Stefanie Mauerer

High-Level Fluorescence Labeling of Gram-Positive Pathogens

Fluorescence labeling of bacterial pathogens has a broad range of interesting applications including the observation of living bacteria within host cells. We constructed a novel vector based on the E. coli streptococcal shuttle plasmid pAT28 that can propagate in numerous bacterial species from different genera. The plasmid harbors a promoterless copy of the green fluorescent variant gene egfp under the control of the CAMP-factor gene (cfb) promoter of Streptococcus agalactiae and was designated pBSU101. Upon transfer of the plasmid into streptococci, the bacteria show a distinct and easily detectable fluorescence using a standard fluorescence microscope and quantification by FACS-analysis demonstrated values that were 10–50 times increased over the respective controls. To assess the suitability of the construct for high efficiency fluorescence labeling in different gram-positive pathogens, numerous species were transformed. We successfully labeled Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus dysgalactiae subsp. equisimilis, Enterococcus faecalis, Enterococcus faecium, Streptococcus mutans, Streptococcus anginosus and Staphylococcus aureus strains utilizing the EGFP reporter plasmid pBSU101. In all of these species the presence of the cfb promoter construct resulted in high-level EGFP expression that could be further increased by growing the streptococcal and enterococcal cultures under high oxygen conditions through continuous aeration.

Human Serum Induces Streptococcal C5a Peptidase Expression

ABSTRACT Streptococcus agalactiae is a major pathogen in humans and animals. Virulence factors are often associated with mobile genetic elements, and their expression can be modulated by host factors. S. agalactiae harbors the genes for C5a peptidase (scpB) and Lmb on a composite transposon structure which is absent in many bovine isolates. To investigate whether these genes participate in the adaptation to human hosts, we determined the influence of human and bovine serum on the promoter activity of scpB and lmb by using fluorescence-activated cell sorter analysis. Culture in the presence of 1 to 50% human serum resulted in a dose-dependent induction of reporter gene activity for scpB but not lmb. Reporter gene activity was, however, unchanged following growth in fetal calf serum. Interestingly, a bovine strain did not display any induction of scpB by either bovine or human serum. Reverse transcription-PCR analysis was used to confirm differential induction of scpB in S. agalactiae and showed a similar induction of the Streptococcus pyogenes C5a peptidase gene scpA by human but not bovine serum. The specific induction of the streptococcal C5a peptidase by human serum corresponds to the absence of scpB in many bovine S. agalactiae isolates and underlines the importance of this virulence factor for human infections.

The β-Hemolysin and Intracellular Survival of Streptococcus agalactiae in Human Macrophages

S. agalactiae (group B streptococci, GBS) is a major microbial pathogen in human neonates and causes invasive infections in pregnant women and immunocompromised individuals. The S. agalactiae β-hemolysin is regarded as an important virulence factor for the development of invasive disease. To examine the role of β-hemolysin in the interaction with professional phagocytes, the THP-1 monocytic cell line and human granulocytes were infected with a serotype Ia S. agalactiae wild type strain and its isogenic nonhemolytic mutant. We could show that the nonhemolytic mutants were able to survive in significantly higher numbers than the hemolytic wild type strain, in THP-1 macrophage-like cells and in assays with human granulocytes. Intracellular bacterial multiplication, however, could not be observed. The hemolytic wild type strain stimulated a significantly higher release of Tumor Necrosis Factor-α than the nonhemolytic mutant in THP-1 cells, while similar levels of the chemokine Interleukin-8 were induced. In order to investigate bacterial mediators of IL-8 release in this setting, purified cell wall preparations from both strains were tested and found to exert a potent proinflammatory stimulus on THP-1 cells. In conclusion, our results indicate that the β-hemolysin has a strong influence on the intracellular survival of S. agalactiae and that a tightly controlled regulation of β-hemolysin expression is required for the successful establishment of S. agalactiae in different host niches.

Chromosomally and Extrachromosomally Mediated High-Level Gentamicin Resistance in Streptococcus agalactiae

ABSTRACT Streptococcus agalactiae (group B Streptococcus [GBS]) is a leading cause of sepsis in neonates. The rate of invasive GBS disease in nonpregnant adults also continues to climb. Aminoglycosides alone have little or no effect on GBS, but synergistic killing with penicillin has been shown in vitro. High-level gentamicin resistance (HLGR) in GBS isolates, however, leads to the loss of a synergistic effect. We therefore performed a multicenter study to determine the frequency of HLGR GBS isolates and to elucidate the molecular mechanisms leading to gentamicin resistance. From eight centers in four countries, 1,128 invasive and colonizing GBS isolates were pooled and investigated for the presence of HLGR. We identified two strains that displayed HLGR (BSU1203 and BSU452), both of which carried the aacA-aphD gene, typically conferring HLGR. However, only one strain (BSU1203) also carried the previously described chromosomal gentamicin resistance transposon designated Tn3706. For the other strain (BSU452), plasmid purification and subsequent DNA sequencing resulted in the detection of plasmid pIP501 carrying a remnant of a Tn3 family transposon. Its ability to confer HLGR was proven by transfer into an Enterococcus faecalis isolate. Conversely, loss of HLGR was documented after curing both GBS BSU452 and the transformed E. faecalis strain from the plasmid. This is the first report showing plasmid-mediated HLGR in GBS. Thus, in our clinical GBS isolates, HLGR is mediated both chromosomally and extrachromosomally.

