Bruna Facinelli

Human Milk Oligosaccharides Inhibit the Adhesion to Caco-2 Cells of Diarrheal Pathogens: Escherichia coli , Vibrio cholerae , and Salmonella fyris

Breast-fed children, compared with the bottle-fed ones, have a lower incidence of acute gastroenteritis due to the presence of several antiinfective factors in human milk. The aim of this work is to study the ability of human milk oligosaccharides to prevent infections related to some common pathogenic bacteria. Oligosaccharides of human milk were fractionated by gel-filtration and characterized by thin-layer chromatography and high-performance anion exchange chromatography. Fractions obtained contained, respectively, 1) acidic oligosaccharides, 2) neutral high-molecular-weight oligosaccharides, and 3) neutral low-molecular-weight oligosaccharides. Experiments were carried out to study the ability of oligosaccharides in inhibiting the adhesion of three intestinal microorganisms (enteropathogenic Escherichia coli serotype O119, Vibrio cholerae, and Salmonella fyris) to differentiated Caco-2 cells. The study showed that the acidic fraction had an antiadhesive effect on the all three pathogenic strains studied (with different degrees of inhibition). The neutral high-molecular-weight fraction significantly inhibited the adhesion of E. coli O119 and V. cholerae, but not that of S. fyris; the neutral low-molecular-weight fraction was effective toward E. coli O119 and S. fyris but not V. cholerae. Our results demonstrate that human milk oligosaccharides inhibit the adhesion to epithelial cells not only of common pathogens like E. coli but also for the first time of other aggressive bacteria as V. cholerae and S. fyris. Consequently, oligosaccharides are one of the important defensive factors contained in human milk against acute diarrheal infections of breast-fed infants.

Dysbiosis contributes to fibrogenesis in the course of chronic liver injury in mice

Nonalcoholic fatty liver disease (NAFLD) may lead to hepatic fibrosis. Dietary habits affect gut microbiota composition, whereas endotoxins produced by Gram‐negative bacteria stimulate hepatic fibrogenesis. However, the mechanisms of action and the potential effect of microbiota in the liver are still unknown. Thus, we sought to analyze whether microbiota may interfere with liver fibrogenesis. Mice fed control (CTRL) or high‐fat diet (HFD) were subjected to either bile duct ligation (BDL) or CCl4 treatment. Previously gut‐sterilized mice were subjected to microbiota transplantation by oral gavage of cecum content obtained from donor CTRL‐ or HFD‐treated mice. Fibrosis, intestinal permeability, bacterial translocation, and serum endotoxemia were measured. Inflammasome components were evaluated in gut and liver. Microbiota composition (dysbiosis) was evaluated by Pyrosequencing. Fibrosis degree was increased in HFD+BDL versus CTRL+BDL mice, whereas no differences were observed between CTRL+CCl4 and HFD+CCl4 mice. Culture of mesenteric lymph nodes showed higher density of infection in HFD+BDL mice versus CTRL+BDL mice, suggesting higher bacterial translocation rate. Pyrosequencing revealed an increase in percentage of Gram‐negative versus Gram‐postive bacteria, a reduced ratio between Bacteroidetes and Firmicutes, as well as a dramatic increase of Gram‐negative Proteobacteria in HFD+BDL versus CTRL+BDL mice. Inflammasome expression was increased in liver of fibrotic mice, but significantly reduced in gut. Furthermore, microbiota transplantation revealed more liver damage in chimeric mice fed CTRL diet, but receiving the microbiota of HFD‐treated mice; liver damage was further enhanced by transplantation of selected Gram‐negative bacteria obtained from cecum content of HFD+BDL‐treated mice. Conclusions: Dietary habits, by increasing the percentage of intestinal Gram‐negative endotoxin producers, may accelerate liver fibrogenesis, introducing dysbiosis as a cofactor contributing to chronic liver injury in NAFLD. (Hepatology 2014;59:1738–1749)

Therapeutic Failures of Antibiotics Used To Treat Macrolide-Susceptible Streptococcus pyogenes Infections May Be Due to Biofilm Formation

