authored by: Renu Agrawal
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Language: English | Imprint: NIPA

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ISBN: 9789395319140 | Year of Publication: 2023 | Pages: 154
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This book deals with biofilm formation in food, agriculture, soil and plant health, fruits and vegetables, seafood, dairy and milk products, meat and meat products and beverage industries. The mechanism and assaying of Biofilm Formation have been explained well. There are many techniques and approaches which interfere with bacterial attachment, bacterial communication systems (quorum sensing, QS), and biofilm matrixs. This book has discussed the role of biofilm communication and Quorum sensing, the role of antibiotics on biofilm formation, biofilm inhibitory peptides and biofilm inhibition by novel methods.

The book deals both with the negative and positive aspects of biofilms, the strategies used to regulate harmful bacterial biofilms and mechanisms of formation and highlights the future perspectives of bacterial biofilms. It deals with the ways of controlling biofilms by the dietary phytochemicals and removal of bacterial adhesion to inhibit biofilms. Another very important aspect in this book is on the release of antimicrobial compounds, role of bacteriocins, bacteriophages, enzymes, essential oils, nano emulsions and nano particles to prevent biofilms formation. Utilization of newer techniques to remove biofilms have been discussed which include Cold Plasma Technology, Ultra Sound, Light assisted Technologies, Pulse Electric Field and High-Pressure Processing.

The book also discusses the impact of genetic diversity in biofilm formation. This book will help the readers, industrialists, students, society activists in keeping the environment green and clean along with safe food and good health. This book will ultimately improve the human health and environment. The book gives a very comprehensive review starting from introduction to the future implications on biofilms. The book provides the most recent updates in the area with a holistic approach. The purpose of this book is to provide information to manage biofilm and to ensure hygienic practices and food safety.

Renu Agrawal is the Ex. Chief Scientist at CSIR-CFTRI, Mysuru, India and Rural Program Coordinator for outreach activities at CFTRI. She has over 20 patents to her name, with 67+ publications in International and National Journals. She has presented more than 160 papers in various national and international conferences and delivered more than 700 talks. She has guided students for M.Sc, M. Phil and PhD in Biotechnology and Microbiology. She has represented India as a team leader taking scientific delegation for the Asia meet at Indonesia, Bali nominated by DST, Government of India. She had her PhD from University of Rajasthan, Jaipur. Her areas of specialization include Biotransformation and Probiotics. Her research involves lactic acid bacteria, yeasts and Bacillus as probiotic properties inhibiting harmful diseases and reduce the use of antibiotics improving health in the society. Her group has developed greener probiotic technologies used by the common man. As CSIR-800 Rural Program coordinator she led CSIR-CFTRI for CSIR 800 to give a better quality of life to 800 million people in India through scientific and technological intervention. Many awareness camps, demonstrations of technologies were dissipated around the country. She has been passionate about farmers and has organized many national level conferences for all India farmers and for women farmers. She has also been proactive to show them alternate cropping with high nutrient crops. The microbial processes of her team on Ready to make idli and dosa batter are the most downloaded processes from CFTRI.


