|Year : 2012 | Volume
| Issue : 4 | Page : 431-436
Effect of biotherapeutics on antitoxin IgG in experimentally induced Clostridium difficile infection
S Kaur1, C Vaishnavi1, R Kochhar1, KK Prasad1, P Ray2
1 Department of Gastroenterology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
2 Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
|Date of Submission||29-Feb-2012|
|Date of Acceptance||29-Apr-2012|
|Date of Web Publication||24-Nov-2012|
Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh
Source of Support: The study was carried out by the internal grants of the Institute., Conflict of Interest: None
Purpose: Recurrent diarrhoea after successful treatment of primary Clostridium difficile associated disease (CDAD) occurs due to bowel flora alterations and failure to mount an effective antibody response. Apart from antibiotics, risk factors include immunosuppressive and acid-suppressive drug administration. Biotherapeutics such as probiotic and epidermal growth factor (EGF) may offer potential effective therapy for CDAD. Materials and Methods: The effect of biotherapeutics in mounting an antibody response against C. difficile toxins was studied in BALB/c mice challenged with C. difficile after pre-treatment with ampicillin, lansoprazole or cyclosporin. Sera from sacrificed animals were estimated for antitoxin IgG by enzyme linked immunosorbent assay. Results: Antitoxin IgG was significantly higher (P<0.05) in C. difficile challenged groups compared to unchallenged controls, but insignificant (P>0.05) in animals in which C. difficile was given after pre-treatment with cyclosporin compared to those without any pre-treatment, or pre-treatment with antibiotic or lansoprazole. In inter-subgroup comparisons also significant anomaly in production of antitoxin IgG was found. The antitoxin IgG levels were raised in animals administered C. difficile after pre-treatment with ampicillin, but lower in animals administered cyclosporin. High levels of antitoxin IgG were also found in the serum samples of animals receiving lansoprazole and C. difficile. Conclusions: Probiotics showed their beneficial effect by boosting the immune response as seen by production of antitoxin IgG. Oral administration of EGF did not affect the immune response to C. difficile toxins as significant increase was not observed in the serum antitoxin IgG levels in any of the groups investigated.
Keywords: C. difficile, antitoxin IgG, biotherapeutics, ampicillin, cyclosporin, lansoprazole
|How to cite this article:|
Kaur S, Vaishnavi C, Kochhar R, Prasad K K, Ray P. Effect of biotherapeutics on antitoxin IgG in experimentally induced Clostridium difficile infection. Indian J Med Microbiol 2012;30:431-6
|How to cite this URL:|
Kaur S, Vaishnavi C, Kochhar R, Prasad K K, Ray P. Effect of biotherapeutics on antitoxin IgG in experimentally induced Clostridium difficile infection. Indian J Med Microbiol [serial online] 2012 [cited 2020 Jan 26];30:431-6. Available from: http://www.ijmm.org/text.asp?2012/30/4/431/103764
| ~ Introduction|| |
Clostridium difficile is currently recognized as the most common cause of nosocomial infectious diarrhoea in hospitals and has become a major issue in many countries. Overall mortality associated with C. difficile infectious diarrhoea is estimated to be 17% but is even higher in the older adult population.  C. difficile is now recognized as the primary cause of hospital acquired colitis in patients who receive antibiotics and other medication. Recurrent diarrhoea after successful treatment of primary episode is also the main complication of C. difficile associated disease (CDAD). Persistent alterations in the indigenous bowel flora and failure to mount an effective antibody response to C. difficile toxins are the main mechanisms whereby recurrent CDAD occur.
C. difficile, causes a spectrum of conditions ranging from asymptomatic carriage to full-blown clinical expression, with pseudomembranous colitis as the most characteristic feature of advanced disease.  C. difficile infection has also been reported to be involved in the exacerbation of ulcerative colitis.  The basis for this variability in response is not entirely clear, but host factors appear to be more important than bacterial virulence factors.
The main virulence factors for CDAD are the two potent toxins: Toxin A and toxin B. The ability of the immune system of the host to produce protective antibodies against the toxins of C. difficile plays an important role in reducing the severity of disease and preventing further recurrences.  Thus antibody response is a major determinant of the disease outcome and may be independent of the infecting strain. Prior studies have reported that an inadequate immune response to C. difficile toxin exposure is associated with severe and recurrent disease. 
