Theory of antimicrobial combinations: biocide mixtures - synergy or addition?

AIMS: To demonstrate the effect that non-linear dose responses have on the appearance of synergy in mixtures of antimicrobials. METHODS AND RESULTS: A mathematical model, which allows the prediction of the efficacy of mixtures of antimicrobials with non-linear dose responses, was produced. The efficacy of antimicrobial mixtures that would be classified as synergistic by time-kill methodology was shown to be a natural consequence of combining antimicrobials with non-linear dose responses. CONCLUSIONS: The effectiveness of admixtures of biocides and other antimicrobials with non-linear dose responses can be predicted. If the dose response (or dilution coefficient) of any biocidal component, in a mixture, is other than one, then the time-kill methodology used to ascertain the existence of synergy in antimicrobial combinations is flawed. SIGNIFICANCE AND IMPACT OF THE STUDY: The kinetic model developed allows the prediction of the efficacy of antimicrobial combinations. Combinations of known antimicrobials, which reduce the time taken to achieve a specified level of microbial inactivation, can be easily assessed once the kinetic profile of each component has been obtained. Most patented cases of antimicrobial synergy have not taken into account the possible effect of non-linear dose responses of the component materials. That much of the earlier literature can now be predicted, suggests that future cases will require more thorough proof of the alleged synergy.     

Membrane damage to bacteria caused by single and combined biocides

AIMS: To examine the effect on the leakage of low molecular weight cytoplasmic constituents from Staphylococcus aureus using phenolics singly and in combination, and to see if the observations could be modelled using a non-linear dose response. METHODS AND RESULTS: The rate of potassium, phosphate and adenosine triphosphate leakage was examined in the presence of chlorocresol and m-cresol. Individually, leakage was observed only at long contact times or high concentrations. Combined at these ineffective concentrations, the cytoplasmic pool of all constituents studied was released within minutes. Both chlorocresol and m-cresol were shown to have non-linear dose responses. A rate model for the combinations, which takes account of these non-linear responses, accurately predicted the observations. CONCLUSIONS: Antimicrobials, which when used alone exhibit a non-linear dose response, will also give a non-linear dose response in combination. The simple linear-additive model ignores the concept of the dilution coefficient and will always describe the phenomenon of synergy for combinations where one or more of the components has a dilution coefficient greater than one. This has been borne out by examination of the purported prime lesion of chlorocresol and m-cresol, alone and in combination. SIGNIFICANCE AND IMPACT OF THE STUDY: Studies aimed at producing synergistic mixtures of antimicrobials, which ignore the non-linear additive effect, may waste valuable research effort looking for a physiological explanation for an apparent synergy, where none, in-fact, exists. Patents granted on the basis of analyses using the linear-additive model for combinations of compounds with non-linear dose responses may no longer be supportable.     

The synergistic effect of EDTA/antimicrobial combinations on Pseudomonas aeruginosa

AIMS: To demonstrate that the nonlinear concentration-dependent inhibition of Pseudomonas aeruginosa to EDTA can be used to successfully model and predict the potentiation of antimicrobials by EDTA. METHODS AND RESULTS: A model used successfully to describe the concentration-dependent inhibition of bacterial growth caused by many antimicrobials was unable to describe the inhibition of P. aeruginosa by EDTA. Examination of the inhibition profiles for EDTA against P. aeruginosa revealed a biphasic inhibitory pattern suggesting different mechanisms of action at different concentrations. A modelled, two-stage inhibitory process was shown to fit the observations. This model was then used to examine the effect of combining EDTA with other antimicrobials. The apparent synergy of mixtures of EDTA with quaternary ammonium surfactants (QAC) and specific antibiotics was successfully modelled. Minimum inhibitory concentrations (MIC) of the QAC and that of oxacillin and cefamandole were reduced by a factor of 3-10, whereas ampicillin was reduced by a factor of 70 from an MIC of 1524 to 21 mg l(-1) in the presence of 500 mg l(-1) of EDTA. CONCLUSIONS: A nonlinear concentration-dependent inhibition of P. aeruginosa by EDTA gives rise to apparent observation of synergy with other antimicrobials. SIGNIFICANCE AND IMPACT OF THE STUDY: This is a further example where the current methodology for the examination of antimicrobial synergy (the summed fractional inhibitory concentrations) leads to false conclusions.     

