MiCId GUI: The Graphical User Interface for MiCId, a Fast Microorganism Classification and Identification Workflow with Accurate Statistics and High Recall.

Although many user-friendly workflows exist for identifications of peptides and proteins in mass-spectrometry-based proteomics, there is a need of easy to use, fast, and accurate workflows for identifications of microorganisms, antimicrobial resistant proteins, and biomass estimation. Identification of microorganisms is a computationally demanding task that requires querying thousands of MS/MS spectra in a database containing thousands to tens of thousands of microorganisms. Existing software can't handle such a task in a time efficient manner, taking hours to process a single MS/MS experiment. Another paramount factor to consider is the necessity of accurate statistical significance to properly control the proportion of false discoveries among the identified microorganisms, and antimicrobial-resistant proteins, and to provide robust biomass estimation. Recently, we have developed Microorganism Classification and Identification (MiCId) workflow that assigns accurate statistical significance to identified microorganisms, antimicrobial-resistant proteins, and biomass estimation. MiCId's workflow is also computationally efficient, taking about 6-17 minutes to process a tandem mass-spectrometry (MS/MS) experiment using computer resources that are available in most laptop and desktop computers, making it a portable workflow. To make data analysis accessible to a broader range of users, beyond users familiar with the Linux environment, we have developed a graphical user interface (GUI) for MiCId's workflow. The GUI brings to users all the functionality of MiCId's workflow in a friendly interface along with tools for data analysis, visualization, and to export results.

Metataxonomic and metaproteomic profiling of the oral microbiome in oral lichen planus - a pilot study.

Background: A growing body of evidence demonstrates a different bacterial composition in the oral cavity of patients with oral lichen planus (OLP). Patients and methods: Buccal swab samples were collected from affected and non-affected sites of six patients with reticular OLP and the healthy oral mucosa of six control subjects. 16S rRNA gene MiSeq sequencing and mass spectrometry-based proteomics were utilised to identify the metataxonomic and metaproteomic profiles of the oral microbiome in both groups. Results: From the metataxonomic analysis, the most abundant species in the three subgroups were Streptococcus oralis and Pseudomonas aeruginosa, accounting for up to 70% of the total population. Principal Coordinates Analysis showed differential clustering of samples from the healthy and OLP groups. ANCOM-BC compositional analysis revealed multiple species (including P. aeruginosa and several species of Veillonella, Prevotella, Streptococcus and Neisseria) significantly over-represented in the control group and several (including Granulicatella elegans, Gemella haemolysans and G. parahaemolysans) in patients with OLP. The metaproteomic data were generally congruent and revealed that several Gemella haemolysans-belonging peptidases and other proteins with inflammatory and virulence potential were present in OLP lesions. Conclusion: Our data suggest that several bacterial species are associated with OLP. Future studies with larger cohorts should be conducted to determine their role in the aetiology of OLP and evaluate their potential as disease biomarkers.

Knockout of Targeted Plasmid-Borne ß-Lactamase Genes in an Extended-Spectrum-ß-Lactamase-Producing Escherichia coli Strain: Impact on Resistance and Proteomic Profile.

Resistance to ß-lactams is known to be multifactorial, although the underlying mechanisms are not well established. The aim of our study was to develop a system for assessing the phenotypic and proteomic responses of bacteria to antibiotic stress as a result of the loss of selected antimicrobial resistance genes. We applied homologous recombination to knock out plasmid-borne ß-lactamase genes (blaOXA-1, blaTEM-1, and blaCTX-M15) in Escherichia coli CCUG 73778, generating knockout clone variants lacking the respective deleted ß-lactamases. Quantitative proteomic analyses were performed on the knockout variants and the wild-type strain, using bottom-up liquid chromatography tandem mass spectrometry (LC-MS/MS), after exposure to different concentrations of cefadroxil. Loss of the blaCTX-M-15 gene had the greatest impact on the resulting protein expression dynamics, while losses of blaOXA-1 and blaTEM-1 affected fewer proteins' expression levels. Proteins involved in antibiotic resistance, cell membrane integrity, stress, and gene expression and unknown function proteins exhibited differential expression. The present study provides a framework for studying protein expression in response to antibiotic exposure and identifying the genomic, proteomic, and phenotypic impacts of resistance gene loss. IMPORTANCE The critical situation regarding antibiotic resistance requires a more in-depth effort for understanding underlying mechanisms involved in antibiotic resistance, beyond just detecting resistance genes. The methodology presented in this work provides a framework for knocking out selected resistance factors, to study the adjustments of the bacterium in response to a particular antibiotic stress, elucidating the genetic response and proteins that are mobilized. The protocol uses MS-based determination of the proteins that are expressed in response to an antibiotic, enabling the selection of strong candidates representing putative resistance factors or mechanisms and providing a basis for future studies to understand their implications in antibiotic resistance. This allows us to better understand how the cell responds to the presence of the antibiotic when a specific gene is lost and, consequently, identify alternative targets for possible future treatment development.

