Mastitis u krava prouzročen brzorastućim mikobakterijama iz okoliša

Rapid-growth mycobacteria were isolated from two cases of cow mastitis with similar clinical appearance and within a narrow time frame. Mycobacteria were isolated on blood esculine agar. The isolated mycobacteria were Gram stained, Ziehl-Nielsen stained and tested for growth at 25°C, 37°C and 42°C, iron uptake, growth on Löwenstein-Jensen (LJ) agar with and without 5% NaCl, arylsulphatase (3 days), tween 80 hydrolysis, tellurite reduction, nitrate reductase and niacin synthesis. Molecular identification was performed using the Mycobacteria GenoType CM and AS tests (Hain Diagnostika, Nehren, Germany). One isolate was additionally sequenced for the hsp65, rpoB, 16S rRNA gene sequence and transcribed spacer sequence (ITS) DNA. Susceptibility testing of isolates was performed on the Sensititre Rapmycol plate (TREK Diagnostic Systems Ltd.) for trimethoprim/sulfamethoxasole, linezolid, ciprofloxacin, imipenem, moxifloxacin, cefepime, cefoxitin, amoxicillin / clavulanic acid, amikacin, ceftriaxone, doxycycline, minocycline, tigecycline, tobramycine and clarythromycine. Gram-positive acid-resistant rods were observed in stained smears. Both strains grew at 25°C, 37°C and 42°C on LJ medium, and on LJ medium containing 5 % NaCl. The conventional biochemical tests for iron uptake, arylsulphatase (3 days), Tween 80 hydrolysis, tellurite reduction and nitrate reductase were positive, while the niacin test was negative. Both isolates were identified by the GenoType Mycobacterium CM as Mycobacterium fortuitum II/ Mycobacterium mageritense, while application of the GenoType Mycobacterium AS kit identified both isolates as belonging to the species Mycobacterium smegmatis. Analysis of the isolate sequences (strain DS) for 16S ribosomal RNA confirmed a 100% identical result with Mycobacterium smegmatis strain INHR2. According to the CLSI criteria, both strains were sensitive to sulfametoxazole/trimethoprim, linezolid, doxicycline, amikacin and tobramycin. The strains differed in their sensitivity to cefoxitim, and both strains were resistant to clarithromycin. There was a strong difference between the isolates in sensitivity toward cefoxitime and tigecycline.Brzo-rastuće bakterije iz roda Mycobacterium izdvojene se iz dva klinički slična slučaja mastitisa krava u relativno kratkom vremenu. Mikobakterije su izdvojene na krvnom agaru s dodatkom eskulina, obojene po Grammu i Ziehl-Nielsenu te im je provjerena sposobnost rasta pri 25 °C, 37 °C i 42 °C, korištenje željeza, sposobnost rasta na Löwenstein-Jensen agaru bez i s dodatkom 5 % NaCl, tvorba arilsulfataze, sposobnost hidrolize tween 80, redukcije telurita i tvorbe nitrat reduktaze i niacina. Molekularna identifikacija izolata provedena je korištenjem testova Mycobacteria GenoType CM i AS. Odsječci gena hsp65, rpoB, 16S rRNK te tzv. (engl. transcribed spacer sequence (ITS)) jednog izolata su sekvencirani. Osjetljivost izolata provjerili smo pomoću SENSITITRE RAPMYCOL ploča (TREK Diagnostic Systems Ltd.) prema trimetoprim/sulfametoksazolu, linezolidu, ciprofloksacinu, imipenemu, moksifloksacinu, cefepimu, cefoksitinu, amoksicilinu s klavulanskom kiselinom, amikacinu, ceftriaksonu, doksiciklinu, minociklinu, tigeciklinu, tobramicinu i klaritromicinu. Gram-pozitivne acido-rezistentne štapićaste bakterije uočili smo u obojenim razmascima. Oba soja rasla su pri temperaturama 25 °C, 37 °C i 42 °C te na podlozi LJ s i bez dodatka 5  % NaCl. U oba soja utvrdili smo sposobnost korištenja željeza, tvorbe arilsulfataze, hidrolize Tween 80, redukcije telurita i tvorbe reduktaze nitrata. Sojevi nisu tvorili niacin. Oba izolata su testom GenoType Mycobacterium CM identificirana kao Mycobacterium fortuitum II/ Mycobacterium mageritense dok su GenoType Mycobacterium AS testom identificirana kao Mycobacterium smegmatis. Sekvencijskom analizom odsječka gena 16S ribosomske RNK soja DS i usporedbom s javno dostupnim izolatima, utvrdili smo potpunu podudarnost sa sojem Mycobacterium smegmatis soj INHR2. Prema CLSI kriterijima, oba izolata su osjetljiva prema sulfametoksazolu s trimetoprimom, linezolidu, doksiciklinu, amikacinu i tobramicinu, a rezistentna prema klaritromicinu. Razlike između sojeva očitovale su se u osjetljivosti prema cefoksitimu i tigeciklinu

