Bovine mastitis, an infection of the cow’s mammary gland, is frequently caused by Streptococcus uberis and causes major economic losses in the dairy industry. The intramammary administration of antibiotics currently remains the predominant preventive and therapeutic measure. These antimicrobial compounds, of which some are considered critical in human health care, are frequently applied as dry therapy resulting in their consistent overuse. Therefore, the use of antibiotics in the dairy sector is being questioned. We here identified two endolysins, i.e., PlySs2 and PlySs9, respectively derived from Streptococcus suis serotype-2 and -9 prophages, with lytic activity against S. uberis in an in vitro setting. Both endolysins gave clear lysis zones in spot-on-plate assays and caused a reduction of the optical density in a turbidity reduction assay. In depth characterization identified PlySs9 as the more potent endolysin over PlySs2 with a lower MIC value and about one additional log of killing. PlySs2 and PlySs9 were challenged to a panel of subclinical and clinical S. uberis milk isolates and were both able to lyse all strains tested. Molecular dissection of these endolysins in catalytic and cell wall binding subdomains resulted in major loss of killing and binding activity, respectively. Taken together, we here propose PlySs2 and PlySs9 as candidate compounds to the current antimicrobial arsenal known against bovine mastitis-causing S. uberis as future add-on or replacement strategy to the currently used intramammary antibiotics.

The transition of ductal carcinoma in situ (DCIS) to invasive carcinoma (IC) in breast cancer can be faithfully reproduced by the intraductal mouse model. Envisaging to use this model for therapeutic testing, we aimed to in-depth characterize the tumor immunity associated with the differential progression of two types of intraductal tumors. More specifically, we focused on triple-negative breast cancer (TNBC) and intraductally inoculated luciferase-expressing metastatic 4T1 and locally invasive Py230 cells in lactating mammary glands of syngeneic BALB/c and C57BL/6 female mice, respectively. Although the aggressive 4T1 cells rapidly formed solid tumors, Py230 tumors eventually grew to a similar size through enhanced proliferation. Yet, ductal tumor cell breakthrough and metastasis occurred earlier in the 4T1- compared to the Py230-based intraductal model and was associated with high expression of matrix metalloproteinase (MMP)-9, vascular endothelial growth factor (VEGF), chitinase 3-like 1 (CHI3L1) and lipocalin 2 (LCN2) as well as an increased influx of immune cells (mainly macrophages, neutrophils and T-cells). Moreover, activated cytotoxic T-cells, B-cells and programmed death-1 (PD-1)-positive cells were more prominent in the 4T1-based intraductal model in line with enhanced pro-inflammatory cytokine and gene expression profiles. Py230-based tumors showed a more immunosuppressed anti-inflammatory profile with a high amount of regulatory T-cells, which may account for the decreased T-cell activation but increased proliferation compared to the 4T1-based tumors. Taken together, our results highlight the differential immunological aspects of aggressive metastatic and non-aggressive intraductal progression of 4T1- vs. Py230-based tumors, providing a base for future studies to explore therapy using these intraductal TNBC models.

Bacteriophage‐derived endolysins are a novel class of antimicrobials known to rapidly kill bacteria, including antibiotic‐resistant strains. We here engineered endolysins against the bovine mastitis pathogens Streptococcus uberis , Streptococcus agalactiae and Streptococcus dysgalactiae , also targeting intracellular survival and biofilm formation. For this purpose, high‐throughput DNA assembly was used to create a library with >80,000 theoretical endolysin variants for screening of their bacteriolytic activity against Gram‐positive isolates from (sub)clinically affected cows. This lytic activity was evaluated by turbidity reduction and time‐kill assays in phosphate‐buffered saline and pasteurized whole cow's milk to allow a rank up of the most potent leading candidates. A top candidate was selected with a 4.0 log killing efficacy against S. uberis , also showing similar activity against S. agalactiae and S. dysgalactiae . This top candidate eradicated S. uberis biofilm and showed intracellular activity in two bovine mammary epithelial cell lines as was confirmed by confocal microscopy. A potentiating effect on cloxacillin, a beta‐lactam penicillin used to intramammarily treat bovine Gram‐positive mastitis, was observed for this top candidate endolysin in raw cow's milk from (sub)clinically infected udders. Our in vitro results indicate that engineered endolysins may have a future role as add‐on in the treatment of bovine streptococcal mastitis.

