Effect of Buffer Room Configuration on Isolation of Bacteriophage phi6 and Micrococcus Luteus Emissions

Abstract

In light of the growing need to mitigate viral infections, such as SARS-CoV-2, which has caused over 770 million infections and 7 million deaths worldwide, the role of architectural design in reducing pathogen spread has become paramount. This study explores how different buffer room configurations impact microbial isolation using the bacterium Micrococcus luteus and bacteriophage phi6 as test organisms. Through a comprehensive approach integrating architecture, technology, and scientific methodologies, we developed strategies to optimise safe buffer environments. Comparative analyses of various isolation systems highlighted significant variations in their effectiveness at reducing bioaerosol concentrations, directly influenced by the design and layout of buffer rooms. These findings are crucial for the effective design of medical and public spaces, particularly during pandemics, where preventing virus transmission is critical for public health.

1. Introduction

Personal protections are an issue currently hotly debated among medical personnel working on the first front line with patients suffering from viral diseases derived from SARS-CoV-2 [1,2,3,4,5,6,7]. Two inter-related problems are involved. One is medical equipment shortage and the other is its improper use. The viruses responsible for COVID-19 spread mainly through droplet, contact and aerosol transmission [8,9,10,11,12,13,14,15]. Studies have shown that the main route of SARS-CoV-2 transmission is contact with droplets exhaled by an infected person. Droplets can be inhaled by persons being in the direct vicinity of an infected person. The virus can also deposit on surfaces, which can lead to infections after a person touches the contaminated surface and then her/his face. Besides by droplets, SARS-CoV-2 can also be transmitted by aerosols, i.e., very fine suspension particles in the air. These particles can remain in the air for a longer time and be inhaled by persons finding themselves in the given room. Droplets are transmitted by larger particles, usually over 5 µm in diameter, which are subject to the action of gravitational forces. They usually travel for a distance not larger than 1 m. Therefore, limiting contact to a distance of 2 m is a preventive measure [16,17]. The use of buffer rooms in viral infection prevention is not a new thing, and its effectiveness has been confirmed even by artificial intelligence [18]. An analysis of the design of spaces, such as hospitals and medical laboratories, aimed at reducing the virus transmission risk, seems to be a right step. But, before new hospitals equipped with proper architectonic solutions are built, we must know which solutions are the optimal ones.

Such a solution can be a buffer room, i.e., a specially designed space or zone, the main purpose of which is to separate two areas characterised by different degrees of cleanliness or contamination risk. The main function of a buffer room is to minimise the risk of transmission of contaminants or microorganisms from one area to the other. This ensures that a clean or controlled environment is not contaminated. Buffer rooms are usually equipped with advanced ventilation systems, air filters and other technologies whose purpose is to maintain a specific air purity level [19,20]. The effectiveness of an existing buffer room is determined in various ways, e.g., using numerical methods [21,22], by observing the spread of smoke and measuring its concentration [23], by examining the flow of air and the effectiveness of its filtration [22,24] and by determining the survival rate of microorganisms [25].

This study presents an integrated approach to the use of various buffer spaces preventing viral infections and to the evaluation of the microbiological efficacy of an adopted solution. The study combines different aspects, such as architecture, technology, management and research. The aim is to develop comprehensive strategies to ensure safe buffer spaces, especially strategies aimed at urgent temporary adaptation of an infrastructure to isolation in the days of fight against viruses in public spaces. As part of this study, a universal methodology for testing the effectiveness of bioaerosol concentration reduction in various configurations of buffer rooms has been developed.

2. Materials and Methods

2.1. Layout of the Tested Rooms

Tests were carried out to determine the effectiveness of different buffer room versions. The tested system constituted a vestibule to a room, in which there was a source of microorganism emission (a patient). The effectiveness of reducing the emission of microorganisms to the outside of the room was to be assessed. For this purpose, a test facility and a test procedure, making it possible to obtain comparative results for different buffer room versions, were defined. As shown in Figure 1, the test facility consists of an infective room, different buffer room (tested system) versions and a test room. The infective room simulates a hospital ward (with defined dimensions, necessary equipment and an entrance door) containing an emission source. Buffer rooms are hermetically located between the infective room (with an emission source) and the test room, which simulates a hospital corridor. This configuration of rooms makes it possible to control the emission of microorganisms to the outside of the buffer zone. Microorganisms that escaped from the infective room gather in the hermetic test room. The comparative method was used in the investigations. The effectiveness of the tested system is defined as a percentage reduction in bioaerosol concentration between the test room and the infective room.

