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Minimum inhibitory concentrations (MICs) are defined as the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism after overnight incubation. MICs are used by diagnostic laboratories mainly to confirm unusual resistance, to give a definitive answer when a borderline result is obtained by other methods of susceptibility testing or when disc diffusion methods are not appropriate. Several species, including Proteus, Morganella, and Providencia have natural elevated imipenem (IPM) MICs.
The increasing numbers of multidrug-resistant (MDR) pathogens that cause health-care-associated infections complicate the treatment strategy.
Carbapenems have been used extensively for severe infections arising from ESBL-producing Enterobacteriaceae, thereby imposing selection pressure for carbapenem resistance [Tzouvelekis L. S.et al., 2012]. In 2007, the World Health Organization highlighted in MDR Gram-negative bacteria (GNB), the carbapenem-resistant Enterobacteriaceae, Acinetobacter baumannii complex and Pseudomonas aeruginosa as critical priority pathogens. Several resistance mechanisms of Gram-negative bacteria including extended-spectrum β-lactamases, AmpC, carbapenemase production for carbapenem resistance, and mcr genes for colistin resistance are reported.
Over the past decade, the prevalence of carbapenem resistance among GNB has increased dramatically worldwide and the outcome is treatment failure and increased mortality.
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The mortality attributable to infection by carbapenem-resistant Gram-negative bacilli varies between 26% and 44%, with the highest mortality reported in patients with bacteremia [Falagas M E et al., 2014]. MIC of carbapenem could guide the treatment strategy and thereby it would be a good predictor for outcome of infections caused by carbapenem non- susceptible isolates. That’s why this observational study was aimed to determine the MIC values of carbapenem against commonly encountered CRGNB, including CRE and CR-non fermentative GNB (NFGNB) isolated from patients admitted to the tertiary care hospital.
MIC of carbapenems in 539 genotypically characterised carbapenem resistant isolates (CRGNB) was determined by agar dilution method.
Of these 539 isolates, 397 were carbapenemase producing and 142 were non carbapenemase producing GNB.
Preparation of Ertapenem, Imipenem and Meropenem (Sigma-Aldrich) stock solution was done following the instructions of their solvent, diluent and storage conditions (40C with protection against light and moisture). Solvent used for meropenem trihydrate was dimethyl sulfoxide (DMSO), for imipenem monohydrate and ertapenem the solvent was water.
Stock solutions were prepared using the formula:
1000 x V x C=W
where P= potency given by the manufacturer (µg/mg), V=volume required (mL), C=final concentration of the solution (multiples of 1000 to make millilitre to Litre) (mg/L) and W=weight of the antibiotic (mg) to be dissolved in volume(mL). For example, 1000/980 x 20 x 1=20.40 mg powder (20.40 mg at a potency of 980 µg/mg) dissolved in 20mL of solvent= 1000mg/L stock solution.
So, final concentration of antibiotic (meropenem/ imipenem/ ertapenem) stock solution is 1mg/ml and to make 11ml antibiotic stock solution, 11mg power has to be dissolved in solvent. Applying the formula V1S1=V2S2 where V2=20ml molten Mueller Hinton Agar (MHA), S2=1µg/ml, S1=1mg/ml and so V1=1µg/ml X 20ml = 0.02 ml
1mg (1000 µg) /ml
=20 µl
Antibiotic dilution range was 1µg/ml, 2µg/ml, 4µg/ml, 8µg/ml, 16µg/ml, 32µg/ml, 64µg/ml, 128µg/ml, 256µg/ml. As 20 µl of stock solution makes 1µg/ml, 40 µl of stock solution makes 2µg/ml, 80 µl of stock solution makes 4µg/ml and so on. Total stock solution required was (20+40+80+160+320+640+1280+2560+5120) µl= 10220 µl (~11ml).
Antibiotic dilution plates were prepared by taking 20µl from molten MHA in McCartney bottle (cooled to 500C in water bath) and adding 20 µl stock solution for antibiotic concentration 1 µg/ml, by taking 40µl from molten MHA and adding 40 µl stock solution for antibiotic concentration 2 µg/ml, by taking 80µl from molten MHA and adding 80 µl stock solution for antibiotic concentration 4 µg/ml, by taking 160µl from molten MHA and adding 160 µl stock solution for antibiotic concentration 8 µg/ml and so on. It was mixed, poured into 90mm Petri dishes and allowed to set. One antibiotic free control plate was also taken.
The inoculum (1-2µl per spot) was adjusted to 0.5 McFarland so that 104 cfu/spot were applied to the plates.
