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Article

Detection of Genes Associated with Polymyxin and Antimicrobial Peptide Resistance in Isolates of Pseudomonas aeruginosa

by
Meseret Alem Damtie
1,
Ajay Kumar Vijay
1,2 and
Mark Duncan Perry Willcox
1,*
1
School of Optometry and Vision Science, University of New South Wales, Sydney, NSW 2052, Australia
2
Centre for Vision Research, Westmead Institute for Medical Research, The University of Sydney, Sydney, NSW 2145, Australia
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2025, 26(21), 10499; https://doi.org/10.3390/ijms262110499
Submission received: 21 September 2025 / Revised: 26 October 2025 / Accepted: 27 October 2025 / Published: 29 October 2025
(This article belongs to the Section Molecular Microbiology)
Browse Figure
Versions Notes

Abstract

Pseudomonas aeruginosa causes ocular and other infections and quickly acquires antimicrobial resistance. Polymyxin B and colistin are last-line agents against resistant P. aeruginosa, yet even resistance to these is increasing. Antimicrobial peptides (AMPs) are also being developed as new antibiotics, but resistant mechanisms to polymyxins might also cause resistance to these AMPs. This study evaluated whether isolates with differing polymyxin resistances also showed elevated minimum inhibitory concentrations (MICs) to the human cathelicidin LL-37 and a synthetic AMP, Mel4. Forty isolates of P. aeruginosa, mostly collected in India and Australia, were assessed for minimum inhibitory concentrations (MICs) by broth microdilution in cation-adjusted Mueller–Hinton broth. Whole genome sequences were analyzed using NCBI BLAST (version 2.17.0). SNPs vs. MIC associations were evaluated with Fisher’s exact test. Sixty-five percent of isolates were resistant to polymyxin B, and 80% to colistin. Polymyxin B MICs ranged from 0.5 to 512 µg/mL, with 32.5% showing intermediate resistance and 22.5% being highly resistant (MIC ≥ 256 µg/mL). MICs for polymyxin B and colistin were strongly correlated with each other (Spearman’s R ≥ 0.6; n = 40; p ≤ 0.001). LL-37 showed moderate correlations with polymyxin B, colistin, and Mel4, whereas Mel4 showed weaker correlations with polymyxin B or colistin (R < 0.4). Genomic analysis identified SNPs in mipB (V469M, G441S) as being associated with the MICs to all the antimicrobials. Strains with MICs between 64 and 512 µg/mL were significantly more likely to harbor nalC (E153Q/D) or the mipB variants (p < 0.05). Higher polymyxin MICs were associated with elevated MICs to LL-37 and, to a lesser extent, Mel4, suggesting partial shared resistance among membrane active peptides. Defining the effect of the SNPs and clinical relevance of AMP cross-resistance may inform future therapies and safer contact lenses.

1. Introduction

Antimicrobial resistance has emerged as a pressing global health challenge, with approximately 700,000 deaths each year attributed to resistant microbes. This number is projected to reach a staggering 10 million deaths annually, accompanied by economic losses of $100 trillion, by 2025 [1]. Recognizing its global threat, both the Centers for Disease Control and Prevention and the World Health Organization have classified P. aeruginosa as a priority pathogen, highlighting the urgent need for novel antimicrobial strategies.
Pseudomonas aeruginosa is a Gram-negative, opportunistic pathogen and a major cause of numerous kinds of infections. It poses a significant threat, particularly to immunocompromised individuals, including those with cystic fibrosis, burn injuries, cancer, or prolonged hospital stays [2,3]. It can cause pneumonia, bloodstream infections, urinary tract infections, and surgical site infections [4]. It is also the leading cause of sight-threatening corneal disease in otherwise healthy patients who use contact lenses [5,6,7,8].
Its ability to rapidly develop antimicrobial resistance makes it a challenging pathogen in both ophthalmic and systemic infections, necessitating effective surveillance and treatment strategies [9]. It can evade antimicrobial therapy through multiple resistance mechanisms. These include active efflux systems such as the MexAB-OprM efflux pump, enzymatic degradation (e.g., β-lactamases), and modifications of the outer membrane that reduce antibiotic permeability. In recent years, there has been an increasing prevalence and dissemination of multidrug-resistant (MDR) and extensively drug-resistant (XDR) P. aeruginosa, with rates of between 15 and 30% in some geographical areas [10,11,12]. Its high adaptability and extensive resistance means infections caused by P. aeruginosa are associated with prolonged hospital stays, increased morbidity, high mortality rates, and increased cost of healthcare [11,13].
Due to the shortage of novel antimicrobials, the lipopeptide antibiotics polymyxin B and colistin (polymyxin E) are often used as last-resort antibiotics to treat MDR/XDR P. aeruginosa infections [14]. These cationic cyclic lipopeptides disrupt the bacterial outer membrane by binding to lipopolysaccharides (LPS) and displacing divalent cations (Ca2+ and Mg2+) from the phosphate groups, leading to membrane destabilization and cell death [3]. Also, there is development of antimicrobial peptides (AMP) as potential new antibiotics. These have similarities to polymyxins, being generally cationic and amphiphilic and reacting with the membranes of bacteria as their most common mode of action [15,16]. The naturally occurring AMP LL-37, the only human cathelicidin, has probably received the most attention for development, although it has yet to reach the clinic [17].
Unfortunately, there is emergence of polymyxin resistance in P. aeruginosa around the world [14]. This resistance occurs most commonly as a result of modification in the lipid A moiety of LPS [18], as well as from efflux of the drugs via pumps. These mechanisms can act synergistically to enhance drug resistance. LPS of P. aeruginosa consists of three distinct domains, a core oligosaccharide, the lipid A portion and the O-antigen region [19]. Modifications of lipid A reduce the electrostatic binding affinity of polymyxins and occur during exposure to polymyxins. Chemically, it is the incorporation of 4-amino-4-deoxy-L-arabinose (L-Ara4N), phosphoethanolamine, and galactosamine into lipid A that reduces its negative charge and consequently the affinity for binding with positively charged polymyxins [20]. This LPS modification in P. aeruginosa is regulated by several two-component signaling systems, PmrAB (also known as BasRS), PhoPQ, CprRS, ParRS, and ColRS. These systems activate overlapping resistance pathways, particularly the arnBCADTEF operon, which leads to the addition of L-Ara4N to lipid A [21], ensuring a robust resistance response even if one pathway is disrupted (not functional) [22,23].
Efflux pumps play a secondary but significant role in polymyxin resistance by reducing intracellular drug accumulation. The MexAB-OprM efflux pump, a major resistance-nodulation-cell division (RND) transporter in P. aeruginosa, actively expels a broad range of antibiotics, including polymyxins [24,25]. Under normal conditions, MexR represses mexAB-oprM expression. However, mutations in mexR disrupt its function, leading to efflux pump overexpression and increased antibiotic efflux [25]. Regulatory proteins such as CpxR also contribute to resistance by modulating stress adaptation genes, which subsequently enhance efflux pump expression [26]. A polymyxin-induced operon, mipAB, can be activated by the ParRS two-component regulatory system, and these act in concert to activate the synthesis of the MexXY-OprA efflux pump [27].
AMPs form an important part of the human defense system in many body sites and have been shown to be important in controlling ocular infections. The mouse cathelicidin CRAMP (its human LL-37 homolog) as well as β-defensins-2 and 3 protect the eye against P. aeruginosa infections [28,29,30]. Whilst bacteria struggle to develop resistance to AMPs in the laboratory in experiments where they are exposed to increasing concentrations of AMPs [31], resistance mechanisms are known, and these can overlap with mechanisms that bacteria use to become resistant to polymyxins. This includes lipid A modification with 4-amino-L-arabinose and active efflux from cells [32]. This implies that resistance to polymyxins may confer resistance to AMPs, even those of the innate human defense system such as LL-37. If this is the case, this may make polymyxin-resistant strains much more virulent as they could also evade the AMP-based defense system.
The aim of this study was to examine strains of P. aeruginosa for their susceptibility to polymyxin antibiotics and the AMPs LL-37 and Mel4, and to examine whether P. aeruginosa ocular isolates with different minimum inhibitory concentrations (MICs) to polymyxins also showed higher MICs with LL-37 and a synthetic AMP Mel4 which was used as an antimicrobial coating for contact lenses in a Phase III clinical trial [33]. Furthermore, whole genome sequences of the strains were analyzed to identify known polymyxin resistance genes and single nucleotide polymorphisms (SNPs) within these genes and to assess their associations with elevated MICs to polymyxins or AMPs.

