E. coli mastitis is a major production disease in dairy cattle and requires alternative treatments to antibiotics. In this context, phages are of growing interest, but their host specificity remains a challenge in therapy. This study aimed to characterize eight newly isolated phages for the control of E. coli mastitis, to assess their efficacy in milk and to investigate their specificity. Physicochemical characterization of the phages was performed, followed by in vitro stability and lytic activity assays in raw and heat-treated milks. Genome sequencing of phages and bacteria was performed to investigate phage attachment. Phage stability was maintained across physiological pH and temperature ranges, as well as in raw milk. Phage lytic activity demonstrated bacterial decreases below detection level, but regrowth occurred in raw milk after 5 h of incubation with 3/8 phages. A narrow host range was linked to the diversity of the bacterial collection and to the presence of two receptor-binding proteins among Tevenvirinae. Indeed, structural analysis of the proteins revealed a variable region in the long tail fiber and a conserved short tail fiber. In conclusion, phage specificity was mainly associated with the long tail fiber and milk components didn’t hinder the efficacy of phages to control bovine mastitis, although resistance should be investigated.

Escherichia coli is a major cause of deadly infections in calves. As surveillance in livestock is mainly performed on carriage isolates, factors driving the dissemination of E. coli infecting calves remain largely unknown. Here, we analyzed the population of β-lactam-resistant E. coli in sick calves across five calving seasons on 444 farms in Wallonia, Belgium. 99% of the isolates were MDR and enriched in virulence factors, adhesins and toxins. Restrictions on critical antibiotic usage have no impact on population structure and β-lactamase gene content. This resilient population is likely shaped by virulence and resistance. Correlations of phylogeny and geographic origin suggest indirect local transmission with differences between regions east and west of the Meuse. Phylogenetic analyses with isolates from EnteroBase show a close relationship of calf isolates with human isolates, suggesting host jumps. These findings provide new means to further model the spread of E. coli in livestock farming.

Calves are one of the most common carriers of antibiotic-resistant bacteria among farm animals. However, the impact of antibiotic usage on resistance mechanisms, transmission routes between farms, and the transmission of resistant bacteria to humans remain largely unknown. Here we analyzed the population of β-lactam resistant E. coli isolated over five calving seasons on 444 farms scattered throughout Wallonia, Belgium. Restrictions on critical antibiotics usage led to a reduction of resistance to 3rd generation cephalosporins but has no impact on population structure and β-lactamase genes indicating a resilient population. The correlation between short genetic distances and geographic proximity suggests indirect transmission between farms by fomites with differences between regions east and west of the river Meuse. Phylogenetic analysis of calf isolates with isolates from public databases indicates transitions from bovine to human adaptation. These findings provide new means to further model the spread of E. coli in livestock farming. ### Competing Interest Statement The authors have declared no competing interest. Agence Nationale de la Recherche, https://ror.org/00rbzpz17, ANR-10-LABX-62-IBEID, ANR-20-PAMR-003, ANR-20-PAMR-0010 Belgian Federal Public Service Health, Food Chain Safety and Environment, RF 17/6317 RU-BLA-ESBL-CPE Fédération Wallonie-Bruxelles, CHK/MA/JCD/CBV 20-41

The Gram-negative bacterium Aeromonas salmonicida subsp. salmonicida is a major fish pathogen causing furunculosis, one of the main bacterial diseases affecting salmonids. Since this bacterium leads to significant losses in aquaculture worldwide and frequently displays antimicrobial resistance, this study aimed to isolate bacteriophages targeting A. salmonicida, characterize them in vitro, and evaluate their therapeutic potential using the Galleria mellonella larvae model. Four novel phages presenting a myovirus morphotype were isolated from fish farms and aquatic environments in Belgium and France. Genomic analyses revealed that the three lytic phages vB_AsaM_ULASA2 (170,823 bp), vB_AsaM_ULASA3 (164,381 bp), and vB_AsaM_ULASA4 (171,205 bp) belong to the Straboviridae family, whereas no known family could be assigned to the phage vB_AsaM_ULASA1 (47,813 bp). These isolates displayed valuable functional properties, including activity against a broad panel of contemporary multidrug-resistant A. salmonicida strains, as well as high biochemical tolerance. A single phage administration prolonged the survival of G. mellonella larvae previously infected with A. salmonicida, although it did not result in complete elimination of the bacteria. Overall, this study highlights the therapeutic potential of lytic ULASA phages for the management of furunculosis, with implications for reducing antibiotic use and limiting antimicrobial resistance in aquaculture.

