Antimicrobial Resistance of Enterococcus Isolates from Poultry Farms in the Republic of Serbia (Autonomous Province of Vojvodina).

Enterococcus species are significant intestinal commensals of animals, including poultry. However, they have emerged as important opportunistic infective agents in both veterinary and human medicine as well as major nosocomial pathogens, owing to their increasing antimicrobial resistance. This research aimed to investigate the prevalence and antimicrobial resistance patterns of Enterococcus spp. isolated from poultry farms in the north of Serbia. A total of 40 samples of overshoes or feces were collected from 40 poultry farms and analyzed for the presence of Enterococcus spp. using PCR or MALDI-TOF mass spectrometry for their identification. The number of isolates was 40 and included 11 isolates from laying hens, 2 isolates from turkeys, 3 from broiler breeders, and 24 from broilers. The Kirby-Bauer disk diffusion method was used to test for antibiotic susceptibility in accordance with the Clinical and Laboratory Standards Institute and EUCAST guidelines. The results showed that Enterococcus faecalis was isolated from 37.5% farms, and E. faecium from 42.5%. E. hirae was identified in 15% of poultry establishments, and E. durans and E. thialandicus on 2.5%. Notably, resistance to erythromycin, streptomycin, fluoroquinolones, and tetracyclines among the frequently used antibiotics was found. Furthermore, 35% of the isolates had multidrug resistance (MDR). In order to prevent the spread of antibiotic resistance in chicken farming and protect the health of the public and animals alike, our findings highlight the critical need for improved surveillance and control measures. To effectively establish a containment strategy for Enterococcus spp. isolated from poultry farms, more research into the processes behind their antibiotic resistance is required.

Antibacterial Potential of Non-Tailed Icosahedral Phages Alone and in Combination with Antibiotics.

Non-tailed icosahedral phages belonging to families Fiersviridae (phages MS2 and Qbeta), Tectiviridae (PRD1) and Microviridae (phiX174) have not been considered in detail so far as potential antibacterial agents. The aim of the study was to examine various aspects of the applicability of these phages as antibacterial agents. Antibacterial potential of four phages was investigated via bacterial growth and biofilm formation inhibition, lytic spectra determination, and phage safety examination. The phage phiX174 was combined with different classes of antibiotics to evaluate potential synergistic interactions. In addition, the incidence of phiX174-insensitive mutants was analyzed. The results showed that only phiX174 out of four phages tested against their corresponding hosts inhibited bacterial growth for > 90% at different multiplicity of infection and that only this phage considerably prevented biofilm formation. Although all phages show the absence of potentially undesirable genes, they also have extremely narrow lytic spectra. The synergism was determined between phage phiX174 and ceftazidime, ceftriaxone, ciprofloxacin, macrolides, and chloramphenicol. It was shown that the simultaneous application of agents is more effective than successive treatment, where one agent is applied first. The analysis of the appearance of phiX174 bacteriophage-insensitive mutants showed that mutations occur with a frequency of 10-3. The examined non-tailed phages have a limited potential for use as antibacterial agents, primarily due to a very narrow lytic spectrum and the high frequency of resistant mutants appearance, but Microviridae can be considered in the future as biocontrol agents against susceptible strains of E. coli in combinations with conventional antimicrobial agents.

Molecular Investigation of Eimeria Species in Broiler Farms in the Province of Vojvodina, Serbia.

Coccidiosis is a significant poultry disease caused by the Eimeria species. This study aims to determine the prevalence of Eimeria spp. on broiler farms in Vojvodina, along with the identification of parasite species, and assess the implemented biosecurity measures. The study was conducted on 100 broiler chicken farms (28 small-sized; 34 medium-sized; 38 large-sized farms) from June 2018 to December 2021. One pooled sample of faeces was collected from three to six-week-old chickens from each farm, and assessment of biosecurity measures was carried out using a questionnaire. Using the PCR method, DNA of Eimeria was found in 59 samples (59%), while 41 samples (41%) were negative. Four species of Eimeria were identified, and their prevalence was the following: E. acervulina (37%), E. maxima (17%), E. mitis (25%) and E. tenella (48%). A significant difference (p < 0.05) was established in the number of oocysts in flocks from small-sized farms compared to medium-sized farms. It was found that regular implementation of disinfection, disinsection and deratisation measures, as well as all the biosecurity measures, can significantly reduce the occurrence of coccidiosis. These results will help to develop better strategies for the control and prevention of coccidiosis on farms.

Diagnosis of Infectious Laryngotracheitis Outbreaks on Layer Hen and Broiler Breeder Farms in Vojvodina, Serbia.

