599 Journal o f Food Protection, Vol. 77, No. 4, 2014, Pages 599-604 doi: 10.4315/0362-028X.JFP-13-232 Copyright © , International Association for Food Protection
Biofilms Formed by Mycobacterium tuberculosis on Cement, Ceramic, and Stainless Steel Surfaces and Their Controls VICTORIA ADETUNJI,1 ADEREMI KEHINDE,2 OLAYEMI BOLATITO,1 a n d JINRU CHEN3* 1Department o f Veterinary Public Health and Preventive Medicine and; 2Department o f Medical Microbiology and Parasitology, College o f Medicine, University College Hospital, University o f Ibadan, Ibadan, 200284 Nigeria; and 3Department o f Food Science and Technology, The University o f Georgia, Griffin, Georgia 30223-1797, USA MS 13-232: Received 6 June 2013/Accepted 3 December 2013
ABSTRACT This study assessed the biofilms formed by selected strains of Mycobacterium tuberculosis and investigated the efficacy of three different treatments to control the biofilms. Two M. tuberculosis strains were inoculated separately in 150 ml of Middlebrook 7H9-Tween 80 (0.1%) broth with 5% liver extract and 10% oleic albumin dextrose catalase (OADC) supplement, 5% liver extract alone, or 10% OADC alone in sterile jars, each containing a 2-cm2 coupon of cement, ceramic, or stainless steel for biofilm development at 37°C, with agitation for 2, 3, or 4 weeks. Biofilms on the coupons were exposed to 10 ml of 2% sanitizer A or 0.5% sanitizer B at 28 and 45°C and to hot water at 85°C for 5 min. Residual biofilms on treated and untreated coupons were assessed. Both strains of M. tuberculosis formed biofilms on the three surfaces; however, one strain formed more biofilms. More biofilms were formed when media containing 5% liver extract was used. Biofdm mass increased as incubation time increased until the third week. More biofdms were formed on cement than on ceramic and stainless steel coupons. Sanitizing treatments at 45 °C removed more biofilms than those at 28°C. However, neither treatment completely eliminated the biofilms.
Previous studies have shown that several microbial species have evolved to survive in stressful environments by self-assembling in highly organized, surface-attached, and matrix-encapsulated structures known as biofilms (3,9, 33). Cohabitation within a population is a preferred microbial survival strategy, which is achieved through genetic components that regulate surface attachment, intercellular communications, and synthesis of extracellular polymeric substances (16, 20). Microbial tolerance to environmental stress is facilitated by production of extracellular polymeric substances and physiological adaptation to the heteroge neous microenvironments within the complex architecture of biofilms (4). Mycobacterium tuberculosis is a causative agent responsible for tuberculosis (TB), a major threat to global public health, especially in developing countries. Nearly one-third of the world’s population is estimated to have been latently infected by the pathogen. About 9 million new cases of active TB were confirmed in 2009, and 1.7 million patients die of the disease every year (11). Africa, with 11% of the world’s population, carries 29% of the global burden of TB cases and 34% of related deaths (8). The World Health Organization estimates that the average incidence of TB in African countries more than doubled between 1990 and 2005, from 149 to 343 per 100,000 population (8). * Author for correspondence. Tel: +1-770-412-4738; Fax: 1-770-412-4748; E-mail: [email protected].
Although the natural host of M. tuberculosis is humans, occasional cases of M. tuberculosis infection have been reported in dairy and food animals such as cattle (7, 26, 37) and goats (6). Humans are usually infected with Mycobac terium through the inhalation of droplet nuclei; however, a significant proportion of human cases involve extrapulmonary TB, presumably caused by the consumption of raw milk from infected animals or milk and meat products contaminated with M. tuberculosis (15). An earlier study conducted in Nigeria isolated three M. tuberculosis strains from 105 unpasteurized milk samples and six strains from 587 slaughtered beef carcasses (5). Although the current body of literature has scarce information on biofilm formation by mycobacteria in food processing environments, earlier studies did show that these pathogens could colonize various ecological niches suc cessfully, with the production of biofilm and biofilm-like structures (24). Viable cells of Mycobacterium spp. were recovered from biofilms formed on dental-unit water systems (39). These findings suggest the possibility of biofilm production by mycobacteria in food processing environments, especially in Africa where the level of processing hygiene is poor. Failure to control biofilms formed by Mycobacterium spp. could lead to recontamina tion of pasteurized products and cross-contamination of safe foods. Ingestion of contaminated food and milk is the most significant link between bovine and human TB, particularly in children (23, 37). This study assessed the formation of
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biofilm s by selected strains o f M. tuberculosis on different contact surfaces and in various grow th media. It also assessed the effectiveness o f sanitizing treatm ents in rem oval o f the biofilm s form ed by M. tuberculosis.
