Microbial Pathogenesis 82 (2015) 7e14
Contents lists available at ScienceDirect
Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Review
The effect of selected factors on the survival of Bacillus cereus in the human gastrointestinal tract Anna Berthold-Pluta a, *, Antoni Pluta a, Monika Garbowska b a Division of Milk Biotechnology, Department of Biotechnology, Microbiology and Food Evaluation, Faculty of Food Sciences, Warsaw University of Life Sciences e SGGW, Nowoursynowska 159C St, 02-787 Warsaw, Poland b Prof. Wacław Da˛ browski Institute of Agricultural and Food Biotechnology, Inter-Department Problem Group for Dairy Industries, Rakowiecka St 36, 02532 Warsaw, Poland
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 August 2014 Received in revised form 17 October 2014 Accepted 9 March 2015 Available online 17 March 2015
Bacillus cereus is a Gram-positive bacterium widely distributed in soil and vegetation. This bacterial species can also contaminate raw or processed foods. Pathogenic B. cereus strains can cause a range of infections in humans, as well as food poisoning of an emetic (intoxication) or diarrheal type (toxicoinfection). Toxico-infections are due to the action of the Hbl toxin, Nhe toxin, and cytotoxin K produced by the microorganism in the gastrointestinal tract. This occurs once the spores or vegetative B. cereus cells survive the pH barrier of the stomach and reach the small intestine where they produce toxins in sufficient amounts. This article discusses the effect of various factors on the survival of B. cereus in the gastrointestinal tract, including low pH and the presence of digestive enzymes in the stomach, bile salts in the small intestine, and indigenous microflora in the lower parts of the gastrointestinal tract. Additional aspects also reported to affect B. cereus survival and virulence in the gastrointestinal tract include the interaction of the spores and vegetative cells with enterocytes. In vitro studies revealed that both vegetative B. cereus and spores can survive in the gastrointestinal tract suggesting that the biological form of the microorganism may have less influence on the occurrence of the symptoms of infection than was once believed. It is most likely the interaction between the pathogen and enterocytes that is necessary for the diarrheal form of B. cereus food poisoning to develop. The adhesion of B. cereus to the intestinal epithelium allows the bacterium to grow and produce enterotoxins in the proximity of the epithelium. Recent studies suggest that the human intestinal microbiota inhibits the growth of vegetative B. cereus cells considerably. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Bacillus cereus Simulated gastrointestinal conditions Pathogenesis
1. Introduction Bacillus cereus is a Gram-positive spore-forming bacterium commonly found in the environments. This bacterium is also a major contaminant of raw or processed foods of plant or animal origin [1e3]. B. cereus exists as a soil saprophyte that can adapt and proliferate in the lower sections of the gastro-intestinal tract (GIT) [4]. It is also an opportunistic pathogen responsible for local and systemic infections [5]. While certain B. cereus strains have been used as probiotics [6e8], others may cause food poisoning in humans [9]. The pathogenicity of B. cereus is attributed to the species' production of extracellular factors such as phospholipase,
* Corresponding author. E-mail address: [email protected] (A. Berthold-Pluta). http://dx.doi.org/10.1016/j.micpath.2015.03.015 0882-4010/© 2015 Elsevier Ltd. All rights reserved.