Extracellular membrane vesicles from Limosilactobacillus reuteri strengthen the intestinal epithelial integrity, modulate cytokine responses and antagonize activation of TRPV1.

Bacterial extracellular membrane vesicles (MV) are potent mediators of microbe-host signals, and they are not only important in host-pathogen interactions but also for the interactions between mutualistic bacteria and their hosts. Studies of MV derived from probiotics could enhance the understanding of these universal signal entities, and here we have studied MV derived from Limosilactobacillus reuteri DSM 17938 and BG-R46. The production of MV increased with cultivation time and after oxygen stress. Mass spectrometry-based proteomics analyses revealed that the MV carried a large number of bacterial cell surface proteins, several predicted to be involved in host-bacteria interactions. A 5'-nucleotidase, which catalyze the conversion of AMP into the signal molecule adenosine, was one of these and analysis of enzymatic activity showed that L. reuteri BG-R46 derived MV exhibited the highest activity. We also detected the TLR2 activator lipoteichoic acid on the MV. In models for host interactions, we first observed that L. reuteri MV were internalized by Caco-2/HT29-MTX epithelial cells, and in a dose-dependent manner decreased the leakage caused by enterotoxigenic Escherichia coli by up to 65%. Furthermore, the MV upregulated IL-1ß and IL-6 from peripheral blood mononuclear cells (PBMC), but also dampened IFN-γ and TNF-α responses in PBMC challenged with Staphylococcus aureus. Finally, we showed that MV from the L. reuteri strains have an antagonistic effect on the pain receptor transient receptor potential vanilloid 1 in a model with primary dorsal root ganglion cells from rats. In summary, we have shown that these mobile nanometer scale MV reproduce several biological effects of L. reuteri cells and that the production parameters and selection of strain have an impact on the activity of the MV. This could potentially provide key information for development of innovative and more efficient probiotic products.

A structural model of the profilin-formin pacemaker system for actin filament elongation.

The formins constitute a large class of multi-domain polymerases that catalyze the localization and growth of unbranched actin filaments in cells from yeast to mammals. The conserved FH2 domains form dimers that bind actin at the barbed end of growing filaments and remain attached as new subunits are added. Profilin-actin is recruited and delivered to the barbed end by formin FH1 domains via the binding of profilin to interspersed tracts of poly-L-proline. We present a structural model showing that profilin-actin can bind the FH2 dimer at the barbed end stabilizing a state where profilin prevents its associated actin subunit from directly joining the barbed end. It is only with the dissociation of profilin from the polymerase that an actin subunit rotates and docks into its helical position, consistent with observations that under physiological conditions optimal elongation rates depend on the dissociation rate of profilin, independently of cellular concentrations of actin subunits.

Identification of Antibiotic Resistance Proteins via MiCId's Augmented Workflow. A Mass Spectrometry-Based Proteomics Approach.

