- Email: [email protected]
Bactericidal Efficacy of Ultraviolet Irradiation on Staphylococcus aureus
Hardjawinata, Setiawati, Dewi
Asian J Oral Maxillofac Surg 2005;17(3):157-161. ORIGINAL RESEARCH
Bactericidal Efficacy of Ultraviolet Irradiation on Staphylococcus aureus Karlina Hardjawinata, Rina Setiawati, Warta Dewi Department of Oral Biology, Faculty of Dentistry, Padjadjaran University, Bandung, Indonesia
Abstract Objective: To determine the bactericidal effect of ultraviolet radiation on Staphylococcus aureus air contaminant for preventing airborne transmission. Materials and Methods: The air of a dental clinic was sampled on settling plates to isolate Staphylococcus aureus. The suspension of Staphylococcus aureus was irradiated with a 6-W ultraviolet lamp after 5, 10, 15, and 30 minutes, and 1, 2, 3, 4, and 24 hours. After every exposure time, 0.1 ml suspension was taken with a pipette and cultured on a sterile nutrient agar plate. This process was repeated by culturing 0.1 ml of Staphylococcus aureus suspension without ultraviolet radiation for a control. All cultures were incubated overnight at 37ºC, and the developed colonies were counted with a Stuart electrical bacteria colony counter. Results: Ultraviolet radiation inhibited 89.1% of the colonies after 5 minutes, and 89.6% after 10 minutes. The number of colonies undergoing division diminished with longer irradiation times, and all bacteria were destroyed after 3 to 4 hours of radiation. Staphylococcus aureus suspension without radiation showed an increase in 6.7% of the colonies after 5 minutes and an increase of 117.9% after 4 hours. A greater number of colony-forming units developed with longer exposure times. Conclusions: Ultraviolet radiation has a bactericidal effect on Staphylococcus aureus, even after exposure for 5 minutes. Therefore, ultraviolet radiation could be used to control the airborne transmission of Staphylococcus aureus in treatment areas such as dental clinics. Key words: Staphylococcus aureus, Ultraviolet rays
Introduction For much of human history, air has been considered the primary cause of contagion.1,2 There is convincing evidence for airborne transmission of varicella zoster, smallpox, influenza, and measles viruses, Mycobacterium tuberculosis, Staphylococcus aureus, Brucella spp, and other micro-organisms.3 Airborne staphylococci may be shed by carriers and may remain viable for a long time.4,5 Diseases caused by S aureus include superficial infections (boils, carbuncles, pustules, abscesses, conjunctivitis, and wound infections), food poisoning (vomiting and diarrhoea), toxic shock syndrome, and deep infections (osteomyelitis, endocarditis, septicaemia, and pneumonia).6-8 Correspondence: Karlina Hardjawinata, Jl Satrugna 64, Bandung-40172, Indonesia. Fax: (62 22) 607 2982; E-mail: [email protected]
© Asian 2005J Asian Oral Maxillofac Association Surg of Oral Vol 17, andNo Maxillofacial 3, 2005 Surgeons.
Ultraviolet (UV) radiation has a shorter wavelength than visible light and carries more energy. UV radiation has a unique ability to kill micro-organisms with which it comes into contact because of the high concentration of energy. Germicidal UV lamps have been developed for controlling contamination in indoor environments. Studies have shown that these measures may be effective for controlling the spread of disease.9,10 However, little is known about the effectiveness of UV radiation for preventing S aureus contamination. This study was undertaken to determine the effectiveness of germicidal UV radiation to reduce S aureus contamination in a dental clinic to prevent its possible airborne transmission.
