The phage lysin field has done nothing but grow in the last decades. As a result, many different research groups around the world are contributing to the field, often with certain methodological differences that pose a challenge to the interpretation and comparison of results. In this work, we present the case study of three Acinetobacter baumannii-targeting phage lysins (wild-type endolysin LysMK34 plus engineered lysins eLysMK34 and 1D10) plus one lysin with broad activity against Gram-positive bacteria (PlySs2) to provide exemplary evidence on the risks of generalization when using one of the most common lysin evaluation assays: the killing assay with resting cells. To that end, we performed killing assays with the aforementioned lysins using hypo-, iso- and hypertonic buffers plus human serum either as the reaction or the dilution medium in a systematic manner. Our findings stress the perils of creating hypotonic conditions or a hypotonic shock during a killing assay, suggesting that hypotonic buffers should be avoided as a test environment or as diluents before plating to avoid overestimation of the killing effect in the assayed conditions. As a conclusion, we suggest that the nature of both the incubation and the dilution buffers should be always clearly identified when reporting killing activity data, and that for experimental consistency the same incubation buffer should be used as a diluent for posterior serial dilution and plating unless explicitly required by the experimental design. In addition, the most appropriate buffer mimicking the final application must be chosen to obtain relevant results.

Mycobacterium tuberculosis causes 6.4 million cases of tuberculosis and claims 1.6 million lives annually. Mycobacterial adhesion, invasion of host cells, and subsequent intracellular survival are crucial for the infection and dissemination process, yet the cellular mechanisms underlying these phenomena remain poorly understood. This study created a Bacillus Calmette-Guérin (BCG) transposon library using a MycomarT7 phage carrying a Himar1 Mariner transposon to identify genes related to mycobacteria adhesion and invasion. Using adhesion and invasion model screening, we found that the mutant strain B2909 lacked adhesion and invasion abilities because of an inactive fadD18 gene, which encodes a fatty-acyl CoA ligase, although the specific function of this gene remains unclear. To investigate the role of FadD18, we constructed a complementary strain and observed that fadD18 expression enhanced the colony size and promoted the formation of a stronger cord-like structure; FadD18 expression also inhibited BCG growth and reduced BCG intracellular survival in macrophages. Furthermore, FadD18 expression elevated levels of the proinflammatory cytokines IL-6, IL-1β, and TNF-α in infected macrophages by stimulating the NF-κB and MAPK signaling pathways. Overall, the FadD18 plays a key role in the adhesion and invasion abilities of mycobacteria while modulating the intracellular survival of BCG by influencing the production of proinflammatory cytokines.

The primary objective of this investigation was to assess the viability of free and encapsulated Lactobacillus plantarum probiotics in mango juice and under simulated gastrointestinal conditions. Specifically, the probiotics were encapsulated using sodium alginate and alginate-soy protein isolate through the internal gelation method, and the obtained probiotics were characterized for various attributes. Both free and encapsulated probiotics were exposed to challenging conditions, including thermal stress, low temperature, and simulated gastrointestinal conditions. Additionally, both types of probiotics were incorporated into mango juice, and their survival was monitored over a 28-day storage period. Following viability under simulated gastrointestinal conditions, the count of free and encapsulated probiotic cells decreased from initial levels of 9.57 log CFU/mL, 9.55 log CFU/mL, and 9.53 log CFU/mL, 9.56 log CFU/mL to final levels of 6.14 log CFU/mL, 8.31 log CFU/mL, and 6.24 log CFU/mL, 8.62 log CFU/mL, respectively. Notably, encapsulated probiotics exhibited a decrease of 1.24 log CFU and 0.94 log CFU, while free cells experienced a reduction of 3.43 log CFU and 6.24 log CFU in mango juice over the storage period. Encapsulated probiotics demonstrated higher viability in mango juice compared to free probiotics throughout the 28-day storage period. These findings suggest that mango juice can be enriched with probiotics to create a health-promoting beverage.

