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Can Microbials Become Immune To Biocides?

Compiled by James McDonald, PE, CWT

BACTERIA BIOCIDES CHLORINE CHLORINE DIOXIDE CRYPTOSPORIDIUM COOLING TOWERS OXIDIZING BIOCIDES

Abstract

Question of the Day: Can microbes become immune to certain biocides?

Editor’s note: This column is based on recent discussions within the LinkedIn Industrial Water Treatment Group

Dennis: “I don't necessarily believe bacteria become "immune" to biocides. My personal belief is that it is a process of natural selection (i.e., survival of the fittest). Some bacteria will have a higher tolerance of certain biocides than others and will survive a biocide dosage at certain concentrations and contact time. With other less tolerant bacteria succumbing (dying) under the same conditions, the surviving bacteria become a greater percentage of the bacterial population and will continue multiply under consistent biocide conditions (e.g., type of biocide, concentration, and contact time). They may be encouraged to grow because of less competition for food and nutrients with the other bacteria dying off.

By periodically switching the type of biocide to a form the new population of bacteria are less tolerant of, bacterial control may once again be reestablished. Since this new biocide will likely have bacteria that are resistant to it, the periodic alternating of biocides may provide a greater degree of control than the consistent use of a single form of biocide.”

Rolf: “I agree with Dennis that immunity is not an evolved mechanism. If you kill off 99.9% of the little critters that means that 0.1% survive. The ones who do survive may just be lucky and hiding behind a dead layer, or they may have some genetic advantage that allows them to be more resistant. These survivors will multiply over time, rendering the biostats less effective.

Switching to a different biostat will start the process over again, and a slightly different colony of critters. Switching back and forth will usually do an excellent job of killing them. There is always a replenishment system (new bugs getting sucked into the system), so it is very rare to have a group of critters that will be resistant to multiple biostats.

For our purposes, evolutionary processes do not usually come into play. This means that a resistant strain will not evolve through a mutation and selection process. Cooling towers and other open systems are not subject to the specific mutation stressors needed to drive an evolutionary process.”

Chris: “Bacteria do not become resistant to biocides; rather, it is a process of natural selection, as the commenters previous to this post have described. The degree to which bacteria can more easily resist biocides is proportional to their reactivity: thus, oxidizers are the most common solution to microbial control needs in cooling water environments. But, not all oxidizers are created equal! And, using strong oxidizers at levels > 2 parts per million (ppm) will keep your system clean of microbes, while simultaneously corroding it away to uselessness! So, microbial control is always a balance between cleanliness/good heat exchange and corrosivity.”

Rehan: “We can say bacteria can’t get immunity against biocide. The proper selection of biocide is important. On the other hand, bacteria are just like us. The immunization process can occur in us, so why not in bacteria? Sometimes, if we use a single biocide in a system, the process of immunization starts there. We can’t get a 100% result. For example, Laxotanil is a well-known hypertension tablet, and widely recommended by doctors. But one of my relatives uses Laxotanil a lot, which is why its formula doesn't work on him. He can eat 2 to 3 tablets at a time without any result. It can also happen in the case of bacteria and a biocide.”

Rolf: “I am not a microbiologist or doctor. I hope there is one reading this who can confirm/refute me. There is a very large difference in vaccination against organisms and the attempt to kill such organisms.

With vaccination, you are training the body's white blood cells to recognize and prepare in advance to kill intruders. A vaccine granting immunity contains inactivated intruders that your white blood cells can learn to kill. Then when the active intruders appear, the white blood cells immediately recognize and kill them before they can overwhelm the immune system.

With biostats, we are attempting to keep the total number of intruders below a harmful threshold. There is no immune response of the intruders. They either live or they die, depending on what it already in their DNA and/or luck.

As for having to take more medication, that is an entirely different effect. Medication is usually just helping the body do something that it does poorly. That means the medication is just 'assisting' a normal process. Taking too much medication depresses the normal production of that medication by the body, eventually shutting down completely the normal production of that needed molecule. That is why you have to take more, and that is why you should always take the minimum amount of medication.”

