Central role of the ramAR locus in the multidrug resistance in ESBL-Enterobacterales.

The aim of this study was to evaluate the proportion of resistance to a temocillin, tigecycline, ciprofloxacin, and chloramphenicol phenotype called t2c2 that resulted from mutations within the ramAR locus among extended-spectrum ß-lactamases-Enterobacterales (ESBL-E) isolated in three intensive care units for 3 years in a French university hospital. Two parallel approaches were performed on all 443 ESBL-E included: (i) the minimal inhibitory concentrations of temocillin, tigecycline, ciprofloxacin, and chloramphenicol were determined and (ii) the genomes obtained from the Illumina sequencing platform were analyzed to determine multilocus sequence types, resistomes, and diversity of several tetR-associated genes including ramAR operon. Among the 443 ESBL-E strains included, isolates of Escherichia coli (n = 194), Klebsiella pneumoniae (n = 122), and Enterobacter cloacae complex (Ecc) (n = 127) were found. Thirty-one ESBL-E strains (7%), 16 K. pneumoniae (13.1%), and 15 Ecc (11.8%) presented the t2c2 phenotype in addition to their ESBL profile, whereas no E. coli presented these resistances. The t2c2 phenotype was invariably reversible by the addition of Phe-Arg-ß-naphthylamide, indicating a role of resistance-nodulation-division pumps in these observations. Mutations associated with the t2c2 phenotype were restricted to RamR, the ramAR intergenic region (IR), and AcrR. Mutations in RamR consisted of C- or N-terminal deletions and amino acid substitutions inside its DNA-binding domain or within key sites of protein-substrate interactions. The ramAR IR showed nucleotide substitutions involved in the RamR DNA-binding domain. This diversity of sequences suggested that RamR and the ramAR IR represent major genetic events for bacterial antimicrobial resistance.IMPORTANCEMorbimortality caused by infectious diseases is very high among patients hospitalized in intensive care units (ICUs). A part of these outcomes can be explained by antibiotic resistance, which delays the appropriate therapy. The transferable antibiotic resistance gene is a well-known mechanism to explain the high rate of multidrug resistance (MDR) bacteria in ICUs. This study describes the prevalence of chromosomal mutations, which led to additional antibiotic resistance among MDR bacteria. More than 12% of Klebsiella pneumoniae and Enterobacter cloacae complex strains presented mutations within the ramAR locus associated with a dysregulation of an efflux pump called AcrAB-TolC and a porin: OmpF. These dysregulations led to an increase in antibiotic output notably tigecycline, ciprofloxacin, and chloramphenicol associated with a decrease of input for beta-lactam, especially temocillin. Mutations within transcriptional regulators such as ramAR locus played a major role in antibiotic resistance dissemination and need to be further explored.

Early detection of plasma d-lactate: Toward a new highly-specific biomarker of bacteraemia?

Background: Bloodstream infections are a leading cause of mortality. Their detection relies on blood cultures (BCs) but time to positivity is often between tens of hours and days. d-lactate is a metabolite widely produced by bacteria but very few in human. We aimed to evaluate d-lactate, d-lactate/l-lactate ratio and d-lactate/total lactate ratio in plasma as potential early biomarkers of bacteraemia on a strictly biological standpoint. Methods: A total of 228 plasma specimens were collected from patients who had confirmed bacteraemia (n = 131) and healthy outpatients (n = 97). Specific l-lactate and d-lactate analyses were performed using enzymatic assays and analytical performances of d-lactate, d-lactate/total lactate and d-lactate/l-lactate ratios for the diagnosis of bacteraemia were assessed. Results: A preliminary in vitro study confirmed that all strains of Escherichia coli, Klebsiella pneumoniae and Staphylococcus aureus were able to produce d-lactate at significant levels. In patients, plasma d-lactate level was the most specific biomarker predicting a bacteraemia profile with a specificity and predictive positive value of 100% using a cut-off of 131 µmol.L-1. However, sensitivity and negative predictive value were rather low, estimated at 31% and 52%, respectively. d-lactate displayed an Area Under Receiver Operating Characteristic (AUROC) curve of 0.696 with a P value < 0.0001. There was no difference of d-lactate levels between BCs bottles positive for Gram-positive or Gram-negative bacteria (p = 0.55). Conclusion: d-lactate shows promise as a specific early biomarker of bacterial metabolism. The development of rapid automated assays could raise clinical applications for infectious diseases diagnosis including early bacteraemia prediction.

A 10-year microbiological study of Pseudomonas aeruginosa strains revealed the circulation of populations resistant to both carbapenems and quaternary ammonium compounds.

Pseudomonas aeruginosa is one of the leading causes of healthcare-associated infections. For this study, the susceptibility profiles to antipseudomonal antibiotics and a quaternary ammonium compound, didecyldimethylammonium chloride (DDAC), widely used as a disinfectant, were established for 180 selected human and environmental hospital strains isolated between 2011 and 2020. Furthermore, a genomic study determined resistome and clonal putative relatedness for 77 of them. During the ten-year study period, it was estimated that 9.5% of patients' strains were resistant to carbapenems, 11.9% were multidrug-resistant (MDR), and 0.7% were extensively drug-resistant (XDR). Decreased susceptibility (DS) to DDAC was observed for 28.0% of strains, a phenotype significantly associated with MDR/XDR profiles and from hospital environmental samples (p < 0.0001). According to genomic analyses, the P. aeruginosa population unsusceptible to carbapenems and/or to DDAC was diverse but mainly belonged to top ten high-risk clones described worldwide by del Barrio-Tofiño et al. The carbapenem resistance appeared mainly due to the production of the VIM-2 carbapenemase (39.3%) and DS to DDAC mediated by MexAB-OprM pump efflux overexpression. This study highlights the diversity of MDR/XDR populations of P. aeruginosa which are unsusceptible to compounds that are widely used in medicine and hospital disinfection and are probably distributed in hospitals worldwide.

Antimicrobial resistance and genetic diversity of Klebsiella pneumoniae strains from different clinical sources in horses.

Introduction: Klebsiella pneumoniae is a major cause of infections and reproductive disorders among horses, ranked in recent French studies as the sixth most frequently isolated bacterial pathogen in equine clinical samples. The proportion of multidrug-resistant (MDR) K. pneumoniae is therefore significant in a context where MDR K. pneumoniae strains are considered a major global concern by the World Health Organization. Methods: In this study, we used a genomic approach to characterize a population of 119 equine K. pneumoniae strains collected by two laboratories specialized in animal health in Normandy (France). We describe the main antibiotic resistance profiles and acquired resistance genes, and specify the proportion of virulence-encoding genes carried by these strains. The originality of our panel of strains lies in the broad collection period covered, ranging from 1996 to 2020, and the variety of sample sources: necropsies, suspected bacterial infections (e.g., genital, wound, allantochorion, and umbilical artery samples), and contagious equine metritis analyses. Results: Our results reveal a remarkable level of genomic diversity among the strains studied and we report the presence of 39% MDR and 9% hypervirulent strains (including 5% that are both MDR and hypervirulent). Discussion: These findings clearly emphasize the importance of improving the surveillance of K. pneumoniae in routine equine diagnostic tests to detect high-risk MDR-hypervirulent Klebsiella pneumoniae strains. The circulation of these worrisome strains reveals that they are not being detected by the simple K1, K2, and K5 serotype approach currently implemented in the French horse-breeding sector.