Bovine stafylokokker og deres spekter av antimikrobiell resistens og virulensegenskaper

Staphylococci are a group of bacteria capable of colonizing and infecting a variety of host species and causing disease in a range of different tissues. Staphylococcus aureus is the most studied staphylococcal species and can cause both clinical and subclinical mastitis in bovines, as well as infections in a range of other species. This bacterium is associated with an array of virulence factors that contributes to its survival and spread. Non-aureus staphylococci (NAS) have, on the other hand, traditionally been regarded as a uniform group with an uncertain clinical importance, but in recent years they have emerged as the most frequently isolated bacterial group from bovine milk in many countries and they are increasingly associated with bovine udder infections. NAS are also regarded as a potential reservoir for antimicrobial resistance genes. Regarding the virulence of NAS, less is known about their content of virulence genes and their mechanisms for colonizing and infecting their hosts. In addition to their large host and tissue range, staphylococci can acquire resistance to a variety of antimicrobial agents, of which methicillin resistant strains are of particular public health concern, especially methicillin resistant Staphylococcus aureus (MRSA). The term methicillin resistance incorporates resistance to almost all β-lactam antimicrobials, including penicillins and most cephalosporins. This resistance is mediated by the penicillin-binding protein PBP2a, encoded by mec genes, such as mecA. To study the interaction between PBP2a and different β-lactam antimicrobials an IPTG-inducible mecA was introduced into a methicillin sensitive laboratory S. aureus strain. This confirmed that expression of PBP2a protects against β-lactam antimicrobials. By testing the resistance of the strain to a panel of nine different β-lactams, variations in the level of protection against different agents were shown. Microfluidics fluorescence time-lapse microscopy also demonstrated a considerable phenotypic variation between cells exposed to β-lactams. This also showed that mecA-expressing S. aureus can survive β-lactam concentrations considerably higher than the minimal inhibitory concentration. As well as studying mechanisms related to the important methicillin resistance in staphylococci, the antimicrobial resistance and virulence characteristics in a collection of bovine staphylococci were examined, using disc diffusion, PCR and whole genome sequencing. The results showed that there was more antimicrobial resistance in NAS, compared to S. aureus, but the MRSA isolates stood out with a higher proportion of resistance characteristics compared to methicillin sensitive S. aureus. Regarding virulence, S. aureus contained a higher number of virulence genes compared to NAS. There were, however, also differences in virulence gene content between different NAS species, with species such as Staphylococcus chromogenes having a higher content of virulence genes compared to most other NAS species. In summary, the findings in this thesis support the notion that there is great diversity within the genus Staphylococcus, not only between S. aureus and NAS, but also within the NAS and within a population of mecA expressing S. aureus. The results may have consequences for antimicrobial treatment of staphylococcal infections and show the need to tailor the choice of antimicrobial treatment regimen to the bacterial species in question and its antimicrobial resistance and virulence.Stafylokokker er en gruppe bakterier som er i stand til å kolonisere og infisere både mennesker og mange forskjellige dyrearter, og bakteriene kan gi sykdom i flere ulike vev. Staphylococcus aureus er den mest studerte stafylokokkarten og kan forårsake både klinisk og subklinisk mastitt hos kyr. I tillegg kan S. aureus være årsak til ulike infeksjoner hos en rekke andre dyrearter. Dette skyldes ikke minst et stort utvalg av virulensfaktorer som bidrar til bakteriens overlevelse og spredning i verten. Non-aureus stafylokokker (NAS) har, i motsetning til S. aureus, tradisjonelt blitt ansett som en ensartet gruppe med usikker klinisk betydning. De senere år er imidlertid NAS rangert som de vanligste bakteriene isolert fra kumelk i flere land, og de blir stadig oftere satt i forbindelse med infeksjoner i juret hos melkekyr. NAS blir også ansett som et mulig reservoar for antimikrobielle resistensgener. Mindre er kjent om virulensen hos NAS, både når det gjelder innholdet av virulensgener og mekanismene for kolonisering og infeksjon av verten. I tillegg til deres store verts- og vevsspekter kan stafylokokker tilegne seg resistens mot mange forskjellige antimikrobielle midler, hvor meticillinresistente stammer er spesielt utfordrende, særlig meticillinresistente S. aureus (MRSA). Begrepet meticillinresistens innbefatter resistens mot nesten alle β- laktamantibiotika, inkludert penicilliner og de fleste cefalosporiner. Resistensen skyldes det penicillin-bindende proteinet PBP2a, som er kodet av mec gener, blant annet mecA. For å studere interaksjonen mellom PBP2a og forskjellige β-laktamantibiotika ble et IPTG indusertbart mecA gen satt inn i en meticillinsensitiv S. aureus laboratoriestamme. Dette bekreftet at ekspresjon av PBP2a beskytter mot β-laktamantibiotika. Ved å teste laboratoriestammens resistens mot ni forskjellige β-laktamer ble det konkludert med at graden av beskyttelse mot de forskjellige midlene varierte. «Microfluidics fluorescence time lapse»- mikroskopi viste en tydelig fenotypisk variasjon mellom celler som ble eksponert for β-laktamantibiotika. Dette viste også at S. aureus som uttrykker mecA kan overleve β-laktam konsentrasjoner som er betydelig høyere enn minste inhibitoriske konsentrasjon. I tillegg til å studere mekanismene knyttet til meticillinresistens i stafylokokker, ble antimikrobiell resistens og virulensegenskaper hos en samling bovine stafylokokker studert ved hjelp av disk diffusjon, PCR og helgenomsekvensering. Resultatene viste at NAS hadde en høyere andel antimikrobiell resistens sammenlignet med S. aureus, men MRSA isolatene skilte seg ut i S. aureus gruppen med en høyere andel resistens sammenlignet med meticillinsensitive S. aureus. Når det gjelder virulens hadde S. aureus en høyere andel virulensgener sammenlignet med NAS. Det var imidlertid også forskjeller i innholdet av virulensgener i forskjellige NAS arter. For eksempel hadde Staphylococcus chromogenes en høyere andel virulensgener sammenlignet med flere andre NAS arter. Funnene i denne avhandlingen støtter antakelsen om at det et stort mangfold innen genuset Staphylococcus, ikke bare mellom S. aureus og NAS, men også innad i NAS eller i en populasjon av S. aureus som uttrykker mecA. Resultatene kan ha betydning for antimikrobiell behandling av stafylokokkinfeksjoner og viser behovet for skreddersydde antimikrobielle behandlingsregimer som tar hensyn til den aktuelle bakteriearten og dens resistens og virulens

