No description available.

Staphylococci are opportunistic pathogens responsible for a range of infections. Many staphylococcal species are frequently found to be resistant to antibiotics. The environment is considered a potential reservoir of genes conferring antibiotic resistance, which known as the 'resistomes'. Monitoring the dissemination of antibiotic resistant staphylococci is instrumental to mitigating this global health risk. The overall aim of this study was to generate informative data regarding dissemination of antibiotic resistance in environmental and public settings. This included looking into the distribution, epidemiology characteristic and transfer of oxacillin resistant determinant mecA; gaining an insight into genomic features that contribute to multiple antibiotic resistance and pathogenicity of one S. epidermidis isolate; and understanding the stress responses in mediating oxacillin resistance in S. aureus. The use of MALDI-TOF MS allowed identification of staphylococci to species level. MALDI-TOF MS data were used for taxonomic analysis of staphylococci, and taxonomic data were then combined with isolation sites and antimicrobial susceptibility profiles to aid the understanding of dissemination of environmental resistant staphylococci. The widespread dissemination of antibiotic resistant staphylococci in the environment was demonstrated. 12% of staphylococci harboured mecA gene. Community associated SCCmec types IV and V were more prevalent than nosocomial associated SCCmec types I, II, and III in the environment. 52% of SCCmec were non-typable. In addition, 14 new environmental S. epidermidis MLST types were reported. 9 antibiotic resistant determinants that were responsible for the resistant to 7 antimicrobial classes have been identified in environmental S. epidermidis 118 (G6_2). Proteomic analysis revealed that stress responses, including SOS response, stringent response and heat shock response, mediate oxacillin resistance in S. aureus. These results demonstrate widespread multiple drug resistance in different staphylococcal species isolated from non-healthcare environments. This uncontrolled dissemination of multidrug resistant bacteria poses a potential public health threats.

For this work, the decision was made to contrast two serological tests, that of the tube agglutination technique and the fluorescent antibody technique for correlation with the coagulase and other characteristics of Staphylococcus strains. This has been a preliminary survey in the hope that as further knowledge is obtained about the staphylococci, grouping of the organisms will become more routine and relatively less complex.

The Staphylococcus genus comprises a diverse group of Gram-positive bacteria that are opportunistic pathogens of humans and other mammals. S. epidermidis and S. aureus are the most common human pathogens of the staphylococci, causing a variety of infections including biofilm-based medical device infections, skin infections, and pneumonia. Both of these organisms produce proteases whose functions in virulence are not fully characterized. In S. epidermidis, protein-mediated biofilm formation requires a cell wall-anchored adhesin called Aap that must be proteolytically processed in order to allow intercellular adhesion. The S. epidermidis protease(s) responsible for cleaving Aap were unknown. Chapter II describes our findings that the secreted metalloprotease SepA is required for Aap-mediated biofilm formation and cleaves Aap at two different sites. Further, this protease is negatively regulated by the global regulator SarA. Chapter III discusses studies of the S. aureus Spl (serine protease-like) proteases. Although they are produced in vivo, their substrates and role in virulence are unknown. We found that in a rabbit model of pneumonia, a mutant lacking the spl protease operon caused more localized disease compared to wild type S. aureus. Proteomics studies of the secreted and surface proteins in wild type compared to spl mutant S. aureus revealed several changes. We also found that the SplA protease cleaves human Mucin-16, the first identification of a biological substrate of the Spls. Finally, we found that the animal-associated species S. caprae produces an autoinducing peptide (AIP) that is a potent inhibitor of S. aureus quorum sensing. We identified the S. caprae AIP structure as an 8-residue thiolactone ring. A synthetic version of the peptide inhibits S. aureus virulence and quorum sensing induction in a murine skin infection model. This is a novel example of quorum sensing cross talk between staphylococcal quorum sensing systems. These studies are described in Appendix A. On the whole, this work identified two substrates of S. aureus proteases and demonstrated their importance in biofilm formation and infection. We also characterized a novel inhibitor of S. aureus quorum sensing that attenuates virulence. These findings shed light on the importance of staphylococcal secreted proteases and quorum sensing cross talk in the modulation of virulence factor production and the ability to cause disease.

