Antimicrobial activity of HL-60 cells compared to primary blood-derived neutrophils against Staphylococcus aureus.

BACKGROUND: The human leukemia cell line HL-60 is considered an alternative cell culture model to study neutrophil differentiation and migration. The aim of this study was to characterize the suitability of HL-60 cells differentiated to neutrophil-like cells (nHL-60) as substitute for blood-derived human neutrophils to investigate the interaction of neutrophils with Staphylococcus aureus. METHODS: For this purpose, antimicrobial activity, bacterial uptake, production of reactive oxygen species and the release of neutrophil extracellular traps (NETs) by nHL-60 cells were analyzed and compared to primary blood-derived neutrophils using Staphylococcus aureus as important human and animal pathogen. RESULTS: Overall, the antimicrobial activities of nHL-60 cells were distinctly lower compared to blood-derived neutrophils. Furthermore, production of reactive oxygen species as well as NET formation was clearly impaired in nHL-60 cells. CONCLUSION: This study indicates that HL-60 cells are of limited usage as an alternative model to study antimicrobial functions of neutrophils against Staphylococcus aureus.

Effects of SecDF on the antimicrobial functions of cathelicidins against Staphylococcus aureus.

Antimicrobial peptides (AMPs) represent an important part of the innate host immune system. Although they are active against a broad range of pathogens, bacteria have evolved different resistance mechanisms to avoid killing by AMPs. Since not much is known about the impact of efflux pumps on the susceptibility of Gram-positive bacteria to AMPs, especially to the cathelicidins, the aim of this study was to analyze whether Staphylococcus aureus can use efflux pumps to resist the antimicrobial effects of cathelicidins derived from different animal species (human, mouse, rabbit or cattle). For this purpose the minimal inhibitory concentrations (MICs) of S. aureus field isolates for the cathelicidins LL-37, mCRAMP, CAP18, BMAP-27 and BMAP-28 in the presence and absence of different efflux pump inhibitors were determined. Furthermore, the MICs of mutants lacking SecDF, a member of the RND efflux pump family, were determined and compared to the MICs of their respective wildtype and complemented strains. The data demonstrated that after blocking RND-type efflux pumps with 1-(1-naphthylmethyl)-piperazine, the MICs for CAP18, but not those for the other cathelicidins tested, were significantly decreased. In good correlation with these data, significantly decreased MICs for CAP18 and also BMAP-27 have been observed for SecDF knockout mutants as compared to their isogenic wildtype strains. In addition, the MIC values increased again after re-introducing a cloned secDF via plasmid complementation. These results indicated an involvement of SecDF in a reduced efficacy of species-specific cathelicidins against S. aureus.

Testing cathelicidin susceptibility of bacterial mastitis isolates: Technical challenges and data output for clinical isolates.

Bovine mastitis caused by bacterial pathogens, such as Staphylococcus (S.) aureus and Escherichia (E.) coli, is a major economic problem in dairy industry. In order to limit the presence of multi-resistant bacteria in bovine mastitis, alternatives for the treatment with antibiotics are urgently needed. Antimicrobial peptides (AMPs) have recently been discussed as a potential new strategy against bacterial infections. They are key players in the innate immune system, as they can directly act against microorganisms or modulate the immune system. The aim of our study was to test S. aureus and E. coli mastitis isolates for their susceptibility to the bovine cathelicidins, BMAP-27 and BMAP-28. Susceptibility testing was performed in analogy to the broth microdilution criteria described by the Clinical and Laboratory Standard Institute (CLSI) to determine MICs of 50 clinical S. aureus and 50 clinical E. coli isolates for BMAP-27 and BMAP-28. Based on the repetitive testing of four well-selected reference strains, the homogeneity of MIC variances for each peptide as well as the effect of temperature, oxygen level and plastic polymers on MIC testing was determined. Statistical analysis revealed not only strong peptide-specific variances, but also strain-specific variances in the technical procedure. Finally, using this technique, susceptibility testing of the field isolates revealed statistically significant peptide-specific differences in the MICs. While BMAP-27 showed lower MICs for E. coli, BMAP-28 showed lower MICs for S. aureus. However, these results clearly illustrate the need of susceptibility testing of AMPs on several unrelated strains and not only on one selected test organism.

