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Q fever is a zoonotic disease caused by Coxiella burnetii, a gram-negative coccobacillus, which has been detected in a wide range of animal species, mostly domestic ruminants, but also in wild mammals, pets, birds, reptiles, arthropods (especially ticks), as well as in humans. Although the exposure to domestic animals in rural areas is regarded as the most common cause of the disease in humans, recent studies have shown that the role of pets in the epidemiology of Q fever has been increasingly growing. Although the primary route of infection is inhalation, it is presumed that among animals the infection circulates through ticks and that they are responsible for heterospecifi c transmission, as well as spatial dispersion among vertebrates. The aim of this study was to determine the presence and prevalence of C. burnetii in ticks removed from stray dogs, as well as to examine the distribution of tick species parasitizing dogs on the territory of Belgrade city. A PCR protocol targeting IS1111 repetitive transposon-like region of C. burnetii was used for the detection of C. burnetii DNA in ticks and the results were confi rmed by sequence analysis. In total, 316 ticks were collected from 51 stray dogs - 40 females (78.43%) and 11 males (21.57%). Three species of ticks were identifi ed: Rhipicephalus s anguineus (72.15%), Ixodes ricinus (27.53%) and Dermacentor reticulatus (0.32%). Out of 316 examined ticks, C. burnetii DNA was detected only in the brown dog tick R. sanguineus, with a total prevalence of 10.53% (24/228) . The high prevalence of C. burnetii in R. sanguineus, which is primarily a dog tick, indicates the importance of dogs in the epidemiology of Q fever in the territory of Belgrade.

The goal of this study was to compare the results of serological and conventional bacteriological methods with the results obtained using multiplex PCR Bruce-ladder assay. Based on the obtained results, the usability of the assay was assessed in regard to rapid diagnosis of canine brucellosis directly from the samples of reproductive organs of infected dogs. Out of 225 blood samples, 33 (14.67%) had a positive agglutination reaction. In this study, out of the 225 assayed reproductive organs of dogs, B. canis was isolated from 3 samples (1.33%), while the PCR Bruce-ladder assay detected two positive samples (0.88%). Two dogs from which B. canis was isolated, an antibody titer of 1/200 was established in blood serums, and third dog from which B. canis was isolated was negative using the tube agglutination test. From a total of 225 assayed organ samples, a positive PCR reaction was obtained from two samples. The obtained results show that the tube agglutination method remains the first choice for the detection of dogs infected with B. canis. In addition, whenever possible, it is necessary to try isolation. It is desirable to attempt the detection of B. canis in tissues using PCR, but the results may not be treated as definitive and reliable.

The aim of the study was to evaluate and adapt the PCR-based protocol that utilizes the developed serotype-specific primers to identify Salmonella enterica species and its serotypes that are most frequently isolated from poultry samples in Vojvodina. Using the slide agglutination test, 64 and 33 out of 107 Salmonella isolates were identified as S. Infantis and S. Enteritidis, respectively, while ten isolates were identified as eight different Salmonella serovars. Using the same isolates, presence of 993-bp (bcfC gene), 636-bp (steB gene) and 293-bp (sdf locus) amplicons in multiplex PCR unambiguously identified 31 isolates as S. Enteritidis. Two isolates identified as Enteritidis in slide agglutination test were not identified as such in PCR-based approach since they both were missing 293-bp long PCR product. Thirty-nine isolates produced a 727-bp amplicon in the specific simplex PCR, and thus were identified as S. Infantis. The greatest discrepancy in comparison to the results of conventional serotyping has been observed in the case of S. Infantis, since 25 more isolates were noted as S. Infantis by conventional serotyping. Seven isolates, with unexpected PCR profiles stayed unidentified by molecular typing, although they were serotyped as S. Typhimurium (1) and S. Infantis (6). S. Gallinarum serovar has to be additionally confirmed, since it shares the same PCR profile with S. Livingstone. Clearly, PCR-based identification has to be thoroughly checked, verified and adapted if it is to be applied as the routine identification protocol.

