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.
Shortly before the mass mortality event of the noble pen shell (
) population in the south-eastern Adriatic coast, two rapidly growing
strains CVI_P3
(DSM 114013 T, ATCC TSD-295 T) and CVI_P4 were ...obtained from the organs of individual mollusks during the regular health status monitoring.
The strains were identified as members of the genus
using basic phenotypic characteristics, genus-specific PCR assays targeting the
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.
Genome-wide phylogeny and average nucleotide identity (ANI) values confirmed that the studied strains are clearly differentiated from their closest phylogenetic relative
and other validly published
species (ANI ≤ 85.0%). The type strain CVI_P3
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_P3
and CVI_P4 represent a novel species, for which the name
sp. nov. is proposed.
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.
The bacterial species
is the most common causative agent of mastitis in cows in most countries with a dairy industry. The prevalence of infection caused by
ranges from 2% to more than 50%, and it ...causes 10-12% of all cases of clinical mastitis.
The objective was to analyze 237 strains of
isolated from the milk of cows with subclinical mastitis regarding the
,
and
genes and to perform
and multi-locus sequence typing (MLST).
Sequencing amplified gene sequences was conducted at Macrogen Europe. Ridom StaphType and BioNumerics software was used to analyze obtained sequences of
and seven housekeeping genes.
The
fragment was present in 204 (86.1%) of strains, while
and
gene were detected in 10 strains, and the
gene was not detected.
typing successfully analyzed 153 tested isolates (64.3%), confirming 53
types, four of which were new types. The most frequent
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.
Taking the number of confirmed
types and sequence types (STs) into account, it can be concluded that the strains of
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.
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.
•Resistance is prevalent among slow growing nontuberculous mycobacteria from animals.•Nearly all isolates showed multidrug resistance.•Humans are at risk of zoonosis that may be difficult to treat ...with current therapy.
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.
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.
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.
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.
Importance of systematic mastitis control in dairy herds is described through the presentation of data concerning mastitis occurrence and mastitis significance in modern milk production. The research ...was carried out by farm visits and by taking quarter samples from all lactating cows at the time of visit. Samples were taken before evening milking. Each quarter sample was tested by Zagreb mastitis test (ZMT). Furthermore, each sample was examined bacteriologically by inoculation on aesculin blood agar. Identification of grown colonies was carried out using internationally accepted methodology. Obtained results were statistically analyzed using Stata 13.1 statistical package. Quarter samples from 385 cows were analyzed. ZMT -positive reactions were noticed in 13.7% of all quarters. Mastitis pathogens were isolated from 175 (13%) of quarter samples. 145 out of 385 cows (37.3%) included in research had at least one ZMT positive quarter or permanently lost (dried off) quarter. From 106 out of 363 cows (29.8%) with all four functional quarters some of mastitis pathogens were isolated. The most frequently isolated pathogens were Staphylococcus aureus, streptococci, Trueperella (Arcanobacterium) pyogenes and Corynebacterium bovis. There was no statistical difference in mastitis occurrence between front and rear mammary quarters. ZMT results and microbiological examination were moderately correlated (Kappa index = 0.4662).
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.
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
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.
•Bosnia and Herzegovina is an endemic for brucellosis in Southeastern Europe.•B. melitensis shows low rates of resistance to commonly used antimicrobials.•A high rate of resistance was observed with ...azithromycin in all three broth media.•The high rate of Brucella resistance to trimethoprim-sulfamethoxazole (T/S) is abolished in alternative broths.•Thymine- and thymidine-depleted broths should be used to test susceptibility for T/S.
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.
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.
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.
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.
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