Mobile Carbapenemase Genes in Pseudomonas aeruginosa

1Pseudomonas aeruginosa is a non-fermentative and aerobic Gram-negative bacillus that is one of the leading causes of severe health care-associated infections targeting immunocompromised patients. The bacterial species is an opportunistic pathogen not only for humans but also for plants and animals. P. aeruginosa is metabolically versatile, and it has an enormous ability for adaptation to different conditions with genome plasticity. There are diverse opinions whether the pangenome of P. aeruginosa is still open or closed to acquire foreign genes. The accessory genome is often composed of genes involved in virulence to human hosts and antimicrobial resistance, resulting in a high risk of mortality and a high rate of multidrug resistance. P. aeruginosa has a median genome size value of 6.7 Mbp, a median number of 6,016 coding sequences, and 66.1% GC on average.

2Although beta-lactams are one of the most commonly used antimicrobial drug classes for P. aeruginosa infection, antipseudomonal beta-lactam drugs are limited because of the species’ intrinsic resistance due to the interplay of chromosomal beta-lactamases, a low outer membrane permeability, and the constitutive expression of efflux pump systems. The beta-lactam regimens for P. aeruginosa infection include antipseudomonal penicillins in combination with a beta-lactamase inhibitor, i.e., piperacillin–tazobactam and ticarcillin–clavulanic acid; antipseudomonal cephalosporins alone or in combination with beta-lactamase inhibitors, i.e., ceftazidime (with avibactam), ceftolozane (with tazobactam), cefoperazone (with sulbactam), and cefepime; and carbapenems, i.e., imipenem and meropenem. Among those, carbapenems are the preferred choice against multidrug-resistant P. aeruginosa. In recent years, the rate of carbapenem resistance in P. aeruginosa has increased worldwide and has become of great concern since it significantly restricts the therapeutic options for patients. Carbapenem resistance in P. aeruginosa is caused by chromosomal substitutions resulting in membrane permeability alterations through porin loss and efflux pump overexpression, together with intrinsic beta-lactamase derepression, and the acquisition of the genes for carbapenemases.

3In this review, we summarized carbapenem resistance in P. aeruginosa. The first half of this review outlines the worldwide epidemiology and the second half discusses the mechanisms of carbapenem resistance and the mobile genetic elements responsible for the horizontal dissemination of resistance determinants. All of the information in the review was collected and analyzed from the National Database of Antibiotic Resistant Organisms1 using the Reference Gene Catalog ver. 2020-09-22.22 for the carbapenemases and the Genome Database (last updated on 22 September 2020)3 for the complete P. aeruginosa genomes.

4Beta-lactams act by binding to and inactivating the penicillin-binding proteins (PBPs), which have an essential role for the completion of peptidoglycan biosynthesis through their dual activity as a transglycosylase and transpeptidase. Among the beta-lactams, carbapenems are the most effective against Gram-positive and Gram-negative bacteria, presenting a broad spectrum of antibacterial activity. Replacing the sulfur atom at the C-1 position of the penicillin backbone by a carbon atom (the red dot of the carbapenem backbone in the box in Figure 1) allows exceptional stability against most enzymes inactivating beta-lactams.

5The early carbapenem ertapenem has a bulky R3 moiety, resulting in a weak affinity to the PBP3 of P. aeruginosa, and consequently, the drug has little activity against P. aeruginosa (Figure 1). Thus, the late carbapenems imipenem and meropenem are actively used for the treatment of infections. Among the three antipseudomonal carbapenems, imipenem has a distinctive stereographic structure and the lowest minimum inhibitory concentrations (MICs) for wild P. aeruginosa strains (Figure 1). Meropenem presents good affinity to the active site of PBP3 (Figure 2). The recently developed combinations of beta-lactam/beta-lactamase inhibitor, such as aztreonam–avibactam, meropenem–vaborbactam, and imipenem–relebactam, have a limited efficacy against the metallo-beta-lactamase (MBL)-producing carbapenem-resistant P. aeruginosa. However, the siderophore cephalosporin cefiderocol is effective against all the carbapenemase-producing P. aeruginosa, including the MBL producers.

