https://orcid.org/0000-0003-2857-2572
Abstract
To analyse the epidemiology, the resistome and the virulome of ceftolozane/tazobactam-susceptible or -resistant Pseudomonas aeruginosa clinical isolates recovered from surveillance studies in Portugal (STEP, 2017–18) and Spain (SUPERIOR, 2016–17).
P. aeruginosa isolates were recovered from intra-abdominal, urinary tract and lower respiratory tract infections in ICU patients admitted to 11 Portuguese and 8 Spanish hospitals. MICs were determined (ISO-standard broth microdilution, EUCAST 2020 breakpoints). A subset of 28 ceftolozane/tazobactam-resistant P. aeruginosa isolates were analysed and compared with 28 ceftolozane/tazobactam-susceptible P. aeruginosa strains by WGS.
Clonal complex (CC) 235 (27%) and CC175 (18%) were the most frequent, followed by CC244 (13%), CC348 (9%), CC253 (5%) and CC309 (5%). Inter-hospital clonal dissemination was observed, limited to a geographical region (CC235, CC244, CC348 and CC253 in Portugal and CC175 and CC309 in Spain). Carbapenemases were detected in 25 isolates (45%): GES-13 (13/25); VIM type (10/25) [VIM-2 (4/10), VIM-20 (3/10), VIM-1 (2/10) and VIM-36 (1/10)]; and KPC-3 (2/25). GES-13-CC235 (13/15) and VIM type-CC175 (5/10) associations were observed. Interestingly, KPC-3 and VIM-36 producers showed ceftolozane/tazobactam-susceptible phenotypes. However, ceftolozane/tazobactam resistance was significantly associated with GES-13 and VIM-type carbapenemase production. Six non-carbapenemase producers also displayed ceftolozane/tazobactam resistance, three of them showing known ceftolozane/tazobactam resistance-associated mutations in the PBP3 gene, ftsI (R504C and F533L). Overall, an extensive virulome was identified in all P. aeruginosa isolates, particularly in carbapenemase-producing strains.
GES-13-CC235 and VIM type-CC175 were the most frequent MDR/XDR P. aeruginosa clones causing infections in Portuguese and Spanish ICU patients, respectively. Ceftolozane/tazobactam resistance was mainly due to carbapenemase production, although mutations in PBP-encoding genes may additionally be involved.
Introduction
The increasing prevalence of MDR/XDR Pseudomonas aeruginosa isolates is a cause of great concern worldwide. P. aeruginosa is an opportunistic pathogen causing severe infections, particularly in the hospital setting and in immunocompromised patients.1 The ability of P. aeruginosa to develop MDR by the selection of mutations in chromosomal genes and to a lesser extent through the horizontal acquisition of antibiotic resistance genes, such as carbapenemase-encoding genes, compromises the selection of appropriate antibiotic therapy, causing high morbidity and mortality rates.2,3 Furthermore, the dissemination in healthcare settings of well-adapted P. aeruginosa high-risk clones (CC235, CC175, CC244 and CC111) has been extensively described worldwide and also poses a threat to public health.1,2
The development of new antimicrobial agents and the optimization of antibiotics that are currently available are required to reduce the incidence of nosocomial infections caused by MDR/XDR P. aeruginosa strains. The novel β-lactam/β-lactamase inhibitor combination ceftolozane/tazobactam has recently been introduced in clinical practice.4 Ceftolozane/tazobactam displays a greater spectrum of in vitro activity against MDR P. aeruginosa isolates than carbapenems and other available antipseudomonal cephalosporins.5–7 This novel combination has also been shown to be useful in the treatment of complicated infections caused by carbapenem-resistant isolates.8 Nevertheless, several studies have reported limited ceftolozane/tazobactam activity against those P. aeruginosa isolates that produce carbapenemases.4,9
Over recent years, the WGS approach has been providing relevant advantages to characterize the population diversity, pathogenicity and complex resistome of MDR/XDR P. aeruginosa high-risk clones involved in severe nosocomial infections.10–13 However, the usefulness of sequencing technologies and bioinformatics analysis tools in the inference or prediction of antibiotic susceptibility profiles needs to be further analysed.