AIMS:Aeromonas salmonicida subsp. salmonicida is the etiological agent of furunculosis, a fish disease highly aggressive for salmonids and responsible for significant economic losses in aquaculture worldwide. This study aimed to explore genomic and antimicrobial resistance traits of Western European A. salmonicida subsp. salmonicida strains and to develop an adapted infection model using larvae of the greater wax moth Galleria mellonella to assess the pathogenic potential of this psychrophilic subspecies. METHODS AND RESULTS:Three A. salmonicida subsp. salmonicida strains, isolated from salmonids displaying clinical signs of furunculosis, were tested against a panel of antibiotics and sequenced to characterize their genome. Virulence was evaluated in G. mellonella larvae using bacterial doses ranging from 101 to 106 CFU/larva. Two isolates exhibited multidrug resistance to antibiotics commonly used against furunculosis. Although closely related to the reference strain A449, genomic analyses revealed multiple plasmids known to encode antibiotic resistance genes. Virulence assays showed that this subspecies was lethal at doses as low as 101 CFU/larva, and that a fully functional Type III secretion system (T3SS) is not essential for the infection of G. mellonella, likely due to the presence of other virulence factors in T3SS-deficient strains. CONCLUSIONS:These findings enhance the genomic characterization of European A. salmonicida subsp. salmonicida and validate the use of G. mellonella larvae as a relevant alternative infection model for studying this psychrophilic subspecies.

SUMMARYAttaching-effacing (AE) lesion- and Shiga toxin-producing Escherichia (E.) coli (AE-STEC), previously known as "enterohemorrhagic E. coli" (EHEC), are responsible for (hemorrhagic) enterocolitis (HC) and hemolytic uremic syndrome (HUS) in humans. The most frequent and pathogenic AE-STEC belong to a few O:H major serotypes that are responsible for the majority of cases and outbreaks worldwide. From time to time, one or another non-major O:H serotype can emerge, causing either local outbreaks or a a progressive increase in clinical cases. One of these minor serotypes is O80:H2, which has been progressively emerging in Western Europe, especially in France, since 2010. AE-STEC O80:H2 are responsible for not only HC and HUS but also invasive infections with bacteremia and internal organ infection. In parallel to their emergence in humans, AE-STEC and enteropathogenic E. coli (EPEC) O80:H2 have also been emerging in young calves suffering diarrhea and enteritis and, more rarely septicemia, in Belgium since 2009. In this manuscript, an overview of AE-STEC and EPEC O80:H2 infections in humans and calves is presented, with particular focus on the clinical manifestations, the prevalence and incidence in Western Europe, and the identification of the potential reservoir(s). In addition, the results of a large-scale whole genome-based phylogenetic analysis of 417 published and unpublished genome sequences currently available in the literature and in the NCBI and EnteroBase databases are presented with hypotheses on the origin and evolution of this new hybrid AE-STEC and EPEC serotype.

The emergence of antimicrobial resistance renewed the interest in bacteriophages as complementary interventions to the use of antibiotics in veterinary medicine. The IABS workshop entitled "Avoiding Antimicrobial Resistance: Veterinary Use of Phages for Prevention, Therapy and Control of Bacterial Infections" brought together experts to discuss the scientific, regulatory and commercial challenges involved in bringing phage-based products to market. The biological characteristics of phages require innovative approaches for product development and regulatory approval. Dependent on their actual use, phages could be marketed as veterinary medicinal products, magistral preparations, food additives, or biocides, each classification implying different regulatory requirements and challenges, and none of which were originally intended for phage-based products. The meeting highlighted the need for regulatory harmonization to facilitate market access and allow manufacturers to choose the most appropriate regulatory pathway for their products. Recent EMA and EDQM guidelines offer some flexibility to take into account the biological nature of phages, but concerns remain about the feasibility of manufacturing phage-based products following existing rules for veterinary chemotherapeutants at commercially viable costs. Overcoming these regulatory and financial barriers is essential for the integration of phage therapy as a therapeutic option for control of bacterial infection and disease in veterinary medicine.

