As part of the ongoing Antibiotic Resistance Surveillance Program, the Spanish National Center for Microbiology (CNM) receives clinical carbapenem-resistant E. coli isolates for molecular characterization from Spanish hospitals. Participation in this program is voluntary and the criteria to send the isolates to the CNM is having a Minimum Inhibitory Concentration (MIC) to meropenem of over 0.12 mg/L (Giske et al., 2017).

Of the total CP-Eco isolates received in this Surveillance Program between January 2015 and December 2020, a subset was selected for further analysis according to the following criteria: i) one isolate for each type of carbapenemase per year was included from each of the collaborating hospitals, ii) in the event that there was more than one isolate for each carbapenemase type in the same year, the selection was prioritized based on the type of infection in which they were involved according to the following order: 1) invasive infections 2) other infections 3) colonizations; iii) in all cases, the first isolate received per year was selected. These selection criteria allowed for a temporal-spatial representative sample of the different types of carbapenemases circulating in E. coli in Spain. Not all hospitals had isolates from all the five years, and from some hospitals more than one isolate per year was included since they produced different carbapenemase types. The information sent along with the isolate from the participating hospitals included collection date, age and sex of the patients, sample type, whether the isolate was a colonizer or causing infections (as determined by the attending microbiologist), and isolate origin [hospital, healthcare center, or community setting; i.e. where the infection is contracted without interaction with healthcare settings during the last 48 hours (Plachouras et al., 2018)].

All the carbapenem-resistant E. coli isolates received at the CNM from 2015 to 2020 were cultured on MacConkey agar (Becton Dickinson Microbiology Systems, Cockeysville, MD, USA) and incubated overnight at 37°C. DNA was extracted using the QIAamp^® DNA Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. Real-time PCR assays targeting the carbapenemase gene families bla _OXA-48, bla _KPC, bla _VIM, bla _IMP and bla _NDM were performed (Ortega et al., 2016). All the isolates were stored at -80°C until used.

Antibiotic Susceptibility Testing (AST) was determined for the selected isolates with broth microdilution using the YDKMGN Sensititre™ Gram Negative panels (Thermo Fisher, Waltham, MA, USA) (International Organization for Standardization (ISO), 2006). E-tests using meropenem/vaborbactam and imipenem/relebactam and disc diffusion assays for cefiderocol (Liofilchem, Roseto degli Abruzzi, Italy) were performed and interpreted according to the EUCAST guidelines (European Society of Clinical Microbiology and Infectious Diseases (EUCAST), 2022). Additionally, E-tests using plazomicin (Liofilchem, Roseto degli Abruzzi, Italy) and fosfomycin (bioMérieux, Marcy l’Etoile, France) strips were performed and interpreted according to an FDA-approved breakpoint of susceptibility of ≤2mg/L for plazomycin (Cañada-García et al., 2022), and according to the EUCAST cutoff values for urinary tract infections (UTI) for fosfomycin due to lack of clinical breakpoints for other types of samples (European Society of Clinical Microbiology and Infectious Diseases (EUCAST), 2022), respectively.

Resistance to different antibiotic families was analyzed considering 14 categories: nine categories according to the classification proposed by Magiorakos et al. (Magiorakos et al., 2012) (extended spectrum cephalosporins, monobactams, carbapenems, aminoglycosides, fluoroquinolones, fosphonic acid derivatives, folic pathway inhibitors, glycylcyclines and polymyxins); and 5 additional categories: ceftazidime/avibactam (extended spectrum cephalosporin + diazabicycloctane inhibitor), imipenem/relebactam (carbapenem + diazabicycloctane inhibitor), meropenem/vaborbactam (carbapenem + boronic acid inhibitor), cefiderocol (siderophore cephalosporin) and plazomicin (extended spectrum aminoglycoside).

DNA was extracted from the selected isolates as described in section 2.2. Paired-end (2x150) libraries were then prepared using the Nextera DNA Flex Library Preparation Kit and sequenced using Illumina NextSeq 550 (Illumina Inc., San Diego, CA, United States) according to the manufacturer’s instructions. Quality of the reads was assessed using FASTQC (version 0.11.9), followed by de novo assembly using Unicycler (version 0.4.8) (Wick et al., 2017). The quality of the assembly was assessed using QUAST (version 5.2.0) and annotation was performed using Prokka (version 1.14.6) (Seemann, 2014). The obtained assemblies were deposited in the European Nucleotide Archive under the accession number PRJEB70795.

