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ORIGINAL ARTICLE Table of Contents  
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Evaluation of in vitro activity of tigecycline against multidrug-resistant clinical isolates

 Department of Microbiology, Father Muller Medical College, Mangalore, Karnataka, India

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Date of Submission25-Apr-2022
Date of Decision11-May-2022
Date of Acceptance16-May-2022
Date of Web Publication30-Sep-2022


Introduction: Multiple drug resistance (MDR) of the bacteria is an issue across any health-care setting worldwide. This leads to prolonged hospital stay and high cost of treatment. One must be cautious to use reserve drugs such as imipenem, meropenem, colistin, polymyxin, and tigecycline. Tigecycline is a mainstay to treat emerging single or MDR pathogens. The study was conducted to assess the in vitro sensitivity of tigecycline to MDR isolates and to compare the sensitivity of Kirby–Bauer (KB) disc-diffusion method with the Epsilometer (E-test) against various MDR clinical isolates. Materials and Methods: This prospective study was done from January 2019 to 2020 on MDR isolates from clinical specimens. The isolates were tested for their antibiotic sensitivity to tigecycline by KB and E-test method to know the minimum inhibitory concentration (MIC) of tigecycline. Statistical data were analyzed by the percentage method. Results: Tigecycline showed a higher in vitro sensitivity against multidrug-resistant isolates in our study. Among the 89 MDR isolates obtained, 38 (42.7) were found to be sensitive to tigecycline by the KB diffusion and 67 (75.3) were sensitive by the E-test method. Conclusions: Tigecycline is a reserve antibiotic against MDR organisms, especially useful to treat coinfections of Gram-positive and Gram-negative superbugs. The E-test was found to be far superior to the KB method to detect in vitro activity and the results of this test will guide the clinician to judiciously use this antibiotic in their day-to-day practice.

Keywords: Epsilometer test, Kirby–Bauer, multidrug resistance, tigecycline

How to cite this URL:
Wilson LA, Kuruvilla TS. Evaluation of in vitro activity of tigecycline against multidrug-resistant clinical isolates. APIK J Int Med [Epub ahead of print] [cited 2022 Dec 4]. Available from: https://www.ajim.in/preprintarticle.asp?id=357508

  Introduction Top

Antibiotics over the past 80 years have only had a comparatively narrow spectrum of activity.[1] The antibiotic sensitivity testing is a critical component of the microbiology laboratory.[2] The ongoing global problem has certainly been multidrug resistance among bacterial pathogens making it difficult to treat these bacterial diseases.[3] Tigecycline, is derived from minocycline and acts by time-dependent killing and is a suitable stand by reserve drug for multiple drug resistance (MDR) isolates.[4],[5] MDR Gram-negative pathogens such as Acinetobacter baumannii, Pseudomonas aeruginosa, and certain Gram-negative isolates in the Enterobacteriaceae family are often seen in patients with prolonged hospital stay leading to higher health-care expenses and higher mortality rates.[6]

Tigecycline is a glycylcycline antibiotic with a specific in vitro sensitivity against both Gram-positive and Gram-negative bacteria.[7] A global trial called Tigecycline Evaluation and Surveillance Trial monitors the in vitro sensitivity of tigecycline alongside comparable antibiotics against Gram-positive and Gram-negative isolates.[8]

Tigecycline has a broader spectrum of activity particularly against carbapenem-resistant Gram-negative bacteria and MDR pathogens.[9] Many studies have shown that tigecycline sensitivity may wane due to an increased expression of the mechanism of efflux pumps through resistance-nodulation-cell division type in Enterobacteriaceae.[8]

Tigecycline can overcome tetracycline resistance by a huge steric substituent at the ninth position in the chemical structure.[10] Tigecycline not only permits tet gene-encoded mechanisms of resistance but can also preserve its activity against Gram-negative organisms that carry various extended-spectrum beta-lactamases (ESBL's).[11] Thus, its efficacy in abdominal and diseases of the skin and soft tissue may include Enterobacteriaceae strains that are sensitive or multidrug-resistant strains and also cover certain anaerobes.[12] Resistance mediated by the ribosome protection and tetracycline efflux of the active drug is overcome by tigecycline as it has a steric spatial configuration.[13]