Fast and accurate identifications of pathogenic bacteria along with their associated antibiotic resistance proteins are of paramount importance for patient treatments and public health. To meet this goal from the mass spectrometry aspect, we have augmented the previously published Microorganism Classification and Identification (MiCId) workflow for this capability. To evaluate the performance of this augmented workflow, we have used MS/MS datafiles from samples of 10 antibiotic resistance bacterial strains belonging to three different species: Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. The evaluation shows that MiCId's workflow has a sensitivity value around 85% (with a lower bound at about 72%) and a precision greater than 95% in identifying antibiotic resistance proteins. In addition to having high sensitivity and precision, MiCId's workflow is fast and portable, making it a valuable tool for rapid identifications of bacteria as well as detection of their antibiotic resistance proteins. It performs microorganismal identifications, protein identifications, sample biomass estimates, and antibiotic resistance protein identifications in 6-17 min per MS/MS sample using computing resources that are available in most desktop and laptop computers. We have also demonstrated other use of MiCId's workflow. Using MS/MS data sets from samples of two bacterial clonal isolates, one being antibiotic-sensitive while the other being multidrug-resistant, we applied MiCId's workflow to investigate possible mechanisms of antibiotic resistance in these pathogenic bacteria; the results showed that MiCId's conclusions agree with the published study. The new version of MiCId (v.07.01.2021) is freely available for download at https://www.ncbi.nlm.nih.gov/CBBresearch/Yu/downloads.html.

Cytokine responses to LPS in reprogrammed monocytes are associated with the transcription factor PU.1.

Myeloid-derived suppressor cells (MDSCs) are functionally immunosuppressive cells that arise and expand during extensive inflammatory conditions by increased hematopoietic output or reprogramming of immune cells. In sepsis, an increase of circulating MDSCs is associated with adverse outcomes, but unique traits that can be used to identify increased activity of MDSCs are lacking. By using endotoxin tolerance as a model of sepsis-induced monocytic MDSCs (M-MDSC-like cells), this study aims to identify the mediator and transcriptional regulator profile associated with M-MDSC activity. After analyzing 180 inflammation-associated proteins, a profile of differentially expressed cytokines was found in M-MDSC-like cells versus normal monocytes stimulated with LPS. These cytokines were associated with 5 candidate transcription factors, where particularly PU.1 showed differential expression on both transcriptional and protein levels in M-MDSC-like cells. Furthermore, inhibition of PU.1 led to increased production of CXCL5 and CCL8 in M-MDSC-like cells indicating its role in regulating the ability of M-MDSC-like cells to recruit other immune cells. Taken together, the study identifies a unique profile in the pattern of immune mediators defining M-MDSC activity upon LPS stimulation, which offers a functional link to their contribution to immunosuppression.

Differential cytokine response in endotoxin induced M-MDSC-like cells and their associated regulators.

Responses of carbapenemase-producing and non-producing carbapenem-resistant Pseudomonas aeruginosa strains to meropenem revealed by quantitative tandem mass spectrometry proteomics.

Pseudomonas aeruginosa is an opportunistic pathogen with increasing incidence of multidrug-resistant strains, including resistance to last-resort antibiotics, such as carbapenems. Resistances are often due to complex interplays of natural and acquired resistance mechanisms that are enhanced by its large regulatory network. This study describes the proteomic responses of two carbapenem-resistant P. aeruginosa strains of high-risk clones ST235 and ST395 to subminimal inhibitory concentrations (sub-MICs) of meropenem by identifying differentially regulated proteins and pathways. Strain CCUG 51971 carries a VIM-4 metallo-ß-lactamase or 'classical' carbapenemase; strain CCUG 70744 carries no known acquired carbapenem-resistance genes and exhibits 'non-classical' carbapenem-resistance. Strains were cultivated with different sub-MICs of meropenem and analyzed, using quantitative shotgun proteomics based on tandem mass tag (TMT) isobaric labeling, nano-liquid chromatography tandem-mass spectrometry and complete genome sequences. Exposure of strains to sub-MICs of meropenem resulted in hundreds of differentially regulated proteins, including ß-lactamases, proteins associated with transport, peptidoglycan metabolism, cell wall organization, and regulatory proteins. Strain CCUG 51971 showed upregulation of intrinsic ß-lactamases and VIM-4 carbapenemase, while CCUG 70744 exhibited a combination of upregulated intrinsic ß-lactamases, efflux pumps, penicillin-binding proteins and downregulation of porins. All components of the H1 type VI secretion system were upregulated in strain CCUG 51971. Multiple metabolic pathways were affected in both strains. Sub-MICs of meropenem cause marked changes in the proteomes of carbapenem-resistant strains of P. aeruginosa exhibiting different resistance mechanisms, involving a wide range of proteins, many uncharacterized, which might play a role in the susceptibility of P. aeruginosa to meropenem.

