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.

Risk of zoonoses involving slow-growing non-tuberculous mycobacteria: Survey of antimicrobial resistance among strains from domestic and wild animals.

OBJECTIVES: Non-tuberculous mycobacteria are opportunistic pathogens that cause disease mainly in immunocompromised hosts. The present study assessed the prevalence of antibiotic resistance among such mycobacteria from domestic and wild animals in Croatia sampled during several years within a national surveillance program. METHODS: A total of 44 isolates belonging to nine slow-growing species were genotyped and analyzed for susceptibility to 13 antimicrobials often used to treat non-tuberculous mycobacterial infections in humans. RESULTS: Most prevalent resistance was to moxifloxacin (77.3%), doxycycline (76.9%), and rifampicin (76.9%), followed by ciprofloxacin (65.4%), trimethoprim-sulfamethoxazole (65.4%), and linezolid (61.4%). Few isolates were resistant to rifabutin (7.7%) or amikacin (6.8%). None of the isolates was resistant to clarithromycin. Nearly all isolates (86.4%) were resistant to multiple antibiotics. CONCLUSION: Our findings suggest substantial risk that human populations may experience zoonotic infections with non-tuberculous mycobacteria that will be difficult to treat using the current generation of antibiotics. Future work should clarify how resistance emerges in wild populations of non-tuberculous mycobacteria.

Circulation of SARS-CoV-Related Coronaviruses and Alphacoronaviruses in Bats from Croatia.

Bats are natural hosts of various coronaviruses (CoVs), including human CoVs, via an assumed direct zoonotic spillover or intermediate animal host. The present study aimed to investigate the circulation of CoVs in a bat colony in the Mediterranean region of Croatia. Guano and individual droppings from four bat species were sampled and tested with the E-gene sarbecovirus RT-qPCR, the pan-CoV semi-nested RT-PCR targeting the RdRp gene and NGS. Furthermore, bat blood samples were investigated for the presence of sarbecovirus-specific antibodies with the surrogate virus neutralization test (sVNT). The initial testing showed E-gene Sarebeco RT-qPCR reactivity in 26% of guano samples while the bat droppings tested negative. The application of RdRp semi-nested RT-PCR and NGS revealed the circulation of bat alpha- and betaCoVs. Phylogenetic analysis confirmed the clustering of betaCoV sequence with SARS-CoV-related bat sarbecoviruses and alpha-CoV sequences with representatives of the Minunacovirus subgenus. The results of sVNT show that 29% of bat sera originated from all four species that tested positive. Our results are the first evidence of the circulation of SARS-CoV-related coronaviruses in bats from Croatia.

Description of Mycobacterium pinniadriaticum sp. nov., isolated from a noble pen shell (Pinna nobilis) population in Croatia.

Introduction: Shortly before the mass mortality event of the noble pen shell (Pinna nobilis) population in the south-eastern Adriatic coast, two rapidly growing Mycobacterium strains CVI_P3T (DSM 114013 T, ATCC TSD-295 T) and CVI_P4 were obtained from the organs of individual mollusks during the regular health status monitoring. Methods: The strains were identified as members of the genus Mycobacterium using basic phenotypic characteristics, genus-specific PCR assays targeting the hsp65 and 16S rRNA genes and the commercial hybridization kit GenoType Mycobacterium CM (Hain Lifescience, Germany). MALDI-TOF mass spectrometry did not provide reliable identification using the Bruker Biotyper Database. Results and discussion: Genome-wide phylogeny and average nucleotide identity (ANI) values confirmed that the studied strains are clearly differentiated from their closest phylogenetic relative Mycobacterium aromaticivorans and other validly published Mycobacterium species (ANI ≤ 85.0%). The type strain CVI_P3T was further characterized by a polyphasic approach using both phenotypic and genotypic methods. Based on the phenotypic, chemotaxonomic and phylogenetic results, we conclude that strains CVI_P3T and CVI_P4 represent a novel species, for which the name Mycobacterium pinniadriaticum sp. nov. is proposed.

