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Inactivation of Clostridium difficile spores by microwave irradiation
Anaerobe 38 (2016) 14e20
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Inactivation of Clostridium difficile spores by microwave irradiation Suvash Chandra Ojha a, Surang Chankhamhaengdecha b, c, Sombat Singhakaew b, Puey Ounjai b, Tavan Janvilisri c, d, * a
Graduate Program in Molecular Medicine, Faculty of Science, Mahidol University, Bangkok, 10400 Thailand Department of Biology, Faculty of Science, Mahidol University, Bangkok, 10400 Thailand c Center for Emerging and Neglected Infectious Diseases, Mahidol University, Nakhon Pathom, 73170, Thailand d Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok, 10400 Thailand b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 July 2015 Received in revised form 25 October 2015 Accepted 30 October 2015 Available online 4 November 2015
Spores are a potent agent for Clostridium difficile transmission. Therefore, factors inhibiting spores have been of continued interest. In the present study, we investigated the influence of microwave irradiation in addition to conductive heating for C. difficile spore inactivation in aqueous suspension. The spores of 15 C. difficile isolates from different host origins were exposed to conductive heating and microwave irradiation. The complete inhibition of spore viability at 107 CFU/ml was encountered following microwave treatment at 800 W for 60 s, but was not observed in the conductive-heated spores at the same time etemperature exposure. The distinct patterns of ultrastructural alterations following microwave and conductive heat treatment were observed and the degree of damages by microwave was in the exposure time-dependent manner. Microwave would therefore be a simple and time-efficient tool to inactivate C. difficile spores, thus reducing the risk of C. difficile transmission. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Clostridium difficile Spore Microwave Ultrastructure
1. Introduction Clostridium difficile is considered to be a major nosocomial pathogen causing clinical manifestations ranging from mild diarrhea to severe pseudomembranous colitis [1]. The incidence and severity of C. difficile infection (CDI) has been rising in several countries, expanding its cases to non-hospital and communityacquired settings [2]. Therefore, CDI has a considerable impact on healthcare systems worldwide. C. difficile spores serve as a reservoir for the disease transmission as they are highly resistant to extreme physical conditions including heat, pH, and chemical treatments including alcohol, antimicrobials, and disinfectants [3,4], so they are able to persist in the environments for a long period of time. Recent data speculate that consumption of contaminated food; inter-species transmission from environmental sources and some levels of host adaptation could attribute to changing epidemiology of CDI in humans [5e7]. Microwave radiation has been applied to control bacterial contaminations in hospital instrument and waste sterilization, as well
* Corresponding author. Department of Biochemistry, Faculty of Science, Mahidol University, 272 Rama VI Road, Phayathai, Rajdhevi, Bangkok, 10400, Thailand. E-mail address: [email protected] (T. Janvilisri). http://dx.doi.org/10.1016/j.anaerobe.2015.10.015 1075-9964/© 2015 Elsevier Ltd. All rights reserved.
as domestic and industrial food processing [8e10]. Numerous studies have addressed the influence of microwave irradiation on spore inactivation [9,11e15]. Both thermal and non-thermal effects have been suggested to play parts in antimicrobial activity [16,17], however the actual mechanism of spore inactivation has not yet been understood. In this study, we investigated the effect of microwave irradiation on C. difficile spore viability and outgrowth in the isolates from different host origins. In addition, we also examined the ultra-structural alterations following microwave and conductive heating treatment by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The data presented in this paper provide a basis for further exploration of the microwave appliance under optimized conditions to control CDI. 2. Materials and methods 2.1. C. difficile strains and growth condition A total of 15 C. difficile isolates including 5 human isolates (630, R20291, H203, H204, H205), 6 animal isolates (A121, A122, A123, A124, A125, A126), and 4 food isolates (F101, F102, F103, F104), were used in this study. The C. difficile strains 630 and R20291 were kindly provided by Prof. Nigel Minton, University of Nottingham. All strains were grown at 37 C in an anaerobic workstation (85%
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N2, 10% H2 and 5% CO2; Don Whitley Scientific, UK) in brain heart infusion supplemented with 0.1% L-cysteine (BHIS) (Sigma) and 5 mg/ml yeast extract (Oxoid) broth or agar. 2.2. Preparation and purification of C. difficile spores A single colony was inoculated into BHIS broth supplemented with 0.1% sodium taurocholate (ST) and incubated overnight at 37 C. A 100 ml aliquot of overnight culture were spread onto BHIS agar supplemented with 0.1% ST, 250 mg/ml cycloserine and 8 mg/ml cefoxitin; and incubated anaerobically at 37 C for 10 days to allow efficient sporulation. Sporulation induced lawns were harvested in 1 ml sterile distilled water (dH2O) using cell scrapers. Suspension was then centrifuged at 5000 g for 15 min, and was washed 5 times with sterile dH2O. To inactivate viable vegetative cells, spore suspensions were then treated with 0.3 mg/ml proteinase K at 37 C for 2 h with gentle shaking, followed by incubation at 65 C for 1 h. Subsequently, spore suspensions were washed 5 additional times to remove any residuals from proteinase K. Purified spores were then enumerated and examined by phase-contrast microscopy to ensure that they were free of vegetative cells and debris, and subsequently stored at 4 C. Prior use, the spore viability was assessed to ensure the colony forming efficiency of >90%. 2.3. Evaluation of spore viability using spotting assays Spores were diluted in a sterilized test tube with sterile dH2O to obtain the final volume of 2 ml with the final concentration of 107 CFU/ml. The aliquots were exposed to a microwave oven with the frequency of 2450 MHz and the power output of 800 W (MR8140; Hitachi) at different time intervals by placing a test tube containing spores in a 200-ml Pyrex beaker at the center of microwave rotating plate. The changes in suspension temperature were measured simultaneously with a digital thermometer (CEM, Shenzen, China). As a control for the thermal effect of the microwave, the spore suspensions were exposed to conductive heating at the temperature achieved by microwave for the corresponding time intervals. Following the treatments, the respective cells were 10-fold serially diluted with phosphate buffer saline (PBS) and spotted on BHIS agar supplemented with 0.1% ST. The differences in spore germination efficiency were recorded following 48 h of incubation at 37 C. 2.4. Measurement of spore outgrowth and return to vegetative cells Spore suspensions were heat activated at 65 C for 30 min, vortex mixed to obtain a homogenous suspension and checked for clumping by microscopic observation. The concentrations of inoculum at OD600 of 0.6 were prepared in sterile dH2O. The samples were then subjected to microwave treatment as described above. Microtiter plates containing 100 ml of 2 concentrated BHIS, supplemented with 0.1% ST were previously prepared and placed in an anaerobic workstation overnight. Following the treatment, spore suspensions at OD600 of 0.1 were added to wells and incubated anaerobically at 37 C for 24 h. Vegetative cell growth was examined by measuring OD600 at 1 h time interval for 24 h using a microplate reader (BiotrakII, Amersham Bioscience). The ratio of the OD600 at the time t and the control (t ¼ 0) was then plotted against time.
