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.

To investigate growth of heterotrophic biofilm bacteria, a model biofilm reactor was developed to simulate a drinking water distribution system. Controlled addition of three different carbon sources (amino acids, carbohydrates, and humics) at three different concentrations (500, 1,000, and 2,000 ppb carbon) in the presence and absence of chlorine were used in separate experiments. An additional experiment was run with a 1:1:2 mixture of the above carbon sources. Biofilm and effluent total and culturable cells in addition to total and dissolved organic carbon were measured in order to estimate specific growth rates (SGRs), observed yields, population densities, and bacterial carbon production rates. Bacterial carbon production rates (µg C/L day) were extremely high in the control biofilm communities (range = 295-1,738). Both growth rate and yield decreased with increasing carbon concentrations. Therefore, biofilm growth rates were zero-order with respect to the carbon concentrations used in these experiments. There was no correlation between growth rate and carbon concentration, but there was a significant negative correlation between growth rate and biofilm cell density (r = -0.637, p = 0.001 control and r = -0.57, p = 0.021 chlorinated biofilms). Growth efficiency was highest at the lowest carbon concentration (range = 12-4.5%, amino acids and humics respectively). Doubling times ranged from 2.3-15.4 days in the control biofilms and 1-12.3 days in the chlorinated biofilms. Growth rates were significantly higher in the presence of chlorine for the carbohydrates, humics, and mixed carbon sources (p = 0.004, < 0.0005, 0.013, respectively). The concept of r/K selection theory was used to explain the results with respect to specific growth rates and yields. Humic removal by the biofilm bacteria (78% and 56% for the control and chlorinated biofilms, respectively) was higher than previously reported literature values for planktonic bacteria. A number of control experiments indicated that filtration of drinking water was as effective as chlorination in controlling bacterial biofilm growth.

Effects of carbon source, carbon concentration, and chlorination on growth related parameters of heterotrophic biofilm bacteria

A direct viable count method for the enumeration of attached bacteria and assessment of biofilm disinfection.

Distribution of enteric bacteria in Antarctic seawater surrounding a sewage outfall.

A method for rapid and minimally disruptive embedding and sectioning of bacterial biofilms has been developed and applied to binary population biofilms of Klebsiella pneumoniae and Pseudomonas aeruginosa grown on stainless steel surfaces in continuous flow annular reactors. Biofilms were cryoembedded using a commercial tissue embedding medium. Frozen embedded biofilms could be removed easily from the substratum by gently flexing the steel coupon. Microscopic examination of the substratum surface after biofilm removal indicated that less than a monolayer of attached cells remained. Five μm thick frozen sections were cut with a cryostat and examined by light or fluorescence microscopy. The cryoembedding technique preserved biofilm structural features including an irregular surface, water channels, local protrusions up to 500 μm thick, and a well‐defined substratum interface. The method requires minimal sample processing without dehydration or prolonged fixation, and can be completed in less than 24 h.

Cryosectioning of biofilms for microscopic examination

Experiments were conducted to measure communities of bacteria within operating ultrapure water treatment systems intended for laboratory use. Samples from various locations within Milli-Q Plus and Milli-Q UV Plus systems were analyzed for populations of planktonic bacteria at weekly intervals over 3 months of operation. Relatively high initial densities of planktonic bacteria (10(2) to 10(3) bacteria per ml) were seen within both units when they were challenged with source water of poor quality, although the product water continued to be acceptable with regard to bacterial numbers, resistivity, and endotoxin concentration. Under more normal operating conditions, significant differences were seen in planktonic populations throughout the systems with excellent product water quality. A great deal of variability was observed in biofilm populations analyzed from various system surfaces after 3 months of operation. The concentrations of planktonic bacteria and biofilm densities were much lower in the unit containing a UV lamp. These findings suggest that a range of microenvironmental conditions exist within purified water systems, leading to variable populations of bacteria. However, product water of excellent quality was obtained despite the bacterial communities.

Gordon A. McFeters

Papers

Effects of carbon source, carbon concentration, and chlorination on growth related parameters of heterotrophic biofilm bacteria

A direct viable count method for the enumeration of attached bacteria and assessment of biofilm disinfection.

Distribution of enteric bacteria in Antarctic seawater surrounding a sewage outfall.

Cryosectioning of biofilms for microscopic examination

Distribution of bacteria within operating laboratory water purification systems.