She has been awarded at multiple scientific associations. Some honourable mentions are Scientific fellow awards as FAMI,FNABS,FSAB,FISBT. She has been awarded Karamveer Chakra by REX-UN both bronze and silver, Best woman scientist NABS, Woman achiever from International Guild of woman achievers, Reflections Excellence Award from All India Association of Rotary Inner wheel, Kissan Ratna Puraskar from JCI International, Abdul Kalam Lifetime Achievement Award, Nari Shakti Award from Random Kindness and the Ministry of Women and Child development and Bharat Shiksha Gaurav Puraskar to name a few. She has been selected as the “Best Woman Scientist” by National Academy of biological sciences, felicitated by Rotary Inner wheel for scientific excellence with “Suguna Award” and felicitated at various scientific and societal forums. In Rotary Innerwheel she was awarded the Best secretary for two times and also Best ISO twice in the district 3180. She is the editor of seven and reviewer for sixteen research Journals and has authored scientific books for the welfare and good health of the community. Her books include "Role of Probiotics in improving human health", "Utilization of agricultural wastes to high valued products", "Making Food safe free from pathogens" and "Traditional Foods and Nutritional Security". The books are being published by CRC publications, UK and NIPA, India. She has written many book chapters with Marcell Dekkar, USA and Taylor and Francis, UK. Her work has won many best paper awards at the national and international conferences. She has taught as faculty of M.Sc Food Technology at CFTRI and as Professor to ACSIR students. She has served as the president of ‘Association of Microbiologists of India” and treasurer in "Society of Biological Chemists", Mysore chapter. She has served on the board of many International Technical Advisory committees at Dubai, Bulgaria, Argentina and Indonesia. She has been on the National committees of NCERT of national Science Talent Search, both for moderating the papers and selection of meritorious children for scholarship under NCERT. She has been on the scientist selection boards of UPSC, DST and DBT and many International technical committees. She is an Executive committee board member of “National Academy of Biological Sciences”. She has been nominated as a National Experts Advisory Committee (NEAC) member from Government of India on; Scheme for Young Scientists and Technologists (SYST), National children science congress, Ministry of science and Technology; member in TIASN (technological intervention for addressing societal need),ASAR, CODER, projects to be taken in cold and Desert areas, Pandit Deen Dayal Upadhyay Gram Sankal Pariyojna, AWSAR for augmenting writing skills and‘KIRAN” which involves projects for women all from the Department of Science and Technology, Govt. of India. She has been invited by DST and Vigyan Prasar for a lead talk on "Emerging Technologies with nutrition" under Atma Nirbhar Bharat - science, society setu. She has been nominated to the Consultative Group made by the Principal Scientific Advisor, Dr. Vijay Raghavan, cabinet rank,who reports to the prime minister of India for inputs on science and technological developments in the country. She has been appointed as a committee member of evaluators by DST on Smart village in Karnataka and is a committee member with Karnataka government for Sustainable Development Goals. She has been nominated in the expert committee of Bangalore Chambers of Commerce and Industries. She is an expert in the committee on export and guidelines of vegan foods by APEDA, ministry of commerce and Industry, Government of India.She writes for City Today newspaper and Biostand Ups a science magazine. Her views on women empowerment have been relayed on All India Radio on women's day both at the state and national radio. She has been an excellent speaker on "Gender sensitization" organized by DST, Govt of India, Mysuru University and various colleges. She has been honoured as “SHERO” by Biostandups and Biosetup both online science magazines and is on their editorial board. She an advisor to the International journal IJBST, a publon publication. Dr. Renu has been leading innovations and change in organisations. Her programs and scientific interviews are telecasted on Doordarshan (DD science). She is also involved in the upliftment of rural education of Karnataka through “Pratham” for teachers training workshops and in translating course material to be made into short videos for the children which is helping them to learn science as fun.

She has been a family counsellor at Police helpline, Mysore for last twenty two years and has saved many lives across various age groups. She has served as a Jury member for the selection of I- woman global awards under science and innovation.


Currently she is a science communicator, reviewer, author, visiting professor in many universities and colleges and a National Expert Advisor in many committees with the Ministry of Science and Technology and has been imparting training to KAS and KPS officers for Sustainable Development Goals, in Karnataka. She has written the nutritional policy for Government of Karnataka on Food and Nutrition. Her commitment to work can be seen as she was taken as Project Advisor by CFTRI even after her retirement. She has been serving the society with her work. She has served as the secretary of "CSIR Pensioners Welfare Association, Mysuru centre, after her retirement and organized a national level conference on "Health and Wellness". She is a philanthropist and has been running many fellowships, national level competitions for poor meritorious students, novel writing competitions, constructed a pre fixed auto stand in Mysore for public use, donated serum analyser for rapid detection of HIV and also Paediatric HIV Kits to Asha Kirana hospital at Mysuru.

1. Introduction 2. Biofilm Formation in Food and Pathogens 3. Biofilm Formation and EPS Production 4. Mechanism and Assaying of Biofilm Formation 5. Biofilm Communication and Quorum Sensing 6. Role of Antibiotics on Biofilm Formation 7. Prediction of Sequence Based Biofilm Inhibitory Peptides 8. Biofilm Inhibition by Novel Methods 9. Impact of Genetic Diversity in Biofilm Formation 10. Application of Reactors with Biofilms for Value Added Products 11. Future of Biofilms

0 Start Pages

It is heartening to note that a book entitled ‘Biofilms” has been authored by Dr. Renu Agrawal which aims at bringing awareness on biofilms. A biofilm is an assemblage of microbial cells enclosed in an extracellular polymeric matrix. The book starts with the introduction of biofilms, the ways microbes form communities and adhere to the surfaces and are resistant to many antibiotics. The book deals with the progress made on the various aspects of biofilms. The book has been divided into 11 chapters. Chapter two deals with biofilm formation in foods and what can cause cross-contamination in food products. The sub sections discuss biofilms in agriculture, quorum sensing and how trophic-mediated communication are utilized by microbial communities for improving agricultural productivity. Biofilms in soil and plant health has elaborated on the membrane antifouling methods and alternatives:ultrasound approach. Biofilms in fruits and vegetables discusses on the contact surfaces of the biofilms and where they stay during food processing. It describes how the contamination could take place during harvesting, transport, processing, distribution or even while marketing. There are problems due to large consumption of seafoods which has resulted in a number of diseases around the globe. This has been well discussed by the author and a subsection has been devoted for this. Milk has some pathogenic bacteria which can adhere on stainless steel surfaces in the production chain and can form biofilms in milk storage tanks and also in milk processing lines. The book covers the biofilms in dairy and milk products in depth. The use of disinfectant agents and their efficiency has been explained well. Another serious problem of biofilms is in the meat industry, 

