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IntroductionAntibiotic resistance represents one of the most significant threats to public health in the 21st century. Polyphenols, natural molecules with antibacterial activity produced by plants, are being considered as alternative antimicrobial strategies to manage infections caused by drug-resistant bacteria. In this study, we investigated the antibacterial activity of a polyphenol mixture extracted from citrus fruits, against both antibiotic-susceptible and resistant strains of Staphylococcus aureus and Staphylococcus epidermidis.MethodsBroth microdilution and time-kill curve experiments were used to test the extract anti-staphylococcal activity. Cytotoxicity was assessed by the hemolysis assay. The interaction between the mixture and antibiotics was investigated by the checkerboard assay. The effect of B alone and in combination with oxacillin on the membrane potential was investigated by the 3,3′-dipropylthiadicarbocyanine iodide assay. The ability of the extract to induce the development of resistance was verified by propagating S. aureus for 10 transfers in the presence of sub-inhibitory concentrations.ResultsThe citrus extract was found to be active against all Staphylococcus strains at remarkably low concentrations (0.0031 and 0.0063%), displaying rapid bactericidal effects without being toxic on erythrocytes. In particular, B was found to rapidly cause membrane depolarization. When combined with methicillin, meropenem, and oxacillin, the mixture displayed synergistic activity exclusively against methicillin-resistant strains. We additionally show that the sequential exposure of S. aureus to sub-inhibitory concentrations did not induce the development of resistance against the extract.DiscussionOverall, these findings support the potential use of the citrus extract as promising option to manage staphylococcal infections and suggest that it may counteract the mechanism behind methicillin-resistance.

IntroductionProbiotics are living microorganisms that, when administered in adequate amounts, confer a health benefit on the host. Adequate number of living microbes, the presence of specific microorganisms, and their survival in the gastrointestinal (GI) environment are important to achieve desired health benefits of probiotic products. In this in vitro study, 21 leading probiotic formulations commercialized worldwide were evaluated for their microbial content and survivability in simulated GI conditions.MethodsPlate-count method was used to determine the amount of living microbes contained in the products. Culture-dependent Matrix-Assisted Laser Desorption/Ionization–Time of Flight Mass Spectrometry and culture-independent metagenomic analysis through 16S and 18S rDNA sequencing were applied in combination for species identification. To estimate the potential survivability of the microorganisms contained in the products in the harsh GI environment, an in vitro model composed of different simulated gastric and intestinal fluids was adopted.ResultsThe majority of the tested probiotic products were concordant with the labels in terms of number of viable microbes and contained probiotic species. However, one product included fewer viable microbes than those displayed on the label, one product contained two species that were not declared, and another product lacked one of the labeled probiotic strains. Survivability in simulated acidic and alkaline GI fluids was highly variable depending on the composition of the products. The microorganisms contained in four products survived in both acidic and alkaline environments. For one of these products, microorganisms also appeared to grow in the alkaline environment.ConclusionThis in vitro study demonstrates that most globally commercialized probiotic products are consistent with the claims described on their labels with respect to the number and species of the contained microbes. Evaluated probiotics generally performed well in survivability tests, although viability of microbes in simulated gastric and intestinal environments showed large variability. Although the results obtained in this study indicate a good quality of the tested formulations, it is important to stress that stringent quality controls of probiotic products should always be performed to provide optimal health benefits for the host.

On the worldwide market, a great number of probiotic formulations are available to consumers as drugs, dietary supplements, and functional foods. For exerting their beneficial effects on host health, these preparations should contain a sufficient amount of the indicated living microbes and be pathogen-free to be safe. Therefore, the contained microbial species and their amount until product expiry are required to be accurately reported on the labels. While commercial formulations licensed as drugs are subjected to rigorous quality controls, less stringent regulations are generally applied to preparations categorized as dietary supplements and functional foods. Many reports indicated that the content of several probiotic formulations does not always correspond to the label claims in terms of microbial identification, number of living organisms, and purity, highlighting the requirement for more stringent quality controls by manufacturers. The main focus of this review is to provide an in-depth overview of the microbiological quality of probiotic formulations commercialized worldwide. Many incongruences in the compositional quality of some probiotic formulations available on the worldwide market were highlighted. Even if manufacturers carry at least some of the responsibility for these inconsistencies, studies that analyze probiotic products should be conducted following recommended and up-to-date methodologies.

