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JAC Advance Access originally published online on March 12, 2007
Journal of Antimicrobial Chemotherapy 2007 59(4):676-680; doi:10.1093/jac/dkm009
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© The Author 2007. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Efficacy of microcin J25 in biomatrices and in a mouse model of Salmonella infection

Fabian E. Lopez, Paula A. Vincent, Ana M. Zenoff, Raúl A. Salomón and Ricardo N. Farías*

Departamento de Bioquímica de la Nutrición, Instituto Superior de Investigaciones Biológicas (Consejo Nacional de Investigaciones Científicas y Técnicas — Universidad Nacional de Tucumán), Chacabuco 461, 4000 San Miguel de Tucumán, Tucumán, Argentina

* Corresponding author. Tel/Fax: +54-381-4248921; E-mail: rfarias{at}conicet.gov.ar

Received 1 November 2006; returned 10 December 2006; revised 28 December 2006; accepted 12 January 2007


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Objectives: To study the possible therapeutic utility of microcin J25 (MccJ25), a peptide RNA polymerase inhibitor.

Methods: We subjected the antibiotic to two types of assays. First, with an ex vivo assay, we evaluated the stability and efficacy of MccJ25 in complex fluid biomatrices such as human whole blood, plasma and serum, compared with that in conventional laboratory media. Antimicrobial efficacy of MccJ25 was assessed by quantitative culture 2 h after inoculation of the biomatrices with a Salmonella Newport target organism and compared with that of MccJ25-free controls. Second, the antibiotic was tested in a mouse model of Salmonella infection. The latter was induced by intraperitoneal inoculation of 106 cfu of Salmonella Newport and the treatment with MccJ25 was initiated at 2 h post-infection.

Results: MccJ25 retained full activity after 24 h of incubation in whole blood, plasma or serum. In addition, it did not show any haemolytic activity. In whole blood, homologous plasma and serum, introduction of MccJ25 was associated with a significant reduction in cfu versus the respective peptide-free controls. The counts of viable bacteria in the spleen and liver of mice treated with MccJ25 at a total dosage of 3 mg/mouse during either 24 h (0.5 mg/mouse every 4 h) or 6 days (0.5 mg/mouse every 24 h) significantly decreased by two or three orders of magnitude (P ≤ 0.05) compared with those in control mice.

Conclusions: Collectively, these findings indicate that the biological activity of MccJ25 is not affected in complex biological matrices. The potent in vitro activity of MccJ25 against Salmonella translates into good in vivo efficacy in a mouse infection model.

Keywords: antimicrobial peptides , in vivo activity , stability in biomatrices


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Escherichia coli microcin J25 (MccJ25) is a plasmid-encoded antibiotic peptide consisting of 21 L-amino acid residues, which is primarily active on Gram-negative bacteria related to the producer strain, such as E. coli, Salmonella and Shigella strains.1,2 The structure of MccJ25 consists of an amino acid backbone chain (G1-G-A-G-H5-V-P-E-Y-F10-V-G-I-G-T15-P-I-S-F-Y20-G), containing a lactam linkage between the {alpha}-amino group of Gly1 and the {gamma}-carboxyl of Glu8, forming an eight-residue ring (Gly1-Glu8) denominated the lariat ring.3–5 The ‘tail’ (Tyr9-Gly21) passes through the lariat ring, with Phe19 and Tyr20 straddling each side of the ring, thus sterically trapping the tail within the ring in a non-covalent way. MccJ25 exhibits strong antimicrobial activity in the range from nanomolar to micromolar when tested in artificial media or buffer systems in vitro. However, the efficacy of MccJ25 in complex fluid matrices such as whole blood, plasma and serum has not yet been assessed. This could be of direct relevance to the potential therapeutic applications of this antibiotic. In this connection, it is well known that antimicrobial peptides from several sources often exhibit strong antimicrobial activities when tested in artificial media or simple buffer systems in vitro; however, in complex fluid matrices such as whole blood, plasma and serum, they tend to show relatively poor activity, since they are rapidly inactivated by peptidases or blocked by proteins. Alternatively, blood and blood fractions may contain components that amplify antimicrobial peptide actions against target pathogens, resulting in enhanced pathogen killing. The present investigation tested the antimicrobial peptide MccJ25 to define potential peptide–biomatrix interactions, using a conventional antibiotic, ampicillin, as a comparator. Another objective was to study the in vivo efficacy of MccJ25 in a murine model of infection with Salmonella Newport. The results demonstrated that MccJ25 exerts a remarkable antimicrobial activity in biomatrices and in in vivo conditions.