Infectious laryngotracheitis (ILT) is a respiratory disease of poultry characterized by high morbidity and variable mortality. ILT is caused by Gallid alpha herpesvirus-1 (GaHV-1), which is transmitted horizontally and most susceptible are chickens older than 4 weeks. After almost two decades since last appearance of this disease in Vojvodina, an outbreak occurred from April 2020 to August 2021 on five laying hen farms and one broiler breeder flock farm. Clinical signs were mild to severe respiratory symptoms, unilateral or bilateral head swelling, serous nasal discharge, conjunctivitis and increased tearing. There was a decrease in feed consumption (2.1-40.0%) and egg production (2.7-42.0%), weight loss and mortality increased (0.8-31.5%). Pathomorphological changes were localized in the upper respiratory tract. Total of 200 carcasses were examined; 40 pooled samples were analyzed by PCR, and 40 by bacteriological analysis. ILT virus was confirmed in tracheal tissue samples. Infected flocks were not vaccinated against this disease. Five flocks had coinfection with Avibacterium paragallinarum. Three-to-four weeks after the first reported case in the flock, clinical symptoms had ceased. Future control and prevention strategies will involve the procurement of flocks vaccinated by recombinant vaccine or the registration of live attenuated vaccines and their use during the rearing period.

Genomic Analysis of Multidrug-Resistant Salmonella enterica Serovar Kentucky Isolates from Humans, Turkey, and Food in the Republic of Serbia.

Owing to the emerging resistance to antimicrobials in Salmonella Kentucky isolates around the globe, the genomic comparison of all the registered multidrug-resistant Salmonella Kentucky isolates in Serbia (five from humans, one from turkey flock, and one from meat) was done. Most of the isolates were isolated from patients returning from Egypt or Tunisia or originated from imported turkey flock and turkey meat. The comparative analysis of resistance and virulence genes was done. All isolates belonged to sequence type-ST198 and were resistant to ciprofloxacin (Cip). The resistance to Cip was mediated by target mutations of the gyrA and parC genes, which encode topoisomerase I and II, respectively. Multidrug-resistant phenotype to aminoglycosides, ß-lactam antibiotics, sulfonamides, and tetracyclines was detected in five isolates. However, none of the isolates was pan-resistant to antimicrobials. The number of single nucleotide polymorphisms between isolates varied from 8 to 43 and phylogenomics revealed the genetic proximity of the human isolate 10475/11 and the turkey meat isolate 5264/14, indicating a possible meat-to-human transfer. All isolates belonged to the main Salmonella Kentucky MDR lineage, carrying the Salmonella genomic island 1 (SGI1-K) subtype. The SGI1-K of Serbian isolates showed mosaicism attributed to rapid intraclonal evolution. Many virulence factors were detected in all the isolates, including SPI-1, SPI-2, SPI-3, SPI-4, SPI-5, SPI-9, and C63PI. Although Salmonella Kentucky has rarely been isolated from humans, food, and animals in Serbia, further surveillance is needed to diminish the risk of the spreading of resistant clones and their meat-to-human transmission.

Characterization of antibiotic resistance in Escherichia coli isolates from Black-headed gulls (Larus ridibundus) present in the city of Novi Sad, Serbia.

Despite common resistance to antimicrobials in Escherichia coli isolates from farm animals in Serbia, no data are currently accessible on its occurrence in E. coli isolated from gulls. Therefore, 67 cloacal swabs and 70 fecal samples from black-headed gulls were investigated for the presence of antibiotic-resistant E. coli isolates. Ninety-nine isolates were obtained during the study. Resistotyping and resistance gene typing has shown that 44 isolates harbor resistance to one or more antibiotics. Multidrug resistance was detected in 24 E. coli isolates. Ten isolates were resistant to extended-spectrum cephalosporin antibiotics and were studied in detail including virulence gene typing, phylogenetic and multilocus sequence typing, and mating. These ten isolates belonged to phylogenetic groups B2 (five isolates), D (four isolates) and B1 (one isolate). Five different sequence types (ST38, ST2307, ST224, ST162 and ST34) were detected in E. coli isolates with AmpC phenotype and genotype. One isolate carried the Inc I2/FIB replicon type plasmid with the blaCTX-M-1 gene. Nine isolates had blaCMY-2 genes, which were detected on conjugative plasmids in seven isolates. The virulence genes hly, iroN, iss, ompT and cvaC were detected in one transconjugant. Ten isolates were found to be resistant to ciprofloxacin, whose MIC ranged from 4 to 32 mg/L. Genotyping revealed single or double mutations in the quinolone resistance determining region (QRDR) of the gyrA or gyrA, parC and parE genes, respectively. So, Black-headed gulls from Serbia may be colonized by multidrug-resistant E. coli, some of which are resistant to critically important antibiotics in medicine.

