Infections are becoming difficult to treat with commonly used antibiotics when caused by extended spectrum β-lactamase (ESBL)- and metallo-β-lactamase (MBL)-producing organisms.1 In recent years, the antibiotic resistance against ESBL-producing organisms has increased at an alarming rate.2 To overcome the antibiotic resistance caused by ESBL producers, carbapenems were introduced in clinical settings. However, carbapenem resistance among the members of the Enterobacteriaceae family has been reported globally.3

In view of the increasing antibiotic-resistance problems because of ESBL and MBL, a combination of ceftriaxone with sulbactam and EDTA, altogether termed as CSE1034, was developed, which has been found to be effective against ESBL-producing organisms.4, 5 However, its efficacy against isolates harboring MBL and ESBL+MBL is yet to be explored. Therefore, in vitro susceptibilities of MBL- and ESBL+MBL-positive Escherichia coli and Klebsiella spp. isolates to CSE1034 and other β-lactams were investigated.

A total of 151 isolates of phase-III clinical trial including E. coli (96, 63.57%), Klebsiella spp. (Klebsiella pneumoniae (34, 22.51%) and Klebsiella oxytoca (21, 13.90%)) were obtained from 278 patients suffering from skin and skin structure infections (SSSIs), bone and joint infections (BJIs), lower respiratory infections (LRTIs), urinary tract infections (UTIs), bacterial septicemia (BS) and chronic suppurative otitis media (CSOM) enrolled in 18 centers across India. Each isolate’s identity was confirmed as described earlier.6 All isolates were screened for ESBL and MBL by the Clinical and Laboratory Standards Institute methods (CLSI)7 and were then subjected to characterization for the types of ESBL and MBL genes.8, 9, 10 The susceptibility testing was performed according to the CLSI method.7

The antibiotics included for in vitro study were as follows: CSE1034 (1.5 g), ceftriaxone (1 g), piperacillin plus tazobactam (4.5 g), cefoperazone plus sulbactam (2 g) and imipenem plus cilastatin (0.5 g).

Out of the 96 E. coli received, 75 were from UTIs, 10 from BS, 7 from LRTIs, 3 from BJIs and 1 from CSOMs. Out of the 34 K. pneumoniae, 9 were from CSOMs, 8 from SSSIs, 7 from LRTIs, 4 from UTIs, 3 from BS and 3 from BJIs; of the 21 K. oxytoca, 7 were from CSOMs, 6 from UTIs, 4 from SSSIs, 3 from LRTIs and 1 from BS. The screening results confirmed that 50 isolates were ESBL producers, 15 were MBL producers and 86 had coproduced both ESBL and MBL. In E. coli, TEM-1, TEM-2 and TEM-50 were observed in 27, 17 and 7 isolates, respectively, whereas SHV-1 and SHV-10 were found in 58 and 10 isolates, respectively. NDM-1, VIM-1 and IMP-1 were found in 28, 17 and 16 isolates, respectively. Similarly, in Klebsiella spp., TEM-1, TEM-2 and TEM-4 were observed in 16, 18 and 2 isolates, respectively, whereas SHV-1 and SHV-2 were found in 35 and 2 isolates, respectively. NDM-1, VIM-1 and IMP-1 were found in 7, 28 and 26 isolates, respectively.

In vitro antibacterial susceptibility data are presented in Table 1. CSE1034 was the most active antibacterial agent with the majority of ESBL-producing E. coli displaying 93.02% susceptibility, whereas MBL- and ESBL+MBL-producing isolates exhibiting 100% susceptibility. Imipenem plus cilastatin appeared to be the second most active agent with 67.44% susceptibility, followed by piperacillin plus tazobactam (58.14%), cefoperazone plus sulbactam (55.81%) and ceftriaxone (51.16%). However, E. coli harboring ESBL+MBL exhibited 86.36%, 95.45%, 90.90% and 100% resistance to imipenem plus cilastatin, piperacillin plus tazobactam, cefoperazone plus sulbactam and ceftriaxone, respectively. Klebsiella spp. (K. oxytoca and K. pneumoniae) appeared to be highly susceptible to CSE1034 (100% to each of ESBL- and MBL-producing isolates, and 93–96% to ESBL+MBL-producing isolates). ESBL-producing Klebsiella spp. were equally susceptible to imipenem plus cilastatin and piperacillin plus tazobactam (66–75%), and susceptibilities of cefoperazone plus sulbactam and ceftriaxone varied from 33 to 50% and 25 to 33%, respectively. However, ESBL+MBL-producing Klebsiella spp. showed 100% resistance to ceftriaxone followed by 96–100% to each of the piperacillin plus tazobactam and cefoperazone plus sulbactam, and 84–87% to imipenem plus cilastatin. Interestingly, the most important observation of this study was that all MBL-producing isolates (E. coli and Klebsiella spp.) were almost 100% resistant to all the comparator antibiotics.

Table 1 In vitro activity of CSE1034 and other comparator antibiotics
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With regard to MIC distributions (Table 2), CSE1034 MICs fell within 4–32 μg ml−1 against 97% of E. coli and 96% of Klebsiella spp. The MIC distributions of ceftriaxone, imipenem plus cilastatin, piperacillin plus tazobactam and cefoperazone plus sulbactam for E. coli isolates varied between 8 and >128. Imipenem plus cilastatin and ceftriaxone MICs varied between 16 and >128, and piperacillin plus tazobactam and cefoperazone plus sulbactam MICs were 32 to >128 for Klebsiella spp.

Table 2 MIC distributions for isolates obtained from various clinical specimens
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CSE1034 showed intermediate-to-resistant response to TEM-50-positive isolates. However, it appeared to be highly susceptible to NDM-1-, VIM-1- and IMP-1-positive isolates. Imipenem plus cilastatin, piperacillin plus tazobactam, cefoperazone plus sulbactam and ceftriaxone that were resistant to NDM-1-, VIM-1-, IMP-1- and TEM-50-positive isolates, however, were found to be susceptible to those isolates positive with TEM-1, TEM-2 and SHV-1. The enhanced susceptibility of CSE1034 against E. coli and Klebsiella spp. is likely to be associated with synergistic activity of ceftriaxone plus sulbactam plus EDTA.11, 12 The CSE1034 enhanced the susceptibility by altering the outer-membrane permeability, which in turn increased penetration of drug inside the bacterial cells. Furthermore, EDTA chelates the divalent ions required for the activity of MBL, thus enhancing the susceptibility of CSE1034 toward MBL-producing organisms. Our previous studies also demonstrated the enhanced in vitro efficacy of CSE1034.5, 6, 11, 13 Studies in animal models also demonstrated promising in vivo efficacy of CSE1034.14, 15

The results obtained in the present study confirmed the broad-spectrum activity of CSE1034 against the MBL- and ESBL+MBL-producing organisms. Hence, CSE1034 can be considered as a drug of choice for the treatment of infections caused by these organisms.