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Veterinary Microbiology 110 (2005) 97–103

Leukotoxin family genes in Staphylococcus aureus isolated from

domestic animals and prevalence of lukM–lukF-PV genes by

bacteriophages in bovine isolates

Tomoko Yamada a, Naoko Tochimaru a, Sachiko Nakasuji a, Eiji Hata b,Hideki Kobayashi b, Masashi Eguchi b, Jun Kaneko c, Yoshiyuki Kamio c,

Toshio Kaidoh a, Shotaro Takeuchi a,*

a Department of Bioscience, Faculty of Biotechnology, Fukui Prefectural University, 4-1-1 Kenjyojima Matsuoka, Fukui 910-1195, Japanb Clinical Epidemiology Section, National Institute of Animal Health, 1-5 Kannondai Tsukuba, Ibaraki 305-0856, Japan

c Laboratory of Applied Microbiology, Department of Microbial Technology, Graduate School of Agriculture Science,

Tohoku University, 1-1 Tsutsumi-dori Amamiya-machi, Aoba, Sendai 981-8555, Japan

Received 20 April 2005; received in revised form 29 June 2005; accepted 7 July 2005

Abstract

Leukotoxin family genes in Staphylococcus aureus isolated from domestic animals were examined by polymerase chain

reaction. LukS and lukF genes were detected in all 48 avian and 72 porcine isolates of S. aureus. LukE and lukD genes, located in

a putative staphylococcal pathogenicity island (Sapln3/Saplm3), were recognized in 44 (91.7%) of 48 avian isolates, but these

genes were not detected in porcine isolates. In 297 bovine isolates collected from mastitic cow’s milk and bulk milk from dairy

farms in two regions, lukM and lukF-PV(P83) genes in addition to lukS–lukF and lukE–lukD genes were detected in 100 (62.5%)

of the 160 isolates from Ishikawa and in118 (86.1%) of the 137 isolates from Hokkaido. When the lysogeny of S. aureus bovine

isolates was examined by treatment with mitomycin C, clearing of the culture due to cell lysis was observed in 34 (91.9%) of 37

lukM–lukF-PV(P83) genes – positive isolates. In addition, we isolated a novel lukM–lukF-PV(P83)-carrying (designated

wLukM), and revealed that the lukM–lukF-PV(P83) genes were located very close to an amidase gene on the temperate phage

genomes. These results suggest horizontal transmission of lukM–lukF-PV(P83) genes by temperate bacteriophages in S. aureus

of bovine origin.

# 2005 Elsevier B.V. All rights reserved.

Keywords: Leukotoxin genes; Staphylococcus aureus; Temperate phage; Bovine mastitis

* Corresponding author. Tel.: +81 776616000;

fax: +81 776616015.

E-mail address: takeuchi@fpu.ac.jp (S. Takeuchi).

0378-1135/$ – see front matter # 2005 Elsevier B.V. All rights reserved

doi:10.1016/j.vetmic.2005.07.006

1. Introduction

Leukotoxins, which are produced by Staphylococ-

cus aureus, belong to a family of pore-forming toxins

that is composed of two distinct components. The

.

T. Yamada et al. / Veterinary Microbiology 110 (2005) 97–10398

toxic effect depends on the synergistic action of both

class S (slow elution)-related and class F (fast elution)-

related proteins on polymorphonuclear cells (PMN)

and monocytes (Kaneko and Kamio, 2004). The

staphylococcal leukotoxin family comprises the long-

known Panton-Valentine leukocidin (PVL; LukS-

PV + LukF-PV), g-hemolysin and leukocidin (Hlg

and Luk; HlgA + LukF and LukS + LukF), and the

more recently described LukM/FPV(P83) (LukM + -

LukF-PV(P83)) and LukE/D (LukE + LukD) (Choorit

et al., 1995; Gravet et al., 1998; Kaneko et al., 1997a;

