Biofilm Monitoring Research Articles

Development of super nanoantimicrobials combining AgCl, tetracycline and benzalkonium chloride

In this work, we demonstrate that a simple argentometric titration is a scalable, fast, green and robust approach for producing AgCl/antibiotic hybrid antimicrobial materials. We titrated AgNO3 into tetracycline hydrochloride (TCH) aqueous solution, thus forming AgCl/TCH in a one-step procedure. Furthermore, we investigated the one-pot synthesis of triply synergistic super-nanoantimicrobials, combining an inorganic source of Ag+ ions (AgCl), a disinfecting agent (benzyl-dimethyl-hexadecyl-ammonium chloride, BAC) and a molecular antibiotic (tetracycline hydrochloride, TCH). Conventional antimicrobial tests, industrial biofilm detection protocols, and in situ IR-ATR microbial biofilm monitoring, have been adapted to understand the performance of the synthesized super-nanoantimicrobial. The resulting hybrid AgCl/BAC/TCH nanoantimicrobials are found to be synergistically active in eradicating Salmonella enterica and Lentilactobacillus parabuchneri bacteria and biofilms. This study paves the way for the development of a new class of super-efficient nanoantimicrobials that combine relatively low amounts of multiple active species into a single (nano)formulation, thus preventing the development of antimicrobial resistance towards a single active principle.

Real-time diagnosis and monitoring of biofilm and corrosion layer formation on different water pipe materials using non-invasive imaging methods

Water distribution networks play a crucial role in ensuring a reliable water supply, yet they encounter challenges such as corrosion, scale formation, and biofilm growth due to interactions with environmental elements. Biofilms and corrosion layers are significant contaminants in water pipes, formed by complex interactions with pipe materials. As the structure of these contamination layers varies depending on the pipe material, it is essential to investigate the contamination layer for each material individually. Specifically, biofilm growth is typically investigated concerning organic sources, while the growth of humus layers is examined in relation to inorganic elements such as manganese (Mn), iron (Fe), and aluminum (Al), which are major elements and organic substances found in water pipes. Real-time imaging of recently contaminated layers can provide important insights to improve system performance by optimizing operations and cleaning processes. In this study, cast iron (7.10 ± 0.78 nm) exhibits greater surface roughness compared to PVC (5.60 ± 0.14 nm) and provides favorable conditions for biofilm formation due to its positive charge. Over a period of 425 h, the fouling layer on cast iron and PVC surfaces gradually increased in fouling thickness, porosity, roughness, and density, reaching maximum value of 29.72 ± 3.6 μm, 11.44 ± 1.1%, 41673 ± 1025.6 pixels, and 0.80 ± 0.3 fouling layer pixel/layer pixel for cast iron, and 8.15 ± 0.4 μm, 20.64 ± 0.9%, 35916.6 ± 755.7 pixels, and 0.58 ± 0.1 fouling layer pixel/layer pixel, respectively. Within the scope of the current research, CNN model demonstrates high correlation coefficients (0.98 and 0.91) in predicting biofilm thickness for cast iron and PVC. The model also presented high accuracy in predicting porosity for both materials (over 0.91 for cast iron and 0.96 for PVC). While the model accurately predicted biofilm roughness and density for cast iron (correlation coefficients 0.98 and 0.94, respectively), it had lower accuracy for PVC (correlation coefficients 0.92 for both parameters).

Biofilm monitoring through the detection of cyclic diguanosine-monophosphate with an easy-to-use electrochemical sensor

The identification and monitoring of biofilm is of utmost importance because it ensures the survival of bacteria in a variety of environments, such as nature, medical indwelling devices, hospital surfaces, or the food industry. Here, we describe the novel application of a screen-printed electrode modified with nanomaterials for the in situ detection of bacterial biofilm. This approach enables simple, rapid, and highly sensitive and selective electrochemical detection of cyclic diguanosine-monophosphate (cdGMP). The best electrochemical signal of cdGMP was obtained after testing several electrode materials and screen-printed electrodes modified with different nanomaterials, different electrolyte solutions, pH, and electrochemical technique parameters. Using the anodic peak of the cdGMP on screen-printed electrodes modified with carbon nanotubes (CNT-SPE), the sensor showed excellent sensitivity, with a 25 nM–1 µM linear range, and a limit of detection of 15 nM. The sensor allowed the selective detection of cdGMP, with low interference from other molecules and was successfully applied to real sample analyses (laboratory biofilm produced by various bacterial species over distinct time intervals).

