Veterinary Microbiology 167 (2013) 737741

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Veterinary Microbiology
journal homepage: www.elsevier.com/locate/vetmic

Short Communications

Virulence genes and antimicrobial susceptibility in Pasteurella

multocida isolates from calves
K. Katsuda *, K. Hoshinoo, Y. Ueno, M. Kohmoto, O. Mikami
Pathology and Pathophysiology Research Division, National Institute of Animal Health, NARO, 3-1-5 Kannondai, Tsukuba, Ibaraki 3050856, Japan

A R T I C L E I N F O

A B S T R A C T

Article history:
Received 30 May 2013
Received in revised form 30 August 2013
Accepted 22 September 2013

A total of 378 isolates of Pasteurella multocida from clinically healthy and diseased calves
were characterised for their susceptibility to 9 antimicrobial agents and screened by PCR
for the presence of antimicrobial resistance genes and 22 genes virulence-associated,
including capsule biosynthesis genes.
Of the 378 isolates, 102 (27.0%) were resistant to at least one of the 9 tested
antimicrobial agents. Resistance to oxytetracycline (21.7%) was the most frequently
observed phenotype among the isolates. The tet(H) gene were the primary determinant
detected. The resistance rates for thiamphenicol, ampicillin, kanamycin and orfenicol
were 13.2%, 5.8%, 9.0% and 0.5%, respectively. Cefazolin, ceftiofur, cefquinome and
enrooxacin were effective antimicrobial agents, with no resistant isolates emerging over
the course of the investigation.
Most isolates were identied as capsular type A, only 6.3% belonged to capsular type D
and no other capsular type was identied. Four of the virulence-associated genes (pfhA,
tadD, tbpA and HAS) exhibited associations to the capsular type, and three (pfhA, tbpA and
hgbB) were associated with the disease status of the animals. These virulence genes have
been considered as epidemiological markers and are hypothesised to have a strong
positive association with the outcome of disease in cattle.
2013 Elsevier B.V. All rights reserved.

Keywords:
Pasteurella multocida
Antimicrobial susceptibility
Virulence gene
Cattle

1. Introduction
Bovine respiratory disease (BRD) is the most economically important disease among cattle. Many bacterial
species are associated with the pneumonia seen in BRD.
Pasteurella multocida is agents of BRD (Confer, 2009).
Although improved vaccines are effective tools for the
control of BRD, poor environmental conditions are one
main reason for shortcoming in vaccination success. For
therapy of clinical cases, antimicrobials remain effective
tools for such bacterial infections. The increased prevalence of antimicrobial-resistant bacterial pathogens has
become a major public health and animal health concern.

* Corresponding author. Tel.: +81 29 838 7925; fax: +81 29 838 7925.
E-mail address: katsuda@affrc.go.jp (K. Katsuda).
0378-1135/$ see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.vetmic.2013.09.029

Continuous regional monitoring of antimicrobial susceptibility is important for the selection of effective antimicrobial agents for treatment of bovine pneumonia.
P. multocida possesses various virulence factors, including capsule, lipopolysaccharide, mbriae and adhesins,
toxins, iron-regulated and iron acquisition proteins, sialic
acid metabolism proteins, hyaluronidase and outer membrane proteins (Ewers et al., 2004; Harper et al., 2006).
These virulence factors play important roles in the
pathogenesis of P. multocida. Data on the antibiotic
resistance proles and virulence factors of P. multocida
from cattle differing in disease status may yield a better
understanding of pasteurellosis in cattle.
We investigated 378 isolates from clinically healthy
and diseased calves to discover their distribution of
capsular type, their phenotypic antimicrobial resistant
proles and the presence or absence of 17 virulence genes.

22
34
82
50
2
0
0
0
0
b

ABPC = Ampicillin, KM = Kanamycin, OCT = Oxytetracycline, TP = Thiamphenicol, FF = Florfenicol CEZ = Cefazolin, CTF = Ceftiofur, CQN = Cefquinome, ERFX = Enrooxacin.
The cut-offs (indicated bold) used for the midpoint of the MIC peaks was dened as the resistance cut-off when the MIC distribution of the antimicrobials was bimodal.