After incubation, all the organisms were grown in antibiotic free control plate. The MIC of antibiotics (meropenem, imipenem and ertapenem) were determined by the lowest concentration of antibiotic where there was no visible growth of the inoculated organisms.
MIC50 and MIC90 of carbapenems in carbapenem resistant isolates were determined. The MIC50 represents the MIC value at which ≥50% of the isolates in a test population are inhibited; it is equivalent to the median MIC value. Given n test strains and the values y1, y2 … yn representing a graded series of MICs starting with the lowest value, the MIC50 is the value at position n × 0.5, as long as n is an even number of test strains. If n is an odd number of test strains, the value at position (n + 1) × 0.5 represents the MIC50 value. The MIC90 represents the MIC value at which ≥90% of the strains within a test population are inhibited; the 90th percentile. The MIC90 is calculated accordingly, using n × 0.9.
Table 1: MIC50 and MIC90 of Carbapenems Against CRGNB
| Carbapenem | MIC50 (µg/ml) | MIC90 (µg/ml) |
|---|---|---|
| Ertapenem | 64 | 256 |
| Imipenem | 32 | 256 |
| Meropenem | 32 | 256 |
Distributions of carbapenem MICs against carbapenemase producers (CP-GNB) and non-carbapenemase producer GNB (non-CP-GNB). Most of the CP-GNB (25.65%) isolates had ertapenem MIC 64µg/ml followed by 14.60% isolates with MIC 128µg/ml and 13.63% had MIC 32µg/ml whereas most of the non CP-GNB isolates (20.42%) had ertapenem MIC 8µg/ml followed by 16.19% with MIC 2µg/ml, 14.78% with MIC 4µg/ml and 12.67% with MIC 16µg/ml. Most of the CP-GNB (23.50%) isolates had imipenem MIC 64µg/ml followed by 21.23% with MIC 32µg/ml and 14.42% with MIC 16µg/ml whereas most of the nonCP-GNB (25.35%) isolates had imipenem MIC 8µg/ml followed by 19.01% with MIC 16µg/ml and 11.97% with MIC 4µg/ml.
Again, most of the CP-GNB (25.61%) isolates had meropenem MIC 32µg/ml followed by 19.63% isolates had MIC 64µg/ml and 14.23% isolates had MIC 16µg/ml whereas most of the nonCP-GNB (26.76%) isolates had imipenem MIC 8µg/ml followed by 20.42% had MIC 16µg/ml and 9.86% had MIC 4µg/ml. All the carbapenemase producing (397 GNB) isolates were arranged according to their MICs of ertapenem, imipenem and meropenem. Most of the isolates were clustering in MIC 64µg/ml, 32µg/ml and 128µg/ml.
Table 1: MIC of Carbapenems Among Carbapenemase Producing GNB (CP-CRGNB)
| Carbapenem/MIC range (µg/ml) | 1 | 2 | 4 | 8 | 16 | 32 | 64 | 128 | 256 | ≥256 |
|---|---|---|---|---|---|---|---|---|---|---|
| E. coli | ||||||||||
| Ertapenem | 0.65% | 2.61% | 3.92% | 9.80% | 13.09% | 14.37% | 24.83% | 18.30% | 9.15% | 3.28% |
| Imipenem | 1.97% | 5.24% | 7.85% | 5.24% | 5.24% | 16.33% | 40.52% | 5.24% | 3.27% | 9.10% |
| Meropenem | 3.26% | - | - | 4.57% | 13.72% | 22.87% | 31.37% | 9.15% | 9.15% | 5.91% |
| Klebsiella spp | ||||||||||
| Ertapenem | 1.61% | 3.22% | 4.30% | 8.06% | 7.52% | 10.75% | 33.34% | 9.13% | 7.52% | 14.55% |
| Imipenem | 2.15% | 4.30% | 4.30% | 8.06% | 8.60% | 26.80% | 16.67% | 12.36% | 8.06% | 9.16% |
| Meropenem | 1.61% | 4.30% | 4.30% | 8.06% | 4.30% | 24.73% | 24.73% | 12.36% | 8.06% | 9.06% |
| Enterobacter spp. | ||||||||||
| Ertapenem | 0% | 5.27% | 10.52% | 15.79% | 10.52% | 15.79% | 15.79% | 21.05% | 5.27% | 0% |
| Imipenem | 0% | 5.26% | 5.26% | 10.52% | 15.78% | 10.52% | 26.31% | 15.78% | 5.26% | 5.26% |
| Meropenem | - | 5.26% | 5.26% | 10.52% | 10.52% | 15.78% | 26.31% | 15.78% | 5.26% | 5.26% |
| Pseudomonas spp. | ||||||||||
| Imipenem | - | - | - | 12.50% | 12.50% | 12.50% | 25% | 18.75% | 12.50% | 6.25% |
| Meropenem | - | - | - | 12.50% | 12.50% | 25% | 6.25% | 12.50% | 12.50% | 18.75% |
| Acinetobacter spp. | ||||||||||
| Imipenem | 2% | - | - | 19% | 30% | 40% | 9% | - | - | - |
| Meropenem | - | - | - |
Majority of carbapenemase producing E.coli had MIC of ertapenem, imipenem and meropenem 64 µg/ml, Similarly majority of carbapenemase producing Klebsiella spp. with ertapenem MIC 64 µg/ml whereas MIC of imipenem and meropenem was 32 µg/ml . Again, majority of carbapenemase producing Enterobacter spp. had ertapenem MIC 128 µg/ml whereas MIC of imipenem and meropenem was 64 µg/ml. In case of Pseudomonas and Acinetobacter spp. majority showed imipenem MIC 64 µg/ml and 32 µg/ml respectively and meropenem MIC in both cases were 32 µg/ml.