Design of a millifluidic device for the evaluation of blue-light disinfection on Pseudomonas aeruginosa PA01 biofilm

SignificanceFor the first time to our knowledge, a millifluidic device has been designed to observe the effects of blue-light irradiation in Pseudomonas aeruginosa PA01 bacterial biofilm. ApproachA bacterial biofilm is formed under controlled flow of nutrients in microfabricated channels settled on a microscope stage. The setup is devised to apply defined irradiation and evaluate the photo-killing efficiency in situ using video-microscopy and fluorescent probes. ResultsWe investigated bacterial survival after delivering a precise, controlled and spatially defined blue light dose upon growing biofilm. We demonstrate in this report that the combined use of constitutive GFP and propidium iodide in a millifluidic setup enables to evidence light-induced mortality at the single cell and community level. ConclusionsReal-time monitoring of a bacterial biofilm growing in a millifluidic device after irradiation opens an avenue for a better understanding of the biofilm community response to blue-light, permitting researchers to approach photokilling efficiency of biofilm as a dynamic process.

Non-destructive monitoring of microalgae biofilms

Biofilm-based cultivation systems are emerging as a promising technology for microalgae production. However, efficient and non-invasive monitoring routines are still lacking. Here, a protocol to monitor microalgae biofilms based on reflectance indices (RIs) is proposed. This framework was developed using a rotating biofilm system for astaxanthin production by cultivating Haematococcus pluvialis on cotton carriers. Biofilm traits such as biomass, astaxanthin, and chlorophyll were characterized under different light and nutrient regimes. Reflectance spectra were collected to identify the spectral bands and the RIs that correlated the most with those biofilm traits. Robust linear models built on more than 170 spectra were selected and validated on an independent dataset. Astaxanthin content could be precisely predicted over a dynamic range from 0 to 4% of dry weight, regardless of the cultivation conditions. This study demonstrates the strength of reflectance spectroscopy as a non-invasive tool to improve the operational efficiency of microalgae biofilm-based technology.

Simulation of Electrical Biofilm Impedance to Determine the Sensitivity of Electrode Geometries

Biofilms are ubiquitous at interfaces of natural and technical origin. Depending on type and application, biofilm formation is desired or has to be prevented. Therefore, reliable detection of initial biofilm growth is essential in many areas. One method of biofilm monitoring is the electrochemical impedance spectroscopy. Among other factors, this method is heavily dependent on the electrode geometry. In order to achieve a high measurement sensitivity, the electrode size must be chosen according to the biofilm that is to be measured. This paper presents an approach for simulating and modeling the optimal electrode geometry for a specific biofilm. First, a geometric model of a biofilm with up to 6000 individual bacteria is generated. The simulated impedances are used to calculate which electrode geometry maximizes sensitivity depending on the biofilm height. In the chosen example of an E. coli biofilm in a nutrient solution, the optimum size of an interdigital electrode (bar gap equals width) was 2.5 µm for a biofilm height of up to 2 µm. The used algorithms and models can be simply adapted for other biofilms. In this way, the most sensitive electrode geometry for a specific biofilm measurement can be determined with minimal effort.

Electrochemical techniques for label-free and early detection of growing microbial cells and biofilms

Over the past decades, the misuse or abuse of antimicrobial agents to prevent and/or control infections has led to increased resistance of microbes to treatments, and antimicrobial resistance is now a subject of major global concern. In some cases, microbes possess the capacity to attach to biotic or abiotic surfaces, and to produce a protective polymeric matrix, forming biofilms of higher resistance and virulence compared to planktonic forms. To avoid further excessive and inappropriate use of antimicrobials, and to propose new effective treatments, it is very important to detect planktonic microbes and microbial biofilms in their early growth stage and at the point of need. In this review, we provide an overview of currently available electrochemical techniques, in particular impedimetric and voltamperometric methods, highlighting recent advances in the field and illustrating with examples in antibiotic susceptibility testing and microbial biofilm monitoring.

Electrochemical Impedance Spectroscopy-Based Sensing of Biofilms: A Comprehensive Review.

