Effect of Chlorine Dioxide Gas on Fungi and Mycotoxins Associated with Sick Building Syndrome

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 2005, p Vol. 71, No /05/$ doi: /aem Copyright 2005, Americn Society for Microbiology. All Rights Reserved. Effect of Chlorine Dioxide Gs on Fungi nd Mycotoxins Associted with Sick Building Syndrome S. C. Wilson,* C. Wu, L. A. Andriychuk, J. M. Mrtin, T. L. Brsel, C. A. Jumper, nd D. C. Strus Center for Indoor Air Reserch, Deprtment of Microbiology nd Immunology, Helth Sciences Center, Texs Tech University, Lubbock, Texs Received 28 October 2004/Accepted 28 Mrch 2005 The growth of indoor molds nd their resulting products (e.g., spores nd mycotoxins) cn present helth hzrds for humn beings. The efficcy of chlorine dioxide gs s fumigtion tretment for inctivting sick building syndrome-relted fungi nd their mycotoxins ws evluted. Filter ppers (15 per orgnism) feturing growth of Stchybotrys chrtrum, Chetomium globosum, Penicillium chrysogenum, nd Cldosporium cldosporioides were plced in gs chmbers contining chlorine dioxide gs t either 500 or 1,000 ppm for 24 h. C. globosum ws exposed to the gs both s colonies nd s scospores without sci nd peritheci. After tretment, ll orgnisms were tested for colony growth using n gr plting technique. Colonies of S. chrtrum were lso tested for toxicity using yest toxicity ssy with high specificity for trichothecene mycotoxins. Results showed tht chlorine dioxide gs t both concentrtions completely inctivted ll orgnisms except for C. globosum colonies which were inctivted n verge of 89%. More thn 99% of scospores of C. globosum were nonculturble. For ll scospore counts, men test redings were lower thn the controls (P < 0.001), indicting tht some scospores my lso hve been destroyed. Colonies of S. chrtrum were still toxic fter tretment. These dt show tht chlorine dioxide gs cn be effective to degree s fumignt for the inctivtion of certin fungl colonies, tht the peritheci of C. globosum cn ply slightly protective role for the scospores nd tht S. chrtrum, while ffected by the fumigtion tretment, still remins toxic. Sick building syndrome (SBS) is brod term for rnge of humn helth symptoms ssocited with poor indoor ir qulity. While poor indoor ir qulity cn be relted to number of issues, the one tht hs received the most ttention in the lst 5 to 10 yers hs been the effects of irborne fungi nd their products (2, 6, 12). It hs been cknowledged tht dmp nd moldy structures re potentilly hrmful to the occupnts (16). Therefore, from humn helth stndpoint, it is recommended tht the conditions tht llowed for fungl prolifertion inside humn occupied structures be ddressed nd corrected nd tht existing fungl growth be removed or treted from ny ffected substrtes. However, problem is the detection of hidden mold growth sites. Invsive investigtions cn ssist in this regrd, but these investigtions cn be costly to effect nd there is lso the possibility tht some growth sites cn be overlooked. It is possible tht gseous fungicides my be ble to circumvent this problem becuse the gs cn penetrte into crevices, wll cvities nd other hrd to ccess res, thus treting the inccessible mold colonies. The contents inside contminted structure my lso be ble to be sterilized (10) using this technique s well. A biocide, chlorine dioxide, hs been previously been used in gseous form for bcteril decontmintion (1, 9). It hs been lso shown to hve degree of fungicidl ctivity in solution (15). In this study, we exmined the fungicidl effect of chlorine dioxide gs on four species of mold, Stchybotrys chrtrum, Cldosporium cldosporioides, Penicillium chrysogenum, nd Chetomium globosum. * Corresponding uthor. Miling ddress: Dept. of Microbiology nd Immunology, TTUHSC, th St., Lubbock, TX Phone: (806) Fx: (806) E-mil: stephen.wilson@ttuhsc.edu. The first three belong to the clss hyphomycetes nd the fourth orgnism to scomycetes, which re chrcterized by the presence of n scus, membrne-like structure surrounding the scospores. These orgnisms hve been found in buildings with indoor ir qulity problems (3, 12, 13). We lso looked t the effect of the gs tretment on mycotoxins produced by S. chrtrum. MATERIALS AND METHODS Experimentl design. The four fungl species were grown seprtely on sterile Whtmn no. 3 filter pper (Whtmn, Florhm Prk, NJ) plced on potto dextrose gr medium (PDA; Difco, Sprks, MD). In ddition to growing colonies, the scospores of one of the orgnisms, C. globosum ws lso presented in order to determine if the peritheci nd sci present in