Pojavnost ergot-sklerocija i ergot-alkaloida u pšenici i raži hrvatskih proizvođača

Ergot alkaloids (EAs) are mycotoxins produced by several species of fungi of the genus Claviceps, among which Claviceps purpurea is the most widespread in Europe. This species has been found in many economically important cereal grains, such as rye, wheat, triticale, barley, millet and oats. The distribution of EA contamination has a sporadic incidence, with many factors involved in its occurrence, greatly varying between fungal strains, geographic regions, host plants and regional/local weather conditions. Cool, damp weather favours ergot by enhancing the germination of sclerotia. The aim of this study was to investigate the occurrence of ergot sclerotia and EAs in wheat and rye grain samples (n = 64) collected during 2021 from Croatian cereal producers in central and eastern Croatia. In two rye samples, the presence of ergot sclerotia was detected in the amount of 259 mg/kg and 536 mg/kg, whereas no wheat samples tested positive for ergot sclerotia. A higher contamination with EAs was determined in the rye samples (18% contaminated; max 167.4 μg/kg), while a lower frequency of contamination was determined in wheat, with only one positive sample (1.9%; 68.5 μg/kg). The results indicate low-level EA contamination of wheat and rye cultivated by Croatian producers during the study period. However, despite the low incidence of positive rye samples with EAs, the contents of ergot sclerotia in two samples were higher than permitted by the legislation for foodstuffs. Since the levels of these mycotoxins and ergot sclerotia content can vary depending on a number of factors, further research is required over a longer period of time and under different cereal cultivation and processing conditions.Ergot-alkaloidi (EA) su mikotoksini koje proizvodi nekoliko vrsta gljivica iz roda Claviceps, među kojima je i Claviceps purpurea najraširenija gljivica u Europi. Ova vrsta je pronađena u ekonomski važnim žitaricama, kao što su: raž, pšenica, tritikal, ječam, proso i zob. Distribucija kontaminacije EA ima sporadičnu pojavu s mnogo čimbenika koji su uključeni u njihovu pojavu, uvelike varirajući između sojeva gljivica, geografskih regija, biljaka domaćina i regionalnog vremena, što ukazuje da hladno i vlažno vrijeme pogoduje ergotu tako što potiče klijanje sklerocija. Cilj je ovog istraživanja bio istražiti pojavu ergot- sklerocija i EA u uzorcima pšenice i raži (n=64) uzorkovanih tijekom 2021. godine od hrvatskih proizvođača žitarica u središnjoj i istočnoj Hrvatskoj. U dva uzorka raži utvrđena je prisutnost ergot-sklerocija u količini od 259 mg/kg i 536 mg/kg. Niti u jednom uzorku pšenice nije pronađen ergot-sklerocij. Veća kontaminacija s EA utvrđena je u uzorcima raži (18 % kontaminirano, max 167,4 μg/kg), dok je manja učestalost kontaminacije, sa samo jednim pozitivnim uzorkom (1,9 %), utvrđena u pšenici (68,5 μg/kg). Rezultati su ukazali na nisku razinu kontaminacije s EA pšenice i raži koje su uzgojili hrvatski proizvođači tijekom cijelog istraživanog razdoblja. Međutim, iako je utvrđen nizak postotak pozitivno testiranih uzoraka raži na EA, sadržaj ergot-sklerocija u dva uzorka bio je veći nego što je to za hranu zakonodavstvom dopušteno. Budući da razine tih mikotoksina i ergot-sklerocija mogu varirati ovisno o brojnim čimbenicima, potrebno je njihovo daljnje istraživanje tijekom dužeg vremenskog razdoblja i pod različitim uvjetima uzgoja i obrade žitarica

