Detection of Antibiotic-Resistance by MALDI-TOF Mass Spectrometry: An Expanding Area.

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 family, non-fermenting Gram-negative bacteria, Gram-positive cocci, anaerobic bacteria and mycobacteria, opening this field to further clinically important developments.

Synergistic activity between colistin and the ionic liquids 1-methyl-3-dodecylimidazolium bromide, 1-dodecyl-1-methylpyrrolidinium bromide, or 1-dodecyl-1-methylpiperidinium bromide against Gram-negative bacteria.

Objectives: Ionic liquids have shown potential for applications as antimicrobials. Their antimicrobial activity has been shown to be higher against Gram-positive than Gram-negative bacteria, suggesting a protective role for the outer membrane of Gram-negative microorganisms. Colistin is a last-resort antibiotic often used for treating infections caused by multi-drug resistant Gram-negative bacteria.

A new culture-based method for rapid identification of microorganisms in polymicrobial blood cultures by MALDI-TOF MS.

Background: The application of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) to microbial identification has allowed the development of rapid methods for identification of microorganisms directly in positive, blood cultures (BCs). These methods can yield accurate results for monomicrobial BCs, but often fail to identify multiple microorganisms in polymicrobial BCs. The present study was aimed at establishing a rapid and simple method for identification of bacteria and yeast in polymicrobial BCs from patients with bloodstream infection.

Comparative evaluation of antimicrobial activity of different types of ionic liquids.

In order to identify most suitable ionic liquids (ILs) for potential applications in infection prevention and control, in the present study we comparatively evaluated the antimicrobial potency and hemolytic activity of 15 ILs, including 11 previously described and four newly synthesized ILs, using standard microbiological procedures against Gram-positive and Gram-negative bacteria. ILs showing the lowest minimum inhibitory concentration (MIC) were tested for their hemolytic activity. Three ILs characterized by low MIC values and low hemolytic activity, namely 1-methyl-3-dodecylimidazolium bromide, 1-dodecyl-1-methylpyrrolidinium bromide, and 1-dodecyl-1-methylpiperidinium bromide were further investigated to determine their minimum bactericidal concentration (MBC), and their ability to inhibit biofilm formation by Staphylococcus aureus or Pseudomonas aeruginosa.

A Rapid Procedure for Identification and Antifungal Susceptibility Testing of Yeasts From Positive Blood Cultures.

The development of rapid diagnostic assays for the identification and analysis of antimicrobial resistance of fungal pathogens causing invasive mycoses is of utmost importance to reduce morbidity and mortality. We evaluated the performance of a novel rapid procedure directly applied to monomicrobial blood cultures from patients with bloodstream infection caused by yeast species, including nine and three non- species. For the rapid procedure herein developed, samples of positive blood cultures were transferred into serum separator tubes and treated with sodium dodecyl sulfate; the yeast layer was recovered and directly used for microbial identification by MALDI-TOF mass spectrometry and antifungal susceptibility testing (AFST) by the Sensititre YeastOne Y010 panel.

MALDI-TOF MS Applications to the Detection of Antifungal Resistance: State of the Art and Future Perspectives.

MALDI-TOF MS technology has made possible revolutionary advances in the diagnosis of infectious diseases. Besides allowing rapid and reliable identification of bacteria and fungi, this technology has been recently applied to the detection of antimicrobial resistance. Several approaches have been proposed and evaluated for application of MALDI-TOF MS to antimicrobial susceptibility testing of bacteria, and some of these have been or might be applied also to yeasts.

Recent Advances and Ongoing Challenges in the Diagnosis of Microbial Infections by MALDI-TOF Mass Spectrometry.

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.

Recent advances in the microbiological diagnosis of bloodstream infections.

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.

Comparative evaluation of six chromogenic media for presumptive yeast identification.

Aims: The present study was undertaken to evaluate the discrimination ability of six chromogenic media in presumptive yeast identification.

