Lecture #15: Control, Biofilms Flashcards Preview

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Flashcards in Lecture #15: Control, Biofilms Deck (19):
1

Controlling Microbial Growth

Irradiation – commonly used with foods, surgical instruments and surfaces. UV and radioactivity. We use a UV light to sterilize agar plates. Can ‘disinfect’ this way.

Autoclaving – surfaces, tools, media, contaminated materials. Targets endospores and microbes.

Temperature – low, high extremes. Arrests growth.

Chemicals and Gasses - Detergents, Disinfectants, Antibiotics.

2

Phenol (Lecture)

Joseph Lister and disinfectants during surgery, used phenolics.

Lysol contains phenol.

Phenol denatures proteins, disrupts cell membranes. Industry Standard; effectiveness of others compared to phenol.

3

Phenolics

First widely used antiseptic and disinfectant. Joseph Lister employed it to reduce the risk of infection during surgery.

Today phenol and phenolics (phenol derivatives) such as cresols, xylenols, and orthophenylphenol are used as disinfectants in laboratories and hospitals. The commercial disinfectant Lysol is made of a mixture of phenolics.

Phenolics act by denaturing proteins and disrupting cell membranes.

They have some real advantages as disinfectants: phenolics are tuberculocidal, effective in the presence of organic material, and remain active on surfaces long after application. However, they have a disagreeable odor and can cause skin irritation.

The newer phenolic, tricolsan, is often used in hand sanitizers due to its effective blockage of bacterial fatty acid synthesis.

4

Iodine

Iodine is used as a skin antiseptic and kills by oxidizing cell constituents and iodinating cell proteins. At higher concentrations, it may even kill spores.

Iodine often has been applied as tincture of iodine, 2% of more iodine in a water-ethanol solution of potassium iodide.

Although it's an effective antiseptic, the skin may be damaged, a stain is left, and iodine allergies can result.

Iodine has been complexed with an organic carrier to form an iodophor. Iodophors are water soluble, stable, and nonstaining, and release iodine slowly to minimize skin burns and irritation. They're used in hospitals for cleansing preoperative skin and in hospitals and labs for disinfecting.

Some popular brands are Wescodyne for skin and laboratory disinfection, and Betadine for woulds.

5

Chlorine

Usual disinfectant for municipal water supplies and swimming pools, and is employed int he dairy and food industries. It may be applied as chlorine gas (Cl2), sodium hypochlorite (bleach, NaOCl), or calcium hypochlorite [Ca(OCL)2], all of which yield hypochlorus acid (HOCl). The result is oxidation of cellular materials and destruction of vegetative bacteria and fungi.

6

Halogens

Iodine, Iodophor (iodine with carrier) like Wescodyne, Chorine (10% Bleach). May require time (5-30 minutes). Wescodyne has Iodophor AND detergent.

7

Killing Problems

General Equation is:
LogNt = LogN0 – t/D

Where D is the time required for a log reduction.

If you have 100,000 cells/mL at 8 a.m., and 1,000 cells/mL at 8:20, you have gone through 2 log reductions (105 to 103) in 20 minutes, so a log reduction is 10 minutes.
At what time will the number fall below 10 cells/mL, when the water is safe to drink?
1 = 3 – t/0.17 or t/0.17 = 2
t=.34 t = 20.4 minutes. So, after 8:41 a.m.

Assume:
- Death is proportional (log reduction) over time and the killing agent does not lose effectiveness.
- Temperature is constant.
- Mixing allows full penetration or diffusion.

8

Live By the Sword, Die By the Sword

Organisms that use oxygen generate toxic substances and must protect themselves from oxygen by-products.

Oxygen use generates radicals that destroy cellular components. Cell must render them harmless.

Superoxide Dismutase (Oxygen radicals converted to Hydrogen Peroxide = dangerous to cells as well)
Catalase or Peroxidase to convert H2O2 to water, oxygen.

9

Catalase Test

Obligate anaerobes (can’t live in oxygen) lack catalase, can’t deal with oxygen by-products. Fermenters and anaerobic respirers may lack protection as well.

This organism has catalase, so when you apply Hydrogen Peroxide to the cells, it releases oxygen.

10

Biofilms

Microbes in aquatic environments were found attached to surfaces that were free-floating (planktonic). This attached microbes are members of complex, slime-encased communities called biofilms.