colonies plyed protective role for the scospores. The filter ppers (15 per orgnism) with fungl colonies nd settled scospores were exposed to either 500 or 1,000 ppm of chlorine dioxide gs for 24 h t room temperture. Control colonies were grown up s for the test colonies but were not exposed to chlorine dioxide gs. After tretment, the filter ppers were then plced in 0.15 M phosphte-buffered sline (PBS), ph 7.0 in 50 ml tubes nd vortexed resulting in the conidi being in suspension. The resulting conidi suspension ws then counted nd then serilly diluted onto gr pltes, with dilution up to 10 4 except Penicillium chrysogenum, which ws diluted up to All pltes were incubted for 7 or more dys t 25 C. At this time the pltes were removed from the incubtor nd ll colonies were counted. The following dt were collected. (i) Totl numbers of recovered CFU of tests versus totl numbers of CFU of controls were counted. A tretment efficiency (TE) vlue ws clculted using recovered CFU of tests versus controls. (ii) Overll conidi numbers of both tests nd controls were counted. (iii) Filter ppers with Stchybotrys chrtrum colonies were lso exmined to determine if ny posttretment colonies were still toxic. Orgnisms. Four fungl species, Penicillium chrysogenum (ATCC 9179), Chetomium globosum (ATCC 16021), nontoxic strin of Stchybotrys chrtrum (ATCC 9633), toxic strin of Stchybotrys chrtrum, nd Cldosporium cldosporioides were used. C. cldosporioides nd the toxic strin of S. chrtrum were collected from building mterils nd identified in the Center for Indoor Air Reserch Texs Tech University Helth Science Center using reference texts 5399

2 5400 WILSON ET AL. APPL. ENVIRON. MICROBIOL. (4, 17). Toxicity of the environmentl isolte of S. chrtrum ws determined by yest toxicity ssy (5). All orgnisms were identified nd verified using reference texts (4, 17). Fungl colony preprtion. For ech species, conidi were collected from mture cultures on PDA nd suspended in PBS. The concentrtion of conidi ws determined by verging t lest four counts on hemocytometer nd multiplying the dilution fctor. A totl of conidi were then inoculted onto 2- by 2-cm sterile Whtmn no. 3 filter pper tht hd been plced in the center of 35- by 10-mm PDA medium pltes. The pltes were seled nd then incubted t 25 C nd 40 to 60% reltive humidity until fungl colonies were both sporulting nd estblished over the filter ppers. Ascospore preprtion. For scospore collection, only PDA pltes with mture colonies were used. It ws scertined microscopiclly tht the mjority of the peritheci hd erupted, freeing the scospores. Following tht, 10 to 15 ml of PBS ws dded to the plte nd sterile glss rod ws used to gently remove the spores from the colonies. The spore suspension ws then centrifuged nd the pellets of the spores were resuspended to hve concentrtion of 200,000 spores per microliter. The spore concentrtion of suspension ws determined s described bove. No prts of peritheci or hyphe were seen microscopiclly in the spore suspension. A totl of conidi were then inoculted onto 2- by 2-cm sterile Whtmn filter pper tht hd been plced in the center of 35- by 10-mm plte nd dried inside biohzrd cbinet. Chlorine dioxide tretment. Ech gs tretment consisted of three seprte runs with five replictes of ech fungl species per run. The filter ppers with fungl colonies were then plced seprtely on Petri dish lids inside 20.8-liter gs chmber which ws creted by modifying commercilly vilble pressure cooker. Chlorine dioxide gs t either 500 ppm or 1,000 ppm ws creted ccording to the instructions of the mnufcturer in the gs chmber through the use of 6 g Aseptrol S10-Tb tblets (Engelhrd Corportion, Iselin, New Jersey) dissolved in distilled wter. The gs chmber ws then seled for 24 h t room temperture under fume hood. Control smples, tht is, filter ppers with identicl fungl growth on them were plced in n identicl gs chmber without ClO 2 gs. As n dditionl check on the production of ClO 2 gs in the chmbers, one dy old Escherichi coli cultures (ATCC DH5- ) on sheep blood gr pltes provided by Clinicl Microbiology Deprtment of Texs Tech University Helth Sciences Center (Becton Dickinson nd Compny, Sprks, MD) were plced inside the test nd control gs chmbers t room temperture. After the tretments, the E. coli colonies were streked on to blood gr pltes nd checked for the next 2 dys for bcteril growth t 37 C. This orgnism hs been shown to be sensitive to low ClO 2 gs concentrtions (8). The ctul mesurement of the concentrtion of ClO 2 gs ws conducted s seprte run of experiments. CFU