Susceptibility of clinical Staphylococcus aureus isolates to innate defense antimicrobial peptides

Microbes and Infection 13 (2011) 761e765 www.elsevier.com/locate/micinf

Short communication

Susceptibility of clinical Staphylococcus aureus isolates to innate defense antimicrobial peptides Siegbert Rieg a,b,*, Achim J. Kaasch c, Julian Wehrle a, Silke C. Hofmann b,d, Magdalena Szymaniak-Vits a, Viola Saborowski b,d, Daniel Jonas e, Hubert Kalbacher f, Harald Seifert c, Winfried V. Kern a a

Center for Infectious Diseases and Travel Medicine, Department of Medicine, University Hospital, Hugstetter Strasse 55, D-79106 Freiburg, Germany b IFB-Center for Chronic Immunodeficiency, University Hospital, Hugstetter Str. 55, D-79106 Freiburg, Germany c Institute for Medical Microbiology, Immunology and Hygiene, University of Cologne, Goldenfelsstr, 19e21, D-50935, Germany d Department of Dermatology, University Hospital, Hauptstr. 7, D-79104 Freiburg, Germany e Institute of Environmental Health Sciences, University Hospital, Breisacher Str. 115b, D-79106 Freiburg, Germany f Medical and Natural Sciences Research Center, Eberhard Karls University, Ob dem Himmelreich 7, D-72074 Tu¨bingen, Germany Received 28 October 2010; accepted 25 March 2011 Available online 5 April 2011

Abstract Antimicrobial peptides (AMPs) are effector molecules of innate immunity. To determine whether AMP susceptibility of S. aureus varies according to different types of infection, 102 isolates from patients with S. aureus bacteremia or recurrent skin and soft tissue infection, and colonizing isolates were investigated. Using microbroth dilution assays we found a narrow range of MICs of human b-defensin-3, cathelicidin LL-37 and bovine indolicidin without significant differences between the groups. Colony-forming unit (CFU) assays revealed minor differences in bactericidal activity with slightly but not significantly higher CFU reduction in colonizing isolates. These data do not support a role for differential AMP susceptibility in vitro as a major determinant of S. aureus invasive infection. Ó 2011 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. Keywords: Innate immunity; Antimicrobial peptides; Staphylococcus aureus; INSTINCT

1. Introduction Staphylococcus aureus is a major pathogen causing community-acquired and healthcare-associated infection. The clinical spectrum ranges from asymptomatic colonization of epithelial surfaces and circumscribed skin and soft tissue infections to invasive life-threatening disease [1]. S. aureus colonizes the anterior nares, pharynx and skin. Approximately 20% of individuals are permanent nasal carriers, 30% intermittent carriers, whereas about 50% are non-carriers. Though our knowledge of human innate immunity and of strategies of * Corresponding author. Department of Medicine, Center for Infectious Diseases and Travel Medicine, University Hospital, Hugstetter Strasse 55, D-79106 Freiburg, Germany. Tel.: þ49 761 270 1819; fax: þ49 761 270 1820. E-mail address: [email protected] (S. Rieg).

S. aureus to circumvent host defense mechanisms has expanded over the last years, factors that determine colonization and, more importantly, favor invasive S. aureus infection remain incompletely understood [2]. Antimicrobial peptides (AMPs) are evolutionarily conserved effector molecules of the innate immune system. As part of the first line of defense against pathogens AMPs are produced by leucocytes and epithelial cells along the inner and outer body surfaces. Apart from direct microbicidal effects, these host defense peptides exert a broad range of immunological functions including chemotactic activity and stimulation of cell proliferation and maturation thereby linking innate and adaptive immunity [3]. Within the last years, several S. aureus mechanism to attenuate AMP-mediated killing have been described: degradation of the human cathelicidin LL-37 by S. aureus proteases aureolysin and V8; production of staphylokinase, an exoprotein

1286-4579/$ - see front matter Ó 2011 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. doi:10.1016/j.micinf.2011.03.010