Despite their prevalence in Europe, the source of contamination of humans by Attaching-Effacing Shigatoxigenic Escherichia coli (AE-STEC) O80:H2 remains unidentified. This study aimed to assess a procedure based on non-melibiose fermentation and resistance to tellurite to isolate AE-STEC and enteropathogenic (EPEC) O80:H2 from healthy cattle. The genome sequences of 40 calf and human AE-STEC and EPEC O80:H2 were analyzed: (i) none harbored the mel operon, but the 70mel DNA sequence instead; (ii) the ter-type 1 operon was detected in 16 EPEC and stx1a or stx2a AE-STEC, while no ter-type 1 operon was detected in the remaining 24 EPEC and stx2d AE-STEC. The 21 calf AE-STEC and EPEC O80:H2 were tested phenotypically: (i) none fermented melibiose on melibiose-MacConkey agar plates; (ii) ten of the 11 ter-type 1-positive strains had Minimal Inhibitory Concentrations (MIC) ≥ 128 µg/mL to potassium tellurite; (iii) conversely, the ten ter-negative strains had MIC of two µg/mL. Accordingly, enrichment broths containing two µg/mL of potassium tellurite and inoculated with one high MIC (≥256 µg/mL) stx1a AE-STEC O80:H2 tested positive with the O80 PCR after overnight growth, but not the enrichment broths inoculated with one low MIC (two µg/mL) EPEC. Nevertheless, neither AE-STEC nor EPEC O80:H2 were recovered from 96 rectal fecal samples collected from healthy cattle at one slaughterhouse after overnight growth under the same conditions. In conclusion, this procedure may help to isolate stx1a and stx2a AE-STEC and EPEC O80:H2, but not stx2d AE-STEC that are tellurite sensitive, and new surveys using different procedures are necessary to identify their animal source, if any.

The second symposium of the Belgian Society for Viruses of Microbes (BSVoM) took place on 8 September 2023 at the University of Liège with 141 participants from 10 countries. The meeting program covered three thematic sessions opened by international keynote speakers: two sessions were devoted to "Fundamental research in phage ecology and biology" and the third one to the "Present and future applications of phages". During this one day symposium, four invited keynote lectures, nine selected talks and eight student pitches were given along with thirty presented posters. The president of the Belgian Society for Viruses of Microbes, Prof. Yves Briers, took advantage of this symposium to launch the Phage Valley concept that will put the spotlight on the exceptionally high density of researchers investigating viruses of microbes as well as the successful triple helix approach between academia, industry and government in Belgium.

La diarrhée est l’une des principales pathologies rencontrées dans les élevages de bovins au Bénin. Les veaux, premiers maillons de la chaîne, en sont les plus atteints. L’objectif de cette étude était d’évaluer la présence des gènes de virulence dans les souches d’Escherichia coli susceptibles de provoquer la diarrhée chez les veaux ainsi que leurs profils de résistance aux antimicrobiens usuels. Pour cela, 106 veaux ont fait objet d’un suivi pendant deux mois après leur naissance dans la commune de Nikki. Au total, 33 échantillons de matières fécales ont été prélevés directement du rectum de 33 veaux atteints de diarrhée et soumis à des analyses bactériologiques. Tous les prélèvements réalisés étaient positifs à E. coli. La caractérisation des souches d’E. coli isolées pour la présence des gènes et facteurs de virulence stx1, stx2, eae, sta, F41 et F5, a révélé la présence du gène stx1 uniquement avec un taux de 63,64 %. La résistance des souches d’E. coli aux antibiotiques les plus utilisés au Bénin a été testée : la doxycycline (taux de résistance de 70 %), l’amoxicilline + acide clavulanique (50 %) et la colistine (50 %). Des études ultérieures sont nécessaires afin de procéder au typage sérologique et au séquençage du génome des souches d’E. coli. Il serait également nécessaire d’étendre l’échantillonnage aux autres régions du Bénin, afin de mieux évaluer le statut des élevages bovins vis-à-vis de ces souches d’E. coli isolées chez les veaux et ainsi identifier un éventuel risque zoonotique.

