Topic 5 & 6: Bacterial Biofilms, Quorum Sensing and Bacteria-Bacteria Interactions

Name: Marcus Foo

Table of Contents:

Abstract ................................................................................................................... 2

Introduction ............................................................................................................. 3

Analysis & Discussion ............................................................................................. 3

Bibliography ............................................................................................................ 14

Appendix ................................................................................................................. 14

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A mysterious illness outbreak at a healthcare facility in mid-winter lead to an investigation to determine the source of the Pseudomonas strains found growing at 4 different locations in the facility and suitable methods of eradication and prevention. Their mechanism of growth and biofilm formation was determined as well as the mode of transmission into the patients; this information was used to determine the best method of eradiation of said Pseudomonas. The most probably source of contamination was a contaminated filter of the facility’s primary water filtration system that filtered water leading into the facility. This resulted in biofilm formation along the piping system, which was most prominent at these four sites: hot tubs, showers, humidifiers and water filtration system for drinking water. A few methods of eradication were proposed with the recommended plan of action being the replacement of contaminated parts from each of the locations and the implementation of the Ecasol distribution system to control microbial counts in the water supply. No chemical methods were used as they were inefficient or posed health risk towards patients and staff at the facility.

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Introduction

A mystery illness outbreak has occurred at a healthcare facility in mid-winter and the source of the outbreak has been pinpointed to spread of various strains of Pseudomonas species at several areas of the facility. High levels of Pseudomonas were found in the water filtration system used for the drinking water, in the humidifier used for the heating system, and in the showerheads and hot tubs of the physical therapy unit after an environmental survey of the facility. A summary of all Pseudomonas strains, whose genomic sequences were identical to isolates already found in GenBank, isolated and their respective locations is listed in the table below:

Isolate P1 P4 P5 P6 P7 P25

GenBank Rep

Pseudomonas aeruginosa PAO1

Pseudomonas aeruginosa PA14

Pseudomonas protegens

Pf-5

Pseudomonas fluorescens A506

Pseudomonas aeruginosa PA7

Pseudomonas fluorescens SBW25

Location Hot tub, Shower

Hot tub, Shower

Filters, Humidifier, Shower

Filters, Humidifier, Shower,

Hot tub

Hot tub, Shower

Filters, Humidifier, Shower

Table 1: Summary of isolates identified, their corresponding GenBank entry and location found they were found in.

Methods of removal of these Pseudomonas species from the facility and the prevention of future colonization by these bacteria are the main focus of this report. We first examined the contaminated sites and pinpointed possible sources of contamination in order to determine how these strains got there. For all of the locations above, we found that biofilms had formed on certain components of each site. Thus, proposed eradication methods would focus on the flushing on Pseudomonas biofilms, as opposed to planktonic cells, as well as the prevention of initial biofilm formation. As similar strains of Pseudomonas were found at the same sites (hot tubs, showers, filters or humidifiers) in different parts of the facility, we predicted that the bacteria originated from the same source, as opposed to spread from one site to the other.

Overall, we predict that a contamination at a primary source of water, or at least before or as the main supply reached the healthcare facility, had occurred, causing the initial ‘distribution’ of planktonic Pseudomonas cells in the facility’s water supply. Adhesion and biofilm formation most probably occurred at certain spots in the facility’s piping system but was most prominent at end points, such as the hot tubs, showers, humidifiers and water filtration system. The methods examined focused on different aspects such as the use of certain chemical or molecules, physical flushing of the system or targeting of specific genes or functions of the biofilms. While the report mostly focused on Pseudomonas aeruginosa as the main target, the methods explained is also applicable to Pseudomonas fluorescens.

Analysis & Discussion

Heatlhcare facility conditions in comparison to optimal Pseudomonas growth conditions

Pseudomonas strains, in particular Pseudomonas aeruginosa, are rather versatile bacteria, capable of colonizing areas in which these strains do not exist naturally but are able to adapt to. Typically found soil and outdoor bodies of water, Pseudomonas strains are capable to surviving in ‘unexpected’ locations such as on contact lenses or in healthcare settings, as observed in this case study. For an outbreak of illness caused by Pseudomonas strains to even occur within the healthcare

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facility, the conditions in the healthcare facility, or rather at the specific sites in the facility, must vaguely mimic optimal Pseudomonas growth conditions typically found in the environment as these bacteria must be in rather large amounts for them to case a facility wide outbreak.

