Available online on 15.09.2023 at http://jddtonline.info

Journal of Drug Delivery and Therapeutics

Open Access to Pharmaceutical and Medical Research

Copyright   © 2023 The  Author(s): This is an open-access article distributed under the terms of the CC BY-NC 4.0 which permits unrestricted use, distribution, and reproduction in any medium for non-commercial use provided the original author and source are credited

Open Access  Full Text Article                                                                                                                                                     Research Article

A Prospective, Observational and Comparative Study of In-Vitro Susceptibility of Isepamicin against Clinical Isolates from Various Clinical Sources (Triple I Study - Isepamicin in India)

Dr Niraj Tyagi¹* (MBBS) , Dr Abdul Ansari² (MD) , Dr Bhaskar Narayan Chaudhuri³ (MD) , Dr Bansidhar Tarai⁴ (MD)  , Dr Ami Varaiya⁵ (MD), Dr Aarthi Sundaresan⁶ (MD)  

1. Consultant, Critical Care Medicine, Sir Ganga Ram Hospital, New Delhi, India. 

2. Director, Critical Care Services, Nanavati Max Super Specialty Hospital, Mumbai, India. 

3. Consultant, Clinical Microbiologist & Infection Control Officer, Peerless Hospital & Research Center Ltd., Kolkata, India. 

4. Principal Consultant & Head Microbiology & Infection Control, Max Super Speciality Hospital, New Delhi, India. 

5. Nanavati Max Super Speciality Hospital, Vile Parle, Mumbai, Life Member of Hospital Infection Control Mumbai Forum, India. 

6. Associate Consultant, Microbiology, Nanavati Max Super specialty hospital, India

INTRODUCTION

The emerging antibiotic resistance represents a major concern nowadays. Several Gram-negative bacteria, which are a leading pathogen for a variety of infectious diseases, have developed resistance to a wide range of antibiotics, causing significant morbidity and mortality worldwide. Data from Indian hospitals has shown the prevalence of extended spectrum beta-lactamase (ESBL) producing Gram-negative bacteria in a range between 19% and 60%, and that of Carbapenem-resistant bacteria between 5.3% and 59%. Also, resistance to Gram-negative bacteria increases the financial burden of patients as measured by mortality, length of stay, and hospitalization cost ¹.

The Enterobacteriaceae family, such as Escherichia coli (E. coli), Klebsiella spp., and Enterobacter spp., is the major cause of urinary tract infections (UTIs), of which the most common causative agent is E. coli, followed by K. pneumoniae ^{2, 3}. The treatment of UTIs is becoming more difficult due to the rapid spread of drug resistance, which is primarily related to the production of ESBLs, which leads to multidrug resistance (MDR) ⁴. Hence, substantial effort should be put into developing new antibiotics against Gram-negative bacteria that reduce the suffering associated with UTIs.

Aminoglycosides have been widely used for the treatment of life-threatening infections, including UTIs, despite showing renal and auditory toxicities as major side effects. Isepamicin, a semisynthetic derivative of Gentamicin B, is one of the recently developed aminoglycosides that is prescribed in Asian and certain European countries for the treatment of infections caused by Gram-negative bacteria (Enterobacteriaceae). However, the emergence of resistance to aminoglycosides is mainly attributed to the aminoglycoside-modifying enzymes such as type 1 6'- acetyltransferase produced by the pathogen, Enterobacteriaceae. Isepamicin has been shown to be more beneficial against strains that produce type 1 6′- acetyltransferase and one of the less toxic aminoglycosides ^{5, 6}.

A review of 14 studies, comprising a total of 4901 isolates and examining Isepamicin against infections with Gram-negative bacteria, demonstrated comparable or higher in-vitro activity compared with Amikacin. Isepamicin also appeared to be superior to Amikacin in studies that included MDR bacteria ⁷. Clinical studies have also shown no important difference in the effectiveness and safety profile of Isepamicin compared with those of Amikacin for the treatment of children with UTIs ⁸. There is a lack of in-vitro data for Isepamicin against various pathogens from India, so the purpose of this study was to investigate the in-vitro susceptibility of clinical isolates obtained from patients admitted to tertiary care hospitals against Isepamicin and compare it with Gentamicin and Amikacin.

