ORCID Profile
0000-0001-9915-1420
Current Organisations
Institut Pasteur
,
University of Melbourne
,
Université Pierre et Marie Curie
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Publisher: Microbiology Society
Date: 04-10-2023
Publisher: eLife Sciences Publications, Ltd
Date: 13-04-2023
Publisher: American Society for Microbiology
Date: 16-10-2023
Publisher: Oxford University Press (OUP)
Date: 13-01-2014
DOI: 10.1093/GBE/EVU010
Publisher: American Society for Microbiology
Date: 24-12-2019
Abstract: Staphylococcus epidermidis is a major cause of hospital-acquired infections, especially those related to implanted medical devices. Understanding how S. epidermidis causes disease and devising ways to combat these infections have been hindered by an inability to genetically manipulate clinically significant hospital-adapted strains. Here, we provide the first comprehensive analyses of the barriers to the uptake of foreign DNA in S. epidermidis and demonstrate that these are distinct from those described for S. aureus . Using these insights, we demonstrate an efficient approach for the genetic manipulation of S. epidermidis to enable the study of clinical isolates for the first time.
Publisher: Cold Spring Harbor Laboratory
Date: 22-05-2019
DOI: 10.1101/644856
Abstract: Staphylococcus epidermidis is a significant opportunistic pathogen of humans. Molecular studies in this species have been h ered by the presence of restriction-modification (RM) systems that limit introduction of foreign DNA. Here we establish the complete genomes and methylomes for seven clinically significant, genetically erse S. epidermidis isolates and perform the first systematic genomic analyses of the type I RM systems within both S. epidermidis and Staphylococcus aureus . Our analyses revealed marked differences in the gene arrangement, chromosomal location and movement of type I RM systems between the two species. Unlike S. aureus , S. epidermidis type I RM systems demonstrate extensive ersity even within a single genetic lineage. This is contrary to current assumptions and has important implications for approaching the genetic manipulation of S. epidermidis . Using Escherichia coli plasmid artificial modification (PAM) to express S. epidermidis hsdMS , we readily overcame restriction barriers in S. epidermidis , and achieved transformation efficiencies equivalent to those of modification deficient mutants. With these functional experiments we demonstrate how genomic data can be used to predict both the functionality of type I RM systems and the potential for a strain to be transformation proficient. We outline an efficient approach for the genetic manipulation of S. epidermidis from erse genetic backgrounds, including those that have hitherto been intractable. Additionally, we identified S. epidermidis BPH0736, a naturally restriction defective, clinically significant, multidrug-resistant ST2 isolate as an ideal candidate for molecular studies. Staphylococcus epidermidis is a major cause of hospital-acquired infections, especially those related to implanted medical devices. Understanding how S. epidermidis causes disease and devising ways to combat these infections has been hindered by an inability to genetically manipulate “hospital-adapted” strains that cause clinical disease. Here we provide the first comprehensive analyses of the mechanisms whereby S. epidermidis resists the uptake of foreign DNA and demonstrate that these are distinct from those described for S. aureus. Until now it had been assumed that these are the same. Using these insights, we demonstrate an efficient approach for the genetic manipulation of S. epidermidis to enable the study of clinically relevant isolates for the first time.
Publisher: American Society for Microbiology
Date: 11-02-2020
DOI: 10.1128/MSYSTEMS.00665-19
Abstract: Bacterial small RNAs (sRNAs) are RNA molecules that can have important regulatory roles across gene expression networks. There is a growing understanding of the scope and potential breadth of impact of sRNAs on global gene expression patterns in Staphylococcus aureus , a major human pathogen. Here, transcriptome comparisons were used to examine the roles of sRNA genes with a potential role in the response of S. aureus to antibiotic exposure. Although no measurable impact on key bacterial phenotypes was observed after deleting each of 18 sRNAs identified by these comparisons, this research is significant because it underscores the subtle modes of action of these sometimes abundant molecules within the bacterium.
