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This retrospective study, conducted at Lausanne University Hospital (2015–2021), compared Staphylococcus aureus bacteraemia (SABA) patients with or without concomitant bacteriuria (SABU). Among 448 included bacteraemic patients, 62 (13.8%) had S. aureus concurrently isolated from urine. In multivariate analysis, there was a significant difference in the odds of community-onset bacteraemia (P 0.030), malignancy (P 0.002), > 1 pair of positive blood cultures (P 0.037), and persistent bacteraemia for at least 48 h (P 0.045) in patients with concurrent SABU. No difference concerning mortality was found. On the other hand, SABU was associated with higher rates of SABA recurrence after antibiotic cessation.

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. ispartof: JOURNAL OF INFECTION vol:86 issue:2 pages:123-133 ispartof: location:England status: published

IntroductionEffective treatment of bloodstream infections (BSIs) is relying on rapid identification of the causing pathogen and its antibiotic susceptibility. Still, most commercially available antibiotic susceptibility testing (AST) methods are based on monitoring bacterial growth, thus impacting the time to results. The Resistell AST is based on a new technology measuring the nanomotion caused by physiologically active bacterial cells and detecting the changes in nanomotion caused by the exposure to a drug.Methods and analysisThis is a single-centre, prospective, cross-sectional, single-arm diagnostic accuracy study to determine the agreement of the Resistell AST on Gram-negative bacteria isolated from blood cultures among patients admitted to a tertiary-care hospital with the reference method. Up to 300 patients will be recruited. Starting with a pilot phase, enrolling 10%–20% of the subjects and limited toEscherichia coliBSI tested for ceftriaxone susceptibility, the main phase will follow, extending the study toKlebsiella pneumoniaeand ciprofloxacin.Ethics and disseminationThis study has received ethical approval from the Swiss Ethics Committees (swissethics, project 2020-01622). All the case report forms and clinical samples will be assigned a study code by the local investigators and stored anonymously at the reference centre (Lausanne University Hospital). The results will be broadly distributed through conference presentations and peer-reviewed publications.Trial registration numberClinicalTrials.gov Registry (NCT05002413).

IntroductionMALDI-TOF MS represents a new technological era for microbiology laboratories. Improved sample processing and expanded databases have facilitated rapid and direct identification of microorganisms from some clinical samples. Automated analysis of protein spectra from different microbial populations is emerging as a potential tool for epidemiological studies and is expected to impact public health.AimTo demonstrate how implementation of MALDI-TOF MS has changed the way microorganisms are identified, how its applications keep increasing and its impact on public health and hospital hygiene.MethodsA review of the available literature in PubMED, published between 2009 and 2018, was carried out.ResultsOf 9,709 articles retrieved, 108 were included in the review. They show that rapid identification of a growing number of microorganisms using MALDI-TOF MS has allowed for optimisation of patient management through prompt initiation of directed antimicrobial treatment. The diagnosis of Gram-negative bacteraemia directly from blood culture pellets has positively impacted antibiotic streamlining, length of hospital stay and costs per patient. The flexibility of MALDI-TOF MS has encouraged new forms of use, such as detecting antibiotic resistance mechanisms (e.g. carbapenemases), which provides valuable information in a reduced turnaround time. MALDI-TOF MS has also been successfully applied to bacterial typing.ConclusionsMALDI-TOF MS is a powerful method for protein analysis. The increase in speed of pathogen detection enables improvement of antimicrobial therapy, infection prevention and control measures leading to positive impact on public health. For antibiotic susceptibility testing and bacterial typing, it represents a rapid alternative to time-consuming conventional techniques.

