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Problem of bacteria resistance on antimicrobial agents is not as simple as in the first days of their application. Increased use of antibiotics and chemotherapeutics contributed that a lot of bacteria strains show not only resistance, but also a multiple resistance to a great number, in some cases even to all used antimicrobial agents. Having that in mind a great many of scientific investigations pointed out a continual appearance and spreading of' bacteria resistance sweet as to a justified fear of fatal infectious diseases emerging, without possibility of an efficient antimicrobial therapy. Therefore, the problem becomes very relevant on both local and global levels, especially if it is well known that antibiotics and chemotherapeutics have the effect both on pathogenic and saprophyte bacteria, leading into a growth suppression or elimination of sensitive strains. This characteristic represents both health and ecological problem because of favoring growth of a small number of resistant bacteria strains. Continual and strong selective pressure of antimicrobial agents used in human veterinary medicine, together with genetic abilities of bacteria brings about emergence of greater number of resistant pathogenic bacteria strains. In intensive farming the emergence of greater number of resistant pathogenic bacteria strains is strengthened by antibiotics and chemotherapeutics application in diseases prevention, as well as growth factor, and in healing, but in larger quantities. For these reasons, the prevalence of bacteria resistance in domestic animals, which is in direct correlation with quantity of applied antimicrobial agents, is discussed. This is especially significant in case of domestic animals which products are used in human nutrition. Problem rezistencije bakterija na antimikrobna sredstva nije tako jednostavan kao u pionirskim danima njihove primene. Velika upotreba antibiotika i hemioterapeutika doprinela je da mnogi sojevi bakterija ispoljavaju ne samo rezistenciju već i multipnu rezistenciju na veći broj, a u nekim slučajevima i na sva korišćena antimikrobna sredstva. Imajući to u vidu, brojna naučna istraživanja ukazuju na kontinuirano pojavljivanje i širenje rezistencije kod bakterija i opravdan strah od pojave fatalnih injektivnih oboljenja bez mogućnosti efikasne antimikrobne terapije. Stoga je problem aktuelan ne samo na lokalnom već i na globalnom nivou, posebno ako se zna da antibiotici i hemioterapeutici deluju ne samo na patogene već i na saprofitne bakterije dovodeći do supresije rasta ili eliminisanja osetljivih sojeva. Ova njihova osobina predstavlja ne samo zdravstveni već i ekološki problem zbog favorizovanja rasta malobrojnih rezistentnih sojeva bakterija. Kontinuiran i jak selektivni pritisak antimikrobnih sredstava korišćenih u humanoj veterinarskoj medicini i poljoprivredi, udružen sa genetskim sposobnoslima bakterija uzrokuje pojavu sve većeg broja rezistentnih sojeva patogenih bakterija. U intezivnim uslovima držanja životinja, pojava većeg broja rezistentnih sojeva patogenih bakterija je još vise potencirana primenom antibiotika i hemioterapeutika u preveniranju bolesti, zatim kao faktora rasta i u lečenju, ali u mnogo većim količinama. Iz tih razloga se i govori o prevalenciji rezistencije bakterija kod domaćih životinja koja je u direktnoj korelaciji sa količinom utrošenih antimikrobnih preparata. Ovo posebno ima značaja kada se radi o domaćim životinjama čiji se proizvodi koriste u ishrani ljudi.

