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Protective Effects of Immunization with Formalin Inactivated Oral Whole Cell Vaccine against Multidrug Resistant Citrobacter freundii in Murine Model

Asma Rahman*, S.M. Shamsuzzaman, Nigha Zannat Dola, Modina Ansary Nabonee

Department of Microbiology, Dhaka Medical College & Hospital, Dhaka-1000, Bangladesh

Abstract: Citrobacter freundii, being an opportunistic pathogen is evolving as an important cause of nosocomial infection along with multidrug resistance, leading to a high mortality rate. The emerging range of high antibiotic resistance and the need to prevent infections has led to the development of alternative therapeutic molecules. Immune prophylaxis is considered a safe approach against the pathogen. This study was carried out to see the protective effectiveness of antibodies caused by oral formalin inactivated whole cell vaccine against multidrug resistant C. freundii. In this study, MDR C. freundii was separated from various clinical samples and used for the oral immunization of 9 Swiss albino mice. Seven days following immunization with the third dose of vaccine, the mice were challenged orally with live C. freundii and witnessed for 14 days. Blood from the tail was drawn 14 days after each booster, and lastly, cardiac puncture was done 14 days post challenge. Total antibodies that bind with antigen and functional antibodies having bactericidal activity were assessed by ELISA and serum bactericidal antibody (SBA) assay, respectively. In this study, 14 days after post challenge, 100% survival rates were seen among the immunized mice. ELISA showed all serum from the pre- and post-challenge immunizations had considerably greater optical density values of IgG in comparison to the control mice. On the other hand, SBA assay showed 100% bactericidal activity of the immunized mouse sera using 50% and 25% guinea pig complement following incubation for 3 hours at 1:10 serum dilution. In this study, formalin inactivated oral immunization with MDR C. freundii produced protective antibodies in Swiss albino mice. Safe and tolerable formalin inactivated oral whole cell vaccines may be the most effective and practical way of preventing enteric diseases.

Keywords: citrobacter freundii, formalin, immunization, oral

Received April 11, 2022; Revised May 16, 2022; Accepted May 25, 2022

Citation: Asma Rahman, S.M. Shamsuzzaman, Nigha Zannat Dola, Modina Ansary Nabonee, Protective Effects of Immunization with Formalin Inactivated Oral Whole Cell Vaccine against Multidrug Resistant Citrobacter freundii in Murine Model. American Journal of Infectious Diseases and Microbiology, 10 (3). 92-97. January 2022. doi:10.12691/ajidm-10-3-1

1. Introduction

Citrobacter freundii belongs to the genus Citrobacter, is an extensive environmental contaminant and one of the commensal species in intestinal tract of humans and animals [1]. This opportunistic pathogen is responsible for serious infections of urinary tract, wound swab, respiratory tract, blood stream, endocardium and meningeal infections exclusively in high risk individuals such as immunocompromised adults and infants [2,3].

Emerging range of multidrug resistant () stains along with increasing nosocomial infections strive for attention on C. freundii [2,4]. Due to failure to prevent repeated infection and allergic reactions to some antibiotics, alternative therapeutic approach such as vaccines are being considered as an option in controlling infection caused by C. freundii [2]. Vaccine reduces susceptibility to infection by lowering the need for antibiotic treatment and finally restricts the environmental pressure causing selection of resistant strains [5]. Moreover, by providing more appropriate inflammatory stimulus for the establishment of an immune response against multiple antigens, especially the protective ones, whole cell bacteria help in the construction of vaccines [6,7].

As the alimentary tract is profoundly occupied with bacteria, extensive refinement from bacterial by-products is not needed for oral vaccines. Oral vaccines prevent infections limited to mucosal surfaces or inhibit mucosal invasion of pathogens [8]. Besides the gastrointestinal tract being the prime immunologic structure in the human body, it is safeguarded by specialized constituents of the native and adaptive immune system [9].

The functional antibodies produced by new vaccines are evaluated on the basis of their immunogenicities and potential defensive capacities, for which assays are needed [10]. For determining the capability of vaccine-induced antibody, to kill bacteria, serum bactericidal antibody (SBA) assay is thought to be the “gold standard” [11] whereas ELISA is thought to be the most widely applied tool for measuring total serum antibodies, accounting for the immunogenicity of various formulations [10]. There is still no such report available to fix the endpoint SBA responses made by the inactivated vaccine against multidrug resistant C. freundii in Swiss albino mice. So, the aim of this study was to assess the protective effects of immunization with a formalin-inactivated oral whole cell vaccine against multidrug resistant Citrobacter freundii in murine model, as immune prophylaxis is regarded a safe method against multidrug resistant infections.

