Darla M. Goeres

A method for growing a biofilm under low shear at the air-liquid interface using the drip flow biofilm reactor.

This protocol describes how to grow a Pseudomonas aeruginosa biofilm under low fluid shear close to the air–liquid interface using the drip flow reactor (DFR). The DFR can model environments such as food-processing conveyor belts, catheters, lungs with cystic fibrosis and the oral cavity. The biofilm is established by operating the reactor in batch mode for 6 h. A mature biofilm forms as the reactor operates for an additional 48 h with a continuous flow of nutrients. During continuous flow, the biofilm experiences a low shear as the media drips onto a surface set at a 10° angle. At the end of 54 h, biofilm accumulation is quantified by removing coupons from the reactor channels, rinsing the coupons to remove planktonic cells, scraping the biofilm from the coupon surface, disaggregating the clumps, then diluting and plating for viable cell enumeration. The entire procedure takes 13 h of active time that is distributed over 5 d.

Measuring antimicrobial effects on biofilm bacteria: from laboratory to field.

Publisher Summary This chapter discusses the key issues in the development of new assays for biofilms and provides a prototype method for growing and evaluating biofilms in the laboratory. The chapter includes two case studies to demonstrate the application of this methodology for evaluating antimicrobial efficacy of biofilms in (1) the household and (2) in an oil production field. The approach to develop acceptable screening tests is to start with an unbiased and repeatable laboratory protocol and then systematically improving the method to reduce the expense and time and developing a practical method without sacrificing the unbiased and repeatable. The chapter shows repeatability of a prototype laboratory biofilm growth, sampling, and analytical protocol using the rotating disk reactor and demonstrates how the rotating disk reactor (RDR) can be used to simulate various field applications. Because biofilm organisms appear to have greater resistance than their planktonic counterparts, it is believed that new standard analytical methods developed specifically for evaluating antimicrobials against biofilms are an essential need in the effort to control biofilm-related problems.

Approaches to biofilm-associated infections: the need for standardized and relevant biofilm methods for clinical applications

ABSTRACT Introduction: The concept of biofilms in human health and disease is now widely accepted as cause of chronic infection. Typically, biofilms show remarkable tolerance to many forms of treatments and the host immune response. This has led to vast increase in research to identify new (and sometimes old) anti-biofilm strategies that demonstrate effectiveness against these tolerant phenotypes. Areas covered: Unfortunately, a standardized methodological approach of biofilm models has not been adopted leading to a large disparity between testing conditions. This has made it almost impossible to compare data across multiple laboratories, leaving large gaps in the evidence. Furthermore, many biofilm models testing anti-biofilm strategies aimed at the medical arena have not considered the matter of relevance to an intended application. This may explain why some in vitro models based on methodological designs that do not consider relevance to an intended application fail when applied in vivo at the clinical level. Expert commentary: This review will explore the issues that need to be considered in developing performance standards for anti-biofilm therapeutics and provide a rationale for the need to standardize models/methods that are clinically relevant. We also provide some rational as to why no standards currently exist.

Development of a standardized antibiofilm test.

Publisher Summary Present tests for evaluating antimicrobials are developed for evaluating the efficacy of antimicrobials on suspended bacteria. Because biofilms have unique characteristics and can be more resistant than suspended bacteria, new and more rigorous test methods must be developed. This chapter uses the rotating disk reactor protocol with sodium hypochlorite treatment as a model for developing a standard tier-one antibiofilm test. A tier-one test is a quick, inexpensive method that is relevant to generic biofilm conditions. Because the tier-one test does not represent a specific field application, it may be most applicable to general screening of antibiofilm products. The tier-one test may also be used as the starting point in developing a tier-two test. The tier-two test is a more stringent test designed to represent a particular field system, such as a cooling tower, a swimming pool, or a medical implant device. For tier-two tests, defining parameters of the field system such as water chemistry, water temperature, water filtration, and types of inoculum must be represented in the test apparatus. Methods for evaluating antibiofilm tests must be conducted using relevant biofilm that represents the genetic, transport, and heterogeneity antimicrobial resistance properties of biofilm. Depending on the antibiofilm product claim being tested, the antibiofilm test may need to determine kill, removal, or prevention of regrowth of a biofilm.