Ridom SeqSphere+ (version 8.3.1; Ridom, Münsten, Germany) was used to perform a core-genome Multi-Locus Sequence-Typing analysis (cgMLST) using a built-in scheme for E. coli containing 2,515 core genes, and to construct a minimum spanning tree based on allelic differences. ARIBA (version 2.6.2) (Hunt et al., 2017) was used to determine STs in accordance with the University of Warwick scheme. Diversity of the samples was calculated using a Simple Diversity Index [SDI (Gastmeier et al., 2006)]. Analysis of the fimH gene was done to subclassify ST131isolates using the FimTyper tool [CGE server; https://cge.cbs.dtu.dk; (accessed 23/02/2024)].

ARGs were analyzed by ARIBA (version 2.14.6) (Hunt et al., 2017) using the CARD database (https://card.mcmaster.ca; (accessed 23/02/2024)) and ResFinder (CGE server) with ID thresholds of 100% for β-lactamase variants and 98% for other resistance genes. In isolates resistant to cefiderocol, the sequences of the genes encoding PBP3 and the siderophore CirA, previously related to resistance to this antibiotic (Wang et al., 2022), were analyzed with ARIBA.

The VirulenceFinder tool (CGE server) (accessed 23/02/2024) was used to detect virulence-associated genes (five toxin-coding genes, four adhesin-coding genes, two siderophore-coding genes, one outer membrane protein-coding gene and one invasive protein-coding gene). PlasmidID [https://github.com/BU-ISCIII/plasmidID; (accessed 23/02/2024)] was used to map the reads against a curated plasmid database, perform de novo plasmid assemblies, and determine the presence of resistance and replicon genes (Pérez-Vazquez et al., 2019).

The differences in prevalence of the ARGs, virulence-related genes, STs, and sample characteristics were evaluated using the Fisher’s exact test using GraphPad Prism (version 3.02; GraphPad Software, Inc., San Diego, CA, USA). P values of less than 0.05 were considered statistically significant.

All the data generated for this study is available in the manuscript and its related Supplementary Material . The sequenced raw reads have been deposited in the open-access European Nucleotide Archive database under the accession number PRJEB70795.

From January 2015 until December 2020, a total of 593 CP-E. coli isolates were confirmed as carbapenems producers by the Spanish CNM Reference Lab after screening for MIC to meropenem of over 0.12 mg/L by the collaborating hospitals. Supplementary Table S1 shows the characteristics and origin of the isolates, as well as the information received from the participating hospitals (total and per year). The isolates were received from 25 Spanish provinces and 59 hospitals. Two-hundred-ninety-nine (50.4%) of the isolates were from females and the average patient age was 77.8 years. Three-hundred-one (50.8%) isolates were colonizers, and the rest were causing infections (24 were unspecified). The most common infection was UTI (175; 29.5%), followed by wound/ulcer infections (32; 5.4%), bacteremia (25; 4.2%), and IAI (17; 2.9%). Four-hundred-forty-three (74.7%) were healthcare-associated cases, 391 from hospitals and 52 from other healthcare centers, and 126 (21.2%) were from community settings; 24 were unspecified.

Using real-time PCR carbapenemase genes were detected in all 593 isolates where bla _OXA-48-like was detected in 485 (81.8%), bla _VIM-like in 71 (12%), bla _NDM-like in 17 (2.9%), and bla _KPC-like in 17 (2.9%) isolates. Three isolates harbored two carbapenemases, one of each of the following combinations bla _OXA-48 and bla _KPC, bla _OXA-48 and bla _NDM, and bla _VIM and bla _KPC. In the community setting, the most frequent carbapenemase was bla _{OXA-48-family}, detected in 109 (86.5%) isolates. The relative frequency of these genes per year was maintained throughout the study period and there was no statistically relevant correlation between the types of carbapenemase gene, sample type, location of isolation, infection, colonization, and type of carbapenemase harbored by the isolates (P > 0.05).