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that besides its fibrinolytic activity binds and inactivates human a-defensins; and alteration of bacterial cell wall structures, namely, D-alanylation of teichoic acids or L-lysinylation of phosphatidylglycerol to reduce the net negative charge and, thus, the affinity of cationic AMPs to the bacterial cell wall [4]. Whether clinical S. aureus strains differ with respect to their AMP susceptibility and whether potential differences are related to colonization or invasion or to specific clinical manifestations is not known. Here, we investigated the in vitro susceptibility to human and a control bovine innate defense AMPs in S. aureus isolates representing bacteremia and sepsis, skin and soft tissue infection and asymptomatic carriage.

MRSA 2222 (PVL-positive, clonal complex t044, kindly donated by Hans-Jo¨rg Linde, Regensburg) and MRSA 3839 (clonal complex CC398) were used as reference strains. AMPs tested in our study were human b-defensin-3 (hBD-3, PeptaNova, Sandhausen, Germany), human cathelicidin LL-37 and bovine neutrophil-derived indolicidin, both were synthesized using standard Fmoc/tBu chemistry on a multiple peptide synthesizer Syro II (MultiSynTech, Witten, Germany). a-defensins human neutrophil peptides (HNP) 1e3 were isolated from peripheral neutrophils as previously described [6].

2. Materials and methods

Susceptibility to AMPs was investigated by determining the minimal inhibitory concentration (MIC) after overnight incubation in a modified microbroth dilution assay, and by measuring bactericidal activity after 2 h of incubation in a colony-forming unit (CFU) assay [7]. A minimal nutrient buffer system was used to minimize interference with the peptide’s biological activity. Briefly, 5  104 CFU of midexponential growth phase cultures were incubated for 2 h with respective peptide concentrations in 10 mM sodium phosphate buffer (pH 7.4) containing 1% Mueller Hinton broth (MHB) using polypropylene microtiter plates and a final volume of 25 mL. Then, 75 mL of MHB was added and plates were incubated for 16e18 h. The lowest concentration without any visible growth was considered the MIC. MIC determinations were done in triplicate. For the CFU assay, 2  105 CFU of bacteria were co-incubated for 2 h with AMPs in phosphate buffer containing 1% MHB. Serial dilutions were plated on blood agar plates and visible colonies were counted after 18e24 h. Bactericidal activity was expressed as the percentage of bacteria that were killed after incubation with the peptides compared with incubation with peptide solvent (0.01% acetic acid). AMP concentrations in the CFU assays were 0.125 mg/ ml of hBD-3, 1 mg/ml of LL-37 and indolicidin, and 8 mg/mL of HNP 1e3. CFU assays were performed in duplicates at least.

2.1. Patients The study protocol was approved by the Institutional Review Board of the medical faculty of Freiburg University. All study participants gave written informed consent. S. aureus colonization was determined by means of 5 consecutive nasal and axillary/inguinal swabs, sampled at 2-week intervals according to standard protocols. Participants were classified as permanent carriers when at least 4 of the swab samples (nares or axillary/inguinal skin) were positive for S. aureus, as intermittent carriers when 3 or less were positive. Exclusion criteria were current antibiotic use, diabetes mellitus, endstage renal disease, psoriasis and atopic dermatitis. The patient group of recurrent S. aureus skin and soft tissue infections (SSTI) included patients with S. aureus furunculosis (defined as a minimum of 6 episodes of furuncles within the last 12 months) and patients with recurrent cutaneous or subcutaneous S. aureus abscesses (at least 4 abscesses per year). Patients with S. aureus bacteremia (SAB) were evaluated within the prospective INvasive STaph. aureus INfections CohorT (INSTINCT) registry [5]. Uncomplicated bacteremia was defined as intravascular catheter-related bacteremia without evidence of dissemination and defervescence within 48e72 h after catheter-removal and initiation of therapy. Complicated SAB was defined as non-catheter-related SAB with presence of deep-seated metastatic foci, endocarditis (confirmed by modified Duke criteria) or severe sepsis/septic shock according to definitions of the ACCP/SCCM consensus conference. Patients with polymicrobial bacteremia and a neutrophil count <1.0  109 cells/L were excluded from the study.

