High Prevalence of GES-5 Variant and Co-Expression of VIM-2 and GES-45 among Clinical Pseudomonas aeruginosa Strains in Tunisia

Carbapenem-resistant Pseudomonas aeruginosa (CRPA) are a global health concern. The antimicrobial resistance, virulence, and molecular typing of 57 CRPA isolated from 43 patients who attended a specific Tunisian hospital from September 2018 to July 2019 were analyzed. All but one were multidrug-resistant CRPA, and 77% were difficult-to-treat-resistant (DTR) isolates. The blaVIM-2 gene was detected in four strains (6.9%), and among the 36 blaGES-positive CRPA (62%), the blaGES-5 gene was the predominant variant (86%). Three strains co-harbored the blaVIM-2 and blaGES-45 genes, and seven CRPA carried the blaSHV-2a gene (14%). OprD alterations, including truncations by insertion sequences, were observed in 18 strains. Regarding the 46 class 1 integron-positive CRPA (81%), the blaGES-5 gene was located in integron In717, while the blaGES-29 and blaGES-45 genes were found in two new integrons (In2122 and In4879), and the blaVIM-2 gene was found in In1183 and the new integron In2142. Twenty-four PFGE patterns and thirteen sequence types (three new ones) were identified. The predominant serotype O:11 and exoU (81%) were mostly associated with ST235 and the new ST3385 clones. The seven blaSHV-2a-CRPA from different patients belonged to ST3385 and the same PFGE pattern. The blaGES-5- and blaVIM-2 + blaGES-45-positive CRPA recovered mostly from ICU patients belonged to the high-risk clone ST235. Our results highlight the alarming prevalence of blaGES-5- and ST235-CRPA, the co-existence of blaGES-45 and blaVIM-2, and their location within integrons favoring their dissemination.


