MEndoB, a chimeric lysin featuring a novel domain architecture and superior activity for the treatment of staphylococcal infections.

Bacterial infections are a growing global healthcare concern, as an estimated annual 4.95 million deaths are associated with antimicrobial resistance (AMR). Methicillin-resistant Staphylococcus aureus is one of the deadliest pathogens and a high-priority pathogen according to the World Health Organization. Peptidoglycan hydrolases (PGHs) of phage origin have been postulated as a new class of antimicrobials for the treatment of bacterial infections, with a novel mechanism of action and no known resistances. The modular architecture of PGHs permits the creation of chimeric PGH libraries. In this study, the chimeric enzyme MEndoB was selected from a library of staphylococcal PGHs based on its rapid and sustained activity against staphylococci in human serum. The benefit of the presented screening approach was illustrated by the superiority of MEndoB in a head-to-head comparison with other PGHs intended for use against staphylococcal bacteremia. MEndoB displayed synergy with antibiotics and rapid killing in human whole blood with complete inhibition of re-growth over 24 h at low doses. Successful treatment of S. aureus-infected zebrafish larvae with MEndoB provided evidence for its in vivo effectiveness. This was further confirmed in a lethal systemic mouse infection model in which MEndoB significantly reduced S. aureus loads and tumor necrosis factor alpha levels in blood in a dose-dependent manner, which led to increased survival of the animals. Thus, the thorough lead candidate selection of MEndoB resulted in an outstanding second-generation PGH with in vitro, ex vivo, and in vivo results supporting further development.IMPORTANCEOne of the most pressing challenges of our era is the rising occurrence of bacteria that are resistant to antibiotics. Staphylococci are prominent pathogens in humans, which have developed multiple strategies to evade the effects of antibiotics. Infections caused by these bacteria have resulted in a high burden on the health care system and a significant loss of lives. In this study, we have successfully engineered lytic enzymes that exhibit an extraordinary ability to eradicate staphylococci. Our findings substantiate the importance of meticulous lead candidate selection to identify therapeutically promising peptidoglycan hydrolases with unprecedented activity. Hence, they offer a promising new avenue for treating staphylococcal infections.

Systemic application of bone-targeting peptidoglycan hydrolases as a novel treatment approach for staphylococcal bone infection.

IMPORTANCE: The rising prevalence of antimicrobial resistance in S. aureus has rendered treatment of staphylococcal infections increasingly difficult, making the discovery of alternative treatment options a high priority. Peptidoglycan hydrolases, a diverse group of bacteriolytic enzymes, show high promise as such alternatives due to their rapid and specific lysis of bacterial cells, independent of antibiotic resistance profiles. However, using these enzymes for the systemic treatment of local infections, such as osteomyelitis foci, needs improvement, as the therapeutic distributes throughout the whole host, resulting in low concentrations at the actual infection site. In addition, the occurrence of intracellularly persisting bacteria can lead to relapsing infections. Here, we describe an approach using tissue-targeting to increase the local concentration of therapeutic enzymes in the infected bone. The enzymes were modified with a short targeting moiety that mediated accumulation of the therapeutic in osteoblasts and additionally enables targeting of intracellularly surviving bacteria.

CkP1 bacteriophage, a S16-like myovirus that recognizes Citrobacter koseri lipopolysaccharide through its long tail fibers.

Citrobacter koseri is an emerging Gram-negative bacterial pathogen, which causes urinary tract infections. We isolated and characterized a novel S16-like myovirus CKP1 (vB_CkoM_CkP1), infecting C. koseri. CkP1 has a host range covering the whole C. koseri species, i.e., all strains that were tested, but does not infect other species. Its linear 168,463-bp genome contains 291 coding sequences, sharing sequence similarity with the Salmonella phage S16. Based on surface plasmon resonance and recombinant green florescence protein fusions, the tail fiber (gp267) was shown to decorate C. koseri cells, binding with a nanomolar affinity, without the need of accessory proteins. Both phage and the tail fiber specifically bind to bacterial cells by the lipopolysaccharide polymer. We further demonstrate that CkP1 is highly stable towards different environmental conditions of pH and temperatures and is able to control C. koseri cells in urine samples. Altogether, CkP1 features optimal in vitro characteristics to be used both as a control and detection agent towards drug-resistant C. koseri infections. KEY POINTS: • CkP1 infects all C. koseri strains tested • CkP1 recognizes C. koseri lipopolysaccharide through its long tail fiber • Both phage CkP1 and its tail fiber can be used to treat or detect C. koseri pathogens.

