Retrospective Data Insight into the Global Distribution of Carbapenemase-Producing Pseudomonas aeruginosa

This study aimed to determine the global distribution and molecular characteristics of carbapenemase-producing Pseudomonas aeruginosa isolates. A total of 328 (11.1%, 328/2953) carbapenemase-producing P. aeruginosa isolates from humans were obtained from public databases as of October 2019. Of which, the blaVIM and blaIMP genes were the most prevalent carbapenemases in the P. aeruginosa isolates. These carbapenemase-producing P. aeruginosa isolates possessed 34 distinct sequence types (STs) and six predominated: ST357, ST823, ST308, ST233, ST175 and ST111. The ST357 and ST823 isolates were primarily found detected in Asia and all ST175 isolates were found in Europe. The ST308, ST233 and ST111 isolates were spread worldwide. Further, all ST823 isolates and the majority of ST111, ST233 and ST175 isolates carried blaVIM but ST357 isolates primarily carried blaIMP. ST308 isolates provide a key reservoir for the spread of blaVIM, blaIMP and blaNDM. WGS analysis revealed that ST111 carried a great diversity of ARG types (n = 23), followed by ST357 (n = 21), ST308 (n = 19), ST233 (n = 18), ST175 (n = 14) and ST823 (n = 10). The ST175 isolates carried a more diversity and frequent of aminoglycoside ARGs, and ST233 isolates harbored more tetracycline ARGs. Our findings revealed that different carbapenem resistance genes were distributed primarily in variant STs of P. aeruginosa isolates, these isolates also possessed an extensive geographical distribution that highlights the need for surveillance studies that detect carbapenemase-producing P. aeruginosa isolates in humans.


Introduction
The antimicrobial-resistant ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp.) are a global threat to human health and include four Gram-negative bacteria [1]. In particular, K. pneumoniae ST307 isolates and Salmonella enterica serotype Kentucky ST198 from the USA and Egypt, respectively, have now worldwide dissemination [2,3]. A. baumannii ST195 isolates are distributed widely in eight countries and isolates recovered from different locations may present less genomic sequence similarity [4]. However, there is paucity of data regarding the global distribution of P. aeruginosa isolates.
Carbapenem antibiotics are generally considered last-line agents for the treatment of severe cases of P. aeruginosa infections [5]. However, the recent increases in the prevalence of carbapenem-resistant P. aeruginosa nosocomial isolates is great concern [5,6]. Evidence suggests that patients who are infected by carbapenem-resistant pathogens have an increased likelihood of morbidity and mortality compared with those infected by susceptible pathogens [7]. The clinical use of carbapenems is currently restricted as a result of the prevalence of carbapenem resistance genes. More seriously, mobile genetic elements have facilitated their rapid dissemination. So far, P. aeruginosa isolates producing bla IMP , bla VIM , bla NDM , or bla KPC have been detected in various countries [8]. These cases of carbapenemase-producing P. aeruginosa are currently localized to a hospital or a country. Therefore, a large-scale survey to detect carbapenemase-producing P. aeruginosa worldwide needs to be further explored.
In this study, we employed genomic analysis to investigate the prevalence and global distribution characteristics of carbapenemase-producing P. aeruginosa isolates from human and we characterized their molecular characteristics, diversity, antibiotic resistance genes (ARGs) and virulence genotypes of these pathogens.

Molecular Characterization of the Carbapenemase-Producing P. aeruginosa
This group of 328 carbapenemase-producing P. aeruginosa isolates possessed 34 distinct ST and six predominated: ST357 (15.24%, n = 50), ST823 (13.72% n = 45), ST233 (7.93%, n = 26), ST308 (7.32%, n = 24), ST111 (6.71%, n = 22) and ST175 (6.40%, n = 21). Interestingly we could not find a matching ST for 66 of the database isolates (Table A4). Minimumspanning trees were constructed using 34 distinct STs of 262 carbapenemase-producing P. aeruginosa isolates, and the results showed that ST111 might be an ancestral isolate and differentiated into a large number of STs among carbapenemase-producing P. aeruginosa isolates (see Appendix Figure A1). We further explored the clonal relatedness of these isolates in which 10 countries possessed carbapenemase-producing P. aeruginosa > 5. The ST types were highly diverse such as for the eleven unique STs in USA, nine for Italy and six for China and Spain, respectively. In contrast, Germany (n = 2), India (n = 1) and Singapore (n = 1) possessed a low level of diverse ( Figure 2). More significantly, we identified for the first time that different carbapenem resistance genes were distinctly distributed Within the group of 165 bla VIM -positive P. aeruginosa in this study, bla VIM-2 accounted for almost 75% of the total, from nine possible variants (73.94%, 122/165). Furthermore, within the 129 members of the bla IMP group, bla IMP-7 represented half of the total (50.39%, 65/129). The bla NDM-1 and bla KPC-2 were the most prevalent in bla NDM -and bla NDM -positive isolates respectively ( Figure 1B).