Les staphylocoques sont des bactéries commensales de la peau et des muqueuses. On distingue Staphylococcus aureus qui est un pathogène virulent bien connu, des staphylocoques à coagulase négative (SCoN), qui sont de plus en plus impliqués dans les infections sur matériel et multirésistants aux antibiotiques. Le linézolide (LZD) est un antibiotique de la classe des oxazolidinones qui inhibent la synthèse protéique au niveau de la SU ribosomale 50S. La résistance est liée à des mutations dans l’ADNr23S (rrl) et les protéines ribosomales ainsi qu’à l’acquisition des gènes cfr et optrA. L’objectif de la thèse était d’étudier la cinétique d’acquisition des mécanismes de résistance au LZD chez les staphylocoques. Nous avons montré in vitro chez les SCoN, que la résistance était liée à une accumulation des différents mécanismes de résistance avec une cinétique d’acquisition identique commençant par l’apparition de mutations dans les protéines ribosomales facilitant possiblement l’acquisition secondaire de mutation dans rrl. Nous avons décrit in vivo chez un patient atteint de mucoviscidose sur 5 ans que l’émergence de la résistance au LZD chez S. aureus pouvait être rapide et de haut niveau sous traitement, avec une adaptation du niveau de résistance dans le temps. Nous avons pu décrire les différentes adaptations liées à la résistance au LZD, telle que la perte d’une copie d’opéron ribosomique, ainsi que le développement d’une dépendance partielle au LZD liée à la présence du gène cfr associé à d’autres mécanismes de résistance. En conclusion, nous avons montré le rôle facilitant de certaines mutations dont le dépistage pourrait prévenir l’émergence de résistance sous traitement ainsi que le rôle de cfr dans la dépendance expliquant potentiellement sa dissémination. Enfin le rôle de mutations apparues dans des gènes non liés directement à la résistance lors de l’émergence de celle-ci devra être précisé. / Staphylococci are commensal bacteria from skin and mucous membrane. Staphylococcus aureus a well-known virulent species could be distinguish from coagulase negative staphylococci (CoNS) which are increasingly implied in indwelling devices infections and are frequently multiresistant to antibiotics. Linezolid (LZD) is an antibiotic of the oxazolidinone family inhibiting protein synthesis by binding to the 50S ribosomal SU. Resistance is related to mutations in 23S rDNA (rrl) and in ribosomal protein as well as to the acquisition of the cfr and optrA genes. We aimed to investigate resistance emergence timeline in staphylococci. We reported in vitro that resistance in CoNS was due to accumulation of resistance mechanism with a similar timeline of acquisition including firstly resistance in ribosomal proteins that probably facilitated second mutation in 23S rRNA. Our in vivo study in a cystic fibrosis patient over a 5 years that LZD resistance emergence in S. aureus could be fast with a high level under treatment and with resistance level adaptation over time. We also described different consequences of LZD resistance such as the loss of a copy of the ribosomal operon, but also the presence of a partial LZD dependence linked to the cfr gene associated with others mutations. In conclusion, we showed the facilitating role of ribosomal mutation whose screening could prevent the emergence of resistance as well as the role of the cfr gene in LZD dependence thus potentially explaining its dissemination. Finally, the role of mutations appeared in genes not directly linked to the resistance during the emergence of it should be further studied.

Staphylococcus lugdunensis and Staphylococcus schleiferi, two newly described species, have been isolated from numerous types of human infections. We compared the pathogenicity of 30 strains of S. lugdunensis, S. schleiferi, Staphylococcus epidermidis, Staphylococcus warneri, and Staphylococcus hominis, using a mouse model in which a foreign body preadhered with the test strain was implanted subcutaneously, followed by injection of the test strain. All five species of staphylococci produced abscesses. Staphylococcus epidermidis, S. schleiferi, and S. lugdunensis yielded species means of 76-91% abscess formation; 80-100% of the infected foreign bodies and tissues were culture positive. These three species were more virulent than S. warneri or S. hominis, which produced abscesses in 54 and 65% of mice, respectively; only 10-48% of the infected samples were culture positive. Transmission electron microscopy of pure cultures of selected strains showed that all species possessed glycocalyx. All species produced a variety of possible virulence factors, such as α and δ hemolysins, as well as the aggressins lipase and esterase. The production of exoenzymes did not always correlate with virulence as demonstrated by abscess formation in mice.

