Assessment of in vitro antimicrobial activity of ceftazidime-avibactam and phenotypic synergy testing with aztreonam against carbapenem resistant Gram-negative bacilli in a tertiary care hospital

INTRODUCTION

Carbapenem resistance is on the rise among the Enterobactarales (CRE) and Pseudomonas spp. carbapenem-resistant Pseudomonas aeruginosa (CRPA). These isolates are often resistant to many other classes of antimicrobials, thus lessening the therapeutic options.^[1] Ceftazidime avibactam (CZA) is one of the molecules used against such carbapenemase producing Gram-negative bacterial infections and is often heralded for its in vitro sensitivity and better clinical outcome.^[2] Avibactam (AVI) is a non-beta-lactam-beta-lactamase inhibitor having activity against serine carbapenemases like Klebsiella pneumoniae carbapenemase (KPC) and OXA (oxacillinase)-48-like carbapenemases while it has no action against metallo-β-lactamase (MBL) enzymes. However, this selective activity has created a potential void in areas like India where the MBL carbapenemases account for a vast proportion of about 61% of CRE and 15–30% of CRPA.^[3]

The age-old drug aztreonam is stable against MBLs due to its potent affinity for penicillin binding protein-3 (PBP3), but its therapeutic benefit is impeded by the fact that MBL producers often secrete other class A, C, or D enzymes against whom it is ineffective.^[4,5] Thus, a clever combination of CZA and aztreonam has been designed, where AVI neutralizes class A (Extended Spectrum β Lactamases [ESBL] and KPC), C enzymes, and D (OXA-48-like) enzymes and aztreonam the MBLs. The utility of this drug combination is limited in MBL-producing P. aeruginosa and Acinetobacter spp., as aztreonam may be ejected out of the cell by the efflux pump in these organisms. This drug combination has provided a glimmer of hope in the antibiotic horizon. The Infectious Diseases Society of America recommends that CZA and aztreonam infusion can be used concurrently for MBL organisms.^[6] However, recent studies have also started reporting decreased susceptibility to ATM-CZA that has been noted in studies due to a small insertion into in PBP3 that impacts the binding of ATM, ceftazidime, and other β-lactams.^[7] Thus, it is important to note the susceptibility pattern of this synergistic combination in clinical microbiology laboratories.

There is no clinical and laboratory standard institute (CLSI) approved method for evaluation of synergy testing of the said drugs. However, studies have shown many effective methods, for example, determining aztreonam minimum inhibitory concentration (MIC) by broth microdilution (BMD) when the broth has 4 mg/L of AVI.^[8] MIC determination using E-strips by CZA and ATM strip stacking and strip crossing methods^[9] overlay of aztreonam (30 μg) disc and CZA disk (30/20 μg) disc.^[9] A disk elution method has been proposed, which has a good correlation with the BMD method.

In view of the lack of published literature describing the in vitro susceptibility pattern of CZA and CZA-ATM combination in our area, the present study was undertaken to denote the in vitro susceptibility pattern of CZA and CZAATM combination.

MATERIALS AND METHODS

Phenotypic detection of synergy between CZA and aztreonam

MBL positive Enterobacterale isolates and all carbapenem resistant Pseudomonas isolates were further tested for synergy between CZA and ATM using CZA E-strip (HiMedia Labs) and 30 μg ATM disks (HiMedia Labs). On a mueller hinton agar (MHA) plate inoculated with an MBL positive isolate, a CZA E-strip is placed, and another ATM disc is placed 15 mm away from at center, taking into consideration that center of the disc remains parallel to the cutoff sensitivity mark of CZA (8 μg/mL). After overnight incubation, the results were interpreted as per CLSI breakpoints for CZA and ATM. Augmentation of the ATM zone towards the CZA strip was considered positive for synergy between these two antibiotics [Figure 1].

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Figure 1:

Picture showing augmentation of aztreonam zone toward ceftazidime avibactam E-strip, thus positive for synergy between these two antibiotics.

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All the data obtained were entered into an Excel sheet, and data were interpreted by basic statistical parameters.

RESULTS

During the study period, 150 carbapenem resistant organisms, including Enterobactarales (131, 87.3%) and P. aeruginosa (19, 12.7 %), were isolated from various samples. Enterobactarales isolated included-K. pneumoniae (101, 67.3%), E. coli (26, 17.3%), and Serratia spp. (4, 2.7%). These organisms were commonly isolated from blood 61 (46.6 %), followed by tracheal aspirates 32 (24.4%) [Table 1].

Colistin and polymyxin B were the most sensitive antibiotics for all the carbapenem resistant organisms. Klebsiella spp. showed dismal susceptibility to all the antibiotics, including cefepime, aminoglycosides, chloramphenicol, and tetracycline. E. coli isolates were sensitive to tetracycline (57.7%), doxycycline (53.8%), and netilmicin (65.4%). Pseudomonas spp. was susceptible to netilmicin and tobramycin (42.1%) [Table 2].

Among beta-lactam beta-lactamase combinations, ceftolozane tazobactam (as tested by Vitek-2) was less susceptible antibiotic to the organisms than CZA. Among Enterobactarales, 69.3% of Klebsiella spp., 61.53% of E. coli, and 50% of Serratia spp. were resistant to CZA. MIC50/MIC90 of both Klebsiella spp. and E. coli was 16/16, while that of Serratia spp. was 12/16.

