BACTERIA IN ANIMAL FEED AND THEIR SUSCEPTIBILITY TO ANTIMICROBIAL DRUGS

Control of microbiological safety of animal feed is regulated by a law. The regulation is based on methodological standards for estimation of feed quality. Those methods are permanently amended and strive for continuous improvement of microbiological safety of feed. The question remains whether the presence or absence of some bacterial species is sufficient for proper assessment of feed safety. The answer to this question can be found not only in the examination of the number of bacteria in animal feed, but also in qualitative analysis of the isolated microorganisms. The sensitivity of bacteria from animal feed to antimicrobials is one of the qualitative characteristics of microorganisms that can affect food safety even if the feed meets the standards and is within the maximum permitted level. The subject of our work was to determine the presence of bacteria in animal feed with a purpose to examine if they possess lower sensibility to antibiotics. For testing antibiotic sensitivity the isolates from routine microbiological feed control were used. The animal feed was analysed by the standard methods for the isolation and identification of bacteria and determination of susceptibility was done using disc diffusion method according to CLSI. The following bacteria were detected: Escherichia coli, Enterobacter sp., Staphylococcus sp., Micrococcus sp., Bacillus cereus, Bacillus sp. The results showed that some isolates have reduced susceptibility to certain antimicrobial drugs (The resistance is different for different antibiotics and microorganism from 8,33% to 100%). This finding gives a reason for further examination of certain characteristics of the bacteria found in animal feed and to detect their possible influence on ecology.

not only in the examination of the number of bacteria in animal feeđ, but also in qualitative analysis of the isolated microorganisms. The sensitivity of bacteria from animal feeđ to antimicrobials is one of the qualitative characteristics of microorganisms that can affect foođ safety even if the feeđ meets the stanđarđs anđ is within the maximum permitted level. The subject of our work was to đetermine the presence of bacteria in animal feeđ with a purpose to examine if they possess lower sensibility to antibiotics. For testing antibiotic sensitivity the isolates from routine microbiological feed control were used. The animal feed was analysed by the standard methođs for the isolation and iđentification of bacteria and determination of susceptibility was done using đisc diffusion method according to CLSI. The following bacteria were đetecteđ: Escherichia coli, Enterobacter sp., Staphylococcus sp., Micrococcus sp., Bacillus cereus, Bacillus sp. The results showeđ that some isolates have ređuceđ susceptibility to certain antimicrobial đrugs (The resistance is đifferent for đifferent antibiotics anđ microorganism -from 8,33% to 100%). This finding gives a reason for further examination of certain characteristics of the bacteria founđ in animal feeđ anđ to đetect their possible influence on ecology.

IN T R O D U C TIO N
Different types of microorganisms can be found in feed. The microorganisms can be transmitted in different ways from the ground: by wind, rain and insects or by mechanical treatment (K.G. Maciorowski et al., 2007). Since the feed is the first critical point in the food production chain, it is necessary permanently to control the feed in terms of its safety. A detailed microbiological control of feed is described in the current legislation ("Official Gazette" SFRJ 25/1980). This refers also to the methods of testing the total number of bacteria (TNB). In this way an entry of certain pathogens and zoonotic microorganisms in the feed is prevented. In indirect way, this ensures the microbiological safety of food of animal origin which can become a potential source of human infection. The standard methods for feed control and allowed limits in terms of number of saprophytic microorganisms are available.
The number of saprophytic microorganisms permitted by regulation is different depending on the type of feed and animal category for which the feed is intended. Tegal acts determine only the quantity of saprophytic bacteria but the qualitative characteristics of saprophytes can be overlooked. On the other hand, saprophytes are living organisms that can posses some undesirable properties. They can also transfer some of their properties to microorganisms in the digestive tract of animals. A qualitative analysis of saprophytic bacteria can contribute in preventing the transfer of undesirable features. An example is the transfer of resistance genes to microbes that reside in animals. The increased selection of the resistant bacteria in animals and humans and the spread of genes that саггу these characteristics may affect ecosystem (Teila Soufi et al., 2011). For these reasons, the subject of our research was related to monitoring the presence of bacteria in animal feed in order to determine whether the isolated bacteria posses the decreased sensitivity to antibiotics.

