The antibiotic management of catheter-related infections (CRIs) often fails owing to the emergence of antimicrobial-resistant strains and/or biofilm/persister apparitions. Thus, we investigated the efficacy of two novel antimicrobial agents, i.e., the synthetic peptide SAAP-148 and the novel antibiotic halicin, against Gram-negative bacteria (GNB) colonizing catheters. The antibacterial, anti-biofilm, and anti-persister activities of both agents were evaluated against Acinetobacter baumannii, Escherichia coli, and Klebsiella pneumoniae strains. The enrolled strains were isolated from catheters and selected based on their resistance to at least three antibiotic classes and biofilm formation potential. Furthermore, the hemolysis and endotoxin neutralization abilities of these agents were explored. The bactericidal activity of both agents was reduced in urine and plasma as compared to buffered saline. In a dose-dependent manner, SAAP-148 and halicin reduced bacterial counts in 24 h preformed biofilms on silicone elastomer discs and eliminated persisters originating from antibiotic-exposed mature 7-day biofilms, with halicin being less effective than SAAP-148. Importantly, SAAP-148 and halicin acted synergistically on E. coli and K. pneumoniae biofilms but not on A. baumannii biofilms. The peptide, but not halicin, decreased the production of IL-12p40 upon exposure to UV-killed bacteria. This preliminary study showed that SAAP-148 and halicin alone/in combination are promising candidates to fight GNB colonizing catheters.

Antimicrobial peptides (AMPs) are promising alternatives to antibiotics for treatment of antimicrobial resistant (AMR) bacterial infections. However, their narrow therapeutic window due to in vivo toxicity and limited stability hampers their clinical use. Here, we evaluated encapsulation of two amphiphilic AMPs, SAAP-148 and snake cathelicidin Ab-Cath, into oleyl-modified hyaluronic acid (OL-HA) nanogels to improve their selectivity index. The AMP-loaded OL-HA nanogels ranged 181-206 nm in size with a PDI of 0.2, highly negative surface charge (-47 to -48 mV) and moderate encapsulation efficiency (53-63%). The AMP-loaded OL-HA nanogels displayed similar activity in vitro as AMP solutions against AMR Staphylococcus aureus and Acinetobacter baumannii, with a dose-dependent effect over time. Importantly, the AMP-loaded OL-HA nanogels showed decreased cytotoxicity towards human erythrocytes and primary skin fibroblast, thereby improving the selectivity index of SAAP-148 and Ab-Cath by 2- and 16.8-fold, respectively. Particularly, the selectivity of Ab-Cath-loaded OL-HA nanogels has great clinical potential, with an index that reached ≥ 300 for S. aureus and ≥ 3000 for A. baumannii. These findings indicate that OL-HA nanogels are a promising drug delivery system to reduce the cytotoxicity of AMPs without substantially affecting their antimicrobial activity, thereby increasing their selectivity index and potential as therapeutics to combat AMR bacterial infections.

Chronic wound infections colonized by bacteria are becoming more difficult to treat with current antibiotics due to the development of antimicrobial resistance (AMR) as well as biofilm and persister cell formation. Synthetic antibacterial and antibiofilm peptide (SAAP)-148 is an excellent alternative for treatment of such infections but suffers from limitations related to its cationic peptidic nature and thus instability and possible cytotoxicity, resulting in a narrow therapeutic window. Here, we evaluated SAAP-148 encapsulation in nanogels composed of octenyl succinic anhydride (OSA)-modified hyaluronic acid (HA) to circumvent these limitations. SAAP-148 was efficiently (>98%) encapsulated with high drug loading (23%), resulting in monodispersed anionic OSA-HA nanogels with sizes ranging 204-253 nm. Nanogel lyophilization in presence of polyvinyl alcohol maintained their sizes and morphology. SAAP-148 was sustainedly released from lyophilized nanogels (37-41% in 72 h) upon reconstitution. Lyophilized SAAP-148-loaded nanogels showed similar antimicrobial activity as SAAP-148 against planktonic and biofilm-residing AMR Staphylococcus aureus and Acinetobacter baumannii. Importantly, formulated SAAP-148 showed reduced cytotoxicity against human erythrocytes, primary human skin fibroblasts and human keratinocytes. Additionally, lyophilized SAAP-148-loaded nanogels eradicated AMR S. aureus and A. baumannii colonizing a 3D human epidermal model, without inducing any cytotoxicity in contrast to SAAP-148. These findings indicate that OSA-HA nanogels increase SAAP-148\'s therapeutic potential for treatment of skin wound infections.