Molecular Characterization of the Carbapenemase-Producing P. aeruginosa
This group of 328 carbapenemase-producing P. aeruginosa isolates possessed 34 distinct ST and six predominated: ST357 (15.24%, n = 50), ST823 (13.72% n = 45), ST233 (7.93%, n = 26), ST308 (7.32%, n = 24), ST111 (6.71%, n = 22) and ST175 (6.40%, n = 21). Interestingly we could not find a matching ST for 66 of the database isolates (Table A4). Minimumspanning trees were constructed using 34 distinct STs of 262 carbapenemase-producing P. aeruginosa isolates, and the results showed that ST111 might be an ancestral isolate and differentiated into a large number of STs among carbapenemase-producing P. aeruginosa isolates (see Appendix A Figure A1). We further explored the clonal relatedness of these isolates in which 10 countries possessed carbapenemase-producing P. aeruginosa > 5. The ST types were highly diverse such as for the eleven unique STs in USA, nine for Italy and six for China and Spain, respectively. In contrast, Germany (n = 2), India (n = 1) and Singapore (n = 1) possessed a low level of diverse ( Figure 2). More significantly, we identified for the first time that different carbapenem resistance genes were distinctly distributed among the variant STs ( Figure 3). All ST823 isolates and the majority of ST111, ST233 and ST175 isolates carried bla VIM while the majority of ST357 isolates carried bla IMP . Furthermore, ST308 isolates provided a reservoir for the spread of bla VIM , bla IMP and bla NDM ( Figure 3). In addition, there were distinctive geographical distributions for these variant STs. For instance, ST357 and ST823 isolates were primarily from Asia and all ST175 isolates were from Europe. However, ST308, ST233 and ST111 isolates were globally distributed ( Figure 3).   Geographic distribution and MLST diversity of carbapenemase-producing P. aeruginosa isolates. The presence of the carbapenemase-producing P. aeruginosa isolates is indicated by brown; the pie chart represents MLST diversity.

Other ARGs
We also identified the presence of other ARGs from the carbapenemase-producing P. aeruginosa isolates. Almost all isolates carried aph, blaOXA, blaPAO, cat and fosA4 that conferred resistance to aminoglycosides, β-lactams, chloramphenicol and fosfomycin. Interestingly, drfB that confers resistance to sulfonamides was detected in blaVIM-positive P. aeruginosa at a higher prevalence than in blaIMP-positive P. aeruginosa (Appendix Figure A2). Generally, the ARGs types among the different STs varied greatly. In particular, the largest numbers of ARGs types were present in ST111 (n = 23), ST357 (n = 21), ST308 (n = 19), ST233(n = 18), ST175 (n = 14) and ST823 (n = 10) (Appendix Figure A3). The diversity and frequency of aminoglycoside resistance genes carried by ST175 were significantly higher than for the other STs and tetracycline resistance genes carried by ST233 were more nu-

Other ARGs
We also identified the presence of other ARGs from the carbapenemase-producing P. aeruginosa isolates. Almost all isolates carried aph, bla OXA , bla PAO , cat and fosA4 that conferred resistance to aminoglycosides, β-lactams, chloramphenicol and fosfomycin. Interestingly, drfB that confers resistance to sulfonamides was detected in bla VIM -positive P. aeruginosa at a higher prevalence than in bla IMP -positive P. aeruginosa (Appendix A Figure A2). Generally, the ARGs types among the different STs varied greatly. In particular, the largest numbers of ARGs types were present in ST111 (n = 23), ST357 (n = 21), ST308 (n = 19), ST233(n = 18), ST175 (n = 14) and ST823 (n = 10) (Appendix A Figure A3). The diversity and frequency of aminoglycoside resistance genes carried by ST175 were significantly higher than for the other STs and tetracycline resistance genes carried by ST233 were more numerous than for the other STs (Appendix A Figure A3). These data indicate that the six predominant STs for our group of carbapenemase-producing P. aeruginosa isolates possessed shared ARGs and at the same time carried unique ARGs.

Virulence Factor
In our group of carbapenemase-producing P. aeruginosa isolates, we detected the presence of exoY, exoU and exoT that coexisted in ST357 isolates, while exoU and exoT were found together in ST823 and ST308 isolates (Appendix A Figure A4). Interestingly, ST233 isolates possessed the same complement of virulence factors as the ST111 and ST175 isolates (Appendix A Figure A3). These results revealed that the distribution of these cytotoxin genes was not uniform among carbapenemase-producing P. aeruginosa isolates, none of the isolates possessed all the cytotoxin genes.