Two kinds of room isolation were considered. A single-chamber version consisted of adding a single buffer volume before the entrance to the isolated “infective” room (Figure 2a). This installation version can be adopted, e.g., when there is a wide hospital corridor and the aim is to better isolate a single room. A two-chamber version consisted of installing a buffer volume (airlock) in such a way that it closed the whole passage to, e.g., a hospital module or a part of the corridor (Figure 2b). In this case, besides the airlock volume, an additional volume forms between the airlock exit and the entrance to the infective room(s). Thus, altogether, two zones—a dirty zone and a clean zone—were created (Figure 3).

Two kinds of bioaerosol—one from the group of bacteria and one from the group of bacteriophages (viruses)—were selected for effectiveness testing. On the basis of experiments, Micrococcus luteus ATCC 7468 was selected from the group of bacteria. Microbiological examinations showed that in the case of this microorganism a countable bioaerosol could be obtained from a suspension with turbidity within the McFarland scale, a bacterial colony (its colour, shape and size) could be easily identified and this bacterium could survive in a physiological solution, artificial saliva and air. On the basis of experiments, phage phi6 was selected from the group of bacteriophages. Microbiological examinations showed that in the case of this microorganism a countable bioaerosol could be obtained, viral plaques (clear areas) could be easily identified and this bacteriophage could survive in a physiological solution. While performing the analyses, material for tests was taken from areas of the particular buffer room zones (

Table 1. Sampling materials for tests.

	Bacterium	Bacteriophage
Kind	Micrococcus luteus ATCC 7468	phage phi6
Control samples	bacterial strain streak inoculation	host strain streak inoculation
	surface inoculation of stabilised suspension before nebulisation	surface inoculation of phage before nebulisation
	surface inoculation of stabilised suspension after nebulisation	surface inoculation of phage suspension after nebulisation
Samples of air in test room	after disinfection	after disinfection
	after nebulisation	after nebulisation

).

2.2. Test Apparatus

A dedicated certified test apparatus and certified laboratory materials were used for the tests. All the materials (bottles, flasks, test tubes and pipettes) and media were sterilised in a Tuttnauer 2840EL-D steam autoclave. Each bottle was labelled with indicator tape showing that the required sterilisation temperature and pressure were reached. Once a month, their sterility was checked by carrying the Sporal A test. Disposable sterile materials were used in the case of such materials as automatic measuring pipette ends, ends with a (bacteriophage) filter for automatic measuring pipettes, disposable test tubes, Falcon type tubes, cryotubes, Petri dishes, platinum loops and bacteria spreaders. A Spectroquant^® Prove 300 (KGaA, Darmstadt, Germany) spectrophotometer was used for spectrophotometric measurements for setting or determining suspension turbidity (OD550/OD600), i.e., absorbance at 550 nm and 600 nm. The accuracy of absorbance measurement in the range of 300–900 nm amounted to ±0.005. Two cabinets, i.e., a laminar flow cabinet for work with bacterial strains and a Biosan cabinet for work with bacteriophages, were used in the tests. Each time, after finished work, the surfaces were disinfected with 70% ethyl alcohol, and a UV lamp was switched on for 20 min. The separation of the workstations for work with, respectively, bacteria (including hosts) and bacteriophages reduced the risk of contamination of the samples and the equipment (including the automatic measuring pipettes). Two Pol-eko ST-1 cooled incubators (temperature stability of ±0.3 °C at 37 °C) were used in the tests. A TERMO-1 temperature recorder (Termoprodukt, Bielawa, Poland) with a resolution of 0.01 °C was used to control temperature. Shaking water baths with a stainless steel chamber and holders for laboratory bottles (temperature stability of ±0.2 °C) were used for cultivating bacteriophages.

A 6 Jet Collison, (CH Technologies, Westwood, New Jersey, USA) nebuliser, producing aerosol characterised by MMAD 2.5 μm and GSD 1.8, was used to spray the prepared suspension containing the test microorganism. A compressed air cylinder was connected, using a PTFE tube with a filter with a pore diameter of 0.2 µm, to the nebuliser. The nebuliser was sterilised in the steam autoclave at 121 °C for 15 min. At a pressure of 20 psi, the nebuliser sprayed 0.0254 mL of the suspension (8.262 × 10¹⁰ particles) per minute. As the bacterial suspension and the phage suspension were nebulised, bacterial cells (single cells, cell aggregates, cell packs or cells in the course of their division) or bacteriophage virions were sprayed in drops of a solution (a physiological solution or a broth/broth diluted with a physiological solution). An SAS DUO sampler with two heads and an SAS Isolator (with one head) were used to take air samples. The SAS Isolator consisted of a programmable unit (which remained outside the controlled area) and an independent sampling head with 219 holes, which used 55 mm contact plates (RODAC) or Petri dishes 90 mm in diameter. The sampler head was made of stainless steel. During the tests, it was sterilised in the steam autoclave (121 °C/15 min). Air was sucked into the sampling head at the flow rate of 100 and 180 L/min.

