Effectiveness of Epidermal Growth Factor Loaded Carboxymethylcellulose (EGF-CMC) Hydrogel in Biofilm Formation in Wounds of Diabetic Patients: A Randomized Clinical Trial.

Diabetic patients frequently develop wounds, which can be colonized by bacteria, mainly Staphylococcus aureus and Pseudomonas aeruginosa, with the ability to form biofilms. This study aimed to evaluate the colonization and biofilm formation of Staphylococcus aureus and Pseudomonas aeruginosa in chronic wounds of diabetic patients treated with a bioactive dressing (EGF-CMC), which consisted of a 2% carboxymethylcellulose (CMC) hydrogel loaded with epidermal growth factor (EGF). This randomized clinical trial was conducted with 25 participants: 14 treated with EGF-CMC hydrogel and 11 treated with CMC hydrogel for 12 weeks. Participants with type 2 diabetes mellitus were selected. All had diabetic foot ulcers or chronic venous ulcers. Swab collections were performed on weeks 1, 6, and 12. The laboratory analyses included the identification of strains, microbial quantification, virulence gene investigation, and the evaluation of biofilm formation. In total, 13 S. aureus strains and 15 P. aeruginosa strains were isolated. There were no statistically significant differences regarding bacterial loads and virulence genes. However, EGF-CMC-hydrogel-treated wounds were colonized by strains with lower biofilm formation abilities. The probability of isolating biofilm-producing strains from CMC-hydrogel-treated wounds was 83% greater than the probability of isolating biofilm-producing strains from EGF-CMC-treated wounds.

Antibiofilm activity of bromelain from pineapple against Staphylococcus aureus

Bromelain is a set of proteolytic enzymes usually obtained from pineapple (Ananas comosus). Although bromelain has distinguished therapeutic properties, little is known about its proteolytic potential against opportunistic pathogens related to wound healing complications, such as Staphylococcus aureus. This study aimed toinvestigate the antibiofilm and antibacterial activity of bromelain in 43 clinical strains of S. aureusisolated from chronic wounds and blood cultures. Bromelain's activity against S. aureusbiofilm in vitrowas assessed by analyzing biofilm formation in cultures grownin the presence of 1% bromelain and biofilm destruction after the addition of 1% bromelain to mature biofilms. Proteinase K and sodium metaperiodate were also added to mature biofilms in parallel to compare their activity with that of bromelain and, together with exopolysaccharide and protein production rate assays, to determine the chemical composition of the biofilm extracellular matrix of selected strains of S. aureus. Bromelain was also evaluated for its DNase activity and impact on cellular hydrophobicity and auto-aggregation. Mueller-Hinton agar dilution was used to determine bromelain minimal inhibitory concentration (MIC). Biofilm assays showed that 1% bromelain significantly inhibits S. aureusbiofilm formation (p= 0.0157) by up to 4-fold and destroys its mature biofilms (p < 0.0001) by up to 6.4-fold, both compared to the control grown without bromelain. Biofilms of methicillin-resistant S. aureusstrains isolated from chronic wounds were the most affected by bromelain treatment. No antibacterial activity was detected with bromelain MIC assays and the proteolytic activity of bromelain was identified as the main antibiofilm mechanism of the enzyme, though its DNase activity may also contribute. The epithelial therapeutic properties of bromelain combined with its antibiofilm activity against S. aureusmake it a promising alternative to compose the therapeutic arsenal for the control of S. aureusbiofilms in the context of wound care.(AU)

Bioactive small molecules produced by the human gut microbiome modulate Vibrio cholerae sessile and planktonic lifestyles.

Humans live in symbiosis with a diverse community of microorganisms, which has evolved to carry out many specific tasks that benefit the host, including protection against invading pathogens. Within the chemical diversity of the gastrointestinal tract, small molecules likely constitute chemical cues for the communication between the microbiota and pathogens. Therefore, we sought to investigate if molecules produced by the human gut microbiota show biological activity against the human pathogen Vibrio cholerae. To probe the effects of the gut metabolome on V. cholerae, we investigated its response to small-molecule extracts from human feces, from a complex bacterial community cultivated in vitro, and from culture supernatants of Enterocloster citroniae, Bacteroides thetaiotaomicron, and Bacteroides vulgatus. Using RNA sequencing, we determined the impact of the human gut metabolome on V. cholerae global gene expression. Among the genes downregulated in the presence of the fecal extract, the most overrepresented functional category was cell motility, which accounted for 39% of repressed genes. Repression of V. cholerae motility by the fecal extract was confirmed phenotypically, and E. citroniae extracts reproduced this phenotype. A complex in vitro microbial community led to increased motility, as did extracts from B. vulgatus, a species present in this community. Accordingly, mucin penetration was also repressed by fecal and E. citroniae extracts, suggesting that the phenotypes observed may have implications for host colonization. Together with previous studies, this work shows that small molecules from the gut metabolome may have a widespread, significant impact on microbe-microbe interactions established in the gut environment.

