Antimicrobial Resistance Among Biofilm Forming Bacteria In Domestic Environments

Antimicrobial Resistance Among Biofilm Forming Bacteria In Domestic EnvironmentsByShaikh Faisal ZamanPlan of WorkStudy and understanding of biofilms.Study of the history of biofilms.Research on formation of biofilms.Study of biofilm life cycle and stages of development.Study of biofilm habitat.Research on the infectious diseases in humans caused by biofilms.Antimicrobial resistance in biofilms and introduction of intrinsic and extrinsic factors in brief.

What is a biofilm? A biofilm is any group of microorganisms in which cells stick to each other on a surface. Eg: Plaques that form on teeth causing tooth decay is a type of bacterial biofilm, rocks are coated with biofilm in a stream or river, gunk that clogs drains, etc.Biofilm forms when bacteria adhere to surfaces in moist environments by excreting a slimy, glue-like substance.Sites for biofilm formation include all kinds of surfaces: natural materials above and below ground, metals, plastics, medical implant materialseven plant and body tissue. A combination of moisture, nutrients and a surface leads to biofilm formation.

These adherent cells are frequently embedded within a self-produced matrix ofextracellular polymeric substance(EPS). The cells produce EPS and are held together by these strands, allowing them to develop complex three-dimensional, resilient, attached communities. Biofilms can be as thin as a few cell layers or many inches thick, depending on environmental conditions.Biofilm extracellular polymeric substance, which is also referred to asslime, is apolymericconglomeration generally composed of extracellularDNA,proteins, and polysaccharides.A biofilm community can be formed by a single bacterial species, but in nature biofilms almost always consist of rich mixtures of many species of bacteria, as well as fungi, algae, yeasts, protozoa, other microorganisms, debris and corrosion products.Over 500 bacterial species have been identified in typical dental plaque biofilms.Biofilms are also sites where genetic material is easily exchanged because of the proximity of the cells, thus maintaining a large gene pool. Microbial multicellular communities or biofilms come in a great variety of sizes and shapes, with some of the most common types containing mushroom-like, pillar-like, hilly, or flat multicellular structures, which allow cells to form long-term relationships, interact with each other and establish metabolic cooperation.Many of the underlying processes are interdependent and require cooperation between various bacterial species with different metabolic capacities. Therefore, in biofilms, the participating microbial members are situated in close proximity seems to be advantageous, since metabolites can easily be transferred and metabolized further.The association of bacteria with a surface and the development of a biofilm can be viewed as a survival mechanism, with bacteria benefiting by acquiring nutrients and protection from biocides.In cases of adverse conditions such as desiccation, osmotic shock, or exposure to toxic compounds, UV radiation, or predators, the microbial community as a whole can provide protection.

Staphylococcus aureusbiofilm on an indwellingcatheterSource: https://en.wikipedia.org/wiki/Biofilm#/media/File:Staphylococcus_aureus_biofilm_01.jpg

Biofilm in kitchen drain pipeSource: https://www.biofilm.montana.edu/content/kitchen-drainpipe http://www.asktheecogeeks.com/clean-stinky-drain/ History of BiofilmsAntonie van Leeuwenhoek (1684) was the first to display the "animalcules" (bacteria) found in plaque scraped from his teeth plaque, and described in a report to the Royal Society of London: "The numbers of these animalcules in the scurf of a mans teeth are so many that I believe they exceed the number of men in a kingdom.In1940 H. Heukelekian and A. Heller in an issue of the Journal of Bacteriology wrote a development takes place either as bacterial slime or colonial growth attached to surfaces, and Zobell (1943) noted an "effect" in seawater and described many of the fundamental characteristics of attached microbial communities. Biofilm science and biofilm engineering have been active fields of study since sessile communities were first described and named in 1978. The group of Dr. Costerton, in 1978, used the name biofilms as a more generic term for microorganisms adhering to wet surfaces in freshwater ecosystems.Formation of BiofilmsFormation of a biofilm begins with the attachment of free-floating microorganisms to a surface. These first colonists adhere to the surface initially through weak, reversible adhesion viavan der Waals forces. If the colonists are not immediately separated from the surface, they can anchor themselves more permanently usingcell adhesionstructures such aspili.Hydrophobicityalso plays an important role in determining the ability of bacteria to form biofilms, as those with increased hydrophobicity have reduced repulsion between theextracellular matrixand the bacterium.Some species are not able to attach to a surface on their own but are sometimes able to anchor themselves to the matrix or directly to earlier colonists. It is during this colonization that the cells are able to communicate via quorum sensingusing products such as AHL. Some bacteria are unable to form biofilms as successfully due to their limited motility. Nonmotile bacteria cannot recognize the surface or aggregate together as easily as motile bacteria. Once colonization has begun, the biofilm grows through a combination of cell division and recruitment.Polysaccharidematrices typically enclose bacterial biofilms. In addition to the polysaccharides, these matrices may also contain material from the surrounding environment, including minerals, soil particles, and blood components, such as erythrocytes and fibrin.The development of a biofilm may allow for an aggregate cell colony (or colonies) to be increasingly antibiotic resistant. Cell-cell communication orquorum sensing(QS) has been shown to be involved in the formation of biofilm in several bacterial species.

