WGS characterization of MDR Enterobacterales with different ceftolozane/tazobactam susceptibility profiles during the SUPERIOR surveillance study in Spain

Abstract Objectives To analyse by WGS the ceftolozane/tazobactam (C/T) resistance mechanisms in Escherichia coli and Klebsiella spp. isolates recovered from complicated intra-abdominal and urinary tract infections in patients from Spanish ICUs (SUPERIOR surveillance study, 2016–17). Methods The clonal relatedness, the resistome and the virulome of 45 E. coli and 43 Klebsiella spp. isolates with different C/T susceptibility profiles were characterized. Results In E. coli, two (C/T susceptible) carbapenemase producers (VIM-2-CC23, OXA-48-ST38) were detected. The most relevant clone was ST131-B2-O25:H4-H30 (17/45), particularly the CTX-M-15-ST131-H30-Rx sublineage (15/17). ST131 strains were mainly C/T susceptible (15/17) and showed an extensive virulome. In non-ST131 strains (28/45), CTX-M enzymes [CTX-M-14 (8/24); CTX-M-15 (6/24); CTX-M-1 (3/24); CTX-M-32 (2/24)] were found in different clones. C/T resistance was detected in non-clonal E. coli isolates (13%, 6/45) with ESBL (4/6) and non-ESBL (2/6) genotypes. Among Klebsiella spp., Klebsiella pneumoniae (42/43) and Klebsiella michiganensis (1/43) species were identified; 42% (18/43) were carbapenemase producers and 58% showed a C/T resistance phenotype (25/43). OXA-48-ST11 (12/18), OXA-48-ST392 (2/18), OXA-48-ST15 (2/18), NDM-1-ST101 (1/18) and OXA-48+VIM-2-ST15 (1/18) isolates were found, all C/T resistant. Correlation between carbapenemase detection and resistance to C/T was demonstrated (P < 0.001). In non-carbapenemase-producing K. pneumoniae (25/43), C/T resistance (28%, 7/25) was detected in ESBL (3/7) and AmpC (2/7) producers. Overall, an extensive virulome was found and was correlated with carbapenemase carriage (P < 0.001) and C/T resistance (P < 0.05), particularly in OXA-48-ST11 strains (P < 0.05). Conclusions Prediction of antimicrobial susceptibility profiles using WGS is challenging. Carbapenemase-encoding genes are associated with C/T resistance in K. pneumoniae, but other resistance mechanisms might be additionally involved.


Introduction
The production of ESBLs and carbapenemases is the major mechanism of resistance to b-lactam antibiotics in Enterobacterales isolates. Since 2000, the global prevalence of ESBL enzymes, mostly CTX-M enzymes, has dramatically increased in both community and hospital settings. 1 Additionally, in the last two decades, the V C The Author(s) 2020. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecom mons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com 1 of 9 JAC Antimicrob Resist doi: 10.1093/jacamr/dlaa084 JAC-Antimicrobial Resistance worldwide dissemination of carbapenemase-producing Enterobacterales (CPE) has been recognized as one of the major challenges for public health due to their high mortality and morbidity and the elevated associated economic costs. 2 In Spain, CTX-M enzymes are considered endemic and are usually detected in nosocomial Klebsiella pneumoniae clones and communityacquired Escherichia coli non-clonal isolates. 3,4 Moreover, CPE prevalence is also increasing and is mostly related to OXA-48, VIM and KPC enzymes. 2 The dissemination of these carbapenemases is frequently associated with successful epidemic clones of K. pneumoniae and with healthcare exposure and prolonged hospital stays. 5,6 The therapeutic options available to treat infections caused by MDR Enterobacterales isolates are limited and the development of novel antimicrobial agents is currently a priority. Ceftolozane/ tazobactam is a cephalosporin/b-lactamase inhibitor combination useful in the treatment of complicated intra-abdominal infections (cIAIs) and complicated urinary tract infections (cUTIs) caused by MDR Gram-negative bacteria, with the exception of carbapenemase producers. 7 Several studies have demonstrated a higher in vitro activity of this novel combination against ESBL-producing Enterobacterales compared with currently available cephalosporins. 8,9 Nevertheless, low resistance rates to ceftolozane/ tazobactam have been recently reported in ESBL-producing K. pneumoniae and E. coli isolates. [10][11][12] In recent years, WGS has become a potent tool to characterize and understand the genetic diversity of bacterial populations. The use of genome-based technologies for the prediction of bacterial antibiotic resistance profiles in surveillance studies has also been proposed as a powerful alternative to antibiotic susceptibility testing. 13 However, discordances are still observed and the prediction of susceptibility or resistance to antimicrobial agents based only on the presence or absence of previously known genes is still under discussion. 14 The aim of this work was to characterize, using WGS, the population structure, the resistome and the virulome of MDR E. coli and Klebsiella spp. clinical isolates recovered from patients admitted to eight Spanish ICUs between 2016 and 2017 as a part of the SUPERIOR surveillance study, focusing on the resistance genotype with respect to the ceftolozane/tazobactam antibiotic.