2.3. Test Procedures

Care was taken to reproduce the real work conditions of medical personnel in contact with an infected patient. Micrococcus luteus ATCC 7468 (PCM 1143) bacteria strains and phi6 bacteriophages were used in the tests. Because of the different survival times of bacteria and phages in air, experiment durations proper for the respective tested microorganisms were experimentally determined [26]. After the nebulisation and homogenisation of the microorganisms started, measurements by means of samplers P I, P II and PIII were performed to evaluate the reduction in the concentration of microorganisms in the air in the particular rooms. In order to maintain the universality of the method and to make comparative analyses of all the tested buffer room systems, the following test procedure variants were defined:

• Determining the effectiveness of airlock prototype I assembled as a single-chamber airlock with passage time of 30 s (the basic variant);
• Determining the effectiveness of airlock prototype I as double-chamber airlock with passage time 30 s;
• Determining the effectiveness of airlock prototype I assembled as a single-chamber airlock with passage time of 5 s;
• Determining the effectiveness of airlock prototype I assembled as a single-chamber airlock with passage time of 120 s.

2.3.1. Determining Effectiveness of Airlock Prototype I Assembled as Single-Chamber Airlock with Passage Time of 30 s (Variant a)

An analysis of the effectiveness of bacterial strain M. luteus reduction (Figure 4) consisted of the following:

▪

The nebulisation of the M. luteus suspension in the infective room for 30 s, followed by bioaerosol homogenisation for 5 min;

▪

Collecting 200 L of air from the infective room and simultaneously opening the door to the airlock for 30 s;

▪

Collecting 200 L of air from the airlock and then opening the door to the test room for 30 s;

▪

Collecting 200 L of air in the test room.

An analysis of the effectiveness of bacteriophage phi6 reduction consisted of the following:

▪

The nebulisation of the phi6 suspension in the infective room for 90 s (without homogenisation—without switching on fans in the infective room);

▪

Collecting 200 L of air from the infective room and simultaneously opening the door to the airlock for 30 s;

▪

Collecting 200 L of air from the airlock and then opening the door to the test room for 30 s;

▪

Collecting 200 L of air in the test room.

2.3.2. Determining Effectiveness of Airlock Prototype I Assembled as Double-Chamber Airlock with Passage Time of 30 s (Variant b)

An analysis of the effectiveness of bacterial strain M. luteus reduction (Figure 5) consisted of the following:

▪

The nebulisation of the M. luteus suspension in the infective room for 30 s, followed by bioaerosol homogenisation for 5 min;

▪

Collecting 200 L of air from the infective room and simultaneously opening the door to the airlock for 30 s;

▪

Collecting 200 L of air from the contaminated room and then opening the door to the clean room for 30 s;

▪

Passage to the airlock; then opening the door to the test room;

▪

Collecting 200 L of air in the test room.

An analysis of the effectiveness of bacteriophage phi6 reduction consisted of the following:

▪

The nebulisation of the phage suspension in the infective room for 90 s (without homogenisation);

▪

Collecting 200 L of air from the infective room and simultaneously opening the door to the contaminated room for 30 s;

▪

Collecting 200 L of air from the contaminated room and then opening the door to the clean room for 30 s;

▪

Passage to the airlock; then opening the door to the test room;

▪

Collecting 200 L of air in the test room.

2.3.3. Determining Effectiveness of Airlock Prototype I Assembled as Single-Chamber Airlock Depending on Time of Passage through Door (Variants c and d)

In order to evaluate airlock effectiveness depending on passage time, additional tests were carried out. The procedure was modified as regards passage-through-airlock time, defined as the duration of the opening of the particular doors when passing through the airlock (from the infective room to the test room). The procedures depending on the passage time of 5 s is shown in Figure 6 and for the passage time of 120 s in Figure 7.

3. Results

3.1. Determining Effectiveness of Airlock Prototype I Assembled as Single-Chamber Airlock

Tests for the single-chamber airlock were repeated eight times for both the bacteria and the bacteriophages (

Table 2. Effectiveness of single-chamber airlock prototype I for passage time of 30 s.