Podudarnost mikrobiološke i molekularne metode identifikacije izolata streptokoka iz kravljeg mlijeka

Pathogenic streptococci are implicated in clinical and subclinical mastitis. Most laboratories identify streptococci on the basis of microbiological examination, but molecular diagnostic methods have become the gold standard of mastitis diagnosis in the last few years. Therefore, this study aims to determine the agreement of microbiological and molecular identification of streptococci isolates from bovine milk. Milk samples were taken before the evening milking into sterile tubes. Samples were examined bacteriologically by inoculation on aesculin blood agar. Identification of grown colonies was carried out using internationally accepted methodology. Sequencing of the 16S rRNA gene was the reference method used to confirm Streptococcus sp. in all bacterial isolates. In the study, 54 strains of bacteria isolated from milk samples from the udder quarters of dairy cows with subclinical mastitis were examined using molecular methods. By conventional microbiological examination, the strains were identified to the species (Strep. agalactiae, Strep. dysgalactiae and Strep. uberis) or the genus level (Streptococcus spp.) without final identification of the species. On the basis of 16S rRNA analysis, 47 out of 54 examined streptococcal strains were found to belong to the genus Streptococcus sp. Among the streptococci identified, 6 isolates belonged to Strep. agalactiae, ¸8 isolates to Strep. dysgalactiae, 2 isolates to Strep. canis and 31 isolates belonged to Strep. uberis. Among the seven remaining isolates, three were identified as Enterococcus faecalis and four as Lactococcus lactis. Agreement between the identification procedures used was fair, with a Kappa index of 0.2181 (SE=0.0612; Z=3.56; p=0.0002).Patogeni streptokoki povezuju se sa subkliničkim i kliničkim mastitisima. Većina laboratorija identifikaciju streptokoka temelji na mikrobiološkoj pretrazi, ali posljednjih godina molekularne metode postaju zlatni standard za dijagnostiku mastitisa. Cilj ovog istraživanja bio je odrediti podudarnost rezultata klasične mikrobiološke pretrage i identifikacije streptokoka molekularnim metodama. Uzorci mlijeka uzeti su u sterilne epruvete prije večernje mužnje. Mikrobiološka pretraga provedena je u skladu s opće prihvaćenim međunarodnim preporukama. Primarno izdvajanje i identifikacija streptokoka provedeni su korištenjem podloge eskulin krvni agar. Molekularna identifikacija vrste Streptococcus spp. učinjena je na osnovu 16S rRNA sekvencioniranja. U istraživanju su molekularnim tehnikama pretražena 54 soja bakterija izdvojena iz uzoraka mlijeka pojedinačnih četvrti vimena krava sa subkliničkim mastitisom. Klasičnom mikrobiološkom pretragom izdvojeni sojevi identificirani su do vrste (S. agalactiae, S. dysgalactiae i S. uberis) ili do roda (Streptococcus spp.) bez konačne identifikacije vrste. Metodom identifikacije na osnovi umnažanja gena 16S rRNA, od 54 pretražena bakterijska soja, za njih 47 je potvrđeno da pripadaju rodu Streptococcus spp. i to 6 Streptococcus agalactiae, 8 Streptococcus dysgalactiae, 2 Streptococcus canis i 31 Streptococcus uberis dok su 3 soja pripadnici vrste Enterococcus faecalis, a 4 soja pripadali su vrsti Lactococcus lactis. Podudarnost primijenjenih metoda za identifikaciju streptokoka je blaga, s Kappa indeksom od 0,2181 (SE=0,0612; Z=3,56; p=0,0002)

Uloga oksidacijskog stresa i upalnog odgovora u patogenezi mastitisa u mliječnih krava

