Significant outcomes
• The influence of meningitis by Klebsiella pneumoniae in the creatine kinase (CK) and mitochondrial respiratory chain activity.
• The meningitis by K. pneumoniae is associated with permanent neurological damage; understand the illness pathophysiology is the main factor that contributes to the therapeutic success.
• Our study helps to better understand the pathophysiology of meningitis by K. pneumoniae.
Limitations
• Animal model.
• The results in animal models should be interpreted with caution before correlate with the clinic.
• This study evaluates the animal model of adult rats.
Introduction
Bacterial meningitis remains a major cause of death and long‐term neurologic sequelae worldwide. However, mortality and morbidity vary by causative organism, age and geographical location of the patient Reference Kim1, Reference Klinger, Chin, Beyene and Perlman2. Klebsiella pneumoniae is a capsulated gram‐negative pathogen that is known to cause infection both in community and mainly as a hospital‐acquired infection presenting as pneumonia, septicemia and meningitis in patients with some predisposing factors Reference Hussen and Shafran3, Reference Ko, Paterson and Sagnimeni4. Klebsiella pneumoniae has recently become an increasingly common cause of the meningitis acute Reference Lu, Chang and Chang5 affecting 14–61% of nosocomial meningitis Reference Barichello, Savi and Simões6, being particularly devastating among immunocompromised patients Reference Sahly and Podschun7 with mortality rates ranging between 30 and 40% Reference Liu, Cheng and Lin8, however in Singapore and parts of northern Taiwan the mortality rate is higher. Furthermore, this pathogen was the most frequent causative of the meningitis, bacteremia and septic shock in patients with liver cirrhosis Reference Su, Chang, Tsai, Huang, Wang and Lu9. Furthermore, bacterial meningitis in young adults in south Taiwan, and Reference Tsai, Lu and Huang10 in some Asian areas, there has been an increased incidence in adults Reference Tang, Chen, Hsu and Chen11. Bacterial invasion in the cerebral spinal fluid (CSF) promotes the release of bacterial components like polysaccharide capsule, peptidoglycan, bacterial DNA and lipopolysaccharide Reference Hirst, Kadioglu, O'Callaghan and Andrew12, Reference Leib and Tauber13, leading to the activation of the brain innate immune defence, releasing a cascade of inflammatory mediators and leukocytes recruitment Reference Grandgirard and Leib14, Reference Klein, Koedel and Pfister15. An excessive release of pro‐inflammatory mediators and reactive oxygen species could contribute to interrupt the bioenergetic activity or the metabolic activity in injured neurons Reference Sellner, Täuber and Leib16, Reference Tauber and Moser17. The CK is vital for normal energy homeostasis by exerting some integrated functions, such as temporary energy buffering, metabolic capacity, energy transfer and metabolic control. The brain, like other tissues with high and variable rates of ATP metabolism, presents high phosphocreatine concentration and CK activity Reference Bessman and Carpenter18, Reference Schnyder, Winkler, Gross, Eppenberger and Wallimann19, Reference Wallimann, Wyss, Brdiczka, Nicolay and Eppenberger20. Furthermore, another generating source of ATP is the oxidative phosphorylation, that is the predominant mitochondria physiological function but additional functions include the production and detoxification of reactive oxygen species, which is involved in various forms of apoptosis, cytoplasmic regulation and mitochondrial matrix calcium, synthesis and metabolites catabolism, so, abnormality any of these processes can lead to mitochondrial dysfunction Reference Brand and Nicholls21. Therefore, the main factor that contributes to the therapy success is to understand the pathogenesis and pathophysiology of the bacteria in the central nervous system (CNS) Reference Sellner, Täuber and Leib16. Thus, to clarify a little more the pathophysiology of this illness, the aim of our study was to investigate the energetic metabolism in the rat brain after meningitis induced by K. pneumoniae
Materials and methods
Infecting organism
Klebsiella pneumoniae was cultured overnight in 10 ml of Todd Hewitt broth, diluted in fresh medium and grown to logarithmic phase. The culture was centrifuged for 10 min at (5000 × g) and resuspended in sterile saline to the concentration of 1 × 106 cfu/ml. The size of the inoculum was confirmed by quantitative cultures Reference Barichello, Savi and Silva22, Reference Irazuzta, Pretzlaff, Zingarelli, Xue and Zemlan23.
