E Coli O157 Lactobacillus Spray Feed
Abstract
This study investigated the protective effects of feeding the immunoenhancing probiotic Lactobacillus rhamnosus HN001 against Escherichia coli O157:H7 infection in murine (BALB/c and C57BL/6 mice) challenge infection models. Mice were fed milk-based diets supplemented with L. rhamnosus HN001 (3×108 cfu g−1) for 7 days prior to and following oral challenge with E. coli O157:H7. Morbidity and feed intake were measured for 1 week following challenge; pathogen translocation to spleen, liver and blood, and humoral and cellular immunological responses (specific antibody and phagocytosis) were measured in a sub-sample of ostensibly healthy animals 1 week post-challenge. Results showed that, after challenge, L. rhamnosus HN001-fed mice exhibited lower cumulative morbidity and bacterial translocation rates, compared to non-probiotic-fed control mice. Significantly higher intestinal anti-E. coli IgA responses and blood leucocyte phagocytic activity were recorded among probiotic-fed mice compared to controls. These results demonstrate that feeding the probiotic L. rhamnosus HN001 to mice can reduce the severity of E. coli O157:H7 infection, and suggest that this reduction may be associated with enhanced humoral and cellular immune responses.
1 Introduction
Enteric bacterial pathogens represent a major cause of gastrointestinal disease worldwide. Current measures to control gastrointestinal infections rely heavily on the use of antimicrobial chemotherapeutic and chemoprophylactic agents. However, widespread use of antibiotics in public health is discouraged due to complications including the emergence of drug-resistant strains and the potential for chronic toxicity [1]. There is, therefore, a desire to develop alternative, non-pharmaceutical strategies for controlling gastrointestinal bacterial infection.
It has long been acknowledged that fermented milk-based diets, such as yogurt, can confer enhanced resistance against infection with enteric pathogens to individuals [2,3]. Enhanced resistance is thought to be effected by the presence of probiotic lactic acid bacteria (LAB) [2]. Potential mechanisms to explain the enhanced resistance conferred by antimicrobial LAB, include inter-microbial competition with pathogens for intestinal attachment sites, production of substances (biocins) that are directly microbicidal for pathogens [4], and stimulation of host immune function [3,5]. Several studies have shown that certain LAB strains are capable of enhancing host immunity and conferring protection against enteric pathogens in both animal and human studies [6–11]. Therefore, certain LAB strains may be useful dietary supplement, for combating enteric pathogens in humans.
Recent research in our laboratory has identified new strains of probiotic LAB (Bifidobacterium lactis HN019 and Lactobacillus rhamnosus HN001) with immune-enhancing capabilities in mice and humans [12–19]. Our studies also demonstrated that these two probiotic strains have potent anti-infection properties, which are significantly associated with enhanced humoral and cellular immune responses (such as microbe-specific intestinal IgA and blood leucocyte phagocytosis responses) [17–19]. The present study was designed to determine whether L. rhamnosus HN001 has protective effects against another significant intestinal pathogen, Escherichia coli O157:H7. This organism is recognised as an important food-borne pathogen inducing haemorrhagic colitis, hemolytic uremic syndrome, and/or diarrhoea in humans and animals [1,20–24]. The results of this trial indicate that L. rhamnosus HN001 is able to protect mice against E. coli O157:H7.
2 Materials and methods
2.1 Microorganisms
L. rhamnosus HN001 (DR20™), originally isolated from yoghurt and maintained as lyophilised seed stock at the New Zealand Dairy Research Institute (Palmerston North, New Zealand) was supplied as a freeze-dried culture. For feeding, L. rhamnosus HN001 was mixed in a skim milk powder (SMP)-based diet prepared by mixing the dry culture to the desired concentration (3×108 cfu g−1).