Materials and Methods Air contaminant bacteria were sampled at 5 different sites in the outpatient clinic at the Faculty of Dentistry, 157
Effect of Ultraviolet Irradiation on Staphylococcus aureus
Padjadjaran University, Bandung, Indonesia, by opening the covers of mannitol-salt agar (MSA) plates (Oxoid CM 85; Oxoid Ltd, Basingstoke, UK) to enable direct contact with the air. After 1 hour of exposure, the covers were replaced. All plates were immediately transported to the laboratory for incubation for 18 to 24 hours at 37ºC. The S aureus colony was isolated from the plate culture and identified on the basis of the colonial and cellular morphology, Gram’s stain, and biochemical reactions.11,12 The isolated colony was heavily inoculated on a nutrient agar slant (Oxoid CM 3) to obtain a subculture and then incubated for 18 to 24 hours at 37ºC. To test for the presence of catalase, the slant was positioned on an incline and 1 ml of a 3% hydrogen peroxide solution was poured to flow over the surface of the S aureus colony.11,12 A coagulase test to indicate the presence of S aureus was performed on a clean glass slide by emulsifying the colony in a drop of water to produce a dense uniform suspension. One drop of fresh human plasma was added to the suspension and mixed in a continuous circular motion for 5 seconds. If any evidence of autoagglutination was noted before adding the plasma, the culture was not considered suitable for the slide test.11 As a stock culture represents the growth of a single bacterial species, each of these isolates was grown on a nutrient agar slant for purification. To obtain a standardised S aureus suspension, an inoculating needle or loop was used to contact the stock culture and to aseptically inoculate a 5-ml nutrient broth (Oxoid CM 1) to match the turbidity standard of McFarland 1. Plates suitable for counting must contain between 30 and 300 colonies.11 With the use of a serial dilution technique,11 the suspension was prepared to achieve 2 x 10-4 concentration as quantitative viable S aureus cells. To achieve the McFarland 0.5 standardised S aureus suspension, 5 ml of sterile nutrient broth was added to the suspension to match the turbidity of McFarland 1. This tube was labelled as tube number 1. Three tubes of 9-ml nutrient broth were labelled as 158
numbers 2, 3, and 4. The labelled tubes were placed in a test tube rack. To achieve even dispersal of bacterial cells in the culture, tube number 1 was mixed by rolling it between the palms of the hands. Then, using a sterile pipette, 1 ml of the bacterial suspension in tube number 1 was aseptically transferred into tube number 2 containing 9 ml nutrient broth and the pipette was discarded into a beaker containing disinfectant. The culture was diluted 10 times to 10-1 concentration. The culture in tube number 2 was mixed and, with a new sterile pipette, 1 ml of the mixture was transferred into tube number 3, the pipette was again discarded. The culture was diluted 100 times to 10-2. The same action was also used for tube number 3 to obtain a dilution of 10-3, and for tube number 4 to obtain a dilution of 10-4. Finally, 10 ml of prepared suspension of S aureus from tube number 4 was placed into an uncovered sterile petri dish (100-mm diameter) for light irradiation. Ultraviolet Irradiation According to the UV technical bulletin,13 ultraviolet energy at a wavelength of 2537 Å (253.7 nm) required for 99% destruction of bacteria was 5800 μW second/ cm2 for Staphylococcus epidermidis and 7000 μW second/cm2 for S aureus. The suspension was irradiated after 5, 10, 15, and 30 minutes, and 1, 2, 3, 4, and 24 hours under a germicidal 6-W UV lamp (Meditec, Bandung, Indonesia). Irradiation was conducted by placing the plate 12 inches below the UV light source in a special box to prevent environmental air contamination. After each exposure time, 0.1 ml suspension was taken by sterile pipette and distributed following the spread-plate technique by means of a bent glass rod held over the surface of a nutrient agar plate.11 Finally, the cover of each inoculated plate was labelled according to the number of S aureus isolated and the exposure time to UV radiation. The process was repeated without UV radiation for the control group. All plate cultures were incubated overnight in an inverted position at 37ºC. For greater accuracy, this method was also applied to S aureus isolates sampled at 4 other clinic sites. Asian J Oral Maxillofac Surg Vol 17, No 3, 2005
Hardjawinata, Setiawati, Dewi
Results Staphylococcus aureus Identification The settling of nutrient agar plates during 1 hour in the dental clinic showed no visible colony on the agar. On the MSA cultures, the S aureus colony was surrounded by a yellow zone, especially when the reading was made after incubation. Well-isolated colonies of staphylococci would be 1 to 3 mm in diameter if incubated within 24 hours. The colony of S aureus was circular, smooth, glistening, and raised to slightly convex. Gram-staining was observed for typical coccus gram-positive morphology and aggregation patterns arranged in grape-like irregular clusters. When 2 to 3 drops of 3% hydrogen peroxide solution were applied over the entire surface of S aureus slant culture, gas bubble formation of free oxygen gas appeared. This indicated that catalase was present. This result distinguishes Staphylococcus spp from Streptococcus spp, which has a negative catalase test.14-17 Production of coagulase was shown by ‘clumping’ within 5 to 10 seconds. This activity showed plasmaclotting potential and was considered coagulaseExposure time (minutes)
positive. The presence of coagulation indicates the presence of a virulent S aureus strain, rather than S epidermidis or S saprophyticus. 14-17 Colony-forming Units The measurements of S aureus CFUs that did not undergo UV irradiation are shown in Table 1 and the increase of colony numbers versus incubation time is shown as an organism population growth curve in Figure 1. Within 5 minutes, there was a slight increase of CFU numbers (6.7%) and, CFU numbers increased to 9.0% after 10 minutes, 16.3% after 15 minutes, 27.3% after 30 minutes, and 49.3% after 60 minutes. A rapid exponential increase in population to the maximum number occurred after 3 to 4 hours of incubation. The generation time in this study (time required by a microbial population to double) was reached after 3 to 4 hours incubation.
