Eur J Clin Microbiol Infect Dis DOI 10.1007/s10096-015-2399-5
ARTICLE
Application of immobilized synthetic anti-lipopolysaccharide peptides for the isolation and detection of bacteria N. Sandetskaya 1 & B. Engelmann 1 & K. Brandenburg 2 & D. Kuhlmeier 1
Received: 22 December 2014 / Accepted: 3 May 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract The molecular detection of microorganisms in liquid samples generally requires their enrichment or isolation. The aim of our study was to evaluate the capture and preconcentration of bacteria by immobilized particular cationic antimicrobial peptides, called synthetic antilipopolysaccharide peptides (SALP). For the proof-ofconcept and screening of different SALP, the peptides were covalently immobilized on glass slides, and the binding of bacteria was confirmed by microscopic examination of the slides or their scanning, in case of fluorescent bacterial cells. The most efficient SALP was further tethered to magnetic beads. SALP beads were used for the magnetic capture of Escherichia coli in liquid samples. The efficiency of this strategy was evaluated using polymerase chain reaction (PCR). Covalently immobilized SALP were capable of capturing bacteria in liquid samples. However, PCR was hampered by the unspecific binding of DNA to the positively charged peptide. We developed a method for DNA recovery by the enzymatic digestion of the peptide, which allowed for a successful PCR, though the method had its own adverse impact on the detection and, thus, did not allow for the reliable quantitative analysis of the pathogen enrichment. Immobilized SALP can be used as capture molecules for bacteria in liquid samples and can be recommended for the design of the assays or decontamination of the fluids. For the accurate subsequent detection of bacteria, DNA-independent methods should be used.
* N. Sandetskaya [email protected] 1
Nanotechnology Unit, Fraunhofer Institute for Cell Therapy and Immunology, Perlickstrasse 1, 04103 Leipzig, Germany
2
Division of Biophysics, Research Center Borstel, Leibniz-Center for Medicine and Biosciences, Parkallee 10, 23845 Borstel, Germany
Introduction Modern diagnostics of infectious diseases, as well as food and environmental analytics, is often based on molecular methods of pathogen detection. Indeed, such techniques, e.g., polymerase chain reaction (PCR), possess clinically relevant sensitivity and can provide results much faster than culture-based methods [1–3]. However, PCR, as a rule, is not suitable for the direct analysis of crude clinical or environmental samples without their pre-analytical preparation [1, 4–8]. Selective capture and isolation of bacteria from complex liquid samples can strongly facilitate pre-analytical steps. With this approach, target microorganisms can be collected from a large volume of a crude specimen and easily separated from inhibitors, which permits prompt effective PCR or other methods of detection. The most popular variant of the selective isolation of bacteria is immunomagnetic separation, which exploits bacteriaspecific antibodies immobilized on magnetic particles [9–14]. After the incubation with the sample, the particles with the captured bacteria can be collected with a magnet, washed, and resuspended in a desired volume of liquid for further analysis. Besides the mentioned advantages, immunomagnetic separation also has some drawbacks. In the case of an unknown presumed pathogen, a broad spectrum of speciesspecific antibodies is required, which makes the assay platform redundant and expensive. The antibodies also have limited stability, which restricts their application in some diagnostic systems [15, 16]. Therefore, investigation of alternative molecules for targeted capture of microorganisms can contribute to the development of fast assays for bacteria detection in different samples. Antimicrobial peptides (AMPs) form a class of bactericidal molecules known for their broad-range affinity towards microbial cell walls [17–19]. This allows suggesting them as
Eur J Clin Microbiol Infect Dis
potential capture agents for a broad spectrum of microorganisms with an advantage over the species-specific immunomagnetic isolation of bacteria. In previous papers, it was shown that particular AMPs, called synthetic antilipopolysaccharide peptides (SALP), were able to bind to bacteria as well as neutralize their toxins, lipopolysaccharide (LPS), in the case of Gram-negative bacteria [19–21]. The SALP were constructed as polycationic agents with a polar moiety at the N-terminal and a hydrophobic moiety at the Cterminal end in a way that an optimal binding to bacterial LPS can take place. Furthermore, it was also shown that a particular SALP, Pep19-2.5, also called Aspidasept®, does not only bind to Gram-negative but also to Gram-positive bacteria, such as to methicillin-resistant Staphylococcus aureus (MRSA) [22]. Here, we investigate an application of immobilized SALP for the isolation of bacterial cells from liquid samples, followed by PCR detection.
of the green fluorescent protein (GFP)-producing E. coli cells was performed directly in a microarray scanner GenePix 4200A (Molecular Devices, USA). The detection of S. aureus was done under a light microscope (Leica, Germany) after the Gram staining of bacteria. AMP-conjugated magnetic beads
Escherichia coli DH5α cells and MRSA t008, kindly provided by J. Knobloch from the University of Lübeck, Institute for Medical Microbiology, were cultivated overnight in LB medium and washed in phosphate-buffered saline (PBS). Preliminary approximate quantification of bacteria was done by measuring the optical density of the suspension at 600 nm (OD600). A precise quantification was done by counting the colonies after plating of the bacterial suspension on LB agar.
50 μg of Pep19-2.5OH was immobilized on 1 mg of magnetic beads via carbodiimide crosslinking. For all experiments followed by PCR detection, MagPrep® P-25 carboxylated magnetic nanobeads (25 nm size; Merck Millipore, Germany) were used; for the primary proof of bacteria binding in solution, Dynabeads® MyOne™ Carboxylic Acid (1 μm size; Life Technologies, Germany) were exploited. The beads were washed with 25 mM morpholinoethanesulfonic buffer (MES)+0.05 % Tween 20 (MEST), pH 5.3, and activated by 30 min of incubation with 50 μl of 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide (EDC; Sigma Aldrich, Germany; 50 mg/ml in cold 100 mM MES, pH 5.3) and 50 μl of NHS (Sigma Aldrich, Germany; 50 mg/ml in cold 100 mM MES). Then the particles were washed once with 25 mM MEST and once with PBS + 0.05 % Tween (PBST), resuspended in 100 μl PBST, and 50 μl of the peptide (1 mg/ml in PBS) was added. The conjugation was done at room temperature and constant shaking for 2 h. The supernatant was removed and replaced with 100 μl 1 M ethanolamine (pH 8.5), followed by 30 min of incubation to quench the remaining activated carboxyl groups. The beads were washed twice with PBST and resuspended in PBST to a final concentration of the beads of 10 mg/ml.
Antimicrobial peptides
Peptide-based isolation of bacteria
Antimicrobial peptides were synthesized at the Borstel Research Institute by applying solid phase synthesis, as previously described [20, 23]. The following peptides were evaluated: Pep19-2.5 (GCKKYRRFRWKFKGKFWFWG), Pep192.5OH (GCKKYRRFRWKFKGKFWFWG-OH), Pep198Acyl (GRRYKKFRWKFKGRWFG-Hexan-CONH2), and Pep19-8.3Acyl (SRRYKKFRWKFKGRWFWFG-HexanCONH2) [23].
100 μl PBS was spiked with different concentrations of bacterial cells (102–108 CFU/ml) and 100 μg peptide-conjugated beads. The samples were incubated for 30 min at room temperature. The supernatant was removed, and then the beads were washed twice with PBST and resuspended in 50 μl of water. The bound cells were thermolysed at 95 °C for 10 min.