6The rate of carbapenem resistance in P. aeruginosa varies worldwide (Figure 3). According to the Antimicrobial Testing Leadership and Surveillance program by Pfizer in 2018 (last updated on September 14, 2020), the rate of resistant clinical strains (-R) of P. aeruginosa by continent was the lowest in Oceania (imipenem-R in 7.1% and meropenem-R in 5.1% of 99 isolates from Australia), and the highest was in the Middle East (imipenem-R in 27.9% and meropenem-R in 19.5% of 226 isolates from four participating countries). In descending order, the median resistance rates were 30.7% in South America (the lowest imipenem-R and meropenem-R both in 12.5% of 24 isolates from the Dominican Republic and the highest imipenem-R in 49.3% and meropenem-R in 75.3% in 75 isolates from Chile, among nine participating countries), 28.0% in Europe (the lowest 0.0% of 19 isolates from Finland and the highest imipenem-R in 48.5% and meropenem-R in 44.8% of 194 isolates from Russia, among 24 participating countries), 24.4% in North America (imipenem-R in 21.4% and meropenem-R in 18.3% of 197 isolates from Canada and imipenem-R in 27.4% and meropenem-R in 15.5% of 588 isolates from the United States), 22.8% in Africa (the lowest imipenem-R in 13.2% and meropenem-R in 15.8% of 38 isolates from Nigeria and the highest imipenem-R in 21.4% and meropenem-R in 19.4% of 98 isolates from South Africa, among three participating countries), and 18.1% in Asia (the lowest imipenem-R and meropenem-R both in 8.0% of 75 isolates from Japan and the highest imipenem-R in 33.2% and meropenem-R in 25.1% of 386 isolates from China, among 10 participating countries).

7The current P. aeruginosa high-risk clones, which meet both requirements of global dominance and association with the multidrug-resistant phenotype, include 10 P. aeruginosa lineages belonging to ST111, ST175, ST233, ST235, ST244, ST277, ST298 (CC445), ST308, ST357, and ST654. The multidrug-resistant P. aeruginosa ST111, ST175, and ST235 have been identified to carry genomic islands. While all the 10 high-risk clones are relevant to MBL production, ST235 and ST111 are by far the most worrisome carbapenemase producers, associated not only with class B but also with class A and D carbapenemases. The widespread P. aeruginosa ST235 clone is often associated with poor clinical outcomes due to its multidrug resistance and virulence factors, representatively the cytotoxin ExoU causing necrotic cell death. The second dominant P. aeruginosa clone is ST111, which has been identified in all six continents except Oceania.

8Pseudomonas aeruginosa can acquire resistance to carbapenems by chromosomal mutations. Loss of the outer membrane protein OprD, which is a channel for imipenem penetration, is associated with a reduced susceptibility to carbapenems, mostly imipenem. Early reports have underlined OprD deficiency as the predominant mechanism of carbapenem resistance in P. aeruginosa. The overexpression of efflux pump systems, such as MexAB-OprM, by mutation at the regulatory region contributes directly to the resistance to meropenem and mutational derepression of the chromosomal cephalosporinase AmpC, especially the extended-spectrum cephalosporinases, and plays a part in carbapenem resistance. The combination of porin loss, efflux pump overexpression, and chromosomal cephalosporinase derepression is able to confer high-level resistance to carbapenems, and P. aeruginosa could have elevated imipenem and meropenem MICs up to 256 and 128 mg/L, respectively.

9Before 1990, the only known mechanism of carbapenem resistance was mutations occurring in the chromosome. Following the first identification of an MBL-producing P. aeruginosa clinical strain, a retrospective screening of P. aeruginosa identified the blaIMP–1 gene in 1992 in Japan. Subsequent outbreaks due to the transferable drug resistance conferred by the gene were reported. The blaVIM–1 gene encoding the Verona integron-encoded MBL (VIM) subtype 1 in P. aeruginosa clinical strain was identified in 1997 in Italy in a P. aeruginosa clinical isolate. And the carbapenem-resistant P. aeruginosa spread rapidly through the contribution of mobile genetic elements and high-risk clones. Thus far, class A, B, and D carbapenemases have been identified in P. aeruginosa, and the class B MBL enzyme is the most prevalent.

10The class A beta-lactamases include serine at amino acid (aa) 70 at the active site and the general base Glu-166 is involved in the catalytic process, which makes a difference from the other serine beta-lactamases of classes C and D. In P. aeruginosa, the Klebsiella pneumoniae carbapenemase (KPC) and the Guiana extended-spectrum beta-lactamase (GES) belonging to the class A beta-lactamases with carbapenemase activity have been identified. The class A carbapenemases actively hydrolyze carbapenems and are partially inhibited by clavulanic acid.