Pseudomonas aeruginosa is capable of living in a pH range of 5.6 to 8.0, with an optimal pH level of 6.6 to 7.0. It is capable of withstanding temperatures of up to around 42 oC, with and optimal growth temperature of 37 oC, similar to normal temperature of the human body. They are mostly found in soil and water in the environment but are also common on surfaces in contact with soil or water; with respect to the case study, we predict Pseudomonas biofilms to form on said interfaces which will be elaborated later on. P. aeruginosa is capable of surviving in nutrient limiting conditions and in anaerobic conditions as well, provided nitrate (NO3) is present to act as the respiratory electron acceptor. P. aeruginosa is an opportunistic pathogen that can begin colonization in patients who exhibit excessive mucus production in the lungs, the most common group of patients being cystic fibriosis patients. This strain was attributed to be the main cause of outbreak, as examined in a previous report.

Pseudomonas fluorescens is also quite similar to P. aeruginosa and thus, most information obtained from scientific articles about P. aeruginosa used in this report is relatively applicable to P. fluorescens, unless specifically mentioned. P. fluorescens has an optimal growth pH level of 6.5, slightly lower than that of P. aeruginosa. It is capable of surviving between the temperature range of 4oC up to around 40oC, with an optimal growth temperature range of 28oC to 30 oC. While they are also found in soil and water in the environment, P. fluorescens is also found on plant leaves and roots, as they are closely associated to plants. Certain strains are capable of producing antimicrobial compounds, protecting plants from invasion of certain microbes. Unlike P. aeruginosa, P. fluorescens is not usually considered as an opportunistic pathogen as they rarely cause diseases in humans, unless they are severely ill and truly immunocompromised. As examined in a previous report, P. fluorescens was predicted to be a ‘passenger’ microbe and not a virulent one responsible for the outbreak of illness.

As no information was previously gathered about the healthcare facility, we predict the facility to have the following temperatures and pH at the relevant sites, based on measurements obtained from households, hospitals or buildings that house the same sites or areas in which Pseudomonas strains were found. The hot tub was predicted to have water with a temperature of about 37oC and a pH range of 7.2 to 7.6. Taps or showers in the facility were predicted to have temperatures of 4 oC to 24 oC for cold water and around 60 oC for hot water; tap and shower water were predicted to have a pH range of 6.5 to 9.5. For the water used in humidifiers and the water filtration system, we predict it would have a temperature and pH range similar to cold tap water as they would share a common water supply, which is the main water supply that feeds into the healthcare facility.

Thus, for all locations in which Pseudomonas strains were found, it is very likely that the water temperature and pH levels allowed for suboptimal to close to optimal growth of Pseudomonas as the healthcare facility conditions were relatively close to natural growth conditions for Pseudomonas. While other conditions such as nutrient availability may vary from site to site, the point remains that the healthcare facility was very suitable for Pseudomonas strains to begin colonization and remain there should the water supply have a high enough concentration of cells. The process in which the strains begin colonization would be the same for each site as all the locations had some sort of body of water or water-air interface allowing for the adhesion of biofilms.

Growth and biofilm formation of Pseudomonas strains

Biofilms are sessile, slime-encased community of bacterial cells; as opposed to free-floating, planktonic cells, biofilms can be seen as a relatively non-motile aggregate of bacterial cells that tend to have higher resistance against environmental stress; they have increased tolerance against

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antibiotics and biocides and are more sheltered against attacks by other microbes or even host immune reponse. The physical structures of biofilms range from flat, thin-layered mats to complex stalk and mushroom-like constructions (1). These biofilms form on surfaces, particularly at an air-water interface; most of the infected sites have some sort of air-water interface. Using a combination of extracellular polymeric substances and cellular structures such as the pili and flagella, cells are able to adhere to many types of surfaces. While the physical characteristics of biofilms formed in the different sites of the healthcare facility may differ, all of them would follow similar stages in their development, as shown in the diagram below:

Diagram 1: Proposed steps in biofilm development.

During stages 1 and 2, planktonic cells first loosely associate to surfaces, such as the inner walls of a pipe. This is followed by robust adhesion to said surface. The cells then begin to grow and multiply. During stages 3 and 4, the cells that are now in relatively high amounts aggregate into microcolonies; this is followed by further growth and maturation. At sufficiently high numbers, certain genes of the bacteria are expressed and are regulated through a mechanism called quorum sensing (QS). This system allows bacteria to estimate its current total population and is important in controlling its virulence in a host or further development of the biofilm such as the formation of the exopolysaccharide matrix that acts as a barrier for the biofilm. These genes and their functions are potential targets in controlling or eradicating biofilm formation. Once the biofilm is of large enough size or depending on environmental or chemical signals, cells of the biofilm are sloughed or shed through various dispersal methods; this is depicted by stage 5 in the diagram. Planktonic cells are released into the surrounding environment and will eventually adhere to another surface and repeat the entire cycle.

