Several MALDI-TOF MS-based methods have been proposed for rapid detection of antimicrobial resistance. The most widely studied methods include assessment of β-lactamase activity by visualizing the hydrolysis of the β-lactam ring, detection of biomarkers responsible for or correlated with drug-resistance/non-susceptibility, and the comparison of proteomic profiles of bacteria incubated with or without antimicrobial drugs. Antimicrobial-resistance to a number of antibiotics belonging to different classes has been successfully tested by MALDI-TOF MS in a variety of clinically relevant bacterial species including members of Enterobacteriaceae family, non-fermenting Gram-negative bacteria, Gram-positive cocci, anaerobic bacteria and mycobacteria, opening this field to further clinically important developments. Early detection of drug-resistance by MALDI-TOF MS can be particularly helpful for clinicians to streamline the antibiotic therapy for a better outcome of patients with systemic infection, in all cases where a prompt and effective antibiotic treatment is essential to preserve organ function and/or patient survival.

Timeliness and accuracy in the diagnosis of microbial infections are associated with decreased mortality and reduced length of hospitalization, especially for severe, life-threatening infections. A rapid diagnosis also allows for early streamlining of empirical antimicrobial therapies, thus contributing to limit the emergence and spread of antimicrobial resistance. The introduction of matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) for routine identification of microbial pathogens has profoundly influenced microbiological diagnostics, and is progressively replacing biochemical identification methods. Compared to currently used identification methods, MALDI-TOF MS has the advantage of identifying bacteria and yeasts directly from colonies grown on culture plates for primary isolation in a few minutes and with considerable material and labor savings. The reliability and accuracy of MALDI-TOF MS in identification of clinically relevant bacteria and yeasts has been demonstrated by several studies showing that the performance of MALDI-TOF MS is comparable or superior to phenotypic methods currently in use in clinical microbiology laboratories, and can be further improved by database updates and analysis software upgrades. Besides microbial identification from isolated colonies, new perspectives are being explored for MALDI-TOF MS, such as identification of pathogens directly from positive blood cultures, sub-species typing, and detection of drug resistance determinants. In this review, we summarize the state of the art in routine identification of microbial pathogens by MALDI-TOF MS, and highlight recent advancements of this technology in special applications, such as strain typing, assessment of drug susceptibility, and detection of virulence factors.

Rapid identification (ID) and antimicrobial susceptibility testing (AST) of the causative agent(s) of bloodstream infections (BSIs) are essential for the prompt administration of an effective antimicrobial therapy, which can result in clinical and financial benefits. Immediately after blood sampling, empirical antimicrobial therapy, chosen on clinical and epidemiological data, is administered. When ID and AST results are available, the clinician decides whether to continue or streamline the antimicrobial therapy, based on the results of the in vitro antimicrobial susceptibility profile of the pathogen. The aim of the present study is to review and discuss the experimental data, advantages, and drawbacks of recently developed technological advances of culture-based and molecular methods for the diagnosis of BSI (including mass spectrometry, magnetic resonance, PCR-based methods, direct inoculation methods, and peptide nucleic acid fluorescence in situ hybridization), the understanding of which could provide new perspectives to improve and fasten the diagnosis and treatment of septic patients. Although blood culture remains the gold standard to diagnose BSIs, newly developed methods can significantly shorten the turnaround time of reliable microbial ID and AST, thus substantially improving the diagnostic yield.

Rapid identification of the causative agent(s) of bloodstream infections using the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) methodology can lead to increased empirical antimicrobial therapy appropriateness. Herein, we aimed at establishing an easier and simpler method, further referred to as the direct method, using bacteria harvested by serum separator tubes from positive blood cultures and placed onto the polished steel target plate for rapid identification by MALDI-TOF. The results by the direct method were compared with those obtained by MALDI-TOF on bacteria isolated on solid media. Identification of Gram-negative bacilli was 100 % concordant using the direct method or MALDI-TOF on isolated bacteria (96 % with score > 2.0). These two methods were 90 % concordant on Gram-positive cocci (32 % with score > 2.0). Identification by the SepsiTyper method of Gram-positive cocci gave concordant results with MALDI-TOF on isolated bacteria in 87 % of cases (37 % with score > 2.0). The direct method herein developed allows rapid identification (within 30 min) of Gram-negative bacteria and Gram-positive cocci from positive blood cultures and can be used to rapidly report reliable and accurate results, without requiring skilled personnel or the use of expensive kits.

The aim of this study was to evaluate the in vitro activity of the synthetic peptide hLF1-11 against biofilm produced by clinical isolates of Candida albicans with different fluconazole susceptibility. The antibiofilm activity of the peptide hLF1-11 was assessed in terms of reduction of biofilm cellular density, metabolic activity and sessile cell viability. The extent of morphogenesis in hLF1-11 treated and untreated biofilms was also investigated microscopically. Transcription levels of genes related to cell adhesion, hyphal development and extracellular matrix production were analysed by qRT-PCR in hLF1-11 treated and untreated biofilms. Exogenous dibutyryl-cAMP (db-cAMP) was used to rescue morphogenesis in cells exposed to the peptide. The results revealed that hLF1-11 exhibited an inhibitory effect on biofilm formation by all C. albicans isolates tested in a dose-dependent manner, regardless of their fluconazole susceptibility. Visual inspection of treated or untreated biofilm cells with an inverted microscope revealed a significant reduction in hyphal formation by hLF1-11 treated cells, as early as 3 hours of incubation. Moreover, hLF1-11 showed a reduced activity on preadherent cells. hLF1-11 induced the down-regulation of biofilm and hyphal-associated genes, which were predominantly regulated via the Ras1-cAMP-Efg1 pathway. Indeed, exogenous db-cAMP restored morphogenesis in hLF1-11 treated cells. The hLF1-11 peptide significantly inhibited biofilm formation by C. albicans mainly at early stages, interfering with biofilm cellular density and metabolic activity, and affected morphogenesis through the Ras1-cAMP-Efg1 pathway. Our findings provide the first evidence that hLF1-11 could represent a potential candidate for the prevention of biofilm formation by C. albicans.