cereulide (emetic toxin), enterotoxin Hbl, non-haemolytic toxin (Nhe), haemolysin IV, which has a strong disruptive effect on cellular membranes, and associated with the induction of necrotic enterocolitis cytotoxin (CytK) [10e14]. B. cereus may cause two different types of food poisoning: the emetic or the diarrheal types. The first one is a form of intoxication caused by the ingestion of food containing the toxin cereulide, whereas the diarrheal type of food poisoning depends to a large extent on the ingestion of B. cereus followed by the production of toxins in the human GIT. The estimated infective dose required for causing the diarrheal type of food poisoning ranges from around 105 to 108 B. cereus vegetative cells or spores [9]. Among the factors that most significantly inhibit the survival of B. cereus in the human GIT are the low pH and presence of digestive enzymes (pepsin) in the stomach, oxygen deficiency and presence of bile in the small intestine, and the indigenous microflora in the
8
A. Berthold-Pluta et al. / Microbial Pathogenesis 82 (2015) 7e14
lower part of the GIT. The ability of B. cereus spores/vegetative cells to adhere to enterocytes and the possible interaction between this species' vegetative cells and the intestine epithelial cells are also aspects contributing to the outbreak of B. cereus food poisoning. 2. The effects of low pH and pepsin on B. cereus The pH of an empty stomach is around 2.0 and it rises to 4.5e5.0 during food intake [15]. B. cereus spores and less than about 10% of vegetative cells can survive at these low pH values [16,17]. Consequently, they can reach further parts of the GIT. A shorter time of digestion in the stomach or the presence of protective compounds in food, e.g., fat, can further enhance the tolerance of B. cereus vegetative cells to the low pH of gastric fluid [16,18]. A factor that plays a role in acid resistance is the mechanism of cross-protection between the different stresses microorganisms are exposed to. For example, exposure of the microorganism to the various stresses they encounter in the course of food production, e.g., heat processing, dehydration, or acidification, can elicit a higher tolerance to stresses encountered passing through the stomach. A study of the response of B. cereus to low pH revealed that the strains which were initially incubated for 40 min at pH 6.3, exhibited a marked increase in survival when exposed later to pH 4.6 for 20 min [19]. Indeed, a sublethal acidic environment can trigger an adaptive response that protects the bacterium during subsequent incubations at lethal acidic pH. This mechanism is known as acid tolerance response (ATR) and plays an important role in the adaptation of intestinal pathogens to the pH of the stomach [20e22]. B. cereus vegetative cells are also able to induce ATR [23,24]. The ATR of B. cereus may involve (i) F0F1 ATPase and/or glutamate decarboxylase (implicated in pHi homeostasis), (ii) modifications of metabolism and (iii) synthesis of proteins which act as protect and/or repair factors [24]. The pH homeostasis system helps to maintain the internal pHi of the cell at higher levels than the external pH0 [19]. The mechanism of pHi homeostasis in B. cereus was induced at pH0 below 6.0 [24], similar to other Gram-positive bacteria [25,26]. B. cereus TZ415, initially grown at pH 6.0, 5.5, and 5.0, respectively, resulted in 101-, 103- i 104-fold greater survival of the cells at pH 4.0 when compared with cultures initially grown at a neutral pH [23]. In reaction to low pH levels, the cells increased the expression of genes coding shock proteins and chaperones [19,25]. Increased acid tolerance was related to an increased synthesis of enzymes providing the cell with CO2 (arginine decarboxylase and glutamate decarboxylase) [27]. Protein neosynthesis contributed to an increase in the acid resistance of B. cereus. The addition of 70 mg/ml chloramphenicol, an antibiotic inhibiting bacterial protein synthesis, resulted in a decrease in the acid resistance of B. cereus (pH 4.0), regardless of pre-treatment (pH 5.5) [24]. The growth phase was also shown to affect the survival of B. cereus at low pH. B. cereus vegetative cells in the early and midexponential phase were the most sensitive to acid exposure (at pH 4.6e5.0, survival ~0.02%), whereas in the stationary phase, their resistance rapidly increased (even up to 100% survival) [19,23]. Ceuppens et al. [28] reported similar results in their studies of B. cereus NVH 1230-88 cultivated in a medium at pH 4.0, which simulated the environment of a human stomach after food ingestion. The number of vegetative cells, which were in the exponential phase of growth at the beginning of the study, decreased by about 2 log CFU/mL after 2 h of incubation, whereas the count of vegetative cells in the stationary phase decreased by only 4 log CFU/mL) in the bacteria population was observed irrespective of the nature of the GM. Both the vegetative cells and the spores showed a similar survival in GM-milk at pH > 4.5 [16]. The protective effect of milk can probably be attributed to its content of lipids and proteins, which can “trap” the bacteria by forming protein-lipid complexes. As a result, the bacteria are not directly exposed to the influence of low pH levels. Similar results were obtained in the studies of B. cereus strains isolated from milk [37,39] and from the cases of food poisoning and food contamination [40]. The number of vegetative cells (B. cereus ATCC 14579) in the simulated gastric fluid (SGF), which had a pH 4.4 value and contained fresh cheese, decreased by 3 log CFU/mL during a 60-minute exposure time. In the case of SGF with chicken and rice baby meal at the same pH value and exposure time, the decrease observed in the B. cereus count was higher by over 1 log CFU/mL. The examined strain showed an even lower survival rate when cultivated in the SGF with food and 16% red wine [39]. Similar results were obtained for the B. cereus NVH 1230-88 strain. About 14% (±9%) of the vegetative cells could survive in the medium simulating the stomach environment at pH values > 4.0 [17]. According to the mathematical model developed by Wijnands et al. [18] which describes the survival of vegetative B. cereus cells during passage through the human stomach, 3e12% of ingested cells can survive exposure to simulated gastric conditions in young adults, while 6e26% can survive exposure to simulated gastric conditions in elderly individuals. The results vary depending on the strain and growth phase of the cells. 