From this ‘lifecycle’, we can focus eradication efforts on different parts of the cycle, depending on the maturity of biofilms in the facility. However, we expect most biofilms to be in stage 5 as this would explain how patients are becoming infected by Pseudomonas strains and are experiencing related symptoms. An effective plan of action would include methods to flush existing biofilms out of the system, as well as reduce the chances of adhesion of planktonic cells in the piping system. Different stages of biofilm development could also be targeted as certain characteristics,

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such as the main component of the exopolysaccharide matrix, differs depending on the biofilm maturity.

Colonization and biofilm formation at specific sites

One of the main reasons biofilms are capable of forming within the healthcare facility is due to the sudden increase in complexity when moving from the main water source to the hospital piping system. Water supply from the common source is transported through large diameter pipes; while these pipes have large surface areas for adhesion, the constant, rapid flow of water reduces the probability of cells attaching to the pipes, inhibiting biofilm formation. However, at the points of entrance into the building, the piping system becomes more complicated. The now narrow pipes have reduced flow rate and may even have dead ends; in other parts of the system, such at faucets and shower heads, water is allowed to stagnate, increasing the chances of planktonic Pseudomonas cells adhering to the pipes and surrounding structures.

While the method in which biofilm forms at each of the sites is relatively similar, the exact location or structure at the four sites in which Pseudomonas biofilms form differs from one site to the other as they all consist of different complex components; once again, the main similarity between the sites is the presence of an air-water interface and/or a large surface area for adhesion. We examined these specific structures as one of the proposed methods of eradication is replacement of the infected site; it would be costly to replace the entire site and thus, identification and replacement of only infected structures is key is eradicating Pseudomonas biofilms and reducing the total cost of the proposed plan of action. As no information was obtained about the actual water filtration system, humidifier, shower heads and hot tubs prior, we obtained information about these commonly used systems from various website and articles.

Water filtration system

Using a typical water purification system (2) as the model of the water filtration system used in the healthcare facility (flow sheet listing all components is included in the appendix), P. aeruginosa was found to be in highest concentration at the filters (points 4, 5, 7 and 10; see appendix), in particular, point 4 which was an activated carbon filter; this was likely due to it acting as a nutrient source for the biofilms. The storage tank of the system (point 8) did not have as high of a concentration as compared to the filters but gram-negative non-fermenting bacteria, including P. aeruginosa (2). It was also found that the ultraviolet (UV) light and 0.05 μm filter at point 10 did not alter the Pseudomonas species found in the preceding points of the flow sheet; both components showed no efficiency over the gram-negative non-forming bacteria, including Pseudomonas (2). It is plausible that these filters are the main site of biofilm formation due to the large surface area and that they filter certain particles from the water as it passes through.

However, it is important to note that certain parts of the purification system, such as the filter, would be replaced on a regular basis, regardless of biofilm formation or not. In this particular case study, it is possible that an excessive accumulation of biofilm on the filter occurred due to irregular maintenance or a huge spike in Pseudomonas concentrations in the water supply contributed to the outbreak of illness at the facility.

Humidifier

As the healthcare facility was assumed to be of decently large size, it would be likely that a humidifier large enough to cater the entire facility in general would be used; thus, we based our study on a whole house humidifier, the Aprilaire model 400 humidifier. Multiple humidifiers would be required for the entire facility, but it would be sufficient to examine one humidifier for parts suitable for biofilm formation. Based on the diagrams of the model listed in the appendix (Diagrams 3 and 4), we predicted that biofilms would form on the water panel and water distribution tray; reasons for this

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are the same for the other sites. The water distribution tray has a relatively large surface area for adhesion and would allow for some stagnation of water, leading to cell adhesion and biofilm formation. The water panel is exposed to some air allowing for the formation of an air-water formation, again promoting biofilm formation. Aerosolization of contaminated water due to biofilm formation in said parts would lead to infection of patients whose medical conditions allow for the growth of opportunistic Pseudomonas strains.

Showerheads

For the analysis of the showerheads, we used a typical faucet head as our basis for determining the likely site of biofilm formation. In the study of taps in neonatal units of Northern Ireland after healthcare-acquired infections, it was found that of all components of the faucet, the highest P. aeruginosa colony counts were found from flow straighteners and metal support collars (see Appendix) (3). The more complex flow straighteners found in sensor taps had higher counts of P. aeruginosa as compared to flow straighteners in non-sensor taps (3). These flow straighteners were present as the final component in the tap body to produce a non-splashing delivery of water; thus, this component was the air-water interface that had a sufficient surface area for biofilm formation, in which water would be left stagnant should the tap not be used.

