Comparison of phenotypic and WGS-derived antimicrobial resistance profiles of Salmonella enterica serovars Typhi and Paratyphi.

Objectives: Surveillance of antimicrobial resistance (AMR) in Salmonella enterica serovars Typhi and Paratyphi is essential to provide an evidence base for empirical treatment protocols and to monitor emerging AMR. We sought to compare phenotypic and WGS-based genotypic methods for the detection of AMR in Salmonella Typhi and Salmonella Paratyphi. Methods: WGS data from 603 isolates of Salmonella Typhi (n = 332) and Salmonella Paratyphi (n = 271) were mapped to genes or chromosomal mutations known to be associated with phenotypic AMR and compared with phenotypic susceptibility data interpreted using breakpoints recommended by EUCAST. Results: There were two (0.03%) discordant interpretations out of a possible 6030 isolate/antimicrobial class combinations. MDR (resistant to three or more classes of antimicrobial) was detected in 83/332 (25.0%) Salmonella Typhi isolates, but was not detected in Salmonella Paratyphi. Thirty-six (10.8%) isolates of Salmonella Typhi were resistant to ciprofloxacin (MIC >0.5 mg/L), with 33 (9.9%) of 332 exhibiting mutations in gyrA and parC, and 244 (73.5%) isolates had reduced susceptibility to ciprofloxacin (MIC 0.06-0.25 mg/L). In comparison, 209/227 (92.1%) isolates of Salmonella Paratyphi A exhibited resistance to ciprofloxacin (MIC >0.5 mg/L). No resistance to azithromycin or the third-generation cephalosporins was detected. Conclusions: WGS data provided a robust and informative approach for monitoring MDR and emerging resistance to ciprofloxacin in Salmonella Typhi and Salmonella Paratyphi. Phenotypic antimicrobial susceptibility testing continues to be performed to guide targeted individual patient treatment, but inferred AMR profiles from WGS data may be used for surveillance and to guide empirical therapy.

Comparison of phenotypic and WGS-derived antimicrobial resistance profiles of enteroaggregative Escherichia coli isolated from cases of diarrhoeal disease in England, 2015-16.

OBJECTIVES: Phenotypic and genotypic methods for the detection of antimicrobial resistance (AMR) in enteroaggregative Escherichia coli (EAEC) were compared and evaluated. METHODS: WGS data from 155 isolates of EAEC isolated between June 2015 and December 2016 were mapped to genes known to be associated with phenotypic AMR. RESULTS: Phenotypic and genotypic testing of 155 isolates against 10 antimicrobial classes resulted in a total of 25 (1.6%) discordant results of a possible 1550 isolate/antimicrobial combinations. Twenty-three of the mismatches were observed in streptomycin or sulphonamide resistance profiles. These discrepancies were associated with either insertions or truncations in the genes predicted to confer resistance, or in their promotors, rendering them non-functional, or with the presence of aadA variants associated with reduced expression. The most common resistances detected were to ampicillin (56.1%), the sulphonamides (49.7%) and trimethoprim (48.4%). The presence of CTX-M ESBL variants and/or acquired AmpC was detected in 87 of 155 (56.1%) isolates and 18 of 155 (11.6%) isolates were resistant to ciprofloxacin. Eighty-eight (56.8%) isolates were MDR. CONCLUSIONS: Phenotypic and genome-derived AMR comparisons showed good correlation for EAEC. A better understanding of the role of allelic variants, specific gene combinations and promoter/attenuator mechanisms in the phenotypic manifestation will improve our ability to provide a robust interpretation of the data for surveillance purposes and, ultimately, in the clinical setting.

Comparison of phenotypic and WGS-derived antimicrobial resistance profiles of Shigella sonnei isolated from cases of diarrhoeal disease in England and Wales, 2015.

