Multiple antimicrobial and immune-modulating activities of cysteamine in infectious diseases.

Infectious diseases are a major threat to global health and cause millions of deaths every year, particularly in developing countries. The emergence of multidrug resistance challenges current antimicrobial treatments, inducing uncertainty in therapeutic protocols. New compounds are therefore necessary. A drug repurposing approach could play a critical role in developing new treatments used either alone or in combination with standard therapy regimens. Herein, we focused on cysteamine, an aminothiol endogenously synthesized by human cells during the degradation of coenzyme-A, which is a drug approved for the treatment of nephropathic cystinosis. Cysteamine influences many biological processes due to the presence of the highly reactive thiol group. This review provides an overview of cysteamine-mediated effects on different viruses, bacteria and parasites, with a particular focus on infections caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Mycobacterium tuberculosis, non-tuberculous mycobacteria (NTM), and Pseudomonas aeruginosa. Evidences for a potential use of cysteamine as a direct antimicrobial agent and/or a host-directed therapy, either alone or in combination with other antimicrobial drugs, are described.

Alternative therapies against Mycobacterium abscessus infections.

BACKGROUND: Mycobacterium abscessus (Mab) is considered as the most pathogenic rapid-growing mycobacteria in humans, causing pulmonary and extra-pulmonary diseases, especially in patients with cystic fibrosis. Mab shows intrinsic and acquired resistance to many drugs, leaving limited treatment options that lead to a generally poor prognosis. The standard therapeutic regimen last for more than 6 months and consists of a drug cocktail that ideally includes a macrolide and amikacin. Yet, toxicity and efficacy are suboptimal due also to the high toxicity. There is a need to introduce innovative and out-of-the-box approaches to improve treatments. OBJECTIVES: In this narrative review, we summarize the recent research on the alternative strategies proposed and discuss the importance of using appropriate experimental assays to assess their activity. SOURCES: Included articles were identified by searching PubMed and MEDLINE until June 2023. The search terms were 'Mycobacterium abscessus', 'antimicrobial', and 'alternative therapies'. Additional relevant references were obtained from articles retrieved from the primary search. CONTENT: Therapies against Mab including host directed therapies, repurposed drugs, phage therapy, anti-virulence strategies, essential oils, and inhalation therapies. IMPLICATIONS: Alternative treatments may represent a valid tool to cope the burden of antimicrobial resistance in Mab-caused diseases.

The ubiquitin ligase TRIM32 promotes the autophagic response to Mycobacterium tuberculosis infection in macrophages.

Mycobacterium tuberculosis (Mtb) is known to evade host immune responses and persist in macrophages for long periods. A mechanism that the host uses to combat Mtb is xenophagy, a selective form of autophagy that targets intracellular pathogens for degradation. Ubiquitination of Mtb or Mtb-containing compartments is a key event to recruit the autophagy machinery and mediate the bacterial delivery to the lysosome. This event relies on the coordinated and complementary activity of different ubiquitin ligases, including PARKIN, SMURF1, and TRIM16. Because each of these factors is responsible for the ubiquitination of a subset of the Mtb population, it is likely that additional ubiquitin ligases are employed by macrophages to trigger a full xenophagic response during Mtb infection. In this study, we investigated the role TRIM proteins whose expression is modulated in response to Mtb or BCG infection of primary macrophages. These TRIMs were ectopically expressed in THP1 macrophage cell line to assess their impact on Mtb replication. This screening identified TRIM32 as a novel player involved in the intracellular response to Mtb infection, which promotes autophagy-mediated Mtb degradation. The role of TRIM32 in xenophagy was further confirmed by silencing TRIM32 expression in THP1 cells, which causes increased intracellular growth of Mtb associated to impaired Mtb ubiquitination, reduced recruitment of the autophagy proteins NDP52/CALCOCO2 and BECLIN 1/BECN1 to Mtb and autophagosome formation. Overall, these findings suggest that TRIM32 plays an important role in the host response to Mtb infection through the induction of autophagy, representing a promising target for host-directed tuberculosis therapies.