Introduction
Pseudomonas aeruginosa is a common Gram-negative pathogen that is recognized as a cause of several hospital-acquired infections ranging from urinary tract infections to life-threatening pneumonia and septicemia, especially among patients with cystic fibrosis, immunocompromised patients, burn patients, and those with indwelling devices.Carbapenems are considered as one of the last resort treatments of infections caused by P. aeruginosa [1,2].Compared to other β-lactams, these potent compounds provide better Gram-negative coverage and exhibit stability against the action of extended spectrum β-lactamases (ESBL) and chromosomal cephalosporinase (AmpC), along with safety and efficiency [3,4].However, this efficiency is increasingly impeded by the emergence of carbapenem-resistant P. aeruginosa (CRPA) isolates.Subsequently, the lack of therapeutic alternatives, together with the spread of CRPA involve that infections caused by this pathogen, have become difficult to treat and a cause of great concern, with significant consequences for clinical and economic outcomes [5,6].Indeed, in 2017, the World Health Organization ranked CRPA in the critical group on the priority pathogens list, that imperatively requires the development of new antibiotics [7].
The mechanisms of resistance to carbapenem in P. aeruginosa include decreased drug uptake, loss of the OprD outer membrane porin, hyperexpression of efflux pump systems, overexpression of AmpC, and acquisition of carbapenemases.Furthermore, the combination of two or more of these carbapenem resistance mechanisms determines the level of carbapenem resistance [8][9][10][11].Among the carbapenemases acquired by P. aeruginosa, the most important ones are metallo-beta-lactamases (MBL), mainly VIM, IMP, and NDM, but class A carbapenemases, including GES-type enzymes, less frequently KPC, and rarely class D oxacillinases (OXA-type), are also responsible for carbapenem resistance [5,[12][13][14].The 59 known GES variants (http://www.bldb.eu,accessed on 28 August 2023) [15] are categorized as minor ESBLs, with the exception of the variants that possess amino acid substitutions within their active sites (Gly170Ser or Gly170Asn) (such as GES-5) because they can expand their spectrum of activity against carbapenems and are categorized as carbapenemases [16].GES-5-producing P. aeruginosa was first described in China [17] and has been increasingly detected worldwide (e.g., South Africa, Brazil, Mexico, Spain, Turkey, Saudi Arabia, and Japan), with it even being associated with the P. aeruginosa ST235 high-risk clone [14,18].Carbapenemase-encoding genes are generally located on mobilized genetic elements such as plasmids, transposons, and integrons; they include numerous antimicrobial resistance determinants, thus promoting the dissemination of multidrug-resistance [14,19].
In addition to its great antimicrobial resistance, the importance of P. aeruginosa is marked by the presence of multiple virulence factors [20], most of them under the control of the quorum-sensing system (mainly LasI/LasR and RhlI/RhlR systems), which is a cell density recognition mechanism.The Type 3 Secretion System (T3SS) and its toxins, termed effectors (ExoU, ExoS, ExoT, ExoY), are the major virulence determinants in P. aeruginosa.These systems enable P. aeruginosa to invade and infect the host, thereby increasing pathogenicity [21].ST235 is the most globally disseminated of the three major international high-risk clones (ST111, ST175, and ST235), and it exhibits a highly virulent phenotype with a high mortality rate, which is most likely due to the production of the ExoU effector [22].
Based on the alarming aspect of multidrug-resistant P. aeruginosa strains, molecular typing is essential to investigate the diversity of P. aeruginosa collections and to confirm or deny the genetic relationship between the strains during outbreaks.In fact, methods such as Pulsed field gel electrophoresis (PFGE) and Multilocus sequence typing (MLST) are valuable tools for identifying the sources of infection and putative reservoirs and tracking the transmission routes of high-risk clones within and across wards, hospitals, cities, and even countries [23,24].
In Tunisia, rates of carbapenem-resistant Gram-negative bacteria are steadily increasing.This country was the second largest consumer of antibiotics in the world in 2015, and according to the Pharmacy and Medicines Directorate, carbapenem consumption doubled between 2011 and 2015 because of the overuse and misuse of antibiotics in human medicine and the livestock, agricultural and industrial sectors.Thus, the CRPA rates reached 46% of P. aeruginosa strains isolated in the ICU, and nearly 42% of these strains were extensively drug-resistant (http://www.dpm.tn/,accessed on 1 July 2023).Regarding CRPA studies in Tunisia, outbreaks of VIM-producing P. aeruginosa strains and, more recently, GES-5producing CRPA have been reported [25][26][27][28][29][30][31].Additionally, the successful worldwide spread of high-risk clones of P. aeruginosa poses a threat to global public health that needs to be studied and managed.However, few reports have been conducted on the molecular epidemiology or molecular typing of CRPA in Tunisia [29][30][31].The aim of the present study was to analyze the characteristics of clinical CRPA isolates recovered from the Military Hospital of Tunis, including their antimicrobial resistance, integrons, virulence factors, and molecular typing.

Characterization of Carbapenem Resistance
The molecular characterization of CRPA showed that 35 of them were carbapenemase producers with a predominance of GES-5 (31/35) against a low prevalence of VIM-2 (4/35).Likewise, the following non-carbapenemase GES-type enzymes were detected in five isolates: GES-1, GES-29, and GES-45 (Table 2).As Table 1 shows, the patients with carbapenemase-CRPA carriers were all admitted to the ICU, except for one who required orthopedic surgery, whereas the remaining patients were distributed in different hospital wards.
The oprD gene was amplified from all but two of the fifty-seven CRPA (P22 and P70), and sixteen amplicons were randomly selected for sequencing.Table 3 shows the high polymorphism in OprD amino acid sequences.Amino acid substitutions and nucleotide insertions and deletions were detected, highlighting the presence of the insertion sequences ISPa33 and ISPa26, which disrupt the oprD gene of P4 and P569, respectively (Table 3).
a Shortening of putative Loop L7: 372V-DSSSSYAGL-L383. b No amplicon of the oprD gene was detected in these strains.

