Synergistic Activity of the Human Lactoferricin-Derived Peptide hLF1-11 in Combination with Caspofungin against Candida Species

The present study describes a synergistic effect between a conventional antifungal drug, caspofungin, and a synthetic peptide derived from human lactoferrin, hLF1-11, against Candida species. These yeasts are able to cause severe systemic fungal infections in immunocompromised hosts.Candida species are the main fungal opportunistic pathogens causing systemic infections that are often associated with drug resistance and biofilm production on medical devices. The pressing need for new antifungal agents led to an increased interest in the use of combination therapies. The present study was aimed at investigating potential synergistic activity of the human lactoferrin-derived hLF1-11 peptide with caspofungin against caspofungin-resistant or -susceptible C. albicans, C. parapsilosis, and C. glabrata strains. Synergism was evaluated by the checkerboard assay, measuring cellular metabolic activity against Candida planktonic and sessile cells. A fractional inhibitory concentration (FIC) index of <= 0.5 was interpreted as synergy. Synergism was evaluated by killing assays on planktonic cells. A cell viability assay was performed with biofilm formation inhibition and preformed biofilm. Synergy for killing and viability assays was defined as a >= 2-log-CFU/mL reduction in comparison with the most active constituent. hLF1-11 and caspofungin exerted (i) synergistic effects against planktonic cells of all the tested strains, yielding drastic caspofungin MIC reduction, (ii) synergistic effects on the inhibition of biofilm formation against biofilm producer strains, yielding caspofungin BIC reduction, and (iii) synergistic effects on preformed biofilm assessed by measuring metabolic activity (FIC range, 0.28 to 0.37) against biofilm-producing strains and by cell viability assay in C. albicans SC5314. The synergistic effect observed between caspofungin and hLF1-11 against Candida spp. is of potential clinical relevance, representing a promising novel approach to target caspofungin-resistant Candida species infections. Further studies elucidating the mechanisms of action of such a synergistic effect are needed. IMPORTANCE The present study describes a synergistic effect between a conventional antifungal drug, caspofungin, and a synthetic peptide derived from human lactoferrin, hLF1-11, against Candida species. These yeasts are able to cause severe systemic fungal infections in immunocompromised hosts. In addition, they can form biofilms in medical implanted devices. Recently, caspofungin-resistant Candida strains have emerged, thus highlighting the need to develop different therapeutic strategies. In in vitro studies, this drug combination is able to restore sensitivity to caspofungin in caspofungin-resistant strains of Candida species, both in free-living cells and in cells organized in biofilms. This synergism could represent a promising novel approach to target infections caused by caspofungin-resistant Candida species

Comparison of different microbiological procedures for the diagnosis of Pneumocystis jirovecii pneumonia on bronchoalveolar-lavage fluid

Background: The current diagnostic gold standard for Pneumocystis jirovecii is represented by microscopic visualization of the fungus from clinical respiratory samples, as bronchoalveolar-lavage fluid, defining "proven" P. jirovecii pneumonia, whereas qPCR allows defining "probable" diagnosis, as it is unable to discriminate infection from colonization. However, molecular methods, such as end-point PCR and qPCR, are faster, easier to perform and interpret, thus allowing the laboratory to give back the clinician useful microbiological data in a shorter time. The present study aims at comparing microscopy with molecular assays and beta-D-glucan diagnostic performance on bronchoalveolar-lavage fluids from patients with suspected Pneumocystis jirovecii pneumonia. Bronchoalveolar-lavage fluid from eighteen high-risk and four negative control subjects underwent Grocott-Gomori's methenamine silver-staining, end-point PCR, RT-PCR, and beta-D-glucan assay.Results: All the microscopically positive bronchoalveolar-lavage samples (50%) also resulted positive by end-point and real time PCR and all, but two, resulted positive also by beta-D-glucan quantification. End-point PCR and RT-PCR detected 10 (55%) and 11 (61%) out of the 18 samples, respectively, thus showing an enhanced sensitivity in comparison to microscopy. All RT-PCR with a Ct <27 were confirmed microscopically, whereas samples with a Ct >= 27 were not.Conclusions: Our work highlights the need of reshaping and redefining the role of molecular diagnostics in a peculiar clinical setting, like P. jirovecii infection, which is a rare but also severe and rapidly progressive clinical condition affecting immunocompromised hosts that would largely benefit from a faster diagnosis. Strictly selected patients, according to the inclusion criteria, resulting negative by molecular methods could be ruled out for P. jirovecii pneumonia