ABSTRACT Streptococcus pyogenes infections often fail to respond to antibiotic therapy, leading to persistent throat carriage and recurrent infections. Such failures cannot always be explained by the occurrence of antibiotic resistance determinants, and it has been suggested that S. pyogenes may enter epithelial cells to escape antibiotic treatment. We investigated 289 S. pyogenes strains isolated from different clinical sources to evaluate their ability to form biofilm as an alternative method to escape antibiotic treatment and host defenses. Up to 90% of S. pyogenes isolates, from both invasive and noninvasive infections, were able to form biofilm. Specific emm types, such as emm6, appeared to be more likely to produce biofilm, although variations within strains belonging to the same type might suggest biofilm formation to be a trait of individual strains rather than a general attribute of a serotype. Interestingly, erythromycin-susceptible isolates formed a significantly thicker biofilm than resistant isolates (P < 0.05). Among resistant strains, those carrying the erm class determinants formed a less organized biofilm than the mef(A)-positive strains. Also, prtF1 appeared to be negatively associated with the ability to form biofilm (P < 0.01). Preliminary data on a selection of strains indicated that biofilm-forming isolates entered epithelial cells with significantly lower efficiency than biofilm-negative strains. We suggest that prtF1-negative macrolide-susceptible or mef(A)-carrying isolates, which are poorly equipped to enter cells, may use biofilm to escape antimicrobial treatments and survive within the host. In this view, biofilm formation by S. pyogenes could be responsible for unexplained treatment failures and recurrences due to susceptible microorganisms.

Association between erythromycin resistance and ability to enter human respiratory cells in group A streptococci

BACKGROUND An increase in erythromycin resistance rates among group A streptococci has been reported in some European countries. These bacteria, long thought to be extracellular pathogens, can be efficiently internalised by, and survive within, human cells of respiratory-tract origin. Macrolide antibiotics enter eukaryotic cells, whereas beta-lactams are essentially confined to the extracellular fluid. A protein encoded by gene prtF1 is required for efficient entry of group A streptococci into epithelial cells. We investigated isolates of group A streptococci from children with pharyngitis in Italy for the presence of prtF1 and cell-invasion efficiency. METHODS We investigated 74 erythromycin-resistant and 52 erythromycin-susceptible isolates collected throughout Italy in 1997-98 from children with pharyngitis. Erythromycin-resistance phenotypes (constitutive, inducible, and M) were assessed by the triple-disc test and resistance determinants (ermB, ermTR, and mefA) by PCR. All strains were examined for the presence of prtF1 by PCR and for their ability to enter cultured human respiratory cells. FINDINGS The proportion of prtF1-positive strains was significantly higher among erythromycin-resistant than susceptible strains (66 [89%] vs 11 [21%]; difference 68% [95% CI 52-84]). All erythromycin-resistant strains without prtF1 were of the M phenotype. The proportion of highly cell-invasive isolates (invasion efficiency >10%) was significantly higher among erythromycin-resistant than among susceptible strains (59 [80%] vs five [10%]; difference 70% [57-83]). INTERPRETATIONS The unsuspected association between erythromycin resistance and cell invasiveness in group A streptococci raises serious concern. Strains combining erythromycin resistance and ability to enter human respiratory-tract cells may be able to escape both beta-lactams by virtue of intracellular location and macrolides by virtue of resistance.

VanA-Type Enterococci from Humans, Animals, and Food: Species Distribution, Population Structure, Tn1546 Typing and Location, and Virulence Determinants

ABSTRACT 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.

