Diversity and Distribution of Resistance Markers in Pseudomonas aeruginosa International High-Risk Clones

Pseudomonas aeruginosa high-risk clones are disseminated worldwide and they are common causative agents of hospital-acquired infections. In this review, we will summarize available data of high-risk P. aeruginosa clones from confirmed outbreaks and based on whole-genome sequence data. Common feature of high-risk clones is the production of beta-lactamases and among metallo-beta-lactamases NDM, VIM and IMP types are widely disseminated in different sequence types (STs), by contrast FIM type has been reported in ST235 in Italy, whereas GIM type in ST111 in Germany. In the case of ST277, it is most frequently detected in Brazil and it carries a resistome linked to blaSPM. Colistin resistance develops among P. aeruginosa clones in a lesser extent compared to other resistance mechanisms, as ST235 strains remain mainly susceptible to colistin however, some reports described mcr positive P. aeurigonsa ST235. Transferable quinolone resistance determinants are detected in P. aeruginosa high-risk clones and aac(6′)-Ib-cr variant is the most frequently reported as this determinant is incorporated in integrons. Additionally, qnrVC1 was recently detected in ST773 in Hungary and in ST175 in Spain. Continuous monitoring and surveillance programs are mandatory to track high-risk clones and to analyze emergence of novel clones as well as novel resistance determinants.


Introduction
Pseudomonas aeruginosa is a Gram-negative ubiquitous rod-shaped bacterium and one of the most common opportunistic human pathogens with the ability of causing a wide range of severe infections, including ventilator-associated pneumonia, otitis externa, burn wound infection, contact lense-related keratitis, and bloodstream infection [1][2][3][4]. Various antibiotic resistance mechanisms can occur in P. aeruginosa that enable it to develop multiresistance, therefore, it can cause difficult to treat infections. P. aeruginosa is a member of the "ESKAPE" group, and this group of pathogens comprises 6 multidrug resistant bacteria that are common nosocomial pathogens [5]. Moreover, infections caused by ESKAPE pathogens are usually associated with significantly high morbidity and mortality rates as limited number of effective antimicrobial agents are available against these pathogens [6][7][8][9].
Recently, the World Health Organization released a priority list for development of novel antibiotics against certain pathogens and carbapenem resistant P. aerigunosa is listed among 'critical' category pathogens, as research and development of antibacterial agents are urgently needed [10]. The need for novel antibiotics against P. aeruginosa is seen worldwide, as both intrinsic and acquired resistance mechanisms are detected in this pathogen [7,11,12].
P. aeruginosa exhibits intrinsic resistance to a variety of antibacterial agents due to its unusually decreased outer-membrane permeability as well as by upregulation of efflux pumps [13][14][15]. Furthermore, acquired resistance to beta-lactams and aminoglycosides can develop through acquisition of resistance genes [7,15,16]. The most common resistance to beta-lactams occurs through production of different beta-lactamases, namely AmpC isolated from a patient with acute pyelonephritis in France in 2012, and from a stem cell transplant recipient in Italy in 2013. Both patients were hospitalized previously in Serbia [11,60]. Subsequently, an another bla NDM-1 positive strain was reported in Vietnam in 2016 [52]. Later, in 2019, a bla NDM-1 positive P. aeruginosa was isolated in Italy from a patient who developed sepsis as a consequence of urinary tract infection caused by P. aeruginosa ST235. This strain carried different virulence determinants, including ExoU phospholipase that is an effector of type III secretion system and it is responsible for cytotoxic activity [61]. In P. aeuginosa ST235, a bla FIM-1 MBL gene was described in 2012 in Florence, Italy. FIM-1 beta-lactamase showed approximately 40% identity of amino acid sequence of NDMtype beta-lactamases. The bla FIM-1 was located on chromosome of P. aeuriginosa ST235 and flanked by ISCR19, that explains its insertion into the chromosome. However, mobilization and dissemination of this determinant is unknown. It seems that bla FIM is endemic in Florence, as no distribution in other geographical regions has been recorded, yet [42].
In most cases, aac(6')-Ib aminoglycoside acetyltransferase was reported in ST235 and it is responsible for resistance to kanamycin, tobramycin, and amikacin [12,51]. Strains of ST235 in Europe usually contained aacA7 that encodes aminoglycoside 6'-N-acetyltransferase and some of them carried aph(6)-Id aminoglycoside O-phosphotransferase or aadA1 adenylyltransferase. In case of plasmid-mediated quinolone resistance determinants, aac(6 )-Ib-cr was first described in P.aeruginosa ST235 in isolates from Serbia and Hungary [33].