The N-Terminus of Human Lactoferrin Displays Anti-biofilm Activity on Candida parapsilosis in Lumen Catheters

Candida parapsilosis is a major cause of hospital-acquired infection, often related to parenteral nutrition administered via catheters and hand colonization of health care workers, and its peculiar biofilm formation ability on plastic surfaces. The mortality rate of 30% points to the pressing need for new antifungal drugs. The present study aimed at analyzing the inhibitory activity of the N-terminal lactoferrin-derived peptide, further referred to as hLF 1-11, against biofilms produced by clinical isolates of C. parapsilosis characterized for their biofilm forming ability and fluconazole susceptibility. hLF 1-11 anti-biofilm activity was assessed in terms of reduction of biofilm biomass, metabolic activity, and observation of sessile cell morphology on polystyrene microtiter plates and using an in vitro model of catheter-associated C. parapsilosis biofilm production. Moreover, fluctuation in transcription levels of genes related to cell adhesion, hyphal development and extracellular matrix production upon peptide exposure were evaluated by quantitative real time RT-PCR. The results revealed that hLF 1-11 exhibits an inhibitory effect on biofilm formation by all the C. parapsilosis isolates tested, in a dose-dependent manner, regardless of their fluconazole susceptibility. In addition, hLF 1-11 induced a statistically significant dose-dependent reduction of preformed-biofilm cellular density and metabolic activity at high peptide concentrations only. Interestingly, when assessed in a catheter lumen, hLF 1-11 was able to induce a 2-log reduction of sessile cell viability at both the peptide concentrations used in RPMI diluted in NaPB. A more pronounced anti-biofilm effect was observed (3.5-log reduction) when a 10% glucose solution was used as experimental condition on both early and preformed C. parapsilosis biofilm. Quantitative real time RT-PCR experiments confirmed that hLF 1-11 down-regulates key biofilm related genes. The overall findings suggest hLF 1-11 as a promising candidate for the prevention of C. parapsilosis biofilm formation and to treatment of mature catheter-related C. parapsilosis biofilm formation

Eco-Friendly photocatalytic treatment of dyes with Ag nanoparticles obtained through sustainable process involving Spirulina platensis

The development of efficient photocatalysts is crucial in addressing water pollution concerns, specifically in the removal of organic dyes from wastewater. In this context, the use of silver nanoparticles (Ag NPs) might represent a method to achieve high dye degradation efficiencies. On the other hand, the classical Ag NP production process involves several reactants and operating conditions, which make it poorly sustainable. In the present work, Ag NPs were synthesized according to a new sustainable process involving the use of natural extracts of Spirulina platensis and milder operating conditions. The material was also calcined to determine the influence of organic content on the properties of Ag NPs. The X-ray diffraction (XRD) analysis displayed the AgCl and Ag phases with a crystalline size of 11.79 nm before calcination. After calcination, only the Ag phase was present with an increased crystalline size of 24.60 nm. Fourier Transform Infrared Spectroscopy (FTIR) confirmed the capping role of the metabolites from the extract. Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) revealed the spherical or quasi-spherical morphologies with agglomeration due to the calcination. Energy-dispersive X-ray spectroscopy (EDX) and Thermogravimetric (TGA) analyses further confirmed the involvement of metabolites in the synthesis of Ag NPs. The optical changes in the products were observed in a UV-Vis analysis. The Ag NPs were tested for their photocatalytic activity against the laboratory dye brilliant blue r invisible light in various conditions. The highest degradation efficiency of 81.9%, with a kapp value of 0.00595 min−1, was observed in alkaline medium after 90 min of light irradiation

Efficacy of ozonated water as a PS in photodynamic therapy: A tool for dental caries management? An in vitro study

Background: The most prevalent noncommunicable disease in the world is dental caries; and when it is not adequately treated, it is usually associated with tooth loss or severe dental lesions. In fact, expensive care or tooth extraction may be necessary due to the negative effects dental caries have on general health. This is due to its frequent pain and secondary bacterial infections. The aim of this study was to investigate the activity of ozonated water as such and in combination with appropriate light radiation so as to perform a photodynamic treatment (PDT) against the cariogenic bacterium Streptococcus mutans. Design and methods: This work has been performed in vitro by using an S. mutans strain mainly structured in a biofilm status, reproducing the natural condition of the tooth infection. The ozone was tested at three different concentrations by using a commercial device able to generate different O3 formulations in water. The PDT treatment requires an appropriate light wavelength, evaluated in this work through the UV-Vis adsorption spectrum of the ozonated water. Results: The obtained results suggested an effective and synergic property of O3 and light at 460–470nm against this microorganism. The most antibiofilm activity was observed using a concentration of ozone of 0.06mg/L alone as well as with PDT treatment. Conclusions: The results are encouraging for additional research and in vitro/in vivo fresh experimental investigations to perform an exhaustive antimicrobial treatment protocol against the S. mutans tooth infection

