Information

4.E: Using Antibiotics and Chemical Agents to Control Bacteria (Exercises) - Biology

4.E: Using Antibiotics and Chemical Agents to Control Bacteria (Exercises) - Biology


We are searching data for your request:

Forums and discussions:
Manuals and reference books:
Data from registers:
Wait the end of the search in all databases.
Upon completion, a link will appear to access the found materials.

These are homework exercises to accompany Kaiser's "Microbiology" TextMap. Microbiology is the study of microorganisms, which are defined as any microscopic organism that comprises either a single cell (unicellular), cell clusters or no cell at all (acellular). This includes eukaryotes, such as fungi and protists, and prokaryotes. Viruses and prions, though not strictly classed as living organisms, are also studied.

4.1: An Overview to Control of Microorganisms

Study the material in this section and then write out the answers to these questions. Do not just click on the answers and write them out. This will not test your understanding of this tutorial.

  1. Matching:

    _____ An agent that kills the organism. (ans)

    _____ An agent that inhibits the organism's growth long enough for body defenses to remove it. (ans)

    _____The chemical agent being used should inhibit or kill the intended pathogen without seriously harming the host. (ans)

    _____ A chemical agent that generally works against just gram-positives, gram-negatives, or only a few bacteria. (ans)

    _____ A chemical agent that is generally effective against a variety of gram-positive and gram-negative bacteria. (ans)

    _____ Antimicrobial drugs synthesized by chemical procedures in the laboratory. (ans)

    _____ Metabolic products of one microorganism that inhibit or kill other microorganisms. (ans)

    _____ The process of destroying all living organisms and viruses. (ans)

    _____ The elimination of microorganisms, but not necessarily endospores, from inanimate objects or surfaces. (ans)

    _____ An agent that kills or inhibits growth of microbes but is safe to use on human tissue. (ans)

    1. selective toxicity
    2. broad spectrum agent
    3. narrow spectrum agent
    4. cidal
    5. static
    6. sterilization
    7. antibiotic
    8. chemotherapeutic synthetic drug
    9. antiseptic
    10. disinfection
    11. disinfectant

4.2: Ways in which Chemical Control Agents Affect Bacteria

Study the material in this section and then write out the answers to these questions. Do not just click on the answers and write them out. This will not test your understanding of this tutorial.

  1. Matching:

    _____ Alter bacterial 30S ribosomal subunits blocking translation. (ans)

    _____ Inhibit peptidoglycan synthesis causing osmotic lysis. (ans)

    _____ Alter bacterial 50S ribosomal subunits blocking translation. (ans)

    _____ Inhibit nucleic acid synthesis. (ans)

    1. macrolides(erythromycin, azithromycin, clarithromycin, dirithromycin, troleandomycin, etc.), oxazolidinones (linezolid), and streptogramins
    2. penicillins, monobactams, carbapenems, cephalosporins, and vancomycin
    3. fluoroquinolones (norfloxacin, lomefloxacin, fleroxacin, ciprofloxacin, enoxacin, trovafloxacin, etc.), sulfonamides and trimethoprim, and metronidazole
    4. aminoglycosides (streptomycin, neomycin, netilmicin, tobramycin, gentamicin, amikacin, etc.) and tetracyclines (tetracycline, doxycycline, demeclocycline, minocycline, etc.)
  2. Describe 4 different ways antibiotics or disinfectants may affect bacterial structures or macromolecules and state how this ultimately causes harm to the cell.
    1. (ans)
    2. (ans)
    3. (ans)
    4. (ans)
  3. Multiple Choice (ans)

4.3: Ways in which Bacteria May Resist Chemical Control Agents

Study the material in this section and then write out the answers to these question. This will not test your understanding of this tutorial.