Prevost et al., 1995a, 1995b, 2001). Cloning of the

genes for five components provided evidence for the

presence of two genetic clusters, an Hlg/Luk cluster

and a PVL cluster. The former consists of hlg2, lukS,

and lukF genes, and the latter consists of lukS-PV (or

lukM) and lukF-PV genes (Rahman et al., 1991, 1992,

1993). Kaneko et al. (1997b, 1998) isolated a

temperate phage, wPVL carrying PVL genes from a

lysate of mitomycin C-treated S. aureus V8 and

suggested horizontal transmission of the PVL gene

cluster via the bacteriophage. Moreover, they reported

that the PVL-like genes, lukM and lukF-PV(P83) were

found on the prophage wPV83-pro genome in S.

aureus strain P83 which was originally isolated from

bovine mastitis (Zou et al., 2000). LukE and lukD

genes are located in a putative staphylococcal

pathogenicity island (Sapln3/Saplm3) (Fitzgerald

et al., 2001; Kuroda et al., 2001).

Rainard et al. (2003) reported that only some of the S.

aureus strains isolated from cows, ewes, and goats with

mastitis possess lukM and lukF-PV (they called lukF-

PV(P83) as lukF0-PV) genes. In the present study, genes

for the leukotoxin family in avian, porcine, and bovine

isolates ofS.aureuswere examinedby PCR. Inaddition,

we investigated the existence of a novel prophage

carrying lukM and lukF-PV(P83) genes in bovine

isolates of S. aureus from mastitic cow’s milk and bulk

milk in order to examine the horizontal transmission of

the LukM gene cluster via the bacteriophage.

2. Materials and methods

2.1. Bacterial isolates

A total of 297 bovine isolates of S. aureus were

isolated from mastitic cow’s milk and bulk milk

obtained from dairy farms in Ishikawa and Hokkaido

prefectures in Japan. In addition, 48 avian and 72

porcine isolates of S. aureus were used. These avian

isolates and porcine isolates were obtained from the

skins of chickens and tonsils of pigs, respectively. S.

aureus ATCC 49775, Newman, ATCC 31890(P83),

and N65 were used as reference strains.

Escherichia coli DH5a and pGEM-T vector

(Promega Corporation, Madison, WI, USA) were

used for cloning and sequencing the PCR products.

Heart infusion broth (Difco Laboratories, Detroit, MI,

USA) and Luria–Bertani medium were used for

routine culture of staphylococcal cells and E. coli,

respectively.

2.2. Polymerase chain reaction

For the detection of the lukS and lukF genes, lukS-

PV and lukF-PV genes, two pairs of primers (hlg1 and

hlg2, lukPV-1 and lukPV-2), designed by Lina et al.

(1999), were synthesized. Primers (lukE, lukD, M1,

and M2) for lukE and lukD genes and the lukM gene

were designed according to the nucleotide sequences

of GenBank accession numbers Y13225 and D83951,

respectively. The sequences of synthesized primers

lukE, lukD, M1, and M2 were 50-CAGAACTT-

CATTTTCGGATGTGAAGGG-30 (nucleotides 762–

788), 50-CTCCAGGATTAGTTTCTTTAGAATCCG-

30 (complementary to nucleotides 2408–2435), 50-AACTTTCAATGATGTTAAACAAAATAGAG-30

(nucleotides 1388–1417), and 50-AAAATAGTCTC-

TAGCATTAGGTCC-30 (complementary to nucleo-

tides 1767–1790). Moreover, five primers (M-ami,

M3, F1, hol1, and M-ami2) were designed for the

detection of LukM–LukF-PV genes, the holin gene,

and the amidase gene on the phage genome according

to the nucleotide sequence of wPV83-pro (GenBank

accession number AB044554). The sequences of

synthesized primers M-ami, M3, F1, hol1, and M-

ami2 were 50-ATATATGCGCCTGGAACATTAAT-

ATATG-30 (nucleotides 39808–39836), 50-ATC-

GATGGTCTGCTTACGATG-30 (nucleotides 40864–

40884), 50-AACTGATTTCTACCCATAAGTCACC-

30 (complementary to nucleotides 42246–42270), 50-TGCACTAAATT TA AT G T T TAGT T T C G G-30