MATERIALS AND METHODS The study was carried out in 2012 at the Tuberculosis Reference Laboratory of the Department of Medical Microbiology and Parasitology, University College Hospital, Ibadan, Nigeria. Bacterial strains and culture conditions. Two M. tubercu losis strains of human origin were used for biofilm development. H37Rv was a reference strain (RIVM, Bilthoven, The Nether lands), whereas MTB1 was isolated from the sputum of an individual with chronic TB infection in Nigeria. The strains were stored on Loewenstein-Jensen agar slants at —20°C and were subcultured, immediately prior to the experiment, in Middlebrook 7H9 broth (Remel, Lenexa, KS) containing 10% Middlebrook growth supplement (oleic, albumin, dextrose, and catalase [OADC; Remel], 0.1% Tween 80 [Fisher Scientific, Pittsburgh, PA], and 0.2% glycerol [AnalaR, BDH Chemical Ltd., Poole, UK]). Inoculated samples were incubated at 37°C aerobically with agitation for 14 days. Contact surfaces for biofilm development. Coupons (2 cm2) of stainless steel (West Gate, Tokyo, Japan), cement (Purechem Industries Ltd., Lagos, Nigeria), and ceramic (Virony Building Materials Company, Ltd., Guangzhou, China) were used as contact surfaces in the study. Prior to biofilm development, the coupons were soaked for 24 h in 5% Ariel detergent (13.5% sodium carbonate, 3.0% sodium hydroxide, 30.0% sodium sulfate, 10.0% pentasodium tripolyphosphate, 13.5% alkyl benzenesulfonic acid sodium salt, and 7.5% sodium fatty alcohol sulfate; Unilever, Lagos, Nigeria) and then were rinsed three times with sterile distilled water each for 15 min. The coupons were later sterilized in a hot air oven (Elektro-Helios, Stockholm, Sweden) at 120°C for 30 min. Biofilm development. Biofilm development on various surfaces took place in three different microbiological media. The base medium was Middlebrook 7H9 broth with 0.1% Tween 80, which was supplemented with 5% liver extract (LE) alone, 10% OADC alone, or 5% LE plus 10% OADC. The media were prepared according to the manufacturer’s instructions. The 5% LE was prepared by adding 5 g of aseptically homogenized fresh bovine liver tissue (Bodija Abattoir, Ibadan, Nigeria) to 10 ml of 0.9% physiologic saline (Dana Pharmaceuticals, Lagos, Nigeria). The homogenates were aseptically sieved into a sterile container, and penicillin G (50 IU/ml), polymyxin B (2,000 U/ml), amphotericin B (200 mg/ml), nalidixic acid (800 mg/ml), and trimethoprim (200 mg/ml) were then added. For biofdm formation, individual stainless steel, cement, or ceramic coupons were placed in separate screw-cap glass jars (Jirui Glass Products Co. Ltd., Xuzhou, China), each containing 150 ml of one of the previously described test media inoculated with either H37Rv or MTB1. Biofilm development was permitted at 37°C during a period of 2, 3, or 4 weeks. Biofilm quantification. After 2, 3, and 4 weeks of incubation, developed biofilms were quantified using the crystal violet binding assay as described by Stepanovic et al. (38) and Adetunji and Adegoke (1), with some modifications. At each sampling point, coupons were collected and washed three times, each with 5 ml of sterile distilled water. Biofilm mass was fixed
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with 1 ml of 95% ethanol (AnalaR, BDH Chemical Ltd.) for 15 min at room temperature. The fixed samples were air dried for 10 min and then stained for 5 min with 2% crystal violet (AnalaR, BDH Chemical Ltd.). Excess stain was rinsed with running tap water, and the coupons were then air dried. Each of the dried coupons was placed on a sterile petri dish, and 3 ml of 33% glacial acetic acid (AnalaR, BDH Chemical Ltd.) was used to solubilize the crystal violet. The solubilized stain was then pipetted into a cuvette (Bibby Scientific Limited, Staffordshire, UK). Absorbance (OD) readings at 600 nm were measured using a spectrophotometer (Surgienfield Instruments, Springfield, UK). Biofilm control. At weeks 2, 3, and 4, sampling intervals test coupons were collected from glass jars and washed three times with 5 ml of sterile distilled water to remove loosely attached cells. The coupons were then treated for 5 min in a 10-ml solution of 2% sanitizer A (55 mg/100 ml active iodine; Crosley Sinbad and Co., Ltd., Lagos, Nigeria) or 0.5% sanitizer B (170.6 g/liter alkyl dimethylbenzyl ammonium chloride, 78 g/liter didecyldimethyl ammonium chloride, 107.25 g/liter glutaraldehyde, 146.25 g/liter isopropanol, 20 g/liter Pineoil; CID Lines, Ypres, Belgium) according to the manufacturer’s instructions at 28 or 45°C. Mycobacterial biofilms on tested surfaces were also treated in 10 ml of water at 85 °C. Thereafter, residual sanitizers were neutralized with double-strength Dey-Engley solution at room temperature for 10 min followed by air drying at 60°C for 2 h, and biofilm mass on various coupons was quantified using the crystal violet binding assay described in “ Biofilm quantification.” Statistical analysis. Each experiment was replicated once, and every test was duplicated. Separation of means was accomplished using a three-way analysis of variance F test and general linear model of statistical analysis software (version 8, SAS Institute Inc., Cary, NC). At a confidence level of 95%, Fisher’s least significant difference design was used to compare the significance of differences among the amounts of biofilm formed by different Mycobacterium strains, on different cell contact surfaces, in different microbiological media, and at different sampling intervals.
RESULTS Biofilm formation. It was observed that, after a 2-week incubation period, the tw o M. tuberculosis strains form ed m ore biofilm s on cem ent than on ceram ic and stainless steel surfaces (Table 1) (P < 0.05). The influence o f growth m edia on biofilm form ation show ed no clear trend at this sam pling point. Strain H 37Rv form ed m ore biofilm s than MTB1 (P < 0.05) on stainless steel and ceram ic surfaces in broth containing 10% OA DC alone or 10% O A D C plus 5% L E and on a cem ent surface in broth containing 10% O A D C (Table 1). Interestingly, MTB1 form ed m ore biofilm s than H 37Rv (P < 0.05) on cem ent and ceram ic surfaces in broth containing 5% LE. Biofilm m ass produced by the two strains under other experim ental conditions w ere statistically insignificant (Table 1). W hen the incubation tim e was extended to 3 w eeks, H 37Rv form ed m ore biofilm s than MTB1 on stainless steel in all grow th m edia and on ceram ic surfaces in broth contam ination in 10% O A D C plus 5% LE (Table 2) (P < 0.05). M T B 1 form ed m ore biofilm s than H 37Rv on ceramic surfaces in broth containing 5% LE or 10% O ADC. Biofilm mass totals produced by the tw o strains under other
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TABLE 1. Biofilms (OD 600 nm) formed by Mycobacterium tuberculosis strains after 2 weeks of incubation in three different microbiological media at 37°Ca Biofilm mass (Agoonm) (« = 4) Base growth media (7H9 with 0.1% T80) and supplements 5% liver extract 10% OADC and 5% liver extract 10% OADC
M. tuberculosis strains
Stainless steel
Cement
Ceramic
H37Rv MTB1 H37Rv MTB1 H37Rv MTB1
0.543 c E 0.540 c e 0.875 b a 0.644 c c 0.746 c b 0.586 b d
0.914 a c 1.007 aB 0.980 a b c 1.055 a b 1.148 a a 0.794 a d
0.661 b f 0.970 b b 0.978 a a 0.763 b d 0.784 b c 0.675 a b e
a OADC, a growth supplement containing oleic, albumin, dextrose, and catalase. Means in the same row not followed by the same lowercase letter are statistically significant (P < 0.05). Means in the same column not followed by the same uppercase letter are statistically significant (P < 0.05).
