Plasmid-Chromosome Crosstalk in Staphylococcus aureus: A Horizontally Acquired Transcription Regulator Controls Polysaccharide Intercellular Adhesin-Mediated Biofilm Formation

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) of clonal complex CC398 typically carry various antimicrobial resistance genes, many of them located on plasmids. In the bovine LA-MRSA isolate Rd11, we previously identified plasmid pAFS11 in which resistance genes are co-localized with a novel ica-like gene cluster, harboring genes required for polysaccharide intercellular adhesin (PIA)-mediated biofilm formation. The ica genes on pAFS11 were acquired in addition to a pre-existing ica locus on the S. aureus Rd11 chromosomal DNA. Both loci consist of an icaADBC operon and icaR, encoding a corresponding icaADBC repressor. Despite carrying two biofilm gene copies, strain Rd11 did not produce PIA and transformation of pAFS11 into another S. aureus strain even slightly diminished PIA-mediated biofilm formation. By focusing on the molecular background of the biofilm-negative phenotype of pAFS11-carrying S. aureus, we identified the pAFS11-borne ica locus copy as functionally fully active. However, transcription of both plasmid- and core genome-derived icaADBC operons were efficiently suppressed involving IcaR. Surprisingly, although being different on the amino acid sequence level, the two IcaR repressor proteins are mutually replaceable and are able to interact with the icaA promoter region of the other copy. We speculate that this regulatory crosstalk causes the biofilm-negative phenotype in S. aureus Rd11. The data shed light on an unexpected regulatory interplay between pre-existing and newly acquired DNA traits in S. aureus. This also raises interesting general questions regarding functional consequences of gene transfer events and their putative implications for the adaptation and evolution of bacterial pathogens

Silence as a way of niche adaptation: mecC-MRSA with variations in the accessory gene regulator (agr) functionality express kaleidoscopic phenotypes

Functionality of the accessory gene regulator (agr) quorum sensing system is an important factor promoting either acute or chronic infections by the notorious opportunistic human and veterinary pathogen Staphylococcus aureus. Spontaneous alterations of the agr system are known to frequently occur in human healthcare-associated S. aureus lineages. However, data on agr integrity and function are sparse regarding other major clonal lineages. Here we report on the agr system functionality and activity level in mecC-carrying methicillin resistant S. aureus (MRSA) of various animal origins (n = 33) obtained in Europe as well as in closely related human isolates (n = 12). Whole genome analysis assigned all isolates to four clonal complexes (CC) with distinct agr types (CC599 agr I, CC49 agr II, CC130 agr III and CC1943 agr IV). Agr functionality was assessed by a combination of phenotypic assays and proteome analysis. In each CC, isolates with varying agr activity levels were detected, including the presence of completely non-functional variants. Genomic comparison of the agr I-IV encoding regions associated these phenotypic differences with variations in the agrA and agrC genes. The genomic changes were detected independently in divergent lineages, suggesting that agr variation might foster viability and adaptation of emerging MRSA lineages to distinct ecological niches

Comparison between hospitalized patients affected or not by COVID-19 (RESILIENCY study)