2.3. Susceptibility testing

2.4. Statistics Proportions (isolates with MIC of 16 mg/ml [hBD-3 and indolicidin] and 128 mg/ml [LL-37] versus isolates with lower MIC values) were compared using Fisher’s exact test. A (two-sided) p-value of <0.05 was considered significant. 3. Results

2.2. S. aureus clinical isolates and reference strains, antimicrobial peptides S. aureus colonizing strains, lesional isolates of furuncles or abscesses and blood culture isolates were identified by conventional methods using Gram staining and Slidex Staph Plus (bioMe´rieux, France). S. aureus ATCC 12598, S. aureus Mu-50 (kindly donated by Stefan Monecke, Dresden), S. aureus USA300 (kindly donated by Alexander Friedrich, Muenster) and two clinical methicillin-resistant isolates community-acquired

3.1. Narrow MIC range of investigated antimicrobial peptides The MICs of hBD-3 ranged between 4 and 16 mg/mL (Table 1). An hBD-3 MIC of 16 mg/mL was observed in 17/27 colonizing isolates compared with 61/75 isolates from patients with SAB and skin infection (63% versus 81%, p ¼ 0.067). The MICs of LL-37 ranged between 32 and 256 mg/mL. A non-significant trend towards higher LL-37 MICs of the

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Table 1 Distribution of minimum inhibitory concentrations (MICs) of AMPs in different clinical S. aureus isolates and selected reference strains. Number of isolates with indicated AMP MIC (mg/mL) hBD-3 4 SAB (n ¼ 59) Complicated SAB (n ¼ 29) Uncomplicated SAB (n ¼ 30) Recurrent SSTI (n ¼ 16) Furunculosis (n ¼ 10) Abscess (n ¼ 6) Colonizing isolates (n ¼ 27) Permanent carriers (n ¼ 16) Intermittent carriers (n ¼ 11)

1 1

LL-37

Indolicidin

8

16

32

64

128

10 4 6 4 3 1 9 4 5

49 25 24 12 7 5 17 11 6

1

23 12 11 2 1 1 6 4 2

35 17 18 14 9 5 19 10 9

1

256

2 2

4

1 1

8

16

7 3 4 3 1 2 4 1 3

52 26 26 13 9 4 22 14 8

MICs of innate defense AMPs human b-defensin-3 (hBD-3), human cathelicidin LL-37 and bovine indolicidin were determined by a microbroth dilution assay: shown is the distribution of the MICs of the various AMPs in different groups of clinical S. aureus isolates (bacteremia [SAB], recurrent skin and soft tissue infection [SSTI] and carriage without disease). The AMP MICs for S. aureus reference strains were: ATCC 12598 and MRSA 3839: 16 mg/ml for hBD-3, 64 mg/ml for LL-37, 16 mg/ml for indolicidin, respectively; corresponding values for MRSA USA300 and CA-MRSA 2222 were: 8, 128, and 16 mg/ml, respectively; for VISA strain Mu-50 they were: 8, 128, and 8 mg/ml, respectively.

