The European Committee on Antimicrobial Susceptibility Testing (EUCAST) guideline “Expected Resistant Phenotypes” (http://www.eucast.org accessed on 12 May 2022) reports that non-fermentative gram-negative bacteria are intrinsically resistant to benzylpenicillin, first- and second-generation cephalosporins, glycopeptides, lipoglycopeptides, fusidic acid, macrolides, lincosamides, streptogramins, rifampicin, and oxazolidinones. Concerning other antimicrobials not cited above, the same document stated that P. aeruginosa is expected to be resistant to ampicillin and amoxicillin, as well as their combinations with β-lactamase inhibitors, ceftriaxone, cefotaxime, ertapenem, chloramphenicol, selected aminoglycosides such as kanamycin and neomycin, trimethoprim, tetracycline, and tigecycline ^[1][25]. Knowledge of the intrinsic resistance of P. fluorescens is still limited; however, the trends appear to be similar to those of P. aeruginosa.

Regarding β-lactams, Rocha et al. evaluated 39 endophytic strains of Pseudomonas sp. isolated from a metal-accumulating plant, after which they correlated the resistance phenotypes with the distribution of strains in a dendrogram ^[2][26]. The authors recognized three clusters. The first cluster, composed of a few isolates from five different species, was associated with resistance to ampicillin and amoxicillin, and was susceptible to most of the β-lactams tested. The second cluster, formed mainly by P. koreensis (P. koreensis group, n = 10) and Pseudomonas simiae (P. fluorescens group, n = 4), showed resistance to ampicillin, amoxicillin, amoxicillin–clavulanic acid, and cefotaxime. Many isolates were also resistant to aztreonam, sulfamethoxazole–trimethoprim, and chloramphenicol. In contrast, the third cluster formed mainly by Pseudomonas sabulinigri (n = 11) included isolates that were resistant to the same β-lactams as in the second cluster, in addition to piperacillin and piperacillin–tazobactam. Some isolates belonging to the third cluster also displayed resistance to cefepime and ceftazidime but were susceptible to other classes of antimicrobial agents. Acquired resistance genes for β-lactams (bla_SHV, bla_TEM, bla_CTX-M, bla_GES, bla_KPC, bla_VIM, bla_IMP, bla_OXA-2-like, bla_OXA-10-like, bla_OXA-30-like), sulfonamides (sul1), and chloramphenicol (cat), as well as the integrase genes intI1 and intI2, were not detected in these strains, supporting the hypothesis that the phenotypes were associated with intrinsic features ^[2][26].

The production of an inducible chromosomal β-lactamase called AmpC contributes to Pseudomonas resistance to most penicillins and to first- and second-generation cephalosporins (such as cefoxitin and cefuroxime). In P. aeruginosa, de-repression of this gene can result in resistance to antipseudomonal penicillins, oxyiminocephalosporins, and cefepime ^[3][4][27,28]. Accordingly, among several β-lactamase genes searched, only the chromosomal class C β-lactamase gene bla_AmpC was detected in two P. koreensis isolates from urban wastewater treatment plants in Italy, showing low susceptibility to several β-lactams ^[5][29].

Recently, acquired β-lactamase genes have not been identified among isolates belonging to the P. fluorescens complex resistant to aztreonam and carbapenems isolated from chicken meat in Norway. Besides genes encoding efflux pumps, the isolates carried bla_AmpC and the penicillin-binding protein gene mrcA; however, mutations in these genes or their promoters have not been addressed. In some isolates, the authors also reported the detection of the pbpC gene, which encodes a PBP3 homolog ^[6][30]. PBP3, a penicillin-binding protein encoded by ftsI, is the target of aztreonam, and mutations in this gene may affect the activity of the drug ^[7][31]. Further studies are necessary to investigate the relevance of this gene in aztreonam resistance.