Publisher: Oxford University Press (OUP)
Date: 23-05-2020
DOI: 10.1093/CID/CIAA609
Abstract: In a post hoc analysis of s les from an intrapartum azithromycin randomized clinical trial, we found that children whose mothers had been treated with the drug had higher prevalence of macrolide-resistance genes msr(A) and ermC at 28 days but not at 12 months. The 2 genes were positively associated in the nasopharynx. NCT1800942.
Publisher: Wiley
Date: 16-08-2013
DOI: 10.1111/MMI.12341
Abstract: Horizontal gene transfer (HGT) is a major force of microbial evolution but was long thought to be marginal in mycoplasmas. In silico detection of exchanged regions and of loci encoding putative Integrative Conjugative Elements (ICE) in several mycoplasma genomes challenged this view, raising the prospect of these simple bacteria being able to conjugate. Using the model pathogen Mycoplasma agalactiae, we demonstrated for the first time that one of these elements, ICEA, is indeed self-transmissible. As a hallmark of conjugative processes, ICEA transfers were DNase resistant and required viable cells. ICEA acquisition conferred ICE-negative strains with the new ability to conjugate, allowing the spread of ICEA. Analysis of transfer-deficient mutants indicated that this process requires an ICEA-encoded lipoprotein of unknown function, CDS14. Formation of a circular extrachromosomal intermediate and the subsequent chromosomal integration of ICEA involved CDS22, an ICEA-encoded product distantly related to the ISLre2 transposase family. Remarkably, ICEA has no specific or no preferential integration site, often resulting in gene disruptions. Occurrence of functional mycoplasma ICE offers these bacteria with a means for HGT, a phenomenon with far-reaching implications given their minute-size genome and the number of species that are pathogenic for a broad host-range.
Publisher: PeerJ
Date: 04-05-2018
DOI: 10.7717/PEERJ.4784
Abstract: Plasmid vectors based on bacteriophage integrases are important tools in molecular microbiology for the introduction of foreign DNA, especially into bacterial species where other systems for genetic manipulation are limited. Site specific integrases catalyze recombination between phage and bacterial attachment sites ( attP and attB , respectively) and the best studied integrases in the actinomycetes are the serine integrases from the Streptomyces bacteriophages ΦC31 and ΦBT1. As this reaction is unidirectional and highly stable, vectors containing phage integrase systems have been used in a number of genetic engineering applications. Plasmids bearing the ΦBT1 integrase have been used to introduce DNA into Streptomyces and Amycolatopsis strains however, they have not been widely studied in other actinobacterial genera. Here, we show that vectors based on ΦBT1 integrase can stably integrate into the chromosomes of a range of Nocardia species, and that this integration occurs despite the absence of canonical attB sites in these genomes. Furthermore, we show that a ΦBT1 integrase-based vector can insert at multiple pseudo- attB sites within a single strain and we determine the sequence of a pseudo- attB motif. These data suggest that ΦBT1 integrase-based vectors can be used to readily and semi-randomly introduce foreign DNA into the genomes of a range of Nocardia species. However, the precise site of insertion will likely require empirical determination in each species to avoid unexpected off-target effects.
Publisher: American Society for Microbiology
Date: 19-04-2021
DOI: 10.1128/AAC.02048-20
Abstract: Topical antibiotic preparations, such as fusidic acid (FA) or mupirocin, are used in the prevention and treatment of superficial skin infections caused by staphylococci. Previous genomic epidemiology work has suggested an association between the widespread use of topical antibiotics and the emergence of methicillin-resistant Staphylococcus aureus in some settings.
