•We have genotyped Coxiella burnetii strains for the first time in Croatia.•For the genotyping we used MLVA.•We have identified 13 novel C. burnetii genotypes that appear to be unique to Croatia.
...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.
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•One of the highest prevalence of dleptospirosis in Europe with human incidence rate 1.53/100000.•Increased risk for human infections was related to defined parameters.•Variations in ...seroprevalence and changing trends in prevailing serogroups were recorded.•Importance of continuous monitoring with constant multidisciplinary communication.
Leptospirosis is a geographically widespread and globally underestimated zoonosis that affects humans and variety of animals. To identify trends and possible risk factors, joined medical and veterinary teams investigated epidemiology and epizootiology of leptospirosis in Croatia. Retrospective analysis of data obtained from referent diagnostic laboratories included a total of 1917 human and 123964 animal sera tested in the period from 2009 to 2014. We found high human leptospirosis average incidence rate of 1.53/100000 with clear predominance of male patients older than 40 years (sex ratio M/F:3.2; median age 51±15.1years). Statistical analysis revealed seasonal and annual variations of incidence in humans that were primarily associated with favourable weather conditions (temperature 10–19, 9°C and precipitation above 100mm/m2). Majority of infections in humans were caused by serogroups Sejroe, Australis and Icterohaemorrhagiae. Notable variations in seroprevalence and changing trends in prevailing serogroups were recorded in most of the domestic animals and during the entire period of investigation. All of the observed findings underline leptospirosis as a significant human and veterinary public health threat and emphasize the importance of continuous multidisciplinary surveillance. We also argue that only input from both professions improves our overall knowledge on leptospirosis and leads to better and more efficient prevention and control strategies.
Rod Brucella biološki je iznimno raznolik, ali genetski vrlo homogen rod bakterija te je već desetljećima znanstvenicima nepoznanica. Ove bakterije su veliki javno-zdravstveni problem, a osobito na ...Balkanu. Pravilno prepoznavanje i razumijevanje patogena ključan je korak u epidemiologiji i epizootiologiji bilo koje bakterijske vrste, čija identifikacija može biti izazovna, osobito u slučaju zoonotskih vrsta. Cilj je ovog rada bio implementirati sekvenciranje četvrte generacije u tipizaciji 11 sojeva Brucella suis koje se čuvaju u našoj arhivi te ovu metodu usporediti s klasičnim i molekularnim metodama koje se trenutačno primjenjuju, a ne zasnivaju se na sekvenciranju. Klasično je biotipiziranje vrlo subjektivno i dalo je podvojene rezultate za 3 soja. Od molekularnih metoda koristili smo višestruku lančanu reakciju polimerazom (engl. Polymerase Chain Reaction, PCR) i polimorfizam duljine restrikcijskih fragmenata (engl. Restriction Fragment Lenght Polymorphism, RFLP) budući da niti jedna od metoda ne može zasebno identificirati i vrstu i biovar, a što je važno u slučaju Brucella suis infekcije. Vrsta i biovar svih sojeva uspješno su potvrđene i u skladu s rezultatima biotipizacije. Sekvenciranje sljedeće generacije (engl. Next Generation Sequencing, NGS) provodili smo na Oxford Nanopore MinION uređaju koji sekvencira duge lance DNK. Za sastavljanje genoma rabljeni su različiti algoritmi, a za identifikaciju i analizu rezultata MLST-a korišten je softver BioNumerics 8.0. MLST 21 je korišten za identifikaciju biovara i epidemiološku usporedbu ispitivanih sojeva. Genomi su bili veličine 3,2 Mb i sastavljeni u dva kromosoma. Analiza MLST 21 smjestila je naše sojeve u vrsne i biovarne skupine u skladu s drugim korištenim molekularnim testovima. Koliko je nama poznato, ovo je prva dokumentirana uporaba sekvenciranja dugih lanaca DNK u identifikaciji Brucella suis u jugoistočnoj Europi. Zaključujemo da je bakteriološka biotipizacija zastarjela i da je identifikacija biovara u ovom rodu, ovisno o domaćinu, netočna te da je molekularna karakterizacija uvijek sigurnija, brža i prikladnija opcija. MinION sekvenciranje pokazalo se kao vrlo pristupačno rješenje za određivanje vrste i biovara Brucella suis. Daljnja su istraživanja potrebna da bi se ustvrdilo koliko detaljne informacije o genomu može dati, imajući u vidu značajniji postotak pogreške prilikom sekvenciranja.
A novel Brucella strain closely related to Brucella (B.) melitensis biovar (bv) 3 was found in Croatian cattle during testing within a brucellosis eradication programme.
Standardised serological, ...brucellin skin test, bacteriological and molecular diagnostic screening for Brucella infection led to positive detection in one dairy cattle herd. Three isolates from that herd were identified to species level using the Bruce ladder method. Initially, two strains were typed as B. melitensis and one as B. abortus, but multiplex PCR based on IS711 and the Suis ladder showed that all of them to belong to B. melitensis, and the combination of whole-genome and multi-locus sequencing as well as Multi-Locus Variable numbers of tandem repeats Analysis (MLVA) highlighted a strong proximity within the phylogenetic branch of B. melitensis strains previously isolated from Croatia, Albania, Kosovo and Bosnia and Herzegovina. Two isolates were determined to be B. melitensis bv. 3, while the third showed a unique phylogenetic profile, growth profile on dyes and bacteriophage typing results. This isolate contained the 609-bp omp31 sequence, but not the 723-bp omp31 sequence present in the two isolates of B. melitensis bv. 3.
