Introduction
Pseudomonas aeruginosa, a gram-negative bacterium, is a significant cause of nosocomial infections in patients. It is common among immunocompromised patients with an extended period of hospitalization.1 For pathogens to infect individuals, they require exposed skin surfaces and delicate internal tissues. Wounds, abdomen, urinary tract, and respiratory tracts are primary sites of P. aeruginosa.1 Patient with third-degree burns is highly susceptible to infections because they have exposed subcutaneous layer after losing both epidemic and dermis layers.2 In this case, burn patients are vulnerable to infections since they lack protective skin to prevent penetration and invasion of pathogens into the bloodstream.
Bacteraemia occurs when P. aeruginosa infects and colonizes burn wounds and subsequently invades the bloodstream. Since P. aeruginosa is infectious and resistant to antibiotics, it causes bacteremia among immunocompetent individuals.1,2 Once the bacterium enters the blood through wounds, it multiplies rapidly and triggers the systemic infection. Pseudomonal infection is life-threatening because the bacteria is resistant to most antibiotics, causes sepsis, and triggers the occurrence of septic shock in individuals.3 Pathogenesis indicates that P. aeruginosa infection causes bacteremia and leads to death within a short period. Usually, P. aeruginosa has a poor prognosis and exhibits mortality rates of between 26-39%, which is higher than that of related gram-negative pathogens.1 In this view, medical researchers have focused on understanding the mechanism of pathogenesis to prevent and manage the occurrence of bacteremia due to P. aeruginosa.
The study of the progression of infection caused by P. aeruginosa to bacteremia is essential because it provides additional information that is critical in elucidating the mechanism of pathogenesis. As one of the nosocomial infections in healthcare settings, P. aeruginosa threatens the lives of patients and contributes significantly to their comorbidities.4 Healthcare providers experience the challenges of treating nosocomial infections associated with P. aeruginosa due to their ability to colonize infection sites, penetrate tissues and resist most antibiotics. Patients with severe burns are susceptible to bacteremia because they have low immunity and burn sites that allow pathogens to colonize and penetrate their blood easily.2 As reliable animal models, mice allow effective monitoring of P. aeruginosa and its progression to bacteremia in immuno-suppressed mice. The study seeks to establish how health status and immune response vary following infection of healthy and immuno-compromised mice by P. aeruginosa. The hypothesis of the study is that P. aeruginosa infection decreases health status as indicated by the loss of body weight and diminishes immune response among immuno-suppressed mice.
Methods
The experiment was set up to examine the influence of bacteremia on body weight and leukocyte production on normal and immunocompromised mice. Ten healthy mice (N = 10) were selected and randomly assigned to control group (n = 5) and test group (n = 5). The method used in this experiment is appropriate because it uses mice, which are model animals for in-vitro studies of human infections, and it complies with the ethical principles of replacement, refinement, and reduction.5 At the commencement of the experiment, mice in the test group were treated with cyclophosphamide (CY) at the dosage of 200mg/kg to subdue their immunity. In contrast, mice in the control group were not given any treatment for their immune system to function normally. On day 0, mice in both the control and test groups were infected with P. aeruginosa through subcutaneous injection. For nine consecutive days of the experiment, the body weight was measured in grams as an indicator of the health status of mice. The blood sample of each mouse was obtained daily during the first six days and the level of the immune response was assessed using the number of white blood cells produced in millions per milliliter.
Means and standard deviations were used to explore the collected data and establish patterns of variations. Measures of central tendency and dispersion of data provide a preliminary and meaningful statistical summary of raw data.5 Subsequently, means of both body weight and the level of leukocytes were depicted in a clustered graph using lines, bars, and error bars. In the hypothesis testing, paired t-test6 was also used to determine if the apparent differences in body weight and the level of leukocytes were statistically significant.
Results
Figure 1 below summarises trends of variation in means and standard deviations of body weight and leucocyte levels in CY and control mice. Regarding the immune response, the primary axis of the bar graph shows a diminishing trend of leukocyte levels in CY mice relative to the control mice. Mice in both control and test groups exhibited the same level of leukocytes during the first three days. Comparatively, while the level of leukocytes rapidly diminished in CY mice, it increased to about 100 during the fourth day but stagnated at about 60 million white cells per milliliter of blood. Error bars of standard deviations depict that variation in leukocyte levels in both CY mice and control were the same during the first three days. In contrast, during the fourth day through the sixth day, variations in leukocyte levels in control mice increased, while those of CY mice decreased.
The secondary axis of the line graph shows the decreasing trend of body weight in CY mice when compared to control mice. The bodyweight of mice in both the test and control groups remained almost the same for the first four days. However, the means of bodyweight of CY mice declined gradually during the fourth day to the ninth day from about 26 grams to 20 grams, but that of control mice was relatively constant. Error bars of the standard deviations show that weights of both CY mice and control mice had small and constant variations during the nine days of the experiment.
In hypothesis testing, paired sample t-tests (Table 1) confirmed that the means of bodyweight of CY mice decreased statistically significantly in the fifth day through the ninth day (p < 0.000), while the level of leukocytes reduced significantly during the fourth day to the sixth day (P < 0.000).
Table 1. P-Values of Body Weight and Leukocytes Levels.
Discussion
The purpose of the experiment was to establish how the infection of P. aeruginosa decreases health status and immune response in immunocompromised mice. A comparative analysis of means of body weights demonstrated that the health status of CY mice started to decline from the fourth day of infection to the ninth day. The declines in body weights are statistically significant, suggesting that P. aeruginosa affects the health status of CY mice. This finding is consistent with the literature because weight loss is one of the symptoms that patients with bacteremia present due to the infection of P. aeruginosa.3, 4,7 Bacteraemia destabilizes metabolism in mice, leading to weight loss.
Further comparison of means indicated that leukocyte levels of CY mice diminished from the fourth day to the sixth day. Similarly, the diminished levels of leukocytes are statistically significant, which means that CY mice do not have the required immunity to fight infections of P. aeruginosa. Low immunity contributes to a higher level of mortality among immunocompromised patients when compared to immunocompetent individuals.1,8 Insufficient immunity permits an uncontrolled proliferation of bacteria and causes speedy progression to bacteremia.
The findings of the research have great significance to both in-vitro studies and clinical treatment of bacteremia caused by P. aeruginosa. A notable significance of the findings is that weight loss is a pathogenic effect of P. aeruginosa infection and bacteremia. Another significance of the findings is that diminished immune response promotes bacteremia and increases the pathogenicity of P. aeruginosa. Hence, urgent treatment of P. aeruginosa is necessary to avert its progression to bacteremia, resulting in sudden death.
A critical analysis of the methodology reveals a small sample size and a surrogate test as limitations of the experiment. The small sample size reduces the external validity of findings, whereas the surrogate test of bacteremia using bodyweight and leukocyte levels is prone to compounding factors. Therefore, increasing the sample test and using bacteremia as a marker are two ways that would improve the experiment. To contribute to the body of knowledge, future research should examine the progression and pathogenicity of bacteremia in in-vivo and clinical studies.
Conclusion
The experiment demonstrated that P. aeruginosa causes a higher level of bacteremia among CY mice than control mice. Significant weight loss and diminished leukocytes suggest that P. aeruginosa has deadly effects on CY mice due to low immunity. Therefore, future research should use a large sample size, measure bacteremia and undertake clinical studies to validate these findings.
References
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