  1. Name 2 bacteria that have low-permeability membrane barriers and are thereby intrinsically resistant to many antibiotics. (ans)
  2. Briefly describe 3 different mechanisms as a result of genetic changes in a bacterium that may enable that bacterium to resist an antibiotic.
    1. (ans)
    2. (ans)
    3. (ans)
  3. State what the following stand for:
    1. MRSA (ans)
    2. VRE (ans)
    3. CRE (ans)
  4. Briefly describe R plasmids and state their significance in our attempts to treat infections with antibiotics. (ans)
  5. Multiple Choice (ans)

Science of Resistance: Antibacterial Agents

When Louis Pasteur demonstrated that bacteria are the agents that cause many infections, people were better able to understand how diseases begin and spread. Ironically, however, even though Pasteur's findings demystified infectious disease, they also led to fear of "germ" contamination. This fear was not allayed until the last half of the twentieth century when antibiotics were discovered and used clinically.

Antibiotics were considered miracle drugs when they were first introduced. Many people felt that diseases caused by bacteria were conquered and soon would no longer exist. Unfortunately, however, because antibiotics were adopted as "wonder drugs," they were often used in indiscriminate and improper ways. Resistant strains of bacteria began to emerge. Today, roughly eighty years after antibiotics were introduced, antibiotic resistance is a serious problem and antibiotics are losing their effectiveness. In health facilities, epidemics of antibiotic-resistant infections are serious threats to those whose health is already compromised. Diseases such as tuberculosis, once felt to be under control, are often resistant to many antibiotics and frequently do not respond to treatment. Public concern about infection has been heightened people are once again afraid of "germs". One response to this fear is greater public use of a variety of antibacterial agents designed to remove disease-causing organisms from external surfaces before they can enter the body. Although first introduced into soaps, detergents and other cleaning and health care products, today antibacterials may also be impregnated into sponges, cutting boards, carpeting, upholstery, and even children's toys.

Using antibacterial agents to destroy many organisms before they enter the body may not always be the best way to stop the spread of infectious disease. This is because we need "good" bacteria to control and compete with "bad" bacteria. We frequently encounter and touch disease-causing or "bad" organisms (as, for example when we touch the nose, the mouth, an open wound, or fecal matter—all sources of infectious agents). However, these bacteria must compete for space with the variety of "good" bacteria that we also carry on and in our bodies and that we encounter in the environment. This means that not all contact we have with bacteria, even "bad" bacteria, result in disease. If we destroy all the bacteria on a surface, we will destroy both kinds, removing the "good" bacteria along with the "bad". Chemical agents don't discriminate between "good" and "bad" bacteria, and can remove all bacteria. However, if bacteria do remain, these may be resistant to the effects of the chemical agents.

In certain settings, antibacterial agents are essential to fight against infection. However, if used too frequently and indiscriminately, certain antibacterial agents—those that leave trace chemical residues and that target particular processes in the life cycle of bacteria—may, like antibiotics, select for resistant strains. To insure that these agents continue to be effective when they are needed, products containing these antibacterials should only be used when they are essential to fight against infection. In other situations, when more information is not available, it is wiser to disinfect with agents that are unlikely to select for resistant strains of bacteria.

Generally, the best way to remove "bad" bacteria is through good hand-washing practice using a non-bactericidal soap and water. Proper hand-washing will remove 99.9% of bacteria, and normally, few other control measures are needed. When susceptible populations are likely to be exposed to "bad" bacteria, other more effective control measures may be required. Settings involving susceptible populations: young children, the elderly, or those whose health is compromised because of AIDS infection, use of immunosuppressive drugs, illnesses requiring hospitalisation, or chemotherapy require additional disinfection measures. Antibacterial agents should be reserved for these settings.


Biological control

Biological control of plant diseases involves the use of organisms other than humans to reduce or prevent infection by a pathogen. These organisms are called antagonists they may occur naturally within the host’s environment, or they may be purposefully applied to those parts of the potential host plant where they can act directly or indirectly on the pathogen.

Although the effects of biological control have long been observed, the mechanisms by which antagonists achieve control is not completely understood. Several methods have been observed: some antagonists produce antibiotics that kill or reduce the number of closely related pathogens some are parasites on pathogens and others simply compete with pathogens for available food.