Lactoferrin and lactoferricin B reduce adhesion and biofilm formation in the intestinal symbionts Bacteroides fragilis and Bacteroides thetaiotaomicron.

Several factors affect the composition of species that inhabit our intestinal tract, including mode of delivery, genetics and nutrition. Antimicrobial peptides and proteins secreted in the gastrointestinal tract are powerful tools against bacteria. Lactoferrin (LF) inhibits the growth of several bacterial species, such as Enterobacteriaceae, but may stimulate probiotic bacteria. Activity of LF against gut symbiotic species of the Bacteroides genus could give us insights on how these species colonize the gut. We investigated the effects of the antimicrobial protein lactoferrin and its derived peptide, lactoferricin B on two species of strict anaerobes, opportunistic pathogens that cause diseases in both adults and children, commonly found in the microbiota of the human gastrointestinal tract, Bacteroides fragilis and B. thetaiotaomicron., In vitro biofilm formation and binding to laminin were strongly inhibited by a low concentration of lactoferrin (12.5 µg/ml). Conversely, the growth of the strains in a micro-dilution assay in minimal media with different iron sources was not affected by physiological concentrations (2 mg/ml) of apo-lactoferrin or holo-lactoferrin. The combination of lactoferrin with antibiotics in synergism assays was also negative. The lactoferricin B fragment was also unable to inhibit growth in a similar test with concentrations of up to 32 µg/ml. Resistance to lactoferrin could confer an advantage to these species, even when high amount of this protein is present in the gastrointestinal tract. However, colonization is hampered by the binding and biofilm inhibitiory effect of lactoferrin, which may explain the low prevalence of Bacteroides in healthy babies. Resistance to this antimicrobial protein may help understand the success of these opportunistic pathogens during infection in the peritoneum.

Impact of violacein from Chromobacterium violaceum on the mammalian gut microbiome.

Violacein is a violet pigment produced by Chromobacterium violaceum that possesses several functions such as antibacterial, antiviral, antifungal, and antioxidant activities. The search for potential compounds and therapies that may interfere with and modulate the gut microbial consortia without causing severe damage and increased resistance is important for the treatment of inflammatory, allergic, and metabolic diseases. The aim of the present work was to evaluate the ability of violacein to change microbial patterns in the mammalian gut by favoring certain groups over the others in order to be used as a therapy for diseases associated with changes in the intestinal microflora. To do this, we used male Wistar rats, and administered violacein orally, in low (50 µg/ml) and high (500 µg/ml) doses for a month. Initially, the changes in the microbial diversity were observed by DGGE analyses that showed that the violacein significantly affects the gut microbiota of the rats. Pyrosequencing of 16S rDNA was then employed using a 454 GS Titanium platform, and the results demonstrated that higher taxonomic richness was observed with the low violacein treatment group, followed by the control group and high violacein treatment group. Modulation of the microbiota at the class level was observed in the low violacein dose, where Bacilli and Clostridia (Firmicutes) were found as dominant. For the high violacein dose, Bacilli followed by Clostridia and Actinobacteria were present as the major components. Further analyses are crucial for a better understanding of how violacein affects the gut microbiome and whether this change would be beneficial to the host, providing a framework for the development of alternative treatment strategies for intestinal diseases using this compound.

