Comparing with previously used rodent models [3, 4], the sepsis model used in the present study takes advantage of the porcine similarities to the human inflammatory response . In addition, the model was further developed to resemble the situation for patients with sepsis admitted to an intensive care unit, where therapeutic actions such as sedation, mechanical ventilation and vasopressors may modify the inflammatory response [20, 29, 30]. Despite the mitigating effects of these intensive care measures and in the absence of the free endotoxin that has been shown to be liberated during the antibiotic-induced killing process in vitro , bacteria killed by the PBP-3-active antibiotic cefuroxime resulted in increased TNF-α and IL-6 responses compared with those caused by live, untreated bacteria. There were also significant cytokine-associated differences in leukocyte activation and capillary leakage. Although significant correlations do not establish causal relationships, the differences observed in the inflammatory response might be further linked to changes in cellular metabolism and organ function.
Because one dose of PBP-3-active antibiotics does not kill all bacteria [5, 7], some remaining live bacteria were also included in the infusate. Because of the low number of live bacteria relative to the number of killed bacteria and because of the extra dose of antibiotics administered after 2 h, it is unlikely that these live bacteria affected the cefuroxime-induced response. Moreover, a mixture of killed and live bacteria is an obvious phenomenon in vivo. Animals that were given cefuroxime-killed bacteria also demonstrated a higher inflammatory response than animals given heat-killed bacteria, which resulted in a response similar to that caused by live bacteria. This finding indicates that the cefuroxime-induced inflammatory increase was not elicited simply because the bacteria were not alive.
IL-10, an early marker associated with the initiation of anti-inflammatory reactions, increased in parallel with TNF-α and IL-6, supporting initial activation of both pro- and anti-inflammatory responses in this model, which is similar to early clinical responses shown in intensive care patients with sepsis [31, 32]. IL-10 was consistently higher in the cefuroxime group as compared with the other groups.
We hypothesize that the bacterial morphological appearance with elongation and formation of filamentous forms associated with PBP-3 activity described previously [16, 17, 33] and seen in the present experiment plays an important role in the induction of inflammation and organ dysfunction. Larger bacterial areas due to elongation most certainly increase the amount of pathogen-associated molecular patterns that can bind with extracellular domains on host inflammatory cells. Because the supernatants after treatment with antibiotics or heat were removed before infusion into the animals, the free endotoxin in this study probably originated from killed bacteria in vivo during the further process of fragmentation. The correlations between free endotoxin peak levels and TNF-α, IL-6 and leukocyte responses suggest that liberated endotoxin might contribute to the inflammatory response. However, a study on the porcine dose-response of cytokines to endotoxin  indicates that the limited concentration differences between the treatment groups would only result in a minor modification of the cytokine response.
Live bacteria are quickly eliminated from the circulation because of an effective immune system and the process of bacterial killing takes mainly place in the spleen and liver . Using this intensive care model in a previous study, animals with live E. coli sepsis treated with cefuroxime demonstrated a more pronounced cytokine increase, leukocyte activation and organ dysfunction than control animals. These events occurred without any increase in plasma free endotoxin . The increased inflammatory response might have been the result of the killing process with or without local endotoxin liberation in the liver and the spleen or the result of the antibiotic-killed bacteria per se. The present results support the findings of that study and further demonstrate that killed bacteria alone can elicit the inflammatory response and that the mechanism of the preceding killing results in inflammatory responses of different magnitudes.
Our results support Mock et al.’s clinical findings in which patients with Gram-negative sepsis treated with PBP-3-active antibiotics showed higher mortality than patients treated with antibiotics with little or no PBP-3 activity . Randomized clinical studies comparing the effect of a PBP-3 active antibiotic with a non-PBP-3 active antibiotic have shown no or only trends towards differences in endotoxin levels, cytokines and outcomes [35–38]. However, considering the mild severity of sepsis and the low number of patients included in these studies, the chance of detecting a difference was minimal. A randomized clinical study specifically on patients with septic shock would be of value but has not yet been performed, probably because of the inherent difficulties in conducting such a study. Nevertheless, the phenomenon of an antibiotic-induced inflammatory response and deterioration is in agreement with Mignon et al.’s study on intensive care patients with sepsis in which deterioration occurred in almost half of the patients within 4 h after the start of antibiotic treatment .
As has previously been shown in vitro [18, 19], this large animal study demonstrated that the addition of tobramycin reduced the cefuroxime-induced inflammatory response also in vivo. This effect seems to be mainly mediated by non-endotoxic effects. The morphologic elongation of bacteria seen after single PBP-3 active treatment was absent when tobramycin was added, which might explain this result. However, in agreement with several in vitro studies [7, 39, 40] the levels of endotoxin were lower in the combination group, suggesting that differences in endotoxin levels might have contributed to the reduced inflammatory response in the combination group. In addition to its binding to the ribosome causing mistranslation and misfolded membrane proteins as well as changes in the bacterial surface, the aminoglycoside-induced inhibition of endotoxin synthesis may play a role [13, 41]. These results, in conjunction with the finding of a more rapid killing , indicate that the beneficial effects of adding an aminoglycoside to a β-lactam antibiotic found in vitro also are seen in vivo. A more rapid killing and a decreased inflammatory response thus offer possible explanations to Kumar et al.’s clinical findings in which patients treated with the combination of a β-lactam antibiotic and an aminoglycoside demonstrated improved survival in comparison with propensity-matched control patients . Although a theoretical experimental model, the results from this large animal intensive care model lend some support to the hypothesis that a combination of antibiotics may arrest inflammation more rapidly and effectively than single treatment which was one of the questions raised by IDSA [22, 23] in their criticism of the Surviving Sepsis Campaign’s recommendation to combine antibiotics for the treatment of patients in septic shock .