Aims The purpose of this work was to study extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli (ESBL-EC) in freshwaters, hospital effluents, and wastewaters during two sampling campaigns in 2021.Methods and results Water sampling was performed at 24 stations in the Ourthe watershed in Belgium. A total of 644 ESBL (n = 642) and AmpC (n = 2) E. coli strains were isolated. Disk-diffusion assays were performed following the EUCAST's recommendations. All strains were tested for the presence of blaCTX-M-1, blaCTX-M-2, and blaCTX-M-9 gene groups by PCR. Genes belonging to blaCTX-M-1 and blaCTX-M-9 groups were detected, respectively, in 73.6% and 14.9% of the strains. No blaCTX-M-2 group's gene was found. A subset of strains (n = 40) was selected for whole genome sequencing. Escherichia coli serotype O18: H7 ST 1463 was predominant (n = 14) in the sequenced strains and showed pathogenicity in the Galleria mellonella larvae model. beta-lactamase genes identified were blaCTX-M (n = 21), with blaCTX-M-15 mostly represented (n = 15), as well as blaTEM (n = 11), blaOXA (n = 7), blaSHV (n = 9), and carbapenemase (CP) genes were observed in several strains-blaKPC-3 (n = 19), blaNDM-1 (n = 1), blaVIM-1 (n = 2), and blaOXA-244 (n = 2)-even from freshwaters.Conclusions ESBL-EC are widely distributed in the aquatic environment in Belgium and contain a variety of ESBL and CP genes.

AIMS:Understanding bacterial phage resistance mechanisms has implications for developing phage-based therapies. This study aimed to explore the development of phage resistance in Escherichia coli K1 isolates' to K1-ULINTec4, a K1-dependent bacteriophage.METHODS AND RESULTS:Resistant colonies were isolated from two different strains (APEC 45 and C5), both previously exposed to K1-ULINTec4. Genome analysis and several parameters were assessed, including growth capacity, phage adsorption, phenotypic impact at capsular level, biofilm production, and virulence in the in vivo Galleria mellonella larvae model. One out of the six resistant isolates exhibited a significantly slower growth rate, suggesting the presence of a resistance mechanism altering its fitness. Comparative genomic analysis revealed insertion sequences in the region 2 of the kps gene cluster involved in the capsule biosynthesis. In addition, an immunoassay targeting the K1 capsule showed a very low positive reaction compared to the control. Nevertheless, microscopic images of resistant strains revealed the presence of capsules with a clustered organization of bacterial cells and biofilm assessment showed an increased biofilm production compared to the sensitive strains. In the G. mellonella model, larvae infected with phage-resistant isolates showed better survival rates than larvae infected with phage-sensitive strains.CONCLUSIONS:A phage resistance mechanism was identified at the genomic level and had a negative impact on the K1 capsule production. The resistant isolates showed an increased biofilm production and a decreased virulence in vivo.

The COVID-19 pandemic due to the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has been plaguing the world since late 2019/early 2020 and has changed the way we function as a society, halting both economic and social activities worldwide. Classrooms, offices, restaurants, public transport, and other enclosed spaces that typically gather large groups of people indoors, and are considered focal points for the spread of the virus. For society to be able to go “back to normal”, it is crucial to keep these places open and functioning. An understanding of the transmission modes occurring in these contexts is essential to set up effective infection control strategies. This understanding was made using a systematic review, according to the Preferred Reporting Items for Systematic reviews and Meta-Analyses statement (PRISMA) 2020 guidelines. We analyze the different parameters influencing airborne transmission indoors, the mathematical models proposed to understand it, and discuss how we can act on these parameters. Methods to judge infection risks through the analysis of the indoor air quality are described. Various mitigation measures are listed, and their efficiency, feasibility, and acceptability are ranked by a panel of experts in the field. Thus, effective ventilation procedures controlled by CO2-monitoring, continued mask wearing, and a strategic control of room occupancy, among other measures, are put forth to enable a safe return to these essential places.

