the antibacterial activity against mrsa strains and other bacteria of a 500 da fraction from magg

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Microbes and Infection xx (2008) 1e9www.elsevier.com/locate/micinf

Original article

The antibacterial activity against MRSA strains and otherbacteria of a <500 Da fraction from maggot excretions/secretions

of Lucilia sericata (Diptera: Calliphoridae)

Alyson Bexfield a,*, A. Elizabeth Bond a,b, Emily C. Roberts a, Edward Dudley a,b,Yamni Nigam c, Stephen Thomas d, Russell P. Newton a,b, Norman A. Ratcliffe a

a Department of Biological Sciences, SOTEAS, Swansea University, Singleton Park, Swansea, SA2 8PP, UKb BAMS Facility, Grove Building, Swansea University, SA2 8PP, UK

c School of Health Science, Swansea University, Singleton Park, Swansea, SA2 8PP, UKd Medetec (Medical Device Technical Consultancy Services), Radyr, Cardiff, UK

Received 4 October 2007; accepted 17 December 2007

Abstract

The application of Lucilia sericata larvae to chronic, infected wounds results in the rapid elimination of infecting microorganisms, includingMRSA. Previously, we demonstrated in vitro antibacterial activity of native excretions/secretions (nES) from L. sericata and partially purifiedtwo low mass antibacterial compounds with masses of 0.5e10 kDa and <500 Da. The present study reports the antibacterial effects of the<500 Da fraction (ES<500) on the growth and morphology of a range of bacteria, including 12 MRSA strains. Distinct morphological changeswere observed in Bacillus cereus and Escherichia coli following exposure to ES<500. Flow cytometry and confocal microscopy analyses, inconjunction with turbidometric and CFU assays, revealed bacteriostatic activity of nES against S. aureus and E. coli. ES<500 also demonstratedbacteriostatic activity against S. aureus, however, bactericidal activity and the induction of a viable but non-culturable state were observed withES<500-treated E. coli.� 2008 Elsevier Masson SAS. All rights reserved.

Keywords: Maggot therapy; MRSA; Antibacterial; Bacterial morphology

1. Introduction

Sterile maggots of Lucilia sericata were reintroduced intoEurope in 1995 to treat infected non-healing wounds [1].

Abbreviations: CFU, colony-forming unit; CSLM, confocal scanning laser

microscopy; Da, dalton; ES, excretions/secretions; ES<500, <500 Da ultrafil-

tration fraction of excretions/secretions; IC, indigo carmine; LM, light micros-

copy; MRSA, methicillin-resistant Staphylococcus aureus; MWCO, molecular

weight cut-off; nES, native excretions/secretions; PBS, phosphate buffered sa-

line; PI, propidium iodide; PW, peptone water; SEM, standard error of the

mean; SI, survival index; TB, turbidometric; TSB, tryptic soy broth.

* Corresponding author. Tel.: þ44 (0) 1792 205678; fax: þ44 (0) 1792

295447.

E-mail address: [email protected] (A. Bexfield).

1286-4579/$ - see front matter � 2008 Elsevier Masson SAS. All rights reserved.

doi:10.1016/j.micinf.2007.12.011

Please cite this article in press as: A. Bexfield et al., The antibacterial activity a

excretions/secretions of Lucilia sericata (Diptera: Calliphoridae), Microb Infect (

Subsequently, over 20 million maggots have been suppliedto ca. 30,000 patients throughout the United Kingdom andbeyond [2].

Most wound infections are polymicrobial, involving a rangeof both aerobic and anaerobic bacteria [3]. The mostfrequently isolated pathogen from acute and chronic woundsis Staphylococcus aureus [3]. S. aureus is carried innocuouslyby approximately 30% of the population [4], but can be path-ogenic upon entry into wounds. S. aureus infections causegreat concern due to their acquisition of resistance to a rangeof antimicrobials. Methicillin-resistant S. aureus (MRSA) hasrapidly spread causing worldwide hospital and community in-fections [5], resulting in increasing numbers of patient mortal-ities [6]. Of particular concern, is the appearance of newstrains of MRSA, resistant to all commonly used antibiotics,

gainst MRSA strains and other bacteria of a <500 Da fraction from maggot

2008), doi:10.1016/j.micinf.2007.12.011

mailto:[email protected]

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2 A. Bexfield et al. / Microbes and Infection xx (2008) 1e9

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including vancomycin [7]. Consequently, the discovery anddevelopment of novel antimicrobials against MRSA and other‘‘superbugs’’ are urgently required.