11Klebsiella pneumoniae carbapenemase was first discovered in a K. pneumoniae clinical isolate from North Carolina, United States, in 1996, presenting a specific pattern of resistance to penicillins, extended-spectrum cephalosporins, and aztreonam. The first KPC-producing P. aeruginosa isolate was identified in Colombia in, and subsequent reports of the pathogen followed all over the world, including America, Asia, and Europe (Figure 4).

12Among the GES-type class A beta-lactamases, only the variants with alterations at aa 170 from glycine to asparagine or serine with a polar uncharged side chain are able to hydrolyze carbapenems. Among the 43 GES variants, subtypes 4–6, 14–16, 18, 20, 21, 24, 25, 27–30, 33, 34, 36, 37, 39, 40–42, and 43 with serine at aa 170 and variants 2 and 13 with asparagine at aa 170 have carbapenem-hydrolyzing activity. Of note is that variant 22, having a substitution to leucine with a hydrophobic side chain at aa 170, does not confer resistance to carbapenems. GES-type carbapenemase-producing P. aeruginosa has been identified in North and South America [Canada, Mexico, and Brazil], in Europe [including Belgium, Turkey, Spain, and Russia], in Africa [South Africa; in the Middle East of Lebanon], in Asia [including Japan, India, China, and South Korea], and in Oceania [Australia] (Figure 4).

13The carbapenem-hydrolyzing class A beta-lactamase producers mostly belong to ST235, ST111, ST357, and ST463. The KPC-, and GES-5-producing P. aeruginosa clones have been identified in Europe [Spain, Italy, Lithuania, Turkey, Germany, Belarus, and Russia] and in Asian countries [Kazakhstan, Thailand, China, and South Korea] (Figure 5).

14The class B beta-lactamases are also known as “metallo-” beta-lactamases because they need divalent cations, usually Zn2+ ions, as a metal cofactor to hydrolyze beta-lactams. Although class B beta-lactamases are subclassified as B1, B2, and B3 based on structural and functional points, we will discuss subclass B1, the only dominant subclass in P. aeruginosa. The B1 subclass contains the largest number of clinically relevant acquired MBLs, not only in P. aeruginosa but also in Enterobacterales and other Gram-negative non-fermenters. MBLs bind two Zn2+ atoms for optimal hydrolysis. Zn2+ ion ligands bind at 3H (His–His–His) and DCH (Asp–Cys–His) sites, and the binding of di-Zn2+ plays a critical role in hydrolyzing beta-lactam substrates. Consequently, the carbapenemase activity of MBLs is diminished in the presence of a chelator of Zn2+ and other divalent cations, i.e., ethylenediaminetetraacetic acid (EDTA). The substrate profile of the MBLs includes penicillins, cephalosporins, and carbapenems, but excludes monobactams. The acquired MBL genes, located mostly within a class 1 integron as gene cassettes, have been found in various bacterial species, including P. aeruginosa.

15Metallo-beta-lactamase are the most prevalent type of carbapenemases produced by P. aeruginosa clinical isolates. VIMs are the most disseminated, followed by imipenemases (IMPs). New Delhi MBLs (NDMs) have also been identified. Regional dissemination of P. aeruginosa clinical strains producing Australian imipenemase (AIM), Central Alberta MBL (CAM), Dutch imipenemase (DIM), Florence imipenemase (FIM), German imipenemase (GIM), Hamburg MBL (HMB), São Paulo MBL (SPM), and Seoul imipenemase (SIM) was also reported.

16A VIM-1-producing P. aeruginosa strain was first isolated in Verona, Italy, in. Together with subtype VIM-2, the first two VIM enzymes were originally found in P. aeruginosa as gene cassettes of class 1 integrons. Notably, VIM-2 has a 10-fold more efficient hydrolyzing activity to both imipenem and meropenem compared to VIM-1, and in most of the countries, VIM-2 is the dominant carbapenemase produced by P. aeruginosa. Thus far, a total of 66 variants of VIM have been identified, and subtype 2-producing P. aeruginosa has been extensively spread, with a few regional exceptions by specific outbreaks. VIM-producing P. aeruginosa clinical strains have been identified in almost all countries reporting surveillance data in six continents: in Europe, in Asia, in North and South America, in Africa, and in Oceania (; Figure 4). P. aeruginosa ST111 mostly produces VIM-2 and has been identified in European countries [the Czech Republic, Portugal, Italy, Greece, and Belgium] and in Latin American countries [Colombia]. Less frequently, VIM-producing P. aeruginosa ST244 was identified in China, Brazil, and Africa; VIM-producing P. aeruginosa ST175 was identified in Europe [Spain and Germany].