Mouthwash Based on Ozonated Olive Oil in Caries Prevention: A Preliminary In-Vitro Study

(1) Background: Ozone (O3) proved to oxidize organic and inorganic compounds, and its efficacy against bacteria, viruses and fungi plasma membranes was of interest. Ozone vehicle can be a gaseous form, ozonated water or ozonized oil. The aim of this in-vitro study was to evaluate the efficacy of ozonated olive oil against Streptococcus mutans. (2) Methods: Two different commercial mouthwashes were tested: Ialozon Blu (IB) (Gemavip, Cagliari, Italy), with ozonated olive oil, and Ialozon Rose (IR) (Gemavip, Cagliari, Italy), with ozonated olive oil, hyaluronic acid and vitamin E. All formulates were analyzed in a dilution range from 2- to 256-folds in saline solution, as to reproduce the salivary dilution. Streptococcus mutans CIP103220 strain was used for the antimicrobial susceptibility test, and the Kirby-Bauer inhibition method was performed to evaluate the Minimum Inhibitory (MIC), Minimum Bactericidal (MBC), and Minimum Biofilm Inhibitory Concentration (MBIC). (3) Results: Both formulates showed the same antimicrobial activity. MIC, MBC, and MBIC were observed for dilution factors of 1/32, 1/8 and 1/8, respectively. The mean value of inhibition zone diameter was 16.5 mm for IB, and 18 mm for IR. (4) Conclusions: The results suggested that ozonized olive oil formulates were able to inactivate Streptococcus mutans avoiding the salivary dilution effect in the oral cavity

Validation of Two Commercial Multiplex Real-Time PCR Assays for Detection of SARS-CoV-2 in Stool Donors for Fecal Microbiota Transplantation

Recurrent infection by Clostridioides difficile has recently been treated by fecal microbiota transplantation (FMT). As viable SARS-CoV-2 was recovered from stool of asymptomatic individuals, the FMT procedure could be a potential risk of SARS-CoV-2 transmission, thus underlying the need to reliably detect SARS-CoV-2 in stool. Here, we performed a multicentric study to explore performances of two commercially available assays for detection of SARS-CoV-2 RNA in stool of potential FMT donors. In three hospitals, 180 stool samples were spiked with serial 10-fold dilutions of a SARS-CoV-2 inactivated lysate to evaluate the Seegene Allplex ™ SARS-CoV-2 (SC2) and SARS-CoV- 2/FluA/FluB/RSV (SC2FABR) Assays for the detection of viral RNA in stool of FMT donors. The results revealed that both assays detected down to 2 TCID50/mL with comparable limit of detection values, SC2 showing more consistent target positivity rate than SC2FABR. Beyond high amplification efficiency, correlation between CT values and log concentrations of inactivated viral lysates showed R2 values ranging from 0.88 to 0.90 and from 0.87 to 0.91 for the SC2 and SC2FABR assay, respectively. The present results demonstrate that both methods are highly reproducible, sensitive, and accurate for SARS-CoV-2 RNA detection in stool, suggesting a potential use in FMT-donor screening

Studio dell'attivita del peptide hLF (1-11) sulla produzione di biofilm in isolati clinici di Candida albicans

