The issues of antimicrobial resistance specifically towards carbapenems require immediate attention. Developing a holistic model to visualize and decipher the role of genes of secondary resistome along with the primary resistance genes is explicit (4, 22). Genes associated with CRE is considered in this study. Reports of association of SRGs with carbapenem resistance are not found yet.
From the 21 genes that had been streamlined from in silico analysis(22) two genes viz. ccdB (cluster 19) involved with plasmid maintenance protein and repA2 (cluster 33) associated with replication regulatory protein have been filtered out for in vitro transcriptional analysis. There are no reports of these genes of secondary resistome to be involved with carbapenem resistance owing to the presence of blaNDM genes.
The response patterns of expression profiles of repA2 and ccdB genes on carbapenem and cephalosporin exposure on clinical isolates of E.coli discretely harboring blaNDM−1, blaNDM−4, blaNDM−5, blaNDM−7 and blaCTX−M−15 were interesting. Elimination of these SRGs is essential for prediction of their role in a system (23).
repA2 gene in the clinical samples was down regulated on exposure to imipenem, meropenem and ertapenem and under ertapenem pressure maximal expression was seen in the isolate harbouring blaNDM−1. Similarly, on various cephalosprins exposure repA2 gene was down regulated. Eliminating repA2 genes also showed changes in the susceptibility pattern against the antibiotics in concern. repA21 is a replication initiation protein that controls replication of the IncFII plasmids group. Promoters at the upstream region of repA21 gene control its transcription and translation, thereby regulating its expression. RNA-CX transcript of repA21 is produced constitutively. The 5' end of RNA-CX encodes a repressor protein, repA2, which regulates the expression of another transcript, RNA-A. Translation of both RNA-CX and RNA-A is regulated by RNA-E, a small transcript produced from the antiparallel DNA strand. RNA-E interacts directly with both the RNAs and inhibits their translation, thus, repA2 regulates plasmid copy number (24, 25). Also reports suggests that mutations in repA2 gene increases the plasmid copy number (26) while its disruption stops the plasmid replication (27).
Congruent to literature, change in transcriptional response pattern of repA2 on antibiotic exposure as well as change in antibiotic susceptibility pattern on its elimination was seen in this study. These changes in expression pattern by repA2 can be utilized to indicate of antibiotic stress(28), thus making repA2 a gene marker (29). Since, plasmids play a pivotal role in spreading antibiotic resistance and increase in the plasmid copy number gives bacteria upper hand to adapt to antibiotic stress (30), therefore, disrupting repA2 gene activity (27) might inactivate replication. The increase in diameters of zone of inhibition upon elimination of repA2 as seen in this study indicates that elimination of this gene from E.coli system leads to loss of plasmid thereby rendering E. coli of clinical relevance non-pathogenic. From this finding it can be considered that repA2 gene plays a crucial role as helper to the primary carbapenem resistance genes and its elimination might also be able to revoke carbapenem resistance and thus help in solving the problem of antibiotic resistance (31).
The ccdB gene, associated with plasmid maintenance, was down regulated when all the above mentioned clinical isolates were put under meropenem, ertapenem and all the three cephalosporins pressure. However, imipenem stress showed over expression of ccdB gene. Elimination of ccdB genes showed increase in the zone of inhibition indicating changes in the susceptibility pattern against the antibiotics in concern The ccdB operon (control of cell death), a type of plasmid addiction system (PAS), is encoded by IncF plasmid to maintain plasmid stability in E. coli (32). The operon consists of ccdBA and ccdB genes that codes for a toxin-antitoxin system which work in unison to maintain plasmid replication during cell division in host (33, 34). The ccdB gene encodes DNA gyrase poison that can induce double strand breaks in E. coli, ultimately killing it (35). This mechanism is activated only when the plasmid copy numbers decreases. Typically, gene ccdBA binds tightly to ccdB and encodes an antitoxin that inhibits the toxic activity of ccdB gene. On losing F-plasmid, Lon protease, a substrate for ccdBA, degrades it leaving ccdB free to act upon DNA gyrase (32, 35). GyrA subunit is an antibiotic target for quinolones, however, quinolone resistant bacteria have no effect on ccdB indicating that ccdB and GyrA subunits interact at different sites (35). Increase in the diameter of zone of inhibition upon elimination of ccdB genes as seen in this study hints that this gene supports the survival of E. coli under therapeutic stress condition and on its elimination the toxic function of this gene activates in order to maintain the PAS. All these findings make ccdB gene, a member of PAS, an interesting antibiotic target that could yield desirable results against carbapenem resistance (32, 36).