SEROLOGICAL CHARACTERISTICS AND ANTIMOCROBIAL RESISTANCE OF SALMONELLA ISOLATES , RECOVERED FROM ANIMAL RAW MATERIAL

INTRODUCTION Currently, Salmonellosis is widely spread in many coun­ tries around the world. It is one of the major infectious diseases and is of a great veterinary and medical concern due to the risk of infection transmission to humans from sick animals and through food. Salmonellosis is an infec­ tious disease caused by numerous serotypes of bacteria of the genus Salmonella, characterized by a variety of clini­ cal manifestations from asymptomatic carrier and mild forms of gastroenteritis to severe generalized forms of the disease, occurring with pronounced intoxication and prolonged fever [1, 2]. Phylogenetic analysis shows that Salmonella belongs to the family of enterobacteria (Enterobacteriacea), the γ class of Proteobacteria, the genus Salmonella, which consists of phenotypically and genotypically related micro organisms. Based on the genomic analysis, two spe­ cies are distinguished in modern classification – S. bongori and S. enterica. S. bongori is small and is composed of only 10 rarely encountered serovars (serotypes); S. enterica in­ cludes about 2,500 serovars. Each Salmonella serovar is further classified into biovars and phage types. Herewith, new Salmonella serotypes are isolated annually in national reference centers (40–60 per year) and their epidemiology is studied [4, 18]. Divergence in the nucleotide sequence of ortho­ logous genes ranges between 3.8 and 4.6% and diffe­ rences in their deduced amino acid sequences range be­ tween 0.7 and 1.3%. This close DNA relatedness among Salmonella serotypes is evidence for their clonal origin, and based on the degree of sequence divergence, it can SUMMARY Salmonella continues to be the primary cause of foodborne intestinal infections in many countries around the world. According to the official data, 47% of the infection outbreaks in the world are associated with salmonellosis, while chicken meat (34%) plays a significant role in the infection transmission to humans through food. Since the early 90’s of the last century, with the massive use of antibiotics, Salmonella strains resistant to a number of antimicrobials began to appear and currently pose a serious public health problem. Resistant strains persistent in animals can be transmitted to humans through the food chain. The paper presents results of studies of morphological, biochemical, serological properties of Salmonella bacteria recovered from animal raw material: beef, pork, poultry meat, tallow, offal derived from broiler chickens and pig slaughter products. In 2018 the FGBI “ARRIAH” Microbiological Laboratory performed 1,204 tests of animal raw material for Salmonella bacteria and recovered 45 Salmonella isolates. Salmonella bacteria were isolated in accordance with GOST 31659-2012 (ISO 6579:2002). Most Salmonella isolates (56%) were recovered from poultry meat. Biological properties of all the studied isolates were quite typical: they formed hydrogen sulfide, fermented glucose and mannitol with the formation of gas and acid, did not utilize sucrose, lactose and urea; reaction to indole was negative. It was established that the recovered Salmonella isolates belong to serogroups O7, O9, O5, O4. The frequency of recovering Salmonella group B was 8.9%, group C – 51.1%, group D – 40.0%. Among Salmonella group B, S. derby (4.4%) and S. typhimurium (2.2%) were more common; group C – S. infantis (29.0%), S. virchow (17.8%); group D – S. enteritidis (40.0%). Isolated cases of S. reading (2.2%) and S. oranienburg (4.4%) were observed. All Salmonella isolates recovered from raw material of animal origin demonstrated sensitivity to ciprofloxacin, chloramphenicol, amoxicillin, amikacin, azithromycin, meropenem, gentamicin, ceftriaxone, kanamycin; were less sensitive to cefotaxime, ampicillin, levofloxacin and had low sensitivity to nalidixic acid, doxycycline, streptomycin, tetracycline. The phenomenon of multiresistance is characteristic of 44.4% of the isolated Salmonella isolates.