RIASSUNTO: Candida albicans è un lievito comunemente presente come commensale nella flora microbica dell’uomo, ma capace di comportarsi da patogeno opportunista in individui con alterata reattività immunitaria. È il più comune agente eziologico di micosi nell’uomo, responsabile di infezioni sia localizzate, che sistemiche. Tra i fattori di virulenza posseduti da questo microrganismo, uno dei più rilevanti in ambito ospedaliero è la capacità di produrre biofilm, definito come una comunità di cellule microbiche adese a un substrato e inglobate in una matrice extracellulare polisaccaridica. Le cellule sessili acquisiscono un fenotipo di farmaco-resistenza, dovuto in ampia parte alla struttura stessa del biofilm, dato che la matrice polisaccaridica funge da barriera fisica che impedisce il passaggio della maggior parte degli agenti antifungini tradizionali. Grazie alla capacità di produrre biofilm, C. albicans è spesso responsabile di infezioni associate a dispositivi medici quali cateteri, protesi e valvole cardiache, molto difficili da eradicare. Negli ultimi anni, si è assistito ad un sviluppo nello studio di approcci terapeutici alternativi all’uso dei comuni farmaci antimicrobici per contrastare infezioni biofilm-correlate. Tra questi, di particolare interesse sono i peptidi antimicrobici (AMPs), piccole molecole (10-50 aminoacidi) prodotte da tutti gli organismi della scala evolutiva, di cui costituiscono un meccanismo di difesa innata. Tra i vari AMPs, il peptide sintetico hLF (1-11), costituito dai primi 11 aminoacidi del dominio N-terminale della lattoferricina umana, ha mostrato una spiccata attività antimicrobica nei confronti di diverse specie batteriche e fungine, inclusa C. albicans. Tuttavia, il ruolo esercitato dal peptide sul biofilm prodotto da C. albicans non è stato ancora delucidato. In tale ottica, il presente lavoro di tesi si prefigge di valutare l’attività inibitoria del peptide hLF (1-11) nei confronti di biofilm prodotto da diversi ceppi clinici di C. albicans. Dieci ceppi di C. albicans isolati presso l’Azienda Ospedaliera-Universitaria Pisana e il ceppo di rifermento di C. albicans SC5314, tutti produttori di biofilm, sono stati sottoposti a genotipizzazone mediante RAPD-PCR, allo scopo di selezionare quattro ceppi rappresentativi per gli esperimenti di inibizione del biofilm. Il saggio di inibizione del biofilm è stato effettuato su piastra da 96 pozzetti a fondo piatto utilizzando come terreno di coltura RPMI 1640 addizionato con il 2% di glucosio, diluito 1:4 con il tampone NaPB (sodium phosphte buffer). La sospensione cellulare di lievito utilizzata per il saggio conteneva 1x106 CFU/ml e veniva incubata per 24 ore a 37°C in presenza di diverse concentrazioni del peptide hLF (1-11). Per ogni piastra, il biofilm prodotto era quantificato mediante: (i) valutazione della biomassa, misurando la densità ottica a 490 nm; (ii) analisi dell’attività metabolica tramite il saggio XTT/menadione e (iii) valutazione della vitalità delle cellule presenti nel biofilm, tramite scraping del pozzetto, semina su agar Sabouraud e conta delle unità formanti colonia (CFU/ml) dopo 24 ore di incubazione a 37°C. I risultati ottenuti dagli esperimenti di co-incubazione indicano che il peptide esercita a 24 ore una significativa riduzione del biofilm sia in termini di biomassa, che attività metabolica e numero di cellule presenti, per tutti e 4 i ceppi analizzati (P<0.05; P<0.001). E’ stato quindi valutato se il peptide esercitasse una azione anti-biofilm anche a tempi più precoci, ripetendo il saggio di co-incubazione a 90 minuti, 3 ore e 6 ore a 37°C. I risultati ottenuti indicano che l’azione del peptide inibisce significativamente la formazione di biofilm a partite da 3 ore di co-incubazione. Questi risultati suggeriscono l’ipotesi che il peptide possa agire inibendo le prime fasi di formazione del biofilm, in particolare la fase di adesione cellulare. Infatti, quando il peptide è stato saggiato su cellule pre-adese ai pozzetti per 90 minuti e 6 ore, ha mostrato una ridotta attività inibitoria dopo 24 di incubazione, rispetto alla co-incubazione con le cellule fungine, confermando l’ipotesi che il peptide eserciti la sua attività anti-biofilm principalmente inibendo l’adesione cellulare. Inoltre, i pozzetti contenenti biofilm trattato con il peptide hLF (1-11) e non trattato sono stati osservati al microscopio invertito dopo 24 ore di co-incubazione, allo scopo di valutare gli eventuali cambiamenti nella architettura strutturale del biofilm fungino indotti dal peptide. L’analisi delle immagini ottenute ha indicato chiaramente una inibizione della morfogenesi di C. albicans, con una riduzione dose-dipendente del numero di ife osservate nei pozzetti del biofilm trattato con il peptide, per tutti e 4 i ceppi di C. albicans analizzati rispetto al controllo. Infine, allo scopo di verificare il ruolo del peptide nella regolazione genetica del biofilm prodotto da C. albicans, è stata effettuata una analisi dei profili di trascrizione di geni associati alla fase ifale e coinvolti nella sintesi di matrice