A statistical genomics framework to trace bacterial genomic predictors of clinical outcomes in Staphylococcus aureus bacteremia.

Outcomes of severe bacterial infections are determined by the interplay between host, pathogen, and treatments. While human genomics has provided insights into host factors impacting Staphylococcus aureus infections, comparatively little is known about S. aureus genotypes and disease severity. Building on the hypothesis that bacterial pathoadaptation is a key outcome driver, we developed a genome-wide association study (GWAS) framework to identify adaptive mutations associated with treatment failure and mortality in S. aureus bacteremia (1,358 episodes). Our research highlights the potential of vancomycin-selected mutations and vancomycin minimum inhibitory concentration (MIC) as key explanatory variables to predict infection severity. The contribution of bacterial variation was much lower for clinical outcomes (heritability <5%); however, GWASs allowed us to identify additional, MIC-independent candidate pathogenesis loci. Using supervised machine learning, we were able to quantify the predictive potential of these adaptive signatures. Our statistical genomics framework provides a powerful means to capture adaptive mutations impacting severe bacterial infections.

Case Commentary: The hidden side of oxacillin resistance in Staphylococcus aureus.

Acquisition of PBP2a (encoded by the mec gene) is the key resistance mechanism to ß-lactams in Staphylococcus aureus. The mec gene can be easily detected by PCR assays; however, these tools will miss mec-independent oxacillin resistance. This phenotype is mediated by mutations in cell wall metabolism genes that can be acquired during persistent infections under prolonged antibiotic exposure. The complex case presented by Hess et al. (Antimicrob Agents Chemother 67:e00437-23, 2023, https://doi.org/10.1128/aac.00437-23) highlights the diagnostic and therapeutic challenges in the management of mec-independent oxacillin resistance.

A high-throughput cytotoxicity screening platform reveals agr-independent mutations in bacteraemia-associated Staphylococcus aureus that promote intracellular persistence.

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 diversity of intracellular bacteria. Using a well-established epithelial cell line model, we have developed a platform called InToxSa (intracellular toxicity of S. aureus) 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.

A multi-site, international laboratory study to assess the performance of penicillin susceptibility testing of Staphylococcus aureus.

OBJECTIVES: There is clinical uncertainty over the optimal treatment for penicillin-susceptible Staphylococcus aureus (PSSA) infections. Furthermore, there is concern that phenotypic penicillin susceptibility testing methods are not reliably able to detect some blaZ-positive S. aureus. METHODS: Nine S. aureus isolates, including six genetically diverse strains harbouring blaZ, were sent in triplicate to 34 participating laboratories from Australia (n = 14), New Zealand (n = 6), Canada (n = 12), Singapore (n = 1) and Israel (n = 1). We used blaZ PCR as the gold standard to assess susceptibility testing performance of CLSI (P10 disc) and EUCAST (P1 disc) methods. Very major errors (VMEs), major error (MEs) and categorical agreement were calculated. RESULTS: Twenty-two laboratories reported 593 results according to CLSI methodology (P10 disc). Nineteen laboratories reported 513 results according to the EUCAST (P1 disc) method. For CLSI laboratories, the categorical agreement and calculated VME and ME rates were 85% (508/593), 21% (84/396) and 1.5% (3/198), respectively. For EUCAST laboratories, the categorical agreement and calculated VME and ME rates were 93% (475/513), 11% (84/396) and 1% (3/198), respectively. Seven laboratories reported results for both methods, with VME rates of 24% for CLSI and 12% for EUCAST. CONCLUSIONS: The EUCAST method with a P1 disc resulted in a lower VME rate compared with the CLSI methods with a P10 disc. These results should be considered in the context that among collections of PSSA isolates, as determined by automated MIC testing, less than 10% harbour blaZ. Furthermore, the clinical relevance of phenotypically susceptible, but blaZ-positive S. aureus, remains unclear.