Publisher: Cold Spring Harbor Laboratory
Date: 28-02-2023
DOI: 10.1101/2023.02.27.530350
Abstract: Among the 16 two-component systems (TCSs) in the opportunistic human pathogen Staphylococcus aureus , only WalKR is essential. Like orthologous systems in other Bacillota, S. aureus WalKR controls autolysins involved in peptidoglycan remodelling and is therefore intimately involved in cell ision. However, despite the importance of WalKR in S. aureus , the basis for its essentiality is not understood and the regulon poorly defined. Here, we defined a consensus WalR DNA-binding motif and the direct WalKR regulon by using functional genomics, including ChIP-seq, with a panel of isogenic walKR mutants that had a spectrum of altered activities. Consistent with prior findings, the direct regulon includes multiple autolysin genes. However, this work also revealed that WalR directly regulates at least five essential genes involved in lipoteichoic acid synthesis ( ltaS ) translation (rplK ) DNA compaction ( hup ) initiation of DNA replication ( dnaA, hup ) and purine nucleotide metabolism ( prs ). Thus, WalKR in S. aureus serves as a polyfunctional regulator that contributes to fundamental control over critical cell processes by co-ordinately linking cell wall homeostasis with purine biosynthesis, protein biosynthesis, and DNA replication. Collectively, our findings address the essentiality of this locus and highlight the importance of WalKR as a bona fide target for novel anti-staphylococcal therapeutics.
Publisher: American Society for Microbiology
Date: 05-2013
DOI: 10.1128/JB.01745-12
Abstract: Integrative and conjugative elements (ICEs) have a major impact on gene flow and genome dynamics in bacteria. The ICEs Tn GBS1 and Tn GBS2 , first identified in Streptococcus agalactiae , use a DDE transposase, unlike most characterized ICEs, which depend on a phage-like integrase for their mobility. Here we identified 56 additional Tn GBS -related ICEs by systematic genome analysis. Interestingly, all except one are inserted in streptococcal genomes. Sequence comparison of the proteins conserved among these ICEs defined two subtypes related to Tn GBS1 or Tn GBS2 . We showed that both types encode different conjugation modules: a type IV secretion system, a VirD4 coupling protein, and a relaxase and its cognate oriT site, shared with distinct lineages of conjugative elements of Firmicutes . Phylogenetic analysis suggested that Tn GBS s evolved from two conjugative elements of different origins by the successive recruitment of a transposition module derived from insertion sequences (ISs). Furthermore, Tn GBS s share replication modules with different plasmids. Mutational analyses and conjugation experiments showed that Tn GBS1 and Tn GBS2 combine replication and transposition upstream promoters for their transfer and stabilization. Despite an evolutionarily successful horizontal dissemination within the genus Streptococcus , these ICEs have a restricted host range. However, we reveal that for Tn GBS1 and Tn GBS2 , this host restriction is not due to a transfer incompatibility linked to the conjugation machineries but most likely to their ability for transient maintenance through replication after their transfer.
Publisher: Cold Spring Harbor Laboratory
Date: 28-02-2018
DOI: 10.1101/273904
Abstract: Large-scale genomic studies of within-host evolution during Staphylococcus aureus bacteraemia (SAB) are needed to understanding bacterial adaptation underlying persistence and thus refining the role of genomics in management of SAB. However, available comparative genomic studies of sequential SAB isolates have tended to focus on selected cases of unusually prolonged bacteraemia, where secondary antimicrobial resistance has developed. To understand the bacterial genomic evolution during SAB more broadly, we applied whole genome sequencing to a large collection of sequential isolates obtained from patients with persistent or relapsing bacteraemia. We show that, while adapation pathways are heterogenous and episode-specific, isolates from persistent bacteraemia have a distinctive molecular signature, characterised by a low mutation frequency and high proportion of non-silent mutations. By performing an extensive analysis of structural genomic variants in addition to point mutations, we found that these often overlooked genetic events are commonly acquired during SAB. We discovered that IS 256 insertion may represent the most effective driver of within-host microevolution in selected lineages, with up to three new insertion events per isolate even in the absence of other mutations. Genetic mechanisms resulting in significant phenotypic changes, such as increases in vancomycin resistance, development of small colony phenotypes, and decreases in cytotoxicity, included mutations in key genes ( rpoB, stp, agrA ) and an IS256 insertion upstream of the walKR operon. This study provides for the first time a large-scale analysis of within-host evolution during invasive S. aureus infection and describes specific patterns of adaptation that will be informative for both understanding S. aureus pathoadaptation and utilising genomics for management of complicated S. aureus infections.
