xenorhabdus antibiotics: a comparative analysis and potential utility for controlling mastitis...

ORIGINAL ARTICLE

Xenorhabdus antibiotics: a comparative analysis andpotential utility for controlling mastitis caused by bacteriaG. Furgani1, E. Boszormenyi1, A. Fodor1,2, A. Mathe-Fodor1, S. Forst3, J.S. Hogan2, Z. Katona4,M.G. Klein5, E. Stackebrandt6, A. Szentirmai7, F. Sztaricskai4 and S.L. Wolf2

1 Department of Genetics, Faculty of Natural Sciences, Eotvos University, Budapest, Hungary

2 Department of Animal Sciences, The Ohio State University, Wooster, OH, USA

3 Department of Biology, University of Wisconsin-Milwaukee, Milwaukee, WI, USA

4 Research Group for Antibiotics of the Hungarian Academy of Science and Department of Pharmaceutical Chemistry, University of Debrecen,

Debrecen, Hungary

5 Department of Entomology, The Ohio State University, Wooster, OH, USA

6 DSMZ, Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Brauschweig, Germany

7 Department of Genetics and Applied Microbiology, Faculty of Science, University of Debrecen, Debrecen, Hungary

Introduction

The practical use of entomopathogenic nematode (EPN) ⁄bacterium (EPB) symbiotic complexes have spurred devel-

opments across a broad scientific front. The use of EPNs

as biological pesticides expanded in the 1980s (Gaugler

and Kaya 1990). Then, primarily taxonomic descriptions

of EPN strains, and their efficacy for controlling insect

pests, were investigated. Because of its commercial avail-

ability, Steinernema carpocapsae was widely evaluated as a

biocontrol agent. Later, a variety of new Steinernema

(hereafter referred to as S.) and Heterorhabditis species

and strains were discovered, and their corresponding

symbiotic EPB, Xenorhabdus spp. and Photorhabdus spp.,

respectively, received more attention (Forst and Nealson

1996; Gaugler 2002; Grewal et al. 2005). The genome of

Keywords

entomopathogenic nematodes ⁄ bacteria,

Escherichia coli, Klebsiella pneumoniae,

mastitis isolates, nematophin, Staphylococcus

aureus, Xenorhabdus antibiotics.

Correspondence

M.G. Klein, Department of Entomology, The

Ohio State University, OARDC, 1680 Madison

Avenue, Wooster, OH 44691, USA.

E-mail: [email protected]

2007 ⁄ 0379: received 10 March 2007, revised

24 August 2007 and accepted 25 August

2007

doi:10.1111/j.1365-2672.2007.03613.x

Abstract

Aims: The role of antibiotics produced by bacterial symbionts of entomopatho-

genic nematodes is to suppress growth of microbes in the soil environment.

These antibiotics are active against Gram-positive and Gram-negative bacteria,

and were tested against mastitis isolates from dairy cows.

Methods and Results: Two bioassays were adapted for Xenorhabdus antibiot-

ics; an overlay method on agar plates, and serially diluted, cell-free, Xenorhab-

dus cultures. The antimicrobial activities of the liquid cultures of 13 strains

from five Xenorhabdus species were further evaluated. Antimicrobial activities

of the type strains of X. nematophila, X. budapestensis and X. szentirmaii were

tested on mastitis isolates of Staphylococcus aureus, Escherichia coli and Klebsiel-

la pneumoniae with both bioassays. A previously reported antibiotic from X.

nematophila, nematophin, was synthesized in three steps from tryptamine and

4-methyl-2-oxovaleric acid sodium salt.

Conclusions: The antibiotics of all three Xenorhabdus strains were powerful in

either bioassay, but the sensitivity of the isolates differed from each other.

While Kl. pneumoniae was the least susceptible, Staph. aureus had the highest

sensitivity to each Xenorhabdus strain. Xenorhabdus szentirmaii and X. buda-

pestensis were more potent antibiotic producers than X. nematophila, and

raceme nematophin was ineffective against all mastitis isolates.

Significance and Impact of the Study: These results indicate that Xenorhabdus

antibiotics are effective against mastitis isolates and should be further evaluated

for their potential in mastitis control or prevention.

Journal of Applied Microbiology ISSN 1364-5072

ª 2007 The Authors

Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 104 (2008) 745–758 745

H. bacteriophora is presently being sequenced (Sandhu

et al. 2006) and the genome of its bacterial symbiont,

P. luminescens, has been published (Duchaud et al. 2003).

In addition, the genomic sequences of two Xenorhabdus

species have been completed and are being analysed

(http://maizeapache. ddpsc.org ⁄ bact_db). All of this has

led to an expansion of our understanding of the

EPN ⁄ EPB symbiotic association such that it is now con-

sidered a model system to address broad biological ques-

tions in mutualism, co-evolution and pathogenesis.

One area of research that has important implications in

agriculture, animal husbandry and human health is the

production of antibiotics by EPB. Akhurst (1982) first

demonstrated the antibiotic activity of cultures of Xenor-

habdus spp. against a wide variety of micro-organisms,

including fungi; Gram-positive Micrococcus, Staphylococcus

and Bacillus, as well as Gram-negative bacteria. Several

bioactive secondary metabolites have been reported from

cultures of Xenorhabdus species (Paul et al. 1981;

reviewed by Nealson et al. 1990; Sztaricskai et al. 1992;

Sundar and Chang 1993). The first paper from the Web-

ster laboratory appeared a few years later (Li et al. 1995).

Some strains, such as X. bovienii A2, have a remarkable

diversity of small molecule, antimicrobial compounds.

Xenomins and xenorxids, several xenorhabdins, including

three novel molecules and four indoles have been isolated

from this strain (Chen 1996). Several promising mole-

cules, such as xenorhabdins, commonly produced by

X. bovienii (McInerney et al. 1991a), and xenocoumacins

produced mainly by X. nematophila, symbionts of

S. feltiae (McInerney et al. 1991b), have been reported.

Later, other secondary metabolites with antibiotic activity

have been discovered. These include nematophin (Li et al.

1997) xenorxids (Li et al. 1998) and xenomins (reviewed

by Webster et al. 2002). Isaacson (2000) was the first to

study the antibiotics of the symbiont of S. riobrave. These

compounds were reported as showing in vitro activity

against Gram-positive bacteria, including multi-drug

resistant strains of Staph. aureus (Webster et al. 2002).

Recently, the antibiotic potential of the Hungarian

EPB stock collection consisting of 103 symbiotic bacte-

rial (Xenorhabdus and Photorhabdus) strains from

EPN has been compared (Szallas et al. 1997, 2001; M.

Hevesi, personal communication). Furthermore, the tax-

onomic status of four novel Xenorhabdus strains, iso-

lated from four Steinernema hosts from different

countries have been described as X. budapestensis, type

strain DSM 16342T, isolated from S. bicornutum; X.

ehlersii, type strain DSM 16337T, isolated from S. longi-

caudum (=serratum); X. innexi, type strain DSM 16336T

isolated from S. scapterisci; and X. szentirmaii,

type strain DSM 16338T, isolated from S. rarum (Leng-

yel et al. 2005). Two of the new species, X. budapesten-

sis and X. szentirmaii gave the largest inhibition zones

in preliminary tests. These findings provide the possibil-

ity of (i) a comparative analysis of the antibiotics pro-

duction of different Xenorhabdus species and strains,

with special attention to taxonomic heterogeneity and

(ii) determining whether the most promising Xenorhab-

dus antibiotics could be potential tools for controlling

Gram-positive and Gram-negative mastitis pathogens.

