Wed, Jul 9, 2025
Volume 18, Issue 4 (Jul-Aug 2024)
mljgoums 2024, 18(4): 17-21 |
Back to browse issues page
Download citation:
BibTeX | RIS | EndNote | Medlars | ProCite | Reference Manager | RefWorks
Send citation to:
BibTeX | RIS | EndNote | Medlars | ProCite | Reference Manager | RefWorks
Send citation to:
Lall S, Bhat V, Biswas S, Khattry N. Comparative evaluation of in vitro activity of tigecycline using the disc diffusion method and the VITEK-2 COMPACT in clinical isolates at a tertiary care cancer center. mljgoums 2024; 18 (4) :17-21
URL: http://mlj.goums.ac.ir/article-1-1779-en.html
URL: http://mlj.goums.ac.ir/article-1-1779-en.html
1- Department of Microbiology, HBNI ACTREC TMC, NAVI Mumbai, India
2- Department of Microbiology, HBNI ACTREC TMC, NAVI Mumbai, India ,vivekbhat2005@yahoo.com
3- Department of Microbiology, HBNITATA Memorial Hospital, Mumbai, India
4- Department of Medical Oncology HBNI ACTREC, TMC, NAVI Mumbai, India
2- Department of Microbiology, HBNI ACTREC TMC, NAVI Mumbai, India ,
3- Department of Microbiology, HBNITATA Memorial Hospital, Mumbai, India
4- Department of Medical Oncology HBNI ACTREC, TMC, NAVI Mumbai, India
Keywords: Tigecycline, Disk diffusion antimicrobial tests, Microbial drug resistance, Multiple drug resistance, Klebsiella pneumoniae, Broth microdilution test
Full-Text [PDF 456 kb]
(404 Downloads)
| Abstract (HTML) (1920 Views)
Conclusion
The disc diffusion method did not perform well compared to VITEK-2 COMPACT. Clinicians and laboratory personnel should be made aware of the discrepancies in reporting this drug. In the absence of appropriate breakpoints or standardized international guidelines from CLSI/EUCAST, AST guidelines for TST should be formulated at the national level to maintain uniformity in reporting.
Acknowledgement
We acknowledge Neha Jewrak for her contribution in collecting data for the manuscript and the technical staff of the microbiology department.
Funding sources
No funding was taken for this study.
Ethical statement
Institutional Ethics Committee permission was obtained (IEC-3/900967). A waiver of patient consent was granted as the study did not involve any direct contact with the patients, and no personal details of the patients were used in the analysis.
Conflicts of interest
There are no conflicts of interests to declare by any of the authors.
Author contributions
SL: conceptualized the study, supervised test performance, examined results, performed data analysis, and wrote the manuscript. VB: supervised test performance, examined results, supervised the literature search, and reviewed the manuscript writing. SB: reviewed the manuscript writing process. NK: encouraged SL and VB to investigate the clinical aspect of reporting Tigecycline in their patient population and conceived this original idea.
Full-Text: (289 Views)
Introduction
With the apparent rise in multidrug-resistant organisms, tigecycline (TGC) and colistin are often considered last-resort antibiotics in the pipeline. TGC, a minocycline derivative, overcomes major tetracycline resistance mechanisms (1). Tigecycline susceptibility testing (TST) and reporting remain enigmatic due to the lack of established guidelines by either the Clinical and Laboratory Standards Institute (CLSI) or the European Committee on Antimicrobial Susceptibility Testing (EUCAST). EUCAST provides interpretive breakpoints for Escherichia coli and Citrobacter koseri, whereas CLSI mentions TGC only as part of quality control. According to CLSI 2023, rare cases may arise where an agent is appropriate for an isolate but lacks CLSI breakpoints (e.g., TGC) (2,3). In such cases, the FDA Susceptibility Test Interpretive Criteria (FDA STIC) should be consulted. The Food and Drug Administration (FDA) has provided interpretive criteria for TGC against Enterobacteriaceae for both disk diffusion and MIC testing (4). The outcomes of in vitro TST-classified as Susceptible (S), Intermediate (I), or Resistant (R)-depend on the testing method used. Disk diffusion, as a method of susceptibility testing, remains widely accepted worldwide due to its ease of use. However, limited published literature is available from India on the utility of this method for tigecycline susceptibility testing, particularly in a cancer care setting. Therefore, the study aimed to detect TST of gram-negative isolates from blood and stool cultures and evaluate the performance characteristics of disk diffusion by comparing its results with those from VITEK-2 COMPACT, considering the latter as the standard. The agreement between the interpretations of disk diffusion breakpoints and MIC results from VITEK-2 COMPACT, using FDA breakpoints for Enterobacterales isolates, was assessed. In addition, the study sought to analyze the changing trends in TST among these clinical isolates.
Methods
After obtaining Ethics Committee approval (IEC-3/900967), the study was conducted over six months, from May 2023 to October 2023, in the Department of Microbiology, ACTREC, TATA Memorial Centre, Navi Mumbai, I ndia. A retrospective analysis of a prospectively maintained laboratory database, including electronic systems and manual registers of TST results, was performed for a period of two years and two months (31.01.2021 to 31.03.2023). Isolates for which TST had been conducted concurrently by disc diffusion (DD) and VITEK-2 COMPACT-either as part of routine antibiotic susceptibility testing per lab protocol or upon request by clinicians-were included. Interpretations from DD were compared with MIC results obtained from VITEK-2 COMPACT.
Bacterial isolates were retrieved from routine bacteriology cultures of clinical specimens, such as blood, stool, urine, tissue, pus, and pus swabs. They were identified using standard microbiological techniques, including Gram’s stain, colony morphology, biochemical tests, and, in some cases, VITEK-2 COMPACT when the results could not be reached manually. The isolates were obtained from carcinoma patients across various Disease Management Groups and Hematopoietic Stem Cell Transplant recipients, encompassing both admitted and outpatient statuses. Isolates for which only one method was performed were excluded from the study.
Disc diffusion was performed using Kirby Bauer’s method on Mueller-Hinton agar and a Hi-media 15 mcg TGC disc, following CLSI guidelines and the manufacturer’s instructions.