MATERIALS AND METHODS

Sample collection

The Gram-negative bacterial isolates evaluated in this study were isolated from clinical specimens of patients treated in three different centres (city hospitals), namely, Peerless Hospital, Kolkata (site A); Nanavati Max Super Speciality Hospital, Mumbai (site B); and Max Super Speciality Hospital, New Delhi (site C), located in the eastern, western, and northern regions of India, respectively.

The bacterial isolates [Klebsiella pneumoniae (K. pneumoniae), Pseudomonas aeruginosa (P. aeruginosa), Acinetobacter baumannii (A. baumannii), Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), Enterobacter aerogenes (E. aerogenes), Serratia, and MRSA] were collected from blood cultures, respiratory secretions, urine cultures, sterile body fluid cultures (bile), frank pus, and tissue culture of the microbiology laboratory. This study assessed the sensitivity of Isepamicin, Gentamicin, and Amikacin and their distributions as susceptible and resistant.

Inclusion Criteria

Both male and female patients, at least 18 years of age, visiting in-patient and out-patient departments and having clinical isolates from urine samples, intra-abdominal samples, broncho-alveolar lavage samples, endotracheal secretion samples, and sterile blood samples were included for sample collection.

Bacterial isolates and identification

After collection, clinical specimens were delivered to the laboratory, where processing and culture were done according to routine laboratory methods. Blood samples were collected in blood culture bottles, while other samples were streaked on MacConkey agar, sheep chocolate agar, and sheep blood agar medium using a calibrated loop (which can hold approximately 0.005 mL of the samples). Blood and chocolate agar were incubated in a Candle jar at 37°C for 24-48 hours. After incubation, the plates were observed for bacterial growth. The samples that showed significant bacterial growth were processed for the identification of the bacterial species using a VITEK 2 compact, fully automated system.

Determination of the sensitivity of bacterial Isolates

The E-test was used to quantify the minimal inhibitory concentration (MIC) for Isepamicin, Gentamicin, and Amikacin. This test was performed using a plastic strip containing a predefined antibiotic gradient, which was imprinted with the MIC reading scale in μg/mL. This strip was directly transferred to the agar matrix when applied to the inoculated agar plate. After incubation, a symmetrical elliptic inhibition zone was visible along the strip as bacterial growth was prevented. MIC was defined by the area of inhibition where the ellipse intersects the strip.

Antimicrobial susceptibility testing

Strain identification and MIC determination for the bacterial isolates were done using a VITEK 2 compact automated system (BioMerieux) against a set of antibiotics (Isepamicin, Amikacin, and Gentamicin), and the results were interpreted as per the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. The CLSI breakpoints (6a) were used for the interpretation of susceptibility to all antimicrobial agents except for Isepamicin. For Isepamicin, the breakpoints proposed in 2003 by the Comité de l'Antibiogramme de la Société Française de Microbiologie (CA-SFM) were used, and susceptibility was defined by an MIC ≤ 8 mg/L and resistance was defined by an MIC ≥16 mg/L.

The percentages of bacterial isolates were categorized as susceptible, intermediate, and resistant according to EUCAST guidelines (as per Comité de l’Antibiogramme de la Société Française de Microbiologie recommendation 2003).

RESULTS

Characteristics of the bacterial isolates

A total of 8 different bacterial isolates were prospectively collected from 150 clinical samples (male: 100, female: 50) at the microbiological laboratories of three centres during the year 2021. The samples were collected from 50 patients with a mean age range of 63 (18-96) years. The demographic characteristics of patients as per different sites are presented in Table 1.

Table 1: Demographic characteristics of patients

The types of culture specimens from which bacterial strains were most frequently isolated included respiratory (63 [42%]) and urine (44 [29.3%]). The most identified bacterial isolates were K. pneumoniae (40 [26.6%]), followed by P. aeruginosa (38 [25%]), E. coli (30 isolates [20%]), and A. baumannii (33 [22%]), which accounted for nearly 94% (Table 2).