E. coli O157:H7 strain 2988, obtained from New Zealand Reference Culture Collection (Communicable Disease Centre), was grown overnight in a brain heart infusion broth (BHI, Difco) at 37°C, washed in sterile phosphate-buffered saline (PBS) and resuspended to the desired concentration (109 cfu ml−1).
2.2 Experimental procedures and measurements
Six- to 8-week-old BALB/c and C57BL/6 male mice were housed in pairs at a controlled temperature (22±2°C) with a 12-h light/dark cycle. Animals were fed standard mouse chow ad libitum with free access to water at all times. For an acclimatisation period of 7 days, prior to commencement of feeding experiments, the mice were fed ad libitum on a SMP-based diet (replenished daily). The SMP-based diet contained SMP (53%), corn oil (8%), vitamin (5%), minerals (5%), corn flour (28%), and cellulose (1%) with a total aerobic microorganism count<104 cfu g−1 (no coliforms, salmonella, or coagulase positive staphylococci was found). Mice were randomly allocated into two groups (L. rhamnosus group and control group). Forty control mice (20 BALB/c mice, and 20 C57 mice) continued to be fed the SMP-based diet as before, while 44 mice in the L. rhamnosus group (24 BALB/c mice, and 20 C57 mice) were fed the SMP-based diet containing L. rhamnosus HN001 (3×108 cfu g−1). After 7 days on test diets, animals were challenged orally with E. coli O157:H7 suspension [0.1 ml/mouse (108 cfu), administered intragastrically]. Mice were maintained on the HN001-supplemented diet following challenge until the end of experiment. Throughout the experiment, feed intake was assessed daily by measuring the remaining (unconsumed) food.
The health appearances (ostensibly normal/abnormal) of all mice were carefully monitored twice daily over the period of experimentation. Morbidity recordings were blinded during the first 6 days post-challenge; group allocations were made known on day 7 for the purpose of collection of blood, intestinal fluid and tissue samples. The criteria used for normal and abnormal appearance were: (1) Normal appearance: mouse bright-eyed and alert, has a smooth coat with a sheen, responds to stimulus, shows interest in its environment and without diarrhoea. (2) Abnormal appearance: fur ruffled, a loss of sheen to the coat, less alert or active, and less interested in environment outside of cage, signs of hyperventilating when handled, hunched over and lethargic, non-reactive to stimulus, agitated or displaying diarrhoea. Morbidity was calculated based on the relative proportion of animals with abnormal appearance in each group. It was pre-determined that, after challenge with E. coli O157:H7, animals would be withdrawn from the trial and killed by isofluorane overdose for reasons of welfare if terminal morbidity became apparent, as gauged by the following criteria: mouse non-reactive to stimulus, fur has a 'bottle brush' appearance, mouse hunched over preferring to sleep than react to environment.
At the end of the trial (1 week post-challenge), twenty mice (10 from each group; 5 BALB/c and five C57 mice) were randomly selected for measuring the translocation of E. coli O157 to blood, spleen and liver, and for determining specific and non-specific immune responses. Mice were killed by isofluorane overdose. Approximately 1 ml of blood was withdrawn via cardiac puncture, and used for assessing bacterial translocation and leukocyte phagocytic activity. The spleen and liver were removed from each mouse aseptically to assess bacterial translocation. The small intestine was recovered, and the contents flushed with 1 ml PBS; particulate material was removed by centrifugation and the remaining supernatant fluid was used to measure mucosal antibody response (Ig A and IgG) to E. coli.
2.3 Culture of E. coli O157:H7 from blood, spleen and liver
Immediately after sampling, the liver and spleen of each mouse were homogenised individually in 0.1% peptone water. The tissue homogenates were serially diluted in peptone water, and then plated in triplicate on E. coli O157 selective agar (CHROMagar O157, Fort Richard Laboratories Ltd, New Zealand). After incubation at 37°C for 48 h, the colonies on agar were enumerated. Blood samples were cultured in BHI broth overnight and then plated on the E. coli O157 selective agar. Representative colonies from each plate were examined for the presence of 0157 antigen using E. coli O antiserum O157 and E. coli H antiserum H7.