Animal model of meningitis
Adult male Wistar rats (250–300 g body weight), from our breeding colony were used for the experiments. All procedures were approved by the Animal Care and Experimentation Committee of UNESC, Brazil, and followed in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80–23) revised in 1996. All surgical procedures and bacterial inoculations were performed under anaesthesia, consisting of an intraperitoneal administration of ketamine (6.6 mg/kg), xylazine (0.3 mg/kg) and acepromazine (0.16 mg/kg) Reference Grandgirard, Schürch, Cottagnoud and Leib24, Reference Hoogman, van de Beek, Weisfelt, de Gans and Schmand25. Rats underwent a cisterna magna tap with a 23‐gauge needle. The animals received either 10 µl of sterile saline as a placebo or an equivalent volume of K. pneumoniae suspension. At the time of inoculation, animals received fluid replacement (2 ml of saline subcutaneously) and were subsequently returned to their cages. The animals were killed in different times at 6, 12, 24 and 48 h after meningitis induction by K. pneumoniae (n = 6 in each group). Another group was treated with antibiotic (ceftriaxone 100 mg/Kg twice a day, i.p.) starting at 16 h and continuing daily until their decapitation at 24 and 48 h after meningitis induction, n = 6 in each group Reference Barichello, Savi and Silva22, Reference Irazuzta, Pretzlaff, Zingarelli, Xue and Zemlan23. The control group did not receive antibiotic treatment Reference Barichello, Silva and Savi26.
Tissue assessment and homogenate preparation
Hippocampus and cortex were homogenised (1:10, w/v) in SETH buffer, pH 7.4 (250 mM sucrose, 2 mM EDTA, 10 mM Trizma base, 50 IU/ml heparin). The homogenates were centrifuged at 800 × g for 10 min and the supernatants kept at −70 °C until used for enzymes activity determination. The maximal period between homogenate preparation and enzyme analysis was always less than 5 days. Protein content was determined by the method described by Lowry et al. Reference Lowry, Rosebough, Farr and Randall27 using bovine serum albumin as standard.
Activities of mitochondrial respiratory chain enzymes
NADH dehydrogenase (complex I) was evaluated according to the method described by Cassina and Radi Reference Cassina and Radi28 by the rate of NADH‐dependent ferricyanide reduction at 420 nm. The activities of succinate: DCIP oxidoreductase (complex II) and succinate: cytochrome c oxidoreductase (complex III) was determined according to the method of Fischer et al. Reference Fischer, Ruitenbeek and Berden29. Complex II activity was measured by following the decrease in absorbance due to the reduction of 2,6‐DCIP at 600 nm. Complex III activity was measured by cytochrome c reduction from succinate. The activity of cytochrome c oxidase (complex IV) was assayed according to the method described by Rustin et al. Reference Rustin, Chretien and Bourgeron30, measured by following the decrease in absorbance due to the oxidation of previously reduced cytochrome c at 550 nm. The activities of the mitochondrial respiratory chain complexes were expressed as nmol/min × mg protein.
CK activity assay
CK was measured in brain homogenates pre‐treated with 0.625 mM lauryl maltoside. The reaction mixture consisted of 60 mM Tris–HCl, pH 7.5, containing 7 mM phosphocreatine, 9 mM MgSO4 and approximately 0.4–1.2 µg protein in a final volume of 100 µl. After 15 min of pre‐incubation at 37 °C, the reaction was started by the addition of 0.3 µmol of ADP plus 0.08 µmol of reduced glutathione. The reaction was stopped after 10 min by the addition of 1 µmol of p‐hydroxymercuribenzoic acid. The creatine formed was estimated according to the colorimetric method of Hughes Reference Hughes31. The colour was developed by the addition of 100 µl 2% μ‐naphtol and 100 µl 0.05% diacetyl in a final volume of 1 ml and read spectrophotometrically after 20 min at 540 nm. Results were expressed as units/min × mg protein.