900 800 Staphylococcus aureus colony-forming units
Quantification of Viable Cells and Statistical Analysis The number of colonies that developed after incubation on all plates are referred to as colonyforming units (CFUs) because each single cell on the plate became visible as a colony, which could then be counted macroscopically. The CFUs were observed and recorded by a Stuart electrical bacteria colony counter. All of the CFUs were statistically calculated according to a regression analysis.
700 600 500 400 300
0
60
120
180
240
300
Time (minutes)
Figure 1. Population growth curve for Staphylococcus aureus not irradiated with ultraviolet light.
Staphylococcus aureus isolates
Increase in CFUs
Site 1
Site 2
Site 3
Site 4
Site 5
Mean
Total
%
0
376
397
402
389
381
389.0
5
417
418
421
411
409
415.2
26.2
6.7
10
423
427
434
419
15
445
451
463
458
417
424.0
35.0
9.0
446
452.6
63.6
16.3
30
480
503
497
516
481
495.4
106.4
27.3
60
564
598
584
573
585
580.8
191.8
49.3
120
674
180
781
686
693
679
698
686.0
297.0
76.3
767
734
756
744
756.4
367.4
94.4
Table 1. Increase in Staphylococcus aureus and colony-forming units (CFUs) not irradiated with ultraviolet light.
Asian J Oral Maxillofac Surg Vol 17, No 3, 2005
159
Effect of Ultraviolet Irradiation on Staphylococcus aureus Exposure time (minutes)
Staphylococcus aureus isolates
0
Increase in CFUs
Site 1
Site 2
Site 3
Site 4
Site 5
Mean
373
391
384
369
347
372.8
Total
%
5
37
42
40
43
41
40.6
332.2
89.1
10
35
39
41
40
38
38.6
334.2
89.6
15
32
37
33
35
32
33.8
339.0
90.9
30
25
31
29
27
30
28.4
344.4
92.4
60
14
17
12
18
13
14.8
358.0
96.0
120
6
9
5
6
8
6.8
366.0
98.2
180
1
3
0
0
2
1.2
371.6
99.7
240
0
0
0
0
0
0.0
372.8
100.0
1440
0
0
0
0
0
0.0
372.8
100.0
Table 2. Decrease in Staphylococcus aureus and colony-forming units (CFUs) irradiated with ultraviolet light.
In contrast, after UV irradiation the CFUs decreased after exposure by 89.1% after 5 minutes and 89.6% after 10 minutes. Longer irradiation times decreased the number of colonies (Table 2). The micro-organisms died at a rapid and uniform rate of 90.9% after 15 minutes, 92.3% after 30 minutes, 96.0% after 60 minutes, and 98.2% after 120 minutes. After 3 hours of exposure to UV irradiation, 2 of 5 plates showed no CFUs, and after 4 hours of UV irradiation there were no colonies on any of the culture plates. The decline in population growth after 3 to 4 hours exposure time is shown in Figure 2. On the basis of statistical evaluation by regression analysis, 198 minutes 42 seconds (3 to 4 hours) of exposure to UV irradiation was required to kill all S aureus organisms.
Staphylococcus aureus colony-forming units
40 30 20 10
60
120
180
240
Time (minutes)
Figure 2. Population growth curve for Staphylococcus aureus irradiated with ultraviolet light.
160
MSA is selected for salt-tolerant organisms such as staphylococci. Differentiation among staphylococci is predicated on their ability to ferment mannitol. S aureus isolates obtained from samples on settling plates in the dental clinic provided evidence that there was air contamination that could be considered as a source of staphylococcal infection. A positive result for coagulase production is considered synonymous with the invasive pathogenic potential of S aureus. The enzyme acts within host tissues to convert fibrinogen to fibrin. It has been suggested that the fibrin meshwork formed by this conversion surrounds the bacterial cells of infected tissues, protecting the organism from non-specific host resistance mechanisms such as phagocytosis and the antistaphylococcal activity of normal serum.6,12,16 The measurement of S aureus CFUs on the agar plates without UV irradiation indicated the increase in CFU numbers. This result was achieved by inoculating and incubating the samples under optimum conditions of nutrition, temperature, and pH of the medium so that the cell division increased.