However, there are likely to be many other influential risk factors for C. difficile infection. Many studies have reported the association of administration of immunosuppressive drugs and development of CDAD. , Exposure to immunosuppressants may reduce the immune response and predispose the patients to CDAD. Proton pump inhibitor (PPI) may also contribute to the pathogenesis of CDAD by inhibiting the gastric acid secretion which acts as barrier for any gastrointestinal pathogen. Previous studies have examined the association between PPI use and risk of CDAD, with conflicting results. ,
There is an increasing interest in biotherapeutic approaches such as employing probiotic and epidermal growth factor (EGF) to the management of gastrointestinal diseases - both for prevention as well as for treatment. Probiotics may offer potential effective therapy for CDAD by re-establishing the disrupted microflora, enhancing immune responses and clearing pathogens and their toxins from the host. Similarly EGF, a potent mitogenic peptide that reduces bacterial colonization and heals gastric ulcers  could be investigated as an alternative approach. Thus use of innovative approaches such as probiotics and EGF as therapeutic agents in the management of CDAD could be highly beneficial.
The aim of the present study was to investigate in an experimental model the antitoxin IgG levels in drug induced CDAD and the effect of biotherapeutics in mounting an antibody response against C. difficile toxins.
| ~ Materials and Methods|| |
The study was approved by the Institute Research Ethics Committee and Institutional Animal Ethics Committee. The investigations were conducted on 4-6-week-old BALB/c mice (n=78) weighing approximately 25 g each. The animals in the study were divided into 5 groups (Group1-5). Group 1 comprised of 6 healthy mice that were not given any inoculum and served as controls for all other groups. All the animals in Groups 2-5 received C. difficile inoculum and were further divided into three subgroups (a, b and c - each comprising of six animals) based on the other inocula administered. Thus Group 2 comprised of subgroups which received C. difficile but no drug. Animals in Group 3 received ampicillin (66 mg/kg body weight) for one week before C. difficile challenge. In Group 4, animals received cyclosporin (10 mg/kg body weight) for one week before C. difficile inoculation. Similarly in Group 5, animals received lansoprazole (0.5 mg/kg body weight) for two weeks before C. difficile administration. Apart from this, animals in subgroups 'b' and 'c' in Groups 2-5 also received probiotic Lactobacillus acidophilus (2b, 3b, 4b and 5b) and EGF (2c, 3c, 4c and 5c) inocula respectively for one week after C. difficile challenge [Table 1].
|Table 1: Study design, inocula given and period of sacrifice of experimental animals|
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| ~ Preparation of Various Inocula|| |
A toxigenic strain of C. difficile serogroup A (W 1194, ATCC 43594) positive for both toxins A and B (kindly provided by Dr. M. Delmee, Belgium) was administered at a dose of 10 8 CFU/ml. Ampicillin inoculum was prepared by dissolving the content of a 500 mg ampicillin capsule (Ranbaxy Laboratories Limited, New Delhi, India) in sterile distilled water. The suspension was administered to animals in Group 3 (3a, 3b and 3c) orogastrically in two divided doses (66 mg/kg body weight) for one week before challenging the animals with C. difficile. Similarly the content of 50 mg cyclosporin capsules (Pharmacia, Hyderabad, India) dissolved in sterile distilled water was administered orogastrically to animals in Group 4 (4a, 4b and 4c) in two divided doses (10 mg/kg of body weight) for 1 week before C. difficile inoculation. For PPI administration, content of 30 mg lansoprazole capsule (Brown & Burk Pharmaceutical Limited, Tamil Nadu, India) was dissolved in sterile distilled water and administered orogastrically as a single dose (0.5 mg/kg body weight) daily for two weeks before C. difficile inoculation to animals in Group 5 (5a, 5b and 5c).
