Evolution ofStaphylococcus aureus

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Introduction

For many decades, the clinical challenge due toStaphylococcus aureus infections in the hospital,community, and also in veterinary medicine hasled to intensive investigations. While S. aureus cancolonize 20-30% of the general population withoutcausing any clinical manifestation, it is also capableof causing a wide spectrum of diseases in humansranging from benign skin infections, through moreserious diseases such as food poisoning, to life-threatening infections such as endocarditis, necro-tizing pneumonia, osteomyelitis or septicemia. Inmilk-producing ruminants, mastitis is the majorcause of economic loss worldwide and a majorconcern in milk transformation. Mastitis is currentlyhard to tackle in dairy cows and goats as this infection is refractory to antibiotic treatment inpart due to the anatomic barrier and an aberrantimmune response. In ewes in particular, severityof mastitis may vary from subclinical to lethal gangrenous diseases.

The versatility of this pathogen could be explainedby different adaptative strategies and virulenceproperties. For instance S. aureus is capable to survive in various environmental conditions; acidic,

oxidative, high temperature variations, as well asin nutrient-limited medium. In addition, S. aureusshow particular capacity to resist to the presenceof chemicals including antibiotics or antisepticmolecules. Some of the properties required to resist to these different conditions are intrinsic to the bacterium but others require adaptationand are linked to genomic evolution, in particularthe acquisition of nucleic acid from various origins.Our purpose will be to evaluate the differentstrategies used by S. aureus to evolve and adapt itsgenome composition and content following naturalevents or after exposition to selective pressure.

Alteration of genome integrity

During the lifetime of a bacterial cell, variousevents contributed to modifications in genomecontent. DNA transposons are frequently found inclinical isolates. They are small linear mobile ele-ments encoding generally for a limited number ofgenes and flanked by insertion sequences. Theseelements are able to jump within the bacterialgenome. The integration could result in the dis-ruption of a coding sequence but also bring newgenes to the transposed bacteria such as antibioticresistance genes. Plasmids are generally larger in

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Evolution of Staphylococcu Importance of mobile gene on bacterial virulence and h

term of size and harbour frequently dozen ofgenes encoding potentially for antibiotic or heavymetal resistance. Most of the time, plasmids foundin clinical isolates are circular autonomous mole-cules, able to replicate in the cytoplasm of the bacterial cells. Finally, lysogenic or temperate phagesare also important partners of clinical isolates. Numerous infections are due to toxins encoded bybacteriophages (e.g. diphtheria toxin gene har-boured by Corynebacterium diphtheria or scarletfever toxin harboured by Streptococcus pyogenes).

In the clinic or in the environment, the frequencyof genomic alterations is difficult to predict and isdependent on bacterial density and lifestyle. Considering populations of S. aureus strains andparticularly of MRSA (methicillin resistant Staphy-lococcus aureus) – present in the clinic, almost allisolates showed in their genome traces of trans-poson, bacteriophage insertion or plasmids eitherresiding autonomously in cellular cytoplasm or integrated in the bacterial chromosome. Notehowever that the most precious good for all cellsis the protection of cellular integrity and particu-larly the protection of its genetic patrimony. Thisprotection is the role of restriction modificationsystems. These systems are able to sense foreign

DNA through specific biochemical motives and to decide to degrade this material with dedicateddeoxyribonuclease. These systems are essential toprotect integrity of the bacterial genome but inclinical isolates, non-functional restriction modifi-cation systems are not rare. In theory, acquisitionof foreign material in deficient bacteria should increase genome plasticity.

The life of a bacterial cell

In the environment, bacteria from various speciesare in close contact and could exchange informa-tion, nutrients and genetic material. Bacterialdeath results in cellular envelop disruption and release of bacterial chromosome. These nucleicacids could be integrated by bacterium yielding tothe acquisition of new genes. This process is namednatural transformation. Considering bacterial populations, numerous mobile genetic elementscould contribute to genome evolution. Plasmids,transposons or prophages represent efficientprocesses for the acquisition of new genomic features. In S. aureus, plasmids are frequently observed in clinical strains and encode generallyfor antimicrobial resistance determinants. S. aureusis also able to produce a plethora of toxins

us aureus : tic elements

host tropism

contributing to its virulence such as toxic shock syndrome toxin, Penton Valentin leucocidin, exfoliatin toxins A and B and more than 20 entero-toxins which are generally acquired through acquisition of integrative elements (e.g. lysogenbacteriophages).