Applying this information to showerheads, the parts that would most likely be colonized by Pseudomonas strains are the membranes that have similar functions and are approximately in the same location as flow straighteners in taps; these parts would be rubber mouldings in the showerhead as well as the face that closes the head (parts 2 and 5 of Diagram 5; see Appendix). Both have relatively large surface areas and form air-water interfaces suitable for biofilm formation; for the plastic face, it is more likely that the biofilm will be on the inner surface as opposed to the exposed one. As with the taps, when the shower is not in use, stagnation of water promotes adhesion of Pseudomonas cells to said surfaces of the showerhead.

Hot tubs

The hot tub can be seen as a combination of the water filtration system, as most have their own filters, and showerheads, because there are components that create an air-water interface. As mentioned before, the use of carbon filters supplies nutrients for biofilms as well as acting as a surface of adhesion. Air induction jets of the hot tub mix warm water with air, creating a suitable environment of growth for Pseudomonas strains (See Appendix). In addition to these two components, a case study of an outbreak of P. aeruginosa infections caused by a contaminated drain in a whirlpool bathtub also suggests the possibility of the formation of biofilms in the drain of hot tubs (4). In the case study, the faucet of the whirlpool tub did not yield Pseudomonas; instead, the drain of the bathtub that closed approximately 2.45 cm below the surface of the tub was found to contain a biofilm. When water was added to the tub, it was found that the water yielded strains of P. aeruginosa found from direct cultures from the drain (4). Thus, it is also likely that drain may also be the main site of biofilm formation. It is worth noting that water in hot tubs tends to be filtered and recirculated; this implies that as soon as a biofilm has formed within the piping of the hot tub, it is now effectively circulating contaminated water throughout the hot tub, increasing the likelihood of contamination of other hot tub components.

Source of Pseudomonas contamination

All the contaminated sites in the healthcare facility essentially derive water from the same source; thus, it is highly probable that the common water source was already contaminated, leading to the formation of biofilms at the very end of plumbing system. In this case study, we would expect a common source contamination to be a filter problem at the primary healthcare facility filtration system, as shown in a simplified diagram of water distribution in the facility below. Irregular

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maintenance or increased concentrations of Pseudomonas strains in the incoming water supply would lead to contamination of all mentioned sites in the facility.

Diagram 2: Simplified flowchart of water distribution system in the healthcare facility.

An alternate source of Pseudomonas contamination would be from localized infection; one contaminated faucet may lead to the contamination of other faucets, hot tubs and showers. For example, a patient or staff that uses the contaminated faucet may inadvertently spread Pseudomonas strains to other parts of the facility; contamination of shared facilities such as showers and hot tubs will rapidly lead to the spread of Pseudomonas strains onto other patients, causing an outbreak.

However, in this report, the methods to eradicate the existing Pseudomonas biofilms are more important than identifying what was the main source of contamination in the first place. This is because these methods emphasize the removal of existing biofilms as well as the prevention of the formation of new biofilms as to avoid another outbreak. Should proper protocols and method be implemented at the facility, it would be enough to prevent the growth of opportunistic pathogens regardless of their source.

Modes of transmission from site to patients

It is likely that patients were exposed to the Pseudomonas strains through more than one site in the healthcare facility; the probability of getting infected increased as total exposure time in contaminated sites increased, especially if patients had no other choice but to use contaminated facilities. By examining the most probable mode of transmission to the patients, we could identify the site most likely to cause infection of patients are focus eradication efforts there first.

Ingestion

While Pseudomonas strains were found in the water filtration system used for drinking water, it is actually unlikely that patients were infected through the consumption of contaminated drinking water. As most of the symptoms observed in a previous report were respiratory related, infection via ingestion would be highly unlikely. This would only be possible if patients were undergoing antibiotic treatments that caused the suppression of normal host microflora, allowing for colonization of pathogenic Pseudomonas strain along the digestive tract. This mode of transmission would be only applicable to the contaminated water filtration system as we assume that the other sites were not used as a source of drinking water.

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Inhalation/Aerosolization

The most probable mode of transmission of Pseudomonas strains into patients is through aerosolization of contaminated water; this would be observed for the showers, hot tubs and humidifiers. Inhalation of aerosolized opportunistic, pathogenic bacteria may lead to respiratory infections and dissemination of organisms from the lungs into the bloodstream (5). From a study that quantified the amount of bacteria found in shower water and shower aerosol (mist), the average total bacterial counts were 3.4 x 104 cells/m3 in shower aerosol; as the researchers were conducting the study in a ‘normal’ environment, we would expect the bacterial count in shower aerosol in the current case study to be higher than said amount (5). Patients would also have a much higher exposure time to aerosolized bacteria as the humidifiers would be constantly running and we would expect them to use the shower frequently; those using the hot tubs would also spend a relatively long time exposed to contaminated mist from the hot tub water. Thus, we view aerosolization of Pseudomonas strains as the main method of transmission that caused the illness outbreak in the healthcare facility. An effective plan of action should include methods to reduce the exposure of patients to aerosolized bacteria at the three relevant sites in the facility.