(nucleotides 38621–38647), and 50-ATTATGGTTGT-

TCCAGTAAATACGG-30 (complementary to nucleo-

tides 39861–39885). PCR amplification was

T. Yamada et al. / Veterinary Microbiology 110 (2005) 97–103 99

Table 1

Leukotoxin genes in avian, porcine, and bovine isolates of S. aureus

Leukotoxin genes Avian isolates

(n = 48)

Porcine isolates

(n = 72)

lukS–lukF (g-hemolysin) 48 (100%) 72 (100%)

lukE–lukD 44 (91.7%) 0 (0%)

lukM–lukF-PV(P83) 0 (0%) 0 (0%)

lukS-PV–lukF-PV (PVL) 0 (0%) 0 (0%)

Bovine isolates

Ishikawa

(n = 160)

Hokkaido

(n = 137)

lukS–lukF (g-hemolysin) 157 (98.1%) 135 (98.5%)

lukE–lukD 154 (96.3%) 131 (95.6%)

lukM-lukF–PV(P83) 100 (62.5%) 118 (86.1%)

lukS-PV–lukF-PV (PVL) 0 (0%) 0 (0%)

performed in a total of 20 ml containing 10 ng of the

DNA template, 20 pmol of each primer, 2.5 mM of the

four deoxynucleotides, and 2.5 U of Taq polymerase

(Takara Shuzo, Shiga, Japan). The reaction mixtures

were subjected to 30 cycles of amplification in

GeneAmp 2400 (PE Apllied Biosystems, Foster City,

CA, USA). After amplification, the PCR products

were analyzed by agarose gel electrophoresis.

2.3. Cloning and sequencing of PCR products

The PCR products were cloned into a pGEM-T

vector according to the manufacturer’s instructions.

The recombinant plasmids were transfected into E.

coli DH5a, and the resulting recombinants were

screened by the colony PCR method with the vector

and insert DNA primers. Nucleotide sequencing of the

insert DNA was performed by a model 3100 Gene

Analyzer (PE Applied Biosystems) with a Big Dye

Terminator v3.1 Cycle Sequencing Kit.

2.4. Mitomycin C treatment and purification of

phage particles

The bacterial cells were inoculated into heart

infusion broth and cultivated on a shaker at 37 8C for

3 h. At the mid-exponential phase, mitomycin C was

added at a final concentration of 1 mg ml�1, and

cultivation was continued at 30 8C. After incubation

for 5 h, the resulting lysate was treated with DNase I

and RNase A (Sigma, St. Louis, MO; 1 mg/ml each) at

room temperature for 1 h, and then 0.7 M NaCl was

added and incubation continued at 4 8C for 1 h. After

centrifugation at 11,000 � g for 10 min, polyethylene

glycol 6000 was added to the supernatant at a final

concentration of 10% (w/v). After incubation for 12 h

at 4 8C, phage particles were recovered by centrifuga-

tion at 11,000 � g for 10 min and suspended in

50 mM Tris–HCl buffer (pH 7.0) containing 0.1 M

NaCl, 8 mM MgSO4, 1 mM CaCl2, and 0.01% gelatin

(SMC buffer). After chloroform treatment to remove

remaining polyethylene glycol, the phage particles

were again recovered by centrifugation at 3000 � g

for 15 min and resuspended in TE buffer. Further

purification of phage particles was performed by

isopycnic centrifugation through CsCl gradients.

2.5. Electron microscope examination

Phage particles were negatively stained with 2%

phosphotungstic acid and examined using a transmis-

sion electron microscope (Tecnai 10, Philps).

