Bacteria that attach to surfaces aggregate in a hydrated polymeric matrix of their own synthesis to form biofilms. Formation of these sessile communities and their inherent resistance to antimicrobial agents are at the root of many persistent and chronic bacterial infections. Studies of biofilms have revealed differentiated, structured groups of cells with community properties. Recent advances in our understanding of the genetic and molecular basis of bacterial community behavior point to therapeutic targets that may provide a means for the control of biofilm infections.

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Bacterial biofilms : A common cause of persistent infections

https://www.thelancet.com/pdfs/journals/lancet/PIIS0140-6736(01)05321-1.pdf

Antibiotic resistance of bacteria in biofilms

https://www.cell.com/trends/microbiology/pdf/S0966-842X(00)01913-2.pdf

Mechanisms of biofilm resistance to antimicrobial agents

The study of microbial communities has been revolutionised in recent years by the widespread adoption of culture independent analytical techniques such as 16S rRNA gene sequencing and metagenomics. One potential confounder of these sequence-based approaches is the presence of contamination in DNA extraction kits and other laboratory reagents. In this study we demonstrate that contaminating DNA is ubiquitous in commonly used DNA extraction kits and other laboratory reagents, varies greatly in composition between different kits and kit batches, and that this contamination critically impacts results obtained from samples containing a low microbial biomass. Contamination impacts both PCR-based 16S rRNA gene surveys and shotgun metagenomics. We provide an extensive list of potential contaminating genera, and guidelines on how to mitigate the effects of contamination. These results suggest that caution should be advised when applying sequence-based techniques to the study of microbiota present in low biomass environments. Concurrent sequencing of negative control samples is strongly advised.

/pdf/reagent-and-laboratory-contamination-can-critically-impact-3f8o71vvck.pdf

Reagent and laboratory contamination can critically impact sequence-based microbiome analyses

According to a public announcement by the US National Institutes of Health
 , “Biofilms are medically important, accounting for over 80% of microbial infections in the body”. Yet bacterial biofilms remain poorly understood and strategies for their control remain underdeveloped. Standard antimicrobial treatments typically fail to eradicate biofilms, which can result in chronic infection and the need for surgical removal of afflicted areas. The need to create effective therapies to counter biofilm infections presents one of the most pressing challenges in anti-bacterial drug development. In this article, the mechanisms that underlie biofilm resistance to antimicrobial chemotherapy will be examined, with particular attention being given to potential avenues for the effective treatment of biofilms.

/pdf/understanding-biofilm-resistance-to-antibacterial-agents-pnlgfboaq5.pdf

Understanding biofilm resistance to antibacterial agents.

Chambers with membrane-filter side walls were designed for studies of the survival of coliform bacteria in natural and artificial waters. Experiments were carried out in the field and in the laboratory. The initial uptake rate of inorganic ions, total carbon, and glucose into the chamber was greater than twice as fast as the accumulation of each into dialysis tubing. When the survival of a water-isolated fecal coliform bacterium was examined in two adjacent mountain streams, it was found that the organism persisted longer in Bozeman Creek than in Middle Creek. These data may be a reflection of the water chemistry because the concentration of inorganic constituents of the former was greater. Laboratory studies of the survival of a fecal coliform bacterium in artificial and natural water with continuous flow were used to determine the effect of chemical composition, temperature, and pH. The relation of this type of data to the use of fecal coliform bacteria as indicators of health hazard in water is discussed.

/pdf/survival-of-coliform-bacteria-in-natural-waters-field-and-38ebx1z7a6.pdf

Survival of Coliform Bacteria in Natural Waters: Field and Laboratory Studies with Membrane-Filter Chambers

Biofilm resistance to antimicrobial agents.