Mastitis is one of the most frequent diseases of dairy cows throughout the world, therefore it causes the greatest economic losses in dairy cattle industry. These losses are reflected through: reduced milk production, increased costs of medication and the other animal health services, reduced fertility, early culling of animals and the value of discarded milk. Mastitis is also important from the aspects of public health, milk processing and animal welfare. In the pathogenesis of mastitis the key role plays the innate immune response which is the first line of defence against the pathogen invasion of the udder. The innate immune response generates an inflammatory reaction which is the elementary response of an organism to the tissue trauma induced by any physical, chemical or biological causative agent, but primarily it is the protective mechanism of a vital significance which includes increased phagocytic activity, secretion of antimicrobial substances, fibrosis as well as the alterations in tissue structure of affected organ or body cavity. The release of a number of inflammatory mediators as well as reactive oxygen species (ROS) is an important part of inflammatory response. In dairy cows, the metabolic challenge that occurred during the transition from dry period to early lactation may additionally increase the release of ROS which may contribute to development of oxidative stress and inflammatory response. Oxidative stress is defined as a shift in the balance from cellular oxidation-reduction reactions towards oxidation, i.e. to the state of excessive release of oxidants when their removal by antioxidants is impaired and even insufficient. During peripartum period antioxidantive status of dairy cows is seriously impaired and consequently both the oxidative stress and inflammatory response may present the predisposing factors to their higher susceptibility to intramammary infections (IMI) and mastitis. This association between oxidative stress and inflammation during IMI and mastitis indicates their role in the pathogenesis of mastitis. Thus, a better understanding of such a synergism could contribute to development of new approaches to prevention and therapy of IMI and mastitis.Mastitis je jedna od najčešćih bolesti mliječnih goveda u cijelom svijetu, jer uzrokuje najveće gubitke u mliječnom gospodarstvu. Ima značajan utjecaj sa stanovišta javnog zdravstva, prerade mlijeka i dobrobiti životinja. Ekonomski gubitci se očituju kroz: smanjenu proizvodnju mlijeka, velike troškove liječenja, prijevremeno izlučivanje životinja, vrijednost odbačenog mlijeka te smanjenu plodnost. U patogenezi mastitisa ključnu ulogu ima urođeni imunosni odgovor koji je prva linija obrane nakon što patogen prodre u mliječnu žlijezdu. Upala je standardna reakcija organizma na ozljedu bilo kakvog fizičkog, kemijskog ili biološkog uzroka, no prije svega to je vitalni zaštitni mehanizam, koji uključuje proizvodnju reaktivnih kisikovih spojeva (ROS), fagocitne mehanizme, izlučivanje protumikrobnih tvari, stvaranje ožiljkastog tkiva i neovaskularizaciju te promjene tkivne strukture zahvaćenog organa ili tjelesnog prostora. Metaboličko opterećenje nastalo prilikom prelaska iz perioda zasušenja u period rane laktacije je dodatno potencijalni izvor oksidansa i ROS-a u mliječnih krava i pogodovni čimbenik za nastanak oksidacijskog stresa i upalnog odgovora. Oksidacijski stres definiran je kao pomak ravnoteže u staničnim oksido-redukcijskim reakcijama prema oksidaciji, odnosno to je stanje prekomjernog stvaranja slobodnih radikala kisika i ROS pri čemu se nadilazi mogućnost njihova uklanjanja. Antioksidacijski status krava u peripartalnom periodu je stoga oslabljen i posljedično oksidacijski stres i upalni odgovor mogu predisponirati osjetljivosti krave za intramamarne infekcije (IMI) i mastitis. Povezanost između upalnog odgovora i oksidacijskog stresa tijekom IMI i mastitisa ukazuje na njihovu važnu ulogu u patogenezi bolesti mliječne žlijezde te stoga bolje razumijevanje takvog sinergizma može doprinijeti razvoju novih pristupa u prevenciji i liječenju IMI i mastitisa

Praćenje aflatoksina M1 u sirovom kravljem mlijeku u Hrvatskoj tijekom zime 2015.

