: A few easily affectable risk factors could be identified. Perhaps not all of the identified risk factors are generalizable to a Swedish context. Previous research demonstrates that community acquired methicillin resistant staphylococcus aureus usually affects healthy individuals, which the findings in this essay partly contradict: comorbidity, for instance with HIV, diabetes or obesity, is a risk factor associated with community acquired methicillin resistant staphylococcus aureus.

The present study aimed to evaluate bacterial isolates and the microbiome of dairies. The specific aims were: to characterize Staphylococcus spp. isolated from mastitic milk, to evaluate the presence of Lactococcus in mastitic milk as a potential causative agent of mastitis, to evaluate the association between microbiome and milk quality parameters, and to characterize Staphylococcus spp. isolated from production lines of Minas Frescal cheese. The detection of genes encoding virulence factors (enterotoxins (sea, seb, sec, sed, see, seg, seh, sei, selj, selk, sell, selm, seln, selo, selp, seIq, ser, ses, set, selu, selv, and selx), hemolysins (hla, hlb, hld, hlg, and hlgv), exfoliative toxins (eta, etb, and etd), Panton-Valentine leukocidin (pvl), and toxic shock syndrome toxin (tst)), genes encoding antibiotic resistance (resistance to tetracycline (tetK, tetL, and tetM), erythromycin (ermA, ermB, and ermC), methicillin (mecA and mecC), and tobramycin (ant(4\')-Ia)), molecular typing (spa, SCCmec, and agr types), and phenotyping regarding antibiotic resistance were performed in staphylococci isolates from mastitic milk, and from cheese processing plant samples. Staphylococcus aureus was identified in the majority of isolates from both origins. Several virulence factor genes were detected. The distribution of genes encoding staphylococcal enterotoxins (85.0% - 85.7% of isolates were positive for one or more enterotoxin gene) was highlighted and the gene related to H toxin was the most prevalent. Methicillin-resistant Staphylococcus aureus were identified in isolates from mastitic milk (4.1%) and cheese processing (6.0%); the genotyping and phenotyping of these isolates were described. t605 had the highest frequency in the S. aureus population studied. In mastitic milk, Lactococcus was suggested as the causative agent of an outbreak of mastitis in a dairy farm. Using next generation sequencing, the abundance of Lactococcus was observed in microbiome samples. Bacterial isolation and DNA sequencing confirmed the presence of Lactococcus lactis and Lactococcus garvieae. The microbiome of environmental samples and bulk tank milk from the dairy farm showed the Lactococcus genus among the most common bacterial taxa, suggesting other sources of this genus. Regarding milk quality parameters, the microbiome of bulk tank milk from several dairy farms was associated with somatic cell count and bacterial count. The core microbiome was described and many genera of importance were identified. Among the associations performed between microbiome and milk quality parameters, the identification of Streptococcus in samples classified with high somatic cell count and high bacterial count was highlighted. Several bacterial taxa with relative abundance significantly higher in samples classified as high and low cell count and bacterial count were shown. Real-time polymerase chain reaction was also performed associated with bacterial diversity, bacterial taxa, and bacterial count. These findings highlight the need to control and prevent bacterial contamination in the dairy industry, from herd to consumers. / O presente estudo apresentou como objetivo avaliar isolados bacterianos e microbioma de lácteos. Os objetivos específicos foram: caracterizar Staphylococcus spp. isolados de leite de vacas com mastite, avaliar a presença de Lactococcus em leite de vacas com mastite como um potencial agente causador de mastite, avaliar a associação entre microbioma de leite de tanque e parâmetros da qualidade de leite, e caracterizar Staphylococcus spp. isolados de linhas de processamento de queijo Minas frescal. A detecção de genes codificadores de fatores de virulência (enterotoxinas (sea, seb, sec, sed, see, seg, seh, sei, selj, selk, sell, selm, seln, selo, selp, seIq, ser, ses, set, selu, selv, e selx), hemolisinas (hla, hlb, hld, hlg, e hlgv), toxinas exfoliativas (eta, etb e etd), leucocidina de Panton-Valentine (pvl), toxina da síndrome do choque tóxico (tst)), genes codificadores de resistência a antibióticos (resistência a tetraciclina (tetK, tetL e tetM), eritromicina (ermA, ermB e ermC), meticilina (mecA e mecC) e tobramicina (ant(4\')-Ia)), tipagem molecular (spa, SCCmec e agr types), e fenotipagem quanto à resistência a antibióticos foram realizadas em estafilococos isolados de leite de vacas com mastite e de amostras de planta de processamento de queijo. Staphylococcus aureus foi identificado na maioria