Comparison of Molecular Subtyping and Antimicrobial Resistance Detection Methods Used in a Large Multistate Outbreak of Extensively Drug-Resistant Campylobacter jejuni Infections Linked to Pet Store Puppies

Abstract

Campylobacter jejuni is a leading cause of enteric bacterial illness in the United States. Traditional molecular subtyping methods, such as pulsed-field gel electrophoresis (PFGE) and 7-gene multilocus sequencing typing (MLST), provided limited resolution to adequately identify C. jejuni outbreaks and separate out sporadic isolates during outbreak investigations. Whole genome sequencing (WGS) has emerged as a powerful tool for C. jejuni outbreak detection. In this investigation, 45 human and 11 puppy isolates obtained during a 2016-2018 outbreak linked to pet store puppies were sequenced. Core genome multilocus sequence typing (cgMLST) and high-quality single nucleotide polymorphism (hqSNP) analysis of the sequence data separated the isolates into the same two clades containing minor within clade differences; however, cgMLST analysis does not require selection of an appropriate reference genome making this method preferable to hqSNP analysis for Campylobacter surveillance and cluster detection. The isolates were classified as ST2109—a rarely seen MLST sequence type. PFGE was performed on 38 human and 10 puppy isolates; PFGE patterns did not reliably predict clustering by cgMLST analysis. Genetic detection of antimicrobial resistance determinants predicted that all outbreak-associated isolates would be resistant to six drug classes. Traditional antimicrobial susceptibility testing (AST) confirmed a high correlation between genotypic and phenotypic antimicrobial resistance determinations. WGS analysis linked C. jejuni isolates in humans and pet store puppies even when canine exposure information was unknown, aiding the epidemiological investigation during this outbreak. WGS data were also used to quickly identify the highly drug-resistant profile of these outbreak-associated C. jejuni isolates.

Ongoing Outbreak of Extensively Drug-ResistantCampylobacter jejuniInfections Associated With US Pet Store Puppies, 2016-2020

“…Pulsed-field gel electrophoresis, the standard subtyping method used by PulseNet for more than 2 decades for detection of multistate outbreaks caused by Salmonella , Listeria , and Escherichia coli , has not worked as well for Campylobacter . 7 , 33 , 37 Whole-genome sequencing could help improve Campylobacter surveillance and outbreak detection, but Campylobacter isolates have been a lower priority for state public health laboratories with limited sequencing capacity. Limited implementation of WGS for Campylobacter affected the timeliness and completeness of case identification across study periods.…”

“…We compared sequences uploaded to PulseNet by cgMLST and estimated isolate relatedness by number of allele differences. 7 We generated de novo assemblies using Shovill software, version 1.0.9, 8 analyzed them for resistance determinants using the ResFinder database (90% identity and 50% cutoff), and screened for gyrA mutations using the PointFinder scheme for Campylobacter species implemented in Staramr software, version 0.4.0. 9 We identified mutations in the 23S ribosomal RNA region using ARIBA (Sanger Pathogens), version 2.12.0.…”

“… 16 We defined extensively drug resistant as resistant to macrolides and fluoroquinolones (the antibiotic classes recommended for treatment of Campylobacter ) 4 and 3 or more additional antibiotic classes. 7 , 17 …”

“…For this report, we defined a case as culture-confirmed C jejuni infection in a patient with (1) an epidemiologic association with a pet store puppy (defined as contact with a pet store puppy before JAMA Network Open | Public Health US Pet Store Puppies and Extensively Drug-Resistant Campylobacter jejuni or after purchase, including contact resulting from pet store employment or during pet store visitation) or (2) an isolate highly related by core genome multilocus sequence typing (cgMLST) 7 to an isolate from a patient with an epidemiologic association. State and local public health officials interviewed patients with cases from January 1, 2016, to February 12, 2018, using a focused questionnaire that included demographic characteristics (age, sex, race, ethnicity, and state of residence), outcomes (hospitalization or death), and exposures 7 days before illness began (contact with a dog or puppy, type of exposure, pet store, or breeder affiliation).…”

Antimicrobial susceptibility testing and successful treatment of hospitalised patients with extensively drug-resistant Campylobacter jejuni infections linked to a pet store puppy outbreak

“…This resistance profile raised urgent concerns about the optimal management of patients infected with this extensively drug-resistant (XDR) Campylobacter strain [ 11 ]. Multiple small studies have previously described in vitro susceptibility of C. jejuni to carbapenems [ 12 – 15 ].…”