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 ~  Abstract
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 ~ Results
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BRIEF COMMUNICATION
Year : 2018  |  Volume : 36  |  Issue : 1  |  Page : 127-130
 

In silico and In vitro activity of ceftolozane/tazobactam against pseudomonas aeruginosa collected across Indian hospitals


1 Department of Clinical Microbiology, Christian Medical College, Vellore, Tamil Nadu, India
2 School of Bioscience and Technology, Vellore Institute of Technology, Vellore, Tamil Nadu, India
3 Department of Medicine, Christian Medical College, Vellore, Tamil Nadu, India
4 Department of Microbiology, PD Hinduja Hospital and Medical Research Centre, Mumbai, Maharashtra, India
5 Department of Microbiology, Manipal Hospital, Bengaluru, Karnataka, India
6 Department of Microbiology, Calcutta Medical Research Institute, Kolkata, West Bengal, India
7 Department of Microbiology, Fortis Hospital, Anandapur, Kolkata, West Bengal, India
8 Department of Microbiology and Immunology, Choithram Hospital, Indore, Madhya Pradesh, India
9 Department of Microbiology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India

Date of Web Publication2-May-2018

Correspondence Address:
Dr. Balaji Veeraraghavan
Department of Clinical Microbiology, Christian Medical College, Vellore, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmm.IJMM_17_349

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 ~ Abstract 

Ceftolozane/tazobactam is a novel antimicrobial agent with activity against Pseudomonas aeruginosa and other common Gram-negative pathogens. In this study, we determined the antimicrobial susceptibility for a total of 149 clinical isolates of P. aeruginosa for the most commonly used antimicrobials including the new agent ceftolozane/tazobactam (C/T). Broth microdilution was performed to determine the minimum inhibitory concentration against various antimicrobials including C/T. Among the β-lactam/β-lactamase inhibitor, overall susceptibility was 67%, 55% and 51% for C/T, Piperacillin/Tazobactam (P/T) and Cefoperazone/Sulbactam, respectively. The variations in the susceptibility rates were noted among the three different β-lactam/β-lactamase inhibitors. Interestingly, 33% susceptibility was noted for C/T against isolates that were resistant to P/T, indicating the higher activity of C/T. This finding suggests about 33% of the P/T-resistant isolates can still be treated effectively with C/T. C/T could be a better alternative for the treatment of ESBL-producing organism, and thereby usage of higher antimicrobials can be minimised.


Keywords: Antimicrobials, ceftolozane/tazobactam, India, Pseudomonas aeruginosa, susceptibility


How to cite this article:
Pragsam AK, Kumar D T, Doss C G, Iyadurai R, Satyendra S, Rodrigues C, Joshi S, Roy I, Chaudhuri BN, Chitnis D S, Tapan D, Veeraraghavan B. In silico and In vitro activity of ceftolozane/tazobactam against pseudomonas aeruginosa collected across Indian hospitals. Indian J Med Microbiol 2018;36:127-30

How to cite this URL:
Pragsam AK, Kumar D T, Doss C G, Iyadurai R, Satyendra S, Rodrigues C, Joshi S, Roy I, Chaudhuri BN, Chitnis D S, Tapan D, Veeraraghavan B. In silico and In vitro activity of ceftolozane/tazobactam against pseudomonas aeruginosa collected across Indian hospitals. Indian J Med Microbiol [serial online] 2018 [cited 2018 Nov 15];36:127-30. Available from: http://www.ijmm.org/text.asp?2018/36/1/127/231670



 ~ Introduction Top


The increasing antimicrobial resistance is a worrisome scenario in developing countries, especially in India. Around 30% of the infections caused by Pseudomonas aeruginosa are resistant to multiple classes of antibiotics. Most of these infections are treated by the higher antimicrobial agents such as carbapenem and colistin. To minimise the use of higher agents, β-lactam/β-lactamase inhibitor combinations are alternative for treating extended spectrum beta lactamase (ESBL) producers. Of these, the frequently used combinations for P. aeruginosa at present are piperacillin/tazobactam (P/T) and cefoperazone/sulbactam (C/S). Recently, in 2014 and 2015, two novel combinations were approved by the Food and Drug Administration (FDA) are ceftolozane/tazobactam (C/T) and ceftazidime/avibactam (C/A), respectively.[1] While aztreonam/avibactam and Imipenem/relebactam are undergoing clinical trials (clinicaltrials. Gov)

Ceftolozane is a novel cephalosporin, active against pseudomonal ampC enzymes, efflux system and membrane impermeability. However, it can be hydrolysed by ESBLs and carbapenemases.[2] The addition of tazobactam to ceftolozane broadens the spectrum of activity against ESBL producers. FDA licenses this novel combination for use in adults for the treatment of complicated intra-abdominal infections (cIAI) and complicated urinary tract infections (cUTI) including pyelonephritis. This combination covers infections caused by Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae, P. aeruginosa, Proteus mirabilis, Bacteroides fragilis and Streptococcus spp. C/T has been shown to demonstrate better in vitro activity against isolates resistant to ceftazidime, cefepime and P/T studied across the world.[2] In this study, we evaluated the in vitro efficacy of C/T against P. aeruginosa collected from Indian hospitals. Further in silico analysis was performed to support the in vitro findings.