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for electron microscopy. The spores were fixed in 0.1 M sodium cacodylate buffer (pH 7.4) with 2.5% glutaraldehyde and 2% paraformaldehyde for 12 h, followed by post-fixation with 1% osmium tetroxide in the same buffer for 1 h at 4 C. For SEM, the samples were dehydrated with series of graded ethanol to 100%, followed by critical point drying and sputter coating with platinum-palladium as described previously [18]. Specimens were visualized using a scanning electron microscope (Hitachi S-2500). For TEM, the samples were dehydrated in graded series of ethanol (30%, 50%, 70%, 90%, and 100%) twice for 15 min at 4 C before solvent substituted with acetone for 2 h. The pellets were then infiltrated with Aradite resin 502 (Electron Microscopy Sciences, Hatfield, PA). Subsequently, the blocks were incubated at 45 C for 2 days and then 60 C for another 2 days. The resin blocks were sectioned at the thickness of ~80 nm using Leica EM UC6 ultramicrotome (Leica Microsystem; Austria). Thin sections were picked up on formvar coated 200-mesh Cu grid before negatively stained with uranyl acetate and lead citrate. The samples were examined in an FEI Tecnai G2 T20 TEM operated at 120 keV. Images were recorded on a Gatan CCD camera. 2.6. Statistical analysis All data presented herein were of at least three independent experiments. Statistical analyses were analyzed by one-way analysis of variance (ANOVA) to compare each condition with the corresponding controls without the treatment. P-values less than 0.05 indicated statistically significant difference. 3. Results Prior to analyzing the spore viability following microwave irradiation, we initially evaluated the changes in temperature with microwave irradiation compared to the conductive heating using hot plate. As shown in Fig. 1, the temperature of the spore suspensions increased from the initial ~25 C to boiling within 30 s with the microwave treatment. However, the temperature of the suspension only reached up to ~80 C with the hot plate at the maximal time interval of the tested conditions. A conductive heating by a thermocycler was therefore favored over hot plate to set the comparable experimental conditions. The spore suspensions were then either microwave irradiated or conductively heated at the same reaction temperature as monitored during the microwave experiments to assess the C. difficile spore viability. As shown in Fig. 2, the microwave irradiation demonstrated significant reduction in the colony forming efficiency of C. difficile
2.5. Electron microscopy Spores were exposed to either microwave irradiation or conductive heating as described above. Spore suspensions were then centrifuged at 15,000 g for 5 min and immediately process
Fig. 1. Changes in the temperature of suspensions respect to exposure time following microwave irradiation and conductive heating using hot plate. Circles and triangles represent the temperature of the suspensions over time following the microwave and hot plate conditions, respectively. All data are the averages of triplicate measurements and bars indicate standard errors of the mean.
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Fig. 2. Survival of C. difficile spores following microwave irradiation and conductive heating. Representative efficiencies of colony formation of a food isolate, F102, after (A) microwave irradiation and (B) conductive heating. The spores were either microwave irradiated for 0e60 s or conductively heated at the indicated temperatures for the indicated time. Spores were then spotted on BHIS agar plates in serial tenfold dilutions and incubated at 37 C for 48 h. Note that RT stands for room temperature.