The intrinsic resistance of P. aeruginosa to chloramphenicol, trimethoprim, and tetracyclines can be ascribed to the presence of chromosomally expressed resistance-nodulation-division (RND)-type multidrug efflux systems on the cell surface. Notably, multidrug-resistance (MDR) efflux pumps are conserved in different microorganisms, which are likely involved in the extrusion of many toxic compounds ^[8][32]. For example, environmental P. aeruginosa strains isolated prior to the discovery of quinolones can extrude this class of antimicrobial agents, suggesting that antimicrobial extrusion is not the primary function of some efflux pumps ^[9][10][33,34]. Likewise, the RND-type efflux pump MexAB–OprM has been detected in a β-lactam resistant Pseudomonas  strain submitted to whole-genome sequencing (WGS)WGS ^[6][30].

In this context, genes encoding an RND efflux pump for polycyclic aromatic hydrocarbons (PAHs) termed EmhABC have been described in the P. fluorescens strain cLP6a ^[11][35]. Disruption of the emhB gene increased the activity of chloramphenicol and nalidixic acid, but not tetracycline, erythromycin, trimethoprim, or streptomycin, suggesting a more limited spectrum of substrates compared to other RND pumps ^[11][12][35,36]. Later, it was suggested that an alternative EmhABC efflux pump conferred resistance to ampicillin, chloramphenicol, tetracycline, ethidium bromide, and crystal violet in the P. fluorescens strain 2P24, which was isolated from wheat roots ^[13][37]. Complementary studies showed that incubation temperature and other physicochemical factors may affect EmhABC activity in P. fluorescens cLP6a ^[14][15][38,39].

Likewise, the knockout of some putative transporters increased the susceptibility of Pseudomonas protegens (P. protegens group) to rifampicin, among other toxic compounds ^[16][40]. Also, genes encoding many efflux-pump proteins, β-lactamases, and a macrolide glycosyltransferase have been described in the genome of the plant growth-promoting bacterium Pseudomonas sp. UW4 (P. jessenii, according to the authors; P. jessenii group). This strain was resistant to ampicillin, erythromycin, and novobiocin ^[17][41].

Aminoglycosides are cationic drugs, and the incubation temperature might affect their activity. Papapetropoulou et al. reported that temperatures higher than 37 °C lowered the minimum inhibitory concentration (MIC) of P. fluorescens to gentamicin and amikacin, suggesting that higher temperatures promote changes in cell-wall lipids, which increases the permeability to these aminoglycosides ^[18][42]. P. aeruginosa harbors the chromosomally encoded aminoglycoside phosphotransferase APH(3′)-IIb, having kanamycin and neomycin as substrates ^[1][18][19][25,42,43]. However, in a search for “APH(3′)” in the NCBI database, only a few results report this gene associated with other Pseudomonas species, including P. fluorescens.

Colistin is a cationic drug that is used to treat P. aeruginosa infections ^[1][20][25,44]. Still, all five P. koreensis isolates obtained from urban wastewater-treatment plants in Italy were resistant to this polymyxin ^[5][29]. Moreover, many Pseudomonas species with plant-beneficial properties, such as P. protegens and P. chlororaphis, have been reported as intrinsically resistant to cationic compounds ^[21][45]. According to the authors, the phenotype is dependent on the presence of O-specific side chains on the cell surface ^[21][45]. Among clinical isolates of P. aeruginosa, adaptive resistance to polymyxins can occur due to the addition of 4-amino-4-deoxy-L-arabinose (Ara4N) to the lipid A moiety of lipopolysaccharide through induction of the arn operon under the control of two-component regulatory systems ^[22][46]. Recently, six genes related to colistin resistance (emrA, lpxA, lpxD, pgsA, phoP, phoQ), but not the plasmid-mediated mcr, have been detected in the genome of colistin-resistant Pseudomonas spp. obtained in the Norwegian food chain ^[6][30].

