Adaptation of IncX3 plasmid encoding blaNDM-4 within broad host range: A study from India

Background: Different variants of bla NDM have been reported across Indian subcontinent within diverse host range of gram negative bacilli. Most of their transferability depends on types of plasmid that encodes resistance determinant. These plasmids with external sub-inhibitory stress of antibiotics facilitate their propagation and maintenance within broad host range. IncX type plasmid is more recently known for carrying carbapenem resistance and NDM-4 variant is considered to have better hydrolytic property than other variants. Both of these factors interferes with therapeutic outcome in clinical setting. The current study was aimed to investigate the maintenance and stability of bla NDM-4 within broad host range and transcriptional response. Results: IncX3 plasmid encoding bla NDM-4 was successfully transferred in six different host when imipenem (0.5µg/ml) screen agar was used for selection of transformants. It was also found to coharbour resistance for aminoglycosides and quinolone. When checked for stability, it was observed that the plasmid was successfully expanded within all the six recipients for 55 th serial passages. Transcriptional expression with IncX3 was random but at consistent level for wild type and without concentration gradient stress of imipenem. Transcriptional expression with NDM gene was variable for parent isolates though for new hosts it was showing randomly increased pattern in proteus, E.coli , and DH5α Conclusion: the present study could highlight that external carbapenem pressure helps in maintenance and expression of bla NDM-4 within different host range. This study is of epidemiological significance and will help in tracking the genetic vehicle responsible for their transmission theirby restricting their spread. Minimum inhibitory concentration agar dilution against imipenem, meropenem, cefepime aztreonam, cotrimoxazole, ampicillin, ciprofloxacin CLSI antibiotic threshold cycle, ESBL-extended spectrum beta lactamases, CLSI- clinical and laboratory standard institute

Transcriptional expression with IncX3 was random but at consistent level for wild type and without concentration gradient stress of imipenem. Transcriptional expression with NDM gene was variable for parent isolates though for new hosts it was showing randomly increased pattern in proteus, E.coli, and DH5α Conclusion: the present study could highlight that external carbapenem pressure helps in maintenance and expression of bla NDM-4 within different host range. This study is of epidemiological significance and will help in tracking the genetic vehicle responsible for their transmission theirby restricting their spread. Background IncX type plasmids were previously known to be of narrow host range. Based on their restriction profile subtypes X1, X2 were formed (1). Later, based on phylogenetic differences, Johnsen et al., 2012 reported another two subtypes X3 and X4 (2). Inc X3 is better known for its ability to carry diverse types of resistance genes. Recently this subtype is more linked with bla NDM−4 and bla  across the globe with a number of cases from India (3)(4)(5). Thus this plasmid type is probably emerging as a potential genetic vehicle for lateral expression of New Delhi Metalo beta lactamases in this subcontinent. Although most reports of this plasmid type is within enterobacteriaceae, they may have potential chance to be disseminated to broader host range within hospital settings. The ability of plasmids to transfer, replicate and persist within a new host makes it most adapted and beneficial for the host. These adapted plasmids must help bacteria to survive against antibiotic exposure. Thus it would be interesting to know how these plasmids responds and when carbapenem therapy is initiated. Also how the resistance gene are expressed within different hosts when carbapenem stress is given. Therefore the present study was designed to investigate the transferability, stability, transcriptional response and copy number alteration of IncX3 type plasmids against carbapenem stress.

Result
Plasmids were obtained from 6 positive isolates which were showing Inc X3 incompatible type in their genetic characteristics. Transformation assay was done with all those 6 isolates and found to be transferable in ampicillin screen agar as well as ciprofloxacin and gentamicin screen agar, and transformants were targeted to have the incompatible type i.e Inc X3. Plasmid stability was checked by serial passage in 1:1000 dilution and was found that inc X3 type was carried till 55th passage.
IncX3 type is linked encoding of different NDM variants across the globe (3)(4)14). This study also reports the presence of bla NDM4 within IncX3 type plasmid. Thus, analysis of this plasmid by determining the copy number alteration is of utmost importance. The study could highlight that plasmid copy number of IncX3 type is maintained under carbapenem stress in diverse host range. The finding is quite unique to the earlier studies where IncX is regarded as having narrow host range (15).
Plasmid copy number is dependent on the type of organism which acts as host of that plasmid and the origin of replication. It is also reported that mutation can bring high copy number (16). The current study showed antibiotic pressure helps in maintenance and adaptation of Inc X type plasmid within diverse host range although there was no significant alteration of plasmid copy number. The study establishes a linkage among selection pressure, stability and copy number of plasmids encoding resistance genes.
The study also investigates analysis of transcriptional expression of bla NDM encoded within Inc X3 type within different hosts and it was observed that the gene was transcriptionally expressed in all the host ranges. This could be due to the adaptation of this plasmid in an unknown host machinery.  (7). Transformants were selected on LB Agar plates containing ampicillin (100 µg/ml). Plasmid copy number alteration and transcriptional expression of bla NDM−4 within broad host range against concentration gradient imipenem stress: Single colony of each host isolate was inoculated into LB broth with 1 µg/ml, 2 µg/ml, 4 µg/ml and 8 µg/ml of imipenem and also without any antibiotic (considered as a control for the reaction) and was incubated at 37•C for 4-7 hour till the OD reaches 0.9 at A 600 . cDNA was extracted from each condition, the reaction was performed using 10 µl of SYBR® Green PCR Master Mix (Applied Biosystem, Warrington, UK), 4 ng plasmid DNA as template and 3 µl of each primer (Table 2) (10 pmol) in a 20 µl reaction and the relative fold change was measured by ∆∆CT method and was normalized against a housekeeping gene rpsl of E. coli (9). The each set of reaction was run in triplicate and the experiment was repeated thrice. Quantitative Real Time PCR was done to determine the level of alteration of the plasmid encoding bla NDM− 4 using Step One Plus real time detection system (Applied biosystem, Warrington). Availability of data and material: All the datas are available in the manuscript.
Competing interests: No competing interest exists.

Funding information
The authors received no specific grant from any funding agency Authors' contributions: NAC has conceived the study plan and performed all the experimental work and prepared manuscript. DP and BJD has participated in real time-based experiment and helped in manuscript preparation. DDC has provided the samples for study and helped in data analysis and preparation of the manuscript. AB has designed study protocol and corrected the manuscript and overall supervised the work.  Transcriptional expression of IncX3 type plasmids under concentration gradient carbapenem (imipenem) exposure