INTRODUCTION
Currently, Salmonellosis is widely spread in many coun tries around the world. It is one of the major infectious diseases and is of a great veterinary and medical concern due to the risk of infection transmission to humans from sick animals and through food. Salmonellosis is an infec tious disease caused by numerous serotypes of bacteria of the genus Salmonella, characterized by a variety of clini cal manifestations from asymptomatic carrier and mild forms of gastroenteritis to severe generalized forms of the disease, occurring with pronounced intoxication and prolonged fever [1,2].
Phylogenetic analysis shows that Salmonella belongs to the family of enterobacteria (Enterobacteriacea), the γ class of Proteobacteria, the genus Salmonella, which consists of phenotypically and genotypically related micro organisms. Based on the genomic analysis, two spe cies are distinguished in modern classification -S. bongori and S. enterica. S. bongori is small and is composed of only 10 rarely encountered serovars (serotypes); S. enterica in cludes about 2,500 serovars. Each Salmonella serovar is further classified into biovars and phage types. Herewith, new Salmonella serotypes are isolated annually in national reference centers (40-60 per year) and their epidemiology is studied [4,18].
Divergence in the nucleotide sequence of ortho logous genes ranges between 3.8 and 4.6% and diffe rences in their deduced amino acid sequences range be tween 0.7 and 1.3%. This close DNA relatedness among Salmonella serotypes is evidence for their clonal origin, and based on the degree of sequence divergence, it can be estimated that a common ancestor of the genus existed about 25 to 40 million years ago [4,13].
Salmonella species are facultative intracellular parasites capable of penetrating (invading) and surviving within different cell types, escaping from the destructive power of phagocytosis, and spreading throughout the body via the systemic circulation. After phagocytosis by neutrophils and macrophages, Salmonella survive and replicate within special vacuoles. Most Salmonella serovars do not contain virulence plasmids, while the most medically important ones (including Typhimurium, Enteritidis, and Cholera e suis) do [13].
Salmonella has factors of adhesion and colonization, factors of invasion; they have endotoxin and S. typhimu r ium and some other serotypes can synthesize two types of exotoxins: heatlabile (LT) and heatstable (ST) entero toxins, shigalike cytotoxins. A specific feature of toxins is intracellular localization and isolation after the destruction of bacterial cells [6].
Adhesion is mediated by fimbriae (pili) found on the outer membrane of bacteria. Salmonella genome en codes acid shock proteins that are important for survival at low pH values, so that Salmonella remains viable in the acidic environment of the stomach, before reaching the areas in the gastrointestinal tract suitable for colonization. Salmonella causes three forms of food poisoning in hu mans: gastroenteric, choleralike, and flulike. Thus, human is the only natural host and reservoir for Typhi and Para typhi A serovars. These serovars cause systemic infections infections in humans -typhoid and paratyphoid. The Galli narum and Pullorum serovars are isolated from birds; the Dublin serovar causes severe systemic infection in cattle and can cause illness in humans. The situation is similar with the Choleraesuis and Typhisuis serovars isolated from pigs, and with the Abortusovis serovar -the causative agent of sheep Salmonellosis. The factors that contribute to the establishment of the carrier have not been studied much, but their dependence on the serovar is observed. From the total number of typhoid fever cases not treated with antibiotics, 10% of patients secrete S. typhi with fe ces for 1-3 months and 2-5% of patients become chronic carriers of Salmonella. Nontyphoid serovars persist in the gastrointestinal tract of warmblooded animals for an ave rage of 1.5-3.0 months, however, carriers are detected only in 0.1% of cases. A characteristic feature of the outbreaks epidemiologically related to poultry products is that the pathogen belongs to the "avian" serovars Pullorum and Gallinarum [8,13].
Of a particular concern is that Salmonellosis often causes latent infection in poultry. However, meat and other products from infected poultry are a source of Salmonella and can pose a risk to human health [5].
According to the Reference Centre for Salmonellosis Monitoring and WHO's global tenyear monitoring of foodborne infections, 47% of outbreaks worldwide are related to Salmonellosis, with chicken meat playing a significant role in the infection transmission to humans through food (34%). In the Russian Federation, Salmonel losis associated food products include: meat and meat products -63%, chicken -28%, eggs -5.5%. In 49.6% of cases Salmonella strains isolated from animals are found in birds [12].
Salmonella continues to be the primary cause of food borne intestinal infections in many countries around the world. In the United States alone, 1.4 million people get infected with Salmonellosis every year, of which about 400 cases are fatal [10].
Analysis of data published by the World Health Organi zation on the detection of pathogens of this acute intesti nal infection in 2009-2011 showed that the most common cause of human disease in different regions of the world (Europe, North and South America, Asia, Africa, Oceania) is S. enteritidis, S. typhimurium, S. virchow, S. panama. Data for 2012-2013 confirmed this trend [7].
According to the Reference Centre for Salmonellosis Monitoring, the etiological structure of Salmonella in hu mans and animals continues to be dominated by S. ente ritidis -80.6% of Salmonella is isolated from humans, and 26.8% from animals. In 2011, in contrast to 2010, S. typhi murium held the leading position in the serovariant diver sity of Salmonella isolated in food products (31.9%). The percentage of S. infantis isolates isolated from food is quite significant and is 14.6% [15].
Salmonella is mainly transmitted through such food products as meat, milk, and eggs. The peculiarity of Sal monellainfected products is the absence of sensory changes: their appearance, color, smell, and taste remain unchanged [17].
In the Russian Federation, the absence of Salmonel la bacteria in raw animal materials is regulated by the Technical Regulations of the Customs Union "On food safety" (TR CU 021/2011). Safety control of poultry meat and products thereof is carried out in accordance with SanPiN 2.3.2.107801. According to this regulation, the presence of Salmonella in meat (25 g from deep layers) as well as in mechanically deboned poultry meat and other meat products is not admissible [11,14,16].
Since the early 90's of XX century with mass use of an tibiotics Salmonella strains resistant to a number of anti microbial drugs have emerged. Today they pose a serious problem for public health. Resistant strains that persist in animals can be transmitted to humans by alimentary route through the entire food chain [3].
Purpose of work: to study biological properties of Sal monella isolates recovered from raw animal materials at the microbiological laboratory of the FGBI "ARRIAH" in 2018.

