Raising the alarm: fosfomycin resistance associated with non-susceptible inner colonies imparts no fitness cost to the primary bacterial uropathogen.

IMPORTANCE: While fosfomycin resistance is rare, the observation of non-susceptible subpopulations among clinical Escherichia coli isolates is a common phenomenon during antimicrobial susceptibility testing (AST) in American and European clinical labs. Previous evidence suggests that mutations eliciting this phenotype are of high biological cost to the pathogen during infection, leading to current recommendations of neglecting non-susceptible colonies during AST. Here, we report that the most common route to fosfomycin resistance, as well as novel routes described in this work, does not impair virulence in uropathogenic E. coli, the major cause of urinary tract infections, suggesting a re-evaluation of current susceptibility guidelines is warranted.

Development of a multiplex real-time PCR assay for phylogenetic analysis of Uropathogenic Escherichia coli.

Uropathogenic Escherichia coli (UPEC) is among major pathogens causing 80-90% of all episodes of urinary tract infections (UTIs). Recently, E. coli strains are divided into eight main phylogenetic groups including A, B1, B2, C, D, E, F, and clade I. This study was aimed to develop a rapid, sensitive, and specific multiplex real time PCR method capable of detecting phylogenetic groups of E. coli strains. This study was carried out on E. coli strains (isolated from the patient with UTI) in which the presence of all seven target genes had been confirmed in our previous phylogenetic study. An EvaGreen-based singleplex and multiplex real-time PCR with melting curve analysis was designed for simultaneous detection and differentiation of these genes. The primers were selected mainly based on the production of amplicons with melting temperatures (Tm) ranging from 82°C to 93°C and temperature difference of more than 1.5°C between each peak.The multiplex real-time PCR assays that have been developed in the present study were successful in detecting the eight main phylogenetic groups. Seven distinct melting peaks were discriminated, with Tm value of 93±0.8 for arpA, 89.2±0.1for chuA, 86.5±0.1 for yjaA, 82.3±0.2 for TspE4C2, 87.8±0.1for trpAgpC, 85.4±0.6 for arpAgpE genes, and 91±0.5 for the internal control. To our knowledge, this study is the first melting curve-based real-time PCR assay developed for simultaneous and discrete detection of these seven target genes. Our findings showed that this assay has the potential to be a rapid, reliable and cost-effective alternative for routine phylotyping of E. coli strains.

Low biological cost of carbapenemase-encoding plasmids following transfer from Klebsiella pneumoniae to Escherichia coli.

OBJECTIVES: The objective of this study was to determine the biological cost, stability and sequence of two carbapenemase-encoding plasmids containing blaKPC-2 (pG12-KPC-2) and blaVIM-1 (pG06-VIM-1) isolated from Klebsiella pneumoniae when newly acquired by uropathogenic Escherichia coli clinical isolates of different genetic backgrounds. METHODS: The two plasmids were transferred into plasmid-free E. coli clinical isolates by transformation. The fitness effect of newly acquired plasmids on the host cell was assessed in head-to-head competitions with the corresponding isogenic strain. Plasmid stability was estimated by propagating monocultures for ∼312 generations. Plasmid nucleotide sequences were determined using next-generation sequencing technology. Assembly, gap closure, annotation and comparative analyses were performed. RESULTS: Both plasmids were stably maintained in three of four E. coli backgrounds and resulted in low to moderate reductions in host fitness ranging from 1.1% to 3.6%. A difference in fitness cost was observed for pG12-KPC-2 between two different genetic backgrounds, while no difference was detected for pG06-VIM-1 between three different genetic backgrounds. In addition, a difference was observed between pG12-KPC-2 and pG06-VIM-1 in the same genetic background. In general, the magnitude of biological cost of plasmid carriage was both host and plasmid dependent. The sequences of the two plasmids showed high backbone similarity to previously circulating plasmids in K. pneumoniae. CONCLUSIONS: The low to modest fitness cost of newly acquired and stably maintained carbapenemase-encoding plasmids in E. coli indicates a potential for establishment and further dissemination into other Enterobacteriaceae species. We also show that the fitness cost is both plasmid and host specific.

Metabolomic analysis of siderophore cheater mutants reveals metabolic costs of expression in uropathogenic Escherichia coli.

Bacterial siderophores are a group of chemically diverse, virulence-associated secondary metabolites whose expression exerts metabolic costs. A combined bacterial genetic and metabolomic approach revealed differential metabolomic impacts associated with biosynthesis of different siderophore structural families. Despite myriad genetic differences, the metabolome of a cheater mutant lacking a single set of siderophore biosynthetic genes more closely approximate that of a non-pathogenic K12 strain than its isogenic, uropathogen parent strain. Siderophore types associated with greater metabolomic perturbations are less common among human isolates, suggesting that metabolic costs influence success in a human population. Although different siderophores share a common iron acquisition function, our analysis shows how a metabolomic approach can distinguish their relative metabolic impacts in E. coli.

Virulence characteristics and genetic affinities of multiple drug resistant uropathogenic Escherichia coli from a semi urban locality in India.

Extraintestinal pathogenic Escherichia coli (ExPEC) are of significant health concern. The emergence of drug resistant E. coli with high virulence potential is alarming. Lack of sufficient data on transmission dynamics, virulence spectrum and antimicrobial resistance of certain pathogens such as the uropathogenic E. coli (UPEC) from countries with high infection burden, such as India, hinders the infection control and management efforts. In this study, we extensively genotyped and phenotyped a collection of 150 UPEC obtained from patients belonging to a semi-urban, industrialized setting near Pune, India. The isolates representing different clinical categories were analyzed in comparison with 50 commensal E. coli isolates from India as well as 50 ExPEC strains from Germany. Virulent strains were identified based on hemolysis, haemagglutination, cell surface hydrophobicity, serum bactericidal activity as well as with the help of O serotyping. We generated antimicrobial resistance profiles for all the clinical isolates and carried out phylogenetic analysis based on repetitive extragenic palindromic (rep)-PCR. E. coli from urinary tract infection cases expressed higher percentages of type I (45%) and P fimbriae (40%) when compared to fecal isolates (25% and 8% respectively). Hemolytic group comprised of 60% of UPEC and only 2% of E. coli from feces. Additionally, we found that serum resistance and cell surface hydrophobicity were not significantly (p = 0.16/p = 0.51) associated with UPEC from clinical cases. Moreover, clinical isolates exhibited highest resistance against amoxicillin (67.3%) and least against nitrofurantoin (57.3%). We also observed that 31.3% of UPEC were extended-spectrum beta-lactamase (ESBL) producers belonging to serotype O25, of which four were also positive for O25b subgroup that is linked to B2-O25b-ST131-CTX-M-15 virulent/multiresistant type. Furthermore, isolates from India and Germany (as well as global sources) were found to be genetically distinct with no evidence to espouse expansion of E. coli from India to the west or vice-versa.