Background: The bacterial species S. aureus is the most common causative agent of mastitis in cows in most countries with a dairy industry. The prevalence of infection caused by S. aureus ranges from 2% to more than 50%, and it causes 10–12% of all cases of clinical mastitis. Aim: The objective was to analyze 237 strains of S. aureus isolated from the milk of cows with subclinical mastitis regarding the spa, mecA, mecC and pvl genes and to perform spa and multi-locus sequence typing (MLST). Methods: Sequencing amplified gene sequences was conducted at Macrogen Europe. Ridom StaphType and BioNumerics software was used to analyze obtained sequences of spa and seven housekeeping genes. Results: The spa fragment was present in 204 (86.1%) of strains, while mecA and mecC gene were detected in 10 strains, and the pvl gene was not detected. Spa typing successfully analyzed 153 tested isolates (64.3%), confirming 53 spa types, four of which were new types. The most frequent spa type was t2678 (14%). MLST typed 198 (83.5%) tested strains and defined 32 different allele profiles, of which three were new. The most frequent allele profile was ST133 (20.7%). Six groups (G) and 15 singletons were defined. Conclusion: Taking the number of confirmed spa types and sequence types (STs) into account, it can be concluded that the strains of S. aureus isolated from the milk of cows with subclinical mastitis form a heterogenous group. To check the possible zoonotic potential of isolates it would be necessary to test the persons and other livestock on the farms.

This study’s objective was to estimate the seasonal occurrence of aflatoxin M₁ (AFM₁) in cow’s milk between winter 2016 and winter 2022 and to assess dietary exposure and risk assessment for the adult Croatian population. In total, 5817 cow milk samples were screened for AFM₁ concentrations using the enzyme immunoassay assay (ELISA). For confirmation purposes of AFM₁ concentration above the European Union maximum permitted level (MRL), ultra high-performance liquid chromatography with tandem mass spectrometry was performed. In 94.7% of milk samples, AFM₁ levels were below the detection limit (LOD) of the ELISA test. For 3.47% of samples, the AFM₁ was between the LOD and MRL values. Only 1.87% of all samples exceeded the MRL. The mean value of elevated AFM₁ in different seasons ranged between 59.2 ng/kg (autumn 2017) and 387.8 ng/kg (autumn 2021). The highest incidences of positive AFM₁ were determined in autumn and winter and the maximum (6.4%) was in winter 2019/2020. The largest percentage of positive samples (69.7%) was found in central Croatia. The estimated daily intakes for positive samples ranged between 0.17 and 2.82 ng/kg body weight/day. Risk assessment indicated a high level of concern during autumn and winter, especially for consumers of large amounts of milk.

Honeybees’ gut microbiota can provide new valuable access into the pathogenesis-related factors included in infections. Hence, we researched the presence and comparison of gut microbiota groups in control and Nosema spp.-infected honeybee colonies through high-throughput sequencing of the 16S rRNA. As the newest approach in apiary management, we hypothesize that the EM^® probiotic for bees could have an important role in therapeutic and immunomodulatory effects on honeybee colonies. The aim of this study was to estimate its impact on the gut microbiota composition of adult honeybees. The major genera were detected, where Lactobacillus was the most abundant genus, followed by Gilliamela, Snodgrassella, and Bifidobacterium. Inoculation with Nosema spp. spores made the relative proportions of Bifidobacterium lower, which was ameliorated by EM^® for bees’ application. In addition, EM^® for bee applied treatments suppressed the increase in the number of Nosema spp. spores. This result points out that continuous EM^® for bees treatment shall change bees’ gut microbiome composition and mitigate the influence of Nosema spp. infection. Snodgrassella alvi was a major member of the honeybee gut microbiota and may be significantly increased by long-term treatment with EM^® for bees. Toward these results, it is possible that EM^® for bees treatment will protect honeybees from herbicide glyphosate negative effects in agricultural fields by improving microbiome and immune functions.

