AcrAB-tolC efflux Pump Mediated Resistance to Carbapenems among Clinical isolates of Enterobacteriaceae

AcrAB-tolC is a resistance nodulation division type of efflux pump present in Enterobacteriaceae . it non-specifically effluxes antibiotics out of the bacterial cell, thus conferring drug resistance. increase in the expression of the AcrAB-tolC efflux pump increases resistance to antibiotics. We aimed to study the expression levels of acrA and acrB that encodes AcrAB-tolC efflux pump, to understand efflux pump mediated resistance to carbapenem among clinical isolates of Enterobacteriaceae obtained from various clinical samples. Additionally, co -production of carbapenemase was also detected in the isolates demonstrating efflux pump mediated resistance to carbapenems. A total of 127 carbapenem resistant clinical isolates of Enterobacteriaceae , isolated from a tertiary care hospital were included in the study. An efflux pump inhibition (ePi) assay with reserpine, an efflux pump inhibitor, was performed to screen for isolates exhibiting efflux pump activity. Real time Reverse transcriptase qPCR was performed to detect the mRNA over expression levels of acrA and acrB that encodes AcrAB-tolC efflux pump. the control strains K. pneumoniae BAA2146 and E. coli AcrB were used. ePi assay with carbapenem showed that 56 /127(44%) isolates were screen positive indicating efflux pump activity. A total of 12 isolates showed 10 1 to 10 7 increase in the expression of both acrA and acrB when compared with the controls indicating a strong efflux pump activity, in addition to producing carbapenemase. the study highlights the role of efflux pump AcrAB-tolC in conferring resistance to carbapenem among clinical isolates of Enterobacteriaceae .


iNtRODuCtiON
Enterobacteriaceae are a large family of Gram negative bacilli that often cause a variety of clinical infections -urinary tract infections, wound infections, sepsis, respiratory infections, etc. 1 Infections caused by Enterobacteriaceae often require treatment with antibiotics. The efficient antimicrobial agents against this group of bacteria are cephalosporins, carbapenems, aminoglycosides and fluoroquinolones. 2,3 Antibiotic resistance (including that related to Enterobacteriaceae) to the commonly used antimicrobials is a growing problem in both developed and developing countries and especially in ICUs.
Resistance to carbapenems is often mediated by enzymes called as carbapenemase and also by non-enzyme mediated methods like over expression of efflux pumps and loss of porin proteins. Efflux pump mediated resistance to carbapenems is one of the few other studied mechanisms of resistance among clinical isolates of Enterobacteriaceae. AcrAB-TolC is a multidrug Resistance Nodulation Division (RND) type of efflux pump, which is present among Enterobacteriaceae. This is made up of three components AcrA -the periplasmic adaptor protein that connects AcrB and TolC, AcrB -the active pump that pushes the substrate from within the bacterial cell and TolC -the outer membrane channel that efflux out the substrate. 4,5 Over expression of the AcrA and AcrB, contributes to drug resistance by effective effluxing out of carbapenems, cephalosporins, fluoroquinolones and aminoglycosides. The AcrAB-TolC efflux pump plays an important role in producing multidrug resistance in Gram-negative bacteria since it is not substrate specific. 6 Genes for efflux pumps are present on plasmids as well as chromosome. Mutations in the local repressor genes or activation of a regulon regulated by a global transcriptional regulator such as MarA or SoxS of E. coli, results in over expression of efflux pumps. 7-10 Since efflux pumps have a broad substrate range, over expression of a pump will confer resistance to more than one class of antibiotics, as well as to some dyes and detergents.
Even though most of the bacteria carry genes that encode MDR efflux pumps on their chromosomes, some of these efflux pump encoding genes have been mobi¬lized onto plasmids that may have the potential to transfer between bacteria. This situation is disquieting, since it would result in dissemination of multidrug resistance, more so because efflux pumps have a broad substrate profile. A recent study reported an IncH1 plasmid that was isolated from a Citrobacter freundii strain that contained the RND MDR efflux pump and also carried the gene for the beta lactamase NDM. 11 In order to understand enterobacterial resistance to carbapenems, it is imperative to comprehend different mechanisms of drug resistance. Enterobacteriaceae that over expresses efflux pumps have been isolated from patients earlier. 12 Understanding the mechanism of over expression may help in designing novel therapeutics to prevent the production of effluxpump proteins. A study from Turkey reported efflux pumps as the causative mechanism for multidrug resistant K. pneumoniae isolates. 13 Tsai et al. 14 from Taiwan showed that the over expression of AcrAB-TolC by ramR deletion in K. pneumoniae resulted in increased antibiotic resistance and reduced virulence at the same time. There are limited studies that have explored both beta lactamase as well as efflux pump mediated resistance, among clinical isolates of Enterobacteriaceae cultured from subjects in different tertiary care hospitals. Here we have demonstrated AcrAB-TolC efflux pump mediated resistance to carbapenems among clinical isolates of Enterobacteriaceae. 15,16

