2025 Volume 48 Issue 3 Pages 230-233
The rapid emergence of drug-resistant microbes has recently become a major concern in the medical field. In Pseudomonas aeruginosa, one of the most important mechanisms underlying antibiotic resistance is MexAB-OprM system, increases in this efflux system result in greater resistance to a wide range of drugs, and genetic mutations have been identified as a contributing factor. Thus, this study characterized point mutations in the mexB gene that are common to 39 clinical P. aeruginosa isolates obtained from The Pseudomonas Genome Database. Basic Local Alignment Search Tool (BLAST) was used to compare the mexB gene sequences of those 39 strains with PAO1. The majority of these point mutations were silent mutations without amino acid mutations. Mutations 2730, 495, and 2280, which were abundant in the strains examined, were characterized by greater codon usage after the mutation. A positive correlation has been reported between tRNA levels and codon usage in Escherichia coli, and the same relationship may be present in P. aeruginosa. In this study, the silent mutations observed in many strains mainly involved the substitution of C or G, which resulted in a higher codon usage and stronger binding power after than before the mutation. This change is considered advantageous for survival in the human body by increasing the translation efficiency of the MexB protein. Thus, combining the silent mutation identified in this study with information on the expression level of mexB is expected to be used as an indicator to identify multidrug-resistant P. aeruginosa.
Pseudomonas aeruginosa is a Gram-negative bacterium that causes opportunistic infections. It is abundant in nature, including in the human intestinal tract, and may grow in low-nutrient content environments. It also causes respiratory and urinary tract infections and septicemia in sick, elderly, and immunocompromised individuals. Recently, many multidrug-resistant P. aeruginosa (MDRP) strains have been identified in clinical settings.
P. aeruginosa and multidrug resistance in bacteria have become a major concern in the medical field recently. According to an estimate by a British government agency, the annual number of deaths from infectious diseases caused by resistant bacteria is expected to exceed 10 million by 2050 if no measures are taken to address it.1) There are four primary mechanisms by which bacteria acquire drug resistance, which are as follows: change in the target site of the drug, inactivation of the drug, decrease in membrane permeability, and expulsion of the drug from the body using drug efflux pumps.2) Of these, the “drug efflux pump,” which effluxes a substantial amount of antimicrobial agents and is inherent in all bacteria, is an important factor contributing to the acquisition of multidrug resistance. There are several types of efflux pumps and their mechanisms of action, expression levels, and the types of drugs they expel vary drastically. In P. aeruginosa, the resistance nodulation cell division (RND) type MexAB-OprM system is the main type of drug efflux pump, which consists of 3 subunits: an inner transmembrane subunit (MexB in P. aeruginosa), outer transmembrane subunit (OprM in P. aeruginosa), and periplasmic subunit (MexA in P. aeruginosa).3,4) It functions by forming a trimer and its driving force is the proton motive force.5,6) In Gram-negative bacteria, the resistance nodulation cell division-type efflux pump makes a significant contribution to drug resistance in terms of its ability to efflux drugs.7) MexAB-OprM may efflux a wide range of antimicrobial agents and is typically expressed at high levels.8) It also effluxes acylated homoserine lactones, self-produced autoinducers, and plays a role in quorum sensing.9) Therefore, further studies on MexAB-OprM are warranted.
Furthermore, higher expression levels of MexAB-OprM are associated with a greater efflux capacity and stronger drug resistance, and point mutations in genes may be a contributing factor. A previous study suggested that point mutations in the mexR gene, a protein that regulates the expression of the P. aeruginosa drug efflux pump mexB, are involved in the acquisition of drug resistance.10) In this study, we attempted to identify and characterize point mutations in the mexB gene that are common to clinical isolates of P. aeruginosa for rapid evaluation of their mexB expression levels and drug resistance.
We obtained the genome sequences of P. aeruginosa clinical isolates from The Pseudomonas Genome Database (https://pseudomonas.com).11) As a specific procedure, we initially searched the site using the keyword “mexB” as “Exact Name” under the condition of “Complete genomes only.” Based on the search results, we selected all human-derived clinical isolates. Moreover, P. aeruginosa PAO1 was used as the reference strain.12) The mexB sequences of clinical isolates and PAO1 were then compared using the Basic Local Alignment Search Tool (BLAST) (https://blast.ncbi.nlm.nih.gov/Blast.cgi).
