SYNERGY OF A NOVEL ANTIBIOTIC ADJUVANT ENTITY AGAINST MULTI DRUG RESISTANT ENTEROBACTERIACEAE

In the present investigation, we investigated the i n vitro interaction of ceftriaxone plus sulbactam w ith disodium edetate, a Non Antibiotic Adjuvant (NAA) a gainst selected clinical isolates and in vitro susceptibility studies were also performed. The iso lates were tested against a range of ratios of ceft riaxone and sulbactam using a microdilution checkerboard me thod. Having determined the appropriate ratios of ceftriaxone plus sulbactam, effect of various conce ntration of disodium edetate were also studied usin g the microdilution checkerboard method. All the results were analysed with the Fractional Inhibitory Concentration (FIC) indices. Susceptibility studies were carried out according to the Clinical and Laboratory Standards Institute (CLSI) methods. Resu lts of this study demonstrated that 2:1 ratio of ceftriaxone and sulbactam was the more synergistic wi h FIC index values 0.4281, 0.4023, 0.4124 and 0.4325 for E. coli, A. baumannii, P. aeruginosa and K. pneumoniae. The synergicity of ceftriaxone and sulbactam was enhanced significantly with increasin g concentration of disodium edetate and produced the lowest FIC index (<0.2) at 10 mM of disodium ed etate in all positive controls as well as clinical isolates. Further, the synergy between ceftriaxone plus sulbactam with disodium edetate (Elores) was confirmed by broth dilution, time kill curve and ag ar diffusion methods. In broth dilution method, Elo res (ceftriaxone+sulbactam+disodium edetate) produced 4 to 5 fold lower MIC when compared with ceftriaxone plus sulbactam. Approximately 10 4 log of killing reduction was observed with synergi stic ratio of Elores in time kill curve study. This stud y suggest that Elores could be an alternative regim n in combating antibiotic resistance among multi drug re sistant Enterobacteriaceae.


INTRODUCTION
Increasing resistance to 3rd generation cephalosporins particlularly due to extended spectrum beta lactamase production has become a major concern especially among Enterobacteriaceae that cause nosocomial infections (Rawat and Nair, 2010). Approximately 20% of Klebsiella pnenumoniae infections and 31% of Enterobacter species infections in intensive care unit in the United States now involve strains not susceptible to 3rd-generation cephalosporins.
Salmonella species also getting resistant to expandedspectrum cephalosporins have been reported in several countries, including Argentina, Turkey, Algeria, Saudi Arabia, Greece, Tunisia and France (Dutil et al., 2010). In addition, most of the bacteria, responsible for community-acquired infections have developed resistance to many antibacterial agents particularly betalactams which are being used in over 50% of all systemic antibiotics (Acevedo et al., 2009). Besides that, several evidences pointed towards the development of resistance to extended-spectrum cephalosporins in bacteria isolated from patients with nosocomial infections (Rawat and Nair, 2010). An increasing number of reports have indicated the steady rise in resistance for ceftriaxone (Unemo et al., 2010;Ohnishi et al., 2011). In addition, aminoglycosides, fluroquinolones and carbapenems have usually been used for the treatment of infections caused by Enterobacteriaceae (Howard et al., 2012;Tam et al., 2010). However, in recent years, these organisms have been reported to be resistant to these commonly used antimicrobial agents worldwide Memish et al., 2012;Muthusamy and Boppe, 2012). Acquisition or expression of Metallo-β-Lactamases (MBLs), Extended-Spectrum β-Lactamases (ESBLs), decreased permeability, overexpression of efflux pump are thought to be the main factors contributing to antibiotic resistance development (Chaudhary and Payasi, 2012;Zavascki et al., 2010;Karthika et al., 2009).
The treatment of infections caused by these microorganisms impose a major challenge to health care system due to failure of monotherapy and lacking of effective regimens. Combination antibiotics have been used frequently in clinical practice, but not all of them work synergistically.
Considering the above background, a team of Venus Medicine Research Centre (VMRC), India has developed a novel Antibiotic Adjuvant Entity (AAE) combination of ceftriaxone with a beta lactamase inhibitor sulbactam and a nonantibiotic adjuvant disodium edetate naming Elores. This AAE can be used successfully for the therapy of infections caused by resistant organisms.
The checkerboard titration method was used to test synergy of various ratios of ceftriaxone and sulbactam against selected clinical isolates and results have been presented in term of the Fractional Inhibitory Concentration Index (FICI). The present study was aimed to differentiate the performance of product on Extended-Spectrum Beta-Lactamase (ESBL), Metallo-Beta-Lactamase (MBL) and efflux positive strains. We investigated the in vitro interactions between ceftriaxone and sulbactam with a Non Antibiotic Adjuvant (NAA) disodium edetate using a checkerboard method. Further, the effect of different concentrations of disodium edetate on the double combination of ceftriaxone and sulbactam was studied in detail to determine whether the apparent synergistic interaction between ceftriaxone and sulbactam is enhanced or diminished by the addition of disodium edetate. Furthermore, we studied the in vitro susceptibilities of these isolates to combinations of ceftriaxone and sulbactam and disodium edetate by the use of broth dilution, disk diffusion and time-kill methods.