Luca: “I agree with Dennis. Bacteria are not able to become ‘immune’ to a biocide. You have to choose the correct mixture of active principles to apply and then you have to follow what happened in the circuit during the time, repeating some killing test (better on the deposits) to know what you have to do (or better what you have probably to do).”

David: “Think about bio augmentation products. Disinfectants are rated based on a phenol coefficient (phenol being a powerful biocide), how well does the disinfectant compare to phenol), yet we (I) sell bacteria for wastewater bio augmentation of phenol. I go with natural selection.”

Fernando: “Here are some observations:

1. Microbiologists recognize two kind of different populations of microorganisms: a. those that are free-floating (planktonic), which are found in the bulk water; and b. Others are attached (sessile), which are populations colonized on surfaces.

2. The same kinds of microorganisms can be found in either population, but the sessile population is responsible for biofouling.

3. Much is known about the formation of biofilms on wetted surfaces such as the tubes of heat exchangers.

4. Those microorganisms on submerged surfaces secrete certain polymers (predominantly polysaccharides, but also proteins), which adhere firmly even to clean surfaces and prevent cells that can be swept away by the normal flow of cooling water.

5. These extracellular polymeric substances are hydrated in the natural state, forming a gel-like network around sessile microorganisms.

6. This polymer network contributes to the integrity of the biofilms and acts as a physical barrier, blocking the entry to the toxic materials and predatory organisms from reaching the living cell.

7. Those polymers can also consume oxidizers before they reach and destroy those microorganisms. As a result, control of sessile microorganisms requires dosages many times greater than biocide required to control planktonic organisms.

8. Biofilms develop slowly at first, because only a few organisms can attach, survive, grow, and multiply. So that populations increase exponentially, the depth of the biofilms increases rapidly.

9. Polymers of these biofilms are sticky and aid in the attachment of new cells to the colonized surface as well as the accumulation of nonliving debris from the bulk water. Such debris may consist of various inorganic chemical precipitates (phosphates), organic flocks, and dead cell masses.

10. Knowledge of how different antimicrobials affect microorganisms is also useful in choosing the appropriate treatment. Some may kill the organisms that they contact. Others may inhibit growth of organisms but do not necessarily kill them. These biostats can be effective if a suitable concentration is maintained in a system for a sufficient time (a continuous concentration is ideal).

11. A laboratory evaluation of the relative effectiveness of antimicrobials should be performed. This helps to identify those likely to work against the fouling organisms in the system and to eliminate those with little chance of success.

12. Because the goal of antimicrobial treatment is control or elimination of biofilm organisms, it is helpful to conduct the evaluation with sessile organisms found in deposits, as well as planktonic organisms in the flowing water.

13. The objective of any treatment program should be to expose the attached microbial population to an antimicrobial dosage sufficient (biocide) to penetrate and disrupt the biofilms.

In summary, microbes originating in the natural environment and are colonized into cooling systems by capitalizing on favorable environmental conditions. Cooling systems are favorable environments for microorganisms because they contain water, operating with acceptable temperature and pH ranges, and provide nutrients for growth. The microbial attachment to surfaces in untreated systems produces deposits that reduce equipment efficiency and can be highly destructive to cooling equipment.”

John: “The biocide has a swath of the microflora it is most effective against and then less and less effective on the fringe areas. You kill the competitors and the organisms that take over are not as affected by the biocide or perhaps the dose. With the competition gone, they double every 20 minutes or so! Halogen biocides are true contact killers; there is nothing immune to chlorine, including us. It is only a matter of dosage.”

Scott: “Bacteria in certain types of water systems can become immune to certain non-oxidizing biocides. A susceptibility study should be performed to determine which non-oxidizers have the highest kill ratios. You may want to consider some of the oxidizers that are available such as chlorine dioxide, chlorine, bromine and hydrogen peroxide. Of course, metallurgy may need to be considered when looking at oxidizers. Other issues to consider will be safety, environmental matters, and cost effectiveness.”

