Micrococcal Nuclease stimulates Staphylococcus aureus Biofilm Formation in a Murine Implant Infection Model

Advancements in contemporary medicine have led to an increasing life expectancy which has broadened the application of biomaterial implants. As each implant procedure has an innate risk of infection, the number of biomaterial-associated infections keeps rising. Staphylococcus aureus causes 34% of such infections and is known as a potent biofilm producer. By secreting micrococcal nuclease S. aureus is able to escape neutrophil extracellular traps by cleaving their DNA-backbone. Also, micrococcal nuclease potentially limits biofilm growth and adhesion by cleaving extracellular DNA, an important constituent of biofilms. This study aimed to evaluate the impact of micrococcal nuclease on infection persistence and biofilm formation in a murine biomaterial-associated infection-model with polyvinylidene-fluoride mesh implants inoculated with bioluminescent S. aureus or its isogenic micrococcal nuclease deficient mutant. Supported by results based on in-vivo bioluminescence imaging, ex-vivo colony forming unit counts, and histological analysis it was found that production of micrococcal nuclease enables S. aureus bacteria to evade the immune response around an implant resulting in a persistent infection. As a novel finding, histological analysis provided clear indications that the production of micrococcal nuclease stimulates S. aureus to form biofilms, the presence of which extended neutrophil extracellular trap formation up to 13 days after mesh implantation. Since micrococcal nuclease production appeared vital for the persistence of S. aureus biomaterial-associated infection, targeting its production could be a novel strategy in preventing biomaterial-associated infection.

Pseudomonas aeruginosa Oligoribonuclease Controls Susceptibility to Polymyxin B by Regulating Pel Exopolysaccharide Production

Polymyxins are considered as the last resort antibiotics to treat infections caused by multidrug-resistant Gram negative pathogens. Pseudomonas aeruginosa is an opportunistic pathogen that causes various infections in humans. Proteins involved in lipopolysaccharide modification and maintaining inner and outer membrane integrities have been found to contribute to the bacterial resistance to polymyxins. Oligoribonuclease (Orn) is an exonuclease that regulates the homeostasis of intracellular (3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP), thereby regulating the production of extracellular polysaccharide in P. aeruginosa . Previously, we demonstrated that Orn affects the bacterial resistance to fluoroquinolone, β-lactam and aminoglycoside antibiotics. In this study, we found that mutation of orn increased the bacterial survival following polymyxin B treatment in a wild type P. aeruginosa strain PA14. Overexpression of c-di-GMP degradation enzymes in the orn mutant reduced the bacterial survival. By using a fluorescence labeled polymyxin B, we found that mutation of orn increased the bacterial surface bound polymyxin B. Deletion of the Pel synthesis genes or treatment with a Pel hydrolase reduced the surface bound polymyxin B and bacterial survival. We further demonstrated that Pel binds to extracellular DNA (eDNA), which traps polymyxin B and thus protects the bacterial cells. Collectively, our results revealed a novel defense mechanism against polymyxin in P. aeruginosa .

Core Microbiota Drive the Prevalence of Extracellular Antibiotic Resistome in the Water Compartments

Unlike intracellular chromosome, extracellular DNA (eDNA) may accelerate the spreading of antibiotic resistance genes (ARGs) through natural transformation, but one of the core issues regarding to the taxonomic characterization of eDNA in the complex water environments is largely unknown. Hence, Illumina Miseq sequencing was used to identify the genotype of eDNA from wastewater (WW), river water (RW) and stormwater (SW) runoff. High-throughput qPCR targeting 384 genes was implemented to detect extracellular ARGs (eARGs) and mobile genetic elements (eMGEs). We obtained 2,708,291 high quality sequences from 66 eDNA samples. The SW exhibited the significant higher Shannon Index. Subsequently, we identified 34 core bacteria sources of eDNA widely distributed in the three water compartments. Among which, Pseudomonas, Flavobacterium, Limnohabitans, Burkholderiaceae_unclassified, Methylotenera and Acinetobacter were the most prevalent. A total of 302 eARGs and eMGEs were detected, suggesting that eDNA is an important antibiotic resistance reservoir. Among the 127 shared genes of the three groups, 15 core resistance genes were filtered, including IS6100, sul1 NEW, intI1, ISPps1-pseud, aac3-Via, qacH_351 and ISSm2-Xanthob. The Procrustes analysis and Variance Partitioning Analysis (VPA) demonstrated that core bacteria and MGEs were significantly correlated with eARGs. These results suggested that the occurrence and changes of eARGs in the water compartments may be largely attributed to the core microbiota and eMGEs.