 ~ Materials and Methods Top


In vitro analysis

In this study, we determined the antimicrobial susceptibility for a total of 149 invasive clinical isolates of P. aeruginosa for the most commonly used antimicrobials including C/T. Isolates collected across various centers in India were tested. The participating center includes Christian Medical College, Vellore, Tamil Nadu; Manipal Hospital, Karnataka; Fortis Hospital, Kolkata, West Bengal; The Calcutta Medical Research Institute, West Bengal; Choithram hospital, Madhya Pradesh; Hinduja Hospital, Mumbai and Sanjay Gandhi Postgraduate Institute of medical science, Lucknow, Uttar Pradesh.

These isolates were sourced from the bloodstream, intra-abdominal and UTIs collected between the year 2013 and 2014. Broth microdilution was performed to determine the minimum inhibitory concentration (MIC) against various antimicrobials and results were interpreted according to the CLSI 2016 breakpoint interpretative criteria.[3] E. coli ATCC 35218 and P. aeruginosa ATCC 27853 were used as quality control organisms.

In silico analysis

The 3D structure of P. aeruginosa ampC was retrieved from the Protein Data Bank (PDB) with the PDB ID 4GZB.[4] The SMILES of C/T, and Piperacillin were retrieved from PubChem database with CIDs 53234134, 123630 and 43672, respectively. The 3D structures were converted to PDB format structure using OpenBabel. Molecular docking was performed using AutoDock 4.2. Hydrogen and necessary charges were charges added to the protein followed by torsion to the drugs. The grid was fixed around the active site, and AutoGrid was performed. Finally, AutoDock was performed using Lamarckian Genetic Algorithm in 10 runs. The docking was performed thrice, and average binding energy was calculated.


 ~ Results Top


In vitro analysis

Antimicrobial susceptibility profiles of the study isolates against the tested agents are summarised in [Table 1]. MIC50 and MIC90 for P. aeruginosa were 1/4 μg/ml and ≥16/4 μg/ml, respectively. Among the β-lactam/β-lactamase inhibitor, overall susceptibility was 67%, 55% and 51% for C/T, P/T and C/S, respectively. The variations in the susceptibility rates were 16% for C/T versus C/S and 12% for C/T versus P/T, respectively. Interestingly, 33% susceptibility was noted for C/T against isolates that were resistant to P/T, indicating the higher activity of C/T. However, the number of isolates analysed are less. Hence, a large number of isolates needs to be tested to prove the superiority of C/T over other agents.
Table 1: Antimicrobial Susceptibility profile to various agents as determined by Broth microdilution for Pseudomonas aeruginosa

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In silico analysis

The docking analysis has shown higher binding affinity for ampC with piperacillin and least binding affinity for ampC with ceftolozane. However, the binding affinity of-6.47 kcal/mol for ampC and tazobactam was noted, which was found to be in between the ampC and ceftolozane (−4.38) and ampC and piperacillin (−7.22). AmpC found to interact with ceftolozone through 13 amino acids (GLN120, THR290, SER289, ASN288, SER64, THR320, ASN321, SER319, ASN344, ASN347, PRO346, ALA348 and ARG350) and seven polar contacts. While for piperacillin, ampC found to react with 12 amino acids (TYR223, GLN129, ASN153, SER64, LYS67, LEU119, ASN344, TYR131, ALA293, ARG350, ASN347 and THR290) and three polar contacts. However, ampC with tazobactam showed interaction with 12 amino acids (SER319, ALA293, THR317, ASN344, TYR345, THR290, PRO346, ASN347, ASN288, SER289, ARG350 and ALA348) and eight polar contacts [Figure 1]a, [Figure 1]b, [Figure 1]c.
Figure 1: (a-c) PyMOL visualization showing the interaction between AmpC and antimicrobials. (a) Interaction with ceftolozone (b) Interaction with piperacillin (c) Interaction with tazobactam

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 ~ Discussion Top


This study finding is in concurrence with the previously published in vitro studies; wherein, the susceptibility rates and MIC50 were almost similar as summarised in [Table 2].
Table 2: Susceptibility of Pseudomonas aeruginosa to ceftolozane/tazobactam reported by various studies

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Further, this study data suggest that C/T will be effective than P/T and C/S, especially for managing infections due to drug-resistant P. aeruginosa. Buehrle et al. have attributed improved C/T activity, as it evades ampC-mediated hydrolysis in P. aeruginosa.[8] Further, ceftolozane escapes from the efflux mechanisms. It is well known that majority of the drug resistance in P. aeruginosa is due to over expression of efflux pumps. The notable difference in the activity of C/T over P/T and C/S could be due to overcoming these resistance mechanisms such as ampC and efflux. This finding has to be validated with testing a large number of isolates with known resistance mechanisms. However, the studies have shown mutations in the ampC confer resistance to C/T. Although it is intrinsic, mutations in the ampC have variable effects in the MIC of other cephalosporin agents as well, including C/T.[9],[10]