Accuracy of p16 IHC in Classifying HPV-Driven OPSCC in Different Populations.

High-risk human papillomavirus (HPV) infection is a defined etiopathogenetic factor in oropharyngeal carcinogenesis with a clear prognostic value. The P16 IHC (immunohistochemistry) is a widely accepted marker for HPV-driven carcinogenesis in oropharyngeal squamous cell carcinoma (OPSCC); in the present paper, we discuss its reliability as a standalone marker in different populations. The literature suggests that rates of p16 IHC false positive results are inversely correlated with the prevalence of HPV-driven carcinogenesis in a population. We propose a formula that can calculate such a false positive rate while knowing the real prevalence of HPV-driven OPSCCs in a given population. As it has been demonstrated that p16 positive/HPV negative cases (i.e., false positives at p16 IHC) have the same prognosis as p16 negative OPSCC, we conclude that despite the valuable prognostic value of p16 IHC, relying only on a p16 IHC positive result to recommend treatment de-intensification could be risky. For this aim, confirmation with an HPV nucleic acid detection system, especially in areas with a low prevalence of HPV-related OPSCCs, should be pursued.

Evaluation of Everolimus Activity against Mycobacterium tuberculosis Using In Vitro Models of Infection.

Even though Everolimus has been investigated in a phase II randomized trial as a host-directed therapy (HDT) to treat tuberculosis (TB), an oncological patient treated with Everolimus for a neuroendocrine pancreatic neoplasia developed active TB twice and a non-tuberculous mycobacterial (NTM) infection in a year and a half time span. To investigate this interesting case, we isolated and genotypically characterized the Mycobacterium tuberculosis (Mtb) clinical strain from the patient and tested the effect of Everolimus on its viability in an axenic culture and in a peripheral blood mononuclear cell (PBMCs) infection model. To exclude strain-specific resistance, we tested the activity of Everolimus against Mtb strains of ancient and modern lineages. Furthermore, we investigated the Everolimus effect on ROS production and autophagy modulation during Mtb infection. Everolimus did not have a direct effect on mycobacteria viability and a negligible effect during Mtb infection in host cells, although it stimulated autophagy and ROS production. Despite being a biologically plausible HDT against TB, Everolimus does not exert a direct or indirect activity on Mtb. This case underlines the need for a careful approach to drug repurposing and implementation and the importance of pre-clinical experimental studies.

Cysteamine/Cystamine Exert Anti-Mycobacterium abscessus Activity Alone or in Combination with Amikacin.

Host-directed therapies are emerging as a promising tool in the curing of difficult-to-treat infections, such as those caused by drug-resistant bacteria. In this study, we aim to test the potential activity of the FDA- and EMA-approved drugs cysteamine and cystamine against Mycobacterium abscessus. In human macrophages (differentiated THP-1 cells), these drugs restricted M. abscessus growth similar to that achieved by amikacin. Here, we use the human ex vivo granuloma-like structures (GLS) model of infection with the M. abscessus rough (MAB-R) and smooth (MAB-S) variants to study the activity of new therapies against M. abscessus. We demonstrate that cysteamine and cystamine show a decrease in the number of total GLSs per well in the MAB-S and MAB-R infected human peripheral blood mononuclear cells (PBMCs). Furthermore, combined administration of cysteamine or cystamine with amikacin resulted in enhanced activity against the two M. abscessus morpho variants compared to treatment with amikacin only. Treatment with cysteamine and cystamine was more effective in reducing GLS size and bacterial load during MAB-S infection compared with MAB-R infection. Moreover, treatment with these two drugs drastically quenched the exuberant proinflammatory response triggered by the MAB-R variant. These findings showing the activity of cysteamine and cystamine against the R and S M. abscessus morphotypes support the use of these drugs as novel host-directed therapies against M. abscessus infections.

Low Prevalence of HPV Related Oropharyngeal Carcinogenesis in Northern Sardinia.