Antimicrobial resistance profiles of human Brucella melitensis isolates in three different microdilution broths: the first multicentre study in Bosnia and Herzegovina.

OBJECTIVES: Brucellosis is a ubiquitous emergent bacterial zoonotic disease causing significant human morbidity in Bosnia and Herzegovina. So far, a high rate of resistant Brucella has been found worldwide. This study prospectively analysed the rates of resistance among human Brucella melitensis strains isolated in Bosnia and Herzegovina. METHODS: This study included 108 B. melitensis isolates from 209 patients diagnosed at five medical centres in Bosnia and Herzegovina. The resistance profiles of the B. melitensis isolates for the 13 most commonly used antimicrobials were studied in standard Brucella broth (BB) and cation-adjusted Mueller-Hinton broth (CAMHB) supplemented with 4% lysed horse blood or 5% defibrinated sheep blood. RESULTS: Of the 209 patients, B. melitensis blood cultures were positive for 111 (53.1%). Among the 108 isolates investigated, 91 (84.3%) were resistant to trimethoprim-sulfamethoxazole on BB, but not on either CAMHB. Nearly all isolates (>90%) were resistant to azithromycin on BB and both CAMHBs. CONCLUSION: We observed a high rate of B. melitensis resistance to azithromycin. The high rate of resistance to trimethoprim-sulfamethoxazole that we observed was related to BB, so an alternative broth should be used, such as the enriched CAMHBs in this study, for evaluating resistance to trimethoprim-sulfamethoxazole. Whole-genome sequencing studies are needed to understand the development of antimicrobial resistance in B. melitensis strains isolated from humans.

SARS-CoV-2 circulation in Croatian wastewaters and the absence of SARS-CoV-2 in bivalve molluscan shellfish.

The circulation of SARS-CoV-2 in the environment has been confirmed numerous times, whilst research on the bioaccumulation in bivalve molluscan shellfish (BMS) has been rather scarce. The present study aimed to fulfil the knowledge gap on SARS-CoV-2 circulation in wastewaters and surface waters in this region and to extend the current knowledge on potential presence of SARS-CoV-2 contamination in BMS. The study included 13 archive wastewater and surface water samples from the start of epidemic and 17 influents and effluents from nine wastewater treatment plants (WWTP) of different capacity and treatment stage, sampled during the second epidemic wave. From that period are the most of 77 collected BMS samples, represented by mussels, oysters and warty venus clams harvested along the Dalmatian coast. All samples were processed according to EN ISO 15216-1 2017 using Mengovirus as a whole process control. SARS-CoV-2 detection was performed by real-time and conventional RT-PCR assays targeting E, N and nsp14 protein genes complemented with nsp14 partial sequencing. Rotavirus A (RVA) real-time RT-PCR assay was implemented as an additional evaluation criterion of virus concentration techniques. The results revealed the circulation of SARS-CoV-2 in nine influents and two secondary treatment effluents from eight WWTPs, while all samples from the start of epidemic (wastewaters, surface waters) were negative which was influenced by sampling strategy. All tertiary effluents and BMS were SARS-CoV-2 negative. The results of RVA amplification were beneficial in evaluating virus concentration techniques and provided insights into RVA dynamics within the environment and community. In conclusion, the results of the present study confirm SARS-CoV-2 circulation in Croatian wastewaters during the second epidemic wave while extending the knowledge on wastewater treatment potential in SARS-CoV-2 removal. Our findings represent a significant contribution to the current state of knowledge that considers BMS of a very low food safety risk regarding SARS-CoV-2.

The Expression Pattern of Surface Markers in Canine Adipose-Derived Mesenchymal Stem Cells.

