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Matrix-assisted laser desorption/ionization time-of-flight-mass spectrometry (MALDI-TOF MS)-based direct-on-target microdroplet growth assay (DOT-MGA) was recently described as a novel method of phenotypic antimicrobial susceptibility testing (AST). Here, we developed the application of MALDI-TOF MS-based DOT-MGA for Gram-positive bacteria including AST from agar cultures and directly from positive blood cultures (BCs) using the detection of methicillin resistance as example. Consecutively collected, a total of 14 methicillin-resistant Staphylococcus aureus (MRSA) and 14 methicillin-susceptible S. aureus (MSSA) clinical isolates were included. Furthermore, a collection of MRSA challenge strains comprising different SCCmec types, mec genes, and spa types was tested. Blood samples were spiked with MRSA and MSSA and positive BC broth processed by three different methods: serial dilution of BC broth, lysis/centrifugation, and differential centrifugation. Processed BC broth was directly used for rapid AST using DOT-MGA. Droplets of 6 μl with and without cefoxitin at the EUCAST breakpoint concentration were spotted in triplicates onto the surface of a MALDI target. Targets were incubated in a humidity chamber, followed by medium removal and on-target protein extraction with formic acid before adding matrix with an internal standard as a quality control (QC). Spectra were acquired and evaluated using MALDI Biotyper software. First, tests were considered as valid, if the growth control achieved an identification score of ≥1.7. For valid tests, same score criterion was used for resistant isolates when incubated with cefoxitin. An identification score <1.7 after incubation with cefoxitin defined susceptible isolates. On-target protein extraction using formic acid considerably improved detection of methicillin resistance in S. aureus and DOT-MGA showed feasible results for AST from agar cultures after 4 h incubation time. Comparing the different processing methods of positive BC broth, lysis/centrifugation method with a final dilution step 10–1 of the 0.5 McFarland suspension resulted in best test performance after 4 h incubation time. Overall, 96.4% test validity, 100% sensitivity, and 100% specificity were achieved for detection of methicillin resistance in clinical isolates. All strains of the MRSA challenge collection were successfully tested as methicillin-resistant. This first study on Gram-positive organisms showed feasibility and accuracy of MALDI-TOF MS-based DOT-MGA for rapid AST of S. aureus from agar cultures and directly from positive BCs.
Staphylococcus aureus is a pathogenic bacterium infecting the human host. It’s multifaced adaptation to various environmental conditions is mediated by a tight regulation of the virulence factors influencing the host’s immune system. In this thesis two regulators of gene expression were analysed: (i) the global influence of the two-component system SaePQRS and (ii) the regulation of superantigen gene expression by the alternative sigma factor σB. At the outset of this thesis, single target genes induced by SaeRS were known (hla, hlb, cap5, fnbA, coa). In order to get a general idea of the Sae-regulon, the influence of SaePQRS on gene-expression was analysed in two strain backgrounds by proteomics and transcriptomics aproaches. Recapitulatory, expression of at least 18 secreted and two covalently cell-wall bound proteins was decreased following inactivation of the Sae-system. Sae-dependently expressed were, amongst others, well decribed virulence factors like the y-hemolysins HlgA, HlgB, HlgC, LukM and LukF, the innate immune system modulating proteins Efb, CHIPS and SCIN-B as well as the enterotoxin SEB. SaeR acts as an activator of its target genes. Some proteins were detected in increased amounts in the extracellular proteome of the Sae-deficient strain. However, these changes did not occur at the transcriptional level. The expression of virulence factors is determined by other global regulators. No influence of SaePQRS on the transcription of five substancial regulators, namely the Agr-system and its effector molecule RNAIII, the alternative sigma factor σB, the two-component system ArlRS and the DNA-binding protein SarA, could be shown. In the second part of this thesis the issue was broached to the regulation of gene-expression of a subgroup of virulence factors, the superantigens (SAgs) of S. aureus by SaePQRS and σB. In contrast to their well described molecule structure and function, the regulation of their gene expression was largely unknown. Six different S. aureus strains (two laboratory strains and four clinical isolates) encoding one to seven SAg-genes each, were used for analysis of a total of twelve SAgs regarding their transcription and mitogenic activity. The transcriptional units were characterized using Northern-Blotting. The expression of SAgs could be correlated to the respective growth phase. While egc-SAgs were expressed mainly at low optical densities, seb was induced during late growth phase. In contrast, the transcription of sea, seh, sek, tst and sep remained constant and growth-phase independent. The transcriptional dataset was verified using T-cell proliferation assays. The expression of seh, tst and the egc-operon was dependent on σB. A potential σB-dependent promotor could be identified preceeding seo, the first gene of the egc-operon. In contrast, the expression of seb was increased in sigB-deficient background. This might be due to indirect effects. Expression of seb required SaePQRS. Transcriptional datasets were verified by Immuno-Blotting and T-cell-proliferation assays. In conclusion, the same mutation in sigB but in different strain backgrounds could result in opposite phenotypes with respect to their mitogenic activity. Besides well characterized virulence factors, some secreted proteins with so far unknown function belong to the Sae-regulon. Given that the influence of SaePQRS was restricted to virulence factors and induced especially modulators of the innate immune system, it can be assumed, that these proteins potentially play a role in virulence of S. aureus. In the third part of this thesis, one of these potential new virulence factors, namely SACOL0908, was analysed in detail. In cooperation with the group of Prof. Stehle, Tübingen, the crystal structure was solved. The protein folding of SACOL0908 is new with only minor similarities to described protein structures. Recombinantly expressed SACOL0908 binds to granulocytes. These cells belong to the innate immune system, incorporate bacteria by phagocytosis and kill them. The receptor for SACOL0908 on the surface of granulocytes could not be identified using immunoprecipitation, antibody-blocking assays and functional assays in cooperation with the group of Prof. Peschel, Tübingen. The gene encoding SACOL0908 was deleted in two S. aureus strain backgrounds (COL and Newman). These mutants are currently in use to characterize their phenotype in mouse-infection studies.
Population-based studies of Staphylococcus aureus contribute to understanding the epidemiology of S. aureus infection. We enrolled surgical inpatients admitted to an African tertiary-care hospital in order to prospectively analyze the nosocomial impact of S. aureus. Data collection included an active sampling of the anterior nares and infectious foci within 48 h after admission and subsequently when clinically indicated. All S. aureus isolates were spa and agr genotyped. Possession of Panton-Valentine leukocidin (PVL) and other toxin genes was determined. We analyzed antibiotic susceptibility profiles by VITEK 2 systems and verified methicillin-resistant S. aureus (MRSA) by mecA/C PCR. Among 325 patients, 15.4% carried methicillin-susceptible S. aureus (MSSA) at admission, while 3.7% carried MRSA. The incidence densities of nosocomial infections due to MSSA and MRSA were 35.4 and 6.2 infections per 10,000 patient-days, respectively. Among all 47 nosocomial infections, skin and soft-tissue (40.4%) and bones or joints’ (25.5%) infections predominated. Six (12.7%) infection-related S. aureus isolates harbored PVL genes including two (4.2%) MRSA: overall, seventeen (36.2%) isolates carried pyrogenic toxin superantigens or other toxin genes. This study illustrates the considerable nosocomial impact of S. aureus in a Nigerian University hospital. Furthermore, they indicate a need for effective approaches to curtail nosocomial acquisition of multidrug-resistant S. aureus.