 
1 Introduction

There are many bacteria and fungi which produce biofilms. These are made up of living, reproducing microorganisms. These microbes form communities and adhere tightly to the surfaces and are resistant to many antibiotics. Many pathogenic microorganisms form biofilms which are difficult to be removed and are harmful.Such microbial colonies produce an extracellular polymeric substance (EPS) to protect them. The dental plaque was the first documented evidence of microbial biofilms. When such microorganisms get a moist environment they attach themselves to any object and reproduce forming an attachment by secreting a slimy, glue-like substance. It is possible with metals, plastics, natural materials, medical implants, kitchen counters, contact lenses, walls of a hot tub or swimming pool. Initially it was the investigation of many scientists to show the presence of biofilms Beneficial effects of biofilms have also been shown (Rosche et al., 2009) especially in agriculture (Bogino et al., 2013; Berlanga and Guerrero, 2016) where it can be utilized as a biological control agents. They are used as biofertilizers to enhance crop production (Timmusk et al., 2017), hazardous pollutants can be removed (Irankhah et al., 2019). 

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2 Biofilm Formation in Food and Pathogens

Microbial life abounds on a solid substrate. Water and some nutrients are sufficient to allow the microbial construction called biofilm. Biofilm-associated infections (BAI) can enhance chronic infections in which the bacterial pathogens can persist causing cross-contamination of the food products. Giaouris et al. (2014) worked on the meat processing and biofilm formation by foodborne bacteria. Work on seafoods has been well worked by Mizan et al. (2015). Habimana et al. (2010) studied them in feed industry on Salmonella versus host resident flora strains. Lin et al. (2017) have studied the characteristics of some food borne pathogens. In a study by Rao et al. (2020) cold atmospheric plasma can be a solution to prevent biofilms.Ferriol-Gonzalez et al. (2020) worked out that bacteriophages can be helpful to remove biofims. According to Przekwas et al. (2020) in early stage of growth of the biofilm use of ascorbic acid can be helpful for the removal of biofilms. Apples can be protected from pathogenic bacteria by protecting them using harmless bacteria. It leads towards the development of alternative foodborne-pathogen-control strategies. Listeria monocytogenes is a big concern in the NE part of India.

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3 Biofilm Formation and EPS Production

Biofilms play an important part in biotechnology, immunology, biofouling and biodeterioration (Fleming and Wingender, 2001; Dunne, 2002). When the pathogenic cells adhere to the surface, it has been found that the transcription of the EPS genes get stimulated (Dunne, 2002). If EPS gene expression are monitored it can provide a good understanding of biofilm phenotypes (Allison and Sutherland, 1997; Helm and Naumann, 1995; Petersen et al., 2005). EPS serves many functions. It also enables the bacteria to capture nutrients (Pontefract,1991).The formation of biofilm goes through a mechanism which is different from species to species. Even the matrix thickness can be different from 0.2 to 1.0pm depending on the species and doubling time of the cells as thickness of 10 to 30 nm has been found in some bacteria species (Sleytr,1997). The cells secrete polysaccharides, proteins, nucleic acids and glycoproteins. Adherence on solid surfaces by microorganisms is a very complicated process that depends mostly on extracellular molecule production (Czaczyk and Myszka, 2007). The biosynthesis of EPS provides an optimal environment in which genetic material between the cells can be exchanged. This is needed for cell identification and the antigenic properties are helpful in serological characterization of the cells. Among the various compounds of extracellular carbohydrates are acetate esters, pyruvtes, formates, succinates, polysaccharides and proteins (Sutherland, 2001a).