experimental conditions were statistically insignificant (Table 2). At the 4-week sampling point, it was clear that significantly more biofilms were formed by the two M. tuberculosis strains on cement than on ceramic and stainless steel surfaces, except for the biofilms formed by MTB1 on ceramic surfaces in 10% OADC, which were insignificantly different from the biofilm formed by the same strain on a cement surface (Table 3). Results of overall statistical analysis revealed that the amount of biofilm mass increased as the incubation time increased from weeks 2 to 3. However, the biofilm mass significantly (P < 0.05) decreased when the incubation time was extended to week 4 (Table 4). The two M. tuberculosis strains formed more biofilms on cement than on ceramic and stainless steel surfaces (Table 4). Furthermore, broth with LE and OADC better supported biofilm formation than broth with a single supplement. Between the two media that contained a single supplement, the amounts of biofilms formed in broth with 5% LE were significantly (P < 0.05) higher than those in broth with 10% OADC (Table 4). Overall, strain H37Rv was a better biofilm former than MTB1 (Table 4). Effect of sanitizing treatments on M. tuberculosis biofilms. A significant reduction (P < 0.05) in biofilm mass was observed when coupons were treated with the two sanitizers used in the study (Table 5). Although treatment with hot water also significantly reduced the amount of
biofilm on coupons, greater reductions in biofilm mass were observed when coupons were treated with 2% sanitizer A and 0.5% sanitizer B (Table 5). Treatments performed at 45 °C were more effective than those at 28 °C for the removal of biofilms from tested coupon surfaces (Table 5). Interest ingly, treatments with 0.5% sanitizer B were more effective than those with 2% sanitizer A at 28°C. However, treatments with 2% sanitizer A were more effective than those with 0.5% sanitizer B at 45°C (Table 5). DISCUSSION Both strains of M. tuberculosis used in the present study were capable of forming biofilms. This finding is in agreement with earlier studies in which the biofilm-forming abilities of M. tuberculosis were reported (22, 29). To our knowledge, mycobacterial biofilms developed on cement, stainless steel, and ceramic surfaces have not been reported previously. However, Ojha et al. (29) found that M. tuberculosis strains could develop biofilms on a polystyrene surface. In general, a greater amount of biofilm mass was observed on a cement surface in the present study, with only two exceptions among samples analyzed at week 3 (Tables 1 to 3). The topography of cement surfaces has been shown to facilitate higher levels of bacterial adhesion and biofilm formation (10, 12, 19, 21, 35). Studies have pointed out that the roughness of a contact surface contributes to bacterial attachment (31, 32). A study by
TABLE 2. Biofilms (OD 600 nm) formed by Mycobacterium tuberculosis strains after 3 weeks of incubation in three different microbiological media at 37°Ca Biofilm mass (A60o„m) (« = 4) Base growth media (7H9 with 0.1% T80) and supplements
5% liver extract 10% OADC and 5% liver extract 10% OADC
M. tuberculosis strains
Stainless steel
Cement
Ceramic
H37Rv MTB1 H37Rv MTB1 H37Rv MTB1
1.640 a b 1.050 b d 1.690 a a 0.740 c e 1.361 c c 0.744 b e
1.571 b a 1.481 a ab 1.479 b ab 1.500 a a b 1.448 a ab 1.302 a b
1.483 c b 1.486 a a 1.443 c c 1.046 b e 0.844 b f 1.139 3 D
° OADC, a growth supplement containing oleic, albumin, dextrose, and catalase. Means in the same row not followed by the same lowercase letter are statistically significant (P < 0.05). Means in the same column not followed by the same uppercase letter are statistically significant (P < 0.05).
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TABLE 3. Biofilms (OD 600 nm) formed by Mycobacterium tuberculosis strains after 4 weeks of incubation in three different microbiological media at 37°Ca Base growth media (7H9 with 0.1% T80) and supplements
Biofilm mass (A600nm) (n = 4) M. tuberculosis strains
Stainless steel
Cement
Ceramic
H37Rv MTB1 H37Rv MTB1 H37Rv MTB1
0.727 c b 0.366 c f 0.592 c d 0.786 c a 0.629 c c 0.579 b e
1.814 a a 1.843 a a 1.813 a a 1.848 a a 1.819 a a 1.593 a a
0.823 b f 0.935 b e 1.750 b a 1.051 b c 1.291 b b 0.998 a b d
5% liver extract 10% OADC and 5% liver extract 10% OADC
“ OADC, a growth supplement containing oleic, albumin, dextrose, and catalase. Means in the same row not followed by the same lowercase letter are statistically significant (P < 0.05). Means in the same column not followed by the same uppercase letter are statistically significant (P < 0.05).
Wingender and Flemming (40) found that significantly higher bacterial densities were associated with biofilms formed on cement-lined pipes than on galvanized steel pipes. Both strains of M. tuberculosis used in the present study were from clinical sources. Note that the lack of food isolates of M. tuberculosis in the study is an important limitation. However, M. tuberculosis is a zoonotic organ ism, and food isolates are likely to have originated from infected animals. Earlier studies showed the presence of M. tuberculosis in milk and animal tissue samples in Nigeria (5, 6). Furthermore, most cases of M. tuberculosis infection presented at the University of Ibadan Teaching Hospital, where one of the clinical strains was isolated, was linked to consumption of unpasteurized milk and infected offal (a delicacy in the Nigerian population). Reports on biofilm formation by food isolates of M. tuberculosis are scarce in the current body of literature. Note that there are possible
TABLE 4. Influence of various factors on the formation of biofilms by strains of Mycobacterium tuberculosis" Biofilm mass (A600nm)
Incubation time (n = 72) Week 2 Week 3 Week 4
0.818 c 1.310 a 1.195 b
Cell contact surface (n = 72) Cement Ceramic Stainless steel
1.404 a 1.062 b 0.833 c
Growth supplement (n = 72) Liver extract and OADC Liver extract alone OADC alone
1.1690 a 1.103 ab 1.051 b
Bacterial strain (n = 108) H37Rv MTB1
1.180 1.036
variations regarding the biofilm-forming ability of clinical versus food isolates of M. tuberculosis. Results of the present study showed that extending incubation time from 2 to 3 weeks provided time for more biofilm mass to accumulate. However, the mass of biofilm decreased from weeks 3 to 4. The reason for the decrease is unknown but is possibly due to nutrient depletion and accumulation of bacterial waste and metabolic products in the growth media. Nutrient-induced biofdm dispersion has been observed in Pseudomonas aeruginosa PAOl in earlier studies (34). Results of the present study indicated that M. tuberculosis strain H37Rv was better at forming biofilm than was MB1, with a few exceptions (Tables 1 to 3). The precise reasons for this phenomenon are currently unknown. A study on Mycobacterium smegmatis, a nonpathogenic mycobacterium, found that an acetylated derivative of glycopeptidolipids is required for surface attachment, sliding motility, and biofilm formation (28). Glycopeptidolipid biosynthesis was induced during multicellular growth of Mycobacterium avium, a physiological phenomenon associated with bacterial growth within biofilms (41). In addition to glycopeptidolipids, mycolyl-diacylglycerol and mycolic acids are the other two mycobacterial surface molecules known to play an important role in biofilm development by M. smegmatis (27, 29, 30). The biofilm forming ability of H37Rv could be attributed to its ability in the formation of cording and the synthesis of mycolic acid cyclopropane (14, 29). M. tuberculosis MTB1 is, neverthe less, an uncharacterized clinical isolate from Nigeria. TABLE 5. Efficacy of sanitizing treatments on biofilm removal Residual biofilm mass Treatment Control (n = 216) Water (n = 216) Sanitizer A (2%; n — 432)
a b
a OADC, a growth supplement containing oleic, albumin, dextrose, and catalase. Means not followed by the same letter are statistically significant (P < 0.05).