Percorsi adriatici. Mobilità studentesca e dinamiche sociali tra le universitates della Puglia, Padova e Venezia (XV-XVI secolo)

Questa ricerca si concentra sulla mobilit\ue0 di studenti che, muovendo dai territori della Puglia storica tra il XV e il XVI secolo, scelsero lo Studio di Padova come sede privilegiata per la propria formazione accademica, facendo tappa anche presso quelli di Ferrara e Bologna. Tale fenomeno si inscrive nel quadro di secolari e costanti rapporti politici, commerciali e culturali lungo l\u2019area adriatica. L\u2019incrocio della variegata documentazione rintracciata - documentazione di carattere universitario, registri di notai, raccolte documentarie come i Codici Diplomatici e i Libri Rossi, Cronache locali - unita alla bibliografia sul tema, ha fatto emergere una vasta gamma di notizie, che sono state analizzate e sviluppate nel corso dei capitoli. La prima parte della ricerca costituisce una presentazione del caso di studio, delle questioni storiografiche che sono alla base, delle metodologie adottate. La seconda parte prende in esame i contesti sociali e culturali di provenienza degli studenti, i luoghi e le modalit\ue0 della formazione in patria e quelli della successiva formazione universitaria. La terza parte si concentra esclusivamente sugli attori di questa particolare forma di mobilit\ue0: si ricostruiscono le estrazioni sociali, i casi di tradizioni familiari di studi, le reti di relazioni costruite, le carriere intraprese con il titolo di dottore. La quarta ed ultima parte si concretizza in un catalogo di studenti e dottori pugliesi, dove sono raccolte tutte le notizie di carattere biografico rintracciate. La ricerca si conclude con una appendice documentaria e con l\u2019elenco delle fonti e la bibliografia.This research concentrate on student mobility from Apulian cities during the XV and the XVI century, in particular towards Padua\u2019s Studium, but also towards Ferrara and Bologna. This phenomenon belongs in the secular and political, economic, cultural relationships in the Adriatic area. Sources utilized \u2013 universitary documentation, notarial acts, Codici Diplomatici and Libri Rossi, local chronicles \u2013 and bibliography, are important for informations analised during the research. The first part is a presentation of the case-study, historiographical questions and methodologies. The second part analise social and cultural context of students and their local and universitary studies. The third part is concentrated on students: their social origin, family traditions of studies, social relationships, careers. The fourth part is a catalogue of Apulian students and doctors, with biographical informations. The last part contained documentation and the list of sources and the bibliography

Penicillin‐binding protein PBP2a provides variable levels of protection toward different β‐lactams in Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) is resistant to most β-lactams due to the expression of an extra penicillin-binding protein, PBP2a, with low β-lactam affinity. It has long been known that heterologous expression of the PBP2a-encoding mecA gene in methicillin-sensitive S. aureus (MSSA) provides protection towards β-lactams, however, some reports suggest that the degree of protection can vary between different β-lactams. To test this more systematically, we introduced an IPTGinducible mecA into the MSSA laboratory strain RN4220. We confirm, by growth assays as well as single-cell microfluidics time-lapse microscopy experiments, that PBP2a expression protects against β-lactams in S. aureus RN4220. By testing a panel of ten different β-lactams, we conclude that there is also a great variation in the level of protection conferred by PBP2a. Expression of PBP2a resulted in an only fourfold increase in minimum inhibitory concentration (MIC) for imipenem, while a 32-fold increase in MIC was observed for cefaclor and cephalexin. Interestingly, in our experimental setup, PBP2a confers the highest protection against cefaclor and cephalexin—two β-lactams that are known to have a high specific affinity toward the transpeptidase PBP3 of S. aureus. Notably, using a single-cell microfluidics setup we demonstrate a considerable phenotypic variation between cells upon β-lactam exposure and show that mecA-expressing S. aureus can survive β-lactam concentrations much higher than the minimal inhibitory concentrations. We discuss possible explanations and implications of these results including important aspects regarding treatment of infection