Biofilms are complex communities of microorganisms that can form on various surfaces, including medical devices, industrial equipment, and natural environments. The presence of biofilms can lead to a range of problems, including infections, reduced efficiency and failure of equipment, biofouling or spoilage, and environmental damage. As a result, there is a growing need for tools to measure and monitor levels of biofilms in various biomedical, pharmaceutical, and food processing settings. In recent years, electrochemical impedance sensing has emerged as a promising approach for real-time, non-destructive, and rapid monitoring of biofilms. This article sheds light on electrochemical sensing for measuring biofilms, including its high sensitivity, non-destructive nature, versatility, low cost, and real-time monitoring capabilities. We also discussed some electrochemical sensing applications for studying biofilms in medical, environmental, and industrial settings. This article also presents future perspectives for research that would lead to the creation of reliable, quick, easy-to-use biosensors mounted on unmanned aerial vehicles (UAVs), and unmanned ground vehicles (UGVs), utilizing artificial intelligence-based terminologies to detect biofilms.

The association between biofilm formation ability and antibiotic resistance phenotype in clinical isolates of gram-negative bacteria: a cross-sectional study

Backgrounds: Antimicrobial-resistant pathogens that form biofilms cause treatment failure, increased mortality, and morbidity. Gram-negative bacteria (GNB) infections have become an emerging issue in antimicrobial resistance. This study analyzes the association between GNB biofilm formation ability and the antibiotic resistance phenotype in GNB clinical isolates. Methods: From June to August 2022, this cross-sectional study was conducted at Dr. Soetomo General Academic Hospital. Isolates derived from clinical specimens, including blood, urine, and respiratory tract specimens, were identified for antibiotic resistance phenotypes along with a quantitative biofilm formation assay. The antibiotic resistance phenotype was studied in relation to the GNB isolates' ability for biofilm formation. Results: Of the 271 isolates studied, 95 (35.1%) were non-MDR, 143 (52.8%) were MDR, and 33 (12.2%) were XDR. On the biofilm formation assay, 34 (12.5%) were non-biofilm producers, 237 (87.5%) were biofilm producers, consisting of 101 (37.3%) weak, 131 (48.3%) moderate, and 5 (1.8%) strong biofilm producers. Biofilm formation and antibiotic resistance in Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii were not significantly associated (p > 0.05). However, Escherichia coli isolates resistant to the antibiotics amikacin, imipenem, meropenem, and amoxicillin-clavulanate were found to have a higher predisposition to form biofilms. Klebsiella pneumoniae isolates resistant to gentamycin were also positively associated with biofilm formation. Conclusions: Gram-negative bacteria's ability to form biofilms is not associated with their antibiotic resistance phenotype; therefore, routine biofilm monitoring is critical for enhancing the quality of treatment strategy for biofilm-associated infection.

A graphene microelectrode array based microfluidic device for in situ continuous monitoring of biofilms.

In situ continuous monitoring of bacterial biofilms has been a challenging job so far, but it is fundamental to the screening of novel anti-biofilm reagents. In this work, a microfluidic system utilizing a graphene-modified microelectrode array sensor was proposed to realize the dynamic state of bacterial biofilm monitoring by electrochemical impedance. The results illustrated that the observation window period of the biofilm state is significantly prolonged due to the increment of bacterial cell load on the sensing interface, thereby greatly improving the sensing signal quality. Simulation of anti-biofilm drug screening demonstrated that the performance of this method manifestly exceeded that of its endpoint counterparts.

Real-time Assessment of Interspecies Candida Biofilm Formation

Background: Candida infections are responsible for increased morbidity and mortality in immunocompromised patients, with Candida having the ability to form highly structured biofilms that protect them from the external environment and the action of antifungals. Few studies have reported on real-time interactions between Candida species. Objective: This study aimed to investigate the interspecies biofilm changes of oral Candida species using the xCELLigence system and the CV staining technique. Methods: This study demonstrated the mixed biofilm interactions of Candida species using an impedance-based biofilm monitoring system and crystal violet staining. Results: When using the xCELLigence system, the maximum cell index increased in most mixed biofilms, except for the C. glabrata/C. parapsilosis and C. albicans combinations. Bulk biofilm formation measured by CV staining was the highest in C. albicans and C. tropicalis combinations and the lowest in the C. glabrata/C. parapsilosis combination. Pseudohyphae were observed in C. albicans and C. glabrata in combination with C. tropicalis or C. parapsilosis. Conclusion: This study is the first to report on the real-time interactions of Candida species using the xCELLigence system and suggests that the presence of specific species influences the biofilm formation of commonly isolated Candida species.