3. The effect of bile salts and other components of intestinal fluid on B. cereus Once bacteria pass through the acid barrier of the stomach, they encounter another antimicrobial compound in the GIT in the form of bile acids. The concentration of bile salts in the small intestine is not constant. It varies from 0.2 to 2.0% (w/v) depending on the individual properties of a given person and the type, composition, and quantity of the food ingested [41]. The main function of bile is to facilitate the decomposition and absorption of lipids. The constituents of bile are also important to the immune system due to their strong antimicrobial activity [42]. The antimicrobial action of bile depends, among other things, on its concentration. Bile salts at high concentrations can rapidly dissolve membrane lipids and cause the dissociation of integral membrane proteins. They can also cause damage to RNA, DNA, and the enzymes involved in DNA repair [42], resulting in almost instantaneous cell death. Bile salts at low concentrations can disrupt membrane stability and integrity, affect the activity of some
9
enzymes, disrupt the transmembrane flux of cations [43,44], and change cell hydrophobicity and the zeta potential of cells [45]. Bile may bring about the oxidative stress of bacteria through the generation of oxygen free radicals [42,46,47]. Bile acid may also chelate calcium and iron, which, consequently, limits microorganisms' access to these macroelements [48e51]. Despite the strong antimicrobial properties of bile salts, some bacterial species can still tolerate their high concentrations. Generally, Gram-positive bacteria seem to be more sensitive to the activity of bile than Gram-negative bacteria [42]. Like Listeria monocytogenes or Enterococcus faecalis, Gram-positive pathogens survive bile salt concentrations as high as 0.3%, which is comparable to the concentration found in the human small intestine [52,53]. According to the results obtained by Kristoffersen et al. [41], B. cereus spores were not affected by exposure to broth containing 0.1 g/L bile salts, whereas a 100-fold lower concentration inhibited the growth of vegetative B. cereus cells. However, other studies showed that B. cereus spores can germinate and its vegetative cells can grow in conditions with a 1.5 g/L concentration of bile salts [40]. The spores were found to germinate after 2 h in a medium simulating intestinal fluid with a bile concentration of 1.0 g/L [28]. According to Ceuppens et al. [54], bile salt concentrations
Contents lists available at ScienceDirect
Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Review
The effect of selected factors on the survival of Bacillus cereus in the human gastrointestinal tract Anna Berthold-Pluta a, *, Antoni Pluta a, Monika Garbowska b a Division of Milk Biotechnology, Department of Biotechnology, Microbiology and Food Evaluation, Faculty of Food Sciences, Warsaw University of Life Sciences e SGGW, Nowoursynowska 159C St, 02-787 Warsaw, Poland b Prof. Wacław Da˛ browski Institute of Agricultural and Food Biotechnology, Inter-Department Problem Group for Dairy Industries, Rakowiecka St 36, 02532 Warsaw, Poland
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 August 2014 Received in revised form 17 October 2014 Accepted 9 March 2015 Available online 17 March 2015
Bacillus cereus is a Gram-positive bacterium widely distributed in soil and vegetation. This bacterial species can also contaminate raw or processed foods. Pathogenic B. cereus strains can cause a range of infections in humans, as well as food poisoning of an emetic (intoxication) or diarrheal type (toxicoinfection). Toxico-infections are due to the action of the Hbl toxin, Nhe toxin, and cytotoxin K produced by the microorganism in the gastrointestinal tract. This occurs once the spores or vegetative B. cereus cells survive the pH barrier of the stomach and reach the small intestine where they produce toxins in sufficient amounts. This article discusses the effect of various factors on the survival of B. cereus in the gastrointestinal tract, including low pH and the presence of digestive enzymes in the stomach, bile salts in the small intestine, and indigenous microflora in the lower parts of the gastrointestinal tract. Additional aspects also reported to affect B. cereus survival and virulence in the gastrointestinal tract include the interaction of the spores and vegetative cells with enterocytes. In vitro studies revealed that both vegetative B. cereus and spores can survive in the gastrointestinal tract suggesting that the biological form of the microorganism may have less influence on the occurrence of the symptoms of infection than was once believed. It is most likely the interaction between the pathogen and enterocytes that is necessary for the diarrheal form of B. cereus food poisoning to develop. The adhesion of B. cereus to the intestinal epithelium allows the bacterium to grow and produce enterotoxins in the proximity of the epithelium. Recent studies suggest that the human intestinal microbiota inhibits the growth of vegetative B. cereus cells considerably. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Bacillus cereus Simulated gastrointestinal conditions Pathogenesis
1. Introduction Bacillus cereus is a Gram-positive spore-forming bacterium commonly found in the environments. This bacterium is also a major contaminant of raw or processed foods of plant or animal origin [1e3]. B. cereus exists as a soil saprophyte that can adapt and proliferate in the lower sections of the gastro-intestinal tract (GIT) [4]. It is also an opportunistic pathogen responsible for local and systemic infections [5]. While certain B. cereus strains have been used as probiotics [6e8], others may cause food poisoning in humans [9]. The pathogenicity of B. cereus is attributed to the species' production of extracellular factors such as phospholipase,
* Corresponding author. E-mail address: [email protected] (A. Berthold-Pluta). http://dx.doi.org/10.1016/j.micpath.2015.03.015 0882-4010/© 2015 Elsevier Ltd. All rights reserved.