Table 2: Identification of bacterial isolates from the clinical specimens

Antimicrobial sensitivity and resistant strain evaluation

Among the bacterial isolates which were most frequently isolated, E. coli was most sensitive to Isepamicin (93.3%), followed by P. aeruginosa (57.9%) and K. pneumoniae (55.0%) (Table 3). Sample-wise organism sensitivity data showed a similar trend, as E. coli was found to be sensitive to Isepamicin in ≥90% of samples obtained from respiratory, urine, intra-abdominal, blood, and CSF (Table 4).

Table 3: Combined sensitivity of Isepamicin against all clinical isolates

Table 4: Isepamicin susceptibility testing among the bacterial isolates

Comparative evaluation of the susceptibility

In a comparative evaluation, antimicrobial sensitivity was found to be highest for Isepamicin (60/108 [56%]), followed by Gentamicin (23/108 [21%]) and Amikacin (4/108 [4%]) at MIC≤1 mg/L. However, Isepamicin, Gentamicin, and Amikacin showed 69%, 41%, and 52% susceptibility up to MIC ≤8 mg/L, respectively.

Isepamicin was found to be most effective against Gram-negative bacterial isolates from the ICUs of all three centres (Figure 1A, B, and C). Isepamicin was also found to be highly effective in urine samples, followed by respiratory samples, and showed higher susceptibility against E. coli, followed by K. pneumoniae and P. aeruginosa bacterial isolates comparable to Gentamicin and Amikacin (Figure 2A, B, C, and D).

Figure 1: Comparative antimicrobial susceptibility ward wise (A-IP Ward; B-OPD; C-ICU)

Figure 2: Comparative evaluation of the susceptibility of Isepamicin, Gentamicin, and Amikacin in different collected samples (A-Blood; B-Intra abdominal; C-Urine; D-Respiratory)

The most susceptible antibiotic to E. coli is Isepamicin (MIC≤1 mg/L), followed by Gentamicin and Amikacin (Table 5). In the urine sample, only 10% of E. coli was found to be Isepamicin-resistant.

Table 5: Comparative susceptibility evaluation of Isepamicin, Amikacin, and Gentamicin on highly sensitive bacterial isolates

DISCUSSION

The infections caused by gram-negative bacilli are important to treat to avoid complications and reduce morbidity and mortality, as they show high resistance to antibiotics. In this TRIPLE I Study, we evaluated the in-vitro activity of Isepamicin against 8 different bacterial isolates from 150 different clinical samples, prospectively collected from patients with different ailments and from 3 participating centres in India ⁹.

In our study, Isepamicin exhibited very high in-vitro activity against Gram-negative pathogens, including E. coli, K. pneumoniae, and P. aeruginosa, collected from different specimens of unique patients. Also, Isepamicin was the most susceptible in-vitro agent against all isolates among all the antibiotics tested. High susceptibility to Isepamicin was also observed for the isolates collected from patients hospitalized in the ICU ¹⁰.

E. coli was found to be 90-100% sensitive to Isepamicin in all collected specimens. Likewise, 67-88% of P. aeruginosa were sensitive to Isepamicin in intra-abdominal and blood specimens, and 59-63% of K. pneumoniae were sensitive to Isepamicin in urine and intra-abdominal samples. In line with the present study, Maraki et al. also reported superior Isepamicin susceptibility (96.9%) when given with Colistin, with E. coli (99.9%) and K. pneumoniae (95.3%) being the most susceptible isolates ⁷. They further found Carbapenem-nonsusceptible isolates (89.6%) to be susceptible to Isepamicin. In another study, Tsai et al. reported that Isepamicin and other aminoglycosides (Gentamicin, Amikacin, and Tobramycin) were found effective against 95% of bacterial isolates ¹¹. Karakullukçu et al. also reported that Isepamicin was effective against 95.1% of Carbapenem-resistant enterobacterales ¹².

Overall, Isepamicin susceptibility was found to be highest, i.e., 69% at MIC values of ≤1mg/l, amongst E. coli, K. pneumoniae, and P. aeruginosa, comparable to Gentamicin (21%), and Amikacin (4%). Isepamicin was also found to be highly effective in urine samples and showed higher sensitivity against E. coli, indicating its potential clinical use in the treatment of severe UTIs.

