Antibacterial activity of Ionic Liquids based on ampicillin against resistant bacteria

Abstract

Antibacterial activities of novel Active Pharmaceutical Ingredient Ionic Liquids (API–ILs) based on ampicillin anion [Amp] have been evaluated. They showed growth inhibition and bactericidal properties on some sensitive bacteria and especially some Gram-negative resistant bacteria when compared to the [Na][Amp] and the initial bromide and chloride salts. For these studies were analysed the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBIC) against sensitive Gram-negative bacteria Escherichia coli ATCC 25922 and Klebsiella pneumoniae (clinically isolated), as well as sensitive Gram positive Staphylococcus Aureus ATCC 25923, Staphylococcus epidermidis and Enterococcus faecalis and completed using clinically isolated resistent strains such as E. coli TEM CTX M9, E. coli CTX M2 and E. coli AmpC MOX. From the obtained MIC values of studied API–ILs and standard [Na][Amp] were derived RDIC values (relative decrease of inhibitory concentration). High RDIC values of [C₁₆Pyr][Amp] especially against two resistant Gram-negative strains E. coli TEM CTX M9 (RDIC >1000) and E. coli CTX M2 (RDIC >100) point clearly to a potential promising role of API–ILs as antimicrobial drugs in particular against resistant bacterial strains.

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Introduction

Ionic liquids (ILs) are usually defined as organic salts with melting points lower than 100 °C (several of them are liquid at room temperature).¹ They became popular due to the large number of possible cation/anion combinations allowing different tunable interactions and potential applications. Some IL properties such as their high thermal and chemical stability, negligible vapor pressure,² high ionic conductivity, lack of inflammability and adjustable solubility have attracted numerous applications across an extensive variety of research areas³ in particular related with organic chemistry, chemical engineering, material science, physical chemistry, analytical chemistry and biotechnology, among others.

One of the most promising applications of ILs seems to be the so-called third generation of ILs (their arrangement with active pharmaceutical ingredients, APIs) or API–ILs.⁴ It is suggested that these compounds can solve problems associated to pharmaceutical industry related with polymorphism and drug solubility.⁴ At the beginning, the uses of ILs in the biosciences were difficult because of ILs toxicity,⁵ however more recently it was shown lower inherent toxicity and thus a lower impact on human health and the environment from some hydrophobic ILs derived from toxic herbicides.⁵ Presently biocidal properties of large cations, such as benzalkonium and imidazolium species, are also largely used as an advantage, to kill or inhibit bacteria⁶ or yeast⁷ growth. In respect to this area, different publications reported antimicrobial activity studies using microorganisms or cell culture for long alkyl chain quaternary ammonium.⁸ Recently, Cole and co-workers⁹ reported the use of metathesis reaction to produce ILs with long alkyl chain quaternary ammonium and ampicillin anion. Some ILs have been efficiently tested with clinically significant microbial pathogens, including Methicillin-resistant Staphylococcus aureus (MRSA).¹⁰

Bacterial resistance to different antibiotics that are commercially available is one of the major public health problems.¹¹ Recent outbreaks of E. coli O104 (ref. 12) in Germany as well as the emergence of multi-drug resistant organisms such as Gram-negative Enterobacteriaceae associated to the New Delhi metallo β-lactamase¹³ confirm that this is a remarkable problem for public health, but also at an economic and social point of view. Besides the fact that bacterial resistance increases the mortality and the morbidity some recent publications have reported the financial burden of health care-associated infections (HAIs) in the USA.¹⁴

Innovative therapies involving the use of ILs as a drug delivery platform suggest others attractive opportunities for exploration. The possibility to eliminate or reduce the negative side effects of a given active compound by delivering it as an API–IL is extremely attractive for pharmaceutical, environmental technologies and other medical applications. It is suggested that the appropriate combination of counter ion and the specific drug as API–IL can influence the final biopharmaceutical drug classification (BCS), toxicity and biodegradability behavior,^7,15 water solubility, permeability as well as their drug formulation process and can change some biological properties.