S. aureus is a common bacterium able to multiplyand survive independently, thus, this microorgan-ism is not an obligatory parasite. In laboratory, inrich medium and in optimal conditions, S. aureuspopulation density doubles every 30 minutes. Itmeans that all the bacterial compounds or mole-cules could be duplicated in 30 minutes to gener-

ate a daughter cell “identical” to its mother. Thisphenomenon requires billions of enzymatic andchemical reactions in a limited times catalysed bythe 2800 genes present in the bacterial genome.Even if protection mechanisms exist, punctual errors are possible and mutation occurs. This typeof mutation appears spontaneously more frequentlyin non-coding sequences but could also yield tothe modification of the sequence of a protein withunpredictable consequences. Note that if a muta-tion occurs in a vital gene, the cell could be non-viable. On the opposite, a mutation appearing ina target of an antimicrobial molecule could resultin an increase resistance against this molecule. To

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Evolution of Staphylococcus aureus through integration of mobile genetic elements modulates baterial virulence and modifies host tropism

Pathways driving the evolution. Each circle corresponds to the circularized genome of a Staphylococcus aureus strain. The purple circles aregenomes of animal colonizers only able to reside in animals (purple square) but not found in human (red square). During its lifetime the

colonizer strain will be exposed to DNA transposons (1) often encoding resistance genes, acquisition of plasmid DNA (2) containing metabolic or resistance genes, to the integration of the SCCmec element (3) containing the genetic basis of methicillin resistance and will

accumulate some punctual mutations (4) symbolized by * on the circular genomes. In our works, we showed that following these potentialevents triggering alteration of the genome integrity of the original strain, the ancestral susceptible colonizer strain remains a colonizer of

animal but is now resistant to several antibiotics. It is only after integration of prophage nucleic acid (5) that the strain changes its tropismfor host and is now able to infect human (red circular genome infecting human).

SCCmec element3

Punctualmutations4

Transposon1

Phage5

Phage5

Plasmidacquisition2

illustrate this evolution process, different groupsobserved emergence of spontaneous mutationsin metabolic pathways yielding to optimization ofthe utilization of carbon source present in thegrowth medium. Another example is the mutationof a single base at specific location among the 2.8million bases of the chromosome that triggers resistance against specific classes of antimicrobialssuch as quinolones or mupirocin. My group studied this phenomenon recently in a variety ofclinical isolates of MRSA. We incubated MRSAstrains from 10 different lineages frequently foundin human clinic, in the presence of low concentra-tions of mupirocin. Typically, the working concen-trations were sufficient to alter the growth rate ofthe bacteria but not sufficient to kill cells. Concen-trations were increased, generally by 50% after48h of incubation until reaching lethal levels. Then,DNA of strains recovered in the medium with high-est concentrations allowing growth, were purifiedfor sequencing purposes. In almost all strainsshowing increase level of resistance, we were ableto identify mutations at specific site of the ileSgene, the target gene conditioning the resistanceagainst mupirocin. In another study, we performedsimilar incubations of MRSA strains with biocidesused for patient decolonization (e.g. polyhexanideand chlorhexidine). Surprisingly we observed inbacteria showing reduced susceptibility to the bio-cide, emergence of punctual mutations in a gene(mprF) known to mediate the resistance againstdaptomycin; an antibiotic used in the treatment ofMRSA infection. This category of resistances, induced by the presence of antimicrobial is thendependent on the selection pressure.

Numerous antibiotic resistances are related to the acquisition of a specific gene of resistance. Itis the case for numerous molecules currently usedin the human clinic. For example, shortly after theintroduction of penicillin in clinic, penicillin-resis-tant bacteria were identified. These resistant or-

ganisms had acquired a gene encoding for an enzyme conditioning the degradation of penicillin;a beta-lactamase. The resistance against methi-cillin is encoded by a mobile element; the SCCmeclocus inserted at a specific site in the genome ofthe bacterium (see also figure legend on page 4).This element contains the gene mecA, responsiblefor the resistance against methicillin. Cyclines andglycopeptides are also families of molecules currently used in human clinic. Resistance genesare known against these families of drugs and different studies about the mechanisms triggeredby these genes showed that they contribute toalter the structure of the target of the antibiotic(the ribosome and the bacterial cell wall, respec-tively). The question of the origin of these genesis of outstanding importance. Most of antibioticsused to treat infectious diseases are natural mol-ecules produced by bacteria or fungi to limit com-petition for nutrients and protect their ecological

Glass surface covered by S. aureus biofilm. Fluorescence microscopy image showing bacterial cells that are actively

multiplying onto the surface of glass lenses in a 3D structure.The picture shows a low content in dead cells (red coloured by

propidium iodide) and a vast majority of multiplying cells(green, coloured by SYTO dye).

