Dysregulated inflammasome activation contributes to respiratory infections and pathologic airway inflammation. Through basic and translational approaches involving murine models and human genetic ...epidemiology, we show here the importance of the different inflammasomes in regulating inflammatory responses in mice and humans with cystic fibrosis (CF), a life-threatening disorder of the lungs and digestive system. While both contributing to pathogen clearance, NLRP3 more than NLRC4 contributes to deleterious inflammatory responses in CF and correlates with defective NLRC4-dependent IL-1Ra production. Disease susceptibility in mice and microbial colonization in humans occurs in conditions of genetic deficiency of NLRC4 or IL-1Ra and can be rescued by administration of the recombinant IL-1Ra, anakinra. These results indicate that pathogenic NLRP3 activity in CF could be negatively regulated by IL-1Ra and provide a proof-of-concept evidence that inflammasomes are potential targets to limit the pathological consequences of microbial colonization in CF.
Stenotrophomonas maltophilia and Burkholderia cepacia complex (Bcc) have been increasingly recognized as relevant pathogens in hospitalized, immunocompromised and cystic fibrosis (CF) patients. As a ...result of complex mechanisms, including biofilm formation and multidrug resistance phenotype, S. maltophilia and Bcc respiratory infections are often refractory to therapy, and have been associated with a worse outcome in CF patients. Here we demonstrate for the first time that N-acetylcysteine (NAC), a mucolytic agent with antioxidant and anti-inflammatory properties, may exhibit antimicrobial and antibiofilm activity against these pathogens. The antimicrobial and antibiofilm activity of high NAC concentrations, potentially achievable by topical administration, was tested against a collection of S. maltophilia (n = 19) and Bcc (n = 19) strains, including strains from CF patients with acquired resistance traits. Minimum Inhibitory Concentrations (MICs) and Minimum Bactericidal Concentrations (MBCs) ranged from 16 to 32 mg/ml and from 32 to >32 mg/ml, respectively. Sub-MIC concentrations (i.e., 0.25 × MIC) slowed down the growth kinetics of most strains. In time-kill assays, 2-day-old biofilms were more affected than planktonic cultures, suggesting a specific antibiofilm activity of NAC against these pathogens. Indeed, a dose- and time-dependent antibiofilm activity of NAC against most of the S. maltophilia and Bcc strains tested was observed, with a sizable antibiofilm activity observed also at 0.5 and 1 × MIC NAC concentrations. Furthermore, at those concentrations, NAC was also shown to significantly inhibit biofilm formation with the great majority of tested strains.
ObjectivesTo assess the seroprevalence of anti-SARS-CoV-2 IgG among health careworkers (HCWs) in our university hospital and verify the risk of acquiring the infection according to work ...area.DesignCross-sectional study.SettingMonocentric, Italian, third-level university hospital.ParticipantsAll the employees of the hospital on a voluntary base, for a total of 4055 participants among 4572 HCWs (88.7%).Primary and secondary outcome measuresNumber of anti-SARS-CoV-2 positive serology according to working area. Association of anti-SARS-CoV-2 positive serology to selected variables (age, gender, country of origin, body mass index, smoking, symptoms and contact with confirmed cases).ResultsFrom 27 April 2020 to 12 June 2020, 4055 HCWs were tested and 309 (7.6%) had a serological positive test. No relevant difference was found between men and women (8.3% vs 7.3%, p=0.3), whereas a higher prevalence was observed among foreign-born workers (27/186, 14.5%, p<0.001), employees younger than 30 (64/668, 9.6%, p=0.02) or older than 60 years (38/383, 9.9%, p=0.02) and among healthcare assistants (40/320, 12.5%, p=0.06). Working as frontline HCWs was not associated with an increased frequency of positive serology (p=0.42). A positive association was found with presence and number of symptoms (p<0.001). The symptoms most frequently associated with a positive serology were taste and smell alterations (OR 4.62, 95% CI: 2.99 to 7.15) and fever (OR 4.37, 95% CI: 3.11 to 6.13). No symptoms were reported in 84/309 (27.2%) HCWs with positive IgG levels. Declared exposure to a suspected/confirmed case was more frequently associated (p<0.001) with positive serology when the contact was a family member (19/94, 20.2%) than a patient or colleague (78/888, 8.8%).ConclusionsSARS-CoV-2 infection occurred undetected in a large fraction of HCWs and it was not associated with working in COVID-19 frontline areas. Beyond the hospital setting, exposure within the community represents an additional source of infection for HCWs.