Statistics
Data about CK and mitochondrial respiratory chain complexes were analysed by Student's t‐test and are expressed as mean ± SME of five to six animals in each group. All analyses were performed using the Statistical Package for the Social Science (SPSS) software version 16.0.
Results
In this study, we evaluated the CK activities and mitochondrial respiratory chain complexes I, II, III and IV in hippocampus and cortex of rats submitted to meningitis by K. pneumoniae.
Survival was analysed by Kaplan–Meier curves (Fig. 1), including all infected animals from the time of infection up to 120 h (n = 38). The animals started dying at 12 h (28.571%) and 24 h (57.143%), after that time surviving animals were killed until 120 h.
Figure 1 Kaplan–Meier survival curves of adults Wistar rats infected by intracisternal inoculation with Klebsiella pneumoniae. Results are expressed as percentage of survival over time with spontaneous death. Rats had a mortality rate of 57.143%.
We verified that complex I was increased at 24 and 48 h in hippocampus in meningitis group without antibiotic treatment (Fig. 2a; p < 0.05); complex II was increased in hippocampus at 48 h in meningitis group without antibiotic treatment (Fig. 2b; p < 0.05); complex III was inhibited at 6, 12, 24 and 48 h in hippocampus in meningitis group without antibiotic treatment (Fig. 2c; p < 0.05) and in complex IV all groups were inhibited in hippocampus after meningitis induced by K. pneumoniae (Fig. 2d; p < 0.05). We also verified that there was no change in CK activity in both structures (Fig. 3).
Figure 2 Activity of the mitochondrial respiratory chain complexes I (a), II (b), III (c) and IV (d) in hippocampus and cortex of rats after meningitis by Klebsiella pneumoniae. Results are expressed as mean ± SD (n = 6) (nmol/min × mg protein). *Statistically significant when compared with sham group, p < 0.05. #Statistically significant when compared between meningitis groups with and without antibiotic in the same period.
Figure 3 CK activity in hippocampus and cortex of rats after meningitis by Klebsiella pneumoniae. Results are expressed as mean ± SD (n = 6) (nmol/min × mg protein). *Statistically significant when compared with sham group, p < 0.05. #Statistically significant when compared between meningitis groups with and without antibiotic in the same period, p < 0.05.
Discussion
There has been an increased incidence of meningitis by Klebsiella sp in adults Reference Tang, Chen, Hsu and Chen11, especially in Asian countries. Among the gram‐negative pathogens implicated in bacterial meningitis, in Taiwan, K. pneumoniae most common in adults Reference Chang, Lu and Huang32, in great part this increase in the number of cases is related to the frequency of neurosurgical procedures, and the large number of patients with head injury from motorcycle accidents Reference Lu, Chang and Lin33; however, chronic diseases like diabetes and liver cirrhosis predispose to meningitis Reference Lu, Chang and Chang5. Bacterial invasion of the meninges induces a complex immune response Reference Coimbra, Voisin and Saizieu Ab34, being that glial cells are an important early source of pro‐inflammatory cytokines during the CNS infection by K. pneumoniae Reference Wen, Chiu, Huang, Chang and Wang35. The complex host inflammatory response from the white blood cells leads to mitochondrial damage initiating the release of the cytochrome c into the cytosol. There are many evidences that mitochondria participates in the caspase‐dependent pathway resulting caspase activation and neuronal damage development in the bacterial meningitis Reference Sellner, Täuber and Leib16, Reference Mitchell, Smith, Braun, Herzog, Weber and Tuomanen36. Furthermore, the brain is highly dependent on ATP; most cell energy is obtained through oxidative phosphorylation, a process that requires the action of various respiratory enzyme complexes situated in a special structure of the inner mitochondrial membrane, the mitochondrial respiratory chain Reference Grimwood, Anderson, Anderson, Tan and Nolan37. Another way to get energy is through