The influence of cultivation on the expression pattern of canine adipose-derived mesenchymal stem cells (cAD-MSCs) surface markers, contributing to, among others, the promotion of growth, proliferation, differentiation and immunomodulatory mechanisms of an excellent therapeutic, is still unknown. To fill the gap, we investigated CD90, CD44, CD73, CD29, CD271, CD105, CD45 and CD14 patterns of expression at the protein level with flow cytometry and mRNA level using a real-time polymerase chain reaction array. Gentle variations of expression occurred during cultivation, along with increased CD90, CD44 and CD29 expression, low and decreasing CD271 and CD73 expression and a decrease of initially high CD105. As expected, CD45 and CD14 were not expressed by cAD-MSCs. Interestingly, we discovered a significant decrease of CD73 expression, compared to early (P1-P3) to late (P4-P6) passages, although the CD73 gene expression was found to be stable. The percentage of positive cells was found to be higher for all positive markers up to P4. As CD73's one important feature is a modulation from a pro-inflammatory environment to an anti-inflammatory milieu, the expression of CD73 in our conditions indicate the need to consider the time cells spend in vitro before being transplanted into patients, since it could impact their favourable therapeutical properties.

Investigating the Presence of SARS CoV-2 in Free-Living and Captive Animals.

Due to SARS CoV-2 recombination rates, number of infected people and recent reports of environmental contamination, the possibility of SARS CoV-2 transmission to animals can be expected. We tested samples of dominant free-living and captive wildlife species in Croatia for the presence of anti-SARS CoV-2 antibodies and viral RNA. In total, from June 2020 until February 2021, we tested blood, muscle extract and fecal samples of 422 free-living wild boars (Sus scrofa), red foxes (Vulpes vulpes) and jackals (Canis aureus); blood and cloacal swabs of 111 yellow-legged gulls (Larus michahellis) and fecal samples of 32 zoo animals. A commercially available ELISA (ID.Vet, France) and as a confirmatory test, a surrogate virus neutralization test (sVNT; GenScript, Netherlands) were used. Fecal samples were tested for the presence of viral RNA by a real-time RT-PCR protocol. Fifteen out of 533 (2.8%) positive ELISA results were detected; in wild boars (3.9%), red foxes (2.9%) and jackals (4.6%). However, the positive findings were not confirmed by sVNT. No viral RNA was found. In conclusion, no spillover occurred within the investigated period (second COVID-19 wave). However, further investigation is needed, especially regarding wildlife sample features for serological tests.

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.

First evidence of hepatitis E virus infection in a small mammal (yellow-necked mouse) from Croatia.

Since the role of wild rodents/small mammals in hepatitis E virus (HEV) epidemiology has been a subject of considerable debate, this study was conducted to investigate the potential presence of HEV RNA in small rodents collected within their natural habitats and to detect if they can be potential reservoirs of the virus. A total of 483 small rodents were captured using snap traps placed at 11 regions in Croatia. Sampling was undertaken in 2008 and repeated from 2010 to 2014. Liver samples were tested for the presence of HEV RNA. HEV RNA was detected in only one liver sample (0.21%) originated from Apodemus flavicollis from the location Medvednica, nearby Zagreb collected in 2014. According to the sequence analysis, the isolate has shown to be a member of Orthohepevirus A species, genotype HEV-3. The genotyping results confirmed grouping into subtype 3a, general cluster 3abchij.The detected HEV strain showed to be genetically highly related to strains found in humans and/or domestic pigs and wild boars from Croatia. Our finding indicates that wild small mammals could play a role in the epidemiology of HEV-3 infection and therefore should be taken under consideration as potential reservoirs or/and transmitters of the disease. However, further investigation is needed to recognize their potential for maintaining the infection in natural conditions.

Melitococcosis in the republic of croatia.