2. Results

2.1. Antimicrobial Susceptibility to the Peptide Antibiotics

The MICs of polymyxin B, colistin, Mel4, and LL-37 against P. aeruginosa are shown in Table 1. For polymyxin and colistin, the Clinical and Laboratory Standards Institute defines an intermediate break point at (MIC ≤ 2 µg/mL) and a resistant breakpoint of (MIC ≥ 4 µg/mL). No susceptible breakpoint has been established. Among the 40 P. aeruginosa clinical and reference isolates tested, 65% were resistant to polymyxin B and 80% to colistin. The MIC of polymyxin B ranged from 0.5 to 512 µg/mL, with the geometric mean MIC being 19.0 µg/mL, while the median MIC was 40.0 µg/mL. A total of 13 strains showed intermediate resistance to polymyxin B (32.5% of strains) and 9 isolates had very high levels of resistance (MIC ≥ 256 µg/mL; 22.5% of strains). The MIC values for colistin ranged from 0.5 to 512 µg/mL, with the geometric mean MIC being 20.4 µg/mL and the median being 16.0 µg/mL. A total of eight strains showed intermediate resistance (20%) and six strains (15%) had very high levels of resistance (MIC ≥ 256 µg/mL). Mel4 had a geometric mean MIC of 50.2 µg/mL, with a median of 48 µg/mL. The MIC ranged between 0.5 and 256 µg/mL. For LL-37, the geometric mean MIC was 51.1 µg/mL, with a median of 48 µg/mL and range of 0.5 to 256 µg/mL.
There was a strong ( r s ≥ 0.6) positive correlation between polymyxin and colistin MIC values (Table 2); this suggests clinical isolates that are resistant to polymyxin were likely to also be resistant to colistin. There were moderate positive correlations ( r s ≥ 0.4) between the MIC for LL-37 and those for polymyxin B, colistin, and Mel4. For Mel4, there was a weak ( r s < 0.4) positive correlation with MICs for polymyxin B and colistin (Table 2).
The correlations between MICs to the four AMPs indicated that there was the possibility of shared genetic determinants of resistance.

2.2. Possession of Genes and SNPs Associated with Polymxin Resistance

Table 3 gives the data for genes involved in polymyxin resistance associated with the arnBCADTEF operon and its regulation (pmrAB, phoPQ, cprRS, parRS, and colRS). All strains possessed every gene, but strains were distinct in the possession of single nucleotide polymorphisms (SNPs) within these genes. For example, strains PA189, PA182, PA224, PA206, PA126, PA193, PA225, PA198, ATCC 19660, PA216, PA220, and PA123 possessed the SNP that led to the L71R amino acid change in PmrA, whereas strains PA33, PA223, PA31, 6206, PA221, PA55, PA217, and PA219 did not possess any SNPs in this gene. Also, strains PA206, PA193, PA225, and PA198 possessed the SNP leading to the amino acid change D61E, strain PA126 possessed the SNP leading to V34L, and strain PA227 possessed the SNP leading to the T31I amino acid change in PmrA. Table 4 gives data for genes involved in efflux pumps and their regulation (mipA, mipB, armR, mexR, mexA, mexB, oprM, mexX, mexY, cpxR, nalC, and nalD). All strains possessed all genes, except for mipA, which was not detected in strains PA206 and PA216. SNPs were detected in most genes in most strains, with the exceptions of mexA (SNP in PA216 only), aprM (no SNP in any strain), cpxR (SNPs in 6206 only), and nalD (SNPs in PA221, PA217, PA220, and PA219 only). Table 5 gives data for genes associated with changes in lipopolysaccharide (LPS) and other factors involved in polymyxin resistance (oprH, papP, mpl, slyB, ppgS, ppgH, speD2, speE2, waaL, PA5005, rsmA, and mprF). Again, most strains possessed SNPs in most genes, with the exceptions of oprH, slyB, speD2, and rsmA, which had no SNPs for any strains. Strains were also examined for possession of any of the mcr genes that have been shown to be involved in polymyxin resistance, but no strain possessed these genes. All the strains that had their whole genomes available possessed all the genes that had been previously associated with resistance to polymyxins, apart from the mcr genes which no strain possessed, and two strains that did not possess mipA.
When analyzing associations with polymyxin B MICs, strain PA123, which had the highest MIC of 512 µg/mL, was found to carry a unique combination of SNPs in the following genes: arnA (Q661L), arnB (Q336L), mpl (V358I), parR (I93T), speE2 (A3V), and waaL (A110G). Additionally, the arnT G156R SNP was shared exclusively between PA123 and strain PA216. PA216, which had an MIC of 256 µg/mL, also harbored mipB R401del, mpl A303V, and mprF N553D variants. The other strains with MIC of 256 μg/mL (PA217, PA219 and PA220) all possessed the SNPs nalC E153Q, nalD R38W, and pmrB V6A, as well as either mprF R188H or mprF N553D (strain PA219 contained both). Strain PA55, with an MIC of 128 μg/mL only and uniquely possessed pmrB H345Y. Strain PA193, the only isolate with an MIC of 8 µg/mL, carried the waaL L281F variant, which was also present in strain PA227. Gene mipB V469M (p = 0.0047) SNP was more common in strains with MIC ≥ 64 but <512 μg/mL than those with MICs of ≤8 μg/mL. Also, strains with MIC ≥ 64 but <512 μg/mL more commonly possessed mipB V469M or G441S (p = 0.0055) SNPs than those strains with MICs of ≤8 μg/mL.
When examined for associations with colistin resistance, the strain with the highest MIC of 512 μg/mL, PA123, had a unique collection of SNPs in several genes: arnA Q661L, arnB Q336L, mpl V358I, parR I93T, speE2 A3V, and waaL A110G (the same as those of this strain in relation to polymyxin resistance). The SNP arnT G156R was shared only between strain PA123 and PA216. Strain PA55, with an MIC of 256 μg/mL, only and uniquely possessed pmrB H345Y. The other strains with MIC of 256 μg/mL, ATCC 19660 and PA219, also both possessed mprF N553D, which was shared with two other strains (PA216 and PA217) with MIC ≥ 64 μg/mL, but no strain with MIC < 64 μg/mL. Strains with MIC ≥ 64 but <512 μg/mL were significantly more likely to possess either nalC E153Q/D (p = 0.0174), mipB V469M (p = 0.0174), or mipB V469M or G441S (p = 0.0007) than strains with MIC ≤ 4 μg/mL.
For Mel4, there were several SNPs that were uniquely possessed by strains with MICs of ≥64 μg/mL compared to strains with MICs of ≤32 μg/mL. These included arnF A125T (3 strains), arnT A267V (4 strains), ppgS N670S (3 strains), ppgH R110H (6 strains), speE2 V217I (3 strains), mipA deletion after N34 (3 strains), waaL G330A (9 strains), and PA5005 D23E (3 strains). Strains with MICs of ≥64 μg/mL were more likely to possess waaL G330A (p = 0.0237) or mipB V469M or G441S (p = 0.0237).
For LL-37, several SNPs (or lack thereof) were uniquely possessed by strains with MICs of ≥64 μg/mL compared to strains with MICs of ≤32 μg/mL. These included a lack of SNPs in pmrA (6 strains), pmrB S2P (7 strains), arnA F80Y (4 strains), arnE R28H (3 strains), arnT A267V (3 strains), armR Y32C (5 strains), mexB S1041E and V1042A (5 strains), mexX A30T (6 strains), mexY I536V (6 strains), nalC E153Q (6 strains), nalD R38W (3 strains), mipA A32V, S33Q and N34-del (3 strains), and mipB V469M (6 strains) or G441S (3 strains). Strains with MICs ≥ 64 μg/mL were significantly more likely to possess mipB V469M or G441S (p = 0.0075). Figure 1 shows a representation of the genes studied and the amino acid changes that were associated with at least one strain having high MICs to the polymyxins of the AMPs.

