Type E Botulinum Neurotoxin-Producing Clostridium butyricum Strains Are Aerotolerant during Vegetative Growth

Botulinum neurotoxins, the causative agents of the potentially fatal disease of botulism, are produced by certain Clostridium strains during vegetative growth, usually in anaerobic environments. Our findings indicate that, contrary to current understanding, the growth of neurotoxigenic C. butyricum strains and botulinum neurotoxin type E production can continue upon transfer from anaerobic to aerated conditions and that adaptation of strains to oxygenated environments requires global changes in proteomic and metabolic profiles. We hypothesize that aerotolerance might constitute an unappreciated factor conferring physiological advantages on some botulinum toxin-producing clostridial strains, allowing them to adapt to otherwise restrictive environments.

However, while the germination of clostridial spores is rare in the presence of low oxygen concentrations, evidence suggests that vegetative cells may display considerable ability to tolerate oxygen (2,3).
Oxygen tolerance among clostridia has been attributed to the enzymatic ability of strains to consume oxygen from the medium and to defend themselves against the toxic effects of the reactive oxygen species (ROS) (2,4,16). Recently, a strategy based on the synthesis of aromatic polyketides (clostrubins) has been proposed for the plant pathogen C. puniceum to survive and grow in aerated environments; nonetheless, the antioxidant role of clostrubins has not been determined (9).
Certain clostridial strains produce the botulinum neurotoxin (BoNT): this protein toxin usually causes severe paralysis in humans when it is synthesized by BoNTproducing clostridia in the colonized intestine, especially in infants younger than 1 year (infant botulism); in infected wounds (wound botulism); or in contaminated food products before consumption (foodborne botulism) (17). Oxygen may not be completely absent from these environments. The newborn intestine is known to be aerobic until it is made anaerobic by oxygen-reducing aerobes (18), wounds come in contact with ambient air, and even the contaminated food products at risk of botulism may be subject to air infiltration. Therefore, studying the responses of BoNT-producing clostridia to oxygen exposure, especially in the vegetative growth phase when they produce BoNT, is imperative for better understanding the within-host dynamics and implementing food safety control measures.
Type E BoNT (BoNT/E), i.e., one of the antigenically different BoNT types causing human botulism, is usually synthesized by C. botulinum type E strains but can also be produced by atypical neurotoxigenic C. butyricum type E strains (17). In Italy, where neurotoxigenic C. butyricum type E strains were first isolated from infants with botulism and then repeatedly recovered from cases of human botulism, these strains appear to be clinically more relevant than C. botulinum type E strains (19). Moreover, neurotoxigenic C. butyricum type E strains have been associated with human botulism in Asia, the United Kingdom, and the United States, contributing to the reemergence of this microorganism as a causative agent of botulism (20)(21)(22)(23).
Although it has been reported that nonneurotoxigenic C. butyricum strains grow in oxygen-containing environments, little is known about the behavior of neurotoxigenic C. butyricum type E strains upon oxygen exposure. The present study aimed to investigate the effects of atmospheric oxygen exposure on the vegetative growth of neurotoxigenic C. butyricum type E strains and BoNT/E production and characterize the strategic defense mechanisms adopted by these microorganisms upon air exposure.

RESULTS
Effects of air exposure on the vegetative growth of neurotoxigenic C. butyricum type E strains and BoNT/E production. The broth cultures of the C. butyricum type E strains ISS-21 and ISS-190 in the mid-exponential-growth phase were analyzed in parallel during a 5-h incubation either under anaerobic (AN) conditions or following ambient air exposure (aerated [AE] conditions).
At the end of the experiments, the average concentrations of dissolved oxygen in AE cultures were 5.2 Ϯ 1 ppm, whereas oxygen was not detectable in AN cultures. The average pH was 5.35 Ϯ 0.15 in both AN and AE cultures.
