The rising prevalence of carbapenem-resistant Gram-negative (CR-GNB) infections is a global threat, which leads to increasing morbidity and mortality.^1,2 There are limited numbers of antibiotics to treat extensively drug resistant (XDR) infections. Colistin in combination with various other drugs like meropenem, tigecycline and fosfomycin has been studied for the treatment of infections with these extensively resistant organisms. Combination of meropenem and colistin has been found to be effective in treating infections caused by carbapenem-resistant Acinetobacter baumannii especially if carbapenem minimum inhibitory concentration (MIC) is <32 mg/L.³ However, there is a scarcity of newer and effective antibiotics for these extensively resistant Gram-negative organisms. Minocycline, one of the older antibiotics that still retain activity against many of the XDR Gram-negatives, is being reintroduced into clinical practice.

Minocycline is a second-generation tetracycline which was introduced in 1960. It is available both in oral and intravenous forms and it is FDA-approved for treatment of infections caused by Acinetobacter baumannii. Compared to tigecycline, minocycline has better pharmacokinetic/pharmacodynamic (PK/PD) properties with longer half-life, better oral absorption, superior tissue penetration and ease of switching to oral therapy.⁴ Minocycline susceptibility data among GNB isolates from India is scarce, however a recently published study showed that minocycline susceptibility was 42% to 55% among CR-GNB isolates.⁵ We hence sought to compare minocycline and tigecycline susceptibilities of carbapenem-resistant isolates of Acinetobacter species and Enterobacteriaceae.

This was a retrospective analysis of in vitro susceptibilities to minocycline and tigecycline in Acinetobacter and Enterobacteriaceae isolates from various sites including blood, urine and tissue between January 2014 and April 2015 from a tertiary care oncology, bone marrow transplant and neurosurgical center in South India. Preexistent laboratory results were reviewed and analyzed for the purpose of the study.

Identification of isolates and antibiotic susceptibility testing was done using Vitek 2 compact analyzer (BioMérieux, Marcy l'Étoile, France), using Clinical and Laboratory Standards Institute (CLSI) 2014 breakpoints.⁶ All Acinetobacter, E. coli and Klebsiella isolates that were identified during this study period were recruited for the study. Carbapenem resistance was defined as ertapenem MIC ≥ 2 mg/L for E. coli and Klebsiella as per CLSI and meropenem MIC of ≥ 8 mg/L for Acinetobacter.Because there are no CLSI EnterobacteriaceaeMIC breakpoints available for tigecycline, the FDA breakpoints for susceptible (MIC ≤ 2 mg/L), intermediate (2-4 mg/L), and resistant (MIC ≥ 8 mg/L) were used. No MIC susceptibility breakpoint for tigecycline against Acinetobacter is available, hence the FDA breakpoint for Enterobacteriaceae was used (<2 mg/L). CLSI minocycline breakpoints for Acinetobacter and Enterobacteriaceae (≤4 µg/mL for susceptible, 8 µg/mL intermediate, and ≥16 µg/mL for resistance) were used.

Approval for the study was taken from the institute’s ethical committee.

A total of 1101 Gram-negative isolates were analyzed (Acinetobacter – 161, E. coli – 825, and Klebsiella – 115). Out of these, carbapenem-resistant isolates were Acinetobacter– 122 (75.14%), E. coli – 107 (12.96%) and Klebsiella – 50 (43.40%).

Susceptibilities to tigecycline

Overall, 118 (73.29%) Acinetobacter isolates were susceptible to tigecycline; among carbapenem-susceptible ones all were susceptible to tigecycline, whereas only 79 (64.75%) isolates retained susceptibility to tigecycline among carbapenem-resistant ones (p<0.001). For E. coli, 823 (99.75%) isolates were susceptible to tigecycline, all retained susceptibility to tigecycline among carbapenem-susceptible ones but among carbapenem-resistant ones 105 (98.1%) isolates were susceptible to tigecycline (p<0.001). For Klebsiella, 38 (66.08%) isolates were susceptible to tigecycline, 59 (90.7%) retained susceptibility to tigecycline among carbapenem-susceptible ones while among carbapenem-resistant ones 17 (34.0%) isolates were susceptible to tigecycline (OR=19.08, p<0.001) – Table 1.

 

 

Susceptibilities to minocycline

Overall 120 (74.53%) Acinetobacter isolates were susceptible to minocycline; among carbapenem-susceptible ones all were susceptible to minocycline, whereas only 81 (66.39%) isolates retained susceptibility to minocycline among carbapenem-resistant ones (p<0.001). For E. coli, 457 (55.39%) isolates were susceptible to minocycline, 429 (59.7%) retained susceptibility to minocycline among carbapenem-susceptible ones but among carbapenem-resistant ones 28 (26.16%) isolates were susceptible to minocycline (OR=4.8, p<0.001). For Klebsiella, 38 (33.04%) isolates were susceptible to minocycline, 34 (52.3%)

retained susceptibility to minocycline among carbapenem-susceptible ones while among carbapenem-resistant ones only 4 (8%) isolates were susceptible to minocycline (OR=12.6, p<0.001).