Samples (30) of feces of calves, piglets, guilds and pregnant sows with signs of diarrhea, as well as rectal smears, received from farms, where related problems were followed for three months, were used in these investigations. The material was processed so as to isolate microorganisms using the usual microbiological methods (microscopy, sowing cultures on bouillon, Triptose agar gel with 6% sheep's blood, MacConkey agar and Saburaud dextrose agar). After clean cultures were obtained, biochemical identification was performed using commercial ID kits (BBL Crystal Entero/nonfermenter ID kit, Bee-ton Dickinson, USA), as well as standard biochemical series. Resistance, or sensitivity of isolated strains of E.coli, were examined using the disk diffusion method according to Kirby Bauer on Mueller Hinton agar, using antibiogram tablets (Torlak, Belgrade) and antibio-gram discs (Becton Dickinson, USA). Investigations covered'17 antimicrobial preparations ampicillin, amoxicillin with clavulonic acid, tetracycline, chloramphenicol neomycin, streptomycin, gentamycin, trimetoprim, sulphamethoxasole trimetoprin + sufametoxasole, cephalexin, cephachlor, cephtriaxone spectinomycin, cholistin, nalidixin acid, ciprofloxacin. The efficacy and choice of the used antibiotic againstthe isolated strain was performed according to the standard recommendations of the National Committee for Clinical Labora- tory Standards (NCCLS), USA, as well as on the grounds of the instructions of the disc manufacturers. The established prevalence of resistant strains of E.coli to certain antibiotics and chemotherapeutics differed depending on animal age and specie. On the grounds of the analyses of the obtained prevalence of resistant strains in diseased, healthy, young and old animals, it is possible to detect the application of a certain antibiotic in prophylaxis and therapy. At the same time, the obtained results indicate the inadequate and uncontrolled administration of antibiotics to both claves (85%) and piglets (95%), which leads to the high prevalence not only of the resistant, but also of multiresistant strains. U ispitivanjima kao materijal korišćeni su uzorci (30 uzoraka) fecesa teladi, prasadi, nazimica i suprasnih krmača sa znacima dijareje, kao i rektalni brisevi dopremani sa farmi na kojima je problematika praćena tokom tri meseca. Materijal je obrađivan radi izolovanja mikro-organizama uobičajenim mikrobiološkim metodama (mikroskopski, kulturelno-zasejavanjem na hranljivi bujon, Triptozni agar sa 6 % ovčje krvi, MacConkey agar i Saburaud dekstrozni agar). Nakon dobijanja čistih kultura obavljena je biohemijska identifikacija uz primenu komercijalnih ID kitova (BBL Crystal Entero/nonfermenter ID kit, Becton Dickinson, USA), kao i standardnih biohemijskih nizova. Ispitivanje rezistencije, odnosno osetljivosti izolovanih sojeva E. coli obavljeno je disk difuzionom metodom po Kirby Baueru na Mueller Hinton agaru uz korišćenje antibiogram tableta (Torlak Beograd) i antibiogram diskova (Becton Dickinson, USA). Ispitivanjima je bilo obuhvaćeno 17 antimikrobnih sredstava: ampicllin, amoksicilin sa klavulonskom kiselinom, tetraciklin, hloramfenikol, neomicin, streptomicin gentamicin, trimetoprim, sulfametoksazol, trimetoprim + sufametoksazol cefaleksin, cefahlor, ceftriakson, spektinomicin, kolistin, nalidiksinska kiselina i ciprofloksacin. Procena efikasnosti i izbor korišćenog antibiotika u odnosu na izolovani soj izvedeni su prema standardnim preporukama NCCLS (The National Committee for Clinical Laboratory Standards USA), kao i na osnovu uputstva proizvođača diskova. Ustanovljena prevalencija rezistentnih sojeva E. coli prema određenim antibioticima i hemoterapeuticima razlikovala se zavisno od starosti i vrste životinja. Na osnovu analize dobijene prevalencije rezistentnih sojeva kod bolesnih, zdravih, mladih i starijih životinja može da se otkrije primena određenog antibiotika u profilaksi i terapiji. Istovremeno dobijeni rezultatii ukazuju na neadekvatnu i nekontrolisanu primenu antibiotika kako kod teladi (85%) tako i kod prasadi (95%) što dovodi do visoke prevalencije ne samo rezistentnih, već i multirezistentnih sojeva.