The two strains exhibited similar OD 600 growth curves under AN and AE conditions over the 5-h culture period, with an overall ϳ2-fold increase in the OD 600 values and no significant differences between each tested time point (Fig. 1A and B). As the OD 600 values measure the turbidity of bacterial suspensions regardless of cell viability, the viable cells in the starting (mid-exponential-growth-phase) cultures and final cultures in AN and AE environments were counted. The average cell count of strains ISS-190 and ISS-21 in the starting cultures was ϳ10 3 and 10 4 cells/ml, respectively ( Fig. 1A and B). After 5 h of incubation under AN conditions, the average viable cell count in strains ISS-190 and ISS-21 was 9.4 ϫ 10 5 cells/ml and 1.5 ϫ 10 7 cells/ml, respectively; in contrast, the average viable cell count in strains ISS-190 and ISS-21 after a 5-h incubation under AE conditions was 6.5 ϫ 10 4 and 1.2 ϫ 10 6 cells/ml, respectively. Therefore, the number of viable cells at the end of the experiments was higher in AN cultures than in AE cultures, and the difference in the average viable cell count under AN and AE conditions was significant only for strain ISS-21 (P Ͻ 0.05) ( Fig. 1A and B). In addition, the average cell count at the end of the experiments was significantly higher in strain ISS-21 than in strain ISS-190 in the AN environment, whereas there was no statistically significant difference between the strains in the AE environment.
Of note is that, at the end of the growth period, gas bubbles were visible in strains cultivated under AN conditions but were not visible in strains grown under AE conditions (Fig. 1C). Importantly, no spores were detected by staining or heat shock in both neurotoxigenic C. butyricum type E strains in the AE environment (data not shown).
Since it is known that bacteria respond to changes in environmental conditions in a cell-density-dependent manner (24), we determined the minimum initial bacterial density necessary to promote growth under AE conditions. The results indicated that   were generated by measuring the OD 600 values. The number of viable cells (symbols) was determined in the starting (mid-exponential-phase) broth cultures and after a 5-h incubation under AN or AE conditions. (C) At the end of the experiments, foam was visible in the AN broth cultures but not in the AE broth cultures. (D) Net growth of the C. butyricum type E strains ISS-21 and ISS-190 at different initial bacterial densities after a 5-h incubation under AN or AE conditions. Under AE conditions, net growth was observed only when the initial densities were Ͼ10 3 cells/ml. The errors were calculated by determining the standard deviation from the mean for three independent experiments for each strain. *, P Ͻ 0.05 according to Student's t test.
both C. butyricum type E strains could grow under AE conditions when the initial density was at least 10 3 cells/ml of bacteria growing exponentially (Fig. 1D).
With regard to BoNT/E production, BoNT/E protein and toxicity levels were measured in the starting cultures of both strains and in the cultures maintained in the AN environment for 24 h (when the BoNT/E levels should be highest) (25) or incubated in the AE environment for the same time. As expected, BoNT/E protein levels determined by an immuno-ELISA were low in the starting cultures, with no significant differences between the two strains (Fig. 2). The production of BoNT/E protein was increased approximately 58-fold and 36-fold in strains ISS-21 and ISS-190, respectively, after culturing under AN conditions for 24 h and approximately 25-fold and 20-fold in the same strains under AE conditions for 24 h. Therefore, the increase in BoNT/E protein levels in the AE environment was significantly lower than in the AN environment for both strains (P Ͻ 0.05). Furthermore, while the BoNT/E protein levels were significantly higher in strain ISS-21 than in strain ISS-190 under AN conditions for 24 h (P Ͻ 0.05), the BoNT/E protein levels were similar between the two strains under AE conditions for 24 h (Fig. 2). The BoNT/E toxicity levels measured using a mouse bioassay were increased 32-fold and 8-fold in strains ISS-21 and ISS-190 cultured under AN conditions for 24 h, respectively, and 4-fold and 2-fold in the same strains under AE conditions for 24 h, respectively (Fig. 2). The increase in BoNT/E protein and toxicity levels was more evident in strain ISS-21 than in strain ISS-190, especially in the AN environment, and this result is consistent with the significantly higher growth rate of the former strain under these culture conditions.
Effects of air exposure on protein expression. As C. butyricum type E strain ISS-190 appeared less affected by air exposure than strain ISS-21, considering the more similar growth characteristics and BoNT/E production under AE and AN conditions, strain ISS-190 was selected for the comparative proteomics analysis. The C. butyricum type E strains ISS-190 and ISS-21 are clonally related, with most of the genetic diversity between the strains consisting of an ϳ168-kb genetic region that is present in the ISS-190 genome but missing from the ISS-21 genome (26).
To identify differentially expressed proteins (DEPs) following air exposure, proteins from cells grown under AN or AE conditions were analyzed by proteomic analysis. A total of 953 proteins were detected; however, only 598 proteins were consistently identified in at least 3 of 5 replicates and were therefore selected for further analyses (see Table S1 in the supplemental material). Among them, 8 and 11 proteins were uniquely expressed in either the AE or AN environment, whereas 579 were identified in both environments and were subjected to quantitative analysis. Of these, 76 proteins were upregulated and 24 were downregulated under AE conditions compared to AN conditions (Fig. 3). The analysis of proteins modulated by air exposure indicated that protein-protein interactions were enriched (P value of 6.3eϪ10), suggesting that specific protein complexes and/or networks were likely affected by oxygen (Fig. S1).
