Presence of Chromosomal crpP-like Genes Is Not Always Associated with Ciprofloxacin Resistance in Pseudomonas aeruginosa Clinical Isolates Recovered in ICU Patients from Portugal and Spain

CrpP enzymes have been recently described as a novel ciprofloxacin-resistance mechanism. We investigated by whole genome sequencing the presence of crpP-genes and other mechanisms involved in quinolone resistance in MDR/XDR-Pseudomonas aeruginosa isolates (n = 55) with both ceftolozane-tazobactam susceptible or resistant profiles recovered from intensive care unit patients during the STEP (Portugal) and SUPERIOR (Spain) surveillance studies. Ciprofloxacin resistance was associated with mutations in the gyrA and parC genes. Additionally, plasmid-mediated genes (qnrS2 and aac(6′)-Ib-cr) were eventually detected. Ten chromosomal crpP-like genes contained in related pathogenicity genomic islands and 6 different CrpP (CrpP1-CrpP6) proteins were found in 65% (36/55) of the isolates. Dissemination of CrpP variants was observed among non-related clones of both countries, including the CC175 (Spain) high-risk clone and CC348 (Portugal) clone. Interestingly, 5 of 6 variants (CrpP1-CrpP5) carried missense mutations in an amino acid position (Gly7) previously defined as essential conferring ciprofloxacin resistance, and decreased ciprofloxacin susceptibility was only associated with the novel CrpP6 protein. In our collection, ciprofloxacin resistance was mainly due to chromosomal mutations in the gyrA and parC genes. However, crpP genes carrying mutations essential for protein function (G7, I26) and associated with a restored ciprofloxacin susceptibility were predominant. Despite the presence of crpP genes is not always associated with ciprofloxacin resistance, the risk of emergence of novel CrpP variants with a higher ability to affect quinolones is increasing. Furthermore, the spread of crpP genes in highly mobilizable genomic islands among related and non-related P. aeruginosa clones alert the dispersion of MDR pathogens in hospital settings.


Introduction
Quinolones are an important broad-spectrum antimicrobial group and represent one of the most frequently used classes of antibacterial drugs. Nevertheless, due to their increasing use, resistance to this group of antimicrobials has progressively being increased over the last thirty years, and quinolone resistance is included in the definition of multi-drug resistant (MDR) microorganisms and difficult to treat resistant (DTR) pathogens [1,2]. Resistance to quinolones usually results from mutational alterations in drug target affinity, efflux pumps and/or porin channels overexpression, and acquisition of resistance-conferring genes [3]. Chromosomal mutational resistance affecting fluroquinolones mainly occurs in the so-called quinolone resistance determining region (QRDR) of topoisomerases [4]. Moreover, plasmid-mediated quinolone resistance (PMQR) has been also described and is generally due to Qnr-type proteins, the aminoglycoside-modifying enzyme AAC(6 )-Ib-cr acetyltransferase and mobile efflux systems such as QepA or OqxAB [5]. Increasing attention to these plasmid-mediated resistance mechanisms has been given in the last year as they are commonly presented in extended spectrum beta-lactamase (ESBL) and carbapenemases producing microorganisms [6].
Ciprofloxacin is the quinolone most frequently used to treat infections caused by Pseudomonas aeruginosa either orally or intravenously [7]. Resistance to ciprofloxacin in this pathogen is mainly due to QRDR mutations, but PMQR has also been encountered [8]. CrpP is a recently described 65-amino-acid ciprofloxacin-modifying enzyme that confers decreased susceptibility to ciprofloxacin, norfloxacin and moxifloxacin, but not levofloxacin, by enzymatic phosphorylation [9]. crpP gene was first detected in 2018 as encoded within the pUM505 plasmid in a P. aeruginosa clinical isolate from Mexico [9]. Since then, ciprofloxacin-modifying crpP genes have been identified in P. aeruginosa as part of different mobile integrative and conjugative elements (ICEs) that frequently transfer horizontally [10]. However, homologous crpP genes have also been located in chromosomal pathogenicity genomic islands (PAGI) of P. aeruginosa clinical isolates recovered in France and Switzerland between 2000 and 2015 [11]. Additionally, a recent study has supported that mutations in the codons encoding Gly7, Asp9, Lys33 and Cys40 of CrpP protein generate modified CrpP variants that restore ciprofloxacin susceptibility [12].
According to recent literature, the novel crpP-like gene is widespread in P. aeruginosa clinical isolates, but it is also easily transferable to other bacterial species, including ESBLproducing Enterobacterales, contributing to the emergence of other multidrug resistance pathogens [13]. In Mexico, it was found in 5.2% of a collection of ESBL producing Enterobacterales. Nevertheless, the role of ciprofloxacin-modifying crpP genes and its prevalence among the hospital circulating P. aeruginosa isolates remain undetermined, particularly in hospital settings such as intensive care units (ICUs), where fluoroquinolones are heavily used. Our aim was to investigate the dissemination of crpP-like genes in MDR or extensively resistant (XDR) P. aeruginosa clinical isolates recovered in patients admitted in Spanish and Portuguese ICUs between 2016 and 2018. These isolates were obtained during surveillance studies to monitor the activity of ceftolozane-tazobactam, a novel β-lactam-βlactamase combination with enhanced antimicrobial activity against P. aeruginosa, including MDR/XDR isolates [14][15][16].

