High prevalence of Merkel cell polyomavirus is associated with dysregulation in transcript levels of TLR9 and type I IFNs in a large cohort of CF patients from the Italian (Lazio) reference center for cystic fibrosis.

Merkel cell polyomavirus (MCPyV) has been detected in respiratory specimens including those from Cystic Fibrosis (CF) patients, raising questions about its immunological and clinical relevance in the respiratory tract. MCPyV might promote an inappropriate antiviral response contributing to a chronic inflammatory response and resulting in detrimental effects in CF. Respiratory samples (n = 1138) were randomly collected from respiratory tract of CF patients (n = 539) during July 2018-October 2019. MCPyV-DNA detection was performed by real time PCR and positive samples were characterized by sequencing of the NCCR genomic region. The transcript levels of Toll-like receptor 9 (TLR9) and type I interferon (IFN-I) genes (IFNα, IFNß and IFNε) were examined by real-time RT-PCR assays. MCPyV-DNA was detected in 268 out of 1138 respiratory specimens (23.5%) without any difference in the prevalence of MCPyV-DNA according to age, gender or bacteriological status of CF individuals. Thirteen out of 137 CF patients remained positive for MCPyV-DNA over the time (a median follow-up period of 8.8 months). Detection of MCPyV-DNA in respiratory specimens was not associated with the occurrence of exacerbation events. Both MCPyV positive adolescents (11-24 years) and adults (≥25 years) had lower mRNA levels of TLR9, IFNß, IFNε and IFNα than the negative patients of the same age group, while MCPyV positive children produced increased levels of TLR9 and IFN-I genes (p < 0.05 for TLR9, IFNß, IFNε) with respect to the negative ones. There were significant differences in TLR9 levels (p < 0.01), but not in those of IFNs, between MCPyV-DNA positive and negative patients with S. aureus, P. aeruginosa or both. Overall, these results indicate that MCPyV-DNA is frequently detected in the respiratory samples of CF patients and might influence the expression levels of IFN-related genes in an age dependent manner. The concomitant detection of MCPyV together with S. aureus and/or P. aeruginosa correlated with alterations in TLR9 levels suggesting that virus-bacteria coinfections might contribute to affect antiviral immunity in CF patients.

SARS-CoV-2 Entry Genes Expression in Relation with Interferon Response in Cystic Fibrosis Patients.

The expression rate of SARS-CoV-2 entry genes, angiotensin-converting enzyme 2 (ACE2), the main viral receptor and the proteases, furin and transmembrane serine protease 2 (TMPRSS2) in cystic fibrosis (CF) individuals is poorly known. Hence, we examined their levels in upper respiratory samples of CF patients (n = 46) and healthy controls (n = 45). Moreover, we sought to understand the interplay of type I interferon (IFN-I) with ACE2, furin and TMPRSS2 by evaluating their gene expression with respect to ISG15, a well-known marker of IFN activation, in upper respiratory samples and after ex vivo IFNß exposure. Lower ACE2 levels and trends toward the reduction of furin and TMPRSS2 were found in CF patients compared with the healthy controls; decreased ACE2 amounts were also detected in CF individuals with pancreatic insufficiency and in those receiving inhaled antibiotics. Moreover, there was a strong positive correlation between ISG15 and ACE2 levels. However, after ex vivo IFNß stimulation of nasopharyngeal cells, the truncated isoform (dACE2), recently demonstrated as the IFN stimulated one with respect to the full-length isoform (flACE2), slightly augmented in cells from CF patients whereas in those from healthy donors, dACE2 levels showed variable levels of upregulation. An altered expression of SARS-COV-2 entry genes and a poor responsiveness of dACE2 to IFN-I stimulation might be crucial in the diffusion of SARS-CoV-2 infection in CF.

Differential toll like receptor expression in cystic fibrosis patients' airways during rhinovirus infection.

OBJECTIVES: Since an inappropriate and sustained activation of TLRs may contribute to a chronic inflammatory response resulting in detrimental effects in cystic fibrosis (CF) patients, we sought to examine whether HRV infection might alter the respiratory expression of TLRs according to the microbiological status of CF patients. METHODS: Respiratory samples were collected from the respiratory tract of CF patients (n = 294) over a period of 12 months. In addition to the usual microbiological investigation, HRV-RNA detection and typing were performed by RT-PCR and sequencing. HRV viral load and TLRs levels were measured by RT-Real Time PCR. RESULTS: HRV-RNA was detected in 80 out of 515 respiratory samples (15.5%) with a similar rate in all age groups (0-10 years, 11-24 years, ≥ 25 years). Patients infected with different HRV A, B and C species exhibited higher levels of TLR2, TLR4 and TLR8 as compared to HRV negative patients. Moreover, the expression level of TLR2, TLR4 and TLR8 correlated with high level of HRV viral load. HRV positive patients co-colonized by Staphylococcus aureus or Pseudomonas aeruginosa showed also enhanced amounts of TLR2 and TLR2/4-mRNAs expression respectively. In the case of presence of both bacteria, TLR2, TLR4, TLR8 and TLR9 levels are elevated in positive HRV patients. CONCLUSIONS: TLRs, especially TLR2 and TLR4, increased in HRV positive CF individuals and varies according to the presence of S. aureus, P. aeruginosa and both bacteria.