Aggressive triple-negative breast cancer (TNBC) is classically treated with chemotherapy. Besides direct tumor cell killing, some chemotherapeutics such as cisplatin provide additional disease reduction through stimulation of anti-tumor immunity. The cisplatin-induced immunomodulation in TNBC was here investigated in-depth using immunocompetent intraductal mouse models. Upon primary tumor transition to invasive carcinoma, cisplatin was injected systemically and significantly reduced tumor progression. Flow cytometric immunophenotyping was corroborated by immunohistochemical analyses and revealed both differential immune cell compositions and positivity for their programmed death (PD)-1 and PD-ligand (L)1 markers across body compartments, including the primary tumor, axillary lymph nodes and spleen. As key findings, a significant decrease in immunosuppressive and a concomitant increase in anti-tumor lymphocytic cell numbers were observed in the axillary lymph nodes and spleen, highlighting their importance in cisplatin-stimulated anti-tumor immunity. These immunomodulatory effects were already established following the first cisplatin dose, indicating that early cisplatin-mediated events may determine (immuno)therapeutic outcome. Furthermore, a single cisplatin dose sufficed to alleviate anti-PD-1 resistance in a 4T1-based model, providing add-on disease reduction without toxic side effects as seen upon multiple cisplatin dosing. Overall, these results highlight cisplatin as immunotherapeutic ally in TNBC, providing durable immunostimulation, even after a single dose.

Bacteriophage-encoded endolysins, peptidoglycan hydrolases breaking down the Gram-positive bacterial cell wall, represent a groundbreaking class of novel antimicrobials to revolutionize the veterinary medicine field. Wild-type endolysins exhibit a modular structure, consisting of enzymatically active and cell wall-binding domains, that enable genetic engineering strategies for the creation of chimeric fusion proteins or so-called ‘engineered endolysins’. This biotechnological approach has yielded variants with modified lytic spectrums, introducing new possibilities in antimicrobial development. However, the discovery of highly similar endolysins by different groups has occasionally resulted in the assignment of different names that complicate a straightforward comparison. The aim of this review was to perform a homology-based comparison of the wild-type and engineered endolysins that have been characterized in the context of bovine mastitis-causing streptococci and staphylococci, grouping homologous endolysins with ≥ 95.0% protein sequence similarity. Literature is explored by homologous groups for the wild-type endolysins, followed by a chronological examination of engineered endolysins according to their year of publication. This review concludes that the wild-type endolysins encountered persistent challenges in raw milk and in vivo settings, causing a notable shift in the field towards the engineering of endolysins. Lead candidates that display robust lytic activity are nowadays selected from screening assays that are performed under these challenging conditions, often utilizing advanced high-throughput protein engineering methods. Overall, these recent advancements suggest that endolysins will integrate into the antibiotic arsenal over the next decade, thereby innovating antimicrobial treatment against bovine mastitis-causing streptococci and staphylococci.

Streptococcus pneumoniae , the pneumococcus, is a cause of major illness globally. Invasive pneumococcal disease (IPD) is characterized by pneumococci invading blood (bacteremia), lungs (pneumonia), or brain and cerebrospinal fluid (meningitis). Meningitis remains an important global health concern because half of the survivors experience long-term neurological damage. The antibiotics commonly used to treat pneumococcal infections are β-lactams and macrolides, however, S. pneumoniae is nowadays often resistant to one or several antibiotics, therefore novel antimicrobials are needed. Here, we found that the bacteriophage-derived cpl-1 endolysin showed consistent antibacterial activity against β-lactam- and macrolide-resistant pneumococcal clinical strains grown in human blood and human cerebrospinal fluid. Exploiting synergistic and additive mechanisms, supplementation of cpl-1 to either penicillin or erythromycin, as representatives for β-lactam and macrolide antibiotics, rescued human neuronal cells from the cytotoxicity of antibiotic-resistant pneumococcal infections. Finally, systemic administration of cpl-1 supplemented to penicillin in mice infected with penicillin-resistant pneumococci successfully reduced bacteremia, and, thanks to the efficient penetration across the blood–brain barrier, abolished bacterial load in the brain, resulting in increased (89%) survival accompanied by an asymptomatic course of infection. These findings strongly suggest that cpl-1 can enhance antibiotic susceptibility in β-lactam- and macrolide-resistant S. pneumoniae , serving as a valuable adjunct therapy to standard-of-care antibiotics for multidrug-resistant IPD.