The primary mastitis pathogens include Staph. aureus,

Escherichia coli and Klebsiella pneumoniae (Smith and

Hogan 1993; Hogan and Smith 2003). Control of intra-

mammary infections and clinical mastitis on dairy farms

is often accomplished employing strict hygiene practices

to reduce exposure to potential pathogens, and the judi-

cious use of antibiotics to eliminate infections that do

occur. Mastitis is the most common reason for antibiotic

use in dairy herds, and thus antimicrobial resistance

of mastitis pathogens has received recent attention

(Woolford et al. 2001; Hogan and Smith 2003; Rajala-

Schultz et al. 2004; Robert et al. 2006; Berry and Hillerton

2007).

The discovery and successful application of new natu-

ral antibiotics, such as those produced by EPNs and

their symbiont bacteria, may provide an alternative way

of mastitis control, if they do not exert serious adverse

effects, or induce immediate resistance. Despite the

impressive data and patents available, there seems to be

more speculation than facts concerning Xenorhabdus

antibiotics, and nothing has been published about their

practical use. Therefore, we decided to carry out a sys-

tematic genus-wide search for antibiotics with potential

for use in mastitis control, based upon (i) reproducible

cell-to-cell competition in an overlay-based bioassay and

(ii) a direct bioassay of serially diluted, cell-free Xenor-

habdus cultures. We also started a systematic search for

natural antibiotics, especially in the recently described

Xenorhabdus species. This study is the first step towards

achieving those goals.

Materials and methods

Media and chemicals

Luria-Broth (LB), Luria Broth Agar (LBA), Tryptone-Soy-

Yeast (TSY) broth and agar, Blood Agar (BA) and Nutri-

ent Agar (NA) plates were used as described by Ausubel

et al. (1999). The components were obtained from Difco.

The 2· LB liquid media contained double the amount of

each component of LB and was used for diluting the

cell-free media in some bioassays (below). Antibiotics

(rifampicin, chloramphenicol, carbenicillin, kanamycin

and ampicillin) were obtained from Sigma and were also

used as described by Ausubel et al. (1999).

Xenorhabdus antibiotics – mastitis bacteria G. Furgani et al.

746 Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 104 (2008) 745–758

ª 2007 The Authors

Chemical synthesis of nematophin [3-indolethyl-(3¢-methyl-

2¢-oxo)-pentanamide]

The synthesis scheme is summarized in Fig. 1. Five

millilitres of 10% hydrochloric acid was added to

4-methyl-2-oxovaleric acid sodium salt (1Æ32 g in 10 ml

of distilled water). Compounds from the solution

(pH � 2) were extracted with ether (3 · 15 ml). The

combined organic phase was washed with saturated NaCl

(2 · 5 ml), dried over Na2SO4, and concentrated under

diminished pressure at <25�C giving a pale-yellow syrup

(1Æ32 g). To this syrup, 2Æ0 ml of thionyl chloride was

added at room temperature, and stirred at 50�C for 2 h.

Excess reagent was removed under reduced pressure, and

the resulting dark-yellow syrup, 4-methyl-2-oxovaleric-

acid-chloride was used. A solution of tryptamine [2-(3-

indolyl-(ethylamine)], 1Æ6 g in an excess (22 ml) of dry

pyridine was added, and the mixture was stirred at room

temperature for 2 h (after 40 min, a precipitate was

observed). The mixture was then diluted with water

(the precipitate dissolved) and extracted with ether

(3 · 15 ml). The organic phase was re-extracted with

saturated NH4Cl (2 · 10 ml), 5% NaOH (2 · 5 ml) and

saturated NaCl (2 · 10 ml). It was dried over Na2SO4,

filtered and concentrated (bath <30�C). The crude, dark-

brown residue was purified by thin-layer chromato-

graphy (TLC), on a Kieselgel G column (eluent: 1 : 2

ethyl acetate : hexane), to obtain asyrup (Ninhydrin Rf =

0Æ38), which was triturated with hexane. The solidified

product (raceme nematophin) was filtered (360 mg,

15Æ2%), mp 76–77�C.

Bacteria, culture and storage

Frozen bacterial stocks were plated and single colonies

were used for each experiment. Both the Xenorhabdus

and the mastitis causing bacteria were cultured on LBA

plates, the former at 25�C and the latter at 37�C. The

mastitis bacteria plates were stored at 4�C and the

Xenorhabdus plates at room temperature, never longer

than 3 weeks.

Antibiotic producing Xenorhabdus strains

Designations, names and origin of the strains are listed

in Table 1. They are all from the stock collection of

Dr A. Fodor. Previously, strain AN6 ⁄ I and ATTC 19061

were thought to be identical because of their close simi-

larity (Brunel et al. 1997; Volgyi et al. 2000). AN6 ⁄ I was

discovered by R. Akhurst, sent to ATTC where it was

given a Type number. However, many researchers

worked on the original AN6 ⁄ I received directly from

CH3

CH3OH

O

O

+ SOCl2

Cl

O

O

CH3

CH3+

NH

NH2

– HClAbsolute Pyridin

NH

NH

O

O

CH3

CH3

4-methyl-2-oxovaleric acid sodium salt

4-methyl-2-oxovaleric acid chloride Tryptamin

Racem nematophin [3-indolethyl- (3′-methyl-2′-oxo)-pentanamide]

*

*

*

HCl+

Figure 1 Scheme of chemical total synthesis for racem nematophin. The structure was validated by 1H and 13C-NMR, which showed the exis-

tence of four aromatic protons, three quaternary C, one vinyl-proton and two –CH2- as well.

G. Furgani et al. Xenorhabdus antibiotics – mastitis bacteria

ª 2007 The Authors


Dr Akhurst causing confusion between ATTC 19061 and

AN6 ⁄ I.

Test organisms (mastitis isolates and control bacteria)

Clinical isolates of mastitis-associated bacteria: six Staph.

aureus (Staph 1–6), six E. coli (E.c. 471, 683, 707, 727, 884

and 902), and one Kl. pneumoniae (696) (hereafter referred

to as Kl. pneumoniae) were used in this study. All were iso-

lated and identified at the Mastitis Laboratory of the

Department of Animal Sciences, The Ohio State University,

Wooster, OH, USA. Although, all are partially character-

ized and kept frozen, each is considered an isolate rather

than a strain. An E. coli, S17 kpir pKNOCK (Alexeyev 1999)

carrying a chloramphenicol-resistant plasmid, was pro-

vided by Dr Eric Martens while in the Laboratory of Dr

Heidi Goodrich-Blair (University of Wisconsin-Madison,

Madison, WI, USA) and used as a control. Another control

bacterium, Serratia entomophila Mo1 strain, was provided

by Dr Trevor Jackson, New Zealand. This control was used

since Serratia have been isolated from mastitis infections in

OH, USA (S. Wolf, personal communication).

