Understanding the Importance of Microbial Water Quality in Reprocessing RMD

Date: 29/07/2015
Read Time: 10 min
Author: Dr Surani McCaw, B.E. (Chemical), Ph. D.

Key points:

• Municipal authorities only monitor water parameters that affect the wellbeing of the human gut, not the presence of contaminants which can be fatal when in contact with the blood stream.
• To destroy the most resistant types of microorganisms, the user must employ exposure times and a concentration of germicide that achieves complete destruction.
• In disregarding the importance of microbial water quality, we are playing Russian roulette in Healthcare.

The importance of the quality of water used in all stages of reprocessing of Reusable Medical Devices (RMDs) is emphasised in numerous scientific research papers for decades.

It is a well published and well-known fact (not just from scientific research but also due to evidence-based practices) that physical, chemical and microbial impurities present in our water supply can affect the efficacy of disinfection and sterilisation of RMDs.

Due to the inconsistency in town water supply and quality from a single / previously used source, a direct consequence of the change in weather patterns, Municipal Authorities are forced to use a mixture of water sources to meet their contractual obligations.  Understandably, their commercial obligation is to produce quantity of water which meets the drinking water standard.  Therefore, they only monitor and measure water parameters that affect the wellbeing of the human gut, not the presence or the concentration of contaminants such as endotoxins and other pathogens which can be fatal when in contact with the blood stream.

As per the literature in Centers for Disease Control and Prevention [1], the number and the location of microorganisms and their resistance to chemical germicides and sterilisation processes are critical factors affecting the efficacy of disinfection and sterilisation.

This document highlights a number of relevant points;

• The concentration of microorganisms can affect the disinfectant exposure time. Spaulding illustrated that with only the concentration as a variable, 10 mins exposure time was required to kill 10 Bacillus atrophaeus (formerly Bacillus subtilis) spore in comparison to 3 hours to kill 100,000 Bacillus atrophaeus spores;
• Depending on type / nature of microorganisms, their intrinsic resistance mechanisms to disinfection vary. For example, spores are resistant to disinfectants because the spore coat and cortex act as a barrier, mycobacteria have a waxy cell wall that prevents disinfectant entry, and gram-negative bacteria possess an outer membrane (lipopolysaccharides) that acts as a barrier to the uptake of disinfectants.

To destroy the most resistant types of microorganisms (i.e., bacterial spores), the user needs to employ exposure times and a concentration of germicide needed to achieve complete destruction.

• Except for prions, bacterial spores possess the highest innate resistance to chemical germicides, followed by coccidia (e.g., Cryptosporidium), mycobacteria (e.g., M. tuberculosis), nonlipid or small viruses (e.g., poliovirus, and coxsackievirus), fungi(e.g., Aspergillus, and Candida), vegetative bacteria (e.g., Staphylococcus, and Pseudomonas) and lipid or medium-size viruses (e.g., herpes, and HIV);
• Water hardness reduces the rate of kill of certain disinfectants as divalent cations such as Calcium and Magnesium, in hard water interacts with the disinfectant to form insoluble precipitates;
• Inorganic contaminants in water can protect microorganisms from disinfection and sterilisation by acting as a physical barrier;
• Microorganisms may be protected from disinfectants by production of thick masses of cells and extracellular materials, or biofilms. Biofilms are microbial communities that are tightly attached to surfaces and cannot be easily removed.  Once these masses form, microbes within them can be resistant to disinfectants by multiple mechanisms, including physical characteristics of older biofilms, genotypic variation of the bacteria, microbial production of neutralizing enzymes, and physiologic gradients within the biofilm (e.g., pH). Bacteria within biofilms are up to 1,000 times more resistant to antimicrobials than are the same bacteria in suspension (planktonic phase);

Disrepute to credential literatures such as the one above [1], there are some articles which state that water does not appear to be a vehicle of contamination.

One such article [2] states that endotoxins were negative on surgical instruments depyrogenated in steam steriliser where the steam generator was artificially contaminated with endotoxins.  Hence, concluding that water does not appear to be a vehicle of contamination, neither when cleaning surgical instruments nor when sterilizing them with steam.

Research paper by Lixiong Li. et al. [3] states that sterilisation and endotoxin inactivation can be achieved by dry heat or moist heat but they use different mechanisms.  While dry heat sterilisation is thought to occur by the oxidation of essential cell constituents, moist heat kills microorganisms by coagulating and denaturing their enzymes and structural protein.  The precise mechanistic steps of endotoxin inactivation are unknown.

There are several research papers such as the one by Michael E. Dawson [4], which state that temperatures in excess of 180°C is required to effectively destroy endotoxin.  This paper states that below 180°C, depyrogenation may be incomplete even after extended periods.  It also states that the time required to achieve a given level of endotoxin destruction decreases as temperature increases, e.g. a 3 log reduction requires 65.4 minutes at 190°C but only 1.5 minutes at 250°C.

