Veterinary Research Communications

ALTERED ELECTROLYTE HOMEOSTASIS ASSOCIATED WITH EXPERIMENTAL SALMONELLOSIS TREATED WITH AMOXICILLIN AND PEFLOXACIN
--Manuscript Draft-Manuscript Number: Full Title: Article Type: Keywords: Corresponding Author: ALTERED ELECTROLYTE HOMEOSTASIS ASSOCIATED WITH EXPERIMENTAL SALMONELLOSIS TREATED WITH AMOXICILLIN AND PEFLOXACIN Original Article Salmonellosis, Electrolyte homeostasis, Pefloxacin, Amoxicillin Solomon Rotimi Ota, NIGERIA Corresponding Author Secondary Information: Corresponding Author's Institution: Corresponding Author's Secondary Institution: First Author: First Author Secondary Information: Order of Authors: Solomon Rotimi David Ojo, PhD Olusola Talabi Elizabeth Balogun, PhD Oladipo Ademuyiwa, PhD Order of Authors Secondary Information: Abstract: In order to investigate the effects of salmonellosis and its chemotherapy on tissue electrolyte handling, experimental salmonellosis was induced by oral infection of rats with Salmonella typhimurium. Infected animals were treated intraperitoneally with pefloxacin (5.71 mg/kg body weight, 12hourly) and amoxicillin (7.14mg/kg body weight, 8hourly) for 5 and 10 days respectively. Blood and organ electrolyte concentrations were determined photometrically 24 hours and 5 days after the last drug administration. Salmonellosis resulted in ionoregulatory disturbances in the tissues of the animals. This ionoregulatory disturbances were characterised by hyponatremia, hypokalemia, hypocalcemia and hypomagnesemia with concomitant increase in the magnesium concentration with erythrocytes (0.890.02mmol/L to 1.260.11mmol/L, p<0.05), spleen (4.100.03mol/g to 5.080.11mol/g, p>0.05) and heart (6.000.18mol/g to 6.750.32mol/g, p<0.05). The antibiotics further modulated the alterations but a total reversal was not attained. The findings of this study suggest that salmonellosis and its therapy with pefloxacin and amoxicillin perturb electrolyte metabolism and this perturbation could be part of the host-response mechanisms. Solomon Rotimi

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Altered electrolyte homeostasis associated with experimental salmonellosis treated with amoxicillin and

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pefloxacin in rats Rotimi S. O.a*, Ojo D. A.b, Talabi O. A.c, Balogun E. A.d And Ademuyiwa O.d a Biochemistry Unit, Department of Biological Sciences, Covenant University, Ota, Nigeria, b Department of Microbiology, University of Agriculture, Abeokuta, Nigeria, c Medical Centre, University of Agriculture Abeokuta, Nigeria and dDepartment of Biochemistry, University of Agriculture Abeokuta, Nigeria.

Correspondence:

Dr Solomon O. Rotimi Biochemistry Unit, Department of Biological Sciences, Covenant University, Canaan Land, Ota, Ogun State, Nigeria. Ola.rotimi@covenantuniversity.edu.ng Phone: +2348034993065

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Abstract In order to investigate the effects of salmonellosis and its chemotherapy on tissue electrolyte handling, experimental salmonellosis was induced by oral infection of rats with Salmonella typhimurium. Infected animals were treated intraperitoneally with pefloxacin (5.71 mg/kg body weight, 12hourly) and amoxicillin (7.14mg/kg body weight, 8hourly) for 5 and 10 days respectively. Blood and organ electrolyte concentrations were determined photometrically 24 hours and 5 days after the last drug administration. Salmonellosis resulted in ionoregulatory disturbances in the tissues of the animals. This ionoregulatory disturbances were characterised by hyponatremia, hypokalemia, hypocalcemia and hypomagnesemia with concomitant increase in the magnesium concentration with erythrocytes (0.890.02mmol/L to 1.260.11mmol/L, p<0.05), spleen (4.100.03mol/g to 5.080.11mol/g, p>0.05) and heart (6.000.18mol/g to 6.750.32mol/g, p<0.05). The antibiotics further modulated the alterations but a total reversal was not attained. The findings of this study suggest that salmonellosis and its therapy with pefloxacin and amoxicillin perturb electrolyte metabolism and this perturbation could be part of the host-response mechanisms. Keywords: Salmonellosis, Electrolyte homeostasis, Pefloxacin, Amoxicillin.