All Staphylococcus aureus bacteraemia-inducing strains can cause infective endocarditis: Results of GWAS and experimental animal studies.

OBJECTIVES: We aimed at determining whether specific S. aureus strains cause infective endocarditis (IE) in the course of Staphylococcus aureus bacteraemia (SAB). METHODS: A genome-wide association study (GWAS) including 924 S. aureus genomes from IE (274) and non-IE (650) SAB patients from international cohorts was conducted, and a subset of strains was tested with two experimental animal models of IE, one investigating the early step of bacterial adhesion to inflamed mice valves, the second evaluating the local and systemic developmental process of IE on mechanically-damaged rabbit valves. RESULTS: The genetic profile of S. aureus IE and non-IE SAB strains did not differ when considering single nucleotide polymorphisms, coding sequences, and k-mers analysed in GWAS. In the murine inflammation-induced IE model, no difference was observed between IE and non-IE SAB strains both in terms of adhesion to the cardiac valves and in the propensity to cause IE; in the mechanical IE-induced rabbit model, there was no difference between IE and non-IE SAB strains regarding the vegetation size and CFU. CONCLUSION: All strains of S. aureus isolated from SAB patients must be considered as capable of causing this common and lethal infection once they have accessed the bloodstream.

Staphylococcus aureus host interactions and adaptation.

Invasive Staphylococcus aureus infections are common, causing high mortality, compounded by the propensity of the bacterium to develop drug resistance. S. aureus is an excellent case study of the potential for a bacterium to be commensal, colonizing, latent or disease-causing; these states defined by the interplay between S. aureus and host. This interplay is multidimensional and evolving, exemplified by the spread of S. aureus between humans and other animal reservoirs and the lack of success in vaccine development. In this Review, we examine recent advances in understanding the S. aureus-host interactions that lead to infections. We revisit the primary role of neutrophils in controlling infection, summarizing the discovery of new immune evasion molecules and the discovery of new functions ascribed to well-known virulence factors. We explore the intriguing intersection of bacterial and host metabolism, where crosstalk in both directions can influence immune responses and infection outcomes. This Review also assesses the surprising genomic plasticity of S. aureus, its dualism as a multi-mammalian species commensal and opportunistic pathogen and our developing understanding of the roles of other bacteria in shaping S. aureus colonization.

Niche-specific genome degradation and convergent evolution shaping Staphylococcus aureus adaptation during severe infections.

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 2590 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.

The bacterium Staphylococcus aureus lives harmlessly on our skin and noses. However, occasionally, it gets into our blood and internal organs, such as our bones and joints, where it causes severe, long-lasting infections that are difficult to treat. Over time, S. aureus acquire characteristics that help them to adapt to different locations, such as transitioning from the nose to the blood, and avoid being killed by antibiotics. Previous studies have identified changes, or 'mutations', in genes that are likely to play an important role in this evolutionary process. One of these genes, called accessory gene regulator (or agr for short), has been shown to control the mechanisms S. aureus use to infect cells and disseminate in the body. However, it is unclear if there are changes in other genes that also help S. aureus adapt to life inside the human body. To help resolve this mystery, Giulieri et al. collected 2,500 samples of S. aureus from almost 400 people. This included bacteria harmlessly living on the skin or in the nose, as well as strains that caused an infection. Gene sequencing revealed a small number of genes, referred to as 'adaptive genes', that often acquire mutations during infection. Of these, agr was the most commonly altered. However, mutations in less well-known genes were also identified: some of these genes are related to resistance to antibiotics, while others are involved in chemical processes that help the bacteria to process nutrients. Most mutations were caused by random errors being introduced in to the bacteria's genetic code which stopped genes from working. However, in some cases, genes were turned off by small fragments of DNA moving around and inserting themselves into different parts of the genome. This study highlights a group of genes that help S. aureus to thrive inside the body and cause severe and prolonged infections. If these results can be confirmed, it may help to guide which antibiotics are used to treat different infections. Furthermore, understanding which genes are important for infection could lead to new strategies for eliminating this dangerous bacterium.