Biotherapeutic administration comprised of probiotic L. acidophilus and EGF. For preparation of L. acidophilus inoculum, LactoBacil capsule (Organon India Limited, Gurgaon, India) containing lyophilized L. acidophilus (10 6 CFU/capsule) were used. The content of each capsule was suspended in 1 ml of sterile saline and administered orogastrically daily for one-week post C. difficile inoculation to the animals in subgroups 2b, 3b, 4b and 5b as mentioned in the experimental design. Epidermal growth factor (Sigma, USA) was suspended in sterile distilled water and administered orogastrically daily at a dose of 100 μg/kg body weight to animals in subgroups 2d, 3d, 4d and 5d for one week post C. difficile inoculation.
The animals in Groups 2-5 were sacrificed one-week post C. difficile inoculation. The animals in control group (Group1) were sacrificed along with animals in Group 2. Immediately after the sacrifice, blood from each animal was collected by cardiac puncture. The serum was separated and kept at -20 °C for estimation of antitoxin IgG.
| ~ Preparation of C. difficile toxins|| |
C. difficile serogroup A strain used for the inoculum was also used to prepare C. difficile toxins. Briefly the organism was inoculated into 250 ml of Columbia broth (HiMedia, Mumbai, India) in a conical flask and incubated anaerobically for 72 h. The culture was then centrifuged at 3000 rpm for 30 min and the supernatant was transferred to a fresh flask under sterile conditions. The toxins were purified by the standard ammonium sulphate precipitation technique and dialysis done as described by Sullivan et al. with some modifications. The dialysate was centrifuged for 10 min at 3000 rpm and the precipitate was discarded. The supernatant was estimated for proteins. The presence of high titers of C. difficile toxin A and B in the supernatant was confirmed by coating latex beads by purified antitoxin A and B separately (kindly provided by Dr. M. Warny) by the procedure described by Vaishnavi et al. which gave separate information of both the toxins. The two toxins were not purified, as it was not required for the investigation.
| ~ Antitoxin IgG Assay|| |
Antitoxin IgG was estimated by an enzyme linked immunosorbent assay (ELISA) as described by Warny et al. with some modifications. Optimum dilutions for C. difficile toxins and the antibodies were carried out by a checker board titration. Microtiter ELISA plate was taken as the solid phase for coating the C. difficile toxins. 100 μl of C. difficile toxins (30 μg of protein/100 μl) comprising of a mixture of toxin A and B was put in each well of the plate and incubated overnight at room temperature. Individual concentration of toxins A and B was not done as the presence of toxins was confirmed qualitatively by agglutination assay.  The coated plate was washed three times with phosphate buffered saline (pH 7.2) containing 0.05% of Tween 20 (PBST). 100 μl of the serum samples (diluted 1 in 20) in PBST containing 1% bovine serum albumin (BSA) was added to the wells. Both positive and negative controls were also included. Antitoxin A and B diluted 1 in 20 in PBST-BSA were used as positive controls. The negative control comprised of PBST-BSA. The ELISA plate was incubated for 2 h at room temperature then washed three times with PBST. One hundred microliters of 1 in 1000 horseradish peroxidase-labeled antimouse IgG (Sigma, USA) in PBST-BSA was added to each well and the plate incubated for 2 h at room temperature. After washing three times with PBST, 100 μl of 1 mg/ml O-dianisidine was added as the substrate. The plate was incubated for half an hour and optical density of the reaction was read at 405 nm with a microplate reader (Multiskan, Finland).
| ~ Statistical Analysis|| |
Data was analysed using the SPSS version 10 software program.
Analysis of variance test was applied for analysing significant differences for antitoxin IgG levels. Dunnett t-test was applied to compare all the groups against the control. Student-Newman-Keuls test was applied for inter-subgroup comparisons. A probability value of P<0.05 was considered to indicate significant differences.
| ~ Results|| |
The absorbance values for antitoxin IgG in all the groups are shown in [Figure 1]. Values for antitoxin IgG were significantly higher (P<0.05) in the four groups given C. difficile (Groups 2, 3, 4 and 5) when compared with controls (Group 1). The antitoxin IgG level was lower although insignificantly (P>0.05) in animals in which C. difficile was given after pre-treatment with cyclosporin (subgroup 4a) when compared with animals given C. difficile without pre-treatment (subgroup 2a), pre-treatment with antibiotic (subgroup 3a) or pre-treatment with lansoprazole (subgroup 5a).
|Figure 1: Mean antitoxin IgG in different groups of experimental animals. CD = C. difficile; PB = probiotic; EGF=epidermal growth factor; CY = cyclosporine; PPI = proton pump inhibitor|
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In inter-subgroup comparison, the mean antitoxin IgG absorbance was significantly higher (P<0.05) in animals receiving probiotic (subgroup 2b) in Group 2. But in those receiving EGF, there was a slight increase in antitoxin IgG levels compared with subgroup 2a, though the increase was not statistically significant (P>0.05).