Atypical antimicrobial resistance profiles in Pseudomonas sp. obtained from pristine sites have been reported in many studies. Shivaji et al. (1989) obtained 10 isolates of Pseudomonas spp. (including P. fluorescens) from Antarctic soil samples, which were susceptible to kanamycin, gentamicin, tobramycin, polymyxin B, tetracycline, rifamycin, colistin, streptomycin, and nalidixic acid ^[23][47]. At that time, considering that these strains grow at low temperatures (4 °C), authors suggested that such a distinct phenotype compared to mesophilic Pseudomonas strains may have resulted from adapting to harsh conditions ^[23][47]. Later, the psychrophilic species P. antarctica, P. meridiana, and P. proteolytica (P. gessardii group) obtained from cyanobacterial mats in Antarctica were characterized ^[24][48]. Curiously, P. antarctica was also susceptible to antimicrobial agents considered ineffective against P. aeruginosa due to intrinsic resistance, such as penicillin, ampicillin, chloramphenicol, sulfamethoxazole–trimethoprim, erythromycin, kanamycin, and tetracycline. In contrast, P. meridiana and P. proteolytica were resistant to all the antimicrobials mentioned above, except for kanamycin and tetracycline. Similar to P. aeruginosa, the three Pseudomonas strains tested were resistant to trimethoprim ^[1][24][25,48]. More recently, Orellana-Saez et al. isolated the Pseudomonas sp. strain MPC6 (closely related to P. fluorescens, according to the authors) of a soil sample from Deception Island (Antarctica) ^[25][49]. This isolate was susceptible to antimicrobials that are ineffective against most of the Pseudomonas analyzed and had a similar resistance profile to the environmental isolates P. putida KT2440 and P. antarctica (P. fluorescens subgroup). Genes encoding antibiotic-inactivation enzymes found in the genome of reference strains P. aeruginosa PA7 and P. aeruginosa PAO1 such as aminoglycoside phosphotransferases (APHs), chloramphenicol acetyltransferases (CATs), bleomycin-binding proteins, and β-lactamases, were absent in the genome of Pseudomonas sp. MPC6. The genome of Pseudomonas sp. MPC6 also lacked genes encoding modifications in cell-wall charges that are reported as determinants of antimicrobial resistance. However, the genome was well equipped with efflux pumps ^[25][49]. An uncommon susceptible phenotype to penicillin, kanamycin, neomycin, and tetracycline was also detected in Pseudomonas sp. strain AHD-1 (closely related to P. azotoformans, P. gessardii, and P. libanensis, according to the authors), which was isolated from wastewater in Tunisia ^[26][50].

Furthermore, recent studies have reported P. fluorescens members that are resistant to clinically relevant antibiotics such as piperacillin, aztreonam, ceftazidime, carbapenems, and colistin. These strains were isolated from diverse environments such as Antarctica soil samples, rhizosphere of desert plants in Atacama, and calcite moonmilk deposits from caves in the Czech Republic ^[27][28][29][51,52,53]. Known acquired resistance mechanisms associated with these unexpected phenotypes have not been detected, although genomic islands and other likely acquired mobile genetic elements have been reported in P. fildesensis (P. fluorescens group) ^[27][51]. Therefore, further studies are needed to characterize the pathogenic potential and the presence of transmissible acquired antimicrobial resistance genes (ARGs)ARGs in such  Pseudomonas sp. strains.

