Antimicrobial Resistance in Rapidly Growing Nontuberculous Mycobacteria among Domestic and Wild Animals Emphasizing the Zoonotic Potential.

Non-tuberculous mycobacteria (NTM) are opportunistic pathogens capable of causing infections in humans and animals. The aim of this study was to demonstrate the potential role of domestic and wild animals as a reservoir of multiple resistant, rapidly growing NTM strains representing a potential zoonotic threat to humans. A total of 87 animal isolates belonging to 11 rapidly growing species (visible colonies appear within three to seven days) were genotyped and tested for susceptibility to the 15 most commonly used antibiotics in the treatment of such infections in a human clinic. By determining the antimicrobial susceptibility, the most prevalent resistance was found to cephalosporins (>50%), followed by amoxicillin-clavulanate (31.0%), clarithromycin (23.0%), tobramycin (14.9%) and doxycycline (10.3%). Resistance to imipenem, ciprofloxacin, minocycline and linezolid was notably lower (<7.0%). All tested isolates were susceptible to amikacin and moxifloxacin. The most frequent resistance was proved in the most pathogenic species: M. fortuitum, M. neoaurum, M. vaccae and M. porcinum. Meanwhile, other species displayed a higher sensitivity rate. No significant resistance differences between domestic and wild animals were found. The established significant frequency of resistance highlights the significant zoonotic potential posed by circulating rapidly growing NTM strains, which could lead to challenges in the treatment of these infections.

Distinguishing Natural Infections of the Bovine Mammary Gland by Staphylococcus from Streptococcus spp. Using Quantitative Milk Proteomics.

Bovine mastitis is the most frequent disease on dairy farms, which leads to a decrease in the health welfare of the animals and great economic losses. This study was aimed at determining the quantitative variations in the milk proteome caused by natural infection by Staphylococcus and Streptococcus species in order to gain further understanding of any discrepancies in pathophysiology and host immune responses, independent of the mastitis level. After identification of Staphylococcus (N = 51) and Streptococcus (N = 67) spp., tandem mass tag (TMT)-labeled quantitative proteomic and liquid chromatography-mass spectrometry (LC-MS/MS) techniques on a modular Ultimate 3000 RSLCnano system coupled to a Q Exactive Plus was applied on aseptically sampled milk from Holstein cows. Proteome Discoverer was used for protein identification and quantitation through the SEQUEST algorithm. Statistical analysis employing R was used to identify differentially abundant proteins between the groups. Protein classes, functions and functional-association networks were determined using the PANTHER and STRING tools and pathway over-representation using the REACTOME. In total, 156 master bovine proteins were identified (two unique peptides, p < 0.05 and FDR < 0.001), and 20 proteins showed significantly discrepant abundance between the genera (p < 0.05 and FDR < 0.5). The most discriminatory proteins per group were odorant-binding protein (higher in staphylococci) and fibrinogen beta chain protein (higher in streptococci). The receiver operating characteristic (ROC) curve showed that protein kinase C-binding protein NELL2, thrombospondin-1, and complement factor I have diagnostic potential for differentiating staphylococci and streptococci intramammary infection and inflammation. Improved understanding of the host response mechanisms and recognition of potential biomarkers of specific-pathogen mastitis, which may aid prompt diagnosis for control implementation, are potential benefits of this study.

Body burdens and distribution of mercury and selenium in bottlenose, striped and Risso's dolphins along the Adriatic coast: A 20-year retrospective.

Top marine predators present high mercury concentrations in their tissues due to biomagnification in the marine food chain. This study reports mercury (Hg) and selenium (Se) status, and the Hg:Se molar ratio assessment in bottlenose (Tursiops truncatus), striped (Stenella coeruleoalba) and Risso's dolphins (Grampus griseus). Total Hg and Se concentrations were determined in muscle, liver, kidney, lung, spleen, adipose tissue and skin collected from 186 specimens stranded in the Croatian part of Adriatic Sea from 1995 to 2014. Total Hg concentrations in tissue samples ranged from 0.001 in the spleen to 2238 mg/kg wet weight in liver. Se concentrations in dolphin samples ranged from 0.010 to 2916 mg/kg ww. Minimum Se concentration was found in muscle and maximum Se concentration were found in liver of bottlenose dolphin. Hg and Se levels in Risso's dolphins showed higher concentrations in all tissues in comparison to bottlenose and striped dolphins. Significant and positive correlations were observed between age and Hg concentrations (P < 0.05). In 66.6 % of Risso's, 15.3 % of bottlenose dolphins and one stranded striped dolphin in this study, the hepatic concentration of Hg exceeded the higher toxic thresholds (400 mg/kg w.w.) previously defined as evidence of liver damage in marine mammals. The Hg:Se molar ratio in the liver of Risso's dolphin was 0.670. The liver of adult bottlenose dolphins showed expected values (0.870), while the liver of young dolphins had a high ratio (0.750), non-specific for the age group. The Hg:Se molar ratio in the liver of striped dolphins was 0.390, which is lower than the literature values.