Assessing Toxicity of Mixtures: The Search for Economical Study Designs

Toxicity screening and testing of chemical mixtures for interaction effects is a potentially onerous task due to the sheer volume of combinations that may be of interest. We propose an economical approach for assessing the interaction effects of chemical mixtures that is guided by risk-based considerations. We describe the statistical underpinnings of the approach and use examples from the published literature to illustrate concepts of local versus global mixture assessment. Our approach employs a sequential testing procedure to find the dose combinations that define the dose boundary for a specified acceptable risk level. The first test is conducted for a dose combination consisting of the acceptable doses of each individual chemical in the mixture. The outcome of this first test indicates the dose combination that should be tested next. Continuing in this manner, the boundary of dose combinations for the specified acceptable risk level can be approximated based on measurements for relatively few dose combinations. Dose combinations on one side of the boundary would have responses less than the response associated with the acceptable risk level, and dose combinations on the boundary would be acceptable levels of exposure for the mixture.     

A generalized response surface model with varying relative potency for assessing drug interaction

When multiple drugs are administered simultaneously, investigators are often interested in assessing whether the drug combinations are synergistic, additive, or antagonistic. Based on the Loewe additivity reference model, many existing response surface models require constant relative potency and some of them use a single parameter to capture synergy, additivity, or antagonism. However, the assumption of constant relative potency is too restrictive, and these models using a single parameter to capture drug interaction are inadequate to describe the phenomenon when synergy, additivity, and antagonism are interspersed in different regions of drug combinations. We propose a generalized response surface model with a function of doses instead of one single parameter to identify and quantify departure from additivity. The proposed model can incorporate varying relative potencies among multiple drugs as well. Examples and simulations are given to demonstrate that the proposed model is effective in capturing different patterns of drug interaction.     

Susceptibility of food-borne bacteria to binary combinations of antimicrobials at selected a(w) and pH

AIM: To evaluate the antibacterial susceptibilities of food-borne bacteria to individual and binary mixtures of a synthetic antimicrobial agent with a natural phenolic compound. METHODS AND RESULTS: Antibacterial susceptibilities of Escherichia coli, Listeria innocua, Salmonella Typhimurium and Staphylococcus aureus to individual and binary mixtures of potassium sorbate with a phenolic compound (thymol, carvacrol, or eugenol) were evaluated, at selected water activity (a(w); 0.99 or 0.97) and pH (5.5 or 4.5). The bacteria studied were susceptible to the action of the antimicrobials individually with minimal inhibitory concentrations that varied from 800-ppm potassium sorbate for Staph. aureus at a(w) 0.99, and pH 5.5 to 100-ppm thymol or carvacrol for the four studied bacteria at a(w) 0.97 and pH 4.5. Several binary mixtures of potassium sorbate with thymol, carvacrol or eugenol inhibited bacterial growth. Antimicrobial agent inhibitory concentrations in the mixture varied among bacteria, additionally depending on the a(w) and the pH tested. CONCLUSIONS: Synergistic binary mixtures with fractional inhibitory concentration index <0.6 include 100- or 200-ppm potassium sorbate with 50- or 100-ppm thymol, carvacrol or eugenol. SIGNIFICANCE AND IMPACT OF THE STUDY: The synergistic combinations could be useful in reducing the amounts of antimicrobials needed to inhibit growth, thus diminishing consumer concerns regarding chemical preservatives.     

