Human breast milk (HBM) is a source of essential nutrients for infants and is particularly recommended for preterm neonates when their own mother’s milk is not available. It provides protection against infections and decreases necrotizing enterocolitis and cardiovascular diseases. Nevertheless, HBM spoilage can occur due to contamination by pathogens, and the risk of a shortage of HBM is very often present. B. cereus is the most frequent ubiquitous bacteria responsible for HBM being discarded. It can contaminate HBM at all stages, from its collect point to the storage and delivery. B. cereus can induce severe infection in newborns with very low birth weight, with sometimes fatal outcomes. Although the source of contamination is rarely identified, in some cases, HBM was suspected as a potential source. Even if the risk is low, as infection due to B. cereus in preterm infants should not be overlooked, human milk banks follow strict procedures to avoid contamination, to accurately identify remaining bacteria following pasteurization and to discard non-compliant milk samples. In this review, we present a literature overview of B. cereus infections reported in neonates and the suspected sources of contamination. We highlight the procedures followed by the human milk banks from the collection of the milk to its microbiological characterization in Europe. We also present improved detection and decontamination methods that might help to decrease the risk and to preserve the public’s confidence in this vital biological product for infants whose mothers cannot breastfeed.

The emergence of B. cereus as an opportunistic food-borne pathogen has intensified the need to distinguish strains of public health concern. The heterogeneity of the diseases associated with B. cereus infections emphasizes the versatility of these bacteria strains to colonize their host. Nevertheless, the molecular basis of these differences remains unclear. Several toxins are involved in virulence, particularly in gastrointestinal disorders, but there are currently no biological markers able to differentiate pathogenic from harmless strains. We have previously shown that CwpFM is a cell wall peptidase involved in B. cereus virulence. Here, we report a sequence/structure/function characterization of 39 CwpFM sequences, chosen from a collection of B. cereus with diverse virulence phenotypes, from harmless to highly pathogenic strains. CwpFM is homology-modeled in silico as an exported papain-like endopeptidase, with an N-terminal end composed of three successive bacterial Src Homology 3 domains (SH3b_1–3) likely to control protein–protein interactions in signaling pathways, and a C-terminal end that contains a catalytic NLPC_P60 domain primed to form a competent active site. We confirmed in vitro that CwpFM is an endopeptidase with a moderate peptidoglycan hydrolase activity. Remarkably, CwpFMs from pathogenic strains harbor a specific stretch of twenty residues intrinsically disordered, inserted between the SH3b₃ and the catalytic NLPC_P60 domain. This strongly suggests this linker as a marker of differentiation between B. cereus strains. We believe that our findings improve our understanding of the pathogenicity of B. cereus while advancing both clinical diagnosis and food safety.

Mfd est une protéine bactérienne fortement conservée et impliquée dans de nombreux processus comme la réparation de l'ADN, la mutagénèse ou encore la virulence.Bacillus cereus est une bactérie responsable d'intoxications alimentaires, mais également un pathogène opportuniste pouvant causer des infections non gastro-intestinales sévères chez les personnes immunodéprimées comme les nouveau-nés prématurés. La pathogénie de B. cereus est multifactorielle, un des facteurs impliquée dans sa virulence est la protéine Mfd. Par l'étude de souches cliniques et environnementales de B. cereus, nous avons pu montrer que Mfd joue un rôle majeur dans la virulence des souches cliniques et que son implication semble être étroitement liée à sa séquence / structure fonctionnelle. Une modification de cette dernière pourrait engendrer une variation dans la virulence d'une souche. Ces données contribuent à une meilleure compréhension des mécanismes de virulence de B. cereus et permettent de suggérer Mfd comme un facteur global de virulence.Grâce à son rôle dans la virulence et la mutagénèse, Mfd est une cible prometteuse pour le développement de nouveaux antibiotiques. Nous avons montré qu'un inhibiteur de Mfd, le NM102, a une activité antimicrobienne sur K. pneumonia et P. aeruginosa exclusivement dans un contexte d'infection sans induire de toxicité dans l'hôte. En plus de cette activité, le NM102 est aussi capable d'inhiber la fonction de Mfd dans la mutagénèse ce qui réduit la fréquence d'apparition de résistance aux antibiotiques. Ces résultats permettent de proposer une alternative prometteuse pour lutter contre la résistance des bactéries aux antibiotiques qui constituent un problème de santé global et alarmant.

Bacterial infections remain a major global health burden, amplified by the rapid emergence of antimicrobial resistance (AMR). Among priority pathogens, the ESKAPEE group represents a critical threat due to their high prevalence in healthcare-associated infections, and their capacity to acquire multidrug resistance. Antivirulence strategies, aiming at disarming pathogens rather than inhibiting their growth, constitute a promising alternative to conventional antibiotics. Their development, however, is hindered by the lack of suitable experimental models to evaluate virulence and antivirulence efficacy. Here, we establish complementary in vitro and in vivo infection models to characterize pathogenicity and assess antivirulence activity against clinically relevant ESKAPEE bacteria ( Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli ). We developed a robust nitric oxide (NO)-based in vitro assay that mimics host immune nitrosative stress and enables quantitative evaluation of bacterial resistance to innate immune defenses. In parallel, we established comparative insect infection models using Galleria mellonella and Bombyx species, allowing reproducible assessment of bacterial virulence across multiple hosts and pathogens. These models highlighted marked differences in pathogenic potential depending on both bacterial species and host background. As a proof of concept, we applied these systems to characterize NM102, a first-in-class antivirulence compound targeting the DNA repair protein Mfd. NM102 significantly reduced bacterial survival under NO stress in vitro and decreased bacterial burden in vivo in insect infection models. Together, our results demonstrate that combining immune-mimicking in vitro assays with complementary insect models provides a powerful and ethically acceptable platform to study bacterial pathogenicity and to accelerate the development of innovative antivirulence therapeutics to combat AMR. IMPORTANCE: Bacterial infections remain a major public health challenge, worsened by the rapid rise of antimicrobial resistance. Among the most concerning pathogens are the ESKAPEE bacteria, which frequently cause hospital-acquired infections and are often resistant to multiple antibiotics. Antivirulence strategies offer promising alternatives to traditional treatments by disarming bacteria rather than killing them, potentially reducing the risk of resistance. However, progress in this field has been limited by the lack of reliable models to measure bacterial virulence and evaluate antivirulence compounds. In this study, we developed complementary models to better assess bacterial pathogenicity and treatment efficacy. We created an in vitro assay that mimics immune defenses and established insect infection models to compare virulence across pathogens and hosts. Using these systems, we tested NM102, a first-in-class antivirulence compound targeting bacterial DNA repair. NM102 reduced bacterial survival and infection severity, highlighting the potential of antivirulence approaches as innovative therapies against drug-resistant infections.