Mutational analyses of regulatory genes, mexR, nalC, nalD and mexZ of mexAB-oprM and mexXY operons, in efflux pump hyperexpressing multidrug-resistant clinical isolates of Pseudomonas aeruginosa

Abstract

The present study deals with membrane-bound efflux pumps, MexAB-OprM and MexXY and their respective regulatory genes mexR, nalC, nalD and mexZ in multidrug resistant (MDR) Pseudomonas aeruginosa. Following antibiotic sensitivity testing and detection of various beta-lactamases, hyperexpression of efflux pump genes, mexB and mexY in the isolates was investigated using semi-quantitative and real-time reverse transcription-PCR. Amplicons from regulatory genes were sequenced and subjected to mutational and phylogenetic analysis. Twenty-nine clinical isolates of P. aeruginosa were obtained from a total of 144 MDR gram-negative bacteria collected from Kerala State, South India. All strains were found to be resistant to ampicillin and nalidixic acid with 13.8, 44.8 and 31% testing positive for extended-spectrum beta-lactamases, metallo-beta-lactamases and AmpC producers respectively. Increased mexB and mexY transcription was detected respectively in 10.3 and 20.7% of the isolates in comparison with P. aeruginosa reference strain, PAO (MTCC). Co-expression of MexY was also observed in MexB overproducers. Various synonymous/and non-synonymous mutations in regulatory gene sequences of efflux pump operons were detected. In the strain designate Pa16, mexR was found to harbour four novel point mutations with one transversion and three transitions which included a substitution of an ochre codon with that for serine. The gene also displayed a novel mutation involving insertion of a cysteine at the 444th base position, followed by an opal codon. The genetic divergence and homogeneity of the concatenated (mexR, nalC and nalD) regulatory gene sequences of mexAB-oprM operon was apparent in the phylogram generated with similar sequences retrieved from public database.

Genetic Variations in MexAB-OprM Efflux Pump Regulators and Their Association with Antibiotic Resistance and Sequence type in Clinical and Epidemiologically High-risk Clones of Pseudomonas Aeruginosa.

“…The contribution of the MexAB-OprM e ux pump to the resistance of P. aeruginosa strains was evident, as 17 out of 19 statistically signi cant mutations associated with MexAB-OprM e ux pump activity also correlated with resistance of the studied strains (p < 0.05). Some authors suggest that mutations in the mexR, nalC, and nalD genes can impair their function, favoring MexAB-OprM e ux pump overexpression with a consequent increase in bacterial resistance [6,12,13], however, in our study, same haplotypes (mutations) lead to different MexAB-OprM e ux pump phenotypic behavior and resistance.…”

“…Ozer et al, (2012) reported a consistent positive correlation between MexAB-OprM expression and multidrug resistance in 50 P. aeruginosa clinical strains [46]. However, in our study, of the P. aeruginosa strains that showed MexAB-OprM activity (phenotypic positive-strains), only in 52.9% the MexAB-OprM e ux pump was the most likely cause of resistance (+), in the remaining 47% of the strains the MexAB-OprM e ux pump was contributing to the resistance (*), which demonstrated that although the MexAB-OprM pump shows positive activity and contributes to resistance, this is not the only mechanism of resistance in these strains [13]. For example, in the remaining phenotypic negative-strains (n = 40) (3 S, 14 MDR, 16 XDR and 7 PDR), where the MexAB-OprM e ux pump was unlikely to be the cause of the resistance (Table 1); the high-drug resistance can be attributed in part to the production of carbapenemases (1 MDR, 3 XDR, and 2 PDR: serine carbapenemases, 8 XDR: metallo-.βlactamases).…”

“…While recent studies have identi ed point mutations in the mexR, nalC, and nalD repressor genes [13,25], few have investigated the relationship between these mutations and the occurrence of multidrug resistance and ST. Here, we observed 27 different haplotypes in the 3 repressor genes.…”

Mutational and Phylogenetic Analysis of &lt;i&gt;nfxB&lt;/i&gt; Gene in Multidrug-Resistant Clinical Isolates of &lt;i&gt;Pseudomonas aeruginosa&lt;/i&gt; Hyperexpressing MexCD-OprJ Efflux Pump

“…Semi-quantitative RT-PCR using primers (Eurofins Genomics India Pvt. Ltd., Bangalore) for genes such as rpsL and mexD [5] [6] were performed as described by us in a related study [7]. The RT-PCR reactions were performed using a mi-…”

“…The regulatory nfxB gene primer [8] was used for DNA amplifications and sequencing [7]. The ampliconic sequences were analyzed with NCBI BLAST tool (https://www.ncbi.nlm.nih.gov/) by comparison with sequences of the reference strain, P. aeruginosa PAO1, retrieved from the GenBank database.…”