dos isolados de ambas as origens. Diversos genes de fatores de virulência foram detectados, com destaque para a distribuição de genes codificadores de enterotoxinas estafilocócicas (85,0%-85,7% dos isolados foram positivos para um ou mais genes codificadores de enterotoxinas), sendo o gene relacionado com a toxina H o mais frequente. Staphylococcus aureus meticilina resistente foram identificados em isolados de leite de vacas com mastite (4.1%) e em processamento de queijo (6.0%); o perfil genotípico e fenotípico destes isolados foram descritos. t605 foi o mais freqüente na população de S. aureus estudada. Em leite de vacas com mastite, Lactococcus foi sugerido como o agente causador de um surto de mastite numa fazenda leiteira. Usando sequenciamento de nova geração, a abundância de Lactococcus foi observada no microbioma das amostras. O isolamento e sequenciamento de DNA confirmaram a presença de Lactococcus lactis e Lactococcus garvieae. O microbioma de amostras ambientais e de leite de tanque da fazenda mostrou o gênero Lactococcus entre os mais comuns, sugerindo outras fontes deste gênero. Contemplando parâmetros da qualidade de leite, o microbioma de leite de tanque de várias fazendas leiteiras foi relacionado com contagem de células somáticas e contagem bacteriana. O core microbiome foi descrito e muitos gêneros bacterianos de importância foram identificados. Dentre as análises realizadas associando microbioma com parâmetros da qualidade de leite, foi destacada a identificação de Streptococcus em amostras classificadas com alta contagem de células somáticas e alta contagem bacteriana. Diversos táxons bacterianos com abundância relativa significativamente maior em amostras classificadas com alta e baixa contagem de células somáticas e contagem bacteriana foram mostrados. Reação em cadeia da polimerase em tempo real também foi realizada e associada com diversidade bacteriana, táxons bacterianos e contagem bacteriana. Estes levantamentos confirmam a necessidade de controlar e prevenir a contaminação bacteriana na indústria de lácteos, do rebanho leiteiro até os consumidores.

Tee PhD thesis presents the study of the response of Mycobacterium tuberculosis, the causative agent of tuberculosis, upon prolonged exposure to lethal concentrations of the first line anti-tuberculosis drug, rifampicin. The study shows that prolonged exposure to lethal concentration of rifampicin causes cell death initially by several log orders of cells, followed by a persistence phase, from where rifampicin resisters emerge carrying mutation in the RRDR locus of the rpoB gene. This phenomenon was found to occur even when the drug concentration is well above MBC levels. Luria-Delbruck experiment, in a modified format, showed that the resisters emerged as fresh mutants, and not due to the growth of pre-existing natural resisters to rifampicin. The per sister cells, which showed high levels of hydroxyl radical generation, were found to have thickened outer layer, unlike the mid-log phase cells, which restricts the permeability of a fluorescent-conjugate of rifampicin, 5-FAM-rifampicin, 10-fold less in per sister cells, as compared to mid-log phase cells. The thickened outer layer has high negative surface charge and is hydrophilic in nature. It is proposed that the hydrophilic-natured thickened outer layer might have restricted the permeability of lethal concentrations of hydrophobic-natured rifampicin. This, in turn, might have ensured the presence of sub-lethal concentration rifampicin inside the per sisters, which in turn might have generated the hydroxyl radical that caused mutagenesis to generate rifampicin resisters. The Chapter 1 is the Introduction to the thesis presented in 4 parts – Part 1.1, 1.2, 1.3 and 1.4, introducing briefly the history of antibiotics, antibiotic resistance, antibiotic persistence and a brief history of tuberculosis respectively. It is concluded with a rationale behind the present study. The Chapter 2 presents the entire Materials and Methods used in the experiments described in the thesis. The Chapter 3 presents the data on the response of M. tuberculosis to rifampicin upon extended exposure. Rifampicin exposure of susceptible M. tuberculosis H37Ra cells showed a decrease in their CFU/ml followed by a persistence phase wherein the CFU/ml remained constant. However, prolonged exposure of rifampicin even at higher concentrations showed regrowth in the culture, which was found to be due to the emergence of rifampicin-resistant bacteria. Screening of rifampicin-resistant mutants showed point mutations in the rifampicin resistance determining region (RRDR) of the rpoB gene in all the mutants. In parallel, using trans formants of M. tuberculosis expressing unstable GFP under the respective native ribosomal RNA promoter, the metabolic status of the per sister cells was determined. When actively growing highly fluorescing cells were exposed to lethal concentration