3. Discussion

This study tested the hypothesis that reduced susceptibility to polymyxins (polymyxin B and colistin) correlates with higher MICs for human cathelicidin LL-37 and the chimeric peptide Mel4, and that these phenotypes would coincide with the presence of, and SNPs within, genes implicated in polymyxin resistance. The hypothesis was at least partially demonstrated, as there were statistically significant correlations between MICs for the polymyxin antibiotics and those for LL-37 and Mel4. Perhaps not surprisingly, given the close chemical similarities between polymyxin B and colistin (polymyxin E), the correlations between MICs for these two antibiotics were the most robust. The moderate correlations may indicate partial overlap of resistance mechanisms, and this was tested in subsequent analyses.
No strain possessed the mcr genes. The mcr genes have been sporadically reported in P. aeruginosa, with only 11.2% of P. aeruginosa strains isolated from healthcare-associated infections in Nepal possessing mcr-1 [34], and only 1% of P. aeruginosa clinical isolates from a study in Brazil [35]. The mcr-5 gene appears to be extremely rare, detected in only 1 out of 2440 P. aeruginosa isolates from the U.S. Multidrug-Resistant Organism Repository and Surveillance Network [36]. Another study of 116 carbapenem-resistant P. aeruginosa clinical isolates from China also found no evidence for possession of mcr-1–8 or mcr-10 [14].
Changes to the lipopolysaccharide of P. aeruginosa appears to be a relatively common mechanism for resistance to polymyxins. This is often mediated by upregulation of the arnBCADTEF operon by several two-component signaling systems such as ParRS, PmrAB, ColRS, PhoPQ, and CprRS [20,21,23], as well as the sensor MipAB [27]. SNPs in these signaling system genes have previously been associated with increased polymyxin resistance.
MipAB is a membrane-associated sensor for polymyxin that is activated by ParRS and upregulates the efflux pump MexXY-OprA [27]. As MipAB has only recently been characterized as being associated with polymyxin resistance, no reports of SNPs affecting its function have been published. The initial report found that some strains of P. aeruginosa such as PA14 and PAO1 contained a truncated version of MipA [27] and all strains in the current study either also had truncated mipA or did not possess the gene (PA206 and PA216). These differences were not associated with resistance to the polymyxins, LL-37, or Mel4. However, the current study found the mipB SNP V469M to be more common in strains with MICs ≥ 64 but <512 μg/mL than those with lower MICs for polymyxin and colistin. Those strains with higher MICs (≥64 μg/mL) for LL-37 or Mel4 were also more likely to possess the SNPs V469M or G441S in mipB. This suggests that these SNPs may affect either expression of mipB, interaction of MipB with MipA, the interaction with ParRS, or the upregulation of MexXY-OprA, and this should be examined in future studies to determine what the role of these SNPs might be in resistance to the polymyxins, LL-37, and Mel4.
Possession of pmrA L71R has been linked to increased expression of this gene in resistant P. aeruginosa [14]. However, this SNP was present in nearly all isolates in the current study, including the highly polymyxin-B-susceptible isolate PA189 and the highly resistant isolate PA123, suggesting it is not associated directly with polymyxin resistance in the current strains. Possession of pmrB H345Y and parS H398R resulted in overexpression of pmrA and arnA [14]. In the current study, only strain PA55 possessed pmrB H345Y, and this strain possessed a unique cohort of SNPs. Its possession of pmrB H345Y may be the reason it had high MICs for the polymyxins (128–256 μg/mL), LL-37 (64 μg/mL), and Mel4 (128 μg/mL). No strain in the current study possessed parS H398R. Whilst several other specific SNPs in pmrB have been linked to increases in colistin resistance [21], none of those SNPs were present in the isolates in the current study, and most pmrB SNPs in the current study were not associated with polymyxin B or colistin resistance. Similarly, SNPs in pmrB associated with resistance to colistin or the antimicrobial peptide murepavadin [37] were also mostly absent from strains in the current study. The only exception was pmrB V6S, which was possessed by one strain with an MIC of 64 μg/mL in the previous study [37] and all strains with an MIC of 256 μg/mL to polymyxin B in the current study, suggesting a link to resistance. A previous study found pmrB A247T SNP in two colistin-resistant strains [38]. This SNP was also found in three strains in the current study (PA193, 6206, and PA198) which had MICs ranging from 4 to 64 μg/mL, but was not found in other colistin-resistant strains. Another study found the SNPs pmrB V15I and G68S in two colistin-resistant strains [39]. The current study found these SNPs in only one colistin-susceptible strain (PA33) but six colistin-resistant strains (PA217, 6206, PA220, PA221, PA31, and PA219), which, whilst not significantly different, may indicate a role for these SNPs in colistin resistance.
A previous study of highly colistin-resistant (MIC > 512 μg/mL) cystic fibrosis P. aeruginosa isolates found various SNPs in phoQ to be associated with this resistance [40]. The current study found no SNPs in phoQ or phoP for any strain, but as all the current MICs were ≤512 μg/mL this may indicate that SNPs in this signaling system are only associated with very high levels of colistin resistance (>512 μg/mL).
The arnBCADTEF operon is also regulated by CprRS, which responds to a range of antimicrobial peptides including polymyxin B, CRAMP, and LL-37 [23,41]. Upon stimulation by antimicrobial peptides, CprRS upregulates the HigBA toxin–antitoxin system, which in turn promotes the production of type III secretion system effectors. In the current study, there were several SNPs in cprR (E183D) and cprS (E111D, A175V, N221H, and T329S) that were shared between four strains (PA225, PA198, PA206, and PA193) but these did not correlate with MICs to the polymyxins, LL-37, or Mel4. These SNPs were not reported in other studies that identified different SNPs in cprS [14,38,39]. The same four strains also had SNPs in parR (T135A) and parS (A115E, V304I, E343D, and Y407H). Again, these did not correlate with MICs to the polymyxins, LL-37, or Mel4. Also, these SNPs were not reported in other studies [14,38,39]. The other two-component signaling system involved in polymyxin resistance, ColRS, had few SNPs. Indeed, there were no SNPs found in colR and only three random SNPs in colS. This indicates that this system may not be important in polymyxin or antimicrobial peptide resistance in the current collection of strains.
These two-component signaling systems activate the arnBCADTEF operon, and the current study found SNPs within genes in this operon. Whilst there were several SNPs within genes in this operon, none appeared to be closely related to MICs to the polymyxins, LL-37, or Mel4. However, the strain with the highest MIC for polymyxin and colistin, PA123, contained a unique set of SNPs (arnA Q661L, arnB Q336L, parR I93T, mpl V358I, speE2 A3V, and waaL A110G) along with arnT G156R which was shared only with one other strain that also had a high MIC for polymyxin B (PA216). All these SNPs appear to be novel [42] and are worthy of further investigation to determine which mediate higher MICs to the polymyxins.
Efflux pumps can also contribute to polymyxin resistance. As mentioned above, MipAB activates the efflux pump MexXY-OprA [27]. MexXY has also been linked to polymyxin resistance through changes to LPS [43]. MexR is a repressor of mexAB-oprM [44]. ArmR is an anti-repressor of MexR and its activity results in expression of the MexAB-OprM efflux pump [44]. The MexAB-OprM efflux pump may mediate colistin tolerance [45]. CpxR activates the MexAB-OprM efflux pump [46]. NalD is also a repressor for the mexAB-oprM operon, and NalC represses ArmR expression and thus de-represses efflux pump expression [47]. The current study found various SNPs associated with efflux pumps associated with polymyxin B (nalC E153Q and nalD R38W), colistin (nalC E153Q/D), and LL-37 (nalC E153Q, nalD R38W, armR Y32C, mexB S1041E and V1042A, mexX A30T, and mexY I536V) high MICs. The nalC E153Q SNP has been previously found in a strain of P. aeruginosa resistant to ciprofloxacin, cefotaxime, and imipenem [48]. The nalC E153Q/D SNP has been found in antibiotic-resistant strains of P. aeruginosa, with the nalC E153Q SNP linked to patient deaths [47]. The other SNPs appear to be novel and so should be examined for their effects on the expression of efflux pumps and the MICs of polymyxin, LL-37, and Mel4.
Another mechanism for polymyxin resistance is changes to the outer membrane that are independent of the arnBCADTEF operon. In the current study, the waaL G330A SNP was associated with high MICs to Mel4, and waaL A110G to high MICs for polymyxin B and colistin. Whilst these SNPs have not been previously associated with resistance, WaaL is a ligase involved in linking the O-antigen polysaccharide to the core of the P. aeruginosa lipopolysaccharide [49], and so the SNPs may affect this function. This should be followed up in future research. Other SNPs in waaL, Y58F, A354S, L363M, and P364S have been found in a highly polymyxin-resistant P. aeruginosa strain [14]. SpE and SpD are involved in spermidine synthesis [50], and spermidine can bind lipopolysaccharide and stabilize and protect the outer membrane of P. aeruginosa in response to polymyxin [50]. In the current study, speE2 A3V was found in strains with high MICs to polymyxin B and colistin but did not appear to be associated with MICs to LL-37 or Mel4. The mpl gene has been shown to be involved in maintaining inner membrane integrity in P. aeruginosa and has a role in polymyxin resistance [51]. The current study found the mpl A303V SNP to be associated with high polymyxin resistance. MprF is an enzyme that modifies anionic phospholipids with L-lysine or L-alanine to give them positive charges into the membrane surface [52]. This can confer resistance to cationic peptides. In Staphylococcus aureus the S295L, T345A, L826F, and S829L SNPs in mprF have been associated with daptomycin resistance [53,54,55]. These SNPs have also been linked to resistance to cationic host defense peptides [56]. The current study found high polymyxin MICs were linked with mprF R188H or N553D SNPs. Whether these function in similar ways to the S. aureus SNPs in mprF should be examined in future studies.