The VITEK-2 AST-N406 (Biomerieux, Inc., Durham, NC, USA) testing was performed using software version 5.04. The susceptibility card contained tigecycline at concentrations of 1.5.4 and 8 µg/ml, used according to the manufacturer’s recommendations. It employs a miniaturized, abbreviated, and automated version of the doubling dilution technique for determining Minimum Inhibitory Concentrations (MICs) through the microdilution method.
FDA breakpoints were applied, where disc diffusion diameters of ≥19 mm were considered sensitive, 15-18 mm intermediate, and ≤14 mm resistant. MIC recommendations were as follows: ≤2 µg/ml as sensitive, 4 µg/ml as intermediate, and ≥8 µg/ml as resistant (3). For comparing results in case of E. coli, the EUCAST guidelines were used: a zone diameter of ≥18 mm was considered sensitive, and <18 mm was considered resistant. MIC values of ≤0.5 µg/ml were classified as susceptible, while values >0.5 µg/ml were resistant (4).
The misclassification of a resistant strain as susceptible by DD was considered a very major error (VME), whereas the reporting of a susceptible strain as resistant was classified as a major error (ME). The interpretive categories of either susceptible or resistant reported as intermediate, or vice versa, were considered minor errors (mEs). Categorical agreement (CA) was evaluated as the percentage of isolate characterizations produced by the disc diffusion method that were consistent with the results (R, S, or I) reported by the VITEK-2 COMPACT method. According to CLSI criteria, when CA is ≥90%, VME is ≤1.5%, and ME is ≤3%, the disc diffusion breakpoints can be considered acceptable (5).
Descriptive statistics were used to summarize the data. Categorical data were described using counts and percentages. Concordance between the interpretations from FDA disc breakpoints and FDA MIC interpretations of VITEK-2 COMPACT was assessed using Cohen’s Kappa statistic, and its 95% confidence interval was reported. Concordance between interpretations was visualized using a River Plot. A Kappa value of less than 0.4 was considered poor, between 0.4 and 0.75 was considered good, and greater than 0.75 represented excellent agreement. A negative Kappa value indicated agreement worse than expected, or disagreement. Trends in tigecycline susceptibility were assessed using proportions. A p-value of less than 0.05 was considered statistically significant. All data analysis was performed using SPSS software (Version 25.0).
Results
A total of 263 isolates were enrolled in the study. Isolates from stool specimens received for weekly surveillance reporting comprised the majority, 180 (68.44%), followed by blood cultures, 50 (19.01%). Other specimens included CSF, 7 (2.67%); wound swabs, 5 (1.90%); sterile body fluid, 5 (1.90%); sputum, 5 (1.90%); NDBAL, 3 (1.14%); urine, 2 (0.76%); abdominal suture site swabs, 2 (0.76%); drain fluid, 1 (0.38%); liver tissue, 1 (0.38%); pleural fluid, 1 (0.38%); and ventriculoperitoneal shunt fluid, 1 (0.38%).
Table 1 shows the distribution of Gram-negative isolates in the study. Escherichia coli was the most commonly isolated organism, 142 (54%), followed by Klebsiella spp, 102 (38.78%). Acinetobacter spp and Gram-negative non-fermenters were interpreted with Enterobacterales breakpoints, but no comparisons could be made due to the small number of isolates.
Table 1 also shows the mean diameter of inhibition and median MIC along with respective ranges for E. coli, Klebsiella spp, and non-fermenter Gram-negative bacilli. Since these bacilli were few in number, not much statistical correlation could be carried out by considering them as a group.
Table 2 shows the overall distribution of susceptibility and the comparison profile between both methods. Major discordance was observed in the results, as 76.0% of isolates were reported as susceptible by VITEK-2 COMPACT compared to 47.14% by disc diffusion. A significant discordance was noted in the intermediate category, with only six (2.28%) isolates reported as intermediate by VITEK-2 COMPACT, while disc diffusion showed 76 (28.89%). The resistant category did not show much variation between the two methods: 57 (21.67%) vs. 63 (23.9%) by VITEK-2 COMPACT and disc diffusion, respectively. VITEK-2 MIC showed 76% overall susceptibility, which included 50.95% (134/263) E. coli and 18.63% (49/263) Klebsiella pneumoniae. In contrast, disc diffusion testing showed 47.14% (124/263) susceptibility, comprising 36.50% E. coli and 5.70% Klebsiella pneumoniae. Tigecycline susceptibility among E. coli isolates using the FDA breakpoint on VITEK MIC was 94.36% (134/142), while using the EUCAST breakpoint it was 86.61% (123/142).
Table 3 and Figure 1 show the comparison of interpretations of disc diameters and MICs for 263 isolates. Using Cohen’s kappa coefficient, the kappa value was 0.328, with a p-value of <0.05. The agreement percentage was 60.84%. Two strains reported as resistant were misclassified as sensitive by disc diffusion, resulting in a VME rate of 0.76%. MEs were noted at 9.5%, and mEs at 27.7%, as 25 strains reported as susceptible were identified as resistant. Lesser CA was observed in blood culture isolates (58%) compared to stool samples (63.89%), although this trend was not observed in the agreement values for E. coli in blood (77%) and stool (68.75%) or for Klebsiella in blood (60.41%) and stool (57.14%). No comparison showed good agreement, except between EUCAST MIC and FDA MIC, but with a p-value of 0.477, this was not statistically significant. On comparing disc diffusion and VITEK interpretations using EUCAST and FDA breakpoints for E. coli, poor agreement was noted. Since poor agreement was observed and disc diffusion diameters exceeded the acceptable performance rate, they were not deemed acceptable according to CLSI criteria.
