Quantitative suspension tests for the evaluation of bactericidal , fungicidal and sporicidal effects of biocides used in vaccine production facility

The appropriate use of biocides is essential in any vaccine production facility and their proper evaluation using standardized tests marks the first step to ensure their proper use. Quantitative suspension tests against reference and environmental isolates were carried out to evaluate the efficacy of various biocides used in the main vaccine production facility in Egypt. Several use-dilutions of the biocides were evaluated at contact times of up to 5 min in case of antiseptics and 30 min in case of disinfectants to measure the biocide activity against bacteria, fungi and spores. Standard strains were used in addition to the main bacterial isolates identified in an environmental monitoring program carried out in the same facility. Alcohol based biocides showed bactericidal and fungicidal activity but no sporicidal activity. Chlorine based compounds and glutaraldehyde showed bactericidal and fungicidal effect while, the sporicidal effects depended on the used dilution and the contact time. Hydrogen peroxide showed bactericidal, fungicidal and sporicidal activity. Quaternary ammonium compounds tested showed very weak activity in all tests. Evaluation of the biocide is an essential step to guarantee the use of the most appropriate agent in any location.


INTRODUCTION
The proper use of biocides is an essential step in decontaminating personnel and environment in any health care settings like vaccine production facilities (Rutala et al., 1998;Kampf et al., 2003;Pineau et al., 2008).Biocides are classified as either antiseptics or disinfectants.Antiseptics are chemical agents that inhibit or kill microbial growth and are nontoxic when applied to living tissues; they are mainly used for hand washing or for treating mucous membranes and surface wounds (Kramer, 2000).Disinfectants are chemical or physical agents used to destroy or irreversibly inactivate many or all of the pathogenic microorganisms but not necessarily spores and not all viruses on inanimate objects (Madigan et al., 2002).Under certain circumstances, the same compound can be used as antiseptic in low concentration and disinfectant in high concentration.
Depending on the antimicrobial effectiveness expected from chemical agents under set conditions, disinfectants can be classified as "high level" (having sterilization activity) such as peroxides and aldehydes, "intermediate level" (which inactivate Mycobacterium tuberculosis and the most resistant types of viruses but not spores) such as alcohol and hypochlorite or "low level" (reduction of bioburden) such as phenol and quaternary ammonium compounds (QACs) (Gamage, 2003;Rutala et al., 1998).
Hydrogen peroxide works by the production of *Corresponding author.E-mail: aymen.yassin@live.com.
Table 1.Antiseptics and disinfectants used and their dilution and composition (Sheraba et al., 2012).destructive hydroxyl free radicals that can attack membrane lipids, DNA, and other essential cell components.Formaldehyde acts by alkylating the amino and sulfhydryl groups of proteins and ring nitrogen atoms of purine bases (Favero and Bond,1991).Glutaraldehyde interacts with amino acids within proteins of microorganisms (McDonnel and Russell, 1999).Chlorine acts as an oxidizing agent (Russel et al., 1982).Alcohol inactivates microorganisms by denaturation of proteins (Fraise, 1999).Phenol, in high concentrations, acts as a gross protoplasmic poison, in low concentrations cause bacterial death by the inactivation of essential enzyme systems and leakage of essential metabolites from the cell wall while QACs have been attributed to the inactivation of energy-producing enzymes, denaturation of essential cell proteins, and disruption of the cell membrane (Rutala et al., 2002).

Biocidal
To reach the level of hygiene required in a health care facility and before the application of biocides, it is necessary to standardize the use of any biocide and select the test organism that is mostly prevalent or common to the material or environment upon which the particular biocide is to be applied (Croshaw, 1981).In a previous study carried out by the same authors in the same facility, the Staphylococci spp.represented the most common isolate that was detected from an environmental monitoring programme with Staphylococcus hominis representing 51% of all Staphylococcci isolates followed by Stapyhlococccus epidermidis (14%), Stapyhlococccus haemolyticus (12%) and Micrococcus spp.(Sheraba et al., 2010).
The aim of the present study was to test the bactericidal, sporicidal and fungicidal activity of the main antiseptics and disinfectants that are commonly used in the main vaccine production facility in Egypt.The standard microorganisms recommended by the pharmacopeia were used, in addition to the main bacterial isolates that were previously identified in an environmental monitoring program to be the main contaminants likely to be present in the same facility.

MATERIALS AND METHODS
A list of all disinfectants and antiseptics used with their composition and dilutions is listed in Table 1.A list of all neutralizers with their exact composition is listed in Table 2.All culture media were from Bacto, France.

Bacterial and fungal strains
Staphylococcus aureus ATCC 6538, Pseudomonas aeruginosa ATCC 9027, Candida albicans ATCC 10231, Aspergillus niger ATCC 16404 and Bacillus subtilis ATCC 6633 were selected as index organisms representing Gram-positive and Gram-negative bacteria, fungi, and spore forming bacteria respectively.S. hominis, S. epidermidis, S. haemolyticus and Micrococcus spp.isolated from an environmental monitoring program previously carried out in the same facility were also used in the tests.