Although Antarctica is the most remote continent in the world, antimicrobial resistance may be transferred to this region due to the migration of animals and humans. Recently, Na et al. evaluated isolates from animal feces, soil, and sediments with varying human and animal impacts in the Fildes Peninsula, Antarctica ^[30][54]. Pseudomonas was the dominant genus that showed resistance to sulfamethazine, and a strong correlation between mobile genetic elements and antimicrobial resistance genes was recognized, considering isolates of different genera included in the study ^[30][54]. In contrast, in a study that included isolates either from human-impacted or pristine sites in Antarctica, most of the strains displaying multi-resistance were collected from areas without human intervention, suggesting that antimicrobial resistance is likely a natural and ancestral process ^[31][55]. Yet, in the characterization of two multidrug-resistant isolates belonging to the P. fluorescens, Marcoleta et al. (2022) reported that although these strains lack genes found in the reference P. aeruginosa PA7 strain, they showed a higher number of genes associated with ATP-binding cassete (ABC) and small multidrug resistance (SMR) efflux pumps ^[31][55]. Genes for putative β-lactamases have also been detected, including a homolog of LRA-3 β-lactamase, which was previously described in soil metagenomic DNA from Alaskan soil ^[31][32][55,56].

In human-impacted sites, the selective pressure caused by antimicrobial pollution may promote the dissemination and persistence of acquired resistance mechanisms. Chow et al. exposed one strain of P. aeruginosa and one of P. protegens to kanamycin, tetracycline, or ciprofloxacin at 1/10 of the MIC in a serial streaking over 40 passages, thus mimicking environmental pollution with antimicrobial agents. Higher MICs and increased genome changes were detected in P. protegens, suggesting that this type of antimicrobial pollution might generate new resistant strains ^[33][57].

Compared to contemporary samples, ancient and well-conserved samples are powerful tools to measure the degree to which the rates of antimicrobial resistance have changed over the years. Addressing this issue, Lugli et al. characterized a P. veronii (P. fluorescens group) strain isolated from a frozen and mummified human body found in an Italian Alpine glacier ^[34][58]. Screening for ARGs revealed an abundance of putative β-lactamases, glycopeptide-resistance proteins, ABC transporters, and major facilitator superfamily (MFS) efflux pump. Notwithstanding, modern strains of P. veronii harbor 24% more ARGs than the ancient strain P. veronii, which might be due to horizontal gene transfer (HGT), accounting for the rapid spread and persistence of antimicrobial resistance determinants in the environment ^[34][58].

As an example of exceptional resistance phenotypes identified in highly human-impacted sites, P. fluorescens strains resistant to clinically available antimicrobial agents, such as piperacillin-tazobactam, ceftazidime, cefepime, imipenem, meropenem, gentamicin, and ciprofloxacin, were isolated from the multinational Danube River ^[35][36][59,60].

Metal resistance can be accompanied by antimicrobial resistance. Metals are not easily degraded and occur in various environments, especially those receiving hospital and industrial effluents, as well as mining areas ^[37][67]. Furthermore, metal pollution causes a persistent selective pressure that favors the development and transmission of antimicrobial resistance traits ^[38][68]. There are two known mechanisms through which metal and antimicrobial resistance are co-selected. So-called “co-resistance” refers to the presence of metal- and antimicrobial-resistance determinants encoded in the same mobile genetic element, whereas “cross-resistance” refers to the same mechanism conferring resistance to both metals and antimicrobial agents (e.g., efflux pumps) ^[38][39][68,69]. In addition to the data on P. fluorescens  found in these contentsis review, previous studies have evaluated metal and antimicrobial resistance in hospital and environmental isolates of members of the genus  Pseudomonas ^{[37][40][41][42][43]}[67,70,71,72,73].

Ramos et al. detected P. saponiphila (n = 13; P. chlororaphis group), P. humanensis (n = 5) and P. asiatica (n = 2) (P. putida group), and P. aeruginosa (n = 3) in water samples obtained from the state of São Paulo and Brasília (Brazil) ^[43][73]. Most of these isolates were resistant to heavy metals and clinically relevant antimicrobial agents, such as the β-lactams piperacillin-tazobactam, ceftazidime, cefepime, imipenem, meropenem, and aztreonam, as well as the quinolones ciprofloxacin, levofloxacin, and norfloxacin, and the aminoglycosides gentamicin and tobramycin. The ARGs bla_GES (β-lactam resistance), tetB (tetracycline resistance), qnrS and qepA (quinolone resistance), and aac(3′)-IIa and ant(2″)-Ia (aminoglycoside resistance) were identified for the first time in P. saponiphila. Even so, plasmids were not detected, suggesting that the identified genes could be located in the chromosome. These findings suggested that the resistant phenotype for most of the antimicrobial agents and heavy metals analyzed might be attributed to alternative mechanisms that were not evaluated, such as efflux pumps of the RND superfamily ^[43][73].