With the apparent rise in multidrug-resistant organisms, tigecycline (TGC) and colistin are often considered last-resort antibiotics in the pipeline. TGC, a minocycline derivative, overcomes major tetracycline resistance mechanisms (1). Tigecycline susceptibility testing (TST) and reporting remain enigmatic due to the lack of established guidelines by either the Clinical and Laboratory Standards Institute (CLSI) or the European Committee on Antimicrobial Susceptibility Testing (EUCAST). EUCAST provides interpretive breakpoints for Escherichia coli and Citrobacter koseri, whereas CLSI mentions TGC only as part of quality control. According to CLSI 2023, rare cases may arise where an agent is appropriate for an isolate but lacks CLSI breakpoints (e.g., TGC) (2,3). In such cases, the FDA Susceptibility Test Interpretive Criteria (FDA STIC) should be consulted. The Food and Drug Administration (FDA) has provided interpretive criteria for TGC against Enterobacteriaceae for both disk diffusion and MIC testing (4). The outcomes of in vitro TST-classified as Susceptible (S), Intermediate (I), or Resistant (R)-depend on the testing method used. Disk diffusion, as a method of susceptibility testing, remains widely accepted worldwide due to its ease of use. However, limited published literature is available from India on the utility of this method for tigecycline susceptibility testing, particularly in a cancer care setting. Therefore, the study aimed to detect TST of gram-negative isolates from blood and stool cultures and evaluate the performance characteristics of disk diffusion by comparing its results with those from VITEK-2 COMPACT, considering the latter as the standard. The agreement between the interpretations of disk diffusion breakpoints and MIC results from VITEK-2 COMPACT, using FDA breakpoints for Enterobacterales isolates, was assessed. In addition, the study sought to analyze the changing trends in TST among these clinical isolates.
Methods
After obtaining Ethics Committee approval (IEC-3/900967), the study was conducted over six months, from May 2023 to October 2023, in the Department of Microbiology, ACTREC, TATA Memorial Centre, Navi Mumbai, I ndia. A retrospective analysis of a prospectively maintained laboratory database, including electronic systems and manual registers of TST results, was performed for a period of two years and two months (31.01.2021 to 31.03.2023). Isolates for which TST had been conducted concurrently by disc diffusion (DD) and VITEK-2 COMPACT-either as part of routine antibiotic susceptibility testing per lab protocol or upon request by clinicians-were included. Interpretations from DD were compared with MIC results obtained from VITEK-2 COMPACT.
Bacterial isolates were retrieved from routine bacteriology cultures of clinical specimens, such as blood, stool, urine, tissue, pus, and pus swabs. They were identified using standard microbiological techniques, including Gram’s stain, colony morphology, biochemical tests, and, in some cases, VITEK-2 COMPACT when the results could not be reached manually. The isolates were obtained from carcinoma patients across various Disease Management Groups and Hematopoietic Stem Cell Transplant recipients, encompassing both admitted and outpatient statuses. Isolates for which only one method was performed were excluded from the study.
Disc diffusion was performed using Kirby Bauer’s method on Mueller-Hinton agar and a Hi-media 15 mcg TGC disc, following CLSI guidelines and the manufacturer’s instructions.
The VITEK-2 AST-N406 (Biomerieux, Inc., Durham, NC, USA) testing was performed using software version 5.04. The susceptibility card contained tigecycline at concentrations of 1.5.4 and 8 µg/ml, used according to the manufacturer’s recommendations. It employs a miniaturized, abbreviated, and automated version of the doubling dilution technique for determining Minimum Inhibitory Concentrations (MICs) through the microdilution method.
FDA breakpoints were applied, where disc diffusion diameters of ≥19 mm were considered sensitive, 15-18 mm intermediate, and ≤14 mm resistant. MIC recommendations were as follows: ≤2 µg/ml as sensitive, 4 µg/ml as intermediate, and ≥8 µg/ml as resistant (3). For comparing results in case of E. coli, the EUCAST guidelines were used: a zone diameter of ≥18 mm was considered sensitive, and <18 mm was considered resistant. MIC values of ≤0.5 µg/ml were classified as susceptible, while values >0.5 µg/ml were resistant (4).
The misclassification of a resistant strain as susceptible by DD was considered a very major error (VME), whereas the reporting of a susceptible strain as resistant was classified as a major error (ME). The interpretive categories of either susceptible or resistant reported as intermediate, or vice versa, were considered minor errors (mEs). Categorical agreement (CA) was evaluated as the percentage of isolate characterizations produced by the disc diffusion method that were consistent with the results (R, S, or I) reported by the VITEK-2 COMPACT method. According to CLSI criteria, when CA is ≥90%, VME is ≤1.5%, and ME is ≤3%, the disc diffusion breakpoints can be considered acceptable (5).
Descriptive statistics were used to summarize the data. Categorical data were described using counts and percentages. Concordance between the interpretations from FDA disc breakpoints and FDA MIC interpretations of VITEK-2 COMPACT was assessed using Cohen’s Kappa statistic, and its 95% confidence interval was reported. Concordance between interpretations was visualized using a River Plot. A Kappa value of less than 0.4 was considered poor, between 0.4 and 0.75 was considered good, and greater than 0.75 represented excellent agreement. A negative Kappa value indicated agreement worse than expected, or disagreement. Trends in tigecycline susceptibility were assessed using proportions. A p-value of less than 0.05 was considered statistically significant. All data analysis was performed using SPSS software (Version 25.0).
Results
A total of 263 isolates were enrolled in the study. Isolates from stool specimens received for weekly surveillance reporting comprised the majority, 180 (68.44%), followed by blood cultures, 50 (19.01%). Other specimens included CSF, 7 (2.67%); wound swabs, 5 (1.90%); sterile body fluid, 5 (1.90%); sputum, 5 (1.90%); NDBAL, 3 (1.14%); urine, 2 (0.76%); abdominal suture site swabs, 2 (0.76%); drain fluid, 1 (0.38%); liver tissue, 1 (0.38%); pleural fluid, 1 (0.38%); and ventriculoperitoneal shunt fluid, 1 (0.38%).
Table 1 shows the distribution of Gram-negative isolates in the study. Escherichia coli was the most commonly isolated organism, 142 (54%), followed by Klebsiella spp, 102 (38.78%). Acinetobacter spp and Gram-negative non-fermenters were interpreted with Enterobacterales breakpoints, but no comparisons could be made due to the small number of isolates.