The aim of this work was to study the molecular epidemiology, resistome and virulome of a subset of clinical P. aeruginosa isolates by WGS, focusing on the resistance mechanisms involved in ceftolozane/tazobactam susceptibility, as a part of the STEP and SUPERIOR surveillance studies performed in Portugal and Spain, respectively.14,15
Materials and methods
Study design and P. aeruginosa strains
STEP and SUPERIOR are two multicentre studies designed to assess the in vitro activity of ceftolozane/tazobactam and comparators against Enterobacterales and P. aeruginosa clinical isolates collected prospectively from ICU patients admitted to Portuguese (STEP) or Spanish (SUPERIOR) hospitals.14,15 A total of 396 P. aeruginosa isolates causing urinary tract infection (UTI), intra-abdominal infection (IAI) or lower respiratory tract infection (LRTI) were recovered from 11 Portuguese ICUs (June 2017–July 2018, STEP study) while 80 other P. aeruginosa isolates were recovered from UTI and IAI in patients admitted to 8 Spanish ICUs (April 2016–April 2017, SUPERIOR study) (Figure 1).14,15 Bacterial identification and antimicrobial susceptibility testing were performed at the Hospital Universitario Ramón y Cajal (Centre A, Madrid, Spain) (Figure S1, available as Supplementary data at JAC Online). Overall, ceftolozane/tazobactam showed good activity against P. aeruginosa isolates, presenting high susceptibility rates in both STEP (375/396, 94.7%) and SUPERIOR (73/80, 91.3%) studies.14,15
Geographical distribution of participant hospitals. Portuguese hospitals (STEP study) are highlighted in green circles and reported as numbers and Spanish hospitals (SUPERIOR study) in red stars and letters. This figure appears in colour in the online version of JAC and in black and white in the printed version of JAC.
Based on the EUCAST 2020 interpretative criteria (http://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/Breakpoint_tables/v_10.0_Breakpoint_Tables.pdf), ceftolozane/tazobactam-resistant P. aeruginosa isolates of both STEP (n = 21) and SUPERIOR (n = 7) studies were selected for the WGS analysis. For a precise comparative analysis, a subset of 28 P. aeruginosa isolates [STEP (n = 21), SUPERIOR (n = 7)] with a ceftolozane/tazobactam-susceptible phenotype were also randomly selected for the WGS analysis.
DNA extraction and WGS
Genomic DNA of the selected P. aeruginosa isolates was extracted using the commercial Chemagic DNA Bacterial External Lysis Kit (PerkinElmer, PA, USA). WGS was performed using the Illumina HiSeq 4000 platform or the Illumina NovaSeq 6000 platform (Oxford Genomics Centre, Oxford, UK) with 2 × 150 bp paired-end reads. Quality control and sequence filtering included FastQC (v0.11.8) (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and PRINSEQ-lite-0.20.3 (http://prinseq.sourceforge.net/) tools, respectively. Sequence reads were de novo assembled in contigs using SPAdes genome assembler (v3.11.1).16 Assembly metrics were evaluated by QUAST (v5.0.2).17 Bacterial identification was confirmed using the Taxonomic Sequence Classification System Kraken (v1.0).18 All draft genomes were annotated by Prokka (v1.13.3).19
Molecular typing
Mash (v2.1) and the iTOL application (Interactive Tree Of Life, https://itol.embl.de/) were used to generate and trace a similarity tree based on a neighbour-joining algorithm.20 mlst (v2.16.1) (https://github.com/tseemann/mlst) was used for the in silico MLST assignment. Serogroups based on the O-specific antigen (OSA) gene cluster sequences were determined using Blastn (v2.9.0+) (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and the OSA database.
Acquired resistome and virulome characterization
Assembled genomes were screened for acquired antibiotic resistance genes using abricate (v0.8.11) and ARG-ANNOT and ResFinder databases (threshold: 95% identity; 90% coverage) (https://github.com/tseemann/abricate/tree/master/db). Virulence factors were also detected using abricate and the VFDB database (threshold: 95% identity; 90% coverage).