Based on the criteria set in section 2.1, 90 isolates from 59 hospitals across 25 Spanish provinces were selected. Supplementary Table S2 lists all the characteristics of these isolates. Sixty (66.6%) isolates were implicated in clinical infections, as determined by the doctor who treated the patient, and mainly were obtained from urine (27; 45%), blood (15; 25%) and abscesses and wounds (12; 20%). The remaining 30 (33.3%) isolates were considered colonizing and came mainly from rectal swabs and feces (26; 86.7%)

Forty-two (46.7%) of the isolates were obtained from the hospital setting, the majority of which harbored bla _OXA-48 (24; 57.1%), followed by bla _VIM-1 (8; 19%), bla _NDM (3 bla _NDM-1 and 3 bla _NDM-5; 14.3% in total), bla _KPC (1 bla _KPC-2 and 1 bla _KPC-3; 4.8% in total), bla _OXA-162 (1; 2.4%), and both bla _KPC-3 and bla _OXA-48 (1; 2.4%). Twelve isolates were obtained from healthcare centers where 3 harbored bla _OXA-48, 3 bla _VIM-1, 2 bla _NDM (1 bla _NDM-1 and 1 bla _NDM-7), 2 bla _KPC (1 bla _KPC-2 and 1 bla _KPC-3), 1 (8.3%) bla _OXA-162, and 1 both bla _KPC-3 and bla _OXA-48. From the community setting, 18 (20%) of the isolates were collected where 8 had bla _OXA-48, 4 bla _VIM-1, 3 bla _NDM (1 bla _NDM-1 and 2 bla _NDM-5), and 3 bla _KPC (2 bla _KPC-2 and 1 bla _KPC-3). The origin of 18 isolates could not be determined.

The carbapenemase genes detected among the infecting isolates were bla _OXA-48 (30; 50%), bla _VIM-1 (13; 21.7%), bla _NDM (5 bla _NDM-1 and 3 bla _NDM-5; 13.3% in total), bla _KPC (3 bla _KPC-2 and 3 bla _KPC-3; 10% in total), one bla _OXA-162 (1; 1.7%), and both bla _KPC and bla _OXA-48 (2; 3.3%). Among the colonizing isolates, the carbapenemases genes observed were bla _OXA-48 (11; 36.7%), bla _VIM-1 (8; 26.7%), bla _NDM (6; 20%) (3 bla _NDM-5, 2 bla _NDM-1, and 1 bla _NDM-7), bla _KPC (4; 13.3%) (3 bla _KPC-2 and 1 bla _KPC-3), and bla _OXA-162 (1; 3.3%). No statistically significant associations were found between the sample type, location of isolation, infection, colonization, and type of carbapenemase harbored by the isolates (P > 0.05)

MLST analyses showed that the 90 isolates belonged to 49 different STs (SDI=54.4) and only ST131 (14 isolates) and ST88 (6 isolates) were detected in more than five isolates. Population diversity was similar for isolates harboring bla _OXA-48 (SDI=65.1) and bla _NDM (SDI=64.3), and was lower than those harboring bla _KPC (SDI=71.0) and bla _VIM (SDI=81.0). Ridom SeqSphere+ was used to construct a maximum-likelihood phylogenetic tree using the gene-by-gene approach and allelic distance from cgMLST. Figure 1 shows a great genetic diversity of the CP-Eco studied alone and in relation to the types of carbapenemases they had. The average allelic distances in pairwise comparison of all the CP-Eco isolates was 2,120 ± 555, and only four main clusters with ≥ 4 isolates were identified. The first cluster included 14 (15.6%) CP-Eco isolates that belonged to ST131, had an average of 105 allelic differences (range:13-660), were collected from 7 provinces, and all 4 carbapenemase gene families were detected in them (including two with both bla _OXA-48 and bla _KPC-like). The second-largest cluster was formed by 6 (6.7%) isolates belonging to ST88, had an average of 34 allelic differences (range:20-58), were collected from 5 provinces and 5 isolates harbored bla _OXA-48. Cluster 3 contained 4 (4.4%) isolates belonging to ST167 that all harbored bla _NDM, were collected from 3 provinces and had an average of 142 allelic differences (range:79-264). Cluster 4 contained 4 (4.4%) isolates pertaining to ST1193 collected from 2 provinces with an average allelic difference of 24 (range:20-32); two of them harbored bla _OXA-48 and the other two bla _NDM. The rest of the isolates were grouped into STs with a frequency of 3 or less isolates. Applying a ≤ 5 allele differences threshold to identify possible transmission events, only one cluster of two OXA-48-producing ST127 isolates were detected.

