Microbiological Quality of Laboratories Works Stations: Impact of a System of Saturated Dry Spray Steam

Hygiene and sanitation in laboratories are some important focus for the well-being of scientists and workers. Due to the lack of these notions, a new sanitation system named Polti Sani System has been tested to overcome the limitations of traditional methods. For this work, some American Type Culture Collection and Institute Pasteur of Strasbourg reference strains have been used. The biocides activities of the Sani System were assessed by the count of the total aerobic mesophilic bacteria in the working environment before and after disinfection. The Adenosine Triphosphate was also quantified. After a time contact of 30 seconds, Sani System reduced more than five logarithmic levels, the bacterial rate tested. The log reduction achieved with the fungi in the same conditions were greater than four logarithmic levels. The antimicrobial activity was observed both in the environment and on inert supports made of glasses, Plexiglas or tiles with an average reduction rate of 99.13%. This study showed that the Sani System associated with ATP-metry can be successfully used to quickly check the hygiene standards on surfaces or lab environment. It is a real challenge in terms of quality, efficiency and safety for the laboratories. It shows that the no growth. The log reductions highlighted after 30 sec disinfection with dry saturated all higher than


INTRODUCTION
Environment as air, surfaces and water faced a permanent but variable microbiological contamination variable in time and space. Microorganisms belong to the environmental saprophytic floras. It also comes from commensal or pathogenic floras of people [1]. As vectors of contamination, these environmental compartments disseminate microorganisms at some distance and insidiously contribute to progressive contamination of various inert supports.
There are many examples of bioburden that have hit the headlines, whether in the hospital, pharmaceutical or food area. Epidemiological data confirm the reality of these risks. Infections are almost due to bacterial causes and in 13% of cases, it is associated to exposure to aerosols [2].
Causing human and material damage, aerobiocontamination is a fundamental problem which affects many sectors. Biocontamination is a biological contamination which may have an adverse effect on the product, the staff or the patient with regard to health facilities [2].
Due to this ubiquitous contamination, personnel safety and protection of handled products can only be achieved in a controlled microbial environment [3]. Therefore, it is important to assess the risk of contamination and to manage it consistently. The control of bioburden in areas at risk relates to compliance with preventive measures but also on physical, chemical and microbiological tests and finally on correctives measures.
The traditional sanitation methods relate to the use of disinfectant solutions, sometimes with high chemical content, which come in contact with the support surfaces or heat or radiation [4].
The limitations of these methods are generally related to the difficulty in reaching the interstices or penetrate the rough surfaces and uneven, it does not provide a total cleaning of the contaminated area. In addition, these disinfectants have a risk of irritation or hypersensitivity due to their chemical composition. Moreover, traditional chemical disinfectants are not suitable for all types of surface and the phenomena of natural and acquired resistance to disinfectants were observed in bacteria [5]. In addition, it requires a manual contact with the surfaces to sanitize. Technological advances in microbiology and sanitation, combined with the new requirements of work safety, product safety and environmental protection led to a complete revision of disinfection procedures. Thus, some new products such as aerosols and complex synthetic compounds appeared.
Among these procedures, the steam was usually used within controlled enclosures which are capable of withstanding the pressures required for sterilization but its direct spray on surfaces is not yet a reality in the world [6]. The principle of such method is particularly interesting because it uses a simple steam and leads to a destruction of microorganisms by the way of generated heat without pollution.
Within the overall framework of risk prevention, specifically the reduction of avoidable proportion of airborne infections and also to overcome the resistance problems due to the use of traditional disinfectants, this study aimed to assess the microbiological quality of the lab working environment by using a nebulizer system of dry saturated steam.

Materials
Materials used were composed of microbial reference strains, experimental equipment disinfection system (nebulization of saturated steam Sani System) luminometer, culture media and reagents, glasswares and consumables.