M A TERIA L AND M ETH O D S
Animal feed was analyzed for six months in the second half of 2011. The obtained samples originated from feed factories that regularly check their products. A total of 26 samples was examined for different animal categories. Total mix used in poultry fattening and grower of breeding hens (starter, grower, finisher), as well as the mix for calves and food supplements (vitamin and mineral, vitamin and protein) were examined. The samples were processed according to the methodology described in the Ordinance on the methods of performing microbiological analysis and super nutritional support life ("Olficial Gazette" SFRJ 25/1980). The samples were homogenized and weighed to 20 g in the Erlenmeyer flask bottle. A sterile physiological saline solution was added to the volume of 180 mT to obtain abasic dilution 10 '. From this dilution 1 ml was taken and placed directly on blood agar (agar with 5% defibrinated sheep blood) and МсСопкеу agar. Identification of bacterial isolates was done after Gram stain and microscopic examination was performed by determining the physiological characteristics of bacteria through a series of biochemical tests (catalase test, Methyl red, indol producton, urease, citrate, mannitol fermentation) (Quinn et al., 2002). The sensitivity of isolated strains was done as recommended by CLSI (2006). We used antibiotic disks Tetracycline (30 pg), ampicilline (10 pg), streptomycin (10 pg), Ceftriaxone (30 pg), Trimethoprim + sulfamethoxazole (25 pg (trimetoprim 1,25 pg + sulamethox-azole 23.75 mg)), chloramphenicol (30 pg), lincomycin (2 pg), enrofloxacin (5pg), amoxicillin (30 pg (amoxiciline 20 pg + 10 pg clavulonic acid)) (produced by Bioanalyse) while Lincospectin (109 pg (lincomycin 9 pg + Spektinomicin 100 pg) discs were from the producer Bioanalyse, (Bioanalyse Ltd.).

RESULTS A N D DISCUSSION
From a total of 26 processed materials 62 isolates were detected. Within the total number of isolated bacteria six different bacterial species were confirmed. Our study was limited to the obtained isolates and the sensitivity to antimicrobial drugs is given in Table 1.
Among Enterobacteriacae Escherichia coli and Enterobacter sp were most frequently found. These microorganisms were represented with 62.85% of total isolates. Bacillus sp" Staphylococcus sp. and Micrococcus sp. are microorganisms that are widespread in nature and are part of the saprophytic microbiota of humans, animals and the external environment. Flavobacter sp. belongs to a group of microorganisms that are found as commensals in the soil and their findings may indicate that the feed is contaminated and has been, directly or indirectly, in contact with bacteria of the soil.
The results showed that E. coli and Enterobacter sp. as the predominant microorganisms, contaminating feed, саггу a high resistance to certain antimicrobial drugs. These two species showed complete resistance to lincomycin. This may be due to the long use of antimicrobial products as growth promoters or in prophylactic purposes (Frank M. Aarestrup, Henrik C.Wegener 1999; Torrence E. Магу, 2001). An extraordinary high resistance (75-100%) to lincomycin and the fact that 50% or more isolates were resistant to ampicillin, amoxicillin and lincospectin is surprising since it is obvious that the strains isolated from food animals were in contact with the antimicrobial drugs long enough to develop resistance. Equally important would be to determine the sources of microorganisms from animal feed because of their high resistance to certain antimicrobial drugs that can occur only in contact with these agents or through horizontal transfer of the genome responsible for resistance (K.G. Maciorowski et al., 2007, Velhner Maja et ак, 2010. The data obtained are similar to the study from Portugal (Paulo Martins da Costa et al., 2007) where the resistance in E. coli was relatively low (29.9% ampicillin, tetracycline 27.7%) or absent for amoxiclav. The study also shows that the resistance to three and more antibiotics was present in 18.1% of isolates.   Maciorowski et al., 2007). Also it has been observed that some resistance of Flavobacter to antimicrobial drugs may indicate that antibiotic substance came in soil where they have caused the appearance of resistance. On the other hand, it is possible that the emergence of resistance occurred as a result of the spread of bacteria among which the horizontal resistance gene transfer may happen.

CO N CLU SIO N
Control of feed in respect to quantitative microbiological examination meets the standards that provide the limits of permissible and impermissible microorganisms. Our study has shown that the qualitative analysis ofbacterial isolates contributed to a higher degree of food safety since it can provide information about the reduced sensitivity of bacteria to antimicrobial agents, a possible elimination from the food chain which leads to improvement of ecology.