Table 1 also shows the mean diameter of inhibition and median MIC along with respective ranges for E. coli, Klebsiella spp, and non-fermenter Gram-negative bacilli. Since these bacilli were few in number, not much statistical correlation could be carried out by considering them as a group.
Table 2 shows the overall distribution of susceptibility and the comparison profile between both methods. Major discordance was observed in the results, as 76.0% of isolates were reported as susceptible by VITEK-2 COMPACT compared to 47.14% by disc diffusion. A significant discordance was noted in the intermediate category, with only six (2.28%) isolates reported as intermediate by VITEK-2 COMPACT, while disc diffusion showed 76 (28.89%). The resistant category did not show much variation between the two methods: 57 (21.67%) vs. 63 (23.9%) by VITEK-2 COMPACT and disc diffusion, respectively. VITEK-2 MIC showed 76% overall susceptibility, which included 50.95% (134/263) E. coli and 18.63% (49/263) Klebsiella pneumoniae. In contrast, disc diffusion testing showed 47.14% (124/263) susceptibility, comprising 36.50% E. coli and 5.70% Klebsiella pneumoniae. Tigecycline susceptibility among E. coli isolates using the FDA breakpoint on VITEK MIC was 94.36% (134/142), while using the EUCAST breakpoint it was 86.61% (123/142).
Table 3 and Figure 1 show the comparison of interpretations of disc diameters and MICs for 263 isolates. Using Cohen’s kappa coefficient, the kappa value was 0.328, with a p-value of <0.05. The agreement percentage was 60.84%. Two strains reported as resistant were misclassified as sensitive by disc diffusion, resulting in a VME rate of 0.76%. MEs were noted at 9.5%, and mEs at 27.7%, as 25 strains reported as susceptible were identified as resistant. Lesser CA was observed in blood culture isolates (58%) compared to stool samples (63.89%), although this trend was not observed in the agreement values for E. coli in blood (77%) and stool (68.75%) or for Klebsiella in blood (60.41%) and stool (57.14%). No comparison showed good agreement, except between EUCAST MIC and FDA MIC, but with a p-value of 0.477, this was not statistically significant. On comparing disc diffusion and VITEK interpretations using EUCAST and FDA breakpoints for E. coli, poor agreement was noted. Since poor agreement was observed and disc diffusion diameters exceeded the acceptable performance rate, they were not deemed acceptable according to CLSI criteria.
|
Table 1. Distribution of mean, median and ranges for disc diameter and mic of gram-negative isolates
.PNG) Table 2. Overall susceptibility profile of TGC by two methods as per FDA interpretation .PNG) Table 3. Profile of comparison of study isolates under various statistical headings .PNG) .PNG) Figure 1. River plot showing the comparison between the two methods. S=Sensitive, I=Intermediate, R=Resistant, MIC=Minimum Inhibitory Concentration. Susceptibility results on the left side are given by VITEK-2 COMPACT, while the interpretations of disc diffusion are shown on the right side. |
Upon analyzing the yearly trend of tigecycline susceptibility from 2021 to 2023, it was observed that the number of susceptible isolates increased from 96 (70%) in 2021 and 17 (18.68%) in 2022 to 30 (85.7%) in 2023. However, the total isolates collected in 2023 were only up to March, which may account for the smaller sample size. Tigecycline-resistant strains varied from 34 (24.8%) in 2021 to 17 (18.68%) in 2022 and 5 (14.28%) in 2023.
Discussion
TGC has emerged as a salvage drug as it overcomes resistance mechanisms applicable to tetracycline by evading tetracycline-specific efflux pump mechanisms and providing ribosomal protection (6). Although currently approved by the FDA (2005) for the treatment of complicated skin and intra-abdominal infections, there is a rising surge in clinical data supporting its use as monotherapy for empirical coverage of various MDRs (7). However, the fear of developing resistance is not far behind, as the detection of high-level TIG resistance has been observed due to mobile plasmid-mediated transmissible tet(X) and resistance-nodulation-division efflux pump tmexCD-toprJ genes (8).
In our study, disc diffusion exhibited poor performance compared with VITEK-2 COMPACT, showing a CA of 60.84%, VME of 0.76%, and ME of 9.5% when using FDA cutoffs (Table 3). Even when analyzed individually for E. coli (CA 78.17%, VME 0%, ME 3.5%), Klebsiella spp. (CA 33.3%, VME 1.01%, ME 18.18%), stool culture isolates (CA 63.89%, VME 1.5%, ME 9.3%), and blood culture isolates (CA 58%, VME 0%, ME 14%), the disc diffusion breakpoints did not perform well. Furthermore, there was no specimen-specific variation observed when comparing disc diameter performance for E. coli in blood (CA 77%, VME 0%, ME 3.84%), E. coli in stool (CA 68.75%, VME 0%, ME 3.51%), Klebsiella spp. in blood (CA 60.41%, VME 0%, ME 10.41%), and Klebsiella spp. in stool (CA 57.14%, VME 1.01%, ME 1.01%), as poor agreement was noted across all groups. Due to its high volume of distribution, tigecycline achieves very low serum concentrations (0.6 mg/L) with standard dosing, and it is therefore not reported as a drug of choice for bacteremia. However, it is used in centers catering to immunocompromised populations, where last-resort antibiotics may be critically required as lifesaving drugs under compassionate use protocols and cascade reporting protocols (9). In addition, weekly stool sample surveillance for admitted patients from hematolymphoid wards (Both adult and pediatric), as well as pre- and post-bone marrow transplant patients, is conducted at our center to evaluate gut microbiota changes and guide empirical drug selection in cases of gut translocation leading to sepsis (10). While some of the comparisons were statistically significant (p < 0.05), they did not show any significant agreement.