Microbial strains reference
The antibacterial and antifungal activities of the disinfectant were in vitro evaluated on ATCC (

Experimental disinfection equipment
Disinfection equipment distributed by the company MIVA was a fogging system saturated vapour and a sanitizer. Sani System is an electro-medical device which delivers saturated steam at high temperature (180ºC) associated with the sanitizing HPMed. Its uniqueness lies in the fact that the steam is brought to a high temperature in an expansion chamber (Fig. 1).

Fig. 1. A nebulizer with dry saturated steam: Sani System Polti
HPMed is a co-adjuvant in sanitation vapour of Sani System. It consists of an alcohol-based solution of sodium metasilicate containing a nonionic surfactant, which helps the sanitizing activity of Sani System. The maximum flow rate of the saturated steam is 100 g/ min HPMed represents an aid to sanitation carried out by superheated dry saturated steam delivered by the Polti Sani System. The guaranteed minimum consumption HPMED is 0.4 ml / min Table I shows the chemical composition of the sanitizer HPMed.
A 3 M luminometer clean-trace NG was used with 3 M kits reagents for measuring contamination levels in samples of surface and can effectively monitor surface hygiene. The reagent kits were some dry swabs and Lucifer inluciferase complex [7].

Methods
The methodology used was about the evaluation of the biocidal activities of the nebulization Sani System. Then, the numeration of total aerobic mesophilic germs in a working environment (air and hard surfaces) before and after disinfection was done. Finally, the Adenosine Triphosphate from the different types of surface was quantified before and after disinfection.

Evaluation of biocides activities of sani system polti
The preparation of the microbial suspension was made according to AFNOR NF T 72-281 [8] where the dilution which gives a concentration of 10 6 germs per ml was selected as microbial suspension. Biocidal activities of Sani System Polti have been assessed by the test support [9,10]. After contamination and drying, some slides were treated by spraying of dry saturated steam in combination with sanitizer HPMed for 30 seconds. Along with these tests, another slides contaminated by each microorganism and untreated were maintained at room temperature (16ºC) throughout the tests for determining the initial contamination level brackets and validation of experimental conditions. For each strain, the log decimal reduction between the number of microorganisms present on the slide before treatment (N) and after treatment (N') was calculated using the formula: R = N/N'; Where log 10 R = log 10 N -log 10 N'

Numeration of mesophilic aerobic microorganisms suspended in the air
Mesophilic aerobic bacteria were collected and counted in the environment of three rooms before and after disinfection for respectively 360 seconds, 600 seconds and 3600 seconds. The method used was the sedimentation one on Petri dish [11]. Three samples were taken in each room and each sample was assayed five times. The time between two (02) samples was seven days. After cultivation staggered between 30-35ºC for 48 hrs and 20-25ºC for 96 hrs, the result of numeration R was expressed as follows: R is expressed in CFU/4 hrs. N 1 , N 2 , N 3 , N 4 , N 5 are the number of colonies counted for each of the five (5) plates seeded.

Enumeration of total aerobic mesophilic floras from lab surfaces
The samples were collected on three types of surfaces: glass, plexiglas and tile (before and after 30 seconds of disinfection) using the technique of wet swab on an area of 10 cm 2 . Ten samples were taken from the surface and 0.1 mL of each dilution sample was plated in duplicate using Tryptic Soy Agar [12]. After incubation at 30-35ºC for 72 hrs, the results expressed as CFU / ml of the initial product, were converted to counts per cm 2 using the following formula

ATP-metry: Measurement of residual ATP before and after disinfection
Surface samples were collected (before and after 30 sec of disinfection) with dried swabs (ATP test surface). Light emitted by the ATP test surface was measured and the result was displayed on the screen of the luminometer.

Statistical analysis
The results were analysed regarding the disinfection time and the type of surface. To evaluate the influence of the duration of disinfection and the type of surface on the rate of reduction of microorganisms, the test of analysis of variance was used when the conditions of normality and equal variances were met. Otherwise, the Kruskal Wallis was preferred. Student's t test or Wilcoxon test was used to compare the rates of reduction of microorganisms obtained for each factor. The significance level was set at 5%.