Phylogenetic analysis and variant calling
Core genomes were obtained from all P. aeruginosa isolates and from the P. aeruginosa PAO1 reference genome (GenBank accession no. NC_002516.2) using Snippy (v4.4.3). A maximum-likelihood phylogenetic tree was reconstructed and visualized using IQ-TREE (v1.6.9) software and iTOL, respectively.21 SNPs and small insertions and deletions (InDels) from 163 chromosomal genes involved in antimicrobial resistance in P. aeruginosa were also extracted using Snippy.13 Briefly, the Snippy/BWA-MEM (v0.7.17) tool mapped the P. aeruginosa assembled sequences against the P. aeruginosa PAO1 reference genome, Snippy/Freebayes (v1.3.1) software called variants (minimum base quality of 20, minimum read coverage of 10× and 90% read concordance at a locus) and Snippy/SnpEff (v4.3) (http://snpeff.sourceforge.net/index.html) annotated the SNPs and InDels detected. Synonymous SNPs and SNPs distributed among all P. aeruginosa strains were not considered to be correlated with antimicrobial resistance phenotypes. Frameshift mutations and premature stop codons were considered to result in the inactivation of the corresponding product.
Statistical analysis
Fisher’s exact test was used to study differences in frequencies and percentages of dichotomous variables and to establish associations between categorical variables. Statistical analysis was conducted using R software (RStudio Team 2016 v1.0.44, RStudio, Boston, MA, USA). A P value <0.05 was considered as statistically significant.
Accession numbers
Sequence files were deposited at DDBJ/ENA/GenBank under the BioProject accession number PRJNA629475 and accession numbers JABDTR000000000–JABDVT000000000 (Table S1).
Ethics
The STEP study was approved by the Ethics Committees of all participating Portuguese Hospitals and the SUPERIOR study was approved by the Ethics Committee of the Hospital Universitario Ramón y Cajal (Ref. 087–16) and the Spanish Medicines Agency (Ref. MSD-CEF-2016-01).
Results
Genome characteristics
The average genome size of the de novo assembled genomes was 6.4 Mb, with an average G + C content of 65.7%. Information about all genome characteristics is summarized in Table S2. One low-quality sequence was detected in the subset of ceftolozane/tazobactam-susceptible strains from the SUPERIOR study and was excluded from the subsequent genome analysis. Bacterial identification using Kraken confirmed that the remaining 55 strains belonged to the species P. aeruginosa.
Molecular epidemiology
According to the in silico MLST, clonal complex (CC) 235 was the most prevalent clone, accounting for 15 (27.3%) of the 55 studied isolates. Interestingly, CC235 P. aeruginosa isolates were only detected in Portuguese ICUs (Centres 1, 2, 3, 4, 5, 9 and 10). Most of them (86.7%, 13/15) showed a ceftolozane/tazobactam-resistant phenotype (MICC/T range = 16/4–64/4 mg/L) and a significant correlation was established (OR = 0.1; 95% CI = 0.01–0.51; P = 0.0019) (Figure 2). The second most frequent P. aeruginosa clone was CC175 (18.2%, 10/55). CC175 was the predominant high-risk clone identified in Spanish ICUs and was found distributed across five different hospitals (Centres C, E, F, G and H). Ceftolozane/tazobactam (MICC/T range = 16/4 to >64/4 mg/L) showed ‘no susceptibility’ for 50% of CC175 (5/10) isolates (Figure 2). An association between CC175 and the ceftolozane/tazobactam-resistant phenotype was not demonstrated (OR = 1.04; 95% CI = 0.21–5.23; P = 1). Other P. aeruginosa clones encountered in our collection included CC244 (12.7%, 7/55) (Centres 5, 6, 8, 9 and 10), CC348 (9.1%, 5/55) (Centres 3 and 8) and CC253 (5.4%, 3/55) (Centres 4, 6 and 8). All of them were located in Portuguese hospitals and showed a lower rate of ceftolozane/tazobactam resistance (2/7 CC244 and 3/5 CC348) (Figure 2). CC309 (5.4%, 3/55) was also detected in both Portuguese (1/3, Centre 2) and Spanish (2/3, Centre H) ICUs, but only the Spanish CC309 strains displayed ceftolozane/tazobactam resistance (MICC/T = >64/4 mg/L) (Figure 2).