Transmission of Salmonella enterica serotype Typhimurium in poultry with and without antimicrobial selective pressure

AIMS: To determine the effect of antimicrobial selective pressure on the transmission of antimicrobial resistant and sensitive strains of Salmonella in poultry. METHODS AND RESULTS: Eight pens housed 12 broiler chicks each. Two chicks in four of the pens were inoculated with a Salm. Typhimurium strain resistant to 12 antimicrobials (including tetracycline), and two chicks in each of the four other pens were inoculated with a strain sensitive to all antimicrobials tested. Two pens inoculated with each strain were treated with chlortetracycline and two were not. Chicks were killed on day 7 and caeca were cultured for Salmonella. Experiments were performed independently twice. Chicks exposed to pen mates inoculated with the resistant strain and treated with tetracycline were 90% positive for Salmonella; whereas 60% of chicks given no antimicrobials were positive. Chicks exposed to the sensitive strain were 95% positive with tetracycline treatment and 90% positive without treatment. CONCLUSIONS: A multidrug-resistant Salm. Typhimurium strain had significantly increased transmission when chicks were treated with tetracycline. Transmission of a sensitive strain was not inhibited by antimicrobial selective pressure at recommended therapeutic dose. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates that antimicrobial usage may influence the transmission of antimicrobial-resistant pathogens in poultry.     

Generalized concentration addition: A method for examining mixtures containing partial agonists

Environmentally relevant toxic exposures often consist of simultaneous exposure to multiple agents. Methods to predict the expected outcome of such combinations are critical both to risk assessment and to an accurate judgment of whether combinations are synergistic or antagonistic. Concentration addition (CA) has commonly been used to assess the presence of synergy or antagonism in combinations of similarly acting chemicals, and to predict effects of combinations of such agents. CA has the advantage of clear graphical interpretation: Curves of constant joint effect (isoboles) must be negatively sloped straight lines if the mixture is concentration additive. However, CA cannot be directly used to assess combinations that include partial agonists, although such agents are of considerable interest. Here, we propose a natural extension of CA to a functional form that may be applied to mixtures including full agonists and partial agonists. This extended definition, for which we suggest the term “generalized concentration addition,” encompasses linear isoboles with slopes of any sign. We apply this approach to the simple example of agents with dose-response relationships described by Hill functions with slope parameter n=1. The resulting isoboles are in all cases linear, with negative, zero and positive slopes. Using simple mechanistic models of ligand-receptor systems, we show that the same isobole pattern and joint effects are generated by modeled combinations of full and partial agonists. Special cases include combinations of two full agonists and a full agonist plus a competitive antagonist.     

Discovering Anti-platelet Drug Combinations with an Integrated Model of Activator-Inhibitor Relationships,Activator-Activator Synergies and Inhibitor-Inhibitor Synergies

Identifying effective therapeutic drug combinations that modulate complex signaling pathways in platelets is central to the advancement of effective anti-thrombotic therapies. However, there is no systems model of the platelet that predicts responses to different inhibitor combinations. We developed an approach which goes beyond current inhibitor-inhibitor combination screening to efficiently consider other signaling aspects that may give insights into the behaviour of the platelet as a system. We investigated combinations of platelet inhibitors and activators. We evaluated three distinct strands of information, namely: activator-inhibitor combination screens (testing a panel of inhibitors against a panel of activators); inhibitor-inhibitor synergy screens; and activator-activator synergy screens. We demonstrated how these analyses may be efficiently performed, both experimentally and computationally, to identify particular combinations of most interest. Robust tests of activator-activator synergy and of inhibitor-inhibitor synergy required combinations to show significant excesses over the double doses of each component. Modeling identified multiple effects of an inhibitor of the P2Y12 ADP receptor, and complementarity between inhibitor-inhibitor synergy effects and activator-inhibitor combination effects. This approach accelerates the mapping of combination effects of compounds to develop combinations that may be therapeutically beneficial. We integrated the three information sources into a unified model that predicted the benefits of a triple drug combination targeting ADP, thromboxane and thrombin signaling.     