BACKGROUND: Melitococcosis is one of the most widespread zoonoses worldwide. In the period from 2009 to 2013, comprehensive melitococcosis testing was conducted in the Republic of Croatia. METHODS AND RESULTS: During the testing, the Rose Bengal test was applied to 344019 blood samples of sheep and goats, and positive reactions were confirmed in 1143 (0.3%) of samples. The complement fixation test (confirmatory test) was conducted on 43428 samples, with positive reactions confirmed in 768 (1.8%) of samples. The organs and tissues of 336 sheep and goats were inspected bacteriologically, and Brucella sp. was isolated in 15 (4.5%) of samples. Positive serological and bacteriological reactions were confirmed in the Karlovac, Lika-Senj and Split-Dalmatia Counties. Bacteriological and molecular techniques (Bru-up/Bru-low and Bruce-Ladder) in isolates proved the presence of Brucella melitensis biovar 3. CONCLUSION: On the basis of this study, it can be concluded that Croatia has a favourable situation concerning the infection of ruminants with B. melitensis, and that ongoing controls of the disease are necessary.

Identification of Coxiella burnetii genotypes in Croatia using multi-locus VNTR analysis.

Although Q fever affects humans and animals in Croatia, we are unaware of genotyping studies of Croatian strains of the causative pathogen Coxiella burnetii, which would greatly assist monitoring and control efforts. Here 3261 human and animal samples were screened for C. burnetii DNA by conventional PCR, and 335 (10.3%) were positive. Of these positive samples, 82 were genotyped at 17 loci using the relatively new method of multi-locus variable number tandem repeat analysis (MLVA). We identified 13 C. burnetii genotypes not previously reported anywhere in the world. Two of these 13 genotypes are typical of the continental part of Croatia and share more similarity with genotypes outside Croatia than with genotypes within the country. The remaining 11 novel genotypes are typical of the coastal part of Croatia and show more similarity to one another than to genotypes outside the country. Our findings shed new light on the phylogeny of C. burnetii strains and may help establish MLVA as a standard technique for Coxiella genotyping.

The presence of methicillin-resistant Staphylococcus aureus on large pig breeding farms in Croatia.

Methicillin-resistant Staphylococcus aureus (MRSA) have emerged worldwide and have become resistant to a variety of antibiotics. MRSA colonisation in pigs was first reported from the Netherlands in 2005, where pigs were implicated as a source of human MRSA infections (Voss et al., 2005). This paper presents the first report on the presence of MRSA on large pig breeding farms in Croatia, together with the determination of the mecA gene, the results of spa typing and susceptibility to commonly used antimicrobials. Dust samples (7-11 per farm) were collected from eight large pig farms in Croatia. Of the total 68 swabs, the mecA gene was detected in 24 isolates growing on the MRSA agar. All isolates were resistant to oxacillin, tetracycline and streptomycin, and susceptible only to vancomycin, while 92% of the strains were susceptible to ciprofloxacin. Genotyping of the MRSA strains was performed by spa typing, and revealed t011 (n = 17), t034 (n = 5) and t1451 (n = 2). The results presented here predict that MRSA is present on a large number of pig farms in Croatia.

Molecular survey of Babesia microti in wild rodents in central Croatia.

Babesia divergens and B. divergens-like organisms are the main causative agents of human babesiosis in Europe. Recently, the first case of human infection with Babesia microti was confirmed in Germany, implicating the presence of zoonotic isolates. To estimate the presence of zoonotic B. microti in Croatia we analyzed 120 small wild mammals that serve as its reservoir by polymerase chain reaction. Yellow-necked mice (Apodemus flavicollis) and bank voles (Myodes glareolus) were both found to be infected with prevalence of 16.2%. Sequence analysis of the portion of 18S rDNA gene demonstrated that six polymerase chain reaction-positive samples, detected in both rodent species, were identical to that of the human Jena/Germany strain (EF413181). The other two isolates were identical to the nonzoonotic Munich strain (AB071177). The results of this study indicate the presence of zoonotic B. microti in A. flavicollis and M. glareolus in Croatia and a potential risk for human health.