Procedures used in different experiments

Comparison of sensitivities of different mastitis pathogens

to Xenorhabdus antibiotics, and the antibiotic potential

of different Xenorhabdus strains

Experiments 1 and 2. A bioassay based on cell-to-cell

competition in an overlay of test bacteria with the antibi-

otic producer colony on a solid media (hereafter referred

to as the overlay bioassay) was established and standard-

ized. This technique was adapted from Dr Noel Boe-

mare’s method (Akhurst and Boemare 1988; Boemare,

personal communication). The overlay bioassay allowed

us to standardize and compare (i) the sensitivities of the

different pathogens and (ii) the antibiotic production of

the type strains of X. nematophila, X. szentirmii and

X. budapestensis) (Experiment 1). Later on, different

strains of X. nematophila and X. bovienii, as well as one

strain of X. cabanillasii were used (Experiment 2). This

bioassay is based on measuring the diameter of the

inactivation circle, after 5 days on LBA plates, around

the colony from 5 ll of an overnight culture of the

Table 1 Designation, source, hosts, origins and accession numbers of Xenorhabdus used

Species and strains Source Steinernema host Country of origin 16S rDNA accession no.

X. nematophila�

ATCC 19061T ATCC/

R. Hurlbert and S. Forst

S. carpocapsae USA –

AN6 ⁄ 1� R. Akhurst S. carpocapsae USA AY278674

N2-4§ Jim Lindgren–– S. carpocapsae Mexico Z7637

RIO– Byron Adams–– S. riobrave TX, USA Z7638

DSM3370T§ Erko Stackebrandt Czech Republic X82251

X. bovieni§

DSM 4766T Erko Stackebrandt S. feltiae Russia X82252

SF22§ Aana Vainio–– S. feltiae SF22 Finland Z77210

Vije§ Lonne Gerritsen–– S. feltiae Vije Norway Z77211

Sulcatus§ Lonne Gerritsen–– S. feltiae Sulcatus USA Z77212

IS6�� Itamar Glazer–– S. feltiae IS6 Israel (Negev) –

NYH�� Andras Fodor–– S. feltiae Hungary –

X. cabanillasii

RIO–,�� Byron Adams–– S. riobrave TX, USA Z7638

X. beddingii

DSM 4764T§,�� Erko Stackebrandt S. longicaudatum Shen and Wang, China X82254

X. budapestensis

DSM 16342T§§ Bela Tallosi–– S. bicornutum Hungary (Vajdasag) AJB10293

X. szentirmaii

DSM 16338T§§ Byron Adams–– S. rarum Argentina (Cordoba) AJB10295

�ATCC, American Type Culture Collection, Rockville, MD, USA. Xu et al. (1991), Brunel et al. (1997), Volgyi et al. (2000).

�Volgyi et al. (2000).

§Szallas et al. (1997).

–Szallas et al. (2001).

��Unpublished strain.

��New information about its taxonomic position (Tailliez et al. 2006).

§§Lengyel et al. (2005).

––Source of nematode, bacteria was isolated in Hungary.TType strain.



ª 2007 The Authors

antibiotic-producing cells. Since the inactivation zone is

circular and the thickness of the LBA was known, we

could calculate the biologically active volume of the anti-

biotics. The details of the procedure were as follows: For

preparation of the Xenorhabdus cultures, 5 ml of LB were

inoculated with a single colony of the antibiotic-produc-

ing bacterium from an LBA plate, and incubated at 28�C

in a shaker incubator (200 rev min)1) overnight. At least

two replicates of 5 ll from the culture were pipetted onto

the centre of the LBA (or TSY) test plates. After the sus-

pension was dried, bacteria were allowed to grow for

5 days. For preparation of the test organisms, 5 ml of LB

media were inoculated 1 day before each test with a single

colony of mastitis pathogen or control bacteria, and incu-

bated at 28�C as above. Two 100 ll aliquots of the patho-

gen or control culture were mixed with 3Æ5 ml of approx.

54�C soft (0Æ6% w ⁄ v) agar. The aliquots were then lay-

ered over the test plate where the Xenorhabdus colony

had been growing. After the soft agar solidified, the Petri

plates were incubated at 37�C for 48 h.

Experiment 3. A bioassay of cell-free Xenorhabdus liquid

cultures was established, standardized and conducted for

(i) screening Xenorhabdus strains for their antibiotics pro-

duction qualitatively and (ii) determining the maximum

inhibiting dilutions (MID) of the different cell-free cul-

tures on the respective test organisms. In first screen, 50%

of the overnight Xenorhabdus cultures were diluted with

2· LB. Cell-free Xenorhabdus media were prepared as fol-

lows: 5 ml of LB was inoculated with a single colony of

the antibiotic-producing bacterium and incubated as

described for Experiment 1. This culture was either used

directly in screening Xenorhabdus strains, which gave posi-

tive antibiotic activity in the overlay bioassay; or served as

an inoculum in a sterile 100 ml LB culture in an Erlen-

meyer flask, grown at 28�C in an incubated shaker

(200 rev min)1) for 6 days. The optimum time for antibi-

otic production is 5–6 days. These cultures were then used

for MID determination. Each culture was centrifuged

using a Sorvall SW34 Rotor at 13 000 rev min)1 for

35 min (5172 RCF or g). The supernatant was filtered

through a sterile 0Æ22 mm nylon filter and centrifuged

again at the same speed. These preparations were consid-

ered to be cell-free cultures of the antibiotic-producing

Xenorhabdus strains. They were stored at 4�C before use.

To guarantee cell-free preparations, at least two replicates

of each were diluted with sterile 2· LB and incubated with

the experimental samples. This kind of bioassay could be

carried out either in test tubes, or different kinds of

microtitre plates. Test tubes contained 4Æ9 ml of cell-free

culture and 100 ll of a 10· diluted O ⁄ N culture (O ⁄ N)10)

of the test organism. A serial dilution of the cell-free cul-

ture was used: 0, 0Æ5, 1Æ0, 1Æ5, 2Æ0, 2Æ5 and 3Æ0 ml of the

cell-free Xenorhabdus culture were diluted with 4Æ9, 4Æ4,

3Æ9, 3Æ4, 2Æ9, 2Æ4 and 1Æ9 ml of fresh 2· LB media, repre-

senting 0, 10, 20, 30, 40, 50 and 60% v ⁄ v (all dilutions are

v ⁄ v unless otherwise noted) of the cell-free culture, respec-

tively. We used two replicates for each concentration and

repeated all tests in time. These tubes were incubated in

the shaker incubator at 28�C (200 rev min)1).

Evaluation

The tubes were evaluated either visually (+ or ) growth)

or quantitatively by measuring the OD values at 610 nm

in a spectrophotometer. The respective dilution of the

cell-free Xenorhabdus culture was considered as effective if

Table 2 Variability in millimetres of the

diameter of inhibition zone on LBA and TSY

agar plates in overlay bioassays

Tested on

Xenorhabdus species and type strains

X. nematophila

ATCC91061T

X. budapestensis DSM

16342T

X. szentirmaii DSM

16338T

Mean ± SE CV+ Mean ± SE CV+ Mean ± SE CV+

Total Staphs

LB 51Æ11 ± 1Æ15 9Æ56 41Æ28 ± 0Æ76 7Æ79 61Æ28 ± 1Æ88 13Æ0

TSY 38Æ67 ± 5Æ90 37Æ6 37Æ83 ± 3Æ38 21Æ9 64Æ50 ± 4Æ90 18Æ6

Total E.c.

LB 31Æ78 ± 0Æ30 3Æ97 32Æ72 ± 0Æ75 9Æ77 51Æ72 ± 1Æ41 11Æ6

TSY 32Æ33 ± 0Æ92 6Æ96 39Æ17 ± 1Æ42 6Æ96 51Æ50 ± 6Æ02 28Æ6

Total K.p.