Prevalence of Virulence Genes Among Escherichia Coli Strains Isolated from Food and Carcass Swabs of Different Animal Origins in Croatia.

Introduction: Escherichia coli is present in the normal intestinal flora but some strains can cause intestinal and extraintestinal diseases, and research on its presence in food of animal origin is in the interests of public health. This study was designed to characterise E. coli strains according to their origin, their carriage of virulence genes specific for certain pathogroups, and phylogenetic group affiliation. Material and Methods: The study was carried out on 100 E. coli strains isolated from food samples of various animal origin as well as pig and cattle carcass swabs. Isolation of the strains was performed using two methods. One method included colony count and the other an overnight enrichment of the samples. Isolation was followed by DNA extraction and detection of virulence genes and phylogenetic group with conventional and multiplex PCRs. Results: In this study, the most prevalent gene was EAST1 (20%) and strains which carried it were identified as enteroadherent E. coli. Other pathogroups were represented in lower incidences. Phylogenetic group analysis revealed the prevalence of the A and B1 groups, with B1 mainly present in game and cattle strains, while the majority of pig and poultry strains were assigned to group A. Conclusion: This study provides an overview of the presence of potentially pathogenic strains and E. coli phylogenetic groups in Croatia, for which the data are limited. Further microbiological and molecular research is required to examine the epidemiological situation in the country.

Effects of immunostimulators of microbial origin on T cells of pigs vaccinated with attenuated vaccine against Aujeszky's disease.

Aujeszky's disease (AD) is a viral infectious disease caused by Suid herpesvirus 1 (SuHV-1). Vaccination and eradication of AD in domestic pigs is possible using marker vaccines with attenuated or inactivated SuHV-1, or subunit vaccines. However, vaccines with attenuated SuHV-1 have shown to be more potent in inducing strong cell-mediated immune response. The studies have shown that Parapoxvirus ovis, as well as Propionibacterium granulosum with lipopolysacharides (LPS) of Escherichia coli have pronounced immunomodulatory effects and that in combination with the vaccines can induce stronger humoral and cellular immune responses than use of vaccines alone. In our study distribution of peripheral blood T cell subpopulations was analysed after administration of vaccine alone (attenuated SuHV-1), immunostimulators (inactivated Parapoxvirus ovis or combination of an inactivated P. granulosum and detoxified LPS of E. coli) and combinations of vaccine with each immunostimulator to the 12-week old piglets. Throughout the study no significant changes were found in the proportions of γδ and most αß T cell subpopulations analysed. However, on the seventh day of the study combination of an inactivated P. granulosum and LPS of E. coli with vaccine induced transient but significant increase of the proportions of CD4+CD8α+ and CD4-CD8α+ αß T cells, that have been strongly associated with early protection of SuHV-1 infected pigs. Our findings indicate that combination of inactivated P. granulosum and detoxified E. coli LPS could be used for enhancement of a cellular immune response induced by vaccines against AD.

Milk and serum proteomes in subclinical and clinical mastitis in Simmental cows.