Resistant P. fluorescens strains have been found in food products such as chicken and camel meat, salad vegetables, fish and mushroom farms, as well as in cheese ^{[6][44][45][46][47][48][49][50][51]}[30,74,75,76,77,78,79,80,81]. Notably, Pseudomonas spp. is one of the main microorganisms causing food spoilage ^{[6][52][53][54][55]}[30,82,83,84,85]. Likewise, Poirel et al. isolated P. synxantha (P. fluorescens group) from chicken meat, which harbored a likely-acquired chromosomal metallo-β-lactamase PFM-1 ^[50][80]. PFM-1 showed high amino-acid identity with Sfh-1 and CphA-1 carbapenemases, which were initially reported in species of Serratia and Aeromonas, respectively. Variants of PFM-1 were also detected in P. libanensis (PFM-2) and P. fluorescens (PFM-3), suggesting that the P. fluorescens group may behave as reservoir of PFM-like encoding genes ^[50][80]. Recently, one isolate of P. fluorescens harboring the β-lactamase bla_SHV was identified in Benin. The isolate was resistant to amoxicillin, amoxicillin-clavulanic acid, ceftriaxone, cefotaxime, ertapenem, imipenem, aztreonam, gentamicin, and ciprofloxacin ^[56][86].

Members of the P. fluorescens complex have also been recognized as veterinary pathogens, causing infections in bovines, canines, dolphins, fish, wild animals, and even frog oocytes. Common resistance phenotypes observed in these strains include tetracycline, chloramphenicol, sulfamethoxazole-trimethoprim, amoxicillin, amoxicillin–clavulanic acid, cefoxitin, cefotaxime, and ticarcillin resistance. Moreover, most of these animals were living in environments under the human influence ^{[57][58][59][60][61][62][63]}[87,88,89,90,91,92,93].

Due to the clinical importance of P. aeruginosa, breakpoints for effective antimicrobial agents against this pathogen are available in the Clinical and Laboratory Standards Institute (CLSI) and EUCAST guidelines. The β-lactams ticarcillin, piperacillin, ceftazidime, cefepime, cefiderecol, ceftolozane, aztreonam, imipenem, doripenem, and meropenem, some of which are associated with β-lactamase inhibitors, as well as the quinolones ciprofloxacin and levofloxacin, the aminoglycosides gentamicin, amikacin, tobramycin, and polymyxins colistin or polymyxin B, should be effective against this microorganism ^[64][65][94,95]. 

Possible HGT from hospitals and health facilities to the environment is a serious public health concern. Forsberg et al. analyzed the transfer of resistance determinants between soil and clinical bacteria. Resistance genes present in the environmental bacterium Pseudomonas sp. K94.23 (according to the authors, a member of the P. fluorescens complex) shared a complete nucleotide identity with clinical pathogens ^[66][96]. Likewise, Herrick et al. suggested that transmissible plasmids from environmental Pseudomonas that confer resistance to tetracycline (commonly used in agriculture) would cause the persistence of co-carried genes that confer resistance to clinically available antimicrobial agents such as gentamicin, ticarcillin, and ciprofloxacin ^[67][97]. In the 1990s, Chandrasekaran et al. suggested that even viable but non-culturable P. fluorescens can transfer plasmids to other bacteria in marine environments ^[68][98]. The same group also reported the transference of an MDR plasmid (pSCL) from rifampicin-resistant P. fluorescens isolated from polluted soil to E. coli and P. putida. It appears that pSCL conferred rifampicin resistance to the transformants, presumably through an efflux pump ^[69][99].