Mash similarity tree of the P. aeruginosa isolates from STEP and SUPERIOR surveillance studies analysed by WGS. Epidemiological, molecular typing, acquired antibiotic resistance gene content and antimicrobial susceptibility (EUCAST 2020 breakpoints) data are also included.14,15 Branch length is indicative of the Mash distance. LV, locus variant; AGly, aminoglycosides; Bla, β-lactams; Flq, fluoroquinolones; Phe, chloramphenicol; Rif, rifampicin; Sul, sulphonamides; Tmt, trimethoprim; AMC, amoxicillin/clavulanic acid; TZP, piperacillin/tazobactam; C/T, ceftolozane/tazobactam; CAZ, ceftazidime; FOF, cefotaxime; FEP, cefepime; ATM, aztreonam; IMP, imipenem; MEM, meropenem; CIP, ciprofloxacin; GEN, gentamicin; TOB, tobramycin; AMK, amikacin; CST, colistin; FOF, fosfomycin; TGC, tigecycline; R, resistant; S, susceptible, standard dose; I, susceptible, increased exposure. This figure appears in colour in the online version of JAC and in black and white in the printed version of JAC.
In silico serotyping confirmed that all P. aeruginosa isolates typed as CC235 and CC175 belonged to the O11 and O4 serogroups, respectively. Serogroups assigned to the remaining P. aeruginosa isolates showed higher diversity (Figure 2).
Acquired resistance genes
Up to 78.2% (43/55) of the selected P. aeruginosa isolates carried horizontally acquired resistance determinants (Figure 2). Carbapenemase-encoding genes were found in 25 strains (45.4%, 25/55), 18 of them from Portuguese centres (42.9%, 18/42) and 7 from Spanish centres (53.8%, 7/13). Overall, GES-13 was the most frequent carbapenemase (23.6%, 13/55) and was only found in association with the CC235-O11 high-risk clone detected in Portuguese hospitals (Centres 1, 2, 3, 4 and 9). A wide variety of VIM MBLs (18.2%, 10/55) were also encountered, mainly among Spanish strains. Three Portuguese isolates from two different hospitals were VIM-2 producers [CC244, Centre 8 (2/3); CC179, Centre 6 (1/3)]. VIM-1 production (2/10) was only identified in Spanish CC309 isolates (Centre H), while VIM-20 (3/10), VIM-2 (1/10) and VIM-36 (1/10) carbapenemases were associated with CC175 isolates from Spanish hospitals (Centres C, E and H). Additionally, KPC-3 carbapenemase was also detected in two non-clonally related isolates from Portuguese centres (CC253, Centre 2; CC449, Centre 8) (Figure 2). OXA-1 (n = 4) and OXA-2 (n = 4) β-lactamase genes were also found mainly associated with CC348 (4/4) (Centre 3) and CC175 (3/4) (Centre G) clones, respectively (Figure 2).
As many as 88% (22/25) of carbapenemase-producing P. aeruginosa strains displayed a ceftolozane/tazobactam-resistant phenotype (MICC/T range = 16/4 to >64/4 mg/L). Only KPC-3 (MICC/T = 1/4 mg/L) and VIM-36 (MICC/T = 2/4 mg/L) producers showed a ceftolozane/tazobactam-susceptible phenotype. Overall, the presence of carbapenemase genes was significantly correlated with the absence of susceptibility to ceftolozane/tazobactam (OR = 26.8; 95% CI = 5.6–187.9; P < 0.001). An association was also established between the ceftolozane/tazobactam-resistant phenotype and the production of both GES-13 (OR = 0.0; 95% CI = 0.0–0.2; P < 0.001) and VIM-type (OR = 0.08; 95% CI = 0.02–0.7; P = 0.01) carbapenemases (Figure 3). However, 20% of non-carbapenemase producers (20%, 6/30) also displayed ceftolozane/tazobactam MIC values in the resistant category (MICC/T range = 8/4 to >64/4 mg/L). Three of them (10%, 3/30) (CC348, Centre 3) carried the blaOXA-1 gene (Figure 3) and a correlation with the ceftolozane/tazobactam-resistant phenotype was observed (OR = 0.05; 95% CI = 0.0–0.9; P = 0.02).
Distribution of P. aeruginosa isolates according to different ceftolozane/tazobactam MIC values, the origin country (Portugal, STEP study; Spain, SUPERIOR study) and the detection of carbapenemase-encoding genes by WGS.14,15 Dotted line represents the ceftolozane/tazobactam EUCAST 2020 breakpoint (S ≤ 4 mg/L; R > 4 mg/L). CP, carbapenemase. This figure appears in colour in the online version of JAC and in black and white in the printed version of JAC.
A wide number of genes conferring co-resistance to other antimicrobial groups, including aminoglycosides, fluoroquinolones, chloramphenicol, rifampicin, sulphonamides and trimethoprim, were also found (Figure 2).