International audience; Staphylococcus aureus causes various diseases ranging from skin and soft tissue infections to life-threatening infections. Adaptation to the different host niches is controlled by a complex network of transcriptional regulators. Global profiling of condition-dependent transcription revealed adaptation of S. aureus HG001 at the levels of transcription initiation and termination. In particular, deletion of the gene encoding the Rho transcription termination factor triggered a remarkable overall increase in antisense transcription and gene expression changes attributable to indirect regulatory effects. The goal of the present study was a detailed comparative analysis of S. aureus HG001 and its isogenic rho deletion mutant. Proteome analysis revealed significant differences in cellular and extracellular protein profiles, most notably increased amounts of the proteins belonging to the SaeR regulon in the Rho-deficient strain. The SaeRS two-component system acts as a major regulator of virulence gene expression in staphylococci. Higher levels of SaeRS-dependent virulence factors such as adhesins, toxins, and immune evasion proteins in the rho mutant resulted in higher virulence in a murine bacteremia model, which was alleviated in a rho complemented strain. Inhibition of Rho activity by bicyclomycin, a specific inhibitor of Rho activity, also induced the expression of SaeRS-dependent genes, at both the mRNA and protein levels, to the same extent as observed in the rho mutant. Taken together, these findings indicate that activation of the Sae system in the absence of Rho is directly linked to Rho's transcription termination activity and establish a new link between antibiotic action and virulence gene expression in S. aureusIMPORTANCE The major human pathogen Staphylococcus aureus is a widespread commensal bacterium but also the most common cause of nosocomial infections. It adapts to the different host niches through a complex gene regulatory network. We show here that the Rho transcription termination factor, which represses pervasive antisense transcription in various bacteria, including S. aureus, plays a role in controlling SaeRS-dependent virulence gene expression. A Rho-deficient strain produces larger amounts of secreted virulence factors in vitro and shows increased virulence in mice. We also show that treatment of S. aureus with the antibiotic bicyclomycin, which inhibits Rho activity and is effective against Gram-negative bacteria, induces the same changes in the proteome as observed in the Rho-deficient strain. Our results reveal for the first time a link between transcription termination and virulence regulation in S. aureus, which implies a novel mechanism by which an antibiotic can modulate the expression of virulence factors.

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. Electronic supplementary material The online version of this article (10.1186/s13073-018-0574-x) contains supplementary material, which is available to authorized users.

We describe an outbreak of Burkholderia stabilis associated with contaminated washing gloves, a commercially available Class I medical device. Triggered by an increase in Burkholderia cepacia complex (BCC) bacteremias and the detection of BCC in unopened packages of washing gloves, an ad hoc national outbreak committee comprising representatives of a public health organisation, a regulatory agency, and an expert association convened and commissioned an outbreak investigation. The investigation included retrospective case finding across Switzerland and whole genome sequencing (WGS) of isolates from cases and gloves. The investigation revealed that BCC were detected in clinical samples of 46 cases aged 17 to 91 years (33% females) from nine institutions between May 2015 and August 2016. Twenty-two isolates from case patients and 16 from washing gloves underwent WGS. All available outbreak isolates clustered within a span of 1,500 alleles. This BCC outbreak was rapidly identified, communicated, investigated and halted by an ad hoc collaboration of multiple stakeholders. WGS served as useful tool for confirming the source of the outbreak. This outbreak also highlights current regulatory limitations regarding Class I medical devices and the usefulness of a nationally coordinated outbreak response.

BACKGROUND: Early identification of pathogens from blood cultures using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry may optimize the choice of empirical antibiotic therapy in the setting of bloodstream infections. We aimed to assess the impact of this new technology on the use of antibiotic treatment in patients with gram-negative bacteremia. METHODS: We conducted a prospective observational study from January to December 2010 to evaluate the sequential and separate impacts of Gram stain reporting and MALDI-TOF bacterial identification performed on blood culture pellets in patients with gram-negative bacteremia. The primary outcome was the impact of MALDI-TOF on empirical antibiotic choice. RESULTS: Among 202 episodes of gram-negative bacteremia, Gram stain reporting had an impact in 42 cases (20.8%). MALDI-TOF identification led to a modification of empirical therapy in 71 of all 202 cases (35.1%), and in 16 of 27 cases (59.3%) of monomicrobial bacteremia caused by AmpC-producing Enterobacteriaceae. The most frequently observed impact was an early appropriate broadening of the antibiotic spectrum in 31 of 71 cases (43.7%). In total, 143 of 165 episodes (86.7%) of monomicrobial bacteremia were correctly identified at genus level by MALDI-TOF. CONCLUSIONS: In a low prevalence area for extended spectrum betalactamases (ESBL) and multiresistant gram-negative bacteria, MALDI-TOF performed on blood culture pellets had an impact on the clinical management of 35.1% of all gram-negative bacteremia cases, demonstrating a greater impact than Gram stain reporting. Thus, MALDI-TOF could become a vital second step beside Gram stain in guiding the empirical treatment of patients with bloodstream infection.