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niche. The fact that an organism is able to producea molecule able to kill cells implies that this cell isable to resist to the effect of the killing agent. It ismandatory that the producer organisms har-boured the corresponding resistance gene. It isthen easily conceivable that antibiotic producercells represent the potential reservoir of resistancegenes for other bacterial cells. The concept thatresistance genes were present in the nature longtime before the utilisation of antibiotics to treat infections has been demonstrated in the past in afascinating study where the authors discoverednumerous resistance genes in analysing permafrostsediments (Antibiotic resistance is ancient. Nature477, 457–461).

“Some of the properties required toresist to these different conditionsare intrinsic to the bacterium but others require adaptation andare linked to genomic evolution, inparticular the acquisition of nucleicacid from various origins. Our purpose will be to evaluate the dif-ferent strategies used by S. aureusto evolve and adapt its genomecomposition and content following natural events or after expositionto selective pressure.”

In conclusion of these different observations, weshowed that resistance against antimicrobialscould result to exposition to the drug, or to the ac-quisition from another bacterial cell of a specificgene allowing resistance, thus following expositionto selection pressure. The most famous examplewas the introduction of the methicillin (a semi-syn-thetic penicillin resistant to β-lactamases) inhuman medicine in 1960. Only 6 months later, theisolation of the first MRSA was reported. The re-sistance was acquired through the integration of alarge genomic element containing the methicillin

resistance determinant as well as other resistancedeterminants, yielding to multi-resistant strains ofS. aureus which are difficult to eradicate. We ob-served the mobility of the SCCmec element in vivoin our hospital in a patient initially identified as aMSSA carrier over several months. Further assaysindicated a positive result for MRSA and the pa-tient was then decolonized for MRSA by treatmentwith topical agent and biocide (mupirocin andchlorhexidine). Subsequent screening showedthat the patient was still colonized with MSSA only.Molecular assay allowed us to demonstrate thatthe 3 strains (initial MSSA, MRSA and the lastMSSA) were clonal, indicating the mobility of theSCCmec element in this patient. A very recent ex-ample of evolution regarding bacterial tropism hasbeen reported in the veterinary clinic. S. aureusstrains infecting poultry showed increased capac-ity to grow at 42°C and to alter avian cells reflect-ing a “human-to-poultry host transition”. Note thatthis temperature in generally not compatible withgrowth of strains from the human clinic. This ob-servation implies that most of essential proteinsevolved through structure showing increase effi-ciency at 42°C, which is not the optimal growthtemperature of the species.

My group is also active in this domain of adapta-tion of animal S. aureus strains to the human clin-ics. Originally detected exclusively in pig farmersin Europe, S. aureus belonging to the clonal com-plex 398 (CC398) lineage has become a worldwidethreat associated with livestock, their human con-tacts and food products. During collaborationwork with Doctor Nathalie van Der Mee-Marquet(University Hospital of Tours, France), who per-formed an active bloodstream surveillance pro-gram since 2000, we observed constant increasein the prevalence of sequence type 398 (ST398) inpatients living in animal-free environments; from0% in 2007 to 15% of S. aureus isolates responsiblefor severe infections in 2015. Basically, this surveil-

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lance program allowed us to identify: i) ancestralST398 strain only able to colonize animal, ii) pre-evolved ST398 strains able to infect animal,iii) evolved strains infecting or colonizing humanin contact with animals and iv) ST398 isolates in-fecting human even without contact with animal.Important efforts were deployed using wholegenome sequencing approach on these differentpopulations of isolates. Analysis of the genomesof these isolates showed that each populationcontained specific prophage content. Whereas theancestral isolate associated with animal are de-void of prophage, emerging clades are character-ized by the presence of Φ3 prophage variants thatencode two immune-modulating proteins thatalter or prevent chemotaxis, phagocytosis andkilling of S. aureus by human neutrophils. Recently,we mobilized prophages from virulent strains andintroduced them in ancestral non-human patho-genic isolate. Our experiments clearly showed ac-quisition of new features, potentially mediatingthe virulence of the bacterium, accompanying ac-quisition of phages. In vitro experiments showedthat strains containing prophages have increasedcapacity to interact with human extracellular ma-trix proteins and increased ability to penetrateinto the cytoplasm of non-phagocytic cells. Even ifS. aureus is not recognized as an intracellular bac-terium, this capacity allows the bacterium to sur-vive in a protected niche, hidden from cellular orhumoral defences. In addition, in an experimentalmodel of infectious endocarditis, we showed thatST398 isolates containing prophages were moreprone to infect cardiac tissue and to multiplywithin cardiac vegetations, yielding to more severeinfection than the prophage-free parental strains.Phages serve as a driving force in bacterial patho-genesis, contributing both to the evolution of bac-terial hosts through gene transfer, and to bacterialpathogenesis at the time of infection. Temperatebacteriophages play an important role in the pathogenicity and cellular tropism of S. aureus.