Further typing of the fimH gene was conducted on the predominant ST131. Among the 14 ST131 isolates, 7 (50%) had fimH30 indicative of belonging to clade C, 4 (28.6%) had fimH41 (clade A), 1 (7.1%) had fimH22 (clade B), 1 (7.1%) had fimH298 (subtype of clade B), and 1 (7.1%) had fimH1196 (no specific clade). bla _CTX-M-15 and bla _KPC genes were more frequent in ST131 clade C isolates (71.4% and 57.1%, respectively) than in non-clade C isolates (14.3% and 28.6%, respectively) but these differences were not statistically significant (P > 0.05).

Table 1 shows the detected ARGs where an average of 5.6 ARGs were detected (range:1–13; excluding chromosomal genes and mutations). bla _OXA-48-like was detected in 43 (47.8%) isolates, bla _VIM-1 in 21 (23.3%), bla _NDM-like in 14 (15.6%), bla _KPC-like in 10 (11.1%), and both bla _OXA-48 and bla _KPC-like in 2 (2.2%) isolates. Isolates harboring bla _VIM-1 (7.2; range:3-11) and bla _NDM-like (6.7; range:1-12) had more ARGs than those isolates harboring bla _OXA-48 (5; range:1-13) and bla _KPC-like (4.6; range:1-12). Twelve isolates harbored only one ARG, six of them had bla _OXA-48-like, five had bla _KPC-like, and one had bla _VIM-1. Isolates belonging to ST88 had an average of 9.8 ARGs (range:7-13). bla _OXA-48 was most frequently encountered in ST88 isolates (5; 11.6%), bla _NDM-like in ST167 (4; 28.6%), bla _KPC-like in ST131 (6; 54.6%) and bla _VIM-like in ST131 isolates (3; 14.3%). bla _OXA-162 was detected in 2 isolates belonging to ST5968.

Acquired ESBL genes were detected in 27 (30%) isolates where bla _CTX-M-15 was predominant (n=13; 48.1%). bla _PER-2 was detected in a bla _NDM-1-harbouring isolate belonging to ST12. Regarding acquired AmpC genes, bla _CMY was detected in four isolates, three of them belonged to ST167 and all four harbored bla _NDM-like ( Supplementary Table S2 ).

The non-β-lactamase acquired ARGs present in at least 25% of the CP-Eco isolates were the sulfonamide-resistance-encoding sul (n=64, 71.1%; mainly sul1); trimethoprim-resistance-encoding dfrA (n=56, 62.2%; mainly dfrA17); tetracycline-resistance-encoding tet (n=38, 42.2%; mainly tetA); aminoglycoside-resistance-encoding aac(6’) (n=31, 34.4%; mainly aac(6’)-Ib) and aadA (n = 52, 57.8%; mainly aadA1); chloramphenicol-resistance-encoding cat (n=25, 27.8%; mainly catA1); and quinolone-resistance-encoding qnr (n=23, 25.6%; mainly qnrS1) ( Table 1 ; Supplementary Table S2 ). Twenty-five isolates had chromosomal mutations associated with resistance to fosfomycin and 2 additional isolates harbored the fosA7.5 and fosA3 acquired genes. These genes were located in chromosomal and plasmid contigs, respectively. The aminoglycoside methyltransferase gene rmt was detected in five isolates belonging to different STs, where 4 harbored bla _NDM-1 and 1 bla _NDM-5.

