Complete Genome Sequences of Gram-Negative Opportunistic Pathogens Isolated in Hospitals in Almaty, Kazakhstan

ABSTRACT The problem of nosocomial infections is growing due to the introduction of new treatment regimens involving immunosuppressive drugs. The genomes of seven Gram-negative clinical isolates of Escherichia, Klebsiella, and Pseudomonas were sequenced and analyzed in this study to serve as model microorganisms to study drug-induced antibiotic resistance reversion.

E scherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa are the most common agents of nosocomial infections. The emergence of nosocomial pathogens often involves the acquisition of a virulence plasmid, horizontal gene transfer, and adaptive mutations. The dynamics of these processes require constant monitoring.
Seven strains of Gram-negative bacteria were isolated at the Department of Vascular Surgery of the Syzganov National Scientific Center of Surgery in Almaty, Kazakhstan. Isolates were obtained by direct plating from biological material onto selective and differential diagnostic media ( Table 1). The aim of the study was to identify and perform genotyping of the potential agents of nosocomial infections. For more details on the isolates, see BioProject accession number PRJNA754843. This study was approved by the Committee of Institutional Animal Care and Use at the Scientific Center for Anti-Infectious Drugs (SCAID), Almaty.
For DNA extraction, cultures were grown on nutrient agar (HiMedia) for 24 h at 37°C. DNA was extracted using the PureLink genomic DNA minikit (Invitrogen, USA). DNA was sheared using the Megaruptor 3 shearing kit. A library was prepared using the PacBio SMRTbell Express template prep kit v2.0. SMRTbell templates were annealed using the Sequel binding and internal control kit v3.0. The Sequel sequencing kit v3.0 and a single-molecule real-time (SMRT) cell 1M v3 tray were used for sequencing. For each SMRT cell (Pacific Biosciences), 600 min movies were captured by Macrogen (Seoul, South Korea) using the PacBio Sequel I sequencing platform. Peaks smaller than 8 kb were removed using the BluePippin system. The numbers of generated reads and N 50 values for each sample are shown in Table 1. Further processing of the DNA reads was performed using software tools as described below, with default parameter settings if not indicated otherwise. The DNA reads were quality controlled and checked for remaining adapters using LongQC v1.2.0c (1) and assembled using Canu v2.0 (2). Plasmid contigs were identified using Platon v1.6 (3). The contigs were scaffolded and joined using MeDuSa at http://combo.dbe.unifi.it/medusa (4) by comparison with the most closely related reference genomes identified in GenBank by BLASTN ( Table 1). The original DNA reads were mapped to the scaffolds using pbmm2 (SMRT Link v10.10.119588) for error correction, and consensus sequences were generated from c Intermediate resistance. The resistance to antibiotics was determined experimentally. The susceptibility was evaluated by the disk diffusion method in Mueller-Hinton agar (HiMedia, India). The results of the threshold inhibition the alignments using the gcpp Arrow algorithm (SMRT Link v10.10.119588). The consensus sequences were annotated using the RAST server (https://rast.nmpdr.org/) with the RASTtk algorithm (5) and the "Fix frameshifts" setting. The chromosomal sequences were rotated to start with dnaA on the positive strand, and the plasmid sequences were shifted for 50 kb to perform circularization, final error fixation, and genome completion by another round of mapping of the initial PacBio reads using pbmm2. The final consensus sequences of the complete genomes were generated from the alignments and deposited at NCBI ( Table 1). The GenBank annotation robot PGAP was used for annotation of the deposited genomes. Multilocus sequence typing (MLST) was performed using the BIGSdb (https://bigsdb.pasteur.fr/) and CBS (https://www.cbs.dtu.dk/services/MLST) databases (6, 7). The E. coli serotypes were predicted by genotype using SerotypeFinder v2.0.1 (http://cge.cbs.dtu.dk/services/SerotypeFinder/) (8).
The E. coli isolates belonged to MLST ST43 and ST3, which are widely distributed uropathogens (9). K. pneumoniae ST23 and ST380 are abundant hypervirulent and multidrug-resistant variants which emerged due to the acquisition of pLVPK-type virulence plasmids (10)(11)(12). Two P. aeruginosa isolates belong to ST308, which is a common causative agent of nosocomial infections (13).
Data availability. The genome sequences are available from NCBI under BioProject accession number PRJNA754843 and the accession numbers shown in Table 1.

ACKNOWLEDGMENT
Sequencing was funded by grant BR09458960 of the program "Study of reversion of antibiotic resistance of pathogenic microorganisms," provided by the Industrial Development Committee of the Ministry of Industry and Infrastructure Development of the Republic of Kazakhstan.