2.4 Phagocytosis assays
Phagocytosis was assessed via flow cytometric analysis of the uptake of fluoresceinated formalin-killed E. coli by blood leucocytes, as described by Gill et al. [14]. Results were expressed as the phagocytic capacity, i.e. the proportion (percentage) of phagocytically active cells in each sample.
2.5 Enzyme-linked immunosorbent assay (ELISA) for measurement of antibody
The assays for determining antibody titres were similar to those described by Shu et al. [17,25]. Antigen binding onto 96-well ELISA plates utilised whole formalin-killed E. coli O157:H7 (5×107 cells ml−1) in 100 µl carbonate coating buffer (pH 9.6). Anti-E. coli antibody responses were assessed in serially diluted samples of intestinal fluid in triplicate wells; antibody binding was visualised using alkaline phosphatase conjugated sheep anti-mouse immunoglobulin (IgA or IgG) (Serotec, UK) and an alkaline phosphatase substrate (Bio-Rad Laboratories, CA, USA). Results were read at 405 nm on an ELISA reader (CERES 900, Bio-Tec Instrument Inc., USA), and titre end-point calculated as highest titration OD > the mean plus 2 standard deviations of control intestinal fluid (derived from mice that had not been challenged with E. coli).
2.6 Statistical analyses
Differences in morbidity, feed intake, phagocytic activity, and antibody titres between L. rhamnosus HN001-treated and control groups were analysed using ANOVA [SAS(r) Proprietary Software Version 8, SAS Institute Inc., Cary, NC, USA]. The morbidity was analysed by using SAS proc lifetest. The feed intake of mice during the week prior to challenge was used as a covariate for the analysis of post-challenge feed intake. Antibody titre end-points were transformed by log10 prior to statistical analysis.
3 Results
3.1 Effect of L. rhamnosus HN001 treatment on morbidity
Both BALB/c and C57 mice which had been fed a diet containing L. rhamnosus HN001 showed lower cumulative morbidity rates following infection with E. coli O157:H7 in comparison to control group mice (P=0.06). There was no statistical difference in the morbidity between the two strains of mice. The overall morbidity in the L. rhamnosus HN001-fed and control groups is summarised in Fig. 1.
Figure 1
Cumulative morbidity of E. coli O157 H:7-challenged mice in the L. rhamnosus HN001-fed and control groups 1 week post-challenge with E. coli O157:H7. Data are expressed as the cumulative percentage of animals with abnormal appearance, of 44 L. rhamnosus HN001-fed and 40 control mice. Abnormal appearance was expressed as: fur ruffled, a loss of sheen to the coat, less alert or active, and less interested in the external environment, signs of hyperventilating when handled, hunched over and lethargic, non-reactive to stimulus, agitated or showing signs of diarrhoea.
3.2 Effect of L. rhamnosus HN001 treatment on feed intake
There was no difference (P>0.05) in feed intake between the two strains of mice (BALB/c and C57). Significant difference (P<0.01) between the L. rhamnosus HN001-treated and control groups was found in mean feed intake (post-challenge) of animals (Fig. 2).
Figure 2
Total mean feed intake (g/mouse) of mice in the L. rhamnosus HN001-fed and control groups over a period of 1 week post-challenge with E. coli O157:H7. Data are least square mean feed intakes, of 44 L. rhamnosus HN001-fed and 40 control mice. The feed intake of mice during the week prior to challenge was used as a covariate during the statistical analysis. Error bars are the standard errors of the least square mean. **P<0.01.