MAteRiAls AND MethODs study type: Cross sectional inclusion Criteria
Clinical isolates of Enterobacteriaceae obtained from various clinical samples (urine, blood, sputum, body fluids) that were resistant to carbapenem were included in the study.

study isolates
A total of 127 carbapenem resistant clinical isolates of Enterobacteriaceae from clinical samples -urine, blood, sputum, body fluids, obtained from patients attending a tertiary care hospital were included in the study. The bacterial isolates were identified by standard microbiological procedures.

Phenotypic screening of efflux Pump Mediated
Resistance to Carbapenems 17 Reserpine, a non-specific efflux pump inhibitor was used to block the activity of efflux pumps involved in conferring antibiotic resistance. An efflux pump inhibition assay (EPI assay) was performed by determining MIC (agar dilution method) of imipenem in the presence and in the absence of reserpine at a fixed concentration of 25µg/mL. A 2 or more fold decrease in the MIC of imipenem in the presence of Reserpine when compared to the MIC of imipenem alone was considered to indicate the activity of efflux pump in conferring drug resistance.

Real time Reverse transcriptase qPCR to Confirm the Over expression of efflux Pump AcrAB-tolC
Real Time Reverse Transcriptase qPCR (Real Time RTqPCR) (Step One Plus TM Real Time PCR system, Applied Biosystems TM ) was performed to study the mRNA expression levels of acrA and acrB of the AcrAB-TolC efflux pump, that is known to confer resistance to antimicrobial drugs. Representative isolates which were screen positive for efflux pump activity against imipenem by EPI assay were grown overnight in LB broth (Hi Media). Total RNA was extracted using FavorPrep tissue total RNA mini kit and RNA was quantified for purity and concentration using Nanodrop at 260/280 and 260/230 nm. RNA was converted to cDNA by RT-PCR using Thermo scientific Verso cDNA synthesis kit. RT-PCR was performed in a conventional thermal cycler. Real Time RT qPCR was performed with SYBR Premix Ex Taq from Takara Clontech in Step One Plus TM Applied Biosystem TM using Comparative ΔΔCT program. The test was done in triplicates. gapA was used as the housekeeping gene for normalizing the transcription levels of target genes. The mean of the normalized expression (transcription) values were calculated for acrA and acrB for each strain manually as well as by using the Step One Plus TM software. Klebsiella pneumoniae BAA2146 was used as a positive control for studying and comparing the mRNA expression levels of both acrA and acrB among the screen positive isolates. E.coli AcrB, a strain that over expresses acrB, was used as a positive control for studying and comparing the mRNA expression levels of acrB among screen positive isolates.

Results
Phenotypic screening of efflux pump mediated resistance to carbapenems detected by efflux pump inhibition assay: A total of 127 carbapenem resistant clinical isolates of Enterobacteriaceae were phenotypically screened for efflux pump mediated resistance to carbapenem. MIC of imipenem, a carbapenem, was determined for all the 127 isolates by agar dilution method. Concentration of imipenem ranged from 512 µg/mL to 1 µg/mL. MIC of imipenem with reserpine was determined.
Isolates showing ≥ 2 fold decrease in MIC of imipenem with reserpine when compared to MIC of imipenem alone was considered to indicate the activity of efflux pumps in conferring resistance to imipenem (Figure 1).
Out of 127 carbapenem resistant clinical isolates of Enterobacteriaceae, 56 (44%) isolates showed ≥ 2 fold decrease in MIC of imipenem with reserpine when compared to MIC of imipenem alone.
Out of 56 isolates, that showed a decrease in MIC of imipenem with reserpine, 41 isolates (73%) showed a 2 fold decrease in MIC. One isolate showed a 16 fold decrease in MIC, from 128 µg/mL as MIC of imipenem to 8 µg/mL as MIC of imipenem with reserpine. (Table) (Figure 2).

Real time Rt qPCR to study the mRNA Over expression of acrA and acrB among Clinical isolates that were screen Positive for efflux Pump Mediated Resistance to Carbapenem
Out of 56 isolates that were screen positive for efflux pump mediated resistance to imipenem, 12 representative isolates were selected for Real Time RTqPCR to study and compare the mRNA expression levels of acrA and acrB with known positive controls.
The mRNA expression levels of acrA ranged from 10 1 to 10 7 . The mRNA expression levels of acrB ranged from 10 1 to 10 9 . The mRNA expression levels of test isolates were either comparable to the control or highly over expressed when compared to control (Figure 3, Figure 4).