Escherichia coli K1 is a leading cause of neonatal meningitis. The asymptomatic carriage of these strains in the maternal intestinal microbiota constitutes a risk of vertical transmission to the infant at birth. The aim of this work was to evaluate the efficacy of phage therapy against E. coli K1 in an intestinal environment and its impact on the intestinal microbiota. For this purpose, three independent experiments were conducted on the SHIME® system, the first one with only the phage vB_EcoP_K1_ULINTec4, the second experiment with only E. coli K1 and the last experiment with both E. coli K1 and the phage. Microbiota monitoring was performed using metagenetics, qPCR, SCFA analysis and the induction of AhR. The results showed that phage vB_EcoP_K1_ULINTec4, inoculated alone, was progressively cleared by the system and replicates in the presence of its host. E. coli K1 persisted in the microbiota but decreased in the presence of the phage. The impact on the microbiota was revealed to be donor dependent, and the bacterial populations were not dramatically affected by vB_K1_ULINTec4, either alone or with its host. In conclusion, these experiments showed that the phage was able to infect the E. coli K1 in the system but did not completely eliminate the bacterial load.

Phage therapy is recognized to be a promising alternative to fight antibiotic-resistant infections. In the quest for oral dosage forms containing bacteriophages, the utilization of colonic-release Eudragit® derivatives has shown potential in shielding bacteriophages from the challenges encountered within the gastrointestinal tract, such as fluctuating pH levels and the presence of digestive enzymes. Consequently, this study aimed to develop targeted oral delivery systems for bacteriophages, specifically focusing on colon delivery and employing Eudragit® FS30D as the excipient. The bacteriophage model used was LUZ19. An optimized formulation was established to not only preserve the activity of LUZ19 during the manufacturing process but also ensure its protection from highly acidic conditions. Flowability assessments were conducted for both capsule filling and tableting processes. Furthermore, the viability of the bacteriophages remained unaffected by the tableting process. Additionally, the release of LUZ19 from the developed system was evaluated using the Simulator of the Human Intestinal Microbial Ecosystem (SHIME®) model. Finally, stability studies demonstrated that the powder remained stable for at least 6 months when stored at +5 °C.

The present study compared multiplex PCR (mPCR) and Whole Genome Sequencing (WGS) using the SCCmecFinder database to identify the Staphylococcal Cassette Chromosome (SCC) mec in five Staphylococcus aureus (SA) and nine non-aureus staphylococci (NAS) isolated from dairy cattle. mPCR identified an SCCmecIV in four SA and one NAS, but could not differentiate between SCCmecII and IV in the fifth SA, that all harbored the mecA gene and were phenotypically resistant to cefoxitin. SCCmecFinder confirmed the presence of an SCCmecIVc(2B) in four SA and of the SCCmecIVa(2B) in the fifth SA and the one NAS. Both methods also detected one untypeable SCCmec in another cefoxitin-resistant NAS harboring the mecA gene and a pseudo SCCmec in one cefoxitin-sensitive NAS harboring one mecC-related gene. No SCCmec elements were identified either in one cefoxitin-sensitive NAS harboring the mecA2 gene, or in five NAS (one resistant and four sensitive to cefoxitin) harboring the mecA1 gene. SCCmecFinder could even not identify the presence of any mecA1 gene in these five NAS, whose presence was nevertheless confirmed by ResFinder. The conclusions of this study are: (i) mPCR and WGS sequencing using SCCmecFinder are complementary methodologies to identify SCCmec; (ii) SCCmecFinder and ResFinder to a lesser extent cannot identify all mec gene allotypes; (iii) a specific classification of the SCCmec in NAS would be epidemiologically helpful; (iv) presence of a mecA gene and a complete SCCmec is linked to cefoxitin resistance, whereas presence of other mec genes and of pseudo or no SCCmec is not.