Streptolysin S of Streptococcus anginosus exhibits broad-range hemolytic activity

Streptococcus anginosus is a commensal of mucous membranes and an emerging human pathogen. Some strains, including the type strain, display a prominent β-hemolytic phenotype. A gene cluster (sag), encoding a variant of streptolysin S (SLS) has recently been identified as the genetic background for β-hemolysin production in S. anginosus. In this study, we further characterized the hemolytic and cytolytic activity of the S. anginosus hemolysin in comparison with other streptococcal hemolysins. The results indicate that SLS of S. anginosus is a broad-range hemolysin able to lyse erythrocytes of different species, including horse, bovine, rabbit and even chicken. The hemolytic activity is temperature dependent, and a down-regulation of the hemolysin expression is induced in the presence of high glucose levels. Survival assays indicate that in contrast to other streptococcal species, S. anginosus does not require SLS for survival in the presence of human granulocytes. Cross-complementation studies using the sagB and sagD genes of Streptococcus pyogenes and Streptococcus dysgalactiae subsp. equisimilis demonstrated functional similarities to the S. anginosus SLS. Nevertheless, distinct differences to other streptolysin S variants were noted and provide further insights into the molecular mechanisms of SLS pathogen host interactions.

Rapid identification of beta-hemolytic streptococci by fluorescence in situ hybridization (FISH)

Rapid identification of pathogenic, beta-hemolytic streptococci is important for treatment decisions. We evaluated fluorescence in situ hybridization (FISH) for this purpose using 23 reference strains, 157 clinical isolates, and 80 blood cultures showing streptococci in the Gram stain. With a sensitivity and specificity in excess of 99%, FISH proved to be suitable for rapid identification of beta-hemolytic streptococci in a diagnostic laboratory.

A streptococcal NRAMP homologue is crucial for the survival of Streptococcus agalactiae under low pH conditions.

Streptococcus agalactiae or Group B Streptococcus (GBS) is a commensal bacterium of the human gastrointestinal and urogenital tracts as well as a leading cause of neonatal sepsis, pneumonia and meningitis. Maternal vaginal carriage is the main source for GBS transmission and thus the most important risk factor for neonatal disease. Several studies in eukaryotes identified a group of proteins natural resistance‐associated macrophage protein (NRAMP) that function as divalent cation transporters for Fe2+ and Mn2+ and confer on macrophages the ability to control replication of bacterial pathogens. Genome sequencing predicted potential NRAMP homologues in several prokaryotes. Here we describe for the first time, a pH‐regulated NRAMP Mn2+/Fe2+ transporter in GBS, designated MntH, which confers resistance to reactive oxygen species (ROS) and is crucial for bacterial growth and survival under low pH conditions. Our investigation implicates MntH as an important colonization determinant for GBS in the maternal vagina as it helps bacteria to adapt to the harsh acidic environment, facilitates bacterial adherence, contributes to the coexistence with the vaginal microbiota and plays a role in GBS intracellular survival inside macrophages.

Regulation of the β-hemolysin gene cluster of Streptococcus anginosus by CcpA

Streptococcus anginosus is increasingly recognized as an opportunistic pathogen. However, our knowledge about virulence determinants in this species is scarce. One exception is the streptolysin-S (SLS) homologue responsible for the β-hemolytic phenotype of the S. anginosus type strain. In S. anginosus the expression of the hemolysin is reduced in the presence of high glucose concentrations. To investigate the genetic mechanism of the hemolysin repression we created an isogenic ccpA deletion strain. In contrast to the wild type strain, this mutant exhibits hemolytic activity in presence of up to 25 mM glucose supplementation, a phenotype that could be reverted by ccpA complementation. To further demonstrate that CcpA directly regulates the hemolysin expression, we performed an in silico analysis of the promoter of the SLS gene cluster and we verified the binding of CcpA to the promoter by electrophoretic mobility shift assays. This allowed us to define the CcpA binding site in the SLS promoter region of S. anginosus. In conclusion, we report for the first time the characterization of a potential virulence regulator in S. anginosus.