50
0
Discussion
However, the irradiated CFUs decreased at a rapid and uniform rate commensurate with the UV irradiation time. UV radiation ranges in wavelength from 240 nm to 280 nm with a peak at 260 nm. This wavelength is most effectively absorbed by DNA.8,16,17 The primary mechanism of UV damage is the formation of cyclobutane-type dimers, usually thymine dimers. Two adjacent thymines in a DNA Asian J Oral Maxillofac Surg Vol 17, No 3, 2005
Hardjawinata, Setiawati, Dewi
strand are covalently joined to inhibit the normal DNA replication and activation of the microorganisms.8,16,17 This study indicates that germicidal UV irradiation may be an effective approach for reducing S aureus contamination within 3 to 4 hours. It is difficult to eradicate pathogenic staphylococci from infected persons because the organisms rapidly develop resistance to many antimicrobial drugs.18 Contact spread of infection is important in hospitals. Patients in operating rooms, intensive care units, and neonatal units are at high risk for S aureus infection, which may lead to serious clinical disease.16 Few nursery staphylococcal outbreaks have been shown to be caused by staff carriers, but it is more prudent to adopt good aseptic techniques and other standard infection control measures than to treat nasal carriers.18 As environmental organisms pose an infection hazard, the ideal is to aim for a clean protective clinical environment. Clearly, special efforts are warranted to reduce airborne contaminants in the vicinity of highly immunosuppressed patients. It is therefore important to consider the use of air purification systems, possibly involving overnight UV irradiation of dental clinics.19
References 1. Kundsin RB. Airborne contagion. Ann NY Acad Sci 1980;353:1. 2. Muilenberg ML, Burge HA. Aerobiology. Boca Raton: Lewis Publishers; 1996. 3. Eickhoff TC. Airborne nosocomial infection. A contemporary perspective. Infect Control Hosp Epidemiol 1994;15:663. 4. Boyce JM, Opal SM, Potter-Byn G, Medeirose AA. Spread of methicillin-resistant Staphylococcus aureus in a hospital after exposure to a healthcare worker with chronic sinusitis. Clin Infect Dis 1993;17:496-504. 5. Belani A, Sheretz RH, Sullivan ML. Outbreak of staphylococcal infection in two hospital nurseries traced
Asian J Oral Maxillofac Surg Vol 17, No 3, 2005
6. 7. 8. 9.
10.
11. 12.
13. 14.
15.
16.
17.
18.
19.
to a single nasal carrier. Infect Control Hosp Epidemiol 1986;7:487-490. Talaro K, Talaro A. Foundation of microbiology, 2nd ed. Dubuque: Wm C Brown Publishers; 1996. Samaranayake LP. Essential microbiology for dentistry. 2nd ed. Edinburgh: Churchill Livingstone; 2002. Prescott LM, Harley JP, Klein DA. Microbiology. 5th ed. Singapore: McGraw-Hill Higher Education (Asia); 2002. Macher JM, Alevant LE, Chang YL, Liu KS. Effect of ultraviolet germicidal lamps on airborne microorganisms in an outpatient waiting room. Appl Occup Environ Hyg 1992;7:505-513. Macher JM. The use of germicidal lamps to control tuberculosis in healthcare facilities. Infect Control Hosp Epidemiol 1993:14:723-729. Cappuccino JG, Sherman N. Microbiology. A laboratory manual. 6th ed. San Francisco: Benjamin Cummings; 2002. Bagg J, MacFarlane TW, Poxton IR, Miller CH, Smith AJ. Essentials of microbiology for dental students. New York: Oxford University Press; 1999. Aqua Treatment Service. UV technical bulletin. www. onthenetnow.com//uv_tech_bulletin_ats.pdf. 2001. Molinari JA. Sterilization and disinfection. In: Schuster GS, editor. Oral microbiology and infectious disease. 3rd ed. Philadelphia: BC Decker Inc; 1990. Rathbun WE. Sterilization and asepsis. In: Newman MG, Nisengard R, editors. Oral microbiology and immnunology. Philadelphia: WB Saunders Company; 1988. Levinson W, Jawetz E. Medical microbiology and immunology: examination and board review. 5th ed. Connecticut: Appleton & Lange; 1998. Brooks GF, Butel JS, Moore SA. The growth, survival, and death of microorganisms. In: Jawetz, Melnick, & Adelberg’s medical microbiology. 22nd ed. New Delhi: Appleton & Lange; 2001:46-54. Sands KEF, Goldmann DA. Epidemiology of staphylococcus and group A streptococci. In: Bennet JV, Brachman PS, editors. Hospital infections. Philadelphia: LippincottRaven Publishers; 1998:621-636. Rhame FS. The inanimate environment. In: Bennet JV, Brachman PS, editors. Hospital infections. Philadelphia: Lippincott-Raven Publishers; 1998:299-324.
