VanA type enterococci from humans, animals and food: species distribution, population structure, Tn1546-typing and location, and virulence determinants

VanA-type human (n = 69), animal (n = 49), and food (n =36) glycopeptide-resistant enterococci (GRE) from different geographic areas were investigated to study their possible reservoirs and transmission routes. Pulsed-field gel electrophoresis (PFGE) revealed two small genetically related clusters, M39 (n = 4) and M49 (n = 13), representing Enterococcus faecium isolates from animal and human feces and from clinical and fecal human samples. Multilocus sequence typing showed that both belonged to the epidemic lineage of CC17. purK allele analysis of 28 selected isolates revealed that type 1 was prevalent in human strains (8/11) and types 6 and 3 (14/15) were prevalent in poultry (animals and meat). One hundred and five of the 154 VanA GRE isolates, encompassing different species, origins, and PFGE types, were examined for Tn1546 type and location (plasmid or chromosome) and the incidence of virulence determinants. Hybridization of S1- and I-CeuI-digested total DNA revealed a plasmid location in 98% of the isolates. Human intestinal and animal E. faecium isolates bore large (>150 kb) vanA plasmids. Results of PCR-restriction fragment length polymorphism and sequencing showed the presence of prototype Tn1546 in 80% of strains and the G-to-T mutation at position 8234 in three human intestinal and two pork E. faecium isolates. There were no significant associations (P > 0.5) between Tn1546 type and GRE source or enterococcal species. Virulence determinants were detected in all reservoirs but were significantly more frequent (P < 0.02) among clinical strains. Multiple determinants were found in clinical and meat Enterococcus faecalis isolates. The presence of indistinguishable vanA elements (mostly plasmid borne) and virulence determinants in different species and PFGE-diverse populations in the presence of host-specific purK housekeeping genes suggested that all GRE might be potential reservoirs of resistance determinants and virulence traits transferable to human-adapted clusters

Attenuation of Listeria monocytogenes Virulence by Cannabis sativa L. Essential Oil

Anti-virulence strategies are being explored as a novel approach to combat pathogens. Such strategies include inhibition of surface adhesion, tissue invasion, toxin production, and/or interference with the gene regulation of other virulence traits. Listeria monocytogenes, the causative agent of listeriosis, is a facultative intracellular food pathogen characterized by a wide distribution in the environment. Its ability to persist within biofilms and to develop resistance to sanitizers is the cause of significant problems in food processing plants and of steep costs for the food industry. In humans, the treatment of listeriosis is hampered by the intracellular location of listeriae and the poor intracellular penetration of some antibiotics. Eleven L. monocytogenes isolates from patients who were diagnosed with invasive listeriosis in Italy in 2014–2016 were studied. This in vitro and in vivo study explored the antibacterial and anti-virulence properties of a steam-distilled essential oil of Cannabis sativa L., which is being intensively investigated for its high content in powerful bioactive phytochemicals. Susceptibility experiments demonstrated a moderate bactericidal activity of the essential oil (Minimum Bactericidal Concentration &gt; 2048 μg/mL). Assessment of the effects of sublethal concentrations of the essential oil on L. monocytogenes virulence traits demonstrated a significant action on motility. Listeriae were non-motile after exposure to the essential oil. Light and scanning electron microscopy documented aggregates of listeriae with the flagella trapped inside the cluster. Real-time RT-PCR experiments showed downregulation of flagellar motility genes and of the regulatory gene prfA. The ability to form biofilm and to invade Caco-2 cells was also significantly reduced. Galleria mellonella larvae infected with L. monocytogenes grown in presence of sublethal concentrations of the essential oil showed much higher survival rates compared with controls, suggesting that the extract inhibited tissue invasion. Food contamination with L. monocytogenes is a major concern for the food industry, particularly for plants making ready-to-eat and processed food. The present work provides a baseline in the study of the anti-virulence properties of the C. sativa essential oil against L. monocytogenes. Further studies are needed to understand if it could be used as an alternative agent for the control of L. monocytogenes in food processing plants

