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REVIEW ARTICLE
Year : 2017  |  Volume : 35  |  Issue : 4  |  Page : 445-468
 

An update on technical, interpretative and clinical relevance of antimicrobial synergy testing methodologies


Department of Clinical Microbiology, Christian Medical College, Vellore, Tamil Nadu, India

Date of Web Publication1-Feb-2018

Correspondence Address:
Dr. Balaji Veeraraghavan
Department of Clinical Microbiology, Christian Medical College, Vellore - 632 004, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmm.IJMM_17_189

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 ~ Abstract 

Testing for antimicrobial interactions has gained popularity in the last decade due to the increasing prevalence of drug-resistant organisms and limited options for the treatment of these infections. In vitro combination testing provides information, on which two or more antimicrobials can be combined for a good clinical outcome. Amongst the various in vitro methods of drug interactions, time-kill assay (TKA), checkerboard (CB) assay and E-test-based methods are most commonly used. Comparative performance of these methods reveals the TKA as the most promising method to detect synergistic combinations followed by CB assay and E-test. Various combinations of antimicrobials have been tested to demonstrate synergistic activity. Promising results were obtained for the combinations of meropenem plus colistin and rifampicin plus colistin against Acinetobacter baumannii, colistin plus carbapenem and carbapenem plus fluoroquinolones against Pseudomonas aeruginosa and colistin/polymyxin B plus rifampicin/meropenem against Klebsiella pneumoniae. Antagonism was detected in only few instances. The presence of synergy or antagonism with a combination seems to correlate with minimum inhibitory concentration of the agent and molecular mechanism involved in the resistance. Further studies need to be conducted to assess the utility of in vitro testing to predict clinical outcome and direct therapy for drug-resistant organisms.


Keywords: Acinetobacter baumannii, antimicrobial resistance, checkerboard assay, combination testing, Klebsiella pneumoniae, Pseudomonas aeruginosa, time-kill assay


How to cite this article:
Laishram S, Pragasam AK, Bakthavatchalam YD, Veeraraghavan B. An update on technical, interpretative and clinical relevance of antimicrobial synergy testing methodologies. Indian J Med Microbiol 2017;35:445-68

How to cite this URL:
Laishram S, Pragasam AK, Bakthavatchalam YD, Veeraraghavan B. An update on technical, interpretative and clinical relevance of antimicrobial synergy testing methodologies. Indian J Med Microbiol [serial online] 2017 [cited 2018 Feb 20];35:445-68. Available from: http://www.ijmm.org/text.asp?2017/35/4/445/224426



 ~ Introduction Top


In recent times, need for synergy testing has been driven by the following reasons: (i) necessity to extend the antimicrobial spectrum, (ii) possibility of reducing the dosage and toxicity and (iii) possibility of reducing the development of resistance.[1] In addition, the emergence of multidrug resistance (MDR), extensive drug resistance (XDR) and pan-drug resistance (PDR) strains, combined with the lacunae in the development of newer antimicrobial agents, has contributed to the necessity for the synergy testing between various combinations of antimicrobial agents.

The development of drug-resistant organisms is the prime cause for the increase in healthcare-associated infections, especially ventilator-associated pneumonia (VAP) and bacteraemia. Among the hospital-acquired infections (HAIs) due to Gram-negative organisms, MDR-Gram negative bacilli (GNB) infections accounted for 36.8% in a tertiary care centre in Taiwan during a 7-year period (2002–2009).[2] Similar trend was seen in South America, where a tertiary care centre in Brazil recorded 3.7-fold increase in the infection rates due to MDR-GNB during 1999–2008.[3] The development of MDR and carbapenem resistance was increasingly seen, especially for Acinetobacter baumannii.[4] However, good infection control practices were able to decrease the overall HAI rates, and the trend remains unchanged for GNB-HAI contributed by carbapenem resistance organisms.[2]

Alternative therapies or treatment strategies for such XDR and carbapenem-resistant (CR) GNBs are limited. Nevertheless, old drugs such as colistin, fosfomycin and tigecycline can be used in combination with other agents. In the past decades, the use of colistin has been restricted by the concerns of toxicity and problems in optimisation of dosage. Tigecycline use was hampered by its large distribution volume, leading to sub-inhibitory levels and selection of resistant strains with increase in the geometric mean of minimum inhibitory concentration (MIC).[5] Further, the Food and Drug Administration approved the use of tigecycline only for complicated skin and soft tissue infections, intra-abdominal infections and community-acquired pneumonia. However, it was not approved for the use in VAP because of higher mortality rate. Fosfomycin was reported to have superior in vitro activity against CR-Enterobacteriaceae isolates but was restricted for the treatment of urinary tract infection (UTI). Very few reports exist on the use of fosfomycin for other systemic infections. Moreover, fosfomycin must be used in combination with other antimicrobial agents because of high rate of resistance mutation. To overcome the aforementioned concerns, it is essential to test different antimicrobial combinations including the agent to which the organism has developed resistance.