Preparation of working cultures of bacterial, fungal and spore suspension
According to European standard EN 12353 (Jette et al., 1995), test organisms were subcultured from the stock culture by streaking into trypticase soy agar (TSA) media , and incubated at 36°C for bacteria or into sabouraud dextrose agar (SDA) media, and incubated at 20-25°C for fungi (in the case of Aspergillus).After 24 h, a second subculture was prepared from the first one in the same way and incubated again.The test organisms of the second subculture were washed off with 10 ml of a diluent containing 0.1% McFarland standard of 2 units (1.5-5X10 9 CFU/ml).For Aspergillus niger, a subculture was grown on SDA and incubated at 20-25°C for 7-14 days then mycelial mats were harvested from the agar surface of the working culture, homogenized with sterile glass beads in 1% physiological peptone and filtered through sterile cotton gauze to remove hyphae then suspended in 1% physiological peptone with 0.1% Tween 80 and adjusted to McFarland standard of 2 units (1.5-5X10 9 CFU/ml).Spore cultures were developed for a period of 3-8 weeks in a sporulation medium (15 g/L Peptone, 3 g/L yeast extract; 6.0 g/L NaCl, 1.0 g/L D (+)glucose; 0.1 g/L Manganese sulphate) at 37°C then they were harvested, centrifuged (4 times at 1935 × g/ 30 min), heat-shocked 80°C/10 min and the spore suspension was adjusted to McFarland standard of 2 units (1.5-5X10 9 CFU/ml).

Preparation of the biocide dilutions
The procedure was done according to Salo and Wirtanen (2005).Each of the biocide to be tested (mentioned in Table 1) was diluted in sterile distilled water according to the recommendations of the manufacturer.The initial dilution was termed the "use-dilution" with two further dilutions prepared (one above and one below the usedilution) as shown in Tables 3 to 8. The preparations were filtered through 0.22 μm membrane filter, pH was adjusted to 6.8-7.0, and all preparations were made fresh prior to testing.

Challenge test procedure
Quantitative suspension test was used according to Hernandez et al. (2003) as follows; for each microorganism used, 0.1 ml of bacterial or fungal suspension (1X10 9 CFU/ml) was added separately to 10 ml biocide at room temperature.Controls contained 10 ml diluent instead of disinfectant or antiseptic.After contact times (5, 10, 20 and 30 min for disinfectant) or (30 s, 1 min, 2 min and 5 min for antiseptic), 1 ml was added to 9 ml neutralizing solution in order to terminate the activity of the biocide without interfering with survivor growth.The neutralizers chosen for each biocide with each test organism were previously tested and determined in a previous study that was carried out in the same facility as part of the same project (Sheraba et al., 2012).Serial dilutions were prepared (1:10, 1:10 2 , 1:10 3 , 1:10 4 , and 1:10 5 ) and 0.1 ml was taken from each serial dilution tube and plated onto TSA with 0.5% glucose by the spread-plate technique with sterile glass spatula.Plates were incubated for 24-48 h at 37°C for bacteria, spores and bacterial environmental isolates and into SDA and incubated for 3-5 days at 22.5°C for fungi and yeast.For each biocide, the test was repeated three times, and then average colonies count were enumerated and expressed as colony-forming units per millilitre.

Determination of activity
The logarithmic reduction factor (RF) was calculated as the expression of the biocide efficacy, according to the following formula (Russell et al., 1982;James, 1999;MerapÖzalp et al., 2007): RF= log Nc -log Nd RF is the Logarithmic reduction factor, Nc is bacterial colony

Evaluation of bactericidal activity
As shown in Table 3, the antiseptics: Alcohol, AHD 2000 and Hospidermin were highly effective against both Gram-positive and Gram-negative bacteria at all concentrations and contact times tested.Also, Sanigel was highly effective at concentration 50, 75 and 95% at 60 s, 120 s and 300 s contact times, less effective at concentration 50% in 30 s and effective at 50% at 60 s, at 75% at 30 s, 60 s and at 95% at 30 s.For the disinfectants tested as shown in at 1:15 for 30 min.

Evaluation of Fungicidal activity
As shown in Table 5, the antiseptics, Alcohol, AHD 2000 and Hospidermin were highly effective against fungi and yeast at all concentrations and contact times tested.In contrast, Sanigel was less effective at concentration 50% at 30 and 60 scontact times, effective at concentration 75% and highly effective at 95%.For the disinfectants tested as shown in Table 6, Lysoformin 3000, Chlorax and H 2 O 2 25% were the most potent disenfectants against the fungi tested (A.niger and C. albicans) followed by Trichlorol, while Sanipine and Sanismell performed less effectively.

Evaluation of Sporicidal activity
All antiseptics mentioned were found to be ineffective against spores of B. subtilis (Table 7).For the tested disinfectants, H 2 O 2 25% and Chlorax were found to be highly effective against B. subtilis spores at concentration of 2, 3, 5% and 1:8 and 1:16 respectively at 20 and 30 min contact time, while Lysoformin 3000 was found to be less effective at concentration 1 and 2% at 30 min.
Trichlorol was found to be less effective at 3% for 10, 20, 30 min contact time, while Sanipine and Sanismell were shown to be ineffective (Table 8).

