Evaluation of ceftriaxone-sulbactam-disodium edetate adjuvant combination against multi-drug resistant Gram-negative organisms

Background Multi-drug resistant (MDR) Gram-negative bacteria are an emerging threat, both in hospital and community settings. As very few antibiotics are effective against such infections, the need of the hour is a new antibiotic or drug combination which can overcome the effect of extended-spectrum β-lactamases (ESBL) and metallo β-lactamases (MBL). A new antibiotic combination of ceftriaxone, sulbactam and disodium edetate (CSE) has recently been proposed to tackle the MDR organisms. Objective Our study was carried out to assess the susceptibility of ESBL- and MBL-producing Gram-negative organisms to CSE. Methods The study was conducted in a tertiary-care hospital in Delhi, India, from February 2017 to June 2017. A total of 179 MDR (85 ESBL + 94 MBL) Gram-negative isolates from various clinical samples, identified by an automated system (Vitek 2) were tested against CSE using the Kirby-Bauer disc diffusion method. Susceptibility to CSE was recorded based on interpretative zone sizes of ceftriaxone as per 2017 Clinical and Laboratory Standards Institute guidelines. Results The most common isolate was Escherichia coli (76/179; 42.4%) followed by Klebsiella pneumoniae (53/179; 29.6%) and Acinetobacter baumanii (27/179; 15.1%). The in vitro susceptibility of ESBL- and MBL-producing Gram-negative isolates to CSE was found to be 58/85 (68.2%) for ESBL and 37/94 (39.4%) for MBL. Conclusion The in vitro susceptibility results obtained for CSE against ESBL-producing organisms is promising. It has the potential to emerge as a carbapenem-sparing antibiotic, active against ESBL-producing strains. Further clinical studies are required to establish the clinical efficacy of CSE against MDR pathogens.


Introduction
http://www.ajlmonline.org Open Access spectrum enzymes that also hydrolyse most β-lactam antibiotics, except monobactams. 8 Other mechanisms that contribute to resistance are drug efflux systems, outer membrane protein changes, antibiotic-modifying enzymes and antibiotic-target modification. 9 Carbapenems are used against the ESBL-producing organisms because of their stability against hydrolysis by ESBLs and broad-spectrum activity. 10 However, with the emergence of carbapenemhydrolysing enzymes, overexpression of efflux pumps and changes in outer membrane proteins, increases in carbapenem resistance have been reported among the members of enterobacteriaceae and non-fermenter GNB, such as the Acinetobacter and Pseudomonas group of pathogens. 11,12 In India, the prevalence of ESBL and MBL producers among Gram-negative organisms range between 28% -84% and 7.5% -71%, respectively. 13,14 The increasing resistance towards available antibiotics, as well as the lack of development of new antibiotics against GNB, could soon lead to the world experiencing the tough situations of the pre-antibiotic era with an increase in cases with untreatable infections. A newer approach to improving the efficiency of the existing antimicrobials is the use of antibiotic adjuvants. A novel antibiotic adjuvant entity of ceftriaxone, sulbactam with adjuvant disodium edetate (CSE) is being used in Indian hospitals against MDR infections. 15,16 The antibiotic adjuvant entity is a combination of ceftriaxone plus sulbactam with disodium edetate and has undergone Phase III clinical trials under the aegis of the Central Drugs Standard Control Organisation, India. 17 This study aimed to study the in vitro susceptibility to CSE of MDR Gram-negative organisms isolated in our centre. Thus, the present study aimed to study the in vitro susceptibility of ESBL-and MBL-producing GNB to CSE and to explore whether it could be utilised as a carbapenem-sparing drug.

Ethical considerations
Ethical clearance was obtained from the Institutional Ethics Committee, Army Hospital (Research and Referral), New Delhi, India (92/2016).

