Effect of sub-inhibitory concentrations of cefepime on biofilm formation by Pseudomonas aeruginosa

This study investigated the effect of cefepime at sub-minimum inhibitory concentrations (sub-MICs) on in vitro biofilm formation (BF) by clinical isolates of Pseudomonas aeruginosa. The effect of cefepime at sub-MIC levels (½–1/256 MIC) on in vitro BF by six clinical isolates of P. aeruginosa was phenotypically assessed following 24 and 48 h of challenge using the tissue culture plate (TCP) assay. Quantitative real-time polymeric chain reaction (qRT-PCR) was employed to observe the change in expression of three biofilm-related genes, namely, a protease-encoding gene (lasA), fimbrial protein-encoding gene (cupA1), and alginate-encoding gene (algC), in a weak biofilm-producing strain of P. aeruginosa following 24 and 48 h of challenge with sub-MICs of cefepime. The BF morphology in response to cefepime was imaged using scanning electron microscopy (SEM). The TCP assay showed strain-, time-, and concentration-dependent changes in in vitro BF in P. aeruginosa following challenge with sub-MICs of cefepime, with a profound increase in strains with inherently no or weak biofilm-producing ability. RT-PCR revealed time-dependent upregulation in the expression of the investigated genes following challenge with ½ and ¼ MIC levels, as confirmed by SEM. Cefepime at sub-MICs could upregulate the expression of BF-related genes and enhance BF by P. aeruginosa clinical isolates.

Porphyromonas gingivalis fimbrial protein Mfa5 contains a von Willebrand factor domain and an intramolecular isopeptide

The Gram-negative bacterium Porphyromonas gingivalis is a secondary colonizer of the oral biofilm and is involved in the onset and progression of periodontitis. Its fimbriae, of type-V, are important for attachment to other microorganisms in the biofilm and for adhesion to host cells. The fimbriae are assembled from five proteins encoded by the mfa1 operon, of which Mfa5 is one of the ancillary tip proteins. Here we report the X-ray structure of the N-terminal half of Mfa5, which reveals a von Willebrand factor domain and two IgG-like domains. One of the IgG-like domains is stabilized by an intramolecular isopeptide bond, which is the first such bond observed in a Gram-negative bacterium. These features make Mfa5 structurally more related to streptococcal adhesins than to the other P. gingivalis Mfa proteins. The structure reported here indicates that horizontal gene transfer has occurred among the bacteria within the oral biofilm.

A rapid method for the detection of motility in Acinetobacter baumannii and its association to the existence of the virulence-associated genes pilA and algW

Acinetobacter (A.) baumannii is one of the major nosocomial pathogens worldwide. It is associated with bloodstream infection, pneumonia, meningitis, urinary tract, soft tissue, and wound infections. Several factors contribute to its survival and spread as a nosocomial pathogen, and motility is often associated with the virulence, fitness, and tenacity of A. baumannii on surfaces. In the present study, the correlation between the presence of genes encoding for fimbrial protein PilA and periplasmic protease AlgW and motility was investigated in 87 clinical and non-clinical A. baumannii isolates from Germany. A. baumannii exhibited robust swimming, swarming, and twitching movement based on the percentage of agar in the medium, as well as the time and temperature of incubation. The swarm motility medium utilizing 2% agar with tetrazolium salts provided an efficient assay for the phenotypic characterization of A. baumannii and it was more efficient than the classical motility assays in terms of time, visibility, and biosafety. The presence of the pilA gene increased motility of A. baumannii but was not required for motility. The algW gene was found in 18 strains obtained from milk, all of them with proven phenotypic motility. The rapid detection of motility is essential to evaluate the virulence and fitness of A. baumannii. Further studies on the level of genome, transcriptome and proteome are needed to investigate the secrets behind different movement paths in each strain.

Assessment of CafA Targeted BAR-Encapsulated Nanoparticles against Oral Biofilms

Porphyromonas gingivalis adherence to Streptococcus gordonii is a crucial initial event that facilitates the colonization of P. gingivalis, a key pathogen in periodontal disease. As such, blocking these early interactions may present a potential avenue to limit P. gingivalis colonization. Nanoparticles encapsulating a synthetic peptide BAR (BAR-encapsulated NPs) inhibit P. gingivalis/S. gordonii biofilm formation 1.8-fold more potently relative to free BAR. However, BAR-encapsulated NPs, like many orally delivered formulations, may benefit from a strategy that improves their retention in an open flow environment. Here, we sought to enhance the efficacy of BAR-encapsulated NPs by modifying their surfaces with coaggregation factor A (CafA), a fimbrial protein expressed by the early colonizer, Actinomyces oris. We demonstrate that the targeting moiety, CafA, enhances NP binding and exhibits specificity of adherence to S. gordonii, relative to other oral bacterial species. Furthermore, CafA-modified NPs release inhibitory concentrations of BAR for 12 h, a time frame relevant to oral dosage form delivery. Lastly, CafA-modified NPs potently inhibit P. gingivalis/S. gordonii biofilm formation for up to 12 h and are non-toxic at therapeutically-relevant concentrations. These results suggest that CafA-modified NPs represent a novel and efficacious delivery vehicle for localized, targeted delivery of BAR to P. gingivalis preferred niches.