spores, compared to the conductive heating. Complete spore inactivation was observed by microwave irradiation for 60 s at 800 W. In contrast to the microwave treatment, the spore viability was slightly reduced following conductive heating at 98 C for 30 s, and was completely inhibited after 120 s. These results showed that spore preparations could be sterilized by microwave irradiation in a short amount of time. An assessment of C. difficile spore germination, outgrowth and return to vegetative cell growth was also carried out by measuring the changes in OD600 of C. difficile cultures over a 24 h time period. Under the control condition (t ¼ 0), the different patterns in the growth kinetics were observed among 15 tested strains (Fig. 3), however there seemed to be no relationship between growth patterns and host origins. The conductively heated spores at 98 C for 60 s appeared to be able to retain the growth with similar patterns to the controls (data not shown). However, the complete inhibition was observed following 90 s (strains A121, A122, A123, and H203), 120 s (strains F101, F102, F104, A124, H201, H202, H204, and H205), 180 s (strains F103, A125, and A126) of conductive heating. The microwave exposure for 10s did not seem to affect the growth of the cells, except for the human isolate H203. A significant difference (P < 0.001) in the growth rate was found for the spores treated with microwave irradiation at 800 W for 20, 30, and 60s compared to the untreated control. The levels of spore inactivation appeared to be in the exposure time-dependent manner. The complete inhibition was observed with the 60s treatment of microwave irradiation, supporting the results of colony spotting assays. The differences in the viability and growth pattern of C. difficile spores following microwave irradiation and conductive heating prompted us to investigate the spore ultrastructural changes under these experimental settings using SEM and TEM. We selected six
C. difficile isolates including 2 food isolates (F101 and F102); 2 animal isolates (A125 and A126) and 2 human isolates (R20291 and H203) based on their characteristics of the spore germination, outgrowth, and vegetative cell growth pattern. As shown in Fig. 4, while the spores under the control condition retained their normal size displaying ellipsoidal structure and smooth and intact surfaces, the microwave irradiation caused a range of damages including spore shrinkage and deformation, to spore fragmentation and lyses, as well as rupture of spore membrane. The degree of damage was correlated well with the exposure time to microwave irradiation. Spores following conductive heating exhibited the lesser degree of destruction with only minor shrinkage or blisters on their surfaces compared to the microwave treatment. The data from SEM corresponded well with the viability and growth kinetic results. To gain more information in spore inactivation, we performed TEM to further analyze ultrastructural alterations in spores treated with microwave irradiation. The control (untreated) spores exhibited ellipsoidal structure with sequential organization of spore layers including intact spore coat, cortex, well defined inner membrane and granular core (Fig. 5; left panel). Under the conductive heating condition, some disruptive features in the spore ultrastructure were observed (Fig. 5, right panel). In contrast to the untreated spores, the spores exposed to conductive heat showed evident swelling of the outer coat, bubbling inside the core region (A125, A126, and H203), initiation of spot formation or outer wall disruption (A125 and R20291). The severe damages following conductive heating were observed in the food isolate F102. In comparison to the conductive heating, the microwave-associated damages were more evident and the magnitude of damages was correlated with the exposure time. Following the microwave irradiation for 30 s, the spore deformity with outer coat degradation and certain level of core alterations was observed in all strains. The strains F102 and A125, which were able to grow following the microwave exposure of 30 s (Fig. 3B and I), exhibited modest modifications, compared to other strains, whose spores could not return to grow in the vegetative states. The microwave exposure for 60 s resulted in the profound disruption of spore core complexes with heavy aggregation of cytoplasmic proteins, inner membrane fusion and the distortion of outer coat and cortex. Interestingly, the effect of the microwave irradiation on spores was most pronounced in the strain H203. This strain appeared to be very sensitive to the microwave treatment as its conversion of spores to actively growing vegetative cells was inhibited following the exposure to microwave for 10 s. Altogether, the results from SEM and TEM suggested that there might be different mechanisms of spore inactivation from microwave irradiation and conductive heating. 4. Discussion While C. difficile is a major cause of hospital-acquired antibioticassociated infections, recent epidemiological surveys suggest that community-acquired CDI cases become increasingly more common and are linked with considerable morbidity [2]. The contamination of C. difficile in food products [5,6], and farm animals [6,7,19e21], as well as the close genetic relationship among C. difficile isolates from humans, food, and animals [7,22,23], point us to the possibility of interspecies and foodborne transmission of this bacterium. C. difficile is able to form spores, which are well tolerated to harsh environments and disinfectants. It has been shown that the mutant strains that cannot form spores, are also unable to persist in the colonic tract of the host and be horizontally transmitted [24]. Spores are therefore likely to be responsible for dissemination of C. difficile in nosocomial and non-hospital settings [3,4]. Thus, several investigations have been focusing on various approaches to eliminate spore contaminants. One of such approaches includes
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Fig. 3. Vegetative cell growth of C. difficile from spores following microwave irradiation and conductive heating. Spores were microwave irradiated at different time interval and then incubated with BHIS supplemented 0.1% (w/v) ST. Germination and outgrowth were followed at 37 C in an anaerobic environment by measuring changes in OD600 every hour for 24 h. Data points represent the mean of the relative OD600 at the indicated time points normalized to t ¼ 0 (control). All the experiments were performed in triplicates and error bar represents the standard errors. The results include the strains: A) F101; B) F102; C) F103; D) F104; E) A121; F) A122; G) A123; H) A124; I) A125; J) A126; K) 630; L) R20291; M) H203; N) H204; O) H205. Triangle (D) denotes t ¼ 0; circle (B), diamond (◊), cross ( ), square ( ) denotes the microwave treatment for 10s, 20s, 30s, 60s, respectively.