Different steps and conditions for DNA extraction for microbiota analysis in sputum have been reported in the literature. We aimed at testing both dithiothreitol (DTT) and enzymatic treatments of ...sputum samples and identifying the most suitable DNA extraction technique for the microbiota analysis of sputum. Sputum treatments with and without DTT were compared in terms of their median levels and the coefficient of variation between replicates of both DNA extraction yield and real-time PCR for the 16S rRNA gene. Treatments with and without lysozyme and lysostaphin were compared in terms of their median levels of real-time PCR for
. Two enzyme-based and three beads-based techniques for DNA extraction were compared in terms of their DNA extraction yield, real-time PCR for the 16S rRNA gene and microbiota analysis. DTT treatment decreased the coefficient of variation between replicates of both DNA extraction yield and real-time PCR. Lysostaphin (either 0.18 or 0.36 mg/mL) and lysozyme treatments increased
detection. One enzyme-based kit offered the highest DNA yield and 16S rRNA gene real-time PCR with no significant differences in terms of alpha-diversity indexes. A condition using both DTT and lysostaphin/lysozyme treatments along with an enzymatic kit seems to be preferred for the microbiota analysis of sputum samples.
Group A Streptococcus (GAS) causes multiple clinical manifestations, including invasive (iGAS) or even life-threatening (severe-iGAS) infections. After the drop in cases during COVID-19 pandemic, in ...2022 a sharp increase of GAS was reported globally.
GAS strains collected in 09/2022-03/2023 in two university hospitals in Milan, Italy were retrospectively analyzed. Clinical/epidemiological data were combined with whole-genome sequencing to: (i) define resistome/virulome, (ii) identify putative transmission chains, (iii) explore associations between
types and clinical severity.
Twenty-eight isolates were available, 19/28 (67.9%) from adults and 9/28 (32.1%) from pediatric population. The criteria for iGAS were met by 19/28 cases (67.9%), of which 11/19 (39.3%) met the further criteria for severe-iGAS. Pediatric cases were mainly non-invasive infections (8/9, 88.9%), adult cases were iGAS and severe-iGAS in 18/19 (94.7%) and 10/19 (52.6%), respectively. Thirteen
-types were detected, the most prevalent being
1 and
12 (6/28 strains each, 21.4%). Single nucleotide polymorphism (SNP) analysis of
1.0 and
12.0 strains revealed pairwise SNP distance always >10, inconsistent with unique transmission chains.
12.0-type, found to almost exclusively carry virulence factors
H and
I, was mainly detected in children and in no-iGAS infections (55.6 vs. 5.3%,
= 0.007 and 66.7 vs. 0.0%,
< 0.001, respectively), while
1.0-type was mainly detected in severe-iGAS (0.0 vs. 45.5%,
= 0.045).
This study showed that multiple
-types contributed to a 2022/2023 GAS infection increase in two hospitals in Milan, with no evidence of direct transmission chains. Specific
-types could be associated with disease severity or invasiveness. Overall, these results support the integration of classical epidemiological studies with genomic investigation to appropriately manage severe infections and improve surveillance.
Few studies, based on a limited number of patients using non-uniform therapeutic protocols, have analyzed Methicillin-resistant Staphylococcus aureus (MRSA) eradication.
In a randomized multicenter ...trial conducted on patients with new-onset MRSA infection we evaluated the efficacy of an early eradication treatment (arm A) compared with an observational group (B). Arm A received oral rifampicin and trimethoprim/sulfamethoxazole (21 days). Patients' microbiological status, FEV1, BMI, pulmonary exacerbations and use of antibiotics were assessed.
