MexCD-OprJ efflux pump of resistance-nodulation-cell division superfamily, normally quiescent in wild-type strain of P. aeruginosa, does not contribute to intrinsic antimicrobial resistance. This pump is capable of extruding a variety of antimicrobial agents, biocides, organic solvents, dyes and detergents. The mexCD-oprJ operon is regulated by repressors such as NfxB and EsrC. The nfxB gene encoding repressor protein, NfxB, is located upstream of the structural genes mexCD-oprJ with an intergenic regulatory region facilitating divergent transcription. NfxB shows similarity to proteins of the LacI-GalR family. It binds to a site composed of two 39 bp repeats within the aforementioned intergenic sequences between nfxB and mexC which negatively regulates mexCD-oprJ as well as its own expression. Mutations within nfxB gene negatively impact NfxB repressor activity leading to hyperexpression of mexCD-oprJ, and strains harbouring such defects are known as nfxB-type mutants  . EsrC, a second regulator of MexCD-OprJ, encoded by a gene PA4596, is located downstream of mexCD-oprJ operon. EsrC represses transcription of mexCD-oprJ only under “envelope stress” (membrane-damaged) condition and is dependent on NfxB . The present study was undertaken to analyze mutational and phylogenetic relatedness of nfxB regulatory gene in MDR isolates of P. aeruginosa hyperexpressing MexCD-OprJ efflux pump.
2. Materials and Methods
2.1. Bacterial Isolates
Of a total of 144 MDR gram-negative bacteria, collected during the period 2012-2016 from various clinical laboratories in Kerala, 29 isolates of P. aeruginosa (designated as Pa1 - Pa29) were included in this study along with a reference P. aeruginosa MTCC-3541 (Microbial Type Culture Collection) strain, PAO.
2.2. Random Amplified Polymorphic DNA (RAPD)
Random Amplified Polymorphic DNA (RAPD) typing was performed with RBa-D5 primer (GenBank accession. no. AM911680) according to manufacturer’s instructions. Images of DNA banding patterns obtained after agarose gel electrophoresis were analyzed using the PyElph software  to prepare dendrograms using unweighed pair-group method arithmetic mean (UPGMA) method.
2.3. Semi-Quantitative RT-PCR
Semi-quantitative RT-PCR using primers (Eurofins Genomics India Pvt. Ltd., Bangalore) for genes such as rpsL and mexD   were performed as described by us in a related study . The RT-PCR reactions were performed using a minicycler (MJ Research, USA) in a reaction volume of 25 μl containing 12.5 μl of 2x Emerald GT master mix (TaKaRa Inc., Japan), 0.25 μM of each primer and 0.5 μl of cDNA. Amplification products were analyzed on 1% (w/v) agarose gels to detect the presence of the expected amplicons and compare the band intensities with those from the reference strain P. aeruginosa MTCC-PAO.
2.4. PCR Amplification and Sequencing of the nfxB Gene and Its Phylogenetic Analysis
The regulatory nfxB gene primer  was used for DNA amplifications and sequencing . 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. The phylogram construction, employing nfxB gene sequences obtained in this study in combination with similar sequences retrieved from public databases, was carried out by Bayesian inference using Markov Chain Monte Carlo method . The jModelTest software  used for the analysis of nucleotide sequence evolution with respect to each gene was based on Akaike Information Criterion (AIC).
2.5. Nucleotide Sequence Accession Numbers
The nfxB gene nucleotide sequences obtained in the present study were deposited in the GenBank database under the following accession numbers MH346508-MH346510.
3. Result and Discussion
The RAPD profiles generated in our study were found to be distinctive and reproducible (Figure 1). The dendrogram showed two major clusters—A and B, in which major cluster A was found to be subdivided into two sub-clusters A1 and A2 (Figure 2). The mexD was significantly overexpressed in three isolates such as Pa6, Pa7 and Pa13 (Figure 3). Isolates with efflux pump activity were found to be included in the sub-cluster A1 of RAPD dendrogram. Of them, Pa7 and Pa13 belonged to a single clade, whilst strain Pa6 was found grouped into another clade. Mutation analysis of nfxB gene revealed point mutations, T → C at 239th position in Pa6, T → C at 349th position in Pa7 and C → T at 14th position in Pa13 (Table 1). Notably, mutations detected in Pa6 and Pa7 isolates have been found to be novel and are hitherto unreported in GenBank data base, whilst the mutation detected in Pa13 has already been reported earlier . Phylogram (Figure 4) was constructed using three nfxB gene sequences of MexCD-OprJ overproducers—Pa6, Pa7 and Pa13 and nine other selected sequences of P. aeruginosa isolate retrieved from the GenBank database including the reference strain, P. aeruginosa—PAO1 (GenBank accession no. AE004091. 2). For this analysis, Pseudomonas chlororaphis (GenBank accession no. CP011110.1) was taken as
Figure 1. RAPD profiles of reference strain PAO (MTCC) and MDR P. aeruginosa clinical isolates. Lane denoted M represents 100 bp DNA ladder.
Figure 2. RAPD dendrogram showing clonal relatedness amongst P. aeruginosa.
Figure 3. Semi-quantitative RT-PCR. Lane denoted M represents 100 bp DNA ladder. Other lanes show P. aeruginosa MTCC reference strain PAO and clinical isolates. (a) Expression of housekeeping gene, rpsL in PAO and three isolates, Pa7, Pa6 and Pa13. (b) Expression of mexD in PAO and three isolates, Pa7, Pa6 and Pa13.
Table 1. Summary of genetic analyses of mutations in nfxB gene of P. aeruginosa clinical isolates.
“−” represents no amino acid change.
Figure 4. Bayesian phylogenetic tree reconstructed employing nfxB ampliconic sequences from P. aeruginosa isolates. Scale bar denotes probability of nucleotide change. Analysis was run for 1,000,000 generations. Clade credibility values are shown at each node. P. chlororaphis (GenBank accession no. CP011110.1) was used as the outgroup.
the outgroup as this sequence showed better sequence similarity with a higher query coverage. GTR was selected as the best fit model of sequence evolution on the basis of AIC. Potential scale reduction factor (PSRF) and estimated sample size (ESS) value were observed as 1.0 and above 100 respectively. Analysis was run for 1,000,000 generations and clade credibility values represented at each node were found to be >0.5. Bayesian phylogenetic analysis revealed that the sequence from Pa6 exhibited similarity with isolates reported from Taiwan (GenBank accession no. CP004061.1), Mexico (GenBank accession no. CP021999.1) and Brazil (GenBank accession no. CP021380.1). Pa13 isolate showed similarity with that reported from Tamil Nadu with GenBank accession no. CP008739.2  whilst Pa7 was found to be similar to P. aeruginosa sequences deposited from North America with GenBank accession no. CP012901.1 .
Antibiotic resistance is a stark reality across the world including the Indian subcontinent. Antimicrobial selection pressure and spread of resistant organisms are the main leading factors for the emergence of resistance. The present study has been successful in unraveling vital information on the mutational variations of nfxB regulatory gene sequences of MexCD-OprJ efflux pump present in P. aeruginosa. Occurrence of genetic similarities of nfxB gene sequences among the isolates used in this study as well as with those previously reported from other countries warrants the need for implementation of strict control measures to prevent spread of resistance genes.
This study was supported by the Government of India, DST (Department of Science and Technology)—INSPIRE fellowship (Grant No. IF120394) to MS.