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Antimicrobial agents and microorganism

MccJ25 was purified as described previously.6 Pure MccJ25 and ampicillin (Sigma Chem Co.) were suspended in methanol and water, respectively, prior to use. MccJ25 was diluted with sterile distilled water containing 0.01% Tween 80.

An MccJ25-supersusceptible clinical isolate of Salmonella Newport was used as a target organism in these studies [MIC of MccJ25, 0.02 µM (0.042 mg/L)]. For assays described subsequently, the microorganism was cultured to mid-logarithmic phase in Luria broth (LB) at 37°C with agitation, harvested by centrifugation, washed and resuspended in saline (NaCl 0.9%). Inocula were validated by cell viable counts.

Assays with biomatrices

The antimicrobial activity of MccJ25 in human whole blood and homologous plasma and serum fractions was assessed. For a given experiment, 20 mL of fresh whole blood was obtained from a healthy volunteer. Heparin (10 units/mL) was used to anticoagulate a portion of the whole-blood sample. A part of the heparinized whole blood was set aside, and the rest was subjected to low-speed centrifugation (210 g), yielding a plasma supernatant. These preparations comprised the whole-blood and plasma biomatrices, respectively. The remaining non-anticoagulated whole blood was allowed to spontaneously clot for 30 min and centrifuged (300 g) to yield homologous serum (that is, derived from the same donor whose whole blood was also studied). The serum was heated at 56°C for 30 min to obtain heat-inactivated serum.

The extent and durability of MccJ25 activity in human whole blood and homologous plasma and serum fractions were assessed. MccJ25 (1 mg/mL) was incubated for 24 h at 37°C in whole blood, plasma, serum and inactivated serum. Serial double dilutions of each mixture were then prepared. At time zero and after incubation, 10 µL of each dilution was spotted onto LB plates. After the drops had dried, the plates were overlaid with 4 mL of soft agar inoculated with 106 cells of Salmonella Newport. After overnight incubation at 37ºC, the plates were inspected for the size of the inhibition halos. For comparison, the stability of microcin was tested in parallel in a conventional medium (LB).

To compare the antimicrobial efficacy of MccJ25 and ampicillin, the antibiotics were added to 1 mL of each matrix simultaneously with pathogen inoculation (10 µL, yielding a final inoculum of 106 cfu/mL). After 2 h of incubation, the mixtures were vortexed to ensure dispersion, and 10-fold dilutions in saline were quantitatively cultured onto LB agar plates. For comparison, the antimicrobial activities of both antibiotics were tested in LB medium.

Haemolysis assay

Blood was drawn with heparin as anticoagulant, processed and assayed immediately. The erythrocytes were spun at 1000 g, washed three times in saline, and about 108 cells, suspended in 100 µL of saline, were added to glass tubes containing 900 µL of MccJ25 solution (1000 µg/mL) in saline, saline alone (for baseline values) or distilled water (for 100% haemolysis). After incubation at 37°C for 30 min, samples were centrifuged and the haemolytic activity was assessed as a function of haemoglobin release by measuring the absorbance of 500 µL of supernatant (540 nm).

Evaluation of MccJ25 activity in vivo and pharmacokinetic studies

Groups of five mice of ~28–32 g were inoculated intraperitoneally with 106 cells of Salmonella Newport. MccJ25 or vehicle alone (sterile water or saline), as a control, was administered intraperitoneally 2 h following bacterial challenge. After 1 or 6 days, depending on the experimental scheme (Table 1), spleen and liver were excised and homogenized. The number of viable organisms was determined by plating dilutions of the homogenates on LB agar.