MATERIALS AND METHODS
The following raw animal materials were studied: beef, pork, poultry meat, raw fat, offal from broiler chicken, and pig byproducts. The total number of samples was 1,204, the number of recovered isolates -45.
The tinctorial properties of Salmonella isolates were de termined by microscopic examination of gramstained 24 h culture smears (100×1.25 immersion lens magnification).
For biochemical and serological identification and determination of antibiotic resistance, pure cultures of Salmonella bacteria obtained during incubation of typical colonies on FPH slant agar were used.
Biochemical identification was performed in accor dance with GOST 316592012 (ISO 6579: 2002) "Food Pro ducts. Method for Salmonella detection" and GOST 54354 2011 "Meat and meat products. General requirements and methods for microbiological analysis" using semi liquid GISS media with glucose, lactose, mannitol, sucrose, maltose, xylose. Additionally, chromogenic nutrient me dia were used: Rambachagar and Coliform Agar ES (en hanced selectivity).
The ability to decompose urea was determined by streaking the urea agar (Christensen agar) slant surface. To detect indole, the test cultures were introduced into test tubes containing nutrient broth with Ltryptophan using a loop. 1 cm 3 of Kovacs reagent was added to the test tubes with 24 h broth culture. No later than 5 minutes after that, test results were read based on the color of the ring formed in the medium.
The serogroup and serovariant of the obtained isolates was determined by slide agglutination test using polyva lent serum for detection of Salmonella of ABCDE groups and monoreceptor O and Hagglutinating sera.
Antibiotic resistance of Salmonella isolates was deter mined by disc diffusion method using paper disks pro duced by the Saint Petersburg Pasteur Research Institute of Epidemiology and Mictobiology (Russian Federation) according to Methodical Guidelines 4.2.189004 [9].

RESULTS AND DISCUSSION
During the research in 2018, 45 Salmonella isolates were recovered from 1,204 samples of animal raw mate rials. The largest number of isolates was recovered from poultry, beef and pork (Fig. 1).
When studying the morphological properties of Salmo nella bacteria, it was found that they are small, straight gramnegative rods with rounded edges. All isolates showed similar cultural and biochemical properties: smooth convex semitransparent rounded colonies with a diameter of 1-3 mm were formed on the nutrient agar. Uniform turbidity of the medium and graywhite sediment were observed in test tubes with nutrient broth.
In semisolid agar medium motile bacteria gave a diffuse spreading growth throughout the agar column; on XLD agar the colonies had a black centre and a lightly transparent zone of the pinkish color around the colo nies; on Endo agar -round, translucent, slightly pinkish colonies; on bismuthsulfite agar -black rounded colonies with metallic sheen and colouring of the medium under the colonies; on Rambach agar Salmonella stock cultures produced a crimsoncolored growth.
On Coliform Agar ES (Enhanced Selectivity) the colonies of all the recovered isolates appeared as colorless, which means that Salmonella bacteria do not have βgalactosi dase enzyme.
Salmonella isolates exhibited typical biochemical pro perties: they produced hydrogen sulfide, fermented glu cose and mannitol with the production of gas and acid, did not utilize sucrose, lactose and urea; showed negative reaction to indole.
Based on the agglutination test results, the largest number of isolates belonged to the O 7 group. In addition to that, Salmonella from groups O 9 , O 5 , and O 4 were pre sent in the studied samples (Fig. 2).
As a result of the conducted research, 44.4% of the re covered Salmonella isolates were found to be multiresis tant. 92.3% of S. infantis isolates demonstrated resistance to two groups of antibiotics at the same time: fluoroquino lones (nalidixic acid) and tetracyclines (tetracycline). All the studied isolates of S. virchow are resistant to cepha losporins (cefotaxime), penicillins (ampicillin), and ami noglycosides (streptomycin). 76.9% of S. infantis isolates showed high resistance to aminoglycosides (streptomy cin) and 87.5% of S. virchow isolates -to fluoroquinolones (nalidixic acid).

CONCLUSION
A large number of tests (1,240 tests) for Salmonella was performed to assess the microbiological safety of raw animal materials. As a result, 45 Salmonella isolates were recovered.
All isolates showed identical morphological, cultural, and biochemical properties, typical for the genus Salmo nella.