Ruggedness and reproducibility of the MBEC biofilm disinfectant efficacy test

The MBEC™ Physiology & Genetics Assay recently became the first approved ASTM standardized biofilm disinfectant efficacy test method. This report summarizes the results of the standardization process using Pseudomonas aeruginosa biofilms. Initial ruggedness testing of the MBEC method suggests that the assay is rugged (i.e., insensitive) to small changes to the protocol with respect to 4 factors: incubation time of the bacteria (when varied from 16 to 18h), treatment temperature (20-24°C), sonication duration (25-35min), and sonication power (130-480W). In order to assess the repeatability of MBEC results across multiple tests in the same laboratory and the reproducibility across multiple labs, an 8-lab study was conducted in which 8 concentrations of each of 3 disinfectants (a non-chlorine oxidizer, a phenolic, and a quaternary ammonium compound) were applied to biofilms using the MBEC method. The repeatability and reproducibility of the untreated control biofilms were acceptable, as indicated by small repeatability and reproducibility standard deviations (SD) (0.33 and 0.67 log10(CFU/mm(2)), respectively). The repeatability SDs of the biofilm log reductions after application of the 24 concentration and disinfectant combinations ranged from 0.22 to 1.61, and the reproducibility SDs ranged from 0.27 to 1.70. In addition, for each of the 3 disinfectant types considered, the assay was statistically significantly responsive to the increasing treatment concentrations.

Control of Hydrogen Sulfide in Oil and Gas Wells With Nitrite Injection

The downhole injection of nitrite-containing solutions into sour oil and gas wells under controlled conditions has been observed to effectively remove hydrogen sulfide (H 2 S) from aqueous and gas phases. Souring control using nitrite has been successfully applied to a gas well in the San Juan Basin of New Mexico and to an oil well in the Amoco Netherlands Rijnfield. H 2 S removal from topsides water separations equipment was also facilitated using nitrite. Injected nitrite (NO 2 - ) scavenges H 2 S and prevents the activity of sulfate-reducing bacteria while it is present. H 2 S production from oil or gas wells treated with nitrite may be suppressed for much longer periods of time. In a 36-hour downhole squeeze in a gas well, injected NO 2 - removed H 2 S from produced gas for a period of 7 months. SRB populations in produced fluids were reduced for a period of 3 months following treatment. In an oil dehydrator, the addition of NO 2 - during regular operation resulted in a decrease in aqueous-phase H 2 S from 40-60 mg/l to less than 1 mg/l. Corrosion rate data indicated a less corrosive environment during the NO 2 - addition period than prior to the addition. In a downhole squeeze in an oil production well, injected NO 2 - decreased H 2 S in associated gas from 140 ppm to less than 1 ppm for 60 days following treatment. H 2 S levels remained below pre-injection levels for 6 months following treatment. Oil production increased immediately following the treatment, probably due to the dissolution of precipitated iron sulfides in the zone surrounding the well bore. This treatment has proven an effective means of controlling H 2 S in oil and gas wells as well as a method of removing iron sulfide scale from the near wellbore.

Minimum information about a biofilm experiment ( MIABiE): standards for reporting experiments and data on sessile microbial communities living at interfaces

The minimum information about a biofilm experiment (MIABiE) initiative has arisen from the need to find an adequate and scientifically sound way to control the quality of the documentation accompanying the public deposition of biofilm-related data, particularly those obtained using high-throughput devices and techniques. Thereby, the MIABiE consortium has initiated the identification and organization of a set of modules containing the minimum information that needs to be reported to guarantee the interpretability and independent verification of experimental results and their integration with knowledge coming from other fields. MIABiE does not intend to propose specific standards on how biofilms experiments should be performed, because it is acknowledged that specific research questions require specific conditions which may deviate from any standardization. Instead, MIABiE presents guidelines about the data to be recorded and published in order for the procedure and results to be easily and unequivocally interpreted and reproduced. Overall, MIABiE opens up the discussion about a number of particular areas of interest and attempts to achieve a broad consensus about which biofilm data and metadata should be reported in scientific journals in a systematic, rigorous and understandable manner.