Phylogenetic analysis
A core-genome phylogenetic tree of all P. aeruginosa isolates and the P. aeruginosa PAO1 reference strain was reconstructed (Figure 4). Each genome shared between 62.3% and 95.6% (3.9–6.0 Mb) of the content with the P. aeruginosa PAO1 reference genome. An average of 20 672 high-quality variants (range 12 036–53 057) was detected (Table S3). As expected, clustering by the assigned CC was observed (Figure 4).
Core-genome maximum-likelihood phylogenetic tree of all P. aeruginosa isolates and the P. aeruginosa PAO1 reference genome (NC_002516.2). The units of the scale are SNPs by position. Colour of isolate names indicates the origin country: green (Portugal, STEP) and red (Spain, SUPERIOR). CC clusters of more than one isolate are highlighted by coloured circles: CC235 (orange), CC175 (dark green), CC244 (light blue), CC348 (pink), CC253 (light green), CC309 (yellow), CC179 (dark blue) and CC554 (purple). This figure appears in colour in the online version of JAC and in black and white in the printed version of JAC.
Mutational resistome
The complete list of the 163 chromosomal genes investigated in the 55 P. aeruginosa studied isolates and all non-synonymous SNPs and InDels suspected to be related to antimicrobial resistance are reported in Table S4. As many as 144 (88.3%) of the 163 analysed genes showed non-synonymous SNPs, frameshift mutations or a premature stop codon in at least one of the studied isolates. Figure 5 shows a selection of the main genes known to be involved in resistance to β-lactams (ampC gene and regulators, efflux pump-encoding genes and regulators, PBP and Opr porin genes), aminoglycosides (fusA1, efflux pump-encoding genes and regulators), fluoroquinolones [quinolone resistance-determining regions (QRDRs), efflux pump-encoding genes and regulators], polymyxins (LPS modification genes), fosfomycin (glpT) and rifampicin (rpoB). Overall, clustering of mutated genes by CC was observed, mainly in the predominant clones CC235 and CC175, which reinforces their MDR/XDR trait.
Main mutated genes involved in antibiotic resistance in the P. aeruginosa isolates from the STEP and SUPERIOR studies, detected by WGS. Epidemiological, molecular typing and antibiotic susceptibility (EUCAST 2020 breakpoints) data are also included.14,15 Dotted boxes show specific mutations. CP, carbapenemase; AMC, amoxicillin/clavulanic acid; TZP, piperacillin/tazobactam; C/T, ceftolozane/tazobactam; CAZ, ceftazidime; CTX, cefotaxime; FEP, cefepime; ATM, aztreonam; IMP, imipenem; MEM, meropenem; CIP, ciprofloxacin; GEN, gentamicin; TOB, tobramycin; AMK, amikacin; CST, colistin; FOF, fosfomycin; TGC, tigecycline; R, resistant; S, susceptible, standard dose; I, susceptible, increased exposure. This figure appears in colour in the online version of JAC and in black and white in the printed version of JAC.
All P. aeruginosa isolates except CC175 and CC244 clones contained non-synonymous mutations in the ampC gene [69.1% (38/55)]. Substitutions in ampC known to be related to ceftolozane/tazobactam resistance (T96I, F147L, G183D, E247G) were not detected. Up to 54.5% (30/55) of isolates contained mutations in the ampR gene, an AmpC regulator gene linked to AmpC hyperproduction. Only one isolate showed the ampR G154R substitution and other frequent missense mutations related to ampC overexpression and β-lactam resistance (D135G or D135N) were not found. Additionally, mutations in other well-known AmpC regulator genes, such as ampD, mpl and dacB (PBP4), were also evidenced in 80% (44/55), 41.8% (23/55) and 21.8% (12/55) of the isolates, respectively. Associations by clones were observed, but were not in agreement with the ceftolozane/tazobactam MIC values (Figure 5).
Mutations R504C and F533L in ftsI (PBP3-encoding gene), previously reported as a potential resistance mechanism to ceftolozane/tazobactam, were found in four strains belonging to clones CC175 (isolates E16 and E17) and CC348 (isolates 3-38 and 3-41). Note that three of them (isolates 3-38-F533L, 3-41-R504C and E17-R504C) were non-carbapenemase producers but displayed resistance to ceftolozane/tazobactam (Figure 5). Moreover, both CC348 isolates carried the blaOXA-1 gene (Figure 2).