17After the first IMP enzyme found in Japan in, subsequent interspecies spread of the blaIMP gene to Enterobacterales and Acinetobacter baumannii was reported in Japan and Europe, respectively. Currently, up to 79 variants of IMP have been identified, and varied subtypes have been identified with regional inconsistency. The global prevalent subtype is IMP-1, with a few exceptions of IMP-9 in China and IMP-6 in South Korea. Notably, IMP-6 has better hydrolyzing activity to meropenem than to imipenem compared to the other subtypes of IMP enzymes. IMP is the second most common carbapenemase produced by P. aeruginosa regardless of whether the strain was isolated in Europe, in Asia, in North, and South America, in Africa, and in Oceania (; Figure 4). P. aeruginosa ST357 mostly produces IMP. The clone was spread in Europe and Asia. IMP-producing P. aeruginosa ST621 was identified in Europe [Italy and France] (Figure 5).

18The NDM enzyme was first identified in 2008 in K. pneumoniae isolated from a Swedish patient with a history of travel to India. Shortly after, the NDM producers became endemic in South Asian countries located in the Indian subcontinent, such as India, Pakistan, and Bangladesh. The first report of NDM-1-producing P. aeruginosa was in Serbia in, and subsequent dissemination was observed in the Balkans. NDM-producing P. aeruginosa has been identified mostly in Asia, in Europe, and in Africa. And, as a sporadic emergence, it was also identified in North America (; Figure 4). Among a total of 27 variants of NDM that have been identified, NDM-1 seems to be the most prevalent subtype.

19Being different from the intercontinental dissemination of VIM-, IMP-, and NDM-producing P. aeruginosa, other MBL-producing P. aeruginosa outbreaks or regional spreading have occurred, except for SPM-producing P. aeruginosa. SPM-producing P. aeruginosa was first identified in São Paulo, Brazil, and the clone belonged to ST277. The SPM producer was subsequently identified in Switzerland, the United Kingdom, India, China, and Uganda (; Figure 4). DIM-producing P. aeruginosa clinical strains were regionally disseminated mostly in Asia and Europe. GIM-producing P. aeruginosa was identified in Germany and India. SIM-producing P. aeruginosa was identified in China. HMB-producing P. aeruginosa was identified both in Hamburg, Germany, and in the United States. CAM-producing P. aeruginosa was identified in Canada, and no more dissemination was observed. The AIM and FIM producers were restricted in Australia and Italy, respectively (Figure 4).

20Class D beta-lactamases belong to the superfamily of serine beta-lactamases with a unique carboxylated Lys-73 responsible for the beta-lactam hydrolysis activity. The carbapenem-hydrolyzing class D beta-lactamases (CHDLs) were first described in A. baumannii and published by. CHDLs are serine beta-lactamases with a relatively weak activity against carbapenems and are poorly inhibited by EDTA or clavulanic acid. Among a total of 12 groups of CHDLs, three groups—OXA-40-like, OXA-48-like, and OXA-198-like—have been identified in P. aeruginosa.

21OXA-type carbapenemases are rarely identified in P. aeruginosa, and the emergence of the following strains has been reported: OXA-40-producing P. aeruginosa in Spain, OXA-48-producing P. aeruginosa in India, OXA-181-producing P. aeruginosa in the United Kingdom, and OXA-198-producing P. aeruginosa in Belgium (; Figure 4).

22The acquired genes encoding carbapenemases are associated with a plethora of mobile genetic elements, such as plasmids, gene cassettes of integrons, transposons, and genomic islands. Mobile genetic elements have the ability to move from genome to genome by transformation, conjugation, and transduction, presenting intracellular and intercellular mobility.