Stapled Porcine Pericardium Displays Lower Infectivity In Vitro Than Native and Sutured Porcine Pericardium.

BACKGROUND: Biological xenografts using tubulized porcine pericardium are an alternative to replace infected prosthetic graft. We recently reported an innovative technique using a stapled porcine pericardial bioconduit for immediate vascular reconstruction in emergency. The objective of this study is to compare the growth and adherence to grafts of bacteria and yeast incubated with stapled porcine pericardium, sutured or naked pericardium. MATERIAL AND METHODS: One square centimeter of porcine pericardial patches, with or without staples or sutures, was incubated with 105 colony forming units of Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, and Candida albicans for 1, 6, and 24 h. The medium was collected to quantify planktonic microorganisms, while grafts were sonicated to quantify adherent microorganisms. Dacron and Dacron Silver were analyzed in parallel as synthetic reference prostheses. RESULTS: Stapled porcine pericardium reduced the growth and the adherence of E coli (2- to 30-fold; P < 0.0005), S aureus (11- to 1000-fold; P < 0.0006), S epidermidis (>500-fold; P < 0.0001), and C albicans (12- to 50-fold; P < 0.0001) when compared to medium alone (growth) and pericardium or Dacron (adherence). Native and sutured porcine pericardium interfered with the growth and the adherence of E coli and C albicans, and Dacron with that of S epidermidis. As expected, Dacron Silver was robustly bactericidal. CONCLUSIONS: Stapled porcine pericardium exhibited a lower susceptibility to infection by bacteria and yeasts in vitro when compared to the native and sutured porcine pericardium. Stapled porcine pericardium might be a good option for rapid vascular grafting without increasing infectivity.

Comprehensive Genomic Investigation of Adaptive Mutations Driving the Low-Level Oxacillin Resistance Phenotype in Staphylococcus aureus.

Antistaphylococcal penicillins such as oxacillin are the key antibiotics in the treatment of invasive methicillin-susceptible Staphylococcus aureus (MSSA) infections; however, mec gene-independent resistance adaptation can cause treatment failure. Despite its clinical relevance, the basis of this phenomenon remains poorly understood. Here, we investigated the genomic adaptation to oxacillin at an unprecedented scale using a large collection of 503 clinical mec-negative isolates and 30 in vitro-adapted isolates from independent oxacillin exposures. By combining comparative genomics, evolutionary convergence, and genome-wide association analysis, we found 21 genetic loci associated with low-level oxacillin resistance, underscoring the polygenic nature of this phenotype. Evidence of adaptation was particularly strong for the c-di-AMP signal transduction pathways (gdpP and dacA) and in the clpXP chaperone-protease complex. The role of mutations in gdpP in conferring low-level oxacillin resistance was confirmed by allele-swapping experiments. We found that resistance to oxacillin emerges at high frequency in vitro (median, 2.9 × 10-6; interquartile range [IQR], 1.9 × 10-6 to 3.9 × 10-6), which is consistent with a recurrent minimum inhibitory concentration (MIC) increase across the global phylogeny of clinical isolates. Nevertheless, adaptation in clinical isolates appears sporadically, with no stably adapted lineages, suggesting a high fitness cost of resistance, confirmed by growth assessment of mutants in rich media. Our data provide a broader understanding of the emergence and dynamics of oxacillin resistance adaptation in S. aureus and a framework for future surveillance of this clinically important phenomenon.IMPORTANCE 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.

Using genomics to understand meticillin- and vancomycin-resistant Staphylococcus aureus infections.

Resistance to meticillin and vancomycin in Staphylococcus aureus significantly complicates the management of severe infections like bacteraemia, endocarditis or osteomyelitis. Here, we review the molecular mechanisms and genomic epidemiology of resistance to these agents, with a focus on how genomics has provided insights into the emergence and evolution of major meticillin-resistant S. aureus clones. We also provide insights on the use of bacterial whole-genome sequencing to inform management of S. aureus infections and for control of transmission at the hospital and in the community.

