|Year : 2020 | Volume
| Issue : 3 | Page : 307-312
Assessment of antibacterial activity of levonadifloxacin against contemporary gram-positive clinical isolates collected from various Indian hospitals using disk-diffusion assay
Shrikala Baliga1, Dhruv K Mamtora2, Varsha Gupta3, Priyadarshini Shanmugam4, Sanjay Biswas5, DN Mukherjee6, Suchitra Shenoy1
1 Department of Microbiology, Kasturba Medical College, Mangalore, Manipal Academy of Higher Education, Manipal, Karnataka, India
2 Department of Microbiology, S. L. Raheja (A Fortis Associate) Hospital, Mahim, Mumbai, Maharashtra, India
3 Department of Microbiology, Government Medical College, Chandigarh, India
4 Department of Microbiology, Chettinad Hospital and Research Institute, Chettinad Academy of Research and Education, Chennai, Tamil Nadu, India
5 Department of Microbiology, Tata Memorial Hospital, Mumbai, Maharashtra, India
6 Department of Microbiology, Woodlands Multispeciality Hospital, Kolkata, West Bengal, India
|Date of Submission||03-Jul-2020|
|Date of Decision||19-Aug-2020|
|Date of Acceptance||03-Sep-2020|
|Date of Web Publication||4-Nov-2020|
Dr. Shrikala Baliga
Department of Microbiology, Kasturba Medical College, Mangalore - 575 001, Karnataka
Source of Support: None, Conflict of Interest: None
Objectives: Levonadifloxacin is a novel benzoquinolizine subclass of quinolone with broad-spectrum activities against problematic pathogens such as methicillin-resistant Staphylococcus aureus, quinolone-resistant S. aureus, vancomycin intermediate S. aureus, and vancomycin-resistant S. aureus. Levonadifloxacin and its oral prodrug, alalevonadifloxacin, have been recently approved in India for the treatment of acute bacterial skin and skin structure infections, including concurrent bacteraemia and diabetic foot infections. The aim of the study is to assess the activity of levonadifloxacin against Gram-positive clinical isolates collected from various Indian hospitals using the disc-diffusion method. Materials and Methods: Nonduplicate isolates of S. aureus and other Gram-positive isolates collected from June 2019 to March 2020 were subjected to levonadifloxacin susceptibility testing (disk diffusion method) as per the Clinical and Laboratory Standards Institute guidelines (Year 2019). Levonadifloxacin 10 μg impregnated disks were used during the testing. Results: A total of 664 diverse Gram-positive clinical isolates collected from six different hospitals in India were analyzed. Majority (65.5%) of the isolates were S. aureus. All the S. aureus and other Gram-positive isolates were found to be susceptible to levonadifloxacin as per the prespecified interpretive criteria identified based on population pharmacokinetic model and Monte Carlo simulation enabled probability of pharmacodynamic target attainment analysis. Conclusions: The present study showed that levonadifloxacin was highly active against contemporary Gram-positive pathogens and furthermore demonstrated that levonadifloxacin susceptibilities can be reliably determined using the disc-diffusion method.
Keywords: Gram-positives, levonadifloxacin, methicillin-resistant Staphylococcus aureus, quinolone-resistant Staphylococcus aureus, susceptibility testing
|How to cite this article:|
Baliga S, Mamtora DK, Gupta V, Shanmugam P, Biswas S, Mukherjee D N, Shenoy S. Assessment of antibacterial activity of levonadifloxacin against contemporary gram-positive clinical isolates collected from various Indian hospitals using disk-diffusion assay. Indian J Med Microbiol 2020;38:307-12
|How to cite this URL:|
Baliga S, Mamtora DK, Gupta V, Shanmugam P, Biswas S, Mukherjee D N, Shenoy S. Assessment of antibacterial activity of levonadifloxacin against contemporary gram-positive clinical isolates collected from various Indian hospitals using disk-diffusion assay. Indian J Med Microbiol [serial online] 2020 [cited 2021 Jan 27];38:307-12. Available from: https://www.ijmm.org/text.asp?2020/38/3/307/299830
| ~ Introduction|| |
Methicillin-resistant Staphylococcus aureus (MRSA) is a major concern for health-care workers. The World Health Organisation has estimated a 64% higher likelihood of mortality due to MRSA compared to infections with non-resistant Staphylococcal isolates. MRSA prevalence is high across India showing some regional variation with Western India reporting 25% MRSA and Southern India reporting 50%. Furthermore, as per another recent publication, the prevalence of MRSA in India has increased significantly from 29% in 2009 to 37.3% in 2017.
