Biology subjects

Chiarelli, A.

Publications and source records attributed to Chiarelli, A..

2 recordsLinked to original sources

bioRxiv2022

Carbapenem heteroresistance of KPC-producing Klebsiella pneumoniae results from tolerance, persistence and resistance.

Carbapenemase-producing Klebsiella pneumoniae (CPKp) have disseminated globally and represent a major threat in hospitals with few therapeutic options and high mortality rates. Isolates producing the carbapenemase KPC (KPC-Kp) might be classified as susceptible according to clinical breakpoints by antibiotic susceptibility testing (AST), allowing the use of imipenem or meropenem for treatment of infections. However, some KPC-Kp show heteroresistance (HR) to carbapenems, with colonies growing in the inhibition halo of agar-based AST. HR KPC-Kp have been associated with a higher risk of treatment failure. Here, we characterized the diversity of mechanisms behind HR to imipenem of these isolates. By analyzing a diverse collection of CPKp, we showed that HR is frequent among KPC-Kp. By monitoring single HR colony appearance using the ScanLag setup, we discriminated surviving cells in two subpopulations leading to a Gaussian-like distribution of early-appearing colonies, with a delayed emergence compared to colonies arising in the absence of antibiotics, and a long tail of late-appearing colonies. A subset of colonies showed a reduced growth rate. Characterization of surviving populations by AST and whole-genome sequencing of 333 colonies revealed a majority of parental genotypes and a broad landscape of genetic alterations in 28% of the colonies, including gene loss, DNA amplification and point mutations. This unveils the complexity of imipenem HR among KPC-Kp isolates, which involves tolerant and persistent cells, but also resistant bacteria. These observations contribute to a better understanding of reasons behind carbapenem treatment failure of KPC-Kp isolates. IMPORTANCEThe ability of a bacterium to defeat antibiotics not only depends upon resistance, but also on tolerance and persistence, which allow a bacterial population to temporarily survive high drug doses. Carbapenems are antibiotics of last resort and Klebsiella pneumoniae isolates producing the carbapenemase KPC are a threat to hospitals, although they might remain susceptible to carbapenems. However, seemingly homogeneous populations of KPC-K. pneumoniae isolates frequently show varying degrees of susceptibility to carbapenem, i.e., a phenomenon called heteroresistance. We characterized bacteria surviving a high dose of imipenem, progressively degraded by the released carbapenemase, by monitoring the growth of the resulting colonies using the ScanLag system, their genome sequence and carbapenem susceptibility. We show that the observed phenotypic diversity combines tolerance, persistence and resistance making the treatment with high doses of carbapenems frequently inefficient.

microbiology↗

bioRxiv2021

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

Outbreaks of carbapenemase producing Klebsiella pneumoniae (CPKp) represent a major threat for hospitals. We molecularly characterized the first outbreak of VIM-1 producing K. pneumoniae in Spain, that raised fears about the spread of this strain or of the plasmid carrying blaVIM-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 pumps regulators ramR and opxR, deletion of mgrB (colistin resistance) and frameshift mutations in ompK36 ({beta}-lactam resistance) likely selected by antibiotic usage. Comparison with publicly available genome sequences and literature review revealed no sign of dissemination of this CPKp strain. However, the VIM-1 plasmid was found in diverse Enterobacterales species, although restricted to Spain. One isolate became urease negative following IS5075 transposition into ureC. Analysis of 9755 K. pneumoniae genomes showed the same ureC::IS5075 insertion in 14.1% of the isolates and explained why urease activity is a variable identification trait for K pneumoniae. Transposition into ureC results from the similarity of its 3-end and the terminal inverted repeats of Tn21 like transposons, the targets of IS5075 and related ISs. As these transposons frequently carry ARGs, this might explain the frequent chromosomal invasion by these ISs and ureC inactivation in multidrug resistant isolates. IMPORTANCEEvolution 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 only endemic in Spain. We also discovered that urease loss in K. pneumoniae results from the specific transposition of an IS element into the ureC gene and was more frequent in fluoroquinolone resistant isolates and carrying a carbapenemase gene.

evolutionary biology↗