Inactivation of MarR gene homologs increases susceptibility to antimicrobials in Bacteroides fragilis

Bacteroides fragilis is the strict anaerobic bacteria most commonly found in human infections, and has a high mortality rate. Among other virulence factors, the remarkable ability to acquire resistance to a variety of antimicrobial agents and to tolerate nanomolar concentrations of oxygen explains in part their success in causing infection and colonizing the mucosa. Much attention has been given to genes related to multiple drug resistance derived from plasmids, integrons or transposon, but such genes are also detected in chromosomal systems, like the mar (multiple antibiotic resistance) locus, that confer resistance to a range of drugs. Regulators like MarR, that control expression of the locus mar, also regulate resistance to organic solvents, disinfectants and oxygen reactive species are important players in these events. Strains derived from the parental strain 638R, with mutations in the genes hereby known as marRI (BF638R_3159) and marRII (BF638R_3706) were constructed by gene disruption using a suicide plasmid. Phenotypic response of the mutant strains to hydrogen peroxide, cell survival assay against exposure to oxygen, biofilm formation, resistance to bile salts and resistance to antibiotics was evaluated. The results showed that the mutant strains exhibit statistically significant differences in their response to oxygen stress, but no changes were observed in survival when exposed to bile salts. Biofilm formation was not affected by either gene disruption. Both mutant strains however, became more sensitive to multiple antimicrobial drugs tested. This indicates that as observed in other bacterial species, MarR are an important resistance mechanism in B. fragilis.(AU)

Inactivation of MarR gene homologs increases susceptibility to antimicrobials in Bacteroides fragilis

ABSTRACT Bacteroides fragilis is the strict anaerobic bacteria most commonly found in human infections, and has a high mortality rate. Among other virulence factors, the remarkable ability to acquire resistance to a variety of antimicrobial agents and to tolerate nanomolar concentrations of oxygen explains in part their success in causing infection and colonizing the mucosa. Much attention has been given to genes related to multiple drug resistance derived from plasmids, integrons or transposon, but such genes are also detected in chromosomal systems, like the mar (multiple antibiotic resistance) locus, that confer resistance to a range of drugs. Regulators like MarR, that control expression of the locus mar, also regulate resistance to organic solvents, disinfectants and oxygen reactive species are important players in these events. Strains derived from the parental strain 638R, with mutations in the genes hereby known as marRI (BF638R_3159) and marRII (BF638R_3706) were constructed by gene disruption using a suicide plasmid. Phenotypic response of the mutant strains to hydrogen peroxide, cell survival assay against exposure to oxygen, biofilm formation, resistance to bile salts and resistance to antibiotics was evaluated. The results showed that the mutant strains exhibit statistically significant differences in their response to oxygen stress, but no changes were observed in survival when exposed to bile salts. Biofilm formation was not affected by either gene disruption. Both mutant strains however, became more sensitive to multiple antimicrobial drugs tested. This indicates that as observed in other bacterial species, MarR are an important resistance mechanism in B. fragilis.

Inactivation of MarR gene homologs increases susceptibility to antimicrobials in Bacteroides fragilis.

Bacteroides fragilis is the strict anaerobic bacteria most commonly found in human infections, and has a high mortality rate. Among other virulence factors, the remarkable ability to acquire resistance to a variety of antimicrobial agents and to tolerate nanomolar concentrations of oxygen explains in part their success in causing infection and colonizing the mucosa. Much attention has been given to genes related to multiple drug resistance derived from plasmids, integrons or transposon, but such genes are also detected in chromosomal systems, like the mar (multiple antibiotic resistance) locus, that confer resistance to a range of drugs. Regulators like MarR, that control expression of the locus mar, also regulate resistance to organic solvents, disinfectants and oxygen reactive species are important players in these events. Strains derived from the parental strain 638R, with mutations in the genes hereby known as marRI (BF638R_3159) and marRII (BF638R_3706) were constructed by gene disruption using a suicide plasmid. Phenotypic response of the mutant strains to hydrogen peroxide, cell survival assay against exposure to oxygen, biofilm formation, resistance to bile salts and resistance to antibiotics was evaluated. The results showed that the mutant strains exhibit statistically significant differences in their response to oxygen stress, but no changes were observed in survival when exposed to bile salts. Biofilm formation was not affected by either gene disruption. Both mutant strains however, became more sensitive to multiple antimicrobial drugs tested. This indicates that as observed in other bacterial species, MarR are an important resistance mechanism in B. fragilis.

Effect of hospital disinfectants on spores of clinical Brazilian Clostridium difficile strains.

The aim of this study was to evaluate the sporicidal activity of hospital disinfectants against spores of two Brazilian Clostridium difficile ribotypes and the BI/NAP1/027. Our results showed that CloroRio(®) and Cidex Opa(®) were the most efficient agents for eliminating spores of C difficile.