Effectiveness of Sani System Polti
The Table 2  These results confirm those of [6] who have also obtained more than 5 log units reductions for bacteria (Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus hirae) and reductions greater than 4 log units for Aspergillus niger and Candida albicans. Meanwhile, the log reductions obtained in this study for fungi differ from those reported by [16] who highlighted, for a similar system, some reductions discounts greater than 5 log units for Candida albicans.

Numeration of Mesophilic Aerobic Microorganisms in the Air
The floras for the three rooms (S 1 , S 2 and S 3 ) before and after disinfection are mentioned in the Table 3. More disinfection time increases, higher is the reduction of the number of microorganisms.
Microbial quantum in the three rooms (R 1 , R 2 , and R 3 ) after disinfection during 3600 seconds is respectively 02, 04 and 05 CFU/4 hrs. All these rooms belong to Class B as recommended by limits about microbiological contamination [17]. The average reduction of airborne microorganisms in three rooms after 360 sec, 600 sec and 3600 sec nebulization is shown in Fig. 2. It shows that the rate of reduction of airborne germs in three rooms increases with duration of disinfection. Thus, the average reduction rate was 4.57% after 360 sec disinfection. This rate raised to 41.39% after treatment of 600 sec and reached 94.27% after 3600 sec of disinfection.
The results of the analysis of variance of two factors are presented in the Table 4. The duration of disinfection has a significant effect (pvalue ˂ 0.05) on the rate of reduction of microorganisms. This time would probably depend on the surface to be disinfected. The analysis of variance of two factors also indicates that the disinfecting time duration has a highly significant effect on the rate of reduction of airborne germs. Although, disinfection is a onetime treatment effect and does not persist over time (p-value = 0.79).

Fig. 2. Evolution of the average rate of reduction of microorganisms by nebulization time
After 360 sec After 600 sec After 3600 sec

Numeration of Mesophilic Aerobic Microorganisms on Surfaces
The boxplot in Fig. 3 shows the distribution of the average rate of reduction of microorganisms by type of surfaces. The average rate of reduction of microorganisms on glass and plexiglas has a wide variation different from that of the tiles. The reduction rate corresponding to the average numbers were respectively 98.92%, 98.51% and 99.98%. The system with saturated steam is effective on smooth surfaces. These results are similar to those of European Union [18] who obtained a rate reduction between 98-100% for smooth surfaces. Moreover, none of the treated surfaces have been damaged. No fundamental change in colour, shape and general appearance has been noted.
Kruskal Wallis test shows that the nature of the surface significantly influences the rate of reduction of microorganisms after disinfection. Indeed, the rate of reduction of microorganisms after nebulization of ground (tiles) is significantly higher (p ˂ 0.05) than the rate of reduction of the smooth surfaces.

ATP-metry
The average of different values taken by the luminometer before and after disinfection of different types of surfaces (tiles, glass and Plexiglas) and the average reduction are shown in [20]. The rate of disinfection on glass and tile is significantly higher than the rate of disinfection in the plexiglas (p ˂ 0.05). However, there is no statistically significant difference between the rate of reduction of microorganisms on the tile and glass (p = 0.73). Although, whatever the disinfected surface, the reduction rate estimated by ATP-metry method was significant (p ˂ 0.05).
It is often wrongly assumed that the results obtained by Relative Light Unit (RLU) should demonstrate a direct correlation with the numeration on petri dish for the same sample. The amount of ATP present in microbial cells can vary greatly depending on the strain and one CFU may correspond to one or more microorganisms. In addition, the bacteria need to grow with food waste so even if in theory, it is possible to make a correlation between RLU and CFU, in practice, however it is impossible to determine the exact origin of ATP (bacteria, yeast, mold, food residue). The most frequent case is a combination of multiple factors. Thus the ATP-metry is not a direct indicator of the presence of bacteria. Nevertheless, it is the fastest and easiest technology to determine the potential for bacterial growth. Therefore, it is an indicator of the actual surface level of hygiene. There is no easier or faster else way to determine a problem. Thus, with the ATP-metry, it is possible to implement corrective actions upstream to avoid any problem of noncompliance.