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.

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. In this context, the comparison of proteomic profiles of bacteria/yeasts incubated with or without antimicrobial drugs is a very promising method. Another recently proposed MALDI-TOF MS-based approach for antifungal susceptibility testing is the application of the semi-quantitative MALDI Biotyper antibiotic susceptibility test rapid assay, which was originally designed for antimicrobial susceptibility testing of bacteria, to yeast isolates. Increasingly effective and accurate MS tools and instruments as well as the possibility to optimize analytical parameter settings for targeted applications have generated an expanding area in the field of clinical microbiology diagnostics, paving the way for the development and/or optimization of rapid methods for antifungal susceptibility testing in the near future. In the present study, the state of the art of MALDI-TOF MS applications to antifungal susceptibility testing is reviewed, and cutting-edge developments are discussed, with a particular focus on methods allowing rapid detection of drug resistance in pathogenic fungi causing systemic mycoses.

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 Candida and three non-Candida 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. The results were compared with those obtained by the same methods applied to colonies isolated on solid media. Using a score value of 1.700 as cut-off for valid identification, the rapid procedure identified 66 of 124 (53.2%) isolates, all of which concordantly with the reference method. However, adopting a cut-off ≥1.300 and ≥4 consecutive repetitions of the same species in the list of matches would extend concordant identification to 107/124 (86.3%) samples. Importantly, AFST revealed essential agreement between the two methods for all the isolate/antifungal drug combinations tested, including misidentified and not identified isolates. Therefore, the procedure herein developed represents a valid alternative for AFST of yeasts from positive blood cultures, yielding accurate and reliable results at least 24 h earlier than with the routine procedure, thus allowing clinicians to promptly streamline antifungal therapy.

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.

Recent guidelines indicate that oral probiotics, living microorganisms able to confer a health benefit on the host, should be safe for human consumption, when administered in a sufficient amount, and resist acid and bile to exert their beneficial effects (e.g., metabolic, immunomodulatory, anti-inflammatory, competitive). This study evaluated quantitative and qualitative aspects and the viability in simulated gastric and intestinal juices of commercial probiotic formulations available in Italy. Plate counting and MALDI-TOF mass spectrometry were used to enumerate and identify the contained organisms. In vitro studies with two artificial gastric juices and pancreatin–bile salt solution were performed to gain information on the gastric tolerance and bile resistance of the probiotic formulations. Most preparations satisfied the requirements for probiotics and no contaminants were found. Acid resistance and viability in bile were extremely variable depending on the composition of the formulations in terms of contained species and strains. In conclusion, this study indicates good microbiological quality but striking differences in the behavior in the presence of acids and bile for probiotic formulations marketed in Italy.

Besides sporulation, Bacillus cereus can undergo a differentiation process in which short swimmer cells become elongated and hyperflagellated swarmer cells that favor migration of the bacterial community on a surface. The functionally enigmatic flagellar protein FlhF, which is the third paralog of the signal recognition particle (SRP) GTPases Ffh and FtsY, is required for swarming in many bacteria. Previous data showed that FlhF is involved in the control of the number and positioning of flagella in B. cereus. In this study, in silico analysis of B. cereus FlhF revealed that this protein presents conserved domains that are typical of SRPs in many organisms and a peculiar N-terminal basic domain. By proteomic analysis, a significant effect of FlhF depletion on the amount of secreted proteins was found with some proteins increased (e.g. B component of the non-hemolytic enterotoxin, cereolysin O, enolase) and others reduced (e.g. flagellin, L2 component of hemolysin BL, bacillolysin, sphingomyelinase, PC-PLC, PI-PLC, cytotoxin K) in the extracellular proteome of a ΔflhF mutant. Deprivation of FlhF also resulted in significant attenuation in the pathogenicity of this strain in an experimental model of infection in Galleria mellonella larvae.Our work highlights the multifunctional role of FlhF in B. cereus, being this protein involved in bacterial flagellation, swarming, protein secretion, and pathogenicity.