Clinically, applying maggots to infected wounds results inthe rapid elimination of infecting microorganisms, includingMRSA [8e10]. Maggot therapy success is partly due to the in-gestion and killing of living microorganisms [11], but antibac-terial activity of externalised excretions/secretions (ES), thatinclude the salivary gland secretions and faecal waste productsof L. sericata, has also been demonstrated in vitro [12e15].Bexfield et al. [14] reported that native ES (nES) from sterilethird instar larvae of L. sericata has constitutive, heat-stable,protease-resistant, antibacterial activity against a range ofGram-negative and Gram-positive bacteria, includingMRSA. They also separated nES into three molecular massfractions (>10 kDa, 0.5e10 kDa and <500 Da) and detectedtwo discrete antibacterial moieties [14]. Both the 0.5e10 kDa and the <500 Da fractions demonstrated antibacterialactivity against S. aureus, whereas only the <500 Da fractionwas active against MRSA. The present paper investigatesfurther the antibacterial activity of the <500 Da fraction(ES<500) against a range of bacteria including 12 strains ofMRSA.

2. Materials and methods

2.1. Larvae

Sterile third instar larvae of Lucilia sericata were suppliedby ZooBiotic Ltd, Bridgend, UK.

2.2. Microorganisms

Staphylococcus aureus 9518, Escherichia coli 10536,Enterococcus faecalis 19433, Bacillus subtilis 6633, Proteusmirabilis 10975 and Pseudomonas aeruginosa 10662 werepurchased from the National Collections of Industrial andMarine Bacteria (NCIMB), Aberdeen, UK. E. coli K12 waspurchased from the Coli Genetic Stock Center (CGSC), NewHaven, USA. Klebsiella pneumoniae, Serratia marcescensand Bacillus cereus were kindly provided by the NPHS Micro-biology Laboratory, Singleton Hospital, Swansea, UK. MRSAstrains included EMRSA-13, EMRSA-15, EMRSA-16, [16],252, COL, MW2 [17] and NCTC 10442 (SCCmec-type I),N315 (typeII), 85/2082 (typeIII), JCSC 1968 (typeIVa),JCSC 1978 (typeIVb), MR108 (typeIVc) [18]. S. epidermidis8400 was kindly provided by Professor D. Mack, SwanseaUniversity, UK. All bacteria were grown for 17 h in 20 mlof tryptic soy broth (TSB, Difco, Becton Dickinson UKLtd., Oxford, UK), washed twice in phosphate buffered saline(PBS, Oxoid Ltd., Basingstoke, UK), and diluted to 2 � 105

bacteria ml�1 in TSB.

2.3. Chemicals

Chemicals were purchased from Sigma-Aldrich, Dorset,UK, unless otherwise stated.



2.4. Collection of larval secretions

Native excretions/secretions (nES) were collected asdescribed previously [14].

2.5. Ultrafiltration

Pooled nES (10 ml) was sequentially fractionated througha 10 kDa molecular weight cut-off (MWCO) membrane thena 500 Da MWCO membrane in an Amicon stirred ultrafiltra-tion cell (Millipore UK Ltd) [14]. Following filter sterilisation(0.2 mm), ES<500 was tested for antibacterial activity usinga turbidometric assay.

2.6. Turbidometric (TB) assay

The TB assay, a liquid growth-inhibition assay, was modi-fied from Bexfield et al. [14] with 150 ml of either nES orES<500 mixed with 16.7 ml of 10% peptone water (PW), pH8.5, and 50 ml incubated with 10 ml of bacterial suspensionin triplicate wells. In controls, test samples were replacedwith sterile Milli-Q water. For antibacterial standardisation,S. aureus was included in the assay as a reference for relativeactivity. Each bacterial species was tested at least three times.Survival index (SI) was calculated as follows:

SI¼ OD550 of test sample at corresponding time point

OD550 at mid-log of control bacterial growth� 100

For S. epidermidis and P. mirabilis, bacterial concentrationsof 2 � 102 to 2 � 105 bacteria ml�1 were used. For multipledose experiments, 150 ml of nES were mixed with 16.7 ml of10% PW and 30 ml of bacterial suspension in microcentrifugetubes and incubated at 37 �C for 1 h. Samples were thencentrifuged for 7 min at 3300 � g, the pellet resuspended in150 ml of fresh nES, 16.7 ml of 10% PW and 30 ml of TSB,and incubated at 37 �C for a further hour. After the fourth ad-dition of fresh sample mix, 50 ml aliquots were placed in trip-licate in the wells of a 96-well microtitre plate and the TBassay was completed.

