Efficacy of Disinfectants on Common Laboratory Surface Microorganisms at R.S Mangaliso Hospital, NHLS Laboratory, South Africa

Introduction

Safety is mandatory for everyone working environment that includes the clinical laboratory especially when the environment is dealing with bacteria and other microbes. During the COVID-19 period, every household and even transport and shopping complexes were expected to disinfect to reduce the transmission of the infections.  The use of appropriate and reliable disinfectants on surfaces regularly is critical in preventing the transmission of colds, microbial infections, and other sicknesses among health workers [1-3].

In most clinical laboratories hazards such as chemicals, flames, infectious agents, and glassware are present and this inherently makes the working environment dangerous [4, 5]. Exposure to these potential hazards is possible simply through direct contact with contaminated surfaces or through spillages and spattering of hazardous substances on the bench when PPE is not applied [5]. Cleaning plays a huge role in laboratories working with infectious agents or chemicals to minimize risks and improve the quality of results [6]. According to CLSI, a very essential method to ensure the safety of laboratory employees is to utilize good decontamination procedures with the use of effective disinfectants [5, 7, 8].

Decontamination is a process used in laboratories to reduce microbial contamination and minimize infection transmission [9].  Sterilization, disinfection and antiseptic are types of decontamination. Sterilization and disinfection both remove pathogens, but sterilization differs from disinfectants because they completely kill all microorganisms including spores [9-11]. Procedures for sterilization include radiation, heating, steaming, and chemical sterilization; these methods are used to sterilize food, medicine, and surgical instruments [9, 11]. Antiseptics are substances used during surgery or other medical procedure to stop or slow down the growth of microorganisms [12, 13]. The difference between antiseptics and disinfection is that antiseptics are applied to the body whereas disinfectants are applied on non-living objects or surfaces such as cabinets, laboratory work areas, telephones, computer terminals, and equipment [5, 12, 13]. Disinfection is used mostly to decontaminate surfaces or air and for that reason, the purpose of this research will be to study the effectiveness of disinfectants used to decontaminate laboratory surfaces [13]. There are most common disinfectants used to decontaminate laboratory surfaces that include sodium hypochlorite (bleach/jik), alcohols, hydrogen peroxide, chlorine, aldehydes, peroxygenase, and quaternary ammonium compounds [13].

During the early 1960s, 3 categories (non-critical, semi-critical, and critical) of germicidal action to prevent risks of infection associated with the use of equipment or surfaces were suggested [14]. Environmental surfaces were considered noncritical items because they come in contact with intact skin and intact skin serves as a barrier not to acquiring diseases or infection [14]. Therefore, when in contact with noncritical surfaces there is a low risk of transmitting pathogens to health workers. However, surfaces may become contaminated with infectious agents and may serve as a drive to initiate outbreaks for person-to-person transmission [14]. This controversy prompted the study of disinfectants and their effect on environmental surfaces to prevent transmission of microorganisms between surfaces and laboratory staff because they are more exposed to these microorganisms on daily basis.

During the beginning of the 21^st century, a study done in Canada tested nine liquid disinfectants (6% hydrogen peroxide, ammonium hydroxide windshield washer fluid, 70% ethanol, 37%methanol, 6% sodium hypochlorite, 70% isopropanol, and three commercial disinfectants) at room temperature (22 to 24°C) for a period of 4, 13, and 33 min to examine their ability to reduce the infectivity of Cryptosporidium parvum oocysts (ATCC 87665) in cell culture [8]. Susan et al., results of their study indicated that 4 to 13 minutes exposure of to hydrogen peroxide and ammonium hydroxide reduced the infectivity of Cryptosporidium parvum oocysts while other disinfectants did not reduce the infectivity of the above organism after 33 minutes of exposure. According to the results, hydrogen peroxide and ammonium hydroxide disinfectants are suitable laboratory disinfectants against Cryptosporidium parvum oocysts [8].

Another study to assess the Microbiology quality and efficacy of two disinfectants (30% Jik and 2, 5% Lysol-hydrogen peroxide containing disinfectant) was conducted in the year 2017 in the indoor environments of the Medical Microbiology Laboratory Department of Babcock University Teaching Hospital, Ilishan-Remo, Ogun State, Nigeria [15]. According to the researcher’s findings, the two disinfectants passed the sterility test as there was no significant growth of microbial contaminants. The bactericidal activity of the two disinfectants was also examined and only Lysol showed to be more effective than jik at the time of dilution and contact time testing [15]. However, the bactericidal activity of the two disinfectants was dependent on time and therefore periodical assessment is required, and factors such as temperature, higher concentration, and prolonged contact time which may influence the efficacy of these disinfectants had to be investigated [15].