Unstable chromosome rearrangements in Staphylococcus aureus cause phenotype switching associated with persistent infections.

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, hampered by the instability of the SCV phenotype. Here we investigated the genetic basis for an unstable S. aureus SCV that arose spontaneously while studying rifampicin 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 amplicon 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.

Successful treatment with daptomycin and ceftaroline of MDR Staphylococcus aureus native valve endocarditis: a case report.

OBJECTIVES: The best therapeutic approach for treating MRSA endocarditis remains unknown, particularly in cases of high vancomycin MICs. We report here a case of daptomycin-non-susceptible, ceftaroline-resistant and fosfomycin-resistant MRSA native left valve endocarditis that was successfully treated with valve repair and a combination of high-dose daptomycin and ceftaroline. METHODS: Antimicrobial testing of the clinical strain was performed using Etest and microdilution broth methods. Time-kill and chequerboard methodologies were used to test the activity of antibiotic combinations. RESULTS: By Etest, the MIC of vancomycin was 2 mg/L, the MIC of daptomycin was 2 mg/L, the MIC of fosfomycin was 1024 mg/L and the MIC of ceftaroline was 1.5 mg/L. At the standard inoculum (105 cfu/mL), the three combinations of daptomycin plus ceftaroline, cloxacillin or fosfomycin were synergistic and bactericidal. However, when these combinations were tested using a higher inoculum (108 cfu/mL), all combinations were synergistic, but only daptomycin plus ceftaroline had bactericidal activity. CONCLUSIONS: These results confirmed a synergistic effect between daptomycin plus ceftaroline and increased bactericidal activity against MRSA, suggesting that this combination may be effective for the treatment of invasive MRSA infection. Our experience highlights the potential clinical use of synergy testing to guide difficult treatment decisions in patients with MDR MRSA infection.

Comprehensive antibiotic-linked mutation assessment by resistance mutation sequencing (RM-seq).

Mutation acquisition is a major mechanism of bacterial antibiotic resistance that remains insufficiently characterised. Here we present RM-seq, a new amplicon-based deep sequencing workflow based on a molecular barcoding technique adapted from Low Error Amplicon sequencing (LEA-seq). RM-seq allows detection and functional assessment of mutational resistance at high throughput from mixed bacterial populations. The sensitive detection of very low-frequency resistant sub-populations permits characterisation of antibiotic-linked mutational repertoires in vitro and detection of rare resistant populations during infections. Accurate quantification of resistance mutations enables phenotypic screening of mutations conferring pleiotropic phenotypes such as in vivo persistence, collateral sensitivity or cross-resistance. RM-seq will facilitate comprehensive detection, characterisation and surveillance of resistant bacterial populations ( https://github.com/rguerillot/RM-seq ).

Genomic exploration of sequential clinical isolates reveals a distinctive molecular signature of persistent Staphylococcus aureus bacteraemia.

BACKGROUND: Large-scale genomic studies of within-host diversity in 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. METHODS: To understand bacterial genetic diversity during SAB more broadly, we applied whole genome sequencing to a large collection of sequential isolates obtained from patients with persistent or relapsing bacteraemia. After excluding genetically unrelated isolates, we performed an in-depth genomic analysis of point mutations and chromosome structural variants arising within individual SAB episodes. RESULTS: We show that, while adaptation 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. Analysis of structural genomic variants revealed that these often overlooked genetic events are commonly acquired during SAB. We discovered that IS256 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. CONCLUSIONS: This study provides for the first time a large-scale analysis of within-host genomic changes 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.

Convergent Evolution Driven by Rifampin Exacerbates the Global Burden of Drug-Resistant Staphylococcus aureus.