Furthermore, it should be emphasised that the amount of endotoxin present in clean steam depends on the dedicated steam generator’s design in removing endotoxins.  Like all validation processes, the validation process of the clean steam generator design is based on a maximum feed water endotoxin limit.  A dedicated steam generator is not capable of continuously maintaining endotoxin level as per EN285 if we continue to challenge it with endotoxin levels greater than its design limit.

As stated in number of research articles [5], [6], once endotoxin are present on products, it is not reliably destroyed by disinfection, steam sterilisation processes, or ethylene oxide sterilisation.  It is far better to keep finished products and components relatively endotoxin-free rather than have to remove it once present.  Water containing ≤ 100 EU/mL has been determined to leave very little endotoxin residue on instrument surfaces, thereby minimising the potential for a pyrogenic reaction in the patient after surgery [7].

Although, steam can inactivate all microorganisms, the effectiveness of the sterilisation process depends on the clean steam generator design and the bioburden of the load.  As depyrogenation is not reliably achieved at the temperature used in steam sterilisers, it is critical that water used for the final rinsing process and for the generation of steam for the sterilisation process is low in physical and chemical impurities and is microbially safe with low level of endotoxins.  Once endotoxins are generated in the feed, the reliable destruction of these pyrogens are not possible in a conventional steriliser.

Research paper by Goveia VR et al. [8], investigates endotoxins in sterilized surgical instruments used in hip Arthroplasties.

The study was conducted using drinking water in a public teaching Hospital in Brazil prior to the publication of their 2012 regulation for the use of purified water low in bacteria and endotoxins for the rinsing of critical health products.

Endotoxins in quantity ≥0.125 UE/mL (i.e. the sensitivity to detect endotoxins was established at 0.125 UE/mL) were detected in 13.3% of the instruments tested.

This paper cites that in the United States, 500,000 arthroplasties are performed every year.  40,000 of these undergo revisions due to aseptic loosening, often associated with the presence of endotoxins.  Presence of osteolysis around the prosthesis is observed in aseptic loosening, due to the presence of particles generated from the implant.  If endotoxins are present, they adhere to these particles, being probably one more factor which induce the loosening.

These findings highlight the importance of understanding the role of endotoxins in these situations of inflammatory response, where there are no clinical and microbiological signs of infection.  It states that initially, it might seem contradictory to address aseptic loosening without excluding the presence of a subclinical level of bacteria that may colonize orthopaedic implants, form biofilm which is the source of endotoxins.

With credential research papers to support (cited above and below), it can be stated that it is critical that water quality standard in Healthcare quantifies the level of bacteria and endotoxins present in the water used for the reprocessing of RMDs for the below reasons;

1. It is well understood that in water not treated using Reverse Osmosis, the presence of organic and inorganic contaminants found in the municipal water supply would hinder the cleaning, the disinfection and the sterilisation process. However, RO technology has its downfall.

Due to the potential oxidation of RO membranes by the residual disinfectants present in our municipal water supply, the removal of these residual disinfectants is critical for the longevity of the RO plant.  Due to the absence of residual disinfectant and due to the fact that RO membranes are not absolute filters, some microorganisms would enter the purified water.  These microorganisms can then proliferate within the purified water storage tank and the distribution pipework.

2. Most RO systems used in laboratory and healthcare applications operate a flow through system with UV steriliser and endotoxin filter for the control of microorganisms.

Although some rely on UV steriliser as a disinfection process, there are microorganisms such as Bacillus anthracis and Bacillus subtilis which are highly resistant to UV irradiation.  Moreover, UV irradiation is only effective when the organisms are in direct contact with UV light as the process does not general residual disinfectant.  Moreover, high concentration of free swimming (planktonic) bacteria can mask UV irradiation hence making it less effective.  The bacteria that escape the irradiation process will continue to proliferate within the distribution network.  Hence regular routine disinfection using either chemical or high temperature between 80 and 90 ^oC is critical to control microorganism colonisation.

3. Research paper by R. L. Anderson et al. [9] states that the resistance of microorganisms to antimicrobial agents in general and chemical germicides in particular is controlled by a number of factors including the culture history and strain of the microorganism, the nature of the suspending menstruum as well as a variety of physical factors such as temperature, pH, and hardness. Further, microorganisms in their naturally occurring state have been shown to be significantly more resistant to chemical germicides than microorganisms that have been sub cultured on artificial culture media.

Their research indicated that microorganisms were observed to survive and re-establish in PVC pipes previously exposed to chemical germicides.

This re-establishment of microbial contamination suggests a continuous reservoir of organisms adhering to and shedding from the interior PVC wall surface. Organisms showing survival in five PVC test pipes were P. aeruginosa (after exposure to chlorine or a quaternary ammonium compound) and P. pickettii (after exposure to iodophor disinfectant, chlorine, or 70 percent ethanol).  This is strong evidence that these organisms survived within the colonized PVC pipes after exposure for seven days to a variety of chemical treatments.