Marine sponge-associated actinomycetes are reservoirs of diverse natural products with novel biological activities. Their antibiotic potential has been well explored against a range of Gram positive and negative bacteria. However, not much is known about their anti-infective or anti-virulence potential against human pathogens. This Ph.D. project aimed to investigate the anti-infective (anti-Shiga toxin and anti-biofilm) potential of sponge-derived actinobacteria through identification and isolation of their bioactive metabolites produced and characterizing their mechanism of action by transcriptomics. This thesis is divided into three studies with the overall objective of exploring the anti-infective efficacy of actinomycetes-derived extracts and compound(s) that could possibly be used as future therapeutics. The first study deals with investigation on the anti-Shiga toxin effects of sponge-associated actinomycetes. Diarrheal infections pose a huge burden in several developing and developed countries. Diarrheal outbreaks caused by Enterohemorrhagic Escherichia coli (EHEC) could lead to life-threatening complications like gastroenteritis and haemolytic uremic syndrome (HUS) if left untreated. Shiga toxin (Stx) produced by EHEC is a major virulence factor that negatively affects the human cells, leading them to death via apoptosis. Antibiotics are not prescribed against EHEC infections since they may enhance the risk of development of HUS by inducing the production and release of Stx from disintegrating bacteria and thereby, worsening the complications. Therefore, an effective drug that blocks the Stx production without affecting the growth needs to be urgently developed. In this study, the inhibitory effects of 194 extracts and several compounds originating from a collection of marine sponge-derived actinomycetes were evaluated against the Stx production in EHEC strain EDL933 with the aid of Ridascreen® Verotoxin ELISA assay kit. It was found that treatment with the extracts did not lead to significant reduction in Stx production. However, strepthonium A isolated from the culture of Streptomyces sp. SBT345 (previously cultivated from the Mediterranean sponge Agelas oroides) reduced the Stx production (at 80 μM concentration) in EHEC strain EDL933 without affecting the bacterial growth. The structure of strepthonium A was resolved by spectroscopic analyses including 1D and 2D-NMR, as well as ESI-HRMS and ESI-HRMS2 experiments. This demonstrated the possible application of strepthonium A in restraining EHEC infections. VI In the second study, the effect of marine sponge-associated actinomycetes on biofilm formation of staphylococci was assessed. Medical devices such as contact lenses, metallic implants, catheters, pacemakers etc. are ideal ecological niches for formation of bacterial biofilms, which thereby lead to device-related infections. Bacteria in biofilms are multiple fold more tolerant to the host immune responses and conventional antibiotics, and hence are hard-to-treat. Here, the anti-biofilm potential of an organic extract derived from liquid fermentation of Streptomyces sp. SBT343 (previously cultivated from the Mediterranean sponge Petrosia ficiformis) was reported. Results obtained in vitro demonstrated its anti-biofilm (against staphylococci) and non-toxic nature (against mouse macrophage (J774.1), fibroblast (NIH/3T3) and human corneal epithelial cell lines). Interestingly, SBT343 extract could inhibit staphylococcal biofilm formation on polystyrene, glass and contact lens surfaces without affecting the bacterial growth. High Resolution Fourier Transform Mass Spectrometry (HR-MS) analysis indicated the complexity and the chemical diversity of components present in the extract. Preliminary physio-chemical characterization unmasked the heat stable and non-proteinaceous nature of the active component(s) in the extract. Finally, fractionation experiments revealed that the biological activity was due to synergistic effects of multiple components present in the extract. In the third study, anti-biofilm screening of 50 organic extracts generated from solid and liquid