Genomic Epidemiology of Carbapenem- and Colistin-Resistant Klebsiella pneumoniae Isolates From Serbia: Predominance of ST101 Strains Carrying a Novel OXA-48 Plasmid

Klebsiella pneumoniae is a major cause of severe healthcare-associated infections and often shows MDR phenotypes. Carbapenem resistance is frequent, and colistin represents a key molecule to treat infections caused by such isolates. Here we evaluated the antimicrobial resistance (AMR) mechanisms and the genomic epidemiology of clinical K. pneumoniae isolates from Serbia. Consecutive non-replicate K. pneumoniae clinical isolates (n = 2,298) were collected from seven hospitals located in five Serbian cities and tested for carbapenem resistance by disk diffusion. Isolates resistant to at least one carbapenem (n = 426) were further tested for colistin resistance with Etest or Vitek2. Broth microdilution (BMD) was performed to confirm the colistin resistance phenotype, and colistin-resistant isolates (N = 45, 10.6%) were characterized by Vitek2 and whole genome sequencing. Three different clonal groups (CGs) were observed: CG101 (ST101, N = 38), CG258 (ST437, N = 4; ST340, N = 1; ST258, N = 1) and CG17 (ST336, N = 1). mcr genes, encoding for acquired colistin resistance, were not observed, while all the genomes presented mutations previously associated with colistin resistance. In particular, all strains had a mutated MgrB, with MgrBC28S being the prevalent mutation and associated with ST101. Isolates belonging to ST101 harbored the carbapenemase OXA-48, which is generally encoded by an IncL/M plasmid that was no detected in our isolates. MinION sequencing was performed on a representative ST101 strain, and the obtained long reads were assembled together with the Illumina high quality reads to decipher the blaOXA-48 genetic background. The blaOXA-48 gene was located in a novel IncFIA-IncR hybrid plasmid, also containing the extended spectrum β-lactamase-encoding gene blaCTX-M-15 and several other AMR genes. Non-ST101 isolates presented different MgrB alterations (C28S, C28Y, K2∗, K3∗, Q30∗, adenine deletion leading to frameshift and premature termination, IS5-mediated inactivation) and expressed different carbapenemases: OXA-48 (ST437 and ST336), NDM-1 (ST437 and ST340) and KPC-2 (ST258). Our study reports the clonal expansion of the newly emerging ST101 clone in Serbia. This high-risk clone appears adept at acquiring resistance, and efforts should be made to contain the spread of such clone

Isolation and development microsatellite markers in the Pygocentrus nattereri (Kner, 1858) (Characiformes, Serrasalminae), an important freshwater fish in the Amazon

The red-bellied piranha belongs to the Pygocentrus genus, and is a representative of the Serrasalmidae family. The P. nattereri is the most abundant species in the floodplain of the Solimões-Amazonas river system. In the present work, 11 microsatellite loci were developed and used to investigate the genetic variation on 35 wild individuals. The number of alleles per locus ranged from 3 to 15, with an average of 8.2. The observed and expected heterozygosity values varied from 0.300 to 0.824 (mean 0.559) and 0.261 to 0.779 (mean 0.571), respectively. Only one loci (PN11) deviated significantly from Hardy-Weinberg equilibrium after Bonferroni correction. No significant linkage disequilibrium was detected. These polymorphic markers should be useful tools for assessing population genetics of P. nattereri, an overfished species in the Amazon varzea lakes that is most appreciated as a food item. © 2011 Springer Science+Business Media B.V