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Asian J Oral Maxillofac Surg 2005;17(3):157-161. ORIGINAL RESEARCH
Bactericidal Efficacy of Ultraviolet Irradiation on Staphylococcus aureus Karlina Hardjawinata, Rina Setiawati, Warta Dewi Department of Oral Biology, Faculty of Dentistry, Padjadjaran University, Bandung, Indonesia
Abstract Objective: To determine the bactericidal effect of ultraviolet radiation on Staphylococcus aureus air contaminant for preventing airborne transmission. Materials and Methods: The air of a dental clinic was sampled on settling plates to isolate Staphylococcus aureus. The suspension of Staphylococcus aureus was irradiated with a 6-W ultraviolet lamp after 5, 10, 15, and 30 minutes, and 1, 2, 3, 4, and 24 hours. After every exposure time, 0.1 ml suspension was taken with a pipette and cultured on a sterile nutrient agar plate. This process was repeated by culturing 0.1 ml of Staphylococcus aureus suspension without ultraviolet radiation for a control. All cultures were incubated overnight at 37ºC, and the developed colonies were counted with a Stuart electrical bacteria colony counter. Results: Ultraviolet radiation inhibited 89.1% of the colonies after 5 minutes, and 89.6% after 10 minutes. The number of colonies undergoing division diminished with longer irradiation times, and all bacteria were destroyed after 3 to 4 hours of radiation. Staphylococcus aureus suspension without radiation showed an increase in 6.7% of the colonies after 5 minutes and an increase of 117.9% after 4 hours. A greater number of colony-forming units developed with longer exposure times. Conclusions: Ultraviolet radiation has a bactericidal effect on Staphylococcus aureus, even after exposure for 5 minutes. Therefore, ultraviolet radiation could be used to control the airborne transmission of Staphylococcus aureus in treatment areas such as dental clinics. Key words: Staphylococcus aureus, Ultraviolet rays
Introduction For much of human history, air has been considered the primary cause of contagion.1,2 There is convincing evidence for airborne transmission of varicella zoster, smallpox, influenza, and measles viruses, Mycobacterium tuberculosis, Staphylococcus aureus, Brucella spp, and other micro-organisms.3 Airborne staphylococci may be shed by carriers and may remain viable for a long time.4,5 Diseases caused by S aureus include superficial infections (boils, carbuncles, pustules, abscesses, conjunctivitis, and wound infections), food poisoning (vomiting and diarrhoea), toxic shock syndrome, and deep infections (osteomyelitis, endocarditis, septicaemia, and pneumonia).6-8 Correspondence: Karlina Hardjawinata, Jl Satrugna 64, Bandung-40172, Indonesia. Fax: (62 22) 607 2982; E-mail: [email protected]
© Asian 2005J Asian Oral Maxillofac Association Surg of Oral Vol 17, andNo Maxillofacial 3, 2005 Surgeons.
Ultraviolet (UV) radiation has a shorter wavelength than visible light and carries more energy. UV radiation has a unique ability to kill micro-organisms with which it comes into contact because of the high concentration of energy. Germicidal UV lamps have been developed for controlling contamination in indoor environments. Studies have shown that these measures may be effective for controlling the spread of disease.9,10 However, little is known about the effectiveness of UV radiation for preventing S aureus contamination. This study was undertaken to determine the effectiveness of germicidal UV radiation to reduce S aureus contamination in a dental clinic to prevent its possible airborne transmission.