The rationale behind the choice of combination therapy is that the antimicrobials will have a synergistic effect when given together. This review summarises the various methods available to determine synergy between different antimicrobial agents and to provide scientific evidence for utility of such combinations in the clinical setting. In particular, special focus is given on in vitro efficacy of the combined antimicrobials against drug-resistant A. baumannii, Klebsiella pneumoniae and Pseudomonas aeruginosa.


 ~ Technical Performance Of Methods For The Determination Of Interactions Between Antimicrobial Agents Top


Although many test methods are available to determine the interaction between antimicrobial agents, they were not well standardised. Interpretation criteria followed for test results are not defined and remain uncertain. The various testing methods for determining the synergistic activity of antimicrobials are discussed below.

In vitro assay

Time-kill assay

The time-kill assay (TKA) is considered as the standard reference method for the determination of synergy between antimicrobial agents. TKA determines the actual reduction in the viable count of the organism after exposure to the drug combination compared to the most active single agent at different time intervals. This is done by adding a standard inoculum in broths containing the individual antimicrobial agents and its combination. Sub-culturing is done from the broth containing antimicrobials at different time intervals and the bacterial count is done. Colony count is done at shorter time intervals e.g., every 2 h over a 24-h period for drugs having concentration-dependent activity. For drugs having time-dependent killing activity, colony count is done every 3–4 h till 24–48 h. The determination of the synergistic action by TKA is defined as ≥2 log10 CFU/ml reduction in the bacterial growth in the combination when compared to the most active single agent. However, antagonism is defined by an increase of ≥2 log10 CFU/ml in the combination compared to the most active single agent. Less than 2 log10 CFU/ml difference is interpreted as indifference. Bactericidal effects of the combinations are determined by a decrease of ≥3 log10 CFU/ml from the initial inoculum.

Another method of interpretation of the TKA is area under the killing curve (AUKC),[6] where instead of measuring the log10 difference; the result was plotted on a graph with the log10 CFU/ml value in the Y-axis and the time at the X-axis. The AUKC is calculated for single agent and combinations as well. Any statistically significant difference with P < 0.05 is taken as a synergistic interaction.[6] This method of interpretation was found to be robust with high precision and less intra-experimental variation but not widely used.

This method allows the testing of one concentration and one ratio of the antimicrobials at one time. The test has to be repeated to observe interactions at other concentrations and ratios. There is also a lack of consensus as to a standard inoculum of the organism to be used though the inoculum size varied from 1 to 5 × 105. The reported concentration of antimicrobials tested in other studies varies from 0.125 × MIC to 4 × MIC.[7] When drug combinations are tested at the MIC or more than MIC concentrations, the test may be hard to interpret because inhibition of the organism by the single agent may preclude demonstration of synergy.

Some authors prefer testing of drug concentrations that are achievable in human serum when standard dosing regimens are administered.[8] Though this strategy incorporates the pharmacokinetic (PK) property of the tested drugs, it does not implicate the concentration of drug at tissues or other sites of infection. Thus, results may not be extrapolated to particular organ system infection such as VAP where the serum concentration of the drug may not reflect the tissue concentration. The drug concentration in the in vitro test does not vary, while in vivo, there is a variation in the concentration and ratio of the drugs used. This depends on the PK and pharmacodynamic (PD) property of the drugs, dosing interval, strength and route of administration. The drawbacks of TKA include testing of limited antimicrobial concentrations, non-standardised inoculum size and antimicrobial concentration, static concentration of the drug, labour intensive and time-consuming.

