Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 1393

Antibiotic-induced BacterialToxin Release – Inhibition byProtein Synthesis Inhibitors

BY

GUNILLA HJERDT-GOSCINSKI

ACTA UNIVERSITATIS UPSALIENSISUPPSALA 2004

List of papers

This thesis is based on the following papers, which will be referred to in the text by their Roman numerals:

I Sjölin J., Goscinski G., Lundholm M., Bring J. and I. Odenholt. Endotoxin release from Escherichia coli after exposure to tobra-mycin: dose-dependency and reduction in cefuroxime-induced endotoxin release. Clinical Microbiology and Infection. 2000; 6:74-81.

II Goscinski G., Lundholm M., Odenholt I. and J. Sjölin. Variation in the propensity to release endotoxin after cefuroxime exposure in different Gram-negative bacteria. Uniform and dose-dependent reduction by the addition of tobramycin. Scandinavian Journal of Infectious Diseases. 2003; 35:40-46.

III Goscinski G., Lipcsey M., Eriksson M., Larsson A., Tano E. and J. Sjölin. Endotoxin neutralization and anti-inflammatory effects of tobramycin and ceftazidime in porcine endotoxin shock. Criti-cal Care. 2004; 8:35-41.

IV Goscinski G., Tano E., Löwdin E. and J. Sjölin. Propensity to release endotoxin after two repeated doses of cefuroxime in an invitro kinetic model; higher release after the second dose. Submit-ted.

V Goscinski G., Tano E., Thulin P., Norrby-Teglund A. and J. Sjölin. Release of streptococcal SpeA after exposure to penicil-lin; dependency on dose and inhibition by clindamycin. In manu-script.

Contents

Introduction and background ..........................................................................7 Pathogenesis of severe sepsis and septic shock..........................................7

Endotoxins .............................................................................................9 Exotoxins .............................................................................................11

Endotoxin neutralisation and immunomodulation as treatment strategies..................................................................................................................11 Release of toxin after exposure to antibiotics...........................................12

Antibiotic-induced release of endotoxin from gram-negative bacteria12 Release of exotoxin from gram-positive bacteria ................................13

Effects of antibiotics on the immune system............................................14

Aims of the study ..........................................................................................15

Materials and methods ..................................................................................16 Bacterial cultures......................................................................................16 Media........................................................................................................16 Antibiotics ................................................................................................16 Determination of minimum inhibitory concentrations (MIC)..................17 Determination of bacterial killing by viable count...................................17 In vitro kinetic model and experimental procedure..................................18 Morphology..............................................................................................18 Determination of endotoxin in the in vitro experiments .........................19 Determination of possible mechanisms for a tobramycin-caused reduction in cefuroxime-induced endotoxin release ................................................19 Determination of SpeA concentration .....................................................19 Animals, anaesthesia and preparatory procedures....................................20 Administration of endotoxin and antibiotics ............................................21 Measurements ..........................................................................................22 Determination of plasma endotoxin, TNF- and IL-6 concentrations.....22 Calculations and statistical methods.........................................................22

Results and discussion ..................................................................................24 MIC values ...............................................................................................24 Killing rates ..............................................................................................24 Antibiotic-induced endotoxin release.......................................................25

Endotoxin release in relation to the number of killed bacteria.................27 Possible mechanisms for reduction in endotoxin release by tobramycin.30 Possible anti-inflammatory effects by antibiotics ...................................33 Effect of repeated dosing on endotoxin release and morphology ...........33 Exotoxin release ......................................................................................36

Clinical implications .....................................................................................39

Conclusions...................................................................................................42

Acknowledgements.......................................................................................43

References.....................................................................................................45

7

Introduction and background

Sepsis is defined as a condition with fever, takycardia, takypnoe and leuko-cytosis or leukopenia caused by an infection (4). If sepsis leads to organ dysfunction, hypoperfusion or hypotension, it is classified as severe sepsis. When the hypotension persists despite adequate fluid resuscitation and hy-poperfusion or organ dysfunction is present the term septic shock is used (4).

Bacteria are the most common agents causing septic shock but fungi, mainly Candida albicans, as well as parasites and viruses may be responsible in rare cases (101). Among bacterial agents E. coli, group B streptococci and Lis-teria predominate in the neonates whereas pneumococci, meningococci and S. aureus are the most common causative agents in children and younger adults (86). Septic shock in elderly patients is more frequently caused by gram-negative bacteria from the gastrointestinal, urinary and respiratory tracts. Since the 1980s there has been a resurgence of severe group A strep-tococcal (GAS) infections with rapid progression to toxic shock in patients of all ages, also including otherwise healthy young persons (41,88).

Despite adequate antibiotic treatment and optimal intensive care efforts, severe infections caused by gram-negative, as well as gram-positive bacteria still often proceed to shock and multiorgan failure (48,92). Thus, in studies performed on patients with sepsis and septic shock the mortality rate has ranged from 30 to 80 %, depending on severity, duration, underlying condi-tions and on the definitions used (58,70,80,83).

Since the beginning of the antibiotic era, experimental studies and discus-sions have been pursued to find the reasons for and to prevent therapeutic failures in severe sepsis where antibiotic treatment even has been suggested by some authors to be responsible for the aggravating conditions. (25,69,85).

Pathogenesis of severe sepsis and septic shock

It is well known that bacterial products, like endotoxin from the gram-negative and exotoxins from the gram-positive bacteria, in different ways

8

initiate the inflammatory host response in sepsis and that this may further progress to severe sepsis and septic shock (19,43,84). With a high virulence or number of bacteria in the circulation or locally, the proinflammatory reac-tion thus initiated cannot be inhibited by the anti-inflammatory systems that occur somewhat later. Besides bacterial properties, the degree of response in the host also depends on individual factors like underlying conditions, medi-cations and probably also genetic factors.

The initial response to toxin release includes activation of the macrophages and monocytes to produce IL-1, TNF and other cytokines and, through a complicated interaction with the activated coagulation, fibrinolytic, kallik-rein-kinin and complement systems, an activation of the neutrophil granulo-cytes takes place. Through adhesion of the neutrophils to the endothelium in multiple organs there is an increased permeability that might lead to a leak-age from the blood vessels. Simultaneous activation of the coagulation and anti-coagulation may lead to imbalance resulting in microthrombosis or, occasionally, bleeding (86, Fig.1).

Figure 1. Initial activation of inflammatory response.

In septic shock, a general vasodilatation, caused by released substances, such as TNF, PAF, bradykinins, prostaglandins and nitrous oxide, takes place.

9

This, together with tissue oedema and microthrombosis, leads to a lowered oxygen uptake by different organs. Hypovolaemia occurs because of the vasodilatation, as well as dehydration, and the increased capillary leakage. Initially, the circulation is hyperdynamic with a high cardiac output and a low vascular resistence. If the hypovolaemia is not corrected, the cardiac output decreases and a hypodynamic septic shock develops.

A serious inflammatory reaction may also lead to development of acute res-piratory distress syndrome (ARDS) in the lungs which further lowers the oxygen delivery to the organs. Decreased oxygen delivery and organ uptake causes anaerobic metabolism and metabolic acidosis which, together with the generalised inflammation results in multiple organ dysfunction.

Endotoxins

The term endotoxin refers to the biological activity of the lipopolysacharide molecule (LPS) in the cell wall of the gram-negative bacteria. The LPS molecule consists of 3 parts: the outer polysaccharide hydrophilic O-side chain, a centre core oligosaccharide and an inner hydrophobic lipid A por-tion which is the mediator of toxicity (43,58). It has been shown in animals and humans that administration of purified endotoxin can induce the same pathophysiological effects as those in gram-negative sepsis (68).

Endotoxin release from the bacterial cell wall occurs mainly as a conse-quence of death and lysis of the cell and of growth by cell division (46). When the endotoxin is released and appears as free endotoxin, its biological activity increases considerably as compared with the bacterial cell-bound endotoxin (57).

Once released, endotoxin binds to several receptors and carrier proteins, the most important being the LPS binding protein (LBP) (99, Fig.2).

10

Figure 2. Release of free endotoxin from gram-negative bacteria.

The LBP-endotoxin complex interacts with the CD14-receptor on the macrophage where a Toll-like receptor-4 (TLR-4) transduces the endotoxin signal to the cell which responds by cytokine production. LBP is crucial for the interaction with the CD14 receptor and it has been demonstrated that TNF-production is reduced 1000-fold in LBP-depleted plasma (64). Besides binding to the CD14 receptor, endotoxin may also activate the macrophage via other receptors, such as the 2 integrin CD11/CD18 receptor (45). Mem-brane bound CD14 may be released from the cell surface and this soluble CD14 (sCD14) may bind to the LBP-endotoxin complex. LBP is freed and the endotoxin-sCD14 complex may then activate cells that do not express CD14, such as endothelial cells (29).

The effect of endotoxin may be inhibited by binding to a LBP-like protein, the bactericidal/permeability increasing protein (BPI) which is synthesised by the neutrophil granulocytes upon endotoxin stimulation (93). Already at the onset of endotoxin release, LPS can bind to specific antibodies that bind to the O-chain and represent the result of previous endotoxin exposures (28,35). In addition, low- and high-density lipoproteins and apolipoprotein A have been shown to inhibit endotoxin-induced cytokine release (26).

11

Exotoxins

Gram-positive bacteria like staphylococci and streptococci produce exotox-ins with varying biological activities that can be released without bacterial growth or lysis. (92). As an example, in severe group A streptococcal infec-tions the pyrogenic exotoxins together with the phagocytosis-inhibiting M-proteins on the streptococcal surface are the most important virulence fac-tors. In similarity to endotoxin, the exotoxins stimulate monocytes and macrophages to release TNF and other cytokines (37). In addition, the exo-toxins have superantigenic properties, meaning that they are mitogenic for certain T-cell subsets but do not require processing by antigen presenting cells (52). Consequently, release of excessive amounts of inflammatory cy-tokines, such as TNF- , IL-1 and IL-6 can take place. Exotoxins of major importance are the superantigens streptococcal pyrogenic exotoxin A (speA), B , C and F (72).

Endotoxin neutralisation and immunomodulation as treatment strategies

The cornerstones in the treatment of severe sepsis and septic shock include antibiotics and, if necessary, surgery for the killing and removal of causative bacteria, fluid replacement for reversal of hypovolemia, oxygen therapy for the treatment of hypoxia and support of deteriorating vital organs. Despite this, mortality is still high and therefore numerous attempts have been made to interfere with the proinflammatory cascades in severe sepsis and septic shock.

As endotoxin is the major activator in severe gram-negative sepsis, several clinical trials have studied the effect of polyclonal or monoclonal antibodies directed against the inner biological part of the LPS molecule (36,102,103). Positive effects, especially in the most severely sick patients, were seen but in the confirmatory trials all-cause mortality was not significantly affected. Other studies have demonstrated a binding and neutralization of LPS after addition of recombinant BPI that shows higher affinity for endotoxin than LBP (30,98). In a clinical study in patients with meningococcemia, known to have high endotoxin concentrations, there was a trend towards an improved clinical outcome in the group that was treated with rBPI but no statistically significant effects could be demonstrated (59).