Publisher: eLife Sciences Publications, Ltd
Date: 08-06-2023
DOI: 10.7554/ELIFE.84778
Abstract: Staphylococcus aureus infections are associated with high mortality rates. Often considered an extracellular pathogen, S. aureus can persist and replicate within host cells, evading immune responses, and causing host cell death. Classical methods for assessing S. aureus cytotoxicity are limited by testing culture supernatants and endpoint measurements that do not capture the phenotypic ersity of intracellular bacteria. Using a well-established epithelial cell line model , we have developed a platform called InToxSa ( in tracellular tox icity of S. a ureus ) to quantify intracellular cytotoxic S. aureus phenotypes. Studying a panel of 387 S . aureus bacteraemia isolates, and combined with comparative, statistical, and functional genomics, our platform identified mutations in S. aureus clinical isolates that reduced bacterial cytotoxicity and promoted intracellular persistence. In addition to numerous convergent mutations in the Agr quorum sensing system, our approach detected mutations in other loci that also impacted cytotoxicity and intracellular persistence. We discovered that clinical mutations in ausA , encoding the aureusimine non-ribosomal peptide synthetase, reduced S. aureus cytotoxicity, and increased intracellular persistence. InToxSa is a versatile, high-throughput cell-based phenomics platform and we showcase its utility by identifying clinically relevant S. aureus pathoadaptive mutations that promote intracellular residency.
Publisher: Public Library of Science (PLoS)
Date: 10-01-2022
DOI: 10.1371/JOURNAL.PPAT.1010166
Abstract: A hallmark of Listeria (L . ) monocytogenes pathogenesis is bacterial escape from maturing entry vacuoles, which is required for rapid bacterial replication in the host cell cytoplasm and cell-to-cell spread. The bacterial transcriptional activator PrfA controls expression of key virulence factors that enable exploitation of this intracellular niche. The transcriptional activity of PrfA within infected host cells is controlled by allosteric coactivation. Inhibitory occupation of the coactivator site has been shown to impair PrfA functions, but consequences of PrfA inhibition for L . monocytogenes infection and pathogenesis are unknown. Here we report the crystal structure of PrfA with a small molecule inhibitor occupying the coactivator site at 2.0 Å resolution. Using molecular imaging and infection studies in macrophages, we demonstrate that PrfA inhibition prevents the vacuolar escape of L . monocytogenes and enables extensive bacterial replication inside spacious vacuoles. In contrast to previously described spacious Listeria -containing vacuoles, which have been implicated in supporting chronic infection, PrfA inhibition facilitated progressive clearance of intracellular L . monocytogenes from spacious vacuoles through lysosomal degradation. Thus, inhibitory occupation of the PrfA coactivator site facilitates formation of a transient intravacuolar L . monocytogenes replication niche that licenses macrophages to effectively eliminate intracellular bacteria. Our findings encourage further exploration of PrfA as a potential target for antimicrobials and highlight that intra-vacuolar residence of L . monocytogenes in macrophages is not inevitably tied to bacterial persistence.
Publisher: Cold Spring Harbor Laboratory
Date: 31-01-2018
DOI: 10.1101/257915
Abstract: Acquired mutations are a major mechanism of bacterial antibiotic resistance generation and dissemination, and can arise during treatment of infections. Early detection of sub-populations of resistant bacteria harbouring defined resistance mutations could prevent inappropriate antibiotic prescription. Here we present RM-seq, a new licon-based DNA sequencing workflow based on single molecule barcoding coupled with deep-sequencing that enables the high-throughput characterisation and sensitive detection of resistance mutations from complex mixed populations of bacteria. We show that RM-seq reduces both background sequencing noise and PCR lification bias and allows highly sensitive identification and accurate quantification of antibiotic resistant sub-populations, with relative allele frequencies as low as 10 -4 . We applied RM-seq to identify and quantify rif icin resistance mutations in Staphylococcus aureus using pools of 10,000 in vitro selected clones and identified a large number of previously unknown resistance-associated mutations. Targeted mutagenesis and phenotypic resistance testing was used to validate the technique and demonstrate that RM-seq can be used to link subsets of mutations with clinical resistance breakpoints at high-throughput using large pools of in vitro selected resistant clones. Differential analysis of the abundance of resistance mutations after a selection bottleneck detected antimicrobial cross-resistance and collateral sensitivity-conferring mutations. Using a mouse infection model and human clinical s les, we also demonstrate that RM-seq can be effectively applied in vivo to track complex mixed populations of S. aureus and another major human pathogen, Mycobacterium tuberculosis during infections. RM-seq is a powerful new tool to both detect and functionally characterise mutational antibiotic resistance.