Checkerboard assay

The checkerboard (CB) assay utilises a panel of antimicrobial combinations at different concentrations either in the macrobroth (2 ml volume) or microbroth (100 μl volume) method. The range of tested concentrations varies from four to eight times the MIC to at least 1/8–1/16 of the MIC. It is important to include broad range of concentrations because MIC can vary depending on the method used and also within the method (a variation of one/two-fold dilution is allowed within a test system). For the interpretation of result, the fractional inhibitory concentration (FIC) is calculated for each antibiotic at a given concentration combination by the following formula:

FIC of agent A = MIC of agent A in combination/MIC of agent A alone

The cumulative FIC is then calculated by summing up the FIC of both the agents. 'Synergy' is interpreted when the FIC index is ≤0.5, 'indifference' or 'no interaction' corresponds to the FIC index >0.5–4.0 and 'antagonism' when the FIC index is >4.0.[9]

However, in some studies, authors have defined 'partial synergy' for FIC index between >0.5 and <1 and an 'additive interaction' for FIC index of 1. Reporting of such results has to carefully considered because of the acceptance of inherent one tube dilution variation with this method and possibility of reproducibility error.[9] This was addressed by Rand et al., who reported 25% discordance with the CB method and suggested testing in at least five replicates and considering the reading only with ≥80% agreement between the replicates.[10] Another contentious issue with CB assays is the use of different criteria to interpret the test.

E-test

E-test strips containing gradient of antimicrobial agents have been used to determine the synergistic combinations. The different methods are (i) E-test cross method, (ii) E-test fixed ratio method, (iii) E-test agar method and (iv) E-test MIC: MIC method.

E-test cross method

Mueller-Hinton agar (MHA) plate is inoculated with 0.5 McFarland matched inoculum, to which E-test strips are placed one over the other at 90° angle crossing at the MICs of the individual agent of the organism determined earlier [Figure 1]. After incubation for 18 h, the zone of inhibition is read and the FIC index is calculated and interpreted as described for CB assay.[11]
Figure 1: E- test cross method. In this example, minimum inhibitory concentration of A is 6 μg/ml and minimum inhibitory concentration of B is 8 μg/ml. After combination of A and B, minimum inhibitory concentration of A is 0.094 μg/ml and minimum inhibitory concentration of B is 0.75 μg/ml. ∑FIC = 0.1 (synergy). FIC: Fractional inhibitory concentration

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E-test fixed ratio method

In this method, MHA plates are inoculated with 0.5 McFarland matched inoculum. E-test strip of the first agent is placed and incubated at room temperature for 1 h to allow the antimicrobial to diffuse into the medium. After 1 h, it is removed and saved as MIC template. The E-test strip for the second agent is then placed directly over the imprint of the first strip [Figure 2]. The FIC index is again calculated and interpreted as described for CB assay.[12],[13]
Figure 2: E-test fixed ratio method. In this example, minimum inhibitory concentration of A is 16 μg/ml and minimum inhibitory concentration of B is 32 μg/ml. Minimum inhibitory concentration of combination A and B is 1 μg/ml. ∑FIC = 0.09 (synergy). FIC: Fractional inhibitory concentration

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E-test agar method

In this method, MHA plates are incorporated with 0.5 × or 0.125 × MIC of one agent and the E-test strip of the second agent is placed over the inoculated surface [Figure 3]. The MIC obtained is compared with the MIC in drug-free medium. The synergy is interpreted when there is more than three-fold reduction in MIC on the drug-incorporated medium.[13]
Figure 3: E-test agar method. In this example, minimum inhibitory concentration of A is 12 μg/ml and in combination 0.38 μg/ml. Fractional inhibitory concentration of A = 0.03. Minimum inhibitory concentration of B is 96 μg/ml and in combination is 6 μg/ml. Fractional inhibitory concentration of B = 0.06. ∑FIC = 0.09 (synergy). FIC: Fractional inhibitory concentration

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E-test minimum inhibitory concentration: minimum inhibitory concentration method

In this method, one test strip is placed on the inoculated MHA plate and incubated at room temperature for 1 h to allow diffusion of the agent. After 1 h, the agar is marked adjacent to the previously determined MIC of the agent and removed. The second E-test strip is then placed over the imprint of the previous strip such that the mark on the agar corresponds to the MIC of the second agent [Figure 4]. The resulting ellipse of inhibition is read after 20 h of incubation at 37°C. The FIC index is calculated and interpreted as like that of CB assay.
Figure 4: E-test minimum inhibitory concentration: minimum inhibitory concentration method. In this example, minimum inhibitory concentration of A is 12 μg/ml and minimum inhibitory concentration of B is 6 μg/ml. Minimum inhibitory concentration of combination A and B is 0.5 μg/ml. ∑FIC = 0.12 (synergy). FIC: Fractional inhibitory concentration

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Compared to the other commonly used methods such as TKA and CB assay, E-test methods are technically simpler to perform and reproducible. The limitations of E-test methods are the inability to determine interaction of more than two antimicrobial combinations and the limited gradient of antimicrobial on the paper strip. For organisms where the MIC is more than the highest concentration on the strip, difficulties may be encountered with calculation of the FIC index and may result in the false interpretations. In addition, detection of antagonistic combinations will be limited for such isolates. With the E-test cross method, mild degree of antagonism may not be detected because of overlapping of strips.[11]