Polyclonal intravenous immunoglobulin (IVIG) has been shown to neutralise

12

streptococcal toxins (73). However, clinical experience is limited to one retrospective comparative study demonstrating a beneficial effect on mortal-ity in patients with streptococcal toxic shock syndrome (50) and one small prospective study showing a significant decrease in sepsis-related organ failure (20). In patients with severe sepsis or septic shock caused by other bacteria, no beneficial effects have been demonstrated.

Clinical trials with antibodies or other molecules antagonising the effect of several proinflammatory cytokines, such as TNF- , IL-1 platelet activating factor, or coagulation factors, have shown positive results in some subgroups but no significant effect in all-cause mortality (1,2,3,16,22,74,81,97) and therefore these products have not been licensed for clinical use. Recently, beneficial effects on mortality have been demonstrated for corticosteroids (6) and activated protein C (10). However, with these treatments there are al-ways difficulties in establishing the optimal dose because, if doses are too high, the beneficial effect may proceed into an adverse one leading to im-mune paralysis or bleeding.

Release of toxin after exposure to antibiotics

Above their primary bactericidal and bacteriostatic activity, antibiotics have been shown to exert other effects that may be of importance for treatment of infections. One of these is the impact on the liberation of bacterial toxins.

Antibiotic-induced release of endotoxin from gram-negative bacteria

A number of in vitro and in vivo studies have shown an increase in en-dotoxin release after exposure to different antibiotics (5,18,23,44,77,85).

In general, bactericidal antibiotics liberate initially more endotoxin than bac-teriostatic antibiotics and antibiotics active on the cell wall, such as penicil-lins and cephalosporins, release more than antibiotics with other modes of action, such as protein synthesis inhibitors. However, there are wide varia-tions between different antibiotics and even among the -lactam antibiotics, there are great differences in the propensity to free endotoxin (11,24,85,95).

Penicillin-binding proteins (PBP) are enzymes that are located in the bacte-rial cell wall and responsible for the cell wall synthesis. They are also the

13

primary targets for the -lactam antibiotics and, depending on the affinity to these PBPs, varying amounts of endotoxin are liberated from the gram-negative bacteria at exposure to these antibiotics (78). Beta-lactam antibiot-ics with affinity for PBP 1, lead to rapid killing without additional release of endotoxin whereas antibiotics with selective affinity for PBP 2, lead to con-version of the bacteria to round cells, spheroplasts, with loss of viability but without cell wall destruction and excessive endotoxin release. Binding to PBP 3, causes selective inhibition of septation and continuing bacterial elon-gation with formation of long filaments and a subsequent increased en-dotoxin production.

Thus, release of high amounts of endotoxin is mainly associated with PBP 3 binding. Cefuroxime, cefotaxime, piperacillin and aztreonam bind to PBP 3 and are associated with antibiotic-induced endotoxin release (24,78). Cef-tazidime, at high concentrations, binds to PBP 1 and the carbapenems to PBP 2 (24,49,78). At lower concentrations, ceftazidime and meropenem bind predominantly to PBP3 resulting in higher release of endotoxin at lower than at higher doses (49,71,78,94). Simultaneous inhibition of PBP 1a and 3, that may be caused by ceftazidime at mid-concentration levels, has also been demonstrated to result in formation of spheroplasts. These findings have been explained as the sum of inhibitory effects (39).

Among the bactericidal antibiotics, aminoglycosides inhibit protein synthesis by binding to 16S rRNA (32), which results in rapid killing without exces-sive endotoxin release (8,24,27). Limited data also suggest that aminoglyco-sides may even reduce the endotoxin liberation induced by -lactam antibi-otics (24). The quinolones inhibit protein synthesis via inhibition of bacterial DNA synthesis (42). Despite this mode of action, quinolones have in some studies been associated with a substantial endotoxin release, whereas in oth-ers this has not been observed (58,78).

Antibiotic-induced endotoxin release has been shown to be biologically ac-tive and to induce a macrophage TNF response in vitro (23). In animal mod-els, there is strong evidence that soluble endotoxin released from gram-negative bacteria by antibiotics contributes to pathogenesis and mortality in experimental sepsis (70). However, the clinical significance of antibiotic-induced endotoxin release has still not been established.

Release of exotoxin from gram-positive bacteria

Data on exotoxin release from gram-positive bacteria are more limited than those on endotoxin release from gram-negative bacteria but recently it has

14

been shown the liberation of streptococcal toxin may be dependent on the antibiotic treatment (17,62,89). It has been demonstrated in vitro that libera-tion of SpeA is lower after treatment with protein synthesis inhibitors, like clindamycin and linezolid, than the -lactam antibiotic penicillin (17,62). The proposed mechanism has been inhibition of toxin production. In severe group A streptococcal myositis in mice, these results have been supported and in the experiments it has been shown that clindamycin significantly in-creases survival compared with penicillin treatment (91). Inhibition of toxin synthesis as well as inoculum independency and a long post-antibiotic effect of clindamycin were suggested as explanations for the result.

Effects of antibiotics on the immune system

In a considerable number of in vitro and ex vivo experiments, antibiotics have also been found to modulate host inflammatory mechanisms (31,38,51). Graded doses of various antibiotics have been tested for their ability to affect functions such as chemotaxis, phagocytosis as well as bacte-rial killing with the generation of reactive oxygen molecules. This oxidative stress induced by polymorphonuclear activation during septic shock has been shown to trigger an inflammatory response with later cytokine production (55). Several studies have demonstrated that certain -lactam antibiotics and aminoglycosides provide protection from oxygen radicals with subsequent reduction of epithelial damage (15,76). While immune modulation in these in vitro and ex vivo experiments appear to be marked, in vivo data are lim-ited and clinically decisive effects have not been demonstrated (38).

15

Aims of the study

As the endo- and exotoxins are primary important mediators of septic shock and since antitoxic and most anti-inflammatory agents have shown no clear efficacy, an attractive alternative seems to be prevention or minimisation of their release in order to diminish the effects on the inflammatory response. Therefore, it was of interest to further study the antibiotic-induced release of toxins after exposure to antibiotics used for the treatment of the most severe infections, severe sepsis and septic shock and especially if protein synthesis inhibitors could reduce the release induced by PBP 3-specific -lactam anti-biotics.

The aims were: To investigate the inhibitory effects of varying doses of a protein synthe-sis inhibitor, tobramycin, on endotoxin release in vitro from various gram-negative bacteria induced by a PBP 3-specific -lactam antibiotic, cefuroxime.

To study the relationship between endotoxin release and bacterial kill-ing.

To study if tobramycin binds unspecifically to endotoxin in vitro or invivo or affects the binding of the LBP-endotoxin complex to the macro-phages,in vivo

To study the effects on endotoxin release by repeated cefuroxime doses in an in vitro pharmacokinetic model simulating the human concentra-tion time-profile.

To study if tobramycin or ceftazidime can reduce the cytokine and physiological response to endotoxin in vivo.

To investigate the release of SpeA from a GAS strain after exposure to a -lactam antibiotic, penicillin G, a protein synthesis inhibitor, clindamy-

cin or a combination of the two and to relate the toxin release to the ini-tial bacterial concentration, the antibiotic doses and the killing rate.

16

Materials and methods

Bacterial cultures

Clinical isolates of Escherichia coli, B 049-3036 (I,IV), B 940-3012 (II), B 942-1035 (II) and Klebsiella pneumoniae B 541-1003 (II) obtained from the Department of Clinical Microbiology, Uppsala, Sweden, were used. In addi-tion, a clinical isolate of Streptococcus pyogenes group A, 8004 (V) obtained from Mount Sinai Hospital, Toronto, Canada was studied.

The reference strains of Escherichia coli CCUG 12620* (II), Salmonella enteritidis CCUG 35852 (II) and Neisseria meningitidis CCUG 15117 (II) purchased from the Department of Clinical Microbiology, Gothenburg, Sweden were used, as well as the reference strain of Escherichia coli ATCC25922 (IV).

* The CCUG number was discovered to be wrong in the lab. storage. The strain should have been denominated CCUG 17620 which is identical with ATCC 25922.

Media (I, II, IV, V)

The GAS strain (V) was cultured in Todd-Hewitt broth supplemented with 1.5% yeast extract. The Neisseria meningitidis (II) was grown in a serum containing broth (Fildes and horse serum) made from pyrogen-free water and all the other gram-negative strains were cultured in Brain Heart Infusion (BHI) which was also made with pyrogen-free water.

Antibiotics

The antibiotics were obtained as reference powders with known potencies. Cefuroxime and ceftazidime were purchased from Glaxo Wellcome AB,

17

Gothenburg, Sweden, bensylpenicillin from Astra Läkemedel AB, Sö-dertälje, Sweden, clindamycin from Pharmacia Upjohn, Kalamazoo, Michi-gan, USA and tobramycin from Eli Lilly Sweden AB, Stockholm, Sweden.

Determination of minimum inhibitory concentrations (MIC)

MICs were determined in triplicate by two-fold microdilution in broth with an inoculum of approximately 105 cfu of the test strain per ml according to NCCL´s standard (I), by E-test (Biodisk, Solna, Sweden) (II,IV), and the microdilution method (V).

Determination of bacterial killing by viable count (I, II, V)

Strains in exponential growth phase were diluted and transferred into pyro-gen-free glass vials to obtain a starting inoculum of approximately 106

cfu/ml (I,II). The bacteria were then exposed to 0.1 (I), 2 (I,II), 10 (I,II) and 50 (I,II) x MIC of cefuroxime, tobramycin or a combination of the two and further incubated at 35oC. In each experiment, one glass vial served as a control. Samples for viable counts were drawn and diluted in phosphate buffered saline (PBS) immediately before and after addition of antibiotics and at 1, 2, 6, 24 h (I) and 2, 4 h (II). Dilutions of N. meningitidis were seeded on chocolate agar plates and the remaining gram-negative strains on blood-agar plates.

The GAS strain (V) in a log phase culture of approximately 109 cfu/ml was centrifuged and diluted to around 7.0 log10 cfu/ml in the low and 8.6 log10cfu/ml in the high inoculum experiment. The strain was then exposed to 2 and 1000xMIC of bensylpenicillin, 2 and 32xMIC of clindamycin and com-binations of the two at 2xMIC of both and 1000xMIC and 32xMIC, respec-tively. These concentrations were chosen to mimic low and high concentra-tions in the clinical setting. One glass vial without antibiotic served as a con-trol. Samples for viable counts were drawn immediately before and after addition of antibiotics, and, after further incubation at 37oC, at 3, 6, and 24 h. Dilutions were spread on blood-agar plates.

18

In vitro kinetic model and experimental procedure (IV)

The in vitro kinetic model has been described in detail elsewhere (61). It consists of a spinner flask with a total volume of 110 ml with a filter mem-brane (0.45 m) fitted in between the upper and bottom part, impeding elimi-nation of bacteria. A magnetic stirrer ensures homogeneous mixing of the culture and prevents membrane pore blockage. In one of the side arms of the culture vessel, a silicon membrane is inserted to enable repeated sampling. A thin plastic tubing from a vessel containing fresh medium is connected to the other arm. The medium is drawn from the flask at a constant rate by a pump (type P-500, Pharmacia Biotech. Norden, Sollentuna, Sweden), while fresh, sterile medium is sucked into the flask at the same rate by the negative pres-sure built up inside the culture vessel. The flow-rate of the pump was ad-justed to obtain the desired half-life of the drug. The apparatus was placed in a thermostatic room at 37oC during the experiments.