Publisher: Oxford University Press (OUP)
Date: 19-08-2019
DOI: 10.1093/JAC/DKZ341
Abstract: Oral azithromycin given during labour reduces carriage of bacteria responsible for neonatal sepsis, including Staphylococcus aureus. However, there is concern that this may promote drug resistance. Here, we combine genomic and epidemiological data on S. aureus isolated from mothers and babies in a randomized intra-partum azithromycin trial (PregnAnZI) to describe bacterial population dynamics and resistance mechanisms. Participants from both arms of the trial, who carried S. aureus in day 3 and day 28 s les post-intervention, were included. Sixty-six S. aureus isolates (from 7 mothers and 10 babies) underwent comparative genome analyses and the data were then combined with epidemiological data. Trial registration (main trial): ClinicalTrials.gov Identifier NCT01800942. Seven S. aureus STs were identified, with ST5 dominant (n = 40, 61.0%), followed by ST15 (n = 11, 17.0%). ST5 predominated in the placebo arm (73.0% versus 49.0%, P = 0.039) and ST15 in the azithromycin arm (27.0% versus 6.0%, P = 0.022). In azithromycin-resistant isolates, msr(A) was the main macrolide resistance gene (n = 36, 80%). Ten study participants, from both trial arms, acquired azithromycin-resistant S. aureus after initially harbouring a susceptible isolate. In nine (90%) of these cases, the acquired clone was an msr(A)-containing ST5 S. aureus. Long-read sequencing demonstrated that in ST5, msr(A) was found on an MDR plasmid. Our data reveal in this Gambian population the presence of a dominant clone of S. aureus harbouring plasmid-encoded azithromycin resistance, which was acquired by participants in both arms of the study. Understanding these resistance dynamics is crucial to defining the public health drug resistance impacts of azithromycin prophylaxis given during labour in Africa.
Publisher: Cold Spring Harbor Laboratory
Date: 16-05-2016
DOI: 10.1101/053728
Abstract: Alcohol-based hand rubs are international pillars of hospital infection control, restricting transmission of pathogens such as Staphylococcus aureus . Despite this success, health care infections caused by Enterococcus faecium (Efm) - another multidrug resistant pathogen - are increasing. We tested alcohol tolerance of 139 hospital Efm isolates, obtained between 1997 and 2015 and found Efm post-2010 were 10-fold more tolerant to alcohol killing than older isolates. Using a mouse infection control model, we then showed that alcohol tolerant Efm resisted standard 70% isopropanol surface disinfection and led to gastrointestinal colonization significantly more often than alcohol sensitive Efm. We next looked for bacterial genomic signatures of adaptation. Tolerant Efm have independently accumulated mutations modifying genes involved in carbohydrate uptake and metabolism. Mutagenesis confirmed their roles in isopropanol tolerance. These findings suggest bacterial adaptation and complicate infection control recommendations. Additional policies and procedures to prevent Efm spread are required.