LB 26Æ00 ± 1Æ73 11Æ5 38Æ00 ± 0Æ57 2Æ63 51Æ00 ± 0Æ58 1Æ96

TSY 26Æ00 ± 3Æ00 16Æ3 36Æ00 ± 1Æ00 3Æ93 47Æ00 ± 1Æ00 15Æ0

LB, Luria Broth Agar, average of 18 data points; TSY, Tryptone-Soy-Yeast agar, average of six

data points; CV+, coefficient of variation (%) calculated with MiniTab14 program. Staphs,

pooled data of Staphylococcus aureus isolates; E.c., pooled data of Escherichia coli isolates;

Kl.p., pooled data of Klebsiella pneumoniae 696 isolate.


ª 2007 The Authors


there was no growth (labelled with ‘+’ for positive antibi-

otic activity in the tables) of the test organism. Twelve-

wells microtitre plate experiments were performed with

2Æ20 ml of cultures consisting of 2Æ16 ml of cell-free

Xenorhabdus culture and 40 ll of a 10· diluted O ⁄ N cul-

ture of the mastitis-causing bacteria. The dilution series

of the cell-free culture was: 220, 440, 660, 880, 1100 and

1320 ll of cell-free Xenorhabdus media diluted with 1940,

1760, 1540, 1280, 1060 and 840 ll of fresh 2· LB liquid

media, resulting in 10, 20, 30, 40, 50 and 60% of the cell-

free culture. Each organism was tested in a separate mic-

rotitre plate containing two replicates of each antibiotic

concentration. Two separate microtitre plates served as a

control. One of them contained two replicates of 2Æ22 ll

of 10% of the different cell-free cultures without test bac-

teria, while the other two had 2Æ16 ml of 2· LB media

inoculated with 40 ll of O ⁄ N)10 of the respective test

bacteria. Incubation was at 37�C when mastitis pathogens

or any E. coli strains were studied.

After O ⁄ N incubation (at 37�C or 28�C) each well was

examined visually for the propagation of the cells of the

test organism. As with the test tubes, for determination of

the MID values, serial dilutions of 10, 20, 30, 40, 50 and

60% of each respective cell-free culture were used. The

smaller the MID value, the stronger the antibiotic pro-

duction obtained.

Experiment 4. This set of experiments was set up to study

the correlation between the data obtained in overlay, and

direct bioassays of cell-free media. For the latter, we used

the same procedure as in Experiment 3. Samples

(2 · 5 ll) were taken from each inoculum and used for

cell-to-cell competition tests using the same procedure as

described in Experiments 1 and 2.

Determination of the biological activity of synthetic

raceme nematophin in comparison with known antibiotics

Experiment 5. Experiment 5 determined the biological

activity of commercial antibiotics. In the first screen,

100 ll of O ⁄ N cultures from each of the 13 mastitis iso-

lates were plated on LBA plates containing selected

micrograms per millilitre concentration of kanamycin

(Km30), rifampicin (Rif100), carbenicillin (Ca75) and

chloramphenicol (Chl25). To evaluate rifampicin resis-

tance in Ec707 and 902 (the two least tolerant E. coli mas-

titis isolates) on LBA plates, 5 ml of LB media was

inoculated with a single colony of the two isolates, and

also with the nonpathogenic (rifampicin sensitive) E. coli

S17 kpir pKNOCK strain. When the cultures reached their

stationary phases, 100 ll of each was seeded on LBA

plates containing 0, 25, 50, 100 and 200 lg ml)1 rifampi-

cin. The plates were incubated at 37�C for 1 day and the

colony growth was scored. To test the degree of rifampi-

cin resistance of Ec707 and 902 in liquid, a serial dilution

of rifampicin with 0, 25, 50, 100 and 200 lg ml)1 was

made in 5 ml LB and inoculated with 100 ll O ⁄ N cul-

tures of the mastitis isolates as well as with the nonpatho-

genic rifS E. coli S17 kpir pKNOCK strain as a control.

Cultures were incubated in a shaker incubator at 37�C

and examined visually every 4 h for 16 h. To test the rif-

ampicin resistance of single colonies, the same procedure

was followed, except single colonies from plates were

directly used as inoculum.

Experiment 6. Experiment 6 determined the antimicrobial

activity of synthetic raceme nematophin in three test sys-

tems. Nematophin was used directly on NA plates against

Bacillus subtilis. Nutrient agar plates were seeded with

heat-activated B. subtilis spores. A 1 cm diameter hole

was made with a sterile cork borer, and 0Æ5 ml of a

100 mg ml)1 solution of synthetic nematophin (dissolved

in methanol) was added. The plates were incubated at

37�C O ⁄ N. A second experiment used 1 cm diameter

sterile filter paper discs placed in the centre of LBA plates

that were overlaid with 100 ll of O ⁄ N culture of Staph.

aureus (Staph 6) diluted with 3Æ5 ml of soft agar as

described for Experiments 1 and 2. The plates were incu-

bated O ⁄ N at 37�C and then evaluated. The third experi-

ment used 8 · 12-well microtitre plates. Simultaneously,

two replicates of three concentrations of nematophin and

chloramphenicol were tested. The test organisms included

four staphylococcal isolates, two E. coli and one Kl. pneu-

moniae, as well as the chloramphenicol-resistant E. coli

strain (S17 kpir pKNOCK). As a control, 10% methanol

was added to the LB media. The plates were incubated at

37�C O ⁄ N and then evaluated.

Other methods

Statistical analyses (including basic statistics, homogeneity

tests, two-sample t-tests and regression analysis) were

carried out using the MiniTab Release 14 Statistical

Software program (see the Minitab Web site http://www.

minitab.com for a complete list of features available in

Release 14).

Results

The sensitivities of different mastitis isolates to Xenorhab-

dus antibiotics, and the antibiotic potential of different

Xenorhabdus strains in Experiment 1 allowed establish-

ment and standardization of a bioassay for Xenorhabdus

antibiotics based on an overlay of the test bacteria. It also



ª 2007 The Authors

allowed a comparison of the sensitivities of three groups

of mastitis isolates to Xenorhabdus antibiotics in general.

To standardize the overlay bioassay of Xenorhabdus

antibiotics, a medium was chosen on which (i) both the

antibiotic producer (Xenorhabdus) and each mastitis bac-

terium grew well, (ii) the results were reliable and repro-

ducible and (iii) the variability of the data was within a

narrow range. We tested the antibiotic production of X.

nematophila, X. szentirmaii and X. budapestensis on LBA

and TSY. A comparison of coefficients of variation (CV)

in Table 2 showed that LBA was a better media for this

purpose. Therefore, we used LBA routinely thereafter.

Data in Table 3 allowed us to compare the sensitivities

of the three groups of mastitis isolates to the Xenorhabdus

antibiotics studied. Each group reacted differently to the

various Xenorhabdus antibiotics studied. Within each of

the two mastitis isolates represented by more than one

isolate, the reaction was fairly uniform. The CV values

within each group were acceptably low (3Æ33, 12Æ76 and

15Æ46, respectively), and intraspecific variability did

not exceed intrastrain variability. Statistic analysis (two-

sample t-tests) backed up these observations.