Introduction

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Foodborne infections cause a major burden on public health services and represent significant costs in many countries (Baeumner 2008). Salmonellosis, one of the most common and widely distributed foodborne disease, is a collective description of a group of feco-oral diseases with symptoms which vary from severe enteric fever to mild food poisoning caused by Salmonella species. Salmonella enteritidis is the most frequently isolated serotype, causing gastroenteritis in most humans and systemic infection in a subpopulation (Cummings, et al. 2010,Rodenburg, et al. 2007). In experimental animal models, it has been reported to infect internal organs following oral infection (Rodenburg, et al. 2007). The initial host response following infection like salmonellosis is characterized by systemic vasodilation, usually resulting in a hyperdynamic state with an elevated heart rate and normal or slightly decreased blood pressure. In the subsequent phase, a hyperdynamic circulation related to the massive vasodilation is characterized by haemodynamics as decreased or borderline blood pressure, hyperventilation as well as fever (Matthews and Battezzati 1993,Khovidhunkit, et al. 2004). Although the pathophysiology of fever and the mechanisms that control the body temperature are complex and are only partially known, the different mediators, specific cations such as Na +, K+, Ca2+, and Mg2+ exert a clear effect on the body temperature (Melesova, et al. 1993). Sitprija V. (2008) reported that artificially induced hyperthermia and hyperventilation due to pyrexia produces catabolic changes that are similar to those observed during infection. Severe infections, including salmonellosis, have been reported to be associated with septic shock, renal dysfunction as well as changes in fluid compartmentalization, endocrine function and membrane transport (Khovidhunkit, et al. 2004,Wiegand, et al. 2009,Fica, et al. 1997); all of which have a causal relationship with perturbations of electrolyte handling and subsequently resulting in multiple organ failure. As a result of these alterations and their consequence of the host, the management of salmonellosis often involves supportive measures as well as the use of antibiotics which are generally employed as the first-line treatment (Kovalenko, et al. 2011,Makhnev 2003,Ogunlesi, et al. 2011). Floroquinolones (e.g. pefloxacin) are considered to be optimal therapy while amoxicillin is often used as alternative effective therapy (Kovalenko, et al. 2011, Makhnev 2003, Basnyat 2010). Amoxicillin is a -lactam aminopenicillin and acts by inhibiting the synthesis of bacterial cell walls while pefloxacin is a fluoroquinolone and blocks the bacterial DNA synthesis (Istifli and Topaktas 2010). Despite the use of antibiotics, aggressive operative intervention, nutritional support, and even anticytokine therapies, multiple organ failure continues to be a major cause of morbidity and mortality in sepsis (Basnyat 2010,Brun-Buisson, et al. 1995,Levine, et al. 2001,Iperepolu, et al. 2008). Recently, the use

of antibiotics has been reported to be limited by the associated toxic effects which include electrolytes disorders.

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Zietse et al. (2009) reported that these disorders can occur while renal function remains near to normal. Renton (2005) also stated that the expression and activity of cytochrome P 450 is altered during periods of infectious disease and most of the major forms of this enzyme complex are affected in this manner that leads to a decrease in the capacity of the liver and other organs to handle drugs. Thus, it is essential to study the impact of antibiotic treatment on tissue electrolyte handling in experimental salmonellosis. Materials and Methods Chemicals Pefloxacin was a product of Lek Pharmaceutical and Chemical Company, Ljubljana, Slovenia, while amoxillin was obtained from Beecham Pharmaceuticals, Brentford, England. All other chemicals used in this study were of the purest grade available and were obtained from British Drug House (BDH) Chemicals Limited, Poole, England and Sigma-Aldrich, Missouri, U. S. A. Bacteria strain Salmonella enterica serovar Typhimurium strain TA98 (obtained from the Nigerian Institute of Medical Research (NIMR), Yaba, Lagos, Nigeria) was grown for 48hours under static conditions in nutrient broth (CMI, Unipath, UK). The organism was maintained on nutrient agar slant at 4C. Bacteria were harvested from the slant, suspended in 100ml nutrient broth and allowed to grow at 37oC for 12 h (late logarithmic growth phase). The cells were spun at 4,300 x g at 4C for 10 min. Once the supernatant was discarded, the cells were resuspended in 20ml of buffer and the centrifugation was repeated. The final washed pellet of bacteria cells was resuspended in 10 ml of phosphate-buffered saline (PBS) pH 7.4. The stock concentration of bacteria was determined from an optical density curve with a spectrophotometer. Animal handling and experimental protocol Experimental protocols were conducted in accord with guidelines of the Institutional Animal Care and Use Committee and were approved by the Animal Ethical Committee of the Department of Biochemistry, University of Agriculture Abeokuta. Pathogen free male albino rats weighing 230-280g obtained from the NIMR were used for the study. The rats were housed in metabolic cages at room temperature (22-24oC) and had free access to rat chow and autoclaved tap water throughout the period of the experiment. Animals were acclimatized to the housing and dietary conditions for 2 weeks after which they were fasted overnight and infected orally with 0.2ml phosphate-buffered saline (PBS) pH 7.4 containing 1x 1010 colony-forming unit (CFU) live culture of the

bacteria as described Hung and Wang (2004). Five animals that were not infected and received 0.2ml PBS orally