Bovine mastitis causes changes in the milk and serum proteomes. Here changes in both proteomes caused by naturally occurring subclinical and clinical mastitis have been characterised and quantified. Milk and serum samples from healthy dairy cows (n = 10) were compared to those of cows with subclinical (n = 12) and clinical mastitis (n = 10) using tandem mass tag (TMT) proteomics. Proteins that significantly increased or decreased in milk (n = 237) or serum (n = 117) were quantified and classified by the type of change in subclinical and clinical mastitis. A group of the proteins (n = 38) showed changes in both milk and serum a number of which decreased in the serum but increased in milk, suggesting a particular role in host defence for maintaining and restoring homeostasis during the disease. Proteins affected by bovine mastitis included proteins in host defence and coagulation pathways. Investigation of the modified proteomes in milk and serum was assessed by assays for haptoglobin, serum amyloid A and α1 acid glycoprotein validating the results obtained by quantitative proteomics. Alteration of abundance patterns of milk and serum proteins, together with pathway analysis reveal multiple interactions related to proteins affected by mastitis. Data are available via ProteomeXchange with identifier PXD022595. SIGNIFICANCE: Mastitis is the most serious condition to affect dairy cows and leads to reduced animal welfare as well as having a negative economic effect for the dairy industry. Proteomics has previously identified changes in abundance of milk proteins during mastitis, but there have been few investigations addressing changes that may affect proteins in the blood during the infection. In this study, changes in the abundance of proteins of milk and serum, caused by naturally occurring mastitis have been characterised by proteomics using a quantitative approach and both subclinical and clinical cases of mastitis have been investigated. In both milk and serum, change in individual proteins was determined and classified into varying types of altering abundance, such as increasing in subclinical mastitis, but showing no further increase in clinical mastitis. Of special interest were the proteins that altered in abundance in both milk and serum which either showed similar trends - increasing or decreasing in both biological fluids or showed reciprocal change decreasing in serum but increasing in milk. As well as characterising proteins as potential markers of mastitis and the severity of the disease, these results provide insight into the pathophysiology of the host response to bovine mastitis.

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.

Effects of Dietary Vibroactivated Clinoptilolite Supplementation on the Intramammary Microbiological Findings in Dairy Cows.

: The aim of this study was to determine the effects of dietary vibroactivated clinoptilolite supplementation on the intramammary microbiological findings in dairy cows, causative agents of potential intramammary infection, and their sensitivity to antibiotics. Cows (n = 78) were randomly divided into two groups: CPL-treated group that received clinoptilolite (CPL) in-feed (n = 38), i.e., 50 g natural powdered zeolite CPL, twice daily from the seventh month of pregnancy to 75 days after calving, and the control group (CON) of untreated cows (n = 40). Milk samples were taken from each cow on days 7, 25, 45, and 75 postpartum. The following causative pathogens were isolated in 86 udder quarters: Staphylococcus aureus in 5.81% of positive samples, Staphylococcus spp. 9.32%, coagulase-negative Staphylococcus (CNS) 22.09%, Streptococcus uberis 13.95%, Streptococcus agalactiae 1.16%, Streptococcus sp. 3.49%, Escherichia coli 8.13%, Enterococcus spp. 6.98%, Corynebacterium spp. 11.63%, Pasteurella sp. 10.47%, Serratia spp. 2.33%, and Arcanobacterium pyogenes, Citrobacter sp., Prototheca sp., and yeasts each in 1.16% of samples. Additionally, 3.87% of environmental microflora samples (n = 47) and 89.06% of udder samples (n = 1083) were bacteriologically negative. The most effective antibiotics were cefoperazone and amoxicillin-clavulanic acid, while cloxacillin and tetracycline were the least effective antibiotics in both groups. In the clinoptilolite supplemented (CPL) group (n = 38) of 14 cows, nine causative agents of mastitis were isolated in 27 quarters, while in the control (CON) group (n = 40) of 24 cows, 13 causative agents of mastitis were isolated in 59 quarters. Cows from the CON group had a 1.96 times higher risk of intramammary infection than cows from the CPL group during the observation period (odds ratio = 1.96, p = 0.0031; 95% CI = 1.2570-3.0770).

Trichinella spp. in wild boar (Sus scrofa) populations in Croatia during an eight-year study (2010-2017).

Wild animals represent a constant source of Trichinella spp. infections for domestic animals and humans. To date, four species of Trichinella have been isolated in wild boar populations in Europe: T. pseudospiralis, T. spiralis, T. britovi and T. nativa, in addition to several mixed infection types and one hybrid formation between T. britovi and T. spiralis. Meanwhile, insufficiently thermally processed wild boar meat has been reported to be a source of trichinellosis in humans in several European countries. In Croatia, there have been no reported or proven cases of trichinellosis caused by wild boar meat consumption. The aim of this study was to obtain data on the prevalence of Trichinella species present in Croatia and to anticipated the potential risk of infection for humans in specific Croatian regions based on information obtained over an eight-year surveillance period. A veterinary inspection of wild boar carcasses for Trichinella larvae in Croatia has been mandatory since 1989, and the artificial digestion method was introduced as a compulsory test for wild boar samples in 2008. Based on the official data submitted to the Ministry of Agriculture, Directorate of Veterinary Services, in the period 2010-2017, 303 of 183,184 (0.17%) wild boar meat samples tested positive for Trichinella spp. Infected wild boar were found in 18 of 21 counties. Of these positive samples, 85 were submitted by the authorised veterinary inspectors to the National Reference Laboratory for further examination. The intensity of infection in muscle samples was 0.04-152.66 (mean: 23,37) larvae per gram, and Trichinella species were identified as T. spiralis, T. britovi, T. pseudospiralis and T. spiralis + T. britovi. Genetic analysis of T. pseudospiralis isolates demonstrated their belonging to the Palaearctic population.