In vitro pharmacokinetic model

The various in vitro tests for the determination of antimicrobial interactions involve testing of drugs at a static concentration without any change in concentration with time. However, the in vivo drug concentrations and ratio keep changing with time. To better simulate these changing conditions, PK models were designed. In a single-compartment model, a glass apparatus with inlet and outlet is maintained at 37°C. Fresh media with antimicrobial agents are loaded and the media from the apparatus are withdrawn using a peristaltic pump at a constant rate mimicking the elimination kinetics of the drug and the half-life at the standard dosing regimen of the drug tested. The compartment is charged with a standard inoculum and the change in organism load is compared between single agent and the combination.[14]

In a two-compartment model, a similar central compartment as above was used with a constant volume with changing antimicrobial concentration. The compartment is connected to three or four dialyser unit which acts as the peripheral compartment. Each peripheral compartment containing 150 ml of the organism culture is exposed to a changing antimicrobial concentration similar to the central compartment. The whole system was then kept at 37°C. This method enables simultaneous testing of up to four isolates.[15],[16]

The two models mimic the in vivo PK property of the individual agents at the standard dose and regimen. The change in CFU/ml was compared using a standard inoculum for the single-drug administration and the combination regimen at regular time intervals. Synergism is interpreted by decrease of ≥2 log10 CFU/ml compared to the best monotherapy regimen or AUKC analysis can be used to detect synergistic interactions.[15],[16]

Hollow fibre infection model

The hollow fibre bioreactor is an important advancement in the in vitro combination testing. Currently available in vitro testing methods have a drawback of not examining time and concentration of the drug at various exposure concentrations. Hollow fibre model has an advantage of considering PK and PD parameters; thereby it mimics the in vivo conditions with dynamic concentration of drug over time. The bioreactor module contains thousands of filters with 200 μ in diameter. The peripheral chamber containing the bacteria is separated from the central compartment via semi-permeable membranes, which allows the flow of nutrients and other molecules in and out while retaining the bacteria. These fibres are designed in such a way that the fibre acts as barriers for the flow of contents. Drug concentration is adjusted through infusions at different intervals and by supplying fresh medium to promote dilution of the drug. By adjusting the volume of central reservoir, a state of dynamic concentration of the drug is created, without diluting the bacterial load in the peripheral compartment. Sampling is done from the peripheral compartment at different intervals to quantify the drug concentration and the bacterial count [Figure 5]. This phenomenon provides the reliable PK and PD profiles, which could be considered for clinical decision-making. Two-compartment hollow fibre infection models provide advantages over one-compartment model with respect to the variable concentration of drug exposure over time. Such in vitro PK/PD models are cost-effective and resource intensive. Moreover, it permits the investigation over considerable duration, which is not feasible to perform in animal models. However, this method is technically demanding and requires complex instruments and difficult to standardise.[17]
Figure 5: Hollow fibre infection model for in vitro antimicrobial combination testing

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Critical inhibitory concentration

Determination of critical inhibitory concentration (CIC) was shown to help predict in vivo synergistic effect.[18] For the determination of CIC, a pour plate of media inoculated with the organism is prepared. Ten-millimetre holes are made and filled with combinations of the antimicrobials at different concentration ratios and at graded concentrations [Figure 6]. After incubation for 20 h at 37°C, the distance from the edge of the well till the edge of the zone of inhibition is measured (d). The square of d (d2) was then plotted against the concentration of antibiotic at time zero (logem0). A straight line was obtained intercepting the logem0 axis, and antilog of this point of interception gives the CIC value of the combination. A lower CIC indicates a higher killing effect. Using CIC, Chan et al. demonstrated the synergistic activity for the combination of amikacin and piperacillin at the ratio of 70:30 for P. aeruginosa and was confirmed by TKA and in vivo mouse model.[18]
Figure 6: Scheme for critical inhibitory concentration determination. 'd' is the distance between the edge of the well and the edge of zone of inhibition in mm. 'd2' plotted against concentration of drug gives value of critical inhibitory concentration