U ovom ispitivanju vršeno je uzorkovanje briseva poreklom od klinički zdravih riba koje su poticale iz različitih sredina (ribnjaci, akvarijumi, riblje pijace). Izvršena je izolacija bakterija koje su sastavni deo mikrobioma kože, škrga i creva riba i ispitivana je osetljivost ovih bakterija na određeni broj antibiotika koji se koriste u veterinarskoj i humanoj medicinskoj praksi. Precizna identifikacija ispitivanih sojeva bakterija vršena je primenom metoda PCR, sekvenciranje gena za 16S rRNK, MALDI-TOF. Primenom disk difuzionog testa i E testa ispitivano je fenotipsko ispoljavanje rezistencije na karbapeneme, ureidopeniciline sa i bez inhibitora betalaktamaza, cefalospirine III i IV generacije, aminoglikozide, tetraciklin, kolistin, flurohinolone i hloramfenikol. Prisustvo gena rezistencije, njihova lokalizacija (na hromozomu ili na mobilnim genetičkim elementima) vršena je primenom metode PCR. Kod sojeva koji su ispoljili rezistenciju na nabrojane antibiotike traženi su plazmidi i ispitivana je konjugabilnost izolovanih plazmida. Posmatrano na ukupan broj ispitanih sojeva u ovom istraživanju, bez obzira na rod i vrstu bakterija, ukupno je nađeno 55% sojeva koji su bili osetljivi na sve antibiotike, kod 22,8% sojeva nađena je rezistencija na 3 do 16 antibiotika uključujući i antibiotike koji se koriste isključivo kod ljudi (karbapenemi, ureidopenicilini, cefalosporini III i IV generacije). Ukupno 22,2% sojeva bilo je rezistentno na 1 do 2 antibiotika, mada je i među tim sojevima bilo onih koji su bili rezistentni na antibiotike registrovane samo za upotrebu kod ljudi (ceftazidim, piperacilin). Kod soja A. hydrophila izolovanom iz akvarijumske ribice gupi potvrđen je mehanizam rezistencije nalazom gena rmtB koji je bio lokalizovan na transpozonu Tn1548 smeštenom na konjugabilnom plazmidu koji je po tipu replikona bio kategorisan u grupu IncL/M. Kod sojeva Pseudomonas koji su bili rezistentni na karbapeneme, ureidopeniciline sa i bez inhibitora betalaktamaza, kao i na cefalosporine III i IV generacije, nisu nađeni geni za, karbapenemaze, MßL, ESBL, OXA i AmpC beta-laktamaze (KPC, OXA-23, OXA- 24, OXA-40, OXA-58, VIM, IMP, SPM, GIM, NDM, TEM, SHV, CTX-M-1, CTXM- 9, OXA-1, OXA-9, AmpC grupni kao i pojedinačni-MOXM, CITM, ACCM, EBCM, FOXM, DHAM).Na osnovu dobijenih rezultata primenom E testa, kod 3 soja iz roda Pseudomonas izolovanih od šarana nađena je rezistencija na kolistin sa dobijenim vrednostima MIK 4 μg/mL. During this research, a series of microbiological smears was collected from clinically healthy fish found in different environments (aquaculture ponds, aquariums, and fish markets) has been done. Bacteria which belong to skin microbiome, gills, and fish intestines have been isolated, and their sensitivity to several antibiotics used in veterinary and human practice has been tested. Precise identification of tested strains of bacteria has been performed with PCR method, 16S rRNA gene sequencing and MALDI-TOF. Phenotypical manifestation of resistance to carbapenems, ureidopenicillins (with and without inhibitors of beta-lactamase), cephalosporins of the third and fourth generation, aminoglycosides, tetracyclines, colistin, fluoroquinolones and chloramphenicol has been tested using disc diffusion method and E test. Presence of resistant genes, their localization (on chromosome or on mobile genetic elements) has been conducted with PCR method. For strains showing resistance to the antibiotics mentioned above, plasmids have been searched and conjugation of isolated plasmids has been tested. Observing the total number of tested strains in this research, regardless of the genus and species of bacteria, 55% of examined strains were found to be sensitive to all antibiotics, and in 22.8% of strains resistance was observed to 3-16 antibiotics, including antibiotics used exclusively in human medicine (carbapenems, ureidopenicillins, cephalosporins of third and fourth generation). In additional 22.2% of strains the resistance to 1 or 2 antibiotics was recorded, including resistance to antibiotics registered for exclusive use in human medicine (ceftazidime, piperacillin). In A. hydrophila strain isolated from aquarium fish guppy that showed resistance to all 16 antibiotics, a mechanism of resistance has been confirmed by identifying gene rmtB which has been localized on transposon Tn1548 located on conjugal plasmid which belongs to group IncL/M type of replicon. In Pseudomonas strains resistant to carbapenems, ureidopenicillins (with and without inhibitors of beta-lactamase), cephalosprins of third and fourth generation, the genes for carbapenemases MßL, ESBL, OXA and AmpC beta-laktamases (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, group AmpC and specific MOXM, CITM, ACCM, EBCM, FOXM, DHAM) have not been found. Based on results obtained with use of E test, resistence to colistin has been found in 3 strains of Pseudomonas spp. isolated from carp with MIC values of 4 μg/mL.