The median size of the detected plasmids was 63,598 bp with a median coverage of 98% and a median similarity of 100% with the respective entries in Genbank. IncL plasmids were the most frequently detected (n=42; 46.7%). Thirty-two of these harboured bla _OXA-48, eight harboured bla _VIM-1, and two harboured bla _OXA-162. The second most frequent plasmid type was IncFII detected in 12 (13.3%) isolates, eight of which harbored bla _OXA-48 and four harbored bla _NDM. Five of the IncF-harboring isolates also belonged to clade C of ST131. IncN was detected in five (5.3%) isolates where four harbored bla _VIM-1 and one harbored bla _KPC-2. IncP6 was detected in four isolates harboring bla _KPC-2, and IncFIB was detected in four of the five isolates harboring bla _KPC-3. Other plasmid types were detected with a frequency of two or less and it was not possible to fully reconstruct the carbapenemase-harboring plasmids in 20 isolates ( Supplementary Table S2 ).

No other ARG was detected on the same plasmid harboring bla _OXA-48-like nor for those harboring bla _KPC-like, except for one isolate harboring bla _KPC-2 that also harbored a class 1 integron with the sequence Int1-aac(6’)-Ib-arr3-dfrA27-aadA16-qacEΔ1-sul1. bla _VIM-1 in IncL plasmids was found in a class 1 integron with the sequence Int1-bla_VIM-1-aac(6’)-Ib-dfrB1-aadA1-catB2-qacEΔ1-sul1. In four other isolates, IncN plasmids harbored class 1 integron with bla _VIM-1 but with a different combination of genes. Six isolates harbored bla _NDM-5 (n=4), bla _NDM-1 (n=1) or bla _NDM-7 (n=1) in complex class 1 integrons with different combinations of aminoglycoside-resistance genes, including rmtC. Another isolate had bla _NDM-1 and rmtC on the same plasmid, but they did not form part of an integron ( Supplementary Table S2 ).

Table 2 shows the AST results that were analyzed according to the categories described in section 2.3. All isolates had MICs>0.12mg/L to meropenem, harbored at least one gene of carbapenem resistance, and were resistant to at least one carbapenem ( Table 2 ). The two isolates producing two carbapenemases presented relatively low MICs of 4 mg/L to imipenem (both) and 4 and 1 mg/L to meropenem (each). Antibiotics with in-vitro activity against more that 90% of the isolates were colistin, plazomicin, cefiderocol, and meropenem/vaborbactam. The mcr genes and known mutations in the PhoPQ and PmrAB encoding genes related to colistin resistance were not detected in the colistin-resistant isolate.

Forty isolates (44.4%) were resistant to more than six out of the 14 antibiotic categories considered. These isolates harbored bla _VIM-1 (n=15; 37.5%), bla _NDM-like (n=13; 32.5%), bla _OXA-48-like (n=7; 17.5%), bla _KPC-like (n=4; 10%), and both bla _KPC-3 and bla _OXA-48 (n=1; 2.4%). Nine isolates (7 NDM-producers, 1 VIM-producer, and 1 OXA-48-producer) were only susceptible to three or four antibiotic categories. The most active agents against these isolates were colistin (9/9) and tigecycline (6/9).

Table 3 shows the antibiotic resistance rates of the CP-Eco isolates, grouped by the type of carbapenemase gene harbored. Isolates harboring bla _OXA-48 had lower resistance rates towards meropenem and imipenem as compared to non-bla _OXA-48-harboring isolates (4.7% and 7% versus 21.3% and 46.2%, respectively; P = 0.0003). OXA-48 and ESBL co-producers (n=15) were significantly more resistant to ceftazidime (46.7%), cefotaxime (100%), and aztreonam (100%) than OXA-48 producers without ESBL (n=26, with 3.8%, 15.4%, and 3.8% resistance rates, respectively (P<0.05)). No other β-lactamases combinations could be statistically studied in relation to antibiotic resistance profiles due to the very low number of isolates for each combination. All the isolates resistant to imipenem/relebactam (n=29), ceftazidime/avibactam (n=26) or meropenem/vaborbactam (n=8) were metallo-β-lactamase-(MBL)-producers, except for one OXA-48-producer that was resistant to meropenem/vaborbactam and imipenem/relebactam, one OXA-48-producer resistant to imipenem/relebactam and ceftazidime/avibactam, and two isolates resistant to ceftazidime/avibactam producers of KPC-3/OXA-48 and OXA-48 carbepenemases, respectively. Three of these isolates presented insertions in the ompC gene in comparison with ertapenem-resistant isolates from this same study but susceptible to the rest of the carbapenems and to 3rd generation cephalosporins and their combinations. Insertions detected were E87_N88insD in one OXA-48 producer, and T225_A226insGLNGYG in one OXA-48- and one KPC-3/OXA-48-producing isolates. However, these insertions do not generate a premature stop codon. ompF mutations were not detected.