Mutations in the beta-subunit of bacterial RNA polymerase (RpoB) cause resistance to rifampin (Rifr), a critical antibiotic for treatment of multidrug-resistant Staphylococcus aureus. In vitro studies have shown that RpoB mutations confer decreased susceptibility to other antibiotics, but the clinical relevance is unknown. Here, by analyzing 7,099 S. aureus genomes, we demonstrate that the most prevalent RpoB mutations promote clinically relevant phenotypic plasticity resulting in the emergence of stable S. aureus lineages, associated with increased risk of therapeutic failure through generation of small-colony variants (SCVs) and coresistance to last-line antimicrobial agents. We found eight RpoB mutations that accounted for 93% (469/505) of the total number of Rifr mutations. The most frequently selected amino acid substitutions affecting residue 481 (H481N/Y) were associated with worldwide expansions of Rifr clones spanning decades. Recreating the H481N/Y mutations confirmed no impact on S. aureus growth, but the H481N mutation promoted the emergence of a subpopulation of stable Rifr SCVs with reduced susceptibility to vancomycin and daptomycin. Recreating the other frequent RpoB mutations showed similar impacts on resistance to these last-line agents. We found that 86% of all Rifr isolates in our global sample carried the mutations promoting cross-resistance to vancomycin and 52% to both vancomycin and daptomycin. As four of the most frequent RpoB mutations confer only low-level Rifr, equal to or below some international breakpoints, we recommend decreasing these breakpoints and reconsidering the appropriate use of rifampin to reduce the fixation and spread of these clinically deleterious mutations. IMPORTANCE 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 rifampin 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 rifampin-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 rifampin to allow phenotypic detection of critical rpoB mutations conferring low resistance to rifampin and reconsidering the appropriate use of rifampin to reduce the fixation and spread of these deleterious mutations globally.

Genomic analysis of ST88 community-acquired methicillin resistant Staphylococcus aureus in Ghana.

BACKGROUND: The emergence and evolution of community-acquired methicillin resistant Staphylococcus aureus (CA-MRSA) strains in Africa is poorly understood. However, one particular MRSA lineage called ST88, appears to be rapidly establishing itself as an "African" CA-MRSA clone. In this study, we employed whole genome sequencing to provide more information on the genetic background of ST88 CA-MRSA isolates from Ghana and to describe in detail ST88 CA-MRSA isolates in comparison with other MRSA lineages worldwide. METHODS: We first established a complete ST88 reference genome (AUS0325) using PacBio SMRT sequencing. We then used comparative genomics to assess relatedness among 17 ST88 CA-MRSA isolates recovered from patients attending Buruli ulcer treatment centres in Ghana, three non-African ST88s and 15 other MRSA lineages. RESULTS: We show that Ghanaian ST88 forms a discrete MRSA lineage (harbouring SCCmec-IV [2B]). Gene content analysis identified five distinct genomic regions enriched among ST88 isolates compared with the other S. aureus lineages. The Ghanaian ST88 isolates had only 658 core genome SNPs and there was no correlation between phylogeny and geography, suggesting the recent spread of this clone. The lineage was also resistant to multiple classes of antibiotics including ß-lactams, tetracycline and chloramphenicol. DISCUSSION: This study reveals that S. aureus ST88-IV is a recently emerging and rapidly spreading CA-MRSA clone in Ghana. The study highlights the capacity of small snapshot genomic studies to provide actionable public health information in resource limited settings. To our knowledge this is the first genomic assessment of the ST88 CA-MRSA clone.

Use of bacterial whole-genome sequencing to understand and improve the management of invasive Staphylococcus aureus infections.