HPV infection is a clear etiopathogenetic factor in oropharyngeal carcinogenesis and is associated with a markedly better prognosis than in smoking- and alcohol-associated cases, as specified by AJCC classification. The aim of the present work is to evaluate the prevalence of HPV-induced OPSCC in an insular area in the Mediterranean and to assess the reliability of p16 IHC (immunohistochemistry) alone, as accepted by AJCC, in the diagnosis of HPV-driven carcinogenesis in such a setting. All patients with OPSCC consecutively managed by the referral center in North Sardinia of head and neck tumor board of AOU Sassari, were recruited. Diagnosis of HPV-related OPCSS was carried out combining p16 IHC and DNA testing on FFPE samples and compared with the results of p16 IHC alone. Roughly 14% (9/62) of cases were positive for HPV-DNA and p16 IHC. Three more cases showed overexpression of p16, which has a 100% sensitivity, but only 75% specificity as standalone method for diagnosing HPV-driven carcinogenesis. The Cohen's kappa coefficient of p16 IHC alone is 0.83 (excellent). However, if HPV-driven carcinogenesis diagnosed by p16 IHC alone was considered the criterion for treatment deintensification, 25% of p16 positive cases would have been wrongly submitted to deintensified treatment for tumors as aggressive as a p16 negative OPSCC. The currently accepted standard by AJCC (p16 IHC alone) harbors a high rate of false positive results, which appears risky for recommending treatment deintensification, and for this aim, in areas with a low prevalence of HPV-related OPSCC, it should be confirmed with HPV nucleic acid detection.

Proinflammatory and Cancer-Promoting Pathobiont Fusobacterium nucleatum Directly Targets Colorectal Cancer Stem Cells.

Intestinal bacterial communities participate in gut homeostasis and are recognized as crucial in bowel inflammation and colorectal cancer (CRC). Fusobacterium nucleatum (Fn), a pathobiont of the oral microflora, has recently emerged as a CRC-associated microbe linked to disease progression, metastasis, and a poor clinical outcome; however, the primary cellular and/or microenvironmental targets of this agent remain elusive. We report here that Fn directly targets putative colorectal cancer stem cells (CR-CSCs), a tumor cell subset endowed with cancer re-initiating capacity after surgery and chemotherapy. A patient-derived CSC line, highly enriched (70%) for the stem marker CD133, was expanded as tumor spheroids, dissociated, and exposed in vitro to varying amounts (range 100-500 MOI) of Fn. We found that Fn stably adheres to CSCs, likely by multiple interactions involving the tumor-associated Gal-GalNac disaccharide and the Fn-docking protein CEA-family cell adhesion molecule 1 (CEACAM-1), robustly expressed on CSCs. Importantly, Fn elicited innate immune responses in CSCs and triggered a growth factor-like, protein tyrosine phosphorylation cascade largely dependent on CEACAM-1 and culminating in the activation of p42/44 MAP kinase. Thus, the direct stimulation of CSCs by Fn may contribute to microbiota-driven colorectal carcinogenesis and represent a target for innovative therapies.

Cysteamine exerts in vitro antiviral activity against the SARS-CoV-2 Delta and Omicron variants.