polisaccaridica, mediante qRT-PCR. I dati ottenuti hanno evidenziato una sotto-regolazione dei geni in studio indotta dal peptide, confermando quanto visto all’analisi microscopica. In conclusione, i risultati ottenuti in questo studio indicano che il peptide hLF (1-11) potrebbe essere un ottimo candidato per prevenire la formazione di biofilm da parte di C. albicans in ambito nosocomiale. ABSTRACT: Candida albicans is an opportunistic fungal pathogen of humans with an increasing medical relevance, causing superficial as well as systemic infection in individuals with impaired immune response. One of the most relevant characteristic of C. albicans is the ability to produce biofilm, defined as a structured microbial community that is attached to a surface and embedded in an exopolymeric extracellular matrix. This polysaccharide matrix acts as a barrier for the sessile cells, preventing the entrance of most commonly used antifungal agents and, therefore, conferring drug resistance. Consequently, the ability to produce biofilm makes C. albicans one of the most common causative agent of infections associated with medical devices such as catheters, implants and heart valves. These infections are very difficult to eradicate. Recently, there is an increasing effort in developing alternative strategies aimed at eradicate biofilm-associated infections. In this regard, antimicrobial peptides (AMPs), small molecules (10-50 amino acids) conserved in several organisms as part of natural immunity, have widely been investigated as novel potential therapeutic agents. Among AMPs, the synthetic peptide hLF (1-11), derived from the first 11 amino acids of the N-terminal domain of human lactoferricin, showed a strong antimicrobial activity against various bacterial and fungal species, including C. albicans. However, the role played by this peptide on biofilm production by C. albicans remains to be elucidated. For these reasons, the present work aims to evaluate the inhibitory activity of the synthetic peptide hLF (1-11) against biofilm produced by different clinical strains of C. albicans. Ten C. albicans clinical isolates, obtained from the Azienda Ospedaliera-Univerisitaria Pisana, and the reference strain SC5314 were genotyped by RAPD PCR, in order to select four different strains for biofilm-inhibition experiments. The anti-biofilm assay was performed in a flat bottom 96-well plate using RPMI 1640 as a medium supplemented with 2% glucose, diluted 1 in 4 in NaPB (sodium phosphate buffer). A C. albicans suspension containing 1x106 cells/ml was co-incubated for 24 hours at 37 ° C in the presence of different concentrations of hLF (1-11). For each plate, quantification of biofilm was assessed by: (i) evaluating the biofilm biomass, at490 nm; (ii) measuring biofilm metabolic activity using the XTT/menadione assay and (iii) evaluating the viability of sessile cells, by scraping biofilm embedded cells from each well, plating them on Sabouraud agar and counting the colony forming units (CFU/ml) following a 24 hour incubation at 37°C. The results obtained show that, after 24 hours, the peptide induces a significant reduction of biofilm, both in terms of biomass, metabolic activity or cell viability, for all 4 strains analysed (P <0.05; P <0.001). Next, in order to investigate whether the peptide could also exert its anti-biofilm activity at earlier stages, we repeated co-incubation assay for 90 minutes, 3 hours and 6 hours at 37°C. The results obtained indicate that the peptide significantly inhibits the formation of biofilm after 3 hours of co-incubation. These findings suggest that the peptide may act by inhibiting the biofilm formation at early stages, when C. albicans adhesion to the abiotic surface occurs. Indeed, when the peptide was tested on cells pre-adhered to the wells for 90 minutes and 6 hours, it showed a reduced inhibitory activity after 24 hours of incubation, compared to the co-incubation experiments. This result confirms the hypothesis that the peptide exerts its anti-biofilm activity mainly by inhibiting cell adhesion. Furthermore, biofilm treated/untreated with the peptide hLF (1-11) were observed at the inverted microscope, after 24 hours of co-incubation, in order to assess potential changes in the architecture of the fungal biofilm induced by the peptide. The images clearly revealed an inhibition in C. albicans morphogenesis: all four strains treated with the peptide exhibited a dose-dependent reduction in the number of hyphae, compared to untreated control. Finally, in order to assess the effect of hLF (1-11) on biofilm-related gene expression, we investigated the transcript levels of genes linked to hyphal development or polysaccharide matrix synthesis, by qRT-PCR. The data obtained showed a marked down-regulation of these genes, induced by the peptide, thus confirming previous microscopic analysis. In conclusion, despite further studies candidate for a potential use as C. albicans anti-biofilm agent, our results suggest that the peptide hLF (1-11) could be an excellent candidate to prevent biofilm formation by C. albicans