Sixty-one patients were randomized. Twenty-nine (47.5%) patients were assigned to active arm A and 32 (52.5%) patients to observational arm B. Twenty-nine (47.5%) patients, 10 patients in arm A and 19 in arm B, dropped out of the study. At 6 months MRSA was eradicated in 12 (63.2%) out of 19 patients in arm A while spontaneous clearance was observed in 5 (38.5%) out of 13 patients in arm B. A per-protocol analysis showed a 24.7% difference in the proportion of MRSA clearance between the two groups (z = 1.37, P(Z>z) = 0.08). Twenty-seven patients, 15 (78.9%) out of 19 in arm A and 12 (92.3%) out of 13 in arm B, were able to perform spirometry. The mean (±SD) FEV1 change from baseline was 7.13% (±14.92) in arm A and -1.16% (±5.25) in arm B (p = 0.08). In the same period the BMI change (mean ±SD) from baseline was 0.54 (±1.33) kg/m2 in arm A and -0.38 (±1.56) kg/m2 in arm B (p = 0.08). At 6 months no statistically significant differences regarding the number of pulmonary exacerbations, days spent in hospital and use of antibiotics were observed between the two arms.
Although the statistical power of the study is limited, we found a 24.7% higher clearance of MRSA in the active arm than in the observational arm at 6 months. Patients in the active arm A also had favorable FEV1 and BMI tendencies.
Small non-coding RNAs (sRNAs) are post-transcriptional regulators of gene expression that have been recognized as key contributors to bacterial virulence and pathogenic mechanisms. In this study, we ...characterized the sRNA PesA of the opportunistic human pathogen Pseudomonas aeruginosa. We show that PesA, which is transcribed within the pathogenicity island PAPI-1 of P. aeruginosa strain PA14, contributes to P. aeruginosa PA14 virulence. In fact, pesA gene deletion resulted in a less pathogenic strain, showing higher survival of cystic fibrosis human bronchial epithelial cells after infection. Moreover, we show that PesA influences positively the expression of pyocin S3 whose genetic locus comprises two structural genes, pyoS3A and pyoS3I, encoding the killing S3A and the immunity S3I proteins, respectively. Interestingly, the deletion of pesA gene results in increased sensitivity to UV irradiation and to the fluoroquinolone antibiotic ciprofloxacin. The degree of UV sensitivity displayed by the PA14 strain lacking PesA is comparable to that of a strain deleted for pyoS3A-I. These results suggest an involvement of pyocin S3 in DNA damage repair and a regulatory role of PesA on this function.
Resistance and tolerance mechanisms participate to the interplay between host and pathogens. IL-17-mediated response has been shown to be crucial for host resistance to respiratory infections, ...whereas its role in host tolerance during chronic airway colonization is still unclear. Here, we investigated whether IL-17-mediated response modulates mechanisms of host tolerance during airways chronic infection by P. aeruginosa. First, we found that IL-17A levels were sustained in mice at both early and advanced stages of P. aeruginosa chronic infection and confirmed these observations in human respiratory samples from cystic fibrosis patients infected by P. aeruginosa. Using IL-17a(-/-) or IL-17ra(-/-) mice, we found that the deficiency of IL-17A/IL-17RA axis was associated with: i) increased incidence of chronic infection and bacterial burden, indicating its role in the host resistance to P. aeruginosa; ii) reduced cytokine levels (KC), tissue innate immune cells and markers of tissue damage (pro-MMP-9, elastin degradation, TGF-β1), proving alteration of host tolerance. Blockade of IL-17A activity by a monoclonal antibody, started when chronic infection is established, did not alter host resistance but increased tolerance. In conclusion, this study identifies IL-17-mediated response as a negative regulator of host tolerance during P. aeruginosa chronic airway infection.