Fernando: “An understanding of the chemistry and modes of action of antimicrobials are needed to ensure us their proper use. We also need an appreciation of their limitations do so that microbes aren´t becoming immunes.”

A. Ric: “We have always assumed that this can happen, and it is the basis for alternating the non-oxidizing biocide in cooling systems. The biological demands of a cooling system tend to change, and I do think it makes sense to alternate non-oxidizing biocides.”

David:  “Yes, they can develop immunity.”

Nikhilesh:  “In cooling towers, the circulation of oxygen-saturated aerated water with a constant supply of organic nutrients via make-up water, suitable temperatures, and high surface-to-volume ratio favor the growth of the microbial clusters or layers known as biofilm.

The load of organic matter nutrients is unpredictable and can cause rapid multiplication of microbes in cooling tower water. These organic matters also cause a biocide demand for some antimicrobial agents and inactivate them. While we dose a certain quantity of biocide on certain assumptions of biocide demand, it may turn out to be completely inadequate.

The efficacy of pathogen kill by a biocide depends on the CT factor for that particular pathogen. C= Concentration of biocide (milligrams [mg]/liter [L]) and T = Time of contact (minutes [min]). Let us take the example of chlorine. Chlorine inactivates most pathogens, but not all.

The CT factor can be used to compare the effectiveness of chlorine against different pathogens. Higher CT factors indicate relatively higher tolerance to chlorine, while lower CT factors indicate relatively low tolerance to chlorine.

The efficacy of disinfection using chlorine is dependent not only on the pathogen itself, but also on the pH , temperature of the water, extent of turbidity in water that leads to aggregation, and encapsulation of pathogens, which become a barrier for chlorine to reach pathogens.

The CT factor for some protozoa cysts is very high. For example, CT for Cryptosporidium parvum Oocysts is >15,000. Chlorine cannot kill Cryptosporidium parvum Oocysts. Cryptosporidium parvum Oocysts is a typical example of a chlorine-resistant pathogen.

Some anaerobic bacteria, such as sulfate reducers, can develop resistance to chlorine. Other organisms can encapsulate and resist chlorine by moving to other locations in the system.

There are advantages to rotating the use of oxidizing and non-oxidizing biocides.

The U.S. EPA has limited free available chlorine (FAC) to mass limitations of 0.2 mg/L daily average concentration and 0.5 mg/L daily maximum concentrations for cooling systems— so there is a limitation on the use of chlorine.

Oxidizing biocides with contaminants such as ammonia, organic material, or nitrites, which are very common in cooling tower water, can cause chlorine demand for oxidizing biocide but not affect a properly applied non-oxidizing biocide program. Also, these are environmentally degradable, providing minimal environmental discharge problems.

Oxidizing biocides with contaminants such as ammonia, organic material, or nitrites, very common in cooling tower water, can cause a chlorine demand for oxidizing biocide but do not affect a properly applied non-oxidizing biocide program.

Since all organisms are not equally affected by each of the classes of biocide kill mechanisms, some organisms cannot be controlled for extended periods by any one biocide, so alternating chlorination with non-oxidizing biocide treatment provides the ultimate microbial control with minimum treatment costs.”

Nikhilesh: “When we say immunity of pathogens to a biocide, it is either insufficient CT not killing them like Cryptosporidium parvum Oocysts, or the reaction mechanism of a biocide with the pathogen– like sulfate reducing bacteria (SRBs) are chlorine resistant when the source of chlorine is hypochlorite.

SRBs are, however, vulnerable to chlorine dioxide oxidation because the chemistry of ClO₂ oxidation is totally different from hypochlorite. Chlorine dioxide does not hydrolyze in water like hypochlorite and remains a truly dissolved gas in water, making it much more effective to diffuse into the cell membrane of pathogens. Also, it is not pH dependent.

Thus, chlorine dioxide has a lower oxidative potential than chlorine but has 2.5 times the capacity for kill.

 

Ramanathan: “Good morning, Nikhilesh. Thank you very much for the good explanation. I read your comments. Your explanation is always very simple yet logical and easy to understand. It helps us to improve our knowledge and implement accordingly. Thank you once again.”

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