Disc diffusion reported lower susceptibility rates compared to VITEK-2 COMPACT (Table 2). This could be attributed to the higher manganese content in the media used for disc diffusion testing. J. Veenemans et al. have suggested that manganese concentrations in the test medium above 8 mg/L can affect in vitro TST results, whereas tigecycline's activity remains unaffected in human serum, which contains lower manganese concentrations (11). In addition, variations in divalent cation concentrations have been reported to affect the results of antibiotics such as piperacillin, gentamicin, amikacin, tobramycin, and tetracycline in Mueller-Hinton broth or agar from different manufacturers, with manganese concentrations above 500 mg/L being particularly notable (12-15). Various studies have investigated differences in inhibition zone diameters and E test results based on manganese content, with smaller zones observed at higher manganese concentrations (16-18). The Mueller-Hinton agar used in our study from HiMedia contained 210 μg/L of manganese, which is significantly higher than the normal range reported in human serum (0.8-1.2 μg/L). This elevated manganese content contributed to the lower susceptibility rates reported by disc diffusion (19,20).
A major discrepancy noted in our study pertains to the reporting of the Intermediate category. The "Intermediate" category indicates an equivocal result and should be reported if the organism is not susceptible to other alternative drugs. This category also serves as a buffer zone, accommodating technical variations. VITEK-2 COMPACT reported six strains as intermediate, compared to 76 reported by disc diffusion diameter. Among the 76 intermediate strains, only three were concordantly intermediate by both methods. Fifty-five of the 76 intermediate strains were reported as sensitive by VITEK-2 COMPACT. This contrasts with the phenomenon of false resistance reported by Lat et al. (21). The VITEK-2 COMPACT AST card N-406 contains tigecycline concentrations of 1.5, 4, and 8 µg/mL, with a calling range of ≤0.5 to ≥8. Whether VITEK-2 COMPACT falsely reported these strains as sensitive cannot be conclusively determined without comparison to the reference Broth Microdilution test. Nonetheless, the influence of high manganese content on the reporting of these strains as intermediate also cannot be overlooked. In summary, MIC values of 1.5-2 from VITEK-2 COMPACT require careful assessment with BMD before reporting. Overlapping MICs in this range should be meticulously evaluated through clinical trials in the future to establish appropriate in vivo correlations.
Susceptibility to tigecycline varies from 97-98% in E. coli and 82-90% in Klebsiella pneumoniae as reported by various studies (22-30). Recent studies from India have shown a decrease in susceptibility rates compared to previous years (25). In our study, no such trend was observed, as 70.58% of strains were reported as sensitive in 2021, while 79.2% were reported in 2022. However, a larger number of isolates followed over a longer time period is needed to generate more data for evaluating susceptibility rate trends over years.
There was also discordance in the susceptibility pattern of E. coli reported by the FDA and EUCAST guidelines, but a good agreement was observed between them (Table 3). Since 2019, a grey zone has emerged for isolates with MICs of 0.5-8 µg/ml. These may be termed resistant according to EUCAST but susceptible or intermediate according to the FDA, as EUCAST considers an isolate with an MIC > 0.5 mg/L resistant. Despite the sixteen-fold difference in MIC cutoffs for reporting resistance between the two methods, no major discrepancies were noted in our study. This could be attributed to the mean MIC of E. coli isolates in our study, which was 1.07. This value might explain the lack of significant discrepancies, as no extreme values were observed in the data that could influence interpretation according to EUCAST (31).
Table 4 highlights different studies conducted in various locations to compare interpretations of different methods for TST. No single method demonstrates complete agreement with BMD, and the studies do not unanimously endorse a specific method. This aligns with the findings of our study.
We could not perform a comparison with the broth microdilution test, which could have served as the gold standard for both tests. We will continue to collect isolates and perform TST using various other methods to gain a panoramic view of this subject.
To summarize, center-specific protocols can be developed by making appropriate comparisons with various methods to combat this enigma. Furthermore, microbiologists should abstain from unscientific and irrational reporting of TST. If such reporting is necessary on a compassionate basis, it should always be accompanied by microbiology-specific remarks stating the mode of reporting in your lab and the extent of its relevance.
Discussion
TGC has emerged as a salvage drug as it overcomes resistance mechanisms applicable to tetracycline by evading tetracycline-specific efflux pump mechanisms and providing ribosomal protection (6). Although currently approved by the FDA (2005) for the treatment of complicated skin and intra-abdominal infections, there is a rising surge in clinical data supporting its use as monotherapy for empirical coverage of various MDRs (7). However, the fear of developing resistance is not far behind, as the detection of high-level TIG resistance has been observed due to mobile plasmid-mediated transmissible tet(X) and resistance-nodulation-division efflux pump tmexCD-toprJ genes (8).
In our study, disc diffusion exhibited poor performance compared with VITEK-2 COMPACT, showing a CA of 60.84%, VME of 0.76%, and ME of 9.5% when using FDA cutoffs (Table 3). Even when analyzed individually for E. coli (CA 78.17%, VME 0%, ME 3.5%), Klebsiella spp. (CA 33.3%, VME 1.01%, ME 18.18%), stool culture isolates (CA 63.89%, VME 1.5%, ME 9.3%), and blood culture isolates (CA 58%, VME 0%, ME 14%), the disc diffusion breakpoints did not perform well. Furthermore, there was no specimen-specific variation observed when comparing disc diameter performance for E. coli in blood (CA 77%, VME 0%, ME 3.84%), E. coli in stool (CA 68.75%, VME 0%, ME 3.51%), Klebsiella spp. in blood (CA 60.41%, VME 0%, ME 10.41%), and Klebsiella spp. in stool (CA 57.14%, VME 1.01%, ME 1.01%), as poor agreement was noted across all groups. Due to its high volume of distribution, tigecycline achieves very low serum concentrations (0.6 mg/L) with standard dosing, and it is therefore not reported as a drug of choice for bacteremia. However, it is used in centers catering to immunocompromised populations, where last-resort antibiotics may be critically required as lifesaving drugs under compassionate use protocols and cascade reporting protocols (9). In addition, weekly stool sample surveillance for admitted patients from hematolymphoid wards (Both adult and pediatric), as well as pre- and post-bone marrow transplant patients, is conducted at our center to evaluate gut microbiota changes and guide empirical drug selection in cases of gut translocation leading to sepsis (10). While some of the comparisons were statistically significant (p < 0.05), they did not show any significant agreement.