3. Results

3.1. Leukotoxin family genes in avian and porcine

isolates

Leukotoxin family genes in avian and porcine

isolates of S. aureus were amplified by the PCR with

six primers and examined by agarose gel electrophor-

esis. S. aureus strains ATCC49775 (V8), Newman,

and ATCC31890 (P83) were used as reference strains

for lukS-PVand lukF-PV, lukE and lukD, and lukM and

lukF-PV genes, respectively. In the PCR method using

primers hlg1 and hlg2 for lukS and lukF genes, one

band of the expected length (937 bp) was detected in

all 48 avian and 72 porcine isolates (Table 1). LukE

and lukD genes, located in a putative staphylococcal

pathogenicity island (Sapln3/Saplm3), were recog-

nized in 44 (91.7%) of 48 avian isolates by the PCR

method with primers lukE and lukD, but these genes

were not detected in porcine isolates. No lukM–lukF-

PV genes or lukS-PV–lukF-PV genes were detected in

any avian or porcine isolates. The PCR products were

cloned into a pGEM-T vector and DNA inserts were

sequenced. The nucleotide sequences of PCR products

T. Yamada et al. / Veterinary Microbiology 110 (2005) 97–103100

from two avian and two porcine isolates and reference

strains revealed lukS–lukF and lukE–lukD-gene

sequences that were 100% identical to the published

sequences (data not shown).

3.2. Leukotoxin family genes in bovine isolates

LukS–lukF genes and lukE–lukD genes were detect-

ed in almost all of the bovine isolates of S. aureus that

were collected from mastitic cow’s milk and bulk milk

from dairy farms in Ishikawa and Hokkaido prefectures,

Japan(Table1). In thePCRmethodwithprimersM1and

M2, the band (405 bp) for lukM–lukF-PV(P83) genes

was amplified in 100 (62.5%) of the 160 isolates from

Ishikawa and in 118 (86.1%) of the 137 isolates from

Hokkaido. LukS-PV–lukF-PV genes were detected in

the reference strain ATCC 49775, but were not detected

in the bovine isolates. The percentage of lukM–lukF-

PV(P83)geneswere almost thesame in the isolates from

mastitic cow’s milk (57.9% in Ishikawa, 91.5% in

Hokkaido) and bulk milk (65.5% in Ishikawa, 94.0% in

Hokkaido) (Table 2). The nucleotide sequences of PCR

products (405 bp) from four bovine isolates corre-

sponded to those of lukM–lukF-PV genes of S. aureus

ATCC31890 (P83) (Zou et al., 2000) (data not shown).

3.3. Isolation of phage particles

To examine the lysogeny of S. aureus bovine

isolates, bacterial cells in the logarithmic phase of

Table 2

Relationship between leukotoxin genes and S. aureus isolates from

bulk milk or mastitic cow’s milk

Leukotoxin gene by region Bulk milk

isolates

(n = 84a, 100b)

Mastitic milk

isolates

(n = 76a, 47b)

Ishikawa

lukS–lukF (g-hemolysin) 82 (97.6%) 75 (98.7%)

lukE–lukD 80 (95.2%) 74 (97.4%)

lukM–lukF-PV(P83) 55 (65.5%) 44 (57.9%)

Hokkaido

lukS–lukF (g-hemolysin) 99 (99.0%) 46 (97.9%)

lukE–lukD 98 (98.0%) 34 (72.3%)

lukM–lukF-PV(P83) 94 (94.0%) 43 (91.5%)a Isolates obtained from bulk milk or mastitic cow’s milk in

Ishikawa.b Isolates obtained from bulk milk or mastitic cow’s milk in

Hokkaido.

growth were treated with mitomycin C. After

incubation for 5 h, clearing of the culture as a result

of cell lysis was observed in 34 (91.9%) of 37 lukM–

lukF-PV(P83) genes-positive isolates, but not in 49

(98.0%) of 50 lukM–lukF-PV(P83) genes-negative

isolates, suggesting that there was a definite associa-

tion between possession of lukM–lukF-PV(P83) genes

and lysogenization of phage. The cultures of eight

lukM–lukF-PV(P83) genes-positive isolates that

induced lysogenic phages were treated with DNase

I and RNase A. Subsequently, phage particles were

collected by precipitation with NaCl and polyethylene

glycol 6000, and purified by iopycnic centrifugation

through CsCl gradients. The phages, purified from

eight isolates, had the same morphological character.