Publisher: Cold Spring Harbor Laboratory
Date: 12-02-2022
DOI: 10.1101/2022.02.11.480068
Abstract: During severe infections, Staphylococcus aureus moves from its colonising sites to blood and tissues, and is exposed to new selective pressures, thus potentially driving adaptive evolution. Previous studies have shown the key role of the agr locus in S. aureus pathoadaptation, however a more comprehensive characterisation of genetic signatures of bacterial adaptation may enable prediction of clinical outcomes and reveal new targets for treatment and prevention of these infections. Here, we measured adaptation using within-host evolution analysis of 2,590 S. aureus genomes from 396 independent episodes of infection. By capturing a comprehensive repertoire of single-nucleotide and structural genome variations, we found evidence of a distinctive evolutionary pattern within the infecting populations compared to colonising bacteria. These invasive strains had up to 20-fold enrichments for genome degradation signatures and displayed significantly convergent mutations in a distinctive set of genes, linked to antibiotic response and pathogenesis. In addition to agr -mediated adaptation we identified non-canonical, genome-wide significant loci including sucA - sucB and stp1 . The prevalence of adaptive changes increased with infection extent, emphasising the clinical significance of these signatures. These findings provide a high-resolution picture of the molecular changes when S. aureus transitions from colonisation to severe infection and may inform correlation of infection outcomes with adaptation signatures.
Publisher: Springer Science and Business Media LLC
Date: 18-03-2023
DOI: 10.1038/S41467-023-37200-W
Abstract: Even in the setting of optimal resuscitation in high-income countries severe sepsis and septic shock have a mortality of 20–40%, with antibiotic resistance dramatically increasing this mortality risk. To develop a reference dataset enabling the identification of common bacterial targets for therapeutic intervention, we applied a standardized genomic, transcriptomic, proteomic and metabolomic technological framework to multiple clinical isolates of four sepsis-causing pathogens: Escherichia coli , Klebsiella pneumoniae species complex, Staphylococcus aureus and Streptococcus pyogenes . Exposure to human serum generated a sepsis molecular signature containing global increases in fatty acid and lipid biosynthesis and metabolism, consistent with cell envelope remodelling and nutrient adaptation for osmoprotection. In addition, acquisition of cholesterol was identified across the bacterial species. This detailed reference dataset has been established as an open resource to support discovery and translational research.
Publisher: American Society for Microbiology
Date: 28-02-2018
Abstract: Increasing antibiotic resistance in the major human pathogen Staphylococcus aureus is threatening the ability to treat patients with these infections. Recent laboratory studies suggest that mutations in the gene commonly associated with rif in resistance may also impact susceptibility to other last-line antibiotics in S. aureus however, the overall frequency and clinical impact of these mutations are unknown. By mining a global collection of clinical S. aureus genomes and by mutagenesis experiments, this work reveals that common rif in-induced rpoB mutations promote phenotypic plasticity that has led to the global emergence of stable, multidrug-resistant S. aureus lineages that are associated with increased risk of therapeutic failure through coresistance to other last-line antimicrobials. We recommend decreasing susceptibility breakpoints for rif in to allow phenotypic detection of critical rpoB mutations conferring low resistance to rif in and reconsidering the appropriate use of rif in to reduce the fixation and spread of these deleterious mutations globally.
Publisher: American Association for the Advancement of Science (AAAS)
Date: 08-2018
DOI: 10.1126/SCITRANSLMED.AAR6115
Abstract: Alcohol-based disinfectants and particularly hand rubs are a key way to control hospital infections worldwide. Such disinfectants restrict transmission of pathogens, such as multidrug-resistant
Publisher: Cold Spring Harbor Laboratory
Date: 13-12-2022
DOI: 10.1101/2022.12.11.519971
Abstract: Staphylococcus aureus infections are associated with high mortality rates. Often considered an extracellular pathogen, S. aureus can persist and replicate within host cells, evading immune responses and causing host cell death. Classical methods for assessing S. aureus cytotoxicity are limited by testing culture supernatants and endpoint measurements that do not capture the phenotypic ersity of intracellular bacteria. Using a well-established epithelial cell line model, we have developed a platform called InToxSa ( In tracellular Tox icity of S. a ureus ) to quantify intracellula cytotoxic S. aureus phenotypes. Studying a panel of 387 S. aureus bacteraemia isolates, and combined with comparative, statistical and functional genomics, our platform identified mutations in S. aureus clinical isolates that reduced bacterial cytotoxicity and promoted intracellular persistence. In addition to numerous convergent mutations in the Agr quorum sensing system, our approach detected mutations in other loci that also impacted cytotoxicity and intracellular persistence. We discovered that clinical mutations in ausA, encoding the aureusimine non-ribosomal peptide synthetase, reduced S. aureus cytotoxicity and increased intracellular persistence. InToxSa is a versatile, high-throughput cell-based phenomics platform and we showcase its utility by identifying clinically relevant S. aureus pathoadaptive mutations that promote intracellular residency.