Pseudomonas aeruginosa is the most common pathogen infecting cystic fibrosis (CF) lungs, causing acute and chronic infections. Intrinsic and acquired antibiotic resistance allow P. aeruginosa to ...colonize and persist despite antibiotic treatment, making new therapeutic approaches necessary. Combining high-throughput screening and drug repurposing is an effective way to develop new therapeutic uses for drugs. This study screened a drug library of 3,386 drugs, mostly FDA approved, to identify antimicrobials against P. aeruginosa under physicochemical conditions relevant to CF-infected lungs. Based on the antibacterial activity, assessed spectrophotometrically against the prototype RP73 strain and 10 other CF virulent strains, and the toxic potential evaluated toward CF IB3-1 bronchial epithelial cells, five potential hits were selected for further analysis: the anti-inflammatory and antioxidant ebselen, the anticancer drugs tirapazamine, carmofur, and 5-fluorouracil, and the antifungal tavaborole. A time-kill assay showed that ebselen has the potential to cause rapid and dose-dependent bactericidal activity. The antibiofilm activity was evaluated by viable cell count and crystal violet assays, revealing carmofur and 5-fluorouracil as the most active drugs in preventing biofilm formation regardless of the concentration. In contrast, tirapazamine and tavaborole were the only drugs actively dispersing preformed biofilms. Tavaborole was the most active drug against CF pathogens other than P. aeruginosa, especially against Burkholderia cepacia and Acinetobacter baumannii, while carmofur, ebselen, and tirapazamine were particularly active against Staphylococcus aureus and B. cepacia. Electron microscopy and propidium iodide uptake assay revealed that ebselen, carmofur, and tirapazamine significantly damage cell membranes, with leakage and cytoplasm loss, by increasing membrane permeability.
Antibiotic resistance makes it urgent to design new strategies for treating pulmonary infections in CF patients. The repurposing approach accelerates drug discovery and development, as the drugs' general pharmacological, pharmacokinetic, and toxicological properties are already well known. In the present study, for the first time, a high-throughput compound library screening was performed under experimental conditions relevant to CF-infected lungs. Among 3,386 drugs screened, the clinically used drugs from outside infection treatment ebselen, tirapazamine, carmofur, 5-fluorouracil, and tavaborole showed, although to different extents, anti-P. aeruginosa activity against planktonic and biofilm cells and broad-spectrum activity against other CF pathogens at concentrations not toxic to bronchial epithelial cells. The mode-of-action studies revealed ebselen, carmofur, and tirapazamine targeted the cell membrane, increasing its permeability with subsequent cell lysis. These drugs are strong candidates for repurposing for treating CF lung P. aeruginosa infections.
Nontypeable
(NTHi) is commonly isolated from airways of patients suffering from chronic respiratory diseases, such as COPD or cystic fibrosis (CF). However, to what extent NTHi long-term infection ...contributes to the lung inflammatory burden during chronic airway disease is still controversial. Here, we exploited human respiratory samples from a small cohort of CF patients and found that patients chronically infected with NTHi had significantly higher levels of interleukin (IL)-8 and CXCL1 than those who were not infected. To better define the impact of chronic NTHi infection in fuelling inflammatory response in chronic lung diseases, we developed a new mouse model using both laboratory and clinical strains. Chronic NTHi infection was associated with chronic inflammation of the lung, characterised by recruitment of neutrophils and cytokine release keratinocyte-derived chemokine (KC), macrophage inflammatory protein 2 (MIP-2), granulocyte colony-stimulating factor (G-CFS), IL-6, IL-17A and IL-17F) at 2 and 14 days post-infection. An increased burden of T-cell-mediated response (CD4
and γδ cells) and higher levels of pro-matrix metalloproteinase 9 (pro-MMP9), known to be associated with tissue remodelling, were observed at 14 days post-infection. Of note we found that both CD4
IL-17
cells and levels of IL-17 cytokines were enriched in mice at advanced stages of NTHi chronic infection. Moreover, by immunohistochemistry we found CD3
, B220
and CXCL-13
cells localised in bronchus-associated lymphoid tissue-like structures at day 14. Our results demonstrate that chronic NTHi infection exerts a pro-inflammatory activity in the human and murine lung and could therefore contribute to the exaggerated burden of lung inflammation in patients at risk.
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