Cultural practices that favour a naturally occurring antagonist and exploit its beneficial action often are effective in reducing disease. One technique is to incorporate green manure, such as alfalfa, into the soil. Saprotrophic microorganisms feed on the green manure, depriving potential pathogens of available nitrogen. Another practice is to make use of suppressive soils—those in which a pathogen is known to persist but causes little damage to the crop. A likely explanation for this phenomenon is that suppressive soils harbour antagonists that compete with the pathogen for food and thereby limit the growth of the pathogen population.

Other antagonists produce substances that inhibit or kill potential pathogens occurring in close proximity. An example of this process, called antibiosis, is provided by marigold (Tagetes species) roots, which release terthienyls, chemicals that are toxic to several species of nematodes and fungi.

Only a few antagonists have been developed specifically for use in plant-disease control. Citrus trees are inoculated with an attenuated strain of tristeza virus, which effectively controls the virulent strain that causes the disease. An avirulent strain of Agrobacterium radiobacter (K84) can be applied to plant wounds to prevent crown gall caused by infection with Agrobacterium tumefaciens. Many more specific antagonists are being investigated and hold much promise for future control of disease.


Programming DNA to reverse antibiotic resistance in bacteria

At its annual assembly in Geneva last week, the World Health Organization approved a radical and far-reaching plan to slow the rapid, extensive spread of antibiotic resistance around the world. The plan hopes to curb the rise caused by an unchecked use of antibiotics and lack of new antibiotics on the market.

New Tel Aviv University research published in PNAS introduces a promising new tool: a two-pronged system to combat this dangerous situation. It nukes antibiotic resistance in selected bacteria, and renders other bacteria more sensitive to antibiotics. The research, led by Prof. Udi Qimron of the Department of Clinical Microbiology and Immunology at TAU's Sackler Faculty of Medicine, is based on bacterial viruses called phages, which transfer "edited" DNA into resistant bacteria to kill off resistant strains and make others more sensitive to antibiotics.

According to the researchers, the system, if ultimately applied to pathogens on hospital surfaces or medical personnel's hands, could turn the tide on untreatable, often lethal bacterial infections. "Since there are only a few pathogens in hospitals that cause most of the antibiotic-resistance infections, we wish to specifically design appropriate sensitization treatments for each one of them," Prof. Qimron says. "We will have to choose suitable combinations of DNA-delivering phages that would deliver the DNA into pathogens, and the suitable combination of 'killing' phages that could select the re-sensitized pathogens."

Reprogramming the system

"Antibiotic-resistant pathogens constitute an increasing threat because antibiotics are designed to select resistant pathogens over sensitive ones," Prof. Qimron says. "The injected DNA does two things: It eliminates the genes that cause resistance to antibiotics, and it confers protection against lethal phages.

"We managed to devise a way to restore antibiotic sensitivity to drug-resistant bacteria, and also prevent the transfer of genes that create that resistance among bacteria," he continues.

Earlier research by Prof. Qimron revealed that bacteria could be sensitized to certain antibiotics -- and that specific chemical agents could "choose" those bacteria more susceptible to antibiotics. His strategy harnesses the CRISPR-Cas system -- a bacterial DNA-reprogramming system Prof. Qimron pioneered -- as a tool to expand on established principles.

According to the researchers, "selective pressure" exerted by antibiotics renders most bacteria resistant to them -- hence the epidemic of lethal resistant infections in hospitals. No counter-selection pressure for sensitization of antibiotics is currently available. Prof. Qimron's strategy actually combats this pressure -- selecting for the population of pathogens exhibiting antibiotic sensitivity.

"We believe that this strategy, in addition to disinfection, could significantly render infections once again treatable by antibiotics," said Prof. Qimron.

Prof. Qimron and his team are now poised to apply the CRISPR/phage system on pseudomonas aeruginosa -- one of the world's most prevalent antibiotic-resistant pathogens involved in hospital-acquired infections -- and to test whether bacterial sensitization works in a more complex microbial environment: the mouse cage.


Irreversible Inhibition: Poisons

An irreversible inhibitor inactivates an enzyme by bonding covalently to a particular group at the active site. The inhibitor-enzyme bond is so strong that the inhibition cannot be reversed by the addition of excess substrate. The nerve gases, especially Diisopropyl fluorophosphate (DIFP), irreversibly inhibit biological systems by forming an enzyme-inhibitor complex with a specific OH group of serine situated at the active sites of certain enzymes. The peptidases trypsin and chymotrypsin contain serine groups at the active site and are inhibited by DIFP.