fermentation of 25 different previously characterized sponge-derived actinomycetes was carried out. This led to identification of the anti-biofilm organic extract derived from the solid culture of Streptomyces sp. SBT348 (previously cultivated from the Mediterranean sponge Petrosia ficiformis). Bioassay-guided fractionation was employed to identify the active fraction Fr 7 in the SBT348 crude extract. Further purification with semi-preparative HPLC led to isolation of the bioactive SKC1, SKC2, SKC3, SKC4 and SKC5 sub-fractions. The most active sub-fraction SKC3 was found to be a pure compound having BIC90 and MIC values of 3.95 μg/ml and 31.25 μg/ml against S. epidermidis RP62A. SKC3 had no apparent toxicity in vitro on cell lines and in vivo on the greater wax moth Galleria melonella larvae. SKC3 was stable to heat and enzymatic treatments indicating its non-proteinaceous nature. HR-MS analysis revealed the mass of SKC3 to be 1258.3 Da. Structure elucidation of SKC3 with the aid of 1D and 2D-NMR data is currently under investigation. Further, to obtain insights into the mode of action of SKC3 on S. epidermidis RP62A, RNA sequencing was done. Transcriptome data revealed that SKC3 was recognized by RP62A at 20 min and SKC3 negatively interfered with the central metabolism of staphylococci at 3 h. Taken VII together, these findings suggest that SKC3 could be a lead structure for development of new anti-staphylococcal drugs. Overall, the results obtained from this work underscore the anti-infective attributes of actinomycetes consortia associated with marine sponges, and their applications in natural product drug discovery programs. / Meeresschwamm-assoziierte Actinomyceten stellen ein Reservoir für verschiedene natürliche Produkte mit neuartigen biologischen Aktivitäten dar. Ihr antibiotisches Potenzial gegenüber einer Reihe von Gram-negativen und -positiven Bakterien ist bereits intensiv erforscht worden. Wenig ist allerdings über ihre antiinfektive und antivirulente Wirksamkeit gegenüber menschlichen Pathogenen bekannt. Ziel dieser Doktorarbeit war es, die antiinfektiven Fähigkeiten (anti-Shiga-Toxin und anti-Biofilm) der aus Schwämmen isolierten Actinobakterien zu untersuchen. Hierfür wurden bioaktive Metabolite der Actinobakterien identifiziert und isoliert und abschließend wurde ihr Wirkmechanismus mit Hilfe einer Transkriptomanalyse charakterisiert. Diese Arbeit ist in drei Studien gegliedert, welche alle zum Ziel hatten die antiinfektive Wirksamkeit von aus Actinomyceten gewonnenen Extrakten und Komponente(n), welche möglicherweise als zukünftige Therapeutika dienen könnten, zu untersuchen. Die erste Studie befasst sich mit den anti-Shiga-Toxin Effekten der Meeresschwamm- assoziierten Actinomyceten. Durchfallinfektionen stellen in vielen Entwicklungsländern aber auch in Industrieländern eine große Gefahr dar. Durchfallerkrankungen die durch enterohämorrhagische Escherichia coli (EHEC) hervorgerufen werden, können sich zu lebensbedrohlichen Komplikationen wie Gastroenteritis oder dem hämolytisch urenischen Syndrom (HUS) weiterentwickeln. Das von den EHEC Stämmen produzierte Shiga-Toxin (Stx) stellt hierbei den Haupt Virulenz Faktor dar, welcher die eukaryotische Proteinsynthese menschlicher Zellen negativ beeinflusst, was wiederum den Zelltod durch Apoptose zur Folge hat. Die Behandlung der EHEC-Patienten mit Antibiotika wird nicht empfohlen, da dies zu einem Anstieg von freigesetztem Stx der zersetzen Bakterien führen könnte, wodurch das Risiko für die Entwicklung des HUS ansteigt. Aus diesem Grund werden effektive Medikamente dringen benötigt, welche die Stx Produktion blockieren ohne das Wachstum der Bakterien zu beeinflussen. In dieser Studie wurden 194 Extrakte und einige isolierte Komponenten von aus Schwämmen gewonnenen Actinomyceten auf ihren negativen Einfluss auf die Stx Produktion des EHEC Stammes EDL933 mit der Hilfe des Ridascreen® Verotoxin ELISA Kits untersucht. Es konnte gezeigt werden, dass die Zugabe der Extrakte keinen signifikanten Einfluss auf die Stx Produktion hatte. Strepthonium A auf der anderen Seite, welches aus Streptomyces sp. SBT345 isoliert