Moreover, Isepamicin can also be used as an adjuvant therapy for the treatment of intra-abdominal and blood infections that occur due to Gram-negative bacilli. These findings are further supported by two multicentre studies ¹³, in which E. coli (97%) and Enterobacteriaceae (93.6%) were found susceptible to Isepamicin in clinical samples. However, for P. aeruginosa isolates, Isepamicin has shown low susceptibility (36%), and other aminoglycosides tested in this study (Gentamicin and Amikacin) also have not shown any potential antimicrobial effect against P. aeruginosa.

The high susceptibility and more effective in-vitro potential of Isepamicin against gram- negative bacterial isolates, as compared with other aminoglycosides, could be attributed to the lower impact of 1 6’-N-acetyltransferase enzyme on Isepamicin ¹⁴. This enzyme is the most common aminoglycoside-modifying enzyme present in these pathogens and causes resistance to aminoglycosides. However, K. pneumoniae and P. aeruginosa bacterial isolates were found to be resistant in urine (69% and 70%, respectively) and respiratory (58% and 46%, respectively) clinical samples against Isepamicin. This resistance to Isepamicin could result from the combination of more than one aminoglycoside-modifying enzyme or decreased permeability or efflux mechanisms accompanying such an enzyme ^{15, 16}. However, these mechanisms should be explored in future studies. Despite the beneficial role of Isepamicin in our in vitro study, the clinical effectiveness of Isepamicin in patients is difficult to interpret because it was used in combination with other agents in a few studies, and thus the outcome cannot be attributed to Isepamicin per se.

Isepamicin could be used as an alternative to Amikacin and Gentamicin based on historical data, as the sensitivity pattern has not significantly changed much compared to older data. Also, there is a need to emphasize a local antibiogram with sensitivity to Isepamicin while deciding about empirical therapy. Though aminoglycosides can cause nephrotoxicity, vestibular toxicity, and ototoxicity, Isepamicin is one of the less toxic aminoglycosides. Nevertheless, the current data support the use of Isepamicin over other aminoglycosides in patients with drug resistant Gram-negative bacterial infections.

CONCLUSION

We found that Isepamicin has the potential to treat infections caused by E. coli and could be an effective therapy in the treatment of UTIs. It is further found to be moderately sensitive to Klebsiella and Pseudomonas. Moreover, it could be used as an alternative to Gentamicin and Amikacin and against resistant cases. To validate these findings, future research is warranted to explore Isepamicin’s potential in adequately designed clinical studies.

Conflicts of Interest

There are no conflicts of interest.

Author contributions      

This work was carried out in collaboration among all authors. Dr. Ami Varaiya, Dr. Bansidhar Tarai, and Dr. Bhaskar Narayan Chaudhuri participated in study design, study execution, data collection, data analysis, and manuscript development. Dr. Abdul Ansari and Dr. Niraj Tyagi supervised the study. All the authors read and approved the final version of the manuscript.

Acknowledgement: We thank NeoCrest Life Science Consulting Pvt. Ltd. for providing medical writing support. 

REFERENCES

1. Nagvekar V, Sawant S, Amey S. Prevalence of multidrug-resistant Gram-negative bacteria cases at admission in a multispeciality hospital. J of global antimicro resist. 2020; 22:457-61. https://doi.org/10.1016/j.jgar.2020.02.030 PMid:32165287

2. Flores-Mireles AL, Walker JN, Caparon M. et al. Urinary tract infections: epidemiology, mechanisms of infection and treatment options. N reviews microb. 2015; 13(5):269-84. https://doi.org/10.1038/nrmicro3432 PMid:25853778 PMCid:PMC4457377

3. Venkataraman R, Divatia JV, Ramakrishnan N et al. Multicenter observational study to evaluate epidemiology and resistance patterns of common intensive care unit-infections. Ind J of Crit Care Medi: Peer-reviewed, Official Publication of Indian Soci of Crit Care Medi. 2018; 22(1):20. https://doi.org/10.4103/ijccm.IJCCM_394_17 PMid:29422728 PMCid:PMC5793017