Clinical Isolates Collection and Their Identification
A total of 140 clinical isolates 35 of each E. coli, K. pneumoniae, A. baumannii and P. aeruginosa were included in the study. The re-identification of clinical isolates were done according to standard microbiological procedures (Khan et al., 2011

ESBL and MBL Characterization
All these isolates were subjected to ESBL and MBL characterization as previously described (CLSI, 2011;Yong et al., 2002).

Fractional Inhibitory Concentration (FIC) Study
In vitro drug interaction was determined by the checkerboard method as described by Wijayanti et al. (2010) and results were analyzed with the FIC indices. For each ratio, a two-dimensional checkerboard with twofold dilutions was used for the study. Growth control wells containing medium were included in each plate. Each test was performed in triplicate. The concentration of antibiotics needed to inhibit growth was recorded. The following formula was used to calculate FIC: The FIC index (∑ FIC) calculated as the sum of each FIC, was interpreted as follows: Synergy is defined as an FIC index of ≤0.5. Antagonism is defined as an FICindex of ≥2. An indifferent/additive effect is defined as an FIC index of >0.5 to 2 or a micro dilution decrease of 1 dilution in the MIC of the one or the other drug or no change in the MIC of either of the drugs.

Effect of Non-Antibiotic Adjuvant (NAA) on Double Combinations
Effect of NAA on ∑ FIC of double combinations, ceftriaxone plus sulbactam was also conduced using checkerboard method (Wijayanti et al., 2010) in the absence and presence of increasing concentration of disodium edetate in all positive controls as well as clinical isolates.

Determination of Minimum Inhibitory Concentration (MIC)
MICs were determined by broth dilution method following the guidelines of the CLSI (2011) using cation-adjusted Mueller-Hinton Broth (MHB) [Hi-Media, India]. MIC was defined as the lowest concentration of antibiotic that completely inhibited the growth of the organism as detected with the naked eye.

Determination of Antimicrobial Susceptibility Test (AST)
AST was determined according to the cup plate method described by Chaudhary et al. (2012b). The cups were made in the agar plate using a sterile cork borer (6.5 mm). Then, 30 µL of the drug preparation Elores (ceftriaxone+sulbactam+disodium edetate (30:15 µg), ceftriaxone+sulbactam (30:15 µg) and ceftriaxone (30 µg) were placed into the wells using a micro-pipette and allowed the plates to incubate at 37°C for 18 h in the upright position. After incubation the zone of inhibition around the wells was measured in mm (millimeter), averaged and the mean values were recorded.

Determination of Time Kill Curve (TKC)
TKC study was performed according to CLSI (2011) guidelines.
Twice the MIC of ceftriaxone, ceftriaxone+sulbactam and Elores (ceftriaxone+sulbactam+disodium edetate) was used for this study. For TKC study, two randomly selected clinical isolate of each E. coli, K. pneumoniae, P. aeruginosa and A. baumannii and all positive controls were used. Overnight grown bacterial suspension was diluted to approximately 10 6 to 10 7 cfu mL −1 in MHB containing antibiotics or no antibiotics. The samples were removed at 2, 4, 6, 8, 10 and 12 h and were diluted and plated on MHA. The agar plates were incubated at 37°C for 24 h and colony forming unit (cfu) were counted.

