Short-chain fatty acids (SCFAs) are the main by-products of microbial fermentations occurring in the human intestine and are directly involved in the host's physiological balance. As impaired gut ...concentrations of acetic, propionic, and butyric acids are often associated with systemic disorders, the administration of SCFA-producing microorganisms has been suggested as attractive approach to solve symptoms related to SCFA deficiency.
In this research, nine probiotic strains (
NR, OC, SIN, and T,
ATCC 7050,
DSM 16604,
DSM 17938,
ATCC 53103, and
CNCM I-745) commonly included in commercial formulations were tested for their ability to secrete SCFAs by using an improved protocol in high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS-MS).
The developed method was highly sensitive and specific, showing excellent limits of detection and quantification of secreted SCFAs. All tested microorganisms were shown to secrete acetic acid, with only
and
additionally able to produce propionic and butyric acids. Quantitative differences in the secretion of SCFAs were also evidenced.
The experimental approach described in this study may contribute to the characterization of probiotics as SCFA-producing organisms, a crucial stage toward their application to improve SCFA deficiency.
The mechanisms of action and resistance of cefiderocol (FDC) in
are still not fully elucidated, but iron transport systems have been evoked in its entry into the cell to reach the penicillin-binding ...proteins (PBPs). To capture the dynamics of gene expression related to FDC action in various conditions, we report on the genomic and transcriptomic features of seven
strains with different FDC susceptibility, focusing on the variants in genes associated with β-lactam resistance and the expression of the siderophore biosynthesis and transport systems acinetobactin and baumannoferrin. We also investigated the expression of the TonB energy transduction system (ETS) and siderophore receptors
A and
A. The four clinical samples belonged to the same clonal complex (CC2), and the two strains with the highest FDC MICs showed peculiar variants in PBP2 and
C. Similarly, the two clinical strains with the lowest MICs shared variants in an outer membrane protein as well as
C. Gene expression analyses highlighted the up-regulation of the acinetobactin and baumannoferrin genes in response to iron depletion and a down-regulation in the presence of high iron concentrations. In response to FDC, gene expression seemed strain-dependent, probably due to the different metabolic features of each strain. Overall, FDC activates the ETS, confirming the active import of the drug; baumannoferrin, more than acinetobactin, appeared stimulated by FDC in an iron-depleted medium. In conclusion, iron transport systems play a clear role in the FDC uptake, and their expression likely contributes to MIC variation together with β-lactam resistance determinants.IMPORTANCE
poses a threat to healthcare due to its ability to give difficult-to-treat infections as a consequence of our shortage of antibiotic molecules active on this multidrug-resistant bacterium. Cefiderocol (FDC) represents one of the few drugs active on
and to preserve its activity, this study explored the transcriptomic and genomic features of seven strains with varying susceptibility to FDC. Transcriptomic analyses revealed the different effects of FDC on iron transport systems, promoting mainly baumannoferrin expression-thus more likely related to FDC entry-and the energy transduction systems. These findings suggest that not all iron transport systems are equally involved in FDC entry into
cells. Finally, mutations in PBPs and β-lactamases may contribute to the resistance onset. Overall, the study sheds light on the importance of iron availability and metabolic differences in FDC resistance, offering insights into understanding the evolution of resistance in
strains.
This study investigated
presence in 122 samples belonging to 34 typologies of fresh or short-ripened cheeses made from cow, sheep, goat, or buffalo pasteurized milk, and sold on the Italian market.
...was isolated at a prevalence of 9.8%, with a marked variability among cheese categories, and at low counts (always below 2.26 Log CFU/g). Twelve isolates were identified by MALDI-TOF analysis and typified by RAPD PCR as belonging to different
strains. All the strains were tested for the production of hemolysin BL, phosphatidylcholine-specific phospholipase C, proteases, and biofilm formation, and for the presence of chromosomal toxin-encoding genes (
,
,
,
,
,
,
,
). Overall, 92% of strains harbored
, 75% the three genes
,
, and
as well as
and
, 67%
, and 33%
. All strains showed biofilm-forming ability. A chemical-physical characterization of the cheeses was also performed to show their suitability as substrates for
growth, showing high heterogeneity in terms of pH, aw, salt content, and concentration of organic acids. Finally, the ability to support spore germination and vegetative cell growth of a selected cheese was investigated in spores-inoculated samples maintained at 10 °C and 15 °C, showing the inhibitory effect of low storage temperatures.