On phenotypic testing of the Enterobacterale strains, 72 (54.9%) produced MBL type of carbapenemase. All the Serratia spp. and 88.5% of E. coli were MBL producers. Coproduction of ESBL enzyme was noticed in only 12.2% of tested isolates. There was a striking higher (82.4%) coproduction of Amp C type of beta-lactamases along with the carbapenemase production. Klebsiella spp. was highest producers of Amp C (85.14%) [Table 3].

Among the isolated organisms, 78 (77.28%) of Klebsiella spp., 12 (46.15%) of E. coli, and 9 (47.7%) of Pseudomonas spp. were multidrug-resistant (MDR) organisms. These MDR organisms also showed a high degree of resistance to CZA. Apart from 17 (21.8%) of Klebsiella spp., the rest were resistant to CZA.

Synergistic activity of CZA with ATM was determined by phenotypic method in MBL positive Enterobactarales and P. aeruginosa isolates. All the Serratia isolates showed in vitro synergistic activity of these drugs. Among Klebsiella and E. coli, 88.9% and 65.2% of MBL isolates showed in vitro synergy [Table 3].

DISCUSSION

Resistance to carbapenems is common worldwide and often mediated due to one of these mechanisms-porin mutation, overexpression of the efflux pump, and carbapenemase production. Carbapenemase production is the predominant among them and is often mediated by plasmid encoded serine enzymes such as KPC and OXA 48 of Ambler class A and D, respectively. MBLs, Ambler class B including New Delhi metallo beta- lactamase (NDM), verone integron-encoded metallo-beta-lactamase (VIM), and imipenemase (IMP) are also implicated. India has a high preponderance of NDM producing Enterobacterales. Tigecycline, polymyxin, and CZA are limited antimicrobial options available at our end for tackling CRE strains. The antibiotics such as tigecycline and colistin are associated with the emergence of drug resistance during treatment and are toxic as well and contribute to high mortality and morbidity. Tigecycline also lacks in vitro synergy, limiting its use in many situations.^[11]

CZA was launched in September 2015, and it contained a novel diazabicyclooctane β-lactamase inhibitor AVI which had promising activity against Enterobacterales producing serine enzymes, that is, ESBL enzymes, KPC and OXA 48 as well as against the class C cephalosporinases of P. aeruginosa. CZA is being used for these organisms in adults with limited treatment options as seen in various hospital acquired infections.^[12,13] CZA in many studies has been shown to fare superiorly than colistin the last resort drug till date in terms of amelioration of clinical symptoms and low mortality in CRE infection.^[14] In vitro studies have found CZA as having the highest percentage of susceptibility against clinical isolates of Enterobacterales.^[15] However, in the present study among the carbapenem resistant GNBs, 69.3% of Klebsiella spp., 61.53% of E. coli and 50% of Serratia spp. and 62.5% of P. aeruginosa were resistant to CZA. Similarly in another study,^[16] the resistance to CZA was shown in 90% of CRE. This is higher in comparison to a recent study^[17] where the CZA susceptibility for CRE and CRPA cases was 34% and 32%, respectively. The resistance to CZA closely correlated with MBL positivity (54.9%) in CRE cases. Although serine carbapenemase are predominant in other parts of the world, MBLs are common in Southeast Asia including India thus limiting the usefulness of this novel antibiotic.^[18,19] In contrast to studies from African subcontinent,^[17] the susceptibility of our isolates to carbapenem resistant P. aeruginosa is low (36.8%). This is probably due to presence of many MBL genes but there may be many other unexplored factors. We could not perform the genotypic assay to detect the MBL genes which are a limitation of the present study.

A solution to the CZA resistance was to cleverly combine CZA and ATM, which can prove as a cost-effective as well as clinically effective solution.^[20] Marshall et al. had first shown in 2017 that among 21 MBL positive CZA resistant isolates, 17 responded to combination of ATM and CZA.^[21] Karlowsky et al. had also demonstrated that 99.9% of isolates collected from 40 different countries were inhibited by the said combination.^[22] In the present study, synergy was noted in 89% Klebsiella isolates while it was markedly less in E. coli strains (65.2%). This is much less than that noted by Taha et al.^[16] where CZA and aztreonam combination showed a synergistic effect in 98.8% of Klebsiella spp. and 95% of E coli. Sreenivasan et al.^[23] also showed 100% in vitro synergy of this combination in MBL positive Enterobactarales. Few recent studies had also demonstrated the synergy between CAZ/AVI and ATM in MDR, extentensive drug resistant (XDR) and pan drug resistant (PDR) isolates using different phenotypic techniques.^{[8,9,16,21,23,24]} The previous studies^[7,25] have also reported the resistance to this combination due to change in PBP3 protein.

Our study has several limitations like-not performing the genotypic evaluation of the carbapenem resistant isolates. Inclusion of samples from a single tertiary care set up, no data regarding the clinical effectiveness of this drug combination could be collected. We also did not perform the checkerboard method the current method of choice for in vitro testing of combination regimens.

CONCLUSIONS

This is one of the foremost studies depicting the resistance pattern of CZA, a commonly used drug in ICU setting and highlights the present state of synergistic combination with aztreonam in our area. MBL positive Klebsiella and E. coli show 88.9% and 65.2% in vitro sensitivity, respectively, to this combination which is lesser than many previous studies. Further, evaluation to denote the genetic basis of this non-synergistic interaction should be looked into in.