In order to detect thermotolerant Campylobacter spp., 241 samples of fresh chicken meat, at retail in Croatia, were analysed according to a standard method, followed by biochemical test and molecular polymerase chain reaction/restriction enzyme analysis for exact species determination. Campylobacter spp. prevalence was 73.86%. Campylobacter jejuni and Campylobacter coli were isolated from 53.53 and 15.35% of the samples, respectively. In 4.98% of isolates thermotolerant Campylobacter spp. were not determined. The multilocus sequence typing method was used to evaluate genetic diversity of eight Campylobacter jejuni and four Campylobacter coli isolates. To our knowledge, these results of genotyping provided the first data on the presence of sequence types (STs) and clonal complexes (CCs) of Campylobacter jejuni and C. coli isolates in Croatia. By applying the multilocus sequence typing, a new allele of tkt gene locus was discovered and marked tkt508. The C. jejuni ST 6182 and C. coli ST 6183 genotypes were described for the first time, and all other identified genotypes were clustered in the previously described sequence types and clonal complexes. These findings provide useful information on the prevalence and epidemiology of Campylobacter jejuni and C. coli in Croatia.

Campylobacter lari is a thermotolerant bacterium that sporadically causes gastrointestinal diseases in humans and can be found in wildlife and the environment. C. lari is an understudied species, especially in wild birds such as gulls. Gulls are potentially good carriers of pathogens due to their opportunistic behavior and tendency to gather in large flocks. During winter and their breeding period, 1753 gulls were captured, and cloacal swabs were taken to be tested for the presence of C. lari. From isolated bacteria, the DNA was sequenced, and sequence types (ST) were determined. Sixty-four swabs were positive for C. lari, and from those, forty-three different STs were determined, of which thirty-one were newly described. The whole genome was sequenced for 43 random isolates, and the same isolates were tested for antimicrobial susceptibility using the broth microdilution method to compare them to WGS-derived antimicrobial-resistant isolates. All the tested strains were susceptible to erythromycin, gentamicin, and chloramphenicol, and all were resistant to ciprofloxacin. Resistance to ciprofloxacin was attributed to a gyrA_2 T86V mutation. Genes connected to possible beta-lactam resistance (blaOXA genes) were also detected.

This study aimed to assess the presence of thermotolerant Campylobacter spp., as one of the most important foodborne zoonotic pathogens, in three shellfish species: mussels (Mytilus galloprovincialis), oysters (Ostrea edulis) and queen scallops (Aequipecten opercularis). The samples were collected from nine locations in the Istrian aquatory, Croatia. Isolation of Campylobacter was done according to standard ISO method, and species were identified using multiplex PCR. Isolates identified as C. jejuni and C. lari were genotyped using multilocus sequence typing (MLST) to determine the potential source of contamination. Among 108 examined samples of bivalve molluscs, mussels dominated and were the only ones found positive for the presence of Campylobacter (25.6%). In total, 19 C. lari and 1 C. jejuni strains were isolated. C. lari isolates found in this study belong to 13 sequence types (STs), and 9 of them are newly described in this paper. Two out of the four previously described C. lari STs that were found in this study were previously found in human stool. The only C. jejuni isolate was found to be sequence type 1268, which belongs to ST-1275 clonal complex that is almost exclusively found in seabirds and can sporadically cause infection in humans. Regarding the obtained results, introducing surveillance of thermotolerant Campylobacter in shellfish in the Republic of Croatia is advised as an improvement for public health safety.

Campylobacteriosis is the most common gastrointestinal bacterial disease in the European Union (EU). Wild birds are one of the natural reservoirs of these pathogens. In this study we tested cloacal swabs of 643 gulls captured on rubbish tip in Zagreb, Croatia for the presence of Campylobacter spp. and found 168 Campylobacter positive samples. We used multilocus sequence typing (MLST) to genotype 62 random C. jejuni isolates from gulls, 24 isolates from broiler caeca, 27 isolates from broiler neck skins and 23 human isolates. Altogether, we identified 44 different STs, from which 19 were newly described. Most of the new STs (14) originate from gulls. Although humans and broilers share the majority of STs and isolates from gulls are separated from these, there was one ST present in all three hosts: 45. Additionally antimicrobial susceptibility to six antimicrobials was performed on 123 C. jejuni strains isolated from broiler caeca (n = 22), neck skins of broilers (n = 20), gulls cloacal swabs (n = 50) and human faeces (n = 31) by the broth microdilution method. Results show lower resistance of gull isolates to NAL and CIP, while resistance to TET was as high as in human and broiler isolates.

Rapid and accurate serotyping of Salmonella (S.) enterica subsp. enterica serovars is essential for effective public health surveillance, outbreak control, and food safety management. Traditional serotyping, although considered the gold standard, is time-consuming, technically demanding, and costly. This study aimed to evaluate the applicability of artificial intelligence (AI)-enhanced Fourier-transform infrared (FT-IR) spectroscopy using an IR Biotyper (Bruker Daltonics, Bremen, Germany) for the rapid and accurate serotyping of Salmonella enterica subsp. enterica isolates in Croatia. Materials and Methods: A total of 143 isolates representing different S. enterica serovars of human and food origin were analysed using the IR Biotyper. Each strain was tested in three biological and at least three technical replicates. The obtained results were compared with traditional serotyping according to the Kauffmann–White–Le Minor scheme. Isolate identification at the genus level was further confirmed by MALDI-TOF mass spectrometry. Results: The IR Biotyper demonstrated high reproducibility and complete concordance with standard serotyping methods, enabling accurate differentiation of the most prevalent S. enterica serovars in Croatia. Conclusions: Our findings demonstrate the applicability of FT-IR in routine laboratory work, with the potential to reduce typing time, reduce the number of strains, and lower overall costs required for epidemiological surveillance within the One Health approach.

The aim of our study was to compare the composition of polyphenolic compounds between the Croatian acacia (Robinia pseudoacacia L.) and chestnut (Castanea sativa Mill.) honey from several aspects: production season, hive position (on the edge and in the middle of a series of hives), part of the hive (small or normal extension), and honey extraction method (centrifuging or draining honey combs). Additionally, in acacia honey, we also monitored the influence of different storage temperatures (room temperature (RT) and 4 °C) on the content of phenolic compounds. To separate, identify and quantify individual flavonoids and phenolic acids from the honey, we used the HPLC method. The total polyphenols and antioxidant activity of the samples, their antimicrobial activity and their elemental content were also measured. The significant influence of the season, hive position, and extraction method on the total identified phenolic compounds, phenolic acids, flavonoids, total phenols and antioxidant activity was detected in almost all the acacia and chestnut honey samples. Chestnut honey from 2013 had more total phenolics (TPs) and antioxidant capacity (FRAP) than chestnut from 2014 and 2015. Honey collected from smaller extensions of hives had significantly higher TPs and FRAP compared to normal hive extensions. Centrifugation reduced the TPs and FRAP in most cases, but not always uniformly. Storage at RT led to the predominance of gallic, p-coumaric and benzoic acid in acacia honey, while storage at 4 °C maintained p-coumaric acid as the dominant phenolic acid. Flavonoids, particularly pinobanksin in acacia honey and hesperetin/pinobanksin in chestnut honey, were less affected by the storage conditions compared to phenolic acids. The non-centrifuged chestnut sample from 2015 showed the lowest MIC values against the most tested pathogenic bacteria. All the honey samples showed an extremely low concentration of heavy metals and relatively high concentrations of potassium and calcium.