Determination of MIC Distribution of Arbekacin, Cefminox, Fosfomycin, Biapenem and Other Antibiotics against Gram-Negative Clinical Isolates in South India: A Prospective Study

Objectives To determine the in vitro activity of antibiotics, including arbekacin, cefminox, fosfomycin and biapenem which are all still unavailable in India, against Gram-negative clinical isolates. Methods We prospectively collected and tested all consecutive isolates of Escherichia coli, Klebsiella spp., Pseudomonas aeruginosa and Acinetobacter spp. from blood, urine and sputum samples between March and November 2012. The minimum inhibition concentration (MIC) of 16 antibiotics was determined by the broth micro-dilution method. Results Overall 925 isolates were included; 211 E. coli, 207 Klebsiella spp., 153 P. aeruginosa, and 354 Acinetobacter spp. The MIC50 and MIC90 were high for cefminox, biapenem and arbekacin for all pathogens but interpretative criteria were not available. The MIC50 was categorized as susceptible for a couple of antibiotics, including piperacillin/tazobactam, carbapenems and amikacin, for E. coli, Klebsiella spp. and P. aeruginosa. However, for Acinetobacter spp., the MIC50 was categorized as susceptible only for colistin. On the other hand, fosfomycin was the only antibiotic that inhibited 90% of E. coli and Klebsiella spp. isolates, while 90% of P. aeruginosa isolates were inhibited only by colistin. Finally, 90% of Acinetobacter spp. isolates were not inhibited by any antibiotic tested. Conclusion Fosfomycin and colistin might be promising antibiotics for the treatment of infections due to E. coli or Klebsiella spp. and P. aeruginosa, respectively, in India; however, clinical trials should first corroborate the in vitro findings. The activity of tigecycline should be evaluated, as this is commonly used as last-resort option for the treatment of multidrug-resistant Acinetobacter infections.


Introduction
Antimicrobial resistance has risen alarmingly worldwide during the last decade. The widespread of Gram-negative organisms producing extended-spectrum beta-lactamases (ESBLs) conferring resistance to penicillins, cephalosporins, and fluoroquinolones, or carbapenemases conferring resistance even to carbapenems limits significantly the treatment armamentarium against infections. India is one of the countries facing the greatest burden of antimicrobial resistance around the world. The high availability of antibiotics over the counter in the country is major contributor in the high antimicrobial resistance observed. New-Delhi Metallo-blactamase (NDM)-producing Enterobacteriaceae, which were first detected here, [1] are now endemic in India. [2] Carbapenemase-producing Enterobacteriaceae cause difficult-to-treat infections usually characterized by high mortality. [3,4] Furthermore, high prevalence of infections caused by carbapenemase-producing Acinetobacter baumannii [5,6,7] and Pseudomonas aeruginosa, [6,8] as well as of infections caused by ESBL-producing Enterobacteriaceae has been observed in India [9,10,11].
The introduction into clinical practice of antibiotics that are still unavailable in India could be a solution to the problem of the antimicrobial resistance. Arbekacin, the cephamycin cefminox, and the group 2 carbapenem biapenem are effective antibiotics, mainly used in Japan and South Korea. Arbekacin is primarily used for the treatment of infections caused by methicillin-resistant Staphylococcus aureus [12] and few in vitro data suggest that this antibiotic might be also considered as an adjunct treatment for infections due to multidrug-resistant (MDR) Gram-negative pathogens., [13,14] Cefminox is active against anaerobic bacteria [15] as well as ESBL-producing Enterobacteriaceae. [16] As regards biapenem, this is active against Gram-negative and Grampositive anaerobic bacteria, [17], [18] but also against aerobic bacteria, both alone [19] and in combination with other agents against MDR pathogens. [20] Finally, fosfomycin, which is also not marketed in India, is an ''old'' antibiotic, discovered in the late 60's which has been re-evaluated the last years and re-introduced successfully into clinical practice in many countries of the world. [21,22,23] Fosfomycin has broad antimicrobial spectrum against MDR pathogens, both Gram-negative [24] and Gram-positive ones [25].
In this context, we aimed to determine the minimum inhibition concentration (MIC) 50 (the antibiotic concentration required to inhibit the growth of 50% of the pathogens) and MIC 90 (the antibiotic concentration required to inhibit the growth of 90% of the pathogens) of antibiotics, including arbekacin, cefminox, biapenem and fosfomycin for common Gram-negative clinical isolates collected from patients with hospital-or communityacquired infections in a tertiary care hospital, in South India.