A total of 548 raw milk samples were collected in the western, central and eastern regions of Croatia during February and March 2015. Aflatoxin M1 (AFM1) concentrations were quantified by the enzyme immunoassay method. The method limits of detection (LOD) and quantification (LOQ) were 22.2 and 34.2 ng/kg, respectively. The mean AFM1 levels measured in the three regions were (ng/kg) as follows: western 3.69, central 3.11 and eastern 4.14. In total, the 548 samples analysed concentrations were below the LOD value and accordingly below the European Union maximum residue level (EU MRL) of 50 ng/kg. The results suggest an absence of use of contaminated with aflatoxin B1 supplementary feedstuff for lactating cows in winter 2015. Such results might be related to the improved storage conditions for feed as well as to the enhanced and more stringent feed control system for mycotoxins in Croatia.Ukupno 548 uzoraka sirovog mlijeka prikupljeno je u zapadnoj, središnjoj i istočnoj regiji Hrvatske tijekom veljače i ožujka 2015. Koncentracije aflatoksina M1 (AFM1) su određivane primjenom imunoenzimskog testa. Granice detekcije (LOD) i kvantifikacije (LOQ) testa su 22,2 i 34,2 ng/kg. Prosječne razine AFM1 izmjerene u tri regije su (ng/kg): zapadna 3,69, središnja 3,11 i istočna 4,14. U ukupno 548 uzoraka analizirane koncentracije su bile ispod LOD vrijednosti i sukladno tome ispod maksimalne dozvoljene koncentracije propisane u Europskoj uniji (EU MDK) od 50 ng/kg. Rezultati upućuju na odsutnost upotrebe dopunske hrane za krave muzare koja je kontaminirana s aflatoksinom B1 tijekom zime 2015. To može biti rezultat poboljšanih uvjeta držanja hrane te poboljšanog i postroženog sustav kontrole mikotoksina u hrani za životinje u Hrvatskoj

Detection of pathogenic bacteria in raw milk and dairy products with special regard to Yersinia enterocolitica

U sjeverozapadnoj Hrvatskoj meki svježi sir se tradicionalno proizvodi po nepisanoj recepturi od sirovog kravljeg mlijeka. Mlijeko predstavlja idealan supstrat za rast i razmnožavanje brojnih mikroorganizama, mnogih uzročnika zoonoza pa tako i vrste Y. enterocolitica. Tijekom naših istraživanja pretraženo je 159 uzoraka, od toga 109 uzoraka sirovog kravljeg mlijeka i 50 uzoraka svježeg kravljeg sira i vrhnja. Y. enterocolitica izdvojena je iz 2 (1,8 %) uzorka sirovog mlijeka. Od 50 uzoraka mliječnih proizvoda (sir i vrhnje) u 16 (32 %) njih je dokazana E. coli, u 12 (24 %) uzoraka S. aureus te u 1 (2 %) uzoraka L. innocua. Osim njih u 15 (13,8 %) uzoraka sirovog mlijeka i 24 (48 %) uzoraka svježeg sira i vrhnja izdvojene su enterobakterije, a biokemijski su identificirane vrste: S. marcescens, S. liquefaciens, M. morganii, C. freundii i C. braakii. Bakterije iz roda Salmonella i L. u istraživanim uzorcima nisu dokazane. Nakon provedene biokemijske identifikacije, dva soja identificirana kao vrsta Y. enterocolitica pretraženi su na molekularnoj razini, molekularnom pretragom potvrdni rezultat dobiven je rezultat samo kod jednog uzorka, što je usporedivo s rezultatima drugim istraživanja. Zbog zahtjevne identifikacije bakterijske vrste Y. enterocolitica, kombiniranom primjenom uzgojnih i molekularnih metoda dobivaju se pouzdaniji i brži rezultati identifikacije. Sirovo mlijeko i vrhnje od sirovog mlijeka izvor su patogenih mikroorganizama (E. coli i S. aureus) i drugih enterobakterija, a našim istraživanjem dokazali smo i patogenu bakterijsku vrstu Y. enterocolitica koja predstavlja potencijalni rizik za zdravlje potrošača i u Republici Hrvatskoj. Y. enterocolitica je znatno otporna na procese u proizvodnji hrane (dezinfekcija, niski pH, salinitet, preživljavanje u hladnijim uvjetima) te u budućnosti zahtijeva veću pažnju i nadzor u proizvodnji hrane.In northwestern Croatia, soft fresh cheese is produced using traditional, unwritten recipes of raw cow milk. Milk is an ideal substrate for the growth and reproduction of numerous microorganisms, many zoonotic agents and the species Y. enterocolitica. In this study, 159 samples were tested: 109 samples of raw cow milk and 50 samples of fresh cow cheese and cream. Y. enterocolitica was detected in 2 (1.8%) samples of raw milk. E. coli was detected in 16 (32%) of 50 samples of dairy products (cheese and cream). S. aureus was detected in 12 (24%) samples and L. innocua in 1 (2%) sample. Additionally, 15 (13.8%) samples of raw milk and 24 (48%) samples of fresh cheese and cream also harboured enterobacteria, identified biochemically as: S. marcescens, S. liquefaciens, M. morganii, C. freundii and C. braakii. Salmonella and L. monocytogenes were detected in any of the samples. After the biochemical identification, two strains identified as Y. enterocolitica were examined at the molecular level, and the molecular test confirmed the result in only one sample, which is comparable to the results of other studies. Due to the demanding identification of the bacterial species Y. enterocolitica, the combined application of breeding and molecular methods produces more reliable and faster identification results. Raw milk and crude milk cream are a source of pathogenic microorganisms (E. coli and S. aureus) and other enterobacteria, while this study also found the presence of the pathogenic bacterial species Y. enterocolitica, presenting a potential consumer health risk in the Republic of Croatia. Y. enterocolitica is considerably resistant to food production processes (disinfection, low pH, salinity, survival in cold conditions) and deserves greater attention and supervision in food production in the future