Intravenous Access Device

It is also possible, for certain patients like stem cell transplant patients and many other immunocompromised patients, to undergo treatments that require the use of a central venous access device; this would include the administration of chemotherapy and other medications. Opportunistic microbes in shower water may contaminate the central venous catheter and provide a mechanism for bacterial invasion into the bloodstream; this combined with the possibility of minor unhygienic protocol greatly increases the possibility of infection via intravenous access devices, particularly in patients whose immune systems are not functioning at normal conditions. However, with respect to the current case study, we do not expect this mode of transmission to be the main cause as there was no reports of Pseudomonas strains on these devices.

Cross-infection through ventilators

Ventilated patients in intensive therapy units are particularly susceptible to respiratory infections such as those caused by P. aeruginosa; this is mainly due to the administration of broad-spectrum antibiotics and pre-existing immunosuppression (6). In a case study at an intensive therapy unit, it was found that the water used in humidifiers had caused the colonization of P. fluorescens in patients (6). Unhygienic refilling protocols of the humidifier water caused the plastic containers used to refill the ventilators to act as a reservoir for the Pseudomonas strain. The allocation of humidifier water to each patient, combined with more strict maintenance protocol, allowed for the significant reduction of infection of patients admitted to the unit. Thus, the sharing of contaminated ventilator or personal humidifiers is another likely method of transmission in this case study.

Methods of eradication of Pseudomonas strains

The method of eradication of Pseudomonas strains from the healthcare facility were examined, as best as possible based on the following criteria: health/ safety issues, effectiveness, advantages and disadvantages. Cost is also an important aspect but will only be included if said information is found. While certain information was unattainable or estimated for some of the methods, we examined various techniques separately in removing existing biofilms, preventing initial biofilm formation, and interfering with further biofilm formation. An effective plan of action would require a combination of methods to ensure thorough removal.

Physical flushing/ Replacement of parts

The most direct way to remove biofilms from the infected sites is to remove the entire site itself; however, to replace all the showerheads, hot tubs, humidifiers and the entire water filtration

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system would be too costly. Thus, it would be more efficient to replace the parts that were predicted to be key sites for biofilm formation, as explained in a previous section (see Colonization and biofilm formation at specific sites of Analysis and Discussion). A summary of parts to replace, excluding costs as parts vary greatly in price, as well as any required maintenance or necessary protocols is as shown below:

Contaminated site Key parts to replace Other notes Water filtration system All filters Filters should be replaced on a

regular basis due to quick buildup of particles including microbes

Humidifier Water distribution tray; Water panel Water panels should be replaced more frequently as it acts as the main air-water interface

Showerheads Rubber moulding; Plastic face - Hot tub Filters; Jets; Drain Filters should be replaced on a

regular basis, while drains should be thoroughly cleaned

Table 1: Summary of key parts to replace in each infected site.

It should also be noted that any structures surrounding these key parts should also be checked for the presence of biofilms; a newly replaced part may be quickly colonized should biofilms from nearby parts spread onto it. However, should all contaminated parts be replaced efficiently, it would be much easier to now prevent the formation of new biofilms as opposed to constantly removing repeatedly forming biofilms. This assumes that parts would be only replaced once (for parts that do not require regular maintenance) and maintained in good conditions. In terms of health and safety, the new parts should not have any impact on patients who use these sites.

Antibiotic Application

Another potential method to rid biofilms from the infected sites is the application of antibiotic in the water supply. For example, with the right concentrations, azithronycin (AZM), an inhibitor of protein synthesis, as well as two other antibiotics, ceftazidine and ciprofloxacin can affect the quorum sensing of the biofilms, altering the membrane permeability (1). Other types of antibiotics that target different structures and function of bacteria could also be used. However, there are many disadvantages in antibiotic treatment, the main problem being selective pressure and antibiotic resistance. Biofilms are naturally more resistant towards antibiotic due to structure of the biofilm; the antibiotic might not be able to penetrate through the matrix of the biofilm due to the formation of stationary phase ‘dormant zones’ that exhibit less of the cellular activities that many antibiotics target. If the biofilm is not inherently resistant to the antibiotic but is not completely cleared, a mutant cell may be able to survive the treatment and pass the gene responsible for resistance to other cells; the fact that the biofilm is an aggregate of cells allows for the rapid spread of this resistance gene. Another disadvantage to antibiotic application is the potential impact on patients who use these sites, in particular, those who consume drinking water. Consumption of water containing antibiotics would suppress natural gut flora, increasing the possibility of an opportunistic pathogen infection. Moreover, it will again induce selective pressure and cause antibiotic resistance should the patient be suffering from an existing bacterial infection that is not cured. The waste water generated would also be potentially dangerous to be released into the environment, for the same reasons mentioned above.

