Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for the Identification of Clinically Relevant Bacteria

Background: Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) allows rapid and reliable identification of microorganisms, particularly clinically important pathogens. Methodology/Principal Findings: We compared the identification efficiency of MALDI-TOF MS with that of PhoenixH, APIH and 16S ribosomal DNA sequence analysis on 1,019 strains obtained from routine diagnostics. Further, we determined the agreement of MALDI-TOF MS identifications as compared to 16S gene sequencing for additional 545 strains belonging to species of Enterococcus, Gardnerella, Staphylococcus, and Streptococcus. For 94.7 % of the isolates MALDI-TOF MS results were identical with those obtained with conventional systems. 16S sequencing confirmed MALDI-TOF MS identification in 63 % of the discordant results. Agreement of identification of Gardnerella, Enterococcus, Streptococcus and Staphylococcus species between MALDI-TOF MS and traditional method was high (Crohn’s kappa values: 0.9 to 0.93). Conclusions/Significance: MALDI-TOF MS represents a rapid, reliable and cost-effective identification technique for clinically relevant bacteria

Global Analysis of Circulating Immune Cells by Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry

Background: MALDI-TOF mass spectrometry is currently used in microbiological diagnosis to characterize bacterial populations. Our aim was to determine whether this technique could be applied to intact eukaryotic cells, and in particular, to cells involved in the immune response. Methodology/Principal Findings: A comparison of frozen monocytes, T lymphocytes and polymorphonuclear leukocytes revealed specific peak profiles. We also found that twenty cell types had specific profiles, permitting the establishment of a cell database. The circulating immune cells, namely monocytes, T lymphocytes and polymorphonuclear cells, were distinct from tissue immune cells such as monocyte-derived macrophages and dendritic cells. In addition, MALDI-TOF mass spectrometry was valuable to easily identify the signatures of monocytes and T lymphocytes in peripheral mononuclear cells. Conclusions/Significance: This method was rapid and easy to perform, and unlike flow cytometry, it did not require any additional components such as specific antibodies. The MALDI-TOF mass spectrometry approach could be extended t

Importance of the Collagen Adhesin Ace in Pathogenesis and Protection against Enterococcus faecalis Experimental Endocarditis

Ace is an adhesin to collagen from Enterococcus faecalis expressed conditionally after growth in serum or in the presence of collagen. Here, we generated an ace deletion mutant and showed that it was significantly attenuated versus wild-type OG1RF in a mixed infection rat endocarditis model (P<0.0001), while no differences were observed in a peritonitis model. Complemented OG1RFΔace (pAT392::ace) enhanced early (4 h) heart valve colonization versus OG1RFΔace (pAT392) (P = 0.0418), suggesting that Ace expression is important for early attachment. By flow cytometry using specific anti-recombinant Ace (rAce) immunoglobulins (Igs), we showed in vivo expression of Ace by OG1RF cells obtained directly from infected vegetations, consistent with our previous finding of anti-Ace antibodies in E. faecalis endocarditis patient sera. Finally, rats actively immunized against rAce were less susceptible to infection by OG1RF than non-immunized (P = 0.0004) or sham-immunized (P = 0.0475) by CFU counts. Similarly, animals given specific anti-rAce Igs were less likely to develop E. faecalis endocarditis (P = 0.0001) and showed fewer CFU in vegetations (P = 0.0146). In conclusion, we have shown for the first time that Ace is involved in pathogenesis of, and is useful for protection against, E. faecalis experimental endocarditis

Lysozyme M deficiency leads to an increased susceptibility to Streptococcus pneumoniae-induced otitis media

<p>Abstract</p> <p>Background</p> <p>Lysozyme is an antimicrobial innate immune molecule degrading peptidoglycan of the bacterial cell wall. Lysozyme shows the ubiquitous expression in wide varieties of species and tissues including the tubotympanum of mammals. We aim to investigate the effects of lysozyme depletion on pneumococcal clearance from the middle ear cavity.</p> <p>Methods</p> <p>Immunohistochemistry was performed to localize lysozyme in the Eustachian tube. Lysozyme expression was compared between the wild type and the lysozyme M^-/-mice using real time quantitative RT-PCR and western blotting. Muramidase activity and bactericidal activity of lysozyme was measured using a lysoplate radial diffusion assay and a liquid broth assay, respectively. To determine if depletion of lysozyme M increases a susceptibility to pneumococal otitis media, 50 CFU of <it>S. pneumoniae </it>6B were transtympanically inoculated to the middle ear and viable bacteria were counted at day 3 and 7 with clinical grading of middle ear inflammation.</p> <p>Results</p> <p>Immunolabeling revealed that localization of lysozyme M and lysozyme P is specific to some/particular cell types of the Eustachian tube. Lysozyme P of lysozyme M^-/-mice was mainly expressed in the submucosal gland but not in the tubal epithelium. Although lysozyme M^-/-mice showed compensatory up-regulation of lysozyme P, lysozyme M depletion resulted in a decrease in both muramidase and antimicrobial activities. Deficiency in lysozyme M led to an increased susceptibility to middle ear infection with <it>S. pneumoniae </it>6B and resulted in severe middle ear inflammation, compared to wild type mice.</p> <p>Conclusion</p> <p>The results suggest that lysozyme M plays an important role in protecting the middle ear from invading pathogens, particularly in the early phase. We suggest a possibility of the exogenous lysozyme as an adjuvant therapeutic agent for otitis media, but further studies are necessary.</p

Genetic Pathway in Acquisition and Loss of Vancomycin Resistance in a Methicillin Resistant Staphylococcus aureus (MRSA) Strain of Clonal Type USA300

