Ability of Three Lactic Acid Bacteria to Grow in Sessile Mode and to Inhibit Biofilm Formation of Pathogenic Bacteria.

In this study, we explored the effect of three lactic acid bacteria (LAB), i.e. Enterococcus sp CM9, Enterococcus sp CM18 and Enterococcus faecium H3, and their supernatants, on seven biofilm-forming pathogenic strains isolated from human urinary tract or nose infections. By quantitative biofilm production assay, a strong adherence ability of Enterococcus sp CM9 and Enterococcus sp CM18 was revealed while E. faecium H3 resulted to be moderately adherent. Inhibition tests demonstrated an antimicrobial activity of LAB against pathogens.The presence of cell free supernatant (CFS) of CM9 and CM18 strains significantly decreased the adhesion of S. aureus 10,850, S. epidermidis 4,296 and E. coli FSL24. The CFS of H3 strain was effective against S. epidermidis 4,296 and P. aeruginosa PA1FSL biofilms only. Biofilm formation of K. pneumoniae Kp20FSL, A. baumannii AB8FSL and ESBL+ E. coli FS101570 have not been affected by any CSF while P. aeruginosa PA1FSL biofilm increase in presence of CM9 and CM18 CFS.Confocal Laser Scanning Microscopy revealed that K. pneumoniae Kp20FSL biofilm was inhibited by Enterococcus sp CM9, when grown together.Our results suggest that the LAB strains and/or their bacteriocins can be considered as potential tools to control biofilm formation of some bacterial pathogens.

Biofilm formation and antibiotic resistance in Klebsiella pneumoniae urinary strains.

AIMS: Multidrug-resistant Klebsiella pneumoniae has become a relevant healthcare-associated pathogen. Capsule, type 1 and 3 fimbriae (mrkA gene), type 2 quorum-sensing system (luxS), synthesis of D-galactan I (wbbM), LPS transport (wzm) and poly-beta-1,6-N-acetyl-D-glucosamine (pgaA) seem involved in K. pneumoniae biofilm. Nonenzymatic antibiotic resistance is related to nonexpression or mutation of porins (OmpK35 and OmpK36), and efflux pump (acrB) overexpression. The aim of this study was to analyse some virulence factors of K. pneumoniae isolates, and to evaluate possible correlations between their antibiotic resistance profile and ability to form biofilm. METHODS AND RESULTS: Quantitative biofilm production assay, congo red agar test and string test were performed on 120 isolates clustered in 56 extensively drug-resistant (XDR), 40 MDR and 24 susceptible (S) strains. Nine representative strains were analysed by real-time RT-PCR for the expression of antibiotic resistance (OmpK35, OmpK36, acrB) and biofilm production genes (mrkA, luxS, pga, wbbM, wzm) during planktonic and sessile growth. XDR isolates showed a higher ability to form biofilm (91·07%) and to produce polysaccharides (78·57%) when compared to MDR and S strains. In biofilm-growing XDR strains, seven of eight genes were upregulated, with the only exception of OmpK36. CONCLUSIONS: XDR strains exhibited phenotypic and genotypic features supporting a significant growth as biofilm. SIGNIFICANCE AND IMPACT OF THE STUDY: This study produces new findings that highlight a positive correlation between antibiotic resistance profile and biofilm-forming ability in XDR K. pneumoniae strains. These new evidences might contribute to the progress in selection of therapeutic treatments of infections caused by K. pneumoniae resistant also to the 'last line of defence' antibiotics, that is, carbapenems.

Lactobacillus brevis CD2 inhibits Prevotella melaninogenica biofilm.

OBJECTIVE: To evaluate the ability of the probiotic strain Lactobacillus brevis CD2 to inhibit the opportunistic anaerobe Prevotella melaninogenica (PM1), a well-known causative agent of periodontitis. MATERIALS AND METHODS: The inhibitory effect of Lactobacillus CD2 on Prevotella PM1 biofilm was assessed both by exposing the anaerobe to the supernatant of the probiotic strain and by growing the two strains to obtain single or mixed biofilms. The inhibitory effect of CD2 on PM1 was also checked by the agar overlay method. RESULTS: The development of PM1 biofilm was strongly affected (56% decrease in OD value) by the CD2 supernatant after 96 h. A dose-dependent biofilm reduction was also observed at 1/10 and 1/100 dilutions of supernatant. Confocal microscopy on the mixed biofilms revealed the ability of CD2 to prevail on PM1, greatly reducing the biofilm of the latter. CONCLUSIONS: It has been hypothesized a multifactorial nature of the inhibition mechanism, the strong adherence ability of CD2 strain together with the released metabolites presumably contributing to the reduction in the PM1 biofilm detected by confocal microscopy.

Antibiotic pressure can induce the viable but non-culturable state in Staphylococcus aureus growing in biofilms.

OBJECTIVES: Staphylococcal biofilms are among the main causes of chronic implant-associated infections. We have recently suggested that their transformation into viable but non-culturable (VBNC) forms (i.e. forms capable of resuscitation) could be responsible for the recurrent symptoms. This work aims to establish whether Staphylococcus aureus biofilms can give rise to VBNC forms capable of being resuscitated in suitable environmental conditions, the role of different stressors in inducing the VBNC state and the conditions favouring resuscitation. METHODS: S. aureus 10850 biofilms were exposed to different concentrations of antibiotic (vancomycin or quinupristin/dalfopristin) and/or to nutrient depletion until loss of culturability. The presence of viable cells and their number were examined by epifluorescence microscopy and flow cytometry. Gene expression was measured by real-time PCR. Resuscitation ability was tested by growth in rich medium containing antioxidant factors. RESULTS: Viable subpopulations were detected in all non-culturable biofilms. However, viable cell numbers and gene expression remained constant for 150 days from loss of culturability in cells from antibiotic-exposed biofilms, but not in those that had only been starved. Resuscitation was obtained in rich medium supplemented with 0.3% sodium pyruvate or with 50% filtrate of a late-log culture. CONCLUSIONS: Our findings demonstrate that S. aureus can enter the VBNC state in infectious biofilms. The presence of vancomycin or quinupristin/dalfopristin can inadvertently induce a true VBNC state or its persistence in S. aureus cells embedded in biofilms, supporting previous findings on the role of staphylococcal biofilms in recurrent infections.