Bacteria binding on a glass slide
Enzymatic digestion of the peptides
The peptides (1 mg/ml in 5 mM acetate buffer, pH 5.0) were immobilized on an N-hydroxysuccinimide (NHS)-activated hydrogel slide (Xantec, Germany), according to the slide manufacturer’s recommendations. Bacteria suspension (10 9 CFU/ml in PBS) was applied all over the surface. The glass was incubated for 30 min at room temperature, rinsed in double-distilled water, and air-dried. Detection
Enzymatic digestion of the peptides was done after the bacteria isolation by the addition of 50 μl trypsin solution (0.05 %/ 0.02 % w/v trypsin/EDTA; Biochrom AG, Germany). The sample was incubated for 30 min at 37 °C and then for 15 min at 95 °C for trypsin denaturation. 25 μl of the obtained lysate was investigated in conventional PCR; 8 μl was applied to real time-PCR (RT-PCR).
Materials and methods Bacterial cells
Eur J Clin Microbiol Infect Dis
Conventional and RT-PCR Conventional and RT-PCR were performed with the pair of primers TEcol553 (5′-TGGGAAGCGAAAATCCTG-3′) and TEcol754 (5′-CAGTACAGGTAGACTTCTG-3′) [24]. Conventional PCR was performed in a TProfessional Thermocycler (Biometra, Germany) in a 50-μl reaction volume. The reaction mix contained 10 pmol of each primer (Sigma-Aldrich, Germany), 10 nmol of each dNTP (Peqlab, Germany), 50 nmol MgCl2 (in addition to that already included in the PCR buffer), 0.05 % BSA, 0.1 % Tween 20 (Carl Roth, Germany), and 2.5 units Taq DNA Polymerase (Peqlab, Germany) in 1×PCR Buffer Y [20 mM Tris–HCl, pH 8.55, 16 mM (NH4)2SO4, 0.001 % Tween 20, 2 mM MgCl2] (Peqlab, Germany). Initial denaturation at 95 °C for 10 min was followed by 40 cycles of 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 30 s, and then the final elongation at 72 °C for 3 min. The PCR product was detected in electrophoresis on 2 % agarose gel. RT-PCR was performed in the LightCycler® 480 RealTime PCR System (Roche, Germany) in a 20-μl reaction volume containing 5 pmol of each primer in 1× LightCycler® 480 SYBR Green I Master Mix (Roche, Germany). The initial template denaturation was done at 95 °C for 10 min, and then 40 cycles of 95 °C for 15 s, 58 °C for 20 s, and 72 °C for 20 s were performed. Electrophoretic mobility shift assay (EMSA) 3 μl (1.5 μg) of Low Range DNA Ruler (Fermentas, Germany) was mixed with 10 μl peptide solution (1 mg/ml) and 2 μl 1×PCR Buffer Y. The sample was incubated for 15 min at room temperature, mixed with 0.5 μl of 6×loading dye (Thermo Scientific, Germany), and investigated in electrophoresis on 2 % agarose gel.
Results We screened several SALP to reveal the best candidate for application as a capture element in a solid-phase assay. The binding ability towards bacteria was first tested on a planar surface of a glass slide. All four investigated peptides bound GFP-producing E. coli cells, which resulted in bright fluorescent spots at the SALP-functionalized positions (Fig. 1). In a comparative visual evaluation, the most distinct and intensive fluorescent spots were demonstrated by the SALP Pep192.5OH; therefore, we have chosen this peptide for further detailed characterization. In order to confirm the ability of the peptide to bind other microorganisms, we performed a similar experiment on a glass slide, where Pep19-2.5OH was exposed to a methicillin-resistant strain of S. aureus. After Gram staining,
Fig. 1 Binding of fluorescent Escherichia coli cells by different synthetic anti-lipopolysaccharide peptides (SALP), immobilized on a glass slide (in triplicate). 1 Pep19-2.5; 2 Pep19-2.5OH; 3 Pep19-8Acyl; 4 Pep198.3Acyl
a significantly higher density of the bound bacteria was observed on the peptide-treated surface in comparison to a reference (Fig. 2). We further investigated the application of the immobilized SALP for the pre-enrichment of bacteria in liquid samples. For that purpose, we produced magnetic particles coated with the covalently immobilized Pep19-2.5OH. Initially, an attempt to detect E. coli cells that were captured by the SALP-conjugated beads was done in PCR straight after the bacteria isolation. However, the results were negative (not shown). In order to confirm the functional sustainability of the beads, we roughly evaluated the concentration of E. coli after the incubation of bacterial suspensions (OD600 = 1.043 ± 0.026) with different amounts of the SALP-conjugated magnetic particles (Fig. 3). We observed that the amount of bacteria (assessed via the OD600 value) clearly decreases after the sample introduction to the SALP beads. It was also demonstrated that the reduction of the pathogen concentration significantly depends on the amount of the applied beads: in 100-μl samples, the OD600 dropped for approximately 0.2 OD units per 50 μg of SALP beads added to the sample (see Fig. 3). The binding of bacteria to the plain beads without SALP was significantly lower than that to the functionalized particles, which proves the active role of the immobilized peptide in the capture of E. coli. Because the PCR results were negative despite the proven capture of bacteria, we suspected a peptide-mediated PCR failure. The cationic peptide may cause an unspecific binding of bacterial DNA, therefore making it unavailable for
Fig. 2 Binding of bacteria on an SALP-treated surface (glass slide; Gram staining). a SALP, b BSA (reference)
Eur J Clin Microbiol Infect Dis
Fig. 5 Detection of different E. coli concentrations in buffer samples after the SALP-based isolation and trypsin treatment. M DNA length marker; NTC no template control; 1 108 CFU/ml; 2 107 CFU/ml; 3 106 CFU/ml; 4 105 CFU/ml; 5 104 CFU/ml; 6 103 CFU/ml; 7 102 CFU/ml
The limit of the RT-PCR detection of bacteria after their capture was the same as with conventional PCR: 104 CFU/ml.
Fig. 3 Bacteria binding to the SALP-conjugated beads, evaluated as a reduction of the optical density (OD600) of the sample (n=3). A decrease of the OD600 value indicates lowered concentration of E. coli cells in the sample after incubation with the defined amounts of beads
amplification. This hypothesis was confirmed in EMSA: 1.5 μg of DNA was completely scavenged by the tested peptide (Fig. 4). Thermal denaturation of the SALP (95 °C for 10 min) prior to PCR did not lead to successful results (not shown). As another strategy for the destruction of the peptide and DNA release, an enzymatic digestion of the SALP was developed. The effect of trypsin digestion was initially tested in conventional PCR. It was possible to obtain clear positive PCR results in comparison to the negative outcomes of the experiments without the trypsin treatment. The limit of E. coli detection with the peptide-based pathogen pre-enrichment was 104 CFU/ml in the current experimental setup (Fig. 5). The efficiency of the bacteria detection after the adjusted SALP-based isolation was further assessed in a quantitative test. Buffer samples were spiked with different concentrations of E. coli and analyzed in RT-PCR with and without the described bacteria isolation (Fig. 6). No statistically significant difference was observed between these two series (p>0.05).