Quality of Antimicrobial Products Used in Striped Catfish (Pangasianodon hypophthalmus) Aquaculture in Vietnam

Antimicrobial usage is common in Asian aquaculture. This study aimed to determine the quality of antimicrobial products used by Vietnamese striped catfish (Pangasianodon hypophthalmus) farmers. Twenty one antimicrobial products (11 products contained a single antimicrobial and 10 products contained a mixture of two different antimicrobials) commonly used by catfish farmers were obtained from so-called chemical shops located in the Mekong Delta, Vietnam. Ultra High Performance Liquid Chromatography Mass Spectrometry was used to analyze concentration of sulfonamides, trimethoprim, amoxicillin, cefalexin and ciprofloxacin whereas concentrations of florfenicol and doxycycline were analyzed by High Performance Liquid Chromatography with UV detection. Results revealed that only 4/11 products with a single antimicrobial and 2/10 products with a mixture of antimicrobials contained active substances within ±10% of the concentration declared on the product label. Two products with antimicrobial mixtures did not contain any of the declared antimicrobials. Comparing two batches, analysis of 11 products revealed that only one product contained a concentration of active compound that varied with less than 10% in both batches. Several product labels provided inadequate information on how to calculate therapeutic dosage and further stated withdrawal time despite lack of pharmacokinetic data on the antimicrobials in catfish. There is an urgent need to strengthen approval procedures and in particular regularly to monitor the quality of antimicrobials used in Vietnamese aquaculture.     

Enhanced inactivation of Listeria monocytogenes by nisin in the presence of ethanol

AIMS: The effect of combinations of nisin and ethanol on the survival of Listeria monocytogenes was investigated. METHODS AND RESULTS: Killing by nisin was enhanced during simultaneous exposure to ethanol (2-7% v/v). For example, while 10 IU ml(-1) nisin reduced viability by 1 log unit in 20 min, a combination of this antimicrobial peptide and 5% ethanol, reduced numbers of surviving cells by 3 log units. Increasing the concentrations of either ethanol (2-7%) or nisin (10-50 IU ml(-1)) led to increased cell death with synergy being demonstrated for all combinations tested and at a range of temperatures from 5 to 37 degrees C. CONCLUSIONS: Ethanol can act synergistically with nisin to reduce the survival of L. monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: Combinations of ethanol and nisin may be feasible as an effective way of controlling this pathogen in the food processing environment.     

Determination of Antioxidant Activity of Various Bioantioxidants and Their Mixtures by the Amperometric Method

To date, simple and operative electrochemical methods are widely used to study antioxidant properties of various biological objects. One of them is the amperometric method, which is used in various fields of science, technology and medicine to determine the antioxidant activity (AOA) of various beverages, extracts and biological fluids, as well as the total content of antioxidants (AOs) present in them. Measurements of AOA (electrochemical oxidability) of some known individual AO and their mixtures by the amperometric method was performed in the present work. The coefficients of their oxidability were determined. The AOA values obtained correlate well with the AOA of these compounds measured by other methods. The coincidence of the experimental values of AOA and the values calculated with the use of the coefficients of oxidability for each component of the mixture was observed for most combinations of binary mixtures of used AOs. This indicates the absence of interaction between them (synergism or antagonism) during the oxidation. Several mixtures with a lower value of the measured AOA compared with the calculated value (antagonism) represented the exception. During the electrochemical oxidation of the “oxidized glutathione + ascorbic acid” mixture, partial reduction of glutathione by ascorbic acid is likely to proceed, which results in an excess of the measured AOA value over the calculated value. The results obtained may be useful when working with devices based on the amperometric method.     

Concentration Addition,Independent Action and Generalized Concentration Addition Models for Mixture Effect Prediction of Sex Hormone Synthesis In Vitro

Humans are concomitantly exposed to numerous chemicals. An infinite number of combinations and doses thereof can be imagined. For toxicological risk assessment the mathematical prediction of mixture effects, using knowledge on single chemicals, is therefore desirable. We investigated pros and cons of the concentration addition (CA), independent action (IA) and generalized concentration addition (GCA) models. First we measured effects of single chemicals and mixtures thereof on steroid synthesis in H295R cells. Then single chemical data were applied to the models; predictions of mixture effects were calculated and compared to the experimental mixture data. Mixture 1 contained environmental chemicals adjusted in ratio according to human exposure levels. Mixture 2 was a potency adjusted mixture containing five pesticides. Prediction of testosterone effects coincided with the experimental Mixture 1 data. In contrast, antagonism was observed for effects of Mixture 2 on this hormone. The mixtures contained chemicals exerting only limited maximal effects. This hampered prediction by the CA and IA models, whereas the GCA model could be used to predict a full dose response curve. Regarding effects on progesterone and estradiol, some chemicals were having stimulatory effects whereas others had inhibitory effects. The three models were not applicable in this situation and no predictions could be performed. Finally, the expected contributions of single chemicals to the mixture effects were calculated. Prochloraz was the predominant but not sole driver of the mixtures, suggesting that one chemical alone was not responsible for the mixture effects. In conclusion, the GCA model seemed to be superior to the CA and IA models for the prediction of testosterone effects. A situation with chemicals exerting opposing effects, for which the models could not be applied, was identified. In addition, the data indicate that in non-potency adjusted mixtures the effects cannot always be accounted for by single chemicals.     