Discussion
Carbapenems are the most effective antimicrobial agents against serious infections caused by multidrug-resistant Gram-negative bacilli [9]. However, carbapenem-resistant P. aeruginosa are emerging worldwide with increasing reports of carbapenemase-producing isolates, such as China, Singapore, Canada, Germany, Spain, US and so on [8,[10][11][12][13][14]. In this study, 328 carbapenemase-producing P. aeruginosa isolates were collected from public database, and distributed across from 40 countries. Of which, the countries possessing the greatest numbers of carbapenemase-producing P. aeruginosa isolates were Indonesia, India, Italy, China, Germany and Spain, further demonstrate that Asia has been severely contaminated by carbapenemase-producing P. aeruginosa isolates.
Among the carbapenemase-producing P. aeruginosa isolates, the most prevalent were bla VIM , bla IMP , bla NDM and bla KPC . The VIM family contains the most important carbapenemases among P. aeruginosa strains, while bla NDM and bla KPC are the most prevalent for E. coli and K. pneumoniae, respectively [15][16][17]. There is a distinct species preference associated with possession of particular carbapenemase variants. In addition, the high incidence of both bla VIM -and bla IMP -positive P. aeruginosa from Indonesia and Italy compared with other countries. This is consistent with previous research; Indonesia and Italy have been severely contaminated by carbapenemase-producing P. aeruginosa isolates [18,19]. Within the group of 165 bla VIM -positive P. aeruginosa in this study, bla VIM-2 accounted for almost 75% of the total from nine possible variants. Furthermore, within the 129 members of the bla IMP group, bla IMP-7 represented half of the total. Consistent with previous studies, bla IMP-7 and bla VIM-2 were the dominant variants in carbapenemase-producing P. aeruginosa isolates from the Czech Republic and Spain, respectively [6,20].
This group of 328 carbapenemase-producing P. aeruginosa isolates possessed 34 distinct ST and six predominated: ST357, ST823, ST233, ST308, ST111 and ST175. Core-genome analysis showed that ST111 might be an ancestral isolate and differentiated into a large number of STs among carbapenemase-producing P. aeruginosa isolates. This is consistent with previous studies, the international high-risk clone ST111 is the founder of a subgroup from which an important number of STs derive [21]. Interestingly, we identified for the first time that different carbapenem resistance genes were distinctly distributed among the variant STs. All ST823 isolates and the majority of ST111, ST233 and ST175 isolates carried bla VIM while the majority of ST357 isolates carried bla IMP . Furthermore, ST308 isolates provided a reservoir for the spread of bla VIM , bla IMP and bla NDM . In addition, there were distinctive geographical distributions for these variant STs. For instance, ST357 and ST823 isolates were primarily from Asia and all ST175 isolates were from Europe. However, ST308, ST233 and ST111 isolates were globally distributed. Consistent with these results, ST823 isolates that harbored bla VIM-2 have been identified in multiple Asian countries [18,22]. ST233 is an internationally recognized high-risk clone and frequently associated with carbapenemase production as well as exhibiting resistance to all antimicrobial drugs, and is present in at least 12 countries [21,23]. Additionally, 90% of multidrug resistant isolates belonged to only three clones and classified as the major international MDR/XDR high-risk clones: ST175, ST111 and ST235 [24]. In particular, the ST111 high-risk clonal isolates have also been found associated with bla VIM globally distribution and bla VIM -positive ST175 isolates have been detected in Germany and Spain. ST357 is also frequently but not exclusively associated with possession of the carbapenemase IMP gene in Asia [6,23]. The bla NDM , bla VIM and bla IMP positive ST308 isolates have previously identified in Asian countries [25], we found bla VIM -and bla IMP-positive ST308 P. aeruginosa isolates in Europe.
We also identified the presence of other ARGs from the carbapenemase-producing P. aeruginosa isolates. Almost all isolates carried aph, bla OXA , bla PAO , cat and fosA4 that conferred resistance to aminoglycosides, β-lactams, chloramphenicol and fosfomycin [23,26]. It is similar to carbapenemase-producing E. coli isolates: carbapenem resistance genes often co-existed with other antibiotic resistance genes, conferring resistance to multiple antimicrobials [27,28].
The possession of specific virulence genes by a pathogen including P. aeruginosa, is a relevant independent marker of potential disease-causing potential [29]. P. aeruginosa possesses numerous virulence factors and one of the most important is its possession of a type III secretion system (TTSS). This system functions to inject effector cytotoxins encoded by exoS, exoT, exoU and exoY into host cells [24]. In our group of carbapenemase-producing P. aeruginosa isolates, we detected the presence of exoY, exoU and exoT that co-existed in ST357 isolates, while exoU and exoT were found together in ST823 and ST308 isolates. The exoU genotype has been associated with increased early mortality and is a potential prognostic biomarker in P. aeruginosa infections as an indicator of predicated disease severity [24]. In Antibiotics 2021, 10, 548 6 of 13 agreement with this, we found that the ST111 and ST175 high-risk clones possessed exoY, exoT and exoS [6]. Interestingly, ST233 isolates possessed the same complement of virulence factors as the ST111 and ST175 isolates. These results revealed that the distribution of these cytotoxin genes was not uniform among carbapenemase-producing P. aeruginosa isolates, none of the isolates possessed all the cytotoxin genes and possession of exoS and exoU appeared to be mutually exclusive. Our findings are therefore in agreement with previous studies showing that exoT and exoY were present in the vast majority of strains but exoS and exoU were nearly mutually exclusive among our P. aeruginosa clinical isolates [29].
We acknowledge that our study mainly has two limitations. Firstly, the isolates collection was only dependent on the public repository that could not harbor all the P. aeruginosa in the world. Secondly, we could not obtain strains for further research. For instance, the levels of antimicrobial susceptibility and evaluated the transfer of carbapenem resistance genes.