Impact of clonally-related Burkholderia contaminans strains in two patients attending an Italian cystic fibrosis centre: a case report.

BACKGROUND: Burkholderia contaminans is one of the 20 closely related bacterial of the Burkholderia cepacia complex, a group of bacteria that are ubiquitous in the environment and capable of infecting people with cystic fibrosis (CF). This species is an emerging pathogen and it has been widely isolated from CF patients in Argentina, Spain, Portugal, Australia, Canada, USA with a low prevalence in Ireland, France, Russia, Switzerland, Czech Republic, and Italy. This is the first report of B. contaminans affecting two Italian CF patients attending the same CF Centre. We correlate B. contaminans colonisation with lung function decline and co-infection with other clinically relevant CF pathogens. CASE PRESENTATION: B. contaminans was identified by Multi Locus Sequence Typing in routine sputum analysis of two Caucasian CF women homozygous for Phe508del CFTR mutation. Sequence Type 102 was detected in both strains. It is known that B. contaminans ST102 was isolated both from CF and non-CF patients, with an intercontinental spread across the world. Random Amplified Polymorphic DNA analysis revealed the genetic relatedness between the two strains. We examined their susceptibility to antimicrobial agents, comparing the latter with that recorded for other B. contaminans isolated from different countries. We also described key virulence factors possibly linked with a clinical outcome. Specifically, we attempted to correlate colonization with the incidence of acute exacerbation of symptoms and lung function decline. CONCLUSIONS: This case presentation suggests that acquisition of B. contaminans ST102 is not directly associated with a lung function decline. We retain that the presence of other CF pathogens (i.e. MRSA and Trichosporon) along with B. contaminans ST102 might have contributed to the worsening of clinical conditions in our CF patients. The circumstances leading to the establishment of B. contaminans ST102 infections are still unknown. We highlight the importance to proper detect and typing bacteria implicated in CF infection by using molecular techniques.

Merkel Cell Polyomavirus DNA Detection in Respiratory Samples: Study of a Cohort of Patients Affected by Cystic Fibrosis.

BACKGROUND: The role of Merkel cell polyomavirus (MCPyV) as a respiratory pathogen is controversial, and it is still unclear in patients with cystic fibrosis (CF). The aim of this study was to define the MCPyV prevalence and epidemiology in CF patients in order to gain new insights into the association between MCPyV infection and respiratory diseases. METHODS: A one-year study was conducted testing oropharyngeal aspirate samples from 249 and 124 CF and non-CF patients, respectively. Detection of MCPyV was carried out by nested polymerase chain reaction (PCR). Moreover, a sequence alignment to examine viral capsid protein 1 (VP1) and a phylogenetic analysis were performed. RESULTS: MCPyV DNA was detected in 65 out of 249 samples analyzed CF (26%), a percentage that was higher than that recorded in non-CF patients (0.8%). There were no statistically significant differences in MCPyV prevalence according to gender, while there was a correlation between MCPyV detection and age. Interestingly, an association between the presence of MCPyV and the concurrent isolation of Staphylococcus aureus was found. Sequence analysis of MCPyV VP1 and phylogenetic analysis revealed a 99% homology with the published sequences of these viruses in GenBank. CONCLUSIONS: Detection of MCPyV in CF patient specimens pointed out a possible interaction between the virus and CF. Further studies are necessary to fully understand the involvement of MCPyV in the pathogenesis of respiratory disorders.

Yersinia enterocolitica in Italy: A Case of Septicemia and Abdominal Aortic Aneurysm Infection.