Objectives: Phenotypic and genotypic methods for the detection of antimicrobial resistance (AMR) in Shigella sonnei in England and Wales were compared and evaluated. Methods: WGS data from 341 isolates of S. sonnei isolated between June 2015 and January 2016 were mapped to genes known to be associated with phenotypic AMR. Antimicrobial susceptibility testing was performed on all viable isolates (n = 335). Results: Fifteen of 335 isolates had a discrepancy between phenotypic and genotypic testing for 1 of the 10 antimicrobial classes tested, equating to 15 (0.45%) discordant results out of a possible 3350 isolate/antimicrobial combinations. All 15 mismatched results were genotypically resistant but phenotypically susceptible. Eleven of the 15 discrepancies were observed in streptomycin resistance profiles. The most common resistance profile was trimethoprim, sulphonamides, tetracyclines and streptomycin, occurring in 97 (28.4%) isolates. Resistances to ciprofloxacin and the third-generation cephalosporins, not detected in England and Wales prior to 2002, were identified in 18.2% and 12% of isolates, respectively. Three hundred and four (89.1%) isolates were MDR. There was no significant association between any of the AMR determinants tested and recent foreign travel in male or female cases. The number of isolates of S. sonnei harbouring blaTEM-1 and ermB/mphA was significantly higher in men who reported no recent travel outside the UK. Conclusions: The use of WGS for routine public health surveillance is a reliable method for rapid detection of emerging AMR in isolates of S. sonnei.

Antimicrobial resistance in Shiga toxin-producing Escherichia coli other than serotype O157 : H7 in England, 2014-2016.

Introduction. Despite many ongoing surveillance projects and the recent focus on the veterinary and clinical 'One Health' aspects of antimicrobial resistance (AMR), evidence of the extent of any public health risk posed by animal reservoirs with respect to the transmission of resistant strains of Escherichia coli to humans remains varied and contentious. In the UK, the main zoonotic reservoir for the foodborne pathogen Shiga toxin-producing E. coli (STEC) is cattle and sheep. In this study, we adopt an alternative approach to the risk assessment of transmission of AMR E. coli from animals to humans, involving monitoring AMR in isolates of STEC, an established zoonotic, foodborne pathogen, from human cases of gastrointestinal disease.Aim. The aim of this study was to determine the genome-derived AMR profiles for STEC from human cases to assess the risk of transmission of multidrug-resistant STEC from ruminants to humans.Methodology. STEC belonging to 10 different clonal complexes (CCs) (n=457) isolated from human faecal specimens were sequenced and genome-derived AMR profiles were determined. Phenotypic susceptibility testing was undertaken on all isolates (n=100) predicted to be resistant to at least one class of antimicrobial.Results. Of the 457 isolates, 332 (72.7 %) lacked identifiable resistance genes and were predicted to be fully susceptible to 11 classes of antimicrobials; 125/332 (27.3 %) carried 1 or more resistance genes, of which 83/125 (66.4 %) were resistant to 3 or more classes of antibiotic. The percentage of isolates harbouring AMR determinants varied between CCs, from 4% in CC25 to 100% in CC504. Forty-six different AMR genes were detected, which conferred resistance to eight different antibiotic classes. Resistance to ampicillin, streptomycin, tetracyclines and sulphonamides was most commonly detected. Four isolates were identified as extended-spectrum ß-lactamase producers. An overall concordance of 97.7 % (n=1075/1100) was demonstrated between the phenotypic and genotypic methods.Conclusion. This analysis provided an indirect assessment of the risk of transmission of AMR gastrointestinal pathogens from animals to humans, and revealed a subset of human isolates of the zoonotic pathogen STEC were resistant to the antimicrobials used in animal husbandry. However, this proportion has not increased over the last three decades, and thismay provide evidence that guidancepromoting responsible practice has been effective.

Antimicrobial resistance profiles of diarrhoeagenic Escherichia coli isolated from travellers returning to the UK, 2015-2017.