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  		Table 1.. Effect of MccJ25 on viable counts in liver and spleen of mice infected with Salmonella Newport

 
Studies of MccJ25 pharmacokinetics in plasma after administration of a single dose were performed with mice treated with three different intraperitoneal doses of the antibiotic (7, 18.18 and 36.36 mg/kg). Blood samples were obtained from the tail at different times. Microcin concentration in plasma was determined by the critical dilution method, by comparison with a reference purified sample of the antibiotic titrated in parallel with the unknown samples. The pharmacokinetic parameters determined were half-life (t1/2) and maximum concentration of drug in serum (Cmax) by using the TopFit software. Half-life was calculated as the average of the half-lives obtained from the curves for each of the doses tested.


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Stability of MccJ25 activity in biomatrices

MccJ25 has been shown previously to be stable upon protease treatment and heating for 30 min at 120°C.1 In the present work, by using an ex vivo assay, we evaluated the extent and durability of MccJ25 activity in complex fluid biomatrices. To this end, the antibiotic was incubated for 24 h in whole blood, plasma and serum, as well as in LB medium. As shown in Figure 1, the inhibition zones produced by 1/1024 dilutions of the incubation mixtures had the same size as those obtained at time zero. We conclude that MccJ25 activity is not affected by any component of blood-derived biomatrices. In addition, MccJ25 did not show any haemolytic activity on human erythrocytes.


Figure 1
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  		Figure 1.. Stability of MccJ25 in LB and different biomatrices. MccJ25 (1 mg/mL) was incubated for 24 h at 37°C in LB (1), plasma (2), whole blood (3), inactivated serum (4) and serum (5), and the biological activity was assayed by a spot-on-lawn test. For comparison, the halos given by a 1/1024 dilution of each mixture at time zero (T0) and after 24 h of incubation (T24) are shown.

 
Antimicrobial activity of MccJ25 in different biomatrices

Incubation of Salmonella Newport in whole blood and plasma did not cause a significant change in cfu/mL values, suggesting that these biomatrices have minimal inherent antibacterial activity (Figure 2). On the contrary, a decrease from 106 to 103 cfu/mL was observed in intact serum. This reduction was not observed in heat-inactivated serum, indicating the presence of an intrinsic activity against Salmonella Newport in intact serum. A similar antibacterial activity in serum has been described for other microorganisms.7 In LB medium, an inoculum of 106 cfu/mL of Salmonella Newport yielded a mean of 108 cfu/mL after 2 h of incubation at 37°C. When 106 cfu/mL of Salmonella Newport was inoculated into LB containing either 0.1 or 1 µM MccJ25, a decrease to 3 log10 and 2 log10 cfu/mL, respectively, was observed. Similar results were obtained when the inoculation was done in biomatrices containing the same concentrations of MccJ25. These results indicated that MccJ25 exerts a potent antimicrobial activity in the biomatrices assayed. For comparison, we tested the activity of ampicillin [MIC for Salmonella Newport, 8.41 µM (3.12 mg/L)] under the same conditions. As shown in Figure 2, as low a concentration of MccJ25 as 0.1 µM has an activity higher than that of 1 and 130 µM ampicillin in both whole blood and plasma. Thus, on a molar basis, MccJ25 is considerably more potent than a conventional antibiotic.


Figure 2
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  		Figure 2.. Antimicrobial activity of MccJ25 in different biomatrices. LB, whole blood (WB), plasma (P) and inactivated serum (IS) were inoculated with about 106 Salmonella Newport cells. Serum (S) was inoculated with about 108 cells. Inocula were validated by quantitative culture at time zero (black bars). Cultures were incubated for 2 h in the absence (control) and presence of two different concentrations of MccJ25 and ampicillin. Results are expressed as the mean ± SD of the log cfu (n = 5). The final response was analysed by logarithmic transformation of the cfu data followed by ANOVA test (Software Infostat 2.0).

 
MccJ25 reduces cfu counts in spleen and liver

Sixty minutes after intraperitoneal injection of MccJ25 doses of 7, 18.18 and 36.36 mg/kg, the plasma antibiotic concentration reached a maximum (Cmax) of 1.96, 7.84 and 9.03 µg/mL, respectively. MccJ25 started to be removed from the circulation after 60 min, with a half-life of 77.82 ± 11.3 min (Figure 3).


Figure 3
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  		Figure 3.. Determination of MccJ25 half-life. Mice were given MccJ25 doses of 7 (squares), 18.18 (circles) and 36.36 (triangles) mg/kg. At different times, blood samples were taken and MccJ25 concentrations in plasma were measured as indicated in the Materials and methods section.