Chloramphenicol resistance genes, cmlA7 and catB7 were also described in Hungary, Serbia, and Nigeria [33,52]. In a Nigerian sample, other resistance determinants were also confirmed, fosA for fosfomycin, tetA, tetG for tetracycline, and qnrVC1 for fluoroquinolone resistance [52]. Compared to each other, strains of ST235 were characterized by quite similar resistance patterns however, they showed diversity based on the produced enzyme types and in the linkage to their geographic distribution.
A comprehensive study conducted in Madrid, revealed that MDR P. aeuginosa ST235 causes high mortality rate in patients in invasive infections, especially in patients with chronic respiratory tract infections [62].

ST111
This international successful clone is also responsible for numerous MDR P. aeruginosa cases around the world [63]. Reports from various European countries described ST111 in Portugal, Spain, Greece, France, Croatia, [12,64] Czech Republic [65], Sweden, and Norway [47]. (Figure 1) MDR ST111 isolates from Bulgarian hospitals showed a combination of ESBL production (VEB-1 and OXAs) with the lack of OprD carbapenem porin and overexpression of MexXY-OprM efflux pump resulting carbapenem resistance without harboring any encoded MBLs [66]. In The Netherlands, a nationwide emergence of this sequence type was also detected in 2012 [67]. The first full genome sequence of ST111 P. aeruginosa was described in the United Kingdom in 2014 and was deposited in the European Nucleotide Archive with accession number PRJEB4573 [63] (Table 1). ST111 strains were reported from Colombia [75], India, Venezuela, Canada, and United States [12]. In Columbia, a report detailed detection of co-carriage of bla VIM-2 and bla KPC-2 in a single P. aeruginosa strain that belonged to ST111. This strain was resistant to amikacin, ciprofloxacin, polymyxin, and to all beta-lactams, including carbapenems and aztreonam [75]. In 2015, almost 90 isolates of P. aeruginosa ST111 were analyzed in the United Kingdom. Most tested strains carried bla VIM-2 , a few had bla IMP-1 or bla IMP-13 , and one presented coexistence of bla VIM-2 and bla IMP-18 , whereas another strain presented bla NDM-1 [68].
GIM type metallo-beta-lactamase was reported as an integron-encoded gene [76]. This MBL type shares 40% amino acid sequence similarity to IMP type MBLs. So far, bla GIM was detected only in P. aeruginosa ST111, and seems endemic in Germany [77].

ST175
This epidemic high-risk clone was first identified in 2005 in the United Kingdom and Canada. Since then, it was reported in Hungary, the Czech Republic, Poland, the United States, China, Spain, and France [69,78]. (Figure 1) In case of ST175, P. aeruginosa infections the predominantly detected resistance mechanisms are associated to specific mutations in chromosomally encoded versatile resistome including oprD for inhibition of carbapenems, ampR for AmpC overexpression, mexZ efflux pumps, gyrA and parC as fluoroquinolone resistance markers [7,70,79]. Furthermore, presence of bla CARB-12 , catB7, bla IMP-1 , aacA4 -3 in Japan [50], just as bla VIM-2 , bla IMP-22 , aac(6)-Ib in Spain were also confirmed associated to class I integron [78]. In a study conducted in Spain, a VIM-47 producing P. aerugonosa ST175 strain was reported [59].
In 2019, an MDR P. aeruginosa ST175 was identified in Spain. It carried bla VIM-20 and bla OXA-681 in class I integron on a plasmid. OXA-681 is a variant of OXA-2, and specific amino acid differences at position 159 and 160 distinguish the two beta-lactamases. OXA-681 is able to hydrolyze cephalosporins, including ceftolozane and induces cross-resistance to ceftolozane-tazobactam and ceftazidime-avibactam. Thereby it may cause serious clinical problems, because new combinations with antipseudomonal effect like ceftolozanetazobactam and ceftazidime-avibactam are the most effective and recommended options for therapy of MDR P. aeruginosa infections. On the other hand, bla OXA-681 is located on a plasmid in a class I integron thus, it enables this resistance determinant for further dissemination. Gene accession numbers are available: ST175 genome sequence SRX5389644, bla OXA-681 MH986647.1 [69] (Table 1).