The Determination, Monitoring, Molecular Mechanisms and Formation of Biofilm in E. coli.

Biofilms are cell assemblies embedded in an exopolysaccharide matrix formed by microorganisms of a single or many different species. This matrix in which they are embedded protects the bacteria from external influences and antimicrobial effects. The biofilm structure that microorganisms form to protect themselves from harsh environmental conditions and survive is found in nature in many different environments. These environments where biofilm formation occurs have in common that they are in contact with fluids. The gene expression of bacteria in complex biofilm differs from that of bacteria in the planktonic state. The differences in biofilm cell expression are one of the effects of community life. Means of quorum sensing, bacteria can act in coordination with each other. At the same time, while biofilm formation provides many benefits to bacteria, it has positive and negative effects in many different areas. Depending on where they occur, biofilms can cause serious health problems, contamination risks, corrosion, and heat and efficiency losses. However, they can also be used in water treatment plants, bioremediation, and energy production with microbial fuel cells. In this review, the basic steps of biofilm formation and biofilm regulation in the model organism Escherichia coli were discussed. Finally, the methods by which biofilm formation can be detected and monitored were briefly discussed.

Real‐Time Monitoring of Wound States via Rationally Engineered Biosensors

AbstractCurrent strategies for real‐time wound monitoring have limited applicability and therapeutic efficiency due to the reliance on clinician experience and time‐consuming laboratory analysis requirements. In this article, an educational review is provided on the accumulated knowledge of wound healing, such as the characterization of acute and chronic wounds, the four main stages of wound healing, and the factors that influence wound healing. The current state‐of‐the‐art wound state sensing methods are presented and reviewed, with particular attention paid to the real‐time monitoring of pH, biofilms, electrophysiological signals, temperature, and reactive oxygen species. It is expected that the presentation of this evolving field will contribute to deepen the existing understanding of wounds and steer wound treatment toward real‐time precision to drive further and faster development of clinically valuable technologies.

Monitoring Aerobic Marine Bacterial Biofilms on Gold Electrode Surfaces and the Influence of Nitric Oxide Attachment Control.

Detection of aerobic marine bacterial biofilms using electrochemical impedance spectroscopy has been done to monitor the interfacial response of Pseudoalteromonas sp. NCIMB 2021 attachment and growth in order to identify characteristic events on a 0.2 mm diameter gold electrode surface. Uniquely, the applicability of surface charge density has been proven to be valuable in determining biofilm attachment and cell enumeration over a 72 h duration on a gold surface within a modified continuous culture flow cell (a controlled low laminar flow regime with Reynolds number ≈ 1). In addition, biofilm dispersal has been evaluated using 500 nM sodium nitroprusside, a nitric oxide donor (nitric oxide is important for the regulation of several diverse biological processes). Ex situ confocal microscopy studies have been performed to confirm biofilm coverage and morphology, plus the determination and quantification of the nitric oxide biofilm dispersal effects. Overall, the capability of the sensor to electrochemically detect the presence of initial bacterial biofilm formation and extent has been established and shown to have potential for real-time biofilm monitoring.

Flexible biofilm monitoring device.

Biofilms and their analysis are increasingly attracting the attention of the scientific community due to the immense importance and impact of biofilms in various natural, technical and medical fields. For these purposes, an optimized and extended antibiofilm assay system based on the Calgary Biofilm Device (MBEC Assay® system) consisting of microtiter plate and PCR tubes was established. Its implementation was used to study the growth characteristics of the sessile phenotype of Pseudomonas fluorescens exposed to antimicrobial peptides. Inhibitory effects of an antimicrobial peptide on P. fluorescens biofilm formation could be determined at a concentration of 250μg/ml (biofilm prevention concentration (BPC)) using the modified biofilm assay. Similarly, the biofilm bactericidal concentration (BBC) at 125μg/ml and the minimum biofilm elimination concentration to remove 90% of the total biofilm mass (MBEC90) were measured at a concentration range of 15.625-1.95μg/ml. In conclusion, this optimized system provides a highly variable, simple, and cost-effective alternative to high-throughput screening based on the Calgary Biofilm Device (CBD).