Efficient transference of plasmids carrying resistance genes to chloramphenicol (cat) ^[70][100] and colistin (mcr-variants) have been reported for P. putida and/or P. aeruginosa ^[71][72][73][101,102,103]. Regarding P. fluorescens, the gene mcr-1 was detected in one isolate from a community/household environment in the Republic of Congo ^[74][104]. A gene encoding BIC-1, a class A carbapenemase capable of hydrolyzing penicillins, cephalosporins (except ceftazidime), and carbapenems, was detected in the chromosome of a P. fluorescens isolate from the Seine River (France). Three months later, the β-lactamase gene was also found in the chromosomes of two other P. fluorescens isolates from the same site ^[75][105]. Furthermore, the class B metallo-β-lactamase IMP-22, encoded by the bla_IMP-22 gene, located in a class 1 integron and capable of hydrolyzing narrow and extended-spectrum β-lactams, was detected in two strains of P. fluorescens from urban wastewater, as well as in a clinical isolate of P. aeruginosa in Italy ^[76][64].

The risk of human infection by exposure to contaminated rivers was illustrated by a case of a patient admitted to an intensive care unit for near-drowning in a river in France, who was colonized and infected by carbapenem-resistant bacteria of probable environmental origin. Among the isolates characterized, six strains belonging to the P. fluorescens complex collected from the river had the same carbapenem-resistant phenotype as a P. fluorescens strain colonizing the patient’s respiratory tract ^[77][106]. In another country, an isolate of P. cedrina (P. fluorescens group) was identified in water bodies in Los Angeles, which was resistant to cefotaxime, meropenem, and imipenem ^[78][107]. However, none of these studies reported carbapenemase production, suggesting that other genotype features, transmissible or not, might be responsible for the observed phenotype.

In 2012, Maravić et al. published the first report of a TEM-type ESBL in P. fluorescens ^[79][108]. The blaTEM-116 gene was present in the chromosome of isolates from a Croatian bay highly impacted by agricultural, industrial, and municipal effluents. In the same study, out of 185 P. fluorescens isolates investigated, 70 presented a multidrug resistant phenotype, with the highest resistance rates for cefotaxime, ceftazidime, meropenem, aztreonam, and tetracycline ^[79][108]. Later studies have reported the presence of blaTEM in isolates belonging to the P. fluorescens group in South Africa and India ^[80][81][109,110].

Some results from studies whose focus was not on the genus Pseudomonas are worth mentioning. Pseudomonas was one of the most abundant genera recovered under selective pressure with cefotaxime or imipenem from Lake Bolonha in the Brazilian Amazon. Among 37 Pseudomonas strains displaying the above-mentioned resistance phenotypes (species were not determined), 25 carried the likely acquired β-lactamase genes blaTEM, blaSHV, blaCTX, blaIMP, and blaVIM either alone or in combination. Many of these isolates were also resistant to non-β-lactams such as gentamicin ^[82][111]. Similarly, Chakraborty et al. reported that Pseudomonas was the most abundant genus contributing to the occurrence of ARGs, including multidrug-efflux pumps, glycopeptide, bacitracin, tetracycline, and aminoglycoside-resistance genes, in the Lonar soda lake (India) ^[83][112].

54.1. Research of Resistance Genes in Genome Public Databases

Congruent with the data collected in the scientific literature, the analysis conducted in abovhe contentsrein identified a small number of genomes of species belonging to the P. fluorescens complex (n = 17, 2.7%) carrying one or more transferable ARG. Genes associated with resistance to β-lactams, aminoglycosides, phenicol, fosfomycin, sulfamethoxazole, or tetracycline classes have been found in isolates of different species, which were recovered from variable sources and countries.