4. Materials and Methods

4.1. Bacterial Strains

Clinical isolates of Pseudomonas aeruginosa, mostly from cases of microbial keratitis in Australia and India, were randomly selected and retrieved from the microbiology biobank (School of Optometry and Vision Science, UNSW Sydney, Australia), where they had been stored at −80 °C. Isolates were revived in nutrient broth (Oxoid Ltd., Basingstoke, Hampshire, UK) prior to testing.
Table 6 provides details of the 40 P. aeruginosa strains used in the current investigation. Strains, randomly selected from the biobank at the School of Optometry and Vision Science, UNSW Sydney, Australia, came from either cases of keratitis (32/40) or cystic fibrosis (4/40) or from other sources. Fifty percent of strains were from Australia; others were from India (16/40) or the USA (1/40) or were of unknown origin. Eighty percent of strains had previously been screened for susceptibility to traditional antibiotics [57,58,59,60,61,62,63,64,65], and the antibiograms are given in Table 6. Whole genome sequences were downloaded, for those that were available, from the NCBI database, and the biosample numbers are given in Table 6.

4.2. Antimicrobial Susceptibility Testing

The minimum inhibitory concentrations (MICs) of colistin, polymyxin B, Mel4, and LL-37 against P. aeruginosa were determined by the broth microdilution method in cation-adjusted Mueller–Hinton broth (CAMHB, Becton Dickinson and Company, Franklin Lakes, NJ, USA). A standardized bacterial inoculum was prepared by adjusting an overnight culture to a 0.5 McFarland standard (~1.5 × 108 colony forming units (CFU)/mL), followed by further dilution in CAMHB to achieve a final concentration of approximately 1.5 × 106 CFU/mL. Serial two-fold dilutions of the antimicrobial working solutions were prepared across a 96-well microtiter plate, with concentrations ranging from 0.125 to 512 µg/mL. Bacterial growth and sterility controls were included to ensure assay validity. The wells were inoculated with 100 µL of the prepared bacterial suspension and incubated at 37 °C for 18–24 h. MICs were determined by visual inspection and confirmed by measuring their absorbance at 660 nm (OD660) using a FLUOstar Omega microplate reader (BMG LABTECH, Ortenberg, Germany). The MIC was defined as the lowest antibiotic concentration producing 90% growth inhibition relative to the untreated growth control. The reference strain P. aeruginosa PA01 was used to validate test accuracy, ensuring reproducibility and alignment with the clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines.

4.3. Detection of Polymyxin Resistance Genes and SNPs

The available whole genome sequences of strains (21 in total; Table 6) were downloaded from the NCBI database from bioprojects PRJNA590804 and PRJNA431326, as well as by searching for individual sequences (ATCC 19660 [biosample SAMN01918025] and 6206 [biosample SAMN12437401]), and details of the genomes can be found in the NCBI database and the original publications [58,65]. Genes in these sequences were compared to the sequence of polymyxin-resistance genes in P. aeruginosa PA01 (accession number NC_002516.2) and other strains (Supplementary Table S1) also downloaded from the NCBI database in BLAST (version 2.17.0) searches. Single nucleotide polymorphism (SNP) calling using BLASTn (version 2.17.0) and amino acid sequences were compared, after translation of the gene sequence using the Expasy Translate tool (https://web.expasy.org/translate/, accessed on 14 September 2025), to protein sequences in the Pseudomonas genome database (https://pseudomonas.com, accessed on 14 September 2025) (or if not present there, from NCBI) using the SIM—Alignment Tool for Protein Sequences in Expasy (https://web.expasy.org/sim/, accessed on 14 September 2025). The genes to be examined were selected from published studies demonstrating them to have associations with polymyxin or AMP resistance, including mcr genes [3,14,20,21,23,27,32,37,38,39,40,41,42,43,44,45,46,47,49,50,51,52,56,68,69].

4.4. Statistical Analysis

The MIC data was tested for normality of its distribution using the Kolmogorov–Smirnov test; as the data were found to not be normally distributed (p < 0.025), assessment of correlations between MICs of the compounds was performed using the Spearman rank test. Fisher’s exact test was used to determine whether there were associations between two categorical variables, MIC and SNP. The null hypothesis was that the SNP distribution was independent of the MIC. As this was the first time, as far as the authors are aware, that such analyses have been conducted with P. aeruginosa strains mostly isolated from ocular infections, corrections for multiple testing were not used in order to give directions for future in depth evaluations.

5. Conclusions

In conclusion, the current study found correlations between high MICs to polymyxin antibiotics, the human cathelicidin LL-37, and a chimeric antimicrobial peptide Mel4. Analysis of genes associated with polymyxin resistance in the P. aeruginosa found several SNPs that could be associated with the cross-resistance. The most significant of these were SNPs in mipB, particularly V469M or G441S. The functional effect of these SNPs on MipB should be investigated in future studies. Also, increasing the number of strains examined will help to make the results more generalizable. As no corrections for multiple associations were made in the current study, some of the associations may have occurred by chance, and so focused research on these initial significant associations should be undertaken in future studies.
This may be the first study focused on ocular P. aeruginosa isolates showing that elevated polymyxin MICs are moderately but consistently associated with increased MICs of both a host (LL-37) and a synthetic (Mel4) AMP, while suggesting specific SNPs in genes, such as mipB V469M and G441S, as candidate genomic markers of cross-resistance. Prior studies typically examined non-ocular isolates or single AMPs or did not analyze phenotype–genotype associations. Whilst polymyxin antibiotics are not currently used to treat P. aeruginosa ocular infections, this may change in the future due to increasing reports of strains being resistant to commonly used antibiotics such as fluoroquinolones, aminoglycosides, and cephalosporins [62,63,64,65]. Polymyxins are increasingly used to treat other infections caused by antibiotic-resistant P. aeruginosa [70]. The current results indicate a potential risk as polymyxin resistance in these isolates was surprisingly high. Also, the findings may have implications for the development of AMPs to produce antimicrobial contact lens coatings [33], where the cross-resistance might allow strains to colonize these types of lenses. This should be followed up in future studies. Confirmatory studies of the associations found in the current study, focused on, for example, mipB V469M or G441S in separate strains, as well as mutating the parent strain PAO1 with these SNPs to determine the effect on resistance, are needed.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms262110499/s1.

