A. Philippon

OBJECTIVES: WGS-based antimicrobial susceptibility testing (AST) is as reliable as phenotypic AST for several antimicrobial/bacterial species combinations. However, routine use of WGS-based AST is hindered by the need for bioinformatics skills and knowledge of antimicrobial resistance (AMR) determinants to operate the vast majority of tools developed to date. By leveraging on ResFinder and PointFinder, two freely accessible tools that can also assist users without bioinformatics skills, we aimed at increasing their speed and providing an easily interpretable antibiogram as output. METHODS: The ResFinder code was re-written to process raw reads and use Kmer-based alignment. The existing ResFinder and PointFinder databases were revised and expanded. Additional databases were developed including a genotype-to-phenotype key associating each AMR determinant with a phenotype at the antimicrobial compound level, and species-specific panels for in silico antibiograms. ResFinder 4.0 was validated using Escherichia coli (n = 584), Salmonella spp. (n = 1081), Campylobacter jejuni (n = 239), Enterococcus faecium (n = 106), Enterococcus faecalis (n = 50) and Staphylococcus aureus (n = 163) exhibiting different AST profiles, and from different human and animal sources and geographical origins. RESULTS: Genotype-phenotype concordance was ≥95% for 46/51 and 25/32 of the antimicrobial/species combinations evaluated for Gram-negative and Gram-positive bacteria, respectively. When genotype-phenotype concordance was <95%, discrepancies were mainly linked to criteria for interpretation of phenotypic tests and suboptimal sequence quality, and not to ResFinder 4.0 performance. CONCLUSIONS: WGS-based AST using ResFinder 4.0 provides in silico antibiograms as reliable as those obtained by phenotypic AST at least for the bacterial species/antimicrobial agents of major public health relevance considered.

Before 1985 at the Pitié-Salpêtrière Hospital in Paris (2,400 beds), resistance to cefotaxime in clinical isolates of Enterobacteriaceae involved only species producing inducible class 1 beta-lactamase. Between November 1985 and April 1987, however, 62 isolates (57 of Klebsiella pneumoniae and five of Escherichia coli) showed decreased susceptibility to cefotaxime (mean MIC, 8-16 micrograms/mL). The transferability of cefotaxime resistance in E. coli K12 was demonstrated for 15 of 16 selected isolates. By isoelectric focusing using iodometric detection with 20 mg of ceftriaxone/100 mL and determination of substrate and inhibition profiles, three beta-lactamases mediating cefotaxime resistance were identified as SHV-2 (isoelectric point [pI] 7.6), CTX-1 (pI 6.3), and "SHV-2-type" or SHV-3 (pI 6.98). The three beta-lactamases hydrolyzed penicillins and cephalosporins (including cefotaxime and ceftriaxone) and were therefore designated "extended broad-spectrum beta-lactamases" (EBS-Bla). The enzymes conferred to derivatives a high level of resistance to amoxicillin, ticarcillin, piperacillin, and cephalothin and a decreased degree of susceptibility (i.e., MICs increased by 10- to 800-fold) to cefotaxime, ceftriaxone, ceftazidime, and aztreonam. These beta-lactamases did not affect the activity of cephamycins (cefoxitin, cefotetan, moxalactam) or imipenem. Synergy between clavulanate or sulbactam (2 micrograms/mL) and amoxicillin was greater against derivatives producing EBS-Bla than against those producing TEM-1, TEM-2, or SHV-1; this synergy was greater with clavulanate than with sulbactam against derivatives producing SHV-2 and the SHV-2-type enzyme but was similar with clavulanate and sulbactam against those producing CTX-1. A double-disk synergy test performed with cefotaxime and Augmentin disks (placed 30 mm apart, center to center) seemed a useful and specific test for the detection of strains producing EBS-Bla.

The predominant mechanism for resistance to β-lactam antibiotics in gram-negative bacteria is the synthesis of β-lactamase. To meet this challenge, β-lactams with greater β-lactamase stability, including cephalosporins, carbapenems, and monobactams, were introduced in the 1980s. Resistance