Quinolone-resistant Salmonella Infantis (n = 64) isolated from human stool samples, food and poultry during the years 2006-2011 were analysed for their resistance phenotypes, macrorestriction patterns and molecular mechanisms of decreased susceptibility to fluoroquinolones. Minimum inhibitory concentrations (MICs) of nalidixic acid (NAL) and ciprofloxacin (CIP) were determined by the agar dilution procedure, and the susceptibility to additional antimicrobial agents was determined by the disc diffusion method. To assess the influence of enhanced efflux activity, MICs were determined in the presence and absence of the inhibitor PAβN. The results of pulsed-field gel electrophoresis (PFGE) typing revealed that quinolone-resistant S. Infantis in Serbia had similar or indistinguishable PFGE profiles, suggesting a clonal spread. All S. Infantis showed combined resistance to NAL and tetracycline, whereas multiple drug resistance to three or more antibiotic classes was rare (2 isolates of human origin). The MICs ranged between 512 and 1024 μg/mL for NAL and 0.125-2 μg/mL for CIP. A single-point mutation in the gene gyrA leading to a Ser83→Tyr exchange was detected in all isolates, and a second exchange (Ser80→Arg) in the gene parC was only present in eight S. Infantis isolates exhibiting slightly higher MICs of CIP (2 μg/mL). The inhibitor PAβN decreased the MIC values of CIP by two dilution steps and of NAL by at minimum 3-6 dilution steps, indicating that enhanced efflux plays an important role in quinolone resistance in these isolates. The plasmid-mediated genes qnr, aac(6')-lb-cr and qepA were not detected by PCR assays.
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.
Bacteria of the genus Salmonella, some Escherichia coli strains as well as other species of the family Enterobacteriaceae, manifest the ability to express a specific phenotype, which is commonly termed as the "rdar" (red, dry and rough) morphotype. Both rdar and non-rdar phenotypes are commonly found in the nature; however, rdar is of essential importance for bacterial survival in the environment, outside of the host organisms. In that respect, it is comparable with the process of spore formation in Gram-positive bacteria. In laboratory conditions, rdar morphotype demonstrates growth of aforementioned organisms on agar containing diazo dye Congo Red, appearing as dark red (violet), rough and irregularly margined colonies. A coordinated expression of the number of genes mediated by major transcriptional regulators CsgD (curli subunit gene D) is the prerequisite for the formation of rdar morphotype colonies. The key point in this process is the activation of csgBAC operon transcription, which encodes the synthesis of curli fimbriae and activates the AgfD regulated gene (AdrA) involved in the cellulose biosynthesis. Curli fimbriae and cellulose are two basic structural components of the rdar morphotype. Curli fimbriae enable initial surface adhesion and intercellular aggregation of bacteria, whilst the cellulose (and other exopolysaccharides) promotes intercellular interactions. CsgD affects the overall cell physiology towards a "bacterial multicellular behavior" pattern, such as biofilm formation. The colony growth on Congo Red agar is a widely accepted laboratory method used for bacteria from the family Enterobacteriaceae and highly conserved between Salmonella. However, repeated laboratory subpassaging results in the substantial loss of rdar morphotype and the formation of smooth mutants, suggests the necessity for using wild isolates. In this article, the characteristics of rdar and non-rdar morphotypes of diverse Salmonella serotypes isolated from animal feed, as well as Escherichia coli isolates originating from cows milk with clinical mastitis, were presented. The research indicates substantial importance of the rdar phenotype for the persistence of Salmonella in the environment and its entering the food chain as well as the potential role of rdar phenotype of Escherichia coli in the pathogenesis of recurrent coli mastitis in dairy cows.
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.
ABSTRACT. The knowledge about virulence mechanisms, resistance to antimicrobial agents and the biofilm formation ability of Salmonella spp. in poultry industry has been expanded over the years. However, in spite of the research efforts and significant investments to improve management systems in poultry industry, it has become evident that none of the methods applied in all stages of food production chain are 100% effective in eliminating Salmonella spp. Different serovars are manifesting different mechanisms of invasiveness which depend on their ability to invade lower zones of the lamina propria, their ability to gain accesses to parenchymatous organs and survive in macrophages. The ubiquitous nature of Salmonella spp. due to their adaptation to animal and plant hosts, as well as their survival in hostile environments and their enhanced capacity to produce biofilms, contribute to a long lasting contamination of the environment, feed and animals. The emergency and spread of antimicrobial resistances in Salmonella spp. raise additional concerns.
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