The antitoxin IgG values were significantly raised (P<0.05) in animals in Group 3 given C. difficile after antibiotic treatment (subgroups 3a, 3b and 3c) when compared with controls (Group 1). When inter-group comparison between animals in subgroups 3a (ampicillin and C. difficile) and 3b (ampicillin, C. difficile and probiotic) was made, the antitoxin IgG values were found to be higher in the probiotic receiving animals, but not significantly (P>0.05). In the animals receiving EGF, there was an insignificant decrease (P>0.05) in antitoxin IgG levels compared to subgroup 3a where animals were administered with ampicillin and C. difficile.
The antitoxin IgG levels were significantly raised in the three subgroups 4a, 4b and 4c given C. difficile after cyclosporin administration compared to controls (Group 1). In inter-subgroup analysis there was a significant increase (P<0.05) in the IgG response in animals receiving probiotic (subgroups 4b). However, there was insignificant increase in IgG in EGF receiving animals (subgroups 4c) compared to those not receiving the biotherapeutics (subgroup 4a).
The values for antitoxin IgG were significantly increased (P<0.05) in the three subgroups (5a, 5b and 5c) given C. difficile after lansoprazole administration compared to controls (Group 1). In inter-subgroup comparison an insignificant (P>0.05) decrease in the absorbance values was observed in animals receiving probiotic (subgroup 5b) and EGF (subgroup 5c) compared to animals in subgroup 5a.
| ~ Discussion|| |
Toxigenic strains of C. difficile produce toxins A and B which are related to each other and belong to a group of large clostridial toxins. The epidemic hypervirulent mutant has the potential to cause more severe disease by production of increased levels of toxins.  The ability of the host immune system to produce protective antibodies against C. difficile toxins plays an important role in reducing the severity of disease and preventing further recurrences,  and may be independent of the infecting strain. Inadequate immune response to C. difficile toxins predisposes patients to severe, prolonged and recurrent C. difficile diarrhoea.
In the present study significantly higher levels of antitoxin IgG were found in the animals in all the Groups administered C. difficile. The antitoxin IgG levels were raised in animals administered C. difficile after pretreatment with ampicillin. The observation of raised titers of specific antibodies in the ampicillin treated animals is in agreement with previous studies. ,
Kaur et al. reported mild to moderate inflammation in the colonic segments of animals colonized with C. difficile. In our study, animals given C. difficile without any pretreatment (Group 2) showed significantly raised levels of antitoxin IgG. It has also been established that patients with C. difficile colonization and a serum IgG response to C. difficile enterotoxin usually become asymptomatic carriers. On the other hand, individuals who mount a significant serum IgG response to toxin A are protected against recurrent CDAD. 
Impaired immune response may be responsible for recurrent CDAD. A defective antibody response to toxin A has been found in patients with relapsing and prolonged CDAD.  Although the ability to mount an immune response is not protective against C. difficile colonization, it is associated with decreased morbidity, mortality, and recurrence of CDAD.  Kyne et al. found no evidence of immune protection against colonization by C. difficile. However, after colonization there was an association between a systemic anamnestic response to toxin A, as evidenced by increased serum levels of IgG antibody against toxin A, and asymptomatic carriage of C. difficile.  Most patients convalescent from C. difficile diarrhoea demonstrate systemic and mucosal antibodies to toxin A, but these antibodies following natural infection do not appear to alter the clinical course of C. difficile infection. 