Detection of Virulence Factors
The presence of the seven genes involved in the virulence and quorum-sensing system was investigated among the 57 P. aeruginosa strains, and six different profiles were obtained (Table 4).The T3SS exoU + /exoS -genotype was detected in 46 isolates (81%), and the exoU -/exoS + genotype was detected in the remaining 11 isolates (19%).On the other hand, no isolate produced the exolysin-encoding gene exlA.All strains amplified the lasI gene, but the lasR gene was amplified in 52 CRPA (91%).P6 and P74 CRPA showed a <400 bp lasR amplicon.On the other hand, the lasR amplicon of the P38 strain was larger than 2 kb, and sequencing analysis showed that the insertion sequence ISPa26 truncated the lasR gene.The rhlI gene was absent in six strains, and the rhlR was absent in 11 CRPA.
a truncated by ISPa26; b an amplicon of only 340 bp was detected.

Detection and Characterization of Integron Structures
Class 1 integrons were detected in 46 of the 57 CRPA isolates (81%), but class 2 and class 3 integrons were not found (Supplementary Table S1).Four new class 1 integrons were detected in this study, namely In2115, In2122, In2142, and In4879 (named by INTE-GRALL), and were submitted to GenBank with the following respective accession numbers: OM831263, OM831264, OM863779, and OQ858935.
In the current study, most of the CRPA were found in respiratory specimens from ICU patients.Typically, MDR bacteria are mostly recovered in the ICU, where the extensive use of antibiotics, particularly carbapenems, creates selection pressure, that promotes the emergence of MDR strains [39,40].In addition, our data highlighted the high percentage of DTR-P.aeruginosa (77%), whose treatment requires new options such as ceftolozane-tazobactam, ceftazidime-avibactam, or imipenem-relebactam, as international guidelines currently recommend [41].
Our analysis of the carbapenem resistance mechanisms in the 57 CRPA revealed that carbapenemases (GES-5 and VIM-2), active efflux pumps, the inducible expression of AmpC, and the overexpression of AmpC β-lactamase were found in 61%, 89%, 67%, and 7% of the P. aeruginosa isolates, respectively.Additionally, a high polymorphism in the porin OprD was observed in the 18 CRPA analyzed.As previously reported [42][43][44][45][46], the detected insertions, deletions, premature stop codons, and insertion sequences ISPa33 and ISPa26 truncating the oprD gene would suppose a direct correlation with car-
In the current study, most of the CRPA were found in respiratory specimens from ICU patients.Typically, MDR bacteria are mostly recovered in the ICU, where the extensive use of antibiotics, particularly carbapenems, creates selection pressure, that promotes the emergence of MDR strains [39,40].In addition, our data highlighted the high percentage of DTR-P.aeruginosa (77%), whose treatment requires new options such as ceftolozanetazobactam, ceftazidime-avibactam, or imipenem-relebactam, as international guidelines currently recommend [41].
Our analysis of the carbapenem resistance mechanisms in the 57 CRPA revealed that carbapenemases (GES-5 and VIM-2), active efflux pumps, the inducible expression of AmpC, and the overexpression of AmpC β-lactamase were found in 61%, 89%, 67%, and 7% of the P. aeruginosa isolates, respectively.Additionally, a high polymorphism in the porin OprD was observed in the 18 CRPA analyzed.As previously reported [42][43][44][45][46], the detected insertions, deletions, premature stop codons, and insertion sequences ISPa33 and ISPa26 truncating the oprD gene would suppose a direct correlation with carbapenem resistance, mainly imipenem resistance, by disrupting the coding region of the oprD gene or by downregulating its expression in our CRPA.The combination of two or more of these carbapenem resistance mechanisms determined the carbapenem resistance in the studied CRPA.