 
The counts of viable Salmonella Newport in the spleen and liver of mice treated with MccJ25 at a total dosage of 3 mg/mouse, either every 4 h for 1 day or every 24 h for 6 days, decreased significantly (P ≤ 0.05) compared with those in control mice (Table 1).

Three days after inoculation with 106 cells of Salmonella Newport, the animals showed the same signs of disease as those described for Salmonella Typhimurium infection.8 No mortality was observed under these conditions up to day 7. However, when mice were inoculated with 108 cells of Salmonella Newport, 70% of the animals died over the same period.


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Cationic antimicrobial peptides have in recent years been attracting increasing interest from both the scientific community and the pharmaceutical industry for their potential as new therapeutic agents. For example, the 55 kDa bactericidal/permeability-increasing protein (BPI) is a neutrophil-derived polypeptide belonging to a family of lipid and endotoxin-binding proteins. In multiple animal models, a recombinant amino-terminal fragment of BPI, rBPI(21), is non-toxic and protects against Gram-negative bacteria and endotoxin. In humans, rBPI(21) is also non-toxic and non-immunogenic and has undergone Phase II/III clinical trials with apparent therapeutic benefit.9 However, most of these peptide agents lack specificity and might be too toxic for systemic treatment.10,11 Therefore, topical use has been chosen for various antimicrobial peptides that are currently undergoing clinical trials.12,13 The Food and Drug Administration recently approved the use of daptomycin, a cyclic lipodepsipeptide antibiotic, for the treatment of complicated skin and skin structure infections caused by several Gram-positive bacteria.14

The potential interactions between a given antimicrobial peptide and blood components are multifactorial. For example, whole blood and other fluid biomatrices may contain binding or blocking proteins that may inactivate such a peptide. It is well recognized that antibiotics which are highly bound to serum proteins have reduced antibacterial activity when they are tested for in vitro activity in the presence of serum proteins, since only free drug is available for antibacterial activity.15 On the other hand, in vitro synergy between antibiotics and serum components, including antibody and complement, has been reported16–18 and may also be expressed in the responses of patients during antimicrobial chemotherapy. Few MIC studies have attempted to include host factors, although it is known that several variables, including serum proteins, phosphates, osmolarity, divalent cations and pH, can affect the activity of certain antimicrobial agents.19 Similarly, blood may contain peptidases that degrade the peptide over time.

In this paper, using an ex vivo assay, we evaluated the extent and durability of the peptide antibiotic MccJ25 in complex fluid biomatrices such as human whole blood, plasma and serum compared with those in conventional laboratory media. The antibiotic was shown to be as effective in biomatrices as in artificial media, indicating that its antimicrobial activity was not affected by any blood component. This constitutes an important advantage which distinguishes it from other peptides with therapeutic potential. Besides, the antimicrobial activity in biomatrices appeared to be significantly higher than that of a conventional antibiotic, ampicillin, at least for Salmonella Newport.

Many antimicrobial peptides have the disadvantage of being cytotoxic and/or erythrolytic, and this could limit their potential therapeutic utility. In the present study, we show that MccJ25 has no haemolytic activity, which suggests that it could be harmless for other mammalian cell types.

Another interesting finding of this study is that MccJ25 displays a prolonged systemic antimicrobial activity in a mouse infection model. Intraperitoneal administration of the antibiotic resulted in a significant reduction in the bacterial number in both the spleen and liver of mice infected with Salmonella Newport. Although additional studies are required to improve the therapeutic window and potency of MccJ25, our results suggest that this antibiotic has potential for systemic administration and treatment of otherwise antibiotic-resistant infections.


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None to declare.


   Acknowledgements
 
We are indebted to Dr M. C. Rubio for help with pharmacokinetic studies and Dr G. del V. Perdigón for her assistance with the murine systemic infection model. This work was supported by grants from Agencia Nacional de Promoción Científica y Tecnológica (PICT 2003-17819 and PICTO 2004-843). F. E. L. was a recipient of a CONICET fellowship. P. A. V., R. A. S. and R. N. F. are career investigators of CONICET.