Disinfectants are constituents of the disinfection process that destroys bacteria, viruses, fungi, and mould living on objects or surfaces, but they do not all remove endospores [3, 10, 16]. The antimicrobial activity of disinfectants occurs by inhibiting microbial growth e.g. bacteriostatic and fungistatic effects or through lethal activity e.g. sporicidal, bactericidal, fungicidal, and virucidal effects [16]. The active ingredients that are generally available are alcohols, chlorine, aldehydes, peroxygenase, and quaternary ammonium compounds [13]. Since there are many types of disinfectants on the market, it is important to understand the mode of action of each disinfectant, including their advantage and disadvantages to decide how to best disinfect and protect the work environment and its employees [3, 17].

Factors affecting the efficacy of disinfection

Disinfectant effectiveness depends on many factors, the following factors are explained:

Concentration of disinfectants

It is important to choose a suitable concentration of disinfectant that is best suited for each situation. To achieve the lethal effect of microorganisms, the concentration of disinfectant must be increased to increase its efficacy and shorten the time for microbial killing [18, 19]. However, some disinfectants such as quaternary ammonium compounds and phenol are not similarly affected by concentration adjustments.

Physical and chemical factors

Temperature, pH of the environment, humidity, and water hardness are physical and chemical factors that influence the disinfectant procedure.

Temperature

Disinfectant efficacy increases when temperature increase but when the temperature is too high it may cause the disinfectant to decrease and cause potential health hazard [19].

pH

Influences antimicrobial activity of disinfectants by changing their molecule or the cell surface [20]. An increase in pH can either increase some disinfectant's antimicrobial activity (such as glutaraldehyde and quaternary ammonium compounds) or decrease the antimicrobial activity of others, for example, phenols, hypochlorite, and iodine [19].

Humidity

Influences the activity of gaseous disinfectants such as chlorine dioxide and formaldehyde.

Water hardness

Reduces the rate of antimicrobial activity of certain disinfectants because cations such as magnesium and calcium in hard water interact with the disinfectant to form insoluble precipitates [21, 22].

Contact time

Sufficient time of contact must be allowed for the disinfectant to act efficiently on microbes. For different microorganisms, different time is needed; longer contact times are more effective than shorter contact times [19].

Types and number of microorganism present

Most disinfectants except aldehyde are not effective against bacterial spores, knowing what type of microorganism is present, will help select a suitable and effective disinfectant [23, 24]. In addition, the greater the amount of microorganism present, the more disinfectants are needed to kill the microbes [19].

Interfering substances (organic and inorganic) in the environment

Organic matter such as serum, blood, pus, fecal, or lubricant material can interfere with the antimicrobial activity of disinfectants. Interference occurs by a chemical reaction between the active ingredient and the organic matter resulting in a complex that is less effective to kill microorganisms [25, 26]. Sometimes organic matter acts as a physical barrier to protect microorganisms from being attacked by disinfectants [25, 26]. Chlorine and iodine are disinfectants interfered with by organic substances [25, 26]. Inorganic matter protects microorganisms from all disinfection processes resulting from occlusion in salt crystals [27, 28]. Hence it is important that cleaning should be done prior disinfection procedure [27].

Common microbes present on surfaces

Nosocomial pathogens are the most persistent microbes found on surfaces and increase risks of healthcare-acquired infection [29]. That is why it's important to use disinfectants that have broad antimicrobial killing efficacy. Table 1 below shows the common pathogens and the survival period if left not decontaminated on the various surfaces [30].

[29, 30]

Selection of disinfectants

Selecting a disinfectant to best meet the needs of your facility is important to keep the working environment safe [3]. An ideal disinfectant should be broad spectrum, non-irritating, non-toxic, non-corrosive, and inexpensive [14]. When choosing a disinfectant, consider the effectiveness against the potential pathogenic agent, safety to people, impact on equipment, environment, and expense [14]. Disinfectants have been also categorized into high-level, intermediate-level, and low-level disinfection according to the anti-microbial activity of the disinfectant [3, 14, 31].