The DEPs were categorized by function (Table 1 and Fig. 4). The results indicate that the modulation of biological processes depends on the AE/AN growth conditions (Fig. 5). The most affected biological processes were membrane transport, redox homeostasis, carbohydrate metabolism, sulfur metabolism, and protein translation. The expression of ribosomal proteins was decreased under AE conditions. On the other hand, proteins involved in cell redox homeostasis, antioxidant defense, and sulfur metabolism and some proteins involved in the transport of solutes across membranes, particularly sugars and proteins, were overexpressed after air exposure. Similarly, several proteins involved in DNA damage responses and flagellum-associated proteins were more abundant under AE conditions.
With respect to carbohydrate metabolism, some proteins related to polysaccharide catabolic processes were upregulated in the AE environment, whereas three enzymes responsible for converting acetoacetyl-CoA to butyryl-CoA (3-hydroxybutyryl-CoA dehydrogenase, acyl-CoA dehydrogenase, and electron transfer flavoprotein subunit beta) were downregulated in the AE environment. Of note, the pyruvate formate lyase (PFL)-activating protein was also decreased in the AE environment.
Effects of air exposure on extracellular metabolism evaluated by 1 H NMR spectroscopy. Nuclear magnetic resonance (NMR) spectroscopy was used to identify and quantify fermentation products in the extracellular medium for both strains after a 5-h incubation under either AN or AE conditions (Fig. 6).
The formate levels were significantly lower (P ϭ 0.004) in the AE extracellular medium than in the AN extracellular medium, whereas the acetate content was significantly higher in the AE medium than in the AN medium (P ϭ 0.003). Moreover, there was a significant increase in the acetate/butyrate ratio (P ϭ 0.004) in the media of both strains under AE conditions compared to the strains grown under AN conditions. There were no other significant differences in the levels of metabolites linked to pyruvate metabolism (lactate, ethanol, and alanine).

DISCUSSION
Anaerobically cultured C. butyricum type E strains continued to grow and produce BoNT/E in liquid medium during the transition from anaerobic to aerated conditions,     (27) but is not the oxygen-free conditions required for vegetative growth of anaerobic clostridia. While the OD 600 data indicated similar growth rates under AN and AE conditions over 5 h, viable cell counts were lower in the AE environment than in the AN environment at the end of the culture period, suggesting that bacterial growth was decreased under the stress conditions of aeration compared to the ideal conditions of anaerobiosis. The lack of complete correlation between OD and viable cell count data under stress conditions has been reported for other bacteria (28) and may be because OD values are affected by light scattering due to cell debris and stress-induced bacterial cell damage.
C. butyricum type E strains continued to grow upon air exposure only when the starting exponentially growing cultures contained at least 10 3 cells/ml. The absence of growth at lower initial bacterial densities could be a result of either oxygen toxicity or decreased cell-to-cell signaling (24). The absence of spores in the cultures of C. butyricum type E strains after a 5-h aeration indicates that sporulation did not occur in a FC is reported as log 2 AE/AN ratio, as described in Materials and Methods. * indicates proteins for which FC has not been calculated because they have been detected only in aerobic or anaerobic conditions. b Notes column reports some specific relevant functional protein features discussed in the text.