Aniridescentbiofilm on the surface of a fishtankSource: https://en.wikipedia.org/wiki/Biofilm#/media/File:Iridescent_biofilm_on_a_fishtank.JPGLife Cycle of a BiofilmFree-floating, or planktonic, bacteria encounter a submerged surface and within minutes can become attached. They begin to produce slimy extracellular polymeric substances (EPS) and to colonize the surface.EPS production allows the emerging biofilm community to develop a complex, three-dimensional structure that is influenced by a variety of environmental factors. Biofilm communities can develop within hours.Biofilms can propagate through detachment of small or large clumps of cells, or by a type of "seeding dispersal" that releases individual cells. Either type of detachment allows bacteria to attach to a surface or to a biofilm downstream of the original community.

Source: http://www.biofilm.montana.edu/node/2390Stages of Biofilm DevelopmentThere are five stages of biofilm development:Initial attachmentIrreversible attachmentMaturation IMaturation IIDispersion

Five stages of biofilm development: (1) Initial attachment, (2) Irreversible attachment, (3) Maturation I, (4) Maturation II, and (5) Dispersion.Source: https://en.wikipedia.org/wiki/Biofilm#/media/File:Iridescent_biofilm_on_a_fishtank.JPGThe first step is the adhesion of pioneer bacteria, with some of the planktonic or free-floating bacteria approaching the surface (live or alive) and becoming attached to the boundary layer, the quiescent zone at the surface where the flow velocity falls to zero. Some of these cells strike and are adsorbed to the surface for only a finite time, before being deadsorbed, in a process called reversible adsorption.This initial attachment is based on electrostatic attraction and physical forces, but due to not any chemical attachments. Some of these reversibly adsorbed cells begin to make preparations for a lengthy stay by forming structures which may then permanently bind then to the surface within the next few hours, the pioneer cells proceed to reproduce and the daughter cells, which form microcolonies on the surface and begin to produce a polymer matrix around the microcolonies, in an irreversible steps. In the next stage, focal areas of the biofilm dissolve and the liberated bacterial cells are then able to spread to other locations where new biofilms can be formed, and the mature biofilm may contain water-filled channels and thereby resemble primitive, multicellular organisms and the attachment is mediated by extracellular polymers that extend outward from the bacterial cell wall. This polymeric material, or glycocalyx, consists of charged and neutral polysaccharides groups that not only facilitate attachment, but also act as an ion-exchange system for trapping and concentrating trace nutrients from the overlying water. The glycocalyx also acts as a protective coating for the attached cells, thereby mitigating the effects of biocides and other toxic substances.As well as trapping nutrient molecules, the glycocalyx net also snares other types of microbial cells through physical restraint and electrostatic interaction.In a mature biofilm, more volume is occupied by the loosely organized glycocalyx matrix (75-95%) than by bacterial cells (5-25%).The base of the biofilm is a bed of dense, with thickness up 5 to 50 m, composed of a sticky mix of polysaccharides, other polymeric substances and water, all produced by the bacteria. Importance of Biofilm StudyBiofilms in nature are generally beneficial and are frequently established on hydrous solid and semi-solid surfaces, such as soil, rock material, or the surfaces of animals and plants. Microbial communities natively populate human mucous membranes and epithelial surfaces, for example, the gastrointestinal tract, oral cavity, and skin. Despite our bodies being colonized with a mixed microbial community of characteristic compositions and they are important and beneficial to us as they can degrade nutrients and thereby make these