They had an elongated head, which was about

100 nm � 50 nm, and a flexible 300 nm tail

(Fig. 1). A small protuberance was observed at the

end of the tail. This morphological character is the

same as that of the PVL-converting phage wSLT

reported by Narita et al. (2001).

3.4. Detection and sequencing of lukM–lukF-

PV(P83) genes from the phage genome

The existence of lukM–lukF-PV(P83) genes on

eight temperate phages induced by mitomycin C

treatment was examined by the PCR method with

three pairs of primers (M1 and M2, M-ami and M2,

M3 and F1). Bands of 403, 1130, and 1374 bp were

Fig. 1. Electron micrograph of the wLukM negatively stained with

phosphotungstic acid.

T. Yamada et al. / Veterinary Microbiology 110 (2005) 97–103 101

Table 3

Plaque formation of purified phages on LukM–lukF-PV genes-

negative isolates (n = 33)

Phages Plaque formationa Sensitivity

isolates (%)+++ + + �

wLukM17 2 4 18 9 24 (72.7)

wLukM18 1 5 22 5 28 (84.8)

wLukM21 2 31 2 (6.1)

wLukM23 1 32 1 (3.0)

wLukM26 4 29 4 (12.1)

wLukM27 2 6 14 11 22 (66.7)

wLukM38 2 8 23 10 (30.3)

wLukM47 3 18 12 21 (63.6)a (+++) clear; (++) semi-clear; (+) opacity plaque; (�) no plaque.

detected in all of the phage genomes. The nucleotide

sequences of the PCR products for the lukM–lukF-

PV(P83) genes of eight phages were identical to one

another, but the sequences of lukM and lukF-

PV(P83)genes differed at 6 and 5 positions, respec-

tively, from those for the lukM and lukF-PV(P83)

genes of the wPV83-pro (Zou et al., 2000) (data not

shown). The deduced amino acid sequences for the

LukM and LukF-PV of eight phages differed at three

(V148A, L273Q, Y300H) and three (F3I, L17F,

R274T) positions, respectively, from those for the

genes of thewPV83-pro, respectively.

The holin gene and part of the amidase gene on the

phage genomes were amplified by the PCR method

with primers hol1 and M-ami2, and sequenced. The

amidase gene for a lytic enzyme (N-acetylmuramyl-

lalanine amidase) located upstream of the lukM gene

and its nucleotide sequence differed at one position

from that for the amidase gene of the wPV83-pro. It is

interesting that a mutation of G12A in the holin gene

changed the fourth tryptophan (TGG) to a stop codon

(TGA). This mutation was recognized in all eight

phages of the holin gene and suggested that the second

atg codon in the orf58 (holin) of wPV83-pro may act

as start codon. These results suggest that lukM and

lukF-PV-converting phages are heterogeneous. We

tentatively designated these phages as wLukM.

The sequence of the lukM–lukF-PV genes, amidase

and holing genes inwLukM has been deposited in

DDBJ/EMBL/GenBank nucleotide sequence data-

bases with accession number AB218700.

3.5. Plaque formation of the purified phages

We examined 33 lukM–lukF-PV genes-negative

isolates of S. aureus for their sensitivity to eight

purified phages in order to study the conversion of the

lukM–lukF-PV genes by temperate bacteriophages. In

this experiment, about 5 ml of the phage solutions was

placed directly onto agar plates on which the isolates

tested were smeared, and the plates incubated at

37 8C for 18 h. wLukM18, wLukM17, wLukM27, and

wLukM47 formed plaques on 28 (84.8%), 24 (72.7%),

22 (66.7%) and 21 (63.6) of 33 lukM–lukF-PV genes-

negative isolates, respectively (Table 3), indicating

that these phages possess strong infectivity to most of

lukM–lukF-PV genes-negative isolates of S. aureus.

Moreover, these results suggest the possibility of

conversion of the lukM–lukF-PV genes by these

temperate bacteriophages. Therefore, we conjectured

that the horizontal transmission of the lukM and lukF-

PV genes by temperate bacteriophages occurs among

bovine isolates of S. aureus, such as PVL-carrying

phages.