Dear Editor, in the recent report of Munblit and coworkers [1], authors observed that the combination of clinical features was sufficient to diagnose COVID-19 indicating that laboratory testing is not critical in real-life clinical practice. To date, all patients admitted to Emergency Department with acute respiratory failure and/or fever should be considered as a suspected SARS-CoV-2 infection [2-3], and an early recognition of etiology and the prompt therapeutic management are crucial to improve survival [4-5]. From March to July 2020, we performed a prospective, multicenter study (RESILIENCY study). During the study period, all patients hospitalized for suspected or confirmed COVID-19 were prospectively recruited in 3 large hospitals in Rome, Italy. All patients with suspected SARS-CoV-2 infection, admitted to the hospital in case of fever and/or hypoxemic respiratory failure (PaO2 <60 mmHg at rest in ambient air) or of exacerbation of underlying diseases or severe symptoms not manageable outside the hospital, were evaluated according to a predefined protocol (see Figure 1). Overall, 653 patients were included in the study: 309 (47.3%) patients with confirmed COVID-19 and 344 (52.7%) without COVID-19, hospitalized for other causes. Baseline characteristics and outcomes of the study population showed that the main causes of hospitalization among patients without COVID-19 were: acute heart failure (47%), bacterial pneumonia (38.5%), and pulmonary embolism (9.2%). Overall, 67 (21.7%) patients of COVID-19 group and 45 (13.1%) hospitalized for other causes were admitted to intensive care unit; 30-day mortality was observed in 59 (19%) patients of COVID-19 group and 62 (18%) of non-COVID-19 group. The multivariate analysis about risk factors for COVID-19 etiology at time of hospitalization showed that dry cough (OR 3.76, CI 95% 1.98-7.92, P<0.001), duration of fever>3 days (OR 5.21, CI 95% 2.34-9.21, P<0.001), lymphocytopenia (OR 1.98, CI 95% Downloaded from https://academic.oup.com/cid/advance-article/doi/10.1093/cid/ciaa1745/5989494 by Sapienza Università di Roma user on 01 December 2020 Accepted Manuscript 3 1.27-4.22, P=0.002) and PaO2/FiO2 ratio<250 (OR 4.98, CI 95% 2.22-9.71, P<0.001) were independently associated with COVID-19 etiology, while procalcitonin value>1 ng/ mL (OR 0.21, CI 95% 0.08-0.82, p<0.001), and lactate>2 mmol/L (OR 0.41, CI 95% 0.15-0.77, p<0.001) were associated with non-COVID-19 etiology. Finally, analysis about predictors of 30-day mortality showed that age (per-year increase OR 1.33; CI 95% 1.11-2.10; p<0.001), cardiovascular disease (OR 4.58; CI 95% 2.07-8.25; p<0.001), and ICU admission (OR 2.1; CI 95% 1.48-4.4; p<0.001) were independently associated with all-cause 30-day mortality, while the use of low-molecularweight heparin (OR 0.22, CI 95% 0.03-0.45, p=0.002) was associated with survival. The findings of the present study can be summarized as follows:1) the prompt identification of specific clinical characteristics (like dry cough or duration of fever>3 days), and laboratory findings (like lymphocytopenia, PaO2/FiO2 ratio<250, procalcitonin value>1 ng/ mL, and lactate>2 mmol/L) can help physicians to distinguish rapidly between COVID19 or other etiologies [6]; 2) the application of a standard approach to management of patients with acute respiratory failure and/or fever associated with the knowledge of clinical and laboratory characteristics of COVID-19 can early drive physicians to therapeutic choices; and 3) age, cardiovascular disease, and ICU admission show an independent association with all-cause 30-day mortality [7], while the use of low-molecular-weight heparin was associated with survival [8]. In conclusion, COVID-19 syndrome is characterized by a heterogeneous clinical, laboratoristic, and radiological presentation, especially at its onset [9]. However, the application of a standard approach to management of patients with acute respiratory failure and/or fever and the knowledge of clinical and laboratory characteristics of COVID-19 can early drive therapeutic choic

Silence as a way of niche adaptation: mecC-MRSA with variations in the accessory gene regulator (agr) functionality express kaleidoscopic phenotypes

Functionality of the accessory gene regulator (agr) quorum sensing system is an important factor promoting either acute or chronic infections by the notorious opportunistic human and veterinary pathogen Staphylococcus aureus. Spontaneous alterations of the agr system are known to frequently occur in human healthcare-associated S. aureus lineages. However, data on agr integrity and function are sparse regarding other major clonal lineages. Here we report on the agr system functionality and activity level in mecC-carrying methicillin resistant S. aureus (MRSA) of various animal origins (n = 33) obtained in Europe as well as in closely related human isolates (n = 12). Whole genome analysis assigned all isolates to four clonal complexes (CC) with distinct agr types (CC599 agr I, CC49 agr II, CC130 agr III and CC1943 agr IV). Agr functionality was assessed by a combination of phenotypic assays and proteome analysis. In each CC, isolates with varying agr activity levels were detected, including the presence of completely non-functional variants. Genomic comparison of the agr I–IV encoding regions associated these phenotypic differences with variations in the agrA and agrC genes. The genomic changes were detected independently in divergent lineages, suggesting that agr variation might foster viability and adaptation of emerging MRSA lineages to distinct ecological niches.Peer Reviewe

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Altering gene expression by aminocoumarins: the role of DNA supercoiling in Staphylococcus aureus

BACKGROUND: It has been shown previously that aminocoumarin antibiotics such as novobiocin lead to immediate downregulation of recA expression and thereby inhibit the SOS response, mutation frequency and recombination capacity in Staphylococcus aureus. Aminocoumarins function by inhibiting the ATPase activity of DNA gyrase subunit B with a severe impact on DNA supercoiling. RESULTS: Here, we have analysed the global impact of the DNA relaxing agent novobiocin on gene expression in S. aureus. Using a novobiocin-resistant mutant, it became evident that the change in recA expression is due to gyrase inhibition. Microarray analysis and northern blot hybridisation revealed that the expression levels of a distinct set of genes were increased (e.g., recF-gyrB-gyrA, the rib operon and the ure operon) or decreased (e.g., arlRS, recA, lukA, hlgC and fnbA) by novobiocin. The two-component ArlRS system was previously found to decrease the level of supercoiling in S. aureus. Thus, downregulation of arlRS might partially compensate for the relaxing effect of novobiocin. Global analysis and gene mapping of supercoiling-sensitive genes did not provide any indication that they are clustered in the genome. Promoter fusion assays confirmed that the responsiveness of a given gene is intrinsic to the promoter region but independent of the chromosomal location. CONCLUSIONS: The results indicate that the molecular properties of a given promoter, rather than the chromosomal topology, dictate the responsiveness to changes in supercoiling in the pathogen Staphylococcus aureus