Therapeutic options for infections due to carbapenem-resistant Gram-negative organisms are limited to a few drugs like colistin, fosfomycin and tigecycline. Combination therapy does appear as an attractive option. Meropenem-colistin combination has been found to have synergistic effects especially for carbapenem resistant Acinetobacter.³ However, when treatment of Enterobacteriaceae is considered, the combination of another agent with colistin was found to be superior in patients with high predictive mortality score but not in patients with low predictive mortality score.⁷

Tetracyclines, introduced in 1940, became the first broad spectrum antibiotics to be made available. Among these, tetracycline is considered short-acting, whereas doxycycline and minocycline are long-acting. While most other antibiotics have been rendered ineffective or have turned into narrow spectrum agents due to multiple modes of resistance, long lasting tetracyclines still remain active against a variety of microorganisms, especially the ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species), including multidrug-resistant strains.^8,9 Tetracyclines enter the bacterial cell through energy-dependent outer membrane porin channels and bind to the 30S unit of the ribosome, thus blocking the entry of aminoacyl transfer-RNA and inhibiting protein synthesis.¹⁰ Resistance to tetracycline occurs either through efflux proteins or through ribosomal protection.⁸ However, second generation tetracyclines (minocycline and tetracycline) are able to evade these protective mechanisms because they require different efflux proteins.¹¹

Although tigecycline displays good in vitro activity against CR-GNBs, its use in invasive infections is limited by its pharmacokinetics, as its high lipophilicity may lead to poor serum concentrations. A double blinded randomized control trial showed that tigecycline failed to demonstrate non-inferiority to imipenem in the treatment of hospital-acquired pneumonia, owing to poor drug levels in epithelial fluid.^12,13 In contrast to tigecycline, minocycline achieves higher concentration in blood and urine.¹⁴Besides, the availability of an oral formulation for minocycline makes it an attractive choice for de-escalation of invasive infections by CR-GNBs.

Various studies have demonstrated in vitro activity of minocycline against carbapenem-resistant strains of A. baumannii (CRAB) ranging from 70% to 89%.^15-17 A recently published study from India also showed that minocycline is active against 42% of CRAB isolates.⁵ In our study, minocycline was active against 66.39% isolates of CRAB. In vitro activity of minocycline was poor against carbapenem-resistant strains of K. pneumoniae, ranging from 12% to 45% in studies done worldwide.^15-18 In the present study, the susceptibility to minocycline seen in CR E. coli and CR K. pneumoniae was 26.16% and 8%, respectively. However, another Indian study by Veeraraghavan B et al. reported higher susceptibility among CR E. coli (52%)and Klebsiella (55%),which was higher than that identified in Acinetobacter isolates (42%).⁵

Various clinical studies demonstrated clinical usefulness of minocycline. Wood et al. and Griffith et al. demonstrated clinical cure rates of 88% and 100% respectively using minocycline as monotherapy against wound infections and ventilator‐associated pneumonia due toA. baumannii.^19,20Chan et al. reported a clinical cure rate of 81% for pneumonia due to A. baumannii using minocycline in combination with other agents.²¹Pogue et al. reported 69% clinical cure for bloodstream infection and pneumonia due to A. baumannii and CR K. pneumoniae.²² None of these studies reported untoward side effects with the use of minocycline. Many in vitro studies have shown the synergistic efficacy of combining minocycline with polymyxin.^23,24 The mechanism proposed for this synergy is that polymyxin increases the intracellular concentration of minocycline, potentially through efflux pump disruption.²³ The clinical effectiveness of the combination of minocycline and colistin was found to be comparable with that of meropenem and colistin against CRAB isolates.²⁴

There are several limitations to our study. First, as it is an in vitro study we did not analyze the clinical efficacy of minocycline or tigecycline at our center. Second, we could not perform in vitro synergy tests for colistin/polymyxin and minocycline along with verification of Vitek MIC values by broth microdilution, due to the lack of infrastructure. Finally, as proved by Marchaimet al.,Vitek-2 may not be an accurate method to determine the MICs ofAcinetobacter to tigecycline and correlation with the broth microdilution method is needed,²⁵ which was not done in our study.

Acinetobacter isolates, including carbapenem-resistant ones showed comparable susceptibility to both minocycline and tigecycline. However the susceptibility of Enterobacteriaceae isolates is higher for tigecycline than for minocycline. Hence, minocycline may be an attractive alternative option for the management of carbapenem-resistant Acinetobacterinfections.

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