Publisher: Elsevier BV
Date: 12-2013
DOI: 10.1016/J.RESMIC.2013.09.002
Abstract: Streptococcus gallolyticus is an emerging cause of infective endocarditis that has been epidemiologically linked to colorectal cancer. S. gallolyticus is poorly transformable using electroporation and no defined mutant has been published yet. Hence, we used mobilization to introduce plasmid DNA from Streptococcus agalactiae into S. gallolyticus using the transfer origin of the conjugative element TnGBS1 (oriTTnGBS1), followed by a classical homologous recombination technique. Two isogenic mutants of S. gallolyticus UCN34, one deleted for the pil1 pilus operon and another for the sortase A gene, were constructed and characterized. This genetic tool should help in unravelling virulence mechanisms of this bacterium.
Publisher: Springer Science and Business Media LLC
Date: 23-08-2018
Publisher: Springer Science and Business Media LLC
Date: 31-08-2018
Publisher: Proceedings of the National Academy of Sciences
Date: 16-09-2019
Abstract: Staphylococcus aureus small-colony variants (SCVs) are associated with unusually chronic and persistent infections despite active antibiotic treatment. The molecular basis for this clinically important phenomenon is poorly understood, h ered by the instability of the SCV phenotype. Here we investigated the genetic basis for an unstable S. aureus SCV that arose spontaneously while studying rif icin resistance. This SCV showed no nucleotide differences across its genome compared with a normal-colony variant (NCV) revertant, yet the SCV presented the hallmarks of S. aureus linked to persistent infection: down-regulation of virulence genes and reduced hemolysis and neutrophil chemotaxis, while exhibiting increased survival in blood and ability to invade host cells. Further genome analysis revealed chromosome structural variation uniquely associated with the SCV. These variations included an asymmetric inversion across half of the S. aureus chromosome via recombination between type I restriction modification system (T1RMS) genes, and the activation of a conserved prophage harboring the immune evasion cluster (IEC). Phenotypic reversion to the wild-type–like NCV state correlated with reversal of the chromosomal inversion (CI) and with prophage stabilization. Further analysis of 29 complete S. aureus genomes showed strong signatures of recombination between hsdMS genes, suggesting that analogous CI has repeatedly occurred during S. aureus evolution. Using qPCR and long-read licon deep sequencing, we detected subpopulations with T1RMS rearrangements causing CIs and prophage activation across major S. aureus lineages. Here, we have discovered a previously unrecognized and widespread mechanism of reversible genomic instability in S. aureus associated with SCV generation and persistent infections.
Publisher: Elsevier BV
Date: 08-2020
Publisher: American Society for Microbiology
Date: 22-12-2020
Abstract: The majority of Staphylococcus aureus strains causing human disease are methicillin-susceptible (MSSA) and can be treated with antistaphylococcal penicillins (such as oxacillin). While acquisition of the mec gene represents the main resistance mechanism to oxacillin, S. aureus can acquire low-level resistance through adaptive mutations in other genes. In this study, we used genomic approaches to understand the basis of S. aureus adaption to oxacillin and its dynamic at the population level. By combining a genome analysis of clinical isolates from persistent MSSA infections, in vitro selection of oxacillin resistance, and genome-wide association analysis on a large collection of isolates, we identified 21 genes linked to secondary oxacillin resistance. Adaptive mutations in these genes were easy to select when S. aureus was exposed to oxacillin, but they also came at a substantial cost in terms of bacterial fitness, suggesting that this phenotype emerges preferentially in the setting of sustained antibiotic exposure.
No related grants have been discovered for Romain Guerillot.