Interaction of Bacterial Exotoxins with Neutrophil Extracellular Traps: Impact for the Infected Host.

Since their discovery in 2004, neutrophil extracellular traps (NETs) have been characterized as a fundamental host innate immune defense against various pathogens. Released in response to infectious and pro-inflammatory stimuli, NETs can immobilize invading pathogens within a fibrous matrix consisting of DNA, histones, and antimicrobial peptides. Conversely, excessive or dysregulated NET release may hold a variety of detrimental consequences for the host. A fine balance between NET formation and elimination is necessary to sustain a protective effect during infectious challenge. In recent years, a number of microbial virulence factors have been shown to modulate formation of NETs, thereby facilitating colonization or spread within the host. In this mini-review we summarize the contemporary research on the interaction of bacterial exotoxins with neutrophils that modulate NET production, focusing particular attention on consequences for the host. Understanding host-pathogen dynamics in this extracellular battlefield of innate immunity may provide novel therapeutic approaches for infectious and inflammatory disorders.

In vitro activity of human and animal cathelicidins against livestock-associated methicillin-resistant Staphylococcus aureus.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) is an important zoonotic pathogen. An emerging problem in treating S. aureus infections is the increasing resistance against antibiotics. A possible way to overcome this issue is to boost the host immune system and one target are antimicrobial peptides (AMPs), especially cathelicidins. The aim of this study was to characterize the antimicrobial activity of cathelicidins from different animal species against LA-MRSA and to reveal whether major antimicrobial resistance mechanisms influence the bactericidal activity of these peptides. The MICs of 153 LA-MRSA field isolates for different cathelicidins (LL-37, mCRAMP, CAP18, BMAP-27 and BMAP-28) were analysed. The cathelicidin MICs of S. aureus RN4220 and isogenic transformants, that carried 14 functionally active antimicrobial resistance genes, were determined. These resistance genes have been identified in LA-MRSA and specify the resistance mechanisms active efflux, enzymatic inactivation and modification/protection/replacement of target sites. The data showed that mode MIC values for the cathelicidins did not differ among the LA-MRSA isolates of different animal origin. However, distinct differences were detected between the MIC values for the different cathelicidins. MIC values were lowest for bovine cathelicidins (BMAP-27 and BMAP-28) and highest for the human and mouse cathelicidins (LL-37 and mCRAMP). None of the tested antimicrobial resistance genes affected the antimicrobial activity of the cathelicidins. The findings obtained in this study support the hypothesis that cathelicidins might be a promising target to support the host defense against LA-MRSA, especially since the antimicrobial activity of these peptides is not affected by common staphylococcal antimicrobial resistance genes.

Formation of Neutrophil Extracellular Traps under Low Oxygen Level.