Methods: We analysed 108 clinical isolates and reference strains belonging to eight different species: ,, , , , , and .

Results: , and could be distinguished from one another in all the tested chromogenic media, as predicted by the manufacturers.

A new rapid method for direct antimicrobial susceptibility testing of bacteria from positive blood cultures.

Background: Rapid identification and antimicrobial susceptibility testing (AST) of the causative agent(s) of bloodstream infections can lead to prompt appropriate antimicrobial therapy. To shorten species identification, in this study bacteria were recovered from monomicrobial blood cultures by serum separator tubes and spotted onto the target plate for direct MALDI-TOF MS identification. Proper antibiotics were selected for direct AST based on species identification.

The BCG1619c gene is not essential for invasion and intracellular persistence of Mycobacterium bovis BCG in human THP-1 and A549 cell lines.

The BCG1619c gene of Mycobacterium bovis bacillus Calmette-Guérin (BCG) encodes for a 24 kDa invasin-like protein and is identical to the Rv1566c gene of Mycobacterium tuberculosis. To assess whether this protein was necessary for entry and (or) intracellular persistence in professional phagocytes and (or) in lung epithelial cells, a BCG1619c knockout mutant of M. bovis BCG was generated and compared with the parental BCG strain for its ability to infect and multiply in human monocyte derived THP-1 cells and in the lung epithelial cell line A549.

Direct binding of human NK cell natural cytotoxicity receptor NKp44 to the surfaces of mycobacteria and other bacteria.

Our previous studies demonstrated that Mycobacterium bovis bacillus Calmette-Guérin (BCG) can directly interact with human NK cells and induce the proliferation, gamma interferon production, and cytotoxic activity of such cells without the need for accessory cells. Thus, the aim of the present study was to identify the putative receptor(s) responsible for the recognition of BCG by human NK cells and potentially involved in the activation of NK cells. To this end, we first investigated the surface expression of three NK cell-activating receptors belonging to the natural cytoxicity receptor (NCR) family on highly purified human NK cells upon in vitro direct stimulation with BCG.

Evaluation of the inhibitory effects of human serum components on bactericidal activity of human beta defensin 3.

Naturally occurring cationic antimicrobial peptides (CAPs) are an essential component of the innate immune system of multicellular organisms. At concentrations generally higher than those found in vivo, most CAPs exhibit strong antibacterial properties in vitro, but their activity may be inhibited by body fluids, a fact that could limit their future use as antimicrobial and/or immunomodulatory agents. In the present study, we evaluated the effects of human serum components on bactericidal activity of the human beta-defensin 3 (hBD-3), a CAP considered particularly promising for future therapeutic employment.

Comparative analysis of the bactericidal activities of amphibian peptide analogues against multidrug-resistant nosocomial bacterial strains.

Due to the widespread resistance of bacteria to the available drugs, the discovery of new classes of antibiotics is urgently needed, and naturally occurring antimicrobial peptides (AMPs) are considered promising candidates for future therapeutic use. Amphibian skin is one of the richest sources of such AMPs. In the present study we compared the in vitro bactericidal activities of five AMPs from three different species of anurans against multidrug-resistant clinical isolates belonging to species often involved in nosocomial infections (Staphylococcus aureus, Enterococcus faecium, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Acinetobacter baumannii).

The secretion antigen SA5K has a role in the adaptation of Mycobacterium bovis bacillus Calmette-Guérin to intracellular stress and hypoxia.

The Mycobacterium tuberculosis TB8.4 (Rv1174c) gene encodes a secreted protein of 8.4 kDa (TB8.

In vitro bactericidal activity of human beta-defensin 3 against multidrug-resistant nosocomial strains.

The antimicrobial activity of human beta-defensin 3 (hBD-3) against multidrug-resistant clinical isolates of Staphylococcus aureus, Enterococcus faecium, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Acinetobacter baumannii was evaluated. A fast bactericidal effect (within 20 min) against all bacterial strains tested was observed. The presence of 20% human serum abolished the bactericidal activity of hBD-3 against gram-negative strains and reduced the activity of the peptide against gram-positive strains.