2. Materials and Methods

This study was conducted from July 2019 to June 2020 at the Department of Microbiology at Dhaka Medical College.

2.1. Culture of Bacteria

Culture of organisms from different samples was done in MacConkey agar media at 37°C for 24 hrs to prepare vaccine solutions.

2.2. Immunization of Mice

2.2.1. Animals

Nine Swiss-albino female mice of 5-6 weeks of age and two guinea pigs were collected from Animal Resources Facility of icddr,b and were retained under precise pathogen free environments in the animal house of Dhaka Medical College.

2.2.2. Preparation of Formalin Inactivated Whole Cell C. freundii Vaccine

Group-1 was vaccinated against formalin inactivated solution prepared from a mixed solution of C. freundii isolated from urine, stool, blood, endotracheal aspirate, sputum and wound swab. For this, a loop full of organisms was inoculated into tryptica soya broth (TSB) and was incubated at 37°C overnight. Following incubation, centrifugation was done at 2,000 g for 20 min at 4°C and the supernatant was discarded. Phosphate buffered saline (PBS) was then used to wash twice the pelleted bacteria. To formulate formalin inactivated C. freundii, 37% formalin was included to the suspension to achieve a final conc. of 3% (v/v) and incubated for 2 hours at 37°C. After incubation, the suspension was again two times washed with sterile PBS and resuspended in PBS to achieve conc. of 1.5 X 10⁸ CFU/ml. 134 µl of inoculum was mixed with 866 µl of sterile PBS to achieve a conc. of 2 X10⁷ CFU/ml [12,13].

2.2.3. Immunization Schedule

Four doses of oral immunizations were performed with 250 µl of bacterial solution (2X10⁷ CFU/ml) on day 0, 7, 14 and 21 with the help of 22Fr feeding tube in group-1 experimental group. The mice were sedated with intraperitoneal ketamine injection (100mg/kg) prior to each immunization and appropriate anesthetic condition was maintained by chloroform [13]. Also, sterile PBS of 250 µl was also given to the control group-2 on same days.

2.2.4. Blood Collection for ELISA and SBA

Blood from the tail of mice was collected 14 days following each immunization. The tail was stretched and 70% alcohol was used to clean it. The tail was cut before its rounded end by a sterile scalpel (22 FR) and 10µl of fresh blood was kept in a micro centrifuge tube containing 40µl of phosphate buffer saline to maintain a dilution of 1:5.The diluted sera was retained at vertical position for 2 hours, after which centrifugation at for 10 minutes was done and clear sera was moved into a separate micro centrifuge tube [13].

2.2.5. Oral Challenge

To challenge mice, inoculums were prepared with strains same as used in vaccine formulation and were not formalin inactivated. Two weeks afterwards last immunization, challenge of group-1 and 2 mice were done orally with 2X10⁸ CFU/ml of bacteria in 300 µl PBS. All mice were kept on observation for 14 days post challenge for any clinical expressions such as rise of temperature, unable of movement, reluctance to feed etc [13].

2.2.6. Collection of Blood By Cardiac Puncture

Sera from blood were drawn by cardiac puncture 14 days post challenge from group-1 and 3. At an angle of 45° with an insulin syringe, cardiac blood was drained by feeling cardiac pulsation after shaving, followed by washing with povidone iodine and 70% alcohol. In a sterile test tube, collection of about 1 ml of blood was done and was set aside without dilution [13].

2.3. Antibody Detection by ELISA

To determine the optical density (OD) by ELISA for the existence of immunoglobulin G (IgG) specific for C. freundii antigen, mice sera were used.

2.3.1. Sonication of Whole-Cell C. freundii

About 100 μL of distilled water was used to dilute the bacterial pellets and set aside for 30 mins on ice and sonicated at 20 kHz for 2 × 10 s (reliant on samples and thickness of the samples), and retained on ice for 5 min. Then, this solution was centrifuged at a 10,000 g for 20 min to pellet debris (debris may contain unbroken cells or organelles and nuclei). Then, to a fresh microcentrifuge tube supernatants were transferred and were stored at −20°C for usage as antigen. Checkerboard titration was done to optimize the antigen, and for ELISA 10 μg of antigen was used [13].