Genes involved in the expression and regulation of efflux pumps, also contributing to some β-lactam resistance, were frequently mutated. Up to 36 isolates (65.4%) presented non-synonymous SNPs in the regulator gene mexZ and 9 of them (all assigned as CC175) showed the previously identified G195E substitution. Mutations in other efflux pump regulators such as mexR (nalB) and nalD genes were also detected in 30 (54.5%) and 12 (21.8%) isolates, respectively (Figure 5).
Mutations suggestive of OprD deficiency and resistance to carbapenems were also detected in 49 of the 55 (89.1%) P. aeruginosa isolates. Frameshift mutations were encountered in 31 isolates (63.3%), non-synonymous SNPs in 10 (20.8%) and premature stop codons in 8 others (16.3%) (Table S4). Premature stop codons included the known inactivating W277X and Q142X mutations that were only found in one and four isolates, respectively. All isolates containing Q142X belonged to the CC175 clone (Figure 5). The previously described QRDR mutations gyrA T83I, gyrA D87N and parC S87W were found in 36 (65.4%), 9 (16.4%) and 10 (18.2%) P. aeruginosa isolates, respectively. Both gyrA D87N and parC S87W substitutions were only detected in the CC175 isolates, while gyrA T83I was encountered among all the predominant clones, including CC235, CC175, CC244 and CC348. Almost all of these isolates were resistant to ciprofloxacin. Additionally, the parC S87L substitution was also found in 21 (38.2%) strains (Figure 5).
Mutations in parS and parR genes, as well as in PmrAB or PhoPQ two-component regulators, involved in LPS modification and resistance to polymyxins, were also frequently detected, mainly in CC235 (Figure 5).
Virulome characterization
Overall, an extensive number of genes related to virulence were found among the studied P. aeruginosa isolates. Clustering by CC was also observed (Figure S2). An average of 163 (range 82–222) virulence factors (VFs) belonging to 26–47 virulence loci (VLs) were detected. A higher virulence gene content was identified in the GES-13-CC235 high-risk clone (147–211 VFs, 35–43 VLs) from Portuguese ICUs and among VIM-type producers (119–206 VFs, 31–44 VLs) from all centres. As expected, P. aeruginosa isolates displaying a ceftolozane/tazobactam-resistant phenotype also showed an extensive virulome (119–213 VFs, 31–45 VLs).
Discussion
MDR/XDR P. aeruginosa is frequently associated with difficult-to-treat hospital-acquired infections and high mortality rates, mainly in immunocompromised patients.1,2 In 2016, the ECDC reported P. aeruginosa as the most frequent microorganism causing hospital-acquired pneumonia episodes and one of the most frequently represented microorganisms in UTIs and bloodstream infections in ICU patients.22 Furthermore, this species has been categorized by the WHO as a priority pathogen for research and development of new antibiotics.23 In the present work we used WGS to analyse the distinct genomic characteristics of a subset of P. aeruginosa clinical isolates recovered from ICU patients admitted to Portuguese and Spanish hospitals, as a part of the STEP and SUPERIOR surveillance studies, respectively. We also focused on the study of resistance mechanisms affecting the novel ceftolozane/tazobactam antibiotic combination.14,15
From an epidemiological point of view, we observed a wide inter-hospital spread of P. aeruginosa isolates recovered from both studies, mostly linked to the globally disseminated CC235 and CC175 lineages. However, inter-regional differences were observed in the geographical distribution and the acquired resistance gene content of these clones. CC235 was the most frequent clone in Portuguese hospitals and was related to GES-13 production. The increased carbapenemase activity of the GES-13 enzyme has been previously associated with the Gly-170→Asn substitution.24 The association of the ST235 high-risk clone with other GES class A β-lactamases with carbapenemase activity has been previously described in clinical samples from Portuguese patients.25,26 Nevertheless, to the best of our knowledge, this is the first description of GES-13-producing P. aeruginosa isolates in Portugal. Additionally, blaVIM-2 has frequently been reported in P. aeruginosa clinical isolates from Portuguese hospitals in a wide variety of clones, including ST179, ST244, ST235 and ST175.27 In our study, VIM-2 was also encountered in CC244 and CC179 clones from the STEP (Portugal) collection.