Since their discovery, neutrophil extracellular traps (NETs) have been characterized as a fundamental host innate immune defense mechanism. Conversely, excessive NET-release may have a variety of detrimental consequences for the host. A fine balance between NET formation and elimination is necessary to sustain a protective effect during an infectious challenge. Our own recently published data revealed that stabilization of hypoxia-inducible factor 1α (HIF-1α) by the iron chelating HIF-1α-agonist desferoxamine or AKB-4924 enhanced the release of phagocyte extracellular traps. Since HIF-1α is a global regulator of the cellular response to low oxygen, we hypothesized that NET formation may be similarly increased under low oxygen conditions. Hypoxia occurs in tissues during infection or inflammation, mostly due to overconsumption of oxygen by pathogens and recruited immune cells. Therefore, experiments were performed to characterize the formation of NETs under hypoxic oxygen conditions compared to normoxia. Human blood-derived neutrophils were isolated and incubated under normoxic (21%) oxygen level and compared to hypoxic (1%) conditions. Dissolved oxygen levels were monitored in the primary cell culture using a Fibox4-PSt3 measurement system. The formation of NETs was quantified by fluorescence microscopy in response to the known NET-inducer phorbol 12-myristate 13-acetate (PMA) or Staphylococcus (S.) aureus wild-type and a nuclease-deficient mutant. In contrast to our hypothesis, spontaneous NET formation of neutrophils incubated under hypoxia was distinctly reduced compared to control neutrophils incubated under normoxia. Furthermore, neutrophils incubated under hypoxia showed significantly reduced formation of NETs in response to PMA. Gene expression analysis revealed that mRNA level of hif-1α as well as hif-1α target genes was not altered. However, in good correlation to the decreased NET formation under hypoxia, the cholesterol content of the neutrophils was significantly increased under hypoxia. Interestingly, NET formation in response to viable S. aureus wild-type or nuclease-deficient strain was retained under hypoxia. Our results lead to the conclusion that hypoxia is not the ideal tool to analyze HIF-1α in neutrophils. However, the data clearly suggest that neutrophils react differently under hypoxia compared to normoxia and thereby highlight the importance of the usage of physiological relevant oxygen level when studying neutrophil functions.

Yersinia enterocolitica-mediated degradation of neutrophil extracellular traps (NETs).

Neutrophil extracellular trap (NET) formation is described as a tool of the innate host defence to fight against invading pathogens. Fibre-like DNA structures associated with proteins such as histones, cell-specific enzymes and antimicrobial peptides are released, thereby entrapping invading pathogens. It has been reported that several bacteria are able to degrade NETs by nucleases and thus evade the NET-mediated entrapment. Here we studied the ability of three different Yersinia serotypes to induce and degrade NETs. We found that the common Yersinia enterocolitica serotypes O:3, O:8 and O:9 were able to induce NETs in human blood-derived neutrophils during the first hour of co-incubation. At later time points, the NET amount was reduced, suggesting that degradation of NETs has occurred. This was confirmed by NET degradation assays with phorbol-myristate-acetate-pre-stimulated neutrophils. In addition, we found that the Yersinia supernatants were able to degrade purified plasmid DNA. The absence of Ca(2+) and Mg(2+) ions, but not that of a protease inhibitor cocktail, completely abolished NET degradation. We therefore postulate that Y. enterocolitica produces Ca(2+)/Mg(2+)-dependent NET-degrading nucleases as shown for some Gram-positive pathogens.

[Antimicrobial resistance, ESBL and MRSA--definitions and laboratory diagnostics]. / Antimikrobielle Resistenz, ESBL und MRSA--Definitionen und Labordiagnose.

In the light of frequent discussions about the correct performance of in vitro susceptibility testing and the interpretation of the results obtained, the aim of the present report is to summarize basic facts that may facilitate the understanding of this complex topic. For this, the terms "antimicrobial resistance", "ESBL", and "MRSA" are defined. Besides the statements on antimicrobial resistance, information on intrinsic and acquired resistance properties as well as basic rules for the correct performance of antimicrobial susceptibility testing in routine diagnostics are presented. Moreover, the two groups of interpretive criteria--clinical breakpoints and epidemiological cut-off values--including their applications are explained in detail. Furthermore, currently valid diagnostic procedures--as published by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) and the Clinical and Laboratory Standards Institute (CLSI)--for the screening of ESBL-producing Enterobacteriaceae and MRSA as well as for the confirmation of suspicious isolates are presented and compared. Based on the information given, it becomes obvious that the correct performance of the diagnostic tests, which includes strict following the performance standards and the detailed information given therein, is an indispensable prerequisite for a standardized and harmonized in vitro susceptibility testing and--as a consequence--for the determination of valid and reliable susceptibility data in routine diagnostics. This is of utmost importance since the susceptibility data based on the use of clinical breakpoints often represent the basis for therapeutic interventions.