SARS-CoV-2 susceptibility and COVID-19 disease severity are associated with genetic variants affecting gene expression in a variety of tissues

Variability in SARS-CoV-2 susceptibility and COVID-19 disease severity between individuals is partly due to genetic factors. Here, we identify 4 genomic loci with suggestive associations for SARS-CoV-2 susceptibility and 19 for COVID-19 disease severity. Four of these 23 loci likely have an ethnicity-specific component. Genome-wide association study (GWAS) signals in 11 loci colocalize with expression quantitative trait loci (eQTLs) associated with the expression of 20 genes in 62 tissues/cell types (range: 1:43 tissues/gene), including lung, brain, heart, muscle, and skin as well as the digestive system and immune system. We perform genetic fine mapping to compute 99% credible SNP sets, which identify 10 GWAS loci that have eight or fewer SNPs in the credible set, including three loci with one single likely causal SNP. Our study suggests that the diverse symptoms and disease severity of COVID-19 observed between individuals is associated with variants across the genome, affecting gene expression levels in a wide variety of tissue types

Whole-genome sequencing reveals host factors underlying critical COVID-19

Critical COVID-19 is caused by immune-mediated inflammatory lung injury. Host genetic variation influences the development of illness requiring critical care1 or hospitalization2–4 after infection with SARS-CoV-2. The GenOMICC (Genetics of Mortality in Critical Care) study enables the comparison of genomes from individuals who are critically ill with those of population controls to find underlying disease mechanisms. Here we use whole-genome sequencing in 7,491 critically ill individuals compared with 48,400 controls to discover and replicate 23 independent variants that significantly predispose to critical COVID-19. We identify 16 new independent associations, including variants within genes that are involved in interferon signalling (IL10RB and PLSCR1), leucocyte differentiation (BCL11A) and blood-type antigen secretor status (FUT2). Using transcriptome-wide association and colocalization to infer the effect of gene expression on disease severity, we find evidence that implicates multiple genes—including reduced expression of a membrane flippase (ATP11A), and increased expression of a mucin (MUC1)—in critical disease. Mendelian randomization provides evidence in support of causal roles for myeloid cell adhesion molecules (SELE, ICAM5 and CD209) and the coagulation factor F8, all of which are potentially druggable targets. Our results are broadly consistent with a multi-component model of COVID-19 pathophysiology, in which at least two distinct mechanisms can predispose to life-threatening disease: failure to control viral replication; or an enhanced tendency towards pulmonary inflammation and intravascular coagulation. We show that comparison between cases of critical illness and population controls is highly efficient for the detection of therapeutically relevant mechanisms of disease

