In-vitro activity of tigecycline and comparator agents against common pathogens : Indian experience

Introduction: Tigecycline Evaluation and Surveillance Trail (TEST) study is an on-going global surveillance. The study was performed to determine the susceptibility of common pathogens to tigecycline and comparator antibiotics by broth microdilution (BMD) at two tertiary care centres in India from 2015 to 2017. Methodology: Total of 989 isolates collected from various clinical specimens between January 2015 and September 2017 from two centres in India were included. BMD was performed to determine the minimum inhibitory concentration (MIC) for tigecycline and comparator antibiotics. Results: Among Gram-negative bacteria, susceptibility to tigecycline was lowest among Klebsiella spp. being 84% while others such as E. coli, Enterobacter spp., Serratia spp. and H. influenzae showed susceptibility of 98%, 95%, 98% and 100% respectively. Overall, 99 isolates among Enterobacteriaceae (E. coli, Klebsiella spp. and Enterobacter spp.) were ESBL producers, susceptible to tigecycline. Among the 101 meropenem resistant Enterobacteriaceae, 85 were susceptible to tigecycline (84%). Among the Gram-positive bacteria, S. aureus and Enterococcus spp. were 99% and 98% susceptible to tigecycline respectively. Among 68 MRSA isolates in the study, 66 (97%) were susceptible to tigecycline. Seven vancomycin resistant E. faecalis were isolated and all were susceptible to tigecycline. Conclusion: Tigecycline has retained activity over both Gram-positive and Gram-negative organisms with MIC values comparable to global reports. About 98% of the MDR Gram-positive and Gram-negative bacteria in the study are susceptible to tigecycline. With increased incidence of extensively drug resistant organisms, tigecycline is a potential reserve drug.


Introduction
Tigecycline is a glycylcycline which is a derivative of minocycline and is structurally better in overcoming the ribosomal protection proteins and efflux pumps which confer resistance to other tetracyclines [1,2].It is often used in the treatment of multidrug resistant organisms as a last resort apart from colistin.It is active against both Gram-positive and Gram-negative bacteria.Studies focusing on the usage and prevalence of tigecycline resistance are crucial in judicious use of the reserved drug in order to prevent resistance.Though tigecycline has been licensed only for skin and soft tissue infection (SSTI) and intra-abdominal infections (IAI) [3], it is used for the treatment of other infections such as bacteraemia secondary to SSTI and IAI.Tigecycline Evaluation and Surveillance Trail (TEST) study was performed to determine the susceptibility of common pathogens to tigecycline and comparator antibiotics.The present study details the observation made at two tertiary care centres in India as a part of TEST study from 2015 to 2017.This study determines the susceptibility to tigecycline by broth-micro dilution and MIC 50 and MIC 90 have been calculated to determine tigecycline susceptibility.

Methodology
Total of 989 isolates collected from various clinical specimens between January 2015 and September 2017 from two centres in India namely Christian Medical College, Vellore, and Breach Candy Hospital Trust, Mumbai, were included in the study.Only the first isolate from each patient was included in the study.Identification was performed by standard biochemical methods [4].Antimicrobial susceptibility was performed by Kirby Bauer disc diffusion method for preliminary screening as per CLSI guidelines [5].The number of isolates for each organism included in the study from the study centres is mentioned in Table 1.Minimum inhibitory concentration (MIC) of antibiotics was determined by broth micro dilution and the results were interpreted according to CLSI guidelines for all antibiotics except tigecycline for which the US Food and Drug Administration prescribed breakpoints were used [6,7].The panel of antibiotics for Gram-negative bacteria includes ampicillin, amoxicillin/clavulanic acid, piperacillin/tazobactam, ceftriaxone, cefepime, ceftazidime, meropenem, levofloxacin, amikacin, minocycline and tigecycline.Gram-positive panel includes ampicillin, penicillin, amoxicillin/clavulanic acid, piperacillin/ tazobactam, ceftriaxone, meropenem, vancomycin, linezolid, minocycline and tigecycline.Etest (Liofilchem, Roseto Degli Abruzzi, Italy) was performed to determine MIC for ampicillin/sulbactam and cefoperazone/ sulbactam for all the study isolates.β-lactamase production in H. influenzae was performed using nitrocefin disc.Currently, there are no breakpoints described for interpretation of cefoperazone/ sulbactam susceptibility and hence the MIC range was determined.E. coli ATCC 25922 and P. aeruginosa ATCC 27853 were used as the controls for susceptibility testing.