Study design and setting
This prospective study was performed at the Christian Medical College, Vellore, South India, at the Department of Clinical Microbiology, between March and November 2012. All consecutive isolates of E. coli, Klebsiella spp., P. aeruginosa, and Acinetobacter spp. isolated from the urine, blood, and sputum were included in the study. Only one isolate per patient was included in the study. Data on the demographic details of patients whose isolates were studied was not included.

Ethics considerations
The study was approved by the Institutional review board and ethics committee of Christian Medical College, Vellore, South India. A written or oral informed consent was not obtained by the patients whose isolates were included in this study due to the noninterventional study design and this consent procedure was approved by the ethics committee of our institution.

Microbiological methods
Isolation and identification of the isolates from the specimens was performed using a semi-quantitative culture method and biochemically characterized using the mannitol motility medium, triple sugar iron agar medium, peptone water and Simmons citrate medium. [26,27] Identification up to the genus level for Klebsiella and Acinetobacter isolates was performed. An oxidase test was also performed for Acinetobacter spp. and P. aeruginosa and an indole test was performed in order to differentiate between Klebsiella spp. and E. coli. Standard American type culture collection (ATCC) control strains within acceptable limits were used as quality control strains for the drugs tested. E. coli ATCC 25922, Staphylococcus aureus ATCC 29213, P. aeruginosa ATCC 27853 and Enterococcus faecalis ATCC29212 were used for susceptibility testing to ampicillin/sulbactam, piperacillin/tazobactam, cefminox, cefmetazole, ceftazidime, ceftriaxone, aztreonam, fosfomycin, imipenem, meropenem, doripenem, biapenem, amikacin, arbekacin, gentamicin and colistin.
The MIC was determined by the broth micro-dilution method (Meiji Co., Japan) using cation-adjusted Muller-Hinton broth. The inoculum was prepared by the growth method with which the test bacteria were grown on non-selective culture media and incubated overnight. On the following day, 4-5 colonies were taken from that plate and suspended into 2 ml of nutrient broth and incubated for 2 hours. The bacterial inoculum was adjusted to 1 McFarland Standard by sterilized physiological saline. Then, 25 mL of the inoculum was added into 12 mL of cation-adjusted Mueller-Hinton broth (CAMHB) and 50 mL of the mixture was inoculated into each plate. The final inoculum size was approximately 2.5610 4 CFU of bacteria in each plate. Finally, the inoculated plates were incubated at 3562uC in ambient air for 20-24 hours for Acinetobacter spp. or 16-20 hours for the other bacteria.
[28] The MICs for fosfomycin were reported as susceptible (#64 mg/ml), intermediate (128 mg/ml), or resistant ($256 mg/ml). Interpretative criteria of the MIC are not available by CLSI for cefminox, biapenem and arbekacin for any of the four pathogens tested and for fosfomycin for P. aeruginosa and Acinetobacter spp.

Results
A total of 925 isolates were collected and tested during the study period; 211 isolates of E. coli, 207 of Klebsiella spp., 153 of P. aeruginosa, and 354 of Acinetobacter spp. With regard to the source of isolation, 363 originated from the sputum, 362 isolates from the blood, and 200 isolates from the urine. 74% and 75% of the isolates identified in the blood and urine, respectively, were Enterobacteriaceae, while 87.9% and 12.1% of the isolates identified in the sputum were Acinetobacter and P. aeruginosa, respectively. The pathogens by source of isolation are presented in Table 1. The isolates originated from medical, surgical, and critical care departments of the hospital.

Acinetobacter spp
The MIC range, MIC 50 , and MIC 90 of all antibiotics tested are presented in Table 5. The MIC 50 was low (0.5 mg/ml) and within the susceptible range only for colistin, while the MIC 90 value was not low for any antibiotic.
In Table 6 the resistance profile of the included isolates to cefminox, biapenem, and arbekacin is presented in detail.