Endolysin Inhibits Skin Colonization by Patient-Derived Staphylococcus Aureus and Malignant T-Cell Activation in Cutaneous T-Cell Lymphoma.

Staphylococcus aureus is suspected to fuel disease activity in cutaneous T-cell lymphomas. In this study, we investigate the effect of a recombinant, antibacterial protein, endolysin (XZ.700), on S. aureus skin colonization and malignant T-cell activation. We show that endolysin strongly inhibits the proliferation of S. aureus isolated from cutaneous T-cell lymphoma skin and significantly decreases S. aureus bacterial cell counts in a dose-dependent manner. Likewise, ex vivo colonization of both healthy and lesional skin by S. aureus is profoundly inhibited by endolysin. Moreover, endolysin inhibits the patient-derived S. aureus induction of IFNγ and the IFNγ-inducible chemokine CXCL10 in healthy skin. Whereas patient-derived S. aureus stimulates activation and proliferation of malignant T cells in vitro through an indirect mechanism involving nonmalignant T cells, endolysin strongly inhibits the effects of S. aureus on activation (reduced CD25 and signal transducer and activator of transcription 5 phosphorylation) and proliferation (reduced Ki-67) of malignant T cells and cell lines in the presence of nonmalignant T cells. Taken together, we provide evidence that endolysin XZ.700 inhibits skin colonization, chemokine expression, and proliferation of pathogenic S. aureus and blocks their potential tumor-promoting effects on malignant T cells.

A novel surface functionalization platform to prime extracellular vesicles for targeted therapy and diagnostic imaging.

Extracellular vesicles (EVs), nanovesicles released by cells to effectively exchange biological information, are gaining interest as drug delivery system. Yet, analogously to liposomes, they show short blood circulation times and accumulation in the liver and the spleen. For tissue specific delivery, EV surfaces will thus have to be functionalized. We present a novel platform for flexible modification of EVs with target-specific ligands based on the avidin-biotin system. Genetic engineering of donor cells with a glycosylphosphatidylinositol-anchored avidin (GPI-Av) construct allows the isolation of EVs displaying avidin on their surface, functionalized with any biotinylated ligand. For proof of concept, GPI-Av EVs were modified with i) a biotinylated antibody or ii) de novo designed and synthesized biotinylated ligands binding carbonic anhydrase IX (CAIX), a membrane associated enzyme overexpressed in cancer. Functionalized EVs showed specific binding and uptake by CAIX-expressing cells, demonstrating the power of the system to prepare EVs for cell-specific drug delivery.

Chimeric Peptidoglycan Hydrolases Kill Staphylococcal Mastitis Isolates in Raw Milk and within Bovine Mammary Gland Epithelial Cells.

Staphylococcus aureus is a major causative agent of bovine mastitis, a disease considered one of the most economically devastating in the dairy sector. Considering the increasing prevalence of antibiotic-resistant strains, novel therapeutic approaches efficiently targeting extra- and intracellular bacteria and featuring high activity in the presence of raw milk components are needed. Here, we have screened a library of eighty peptidoglycan hydrolases (PGHs) for high activity against S. aureus in raw bovine milk, twelve of which were selected for further characterization and comparison in time-kill assays. The bacteriocins lysostaphin and ALE-1, and the chimeric PGH M23LST(L)_SH3b2638 reduced bacterial numbers in raw milk to the detection limit within 10 min. Three CHAP-based PGHs (CHAPGH15_SH3bAle1, CHAPK_SH3bLST_H, CHAPH5_LST_H) showed gradually improving activity with increasing dilution of the raw milk. Furthermore, we demonstrated synergistic activity of CHAPGH15_SH3bAle1 and LST when used in combination. Finally, modification of four PGHs (LST, M23LST(L)_SH3b2638, CHAPK_SH3bLST, CHAPGH15_SH3bAle1) with the cell-penetrating peptide TAT significantly enhanced the eradication of intracellular S. aureus in bovine mammary alveolar cells compared to the unmodified parentals in a concentration-dependent manner.

Linker-Improved Chimeric Endolysin Selectively Kills Staphylococcus aureus In Vitro, on Reconstituted Human Epidermis, and in a Murine Model of Skin Infection.