The impact of Mediterranean diet on coronary plaque vulnerability, microvascular function, inflammation and microbiome after an acute coronary syndrome: study protocol for the MEDIMACS randomized, controlled, mechanistic clinical trial.

BACKGROUND: Primary prevention trials have demonstrated that the traditional Mediterranean diet is associated with a reduction in cardiovascular mortality and morbidity. However, this benefit has not been proven for secondary prevention after an acute coronary syndrome (ACS). We hypothesized that a high-intensity Mediterranean diet intervention after an ACS decreases the vulnerability of atherosclerotic plaques by complex interactions between anti-inflammatory effects, microbiota changes and modulation of gene expression. METHODS: The MEDIMACS project is an academically funded, prospective, randomized, controlled and mechanistic clinical trial designed to address the effects of an active randomized intervention with the Mediterranean diet on atherosclerotic plaque vulnerability, coronary endothelial dysfunction and other mechanistic endpoints. One hundred patients with ACS are randomized 1:1 to a monitored high-intensity Mediterranean diet intervention or to a standard-of-care arm. Adherence to diet is assessed in both arms using food frequency questionnaires and biomarkers of compliance. The primary endpoint is the change (from baseline to 12 months) in the thickness of the fibrous cap of a non-significant atherosclerotic plaque in a non-culprit vessel, as assessed by repeated optical coherence tomography intracoronary imaging. Indices of coronary vascular physiology and changes in gastrointestinal microbiota, immunological status and protein and metabolite profiles will be evaluated as secondary endpoints. DISCUSSION: The results of this trial will address the key effects of dietary habits on atherosclerotic risk and will provide initial data on the complex interplay of immunological, microbiome-, proteome- and metabolome-related mechanisms by which non-pharmacological factors may impact the progression of coronary atherosclerosis after an ACS. TRIAL REGISTRATION: ClinicalTrials.gov NCT03842319 . Registered on 13 May 2019.

Mass Spectrometry Proteotyping-Based Detection and Identification of Staphylococcus aureus, Escherichia coli, and Candida albicans in Blood.

Bloodstream infections (BSIs), the presence of microorganisms in blood, are potentially serious conditions that can quickly develop into sepsis and life-threatening situations. When assessing proper treatment, rapid diagnosis is the key; besides clinical judgement performed by attending physicians, supporting microbiological tests typically are performed, often requiring microbial isolation and culturing steps, which increases the time required for confirming positive cases of BSI. The additional waiting time forces physicians to prescribe broad-spectrum antibiotics and empirically based treatments, before determining the precise cause of the disease. Thus, alternative and more rapid cultivation-independent methods are needed to improve clinical diagnostics, supporting prompt and accurate treatment and reducing the development of antibiotic resistance. In this study, a culture-independent workflow for pathogen detection and identification in blood samples was developed, using peptide biomarkers and applying bottom-up proteomics analyses, i.e., so-called "proteotyping". To demonstrate the feasibility of detection of blood infectious pathogens, using proteotyping, Escherichia coli and Staphylococcus aureus were included in the study, as the most prominent bacterial causes of bacteremia and sepsis, as well as Candida albicans, one of the most prominent causes of fungemia. Model systems including spiked negative blood samples, as well as positive blood cultures, without further culturing steps, were investigated. Furthermore, an experiment designed to determine the incubation time needed for correct identification of the infectious pathogens in blood cultures was performed. The results for the spiked negative blood samples showed that proteotyping was 100- to 1,000-fold more sensitive, in comparison with the MALDI-TOF MS-based approach. Furthermore, in the analyses of ten positive blood cultures each of E. coli and S. aureus, both the MALDI-TOF MS-based and proteotyping approaches were successful in the identification of E. coli, although only proteotyping could identify S. aureus correctly in all samples. Compared with the MALDI-TOF MS-based approaches, shotgun proteotyping demonstrated higher sensitivity and accuracy, and required significantly shorter incubation time before detection and identification of the correct pathogen could be accomplished.