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microwave irradiation, which has been used in sterilization of foods, hospital waste, and medical appliances [8e10]. The penetration capacity of the microwave depends on the materials having low or high loss factor and the type of microwave used. Most household appliances use 2450 MHz microwave as they are smaller and easier to work with, however, deeper penetration into the materials can be achieved with microwave having 915 MHz power generator [25]. It has been shown to inhibit the growth of several microbial pathogens such as Escherichia coli, Streptococcus faecalis, and Staphyloccocus aureus [10,15,16,18], as well as inactivate spores of Bacillus subtilis [9], Bacillus atrophaeus [11], Bacillus licheniformis [13], and Clostridium sporogenes [14]. Microwave radiation generates heat, which is thought to be responsible for the destruction of these pathogens [16]. However, nonthermal effects on microorganisms have also been proposed [17]. It is therefore of interest to investigate the effect of microwave irradiation on C. difficile spore viability, outgrowth, and return to vegetative cells. In this study, the exposure time to microwave irradiation was kept short to minimize non-specific side reactions. Conductive heating at the same-temperature reaction time was also performed as a control for the thermal effect of the microwave. Our results revealed that the colony forming efficiency of microwave irradiated spores was found to dramatically decline relative to an increase in the exposure time and temperature. However, the spores that were conductively heated at the same temperature for the same exposure time, were found viable. These data indicate the existence of nonthermal microwave effect of spore inactivation. Consistently, it has been reported that C. difficile spores retain viability following the heat treatment at 71 C for at least 2 h [26], however the extended heating at 85 C noticeably reduces cell division, but not spore germination [27]. The marked inhibitory effect of 96 C on C. difficile spores has also been documented, but it could not completely abrogate the germination of a population of superdormant spores [27]. Furthermore, the microwave inactivation of C. difficile spores to return to vegetative states was demonstrated. All isolates exhibited substantially different outgrowth rates, reaching late exponential phase at different time frame when the
starting inoculum was standardized. The differences in growth characteristics among C. difficile isolates from humans, food, and animals, and the time taken to reach maximal growth were apparent from strains to strains, regardless of the host origins. Microwave irradiation was shown to inhibit the vegetative cell growth from spores in all isolates and the level of spore inactivation was shown to be exposure time-dependent. Altogether, the stresses generated through microwave irradiation may cause various mechanistic deterioration to the spore internal organization, thus resulting in time taken to retain their viability. This suggests that the microwave power and exposure time for sterilization should be taken into consideration to completely inhibit spore germination and return to vegetative growth, thereby reducing the chance of gut colonization under favorable conditions to initiate CDI. Moreover, in the case of food processing, microwave radiation generates high temperature in a very short time, resulting in nutritional and sensorial advantages compared to heating method. Ultrastructural analysis of treated spores indicated that the spore damage induced by microwave irradiation was significantly different from that attributable to conductive heating. SEM analysis of spore surface structure revealed that the untreated spores possessed smooth surface, while the microwave irradiated or conductively heated spores exhibited disruption on their surfaces. The microwave irradiated spores seemed to undergo spore lysis, which was more pronounced compared to the heating condition, and was related to the exposure time. The effects exerted by microwave radiation on the spore ultrastructure was clearly distinguishable from that due to conductive heating as seen by TEM. An altered organization of intracellular components and aggregation of cytoplasmic proteins became more prominent with longer exposure times. A number of studies have argued that microwave irradiation has identical lethality to bacterial spores as compared to conductive heating at a given temperature [13,14]. Similar results have been also reported on B. subtilis spores; but the damage induced by microwave treatment was intrinsically different from that observed for heating [9]. However, we noticed that the degree of lethality varied among the strains in this study. Microwave
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Fig. 4. Scanning electron micrographs of C. difficile spores following microwave irradiation and conductive heating. The selected isolates included F101, F102, A125, A126, R20291, and H203. Control (untreated) spores displayed ellipsoidal structure with smooth surfaces as shown in the left panel. Spores were treated either with microwave radiation for 30s and 60s, or with 60s of 98 C, and were then subjected to SEM. All the micrographs were taken at a magnification of 11,000. Scale bars represent 2 mm.
irradiation disrupted the spores at the greater extent compared to conductive heating at a given temperature. The damage of C. difficile spores by microwave irradiation was probably attributed to generation of internal pressure in core region, leading to sequential events such as bubbling, protein aggregation, inner membrane fusion, finally resulting in the hydrolysis of spore cortex and spore coat [12]. In contrast, conductive heating generates external pressure so that spores seem to withstand in various degrees [24]. The formation of bubbling indicates an early sign of damage that spores may be able to retain their germination characteristics by an unknown mechanism. The dark spot formation in the core region following microwave and conductive heat treatments could probably be due to the condensation of nucleoprotein complexes within the core region, as advocated for cytoplasmic proteins of vegetative bacterial cells following both treatments [18,28], which may contribute to partial or full spore inactivation. The disruption of
spore inner membrane and release of the core components are possibly correlated with spore viability [29]. Our results suggest that there is a marked difference in ultrastructural alterations following microwave irradiation and conductive heating, which hints us upon the nonthermal influence on spores. In summary, our study thus highlights the possible use of microwave irradiation to inactivate C. difficile spores. Fifteen C. difficile isolates from different host origins exhibiting distinct germination and growth characteristics were found sensitive to microwave treatment, while all were well tolerated to conductive heating at the same exposure time. A combined assessment by SEM and TEM revealed dynamical changes in spore ultrastructure caused by these treatments. The degree of damage caused by microwave irradiation was markedly more pronounced than conductive heating. Taken together, the results herein demonstrate the effectiveness of microwave irradiation against C. difficile spores. Further studies to
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Fig. 5. Transmission electron micrographs of C. difficile spores following microwave irradiation and conductive heating. The selected isolates included F101, F102, A125, A126, R20291, and H203. Control (untreated) spores displayed typical organization of spore layers as shown in the left panel. Spores were treated either with microwave radiation for 30s and 60s, or with 60s of 98 C, and were then subjected to TEM. All the micrographs were taken at a magnification of 19,000. Scale bars represent 200 nm.
understand the mechanism of spore inactivation and to identify the optimal conditions for microwave sterilization will certainly lower the transmission risk of C. difficile. Acknowledgments This work is supported by the Faculty of Science, Mahidol University; and grants from the International Society for Infectious Diseases to TJ, Office of Higher Education Commission and Mahidol University under the National Research Universities Initiative to SC, and a research assistant scholarship from the Faculty of Graduate Studies, Mahidol University to SCO. References [1] S. Johnson, D.N. Gerding, Clostridium difficileeassociated diarrhea, Clin. Infect. Dis. 26 (1998) 1027e1034.