Clinical Isolate Identification and Characterization
All of the clinical isolates obtained from isolate banks were identified as A. baumannii, E. coli, P. aeruginosa and K. pneumoniae based on their morphological and biochemical characterization. Out of the 140 isolates, 60 were found to be ESBL positive (A.  Figure 1 summarizes the results of the FIC index analysis of the various ratios of ceftriaxone and sulbactam tested against E. coli, A. baumannii, P. aeruginosa and K. pneumoniae. The results demonstrated that 2:1 ratio of ceftriaxone and sulbactam was the most synergistic. Further increasing the ratio of either ceftriaxone or sulbactam synergistic activity was either lost or no further potentiation was observed. This study was conducted in all selected clinical isolates as well as positive controls and synergistic activity was noted at 2:1 ratio of ceftriaxone and sulbactam. The results of one clinical isolate of each E. coli, A. baumannii, P. aeruginosa and K. pneumoniae positive with both MBL and efflux is presented here only.

Effect of NAA on Double Combinations
Effect of NAA, disodium edetate on FIC indices of double combinations ceftriaxone plus sulbactam in the absence and presence of increasing concentration of disodium edetate was done in all 140 isolates of E. coli, A. baumannii, P. aeruginosa and K. pneumoniae and positive controls. Results of this study showed that ∑FIC decreased with increasing concentration of disodium edetate and maximum decrease was found 10 mM of disodium edetate. Further increasing the concentration of disodium edetate ∑FIC remained constant. The FIC analysis for four selected clinical isolates which were used for FIC study are presented in Fig. 2. From ∑FIC analysis of all clinical isolates, FICI min and FICI max were calculated and results are presented in Fig. 3. The FICI min and FICI max were significantly lower equal to less than 0.5, which indicates the presence of synergistic interactions among the three combinations.

MIC
Synergism between ceftriaxone and sulbactam along with NAA was also performed by a broth dilution method against selected clinical isolates and positive isolates. The MICs for positive controls ranged 2-4 µg mL −1 for ceftriaxone+sulbactam+disodium edetate (Elores), whereas it was ranged between 512->1024 and 256-512 for ceftriaxone and ceftriaxone+sulbactam, respectively ( Table  1). MICs for Elores were 4-32 µg mL −1 for clinical isolates of A. baumannii and 4-16 µg mL −1 for each of E. coli, K. pneumoniae and P. aeruginosa positive with ESBL. MICs for Elores to MBL positive isolates of A. baumannii and E. coli were 2-16 and 1-8 µg mL −1 , respectively whereas it was ranged 2-8 µg mL −1 for K. pneumoniae and P. aeruginosa isolates. Similarly, MICs for Elores against efflux positive isolates were 2-16 µg mL −1 for each of A. baumannii, K. pneumoniae and P. aeruginosa and 1-8 µg mL −1 for E. coli. Contrary to this, ceftriaxone MICs were >1024 to all the isolates except E. coli and K. pneumoniae positive with efflux. Ceftriaxone+sulbactam demonstrated MICs values 4-6 fold higher than Elores in all isolates (Table 1). MIC studies were also conducted using other ratios (1:1, 1:2, 3:1 and 4:1) of ceftriaxone plus sulbactam but significant results were obtained only with 2:1 ratio.

AST
Synergism of ceftriaxone and sulbactam against A. baumannii, E. coli, K. pneumoniae and P. aeruginosa were also demonstrated by a cup-plate agar diffusion method. For positive controls of E. coli, K. pneumoniae and P. aeruginosa inoculated onto a MHA plate containing Elores produced a ≥5 mm enhanced zone of inhibition 25.78±1.4, 26.24±1.8 and 25.53±1.6 mm, respectively compared to ceftriaxone alone and ceftriaxone plus sulbactam, indicating enhanced synergistic activity between the ceftriaxone and sulbactam in presence of non antibiotic adjuvant disodium edetate (Table 1). Similarly for clinical isolates of A. baumannii, E. coli, K. pneumoniae and P. aeruginosa positive with ESBL, MBL and efflux, ceftriaxone plus sulbactam with disodium edetate combination (Elores) produced a greater zone of inhibition ≥5 mm when compared with other two groups ( Table 1). AST studies were also conducted using other ratios (1:1, 1:2, 3:1 and 4:1) of ceftriaxone and sulbactam but did not show significant synergy (data not shown).