On the other hand, in agreement with previous surveillance studies performed in Spain, CC175 was the predominant P. aeruginosa clone in Spanish hospitals.10–12,28,29 In concordance with these surveys, VIM-type MBLs were the carbapenemases most frequently detected among the SUPERIOR P. aeruginosa isolates.11,12 In Spain, the inter-hospital dissemination of blaVIM-20 has been previously described among healthcare-associated XDR P. aeruginosa clinical strains, mainly belonging to the ST175 lineage.11 Coincidentally, VIM-20 was the most frequent carbapenemase in the SUPERIOR collection and was also linked to the CC175 high-risk clone.
In addition, a high number of VFs were found in both the STEP and SUPERIOR P. aeruginosa collections, particularly in carbapenemase-producing isolates, including both GES-13-CC235 and VIM type-CC175 lineages. The presence of an extensive virulome in MDR/XDR P. aeruginosa has also been demonstrated to be significantly involved in a higher severity of infections and an increase in the mortality rate.1
The increasing prevalence of hypervirulent MDR/XDR P. aeruginosa high-risk clones in hospital environments reduces the chances of appropriate empirical therapy for treating severe infections. Ceftolozane/tazobactam is indicated for the treatment of complicated IAIs and UTIs caused by β-lactam-resistant P. aeruginosa strains, including those that display resistance to carbapenems.4,8 Nevertheless, ceftolozane/tazobactam resistance has been reported in carbapenemase-producing P. aeruginosa isolates.4,9 In agreement with these studies, in our collection, the ceftolozane/tazobactam-resistant phenotype was significantly represented among carbapenemase producers (88%), particularly those strains carrying blaGES-13 (CC235) or blaVIM (CC175, CC244 and CC309) genes. Nevertheless, absence of ceftolozane/tazobactam susceptibility was also encountered in a low proportion of non-carbapenemase producers (20%). Three of them carried the blaOXA-1 gene and a statistical association could be determined. However, due to the low number of strains this correlation requires further studies.
Note that in addition to horizontally acquired resistance determinants, a complex intrinsic and mutational resistome has been widely described to be involved in antimicrobial resistance in MDR/XDR P. aeruginosa isolates.2,3 In our collection, we found a large number of mutations that could have affected the expression and/or function of chromosomal genes conferring resistance to several antimicrobial groups, including ceftolozane/tazobactam. The presence of a mutated ampC gene was not exclusive to ceftolozane/tazobactam-resistant strains, as in previous studies.30 Missense mutations in the ampC gene known to cause ceftolozane/tazobactam resistance in P. aeruginosa were not detected.10,11 However, the previously described R504C and F533L substitutions in ftsI (PBP3 gene) were found in three non-carbapenemase-producing P. aeruginosa isolates displaying ceftolozane/tazobactam resistance, two of them also carrying blaOXA-1.10,11 Nevertheless, an association between known resistance-associated mutations and the absence of ceftolozane/tazobactam activity could not be established in all those isolates in which carbapenemase-encoding genes were not detected.
In conclusion, during the STEP and SUPERIOR surveillance studies, GES-13-CC235 and VIM type-CC175 were the most frequent MDR/XDR P. aeruginosa high-risk clones involved in complicated infections in Portuguese and Spanish ICUs, respectively. Despite the close geographic proximity of Portugal and Spain, our study highlights distinct epidemiology of P. aeruginosa and resistance mechanisms when surveillance studies were performed and WGS was an excellent tool to depict this situation. Furthermore, carbapenemase-encoding genes were the major drivers of ceftolozane/tazobactam resistance in P. aeruginosa isolates causing infections in ICU patients from the Iberian Peninsula. However, in the absence of horizontally acquired carbapenemases/β-lactamases, mutations leading to overexpression of AmpC β-lactamase or those affecting the PBP3-encoding gene (ftsI) may also be a resistance mechanism against ceftolozane/tazobactam in P. aeruginosa and need to be further studied.
Acknowledgements
We thank Mary Harper for correction of the English used in the manuscript.