Profilin-A master coordinator of actin and microtubule organization in mammalian cells.

The last two decades have witnessed a tremendous increase in cell biology data. Not least is this true for studies of the dynamic organization of the microfilament and microtubule systems in animal cells where analyses of the molecular components and their interaction patterns have deepened our understanding of these complex force-generating machineries. Previous observations of a molecular cross-talk between the two systems have now led to the realization of the existence of several intricate mechanisms operating to maintain their coordinated cellular organization. In this short review, we relate to this development by discussing new results concerning the function of the actin regulator profilin 1 as a control component of microfilament-microtubule cross-talk.

Rational antibody design for undruggable targets using kinetically controlled biomolecular probes.

Several important drug targets, e.g., ion channels and G protein-coupled receptors, are extremely difficult to approach with current antibody technologies. To address these targets classes, we explored kinetically controlled proteases as structural dynamics-sensitive druggability probes in native-state and disease-relevant proteins. By using low-Reynolds number flows, such that a single or a few protease incisions are made, we could identify antibody binding sites (epitopes) that were translated into short-sequence antigens for antibody production. We obtained molecular-level information of the epitope-paratope region and could produce high-affinity antibodies with programmed pharmacological function against difficult-to-drug targets. We demonstrate the first stimulus-selective monoclonal antibodies targeting the transient receptor potential vanilloid 1 (TRPV1) channel, a clinically validated pain target widely considered undruggable with antibodies, and apoptosis-inducing antibodies selectively mediating cytotoxicity in KRAS-mutated cells. It is our hope that this platform will widen the scope of antibody therapeutics for the benefit of patients.

The actin regulator profilin 1 is functionally associated with the mammalian centrosome.

Profilin 1 is a crucial actin regulator, interacting with monomeric actin and several actin-binding proteins controlling actin polymerization. Recently, it has become evident that this profilin isoform associates with microtubules via formins and interferes with microtubule elongation at the cell periphery. Recruitment of microtubule-associated profilin upon extensive actin polymerizations, for example, at the cell edge, enhances microtubule growth, indicating that profilin contributes to the coordination of actin and microtubule organization. Here, we provide further evidence for the profilin-microtubule connection by demonstrating that it also functions in centrosomes where it impacts on microtubule nucleation.

Staphylococcus borealis sp. nov., isolated from human skin and blood.

When analysing a large cohort of Staphylococcus haemolyticus, using whole-genome sequencing, five human isolates (four from the skin and one from a blood culture) with aberrant phenotypic and genotypic traits were identified. They were phenotypically similar with yellow colonies, nearly identical 16S rRNA gene sequences and initially speciated as S. haemolyticus based on 16S rRNA gene sequence and MALDI-TOF MS. However, compared to S. haemolyticus, these five strains demonstrate: (i) considerable phylogenetic distance with an average nucleotide identity <95 % and inferred DNA-DNA hybridization <70  %; (ii) a pigmented phenotype; (iii) urease production; and (iv) different fatty acid composition. Based on the phenotypic and genotypic results, we conclude that these strains represent a novel species, for which the name Staphylococcus borealis sp. nov. is proposed. The novel species belong to the genus Staphylococcus and is coagulase- and oxidase-negative and catalase-positive. The type strain, 51-48T, is deposited in the Culture Collection University of Gothenburg (CCUG 73747T) and in the Spanish Type Culture Collection (CECT 30011T).

Acinetobacter portensis sp. nov. and Acinetobacter guerrae sp. nov., isolated from raw meat.

