Insights into selected genomic adaptation strategies of Staphylococcus aureus

Staphylococcus (S.) aureus is a ubiquitous Gram-positive, spherical bacterium commonly isolated from humans, domestic-, livestock-, wildlife-, and even aquatic animals. The bacteria also survive and continue their growth on abiotic material in natural and artificial environments. Transmission of the immobile bacterium often occurs through direct contact with infected living beings or contaminated objects, whereby S. aureus is continuously forced to adapt to altered environmental conditions, including various host species. To overcome these challenges, members of the Genus Staphylococcus possess numerous strategies that include genomic alterations as well as the uptake or loss of mobile genetic elements (MGEs). The enormous adaptive capabilities and tendencies to accumulate genes encoding resistance traits define S. aureus as a pathogen of great clinical importance in human and veterinary medicine. Further insights into the adaptive strategies of this notorious pathogen are crucial, especially regarding the development of novel treatment options in case of infection. Against this background, changes in the essential regulatory quorum sensing (QS) system that promote bacterial niche adaptation were identified and characterised in this cumulative work. In addition, the genetic structure and putative role of the MGE mediating broad-spectrum β-lactam resistance in methicillin resistant S.aureus (MRSA) was investigated in detail using livestock-associated (LA)-MRSA as a prime example for adaptation to challenging environments. The data presented in Publication I revealed core genome alterations within the accessory gene regulator (agr) system, especially in the agrA and agrC genes, that led to variation or even silencing of the bacterial QS capabilities. Moreover, these changes occurred independently in strains with completely different genomic backgrounds of various animal origins across Europe as well as in closely related human isolates, clearly indicating the importance of QS regarding adaptive changes and not at least bacterial evolution. These adaptive modulatory changes enable the bacteria, beyond others, to survive among competing staphylococci belonging to the resident microbiota, simultaneously reducing their perceptibility by the host’s immune system while enhancing their biofilm formation capabilities. Consequently, S. aureus, lacking a functional agr system, saves the energy otherwise spend on QS and can conquer challenging environments since this deficit obviously promotes inter-species spread between different animal species, and, not at least, humans. The second part of this cumulative thesis focuses on a specific kind of MGEs, the Staphylococcal Cassette Chromosome (SCC) mec elements. A mechanism leading to the loss of a complete SCCmec element from its genomic integration site was reconstructed in detail for an MRSA sequence type (ST) 398 strain and described in Publication II. The investigated excision of the complete SCCmecVc element is suggested to be the result of homologous recombination within a repetitive sequence between the end of intergenic spacer region 1 and the first 48 bp of the large serine recombinases (ccr). Furthermore, the presence of an operon that encodes the machinery for autonomous replication of the deleted SCCmecVc element was revealed. Livestock-associated environmental conditions that are in particular associated with industrial pig farming were previously discussed as a potential trigger for the loss of SCCmec elements. To mimic conditions commonly associated with pig farming, LA-MRSA were exposed to porcine serum, an increased ammonia concentration and a combination of both for 10 min or 60 min. Since ccr genes are known for their capability to induce the elements’ uptake and loss, the growth conditions evaluated here did not significantly induce ccr transcription compared to the unchallenged controls. A detailed study of the deletion leading to the loss of a complete and functional SCCmec at defined genomic sites, on the other hand, strongly suggests a complex biological role of this particular event. While the induction of the ccr genes seems to depend on a massive cell- or DNA damage that leads to a strong activation of the SOS response, a far more efficient pathway for the horizontal spread of SCCmec is the occasional loss of complete elements at certain redundant “breaking points”, as described in Publication II. Since SCCmec elements possess the machinery for autonomous replication, this pathway might be of utmost epidemiological importance for the emergence, maintenance and spread of methicillin resistance. Both adaptation strategies investigated in this cumulative work highlight that the evolution of S. aureus will continue unabated. However, the unwearyingly evolution of these bacteria will lead to further problems in combating infections caused by S. aureus. The development of completely new treatment options, which consider the current knowledge on adaptation strategies of S. aureus, is therefore of clinical importance. Additionally, a holistic one-health approach that engages scientists from human and veterinary medicine as well as agricultural and environmental sciences is necessary to prevent further trans-sectoral transmission and spread by reducing the anthropogenic selective pressure that leads to resistance accumulation in bacteria.Staphylococcus (S.) aureus ist ein ubiquitär vorkommendes Gram-positives, kugelförmiges Bakterium, welches vielfach aus Proben von Menschen, Haus-, Nutz- und Wildtieren sowie von Meeressäugern isoliert wird. Das Bakterium kann auch auf abiotischem Material in natürlicher und künstlicher Umgebung überleben und dort sein Wachstum fortsetzen. Die Übertragung des unbeweglichen Bakteriums erfolgt häufig durch den