Jelena Asanin

ISOLATION OF ETEC STRAINS FROM PIGLETS WITH DIARRHEA IN THE NEONATAL PERIOD AND THEIR TYPIZATION BASED ON SOMATIC AND FIMBRIAL ANTIGENS

Among different pathogens, enterotoxic E. coli (ETEC) has been for many years an important etiological agent in the occurrence of digestive system disease of newborn animals. In counties with developed pig breeding (farming), including our country, diarrhea in the neonatal period, caused by ETEC strains is one of the most present and economically most significant diseases. The aim of this investigation wais to determine the prevalence of ETEC strains in piglets (weaning pigs), originated from 5 (five) pig farms in the Republic of Serbia, as well as their serological typization based on characteristics of somatic O antigens, presence of fimbrial antigensadhesins and hemolytic activity. The material for this investigation was targeted and sampled from piglets that have shown clinical signs of neonatal diarrhea or pathoanatomical changes characteristic for enteritis caused by ETEC strains. The total number of isolated ETEC strains were 148, of which 91 (61.48 %) were determined on the basis of somatic O antigen characteristics. The largest number of strains, 42 (46.15 %) belonged to serotype O149. Serological types O8 and O147 were represented, each with 15 strains (16.48 %). In 13 (14.28 %) strains the somatic antigen which belonged to serotype O138 was determined and in 6 (6.59 %) strains the antigen belonged to serotype O157. No strain agglutinated with hyperimmune O139 serotype serum. The presence of fimbrial adhesins was determined in 47 (51.64%) strains and of that number the F4 type of fimbrial adhesins was detected in 38 (80.85 %) strains. The presence of F5 adhesins was determined in 4, and F6 in 3. In 2 strains, the paralell presence of two adhesin types, F4 and F6 was detected. The greatest number of strains 30 (71.42 %) with adhesin F4 belonged to O149 serotype, a considerably smaller number, 4 (26.66%) to serotype O8, 2 strains to serotype O157 and to each serotype O147 and O138 1 strain. The fimbrial adhesin of F5 type was detected in 3 strains which belonged to serotype O8 and in 1 strain of serotype O149. All 3 strains with F6 adhesin, belonged to serotype O8. From 2 strains which had, at the same time, adhesins F4 and F6 one belonged to serotype O8 and the other to serotype O138. Hemolytic activity was present in 42 (46.15 %) strains, of which 34 strains belonged to O149 serotype, 6 strains to O157 serotype and 2 strains to O147 serotype.

Biochemical parameters in rats with an applied model of sepsis (cecal ligation and puncture) with pure and mixed bacterial culture.

The goal of this work was to induce clinical sepsis in rats in order to measure the following biochemical parameters (glucoses, triglycerides, cholesterol, total proteins, albumins and creatinine). The experiments were done on 104 male Wistar rats, body weight 190 to 240 g. The rats were divided in four groups. Treated groups consisted of 28 animals while in the control groups there were 20 animals. Animals were observed and sacrificed at 12, 24, 72 and 120 hours after surgery. In this model of sepsis (cecal ligation and puncture - CLP) with pure and mixed bacterial cultures, significant changes were noticed in all biochemical parameters. Significant hypoglycemia, hypercholesterolemia, decreased concentration of urea and increased concentration of creatinine were found in the first half of the experiment in all groups of rats with sepsis (at 12 and 24 hours). Hyperproteinemia and hypotriglyceridemia reached statistically significant values at 24 h in the groups of rats in which sepsis was induced with pure bacteria culture. In the other half of the experiment significant hypoalbuminemia was found in all rats with sepsis (at 71 and 120 h). [Projekat Ministarstva nauke Republike Srbije, br. TR 31071]

DETERMINATION OF PRESENCE OF RESISTANCE TO ERYTHROMYCIN AND TETRACYCLINE ANTIBIOTICS IN ISOLATED STREPTOCOCCUS SPECIES FROM GROUP C AND GROUP G