Fig. 4 SALP-mediated DNA scavenge [electrophoretic mobility shift assay (EMSA) results]. Each lane was loaded with 1.5 μg of Low Range DNA Ruler. 1 Control lane: no antimicrobial peptides (AMP) added; 2 reference lane: DNA ruler+10 μg BSA; 3 DNA ruler+10 μg of the SALP
Discussion AMPs belong to a diverse group of molecules which possess bactericidal activity [17–19]. Intensive studies of AMPs are invoked by their high potential applicability as therapeutic agents for a broad spectrum of infections, including antibiotic-resistant cases and septic shock [19, 20, 22, 25, 26]. In a prevalent number of publications, only the activity of soluble AMPs was studied. In recent years, there has been a growing interest in the properties of the immobilized peptides due to their potential for the design of antimicrobial surfaces [27, 28]. However, most of the researchers were interested in the investigation of the entire antibacterial effect, which includes binding of a pathogen, pore formation, and interruption of the intracellular processes, leading to the death of cells [27–29]. At the same time, the binding activity alone can contribute significantly to the development of such applications as diagnostic assays, decontamination of liquids, etc. A highly selective binding towards prokaryotic cells in combination with a broad binding spectrum (Gram-negative and Gram-positive bacteria, fungi, viruses) and high stability of
Fig. 6 Efficiency of E. coli isolation from buffer samples using SALPconjugated magnetic particles. n=4
Eur J Clin Microbiol Infect Dis
the peptides make them a very promising basis for various approaches and platforms [15–19, 30, 31]. Usually, AMPs have a sequence of less than 100 amino acids, which is often (however, not exclusively) positively charged [17, 26, 32]. Positive charge plays an important role in the initial binding of the microorganisms, being the key factor of the electrostatic interaction with the negatively charged cell wall of a microorganism [17, 33]. At the Research Center Borstel, a new class of antilipopolysaccharide cationic peptides (SALP) was developed, possessing an optimized structure for binding to the surface structures of bacterial cells [20–22]. Their complex antimicrobial activity was successfully proven in a bacterial growth suppression test [20]. In the current work, we evaluated some aspects of a particular SALP application for the isolation of bacteria and their further PCR-based detection. For the proposed application of the peptides for microorganism capture, it is essential that the SALP retains its function while being immobilized on a solid surface. We used carbodiimine chemistry for the immobilization, which involves amino groups of the molecules for the crosslinking. Because the same moieties (in K and R) are likely the key functional groups which determine the charge-mediated binding activity of the peptide, the latter may be affected after the peptide conjugation to a carrier. However, we proved that, after the covalent tethering to the surface, the peptides effectively maintain bacteria binding. Our further work was focused on a particular peptide, Pep19-2.5OH, which demonstrated a slightly greater affinity to bacteria in a screening test on a glass slide. This SALP was also capable of the binding of both Gram-negative and Gram-positive microorganisms, at least in the tests with the two widely distributed representatives of these groups, E. coli and S. aureus. A presumed broad-range affinity towards the pathogens makes AMPs attractive capture agents for the enrichment of bacteria prior to their molecular detection. Although Pep19-2.5OH bound bacteria, it was difficult to prove it via PCR due to the peptide-associated DNA binding. This phenomenon can be explained by the electrostatic interactions between the cationic molecules of the peptide and negatively charged DNA. Using an anionic AMP instead of a cationic AMP may eliminate this problem; however, precise mechanisms of the interaction of anionic peptides with microbial cells are, in many cases, still unclear [34]. Therefore, without a detailed study, it is difficult to predict if they can be used as immobilized capture molecules. In our case, release of the DNA from the cationic peptide can potentially be provided by denaturation or digestion of the SALP. Heating of the SALP-conjugated magnetic particles to 95 °C did not allow DNA recovery. Some researchers also mention an extreme stability of AMPs under various thermal and chemical conditions [15, 35].
Treatment of the beads with trypsin allowed for the digestion of the immobilized SALP and facilitated amplification and detection of bacterial DNA down to an E. coli concentration of 104 CFU/ml. It is necessary to take into account that this method of sample preparation itself can lead to PCR disturbance and decrease the sensitivity of the assay, making it suboptimal. First, trypsin formulation contained EDTA, which can hamper PCR due to the scavenge of Mg2+. We empirically adjusted the concentration of magnesium in the reaction; however, an EDTA-free preparation protocol may provide better assay efficiency. Second, assuming that the thermal inactivation of the trypsin before PCR can be insufficient, this protease may also cause partial digestion of the polymerase. Thus, despite the adjustments, the developed method for the DNA release may still lead to an insufficient PCR performance. In this case, PCR cannot be considered an appropriate quantitative detection method. We could demonstrate no significant improvement in the RT-PCR detection of E. coli after its isolation with SALP beads; however, that may be conditioned by the suboptimal detection approach rather than ineffective bacteria capture. The clearly demonstrated ability of the peptides to bind bacteria on surfaces encourages seeking alternative, DNAindependent approaches for the downstream analysis of the samples. Direct detection of the captured bacteria via appropriate biosensors or sensitive visualization techniques may significantly raise the value of the AMP-based enrichment of the microorganisms. Currently, there are only a few reports on the application of AMPs for the binding and detection of bacteria [15, 16, 30, 36, 37]. Arcidiacono et al. studied another peptide, cecropin P1, in a sandwich assay with fluorescent detection and reached a limit of detection corresponding to ours [36]. Kulagina et al. reported the detection limits of 5× 104–5×106 CFU/ml obtained with three other cationic peptides in a principally similar sandwich assay [37]. Mannoor et al. used direct electrical detection of E. coli instead of optical, and were able to detect 1,000 cells/ml [15]. Although available records did not demonstrate detection below 103 CFU/ml achieved with AMP, existing data have also confirmed a major influence of the detection strategy on the assay performance. A diverse behavior of the various AMPs does not allow extrapolating the data to all the pathogens or peptides; hence, further studies and optimization of the detection methods will allow to find the best niche for the application of SALP in the assays.
Conclusion We have demonstrated effective capture of microorganisms on synthetic anti-lipopolysaccharide peptides (SALP)-coated surfaces. The investigated cationic peptides retained their affinity towards bacteria after their covalent immobilization. We
Eur J Clin Microbiol Infect Dis
proposed the application of SALP for the magnetic beadbased enrichment of prokaryotic pathogens in liquid samples and evaluated it with the peptide Pep19-2.5OH. The unspecific electrostatic binding of bacterial DNA to the peptide strongly affected polymerase chain reaction (PCR) and, thus, hampered the quantitative assessment of the bacteria enrichment. The use of DNA-independent approaches may allow for a better characterization of the peptide-based pathogen pre-concentration. Relying on our observations, we can recommend cationic antimicrobial peptides (AMPs) for application in assays with the direct detection of microorganisms, and, potentially, also for the decontamination of liquid samples.
12.
13.
14.
15.
16. Acknowledgments The published work was funded by the German Federal Ministry of Education and Research [BMBF; project MinoLab (number 16SV4030) and project number 01GU0824]. Conflict of interest Klaus Brandenburg is also a founder and CSO of the company Brandenburg Antiinfektiva GmbH. Development and investigation of the described peptide Pep19-2.5, also called Aspidasept®, is one of the ongoing activities of this company. Here, we declare that the research work described in this manuscript was neither sponsored nor had any other commercial relation to the aforementioned enterprise. Thus, we declare no conflicts of interest in regard to this publication.
References 1.
2.
3. 4.
5.
6.
7.
8.
9. 10.
11.