Staphylococcus aureus causes a broad spectrum of diseases in humans and animals. It is frequently associated with inflammatory skin disorders such as atopic dermatitis, where it aggravates symptoms. Treatment of S. aureus-associated skin infections with antibiotics is discouraged due to their broad-range deleterious effect on healthy skin microbiota and their ability to promote the development of resistance. Thus, novel S. aureus-specific antibacterial agents are desirable. We constructed two chimeric cell wall-lytic enzymes, Staphefekt SA.100 and XZ.700, which are composed of functional domains from the bacteriophage endolysin Ply2638 and the bacteriocin lysostaphin. Both enzymes specifically killed S. aureus and were inactive against commensal skin bacteria such as Staphylococcus epidermidis, with XZ.700 proving more active than SA.100 in multiple in vitro activity assays. When surface-attached mixed staphylococcal cultures were exposed to XZ.700 in a simplified microbiome model, the enzyme selectively removed S. aureus and retained S. epidermidis. Furthermore, XZ.700 did not induce resistance in S. aureus during repeated rounds of exposure to sublethal concentrations. Finally, we demonstrated that XZ.700 formulated as a cream is effective at killing S. aureus on reconstituted human epidermis and that an XZ.700-containing gel significantly reduces bacterial numbers compared to an untreated control in a mouse model of S. aureus-induced skin infection.

Inflammatory Response of Primary Cultured Bovine Mammary Epithelial Cells to Staphylococcus aureus Extracellular Vesicles.

In dairy cows, Staphylococcus aureus (S. aureus) is among the most prevalent microorganisms worldwide, causing mastitis, an inflammation of the mammary gland. Production of extracellular vesicles (EVs) is a common feature of S. aureus strains, which contributes to its pathogenesis by delivering bacterial effector molecules to host cells. In the current study, we evaluated the differences between five S. aureus mastitis isolates regarding their EV production. We found that different mastitis-related S. aureus strains differ in their behaviour of shedding EVs, with M5512VL producing the largest amount of EVs containing alpha-haemolysin, a strong cytotoxic agent. We stimulated primary cultured bovine mammary epithelial cells (pbMECs) with EVs from the S. aureus strain M5512VL. After 24 h of incubation, we observed a moderate increase in gene expression of tumour necrosis factor-alpha (TNF-α) but, surprisingly, a lack of an associated pronounced pro-inflammatory response. Our results contribute to understanding the damaging nature of S. aureus in its capacity to effectively affect mammary epithelial cells.

An Enzybiotic Regimen for the Treatment of Methicillin-Resistant Staphylococcus aureus Orthopaedic Device-Related Infection.

Orthopaedic device-related infection (ODRI) presents a significant challenge to the field of orthopaedic and trauma surgery. Despite extensive treatment involving surgical debridement and prolonged antibiotic therapy, outcomes remain poor. This is largely due to the unique abilities of Staphylococcus aureus, the most common causative agent of ODRI, to establish and protect itself within the host by forming biofilms on implanted devices and staphylococcal abscess communities (SACs). There is a need for novel antimicrobials that can readily target such features. Enzybiotics are a class of antimicrobial enzymes derived from bacteria and bacteriophages, which function by enzymatically degrading bacterial polymers essential to bacterial survival or biofilm formation. Here, we apply an enzybiotic-based combination regimen to a set of in vitro models as well as in a murine ODRI model to evaluate their usefulness in eradicating established S. aureus infection, compared to classical antibiotics. We show that two chimeric endolysins previously selected for their functional efficacy in human serum in combination with a polysaccharide depolymerase reduce bacterial CFU numbers 10,000-fold in a peg model and in an implant model of biofilm. The enzyme combination also completely eradicates S. aureus in a SAC in vitro model where classical antibiotics are ineffective. In an in vivo ODRI model in mice, the antibiofilm effects of this enzyme regimen are further enhanced when combined with a classical gentamicin/vancomycin treatment. In a mouse model of methicillin-resistant S. aureus (MRSA) ODRI following a fracture repair, a combined local enzybiotic/antibiotic treatment regimen showed a significant CFU reduction in the device and the surrounding soft tissue, as well as significant prevention of weight loss. These outcomes were superior to treatment with antibiotics alone. Overall, this study demonstrates that the addition of enzybiotics, which are distinguished by their extremely rapid killing efficacy and antibiofilm activities, can enhance the treatment of severe MRSA ODRI.