Sanitizer B (0.5%; n == 432)
Temp (°C)
85 28 45 28 45
0 ^ 6 0 0 nm )
1.272 a 0.864 b 0.821 c 0.469 f 0.798 d 0.487 e
a Means not followed by the same letter are statistically significant (P < 0.05).
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Further studies are needed to identify the genotypic and phenotypic characteristics that have contributed to the differences in biofilm-forming ability of the two Mycobac terium strains. Results of the present study suggest that cells of M. tuberculosis formed more biofilms in broth containing the iron-rich LE (Table 4). Similar findings have been observed with M. avium (18). Iron is an important cofactor that plays a central role in biofilm formation (2, 28). It has been shown that supplemental iron above 1 pM is necessary for proper biofilm development by M. smegmatis, although it is not needed for planktonic growth (28). The enhancement of mycobacterial biofilm formation by elevated iron concen tration has been noticed in several previous studies (2, 28, 29). Biofilm mass has been reported as an indicator for microbial cell growth. Biofilm mass should be directly proportional to the number of bacterial cells within the biofilm. The crystal violet binding assay, used in this and numerous other studies (1,36,42) to quantify biofilms, was selected over standard plate count assay because biofilm is difficult to remove from cell contact surfaces and because biofilm-embedded cells are often clumped together and are difficult to separate, causing variations and inaccuracies in plant count results. Results showed that treatments with the two sanitizers significantly (P < 0.05) reduced the biofilm mass on the solid surfaces used in the present study (Table 5). Sanitizer B is more effective in biofilm removal than sanitizer A at 28 °C. However, sanitizer A was more effective than sanitizer B at 45°C. Sanitizer B is a polyvalent sanitizer composed of two quaternary ammonium compounds, alkyl dimethylbenzyl ammonium chloride and didecyldimethyl ammonium chloride, as well as an aldehyde and pineoil. The bactericidal action of the quaternaries has been attributed to the inactivation of energy-producing enzymes, denaturation of essential cell proteins, and disruption of cell membranes (25). Sanitizer A is a monovalent sanitizer composed of active iodine, whose use as an antimicrobial agent dates back to the 1800s (35). The bactericidal activity of iodine is accomplished by direct halogenation of proteins, damage of cell walls, and destruction of enzyme activities (35). Similar to what was observed in the present study, previous reports indicated that the effectiveness of iodine increased at higher temperatures (13, 17). In general, sanitizing treatments at 45 °C were more effective than those at 28°C (Table 5). At higher temper ature, the sanitizers probably potentiated their disinfection and dislodging effects on the biofilms, thus producing a drastic reduction in biofilm mass on their contact surfaces. Similar to the present study, an earlier report showed that biofilms were completely removed from the surfaces of a model copper water plumbing system operated at 60°C (31). In another study, a mycobacterial biofilm mass on plastic surfaces was not affected by a treatment with water at 22 to 30°C (34). Neither treatment used in the present study completely removed the biofilms from the solid surfaces (Table 5). Earlier studies have shown that planktonic cells are more
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easily destroyed than those in biofilms (38). The macro molecules within the biofilms could reduce the rate of diffusion of the sanitizers into the biofilms. Perhaps only the parts of the biofilm mass close to the surface were reached by these sanitizers, whereas the deeper layers of the biofilms were unaffected. This study showed that pathogenic M. tuberculosis strains could develop biofilms on cement, ceramic, and stainless steel surfaces. In general, cells formed more biofilms on cement than on ceramic and stainless steel surfaces. This information is of particular importance in developing countries, where cement is still a major food contact surface. Growth media containing the iron-rich LE significantly (P < 0.05) enhanced biofilm formation. This implies that the presence of iron in the environment, especially the presence of denatured blood in food processing environments such as abattoirs, may enhance biofilm formation by bacterial pathogens such as mycobac teria. Study results indicate that careful selection of food processing materials and good environmental hygiene are important considerations for biofilm control and that raising the temperature of sanitizing treatments will generate a greater biofilm reduction. Results of this study will be useful in the design of an effective approach for control of M. tuberculosis biofilms. ACKNOWLEDGMENT Laboratory scientists at the Tuberculosis Reference Laboratory at the University College Hospital, Ibadan, Nigeria, are graciously acknowledged with thanks.