Endogenous antimicrobial peptides (AMPs) play a key role in the host defense against pathogens. AMPs attack pathogens preferentially at the site of entry to prevent invasive infection. Mycobacterium tuberculosis (Mtb) enters its host via the airways. AMPs released into the airways are therefore likely candidates to contribute to the clearance of Mtb immediately after infection. Since lysozyme is detectable in airway secretions, we evaluated its antimicrobial activity against Mtb. We demonstrate that lysozyme inhibits the growth of extracellular Mtb, including isoniazid-resistant strains. Lysozyme also inhibited the growth of non-tuberculous mycobacteria. Even though lysozyme entered Mtb-infected human macrophages and co-localized with the pathogen we did not observe antimicrobial activity. This observation was unlikely related to the large size of lysozyme (14.74 kDa) because a smaller lysozyme-derived peptide also co-localized with Mtb without affecting the viability. To evaluate whether the activity of lysozyme against extracellular Mtb could be relevant in vivo, we incubated Mtb with fractions of human serum and screened for antimicrobial activity. After several rounds of sub-fractionation, we identified a highly active fraction-component as lysozyme by mass spectrometry. In summary, our results identify lysozyme as an antimycobacterial protein that is detectable as an active compound in human serum. Our results demonstrate that the activity of AMPs against extracellular bacilli does not predict efficacy against intracellular pathogens despite co-localization within the macrophage. Ongoing experiments are designed to unravel peptide modifications that occur in the intracellular space and interfere with the deleterious activity of lysozyme in the extracellular environment.

Vibrio fluvialis is an emerging foodborne pathogenic bacterium that can cause severe cholera-like diarrhea and various extraintestinal infections, posing challenges to public health and food safety worldwide. The arginine deiminase (ADI) pathway plays an important role in bacterial environmental adaptation and pathogenicity. However, the biological functions and regulatory mechanisms of the pathway in V. fluvialis remain unclear. In this study, we demonstrate that L-arginine upregulates the expression of the ADI gene cluster and promotes the growth of V. fluvialis. The ADI gene cluster, which we proved to be comprised of two operons, arcD and arcACB, significantly enhances the survival of V. fluvialis in acidic environments both in vitro (in culture medium and in macrophage) and in vivo (in mice). The mRNA level and reporter gene fusion analyses revealed that ArgR, a transcriptional factor, is necessary for the activation of both arcD and arcACB transcriptions. Bioinformatic analysis predicted the existence of multiple potential ArgR binding sites at the arcD and arcACB promoter regions that were further confirmed by electrophoretic mobility shift assay, DNase I footprinting, or point mutation analyses. Together, our study provides insights into the important role of the ArgR-ADI pathway in the survival of V. fluvialis under acidic conditions and the detailed molecular mechanism. These findings will deepen our understanding of how environmental changes and gene expression interact to facilitate bacterial adaptations and virulence.

The availability of microbial biobanks for the storage of individual gut microbiota members or their derived and artificially assembled consortia has become fundamental for in vitro investigation of the molecular mechanisms behind microbe-microbe and/or microbe-host interactions. However, to preserve bacterial viability, adequate storage and processing technologies are required. In this study, the effects on cell viability of seven different combinations of cryoprotective agents were evaluated by flow cytometry for 53 bacterial species representing key members of the human gut microbiota after one and 3 months of cryopreservation at -80°C. The obtained results highlighted that no universal cryoprotectant was identified capable of guaranteeing effective recovery of intact cells after cryopreservation for all tested bacteria. However, the presence of inulin or skimmed milk provided high levels of viability protection during cryoexposure. These results were further corroborated by cryopreserving 10 artificial gut microbiota produced through in vitro continuous fermentation system technology. Indeed, in this case, the inclusion of inulin or skimmed milk resulted in a high recovery of viable cells, while also allowing consistent and reliable preservation of the artificial gut microbiota biodiversity. Overall, these results suggest that, although the efficacy of various cryoprotective agents is species-specific, some cryoprotectants based on glycerol and the addition of inulin or skimmed milk are preferable to retain viability and biodiversity for both single bacterial species and artificial gut microbiota.

Enzymatic browning in fruits and vegetables, driven by polyphenol oxidase (PPO) activity, results in color changes and loss of bioactive compounds. Emerging technologies are being explored to prevent this browning and ensure microbial safety in foods. This study assessed the effectiveness of pulsed light (PL) and ultraviolet light-emitting diodes (UV-LED) in inhibiting PPO and inactivating Escherichia coli ATTC 25922 in fresh apple juice (Malus domestica var. Red Delicious). Both treatments\' effects on juice quality, including bioactive compounds, color changes, and microbial inactivation, were examined. At similar doses, PL-treated samples (126 J/cm2) showed higher 2,2- diphenyl-1-picrylhydrazyl inhibition (9.5%) compared to UV-LED-treated samples (132 J/cm2), which showed 1.06%. For microbial inactivation, UV-LED achieved greater E. coli reduction (>3 log cycles) and less ascorbic acid degradation (9.4% ± 0.05) than PL. However, increasing PL doses to 176 J/cm2 resulted in more than 5 log cycles reduction of E. coli, showing a synergistic effect with the final temperature reached (55 °C). The Weibull model analyzed survival curves to evaluate inactivation kinetics. UV-LED was superior in preserving thermosensitive compounds, while PL excelled in deactivating more PPO and achieving maximal microbial inactivation more quickly.