2.7. Indigo carmine quantificationof ES<500 dilution [19]

Dilution of ES<500 incurred during ultrafiltration was inves-tigated using indigo carmine (IC; 466 Da), as a recoverymarker. Ultrafiltration of nES (1.5 ml) was performed sixtimes in the presence (IC-positive) and absence (IC-negative)of freshly prepared IC in Milli-Q water (final concentration:100 mg ml�1). Standards for IC quantification (100, 50, 10, 5and 1 mg ml�1) were simultaneously prepared from the ICstock solution. The IC-negative fractions generated werebioassayed using the TB assay while the optical density ofIC-positive samples was measured at 620 nm and the IC con-tent determined from the standards. IC-positive fractions wereblanked against IC-negative fractions to account for the


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natural ES colour. IC was replaced with Milli-Q water in IC-negative controls.

2.8. Light microscopy (LM) assay

Bacteria were examined for morphological changes follow-ing incubation with ES<500 at 37 �C using phase contrast in anOlympus BX41 microscope. Bacteria were prepared as for theTB assay and resuspended in TSB to 2 � 107 bacteria ml�1.ES<500 (135 ml) was mixed with 15 ml of 10% PW, pH 8.5, in-cubated with 15 ml of bacterial suspension at 37 �C and 10 mlaliquots examined hourly. In controls, ES<500 was replacedwith sterile Milli-Q water. Dilutions of 50% and 25%ES<500 in sterile Milli-Q water were also tested.

2.9. Investigation into bacterial viability

Fig. 1. Growth inhibition of ES<500. (a) Inhibition against Gram-positive and

Gram-negative bacteria; (b) growth inhibition of ES<500 against a range of

MRSA strains (red bars), and the activity of nES against strains EMRSA-15

and 252 (cross-hatched). Bacterial susceptibility was assessed using the TB

assay. Error bars ¼ SEM. *P < 0.05, **P < 0.001. #SI > 100.

The effects of nES and ES<500 on bacterial growth and vi-ability were investigated using three methods, namely, (1) theTB assay, (2) the colony-forming units (CFU) assay and (3)flow cytometry. S. aureus and E. coli represented Gram-posi-tive and Gram-negative bacteria, respectively. Bacterial viabil-ity was investigated at 6 h (i.e. mid-log of control bacterialgrowth, see TB assay, detailed in [14]).

Bacteria were prepared as in Section 2.2 and adjusted to2 � 106 bacteria ml�1 in fresh TSB. Aliquots (150 ml) ofnES or ES<500 were mixed with 16.7 ml of 10% PW pH 8.5.Samples were then prepared for the TB assay and flow cytom-etry. For the TB assay, 50 ml of sample were incubated with5 ml of bacteria, while for flow cytometry, 150 ml of samplewere incubated with 15 ml of bacteria, in triplicate, in 1.5 mlmicrocentrifuge tubes. The flow cytometry preparations wereincubated at 37 �C for 6 h before analysis. Subsequently,2 ml aliquots from each tube were serially diluted with PBS,10 ml aliquots spread on nutrient agar plates in triplicate, incu-bated overnight at 37 �C and CFUs counted after 24 h. Theremainder of the samples were centrifuged at 6600 � g for7 min, resuspended in 90 ml of PBS and incubated with10 ml of Syto-9 (20 nM final concentration for S. aureus,2 mM final concentration for E. coli) and 10 ml of propidiumiodide (PI; final concentration of 5 mg ml�1) for 30 min atroom temperature in the dark. Syto-9 and PI are componentsof the BacLite Live/Dead viability staining kit (InvitrogenLtd, Paisley, UK). Syto-9, a green fluorescent stain, labels nu-cleic acids irrespective of whether the bacterial cell is alive ordead. In contrast, PI is a red fluorescent nucleic acid stain thatcan only penetrate bacteria with compromised membranes,thus selectively labelling non-viable cells.