Study design and bacterial isolates
SUPERIOR is a prospective multicentre surveillance study designed to evaluate the in vitro activity of ceftolozane/tazobactam and comparator antimicrobials against Enterobacterales and Pseudomonas aeruginosa clinical isolates. A total of 400 Enterobacterales [52.3% E. coli (n " 209); 23.8% Klebsiella spp. (n " 95)] isolates were collected from cUTIs and cIAIs from patients (one isolate per patient) admitted to ICUs at eight Spanish hospitals between April 2016 and April 2017. 15

Statistical analysis
Associations between categorical variables were analysed by Fisher's exact test. The kappa index (j) index was used to evaluate the concordance between genotypes based on WGS analysis and the in vitro antibiotic activity results (j , 0.40, weak agreement; j " 0.41-0.60, moderate; j " 0.61-0.80, accurate; and j . 0.81, very accurate). Statistical analysis was carried out using R software (RStudio Team 2016 version 1.0.44, RStudio, Boston, MA, USA). A P value ,0.05 was considered statistically significant.

Accession numbers
All complete sequences were deposited at DDBJ/ENA/GenBank under the BioProject accession number PRJNA609897 (Table S1, available as Supplementary data at JAC-AMR Online).

Genome characteristics and taxonomic classification
Genomic information about all isolates is shown in

Virulence gene content
A high number of genes associated with the virulome was identified in the E. coli collection [14-110 virulence factors (VFs) belonging to 6-29 virulence loci (VL)], particularly in CC131 E. coli strains (53-88 VFs, 17-21 VL). Overall, E. coli strains were clustered by MLST clones according to the virulome detected ( Figure S3). Furthermore, the E. coli isolates with a ceftolozane/tazobactamresistant phenotype also presented a high virulence gene content (17-110 VFs, 10-29 VL), although similarities in the virulence profile were not found ( Figure S3).