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Inactivation of Clostridium difficile spores by microwave irradiation Suvash Chandra Ojha a, Surang Chankhamhaengdecha b, c, Sombat Singhakaew b, Puey Ounjai b, Tavan Janvilisri c, d, * a
Graduate Program in Molecular Medicine, Faculty of Science, Mahidol University, Bangkok, 10400 Thailand Department of Biology, Faculty of Science, Mahidol University, Bangkok, 10400 Thailand c Center for Emerging and Neglected Infectious Diseases, Mahidol University, Nakhon Pathom, 73170, Thailand d Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok, 10400 Thailand b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 July 2015 Received in revised form 25 October 2015 Accepted 30 October 2015 Available online 4 November 2015
Spores are a potent agent for Clostridium difficile transmission. Therefore, factors inhibiting spores have been of continued interest. In the present study, we investigated the influence of microwave irradiation in addition to conductive heating for C. difficile spore inactivation in aqueous suspension. The spores of 15 C. difficile isolates from different host origins were exposed to conductive heating and microwave irradiation. The complete inhibition of spore viability at 107 CFU/ml was encountered following microwave treatment at 800 W for 60 s, but was not observed in the conductive-heated spores at the same time etemperature exposure. The distinct patterns of ultrastructural alterations following microwave and conductive heat treatment were observed and the degree of damages by microwave was in the exposure time-dependent manner. Microwave would therefore be a simple and time-efficient tool to inactivate C. difficile spores, thus reducing the risk of C. difficile transmission. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Clostridium difficile Spore Microwave Ultrastructure
1. Introduction Clostridium difficile is considered to be a major nosocomial pathogen causing clinical manifestations ranging from mild diarrhea to severe pseudomembranous colitis [1]. The incidence and severity of C. difficile infection (CDI) has been rising in several countries, expanding its cases to non-hospital and communityacquired settings [2]. Therefore, CDI has a considerable impact on healthcare systems worldwide. C. difficile spores serve as a reservoir for the disease transmission as they are highly resistant to extreme physical conditions including heat, pH, and chemical treatments including alcohol, antimicrobials, and disinfectants [3,4], so they are able to persist in the environments for a long period of time. Recent data speculate that consumption of contaminated food; inter-species transmission from environmental sources and some levels of host adaptation could attribute to changing epidemiology of CDI in humans [5e7]. Microwave radiation has been applied to control bacterial contaminations in hospital instrument and waste sterilization, as well
* Corresponding author. Department of Biochemistry, Faculty of Science, Mahidol University, 272 Rama VI Road, Phayathai, Rajdhevi, Bangkok, 10400, Thailand. E-mail address: [email protected] (T. Janvilisri). http://dx.doi.org/10.1016/j.anaerobe.2015.10.015 1075-9964/© 2015 Elsevier Ltd. All rights reserved.
as domestic and industrial food processing [8e10]. Numerous studies have addressed the influence of microwave irradiation on spore inactivation [9,11e15]. Both thermal and non-thermal effects have been suggested to play parts in antimicrobial activity [16,17], however the actual mechanism of spore inactivation has not yet been understood. In this study, we investigated the effect of microwave irradiation on C. difficile spore viability and outgrowth in the isolates from different host origins. In addition, we also examined the ultra-structural alterations following microwave and conductive heating treatment by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The data presented in this paper provide a basis for further exploration of the microwave appliance under optimized conditions to control CDI. 2. Materials and methods 2.1. C. difficile strains and growth condition A total of 15 C. difficile isolates including 5 human isolates (630, R20291, H203, H204, H205), 6 animal isolates (A121, A122, A123, A124, A125, A126), and 4 food isolates (F101, F102, F103, F104), were used in this study. The C. difficile strains 630 and R20291 were kindly provided by Prof. Nigel Minton, University of Nottingham. All strains were grown at 37 C in an anaerobic workstation (85%
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N2, 10% H2 and 5% CO2; Don Whitley Scientific, UK) in brain heart infusion supplemented with 0.1% L-cysteine (BHIS) (Sigma) and 5 mg/ml yeast extract (Oxoid) broth or agar. 2.2. Preparation and purification of C. difficile spores A single colony was inoculated into BHIS broth supplemented with 0.1% sodium taurocholate (ST) and incubated overnight at 37 C. A 100 ml aliquot of overnight culture were spread onto BHIS agar supplemented with 0.1% ST, 250 mg/ml cycloserine and 8 mg/ml cefoxitin; and incubated anaerobically at 37 C for 10 days to allow efficient sporulation. Sporulation induced lawns were harvested in 1 ml sterile distilled water (dH2O) using cell scrapers. Suspension was then centrifuged at 5000 g for 15 min, and was washed 5 times with sterile dH2O. To inactivate viable vegetative cells, spore suspensions were then treated with 0.3 mg/ml proteinase K at 37 C for 2 h with gentle shaking, followed by incubation at 65 C for 1 h. Subsequently, spore suspensions were washed 5 additional times to remove any residuals from proteinase K. Purified spores were then enumerated and examined by phase-contrast microscopy to ensure that they were free of vegetative cells and debris, and subsequently stored at 4 C. Prior use, the spore viability was assessed to ensure the colony forming efficiency of >90%. 2.3. Evaluation of spore viability using spotting assays Spores were diluted in a sterilized test tube with sterile dH2O to obtain the final volume of 2 ml with the final concentration of 107 CFU/ml. The aliquots were exposed to a microwave oven with the frequency of 2450 MHz and the power output of 800 W (MR8140; Hitachi) at different time intervals by placing a test tube containing spores in a 200-ml Pyrex beaker at the center of microwave rotating plate. The changes in suspension temperature were measured simultaneously with a digital thermometer (CEM, Shenzen, China). As a control for the thermal effect of the microwave, the spore suspensions were exposed to conductive heating at the temperature achieved by microwave for the corresponding time intervals. Following the treatments, the respective cells were 10-fold serially diluted with phosphate buffer saline (PBS) and spotted on BHIS agar supplemented with 0.1% ST. The differences in spore germination efficiency were recorded following 48 h of incubation at 37 C. 2.4. Measurement of spore outgrowth and return to vegetative cells Spore suspensions were heat activated at 65 C for 30 min, vortex mixed to obtain a homogenous suspension and checked for clumping by microscopic observation. The concentrations of inoculum at OD600 of 0.6 were prepared in sterile dH2O. The samples were then subjected to microwave treatment as described above. Microtiter plates containing 100 ml of 2 concentrated BHIS, supplemented with 0.1% ST were previously prepared and placed in an anaerobic workstation overnight. Following the treatment, spore suspensions at OD600 of 0.1 were added to wells and incubated anaerobically at 37 C for 24 h. Vegetative cell growth was examined by measuring OD600 at 1 h time interval for 24 h using a microplate reader (BiotrakII, Amersham Bioscience). The ratio of the OD600 at the time t and the control (t ¼ 0) was then plotted against time.