Sinergia con antibiotici ed azione anti-virulenza di fitocomposti bioattivi

L’aumentata diffusione di patogeni antibiotico-resistenti e la mancanza di nuove molecole antibiotiche ha risvegliato l'interesse nei confronti di fitocomposti come possibili agenti antimicrobici. Vengono esposti i risultati di nostri studi recenti riguardanti l'azione sinergica con antibiotici e le proprietà anti-virulenza di alcuni fitocomposti nei confronti di ceppi clinici di patogeni umani multi-resistenti, produttori di biofilm e invasivi per cellule. Gli studi sull’attività antimicrobica di carvacrolo (monoterpene fenolico presente negli oli essenziali di Thymus vulgaris e Origanum vulgare) e di capsaicina (alcaloide presente in piante del genere Capsicum), nei confronti di Streptococcus pyogenes, eritromicino-resistenti ed invasivi per cellule respiratorie umane, hanno dimostrato che, a concentrazioni sub-MIC, il carvacrolo agiva in sinergia (FICI≤0.5) con eritromicina e che la capsaicina, riduceva l’espressione della streptolisina O e la capacità di invadere cellule respiratorie A549. Gli studi sull’attività antimicrobica di curcumina (composto polifenolico presente nel rizoma di Curcuma longa) nei confronti di Mycobacterium abscessus, multi-resistente e produttore di biofilm, hanno dimostrato che, a concentrazioni sub-MIC, la curcumina agiva in sinergia con amikacina, claritromicina, ciprofloxacina, e linezolid ed era in grado di disgregare il biofilm maturo. Studi più recenti sull’attività anti-virulenza di olio essenziale di Cannabis sativa L. [varietà di canapa a basso contenuto di THC (<0.2%), ad uso industriale] nei confronti di Listeria monocytogenes da casi di listeriosi umana invasiva, hanno dimostrato che, a concentrazioni sub-letali, l’olio essenziale era in grado di ridurre la produzione di biofilm, la motilità, la capacità di invadere cellule epiteliali intestinali umane Caco-2 e di aumentare la sopravvivenza di larve infettate di Galleria mellonella. In un momento storico in cui l’aumento dei patogeni multi-resistenti costituisce una vera e propria emergenza sanitaria, per combattere la quale gli antibiotici a disposizione sono sempre gli stessi, una strategia che sfrutti la sinergia con antibiotici tradizionali oppure l’azione anti-virulenza di fitocomposti offre nuove prospettive alla lotta contro l’antibiotico resistenza. In particolare, l’azione anti-virulenza presenta dei vantaggi rispetto agli antibiotici in quanto non esercita una pressione selettiva verso l’antibiotico-resistenza

Attenuation of Listeria monocytogenes virulence by Cannabis sativa L. essential oil

Introduction. In recent years, the use of plant products as alternative/adjunct antimicrobial agents to control pathogenic microorganisms has been attracting mounting interest. A major group of plant antimicrobial compounds is represented by essential oils (EOs), complex mixtures of volatile secondary metabolites belonging to different chemical families. Cannabis sativa L. has been grown for thousands of years for a multiplicity of purposes; in recent years, some genotypes containing low cannabinoid concentrations have been selected and used for research purposes. Listeria monocytogenes is a facultative intracellular food pathogen able to persist within biofilms and to develop resistance to sanitizers. In humans, the treatment of listeriosis is hampered by the intracellular location of listeriae and the poor intracellular penetration of some antibiotics. The purpose of this study was to investigate the in vitro and in vivo antibacterial and anti-virulence properties of an EO extracted from a legal C. sativa L. variety against L. monocytogenes isolates collected from patients with invasive listeriosis. Materials and Methods. Eleven L. monocytogenes strains isolated in Italy in 2014-2016 were studied. The antibacterial and anti-virulence activity of the EO, extracted from C. sativa Futura 75 by steam-distillation, was determined by susceptibility tests, biofilm formation assays, Caco-2 invasion assays, motility assays, optical and scanning electron microscopy, Real-time RT-PCR experiments, and Galleria mellonella survival assays. Results. A moderate bactericidal activity of the C. sativa EO was detected (Minimum Bactericidal Concentration >2048 mg/L). In presence of EO at sub-lethal concentrations, the ability to form biofilm and to invade Caco-2 cells was significantly reduced (up to 15% and 73%, respectively). Motility tests demonstrated that, listeriae became non-motile i.e. they didn’t show the typical umbrella-like growth. Real-time RT-PCR assay demonstrated a significant downregulation of motility genes (flaA, motA, and motB) in L. monocytogenes exposed to the EO. In survival experiments with Galleria mellonella, larvae infected with L. monocytogenes grown in presence of EO showed much higher (93%) survival rates compared with controls (50%). Discussion and Conclusions. Anti-virulence strategies are being explored among plant products as a novel approach to combat bacterial pathogens. The significant anti-virulence properties of C. sativa EO against L. monocytogenes suggests that it could be employed as an alternative agent to control L. monocytogenes infectio