This is the first report of testing C/T against clinical isolates from India showing 30% incremental increase in the susceptibility for beta-lactamase producers. This finding suggests about 33% of the P/T-resistant isolates can still be treated effectively with C/T. From the docking analysis, hydrolysis rate of piperacillin by ampC was higher due to the higher binding affinity, leading to hydrolysis of piperacillin. However, ceftolozane is less prone to hydrolysis by ampC due to the less binding affinity, results in the effective activity of ceftolozane. This concurs with the in vitro susceptibility testing, wherein ceftolozane activity is superior to piperacillin. To conclude, as the development of newer agents are minimal, the utility of higher antibiotics (carbapenems and colistin) can be minimised using C/T when available in India and found susceptible against the beta-lactamase producing P. aeruginosa.


 ~ Conclusion Top


Antimicrobial resistance has become a major concern across the world. C/T is a new beta-lactam/beta-lactamase inhibitor-based antimicrobial agent, which has shown to have an excellent antipseudomonal activity, as it by passes ampC-mediated resistance. Among the β-lactam/β-lactamase inhibitor, the activity of C/T was observed to be superior to P/T and C/S. This overall in vitro and in silico analysis elucidates that the C/T could be a better alternative to P/T for the treatment of infections due to P. aeruginosa.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
 ~ References Top

1.
Papp-Wallace KM, Bonomo RA. New β-lactamase inhibitors in the clinic. Infect Dis Clin North Am 2016;30:441-64.  Back to cited text no. 1
[PUBMED]    
2.
Zhanel GG, Chung P, Adam H, Zelenitsky S, Denisuik A, Schweizer F, et al. Ceftolozane/tazobactam: A novel cephalosporin/β-lactamase inhibitor combination with activity against multidrug-resistant gram-negative bacilli. Drugs 2014;74:31-51.  Back to cited text no. 2
[PUBMED]    
3.
Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing; Twenty Sixth Informational Supplement. CLSI Document M100-S26. Wayne, PA: Clinical and Laboratory Standards Institute; 2016.  Back to cited text no. 3
    
4.
Lahiri SD, Mangani S, Durand-Reville T, Benvenuti M, De Luca F, Sanyal G, et al. Structural insight into potent broad-spectrum inhibition with reversible recyclization mechanism: Avibactam in complex with CTX-M-15 and Pseudomonas aeruginosa AmpC β-lactamases. Antimicrob Agents Chemother 2013;57:2496-505.  Back to cited text no. 4
[PUBMED]    
5.
Farrell DJ, Flamm RK, Sader HS, Jones RN. Antimicrobial activity of ceftolozane-tazobactam tested against Enterobacteriaceae and Pseudomonas aeruginosa with various resistance patterns isolated in U.S. Hospitals (2011-2012). Antimicrob Agents Chemother 2013;57:6305-10.  Back to cited text no. 5
    
6.
Sader HS, Farrell DJ, Castanheira M, Flamm RK, Jones RN. Antimicrobial activity of ceftolozane/tazobactam tested against Pseudomonas aeruginosa and enterobacteriaceae with various resistance patterns isolated in european hospitals (2011-12). J Antimicrob Chemother 2014;69:2713-22.  Back to cited text no. 6
[PUBMED]    
7.
Farrell DJ, Sader HS, Flamm RK, Jones RN. Ceftolozane/tazobactam activity tested against Gram-negative bacterial isolates from hospitalised patients with pneumonia in US and European medical centres (2012). Int J Antimicrob Agents 2014;57:2496-505.  Back to cited text no. 7
    
8.
Buehrle DJ, Shields RK, Chen L, Hao B, Press EG, Alkrouk A, et al. Evaluation of the in vitro activity of ceftazidime-avibactam and ceftolozane-tazobactam against meropenem-resistant Pseudomonas aeruginosa isolates. Antimicrob Agents Chemother 2016;60:3227-31.  Back to cited text no. 8
[PUBMED]    
9.
Cabot G, Bruchmann S, Mulet X, Zamorano L, Moyà B, Juan C, et al. Pseudomonas aeruginosa ceftolozane-tazobactam resistance development requires multiple mutations leading to overexpression and structural modification of AmpC. Antimicrob Agents Chemother 2014;58:3091-9.  Back to cited text no. 9
    
10.
Berrazeg M, Jeannot K, Ntsogo Enguéné VY, Broutin I, Loeffert S, Fournier D, et al. Mutations in β-lactamase AmpC increase resistance of Pseudomonas aeruginosa isolates to antipseudomonal cephalosporins. Antimicrob Agents Chemother 2015;59:6248-55.  Back to cited text no. 10
    


    Figures

  [Figure 1]
 
 
    Tables

  [Table 1], [Table 2]



 

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