In this study, the frequency of aflatoxin M1 (AFM1) occurrence in raw milk was investigated across different seasons over a three-year period from 2022 to 2024 in Croatia. Risk assessment was conducted using estimated daily intake (EDI), hazard index (HI), and margin of exposure (MOE) for various age groups and both genders. The frequency of AFM1 detection above the maximum level (ML) ranged from 1.60% to 15.1%. The average incidence of AFM1 exceeding the ML was 5.67%, with the highest incidence recorded in autumn 2024. AFM1 levels within the limit of detection (LOD) and ML were found in 13% of the samples. The average mean value of AFM1 over the three-year period was 19.2 ng/kg. The highest mean AFM1 EDI values were determined for toddlers (0.61–0.67 ng/kg bw/day) and children (0.41–0.43 ng/kg bw/day). The lowest EDI values were observed in elderly females and males (0.058–0.074 ng/kg bw/day). The EDI values for females and males were slightly different. The risk assessment, based on the HI and MOE, indicated that toddlers and children are at the highest risk of exposure to AFM1, which raises significant health concerns. Additionally, consumers of large quantities of milk face a high risk of exposure, particularly during the spring and autumn seasons. For adults and the elderly, milk consumption does not pose a serious health risk.

Bacillus anthracis, the causative agent of anthrax disease, is a worldwide threat to livestock, wildlife and public health. It is also considered one of the most important pathogens of bioterrorism. Rapid and reliable diagnosis and administration of antimicrobials are essential for effective anthrax treatment. In this study, we determined the in vitro susceptibilities of 40 isolates of B. anthracis isolated in Croatia over the recent two decades to 18 antimicrobials. Whole-genome sequencing was performed, and bioinformatics tools were used to determine virulence factors and antimicrobial resistance genes. Core genome-based multilocus sequence typing was used for isolate comparison and phylogenetic analysis. All isolates were susceptible to all antimicrobials recommended for post-exposure prophylaxis or anthrax therapy. Susceptibility was found to all other tested antimicrobials that are an alternative for primary therapy. We found two beta-lactamase genes, but their expression is not sufficient to confer resistance. In all isolates used in this study, we found 21 virulence genes, 8 of which are responsible for toxin and capsule production. As far as phylogenetic analysis is concerned, the B. anthracis isolates from Croatia are categorised into two clades. The first is clade A, subclade Trans Eurasia, and the other is clade B, subclade B2.

The emergence and rapid spread of the plasmid-mediated colistin-resistant mcr-1 gene introduced a serious threat to public health. In 2021, a multi-drug resistant, mcr-1 positive Escherichia coli EC1945 strain, was isolated from pig caecal content in Croatia. Antimicrobial susceptibility testing and whole genome sequencing were performed. Bioinformatics tools were used to determine the presence of resistance genes, plasmid Inc groups, serotype, sequence type, virulence factors, and plasmid reconstruction. The isolated strain showed phenotypic and genotypic resistance to nine antimicrobial classes. It was resistant to colistin, gentamicin, ampicillin, cefepime, cefotaxime, ceftazidime, sulfamethoxazole, chloramphenicol, nalidixic acid, and ciprofloxacin. Antimicrobial resistance genes included mcr-1, bla_TEM-1B, bla_CTX-M-1, aac(3)-IId, aph(3’)-Ia, aadA5, sul2, catA1, gyrA (S83L, D87N), and parC (A56T, S80I). The mcr-1 gene was located within the conjugative IncX4 plasmid. IncI1, IncFIB, and IncFII plasmids were also detected. The isolate also harbored 14 virulence genes and was classified as ST744 and O101:H10. ST744 is a member of the ST10 group which includes commensal, extraintestinal pathogenic E. coli isolates that play a crucial role as a reservoir of genes. Further efforts are needed to identify mcr-1-carrying E. coli isolates in Croatia, especially in food-producing animals to identify such gene reservoirs.