Immunosuppressive drugs have also been reported to be associated with the development of CDAD. , Patients receiving immunosuppressive drugs are debilitated and are therefore unable to mount an effective IgG antibody response against C. difficile toxin A, thereby increasing the risk for CDAD. In the current study the antitoxin IgG level was lower in animals administered cyclosporin, an immunosuppressive drug, compared to the ampicillin-receiving animals, though the decrease was not statistically significant. A high myeloperoxidase activity and inflammatory changes in the colon inclusive of epithelial damage and cryptitis have been reported in animals.  Thus patients at highest risk for CDAD fulminant disease include those who have recently received immunosuppressive therapy.
Viscidi et al. tested serum samples from 340 patients for determination of the age-related prevalence of antitoxin and reported that antibodies to toxins A and B are present in the majority of older children and adults and that CDAD patients develop serologic response to one or both toxins. In a study of consecutive serum samples from 61 patients with CDAD and 64 control samples, immune response was detected in approximately half of the CDAD patients and a correlation was found between clinical recovery without relapse of CDAD and high IgG titers to toxin B.  Katchar et al. compared the clinical characteristics and humoral immune responses of 13 patients with recurrent CDAD with an equal number of those with a single CDAD episode. They observed that subjects with recurrent CDAD did not show evidence of widespread humoral immune deficiency or IgG subclass deficiency even though low serum IgG antitoxin A concentrations reflected selectively reduced IgG2 and IgG3 subclass responses.
Patients who are symptomatic respond to C. difficile in a manner typical of secondary antibody response with no evidence that an inability to respond predisposes to appearance of symptoms.  Aboudala et al. reported induction of very high level responses to antitoxin A IgG in sera of healthy volunteers after parenteral vaccination with C. difficile toxoid vaccine and found a strong association between serum antibody responses to toxin A and protection against C. difficile diarrhoea. It is likely that antibody levels begin to be boosted following colonization and is further enhanced by disease itself as a secondary antibody response.
We did not look at local or systemic IgA because C. difficile is no longer regarded as a non-invasive micro-organism as it is also known to cause bacteremia and local IgA antibodies just give information about enteric response. Earlier studies have shown the importance of serum IgG in C. difficile infection. Warny et al. reported that serum IgG antibody to toxins were higher in patients with mild CDAD than in those suffering from prolonged diarrhoea. Kyne et al. found that patients with high serum IgG become asymptomatic carriers while patients who lacked active immunity suffer from diarrhoea and colitis. It has been postulated that serum IgG may reflect systemic infection and serum IgA may preferentially be stimulated in response to enteric infection in which systemic exposure to toxins is relatively limited. Patients with C. difficile diarrhoea develop intestinal IgA response to toxin A that parallel serum IgG responses to toxin A.
Results from studies have suggested that there might be an association between the C. difficile strains, production of toxins, and clinical manifestation of the infection. However one cohort study employing an immunoblot typing method in a unit where C. difficile was not epidemic showed that there was no difference between strains resulting in symptomatic cases and asymptomatic carriage.  In another study also no strain-specific difference for asymptomatic carrier versus patients with CDAD were seen and most strains from both the groups were toxigenic in vitro. The antibody response before infection with C. difficile might also be very important in stopping the development of symptoms.
In the present study animals which received L. acidophilus in addition to C. difficile (subgroup 2b) had significantly higher levels of antitoxin IgG than those not given L. acidophilus (subgroup 2a). It is likely that L. acidophilus may be able to stimulate the peripheral immune response also. An increase in antitoxin IgG level was also found in animals administered C. difficile and cyclosporin in addition to L. acidophilus. It is known that lactic acid bacteria stimulate the local immune response of the animals in the gut  and could be the reason of improved antitoxin IgG level in the present study also. In conclusion probiotics showed their beneficial effect by boosting the immune response by production of antitoxin IgG. Oral administration of EGF did not affect the immune response to C. difficile toxins as significant increase was not observed in the serum antitoxin IgG levels in any of the groups in the present study.
| ~ Acknowledgments|| |
The study was carried out by the internal grants of the Institute. Dr. Sukhminderjit Kaur received the Senior Research Fellowship from the Indian Council of Medical Research, New Delhi, India. We are indebted to Dr. M. Delmee, Belgium, for the standard C. difficile strain, Dr. M. Warny, USA, for donating antisera for C. difficile toxin A and B.
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