Disc diffusion tests are the most widely used method to detect tigecycline sensitivity. Just as with MIC values from macro broth and micro broth dilutions, zone sizes also have break points as defined by Clinical and Laboratory Standards Institute (CLSI) that can identify if the organism is sensitive, intermediate sensitive, or resistant to tigecycline.[14] A recent modification and a more significant method than KB is the use of a strip with graded antibiotic for quantitative minimum inhibitory concentration (MIC) test method also called the Epsilometer or E-test. The elliptical edge where the antibiotic concentration meets with the strip is taken as the MIC value.[15] The break point MIC for tigecycline is read as sensitive, intermediate sensitive, or resistant at ≤2, 4, and ≥8 mg/L, respectively.[16] The reference range of tigecycline (15 μg) against Enterobacteriaceae is ≥18mm as sensitive and ≤15mm is taken as resistant, whether or not ESBL's are produced.[17]

It is observed that the MIC of the antibiotics such as doxycycline and minocycline are higher than tigecycline. Because they overexpress genes such as Mex AB-OprM and MexCD-OprJ. Acinetobacter species and Stenotrophomonas maltophilia shows resistance against tigecycline doses.[18] The objective of our study was to analyze the in vitro action of tigecycline to isolates that show multidrug resistance and compare the sensitivity using conventional Kirby–Bauer (KB) and the Epsilometer method against various multidrug-resistant isolates.

  Materials and Methods Top

This prospective, analytical, observational study was done at the Department of Microbiology of a tertiary care institute, for a period of 1 year from January 2019 to January 2020. Ethical clearance was obtained from the Institutional Ethics Committee. All multidrug-resistant isolates that were resistant to one or more antibiotics were included in the study and tested. All fully sensitive clinical isolates were not a part of the study. With P = 35.9% (reference value), confidence level 95%, and absolute error 10%, the estimated sample size was calculated as 89.

A 0.5 McFarland standard suspension of the resistant test strains were lawn cultured onto Mueller–Hinton agar using a sterile cotton swab. After the plate dried, a tigecycline disk (15 μg) was placed in the center of the plate using a sterile forceps. After incubating the plate at 37°C overnight, the zone of inhibition around the disk was measured.

Then, the Epsilometer containing a predefined gradient of antibiotic concentrations was used as a quantitative method of detecting MIC using the principles of both dilution and diffusion of antibiotic into the medium. The MDR isolates were lawn cultured onto Mueller–Hinton agar and the tigecycline E strip was placed in the center of the plate. The plate was then incubated at 37°C overnight. An elliptical zone of inhibition was produced surrounding the strip and the MIC determined. The statistical analysis was done by frequency and percentage.

  Results Top

The number and type of organisms isolated showing multidrug resistance are shown in [Table 1] and [Figure 1]. The patients selected were from 1 to 85 years of age, and a greater percentage of them were between 51 and 70 years. In the KB disc diffusion method, 15 (16.9%) isolates were resistant, 36 (40.4%) isolates were intermediate sensitive, and 38 (42.7%) were found to be sensitive. In MIC E-test method, 16 (18%) isolates were resistant, 6 (6.7%) isolates were intermediate sensitive, and 67 (75.3%) isolates were sensitive. The organisms isolated and their tigecycline MIC ranges are depicted in [Table 2] and [Figure 2].
Figure 1: Graphic representation of pathogens showing multidrug resistance

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Figure 2: Tigecycline minimum inhibitory concentration range for the isolated pathogens

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Table 1: Percentage of pathogens showing multidrug resistance

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Table 2: Organisms isolated and their tigecycline minimum inhibitory concentrations range

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Among these, E-test MIC-resistant isolates were 9 (25%), intermediate sensitive were 4 (11.1%), and sensitive isolates were 23 (63.9%). In the KB method, the total number of resistant isolates were 15. Among these, E-test MIC of the resistant isolates was 7 (46.7%), intermediate isolates was 1 (6.7%), and sensitive isolates were 7 (46.7%). In the KB method, the total number of sensitive isolates was 38. Among these the E-test MIC showed no resistant isolates, although intermediate sensitive isolate was 1 (2.6%), and sensitive isolates were 37 (97.4%).

