ISSN2423-9143

福岡県農林業総合試験場特別報告

第 10 号

In vitro synergistic activities ofcefazolin and nisin A against mastitis pathogens

平成 31 年３月

福岡県農林業総合試験場（福岡県筑紫野市大字吉木）

SPECIAL BULLETIN
OF
THE FUKUOKA AGRICULTURE AND FORESTRYRESEARCH CENTER
NO.10

In vitro synergistic activities of cefazolin and nisin A
against mastitis pathogens
byKohei KITAZAKITHE FUKUOKA AGRICULTURE AND FORESTRYRESEARCH CENTER
Chikushino, Fukuoka 818-8549, Japan

March

2019

In vitro における cefazolin と nisin A の乳房炎原因菌に対する相乗的殺菌効果

北﨑宏平
２０１９

* 宮崎大学 審査学位論文

CONTENTSINTRODUCTION

・・・・・・・・・・・・・・・・・・・・・・・・

MATERIALS AND METHODS

1・・・・・・・・・・・・・・・・・・・・

2

Bacterial strains ・・・・・・・・・・・・・・・・・・・・・・・・・・・・

2

Antimicrobial substance・・・・・・・・・・・・・・・・・・・・・・・・

2

Determination of minimum inhibitory concentration (MIC) ・・・・・・・・・・・

2

Checkerboard assay

・・・・・・・・・・・・・・・・・・・・・・・・

3

Time-kill assay ・・・・・・・・・・・・・・・・・・・・・・・・・・・・

3

RESULTS・・・・・・・・・・・・・・・・・・・・・・・・・・・・

MIC and checkerboard assay

5・・・・・・・・・・・・・・・・・・・・

5

Time-kill assay ・・・・・・・・・・・・・・・・・・・・・・・・・・・・

5

Figures and Tables

・・・・・・・・・・・・・・・・・・・・・・・・

7

DISCUSSION

・・・・・・・・・・・・・・・・・・・・・・・・・・・・

16

ABSTRACT・・・・・・・・・・・・・・・・・・・・・・・・・・・・

19

ACKNOWLEDGEMENTS・・・・・・・・・・・・・・・・・・・・

20

・・・・・・・・・・・・・・・・・・・・・・・・・・・・

21

和文摘要 ・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・

26

REFERENCESINTRODUCTION
Bovine mastitis is an inflammatory condition of the mammary gland, most often caused byintramammary microbial infection. It might necessitate treatment, culling, discarding of milk, andreduced milk quality and yield, which can lead to increased production costs and impose a hugeeconomic strain on the industry [1, 38]. Over 150 different types of bacteria have been isolated fromanimals with bovine mastitis [6]. On the basis of the bacterial etiologic agents, bovine mastitis can beclassified as the contagious or environmental type. While Staphylococcus aureus and Streptococcus
agalactiae are categorized as contagious pathogens [37], coagulase-negative staphylococcus (CNS)[16], Escherichia coli [37], S. dysgalactiae [37], and Enterococcus faecalis [39] are categorized asenvironmental pathogens. Many intramammary infections caused by these pathogens lead to
subclinical and chronic mastitis. Intramammary infection caused by CNS is especially common in
many dairy farms around the world; it has caused herd problems such as elevated bulk milk somatic
cell count (SCC) and decreased milk quality [35, 44]. In the dairy industry, mastitis is one of the major
reasons for the use of antibiotics [29, 33]. Intramammary infusion of antibiotics is the most common
therapy for mastitis in dairy farms worldwide [6, 32].
Cephalosporins, a type of β-lactam antibiotics, are considered to be the most importantsemisynthetic antibiotics for mastitis treatment in dairy cattle [6, 15]. Cephalosporins exert
bacteriocidal effects by inhibiting bacterial cell wall synthesis [11, 15]. First-generation
cephalosporins are most frequently used for intramammary treatment of mastitis [6, 15], where theygenerally exhibit good activity [15, 17, 19]. However, the emergence of antibiotic-resistant bacteria
due to overuse of antibiotics against mastitis pathogens has been reported previously [3, 34]. Moreover,antibiotic usage results in the presence of antibiotic residues in milk, which is used for humanconsumption [2, 20, 29]. Therefore, in order to minimize the use of antibiotics, there is an urgent needfor alternative antibiotic therapy approaches for bovine mastitis.
Nisin A, a class-I bacteriocin produced by Lactococcus lactis, is an antibacterial peptide
comprising 34 amino acids. It has been used as a food preservative for over 50 years [23]. Nisin Z,
variant of nisin A possessing a different amino acid residue at position 27, has recently been revealed
to be a possible alternative for treatment of bovine mastitis caused by Gram-positive bacteria [10, 46].
However, nisin A and Z are generally more active against Gram-positive bacteria than Gram-negative
bacteria and exert their bactericidal effect at the cytoplasmic membrane of the target organism [7, 25].
Therefore, the combination of cephalosporin and nisin A might provide an extended activity spectrum
against mastitis pathogens and reduce the antibiotic dose for mastitis treatment.
This study aimed to evaluate the combined effect of a first-generation cephalosporin and nisin A
against mastitis pathogens using the checkerboard and time-kill assays.