Statistical Analysis
Fisher's exact test was used to analyze associations between categorical variables. Statistical analysis was performed using R software (RStudio Team 2016 v1.0.44, RStudio, Boston, MA, USA). A p-value < 0.05 was considered as statistically significant.

Acquired Resistance Genes
PMQR genes were detected in 6 P. aeruginosa isolates (10.9%). qnrS2 gene was identified in two clonally related P. aeruginosa strains belonging to the CC309 from the same Spanish center. Moreover, both CC309 strains and four other isolates belonging to the CC348 and recovered in the same Portuguese hospital carried the aac(6 )-Ib-cr gene. All these isolates displayed a ciprofloxacin resistance phenotype (MIC CIP > 2 mg/L), although statistical significance was not observed ( Table 2, Table S2).

Mutational Resistome
Single nucleotide polymorphisms (SNPs) and small insertions and deletions (InDels) were found in 91.2% (31/34) of the chromosomal genes known to be involved in fluoroquinolone resistance (Table S2).
Mutations in efflux pumps regulatory genes such as nalC and nfxB were also detected in 87.3% (48/55) and 7.3% (4/55) of the P. aeruginosa isolates, but a statistically significant correlation with a decreased ciprofloxacin susceptibility was not observed (Table 2).
The overall prevalence of crpP genes in MDR/XDR P. aeruginosa isolates was 59. 5% (25/42) and 84.6% (11/13) in Portugal and Spain, respectively. Moreover, crpP-like genes were found in a wide variety of P. aeruginosa clones (13/16) in both countries (11/14 in Portugal and 2/3 in Spain) (Table S2). CrpP1 (G7D), CrpP2 (G7H, I26L), CrpP3 (G7H, F16Y, I26L) and CrpP4 (K4R, G7D) variants were the most represented in our collection (CrpP1 (23/36), CrpP2 (4/36), CrpP3 (4/36) and CrpP4 (3/36) and were mostly linked to certain clones (Table 3). CrpP1 was the predominant variant and was identified among non-related clones of both countries including the CC175 (Spain) high-risk clone and the CC348 (Portugal) clone (Table 3, Table S2). Note that crpP-like gene was only found in one P. aeruginosa isolate belonging to the CC235 (1/15), the most frequent clone during this study (Table S2). Table 3. CrpP enzymes detected in the P. aeruginosa isolates from STEP and SUPERIOR surveillance studies.  (purple), I26L (green) and I26fs (gray). * crpP5 and crpP6 variants were described during this study. Fs = frameshift mutation. Table 3. CrpP enzymes detected in the P. aeruginosa isolates from STEP and SUPERIOR surveillance studies.  Correlation between the detection of crpP genes and the resistance to ciprofloxacin could not be precisely established (Table 2). In fact, 8 of 9 ciprofloxacin susceptible isolates (MIC CIP = 0.25-0.5 mg/L) contained a CrpP protein (CrpP2 (n = 3), CrpP1 (n = 2), CrpP4 (n = 2) and CrpP5 (n = 1)). Furthermore, CrpP2 variant was detected in CC244 isolates in which QRDR mutations were not found and statistical significance could be established with the ciprofloxacin susceptible phenotype (Table 2). Interestingly, with the exception of CrpP6, all CrpP1-crpP5 variants carried a missense mutation in the G7 amino acid position, essential to the function of the protein, and a correlation with a decreased susceptibility to ciprofloxacin was not determined (Table 3) (Figure 1). Additionally, mutations in the I26 amino acid position were also found in 3 of 6 variants (CrpP2, CrpP3 and CrpP5) (Figure 1). Note that the CrpP6 producing P. aeruginosa isolate (CC449, center 2) showed a ciprofloxacin resistant phenotype (MIC CIP = 1 mg/L), but mutations in QRDR or the presence other acquired resistance genes related to decreased susceptibility to fluoroquinolones were not found (Table S2).

crpP-Carrying Pathogenicity Genomic Islands (PAGI)
WGS analysis confirmed that all crpP-like genes were located in a large chromosomal region flanked by attL and attR sequences. xerD and parA genes involved in the mobilization and integration of pathogenicity genomic islands (PAGI) structures were also identified flanking this region. Ten PAGIs were reconstructed using one representative isolate harboring a different crpP gene (Figure 2). Our crpP-carrying P. aeruginosa strains contained related PAGIs varying in size (ca. 85-150 Kb). Moreover, common regions involved in mobilization and maintenance such as the pil operon were found in all PAGIs ( Figure 2). PAGI-crpP1.1 (ca. 85 Kb) was the most frequent genomic island (20/55) and was found in different clones in 5 centers from Portugal (CC348, CC253, CC308 and CC179) and 5 centers from Spain (CC175) ( Table 2, Table S2).