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for electron microscopy. The spores were fixed in 0.1 M sodium cacodylate buffer (pH 7.4) with 2.5% glutaraldehyde and 2% paraformaldehyde for 12 h, followed by post-fixation with 1% osmium tetroxide in the same buffer for 1 h at 4 C. For SEM, the samples were dehydrated with series of graded ethanol to 100%, followed by critical point drying and sputter coating with platinum-palladium as described previously [18]. Specimens were visualized using a scanning electron microscope (Hitachi S-2500). For TEM, the samples were dehydrated in graded series of ethanol (30%, 50%, 70%, 90%, and 100%) twice for 15 min at 4 C before solvent substituted with acetone for 2 h. The pellets were then infiltrated with Aradite resin 502 (Electron Microscopy Sciences, Hatfield, PA). Subsequently, the blocks were incubated at 45 C for 2 days and then 60 C for another 2 days. The resin blocks were sectioned at the thickness of ~80 nm using Leica EM UC6 ultramicrotome (Leica Microsystem; Austria). Thin sections were picked up on formvar coated 200-mesh Cu grid before negatively stained with uranyl acetate and lead citrate. The samples were examined in an FEI Tecnai G2 T20 TEM operated at 120 keV. Images were recorded on a Gatan CCD camera. 2.6. Statistical analysis All data presented herein were of at least three independent experiments. Statistical analyses were analyzed by one-way analysis of variance (ANOVA) to compare each condition with the corresponding controls without the treatment. P-values less than 0.05 indicated statistically significant difference. 3. Results Prior to analyzing the spore viability following microwave irradiation, we initially evaluated the changes in temperature with microwave irradiation compared to the conductive heating using hot plate. As shown in Fig. 1, the temperature of the spore suspensions increased from the initial ~25 C to boiling within 30 s with the microwave treatment. However, the temperature of the suspension only reached up to ~80 C with the hot plate at the maximal time interval of the tested conditions. A conductive heating by a thermocycler was therefore favored over hot plate to set the comparable experimental conditions. The spore suspensions were then either microwave irradiated or conductively heated at the same reaction temperature as monitored during the microwave experiments to assess the C. difficile spore viability. As shown in Fig. 2, the microwave irradiation demonstrated significant reduction in the colony forming efficiency of C. difficile
2.5. Electron microscopy Spores were exposed to either microwave irradiation or conductive heating as described above. Spore suspensions were then centrifuged at 15,000 g for 5 min and immediately process
Fig. 1. Changes in the temperature of suspensions respect to exposure time following microwave irradiation and conductive heating using hot plate. Circles and triangles represent the temperature of the suspensions over time following the microwave and hot plate conditions, respectively. All data are the averages of triplicate measurements and bars indicate standard errors of the mean.
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Fig. 2. Survival of C. difficile spores following microwave irradiation and conductive heating. Representative efficiencies of colony formation of a food isolate, F102, after (A) microwave irradiation and (B) conductive heating. The spores were either microwave irradiated for 0e60 s or conductively heated at the indicated temperatures for the indicated time. Spores were then spotted on BHIS agar plates in serial tenfold dilutions and incubated at 37 C for 48 h. Note that RT stands for room temperature.