Materials and Methods Air contaminant bacteria were sampled at 5 different sites in the outpatient clinic at the Faculty of Dentistry, 157
Effect of Ultraviolet Irradiation on Staphylococcus aureus
Padjadjaran University, Bandung, Indonesia, by opening the covers of mannitol-salt agar (MSA) plates (Oxoid CM 85; Oxoid Ltd, Basingstoke, UK) to enable direct contact with the air. After 1 hour of exposure, the covers were replaced. All plates were immediately transported to the laboratory for incubation for 18 to 24 hours at 37ºC. The S aureus colony was isolated from the plate culture and identified on the basis of the colonial and cellular morphology, Gram’s stain, and biochemical reactions.11,12 The isolated colony was heavily inoculated on a nutrient agar slant (Oxoid CM 3) to obtain a subculture and then incubated for 18 to 24 hours at 37ºC. To test for the presence of catalase, the slant was positioned on an incline and 1 ml of a 3% hydrogen peroxide solution was poured to flow over the surface of the S aureus colony.11,12 A coagulase test to indicate the presence of S aureus was performed on a clean glass slide by emulsifying the colony in a drop of water to produce a dense uniform suspension. One drop of fresh human plasma was added to the suspension and mixed in a continuous circular motion for 5 seconds. If any evidence of autoagglutination was noted before adding the plasma, the culture was not considered suitable for the slide test.11 As a stock culture represents the growth of a single bacterial species, each of these isolates was grown on a nutrient agar slant for purification. To obtain a standardised S aureus suspension, an inoculating needle or loop was used to contact the stock culture and to aseptically inoculate a 5-ml nutrient broth (Oxoid CM 1) to match the turbidity standard of McFarland 1. Plates suitable for counting must contain between 30 and 300 colonies.11 With the use of a serial dilution technique,11 the suspension was prepared to achieve 2 x 10-4 concentration as quantitative viable S aureus cells. To achieve the McFarland 0.5 standardised S aureus suspension, 5 ml of sterile nutrient broth was added to the suspension to match the turbidity of McFarland 1. This tube was labelled as tube number 1. Three tubes of 9-ml nutrient broth were labelled as 158
numbers 2, 3, and 4. The labelled tubes were placed in a test tube rack. To achieve even dispersal of bacterial cells in the culture, tube number 1 was mixed by rolling it between the palms of the hands. Then, using a sterile pipette, 1 ml of the bacterial suspension in tube number 1 was aseptically transferred into tube number 2 containing 9 ml nutrient broth and the pipette was discarded into a beaker containing disinfectant. The culture was diluted 10 times to 10-1 concentration. The culture in tube number 2 was mixed and, with a new sterile pipette, 1 ml of the mixture was transferred into tube number 3, the pipette was again discarded. The culture was diluted 100 times to 10-2. The same action was also used for tube number 3 to obtain a dilution of 10-3, and for tube number 4 to obtain a dilution of 10-4. Finally, 10 ml of prepared suspension of S aureus from tube number 4 was placed into an uncovered sterile petri dish (100-mm diameter) for light irradiation. Ultraviolet Irradiation According to the UV technical bulletin,13 ultraviolet energy at a wavelength of 2537 Å (253.7 nm) required for 99% destruction of bacteria was 5800 μW second/ cm2 for Staphylococcus epidermidis and 7000 μW second/cm2 for S aureus. The suspension was irradiated after 5, 10, 15, and 30 minutes, and 1, 2, 3, 4, and 24 hours under a germicidal 6-W UV lamp (Meditec, Bandung, Indonesia). Irradiation was conducted by placing the plate 12 inches below the UV light source in a special box to prevent environmental air contamination. After each exposure time, 0.1 ml suspension was taken by sterile pipette and distributed following the spread-plate technique by means of a bent glass rod held over the surface of a nutrient agar plate.11 Finally, the cover of each inoculated plate was labelled according to the number of S aureus isolated and the exposure time to UV radiation. The process was repeated without UV radiation for the control group. All plate cultures were incubated overnight in an inverted position at 37ºC. For greater accuracy, this method was also applied to S aureus isolates sampled at 4 other clinic sites. Asian J Oral Maxillofac Surg Vol 17, No 3, 2005
Hardjawinata, Setiawati, Dewi
Results Staphylococcus aureus Identification The settling of nutrient agar plates during 1 hour in the dental clinic showed no visible colony on the agar. On the MSA cultures, the S aureus colony was surrounded by a yellow zone, especially when the reading was made after incubation. Well-isolated colonies of staphylococci would be 1 to 3 mm in diameter if incubated within 24 hours. The colony of S aureus was circular, smooth, glistening, and raised to slightly convex. Gram-staining was observed for typical coccus gram-positive morphology and aggregation patterns arranged in grape-like irregular clusters. When 2 to 3 drops of 3% hydrogen peroxide solution were applied over the entire surface of S aureus slant culture, gas bubble formation of free oxygen gas appeared. This indicated that catalase was present. This result distinguishes Staphylococcus spp from Streptococcus spp, which has a negative catalase test.14-17 Production of coagulase was shown by ‘clumping’ within 5 to 10 seconds. This activity showed plasmaclotting potential and was considered coagulaseExposure time (minutes)
positive. The presence of coagulation indicates the presence of a virulent S aureus strain, rather than S epidermidis or S saprophyticus. 14-17 Colony-forming Units The measurements of S aureus CFUs that did not undergo UV irradiation are shown in Table 1 and the increase of colony numbers versus incubation time is shown as an organism population growth curve in Figure 1. Within 5 minutes, there was a slight increase of CFU numbers (6.7%) and, CFU numbers increased to 9.0% after 10 minutes, 16.3% after 15 minutes, 27.3% after 30 minutes, and 49.3% after 60 minutes. A rapid exponential increase in population to the maximum number occurred after 3 to 4 hours of incubation. The generation time in this study (time required by a microbial population to double) was reached after 3 to 4 hours incubation.