Identification of a novel Brucella variant in this geographic region is predictable given the historic endemicity of brucellosis. The emergence of a new variant may reflect a combination of high prevalence among domestic ruminants and humans as well as weak eradication strategies. The zoonotic potential, reservoirs and transmission pathways of this and other Brucella variants should be explored.
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.
To present the surveillance data on Brucella melitensis, B. suis, and B. ovis infection in cattle, sheep, goats, and swine in Croatia obtained in 2008 by serological, bacteriological, and molecular ...methods for diagnostics of brucellosis in domestic animals.
We serologically tested 42,785 cattle serums, 22,686 sheep and goat serums, and 28520 swine serums using the Rose Bengal test, complement fixation test, and various immunosorbent assays. We also tested 10,173 ram blood samples for B. ovis infection using the complement fixation test. Bacteriological examination was conducted on 214 samples collected from 34 serologically positive animals. Different molecular methods were employed in the identification and typing of 20 isolates from the samples.
B. melitensis biovar (bv.) 3 was confirmed with different identification methods in 2 flocks in 2 Croatian counties and B. suis bv. 2 in 3 flocks in 3 counties. B. melitensis in cows was confirmed for the first time in Croatia. Infection with B. ovis was serologically confirmed in 202 rams in 12 counties.
In 2008, the size of the brucellosis-affected area in Croatia and the efficiency of detection and prevention of brucellosis in sheep, goats, and swine were satisfactory. Infection with B. melitensis in cattle was confirmed for the first time and possible links for infection in humans were detected. More efficient measures for suppression and control of ovine epididymitis are required and a new strategy may be necessary for complete eradication of this disease.
Bakterijske su infekcije urinarnog trakta (IUT) u pasa učestale. Porast antimikrobne rezistencije i uporaba kritično-važnih antimikrobnih lijekova, osobito u liječenju IUT pasa, zabrinjavajuća je ...zbog pojave multiplo rezistentnih bakterijskih sojeva. Cilj je ovog rada bio prikazati prevalenciju i antimikrobnu rezistenciju najčešćih bakterijskih uzročnika IUT pasa i trendove antimikrobne rezistencije izdvojenih bakterijskih sojeva u Republici Hrvatskoj tijekom 11 godina. Od ukupno 2721 pretraženih uzoraka urina, pozitivno je bilo 1363 (50,1 %). Bakterijska vrsta Escherichia coli bila je najčešće izdvojeni mikroorganizam (47,3 %), nakon čega slijede: koagulaza pozitivni Staphylococcus sp. (20,7 %), Proteus sp. (14,7 %), Enterococcus sp. (13,6 %), beta hemolitični Streptococcus sp. (11,7 %), Pseudomonas aeruginosa (4,5 %) i meticilin rezistentni koagulaza pozitivni Staphylococcus sp. (3,4 %). Kod gram-negativnih bakterija uočen je visok stupanj osjetljivosti na: gentamicin, amikacin (77,9 %-97,3 %), marbofloksacin i ciprofloksacin (69,7 %-98,4 %). Kod bakterijskog roda Proteus utvrđen je i visok stupanj osjetljivosti na beta-laktame (70,5 %-86,7 %), a kod Escherichia coli i na kloramfenikol (73,3 %) i trimetoprim/sulfametoksazol (72,7 %). Neznatan postotak koagulaza pozitivnih Staphylococcus sp. bila je osjetljiva na penicilin (10,6 %) i ampicilin (16,7 %), dok je mala osjetljivost zamijećena na streptomicin, neomicin i tetracikline (56,0 %-66,0 %). Najmanji postotak beta hemolitičnih Streptococcus sp. bio je osjetljiv na aminoglikozide, tetracikline (8,2 %-31,9 %) i fluorokinolone (31,5 %-61,6 %). Kod bakterijskih izolata Enterococcus sp. najveća osjetljivost prisutna je na amoksicilin/klavulansku kiselinu (78,0 %) i ampicilin (70,4 %). Amikacin i kloramfenikol, s postotkom osjetljivosti od 89,3 %, odnosno 80,9 %, bili su jedini antimikrobni lijekovi učinkoviti na meticilin rezistentne koagulaza pozitivne Staphylococcus sp. Tijekom 11 godina istraživanja zamijećen je trend porasta antimikrobne rezistencije bakterija Escherichia coli i Proteus sp., izdvojenih iz IUT pasa, na fluorokinolone, ampicilin i amoksicilin/klavulansku kiselinu, dok je kod bakterijske vrste Escherichia coli uočen porast antimikrobne rezistencije i na cefalosporine. Ovi rezultati će pomoći pri empirijskom odabiru antimikrobnih lijekova za terapiju IUT pasa te podizanju svijesti o razumnoj uporabi antimikrobnih lijekova.
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