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Double disc synergy

Double disc synergy test was conventionally used for the detection of extended-spectrum beta-lactamase (ESBL) production and can also be used for the detection of synergy between antimicrobial combinations. In this method, discs containing the antimicrobials are placed 20 mm (or sum of radii of the zone of inhibition of each drug separately) apart over a lawn culture of the organism and incubated at 37°C. Synergy was indicated by an increase in the zone diameter of ≥2 mm compared to the single agent or bridging of the zone of inhibition [Figure 7].[19],[20] An increase of <2 mm in the zone of inhibition is classified as weak synergy, and antagonism is indicated by truncation of the zone of inhibition at the junction of the two antimicrobials. For P. aeruginosa, this method was shown to give more synergism for a combination of antimicrobials than CB assay.[19] In addition, double disc synergy test was observed to show more synergy than other methods such as agar-based and broth-based dilution method.[21] Despite the simplicity and easy interpretation of results, this method has not been widely used because of its qualitative nature and subjective interpretation.
Figure 7: Double disk synergy test. (a) Synergy (bridging of zone of inhibition); (b) synergy (appearance of zone of inhibition in between agent A and B); (c) antagonism (flattening of zone of inhibition); (d) indifference/additive (no effect on zone of inhibition)

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Paper strip diffusion

In this method, filter paper strips soaked in different antimicrobial solutions at or above MIC were placed at right angles on the MHA plate inoculated with the test organism. Antibiotics in the filter paper strips are allowed to diffuse in the medium and are removed after several hours and the plates are incubated for 18–24 h at 37°C. Alternately, the antibiotic soaked strips can be overlaid onto un-inoculated plate media for 24 h for diffusion and the organism inoculated using a membrane transfer technique [Figure 8].[22] The pattern of growth of the organism was interpreted as follows: indifferent (additive) effect is considered as two oval area of inhibition joining at right angles, synergism is indicated by broadening of the inhibition around the angle and antagonism is indicated by indentation or narrowing around the angle.[23] This method provides qualitative result and has not been widely evaluated.
Figure 8: Paper strip diffusion test, (a) synergy (broadening of zone of inhibition at the angle); (b) synergy (appearance of zone of inhibition at the angle); (c) antagonism (indentation and narrowing of zone of inhibition at the angle); (d) indifference/additive (no effect in the zone of inhibition)

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Multiple-combination bactericidal test

Multiple-combination bactericidal test is done in 96-well microtiter plates. Different combination of antimicrobials with a standard inoculum is added into each well and incubated for 48 h. All the non-turbid wells following incubation is sub-cultured onto antimicrobial-free medium and checked for 99.9% killing. Antagonism is defined as growth of the organism on addition of a second antibiotic to a single agent which was bactericidal when tested alone. Though this method detects the extent of bacterial killing, the outcome is not clearly defined. Enhancement of bactericidal activity of a previous non-bactericidal drug in combination can only be made out in terms of a synergistic combination. Its use is limited to the detection of antagonistic combinations rather than a synergistic combinations for agents used for the treatment of respiratory infection in cystic fibrosis patients.[24],[25]

Overlay inoculum susceptibility disc method

In this method, solid media incorporated with half the MIC of one agent were used as an agar base over which molten antibiotic-free agar with a standard inoculum of the organism is poured to obtain an overlay inoculum layer. Similar control plates are prepared without antibiotic containing base. Antimicrobial discs are placed over the plate and incubated [Figure 9]. An increase in the inhibition zone diameter (IZD) by 19% corresponds to synergy, <19% synergy corresponds to additive effect and no variation in IZD is an indicative of indifference.[26] Nworu and Esimone demonstrated agreement of this technique with CB with both techniques, showing synergistic interaction between ampicillin and ciprofloxacin for Staphylococcus aureus and  Escherichia More Details coli.[27] However, this method has not been widely evaluated.
Figure 9: Overlay inoculum susceptibility disk method for determination of synergy. Increase in inhibition zone diameter >19% indicates synergy

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Serum bactericidal titre

For the better prediction of the PK property of the antimicrobials tested, synergy can be tested using the serum bactericidal titre (SBT) method. This method takes into account not just the drug elimination kinetics but also the protein binding and the effect of metabolic congeners of the antimicrobial agents.[22] Here, serum from patient or volunteer is collected to get the peak and the trough level of the antimicrobial in single doses and in combination. The serum is serially diluted and standard inoculum of the organism inoculated. The highest dilution of the patient serum which results in 99.9% killing is designated as the SBT. The minimum bactericidal concentrations of the antimicrobials are determined in Mueller-Hinton broth and the free drug concentrations (drug-f) in serum are determined. The drug interaction is determined using the formula given below:

Drug-A-f/(MBC-A)(SBT) + Drug-B-f/(MBC-B)(SBT)

A value of ≤0.25 indicates synergy, 0.25–4 indicate additive effect and ≥4 indicate antagonism.