Results
Table 2 mentions the MIC 50 and MIC 90 of all the antibiotics tested for E. coli, Klebsiella spp., Enterobacter spp.and P. aeruginosa. Figure 1 and Figure 2   Amp: Ampicillin; Aug: Amoxicillin/clavulanic acid; A/S: Ampicillin/sulbactam; P/T: Piperacillin/tazobactam; CTX: Ceftriaxone; CPE: Cefepime; Czd: Ceftazidime; Mero: Meropenem: Levo: levofloxacin; Amik: Amikacin; Mino: Minocycline; Tig: Tigecycline.(n = 13) and Serratia spp.(n = 1)] isolates, 85 were susceptible to tigecycline (84%).Tigecycline is found to be effective against multi-drug and extensively drug resistant isolates.Figure 3 describes the susceptibility profile of Gram-positive bacteria such as S. aureus, S. pneumoniae, Enterococci and S. agalactiae.MIC 50 and MIC 90 of tigecycline for E. coli, Klebsiella spp.and Enterobacter spp.are within the susceptible range indicating the effectiveness of tigecycline for these commonly encountered organisms.For meropenem, MIC 50 (0.5 µg/mL) is within the susceptible range (≤ 1 µg/mL) while MIC 90 (> 16 µg/mL) falls in the resistance range (> 1 µg/mL).Though there are large number of MDR organisms, tigecycline retains its activity over these bacteria.MIC 50 and MIC 90 for all the antimicrobials included in the study for E. coli, Klebsiella spp., Enterobacter spp., P. aeruginosa and Acinetobacter spp.are mentioned in Table 2.Among Gram-negative bacteria, susceptibility to tigecycline was lowest among Klebsiella spp.being 84% while others such as E. coli, Enterobacter spp., Serratia spp.and H. influenzae showed susceptibility of 98%, 95%, 98% and 100% respectively.Currently, there is no tigecycline susceptibility breakpoint for Acinetobacter spp.P. aeruginosa is intrinsically resistant to tigecycline.MIC 50 and MIC 90 for Acinetobacter spp.for tigecycline were found to be 1 µg/mL and 2 µg/mL respectively.The MIC for Acinetobacter spp.ranged from 0.06 to 2 µg/mL for tigecycline.Meropenem, commonly used in  the Indian setting, shows significantly decreased susceptibility to Klebsiella spp., P. aeruginosa and Acinetobacter spp. with 51%, 58% and 12% respectively.Among the non-fermenters, P. aeruginosa retains 60% susceptibility to most antibiotics while Acinetobacter spp.shows 20% susceptibility to the antimicrobials tested.MIC 50 and MIC 90 for all the antimicrobials for S. aureus in mentioned in Table 3.Among the Grampositive bacteria, S. aureus and Enterococcus spp.were 99% and 60% susceptible respectively to tigecycline.Among 68 MRSA isolates in the study, 66 (97%) were susceptible to tigecycline.MIC 50 and MIC 90 of vancomycin for S. aureus were 0.5 µg/mL and 1 µg/mL respectively.Seven vancomycin resistant E. faecalis were isolated and all were susceptible to tigecycline.A total of 15 vancomycin resistant Enterococcus spp.were seen which were susceptible to linezolid.Among MDR Gram-positive bacteria, tigecycline is a preferable therapeutic agent.However, only one isolate of S. agalactiae was susceptible to tigecycline while none of the S. pneumoniae included in the study was susceptible to tigecycline.Vancomycin resistant Enterococci (VRE) was found to be 11% (n = 15) among the study isolates.For Enterococcus spp., MIC 50 and MIC 90 of vancomycin were 1 µg/mL and 4 µg/mL respectively.
In the present study, tigecycline retains activity against 90% of the Enterobacteriaceae and 100% of H. influenzae, 98% of S. aureus and E. faecalis.The current FDA breakpoints do not describe the criteria for E. faecium.Among the organisms tested, Klebsiella spp.has susceptibility of 84% to tigecycline with good activity compared to carbapenems which is 51% susceptible.The main mechanism of resistance to tigecycline is by mutations in the regulators of efflux pumps such as acrAB and oqxAB, which also contribute to resistance to other antimicrobials in Klebsiella spp.[11].Tigecycline has retained good activity against H. influenzae over the years with 98 to 100% susceptibility as reported in various studies [2,12].
Similarly, TEST studies conducted in various countries, determined MIC 90 for tigecycline as 2 µg/mL which was also observed in the present study [12][13][14].For Acinetobacter baumannii, none of the organisation (EUCAST, USCAST, FDA) has suggested tigecycline breakpoints.In this scenario, most of the clinical laboratories are extrapolating tigecycline FDA breakpoints of Enterobacteriaceae for A. baumannii as well (MIC, ≥ 8 µg/mL as non-susceptible).