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served as the normal control. In previous Salmonella infection studies in rats (Havelaar, et al. 2001,Naughton, et al. 1996) it was established that monitoring functional infection outcomes like Salmonella colonisation, translocation and infection induced changes, follow-up of infected rats for at least 3 to 4 days is needed. Therefore the infected rats were left for four days after which fresh faecal samples were collected to quantify Salmonella colonisation daily, as described by van Ampting (2009). Infected animals were divided into 7 groups of 5 animals each. While 1 group served as infection control group, three groups were treated with amoxillin (7.14mg/kg body weight, 8 hourly) and the remaining three groups with pefloxacin (5.71mg/kg body weight, 12 hourly) for 5 and 10 days respectively. The antibiotics were constituted in 5% dextrose and were prepared fresh before each administration. They were administered in a total volume of 0.1ml. Control animals received equivalent volume of 5% dextrose. All drug administration was by the intraperitoneal route. At the end of the antibiotic treatment and 5 days after the discontinuation of the antibiotics, blood was collected from the animals into heparinised tubes by cardiac puncture under light ether anaesthesia after an overnight fast. Liver, kidney, brain, heart and spleen were removed, rinsed in ice-cold saline, blotted dry and kept frozen at 20oC. Sample preparation Blood samples were centrifuged to separate plasma and red blood cells. The erythrocytes were washed twice with isotonic buffer. Erythrocyte membranes were prepared according to the method described by Hanahan and Ekholm (1974). Briefly, aliquots of the erythrocytes suspended in isotonic Tris-HCl buffer were recentrifuged at 5000rpm for 15 minutes at 4C. The supernatant was again removed by careful suction and a few red cells were sacrificed to remove any remaining buffy layer. This washing procedure was repeated twice. The washed cells were then suspended in isotonic Tris-HCl buffer pH 7.6 to an approximate hematocrit of 50% and were kept on ice. The samples were mixed gently by inversion for about 1 minute before membrane preparation. 5ml aliquots of the 50% cell suspensions were transferred to 50ml polyethylene tubes. 30ml of hypotonic Tris-HCl buffer pH 7.6 were added to the cell suspension for osmotic lysis. After allowing the tubes to stand for about 10 minutes, they were centrifuged at 20,000rpm for 20 minutes at 4C. The supernatants were discarded and the pellets resuspended in 10 ml Tris-HCl and centrifuged for 20 minutes at 20,000rpm at 4C. The pellets were washed four times until the membranes were colourless. Finally, the resultant pellets were rinsed twice with 100l cold

Tris-HCl buffer and poured into Eppendorf tubes. The membrane suspensions were kept frozen in this latter

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buffer at -20C. A portion of the tissue (0.25g) was transferred to 2.0ml chilled 0.25mol/L sucrose and homogenized and digested following the method of Chwelatiuk et al., (2006). In brief, 1ml of the whole homogenate (or 100l in case of erythrocytes) was placed in a Pyrex tube with 2.0ml of concentrated nitric acid. After 20hours of sample digestion at room temperature, 72% perchloric acid (0.5ml) was added and the mixture was heated at 150oC until a clear digest was obtained. The digest was then cooled to room temperature and analyzed for sodium, potassium, calcium and magnesium content. Analyses Sodium and potassium concentrations in the plasma, erythrocyte ghost as well as the digests of erythrocytes and the organs were determined by flame photometry (Mahboob, et al. 1996). Calcium and magnesium concentrations in the samples were determined spectrophotometerically according to the procedures described by Abam et al., (2008) using kits supplied by Quimica Clinical aplicada S. S., Amposta, Spain. Statistical analysis Data are expressed as meanS.E.M. One way analysis of variance (ANOVA) followed by Duncan Multiple Range Test was used to analyse the results with p < 0.05 considered significant. Associations among the parameters and their magnitudes were tested for using Multiple Linear Regression analysis.