As mentioned before the bacterial resistance to antibiotics is a serious public health threat and needs novel and urgent actions. In this context, we studied the bacterial activity of different API–ILs based on ampicillin¹⁰ against a panel of sensitive Gram-negative and Gram-positive bacteria as well as a panel of resistant bacteria were clinically isolated.^11,16 In particular, sensitive Gram-negative bacteria Escherichia coli ATCC 25922 and Klebsiella pneumoniae (clinically isolated); the sensitive Gram positive S. aureus ATCC 25923, Staphylococcus epidermidis (clinically isolated) and Enterococcus faecalis (clinically isolated) and the resistant bacteria (clinically isolated strains) E. coli TEM CTX M9, E. coli CTX M2, E. coli AmpC MOX2 were selected.

Results and discussion

In this work, API–ILs or pharmaceutical organic salts were produced by the neutralization method^4c from ampicillin. The toxic effect of cationic counter ion of API–ILs should not be neglected.¹⁷ All prepared ampicillin based ILs are considered non-toxic, except in the case of [P_6,6,6,14] cation which is toxic according to standard toxicity test against human colon carcinoma cell line (CaCo-2).¹⁸ Contrarily, choline cation ([cholin]⁺) is used as essential nutrient; low molecular substituted ammonium or imidazolium cations ([TEA]⁺ or [C_nMIM]⁺) as alternative low-toxic cations and cetylpyridinium cation ([C₁₆Pyr]⁺) already applied in pharmaceutical applications.¹⁹ For comparative studies, the activity tests were also performed using starting materials (bromide and chloride salts) while ampicillin sodium salt was always used as standard API. The purity of compounds was checked by ¹H and ¹³C NMR and mass spectra analysis. Fig. 1 summarizes all prepared API–ILs based on ampicillin anion ([Amp]).

Fig. 1 Representation of the prepared API–ILs based on ampicillin anion.

Table 1 shows the MICs for the API–ILs based on ampicillin sensitive bacteria and Table 2 presents the MICs for resistant strains. The analysis from Table 1 suggests that in case of sensitive bacteria, the [C₁₆Pyr][Amp] exhibits the highest activity and significantly lower MIC values when compared to [Na][Amp]. In the case of K. pneumoniae, S. epidermidis and E. faecalis suggests that this could be a promising antibacterial compound for this kind of bacteria.

Table 1 Minimum inhibitory concentrations (mM) on the ampicillin sensitive bacterial strains tested

Comp.	Strains
	Gram-negative		Gram-positive
	E. coli ATCC 25922	K. pneumoniae	S. aureus ATCC 25923	E. faecalis	S. epidermidis
a The [Na][Amp] was used as control.
[Na][Amp]^a	0.05	2.5	0.005	0.05	0.05
[TEA][Amp]	>5	>5	>5	>5	>5
[TEA][Br]	>5	>5	2.5	>5	>5
[P6,6,6,14][Amp]	2.5	5	0.05	0.05	0.05
[P6,6,6,14][Cl]	2.5	2.5	2.5	>5	2.5
[C16Pyr][Amp]	0.5	0.05	0.005	0.005	0.005
[C16Pyr][Cl]	0.5	2.5	0.5	0.5	2.5
[cholin][Amp]	>5	>5	>5	>5	>5
[cholin][Cl]	>5	>5	2.5	>5	>5
[EMIM][Amp]	>5	>5	>5	>5	>5
[EMIM][Br]	>5	>5	0.05	>5	5
[C2OHMIM][Amp]	5	>5	>5	5	2.5
[C2OHMIM][Cl]	5	>5	>5	5	5

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Table 2 Minimum inhibitory concentrations (mM) on the ampicillin resistant bacterial strains tested

Comp.	Strains
	E. coli TEM CTX M9	E. coli CTX M2	E. coli AmpC MOX2
a The [Na][Amp] was used as control.
[Na][Amp]^a	>5	>5	>5
[TEA][Amp]	>5	>5	>5
[TEA][Br]	>5	2.5	>5
[P6,6,6,14][Amp]	0.5	0.5	>5
[P6,6,6,14][Cl]	2.5	5	>5
[C16Pyr][Amp]	0.005	0.05	>5
[C16Pyr][Cl]	0.5	>5	>5
[cholin][Amp]	>5	>5	>5
[cholin][Cl]	>5	>5	>5
[EMIM][Amp]	>5	>5	>5
[EMIM][Br]	5	>5	>5
[C2OHMIM][Amp]	>5	>5	5
[C2OHMIM][Cl]	>5	2.5	5