Valutazione dell'effetto sinergico di hLF 1-11 e caspofungina su cellule planctoniche e biofilm di Candida spp.

Candida species are the main fungal opportunistic pathogens causing mucosal and systemic infections often associated with drug resistance and biofilm production on medical device. Biofilm associated infection are difficult to treat and could evolve in Candidaemia, often associated with an increase of morbidity and mortality. The difficulty to find new antifungal drugs has led to an increased interest in the use of antimicrobial peptides, alone or in combination with conventional drugs. The present study was aimed at investigating the possible synergistic activity of a synthetic peptide hLF1–11 and caspofungin against different strains of C. albicans , C. parapsilosis and C. glabrata planctonic cells and biofilm in vitro. The interaction between caspofungin and hLF1–11 and was first evaluated by the checkerboard assay using 96-well bottom polystyrene microtiter plates and a killing assay on planctonic cells. Then, synergistic activity was evaluated in biofilm producer strains by XTT reduction metabolic assay in the checkerboard plate and then, reduction of cell viability was evaluated in selected concentrations with low metabolic activity. Live/dead staining was performed by combining the green-fluorescent nucleic acid stain SYTO 9 and the red-fluorescent nucleic acid stain propidium iodide in PBS, then epifluorescence images were acquired using the Operetta CLS High-Content Imager and the Harmony software. A strong synergistic effect in biofilm inhibition was observed against all the tested C. albicans, C. parapsilosis and C. glabrata strains. A fractional inhibitory concentration (FIC) index ≤0.5 was interpreted as synergy as a decrease in CFU/ml of ≥2 Log by the combination of hLF1–11 and caspofungin in comparison with the most active constituent. Checkerboard assay shows a strong synergistic effect against all the tested C. albicans, C. parapsilosis and C. glabrata strains, in the planktonic cells, biofilm inhibition and biofilm eradication. On C. albicans caspofungin-resistant strains (CACR) biofilm inhibitory concentration (BIC) of drugs in combination was reduced up to 5 serial dilution, and comparable results were obtain for other strains. In viability assay performed on planktonic cells and sessile cells, synergy was found with various combinations of concentrations. Fluorescence imaging analysis confirmed the synergistic effect observed between caspofungin and hLF1-11 against biofilm, interesting the combinations of two drugs show the ability to inhibit hyphae and pseudohyphae formation in all strains tested. In conclusion, synergistic effect observed between caspofungin and hLF1-11 against Candida biofilm representing a possible interesting novel approach to target drug resistant fungal biofilm infections

Rapid identification of nontuberculous mycobacteria directly from positive primary MGIT coltures by MALDI-TOF MS

Introduction: Over the last years, nontuberculous mycobacteria (NTM) have emerged as important human pathogens. Accurate and rapid mycobacterial species identification is needed for successful diagnosis, treatment, and management of infections caused by NTM. Matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS) was demonstrated to effectively identify mycobacteria isolates subcultured from solid or liquid media rather than new positive cultures. The aim of the present study is to develop a rapid method for direct identification of NTM from primary MGIT cultures by MALDI-TOF MS. Materials and Methods: A total of 20 positive MGIT broths, collected from February to July 2021, were examined by the Bruker Biotyper system with Mycobacteria Library v 2.0 (Bruker Daltonics). Extraction was performed within 24-72 h after automated growth detection by MGIT. Protein extraction was carried out by the manufacturer’s MycoEx protocol. Results were compared with those obtained by the Line probe assay GenoType Mycobacterium CM/AS/NTM-DR (Hain LifeScience). Results: Our results showed concordant identification for all the mycobacteria isolated. In particular, the molecular test identified the mycobacteria as M. avium (n. 5), M.intracellulare (n. 3), M. chimaera (n. 3), M. gordonae (n. 2), M. fortuitum (n. 2), M. tuberculosis complex (n. 2), M. chelonae (n. 1), M. lentiflavum (n. 1) and M. celatum (n. 1). All identifications based on MALDI-TOF MS had scores &gt;1.7 and were concordant with the molecular identifications. MALDI-TOF MS cannot differentiate between M. intracellulare and M. chimaera, two closely related potentially pathogenic species of NTM that are members of the M. avium complex. Discussion and conclusions: Although a small number of strains and a limited diversity of mycobacterial species were analysed, our results indicate that MALDI-TOF MS could represent a useful routine diagnostic tool for identification of mycobacterial species directly from primary liquid culture