Quantitative proteogenomics of human pathogens using DIA-MS.

The increasing number of bacterial genomes in combination with reproducible quantitative proteome measurements provides new opportunities to explore how genetic differences modulate proteome composition and virulence. It is challenging to combine genome and proteome data as the underlying genome influences the proteome. We present a strategy to facilitate the integration of genome data from several genetically similar bacterial strains with data-independent analysis mass spectrometry (DIA-MS) for rapid interrogation of the combined data sets. The strategy relies on the construction of a composite genome combining all genetic data in a compact format, which can accommodate the fusion with quantitative peptide and protein information determined via DIA-MS. We demonstrate the method by combining data sets from whole genome sequencing, shotgun MS and DIA-MS from 34 clinical isolates of Streptococcus pyogenes. The data structure allows for fast exploration of the data showing that undetected proteins are on average more amenable to amino acid substitution than expressed proteins. We identified several significantly differentially expressed proteins between invasive and non-invasive strains. The work underlines how integration of whole genome sequencing with accurately quantified proteomes can further advance the interpretation of the relationship between genomes, proteomes and virulence

High Genetic Diversity among Community-Associated Staphylococcus aureus in Europe: Results from a Multicenter Study

Background: Several studies have addressed the epidemiology of community-associated Staphylococcus aureus (CA-SA) in Europe; nonetheless, a comprehensive perspective remains unclear. In this study, we aimed to describe the population structure of CA-SA and to shed light on the origin of methicillin-resistant S. aureus (MRSA) in this continent. Methods and Findings: A total of 568 colonization and infection isolates, comprising both MRSA and methicillin-susceptible S. aureus (MSSA), were recovered in 16 European countries, from community and community-onset infections. The genetic background of isolates was characterized by molecular typing techniques (spa typing, pulsed-field gel electrophoresis and multilocus sequence typing) and the presence of PVL and ACME was tested by PCR. MRSA were further characterized by SCCmec typing. We found that 59 % of all isolates were associated with community-associated clones. Most MRSA were related with USA300 (ST8-IVa and variants) (40%), followed by the European clone (ST80-IVc and derivatives) (28%) and the Taiwan clone (ST59-IVa and related clonal types) (15%). A total of 83 % of MRSA carried Panton-Valentine leukocidin (PVL) and 14 % carried the arginine catabolic mobile element (ACME). Surprisingly, we found a high genetic diversity among MRSA clonal types (ST-SCCmec), Simpson’s index of diversity = 0.852 (0.788–0.916). Specifically, about half of the isolates carried novel associations between genetic background and SCCmec. Analysis by BURP showed that some CA-MSSA and CA-MRS

Species specific susceptibility testing for ß-lactam antibiotics. With special reference to staphylococci.

The main objective of this thesis was to identify methods for the detection of ß-lactam resistance in staphylococci, bacteria often causing nosocomial infections. Detection of ß-lactam resistance in these species is difficult due to strong regulation of genes encoding for the two main resistance mechanisms, ß-lactamase production and the penicillin-binding protein (PBP) 2?. ß-lactamase has high affinity to penicillins and hydrolyse these drugs. In total ten applications were evaluated with respect to ability to identify ß-lactamase producing strains of staphylococci. Three applications, the starch-iodine plate test using induced and uninduced strains, and the nitrocefin spot test using induced strains grown on unsupplemented agar, efficiently identified ß-lactamase producing strains. The nitrocefin spot test proved to be the most appropriate test for the routine laboratory. Novobiocin-resistant coagulase-negative species, showed a high rate of false positive reactions in several tests and should not be tested for ß-lactamase production. PBP2? is encoded by the mecA gene and is only found in methicillin-resistant staphylococci, where it supersedes the regular PBPs in the presence of ß-lactam antibiotics. An appli- cation for detection of the mecA gene with polymerase chain reaction (PCR) was developed and used as reference method. The effect of the inoculum density, the incubation time and temperature, supplementation of the growth medium, and the disk content or drug, on the expression of methicillin-resistance with the disk diffusion method and MIC determination was evaluated. The disk diffusion method proved to be by far the most reliable method for the identification of methicillin-resistant strains if an inoculum of 108 cfu/ml, incubation at 30°C for 24 h, agar with no more than 0.5% NaCl, and disks with 1 and 5 µg oxacillin were applied. MIC determination as a tool for identification of methicillin- resistant strains is unsuitable, due to a poor discrimination between mecA+ and mecA- strains or a poor correlation to MIC limits