The purpose of this study was to determine the presence of resistance to macrolide and tetracycline in -haemolytic streptococci which belong to group C (GCS) and group G (GGS), isolated from variuos clinical specimens collected at the Institute of Public Health of Serbia during the period 2006-2008. After determination of resistance in isolated streptococci to tested antibiotics, their phenotypic and genotypic characteristics were investigated. Resistance to erythromycin and tetracycline were evaluated in a total of 112 GGS and 29 GCS isolates. Resistance to erythromycin was determined in 6 (6.9%) GGS isolates and 4 of them were also resistant to tetracycline. Resistance to erythromycin was determined in 2 (5.4%) GCS isolates, but both isolates were sensitive to tetracycline. The erythromycinresistance phenotypes were determined by the double-disk test with erythromycin and clindamycin disks. All 8 isolates showed the MLSB macrolide resistance phenotype leading to macrolide, lincosamide and streptogramin B resistance. These 8 isolates were genotyped for the presence of the erm(TR), erm(B), mef(A) and tet(M) genes and transposon of the Tn916-Tn1545 family by polymerase chain reaction. The presence of erm(TR) gene was detected in 3 GGS isolates and in both GCS isolates, while the presence of erm(B) gene was detected in other 3 GGS isolates. The presence of tet(M) gene with transposon of the Tn916-Tn1545 family was detected in all 4 tetracycline-resistant GGS isolates. The results of this study indicate that continued monitoring of macrolide- and tetracycline- resistance in tested groups of streptococci in Belgrade and in Republic of Serbia is necessary.

Enterotoxin production and antimicrobial susceptibility in Staphylococci isolated from traditional raw milk cheeses in Serbia

ABSTRACT This study was undertaken to determine the prevalence of coagulase positive staphylococci (CPS) by examining a total of 71 raw milk cheeses. Additionally, enterotoxigenicity, antimicrobial susceptibility and the presence of mecA and mecC genes in the staphylococcal isolates were investigated. The isolation and enumeration procedure of CPS followed the International Organization for Standardization (ISO) standard. The presumptive staphylococci were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) using the VITEK MS system. VIDAS® Staph enterotoxin II assay was used for the detection of classical enterotoxins. Antimicrobial susceptibility testing (AST) was accomplished performing the disk diffusion method. All suspected methicillin resistant staphylococci were investigated for the presence of mecA and mecC genes by PCR assay. A high prevalence (87.32%) of CPS was detected in the cheeses at contamination levels up to 5.58 log CFU g−1. Among 47 staphylococcal isolates screened for enterotoxin production, only one isolate, identified as S. hyicus, was confirmed as being enterotoxigenic. Resistance to penicillin (63.70%) was the most common resistance among the tested Staphylococcus aureus isolates. The dominant phenotypic resistance patterns in coagulase negative staphylococci (CNS) were resistance to ofloxacin and fusidic acid. All CNS isolates were susceptible to the clinically important antibiotics clindamycin, chloramphenicol, gentamicin, linezolid, rifampicin and trimethoprim-sulfamethoxazole. The mecA and mecC genes were not detected. To the best of our knowledge, this is the first study concerning evaluation of the presence of methicillin resistant staphylococci (MRS) in dairy products in Serbia.

Occurrence of methicillin-resistant S taphylococcus aureus in milk samples from Serbian cows with subclinical mastitis

The objective of this study was to analyze the occurrence of methicillin-resistant Staphylococcus aureus (MRSA) from cattle with subclinical mastitis in two dairy herds in the northwest of Serbia. All quarters reacting positive in the California Mastitis Test were sampled for bacteriological analysis. Nasal and vaginal swabs from MRSA positive cattle, and nasal swabs from humans working on farms were also collected. From 1026 cows, 212 (20.7%) suffered from subclinical mastitis. S. aureus was detected in milk samples from 84 (39.6%) cows with subclinical mastitis. Among them, MRSA were isolated from 5 (5.9%) cows. Three out of five positive cows harboured MRSA in their nose and 1 cow harboured MRSA in the vagina. No MRSA was found in human nasal swabs. Seven out of 10 MRSA isolates in farm A, and two MRSA isolates in farm B, were resistant to gentamicin and tobramycin and exhibited SCCmec type IV. The three other isolates in farm A were resistant to tetracycline and carried SCCmec elements of type V. To our knowledge, this is the first description of MRSA from bovine subclinical mastitis in Serbia. The results of our study herald the emergence of MRSA in dairy farms in Serbia, so continued surveillance is recommended.