The novel SARS-CoV-2 variants of concern (VOC) represent a considerable global alarm because their mutations are known to affect transmissibility and cause immune escape. While preventing severe disease and deaths, the available vaccines do not avoid infection; therefore, COVID-19 disease management still requires effective therapies. We have recently reported that the aminothiol cysteamine, a drug already applied to humans, exerts direct antiviral activity against SARS-CoV-2 and has in vitro immunomodulatory effect. To evaluate whether this compound exerts antiviral effects also against SARS-CoV-2 variants, we performed different infected cell-based assays using Wild type, Delta, or Omicron VOC. We found that cysteamine significantly reduces the cytopathic effect induced by SARS-CoV-2 Wild type strain and Delta variant in Vero E6 cells. On the other hand, cysteamine had no effects on the survival of cells infected with the Omicron variant, due to the lack of cytotoxicity on Vero E6 cells, at least when infected at MOI = 0.001 for 72 h. Moreover, cysteamine significantly reduced the production of Wild type, Delta, and Omicron variants as measured by the virus released in the culture media (Vero E6 and Calu-3 cells) and by transmission electron microscopy analysis (Vero E6 cells). Notably, cysteamine is more effective in inhibiting the Omicron rather than Delta or Wild type viruses, with an 80% inhibition of Omicron production compared to 40% of Wild type and Delta variant. Overall, our findings demonstrate that cysteamine exerts direct antiviral actions against SARS-CoV-2 Delta and Omicron variants, in addition to the Wild type virus. Our data further demonstrate that cysteamine is a good candidate as repurposing drug for the treatment of SARS-CoV-2 infection for the present and, likely, the future VOC and, therefore, it would be important to investigate its clinical relevance in randomized clinical trials.

Prevalence of HPV Infection and p16INK4a Overexpression in Surgically Treated Laryngeal Squamous Cell Carcinoma.

Background: The mucosal high-risk (HR) human papillomavirus (HPV) is associated with oropharyngeal carcinogenesis. Aims of this study were to evaluate the prevalence of HR-HPV infection in laryngeal squamous cell carcinoma (LSCC) from different subsites, and the clinico-biological meaning of p16 overexpression. Methods: Ninety-seven LSCCs submitted to primary surgery (n = 75) or to post-irradiation salvage laryngectomy (n = 22) were evaluated for HR-HPV DNA and RNA using Luminex-based assays. p16 immunohistochemistry was performed. Results: HR-HPV DNA from HPV16 was detected in seven cases (8.75%), without significant differences between supraglottic and glottic lesions. HPV RNA was never detected. p16 overexpression correlated with HR-HPV DNA, but the kappa agreement score was poor. HPV DNA showed no impact on prognosis. p16 overexpression was associated with a better survival (OS, RFS) in primarily operated cases, while an inverse association with OS was observed in the salvage surgery group. Conclusions: HR-HPV infection appears to have a marginal role in LSCC independent of the anatomical subsite. p16 expression is deregulated in LSCC independent of HPV but displays a prognostic role in patients submitted to primary surgery. The negative predictive role of p16 overexpression in patients undergoing salvage surgery deserves more investigations for validation and elucidation of its clinical relevance.

Characterization of gut microbiota in patients with metabolic syndrome candidates for bariatric/metabolic surgery: Preliminary findings of a multi-center prospective study.

INTRODUCTION: gut microbiota (GM) seems to be involved in the pathophysiology and progression of both metabolic syndrome (MS) and obesity. The aim was to investigate GM's composition in patients with severe obesity, candidates for bariatric/metabolic surgery BMS. MATERIALS AND METHODS: Multicentre, prospective, cohort study, enrolling 84 patients with BMI 40-55 kg/m2, divided bymetabolic status (MS) inhealthy(group A), pre-MS (B), or MS (C). RESULTS: No differences were foundregarding anthropometric,nutritional parameters, except for vitamin D.As a whole the alpha and beta diversity examinations showed no statistical differences in GM profile. A total of 5/7 phyla with relative frequencies were identified above 0.1% (Actinobacteria,Bacteroidetes,Firmicutes,Proteobacteria,Verrucomicrobia).FusobacteriaandPatescibacteriarepresented the less abundant. There were no significant differences in the top ten genera.Data onBacteroidetes(inversely related to triglycerides and LDL and directly related to HDL levels) and onFirmicutes(opposite trend) relative abundances suggest no differences among the three conditions.No correlation between the relative abundance of themain phylaand plasmatic glucose levels was observed. CONCLUSIONS: In a selected cohort of patients with obesity, MS did not affect the preoperative GM's profile. Severe obesity, per se, seems to be an independent condition affecting GM.