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Results In fig. 1 is the representation of the effects of pefloxacin and amoxicillin on the fecal bacteria load of the rats. The oral infection of the rats with S. typhimurium resulted in salmonellosis as observed by the apperance of S. typhimurium in the feces of the rats. The concentration of S. typhimurium cultured in the feces was significantly (p<0.05) decreased upon the administration of pefloxacin and amoxicillin for 5 days. The decrease was sustained even 5 days after cessation of antibiotic administration. Table 1 shows the summary of the effects of pefloxacin and amoxicillin on Na +, K+, Ca2+ and Mg2+ concentrations in plasma, erythrocyte and erythrocyte ghost of rats infected with S. typhimurium. Salmonellosis caused a significant (p<0.05) decrease in plasma Na +, K+, Ca2+ and Mg2+ concentrations. After 5 days of treatment with pefloxacin and amoxicillin, there was a rebounce to values close to control in the level of plasma sodium. This elevation was maintained on day 15 in rats treated with both drugs. The decrease in plasma potassium concentration occasioned by salmonellosis was not significantly (p<0.05) altered by any of the treatments. Treatment of salmonellosis with both antibiotics for 5 days led to a reversal of the decrease in the plasma calcium level. This was however followed by a significant (p<0.05) decrease on days 10 and 15 in rats treated with both drugs. The plasma magnesium level was only significantly (p<0.05) elevated in infected rats treated with pefloxacin for 10 days. In the erythrocyte, salmonellosis led to a significant (p<0.05) decrease in sodium, potassium and calcium concentration as well as a significant (p<0.05) increase in magnesium concentration. Pefloxacin and amoxicillin therapy reversed this trend in the level of sodium and potassium with amoxicillin day 5 group having its sodium level not significantly (p>0.05) different from control. The erythrocyte ghost sodium and potassium concentrations were not significantly (p>0.05) altered by salmonellosis and its chemotherapy. Salmonellosis, however, induced a significant (p<0.05) increase in

erythrocyte ghost calcium and magnesium concentrations. Chemotherapy did not seem to reverse this increase except for calcium whose concentration returned to control values after 5 days of pefloxacin withdrawal. Table 2 summarizes the effects of pefloxacin and amoxicillin on Na +, K+, Ca2+ and Mg2+ concentrations in liver, kidney and brain of rats infected with S. typhimurium. Salmonellosis led to significant (p<0.05) decrease in hepatic potassium and magnesium concentrations with significant (p<0.05) increase in hepatic calcium level. None of the treatments significantly altered the levels of sodium whereas there was a slight

decrease in hepatic potassium. After 10 days of pefloxacin and amoxicillin therapy, the level of calcium was

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significantly (p<0.05) decreased. The decrease in hepatic magnesium concentration was reversed by pefloxacin on day 15 and amoxicillin on day 5. Salmonellosis led to a significant (p<0.05) increase in renal sodium concentration as well as significant (p<0.05) decreases in potassium, calcium and magnesium concentrations. In rats treated with pefloxacin the increase in sodium level was reversed on day 5 and by day 15 an increase to a level not significantly (p>0.05) different from the infection control was observed. Although rats treated with amoxicillin had a significantly (p<0.05) reduced level on day 10, a further increase to a level significantly (p<0.05) higher than the infection control was observed. The reduced renal levels of potassium and calcium were significant (p<0.05) increased by all the treatments although at the end of chemotherapy, control values were not attained. The decreased renal magnesium levels were reversed in rats treated with both antibiotics. Salmonellosis caused a significant (p<0.05) decrease in brain sodium and magnesium concentrations as well as a significant (p<0.05) increase in potassium concentration. Neither salmonellosis nor its chemotherapy seems to alter the levels of calcium in the brain. A significant (p<0.05) increase was however observed in level sodium in the brain after treating salmonellosis with pefloxacin and amoxicillin for 5 days and by day 15, amoxicillin treated rats brain sodium level was not significantly (p<0.05) different from the normal control. Both antibiotics caused a reversal of the increased brain potassium to a level not significantly (p<0.05) different from each other but significantly (p<0.05) higher than the normal control. On day 15, rats treated with both antibiotics had decreased brain magnesium levels reversed though to a level still significantly (p<0.05) lower than the normal control. In the spleen, the level of sodium was significantly (p<0.05) decreased while magnesium was significantly (p<0.05) increased. While both drugs reversed this trend in sodium level, the levels of potassium, calcium and magnesium were not significantly (p>0.05) altered by any of the treatments. Cardiac sodium and magnesium levels were significantly (p<0.05) increased while calcium and magnesium levels were significantly (p<0.05) decreased in the rats as a result of salmonellosis. Although treatment of murine salmonellosis with both drugs reversed the increased cardiac sodium and magnesium levels. The levels for sodium were still significantly (p<0.05) higher than the normal control. Pefloxacin and amoxicillin therapy of murine salmonellosis caused a further significant (p<0.05) decrease in cardiac potassium. The decrease in cardiac calcium concentration was reversed by pefloxacin and amoxicillin on day 15.