Reports of various carbapenemase types among P. aeruginosa strains have increased worldwide over the last decade [14,15,47].GES-5 class A carbapenemase (54%) appears to be the leading carbapenemase in this study.Moreover, four isolates (7%) were VIM-2 MBL producers.This result aligns with the recent studies reporting the detection of GES-5 and VIM-2 in the CRPA of Tunisia [30,31], and it is worth mentioning that this GES-5 prevalence in CRPA might be a sign of the start of the expansion of these strains in Tunisia.On the other hand, the predominance of VIM-2 among P. aeruginosa was previously revealed in studies performed in Tunisia [25,28,29,31] and in other countries such as the United Arab Emirates [34], Spain [32,43,44,48], Russia, the United States [14], Lebanon, and Egypt [49,50].
Along with the detected carbapenemases, GES-1, GES-29, and GES-45 class A βlactamases and the SHV-2a ESBLs were also found in our study.Indeed, three bla VIM-2producing isolates also harbored the bla GES-45 gene.The co-existence of VIM and GES enzymes in P. aeruginosa has been previously demonstrated in Tunisia [30,31] as well as elsewhere [34,51,52].However, to our knowledge, this is the first work to describe the co-existence of the bla GES-45 and bla VIM-2 genes in CRPA.
P. aeruginosa can also exhibit resistance to cephalosporins mediated by ESBLs embedded in plasmids and/or integrons.In fact, GES-and SHV-type ESBLs mainly possess ceftazidimase activity, affecting cefepime to a lesser extent [56].In our work, particularly noteworthy was the existence of seven MDR bla SHV-2a -producing CRPA belonging to the new exoU-positive ST3385 and to the same E7 PFGE pattern, which spread in different wards of the hospital.These results suggest an intra-hospital outbreak due to a SHV-2aproducing CRPA, as previously described by other authors in France and Tunisia [25,57].
Along with the ST235 high-risk clone, ST244 and ST274 are of particular significance as they are frequently detected worldwide but not always linked to MDR/XDR profiles, as our results show [58].
Several studies have revealed that high levels of antimicrobial resistance are represented by a relatively low genetic diversity, as demonstrated by the current work [14,32,58].However, the horizontal gene transfer of carbapenemase genes through mobilizable genetic elements such as integrons requires special attention because it can accelerate the dissemination of MDR CRPA [14,62].Class 1 integrons were detected in 81% of our CRPA isolates, with 9 different gene cassette arrangements containing genes encoding resistance to βlactams, aminoglycosides, rifampicin, and fluoroquinolones.More than one integron per isolate was detected in 78% of the CRPA, as described in other studies [43], and all of them were ascribed to ST235.Four new genetic arrangements were detected in the study (In2115, In2122, In2142, In4879), and all of the carbapenemase genes were detected as gene cassettes embedded in class 1 integron structures.Three of the four bla VIM-2 -positive CRPA harbored the In1183 integron (bla OXA-10 + aadB + bla VIM-2 + aadB + bla OXA-10 ), an arrangement first described in Tunisia [29].Interestingly, a new arrangement (aadB + bla VIM-2 + aadB + arr-4 + qnrVC1 + aadA1a + intronIIC + bla OXA-2 ) was identified in the exoS-VIM-2-positive isolate belonging to ST267.This integron partially shared the arrangement of a class 1 integron also first described in Tunisia (aadB + bla VIM-2 + aadB + arr-6 + qnrVC1 + aadA1c + intronIIC + bla OXA-2 ) [63].Additionally, all 31 GES-5-positive isolates carried a previously described arrangement (bla GES-5 + aacA4 + gcuE15 + aphA15 + ISPa21e) [18].This is the first study to show the coexistence of VIM-2 and GES-45 and elucidate the phylogenetic relationship (by PFGE and MLST) as well as the main virulence determinants among circulating CRPA strains in Tunisia.However, a limitation of our study is that it was performed using CRPA from a single hospital.Thus, the prevalence and molecular characteristics of the CRPA strains may not be nationally representative.Moreover, the absence of susceptibility testing to last-line and novel antimicrobials (e.g., ceftolozanetazobactam, ceftazidime-avibactam, and imipenem-relebactam), together with the several difficulties experienced in obtaining more clinical patient data, such as length of stay, comorbidities, outcome (recovery or death), etc., meant that we were unable to analyze the impact of the high rates of DTR P. aeruginosa and CRPA on the clinical outcomes of patients.