We report a case of Yersinia enterocolitica septicemia in a 63-year-old patient admitted to the Vascular Surgery Department of Umberto I Hospital (Rome, Italy) for an abdominal aortic aneurysm. The microorganism, recovered from both peripheral blood cultures and aneurysmatic aortic wall specimens, was identified as Y. enterocolitica using matrix-assisted laser desorption ionization-time of flight analysis (MALDI-TOF MS) and 16S rDNA gene sequencing. The isolate responsible for septicemia belonged to the O:9 serotype (biogroup 2). A genetic screening of the isolate made it possible to detect the presence of both the yst and ail genes, encoding a heat-stable enterotoxin and a protein involved in invasion/adherence and serum resistance, respectively. Our case contributes in enriching epidemiological data concerning Y. enterocolitica infections, which might represent severe complications in patients suffering from cardiovascular diseases. Moreover, this study, together with the others, should be regarded as valuable and useful tools for monitoring the rate of infections worldwide.

Efficient propagation of archetype JC polyomavirus in COS-7 cells: evaluation of rearrangements within the NCCR structural organization after transfection.

John Cunningham virus (JCPyV) is an ubiquitous human pathogen that causes disease in immunocompromised patients. The JCPyV genome is composed of an early region and a late region, which are physically separated by the non-coding control region (NCCR). The DNA sequence of the NCCR distinguishes two forms of JCPyV, the designated archetype and the prototype, which resulted from a rearrangement of the archetype sequence. To date, the cell culture systems for propagating JCPyV archetype have been very limited in their availability and robustness. Prior to this study, it was demonstrated that JCPyV archetype DNA replicates in COS-7 simian kidney cells expressing SV40 TAg and COS-7 cells expressing HIV-1 Tat. Based on these observations, the present study was conducted to reproduce an in vitro model in COS-7 cells transfected with the JCPyV archetype strain in order to study JCPyV DNA replication and analyze NCCR rearrangements during the viral life cycle. The efficiency of JCPyV replication was evaluated by quantitative PCR (Q-PCR) and by hemagglutination (HA) assay after transfection. In parallel, sequence analysis of JCPyV NCCR was performed. JCPyV efficiently replicated in kidney-derived COS-7 cells, as demonstrated by a progressive increase in viral load and virion particle production after transfection. The archetypal structure of NCCR was maintained during the viral cycle, but two characteristic point mutations were detected 28 days after transfection. This model is a useful tool for analyzing NCCR rearrangements during in vitro replication in cells that are sites of viral persistence, such as tubular epithelial cells of the kidney.

Increased Prevalence of Human Polyomavirus JC Viruria in Chronic Inflammatory Rheumatic Diseases Patients in Treatment with Anti-TNF α: A 18 Month Follow-Up Study.

Chronic inflammatory rheumatic diseases (CIRDs) are immune-mediated pathologies involving joints. To date, TNFα-blocking agents administration is the most promising therapy, although these treatments are associated with an increased Polyomavirus JC (JCPyV) reactivation, the etiological agent of the Progressive Multifocal Leukoencephalopathy (PML). The aim of this study was the recruitment and the analysis of a CIRDs cohort in order to investigate a possible correlation between JCPyV presence and the influence of anti-TNF-α agents on viral loads. Blood and urine samples were collected from 34 CIRDs subjects prior the first anti-TNF-α infusion (T0) and after 3 (T3), 6 (T6), 12 (T12), and 18 (T18) months. Results showed persistent JC viruria significantly higher than JC viremia throughout the 18 month follow-up study (p = 0.002). In JCPyV positive samples, the non-coding control region (NCCR) was analyzed. Results evidenced archetypal structures (type II-S) in all isolates with the exception of a sequence isolated from a plasma sample, that corresponds to the type II-R found in PML subjects. Finally, the viral protein 1 (VP1) genotyping was performed and results showed the prevalence of the European genotypes 1A, 1B, and 4. Since only few studies have been carried out to understand whether there is a PML risk in CIRDs population infected by JCPyV, this study contributes to enrich literature insight on JCPyV biology in this cluster. Further investigations are necessary in order to recognize the real impact of biologics on JCPyV life cycle and to identify possible and specific viral variants related to increased virulence in CIRDs patients.

Bdellovibrio bacteriovorus directly attacks Pseudomonas aeruginosa and Staphylococcus aureus Cystic fibrosis isolates.

Bdellovibrio bacteriovorus is a predator bacterial species found in the environment and within the human gut, able to attack Gram-negative prey. Cystic fibrosis (CF) is a genetic disease which usually presents lung colonization by Pseudomonas aeruginosa or Staphylococcus aureus biofilms. Here, we investigated the predatory behavior of B. bacteriovorus against these two pathogenic species with: (1) broth culture; (2) "static" biofilms; (3) field emission scanning electron microscope (FESEM); (4) "flow" biofilms; (5) zymographic technique. We had the first evidence of B. bacteriovorus survival with a Gram-positive prey, revealing a direct cell-to-cell contact with S. aureus and a new "epibiotic" foraging strategy imaged with FESEM. Mean attaching time of HD100 to S. aureus cells was 185 s, while "static" and "flow" S. aureus biofilms were reduced by 74 (at 24 h) and 46% (at 20 h), respectively. Furthermore, zymograms showed a differential bacteriolytic activity exerted by the B. bacteriovorus lysates on P. aeruginosa and S. aureus. The dual foraging system against Gram-negative (periplasmic) and Gram-positive (epibiotic) prey could suggest the use of B. bacteriovorus as a "living antibiotic" in CF, even if further studies are required to simulate its in vivo predatory behavior.