Ubiquitous in nature, where they're most often seen as layers of slime on rocks or other objects in water or at water-air interfaces. When they form on the hulls of boats and ships, they cause corosion, which limits the life of ships and results in economic losses. Of major concern is formation of biofilms on medical devices such as hip and knee implants. Often cause serious illness and failure of medical device.

11

Surfaces with Biofilms & EPS

Biofilms can form on virtually any surface, once it has been conditioned by proteins and other molecules present in the environment. Initially microbes attach to the conditioned surface but can readily detach.

Eventually they form a matrix made up of polysaccharides, proteins, glycoproteins, glycolipids, and DNA. These are collectively called extracellular polymeric substances (EPS).

The EPS matrix allows the microbes to stick more stably to the surface. As the biofilm thickens and matures, the microbes reproduce and secrete additional polymers.

12

Within Biofilm

A mature biofilm is a complex, dynamic community of microbes. It exhibits considerable heterogeneity due to differences in the metabolic activity of microbes at various locations within the biofilm. The microbes interact in a variety of ways. For instance, the waste products of one microbe may be the energy source for another. The cells also communicate with each other. Finally, DNA present int he EPS can be taken up by members of the biofilm communtiy. Thus genes can be transferred from one cell/species to another.

While in the biofilm, microbes are protected from numerous harmful agents. This is due in part to eh EPS in which they are embedded. But it's also due to physiological changes. Indeed, numerous proteins are synthesized or activated in biofilm cells are not observed when these cells are free-living, planktonic cells, and vice versa.

Conditions vary, especially edges vs. internal areas. Layering of species and metabolic types/environmental types.

Sort out different species by tolerances and preferences.

13

A Rock in a Stream

Rock in a river with moderate flow. Thin film develops on rock surface. The rock is a source of iron, and the first microbe in the film reduces iron.

Other cells colonize the matrix. Aerobic ones are at the fringes, iron dependent ones are basal and moderate anaerobes fill the middle layers.

As matrix grows thicker, cells in the middle slow their metabolism. Those toward the edges are more active. Stratification. With time, more complexity with new species joining in and greater habitat variation due to internal channels, increasing thickness, varied waste products.

14

Shelter & Insulation

Cells deep within the matrix can be dormant. This is one reason why antibiotics and other antimicrobial agents have little effect – if they target actively growing, actively metabolizing or dividing cells.

Matrix itself is protective. Resists drying and may not be easily penetrated by antibiotics, other antimicrobial compounds or even UV light. Polysaccharide or protein or a mix—difficult to penetrate by larger molecules.

15

Where Biofilms Form

Form wherever its moist. Species diversity in a biofilm, just as in natural areas we know, varies with types and extent of resources.

Dryness is a key anti-bacterial strategy.

16

Matrix and Its Recesses are Protective

Antimicrobial agents can’t penetrate the matrix. Further, cells within the matrix may produce protective compounds; build up.

Cells deep within biofilm may be resting, slow-growing, not actively dividing. Most antibiotics target cell wall or protein synthesis.

17

Biofilms & Conditioning of the Soil

Microbial communities condition the soil—provide structuring, bind ions and filter flow, enhance nutrients, stabilize particles. A mature soil supports climax species.

Effective groundwater filter that regenerates clean drinking water naturally. Linda Handley.

18

Quorum Sensing

Bacterial cells use molecular signals to communicate with each other in a density dependent manner. This is called quorum sensing; a quorum usually refers to the min number of members in an organization, such as a legislative body, needed to conduct business.

Behavior in a biofilm suggests co-ordination, communication.

Actions relate to cell density. Quorum. For example, opportunistic bacteria within a host cause no harm until they reach a critical density and then behavior changes – all at once, all cells. New proteins transcribed, as if on cue. Many of these are related to virulence. Critical density required to overwhelm a host’s immune system and cause disease. Myxobacterium, Streptomyces.

Bacteria produce chemicals that function as signals. The signals are released into the medium. They also have receptors for these signaling compounds. When they bind them, induces the production of more.

If few cells in the vicinity, bind few signals and produce signaling compounds at low levels. If a quorum reached (critical density of cells), bind enough and frequently enough that produce many themselves. Triggers secondary response as transcription of particular genes. So more gets you more (positive feedback).

19

Communication Can be Deadly…

Costerton & Stewart suggest using the very same signaling compounds to disrupt biofilms, and render the cells vulnerable.

Many examples of biofilms with application to medicine and industry. Water pipes in houses, heart valves…

Problems attempting to use signaling compounds to break-up biofilms in the body. Unintended side effects as the same compounds, or related ones, also direct processes in the native microbes.