Both bacterial and host factors contribute to complicated bloodstream infection (BSI) caused by Staphylococcus aureus including methicillin-resistant S. aureus (MRSA). One bacterial factor that may affect the persistence of S. aureus in complicated BSI is reduced susceptibility to the innate immune defence peptide LL-37. LL-37 susceptibility among S. aureus isolates causing uncomplicated and complicated BSI was investigated. Following incubation with 2.5 μg/ml LL-37 for 1 h, the mean percentage survival was 67.6% and 74.9% for isolates causing uncomplicated and complicated BSI, respectively. Reduced LL-37 susceptibility may contribute to the persistence of S. aureus in complicated BSI.
The clinical outcome of bloodstream infection (BSI) caused by Staphylococcus aureus is variable and both bacterial and host innate immune factors may contribute to the clinical course of infection. The innate immune system acts as the first line of defence against microorganisms such as S. aureus and cationic anti-microbial peptides (CAMPs) are an important component of the innate immune response [
Twenty S. aureus isolates that caused BSI in Beaumont Hospital Dublin between the years 2008-2011 were selected for this study. Ten isolates had caused uncomplicated BSI and 10 had caused a complicated infection, defined as a persistent (lasting >3 days despite appropriate antibiotics) or disseminated infection (e.g. osteomyelitis). Isolate details are summarized in
A combination of DNA microarray and spa typing was used to determine the clonal complex (CC) to which each isolate belonged. DNA microarray was also used to determine the presence or absence of genes that may contribute to LL-37 susceptibility. Genomic DNA was
aPresence or absence of gene determined by DNA microarray; bClonal lineage determined by a combination of DNA microarray and spa typing as described in the methods. Mprf, encodes multiple peptide resistance factor, aur encodes aureolysin, ica encodes intracellular adhesion, fnBPA encodes fibrinogen binding protein.
extracted using a DNeasy® blood and tissue kit (Qiagen, Crawley, UK). Spa typing, which involves PCR amplification and sequencing of the polymorphic 24 base pair variable number tandem repeat region within the 3’ end of the protein A gene spa, was carried out according to the SeqNet website (http://www.seqnet.org). Sequencing was performed by Beckman Coulter Genomics (Takeley, UK) and Source BioScience (Dublin, Ireland). Genetic characterization of isolates was undertaken using the StaphyType Kit (Alere Technologies Germany) as previously described [
A modification of the method described by Ouhara et al. (2008) [
(1/10) with 0.95% w/v NaCl. Percentage survival was calculated from viable counts (CFU/ml) from assays containing LL-37 compared to control assays containing no LL-37. The assay was performed in duplicate on three occasions for each isolate and the reference strains. Preliminary investigation of laboratory strains of S. aureus demonstrated concentration-dependent susceptibility to LL-37. The lowest concentration tested (2.5 µg/ml) allowed the observation of susceptibility differentials between strains (24%, 54%, 92% survival for COL, SH 1000 and 8325-4 respectively) and this concentration was therefore chosen for investigation of clinical isolates of S. aureus.
Mean survival following incubation with LL-37 was compared between isolates from uncomplicated Vs complicated infection using the Student’s t-test for statistical significance.
Among S. aureus clinical isolates causing complicated and uncomplicated BSI there was a wide variation in susceptibility to killing by LL-37 under the assay conditions used (uncomplicated BSI isolates, % survival; range = 24.2% - 100%, mean = 67.6%, complicated S. aureus BSI isolates, % survival; range = 52.8% - 100%, mean = 74.9%. However, overall, S. aureus isolates causing uncomplicated BSI were more susceptible to killing by LL- 37 than isolates causing complicated BSI but this difference was not statistically significant (p = 0.48, Student’s t-test) (
S. aureus isolates causing both complicated and uncomplicated infections belonged to a variety of CCs (
Genes that encode virulence factors that may influence LL-37 susceptibility among S. aureus, include mprf, aur, ica and fnBPA. Microarray analysis revealed the carriage of mprf in 18/20 S. aureus isolates (9/10 complicated and 9/10 uncomplicated) and the other three genes were found in all isolates investigated (
There was marked variation in the in-vitro susceptibility of S. aureus isolates to LL-37 and high variation has been previously reported among other collections of S. aureus clinical isolates [
It has previously been shown that MRSA isolates are more susceptible to killing by LL-37 than MSSA isolates [5,8,10] . Although the number of isolates studied here were relatively low, this pattern of MRSA/MSSA comparative susceptibility was not found in S. aureus causing BSI. Removing the four MRSA isolates from the previous comparison of uncomplicated Vs complicated infections did not result in a significant change in the statistical comparison of LL-37 susceptibility (percentage survival 64.1 % Vs 75.3 %, p = 0.39).
LL-37 has been shown to play an important role in the innate immune response to S. aureus infection However, S. aureus has evolved mechanisms that confer reduced susceptibility to LL-37 (e.g. increasing surface charge, inactivation of LL-37 by aureolysin) [4-6,10,11]. A number of other factors (PIA production, fnbP and the presence of a capsule) have also been shown to influence susceptibility of S. aureus isolates to killing by CAMPs, including LL-37 although the exact mechanisms are unknown [4,5]. The ability of certain S. aureus isolates to evade and inactivate this important antimicrobial peptide may contribute to their persistence in complicated BSI. However resistance to β-lactam antibiotics, capsule type or clonal type of the isolate did not significantly influence in-vitro susceptibility to LL-37.
Variations in LL-37 susceptibility among the isolates were also independent of the presence of genes encoding lysylphosphatidylglycerol synthetase (mprf), polysaccharide intracellular adhesin (ica), fibronectin binding protein (fnbAB) and aureolysin (aur) as all isolates investigated, contained them (except two which did not contain mprf). S. aureus causing BSI may produce other as yet unidentified virulence factors that may protect against innate immune defences. It is also possible that the expression of these genes varies among S. aureus isolates, which may contribute to the variations found. Expression of virulence genes is highly dependent on the dynamic environment encountered by the organism during infection and the killing assay used here does not facilitate investigation of the effects of these factors on LL-37 susceptibility.
In conclusion, LL-37 susceptibility may play a role in the persistence or potential for complications of S. aureus BSI, but the complex roles of LL-37 in the immune response against S. aureus and the significance of LL-37 resistance among S. aureus isolates, warrants further study.
We are grateful to the staff of the Microbiology Department, Beaumont Hospital, who provided S. aureus isolates. The authors acknowledge financial support from Pfizer Ireland through an Educational Award, No. WS 376235.