d-Alanylation of Lipoteichoic Acids in Streptococcus suis Reduces Association With Leukocytes in Porcine Blood.

Streptococcus suis (S. suis) is a common swine pathogen but also poses a threat to human health in causing meningitis and severe cases of streptococcal toxic shock-like syndrome (STSLS). Therefore, it is crucial to understand how S. suis interacts with the host immune system during bacteremia. As S. suis has the ability to introduce d-alanine into its lipoteichoic acids (LTAs), we investigated the working hypothesis that cell wall modification by LTA d-alanylation influences the interaction of S. suis with porcine blood immune cells. We created an isogenic mutant of S. suis strain 10 by in-frame deletion of the d-alanine d-alanyl carrier ligase (DltA). d-alanylation of LTAs was associated with reduced phagocytosis of S. suis by porcine granulocytes, reduced deposition of complement factor C3 on the bacterial surface, increased hydrophobicity of streptococci, and increased resistance to cationic antimicrobial peptides (CAMPs). At the same time, survival of S. suis was not significantly increased by LTA d-alanylation in whole blood of conventional piglets with specific IgG. However, we found a distinct cytokine pattern as IL-1ß but not tumor necrosis factor (TNF)-α levels were significantly reduced in blood infected with the ΔdltA mutant. In contrast to TNF-α, activation and secretion of IL-1ß are inflammasome-dependent, suggesting a possible influence of LTA d-alanylation on inflammasome regulation. Especially in the absence of specific antibodies, the association of S. suis with porcine monocytes was reduced by d-alanylation of its LTAs. This dltA-dependent phenotype was also observed with a non-encapsulated dltA double mutant indicating that it is independent of capsular polysaccharides. High antibody levels caused high levels of S. suis-monocyte-association followed by inflammatory cell death and strong production of both IL-1ß and TNF-α, while the influence of LTA d-alanylation of the streptococci became less visible. In summary, the results of this study expand previous findings on d-alanylation of LTAs in S. suis and suggest that this pathogen specifically modulates association with blood leukocytes through this modification of its surface.

Porcine iucA+ but rmpA- Klebsiella pneumoniae strains proliferate in blood of young piglets but are killed by IgM and complement dependent opsonophagocytosis when these piglets get older.

Klebsiella (K.) pneumoniae causes different diseases in humans and animals including the life-threatening liver abscess syndrome and septicemia, respectively. However, host-pathogen interactions of K. pneumoniae in porcine blood have not been studied. We investigated the working hypothesis that only distinct K. pneumoniae strains have the capacity to survive in porcine blood and that this feature is associated with specific molecular markers such as sequence type, profile of siderophore genes and the regulator of the mucoid phenotype (rmp). Furthermore, we characterize the immune response in growing piglets leading to killing of an invasive K. pneumoniae strain. The veterinary isolates showed great diversity in sequence types and profile of siderophore genes. Porcine isolates were mainly positive for the aerobactin gene iucA but did not carry rmpA and this genotype was associated with proliferation in blood of 4-week-old piglets. Supernatants of an iucA+ but not an iucA- strain boosted growth in porcine serum. Between four and eight weeks of age, piglets showed a prominent increase of IgM binding to K. pneumoniae. Immunglobulin M and complement were crucial for killing of a serum-resistant iucA+ porcine K. pneumoniae strain at eight weeks of age. Flow cytometry analysis confirmed induction of phagocytosis and oxidative burst mediated by serum samples of 8-week-old piglets. Based on our in vitro findings we propose that many porcine iucA+ rmpA- K. pneumoniae strains have the ability to cause bacteremia in young piglets in association with aerobactin-mediated iron acquisition and that this phenotype is lost as specific IgM increases after weaning.

Proline-rich antimicrobial peptide Api137 is bactericidal in porcine blood infected ex vivo with a porcine or human Klebsiella pneumoniae strain.

OBJECTIVES: Klebsiella pneumoniae is an emerging invasive pathogen in humans and pigs. Resistance against multiple antibiotics in this species is a major health concern and the development of new antibiotics is urgently needed. The objective of this study was to investigate the effects of proline-rich antimicrobial peptides (PrAMPs) on the survival of K. pneumoniae strains in porcine blood. METHODS: We established a bactericidal assay with K. pneumoniae in fresh blood drawn from 4-week-old piglets. PrAMPs, namely the apidaecins Api137 and Api802 as well as the oncocin Onc112, were added to ex vivo-infected whole blood samples in order to study their bactericidal effects and, in the case of Api137, also immune responses. RESULTS: A porcine invasive and a human iucA+rmpA+ K. pneumoniae strain showed prominent proliferation in porcine blood. Application of Api137 resulted in a dose-dependent prominent bactericidal effect killing the invasive porcine K. pneumoniae strain. Addition of 8 µg/mL Api137 also resulted in complete killing of the human iucA+rmpA+ strain. Cytotoxicity, haemolysis and induction of the pro-inflammatory cytokine tumour necrosis factor-alpha (TNFα) in K. pneumoniae-infected porcine blood treated with Api137 was comparable with values obtained after application of 10 µg/mL cefquinome. CONCLUSION: We describe a new non-rodent model for invasive K. pneumoniae bacteraemia and present promising data for the PrAMP Api137 for the control of infection with hypervirulent K. pneumoniae strains.