The bacteria were added to the antibiotic suspension immediately before it was transferred to the culture vessel at time zero, resulting in a starting in-oculum of 5x106 cfu/ml . The initial cefuroxime concentration in the vessel was 60 g/ml. The half-time of 1.5 h for cefuroxime was chosen in order to simulate the human pharmacokinetics. The same dose was placed into the vessel after about 12 h when the inoculum had reached a level approximat-ing that of the starting inoculum. Six experiments were performed with each strain.

In an additional experiment, the same initial dose was given, but at the time of the second dose, a combination of cefuroxime and tobramycin to result in final concentrations of 60 g/ml and 20 g/ml, respectively, was given. Two experiments with each strain were performed.

Samples for viable counts were drawn before addition of antibiotics and at 2 h, 4 h, 14 h and 16 h. After dilution in PBS, the samples were seeded on Columbia agar plates and incubated at 35oC.

Morphology (IV)

The morphology of E. coli was examined with scanning electron micros-copy. For this purpose a separate experiment was performed with the ATCC 25922 strain given the same antibiotic concentrations as those used in the preceding experiments and with samples drawn before the antibiotic doses

19

and at 1 h and 2 h after each dose. The examinations were made with a LEO Gemini 1530 scanning electron microscope at an accelerating voltage of 2kV.

Determination of endotoxin in the in vitro experiments (I, II, IV)

For all endotoxin assays endotoxin-free glass tubes and vials, preheated at 180o for 4 h, were used. Sterile, pyrogen-free plastic syringes and pipettes were used, and to analyse the free endotoxin, non-pyrogenic filters of 0.45

m were employed. Samples for endotoxin analysis were drawn at the same time points as those for viable counts. The samples were kept on ice, diluted in pyrogen-free water, filtered and frozen at –20o pending analysis.

Analyses of endotoxin were performed in duplicate with the chromogenic limulus amoebocyte (LAL) assay (Bio Whittaker, Walkersville, MD, USA).

Determination of possible mechanisms for a tobramycin-caused reduction in cefuroxime-induced endotoxin release (I)

Tobramycin at concentrations of 0 (control), 10, 50 and 100xMIC was added to BHI spiked with endotoxin from E. coli O55:B5 to concentrations of 500 and 2000 EU/mL, respectively. Samples for endotoxin analysis were taken immediately before addition of tobramycin and after 0.5 h, 2 h, and 4 h of incubation at 35oC.

In another experiment, tobramycin at similar concentrations as above was added to bacteria pre-exposed to cefuroxime 10xMIC for 2 h. Samples for endotoxin analyses were taken before and after addition of tobramycin and the reduction in the cefuroxime-induced endotoxin release was analyzed and compared with the control after an additional 2 h.

Determination of SpeA concentration (V)

Samples for SpeA analysis were drawn concomitantly with those for viable

20

counts. At each time point, centrifugation was performed and 5 ml of the supernatants were transferred to Falcon tubes containing 15 ml of ethanol and kept at –20oC pending analysis.

The levels of the SpeA present in the concentrated bacterial culture super-natants were quantified by a competitive ELISA described in detail else-where (100).

Animals, anaesthesia and preparatory procedures (III)

The study included 13 domestic breed piglets with a median weight of 24 kg. The animals were 12-14 weeks old and without evidence of illness. Further-more, the animals had to have a PaO2 >10 kPa (75 mmHg) in arterial blood and a mean pulmonary arterial pressure (MPAP) of <2.7 kPa (20 mmHg) at baseline 20 minutes after completed preparatory procedure (see below). The Ethics committee of Uppsala University, Sweden, approved the experiment. The piglets were treated according to the European Convention on Animal Care.

Anaesthesia was induced with the mixture of 6 mg/kg tilétamin-zolazepam (Zoletil forte vet , Boehringer Ingelheim Vetmedica, Ingelheim, Germany), mixed with 2.2 mg/kg of xylazin (Rompun Vet , Bayer, Leverkusen, Ger-many) and atropine 0.04 mg/kg (Atropin Pharma, Stockholm, Swe-den). Anesthesia was maintained by a continuous infusion of sodium pento-barbital 8 mg/kg per hour (Pentobarbitalnatrium Apoteket, Umeå, Swe-den), pancuronium bromide 0.26 mg/kg per hour (Pavulon , Organon, Oss, the Netherlands) and morphine 0.48mg /kg per hour (Morphine , Phar-macia, Uppsala, Sweden) dissolved in a 2.5% glucose-electrolyte solution. During the experiment, sodium chloride infusion was given resulting in a total fluid administration rate of 30 mL/kg per hour.

A bolus dose of 20 mg morphine was given intravenously before the per-formance of a tracheotomy, which was done in order to secure a free airway during the experiment and to administer nitrous oxide (N2O) during surgical preparation. The animals were artificially ventilated throughout the experi-mental procedure (Servo 900CTM, Siemens Elema, Stockholm, Sweden). During catheter insertions, 30% oxygen in N2O was given. The gas mixture was switched to 30% oxygen in air during the rest of the experiment. The ventilation after preparation was set to yield a PaCO2 between 5.0 and 5.5 kPa. The respiratory rate was 25 per min and the inspiratory-expiratory ratio was 1:3. Ventilator settings were then kept constant throughout the experi-

21

ment. Atelectasis was prevented by placing the piglets into prone position after preparatory procedures and by maintaining a positive end expiratory pressure of 5 cm H2O.A central venous line as well as a 7 F Swan-Ganz catheter equipped with thermistor were inserted through the internal jugular vein into the superior caval vein and the pulmonary artery, respectively. In addition, a cervical artery was catheterised with an arterial line used for blood sampling and continuous measurement of the arterial blood pressure. A minor vesicotomy was performed for the urinary catheter and a heating pad (Operatherm 200WTM, KanMed, Bromma, Sweden) was used to keep the animals at a constant body temperature.

Administration of endotoxin and antibiotics (III)

As the preparation was completed, at least 20 minutes of stabilisation time passed before baseline values were registered and baseline blood samples were taken prior to administration of antibiotics and endotoxin. All animals were subjected to endotoxin infusion (Escherichia Coli O111:B4; Sigma Chemical, St. Louis, MO, USA) with an initial infusion rate of 4 g/kg per hour. After 30 minutes the infusion rate was reduced to 1 g/kg per hour which was continued during the rest of the experiment. At least a two-fold increase in MPAP (mm Hg) was taken as a sign of severe endotoxemic shock.

If the mean arterial pressure (MAP) decreased to the level of the MPAP dur-ing the first hour of the experiment, a single dose of 0.2 mg adrenaline was given intravenously. Values were registered hourly after baseline for six hours after which all surviving piglets were killed by an intravenously in-jected overdose of potassium chloride.

The animals were randomised by the sealed envelope method to receive either tobramycin, ceftazidime or saline solution. Tobramycin was given as an iv infusion of 140 mg in 100 ml saline solution for 20 minutes, starting 10 minutes before initiation of endotoxin infusion. Ceftazidime was injected as an iv bolus dose of 1 g in 10 ml saline 5 minutes before the start of en-dotoxin infusion and followed by 90 ml of saline for a total infusion time of 10 minutes. In the control group, an infusion of 100 ml saline was initiated 5 minutes before the start of endotoxin administration and given for 10 min-utes.

22

Measurements (III)

MAP and MPAP were continuously monitored through the arterial and the Swan-Ganz catheter, respectively. The central line was used for injection of 10 mL cold saline for determining the cardiac output (CO). CO was ex-pressed as cardiac index (CI) and body surface area was calculated as weight0.67 x 0.112. CI was assessed hourly by the thermodilution method using the thermistor in the Swan Ganz catheter. The average value of at least three serial measurements was registered. Heart rate (HR) was continuously monitored. Blood samples for analysis of endotoxin, TNF- and IL-6 were drawn immediately before addition of antibiotics and hourly until the end of the experiment. At the same time points, arterial blood gas and base deficit were analyzed (ABLTM 300, Radiometer, Brønhøj, Denmark).

Determination of plasma endotoxin, TNF- and IL-6 concentrations (III)

Blood samples for analysis of plasma endotoxin, TNF- , and IL-6 concen-trations were drawn immediately before addition of antibiotics and hourly until the end of the experiment at 6 h. Endotoxin-free, heparinized tubes were used for the endotoxin samples and EDTA tubes for the TNF- and IL-6 samples. After centrifugation the supernatants were transferred to en-dotoxin-free tubes and subsequently kept at –70oC until analysis.

Analyses of endotoxin were performed in duplicate with the limulus amoe-bocyte lysate assay (CoatestTM Plasma ChromoLAL; Charles River En-dosafe, Charleston, SC, USA). Commercial sandwich enzyme-linked immu-nosorbent assays were used for detection of IL-6 (QuantikineTM porcine IL-6, P6000; R&D Systems, Minneapolis, MN, USA) and TNF- (KSC3012; Biosource International, Nivelles, Belgium).

Calculations and statistical methods

Reduction in cfu at various time points was calculated by subtraction of the remaining number of cfu from that at time zero (I,II,IV,V) and from that at the time for the second antibiotic dose (IV). The number of cfu at time zero was calculated as the mean of the values obtained immediately before and after addition of antibiotics (I, II, V). The endotoxin and SpeA release was

23

calculated similarly (I, II, IV,V). The bacterial killing rate in papers I and II was calculated as the time needed for the antibiotic to kill 2 log10 cfu and in the same studies analysis of variance (ANOVA) with repeated measurements was used to compare propensities to release endotoxin and bacterial killing rates. Regression analysis was used to determine the relationship between the number of killed bacteria and the increase in endotoxin (I,II). In papers I and II, the propensity to release endotoxin was expressed as endotoxin re-lease per killed bacterium, whereas in paper IV, in which the variation in the viable count was greater, this was expressed as the log10 endotoxin release per log10 killed bacteria.

To compare the differences in killing rates and SpeA release between antibi-otic regimens, a repeated measures ANOVA was performed (V). A Spear-man rank correlation test was employed to test relationships between SpeA release, inoculum size and killing rate.

In the in vivo study (III), the TNF- response at 1 h and the IL-6 response at 2 h were compared in the primary analyses of the immediate initial cytokine response. Since there was a variation in the immediate cytokine response, the reduction in the concentrations of the TNF- and IL-6 from 1 h and 2 h, respectively, to 6 h, as well as the concentrations at 6 h, were considered as late cytokine responses. In order to be able to detect a difference in the initial log concentration of the cytokines of at least 20 % with an -error of 0.05, a

-error of 0.2, a power of 0.8, and a calculated interindividual variation of 14 %, eight evaluable animals should be included in each treatment group. In this exploratory trial, an interim analysis was planned when there were at least four animals in each group that had survived for 6 h. The distribution of the logarithm of the initial cytokine response approximated normal distribu-tion in this model and was therefore analysed by an unpaired t-test. Other differences between the treatment groups in this study were calculated by the non-parametric Mann-Whitney U-test.