Genomic Characteristics of Colistin-Resistant Salmonella enterica subsp. enterica Serovar Infantis from Poultry Farms in the Republic of Serbia.

The antimicrobial susceptibility testing was conducted on 174 single isolates from poultry farms in Serbia and it was determined that seven Salmonella spp. were multidrug resistant. Sixteen serotypes were detected, but only serotype Infantis confirmed reduced susceptibility to colistin. Seven colistin resistant Salmonella Infantis were studied in detail using the WGS approach. Three sequence types were identified corresponding to different epizootiology region. The isolate from the Province of Vojvodina 3842 and isolates from Jagodina (92 and 821) are represented by the sequence type ST413 and ST11, respectively. Four isolates from Kraljevo are ST32, a common S. Infantis sequence type in humans, poultry and food. The fosfomycin resistance gene fosA7 in isolate 3842 and the vgaA gene in isolate 8418/2948 encoding resistance to pleuromutilins were reported for the first time in serovar Infantis. The changes in relative expression of the phoP/Q, mgrB and pmrA/B genes were detected. Single nucleotide polymorphisms of the pmrB gene, including transitions Val164Gly or Val164Met, and Arg92Pro are described. Analyses of quinolone resistance determining region revealed substitutions Ser83Tyr in GyrA protein and Thr57Ser and Ser80Arg in ParC protein. Based on WGS data, there are two major clusters among analyzed Salmonella Infantis isolates from central Serbia.

Fluoroquinolone-resistant and extended-spectrum beta-lactamase producing Escherichia coli isolates from free-living wild animals.

During the hunting season 2013-2014, fecal samples collected from hare, roe deer, deer and wild boars were sent to the bacteriology laboratory for the isolation of Escherichia coli and multidrug resistant isolates were characterized phenotypically and genotypically. Out of 106 fecal samples, E. coli was isolated from 101 samples. Although the majority of isolates belonged to phylogenetic groups A and B1, 14 out of 101 isolates were affiliated to group B2. A multidrug resistance phenotype was determined in 7 isolates, all of which had distinguishable genomic macrorestriction profiles. PCR analysis and sequencing revealed a variety of resistance genes, gene cassettes and cassette arrays in these multidrug resistant isolates. Resistance to fluoroquinolones was found in five E. coli isolates (two from a roe deer, one from a deer and two from a wild boar) and multiple mutations in the chromosomal topoisomerase genes were identified. In an E.coli isolate from a hare, the qnrB19 gene was detected. The same isolate carried an aadA23 gene cassette in class 1 integron. In addition, an extended- spectrum beta-lactamase blaCTX-M-1 gene was detected in an E. coli isolate from a roe deer. The gene was located on a conjugative multi resistance plasmid, which was transferable to a plasmid free E. coli recipient. In conclusion, a number of resistance genes and mobile genetic elements were detected in E. coli isolates from wildlife in Vojvodina, emphasizing the role of environmental pollution in spreading resistant bacteria.

Molecular Characterization of Multidrug-Resistant Escherichia coli Isolates from Bovine Clinical Mastitis and Pigs in the Vojvodina Province, Serbia.

The aim of the study was to characterize multidrug-resistant (MDR) Escherichia coli isolates collected in Serbia from bovine clinical mastitis cases and diseased pigs, mainly with molecular methods. A total of 48 E. coli isolates was collected during the years 2013-2014, of which 22 were MDR and were included in further analysis. Phylogenetic typing showed that 17 isolates belonged to group A, while two isolates were classified in group B1 and a single one in group D. All isolates showed unique macrorestriction patterns. Phenotypic susceptibility testing revealed resistances of the isolates against up to 13 antimicrobial agents, including resistance to fluoroquinolones. A wide variety of resistance genes was detected by PCR amplification and sequencing of amplicons. Sequence analysis of the quinolone resistance determining regions of topoisomerase genes revealed mutations in gyrA, parC, and/or parE. Plasmid-mediated quinolone resistance genes were detected in two porcine (aac-6'-Ib-cr and qnrS, respectively) isolates and a single bovine (aac-6'-Ib-cr) isolate. Resistance genes were found to be located on conjugative plasmids in 16 cases, many of which conferred a multidrug resistance phenotype. In conclusion, the plentitude of resistance genes located on conjugative plasmids and integrons in E. coli from cows and pigs in Vojvodina, Serbia, pose a high risk for horizontal gene transfer in bacteria from livestock husbandry.