Discussion
In this study we used genome-based technology to analyse the genetic diversity, the resistome and the virulome of a subset of E. coli and Klebsiella spp. isolates displaying different MDR profiles, including ceftolozane/tazobactam-resistant and -susceptible phenotypes, recovered from ICU patients admitted to eight hospitals distributed throughout Spain as a part of the SUPERIOR surveillance study. 15 Among the sequenced E. coli isolates, the high-risk clone ST131 was the most prevalent. ST131 is the most frequent E. coli clone related to ESBL production and fluoroquinolone resistance and is considered worldwide as a major cause of urinary tract and bloodstream infections. 24 In Spain, the CTX-M-15-ST131-H30-Rx subclade has been reported as the main pathogen causing healthcare-associated ESBL-producing E. coli bacteraemia of urinary origin. 25 We found that the CTX-M-15-ST131-H30-Rx subclone is also predominant among the E. coli isolates causing cUTI and cIAI in patients admitted to Spanish ICUs. Moreover, we detected an extensive virulence gene content among all E. coli isolates, but particularly in the ST131 high-risk clone. The acquisition of virulence factors has been defined as a precursor in the evolution and global dissemination of this successful ST131 clone, conferring an increased capacity to cause human infections and to gain antibiotic resistance genes. 26 Note that the ST131 E. coli isolates detected in this collection came from five hospitals, although the hypervirulent H30-Rx subclade was only found in three centres, two of them located on the Mediterranean coast (Barcelona and Valencia).
A high clonal diversity was found in non-ST131 E. coli isolates. An association with different bla CTX-M genes was observed and bla CTX-M-14 was the most frequent ESBL, followed by bla CTX-M-15 . Both CTX-M-14 and CTX-M-15 have been reported as the most prevalent ESBL enzymes in our country in the last decade and have usually been related to community-acquired E. coli isolates. 1 In this work, the inter-hospital distribution of E. coli clones previously described in Spanish healthcare centres, such as ST10, ST23, ST38 or ST410, was also found to be related to CTX-M production. 27 Furthermore, carbapenemase-encoding genes were only detected in two E. coli isolates and both strains were typed as ST38 (bla OXA-48 ) and CC23 (bla VIM-2 ). The association of some of these clones with carbapenemase production, particularly OXA-48 type, is globally increasing and is currently a cause of great concern for public health. 28 Among the K. pneumoniae isolates, carbapenemase production was frequently detected in ceftolozane/tazobactam-resistant isolates and bla OXA-48 was predominant. Most OXA-48-producing isolates belonged to the ST11 high-risk clone and were also CTX-M-15 and OXA-1 (Figure 2) co-producers. CTX-M-15!OXA-48-ST11 is considered the most prevalent K. pneumoniae clone among both infected and colonized patients and has recently been involved in different healthcare-associated outbreaks in Spain. 5,6 In this work, the OXA-48-ST11 clone was found in three participating ICUs, two of them from the same geographical region (Madrid). Additionally, other K. pneumoniae epidemic clones previously reported in Spanish hospitals were identified among OXA-48 producers (ST15, ST392) and were also related to the production of ESBL enzymes (ST15, ST307, ST101, ST405). 5,6,29 Most of them were detected in at least two hospitals, showing the inter-hospital clonal dispersion among Spanish ICUs. The risk of endemicity of the OXA-48 enzyme is increasing and has been related to the successful dissemination and establishment of K. pneumoniae high-risk clones in the healthcare setting. 28 The role of certain K. pneumoniae clones such as ST11 in the spread of OXA-48 among different K. pneumoniae clones and also to other Enterobacterales species has been demonstrated recently. 30,31 In this study, we highlight that the OXA-48-ST11 K. pneumoniae clone is also predominant in ICU patients with cUTI and cIAI and that the risk of OXA-48 transmission to other Enterobacterales members may also be increasing in these units.
The key to controlling and avoiding the spread of resistance determinants such as carbapenemase-encoding genes among clinical isolates is their accurate and rapid identification and characterization. The contribution of WGS to the study of the emergence and evolution of resistant microbial populations has been clearly demonstrated. 32 Nevertheless, the surveillance of MDR microorganisms and the prediction of antimicrobial resistance profiles using DNA sequencing technology is still challenging for microbiology laboratories. 33,34 In this collection, we found discrepancies in both E. coli and Klebsiella spp. subsets between the genomic detection of carbapenemase-encoding genes and the antibiotic susceptibility testing results obtained for carbapenems in our previous work. 15 The detection of carbapenemases among the E. coli isolates was scarce and conclusive results could not be obtained. However, in the Klebsiella spp. collection, genotype/ phenotype discrepancies could be explained by the predominance of the OXA-48 enzyme. OXA-48-type carbapenemases weakly hydrolyse carbapenems and their recognition and detection based on phenotypic profiles can be difficult. 35 In the same way, colistin resistance was identified in 7% and 21% of E. coli and K. pneumoniae isolates, respectively, although acquired resistance mechanisms such as the mcr-1 gene were only detected in one E. coli isolate.
During the SUPERIOR study, good activity of ceftolozane/ tazobactam was demonstrated in both E. coli (95%) and Klebsiella spp. (66%) populations. This novel combination is proposed as a therapeutic option for the treatment of infections caused by ESBL-producing Enterobacterales isolates in order to spare carbapenems. 7 In our collection, resistance to ceftolozane/tazobactam was detected among carbapenemase producers, but only in K. pneumoniae isolates, since both carbapenemase-producing E. coli isolates were susceptible to this antibiotic. On the other hand, in agreement with other studies, a low resistance rate to WGS of ceftolozane/tazobactam-resistant Enterobacterales JAR ceftolozane/tazobactam was also detected among ESBL-and/or AmpC-producing K. pneumoniae and E. coli strains, although correlation with these resistance mechanisms could not be established. [10][11][12] In conclusion, OXA-48!CTX-M-15-ST11-K. pneumoniae and CTX-M-15-E. coli-ST131-H30-Rx are the most frequent clones causing cUTI and cIAI in patients admitted to ICUs from Spanish hospitals. As expected, in vitro activity of ceftolozane/tazobactam was not demonstrated in carbapenemase-producing K. pneumoniae isolates, but this could be a therapeutic option to treat complicated infections caused by the hypervirulent ST131 E. coli high-risk clone. In addition, although further analysis should be performed, according to our results, the presence of carbapenemase genes in E. coli does not always correlate to ceftolozane/tazobactam resistance. This correlation was present in K. pneumoniae, but other resistance mechanisms might be involved when resistance to this b-lactam/b-lactamase inhibitor combination is present. The genome-based approach is a powerful tool to study the molecular epidemiology of circulating nosocomial pathogens, but the implementation of this technology to infer genotype/phenotype relationships is still a challenge for diagnostic laboratories.