We obtained the genome sequences of 39 clinical isolates. The countries in which these strains are registered belong to different regions: China (7), India (6), U.S.A. (6), Kazakhstan (3), Thailand (2), Australia (2), others (10), and unknown (3) (Supplementary Table 1). These strains were also isolated from a variety of specimens. Among the 33 isolates whose collection sites were identified, sputum (16) was the most common, while the remainder were collected from blood (5), wound (4), urine (3), and others (5) (Supplementary Table 1). Regarding multilocus sequence typing (MLST) information, ST235 (2), ST111 (1), ST233 (1), ST357 (6), ST298 (1), ST175 (1), and ST277(1), which are global high-risk clones associated with multidrug resistance and extensive drug resistance, were also included13) (Table 1).
| Strain | MLST | Mismatches | Strain | MLST | Mismatches |
|---|---|---|---|---|---|
| PAO1 (Reference) | ST549 | BA7823 | ST357 | 20 | |
| 1334/14 | ST234 | 6 | SP2230 | ST357 | 20 |
| 97 | ST234 | 6 | SP4371 | ST357 | 20 |
| 1811-13R031 | ST395 | 6 | UCBPP-PA14 | ST253 | 17 |
| 1811-18R001 | ST395 | 6 | PAK | ST693 | 15 |
| CCUG 70744 | ST395 | 7 | PA7 | ST1195 | 82 |
| LESB58 | ST146 | 6 | AZPAE15042 | ST2211 | 85 |
| 243931 | ST235 | 20 | CF39S | ST175 | 6 |
| CCUG 51971 | ST235 | 20 | E90 | ST282 | 6 |
| 519119 | ST360 | 5 | HOU1 | ST2855 | 9 |
| WCHPA075019 | ST277 | 5 | INP-43 | – | 8 |
| 60503 | ST773 | 16 | SCAID WND1-2019 | – | 7 |
| ST773 | ST773 | 15 | SCAID WND2-2019 | – | 7 |
| T2436 | ST1121 | 15 | SCAID WND3-2019 | ST1031 | 8 |
| AES1M | ST649 | 8 | T2101 | ST708 | 6 |
| AES1R | ST649 | 8 | PABL048 | ST298 | 12 |
| AG1 | ST111 | 9 | C7447m | – | 5 |
| B14130 | ST357 | 20 | A681 | ST274 | 4 |
| B41226 | ST357 | 20 | K34-7 | ST233 | 11 |
| BA15561 | ST357 | 20 | MRSN12280 | – | 20 |
MLST, multilocus sequence typing.
The number of mutations in each strain is presented in Table 1. Strains belonging to the same MLST had a similar number and position of point mutations (Table 1, Supplementary Table 1). Most of these point mutations were silent and without amino acid mutations (Supplementary Table 2).
In the genomic data obtained, 32 silent mutations without amino acid mutations and five nonsynonymous mutations with amino acid mutations in the mexB gene were detected in 3 or more of the 39 strains (Supplementary Table 2). Four of these 5 nonsynonymous mutations were found in the same 11 strains. Furthermore, the top 9 mutations with the highest number of confirmed strains are summarized in Table 2. These top 9 mutations were all silent mutations. The positions of these 9 mutations were dispersed throughout the MexB protein and no particular bias was confirmed (Supplementary Fig. 1). Eight of the top 9 had greater codon usage after the mutation. Codon usage refers to the ratio of the number of occurrences of a particular codon to that of all codons that code for the same amino acid. It uses the numbers as previously reported by West and Iglewski.14) Additionally, these 8 mutations were all mutations to C or G. The top 3 mutations, 2730, 495, and 2280, were T→C, A→G, and T→C mutations, respectively, and the codon usage increased significantly from 0.11 to 0.79, 0.03 to 0.62, and 0.14 to 0.86, respectively, which were confirmed in most of the 39 strains (39, 38, and 37 strains, respectively). We introduced these 3 mutations into the MexB expression plasmid and found that they increased resistance to ciprofloxacin (Supplementary Fig. 2). Ciprofloxacin is the substrate drug for MexB among the three drugs used to detect MDRP in Japan (imipenem, amikacin, and ciprofloxacin).
| Mutation | Pre-mutation base |
Post-mutation base |
Pre-mutation codon |
Post-mutation codon |
Comparison of codon usage before and after the mutation |
Number of samples |
|---|---|---|---|---|---|---|
| 2730 | T | C | GGT | GGC | 0.11→0.79 | 39 |
| 495 | A | G | CCA | CCG | 0.03→0.62 | 38 |
| 2280 | T | C | GAT | GAC | 0.14→0.86 | 37 |
| 3117 | G | A | CAG | CAA | 0.86→0.14 | 23 |
| 2067 | T | C | GGT | GGC | 0.11→0.79 | 21 |
| 2226 | T | C | CTT | CTC | 0.02→0.24 | 17 |
| 2892 | C | G | GAA | GAG | 0.38→0.62 | 16 |
| 1749 | T | C | AGT | AGC | 0.04→0.40 | 15 |
| 2079 | A | G | GAA | GAG | 0.38→0.62 | 15 |
In P. aeruginosa, MexAB-OprM is an important factor that significantly contributes to the acquisition of drug resistance. The spread of strains that highly express these factors in clinical settings increases the treatment difficulty. Therefore, the functions of these drug efflux pumps in clinical isolates need to be closely monitored. As previously mentioned, point mutations in the mexR gene, a protein that regulates the expression of the P. aeruginosa drug efflux pump, have been suggested to contribute to drug resistance, and mutations in genes related to drug efflux pumps warrant further study.10) Therefore, it is crucial to investigate point mutations in the mexB gene, as performed in the present study. If many P. aeruginosa strains with the point mutations identified in this study acquire multidrug resistance by highly expressing mexB, it can be used as an indicator to identify MDRP.