The physical thickness of cellular and extracellular material that form on PVC pipe surfaces could protect organisms from the action of germicidal chemicals and serve as a continuous reservoir for microbial contamination in test pipes and of water flowing through pipes.

In 1984, during an investigation of a manufacturing plant which produced iodophor antiseptics, P. aeruginosa contamination was recovered throughout the PVC water distribution system.  Subsequently it was found that intrinsic contamination of a poloxamer-iodine antiseptic solution resulted when the formulated iodophor was allowed to stand in contaminated PVC pipes (pipes between the mixing tank and storage tank and between the storage tank and the bottling area) prior to bottling.  As remedial measures, the company removed all PVC pipes, installed new stainless pipe, and initiated a hot water sanitizing procedure of all process water and product distribution pipes.  In addition, better and more frequent microbiological quality control procedures on municipal and process water were instituted.

These types of laboratory studies are important in identifying possible mechanisms by which microorganisms could survive in disinfectant solutions for extended periods of time and to determine how bacterial organisms could continuously contaminate the various parts within a water distribution system. The best way to maintain a water distribution system in a safe condition is to limit the number of microorganisms by proper and scheduled maintenance and to routinely sanitize pipes or tubing that transport water. The laboratory findings presented here add to development of effective strategies for disinfecting water or product distribution lines and holding tanks in manufacturing plants and in laboratory, medical, and pharmaceutical facilities where microbial contamination by pseudomonads or other naturally occurring gram-negative water bacteria is a problem.

4. Literature by Tim Sandle [10], states that vast majority of aquatic bacteria found in water are gram-negatives and that the risk increases as water undergoes greater processing, where bacteria are destroyed, thereby increasing the potential risk of endotoxins. The environmental endotoxin produced by the gram-negative bacteria in water is highly heterogeneous and the potency varies according to bacterial species and strain; and by solubility and molecular weight.

The more potent endotoxins are those of the highest molecular Lipid A weight and those which are most disaggregated.  In water, endotoxin has a tendency to aggregate to form vesicles (membranous structures).  The size of these vesicles is dependent upon the type of the LPS structure and the pH, salt concentration and purity of the water. In pure water, the size is typically between 20,000 and 100,000 Daltons. Such environmental aggregates of endotoxin have a high affinity to surfaces.

The importance of physical, chemical and microbial water quality is highlighted in the literatures presented in this document.

Achieving good healthcare outcomes is a universal responsibility, which starts from the government bureaucrats, healthcare personnel in hospitals and the general public.  Recognising the effort of international healthcare experts in setting ISO and standards in individual countries, based on their experience and evidence-based practices is paramount in securing the universal healthcare system.  This is even more critical due to the increased human migration and frequent air travel we are experiencing which reduces the overall footprint of our planet.

Disregarding the important of microbial water quality without quantifying their limits and without routine sampling and analysis (more frequent than annual) to check the performance of the purified water plant, we are entering the danger zone and playing Russian Roulette with our Health.

1. “Factors Affecting the Efficacy of Disinfection and Sterilization”, Centers for Disease Control and Prevention, National Center for Emerging and Zoonotic Infectious Diseases (NCEZID), Division of Healthcare Quality Promotion (DHQP), 2016.
2. Flocard, V & Goullet, D & Freney, J., “Evaluation of the endotoxin risk posed by use of contaminated water during sterilization of surgical instruments”, Zentralsterilisation – Central Service., 14. 93-102, 2006.
3. Lixiong Li, Chris L. Wilbur, and Kathryn L. Mintz, “Kinetics of Hydrothermal Inactivation of Endotoxins”, Applied Research Associates, Inc., 2010.
4. Michael E. Dawson, Ph.D., “Depyrogenation”, LAL UPDATE Vol. II. Number 5, 1993.
5. “Bacterial Endotoxins/Pyrogens”, Dept. of Health, Education, and Welfare Public Health Service Food & Drug Administration, No. 4, 1985.
6. Lerouge, “Sterilisation and cleaning of metallic biomaterials”, Metals for Biomedical Devices, 2010.
7. “Water for the Reprocessing of Medical Devices”, AAMI, 2007.
8. Goveia VR, Mendoza IYQ, Guimarães GL, Ercole FF, Couto BRGM, Leite EMM, et al., “Endotoxins in surgical instruments of hip arthroplasty”, Rev Esc Enferm USP., 2016.
9. L. Anderson, Ph.D, B. W. Holland, J. K. Carr, BS, W. W. Bond, MS, and M. S. Favero, Ph.D, “The Effect of Disinfectants on Pseudomonads Colonized on the Interior Surface of PVC Pipes”, AJPH, Vol. 80, No. 1, 1990.
10. Tim Sandle, “Depyrogenation and endotoxin”, Sterility, Sterilisation and Sterility Assurance for Pharmaceuticals: Technology, Validation and Current Regulations, 2013.