Influence of season, rainfall and air temperature on the reproductive efficiency in Romanov sheep in Croatia.

The aim of this study was to investigate the influence of season, rainfall and air temperature on the reproductive efficiency in the Romanov breed of sheep in continental part of Croatia during five consecutive years (2012-2016). During this period, 5379 matings resulted in 5046 successful conceptions, i.e. lambings at eight medium-scale Romanov breed sheep farms. The conception rate was 93.81%, fecundity was 195% and average preweaning mortality until 90 days of age was 12.41%. The seasonal distribution of lambings was 47.64% for ewes that delivered in winter (n = 2422), 23.37% in spring (n = 1179), 18.82% in summer (n = 950) and 9.81% in autumn (n = 495). Sexual activity was lowest during spring and early summer when air temperatures were above average (very and extremely warm), and sexual activity peaked from August to September, especially during extremely wet and very wet seasons. Litter size was greater during winter than in other seasons (1.70 vs. 1.54) and was significantly different as compared to each of selected years of the study period. There was a statistically significant difference in the number of pregnant ewes between mating seasons. Most female Romanov lambs born during winter and early spring mated in late summer or autumn and delivered at the age of 1 year or earlier. The seasonal distribution of matings and lambing was not uniform throughout the seasons over five consecutive years. Thus, it can be assumed that air temperature and rainfall during different seasons could affect the reproductive efficiency in Romanov breed of sheep in continental part of Croatia.

The effects of monthly air temperature and rainfall variations on the reproductive performance and lambing distribution of the Jezersko-Solcava sheep.

The aim of this study was to investigate the effects of monthly air temperature and rainfall variations on the reproductive performance and lambing distribution of the Jezersko-Solcava breed in northwestern Croatia during 7 years period from 2010 until 2016. At 11 medium-scale sheep farms, the sheep were kept on a pasture in a semi-intensive environment with access to the stables. The anomalies of rainfall observed on a monthly basis during monitoring were statistically significant. Non-parametric test showed that the percentages of lambing differ among the months during the year (P = 0.0001). By using regression analysis, it was found that the percentages of pregnant sheep were linearly associated with the sequential number of the month. The seasonal distribution of lambings were 53.85% of ewes delivered in winter (n = 1296), 21.13% in spring (n = 509), 8.91% in summer (n = 215) and 16.11% in autumn (n = 387). Sexual activity was lowest during spring and early summer (from March to May) with a peak from the end of July to October. The conception rate during year 2011 was significantly lower (89.74%), and the litter size was the lowest (1.05) than in other years of the observation. Average conception rate was 92.85% and average litter size 1.21. Despite being a seasonally polyoestrous, the distribution of matings and lambings of this breed of sheep was not uniform throughout the seasons. There were established a positive or negative correlations between the air temperature and rainfall precipitation variations with tested reproductive performances of Jezersko-Solcava breed sheep in the Northwestern part of Croatia.

Estimation of the Withdrawal Time of Levamisole in Eggs after Oral Administration to Laying Hens.

This study was conducted to evaluate withdrawal time of levamisole in eggs after oral administration in laying hens at different doses. Sampling of eggs was conducted for 37 days after the end of treatment, and levamisole concentrations were measured by liquid chromatography-tandem mass spectrometry validated according to the Commission Decision 2002/657/EC. Estimated validation parameters were as follows: decision limit, 0.54 µg/kg; detection capability, 0.56 µg/kg; limit of detection, 0.04 µg/kg; limit of quantification, 0.15 µg/kg; accuracy (recovery), between 92.9 and 102.3%; precision (relative standard deviation), ≤4.62%; and within-laboratory precision (relative standard deviation), ≤5.19%. Levamisole residue levels were significantly higher in egg yolks than in egg whites. The highest levels of levamisole were detected on day 2 posttreatment in groups receiving 50 mg/kg of body weight (556.2 µg/kg in egg yolks and 166.5 µg/kg in egg whites). Significant elimination occurred within 5 days after the cessation of treatment in all groups, with an elimination half-life of 1.3 days. Levamisole was still detectable on day 30 after the end of treatment in egg whites (0.06 µg/kg) and on day 37 in egg yolks (0.06 µg/kg). The longest withdrawal time for levamisole in eggs (14.9 days) was determined in a group treated with 25 mg of levamisole per kg of body weight for two consecutive days. According to the results, oral treatment of laying hens with levamisole may result in noncompliant egg samples even 14 days after treatment.