Differentiation between Pseudomonas and Stenotrophomonas Species Isolated from Fish Using Molecular and MALDI-TOF Method

Abstract 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 results of the standard PCR test, 16S rRNA gene sequencing and MALDI-TOF analysis confirmed 35 strains to belong to the Pseudomonas genus. Standard PCR test and VITEK MS device confirmed that 10 strains belong to Stenotrophomonas maltophilia species. Three strains were positive in both standard PCR tests for Pseudomonas and Stenotrpohomonas. 16S rRNA gene sequencing identified these 3 strains to be 99% Pseudomonas sp. and 99% Stenotrophomonas sp. VITEK MS first identified these three strains as 99% Stenotrophomonas, and in the repeated identification it identified them as 99% Pseudomonas. MALDI TOF/TOF 4800 Plus device identified these strains as Stenotrophomonas. Three strains were negative in both standard PCR tests for Pseudomonas and Stenotrpohomonas. 16S rRNA gene sequencing identified these 3 strains to be 99% Pseudomonas sp. and 99% Stenotrophomonas sp. VITEK MS first identified these three strains as 99% Stenotrophomonas, and in the repeated identification it identified them as 99% Pseudomonas. MALDI TOF/TOF 4800 Plus device identified these strains as Stenotrophomonas. Although modern test methods that have very high specificity (PCR, 16S rRNA gene sequencing, MALDI TOF) were used in this study, precise differentiation between Pseudomonas and Stenotrophomonas species for 6 isolates could not be reached using the above mentioned methods.

DETECTION OF PBP2a (PENICILLIN-BINDING PROTEIN 2a) AND mecA GENE IN METHICILLIN RESISTANT STAPHYLOCOCCI ORIGINATED FROM ANIMALS

For the purpose of detecting methicillin (oxacillin) resistance in staphylococcal strains, in a number of microbiological laboratories only disc diffusion method with cefoxitin and/or oxacillin discs is used. Besides this method, it is desirable to determine MIC values for cefoxitin and/or oxacillin. After examination by disc diffusion and dilution methods, latex agglutination is used for the detection of PBP2a and PCR is used for the detection of mecA gene. Use of PCR is not possible in a large number of diagnostic laboratories and as method of choice, latex agglutination test for rapid detection of PBP2a is recommended. In this investigation, as confirmatory methods, latex agglutination and PCR were used for strains that were resistant to oxacillin and/or cefoxitin by disc diffusion and broth microdilution methods. In total, 14 strains of coagulase-negative staphylococci originating from clinical specimens of cats, dogs and chicken were examined. Among isolated strains, it was established that the dominating species was Staphylococcus haemolyticus with 11 isolated strains. Other isolated species were Staphylococcus epidermidis, Staphylococcus capitis and Staphylococcus vitulinus, each with one isolated strain. For all 14 strains, oxacillin MIC values ranged from 0.5 g/mL to >64 g/mL and cefoxitin MIC values ranged from 1 g/mL to >256 g/mL. Positive agglutination reaction by latex agglutination test was recorded in 13 out of 14 strains. The PCR assay for mecA gene was positive in 12 investigated strains.

Implementation of polymerase chain reaction (PCR) and Real-Time PCR in quick identification of bovine herpesvirus 1.

Examinations were performed on 65 samples of nasal smeas taken from calves and young cows with clinical symptoms of respiratory infection to determine the presence of the bovine herpes virus 1 using parallel implementation of molecular and standard methods of virological diagnostics. The appearance of a cytopathogenic effect (CPE) was not established in inoculated cell lines 24h, 48h and 72h following inoculation, or after two successive passages of the examined material sample through these cell lines. The application of polymerize chain reaction (PCR) using a primer for glucoprotein B and thymidine - kinasis, established the presence of bovine herpes virus 1 nucleic acid in one sample of a bovine nasal smear, while the presence of this virus was established in three samples in an examination of the nasal smear samples using the Real-Time PCR method.