-1-

MATERIALS AND METHODS
Bacterial strains
All mastitis pathogen isolates including S. aureus (n = 20), S. intermedius (n = 20), S. agalactiae (n =10), S. dysgalactiae (n = 18), E. faecalis (n = 18), and E. coli (n = 18) were obtained from clinical or
subclinical cases in commercial dairy herds located in different geographical areas of Fukuoka
Prefecture, Japan, between 2006 and 2012. These isolates were identified on the basis of colonymorphology, Gram-staining properties, and catalase and coagulase test findings and by using
commercial kits (API® STAPH, API® 20 STREP, and API® 20 E, bioMérieux Industry, Marcy-l'Étoile,
France). Isolates maintained at –80°C in tryptic soy broth with 20% glycerol were retrieved and plated
onto Mueller–Hinton agar (MHA) plates supplemented with 5% sheep blood and incubated at 37°C
for 24 hr [15].

Antimicrobial substanceCefazolin (CEZ), which is a first-generation cephalosporin, was purchased from Meiji Seika PharmaCo., Ltd. (Tokyo, Japan). For use in this study, CEZ stock solutions were prepared at a concentrationof 10 mg/ml.
Nisin A was manufactured by Omu Milk Products Co., Ltd. (Fukuoka, Japan). The nisin Aproducing L. lactis strain was grown in MRS broth (Oxoid Ltd., Basingstoke, UK) for 16 hr at 30°C.
Nisin A was recovered from the culture supernatant and purified as previously described, with somemodifications [47]. Briefly, the culture supernatant was applied to a hydrophobic resin column, andthe adsorbed nisin A was eluted with 40–70% ethanol and separated by evaporation. The compound
was further purified by reverse-phase high-performance liquid chromatography (HPLC). The
concentration of purified nisin A was determined from the HPLC peak area with a standard curve
prepared by commercial nisin preparation (Sigma-Aldrich, St. Louis, MO, U.S.A.) [13].

Determination of minimum inhibitory concentration (MIC)
The MICs of CEZ and nisin A against mastitis pathogens were determined by the microdilution method,in accordance with the Clinical and Laboratory Standards Institute guidelines [14]. Briefly, target
strains were inoculated onto MHA plates supplemented with 5% sheep blood and incubated overnight-2-

at 37°C. The cells were diluted in Mueller–Hinton broth (MHB) to an approximate final concentrationof 5 × 105 colony forming units (CFUs) per ml. Fifty microliters each of the antimicrobial agent
dilutions and bacterial inocula were dispensed into individual wells of a 96-well microtiter plate. The
plate was then incubated at 37°C for 24 hr. Minimum inhibitory concentration was defined as thelowest concentration of each antibiotic that completely inhibited bacterial growth, as apparent to theunaided eye.

