Resistance to cephalosporins in Enterococcus spp.
Laura Carrilero Aguado defended the PhD Thesis at the Faculty of Veterinary Medicine of the Complutense University of Madrid
January 25th, 2016
Enterococcus spp. are facultatively anaerobic, catalase-negative Gram-positive cocci, arranged individually, in pairs, or short chains. Enterococci include one of the most important nosocomial pathogens, Enterococcus faecalis. E. faecalis can be responsible for infections of wounds, soft tissue and the urinary tract. In some cases they produce bacteremia which can lead to endocarditis in previously damaged cardiac valves. Several factors have been identified as important in the pathogenesis of the bacteria. Nevertheless, it remains unclear how this commensal bacteria evolved into an insidious pathogen. In addition, Enterococcus faecalis harbours a large amount of both intrinsic and adquired antibiotic resistance determinants. In fact, the administration of cephalosporins is a risk factor for the adquisition of an enterococcal infection.
Among the different antibiotic resistance phenotypes that this bacterium displays, one of the most fascinating resistance mechanism is the intrinsic resistance to cephalosporins. Various important factors important for this intrinsic resistance have been identified, but the relationships between them and their upstream and downstream effectors remain unknown.
The SOS regulon is a global response of the bacteria to damaged DNA. It is a highly coordinated response to address the DNA damage mediated through recombination, DNA repair, translesion synthesis and cell division arrest, in order to avoid the cell division until the genetic material is repaired.
The objectives of this work are to characterize the SOS response of E. faecalis, study the implication of the SOS response on the antibiotic resistance profile and to elucidate the reversion of the intrinsic resistance to cephalosporins upon the induction of the SOS response.
Mutants for LexA, RecA and an uncleavable LexA variant in E. faecalis demonstrated a phenotype compatible with a typical SOS response in this bacterium, mediated by the two main proteins LexA and RecA. Interestingly, the LexA mutant was susceptible to cephalosporins. We saw that under the effect of mitomycin C, a potent DNA crosslinker that leads to breaks in the DNA inducing the SOS response, E. faecalis, E. faecium and L. ivanovii became sensitive methoxyimino cephalosporins.
As this phenomenom is caused by the SOS response, two LexA defective mutants in two different genetic backgrounds in E. faecalis result in an identical phenotype. In vivo, we mimic this effect by treating mice with a combination of levofloxacin, an SOS response inducing fluoroquinolone, and ceftriaxone, a Methoxymino cephalosporin.
In order to identify the SOS regulon and to determine the mechanism by wich the resistance is reverted we conducted a transcriptomic study of a wild type E. faecalis and its LexA- mutant. We demonstrated that the SOS response involved some known genes also described in the SOS response of other bacteria, such as genes involved in repair and recombination of DNA. It also displays peculiarities, such as the lack of both a cell division inhibitor in the genome and a single strand binding protein (SSB) in the regulon.
None of the genetic determinants thought to be required for the intrinsic resistance to cephalosporins, e. g., murAA, CroRS, IreK/P, low affinity pbp or rpoB, showed significant variations in their expression levels. These results were validated by quantitative PCR. To rule out the involvement of phages we also conduct resistance profiles in a phage-free bacteria (bacteria lacking phages).
To decipher the mechanism of the resistance reversion, we performed a random insertion library by transposition. Subsequently we selected the mutants that remain resistant in the presence of mytomicin C, whereby we identified several interesting transcriptional units (e. g. several transporters).
With these results we proceeded to disrupt two ABC type amino acid transporters implicated in the uptake of glutamine. Intriguingly, one of these transporters is regulated by the CroRS two component system implicated in the intrinsic resistance to cephalosporins, while the other transporter is a component of the SOS regulon. Additionally we identified both a cation and a peptide transporter system from the random insertion library. This peptide transporter has homology with the Opp family, implicated in the transport of cell wall peptides. In another mutant, the expression of the gen that codifies for the RecO protein, required for the induction of the SOS response, is affected by an upstream transposon insertion within its operon.
Finally we disrupted the first gene of the Cop operon, the CopY repressor. This operon is involved in copper homeostasis. There is a copper box upstream of the ssb-2 gene, a protein involved in DNA metabolism that binds single stranded DNA and therefore participates in the SOS system induction.
Strikingly, the presence of copper in wild type E. faecalis and E. faecium produces the same susceptibility profile to cephalosporins as mitomycin C. In an insertion mutant in the second gene of the operon, in which the CopY repressor should be expressed but not CopA and CopZ, the susceptibility to cephalosporins is induced only by mitomycin C but not by copper. This suggests that the effect of the SOS response on the intrinsic resistance to cephalosporins depends on the CopY repressor.
Further work should be performed to unravel the underlying mechanisms of this phenomenon.