3.3 Effect of L. rhamnosus HN001 treatment on bacterial translocation
All the blood samples collected from the animals in the L. rhamnosus HN001-fed group proved negative for E. coli O157. However, a blood sample from one of the C57 control mice was found to be positive for E. coli O157. E. coli O157 was also detected from two spleen and two liver samples from the L. rhamnosus HN001 treatment group, and from three spleens and five livers collected from the control group (Table 1). Mean log bacterial burdens per positive organ were 1.2 and 2.3 in the L. rhamnosus HN001-fed mice, compared to 3.1 and 4.2 in the controls (values for spleen and liver, respectively).
Table 1
Translocation of E. coli O157:H7 to blood, spleen and liver in mice in L. rhamnosus HN001-fed and control groups
| Number of animals with E. coli O157-positive culture (mean log10 cfu/positive organ) | Total number of mice with E. coli O157-positive culture (%) | |||
| Blood | Spleen | Liver | ||
| L. rhamnosus HN001 (n=10) | 0 | 2 (1.2) | 2 (2.3) | 2 (20%) |
| Control (n=10) | 1 | 3 (3.1) | 5 (4.2) | 5 (50%) |
| Number of animals with E. coli O157-positive culture (mean log10 cfu/positive organ) | Total number of mice with E. coli O157-positive culture (%) | |||
| Blood | Spleen | Liver | ||
| L. rhamnosus HN001 (n=10) | 0 | 2 (1.2) | 2 (2.3) | 2 (20%) |
| Control (n=10) | 1 | 3 (3.1) | 5 (4.2) | 5 (50%) |
Numbers of E. coli O157 were expressed as log10 value of cfu.
Table 1
Translocation of E. coli O157:H7 to blood, spleen and liver in mice in L. rhamnosus HN001-fed and control groups
| Number of animals with E. coli O157-positive culture (mean log10 cfu/positive organ) | Total number of mice with E. coli O157-positive culture (%) | |||
| Blood | Spleen | Liver | ||
| L. rhamnosus HN001 (n=10) | 0 | 2 (1.2) | 2 (2.3) | 2 (20%) |
| Control (n=10) | 1 | 3 (3.1) | 5 (4.2) | 5 (50%) |
| Number of animals with E. coli O157-positive culture (mean log10 cfu/positive organ) | Total number of mice with E. coli O157-positive culture (%) | |||
| Blood | Spleen | Liver | ||
| L. rhamnosus HN001 (n=10) | 0 | 2 (1.2) | 2 (2.3) | 2 (20%) |
| Control (n=10) | 1 | 3 (3.1) | 5 (4.2) | 5 (50%) |
Numbers of E. coli O157 were expressed as log10 value of cfu.
3.4 Effect of L. rhamnosus HN001 treatment on key immune response parameters — intestinal IgG and IgA antibody titres, and blood phagocytic capacity
Although the difference in IgG titres between the L. rhamnosus HN001 and control groups was not statistically significant (P>0.05), mice fed L. rhamnosus HN001 had significantly higher mean anti-E. coli IgA titre end-points in comparison to the control mice (P<0.05; Fig. 3). In addition, mice in the L. rhamnosus HN001 treatment group exhibited significantly greater phagocytic capacity (i.e. percentage of phagocytically active blood leukocytes) in comparison to non-L. rhamnosus HN001 controls (P<0.05; Fig. 4). There was no difference in either antibody titre or phagocytic capacity between BALB/c and C57 mice (P>0.05).
Figure 3
Anti-E. coli mucosal antibody IgA response of mice in the L. rhamnosus HN001-fed and control groups 1 week post-challenge with E. coli O157:H7. Data are least square mean anti-E. coli antibody IgA titre end-points (inverse titre end-point, log10-transformed) of 10 L. rhamnosus HN001-fed and 10 control mice. Error bars are the standard errors of the least square mean. *P<0.05.
Figure 4
Phagocytic activities of blood leukocytes in mice in the L. rhamnosus HN001-fed and control groups 1 week post-challenge with E. coli O157:H7. Data are least square mean percentages of cells showing phagocytic activity, of 10 L. rhamnosus HN001-fed and 10 control mice. *P<0.05.