Introduction. Bacteria isolated from skin, gills, and fish intestines from aquaculture ponds, ornamental fish stores/aquariums and live fish markets were investigated. Materials and Methods. Disk diffusion and E-test were used for susceptibility testing to carbapenems, ureidopenicillins with or without ß-lactamase inhibitor, 3rd and 4th generation cephalosporins, aminoglycosides, colistin, fluoroquinolones, and chloramphenicol. PCR was used to detect resistance genes in the bacterial isolates. Results and Conclusions. Among the total number of bacteria tested, regardless of the genus and species, 56.7% of isolates were found to be sensitive to all antibiotics, 23.1% of isolates were resistant to one or two antibiotics, and 20.2% of isolates were resistant to three and up to 16 antibiotics. In A. hydrophila isolated from a guppy (Poecilia reticulata) sampled in an ornamental fish store aquarium, 16S rRNA methyltransferase was confirmed by finding the rmtB gene. Pseudomonas isolates showing phenotypic resistance to carbapenems, ureidopenicillins with or without ß-lactamase inhibitor and 3rd and 4th generation cephalosporins were tested and found negative for different resistance genes by PCR (MßL, ESBL, KPC, OXA-23, OXA-24, OXA-40, OXA-58, VIM, IMP, SPM, GIM, NDM, TEM, SHV, CTX-M-1, CTX-M-9, OXA-1, OXA-9 and the AmpC group, as well as single genes, MOXM, CITM, ACCM, EBCM, FOXM, DHAM). Based on E-test results, three Pseudomonas isolates from common carp (Cyprinus carpio) were found to be resistant to colistin with MIC values of 4 μg/mL. [Project of the Serbian Ministry of Education, Science and Technological Development, Grant no. TR 31079 and Grant no. III 45017]

For the purpose of precise antibiotic susceptibility testing it is necessary to clearly distinguish Pseudomonas and Stenotrophomonas genera, considering acquired resistance of Pseudomonas species, as well as the intrinsic resistance of Stenotrophomonas species. This is why in the identification of the 51 isolates originated from fish, the following methods were used: standard PCR, 16S rRNA gene sequencing, and MALDI-TOF.

The objective of this work was to identify isolated Staphylococcus species and to investigate their sensitivity to some antibacterial drugs. The material used for these investigations were Staphylococcus isolates originating from milk samples. A total of 25 strains of Staphylococcus isolates were examined, including 24 from milk samples from cows with mastitis, and one strain was isolated from a milk sample from a cow following treatment for mastitis. For primary identification, catalase and oxidase tests were used, as well as the free coagulase test. Following the preliminary tests, the isolated strains were identified using commercial systems ID32 STAPH (bioMérieux, France) and the BBL Crystal Gram-Positive ID Kit (Becton Dickinson, USA) according to the enclosed instructions. The Staphylococcus isolates were examined for sensitivity to the following: oxacillin, penicillin, cefoxitin, gentamicin, erythromycin, chloramphenicol, tetracycline, ciprofloxacin, sulfametoxazol/trimetoprim, and vacomycin using the disk diffusion method and the broth microdilution method as recommended by the Clinical and Laboratory Strandards Institute - CLSI(2003), and the results were interpreted according to CLSI recommendations from 2008 and 2010. Antibiogram disks manufactured by Becton Dickinson (USA) were used, and the broth microdilution method was applied using pure antibiotic substances from different manufacturers: erythromycin, chloramphenicol, cefoxitin, gentamicin, oxacillin, tetracycline (Sigma Aldrich, USA), sulfametoxazol (Fluka, USA), penicillin (Calbiochem, Germany), vancomycin (Abbott laboratories, USA), ciprofloxacin and trimetoprim (Zdravlje A.D., Serbia). All 25 strains were catalase positive and oxidase negative. Of the 25 strains, 19 were coagulase positive and 6 were coagulase negative.With the implementation of the disk diffusion method on 19 strains of S. aureus, 17 were established to be resistant to penicillin (89.5%), and 2 strains to gentamicin (10.5%). The investigation of 3 strains of S. xylosus using the disk diffusion method showed that one strain was resistant to tetracycline (33.3%) and to oxacillin (33.3%), while another strain was found to be resistant to penicillin (33.3%). The third strain of S. xylosus was sensitive to all the examined antibiotics. Two strains of S. simulans and one strain of S. haemolyticus were not found to be resistant to any of the examined antibiotics using the disk diffusion method. The implementation of the broth microdilution method yielded in 13 strains of S. aureus resistance to penicillin (68.4%) with MIC values from 0.5 to 4 μg/m, in 2 strains to gentamicin (10.5%) with MIC values of 32 μg/ml, and intermediary sensitivity to chloramphenicol was established in 9 strains of S. aureus (47.4%) with MIC values of 16 μg/ml and to vancomycin in 1 strain of S. aureus (5.3%) with MIC values of 4 μg/ml. [Projekat Ministarstva nauke Republike Srbije, br. br 31079]