4. Discussion

The hlg2, lukS, and lukF genes encoding g-

hemolysin and leucocidin are located on the chromo-

some of S. aureus (Kaneko and Kamio, 2004; Kuroda

et al., 2001). This gene cluster was detected in most of

the isolates from nasal, blood, primary skin infection,

and pneumonia in humans (Lina et al., 1999; Von Eiff

et al., 2004). In the present study, the gene cluster was

recognized in most of the isolates from domestic

animals, including chicken, pigs, and cows, suggest-

ing that the hlg2, lukS, and lukF genes are conserved

equally in human and domestic animal isolates of S.

aureus.

The lukE and lukD genes are located in a putative

staphylococcal pathogenicity island (Sapln3/Saplm3)

on the chromosome of S. aureus (Kuroda et al., 2001).

The pathogenicity island contains a serine protease

cluster and an enterotoxin gene cluster. Von Eiff et al.

(2004) reported that the lukE and lukD genes were

found at high prevalence in S. aureus isolates from

humans, significantly more so in blood (82%) than in

nasal isolates (60.5%). In domestic animal isolates of

S. aureus, these genes were detected in all the S.

aureus isolates from ruminants with mastitis by PCR

T. Yamada et al. / Veterinary Microbiology 110 (2005) 97–103102

amplification (Poutrel et al., unpublished data). Our

study also demonstrated that the lukE and lukD genes

were recognized in almost all (96.0%) of the bovine

isolates of S. aureus that were collected from mastitic

cow’s milk and farm bulk milk. Moreover, 91.7% of

avian isolates possessed these genes. In contrast, lukE

and lukD genes were not detected in the porcine

S. aureus isolates. This may reflect a target cell- and

species-specificity of LukED.

Zou et al. (2000) reported that PVL-like genes,

lukM and lukF-PV, were found on the genome of a

prophage, which was designated wPV83-pro. This

prophage shares genes for packaging and morphogen-

esis with wPVL, whose head is isometric. Unfortu-

nately, wPV83-pro could not be induced as a phage

particle because its attachment site was separated by

an insertion sequence (Zou et al., 2000). When we

examined the lukM and lukF-PV genes in S. aureus

bovine isolates that were collected from mastitic cow’s

milk and bulk milk from dairy farms in two regions,

the genes were detected in 100 (62.5%) of the 160

isolates from Ishikawa and in118 (86.1%) of the 137

isolates from Hokkaido. The percentage of lukM and

lukF-PV gene-positive isolates was much higher than

the 35.4% (17/48) of S. aureus isolates from cows with

mastitis reported by Rainard et al. (2003), suggesting

that the conservation of lukM and lukF-PV genes in

S. aureus isolates is different in different countries or

regions. Therefore we tried to isolate the LukM/F-PV

carrying phage from our lukM–lukF-PV(P83) positive

isolates. The lysogeny of S. aureus was demonstrated

in 34 (91.9%) of 37 lukM–lukF-PV genes-positive

isolates, but not in 49 (98.0%) of 50 lukM–lukF-PV

genes-negative isolates. As a result, we isolated eight

LukM/F-PV carrying phages (wLukM). Interestingly,

the morphology of these phages was very close to that

of wSLT, rather than to wPVL. Moreover, PCR

amplification and sequence analysis revealed that the

lukM–lukF-PV genes are located very close to an

amidase gene on the temperate phage genomes, like

wPV83-pro. From the results, we conjectured that at

least two types of temperate bacteriophages involved

in horizontal transmission of the lukM and lukF-PV

genes by temperate bacteriophages occur in bovine

isolates of S. aureus, like PVL-carrying phages.

Further investigations to characterize the novel

LukM/F-PV-carrying phages at the molecular level are

in progress.

Acknowledgments

We are grateful to Dr. Gerard Lina for the gift of

S. aureus ATCC 49775, Newman, and N65, and to

Dr. Kazuo Matsumoto for technical assistance with

electron microscopy.

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