ST233
Initially, ST233 was reported as a high-risk clone in Mexico, in the United States, in Japan, and in South Africa [81][82][83] (Figure 1). Earlier, this clone was described as colistin susceptible but in case of an extensively drug-resistant (XDR) strain resistance to colistin in 2013 was reported [81]. The first whole genome sequence of this clone was reported in Germany. The detected resistance genes sul1, tet(G), bla VIM-2 , bla OXA-4 , bla OXA-50 , bla PAO , aac(3)-Id, aadA2, aph(3 )-IIb, dfrB5, fosA, catB7 were all located on bacterial chromosome. Assigned accession numbers CP017293 and CP017294 are added to GenBank (Table 1). In this study, the ST233 strains was exposed to colistin and colistin resistant strains were successfully selected through developing mutations in active genes contributing in lipopolysaccharide synthesis [71].
Further studies demonstrated the presence of bla IMP-1 in Singapore [84], the copresence of bla VIM-2 , bla OXA-4 with chromosomal mutations in gyrA and parC in Germany, France, Romania, US, and India [12]. Along with ST235 and ST111, bla NDM-1 was also detected in ST233 in tertiary care hospitals in Egypt in 2015 [85].
An interesting aspect of this clone is the confirmed case of reverse zoonotic effect in Brazil, called as zoo anthroponotic transmission. A dog was suffering from severe ear infection and by laboratory tests carbapenem-resistant ST233 P. aeruginosa was identified from this infected ear, oral, and rectal swab and later from the pet owner's feces and also on the household settings. Interestingly, the pet owner, a 50-year-old man had 1-month long hospital stay in the ICU and after the successful recovery he was released from the hospital a month before the onset of the dog's symptoms. This strain of ST233 carried chromosomally encoded beta-lactamases, namely bla VIM-2 , bla PAO , bla OXA-4 , bla OXA-50 , and other different resistance determinants such as aadA2, aac(3)-Id, aph(3)-IIb), sul-1, tet(G), fosA, catB7, and cmlA1. These findings made attention for the colonization possibility in the household environments and further transmission to pets of hospital-acquired human pathogens, such as MDR P. aeruginosa [86].
Interestingly, scarce reports detail ST277 out of Brazil however, in the UK and Japan, ST277 has been already described. In Japan, this clone was identified with quite different genomic properties, it carried bla IMP-1 , bla IMP-10 , aacA1 genes in urine samples [50]. In 2016, a SPM-1 producing P. aeruginosa ST277 that was only susceptible to colistin was reported in the UK from a patient who was hospitalized earlier in Brazil [88].
According to a study, which performed a phylogenetic analysis of almost fifty ST277 strains, the Brazilian isolates have a big number of single nucleotide polymorphisms and distinctively bear genomic islands (In163 and PAGI-25) compared to other ST277 genomes described in Asia, Europe, or North America. The presence of these special elements could explain the widespread dissemination of this endemic clone in Brazil [89].
Additionally, P. aeruginosa ST277 carried a 763,863 bp chromosomally coded unique region comprising two new genomic islands (PAGI-13 and PAGI-14), a mobile element, an integrative and conjugative element (ICE) associated to bla SPM-1 . Furthermore, rmtD and In163 are inserted in PAGI-13 while PAGI-14 has genes encoding proteins related to type III restriction system and phages [87].
Strains of ST277 were isolated from microbiota of migratory birds in Brazil, suggesting that these birds might have played an important role in the widespread dissemination in this territory [38,90]. In this geographical region, the most commonly detected carbapenem resistance mechanism is bla SPM-1 (São Paulo metallo-β-lactamase) in clinical samples of MDR P. aeruginosa. Migratory bird-associated ST277 strains carried also bla SPM , and these strains were susceptible only to colistin and contained aac(6 )-Ib-cr, rmtD1, aacA4 [90]. Furthermore, P. aeruginosa ST277 strains were also recovered from environmental water samples in Brazil [91].
ST654 was also reported as a bla IMP carrier, and bla IMP-1 and bla IMP-26 were reported from Singapore [84]. In Sweden, bla VIM-2 positive ST654 was reported in a patient who was hospitalized earlier in Tunisia [47]. Recently, VIM-producing P. aeruginosa ST654 strains were reported from a tertiary and quaternary hospital in Saudi Arabia [95]. ST654 was reported as bla KPC-2 positive in Argentina [96]. Furthermore, bla NDM-1 carriage was also reported in the UK [19].