Intensity of associations between fecal bacteria load and Na+, K+, Ca2+ and Mg2+ concentrations in rats infected

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with S. typhimurium was shown in Table 3. The fecal bacteria load was positively correlated with liver calcium (r= 0.542, p<0.01), kidney sodium (r=0.420, p<0.01), spleen magnesium (r=0.433, p<0.01), heart sodium (r= 0.474, p<0.01), erythrocyte ghost calcium (r=0.540, p<0.01) and erythrocyte ghost magnesium (r= 0.557, p<0.01). However, a negative correlation was observed with liver potassium (r= -0.549, p<0.01), liver

magnesium (r= -0.694, p<0.01), kidney calcium (r= -0.333, p<0.05), kidney magnesium (r= -0.424, p<0.01), brain sodium (r= -0.484, p<0.01), brain magnesium (r= -0.465, p<0.01), heart potassium (r= -0.339, p<0.05), heart calcium (r= -0.536, p<0.01). Plasma sodium (r= -0.476, p<0.01), plasma magnesium (r= -0.493, p<0.01), erythrocyte potassium (r= -0.638, p<0.01), erythrocyte calcium (r= -0.353, p<0.05).

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Discussion This study shows a profound appearance of Salmonella in the feces of the rats infected with S. typhimurium after 3days of infection. This is consistent with other studies (Rodenburg, et al. 2007,Islam, et al. 2000,Mehta, et al. 1999) in which gene expression changes in the rat colon upon colonization following oral Salmonella infection was also reported. Rodenburg et al. (2007) reported that the earliest responses were on the mucosal transports like chloride channel calcium activated 6, H+/K+ transport ATPase as well as oxidative stress related genes. The alterations in transport of chloride through the apical border of the intestinal epithelial cells are known to be followed by the efflux of water as well as electrolytes into the intestinal lumen, resulting in diarrhea (Sitprija 2008). One consequence of the upregulation of the oxidative stress related genes upon colonization of the colon by Salmonella is a concomitant increase in the synthesis of nitric oxide by the vascular endothelial cells (Henard and Vazquez-Torres 2011). Izzo et al. (1998) reported that under pathophysiological conditions, nitric oxide may be produced at higher concentrations that are capable of evoking net secretion thereby contributing to loss of water and electrolyte. These pathophysiological changes in the intestine could have result in the hyponatremia, hypocalcemia, hypokalemia and hypomagnesemia observed in these study. Similar fluid and electrolyte alterations have been reported in other febrile conditions and infectious disease (Zaloga and Chernow 1987,Sankaran, et al. 1997,Chesney, et al. 1981). The results of this study indicated that the efflux of the electrolyte from the plasma was accompanied with a corresponding decrease in the levels of sodium and potassium in the erythrocytes while their levels remained unchanged in the erythrocyte membrane following salmonella infection. While the level of calcium in the erythrocytes also decreased, there was an increase in magnesium concentration in the erythrocytes and erythrocyte membrane as a result of salmonellosis. Following bacterial invasion of the host system, a wide of changes are triggered in order for the host to protect itself and facilitate repair process. Elevation of magnesium in the erythrocytes could have a part of the host response salmonellosis. During infection there is an upregulation of energy production pathways. Increase in oxidation of glucose to form lactate has been reported in septic patients (Gutierrez and Wulf 2005,Gore, et al. 1996). Erythrocytes only depend on glycolysis for energy for energy production; hence, enhancement of this pathway is essential during infection like salmonellosis. One mechanism of increasing cellular glucose utilization is by elevation of magnesium concentration (Barbagallo, et al. 1999). Magnesium is known to directly influence insulin function and is also important for the rate limiting enzyme in glycolysis (Barbagallo, et al. 1999). Although erythrocytes are not

insulin-sensitive cells, they are insulin and glucose responsive in ionic terms. Barbagallo et al. (1999) also