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Some toxicity studies indicated that the least toxic anions are Cl and BF₄ (ref. 20) and a possible relation between toxicity and the length of alkyl chains in the case of imidazolium, pyridinium and quaternary ammonium cations to several microorganisms such as rods, cocci and fungi.²¹

In our work, the combination effect of ampicillin anion (API) with selected organic cation has been evaluated. Also, the cation alone contribution or synergetic effect from cation/anion were checked by measuring MIC values of [Cat][Cl] and [Cat][Br] (shown in Tables 1 and 2) that were used in synthesis of [Cat][Amp].^4c

The results showed that the toxic effect of the chloride and bromide is almost negligible in diluted solutions as already described in the literature.^20,22 With the exception of [C₂OHMIM][Cl] providing lower MIC value against resistant E. coli CTX M2 than [C₂OHMIM][Amp], all other [Cat][Cl] and [Cat][Br] provided higher MIC values when compared to values of [Na][Amp] (control) and/or correspondent [Cat][Amp].

MIC values of ionic liquids and salts containing highly polar cations in particular [cholin][Amp] or [C₂OHMIM][Amp] are always higher than [Na][Amp]. Contrarily, the ampicillin ILs containing apolar cations such as [P_6,6,6,14][Amp] and [C₁₆Pyr][Amp] presented MIC values lower than [Na][Amp]. These observations can be explained with the highly polar cations are more prone to stay in aqueous solution instead of hydrophobic cell membranes and therefore anchor themselves to ampicillin anions (ion trapping effect).²³ The correspondent ampicillin salts thus stay trapped to aqueous solution where eventually became hydrolyzed.

Unlike penicillin G which is active only against Gram-positive bacteria, ampicillin possesses an additional amino group and it is active against both Gram-positive and some Gram-negative bacteria. Bacteria may be resistant (or develop) resistance on β-lactam antibiotics by developing very efficient β-lactamase enzymes (“penicillinases”), by developing change (resistance gene) in penicillin-binding proteins (PBPs) and therefore providing poor affinity for antibiotic or development of porin channel blockage (in case of Gram negative bacteria).²⁴

As observed from Tables 3 and 4, [C₁₆Pyr][Amp] exhibits the most noticeable Relative Decrease of Inhibitory Concentration (RDIC). Among sensitive Gram positive bacteria [C₁₆Pyr][Amp] shows no effect on S. aureus ATCC 25923 (RDIC = 1) or mild positive effect against E. faecalis and S. epidermidis (RDIC = 10 for each). Among sensitive and resistant Gram-negative strains (Tables 3 and 4) [C₁₆Pyr][AMP] IL presents slight increase of the MIC value on E. coli ATCC 25922 (RDIC = 0.1). MIC decreases in case of K. pneumoniae (RDIC = 50) and regarding the resistant strains E. coli TEM CTX M9 (RDIC >1000) and E. coli CTX M2 (RDIC >100) the decrease was remarkable.

Table 3 Relative decrease of inhibitory concentration (RDIC) of ampicillinate anion in [Cat][Amp] (RDIC) comparing with sodium ampicillin for ampicillin sensitive bacteria^a

Comp.	Strains
	Gram-negative		Gram-positive
	E. coli ATCC 25922	K. pneumoniae	S. aureus ATCC 25923	E. faecalis	S. epidermidis
a For explanation of RDIC values see Experimental section.
[Na][Amp]^a	1	1	1	1	1
[P6,6,6,14][Amp]	0.02	0.5	0.1	1	1
[C16Pyr][Amp]	0.1	50	1	10	10
[C2OHMIM][Amp]	0.01	—	—	0.01	0.02

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Table 4 Relative decrease of inhibitory concentration (RDIC) of ampicillinate anion in [Cat][Amp] comparing with sodium ampicillin for ampicillin resistant bacteria

Comp.	Strains
	E. coli TEM CTX M9	E. coli CTX M2	E. coli AmpC MOX2
a For explanation fo RDIC values see Experimental section.
[Na][Amp]^a	1	1	1
[P6,6,6,14][Amp]	>10	>10	—
[C16Pyr][Amp]	>1000	>100	—