Disc diffusion reported lower susceptibility rates compared to VITEK-2 COMPACT (Table 2). This could be attributed to the higher manganese content in the media used for disc diffusion testing. J. Veenemans et al. have suggested that manganese concentrations in the test medium above 8 mg/L can affect in vitro TST results, whereas tigecycline's activity remains unaffected in human serum, which contains lower manganese concentrations (11). In addition, variations in divalent cation concentrations have been reported to affect the results of antibiotics such as piperacillin, gentamicin, amikacin, tobramycin, and tetracycline in Mueller-Hinton broth or agar from different manufacturers, with manganese concentrations above 500 mg/L being particularly notable (12-15). Various studies have investigated differences in inhibition zone diameters and E test results based on manganese content, with smaller zones observed at higher manganese concentrations (16-18). The Mueller-Hinton agar used in our study from HiMedia contained 210 μg/L of manganese, which is significantly higher than the normal range reported in human serum (0.8-1.2 μg/L). This elevated manganese content contributed to the lower susceptibility rates reported by disc diffusion (19,20).
A major discrepancy noted in our study pertains to the reporting of the Intermediate category. The "Intermediate" category indicates an equivocal result and should be reported if the organism is not susceptible to other alternative drugs. This category also serves as a buffer zone, accommodating technical variations. VITEK-2 COMPACT reported six strains as intermediate, compared to 76 reported by disc diffusion diameter. Among the 76 intermediate strains, only three were concordantly intermediate by both methods. Fifty-five of the 76 intermediate strains were reported as sensitive by VITEK-2 COMPACT. This contrasts with the phenomenon of false resistance reported by Lat et al. (21). The VITEK-2 COMPACT AST card N-406 contains tigecycline concentrations of 1.5, 4, and 8 µg/mL, with a calling range of ≤0.5 to ≥8. Whether VITEK-2 COMPACT falsely reported these strains as sensitive cannot be conclusively determined without comparison to the reference Broth Microdilution test. Nonetheless, the influence of high manganese content on the reporting of these strains as intermediate also cannot be overlooked. In summary, MIC values of 1.5-2 from VITEK-2 COMPACT require careful assessment with BMD before reporting. Overlapping MICs in this range should be meticulously evaluated through clinical trials in the future to establish appropriate in vivo correlations.
Susceptibility to tigecycline varies from 97-98% in E. coli and 82-90% in Klebsiella pneumoniae as reported by various studies (22-30). Recent studies from India have shown a decrease in susceptibility rates compared to previous years (25). In our study, no such trend was observed, as 70.58% of strains were reported as sensitive in 2021, while 79.2% were reported in 2022. However, a larger number of isolates followed over a longer time period is needed to generate more data for evaluating susceptibility rate trends over years.
There was also discordance in the susceptibility pattern of E. coli reported by the FDA and EUCAST guidelines, but a good agreement was observed between them (Table 3). Since 2019, a grey zone has emerged for isolates with MICs of 0.5-8 µg/ml. These may be termed resistant according to EUCAST but susceptible or intermediate according to the FDA, as EUCAST considers an isolate with an MIC > 0.5 mg/L resistant. Despite the sixteen-fold difference in MIC cutoffs for reporting resistance between the two methods, no major discrepancies were noted in our study. This could be attributed to the mean MIC of E. coli isolates in our study, which was 1.07. This value might explain the lack of significant discrepancies, as no extreme values were observed in the data that could influence interpretation according to EUCAST (31).
Table 4 highlights different studies conducted in various locations to compare interpretations of different methods for TST. No single method demonstrates complete agreement with BMD, and the studies do not unanimously endorse a specific method. This aligns with the findings of our study.
We could not perform a comparison with the broth microdilution test, which could have served as the gold standard for both tests. We will continue to collect isolates and perform TST using various other methods to gain a panoramic view of this subject.
To summarize, center-specific protocols can be developed by making appropriate comparisons with various methods to combat this enigma. Furthermore, microbiologists should abstain from unscientific and irrational reporting of TST. If such reporting is necessary on a compassionate basis, it should always be accompanied by microbiology-specific remarks stating the mode of reporting in your lab and the extent of its relevance.
|
Table 4. Various studies highlighting the comparison done between different methods of tigecycline reporting
.PNG) |
Conclusion
The disc diffusion method did not perform well compared to VITEK-2 COMPACT. Clinicians and laboratory personnel should be made aware of the discrepancies in reporting this drug. In the absence of appropriate breakpoints or standardized international guidelines from CLSI/EUCAST, AST guidelines for TST should be formulated at the national level to maintain uniformity in reporting.
Acknowledgement
We acknowledge Neha Jewrak for her contribution in collecting data for the manuscript and the technical staff of the microbiology department.
Funding sources
No funding was taken for this study.
Ethical statement
Institutional Ethics Committee permission was obtained (IEC-3/900967). A waiver of patient consent was granted as the study did not involve any direct contact with the patients, and no personal details of the patients were used in the analysis.
Conflicts of interest
There are no conflicts of interests to declare by any of the authors.
Author contributions
SL: conceptualized the study, supervised test performance, examined results, performed data analysis, and wrote the manuscript. VB: supervised test performance, examined results, supervised the literature search, and reviewed the manuscript writing. SB: reviewed the manuscript writing process. NK: encouraged SL and VB to investigate the clinical aspect of reporting Tigecycline in their patient population and conceived this original idea.