Introduction. Diarrhoeagenic Escherichia coli (DEC) are difficult to distinguish from non-pathogenic commensal E. coli using traditional culture methods. The implementation of PCR targeting specific virulence genes characteristic of the five DEC pathotypes, has improved the detection of DEC in faecal specimens from patients with symptoms of gastrointestinal disease.Aim. Antimicrobial resistance (AMR) profiles of 660 strains of DEC isolated between 2015 and 2017 from UK travellers reporting symptoms of gastrointestinal disease were reviewed to look for evidence of emerging AMR associated with travellers' diarrhoea.Methodology. All isolates of DEC were sequenced, and sequence type, serotype, pathotype markers and AMR profiles were derived from the genome data.Results. A travel history was provided for 54.1 % (357/660) of cases, of which 77.0 % (275/357) reported travel outside the UK within 7 days of onset of symptoms, and 23.0 % (82/357) reported no travel in that time frame. Of the 660 strains of DEC in this study, 265 (40.2 %) samples were identified as EAEC, 48 (7.3 %) as EIEC, 61 (9.2 %) were ETEC and 286 (43.3 %) were EPEC. EPEC caused the highest percentage of infections in children (40.6 %) whilst the highest proportion of cases reporting recent travel were infected with ETEC (86.1 %). There were 390/660 (59.0 %) isolates resistant to at least one antimicrobial on the panel tested (EIEC, 81.3 %; ETEC, n=65.6 %; EAEC, n=73.2 %; EPEC, 40.9 %) and 265/660 (40.2 %) were multidrug-resistant (EIEC, 33.3 %; ETEC, 32.8 %; EAEC, 56.2 %; EPEC, 28.0 %). Genes conferring resistance to the beta-lactams and fluroquinolones were highest in the EAEC pathotype, 56.6 and 60.7%, respectively.Conclusions. Increasing MDR, along with resistance to the fluroquinolones and the third-generation cephalosporins, in DEC causing travellers' diarrhoea provides further evidence for the need to restrict the use of antimicrobial agents and continuous monitoring.

Antimicrobial resistance profiles of Shigella dysenteriae isolated from travellers returning to the UK, 2004-2017.

PURPOSE: Antimicrobial resistance (AMR) profiles of 754 strains of Shigella dysenteriae isolated between 2004 and 2017 from UK travellers reporting symptoms of gastrointestinal (GI) disease were reviewed to look for evidence of emerging AMR associated with travellers' diarrhoea. METHODOLOGY: A travel history was provided for 72.7 % (548/754) of cases, of which 90.9 % (498/548) reported travel outside the UK within 7 days of onset of symptoms, and 9.1 % (50/498) reported no travel in that time frame. During the course of this study, whole genome sequencing (WGS) was implemented for GI disease surveillance, and we compared phenotypic AMR profiles with those derived from WGS data (n=133).Results/Key findings. The phenotypic and genotypic AMR results correlated well, with 90.1 % (121/133) isolates having concordant results to 10 classes of antimicrobials. Resistance to the first-line drugs commonly used in the treatment of shigellosis was observed throughout the study (ampicillin, 54.1%; chloramphenicol, 33.7 %; sulphonamides, 76.0 %; trimethoprim, 80.0%). Between 2004 and 2017, resistance to all classes of antimicrobials (except the phenicols) increased. The proportion of isolates exhibiting reduced susceptibility to ciprofloxacin increased from 3.8 % in 2004 to 75.7 % in 2017, and this was significantly associated with cases reporting travel to Asia compared to Africa (P<0.001). Of the 201 sequenced isolates, 3.0 % (20/201) had either blaCTX-M-15 or blaCMY-4. CONCLUSIONS: Increasing MDR, along with resistance to the fluroquinolones and the third generation cephalosporins, in Shigella dysenteriae causing travellers' diarrhoea provides further evidence for the need to regulatethe use of antimicrobial agents and continuous monitoring of emerging AMR.