A high sensitive microwave sensor to monitor bacterial and biofilm growth

Bacterial biofilms have a significant economic and health impact in many different domains. In such films, the extracellular matrix prevents the diffusion of biocides, so antibiotic treatments require a concentration 500 to 1000 times higher than that used to eliminate the same bacteria when present as planktonic stage. Early detection of biofilms is therefore essential for effective eradication. In this paper, we present the development of a real-time and label-free radiofrequency biosensor dedicated to the monitoring of bacteria and biofilm growth. Its principle relies on an open-ended coaxial probe sensitive to the variation of the electrical conductivity of the probed medium in the microwave range. As shown, between 0.3 and 1 GHz, the high sensitivity of the method (2.3 × 104 CFU/mL) highlights the biofilm growing at the early stage of its formation. To demonstrate experimentally such effects, two model bacteria, Vibrio natriegens and P. aeruginosa, are considered. The proposed method should therefore be considered as a promising technique for biofilm monitoring in batch bioreactors or flow cells experiments.

Novel Diagnostic Technologies and Therapeutic Approaches Targeting Chronic Wound Biofilms and Microbiota.

To provide an up-to-date overview of recent developments in diagnostic methods and therapeutic approaches for chronic wound biofilms and pathogenic microbiota. Biofilm infections are one of the major contributors to impaired wound healing in chronic wounds, including diabetic foot ulcers, venous leg ulcers, pressure ulcers, and nonhealing surgical wounds. As an organized microenvironment commonly including multiple microbial species, biofilms develop and persist through methods that allow evasion from host immune response and antimicrobial treatments. Suppression and reduction of biofilm infection have been demonstrated to improve wound healing outcomes. However, chronic wound biofilms are a challenge to treat due to limited methods for accurate, accessible clinical identification and the biofilm's protective properties against therapeutic agents. Here we review recent approaches towards visual markers for less invasive, enhanced biofilm detection in the clinical setting. We outline progress in wound care treatments including investigation of their antibiofilm effects, such as with hydrosurgical and ultrasound debridement, negative pressure wound therapy with instillation, antimicrobial peptides, nanoparticles and nanocarriers, electroceutical dressings, and phage therapy. Current evidence for biofilm-targeted treatments has been primarily conducted in preclinical studies, with limited clinical investigation for many therapies. Improved identification, monitoring, and treatment of biofilms require expansion of point-of-care visualization methods and increased evaluation of antibiofilm therapies in robust clinical trials.

Application of proper orthogonal decomposition for evaluation of coherent structures and energy contents in microbial biofilms

Microbial biofilms are structured communities of surface-associated microbial populations embedded in a matrix of extracellular polysaccharides that provide protection for biofilm cells. Among the wide plethora of microbial species adept at forming biofilms, the fungal pathogen Candida albicans (C. albicans), is one of the most notable. C. albicans biofilm development occurs in a series of sequential steps over a period of 24 h. Various quantitative and microscopic methods are available for the monitoring and evaluation of biofilms, including several innovative real-time methods for the evaluation of the cell-to-cell dynamics occurring during biofilm formation. These methods utilize biosensors which capture electrical, acoustic, and reflectance signals in bacterial populations (Li et al., 2021; Li et al., 2020; Kim et al., 2021; Paredes et al., 2021; Reipa et al., 2021). Additionally, machine learning, deep learning and other computational approaches have progressively been incorporated in the field of microbiology (Qu et al., 2019; Goodswen et al., 2021; Zhang et al., 2021; Ghannam and Techtmann, 2021; Rani et al., 2021; Berg et al., 2019) including some studies in biofilms (Buetti-Dinh et al., 2019; Srivastava et al., 2020; Hartmann et al., 2021; Dimauro et al., 2021) but given the potential of machine and deep learning, this niche is in large need of collaborative work between microbiology and engineering or physics experts to propel machine learning to a higher level. Therefore, whilst promising advances have been made, there is an urgent need for extensive development to take place to study and comprehend the complex interaction of microbial pathogens during biofilm formation. Specifically, there is a lot left to be understood about biofilm energy kinetics, and who the active microbial populations are. We infer that biofilm formation is an extremely diverse phenomenon and that each microorganism exerts different pathways to form a biofilm. Thus, we reasoned on the need for a model that would allow us to study the energy kinetics during C. albicans biofilm development. Modal decomposition techniques (MDTs) commonly used in fluid mechanics are gaining popularity outside their original field and might help decipher some of the dynamically relevant structures of biofilm formation. MDTs permit the identification of coherent structures in fluids and have been used in complex applications of information obtained during a particular time-lapse. A common MDT, Proper Orthogonal Decomposition (POD), can be used in reduced order modelling and machine learning applications. POD allows decomposition of a physical field influenced by different variables that may affect its physical properties. We aimed to evaluate the applicability of this technique in the analysis of energy kinetics during microbial biofilm formation, more specifically C. albicans biofilms. Using POD, we were able to easily distinguish visually distinct modes of growth of C. albicans cells in PBS and RPMI in terms of energy accumulation during the kinetic experiment. Comparing both PBS and RPMI, RPMI contains more energetic and dynamically relevant structures than PBS.