Barken KB, Haagensen JA, Tolker-Nielsen T (2007) Advances in nucleic acid-based diagnostics of bacterial infections. Clin Chim Acta 384:1–11 Jannes G, De Vos D (2006) A review of current and future molecular diagnostic tests for use in the microbiology laboratory. Methods Mol Biol 345:1–21 Picard FJ, Bergeron MG (2002) Rapid molecular theranostics in infectious diseases. Drug Discov Today 7:1092–1101 Yang S, Rothman RE (2004) PCR-based diagnostics for infectious diseases: uses, limitations, and future applications in acute-care settings. Lancet Infect Dis 4:337–348 Rådström P, Knutsson R, Wolffs P, Lövenklev M, Löfström C (2004) Pre-PCR processing: strategies to generate PCRcompatible samples. Mol Biotechnol 26:133–146 Lantz PG, Abu Al-Soud W, Knutsson R, Hahn-Hägerdal B, Rådström P (2000) Biotechnical use of polymerase chain reaction for microbiological analysis of biological samples. Biotechnol Annu Rev 5:87–130 Hansen WL, Bruggeman CA, Wolffs PF (2009) Evaluation of new preanalysis sample treatment tools and DNA isolation protocols to improve bacterial pathogen detection in whole blood. J Clin Microbiol 47:2629–2631 Mancini N, Carletti S, Ghidoli N, Cichero P, Burioni R, Clementi M (2010) The era of molecular and other non-culture-based methods in diagnosis of sepsis. Clin Microbiol Rev 23:235–251 Brewster JD (2003) Isolation and concentration of Salmonellae with an immunoaffinity column. J Microbiol Methods 55:287–293 Enroth H, Engstrand L (1995) Immunomagnetic separation and PCR for detection of Helicobacter pylori in water and stool specimens. Clin Microbiol 33:2162–2165 Fernandes CP, Seixas FK, Coutinho ML, Vasconcellos FA, Moreira AN, Conceição FR, Dellagostin OA, Aleixo JA (2008) An immunomagnetic separation-PCR method for detection of
17. 18.
19.
20.
21.
22.
23. 24.
25. 26.
27.
28.
29.
pathogenic Leptospira in biological fluids. Hybridoma (Larchmt) 27:381–386 Fu Z, Rogelj S, Kieft TL (2005) Rapid detection of Escherichia coli O157:H7 by immunomagnetic separation and real-time PCR. Int J Food Microbiol 99:47–57 Qiu J, Zhou Y, Chen H, Lin JM (2009) Immunomagnetic separation and rapid detection of bacteria using bioluminescence and microfluidics. Talanta 79:787–795 Wright DJ, Chapman PA, Siddons CA (1994) Immunomagnetic separation as a sensitive method for isolating Escherichia coli O157 from food samples. Epidemiol Infect 113:31–39 Mannoor MS, Zhang S, Link AJ, McAlpine MC (2010) Electrical detection of pathogenic bacteria via immobilized antimicrobial peptides. Proc Natl Acad Sci U S A 107:19207–19212. doi:10.1073/ pnas.1008768107 Soares JW, Morin KM, Mello CM (2004) Antimicrobial peptides in biosensing applications. Report of US Army Research, Development, & Engineering Command. Available online at: http://www.dtic.mil/cgi-bin/GetTRDoc?AD=ADA433515. Retrieved 11 Dec 2014 Brogden KA (2005) Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nat Rev Microbiol 3:238–250 Friedrich CL, Moyles D, Beveridge TJ, Hancock RE (2000) Antibacterial action of structurally diverse cationic peptides on gram-positive bacteria. Antimicrob Agents Chemother 44:2086– 2092 Schuerholz T, Brandenburg K, Marx G (2012) Antimicrobial peptides and their potential application in inflammation and sepsis. Crit Care 16:207 Gutsmann T, Razquin-Olazarán I, Kowalski I, Kaconis Y, Howe J, Bartels R, Hornef M, Schürholz T, Rössle M, Sanchez-Gómez S, Moriyon I, Martinez de Tejada G, Brandenburg K (2010) New antiseptic peptides to protect against endotoxin-mediated shock. Antimicrob Agents Chemother 54:3817–3824 Kaconis Y, Kowalski I, Howe J, Brauser A, Richter W, RazquinOlazarán I, Iñigo-Pestaña M, Garidel P, Rössle M, Martinez de Tejada G, Gutsmann T, Brandenburg K (2011) Biophysical mechanisms of endotoxin neutralization by cationic amphiphilic peptides. Biophys J 100:2652–2661 Heinbockel L, Sánchez-Gómez S, Martinez de Tejada G, Dömming S, Brandenburg J, Kaconis Y, Hornef M, Dupont A, Marwitz S, Goldmann T, Ernst M, Gutsmann T, Schürholz T, Brandenburg K (2013) Preclinical investigations reveal the broad-spectrum neutralizing activity of peptide Pep19-2.5 on bacterial pathogenicity factors. Antimicrob Agents Chemother 57:1480–1487 Brandenburg K (2009) Novel antimicrobial peptides. Patent no. PCT/EP2009/002565 Maheux AF, Picard FJ, Boissinot M, Bissonnette L, Paradis S, Bergeron MG (2009) Analytical comparison of nine PCR primer sets designed to detect the presence of Escherichia coli/Shigella in water samples. Water Res 43:3019–3028 Hancock RE, Lehrer R (1998) Cationic peptides: a new source of antibiotics. Trends Biotechnol 16:82–88 Peters BM, Shirtliff ME, Jabra-Rizk MA (2010) Antimicrobial peptides: primeval molecules or future drugs? PLoS Pathog 6: e1001067. doi:10.1371/journal.ppat.1001067 Costa F, Carvalho IF, Montelaro RC, Gomes P, Martins MC (2011) Covalent immobilization of antimicrobial peptides (AMPs) onto biomaterial surfaces. Acta Biomater 7:1431–1440 Siedenbiedel F, Tiller JC (2012) Antimicrobial polymers in solution and on surfaces: overview and functional principles. Polymers 4: 46–71 Li Y, Kumar KN, Dabkowski JM, Corrigan M, Scott RW, Nüsslein K, Tew GN (2012) New bactericidal surgical suture coating. Langmuir 28:12134–12139
Eur J Clin Microbiol Infect Dis 30.
31.
32. 33.
Kulagina NV, Shaffer KM, Ligler FS, Taitt CR (2007) Antimicrobial peptides as new recognition molecules for screening challenging species. Sensors Actuators B Chem 121:150–157 Scott MG, Gold MR, Hancock RE (1999) Interaction of cationic peptides with lipoteichoic acid and gram-positive bacteria. Infect Immun 67:6445–6453 Yeaman MR, Yount NY (2003) Mechanisms of antimicrobial peptide action and resistance. Pharmacol Rev 55:27–55 Epand RM, Vogel HJ (1999) Diversity of antimicrobial peptides and their mechanisms of action. Biochim Biophys Acta 1462:11–28
34.