Staphylococci were much more sensitive than E. coli iso-

lates, and the Kl. pneumoniae was more tolerant than E.

coli. The differences between Staphylococcus and E. coli

strains, as well as between Staphylococcus and Kl. pneumo-

niae, were highly significant (P < 0Æ001). With X. nemato-

phila, a smaller, but still significant difference was found

between E. coli isolates and Kl. pneumoniae. Data in

Table 3 also allow comparison of the relative strengths of

the antibiotics from the three Xenorhabdus type strains

with each other. With staphylococci, the order is X. szen-

tirmaii (P < 0Æ05), X. nematophila (P < 0Æ001), X. buda-

pestensis (P < 0Æ001). With the E. coli, the order is

X. szentirmaii (P < 0Æ05), X. budapestensis (P < 0Æ05),

X. nematophila (P < 0Æ05). The order with Kl. pneumoniae

was the same as for E. coli, but the differences were all

highly significant (P < 0Æ001).

The biologically active quantity of antibiotics produced

by each Xenorhabdus colony was calculated as the volume

(cm3) of LBA within the inactivation zone (Table 4). The

statistical analysis of the distribution of these data con-

firms that the differences between the data sets from

Staphylococci, E. coli and Kl. pneumoniae were highly sig-

nificant (P < 0Æ01).

Intraspecific homogeneity in X. nematophila, intraspecific

heterogeneity in X. bovienii, antibiotic production based

on two different bioassays

In Experiment 2, the same overlay bioassay as in Experi-

ment 1 was used to compare antibiotic production of dif-

ferent strains of X. nematophila and X. bovienii, and one

strain of X. cabanillasii. Table 5 shows the antibiotic pro-

duction of the different X. nematophila strains to be

rather similar to each other. The pooled data have a nor-

mal distribution. The antibiotics of the type strain and

the other three X. nematophila strains were significantly

(P < 0Æ01) less effective on Kl. pneumoniae than on Staph.

aureus. The antibiotics from the Staph. riobrave symbiont,

X. cabanillasii, did not differ significantly from the other

three. However, the antibiotic production of X. bovienii

(Table 5) differed greatly between strains. Twenty-five test

plates were used for each strain and measurable antibiotic

activity could be detected on all of the X. nematophila

plates. Of the six X. bovienii strains, only NYH produced

antibiotics on all plates. Table 5 also presents the

Table 3 Effects of antibiotics in overlay bioassay from three Xenor-

habdus type strains on mastitis isolates

Tested on


X. nematophila

ATCC91061T

X. budapestensis

DSM 16342T

X. szentirmaii

DSM 16338T

Staph 1 50Æ33 ± 0Æ33� 44Æ33 ± 2Æ96 51Æ67 ± 3Æ84

Staph 2 50Æ00 ± 1Æ15 40Æ33 ± 0Æ88 64Æ00 ± 0Æ58

Staph 3 53Æ33 ± 5Æ30 40Æ33 ± 0Æ33 65Æ00 ± 0Æ57

Staph 4 50Æ00 ± 0Æ58 38Æ33 ± 1Æ67 62Æ00 ± 0Æ57

Staph 5 49Æ67 ± 3Æ20 43Æ67 ± 1Æ86 71Æ00 ± 0Æ58

Staph 6 53Æ33 ± 4Æ37 40Æ67 ± 0Æ67 54Æ00 ± 10Æ0

E.c. 471 37Æ33 ± 0Æ33 31Æ33 ± 1Æ33 51Æ33 ± 1Æ45

E.c. 673 32Æ00 ± 0Æ57 36Æ33 ± 0Æ88 41Æ00 ± 0Æ58

E.c. 707 31Æ00 ± 0Æ58 34Æ00 ± 0Æ58 55Æ00 ± 0Æ58

E.c. 727 31Æ00 ± 0Æ58 31Æ00 ± 0Æ58 52Æ00 ± 0Æ58

E.c. 884 33Æ67 ± 0Æ33 30Æ00 ± 0Æ57 60Æ00 ± 0Æ57

E.c. 902 30Æ67 ± 0Æ33 37Æ00 ± 0Æ58 51Æ00 ± 0Æ57

K.p. 696 26Æ00 ± 1Æ73 38Æ00 ± 0Æ57 51Æ00 ± 0Æ58

Staph, clinical isolate of Staphylococcus aureus; E.c., clinical isolate of

Escherichia coli; K.p., clinical isolate of Klebsiella pneumoniae.

�Mean ± SE diameters of the inhibition zones (mm) around the antibi-

otic-producing colonies on LBA plates (n = 3).

Table 4 Calculated amount of antibiotics produced on plates in over-

lay bioassays

Tested on


X. nematophila

ATCC91061T

X. budapestensis

DSM 16342T

X. szentirmaii

DSM 16338T

Staphylococcus

aureus�

9Æ55 ± 0Æ40§ 6Æ72 ± 0Æ72 15Æ37 ± 0Æ42

Escherichia coli� 4Æ21 ± 0Æ78 5Æ92 ± 1Æ16 10Æ76 ± 2Æ68

Klebsiella

pneumoniae�

2Æ74 ± 0Æ65 5Æ30 ± 0Æ68 10Æ24 ± 0Æ32

� n = 18 for each Xenorhabdus strain.

� n = 3 for each Xenorhabdus strain.

§ Mean ± SD of cm3 of saturated Luria Broth Agar in the inhibition

zone.


ª 2007 The Authors


mean ± SE of X. bovienii test plates. The X. bovienii DSM

4766 type strain produced antibiotics on 14 plates, Vije

produced on six and SF22 produced on only four of the

25 test plates. Neither IS6 nor Sulcatus produced any

detectable antibiotics in our bioassays. However, on the

basis of (i) colony morphology and dye uptake on LBTA,

NBTA (Luria Broth and Nutrient Brome Thymol-blue

Agar) and Mac Conkey indicator plates; (ii) phospholi-

pase-lecitinase activity, tested on yolk agar; and (iii) pro-

teolytic activity, tested on gelatin agar by Frazier’s

method (Akhurst and Boemare 1988; Boemare et al.

1996, personal communication; Somogyi et al. 2002), they

both showed all of the Xenorhabdus phenotype character-

istics.

Experiment 3 compared the antibiotics from cell-free

cultures of different strains of X. nematophila and X. bovi-

enii, as well as the one strain of X. cabanillasii. Cell-free

Xenorhabdus cultures were used for screening, and also to

determine the MID values. Only those Xenorhabdus

strains were tested which showed some antibiotic activity

against the mastitis isolates in Experiment 2. Asall isolates

of staphylococci were sensitive to Xenorhabdus antibiotics,

each Xenorhabdus strain was tested first on (at least) one

Staph. aureus isolate. If the test 23 was positive, the strain

was tested on (at least) one E. coli isolate, and if positive,

on Kl. pneumoniae as well. Results from X. nematophila,

X. cabanillasii and X. bovienii strains are presented in

Table 6. The MID values of the strains, which were not

Table 6 Antibiotic activity of 50% (v ⁄ v) of

cell-free O ⁄ N cultures of X. nematophila,

X. cabanillasii and X. bovienii strains

50% v ⁄ v of cell-free

O ⁄ N culture of

Tested on

Staph. aureus E. coli Kl. pneumoniae

Staph 1 Staph 6 E.c.471 E.c.727 E.c.884 696

X. nematophila

ATTC61061 ) + ) + + 0

AN6 ⁄ 1 + + + + + 0

DSM3370 + ) 0 ) + 0

N2-4 + + + ) + 0

X. cabanillasii

RIO + + + + + +

X. bovienii�

DSM 4466 + 0 0 0 0 0

Hungary, NYH + + + + + +

+, 50% cell-free O ⁄ N Xenorhabdus culture completely inhibited test organism growth; 0, 50%

cell-free culture did not exert any detectable antimicrobial activity; -, not tested. Staph. aureus,

Staphylococcus aureus, E. coli, Escherichia coli, and Kl. pneumoniae, Klebsiella pneumoniae.