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Ojha, A. K., A. D. Baughn, D. Sambandan, T. Hsu, X. Trivelli, Y. Guerardel, A. Alahari, L. Kremer, W. R. Jacobs, Jr., and G. F. Hatfull. 2008. Growth of Mycobacterium tuberculosis biofilm containing free mycolic acids and harbouring drug-tolerant bacteria. Mol. Microbiol. 69:164-174. Ojha, A. K., X. Trivelli, Y. Guerardel, L. Kremer, and G. F. Hatfull. 2010. Enzymatic hydrolysis of trehalose dimycolate releases free mycolic acids during mycobacterial growth in biofilms. J. Biol. Chem. 285:17380-17389. Pedersen, K. 1990. Biofilm development on stainless steel and PVC surface in drinking water. Water Res. 24:239-243. Percival, S. L., J. S. Knapp, R. Edyvean, and D. S Wales. 1998. Biofilm development on stainless steel in mains water. Water Res. 32: 243-253. Saltini, C. 2006. Chemotherapy and diagnosis of tuberculosis. Respir. Med. 100:2085-2097. Sauer, K„ M. C. Cullen, A. H. Rickard, L. A. H. Zeef, D. G. Davies, and P. Gilbert. 2004. Characterization of nutrient-induced dispersion in Pseudomonas aeruginosa PAOl biofilm. J. Bacteriol. 186:7312— 7326. Schmidt, R. H. 2013. Basic elements of equipment cleaning and sanitizing in food processing and handling operations, FS14, p.1-11. University of Florida, Institute of Food and Agricultural Sciences Extension, Gainesville. Available at: http://edis.ifas.ufl.edu/pdffiles/ FS/FS07700.pdf. Accessed 10 May 2013. Simoes, M., L. C. Simoes, and M. J. Vieira. 2010. A review of current and emergent biofilm control strategies. LWT Food Sci. Technol. 43:573-583. Srivastava, K., D. S. Chauhan, P. Gupta, H. B. Singh, V. D. Sharma, V. S. Yadav, T. Sreekumaran, S. S. Thakral, J. S. Dharamdheeran, P. Nigam, H. K. Prasad, and V. M. Katoch. 2008. Isolation of Mycobacterium bovis & M. tuberculosis from cattle of some farms in north India—possible relevance in human health. Indian J. Med. Res. 128:26-31. Stepanovic, S., I. Cirkovic, L. Ranin, and M. Svabic-Vlahovic. 2004. Biofilm formation by Salmonella sp. and Listeria monocytogenes on plastic surface. Lett. Appl. Microbiol. 38:428^432. Walker, J. T., D. J. Bradshaw, A. M. Bennett, M. R. Fulford, M. V. Martin, and P. D. Marsh. 2000. Microbial biofilm formation and contamination of dental-unit water systems in general dental practice. Appl. Environ. Microbiol. 66:3363-3367. Wingender, J., and H. C. Flemming. 2004. Contamination potential of drinking water distribution network biofilms. Water Sci. Technol. 49:277-286. Yamazaki, Y., L. Danelishvili, M. Wu, M. MacNab, and L. E. Bermudez. 2006. Mycobacterium avium genes associated with the ability to form a biofilm. Appl. Environ. Microbiol. 72:819-825. Zmantar, T., B. Kouidhi, H. Miladi, K. Mahdouani, and A. Bakhrouf. 2010. A microtiter plate assay for Staphylococcus aureus biofilm quantification at various pH levels and hydrogen peroxide supple mentation. New Microbiol. 33:137-145.
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Biofilms Formed by Mycobacterium tuberculosis on Cement, Ceramic, and Stainless Steel Surfaces and Their Controls VICTORIA ADETUNJI,1 ADEREMI KEHINDE,2 OLAYEMI BOLATITO,1 a n d JINRU CHEN3* 1Department o f Veterinary Public Health and Preventive Medicine and; 2Department o f Medical Microbiology and Parasitology, College o f Medicine, University College Hospital, University o f Ibadan, Ibadan, 200284 Nigeria; and 3Department o f Food Science and Technology, The University o f Georgia, Griffin, Georgia 30223-1797, USA MS 13-232: Received 6 June 2013/Accepted 3 December 2013
ABSTRACT This study assessed the biofilms formed by selected strains of Mycobacterium tuberculosis and investigated the efficacy of three different treatments to control the biofilms. Two M. tuberculosis strains were inoculated separately in 150 ml of Middlebrook 7H9-Tween 80 (0.1%) broth with 5% liver extract and 10% oleic albumin dextrose catalase (OADC) supplement, 5% liver extract alone, or 10% OADC alone in sterile jars, each containing a 2-cm2 coupon of cement, ceramic, or stainless steel for biofilm development at 37°C, with agitation for 2, 3, or 4 weeks. Biofilms on the coupons were exposed to 10 ml of 2% sanitizer A or 0.5% sanitizer B at 28 and 45°C and to hot water at 85°C for 5 min. Residual biofilms on treated and untreated coupons were assessed. Both strains of M. tuberculosis formed biofilms on the three surfaces; however, one strain formed more biofilms. More biofilms were formed when media containing 5% liver extract was used. Biofdm mass increased as incubation time increased until the third week. More biofdms were formed on cement than on ceramic and stainless steel coupons. Sanitizing treatments at 45 °C removed more biofilms than those at 28°C. However, neither treatment completely eliminated the biofilms.
Previous studies have shown that several microbial species have evolved to survive in stressful environments by self-assembling in highly organized, surface-attached, and matrix-encapsulated structures known as biofilms (3,9, 33). Cohabitation within a population is a preferred microbial survival strategy, which is achieved through genetic components that regulate surface attachment, intercellular communications, and synthesis of extracellular polymeric substances (16, 20). Microbial tolerance to environmental stress is facilitated by production of extracellular polymeric substances and physiological adaptation to the heteroge neous microenvironments within the complex architecture of biofilms (4). Mycobacterium tuberculosis is a causative agent responsible for tuberculosis (TB), a major threat to global public health, especially in developing countries. Nearly one-third of the world’s population is estimated to have been latently infected by the pathogen. About 9 million new cases of active TB were confirmed in 2009, and 1.7 million patients die of the disease every year (11). Africa, with 11% of the world’s population, carries 29% of the global burden of TB cases and 34% of related deaths (8). The World Health Organization estimates that the average incidence of TB in African countries more than doubled between 1990 and 2005, from 149 to 343 per 100,000 population (8). * Author for correspondence. Tel: +1-770-412-4738; Fax: 1-770-412-4748; E-mail: [email protected].
Although the natural host of M. tuberculosis is humans, occasional cases of M. tuberculosis infection have been reported in dairy and food animals such as cattle (7, 26, 37) and goats (6). Humans are usually infected with Mycobac terium through the inhalation of droplet nuclei; however, a significant proportion of human cases involve extrapulmonary TB, presumably caused by the consumption of raw milk from infected animals or milk and meat products contaminated with M. tuberculosis (15). An earlier study conducted in Nigeria isolated three M. tuberculosis strains from 105 unpasteurized milk samples and six strains from 587 slaughtered beef carcasses (5). Although the current body of literature has scarce information on biofilm formation by mycobacteria in food processing environments, earlier studies did show that these pathogens could colonize various ecological niches suc cessfully, with the production of biofilm and biofilm-like structures (24). Viable cells of Mycobacterium spp. were recovered from biofilms formed on dental-unit water systems (39). These findings suggest the possibility of biofilm production by mycobacteria in food processing environments, especially in Africa where the level of processing hygiene is poor. Failure to control biofilms formed by Mycobacterium spp. could lead to recontamina tion of pasteurized products and cross-contamination of safe foods. Ingestion of contaminated food and milk is the most significant link between bovine and human TB, particularly in children (23, 37). This study assessed the formation of
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biofilm s by selected strains o f M. tuberculosis on different contact surfaces and in various grow th media. It also assessed the effectiveness o f sanitizing treatm ents in rem oval o f the biofilm s form ed by M. tuberculosis.