The management of MRSA infections in large inpatient and outpatient settings relies on the availability of well-tolerated, anti-MRSA intravenous (IV) and oral antibiotics, respectively. At present, vancomycin and linezolid are the two most common antibiotics deployed to treat MRSA infections (teicoplanin and daptomycin to some extent); however, both drugs suffer from several limitations often complicating their clinical usage. Although vancomycin has been the drug of choice for years to treat MRSA infections, it is considered a suboptimal option in critically ill patients due to its weak bactericidal activity, poor penetration to tissues (such as lung), renal toxicity and risk of clinical failure due to minimum inhibitory concentration (MIC) creep.,, On the other hand, the clinical use of linezolid is limited by its bacteriostatic activity, and therefore, is not recommended to be used in blood stream infections. Moreover, the development of adverse side effects such as bone marrow suppression leading to thrombocytopenia requires linezolid to be used for shorter duration and necessitates concomitant monitoring of safety parameters. Due to these reasons, to treat MRSA, clinicians need access to improved antibiotics that are bactericidal, have adequate tissue penetration and is safe especially for the longer duration of treatment which may be necessary in diabetic foot and bone and joints infections.
Levonadifloxacin (WCK 771) is a novel antibiotic belonging to the benzoquinolizine subclass of fluoroquinolones with potent activity against MRSA and quinolone-resistant S. aureus (QRSA). IV levonadifloxacin [Figure 1] and its oral formulation, alalevonadifloxacin, have recently been approved in India for the treatment of acute bacterial skin and skin structure infections with concurrent bacteraemia and diabetic foot infections. The approval is based on a successfully conducted Phase 3 clinical study comparing levonadifloxacin with linezolid (Clinical Trial Registry India, CTRI/2017/06/008843). The potent activity of levonadifloxacin against MRSA, QRSA and hetero-vancomycin-intermediate S. aureus is an outcome of its well-differentiated mechanism of action involving preferential targeting to DNA gyrase while retaining high affinity toward topoisomerase IV as well. Levonadifloxacin also shows clinically relevant activities against resistant respiratory pathogens such as macrolide- and penicillin-resistant Streptococcus pneumoniae, Streptococcus pyogenes, Haemophilus influenzae and Moraxella catarrhalis. It is also active against anaerobic pathogens as well as atypical respiratory pathogens such as Chlamydia pneumoniae, Legionella pneumophila and Mycoplasma pneumoniae. Recently, the potentin vitro activity (MIC) of levonadifloxacin against contemporary Indian MRSA isolates, including the Bengal Bay clones, has been reported. In another report, the clinical, pharmacological and antimicrobial profile of levonadifloxacin has been elaborated in comparison with other anti-MRSA antibiotics.
|Figure 1: Levonadifloxacin is S-(-)-9-fluoro-6,7-dihydro-8- (4-hydroxypiperidin-1-yl)-5-methyl-1-oxo-1H,5H-benzo [i, j] quinolizine-2-carboxylic acid L-arginine salt tetrahydrate. The molecular formula of levonadifloxacin is C25H35FN6O60.4H2O and the molecular weight is 606.6 g/mol|
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Ahead of market launch of both IV and oral formulations of levonadifloxacin in India, laboratories need to gain experience in susceptibility testing of this novel drug. A 10 μg levonadifloxacin disk has been developed and approved by the Clinical and Laboratory Standards Institute (CLSI) in 2016. In the present study, activity of levonadifloxacin was assessed using the Kirby–Bauer disk diffusion assay against contemporary Gram-positive clinical isolates collected from multiple tertiary care hospitals in India.
| ~ Materials and Methods|| |
From June 2019 to March 2020, specimens were collected from patients in cardiology, surgery, oncology, paediatrics, medicine, gynaecology, ear-nose-throat and orthopaedic wards and intensive care unit of six hospitals located at different cities (Mumbai: Tata Memorial Hospital, S. L. Raheja Hospital; Chennai: Chettinad Hospital and Research Institute; Chandigarh: Government Medical College; Mangalore: Kasturba Medical College; Kolkata: Woodlands Hospital) in India. Clinical specimens collected were tissues, pus, sputum, urine, blood, pleural fluid, bone, vaginal swab, abscess, placental membrane, wound swab, bronchioalveolar lavage, endotracheal tube suction and peritoneal fluid from the various clinical sites of infection.