During this research, a series of microbiological smears was collected from clinically healthy fish found in different environments (aquaculture ponds, aquariums, and fish markets) has been done. Bacteria which belong to skin microbiome, gills, and fish intestines have been isolated, and their sensitivity to several antibiotics used in veterinary and human practice has been tested. Precise identification of tested strains of bacteria has been performed with PCR method, 16S rRNA gene sequencing and MALDI-TOF. Phenotypical manifestation of resistance to carbapenems, ureidopenicillins (with and without inhibitors of beta-lactamase), cephalosporins of the third and fourth generation, aminoglycosides, tetracyclines, colistin, fluoroquinolones and chloramphenicol has been tested using disc diffusion method and E test. Presence of resistant genes, their localization (on chromosome or on mobile genetic elements) has been conducted with PCR method. For strains showing resistance to the antibiotics mentioned above, plasmids have been searched and conjugation of isolated plasmids has been tested. Observing the total number of tested strains in this research, regardless of the genus and species of bacteria, 55% of examined strains were found to be sensitive to all antibiotics, and in 22.8% of strains resistance was observed to 3-16 antibiotics, including antibiotics used exclusively in human medicine (carbapenems, ureidopenicillins, cephalosporins of third and fourth generation). In additional 22.2% of strains the resistance to 1 or 2 antibiotics was recorded, including resistance to antibiotics registered for exclusive use in human medicine (ceftazidime, piperacillin). In A. hydrophila strain isolated from aquarium fish guppy that showed resistance to all 16 antibiotics, a mechanism of resistance has been confirmed by identifying gene rmtB which has been localized on transposon Tn1548 located on conjugal plasmid which belongs to group IncL/M type of replicon. In Pseudomonas strains resistant to carbapenems, ureidopenicillins (with and without inhibitors of beta-lactamase), cephalosprins of third and fourth generation, the genes for carbapenemases MßL, ESBL, OXA and AmpC beta-laktamases (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, group AmpC and specific MOXM, CITM, ACCM, EBCM, FOXM, DHAM) have not been found. Based on results obtained with use of E test, resistence to colistin has been found in 3 strains of Pseudomonas spp. isolated from carp with MIC values of 4 μg/mL. U ovom ispitivanju vršeno je uzorkovanje briseva poreklom od klinički zdravih riba koje su poticale iz različitih sredina (ribnjaci, akvarijumi, riblje pijace). Izvršena je izolacija bakterija koje su sastavni deo mikrobioma kože, škrga i creva riba i ispitivana je osetljivost ovih bakterija na određeni broj antibiotika koji se koriste u veterinarskoj i humanoj medicinskoj praksi. Precizna identifikacija ispitivanih sojeva bakterija vršena je primenom metoda PCR, sekvenciranje gena za 16S rRNK, MALDI-TOF. Primenom disk difuzionog testa i E testa ispitivano je fenotipsko ispoljavanje rezistencije na karbapeneme, ureidopeniciline sa i bez inhibitora betalaktamaza, cefalospirine III i IV generacije, aminoglikozide, tetraciklin, kolistin, flurohinolone i hloramfenikol. Prisustvo gena rezistencije, njihova lokalizacija (na hromozomu ili na mobilnim genetičkim elementima) vršena je primenom metode PCR. Kod sojeva koji su ispoljili rezistenciju na nabrojane antibiotike traženi su plazmidi i ispitivana je konjugabilnost izolovanih plazmida. Posmatrano na ukupan broj ispitanih sojeva u ovom istraživanju, bez obzira na rod i vrstu bakterija, ukupno je nađeno 55% sojeva koji su bili osetljivi na sve antibiotike, kod 22,8% sojeva nađena je rezistencija na 3 do 16 antibiotika uključujući i antibiotike koji se koriste isključivo kod ljudi (karbapenemi, ureidopenicilini, cefalosporini III i IV generacije). Ukupno 22,2% sojeva bilo je rezistentno na 1 do 2 antibiotika, mada je i među tim sojevima bilo onih koji su bili rezistentni na antibiotike registrovane samo za upotrebu kod ljudi (ceftazidim, piperacilin). Kod soja A. hydrophila izolovanom iz akvarijumske ribice gupi potvrđen je mehanizam rezistencije nalazom gena rmtB koji je bio lokalizovan na transpozonu Tn1548 smeštenom na konjugabilnom plazmidu koji je po tipu replikona bio kategorisan u grupu IncL/M. Kod sojeva Pseudomonas koji su bili rezistentni na karbapeneme, ureidopeniciline sa i bez inhibitora betalaktamaza, kao i na cefalosporine III i IV generacije, nisu nađeni geni za, karbapenemaze, MßL, ESBL, OXA i AmpC beta-laktamaze (KPC, OXA-23, OXA- 24, OXA-40, OXA-58, VIM, IMP, SPM, GIM, NDM, TEM, SHV, CTX-M-1, CTXM- 9, OXA-1, OXA-9, AmpC grupni kao i pojedinačni-MOXM, CITM, ACCM, EBCM, FOXM, DHAM).Na osnovu dobijenih rezultata primenom E testa, kod 3 soja iz roda Pseudomonas izolovanih od šarana nađena je rezistencija na kolistin sa dobijenim vrednostima MIK 4 μg/mL.