MATERIALS AND METHODS The study was carried out in 2012 at the Tuberculosis Reference Laboratory of the Department of Medical Microbiology and Parasitology, University College Hospital, Ibadan, Nigeria. Bacterial strains and culture conditions. Two M. tubercu losis strains of human origin were used for biofilm development. H37Rv was a reference strain (RIVM, Bilthoven, The Nether lands), whereas MTB1 was isolated from the sputum of an individual with chronic TB infection in Nigeria. The strains were stored on Loewenstein-Jensen agar slants at —20°C and were subcultured, immediately prior to the experiment, in Middlebrook 7H9 broth (Remel, Lenexa, KS) containing 10% Middlebrook growth supplement (oleic, albumin, dextrose, and catalase [OADC; Remel], 0.1% Tween 80 [Fisher Scientific, Pittsburgh, PA], and 0.2% glycerol [AnalaR, BDH Chemical Ltd., Poole, UK]). Inoculated samples were incubated at 37°C aerobically with agitation for 14 days. Contact surfaces for biofilm development. Coupons (2 cm2) of stainless steel (West Gate, Tokyo, Japan), cement (Purechem Industries Ltd., Lagos, Nigeria), and ceramic (Virony Building Materials Company, Ltd., Guangzhou, China) were used as contact surfaces in the study. Prior to biofilm development, the coupons were soaked for 24 h in 5% Ariel detergent (13.5% sodium carbonate, 3.0% sodium hydroxide, 30.0% sodium sulfate, 10.0% pentasodium tripolyphosphate, 13.5% alkyl benzenesulfonic acid sodium salt, and 7.5% sodium fatty alcohol sulfate; Unilever, Lagos, Nigeria) and then were rinsed three times with sterile distilled water each for 15 min. The coupons were later sterilized in a hot air oven (Elektro-Helios, Stockholm, Sweden) at 120°C for 30 min. Biofilm development. Biofilm development on various surfaces took place in three different microbiological media. The base medium was Middlebrook 7H9 broth with 0.1% Tween 80, which was supplemented with 5% liver extract (LE) alone, 10% OADC alone, or 5% LE plus 10% OADC. The media were prepared according to the manufacturer’s instructions. The 5% LE was prepared by adding 5 g of aseptically homogenized fresh bovine liver tissue (Bodija Abattoir, Ibadan, Nigeria) to 10 ml of 0.9% physiologic saline (Dana Pharmaceuticals, Lagos, Nigeria). The homogenates were aseptically sieved into a sterile container, and penicillin G (50 IU/ml), polymyxin B (2,000 U/ml), amphotericin B (200 mg/ml), nalidixic acid (800 mg/ml), and trimethoprim (200 mg/ml) were then added. For biofdm formation, individual stainless steel, cement, or ceramic coupons were placed in separate screw-cap glass jars (Jirui Glass Products Co. Ltd., Xuzhou, China), each containing 150 ml of one of the previously described test media inoculated with either H37Rv or MTB1. Biofilm development was permitted at 37°C during a period of 2, 3, or 4 weeks. Biofilm quantification. After 2, 3, and 4 weeks of incubation, developed biofilms were quantified using the crystal violet binding assay as described by Stepanovic et al. (38) and Adetunji and Adegoke (1), with some modifications. At each sampling point, coupons were collected and washed three times, each with 5 ml of sterile distilled water. Biofilm mass was fixed
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with 1 ml of 95% ethanol (AnalaR, BDH Chemical Ltd.) for 15 min at room temperature. The fixed samples were air dried for 10 min and then stained for 5 min with 2% crystal violet (AnalaR, BDH Chemical Ltd.). Excess stain was rinsed with running tap water, and the coupons were then air dried. Each of the dried coupons was placed on a sterile petri dish, and 3 ml of 33% glacial acetic acid (AnalaR, BDH Chemical Ltd.) was used to solubilize the crystal violet. The solubilized stain was then pipetted into a cuvette (Bibby Scientific Limited, Staffordshire, UK). Absorbance (OD) readings at 600 nm were measured using a spectrophotometer (Surgienfield Instruments, Springfield, UK). Biofilm control. At weeks 2, 3, and 4, sampling intervals test coupons were collected from glass jars and washed three times with 5 ml of sterile distilled water to remove loosely attached cells. The coupons were then treated for 5 min in a 10-ml solution of 2% sanitizer A (55 mg/100 ml active iodine; Crosley Sinbad and Co., Ltd., Lagos, Nigeria) or 0.5% sanitizer B (170.6 g/liter alkyl dimethylbenzyl ammonium chloride, 78 g/liter didecyldimethyl ammonium chloride, 107.25 g/liter glutaraldehyde, 146.25 g/liter isopropanol, 20 g/liter Pineoil; CID Lines, Ypres, Belgium) according to the manufacturer’s instructions at 28 or 45°C. Mycobacterial biofilms on tested surfaces were also treated in 10 ml of water at 85 °C. Thereafter, residual sanitizers were neutralized with double-strength Dey-Engley solution at room temperature for 10 min followed by air drying at 60°C for 2 h, and biofilm mass on various coupons was quantified using the crystal violet binding assay described in “ Biofilm quantification.” Statistical analysis. Each experiment was replicated once, and every test was duplicated. Separation of means was accomplished using a three-way analysis of variance F test and general linear model of statistical analysis software (version 8, SAS Institute Inc., Cary, NC). At a confidence level of 95%, Fisher’s least significant difference design was used to compare the significance of differences among the amounts of biofilm formed by different Mycobacterium strains, on different cell contact surfaces, in different microbiological media, and at different sampling intervals.
RESULTS Biofilm formation. It was observed that, after a 2-week incubation period, the tw o M. tuberculosis strains form ed m ore biofilm s on cem ent than on ceram ic and stainless steel surfaces (Table 1) (P < 0.05). The influence o f growth m edia on biofilm form ation show ed no clear trend at this sam pling point. Strain H 37Rv form ed m ore biofilm s than MTB1 (P < 0.05) on stainless steel and ceram ic surfaces in broth containing 10% OA DC alone or 10% O A D C plus 5% L E and on a cem ent surface in broth containing 10% O A D C (Table 1). Interestingly, MTB1 form ed m ore biofilm s than H 37Rv (P < 0.05) on cem ent and ceram ic surfaces in broth containing 5% LE. Biofilm m ass produced by the two strains under other experim ental conditions w ere statistically insignificant (Table 1). W hen the incubation tim e was extended to 3 w eeks, H 37Rv form ed m ore biofilm s than MTB1 on stainless steel in all grow th m edia and on ceram ic surfaces in broth contam ination in 10% O A D C plus 5% LE (Table 2) (P < 0.05). M T B 1 form ed m ore biofilm s than H 37Rv on ceramic surfaces in broth containing 5% LE or 10% O ADC. Biofilm mass totals produced by the tw o strains under other
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TABLE 1. Biofilms (OD 600 nm) formed by Mycobacterium tuberculosis strains after 2 weeks of incubation in three different microbiological media at 37°Ca Biofilm mass (Agoonm) (« = 4) Base growth media (7H9 with 0.1% T80) and supplements 5% liver extract 10% OADC and 5% liver extract 10% OADC
M. tuberculosis strains
Stainless steel
Cement
Ceramic
H37Rv MTB1 H37Rv MTB1 H37Rv MTB1
0.543 c E 0.540 c e 0.875 b a 0.644 c c 0.746 c b 0.586 b d
0.914 a c 1.007 aB 0.980 a b c 1.055 a b 1.148 a a 0.794 a d
0.661 b f 0.970 b b 0.978 a a 0.763 b d 0.784 b c 0.675 a b e
a OADC, a growth supplement containing oleic, albumin, dextrose, and catalase. Means in the same row not followed by the same lowercase letter are statistically significant (P < 0.05). Means in the same column not followed by the same uppercase letter are statistically significant (P < 0.05).