Disk-diffusion susceptibility assay
Levonadifloxacin disc-diffusion assay-based pathogen susceptibilities were determined following methods as recommended by CLSI. Levonadifloxacin disks were prepared as follows: a stock solution of levonadifloxacin (synthesized by Wockhardt) was prepared in L-arginine (SRL, Mumbai) (27.5 mg/L) and diluted in water. Commercial paper disks were manufactured by leading diagnostic company-Mast Group Ltd, UK, by loading 10 μg of levonadifloxacin. Non-fastidious clinical isolates were tested on Mueller-Hinton Agar (MHA) at 35°C ± 2°C, ambient air, 16–18 h, while MHA with 5% sheep blood was used for disc-susceptibility testing of S. pneumoniae (35°C ± 2°C with 5% CO2 for 20–24 h). Depending on the zone diameter criteria for an organism group, susceptibilities were determined by following the interpretive criteria identified for levonadifloxacin [Table 1]. The interpretive criteria were identified based on probability of (pharmacodynamic [PD]) target attainment (PTA) analysis undertaken at University of Maryland, USA. The PD target is the minimum antibiotic exposure in plasma, generally expressed in terms of PD index (either free area under the curve/MIC or %f T > MIC) required to exert antibacterial action against the infecting pathogen relative to its MIC. In general, for a new drug, the PD index and PD targets are identified using standardin vitro orin vivo pharmacokinetic PK/PD models. Attainment of specific PK/PD target is contingent to the adequate exposure of antibacterial agent to the microorganism which is dependent on the dose and PK properties of the drug. In line with this approach, robust PD targets were identified for levonadifloxacin from nonclinicalin vivo PK/PD studies (standard neutropenic mice lung infection model) by employing S. aureus expressing diverse resistance mechanisms such as, norA efflux expressing, MRSA and QRSA strains with levonadifloxacin MICs up to 2 mg/L (levofloxacin and moxifloxacin MIC up to >32 and 16 mg/L, respectively). In order to assess whether the identified PD target exposure relative to infecting pathogen's MIC is attainable in the large patient population receiving the standard dose of levonadifloxacin (800 mg, q12 h), PTA analyses were undertaken by Monte Carlo simulation (PK simulated for 2000 patients to capture PK variability usually observed in the patient population). This simulation is undertaken using a population PK model for levonadifloxacin developed using Phase 1 and Phase 2 (patients) PK data. A population PK model typically characterises population parameter values, between-subject and between-occasion PK variability, and influence of covariate on PKs. The PTA analyses revealed a high >90% attainment of levonadifloxacin PD target exposures with levonadifloxacin dose of 800 mg BID and alalevonadifloxacin dose of 1000 mg BID for strains with levonadifloxacin MIC up to 4 mg/L, suggesting high probability of consistent clinical cure in vast majority of patients despite imminent PK variability. Even though the identified doses of levonadifloxacin have demonstrated potential to cover strains with MIC up to 4 mg/L, a conservative susceptibility breakpoint of 2 mg/L (one-fold lower than PK/PD analysis-supported breakpoint) was chosen for S. aureus. This approach imparts a significant PK/PD leeway, as several studies have established a levonadifloxacin MIC90 of 0.5–1 mg/L for S. aureus. A similar approach was followed for deriving levonadifloxacin susceptibility interpretive criteria for other target pathogens as well. The disk-susceptibility criteria were evolved based on data showing excellent correlation between MIC and zone diameter for levonadifloxacin., The optimal performance of the disks during susceptibility testing was ensured by employing quality control strains [Table 2]. The ethics committee clearance was obtained vide letter MAHE EC/014/2019.