The emergence of carbapenem-resistant gram-negative bacilli poses a great threat to human health. Currently, antibiotic options for the treatment of CRGNB are very limited. In that case, determination of exact MIC value of carbapenems is helpful to optimize the dose to maximize therapeutic efficacy. Based on several animal infection model studies, Daikos and Markogiannakis (2011) proposed, that high dose and prolonged-infusion carbapenems can achieve bactericidal effects in immunocompetent animals infected by KPC-producing K. pneumoniae isolates with MICs up to 8 mg/L.
Moreover, Giannella et al. 2018 evaluated the efficacy of high-dose carbapenem-based combination therapy among 595 patients with CRKP BSI and studied the benefits of high-dose carbapenem in strains with meropenem MIC ≥ 16 mg/L, which comprised 77% of all isolates in the study. These clinical observations showed that a high-dose prolonged-infusion carbapenem regimen can reach attainable bactericidal targets in isolates with meropenem MICs up to 32–64 mg/L, though failure was observed with very high MICs of 256–1024 mg/L.
The researchers found that there was a carbapenem MIC shift to high level from 2001 [Codjoe FS et al. 2017]. In 2001 and 2002, 77.4% of Enterobacteriaceae had low-MIC values (≤0.25 µg/mL) for imipenem (IPM), and 21.1% of isolates had elevated-MIC values (ranging from 0.5 µg/mL to 1 µg/mL). In 2013 and 2014, the percentage of low-MICs decreased to 68.8%, and elevated-MICs increased to 27.8% and Klebsiella pneumoniae isolates were particularly affected by the IMP MIC shift, with 95.8% of isolates having low-MIC values in 2001 and 2002. That value dropped to 77.7% in 2013 and 2014 (P = .0002) [Lee MJ, et al.,2016].
In this study, most of the carbapenemase producing GNB had MIC 64 µg/mL or 32 µg/mL whereas non carbapenemase producing GNB had MIC 8µg/mL or 16µg/mL. Higher MIC meant that there was a high chance of carbapenemase production and thereby outcome was guarded [Wu, P., Chuang et al., 2016]. However, the MIC value of CR Pseudomonas and CR Acinetobacter was lower than that of other CRGNB.
MIC value of carbapenems can predict the synergistic activity of meropenem/imipenem+ertapenem and gave an idea whether double-carbapenem (DC) regimen would be more effective in comparison with the best available treatment (BAT) in infections caused by CP-CRGNB by fractional inhibitory concentration index (FICI) calculation. The FICI of each combination was defined as follows: ∑FIC: FICA + FICB= MICA+B/MICA alone + MICB+A/MICB alone. Complete synergy was defined as FICI ≤ 0.5, indifference as FICI > 0.5–4.0, and antagonism as FICI > 4.0. The subjects with meropenem MIC ≤128 μg/mL were found to respond DC therapy.
Determining accurate MIC values for carbapenems among carbapenem-resistant GNB is essential for optimizing antibiotic therapy and combating resistance. This study provides valuable insights into the susceptibility patterns of CRGNB, aiding in the development of more effective treatment strategies against these formidable pathogens.
Carbapenem MIC in Carbapenemase-Producing GNB: A Study. (2024, Feb 23). Retrieved from https://studymoose.com/document/carbapenem-mic-in-carbapenemase-producing-gnb-a-study
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