Following staining, samples were diluted to 1.4 ml withPBS and bacterial viability was determined using a FACSAria (BD Biosciences, NJ, USA). Bacteria were initially gatedusing forward scatter. Cells of the appropriate size were thenanalysed for red (610 � 20 nm) and green (530 � 30 nm) fluo-rescence, excited by the 488 nm argon laser. All experimentswere conducted in triplicate and for each sample 10,000stained bacteria were recorded. Position of the ‘live’ and‘dead’ gates were determined using triplicate samples of



stained dead (treated with 70% isopropanol for 30 min),stained live and unstained bacteria. For bacterial enumeration,standards containing 1 mm yellow-green microspheres (Invi-trogen Ltd) at 5 � 105 beads ml�1 were used to quantifyflow rate.

2.10. Confocal laser scanning microscopy (CLSM)

Samples remaining from flow cytometry, were filtered onto0.2 mm pore black polycarbonate filters (Millipore, Watford,UK) and viewed using a Zeiss LSM 510 META laser scanningmicroscope (Carl Zeiss Ltd., Hertfordshire, UK). The 488 nmargon laser and 543 nm He/Ne lasers were used to excite liveand dead cells, respectively. Band-pass 505e530 nm and long-pass 560 nm filters were used to observe green and red fluores-cence, respectively.

2.11. Statistical analysis

Data are expressed as arithmetic means � SEM. The signif-icance of differences between sample values was assessed us-ing two-tailed unpaired Student’s t-tests with significance setat P � 0.05. For the survival index (SI) analyses, raw blankedoptical density values at mid-logarithmic phase of the controlswere compared with the corresponding data for test samples.


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3. Results

3.1. Antibacterial activity

ES<500 demonstrated growth inhibition against a range ofGram-positive and Gram-negative bacteria (Fig. 1a). B. subtilis(SI ¼ �2.15 � 1.54) and K. pneumoniae (SI ¼ �0.34 � 2.72)were most sensitive to the antibacterial effects of ES<500,while P. mirabilis (SI ¼ 181 � 11.34) and S. epidermidis(SI ¼ 214 � 30.71) were the most resistant. Ten of the 12 ofthe MRSA strains tested were significantly inhibited(P < 0.001) by ES<500 (Fig. 1b). Growth of MRSA strains252 and EMRSA-15 was initially unaffected by ES<500. How-ever, both MRSA 252 (SI ¼ 23 � 14.53) and EMRSA-15(SI ¼ 21 � 24.16) were significantly (P < 0.001) inhibited bynES (Fig. 1b). Further testing revealed that storage of nES at�80 �C instead of �20 �C prior to ultrafiltration, restoredES<500 activity against MRSA 252 and EMRSA-15(SI ¼ 13 � 4.74 and 36 � 9.07, respectively). Incubation ofP. mirabilis or S. epidermidis with multiple nES doses failed

Fig. 2. Phase contrast microscopy of Bacillus cereus. (a) and (b) Control bacteria

bacteria following incubation with ES<500 for 4 h. *An example of a bacterium w

Scale bar ¼ 10 mm.



to result in antibacterial activity as did decreasing the bacterialinoculum to 2 � 102 ml�1.

3.2. Indigo carmine quantification of ES<500 dilution

The addition of IC to nES before ultrafiltration revealeda 65% reduction in IC concentration in the ES<500. This wasparalleled by a 65% loss of antibacterial activity against S. au-reus (data not shown).

3.3. Light microscopy (LM) assay

Hourly examination of bacteria following incubation withES<500 revealed distinct morphological changes in B. cereusand E. coli, visible after 3 h. Incubation of B. cereus withES<500 resulted in bacterial filamentation compared withcontrols, the extent of which varied between individual cells(Fig. 2aee). A bacterium exhibiting a 17-fold increase inlength is shown in Fig. 2e.

following incubation with 1% peptone water for 4 h; (c)e(e) filamentation of

hich has undergone a 17-fold increase in length compared with control cells.


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Microscopic examination of E. coli K12 and 10536 incu-bated with ES<500 revealed concentration-dependent mor-phological changes. All concentrations from 25 to 100%inhibited division of E. coli within 1 h. However, after 5 hincubation, morphological changes observed dependedupon the concentration of ES used. With 100%, 50% and25% ES<500, 90, 13 and 20% of bacteria underwent nochanges, 8, 46 and 17% became lemon-shaped, 2, 38, and44% racket shaped and 0, 3 and 19% spherical, respectively(Fig. 3). In contrast to E. coli, dilution of ES<500 did notaccentuate the effect on B. cereus but rather reduced andthen abolished filamentation. Bacterial cell ghosts were pre-dominant for B. subtilis and no morphological changes wereobserved for S. aureus, E. faecalis, S. marcescens, K. pneu-moniae or Ps. aeruginosa with 100%, 50% or 25% dilutionsES<500.