900 800 Staphylococcus aureus colony-forming units
Quantification of Viable Cells and Statistical Analysis The number of colonies that developed after incubation on all plates are referred to as colonyforming units (CFUs) because each single cell on the plate became visible as a colony, which could then be counted macroscopically. The CFUs were observed and recorded by a Stuart electrical bacteria colony counter. All of the CFUs were statistically calculated according to a regression analysis.
700 600 500 400 300
0
60
120
180
240
300
Time (minutes)
Figure 1. Population growth curve for Staphylococcus aureus not irradiated with ultraviolet light.
Staphylococcus aureus isolates
Increase in CFUs
Site 1
Site 2
Site 3
Site 4
Site 5
Mean
Total
%
0
376
397
402
389
381
389.0
5
417
418
421
411
409
415.2
26.2
6.7
10
423
427
434
419
15
445
451
463
458
417
424.0
35.0
9.0
446
452.6
63.6
16.3
30
480
503
497
516
481
495.4
106.4
27.3
60
564
598
584
573
585
580.8
191.8
49.3
120
674
180
781
686
693
679
698
686.0
297.0
76.3
767
734
756
744
756.4
367.4
94.4
Table 1. Increase in Staphylococcus aureus and colony-forming units (CFUs) not irradiated with ultraviolet light.
Asian J Oral Maxillofac Surg Vol 17, No 3, 2005
159
Effect of Ultraviolet Irradiation on Staphylococcus aureus Exposure time (minutes)
Staphylococcus aureus isolates
0
Increase in CFUs
Site 1
Site 2
Site 3
Site 4
Site 5
Mean
373
391
384
369
347
372.8
Total
%
5
37
42
40
43
41
40.6
332.2
89.1
10
35
39
41
40
38
38.6
334.2
89.6
15
32
37
33
35
32
33.8
339.0
90.9
30
25
31
29
27
30
28.4
344.4
92.4
60
14
17
12
18
13
14.8
358.0
96.0
120
6
9
5
6
8
6.8
366.0
98.2
180
1
3
0
0
2
1.2
371.6
99.7
240
0
0
0
0
0
0.0
372.8
100.0
1440
0
0
0
0
0
0.0
372.8
100.0
Table 2. Decrease in Staphylococcus aureus and colony-forming units (CFUs) irradiated with ultraviolet light.
In contrast, after UV irradiation the CFUs decreased after exposure by 89.1% after 5 minutes and 89.6% after 10 minutes. Longer irradiation times decreased the number of colonies (Table 2). The micro-organisms died at a rapid and uniform rate of 90.9% after 15 minutes, 92.3% after 30 minutes, 96.0% after 60 minutes, and 98.2% after 120 minutes. After 3 hours of exposure to UV irradiation, 2 of 5 plates showed no CFUs, and after 4 hours of UV irradiation there were no colonies on any of the culture plates. The decline in population growth after 3 to 4 hours exposure time is shown in Figure 2. On the basis of statistical evaluation by regression analysis, 198 minutes 42 seconds (3 to 4 hours) of exposure to UV irradiation was required to kill all S aureus organisms.
Staphylococcus aureus colony-forming units
40 30 20 10
60
120
180
240
Time (minutes)
Figure 2. Population growth curve for Staphylococcus aureus irradiated with ultraviolet light.
160
MSA is selected for salt-tolerant organisms such as staphylococci. Differentiation among staphylococci is predicated on their ability to ferment mannitol. S aureus isolates obtained from samples on settling plates in the dental clinic provided evidence that there was air contamination that could be considered as a source of staphylococcal infection. A positive result for coagulase production is considered synonymous with the invasive pathogenic potential of S aureus. The enzyme acts within host tissues to convert fibrinogen to fibrin. It has been suggested that the fibrin meshwork formed by this conversion surrounds the bacterial cells of infected tissues, protecting the organism from non-specific host resistance mechanisms such as phagocytosis and the antistaphylococcal activity of normal serum.6,12,16 The measurement of S aureus CFUs on the agar plates without UV irradiation indicated the increase in CFU numbers. This result was achieved by inoculating and incubating the samples under optimum conditions of nutrition, temperature, and pH of the medium so that the cell division increased.