Streptococcus suis, an Emerging Drug-Resistant Animal and Human Pathogen

Streptococcus suis, a major porcine pathogen, has been receiving growing attention not only for its role in severe and increasingly reported infections in humans, but also for its involvement in drug resistance. Recent studies and the analysis of sequenced genomes have been providing important insights into the S. suis resistome, and have resulted in the identification of resistance determinants for tetracyclines, macrolides, aminoglycosides, chloramphenicol, antifolate drugs, streptothricin, and cadmium salts. Resistance gene-carrying genetic elements described so far include integrative and conjugative elements, transposons, genomic islands, phages, and chimeric elements. Some of these elements are similar to those reported in major streptococcal pathogens such as Streptococcus pyogenes, Streptococcus pneumoniae, and Streptococcus agalactiae and share the same chromosomal insertion sites. The available information strongly suggests that S. suis is an important antibiotic resistance reservoir that can contribute to the spread of resistance genes to the above-mentioned streptococci. S. suis is thus a paradigmatic example of possible intersections between animal and human resistomes.

Characterization of a Streptococcus suis tet(O/W/32/O)-Carrying Element Transferable to Major Streptococcal Pathogens

ABSTRACT Mosaic tetracycline resistance determinants are a recently discovered class of hybrids of ribosomal protection tet genes. They may show different patterns of mosaicism, but their final size has remained unaltered. Initially thought to be confined to a small group of anaerobic bacteria, mosaic tet genes were then found to be widespread. In the genus Streptococcus, a mosaic tet gene [tet(O/W/32/O)] was first discovered in Streptococcus suis, an emerging drug-resistant pig and human pathogen. In this study, we report the molecular characterization of a tet(O/W/32/O) gene-carrying mobile element from an S. suis isolate. tet(O/W/32/O) was detected, in tandem with tet(40), in a circular 14,741-bp genetic element (39.1% G+C; 17 open reading frames [ORFs] identified). The novel element, which we designated 15K, also carried the macrolide resistance determinant erm(B) and an aminoglycoside resistance four-gene cluster including aadE (streptomycin) and aphA (kanamycin). 15K appeared to be an unstable genetic element that, in the absence of recombinases, is capable of undergoing spontaneous excision under standard growth conditions. In the integrated form, 15K was found inside a 54,879-bp integrative and conjugative element (ICE) (50.5% G+C; 55 ORFs), which we designated ICESsu32457. An ∼1.3-kb segment that apparently served as the att site for excision of the unstable 15K element was identified. The novel ICE was transferable at high frequency to recipients from pathogenic Streptococcus species (S. suis, Streptococcus pyogenes, Streptococcus pneumoniae, and Streptococcus agalactiae), suggesting that the multiresistance 15K element can successfully spread within streptococcal populations.

Antimicrobial activity of essential oils and carvacrol, and synergy of carvacrol and erythromycin, against clinical, erythromycin-resistant Group A Streptococci

In the present study, we have evaluated the in vitro antibacterial activity of essential oils from Origanum vulgare, Thymus vulgaris, Lavandula angustifolia, Mentha piperita, and Melaleuca alternifolia against 32 erythromycin-resistant [Mininum Inhibitory Concentration (MIC) ≥1 μg/mL; inducible, constitutive, and efflux-mediated resistance phenotype; erm(TR), erm(B), and mef(A) genes] and cell-invasive Group A streptococci (GAS) isolated from children with pharyngotonsillitis in Italy. Over the past decades erythromycin resistance in GAS has emerged in several countries; strains combining erythromycin resistance and cell invasiveness may escape β-lactams because of intracellular location and macrolides because of resistance, resulting in difficulty of eradication and recurrent pharyngitis. Thyme and origanum essential oils demonstrated the highest antimicrobial activity with MICs ranging from 256 to 512 μg/mL. The phenolic monoterpene carvacrol [2-Methyl-5-(1-methylethyl) phenol] is a major component of the essential oils of Origanum and Thymus plants. MICs of carvacrol ranged from 64 to 256 μg/mL. In the live/dead assay several dead cells were detected as early as 1 h after incubation with carvacrol at the MIC. In single-step resistance selection studies no resistant mutants were obtained. A synergistic action of carvacrol and erythromycin was detected by the checkerboard assay and calculation of the Fractional Inhibitory Concentration (FIC) Index. A 2- to 2048-fold reduction of the erythromycin MIC was documented in checkerboard assays. Synergy (FIC Index ≤0.5) was found in 21/32 strains and was highly significant (p < 0.01) in strains where resistance is expressed only in presence of erythromycin. Synergy was confirmed in 17/23 strains using 24-h time-kill curves in presence of carvacrol and erythromycin. Our findings demonstrated that carvacrol acts either alone or in combination with erythromycin against erythromycin-resistant GAS and could potentially serve as a novel therapeutic tool.