Culturing the gut microbiota in
models that mimic the intestinal environment is increasingly becoming a promising alternative approach to study microbial dynamics and the effect of perturbations on ...the gut community. Since the mucus-associated microbial populations in the human intestine differ in composition and functions from their luminal counterpart, we attempted to reproduce
the microbial consortia adhering to mucus using an already established three-dimensional model of the human gut microbiota. Electrospun gelatin structures supplemented or not with mucins were inoculated with fecal samples and compared for their ability to support microbial adhesion and growth over time, as well as to shape the composition of the colonizing communities. Both scaffolds allowed the establishment of long-term stable biofilms with comparable total bacterial loads and biodiversity. However, mucin-coated structures harbored microbial consortia especially enriched in
,
, and
, being therefore able to select for microorganisms commonly considered mucosa-associated
.
These findings highlight the importance of mucins in shaping intestinal microbial communities, even those in artificial gut microbiota systems. We propose our
model based on mucin-coated electrospun gelatin structures as a valid device for studies evaluating the effects of exogenous factors (nutrients, probiotics, infectious agents, and drugs) on mucus-adhering microbial communities.
Biofilms are surface-associated microbial communities embedded in a matrix that is almost impenetrable to antibiotics, thus constituting a critical health threat. Biofilm formation on the cornea or ...ocular devices can lead to serious and difficult-to-treat infections. Nowadays, natural molecules with antimicrobial activity and liposome-based delivery systems are proposed as anti-biofilm candidates. In this study, the anti-biofilm activity of a formulation containing citrus polyphenols encapsulated in liposomes was evaluated against
and
, the most common agents in ocular infections. The formulation activity against planktonic staphylococci was tested by broth microdilution and sub-inhibitory concentrations were used to evaluate the effect on biofilm formation using the crystal violet (CV) assay. The eradicating effect of the preparation on mature biofilms was investigated by the CV assay, plate count, and confocal laser scanning microscopy. The product was bactericidal against staphylococci at a dilution of 1:2 or 1:4 and able to reduce biofilm formation even if diluted at 1:64. The formulation also had the ability to reduce the biomass of mature biofilms without affecting the number of cells, suggesting activity on the extracellular matrix. Overall, our results support the application of the used liposome-encapsulated polyphenols as an anti-biofilm strategy to counter biofilm-associated ocular infections.
In vitro models for culturing complex microbial communities are progressively being used to study the effects of different factors on the modeling of in vitro-cultured microorganisms. In previous ...work, we validated a 3D in vitro model of the human gut microbiota based on electrospun gelatin scaffolds covered with mucins. The aim of this study was to evaluate the effect of
, a pathogen responsible for food poisoning diseases in humans, on the gut microbiota grown in the model. Real-time quantitative PCR and 16S ribosomal RNA-gene sequencing were performed to obtain information on microbiota composition after introducing
ATCC 14579 vegetative cells or culture supernatants. The adhesion of
to intestinal mucins was also tested. The presence of
induced important modifications in the intestinal communities. Notably, levels of
(particularly
),
, and
were reduced, while abundances of
and
increased. In addition,
was able to adhere to mucins. The results obtained from our in vitro model stress the hypothesis that
is able to colonize the intestinal mucosa by stably adhering to mucins and impacting intestinal microbial communities as an additional pathogenetic mechanism during gastrointestinal infection.