P-values <0.01 were considered significant in study I due to multiple statisti-cal analyses, in the remaining studies p-values <0.05 were significant. Re-sults were expressed as mean + SE or median ± range.

24

Results and discussion

MIC values (I II, IV,V)

The MICs for the gram-negative strains are shown in Table 1. The difference in MIC-values for the B049 strain against cefuroxime in papers I (MIC=8 mg/L) and V (MIC=2 mg/L) may be explained by differences in methods and a method error of plus one step.

Table 1. MICs against cefuroxime and tobramycin for the gram-negative strains.

Bacterial strain Cefuroxime Tobramycin E. coli B 049-3036 (I) 8,0 0,25 E. coli B 049-3036 (IV) 2,0 0,5 E.coli CCUG 17620 (II) 2,0 1,5 E. coli ATCC 25922 (IV) 4,0 1,0 E. coli B 940-3012 (II) 2,0 1,0 E. coli B 942-1035 (II) 1,5 1,0 Klebsiella pneumoniae B 541-1003 (II) 2,0 1,0 Salmonella enteritidis CCUG 35852 (II) 8,0 1,0 Neisseria meningitidis CCUG 15117 (II) 0,05 2,0

The group A streptococcus strain used was highly sensitive to penicillin and clindamycin, with MIC values of 0.008 mg/L and 0.125 mg/L against peni-cillin G and clindamycin, respectively.

Killing rates (I, II)

The killing rate was expressed as the time needed for 2 log10 killing. In all the tested bacteria, including the four E. coli strains, the Salmonella enteriti-

25

dis, Klebsiella pneumoniae and N. meningitidis strains, there was an in-creased killing rate after exposure to tobramycin and cefuroxime in combi-nation with tobramycin as compared with cefuroxime alone (p<0.001). At higher concentrations within the same treatment, the killing rate was signifi-cantly greater for all three regimens. The concentration-dependency of the killing rate was greater for tobramycin and tobramycin plus cefuroxime than for cefuroxime alone, which was confirmed by using interaction terms in the multivariate analysis (p<0.01).

Antibiotic-induced endotoxin release (I, II)

The PBP-3 specific cefuroxime led to a considerably greater endotoxin re-lease than tobramycin or the combination of the two at all concentration levels, even if there was a large species variation (Table 2).

26

Table 2. Increase in endotoxin release after 2 and 4 hours. EU/ml + SE.

E.coli CCUG E.coli B940 E.coli B942 Salmonella Klebsiella Neisseria 2h 4h 2h 4h 2h 4h 2h 4h 2h 4h 2h 4h

Cefuroxime 2xMIC 141+80 3006+1484 843+272 2699+534 791+296 6563+769 97+50 1163+79 1848+1288 3997+1724 2+2 83+5110xMIC 414+83 4186+2470 452+181 3727+654 1038+283 7060+882 1064+62 1421+145 2754+1953 4653+2011 6+4 76+5250xMIC 292+50 5027+3602 520+217 2656+587 1312+478 5876+858 426+61 477+26 1957+871 3126+1275 24+17 49+36

Tobramycin 2xMIC 140+81 250+13 242+31 199+61 200+51 300+69 20+2 41+13 645+231 851+220 2+1 14+8

10xMIC 197+58 197+34 142+15 124+48 122+45 182+34 21+10 41+16 348+88 590+160 6+3 6+3 50xMIC 134+26 139+65 144+22 168+78 156+10 160+37 19+7 29+8 296+68 383+103 4+2 5+3

Cefuroxime + 2xMIC 160+11 202+9 216+32 238+43 199+21 263+28 108+26 190+59 823+115 1165+288 10+4 35+8Tobramycin 10xMIC 130+29 193+17 184+47 218+49 157+25 204+30 81+21 119+32 467+63 589+88 11+3 20+5

50xMIC 97+18 142+11 194+49 238+81 205+17 215+6 89+21 112+29 420+47 459+88 8+1 21+5

27

Most of the endotoxin was liberated within 2 h after exposure to tobramycin or the combination, whereas an important additional increase was seen be-tween 2 and 4 h from most of the strains exposed to cefuroxime alone (I,II). The prolonged and high endotoxin liberation by cefuroxime can be ex-plained by the PBP 3-associated filamentous elongation of the bacteria with continuing endotoxin production and liberation (24,78).

Endotoxin release in relation to the number of killed bacteria (I, II)

For the E. coli B049-3036, investigated in paper I, there was a linear rela-tionship between the endotoxin release and the number of killed bacteria during the first 4 h for each antibiotic regimen at each dosage level, with r2

values 0.61-0.96. This indicated a proportionality, and that the propensity to release endotoxin could be expressed as endotoxin per killed bacterium.

At 4 h, there was a significant difference between the antibiotics, with the highest propensity to release endotoxin for cefuroxime, lower for tobramycin and the lowest for the combination of the two (p<0.001) (Fig.3).

Figure 3. Endotoxin release per killed bacterium 4h after exposure to antibiotics at 2xMIC, 10xMIC, and 50xMIC.

28

With increasing doses, there was a significant reduction (p<0.001) in en-dotoxin release per killed bacterium at 10xMIC and 50xMIC for tobramycin and the combination and at 50xMIC also for cefuroxime alone. It seems reasonable to assume that the reduction in the propensity to release en-dotoxin by cefuroxime at the highest doses is due to changes in PBP-affinity, as has earlier been shown for ceftazidime (78). Thus, the affinity for PBP 3 decreases in favour of PBP 1, resulting in bacterial lysis without filament formation and with less endotoxin release. When the bacterial killing rate was correlated to the propensity to release endotoxin in bacteria exposed to tobramycin or the combination of tobramycin and cefuroxime, a significant negative correlation was found.

The intention with paper II was to see if the demonstrated reduction in the propensity to release endotoxin by tobramycin in one strain was applicable to other E. coli-strains and to other gram-negative species. Moreover, the proportionality between endotoxin release and number of killed bacteria, observed in our previous study, was studied over the wider range of bacteria often seen in clinical practice.

After 4 h of antimicrobial exposure, large species variations in the propen-sity to release endotoxin were demonstrated. After exposure to cefuroxime, the Klebsiella strain liberated 6.3 – 9.9 x 10-3 EU/bacterium, whereas the N. meningitidis strain released considerably less, 0.1 - 0.4 x 10-3 EU/bacterium. Even among the E. coli strains, there was a difference in cefuroxime-induced liberation, the B 940 strain releasing only half of that of the B 942 strain. Corresponding variations were seen after exposure to tobramycin or the combination of cefuroxime and tobramycin, but these were of smaller mag-nitude. The propensity to release endotoxin upon antimicrobial exposure seems to be a strain-specific characteristic, since there was a significant cor-relation between the release caused by tobramycin and that by cefuroxime (p<0.05 at each concentration level). The endotoxin released by the N. men-ingitidis strain was remarkably low in comparison with that of the other strains, especially when the exposure to cefuroxime was compared. The findings are in agreement with the results from an ex vivo study by Prins et al. (79). It may also be the result of the large intraspecies variation that has earlier been described for meningococci (5). In addition, PBP 1, 2 and 3 have all been found in the meningococcal cell wall and varying affinities to

-lactam antibiotics have been described for all of them (67,75,82).

In the endotoxin release per killed bacterium after 4 h, there was a signifi-cant treatment effect in the E.coli CCUG strain (p<0.001), the B940-3012 strain (p<0.001), the B942-1035 strain (p<0.001), the Klebsiella strain (p<0.001), and the Salmonella strain (p<0.01). Paired comparisons demon-

29

strated highly significant differences between cefuroxime and the combina-tion of tobramycin and cefuroxime. The reduction was 96%, 93%, 97%, 86%, and 85%, respectively, from that caused by cefuroxime alone. Simi-larly, the difference between cefuroxime and tobramycin alone was large and strongly significant. For the N. meningitidis strain, these differences did not reach statistical significance but a similar trend was seen with a reduction of 52% when tobramycin was added to cefuroxime. In none of these strains there was a statistical difference between the combination and tobramycin alone.

After 4 h, there was a significant dose-dependent reduction in endotoxin release per killed bacterium in the Salmonella, the E. coli CCUG, the E.coli B942, and the Klebsiella strains (p<0.05 for each strain). The reductions in tobramycin-induced endotoxin release from 2xMIC to 50xMIC were 67%, 48%, 46% and 29%, respectively. Corresponding values for the combination were 44%, 33%, 16%, and 41%, respectively. In the N. meningitidis strain, a similar trend was demonstrated (p=0.05), with a mean reduction of 71% and 29% by tobramycin and the combination, respectively. No dose-dependency at all was seen in the E. coli B940 strain.

There was no significant dose-dependency in the cefuroxime-induced release in any of the strains.

Despite large variations in antibiotic-induced endotoxin release, there was in all strains a considerable reduction in the cefuroxime-induced endotoxin release per killed bacterium when tobramycin was given concomitantly. This indicates that the effect observed in the previously investigated E. coli strain seems to be applicable to many other gram-negative bacteria. Similarly, dose-dependency with a reduction in endotoxin release at higher tobramycin concentrations was seen in all strains with the exception of the E. coli B940 strain. This response constitutes another advantage besides the increase in bacterial killing rate and the reduction in toxicity associated with the once daily high-dose regimen of aminoglycosides.

In a separate experiment, the endotoxin release of K. pneumoniae with dif-ferent starting inoculae (103, 105 and 107 cfu/ml) after exposure to 2x MIC of cefuroxime, tobramycin or a combination of cefuroxime and tobramycin, was studied in relation to the number of killed bacteria (Fig 4).

30

Figure 4. Klebsiella pneumoniae. Correlation between endotoxin release and num-ber of killed bacteria after 4h at starting inoculae of 103, 105 and 107 cfu/ml, respec-tively.

The correlations between logarithmic values of endotoxin release and num-ber of killed bacteria were highly significant for cefuroxime (r=0.99; p<0.001), tobramycin (r=0.93; p<0.001), and the combination (r=0.99; p<0.001). The slopes of the regression lines did not differ significantly, the mean value being 0.75+0.04. This implies that there is a considerably higher release at higher bacterial concentrations but per killed bacterium the en-dotoxin release is somewhat lower. The reason for this is not known but it may be speculated that a high environmental endotoxin concentration may lead to formation of endotoxin aggregates and a reduction in the biologial activity. The magnitude of the slope demonstrates that within 1 log10 the deviation from the straight line leads to only a small systematic error when expressing the endotoxin release in terms of release per killed bacterium and in comparison with the effect on endotoxin release due to variations in in-oculum size, this error is negligible.

Possible mechanisms for reduction in endotoxin release by tobramycin (I,III)

The initial hypothesis to explain the reduction in endotoxin release by the aminoglycosides was by an unspecific binding similar to that demonstrated

31

for polymyxin B (47,78,85). However, in paper I it was shown that tobramy-cin at 2.5, 12.5 and 25mg/L added to BHI spiked with endotoxin did not reduce the endotoxin activity in the solution as compared with the control

In a separate experiment with cefuroxime at a concentration of 10xMIC in a bacterial solution, it was demonstrated that if the addition of tobramycin was delayed for 2 h, no reduction in the cefuroxime-induced endotoxin release took place, neither immediately nor after 4 h. Thus, neutralisation can be excluded as the principal explanation of the reduction in cefuroxime-induced endotoxin release.

The absence of in vitro neutralisation indicates that there is no aminoglyco-side binding that affects the biological activity in the limulus amoebocyte lysate assay but, on the other hand, a tobramycin binding that in vivo neutral-ises the endotoxin effect cannot be excluded. Inhibition of the binding of endotoxin to LBP, the most important carrier protein in the body which is crucial for the attachment to the CD14 receptor on the macrophages, repre-sents one possible mode of action. Another might be that binding with LBP is unaffected, whereas the tobramycin-endotoxin-LBP complex has a re-duced affinity for the CD14 receptor or a lower potential to elicit a signal via TLR 4 for a subsequent TNF- - and IL-6- production.

In the porcine in vivo model (III), the primary endpoints were therefore to analyse the immediate TNF- and IL-6 responses after 1 h and 2 h, respec-tively, in relation to the endotoxin concentration. These time points were chosen since cytokine values obtained later might be influenced by an ami-noglycoside-induced protection against oxidative injury, which has been demonstrated in vitro (15). The plasma TNF- levels reached peak values 1 h after the start of endotoxin infusion with a median value of 7265 ng/L (range 2492 – 10625 ng/L). There were no significant differences in peak TNF- values between the treatment groups. TNF- concentration de-creased to baseline levels within 3 h in most animals.

Plasma IL-6 levels peaked after 2 h or 3h with a median maximum IL-6 value of 2372 ng/L (range 1544–7138 ng/L). There were no significant dif-ferences in IL-6 concentration at 2 h between treatment groups (Fig. 5).

32

Figure 5. Plasma IL-6 concentrations 2-6 hours after start of endotoxin infusion (n=4 in each group).

These results indicate that there is no in vivo neutralisation of endotoxin-induced cytokine production and consequently that there is no interaction with the binding to LBP or the CD14 receptor.

In order to maximise a possible neutralising effect, the tobramycin dose cho-sen was in the highest range of that used clinically. Nonetheless, neither tobramycin nor ceftazidime had any endotoxin neutralising effect in the LAL assay in comparison with that caused by saline. These results are thus in agreement with the in vitro results described in paper I.

An alternative hypothesis to endotoxin neutralisation may be that the mode of action of tobramycin is responsible for the reduced release of endotoxin. The significant negative correlation between the bacterial killing rates of tobramycin and the combination of tobramycin and cefuroxime, on the one hand, and the endotoxin release on the other, supports this hypothesis. Fail-ure of tobramycin to reduce cefuroxime-induced endotoxin release if added after a 2 h pre-exposure to cefuroxime, indicates that initial mechanisms probably are the most important. Previously, Kusser et al. have shown that aminoglycosides are able to inhibit the synthesis of endotoxin (53). Our re-sults strongly support that such a mechanism is quantitatively more impor-tant in comparison with neutralization for the reduction in endotoxin release.

33

Possible anti-inflammatory effects by antibiotics (III)

The inflammatory damage to endothelial cells and other tissue cells medi-ated by oxygen radicals induces cytokine production (55,76). In several in vitro experiments, it has been demonstrated that ceftazidime and some of the other -lactam antibiotics inactivate hypochlorous acid (HOCl) (21) and, in the case of ceftazidime, also singlet oxygen (66). In clinically relevant con-centrations, ceftazidime has also been shown to partially protect endothelial cells from the oxidative stress of activated neutrophils in vitro (55). The documentation for aminoglycosides is less extensive but both tobramycin and gentamicin have been shown to protect lung epithelial cells against mye-loperoxidase-dependant oxidant injury by binding to anionic cell surfaces and neutralising HOCl (15). These findings led to the hypothesis that the anti-inflammatory effects might affect the cytokine response during the last phase of our porcine model. At the end of the experiment, at 6 h, there was a treatment effect expressed as a significantly greater reduction in IL-6 by ceftazidime as well as tobramycin (p<0.05) and a lower absolute IL-6 level in the ceftazidime group as compared with placebo (p<0.05). Since there are in vitro data that ceftazidime neither directly affects IL-6 production (66) nor neutrophil function (34). this result may have been caused by its antioxida-tive properties. For tobramycin, in vitro data indicate that neutrophil function and oxidative metabolism might be influenced but these results have not been reproduced in vivo (34). Whether our findings are due to antioxidative properties of the antibiotics or to other mechanisms cannot be concluded from data presented, but the result indicates that in vitro reported anti-inflammatory properties may be demonstrable in an animal model.

Effect of repeated dosing on endotoxin release and morphology (IV)

In an in vitro kinetic model, simulating the human concentration time-profile, the endotoxin release from two E. coli strains after exposure to two repeated doses of cefuroxime was investigated. In addition, it was studied whether the combination with tobramycin at the second dose resulted in a reduction similar to that previously shown when combined with the first dose.

34

Figure 6. Bacterial killling rate (mean + SE) and endotoxin release (geometric mean + antilog of the SE of the logaritmic values) after the first and second dose of cefu-roxime in the ATCC strain (a) and the B049 strain (b). Note that a considerably lower number of bacteria were killed after the second dose.

35

There was a large variation in endotoxin concentrations, mainly due to the variation in the number of killed bacteria. Median endotoxin release after correction for elimination via the outflow of the pump was 2 h and 4 h after the first dose 1720 and 12600 EU/ml, respectively, for the ATCC strain and 8570 and 21100 EU/ml, respectively, for the B049 strain. Corresponding values after the second dose were 102000 and 124000 EU/ml for the ATCC strain (p<0.01) and 128000 and 149000 EU/ml (NS) for the B049 strain, respectively. The geometric mean of the endotoxin release is shown in Fig. 6.

When the endotoxin release was related to the number of killed bacteria, there was a marked reduction in the variation in both strains. The increase in the tendency to release endotoxin after the second dose in comparison with that after the first was highly significant (p<0.001 for both strains). The dif-ference between the doses ranged from 0.15 to 0.26 log10 EU/log10 number of killed bacteria. This implies that the difference between the doses will be greater at higher numbers of killed bacteria and this will, in effective treat-ment, covariate with the bacterial concentration at the time of the dose. The time course was also significantly different, with an earlier release after the second dose (p<0.001 for both strains).

One possible mechanism behind the higher endotoxin release after the sec-ond dose of cefuroxime might be the continued release from remaining fila-ments caused by the first dose. This hypothesis is supported by Jackson and Kropp, who found an increased endotoxin release at sub-MIC concentrations of several -lactam antibiotics (48). Our electron microscopy findings just before the second dose with a few elongated forms may also be in agreement with this. However, at that time, the endotoxin concentration was relatively low, indicating that even if there may be some sustained release, the contri-bution of this to the total release after the second must be limited. A change in PBP affinity with a higher binding to PBP 3 than to PBP 1 represents an-other possibility, but the presence of spheroplasts together with filaments after the second dose does not favour this hypothesis. Thus, the mechanism is not clear but it might be speculated that there is an enduring antibiotic effect on cell wall synthesis after the first dose resulting in a quicker and more extensive bacterial elongation after the second dose.

After addition of tobramycin to the second dose of cefuroxime, the propen-sity to release endotoxin was significantly reduced in comparison with that after cefuroxime alone (p<0.001), even if the bacteria had previously been exposed to cefuroxime. In addition, the filamentation was less pronounced. The effect of a repeated combined aminoglycoside -lactam antibiotic treat-ment could not be studied, because, at the clinical concentrations used all

36

bacteria were killed already after the first dose at the inoculum sizes possible to use in our in vitro kinetic model.

Exotoxin release (V)

The purpose was to investigate the amount and time course of SpeA libera-tion from a GAS strain, at different starting inoculae, after exposure to dif-ferent doses of penicillin, clindamycin or a combination of the two, and to relate the release to the bacterial concentration and the killing rate.

The starting inoculum sizes in the low and high inoculum experiments were 7.0 + 0.1 and 8.6+ 0.2 log10 cfu/ml, respectively. At the low, as well as the high initial inoculum, a considerably higher killing rate was seen for penicil-lin than for the combination of penicillin and clindamycin (p<0.01 and p<0.001, respectively), but there was no difference in bacterial killing rate between low and high antibiotic doses within any of the treatment regimens. A similar result was seen in the high inoculum experiment when penicillin was compared with clindamycin alone (p<0.01), whereas in the low inocu-lum experiment, only a trend in the similar direction was observed (p=0.09).

In contrast to antibiotic-induced endotoxin liberation from gram-negative bacteria which, after exposure to -lactam antibiotics, initially leads to higher endotoxin concentration than in the control (24), all treatment regi-mens in the present study resulted in a SpeA release, which was many times lower than that of the control. In the high inoculum experiments (Fig.7), there was a large variation in the SpeA production, from 39+9 ng/ml for low dose penicillin to -2+3 ng/ml for the high dose of the combination. The toxin release was significantly higher at low than at high concentrations of penicil-lin (p<0.05). This dose-dependency was also seen for the combination (p<0.05) but not for clindamycin alone. Since there was no difference in killing rate between the two doses this cannot explain the result. For certain

-lactam antibiotics, such as ceftazidime or cefuroxime, binding to different E.coli PBPs has been demonstrated at varying concentrations, which results in a higher endotoxin release after low than after high doses (71).It may be speculated that one possible mechanism is that penicillin has a similar dose-dependent varying affinity to different PBPs. This would imply a different binding pattern at low and high concentrations, which then may lead to a divergent effect on cellular activity prior to lysis. Varying clindamycin con-centrations did not influence the SpeA release but addition of clindamycin could not abolish the dose-effect of penicillin in the combination.

37

Figure 7. SpeA production in the high inoculum experiment, at low (a) and high (b) antibiotic doses, g/L + SE.

38

In the low inoculum experiments, the highest release of SpeA was seen at low penicillin concentration. However, the release was >1 log10 lower than that seen in the high inoculum experiment and no statistically significant differences between treatments could be demonstrated.

Clindamycin alone released less SpeA than penicillin, which, however, was significant only at low concentrations (p<0.01). When clindamycin was added to penicillin, there was a significant reduction in SpeA release both at low and high concentrations (p<0.01 for both comparisons)which is in ac-cordance with an in vitro study by Coyle et al. (17). The most probable ex-planation of this is that clindamycin, as a protein synthesis inhibitor, reduces the production of toxins or proteins responsible for their release to the envi-ronment (52,89).

Most of the speA was released within the first 3 h. The SpeA production during this period correlated significantly to the number of killed bacteria during the same period (n=18, r=0.50; p<0.05). When analysed separately for each treatment regimen, a positive correlation could be demonstrated after treatment with penicillin and the combination, the latter being signifi-cant (p<0.01), whereas no similar correlation was seen for clindamycin. Within the limited range of the inoculum size of 0.8 log10 cfu/ml, there was a significant positive correlation between this and the speA concentration at time zero (r=0.54; p<0.05). In contrast, there was a trend towards a negative correlation to the increase during the following hours (r=-0.42; NS), being negative for all treatment regimens. Therefore, variation in the initial inocu-lum size does not explain the correlation between toxin release and number of killed bacteria. The reason for this is not clear, but the killing rate of -lactam antibiotics has been associated with growth rate and hence the meta-bolic activity of the bacteria (90), and it may therefore be speculated that an initially higher metabolism also results in an increased toxin production. When a protein synthesis inhibitor, such as clindamycin, shuts this off, the relationship is no longer seen.

In several experimental infections of animals with GAS, clindamycin has turned out to be more effective than penicillin (12,91). Therefore, the com-bination of penicillin and clindamycin has often been recommended for the treatment of severe GAS infections (41,87). Even if there is a limited reduc-tion in the killing rate, this seems to be outweighed by the reduced inoculum effect (90,91), longer post-antibiotic effect (89), suppressed synthesis of virulence factors, such as M-protein and capsule (12,32), enhanced opsonisa-tion and phagocytosis (32) and decreased toxin release by clindamycin.

39

Clinical implications

In animal models, there is strong evidence that soluble endotoxin released from gram-negative bacteria by antibiotics contributes to pathogenesis and mortality of the sepsis (70). However, the clinical significance of antibiotic-induced endotoxin release is still not known (78). Anecdotal clinical data from the studies by Dofferhoff et al. and Arditi et al. demonstrate that in-creases in endotoxin concentration may be associated with clinical deteriora-tion (7,24) and the results from a prospective clinical study in patients with gram-negative urosepsis by Prins et al. suggest that the difference between imipenem and ceftazidime in endotoxin release may also be reflected by differences in the systemic inflammatory response (77). Furthermore, a ret-rospective study by Mock et al. has demonstrated that mortality was higher in trauma patients treated with endotoxin-releasing PBP 3-specific antibiot-ics than in those given other antibiotics, which lends some support to a pos-sible clinical significance (69).

On the other hand, there are clinical studies that do not find any differences in the concentrations of endotoxin and cytokines or in clinical outcome be-tween PBP 2-specific imipenem and PBP 3-specific ceftazidime treatment of various gram-negative infections (14,60,63). In a study by Maury et al., in which patients had both a verified gram-negative bacteremia and a severe infection in the form of severe sepsis or septic shock, endotoxin concentra-tion was significantly decreased after one and four hours of antibiotic treat-ment, suggesting that antibiotic-induced endotoxin release is not a clinically relevant phenomenon (65). However, in this non-comparative study, the total endotoxin concentration was measured, also including bacterial cell-bound endotoxin. As has been shown by Arditi et al. in studies on children with gram-negative meningitis, there is a fall in total endotoxin concentration that parallels the reduction in bacterial count, whereas the free endotoxin level concomitantly increases (7). In another study, Hurley et al. found that free endotoxin concentrations increased when chronically bacteriuric patients were treated with different antibiotics (44) Thus, if measured as free en-dotoxin, which is the most biologically active form, antibiotic-induced en-dotoxin liberation has been demonstrated to occur also in patients.

40

In clinical trials, there are difficulties to prove an effect between treatments, even if there is one, due to the large variations in the endotoxin release. As shown in our study, endotoxin liberation is highly dependent on the number of bacteria which may clinically vary considerably. If the release of en-dotoxin is related to the number of bacteria, it may be possible to reduce the interindividual variation and consequently increase the chance in clinical trials to detect a difference between treatments. However, even if this varia-tion is reduced, the strain specific propensity to release endotoxin still repre-sents an important confounding factor.

Taken together with the similarity between the responses of human volun-teers and animals to administration of endotoxin, the resemblance of this response to the symptoms seen in sepsis and septic shock (96), and the re-duction in systemic inflammatory response demonstrated in at least one pro-spective and controlled study (77), available data suggest that reduction in endotoxin release may be of clinical importance. In patients with uncompli-cated gram-negative infections, antibiotic-induced endotoxin release is most probably of limited value. On the other hand, in patients with progressing severe sepsis or who have developed septic shock, an additional dose of endotoxin may be deleterious (58).

Despite a great inter- and intraspecies variation among the gram-negative bacteria, it was possible in our in vitro endotoxin studies to demonstrate significant reductions in endotoxin release when the combination of the PBP 3-specific -lactam antibiotic, cefuroxime, and the protein synthesis inhibi-tor, tobramycin, was compared with cefuroxime alone. Furthermore, increas-ing doses of tobramycin led to a lower endotoxin release and a higher bacte-rial killing rate. If tobramycin was added as late as 2 h after cefuroxime ad-ministration, no inhibitory effect on the endotoxin release was registered.

These findings indicate that it is probably an advantage to add an aminogly-coside to the -lactam antibiotic for the treatment of severe infections, such as progressing severe sepsis and septic shock, a combination that has been recommended for a long time (101). Besides a broad spectrum and a high killing rate, also the release of endotoxin is diminished. If the treatment is initiated with a PBP 3-specific antibiotic but the clinical condition of the patient deteriorates, an aminoglycoside dose may be given concomitantly or before the second dose. Our data also indicate that it is an advantage to give a high dose of the aminoglycoside. High doses have also been recommended in critically ill patients due to the increase in the volume of distribution (13,40). However, the beneficial effects should be weighed against the nephro- and neurotoxic effects of the aminoglycosides. In a study in critical care patients with a creatinine clearance of >70 ml/min, very high doses

41

were given for six days without impairment of renal function, provided that the aminoglycoside concentrations were followed (54). In these patients, kidney function was more or less preserved but, a recent study in patients with poor renal function and peritoneal dialysis has demonstrated that ami-noglycoside treatment did not affect residual renal function (9). The use of aminglycosides in a high dose regimen in oliguric or anuric patients or pa-tients who present with a rapidly decreasing renal function needs further investigation. However, taken together, these data indicate that one or a few doses of an aminoglycoside in the initial phase of the treatment of severe sepsis or septic shock may have beneficial effects and that the risk of renal injury, with the exception of the patients with the poorest function, seems to be very low.

In some similarity with the discussion above, there seems to be an advantage to combine penicillin with the protein synthesis inhibitor, clindamycin, for the treatment of severe group A streptococcal infections.

In animal studies clindamycin alone, or in combination with penicillin, has shown beneficial effects in the treatment of invasive group A streptococcal infections (56,91). This is in part due to inhibited expression of different streptococcal virulence factors, such as SpeA (62,90).

The findings in our in vitro study that clindamycin released less SpeA from a GAS strain than penicillin and that the addition of clindamycin to penicillin resulted in less toxin production than treatment with penicillin alone, are in agreement with earlier in vitro and animal experiments. The study also dem-onstrated that the SpeA concentration was dependent on the number of bac-teria prior to antibiotic exposure and the dose of penicillin. In the clinical setting, early treatment seems to be very important and, in addition, combi-nation therapy from the start with high doses of penicillin may offer an ad-vantage.

42

Conclusions

The in vitro release of endotoxin is associated with the number of killed bacteria, the logarithm of the release being proportional to the logarithm of the number of killed bacteria.

The protein synthesis inhibitor, tobramycin, reduces endotoxin release in vitro from various gram-negative strains induced by the PBP 3-specific -lactam antibiotic, cefuroxime. Increasing doses of tobramycin lead to less endotoxin liberation.

There is no unspecific binding between endotoxin and tobramycin in vitro.Nor does tobramycin affect the binding of the LBP-endotoxin complex to the macrophages in vivo.

The reduction in endotoxin release is associated with the killing rate of to-bramycin.

The propensity to liberate endotoxin in vitro is higher after the second dose of cefuroxime than after the first, which results in a higher release of en-dotoxin than expected from the bacterial count. The release of the second dose can be reduced by the addition of tobramycin

There is a possible anti-inflammatory effect given by ceftazidime and to-bramycin expressed as a late cytokine inhibition in porcine endotoxin shock.

The SpeA release from a GAS strain in vitro is correlated to the initial num-ber of bacteria and the killing rate. Addition of a protein synthesis inhibitor, clindamycin, reduces the toxin release in comparison with that of the -lactam antibiotic, penicillin G, alone. The SpeA release is also dependent on the dose of penicillin, with high doses leading to lower SpeA levels as com-pared with low doses.

43

Acknowledgements

I would like to express my sincere gratitude to all the people who made this thesis possible. In particular I would like to thank:

Jan Sjölin, my tutor, who persuaded me to undertake this project and since then, has always been available for questions and discussions, despite an immense work load. I am most grateful for your never ending support and enthusiasm, your enormous knowledge and your efforts to find new solu-tions when everything looked hopeless, especially the statistics.

Monica Lundholm, my co-tutor, unfortunately no longer with us. Monica introduced me to the endotoxin studies in the Laboratory of Microbiology and were most helpful and supporting during my first not so skilful activities in the lab. R.I.P.

Inga Odenholt, my co-tutor, always optimistic and encouraging. Your pharmacokinetic knowledge, advices and critical comments were most valu-able, especially before you left us for Malmö.

Eva Tano, co-author, my support in the laboratory during the last years, as well as my talking partner when problems arose. Without your skill in plan-ning and performing the laboratory work, day or night, there would have been nothing. You are a real guarantee of quality.

Caroline Stenbom-Johansson and Christina Nylander, skilful laboratory technicians performing the analyses of the first two studies. I will remember our sports discussions and other fun during work.

Anders Nordgren, intensive care nurse, for giving anaesthesia to the pigs, supervising them as if they were human patients and taking care of every-thing.

All my co-authors in the papers. Especially I want to thank Miklos Lip-csey for doing the invasive operations on the pigs, Anna Norrby-Teglund for introducing me into the “superantigenic” world and Pontus Thulin for

44

the time-consuming work with the SpeA-analyses.

Professor Göran Friman for allocating research time during the years.

Elisabeth Löwdin, friend and loyal room-mate since 1986, lately also co-author and always willing to take on my duties in the clinic when necessary. In our office, where visitors have to find space between desks and bikes there has been time for laughter as well as gossip talk.

Anders Lannergård, Cecilia Ryden and Erik Torell, my colleagues, for doing my clinical work during the last crucial weeks of the thesis.

Birgitta Sembrant, you never stop impressing me with your technical skills with figures and tables in any document form. A life-line during the final stress.

Osvaldo, my husband and life time supporter. Thank you for your patience, consideration and for serving coffee, porridge and dinners during the breaks.

Tomas and Pia, Jens, Hannes, Jan and Nea, all of you remind me of other qualities in life like the travels and outdoor activities we have enjoyed to-gether and hopefully will continue to do.

For financial support I would like to thank: The Olinder-Nielsen Foundation, Eli Lilly Sweden AB, Stockholm, Sweden, Glaxo Wellcome AB, Gothen-burg, Sweden, Laerdal Foundation for Acute Medicine, Swedish Foundation for Strategic Research and the Swedish Research Council.

45

References

1. Abraham E, Laterre PF, Garbino J et al. (2001). Lenercept (p55 tumor necro-sis factor receptor fusion protein) in severe sepsis and early septic shock: a randomized, double-blind, placebo-controlled, multicenter phase III trial with 1,342 patients. Crit Care Med 29(3), 503-10.

2. Abraham E, Reinhart K, Opal S et al. (2003). Efficacy and safety of tifacogin (recombinant tissue factor pathway inhibitor) in severe sepsis: a randomized controlled trial. JAMA 290(2), 256-8.

3. Abraham E, Wunderink R, Silverman H et al. (1995). Efficacy and safety of monoclonal antibody to human tumor necrosis factor alpha in patients with sepsis syndrome. A randomized, controlled, double-blind, multicenter clinical trial. TNF-alpha Sepsis Study Group. JAMA 273(12), 934-41.

4. The ACCP/SCCM Consensus Conference Committee. (1992). Definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. Chest 101, 1644-55.

5. Andersen BM, Solberg O. (1984). Endotoxin liberation and invasivity of Neis-seria meningitidis. Scand J Infect Dis 16, 247-54.

6. Annane D, Sebille V, Charpentier C et al. (2002). Effect of treatment with low doses of hydrocortisone and fludrocortisone on mortality in patients with sep-tic shock. JAMA 288(7), 862-71.

7. Arditi M, Ables L, Yogev R. (1989). Cerebrospinal fluid endotoxin levels in children with H. influenzae meningitis before and after administration of in-travenous ceftriaxone. J Infect Dis 160, 1005-11.

8. Artenstein AW, Cross AS. (1989). Inhibition of endotoxin reactivity by ami-noglycosides. J Antimicrob Chemother 24, 826-8.

9. Baker RJ, Senior H, Clemenger M et al. (2003). Empirical aminoglycosides for peritonitis do not affect residual renal function. Am J Kidney Dis 41, 670-5.

10. Bernard GR, Margolis BD, Shanies HM et al. (2004). Extended evaluation of recombinant human activated protein C in the United States Trial (ENHANCE US): a single-arm, phase 3B, multicenter study of drotrecogin alpha (acti-vated) in severe sepsis. Chest 125, 2206-16.

11. Bingen E, Goury V, Bennani H et al. (1992). Bactericidal activity of ß-lactams and amikacin against Haemophilus influenzae: effect on endotoxin release. JAntimicrob Chemother 30, 165-72.

12. Brook I., Gober A.E, Leyva F. (1995). In vitro and in vivo effects of penicillin and clindamycin on expression of group A beta-hemolytic streptococcal cap-sule. Antimicrob Agents Chemother 39:7, 1565-8.

13. Buijk SE, Mouton JW, Gyssens IC et al. (2002). Experience with a once-daily dosing program of aminoglycosides in critically ill patients. Intensive Care Med 28, 936-42.

14. Byl B, Clevenbergh P, Kentos A et al. (2001). Ceftazidime- and imipenem-induced endotoxin release during treatment of gram-negative infections. Eur J Microbiol Infect Dis 20, 804-7.

46

15. 13. Cantin A, Woods DE: (1993). Protection by antibiotics against myeloper-oxidase-dependent cytotoxicity to lung epithelial cells in vitro. J Clin Invest91, 38-45.

16. 14. Cohen J. (1996). INTERSEPT: an international, multicenter, placebo-controlled study of monoclonal antibody to human necrosis factor-alpha in pa-tients with sepsis. International Sepsis Trial Study Group. Crit Care Med 24(9), 1431-40.

17. Coyle EA, Cha R, Rybak MJ. (2003). Influences of linezolid, penicillin, and clindamycin, alone and in combination, on streptococcal pyrogenic exotoxin A release. Antimicrob Agents and Chemother 47, 1752-5.

18. Crosby HA, Bion JF, Penn CW et al. (1994). Antibiotic-induced release of endotoxin from bacteria in vitro. J Med Microbiol 40, 23-30.

19. Cunningham MW. (2000). Pathogenesis of group A streptococcal infections. Clin Microbiol Rev 13, 470-511.

20. Darenberg J, Ihendyane N, Sjolin J et al. StreptIg Study Group. (2003). Intra-venous immunoglobulin G therapy in streptococcal toxic shock syndrome: a European randomized, double blind, placebo-controlled trial. Clin Infect Dis 37(3), 341-3.

21. Deby-Dupont G, Deby C, Mouithys-Mickalad A et al. (1998). The antibiotic ceftazidime is a singlet oxygen quencher as demonstrated by ultra-weak chemiluminescence and by inhibition of AAP consumption. Biochim Biophys Acta 8:61-8.

22. Dhainault JF, Tenaillon A, Le Tulzo Y et al. (1994). Platelet activating factor receptor antagonist BN 52021 in the treatment of severe sepsis: A randomized, double-blind, placebo-controlled multi-center clinical trial. Crit Care Med 22, 1720-8.

23. Dofferhoff ASM, Esselink MT, de Vries-Hospers HG et al. (1993). The re-lease of endotoxin from antibiotic-treated Escherichia coli and the production of tumor necrosis factor by human monocytes. J Antimicrob Chemother 31,373-84.

24. Dofferhoff ASM, Nijland JH, de Vries-Hospers HG et al. (1991). Effects of different types of combinations of antimicrobial agents on endotoxin release from Gram-negative bacteria: an in vitro and in vivo study. Scand J Inf Dis 23, 745-54.

25. Eagle H. (1952). Experimental approach to the problem of treatment failure with penicillin. 1. Group A streptococcal infection in mice. Am J Med 13, 389-99.

26. Flegel WA, Baumstark MW, Weinstock C et al. (1993). Prevention of en-dotoxin-induced monokine release by human low- and high-density lipopro-teins and by apolipoprotein A-1. Infect Immun. 61, 5140-6.

27. Foca A, Matera G, Iannello D et al. (1991). Aminoglycosides modify the in vitro metachromatic reaction and murine generalized Shwartzman phenome-non induced by Salmonella minnesota R595 lipopolysaccharide. Antimicrob Agents Chemother 35, 2161-4.

28. Freeman R, Gould EK. Prevention of fever and gramnegative infection after open heart surgery by antiendotoxin. (1985). Thorax 40, 846-8.

29. Frey EA, Miller DS, Gullstein Jahr T et al. (1992). Soluble CD14 participates in the response to endotoxin. J Exp Med 176, 1665-71.

30. Gazzano-Santoro H, Parent JB, Grinna L et al. (1992). High-affinity binding of the bactericidal/permeability increasing protein and a recombinant amino-

47

terminal fragment to the lipid A region of lipopolysacharide. Infect Immun 1992, 4754-61.

31. Gemmel CG. (1993). Antibiotics and neutrophil function – potential immu-nomodulating activities. J Antimicrob Chemother 31 Suppl B, 23-33.

32. Gemmel CG., Peterson PK, Schmeling D et al. (1981). Potentiation of opsoni-zation and phagocytosis of Streptococcus pyogenes following growth in the presence of clindamycin. J Clin Invest 67, 1249-56.

33. Gilbert DN. (2000). Aminoglycosides. In: Mandell GL, Bennet JE, Dolin R, eds. Principles and practice of infectious diseases, 5th edn. New York: Chur-chill Livingstone, 690-705.

34. Gnarpe H, Belsheim J, Blomqvist C et al. (1984). The in-vitro influence of ceftazidime on host defence mechanisms. J Antimicrob Chemother 13:369-75.

35. Gould EK, Harvey JA, Dytrych JK. (1989). Antibody to endotoxin is associ-ated with decreased frequency of postoperative infection. Am J Obstet Gyne-col 160, 317-9.

36. Greenman RL, Schein RMH, Martin MA et al. (1991). A controlled clinical trial of E5 murine monoclonal IgM antibody to endotoxin in the treatment of gramnegative sepsis. JAMA 266, 1097-102.

37. Hacket SP, Stevens DL. (1993). Superantigens associated with staphylococcal and streptococcal toxic shock syndrome are potent inducers of tumor necrosis factor-beta synthesis. J Infect Dis 168(1), 232-5.

38. Hamilton-Miller JM. (2001). Immunopharmacology of antibiotics: direct and indirect immunomodulation of defence mechanisms. J Chemother 13, 107-11.

39. Hanberger H, Nilsson LE, Kihlström E et al. (1989). Postantibiotic effect of -lactam antibiotics on Escherichia coli evaluated by bioluminescence assay of bacterial ATP. Antimicr Agents Chemother 34, 102-6.

40. Hansen M, Christrup LL, Jarlov JO et al. (2001). Gentamicin dosing in criti-cally ill patients. Acta Anaesthesiol Scand 45, 734-40.

41. Holm SE. (1989). Invasive group A streptococcal infections. N Engl J Med335, 590-1

42. Hooper DC. (2000). Quinolones. In: Mandell GL, Bennet JE, Dolin R, eds. Principles and practice of infectious diseases, 5th edn. New York: Churchill Livingstone, 404-23.

43. Hurley JC. Antibiotic-Induced Release of Endotoxin: A Reappraisal. (1992). Clin Infect Dis 15, 840-54.

44. Hurley JC, Louis WJ, Tosolini FA et al. (1991) Antibiotic-induced release of endotoxin in chronically bacteriuric patients. Antimicrob Agents Chemother35, 2388-94.

45. Ingalls RR, Heine H, Lien E et al. (1999). Lipopolysacharide recognition, CD14, and lipopolysacharide receptors. Infect Dis Clin North Am 13, 341-53.

46. Ishiguro EE, Vanderwel E, Kusser W. Control of lipopolysacharide biosynthe-sis and release by Escherichia coli and Salmonella typhimurium. (1986). JBacteriol 168, 328-33.

47. Issekutz AC. (1983). Removal of Gram negative endotoxin from solutions by affinity chromatography. J Immunol Methods 61, 275-81.

48. Jackson JJ, Kropp H. Beta-lactam antibiotic-induced release of free endotoxin: in vitro comparison of penicillin-binding protein (PBP) 2-specific imipenem and PBP 3-specific ceftazidime. (1992). J Infect Dis 165, 1033-41.

48

49. Jackson JJ, Kropp H. (1996). Differences in mode of action of -lactam anti-biotics influence morphology, LPS release and in vivo antibiotic efficiency. JEndotoxin Res 3, 201-18.

50. Kaul R, McGeer A, Norrby-Teglund A. (1999). Intravenous immunoglobulin therapy for streptococcal toxic shock syndrome—a comparative observational study. The Canadian Streptococcal Study Group. Clin Infect Dis 28(4), 800-7.

51. Korzeniowski OM. (1989). Effects of antibiotics on the mammalian immune system. Infect Dis Clin North Am 3, 469-78.

52. Kotb M. (1995). Bacterial pyrogenic exotoxins as superantigens. Clin Micro-biol Rev 8:3, 411-26.

53. Kusser WC, Ishiguro EE. (1988). Effects of aminoglycosides and spectinomy-cin on the synthesis and release of lipopolysaccharide by Escherichia coli. An-timicrob Agents Chemother 32, 1247-50.

54. Laczika K, Staudinger T, Hollenstein U et al. (1997). Renal tolerability of four different once-daily dose regimens of netilmicin in critical care patients. WienKlin Wochenschr 109, 840-4.

55. Lamy M, Mathy-Hartert M, Deby-Dupont G. (1996). Neutrophil-induced Oxidative Stress. In: Vincent JL ed. Yearbook of Intensive Care and Emer-gency Medicine. Berlin, Heidelberg: Springer-Verlag. 83-95

56. Lee PK, Schlievert PM. (1989). Quantification and toxicity of group A strep-tococcal pyrogenic exotoxins in an animal model of toxic shock syndrome-like illness. J Clin Microbiol 27, 1890-2.

57. Leeson MC, Morrison DC. (1994). Induction of proinflammatory responses in human monocytes by particulate and soluble forms of lipopolysacharide. Shock 2, 235-45.

58. Lepper PM, Held TK, Schneider EM et al. (2002). Clinical implications of antibiotic-induced endotoxin release in septic shock. Intensive Care Med 28, 824-33.

59. Levin M, Quint PA, Goldstein B. (2000). Recombinant bacteri-cidal/permeability increasing protein (rBPI21) as adjunctive treatment for children with severe meningococcal sepsis: a randomized trial. Lancet356(9234), 961-7.

60. Luchi M, Morrison DC, Opal S et al. (2000). A comparative trial of imipenem versus ceftazidime in the release of endotoxin and cytokine generation in pa-tients with gram-negative urosepsis. J Endotox Res 6, 25-31.

61. Löwdin, E., I. Odenholt, S. Bengtsson, and O. Cars. (1996). Pharmacodynamic effects of sub-MICs of Benzylpenicillin against Streptococcus pyogenes in a newly developed in vitro kinetic model. Antimicrob. Agents. Chemother40,2478-82.

62. Mascini, EM, Jansze M, Shouls LM et al. (2001). Penicillin and clindamycin differentially inhibit the production of pyrogenic exotoxins A and B by group A streptococci. Int J Antimicr Agents 18, 395-8.

63. Maskin B, Fontan PA, Spinedi EG. (2002). Evaluation of endotoxin release and cytokine production induced by antibiotics in patients with gram-negative nosocomial pneumonia. Crit Care Med 30, 349-54.

64. Mathison JC, Tobias PS, Wolfson E et al. (1992). Plasma lipopolysacharide binding protein. A key component in macrophage recognition of gram-negative LPS. J Immunol 149, 200-6.

49

65. Maury E, Barakett V, Blanchard H et al. (1998). Circulating endotoxin during initial antibiotic treatment of severe gram-negative bacteraemic infections. JInfect Dis 178, 270-3.

66. Meloni F, Ballabio P, Bianchi L et al. (1995). Cefodizime modulates in vitro tumor necrosis factor-alpha, interleukin-6 and interleukin-8 release from hu-man peripheral monocytes. Chemotherapy 41, 289-95.

67. Mendelman PM, Campos J, Chaffin DO et al. (1988). Relative penicillin G resistance in Neisseria meningitidis and reduced affinity of penicillin-binding protein 3. Antimicrob Agents Chemother 32, 706-9.

68. Michie HR, Manogue KR, Spriggs DR et al. (1988). Detection of circulating tumor necrosis factor after endotoxin administration. N Engl J Med 318, 1481-6.

69. Mock CN, Jurkovich GJ, Dries DJ et al.(1995). Clinical significance of antibi-otic endotoxin-releasing properties in trauma patients. Arch Surg 130, 1234-41.

70. Morrison DC, Bucklin SE, Leeson MC et al. (1996). Contribution of soluble endotoxin released from gramnegative bacteria by antibiotics to the patho-genesis of experimental sepsis in mice. J Endotox Res 3, 237-43.

71. Neu, HC. (1985). Relation of structural properties of beta-lactam antibiotics to antibacterial activity. Am J Med 79, 2-13.

72. Norrby-Teglund A, Thulin P, Gan BS. (2001). Evidence for superantigen involvement in severe group A streptococcal tissue infections. J Inf Dis 184, 853-60.

73. Norrby-Teglund A, Kaul R, Low DE et al. (1996). Evidence for the presence of streptococcal-superantigen-neutralizing antibodies in normal polyspecific immunoglobulin G. Infect Immun 64(12), 5395-8.

74. Opal SM, Cross AS, Jhung JW et al. (1996). Potential hazards of combination immunotherapy in the treatment of experimental septic shock. J Infect Dis 173(6), 1415-21.

75. Orus P, Vinas M. (2001). Mechanisms other than penicillin-binding protein-2 alterations may contribute to moderate penicillin resistance in Neisseria men-ingitidis. Int J Antimicrob Agents 18, 113-9.

76. Ottonello L, Dallegri F, Dapino P et al. (1991). Cytoprotection against neu-trophil-delivered oxidant attack by antibiotics. Biochem Pharmacology 42,2317-21.

77. Prins JM, van Agtmael MA, Kuijper EJ et al. (1995). Antibiotic-induced en-dotoxin release in patients with Gram-negative urosepsis: a double blind study comparing imipenem and ceftazidime. J Infect Dis 172, 886-91.

78. Prins JM, van Deventer SJH, Kuijper EJ et al. (1994). Clinical relevance of antibiotic-induced endotoxin release. Antimicrob Agents Chemother 38, 1211-8.

79. Prins JM, Speelman P, Kuijper EJ et al. (1997). No increase in endotoxin exposure during antibiotic killing of meningococci. J Antimicrob Chemother39(1), 13-8.

80. Rangel-Frausto MS, Pittet D, Costigan M et al. (1995). The natural history of the systemic inflammatory response syndrome (SIRS): A prospective study JAMA 273, 117-23.

81. Reinhart K, Wiegand-Lohnert C, Grimminger F et al. (1996). Assessment of the safety and efficacy of the monoclonal anti-tumor necrosis factor antibody-fragment, MAK 195F, in patients with sepsis and septic shock : a multicenter,

50

randomized, placebo-controlled, dose-ranging study. Crit Care Med 24(5), 733-42.

82. Ropp PA, Nicholas RA. (1997). Cloning and characterization of the ponA gene encoding penicillin-binding protein 1 from Neisseria gonorrhoeae and Neisseria meningitidis. J Bacteriol 179, 2783-7.

83. Salvo I, de Cian W, Musicco M, et al. The Italian sepsis study: preliminary results on the incidence and evolution of SIRS, sepsis, severe sepsis and septic shock. (1995) Intensive Care Med 21, 244-9.

84. Schlievert PM, Assimacoupoulos AP, Cleary PP. (1996). Severe invasive group A streptococcal disease: Clinical description and mechanisms of patho-genesis. J Lab Clin Med, 127, 13-22.

85. Shenep JL, Barton RP, Mogan KA. (1985). Role of antibiotic class in the rate of liberation of endotoxin during therapy for experimental gram-negative bac-terial sepsis. J Infect Dis 151, 1012-18.

86. Sjölin, J. (1996). Pathophysiology in septic shock. In: Risberg B, ed. Trauma care- an update. Pathophysiology of trauma. Mölndal, Sweden. PR-Offset. 88-103.

87. Stevens DL, Madaras-Kelly KJ, Richards DM. (1998). In vitro antimicrobial effects of various combinations of penicillin and clindamycin against four strains of Streptococcus pyogenes. Antimicrob Agents Chemother 42, 1266-8.

88. Stevens DL, Tanner MH, Winship J et al. (1989). Severe group A streptococ-cal infections associated with a toxic shock-like syndrome and scarlet fever toxin A. N Engl J Med 321, 1-8.

89. Stevens DL, Mayer KA, Mitten JE. (1987). Effects of antibiotics on toxin production and viability of Clostridium perfringens. Antimicrob Agents Che-mother 31, 213-8.

90. Stevens DL, Yan S, Bryant AE. (1993). Penicillin-binding protein expression at different growth stages determines penicillin efficacy in vitro and in vivo: an explanation for the inoculum effect. J Infect Dis 167, 1401-5.

91. Stevens DL, Gibbons AE, Bergstrom R et al. (1988). The Eagle effect revis-ited: Efficacy of clindamycin, erythromycin, and penicillin in the treatment of streptococcal myositis. J Infect Dis 158, 23-8.

92. Stevens DL, Bryant AE, Hackett SP. (1992). Grampositive shock. Curr Op Inf Dis 5, 355-63.

93. Tobias PS, Mathison J, Mintz D et al. (1992). Participation of lipopolysacha-ride-binding protein in lipopolysacharide-dependent macrophage activation. Am J Resp Cell Mol Biol 7, 239-45.

94. Tomasz A.(1986). Penicillin-binding proteins and the antibacterial effective-ness of -lactam antibiotics. Rev Infect Dis 8, 260-78.

95. Van den Berg C, de Neeling AJ, Schot CS et al. (1992). Delayed antibiotic-induced lysis of Escherichia coli in vitro is correlated with enhancement of LPS release. Scand J Infect Dis 24, 619-27.

96. van Deventer SJH, Buller HR, ten Cate JW et al. (1990). Experimental en-dotoxemia in humans: analysis of cytokine release and coagulation, fibri-nolytic and complement pathways. Blood 76,2520-6.

97. Warren BL, Eid A, Singer P et al. (2001). Caring for the critically ill patient. High dose antithrombin III in severe sepsis: a randomized controlled trial. JAMA 286(15), 1869-78.

51

98. Weiss J, Elsbach P, Olsson I et al. (1978). Purification and characterization of a potent bactericidal and membrane active protein from the granules of human polymorphonuclear leukocytes. J Biol Chem 253, 2664-72.

99. Wright SD, Tobias PS, Ulevitch RJ et al. (1989). Lipopolysacharide (LPS) binding protein opsonizes LPS bearing particles for recognition by a novel re-ceptor on macrophages.J Exp Med 170, 1231-41.

100. Xu S, Collins CM. (1996). Temperature regulation of the streptococcal pyro-genic exotoxin A-encoding gene (speA). Infect Immun 64, 5399-402.

101. Young LS. Sepsis syndrome. (1995). In: Mandell GL, Bennet JE, Dolin R, eds. Principles and practice of infectious diseases, 4th edn. New York: Chur-chill Livingstone, 690-705.

102. Ziegler EJ, Mc Cutchan JA, Fierer J et al. (1982). Treatment of gramnegative bacteremia and shock with human antiserum to a mutant E. coli. N Engl J Med 307, 1225-30.

103. Ziegler EJ, Fischer CJ, Sprung CL et al. (1991). Treatment of gramnegative bacteremia and septic shock with HA-1A human monoclonal antibody against endotoxin. N Engl J Med 324, 429-36.

Acta Universitatis UpsaliensisComprehensive Summaries of Uppsala Dissertations

from the Faculty of MedicineEditor: The Dean of the Faculty of Medicine

Distribution:Uppsala University Library

Box 510, SE-751 20 Uppsala, Swedenwww.uu.se, acta@ub.uu.se

ISSN 0282-7476ISBN 91-554-6109-3

A doctoral dissertation from the Faculty of Medicine, Uppsala University,is usually a summary of a number of papers. A few copies of the completedissertation are kept at major Swedish research libraries, while the sum-mary alone is distributed internationally through the series Comprehen-sive Summaries of Uppsala Dissertations from the Faculty of Medicine.(Prior to October, 1985, the series was published under the title “Abstracts ofUppsala Dissertations from the Faculty of Medicine”.)