The Belgian Society for Viruses of Microbes (BSVoM) was founded on 9 June 2022 to capture and enhance the collaborative spirit among the expanding community of microbial virus researchers in Belgium. The sixteen founders are affiliated to fourteen different research entities across academia, industry and government. Its inaugural symposium was held on 23 September 2022 in the Thermotechnical Institute at KU Leuven. The meeting program covered three thematic sessions launched by international keynote speakers: (1) virus–host interactions, (2) viral ecology, evolution and diversity and (3) present and future applications. During the one-day symposium, four invited keynote lectures, ten selected talks and eight student pitches were given along with 41 presented posters. The meeting hosted 155 participants from twelve countries.

The invasiveness properties of Shigatoxigenic and enteropathogenic Escherichia coli (STEC and EPEC) O80:H2 in humans and calves are encoded by genes located on a pS88-like ColV conjugative plasmid. The main objectives of this study in larvae of the Galleria mellonella moth were therefore to compare the virulence of eight bovine STEC and EPEC O80:H2, of two E. coli pS88 plasmid transconjugant and STX2d phage transductant K12 DH10B, of four E. coli O80:non-H2, and of the laboratory E. coli K12 DH10B strains. Thirty larvae per strain were inoculated in the last proleg with 10 μL of tenfold dilutions of each bacterial culture corresponding to 10 to 106 colony-forming units (CFUs). The larvae were kept at 37 °C and their mortality rate was followed daily for four days. The main results were that: (i) not only the STEC and EPEC O80:H2, but also different E. coli O80:non-H2 were lethal for the larvae at high concentrations (from 104 to 106 CFU) with some variation according to the strain; (ii) the Stx2d toxin and partially the pS88 plasmid were responsible for the lethality caused by the E. coli O80:H2; (iii) the virulence factors of E. coli O80:non-H2 were not identified. The general conclusions are that, although the Galleria mellonella larvae represent a useful first-line model to study the virulence of bacterial pathogens, they are more limited in identifying their actual virulence properties.

The objective of this work was to evaluate the antimicrobial resistant (AR) E. coli prevalence in recreational waters in Belgium and to assess the exposure risk for bathers. Nine stations were sampled during the 2021 bathing season. A total of 912 E. coli strains were isolated and tested by the disk diffusion method in accordance with EUCAST recommendations, including Extended-Spectrum Beta-Lactamase (ESBL) production. AR E. coli were counted at each bathing sites, 24% of strains were resistant to at least one antibiotic and 6% were Multi-Drug Resistant (MDR). A Multiple Antibiotic Resistance (MAR) index was calculated to compare the bathing sites. The Lesse river had the highest MAR index as well as the highest E. coli absolute abundance and the largest number of ESBL-producing E. coli. Conversely, the 3 lakes showed lower E. coli contamination levels and AR rates. A human health risk assessment of exposure to AR E. coli, based on the calculation of measured prevalence, was performed considering four different dose-response model scenarios. The human health risk (Pd) ranged from 10-9 to 0.183 (children). The exposure probabilities were low, except for scenario 3 (E. coli O157:H7), which is the most severe.

New control methods are needed to counter antimicrobial resistances and the use of bacteriophages as an alternative treatment seems promising. To that end, the effect of the phage vB_KpnP_K1-ULIP33, whose host is the hypervirulent Klebsiella pneumoniae SA12 (ST23 and capsular type K1), was assessed on intestinal microbiota, using an in vitro model: the SHIME® system (Simulator of the Human Intestinal Microbial Ecosystem). After stabilization of the system, the phage was inoculated for 7 days and its persistence in the different colons was studied until its disappearance from the system. The concentration of short chain fatty acids in the colons showed good colonization of the bioreactors by the microbiota and no significant effect related to the phage treatment. Diversity (α and β), the relative abundance of bacteria, and qPCR analysis targeting different genera of interest showed no significant variation following phage administration. Even if further in vitro studies are needed to assess the efficacy of this phage against its bacterial host within the human intestinal ecosystem, the phage ULIP33 exerted no significant change on the global colonic microbiota.