Fluorometric Paper-Based, Loop-Mediated Isothermal Amplification Devices for Quantitative Point-of-Care Detection of Methicillin-Resistant Staphylococcus aureus (MRSA).

Loop-mediated isothermal amplification (LAMP) has been widely used to detect many infectious diseases. However, minor inconveniences during the steps of adding reaction ingredients and lack of simple color results hinder point-of-care detection. We therefore invented a fluorometric paper-based LAMP by incorporating LAMP reagents, including a biotinylated primer, onto a cellulose membrane paper, with a simple DNA fluorescent dye incubation that demonstrated rapid and accurate results parallel to quantitative polymerase chain reaction (qPCR) methods. This technology allows for instant paper strip detection of methicillin-resistant Staphylococcus aureus (MRSA) in the laboratory and clinical samples. MRSA represents a major public health problem as it can cause infections in different parts of the human body and yet is resistant to commonly used antibiotics. In this study, we optimized LAMP reaction ingredients and incubation conditions following a central composite design (CCD) that yielded the shortest reaction time with high sensitivity. These CCD components and conditions were used to construct the paper-based LAMP reaction by immobilizing the biotinylated primer and the rest of the LAMP reagents to produce the ready-to-use MRSA diagnostic device. Our paper-based LAMP device could detect as low as 10 ag (equivalent to 1 copy) of the MRSA gene mecA within 36-43 min, was evaluated using both laboratory (individual cultures of MRSA and non-MRSA bacteria) and clinical blood samples to be 100% specific and sensitive compared to qPCR results, and had 35 day stability under 25 °C storage. Furthermore, the color readout allows for quantitation of MRSA copies. Hence, this device is applicable for point-of-care MRSA detection.

Deimmunization of protein therapeutics - Recent advances in experimental and computational epitope prediction and deletion.

Biotherapeutics, and antimicrobial proteins in particular, are of increasing interest for human medicine. An important challenge in the development of such therapeutics is their potential immunogenicity, which can induce production of anti-drug-antibodies, resulting in altered pharmacokinetics, reduced efficacy, and potentially severe anaphylactic or hypersensitivity reactions. For this reason, the development and application of effective deimmunization methods for protein drugs is of utmost importance. Deimmunization may be achieved by unspecific shielding approaches, which include PEGylation, fusion to polypeptides (e.g., XTEN or PAS), reductive methylation, glycosylation, and polysialylation. Alternatively, the identification of epitopes for T cells or B cells and their subsequent deletion through site-directed mutagenesis represent promising deimmunization strategies and can be accomplished through either experimental or computational approaches. This review highlights the most recent advances and current challenges in the deimmunization of protein therapeutics, with a special focus on computational epitope prediction and deletion tools.

Bacteriophage endolysins - extending their application to tissues and the bloodstream.

The rapid emergence of antibiotic-resistant bacteria and the lack of novel antibacterial agents pose a serious threat for patients and healthcare systems. Bacteriophage-encoded peptidoglycan hydrolases (endolysins) represent a promising new class of antimicrobials. Over the past two decades, research on these enzymes has evolved from basic in vitro characterization to sophisticated protein engineering approaches, including advanced preclinical and clinical testing. In recent years, increasingly specific animal models have shown efficacy of endolysins against bacterial infections of various different organs and tissues of the body. Despite these advances, some challenges with regard to systemic application of endolysins remain to be addressed. These include immunogenicity, circulation half-life, and cell and tissue-specific targeting and penetration properties.

In Situ Nucleic Acid Amplification and Ultrasensitive Colorimetric Readout in a Paper-Based Analytical Device Using Silver Nanoplates.

A rapid, highly sensitive, and quantitative colorimetric paper-based analytical device (PAD) based on silver nanoplates (AgNPls) and loop-mediated isothermal amplification (LAMP) is presented. It is shown that cauliflower-like concatemer LAMP products can mediate crystal etching of AgNPls, with a threefold signal enhancement versus linear dsDNA. Methicillin-resistant Staphylococcus aureus (MRSA), an antimicrobial resistant bacterium that poses a formidable risk with persistently high mortality, is used as a model pathogen. Due to the excellent color contrast provided by AgNPls, the PAD allows qualitative analysis by the naked eye and quantitative analysis using a smartphone camera, with detection limits down to a single copy in just 30 min, and a linear response from 1 to 104 copies (R2 = 0.994). The entire assay runs in situ on the paper surface, which drastically simplifies operation of the device. This is the first demonstration of single copy detection using a colorimetric readout, and the developed PAD shows great promise for translation into an ultrasensitive gene-based point-of-care test for any infectious disease target, via modification of the LAMP primer set.

