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.

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.

Clostridium difficile infection among immunocompromised patients in Rio de Janeiro, Brazil and detection of moxifloxacin resistance in a ribotype 014 strain.

Clostridium difficile is a Gram-positive spore forming anaerobic bacterium, often associated with nosocomial diarrhea and pseudomembranous colitis. The acquisition of this organism occurs primarily in hospitals through accidental ingestion of spores, and its establishment and proliferation in the colon results from the removal of members of the normal intestinal flora during or after antibiotic therapy. In this study, stool samples from patients admitted to the University Hospital Clementino Fraga Filho (HUCCF/UFRJ) were screened for C. difficile toxins with an ELISA test and cultured with standard techniques for C. difficile isolation. A total of 74 stool samples were collected from patients undergoing antibiotic therapy between August 2009 and November 2010, only two (2.7%) were positive in the ELISA test and culture. A third isolate was obtained from a negative ELISA test sample. All cases of CDI were identified in patients with acute lymphoid or myeloid leukemia. Genotypic and phenotypic characterization showed that all strains carried toxins A and B genes, and belonged to PCR-ribotypes 014, 043 and 046. The isolated strains were sensitive to metronidazole and vancomycin, and resistant to ciprofloxacin and levofloxacin. Resistance to moxifloxacin, was present in the strain from PCR-ribotype 014, that showed an amino acid substitution in gyrB gene (Asp 426 â†’ Asn). This is the first time that this mutation in a PCR-ribotype 014 strain has been described in Brazil.