the creatine/phosphocreatine/CK system that is essential for normal energy homeostasis by exerting some integrated functions, such as, temporary energy buffering, metabolic capacity, energy transfer and metabolic control Reference Bessman and Carpenter18, Reference Andres, Ducray, Schlattner, Wallimann and Widmer38. Meningitis caused by K. pneumoniae increased complex I at 24 and 48 h and complex II at 48 h in hippocampus. In a previous study, we also showed increased complex II at 24 and 48 h in hippocampus among surviving rats by pneumococcal meningitis Reference Barichello, Savi and Simões6. The increase of the complexes I and II could be compensation mechanisms, because of the decreased activity of complex III and IV. There was an activity decrease in complex III at 6, 12, 24 and 48 h; however at 24 and 48 h with antibiotic treatment the levels did not change in the hippocampus. In complex IV, there was also an activity decrease in all the times in the hippocampus. Furthermore, complex III deficiencies are among the least common respiratory chain abnormalities identified to date in humans Reference Bénit, Lebon and Rustin39, mutations in the cytochrome b gene constitute a major cause of complex III deficiency, and underlie a variety of disorders, such as encephalopathy, optic neurophathy Reference Bénit, Lebon and Rustin39, encephalomyopathy Reference Keightley, Anitori, Burton, Quan, Buist and Kennaway40 although, there are not clinical findings which are specific for complex III deficiency Reference Mourmans, Wendel and Bentlage41, likewise, meningitis caused by K. pneumoniae inhibited the activity of complex III and IV in the hippocampus. The mitochondrial dysfunction can be responsible of oxidative stress due to the lack of reactive oxygen species detoxification and neurological clinical symptoms Reference Brand and Nicholls21. In autopsy studies on patients who died from bacterial meningitis, injure in the CNS was characterised by tissue necrosis in the cortical hemispheres and by apoptotic cell death in the dentate gyrus Reference Nau, Soto and Bruck42. Hippocampal apoptosis is associated with learning and memory deficits observed in survivors of bacterial meningitis Reference Leib, Heimgartner, Bifrare, Loeffler and Täauber43.
The meningitis by K. pneumoniae also is associated with permanent neurological damage Reference Wu, Mai and Sheu44, moreover, understand the illness pathophysiology is the main factor that contributes to the therapeutic success Reference Sellner, Täuber and Leib16. Although descriptive and with high rate mortality our results show that antibiotic prevented in part the changes of the mitochondrial respiratory chain. White blood cells and oxidative stress are responsible to apoptosis activation; furthermore, treatment with antibiotics decreases immunogenic components in the CSF. The complete sterilisation of Neisseria meningitidis from CSF occurs within 2 h of given a parenteral third‐generation cephalosporin and the beginning of sterilisation of Streptococcus pneumoniae from CSF by 4 h into treatment Reference Kim1. In previous studies, we verified that early antibiotic administration prevented cognitive impairment induced by meningitis in rats Reference Barichello, Silva and Batista45 and prevented in part oxidative stress Reference Barichello, Savi and Silva22.
We believe that the damage by K. pneumoniae meningitis is related to mitochondrial respiratory chain dysfunction. The statistic shows differences between groups; however, the work do not have statistical power to generalise the findings. Although descriptive, our findings suggest that the meningitis model could be a good research tool to study the biological mechanisms involved in the pathophysiology of this illness and the secondary alterations of the K. pneumoniae meningitis.
Acknowledgements
This research was supported by grants from work supported by NENASC project (PRONEX program CNPq/FAPESC), CNPq, FAPESC, UNESC, INCT‐TM and L'Oréal‐UNESCO Brazil Fellowship for Women in Science 2011. The authors declare that they have no conflict of interest.
