Complete Genome Sequences of Four Extensively Drug-Resistant Pseudomonas aeruginosa Strains, Isolated from Adults with Ventilator-Associated Pneumonia at a Tertiary Referral Hospital in Mexico City

ABSTRACT Four extensively drug-resistant Pseudomonas aeruginosa strains, isolated from patients with pneumonia, were sequenced using PacBio RS-II single-molecule real-time (SMRT) technology. Genome sequence analysis identified great variability among mobile genetic elements, as well as some previously undescribed genomic islands and new variants of class 1 integrons (In1402, In1403, In1404, and In1408).

P seudomonas aeruginosa is a Gram-negative bacterium and is considered to be an opportunistic human pathogen associated with infections in immunocompromised patients. The presence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains is associated with high mortality in patients with chronic infections (1,2). Here, we present the complete genome sequences of four P. aeruginosa strains (Pa58, Pa84, Pa124, and Pa127), all isolated from adult patients with pneumonia associated with the use of automatic ventilators in an intensive care unit at a tertiary referral hospital in Mexico City.
Strains Pa58, Pa124, and Pa127 were resistant to different ␤-lactams, including carbapenems, as well as aminoglycosides and fluoroquinolones. Strain Pa84 was resistant to only imipenem, ciprofloxacin, and levofloxacin. All of the strains were susceptible to polymyxin B. Two strains belong to sequence type 309 (ST-309), a high-risk clone previously documented in Mexico (3).
The genomic DNA of the strains was purified with the DNeasy blood and tissue kit (Qiagen) and sent to the Yale Center for Genome Analysis for PacBio RS-II singlemolecule real-time (SMRT) sequencing. A standard library of 20-kb fragments was prepared and sequenced on four SMRT cells with the P4-C2 chemistry. The continuous long reads were assembled using the HGAP/Quiver protocol in SMART portal version 3 (4). The genome features are described in Table 1.
All of the strains have a core genome size averaging 6.15 Mb, with a variable accessory genome. The strains presented different islands inserted at previosly described loci (9). However, these islands present a different gene composition and size than those of the previously reported PAGI-1, PAPI-2, PAGI-4, and pKLC102 strains (10)(11)(12). Strains Pa58, Pa124, and Pa127 presented a new island of 70 open reading frames, which is integrated in the tRNA-arg (PA0256) 3= end. This new island has genes involved in a type I restriction modification system.
Many bacteriophages were detected in all of the strains, most of them having genes involved in phage biogenesis and heavy-metal resistance. Strains Pa58 and Pa84 presented a CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated genes) array in their chromosomes.
All of the strains showed genes of four efflux pump systems, MexAB, MexCD, MexEF, and MexXY. Strain Pa58 also has genes of the AcrAB efflux pump from Enterobacter cloacae. All of the strains carried the OprD, OprJ, and OprM porins, the ␤-lactams OXA-2 and OXA-50, and the genes fosA and aph(3=). GyrA presented a mutation at amino acid position 83 (T ¡ I), conferring resistance to fluoroquinolones.
Accession number(s). These whole-genome projects have been deposited in GenBank under the accession numbers listed in Table 1. The BioProject accession number is PRJNA389181.

ACKNOWLEDGMENTS
We thank José Luis Méndez for performing the antibiotic susceptibility essays, and Guilin Wang from the Yale Center for Genome Analysis, Yale University, for skillful PacBio sequencing.
This report is part of the doctoral research carried out by Luis Fernando Espinosa Camacho at the Programa de Doctorado en Ciencias Biomédicas, Universidad Nacional Autónoma de México (UNAM), and supported by fellowship 619928 from CONACYT. This work was also supported by Dirección General de Asuntos del Personal Académico, Universidad Nacional Autónoma de México (DGAPA/PAPIIT, UNAM) (IN213816 to Rosario Morales-Espinosa). The funders had no role in study design, data collection and interpretation, or decision to submit the work for publication.