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Lehn et al. demonstrated that transport across biological membranes of highly polar anionic compounds can be facilitated if they are paired with lipophilic ammonium ions that act as phase transfer.²⁵ We and others have recently suggested the application of the same principle for penetration and drug delivery in API–ILs with lipophilic counterions.^4,10 The results of [C₁₆Pyr][Amp] on ampicillin sensitive Gram-positive and both sensitive and resistant Gram-negative strains are according to this principle. [C₁₆Pyr][Amp] usually does not influence the RDIC values of sensitive strains as peptidoglycan of Gram positive bacteria is already directly exposed to aqueous solution while outer membrane of sensitive Gram-negative bacteria does not represent a physical barrier for [Na][Amp]. Regarding small fluctuations in the case of E. coli ATCC 25922 (RDIC = 0.1), K. pneumoniae (RDIC = 50), E. faecalis and S. epidermidis (each RDIC = 10) all can be explained in morphological differences of the cell surface of these bacteria, peptidoglycans, outer lipid layer among others and their interaction with API–IL. However, the most remarkable effect of [C₁₆Pyr][AMP] is observed against resistant Gram-negative strains. For E. coli CTX M2 RDIC value is higher than 100 and for E. coli TEM CTX M9 is even higher (RDIC >1000). No activity (and consequently no RDIC value) has been found for E. coli AmpC MOX2. In the case of [P_6,6,6,14][Amp] the MIC values decrease in resistant bacteria E. coli TEM CTX M9 and E. coli CTX M2. In this case the RDIC values are lower than for [C₁₆Pyr][Amp], however the RDICs are in order of tens and no value for E. coli AmpC MOX2 was observed too. These results clearly suggest that antibiotic API–ILs help in delivering antibacterial agent [Amp] to some Gram negative bacteria that developed resistance against β-lactam antibiotics²⁴ most probably acting as lipophilic phase transfer agent across outer membrane of the bacteria.²⁵ If this transportation mechanism is true, API–ILs obviously may resolve some resistance problems – e.g. in case of porin channel blockage they should be fully effective against bacterial resistance but if mutation on PBPs occurred they would show no effect, while in case of resistance based on development of β-lactamases their success may depend on several factors. However as bacteria frequently developed more than one resistance mechanism, the full effect of ILs–APIs on them should be further studied. Regarding sensitive strains, the MIC values of [P_6,6,6,14][Amp] present an opposite effect. In this group of bacteria RDIC values were equal or lower than 1 indicating more complex interaction. The Growth Rate (GR) studies were performed by exposing approximately 0.5 to 2.5 × 10⁴ CFU mL⁻¹ of bacteria to an ampicillin based IL concentration. The selected concentrations were superior, equal or inferior value of the determined MIC.

As expected in the presence of concentrations equal and superior to MIC no growth was observed. In the presence of inferior values of MIC, it was observed a decrease on GR in most of the cases (Table 5, 6 and Fig. 2). These observations indicate that in the presence of ionic liquid at inferior concentration of MIC, a possible growth inhibition occurs in a similar way, specially, in the case of pathogenic strains. These results point to a potential use of these compounds as antibacterial drugs mainly to resistance strains at very low concentrations.

Table 5 Growing rates for the different organisms in the presence and the absence of [P_6,6,6,14][Amp]

Organism	MIC/mM	Conc. tested/mM	Growing rate/min^−1		Decreasing rate
			With [P6,6,6,14][Amp]	No compound
E. coli ATCC 25922	2.5	0.5	0.0007 ± 0.0001	0.0020 ± 0.0001	0.035
S. aureus ATCC 25923	0.005	0.0005	0.0004 ± 0.0003	0.0052 ± 0.0007	0.077
K. pneumoniae	5	0.5	0.00045 ± 0.00007	0.0025 ± 0.0005	0.18
E. faecalis	0.05	0.005	0.0009 ± 0.0004	0.0033 ± 0.0001	0.28
S. epidermidis	0.05	0.005	0.0006 ± 0.0000	0.0047 ± 0.0002	0.13
E. coli TEM CTX M9	0.5	0.05	0.00067 ± 0.00006	0.0033 ± 0.0007	0.20
E. coli AmpC MOX2	0.5	0.05	0.00077 ± 0.00006	0.0032 ± 0.0004	0.24