spores, compared to the conductive heating. Complete spore inactivation was observed by microwave irradiation for 60 s at 800 W. In contrast to the microwave treatment, the spore viability was slightly reduced following conductive heating at 98 C for 30 s, and was completely inhibited after 120 s. These results showed that spore preparations could be sterilized by microwave irradiation in a short amount of time. An assessment of C. difficile spore germination, outgrowth and return to vegetative cell growth was also carried out by measuring the changes in OD600 of C. difficile cultures over a 24 h time period. Under the control condition (t ¼ 0), the different patterns in the growth kinetics were observed among 15 tested strains (Fig. 3), however there seemed to be no relationship between growth patterns and host origins. The conductively heated spores at 98 C for 60 s appeared to be able to retain the growth with similar patterns to the controls (data not shown). However, the complete inhibition was observed following 90 s (strains A121, A122, A123, and H203), 120 s (strains F101, F102, F104, A124, H201, H202, H204, and H205), 180 s (strains F103, A125, and A126) of conductive heating. The microwave exposure for 10s did not seem to affect the growth of the cells, except for the human isolate H203. A significant difference (P < 0.001) in the growth rate was found for the spores treated with microwave irradiation at 800 W for 20, 30, and 60s compared to the untreated control. The levels of spore inactivation appeared to be in the exposure time-dependent manner. The complete inhibition was observed with the 60s treatment of microwave irradiation, supporting the results of colony spotting assays. The differences in the viability and growth pattern of C. difficile spores following microwave irradiation and conductive heating prompted us to investigate the spore ultrastructural changes under these experimental settings using SEM and TEM. We selected six
C. difficile isolates including 2 food isolates (F101 and F102); 2 animal isolates (A125 and A126) and 2 human isolates (R20291 and H203) based on their characteristics of the spore germination, outgrowth, and vegetative cell growth pattern. As shown in Fig. 4, while the spores under the control condition retained their normal size displaying ellipsoidal structure and smooth and intact surfaces, the microwave irradiation caused a range of damages including spore shrinkage and deformation, to spore fragmentation and lyses, as well as rupture of spore membrane. The degree of damage was correlated well with the exposure time to microwave irradiation. Spores following conductive heating exhibited the lesser degree of destruction with only minor shrinkage or blisters on their surfaces compared to the microwave treatment. The data from SEM corresponded well with the viability and growth kinetic results. To gain more information in spore inactivation, we performed TEM to further analyze ultrastructural alterations in spores treated with microwave irradiation. The control (untreated) spores exhibited ellipsoidal structure with sequential organization of spore layers including intact spore coat, cortex, well defined inner membrane and granular core (Fig. 5; left panel). Under the conductive heating condition, some disruptive features in the spore ultrastructure were observed (Fig. 5, right panel). In contrast to the untreated spores, the spores exposed to conductive heat showed evident swelling of the outer coat, bubbling inside the core region (A125, A126, and H203), initiation of spot formation or outer wall disruption (A125 and R20291). The severe damages following conductive heating were observed in the food isolate F102. In comparison to the conductive heating, the microwave-associated damages were more evident and the magnitude of damages was correlated with the exposure time. Following the microwave irradiation for 30 s, the spore deformity with outer coat degradation and certain level of core alterations was observed in all strains. The strains F102 and A125, which were able to grow following the microwave exposure of 30 s (Fig. 3B and I), exhibited modest modifications, compared to other strains, whose spores could not return to grow in the vegetative states. The microwave exposure for 60 s resulted in the profound disruption of spore core complexes with heavy aggregation of cytoplasmic proteins, inner membrane fusion and the distortion of outer coat and cortex. Interestingly, the effect of the microwave irradiation on spores was most pronounced in the strain H203. This strain appeared to be very sensitive to the microwave treatment as its conversion of spores to actively growing vegetative cells was inhibited following the exposure to microwave for 10 s. Altogether, the results from SEM and TEM suggested that there might be different mechanisms of spore inactivation from microwave irradiation and conductive heating. 4. Discussion While C. difficile is a major cause of hospital-acquired antibioticassociated infections, recent epidemiological surveys suggest that community-acquired CDI cases become increasingly more common and are linked with considerable morbidity [2]. The contamination of C. difficile in food products [5,6], and farm animals [6,7,19e21], as well as the close genetic relationship among C. difficile isolates from humans, food, and animals [7,22,23], point us to the possibility of interspecies and foodborne transmission of this bacterium. C. difficile is able to form spores, which are well tolerated to harsh environments and disinfectants. It has been shown that the mutant strains that cannot form spores, are also unable to persist in the colonic tract of the host and be horizontally transmitted [24]. Spores are therefore likely to be responsible for dissemination of C. difficile in nosocomial and non-hospital settings [3,4]. Thus, several investigations have been focusing on various approaches to eliminate spore contaminants. One of such approaches includes
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Fig. 3. Vegetative cell growth of C. difficile from spores following microwave irradiation and conductive heating. Spores were microwave irradiated at different time interval and then incubated with BHIS supplemented 0.1% (w/v) ST. Germination and outgrowth were followed at 37 C in an anaerobic environment by measuring changes in OD600 every hour for 24 h. Data points represent the mean of the relative OD600 at the indicated time points normalized to t ¼ 0 (control). All the experiments were performed in triplicates and error bar represents the standard errors. The results include the strains: A) F101; B) F102; C) F103; D) F104; E) A121; F) A122; G) A123; H) A124; I) A125; J) A126; K) 630; L) R20291; M) H203; N) H204; O) H205. Triangle (D) denotes t ¼ 0; circle (B), diamond (◊), cross ( ), square ( ) denotes the microwave treatment for 10s, 20s, 30s, 60s, respectively.
