Staphylococcus aureus biofilms are extremely difficult to treat. They provide a protected niche for the bacteria, rendering them highly recalcitrant toward host defenses as well as antibiotic ...treatment. Bacteria within a biofilm are shielded from the immune system by the formation of an extracellular polymeric matrix, composed of polysaccharides, extracellular DNA (eDNA), and proteins. Many antibiotics do not readily penetrate biofilms, resulting in the presence of subinhibitory concentrations of antibiotics. Here, we show that subinhibitory concentrations of clindamycin triggered a transcriptional stress response in S. aureus via the alternative sigma factor B (σ(B)) and upregulated the expression of the major biofilm-associated genes atlA, lrgA, agrA, the psm genes, fnbA, and fnbB Our data suggest that subinhibitory concentrations of clindamycin alter the ability of S. aureus to form biofilms and shift the composition of the biofilm matrix toward higher eDNA content. An understanding of the molecular mechanisms underlying biofilm assembly and dispersal in response to subinhibitory concentrations of clinically relevant antibiotics such as clindamycin is critical to further optimize antibiotic treatment strategies of biofilm-associated S. aureus infections.
BCOR
-rearranged sarcomas are rare and belong to the Ewing-like sarcomas (ELS). Their morphology and histopathological features make the diagnosis challenging. We present a case, initially diagnosed ...as an unusual extraskeletal myxoid chondrosarcoma (EMC). A 54-year-old male patient developed an asymptomatic swelling of the lower leg. Imaging showed a 9.5-cm large intramuscular soft tissue mass. Due to its morphological and immunohistochemical profile on biopsy, it was initially diagnosed as an EMC. The patient was treated by complete resection and adjuvant radiotherapy and remained free of tumor at 7 years follow-up. Using next-generation sequencing (NGS), we retrospectively identified
RGAG1-BCOR
gene fusion (confirmed by RT-PCR), which has not been described in somatic soft tissue tumors so far. This finding broadens the spectrum of partner genes in the
BCOR
-rearranged sarcomas in a tumor with a well-documented, long clinical follow-up.
Nonalcoholic steatohepatitis (NASH)-induced hepatocellular carcinoma (HCC) and its precursor, nonalcoholic fatty liver disease (NAFLD) are an unmet health issue due to widespread obesity. We assessed ...copy number changes of genes associated with hepatocarcinogenesis and oxidative pathways at a single-cell level. Eleven patients with NASH-HCC and 11 patients with NAFLD were included. Eight probes were analyzed using multiplex interphase fluorescence in situ hybridization (miFISH), single-cell imaging and phylogenetic tree modelling: Telomerase reverse transcriptase (TERT), C-Myc (MYC), hepatocyte growth factor receptor tyrosine kinase (MET), tumor protein 53 (TP53), cyclin D1 (CCND1), human epidermal growth factor receptor 2 (HER2), the fragile histidine triad gene (FHIT) and FRA16D oxidoreductase (WWOX). Each NASH-HCC tumor had up to 14 distinct clonal signal patterns indicating multiclonality, which correlated with high tumor grade. Changes frequently observed were TP53 losses, 45%; MYC gains, 36%; WWOX losses, 36%; and HER2 gains, 18%. Whole-genome duplications were frequent (82%) with aberrant tetraploid cells evolving from diploid ancestors. Non-tumorous NAFLD/NASH biopsies did not harbor clonal copy number changes. Fine mapping of NASH-HCC using single-cell multiplex FISH shows that branched tumor evolution involves genome duplication and that multiclonality increases with tumor grade. The loss of oxidoreductase WWOX and HER2 gains could be potentially associated with NASH-induced hepatocellular carcinoma.
Healthcare-associated outbreaks of vancomycin-resistant Enterococcus faecium (VREfm) are a worldwide problem with increasing prevalence. The genomic plasticity of this hospital-adapted pathogen ...contributes to its efficient spread despite infection control measures. Here, we aimed to identify the genomic and phenotypic determinants of health care-associated transmission of VREfm. We assessed the VREfm transmission networks at the tertiary-care University Hospital of Zurich (USZ) between October 2014 and February 2018 and investigated microevolutionary dynamics of this pathogen. We performed whole-genome sequencing for the 69 VREfm isolates collected during this time frame and assessed the population structure and variability of the vancomycin resistance transposon. Phylogenomic analysis allowed us to reconstruct transmission networks and to unveil external or wider transmission networks undetectable by routine surveillance. Notably, it unveiled a
, sampled 31 times over a 29-month period. Exploring the evolutionary dynamics of this clone and characterizing the phenotypic consequences revealed the spread of a variant with decreased daptomycin susceptibility and the acquired ability to utilize N-acetyl-galactosamine (GalNAc), one of the primary constituents of the human gut mucins. This nutrient utilization advantage was conferred by a novel plasmid, termed pELF_USZ, which exhibited a linear topology. This plasmid, which was harbored by two distinct clones, was transferable by conjugation. Overall, this work highlights the potential of combining epidemiological, functional genomic, and evolutionary perspectives to unveil adaptation strategies of VREfm.
Sequencing microbial pathogens causing outbreaks has become a common practice to characterize transmission networks. In addition to the signal provided by vertical evolution, bacterial genomes harbor mobile genetic elements shared horizontally between clones. While macroevolutionary studies have revealed an important role of plasmids and genes encoding carbohydrate utilization systems in the adaptation of Enterococcus faecium to the hospital environment, mechanisms of dissemination and the specific function of many of these genetic determinants remain to be elucidated. Here, we characterize a plasmid providing a nutrient utilization advantage and show evidence for its clonal and horizontal spread at a local scale. Further studies integrating epidemiological, functional genomics, and evolutionary perspectives will be critical to identify changes shaping the success of this pathogen.