Antibacterial effects of plant extracts were examined using mixtures of extracts obtained using different technological processes: usnea extract was obtained using the process of supercritical extraction (NKE), angelica extract was obtained through supercritical extraction processes (NKE) and ultrasound extraction using ethanol (UZ), and thyme extract was obtained using the process of hydrodistillation (HD). Mixtures of the listed extracts were examined in various ratios: U (NKE) and T (HD) in a ratio of 1:1, U (NKE) and T (HD) in a ratio of 7:3, U (NKE), T (HD) and A (NKE) in a ratio of 2:2:1, and U (NKE), T (HD) and A (UZ) in a ratio of 2:2:1. The investigations covered 15 strains of bacteria of the genus Staphylococcus, Streptococcus and Enterococus, including the strains MRSA, VRE as well as reference strains of S. pyogenes ATCC 19615, S. agalactiae ATCC 27959 and S. aureus ATCC 11632. The antibacterial action of mixes of plant extracts was examined using the microdilution method in bouillon, and the examined mix concentrations were from 1.25 g/mL to 1280 g/mL. The strongest antibacterial effect was exhibited by mixes of usnea (NKE) and thyme (HD) in ratios of 1:1 and 7:3 with obtained MIC values from 5 g/mL to 160 g/mL, but the MIC value of the listed mixtures for the biggest number of strains amounted to 40 g/mL. A somewhat weaker effect was exhibited by the other examined extract mixtures with obtained MIC values of 10 g/mL to 320 g/mL. Based on the obtained MIC values and the results of previous investigations, it can be concluded that the examined mixtures of plant extracts exhibited a very strong antibacterial effect on the examined bacteria strains. .

The resistance of bacteria poses a significant problem everywhere in the world, and consequently in our country as well. The non-critical use of antibacterial medicines in human and veterinary medicine has contributed to the spreading of this resistance. Due to the topical importance of this problem, large numbers of states in the world are financing projects of which the objective is to follow and monitor bacterial resistance. The objective of this investigation was to isolate and identify pathogenic strains of E. coli from piglets with clinically manifest diarrhea and to examine the sensitivity of the isolated strains to a certain number of selected antibiotics. The material for these investigations were parts of intestines (jejunum, ileum) from piglets that died, rectal smears and feces of diseased piglets sampled pig farms in the vicinity of Belgrade. Conventional methods of microbiological diagnostics were used for isolation, and conventional and commercial tests API 20E (bio Merieux, France) were used for identification. Following biochemical identification using hyper immune serums for certain group (O) antigens: (O8, O138, O139, O147, O149, and O157), the serological typization of the strains was carried out. Commercial antiserums: T K88 (F4), K99 (F5), and 987P (F6), Toxigenic E. coli pili antisera, Denka Seiken Co. Ltd. Tokyo, Japan) were used to establish the presence and to identify fimbrial antigens-adhesions through slide agglutination reaction. The sensitivity of the antibiotics to the isolated strains of E. coli was examined using the disc diffusion method according to Kirby Bauer and the microdillution method in bouillon according to CLSI recommendations (2008). Examinations using the microdillution method in bouillon were performed with pure active antibiotic substances: ampicillin, apramycin, gentamicin, kanamycin, tetracycline, ceftriaxone, and ciprofloxacin (Sigma, USA). A total of 400 E. coli strains were isolated, including 48 E. coli strains that are within the category of pathogenic strains. Out of the total number of pathogenic strains of E. coli, 32 (66.67%) were found to be multi resistant to 3 or more than 3 antibiotics, and 16 (33.33%) pathogenic strains of E. coli were resistant to 2 or 1 of the examined antibiotics. Resistance to tetracycline and ampicillin was established in 89.58% isolated strains of E. coli, and to ciprofloxacin in 37.5% strains. The lowest percentage of resistance to ceftriaxone was established in 4.17% strains of E. coli. The isolated strains of E. coli were also found to be resistant to certain amino glycosides antibiotics. Thus, resistance to apramycin was established in 18.75% strains whose MIC values were higher than 128 µg/mL. Resistance to kanamycin was established in 52.08% strains of E. coli, and in these, 96.00% strains showed MIC kanamycin values of over 256µg/mL. Resistance to the third amino glycosides antibiotic, gentamicin, was established in 33.33% E. coli strains. All the E. coli strains that led to diarrhea in piglets were resistant to at least two antibiotics, and more than 50% strains were found to be resistant to more than 3 antibiotics. It is significant to stress that all isolated strains of E. coli covered by these investigations were sensitive only to amikacyn. .