Among the strains examined herein, PA7 and AZPAE15042 were taxonomic outliers based on comparisons with the housekeeping genes.15,16) These strains had a high number of mexB mutations, which might reflect taxonomic differences. Most of the point mutations identified in this study were silent mutations. Furthermore, the codon usage was often higher after than before the mutation, and a positive correlation has been reported between the frequency of codon usage and the amount of tRNA in Escherichia coli.17) Thus, the higher the codon usage, the greater the amount of tRNA corresponding to it, and this is considered to increase the efficiency of translation. By assuming that the same mechanism exists in P. aeruginosa, these point mutations without amino acid mutations (silent mutations) in clinical isolates are considered to provide individuals with survival advantage. This change may increase the drug efflux capacity, which is likely to be advantageous for survival in the human body, where there are plenty of opportunities for antimicrobial exposure.
Furthermore, previous studies demonstrated that P. aeruginosa preferentially used codons with strong codon–anticodon interactions.14) In the present study, the silent mutations observed in many strains primarily involved the substitution of C or G, which were considered to have a stronger binding power after than before the mutation. In this case also, the translation efficiency of the MexB protein is higher than before the mutation, which increases the drug efflux capacity, a favorable factor for survival against antimicrobial exposure in the human body. Therefore, these mutations can easily occur and promote drug resistance by P. aeruginosa in clinical settings.
The effects of the codon may extend to the mRNA. Previous studies showed a relationship between the codon usage and the mRNA stability in E. coli.18) Hence, these point mutations may stabilize mRNA, resulting in upregulated protein expression. The upregulated expression of mexB can contribute to the acquisition of drug resistance.
Moreover, mexB expression in clinical strains of P. aeruginosa is elevated and it leads to the acquisition of drug resistance.19–21)
In the genomic data obtained, 5 nonsynonymous mutations with amino acid mutations in the mexB gene were also detected in 3 or more of the 39 strains. These 5 nonsynonymous mutations were related to 3 amino acid changes (G957D, S1041E, and V1042A). The nonsynonymous mutations are likely to cause the possible structural changes in RND pumps, inducing drug susceptibility changes. For example, a clinical isolate of Salmonella enterica serovar Typhimurium with a G288D substitution in the distal binding pocket of AcrB had significantly lower ciprofloxacin sensitivity.22) Moreover, in clinical isolates of Salmonella Typhi, reduced sensitivity to azithromycin was caused by the R717Q mutation on the periplasmic cleft of AcrB.23) Regarding MexB, our previous studies revealed that the effect of the efflux pump inhibitor ABI-PP is decreased by amino acid mutations in multiple locations.24) However, the G957D, S1041E, and V1042A mutations in this study were all located in the region that protrudes into the cytoplasm at the edge of the protein, and homology model analysis also suggested that the impact on the whole protein structure was negligible (Supplementary Fig. 3).
Thus, the following studies are required to further examine the hypotheses derived from the present results. The relationship between codon usage and tRNA needs to be clearly demonstrated in P. aeruginosa, as in E. coli. Furthermore, the detailed mechanisms by which the mutation affects the expression level of the efflux pump warrant further study using P. aeruginosa in which the point mutation identified in the present study is artificially inserted.
The authors would like to thank S. Kawakami and T. Yoneda for their assistance with the data analysis.
This work was supported by the Research Program for CORE lab, CORE2-A lab, and Grants-in-Aid of “Dynamic Alliance for Open Innovation Bridging Human, Environment and Materials” in “Network Joint Research Center for Materials and Devices” from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT); KAKENHI (23K27322, 23H02631, JP18K14902, and JP21K16318) from the Japan Society for the Promotion of Science (JSPS); Kowa Life Science Foundation; Japan Antibiotics Research Association; Japanese Society of Chemotherapy; the Nippon Foundation-Osaka University Project for Infectious Disease Prevention; and Japan Agency for Medical Research and Development (AMED).
SY and NK collected the samples and performed bioinformatics analyses. RN visualized the efflux pump structures. SY and NK wrote the manuscript. KN reviewed and edited the manuscript. All authors revised the manuscript. SY, MH-N, and KN contributed to funding acquisition. SY, MH-N, and KN designed and supervised the study. All authors contributed to the article and approved the submitted version.
The authors declare no conflict of interest.
Data on the genome sequences were obtained from The Pseudomonas Genome Database (www.pseudomonas.com).
This article contains supplementary materials.