Mineral oil and aliphatic alcohols: toxicity and analysis of synergistic effects on German cockroaches (Dictyoptera: Blattellidae)

Two mineral oils and 12 linear primary alcohols were studied, alone and in combination, to determine their contact toxicity to adult German cockroaches, Blattella germanica (L.) (Dictyoptera: Blattellidae). The more toxic oil, PD23 (LD50 = 1.45 mg per cockroach) was used for combination studies. Alcohols with carbon chain lengths of C3 and C8 through C12 were the most toxic, with LD50 values ranging from 0.3 to 0.6 mg. C1 (methanol) and C14 (1-tetradecanol) were least toxic, with LD50 values of 2.35 and 1.75 mg, respectively. Eight of the 12 combinations of a nonlethal dose of PD23 oil with an LD10 dose of alcohol produced significantly greater mortality than predicted under the assumption of additive effects. A sample of five synergistic oil + alcohol combinations, covering most of the alcohol carbon chain length range over which synergy occurred, was further studied by calculating LD50 values for three fixed mixture ratios (80:20, 50:50, and 20:80) of each combination. Results were analyzed using both graphical techniques (isobole analysis) and by nonlinear regression. At least one, but not necessarily all, of the three fixed ratio combinations of each oil + alcohol pairing indicated synergy. The conclusions drawn from the isobole and regression analyses were consistent.     

A Response-Additive Model for Analyzing Mixtures of Two Chemicals in the Salmonella Reversion Assay

A statistical model is proposed for analyzing the mutagenic responses produced by mixtures of two chemicals in the Salmonella reversion assay. This model is based on the simplex-lattice design for mixtures with the total concentration fixed. The dose-response relationship is expressed as a function of both the proportions of the two chemicals and the total concentration of the chemicals in the mixture. If the joint action of two chemicals can be predicted by response-additivity, then the response of the mixture at the total concentration T can be represented by the weighted average of the responses produced by the individual chemicals at the same concentration T with the weights for individual responses being equal to the proportions of the chemicals in the mixture. Two non-additive models, synergism and antagonism, are discussed. An example is illustrated by analyzing the joint mutagenic effects of 1-nitrobenzo(a)pyrene (1-NBP), and 3-NBP in the Salmonella reversion assay.     

Synergistic effects of short peptides and antibiotics against bacterial and fungal strains

There is a rising tide of concern about the antibiotic resistance issue. To reduce the possibility of antibiotic-resistant infections, a new generation of antimicrobials must be developed. Antimicrobial peptides are potential alternatives to antibiotics that can be used alone or together with conventional antibiotics to combat antimicrobial resistance. In this work, lead compounds LP-23, DP-23, SA4, and SPO from previously published studies were synthesized by solid-phase peptide synthesis and their antimicrobial evaluation was carried out against various bacterial and fungal strains. Peptide combinations with antibiotics were evaluated by using the checkerboard method and their minimal inhibitory concentration (MIC) in combination was calculated by using the fractional inhibitory concentration (FIC) index. Cytotoxicity evaluations of these peptides further confirmed their selectivity toward microbial cells. Based on the FIC values, LP-23, DP-23, and SPO demonstrated synergy in combination with gentamicin against a gentamicin-resistant clinical isolate of Escherichia coli. For Staphylococcus aureus, Escherichia coli, and Salmonella typhimurium, seven combinations exhibited synergistic effects between peptide/peptoids and the tested antibiotics. Additionally, almost all the combinations of peptides/peptoids with amphotericin B and fluconazole also showed effective synergy against Aspergillus niger and Aspergillus flavus. The synergy found between LP-23, DP-23, SA4, and SPO with the selected antibiotics may have the potential to be used as a combination therapy against various microbial infections.     