PE_PGRS33, an Important Virulence Factor of Mycobacterium tuberculosis and Potential Target of Host Humoral Immune Response.

PE_PGRS proteins are surface antigens of Mycobacterium tuberculosis (Mtb) and a few other pathogenic mycobacteria. The PE_PGRS33 protein is among the most studied PE_PGRSs. It is known that the PE domain of PE_PGRS33 is required for the protein translocation through the mycobacterial cell wall, where the PGRS domain remains available for interaction with host receptors. Interaction with Toll like receptor 2 (TLR2) promotes secretion of inflammatory chemokines and cytokines, which are key in the immunopathogenesis of tuberculosis (TB). In this review, we briefly address some key challenges in the development of a TB vaccine and attempt to provide a rationale for the development of new vaccines aimed at fostering a humoral response against Mtb. Using PE_PGRS33 as a model for a surface-exposed antigen, we exploit the availability of current structural data using homology modeling to gather insights on the PGRS domain features. Our study suggests that the PGRS domain of PE_PGRS33 exposes four PGII sandwiches on the outer surface, which, we propose, are directly involved through their loops in the interactions with the host receptors and, as such, are promising targets for a vaccination strategy aimed at inducing a humoral response.

Cysteamine with In Vitro Antiviral Activity and Immunomodulatory Effects Has the Potential to Be a Repurposing Drug Candidate for COVID-19 Therapy.

The ongoing pandemic of coronavirus disease-2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), needs better treatment options both at antiviral and anti-inflammatory levels. It has been demonstrated that the aminothiol cysteamine, an already human applied drug, and its disulfide product of oxidation, cystamine, have anti-infective properties targeting viruses, bacteria, and parasites. To determine whether these compounds exert antiviral effects against SARS-CoV-2, we used different in vitro viral infected cell-based assays. Moreover, since cysteamine has also immune-modulatory activity, we investigated its ability to modulate SARS-CoV-2-specific immune response in vitro in blood samples from COVID-19 patients. We found that cysteamine and cystamine decreased SARS-CoV-2-induced cytopathic effects (CPE) in Vero E6 cells. Interestingly, the antiviral action was independent of the treatment time respect to SARS-CoV-2 infection. Moreover, cysteamine and cystamine significantly decreased viral production in Vero E6 and Calu-3 cells. Finally, cysteamine and cystamine have an anti-inflammatory effect, as they significantly decrease the SARS-CoV-2 specific IFN-γ production in vitro in blood samples from COVID-19 patients. Overall, our findings suggest that cysteamine and cystamine exert direct antiviral actions against SARS-CoV-2 and have in vitro immunomodulatory effects, thus providing a rational to test these compounds as a novel therapy for COVID-19.

IP-10 contributes to the inhibition of mycobacterial growth in an ex vivo whole blood assay.

Interferon-γ inducible protein 10 (IP-10), is a potent chemoattractant that promotes migration of monocytes and activated T-cells to inflammation foci. IP-10 is elevated in serum of patients with chronic hepatitis C virus (HCV) and tuberculosis (TB) infections, although it remains to be determined the contribution of IP-10 in restricting Mycobacterium tuberculosis (Mtb) replication. Here, we investigated the impact of IP-10 on mycobacteria replication using the ex vivo model of human whole-blood (WB) assay. In particular, we compared the levels of IP-10 upon infection with different Mtb clinical strains and species of non-tuberculous mycobacteria (NTM) and evaluated how IP-10 may contain bacterial replication. Interestingly, we observed that the inhibition of the host enzyme dipeptidyl peptidase IV (DPP-IV), which inactivates IP-10 through cleavage of two amino acids at the chemokine N-terminus, restricted mycobacterial persistence in WB, supporting the critical role of full length IP-10 in mediating an anti-Mtb response. Addition of recombinant IP-10 expressed in eukaryotic cells enhanced the anti-mycobacterial activity in WB, although no differences were observed when IP-10 containing different proportions of cleaved and non-cleaved forms of the chemokine were added. Moreover, recombinant IP-10 did not exert a direct anti-mycobacterial effect. Our results underscore the clinical relevance of IP-10 in mycobacteria pathogenesis and support the potential outcomes that may derive by targeting the IP-10/CXCR3 pathway as host directed therapies for the treatment of Mtb or NTM infections.