Antimicrobial susceptibility and trends in antimicrobial resistance of bacterial pathogens isolated from dogs urinary tract infection i Croatia from 2012-2022

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.Bacterial urinary tract infections (UTIs) occur commonly in dogs. Increase of bacterial resistance and using criticaly–important antimicrobials, especially in UTIs in dogs, concern because of emergence of multidrug resistant bacterial strains. The objectives of this study were to investigate prevalence and antimicrobial resistance of most frequently isolated bacterial isolates causing UTIs in dogs, and to show trends of antimicrobial resistance during 11 year period in Croatia. A total of 2721 dog urine samples were analysed and 1363 were positive for bacterial growth (50.1%). Escherichia coli was the most frequently isolated organism (47.3%), followed by coagulase positive Staphylococcus sp. (20.7%), Proteus sp. (14.7%), Enterococcus sp. (13.6%), beta-hemolitic Streptococcus sp. (11.7%), Pseudomonas aeruginosa (4,5%) and meticillin resistent coagulasa pozitive Staphylococcus sp. (3.4%). In Gram-negative bacteria the susceptibility rates were high for gentamicin, amikacin (77.9%-97.3%), marbofloxacin and ciprofloxacin (69.7%-98.4%). High susceptibility rates in Proteus sp. to beta-lactams (70.5%-86.7%) and in Escherichia coli to chloramphenicol (73.3%) and trimethoprim/sulfamethoxazole (72,7%) were noticed. In coagulase positive Staphylococcus sp. only few isolates were susceptible to penicilin (10.6%) and ampicillin (16.7%), followed by streptomycin, neomycin and tetracyclines (56.0%-66.0%). In beta-hemolitic Streptococcus sp. the lowest susceptibility rates were found for aminoglycosides, tetracyclines (8.2%-31.9%) and fluoroquinolones (31.5%-61.6%). Enterococcus sp. isolates showed highest susceptibility to amoxicillin/clavulanic acid (78.0%) and ampicillin (70.4%). Amikacin and chloramphenicol, with susceptibility rate of 89.3% and 80.9%, were the only drugs effective against meticillin resistant coagulase positive Staphylococcus sp. Trends during eleven years of investigation period showed increase of resistance in Escherichia coli and Proteus sp. to fluoroquinolones, ampicillin and amoxicillin/clavulanic acid, while in Escherichia coli increase of resistance to cephalosporins was also noticed. These results will help in drug selection for empirical treatment of UTIs in dogs and raising awareness of resonable use of antimicrobials