INTRODUCTION: Management of invasive Staphylococcus aureus infections is complex. Dramatic improvements in bacterial whole genome sequencing (WGS) offer new opportunities for personalising the treatment of S. aureus infections. Areas covered: We address recent achievements in S. aureus genomics, describe genetic determinants of antibiotic resistance and summarise studies that have defined molecular characteristics associated with risk and outcome of S. aureus invasive infections. Potential clinical use of WGS for resistance prediction, infection outcome stratification and management of persistent /relapsing infections is critically discussed. Expert commentary: WGS is not only providing invaluable information to track the emergence and spread of important S. aureus clones, but also allows rapid determination of resistance genotypes in the clinical environment. An evolving opportunity is to infer clinically important outcomes and optimal therapeutic approaches from widely available S. aureus genome data, with the goal of individualizing management of invasive S. aureus infections.

A retrospective study of deep sternal wound infections: clinical and microbiological characteristics, treatment, and risk factors for complications.

Deep sternal wound infection (DSWI) is a feared complication following cardiac surgery. This study describes clinical, microbiological, and treatment outcomes of DSWI and determines risk factors for complications. Of 55 patients with DSWI, 66% were male and mean age was 68.2 years. Initial sternotomy was for coronary artery bypass graft in 49% of patients. Sternal debridement at mean 25.4±18.3 days showed monomicrobial (94%), mainly Gram-positive infection. Secondary sternal wound infection (SSWI) occurred in 31% of patients, was mostly polymicrobial (71%), and was predominantly due to Gram-negative bacilli. Risk factors for SSWI were at least 1 revision surgery (odds ratio [OR] 4.8 [95% confidence interval {CI} 1.0-22.4], P=0.047), sternal closure by muscle flap (OR 4.6 [1.3-16.8], P=0.02), delayed sternal closure (mean 27 versus 14 days, P=0.03), and use of vacuum-assisted closure device (100% versus 58%, P=0.008). Hospital stay was significantly longer in patients with SSWI (69 days versus 48 days, P=0.04).

Acquired Multidrug Antifungal Resistance in Candida lusitaniae during Therapy.

Candida lusitaniae is usually susceptible to echinocandins. Beta-1,3-glucan synthase encoded by FKS genes is the target of echinocandins. A few missense mutations in the C. lusitaniae FKS1 hot spot 1 (HS1) have been reported. We report here the rapid emergence of antifungal resistance in C. lusitaniae isolated during therapy with amphotericin B (AMB), caspofungin (CAS), and azoles for treatment of persistent candidemia in an immunocompromised child with severe enterocolitis and visceral adenoviral disease. As documented from restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) analysis, the five C. lusitaniae isolates examined were related to each other. From antifungal susceptibility and molecular analyses, 5 different profiles (P) were obtained. These profiles included the following: profile 1 (P1) (CAS MIC [µg/ml], 0.5; fluconazole [FLC] MIC, 0.25), determined while the patient was being treated with liposomal AMB for 3 months; P2 (FLC MIC [µg/ml], 0.25; CAS MIC, 4), while the patient was being treated with CAS for 2 weeks; P3 (CAS MIC [µg/ml], 0.5; FLC MIC, 32), while the patient was being treated with azoles and CAS initially followed by azoles alone for a week; P4 (CAS MIC [µg/ml], 8; FLC MIC, 8), while the patient was being treated with both drugs for 3 weeks; and P5 (AMB MIC [µg/ml], 0.125; CAS MIC, 8), while the patient was being treated with AMB and FLC for 2 weeks. CAS resistance was associated with resistance not only to micafungin and anidulafungin but also to AMB. Analysis of CAS resistance revealed 3 novel FKS1 mutations in CAS-resistant isolates (S638Y in P2; S631Y in P4; S638P in P5). While S638Y and -P are within HS1, S631Y is in close proximity to this domain but was confirmed to confer candin resistance using a site-directed mutagenesis approach. FLC resistance could be linked with overexpression of major facilitator gene 7 (MFS7) in C. lusitaniae P2 and P4 and was associated with resistance to 5-flurocytosine. This clinical report describes resistance of C. lusitaniae to all common antifungals. While candins or azole resistance followed monotherapy, multidrug antifungal resistance emerged during combined therapy.