  Discussion Top

Any irrational use of the World Health Organization (WHO) classified reserve antibiotics including tigecyclines are the culprits of the menace of drug resistance worldwide. A more accurate in vitro testing gives us a greater picture of true usefulness of any particular third-line reserve antibiotic as emphasized by Yilmaz et al. in her study using MIC values for the treatment.[19] The same principle is applicable for vancomycin-resistant Enterococci and methicillin-resistant Staphylococcus aureus according to Flávia Rossi and Andreazzi.[20]

Majority of the isolates belonged to the patients in the age groups of 51–70 years (47.2%), followed by 71–90 years (23.6%). This data was similar to a study by Mamta Kumari and Soni,[21] who showed that MDR isolates were predominantly seen in patients aged 41–60 years. Isolates from female patients were more common when compared to males with a female-to-male ratio of 2:1 in our study. Our results were comparable with a study by Muralidharan et al.,[22] who also showed MDR isolates were more common in women than in men. In contrast Najotra et al.[23] showed a preponderance among males than females with 52 males and 33 females. Tigecycline, and its increased effectiveness does not vary with age, gender, or the disease in patients according to Pankey et al.[9] Kusuma et al.[24] showed a preponderance of MDR Klebsiella pneumoniae (35) and Acinetobacter spp. (35) when compared to Escherichia coli (15) which was also a finding in our study. We had tested the isolates by both the KB and Epsilometer E-test methods and a better performance was shown by the E-test, and the findings were similar to studies by Soham Gupta et al.[25] In our study, tigecycline in vitro activity by MIC method showed a good sensitivity of (75.3%) against multidrug-resistant isolates. Giammanco et al. also showed tigecycline has the capacity to retain in vitro activity to MDR bacteria.[26] Our study can be closely compared with a report published by Najotra et al. showing a higher percentage of Acinetobacter spp. tested were found sensitive to tigecycline.[23]

The E-test results, however, were not similar to findings by Behera et al., who showed that 58% percentage of Acinetobacter spp. tested were resistant to tigecycline.[27] Thus, certain studies call for caution while testing and interpreting the in vitro susceptibility of tigecycline keeping in mind possible evolving resistant mechanisms.

It also calls for a continuous monitoring and molecular identification of these resistant bugs including A. baumannii according to Mavroidi et al.[28] Xiaoxing Du et al. also demonstrated in vivo evidence that tetA gene may lead to a failure of treatment with resistant strains.[29]

The MIC range among our isolates was 0.19–8 μg/ml. The isolates with an MIC of ≤2 μg/ml showed a KB zone size diameter of ≥18 mm except in one Klebsiella isolate from blood where zone size was 17 mm. The isolates that had an MIC of 4 μg/ml were found to be intermediate (20–21 mm) and resistant (16–19 mm) and those with an MIC of ≥8 μg/ml were found to be intermediate sensitive (20–22 mm). Veeraraghavan et al. says there is wide gray area in MIC breakpoints that differ by four two fold dilutions by two organizations such as Food and Drug Administration (FDA) and EUCAST 2019, so it is better to make use of the EUCAST PK/PD nonspecies-related breakpoints for a useful clinical interpretation.[30]

A study by Babaei and Haeili[31] also confirms that the E-test is a suitable method to test extensively drug-resistant A. baumannii and carbapenem-resistant Klebsiella pneumoniae (CRKP) and E. coli. There are, however, no existing susceptibility breakpoints for some of the most important clinical pathogens including carbapenem-resistant A. baumannii (CRAB) from any breakpoint organization, including the FDA.[32] The CLSI too have not mentioned breakpoints or any preferred method for the tigecycline susceptibility testing of Enterobacteriaceae, and only a few studies have actually validated available methods for testing.[32]

The disk diffusion method still remains a simple, accurate, and inexpensive method even to test CRKP and CRAB.[33] However, our study substantiates that the MIC E-test method was found to be an effective tool by demonstrating a greater percentage of sensitive strains which might otherwise have been declared as resistant by the KB method.

  Conclusion Top

Tigecycline is the viable future option among this menace of antibiotic resistance, especially when treating coinfections of Gram-positive and Gram-negative superbugs. Despite merits and demerits of various methods of testing tigecycline sensitivity, an E-test report will be a better guide to clinicians to judiciously use this antibiotic.

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Conflicts of interest

There are no conflicts of interest.

  References Top

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Correspondence Address:
Thomas S Kuruvilla,
Flat No. 504, Muller Nest, Father Muller Doctor's Quarters, Jeppu, Mangalore - 575 002, Karnataka
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ajim.ajim_53_22


  [Figure 1], [Figure 2]

  [Table 1], [Table 2]


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