Robinson et al. compared the SBT with in vitro TKA and CB in patients receiving multiple antimicrobial combinations for endocarditis, osteomyelitis or severe septicaemia.[28] Compared to CB assay, SBT detected synergy in 3/10 tests while CB detected synergy in 2/10 tests. One antagonistic combination detected by SBT was determined as synergistic by CB, while two of the synergistic combinations by SBT were determined as antagonistic by CB. TKA detected more number of synergy than either SBT or CB (6/10 at 0.5 × MBC and 5/10 at 1 × MBC). There was no concordance among the three methods when strict definitions are used. However, for four additive combinations tested by SBT, results of synergy or indifference were achieved in the TKA and CB.

Technical difficulties encountered with SBT include difficulty in measuring the drug-f concentration and the need to compare SBT following removal of the antimicrobials from the sample to exclude bactericidal effect due to complement or other inhibitors in the sample.

In vivo models

In vivo studies are essential for the translation of in vitro combination testing data to clinical trials for implementation in the clinical setting. The in vitro methods does not consider the following factors: pharmacokinetics of the antimicrobials in combination, difference in the route of delivery, humoral and cellular immunity of the host, site of involvement, inoculum of the organism at the infected site, virulence factors of the organism and continuous changing concentration of the antimicrobials as single agent and relative to one another (changing ratio of drug concentration).[29] Animal model studies may confirm or contradict in vitro findings based on the PD properties of the antimicrobial agents as well as the host immune response. In addition, in vivo models are necessary to determine the optimum dosing strategy.[30]

To better simulate the in vivo conditions, various experimental models of infection have been used. Synergy between different drug combinations is determined by statistically significant survival rate or organism load reduction in the combination therapy compared to the most active single-drug regimen. However, using these criteria, additive effect cannot be differentiated from a synergistic activity. Fantin and Carbon suggested to define in vivo synergy as 'a significant bactericidal effect of the drug combination in comparison with the sum of the bactericidal effect of each agent alone in comparison with the effect in untreated animal'.[30]

For the mouse pneumonia model, the organisms are inoculated intranasal and kept in hyperoxic condition.[31] For a systemic infection model, organisms are inoculated intraperitoneally in neutropenic mice.[18] Due to ethical and technical considerations, invertebrate models of infections have become an attractive option to study pathogenesis.In vivo models involving larva of Galleria mellonella (wax moth) has been used for the study of antimicrobial efficacy as infection in this model is amenable to treatment.[32] Hornsey and Wareham demonstrated combination of colistin and vancomycin to be highly effective (>90%) in protecting the larva against infection with both a susceptible and a blaOXA-23 producing-resistant strain of A. baumannii which showed synergism in vitro by CB assay.[33] On the other hand, combination of colistin and teicoplanin was more effective in controlling infection by the susceptible strain than the resistant strain. Monotherapy with vancomycin also showed in vivo activity. This has been postulated to be due to ability of vancomycin to enhance the immune response in the larva. O'Hara et al. reported a significant improvement in the survival of larva using combinations of doripenem and vancomycin and triple combination of colistin, doripenem and vancomycin in colistin-resistant A. baumannii infection. In the same experiment, TKA did not demonstrate synergy with doripenem and vancomycin combination.[34] Hornsey et al. also demonstrated the synergistic activity of telavancin and colistin against A. baumannii.[35] In spite of its good turnaround time (96 h), simplicity of procedures and clearly defined endpoints, results obtained in the invertebrate model need to be confirmed in vertebrate model as this model may not reflect the exact mammalian in vivo milieu.

Comparison of different methods of detection of synergy

[Table 1] summarises the relative merits and demerits of the different methods for determination of synergy.
Table 1: Relative merits and demerits of methods of determination of antimicrobial interaction

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[Table 2] gives the comparison of commonly used methods of determination of synergy for A. baumannii, P. aeruginosa and K. pneumoniae. Synergy is detected most often by TKA followed by CB. E-test detected least of synergistic interactions.
Table 2: Comparison of different methods for determination of synergy with different antibiotic combinations for Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter spp.

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Concordance rates between TKA, CB and E-test observed were as follows: 33.3%–100% between CB and TKA; 60%–80.6% between E-test and TKA; 83%–84.4% between CB and E-test; 52%–75% with all three methods. In spite of more conservative interpretation of synergy, Wareham and Wareham have argued that E-test methods may be clinically relevant by giving rapid results of combinations with marked synergy only.[36] Clinical relevance of combinations with only weak synergistic interactions missed by the E-test method needs to be studied further by in vivo milieu to give evidence for the recommendation of E-test for rapid reporting of synergistic combinations.