On-Line Monitoring of Biofilm Accumulation on Graphite-Polypropylene Electrode Material Using a Heat Transfer Sensor.

Biofilms growing on electrodes are the heart piece of bioelectrochemical systems (BES). Moreover, the biofilm morphology is key for the efficient performance of BES and must be monitored and controlled for a stable operation. For the industrial use of BES (i.e., microbial fuel cells for energy production), monitoring of the biofilm accumulation directly on the electrodes during operation is desirable. In this study a commercially available on-line heat transfer biofilm sensor is applied to a graphite-polypropylene (C-PP) pipe and compared to its standard version where the sensor is applied to a stainless-steel pipe. The aim was to investigate the transferability of the sensor to a carbonaceous material (C-PP), that are preferably used as electrode materials for bioelectrochemical systems, thereby enabling biofilm monitoring directly on the electrode surface. The sensor signal was correlated to the gravimetrically determined biofilm thickness in order to identify the sensitivity of the sensor for the detection and quantification of biofilm on both materials. Results confirmed the transferability of the sensor to the C-PP material, despite the sensor sensitivity being decreased by a factor of approx. 5 compared to the default biofilm sensor applied to a stainless-steel pipe.

Distribution of Biofilm-associated Genes among Acinetobacter baumannii by in-silico PCR

Background: Acinetobacter baumannii is a coccobacillus that is Gram negative, non motile, non fermentative and oxidase negative. It is the most common and successful nosocomial pathogen recognised by WHO. This dreadful pathogen causes urinary tract infections, ventilator associated pneumonia (VAP), bacteremia, etc., These infections are most common in hospital wards especially Intensive Care Unit (ICU). The infections are due to biofilm formation by the virulent genes of A. baumannii, and the common biofilm-associated genes of A. baumannii were bap, csuE, fimH, epsA, bfmS, ptk, pgaB, ompA, blaPER-1. Among these, bap, epsA and ompA genes are highly prevalent among the clinical strains of A. baumannii.
 Aim: To detect the three vital biofilm-associated genes of A. baumannii by in-silico PCR analysis.
 Materials and Methods: 19 isolates of A. baumannii were selected and 3 target genes, namely epsA, ompA and bap gene were used for the amplification process through in-silico PCR simulation tools. Evolutionary analysis was done for the ompA gene.
 Results: The epsA gene was expressed in 10.52% of the total strains selected with the highest occurrence of ompA gene as 57.89%. bap gene was not observed from the study strains included. From evolutionary analysis based on ompA distributed strains, the Acinetobacter baumannii SDF and Acinetobacter baumannii BJAB0715 might be the parental strains where the evolution of strains would have started. Through successive generations, the Acinetobacter baumannii MDR-ZJ06 and Acinetobacter baumannii TYTH-1 had become the multidrug resistant strains present in the environment.
 Conclusion: The findings of the study confirms the distribution of epsA and ompA genes among the 19 different strains of A. baumannii. The study suggests periodical monitoring of biofilm based virulence genes among the clinical strains and to curtail the A. baumannii infections.