2.3.2. Optimized ELISA

Coating with bicarbonate-coating buffer (pH 9.6) and overnight incubation at room temperature of ELISA plates were done with 100 μl/well of antigen (10 μg/ml) followed by washing two times with PBS and blocking with 200 μl/well skimmed milk in PBS, incubation at 37°C for 30 min, then three times washing with PBS-tween and once with PBS. Samples of serum then were added at 1:100 dilution (100 μl/well) and incubated at 37°C for 90 min, followed by refrigeration at 4°C for the whole night. The next day, plates were washed according to above mentioned procedures, horseradish peroxidase-labeled anti-mouse IgG antibody (Thermo Fisher Scientific, USA), diluted (1:5000) conjugate, was mixed in PBS-Tween (100 μl/well) and added to the plates and incubated at 37°C for 90 min and after washing, 100 μl/well tetramethylbenzidine (50 μl) and urea peroxide (50 μl) substrate were added. Fifty microliter of 1M sulfuric acid was included to these plates to halt the reaction. Measurement of absorbance was done at 450 nm with the help of ELISA plate reader (BioTek Inc., USA). Calculation of cutoff OD value was done by the following formula:

2.4. Serum Bactericidal Antibody Assay

2.4.1. Bacterial Samples and Its Growth

A C.freundii organism separated from urine sample was cultured at 37°C in Mueller-Hinton agar media for overnight. Bacterial suspension was compared (1.5 × 10⁸ CFU/ml) to 0.5 McFarland standard solution and consecutively diluted in PBS to attain various concentrations of ~1 × 10³ CFU/ml [13].

2.4.2. Complement for SBA

Complement was obtained from combined sera of guinea pigs. Blood was drawn in tubes maintaining all sterilized measures after cardiac puncture and centrifugation at 3000 g for 10 min followed by separation of serum in aseptic cryotubes and stockpiled at −20°C. The result of serum resistance was determined by testing the survival of the C. freundii in the presence of only complement at a concentration of 12.5%, 25% and 50% (diluted in PBS). Persistence of the C. freundii strain in the company of immune and nonimmune serum lacking complement was also carried out [13,14].

2.4.3. Measurement of Antibody-Dependent Complement-Mediated Killing by Optimization of SBA

Diluted (1:10, 1:20, 1:40, 1:80, and 1:160 in PBS) and heat inactivated (at 56°C for 30 min) control and immune serum samples were positioned in a micro-titer plate. Then, bacterial suspension of 10 μl (from 1.5 × 10⁸ CFU/ml till 1.5 × 10³ CFU/ml) was added to the identical wells. Lastly, to every well, 30 μl of pooled guinea pig sera (12.5%, 25% and 50%) was included and ultimately 50 μl volume was attained. Then incubation was done at 37°C for 1 h, 2 h, 3 h, and 4 h [10,13].

From every single well, 1 μl of solution was taken and plated in Mueller-Hinton agar media and incubated at 37°C overnight to observe the viability of the bacteria by spotting their presence on agar plates. All of the techniques were completed in duplicate [14].

2.4.4. SBA of Optimized Sera

Log-phase cultures (bacteria grown at 1.5 × 10⁴ CFU/ml) of C. freundii were made in normal saline. Serum samples from immunized and control mice were diluted (1:10 and 1:20). Optimal results were gotten by mixing of 30 μl of guinea pig sera (50% final concentration), 10 μl of diluted bacterial solution (1.5 × 10⁴ CFU/ml) and 10 μl of diluted serum, which were then incubated at 37°C for 3 h. The negative control was confined only in bacteria and complement. Viable CFU counts were determined [15,16].

2.5. Data Processing

Data were assembled and revised precisely by examination and reexamination. All errors and discrepancies were fixed and were aloof methodically.

2.6. Data Analysis

The study results were documented scientifically. Data were evaluated and compared by T test. Statistical analysis of all data was carried out by SPSS 25 version, SPSS Inc. P value of 0.05 was taken as lowest level of significance.

2.7. Ethical Approval

This study was approved by the Research Review Committee (RRC) and the Ethical Review Committee (ERC) of Dhaka Medical College.

3. Results

Immunized and unimmunized mice survival rates in C. freundii following lethal challenge were 100% in group 1 mice which lasted the 14 days scrutiny period following challenge and group 3 control mice that were not challenged. Death of every Group 2 control mice following challenge was also noted.

Figure 1. OD value of serum samples of Group-1 mice after the 1st booster, 2nd booster and 3rd booster by ELISA

Figure 2. OD value of serum samples following lethal challenge by ELISA

OD value in 3 serum samples from group-1, collected 14 days after 1st immunization followed by 14 days after 2nd immunization and 14 days after 3rd immunization (Figure 1) was analyzed. Significant difference was observed among the OD values of experimental and control mice sera (P <0.001 after both first, second and third booster). Mean of negative control was 0.164; standard deviation 0.0214, cutoff value was 0.202. OD value range after the 1st booster was 0.497–0.508, 2nd booster was 1.007-1.136 and after 3rd booster: 2.227-2.354.