(5.9)
(9.2)
(22.3)
(14.7)
(0.8)
(0.0)
(0.0)
(0.0)
(0.0)
14
22
53
35
2
0
0
0
0
(5.7)
(8.6)
(20.7)
(10.7)
(0.0)
(0.0)
(0.0)
(0.0)
(0.0)
8
12
29
15
0
0
0
0
0
4.0
32.0
128.0
128.0
0.5
2.0
0.125
0.125
1.0
0
26
2
0
0
0
0
0
0
0
6
0
0
0
0
0
0
0
0
2
0
4
0
0
0
0
0
6
0
36
44
0
0
0
0
0
4
0
34
2
0
0
0
0
0
2
34
10
0
0
0
0
0
0
ABPC
KM
OTC
TP
FF
CEZ
CTF
CEQ
ERFX

22
0
0
0
10
2
378
378
236

18
0
6
14
64
66
0
0
4

40
2
20
208
286
54
0
0
20

140
2
104
102
16
192
0
0
118

118
22
166
0
0
60
0
0
0

18
32
0
4
0
4
0
0
0

0
102
0
0
2
0
0
0
0

10
150
0
0
0
0
0
0
0

512
256
128
64
32
16
8.0
4.0
2.0
1.0
0.5
0.25

MIC (mg/mL)

Bacterial DNA was extracted using the InstaGene

Matrix (BIO-RAD Laboratories Inc., CA, USA) according to
the manufacturers instructions.
All isolates were analysed with PCR for the presence of
capsule biosynthesis genes and the virulence-associated
genes. PCR analysis of virulence-associated genes for
capsule biosynthesis (capA, capB, capD, capE and capF),
mbriae and adhesins (ptfA, mA, hsf-2, pfhA, tadD, nanB
and nanH), iron-regulated and iron acquisition proteins
(tonB, tbpA, hgbA and hgbB), sialic acid metabolism (sodA
and sodC), hyaluronidase (HAS), and outer membrane
proteins (ompH, omp87 and psl) was conducted as
described previously (Ewers et al., 2006; Tang et al.,
2009; Townsend et al., 1998).

Agentsa

2.3. Virulence genes

Table 1
Antimicrobial susceptibility of P. multocida isolated from cattle.

To determine the susceptibility of the P. multocida

isolates to antimicrobial agents, the agar dilution method
(dilution range: 0.125512.0 mg/ml) recommended by the
Clinical and Laboratory Standards Institute subcommittee
on Veterinary Antimicrobial Susceptibility Testing was
used (CLSI, 2005). The following quality-control strains
were also tested: Staphylococcus aureus ATCC 29213,
Enterococcus faecalis ATCC 29212, Escherichia coli ATCC
25923 and Pseudomonas aeruginosa ATCC 27853. The
antimicrobial agents used in this study are listed in
Table 1. All isolates were analysed with PCR for the presence
of specic resistance genes for orfenicol (oR), thiamphenicol (cat3A), tetracycline (tet(B), tet(G), tet(H), tet(L), tet(M)),
kanamycin (aphA1), and penicillins (blaROB-1, blaTEM-1, blaPSE1) was conducted as described previously (Kehrenberg and
Schwarz, 2001; Mak et al., 2009; White et al., 2000).
Production of lactamase was assayed using the chromogenic
disc method (Becton Dickinson Microbiology Systems)
according to the manufacturers instructions.

>512

2.2. Measurement of MICs and antimicrobial resistance gene

1.0
16.0
2.0
0.5
0.5
1.0
0.125
0.125
0.125

MIC50
(mg/ml)

MIC90
(mg/ml)

No. of resistant isolates (%)

A total of 378 P. multocida eld isolates collected on the

basis of one isolate per herd from cattle were investigated.
Of the 378 isolates, 140 were from nasal swabs of clinically
healthy cattle; the remaining 238 isolates were from lung
lesions of BRD-affected cattle. The isolates were collected
at our institute between 2009 and 2012 and originated
from 30 different regions in Japan. All isolates were
identied using API micro standardised strips (API 20NE;
bioMerieux Japan Ltd., Tokyo, Japan) and by PCR detection
of the species-specic gene fragment kmt (Ewers et al.,
2006; Tang et al., 2009; Townsend et al., 1998). All isolates
were stored in 1 ml of tryptic soy broth (Becton, Dickinson
and Company, Sparks, MD, USA) supplemented with 20%
(v/v) glycerol at 80 8C. Prior to minimum inhibitory
concentration (MIC) determination, the isolates were
cultured on tryptic soy agar plates supplemented with
5% sheep blood (Becton, Dickinson and Company, MD,
USA).