colonizing isolates as compared to the SAB isolates and the skin infection isolates was observed. The MICs of indolicidin ranged between 4 and 16 mg/mL, with most isolates revealing a MIC of 16 mg/mL and without significant differences between the isolates of the three groups. To investigate whether isolates within the different clinical groups differed in their AMP susceptibility, isolates from subgroups of permanently versus intermittently colonized hosts and furunculosis isolates versus abscess-causing isolates were investigated, but we found no significant differences in the distribution of MICs of hBD-3, LL-37 and indolicidin (Table 1). Isolates of uncomplicated catheter-related SAB had MIC values that were comparable to those of isolates from complicated SAB (Table 1). We were also interested in whether peptide MICs correlated with specific clinical manifestations and outcome. No differences in the distribution of MICs, however, were found for endocarditis versus non-endocarditis isolates, for isolates from patients with and without severe sepsis/septic shock, and for isolates from patients with and without deep-seated metastatic foci (data not shown). Of note, 12 out of 13 isolates from patients who died within 30 days after SAB onset had hBD-3 MICs of 16 mg/mL, a proportion which was slightly higher than among isolates from patients who survived. The difference, however, was small and statistically not significant (12/13 [92%] versus 37/46 [80%], p ¼ 0.43). 3.2. Slightly higher CFU reduction by AMPs in colonizing S. aureus isolates Susceptibility of 12 SAB isolates and 12 colonizing isolates to hBD-3, LL-37, indolicidin and HNP 1e3 was tested by means of a CFU assay (Fig. 1). Overall, the AMP susceptibility range, i.e. the differences in bactericidal activity, was greater within the group of colonizing isolates. This effect was observed with each of the investigated peptides, was strongest for LL-37 (range 0e82% in SAB isolates versus 10e39% in colonizing isolates) and indolicidin (0e87% versus 16e60%

respectively), and was mainly due to a greater CFU reduction (of w80%) of AMPs in some of the colonizing isolates. With respect to mean CFU reduction, there was a trend toward higher susceptibility of colonizing isolates as compared to SAB isolates for each of the four AMPs investigated. Noteworthy, cross-susceptibility between the different AMPs could be observed, i.e. isolates that were found to be particularly susceptible against hBD-3 (e.g. colonizing isolates COL2 and COL6) also revealed pronounced susceptibility to LL-37, indolocidin and HNP 1e3. Accordingly, cross-resistance between hBD-3, LL-37 and indolicidin could be observed. 4. Discussion Using microbroth dilution assays the MICs of the investigated AMPs did not show major differences between colonizing and infecting S. aureus isolates although the trend of a possibly enhanced susceptibility of carriage isolates is noteworthy. We found that almost all isolates from SAB patients who died had high hBD-3 MICs, yet we recognize that the difference was small and statistically non-significant. Overall, we found a rather narrow range of MICs, comprising only three (hBD-3 and indolicidin) or four (LL-37) dilutional steps without any indication of a bimodal distribution. This is unlike the ranges usually described in standard drug MIC testing and argues against the existence of true AMP “resistance” among clinical S. aureus as a major virulence factor. We performed MIC/inhibition of growth testing in a minimal nutrient buffer system due to its proximity to growth conditions on skin or mucosal surfaces, yet are aware of the limitations this as any in vitro assay bears. Besides along epithelial surfaces, AMPs are abundantly present in neutrophil primary and secondary granules. Upon contact with pathogens in the phagolysosome (HNP 1e3) or upon secretion and processing of LL-37, bactericidal activity rather than growth inhibition seems important, thus, we decided to complement MIC testing with CFU assays. Corroborating the

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Fig. 1. Colony-forming unit assays of 12 S. aureus bacteremia isolates (left column), 12 colonizing S. aureus isolates (center column) and five S. aureus reference strains (right column). AMPs human b-defensin-3 (hBD-3, 0.125 mg/ml), human cathelicidin LL-37 (1 mg/ml), bovine indolicidin (1 mg/ml) and a-defensins human neutrophil peptides 1e3 (HNP 1e3, 8 mg/ml) were incubated for 2 h with 2  105 CFU S. aureus. CFU reduction, i.e. bactericidal activity, was expressed as percentage of bacteria that were killed after incubation with AMPs as compared to incubation with peptide solvent. Presented data are means  standard deviation of at least two experiments.