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reported that magnesium depletion renders the erythrocytes more sensitive to oxidative damage; thus, magnesium may itself possess antioxidant properties possible by affecting the rate of spontaneous dismutation of superoxide ion (Afanas'ev, et al. 1995) which are abundantly produced by macrophages as part of the host innate immune response to salmonellosis (Janssen, et al. 2003). The data presented in this study showed that the degree of alterations in the electrolyte handling by the tissues investigated is influenced by the fecal bacterial load. As part of the metabolic response of the host to the invading bacterial, substrates are mobilized from the periphery to be utilized by the visceral tissues and immune cells resulting in altered energy balance in the tissues (Hasselgren, et al. 1986). Studies have shown that adjustments in glucose metabolism alter the cellular ionic regulation (Takahashi, et al. 1995,Dixit and Lazarow 1967,Duelli, et al. 1999). More than any other organ, the brain is entirely dependent on a continuous supply of glucose from the circulation since glucose is almost the sole substrate for its energy metabolism. Sodiumdependent isoform of glucose transporter proteins mediate the transport of glucose against a concentration gradient. The driving force is the flux of sodium along an electrochemical gradient that is directed opposite to the transport of glucose (Kumagai 1999). This transporter has recently been reported to be present in rat brain (Yu, et al. 2010). This could, therefore, explain the observed salmonellosis-induced decrease in brain sodium concentration. The need for increased glucose utilization could have also induced the observed elevation in splenic magnesium concentration (Kawai, et al. 2006). Apart from spleen phagocytosing circulating bacteria, it is well known that the germinal centers in splenic white pulp are sites of active production of B lymphocytes during antigenic stimulation (Kawai, et al. 2006). Innate immunity to Salmonella initiates marked splenic erythropoiesis (Jackson, et al. 2010) which is associated with an up-regulation of a distinct transporter, Glut4 resulting in increased glucose transport in mice spleen (Montel-Hagen, et al. 2008). Humphery (2004) reported the diverse expression of glut1, glut2 and glut3 in chicken tissues at different stages of growth. It therefore seems that the mechanism of glucose transport into cells varies from one organism to another and from one organ to another. Conversely, we observed a decrease in the level of magnesium and potassium in the liver which was accompanied with increase in calcium and sodium concentrations as a result of the infection. An important defect in glucose homeostasis during infection is that hepatic glucose metabolism, which include gluconeogenesis (McGuinness 2005) and increased insulin resistance (Gauglitz, et al. 2010,Jia, et al. 2009,Bell 2000). Insulin causes cellular uptake of Mg2+ (Romani 2011). Low concentration can also result in dysfunction

of the Na+-Mg2+ exchanger (Romani 2011,Agus 1999), leading to increased cellular sodium (Romani 2011). If

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there is insufficient magnesium for adequate ATP utilization, then the primarily extracellular cations Na+ and Ca2+ tend to leak into the cells and the primarily intracellular cations K + and Mg2+ tend to leak out. This leakiness disrupts proper gradients and cellular function (Romani 2011,Resnick 1992). It is also worthy of note that the decrease in level of Mg2+ could allow accelerated free radical damage and this, coupled with elevation of Ca2+, is an indication of apoptotic damage in the hepatic tissue (Rosenstock, et al. 2004). Similar to our observation in the liver, the level of sodium was also increased with a concomitant decrease in potassium level in the heart and kidney of the rats following Salmonella infection. It was however interesting to note that the level of calcium reduced. Although the reason for the decrease in calcium levels in these tissues is not clearly known, we observed that is has a direct correlation with the fecal bacterial load. The bacterial antigenic determinant is lipopolysaccharide (LPS). This LPS has been reported to reduce basal Ca 2+ concentration and also impair calcium responses to both thrombin and bradykinin in rat mesandial cells contractile cells that share many characteristics of vascular smooth muscle (Murray, et al. 1997). Using bovine aortic myocytes, Murray et al. (1998), suggested that LPS-induced vascular contractile impairment is at least partly mediated by an NO-dependent impairment of myocyte Ca2+ mobilization. This has been implicated in the vasoconstrictor-resistant systemic vasodilation, as well as in the failure of multiple organ systems; which is the characteristic of septic shock (Umans, et al. 1993). No specific study is available regarding the effects of experimental Salmonellosis on levels of sodium, potassium, calcium and magnesium in rat tissues. However, using rhesus monkeys as model of Salmonella sepsis, Nichol et al. (1981) reported altered serum, muscle and urine sodium and potassium concentrations and suggested that the alterations were the sum of renal and extrarenal factors controlling the electrolyte metabolism, and that some of the most remarkable alterations occur as renal function returned to normal. The findings of this research also demonstrated that pefloxacin and amoxicillin therapy did not reverse the salmonellosis-induced ionoregulatory disruptions in the rat tissues. It is noteworthy that the disruptions still persisted to an appreciable extent even 5days post treatment. Zietse et al. (2009) reported that the use of antibiotics often results in the onset of electrolyte disorders. They stated that renal tubules can be affected by antibiotics at various locations. Penicillins and ciprofloxacin were reported to cause a diverse range of disorders in the collecting ducts resulting in ionoregulatory disruptions which included hyponatremia and hypokalemia (Zietse, et al. 2009). Penicillins, like amoxicillin, might cause high-anion-gap acidosis through disturbance of the -glutamyl cycle, which lead to rising serum concentrations of pyroglutamic acid (Fenves, et al. 2006).