23In general, the plasmids carrying the carbapenemase-encoding genes in P. aeruginosa belong to the distinct incompatibility groups from those in Enterobacterales. Among the 13 known incompatibility types of IncP, IncP-2-type plasmids are classic types frequently identified in P. aeruginosa. Among the 207 complete genomes of P. aeruginosa of the Genome Database, five genomes include a plasmid carrying one or two carbapenemase-encoding genes; two of the five plasmids are of the IncP-2 incompatibility type, while the other three are untypable: two blaKPC–2 genes are harbored by untypable plasmids, which are almost the same (identical nucleotide sequences, except for a 1-bp gap difference between the 57,053- and 57,052-bp plasmids), one blaVIM–1 is harbored by an untypable plasmid, one IncP-2 plasmid harbors blaIMP–45, and one IncP-2 plasmid harbors both the blaVIM–1 and the blaIMP–45 genes.

24Among the plasmids in P. aeruginosa having an incomplete genome, the 31,529- and 38,939-bp IncP-6 plasmids carrying the blaKPC–2 gene have been identified in Colombia and China, respectively, and the 7,995-bp IncU plasmid including the blaKPC–2 gene has been identified in Colombia. An untypable 3,652-bp plasmid harboring the blaKPC–2 gene was identified in Brazil.

25Recently, a Pseudomonas plasmid lineage carrying the MBL genes has been reported. A retrospective analysis revealed that the plasmid lineage has been identified since the 1990s, mostly in Europe. While the plasmid does not belong to a recognized plasmid type, the type 4 secretion system components classified the plasmids as MOBF11 or MPFT plasmid families. The MBL genes in the plasmid were identified as gene cassettes of the class 1 integron In70.

26The carbapenemase-encoding genes are frequently included in transposable elements, which are often associated with the insertion sequences with a common region (ISCRs) being responsible for the rapid transmission of bacterial multidrug resistance (Figure 6; ). Typically, the ISCR element lacks flanking inverted repeats (IRs) and the integration does not produce direct repeat (DR) sequences. Rolling circle has been suggested for the transmission mechanism of ISCRs (Figure 7), and the transposition method allows a polarized transfer of the ISCR elements to mobilize adjacent DNA sequences in varied sizes.

27The blaCAM and blaFIM genes were identified in transposable elements associated with ISCR14 and ISCR19, respectively, located downstream from the gene. The blaSPM gene was identified in P. aeruginosa accompanied with the chaperone groEL gene and flanked by a pair of ISCR4 elements. The blaAIM gene was identified in a transposable element associated with the upstream ISCR15. The blaHMB–1 gene was in a transposable unit, namely, Tn6345 (Supplementary Table 1; ), flanked by IS1595 upstream and ISCR27-like downstream. For the blaNDM gene, the composite transposon Tn125 flanked by a pair of ISAba125 copies at both ends has been identified in A. baumannii. The blaNDM–1 gene-associated transposable element is composed of a truncated Tn125. ISCR is located upstream from the ΔISAba1, and downstream of the blaNDM–1 gene, the ble gene for bleomycin resistance and the trpF gene for phosphoribosylanthranilate isomerase are followed.

28The Tn4401 carrying the blaKPC gene has been found in diverse bacterial hosts, including P. aeruginosa (; Figure 6). Tn4401 is a 10-kb transposon composed of genes encoding a transposase and a resolvase, and the blaKPC gene together with two insertion sequences (ISs), ISKpn6 and ISKpn7 (Figure 6), and transposition of Tn4401 occurs through the mechanism of copy-and-paste replicative transposition (Figure 7; ). Tn4401 includes nine isoforms from a to I differing in the sequences upstream of the blaKPC gene. While both Tn4401a and Tn4401b are prevalent in Enterobacterales, Tn4401b is the only isotype identified in P. aeruginosa. Seven Tn4401 elements were found through the restricted BLAST against the species P. aeruginosa, and all were Tn4401b: two Tn4401b copies in a chromosome (GenBank accession CP029605) and each Tn4401b copy in five plasmids (GenBank accessions MN082782.1, CP027168.1, CP029092.1, KC609323.1, and EU176013.1).