Nonlinearity in optical resolution via distillation applying mixtures of resolving agents

During an optical resolution it is the resolving agent that has the strongest influence on the outcome of the process. Applying a mixture of resolving agents can result either in antagonism or in synergy. We found that using mixtures of tartaric acid and its derivatives chiral selectivity is at least the same, but in several cases markedly better (synergistic effect), than the sum of the effect of the individual resolving agents. Thus, the "Dutch method," reported for the crystallization method, also works for distillation. A calculation method is applied for measuring the synergistic effect. Interestingly, an individually inactive resolving agent can be a useful contributor to the mixture of the resolving agents.     

Effect of selected natural antimicrobials on Baker''s yeast activity

AIMS: To evaluate the responses of Baker's yeast (Saccharomyces cerevisiae) activity to the natural antimicrobials acetic acid, calcium lactate, a lactate-containing cocktail and lactic acid compared to calcium propionate. METHODS AND RESULTS: A dough fermentometer test was used to measure Baker's yeast activity in the presence of these natural antimicrobials and calcium propionate. Yeast activity generally decreased as a function of increasing antimicrobial concentrations, but the lactate-containing cocktail showed no relationship between concentration and yeast activity reduction. At in situ concentrations, calcium propionate resulted in the highest yeast activity reduction, followed by calcium lactate, acetic acid, the lactate-containing cocktail and lactic acid in decreasing order. CONCLUSION: Based on yeast activity reduction, all natural antimicrobials tested showed potential as possible replacements for calcium propionate. SIGNIFICANCE AND IMPACT OF THE STUDY: This has practical implications since calcium propionate inhibits Baker's yeast activity and attracts negative consumer perceptions as a chemical bread preservative.     

Activity of natural antimicrobial compounds against Escherichia coli and Salmonella enterica serovar Typhimurium

AIMS: The objective of this study was to evaluate the inhibitory activity of several natural organic compounds alone or in combination with nisin against Escherichia coli and Salmonella Typhimurium. METHODS AND RESULTS: The minimum inhibitory concentration (MIC) of five natural organic compounds were determined, and the effect of their combinations with nisin was evaluated by the checkerboard assay using the Bioscreen C. As expected, nisin by itself showed no inhibition against either of the Gram-negative bacteria. Thymol was found to be the most effective with the lowest MIC values of 1.0 and 1.2 mmol 1-1 against Salm. Typhimurium and E. coli, respectively. After thymol, the antimicrobial order of the natural organic compounds was carvacrol > eugenol > cinnamic acid > diacetyl. However, the combination of nisin with the natural organic compounds did not result in the enhancement of their antimicrobial activities. On the contrary, combination of nisin with diacetyl against Salm. Typhimurium resulted in an antagonism of diacetyl activity. CONCLUSIONS: While the individual natural organic compounds showed inhibitory activity against the two Gram-negatives, their combinations with nisin showed no improvement of antimicrobial activity. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows the potential of the natural organic compounds to control E. coli and Salm. Typhimurium.     

Modulation of anti-pathogenic activity in canine-derived Lactobacillus species by carbohydrate growth substrate

AIMS: To investigate the effect of various carbon sources on the production of extracellular antagonistic compounds against two Escherichia coli strains and Salmonella enterica serotype Typhimurium by three canine-derived lactobacilli strains. METHODS AND MATERIALS: Cell-free preparations, pH neutralized, were used in antibiotic disc experiments as an initial screening. The bacteria/carbohydrate combinations that showed inhibition of the growth of those pathogens, were further investigated in batch co-culture experiments. The cell-free supernatants of the cultures, that decreased the population number of the pathogens in the co-culture experiments to log CFU ml-1 相似文献