3.4. Investigation into bacterial viability

Flow cytometric analysis of S. aureus and E. coli livecontrols showed that the bacteria were 99% viable (Fig. 4a)and 90% viable (Fig. 5a), respectively, after 6 h incubation

Fig. 3. Phase contrast microscopy of E. coli K12. (a) E. coli controls following incub

a 25% dilution of ES<500 for 5 h. Bacteria incubated with 25% ES<500 exhibit signifi

Examples of lemon-shaped bacteria are shown in (b), racket-shaped bacteria are s



with 1% PW. Isopropanol-killed S. aureus and E. coli controlsare shown for reference in Figs. 4b and 5b, respectively. Acomparison of TB, CFU and flow cytometry results can befound in Table 1.

Incubation of S. aureus with nES for 6 h resulted in 84%inhibition of bacterial growth in the TB assay (SI ¼ 15.8 �16.72; n ¼ 3; P < 0.001) and only 8.5% � 8.3% of the bacte-ria produced colonies on agar plates compared with controls(Table 1). Flow cytometric analysis of viability-stained S.aureus following incubation with nES revealed that71.5% � 2.3% of S. aureus cells were viable and 15% werenon-viable (Table 1, Fig. 4c). The remaining bacteria wereof unconfirmed viability (Table 1, Fig. 4c). When S. aureuswas incubated with ES<500, growth was inhibited by 97%(SI ¼ 2.5 � 2.86; n ¼ 3; P < 0.001) in the TB assay and95% � 5% in the CFU assay, while flow cytometry revealedthat 87% of the bacteria were still viable (Table 1, Fig. 4d).Flow cytometric enumeration of S. aureus revealed significant(P < 0.001) inhibition of bacterial growth following in-cubation with both nES and ES<500 (mean bacterialconcentrations were 4.3 � 106, 1.5 � 105 and 9.6 � 104 bacte-ria ml�1 for the live control, nES and ES<500 samples,

ation with 1% peptone water for 5 h; (b)e(d) E. coli following incubation with

cant rounding of cells, increased volume and different morphological changes.

hown in (c), and spherical bacteria are shown in (d). Scale bar ¼ 10 mm.


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Fig. 4. Flow cytometry of S. aureus. (a) and (b) Live and dead (isopropanol-killed) S. aureus controls following incubation with 1% peptone water for 6 h. Live

bacteria are green, dead bacteria are red, and bacteria of unconfirmed viability are blue; (c) and (d) S. aureus following incubation with nES (c) and ES<500 (d) for

6 h. The majority of bacteria are viable in both samples; (e) and (f) confocal microscopy images of bacteria from the live control (e) and after ES<500 treatment for

6 h (f). Viable bacteria appear green while dead bacteria are red. Scale bar ¼ 10 mm.


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respectively, compared with an initial inoculum of approxi-mately 3 � 104 bacteria ml�1). The same S. aureus samplesin confocal microscopy of live controls and ES<500-treatedbacteria showed differential labelling of viable bacteria ingreen and non-viable bacteria in red (Fig. 4e and f,respectively).

Incubation of E. coli with nES in the TB assay significantlyinhibited bacterial growth by approximately 99% (SI ¼ 1.3 �2.59; n ¼ 3; P < 0.001), and growth of bacterial colonies onagar was significantly reduced (P < 0.001), with only2.6% � 2.58% of bacteria producing CFUs compared with



controls (Table 1). In flow cytometry, however, 85% �0.41% of the bacteria were viable after incubation with nES(Table 1, Fig. 5c). When E. coli were incubated withES<500, the growth of E. coli was inhibited by 100%(SI ¼ �1.38 � 0.39; n ¼ 3; P < 0.001) in the TB assay andvery few colonies were formed on agar plates (3.1 �102 CFU ml�1 for ES<500-treated E. coli vs. 3.7 � 107 CFUml�1 in the control; approximating to 0.0008% � 0.0006%,Table 1). This showed a 100-fold reduction in bacteria fromthe initial inoculum of 3 � 104 ml�1, suggesting bactericidalactivity. Flow cytometry results showed that 40% of the