The widespread use of antibiotics has led to a gradual increase in drug-resistant bacterial infections, which severely weakens the clinical efficacy of antibacterial therapies. In recent decades, ...stilbenes aroused great interest because of their high bioavailability, as well as their manifold biological activity. Our research efforts are focused on synthetic heteroaromatic stilbene derivatives as they represent a potentially new type of antibiotic with a wide antibacterial spectrum. Herein, a preliminary molecular modeling study and a versatile synthetic scheme allowed us to define eight heteroaromatic stilbene derivatives with potential antimicrobial activity. In order to evaluate our compound’s activity spectrum and antibacterial ability, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) tests have been performed on Gram-positive and Gram-negative ATCC strains. Compounds PB4, PB5, PB7, and PB8 showed the best values in terms of MIC and were also evaluated for MBC, which was found to be greater than MIC, confirming a bacteriostatic activity. For all compounds, we evaluated toxicity on colon-rectal adenocarcinoma cells tumor cells (CaCo2), once it was established that the whole selected set was more active than 5-Fluorouracil in reducing CaCo-2 cells viability. To the best of our knowledge, the biological assays have shown for these derivatives an excellent bacteriostatic activity, compared to similar molecular structures previously reported, thus paving the way for a new class of antibiotic compounds.
Starting in 2019, the COVID-19 pandemic is a global threat that is difficult to monitor. SARS-CoV-2 is known to undergo frequent mutations, including SNPs and deletions, which seem to be transmitted ...together, forming clusters that define specific lineages. Reverse-Transcription quantitative PCR (RT-qPCR) has been used for SARS-CoV-2 diagnosis and is still considered the gold standard method. Our Eukaryotic Host Pathogens Interaction (EHPI) laboratory received six SARS-CoV-2-positive samples from a Sicilian private analysis laboratory, four of which showed a dropout of the E gene. Our sequencing data revealed the presence of a synonymous mutation (c.26415 C > T, TAC > TAT) in the E gene of all four samples showing the dropout in RT-qPCR. Interestingly, these samples also harbored three other mutations (S137L-Orf1ab; N439K-S gene; A156S-N gene), which had a very low diffusion rate worldwide. This combination suggested that these mutations may be linked to each other and more common in a specific area than in the rest of the world. Thus, we decided to analyze the 103 sequences in our internal database in order to confirm or disprove our "mutation cluster hypothesis". Within our database, one sample showed the synonymous mutation (c.26415 C > T, TAC > TAT) in the E gene. This work underlines the importance of territorial epidemiological surveillance by means of NGS and the sequencing of samples with clinical and or technical particularities, e.g., post-vaccine infections or RT-qPCR amplification failures, to allow for the early identification of these SNPs. This approach may be an effective method to detect new mutational clusters and thus to predict new emerging SARS-CoV-2 lineages before they spread globally.
Introduction Antibiotic 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 . Methods Broth 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. Results The 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. Discussion Overall, 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.
Purpose
In this study, we aimed to identify prognostic factors of cancer mortality in patients who received radical cystectomy and to identify genomic alterations in a sub-cohort of patients with ...locally advanced (pT3-4) and/or positive lymph nodes bladder cancer (BC).
Methods
We collected 101 BC samples from 2010 to 2018 who previously received radical cystectomy. Immunohistochemical slides were evaluated for PPAR, cAMP, IMP3, Ki67, CDK4, POU5F1, Cyclin E and MDM2, p65, CD3, CD4, CD8, CD20, CD68, CD163, FOXP3, PD-1 and PD-L1 expression. We calculated a prognostic score (PS) based on the positivity to PD-1, PD-L1 and of cAMP (final score ranging from 0 to 3). DNA of each sample have been used for sequencing by NGS in a sub-cohort of 6 patients with locally advanced (pT3-4) and/or positive lymph nodes BC.
Results
PD-1
+
(HR hazard ratio 2.59;
p
= 0.04), PD-L1
+
(HR = 6.46;
p
< 0.01) and cAMP
+
(HR 3.04;
p
= 0.02) were independent predictors of cancer-specific mortality (CSM). Increase of PS (score = 0 as reference) was associated with CSM, 0.81 (
p
= 0.80), 4.72 (
p
= 0.01) and 10.51 (
p
< 0.0) for PS 1, 2 and 3, respectively. ERBB2 was the gene most frequently mutated.
Conclusion
BC exhibited heterogenous protein expression and variable genomic features. Identification of expression of PD-1, PD-L1 and cAMP could help in predicting oncological outcomes.
PDF