OD value of 3 serum samples of group 1 mice, drawn after lethal challenge is shown in (Figure 2). There was significant difference among the OD values of experimental and control mice sera (P <0.001). Here, mean of negative control was 0.157, standard deviation 0.0192 and cutoff value was 0.195. Range after post-challenge was 1.959-2.110.

Different immune response was produced after each booster of formalin inactivated oral vaccine where P-value <0.001 was significant (Table 1).

Table 1. OD value difference in immune response produced after each booster of formalin inactivated whole cell vaccine by ANOVA

SBA assay with both pre-challenge (Figure 3) and post-challenge (Figure 4) sera showed bacterial inhibition with 1:10 dilution, 1.5 × 10⁴ colony-forming unit/ml of bacterial inoculum and 25% and 50% guinea pig complement after 3 hours of incubation.

Figure 3. SBA of prechallenge sera

Figure 4. SBA of post-challenge sera

4. Discussion

About 0.8% of Citrobacter species account for infection among all gram negative infections, and are nosocomially responsible for approximately 3–6% of the Enterobacteriaceae [17]. Patients with invasive devices are more inclined to invasive Citrobacter infections leading to bacteremia [18], yielding to 33% to 48% of mortality rate [19].

Both humoral and cellular immune responses at systemic and mucosal sites are stimulated, probably by oral vaccination. Due to the homing of activated antigen-specific lymphocytes to distant mucosal effector sites, effective immunity to the small intestine, ascending colon, salivary and mammary glands is covered by successful oral vaccination [20].

Current study showed survival rate among the experimental mice were 100% following 14 days postchallenge. No data concerning survival rates of mice after lethal challenge following oral immunization with formalin inactivated MDR C. freundii is available in Bangladesh or any Asian country. But a study in Japan showed 100% survival of mice following immunization with formalin inactivated oral whole cell C. rodentium vaccine [12]. Another study in Iran reported 100% survival of mice after formalin inactivated oral Esch.. coli O157:H7 immunization. Also, whole cell vaccines were formulated as antigens in the usual form provide combination of identified and unidentified immunogens and create a stout and long-term immune response [21]. Formalin inactivation was done because surface antigens can maintain their conformations on the bacteria inactivated with formalin.

In this study, IgG polyclonal antibodies produced in every vaccinated mice sera following 1st booster, 2nd booster, 3rd booster and lethal challenge was measured by ELISA. A study showed immunization with formalin inactivated whole cell vaccine elicited significant levels of IgG in the immunized mice group than the control group [12]. These antibodies play a crucial role in activating the classical pathway of the complement system, causing efficient disruption of the bacteria [22]. The OD values of the IgG antibodies in this current study were highest for pre challenge mice sera after 3rd booster might be due to production of more IgG antibodies by the memory cells after 1st and 2nd booster immunization. But after lethal challenge, IgG antibodies declined slightly, which may be due to cleansing up the offending pathogen from the body.

Complement activation can play a vital part in reducing the chance of escaping immune clearance by C. freundii as its cell complexity confers killing mainly by the complement cascade [14]. The current study revealed that the sera achieved from mice immunized with formalin inactivated oral whole cell vaccine were capable of activating complement mediated lysis in the presence of 50% guinea pig complement after 3 hours of incubation with MDR C.freundii at 10% serum dilution. No data about the SBA assay for estimation of functional capacity of serum immunized with C. freundii to compare with the present study is still available.

5. Conclusion

Prophylactic usage of bacterial vaccines can prevent bacterial infections, resulting in decreasing the requirement for antibiotic prescriptions and a diminishing of the selective drug pressure that can cause resistant strains. Although, the present study provides a clue for developing a vaccine using whole-cell formalin inactivated gram-negative bacteria and its estimation by ELISA and SBA to guard against MDR bacterial infections but it is also essential to establish the immunological protection. Our results propose that formalin inactivated oral whole cell MDR C. freundii forms protective antibodies in Swiss albino mice.

Acknowledgements

Microbiology Department of Dhaka Medical College, Dhaka provided laboratory support to perform this study.

Financial Support and Sponsorship

Nil.

Conflict of Interest

There are no conflicts of interest in this study.

List of Abbreviations

MDR, multidrug resistance; ELISA, Enzyme Linked Immunosorbent Assay; SBA, Serum bactericidal assay; OD, optical density.

References

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