Clinical relevance and prognostic role of preoperative cell-free single-stranded DNA concentrations in colorectal cancer patients

Purpose: Circulating cell-free single-stranded DNA (ccf-ssDNA) is extracellular DNA and it is a useful biomarker for the diagnosis of tumors and predicting the prognosis of tumors. However, the clinical usefulness of ccf-ssDNA in colorectal cancer (CRC) is not well known. Thus, the purpose of this study was to investigate the clinical usefulness of ccf-ssDNA in CRC.Methods: The study was conducted on 44 patients who had undergone surgery for CRC, and ccf-ssDNA level was measured before surgery and statistical analysis was performed on clinical factors.Results: The association between ccf-ssDNA level and clinicopathological factors was analyzed and compared, and these factors included age, sex, body mass index, diabetes mellitus, hypertension, tumor markers (carcinoembryonic antigen and carbohydrate antigen 19-9), tumor location, size, stage (TNM), recurrence, and death. The group with a ccf-ssDNA level of ≥ 7.5 ng/μL had a lower age (P = 0.010), and was associated with diabetes mellitus (P = 0.037) and lymph node metastasis (P = 0.049). Multivariate analysis of disease-free survival showed that lymph node metastasis and ccf-ssDNA level (hazard ratio, 10.011; 95% confidence interval, 2.269–44.175; P = 0.002) were independent prognostic factors for recurrence. In terms of overall survival, there were no statistically significant results except for vascular invasion.Conclusion: This study showed that ccf-ssDNA level in plasma in CRC patients was an independent prognostic factor that could predict recurrence non-invasively. In this regard, further evaluation with a prospective, large sample size study will be needed to obtain additional results.

Extracellular CIRP Induces Macrophage Extracellular Trap Formation Via Gasdermin D Activation

Extracellular cold-inducible RNA-binding protein (eCIRP) is a damage-associated molecular pattern promoting inflammation and tissue injury. During bacterial or viral infection, macrophages release DNA decorated with nuclear and cytoplasmic proteins known as macrophage extracellular traps (METs). Gasdermin D (GSDMD) is a pore-forming protein that has been involved in extracellular trap formation in neutrophils. We hypothesized that eCIRP induces MET formation by activating GSDMD. Human monocytic cell line THP-1 cells were differentiated with phorbol 12-myristate 13-acetate (PMA) and treated with recombinant murine (rm) CIRP. The MET formation was detected by three methods: time-lapse fluorescence microscopy (video imaging), colorimetry, and ELISA. Cleaved forms of GSDMD, and caspase-1 were detected by Western blotting. Treatment of THP-1 cells with rmCIRP increased MET formation as revealed by SYTOX Orange Staining assay in a time- and dose-dependent manner. METs formed by rmCIRP stimulation were further confirmed by extracellular DNA, citrullinated histone H3, and myeloperoxidase. Treatment of THP-1 cells with rmCIRP significantly increased the cleaved forms of caspase-1 and GSDMD compared to PBS-treated cells. Treatment of macrophages with caspase-1, and GSDMD inhibitors z-VAD-fmk, and disulfiram, separately, significantly decreased rmCIRP-induced MET formation. We also confirmed rmCIRP-induced MET formation using primary cells murine peritoneal macrophages. These data clearly show that eCIRP serves as a novel inducer of MET formation through the activation of GSDMD and caspase-1.

The Role of Minor Pilins in Assembly and Function of the Competence Pilus of Streptococcus pneumoniae

The remarkable genomic plasticity of Streptococcus pneumoniae largely depends on its ability to undergo natural genetic transformation. To take up extracellular DNA, S. pneumoniae assembles competence pili composed of the major pilin ComGC. In addition to ComGC, four minor pilins ComGD, E, F, and G are expressed during bacterial competence, but their role in pilus biogenesis and transformation is unknown. Here, using a combination of protein-protein interaction assays we show that all four proteins can directly interact with each other. Pneumococcal ComGG stabilizes the minor pilin ComGD and ComGF and can interact with and stabilize the major pilin ComGC, thus, deletion of ComGG abolishes competence pilus assembly. We further demonstrate that minor pilins are present in sheared pili fractions and find ComGF to be incorporated along the competence pilus by immunofluorescence and electron microscopy. Finally, mutants of the invariant Glu5 residue (E5), ComGDE5A or ComGEE5A, but not ComGFE5A, were severely impaired in pilus formation and function. Together, our results suggest that ComGG, lacking E5, is essential for competence pilus assembly and function, and plays a central role in connecting the pneumococcal minor pilins to ComGC.

