First Report and Characterization of the mcr-1 Positive Multidrug-Resistant Escherichia coli Strain Isolated from Pigs in Croatia.

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, blaTEM-1B, blaCTX-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.

Influence of Probiotic Feed Supplement on Nosema spp. Infection Level and the Gut Microbiota of Adult Honeybees (Apis mellifera L.).

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.

Seasonal Occurrence of Aflatoxin M1 in Raw Milk during a Five-Year Period in Croatia: Dietary Exposure and Risk Assessment.

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.

Multi Locus Sequence Typing and spa Typing of Staphylococcus Aureus Isolated from the Milk of Cows with Subclinical Mastitis in Croatia.

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.

Estimation of the Withdrawal Time of Levamisole in Eggs after Oral Administration to Laying Hens.

This study was conducted to evaluate withdrawal time of levamisole in eggs after oral administration in laying hens at different doses. Sampling of eggs was conducted for 37 days after the end of treatment, and levamisole concentrations were measured by liquid chromatography-tandem mass spectrometry validated according to the Commission Decision 2002/657/EC. Estimated validation parameters were as follows: decision limit, 0.54 µg/kg; detection capability, 0.56 µg/kg; limit of detection, 0.04 µg/kg; limit of quantification, 0.15 µg/kg; accuracy (recovery), between 92.9 and 102.3%; precision (relative standard deviation), ≤4.62%; and within-laboratory precision (relative standard deviation), ≤5.19%. Levamisole residue levels were significantly higher in egg yolks than in egg whites. The highest levels of levamisole were detected on day 2 posttreatment in groups receiving 50 mg/kg of body weight (556.2 µg/kg in egg yolks and 166.5 µg/kg in egg whites). Significant elimination occurred within 5 days after the cessation of treatment in all groups, with an elimination half-life of 1.3 days. Levamisole was still detectable on day 30 after the end of treatment in egg whites (0.06 µg/kg) and on day 37 in egg yolks (0.06 µg/kg). The longest withdrawal time for levamisole in eggs (14.9 days) was determined in a group treated with 25 mg of levamisole per kg of body weight for two consecutive days. According to the results, oral treatment of laying hens with levamisole may result in noncompliant egg samples even 14 days after treatment.

Distribution of sulfamonomethoxine and trimethoprim in egg yolk and white.

The distribution of sulfamonomethoxine (SMM) and trimethoprim (TMP) in egg yolk and white was measured during and after administration of a SMM/TMP combination in laying hens in doses of 8 g l(-)(1) and 12 g l(-)(1) in drinking water for 7 days. The SMM concentration reached maximal levels on day 2 of the post-treatment period for both doses (µg kg(-)(1)): 5920 and 9453 in yolk; 4831 and 6050 in white, in doses 1 and 2, respectively. Significant differences in the SMM and TMP concentrations between yolk and white in post treatment period were found. SMM dropped below the LOD (1.9 µg kg(-1)) in yolk after day 16 and 19 for doses 1 and 2. TMP reached maximal levels on day 3 after drug administration for doses 1 and 2 (µg kg(-)(1)): 6521 and 7329 in yolk, 1370 and 1539 in white. TMP residues were measured above LOD (0.3 µg kg(-)(1)) in yolk for both doses on day 37 post-treatment.

Deposition and depletion of maduramicin residues in eggs after oral administration to laying hens determined by LC-MS.

The coccidiostat maduramicin has been approved as a feed additive for chickens and turkeys, although it is prohibited for use in laying hens. In the present study, laying hens were divided into three groups and fed for 14 days with medicated feed containing maduramicin, at three different concentrations: 50, 100 and 500 µg kg(-1). Eggs were collected during treatment and for 26 days after the end of feeding with medicated feed. Maduramicin residues were found exclusively in egg yolk, with the highest concentration in egg yolk of 459 µg kg(-1) for the highest dose. The maximum concentration of maduramicin in whole egg was 16.6 µg kg(-1) for the group receiving feed containing the maximum permitted level of maduramicin in feed (50 µg kg(-1)). The half-life of elimination of maduramicin, calculated for post-treatment days 1-10, was 6.5 days. Twelve days after drug administration, the concentration of the maduramicin in egg yolk for Group 3 (fed with 500 µg kg(-1) maduramicin) still exceeded 20 µg kg(-1), while the concentrations for Groups 1 and 2 were 1.2 and 2.7 µg kg(-1), respectively.