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4 Mechanism and Assaying of Biofilm Formation

The biofilm formation is usually assayed by using Congo Red Agar (CRA) plate test. According to a method developed by Freeman et al., (1989) the isolates are stored in nutrient agar slants and screened for biofilm formation ability using CRA plates. Biofilm forming strains are observed as dark black colonies while others as red colonies on the CRA plates. Recently, the concept of these biofims have changed with new imaging technologies, biochemical methods and molecular ecosystem biology tools. A biofilm structure can be observed in 3-D form to the nano-scale level (Neu and Lawrence, 2015). A lot of studies are being conducted on the physiology of the biofilm cells, the genotypic and phenotypic variation among the biofilm community, as well as the biofilm metabolome, proteome and transcriptome (Raes and Bork, 2008). This will help in the the development of efficient methods to control deleterious biofilms (clinical biofilms, food contaminants, biofouling on industrial equipment and on ship hulls) and to enhance and modulate beneficial ones

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5 Biofilm Communication and Quorum Sensing

According to Li and Tian (2012) a pure-culture planktonic growth of bacteria is rarely found in natural environments.There is a physical interaction between the bacterial cells in biofilms (Kolenbrander, 2002; Watnick and Kolter,2000).The bacteria can organize into groups and can form well-organized communities. They also communicate for coordinated activities (Davey and O toole,2000; Watnick et al., 2000; Nadell et al., 2009). Quorum-sensing mechanisms are common in prokaryotic and single-celled eukaryotic organisms like fungi (van Bodman et al, 2008; Sordi and Muhlschlegal, 2009). For any biofilm structure to develop a high level of cell density is important (Kolenbrander et al., 2002; Kuramitsu et al., 2007 and Webb et al., 2003) and also for checking the effect of antibiotics against planktonic organisms in exponential growth phase using MIC and MBC.This can predict the antibiotic efficacy against rapidly dividing bacteria in acute infections. Papanna, Shobharani and Agrawal Renu (2010) studied the interception of quorum sensing signal molecule

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6 Role of Antibiotics on Biofilm Formation

Biofilms are resistant to antibiotics even at high concentrations (Costerton et al., 1999).This could happen when the diffusion of the antibiotic within the biofilm matrix is inefficient and results in slow growth rate of biofilm cells (Davey et al., 2000), there could be the presence of persistent cells and antibiotic-resistant small-colonies (Hogan and Kolter, 2002; Kolenbrander, 2000).Studies have proved that antibiotics can inhibit biofilm formation. An antibiotic azithromycin, is known to inhibit Pseudomonas aeruginosa biofilm. Biofilm colonies become resistant at higher concentrations of the antibiotics whereas at low concentrations these can be induced (Mah and OToole, 2001). P.aeruginosa is commonly present in the lungs of patients infested with cystic fibrosis. The clinical isolates show resistance to azithromycin (MIC>64 pg/ ml).However, low doses of azithromycin have shown improvement in lung function in patients with cystic fibrosis (Borriello et al., 2004). If antibiotics are given at low concentrations (below the MIC) than it induces biofilm formation in a variety of bacterial species. Sub-MIC concentrations of tobramycin can induce biofilm formation in P. aeruginosa. The use of antibiotics in agriculture, as growth promoters have shown to expose bacteria to low levels of the drugs and has caused havoc (Smith et al., 2002). Rachid et al. 

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7 Prediction of Sequence Based Biofilm Inhibitory Peptides

According to Fereshteh et al. (2020) microorganisms use biofilm to protect themselves against the harmful effects of the host body immune system and the surrounding environment. This helps them to survive against the anti-microbial agents. Inhibition of bacterial biofilm formation is very time consuming and expensive. Newer computational tools have been found to be a good option to predict the inhibition of biofilm peptides. In a computational prediction tool a large number of peptide sequences can be screened to select the peptides for validation. Vectors are extracted from the peptide primary structure and classification algorithms are carried out which includes SVM, random forest and k-nearest neighbour to evaluate their performance. Using a combination of features, helps in good evaluation. National Institute of Health (NIH) has found out that 80% of bacterial pathogens form biofilms and their multicellular structure makes them resistant to the hosts’ defense systems and traditional antimicrobials than planktonic bacteria. Recently, the use of Antimicrobial peptides

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8 Biofilm Inhibition By Novel Methods

Joana et al.(2016) have written a critical review on biofilms and have discussed newer methods to study biofilms. These have given a deep knowledge on the physiology, structure and composition of biofilms. More polar amino acids were present in ABPs but QSPs were made up of higher number of neutral amino acids.This helps in recognition. Web application WebLogo help in generating sequence logos (Crooks et al., 2004). The uncharged polar amino acid is present at the first, third and fifth positions.The basis of SVM model is on the amino acid composition of the peptides. The Weka model is based on selected eight inputs which is trained on the NT5 dataset and is accurate, sensitive and specific.Peptide drugs are FDA approved and a total of 242 sequences of biotech peptides/proteins are available at the Drug Bank database. To identify potential anti-biofilm activity for characterization the peptide sequences are submitted on the module of dPABBs. The prediction outcomes are displayed under the ‘Putative ABPs’ section(http://ab-openlab.

