Nucleotide substitutions in the mexR, nalC and nalD regulator genes of the MexAB-OprM efflux pump are maintained in Pseudomonas aeruginosa genetic lineages

Abstract

Pseudomonas aeruginosa has different resistant mechanisms including the constitutive MexAB-OprM efflux pump. Single nucleotide polymorphisms (SNPs) in the mexR, nalC, and nalD repressors of this efflux pump can contribute to antimicrobial resistance; however, it is unknown whether these changes are mainly related to genetic lineages or environmental pressure. This study identifies SNPs in the mexR, nalC, and nalD genes in clinical and environmental isolates of P. aeruginosa (including high-risk clones). Ninety-one P. aeruginosa strains were classified according to their resistance to antibiotics, typified by multilocus sequencing, and mexR, nalC, and nalD genes sequenced for SNPs identification. The mexAB-oprM transcript expression was determined. The 96.7% of the strains were classified as multidrug resistant. Eight strains produced serine carbapenemases, and 11 strains metallo-β-lactamases. Twenty-three new STs and high-risk clones ST111 and ST233 were identified. SNPs in the mexR, nalC, and nalD genes revealed 27 different haplotypes (patterns). Sixty-two mutational changes were identified, 13 non-synonymous. Haplotype 1 was the most frequent (n = 40), and mainly identified in strains ST1725 (33/40), with 57.5% pan drug resistant strains, 36.5% extensive drug resistant and two strains exhibiting serin-carbapenemases. Haplotype 12 (n = 9) was identified in ST233 and phylogenetically related STs, with 100% of the strains exhibiting XDR and 90% producing metallo-β-lactamases. Haplotype 5 was highly associated with XDR and related to dead when compared to ST1725 and ST233 (RRR 23.34; p = 0.009 and RRR 32.01; p = 0.025). A significant relationship between the mexR-nalC-nalD haplotypes and phylogenetically related STs was observed, suggesting mutational changes in these repressors are highly maintained within genetic lineages. In addition, phylogenetically related STs showed similar resistant profiles; however, the resistance was (likely or partly) attributed to the MexAB-OprM efflux pump in 56% of the strains (only 45.05% showed mexA overtranscription), in the remaining strains the resistance could be attributed to carbapenemases or mechanisms including other pumps, since same SNPs in the repressor genes gave rise to different resistance profiles.

Fig 1. Phylogenetic network based on the MLST genotyping of the P. aeruginosa strains (ST/haplotypes).

Phylogenetic relationships, evolutionary history, clonal complexes, and relationship between the mexR-nalC-nalD haplotypes and the STs are shown. P. aeruginosa isolates (n = 91, 48 STs, 27 mexR-nalC-nalD haplotypes). Circles represent sequence types (STs); Circumference is based on ST frequency; Two or more strains with the same ST are depicted as fractions in each circle (ST1725, n = 34 strains); Lines connect locus variants; Numbers indicate the number of locus variants among the connected STs. Clonal complexes (CC) formed are highlighted in rectangles and described as I, II, III, IV, V and, VI. STs not grouped into a CC are considered singletons (>3 locus variants with other STs). Numbers inside the circles (1–27) corresponds with the mexR-nalC-nalD haplotype and are colored differently. †: Fatal patient outcome.

Fig 2. Phylogenetic network based on the MLST genotyping of the P. aeruginosa strains (ST/haplotypes/resistance).

Relationship between the mexR-nalC-nalD haplotypes, the STs and, the susceptibility profiles are shown. P. aeruginosa isolates (n = 91, 48 STs, 27 mexR-nalC-nalD haplotypes). Circles represent sequence types (STs); Circumference is based on ST frequency; Two or more strains with the same ST, including ST1725 are depicted as fractions in each circle (n = 34 strains); Lines connect locus variants; Numbers indicate the number of locus variants among the connected STs. Clonal complexes (CC) formed are highlighted in rectangles and described as I, II, III, IV, V and, VI. STs not grouped into a CC are considered singletons (>3 locus variants with other STs). Number inside the circles (1–27) corresponds with the mexR-nalC-nalD haplotype and are colored differently. †: Fatal patient outcome. Susceptibility profiles: S (green), sensitive; MDR (yellow), multidrug resistant; XDR (orange), extensively drug resistant; PDR (red), pan drug resistant. *S: Serine carbapenemase; M*: Metallo-β-lactamase.

Fig 3. Neighbor-net graph based on the MLST genotyping of the P. aeruginosa strains.

P. aeruginosa isolates (48 STs). Rectangular boxes represent the high probability of extensive homologous recombination. The PHI test detected statistically significant evidence of recombination (p = 0.0). Clonal complexes (CC) formed are highlighted in rectangles and described as (I, II, III, IV, V, and VI). The mexR-nalC-nalD haplotype of each CC is shown.

Fig 4. Principal component analysis of the 91 P. aeruginosa strains.

P. aeruginosa isolates (n = 91). Each strain is represented by a colored dot according to mexR-nalC-nalD haplotype (Fig 1). †: Fatal patient outcome. Variables included: Outcome (black vector): living, died; Resistance (purple vector): sensitive (S), multi-drug resistant (MDR), extensively drug resistant (XDR), and pan drug resistant (PDR); ST (blue vector) (n = 48); MexAB-OprM haplotype (mexR-nalC-nalD) (green vector) (n = 27); Isolation site (yellow vector): hospital, environmental; Date (pink vector): 2007–2013; Hospital ward (aqua vector): Stx, surgical therapy; E, emergency room; N, neurology; PICU, pediatric intensive care unit; P, pediatrics; S, surgery; Neph, nephrology; O, oncology; It, internal therapy; C, cardiology; NICU, neonatal intensive care unit; Ne, necropsy; IMed, internal medicine.