29Integrons are assembly platforms comprising an intI gene for a site-specific tyrosine recombinase, an attI for a primary recombination site, the promoter Pc for transcription, and an assemblage of passenger genes composing a gene cassette array (Figure 8; ). The IntI integrase recognizes the attC site of the gene cassette and the promoterless gene cassette is inserted as a linear form in the integron (Figure 8; ). In addition to the catalysis of attC × attC, the integrase catalyzes attI × attC, leading to the gene cassette integration into the attI site. A successive integration of the gene cassettes occurs downstream of the resident Pc promoter. The expression of the gene cassette is dependent on the sole promoter and the level of gene expression depends on the distance from the sole promoter Pc. By nature, the first few cassettes are expressed and the rest of the array exists as a reservoir of standing genetic variation. Rearrangement of the order of gene cassettes affects the resistance phenotype of the bacterial host.

30The class A carbapenemase-encoding gene blaGES, the MBL genes blaVIM, blaIMP, and blaGIM–1, and the class D gene blaOXA–198 were identified as a gene cassette composing a class 1 integron. In the integron database INTEGRALL (4 last updated on 10 December 2020), a total of 812 class 1 integrons of 282 different gene cassette arrays were identified for the organism P. aeruginosa. Among them, 191 class 1 integrons of 148 different arrays carried one or two carbapenemase-encoding gene cassettes (Supplementary Table 2). The most prevalent blaVIM genes were identified as gene cassettes in 98 arrays of the 130 class 1 integrons. Among them, 13 carried only the blaVIM cassette; the others harbored additional gene cassettes encoding aminoglycoside-modifying enzymes, mostly the aacA genes and less frequently the aadA or aadB genes. All but five carried the blaVIM gene cassette in the first position of the array (62.3%) or in the second position of the array (33.1%).

31The second most dominant blaIMP cassettes were identified in 50 class 1 integrons of 40 different arrays, and 14 of the integrons harbored a single gene cassette. All but two harbored the blaIMP gene cassette in the first of the array (70.0%) or in the second of the array (22.9%). Less frequently, the blaGIM–1 gene cassette was identified in six class 1 integrons of five different arrays all in the first of the array, and three blaGES–5 cassette-associated and one of each of the blaGES–8, blaGES–9, and blaGES–15 cassette-associated class 1 integrons were identified.

32Basically, class 1 integrons are immobile per se, and a set of functional transposition modules is needed for transposition. The Tn402 family transposon Tn5090 is a good example of a functional transposon giving mobility to a class 1 integron (Figure 9; ). The Tn402 family transposon is a Mu-related transposon that has been identified in the broad-host IncP plasmid R751 conferring trimethoprim resistance. The transposon family has two modules: a transposition module composed of the transposase TniA, the ATP-binding protein TniB, the transposition auxiliary protein TniQ-resolution site res, the serine resolvase TniC, and a class 1 integron carrying antimicrobial resistance gene cassettes. Transposition of the Tn402 family transposon involves a TniABQ-dependent cointegrate formation and a site-specific serine resolvase-dependent resolution. The transposon has 25-bp inverted repeats, and the integration generates 5-bp DRs. The transposon targets the res site of Tn21 subfamily transposons as well as resolution sites found on plasmids. The Tn21-like Tn1403, Tn6060, Tn6162, and Tn6249, which nest inside the Tn402-like, carry a class 1 integron possessing the blaVIM–1 gene cassette.

33In the transposon repository database, a total of 20 transposons carried the gene encoding carbapenemases (Supplementary Table 1), and of those, 19 transposons are associated with class 1 integrons harboring the blaVIM (n = 9), blaIMP (n = 6), blaGES (n = 2), blaSIM (n = 1), and blaDIM (n = 1) gene cassettes. The 207 complete P. aeruginosa genomes from the Genome Database included a total of 29 chromosomal class 1 integrons with a carbapenemase-encoding gene cassette. Among those, 20 integrons included a part or the entire set of the TniABQR transposition module of the Tn402-like.

34Genomic islands carrying many foreign genes are useful for adaptation by providing multiple fitness-associated elements to P. aeruginosa in a single event of horizontal gene transfer. Genomic islands are often free to move in and out of the chromosome, and pKLC102, a 100-kb plasmid identified in P. aeruginosa C strain, is an example of this. The plasmid pKLC102 was found simultaneously in the chromosome as a genomic island being integrated into the 3′ end of the tRNALys gene in favor of an att site.