Among the Gram-positive bacteria, S. aureus and Enterococci show 98% susceptibility tigecycline unlike S. pneumoniae and S. agalactiae which are resistant to tigecycline.In contrast to the present study, Garrison et al., reported 99.7% susceptibility to tigecycline among S. agalactiae [12].This vast difference in susceptibility rates can be attributed to the time and geographical variation.Earlier study [12] included isolates from 2004 to 2010 collected globally while the present study isolates are from 2015 to 2017 from Indian setting.Also, the usage of antibiotics differs significantly among various countries.Susceptibility of S. pneumoniae was not interpreted in the study by  For S. aureus, MIC 50 and MIC 90 of vancomycin were 0.5 µg/mL and 1 µg/mL respectively in the present study while earlier studies report MIC 50 and MIC 90 to be 1 µg/mL.Globally, there has not been an increase in MIC of vancomycin for S. aureus [12,13].88% of the Enterococci are susceptible to vancomycin while other Gram-positive bacteria in the present study retain 100% susceptibility.For Enterococcus spp., MIC 50 and MIC 90 of vancomycin were 1 µg/mL and 4 µg/mL respectively in the present study while earlier reports show lower values such as 1-2 µg/mL for MIC 50 and MIC 90 [13].The susceptibility to tigecycline among MRSA and VRE was found to be 97% (66/68) and 100% (7/7) respectively.Among MDR Gram-positive bacteria, tigecycline is a potential therapeutic agent.
A recent publication on results of TEST 2016 from regions including North America, Europe, Latin America and Asia Pacific show that tigecycline has retained its activity against both Gram-positive and Gram-negative bacteria worldwide [15].MIC 50 and MIC 90 of tigecycline for carbapenem resistant Enterobacteriaceae is reported as 0.5 µg/mL and 2 µg/mL respectively which is in the susceptible range.Among Gram-positive bacteria such as S. aureus, MIC 50 and MIC 90 of tigecycline was as low as 0.06 µg/mL and 0.12 µg/mL respectively.Similar to the study by Pfaller et al., our present study also finds MDR organisms such as carbapenem resistant Enterobacteriaceae, MRSA and VRE to retain > 95% susceptibility to tigecycline.
Cefoperazone/sulbactam has good activity against some ESBL producers.Its activity varies among Enterobacteriaceae species which include some AmpC β-lactamase producers and is also active against some important non-fermentative Gram-negative bacteria species that are resistant to cefoperazone [16].But currently to determine its efficacy against MDR isolates, no breakpoints are defined for this combination.Determining the MIC range and MIC 50 can aid in establishing breakpoints.This study showed lowest MIC 50 and MIC 90 among H. influenzae and the highest among Acinetobacter spp.Among Enterobacteriaceae, Enterobacter spp.had lowest MIC 50 of 0.75 µg/mL.Gram-positive bacteria had lower MIC 50 and MIC 90 values when compared to Gram-negative bacteria.Jean et al., [16] reported lower MIC 50 and MIC 90 values of ≤8 µg/mL as determined by Vitek2 for Enterobacteriaceae including E. coli, K. pneumoniae, E. cloacae, C. freundii, Salmonella spp.and S. marcescens.However, for Acinetobacter spp.MIC 50 and MIC 90 were ≤8 µg/mL and 32 µg/mL respectively in contrast to the present study where MIC 50 and MIC 90 was found to be > 256 µg/mL (Table 2).For P. aeruginosa Jean et al., report MIC 50 and MIC 90 ≤ 8 µg/mL and > 64 µg/mL respectively while in the present study MIC 50 and MIC 90 were observed to be > 256 µg/mL.
Surveillance of susceptibility to commonly used antimicrobials against various pathogens helps in understanding the antibiogram and optimises the standard therapeutic practices.This in turn helps the use of reserve drugs such as tigecycline, colistin, linezolid and vancomycin with discernment.In India, determining the MIC of antimicrobials across the country in various centres along with monitoring the usage of tigecycline will enable in establishing the susceptibility trend over the years.

Conclusion
Tigecycline has retained activity over both Grampositive and Gram-negative organisms with MIC values comparable to global reports.About 98% of the MDR Gram-positive and Gram-negative bacteria in the study are susceptible to tigecycline.With increased incidence of extensively drug resistant organisms, tigecycline is a potential reserve drug.However, there is need to establish uniformity of testing methodology for tigecycline and also define breakpoints for Acinetobacter spp.for which tigecycline is a potential therapeutic option.

Figure 2 .
Figure 2. Susceptibility of other Gram-negative bacteria to antimicrobials.

Table 1 .
Number of isolates for each organism included in the study.
Table3.MIC50 and MIC90 of tested antibiotics for S. aureus.
[13]ison and colleagues due to lack of breakpoints.Hoban et al., reported 99.7% susceptibility of E. faecalis to tigecycline similar to the present study wherein 53 out of 54 E. faecalis were susceptible to tigecycline[13].