DISCUSSION
In this study, quantitative suspension test was performed to evaluate the bactericidal, fungicidal and sporicidal activity of four chemical antiseptics and six chemical disinfectants at various concentrations, at defined contact times, in the absence of interfering substances.Concerning the bactericidal and fungicidal efficiencies of the tested antiseptics, the results indicate that three of the alcohol based biocides (Alcohol, AHD 2000 and Hospidermin) reduced the viable titers of the suspended vegetative bacteria and fungi by more than 5 log values even at a concentration of 50% and within 30 s.The fourth alcohol based biocide, Sanigel, reached the same efficacy but at the longer contact time of 120 s.Similar findings have been reported by other groups showing that alcohol based antiseptics have a very rapid and effective antimicrobial action that exceeds any other antiseptic at concentration 50-90% for shorter contact times against bacteria and fungi (Van Klingeren et al., 1998;Vieira et al., 2005;Jeng and Severin, 1998).
Among the six disinfectants tested, only the aldehydebased disinfectant (Lysoformin 3000), Chlorine-based: (Chlorax and Trichlorol) and Hydrogen peroxide had an excellent killing activity within 5 min against the suspended vegetative bacteria and fungi at the recommended concentrations.These findings are similar to another study, which showed that 2% Lysoformin 3000 at 30 min was enough for bactericidal and fungicidal activity (Aksen et al., 2004).It was reported that all chlorine-based disinfectants were significantly more effective in killing bacteria and fungi in 5 min and the efficiency was generally increased when the contact time was prolonged from 5 to 15 min (Van Klingeren et al., 1998;Özalp et al., 2007;Salo and Wirtanen, 2005).Other groups reported that 3-25% hydrogen peroxide was able to inactivate bacteria and fungi by > 6 log 10 reduction in a contact time of 5 min, while, Sanipine and Sanismel were effective only at 1:15 for 30 min requiring prolonged exposure time or greater concentration (Russell, 1998;Alfa and Jackson, 2001;Sattar et al., 2002).Quaternary ammonium compounds gained popularity due to its good antibacterial and antifungal at relatively low concentration within shorter contact time (Russell, 1998;Fraise, 1999).
The principal of evaluation of sporicidal activity was the same as for determination of bactericidal activity; the main difference was to achieve sporulation (Russell, 1998).Our results indicate that all alcohol based antiseptics were found to be nonsporicidal.Similar findings have been reported by other groups for these agents (Fraise, 1999;Gupta et al., 2007(. Hydrogen peroxide (25%) was found to the more rapid and effective in killing spores with all its recommended concentrations (Coates, 1996;Jose-Luis Sagripanti and Bonifacino, 1996) which agrees with our findings.Chlorax showed high killing effect at concentration1:8 and 1:16 for 20-30 min, however, Trichlorol requires higher concentration to achieve the same effect of killing as Chlorax which agrees with the study done by Russel in 1998 (Russell, 1998).In contrast, 30 min of 2% Lysoformin 3000 was found to be effective against bacterial spores as reported (Aksen et al., 2004;Holton et al., 1995) who noted that glutaraldehyde based disinfectants have slow sporicidal effect while sanismel and Sanipine were found to be nonsporicidal.
Most previous reports have only analyzed the effect of biocides against reference strains from ATCC (Jang et al., 2008;Sagripanti and Bonifacino, 1996;Crowe et al., 2007).Susceptibility of environmental isolates to biocides is now attracting special attention since ATCC strains that are laboratory adapted, may not be good predictors for the susceptibility of strains extracted from environments.It was interesting that environmental isolates as Micrococcus spp., S. hominis, S. haemolyticus and S. epidermidis also showed the same susceptibility as ATCC strains.

Conclusion
From the tested biocides, hydrogen peroxide showed the strongest effect as it had bactericidal, fungicidal and sporicidal activity.Alcohol or chlorine based compounds, and gluteraldehyde showed bactericidal and fungicidal activity but no spirocidal activity except for some used dilutions at certain contact times in case of chlorine based compounds and gluteraldehyde.Quaternary ammonium compounds had the weakest activity in all tests.
It is essential to constantly monitor the activity of biocides that are frequently used in any health care settings as represented in this work by the vaccine production facility where this work was done.The efficacy of the biocides should be tested with index microorganisms as well as common isolates identified in environmental monitoring programs and known to be possibly present in that particular location.

Table 3 .
Evaluation of bactericidal activity of test antiseptics

Table 4 .
Evaluation of bactericidal activity of test disinfectants

Table 4
, Lysoformin 3000, Chlorax, Trichlorol and H 2 O 2 25% were highly effective against both Gram-positive and Gram-negative bacteria at all concentrations and contact time tested, while Sanipine and Sanismell showed only limited activity

Table 5 .
Evaluation of fungicidal activity of test antiseptics.

Table 6 .
Evaluation of fungicidal activity of test disinfectants.