Genome sequences of four colistin-resistant ESKAPE bacterial strains isolated from patients within the same hospital

ABSTRACT The genomes of four clinical Gram-negative ESKAPE bacterial strains highly resistant to the last-resort antibiotic colistin were sequenced and analyzed. The strains were found to carry multidrug-resistant genes besides colistin-resistant genes.

C olistin (polymyxin E) is a last-resort antibiotic used against multidrug-resistant (MDR) Gram-negative bacteria.However, its efficacy is increasingly compromised by the emergence of colistin-resistant strains.Here, we sequenced the genomes of four colistin-resistant strains isolated from unrelated patients suspected of infection at the Microbiology Department of the Ramón y Cajal University Hospital of Madrid, Spain, with the approval from The Drug Research Ethics Committee of the hospital (CEIm, identification number 2017-163/17).The strains belong to two different species: two are of Pseudomonas aeruginosa and two of Klebsiella pneumoniae.The P. aeruginosa strains were isolated from the sputum of cystic fibrosis patients and K. pneumoniae strains from a patient with a urinary tract infection.Bacterial samples were grown on McConkey growth medium (Difco) at 37°C and identified by MALDI-TOF MS (Burker).
The minimum inhibitory concentration (MIC) for colistin was determined according to the EUCAST procedure, using the microdilution method with the cationic-adjusted Mueller-Hinton broth medium (Difco).The MIC values of all strains were well above the species breakpoint (2 mg/L for K. pneumoniae and 4 mg/L for P. aeruginosa), classifying them as colistin-resistant (Table 1).
For genome sequencing, strains were retrieved from −70°C glycerol stocks and cultured overnight in Lysogenic Broth medium at 37°C with agitation.Genomic DNA was extracted using the GenElute Bacterial Genomic DNA Kits (Sigma-Aldrich), quanti fied using the Qubit dsDNA Kit (Thermo Fisher Scientific) and sent to Eurofins Genom ics Europe Sequencing (Germany) for sequencing.DNA-seq libraries were prepared according to Eurofins Illumina's protocol and sequenced on NovaSeq 6000 with 2 × 150 bp paired-end read mode and output of approximately 5 million paired-end reads per sample.Reads quality was checked by FastQC (version:0.11.9) (1).
In addition to antimicrobial resistant genes annotated by Staramr (Galaxy ver sion:0.8.0+galaxy0) (11), genes known to be associated with colistin tolerance/resistance mechanisms in the annotated genome sequences are listed in Table 1.Colistin is a cationic polypeptide that interacts with phosphate groups of the lipopolysaccharide (LPS) in the outer membrane.The mechanisms of resistance mainly involve modifications of the LPS target (12) or export of the antibiotic by multidrug efflux (Mex) systems (13).The colistin resistance-related genes arnBCADTEF, kdsD, eptA, phoPQ which are involved in the addition of either 4-amino-4-deoxy-L-arabinose (L-Ara4N) or phosphoethanola mine (pEtN) to the LPS to reduce its negative charge and thus its affinity for colistin (12,14,15) are present in all four genomes.The regulatory genes mexAB, emrAB, encoding components of the MDR efflux pumps Mex and MFS (13,16,17) are also present in all four genomes.
Two Pseudomonas aeruginosa and two Klebsiella pneumoniae colistin-resistant clinical strains were isolated from unrelated patients under suspicion of infection at the Microbiology Department of the Ramón y Cajal University Hospital of Madrid, Spain.P. aeruginosa strains were isolated from sputum of cystic fibrosis patients and K. pneumo niae strains were obtained from a patient with urinary tract infection.The four isolates were sequenced by INVIEW Illumina sequencing with 2 × 150 bp paired-end read mode and output of approximately 5 million read pairs.DNAseq libraries were prepared according to Illumina's protocol.

TABLE 1
Genome assembly details and statistics of P. aeruginosa and K. pneumoniae isolated from patients within the same hospital