DisCussiON
C a r b a p e n e m re s i sta n c e a m o n g Enterobacteriaceae is mediated mainly by acquired carbapenemases. 18,19 Resistance to carbapenems is also mediated by a combination of non-carbapenemase beta lactamases or ESBLs and reduced drug influx via loss of outermembrane porins. 20 This study demonstrates, efflux pump mediated resistance to carbapenem group of antibiotics observed in clinical isolates of Enterobacteriaceae and also the potential of reserpine to be used along with carbapenem to treat infections caused by strains of carbapenem resistant Enterobacteriaceae showing efflux pump mediated antibiotic resistance.
A study by Adler et al. 21 showed that mutations in acrB and acrR activates AcrAB-TolC mediated efflux thereby conferring high level carbapenem resistance in E.coli. Another study showed that over expression of efflux pumps expels carbapenems leading to carbapenem resistance associated with multidrug resistance. 22 A study from Taiwan demonstrated that over expression of acrB was observed in 44% of clinical isolates from UTI showing resistance to carbapenem and tigecycline. 23 A study from India reported that 35% clinical isolates of E.coli showed efflux pump mediated resistance to carbapenems by efflux pump inhibition assay using CCCP as an efflux pump inhibitor. The same study reported that 52% of the isolates selected for Real Time qRT PCR showed upregulation of acrA. 24 In the current study, 127 carbapenem resistant clinical isolates of Enterobacteriaceae were phenotypically screened for efflux pump mediated resistance to carbapenem by efflux pump inhibition assay using reserpine as an efflux pump inhibitor.
Of the 127 isolates, 56 (44%) isolates showed ≥ 2 fold decrease in MIC of imipenem with reserpine when compared to MIC of imipenem alone. This indicates role of an efflux pump inhibitor in mediating resistance to imipenem.
It was interesting to note that one isolate showed a 16 fold decrease in MIC from 128 µg/ mL MIC of imipenem to 8 µg/mL MIC of imipenem with reserpine, suggesting the potential use of an efflux pump inhibitor in tandem with an antibiotic to effectively treat infections instead of consorting to higher classes of antibiotics.
Of the 56 isolates that were screen positive for efflux pump mediated resistance to imipenem, 12 representative isolates were selected for Real Time RTqPCR to study the mRNA expression levels of acrA and acrB. All the 12 strains had increased expression levels for acrA that ranged from 10 1 to 10 7 and for acrB that ranged from 10 1 to 10 9 .
Out of 12 isolates, three showed the highest expression of acrA i.e., 10 7 . In addition to the over expression of acrA, these isolates also produced beta lactamases. (a) Isolate no. C65 showed an 8 fold decrease in MIC of imipenem with reserpine, and was also positive for bla NDM .  was also positive for bla NDM and bla OXA -48. All the three isolates were Klebsiella pneumoniae isolated from urine samples of three patients who were above 70 years old suffering from UTI.
Out of 12 isolates, four showed a very high expression of acrB i.e., 10 8 -10 9 . In addition to the over expression of acrB, these isolates also produced beta lactamases. (a) Isolate no. C94 from a drain sample of a 50 year old female patient showed a 16 fold decrease in MIC of imipenem with reserpine, and was also positive for bla NDM . (b) Isolate no. C93 from a urine sample showed a 4 fold decrease in MIC of imipenem with reserpine, and was also positive for bla NDM . (c) Isolate no. C119 from a urine sample showed a 4 fold decrease in MIC of imipenem with reserpine, and was also positive for bla OXA -48 and bla KPC . (d) Isolate no. C157 from a urine sample showed a 4 fold decrease in MIC of imipenem with reserpine, and was also positive for bla NDM . All the four isolates (C94, C93, C119 and C157) were E. coli.
Over expression of the efflux pump AcrAB-TolC contributes to antibiotic resistance. If the genes that encode for over expression of the efflux pump are present on plasmids, along with genes that encode for enzyme mediated resistance, then it would result in rampant dissemination of multi-drug resistance that is mediated by multiple mechanisms. However, the presence of genes for AcrAB-TolC on plasmids is not explored in this study.

CONClusiON
The present study demonstrates efflux pump AcrAB-TolC mediated resistance to carbapenems among clinical isolates of Enterobacteriaceae. Antibiotic resistance, mediated by over expression of acrA and acrB that encodes the efflux pump AcrAB-TolC in Enterobacteriaceae enhances the non-susceptibility of the bacteria to a range of antibiotics, because of the nonspecificity of substrates that could be effluxed out of the efflux pump.