Use of somatic cell count in the diagnosis of mastitis and its impacts on milk quality

Mastitis je upalna reakcija organizma na infekciju sekretornog dijela mliječne žlijezde. Uzročnici mastitisa dijele se bojanjem po Gramu na Gram-pozitivne i Gramnegativne; prema etiologiji na kontagiozne i uvjetovane uzročnike. Kontagiozni se uzročnici najčešće prenose tijekom mužnje i prouzroče dugotrajne, kronične mastitise, uglavnom subkliničkog tijeka. Za razliku od njih, uvjetovani uzročnici u pravilu izazivaju kratkotrajne infekcije, ali s češćim kliničkim manifestacijama. Inficirane se krave otkrivaju dijagnostikom mastitisa, koja se najčešće provodi neizravnim ili izravnim metodama određivanja broja somatskih stanica i bakteriološkom pretragom mlijeka.Zagrebački mastitis test je brza i jednostavna orijentacijska metoda u procjeni broja somatskih stanica koji se provodi u terenskim uvjetima. Somatske stanice čine epitelne stanice i leukociti. Nalaze se u mlijeku, ali se njihov broj i odnos mijenja ovisno o upalnim promjenama u mliječnoj žlijezdi. Kretanje broja somatskih stanica uglavnom ovisi o pojavi mastitisa, ali i o stadiju laktacije, životnoj dobi životinje, godišnjem dobu, ali i ostalim bolestima mliječne krave. Mlijeko s povećanim brojem somatskih stanica ima promijenjenu kakvoću. Mlijeko sadrži niži udio mliječne masti i laktoze, a povećan je i udio proteina i električna provodljivost zbog povišene koncentracija klora i natrija u mlijeku. Osim toga, mlijeko ima i smanjenu termostabilnost te mu je smanjena sposobnost podsiravanja i formiranja gruša. Takvo mlijeko ima užegli okus i nije pogodno za proizvodnju kondenziranog mlijeka, mlijeka u prahu i steriliziranog mlijeka. Rok trajanja sireva proizvedenih od takvog mlijeka zbog zadržavanja veće količine vode je smanjen. Pravilnim i redovitim provođenjem postupaka dezinfekcije sisa vimena prije i poslije mužnje poboljšava se prinos i kakvoća mlijeka, odnosno smanjuju se troškovi liječenja mastitisa, a povećava se i prihod farme.Mastitis is an inflammatory reaction to an infection of the secretory part of the mammary gland. The causes of mastitis are divided by Gram staining into Gram-positive and Gram-negative, and according to aetiology into contagious and environmental causes. Contagious pathogens are most transmitted during milking, and cause long-lasting, chronic mastitis with a mostly subclinical course. In contrast, environmental pathogens typically cause short-term infections, but with more frequent clinical manifestations. Infected cows are detected by the diagnosis of mastitis, which is most often reached through indirect or direct methods of determining the somatic Cell count and by conducting bacteriological examination of milk. The Zagreb mastitis test is a quick and simple orientation method for estimating the number of somatic cells. Its advantage is that can be performed in field conditions. Somatic cells are made up of epithelial cells and leukocytes. They are normally found in milk, but their number and ratio change depending on inflammatory changes in the mammary gland. The somatic cell count can change depending on the occurrence of mastitis, but also on the stage of lactation, animal age, season and other diseases of the dairy cow. Milk with an increased number of somatic cells has an altered quality compared to the milk of a healthy cow. Milk contains a lower proportion of milk fat and lactose, and an increased proportion of protein. Furthermore, electrical conductivity is increased due to elevated concentrations of chlorine and sodium in milk. It also has reduced thermal stability and a reduced ability to curdle and form lumps. Such milk has a rancid taste and is not suitable for the production of condensed milk, milk powder or sterilized milk. The shelf life of cheeses produced from such milk is reduced due to the retention of a larger amount of water. Proper and regular disinfection of udder teats before and after milking raises the yield and quality of milk, and reduces the cost associated with mastitis treatment, thereby increasing both milk quality and farm revenues