experimental conditions were statistically insignificant (Table 2). At the 4-week sampling point, it was clear that significantly more biofilms were formed by the two M. tuberculosis strains on cement than on ceramic and stainless steel surfaces, except for the biofilms formed by MTB1 on ceramic surfaces in 10% OADC, which were insignificantly different from the biofilm formed by the same strain on a cement surface (Table 3). Results of overall statistical analysis revealed that the amount of biofilm mass increased as the incubation time increased from weeks 2 to 3. However, the biofilm mass significantly (P < 0.05) decreased when the incubation time was extended to week 4 (Table 4). The two M. tuberculosis strains formed more biofilms on cement than on ceramic and stainless steel surfaces (Table 4). Furthermore, broth with LE and OADC better supported biofilm formation than broth with a single supplement. Between the two media that contained a single supplement, the amounts of biofilms formed in broth with 5% LE were significantly (P < 0.05) higher than those in broth with 10% OADC (Table 4). Overall, strain H37Rv was a better biofilm former than MTB1 (Table 4). Effect of sanitizing treatments on M. tuberculosis biofilms. A significant reduction (P < 0.05) in biofilm mass was observed when coupons were treated with the two sanitizers used in the study (Table 5). Although treatment with hot water also significantly reduced the amount of
biofilm on coupons, greater reductions in biofilm mass were observed when coupons were treated with 2% sanitizer A and 0.5% sanitizer B (Table 5). Treatments performed at 45 °C were more effective than those at 28 °C for the removal of biofilms from tested coupon surfaces (Table 5). Interest ingly, treatments with 0.5% sanitizer B were more effective than those with 2% sanitizer A at 28°C. However, treatments with 2% sanitizer A were more effective than those with 0.5% sanitizer B at 45°C (Table 5). DISCUSSION Both strains of M. tuberculosis used in the present study were capable of forming biofilms. This finding is in agreement with earlier studies in which the biofilm-forming abilities of M. tuberculosis were reported (22, 29). To our knowledge, mycobacterial biofilms developed on cement, stainless steel, and ceramic surfaces have not been reported previously. However, Ojha et al. (29) found that M. tuberculosis strains could develop biofilms on a polystyrene surface. In general, a greater amount of biofilm mass was observed on a cement surface in the present study, with only two exceptions among samples analyzed at week 3 (Tables 1 to 3). The topography of cement surfaces has been shown to facilitate higher levels of bacterial adhesion and biofilm formation (10, 12, 19, 21, 35). Studies have pointed out that the roughness of a contact surface contributes to bacterial attachment (31, 32). A study by
TABLE 2. Biofilms (OD 600 nm) formed by Mycobacterium tuberculosis strains after 3 weeks of incubation in three different microbiological media at 37°Ca Biofilm mass (A60o„m) (« = 4) Base growth media (7H9 with 0.1% T80) and supplements
5% liver extract 10% OADC and 5% liver extract 10% OADC
M. tuberculosis strains
Stainless steel
Cement
Ceramic
H37Rv MTB1 H37Rv MTB1 H37Rv MTB1
1.640 a b 1.050 b d 1.690 a a 0.740 c e 1.361 c c 0.744 b e
1.571 b a 1.481 a ab 1.479 b ab 1.500 a a b 1.448 a ab 1.302 a b
1.483 c b 1.486 a a 1.443 c c 1.046 b e 0.844 b f 1.139 3 D
° OADC, a growth supplement containing oleic, albumin, dextrose, and catalase. Means in the same row not followed by the same lowercase letter are statistically significant (P < 0.05). Means in the same column not followed by the same uppercase letter are statistically significant (P < 0.05).
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TABLE 3. Biofilms (OD 600 nm) formed by Mycobacterium tuberculosis strains after 4 weeks of incubation in three different microbiological media at 37°Ca Base growth media (7H9 with 0.1% T80) and supplements
Biofilm mass (A600nm) (n = 4) M. tuberculosis strains
Stainless steel
Cement
Ceramic
H37Rv MTB1 H37Rv MTB1 H37Rv MTB1
0.727 c b 0.366 c f 0.592 c d 0.786 c a 0.629 c c 0.579 b e
1.814 a a 1.843 a a 1.813 a a 1.848 a a 1.819 a a 1.593 a a
0.823 b f 0.935 b e 1.750 b a 1.051 b c 1.291 b b 0.998 a b d
5% liver extract 10% OADC and 5% liver extract 10% OADC
“ OADC, a growth supplement containing oleic, albumin, dextrose, and catalase. Means in the same row not followed by the same lowercase letter are statistically significant (P < 0.05). Means in the same column not followed by the same uppercase letter are statistically significant (P < 0.05).