wurde (vom mediterranen Schwamm Agelas oroides) konnte die Stx Produktion von EDL933 bei einer Konzentration von 80 µM reduzieren ohne das Wachstum des EHEC Stammes zu beeinflussen. Die Struktur von Strepthonium A wurde mittels spektroskopischer Analyse (1D- und 2D-NMR), sowie mittels ESI-HRMS und ESI-HRMS2 Experimenten entschlüsselt. Basierend auf diesen Ergebnissen könnte Strepthonium A eine mögliche Alternative oder Zusatz in der Behandlung einer EHEC Infektion darstellen. In der zweiten Studie wurde der Einfluss der Meeresschwamm-assoziierten Actinomyceten auf die Biofilmbildung von Staphylokokken bewertet. Medizinische Produkte wie Kontakt Linsen, metallische Implantate, Katheter, Herzschrittmacher, usw. stellen optimale ökologische Nischen für die Ausbildung von bakteriellen Biofilmen dar, wodurch Infektionen im Menschen hervorgerufen werden können. Bakterien in einem Biofilm sind deutlich toleranter gegenüber der Immunantwort ihres Wirtes sowie gegenüber konventionellen Antibiotika und sind daher schwer zu bekämpfen. In dieser Studie wurde das anti-Biofilm Potential eines organischen Extrakts der flüssigen Fermentation von Streptomyces sp. SBT343 (vom mediterranen Schwamm Petrosia ficiformis) ermittelt. In vitro Ergebnisse zeigten, dass das organische Extrakt anti-Biofilm (gegenüber Staphylococci) Fähigkeiten besitzt und nicht toxisch für Maus Makrophagen (J774.1), Fibroblasten (NIH/3T3) und humane korneale Epithelzellen ist. Zudem konnte gezeigt werden, dass das SBT343 Extrakt die Ausbildung eines Biofilms von Staphylokokken auf den Oberflächen von Polystyrol, Glass und Kontaktlinsen unterbinden konnte ohne das bakterielle Wachstum zu beeinflussen. Die hochauflösende Fouriertransformation-Massenspektrometrie (HR-MS) Analyse konnte die Komplexität sowie die chemische Vielfalt an Komponenten im Extrakt aufzeigen. Eine vorläufige, physio-chemische Charakterisierung deutet darauf hin, dass die aktive Komponente im Extrakt hitzestabil und nicht proteinartiger Natur ist. Abschließend konnte durch Fraktionierungsexperimente gezeigt werden, dass die biologische Aktivität auf synergistischen Effekten mehrerer Komponenten im Extrakt beruht. In einer dritten Studie wurden 50 organische Extrakte, welche aus fester und flüssiger Fermentierung von 25 verschiedenen aus Meeresschwämmen isolierten Actinomyceten gewonnen wurden, auf anti-Biofilm-Aktivität untersucht. Hierbei wurde die anti-Biofilm Aktivität des organischen Extrakts der Festkultur von Streptomyces sp. SBT348 (vom mediterranen Schwamm Petrosia ficiformis) identifiziert. Eine Bioassay gestützte Fraktionierung führte zu der Identifikation der aktiven Fraktion Fr 7 im SBT348 Extrakt. Durch weitere Aufreinigung des Extrakts mit einer semipräparativen HPLC, konnten die bioaktiven Sub-Fraktionen SKC1, SKC2, SKC3, SKC4 und SKC5 isoliert werden. Die Sub- Fraktion SKC3 hatte den stärksten anti-Biofilm Effekt und bestand aus einer reinen Verbindung mit BIC90 und MIC Werten von 3,95 µg/ml und 31,25 µg/ml gegen S. epidermidis RP62A. SKC3 zeigte weder erkennbare Toxizität gegenüber Zelllinien in vitro noch gegenüber den Larven der großen Wachsmotte Galleria melonella in vivo. SKC3 war Hitze- und Enzym-resistent, was auf eine nicht proteinartige Natur hindeutet. Eine HR-MS Analyse ergab, dass die Masse von SKC3 1258,3 Da beträgt. Die Strukturanalyse von SKC3 durch 1D und 2D-NMR ist zurzeit in Bearbeitung. Um weiteres Verständnis über den anti-Biofilm Wirkmechanismus von SKC3 auf S. epidermidis RP62A zu erlangen, wurde eine RNA Sequenzierungsanalyse durchgeführt. Die Transkriptomanalyse zeigte, dass SKC3 von RP62A nach einer 20-minütigen Inkubationszeit erkannt wird und dass SKC3 den zentralen Metabolismus des Staphylokokken Stammes nach 3 h negativ beeinflusst. Zusammengenommen deuten die Ergebnisse darauf hin, dass SKC3 als Leitstruktur für die Entwicklung neuer anti- Staphylokokken Medikamente dienen könnte. Zusammenfassend heben die Ergebnisse dieser Arbeit die antiinfektiven Eigenschaften der Meeresschwamm-assoziierte Actinomyceten hervor und bieten eine Möglichkeit für die Nutzung dieser in Wirkstoffentwicklungsprogrammen.