In this study, different molecular typing tools were applied to characterize 95 Salmonella enterica blood isolates collected between 2008 and 2013 from patients at nine public hospitals in Lima, Peru. Combined results of multiplex PCR serotyping, two- and seven-loci multilocus sequence typing (MLST) schemes, serotyping, IS200 amplification and RAPD fingerprints, showed that these infections were caused by eight different serovars: Enteritidis, Typhimurium, Typhi, Choleraesuis, Dublin, Paratyphi A, Paratyphi B and Infantis. Among these, Enteritidis, Typhimurium and Typhi were the most prevalent, representing 45, 36 and 11% of the isolates, respectively. Most isolates (74%) were not resistant to ten primarily used antimicrobial drugs; however, 37% of the strains showed intermediate susceptibility to ciprofloxacin (ISC). Antimicrobial resistance integrons were carried by one Dublin (dfra1 and aadA1) and two Infantis (aadA1) isolates. The two Infantis isolates were multidrug resistant and harbored a large megaplasmid. Amplification of spvC and spvRA regions showed that all Enteritidis (n = 42), Typhimurium (n = 34), Choleraesuis (n = 3) and Dublin (n = 1) isolates carried the Salmonella virulence plasmid (pSV). We conclude that the classic serotyping method can be substituted by the multiplex PCR and, when necessary, sequencing of only one or two loci of the MLST scheme is a valuable tool to confirm the results. The effectiveness and feasibility of different typing tools is discussed.

Enterococci have recently become important due to their increased isolation rates in community-based and nosocomial infections and resistance to many antibiotics, including glycopeptides. The aim of this study was to evaluate the antimicrobial susceptible patterns and virulence factors of various clinical specimens; urine (n= 149), blood (n= 38), wound (n= 17), stool (n= 13), and other (n= 12) with a total of 229 enterococci including 138 E. faecalis and 91 E. faecium isolates. Aggregation factor (AF), enterococcus surface protein (esp), cytolysins and gelatinase encoding genes (asa1, esp, cylM, cylBcyl A, cylll, cylls, gelE, respectively) were investigated by molecular methods. Haemolysin production and gelatinase were studied phenotypically. A total of 30 isolates, 29 of E. faecium and one of E. faecalis isolates were resistant to vancomycin. High-level gentamicin and high-level streptomycin resistance in E. faecalis were 40.7% and 63.7% however, they were 47.1% and 55.8% in E. faecalis isolates. All strains were susceptible to linezolid. Ampicillin, penicillin and vancomycin resistance in E. faecium isolates were found to be higher than E. faecalis isolates (p= 0.001, p= 0.008 and p< 0.001). Asa1 (p< 0.001), cylll (p= 0.002) and cylls (p< 0.001) as well as gelatinase activity in isolates of E. faecalis were significantly higher than the isolates of E. faecium (p< 0.001). The most common virulence genes in our study were asa1 gene (45%), cyLs gene (33.2%) and esp gene (32.3%). Ciprofloxacin resistance in cylLL and cyLs gene positive isolates of E. faecalis were significantly higher compared to isolates that do not contain these genes (p= 0.035 and p= 0.047). Likewise, haemolysin producing E. faecium isolates were significantly more resistant to vancomycin compared to isolates that do not produce hemolysin (p< 0.001). When the virulence factors of vancomycin resistant and susceptible isolates were compared, the esp gene level in VRE E. faecium isolates was found to be 24.1%, while no esp gene was found in VRE E. faecalis isolates. The existence of asa1was negative in both VRE E. faecium and VRE E. faecalis isolates. The activity of hemolysin was found 42.3% for E. faecalis and 19.3% for E. faecium. In vancomycin-sensitive enterococcus (VSE) species, esp gene activity was 35.1% for E. faecalis, 29.4% for E. faecium, asa1 gene activity was 60.8% for E. faecalis and 47.1% for E. faecium, hemolysin activity was 52.8% for E. faecalis and 23.5% for E. faecium. In our study, it was found that VSE isolates have more virulence genes than VRE isolates. It should be kept in mind that VRE can causeinfections which are difficult-to-treat especially in hospitalized patients and VSE have significant virulence factors that can cause severe infections.

The presence of 25 virulence genes (VGs), genetic phylogroups, quinolone-resistance and Extended Spectrum beta-lactamase (ESBL)-production was assessed in 65 Escherichia coli isolates from blood cultures in children

Comment on Reduced susceptibility to host-defense cationic peptides and daptomycin coemerge in methicillin-resistant Staphylococcus aureus from daptomycin-naive bacteremic patients. [J Infect Dis. 2012]