determintion. After tretment, the filter ppers with fungl colonies were removed from the gs chmber nd plced into 10-ml sterile tube contining 5 ml of PBS. After3hofoccsionl vortexing, the suspension ws diluted up to four times in 10-fold seril dilutions. For ech of five replictes, totl of 100 l of ech dilution ws spred plted on PDA medi in 150- by 10-mm petri dishes. The pltes were seled nd then incubted t 25 C for 5 to 7 dys t which time they were exmined for fungl growth, which ws recorded s CFU. A tretment efficiency (TE) vlue ws clculted for comprison purposes between test nd controls. The vlue ws derived by subtrcting the recovered test CFU (T) from the recovered control CFU (C) nd expressing the difference s percentge, e.g., %TE [(C T)/C)] 100. During the first step of CFU determintion, n liquot ws removed from the suspension nd plced in hemocytometer where the fungl conidi were counted. Totl conidi numbers were then compred to the number of recovered CFU for tests nd control. The rtio of recovered CFU to totl conidi ws lso determined, resulting in percent culturble spores vlue. Toxicity determintion. Kluyveromyces mrxinus (ATCC 8554), yest demonstrted to be prticulrly sensitive to trichothecene mycotoxins (5), ws mintined in YPG-50 (1% yest extrct, 1% bcteriologicl peptone, nd 50 mm glucose) (5) t 80 C in 1-ml liquot. Cultures for inocultion of the biossy were prepred by dding 1 ml liqout to 50 ml of YPG-50 in culture flsk. The flsk ws incubted in rotry incubtor for pproximtely 16 h t 37 C. For the biossy procedure, YPG-50 ws supplemented from stock solution of polymixin B sulfte (PMBS; ICN Biomedicls, Seoul, Kore) to give finl biossy PMBS concentrtion of 15 mg/ml (5). A totl of 136 l of PMBS supplemented YPG-50 medium ws dded to the wells of 96-well polystyrene microtiter plte in triplicte. A totl of 8 l of test smple or control ws dded to ech well, followed by 16 l of yest inoculum to yield n initil cell density of pproximtely cells/ml. Blnk wells contined 152 l of medium nd 8 l of sterile distilled wter. Negtive control wells consisted of 144 l of medium nd 16 l of yest inoculum. Positive control wells were lso included which contined the medium, yest inoculum nd one microgrm of roridin A. The pltes were seled nd incubted on plte shker t 37 C for 8 h. Turbidity ws mesured every two hours by mesuring the bsorbnce t 550 nm in microtiter plte reder. Toxicity of the smple ws bsed on these bsorbnces. Yest growth resulted in high bsorbnce, indicting tht the smple ws not toxic. Conversely, the lower the bsorbnce, the more toxic the smple ws to the yest. Sttisticl nlysis. To determine if the tretments my hve destroyed conidi rther thn compromised them, the men number of C. globosum scospores for the two test tretments were compred to the control men (N 30 control nd 15 test). This nlysis ws not performed on the hyphomycetes orgnisms becuse the pre-tretment conidi numbers could not be determined nd n nlysis on the finl numbers therefore could not be mde. A one-wy nlysis of vrince (ANOVA) ws performed on the scospore test nd control mens followed by Tukey s post hoc test (SigmStt softwre, version 2.0; SPSS Inc., Chicgo, IL). Conditions of normlity nd equl vrince were met for the nlysis. RESULTS No culturble E. coli ws recovered fter chlorine dioxide gs tretment indicting tht the gs ws present in the gs chmbers. For both the 500 nd 1,000 ppm levels of ClO 2 gs tretment, P. chrysogenum, S. chrtrum, nd C. cldosporioides were not ble to be cultured, indicting tht the tretments were successful in completely inhibiting the culturbility of these orgnisms. Tble 1 shows the results for the tril for the hyphomycetes orgnisms. With regrd to C. globosum colonies the tretment of ClO 2 t 500 ppm resulted in TE of 91%. At 1,000 ppm, the TE ws 87%. The C. globosum scospores were virtully inctivted 100% by both concentrtions. The men number of scospores of the control ws significntly higher thn tht for both tretments (P 0.001). Tble 2 shows the results for colonies nd scospores of C. globosum. The yest toxicity ssy results showed tht the ClO 2 gs tretment ws unsuccessful in detoxifying the colonies of the toxic strin of S. chrtrum. Figure 1 nd Tble 3 show the results for this ssy. These results show tht the toxicity of S. chrtrum ws due to the properties of the orgnism rther thn to combintion of ClO 2 nd orgnism. DISCUSSION The results show tht the two dosges of the ClO 2 gs tretment were successful in rendering the ll conidi completely nonculturble of hyphomycetes orgnisms