Materials
A total of 5208 assembled genomes and information of P. aeruginosa isolates were downloaded from the NCBI database as of October 2019. Human-derived isolates (n = 2953) were filtered according to detailed information from the P. aeruginosa database (https: //www.ncbi.nlm.nih.gov/pathogens, accessed on 19 October 2019).

Methods
All the P. aeruginosa isolates were applied to a filter for the presence of at least one of the five major acquired carbapenem resistance genes (bla KPC , bla NDM , bla IMP , bla OXA-48-like and bla VIM ) using ABRicate (https://github.com/tseemann/abricate, accessed on 7 January 2020) The bla KPC , bla NDM , bla IMP , bla OXA-48-like and bla VIM were the most broadly spread carbapenemases [30]. Multilocus sequence types (MLST) were identified using Github (https://github.com/tseemann/mlst, accessed on 7 January 2020). P. aeruginosa isolates virulence factors were identified using the Virulence Factor Database (http://www.mgc. ac.cn/VFs/main.htm, accessed on 7 January 2020). Epidemic ST clones (The STs with the higher detection and distributed in the world) for the carbapenemase-producing P. aeruginosa isolates selections were used to analyze associations between STs, geographic location and carbapenem resistance genes. The map was generated using R 3.6.0 using the rworidmap package (https://cran.r-project.org/web/packages/rworldmap/, accessed on 19 July 2020). Minimum-spanning tree of carbapenemase-producing P. aeruginosa isolates based on a core-genome MLST was constructed using PHYLOViZ software of BIGSdb [31,32].

Conclusions
In conclusion, we identified 328 carbapenemase-producing P. aeruginosa human clinical isolates from public databases and the carbapenem resistance genes bla VIM and bla IMP were the most prevalent and present in~50 and 39% of the isolates, respectively. These strains possessed diverse ST types in most countries and eleven unique STs were identified in the USA, nine in Italy and six in China and Spain, respectively. In contrast, Germany (n = 2), India (n = 1) and Singapore (n = 1) possessed a low level of diverse. We found six prevalent STs and comprised distinct groups. For instance, all ST823 isolates and the majority of ST111, ST233 and ST175 isolates carried bla VIM , while the majority of ST357 isolates carried bla IMP . The ST308 isolates provided a reservoir for the spread of bla VIM , bla IMP and bla NDM . The distribution of the genes encoding these cytotoxins was not uniform among carbapenemase-producing P. aeruginosa isolates, none possessed all cytotoxins, and some of them, particularly exoS and exoU, appeared to be mutually exclusive. These results suggest that surveillance studies for carbapenemase-producing P. aeruginosa isolates in humans are urgently needed.

Data Availability Statement:
The data for this manuscript is available from correspondence author.

Conflicts of Interest:
The authors declare no conflict of interest.   Figure A1. Minimum-spanning tree of carbapenemase-producing P. aeruginosa isolates based on a core-genome MLST (cgMLST). Each node within the tree represents a cgMLST type, with diameters scaled to the number of isolates belonging to that type. Figure A1. Minimum-spanning tree of carbapenemase-producing P. aeruginosa isolates based on a core-genome MLST (cgMLST). Each node within the tree represents a cgMLST type, with diameters scaled to the number of isolates belonging to that type. . Figure A3. ARGs present among the six most prevalent STs in carbapenemase-producing P. aeruginosa isolates. The blue represents positive for ARGs in the carbapenemase-producing P. aeruginosa isolates. The red dotted line represents a special value of worth noting Figure A3. ARGs present among the six most prevalent STs in carbapenemase-producing P. aeruginosa isolates. The blue represents positive for ARGs in the carbapenemase-producing P. aeruginosa isolates. The red dotted line represents a special value of worth noting