Most bacteria live in communities, often with closely related strains and species with whom they must compete for space and resources. Consequently, bacteria have acquired or evolved mechanisms to antagonize competitors through the production of antibacterial toxins. Similar to bacterial systems that combat phage infection and mechanisms to thwart antibiotics, bacteria have also acquired and evolved features to protect themselves from antibacterial toxins. Just as there is a large body of research identifying and characterizing antibacterial proteins and toxin delivery systems, studies of bacterial mechanisms to resist and survive assault from competitors\' weapons have also expanded tremendously. Emerging data are beginning to reveal protective processes and mechanisms that are as diverse as the toxins themselves. Protection against antibacterial toxins can be acquired by horizontal gene transfer, receptor or target alteration, induction of protective functions, physical barriers, and other diverse processes. Here, we review recent studies in this rapidly expanding field.

Antimicrobial resistance (AMR) is an escalating global health crisis, driven by the overuse and misuse of antibiotics. Multidrug-resistant Gram-negative bacteria, such as Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae, are particularly concerning due to their high morbidity and mortality rates. In this context, endolysins, derived from bacteriophages, offer a promising alternative to traditional antibiotics. This study introduces LysJEP8, a novel endolysin derived from Escherichia phage JEP8, which exhibits remarkable antimicrobial activity against key Gram-negative members of the ESKAPE group. Comparative assessments highlight LysJEP8\'s superior performance in reducing bacterial survival rates compared to previously described endolysins, with the most significant impact observed against P. aeruginosa, and notable effects on A. baumannii and K. pneumoniae. The study found that LysJEP8, as predicted by in silico analysis, worked best at lower pH values but lost its effectiveness at salt concentrations close to physiological levels. Importantly, LysJEP8 exhibited remarkable efficacy in the disruption of P. aeruginosa biofilms. This research underscores the potential of LysJEP8 as a valuable candidate for the development of innovative antibacterial agents, particularly against Gram-negative pathogens, and highlights opportunities for further engineering and optimization to address AMR effectively.

OBJECTIVE: MRSA is a major cause of hospital-acquired and community-acquired infections. Treatment options for MRSA are limited because of the rapid development of β-lactam resistance. Combining antibiotics offers an affordable, time-saving, viable and efficient approach for developing novel antimicrobial therapies. Both amoxicillin and cefdinir are oral β-lactams with indications for a wide range of bacterial infections and mild side effects. This study aimed to investigate the in vitro and in vivo efficacy of combining these two β-lactams against MRSA strains.
METHODS: Fourteen representative prevalent MRSA strains with diverse sequence types (STs) were tested with a combination of amoxicillin and cefdinir, using chequerboard and time-kill assays. The Galleria mellonella larvae infection model was used to evaluate the in vivo efficacy of this dual combination against the community-acquired MRSA (CA-MRSA) strain USA300 and the hospital-acquired MRSA (HA-MRSA) strain COL.
RESULTS: The chequerboard assay revealed a synergistic activity of the dual amoxicillin/cefdinir combination against all tested MRSA strains, with fractional inhibitory concentration index (FICI) values below 0.5 and at least a 4-fold reduction in the MICs of both antibiotics. Time-kill assays demonstrated synergistic bactericidal activity of this dual combination against the MRSA strain USA300 and strain COL. Moreover, in vivo studies showed that the administration of amoxicillin/cefdinir combination to G. mellonella larvae infected with MRSA strains significantly improved the survival rate up to 82%, which was comparable to the efficacy of vancomycin.
CONCLUSIONS: In vitro and in vivo studies indicate that the dual combination of amoxicillin/cefdinir demonstrates a synergistic bactericidal efficacy against MRSA strains of various STs. Further research is needed to explore its potential as a treatment option for MRSA infections.