Staphylococci are important opportunistic pathogens often found in the microflora of skin and mucosal surfaces of the upper respiratory tract of man and animals. The coagulase-positive species such as Staphylococcus aureus are capable of causing invasive (eg furuncles and bacteraemia) and non-invasive (food poisoning and toxic shock syndrome) conditions in humans. In animals, S. intermedius and S. aureus have been implicated in a variety of conditions including pyoderma in dogs, mastitis in cows and skin infections in horses with S. intermedius being responsible for more than 95% of staphylococcal infections in dogs. The emergence of antibiotic-resistant bacteria, in humans, animals and the environment, has become a cause for concern following the introduction of antimicrobial agents in clinical practice. Staphylococcal species, in particular, have developed or acquired antibiotic-resistance determinants to almost all the antimicrobial agents in clinical use today. In recent years there has been an increase in reports of the isolation of antibiotic-resistance staphylococci especially methicillin-resistant staphylococci (MRS), from cats and dogs. Cats and dogs are in close contact with humans, especially in advanced countries and therefore the possibility for transfer of antibiotic-resistant staphylococci from these animals to humans or vice versa may exist. The aims of this study were; to determine the species distribution and antibiotic sensitivity of staphylococci obtained from cats and dogs, to investigate the molecular basis of resistance and to examine the genetic relatedness of specific resistant isolates. Many studies have shown that S. intermedius and S. simulans (S. felis) are the predominante species on healthy dogs and cats, respectively, and lesions in these animals are caused by S. aureus and S. intermedius. In this study, a diverse range of coagulase-negative staphylococci was isolated from healthy animals but staphylococci from skin lesions of cats and dogs were identified mainly as S. intermedius as reported in the literature. A limited number of resistant isolates (~20%) were observed in this study and were mostly isolated from dogs. Investigations into the molecular basis of resistance to beta-lactam, macrolide and tetracycline antibiotics were carried out. The resistant isolates were analysed by polymerase chain reaction (PCR) and DNA sequencing techniques. MRS were analysed for the presence of the mecA gene and the staphylococcal cassette chromosome mec (SCCmec). The recombinase genes, cassette chromosome recombinase (ccr) on SCCmec elements were also examined. The SCCmec elements detected were as diverse as those reported in human staphylococcal strains. Comparative analysis of nucleotide and amino acid sequences of mecA and ccr gene complexes revealed that the genes are conserved among MRS of cat and dog orgin. Multilocus sequence typing (MLST) of methicillin-resistant S. aureus (MRSA) and S. epidermidis isolates showed that, the MRSA were of human origin but the S. epidermidis isolates were unique to cats and dogs. In addition, the blaZ gene which codes for ??-lactamases production, the erm genes responsible for erythromycin resistance and the tet genes which encode tetracycline resistances, were found to be identical to those observed in humans and other animal staphylococci and demonstrated similar diversity. The study has provided important information about the molecular basis of resistance in beta-lactamase producing staphylococci as well as the molecular epidemiology of MRS of cat and dog origin and identifies the risk of spread of MRS between humans and pets and vice versa. These findings should form part of a larger surveillance study on staphylococci of cat and dog origin for a better understanding of the epidemiology of antibiotic-resistance genes for improved management and control of resistant staphylococci in the community and in health care settings. / PhD Doctorate

Iron is necessary for many cellular processes such as the electron transport chain and gene regulation. However, most iron on earth is found in insoluble iron-hydroxide complexes. In addition, iron is tightly sequestered in the human body by proteins such as transferrin, making it unavailable for pathogens. In order to overcome these limitations bacteria have evolved siderophores. Siderophores are low molecular weight compounds that bind ferric iron with a high affinity. Staphylococcus aureus is an important human pathogen that is known to produce at least four siderophores, and these siderophores contribute to its virulence. S. aureus strain H372 was found to produce a siderophore that was a carboxylate type, hydrophilic, and contained ornithine. These properties were similar to the known siderophore staphyloferrin A. However, the probable molecular weight was 658, which is different from known staphylococcal siderophores.

No description available.