Lecture 6 Microbial Growth Patterns PDF

Lecture 6-Microbial Growth-Planktonic and Biofilms Chapter 7 (partial) Copyright © 2019 Pearson Education, Inc. All Rights Reserved. At the end of this lecture, you should be able to: Discuss basic differences in features of microbial growth in a laboratory versus in...

Lecture 6-Microbial Growth-Planktonic and Biofilms Chapter 7 (partial) Copyright © 2019 Pearson Education, Inc. All Rights Reserved. At the end of this lecture, you should be able to: Discuss basic differences in features of microbial growth in a laboratory versus in nature. Calculate generation time for a bacterium. Outline the features of the four stages of bacterial growth in a closed batch system. Define the terms optimal, minimum, and maximum as they apply to temperature and pH conditions. Describe the temperatures where psychrophiles, psychrotrophs, mesophiles, thermophiles, and extreme thermophiles would thrive, and state the grouping for most pathogens. Differentiate between obligate aerobes, obligate anaerobes and facultative anaerobes based on their ability to grow in or tolerate oxygen. Differentiate between planktonic (free-living) and biofilm forms of microbial growth. Outline the requirements and features of a biofilm community. Explain the importance and redundant nature of polysaccharides in biofilm formation Differentiate between the five stages of biofilm formation and dissemination. Explain the health consequences of biofilm formation Copyright © 2019 Pearson Education, Inc. All Rights Reserved. Microbial Growth pattern depends on its environment When nutritional requirements are met, a microbe will enlarge in size and eventually divide Microbial growth is cell division that produces new (daughter) cells and increases the total cell population Bacteria in Most of our knowledge comes from studying species laboratory that can be cultured in the laboratory – ~1% of the bacteria species on our planet In the laboratory, bacteria are often studied as single, pure cultures But out in the environment, bacteria exists as biofilms- mixed communities containing eukaryotes, other Bacteria in bacterial species along with other extracellular biofilms components. Copyright © 2019 Pearson Education, Inc. All Rights Reserved. Generation Time Time it takes for a cell to divide Depends on species and nutritional availability E. coli = as little as 20 mins M. tuberculosis = 15-20 hrs. Copyright © 2019 Pearson Education, Inc. All Rights Reserved.  Growth Phases In a closed batch system (such as set volume of media in a flask), bacteria exhibit four distinct growth phases. Lag phase: Cell number stays constant at the low level that was introduced into media. – Delay that occurs while cells added to the media adjust to their new environment. Log phase: Cells number increases in an exponential manner – due to rapid growth as nutrients are available in large amounts Copyright © 2019 Pearson Education, Inc. All Rights Reserved. Growth Phases-continued Stationary phase: Population growth rate levels off – Nutrients are depleted, waste accumulates. Death phase – At a critical point of waste buildup and decreasing nutrients, the cells begin to die Air in – Rate of cell death is exponential Air out Feeder tube In industry, maintaining cells at a specific growth delivers fresh media phase is often necessary Exit tube Chemostat Cells growing removes waste and cells – Fresh growth medium is added in media Stirrer – Waste and excess cells are removed – Cells are maintained at a constant growth rate Chemostat Copyright © 2019 Pearson Education, Inc. All Rights Reserved. Bacteria can grow in different environmental conditions All microbes find a niche by adapting to specific conditions (e.g., temperature, pH, and available nutrients) Every microbe has a minimum, optimum, and maximum range of temperature and pH for growth. Bacteria can be classified into Mesophiles, Psychrophiles, Psychrotrophs, Thermophiles and Extreme thermophiles depending on the temperature at which they grow Copyright © 2019 Pearson Education, Inc. All Rights Reserved. Bacteria can be classified according to their Oxygen needs Many microbes on this planet live either without oxygen or with minimal oxygen Most pathogens thrive in low-oxygen environments within the host. Bacteria can be classified as obligate (strictly) aerobes, obligate anaerobes, facultative anaerobes Obligate aerobes require Oxygen for growth. Obligate anaerobes die in the presence of oxygen Facultative anaerobes can grow in the presence or absence of oxygen Copyright © 2019 Pearson Education, Inc. All Rights Reserved. Microbial growth patterns Microbiology in the lab Bacteria in biofilms Microbiology in nature Adhere to surfaces, Bacteria are free-living (planktonic) non-motile, Can be motile Differentiated All cells have identical functions structure and function Copyright © 2019 Pearson Education, Inc. All Rights Reserved.  