the evaluated period and that aerotolerance was not due to spore formation. Sporulation in clostridia has been reported to be strain dependent (29). Of note, our results indicated that even BoNT/E production was maintained during the transition from AN to AE conditions. BoNT/E protein and toxicity levels were increased in the cultures transferred to AE conditions for 24 h, although the levels were significantly lower than those in cultures maintained under AN conditions for the same period. The significantly lower BoNT/E protein and toxicity levels detected in the AE environment than those in the AN environment can be attributed to the decreased growth rates under the former conditions. Moreover, our proteomics analysis revealed that the abundance of several ribosomal proteins was lower under AE conditions,  evidencing that the overall protein synthesis could be downregulated in the presence of oxygen, which is a general adaptive response to acute stress in bacteria (30). Additional effects due to downregulation of BoNT/E synthesis under AE conditions cannot be excluded. Little is known on the toxin synthesis regulation in BoNT/Eproducing clostridial strains. We recently hypothesized that production of BoNT/E in C. butyricum type E strains might be controlled at the posttranscriptional and/or posttranslational levels (i.e., protein folding, secretion, and degradation) (31); accordingly, since oxygen is known to modify the structure and function of proteins, BoNT/E inactivation by oxidative damage could be expected (32). Recently, it was shown that BoNT/E expression in C. botulinum type E strains is positively regulated by the sporulation regulator Spo0A (33); our proteomics results showed similar Spo0A protein levels in the C. butyricum type E broth cultures incubated for 5 h under AN and under AE conditions (see Table S1 in the supplemental material). The absence of significant differences in the BoNT/E protein levels under AN and AE conditions in the proteomics analysis may be due to only cellular proteins being analyzed whereas BoNT/E protein is an exotoxin secreted by clostridia in the medium (34). Nevertheless, cellular levels of the protein P-47 were decreased under AE conditions (Table 1); this protein is encoded in the bont/e toxin gene cluster and coexpressed with BoNT/E, although the two proteins might not be secreted together (35).
Our proteomics analyses indicated that membrane transport was one of the most affected processes under AE conditions: many proteins overexpressed in AE contain or interact with ATP-binding cassettes (ABC transporters) and are involved in transporting solutes across membranes. Other overexpressed proteins are involved in sugar transport, especially five proteins (Table 1) from the phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS), which is a major mechanism used by bacteria for carbohydrate uptake and conversion into phosphoesters during transport (36). Moreover, the proteomic analysis indicated that acetylmuramoyl-L-alanine amidase and cell wall hydrolase, which are involved in peptidoglycan catabolism, were upregulated under AE conditions.
Other proteins involved in protein transport, including SecD and SecF, which are members of the Sec protein translocase complex, were overexpressed in the AE environment. These results indicate that molecular trafficking through the cellular membrane and carbohydrate metabolism are enhanced under AE conditions, suggesting that energy requirements are higher.
Aeration also induced the expression of proteins involved in redox homeostasis, including NADH oxidase, rubrerythrin, peroxiredoxin, and thioredoxin reductase. In this respect, Kawasaki et al. (4) detected the activity of NAD(P)H peroxidase and superoxide dismutase (SOD) in nonneurotoxigenic C. butyricum strains exposed to air. It is known that rubrerythrin has a strong NAD(P)H peroxidase activity (10). Moreover, enzymes homologous to peroxiredoxin and thioredoxin reductase prevent the inactivation of manganese-SOD (Mn-SOD) of Saccharomyces cerevisiae under oxidative stress, contributing to the antioxidant defense in yeast (37). Since an Mn-SOD gene is carried in the genome of three closely related neurotoxigenic C. butyricum type E strains, we speculate that peroxiredoxin and thioredoxin reductase in these strains may protect Mn-SOD.
The proteomic analysis indicated that proteins involved in sulfur metabolism were upregulated under AE conditions, including two subunits of the bifunctional enzyme CysN/CysC sulfate adenylyltransferase and sulfite reductase: these enzymes are involved in cysteine production, and this sulfur-containing amino acid strongly inhibits toxin production in C. difficile (38). Furthermore, several cysteine-containing proteins, often required for protein folding or involved in cellular response to oxidative stress, and (Fe-S) cluster-containing proteins are sensitive to oxidization, and many of these proteins were overexpressed under AE conditions (Table 1), suggesting the need to restore the pool of these proteins in their active state. In contrast, the PFL-activating enzyme, an oxygen-sensitive Fe-S binding protein, responsible for the conversion of pyruvate into acetyl-CoA and formate in anaerobic metabolism (Fig. 7), was downregulated under AE conditions. In accordance, the formate content was significantly lower in the supernatants from aerated C. butyricum type E cells than in those from anaerobic cells. Formate is ultimately converted to carbon dioxide and hydrogen in the metabolic pathways of clostridial species (Fig. 7). Therefore, the lower levels of formate resulting from the decreased expression of the PFL-activating enzyme, together with the upregulation of hydrogen-consuming enzymes, may partially explain the decreased formation of gas in the cultures upon aeration.