of rifampicin, their metabolism diminished, as illustrated by the decrease in their fluorescence during persistence phase, followed by the emergence of a sub-population of bacteria which were again metabolically active. In order to verify whether the rifampicin resisters are freshly formed mutants or have come from the naturally existing resisters, Luria-Delbruck fluctuation test was performed in a modified manner. The number of rifampicin resisters that emerged from the persistent phase was found to vary amongst different cultures from different days and different times of exposure, showing fluctuation. However, the addition of theorem before persistence phase almost abolished the generation of the rifampicin-resistant bacilli, indicating the role of hydroxyl radical in the emergence of rifampicin resisters. The generation of hydroxyl radical in mycobacteria exposed to rifampicin was confirmed using electron para-magnetic resonance spectrometry (EPR), with the spin trap agent, 5,5- Dimethyl-1-pyrroline N-oxide (DMPO) specific for hydroxyl radical. An increase in the formation DMPO-OH adduct in the persistence phase cells was observed, in comparison to mid-log phase cells. Exposure to theorem significantly diminished the adduct formation. The persistence phase cells also showed significantly high levels of signal specific to the hydroxyl-specific fluorescent dye, hydroxyphenyl fluorescein (HPF), as compared to the mid-log phase cells. In addition we have determined the oxidative stress in the bacilli upon rifampicin exposure using a redox biosensor (Mrx1-roGFP) which also showed high oxidative stress in the persistent phase. These observations confirmed the presence of high levels of oxidative stress and hydroxyl radical in the rifampicin persistent cells, in comparison with mid-log phase cells. Whole genome sequencing of the four independently isolated rifampicin resistant M. tuberculosis showed genome wide mutations having less common mutations with respect to the wild type genome, indicative of the occurrence of random mutagenesis. In addition mutation frequencies were comparable between the samples with respect to the wild type sample. About 69% of the mutations were A-C or T-G, followed by A-T or T-A, which is known to be due to oxidative stress in the cells. Variations in the colony morphology were also observed on the persistent phase cells, indicating the occurrence of mutagenesis in the bacterial genome during rifampicin treatment. The Chapter 4 is on the morphological and ultrastructural studies on rifampicin-exposed M. tuberculosis cells. Transmission electron micrographs of rifampicin per sisters showed significant thickening of the outermost capsular layer (OL), as compared to the mid-log phase cells. This observation was verified by staining the cells with ruthenium red, which specifically stains anionic polysaccharide of the OL. Zeta potential (ZP) measurement of the surface charge of the persistent cells showed high negative ZP, as compared to the mid-log phase cells. The negative ZP value was found to gradually increase during the course of rifampicin treatment and to decrease during the regrowth phase. Hexadecane assay showed larger proportion of per sister cells being retained in the aqueous phase, as compared to the mid-log phase cells. This indicated the higher hydrophilicity of the per sister cells, which was in agreement with the higher surface negative charge of the cells. The permeability of rifampicin per sister cells to 5-FAM-rifampicin (rifampicin conjugated to 5-carboxy fluorescein, which is as hydrophobic as rifampicin) was found to be 10-fold less than that of the mid-log phase cells. However, removal of the thick OL by bead beating (BB) with 4 mm glass beads significantly improved the permeability of per sister cells to 5-FAM-rifampicin. On the contrary, no difference in the 5-FAM-rifampicin uptake was observed in mid-log phase cells, with or without BB. These observations implied that the thick hydrophilic-natured OL with high negative surface charge may be playing a significant role in limiting the permeability of hydrophobic-natured rifampicin entry into the persisted cells. This in turn may ensure the presence of sub-lethal concentration of rifampicin inside the persisted cells that are exposed to lethal concentration of the antibiotic. Exposure of bacteria to sub-lethal concentration of antibiotics has been reported to generate oxidative stress in the bacteria, leading to mutagenesis. A model has been proposed based on these observations in which the persistent mycobacteria are protected from lethal concentrations of the rifampicin by the thick OL which in turn ensures sub-lethal intracellular antibiotic concentration, leading to the generation of hydroxyl radical mediated mutagenesis and thereby emergence of rifampicin resisters. This thesis is concluded with the list of salient findings, publications and references.