Discussion
The main finding of the study is that fosfomycin was the only antibiotic that inhibited 90% of E. coli and Klebsiella spp. isolates, while colistin was the only antibiotic that inhibited 90% of P. aeruginosa isolates. In addition, 90% of Acinetobacter spp. isolates were not inhibited by any antibiotic tested.
Among the four antibiotics that were tested and are still not marketed in India, only fosfomycin seems to be a promising treatment option. However, the development of resistance during treatment with fosfomycin is an issue that has not been clarified yet and thus, fosfomycin should not be administered as monotherapy. [29] Interpretation of the antimicrobial susceptibility testing results could not be performed for arbekacin, cefminox, and biapenem. However, the MIC 90 of these antibiotics was high for all four pathogens. Only biapenem was active against 50% of E. coli and Klebsiella spp. isolates (MIC 50 #0.25 mg/ml for both pathogens) according to a previous study that attempted to suggest rational breakpoints for biapenem. [20] On the contrary, the MIC 50 of arbekacin was very high for Acinetobacter spp. (.128 mg/ml) according to potential breakpoints suggested by one study (, 2 mg/ml) [30].
It is noteworthy that piperacillin/tazobactam, cefmetazole, group 2 carbapenems, fosfomycin and amikacin were active against 50% of E. coli and Klebsiella spp. isolates but only fosfomycin retained this activity against 90% of these pathogens. Likewise, piperacillin/tazobactam, ceftazidime, aztreonam, dor- ipenem, amikacin and gentamicin were active against 50% of P. aeruginosa isolates but colistin was the only antibiotic active against 90% of the isolates. The finding of the high in vitro activity of fosfomycin against E. coli has also been illustrated in one of our previous studies which evaluated the activity of fosfomycin against common uropathogens in India. [31] In general, the published literature suggests that fosfomycin might be an effective antibiotic against infections caused by MDR, including ESBL-producing, Enterobacteriaceae. [24,32] Also, previous studies have shown high in vitro activity of colistin against MDR P. aeruginosa isolates, [33,34] while treatment with colistin resulted in sufficient clinical effectiveness when administered to patients with severe infections due to MDR P. aeruginosa [35,36,37].  Regarding the antimicrobial resistance profile of the isolates included in this study, it is noteworthy that the MIC 50 was high for extended-spectrum cephalosporins and aztreonam for E. coli and Klebsiella spp. isolates implying that these isolates may be possibly producers of ESBLs. Likewise, the MIC 50 for imipenem and meropenem was high for P. aeruginosa and Acinetobacter spp. isolates and thus, these isolates may produce carbapenemases. It arises that high percentages of multidrug-or extensively drugresistant Gram-negative pathogens are prevalent in this area of South India urging clinicians to consider alternative antibiotic options for the treatment of these infections.
Other interesting findings of this study are the high incidence of Acinetobacter spp. compared to other Gram-negative bacteria as well as the high antimicrobial resistance recorded among Acinetobacter spp. isolates. It is actually discouraging that only colistin was active against 50% of the isolates and 90% of the isolates were not inhibited by any antibiotic tested. Multidrug resistance of Acinetobacter spp. in India is a great concern addressed by previous studies, as well. [38,39,40] Tigecycline and  colistin are treatment of choice against severe nosocomial infections due to MDR Acinetobacter spp. but emergence of resistance for both antibiotics has been reported in India leading to treatment deadlock. [41] In our study, antimicrobial susceptibility testing for tigecycline was not performed due to economic reasons. The findings of the present study should be interpreted taking into consideration the limitation that the in vitro activity of tigecycline, which is one the most effective antibiotics used for the treatment of infections caused by Acinetobacter spp., was not tested. In addition, species identification of the Klebsiella and Acinetobacter isolates was not performed and therefore, the incidence and susceptibility of the individual species to the antibiotics tested could not be determined.
In conclusion, fosfomycin and colistin might be effective treatment options against infections caused by E. coli or Klebsiella spp. and P. aeruginosa, respectively, in India. However, clinical trials are needed to confirm the in vitro findings, especially before fosfomycin is introduced into clinical practice. The high antimicrobial resistance observed among Acinetobacter spp. isolates is a great concern which necessitates further investigation through studies evaluating the in vitro activity of tigecycline and antibiotic combinations. With regard to arbekacin, cefminox and biapenem, further microbiological studies are warranted to evaluate the activity of these antibiotics against clinical isolates in India.