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Fig. 5. Flow cytometry of E. coli. (a) and (b) Live and dead (isopropanol-killed) E. coli controls following incubation with 1% peptone water for 6 h. Live bacteria

are green, dead bacteria are red, and bacteria of unconfirmed viability are blue; (c) and (d) E. coli following incubation with nES (c) and ES<500 (d) for 6 h. The

majority of bacteria are viable following treatment with nES (c), whereas an increased number of bacteria are present in the dead region following incubation with

ES<500; (e)e(h) confocal microscopy images of bacteria from the live control (e) and after ES<500 treatment for 6 h (feh). Viable bacteria appear green while dead

bacteria appear red. While the majority of bacteria were still rod-shaped after treatment with ES<500 (f,g), occasional racket-shaped bacteria were observed (h), as

previously described in Fig 3c. Scale bar ¼ 10 mm.


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bacteria were killed by ES<500 (Table 1, Fig. 5d). Confocalmicroscopy images of live controls and ES<500-treated bacte-ria are shown in Fig. 5eeh. Most E. coli maintained theirrod-shape during treatment with ES<500 (Fig. 5f,g), however,occasional racket-shaped bacteria were observed (Fig. 5h),as previously shown in Fig. 3c.

Flow cytometric enumeration revealed significant(P < 0.001) inhibition of E. coli growth after 6 h incubationwith nES and ES<500 (mean bacterial concentrations were1.5 � 107, 1.2 � 106 and 3 � 104 bacteria ml�1 for the livecontrol, nES and ES<500 samples, respectively, from an initialinoculum of approximately 3 � 104 bacteria ml�1). E. coli was



therefore more sensitive to ES<500 than nES, although this wasnon-significant. When the two bacterial species were com-pared, there was no significant difference between the sensitiv-ity of S. aureus and E. coli to nES, however, E. coli wassignificantly more sensitive to ES<500.

4. Discussion

The present study reports the effects of the L. sericataES<500 on the growth and morphology of MRSA strains andother bacteria and investigates the effect of nES and ES<500

on the viability of S. aureus and E. coli.


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Table 1

Comparison of results from methods used to assess bacterial growth and viability following incubation with nES and ES<500

Sample Growth Viability (by flow cytometry)

TB (SI) CFU (%)a Live (%) Dead (%) Unconfirmed (%)

S. aureus vs. nES 15.8 � 16.7 8.5 � 8.26 71.5 � 2.3 14.9 � 1.15 13.6 � 1.22

S. aureus vs. ES<500 2.5 � 2.86 5.2 � 5.02 87 � 0.56 9 � 0.44 4 � 0.13

E. coli vs. nES 1.3 � 2.59 2.6 � 2.58 85 � 0.41 10 � 0.53 5 � 0.18

E. coli vs. ES<500 �1.4 � 0.39 0.0008 � 0.0006 54 � 0.94 40 � 0.11 6 � 0.12

Bacterial growth was measured using the turbidometric (TB) assay and colony forming units (CFU) assay. Bacterial viability was measured using the molecular

probes, Syto-9 and propidium iodide in conjunction with flow cytometry. Data are expressed as arithmetic mean � SEM.a CFUs expressed as a percentage of control growth.


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The TB assay showed that ES<500 was active against Gram-positive and Gram-negative bacteria. Antibacterial activityagainst S. aureus was used as a reference to allow a tentativesensitivity spectrum to be constructed. The growth of S. epi-dermidis and P. mirabilis was not inhibited by ES<500 ornES using the TB assay, however, there is evidence of an im-portant biological effect on S. epidermidis biofilms by nES(Harris and Mack, unpublished). Proteus has been shown inprevious studies to be resistant to the effects of unfractionatedmaggot ES [13,20] and, in the present study, incubation of P.mirabilis and S. epidermidis with multiple doses of nES orwith a reduced bacterial inoculum failed to inhibit growth.