Figure 2.
Map of sequence comparison of crpP-like-carrying PAGI reconstructed from P. aeruginosa isolates recovered during the STEP (Portugal) and SUPERIOR (Spain) surveillance studies. Arrows represent genes and indicate the orientation. Relevant genes are indicated by colored arrows as follows: attL and attR sequences (pink), xerD and parA genes (orange), pil operon (yellow) and crpP-like genes (blue). Blue bands indicate the portions of the PAGI sequences that align to each other.

Discussion
In the last thirty years, quinolones have been widely used to treat infections caused by both Gram-positive and Gram-negative bacteria in different infection sites. However, Figure 2. Map of sequence comparison of crpP-like-carrying PAGI reconstructed from P. aeruginosa isolates recovered during the STEP (Portugal) and SUPERIOR (Spain) surveillance studies. Arrows represent genes and indicate the orientation. Relevant genes are indicated by colored arrows as follows: attL and attR sequences (pink), xerD and parA genes (orange), pil operon (yellow) and crpP-like genes (blue). Blue bands indicate the portions of the PAGI sequences that align to each other.

Discussion
In the last thirty years, quinolones have been widely used to treat infections caused by both Gram-positive and Gram-negative bacteria in different infection sites. However, the high level of use of these antimicrobials has contributed to the parallel selection and dispersion of quinolone-resistant pathogens in both hospital and community settings [6]. During the STEP (Portugal) and SUPERIOR (Spain) surveillance studies, that monitor ceftolozane-tazobactam susceptibility, 55-56% of MDR/XDR P. aeruginosa clinical isolates recovered from ICU patients showed a ciprofloxacin resistant phenotype (MIC CIP > 2 mg/L). Moreover, a statistical association was found between the ciprofloxacin resistance and the resistant ceftolozane-tazobactam phenotype in both STEP and SUPERIOR collections. Ceftolozane-tazobactam is a new cephalosporin-β-lactamase inhibitor combination with a wide antimicrobial spectrum and high activity against MDR Gram-negative bacteria, with the exception of carbapenemase producers [23]. However, ceftolozane-tazobactam resistance has been recently described in ESBL-producing Enterobacterales populations, including those recovered during the STEP and SUPERIOR surveillance studies [24,25]. The selection pressure in the intestinal microbiota caused by the extensive use of antibiotics during decades has led to the emergence of MDR/XDR pathogens carrying multiple antibiotic resistance determinants, including mechanisms involved in quinolone resistance but also β-lactamases genes such as ESBL and carbapenemases.
In consistence with previous studies, the most frequent resistance mechanism to quinolones in our MDR/XDR P. aeruginosa sequenced isolates was the presence of mutations in the gyrA and parC genes, encoding gyrase and topoisomerase IV subunits [4]. Additionally, the horizontally transferred genes qnrS2 and aac(6 )-Ib-cr were eventually detected in relation to certain clones. On the other hand, ten different crpP-encoding genes resulting in six CrpP enzymes were also found in 65.4% of MDR/XDR P. aeruginosa clinical isolates. Four of these CrpP variants (CrpP1, CrpP2, CrpP3 and CrpP4) have been previously found in P. aeruginosa isolates from other European countries [11], but two novel CrpP proteins (CrpP5 and CrpP6) were first identified in our study in two Portuguese centers.
CrpP-like genes have been recently described as a novel resistance mechanism affecting ciprofloxacin in P. aeruginosa and other MDR Gram-negative bacteria [9,13]. However, in our P. aeruginosa collection, decreased susceptibility to ciprofloxacin was not correlated to the presence of crpP genes. In fact, in the absence of mutations in QRDR or other PMQR genes such as qnrS2 and aac(6 )-Ib-cr, only the novel crpP6-encoding gene could be associated with a decreased ciprofloxacin susceptibility. It should be noted that the remaining CrpP variants (CrpP1-CrpP5) carried a missense mutation in the G7 amino acid, previously defined in a recent study as an essential residue for crpP-mediated ciprofloxacin resistance [12]. G7 amino acid (involved in catalysis) and I26 amino acid (involved in ATP binding) are located at the N-terminal region and are suggested to be conserved residues essential for the function of CrpP protein [12]. According to recent literature, mutations in G7 and I26 position are non-commonly detected among the amino acid sequences of CrpP present in GenBank [6,26]. However, in both Portuguese and Spanish collections, CrpP variants carrying mutations in G7 were predominant, as recently described in other European countries [11]. It should be noted that the emergence and dissemination of modified CrpP variants in P. aeruginosa clinical isolates circulating in nosocomial settings such as ICUs could lead to the appearance and dissemination of novel crpP alleles with a higher ability to affect quinolones.
Interestingly, all crpP-encoding genes were located in PAGIs closely related to other genomic islands previously found in Europe between 2000 and 2015 [11]. Previous studies have demonstrated that xerD and parA genes contribute to transfer PAGI through P. aeruginosa strains, contributing to the dissemination of different antibiotic resistance mechanisms and virulence determinants and leading to the evolution and expansion of this species among different habitats [27]. Additionally, homologues of crpP-like gene have also been identified in other Gram-negative bacteria such as ESBL producing Enterobacterales [13]. The spread of crpP-like genes among different species and clones through well-conserved genetic platforms could be also a consequence of the selective pressure caused by the extensive use of ciprofloxacin in the treatment of a large number of infections.
According to our results, PAGI-crpP1.1 was the most widely disseminated genomic island. Furthermore, the dissemination of this conserved PAGI-crpP1.1 occurred among non-clonally related isolates distributed in both countries, including clones broadly disseminated in our region such as CC175 high-risk clone (Spain) and CC348 clone (Portugal). Note that prevalence of crpP genes was higher in the P. aeruginosa isolates recovered in Spain than in those recovered in Portuguese hospitals, and this finding could be related to a successful association between the PAGI-crpP1.1 and the CC175 high-risk clone. It should be noted that the CC175 is the most frequent P. aeruginosa clone in Spanish hospitals and that its extensive resistome has been found to be determined mainly by mutational events [16,28,29]. On the other hand, the presence of crpP genes was scarcely observed in the CC235 (1/15), the most frequent clone in the Portuguese collection. However, CC235-P. aeruginosa harboring homologues of crpP genes has been reported as the most prevalent clone among clinical isolates recovered in France and Switzerland between 2000 to 2015 [11]. In this sense, the presence of chromosomal crpP-like genes with a high capacity of mutation in well-adapted hospital P. aeruginosa lineages such as CC175 and CC235 could contribute to the emergence of novel crpP genes with a higher spectrum of activity against fluoroquinolones.
On the other hand, genomic islands of P. aeruginosa have previously been shown to carry different virulence factors generating hypervirulent strains with a high adaptative capacity [30]. Fortunately, although our collection of P. aeruginosa carried a high virulence gene content, virulence factors were not detected in the crpP-like-carrying PAGIs [16].
According to our results, mutations in gyrase and topoisomerase encoding genes, particularly in gyrA and parC, are the main resistance mechanism to ciprofloxacin in MDR/XDR P. aeruginosa clinical isolates from ICU patients in Portugal and Spain. Additionally, transferable mechanisms of quinolone resistance such as qnrS2 and aac(6 )-Ib-cr genes were also detected in a low proportion of strains. Nevertheless, the spread of different crpP-like genes among non-related P. aeruginosa clones through conserved and wide genomic islands may contribute to the emergence and persistence of MDR/XDR pathogens in hospital settings and should be further explored.