Harris F, Dennison SR, Phoenix DA (2009) Anionic antimicrobial peptides from eukaryotic organisms. Curr Protein Pept Sci 10:585–606 35. Rydlo T, Rotem S, Mor A (2006) Antibacterial properties of dermaseptin S4 derivatives under extreme incubation conditions. Antimicrob Agents Chemother 50:490–497 36. Arcidiacono S, Pivarnik P, Mello CM, Senecal A (2008) Cy5 labeled antimicrobial peptides for enhanced detection of Escherichia coli O157:H7. Biosens Bioelectron 23:1721–1727 37. Kulagina NV, Shaffer KM, Anderson GP, Ligler FS, Taitt CR (2006) Antimicrobial peptide-based array for Escherichia coli and Salmonella screening. Anal Chim Acta 575:9–15
ARTICLE
Application of immobilized synthetic anti-lipopolysaccharide peptides for the isolation and detection of bacteria N. Sandetskaya 1 & B. Engelmann 1 & K. Brandenburg 2 & D. Kuhlmeier 1
Received: 22 December 2014 / Accepted: 3 May 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract The molecular detection of microorganisms in liquid samples generally requires their enrichment or isolation. The aim of our study was to evaluate the capture and preconcentration of bacteria by immobilized particular cationic antimicrobial peptides, called synthetic antilipopolysaccharide peptides (SALP). For the proof-ofconcept and screening of different SALP, the peptides were covalently immobilized on glass slides, and the binding of bacteria was confirmed by microscopic examination of the slides or their scanning, in case of fluorescent bacterial cells. The most efficient SALP was further tethered to magnetic beads. SALP beads were used for the magnetic capture of Escherichia coli in liquid samples. The efficiency of this strategy was evaluated using polymerase chain reaction (PCR). Covalently immobilized SALP were capable of capturing bacteria in liquid samples. However, PCR was hampered by the unspecific binding of DNA to the positively charged peptide. We developed a method for DNA recovery by the enzymatic digestion of the peptide, which allowed for a successful PCR, though the method had its own adverse impact on the detection and, thus, did not allow for the reliable quantitative analysis of the pathogen enrichment. Immobilized SALP can be used as capture molecules for bacteria in liquid samples and can be recommended for the design of the assays or decontamination of the fluids. For the accurate subsequent detection of bacteria, DNA-independent methods should be used.
* N. Sandetskaya [email protected] 1
Nanotechnology Unit, Fraunhofer Institute for Cell Therapy and Immunology, Perlickstrasse 1, 04103 Leipzig, Germany
2
Division of Biophysics, Research Center Borstel, Leibniz-Center for Medicine and Biosciences, Parkallee 10, 23845 Borstel, Germany
Introduction Modern diagnostics of infectious diseases, as well as food and environmental analytics, is often based on molecular methods of pathogen detection. Indeed, such techniques, e.g., polymerase chain reaction (PCR), possess clinically relevant sensitivity and can provide results much faster than culture-based methods [1–3]. However, PCR, as a rule, is not suitable for the direct analysis of crude clinical or environmental samples without their pre-analytical preparation [1, 4–8]. Selective capture and isolation of bacteria from complex liquid samples can strongly facilitate pre-analytical steps. With this approach, target microorganisms can be collected from a large volume of a crude specimen and easily separated from inhibitors, which permits prompt effective PCR or other methods of detection. The most popular variant of the selective isolation of bacteria is immunomagnetic separation, which exploits bacteriaspecific antibodies immobilized on magnetic particles [9–14]. After the incubation with the sample, the particles with the captured bacteria can be collected with a magnet, washed, and resuspended in a desired volume of liquid for further analysis. Besides the mentioned advantages, immunomagnetic separation also has some drawbacks. In the case of an unknown presumed pathogen, a broad spectrum of speciesspecific antibodies is required, which makes the assay platform redundant and expensive. The antibodies also have limited stability, which restricts their application in some diagnostic systems [15, 16]. Therefore, investigation of alternative molecules for targeted capture of microorganisms can contribute to the development of fast assays for bacteria detection in different samples. Antimicrobial peptides (AMPs) form a class of bactericidal molecules known for their broad-range affinity towards microbial cell walls [17–19]. This allows suggesting them as
Eur J Clin Microbiol Infect Dis
potential capture agents for a broad spectrum of microorganisms with an advantage over the species-specific immunomagnetic isolation of bacteria. In previous papers, it was shown that particular AMPs, called synthetic antilipopolysaccharide peptides (SALP), were able to bind to bacteria as well as neutralize their toxins, lipopolysaccharide (LPS), in the case of Gram-negative bacteria [19–21]. The SALP were constructed as polycationic agents with a polar moiety at the N-terminal and a hydrophobic moiety at the Cterminal end in a way that an optimal binding to bacterial LPS can take place. Furthermore, it was also shown that a particular SALP, Pep19-2.5, also called Aspidasept®, does not only bind to Gram-negative but also to Gram-positive bacteria, such as to methicillin-resistant Staphylococcus aureus (MRSA) [22]. Here, we investigate an application of immobilized SALP for the isolation of bacterial cells from liquid samples, followed by PCR detection.
of the green fluorescent protein (GFP)-producing E. coli cells was performed directly in a microarray scanner GenePix 4200A (Molecular Devices, USA). The detection of S. aureus was done under a light microscope (Leica, Germany) after the Gram staining of bacteria. AMP-conjugated magnetic beads
Escherichia coli DH5α cells and MRSA t008, kindly provided by J. Knobloch from the University of Lübeck, Institute for Medical Microbiology, were cultivated overnight in LB medium and washed in phosphate-buffered saline (PBS). Preliminary approximate quantification of bacteria was done by measuring the optical density of the suspension at 600 nm (OD600). A precise quantification was done by counting the colonies after plating of the bacterial suspension on LB agar.
50 μg of Pep19-2.5OH was immobilized on 1 mg of magnetic beads via carbodiimide crosslinking. For all experiments followed by PCR detection, MagPrep® P-25 carboxylated magnetic nanobeads (25 nm size; Merck Millipore, Germany) were used; for the primary proof of bacteria binding in solution, Dynabeads® MyOne™ Carboxylic Acid (1 μm size; Life Technologies, Germany) were exploited. The beads were washed with 25 mM morpholinoethanesulfonic buffer (MES)+0.05 % Tween 20 (MEST), pH 5.3, and activated by 30 min of incubation with 50 μl of 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide (EDC; Sigma Aldrich, Germany; 50 mg/ml in cold 100 mM MES, pH 5.3) and 50 μl of NHS (Sigma Aldrich, Germany; 50 mg/ml in cold 100 mM MES). Then the particles were washed once with 25 mM MEST and once with PBS + 0.05 % Tween (PBST), resuspended in 100 μl PBST, and 50 μl of the peptide (1 mg/ml in PBS) was added. The conjugation was done at room temperature and constant shaking for 2 h. The supernatant was removed and replaced with 100 μl 1 M ethanolamine (pH 8.5), followed by 30 min of incubation to quench the remaining activated carboxyl groups. The beads were washed twice with PBST and resuspended in PBST to a final concentration of the beads of 10 mg/ml.
Antimicrobial peptides
Peptide-based isolation of bacteria
Antimicrobial peptides were synthesized at the Borstel Research Institute by applying solid phase synthesis, as previously described [20, 23]. The following peptides were evaluated: Pep19-2.5 (GCKKYRRFRWKFKGKFWFWG), Pep192.5OH (GCKKYRRFRWKFKGKFWFWG-OH), Pep198Acyl (GRRYKKFRWKFKGRWFG-Hexan-CONH2), and Pep19-8.3Acyl (SRRYKKFRWKFKGRWFWFG-HexanCONH2) [23].
100 μl PBS was spiked with different concentrations of bacterial cells (102–108 CFU/ml) and 100 μg peptide-conjugated beads. The samples were incubated for 30 min at room temperature. The supernatant was removed, and then the beads were washed twice with PBST and resuspended in 50 μl of water. The bound cells were thermolysed at 95 °C for 10 min.