Members of the STEP study group
José Melo-Cristino (Serviço de Microbiologia Centro Hospitalar Lisboa Norte, Lisboa, Portugal); Margarida F. Pinto, Cristina Marcelo, Helena Peres, Isabel Lourenço, Isabel Peres, João Marques, Odete Chantre and Teresa Pina (Laboratório de Microbiologia, Serviço de Patologia Clínica, Centro Hospitalar Universitário Lisboa Central, Lisboa, Portugal); Elsa Gonçalves and Cristina Toscano (Laboratório de Microbiologia Clínica Centro Hospitalar de Lisboa Ocidental, Lisboa, Portugal); Valquíria Alves (Serviço de Microbiologia, Unidade Local de Saúde de Matosinhos, Matosinhos, Portugal); Manuela Ribeiro, Eliana Costa and Ana Raquel Vieira (Serviço Patologia Clínica, Centro Hospitalar Universitário São João, Porto, Portugal); Sónia Ferreira, Raquel Diaz and Elmano Ramalheira (Serviço Patologia Clínica, Hospital Infante Dom Pedro, Aveiro, Portugal); Sandra Schäfer, Luísa Tancredo and Luísa Sancho (Serviço de Patologia Clínica, Hospital Prof. Dr Fernando Fonseca, Amadora, Portugal); Ana Rodrigues and José Diogo (Serviço de Microbiologia, Hospital Garcia de Orta, Almada, Portugal); Rui Ferreira (Serviço de Patologia Clínica—Microbiologia—CHUA – Unidade de Portimão, Portugal); Helena Ramos, Tânia Silva and Daniela Silva (Serviço de Microbiologia, Centro Hospitalar Universitário do Porto, Porto, Portugal); Catarina Chaves, Carolina Queiroz and Altair Nabiev (Serviço de Microbiologia, Centro Hospitalar Universitário de Coimbra, Coimbra, Portugal); and Leonor Pássaro, Laura Paixao, João Romano and Carolina Moura (MSD Portugal, Paço de Arcos, Portugal).
Members of the SUPERIOR study group
Antonio Oliver and Xavier Mulet (Hospital Universitario Son Espases, Palma de Mallorca, Spain); Emilia Cercenado (Hospital General Universitario Gregorio Marañón, Madrid, Spain); Germán Bou and M. Carmen Fernández (Hospital Universitario A Coruña, A Coruña, Spain); Álvaro Pascual and Mercedes Delgado (Hospital Universitario Virgen Macarena, Sevilla, Spain); Concepción Gimeno and Nuria Tormo (Consorcio Hospital General Universitario de Valencia, Valencia, Spain); Jorge Calvo, Jesús Rodríguez-Lozano and Ana Ávila Alonso (Hospital Universitario Marqués de Valdecilla, Santander, Spain); Jordi Vila, Francesc Marco and Cristina Pitart (Hospital Clínic, Barcelona, Spain); and María García del Castillo, Sergio García-Fernández, Marta Hernández-García, Marta Tato and Rafael Cantón (Hospital Universitario Ramón y Cajal, Madrid, Spain).
Funding
The study was funded by MSD Portugal (protocol VP6918) and MSD Spain (protocol MSD-CEF-2016-01). This study was also supported by Plan Nacional de I + D + i 2013–16 and Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía, Industria y Competitividad, Spanish Network for Research in Infectious Diseases [RD16/0016/0001, RD16/0016/0004, RD16/0016/0006, RD16/0016/0007, RD16/0016/0010 and REIPI RD16/0016/0011], co-financed by the European Development Regional Fund ‘A way to achieve Europe’ (EDRF), Operative Program Intelligent Growth 2014–20. M.H.-G. is supported by a research contract from a European Project [IMI-JU-9–2013, Ref. iABC - 115721–2].
Transparency declarations
R.C. has participated in educational programmes organized by MSD, Pfizer and Shionogi. J.M.-C., G.B. and E.C. have participated in educational programmes organized by MSD and Pfizer. M.F.P. had a travel grant for ECCMID 2019 from MSD Portugal. L. Pássaro, L. Paixão and J.R. are MSD Portugal employees and/or may hold stock options in Merck & Co., Inc., Kenilworth, NJ, USA. J.D.-R. and D.L.-M. are employees of MSD Spain. All other authors declare no competing interests.
Supplementary data
Tables S1–S4 and Figure S1 are available as Supplementary data at JAC Online.
References
ECDC. Healthcare-Associated Infections in Intensive Care Units—Annual Epidemiological Report for
Author notes
Members of the STEP and SUPERIOR study groups are listed in the Acknowledgements section.