Research Article: Research Article |
Subject:
Microbiology
Received: 2024/01/30 | Accepted: 2024/07/17 | Published: 2025/03/11 | ePublished: 2025/03/11
Received: 2024/01/30 | Accepted: 2024/07/17 | Published: 2025/03/11 | ePublished: 2025/03/11
References
1. Petersen PJ, Jacobus NV, Weiss WJ, Sum PE, Testa RT. In vitro and in vivo antibacterial activities of a novel glycylcycline, the 9-t-butylglycylamido derivative of minocycline (GAR-936). Antimicrob Agents Chemother. 1999; 43(4): 738-44. [View at Publisher] [DOI] [PMID] [Google Scholar]
2. Clinical and Laboratory Standards Institute (CLSI). The Performance standards for antimicrobial susceptibility testing, M100-S29. 2023. [View at Publisher]
3. The European Committee on Antimicrobial Susceptibility Testing. Breakpoint tables for interpretation of MICs and zone diameters. Version 13.0, 2023. http://www.eucast.org. [View at Publisher]
4. US Food and Drug Administration. FDA-Identified Interpretive Criteria for Tigecycline-Injection products; 2019. [View at Publisher]
5. Clinical and laboratory Standards Institute (CLSI). Verification of Commercial Microbial Identification and Antimicrobial Susceptibility Testing Systems M52-1st Edition. 2015. [View at Publisher]
6. Livermore DM. Tigecycline: what is it, and where should it be used? J AntimicrobChemother. 2005; 56(4): 611-4. [View at Publisher] [DOI] [PMID] [Google Scholar]
7. Yaghoubi S, Zekiy AO, Krutova M, Gholami M, Kouhsari E, Sholeh M, et al. Tigecycline antibacterial activity, clinical effectiveness, and mechanisms and epidemiology of resistance: narrative review. Eur J Clin Microbiol Infect Dis. 2022; 41(7): 1003-1022. [View at Publisher] [DOI:10.1007/s10096-020-04121-1] [PMID] [Google Scholar]
8. Anyanwu MU, Nwobi OC, Okpala COR, Ezeonu IM. Mobile Tigecycline Resistance: An Emerging Health Catastrophe Requiring Urgent One Health Global Intervention. Front Microbiol. 2022; 13: 808744. [View at Publisher] [DOI] [PMID] [Google Scholar]
9. Rodvold KA, Gotfried MH, Cwik M, Korth-Bradley JM, Dukart G, Ellis-Grosse EJ. Ellis-Grosse, Serum, tissue and body fluid concentrations of tigecycline after a single 100 mg dose. J Antimicrob Chemother. 2006; 58(6): 1221-9. [View at Publisher] [DOI] [PMID] [Google Scholar]
10. Hooper LV, Littman DR, Macpherson AJ. Interactions between the microbiota and the Immune System. Science. 2012; 336(6086): 1268-1273. [View at Publisher] [DOI] [PMID] [Google Scholar]
11. Veenemans J, Mouton JW, Kluytmans JA, Donnely R, Verhulst C, van Keulen PH. Effect of manganese in test media on in vitro susceptibility of Enterobacteriaceae and Acinetobacter baumannii to tigecycline Clin Microbiol. 2012; 50(9): 3077-9. [View at Publisher] [DOI] [PMID] [Google Scholar]
12. Fass RJ, Barnishan J. Effect of divalent cation concentrations on the antibiotic susceptibilities of non-fermenters other than Pseudomonas aeruginosa. Antimicrob Agents Chemother. 1979; 16(4): 434-8. [View at Publisher] [DOI] [PMID] [Google Scholar]
13. Reller LB, Schoenknecht FD, Kenny MA, Sherris JC. Antibiotic susceptibility testing of Pseudomonas aeruginosa: selection of a control strain and criteria for magnesium and calcium content in media. J Infect Dis. 1974; 130(5): 454-63. [View at Publisher] [DOI] [PMID] [Google Scholar]
14. D'amato RF, Thornsberry C, Baker CN, Kirven LA. Effect of calcium and magnesium ions on the susceptibility of Pseudomonas species to tetracycline, gentamicin polymyxin B, carbenicillin. Antimicrob Agents Chemother. 1975; 7(5): 596-600. [View at Publisher] [DOI] [PMID] [Google Scholar]
15. Washington JA 2nd, Snyder RJ, Kohner PC, Wiltse CG, Ilstrup DM, McCall JT. Effect of cation content of agar on the activity of gentamicin, tobramycin, and amikacin against Pseudomonas aeruginosa. J Infect Dis. 1978; 137(2): 103-11. [View at Publisher] [DOI] [PMID] [Google Scholar]
16. Fernández-Mazarrasa C, Mazarrasa O, Calvo J, del Arco A, Martínez-Martínez L. High concentrations of manganese in Mueller-Hinton agar increase MICs of tigecycline determined by Etest. J Clin Microbiol. 2009; 47(3): 827-9. [View at Publisher] [DOI] [PMID] [Google Scholar]
17. Thamlikitkul V, Tiengrim S. Effect of different Mueller-Hinton agars on tigecycline disc diffusion susceptibility for Acinetobacter spp. J AntimicrobChemother. 2008; 62(4): 847-8. [View at Publisher] [DOI] [PMID] [Google Scholar]
18. Fernández-Mazarrasa C, Mazarrasa O, Calvo J, del Arco A, Martínez-Martínez L. High concentrations of manganese in Mueller-Hinton agar increase MICs of tigecycline determined by Etest. J Clin Microbiol. 2009; 47(3): 827-9. [View at Publisher] [DOI] [PMID] [Google Scholar]
19. Rahelić D, Kujundzić M, Romić Z, Brkić K, Petrovecki M. Serum concentration of zinc, copper, manganese and magnesium in patients with liver cirrhosis. Coll Antropol. 2006; 30(3): 523-8. [View at Publisher] [PMID] [Google Scholar]
20. Rükgauer M, Klein J, Kruse-Jarres JD. Reference values for the trace elements copper, manganese, selenium, and zinc in the serum/plasma of children, adolescents, and adults. J Trace Elem Med Biol. 1997; 11(2): 92-8 [View at Publisher] [DOI] [PMID] [Google Scholar]
21. Lat A, Clock SA, Wu F, Whittier S, Della-Latta P, Fauntleroy K, et al. Comparison of polymyxin B, tigecycline, cefepime, and meropenem MICs for KPC-producing Klebsiella pneumoniae by broth microdilution, Vitek 2, and Etest. J Clin Microbiol. 2011; 49(5): 1795-8. [View at Publisher] [DOI] [PMID] [Google Scholar]
