Macrocolony of NDM-1 Producing Enterobacter hormaechei subsp. oharae Generates Subpopulations with Different Features Regarding the Response of Antimicrobial Agents and Biofilm Formation

Abstract

Enterobacter cloacae complex has been increasingly recognized as a nosocomial pathogen representing the third major Enterobacteriaceae species involved with infections. This study aims to evaluate virulence and antimicrobial susceptibility of subpopulations generated from macrocolonies of NDM-1 producing Enterobacter hormaechei clinical isolates. Biofilm was quantified using crystal violet method and fimbrial genes were investigated by PCR. Susceptibility of antimicrobials, alone and combined, was determined by minimum inhibitory concentration and checkerboard assays, respectively. Virulence and efficacy of antimicrobials were evaluated in Galleria mellonella larvae. Importantly, we verified that some subpopulations that originate from the same macrocolony present different biofilm production ability and distinct susceptibility to meropenem due to the loss of blaNDM-1 encoding plasmid. A more in-depth study was performed with the 798 macrocolony subpopulations. Type 3 fimbriae were straightly related with biofilm production; however, virulence in larvae was not statistically different among subpopulations. Triple combination with meropenem–rifampicin–polymyxin B showed in vitro synergistic effect against all subpopulations; while in vivo this treatment showed different efficacy rates for 798-1S and 798-4S subpopulations. The ability of multidrug resistant E. hormaechei isolates in generating bacterial subpopulations presenting different susceptible and virulence mechanisms are worrisome and may explain why these infections are hardly overcome.

The membrane depolarization and increase intracellular calcium level produced by silver nanoclusters are responsible for bacterial death

“…blood, urine, feces, and body fluids) 33 . On the other hand, studying the biofilm production of 9 strains of E. hormaechei from clinical origin reported that they formed a moderate biofilm 34 . Other studies have been evidenced the capability of K. pneumoniae to produce weak and moderate biofilm.…”

Cluster Differences in Antibiotic Resistance, Biofilm Formation, Mobility, and Virulence of Clinical Enterobacter cloacae Complex

“…To explore the differences in biofilm formation ability among different species of ECC, biofilm formation assays were performed in a 96-well polystyrene microtiter plate, as previously described with some modifications ( Brust et al, 2019 ). Briefly, the isolates were grown overnight in Luria–Bertani (LB) broth at 37°C.…”

“…In order to investigate the differences in virulence genes among various species of ECC, the genome DNA of all 130 ECC strains was extracted using the Biospin Bacterial Genomic DNA Extraction kit (Bioflux, Tokyo, Japan) according to the manufacturer’s instructions and served as templates for subsequent analysis. Virulence genes, including fimA (type 1 fimbriae gene) ( Brust et al, 2019 ), mrkD (type 3 fimbriae gene) ( Compain et al, 2014 ), csgD (curli gene) ( Brust et al, 2019 ), papC and papD (P pili genes) ( Brust et al, 2019 ), fyuA ( Yersinia siderophore receptor gene) ( Johnson et al, 2015 ), iroNec (salmochelin siderophore receptor gene) ( Johnson et al, 2015 ), entB (enterobactin gene) ( May and Okabe, 2011 ), and ybtS (yersiniabactin gene) ( El Fertas-Aissani et al, 2013 ), were identified by PCR. The primers of all genes for PCR are listed in Supplementary Table 1 .…”

“…Biofilm is an aggregate of sessile microorganisms, which prevents the antibiotics from coming into contact with bacteria, leading to repeated infections. Also, virulence-related genes in ECC, such as siderophore and adhesin, play a specific role in its infection; these include fimA (type 1 fimbriae gene) ( Brust et al, 2019 ), mrkD (type 3 fimbriae gene) ( Compain et al, 2014 ), csgD (curli gene) ( Brust et al, 2019 ), papC and papD (P pili genes) ( Brust et al, 2019 ), fyuA ( Yersinia siderophore receptor gene) ( Johnson et al, 2015 ), iroNec (salmochelin siderophore receptor gene) ( Johnson et al, 2015 ), entB (enterobactin gene) ( May and Okabe, 2011 ), and ybtS (yersiniabactin gene) ( El Fertas-Aissani et al, 2013 ).…”

Antimicrobial resistance, molecular characteristics, virulence and pathogenicity of bla NDM-1-positive Enterobacter cloacae

“…E. cloacae is an important opportunistic pathogen with complex genetic elements that can produce a variety of antibiotic hydrolases, and current studies have shown that carbapenemase, especially NDM-1, is the main resistance mechanism of carbapenem-resistant E. cloacae (CR-ECL) [1,2]. E. cloacae infect hosts and cause disease through various mechanisms, such as colonization, invasion, evasion of the immune response, and release of toxins [3,4], and its pathogenic process is closely related to various virulence factors such as fimbriae, siderophore, toxin secretion system, biofilm, efflux pump, virulence island and lipopolysaccharide [5][6][7]. E. cloacae contains abundant plasmid and chromosomal genomes and has easy access to multiple mobile genetic elements containing antimicrobial resistance genes and virulence genes, both of which play an important role in the evolution of bacteria mainly through gene-level transfer and are interrelated [3].…”