Engineering of Long-Circulating Peptidoglycan Hydrolases Enables Efficient Treatment of Systemic Staphylococcus aureus Infection.

Staphylococcus aureus is a human pathogen causing life-threatening diseases. The increasing prevalence of multidrug-resistant S. aureus infections is a global health concern, requiring development of novel therapeutic options. Peptidoglycan-degrading enzymes (peptidoglycan hydrolases, PGHs) have emerged as a highly effective class of antimicrobial proteins against S. aureus and other pathogens. When applied to Gram-positive bacteria, PGHs hydrolyze bonds within the peptidoglycan layer, leading to rapid bacterial death by lysis. This activity is highly specific and independent of the metabolic activity of the cell or its antibiotic resistance patterns. However, systemic application of PGHs is limited by their often low activity in vivo and by an insufficient serum circulation half-life. To address this problem, we aimed to extend the half-life of PGHs selected for high activity against S. aureus in human serum. Half-life extension and increased serum circulation were achieved through fusion of PGHs to an albumin-binding domain (ABD), resulting in high-affinity recruitment of human serum albumin and formation of large protein complexes. Importantly, the ABD-fused PGHs maintained high killing activity against multiple drug-resistant S. aureus strains, as determined by ex vivo testing in human blood. The top candidate, termed ABD_M23, was tested in vivo to treat S. aureus-induced murine bacteremia. Our findings demonstrate a significantly higher efficacy of ABD_M23 than of the parental M23 enzyme. We conclude that fusion with ABD represents a powerful approach for half-life extension of PGHs, expanding the therapeutic potential of these enzybiotics for treatment of multidrug-resistant bacterial infections.IMPORTANCE Life-threatening infections with Staphylococcus aureus are often difficult to treat due to the increasing prevalence of antibiotic-resistant bacteria and their ability to persist in protected niches in the body. Bacteriolytic enzymes are promising new antimicrobials because they rapidly kill bacteria, including drug-resistant and persisting cells, by destroying their cell wall. However, when injected into the bloodstream, these enzymes are not retained long enough to clear an infection. Here, we describe a modification to increase blood circulation time of the enzymes and enhance treatment efficacy against S. aureus-induced bloodstream infections. This was achieved by preselecting enzyme candidates for high activity in human blood and coupling them to serum albumin, thereby preventing their elimination by kidney filtration and blood vessel cells.

Point-of-care testing system for digital single cell detection of MRSA directly from nasal swabs.

We present an automated point-of-care testing (POCT) system for rapid detection of species- and resistance markers in methicillin-resistant Staphylococcus aureus (MRSA) at the level of single cells, directly from nasal swab samples. Our novel system allows clear differentiation between MRSA, methicillin-sensitive S. aureus (MSSA) and methicillin-resistant coagulase-negative staphylococci (MR-CoNS), which is not the case for currently used real-time quantitative PCR based systems. On top, the novel approach outcompetes the culture-based methods in terms of its short time-to-result (1 h vs. up to 60 h) and reduces manual labor. The walk-away test is fully automated on the centrifugal microfluidic LabDisk platform. The LabDisk cartridge comprises the unit operations swab-uptake, reagent pre-storage, distribution of the sample into 20 000 droplets, specific enzymatic lysis of Staphylococcus spp. and recombinase polymerase amplification (RPA) of species (vicK) - and resistance (mecA) -markers. LabDisk actuation, incubation and multi-channel fluorescence detection is demonstrated with a clinical isolate and spiked nasal swab samples down to a limit of detection (LOD) of 3 ± 0.3 CFU µl-1 for MRSA. The novel approach of the digital single cell detection is suggested to improve hospital admission screening, timely decision making, and goal-oriented antibiotic therapy. The implementation of a higher degree of multiplexing is required to translate the results into clinical practice.

Glycotyping and Specific Separation of Listeria monocytogenes with a Novel Bacteriophage Protein Tool Kit.

