Microscopic Examination of Biofilm Destruction by Atmospheric Pressure Plasma

Objective: The aim of this study is to examine microscopically the destruction of bacterial biofilms mediated by atmospheric pressure non-thermal plasma (APNTP) at cellular level as well as at the level of biofilm structure as a whole. Methods: 3-day old bacterial biofilms were grown on polycarbonate coupons in a dual channel flow cell and were treated with an in-housed designed atmospheric pressure non-thermal plasma jet for up to 4 minutes of exposure before being examined by both confocal laser scanning microscopy (CLSM), preceded by Live/Dead bacterial viability staining, and scanning electron microscopy (SEM). Results: Differential live/dead staining followed by confocal microscopy examination revealed that biofilm eradication by APNTP was mediated by varying levels of both cell killing and physical removal. Relative extent of each mechanism was dependent on plasma operating conditions, bacterial species, growth conditions and biofilm thickness. On the other hand, SEM examination of plasma-exposed biofilms revealed a series of morphological changes exhibited by biofilm cells ranging from increased roughness of cell surface to complete cell lysis. Conclusions: Interesting mechanistic insights have been revealed by microscopic examination of plasma-treated bacterial biofilms that, when coupled with more specific biochemical studies, will not only contribute significantly to our understanding of the mechanism of plasma mediated biofilm destruction but also will help in better application-guided development of this novel anti-biofilm approach.

Treatment of a cochlear implant biofilm infection: a potential role for alternative antimicrobial agents

OBJECTIVE:
This study aimed to investigate antimicrobial treatment of an infected cochlear implant, undertaken in an attempt to salvage the infected device.

METHODS:
We used the broth microdilution method to assess the susceptibility of meticillin-sensitive Staphylococcus aureus isolate, cultured from an infected cochlear implant, to common antimicrobial agents as well as to novel agents such as tea tree oil. To better simulate in vivo conditions, where bacteria grow as microcolonies encased in glycocalyx, the bactericidal activity of selected antimicrobial agents against the isolate growing in biofilm were also compared.

RESULTS:
When grown planktonically, the S aureus isolate was susceptible to 17 of the 18 antimicrobials tested. However, when grown in biofilm, it was resistant to all conventional antimicrobials. In contrast, 5 per cent tea tree oil completely eradicated the biofilm following exposure for 1 hour.

CONCLUSION:
Treatment of infected cochlear implants with novel agents such as tea tree oil could significantly improve salvage outcome.

The In Vitro Susceptibility of Biofilm Forming Medical Device Related Pathogens to Conventional Antibiotics

Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and minimum biofilm eradication concentration (MBEC) and kill kinetics were established for vancomycin, rifampicin, trimethoprim, gentamicin, and ciprofloxacin against the biofilm forming bacteria Staphylococcus epidermidis (ATCC 35984), Staphylococcus aureus (ATCC 29213), Methicillin Resistant Staphylococcus aureus (MRSA) (ATCC 43300), Pseudomonas aeruginosa (PAO1), and Escherichia coli (NCTC 8196). MICs and MBCs were determined via broth microdilution in 96-well plates. MBECs were studied using the Calgary Biofilm Device. Values obtained were used to investigate the kill kinetics of conventional antimicrobials against a range of planktonic and biofilm microorganisms over a period of 24 hours. Planktonic kill kinetics were determined at 4xMIC and biofilm kill kinetics at relative MBECs. Susceptibility of microorganisms varied depending on antibiotic selected and phenotypic form of bacteria. Gram-positive planktonic isolates were extremely susceptible to vancomycin (highest MBC: 7.81 mg L−1: methicillin sensitive and resistant S. aureus) but no MBEC value was obtained against all biofilm pathogens tested (up to 1000 mg L−1). Both gentamicin and ciprofloxacin displayed the broadest spectrum of activity with MIC and MBCs in the mg L−1 range against all planktonic isolates tested and MBEC values obtained against all but S. epidermidis (ATCC 35984) and MRSA (ATCC 43300).

Biomolecular Mechanisms of Pseudomonas aeruginosa and Escherichia coli Biofilm Formation

Pseudomonas aeruginosa and Escherichia coli are the most prevalent Gram-negative biofilm forming medical device associated pathogens, particularly with respect to catheter associated urinary tract infections. In a similar manner to Gram-positive bacteria, Gram-negative biofilm formation is fundamentally determined by a series of steps outlined more fully in this review, namely adhesion, cellular aggregation, and the production of an extracellular polymeric matrix. More specifically this review will explore the biosynthesis and role of pili and flagella in Gram-negative adhesion and accumulation on surfaces in Pseudomonas aeruginosa and Escherichia coli. The process of biofilm maturation is compared and contrasted in both species, namely the production of the exopolysaccharides via the polysaccharide synthesis locus (Psl), pellicle Formation (Pel) and alginic acid synthesis in Pseudomonas aeruginosa, and UDP-4-amino-4-deoxy-l-arabinose and colonic acid synthesis in Escherichia coli. An emphasis is placed on the importance of the LuxR homologue sdiA; the luxS/autoinducer-II; an autoinducer-III/epinephrine/norepinephrine and indole mediated Quorum sensing systems in enabling Gram-negative bacteria to adapt to their environments. The majority of Gram-negative biofilms consist of polysaccharides of a simple sugar structure (either homo- or heteropolysaccharides) that provide an optimum environment for the survival and maturation of bacteria, allowing them to display increased resistance to antibiotics and predation.

Biofilm Eradication Kinetics of the Ultrashort Lipopeptide C12-OOWW-NH2 Utilizing a Modified MBEC Assay™

In this study, we report the antimicrobial planktonic and biofilm kill kinetics of ultrashort cationic lipopeptides previously demonstrated by our group to have a minimum biofilm eradication concentration (MBEC) in the microgram per mL (μg/mL) range against clinically relevant biofilm-forming micro-organisms. We compare the rate of kill for the most potent of these lipopeptides, dodecanoic (lauric) acid-conjugated C₁₂-Orn-Orn-Trp-Trp-NH₂ against the tetrapeptide amide H-Orn-Orn-Trp-Trp-NH₂ motif and the amphibian peptide Maximin-4 via a modification of the MBEC Assay™ for Physiology & Genetics (P&G). Improved antimicrobial activity is achieved upon N-terminal lipidation of the tetrapeptide amide. Increased antimicrobial potency was demonstrated against both planktonic and biofilm forms of Gram-positive micro-organisms. We hypothesize rapid kill to be achieved by targeting of microbial membranes. Complete kill against established 24-h Gram-positive biofilms occurred within 4 h of exposure to C₁₂-OOWW-NH₂ at MBEC values [methicillin-resistant Staphylococcus epidermidis (ATCC 35984): 15.63 μg/mL] close to the values for the planktonic minimum inhibitory concentration (MIC) [methicillin-resistant Staphylococcus epidermidis (ATCC 35984): 1.95 μg/mL]. Such rapid kill, especially against sessile biofilm forms, is indicative of a reduction in the likelihood of resistant strains developing with the potential for quicker resolution of pathogenic infection. Ultrashort antimicrobial lipopeptides have high potential as antimicrobial therapy.