In spite of the availability of different methods to determine interactions between different antimicrobial agents, lack of standardisation has hampered reliable comparison and compilation of results of different studies. It is also difficult to assess the difference in results due to strain difference, and thus, the reproducibility or clinical efficacy of the combination might vary.


 ~ In Vitro Synergy Of Antimicrobial Combinations In Multidrug-Resistant -Gram-Negative Bacilli Top


Emergence of MDR, carbapenem-resistant organism and PDR GNB has triggered the search for synergistic combinations of antimicrobials in the last decade. A. baumannii, P. aeruginosa and K. pneumoniae are the most commonly studied organisms because of their major role as nosocomial pathogen with frequent drug resistance.

Acinetobacter baumannii

Among commonly studied drug combinations, colistin with either meropenem or rifampicin shows high synergy rates of 96.3% and 94.2% by TKA. Imipenem plus sulbactam/colistin shows moderate rate of synergy (66.6% and 59%, respectively, by TKA). There is a paucity of data to allow adequate comparison of differences among the different carbapenems. In general, all the carbapenems gave a wide range of synergy levels at different combinations which may be accounted by strain difference. Antagonism was noted with combinations sulbactam plus colistin/meropenem; colistin plus meropenem and polymyxin B plus meropenem in few studies. The significance of these observations needs to be further validated by in vivo model testing. [Table 3] summarises in vitro studies done on sulbactam-based combinations and [Table 4] summarises polymyxin-based in vitro studies done for A. baumannii.
Table 3: Sulbactam-based in vitro combination study for Acinetobacter baumannii

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Table 4: Polymyxin based in vitro combination study other than sulbactam for Acinetobacter baumannii

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Pseudomonas aeruginosa

Available data are very limited to give meaningful interpretation of the combinations tested. However, combinations of colistin plus carbapenem and carbapenem plus higher fluoroquinolone such as gatifloxacin and levofloxacin seem promising. Except for the combination of colistin with vancomycin and sulphonamides, antagonism was not seen in any of the combinations. [Table 5] summarises in vitro studies done on antimicrobial combinations in P. aeruginosa.
Table 5: In vitro studies on antimicrobial combinations against Pseudomonas aeruginosa

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Klebsiella pneumoniae

Combinations of polymyxin B/colistin plus rifampicin/meropenem give promising results. It may be noted that antagonism was detected in combinations of colistin plus ertapenem/imipenem and was found to be correlating with the high MIC of colistin. This needs to be studied further with characterisation of the isolates to understand the underlying mechanism. [Table 6] summarises in vitro studies done on antimicrobial combinations in K. pneumoniae.
Table 6: In vitro studies on antimicrobial combinations against Klebsiella pneumoniae

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[Table 7] summarises the most commonly studied combinations of antimicrobials for A. baumannii, P. aeruginosa and K. pneumoniae. Combined rates were calculated from total number of synergy observed in different studies against the total number of isolates studied.
Table 7: Combined synergy by different methods for Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella pneumoniae*

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 ~ Correlation Of Synergism With Other Factors Top


Synergy in relation to minimum inhibitory concentration value

There seems to be some degree of relationship between MIC of the combination of antimicrobials tested against a particular organism. Some studies show more synergy in isolates with higher MIC, while other studies have reported contrary findings [Table 8].
Table 8: Correlation of interaction of antimicrobial agents with minimum inhibitory concentration value*

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For A. baumannii, combinations of sulbactam plus meropenem/doripenem and doripenem plus colistin/tigecycline/amikacin/rifampicin exhibited more synergy with isolates with higher MIC for either sulbactam or doripenem.[37],[38] However, the actual MIC for the agents seems to have an effect on the level of synergy. Lee et al. demonstrated synergism for isolates with moderately high MIC of about 16 μg/ml for sulbactam and 64 μg/ml for meropenem. In contrast, no synergism was noted for isolates with very high MIC of about 128 μg/ml and 256 μg/ml for sulbactam and meropenem, respectively.[39] Combination of colistin and rifampicin also showed synergy for isolates with higher MIC for rifampicin,[8] whereas a combination of ampicillin-sulbactam plus colistin/imipenem and imipenem plus amikacin/colistin/tigecycline showed more synergy with isolates which are colistin susceptible or with lower MIC for imipenem.[40],[41]

For K. pneumoniae, the combination of doripenem plus colistin showed higher synergy with isolates having high colistin MIC, whereas Clancy et al. reported higher synergy with isolates having low doripenem MIC.[42],[43] Combination of colistin and imipenem however showed more synergy in isolates with low colistin MIC, with antagonism detected at high MIC.[44]

The variations observed between the tests may be due to the difference in the strain, methodology and geographical area. In particular, the mechanisms of resistance in these isolates were not fully characterised. The question of presence of synergism or antagonism as a function of MIC value for each agent needs to be investigated further to use MIC as a predictor for success of combination therapy. Henceforth, studies must be carried out to decipher the MIC value of individual agents, which is likely to yield synergism or antagonism for a particular combination.