direkten Kontakt mit infizierten Lebewesen oder kontaminierten Gegenständen, wodurch S.aureus ständig gezwungen ist, sich an veränderte Umweltbedingungen, einschließlich verschiedener Wirtsarten, anzupassen. Um diese Herausforderungen zu bewältigen, verfügen die Mitglieder des Genus Staphylococcus über zahlreiche Strategien, zu denen genomische Veränderungen sowie die Aufnahme oder der Verlust von mobilen genetischen Elementen (MGE) gehören. Die enormen Anpassungsfähigkeiten und die Tendenz zur Akkumulation von Genen, die für Resistenzeigenschaften kodieren, machen S. aureus zu einem Erreger von großer klinischer Bedeutung in der Human- und Veterinärmedizin. Weitere Erkenntnisse über die Anpassungsstrategien dieser Bakterien sind im Hinblick auf die Entwicklung neuer Behandlungsmöglichkeiten im Falle einer Infektion von entscheidender Bedeutung. Vor diesem Hintergrund wurden in dieser kumulativen Arbeit Veränderungen im regulatorisch wichtigen „Quorum-Sensing“ (QS)-System identifiziert und charakterisiert, welche die bakterielle Nischenanpassung unterstützen. Ergänzend dazu wurde die genetische Struktur und die mutmaßliche Rolle des MGE, welches die Breitspektrum-β-Lactam-Resistenz bei Methicillin-resistentem S. aureus (MRSA) vermittelt, eingehend untersucht, wobei hier der mit Nutztieren assoziierte (engl. livestock-associated (LA)) MRSA als Paradebeispiel für die Anpassung an schwierige Umgebungen dient. Die in der Publikation I vorgestellten Daten zeigen genomische Varianten innerhalb des akzessorischen Genregulatorsystems (agr), insbesondere in den agrA- und agrC-Genen, die zu einer Veränderung oder sogar Unterdrückung der bakteriellen QS-Fähigkeiten führten. Darüber hinaus traten diese Veränderungen unabhängig voneinander in Stämmen mit völlig unterschiedlichen phylogenetischem und geografischem Hintergrund, bei unterschiedlichen Tierarten sowie in eng verwandten menschlichen Isolaten auf, was eindeutig auf die Bedeutung von QS für adaptive Veränderungen und nicht zuletzt für die bakterielle Evolution hinweist. Diese adaptiven modulatorischen Veränderungen ermöglichen es den Bakterien, unter anderem unter konkurrierenden Staphylokokken der residenten Mikrobiota des Wirtes zu überleben und gleichzeitig ihre Perzeption durch das Immunsystem zu verringern, während die Fähigkeiten zur Biofilmbildung verstärkt werden. Infolgedessen verfügen S. aureus, denen ein funktionsfähiges agr-System fehlt, über einen energetischen Vorteil und können schwierige Umgebungen erobern, da dieses Defizit offensichtlich die Ausbreitung zwischen verschiedenen Tierarten und nicht zuletzt dem Menschen fördert. Der zweite Teil dieser kumulativen Arbeit konzentriert sich auf eine spezielle Art von MGEs, die „Staphylococcal Cassette Chromosome“ (SCC) mec-Elemente. Ein Mechanismus, der zum Verlust eines kompletten SCCmec-Elements von seiner genomischen Integrationsstelle führt, wurde für einen MRSA Sequenztyp (ST) 398-Stamm im Detail rekonstruiert und in Publikation II beschrieben. Die untersuchte Exzision des vollständigen SCCmecVc-Elements ist wahrscheinlich das Ergebnis homologer Rekombination innerhalb einer repetitiven Sequenz zwischen dem Ende der „intergenen Spacer“-Region 1 und den ersten 48 bp der großen Serin-Rekombinasen (ccr). Außerdem wurde das Vorhandensein eines Operons nachgewiesen, welches die Maschinerie für eine autonome Replikation des deletierten SCCmecVc-Elements kodiert. Mit der Tierhaltung einhergehende Umweltbedingungen, die insbesondere mit der industriellen Schweinehaltung assoziiert sind, wurden zuvor als möglicher Auslöser für den Verlust von SCCmec-Elementen diskutiert. Um Bedingungen nachzuahmen, die üblicherweise mit der Schweinehaltung in Verbindung gebracht werden, wurden LA-MRSA jeweils 10 Minuten oder 60 Minuten mit Schweineserum und einer erhöhten Ammoniakkonzentration sowie einer Kombination aus beidem behandelt. Die ccr-Gene sind dafür bekannt, dass sie die Aufnahme und den Verlust von Elementen induzieren können, jedoch führten die hier untersuchten Wachstumsbedingungen im Vergleich zu den unbelasteten Kontrollen zu keiner signifikanten Induktion der ccr-Transkription. Eine detaillierte Untersuchung des Vorgangs, welcher zum Verlust eines vollständigen und funktionsfähigen SCCmec an bestimmten Stellen im Genom führen kann, deutet dagegen auf eine komplexe biologische Rolle dieses Ereignisses hin. Während die Induktion der ccr-Gene von einer starken Aktivierung der SOS-Antwort in Folge einer massiven Zell- oder DNA-Schädigung abzuhängen scheint, ist ein weitaus effizienterer Weg für die horizontale Verbreitung von SCCmec der gelegentliche Verlust kompletter Elemente an bestimmten redundanten „Sollbruchstellen“, wie in Publikation II beschrieben. Da SCCmec-Elemente die Maschinerie zur autonomen Replikation besitzen, könnte dieser Weg von größter epidemiologischer Bedeutung für die Entstehung, Aufrechterhaltung und Verbreitung der Methicillin-Resistenz sein. Beide Anpassungsstrategien, die in dieser kumulativen Arbeit untersucht wurden, machen deutlich, dass die Evolution von S. aureus ungebremst weitergehen wird. Die unermüdliche Evolution dieser Bakterien wird jedoch zu weiteren Problemen bei der Bekämpfung von S. aureus verursachten Infektionen führen. Die Entwicklung völlig neuer Behandlungsmöglichkeiten, die das aktuelle Wissen über die Anpassungsstrategien von S. aureus berücksichtigen, ist daher von klinischer Bedeutung. Darüber hinaus sollte für die Zukunft ein ganzheitlicher „One-Health“-Ansatz in Betracht gezogen werden, der Wissenschaftlerinnen und Wissenschaftler aus der Human- und Veterinärmedizin sowie den Agrar- und Umweltwissenschaften einbezieht, um eine weitere sektorübergreifende Übertragung und Ausbreitung resistenter Bakterien zu vermindern, indem der anthropogene Selektionsdruck, welcher zu einer Resistenzakkumulation führt, verringert wird