Author Contributions

Conceptualization, M.A.D. and M.D.P.W.; methodology, M.A.D., A.K.V., and M.D.P.W.; formal analysis, M.A.D. and M.D.P.W.; investigation, M.A.D.; resources, M.A.D.; writing—original draft preparation, M.A.D. and M.D.P.W.; writing—review and editing, M.A.D., A.K.V., and M.D.P.W.; supervision, A.K.V. and M.D.P.W. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
AMPAntimicrobial peptide
MICMinimum inhibitory concentration
CARDComprehensive Antibiotic Resistance Database
NCBINational Center for Biotechnology Information
BLASTBasic Local Alignment Search Tool
SNPSingle nucleotide polymorphism
MDRMultidrug resistant
XDRExtensively drug resistant
LPSLipopolysaccharide
L-Ara4N4-amino-4-deoxy-L-arabinose

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Ijms 26 10499 g001
Figure 1. Schematic representation of the genes studied and their effects on cellular processes (red boxes) that result in resistance to polymyxins. Red lines represent inhibition; blues lines represent activation. Red and bold letters and numbers represent amino acid changes to that protein significantly associated with high MICs to polymyxins; other letters and numbers in parentheses represent amino acid changes that may be associated with high MICs in certain strains. The red cross represents the fact that no strain contained any of the mcr genes.
Figure 1. Schematic representation of the genes studied and their effects on cellular processes (red boxes) that result in resistance to polymyxins. Red lines represent inhibition; blues lines represent activation. Red and bold letters and numbers represent amino acid changes to that protein significantly associated with high MICs to polymyxins; other letters and numbers in parentheses represent amino acid changes that may be associated with high MICs in certain strains. The red cross represents the fact that no strain contained any of the mcr genes.
Ijms 26 10499 g001
Table 1. Minimum inhibitory concentrations of polymyxin B, colistin, Mel4, and LL37 for the P. aeruginosa isolates.
Table 1. Minimum inhibitory concentrations of polymyxin B, colistin, Mel4, and LL37 for the P. aeruginosa isolates.
StrainsMinimum Inhibitory Concentrations (µg/mL)
Polymyxin BColistinMel4LL37
PA01—resistant12812812864
PA01—sensitive0.50.50.50.5
PA1890.546432
PA122113264
PA9113216
PA17914328
PA214143232
PA33221632
PA182221616
PA223223232
PA2242212832
PA2122825632
PA213283216
PA2062161616
PA1964212864
PA126441616
PA314128128256
PA193843216
PA20916832256
PA124161632128
PA229641283232
PA2256412864128
PA2266412864128
620664128128512
PA2221286432256
PA19812864128128
PA22712864256256
PA22112812812864
ATCC 1966012825664256
PA5512825612864
PA5625643216
PA6525686432
PA2172566416256
PA21625612864256
PA22025612825632
PA542562563216
PA219256256128256
PA8325625625632
PA1235125121664
Table 2. Correlations between MICs for polymyxin B, colistin, Mel4, and LL37.
Table 2. Correlations between MICs for polymyxin B, colistin, Mel4, and LL37.
Statistical
Parameters		Polymyxin B vs. ColistinPolymyxin B vs. Mel4Colistin vs. Mel4Polymyxin B vs. LL37Colistin vs. LL-37Mel4 vs. LL-37
r s 0.7470.2960.3890.4410.5060.419
r s 20.5580.0880.1510.1950.2560.176
p value<0.0010.0630.0130.0050.0010.007
Table 3. Possession of genes associated with the arnBCADTEF operon and its regulation.
Table 3. Possession of genes associated with the arnBCADTEF operon and its regulation.
StrainspmrApmrBphoPphoQcprRcprSparRparScolRcolSarnAarnBarnCarnDarnEarnFarnT
PA189L71R++++E386D, L411M+H398R++C312S, S313GV302A, E376D+++L114FV20A, I509V
PA33+S2P, A4T, V15I, G68S,+++T16SM59I, L153R, S170NH398R++F80Y, C312S, S313G, I388VV302A, E376D++++H151Y, A267S, L337Q, T443A, I509V
PA182L71R++++E386D, L411M+H398R++C312S, S313GV302A+++V14MA267S, R445H, I509V
PA223+++++++H398R++C312S, S313GV302A, E376D++++C7W, H151Y, M274I, T443A, I509V
PA224L71RS2P, A4T+++++H398R+++V302A+E25D, F58L, G208S+V14M, A125TS257R, R502Q, I509V, R521H
PA206D61E, L71RA4T, P369A, A427T++E183DE111D, A175V, N211H, T329S, E386DT135A, L153RA115E, V304I, E343D, H398R, Y407H+H353RT42I, P57A, I138V, S313G, I388V, T636AV302A, E376DE35G, I309V, T316AE25D, F58LA109VV14MC7W, G14A, V89A, A116T, L163F, T166S, A214V, A404G, T443A, R502Q, I509V, S517G
PA126V34L, L71R++++++H398R++C312S, S313G, I388V, L591MK286E, V302A++++V266I, T443A, I509V
PA31+S2P, A4T, V15I, G68S,+++T16SM59I, L153R, S170NH398R++F80Y, C312S, S313G, I388VV302A, E376D+F58L++H151Y, A267V, L337Q, T443A, I509V
PA193D61E, L71RA4T, P369A, A427T++E183DE111D, A175V, N211H, T329S, E386DT135A, L153RA115E, V304I, E343D, H398R, Y407H++T42I, P57A, I138V, S313G, I388V, T636AV302A, E376D+F58L++D154E, V290L
PA225D61E, L71RA4T, P369A, A427T++E183DE111D, A175V, N211H, T329S, E386DT135A, L153RA115E, V304I, E343D, H398R, Y407H++T42I, P57A, I138V, S313G, I388V, T636AA175V, V302A+G206C+V14M, A125TR445H, R502Q, I509V
6206+S2P, A4T, V6A, V15I, G68S,+++T16S, E386DL153R, S170NH398R+G285SC312S, S313G, I388VK286E, V302A, E376DL254F+R28HV14MH151Y, T166I, A267V, I509V
PA198D61E, L71RA4T, P369A, A427T++E183DE111D, A175V, N211H, D220E, T329S, E386DT135A, L153RA115E, V304I, E343D, H398R, Y407H++T42I, P57A, I138V, S313G, I388V, T636AV302A, E376D++++H151Y, A267S, L337Q, T443A, I509V
PA227T31I++++++H398R++A170T, C312S, S313G, I388VA175V, V302A+G206C+V14M, A125TR445H, R502Q, I509V
PA221+S2P, A4T, V6A, V15I, G68S,+++T16S, E386DL153R, S170NH398R++F80Y, C312S, S313G, I388VV302A++R28HV14MH151Y, T166I, A267V, I509V
ATCC 19660L71RS2P, A4T+++T16S, A88V, D153N, V159I, E386DL153R, S170NH398R+A316TI138V, S313G, I388V, V564IG72S, K286E, V302A, E376D+E25D, F58L, V123AA109VV14MC7W, A214V, A225V, L337Q, I509V
PA55+H345Y+++++++++++++++
PA217+S2P, A4T, V6A, V15I, G68S,+++T16S, D386EL153R, S170NH398R++F80Y, C312S, S313G, I388VQ23L, K286E, V302A, E376D+F58LR28H+C7W, H151Y, L337Q, T443A, I509V
PA216L71R++++++H398R++C312S, S313GA259T, V302A, A316V, R340H++++G156R, A267S
PA220L71RS2P, A4T, V6A, V15I, G68S,+++T16S, E386DL153R, S170NH398R++F80Y, C312S, S313G, I388VV302A++R28HV14MH151Y, T166I, A265V, G338E, I509V