The Gram-positive pathogen Listeria monocytogenes can be subdivided into at least 12 different serovars, based on the differential expression of a set of somatic and flagellar antigens. Of note, strains belonging to serovars 1/2a, 1/2b, and 4b cause the vast majority of foodborne listeriosis cases and outbreaks. The standard protocol for serovar determination involves an agglutination method using a set of sera containing cell surface-recognizing antibodies. However, this procedure is imperfect in both precision and practicality, due to discrepancies resulting from subjective interpretation. Furthermore, the exact antigenic epitopes remain unclear, due to the preparation of the absorbed sera and the complex nature of polyvalent antibody binding. Here, we present a novel method for quantitative somatic antigen differentiation using a set of recombinant affinity proteins (cell wall-binding domains and receptor-binding proteins) derived from a collection of Listeria bacteriophages. These proteins enable rapid, objective, and precise identification of the different teichoic acid glycopolymer structures, which represent the O-antigens, and allow a near-complete differentiation. This glycotyping approach confirmed serovar designations of over 60 previously characterized Listeria strains. Using select phage receptor-binding proteins coupled to paramagnetic beads, we also demonstrate the ability to specifically isolate serovar 1/2 or 4b cells from a mixed culture. In addition, glycotyping led to the discovery that strains designated serovar 4e actually possess an intermediate 4b-4d teichoic acid glycosylation pattern, underpinning the high discerning power and precision of this novel technique.IMPORTANCEListeria monocytogenes is a ubiquitous opportunistic pathogen that presents a major concern to the food industry due to its propensity to cause foodborne illness. The Listeria genus contains 15 different serovars, with most of the variance depending on the wall-associated teichoic acid glycopolymers, which confer somatic antigenicity. Strains belonging to serovars 1/2 and 4b cause the vast majority of listeriosis cases and outbreaks, meaning that regulators, as well as the food industry itself, have an interest in rapidly identifying isolates of these particular serovars in food processing environments. Current methods for phenotypic serovar differentiation are slow and lack accuracy, and the food industry could benefit from new technologies allowing serovar-specific isolation. Therefore, the novel method described here for rapid glycotype determination could present a valuable asset to detect and control this bacterium.

Targeting Hidden Pathogens: Cell-Penetrating Enzybiotics Eradicate Intracellular Drug-Resistant Staphylococcus aureus.

Staphylococcus aureus is a major concern in human health care, mostly due to the increasing prevalence of antibiotic resistance. Intracellular localization of S. aureus plays a key role in recurrent infections by protecting the pathogens from antibiotics and immune responses. Peptidoglycan hydrolases (PGHs) are highly specific bactericidal enzymes active against both drug-sensitive and -resistant bacteria. However, PGHs able to effectively target intracellular S. aureus are not yet available. To overcome this limitation, we first screened 322 recombineered PGHs for staphylolytic activity under conditions found inside eukaryotic intracellular compartments. The most active constructs were modified by fusion to different cell-penetrating peptides (CPPs), resulting in increased uptake and enhanced intracellular killing (reduction by up to 4.5 log units) of various S. aureus strains (including methicillin-resistant S. aureus [MRSA]) in different tissue culture infection models. The combined application of synergistic PGH-CPP constructs further enhanced their intracellular efficacy. Finally, synergistically active PGH-CPP cocktails reduced the total S. aureus by more than 2.2 log units in a murine abscess model after peripheral injection. Significantly more intracellular bacteria were killed by the PGH-CPPs than by the PGHs alone. Collectively, our findings show that CPP-fused PGHs are effective novel protein therapeutics against both intracellular and drug-resistant S. aureusIMPORTANCE The increasing prevalence of antibiotic-resistant bacteria is one of the most urgent problems of our time. Staphylococcus aureus is an important human pathogen that has acquired several mechanisms to evade antibiotic treatment. In addition, S. aureus is able to invade and persist within human cells, hiding from the immune response and antibiotic therapies. For these reasons, novel antibacterial strategies against these pathogens are needed. Here, we developed lytic enzymes which are able to effectively target drug-resistant and intracellular S. aureus Fusion of these so-called enzybiotics to cell-penetrating peptides enhanced their uptake and intracellular bactericidal activity in cell culture and in an abscess mouse model. Our results suggest that cell-penetrating enzybiotics are a promising new class of therapeutics against staphylococcal infections.