Prediction of Phenotypic Antimicrobial Resistance Profiles From Whole Genome Sequences of Non-typhoidal Salmonella enterica.

Surveillance of antimicrobial resistance (AMR) in non-typhoidal Salmonella enterica (NTS), is essential for monitoring transmission of resistance from the food chain to humans, and for establishing effective treatment protocols. We evaluated the prediction of phenotypic resistance in NTS from genotypic profiles derived from whole genome sequencing (WGS). Genes and chromosomal mutations responsible for phenotypic resistance were sought in WGS data from 3,491 NTS isolates received by Public Health England's Gastrointestinal Bacteria Reference Unit between April 2014 and March 2015. Inferred genotypic AMR profiles were compared with phenotypic susceptibilities determined for fifteen antimicrobials using EUCAST guidelines. Discrepancies between phenotypic and genotypic profiles for one or more antimicrobials were detected for 76 isolates (2.18%) although only 88/52,365 (0.17%) isolate/antimicrobial combinations were discordant. Of the discrepant results, the largest number were associated with streptomycin (67.05%, n = 59). Pan-susceptibility was observed in 2,190 isolates (62.73%). Overall, resistance to tetracyclines was most common (26.27% of isolates, n = 917) followed by sulphonamides (23.72%, n = 828) and ampicillin (21.43%, n = 748). Multidrug resistance (MDR), i.e., resistance to three or more antimicrobial classes, was detected in 848 isolates (24.29%) with resistance to ampicillin, streptomycin, sulphonamides and tetracyclines being the most common MDR profile (n = 231; 27.24%). For isolates with this profile, all but one were S. Typhimurium and 94.81% (n = 219) had the resistance determinants blaTEM-1,strA-strB, sul2 and tet(A). Extended-spectrum ß-lactamase genes were identified in 41 isolates (1.17%) and multiple mutations in chromosomal genes associated with ciprofloxacin resistance in 82 isolates (2.35%). This study showed that WGS is suitable as a rapid means of determining AMR patterns of NTS for public health surveillance.

Plasmid-mediated quinolone resistance determinant qnrS1 found in Salmonella enterica strains isolated in the UK.

OBJECTIVES: To determine the prevalence of qnr genes in selected Salmonella enterica and Escherichia coli isolated in the UK. METHODS: One hundred and eighteen S. enterica and 103 E. coli were screened for qnrA, qnrB and qnrS by PCR. Transferability of qnr plasmids was assessed and isolated plasmids compared with previously identified qnr plasmids by restriction fragment length polymorphism analysis and hybridization experiments. PCRs and sequencing identified co-transferred beta-lactamase genes and mutations in the quinolone resistance-determining region of gyrA. RESULTS: Only six S. enterica strains belonging to four serotypes (Stanley, Typhimurium, Virchow and Virginia) were positive for qnrS1. qnrS1 was present on plasmids of 13.5 kb (TPqnrS-1a and -1b) in Typhimurium and Virginia isolates, 44 kb (TPqnrS-2) in two Virchow isolates and >148 kb (TPqnrS-3a and -3b) in two Stanley isolates. bla(TEM-1) and a group 9 bla(CTX-M) were co-transferred on TPqnrS-2 and TPqnrS-3b. Hybridization of a qnrS1 probe to digested qnrS1 plasmids suggested qnrS1 on TPqnrS-2 may be located in a similar genetic environment to Shigella qnrS plasmid pAH0376, but in a different environment in the other plasmids. CONCLUSIONS: This is the first report of plasmid-mediated quinolone resistance in a Salmonella isolate from the UK; five isolates were associated with foreign travel to, or food imported from, the Far East. The presence of qnrS1 on different plasmid backbones in several Salmonella serotypes suggests successful dissemination of plasmids or qnrS1. It is of concern that qnrS1 is being identified in Salmonella serotypes that are commonly implicated in human infection in the UK. Coupled with beta-lactam resistance, it may compromise treatment of vulnerable patient groups.