Bacteria binding on a glass slide
Enzymatic digestion of the peptides
The peptides (1 mg/ml in 5 mM acetate buffer, pH 5.0) were immobilized on an N-hydroxysuccinimide (NHS)-activated hydrogel slide (Xantec, Germany), according to the slide manufacturer’s recommendations. Bacteria suspension (10 9 CFU/ml in PBS) was applied all over the surface. The glass was incubated for 30 min at room temperature, rinsed in double-distilled water, and air-dried. Detection
Enzymatic digestion of the peptides was done after the bacteria isolation by the addition of 50 μl trypsin solution (0.05 %/ 0.02 % w/v trypsin/EDTA; Biochrom AG, Germany). The sample was incubated for 30 min at 37 °C and then for 15 min at 95 °C for trypsin denaturation. 25 μl of the obtained lysate was investigated in conventional PCR; 8 μl was applied to real time-PCR (RT-PCR).
Materials and methods Bacterial cells
Eur J Clin Microbiol Infect Dis
Conventional and RT-PCR Conventional and RT-PCR were performed with the pair of primers TEcol553 (5′-TGGGAAGCGAAAATCCTG-3′) and TEcol754 (5′-CAGTACAGGTAGACTTCTG-3′) [24]. Conventional PCR was performed in a TProfessional Thermocycler (Biometra, Germany) in a 50-μl reaction volume. The reaction mix contained 10 pmol of each primer (Sigma-Aldrich, Germany), 10 nmol of each dNTP (Peqlab, Germany), 50 nmol MgCl2 (in addition to that already included in the PCR buffer), 0.05 % BSA, 0.1 % Tween 20 (Carl Roth, Germany), and 2.5 units Taq DNA Polymerase (Peqlab, Germany) in 1×PCR Buffer Y [20 mM Tris–HCl, pH 8.55, 16 mM (NH4)2SO4, 0.001 % Tween 20, 2 mM MgCl2] (Peqlab, Germany). Initial denaturation at 95 °C for 10 min was followed by 40 cycles of 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 30 s, and then the final elongation at 72 °C for 3 min. The PCR product was detected in electrophoresis on 2 % agarose gel. RT-PCR was performed in the LightCycler® 480 RealTime PCR System (Roche, Germany) in a 20-μl reaction volume containing 5 pmol of each primer in 1× LightCycler® 480 SYBR Green I Master Mix (Roche, Germany). The initial template denaturation was done at 95 °C for 10 min, and then 40 cycles of 95 °C for 15 s, 58 °C for 20 s, and 72 °C for 20 s were performed. Electrophoretic mobility shift assay (EMSA) 3 μl (1.5 μg) of Low Range DNA Ruler (Fermentas, Germany) was mixed with 10 μl peptide solution (1 mg/ml) and 2 μl 1×PCR Buffer Y. The sample was incubated for 15 min at room temperature, mixed with 0.5 μl of 6×loading dye (Thermo Scientific, Germany), and investigated in electrophoresis on 2 % agarose gel.
Results We screened several SALP to reveal the best candidate for application as a capture element in a solid-phase assay. The binding ability towards bacteria was first tested on a planar surface of a glass slide. All four investigated peptides bound GFP-producing E. coli cells, which resulted in bright fluorescent spots at the SALP-functionalized positions (Fig. 1). In a comparative visual evaluation, the most distinct and intensive fluorescent spots were demonstrated by the SALP Pep192.5OH; therefore, we have chosen this peptide for further detailed characterization. In order to confirm the ability of the peptide to bind other microorganisms, we performed a similar experiment on a glass slide, where Pep19-2.5OH was exposed to a methicillin-resistant strain of S. aureus. After Gram staining,
Fig. 1 Binding of fluorescent Escherichia coli cells by different synthetic anti-lipopolysaccharide peptides (SALP), immobilized on a glass slide (in triplicate). 1 Pep19-2.5; 2 Pep19-2.5OH; 3 Pep19-8Acyl; 4 Pep198.3Acyl
a significantly higher density of the bound bacteria was observed on the peptide-treated surface in comparison to a reference (Fig. 2). We further investigated the application of the immobilized SALP for the pre-enrichment of bacteria in liquid samples. For that purpose, we produced magnetic particles coated with the covalently immobilized Pep19-2.5OH. Initially, an attempt to detect E. coli cells that were captured by the SALP-conjugated beads was done in PCR straight after the bacteria isolation. However, the results were negative (not shown). In order to confirm the functional sustainability of the beads, we roughly evaluated the concentration of E. coli after the incubation of bacterial suspensions (OD600 = 1.043 ± 0.026) with different amounts of the SALP-conjugated magnetic particles (Fig. 3). We observed that the amount of bacteria (assessed via the OD600 value) clearly decreases after the sample introduction to the SALP beads. It was also demonstrated that the reduction of the pathogen concentration significantly depends on the amount of the applied beads: in 100-μl samples, the OD600 dropped for approximately 0.2 OD units per 50 μg of SALP beads added to the sample (see Fig. 3). The binding of bacteria to the plain beads without SALP was significantly lower than that to the functionalized particles, which proves the active role of the immobilized peptide in the capture of E. coli. Because the PCR results were negative despite the proven capture of bacteria, we suspected a peptide-mediated PCR failure. The cationic peptide may cause an unspecific binding of bacterial DNA, therefore making it unavailable for
Fig. 2 Binding of bacteria on an SALP-treated surface (glass slide; Gram staining). a SALP, b BSA (reference)
Eur J Clin Microbiol Infect Dis
Fig. 5 Detection of different E. coli concentrations in buffer samples after the SALP-based isolation and trypsin treatment. M DNA length marker; NTC no template control; 1 108 CFU/ml; 2 107 CFU/ml; 3 106 CFU/ml; 4 105 CFU/ml; 5 104 CFU/ml; 6 103 CFU/ml; 7 102 CFU/ml
The limit of the RT-PCR detection of bacteria after their capture was the same as with conventional PCR: 104 CFU/ml.
Fig. 3 Bacteria binding to the SALP-conjugated beads, evaluated as a reduction of the optical density (OD600) of the sample (n=3). A decrease of the OD600 value indicates lowered concentration of E. coli cells in the sample after incubation with the defined amounts of beads
amplification. This hypothesis was confirmed in EMSA: 1.5 μg of DNA was completely scavenged by the tested peptide (Fig. 4). Thermal denaturation of the SALP (95 °C for 10 min) prior to PCR did not lead to successful results (not shown). As another strategy for the destruction of the peptide and DNA release, an enzymatic digestion of the SALP was developed. The effect of trypsin digestion was initially tested in conventional PCR. It was possible to obtain clear positive PCR results in comparison to the negative outcomes of the experiments without the trypsin treatment. The limit of E. coli detection with the peptide-based pathogen pre-enrichment was 104 CFU/ml in the current experimental setup (Fig. 5). The efficiency of the bacteria detection after the adjusted SALP-based isolation was further assessed in a quantitative test. Buffer samples were spiked with different concentrations of E. coli and analyzed in RT-PCR with and without the described bacteria isolation (Fig. 6). No statistically significant difference was observed between these two series (p>0.05).