TKC
TKC study was performed on all clinical as well as positive controls and results are presented only for one clinical isolate of each of A. baumannii, E. coli, K. pneumoniae and P. aeruginosa positive with both MBL and efflux. Synergy was defined as ≥10 3 log of killing compared to the starting inoculum. Results of TKC demonstrated an enhancement of killing of selected organisms in the presence of AAE ceftriaxone +sulbactam in a ratio of 2:1 with non antibiotic adjuvant disodium edetate, in comparison to ceftriaxone alone and ceftriaxone plus sulbactam. After 12 h of incubation Elores exhibited approximately 10 4 log reduction in A. baumannii, E. coli, K. pneumoniae and P. aeruginosa positive with both MBL and efflux whereas when ceftriaxone was tested alone against these isolates no killing was observed at any time point and regrowth appeared after 4 h with ceftriaxone plus sulbactam (Fig. 4). TKC studies were also conducted using other ratios (1:1, 1:2, 3:1 and 4:1) of ceftriaxone and sulbactam with non antibiotic adjuvant disodium edetate, but significant results were obtained only with 2:1 ratio.

DISCUSSION
Indiscriminate use of antibiotics, poor patient compliance and improper infection control practices has led to emergence of multi drug resistant strains which transfer resistance through plasmids and confer resistance to commonly used cephaloroporin antibiotics. Combination therapy has been reported to be beneficial for the treatment of infections which fail to respond to single drug therapy because of lacking of efficacy or rapid emergence of resistance (Deresinski, 2009;Kumar et al., 2010). Although there are several data on the inter action of antibacterial agents against gram-positive and gramnegative organisms (Deveci et al., 2012), there is no report on the interaction between ceftriaxone and sulbactam and disodium edetate. Ceftriaxone in combination with sulbactam and disodium edetate show a greater susceptibility against resistant organisms as combination of trio, acting by different mechanisms, is used for the treatment of MDR bacterial infections.
The FIC index is the most commonly used method to determine the interaction between antibacterial drugs. The significant synergy was obtained at ratio 2:1 of ceftriaxone and sulbactam, which enhanced with increasing the concentration of disodium edetate and maximum synergy was found at 10 mM of disodium edetateA, suggesting synergistic activity of ceftriaxone + sulbactam + disodium edetate. This AAE was synergistic for both positive controls as well as selected clinical isolates positive with ESBL, MBL and efflux. Earlier it was demonstrated that ceftriaxone montherapy is ineffective in the treatment ESBL but when it was combined with sulbactam and disodium edetate synergy was enhanced significantly (Chaudhary et al., 2012c). Deveci et al. (2012) studied the combinations of sulbactam with ceftriaxone, ceftazidime and gentamicin against A. baumannii and observed synergy among these. The synergistic interaction between ceftriaxone plus sulbactam with disodium edetate was also demonstrated in animal model where combination therapy resulted in faster recovery in animals (infected with pneumonia) treated with combination compared with mono therapy (Dwivedi et al., 2012). Moreover, the synergistic interaction between ceftriaxone plus sulbactam with disodium edetate was also proven through clinical trials in patients suffering from Lower Respiratory Tract Infections (LTRIs), Urinary Tract Infections (UTIs), skin and skin structure infections (SSSIs) and Bone and Joint Infections (BJIs) (Chaudhary and Payasi, 2013a;2013b).
The synergistic activity of ceftriaxone plus sulbactam with disodium edetate was also reported in efflux positive isolates of E. coli and P. aeruginosa (Chaudhary et al., 2012a;Chaudhary and Payasi, 2012). In addition, TKC, broth dilution, agar diffusion studies also carried out against all clinical isolates and indicated synergy between the ceftriaxone and sulbactam in a ratio of 2:1 with disodium edetate. Earlier, the synergistic activity of ceftriaxone with moxifloxacin was studied and found to be synergistic (Zakaria et al., 2012).

CONCLUSION
The current study revealed that novel AAE a combination of ceftriaxone plus sulbactam with disodium edtate could be the effective solution against the infections caused by A. baumannii, E. coli, K. pneumoniae and P. aeruginosa microorganisms positive with ESBL, MBL and efflux rather than searching for new antibiotics for treatment of infections caused by these organisms.

ACKNOWLEDGEMENT
Researchers are thankful to Venus Medicine Research Centre, Werne, Germany and BFMC, Faridkot, Punjab, India for providing assistance to carry out this study.