However, recently, bla NDM-1 was also confirmed in ST773. This strain developed resistance to tobramycin, ciprofloxacin, and levofloxacin, and showed susceptibility to colistin. This strain of ST773 carried qnrVC1 fluoroquinolone resistance marker localized on class I integron that was originated from Vibrio cholerae. Besides this resistance marker, that ST773 strain carried several other genes, such as bla OXA-50-like , bla PAO , rmtB, aph(3 )-Iib, tetG, fosA, catB7, and sul1. Whole genome sequence of this strain was deposited in GenBank under the accession number RHDU00000000 [20]. A bla NDM-1 positive ST773 strain was also reported in USA from a patient who received medical care earlier in India due to her intraabdominal surgery. Whole genome sequence data are deposited at BioSample: SAMN12307670 [74] (Table 1).

Discussion
Emergence and dissemination of P. aeruginosa international high-risk clones are great challenge worldwide [54,81,98]. MDR and XDR strains of high-risk P. aeruginosa are frequently identified in nosocomial infections and these cause difficult to treat infections as limited treatment options are available [60]. Furthermore, genome of P. aeurginosa has its complexitiy and high variability, therefore various resistance genes can be acquired from non-fermentative bacteria or even from different strains of Enterobacterales [74].
The most commonly detected P. aeruginosa clone is ST235 with a wide variety of betalactamases, including the most frequent bla VIM-2 , bla OXA-2 , bla KPC-2 , bla PAO , and bla NDM-1 . Moreover, bla FIM-1 was also reported in ST235 in Italy, however, this is the only detection of bla FIM so far [42]. A few years ago, bla NDM-1 was the dominant MBL described in ST235, [11,52,60,61] but it has disseminated with an increasing tendency, since it was also detected in ST111 [68], ST233 [85], ST654 [19], and ST773 [20,74] (Table 2). Beside the most frequently isolated ST235, other sequence types have also important features. In ST175, OXA-681 was reported, which hydrolyzes ceftolozane and induces cross-resistance to ceftolozane-tazobactam and ceftazidime-avibactam. Possible transfer of bla OXA-681 contributes to diverse resistance pattern of P. aeruginsa and adds to the disquieting challenges related to antibiotic resistance [69].
ST235 and ST111 strains show mainly positivity for different ESBL production (bla PES-1 , bla BEL-1 , bla GES , bla PER ), these genes are associated to bla OXA variants and in certain cases to distinctive types of bla VIM or bla IMP . Further resistance mechanisms against aminoglycosides, fluoroquinolones (mainly mutations in gyrA and parC), fosfomycin, tetracycline, and sulfonamides were also detected [12]. Recently, a study conducted in Greece reported predominance of VIM producing ST111 amd ST235. Interstingly, bla VIM genes were incorporated into three integron types, namely, In59, In595, and In1760 [99]. The analyzed high-risk clones are worldwide disseminated, but as an exception, the ST277 is detected mostly in Brazil and shows a relevant spreading tendency in that geographical area [27,87]. Strains of ST277 carry distinct resistomes that are linked to bla SPM-1 and interestingly these strains are particularly colistin-sensitive. According to a study, Brazilian ST277 strains have several single nucleotid polymorphisms, and carry distinct genomic islands, phages, and CRISPR-Cas system compared to other ST277 genomes described in other countries, and these features could be the explanation for epidemic spread of ST277 in Brazil [27,87,89,100]. In strains of ST277, specific PAGIs were reported, that belong to ICEs. It must be underlined that ICEs play an important role in mobilization of carbapenemase genes [101]. It also turned out that migratory birds might have played an important role in transmission of ST277 in Brazil, furthermore, in environmental water sources, ST277 could be also isolated [90,91]. Additionally, other studies supported the zoonotic impact of MDR P. aeruginosa ST233, that these high-risk clones are able to be transmitted from human individuals to pets and the bacteria can circulate between them [86].
These features all underline the importance of possible exchange of resistance determinants and clones between animals, humans, environmental, and hospital source. Therefore, the "One health" approach seems the most accurate one to cope with antibiotic resistance.
As a summary: new antimicrobial agents and protocols for therapy are urgently needed to combat infections caused by MDR P. aeruginosa. Early detection and analysis by whole genome sequencing can help to follow evolutionary changes in MDR P. aeruginosa, to track dissemination of novel resistance determinants and emergence of international high-risk clones.