Low-Level disinfectant (0.4-1.6 % Quaternary Ammonium Compounds) is an agent that destroys all vegetative bacteria except tubercle bacilli, lipid viruses, some non-lipid viruses, and some fungi, but not bacterial spores. Intermediate Level disinfectants are an agent that destroys all vegetative bacteria, including tubercle bacilli, lipid-enveloped viruses, some non-lipid enveloped viruses, and fungus spores but not bacterial spores [3]. Examples of these agents are alcohol (ethyl, isopropyl) 70-95%, iodophor compounds, and phenolic compounds (0.4-5 %). A chemical or physical agent or process that can kill some bacterial spores when used in sufficient concentration, temperature, and under suitable conditions are glutaraldehyde 2%, hydrogen peroxide 3-25 %, peracetic acid (variable), and chlorine dioxide [3].

Numerous scientific studies have provided evidence for the transmission of microorganisms between surfaces and health workers or patients. More research is required to gain a better insight into the role of surface disinfection in the laboratory to prevent laboratory-acquired infections [32]. Most importantly, research to decide on the safest and most effective disinfectant to use to protect the work environment and its employees has to be established [33].

The effect of disinfectant depends on the type of organism populations present on surfaces, the concentration of both organism populations, and the duration of exposure of disinfectant to kill or reduce these organisms [33, 34]. Understanding the proper types, concentrations, and contact times of the appropriate laboratory disinfectants is a practice that needs to be implemented to reduce threatening pathogens and make the laboratory a safer place to work. The study of the quality and efficacy of laboratory disinfectants project was therefore an ideal opportunity to perform.

This study aimed to assess the effectiveness of 3 common laboratory surface Disinfectants (5% sodium hypochlorite, 70% alcohol, and 5% Distel (quaternary ammonium compounds).

Objectives of the study were to:

• Test the sterility of the culture medium and saline.
• Identify test microorganism present on the laboratory surface bench (before disinfection).
• evaluate disinfectant efficacy against the test microorganism.

Materials and Methods

The observation and analytical study were carried out at Kimberley NHLS (Robert Mangaliso Sebukwe Hospital) in Northern Cape South Africa. The sampling and analysis were carried out for a period of 9 months (February – November 2020).

Contamination checks of agar plate and saline

Care and caution were exercised to ensure all consumables are not expired. A total of 17 half plates 5% blood and Macconkey agar of the same lot number was used for this project. One agar plate of the lot number in use which has not expired was incubated at 37°C in CO₂ for 2 days to ensure that the plates were not contaminated. A saline sterility test was done by placing 2 drops of saline of lot number in use throughout the entire 5% blood agar and the plate was incubated for 2 days at 37° C in CO₂. Five or more colonies on either plate indicate contamination. After 2 days of incubation, no growth was observed on the plates therefore there was no contamination on each product.

Sample collection

A sampling of the entire work-bench surfaces in the Microbiology (culture bench), Haematology (Rh bench), and chemistry (HBA1C bench) laboratory Department of Kimberley NHLS was collected in duplicate at the end of each shift (before disinfection) using sterile cotton swabs moistened with sterile saline.

Sample culture

Six half plates containing 5% Blood agar (BA) medium and MacConkey agar (MCA) medium were designated as Micro-culture bench 1, Micro-culture bench 2, Haema-RH bench 1, Haema-RH bench 2, Chem-HBA1C bench 1 and Chem-HBA1C bench 2. Swab samples collected were inoculated unto the plates respectively. Using a wire loop working aseptically, the plates were streaked in four- the quadrant streak method as described in SOP TADM0155, and the plates were incubated at 37°C for in CO₂ 18-24 hours as described by Mokhtari et al. in 2011 [35].

Identification of surface test organism isolates

After incubation, plates containing cultured samples were investigated. Colonies were identified by colonial morphology, Gram-stain, DensiCHEK, and Vitek 2 automated system.

Evaluation of disinfectant activity on each test Isolate

In-use disinfectants (5% sodium hypochlorite, 70% alcohol, and 5% Distel- quaternary ammonium compound) were obtained and placed into a 40ml specimen jar from the laboratory department. The efficacy of the disinfectants against the test organisms was evaluated using the agar plate method and the Quantitative Suspension Test.