Ecasol/Electrochemical Activated Solution

Ecasol or rather ECAS (Electrochemical Activated Solution) is a pump system capable of significantly reducing bacterial contamination of taps and output water through generation of hypochlorous acid, HOCl, mimicking human defense against pathogens. The system uses sequential

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filtrations and automated dosing (2.5 ppm) using the pH-neutral electrochemically activated solution, Ecasol (7). Based on a study conducted on the use of Ecasol in controlling bacterial contamination of washbasin taps and output water at Dublin Dental Hospital, mean bacterial counts of 482.5 colony-forming units (cfu)/ml for hot water samples and 5022 cfu/ml for cold water samples were reduced to 1 cfu/ml and 2 cfu/ml respectively during the 54-week study (7).

In terms of health and safety, biosafety studies showed that Ecasol 100 ppm, 40 times higher than the levels used to treat water in the study, had no cytotoxic effect on human epithelium tissue and was readily inactivated by levels of protein found in saliva (7). While a shock-dosage of Ecasol 100 ppm will be initially required for eradication of exisiting biofilms, the 2.5 ppm dosage would be safe for usage and consumption; during the period of Ecasol 100 ppm, it would be recommended to have an external source of water supply should the healthcare facility deemed said concentration to be worrying. The human antioxidant system is also capable of neutralizing HOCl, making Ecasol a viable solution.

In the same study, Ecasol was considered to be a cost-effective long-term solution for the problem of bacterial contamination. The installation costs of Ecasol-generating equipment, pumps and probes was approximately € 35,000 (approximately US$ 43745.28, as of 11/17/2014) while annual running costs were less than € 1000 (approximately US$ 1249.87, as of 11/17/2014); annual maintenance costs were approximately € 4000 (approximately US$ 4999.46, as of 11/17/2014). Overall, Ecasol seems to an effective, cost efficient solution in maintaining a low base line level of bacteria; combining this with other methods would ensure the eradication of Pseudomonas strains from the facility and reduction in new biofilm formation.

Chemical application targeting biofilm formation

At a molecular level, it possible to use specific chemicals to interfere with biofilm formation in order to eradicate the Pseudomonas biofilms at the infected sites. For example, a chemical may impede the formation of a mature biofilm by inducing dispersal of the biofilm or causing the breakdown of the exopolysaccharide matrix. The only drawback to chemical application in the water supply is its potential effects on humans that consume the treated water. Two examples of chemical that target and interfere with biofilm formation are dispersin B and alginate lyase.

Dispersin B is a soluble β-N-acetylglucosaminidase molecule produced by the bacteria Actinobacillus actinomycetemcomitans. It is capable of causing dispersal and detachment of mature biofilms, such as those produced by Staphylococcus epidermidis as well as some other bacterial species (8). Dispersin B catalyzes the breakdown or hydrolysis of linear polymers of N-acetyl-D-glucosamines (a component of the biofilm’s integrity) found in the biofilm matrix, causing the dispersal of the biofilm. It also inhibits biofilm formation and sensitizes pre-formed biofilms to antimicrobials, bacteriophages and host defenses. Moreover, it does not kill or inhibit bacterial growth, reducing the pressures on evolution of resistance against it. In terms of health and safety, an in vitro assay published in the Maerican Society for Microbiology showed that humand cells exposed to dispersin B (at concentration ranging from 40 to 400 mg/ml) did not cause significant shanges in the general morphology of the monolayer or in the major cytoskeletal components that holds the cell together (8).

However, dispersin B works effectively against gram positive bacteria but the Pseudomonas strains that have colonized in the facility are gram negative strains. Moreover, the molecule can be very strain specific so different types would be required to disperse multispecies biofilms. However, dispersin B is still a valid method in flushing out existing biofilms and can be seen as an alternative to replacement of contaminated parts. It would be best that the water supply not be used while this flushing is going on.

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Alginate is an important polysaccharide that is used to anchor the bacteria to a surface, thus influencing the extent of cell detachment from the biofilm. Increased expression of alginate lyase, in a study of the role of alginate lyase in cell detachment of P. aeruginosa, lead to increased cell detachment (9). Alginate lyase also improves the efficacy of various antipseudomonal antibiotics. However, P. aeruginosa has been reported to produce low-molecular-weight polysaccharide other than alginate, which enhances the attachment to precoated surfaces. Thus, the sole application of alginate lyase will not be sufficient to completely flush existing biofilms from the contaminated sites. No information was obtained after the safety of consumption of alginate lyase in humans and thus, should not be the main choice in eradicating the Pseudomonas biofilms.

Chemical application targeting quorum sensing

Similar to chemicals that target biofilm formation, another possible method to remove biofilms from infected sites is the use of chemicals that instead affect quorum sensing systems. This would reduce or halt the expression of genes regulated by quorum sensing, possibly affecting the biofilm’s integrity. Examples of these compounds include quorum sensing signal antagonists that blocks or reduces the extent of quorum sensing regulated gene expression by binding to certain receptors. Specific QS signal antagonists include: PD12, V-06-018, C30, B7, 3oxo-C12-D10, 3oxo-C12-acHone, 3oxo-C12-acPol. This means key genes such as those that produce exopolysaccharides or those that maintain the matrix structure and integrity will be partially shut down; this reduces the biofilm’s resistance towards antibiotics and other compounds. By combining this method with antibiotic treatment, we could theoretically eradicate biofilms from the contaminated sites. An advantage of this method is that it allows for gentler evolutionary pressure towards drug resistance as the quorum sensing system does not control processes essential for cellular survival or growth (1).

However, on its own, these chemicals do not actually cause enough degradation to flush existing biofilms from the piping system. Moreover, bacteria often have more than one type of quorum sensing system and thus, for this method to be effective, all quorum sensing pathways must be disrupted. In terms of health and safety, no information was obtained about the effects on these molecules on human cells and would be best avoided if possible. Overall, it is best to not implement this treatment method as it is reliant on the antibiotic treatment which has its own disadvantages.

Antimicrobial compounds

Other compounds or metals such as ionic silver and gallium have antimicrobial properties that negatively affect biofilms and their formation. For example, ionic silver was shown to clear planktonic P. aeruginosa infections when used in wound dressing; applied on a larger scale, it is theoretically possible to degrade biofilms with the right concentrations (1). Below is a list of antimicrobial compounds and their mechanism of action on biofilms:

Compound Mechanism of action

Ionic silver High concentration of ionic silver disperses biofilms

Gallium (Ga) and Desferrioxamine-gallium (DFO-Ga)

Ga and DFO-Ga compete out iron, an important cue for initiation of biofilm formation

Nitric oxide (NO)-releasing silica nanoparticles

Silica nanoparticles provide a means for rapid diffusion of toxic NO for better biofilm dispersion; NO acts as a signal for dispersal in P. aeruginosa

Table 2: List of antimicrobial compounds and their mechanism of action on biofilms (Adapted from Njoroge, J., and V. Sperandio. 2009. Jamming bacterial communication: new approaches for the treatment of infectious diseases. EMBO Mol Med. 1:201-210.)

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While these compounds are effective on an individual application basis, trying to upscale the concentrations and implementing them into the water distribution system would prove to be difficult and possibly hazardous to health if used in excess.

Other Methods

Overall, general cleaning and hygienic protocols should be implemented in the healthcare facility as to avoid contamination from one site to another, either by patients or staff. Sites such as the hot tubs and showers should be regularly cleaned and tested for microbial counts to ensure it does not act as a site for profuse biofilm formation. For sites that require regular maintenance such as the replacement of filters, it would be better if maintenance were scheduled more frequently as to avoid build up of particles and microbes on filters and other relevant structures. Allocation of specific sites for specific patients or staff would also minimize the spread of microbes; for example, designation of certain sinks, showers and hot tubs for patients suffering from the same infection or illnesses on each floor would be beneficial.

Recommended plan of action

Based on the methods of eradication mentioned above, it would be best to combine the flushing of existing biofilms from the contaminated sites with a long term method to prevent colonization of Pseudomonas strains at new sites in the facility. Thus, we feel that the best combination to achieve this goal is to replace contaminated components of sites as well as implement the Ecasol system in the water distribution system. The replacement of parts allows for immediate removal of biofilms from the facility while Ecasol would be able to target biofilms that might have formed in places not mentioned in this report. During the period of shock-dose Ecasol of 100 pm, it is recommended that the facility find a temporary alternative water supply, should the management be concerned about unreported effects of the usage of Ecasol; at later running concentration of 2.5 ppm, it would be safe to assume that the water supply is safe for consumption. With the exception of minute amounts of hypochlorous acid in the water supply, there would be no other adverse effects on the patients and staff health as no other chemical would be present in the water.