Manganese Concentrations in Tissues and Skin of Three Dolphin Species Stranded in the Croatian Waters of the Adriatic Sea from 1995 to 2013.

Manganese concentrations were determined in muscle, kidney, liver, spleen, lung and fat tissues and skin of three cetacean species, the bottlenose (Tursiops truncatus), striped (Stenella coeruleoalba) and Risso's (Grampus griseus) dolphins, stranded along the Croatian Adriatic coast between 1995 and 2013. Mean ranges determined in tissues were (mg/kg, ww): muscle 0.23-0.27, liver 2.87-4.00, kidney 0.66-1.26, spleen 0.37-0.79, lung 0.18-0.57, skin 0.11-0.97, fat 0.06-0.42. The highest Mn mean levels in tissues were measured in species (mg/kg, ww): T. truncates lung 0.41, skin 0.97, fat 0.42; S. coeruleoalba muscle 0.26; G. griseus kidney 1.26, liver 4.00, spleen 0.64. Significant differences of Mn concentrations in the liver (p = 0.034), spleen (p = 0.037) and skin (p = 0.013) were found among the three dolphin species. Significant differences in Mn levels were found between young and adult T. truncates in kidney (p = 0.042), lung (p = 0.0040) and skin (p = 0.0034).

Copper Levels in Tissues of Dolphins Tursiops truncatus, Stenella coeruleoalba and Grampus griseus from the Croatian Adriatic Coast.

Copper concentrations were determined in muscle, liver, kidney, spleen and lung tissues of three dolphin species. Dolphins of Tursiops truncatus (young and adult), Stenella coeruleoalba and Grampus griseus were stranded along the Croatian coast. Concentrations in tissues of all three dolphin species were highest in the liver (4.92-16.5 µg/g) followed by kidney (2.85-5.29 µg/g). Similar levels were measured in muscle, spleen and lung in range 1.13-3.67 µg/g. Statistics analysis showed significant differences of Cu concentrations for muscle (p = 0.008), kidney (p = 0.04) and liver (p = 0.02) between the three dolphin species. Also, for all three species significant differences between tissue types of the same species were determined (p < 0.001, all). However, there were no significant differences in Cu levels of the same tissues between males and females within same species. Also, significant differences of body length and weight between three dolphin were found (p < 0.001, both).

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.

Toxic element concentrations in the bottlenose (Tursiops truncatus), striped (Stenella coeruleoalba) and Risso's (Grampus griseus) dolphins stranded in eastern Adriatic Sea.

Concentrations of cadmium (Cd), arsenic (As), mercury (Hg) and lead (Pb) were measured in muscle, liver and kidney of three cetacean species, the bottlenose (Tursiops truncatus), striped (Stenella coeruleoalba) and Risso's (Grampus griseus) dolphins from the Croatian waters of the Adriatic Sea. In all three dolphin species Cd levels decreased in tissues in the order: kidney > liver > muscle, while As and Pb decreased in the order: liver > kidney > muscle for striped and Risso's dolphins, but with the order reversed for liver and kidney in the bottlenose dolphin for Pb. Levels of Hg consistently followed the order: liver > muscle > kidney, with mean concentrations in the liver being 11-34 times higher than in the other tissues. The highest mean concentrations of trace elements were measured in Risso's dolphins at 14.9 µg/g wet weight, for Cd in the kidney, and concentrations in the liver of 2.41, 1,115 and 0.63 µg/g for As, Hg and Pb, respectively. Statistically significant differences between the three dolphin species were determined for Cd, Hg and Pb in liver tissues, for As in muscle and for Cd in kidney. Significant correlations of metals between tissues were determined in all three species. The results presented give an indication of the environmental condition with regard to the content of toxic metals along the eastern coast of the Adriatic Sea.

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.