35Genomic islands are typically inserted at the 3′ end of a transfer RNA (tRNA) gene; however, they are also targeted elsewhere in the chromosome. Genomic islands are easily differentiated from the core genome by their atypical G+C contents, differing from the typical P. aeruginosa G+C content of 65–67% and atypical oligonucleotide usage. Genomic islands typically harbor the genes for factors involved in mobility, such as integrases, transposases, ISs, and other components responsible for biological processes. Genomic islands are categorized accordingly to their main characteristics determined by the gene content, such as pathogenicity, symbiosis, metabolic, fitness, or drug resistance. Thus far, 25 genomic islands have been identified: two P. aeruginosa pathogenic islands, PAPI-1 and PAPI-2; 17 P. aeruginosa genomic islands, PAGI-1 to PAGI-17; five Liverpool epidemic strain genomic islands, LESGI-1 to LESGI-5; and a plasmid-origin genomic island, pKLC102. As an effort to group the genomic islands, suggested two families of genomic islands by conserved function and synteny of the backbone genes, and proposed that any known genomic islands originated from an ancestry based on conserved orthologs. However, no scheme is publicly endorsed.

36Genomic islands carrying the antimicrobial resistance genes, so-called resistance islands, specifically provide a great advantage for survival to bacterial hosts in clinical settings. Among the 25 genomic islands, only three—PAGI-13 in P. aeruginosa ST277 and PAGI-15 in ST244 and PAGI-16 in ST235, respectively—were resistance islands carrying antimicrobial resistance determinants, and only two, PAGI-15 (blaGES–24) and PAGI-16 (blaIMP–6, or blaIMP–10), harbored a carbapenemase-encoding gene (Table 1; ). One unnamed PAGI-2-like island in P. aeruginosa ST235 carrying the blaGES–5 gene has also been reported.

37In addition, the PA143/97 genomic island, which was manufactured to include the Tn6249 carrying two class 1 integrons harboring the blaVIM–2 gene cassette, was reported.

38Pseudomonas aeruginosa is able to obtain multidrug resistance at once through resistance islands. Since this review focuses on carbapenem resistance in P. aeruginosa, the exploration is restricted to genomic islands harboring carbapenemase-encoding genes.

39Among the 207 complete genomes of P. aeruginosa extracted from the Genome Database, a total of 38 chromosomes harbor resistance islands carrying the carbapenemase-encoding gene, and eight of those carry two resistance islands. In total, 45 resistance islands, sized between 8,858 and 117,103 bp, were analyzed. The G+C contents varied from 55.5 to 65.5%, which were lower than those of the chromosomes, which were from 65 to 67%, and the integration sites varied.

40The 45 resistance islands carrying the carbapenemase-encoding gene were classified into three groups by the transposition module (Table 2). Group 1 resistance islands furnish the tyrosine-based integrase gene at the 5’ terminal and the conjugative transfer machinery gene cluster at the 3′ terminus (Figure 10). Group 1 can be further grouped into two subgroups: group 1a, with a core structure of ICETn4371 mostly targeting OprD of OccD4/OpdT tyrosine or adenylate cyclase ExoY, and group 1b, which resembles the known multidrug-resistant genomic islands PAGI-13, PAGI-15, and PAGI-16 targeting tRNAGly. Resistance islands belonging to group 1a had G+C contents between 64.5 and 65.1%, and the size was 44–74 kbp. The blaSPM–1 and blaNDM–1 genes carried by the group 1a resistance islands were all associated with IS91-like ISCRs composing unit transposons, and neither the other antimicrobial resistance determinants nor the heavy metal resistance-associated genes were identified in the resistance island. Meanwhile, the G+C contents of group 1b ranged between 60.5 and 64.2%, and the size ranged from 37 to 117 kbp. The upper cluster of group 1b, including four genomic islands (Table 2), targeted tRNAGly at the locus PA0714 of the genome of P. aeruginosa PAO1 (NCBI RefSeq, NC_002516.2). Characteristically, the genomic islands belonging to the cluster always possess the mercury resistance gene cluster, which is likely derived from Tn501, and the carbapenemase-encoding genes are carried by class 1 integrons as a gene cassette. The eight genomic islands belonging to the next cluster of subgroup 1b targeted tRNAGly either at the locus PA2583 or PA2817 of the PAO1 genome. A quarter of the genomic islands possess a class 1 integron, and the acquisition of the carbapenemase-encoding gene is mostly due to ISCR elements. Pieces of the heavy metal gene cluster have been observed.