Disk with High Oxacillin Content Discriminates between Methicillin-Resistant and Borderline Methicillin-Susceptible Staphylococcus aureus Strains in Disk Diffusion Assays Using a Low Salt Concentration

A separation between mecA(+) strains of Staphylococcus aureus and strains lacking mecA was achieved by the disk diffusion assay and the agar dilution method, utilizing disks containing 5 μg of oxacillin and inocula of approximately 5 × 10(5) CFU/spot, respectively, provided that agar with 0 to 0.5% NaCl and incubation at 30°C were employed. The 5-μg oxacillin disks clearly discriminated between borderline methicillin-susceptible and mecA(+) strains. The oxacillin MICs were more affected by the inoculum density and salt concentration than were the methicillin MICs, and oxacillin MICs of 4 to 16 μg/ml were obtained for strains lacking mecA. Significantly higher levels of β-lactamase production and reduced oxacillin susceptibilities were recorded for strains lacking mecA, in particular strains of phage group V, when agar with ≥2% NaCl was used than when agar with 0 to 0.5% NaCl was employed. The results indicate that the borderline methicillin-susceptible phenotype is a salt-dependent in vitro phenomenon of questionable clinical relevance

Epidemiology and antibiotic susceptibility of aerococci in urinary cultures.

In this study, we present population-based data regarding the prevalence of aerococci in clinical urinary samples. During a 3-month period, all aerococcal isolates from urinary samples from 2 clinical microbiology laboratories were collected. We identified 64 Aerococcus urinae isolates and 40 Aerococcus sanguinicola isolates, which correlates with an incidence of 33 cases of aerococcal bacteriuria per 100,000 inhabitants per year. The median age was 83years for all patients with aerococcal bacteriuria, which was significantly higher than for patients with Escherichia coli or Enterococcus faecalis bacteriuria. Sex was almost equally distributed between men and women with aerococcal bacteriuria, whereas females dominated in E. coli bacteriuria. The aerococcal isolates displayed low MICs for ampicillin, cefalotin, mecillinam, and nitrofurantoin. Most A. sanguinicola isolates were resistant to ciprofloxacin, whereas most A. urinae isolates had low MICs. Clinical studies are needed to establish clinical breakpoints and optimal treatment

Disk with high oxacillin content discriminates between methicillin-resistant and borderline methicillin-susceptible Staphylococcus aureus strains in disk diffusion assays using a low salt concentration

A separation between mecA+ strains of Staphylococcus aureus and strains lacking mecA was achieved by the disk diffusion assay and the agar dilution method, utilizing disks containing 5 microg of oxacillin and inocula of approximately 5 x 10(5) CFU/spot, respectively, provided that agar with 0 to 0.5% NaCl and incubation at 30 degrees C were employed. The 5-microg oxacillin disks clearly discriminated between borderline methicillin-susceptible and mecA+ strains. The oxacillin MICs were more affected by the inoculum density and salt concentration than were the methicillin MICs, and oxacillin MICs of 4 to 16 microg/ml were obtained for strains lacking mecA. Significantly higher levels of beta-lactamase production and reduced oxacillin susceptibilities were recorded for strains lacking mecA, in particular strains of phage group V, when agar with >/=2% NaCl was used than when agar with 0 to 0.5% NaCl was employed. The results indicate that the borderline methicillin-susceptible phenotype is a salt-dependent in vitro phenomenon of questionable clinical relevance

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry is a sensitive and specific method for identification of aerococci.

Conventional methods for the species determination of human pathogenic aerococci are not reliable. We show that matrix-assisted laser desorption ionization time-of-flight mass spectrometry correctly identifies aerococci to the species level and that it can be used to identify aerococci with high specificity in the diagnostic clinical microbiology laboratory

Clinical and microbiological features of bacteremia with Streptococcus equi

Streptococcus equi (SE) rarely causes human infections. We identified 18 SE isolates from blood cultures. The focus of infection was unknown (n = 5), arthritis (n = 3), catheter-related (n = 2), pneumonia (n = 2), or other (n = 6). There were no fatalities. Several patients had animal contacts but there were no indications of clonal outbreaks