OBJECTIVES: To determine trends in ciprofloxacin resistance and co-resistance to other antibiotic classes in blood isolates of Escherichia coli, and to investigate if there is an ecological relationship to the community use of fluoroquinolones and other antibiotics.METHODS: Forty-two Spanish hospitals of the European Antimicrobial Resistance Surveillance Network collected ciprofloxacin and other antibiotic susceptibility data for non-duplicate consecutive E. coli isolates from patients with bacteraemia between 2001 and 2009. The nationwide ambulatory use of antibiotics between 1997 and 2008 was determined by WHO methods, and the co-evolution of both parameters was further analysed.RESULTS: Of the 28 307 E. coli blood isolates, 27.9% were ciprofloxacin non-susceptible (CIPNS), increasing from 17.6% in 2001 to 32.7% in 2009. A continuous increase was observed between CIPNS and other resistances, including cephalosporin resistance due to the production of extended-spectrum β-lactamases (ESBLs) and non-susceptibility to both amoxicillin/clavulanic acid and tobramycin. Although the total use of antibiotics did not increase, community use of levofloxacin, moxifloxacin and amoxicillin/clavulanic acid increased by 307.2%, 62.6% and 70.1%, respectively. Yearly rates of CIPNS E. coli strongly correlated with the use of levofloxacin, moxifloxacin and amoxicillin/clavulanic acid (r(2 )> 0.80; P CONCLUSIONS: The rapid increase in CIPNS E. coli causing bacteraemia was closely related to the increase in resistance to amoxicillin/clavulanic acid, production of ESBLs and resistance to aminoglycosides. Community use of fluoroquinolones (mainly moxifloxacin and levofloxacin) and of amoxicillin/clavulanic acid represents a significant driver in the progression of fluoroquinolone resistance in bacteraemic E. coli.

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a rapid identification of the infectious agent to guide optimal antibiotic treatment. The analysis of Gram stains from positive blood culture can be rapidly conducted and already significantly impact the antibiotic regimen. However, the accurate identification of the infectious agent is still required to establish the optimal targeted treatment. We present here a simple and fast bacterial pellet preparation from a positive blood culture that can be used as a sample for several essential downstream applications such as identification by MALDI-TOF MS, antibiotic susceptibility testing (AST) by disc diffusion assay or automated AST systems and by automated PCR-based diagnostic testing. The performance of these different identification and AST systems applied directly on the blood culture bacterial pellets is very similar to the performance normally obtained from isolated colonies grown on agar plates. Compared to conventional approaches, the rapid acquisition of a bacterial pellet significantly reduces the time to report both identification and AST. Thus, following blood culture positivity, identification by MALDI-TOF can be reported within less than 1 hr whereas results of AST by automated AST systems or disc diffusion assays within 8 to 18 hr, respectively. Similarly, the results of a rapid PCR-based assay can be communicated to the clinicians less than 2 hr following the report of a bacteremia. Together, these results demonstrate that the rapid preparation of a blood culture bacterial pellet has a significant impact on the identification and AST turnaround time and thus on the successful outcome of patients suffering from bloodstream infections.

We report for the first time a case of bacteremia caused by Comamonas kerstersii in a 65-year-old patient with sign of diverticulosis. In addition, we review the isolation of Comamonas sp. and related organisms in our hospital over 25 years.

Genotypic and phenotypic tolerance was studied in penicillin treatment of experimental endocarditis due to nontolerant and tolerant Streptococcus gordonii and to their backcross transformants. The organisms were matched for in vitro and in vivo growth rates. Rats with aortic endocarditis were treated for 3 or 5 days, starting 12, 24, or 48 h after inoculation. When started at 12 h, during fast intravegetation growth, 3 days of treatment cured 80% of the nontolerant parent compared with

Objectives: The management of bloodstream infection, a life-threatening disease, largely relies on early detection of infecting microorganisms and accurate determination of their antibiotic susceptibility to reduce both mortality and morbidity. Recently we developed a new technique based on atomic force microscopy capable of detecting movements of biologic samples at the nanoscale. Such sensor is able to monitor the response of bacteria to antibiotic's pressure, allowing a fast and versatile susceptibility test. Furthermore, rapid preparation of a bacterial pellet from a positive blood culture can improve downstream characterization of the recovered pathogen as a result of the increased bacterial concentration obtained. Methods: Using artificially inoculated blood cultures, we combined these two innovative procedures and validated them in double-blind experiments to determine the susceptibility and resistance of Escherichia coli strains (ATCC 25933 as susceptible and a characterized clinical isolate as resistant strain) towards a selection of antibiotics commonly used in clinical settings. Results: On the basis of the variance of the sensor movements, we were able to positively discriminate the resistant from the susceptible E. coli strains in 16 of 17 blindly investigated cases. Furthermore, we defined a variance change threshold of 60% that discriminates susceptible from resistant strains. Conclusions: By combining the nanomotion sensor with the rapid preparation method of blood culture pellets, we obtained an innovative, rapid and relatively accurate method for antibiotic susceptibility test directly from positive blood culture bottles, without the need for bacterial subculture. (C) 2017 The Authors. Published by Elsevier Ltd.