Recently, using a deep learning approach, the novel antibiotic halicin was discovered. We compared the antibacterial activities of two novel bactericidal antimicrobial agents, i.e., the synthetic antibacterial and antibiofilm peptide (SAAP)-148 with this antibiotic halicin. Results revealed that SAAP-148 was more effective than halicin in killing planktonic bacteria of antimicrobial-resistant (AMR) Escherichia coli, Acinetobacter baumannii and Staphylococcus aureus, especially in biologically relevant media, such as plasma and urine, and in 3D human infection models. Surprisingly, SAAP-148 and halicin were equally effective against these bacteria residing in immature and mature biofilms. As their modes of action differ, potential favorable interactions between SAAP-148 and halicin were investigated. For some specific strains of AMR E. coli and S. aureus synergism between these agents was observed, whereas for other strains, additive interactions were noted. These favorable interactions were confirmed for AMR E. coli in a 3D human bladder infection model and AMR S. aureus in a 3D human epidermal infection model. Together, combinations of these two novel antimicrobial agents hold promise as an innovative treatment for infections not effectively treatable with current antibiotics.

OBJECTIVE: Staphylococcus epidermidis is one of the most common Gram-positive cocci in nosocomial infection, which could adhere to the surface of medical apparatus and causes biofilm-related infections. In the present study, we aim to explore the antimicrobial effects of GH12 and SAAP-148 against Staphylococcus epidermidis.
METHODS: Micro-dilution methods were used to detect the minimal inhibitory/bactericidal concentration of peptides on Staphylococcus epidermidis. Biofilm formation positive type strain was used to determine the antibiofilm effects of the peptides. Biofilms were built on the cover slides and fluorescent dye SYTO9 and laser confocal microscope were used to observe the effects of peptides on the three-dimensional structure of Staphylococcus epidermidis biofilms. The cell membrane permeability of Staphylococcus epidermidis was detected by flow cytometry. Expressions of icaA and icaDgenes were analyzed by real-time reverse transcription PCR.
RESULTS: The minimal inhibitory concentrations of GH12 and SAAP-148 against Staphylococcus epidermidis were 8 and 16 μg/mL, respectively, and the minimal bactericidal concentration was 64 μg/mL. GH12 and SAAP-148 significantly inhibited the biofilm formation of Staphylococcus epidermidis at the concentration of 8 μg/mL (t=7.193, P<0.05) and 16 μg/mL (t=7.808, P<0.05), respectively. Similarly, the GH12 and SAAP-148 significantly eradicated the pre-formed biofilms at the concentration of 16 μg/mL (t=5.369, P<0.05) and 32 μg/mL (t=4.474, P<0.05) in a dose-response manner, respectively. Meanwhile, the two peptides broke the structure of biofims and reduce the total biomass. GH12 and SAAP-148 at the concentration of minimal inhibitory concentration significantly disrupted the cell membrane of Staphylococcus epidermidis. The expressions of icaA and icaDgenes were significantly inhibited by antimicrobial peptides at the 1×minimal inhibitory concentration.
CONCLUSIONS: GH12 and SAAP-148 show significantly antimicrobial and anti-biofilm effects against Staphylococcus epidermidis by disruption of cell membrane and inhibition of icaAand icaDgene expression.
目的: 表皮葡萄球菌是院内感染常见的革兰氏阳性球菌之一，它亦黏附于医疗器械表面导致生物膜相关感染。本研究拟探讨两种人工合成的抗菌肽GH12和SAAP-148对表皮葡萄球菌浮游菌生长及其生物膜的影响。方法: 通过微量肉汤稀释法检测抗菌肽GH12和SAAP-148的最低抑菌浓度和最低杀菌浓度；利用生物膜成膜能力阳性标准菌株在微孔板中构建生物膜，分别检测这两种抗菌肽对表皮葡萄球菌生物膜的抑制和分散作用；在盖玻片上构建表皮葡萄球菌生物膜，通过荧光染料SYTO9染色和激光共聚焦显微镜观察，检测两种抗菌肽对表皮葡萄球菌生物膜三维结构的破坏作用；通过流式细胞仪检测抗菌肽对表皮葡萄球菌细胞膜的破坏作用；采用实时反转录聚合酶链反应分析表皮葡萄球菌黏附相关基因icaA和icaD的表达情况。结果: GH12和SAAP-148对表皮葡萄球菌的最低抑菌浓度分别为8和16 μg/mL，最低杀菌浓度均为64 μg/mL；GH12和SAAP-148的质量浓度分别为8 μg/mL(t=7.193，P<0.05)和16 μg/mL(t=7.808，P<0.05)时均可显著抑制表皮葡萄球菌RP62A生物膜的形成，且均呈现出一定的剂量依赖性；GH12和SAAP-148的质量浓度分别为16 μg/mL(t=5.369，P<0.05)和32 μg/mL(t=4.474，P<0.05)时可显著分散RP62A已形成的生物膜，亦呈现剂量依赖性；通过激光共聚焦显微镜观察，GH12和SAAP-148可显著破坏生物膜的三维结构，使生物膜的总量显著减少；流式细胞仪检测发现最低抑菌浓度的GH12和SAAP-148还能显著破坏表皮葡萄球菌的细胞膜；1×最低抑菌浓度的抗菌肽能够显著抑制表皮葡萄球菌黏附相关基因icaA和icaD的表达。结论: 抗菌肽GH12和SAAP-148通过破坏细胞膜对表皮葡萄球菌浮游菌生长具有显著的抑制作用，并能通过抑制黏附相关基因抑制和分散其生物膜。.