Influence of culture medium on the resistance and response of Mycobacterium bovis BCG to reactive nitrogen intermediates.

The aim of the present work was to evaluate the influence of the culture medium on the resistance and response of Mycobacterium bovis BCG to reactive nitrogen intermediates, in vitro. BCG was grown in Sauton, Dubos or Middlebrook 7H9 medium and exposed to sodium nitroprusside (SNP) for up to 7 days. The percentage of bacilli that survived was significantly lower in Middlebrook 7H9 than in Sauton or Dubos medium.

Disruption of the gene encoding for secretion antigen SA5K affects growth of Mycobacterium bovis bacillus Calmette-Guerin in human macrophages and in mice.

An 8.3-kDa secretory antigen of Mycobacterium bovis bacillus Calmette-Guerin (BCG), called SA5K, was previously identified and characterized in our laboratory. Sequence analysis of the BCG sa5k gene, including the corresponding promoter region, showed that it is identical to the homologous gene in Mycobacterium tuberculosis (Rv1174c).

Susceptibility of Streptococcus mutans and Actinobacillus actinomycetemcomitans to bactericidal activity of human beta-defensin 3 in biological fluids.

Bactericidal activity of human beta-defensin 3 (hBD-3) against Streptococcus mutans and Actinobacillus actinomycetemcomitans was inhibited in a dose-dependent manner by the presence of saliva and/or serum. Increasing the concentration of hBD-3 partially overcame this inhibition. A fast bactericidal effect was observed against both bacterial strains, suggesting a potential therapeutic use for hBD-3 in the local treatment of oral infections.

Functional characterization of human natural killer cells responding to Mycobacterium bovis bacille Calmette-Guérin.

The kinetics of activation and induction of several effector functions of human natural killer (NK) cells in response to Mycobacterium bovis bacille Calmette-Guérin (BCG) were investigated. Owing to the central role of monocytes/macrophages (MM) in the initiation and maintenance of the immune response to pathogens, two different experimental culture conditions were analysed. In the first, monocyte-depleted nylon wool non-adherent (NW) cells from healthy donors were stimulated with autologous MM preinfected with BCG (intracellular BCG).

Expression of SA5K, a secretion antigen of Mycobacterium tuberculosis, inside human macrophages and in sputum from tuberculosis patients.

An 8.3 kDa protein (SA5K), secreted by Mycobacterium tuberculosis/Mycobacterium bovis bacillus Calmette-Guérin (BCG) in culture filtrate, has been previously described in our laboratory. In the present study, analysis of the distribution of SA5K gene (Rv1174c) among M.

Activity of human beta-defensin 3 alone or combined with other antimicrobial agents against oral bacteria.

The in vitro activities of human beta-defensin 3 (hBD-3) alone or combined with lysozyme, metronidazole, amoxicillin, and chlorhexidine were investigated with the oral bacteria Streptococcus mutans, Streptococcus sanguinis, Streptococcus sobrinus, Lactobacillus acidophilus, Actinobacillus actinomycetemcomitans, and Porphyromonas gingivalis. hBD-3 showed bactericidal activity against all of the bacterial species tested. The bactericidal effect was enhanced when the peptide was used in combination with the antimicrobial agents mentioned above.

Identification of novel proteins in culture filtrates of Mycobacterium bovis bacillus Calmette-Guérin in the isoelectric point range 6-11.

Two-dimensional gel electrophoresis and mass spectrometry were used to identify proteins in the isoelectric point range 6-11 in culture filtrates of Mycobacterium bovis bacillus Calmette-Guérin (BCG). Twelve proteins were identified, three of which had not been described previously. The expression of the identified proteins was comparatively analyzed in culture filtrates of BCG in different growth phases and culture conditions.