4. Breijyeh Z, Jubeh B, Karaman R. Resistance of gram-negative bacteria to current antibacterial agents and approaches to resolve it. Molecules. 2020; 25(6):1340. https://doi.org/10.3390/molecules25061340 PMid:32187986 PMCid:PMC7144564

5. Kirst, H. A., & Allen, N. E. (2006). Aminoglycosides antibiotics. Comprehensive Medicinal Chemistry II; 629-652, Elsevier. https://doi.org/10.1016/B0-08-045044-X/00281-9

6. Rajni E, Jain A, Garg VK. et al. Providencia Causing Urinary Tract Infections: Are We Reaching a Dead End?. Ind J of Crit Care Medi: Peer-reviewed, Official Publication of Indian Society of Critical Care Medicine. 2022; 26(4):446. https://doi.org/10.5005/jp-journals-10071-24163 PMid:35656046 PMCid:PMC9067475

7. Maraki S, Samonis G, Karageorgopoulos DE. et al. In vitro antimicrobial susceptibility to isepamicin of 6,296 Enterobacteriaceae clinical isolates collected at a tertiary care university hospital in Greece. Anti-agents and chemo. 2012; 56(6):3067-73. https://doi.org/10.1128/AAC.06358-11 PMid:22391548 PMCid:PMC3370768

8. Carbon C. Overview of the efficacy of isepamicin in the adult core clinical trial programme. J of Chemo (Florence, Italy). 1995; 7:79-85.

9. Menon T, Bindu D, Kumar CP. et al. Comparison of double disc and three dimensional methods to screen for ESBL producers in a tertiary care hospital. Ind J of Medi Micro. 2006; 24(2):117-20. https://doi.org/10.4103/0255-0857.25192 PMid:16687862

10. Miller GH, Chiu PJ, Waitz JA. Biological activity of Sch 21420, the 1-NS-α-hydroxy-β- aminopropionyl derivative of gentamicin B. The J of antibio. 1978; 31(7):688-96. https://doi.org/10.7164/antibiotics.31.688 PMid:690004

11. Jones RN, Johnson DM, Barrett MS. et al. Antimicrobial activity of isepamicin (SCH21420, 1-N-HAPA gentamicin B) combinations with cefotaxime, ceftazidime, ceftriaxone, ciprofloxacin, imipenem, mezlocillin and piperacillin tested against gentamicin-resistant and susceptible gram-negative bacilli and enterococci. J of chemo. 1991; 3(5):289-94. https://doi.org/10.1080/1120009X.1991.11739108 PMid:1809808

12. Liu JY, Wang FD, Ho MW. et al. In vitro activity of aminoglycosides against clinical isolates of Acinetobacter baumannii complex and other nonfermentative Gram-negative bacilli causing healthcare-associated bloodstream infections in Taiwan. J of Micro, Immu and Infect. 2016; 49(6):918-23. https://doi.org/10.1016/j.jmii.2015.07.010 PMid:26364729

13. Wang G, Song G, Xu Y. A Rapid Antimicrobial Susceptibility Test for Klebsiella pneumoniae Using a Broth Micro-Dilution Combined with MALDI TOF MS. Infect and dru resist. 2021; 14:1823. https://doi.org/10.2147/IDR.S305280 PMid:34025124 PMCid:PMC8132464

14. Paul D, Anto N, Bhardwaj M. et al. Antimicrobial resistance in patients with suspected urinary tract infections in primary care in Assam, Ind Antimicro Resist. 2021; 3(4):164. https://doi.org/10.1093/jacamr/dlab164 PMid:34917941 PMCid:PMC8669238

15. Falagas ME, Karageorgopoulos DE, Georgantzi GG. et al. Susceptibility of Gram-negative bacteria to isepamicin: a systematic review. Exp rev of anti-infect ther. 2012; 10(2):207-18. https://doi.org/10.1586/eri.11.170 PMid:22339194

16. Yeliz TC, Ali HB, Sarah AF. et al. Investigation of isepamicin in-vitro efficiency in Gram negative bacteria. Ind J of Medi Micro. 2021; 39(1):59-62. https://doi.org/10.1016/j.ijmmb.2020.09.003 PMid:33610258