World Journal of Microbiology and Biotechnology (2018) 34:83
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
· Nithya N.
· Jayasree P. R.
· Vimal K. P.
· Manish Kumar P. R.
Received: 8 January 2018 / Accepted: 25 May 2018 / Published online: 30 May 2018
© Springer Science+Business Media B.V., part of Springer Nature 2018
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 sensitiv-
ity 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 respec-
tively 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.
Keywords Eﬄux pumps · Multidrug-resistant · Mutational analysis · Phylogram · Pseudomonas aeruginosa · Regulatory
Pseudomonas aeruginosa, a ‘priority pathogen’ has now
been included in a list of 12 families of bacteria which pose a
serious health threat to man (WHO 2017). This opportunistic
microbe is also held responsible for nosocomial infections
worldwide. Known to possess a large number of virulence
factors, it causes severe infections with high morbidity and
mortality rate, particularly in immune-compromised patients
or those with underlying disease (Poole 2001; Strateva and
Yordanov 2009; Askoura et al. 2011; Porras-Gómez et al.
2012; Chatterjee et al. 2016). P. aeruginosa continues to
display multidrug-resistant phenotypes through (i) intrinsic
resistance mechanisms such as those mediated by constitu-
tive expression of AmpC beta-lactamase, eﬄux pumps and
porin down-regulation and (ii) acquired resistance caused
by mutational changes or acquisition of resistance mecha-
nisms via horizontal gene transfer. Together, these processes
contribute toward development of overwhelming resistance
against a variety of structurally unrelated antibiotics leading
to diﬃculties or failure in therapy (Poole 2011).
Efflux pumps of clinical relevance in P. aeruginosa
belong to the Resistance-Nodulation-Division (RND) family,
of which MexAB-OprM, MexCD-OprJ, MexEF-OprN, and
* Manish Kumar P. R.
Department of Biotechnology, University of Calicut,
Malappuram, Kerala 673635, India
School of Health Sciences, University of Calicut,
Malappuram, Kerala 673635, India
Department of Botany, University of Calicut, Malappuram,
Kerala 673635, India