The comparative survival of various fecal indicator bacteria and enteric pathogens was studied in a stable well water supply by using membrane chambers There was more variation in the 29 coliform cultures and they died more rapidly, as a group, than the 20 enterococcus cultures that were examined The comparative survival of the organisms tested follows: Aeromonas sp > the shigellae (Shigella flexneri, S sonnei, and S dysenteriae) > fecal streptococci > coliforms = some salmonellae (Salmonella enteritidis ser paratyphi A and D, S enteritidis ser typhimurium) > Streptococcus equinus > Vibrio cholerae > Salmonella typhi > Streptococcus bovis > Salmonella enteritidis ser paratyphi B S bovis had a more rapid die-off than did S equinus, but both had significantly shorter half-lives than the other streptococci The natural populations of indicator bacteria from human and elk fecal material declined similarly to the pure cultures tested, whereas the die-off of fecal streptococci exceeded the coliforms from bovine fecal material

Comparative survival of indicator bacteria and enteric pathogens in well water.

Fluorescent stains in conjunction with cryoembedding and image analysis were applied to demonstrate spatial gradients in respiratory activity within bacterial biofilms during disinfection with monochloramine. Biofilms of Klebsiella pneumoniae and Pseudomonas aeruginosa grown together on stainless steel surfaces in continuous-flow annular reactors were treated with 2 mg of monochloramine per liter (influent concentration) for 2 h. Relatively little biofilm removal occurred as evidenced by total cell direct counts. Plate counts (of both species summed) indicated an average 1.3-log decrease after exposure to 2 mg of monochloramine per liter. The fluorogenic redox indicator 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and the DNA stain 4',6-diamidino-2-phenylindole (DAPI) were used to differentiate respiring and nonrespiring cells in biofilms. Epifluorescence micrographs of frozen biofilm cross sections clearly revealed gradients of respiratory activity within biofilms in response to monochloramine treatment. These gradients in specific respiratory activity were quantified by calculating the ratio of CTC and DAPI intensities measured by image analysis. Cells near the biofilm-bulk fluid interface lost respiratory activity first. After 2 h of biocide treatment, greater respiratory activity persisted deep in the biofilm than near the biofilm-bulk fluid interface.

Nonuniform spatial patterns of respiratory activity within biofilms during disinfection.

W. L. COCHRAN, G. A. McFETERS and P. S. STEWART.2000. Pseudomonas aeruginosa attached to alginate gel beads in sparse, thin biofilms exhibited reduced susceptibility to monochloramine and hydrogen peroxide compared with planktonic cells of the same micro-organism. Disinfection rate coefficients for planktonic bacteria averaged 0·55 l mg−1 min−1 for monochloramine and 3·1 × 10−4 l mg−1 min−1 for hydrogen peroxide. The corresponding values for 24-h-old biofilm cells were 0·29 l mg min−1 and 9·2 × 10−5 l mg−1 min−1 for monochloramine and hydrogen peroxide, respectively. Several pieces of evidence support the interpretation that the reduced susceptibility of biofilm was not due simply to inadequate delivery of the antimicrobial agent to the local environment of the attached cells. No correlation between biofilm susceptibility and biofilm initial areal cell density was observed. Rapid delivery of hydrogen peroxide to the attachment surface, and subsequently to the interior, of the alginate gel beads was visualized by a direct experimental technique. Theoretical analysis of unsteady diffusion and diffusion–reaction interactions also argued against any significant delay or barrier to antimicrobial or oxygen delivery. It was hypothesized that new genes are expressed when bacteria attach to a surface and begin to form a biofilm and that some of the resulting gene products reduce the susceptibility of the cell to antimicrobial agents including oxidative biocides such as monochloramine and hydrogen peroxide.

Gordon A. McFeters

Papers

Survival of Coliform Bacteria in Natural Waters: Field and Laboratory Studies with Membrane-Filter Chambers

Biofilm resistance to antimicrobial agents.

Comparative survival of indicator bacteria and enteric pathogens in well water.

Nonuniform spatial patterns of respiratory activity within biofilms during disinfection.

Reduced susceptibility of thin Pseudomonas aeruginosa biofilms to hydrogen peroxide and monochloramine