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Table 6 Growing rates for the different organisms in the presence and the absence of [C₁₆Pyr][Amp]

Organism	MIC/mM	Conc. tested/mM	Growing rate/min^−1		Decreasing rate
			With [C16Pyr][Amp]	No compound
E. coli	0.5	0.05	0.0025 ± 0.0004	0.0020 ± 0.0001	1.25
S. aureus	0.005	0.0005	0.00325 ± 0.00007	0.0052 ± 0.0007	0.63
K. pneumoniae	0.05	0.0005	0.0010 ± 0.0003	0.0025 ± 0.0005	0.40
E. faecalis	0.05	0.005	0.0035 ± 0.001	0.0033 ± 0.0001	1.06
S. epidermidis	0.005	0.0005	0.0041 ± 0.0001	0.0047 ± 0.0002	0.87
E. coli TEM CTX M9	0.005	0.0005	0.0026 ± 0.0001	0.0033 ± 0.0007	0.79
E. coli CTX M2	0.05	0.005	0.0048 ± 0.0002	0.0051 ± 0.0008	0.94
E. coli AmpC MOX2	0.05	0.005	0.00275 ± 0.00007	0.0032 ± 0.0004	0.86

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Fig. 2 Representation of growth curves from resistant Gram-negative strains bacteria E. coli AmpC MOX2 (a); E. coli TEM CTX M9 (b); E. coli CTX M2 (c) in the case of API–IL [C₁₆Pyr][Amp] for different concentrations. For comparison no compound addition experiments were also performed.

Fig. 2 illustrates resistant Gram-negative strains bacteria E. coli AmpC MOX2 (Fig. 2a); E. coli TEM CTX M9 (Fig. 2b); E. coli CTX M2 (Fig. 2c) growth curves in the case of [C₁₆Pyr][Amp] for different concentrations (0.5, 0.05 and 0.005 mM).

Conclusions

The present work suggested that ILs based on ampicillin can reverse the resistance in some clinical strains previously isolated and tested as resistant. With the appropriate selection of the organic cation, it is possible to tune important biological variations in their antibacterial properties. This report also showed that the ion-pair effect is crucial in ampicillin mechanism of action and the selection of hydrophobic ampicillin counter-ions is required.

[C₁₆Pyr][Amp] demonstrated the highest potential in reversion of resistance. [C₁₆Pyr][Cl] is already used in some types of mouthwashes and toothpastes²⁶ although in higher concentrations it is irritant.²⁷ The highest improvement of activity is obtained against Gram-negative resistant bacteria (E. coli TEM CTX M9 and E. coli CTX M2, RDIC >1000 and 100 respectively). These results are clearly promising and appoint to beneficial effect of drug delivery assisted by API–IL through outer membrane of Gram-negative bacteria, opening possibilities for new similar applications and explorations especially in reversing drug resistance. Drug delivery process never seemed to be a problem, for Gram-positive and Gram-negative sensitive strains and therefore they showed more moderate oscillation in RDIC values (0.1–50) obviously connected to their morphology. RDIC values proved to be good indicator for the measurement of the efficiency effectiveness of the process. In the future they can be used in order to quantify interaction of API–ILs and resistant bacteria in order to determine the influence of API–ILs on various bacterial resistance mechanisms.

Our results also showed that future developments of novel API–ILs must take consideration not only to toxicity and hydrophobicity of the counter ion but to be more focused on it. Example of [P_6,6,6,14][Amp] which generally follows the trend of [C₁₆Pyr][Amp], but with very low RDIC values demonstrates that may be more factors at stake to be considered.

The reversion mechanism of the ampicillin based ILs should be further studied in order to improve infection control within the hospital environment and in community. Nevertheless the use of ILs based antimicrobials will contribute to decrease the nosocomial infections (in terms of morbidity and mortality) and costs associated with them.

Experimental section

Reagents and materials

[Amp] have been used as anion combined with the following organic cations: 1-ethyl-3-methylimidazolium, [emim]; 1-hydroxy-ethyl-3-methylimidazolium, [C₂OHMIM]; choline, [cholin]; tetraethylammonium, [TEA]; cetylpyridinium, [C₁₆Pyr] and trihexyltetradecylphosphonium [P_6,6,6,14]. In addition, in order to be accurate and make comparison, activity tests were performed on starting materials (cation bromide and chloride salts). Sodium salt of ampicillin is always used as standard for comparison.