HPV as a marker for molecular characterization in head and neck oncology: Looking for a standardization of clinical use and of detection method(s) in clinical practice.

BACKGROUND: A consensus about the most appropriate diagnostic method(s) for head and neck human papillomavirus (HPV)-induced carcinogenesis is still lacking because most of the commercially available assays have been designed for the cervix. METHODS: This article summarizes current data and trends concerning HPV diagnostic strategies in oropharyngeal squamous cell carcinoma (OPSCC). Six main approaches are described. RESULTS: The diagnostic gold standard for HPV-related OPSCC, focusing on E6/E7 mRNA detection, requires fresh samples. Because most frequently available samples are formalin-fixed paraffin-embedded (FFPE), the pros and cons of the different approaches were analyzed. CONCLUSIONS: In the FFPE samples, the immunohistochemistry of p16, which is considered appropriate to assess HPV-driven carcinogenesis in OPSCC according to the 8th American Joint Committee on Cancer TNM classification, may not be specific enough to become the diagnostic standard in the perspective of treatment deintensification. p16 may play a safer role in combination with another highly sensible assay. Other promising approaches are based on DNA detection through real-time polymerase chain reaction and RNAscope.

Successful analysis of a 100 years old semen stain generating a complete DNA STR profile.

We performed forensic investigations on a handkerchief containing seminal fluid deposited 100 years earlier. The aim was to verify the possibility to achieve a complete genetic profile exceeding the limit of success reached until today with a partial semen stain profile stored up to 50 years. The current forensic methods were carried out: Alternative Light Source (ALS), Prostate-Specific Antigen (PSA) Test, autosomal and Y-chromosome Short Tandem Repeats (STRs). ALS inspection was a crucial method in the success of the analysis. On a dried semen stain, fluorescence remains constant for a very long time, allowing possibility to minimize the fabric sample to analyze, in order to preserve specimen for future defense guarantees or to protect historical finding. We achieved positive results in 3 of 6 traces, PSA and DNA results were concordant, only those cuttings that tested positive for PSA yielded DNA profiles. A complete male profile was generated by AmpFlSTR Identifiler Plus and 16 loci profile by AmpFLSTR Yfiler. Considering some historical sources that attributed this handkerchief dated 1916, to a famous Italian poet, the DNA profile found on the handkerchief was compared with a living male descendant, and the comparison of the Y-STR between the two gave positive results, confirming the reliability of the outcomes. The findings achieved by the current forensic method empower the application of this methodology to other forensic cases, especially in past unresolved cases.

Inhibition of Transglutaminase 2 as a Potential Host-Directed Therapy Against Mycobacterium tuberculosis.

Host-directed therapies (HDTs) are emerging as a potential valid support in the treatment of drug-resistant tuberculosis (TB). Following our recent report indicating that genetic and pharmacological inhibition of transglutaminase 2 (TG2) restricts Mycobacterium tuberculosis (Mtb) replication in macrophages, we aimed to investigate the potentials of the TG2 inhibitors cystamine and cysteamine as HDTs against TB. We showed that both cysteamine and cystamine restricted Mtb replication in infected macrophages when provided at equimolar concentrations and did not exert any antibacterial activity when administered directly on Mtb cultures. Interestingly, infection of differentiated THP-1 mRFP-GFP-LC3B cells followed by the determination of the autophagic intermediates pH distribution (AIPD) showed that cystamine inhibited the autophagic flux while restricting Mtb replication. Moreover, both cystamine and cysteamine had a similar antimicrobial activity in primary macrophages infected with a panel of Mtb clinical strains belonging to different phylogeographic lineages. Evaluation of cysteamine and cystamine activity in the human ex vivo model of granuloma-like structures (GLS) further confirmed the ability of these drugs to restrict Mtb replication and to reduce the size of GLS. The antimicrobial activity of the TG2 inhibitors synergized with a second-line anti-TB drug as amikacin in human monocyte-derived macrophages and in the GLS model. Overall, the results of this study support the potential usefulness of the TG2-inhibitors cysteamine and cystamine as HDTs against TB.