Fig. 4 SALP-mediated DNA scavenge [electrophoretic mobility shift assay (EMSA) results]. Each lane was loaded with 1.5 μg of Low Range DNA Ruler. 1 Control lane: no antimicrobial peptides (AMP) added; 2 reference lane: DNA ruler+10 μg BSA; 3 DNA ruler+10 μg of the SALP
Discussion AMPs belong to a diverse group of molecules which possess bactericidal activity [17–19]. Intensive studies of AMPs are invoked by their high potential applicability as therapeutic agents for a broad spectrum of infections, including antibiotic-resistant cases and septic shock [19, 20, 22, 25, 26]. In a prevalent number of publications, only the activity of soluble AMPs was studied. In recent years, there has been a growing interest in the properties of the immobilized peptides due to their potential for the design of antimicrobial surfaces [27, 28]. However, most of the researchers were interested in the investigation of the entire antibacterial effect, which includes binding of a pathogen, pore formation, and interruption of the intracellular processes, leading to the death of cells [27–29]. At the same time, the binding activity alone can contribute significantly to the development of such applications as diagnostic assays, decontamination of liquids, etc. A highly selective binding towards prokaryotic cells in combination with a broad binding spectrum (Gram-negative and Gram-positive bacteria, fungi, viruses) and high stability of
Fig. 6 Efficiency of E. coli isolation from buffer samples using SALPconjugated magnetic particles. n=4
Eur J Clin Microbiol Infect Dis
the peptides make them a very promising basis for various approaches and platforms [15–19, 30, 31]. Usually, AMPs have a sequence of less than 100 amino acids, which is often (however, not exclusively) positively charged [17, 26, 32]. Positive charge plays an important role in the initial binding of the microorganisms, being the key factor of the electrostatic interaction with the negatively charged cell wall of a microorganism [17, 33]. At the Research Center Borstel, a new class of antilipopolysaccharide cationic peptides (SALP) was developed, possessing an optimized structure for binding to the surface structures of bacterial cells [20–22]. Their complex antimicrobial activity was successfully proven in a bacterial growth suppression test [20]. In the current work, we evaluated some aspects of a particular SALP application for the isolation of bacteria and their further PCR-based detection. For the proposed application of the peptides for microorganism capture, it is essential that the SALP retains its function while being immobilized on a solid surface. We used carbodiimine chemistry for the immobilization, which involves amino groups of the molecules for the crosslinking. Because the same moieties (in K and R) are likely the key functional groups which determine the charge-mediated binding activity of the peptide, the latter may be affected after the peptide conjugation to a carrier. However, we proved that, after the covalent tethering to the surface, the peptides effectively maintain bacteria binding. Our further work was focused on a particular peptide, Pep19-2.5OH, which demonstrated a slightly greater affinity to bacteria in a screening test on a glass slide. This SALP was also capable of the binding of both Gram-negative and Gram-positive microorganisms, at least in the tests with the two widely distributed representatives of these groups, E. coli and S. aureus. A presumed broad-range affinity towards the pathogens makes AMPs attractive capture agents for the enrichment of bacteria prior to their molecular detection. Although Pep19-2.5OH bound bacteria, it was difficult to prove it via PCR due to the peptide-associated DNA binding. This phenomenon can be explained by the electrostatic interactions between the cationic molecules of the peptide and negatively charged DNA. Using an anionic AMP instead of a cationic AMP may eliminate this problem; however, precise mechanisms of the interaction of anionic peptides with microbial cells are, in many cases, still unclear [34]. Therefore, without a detailed study, it is difficult to predict if they can be used as immobilized capture molecules. In our case, release of the DNA from the cationic peptide can potentially be provided by denaturation or digestion of the SALP. Heating of the SALP-conjugated magnetic particles to 95 °C did not allow DNA recovery. Some researchers also mention an extreme stability of AMPs under various thermal and chemical conditions [15, 35].
Treatment of the beads with trypsin allowed for the digestion of the immobilized SALP and facilitated amplification and detection of bacterial DNA down to an E. coli concentration of 104 CFU/ml. It is necessary to take into account that this method of sample preparation itself can lead to PCR disturbance and decrease the sensitivity of the assay, making it suboptimal. First, trypsin formulation contained EDTA, which can hamper PCR due to the scavenge of Mg2+. We empirically adjusted the concentration of magnesium in the reaction; however, an EDTA-free preparation protocol may provide better assay efficiency. Second, assuming that the thermal inactivation of the trypsin before PCR can be insufficient, this protease may also cause partial digestion of the polymerase. Thus, despite the adjustments, the developed method for the DNA release may still lead to an insufficient PCR performance. In this case, PCR cannot be considered an appropriate quantitative detection method. We could demonstrate no significant improvement in the RT-PCR detection of E. coli after its isolation with SALP beads; however, that may be conditioned by the suboptimal detection approach rather than ineffective bacteria capture. The clearly demonstrated ability of the peptides to bind bacteria on surfaces encourages seeking alternative, DNAindependent approaches for the downstream analysis of the samples. Direct detection of the captured bacteria via appropriate biosensors or sensitive visualization techniques may significantly raise the value of the AMP-based enrichment of the microorganisms. Currently, there are only a few reports on the application of AMPs for the binding and detection of bacteria [15, 16, 30, 36, 37]. Arcidiacono et al. studied another peptide, cecropin P1, in a sandwich assay with fluorescent detection and reached a limit of detection corresponding to ours [36]. Kulagina et al. reported the detection limits of 5× 104–5×106 CFU/ml obtained with three other cationic peptides in a principally similar sandwich assay [37]. Mannoor et al. used direct electrical detection of E. coli instead of optical, and were able to detect 1,000 cells/ml [15]. Although available records did not demonstrate detection below 103 CFU/ml achieved with AMP, existing data have also confirmed a major influence of the detection strategy on the assay performance. A diverse behavior of the various AMPs does not allow extrapolating the data to all the pathogens or peptides; hence, further studies and optimization of the detection methods will allow to find the best niche for the application of SALP in the assays.
Conclusion We have demonstrated effective capture of microorganisms on synthetic anti-lipopolysaccharide peptides (SALP)-coated surfaces. The investigated cationic peptides retained their affinity towards bacteria after their covalent immobilization. We
Eur J Clin Microbiol Infect Dis
proposed the application of SALP for the magnetic beadbased enrichment of prokaryotic pathogens in liquid samples and evaluated it with the peptide Pep19-2.5OH. The unspecific electrostatic binding of bacterial DNA to the peptide strongly affected polymerase chain reaction (PCR) and, thus, hampered the quantitative assessment of the bacteria enrichment. The use of DNA-independent approaches may allow for a better characterization of the peptide-based pathogen pre-concentration. Relying on our observations, we can recommend cationic antimicrobial peptides (AMPs) for application in assays with the direct detection of microorganisms, and, potentially, also for the decontamination of liquid samples.
12.
13.
14.
15.
16. Acknowledgments The published work was funded by the German Federal Ministry of Education and Research [BMBF; project MinoLab (number 16SV4030) and project number 01GU0824]. Conflict of interest Klaus Brandenburg is also a founder and CSO of the company Brandenburg Antiinfektiva GmbH. Development and investigation of the described peptide Pep19-2.5, also called Aspidasept®, is one of the ongoing activities of this company. Here, we declare that the research work described in this manuscript was neither sponsored nor had any other commercial relation to the aforementioned enterprise. Thus, we declare no conflicts of interest in regard to this publication.
References 1.
2.
3. 4.
5.
6.
7.
8.
9. 10.
11.