Conclusion

Even if preservation of the genetic patrimony is essential for all organisms and cells are equippedwith systems dedicated to its protection, genomesare dynamic structures undergoing modifications.Some modifications are discreet (punctual)whereas other events are more important with thetransfer of large genetic elements. Ability to resistto environmental stresses, including the presenceof antimicrobial drugs is dependent on naturaladaptation or direct acquisition of new genomicelements. Natural adaptation could be a “try anderror” phenomena that results in bringing advantages and is more frequently observed inthe presence of selection pressure. Transfer oflarger element encoding for numerous genes isable to modify important phenotypic properties ofthe bacterial cells and modulate its virulence aswell as its host tropism.

My research is supported by the SNF (Grant number31003A_153474/1).

Dr Patrice FrancoisHead of Genomic Research LaboratoryGeneva University HospitalsTel: +41 223794118patrice.francois@genomic.chwww.genomic.chwww.hug-ge.ch

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Basic research is a key area that theSwiss National Science Foundation(SNSF) promotes and funds, as OpenAccess Government’s Ciara Ruane outlines…

The Swiss National Science Foundation (SNSF) primarily supports basic research: i.e., researchthat is not tied to a specific institution or industry,but seeks to improve knowledge overall. The SNSFdescribes the projects they fund as ‘use-inspired’,which means research that aims to provide prac-tical solutions to problems.

Use-Inspired Basic Research (UIBR) differs fromstandard basic research in a number of ways. TheSNSF defines it as dual-purpose, with an aim to“solve or illuminate one or several practical prob-lems, as well as advancing science”. Applicationsmarked UIBR face a more selective process and areless likely to be successful than others. Last yearthis approach was evaluated by an independentconsulting firm called  Technopolis, focusing ontheir definition of UIBR. The report stated that the introduction of the UIBR category in 2011 hadallowed the SNSF to broaden the scope of their research and did not require an overhaul. However,it recommended that the category be defined‘more clearly’. They also stated that the reducedsuccess rate of UIBR applications could be attrib-uted to the complexity of the dual-purpose approach. It also suggested more diverse selectionpanels and a greater emphasis on the ‘broader impact’ criteria outlined under UIBR.

Encouraging young people into research

The SNSF places a great deal of emphasis on encouraging young people to pursue careers in research and development. They outlined a 2013-2016 action plan to best achieve this. Theyemphasise a ‘career friendly’ approach, removingobstacles and restrictions that make it difficult foryoung people to secure a permanent role belowthe level of a professorship. The aim outlined wasto increase doctoral student salaries by 7%, in-crease the potential for promotion, and providesupport for doctorate students with families. The‘120% model’ is aimed at those who have childcareobligations impacting their work, and allows themto work reduced hours with the chance to applyfor childcare funding.

“The SNSF places a great deal ofemphasis on encouraging youngpeople to pursue careers in re-search and development.”

The SNFS has different funding programmes avail-able for researchers at different stages of their career. They have a guide on which programme isbest suited to the individual, detailing the level ofstudy, the nature of the test, and what resourcesare required. The overall aim is to provide com-plete flexibility for applicants, allowing people in amultitude of different circumstances to pursue acareer in research and development. They alsohave international study schemes available, throughpartnerships with countries in Asia and Eastern

Supporting basic researchin Switzerland

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Europe. They hope this allows young researchersthe opportunity to study in a new environmentand build an international network for Switzerlandwithin the scientific community.

Ethical practices

The SNFS makes a strong point of reinforcing eth-ical practices. In 2014 they published a documentdetailing their use of animal testing. The SNFS fol-lows the guidelines laid out by relevant authoritiesand does not get personally involved with deci-sions on the ethics of animal testing in individualresearch projects. The report states that animaltesting makes up for a small part of the researchprocess, and currently there is no effective alter-native available for most experiments. However,they state that animal suffering “must be kept toa minimum”, and researchers often use alterna-tive models such as cell structures. They state thatthey welcome debate on the issue, but distancethemselves from ‘misinformation’.

Gender equality

The SNFS takes a firm stance in favour of genderequality. Their statement on the issue outlines a

‘gender neutral’ atmosphere they hope to create,with a zero-tolerance policy towards any form ofprejudice. They have a number of legislative actions aimed to enforce this goal. They have agender equality grant, specifically targeted atyoung women researchers, a ‘non-discriminatorypay system’, work-at-home options to balancefamily life, and carries out regular evaluations toensure equality is being maintained. These effortsare ongoing, and the SNFS places responsibility onthe highest levels of the organisation. This goesalongside their aim of encouraging all young re-searchers and creating a more forward-thinkingresearch community.

Ciara RuaneCommissioning EditorAdjacent Open Accesscruane@adjacentopenaccess.orgwww.adjacentopenaccess.com www.twitter.com/OpenAccessGov

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