The L. sericata ES<500 was active against 12 MRSAstrains, including the epidemic strains EMRSA-15 andEMRSA-16, and the community-acquired MW2 strain. Theinitial inability of ES<500 to inhibit the growth of EMRSA-15 and MRSA 252 may be due to a dilution effect during prep-aration and/or partial degradation during storage at �20 �C.This was suggested by the sensitivity of strains EMRSA-15and 252 to nES, the demonstration of sample dilution duringultrafiltration by IC quantitation and restored activity againstEMRSA-15 and 252 with ES<500 prepared from nES storedat �80 �C.

Hourly microscopic examinations revealed distinctmorphological changes in B. cereus and E. coli, that becamevisible after approximately 3 h incubation with ES<500. Fila-mentation as observed in B. cereus can be induced by numerousmechanisms, including inhibition or overproduction of the pro-teins involved in septation [21,22], deregulation of proteolyticactivity [23] or indirectly by the SOS stress response, in whichcell division is inhibited until DNA damage is repaired [24].

The SOS response is well documented in E. coli [24], how-ever, the observation of lemon-shaped, racket-shaped andspherical E. coli rather than filaments suggested an alternativemechanism. Similar morphological changes to those observedin E. coli occur in Gram-negative bacilli following treatmentwith b-lactam antibiotics [25e28], which target penicillinbinding proteins in bacterial cell membranes.

B. subtilis was the most sensitive of the bacteria tested andghost formation suggested that the bacteria had been killed byES<500. It was impossible, however, to determine the viabilityof other bacteria exposed to ES<500 using the TB assay andlight microscopy alone. Viability staining in conjunctionwith flow cytometry measured bacterial membrane integrityas a function of viability following the treatment of S. aureus



and E. coli K12 with nES and ES<500. For S. aureus, the TBand CFU results showed significant inhibition of growth com-pared with controls following treatment with nES and ES<500.Flow cytometry revealed that the majority of bacteria treatedwith both nES and ES<500 were still viable, and therefore allthree methods suggested a bacteriostatic effect.

The TB assay, CFU assay and flow cytometry results agreedthat nES also had a bacteriostatic effect against E. coli, whileES<500 almost abolished colony formation in the CFU assay,reducing the initial bacterial inoculum 100-fold. This sug-gested a bactericidal effect and flow cytometry revealed only54% viability of E. coli incubated with ES<500 (comparedwith 85% viability of E. coli incubated with nES). The differ-ent effects on bacterial viability by nES and ES<500 may becaused by nutritional buffering exerted by the high molecularweight molecules in nES [14]. Flow cytometric analysisshowed that E. coli was significantly more sensitive toES<500 than S. aureus, concurring with the sensitivity spec-trum constructed from TB assay data (Table 1, Fig 1a). It isnot unusual for antibiotics to exert both bacteriostatic and bac-tericidal activity depending on the bacterial species targeted,whilst clinically the importance of bacteriostatic versus bacte-ricidal activity is under debate [29].

Traditionally, bacterial viability has been assessed using theability of bacteria to form colonies on agar plates. This is over-simplistic and there are many situations in which bacteria loseculturability but remain viable, as with E. coli followingexposure to nES and ES<500. This state is referred to as viablebut non-culturable and has been shown in many bacteria,including human pathogens, upon exposure to environmentalstresses [30]. Additionally, bacteria exposed to antibiotics donot immediately resume growth following drug removal. Apost-antibiotic effect, due to either temporary persistence ofthe compound at the bacterial target or drug-induced non-lethaldamage, prevents colony formation whilst the bacteria recover[31]. Numerous methods should therefore be used to assessbacterial viability following antibacterial exposure as theCFU assay alone may incorrectly indicate a bactericidal effect.

To summarize, ES<500 has broad-spectrum antibacterialactivity, including activity against a range of MRSA strains.It induces morphological changes in B. cereus and E. coli,and exerts either static or cidal effects depending on thebacterial species tested. Work covered by a UK patent appli-cation is underway to identify the antibacterial compound(s)within ES<500.


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Acknowledgements

Thanks to Dr L.G. Harris for advice and help with collatingbacteria and to Prof. D. Mack for his critical reading of thismanuscript. We thank Dr J. Lindsay and Prof. T. Miethke forkindly providing MRSA strains to our lab and Dr C. Thorntonfor use of the flow cytometer. Thanks to Dr P.J. Dyson for help-ful advice and Action Medical Research, grant AP1010, andthe Rosetrees Trust for financial support.

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2008), doi:10.1016/j.micinf.2007.12.011

the antibacterial activity against mrsa strains and other bacteria of a 500 da fraction from magg

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