Biofilm formation in Staphylococcus aureus under in vitro growth conditions is generally promoted by high concentrations of sugar and/or salts. The addition of glucose to routinely used complex ...growth media triggered biofilm formation in S. aureus strain SA113. Deletion of ccpA, coding for the catabolite control protein A (CcpA), which regulates gene expression in response to the carbon source, abolished the capacity of SA113 to form a biofilm under static and flow conditions, while still allowing primary attachment to polystyrene surfaces. This suggested that CcpA mainly affects biofilm accumulation and intercellular aggregation. trans-Complementation of the mutant with the wild-type ccpA allele fully restored the biofilm formation. The biofilm produced by SA113 was susceptible to sodium metaperiodate, DNase I, and proteinase K treatment, indicating the presence of polysaccharide intercellular adhesin (PIA), protein factors, and extracellular DNA (eDNA). The investigation of several factors which were reported to influence biofilm formation in S. aureus (arlRS, mgrA, rbf, sarA, atl, ica, citZ, citB, and cidABC) showed that CcpA up-regulated the transcription of cidA, which was recently shown to contribute to eDNA production. Moreover, we showed that CcpA increased icaA expression and PIA production, presumably over the down-regulation of the tricarboxylic acid cycle genes citB and citZ.
Staphylococcus aureus
causes chronic and relapsing infections, which may be difficult to treat. So-called small colony variants (SCVs) have been associated with chronic infections and their ...occurrence has been shown to increase under antibiotic pressure, low pH and intracellular localization. In clinics,
S. aureus
isolated from invasive infections often show a dysfunction in the accessory gene regulator (
agr
), a major virulence regulatory system in
S. aureus
. To assess whether intracellular environment and
agr
function influence SCV formation, an infection model was established using lung epithelial cells and skin fibroblasts. This allowed analyzing intracellular survival and localization of a panel of
S. aureus
wild type strains and their isogenic
agr
knock out mutants as well as a natural dysfunctional
agr
strain by confocal laser scanning microscopy (CLSM). Furthermore, bacterial colonies were quantified after 1, 3, and 5 days of intracellular survival by time-lapse analysis to determine kinetics of colony appearance and SCV formation. Here, we show that
S. aureus
strains with an
agr
knock out predominantly resided in a neutral environment, whereas wild type strains and an
agr
complemented strain resided in an acidic environment.
S. aureus agr
mutants derived from an intracellular environment showed a higher percentage of SCVs as compared to their corresponding wild type strains. Neutralizing acidic phagolysosomes with chloroquine resulted in a significant reduction of SCVs in
S. aureus
wild type strain 6850, but not in its
agr
mutant indicating a pH dependent formation of SCVs in the wild type strain. The in-depth understanding of the interplay between intracellular persistence,
agr
function and pH should help to identify new therapeutic options facilitating the treatment of chronic
S. aureus
infections in the future.
Some clinical isolates of Staphylococcus aureus produce the superantigenic toxic shock syndrome toxin 1 (TSST-1), encoded by tst, located on pathogenicity islands. The expression of tst is complex ...and is influenced by environmental conditions such as pH, CO₂, and glucose. We identified a putative catabolite-responsive element (cre) in the promoter regions of all known tst genes, indicating that tst transcription may be regulated by the catabolite control protein CcpA. By introducing tst genes under the control of their native promoters or tst promoter-reporter gene fusions in wild-type strain Newman, we showed that glucose was able to repress tst transcription and TSST-1 production, whereas glucose repression was abolished in the corresponding ΔccpA mutant. Stabilizing the pH ruled out a pH effect due to acid production during glucose catabolism. CcpA thus directly regulates tst transcription, linking carbohydrate utilization to virulence gene expression in S. aureus.
The catabolite control protein A (CcpA) is a member of the LacI/GalR family of transcriptional regulators controlling carbon-metabolism pathways in low-GC Gram-positive bacteria. It functions as a ...catabolite repressor or activator, allowing the bacteria to utilize the preferred carbon source over secondary carbon sources. This study is the first CcpA-dependent transcriptome and proteome analysis in Staphylococcus aureus, focussing on short-time effects of glucose under stable pH conditions.
The addition of glucose to exponentially growing S. aureus increased the expression of genes and enzymes of the glycolytic pathway, while genes and proteins of the tricarboxylic acid (TCA) cycle, required for the complete oxidation of glucose, were repressed via CcpA. Phosphotransacetylase and acetate kinase, converting acetyl-CoA to acetate with a concomitant substrate-level phosphorylation, were neither regulated by glucose nor by CcpA. CcpA directly repressed genes involved in utilization of amino acids as secondary carbon sources. Interestingly, the expression of a larger number of genes was found to be affected by ccpA inactivation in the absence of glucose than after glucose addition, suggesting that glucose-independent effects due to CcpA may have a particular impact in S. aureus. In the presence of glucose, CcpA was found to regulate the expression of genes involved in metabolism, but also that of genes coding for virulence determinants.
This study describes the CcpA regulon of exponentially growing S. aureus cells. As in other bacteria, CcpA of S. aureus seems to control a large regulon that comprises metabolic genes as well as virulence determinants that are affected in their expression by CcpA in a glucose-dependent as well as -independent manner.
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