Investigations of antibacterial effect of supercritical extraction of thyme (TNKE) and thyme extract obtained by hydrodistillation (THD) covered 18 bacteria strains from the genuses Staphylococcus, Streptococcus and Enterococcus, including the strains MRSA, the strains VRE, as well as the reference strains S.pyogenes ATCC 19615, S. agalactiae ATCC 27959, S. aureus ATCC 11632, and S.aureus ATCC 25923. The anibacterial effect of thyme extracts was examined using the microdillution method in bouillon, and the cytotoxic effect of thyme extract was examined on the VERO cell line. The obtained values for MIC THD extract ranged from 160μg/mL to 1280μg/mL for all bacteria strains covered by the investigations. The obtained values for MIC TNKE extract were 1280 μg/mL to 2560 μg/mL for all examined strains except for 1 strain of S. intermedius of 320 μg/mL. The examined extracts revealed a cytotoxic effect on the VERO cell line in concentrations higher than 40 μg/mL.

A total of 1666 samples were examined, of which 512 samples of parenchymatous organs of dead or deliberately sacrtificed animals, 60 samples of non-hatched fertilized eggs, 202 samples of feces, 652 samples of cloacal smears, 221 samples of smears from walls of maintenance objects, incubator stations, and transport vehicles, 19 samples of beddings and shavings. The samples originated from poultry farms and which were taken to a laboratory immediately on sampling and sown the same day. A total of 104 strains of Salmonella were isolated: 94 strains from samples of parenchymatous organs of dead chicks, 1 strain from non-hatched eggs, 3 strains from feces samples, 1 strain from samples of cloacal smears, 4 strains from samples of surface smears of maintenance objects and transport vehicles, and 1 strain from samples of beddings and shavings. Serological typization established the presence of the following serovarieties: Salmonella Enteritidis 79 strains, Salmonella Hartford 17 strains, Salmonella Typohimurium 5 strains, Salmonella Mbandaka 2 strains, and Salmonella Glostrup 1 strain. We examined the sensitivity of Salmonella strains to ampicillin, amoxicillin, gentamycin, streptomycin, neomycin, enrofloxacine, norfloxacine, flumequin, erythromycin, lincospectin, colistin, fluorphenicol, and a combination of sulphamethoxasole and trimethoprim. In S. Enteritidis strains, no resistence was established to colistin, fluorphenicol and sulphamethoxasole+trimethoprim, in fact, the sensitivity to these antibiotics and chemotherapeutics was 100%. Prevalence resitence of 0.96%, in only one strain, was established for enrofloxacine. A high prevalence resistence of 33.6% was established for neomycin, while prevalence resistence of 3.86% was established for the related aminoglycozide antibiotic gentamycin. The highest prevalence resistance in S.Hartford strains was established for erythromycin, 15.38%, and streptomycin, 7.6%. Resistence of S. Tyohimurium was established for flumequin and erythromycin in 1.9% strains. No resistance to antibiotics was established in the strains S. Mbandaka and S. Glostrup.