Barken KB, Haagensen JA, Tolker-Nielsen T (2007) Advances in nucleic acid-based diagnostics of bacterial infections. Clin Chim Acta 384:1–11 Jannes G, De Vos D (2006) A review of current and future molecular diagnostic tests for use in the microbiology laboratory. Methods Mol Biol 345:1–21 Picard FJ, Bergeron MG (2002) Rapid molecular theranostics in infectious diseases. Drug Discov Today 7:1092–1101 Yang S, Rothman RE (2004) PCR-based diagnostics for infectious diseases: uses, limitations, and future applications in acute-care settings. Lancet Infect Dis 4:337–348 Rådström P, Knutsson R, Wolffs P, Lövenklev M, Löfström C (2004) Pre-PCR processing: strategies to generate PCRcompatible samples. Mol Biotechnol 26:133–146 Lantz PG, Abu Al-Soud W, Knutsson R, Hahn-Hägerdal B, Rådström P (2000) Biotechnical use of polymerase chain reaction for microbiological analysis of biological samples. Biotechnol Annu Rev 5:87–130 Hansen WL, Bruggeman CA, Wolffs PF (2009) Evaluation of new preanalysis sample treatment tools and DNA isolation protocols to improve bacterial pathogen detection in whole blood. J Clin Microbiol 47:2629–2631 Mancini N, Carletti S, Ghidoli N, Cichero P, Burioni R, Clementi M (2010) The era of molecular and other non-culture-based methods in diagnosis of sepsis. Clin Microbiol Rev 23:235–251 Brewster JD (2003) Isolation and concentration of Salmonellae with an immunoaffinity column. J Microbiol Methods 55:287–293 Enroth H, Engstrand L (1995) Immunomagnetic separation and PCR for detection of Helicobacter pylori in water and stool specimens. Clin Microbiol 33:2162–2165 Fernandes CP, Seixas FK, Coutinho ML, Vasconcellos FA, Moreira AN, Conceição FR, Dellagostin OA, Aleixo JA (2008) An immunomagnetic separation-PCR method for detection of
17. 18.
19.
20.
21.
22.
23. 24.
25. 26.
27.
28.
29.
pathogenic Leptospira in biological fluids. Hybridoma (Larchmt) 27:381–386 Fu Z, Rogelj S, Kieft TL (2005) Rapid detection of Escherichia coli O157:H7 by immunomagnetic separation and real-time PCR. Int J Food Microbiol 99:47–57 Qiu J, Zhou Y, Chen H, Lin JM (2009) Immunomagnetic separation and rapid detection of bacteria using bioluminescence and microfluidics. Talanta 79:787–795 Wright DJ, Chapman PA, Siddons CA (1994) Immunomagnetic separation as a sensitive method for isolating Escherichia coli O157 from food samples. Epidemiol Infect 113:31–39 Mannoor MS, Zhang S, Link AJ, McAlpine MC (2010) Electrical detection of pathogenic bacteria via immobilized antimicrobial peptides. Proc Natl Acad Sci U S A 107:19207–19212. doi:10.1073/ pnas.1008768107 Soares JW, Morin KM, Mello CM (2004) Antimicrobial peptides in biosensing applications. Report of US Army Research, Development, & Engineering Command. Available online at: http://www.dtic.mil/cgi-bin/GetTRDoc?AD=ADA433515. Retrieved 11 Dec 2014 Brogden KA (2005) Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nat Rev Microbiol 3:238–250 Friedrich CL, Moyles D, Beveridge TJ, Hancock RE (2000) Antibacterial action of structurally diverse cationic peptides on gram-positive bacteria. Antimicrob Agents Chemother 44:2086– 2092 Schuerholz T, Brandenburg K, Marx G (2012) Antimicrobial peptides and their potential application in inflammation and sepsis. Crit Care 16:207 Gutsmann T, Razquin-Olazarán I, Kowalski I, Kaconis Y, Howe J, Bartels R, Hornef M, Schürholz T, Rössle M, Sanchez-Gómez S, Moriyon I, Martinez de Tejada G, Brandenburg K (2010) New antiseptic peptides to protect against endotoxin-mediated shock. Antimicrob Agents Chemother 54:3817–3824 Kaconis Y, Kowalski I, Howe J, Brauser A, Richter W, RazquinOlazarán I, Iñigo-Pestaña M, Garidel P, Rössle M, Martinez de Tejada G, Gutsmann T, Brandenburg K (2011) Biophysical mechanisms of endotoxin neutralization by cationic amphiphilic peptides. Biophys J 100:2652–2661 Heinbockel L, Sánchez-Gómez S, Martinez de Tejada G, Dömming S, Brandenburg J, Kaconis Y, Hornef M, Dupont A, Marwitz S, Goldmann T, Ernst M, Gutsmann T, Schürholz T, Brandenburg K (2013) Preclinical investigations reveal the broad-spectrum neutralizing activity of peptide Pep19-2.5 on bacterial pathogenicity factors. Antimicrob Agents Chemother 57:1480–1487 Brandenburg K (2009) Novel antimicrobial peptides. Patent no. PCT/EP2009/002565 Maheux AF, Picard FJ, Boissinot M, Bissonnette L, Paradis S, Bergeron MG (2009) Analytical comparison of nine PCR primer sets designed to detect the presence of Escherichia coli/Shigella in water samples. Water Res 43:3019–3028 Hancock RE, Lehrer R (1998) Cationic peptides: a new source of antibiotics. Trends Biotechnol 16:82–88 Peters BM, Shirtliff ME, Jabra-Rizk MA (2010) Antimicrobial peptides: primeval molecules or future drugs? PLoS Pathog 6: e1001067. doi:10.1371/journal.ppat.1001067 Costa F, Carvalho IF, Montelaro RC, Gomes P, Martins MC (2011) Covalent immobilization of antimicrobial peptides (AMPs) onto biomaterial surfaces. Acta Biomater 7:1431–1440 Siedenbiedel F, Tiller JC (2012) Antimicrobial polymers in solution and on surfaces: overview and functional principles. Polymers 4: 46–71 Li Y, Kumar KN, Dabkowski JM, Corrigan M, Scott RW, Nüsslein K, Tew GN (2012) New bactericidal surgical suture coating. Langmuir 28:12134–12139
Eur J Clin Microbiol Infect Dis 30.
31.
32. 33.
Kulagina NV, Shaffer KM, Ligler FS, Taitt CR (2007) Antimicrobial peptides as new recognition molecules for screening challenging species. Sensors Actuators B Chem 121:150–157 Scott MG, Gold MR, Hancock RE (1999) Interaction of cationic peptides with lipoteichoic acid and gram-positive bacteria. Infect Immun 67:6445–6453 Yeaman MR, Yount NY (2003) Mechanisms of antimicrobial peptide action and resistance. Pharmacol Rev 55:27–55 Epand RM, Vogel HJ (1999) Diversity of antimicrobial peptides and their mechanisms of action. Biochim Biophys Acta 1462:11–28
34.
Harris F, Dennison SR, Phoenix DA (2009) Anionic antimicrobial peptides from eukaryotic organisms. Curr Protein Pept Sci 10:585–606 35. Rydlo T, Rotem S, Mor A (2006) Antibacterial properties of dermaseptin S4 derivatives under extreme incubation conditions. Antimicrob Agents Chemother 50:490–497 36. Arcidiacono S, Pivarnik P, Mello CM, Senecal A (2008) Cy5 labeled antimicrobial peptides for enhanced detection of Escherichia coli O157:H7. Biosens Bioelectron 23:1721–1727 37. Kulagina NV, Shaffer KM, Anderson GP, Ligler FS, Taitt CR (2006) Antimicrobial peptide-based array for Escherichia coli and Salmonella screening. Anal Chim Acta 575:9–15