�50% cell-free cultures of SF22, Vije, Sulcatus and IS6 strains showed no antimicrobial activity.

Table 5 Inhibition zones (mm) caused by

X. nematophila, X. cabanillasii and X. bovienii

strains in overlay bioassaysXenorhabdus

Species, strain

Tested on

Staph. aureus

Pooled data

E. coli Pooled

data

Kl. pneumoniae

#696

Serratia

Mo1

X. nematophila

AN6 ⁄ I 51Æ8 ± 4Æ5� 47Æ2 ± 5Æ0 30 ± 1Æ1 56Æ3 ± 4Æ5

DSM 3370 54Æ16 ± 1Æ3 49Æ5 ± 1Æ1 34Æ1 ± 3Æ2 60Æ3 ± 3Æ6

N2-4 58Æ25 ± 2Æ1 48Æ0 ± 6Æ1 32Æ0 ± 6Æ4 43Æ6 ± 2Æ8

X. cabanillasii

RIO 60Æ7 ± 0Æ7 54Æ8 ± 3Æ5 32Æ4 ± 3Æ5 58Æ2 ± 1Æ7

X. bovienii

DSM 4766 33Æ9 ± 3Æ8 (n = 6) 29Æ7 ± 1Æ2 (n = 3)� 27Æ3 ± 4Æ4 (n = 4) NT

NYH 42Æ5 ± 4Æ2 (n = 10) 33Æ0 ± 1Æ5 (n = 11) 34Æ3 ± 1Æ9 (n = 6) NT

SF22 37Æ5 ± 8Æ1 (n = 4) 0 0 NT

Vije 29Æ0 (n = 1) 26 ± 2Æ0 (n = 2) 26 ± 2Æ1(n = 3) NT

�Mean ± SE; n = 12 for Staph. aureus and E. coli; n = 6 for Kl. pneumoniae, and n = 3 for

Serratia entomophila. NT ¼ not tested

�For each test bacteria, at least six test plates were used. N = only plates with inactivation zone.



ª 2007 The Authors

dropped after the first screen, were determined using a

serial dilution of each respective cell-free Xenorhabdus

culture, using 12-well plates. Table 7 shows the maximum

dilutions of the respective cell-free Xenorhabdus culture,

which gave complete inactivation of the test organism.

Table 7 also shows the statistical analyses of these data.

The antibiotics from each of the four X. nematophila

strains, as well as those of the X. cabanillasii strain, were

very uniformly effective against Staph. aureus, but less

effective against Kl. pneumoniae. Of the three E. coli

tested, the laboratory strain (S17kpir.pKNOCK) was the

most tolerant. Its sensitivity was similar to that of

Kl. pneumoniae. These data showed a normal distribution

and comprised three sharply distinguishable pools repre-

senting Staphylococci, E. coli and Kl. pneumoniae. The sta-

tistical analysis also showed that the data pools of the

three test organisms were significantly different. On the

other hand, only one of the six X. bovienii strains (NYH)

produced antibiotics that were effective against all the test

organisms, with the results being similar to those in the

overlay bioassays (Table 5). The antibiotics produced by

the type strain (DSM 4466) were effective only against

Staph. aureus.

Experiment 4 comprised an independent set of experi-

ments, including overlay-bioassays and cell-free bioassays

on the same X. nematophila strains. The distribution and

the intra-group homogeneity of the data from the two

bioassays were determined and statistically analysed using

the Mini-Tab program listed above. The distribution of

the data obtained in both bioassays was similar.

Biological activity of synthetic raceme nematophin compared

to known antibiotics

Experiment 5 was an evaluation of some commercial

antibiotics and the synthesized nematophin. All, the mas-

titis bacterial isolates were sensitive to chloramphenicol.

Only the E. coli S17kpir pKNOCK was resistant to chl-

oramphenicol, but it was sensitive to the other three com-

mercial antibiotics. All but Kl. pneumoniae were sensitive

to carbenicillin, and that isolate was resistant to rifampi-

cin and kanamycin. The Staphylococci and E. coli isolates

showed a significant variability in their sensitivity to

Table 7 Antibiotic activity (maximum inhibit-

ing dilution; MID) of 6-day-old cell-free cul-

tures of X. nematophila, X. cabanillasii and X.

bovienii strainsTested on

Staph. aureus E. coli Klebsiella Serratia

Staph 6 S17kpir pKNOCK� E.c. 707 E.c. 902 #696 Mo1

X. nematophila

ATCC 91061 10� 40 30 20 40 20

AN6 ⁄ I 10 40 30 20 40 20

DSM 3370 10 40 30 30 40 20

N2-4 20 50 30 40 50 20

X. cabanillasii

RIO 10 30 30 30 40 10

X. bovienii§

DSM 4466 20 >>60 >>60 >>60 >>60 >>60

Hungary NYH 20 20 20 20 40 20

�Control.

�Each value is an average of two replicates with no difference between the two replicates.

§The 6-day-old cell-free cultures of SF22, Vije, Sulcatus and IS6 strains did not exert detectable

antibiotics activity in this test. Staph. aureus, Staphylococcus aureus; E. coli, Escherichia coli;

Klebsiella, Klebsiella pneumonia; Serratia, Serratia eutomophila.

Staph. aureus,

n = 10

Serratia,

n = 10

E. coli,

n = 30

Klebsiella,

n = 10

Pooled data from Table 7

Mean ± SD� 12Æ00 ± 4Æ47 18Æ00 ± 4Æ47 32Æ67 ± 7Æ99 42Æ00 ± 4Æ47

Staph. aureus P-values 0Æ067 <0Æ0001 <0Æ0001

Serratia P-values <0Æ0001 <0Æ0001

E. coli P-value 0Æ007

There was no significant difference between X. nematophila strains in any of the four tests. The

normal distribution of the data allows treating them as single pools of maximum inhibiting dilu-

tion values of X. nematophila as a species. The analysis based on two-sample t-tests (MiniTab

14). Staph. aureus, Staphylococcus aureus and E. coli, Escherichia coli.

�SD given, as they demonstrate better the low variability of these data than the SE of the

mean.


ª 2007 The Authors


rifampicin. Only Staph 6 was rifampicin resistant of that

group, and only E.c. 727 was completely sensitive. On rif-

ampicin-free plates seeded with 100 ll of O ⁄ N LB cul-

tures of the mastitis isolates, there were bacterial lawns

with uncountable colonies. With 100 lg ml)1 rifampicin

plates, there were bacterial lawns of E.c. 471, 683 and 884.

On the other hand, there were a few colonies of E.c. 707

and 902. Rifampicin-free liquid cultures of both strains

reached their stationary phase within 4 h when inoculated

with single colonies, while there was no growth in

100 lg ml)1 rifampicin-containing cultures by that time.