Outbreak of Achromobacter xylosoxidans in an Italian Cystic fibrosis center: genome variability, biofilm production, antibiotic resistance, and motility in isolated strains.

Cystic fibrosis (CF) patients have chronic airway infection and frequent exposure to antibiotics, which often leads to the emergence of resistant organisms. Achromobacter xylosoxidans is a new emergent pathogen in CF spectrum. From 2005 to 2010 we had an outbreak in A. xylosoxidans prevalence in our CF center, thus, the present study was aimed at deeply investigating virulence traits of A. xylosoxidans strains isolated from infected CF patients. To this purpose, we assessed A. xylosoxidans genome variability by randomly amplified polymorphic DNA (RAPD), biofilm production, antibiotic resistances, and motility. All A. xylosoxidans strains resulted to be biofilm producers, and were resistant to antibiotics usually employed in CF treatment. Hodge Test showed the ability to produce carbapenemase in some strains. Strains who were resistant to ß-lactamics antibiotics, showed the specific band related to metal ß-lactamase (blaIMP-1), and some of them showed to possess the integron1. Around 81% of A. xylosoxidans strains were motile. Multivariate analysis showed that RAPD profiles were able to predict Forced Expiratory Volume (FEV1%) and biofilm classes. A significant prevalence of strong biofilm producers strains was found in CF patients with severely impaired lung functions (FEV1% class 1). The outbreak we had in our center (prevalence from 8.9 to 16%) could be explained by an enhanced adaptation of A. xylosoxidans in the nosocomial environment, despite of aggressive antibiotic regimens that CF patients usually undergo.

Achromobacter xylosoxidans genomic characterization and correlation of randomly amplified polymorphic DNA profiles with relevant clinical features [corrected] of cystic fibrosis patients.

Achromobacter xylosoxidans is an emerging pathogen increasingly being isolated from respiratory samples of cystic fibrosis (CF) patients. Its role and clinical significance in lung pathogenesis have not yet been clarified. The aim of the present study was to genetically characterize A. xylosoxidans strains isolated from CF patients by use of randomly amplified polymorphic DNA (RAPD) profiles and to look for a possible correlation between RAPD profiles and the patients' clinical features, such as their spirometry values, the presence of concomitant chronic bacterial flora at the time of isolation, and the persistent or intermittent presence of A. xylosoxidans strains. A set of 106 strains of A. xylosoxidans were typed by RAPD analysis, and their profiles were analyzed by agglomerative hierarchical classification (AHC) and associated with the patient characteristics mentioned above by factorial discriminant analysis (FDA). The overall results obtained in this study showed that (i) there is a marked genetic relationship between strains isolated from the same patients at different times, (ii) characteristic RAPD profiles are associated with different predicted classes for forced expiratory volume in 1 s (FEV1%), (iii) some characteristic RAPD profiles are associated with different concomitant chronic flora (CCF) profiles, and (iv) there is a significant division of RAPD profiles into "persistent strains" and "intermittent strains" of A. xylosoxidans. These findings seem to imply that the lung habitats found in CF patients are capable of shaping and selecting the colonizing bacterial flora, as seems to be the case for the A. xylosoxidans strains studied.

Emerging cystic fibrosis pathogens: incidence and antimicrobial resistance.

We examined the frequency of isolation and the antimicrobial resistance of Burkholderia cepacia complex, Stenotrophomonas maltophilia and Achromobacter xylosoxidans in cystic fibrosis patients from 2000 to 2004. Strains susceptibility to tobramycin, piperacillin/tazobactam, imipenem, gentamicin, ciprofloxacin and ceftazidime was determined by disc diffusion assay. B. cepacia complex showed a very high resistance also to ciprofloxacin reaching 100% in 2004. S. maltophilia and A. xvylosoxidans showed high rates of antimicrobial resistance both aminoglycoside and ciprofloxacin. It is very important to monitor the percentage of isolation of these species over time to verify strains resistance to antibiotics and also to test new combinations of antimicrobial agents.