findings of the microbroth dilution assay, CFU assays revealed rather small and statistically non-significant differences in bactericidal activity with mean CFU reduction between 5 and 10% higher in the group of colonizing S. aureus strains as compared to bacteremia isolates. So far, data on AMP susceptibility of clinical isolates have been scarce. Escherichia coli strains causing pyelonephritis were found to be moderately more resistant to LL-37 than strains isolated from patients with uncomplicated cystitis (mean MIC 50 vs. 30 mg/ml), providing evidence that AMP “resistance” may contribute to the ability to establish invasive infection [8]. Investigations focusing on AMP susceptibility of S. aureus in the context of methicillin resistance revealed a trend towards higher survival after LL-37 exposure in highly methicillinresistant strains (mean survival after 2 h exposure: 45% in MSSAvs. 60% in MRSAwith methicillin MIC 1024 mg/l) [9]. Similarly, a possible influence of MRSA status on hBD-3 susceptibility in clinical S. aureus isolates had been postulated earlier by Midorikawa and colleagues [10]. In both studies, however, there was no or limited information regarding the

clinical background and origin of the test strains. Gottlieb and colleagues measured the activity of hBD-3 in seven different S. aureus strains from different sources and found very little variation similar to that observed with a number of non-human model-peptides [11]. In a rabbit endocarditis model, enhanced in vitro susceptibility to thrombin-induced platelet microbicidal protein-1 (tPMP-1) was associated with reduced disease progression as measured by lower bacteremia rates and reduced bacterial densities in vegetations and kidneys [12]. Conversely, higher rates of survival (mean survival 22% versus 12%) after exposure to tPMP were observed in isolates of patients with persistent MRSA bacteremia [13]. We believe the current study expands these data by providing information from a larger collection of isolates including a thorough and systematic investigation of the clinical background. We recognize that in this study AMP susceptibility was tested against three major human and one bovine cationic AMPs of epithelial and/or neutrophil origin containing peptides of different chemical and secondary structure. Though to some extent cross-susceptibility/resistance was observed, our findings

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cannot be generalized. Innate defense comprises a range of AMPs, including further cationic, but also anionic peptides, which might influence colonization, invasiveness and severity of S. aureus infection to a greater extent as seen here. Another limitation is that neither of the two assays used here may account for microbial regulation and adaptation e.g. by altered virulence factor expression that might occur under in vivo conditions. The present investigation and previous studies of S. aureus indicate only moderate to minor differences in the in vitro AMP susceptibility between groups of isolates representing different clinical entities and outcomes, thus, pointing towards other factors as major contributing determinants for the wide spectrum of clinical manifestations of S. aureus. These factors may be pathogen- or host-related. With respect to innate defense AMPs, recent evidence suggests that different levels of host AMP expression contribute to the propensity of individuals or certain patient groups to S. aureus colonization and/or infection [14,15]. Notably, investigated reference strains harboring methicillin- or vancomycin-resistance did not exhibit altered AMP susceptibility as documented by comparable MIC and CFU reduction levels. Taken together with the narrow MIC range of the clinical isolates tested, this observation may indicate that AMPs or peptidomimetics may serve as potential candidates or templates for the development and design of new drugs for difficult to treat S. aureus infections. Acknowledgments We thank Raffaele de Luca and Christa Hauser for expert technical assistance. This work was supported in part by BMBF grant 01EO0803 and the Paul-Ehrlich-Society for Chemotherapy. References [1] F.D. Lowy, Staphylococcus aureus infections, N. Engl. J. Med. 339 (1998) 520e532. [2] A. van Belkum, Staphylococcal colonization and infection: homeostasis versus disbalance of human (innate) immunity and bacterial virulence, Curr. Opin. Infect. Dis. 19 (2006) 339e344. [3] Y. Lai, R.L. Gallo, AMPed up immunity: how antimicrobial peptides have multiple roles in immune defense, Trends Immunol. 30 (2009) 131e141. [4] A. Peschel, H.G. Sahl, The co-evolution of host cationic antimicrobial peptides and microbial resistance, Nat. Rev. Microbiol. 4 (2006) 529e536.

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