Wingender and Flemming (40) found that significantly higher bacterial densities were associated with biofilms formed on cement-lined pipes than on galvanized steel pipes. Both strains of M. tuberculosis used in the present study were from clinical sources. Note that the lack of food isolates of M. tuberculosis in the study is an important limitation. However, M. tuberculosis is a zoonotic organ ism, and food isolates are likely to have originated from infected animals. Earlier studies showed the presence of M. tuberculosis in milk and animal tissue samples in Nigeria (5, 6). Furthermore, most cases of M. tuberculosis infection presented at the University of Ibadan Teaching Hospital, where one of the clinical strains was isolated, was linked to consumption of unpasteurized milk and infected offal (a delicacy in the Nigerian population). Reports on biofilm formation by food isolates of M. tuberculosis are scarce in the current body of literature. Note that there are possible
TABLE 4. Influence of various factors on the formation of biofilms by strains of Mycobacterium tuberculosis" Biofilm mass (A600nm)
Incubation time (n = 72) Week 2 Week 3 Week 4
0.818 c 1.310 a 1.195 b
Cell contact surface (n = 72) Cement Ceramic Stainless steel
1.404 a 1.062 b 0.833 c
Growth supplement (n = 72) Liver extract and OADC Liver extract alone OADC alone
1.1690 a 1.103 ab 1.051 b
Bacterial strain (n = 108) H37Rv MTB1
1.180 1.036
variations regarding the biofilm-forming ability of clinical versus food isolates of M. tuberculosis. Results of the present study showed that extending incubation time from 2 to 3 weeks provided time for more biofilm mass to accumulate. However, the mass of biofilm decreased from weeks 3 to 4. The reason for the decrease is unknown but is possibly due to nutrient depletion and accumulation of bacterial waste and metabolic products in the growth media. Nutrient-induced biofdm dispersion has been observed in Pseudomonas aeruginosa PAOl in earlier studies (34). Results of the present study indicated that M. tuberculosis strain H37Rv was better at forming biofilm than was MB1, with a few exceptions (Tables 1 to 3). The precise reasons for this phenomenon are currently unknown. A study on Mycobacterium smegmatis, a nonpathogenic mycobacterium, found that an acetylated derivative of glycopeptidolipids is required for surface attachment, sliding motility, and biofilm formation (28). Glycopeptidolipid biosynthesis was induced during multicellular growth of Mycobacterium avium, a physiological phenomenon associated with bacterial growth within biofilms (41). In addition to glycopeptidolipids, mycolyl-diacylglycerol and mycolic acids are the other two mycobacterial surface molecules known to play an important role in biofilm development by M. smegmatis (27, 29, 30). The biofilm forming ability of H37Rv could be attributed to its ability in the formation of cording and the synthesis of mycolic acid cyclopropane (14, 29). M. tuberculosis MTB1 is, neverthe less, an uncharacterized clinical isolate from Nigeria. TABLE 5. Efficacy of sanitizing treatments on biofilm removal Residual biofilm mass Treatment Control (n = 216) Water (n = 216) Sanitizer A (2%; n — 432)
a b
a OADC, a growth supplement containing oleic, albumin, dextrose, and catalase. Means not followed by the same letter are statistically significant (P < 0.05).
Sanitizer B (0.5%; n == 432)
Temp (°C)
85 28 45 28 45
0 ^ 6 0 0 nm )
1.272 a 0.864 b 0.821 c 0.469 f 0.798 d 0.487 e
a Means not followed by the same letter are statistically significant (P < 0.05).
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Further studies are needed to identify the genotypic and phenotypic characteristics that have contributed to the differences in biofilm-forming ability of the two Mycobac terium strains. Results of the present study suggest that cells of M. tuberculosis formed more biofilms in broth containing the iron-rich LE (Table 4). Similar findings have been observed with M. avium (18). Iron is an important cofactor that plays a central role in biofilm formation (2, 28). It has been shown that supplemental iron above 1 pM is necessary for proper biofilm development by M. smegmatis, although it is not needed for planktonic growth (28). The enhancement of mycobacterial biofilm formation by elevated iron concen tration has been noticed in several previous studies (2, 28, 29). Biofilm mass has been reported as an indicator for microbial cell growth. Biofilm mass should be directly proportional to the number of bacterial cells within the biofilm. The crystal violet binding assay, used in this and numerous other studies (1,36,42) to quantify biofilms, was selected over standard plate count assay because biofilm is difficult to remove from cell contact surfaces and because biofilm-embedded cells are often clumped together and are difficult to separate, causing variations and inaccuracies in plant count results. Results showed that treatments with the two sanitizers significantly (P < 0.05) reduced the biofilm mass on the solid surfaces used in the present study (Table 5). Sanitizer B is more effective in biofilm removal than sanitizer A at 28 °C. However, sanitizer A was more effective than sanitizer B at 45°C. Sanitizer B is a polyvalent sanitizer composed of two quaternary ammonium compounds, alkyl dimethylbenzyl ammonium chloride and didecyldimethyl ammonium chloride, as well as an aldehyde and pineoil. The bactericidal action of the quaternaries has been attributed to the inactivation of energy-producing enzymes, denaturation of essential cell proteins, and disruption of cell membranes (25). Sanitizer A is a monovalent sanitizer composed of active iodine, whose use as an antimicrobial agent dates back to the 1800s (35). The bactericidal activity of iodine is accomplished by direct halogenation of proteins, damage of cell walls, and destruction of enzyme activities (35). Similar to what was observed in the present study, previous reports indicated that the effectiveness of iodine increased at higher temperatures (13, 17). In general, sanitizing treatments at 45 °C were more effective than those at 28°C (Table 5). At higher temper ature, the sanitizers probably potentiated their disinfection and dislodging effects on the biofilms, thus producing a drastic reduction in biofilm mass on their contact surfaces. Similar to the present study, an earlier report showed that biofilms were completely removed from the surfaces of a model copper water plumbing system operated at 60°C (31). In another study, a mycobacterial biofilm mass on plastic surfaces was not affected by a treatment with water at 22 to 30°C (34). Neither treatment used in the present study completely removed the biofilms from the solid surfaces (Table 5). Earlier studies have shown that planktonic cells are more
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easily destroyed than those in biofilms (38). The macro molecules within the biofilms could reduce the rate of diffusion of the sanitizers into the biofilms. Perhaps only the parts of the biofilm mass close to the surface were reached by these sanitizers, whereas the deeper layers of the biofilms were unaffected. This study showed that pathogenic M. tuberculosis strains could develop biofilms on cement, ceramic, and stainless steel surfaces. In general, cells formed more biofilms on cement than on ceramic and stainless steel surfaces. This information is of particular importance in developing countries, where cement is still a major food contact surface. Growth media containing the iron-rich LE significantly (P < 0.05) enhanced biofilm formation. This implies that the presence of iron in the environment, especially the presence of denatured blood in food processing environments such as abattoirs, may enhance biofilm formation by bacterial pathogens such as mycobac teria. Study results indicate that careful selection of food processing materials and good environmental hygiene are important considerations for biofilm control and that raising the temperature of sanitizing treatments will generate a greater biofilm reduction. Results of this study will be useful in the design of an effective approach for control of M. tuberculosis biofilms. ACKNOWLEDGMENT Laboratory scientists at the Tuberculosis Reference Laboratory at the University College Hospital, Ibadan, Nigeria, are graciously acknowledged with thanks.
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