41The group 2 resistance islands contain a transposition module composed of a whole or a partial TniABQR component. The resistance islands had obviously lower G+C contents, between 55.5 and 62.0%, and the sizes were diverse, from 8.8 to 105 kbp. The composition of the resistance islands in group 2 corresponds to that of the Proteus genomic island PGI-1. The resistance islands are constructed through the accumulated assemblage on a Tn402 backbone. Such an assemblage of transposons is commonly observed in the AbaR-type resistance islands in A. baumannii, which evolved from the Tn6019 backbone.

42In the case of the remaining two resistance islands, categorization is unavailable since only two cases of possible clonal relations are available from the GenBank database. Neither a specific tyrosine-based recombinase nor a transposition component has been identified in the resistance islands; however, they are flanked by 20-bp inverted repeats.

43Carbapenems represent a valuable therapeutic option for patients with infections caused by multidrug-resistant P. aeruginosa. It is ironic that carbapenem resistance, especially that conferred by carbapenemase production, is closely related to multidrug resistance, highlighting the role of modular mobile units carrying multiple antimicrobial resistance determinants. The molecular epidemiology of antimicrobial resistance has been studied by traditional methodologies based on PCR and Sanger sequencing to identify the resistance genes and to distinguish the fundamental mobile genetic elements carrying the gene, i.e., gene cassettes of integrons, transposons, and plasmids. The present era of massive next-generation sequencing, mostly the long-read sequencing, allows resolving a wide range of complex genome regions, such as modular mobile units associated with genes for antimicrobial resistance, also known as resistance islands. Such an extensive analysis has been carried out for limited numbers of the P. aeruginosa genome mostly for the genomic islands unrelated to antimicrobial resistance determinants. Among the plenty genomic islands in the genome of P. aeruginosa, resistance islands have a meaning beyond genome plasticity. Such a modular mobile unit harboring antimicrobial resistance determinants is able to disseminate by itself and capture an alien gene for resistance, which means the resistance islands and high-risk clones are the A to Z of acquisition of multidrug resistance. Our trial to classify P. aeruginosa resistance islands needs improvement with more cases for resistance islands.

44The global spread of the carbapenem-resistant P. aeruginosa is one of the major global public health challenges, and the epidemiological scenario is often associated with the circulation of carbapenemase-encoding genes linked with (i) the endemic carbapenemase gene and (ii) the carbapenem usage in clinical settings. The KPC-producing P. aeruginosa in KPC-endemic United States, the SPM-producing P. aeruginosa in SPM-endemic Brazil, and the NDM-producing P. aeruginosa in NDM-endemic India exemplify well the first linkage. The second linkage is illustrated through the dominance of IMP-6-producing P. aeruginosa in South Korea, in which meropenem usage is approximately twice more in clinical settings than that of imipenem. Since IMP-6 has greater hydrolyzing activity to meropenem than to imipenem compared to the other subtypes of IMP enzymes, producing the IMP-6 subtype is favorable to the bacterial host. It emphasizes that the carbapenemase-producing organisms should be controlled regardless of the bacterial host, and control includes both surveillance study and antimicrobial stewardship. Needless to say, more attention is needed to be paid to the emergence and spread of the high-risk P. aeruginosa clones, together with their enzymatic/non-enzymatic carbapenem resistance. Though continuing efforts are being made to develop the beta-lactamase inhibitors in order to preserve the efficacy of beta-lactam drugs including carbapenems, it is fruitful just for the serine beta-lactamases. Development of the inhibitors for MBLs, which are frequently produced by P. aeruginosa, is eager to be accelerated.

45Despite the efforts to control the spread of carbapenem-resistant P. aeruginosa, a conclusive solution to the issue is still far from being accomplished. Surveillance study for the drug-resistant pathogen is essential and global collaboration using harmonized methods is important for a practical comparison of the outputs. In addition, to fight against the drug-resistant pathogen, we need to understand how the pathogens acquire resistance determinants. Taking the advantages of up-to-date techniques, assessment of the bacterial genome should be carried out, not only for the mobile genetic elements carrying a carbapenemase-encoding gene but also for the genomic islands. Furthermore, through the technique, the mobile genetic elements should be investigated extensively, and it would allow a comprehensive grasp of the dissemination of drug resistance.

46SJ conceived and supervised the study, performed the data evaluation and confirmation, and finalized and edited the manuscript. E-JY carried out the analysis and data validation, and drafted the manuscript. Both authors contributed to the article and approved the submitted version.

47The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.