Penicillium chrysogenum, Stchybotrys chrtrum, Cldosporium cldosporioides, nd the mjority of scospores of scomycete Chetomium globosum growing on the filter pper. With regrd to culturble scospore numbers, they were significntly reduced (P 0.001) fter tretment nd this in turn indictes tht some destruction of scospores my hve occurred. It is lso possible tht the scospores nd conidi were compromised. ClO 2 is strong oxidnt (14) nd previous work with bcteril spores hs shown tht it cn ffect the integrity of cell membrnes nd germintion inhibition (20). It is not cler if the nonculturble spores themselves will be helth hzrd in the sme wy tht culturble spores re. However, nonculturble spores will prevent further growth of colonies nd inhibition of fungl growth will prevent further mycotoxin production. In this experiment, filter pper is used to represent the ctul building mterils in terms of food source nd physicl support. Further reserch is needed to show the effectiveness

3 VOL. 71, 2005 CHLORINE DIOXIDE AND FUNGI 5401 TABLE 1. Mens nd stndrd errors for results of tril tht exmined the efficcy of CLO 2 gs over 24 h on the culturbility nd overll conidi numbers of three hyphomycetes mold species Orgnisms nd prmeters Tretment with ClO 2 0 ppm 500 ppm 1,000 ppm S. chrtrum Recovered CFU ( ) 0 (0) 0 (0) Recovered conidi ( ) ( ) ( ) % Culturble spores 48.0 (6.70) 0 (0) 0 (0) C. cldosporioides Recovered CFU ( ) 0 (0) 0 (0) Recovered conidi ( ) ( ) ( ) % Culturble spores 69.0 (7.80) 0 (0) 0 (0) P. chrysogenum Recovered CFU ( ) 0 (0) 0 (0) Recovered conidi ( ) ( ) ( ) % Culturble spores 45.0 (3.6) 0 (0) 0 (0) Vlues in prentheses represent stndrd errors. of the tretment of mold dmged buildings with high concentrtion ClO 2 gs. Chetomium globosum (Chetomicee, Sordriles, Sordriomycetide, Ascomycetes) is n scomycete tht produces conidi known s scospores which re chrcterized by thick multilyered spore cot. Numerous scospores re situted within n scus which is sc-like structure. The sci in turn re situted inside nother enclosure clled peritheci (11). Nothing is known of the function of the scus nd the peritheci with reltion to fungicidl gents. The results of this tril indicte tht these two dditionl lyers plyed slightly protective role for the Chetomium scospores ginst the oxidtive effect of ClO 2. This my possibly be due to the scus nd peritheci providing physicl protective brrier ginst the gs. The tretment of C. globosum with ClO 2 t 500 ppm resulted in TE of 91%, while the 1,000 ppm tretment resulted in TE of 87%. This indictes tht ClO 2 concentrtion of 500 ppm is high enough to inhibit fungl growth. The tretment ws not successful in detoxifying the nonculturble toxic strin of S. chrtrum. Chlorine ws chosen s the ctive gent in this study becuse previous studies hve shown tht sodium hypochlorite cn inctivte trichothecene mycotoxins (18). It hs lso been effective in inctivting fltoxins (19). We hve lso shown tht ClO 2 is effective ginst the trichothecene mycotoxins roridin A nd verrucrin A plced in wter (dt not shown). Strtoxin G is the min trichothecene mycotoxin produced by. S. chrtrum nd it is found predominntly on S. chrtrum conidi, both on the outer membrne nd the inner lyer of the conidi (7) nd it is possible tht becuse of their loction the gs ws not ble to inctivte the trichothecenes. While the gs tretment is successful in inctivting mold nd bcteril colonies, the tretment comes with cvet in tht chlorine dioxide gs hs corrosive properties nd cre hs to be tken tht the tretment does not result in the degrdtion of the contents s well s the components of the structure. It hs been used in the pst, s the disinfection of the offices of the Hrt Sente Office Building by the Environmentl Protection Agency in 2002 ws chieved with the help of ClO 2 gs. This tretment cn be considered where buildings re emptied TABLE 2. Mens nd stndrd errors for results of tril tht exmined the efficcy of ClO 2 gs over 24 h on the culturbility nd overll conidi numbers of the scomycete C. globosum Orgnisms nd prmeters Tretment with ClO 2 0 ppm 500 ppm 1,000 ppm C. globosum colonies Recovered CFU ( ) ( ) ( ) Recovered conidi ( ) ( ) ( ) % Culturble spores 65 (7.1) 7 (1.5) 3 (0.6) Tretment efficiency (%) C. globosum scospores Recovered CFU ( ) 76 (40.0) 30 (15.5) Recovered conidi ( ) A ( ) B ( ) B % Culturble spores 82 (2.5) 0.01 (0.007) (0.001) Tretment efficiency (%) Different superscripts on the rows of recovered conidi indicte significnt difference t the P level. Vlues in prentheses represent stndrd errors.