accessible.They can synthesize some vitamins which we are unable to synthesize on our own.These communities play key roles in the development of our immune systems and in the anatomy of the mucosal surfaces, and also provide protective functions against exogenous pathogens.The relationship between the host and its microbial communities is delicately balanced, but under certain conditions it can break down and result in infectious diseases. According to a recent public announcement from the National Institutes of Health, more than 60% of all microbial infections are caused by biofilms.Although the planktonic form of the bacteria has been very useful in understanding acute infections, chronic ones are more related to the presence of biofilms, with current research indicating an important role for bacterial biofilms in recurrent or chronic infection, including those which are not responsive to a culture-appropriate antibiotic therapy.Biofilm-growing bacteria cause chronic infections, including foreign-body infections, that are characterized by persistent inflammation and tissue damage despite antibiotic therapy and the innate and adaptive immune and inflammatory responses of the host and persisting pathology.Some general features of biofilm infections in humans compared with acute planktonic infections are:Aggregates of bacteria embedded in a self-produced polymer matrix Tolerant to both innate and adaptive immune responses Tolerant to clinically dosing of antibiotics despite susceptibility of planktonic cells Chronic infectionsThe surface-associated microorganisms are responsible for a several chronic infections as: periodontitis, heart valves (endocarditis), in lung infection in patients with cystic fibrosis (CF) causing chronic bronchopneumonia by Pseudomonas aeruginosa, child middle-ear infections (caused by Haemophilus influenzae, for example), in chronic rhinosinusitis, in chronic osteomyelitis and infections caused by a variety of surgical implants, wound infection in burn patients, urinary tract infections (caused by Escherichia coli and other pathogens), in intravenous catheters and stents (caused by Staphylococcus aureus and other gram-positive pathogens), among others.The link between the concept of biofilms and chronic infectious disease is still the subject of a lot of many studies.HabitatBiofilms are ubiquitous. Biofilms will form on virtually every non-shedding surface in a non-sterile aqueous (or very humid) environment.Biofilms can be found on rocks and pebbles at the bottom of most streams orriversand often form on the surface ofstagnantpools of water. In fact, biofilms are important components offood chainsin rivers and streams and are grazed by the aquaticinvertebratesupon which many fish feed.Biofilms can grow in the most extreme environments: from, for example, the extremely hot, briny waters ofhot springsranging from very acidic to very alkaline, to frozenglaciers.

In the human environment, biofilms can grow inshowersvery easily since they provide a moist and warm environment for the biofilm to thrive. Biofilms can form inside water and sewagepipesand cause clogging and corrosion. Biofilms on floors and counters can make sanitation difficult in food preparation areas.Biofilms are present on theteethof most animals asdental plaque, where they may causetooth decayandgum disease.Biofilms are found on the surface of and inside plants. They can either contribute to crop disease or, as in the case of nitrogen-fixing Rhizobium on roots, exist symbiotically with the plant.Examples of crop diseases related to biofilms include Citrus Canker, Pierce's Diseaseof grapes, and Bacterial Spot of plants such as peppers and tomatoes.Recent studies in 2003 discovered that the immune system supports bio-film development in the large intestine. This was supported mainly with the fact that the two most abundantly produced molecules by the immune system also support bio-film production and are associated with the bio-films developed in the gut.