PA219+S2P, A4T, V6A, V15I, G68S,+++T16S, E386DL153R, S170NH398R++F80Y, C312S, S313G, I388VV302A, E376D+ F58L++H151Y, A267S, L337Q, T443A, I509V
PA123L71RS2P, A4T++++I93TH398R++C312S, S313G, Q661LV302A, Q336L++++G156R, A267S, R502Q, I509V
+ = possession of gene but no SNPs found. Capital letters refer to standard single letter amino acid designations, and the numbers between the letters refer to the position in the amino acid sequence where the change has occurred. ins = insertion of one or more amino acids at that position. del = deletion of amino acids after that position.
Table 4. Possession of genes associated with efflux pumps and their regulation.
Table 4. Possession of genes associated with efflux pumps and their regulation.
StrainsmipAmipBarmRmexRmexAmexBoprMmexXmexYcpxRnalCnalD
PA189K2R, T3S, A4P, G14S, T20V, Y26D, L28I, L30P, G31S, A32-delN171S, H195Y, G152_R153insGG, A157_S158insAA, E406K, T413N+E126V, V132A+T90I+K329Q, W358RA254G, Q282R, T543A+G71E, S209R+
PA33K2R, T3S, A4P, G14S, T20V, Y26D, L28I, A32-delD225E, P328L, I350V, S368R, K387R, E406K, I427V, R441S, D494G, T496A, S507PT13I, S21T, Y32CE126V+T90I, S1041E, V1042A+A30T, K329Q, L331V, W358RI536V, T543A, G589A, Q840E+G71E, D79E, S209R+
PA182K2R, T3S, A4P, G14S, T20V, Y26D, L28I, A32-delS8P, H82Q, N171S, H195Y, G152_R153insGG, A157_S158insAA, E406K, I427V, D494G, T496A, S507P, G525S+E126V+T90I+K329Q, L331V, W358RT543A+G71E, S209R+
PA223K2R, T3S, A4P, G14S, T20V, A21S, Y26D, L28I, L30P, G31S, S33-delE40K, N171S, H195Y, H246Q, G255S, A258_S259insAA, I350V, K387R, E406K, P410S, I427V, D494G, T496A, S507P, G525S+++T90I+K329Q, L331V, W358RT543A+G71E, S209R+
PA224K2R, T3S, A4P, G14S, T20V, Y26D, L28I, L30P, G31S, S33-delN171S, H195Y, G152_R153insGG, A157_S158insAA, P343R, E406K+E126V+T90I+K329Q, L331V, W358RT543A, Q840E+G71E, S209R, P210L+
PA206no significant homologyN171S, H195Y, G250S, G58_L59insT, V263T, A341T, K387R, R401P, A402T, E406K, D412E, I427V, F440I, M457A, D494G, T496V, S507P, G550E, T551A, V567I, Q595H, P602H, W603XT5A, S21TA103G, E126V+T90I, N248K+L12P, A30T, V322L, K329Q, L331V, G344D, W358RT543A, Q840E, G1035D, N1036T, Q1039R, I1040T+G71E, Q182K, Q208A, S209D, P210-, A211-, Q212-, G213-+
PA126K2R, T3S, A4P, G14S, T20V, Y26D, L28I, L30P, G31S, A32-delN171S, H195Y, P343R, R401del, E456K++K289RT90I+K329Q, L331V, W358RT543A+G71E+
PA31K2R, T3S, A4P, G14S, T20V, Y26D, L28I, A32-delS8P, H195Y, D125E, P328L, I350V, S368R, K387R, E406K, I427V, G441S, D494G, T496V, S507PT13I, S21T, Y32CE126V+T90I, S1041E, V1042A+A30T, K329Q, L331V, W358RI536V, T543A, G589A+G71E, E153Q, S209R+
PA193K2R, T3S, A4P, G14S, T20V, Y26D, L28I, L30P, G31S, S33-delD494G, T496A, S507PT5A, S21TA103G, E126V+T90I, N248K+K329Q, L331V, W358RQ282R, T543A, V980I+G71E, Q182K, Q208A, S209D, P210-, A211-, Q212-, G213-+
PA225K2R, T3S, A4P, G14S, T20V, Y26D, L28I, A32V, S33Q, N34-delS8P, S368R, K287R, E406K, I427V, Y430H, V469M, D494Q, T496A, S507PT5A, S21TA103G, E126V+T90I, N248K+K329Q, W358RT543A+G71E, Q182K, Q208A, S209D, P210-, A211-, Q212-, G213-+
6206K2R, T3S, A4P, G14S, T20V, Y26D, L28I, A32-delS8P, A28V, H195Y, D225E, I350V, K387R, E406K, I427V, V469M, D494G, T496A, S507PS21T, Y32CE126V+T90I+A30T, K329Q, L331V, W358RI536V, T543AS80A, L92RG71E, E153Q, S209R+
PA198K2R, T3S, A4P, G14S, T20V, Y26D, L28I, A32-delS8P, H195Y, D125E, P328L, I350V, S368R, K387R, E406K, I427V, G441S, D494G, T496A, S507P+A103G, E126V+T90I, N248K+A30T, K329Q, L331V, W358RI536V, T543A, G589A, Q840E, N1036T+G71E, Q182K, Q208A, S209D, P210-, A211-, Q212-, G213-+
PA227K2R, T3S, A4P, G14S, T20V, Y26D, L28I, A32V, S33Q, N34-delS8P, S368R, K287R, E406K, I427V, Y430H, V469M, D494Q, T496A, S507P+++T90I+K329Q, W358RT543A+G71E, E153D, A186T+
PA221K2R, T3S, A4P, G14S, T20V, Y26D, L28I, A32-delS8P, H195Y, D125E, I350V, K387R, E406K, I427V, V469M, D494G, T496A, S507PS21T, Y32CE126V+T90I, S1041E, V1042A+A30T, K329Q, L331V, W358RI536V, T543A+G71E, E153Q, S209RR38W
ATCC 19660K2R, T3S, A4P, G14S, T20V, Y26D, L28I, A32V, S33Q, N34-delS8P, A28V, F47L, H95Y, D225E, Q228M, G255S, R288H, I350V, K387R, E406K, R423H, I427V, V469M, D494G, T496A, S507P, G524SS21T, G23E, Y32CE126V+T90I, I186V, S1041E, V1042A+A30T, K329Q, L331V, W358RI536V, T543A, Q840E+G71E, A145V, S209R+
PA55K2R, T3S, A4P, G14S, T20V, Y26D, L28I, L30P, G31S, S33-del++++T90I++++++
PA217K2R, T3S, A4P, G14S, T20V, Y26D, L28I, S33-delS8P, A28V, D225E, A258_S259insAA, I350V, R356H, S368R, K387R, E406K, V469M, D494G, T496A, S507P+E126V+T90I, S1041E, V1042A+A30T, K329Q, L331V, W358RT543A+G71E, E153Q, S209RR38W
PA216no significant homologyN180S, H195Y, P343R, R401del, E406K+++T90I+K329Q, L331V, W358RT543A+G71E, A186T+
PA220K2R, T3S, A4P, G14S, T20V, Y26D, L28I, A32-delS8P, A28V, H195Y, D225E, I350V, K387R, E406K, I427V, V469M, D494G, T496A, S507PS21T, Y32CE126V+T90I, S1041E, V1042A+A30T, K329Q, L331V, W358RI536V, T543A+G71E, E153Q, S209RR38W
PA219K2R, T3S, A4P, G14S, T20V, Y26D, L28I, A32-delS8P, H195Y, D225E, P328L, I350V, S368R, K387R, E406K, I427V, G441S, D494G, T496A, S507P T13I, S21T, Y32CE126V+T90I, S1041E, V1042A+A30T, K329Q, L331V, W358RI536V, T543A, N1036T+G71E, E153Q, S209RR38W
PA123K2R, T3S, A4P, G14S, T20V, Y26D, L28I, L30P, G31S, A32-delN171S, H195Y, H246Q, A258_S259insAA+++T90I+K329Q, L331V, W358RT543A, Q840E+G71E, S209R+
+ = possession of gene but no SNPs found. Capital letters refer to standard single letter amino acid designations, and the numbers between the letters refer to the position in the amino acid sequence where the change has occurred. ins = insertion of one or more amino acids at that position. del = deletion of amino acids after that position.
Table 5. Possession of genes associated with changes to lipopolysaccharide and other factors related to polymyxin resistance in P. aeruginosa.
Table 5. Possession of genes associated with changes to lipopolysaccharide and other factors related to polymyxin resistance in P. aeruginosa.