Sex Difference in Plasma Deoxyribonuclease Activity in Rats

Extracellular DNA (ecDNA) activates immune cells and is involved in the pathogenesis of diseases associated with inflammation such as sepsis, rheumatoid arthritis or metabolic syndrome. DNA can be cleaved by deoxyribonucleases (DNases), some of which are secreted out of cells. The aim of this experiment was to describe plasma DNase activity in relation to extracellular DNA in adult rats, to analyse potential sex differences and to prove whether they are related to endogenous testosterone. Adult Lewis rats (n=28) of both sexes were included in the experiment. Male rats were gonadectomized or sham-operated and compared to intact female rats. Plasma ecDNA and DNase activity were measured using fluorometry and single radial enzyme diffusion assay, respectively. Concentrations of nuclear ecDNA and mitochondrial ecDNA were determined using real-time PCR. Females had 60% higher plasma DNase activity than males (p=0.03). Gonadectomy did not affect plasma DNase in males. Neither the concentration of total ecDNA, nor nuclear or mitochondrial DNA in plasma differed between the groups. No significant correlations between DNase and ecDNA were found. From previous studies on mice, it was expected, that male rats will have higher DNase activity. In contrast, our study in rats showed the opposite sex difference. This sex difference seems not to be caused by endogenous testosterone. Interestingly, no sex differences were observed in plasma ecDNA suggesting a complex or missing association between plasma ecDNA and DNase. The observed sex difference in plasma DNase should be taken into account in animal models of ecDNA-associated diseases.

Quinolone resistance is transferred horizontally via uptake signal sequence recognition in Haemophilus influenzae

The presence of Haemophilus influenzae strains with low susceptibility to quinolones has been reported worldwide. However, the emergence and dissemination mechanisms remain unclear. In this study, a total of 14 quinolone-low-susceptible H. influenzae isolates were investigated phylogenetically and in vitro resistance transfer assay in order to elucidate the emergence and dissemination mechanisms. The phylogenetic analysis based on gyrA sequences showed that strains with the same sequence type determined by multilocus sequence typing were classified into different clusters, suggesting that H. influenzae quinolone resistance emerges not only by point mutation, but also by the horizontal transfer of mutated gyrA . Moreover, the in vitro resistance transfer assay confirmed the horizontal transfer of quinolone resistance and indicated an active role of extracellular DNA in the resistance transfer. Interestingly, the horizontal transfer of parC only occurred in those cells that harbored a GyrA with amino acid substitutions, suggesting a possible mechanism of quinolone resistance in clinical settings. Moreover, the uptake signal and uptake-signal-like sequences located downstream of the quinolone resistant-determining regions of gyrA and parC , respectively, contributed to the horizontal transfer of resistance in H. influenzae . Our study demonstrates that the quinolone resistance of H. influenzae could emerge due to the horizontal transfer of gyrA and parC via recognition of an uptake signal sequence or uptake-signal-like sequence. Since the presence of quinolone-low-susceptible H. influenzae with amino acid substitutions in GyrA have been increasing in recent years, it is necessary to focus our attention to the acquisition of further drug resistance in these isolates.

Activity of Moxifloxacin Against Biofilms Formed by Clinical Isolates of Staphylococcus aureus Differing by Their Resistant or Persister Character to Fluoroquinolones

Staphylococcus aureus biofilms are poorly responsive to antibiotics. Underlying reasons include a matrix effect preventing drug access to embedded bacteria, or the presence of dormant bacteria with reduced growth rate. Using 18 clinical isolates previously characterized for their moxifloxacin-resistant and moxifloxacin-persister character in stationary-phase culture, we studied their biofilm production and matrix composition and the anti-biofilm activity of moxifloxacin. Biofilms were grown in microtiter plates and their abundance quantified by crystal violet staining and colony counting; their content in polysaccharides, extracellular DNA and proteins was measured. Moxifloxacin activity was assessed after 24 h of incubation with a broad range of concentrations to establish full concentration-response curves. All clinical isolates produced more biofilm biomass than the reference strain ATCC 25923, the difference being more important for those with high relative persister fractions to moxifloxacin, most of which being also resistant. High biofilm producers expressed icaA to higher levels, enriching the matrix in polysaccharides. Moxifloxacin was less potent against biofilms from clinical isolates than from ATCC 25923, especially against moxifloxacin-resistant isolates with high persister fractions, which was ascribed to a lower concentration of moxifloxacin in these biofilms. Time-kill curves in biofilms revealed the presence of a moxifloxacin-tolerant subpopulation, with low multiplication capacity, whatever the persister character of the isolate. Thus, moxifloxacin activity depends on its local concentration in biofilm, which is reduced in most isolates with high-relative persister fractions due to matrix effects, and insufficient to kill resistant isolates due to their high MIC.