Whole-Genome Multilocus Sequence Typing of Extended-Spectrum-Beta-Lactamase-Producing Enterobacteriaceae

Abstract

e Molecular typing has become indispensable in the detection of nosocomial transmission of bacterial pathogens and the identification of sources and routes of transmission in outbreak settings, but current methods are labor-intensive, are difficult to standardize, or have limited resolution. Whole-genome multilocus sequence typing (wgMLST) has emerged as a whole-genome sequencing (WGS)-based gene-by-gene typing method that may overcome these limitations and has been applied successfully for several species in outbreak settings. In this study, genus-, genetic-complex-, and species-specific wgMLST schemes were developed for Citrobacter spp., the Enterobacter cloacae complex, Escherichia coli, Klebsiella oxytoca, and Klebsiella pneumoniae and used to type a national collection of 1,798 extended-spectrum-beta-lactamase-producing Enterobacteriaceae (ESBL-E) isolates obtained from patients in Dutch hospitals. Genus-, genetic-complex-, and species-specific thresholds for genetic distance that accurately distinguish between epidemiologically related and unrelated isolates were defined for Citrobacter spp., the E. cloacae complex, E. coli, and K. pneumoniae. wgMLST was shown to have higher discriminatory power and typeability than in silico MLST. In conclusion, the wgMLST schemes developed in this study facilitate high-resolution WGS-based typing of the most prevalent ESBL-producing species in clinical practice and may contribute to further elucidation of the complex epidemiology of antimicrobial-resistant Enterobacteriaceae. wgMLST opens up possibilities for the creation of a Web-accessible database for the global surveillance of ESBL-producing bacterial clones. The continuing global spread of extended-spectrum-beta-lactamase-producing Enterobacteriaceae (ESBL-E) constitutes a major public health threat in both health care and community settings (1-4). Extended-spectrum beta-lactamases (ESBL) confer resistance to the majority of beta-lactam antibiotics, including third-generation cephalosporins, which limits the options for antimicrobial therapy and results in increased morbidity and mortality and health care costs (5, 6). Infection control guidelines recommend prevention of the spread of ESBL-E in health care settings (7,8). Molecular typing has become an important tool in infection control, as it enables the detection of nosocomial transmission of bacterial pathogens and the identification of sources and routes of transmission in outbreak settings. Different molecular typing methods have been used, ranging from fingerprintbased methods, like pulsed-field gel electrophoresis (PFGE) and amplified fragment length polymorphism (AFLP), to sequencebased methods, like multilocus sequence typing (MLST) (9-11). Although widely adopted, PFGE is labor-intensive and difficult to standardize and has limited interlaboratory reproducibility (12)(13)(14). AFLP is less time-consuming than PFGE but may have lower discriminatory power for typing of Enterobacteriaceae (13, 15). MLST targets 7 or 8 housekeeping genes, depending on the Ent...

ArgO145, a Stx2a prophage of a bovine O145:H- STEC strain, is closely related to phages of virulent human strains

“…MiSeq data were processed with MiSeq control software v2.4.0.4 and MiSeq Reporter v2.4 (Illumina). Quality trimming of reads was performed with CLC Genomics Workbench 7.0.4 (Qiagen) using a minimum Phred (Q) score of 28 (Kluytmans-Van Den Bergh et al, 2016). In addition, a mate-pair DNA library was prepared using the Mate Pair Library Prep Kit v2 (Illumina) according to the manufacturer's instructions followed by running on the Miseq for generating 100-bp reads.…”

Detection of AmpC β-lactamases in Escherichia coli using different screening agars

“…The ESBL-producing E. coli and AmpC/ESBL negative E. coli isolates were confirmed to lack any known alterations in the promoter/attenuator region of the AmpC gene leading to hyperproduction of the β -lactamase, as reported by Tracz et al (7). Isolates were recovered at different study sites or from various clinical specimens, and were isolated from humans (n=141) and animals (n=18) (15–20). More detailed information on the recovery of the isolates is described in the additional file 1.…”

Next-generation sequencing technologies and their application to the study and control of bacterial infections

“…New isolates are compared against a database of all known gene variants (an allele database) which may be used to assess the relatedness of all isolates belonging to the species it was developed for. An increasing number of such databases is being developed with some available in public domain (34–37) making the MLST approach uniquely suitable for multi laboratory outbreak investigations like the ones performed by the PulseNet networks (9). …”

“…A WGS workflow can provide more detailed information than traditional methods in a matter of days. WGS workflows have also been used to successfully identify and characterize strains in clinical settings (11, 37). …”