On the other hand, when the 5 ml LB culture was inocu-

lated with 100 ll of O ⁄ N culture of either of the two iso-

lates, the cells propagated and reached their stationary

phase at 12–16 h. While only Staph 3 and Staph 6 were

resistant to kanamycin, E.c. 727 was strongly resistant and

just E.c. 471 was sensitive.

Bioassays of synthetic raceme nematophin on mastitis

pathogens

Results of the physicochemical analysis and molecular

identification of the structure of the synthetic racem

nematophin [3-indoleethyl-(3¢-methyl-2-oxo-) pentanamide]

The synthesized antibiotic is Ninhydrin positive and was

isolated by TLC. The analytical parameters are presented

in Table 8.

Experiment 6. When nematophin was directly added to

NA plates and tested on B. subtilis, no antimicrobial effect

could be detected. With Staph 6, using sterile filter paper

discs of 1 cm diameter, there was no inactivation ring

around the nematophin at any concentration. The plates,

with chloramphenicol concentrations of 5, 10, 15 and

20 ll of a 150 mg ml)1 stock solution, had inactivation

zones of 13Æ15 (±0Æ10), 14Æ20 (±0Æ13), 15Æ00 (±0Æ12) and

15Æ5 mm (±0Æ11), respectively. The results from 8 ·12-well microtitre plates also showed the synthetic ne-

matophin had no inhibition. On the other hand, chloram-

phenicol, at even the lowest concentration completely

inactivated all the test organisms (four staphylococci, two

E. coli isolates and Kl. pneumoniae), with the exception of

the chloramphenicol-resistant laboratory strain (S17kpir

pKNOCK), which grew at all concentrations.

Discussion

The mastitis pathogen isolates in this study were screened

for their sensitivity to a few commercially available antibi-

otics. Isolates from the same species differed in their anti-

biotic resistance profile, especially to rifampicin. All but

two E. coli isolates (E.c. 707 and 902) were resistant to

100 lg ml)1 rifampicin. These two showed an unusual

behaviour, as described in the results. On plates, the

number of colonies was dose-independent. In liquid, the

cells the two isolates propagated in the presence of rifam-

picin, but reached their stationary phase much later than

the resistant E. coli isolates. If E.c. 707 and 902 were sen-

sitive to rifampicin, no colonies would be seen on the rif-

ampicin plates, but some were always found. If the two

isolates were sensitive, there would be no growth in liquid

with the antibiotic. On the other hand, if the E.c. 707 and

Table 8 Analytical parameters of the synthetic racem nematophin[3-indoleethyl-(3¢-methyl-2-oxo)pentanamide]

TLC

Compound

Nematophin Rf 0Æ38

Solvent system

Ethyl acetate-hexane 1 : 2

Developed by

Ninhydrin

EI-MS 272 m ⁄ z (20%)1H-NMR 500Æ13 MHz, CDCl3, d (ppm)

0Æ9 (C-17, t), 1Æ1 (C-15, d), 1Æ4 (C-16b, m), 1Æ8 (C-16a, m)

3Æ1 (C-10, t), 3Æ5 (C-15, m), 3Æ7 (C-11, q), 7Æ0 (C-2 d),

(C-8, t),

NH-1, s)13C-NMR 125Æ7 MHz, CDCl3, d (ppm)

11Æ92 (C-17), 15Æ57 (C-18), 25Æ57 (C-16), 28Æ85 (C-10),

39Æ93(C-11), 40Æ78 (C-15), 111Æ71 (C-6), 112Æ89 (C-3)

119Æ07 (C-2), 122Æ46 (C-8), 122Æ71 (C-7), 127Æ54 (C-4),

136Æ84 (C-5), 160Æ44 (C-13) 164Æ90 (C-14)

IR (KBr) cm)1

3364 m NH (aromatic NH), 3298 m NH (amid NH),

3100–3000 m CH, 3000–2800 m35 m5

CH(CH2, CH3), 1768 m, 0 = 0, 1682 m amid-I

TCL, thin layer chromatography; IR, infrared spectroscopy; EI-MS, mass spectroscopy.



ª 2007 The Authors

902 strains were resistant to rifampicin, bacterial lawns

would have been found on all rifampicin plates and liquid

cultures would have reached their stationary phases

within 4 h. A possible explanation may be a high fre-

quency of spontaneous rifR mutants in originally sensitive

E. coli 707 and 902 isolates. The differences between the

1-colony-inoculated and 100 ll O ⁄ N inoculated liquid

cultures support this interpretation. These observations

also support the resistance to mastitis therapy with antibi-

otics found by Rajala-Schultz et al. (2004). Inducible

resistance to Xenorhabdus antibiotics was not observed in

either staphylococci or coliforms evaluated so far.

In this study, reliable bioassays were established. The

overlay bioassay, optimized on LBA plates, is based on

measuring the diameter of the inactivation ring around

the antibiotic-producing colony. On LBA plates, both the

Xenorhabdus antibiotic producer and the mastitis test

organism could be grown reliably. The antibiotic produc-

tion was favourable and the results were highly reproduc-

ible demonstrated by the normal distribution and low

SD. A bioassay utilizing a serial dilution of cell-free

Xenorhabdus cultures was also developed. Although con-

siderable antibiotic activity could be demonstrated from

an O ⁄ N Xenorhabdus culture, the optimal time is

5–6 days. The direct bioassays can be used for (i) screen-

ing strains qualitatively and (ii) determining the MID of

the cell-free cultures. The MID is characteristic for spe-

cies, strains and 15 cultures, and was highly reproducible.

The effects of the (ATCC and DSM) type strains of

X. nematophila, X. budapestensis and X. szentirmaii on

isolates from three groups of mastitis isolates, including

six Staph. aureus, six E. coli and one Kl. pneumoniae were

compared. All three groups of mastitis bacteria were sen-

sitive to the antibiotics of each Xenorhabdus, but they

differed in degree. The staphylococci were the most sensi-

tive, and the Kl. pneumoniae isolate the least. Xenorhabdus

szentirmaii and X. budapestensis seem to have the most

potential in mastitis control. As one of the main goals

was to control multi-drug resistant mastitis pathogens, it

is promising that the mastitis pathogen isolates, including

E. coli 727 (Smith et al. 1999), of the same species reacted

uniformly to Xenorhabdus antibiotics, although their sen-

sitivities to commercial antibiotics (such as rifampicin,

kanamycin) were not so uniform. As these mastitis iso-

lates showed variable sensitivities to commercially avail-

able drugs, it will be of interest to determine if the

antibiotics produced by Xenorhabdus spp. are also effec-

tive against isolates with a broader spectrum of resistance.

The mechanisms of antimicrobial compounds pro-

duced by the EPB Xenorhabdus and Photorhabdus are not

well studied because of the diversity of secondary metabo-

lites with potential antimicrobial and ⁄ or toxic effects. Five

related antibiotics, xenorhabdins, were isolated from cul-

tures of Xenorhabdus spp., and were identified as N-acyl

derivatives of either 6-amino-4,5-dihydro-5-oxo-1,2-dithi-

olo [4,3-b] pyrrole, or 6-amino-4,5-dihydro-4-methyl-5-

oxo-1,2-dithiolo[4,3-b] pyrrole. These compounds are

members of the pyrrothine family and have both antimi-

crobial and insecticidal activities (McInerney et al. 1991a).

When entering the hemocoel, Xenorhabdus bacteria

cause septicaemia, which leads to the host insect’s death.