Genetic Diversity of Cell-Invasive Erythromycin-Resistant and -Susceptible Group A Streptococci Determined by Analysis of the RD2 Region of the prtF1 Gene

ABSTRACT The RD2 region of the internalization-associated gene prtF1, which encodes the fibronectin-binding repeat domain type 2 of protein F1, plays a crucial role in the entry of group A streptococci (GAS) into epithelial cells. A molecular study of the variability of the RD2 region was carried out with 77 independent Italian GAS, 66 erythromycin resistant (ER) and 11 erythromycin susceptible (ES), which had previously been investigated for the association between erythromycin resistance and ability to enter human respiratory cells. The amplicons obtained from PCR analysis of the RD2 region were consistent with a number of RD2 repeats ranging from one to five, more frequently four (n = 30), three (n = 27), and one (n = 18). A new method to type cell-invasive GAS (RD2 typing) was developed by combining PCR analysis of the RD2 region and restriction analysis of PCR products with endonucleases HaeIII, DdeI, and HinfI. Overall, 10 RD2 types (a to j) were distinguished (all detected among the 66 ER isolates, four detected among the 11 ES isolates). Comparison and correlation of RD2 typing data with the genotype and phenotype of macrolide resistance and with data from PCR M typing and SmaI macrorestriction analysis allowed us to identify 41 different clones (31 among the 66 ER isolates and 10 among the 11 ES isolates). Three major clones accounted for 40% of the isolates (47% of ER strains). Some ES isolates appeared to be related to ER isolates with identical combinations of RD2 type and emm type. While simultaneous use of different typing methods is essential for a thorough investigation of GAS epidemiology, RD2 typing may be especially helpful in typing cell-invasive GAS.

Interspecies mobilization of an erm(T)-carrying plasmid of Streptococcus dysgalactiae subsp. equisimilis by a coresident ICE of the ICESa2603 family

OBJECTIVES The recently documented presence of almost identical, small, non-self-transmissible, erm(T)-carrying plasmids in clonally unrelated erythromycin-resistant isolates of Streptococcus pyogenes and Streptococcus agalactiae suggests that these plasmids somehow circulate in the streptococcal population. The objective of this study was to characterize the erm(T)-carrying genetic element in a clinical isolate of Streptococcus dysgalactiae subsp. equisimilis (Sde5580) and to provide a possible explanation for the spread of erm(T)-carrying plasmids in streptococci. METHODS The erm(T)-carrying element of Sde5580 was investigated by plasmid analysis, PCR experiments and sequencing. Transfer and retransfer experiments were performed using S. pyogenes, S. agalactiae and Streptococcus suis strains as recipients and by selection in the presence of suitable drug concentrations. Transconjugants were analysed by SmaI-macrorestriction analysis. Genetic studies also included PCR-restriction fragment length polymorphism analysis using HindIII endonuclease. RESULTS Sde5580 contained two mobile genetic elements: a 4950 bp erm(T)-carrying plasmid (p5580) almost identical to the non-self-transmissible erm(T)-carrying plasmids of S. pyogenes and S. agalactiae mentioned above, and an ~63 kb cadC/cadA-carrying integrative and conjugative element (ICESde3396-like) of the ICESa2603 family. p5580 was transferable at high frequency to the recipients of all three species through in trans mobilization by the coresident ICESde3396-like element. p5580 and ICESde3396-like were able to be transferred either separately or together. CONCLUSIONS This is the first evidence of horizontal transfer of an erm(T)-carrying plasmid between streptococci. In trans mobilization by coresident ICEs may be one mechanism for the spread of erm(T)-carrying plasmids in the streptococcal population.