Genotypic resistance determined by whole genome sequencing versus phenotypic resistance in 234 Escherichia coli isolates.

Whole genome sequencing (WGS) enables detailed characterization of bacteria at single nucleotide resolution. It provides data about acquired resistance genes and mutations leading to resistance. Although WGS is becoming an essential tool to predict resistance patterns accurately, comparing genotype to phenotype with WGS is still in its infancy. Additional data and validation are needed. In this retrospective study, we analysed 234 E. coli isolates from positive blood cultures using WGS as well as microdilution for 11 clinically relevant antibiotics, to compare the two techniques. We performed whole genome sequencing analyses on 234 blood culture isolates (genotype) to detect acquired antibiotic resistance. Minimal inhibitory concentrations (MIC) for E. coli were performed for amoxicillin, cefepime, cefotaxime, ceftazidime, meropenem, amoxicillin/clavulanic acid, piperacillin/tazobactam, amikacin, gentamicin, tobramycin, and ciprofloxacin, using the ISO 20776-1 standard broth microdilution method as recommended by EUCAST (phenotype). We then compared the two methods for statistical 'agreement'. A perfect (100%) categorical agreement between genotype and phenotype was observed for gentamicin and meropenem. However, no resistance to meropenem was observed. A high categorical agreement (> 95%) was observed for amoxicillin, cefepime, cefotaxime, ceftazidime, amikacin, and tobramycin. A categorical agreement lower than 95% was observed for amoxicillin/clavulanic acid, piperacillin/tazobactam, and ciprofloxacin. Most discrepancies occurred in isolates with MICs within ± 1 doubling dilution of the breakpoint and 22.73% of the major errors were samples that tested phenotypically susceptible at higher antibiotic exposure and were therefore considered as 'not resistant'. This study shows that WGS can be used as a valuable tool to predict phenotypic resistance against most of the clinically relevant antibiotics used for the treatment of E. coli bloodstream infections.

Molecular characterization of extraintestinal and diarrheagenic Escherichia coli blood isolates.

Pathogenic E. coli strains can be classified into two major groups, based on the presence of specific virulence factors: extraintestinal pathogenic E. coli (ExPEC) and diarrheagenic E. coli (DEC). Several case reports describe that DEC can cause bloodstream infections in some rare cases. This mainly concerns a few specific sequence types that express virulence factors from both ExPEC and DEC. In this study, we retrospectively analysed 234 E. coli blood isolates with whole genome sequencing (WGS). WGS was performed on an Illumina NovaSeq6000. Genotyping was performed using BioNumerics software. The presence of genes was determined with a minimum percentage sequence identity (ID) threshold of 95% and a minimum length for sequence coverage of 95%. Three of the 234 (1.28%) isolates were defined as DEC, 182 (77.78%) as ExPEC, and 49 (20.94%) did not carry pathotype-associated virulence genes. We identified 112 different virulence genes, 48 O-antigens, and 28 H-antigens 82 STs, among the 234 analyzed isolates. ST131 and ST88 were related to healthcare-associated infections. This study provides insight into the prevalence of virulence factors in a large set of E. coli blood isolates from the UZ Brussel. It illustrates high diversity in virulence profiles and highlights the potential of DEC to carry virulence factors associated with extraintestinal infections, making it possible for unusual pathotypes to invade and survive in the bloodstream causing bacteraemia. Diarrheagenic strains causing bacteremia are rare and presently underreported, but modern sequencing techniques will better underscore their importance.

Characteristics of Shiga toxin producing- and enteropathogenic Escherichia coli of the emerging serotype O80:H2 isolated from humans and diarrhoeic calves in Belgium.

OBJECTIVES: Recently a highly virulent Escherichia coli O80:H2 pathotype carrying Shiga toxin genes, the intimin subtype eaeξ, and genes associated with the extraintestinal pathogenic E. coli (ExPEC) pS88 plasmid was described in France. In this study we examine the relatedness of Belgian E. coli O80:H2 isolated from humans and diarrhoeic calves as well their similarities with the French pathotype. METHODS: Eighteen Belgian E. coli O80:H2 strains (nine human Shiga toxin-producing E. coli (STEC) (2008-2016), two bovine STEC (1987) and seven bovine atypical enteropathogenic E. coli (aEPEC) (2009-2015)) were characterized with conventional PCR, disc diffusion susceptibility testing and whole genome sequencing. RESULTS: Only nine sporadic human STEC O80:H2 cases have been detected in Belgium. All patients were female, just two of them suffered from haemolytic uremic syndrome. All studied strains had the eaeξ subtype, belonged to the multi-locus sequence type ST-301, and carried virulence genes associated with the type III secretion system and effectors not encoded by the locus of enterocyte effacement (LEE). Multiple genes of the pS88 plasmid were detected in all but two strains (one human and one calf STEC). The Shiga toxin subtypes stx1a (n = 3; one human, two calf), stx2a (n = 2) and stx2d (n = 6) were detected. All strains were multidrug resistant, two were extended-spectrum ß-lactamase positive. Core genome MLST revealed that some human and calf E. coli differed by only 22 loci. CONCLUSIONS: The STEC/ExPEC O80:H2 pathotype was present in calves in Belgium as early as 1987, but human infections have been rare and mostly mild. The human STEC and bovine aEPEC cluster together and have the potential to be as virulent as the French isolates, as shown by their similar gene content.