22. Behera B, Das A, Mathur P, Kapil A, Gadepalli R, Dhawan B. Tigecycline susceptibility report from an Indian tertiary care hospital. Indian J Med Res. 2009; 129(4): 446-50. [View at Publisher] [PMID] [Google Scholar]
23. Manoharan A, Chatterjee S, Madhan S, Mathai D. Evaluation of tigecycline activity in clinical isolates among Indian medical centers. Indian J PatholMicrobiol. 2010; 53 (4): 734-7. [View at Publisher] [DOI] [PMID] [Google Scholar]
24. Kumar S, Bandyopadhyay M, Mondal S, Pal N, Ghosh T, Bandyopadhyay M, et al. Tigecycline activity against metallo-β-lactamase-producing bacteria. Avicenna J Med. 2013; 3(4): 92-6. [View at Publisher] [DOI] [PMID] [Google Scholar]
25. Veeraraghavan B, Poojary A, Shankar C, Bari AK, Kukreja S, Thukkaram B, et al. In-vitro activity of tigecycline and comparator agents against common pathogens: Indian experience. J Infect Dev Ctries. 2019; 13(3): 245-250. [View at Publisher] [DOI] [PMID] [Google Scholar]
26. Hoban DJ, Reinert RR, Bouchillon SK, Dowzicky MJ. Global in vitro activity of tigecycline and comparator agents: Tigecycline Evaluation and Surveillance Trial 2004-2013. Ann Clin Microbiol Antimicrob. 2015; 14: 27. [View at Publisher] [DOI] [PMID] [Google Scholar]
27. Fernández-Canigia L, Dowzicky MJ. Susceptibility of important Gram-negative pathogens to tigecycline and other antibiotics in Latin America between 2004 and 2010. Ann Clin MicrobiolAntimicrob. 2012; 11: 29. [View at Publisher] [DOI] [PMID] [Google Scholar]
28. Elnasser Z, Elsamarneh R, Obeidat H, Amarin Z, Jaradat S, Kaplan N. In-vitro activity of tigecycline against multidrug-resistant Gram negative bacteria: The experience of a university hospital. J Infect Public Health. 2021; 14(4): 478-483. [View at Publisher] [DOI] [PMID] [Google Scholar]
29. Kenza El Bazi. Tigecycline susceptibility among multi -drug resistant bacteria: A 7-year retrospective study. GSC Advanced Research and Reviews. 2022; 11(01): 079-083 [View at Publisher] [DOI] [Google Scholar]
30. He F, Fu Y, Chen Q, Ruan Z, Hua X, Zhou H, et al. Tigecycline susceptibility and the role of efflux pumps in tigecycline resistance in KPC-producing Klebsiella pneumoniae. PLoS One. 2015; 10(3): e0119064. [View at Publisher] [DOI] [PMID] [Google Scholar]
31. The European Committee on Antimicrobial Susceptibility Testing. Breakpoint Tables for Interpretation of MICs and Zone Diameters. Version 8.0; 2018. [View at Publisher]
32. Li H, Zhou M, Chen X, Zhang Y, Jian Z, Yan Q, et al. Comparative Evaluation of Seven Tigecycline Susceptibility Testing Methods for Carbapenem-Resistant Enterobacteriaceae. Infect Drug Resist. 2021; 14: 1511-1516. [View at Publisher] [DOI] [PMID] [Google Scholar]
33. Saleh M. Evaluation of Colistin and Tigecycline Susceptibility Testing Methods for Klebsiella pneumoniae and Acinetobacter baumannii Clinical Isolates. Egyptian Journal of Medical Microbiology. 2021; 30(2): 35-42. [View at Publisher] [DOI] [Google Scholar]
34. Bedenić B, Cavrić G, Vranić-Ladavac M, Barišić N, Karčić N, Tot T, et al. COMPARISON OF TWO DIFFERENT METHODS FOR TIGECYCLINE SUSCEPTIBILITY TESTING IN ACINETOBACTER BAUMANNII. Acta Clin Croat. 2018; 57(4): 618-623. [View at Publisher] [DOI] [PMID] [Google Scholar]
35. Grandesso S, Sapino B, Amici G, Mazzucato D. Solinas M, Gion M. Are E-test and Vitek 2 good choices for tigecycline susceptibility testing when comparing broth microdilution for MDR and XDR Acinetobacter baumannii? New Microbiol. 2014; 37: 503-8. [View at Publisher] [PMID] [Google Scholar]
36. Zarkotou O, Pournaras S, Altouvas G, Pitiriga V, Tziraki M, Mamali V, et al. Comparative evaluation of tigecycline susceptibility testing methods for expanded-spectrum cephalosporin- and carbapenem-resistant gram-negative pathogens. J Clin Microbiol. 2012; 50(11): 3747-50. [View at Publisher] [DOI] [PMID] [Google Scholar]
37. Marchaim D et al.Major variation in MICs of tigecycline in Gram-negative bacilli as a function of testing method Clin Microbiol. 2014; 52(5):1617-21. [View at Publisher] [DOI] [PMID] [Google Scholar]
38. Zhang J, Zhao C, Chen H, Wang X, Li H, Zhang Y, et al. Comparative evaluation of tigecycline susceptibility testing methods for Acinetobacter baumannii and Enterobacteriaceae. J Glob Antimicrob Resist. 2015; 3(2): 75-79. [View at Publisher] [DOI] [PMID] [Google Scholar]
39. Idelevich EA, Freeborn DA, Seifert H, Becker K. Comparison of tigecycline susceptibility testing methods for multidrug-resistant Acinetobacter baumannii. Diagnostic microbiology and infectious disease. 2018; 91(4):360-362. [View at Publisher] [DOI] [PMID] [Google Scholar]
40. Simsek M, Demir C. Determination of Colistin and Tigecycline Resistance Profile of Acinetobacter Baumannii Strains from Different Clinical Samples in a Territory Hospital in Turkey. Journal of Health Science and Medical Research. 2020; 38(2):81-91. [View at Publisher] [DOI] [Google Scholar]
41. Huang TD, Berhin C, Bogaerts P, Glupczynski Y. In vitro susceptibility of multidrug-resistant Enterobacteriaceae clinical isolates to tigecycline. J Antimicrob Chemother. 2012; 67(11): 2696-9. [View at Publisher] [DOI] [PMID] [Google Scholar]
Send email to the article author
| Rights and permissions | |
 |
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. |
Enamad
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.