Methods for isolation and identification of bacteria of the genus Campylobacter - genotyping methods (Part II)

Različite metode genotipizacije omogućuju razlikovanje bakterijskih sojeva ispod razine vrste ili podvrste. Gel elektroforeza u pulsirajućem polju (PFGE) neprestano se usavršava od 1980-tih godina i do danas je upotrebljavaju mnogi javnozdravstveni laboratoriji. Od ostalih metoda koriste se: metoda analize duljine polimorfizma restrikcijskih odsječaka (RFLP), metoda nasumično umnoženih poliformnih odsječaka (RAPD) i metoda ribotipizacije. Određivanje redoslijeda nukleotidnih sljedova unutar DNK naziva se sekvenciranje. Na osnovi sekvenciranja bazira se rad novih metoda tipizacije određivanjem sljedova nukleotida na više genskih lokusa poput MLST, rMLST i cgMLST. One istražuju odnose između bakterija roda Campylobacter, posebice C. jejuni i C. coli. Korisne su u istraživanju mogućih izvora i rezervoara uzročnika bolesti, njihovih odnosa te molekularne epidemiologije bolesti. Metoda sekvenciranja cijelog genoma (WGS) u mikrobiologiji revolucionarna je metoda za koju se smatra da će postupno zamijeniti ostale metode genotipizacije.Distinction of bacterial strains below the species or subspecies level is made possible by diverse genotyping methods. Many public health laboratories use pulsed field gel electrophoresis (PFGE), which has been constantly improving since the 1980s. Other methods used include restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD) method and ribotyping. The process of determining the order of nucleotides within DNA is called sequencing. Newly evolved methods based on multilocus sequence typing are MLST, rMLST and cgMLST. These examine the relationships between bacteria of the genue Campylobacter, especially C. jejuni and C. coli. They are useful in investigating possible sources and reservoirs of disease, their connection and the molecular epidemiology of diseases. A new revolutionary method in microbiology is whole genome sequencing (WGS), and it is believed that this method will gradually replace other genotyping methods

Methods for the isolation and identification of bacteria of the genus Campylobacter - classical and molecular methods (Part I)

Bakterije mikroorganizmi koji za svoj rast u okolišu, kao i u laboratorijskim uvjetima trebaju mikroaerofilne uvjete. Za rutinsku analitiku klasičnim metodama primjerena je primjena priznatih standardiziranih protokola i propisanih visoko selektivnih tekućih i/ili krutih hranjivih podloga. Nakon porasta karakterističnih kolonija radi određivanja vrste kampilobaktera primjenjuju se mikroskopski, fenotipski, biokemijski, imunozimski testovi, rezistotipizacija i/ili metoda masene spektrometrije. Za razlikovanje sojeva kampilobaktera postoje dvije klasične metode serotipizacije. Do sada je razvijen znatan broj PCR protokola usmjerenih prema otkrivanju Campylobacter roda, vrste ili podvrste, bilo iz uzoraka hrane, okoliša ili izmeta. Glavne prednosti PCR metoda su u njihovoj brzini, osjetljivosti i specifičnosti, a nedostatak PCR metodologije je u tome što ne razlikuje žive od mrtvih stanica, zbog čega se razvijaju novi protokoli bazirani na RT-PCR, NASBA i QRT- PCR molekularnim metodama.Bacteria of the genus Campylobacter require microaerophilic conditions for growth. Use of recognized standardized protocols and prescribed highly selective liquid and/ or solid media is appropriate for routine analysis with classical methods. After the growth of characteristic colonies, microscopic, phenotype and biochemical methods, immunoenzyme tests, resistotypization and/ or mass spectrometry are applied to determine the Campylobacter species. Two classical serotyping methods are used to differentiate Campylobacter strains. There are various PCR protocols intended for the detection of Campylobacter genus, species or subspecies from food samples, environment or faeces. The main advantages of PCR methods are their speed, sensitivity and specificity. The deficiency of this method is that it does not differentiate viable from dead cells, therefore new protocols based on RT-PCR, NASBA and QRT-PRC molecular methods are under development