StrainsoprHpapPmplslyBppgSppgHspeD2speE2waaLPA5005rsmAmprF
PA189+M18L, S56G, M62LM297V+D553NA40D, R110H, A165S, K209Q, Q230K, P388S++S8T, T93A, R147QH399N+S81G, A748V
PA33+M18L, M62L++G81S, H187R, D553N, Q792K A40D, A50V, K209Q, P388S+T271AS8T, T93A, R147Q, L179F, G330A, I398T++R187H, N554D, N670S, A731T, A748V, D772E, K793Q
PA182+M18L, S56G, M62L++G81S, H187R, L199S, D553N, G587S, A731T, E772DA40D, A165S, K209Q, P388S++S8T, R147Q++G587S, A731T, A748V, K793Q
PA223+M18L, S56G, M62L++G81S, H187R, L199S, D553N, G587SA40D, G95S, A165S, K209Q, Q230K++S8T, Y58F, L61I, R64Q, G71R, F75L, F78I, S83A, S90L, T98A, L102F, A109V, F112L, A115V, A116G, E121Q, L124E, K127R, T128N, A129I, I139F, S140A, A143V, L145V, L146V, R147H, Y149H, W150L, D151Q, A152T, N153H, P154_L156insAW, L155M, T158S, A178V, L179V, A182V, P189H, I190L, I197L, L201I, G203C, G204C, I207L, A208S, V216I, G217A, A221C, M223G, V226L, L227V, D230N, R231Q, A234T, A237V, L238I, A239G, L242A, A243L, A245M, L246I, L247V, G248A, L251F, Y252N, V255L, I256V, A261V, A269S, D270E, A271S, S276G, V292K, S294A, I316V, V322S, G330V, S332A, K337R, S338D, A340M, A354S, L363M, P364S, M377L, I385V, Q387KH399N+G587S, A748V, D772E, K793Q
PA224+M18L, S56G, M62LM297V+G81S, H187R, L199S, D553N, G587S, A731T, E772DA40D, A165S, K209Q, Q230K, P388S++S8T, T93A, R147QH399N+G587S, A731T, A748V, K793Q
PA206+M18L, S56G, M62L, S74GM297V+D70E, V103I, H187R, V192I, A545V, G587S, A731EA40D, A109T, S190R, S203T, K209Q, Q230K+T271AS8T, Y58F, L61I, R64Q, G71R, F75L, F78I, S83A, S90L, T98A, L102F, A109V, F112L, A115V, A116G, E121Q, L124E, K127R, T128N, A129I, I139F, S140A, A143V, L145V, L146V, R147H, Y149H, W150L, D151Q, A152T, N153H, P154_L156insAW, L155M, T158S, A178V, L179V, A182V, P189H, I190L, I197L, L201I, G203C, G204C, I207L, A208S, V216I, G217A, A221C, M223G, V226L, L227V, A228V, D230N, R231Q, A234T, A237V, L238I, A239G, L242A, A243L, A245M, L246I, L247A, G248A, L251F, Y252N, V255L, I256V, A261V, A269S, D270E, A271S, S276G, V292K, S294A, I316V, V322S, G330V, S332A, K337R, S338D, A340M, A354S, L363M, P364S, M377L, I385V, Q387KQ396K, H399N, R403Q+D71E, S82G, V104I, V93I, S100L, V546A, N554D, G587S, A731E, A748V, D772E, K793Q
PA126+M18L, S56G, M62L++G81S, D175N, D553N, G587SA40D, G95S, A165S, K209Q++S8T, T93A, R147QH399N+D175N, R187H, S199L, G587S, A748V, D772E, K793Q
PA31+M18L, M62LD411A+G81S, L199S, N670S, A731TA40D, A50V, K209Q, P388S+V217I, T271AS8T, T93A, R147Q, L179F, G330A, I398TH399N+R187H, N554D, N670S, A731T, A748V, D772E, K793Q
PA193+S56G, M62LM297V+H65R, H187R, L199S, G304S, D553N, E772D, Q792KA40D, R110H, A165S, K209Q, Q230K, P388S++S8T, R147Q, L281FH399N+H65R, S81G, G304S, A758V
PA225+S56G, M62LM297V+H187R, L199S, D553N, I740M, E772DA40D, R110H, A165S, K209Q, Q230K, P388S++S8T, T93A, R147Q, L179F, S276G, G330AH399N+S81G, I740M, A748V, K793Q
6206+M18L, M62L++P14L, G81S, L199S, D553N, G587SA40D, R110H, A165S, K209Q, P231A, P388S++S8T, T93A, A129V, R147Q, L179F, I256F, S276G, G330AD23E, H399D+P13L, R187H, G587S, A748V, D772E, K793Q
PA198+M18L, M62L++G81S, L199S, N670S, A731TA40D, A50V, K209Q, P388S+V217I, T271AS8T, T93A, R147Q, L179F, G330A, I398TH399N+R187H, N554D, N670S, A731T, A748V, D772E, K793Q
PA227+M18L, S56G, M62LM297V, A303V+H187R, L199S, D553N, I740M, E772DA40D, R110H, A165S, K209Q, Q230K, P388S++S8T, R147Q, L281F, S276G, G330AH399N+S81G, I740M, A748V, K793Q
PA221+M18L, M62L++G81S, L199S, D553N, G587SA40D, R110H, A165S, K209Q, P231A, P388S++S8T, T93A, A129V, R147Q, L179F, I256F, S276G, G330AD23E, H399D+R187H, G587S, A748V, D772E, K793Q
ATCC 19660++++E616G, A731YK209Q++S8T, T93A, R147Q, L179F, G330AH399N+S81G, R187H, S199L, N553D, E616G, A731T, A748V, D772E, K793Q
PA55+M18L, S56G, M62L++G81S, H187R, L199S, D553N, G587S, E772DA40D, A165S, K209Q, P388S+++++G587S, A748V, K793Q
PA217+M18L, M62L++L199S, A270VA40D, K209Q, P388S+C161S, A165T, V217T, S244A, T271A, E277D, S244A, T271A, E277D, P326A, E235GS8T, R16H, Y58F, L61I, R64Q, G71R, F75L, F78I, S83A, S90L, T98A, L102F, A109V, F112L, A115V, A116G, E121Q, L124E, K127R, T128N, A129I, I139F, S140A, A143V, L145V, L146V, R147H, Y149H, W150L, D151Q, A152T, N153H, P154_L156insAW, L155M, T158S, A178V, L179V, A182V, P189H, I190L, I197L, L201I, G203C, G204C, I207L, A208S, V216I, G217A, A221C, M223G, V226L, L227V, D230N, R231Q, A234T, A237V, L238I, A239G, L242A, A243L, A245M, L246I, L247V, G248A, L251F, Y252N, V255L, I256V, A261V, A269S, D270E, A271S, S276G, V292K, S294A, I316V, V322S, G330I, S332A, K337R, S338D, A340M, A354S, L363M, P364S, M377L, I385V, Q387KH399N, N400D+S81G, R187H, A270V, N553D, A748V, D772E, K793Q
PA216+M18L, S56G, M62LM297V, A303V+A12T, G81S, H187R, L199S, L243MA40D, K209Q++S8T, Y58F, L61I, G71R, F75L, F78I, S83A, S90L, T98A, L102F, A109V, F112L, A116G, E121Q, L124E, K127R, T128A, A129L, V138T, I139L, S140A, A143V, R147H, Y148F, Y149H, W150I, D151Q, A152S, N153P, P154_L156insAW, L155M, T158S, A178V, A182V, P189H, I190A, I197L, L201I, G203C, G204C, I207L, V216I, T219A, A221C, L222M, M223A, V226L, A234T, A237V, L238I, A239G, L240I, A241V, L242V, A245L, L247V, G248V, L251F, L252V, Y253N, V255L, T257I, A261V, A269S, D270E, A271S, S276G, V292K, S294A, I316V, V322S, G330V, S332A, K337R, S338D, A340M, A354S, L363M, P364S, M377L, I385V, Q387KH399N+A12T, L242M, N553D, A731T, A748V, D772E, K793Q
PA220+M18L, M62L++G81S, L199S, D553N, G587SA40D, R110H, A165S, K209Q, P231A, P388S+T271AS8T, T93A, A129V, R147Q, L179F, I256F, S276G, G330AD23E, H399D+R188H, G587S, D772E, K793Q
PA219+M18L, M62L++G81S, L199S, N670S, A731TA40D, A50V, K209Q, P388S+V217I, T271AS8T, T93A, R147Q, L179F, G330A, I398TH399N+R188H, N553D, N670S, A731T, A748V, D772E, K793Q