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microwave irradiation, which has been used in sterilization of foods, hospital waste, and medical appliances [8e10]. The penetration capacity of the microwave depends on the materials having low or high loss factor and the type of microwave used. Most household appliances use 2450 MHz microwave as they are smaller and easier to work with, however, deeper penetration into the materials can be achieved with microwave having 915 MHz power generator [25]. It has been shown to inhibit the growth of several microbial pathogens such as Escherichia coli, Streptococcus faecalis, and Staphyloccocus aureus [10,15,16,18], as well as inactivate spores of Bacillus subtilis [9], Bacillus atrophaeus [11], Bacillus licheniformis [13], and Clostridium sporogenes [14]. Microwave radiation generates heat, which is thought to be responsible for the destruction of these pathogens [16]. However, nonthermal effects on microorganisms have also been proposed [17]. It is therefore of interest to investigate the effect of microwave irradiation on C. difficile spore viability, outgrowth, and return to vegetative cells. In this study, the exposure time to microwave irradiation was kept short to minimize non-specific side reactions. Conductive heating at the same-temperature reaction time was also performed as a control for the thermal effect of the microwave. Our results revealed that the colony forming efficiency of microwave irradiated spores was found to dramatically decline relative to an increase in the exposure time and temperature. However, the spores that were conductively heated at the same temperature for the same exposure time, were found viable. These data indicate the existence of nonthermal microwave effect of spore inactivation. Consistently, it has been reported that C. difficile spores retain viability following the heat treatment at 71 C for at least 2 h [26], however the extended heating at 85 C noticeably reduces cell division, but not spore germination [27]. The marked inhibitory effect of 96 C on C. difficile spores has also been documented, but it could not completely abrogate the germination of a population of superdormant spores [27]. Furthermore, the microwave inactivation of C. difficile spores to return to vegetative states was demonstrated. All isolates exhibited substantially different outgrowth rates, reaching late exponential phase at different time frame when the
starting inoculum was standardized. The differences in growth characteristics among C. difficile isolates from humans, food, and animals, and the time taken to reach maximal growth were apparent from strains to strains, regardless of the host origins. Microwave irradiation was shown to inhibit the vegetative cell growth from spores in all isolates and the level of spore inactivation was shown to be exposure time-dependent. Altogether, the stresses generated through microwave irradiation may cause various mechanistic deterioration to the spore internal organization, thus resulting in time taken to retain their viability. This suggests that the microwave power and exposure time for sterilization should be taken into consideration to completely inhibit spore germination and return to vegetative growth, thereby reducing the chance of gut colonization under favorable conditions to initiate CDI. Moreover, in the case of food processing, microwave radiation generates high temperature in a very short time, resulting in nutritional and sensorial advantages compared to heating method. Ultrastructural analysis of treated spores indicated that the spore damage induced by microwave irradiation was significantly different from that attributable to conductive heating. SEM analysis of spore surface structure revealed that the untreated spores possessed smooth surface, while the microwave irradiated or conductively heated spores exhibited disruption on their surfaces. The microwave irradiated spores seemed to undergo spore lysis, which was more pronounced compared to the heating condition, and was related to the exposure time. The effects exerted by microwave radiation on the spore ultrastructure was clearly distinguishable from that due to conductive heating as seen by TEM. An altered organization of intracellular components and aggregation of cytoplasmic proteins became more prominent with longer exposure times. A number of studies have argued that microwave irradiation has identical lethality to bacterial spores as compared to conductive heating at a given temperature [13,14]. Similar results have been also reported on B. subtilis spores; but the damage induced by microwave treatment was intrinsically different from that observed for heating [9]. However, we noticed that the degree of lethality varied among the strains in this study. Microwave
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Fig. 4. Scanning electron micrographs of C. difficile spores following microwave irradiation and conductive heating. The selected isolates included F101, F102, A125, A126, R20291, and H203. Control (untreated) spores displayed ellipsoidal structure with smooth surfaces as shown in the left panel. Spores were treated either with microwave radiation for 30s and 60s, or with 60s of 98 C, and were then subjected to SEM. All the micrographs were taken at a magnification of 11,000. Scale bars represent 2 mm.
irradiation disrupted the spores at the greater extent compared to conductive heating at a given temperature. The damage of C. difficile spores by microwave irradiation was probably attributed to generation of internal pressure in core region, leading to sequential events such as bubbling, protein aggregation, inner membrane fusion, finally resulting in the hydrolysis of spore cortex and spore coat [12]. In contrast, conductive heating generates external pressure so that spores seem to withstand in various degrees [24]. The formation of bubbling indicates an early sign of damage that spores may be able to retain their germination characteristics by an unknown mechanism. The dark spot formation in the core region following microwave and conductive heat treatments could probably be due to the condensation of nucleoprotein complexes within the core region, as advocated for cytoplasmic proteins of vegetative bacterial cells following both treatments [18,28], which may contribute to partial or full spore inactivation. The disruption of
spore inner membrane and release of the core components are possibly correlated with spore viability [29]. Our results suggest that there is a marked difference in ultrastructural alterations following microwave irradiation and conductive heating, which hints us upon the nonthermal influence on spores. In summary, our study thus highlights the possible use of microwave irradiation to inactivate C. difficile spores. Fifteen C. difficile isolates from different host origins exhibiting distinct germination and growth characteristics were found sensitive to microwave treatment, while all were well tolerated to conductive heating at the same exposure time. A combined assessment by SEM and TEM revealed dynamical changes in spore ultrastructure caused by these treatments. The degree of damage caused by microwave irradiation was markedly more pronounced than conductive heating. Taken together, the results herein demonstrate the effectiveness of microwave irradiation against C. difficile spores. Further studies to
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Fig. 5. Transmission electron micrographs of C. difficile spores following microwave irradiation and conductive heating. The selected isolates included F101, F102, A125, A126, R20291, and H203. Control (untreated) spores displayed typical organization of spore layers as shown in the left panel. Spores were treated either with microwave radiation for 30s and 60s, or with 60s of 98 C, and were then subjected to TEM. All the micrographs were taken at a magnification of 19,000. Scale bars represent 200 nm.
understand the mechanism of spore inactivation and to identify the optimal conditions for microwave sterilization will certainly lower the transmission risk of C. difficile. Acknowledgments This work is supported by the Faculty of Science, Mahidol University; and grants from the International Society for Infectious Diseases to TJ, Office of Higher Education Commission and Mahidol University under the National Research Universities Initiative to SC, and a research assistant scholarship from the Faculty of Graduate Studies, Mahidol University to SCO. References [1] S. Johnson, D.N. Gerding, Clostridium difficileeassociated diarrhea, Clin. Infect. Dis. 26 (1998) 1027e1034.
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