Aligning Discourse and Argumentation Structures using Subtrees and Redescription Mining

International audienceIn this paper, we investigate similarities between discourse and argumentation structures by aligning subtrees in a corpus containing both annotations. Contrary to previous works, we focus on comparing sub-structures and not only relation matches. Using data mining techniques , we show that discourse and argumen-tation most often align well, and the double annotation allows to derive a mapping between structures. Moreover, this approach enables the study of similarities between discourse structures and differences in their expressive power

Lack of antigenic diversification of major outer membrane proteins during clonal waves of Neisseria meningitidis serogroup A colonization and disease

In particular in the ‘meningitis belt' of sub-Saharan Africa, epidemic meningococcal meningitis is a severe public health problem. In the past decades, serogroup A lineages have been the dominant etiologic agents, but also other serogroups have caused outbreaks. A comprehensive vaccine based on subcapsular outer membrane proteins (OMPs) is not available. Here, we have investigated whether meningococcal populations overcome herd immunity by changing antigenic properties of their OMPs. Meningococcal isolates were collected in the context of longitudinal studies in Ghana between 2002 and 2008 and in Burkina Faso between 2006 and 2007. Serogroup A strains isolated during two clonal waves of colonization and disease showed no diversification in the genes encoding their PorA, PorB, and FetA proteins. However, we detected occasional allelic exchange of opa genes, as well as wide variation in the number of intragenic tandem repeats, showing that phase variation of Opa protein expression is a frequent event. Altogether we observed a remarkable antigenic stability of the PorA, PorB and FetA proteins over years. Our results indicate that while herd immunity may be responsible for the disappearance of meningococcal clones over time, it is not a strong driving force for antigenic diversification of the major OMPs analyzed her

Aligning Discourse and Argumentation Structures using Subtrees and Redescription Mining