Prevention of Staphylococcus aureus biofilm formation by antibiotics in 96-Microtiter Well Plates and Drip Flow Reactors: critical factors influencing outcomes

Biofilm formation leads to the failure of antimicrobial therapy. Thus, biofilm prevention is a desirable goal of antimicrobial research. In this study, the efficacy of antibiotics (doxycycline, oxacillin and rifampicin) in preventing Staphylococcus aureus biofilms was investigated using Microtiter Well Plates (MWP) and Drip Flow Reactors (DFR), two models characterized by the absence and the presence of a continuous flow of nutrients, respectively. Planktonic culture of S. aureus was exposed to antibiotics for one hour followed by 24 hours incubation with fresh nutrients in MWP or continuous flow of nutrients in DFR. The DFR grown biofilms were significantly more tolerant to the antibiotics than those grown in MWP without the continuous flow. The differences in log reductions (LR) between the two models could not be attributed to differences in the cell density, the planktonic inoculum concentration or the surface-area-to-volume ratios. However, eliminating the flow in the DFR significantly restored the antibiotic susceptibility. These findings demonstrate the importance of considering differences between experimental conditions in different model systems, particularly the flow of nutrients, when performing anti-biofilm efficacy evaluations. Biofilm antibiotic efficacy studies should be assessed using various models and more importantly, in a model mimicking conditions of its clinical application.

Standardized reactors for the study of medical biofilms: A review of the principles and latest modifications

Abstract Biofilms can cause severe problems to human health due to the high tolerance to antimicrobials; consequently, biofilm science and technology constitutes an important research field. Growing a relevant biofilm in the laboratory provides insights into the basic understanding of the biofilm life cycle including responses to antibiotic therapies. Therefore, the selection of an appropriate biofilm reactor is a critical decision, necessary to obtain reproducible and reliable in vitro results. A reactor should be chosen based upon the study goals and a balance between the pros and cons associated with its use and operational conditions that are as similar as possible to the clinical setting. However, standardization in biofilm studies is rare. This review will focus on the four reactors (Calgary biofilm device, Center for Disease Control biofilm reactor, drip flow biofilm reactor, and rotating disk reactor) approved by a standard setting organization (ASTM International) for biofilm experiments and how researchers have modified these standardized reactors and associated protocols to improve the study and understanding of medical biofilms.

Evaluation and remediation of bulk soap dispensers for biofilm

Recent studies evaluating bulk soap in public restroom soap dispensers have demonstrated up to 25% of open refillable bulk-soap dispensers were contaminated with ∼ 6 log10(CFU ml−1) heterotrophic bacteria. In this study, plastic counter-mounted, plastic wall-mounted and stainless steel wall-mounted dispensers were analyzed for suspended and biofilm bacteria using total cell and viable plate counts. Independent of dispenser type or construction material, the bulk soap was contaminated with 4–7 log10(CFU ml−1) bacteria, while 4–6 log10(CFU cm−2) biofilm bacteria were isolated from the inside surfaces of the dispensers (n = 6). Dispenser remediation studies, including a 10 min soak with 5000 mg l−1 sodium hypochlorite, were then conducted to determine the efficacy of cleaning and disinfectant procedures against established biofilms. The testing showed that contamination of the bulk soap returned to pre-test levels within 7–14 days. These results demonstrate biofilm is present in contaminated bulk-soap dispensers and remediation studies to clean and sanitize the dispensers are temporary.