What are biofilms? An aggregate of microbial cells adherent to a living or nonliving surface, embedded within a matrix of extracellular polymeric substances (EPS) of microbial origin. Many different bacterial species found in natural biofilms. Less than 10% of weight of biofilm EPS made up of – secreted extracellular polysaccharides – extracellular DNA – Proteins such as Type IV pili, curli, flagella etc. – outer membrane vesicles – water channels Highly organized structure with distinct functional roles for bacteria inside biofilms-as secretors, adherent cells, persister cells Copyright © 2019 Pearson Education, Inc. All Rights Reserved. Flemming and Wingender, 2010, Nat. Rev. Microbiol  Stages of biofilm formation Reversible Attachment: Planktonic cells attach to a surface. Can be dislodged by physical means. Irreversible Attachment: Gene expression changes allows bacteria to attach irreversibly to the surface Growth: Adhered bacteria replicate to form microcolonies and secrete extracellular matrix components Maturation: Microcolonies rearrange to develop niches and water channels. Functionally distinct microbes such as wall formers, persister cells also seen at this stage Image from Thelwell, 2017, perfectusbiomed.com Dispersion: Individual or clumps of bacteria are released from biofilms due to nutrient starvation, or fluid shear forces. Allows for transfer of biofilm cells to new environments Copyright © 2019 Pearson Education, Inc. All Rights Reserved. How are biofilms measured in the lab? Biofilms can be readily formed on the surface of plastic when cultures are grown in 96 well plates A short time-point (say 1-2hrs) allows for measurement of early events such as attachment of cells to surface Crystal violet stains bacteria on the surface of plates. To quantitate, crystal violet is solubilized by ethanol/acetic acid and measured in a spectrophotometer More Biofilm=Higher readings Copyright © 2019 Pearson Education, Inc. All Rights Reserved. Understanding the role of polysaccharide Biofilm formation depends on secretion of polysaccharide Pseudomonas aeruginosa is a significant health concern for many cystic fibrosis patients. Two commonly used strains are PAO1 and PA14 Pseudomonas aeruginosa secretes three polysaccharides-Alginate, Pel and Psl. Using the data on the right, answer the following questions: – Which polysaccharide is more important for adherence of PAO1 cells to substrate? – Is Pel important for PA14’s ability to attach to substrate or to form a mature biofilm? – How does PAO1 Δpel mutant continue to adhere and form biofilms? Copyright © 2019 Pearson Education, Inc. All Rights Reserved. Biofilm associated infections Biofilms are a major health concern because pathogens in biofilms are responsible for a wide-range of infections Device associated biofilms form readily on in-dwelling catheters implants replacement heart valves contact lenses dental implants ventilator associated pneumonia Dental Plaque and periodontitis Chronic otitis media Chronic tonsillitis Cystic fibrosis lung infections Urinary tract infections Hall-Stoodley et al, 2004, Nat. Rev. Microbiol Copyright © 2019 Pearson Education, Inc. All Rights Reserved. Hall-Stoodley et al, 2012, FEMS Immunol Med Microbiol Common Features of Biofilm associated infections Adherence to a substratum (manmade or epithelium) Aggregated cells enclosed in a matrix directly visible on surface Make biofilm associated infections Resistance to antibiotics very hard to treat Ineffective host clearance Often culture negative Copyright © 2019 Pearson Education, Inc. All Rights Reserved. Take home message Bacterial growth in pure culture has four distinct growth phases that correlate to nutrient availability and metabolism Microbes have specifically adapted to grow in a niche at a specific temperature, pH and oxygen environment. Biofilms are aggregates of bacteria adhered to a surface, encased in a polymeric extracellular matrix. Bacteria in biofilms are a major health concern because they are inherently more resistant to clearance by the immune system or antibiotics. Copyright © 2019 Pearson Education, Inc. All Rights Reserved.