Graphene oxide coatings prevent Candida albicans biofilm formation with a controlled release of curcumin-loaded nanocomposites.

AIM: Fabrication of graphene oxide (GO)-based medical devices coatings that limit adhesion of Candida albicans, a main issue of healthcare-associated infections. METHODS: The GO composites noncovalently functionalized with curcumin (CU), a hydrophobic molecule with active antimicrobial action, polyethylene glycol (PEG) that hinders the absorption of biomolecules or a combination of CU and PEG (GO-CU-PEG) were drop-casted on surfaces and antifungal efficacy was assessed. RESULTS: We demonstrate that GO-CU-PEG coatings can reduce fungal adhesion, proliferation and biofilm formation. Furthermore, in an aqueous environment, surfaces release curcumin-PEG nanocomposites that have a minimum inhibitory concentration of 9.25 µg/ml against C. albicans. CONCLUSION: Prevention of early cell adhesion and creation of a proximal environment unfavorable for growth make these GO-supported biomaterials attractive for innovative medical device manufacturing.

Vacuum-assisted closure (VAC®) systems and microbiological isolation of infected wounds.

Background: Negative pressure wound therapy is now largely used to treat infected wounds. The prevention and reduction of healthcare-associated infections is a high priority for any Department of Health and great efforts are spent to improve infection control systems. It is assumed that vacuum-assisted closure (VAC®) dressings should be watertight and that all the secretions are gathered in a single container but there is no consistent data on air leakage and possible dispersion of bacteria from the machine. Methods: We have conducted a prospective experimental study on 10 patients with diagnosis of wound infection to verify whether the filtration process is microbiologically efficient. We compared the bacteria population present in the wound to the one present in the air discharged by the VAC® machine. Results: This study shows that the contamination of the VAC® machine is considerably lower than the environment or wound contamination. Conclusions: Negative pressure wound therapy system does not represent a risk factor for healthcare-associated infections.

PE_PGRS3 of Mycobacterium tuberculosis is specifically expressed at low phosphate concentration, and its arginine-rich C-terminal domain mediates adhesion and persistence in host tissues when expressed in Mycobacterium smegmatis.

PE_PGRSs of Mycobacterium tuberculosis (Mtb) represent a family of complex and peculiar proteins whose role and function remain elusive. In this study, we investigated PE_PGRS3 and PE_PGRS4, two highly homologous PE_PGRSs encoded by two contiguous genes in the Mtb genome. Using a gene-reporter system in Mycobacterium smegmatis (Ms) and transcriptional analysis in Mtb, we show that PE_PGRS3, but not PE_PGRS4, is specifically expressed under low phosphate concentrations. Interestingly, PE_PGRS3, but not PE_PGRS4, has a unique, arginine-rich C-terminal domain of unknown function. Heterologous expression of PE_PGRS3 in Ms was used to demonstrate cellular localisation of the protein on the mycobacterial surface, where it significantly affects net surface charge. Moreover, expression of full-length PE_PGRS3 enhanced adhesion of Ms to murine macrophages and human epithelial cells and improved bacterial persistence in spleen tissue following infection in mice. Expression of the PE_PGRS3 functional deletion mutant lacking the C-terminal domain in Ms did not enhance adhesion to host cells, showing a phenotype similar to the Ms parental strain. Interestingly, enhanced persistence of Ms expressing PE_PGRS3 did not correlate with increased concentrations of inflammatory cytokines. These results point to a critical role for the ≈ 80 amino acids long, arginine-rich C-terminal domain of PE_PGRS3 in tuberculosis pathogenesis.