Conclusions
CrpP-encoding genes are frequently detected in the chromosome of non-related clones of P. aeruginosa isolates causing infections in ICU patients from Portugal and Spain. Moreover, crpP-like genes were located in PAGI structures with conserved regions involved in mobilization and integration and related to genomic islands previously found in P. aeruginosa clinical isolates recovered in other European countries. In our collection, inactive or unfunctional CrpP proteins were predominant, and ciprofloxacin resistance was more probably mediated by mutations in the QRDR or eventually due to the presence of horizontally transferred genes other than crpP. Nevertheless, highly mobilizable PAGIs harboring different homologous crpP genes with a high capacity of mutation could play a critical role in the dissemination and transmission of other antimicrobial resistance genes among nosocomial P. aeruginosa high-risk clones in Europe and should be considered for further studies.
Supplementary Materials: The following are available online at https://www.mdpi.com/2076-2 607/9/2/388/s1, Table S1: MDR/XDR-P. aeruginosa clinical isolates analyzed by whole genome sequencing during the SUPERIOR (Spain, 2016-2017) and STEP (Portugal, 2017-2018) surveillance studies; Table S2: Chromosomal genes detected in P. aeruginosa isolates as a part of the mutational resistome affecting fluoroquinolones susceptibility. crpP gene content, epidemiological data, acquired antibiotic resistance genes and ciprofloxacin-susceptibility results are also included. Informed Consent Statement: Patient consent was waived because it was not required by Ethics Committee since patient data was anonymized.

Data Availability Statement:
The sequence data analyzed in this study are openly available at NCBI [see Material and Methods section for Accession numbers].