Clinically healthy
(n = 140)

2.1. Bacterial isolates

Clinically diseased
(n = 238)

Total
(n =378)

2. Materials and methods

(5.8)
(9.0)
(21.7)
(13.2)
(0.5)
(0.0)
(0.0)
(0.0)
(0.0)

K. Katsuda et al. / Veterinary Microbiology 167 (2013) 737741

0.125

738

HAS*

739

2.4. Statistical analysis

97.7
16.7

sodC

100
100

sodA

100
100

HAS
sodC

100
100
100

sodA

100
100
100

92.9
92.4
92.6

K. Katsuda et al. / Veterinary Microbiology 167 (2013) 737741

Statistical analysis was performed with GraphPad

Prism1 software, version 5.0 (MDF Co. Ltd., Tokyo, Japan).
The results were considered statistically signicant if
p  0.05.

Clinically healthy means P. multocida isolated from upper respiratory tract and clinically diseased means P. multocida isolated from lower respiratory tract.
Signicant associations (p  0.01).
b

hgbB

61.3
62.5
95.5
95.8

hgbA
tbpA*

80.5
12.5
100
100

tonB
nanH

88.7
83.3
100
100

nanB
tadD*

94.1
12.5
55.4
8.3
92.7
91.7
100
100
94.6
95.8
100
100
100
100
354
24
A
D

100
100

40.0
74.0
61.4

pfhAb
hsf-2
mA
ptfA
omp87
ompH
psl
No. of isolates
Capsule type by PCR

hgbA

96.4
95.0
95.5
51.4
90.8
76.2

tbpA*
tonB

100
100
100
85.7
89.9
88.4

nanH
nanB

100
100
100
90.0
88.2
88.9

tadD
pfhA*

30.0
65.6
52.4
98.6
89.1
92.6

hsf-2
mA

100
100
100
100
91.6
94.7

ptfA
omp87

100
100
100
100
100
100

ompH
psl

100
100
100
140
238
378

virulence associated genes

No. of isolates
a

Clinical status

Table 2
Percent distribution of virulence associated genes in P. multocida isolates from cattle.

Clinically healthy
Clinically diseased
Total

hgbB*

3. Results
The MICs for 9 antimicrobial agents and the antimicrobial susceptibilities of the P. multocida isolates are
listed in Table 1. Of the 378 isolates, 102 (27.0%) were
resistant to at least one of the 9 antimicrobial agents, and
the resistance rates ranged from 0.5% to 21.7%. Of the 102
resistant isolates, 49 isolates were resistant to 1 class of
antimicrobial (15 isolates were resistant to ampicillin
(ABPC), 32 isolates were resistant to oxytetracycline (OTC),
2 isolates were resistant to thiamphenicol (TP)), 22 isolates
to 2 classes (2 isolates were resistant to TP and kanamycin
(KM), 2 isolates were resistant to KM and OTC, 18 isolates
were resistant to TP and OTC) and 29 isolates to 3 classes (1
isolate resistant to TP, KM and ABPC, 4 isolates resistant to
KM-OTC-ABPC, 24 isolates resistant to TP, KM and OTC).
The nal 2 isolates were resistant to 4 classes of
antimicrobial agents (resistant to TP, ABPC, OTC and
orfenicol (FF)). The resistance rates for ABPC, KM, OTC and
TP were 5.8%, 9.0%, 21.7% and 13.2%, respectively. There
were only 2 isolates resistant to FF (0.3%), and no isolates
were resistant to enrooxacin (ERFX) or cephalosporin
drugs (cefazolin (CEZ), ceftiofur (CTF), and cefquinome
(CEQ)). Of the 82 oxytetracycline-resistant isolates, 75
isolates harboured tet(H) and 4 isolates harboured tet(B).
The remaining 3 isolates were negative for all the tet genes
tested in this study. Of the 22 ampicillin-resistant isolates
that produced b-lactamase when assayed using the
chromogenic disc method, 16 isolates harboured the
blaRob-1 gene; the remaining 6 isolates were negative for
all the bla genes tested in this study. Of the 50
thiamphenicol-resistant isolates, 47 harboured the cat3A
gene. All isolates that showed resistance to kanamycin
were positive for the aphA1 gene. Two orfenicol-resistant
isolates possessed the oR gene. Although we compared
the antimicrobial susceptibility to the clinical status of the
source animals, there is no signicant difference between 2
clinical status; clinically healthy and clinically diseased.
The most prevalent capsule biosynthesis genes
detected in the 378 P. multocida isolates were capA (354
isolates, 93.7%) and capD (24 isolates, 6.3%). None of the
isolates harboured the capB, capE and capF genes. And
there is no association between capsular type and clinical
status (Table 2).
Genes encoding adhesins (ptfA, mA, and hsf-2), iron
acquisition factors (tonB and hgbA), porin and outer
membrane proteins (psl, opmH and omp87), superoxide
dismutases (sodA and sodC), neuraminidase (nanB and
nanH) and hyaluronan synthase gene (HAS) were each
found in more than 88.0% of the isolates. In contrast, 3
genes (pfhA, tbpA and hgbB) were detected from 52.4% to
76.2% (Table 2).
The combination of virulence genes and capsular types
was assessed with a Chi-square test. The most remarkable