La perméabilité membranaire joue un rôle important dans la résistance aux antibiotiques chez lesbactéries à Gram négatif.L’objectif de notre travail était de caractériser la fonction tenue par les deux régulateurs globaux dela perméabilité membranaire chez Enterobacer aerogenes: mar et ram. L’objectif initial futd’identifier le répresseur spécifique de RamA qui manquait en tant qu’élément de la cascade derégulation actuellement définie. La sélection de 60 souches nous a permis de confirmer le rôlecentral joué par RamA dans la régulation, ainsi qu’identifier des mutations pouvant être critiques, auniveau de RamR. Ainsi, l’absence variations observées dans le régulon marRAB et l’expressionmodérée des transcrits montrée par qRT-PCR laisse penser, que RamA a un rôle clef dans larégulation de l’expression des porines et des pompes d’efflux chez E. aerogenes.L’autre partie de notre travail reposait sur l’étude de la translocation des antibiotiques au traversdes porines. L’étude des interactions porine-carbapénèmes s’est faite sur la porine sauvage OmpFd'Escherichia coli et deux mutations. Les résultats indiquent également l'importance de l'aspartateen position 113 dans la sélectivité de translocation des carbapénèmes au sein de la porine OmpF.Ce travail montre ainsi que la translocation des pénicillines est aussi sous la dépendance desinteractions qui se créent entre le substrat et le résidu en position 113 de OmpF et limitent alorsleur passage au niveau du canal porine. Nous avons recherché la contribution attribuée à la porineOmp36 d'E. aerogenes dans la translocation de certaines béta-lactamines. Les mesures ont permisde conclure que les deux beta-lactamin / Genetic permeability plays an important role in antibiotic resistance of Gram-negative bacteria.Our work was to characterize and better understand of the genetic regulation of membranepermeability in E. aerogenes. We focused on two global regulators, mar and ram, in about 60clinical isolates. Alterations in the upstream region of ramA and in ramR but no mutations in marAnor marR were observed. Overexpression of ramA or ramR led to an altered antibiotic susceptibilityassociated to decrease of porins expression and over-expression of efflux-pumps. qRT-PCR pointedout the estimated importance of the ram-regulon in the regulation cascade.Another part of this work was to characterize the translocation of compounds through porins andthe role of porins in drug uptake in general. Measurement of the rate of antibiotic action of threecarbapenems in an E. coli strain solely expressing OmpF as porin clearly indicated the importanceof the aspartate at position 113 in antibiotic translocation. A multi-disciplinary three way approachof computer modeling, black-lipid-bilayer assays and measurement of antibiotic action, suggestedthat interactions with residue D113 of E. coli porin OmpF are rate-limiting for transport throughthe porin channel. Combination of biological and biophysical measurements with E. aerogenesporin Omp36 denoted that interactions between the porin channel and the antibiotic facilitate andaccelerate transport.

Pseudomonas aeruginosa is a Gram-negative, metabolically versatile, opportunistic pathogen that exhibits a multitude of virulence factors, and is extraordinarily resistant to a gamut of clinically significant antibiotics. This ability is in part mediated by two-component systems (TCS) that play a crucial role in regulating virulence mechanisms, metabolism and antibiotic resistance. Our sequence analysis of the P. aeruginosa PAO1 genome revealed the presence of two open reading frames, mifS and mifR, which encodes putative TCS proteins, a histidine sensor kinase MifS and a response regulator MifR, respectively. This two-gene operon was found immediately upstream of the poxAB operon, where poxB encodes a chromosomal ß-lactamase, hinting at the role of MifSR TCS in regulating antibiotic resistance. However, loss of mifSR had no effect on the antibiotic resistance profile when compared to P. aeruginosa parent PAO1 strain. Subsequently, our phenotypic microarray data (BioLOG) and growth profile studies indicated the inability of mifSR mutants to grow in α-ketoglutarate (α-KG), a key tricarboxylic acid (TCA) cycle intermediate, as a sole carbon source. To date, very little is known about the physiology of P. aeruginosa when provided with α-KG as its sole carbon source and the role of MifS and MifR TCS in virulence. Importantly, in the recent years, α-KG has gained notoriety for its newly identified role as a signaling molecule in addition to its conventional role in metabolism. This led us to hypothesize that MifSR TCS is involved in α-KG utilization and virulence in P. aeruginosa. Using mifS, mifR and mifSR clean in-frame deletion strains, our study demonstrates that the MifSR TCS modulates the expression P. aeruginosa kgtP (PA5530) and pcaT (PA0229) genes encoding putative α-KG permeases. In addition, our study shows that the MifSR-regulation of these transporters requires functional sigma factor RpoN (σ54). Loss of mifSR in the presence of α-KG, resulted in differential regulation of P. aeruginosa key virulence determinants including biofilm formation, motility, cell cytoxicity and the production of pyocyanin and pyoverdine. Involvement of multiple regulators and transporters suggests the presence of an intricate circuitry in the transport of α-KG and its importance in P. aeruginosa survival. This is further supported by the α-KG-dependent MifSR regulation of multiple virulence mechanisms. Simultaneous regulation of multiple mechanisms involved in P. aeruginosa pathogenesis suggests a complex mechanism of MifSR action. Understanding the physiological cues and regulation would provide a better stratagem to fight often indomitable P. aeruginosa infections.

No description available.

MSc (Microbiology) / Department of Microbiology / See the attached abstract below