Six of the seven isolates resistant to cefiderocol were NDM-producers (4 bla _NDM-5 and 2 bla _NDM-1) and one had bla _VIM-1. Four NDM-producers belonged to ST167 and had an insertion in the pbp3 gene (Y334insYRIN), in addition to two other mutations in the same gene (E349K and I532L). Three of these isolates additionally had a frameshift mutation in the siderophore gene cirA (V89fs). Another NDM-producer (ST131) had a truncation in cirA (W243trunc), and the final NDM-producer belonged to ST405, had the Y334insYRIN insertion in the pbp3 gene, and a frameshift mutation in cirA (T508fs). The VIM-producer belonged to ST23 and had a frameshift mutation in cirA (T190fs). The median inhibition zone for NDM-producers was significantly lower compared to non-NDM-producers (20.5 versus 29.5; P = 0.00001).

There were six isolates that were resistant to all aminoglycosides, including plazomicin, and that carried rmt methyltransferases (3 rmtC, 1 rmtF1 and 1 rmtB1), with five of them additionally having bla _NDM-like. All 16 amikacin-resistant isolates harbored aac(6’)-Ib and/or methyltransferases genes, while the 46 tobramycin-resistant isolates had aac(3)-II, aac(6’)-Ib, ant(2’’)-Ia or rmtB1 genes ( Supplementary Table S2 ). Two NDM-producers were resistant to aztreonam with no other β-lactamase gene detected, while all other MBL-producers resistant to this antibiotic harbored ESBL and/or AmpC genes ( Supplementary Table S2 ).

Finally, 82 isolates (91.1%) had fosfomycin MICs of ≤ 8 mg/L and were considered susceptible (using EUCAST criteria for UTIs). Of those, 21 isolates (25.6%) had chromosomal mutations associated with resistance to fosfomycin. Of the 8 isolates resistant to this antibiotic, two harbored fosA genes, three had the chromosomal mutations ptsI_V25I/uhpT_E350Q, one had the chromosomal mutation uhpT_E350Q, and in the last two no known genetic mutation or gene was identified. There was a significant correlation between harboring mutations or genes associated with fosfomycin resistance and phenotypic resistance to this antibiotic (P = 0.003).

Table 4 shows the detected virulence-associated genes (13 virulence genes considered), and the number of infection-causing isolates harboring them. The isolates had a median of two virulence-associated genes (range=0-9) where adhesin-coding genes were the most prevalent (68.9%). Thirty-eight isolates (42.2%) had four or more virulence-associated genes, 19 of which were causing infections. The most common infection in this group was UTI (9), followed by bacteremia (5), IAIs (2), wound infections (2), and abscess (1) ( Supplementary Table S2 ). Isolates with 4 or more virulence-associated genes included three isolates belonging to ST131 and two belonging to ST88; and had similar averages of ARGs and susceptibility rates to the different antibiotic categories. Nineteen of these 34 isolates had bla _OXA-48, 8 had bla _VIM-1, 3 had bla _NDM-5, 2 had bla _NDM-1, 1 had bla _KPC-3, and 1 had both bla _OXA-48 and bla _KPC-3. Of the isolates pertaining to ST131, all had fimH and over half of them harbored pap and ompA. Over half of those pertaining to ST88 harbored fimH, iroN, and ompA.

The adhesin-coding pap and the toxin-coding sat genes were found to be more frequent among isolates pertaining to ST131 (P = 0.02 and 0.0004, respectively). Particularly, five of the 9 isolates positive for pap and five out of the six positive for sat belonged to clade C. Additionally, the adhesion-coding fimH gene had almost double the frequency among isolates harboring bla _OXA-48 as compared to isolates harboring other carbapenemase genes (P = 0.04). There were no statistically significant differences in the virulence-associated genes between bloodstream isolates and isolates from other sources.