Evolution of VIM-1-Producing Klebsiella pneumoniae Isolates from a Hospital Outbreak Reveals the Genetic Bases of the Loss of the Urease-Positive Identification Character

Abstract

Outbreaks of carbapenemase-producing Klebsiella pneumoniae (CP<i>Kp</i>) represent a major threat for hospitals. We molecularly characterized the first outbreak of VIM-1-producing K. pneumoniae in Spain, which raised fears about the spread of this strain or of the plasmid carrying <i>bla</i>_VIM-1. Through in-depth genomic analysis of 18 isolates recovered between October 2005 and September 2007, we show that 17 ST39 isolates were clonal, whereas the last isolate had acquired the VIM-1 plasmid from the epidemic clone. The index isolate carried 31 antibiotic resistance genes (ARGs) and was resistant to almost all antibiotics tested. Later isolates further gained mutations in efflux pump regulators <i>ramR</i> and <i>opxR</i>, deletion of <i>mgrB</i> (colistin resistance), and frameshift mutations in <i>ompK36</i> (β-lactam resistance) likely selected by antibiotic usage. Comparison with publicly available genome sequences and literature review revealed no sign of dissemination of this CP<i>Kp</i> strain. However, the VIM-1 plasmid was found in diverse Enterobacterales species, although restricted to Spain. One isolate became urease negative following IS<i>5075</i> transposition into <i>ureC</i>. Analysis of 9,755 K. pneumoniae genomes showed the same <i>ureC</i>::IS<i>5075</i> insertion in 14.1% of the isolates and explained why urease activity is a variable identification trait for K. pneumoniae. Transposition into <i>ureC</i> results from the similarity of its 3' end and the terminal inverted repeats of Tn<i>21</i>-like transposons, the targets of IS<i>5075</i> and related insertion sequences (ISs). As these transposons frequently carry ARGs, this might explain the frequent chromosomal invasion by these ISs and <i>ureC</i> inactivation in multidrug-resistant isolates. <b>IMPORTANCE</b> Evolution of multidrug-resistant bacterial pathogens occurs at multiple scales, in the patient, locally in the hospital, or more globally. Some mutations or gene acquisitions, for instance in response to antibiotic treatment, may be restricted to a single patient due to their high fitness cost. However, some events are more general. By analyzing the evolution of a hospital-acquired multidrug-resistant K. pneumoniae strain producing the carbapenemase VIM-1, we showed a likely environmental source in the hospital and identified mutations contributing to a further decrease in antibiotic susceptibility. By combining the genomic analysis of this outbreak with literature data and genome sequences available in databases, we showed that the VIM-1 plasmid has been acquired by different Enterobacterales but is endemic only in Spain. We also discovered that urease loss in K. pneumoniae results from the specific transposition of an IS element into the <i>ureC</i> gene and was more frequent in fluoroquinolone-resistant isolates and those carrying a carbapenemase gene.