Bacterial Isolates and Identification
A total of fifty-seven non-duplicated CRPA isolates were recovered from forty-three patients of the Military Hospital of Tunis from September 2018 to July 2019.Bacterial identification was firstly performed via the Vitek ® 2 automated system (Biomérieux, Marcyl'Étoile, France), then confirmed via PCR amplification of oprL, the outer cell membrane lipoprotein L-encoding gene [64], and by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) (MALDI Biotyper ® , Bruker Daltonics GmbH & Co. KG, Bremen, Germany) following the manufacturer's guidelines.The results were interpreted according to the MBT Compass Library DB-6903 (V.6).

Molecular Typing
The clonal relationship among the fifty-seven recovered CRPA isolates was determined via PFGE.Agarose plugs containing SpeI-restricted genomic DNA were prepared as previously described [69], and DNA fragments were separated using two ramps at 6 V/cm at 14 • C within a CHEF-DR II system (BioRad).The pulse time ranged from 5 to 15 s during the first 10 h and from 15 to 45 s during the next 10 h.A lambda ladder (Bio-Rad) was used as a DNA size marker.The DNA profiles were analyzed using the GelJ software 2.3 (UPGMA algorithm; Dice coefficient) [70].The isolates showing a Dice coefficient ≥90% were considered genetically related for this study.

Serotyping
Serotypes of the recovered isolates were determined via the slide agglutination test (Bio-Rad, Temse, Belgium), which was conducted according to the International Antigenic Typing scheme and by using 16 type O monovalent antisera specific for P. aeruginosa following the manufacturer's protocol.
OprD porin analysis was carried out via PCR and subsequent sequencing [71].The mutations were determined by comparison with the wild-type P. aeruginosa strain PAO1 sequence (GenBank accession number AE004091) [76,77], and the insertion sequences were characterized using the online tool ISfinder (https://www-is.biotoul.fr/,accessed on 7 November 2019).

Detection and Characterization of Integrons
The presence of genes encoding type 1, 2, and 3 integrases, 3 -conserved segment of class 1 integrons (qacE∆1+ sul1), and Tn402 features was studied via PCR.The characterization of class 1 integron variable regions and gene cassette promoters (Pc) was carried out via PCR mapping and sequencing [78].

Detection of Virulence Factors
All CRPA isolates were tested for the presence of virulence markers such as exoS and exoU exotoxin, exlA exolysin genes, and the quorum-sensing lasR, lasI, rhlR, and rhlI genes [79,80].All the performed PCRs in this work included at least one positive control.

Conclusions
In summary, our findings highlight that GES-5 production, the co-existence of bla GES-45 and bla VIM-2 , and OprD porin alterations were the major causes of carbapenem resistance in the CRPA strains.In our study, the GES-5-producing strains, which were mainly recovered from ICU patients, belonged to clonal clusters and to the international high-risk clone ST235.Moreover, given the pivotal role of integrons in the acquisition and transmission of antibiotic resistance genes, the prevalence of these elements in our isolates is a cause of concern and speaks to the need to implement policies to control the spread of resistance determinants.Based on the above, our findings could be invaluable to future research on the risk factors, origins, and mechanisms underlying outbreaks, thereby also being invaluable to the control of DTR P. aeruginosa.

Figure 2 .
Figure 2. Schematic structure of the different class 1 integrons detected in the 57 CRPA.

Figure 2 .
Figure 2. Schematic structure of the different class 1 integrons detected in the 57 CRPA.

Table 1 .
Clinical characteristics of the CRPA isolates recovered between 2018 and 2019.

Table 4 .
Virulence and quorum-sensing genes detected among the 57 CRPA isolates.