cereulide (emetic toxin), enterotoxin Hbl, non-haemolytic toxin (Nhe), haemolysin IV, which has a strong disruptive effect on cellular membranes, and associated with the induction of necrotic enterocolitis cytotoxin (CytK) [10e14]. B. cereus may cause two different types of food poisoning: the emetic or the diarrheal types. The first one is a form of intoxication caused by the ingestion of food containing the toxin cereulide, whereas the diarrheal type of food poisoning depends to a large extent on the ingestion of B. cereus followed by the production of toxins in the human GIT. The estimated infective dose required for causing the diarrheal type of food poisoning ranges from around 105 to 108 B. cereus vegetative cells or spores [9]. Among the factors that most significantly inhibit the survival of B. cereus in the human GIT are the low pH and presence of digestive enzymes (pepsin) in the stomach, oxygen deficiency and presence of bile in the small intestine, and the indigenous microflora in the
8
A. Berthold-Pluta et al. / Microbial Pathogenesis 82 (2015) 7e14
lower part of the GIT. The ability of B. cereus spores/vegetative cells to adhere to enterocytes and the possible interaction between this species' vegetative cells and the intestine epithelial cells are also aspects contributing to the outbreak of B. cereus food poisoning. 2. The effects of low pH and pepsin on B. cereus The pH of an empty stomach is around 2.0 and it rises to 4.5e5.0 during food intake [15]. B. cereus spores and less than about 10% of vegetative cells can survive at these low pH values [16,17]. Consequently, they can reach further parts of the GIT. A shorter time of digestion in the stomach or the presence of protective compounds in food, e.g., fat, can further enhance the tolerance of B. cereus vegetative cells to the low pH of gastric fluid [16,18]. A factor that plays a role in acid resistance is the mechanism of cross-protection between the different stresses microorganisms are exposed to. For example, exposure of the microorganism to the various stresses they encounter in the course of food production, e.g., heat processing, dehydration, or acidification, can elicit a higher tolerance to stresses encountered passing through the stomach. A study of the response of B. cereus to low pH revealed that the strains which were initially incubated for 40 min at pH 6.3, exhibited a marked increase in survival when exposed later to pH 4.6 for 20 min [19]. Indeed, a sublethal acidic environment can trigger an adaptive response that protects the bacterium during subsequent incubations at lethal acidic pH. This mechanism is known as acid tolerance response (ATR) and plays an important role in the adaptation of intestinal pathogens to the pH of the stomach [20e22]. B. cereus vegetative cells are also able to induce ATR [23,24]. The ATR of B. cereus may involve (i) F0F1 ATPase and/or glutamate decarboxylase (implicated in pHi homeostasis), (ii) modifications of metabolism and (iii) synthesis of proteins which act as protect and/or repair factors [24]. The pH homeostasis system helps to maintain the internal pHi of the cell at higher levels than the external pH0 [19]. The mechanism of pHi homeostasis in B. cereus was induced at pH0 below 6.0 [24], similar to other Gram-positive bacteria [25,26]. B. cereus TZ415, initially grown at pH 6.0, 5.5, and 5.0, respectively, resulted in 101-, 103- i 104-fold greater survival of the cells at pH 4.0 when compared with cultures initially grown at a neutral pH [23]. In reaction to low pH levels, the cells increased the expression of genes coding shock proteins and chaperones [19,25]. Increased acid tolerance was related to an increased synthesis of enzymes providing the cell with CO2 (arginine decarboxylase and glutamate decarboxylase) [27]. Protein neosynthesis contributed to an increase in the acid resistance of B. cereus. The addition of 70 mg/ml chloramphenicol, an antibiotic inhibiting bacterial protein synthesis, resulted in a decrease in the acid resistance of B. cereus (pH 4.0), regardless of pre-treatment (pH 5.5) [24]. The growth phase was also shown to affect the survival of B. cereus at low pH. B. cereus vegetative cells in the early and midexponential phase were the most sensitive to acid exposure (at pH 4.6e5.0, survival ~0.02%), whereas in the stationary phase, their resistance rapidly increased (even up to 100% survival) [19,23]. Ceuppens et al. [28] reported similar results in their studies of B. cereus NVH 1230-88 cultivated in a medium at pH 4.0, which