PA123+M18L, S56G, M62LM297V, V358I+G81S, H187R, L199S, D553N, G587S, E772DA40D, A165S, K209Q, P388S+A3VS8T, Y58F, L61I, R64Q, G71R, F75L, F78I, S83A, S90L, T98A, L102F, A109V, A110G, F112L, A115V, A116G, E121Q, L124E, K127R, T128N, A129I, I139F, S140A, A143V, L145V, L146V, R147H, Y149H, W150L, D151Q, A152T, N153H, P154_L156insAW, L155M, T158S, A178V, L179V, A182V, P189H, I190L, I197L, L201I, G203C, G204C, I207L, A208S, V216I, G217A, A221C, M223G, V226L, L227V, D230N, R231Q, A234T, A237V, L238I, A239G, L242A, A243L, A245M, L246I, L247V, G248A, L251F, Y252N, V255L, I256V, A261V, A269S, D270E, A271S, S276G, V292K, S294A, I316V, V322S, G330V, S332A, K337R, S338D, A340M, A354S, L363M, P364S, M377L, I385V, Q387KH399N+G587S, A748V, K793Q
+ = possession of gene but no SNPs found. Capital letters refer to standard single letter amino acid designations, and the numbers between the letters refer to the position in the amino acid sequence where the change has occurred. ins = insertion of one or more amino acids at that position. del = deletion of amino acids after that position.
Table 6. Details of the Pseudomonas aeruginosa isolates.
Table 6. Details of the Pseudomonas aeruginosa isolates.
Strain NumberInfection Type, Site of Isolation, Country and Date of IsolationKnown Antibiotic
Resistance Characteristics		Biosample Number (NCBI)
PA01—
resistant		Mutant derived from
PAO1-sensitive [57]		Chloramphenicol, Colistin [57]Not available
PA01—
sensitive		Wound, Unknown, Australia, 1954Chloramphenicol [57,66]SAMN02603714
PA189Keratitis, Eye, India, 2017 CIP S, LEV S, GEN S, TOB R, PIP S, IMI R, CFT S [63]SAMN13340385
PA122Keratitis, Eye, Australia, 2006Not knownNot available
PA9Keratitis, Eye, Australia, 1994Not knownNot available
PA179Keratitis, Eye, Australia, 2006CIP R, LEV S, GEN S, TOB S, PIP S, IMI S, CFT S [64]Not available
PA214Keratitis, Eye, India, 2017Not knownNot available
PA33Keratitis, Eye, India, 1998CIP R, LEV R, GEN R, TOB R, PIP R, IMI R, CFF R [63]SAMN08435058
PA182Keratitis, Eye, Australia, 2006CIP S, LEV S, GEN S, TOB S, PIP S, IMI R, CTF S [63]SAMN13340383
PA223Keratitis, Eye, Australia, 2018CIP R, LEV S, GEN S, TOB S, PIP R, IMI S, CFT I [63]SAMN16123412
PA224Keratitis, Eye, Australia, 2018CIP R, LEV S, GEN S, TOB S, PIP S, IMI R, CFT I [63]SAMN16123413
PA212Keratitis, Eye, India, 2017CIP R, LEV S, GEN S, TOB S, PIP S, IMI R, CFT S [58]Not available
PA213Keratitis, Eye, India, 2017CIP R, LEV S, GEN S, TOB S, PIP S, IMI I, CFT I [58]Not available
PA206Keratitis, Eye, India, 2017CIP I, LEV S, GEN S, TOB S, PIP S, IMI S, CFT S [59]SAMN13340389
PA196Keratitis, Eye, India, 2017CIP S, LEV S, GEN S, TOB S, PIP S, IMI R, CFT S [59]Not available
PA126Keratitis, Eye, Australia, 2006CIP S, LEV S, GEN S, TOB S, PIP S, IMI R, CFT R [64]SAMN13340377
PA31Keratitis, Eye, India, 1998CIP R, LEV R, MOX R, GEN R, CFT I, CEFE I, IMI I, TIC I, AZT I [65]SAMN08435056
PA193Keratitis, Eye, India, 2017CIP S, LEV S, GEN S, TOB S, PIP S, IMI S, CFT S [58]SAMN13340386
PA209Keratitis, Eye, India, 2017CIP I, LEV I, GEN S, TOB S, PIP R, IMI S, CFT S [63]Not available
PA124Keratitis, Eye, Australia, 2006CIP I, LEV S, GEN S, TOB S, PIP S, IMI R, CFT S [63]Not available
PA229Keratitis, Eye, Australia, 2018CIP S, LEV S, GEN S, TOB S, PIP S, IMI R, CFT S [63]Not available
PA225Keratitis, Eye, Australia, 2018CIP R, LEV R, GEN S, TOB S, PIP S, IMI R, CEF S [63]SAMN16123414
PA226Keratitis, Eye, Australia, 2018CIP I, LEV I, GEN S, TOB S, PIP R, IMI R, CFT S [63]Not available
6206Keratitis, Eye, USA, 1995CIP S, TOB S [60,61]SAMN12437401
PA222Keratitis, Eye, Australia, 2017CIP S, LEV I, GEN S, TOB S, PIP S, IMI R, CFT R [63]Not available
PA198Keratitis, Eye, India, 2017CIP R, LEV R, MOX R [62]SAMN13340387
PA227Keratitis, Eye, Australia, 2018CIP R, LEV R, GEN S, TOB S, PIP S, IMI R, CFT I [63]SAMN16123415
PA221Keratitis, Eye, India, 2017CIP R, LEV R, GEN R, TOB R, PIP R, IMI R, CFT R [63]SAMN13340395
ATCC 19660Unknown, 1965CIP I, GEN S [61,67]SAMN01918025
PA55Cystic Fibrosis, Sputum, Australia, 2003CIP S, LEV S, MOX S, CFT R, CEFE R, IMI R, TIC R, AZT S [65]SAMN08435068
PA56Cystic Fibrosis, Sputum, Australia, 2003Not knownNot available
PA65Cystic Fibrosis, Sputum, Australia, 2003Not knownNot available
PA217Keratitis, Eye, India, 2017CIP R, LEV R, GEN S, TOB S, PIP R, IMI R, CFF R [63]SAMN13340391
PA216Keratitis, Eye, India, 2017CIP R, LEV R, GEN S, TOB S, PIP R, IMI R, CFT R [63]SAMN13340390
PA220Keratitis, Eye, India, 2017CIP R, LEV S, GEN S, TOB S, PIP S, IMI R, CFT R [63]SAMN13340394
PA54Keratitis, Eye, Australia, 2003Not knownNot available
PA219Keratitis, Eye, India, 2017CIP R, LEV S, GEN S, TOB S, PIP S, IMI R, CFT I [63]SAMN13340393
PA83Cystic Fibrosis, Sputum, Australia, 2003Not knownNot available
PA123Keratitis, Eye, Australia, 2006CIP I, LEV I, GEN S, TOB S, PIP S, IMI I, CFT I [63]SAMN13340376
CIP = ciprofloxacin, LEVO = levofloxacin, MOX = moxifloxacin, CFT = ceftazidime, CEFE = cefepime, TIC = ticarcillin, IMI = imipenem, AZT = Aztreonam, PIP = piperacillin, TOB = tobramycin, GEN = gentamicin. S = susceptible, R = resistant, I = intermediate.
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Damtie, M.A.; Vijay, A.K.; Willcox, M.D.P. Detection of Genes Associated with Polymyxin and Antimicrobial Peptide Resistance in Isolates of Pseudomonas aeruginosa. Int. J. Mol. Sci. 2025, 26, 10499. https://doi.org/10.3390/ijms262110499

AMA Style

Damtie MA, Vijay AK, Willcox MDP. Detection of Genes Associated with Polymyxin and Antimicrobial Peptide Resistance in Isolates of Pseudomonas aeruginosa. International Journal of Molecular Sciences. 2025; 26(21):10499. https://doi.org/10.3390/ijms262110499

Chicago/Turabian Style

Damtie, Meseret Alem, Ajay Kumar Vijay, and Mark Duncan Perry Willcox. 2025. "Detection of Genes Associated with Polymyxin and Antimicrobial Peptide Resistance in Isolates of Pseudomonas aeruginosa" International Journal of Molecular Sciences 26, no. 21: 10499. https://doi.org/10.3390/ijms262110499

APA Style

Damtie, M. A., Vijay, A. K., & Willcox, M. D. P. (2025). Detection of Genes Associated with Polymyxin and Antimicrobial Peptide Resistance in Isolates of Pseudomonas aeruginosa. International Journal of Molecular Sciences, 26(21), 10499. https://doi.org/10.3390/ijms262110499

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