While replacement of parts could be cost ineffective in the short run, we propose that the financial resources from the venture capitalist, that was initially planned to be used for the development of a molecule to flush out biofilms, be redirected to the purchase and replacement of contaminated components in the hospital. Based on the information gathered about potential chemical or molecules that could be used to flush out or degrade biofilms, we believe that the advantages of using any one of these chemical do not outweight the potential disadvantages of using them; many of these chemicals need to be in high concentrations for them to be effective while other chemicals had no significant studies on the potential adverse effects on human cells. The physical replacement of parts ensures that the biofilms are almost completely removed from the most contaminated areas; the use of chemicals does not immediately guarantee the flushing of Pseudomonas biofilms from the water distribution system. Thus, we recommend the replacement of contaminated components at each site and the implementation of Ecasol to eradicate Pseudomonas biofilms from the facility and prevent the reoccurrence of an outbreak.

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Bibliography

1. Njoroge, J., and V. Sperandio. 2009. Jamming bacterial communication: new approaches for the treatment of infectious diseases. EMBO Mol Med. 1:201-210.

2. Penna, V. T. C., Martins, S. A. M., and Mazzola, P. G. 2002. Identification of bacteria in drinking and purified water during the monitoring of a typical water purification system. BMC Public Health. 2: 13.

3. Walker, J. T., A. Jhutty, S. Parks, C. Willis, V. Copley, J. F. Turton, P. N. Hoffman, and A. M. Bennet. 2014. Investigation of healthcare-acquired infections associated with Pseudomonas aeruginosa biofilms in taps in neonatal units in Northern Ireland. J Hosp Infect. 86:16-23.

4. Berrouane, Y. F., L. A. McNutt, B. J. Buschelman, P. R. Rhomberg, M. D. Sanford, R. J. Hollis, M. A. Pfaller, and L. A. Herwaldt. 2000. Outbreak of Severe Pseudomonas aeruginosa Infections Caused by a Contaminated Drain in a Whirlpool Bathtub. Clin Infect Dis. 31: 1331-1337.

5. Perkins, S. D., J. Mayfield, V. Fraser, and L. T. Angenent. 2009. Potentially Pathogenic Bacteria in Shower Water and Air of a Stem Cell Transplant Unit. Appl Environ Microb. 75:5363-5372.

6. Redding, P. J. Pseudomonas fluorescens cross-infection due to contaminated humidifier water. 1980. Br Med J. 281:275.

7. Boyle, M. A., M. J. O’Donnell, A. Miller, R. J. Russell, and D. C. Coleman. 2012. Control of bacterial contamincation of washbasin taps and output water using Ecasol: a one-year study. J Hosp Infect. 80:288-292.

8. Donelli, G, I. Francolini, D. Romoli, E. Guaglianone, A. Piozzi, C. Ragunath, and J. B. Kaplan. 2007. Synergistic Activity of Dispersin B and Cefamandole Nafate in Inhibition of Staphylococcal Biofilm Growth on Polyurathanes. Antimicrob Agents Ch. 51:27332740.

9. Boyd, A, and A. M. Chakrabarty. 1994. Role of Alginate Lyase in Cell Detachment of Pseudomonas aeruginosa. Appl Environ Microb. 60:2355-2359.

Appendix

Flow sheet of a typical water purification system

Point 1: Storage Tank (feed water)

Point 2: Two multimedia Filters (primary filtration)

Point 3: Two water softeners (hardness reduction)

Point 4: One filter of activated carbon (chlorine removal)

Point 5: One 5.0 μm filter (removal of particle materials)

Point 6: One reverse osmosis membrane system (removal of organic and inorganic substances)

Point 7: One continuous deionization column (removal of dissolved minerals and salts)

Point 8: One storage tank (treated water)

Point 9: Light UV: 254 nm (reduce TOC)

Point 10: Three 0.05 μm filters in parallel (removal of particles and bacteria)

Points 11, 12, 13: Loop of distribution of purified water for consumption

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Diagram 3: List of parts of the whole house humidifier used as the model for humidifier used in the healthcare facility. (Note the water distribution tray and water panel)

Diagram 4: Other components of the whole house humidifier used as the model in this case study. (Note the water distribution tray and water panel)

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Diagram 5: Close up image of the water distribution tray of the whole house humidifier

Diagram 6: Close up of the water panel of the whole house humidifier

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Diagram 7: Structural layout of the taps studied said investigation of neonatal units in Northern Ireland (Note the flow straightener)

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Diagram 8: Structural layout of a typical showerhead (Note the rubber moulding and plastic face)

Diagram 9: Cross-sectional diagram of a typical hot tub (Note the airjets and unlabeled filter (next to 3-way valve); this diagram does not include a drain)