simulated the environment of a human stomach after food ingestion. The number of vegetative cells, which were in the exponential phase of growth at the beginning of the study, decreased by about 2 log CFU/mL after 2 h of incubation, whereas the count of vegetative cells in the stationary phase decreased by only 4 log CFU/mL) in the bacteria population was observed irrespective of the nature of the GM. Both the vegetative cells and the spores showed a similar survival in GM-milk at pH > 4.5 [16]. The protective effect of milk can probably be attributed to its content of lipids and proteins, which can “trap” the bacteria by forming protein-lipid complexes. As a result, the bacteria are not directly exposed to the influence of low pH levels. Similar results were obtained in the studies of B. cereus strains isolated from milk [37,39] and from the cases of food poisoning and food contamination [40]. The number of vegetative cells (B. cereus ATCC 14579) in the simulated gastric fluid (SGF), which had a pH 4.4 value and contained fresh cheese, decreased by 3 log CFU/mL during a 60-minute exposure time. In the case of SGF with chicken and rice baby meal at the same pH value and exposure time, the decrease observed in the B. cereus count was higher by over 1 log CFU/mL. The examined strain showed an even lower survival rate when cultivated in the SGF with food and 16% red wine [39]. Similar results were obtained for the B. cereus NVH 1230-88 strain. About 14% (±9%) of the vegetative cells could survive in the medium simulating the stomach environment at pH values > 4.0 [17]. According to the mathematical model developed by Wijnands et al. [18] which describes the survival of vegetative B. cereus cells during passage through the human stomach, 3e12% of ingested cells can survive exposure to simulated gastric conditions in young adults, while 6e26% can survive exposure to simulated gastric conditions in elderly individuals. The results vary depending on the strain and growth phase of the cells. 3. The effect of bile salts and other components of intestinal fluid on B. cereus Once bacteria pass through the acid barrier of the stomach, they encounter another antimicrobial compound in the GIT in the form of bile acids. The concentration of bile salts in the small intestine is not constant. It varies from 0.2 to 2.0% (w/v) depending on the individual properties of a given person and the type, composition, and quantity of the food ingested [41]. The main function of bile is to facilitate the decomposition and absorption of lipids. The constituents of bile are also important to the immune system due to their strong antimicrobial activity [42]. The antimicrobial action of bile depends, among other things, on its concentration. Bile salts at high concentrations can rapidly dissolve membrane lipids and cause the dissociation of integral membrane proteins. They can also cause damage to RNA, DNA, and the enzymes involved in DNA repair [42], resulting in almost instantaneous cell death. Bile salts at low concentrations can disrupt membrane stability and integrity, affect the activity of some
9
enzymes, disrupt the transmembrane flux of cations [43,44], and change cell hydrophobicity and the zeta potential of cells [45]. Bile may bring about the oxidative stress of bacteria through the generation of oxygen free radicals [42,46,47]. Bile acid may also chelate calcium and iron, which, consequently, limits microorganisms' access to these macroelements [48e51]. Despite the strong antimicrobial properties of bile salts, some bacterial species can still tolerate their high concentrations. Generally, Gram-positive bacteria seem to be more sensitive to the activity of bile than Gram-negative bacteria [42]. Like Listeria monocytogenes or Enterococcus faecalis, Gram-positive pathogens survive bile salt concentrations as high as 0.3%, which is comparable to the concentration found in the human small intestine [52,53]. According to the results obtained by Kristoffersen et al. [41], B. cereus spores were not affected by exposure to broth containing 0.1 g/L bile salts, whereas a 100-fold lower concentration inhibited the growth of vegetative B. cereus cells. However, other studies showed that B. cereus spores can germinate and its vegetative cells can grow in conditions with a 1.5 g/L concentration of bile salts [40]. The spores were found to germinate after 2 h in a medium simulating intestinal fluid with a bile concentration of 1.0 g/L [28]. According to Ceuppens et al. [54], bile salt concentrations