Prosthetic joint infection (PJI) is a severe complication of arthroplasty. Due to biofilm and persister formation current treatment strategies often fail. Therefore, innovative anti-biofilm and anti-persister agents are urgently needed. Antimicrobial peptides with their broad antibacterial activities may be such candidates. An in vitro model simulating PJI comprising of rifampicin/ciprofloxacin-exposed, mature methicillin-resistant Staphylococcus aureus (MRSA) biofilms on polystyrene plates, titanium/aluminium/niobium disks, and prosthetic joint liners were developed. Bacteria obtained from and residing within these biofilms were exposed to SAAP-148, acyldepsipeptide-4, LL-37, and pexiganan. Microcalorimetry was used to monitor the heat flow by the bacteria in these models. Daily exposure of mature biofilms to rifampicin/ciprofloxacin for 3 days resulted in a 4-log reduction of MRSA. Prolonged antibiotic exposure did not further reduce bacterial counts. Microcalorimetry confirmed the low metabolic activity of these persisters. SAAP-148 and pexiganan, but not LL-37, eliminated the persisters while ADEP4 reduced the number of persisters. SAAP-148 further eradicated persisters within antibiotics-exposed, mature biofilms on the various surfaces. To conclude, antibiotic-exposed, mature MRSA biofilms on various surfaces have been developed as in vitro models for PJI. SAAP-148 is highly effective against persisters obtained from the biofilms as well as within these models. Antibiotics-exposed, mature biofilms on relevant surfaces can be instrumental in the search for novel treatment strategies to combat biofilm-associated infections.

Antibiotic resistance is a growing public health concern, though the constant development of new antibiotics. The combination of high-throughput screening and drug repurposing is an effective way to develop new therapeutic uses of drugs. In this study, we screened a drug library consisting of 1,573 drugs already approved by the Food and Drug Administration and 903 drugs from the natural product library, to identify antimicrobials against Pseudomonas aeruginosa. A high-throughput screening assay based on microtiter plate was used to screen 39 drugs that inhibit the planktonic or biofilm formation of P. aeruginosa while most of them are antibiotics. The antimicrobial activities of these drugs were evaluated by phenotypic analysis. Further studies showed the combined therapy of tetracycline antibiotics demeclocycline hydrochloride (DMCT) and the novel antimicrobial peptide SAAP-148 has an effective synergistic antibacterial effect on P. aeruginosa PAO1 and P. aeruginosa ATCC27853. Moreover, the time-kill curve assay and murine model of cutaneous abscesses further confirmed the synergistic effect. In addition, the combination of DMCT and SAAP-148 has the potential to combat clinically isolated multidrug-resistant (MDR) P. aeruginosa strains. Our results clearly indicate that DMCT and SAAP-148 combined therapy could be an effective method to combat MDR P. aeruginosa-related infections.