4 Discussion
Consumption of some strains of LAB has been shown to protect animals and humans against a range of gastrointestinal pathogens [4,17,26]. However, there is little information on the protective effects of LAB against E. coli O157:H7 infection in vivo. The results of this study demonstrate that dietary supplementation with L. rhamnosus HN001 can reduce the severity of E. coli O157:H7 infection in mice. A murine model was used, instead of the gnotobiotic pig model commonly used to study EHEC, as the presence of a normal intestinal microflora is considered essential to mimic the microenvironment of human gastrointestinal tract; indigenous microflora plays an important role in preventing pathogenic colonisation. Following challenge infection, L. rhamnosus HN001-fed mice exhibited a lower incidence of bacterial translocation to extra-intestinal tissues and lower mean bacterial burdens among translocation positive animals than the control group. Although the difference between treatment groups failed to reach significant levels, a significantly higher feed intake together with a lower cumulative mortality index in L. rhamnosus-fed mice, compared to the control group, suggests that L. rhamnosus is able to protect mice against E. coli O157 infection. This is consistent with the results of our earlier studies on the efficacy of L. rhamnosus HN001 against Salmonella typhimurium in mice [27]; L. rhamnosus HN001-fed mice showed significantly lower bacterial translocation and mortality rate compared to mice fed a control diet.
Several mechanisms by which probiotics mediate anti-infection effects have been suggested, including competition for adhesion sites, production of antimicrobial substances, competition for nutrients and the stimulation of host immunity. However, little is known about the relative importance of these mechanisms in host protection. In this study, protection against E. coli O157 was accompanied by stimulation of host immune responses that are pertinent to the control of E. coli and other enteropathogenic bacteria. The proportion of peripheral blood leukocytes exhibiting phagocytic activity and the levels of intestinal IgA antibody titres against E. coli in the L. rhamnosus HN001-fed mice were significantly higher, compared with the control mice. It is important to note that whole formalin-killed E. coli O157:H7 cells were used as antigen to detect IgA in this study. Therefore, it is not certain what proportion of the antibody was specific for EHEC or other commensal E. coli. Comparatively, higher IgA responses in L. rhamnosus-fed mice do suggest, however, that a majority of the antibody was specific for E. coli O157. These findings are consistent with the previous observations that some probiotic LAB strains may reduce intestinal infection, and can concomitantly enhance specific mucosal antibody levels (acquired immunity) and/or blood cell phagocytic activities (innate immunity) [17,28–30]. An association between enhanced resistance of L. rhamnosus-fed mice to S. typhimurium and enhanced specific and non-specific host responses has also been reported [30]. Furthermore, this study demonstrates that reduction in the severity of infection and enhancement of potentially protective immune responses can be identified in mice of two different MHC haplotypes (BALB/c [H-2d] and C57 [H-2b]).
The enhanced immunity and reduced disease severity conferred by L. rhamnosus HN001 in this study against E. coli O157, together with evidence from previous studies of immunity-enhancing and protective effects of LAB against microbial pathogens [17,30], suggest that dietary supplementation with defined probiotics may represent an effective biotherapeutic/prophylactic means of countering gastrointestinal infection in humans [31]. This is consistent with the previous reports that probiotic supplementation of the diet is a potentially valuable means of combating diarrhoeal infections [4,18,32,33], and that immunoregulatory LAB have the potential to be incorporated into foodstuffs (e.g. yogurt) and used as a non-pharmaceutical means of boosting immunity and enhancing protection [34,35].
Acknowledgements
We would like to thank Kay Rutherfurd for her help with flow cytometry, Anne Broomfield, Kim Kennedy, Linley Fray, Sarah Blackburn, and Daniel Johnson for their expert technical assistance, Freeman Qu for his help with the ELISA and microbiological analysis, Hugh Morton and Duncan Hedderley for professional statistical advice, and Frank Cross for helpful comments and discussions.
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