Microbiological processing
Samples were processed using conventional methods. Blood culture bottles were incubated in a fully automated blood culture system, the BacT/Alert 3D (bioMérieux SA, Marcyl'Étoile, France). After obtaining a pure bacterial growth, isolate identification and antibiotic sensitivity testing were carried out on a Vitek 2 Compact (bioMérieux SA, Marcy-l'Étoile, France). An MDR isolate was defined as a GNB strain that showed resistance to at least three different categories of antibiotics. 18 A total of 85 ESBL-and 94 MBL-producing GNB were identified by phenotypic tests and confirmed by the Vitek 2 system for inclusion in the study. The confirmatory test for ESBL production was carried out using discs containing ceftazidime (30 μg) alone, along with ceftazidime/clavulanic acid (30/10 μg) discs. Similarly, cefotaxime (30 μg) and cefotaxime/clavulanic acid discs (30/10 μg) were also used. An increase in zone diameter of ≥ 5 mm around the clavulanate disk compared to the zone of inhibition for the ceftazidime and cefotaxime disk alone was used to confirm and isolate as positive for ESBL production as per Clinical and Laboratory Standards Institute guidelines. 19 The modified Hodge test 19 was used for isolates identified as carbapenemase-producing GNB strains. A 10 μg meropenem disc was placed on a Mueller-Hinton agar plate previously inoculated with Escherichia coli American Type Culture Collection 25922 (the indicator organism). Afterwards, the test organisms were streaked out in a straight line, starting from the edge of the meropenem disc, for at least 20 mm -25 mm length. The enhancement of growth of the indicator organism along the test organism's streak line and zone of inhibition of the disk to form a cloverleaf appearance was considered as a positive indicator for carbapenemase production as per Clinical and Laboratory Standards Institute guidelines. 19 For detection of class B carbapenemases (MBL), the doubledisc synergy test using imipenem and imipenem plus ethylenediaminetetraacetic acid disc was done. An organism with a zone size difference of 7 mm between imipenem and imipenem plus ethylenediaminetetraacetic acid discs was considered to be an MBL-producing strain. 20 All 179 isolates were then further tested for antimicrobial susceptibility against CSE ( 19 The zone of inhibition around the disc was measured, and the organism was labelled as sensitive if the zone measured > 23 mm for enterobacteriaceae or > 21 mm for Acinetobacter, intermediate if 20 mm -22 mm (enterobacteriaceae) or 14 mm -20 mm (Acinetobacter), or resistant if < 19 mm for enterobacteriaceae or < 13 mm for Acinetobacter. 19 Non-fermenters such as Pseudomonas aeruginosa and Burkholderia cepacia were not tested against CSE as these are known to be inherently resistant to ceftriaxone and there are no testing standards mentioned in Clinical and Laboratory Standards Institute guidelines. 19

Data analysis
Statistical analysis was done using Graph Pad, a free online software offering by founder Dr Harvey Motulsky (GraphPad Software, San Diego, California, United States). Associations between two factors were drawn through univariate logistic regression using the Fischer exact test. P-values of less than 0.05 were considered statistically significant.

Results
A total of 179 clinical isolates from 168 clinical cases (117 male patients and 51 female patients) were included in the study, with a mean patient age of 43.22 years (range: 4-85 years). Most isolates were from urine, followed by pus and blood specimens, and these accounted for 136 (75.9%) of the total isolates (Table 1). Of the included pathogens, 127 (70.9%) were isolated from inpatients, and 29 (16.2%) were isolated from patients in intensive care units ( Figure 2).

Discussion
This study included only those MDR GNB isolates which were proven to be pathogenic, obtained from clinically established cases of infection. The majority of isolates were from urine samples (n = 84; 47%), followed by pus (n = 34; 19%) and blood (n = 18; 10%  25 However, in the present study, E. coli and K. pneumoniae were implicated in 16 out of 18 (88.8%) Gram-negative bloodstream infection cases, with K. pneumoniae identified in the majority (10/18; 55.5%) of cases as compared to E. coli (6/18; 33.3%), which is in contrast to the National Health Service report. 25 Acinetobacter baumanii is known to be an important pathogen in causing respiratory infections such as hospital-acquired pneumonia and bacteraemia, especially in intensive care patients, followed by skin and soft tissue infections and urinary tract infections. 26,27 The present study has dissimilar findings in terms of isolation of A. baumanii, with maximum isolation from pus, because of skin and soft tissue infection cases (50%); followed by respiratory samples, because of respiratory infections (32.1%); and blood samples, because of bacteraemia (7.1%).
In    One major concern is finding effective treatments for infection with Acinetobacter spp., which is now commonly isolated from critical areas in most of the hospitals worldwide. 24 Our study found CSE to be effective in 48.1% of MBL-producing A. baumanii infections, which is a fair number, and use of CSE could be beneficial in such infections. A similar study conducted in Pune, India found that CSE was a superior antibiotic compared to other commonly used β-lactam antibiotics, including carbapenems, when tested against MDR GNB. 32 A study conducted in Faridabad, Haryana, India, which evaluated the clinical use of CSE on patients, concluded that CSE should be used as a carbapenem-sparing drug and its combination with polymyxins can help to reduce mortality rates by successfully treating complicated MDR cases of intraabdominal, lower respiratory tract and urinary tract infections. 12

Limitations
The limitation of our study was the relatively small number of isolates tested. Larger sample size and diverse health facility level (primary to tertiary) studies would be required to rule out any referral bias that is expected in a tertiary-care hospital. Further, this study can be extended with application to clinical situations to have a clinico-microbiological correlation to guide clinicians for the judicious use of CSE against MDR pathogens.

Conclusion
The results of this study show that CSE can potentially be effective among ESBL-producing bacteria, especially E. coli. The susceptibility of multi-drug resistant Gram-negative microorganisms to CSE suggests that CSE could be a good option as a carbapenem-sparing drug and also against some of the MBL-producing organisms.