Improved Biodistribution and Extended Serum Half-Life of a Bacteriophage Endolysin by Albumin Binding Domain Fusion.

The increasing number of multidrug-resistant bacteria intensifies the need to develop new antimicrobial agents. Endolysins are bacteriophage-derived enzymes that degrade the bacterial cell wall and hold promise as a new class of highly specific and versatile antimicrobials. One major limitation to the therapeutic use of endolysins is their often short serum circulation half-life, mostly due to kidney excretion and lysosomal degradation. One strategy to increase the half-life of protein drugs is fusion to the albumin-binding domain (ABD). By high-affinity binding to serum albumin, ABD creates a complex with large hydrodynamic volume, reducing kidney excretion and lysosomal degradation. The aim of this study was to investigate the in vitro antibacterial activity and in vivo biodistribution and half-life of an engineered variant of the Staphylococcus aureus phage endolysin LysK. The ABD sequence was introduced at different positions within the enzyme, and lytic activity of each variant was determined in vitro and ex vivo in human serum. Half-life and biodistribution were assessed in vivo by intravenous injection of europium-labeled proteins into C57BL/6 wild-type mice. Our data demonstrates that fusion of the endolysin to ABD improves its serum circulation half-life and reduces its deposition in the kidneys in vivo. The most active construct reduced S. aureus counts in human serum ex vivo by 3 logs within 60 min. We conclude that ABD fusions provide an effective strategy to extend the half-life of antibacterial enzymes, supporting their therapeutic potential for treatment of systemic bacterial infections.

Ultrasensitive and Fast Diagnostics of Viable Listeria Cells by CBD Magnetic Separation Combined with A511::luxAB Detection.

The genus Listeria includes foodborne pathogens that cause life-threatening infections in those at risk, and sensitive and specific methods for detection of these bacteria are needed. Based on their unrivaled host specificity and ability to discriminate viable cells, bacteriophages represent an ideal toolbox for the development of such methods. Here, the authors describe an ultrasensitive diagnostic protocol for Listeria by combining two phage-based strategies: (1) specific capture and concentration of target cells by magnetic separation, harnessing cell wall-binding domains from Listeria phage endolysins (CBD-MS); and (2) highly sensitive detection using an adaptation of the A511::luxAB bioluminescent reporter phage assay in a microwell plate format. The combined assay enabled direct detection of approximately 100 bacteria per ml of pure culture with genus-level specificity in less than 6 h. For contaminated foods, the procedure included a 16 h selective enrichment step, followed by CBD-MS separation and A511::luxAB detection. It was able to consistently detect extremely low numbers (0.1 to 1.0 cfu/g) of viable Listeria cells, in a total assay time of less than 22 h. These results demonstrate the superiority of this phage-based assay to standard culture-based diagnostic protocols for the detection of viable bacteria, with respect to both sensitivity and speed.

Tracing back multidrug-resistant bacteria in fresh herb production: from chive to source through the irrigation water chain.

Environmental antibiotic-resistant bacteria (ARB) can be transferred to humans through foods. Fresh produce in particular is an ideal vector due to frequent raw consumption. A major contamination source of fresh produce is irrigation water. We hypothesized that water quality significantly affects loads of ARB and their diversity on fresh produce despite various other contamination sources present under agricultural practice conditions. Chive irrigated from an open-top reservoir or sterile-filtered water (control) was examined. Heterotrophic plate counts (HPC) and ARB were determined for water and chive with emphasis on Escherichia coli and Enterococcus spp. High HPC of freshly planted chive decreased over time and were significantly lower on control- vs. reservoir-irrigated chive at harvest (1.3 log (CFU/g) lower). Ciprofloxacin- and ceftazidime-resistant bacteria were significantly lower on control-irrigated chive at harvest and end of shelf life (up to 1.8 log (CFU/g) lower). Escherichia coli and Enterococcus spp. repeatedly isolated from water and chive proved resistant to up to six or four antibiotic classes (80% or 49% multidrug-resistant, respectively). Microbial source tracking identified E. coli-ST1056 along the irrigation chain and on chive. Whole-genome sequencing revealed that E. coli-ST1056 from both environments were clonal and carried the same transmissible multidrug-resistance plasmid, proving water as source of chive contamination. These findings emphasize the urgent need for guidelines concerning ARB in irrigation water and development of affordable water disinfection technologies to diminish ARB on irrigated produce.