|Table 2: Disk susceptibility analysis of levonadifloxacin and comparators against quality control isolates|
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| ~ Results and Discussion|| |
[Table 3] shows the distribution of 664 diverse Gram-positive clinical isolates collected from six different hospitals. Among them, largest numbers were S. aureus (n = 435) and the proportion of MRSA was 44.8%. Based on the diameter of zone of inhibition observed with 10 μg levonadifloxacin disks, by employing the interpretive criteria provided in [Table 1], all the isolates including MRSA were susceptible to levonadifloxacin. [Table 4] provides the range and mean zone diameter values obtained for each of the Gram-positive organism groups tested in this study. In general, the zone diameter values were ≥20 mm suggesting the potent activity of levonadifloxacin against Gram-positive isolates. In particular, against S. aureus isolates, the mean zone diameter ranged from 22 to 29 mm categorising all the isolates as susceptible to levonadifloxacin. Similarly, other Gram-positive isolates were also levonadifloxacin susceptible with mean zone diameters ranging mostly between 20 and 30 mm. There were differences in mean zone diameters for isolates collected from the different geographical regions which could be ascribed to the differences in the proportions of MRSA versus MSSA as well as quinolone-resistant versus quinolone-susceptible isolates. The activity of levonadifloxacin against Gram-positive isolates observed in this study is consistent with various previous reports., For instance, in a recent report by Appalaraju et al., levonadifloxacin exhibited potent activity against 390 S. aureus isolates (98.7% susceptibility) collected from 15 tertiary hospitals, located in different parts of India. These isolates included MRSA as well as quinolone-resistant phenotypes. In another study, all 793 S. aureus isolates (including Bengal Bay clones) collected at a large tertiary care hospital at Vellore, Tamil Nadu were found to be susceptible to levonadifloxacin.
|Table 3: Distribution of Gram.positive isolates collected from various locations for the determination of disk diffusion susceptibilities|
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The remarkable activity of levonadifloxacin against Gram-positive isolates, especially against MRSA, is attributed to its unique mode of action. The antibacterial action of fluoroquinolones in Gram-positive organisms involves inhibition of bacterial topoisomerases that play a critical role in DNA replication. Older fluoroquinolones, such as ciprofloxacin and levofloxacin, have been shown to possess primary affinity toward topoisomerase IV of S. aureus. As a result, activity of these agents is significantly impacted against those S. aureus isolates that carry mutations in topoisomerase IV. It has been established that majority of clinical QRSA strains owe their quinolone resistance to multiple mutations in topoisomerase IV. In contrast, levonadifloxacin is able to overcome ciprofloxacin- and levofloxacin-resistance in S. aureus due to its preferential affinity towards DNA gyrase, an enzyme highly critical to cell survival. Moreover, it has been established that the primacy of levonadifloxacin towards DNA gyrase imparts a significant potency gain compared to other quinolones such as moxifloxacin, gatifloxacin, sparfloxacin and grepafloxacin., Unlike levonadifloxacin, moxifloxacin lacks the coverage of MRSA which is evident from various previous studies. In a study by Reddy et al. that determined the susceptibilities of Gram-positive isolates to fluoroquinolones using disk-diffusion assay, only 7 out of 97 ciprofloxacin-resistant S. aureus isolates were found to be susceptible to moxifloxacin.
The 100% susceptibility rate observed for levonadifloxacin in this study supports its use as a therapeutic option for MRSA. Further, it could also be used as an empirical therapy. Although vancomycin and linezolid also show similar high susceptibility rates, vancomycin use is often associated with nephrotoxicity and longer duration use of linezolid leads to myelosuppression. Unlike vancomycin, levonadifloxacin can be administered to patient with renal or liver impairment without the need for dose adjustments. Moreover, the availability of oral formulation of levonadifloxacin with comparable PK feature allows easy IV to oral switch.
This study analysis has few limitations such as the lack of comparative data for older isolates to assess change in susceptibility rates, as well as data for other anti-MRSA agents.
In summary, present and pastin vitro studies show consistent activity of levonadifloxacin against Gram-positive isolates including difficult-to-treat MRSA isolates. The 100% susceptibility of isolates to levonadifloxacin observed in this study supports its potential clinical use in the treatment of infections caused by MRSA and other Gram-positive organisms.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4]