Biofilm inYellowstone National ParkSource: https://en.wikipedia.org/wiki/Biofilm#/media/File:Iridescent_biofilm_on_a_fishtank.JPG

Thermophilic bacteria in the outflow ofMickey Hot Springs,Oregon, approximately 20 mm thickSource: https://en.wikipedia.org/wiki/Biofilm#/media/File:Thermophilic_bacteria.jpgBiofilms and Infectious DiseasesDental plaqueDental plaqueis an oral biofilm that adheres to theteethand consists of many species of both fungal and bacterial cells (such asStreptococcus mutansandCandida albicans), salivarypolymersand microbial extracellular products. The accumulation of microorganisms subjects the teeth and gingival tissues to high concentrations of bacterialmetaboliteswhich results in dental disease.The biofilm on the surface of teeth is frequently subject to oxidative stressand acid stress. Dietary carbohydrates can cause a dramatic decrease in pH in oral biofilms to values of 4 and below (acid stress).A pH of 4 at body temperature of 37C causes depurination of DNA, leaving apurinic (AP) sites in DNA,especially loss of guanine.

Dental PlaqueSource: http://www.redrockdental.org/lp/plaque.htmlStreptococcus pneumoniaeS. pneumoniaeis the main cause of community-acquired pneumonia and meningitis in children and the elderly, and of septicemia in HIV-infected persons. WhenS. pneumoniagrows in biofilms, genes are specifically expressed that respond to oxidative stress and induce competence.Formation of a biofilm depends on competence stimulating peptide (CSP). CSP also functions as a quorum-sensing peptide. It not only induces biofilm formation, but also increases virulence in pneumonia and meningitis.LegionellosisLegionellabacteria are known to grow under certain conditions in biofilms, in which they are protected againstdisinfectants. Workers incooling towers, persons working inair conditioned roomsand people taking ashowerare exposed toLegionellaby inhalation when the systems are not well designed, constructed, or maintained.

Source: http://biomedfrontiers.org/infection-2013-dec-2/

Source: http://m2002.tripod.com/legionellosis.htmAntimicrobial resistance in biofilmsDespite decades of research, very little is known about the molecular mechanisms of antibiotic resistance in biofilms. Although several theories have been proposed, the precise mechanism of how this sensitivity is altered has still not been clarified.Nevertheless, it is possible to separate these mechanisms into intrinsic (or innate) and extrinsic (or induced) resistance factors to biofilms.Also, because of the heterogeneous nature of biofilms, it is likely that multiple mechanisms of antimicrobial resistance occur. However, additional mechanisms must also exist to be able account for increased biofilm antibiotic resistance.

In the traditional antibiotic resistance of planktonic bacteria, usually involves inactivation of the antibiotic, modification of targets, and exclusion of the antibiotic. These actions typically require the acquisition of specific genetic factors, such as genes for -lactamase or efflux pumps. One of the most important aspects of bacterial biofilm formation is the increased resistance of the constituent microbes to antibiotics and other stressors.The structural nature of the biofilms and the characteristics of the sessile cells, produce resistance towards the antimicrobial agents, leading to a protected environment against adverse conditions and the hosts defenses.

Video on Bacterial Biofilms

ConclusionDuring their evolution, bacteria have been able to develop successful strategies for survival, which include attachment to surfaces and the development of protective biofilms where bacteria behave very differently to the free-floating types. These successful strategies make it difficult to control biofilm growth, with a biofilm providing bacteria with a 10- to 1,000-fold increase in antibiotic resistance compared to free ones.Due to the heterogeneous nature of biofilms, it is likely that multiple mechanisms of antimicrobial resistance are useful in order to explain biofilm survival in a number of cases, with antibiotic resistance being the result of an intricate mixture of intrinsic and extrinsic factors.

Bibliographyhttp://www.formatex.info/microbiology3/book/736-744.pdfhttp://www.biofilm.montana.edu/node/2390https://en.wikipedia.org/wiki/Biofilmhttp://web.stanford.edu/~amatin/MatinLabHomePage/Biofilm.htmhttp://www.sciencedirect.com/science/article/pii/S0924857910000099http://www.ncbi.nlm.nih.gov/pubmed/20149602http://www.medscape.com/viewarticle/807731_4

Future Plan of WorkFurther research on antimicrobial resistance of bacterial biofilms.Research on intrinsic and extrinsic factors of resistance to antibiotics in biofilms.Study of biofilm formation in domestic environments.Advantages and disadvantages of biofilm in domestic environments.Preventive measures to be taken to prevent growth of biofilms on substances.

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