4 5402 WILSON ET AL. APPL. ENVIRON. MICROBIOL. FIG. 1. Grph showing the representtive result from tril tht exmined the efficcy of ClO 2 gs over 24 h on the toxicity of S. chrtrum (Stchy) colonies using yest toxicity ssy. High bsorbnce redings indicte nontoxicity. NT, nontoxic strin; Tox, toxic strin. of sensitive items nd the construction of the building is such tht there re inccessible mold contminted res. In conclusion, this study hs shown tht chlorine dioxide gs cn be effective to degree s fumignt for the inctivtion of certin fungl colonies, tht the peritheci/sci of C. globosum cn ply protective role for the scospores nd tht S. chrtrum, while inctivted by the fumigtion tretment, still remins toxic t the tested dosges. ACKNOWLEDGMENTS We thnk Assured Indoor Air Qulity Ltd., Dlls, Texs, nd Texs Tech University Helth Sciences Center (TTUHSC) for their finncil support. TABLE 3. Mens nd stndrd errors for results from yest toxicity ssy showing men opticl densities t wvelength of 550 nm tken t 8 h of incubtion for toxic nd nontoxic Stchybotrys chrtrum compred to positive nd negtive controls ClO 2 concn (ppm) Toxic Absorbnce t OD 550 Nontoxic Positive control Negtive control (0.038) (0.016) (0.073) (0.062) 1, (0.055) (0.008) (0.073) (0.062) OD 500, opticl density t 550 nm. Low men bsorbncy indictes toxicity. Vlues in prentheses represent stndrd errors. S. C. Wilson nd D. C. Strus were supported by Center of Excellence grnt from TTUHSC. D. C. Strus nd T. L. Brsel were supported by grnt from the Texs Higher Eduction Coordinting Bord. REFERENCES 1. Buttner, M. P., P. Cruz, L. D. Stetzenbch, A. K. Clim-Komb, V. L. Stevens, nd T. D. Cronin Determintion of the efficcy of two building decontmintion strtegies by surfce smpling with culture nd quntittive PCR nlysis. Appl. Environ. Microbiol. 70: Chpmn, J. A Stchybotrys chrtrum (chrtrum tr lternns) nd other problems cused by llergenic fungi. Allergy Asthm Proc. 24: Cooley, J. D., W. C. Wong, C. A. Jumper, nd D. C. Strus Correltion between the prevlence of certin fungi nd sick building syndrome. Occup. Environ. Med. 55: Domsch, K. H., W. Gms, nd T. Andersen Compendium of soil fungi, vol. 1. IHW-Verlg, Eching, Germny. 5. Engler, K. H., R. D. Coker, nd I. H. Evns A colorimetric technique for detecting trichothecenes nd ssessing reltive potencies. Appl. Environ. Microbiol. 65: Flnnign, B., nd J. D. Miller Helth implictions of fungi in indoor environments: n overview, p In R. A. Smson, B. Flnnign, A. P. Verhoeff, O. C. G Adn, nd E. S. Hoekstr (ed.), Helth implictions of fungi in indoor environments. Elsevier, New York, N.Y. 7. Gregory, L., J. J. Pestk, D. G. Derborn, nd T. G. Rnd Locliztion of strtoxin-g in Stchybotrys chrtrum spores nd spore-impcted mouse lung using immunocytochemistry. Toxicol. Pthol. 32: Hn, Y., J. D. Floros, R. H. Linton, S. S. Nielsen, nd P. E. Nelson Response surfce modeling for the inctivtion of Escherichi coli O157:H7 on green peppers (Cpsicum nnuum L.) by chlorine dioxide gs tretments. J. Food Prot. 64: Hn, Y., B. Applegte, R. H. Linton, nd P. E. Nelson Decontmintion of Bcillus thuringiensis spores on selected surfces by chlorine dioxide gs. J. Environ Helth. 66:16 21.

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