740

K. Katsuda et al. / Veterinary Microbiology 167 (2013) 737741

associations were present between pfhA, tadD, tbpA, and

HAS (p  0.01). These virulence genes revealed a clear
association with capsule type A isolates. The distributions
of these virulence genes among isolates from animals of
differing health status were compared with a Chi-square
test. Three genes (pfhA, tbpA and hgbB) were signicantly
more prevalent (p  0.01) in isolates from animals showing
clinically diseased.
4. Discussion
Monitoring the antimicrobial susceptibility trends of P.
multocida is an important aid to veterinarians in selecting
the most efcacious therapeutic agents. The results of the
present study indicate that CEZ, CTF, CEQ, ERFX and FF are
very active against the P. multocida isolates tested. On the
basis of the MIC breakpoints of these agents, all but two of
the isolates would be considered susceptible to these
antimicrobial agents. OTC showed poor activity against P.
multocida compared with other classes of antimicrobial
agents; this is not unexpected. Tetracycline (19,031.9 kg/
year), penicillin (7,180.4 kg/year) and aminoglycoside
(3,195.0 kg/year) are the main antimicrobial agents used
for cattle in Japan, as tetracyclines account for one-half of
the antimicrobials used for cattle in Japan (Asai et al.,
2005). These ndings agree well with results presented in
previous reports (Portis et al., 2012; Watts et al., 1994). The
results of PCR-based determination of antimicrobial
resistance genes, some isolates were negative for all
known specic resistance genes in this bacterium
(Schwartz, 2008). The genetic background of antimicrobial
resistance remained unclear for these isolates. Furthermore, molecular analyses aimed at uncovering the
resistant mechanisms should be carried out in future
studies.
We have conrmed that P. multocida isolates in capsular
type A are more common than those in capsular type D.
Consistent with this nding, it has been reported that
capsular type A isolates are one of the principal causes of
bovine respiratory diseases (Frank, 1989) and are highly
adapted to bovine hosts (Ewers et al., 2006). In contrast to
all the other virulence genes investigated in this study, 4
virulence genes (pfhA, tadD, tbpA, and HAS) were obviously
associated with certain capsule types (capsule type A).
Although there were only 24 isolates belonging to the
capsule type D in this investigation, similar ndings have
been reported previously (Ewers et al., 2006; Tang et al.,
2009).
Except for the pfhA gene, P. multocida regularly
harboured mbriae- and adhesin-related genes independently of the clinical status of cattle. In this investigation,
the distribution of the pfhA gene was associated with
clinical status. In a past study (Verma et al., 2013), all
isolates possessed the pfhA gene independently of capsule
type and disease status. In contrast, Ewers et al. (2006)
reported a signicant association between the presence of
the pfhA gene in P. multocida isolates and the clinical status
of cattle. We investigated 4 iron-regulated and ironacquisition related genes (tonB, hgbA, tbpA, hgbB). Irrespective of their clinical status, nearly all isolates had the
tonB and hgbA genes. A higher prevalence of the tbpA gene

and hgbB gene were observed in isolates from diseased

cattle than in isolates from healthy cattle. These results
agree with a recent study reporting that these two genes
were signicantly associated with bovine clinical status
(Ewers et al., 2006).
In conclusion, resistance to antimicrobial agents,
including penicillin, chloramphenicol, tetracyclines and
aminoglycosides, was observed in P. multocaida isolates
collected from cattle. Therefore, continuous monitoring of
antimicrobial susceptibility is necessary to determine the
current susceptibility status of P. multocida. Several
virulence genes (tbpA, hgbB, and pfhA) have been considered as epidemiological markers and are hypothesised
to have a strong positive association with the outcome of
disease in cattle. The results of this investigation provide
useful information for understanding the antimicrobial
resistance patterns, capsular types and virulence gene
prevalence of P. multocida isolate from cattle.
Conict of interest
All authors declare that there are no nancial or other
relationships that might lead to a conict of interest.
Acknowledgements
We thank Natsuko Andou and Syuuko Sawajiri for
excellent technical assistance in this study.
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