Alcohols

Alcohols make up another group of chemicals commonly used as disinfectants and antiseptics. They work by rapidly denaturing proteins, which inhibits cell metabolism, and by disrupting membranes, which leads to cell lysis. Once denatured, the proteins may potentially refold if enough water is present in the solution. Alcohols are typically used at concentrations of about 70% aqueous solution and, in fact, work better in aqueous solutions than 100% alcohol solutions. This is because alcohols coagulate proteins. In higher alcohol concentrations, rapid coagulation of surface proteins prevents effective penetration of cells. The most commonly used alcohols for disinfection are ethyl alcohol (ethanol) and isopropyl alcohol (isopropanol, rubbing alcohol).

Alcohols tend to be bactericidal and fungicidal, but may also be viricidal for enveloped viruses only. Although alcohols are not sporicidal, they do inhibit the processes of sporulation and germination. Alcohols are volatile and dry quickly, but they may also cause skin irritation because they dehydrate the skin at the site of application. One common clinical use of alcohols is swabbing the skin for degerming before needle injection. Alcohols also are the active ingredients in instant hand sanitizers, which have gained popularity in recent years. The alcohol in these hand sanitizers works both by denaturing proteins and by disrupting the microbial cell membrane, but will not work effectively in the presence of visible dirt.

Last, alcohols are used to make tinctures with other antiseptics, such as the iodine tinctures discussed previously in this chapter. All in all, alcohols are inexpensive and quite effective for the disinfection of a broad range of vegetative microbes. However, one disadvantage of alcohols is their high volatility, limiting their effectiveness to immediately after application.

Figure 6. (a) Ethyl alcohol, the intoxicating ingredient found in alcoholic drinks, is also used commonly as a disinfectant. (b) Isopropyl alcohol, also called rubbing alcohol, has a related molecular structure and is another commonly used disinfectant. (credit a photo: modification of work by D Coetzee credit b photo: modification of work by Craig Spurrier)

Think About It

  • Name at least three advantages of alcohols as disinfectants.
  • Describe several specific applications of alcohols used in disinfectant products.

Primary Metabolites, Secondary Metabolites and Bioconversions

Primary metabolism, also referred to as trophophase, is characterized by balanced growth of microorganisms. It occurs when all the nutrients needed by the organisms are provided in the medium. Primary metabolism is essential for the very existence and reproduction of cells. In the trophophase, the cells possess optimal concentrations of almost all the macromolecules (proteins, DNA, RNA etc.).

It is during the period of trophophase, an exponential growth of microorganisms occurs. Several metabolic products, collectively referred to as primary metabolites, are produced in trophophase (i.e., during the period of growth).

The primary metabolites are divided into two groups:

1. Primary essential metabolites:

These are the compounds produced in adequate quantizes to sustain cell growth e.g. vitamins, amino acids, nucleosides. The native microorganisms usually do not overproduce essential primary metabolites, since it is a wasteful exercise. However, for industrial overproduction, the regulatory mechanisms are suitably manipulated.

2. Primary metabolic end products:

These are the normal and traditional end products of fermentation process of primary metabolism. The end products may or may not have any significant function to perform in the microorganisms, although they have many other industrial applications e.g. ethanol, acetone, lactic acid. Carbon dioxide is a metabolic end product of Saccharomyces cerevisiae. This CO2 is essential for leavening of dough in baking industry.

Limitations in growth:

Due to insufficient/ limited supply of any nutrient (substrate or even O2), the growth rate of microorganisms slows down. However, the metabolism does not stop. It continues as long as the cell lives, but the formation of products differs.

Overproduction of primary metabolites:

Excessive production of primary metabolites is very important for their large scale use for a variety of purposes.

Overproduction of several metabolites has been successfully accomplished by eliminating the feedback inhibition as briefly described below:

1. By using auxotrophic mutants with a block in one of the steps in the biosynthetic pathway concerned with the formation of primary metabolite (this should be an intermediate and not the final end product). In this manner, the end product (E) formation is blocked, hence no feedback inhibition. But overproduction of the required metabolite (C) occurs as illustrated below.