Penicillin-binding protein PBP2a provides variable levels of protection towards different β-lactams in Staphylococcus aureus RN4220

Methicillin-resistant Staphylococcus aureus (MRSA) is resistant to most β-lactams due to the expression of an extra penicillin-binding protein, PBP2a, with low β-lactam affinity. It has long been known that heterologous expression of the PBP2a-encoding mecA gene in methicillin-sensitive S. aureus (MSSA) provides protection towards β-lactams, however, some reports suggest that the degree of protection can vary between different β-lactams. To test this more systematically, we introduced an IPTGinducible mecA into the MSSA laboratory strain RN4220. We confirm, by growth assays as well as single-cell microfluidics time-lapse microscopy experiments, that PBP2a expression protects against β-lactams in S. aureus RN4220. By testing a panel of ten different β-lactams, we conclude that there is also a great variation in the level of protection conferred by PBP2a. Expression of PBP2a resulted in an only fourfold increase in minimum inhibitory concentration (MIC) for imipenem, while a 32-fold increase in MIC was observed for cefaclor and cephalexin. Interestingly, in our experimental setup, PBP2a confers the highest protection against cefaclor and cephalexin—two β-lactams that are known to have a high specific affinity toward the transpeptidase PBP3 of S. aureus. Notably, using a single-cell microfluidics setup we demonstrate a considerable phenotypic variation between cells upon β-lactam exposure and show that mecA-expressing S. aureus can survive β-lactam concentrations much higher than the minimal inhibitory concentrations. We discuss possible explanations and implications of these results including important aspects regarding treatment of infection

In vitro and in vivo assessment of phage therapy against Staphylococcus aureus causing bovine mastitis.

The aim of this study was to assess the efficacy of lytic bacteriophages on Staphylococcus aureus causing bovine mastitis, by in vitro and in vivo assays using Galleria mellonella and murine mastitis models. Methods: Between May and December 2016, ten S. aureus (five methicillin-resistant and five methicillinsensitive) isolates were isolated from milk samples of cattle with mastitis in Belgium and Norway. The isolates were assessed in vitro for their susceptibility to four lytic bacteriophages (Romulus, Remus, ISP and DSM105264) and subsequently in vivo in G. mellonella larvae and in murine mastitis model. Results: Romulus, Remus and ISP showed a lytic activity against the S. aureus isolates in vitro. A larvae survival rate below 50% was observed at 4 days post-inoculation (DPI) in the groups infected with a methicillin-sensitive S. aureus isolate and treated with these three phages in vivo. An incomplete recovery of the mouse mastitis was observed at 48 h post-inoculation (HPI) in the groups infected and treated with the ISP phage in vivo. Conclusions: The observations are much more pronounced statistically between the infected- phosphate buffered saline (PBS)-treated and infected-phage-treated groups in G. mellonella and the murine mastitis model demonstrating an effect of the phages against S. aureus associated with bovine mastitis

Whole Genome Sequencing of Staphylococci Isolated From Bovine Milk Samples

Staphylococci are among the commonly isolated bacteria from intramammary infections in bovines, where Staphylococcus aureus is the most studied species. This species carries a variety of virulence genes, contributing to bacterial survival and spread. Less is known about non-aureus staphylococci (NAS) and their range of virulence genes and mechanisms, but they are the most frequently isolated bacteria from bovine milk. Staphylococci can also carry a range of antimicrobial resistance genes, complicating treatment of the infections they cause. We used Illumina sequencing to whole genome sequence 93 staphylococcal isolates selected from a collection of staphylococcal isolates; 45 S. aureus isolates and 48 NAS isolates from 16 different species, determining their content of antimicrobial resistance genes and virulence genes. Antimicrobial resistance genes were frequently observed in the NAS species as a group compared to S. aureus. However, the lincosamide resistance gene lnuA and penicillin resistance gene blaZ were frequently identified in NAS, as well as a small number of S. aureus. The erm genes conferring macrolide resistance were also identified in several NAS isolates and in a small number of S. aureus isolates. In most S. aureus isolates, no antimicrobial resistance genes were detected, but in five S. aureus isolates three to six resistance genes were identified and all five of these carried the mecA gene. Virulence genes were more frequently identified in S. aureus, which contained on average five times more virulence genes compared to NAS. Among the NAS species there were also differences in content of virulence genes, such as S. chromogenes with a higher average number of virulence genes. By determining the content of a large selection of virulence genes and antimicrobial resistance genes in S. aureus and 16 different NAS species our results contribute with knowledge regarding the genetic basis for virulence and antimicrobial resistance in bovine staphylococci, especially the less studied NAS. The results can create a broader basis for further research into the virulence mechanisms of this important group of bacteria in bovine intramammary infection