Furthermore, the proteomics analysis showed that three enzymes of the butyrate metabolic pathway were downregulated under AE conditions, suggesting that oxygen induced a decrease in butyrate biosynthesis. In line with this result, butyrate content was decreased, although not significantly, in the aerated supernatants compared to nonaerated supernatants; concomitantly, the acetate levels were significantly higher in the aerated supernatants, suggesting that oxygen caused a shift in electron flow toward acetate formation instead of butyrate formation in these C. butyricum type E strains. The acetate/butyrate ratio in nonneurotoxigenic C. butyricum strains is increased as the hydrogen partial pressure is decreased in the medium (39). Moreover, the higher levels of several proteins putatively involved in DNA replication and repair and nucleotide biosynthesis and flagellum-related proteins under AE conditions suggest that the DNA damage response may be activated and proton-driven bacterial motility enhanced. The improved bacterial motility may increase proton consumption and consequently decrease the generation of ROS (40). Furthermore, our finding that a protein involved in an early stage of sporulation was down-expressed in the AE environment is consistent with the absence of spores in C. butyricum type E cultures after a 5-h exposure to ambient air.
In conclusion, the enhanced aerotolerance of neurotoxigenic C. butyricum type E strains that we report here may have public health significance. First, it may increase the opportunities for these microorganisms to colonize the newborn intestine, because the intestine at birth is aerobic and gradually becomes anaerobic (18); in addition, the ability to inactivate the ROS generated by inflammatory processes in the gut may be advantageous to intestine-colonizing neurotoxigenic C. butyricum type E strains. To date, most neurotoxigenic C. butyricum type E strains have been involved in infant intestinal toxemia botulism (21-23, 41, 42). It is of interest that Cassir et al. (43) recently found a significant association between oxidized gut environment and the presence of cytotoxic (nonneurotoxigenic) C. butyricum strains in preterm neonates with necrotizing enterocolitis. Furthermore, the studies on aerotolerance may improve the isolation and identification of BoNT-producing clostridia, considering that other clostridial strains have been misidentified because of their aerotolerance features (11).
To our knowledge, this study is the first to demonstrate vegetative growth and toxin production upon air exposure for BoNT-producing clostridial strains. The results point to the need for further research on the aerotolerance of other BoNT-producing clostridial strains, especially C. botulinum strains, which are more frequently involved in botulism.

MATERIALS AND METHODS
Bacterial strains and growth conditions. Two neurotoxigenic C. butyricum type E strains (ISS-21 and ISS-190) isolated from distinct infant botulism cases in Italy were used in this study (41,42). The effect of atmospheric oxygen on the vegetative growth of these strains was analyzed by using a culture approach similar to that used by other authors for similar purposes (6,14,15), with modifications. Tryptone-peptone-glucose-yeast extract (TPGY) broth (pH 7.0) (Oxoid) without the addition of reducing agents was used to grow the strains. Clostridial growth was monitored by measuring the optical densities of the cell suspensions at 600 nm (OD 600 ) at regular time intervals (Biophotometer; Eppendorf). The strains were grown to the mid-exponential phase (OD 600 of ϳ1) at 37°C in a jar with an anaerobic gas generator (Anaerogen; Oxoid) (starting cultures). Then, 50% of the exponentially growing cultures were transferred to flasks (flask-to-medium ratio, 1:10) and incubated in ambient air (aerated or AE conditions) in an incubator shaker (New Brunswick Scientific; model G25) at 200 rpm and 37°C for 5 h; the remaining volumes of the starting cultures were overlaid with sterile Vaseline oil to produce oxygen-depleted (anaerobic or AN) conditions and were incubated at 37°C for 5 h. The experimental time of 5 h was empirically chosen in order to assay the differences between the vegetative cultures under the AN and AE conditions before sporulation occurred. Growth curves were generated using the OD 600 values. These experiments were repeated three times.
The AE cultures were streaked on nonselective tryptone soy agar (TSA) plates, and the plates were incubated at 37°C for 24 h to detect contamination with aerobic bacteria at the end of culturing. To assess the presence of spores, slides from the AE cultures were examined microscopically; in addition, aliquots of the AE cultures grown for 5 h were treated at 70°C for 10 min and streaked on egg yolk agar plates, and bacterial growth was assessed after a 48-h incubation at 37°C under anaerobiosis. The pH values of the cultures were determined using a pH meter (Mettler Toledo). The dissolved oxygen levels were measured using a dissolved oxygen meter (model HI9146; Hanna Instruments). Viable bacteria were counted using the three-tube most probable number (MPN) method as previously described (31). The average number of viable cells was measured by combining the data from three separate experiments.
Student's t test was used to perform all pairwise comparisons between OD 600 values or viable cell counts under AN and AE conditions. All calculations were performed using GraphPad Prism software version 6 (GraphPad Software, San Diego, CA), and P values Ͻ 0.05 were considered statistically significant.