An isolate of the methicillin-resistant Staphylococcus aureus (MRSA) clone USA300 with reduced susceptibility to vancomycin (SG-R) (i.e, vancomycin-intermediate S. aureus, VISA) and its susceptible “parental” strain (SG-S) were recovered from a patient at the end and at the beginning of an unsuccessful vancomycin therapy. The VISA phenotype was unstable in vitro generating a susceptible revertant strain (SG-rev). The availability of these 3 isogenic strains allowed us to explore genetic correlates of antibiotic resistance as it emerged in vivo. Compared to the susceptible isolate, both the VISA and revertant strains carried the same point mutations in yycH, vraG, yvqF and lspA genes and a substantial deletion within an intergenic region. The revertant strain carried a single additional frameshift mutation in vraS which is part of two component regulatory system VraSR. VISA isolate SG-R showed complex alterations in phenotype: decreased susceptibility to other antibiotics, slow autolysis, abnormal cell division and increased thickness of cell wall. There was also altered expression of 239 genes including down-regulation of major virulence determinants. All phenotypic properties and gene expression profile returned to parental levels in the revertant strain. Introduction of wild type yvqF on a multicopy plasmid into the VISA strain caused loss of resistance along with loss of all the associated phenotypic changes. Introduction of the wild type vraSR into the revertant strain caused recovery of VISA type resistance. The yvqF/vraSR operon seems to function as an on/off switch: mutation in yvqF in strain SG-R turns on the vraSR system, which leads to increase in vancomycin resistance and down-regulation of virulence determinants. Mutation in vraS in the revertant strain turns off this regulatory system accompanied by loss of resistance and normal expression of virulence genes. Down-regulation of virulence genes may provide VISA strains with a “stealth” strategy to evade detection by the host immune system

Sequencing and Comparative Genome Analysis of Two Pathogenic Streptococcus gallolyticus Subspecies: Genome Plasticity, Adaptation and Virulence

Streptococcus gallolyticus infections in humans are often associated with bacteremia, infective endocarditis and colon cancers. The disease manifestations are different depending on the subspecies of S. gallolyticus causing the infection. Here, we present the complete genomes of S. gallolyticus ATCC 43143 (biotype I) and S. pasteurianus ATCC 43144 (biotype II.2). The genomic differences between the two biotypes were characterized with comparative genomic analyses. The chromosome of ATCC 43143 and ATCC 43144 are 2,36 and 2,10 Mb in length and encode 2246 and 1869 CDS respectively. The organization and genomic contents of both genomes were most similar to the recently published S. gallolyticus UCN34, where 2073 (92%) and 1607 (86%) of the ATCC 43143 and ATCC 43144 CDS were conserved in UCN34 respectively. There are around 600 CDS conserved in all Streptococcus genomes, indicating the Streptococcus genus has a small core-genome (constitute around 30% of total CDS) and substantial evolutionary plasticity. We identified eight and five regions of genome plasticity in ATCC 43143 and ATCC 43144 respectively. Within these regions, several proteins were recognized to contribute to the fitness and virulence of each of the two subspecies. We have also predicted putative cell-surface associated proteins that could play a role in adherence to host tissues, leading to persistent infections causing sub-acute and chronic diseases in humans. This study showed evidence that the S. gallolyticus still possesses genes making it suitable in a rumen environment, whereas the ability for S. pasteurianus to live in rumen is reduced. The genome heterogeneity and genetic diversity among the two biotypes, especially membrane and lipoproteins, most likely contribute to the differences in the pathogenesis of the two S. gallolyticus biotypes and the type of disease an infected patient eventually develops

The Serum Angiotensin Converting Enzyme And Lysozyme Levels In Patients With Ocular Involvement Of Autoimmune And Infectious Diseases

Background Increased serum levels of angiotensin converting enzyme and lysozyme are considered as inflammatory markers for diagnosis of sarcoidosis which is an autoimmune inflammatory disease. The purpose of this study is to evaluate the significance of differences in serum angiotensin converting enzyme and lysozyme levels of patients with ocular involvement of other autoimmune inflammatory and infectious diseases. Methods This is a prospective study involving patients with ankylosing spondylitis, behcet’s disease, presumed sarcoidosis, presumed latent tuberculosis, presumed latent syphilis, and control group. The serum levels of angiotensin converting enzyme and lysozyme were analyzed by enzyme-linked immunosorbent assay. Bonnferoni analysis was used to assess pairwise comparisons between the groups. Results There was a significant increase in serum angiotensin converting enzyme level in patients with presumed sarcoidosis compared to ankylosing spondylitis (p = 0.0001), behcet’s disease (p = 0.0001), presumed latent tuberculosis (p = 0.0001), presumed latent syphilis (p = 0.0001), and control group (p = 0.0001). The increase in serum lysozyme level was significant for patients with presumed sarcoidosis with respect to ankylosing spondylitis (p = 0.0001), behcet’s disease, (p = 0.0001) presumed latent tuberculosis (p = 0.001), presumed latent syphilis (p = 0.033), and control group (p = 0.0001). Conclusion Elevated serum angiotensin converting enzyme levels are significant for patients with presumed sarcoidosis compared to ocular involvement of other autoimmune diseases such as behcet’s disease and ankylosing spondylitis, and ocular involvement of infectious diseases such as presumed latent tuberculosis and presumed latent syphilis. However, elevated serum lysozyme level might be also detected in ocular involvement of infectious diseases such as presumed latent tuberculosis and presumed latent syphilis. Trial registration Trial Registration number: NCT02627209. Date of registration: 12/09/2015.PubMedWoSScopu