   References
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1 Salomón RA and Farías RN. (1992) Microcin 25, a novel antimicrobial peptide produced by. Escherichia coli. J Bacteriol 174:7428–35.[Abstract/Free Full Text]

2 Vincent PA, Delgado MA, Farias RN, et al. (2004) Inhibition of Salmonella enterica serovars by microcin J25. FEMS Microbiol Lett 236:103–7.[CrossRef][ISI][Medline]

3 Bayro MJ, Mukhopadhyay J, Swapna GV, et al. (2003) Structure of antibacterial peptide microcin J25: a 21-residue lariat protoknot. J Am Chem Soc 125:12382–3.[CrossRef][ISI][Medline]

4 Rosengren KJ, Clark RJ, Daly NL, et al. (2003) Microcin J25 has a threaded sidechain-to-backbone ring structure and not a head-to-tail cyclized backbone. J Am Chem Soc 125:12464–74.[CrossRef][ISI][Medline]

5 Wilson KA, Kalkum M, Ottesen J, et al. (2003) Structure of microcin J25, a peptide inhibitor of bacterial RNA polymerase, is a lassoed tail. J Am Chem Soc 125:12475–83.[CrossRef][ISI][Medline]

6 Rintoul MR, de Arcuri BF, Salomon RA, et al. (2001) The antibacterial action of microcin J25: evidence for disruption of cytoplasmic membrane energization in Salmonella newport.. FEMS Microbiol Lett 204:265–70.[CrossRef][ISI][Medline]

7 Miyazaki S, Okazaki K, Tsuji M, et al. (2004) Pharmacodynamics of S-3578, a novel cephem, in murine lung and systemic infection models. Antimicrob Agents Chemother 48:378–83.[Abstract/Free Full Text]

8 Khan SA, Strijbos PJLM, Everest P, et al. (2001) Early responses to Salmonella typhimurium infection in mice occur at focal lesions in infected organs. Microb Pathog 30:29–38.[CrossRef][ISI][Medline]

9 Levy O and Elsbach P. (2001) Bactericidal/permeability-increasing protein in host defense and its efficacy in the treatment of bacterial sepsis. Curr Infect Dis Rep 3:407–12.[Medline]

10 Gura T. (2001) Innate immunity. Ancient system gets new respect. Science 291:2068–71.[Free Full Text]

11 Levy O. (2000) Antimicrobial proteins and peptides of blood: templates for novel antimicrobial agents. Blood 96:2664–72.[Abstract/Free Full Text]

12 Chen J, Falla TJ, Liu H, et al. (2000) Development of protegrins for the treatment and prevention of oral mucositis: structure–activity relationships of synthetic protegrin analogues. Biopolymers 55:88–98.[CrossRef][ISI][Medline]

13 Ge Y, MacDonald D, Henry MM, et al. (1999) In vitro susceptibility to pexiganan of bacteria isolated from infected diabetic foot ulcers. Diagn Microbiol Infect Dis 35:45–53.[CrossRef][ISI][Medline]

14 Jeu L and Fung HB. (2004) Daptomycin: a cyclic lipopeptide antimicrobial agent. Clin Ther 26:1728–57.[CrossRef][ISI][Medline]

15 Craig WA and Welling PG. (1977) Protein binding of antimicrobials: clinical pharmacokinetics and therapeutic implications. Clin Pharmacokinet 2:252–68.[ISI][Medline]

16 Dutcher BS, Reynard AM, Beck ME, et al. (1978) Potentiation of antibiotic activity by normal serum. Antimicrob Agents Chemother 13:820–6.[Abstract/Free Full Text]

17 Marca G, Veronese M, Petrescu D. (1973) Enhancement of the bacteriostatic and bactericidal activities of chloramphenicol and thiamphenicol by normal serum in vitro. Chemotherapy 18:91–8.[ISI][Medline]

18 Pruul H and Reynolds BL. (1972) Interaction of complement and polymyxin with Gram-negative bacteria. Infect Immun 6:709–17.[Abstract/Free Full Text]

19 Stratton CW and Reller LB. (1977) Serum dilution test for bactericidal activity. 1. Selection of a physiological diluent. J Infect Dis 136:187–94.[ISI][Medline]


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