BACKGROUND: Accurate determination of the efficacy of antimicrobial agents requires neutralization of residual antimicrobial activity in the samples before microbiological assessment of the number of surviving bacteria. Sodium polyanethol sulfonate (SPS) is a known neutralizer for the antimicrobial activity of aminoglycosides and polymyxins. In this study, we evaluated the ability of SPS to neutralize residual antimicrobial activity of antimicrobial peptides [SAAP-148 and pexiganan; 1% (wt/v) in PBS], antibiotics [mupirocin (Bactroban) and fusidic acid (Fucidin) in ointments; 2% (wt/wt))] and disinfectants [2% (wt/wt) silver sulfadiazine cream (SSD) and 0.5% (v/v) chlorhexidine in 70% alcohol].
METHODS: Homogenates of human skin models that had been exposed to various antimicrobial agents for 1 h were pipetted on top of Methicillin-resistant Staphylococcus aureus (MRSA) on agar plates to determine whether the antimicrobial agents display residual activity. To determine the optimal concentration of SPS for neutralization, antimicrobial agents were mixed with PBS or increasing doses of SPS in PBS (0.05-1% wt/v) and then 105 colony forming units (CFU)/mL MRSA were added. After 30 min incubation, the number of viable bacteria was assessed. Next, the in vitro efficacy of SAAP-148 against various gram-positive and gram-negative bacteria was determined using PBS or 0.05% (wt/v) SPS immediately after 30 min incubation of the mixture. Additionally, ex vivo excision wound models were inoculated with 105 CFU MRSA for 1 h and exposed to SAAP-148, pexiganan, chlorhexidine or PBS for 1 h. Subsequently, samples were homogenized in PBS or 0.05% (wt/v) SPS and the number of viable bacteria was assessed.
RESULTS: All tested antimicrobials displayed residual activity in tissue samples, resulting in a lower recovery of surviving bacteria on agar. SPS concentrations at ≥0.05% (wt/v) were able to neutralize the antimicrobial activity of SAAP-148, pexiganan and chlorhexidine, but not of SSD, Bactroban and Fucidin. Finally, SPS-neutralization in in vitro and ex vivo efficacy tests of SAAP-148, pexiganan and chlorhexidine against gram-positive and gram-negative bacteria resulted in significantly higher numbers of CFU compared to control samples without SPS-neutralization.
CONCLUSIONS: SPS was successfully used to neutralize residual activity of SAAP-148, pexiganan and chlorhexidine and this prevented an overestimation of their efficacy.

BACKGROUND: We investigated the efficacy of a synthetic antimicrobial peptide SAAP-148, which was shown to be effective against Methicillin-resistant Staphylococcus aureus (MRSA) on tape-stripped mice skin. Unexpectedly, SAAP-148 was not effective against MRSA in our pilot study using rats with excision wounds. Therefore, we investigated factors that might have contributed to the poor efficacy of SAAP-148. Subsequently, we optimised the protocol and assessed the efficacy of SAAP-148 in an adapted rat study.
METHODS: We incubated 100 µL of SAAP-148 with 1 cm2 of a wound dressing for 1 h and determined the unabsorbed volume of peptide solution. Furthermore, 105 colony forming units (CFU)/mL MRSA were exposed to increasing dosages of SAAP-148 in 50% (v/v) human plasma, eschar- or skin extract or PBS. After 30 min incubation, the number of viable bacteria was determined. Next, ex vivo skin models were inoculated with MRSA for 1 h and exposed to SAAP-148. Finally, excision wounds on the back of rats were inoculated with 107 CFU MRSA overnight and treated with SAAP-148 for 4 h or 24 h. Subsequently, the number of viable bacteria was determined.
RESULTS: Contrary to Cuticell, Parafilm and Tegaderm film, < 20% of peptide solution was recovered after incubation with gauze, Mepilex border and Opsite Post-op. Furthermore, in plasma, eschar- or skin extract > 20-fold higher dosages of SAAP-148 were required to achieve a 2-log reduction (LR) of MRSA versus SAAP-148 in PBS. Exposure of ex vivo models to SAAP-148 for 24 h resulted in a 4-fold lower LR than a 1 h or 4 h exposure period. Additionally, SAAP-148 caused a 1.3-fold lower mean LR at a load of 107 CFU compared to 105 CFU MRSA. Moreover, exposure of ex vivo excision wound models to SAAP-148 resulted in a 1.5-fold lower LR than for tape-stripped skin. Finally, SAAP-148 failed to reduce the bacterial counts in an adapted rat study.
CONCLUSIONS: Several factors, such as absorption of SAAP-148 by wound dressings, components within wound exudates, re-colonisation during the exposure of SAAP-148, and a high bacterial load may contribute to the poor antimicrobial effect of SAAP-148 against MRSA in the rat model.