International audienceIn this paper, we investigate similarities between discourse and argumentation structures by aligning subtrees in a corpus containing both annotations. Contrary to previous works, we focus on comparing sub-structures and not only relation matches. Using data mining techniques , we show that discourse and argumen-tation most often align well, and the double annotation allows to derive a mapping between structures. Moreover, this approach enables the study of similarities between discourse structures and differences in their expressive power

Alignement de Structures Argumentatives et Discursives par Fouille de Graphes et de Redescriptions

National audienceIn this paper, we investigate similarities between discourse and argumentation structures by aligning subtrees in a corpus containing both annotations. Contrary to previous works, we focus on comparing sub-structures and not only relation matches. Using data mining techniques , we show that discourse and argumen-tation most often align well, and the double annotation allows to derive a mapping between structures. Moreover, this approach enables the study of similarities between discourse structures and differences in their expressive power.Dans cet article, nous étudions la similarité entre structures argumen-tatives et discursives en alignant des sous-arbres dans un corpus annoté en RST et en structure argumentative. Contrairement aux travaux précédents, nous ne nous intéressons pas uniquement à un alignement relation à relation, mais à un alignement de sous-structures. À l'aide de méthodes de fouille de données, nous montrons que des similitudes existent entre l'argumentation et le discours. L'an-notation multiple du corpus permet également de proposer un alignement entre les structures. De plus, cette approche permet de mettre en évidence les diffé-rences d'expressivité des deux formalismes

Alignement de Structures Argumentatives et Discursives par Fouille de Graphes et de Redescriptions

National audienceIn this paper, we investigate similarities between discourse and argumentation structures by aligning subtrees in a corpus containing both annotations. Contrary to previous works, we focus on comparing sub-structures and not only relation matches. Using data mining techniques , we show that discourse and argumen-tation most often align well, and the double annotation allows to derive a mapping between structures. Moreover, this approach enables the study of similarities between discourse structures and differences in their expressive power.Dans cet article, nous étudions la similarité entre structures argumen-tatives et discursives en alignant des sous-arbres dans un corpus annoté en RST et en structure argumentative. Contrairement aux travaux précédents, nous ne nous intéressons pas uniquement à un alignement relation à relation, mais à un alignement de sous-structures. À l'aide de méthodes de fouille de données, nous montrons que des similitudes existent entre l'argumentation et le discours. L'an-notation multiple du corpus permet également de proposer un alignement entre les structures. De plus, cette approche permet de mettre en évidence les diffé-rences d'expressivité des deux formalismes

Silence as a way of niche adaptation: mecC-MRSA with variations in the accessory gene regulator (agr) functionality express kaleidoscopic phenotypes

Functionality of the accessory gene regulator (agr) quorum sensing system is an important factor promoting either acute or chronic infections by the notorious opportunistic human and veterinary pathogen Staphylococcus aureus. Spontaneous alterations of the agr system are known to frequently occur in human healthcare-associated S. aureus lineages. However, data on agr integrity and function are sparse regarding other major clonal lineages. Here we report on the agr system functionality and activity level in mecC-carrying methicillin resistant S. aureus (MRSA) of various animal origins (n = 33) obtained in Europe as well as in closely related human isolates (n = 12). Whole genome analysis assigned all isolates to four clonal complexes (CC) with distinct agr types (CC599 agr I, CC49 agr II, CC130 agr III and CC1943 agr IV). Agr functionality was assessed by a combination of phenotypic assays and proteome analysis. In each CC, isolates with varying agr activity levels were detected, including the presence of completely non-functional variants. Genomic comparison of the agr I-IV encoding regions associated these phenotypic differences with variations in the agrA and agrC genes. The genomic changes were detected independently in divergent lineages, suggesting that agr variation might foster viability and adaptation of emerging MRSA lineages to distinct ecological niches

How to survive pig farming: Mechanism of SCCmec element deletion and metabolic stress adaptation in livestock-associated MRSA