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9 Impact of Genetic Diversity in Biofilm Formation

Acinetobacter baumannii is an opportunistic pathogen giving rise to nosocomial infections causing threat to public health. Studies of biofilm-forming genes was done in A. baumannii isolates by PCR technique. To study the relationships of genes a technique based on PCR sequences along with multilocus sequence typing (MLST) were utilized (Saeed et al., 2020). Goudarzi, Mehdi et al. (2019) studied the genetic diversity and biofilm formation analysis of Staphylococcus aureus causing urinary tract infections in Tehran, Iran. The biofilm forming and adhesion encoding genes were studied along with distribution of different agr and spa types in S. aureus isolates by PCR screening and multiplex PCR. The workers found that the ability to produce biofilm was higher among MRSA strains as compared to MSSA strains. The most prevalent biofilm forming gene was icaD (77.3%), followed by icaA (76%), icaB (57.3%) and icaC (50.7%). Acinetobacter baumannii is a nosocomial pathogen which is commonly present in hospitals and is a causal organism of many diseases. A. baumannii isolates have been known as multidrug-resistant (MDR). For controlling such serious organism only molecular techniques have been found to be effective. 

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10 Application of Reactors with Biofilms for Value Added Products

Ali Demirci et al., (2014) studied the reactors with biofilms which are used to produce many value added products. These include organic acids, alcohols, enzymes, antibiotics, bacteriocins and many more. Many bioreactor designs favor the biofilm formation in a reactor. The biofilm reactors are utilized for fermentation and bioconversion of large amount of biomass. Recently, the biofilm reactors have been applied for production of many value-added products with novel designs. According to Ercan and Demirci (2015) the biofilms can cause both detrimental and beneficial effects in the environment. Biofilms in bioreactors has many advantages due to the membrane fouling which reduces the cost. Higher biomass density can be utilized and the operation is more stable.Biofilm reactors are commercially being utilized for waste water treatment, bench and pilot-scale production of value-added products. In order to run the biofilm reactor at optimum conditions it is important to understand the fundamentals of biofilm formation, the different properties

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11 Future of Biofims

With recent work it has become clear that bacterial societies are very complex. Gene expression in bacterial communities are heterogenous. The heterogeneous gene expression contributes towards the biofilms in fluctuating environments which needs to be understood.Technique like single-cell analysis can help in the investigation of more complex communities. Recently, Jahid and Ha, (2012) have written a review on microbial biofilms of produce and the future challenges to food safety many outbreaks have been observed involving food- borne pathogens. Therefore, there is an urdent need for the safety of foods especially against microbial attacks and biofilm formation as they cause a risk to public health. Medically, the pathogenic microorganisms form biofilm- associated with the epithelial or endothelial lining which are embedded in the lungs, intestinal or vaginal mucus layer, the teeth or other medical implant surfaces or formed intracellularly (Dewasthale et al., 2018). Teichoic acids are characteristic major components of the cell surface in Gram-positive bacteria.

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12 End Pages

Abdallah M.2014. Biofilm formation and persistence on abiotic surfaces in the context of food and medical environments. Arch Microbiol. 196(7): 453-472. Abedon ST. 2011. Lysis from without. Bacteriophage. 1:46-49. Abu Bakar A, Ali M, Noor N M, Yahaya N, Ismail M and Abdullah A.2017. Bio-corrosion of carbon steel by sulfate reducing bacteria consortium in oil and gas pipelines. J Mech Eng Sci. 11: 2592-2600. Agnolucci M, Turrini A and Giovannetti M.2019.Molecular and functional characterization of beneficial bacteria associated with AMF spores. In:Methods in Rhizosphere Biology Research ed. Reinhardt D and Sharma AK.61-79. Singapore: Springer. Ahmad AL, Che Lah NF, Ismail S and Ooi BS.2012.Membrane antifouling methods and alternatives: ultrasound approach. Sep Purif Rev. 41: 318-346.

 

Microbial communities, Extracellular polymeric substance, Biofilm formation, Quorum sensing, Bacterial adhesion, Biofilm architecture, Antimicrobial resistance, Dental plaque, Medical devices, Biofilm matrix, Biofilm structure, Biofilm development, Biofilm prevention, Biofilm dispersion, Chronic infections, Biofilm inhibitors, Biofilm ecology, Biofilm imaging, Biofilm dynamics, Biofilm resistance, Microbial bioadhesion, Biofilm-associated infections, Biofilm regulation, Biofilm research

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