In the above example, an un-branched pathway is shown. This type of manipulation for overproduction of metabolites can be done for branched metabolic pathways also.

2. Mutant microorganisms with antimetabolite resistance which exhibit a defective metabolic regulation can also overproduce primary metabolites.

Secondary Metabolites:

As the exponential growth of the microorganisms ceases (i.e. as the trophophase ends), they enter idiophase. Idiophase is characterized by secondary metabolism wherein the formation of certain metabolites, referred to as secondary metabolites (idiolites) occurs.

These metabolites, although not required by the microorganisms, are produced in abundance. The secondary metabolites however, are industrially very important, and are the most exploited in biotechnology e.g., antibiotics, steroids, alkaloids, gibberellins, toxins.

Characteristics of secondary metabolites:

1. Secondary metabolites are specifically produced by selected few microorganisms.

2. They are not essential for the growth and reproduction of organisms from which they are produced.

3. Environmental factors influence the production of secondary metabolites.

4. Some microorganisms produce secondary metabolites as a group of compounds (usually structurally related) instead of a single one e.g. about 35 anthracyclines are produced by a single strain of Streptomyces.

5. The biosynthetic pathways for most secondary metabolites are not clearly established.

6. The regulation of the formation of secondary metabolites is more complex and differs from that of primary metabolites.

Functions of secondary metabolites:

Secondary metabolites are not essential for growth and multiplication of cells. Their occurrence and structures vary widely. Several hypotheses have been put forth to explain the role of secondary metabolites, two of them are given below.

1. The secondary metabolites may perform certain (unknown) functions that are beneficial for the cells to survive.

2. The secondary metabolites have absolutely no function. Their production alone is important for the cell, whatever may be the product (which is considered to be useless).

Overproduction of secondary metabolites:

As already stated, the production of secondary metabolites is more complex than primary metabolites. However, the regulatory manipulations employed for excess production of primary metabolites can also be used for the secondary metabolites as well.

Several genes are involved in the production of secondary metabolites. Thus, around 300 genes participate in the biosynthesis of chlortetracycline while 2000 genes are directly or indirectly involved in the production of neomycin. With such complex systems, the metabolic regulation is equally complex to achieve overproduction of secondary metabolites. Some regulatory mechanisms are briefly discussed hereunder.

Addition of methionine induces certain enzymes and enhances the production of cephalosporin. Tryptophan regulates ergot alkaloid biosynthesis.

End product regulation:

Some of the secondary metabolites inhibit their own biosynthesis, a phenomenon referred to as end product regulation e.g. penicillin, streptomycin, puromycin, chloramphenicol. It is possible to isolate mutants that are less sensitive to end product inhibition, and in this manner the secondary metabolite production can be increased.

Catabolite regulation:

In this regulation process, a key enzyme involved in a catabolic pathway is inactivated, inhibited or repressed by adding a commonly used substrate. Catabolic repression can be achieved by using carbon or nitrogen sources. The mechanism of action of catabolite regulation is not very clearly understood.

The most commonly used carbon source is glucose. It is found to inhibit the production of several antibiotics e.g. penicillin, streptomycin, bacitracin, chloramphenicol, puromycin. The nitrogen sources such as ammonia also act as catabolite regulators (i.e. inhibitors) for the overproduction of certain antibiotics.

Inorganic phosphate (Pi) is required for the growth and multiplication of prokaryotes and eukaryotes. Increasing Pi concentration (up to 1 mM) is associated with an increased production of secondary metabolites e.g. antibiotics (streptomycin, tetracycline), alkaloids, gibberellins. However, very high Pi concentration is inhibitory, the mechanism of action is not very clear.

In some microorganisms (particularly actinomycetes), there occurs a self regulation for the production of secondary metabolites. A compound designated as factor A which is analogous to a hormone is believed to be closely involved in auto regulation for the production streptomycin by Streptomyces griseus. More such factors from other organisms have also been identified.