Synergism as a correlate of molecular mechanism of resistance

Another aspect of synergy testing in resistant isolates is its correlation with a particular resistance mechanism involved. [Table 9] gives the correlation of synergy with antimicrobial combinations. Very few studies have further investigated the resistance mechanism for the study isolates, for which combination testing has been done. In case of K. pneumoniae, studies have reported the role of porin channels in determining synergism of the combinations being tested. Similarly, K. pneumoniae-producing blaNDM carbapenemase alone showed significantly more synergy than isolates producing blaOXA-48-like carbapenemases.[78] Such correlations with the specific resistance mechanism involved might help predict synergism for a particular combination of antimicrobials for treatment. Thus, determining molecular mechanisms would help direct combination therapy to improve therapeutic success.[45]
Table 9: Correlation of molecular mechanisms of resistance with the result of in vitro antimicrobial interaction study

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 ~ In Vitro Synergy As A Predictor Of Clinical Response Top


The likelihood of the findings of the in vitro synergy studies to be translated into clinical efficacy still remains debatable. The classical example of in vitro synergy between aminoglycoside and beta-lactam agents has not stood the test of time. Studies have reported no clinical benefit of combination of beta-lactam plus aminoglycoside combination for Gram-negative infection either in the neutropenic or in the non-neutropenic host.[46],[47] Combination therapy may result in adverse effects of nephrotoxicity.

Despite issues of toxicity, combination therapy is the only strategy available for treating infections due to PDR organisms. Very few studies have documented the clinical outcome with combination therapy supported by in vitro synergy. Biancofiore et al. reported successful treatment of multifocal infection of MDR A. baumannii in a 16-year-old female with a combination of colistin, rifampicin and meropenem after synergism between the combinations was proved by CB assay.[48] Lee et al. reported favourable outcome with carbapenem and sulbactam combination in four patients (two patients with VAP and two catheter-related bloodstream infection) caused by A. baumannii. In vitro synergy testing of all four isolates by CB assay showed partial synergy with FIC index ranging from 0.56 to 0.75 for combination of sulbactam and meropenem/imipenem.[49] In one patient with post-neurosurgery bacteraemic meningitis due to CR A. baumannii (CRAB), combination of intravenous meropenem and sulbactam leads to reduction in the colony count in cerebrospinal fluid (CSF) from >50,000 CFU/ml to 10,000 CFU/ml in 4 days.[39] Addition of intravenous and intrathecal colistin resulted in clearance of the organism within 2 days both from CSF and blood. The SBT and CSF bactericidal titre increased from fourfold to 32-fold with the three-drug combination compared to two-drug regimen.In vitro TKA showed synergism with combination of colistin with meropenem, sulbactam or both. TKA with colistin alone and meropenem plus colistin showed re-growth at 24 h. Though the infection was cleared, the patient expired due to hypoxia secondary to respiratory distress. Nastro et al. reported successful treatment for cases with sepsis (n = 1), meningitis (n = 1) and UTI (n = 1) with a combination of colistin and rifampicin against carbapenemase producing GNB.[50] The combination was found to be synergistic for all the isolates by the E-test-agar method. [Table 10] summarises the studies that have correlated in vitro synergy with clinical response.
Table 10: Correlation of in vitro synergy with clinical response

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 ~ Conclusion Top


Combination therapy has gained attention due to increased efficacy and scope for decreasing the toxicity and development of resistance especially against drug-resistant strains. Therefore, it is imperative to investigate the antimicrobials that have to be used in combination for the clinical utility. At present, very few agents are available for treating infections due to PDR pathogens, and combination therapy is found to be the effective strategy to tackle this. Several methods exist for the assessment of synergistic activity of two or more antimicrobial agents. However, wide variation was observed in terms of their technical issues, complexity and interpretation of test results. This signifies the need for global-level standardisation of the various methods for the determination of synergy of antimicrobial combinations. At present, TKA is the reference method which yields considerable level of concordance rate among the various studies. To conclude, majority of the in vitro test methods could not predict the clinical success rates. Therefore, prospective clinical trials with in vitro synergy testing data are needed to improve the clinical outcome.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8], [Table 9], [Table 10]



 

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2004 - Indian Journal of Medical Microbiology
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