50
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Discussion
However, the irradiated CFUs decreased at a rapid and uniform rate commensurate with the UV irradiation time. UV radiation ranges in wavelength from 240 nm to 280 nm with a peak at 260 nm. This wavelength is most effectively absorbed by DNA.8,16,17 The primary mechanism of UV damage is the formation of cyclobutane-type dimers, usually thymine dimers. Two adjacent thymines in a DNA Asian J Oral Maxillofac Surg Vol 17, No 3, 2005
Hardjawinata, Setiawati, Dewi
strand are covalently joined to inhibit the normal DNA replication and activation of the microorganisms.8,16,17 This study indicates that germicidal UV irradiation may be an effective approach for reducing S aureus contamination within 3 to 4 hours. It is difficult to eradicate pathogenic staphylococci from infected persons because the organisms rapidly develop resistance to many antimicrobial drugs.18 Contact spread of infection is important in hospitals. Patients in operating rooms, intensive care units, and neonatal units are at high risk for S aureus infection, which may lead to serious clinical disease.16 Few nursery staphylococcal outbreaks have been shown to be caused by staff carriers, but it is more prudent to adopt good aseptic techniques and other standard infection control measures than to treat nasal carriers.18 As environmental organisms pose an infection hazard, the ideal is to aim for a clean protective clinical environment. Clearly, special efforts are warranted to reduce airborne contaminants in the vicinity of highly immunosuppressed patients. It is therefore important to consider the use of air purification systems, possibly involving overnight UV irradiation of dental clinics.19
References 1. Kundsin RB. Airborne contagion. Ann NY Acad Sci 1980;353:1. 2. Muilenberg ML, Burge HA. Aerobiology. Boca Raton: Lewis Publishers; 1996. 3. Eickhoff TC. Airborne nosocomial infection. A contemporary perspective. Infect Control Hosp Epidemiol 1994;15:663. 4. Boyce JM, Opal SM, Potter-Byn G, Medeirose AA. Spread of methicillin-resistant Staphylococcus aureus in a hospital after exposure to a healthcare worker with chronic sinusitis. Clin Infect Dis 1993;17:496-504. 5. Belani A, Sheretz RH, Sullivan ML. Outbreak of staphylococcal infection in two hospital nurseries traced
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6. 7. 8. 9.
10.
11. 12.
13. 14.
15.
16.
17.
18.
19.
to a single nasal carrier. Infect Control Hosp Epidemiol 1986;7:487-490. Talaro K, Talaro A. Foundation of microbiology, 2nd ed. Dubuque: Wm C Brown Publishers; 1996. Samaranayake LP. Essential microbiology for dentistry. 2nd ed. Edinburgh: Churchill Livingstone; 2002. Prescott LM, Harley JP, Klein DA. Microbiology. 5th ed. Singapore: McGraw-Hill Higher Education (Asia); 2002. Macher JM, Alevant LE, Chang YL, Liu KS. Effect of ultraviolet germicidal lamps on airborne microorganisms in an outpatient waiting room. Appl Occup Environ Hyg 1992;7:505-513. Macher JM. The use of germicidal lamps to control tuberculosis in healthcare facilities. Infect Control Hosp Epidemiol 1993:14:723-729. Cappuccino JG, Sherman N. Microbiology. A laboratory manual. 6th ed. San Francisco: Benjamin Cummings; 2002. Bagg J, MacFarlane TW, Poxton IR, Miller CH, Smith AJ. Essentials of microbiology for dental students. New York: Oxford University Press; 1999. Aqua Treatment Service. UV technical bulletin. www. onthenetnow.com//uv_tech_bulletin_ats.pdf. 2001. Molinari JA. Sterilization and disinfection. In: Schuster GS, editor. Oral microbiology and infectious disease. 3rd ed. Philadelphia: BC Decker Inc; 1990. Rathbun WE. Sterilization and asepsis. In: Newman MG, Nisengard R, editors. Oral microbiology and immnunology. Philadelphia: WB Saunders Company; 1988. Levinson W, Jawetz E. Medical microbiology and immunology: examination and board review. 5th ed. Connecticut: Appleton & Lange; 1998. Brooks GF, Butel JS, Moore SA. The growth, survival, and death of microorganisms. In: Jawetz, Melnick, & Adelberg’s medical microbiology. 22nd ed. New Delhi: Appleton & Lange; 2001:46-54. Sands KEF, Goldmann DA. Epidemiology of staphylococcus and group A streptococci. In: Bennet JV, Brachman PS, editors. Hospital infections. Philadelphia: LippincottRaven Publishers; 1998:621-636. Rhame FS. The inanimate environment. In: Bennet JV, Brachman PS, editors. Hospital infections. Philadelphia: Lippincott-Raven Publishers; 1998:299-324.
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