Previous research on methicillin susceptible Staphylococcus aureus (MSSA) belonging to livestock-associated (LA-) sequence type (ST) 398, isolated from pigs and their local surroundings, indicated that differences between these MSSA and their methicillin resistant predecessors (MRSA) are often limited to the absence of the staphylococcal cassette chromosome mec (SCCmec) and few single nucleotide polymorphisms. So far, our understanding on how LA-MRSA endure the environmental conditions associated with pig-farming as well as the putative impact of this particular environment on the mobilisation of SCCmec elements is limited. Thus, we performed in-depth genomic and transcriptomic analyses using the LA-MRSA ST398 strain IMT38951 and its methicillin susceptible descendant. We identified a mosaic-structured SCCmec region including a putative replicative SCCmecVc which is absent from the MSSA chromosome through homologous recombination. Based on our data, such events occur between short repetitive sequences identified within and adjacent to two distinct alleles of the large cassette recombinase genes C (ccrC). We further evaluated the global transcriptomic response of MRSA ST398 to particular pig-farm associated conditions, i.e., contact with host proteins (porcine serum) and a high ammonia concentration. Differential expression of global regulators involved in stress response control were identified, i.e., ammonia-induced alternative sigma factor B-depending activation of genes for the alkaline shock protein 23, the heat shock response and the accessory gene regulator (agr)-controlled transcription of virulence factors. Exposure to serum transiently induced the transcription of distinct virulence factor encoding genes. Transcription of genes reported for mediating the loss of methicillin resistance, especially ccrC, was not significantly different compared to the unchallenged controls. We concluded that, from an evolutionary perspective, bacteria may save energy by incidentally dismissing a fully replicative SCCmec element in contrast to the induction of ccr genes on a population scale. Since the genomic SCCmec integration site is a hot-spot of recombination, occasional losses of elements of 16 kb size may restore capacities for the uptake of foreign genetic material. Subsequent spread of resistance, on the other hand, might depend on the autonomous replication machinery of the deleted SCCmec elements that probably enhance chances for reintegration of SCCmec into susceptible genomes by mere multiplication

The contemporary "Trojan Horse"

Pathogens frequently associated with multi-drug resistant (MDR) phenotypes, including extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae (ESBL-E) and Acinetobacter baumannii isolated from horses admitted to horse clinics, pose a risk for animal patients and personnel in horse clinics. To estimate current rates of colonization, a total of 341 equine patients were screened for carriage of zoonotic indicator pathogens at hospital admission. Horses showing clinical signs associated with colic (n = 233) or open wounds (n = 108) were selected for microbiological examination of nostril swabs, faecal samples and wound swabs taken from the open wound group. The results showed alarming carriage rates of Gram-negative MDR pathogens in equine patients: 10.7% (34 of 318) of validated faecal specimens were positive for ESBL-E (94%: ESBL-producing Escherichia coli), with recorded rates of 10.5% for the colic and 11% for the open wound group. 92.7% of the ESBL-producing E. coli were phenotypically resistant to three or more classes of antimicrobials. A. baumannii was rarely detected (0.9%), and all faecal samples investigated were negative for Salmonella, both directly and after two enrichment steps. Screening results for the equine nostril swabs showed detection rates for ESBL-E of 3.4% among colic patients and 0.9% in the open wound group, with an average rate of 2.6% (9/340) for both indications. For all 41 ESBL-producing E. coli isolated, a broad heterogeneity was revealed using pulsed-field gel electrophoresis (PFGE) patterns and whole genome sequencing (WGS) -analysis. However, a predominance of sequence type complex (STC)10 and STC1250 was observed, including several novel STs. The most common genes associated with ESBL-production were identified as blaCTX-M-1 (31/41; 75.6%) and blaSHV-12 (24.4%). The results of this study reveal a disturbingly large fraction of multi-drug resistant and ESBL-producing E. coli among equine patients, posing a clear threat to established hygiene management systems and work-place safety of veterinary staff in horse clinics

Genome‑wide insights into population structure and host specifcity of Campylobacter jejuni

The zoonotic pathogen Campylobacter jejuni is among the leading causes of foodborne diseases worldwide. While C. jejuni colonises many wild animals and livestock, persistence mechanisms enabling the bacterium to adapt to host species' guts are not fully understood. In order to identify putative determinants influencing host preferences of distinct lineages, bootstrapping based on stratified random sampling combined with a k-mer-based genome-wide association was conducted on 490 genomes from diverse origins in Germany and Canada. We show a strong association of both the core and the accessory genome characteristics with distinct host animal species, indicating multiple adaptive trajectories defining the evolution of C. jejuni lifestyle preferences in different ecosystems. Here, we demonstrate that adaptation towards a specific host niche ecology is most likely a long evolutionary and multifactorial process, expressed by gene absence or presence and allele variations of core genes. Several host-specific allelic variants from different phylogenetic backgrounds, including dnaE, rpoB, ftsX or pycB play important roles for genome maintenance and metabolic pathways. Thus, variants of genes important for C. jejuni to cope with specific ecological niches or hosts may be useful markers for both surveillance and future pathogen intervention strategies.Peer Reviewe