The taxonomic status of six strains of Acinetobacter obtained from meat samples, collected from supermarkets in Porto, Portugal, was investigated using polyphasic analysis. Partial rpoB sequence similarities lower than 95 % to other Acinetobacter species with validly published names led to the hypothesis that these strains represented novel species. This was confirmed based on comparative multilocus sequence analysis, which included the gyrB, recA and 16S rRNA genes, revealing that these strains represented two coherent lineages that were distinct from each other and from all known species. The names Acinetobacter portensis sp. nov. (comprising four strains) and Acinetobacter guerrae sp. nov. (comprising two strains) are proposed for these novel species. The species status of these two groups was confirmed by low (below 95 %) whole-genome sequence average nucleotide identity values and low (below 70 %) digital DNA-DNA hybridization similarities between the whole-genome sequences of the proposed type strains of each novel species and the representatives of the known Acinetobacter species. Phylogenomic treeing from core genome analysis supported these results. The coherence of each new species lineage was supported by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry differentiation of the species at the protein level, by cellular fatty acid profiles, and by unique and differential combinations of metabolic and physiological properties shared by each novel species. The type strain of A. portensis sp. nov. is AC 877T (=CCUG 68672T=CCM 8789T) and the type strain of A. guerrae sp. nov. is AC 1271T (=CCUG 68674T=CCM 8791T).

Complete genome sequences of Streptococcus pyogenes type strain reveal 100%-match between PacBio-solo and Illumina-Oxford Nanopore hybrid assemblies.

We present the first complete, closed genome sequences of Streptococcus pyogenes strains NCTC 8198T and CCUG 4207T, the type strain of the type species of the genus Streptococcus and an important human pathogen that causes a wide range of infectious diseases. S. pyogenes NCTC 8198T and CCUG 4207T are derived from deposit of the same strain at two different culture collections. NCTC 8198T was sequenced, using a PacBio platform; the genome sequence was assembled de novo, using HGAP. CCUG 4207T was sequenced and a de novo hybrid assembly was generated, using SPAdes, combining Illumina and Oxford Nanopore sequence reads. Both strategies yielded closed genome sequences of 1,914,862 bp, identical in length and sequence identity. Combining short-read Illumina and long-read Oxford Nanopore sequence data circumvented the expected error rate of the nanopore sequencing technology, producing a genome sequence indistinguishable to the one determined with PacBio. Sequence analyses revealed five prophage regions, a CRISPR-Cas system, numerous virulence factors and no relevant antibiotic resistance genes. These two complete genome sequences of the type strain of S. pyogenes will effectively serve as valuable taxonomic and genomic references for infectious disease diagnostics, as well as references for future studies and applications within the genus Streptococcus.

A Pangenome Approach for Discerning Species-Unique Gene Markers for Identifications of Streptococcus pneumoniae and Streptococcus pseudopneumoniae.

Correct identifications of isolates and strains of the Mitis-Group of the genus Streptococcus are particularly difficult, due to high genetic similarity, resulting from horizontal gene transfer and homologous recombination, and unreliable phenotypic and genotypic biomarkers for differentiating the species. Streptococcus pneumoniae and Streptococcus pseudopneumoniae are the most closely related species of the clade. In this study, publicly-available genome sequences for Streptococcus pneumoniae and S. pseudopneumoniae were analyzed, using a pangenomic approach, to find candidates for species-unique gene markers; ten species-unique genes for S. pneumoniae and nine for S. pseudopneumoniae were identified. These species-unique gene marker candidates were verified by PCR assays for identifying S. pneumoniae and S. pseudopneumoniae strains isolated from clinical samples. All determined species-level unique gene markers for S. pneumoniae were detected in all S. pneumoniae clinical isolates, whereas fewer of the unique S. pseudopneumoniae gene markers were present in more than 95% of the clinical isolates. In parallel, taxonomic identifications of the clinical isolates were confirmed, using conventional optochin sensitivity testing, targeted PCR-detection for the "Xisco" gene, as well as genomic ANIb similarity analyses for the genome sequences of selected strains. Using mass spectrometry-proteomics, species-specific peptide matches were observed for four of the S. pneumoniae gene markers and for three of the S. pseudopneumoniae gene markers. Application of multiple species-level unique biomarkers of S. pneumoniae and S. pseudopneumoniae, is proposed as a protocol for the routine clinical laboratory for improved, reliable differentiation, and identification of these pathogenic and commensal species.