Park and Kim (2000) found that this is related to a block

of the insect immune-mediating eicosanoid pathway. The

lethal effect of the bacteria on the infected larvae

decreased with the addition of exogenous arachidonic

acid, a precursor of eicosanoids. However, injections of

dexamethasone, a specific inhibitor of phospholipase, a

specific inhibitor of phospholipase A(2) and other eicosa-

noid inhibitors significantly elevated bacterial pathogenic-

ity. These results indicate that eicosanoids play a role in

the immune response of insects. Whether similar activity

is related with antimicrobial efficacy is not known. In

fact, both Photorhabdus and Xenorhabdus share an inhibi-

tory action against phospholipase A2 that results in

depression of the host immune response (Kim et al.

2005). The mechanism for immunodepression by X. ne-

matophila in insects is rather complex, and seems to be

related to its antimicrobial activities. Exogenous

X. nematophila cells depress haemocyte nodule formation

of target insects by inhibiting eicosanoid biosynthesis. Ji

and Kim (2004) analysed the inhibitory effect of

X. nematophila on the humoral immunity of the target

insects and tested its association with the host eicosanoid

pathway. Heat-killed X. nematophila induced significant

antibacterial activity in the plasma of target insects. The

antibacterial humoral activity was further demonstrated

by examining a specific potent antibacterial peptide, cecr-

opin. These results suggest that X. nematophila inhibits

the antibacterial humoral immune reaction, as well as the

cellular immune reaction, in Spodoptera exigua. In addi-

tion, the inhibition of X. nematophila on the antibacterial

peptide is not associated with inhibition of the eicosanoid

pathway (Ji and Kim 2004). Xenorhabdus nematophila

also inhibited phagocytosis to increase its pathogenicity

(Shrestha and Kim 2007).

From the aspect of Xenorhabdus taxonomy and evolu-

tion, it was surprising to find one species (X. nemato-

phila) comprised a uniform group of antibiotic

producers, while the other (X. bovienii) showed heteroge-

neity (compare Tables 5 and 6). The type strain X. bovie-

nii DSM4467T was a very poor antibiotic producer, and

strains from Finland, Norway, Israel, and the USA pro-

duced almost none. Strain S2, which was reported as an

extremely good antibiotic producer (Webster et al. 2002),

was not available for these tests. Only one strain of X. bo-

vienii (NYH, from Hungary) showed an antibiotic profile


ª 2007 The Authors


comparable to X. nematophila or X. cabanillasii strains.

The bacterial symbiont of S. riobrave (Isaacson 2000) was

first believed to be a strain of X. nematophila (Stacke-

brandt, personal communication). Since then it has been

described as a new species, X. cabanillasii (Tailliez et al.

2006). As the type strain of this species was not available

during our studies, our own isolate was used. We found

that at least in quantity, its antibiotic production is very

similar to those of the X. nematophila strains.

The polymorphism of the antibiotic profile of Xenor-

habdus species and strains suggests that the different spe-

cies, and maybe different strains of the same species,

produce different compounds, and may have different

quantities of antimicrobial activity. This is in agreement

with the literature (Webster et al. 2002).

As the antibiotics of the cell-free cultures of each X.

nematophila strain proved highly efficient against all

three types of mastitis isolates, we wanted to know if

previously discovered and patented compounds were

responsible for this bioactivity. One of these, nematophin

(3-indoleethyl (3¢-methyl-2¢-oxo) pentanamide), was iso-

lated from X. nematophila strain BC1, and detected in all

X. nematophila strains previously studied. The produc-

tion of nematophin is affected by the strain type and

culture conditions (Li et al. 1997; Webster et al. 2002).

These authors found that the compound showed strong

in vitro activity against a series of fungal and bacterial

species and patented it (Webster et al. US Patent

5,569,688; 10 ⁄ 29, 1996). Himmler et al. (1998) synthe-

sized several derivatives, including alkyl- and aryl-substi-

tutions at the 1-position that gave elevated antimicrobial

activity. They declared that the amide NH-group is

essential for bioactivity. In the only other published

study, Kennedy et al. (2000) reported that the 2-phenyl

derivative of nematophin exhibited exceptional activity

against methicillin-resistant Staph. aureus, whereas the

isosteric benzimidazole analogue was much less active.

Synthesized raceme nematophin was produced for this

study, and its structure validated by different techniques

including EI-MS; 1H-NMR, 13C-NMR and IR (Table 8).

No antimicrobial activity was noted in three different test

systems against Gram-positive (B. subtilis, Staph. aureus)

or Gram-negative (E. coli, Kl. pneumoniae) bacteria.

Questions about the chemical purity of the synthesized

compound were ruled out, since the structure was vali-

dated in several ways. No mention was found about if

the racem compound, or either of the two (D,L) antipo-

des, were used in previous studies. If one of them is bio-

logically active, the other may block its activity in the

racemate. Another explanation might be the addition of

protein (blood or serum) to the culture media reduces

the inhibitory activity on bacteria (Himmler et al. 1998),

and the LB media contains peptone. Another problem

might be related to low solubility of the compound in

water. However, the raceme nematophin and chloram-

phenicol are both water insoluble, and stock solutions

were made up in methanol. When the alcohol solutions

were added to filter paper discs over-layered with the

suspension of the Staph 6 isolate, the chloramphenicol

was highly effective, while the nematophin was com-

pletely ineffective. Similarly, when experiments were car-

ried out in 8 · 12-well microtitre plates with eight

different test organisms, the chloramphenicol was active,

but the nematophin was not. It is not known if any of

the antibiotics from the four highly effective X. nemato-

phila strains included nematophin. However, the cell-free

culture of the X. budapestensis type strain does not con-

tain nematophin (Katona, Szentirmai and Sztaricskai,

personal communication).

The results of this study confirm that there may be

new perspectives for the potential of some Xenorhabdus

antibiotics, and for their use as alternative tools of masti-

tis control. In our on-going research, we want to choose,

purify, and develop those Xenorhabdus antibiotics, which

show the best chemical, and thermo-stability, and the

least biological hazard. Strong evidence suggests that we

should focus on the antibiotics of two novel Xenorhabdus

species, X. budapestensis and X. szentirmaii.

Acknowledgements

The authors express thanks and appreciation for consulta-

tions and professional help to Professor K. Larry Smith

(Mastitis Laboratory, OARDC ⁄ OSU) and Dr Christopher

Ranger (USDA, Agricultural Research Service, Wooster,

OH, USA). We also thank Aniko Saskoi, Eszter Somogyi

and Dr Emilia Szallas (Genetics Department, Eotvos Uni-

versity, Budapest, Hungary) and Dr Attila Lucskai (Hun-

garian Ministry of Agriculture) for the isolation of some

Xenorhabdus strains from nematodes. Drs Eric Martens

and Heidi Goodrich-Blair (University of Wisconsin, Mad-

ison, WI, USA) for the chloramphenicol-resistant E. coli

strain S17_pir pKNOCK used in these tests. The strains of

S. riobrave, S. carpocapsae Mexicana, S. feltiae SF22, Vije,

Norway, and IS6, from which the symbiotic bacteria were

isolated were provided by Dr Byron Adams, Dr Jim

Lindegreen, and the COST 850 European Research Com-

munity (Aana Vaino, Dr Ralph-Udo Ehlers, Dr Lonne

Gerritsen and Dr Itamar Glazer).

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