The presence of multiresistance to the effects of antibiotics and chemotherapeutics and extended spectrum beta-lactamase were examined in 45 strains of E. coli and 35 strains of Salmonella. The strains of E. coli originated from several species of domestic animals: dogs, cats, poultry, and cattle, and 30 strains of Salmonella originated from poultry, 4 strains from cattle, and 1 strain from swine. The presence of the following serovarieties was established using serological examinations: Salmonella Enteritidis 17 strains, Salmonella Gallinarum 1 strain, Salmonella Hartford 5 strains, Salmonella Anatum 1 strain, Salmonella Typhimurium 4 strains, Salmonella Agona 1 strain, Salmonella Infantis 1 strain, Salmonella Thompson var. Berlin 1 strain, Salmonella Tennessee 1 strain, Salmonella Senftenberg 1 strain, Salmonella Glostrup 1 strain, and Salmonella Hadar 1 strain. In the examinations of the listed strains we used antibiogram discs of ampicillin, amoxicillin with clavulanic acid, cephalexin, cephtriaxon, cephotaxim, cephtazidime, aztreonam, gentamycin, chloramphenicol, tetracycline, cyprofloxacine, and a combination of sulphamethoxasole and trimethoprim. The lowest prevalence of multiresistance in E. Coli strains to 3 or more antibiotics was established in dogs 20%, and the highest in 60% strains originating from swine. In 62.88% strains of Salmonella we established sensitivity to all applied antibiotics. Resistance was also established in a small number of the examined strains to ampicillin (11 strains), to tetracycline (5 strains), to amoxicillin with clavulanic acid (5 strains), to sulphamethoxasole with trimethoprim (5 strains), to gentamycin (3 strains), and to cloramphenicol (1 strain). Of all the examined strains of Salmonella, 6 strains originating from poultry exhibited multiresistence. The presence of extended spectrum beta-lactamase effects examined using the ESBL test, was not established in strains of E. coli and Salmonella strains.

The examinations covered numerous clinical and referent strain of both bacteria, as well as viruses and fungi. The paper presents only one part of the examinations in which referent strains of E. coli NTCC 8116, Proteus mirabilis ATCC 19086, Staphylococcus aureus ATCC 511, Salmonella Choleraeuis ATCC 10653, Enterococcus faecalis ATCC 6055 and Bacillus cereus ATCC 11778 were used. The effect of Peral-S in concentrations of 0.25%, 0.5% and 1% on the listed strain of bacteria was examined using the suspension method at exposition times of 5, 10, 15 and 20 minutes. Examinations were also performed of the antiviral effects of Peral-S in concentrations of 0.1% to 1% and at exposure times from 30 seconds to 5 minutes on the viruses Aujeszky 257/II, P13, Newcastle virus, Coxackie B6 , Herpes simplex type I. The efficacy of the antiviral effects of Peral-S was checked on tissue cultures MDBK, AUBEK and HEP-2 using the floating technique method. Examinations also covered the fungicidal effect of Peral-S in concentrations of from 0.1% to 1%, and of 3%, 4%, and 5% at exposure times of 5, 15 and 60 minutes and cultures aged 1 to 14 days. The investigations covered fungi from the following genera: Penicillium spp., Aspergillus spp. and Mucor spp. Peral-S showed a bactericidal effect on all the examined bacteria strains, in all the applied concentrations and at all exposure times. Peral-S in a concentration of 0.1% and at an exposure time of 30 seconds had a vurcidal effect on all examined viruses. How-ever, there was no fungicidal effect of Peral-S in concentrations of 0.1% to 1% at all expo-sure times on fungi cultures aged from one to 14 days. The preparation did exhibit a fungicidal effect in a concentration of 3% in all examined fungi cultures after 60 minutes of expo-sure, while it exhibited a fungicidal effect in a concentration of 4% after 5 minutes of expo-sure on all examined fungi cultures, regardless of their age.