Kind of Bioaerosol	Effectiveness without Airlock, %R	Effectiveness with Airlock, %R
M. luteus	77.27% (N = 8; SD = 10%)	95.15% (N = 8; SD = 2.2%)
phage phi6	72.48% (N = 6; SD = 26.3%)	95.22% (N = 8; SD = 3.9%)

). The single-chamber airlock significantly improved the effectiveness of isolation of bioaerosols in the case of both bacterium Micrococcus luteus and bacteriophage phi6, increasing this effectiveness from 72–77% to over 95% for both the microorganisms. The low standard deviation values are indicative of result stability in the different experiment repetitions, which shows that the test methodology is reliable.

3.2. Determining Effectiveness of Airlock Prototype I Assembled as Double-Chamber Airlock in Laboratory Conditions

Tests for the single-chamber airlock were repeated eight times for both the bacteria and the bacteriophages (

Table 3. Summary—effectiveness of double-chamber airlock prototype I for passage time of 30 s.

Kind of Bioaerosol	Effectiveness without Airlock, %R	Effectiveness with Airlock, %R
M. luteus	85.94% (N = 7; SD = 3.2%)	98.17% (N = 7; SD = 1%)
phage phi6	82.99% (N = 8; SD = 8.3%)	98.37% (N = 8; SD = 2%)

). The double-chamber version showed even higher effectiveness, increasing isolation to 98.17% for M. luteus and to 98.37% for phi6, which demonstrates the benefits of additional isolation provided by the second chamber.

3.3. Determining Effectiveness of Airlock Prototype I Assembled as Single-Chamber Airlock, Depending on Passage-through-Airlock Time

The tests showed that shorter passage-through-airlock times (5 s) resulted in higher isolation effectiveness values for both the tested microorganisms, reaching about 98% (

Table 4. Summary—effectiveness of double-chamber airlock prototype I depending on passage time.

Kind of Bioaerosol	Passage-through-Airlock Time	Effectiveness without Airlock, %R	Effectiveness with Airlock, %R
M. luteus	5 s	86.32% (N = 8; SD = 7.1%)	98.31% (N = 8; SD = 0.9%)
	30 s	77.27% (N = 8; SD = 10%)	95.15% (N = 8; SD = 2.2%)
	120 s	59.25% (N = 6; SD = 17.5%)	86.63% (N = 6; SD = 4%)
phage phi6	5 s	67.75% (N = 6; SD = 23.5%)	95.41% (N = 6; SD = 3.1%)
	30 s	72.48% (N = 6; SD = 26.3%)	95.22% (N = 8; SD = 3.9%)
	120 s	65.69% (N = 3; SD = 38.7%)	91.46% (N = 3; SD = 10.1%)

). Longer passage times (120 s) resulted in considerably lower effectiveness, suggesting that longer exposures to a potentially contaminated environment can increase the risk of transmission of microorganisms (Figure 8). The number of repetitions for the particular time variants and the sprayed microorganism is denoted as N, and it amounts to 3–8.

4. Discussion

Through the experiment, it was possible to verify the effectiveness of the different versions of the infective room isolation system. The reduction in transmission by the buffer room system was analysed for two versions of the latter: (1) as a vestibule to the reference room (a single-chamber version) and (2) as whole corridor isolation (a double-chamber version). The isolation effectiveness was determined as a ratio of the bioaerosol concentration at the exit from the airlock (at a distance of less than 2 m and no more than 4 m from the exit) to the bioaerosol concentration in the room after nebulisation, on the basis of at least three samples. The averaged results of the effectiveness analysis for prototype I in two assembly versions (a single-chamber airlock and a double-chamber airlock) are presented in

Table 5. Airlock effectiveness for M. luteus ATCC 7468 and phage phi6.

Version	M. luteus ATCC 7468	Phage phi6
Single-chamber version, passage time 30 s	95.15%	95.22%
Double-chamber version, passage time 30 s	98.17%	98.37%

.

The investigations of the effect of passage-through-airlock door time were aimed at determining the most effective configuration. Conditions that ensured a high reduction in the transmission of microorganisms were regarded as most advantageous. Moreover, a system informing personnel that it is necessary to close the door quicker or stay longer inside the airlock before exiting it has been developed. Even though effectiveness above 80% was achieved in all the versions, it is recommended to open the door for no longer than 30 s and to use the shortest possible door opening times. An optimal configuration of buffer rooms, including the number of chambers and the passage time, plays a key role in ensuring the effective isolation of microorganisms. The presented results have significant implications for the design of hospitals and laboratories, especially in the context of the spread of infections. Implementing double-chamber buffer rooms with strict control over passage times can substantially reduce the risk of airborne pathogen transmission, enhancing patient safety and reducing cross-contamination. These findings advocate for integrating advanced isolation technologies into architectural designs to create more resilient and adaptable healthcare facilities capable of responding effectively to future outbreaks.