Genomic and Proteomic Characterization of the Extended-Spectrum ß-Lactamase (ESBL)-Producing Escherichia coli Strain CCUG 73778: A Virulent, Nosocomial Outbreak Strain.

Escherichia coli strain CCUG 78773 is a virulent extended-spectrum ß-lactamase (ESBL)-producing ST131-O25b type strain isolated during an outbreak at a regional university hospital. The complete and closed genome sequence, comprising one chromosome (5,076,638 bp) and six plasmids (1718-161,372 bp), is presented. Characterization of the genomic features detected the presence of 59 potential antibiotic resistance factors, including three prevalent ß-lactamases. Several virulence associated elements were determined, mainly related with adherence, invasion, biofilm formation and antiphagocytosis. Twenty-eight putative type II toxin-antitoxin systems were found. The plasmids were characterized, through in silico analyses, confirming the two ß-lactamase-encoding plasmids to be conjugative, while the remaining plasmids were mobilizable. BLAST analysis of the plasmid sequences showed high similarity with plasmids in E. coli from around the world. Expression of many of the described virulence and AMR factors was confirmed by proteomic analyses, using bottom-up, liquid chromatography-tandem mass spectrometry (LC-MS/MS). The detailed characterization of E. coli strain CCUG 78773 provides a reference for the relevance of genetic elements, as well as the characterization of antibiotic resistance and the spread of bacteria harboring ESBL genes in the hospital environment.

Identification of surface proteins in a clinical Staphylococcus haemolyticus isolate by bacterial surface shaving.

BACKGROUND: The skin commensal Staphylococcus haemolyticus is an emerging nosocomial pathogen. Despite its clinical relevance, published information about S. haemolyticus virulence factors is scarce. In this study, the adhesive and biofilm forming properties of ten clinical and ten commensal S. haemolyticus strains were examined using standard adhesion and biofilm assays. One of the clinical strains was used to identify expressed surface proteins using bacterial surface shaving. Protein abundance was examined by a comparative analysis between bacterial protein expression after human keratinocyte (HaCaT) colonization and growth in cell culture media supplemented with serum. Relative protein quantification was performed by labeling peptides with tandem mass tags (TMT) prior to Mass Spectrometry analysis. Surface proteins can be used as novel targets for antimicrobial treatment and in diagnostics. RESULTS: Adherence to fibronectin, collagen and plastic was low in all tested strains, but with significantly higher adhesion to fibronectin (p = 0.041) and collagen (p = 0.001) in the commensal strains. There was a trend towards higher degree of biofilm formation in the clinical strains (p = 0.059). By using surface shaving, 325 proteins were detected, of which 65 were classified as surface proteins. Analyses showed that the abundance of nineteen (5.8%) proteins were significantly changed following HaCaT colonization. The bacterial Toll/interleukin-1 like (TIRs) domain containing protein (p = 0.04), the transglycosylase SceD (p = 0.01), and the bifunctional autolysin Atl (p = 0.04) showed a 1.4, 1.6- and 1.5-fold increased abundance. The staphylococcal secretory antigen (SsaA) (p = 0.04) was significantly downregulated (- 1.5 fold change) following HaCaT colonization. Among the 65 surface proteins the elastin binding protein (Ebps), LPXAG and LPXSG domain containing proteins and five LPXTG domain containing proteins were identified; three Sdr-like proteins, the extracellular matrix binding protein Embp and a SasH-like protein. CONCLUSIONS: This study has provided novel knowledge about expression of S. haemolyticus surface proteins after direct contact with eukaryotic cells and in media supplemented with serum. We have identified surface proteins and immune evasive proteins previously only functionally described in other staphylococcal species. The identification of expressed proteins after host-microbe interaction offers a tool for the discovery and design of novel targets for antimicrobial treatment.