Pyroglutamic acidosis during penicillin treatment has been reported (Peter, et al. 2006). During treatment with

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fluoroquinolones, Kushner et al. (2001) observed hyponatremia and these was related to increased vasopressin. We conclude that salmonellosis induced alterations in electrolytes homeostasis in rat tissues and these alterations persisted through and beyond the cause of chemotherapy with pefloxacin and amoxicillin. The insight provided herein, especially on the tissues as most studies have focused on plasma, should pave way for further understanding the pathophysiology of salmonella infection and its treatment.

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49

Figure 1: Effects of pefloxacin and amoxicillin on fecal bacteria load and weight change of rats infected with S. typhimurium

1 2 3 4 Table 1: Effects of pefloxacin and amoxicillin on Na+, K+, Ca2+ and Mg2+ concentrations in plasma, erythrocyte and 5 erythrocyte ghost of rats infected with S. typhimurium 6 7 Plasma Normal Infection Pefloxacin Pefloxacin Pefloxacin Amoxicillin Amoxicillin Amoxicillin 8 control control day 5 day 10 day 15 day 5 day 10 day 15 9 + a d c b c c b 10 Na (mmol/l) 140.960.76 118.384.10 134.171.70 129.081.07 133.320.66 132.470.01 126.535.62 137.571.70e 11 + a b b b b b b K (mmol/l) 5.810.33 5.050.09 5.310.18 5.060.29 5.060.18 5.540.19 5.310.10 4.690.57c 12 2+ Ca (mmol/l) 1.590.02a 1.160.02b 1.600.03a 1.290.03b 1.250.10b 1.490.04a 1.280.07b 1.290.01b 13 14 Mg2+ (mmol/l) 1.260.05a 0.990.04b 0.880.15c 1.100.02d 0.870.02c 0.990.02b 1.030.01b 1.000.08b 15 16 Erythrocyte Na+ (mmol/l) 3.790.00b 3.140.05b 3.580.20c 17 3.260.17c 3.530.12c 3.760.29a 3.130.20b 3.260.17c + b b c d d c d 18 K (mmol/l) 133.891.29 118.971.74 102.964.99 129.340.50 127.466.74 108.990.84 128.250.60 125.193.66d 19 2+ Ca (mmol/l) 0.430.02a 0.330.02b 0.380.03c 0.360.02c 0.360.01c 0.320.02b 0.390.05c 0.330.04b 20 Mg2+ (mmol/l) 21 0.890.02a 1.260.11c 0.880.16a 1.010.05b 1.340.10d 1.330.04c 1.020.15b 1.110.02e 22 Erythrocyte ghost 23 + Na (mol/g) 0.250.03a 0.260.03a 0.300.01a 0.280.03a 0.260.02a 50.270.02a 0.240.04a 0.280.03a 24 + a a a a a a a K (mol/g) 25 0.0300.001 0.0290.001 0.0290.001 0.0290.001 0.0290.001 0.0300.001 0.0290.001 0.0290.001a 26 Ca2+ (mol/g) 0.760.02a 1.380.02c 0.940.02d 0.920.02d 0.790.07a 0.930.02d 1.020.13b 0.880.07d 27 2+ Mg (mol/g) 0.210.01a 0.330.02b 0.350.02b 0.310.01b 0.370.04b 0.330.01b 0.310.01b 0.330.03b 28 Each value represents the meanS.E.M of 5 rats. Values within the same row with different superscripts are significantly different at 29 30 p<0.05. 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49