• Agar plate method

This method test determines the effectiveness of disinfectants on laboratory bench surfaces. Three areas of the surface bench were designated as site 1, site 2, and site 3. Each surface was inoculated with 0.9McFsuspension of the test organisms identified. To prepare 0.9 MCF suspension, a few colonies were emulsified in a 3ml saline in a plastic tube using a vortex mixer. The DensiCHEK™ Plus was used to measure the turbidity value, the ample containing tube was adjusted to give a 0.9McF value. Before disinfection of each surface, swabs were taken on all 3 sites and cultured on half plates with 5% BA and MCA. Site 1 was disinfected with 70% alcohol for 15 minutes then a swab was taken and cultured. Site 2 was disinfected with 5% Distel for 15 minutes then a swab was taken and cultured. Site 3 was disinfected with 5% sodium hypochlorite for 15 minutes swab was taken and cultured. All the plates were incubated overnight at 37°C in CO₂ then colonies were observed, counted, and compared and the results were recorded.

• Quantitative Suspension Test

Eight plastic tubes were used for this analysis, the first 4 tubes contained 3ml of 3.4McF suspension for the test organism identified. The second 4 tubes contained 3.1 McF suspension of the other organism identified. The first tube of each 4 tubes was designated as the control tube which contained suspension of the test organism without disinfectant. The other 3 tubes contained the same suspension of the test organisms with 1ml of each of the 3 types of disinfectants that are being evaluated as explained in the figure above.

In summary, 1mL of the disinfectant being tested was pipetted using a plastic Pasteur pipette and added into a 3ml standardized microbial suspension. This activity was performed at room temperature for contact times of 0, 5, 10, and 15 minutes. The timer was started when the test bacterial suspension and disinfectant are combined. At 0 times, McFarland readings were taken using DensiCHEK™ plus instrument and at 5 minutes intervals, each test sample was re-suspended using a vortex mixture then turbidity readings were taken and recorded. After all, readings were taken and recorded, each test sample was cultured onto half plates with 5% BA and MCA then the plates were incubated overnight at 37°C in CO₂. Turbidity together with colonies on the plates where observed, disinfectant activity was compared, and the results were recorded.

Results and Discussion

The study assessed the efficacy of common laboratory surface disinfectants (70% alcohol, 5% sodium hypochlorite, and 5% Distel- quaternary ammonium compounds). Before the examination was carried out, a sterility check of the medium plate and saline was performed to ensure that the products were not contaminated. Identification of test organisms isolated from the Microbiology, Chemistry, and Haematology surface bench before disinfection is presented in the following table:

The organisms used in the sthudy were identified and verified as summarised in Table 2 prior laboratory testing. Staphylococcus lentus M155 strain was isolated from the microbiology and hematology laboratories' surface benches before disinfection and on the chemistry surface bench, Acinectobacter lwoffi strain ATCC-15309 was isolated pre-disinfection. Staphylococcus lentus is a Gram-positive bacillus, oxidase-positive, coagulase-negative member of the bacterial genus Staphylococcus consisting of clustered cocci. Staphylococcus lentus is a common part of the normal flora of both humans and other animals. However, it can be pathogenic to humans and cause endocarditic peritonitis, septic shock, infections of the urinary tract, a pelvic inflammatory disease most frequently, and wound infections. Acinectobacter lwoffi is a non-fermentative Gram-negative bacillus bacterium that is a member of the genus Acinetobacter. It is considered normal skin flora and can also inhabit the human oropharynx and perineum. It can cause infections in human hosts such as catheter-urinary infections in immunocompromised patients and gastroenteritis.

Acinectobacter lwoffi and Staphylococcus lentus were the two test organisms used to examine the efficacy of the three surface disinfectants used at the laboratory. The following are the results of the agar plate method using Staphylococcus lentus as a test organism:

The above figures show two mediums with growth before and after disinfection with 70% alcohol. Figure 2 shows 2+ growth on both BA and MCA whereas Figure 3 shows 1+ growth on BA and no growth on MCA. This indicates that the test organism survived and recovered from the BA plate even after disinfecting with 70% alcohol.

Figures 4 and 5 show two mediums of before and after disinfection with 5% Distel/ quaternary ammonium compounds. There was a 2+ growth of the test organism on both BA and MCA in Figure 4. In Figure 5 there was 1+ growth of the test organism on BA but there is no growth on the MCA plate. This indicates that there was recovery and survival of the organism on BA but on MCA the organism was killed.

Figures 6 and 7 show two mediums of before and after disinfection with 5% sodium hypochlorite. There was 3+ growth of the test organism on both BA and MCA in Figure 6. In Figure 7 there were less than 5 colonies on BA and no growth on MCA, this indicates that the microorganism was completely killed after disinfection with 5% sodium hypochlorite.

The killing rate of disinfectants was also evaluated; the following line graph is a result of McFarland value versus time using Staphylococcus lentus as a test organism: