Mode of bacterial killing affects the inflammatory response and associated organ dysfunctions in a porcine E. coli intensive care sepsis model.

BACKGROUND: Sepsis is often treated with penicillin-binding protein 3 (PBP-3) acting ß-lactam antibiotics, such as piperacillin-tazobactam, cefotaxime, and meropenem. They cause considerable bacterial structural changes and have in vitro been associated with an increased inflammatory response. In a clinically relevant large animal sepsis model, our primary aim was to investigate whether bacteria killed by a PBP-3-active antibiotic has a greater effect on the early inflammatory response and organ dysfunction compared with corresponding amounts of live or heat-killed bacteria. A secondary aim was to determine whether the addition of an aminoglycoside could mitigate the cefuroxime-induced response. METHOD: Killed or live Escherichia coli were administrated as a 3-h infusion to 16 healthy pigs in a prospective, randomized controlled interventional experimental study. Cefuroxime was chosen as the PBP-3-active antibiotic and tobramycin represented the aminoglycosides. The animals were randomized to receive (I) bacteria killed by cefuroxime, (II) live bacteria, (III) bacteria killed by heat, or (IV) bacteria killed by the combination of cefuroxime and tobramycin. Plasma endotoxin, tumor necrosis factor alpha, interleukin-6, interleukin-10, leukocytes, and organ function were recorded at the start of the experiment and then hourly for 6 h. RESULTS: Differences in dynamics of concentration over time between the four treatment groups were found for the three cytokines (p < 0.001). Animals receiving cefuroxime-killed bacteria demonstrated higher responses than those receiving live (p < 0.05) or heat-killed bacteria (p < 0.01). The addition of tobramycin reduced the cefuroxime-induced responses (p < 0.001). The cytokine responses were associated with leucocyte activation that was further associated with pulmonary dysfunction and increases in lactate (p < 0.01). CONCLUSIONS: In comparison with live or heat-killed bacteria, bacteria killed by a PBP-3-active antibiotic induced an increased inflammatory response that appears to be associated with deteriorated organ and cellular function. The addition of an aminoglycoside to the PBP-3-active antibiotic reduced that response.

Dynamics of Endotoxin, Inflammatory Variables, and Organ Dysfunction After Treatment With Antibiotics in an Escherichia coli Porcine Intensive Care Sepsis Model.

OBJECTIVES: To investigate the dynamics of antibiotic-induced endotoxin liberation and inflammatory response in vivo in a clinically relevant large animal intensive care sepsis model and whether the addition of an aminoglycoside to a ß-lactam antibiotic affects these responses. DESIGN: Prospective, placebo-controlled interventional experimental study. SETTING: University research unit. SUBJECTS: Thirty-six healthy pigs administered Escherichia coli as a 3-hour infusion. INTERVENTIONS: After 2 hours, during E. coli infusion, the animals were exposed to cefuroxime alone, the combination of cefuroxime and tobramycin, or saline. MEASUREMENTS AND MAIN RESULTS: Plasma endotoxin, interleukin-6, tumor necrosis factor-α, leucocytes, and organ dysfunction were recorded for 4 hours after antibiotic treatment, and differences to the values before treatment were calculated. In vitro experiments were performed to ascertain whether endotoxin is released during antibiotic-induced bacterial killing of this E. coli strain. Despite differences between the treatment arms in vitro, no differences in plasma endotoxin were observed in vivo. Antibiotic-treated animals demonstrated a higher interleukin-6 response (p < 0.001), greater leucocyte activation (p < 0.001), and more pronounced deterioration in pulmonary static compliance (p < 0.01) over time than controls. Animals treated with the combination showed a trend toward less inflammation. CONCLUSIONS: Treatment with antibiotics may elicit an increased inflammatory interleukin-6 response that is associated with leucocyte activation and pulmonary organ dysfunction. No observable differences were detected in plasma endotoxin concentrations. The reduction in cefuroxime-induced endotoxin release after the addition of an aminoglycoside in vitro could not be reproduced in this model.

Lactobacilli Reduce Helicobacter pylori Attachment to Host Gastric Epithelial Cells by Inhibiting Adhesion Gene Expression.

The human gastrointestinal tract, including the harsh environment of the stomach, harbors a large variety of bacteria, of which Lactobacillus species are prominent members. The molecular mechanisms by which species of lactobacilli interfere with pathogen colonization are not fully characterized. In this study, we aimed to study the effect of lactobacillus strains upon the initial attachment of Helicobacter pylori to host cells. Here we report a novel mechanism by which lactobacilli inhibit adherence of the gastric pathogen H. pylori In a screen with Lactobacillus isolates, we found that only a few could reduce adherence of H. pylori to gastric epithelial cells. Decreased attachment was not due to competition for space or to lactobacillus-mediated killing of the pathogen. Instead, we show that lactobacilli act on H. pylori directly by an effector molecule that is released into the medium. This effector molecule acts on H. pylori by inhibiting expression of the adhesin-encoding gene sabA Finally, we verified that inhibitory lactobacilli reduced H. pylori colonization in an in vivo model. In conclusion, certain Lactobacillus strains affect pathogen adherence by inhibiting sabA expression and thereby reducing H. pylori binding capacity.

Characterization of motility and piliation in pathogenic Neisseria.

BACKGROUND: The type IV pili (Tfp) of pathogenic Neisseria (i.e., N. gonorrhoeae and N. meningitidis) are essential for twitching motility. Tfp retraction, which is dependent on the ATPase PilT, generates the forces that move bacteria over surfaces. Neisseria motility has mainly been studied in N. gonorrhoeae whereas the motility of N. meningitidis has not yet been characterized. RESULTS: In this work, we analyzed bacterial motility and monitored Tfp retraction using live-cell imaging of freely moving bacteria. We observed that N. meningitidis moved over surfaces at an approximate speed of 1.6 µm/s, whereas N. gonorrhoeae moved with a lower speed (1.0 µm/s). An alignment of the meningococcal and gonococcal pilT promoters revealed a conserved single base pair variation in the -10 promoter element that influence PilT expression. By tracking mutants with altered pilT expression or pilE sequence, we concluded that the difference in motility speed was independent of both. Live-cell imaging using total internal reflection fluorescence microscopy demonstrated that N. gonorrhoeae more often moved with fewer visible retracting filaments when compared to N. meningitidis. Correspondingly, meningococci also displayed a higher level of piliation in transmission electron microscopy. Nevertheless, motile gonococci that had the same number of filaments as N. meningitidis still moved with a lower speed. CONCLUSIONS: These data reveal differences in both speed and piliation between the pathogenic Neisseria species during twitching motility, suggesting a difference in Tfp-dynamics.

Meningococcal resistance to antimicrobial peptides is mediated by bacterial adhesion and host cell RhoA and Cdc42 signalling.

Antimicrobial peptides (AMPs) constitute an essential part of the innate immune defence. Pathogenic bacteria have evolved numerous strategies to withstand AMP-mediated killing. The influence of host epithelia on bacterial AMP resistance is, however, still largely unknown. We found that adhesion to pharyngeal epithelial cells protected Neisseria meningitidis, a leading cause of meningitis and sepsis, from the human cathelicidin LL-37, the cationic model amphipathic peptide (MAP) and the peptaibol alamethicin, but not from polymyxin B. Adhesion to primary airway epithelia resulted in a similar increase in LL-37 resistance. The inhibition of selective host cell signalling mediated by RhoA and Cdc42 was found to abolish the adhesion-induced LL-37 resistance by a mechanism unrelated to the actin cytoskeleton. Moreover, N. meningitidis triggered the formation of cholesterol-rich membrane microdomains in pharyngeal epithelial cells, and host cell cholesterol proved to be essential for adhesion-induced resistance. Our data highlight the importance of Rho GTPase-dependent host cell signalling for meningococcal AMP resistance. These results indicate that N. meningitidis selectively exploits the epithelial microenvironment in order to protect itself from LL-37.

Lactobacilli reduce cell cytotoxicity caused by Streptococcus pyogenes by producing lactic acid that degrades the toxic component lipoteichoic acid.

Lactobacilli are known to prevent colonization by many pathogens; nevertheless, the mechanisms of their protective effect are largely unknown. In this work, we investigated the role of lactobacilli during infection of epithelial cells with group A streptococci (GAS). GAS cause a variety of illnesses ranging from noninvasive disease to more severe invasive infections, such as necrotizing fasciitis and toxic shock-like syndrome. Invasion of deeper tissues is facilitated by GAS-induced apoptosis and cell death. We found that lactobacilli inhibit GAS-induced host cell cytotoxicity and shedding of the complement regulator CD46. Further, survival assays demonstrated that lactic acid secreted by lactobacilli is highly bactericidal toward GAS. In addition, lactic acid treatment of GAS, but not heat killing, prior to infection abolishes the cytotoxic effects against human cells. Since lipoteichoic acid (LTA) of GAS is heat resistant and cytotoxic, we explored the effects of lactic acid on LTA. By applying such an approach, we demonstrate that lactic acid reduces epithelial cell damage caused by GAS by degrading both secreted and cell-bound LTA. Taken together, our experiments reveal a mechanism by which lactobacilli prevent pathogen-induced host cell damage.

Endotoxin, capsule, and bacterial attachment contribute to Neisseria meningitidis resistance to the human antimicrobial peptide LL-37.

Pathogenic bacteria have evolved numerous mechanisms to evade the human immune system and have developed widespread resistance to traditional antibiotics. We studied the human pathogen Neisseria meningitidis and present evidence of novel mechanisms of resistance to the human antimicrobial peptide LL-37. We found that bacteria attached to host epithelial cells are resistant to 10 microM LL-37 whereas bacteria in solution or attached to plastic are killed, indicating that the cell microenvironment protects bacteria. The bacterial endotoxin lipooligosaccharide and the polysaccharide capsule contribute to LL-37 resistance, probably by preventing LL-37 from reaching the bacterial membrane, as more LL-37 reaches the bacterial membrane on both lipooligosaccharide-deficient and capsule-deficient mutants whereas both mutants are also more susceptible to LL-37 killing than the wild-type strain. N. meningitidis bacteria respond to sublethal doses of LL-37 and upregulate two of their capsule genes, siaC and siaD, which further results in upregulation of capsule biosynthesis.

Fitness of Spontaneous Rifampicin-Resistant Staphylococcus aureus Isolates in a Biofilm Environment.

Biofilms of S. aureus accumulate cells resistant to the antibiotic rifampicin. We show here that the accumulation of rifampicin resistant mutants (RifR) in biofilms is not equable but rather is a local event, suggesting that the growth of a few locally emerged mutants is responsible for this. Competition assays demonstrated that, compared to wild-type bacteria, the isolated RifR mutants have a growth advantage in biofilms, but not in planktonic culture. To gain insight into the mechanism of the growth advantage, we tested the involvement of the two-component systems (TCS) that sense and respond to environmental changes. We found that a deletion of SrrAB or NreBC has a drastic effect on the growth advantage of RifR mutants, suggesting the importance of oxygen/respiration responses. All six of the RifR isolates tested showed increased resistance to at least one of the common stresses found in the biofilm environment (i.e., oxidative, nitric acid, and organic acid stress). The RifR mutants also had a growth advantage in a biofilm flow model, which highlights the physiological relevance of our findings.

Meningococcal outer membrane protein NhhA is essential for colonization and disease by preventing phagocytosis and complement attack.

Neisseria meningitidis is a leading cause of meningitis and septicemia worldwide, with a rapid onset of disease and a high morbidity and mortality. NhhA is a meningococcal outer membrane protein included in the family of trimeric autotransporter adhesins. The protein binds to the extracellular matrix proteins heparan sulfate and laminin and facilitates attachment to host epithelial cells. In this study, we show that NhhA is essential for bacterial colonization of the nasopharyngeal mucosa in a murine model of meningococcal disease. Successful colonization depends on bacterial attachment but also to the capacity to overcome innate host immune responses. We found that NhhA protected bacteria from phagocytosis, which is important for the mucosal survival of bacteria. In addition, NhhA mediated extensive serum resistance that increased bacterial survival in blood and promoted lethal sepsis. The presence of NhhA protected bacteria from complement-mediated killing by preventing the deposition of the membrane attack complex. Taken together, the results of this work reveal that NhhA inhibits phagocytosis and protects bacteria against complement-mediated killing, which enhances both nasal colonization and the development of sepsis in vivo.

The Host Cell Transcription Factor EGR1 Is Induced by Bacteria through the EGFR-ERK1/2 Pathway.

The essential first step in bacterial colonization is adhesion to the host epithelial cells. The early host-responses post-bacterial adhesions are still poorly understood. Early growth response 1 (EGR1) is an early response transcriptional regulator that can be rapidly induced by various environmental stimuli. Several bacteria can induce EGR1 expression in host cells, but the involved bacterial characteristics and the underlying molecular mechanisms of this response are largely unknown. Here, we show that EGR1 can be induced in host epithelial cells by different species of bacteria independent of the adherence level, Gram-staining type and pathogenicity. However, bacterial viability and contact with host cells is necessary, indicating that an active interaction between bacteria and the host is important. Furthermore, the strongest response is observed in cells originating from the natural site of the infection, suggesting that the EGR1 induction is cell type specific. Finally, we show that EGFR-ERK1/2 and ß1-integrin signaling are the main pathways used for bacteria-mediated EGR1 upregulation. In conclusion, the increase of EGR1 expression in epithelial cells is a common stress induced, cell type specific response upon host-bacteria interaction that is mediated by EGFR-ERK1/2 and ß1-integrin signaling.

Lactobacilli Interfere with Streptococcus pyogenes Hemolytic Activity and Adherence to Host Epithelial Cells.

Streptococcus pyogenes [Group A streptococcus (GAS)], a frequent colonizer of the respiratory tract mucosal surface, causes a variety of human diseases, ranging from pharyngitis to the life-threatening streptococcal toxic shock-like syndrome. Lactobacilli have been demonstrated to colonize the respiratory tract. In this study, we investigated the interference of lactobacilli with the virulence phenotypes of GAS. The Lactobacillus strains L. rhamnosus Kx151A1 and L. reuteri PTA-5289, but not L. salivarius LMG9477, inhibited the hemolytic activity of S. pyogenes S165. The inhibition of hemolytic activity was attributed to a decrease in the production of streptolysin S (SLS). Conditioned medium (CM) from the growth of L. rhamnosus Kx151A1 and L. reuteri PTA-5289 was sufficient to down-regulate the expression of the sag operon, encoding SLS. The Lactobacillus strains L. rhamnosus Kx151A1, L. reuteri PTA-5289, and L. salivarius LMG9477 inhibited the initial adherence of GAS to host epithelial cells. Intriguingly, competition with a combination of Lactobacillus species reduced GAS adherence to host cells most efficiently. The data suggest that an effector molecule released from certain Lactobacillus strains attenuates the production of SLS at the transcriptional level and that combinations of Lactobacillus strains may protect the pharyngeal mucosa more efficiently from the initial colonization of GAS. The effector molecules released from Lactobacillus strains affecting the virulence phenotypes of pathogens hold potential in the development of a new generation of therapeutics.

Staphylococcus aureus dry stress survivors have a heritable fitness advantage in subsequent dry exposure.

Staphylococcus aureus is a major cause of hospital-acquired infections. The ability to survive on abiotic surfaces is an important characteristic that facilitates transmission between human hosts. We found that S. aureus survivors of dry surface incubation are resistant to subsequent dry stress exposure. Survivors also had reduced sensitivity to the disinfectant chlorhexidine gluconate, but not to ethanol. By using a set of mutants in cardiolipin synthase genes, we further demonstrated that the housekeeping cardiolipin synthase, Cls2, was significant for survival on dry surface. Taken together, this study provides insights into S. aureus survival outside of a host.

Beneficial antimicrobial effect of the addition of an aminoglycoside to a ß-lactam antibiotic in an E. coli porcine intensive care severe sepsis model.

This study aimed to determine whether the addition of an aminoglycoside to a ß-lactam antibiotic increases the antimicrobial effect during the early phase of Gram-negative severe sepsis/septic shock. A porcine model was selected that considered each animal's individual blood bactericidal capacity. Escherichia coli, susceptible to both antibiotics, was given to healthy pigs intravenously during 3 h. At 2 h, the animals were randomized to a 20-min infusion with either cefuroxime alone (n = 9), a combination of cefuroxime+tobramycin (n = 9), or saline (control, n = 9). Blood samples were collected hourly for cultures and quantitative polymerase chain reaction (PCR). Bacterial growth in the organs after 6 h was chosen as the primary endpoint. A blood sample was obtained at baseline before start of bacterial infusion for ex vivo investigation of the blood bactericidal capacity. At 1 h after the administration of the antibiotics, a second blood sample was taken for ex vivo investigation of the antibiotic-induced blood killing activity. All animals developed severe sepsis/septic shock. Blood cultures and PCR rapidly became negative after completed bacterial infusion. Antibiotic-induced blood killing activity was significantly greater in the combination group than in the cefuroxime group (p<0.001). Growth of bacteria in the spleen was reduced in the two antibiotic groups compared with the controls (p<0.01); no difference was noted between the two antibiotic groups. Bacterial growth in the liver was significantly less in the combination group than in the cefuroxime group (p<0.05). High blood bactericidal capacity at baseline was associated with decreased growth in the blood and spleen (p<0.05). The addition of tobramycin to cefuroxime results in increased antibiotic-induced blood killing activity and less bacteria in the liver than cefuroxime alone. Individual blood bactericidal capacity may have a significant effect on antimicrobial outcome.