Bioconversions:

Microorganisms are also used for chemical transformation of unusual substrates to desired products. This process, also referred to as biotransformation, is very important in producing several compounds e.g. conversion of ethanol to acetic acid (in vinegar), sorbitol to sorbose, synthesis of steroid hormones and certain amino acids.

In bioconversion, microorganisms convert a compound to a structurally related product in one or a few enzymatic reactions. The bioconversions can be carried out with resting cells, spores or even killed cells. Non-growing cells are preferred for bioconversions, since high substrate concentration can be used, besides washing the cells easily (to make them free from contamination).

Sometimes, mixed cultures are used for bioconversions to carry out different reactions. In recent years, the yield of bioconversion is increased by using immobilized cells at a lower cost.


Bacillus cereus LMG 6923 T (strain for teaching purposes, BCCM TM /LMG Bacteria Collection) fresh garlic bulbs garlic press 10-mL plastic syringe gauze 250-mL glass containers glass rods gram scale microwave oven petri dishes Pasteur pipettes and 1-mL pipettes microbiological loops agar (bacteriological or available in supermarkets and health food stores) meat (pork or beef) table sugar kitchen salt distilled water 1-L growth-medium flasks (or equivalent microwave glass containers) glass burners discard container with bleach (20%) ethanol (70%) paper towels and Falcon and Eppendorf tubes (or equivalent).

This activity requires handling of bacteria. Therefore, students must act responsibly. They must wash their hands before and after the exercise, and they must not eat or drink in the lab. Work surfaces must be disinfected with ethanol (70%), and the materials used must be previously sterilized. Liquids, plastics, and glassware can be sterilized using a microwave oven. Metallic materials can be sterilized using boiling water. All materials in contact with bacteria must be sterilized prior to disposal.


Abstract

Standard infectious disease practice calls for aggressive drug treatment that rapidly eliminates the pathogen population before resistance can emerge. When resistance is absent, this elimination strategy can lead to complete cure. However, when resistance is already present, removing drug-sensitive cells as quickly as possible removes competitive barriers that may slow the growth of resistant cells. In contrast to the elimination strategy, a containment strategy aims to maintain the maximum tolerable number of pathogens, exploiting competitive suppression to achieve chronic control. Here, we combine in vitro experiments in computer-controlled bioreactors with mathematical modeling to investigate whether containment strategies can delay failure of antibiotic treatment regimens. To do so, we measured the “escape time” required for drug-resistant Escherichia coli populations to eclipse a threshold density maintained by adaptive antibiotic dosing. Populations containing only resistant cells rapidly escape the threshold density, but we found that matched resistant populations that also contain the maximum possible number of sensitive cells could be contained for significantly longer. The increase in escape time occurs only when the threshold density—the acceptable bacterial burden—is sufficiently high, an effect that mathematical models attribute to increased competition. The findings provide decisive experimental confirmation that maintaining the maximum number of sensitive cells can be used to contain resistance when the size of the population is sufficiently large.

Citation: Hansen E, Karslake J, Woods RJ, Read AF, Wood KB (2020) Antibiotics can be used to contain drug-resistant bacteria by maintaining sufficiently large sensitive populations. PLoS Biol 18(5): e3000713. https://doi.org/10.1371/journal.pbio.3000713

Academic Editor: David S. Schneider, Stanford University, UNITED STATES

Received: June 11, 2019 Accepted: April 23, 2020 Published: May 15, 2020

Copyright: © 2020 Hansen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: Data deposited in the Dryad repository: https://doi.org/10.5061/dryad.s4mw6m943.

Funding: This work is supported by the National Science Foundation (NSF No. 1553028 to KBW), the National Institutes of Health (NIH No. 1R35GM124875-01 to KBW NIH No. R01 GM089932 to AFR NIH K08 AI119182 to RJW), the Hartwell Foundation for Biomedical Research (to KBW), and the Eberly Family (to AFR). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Abbreviations: LTEE, long-term evolution experiment OD, optical density Pmax, acceptable burden TA, tetrazolium arabinose


Watch the video: Microbiology Chapter 11 Physical u0026 Chemical Agents for Microbial Control By Dr. Saudi (May 2022).