1 2 3 4Table 2: Effects of pefloxacin and amoxicillin on Na+, K+, Ca2+ and Mg2+ concentrations in liver, kidney and brain of rats infected with 5 6S. typhimurium 7 Normal Infection Pefloxacin day Pefloxacin day Pefloxacin day Amoxicillin day Amoxicillin day 8 Liver control control 5 10 15 5 10 9 + a a a a a a 10 Na (mol/g) 30.570.91 40.761.29 39.780.79 38.322.08 34.652.04 34.247.80 31.792.00a 11 + a b b b b b K (mol/g) 65.964.65 49.360.31 49.652.12 50.102.32 51.132.55 51.610.12 48.613.73b 12 2+ Ca (mol/g) 1.520.01a 1.890.11b 1.890.16b 1.670.03c 1.740.09c 1.920.09b 1.660.02c 13 14 Mg2+ (mol/g) 6.410.29a 4.130.24d 3.990.12d 3.800.10c 4.320.16d 4.490.04b 4.000.23d 15 16 Kidney Na+ (mol/g) 27.551.51a 32.930.08c 29.590.79b 17 30.570.91e 32.611.29c 32.610.00c 30.161.00b + a b c c c c 18 K (mol/g) 37.441.78 29.641.41 36.311.66 32.900.98 32.993.29 34.471.65 35.273.26c 19 2+ Ca (mol/g ) 1.610.10a 1.340.07b 1.440.05c 1.400.10c 1.400.05c 1.420.12c 1.510.02c 20 Mg2+ (mol/g) 7.700.20a 6.030.37b 7.410.57d 21 7.250.58c 7.120.38c 5.650.23b 6.440.47b 22 Brain 23 + Na (mol/g) 26.490.91e 30.490.08a 22.831.00d 28.780.21e 26.901.63c 23.480.79b 23.481.51b 24 + a b c c c a K (mol/g) 55.974.70 63.143.77 57.061.44 60.475.12 61.152.45 49.654.31 59.581.96c 25 26 Ca2+ (mol/g) 1.290.31a 1.420.11a 1.410.21a 1.520.11a 1.600.09a 1.270.03a 1.600.06a 27 2+ Mg (mol/g) 6.610.19a 5.600.37b 5.700.23b 5.500.28b 6.160.11c 5.740.23b 5.530.30b 28 29 Spleen 30 Na+ (mol/g) 29.590.79a 26.091.63c 28.531.29a 26.901.00c 28.530.00a 28.532.23a 28.530.00a 31 + a a a a a a K (mol/g) 52.763.23 54.841.69 57.061.37 54.102.41 54.842.93 58.542.12 56.023.85a 32 33 Ca2+ (mol/g) 0.920.09a 1.060.03a 1.060.11a 1.010.12a 0.940.05a 0.840.03a 1.010.12a 34 Mg2+ (mol/g) 4.100.03a 5.080.11b 4.640.17b 4.700.15b 5.140.25b 4.880.09b 4.790.37b 35 36 Heart 37 Na+ (mol/g) 39.781.51a 45.650.82b 45.900.79b 43.212.08c 42.802.73c 43.860.79c 44.760.24b + 38 a b d a c c K (mol/g) 68.061.50 65.812.25 56.173.71 66.404.79 61.361.24 59.880.94 57.663.02e 39 2+ Ca (mol/g) 1.340.04a 1.030.05b 1.090.09b 1.040.06b 1.200.03c 1.070.10b 1.160.05c 40 41 Mg2+ (mol/g) 6.000.18a 6.750.32c 6.840.23c 7.140.27c 7.400.30b 6.500.23c 7.360.29b 42 Each value represents the meanS.E.M of 5 rats. Values within the same row with different superscripts are significantly different at 43 p<0.05. 44 45 46 47 48 49

Amoxicillin 15 34.730.87a 50.754.20b 1.730.09c 4.320.09d 33.670.79d 32.992.17c 1.390.08c 6.210.26b 31.630.79a 57.802.89c 1.660.07a 5.910.12c 24.460.01b 55.221.71a 1.010.07a 4.840.09b 41.821.51e 62.461.18c 1.210.04c 7.610.79b

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49

Table 3: Intensity of association between fecal bacteria load and Na+, K+, Ca2+ and Mg2+ concentrations in rats infected with salmonella

Parameters Fecal Bacteria load vs. Liver K+ Fecal Bacteria load vs. Liver Ca2+ Fecal Bacteria load vs. Liver Mg2+ Fecal Bacteria load vs. Kidney Na+ Fecal Bacteria load vs. Kidney Ca2+ Fecal Bacteria load vs. Kidney Mg2+ Fecal Bacteria load vs. Brain Na+ Fecal Bacteria load vs. Brain Mg2+ Fecal Bacteria load vs. Spleen Mg2+ Fecal Bacteria load vs. Heart Na+ Fecal Bacteria load vs. Heart K+ Fecal Bacteria load vs. Heart Ca2+ Fecal Bacteria load vs. Plasma Na+ Fecal Bacteria load vs. Plasma Mg2+ Fecal Bacteria load vs. Erythocyte K+ Fecal Bacteria load vs. Erythrocyte Ca2+ Fecal Bacteria load vs. Erythrocyte ghost Ca2+ Fecal Bacteria load vs. Erythrocyte ghost Mg2+ a Correlation is significant at p< 0.01 b Correlation is significant at p< 0.05

Correlation coefficient -0.549a 0.542a -0.694a 0.420a -0.333b -0.424a -0.484a -0.465a 0.433a 0.474a -0.339b -0.536a -0.476a -0.493a -0.638a -0.353b 0.540a 0.557a