43rd NATIONAL CONGRESS OF THE ITALIAN SOCIETY OF MICROBIOLOGY

Introduction: Capsaicin (8-methyl-N-vanillyl- 6-nonenamide) is the active component responsible for the fruit pungency of Capsicum plants, cultivated for food and also for medicinal uses since ancient times. Besides its multiple pharmacological and physiological properties (pain relief, cancer prevention, weight reduction, cardiovascular, and gastrointestinal benefits), capsaicin has recently received attention because of its antimicrobial activity and anti-virulence properties. The aim of the present study was to investigate the effects of capsaicin on Streptococcus pyogenes, the most common cause of acute bacterial pharyngotonsillitis. Materials and Methods: The erythromycin-resistant [erm(B)/cMLS], high cell-invasive, and strong biofilm producer S. pyogenes pharyngeal isolate SP1070 was used throughout the study. Capsaicin was purchased from Sigma-Aldrich and stored (10 mg/mL stock solution) in absolute ethanol at -20°C. The MIC and MBC were determined according to the CLSI guidelines. Survival in presence of capsaicin was studied by the live/dead assay. Biofilm formation was tested by a microtiter assay and quantified by measuring the absorbance at 690 nm. Cell experiments were performed using the human alveolar carcinoma A549 cell line. Results: The MIC and the MBC of capsaicin were both 128 μg/mL. In the live/dead assay, several red cells were detected as early as 15 min after incubation with capsaicin at MIC; all cells were red after 60 min of incubation. At capsaicin sub-MICs (1/2– 1/16 × MIC), a significant increase in biofilm production and in the number of streptococci adherent to A549 cells was observed; whereas a strong reduction in the number of intracellular bacteria was detected. Discussion and Conclusions: Our findings reveal that capsaicin has a dual effect on S. pyogenes. High-level capsaicin exerts a bactericidal effect, probably due to the disruption of the cell membrane, this result being in agreement with previous studies on Gram-positive and Gram-negative pathogens; while sub-lethal capsaicin modifies virulence properties in vitro, such as the ability to form biofilm and to adhere/invade epithelial cells. Capsaicin-induced effects on biofilm formation seem to be similar to those observed for a variety of antibiotics that at sub-lethal concentrations can act as agonists of bacterial biofilm production in vitro. Overall, capsaicin-induced effects on S. pyogenes deserve further studies

Recombination between Streptococcus suis ICESsu32457 and Streptococcus agalactiae ICESa2603 yields a hybrid ICE transferable to Streptococcus pyogenes

Integrative conjugative elements (ICEs) are mobile genetic elements that reside in the chromosome but retain the ability to undergo excision and to transfer by conjugation. Genes involved in drug resistance, virulence, or niche adaptation are often found among backbone genes as cargo DNA. We recently characterized in Streptococcus suis an ICE (ICESsu32457) carrying resistance genes [tet(O/W/32/O), tet(40), erm(B), aphA, and aadE] in the 15K unstable genetic element, which is flanked by two ∼1.3kb direct repeats. Remarkably, ∼1.3-kb sequences are conserved in ICESa2603 of Streptococcus agalactiae 2603V/R, which carry heavy metal resistance genes cadC/cadA and mer. In matings between S. suis 32457 (donor) and S. agalactiae 2603V/R (recipient), transconjugants were obtained. PCR experiments, PFGE, and sequence analysis of transconjugants demonstrated a tandem array between ICESsu32457 and ICESa2603. Matings between tandem array-containing S. agalactiae 2603V/R (donor) and Streptococcus pyogenes RF12 (recipient) yielded a single transconjugant containing a hybrid ICE, here named ICESa2603/ICESsu32457. The hybrid formed by recombination of the left ∼1.3-kb sequence of ICESsu32457 and the ∼1.3-kb sequence of ICESa2603. Interestingly, the hybrid ICE was transferable between S. pyogenes strains, thus demonstrating that it behaves as a conventional ICE. These findings suggest that both tandem arrays and hybrid ICEs may contribute to the evolution of antibiotic resistance in streptococci, creating novel mobile elements capable of disseminating new combinations of antibiotic resistance genes