Bacterial strains

A total of 10 bacterial strains were used in this study. The following reference strains were obtained from the American Culture Collection (ATCC): E. coli ATCC 25922 and S. aureus ATCC 25923. All other strains, including several resistant bacteria are clinical isolates from previous work: K. pneumoniae, S. epidermidis and E. faecalis and the resistant bacteria, E coli TEM CTX M9, E. coli CTX M2, E. coli AmpC MOX2. These resistant strains were tested in Vitek®2 systems from bioMérieux.

Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)

The MIC values were determined in triplicate by the broth micro dilution method in a 96-well microtiter plate using Tryptic Soy Broth (TSB) using adapted methodology from Clinical Laboratory Standard Institute (CLSI)²⁸ (Tables 1 and 2). The organisms tested were grown individually on Tryptic Soy agar for 24 h at 37 °C prior to each antibacterial test. Prior to MIC determination, each inoculum density was adjust in TSB to 0.5 McFarland standard by photometric device,²⁸ this resulted in a suspension containing approximately 1 × 10⁸ to 2 × 10⁸ colony forming units (CFU) mL⁻¹ for E. coli ATCC 25922® similar approach was used for the others strains. Then 0.5 μL of the suspension was added to each well to have 0.5–2.5 × 10⁴ CFU mL⁻¹ as final concentration. Bacteria were exposed to an ampicllin–IL salt in the following concentration: 5 mM, 2.5 mM, 0.5 mM, 0.05 mM, 0.005 mM, 0.0005 mM and 0.00005 mM. With the exception of [P_6,6,6,14][Amp] that was diluted in 1% dimethylsulfoxide (DMSO), all others were dissolved in water and this results were compared with bacteria that had grown in TSB broth in presence of 1% DMSO as a control. The MIC for each Amp–IL was recorded as the lowest concentration that showed no turbidity after 24 h of incubation at 37 °C.²⁸ The presence of turbidity is an indication of microbial growth and the corresponding concentration of antibacterial agent is considered ineffective. To determine whether the Amp–ILs inhibited growth or has bactericidal activity, 5 μL of XTT sodium salt of (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxyanilide inner salt) was added to the non-turbid wells of the MIC assay plate and incubated for 3 h at 37 °C for the bactericidal status determination.²⁹ In the case of viable cells with inhibited growth, mitochondrial dehydrogenases of viable cells cleave the tetrazolium ring of XTT yielding dark orange aqueous soluble formazan crystals, however, a solution containing non-viable cells would remain with the same colour.^29b In this case, MICs values were equal to MBC for each ampicillin based ILs, so the antibacterial activities of these compounds were considered to be bactericidal.

In order to obtain the ratio of MIC values for various sensitive bacteria they were grouped as ratio of MIC values of [Na][Amp] and [Cat][Amp] or so-called RDIC values (relative decrease of inhibitory concentration).

As in case of resistant bacteria the MIC values of [Na][Amp] could not be found experimentally, RDIC values of resistant bacteria are calculated assuming MIC value of [Na][Amp] is at least 5 mM (>5 mM) in all cases (the highest value that can be measured experimentally) providing the numerical RDIC values for all [Cat][Amp] with MIC values (indicated in Table 4).

Growth rate studies

In order to complete the characterization of anti-bacterial activity, the Growth Rate (GR) when exposed to the MICs value was evaluated. Additionally, GR values in the presence of higher and lower concentration of the MICs previously found for each compound was also determined (indicated in Tables 5 and 6).

The GR parameter was determined in triplicate by the broth micro dilution method in a 96-well microtiter plate using Tryptic Soy Broth (TSB).^28,30 Approximately 5000–25 000 CFU mL⁻¹ of bacteria is exposed to an ampicillin based IL concentration superior, equal or inferior value of the determined MIC, performing periodical readings of the optical density (OD) at 620 nm. The growing rate was determined by fitting a linear function to the exponential (log) phase curve.

Acknowledgements

This work was funded by National Funds through FCT – Foundation for Science and Technology under the project PEst-C/EQB/LA0006/2011, PEst-C/EQB/LA0006/2011, PTDC/CTM/103664/2008 and Solchemar Company.

Notes and references

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