Checkerboard assay
The interactions between CEZ and nisin A against mastitis pathogens were evaluated by the
microbroth checkerboard method in 96-well microtiter plates containing MHB, as described inprevious reports [28, 43]. Briefly, CEZ and nisin A were serially diluted along the y and x axes,
respectively. The final antimicrobial substance concentrations (after two-fold dilution) ranged from
1/16 to 4 times the MIC for CEZ and from 1/256 to 4 times the MIC for nisin A. The checkerboard
plates were inoculated with bacteria at an approximate concentration of 5 × 105 CFU/ml and incubatedat 37°C for 24 hr, following which bacterial growth was assessed visually. To evaluate the effect of
the combination treatment, the fractional inhibitory concentration (FIC) index for each combination
was calculated as follows: FIC index = FIC of CEZ + FIC of nisin A, where FIC of CEZ (or nisin A)
was defined as the ratio of MIC of CEZ (or nisin A) in combination and MIC of CEZ (or nisin A)
alone. The FIC index values were interpreted as follows: ≤ 0.5, synergistic; > 0.5 to ≤ 1.0, additive;>1.0 to ≤ 2.0, indifferent; and > 2.0, antagonistic effects [18, 30].

Time-kill assay
The findings of in vitro interaction determined through the checkerboard assay were confirmed
through the time-kill assay, which was performed by the broth dilution method, as described inprevious studies [36, 41]. Briefly, the experiment included the control, CEZ, nisin A, and combination(CEZ–nisin A) groups. For bacterial inoculation, three isolates were arbitrarily selected from the mostdominant FIC index group for each mastitis pathogen. Equal proportions of the three isolates fromeach group were combined to obtain a three-isolate mixture of each mastitis pathogen species [1]. The
MICs of the three-isolate mixtures were measured by the same method as described above. Fivemilliliters of MHB without antimicrobial substances was used as the control, while MHB (5 ml) with
CEZ or nisin A alone at concentrations of 0.5 ×, 1 ×, and 2 × MIC was added separately into the-3-corresponding tubes. The two antimicrobial substances were added in combination to thecorresponding tubes at the following concentrations: 0.5 × MIC CEZ + 0.5 × MIC nisin A; 1 × MIC
CEZ + 1 × MIC nisin A; and 2 × MIC CEZ + 2 × MIC nisin A. The bacterial inocula were diluted toapproximately 5 × 105 CFU/ml in a 5-ml final volume of MHB. At 0, 3, 6, 9, and 24 hr of incubation
with agitation at 37°C, 100-µl aliquots of culture supernatant were collected from each tube, diluted
tenfold, and inoculated onto MHA plates with 5% sheep blood. The plates were incubated at 37°C for
24 hr, following which the colony counts were determined. The lower limit for detection of bacterial
count was 2.3 log10 CFU/ml (i.e., 200 CFU/ml). Experiments were performed in triplicate. A growth
curve was plotted with the average bacterial count at each time point. Bactericidal effect was defined
as a reduction of ≥ 3 log10 CFU/ml relative to the starting inoculum. Synergism was defined as a
reduction of ≥ 2 log10 CFU/ml observed at 24 hr post-incubation with the CEZ–nisin A combination
relative to that observed with either antimicrobial substance. Additive effect was defined as a reductionof 1 to < 2 log10 CFU/ml observed at 24 hr post-incubation with the CEZ–nisin A combination relative
to that observed with either antimicrobial substance. Indifferent effect was defined as an increase or
decrease of < 1 log10 CFU/ml, and antagonism was defined as an increase of > 2 log10 CFU/ml
observed at 24 hr post-incubation with the CEZ–nisin A combination relative to that observed witheither antimicrobial substance [36, 41].

-4-

RESULTS
MIC and checkerboard assay
Table 1 presents a summary of MICs of CEZ and nisin A, alone and in combination, against mastitis
pathogens. Cefazolin was very active against S. aureus, S. intermedius, S. agalactiae, and S.
dysgalactiae, with MIC50 values ranging from 0.13 to 0.5 µg/ml. While CEZ exhibited good activity
against E. coli (MIC50, 2 µg/ml), it was less active against E. faecalis (MIC50, 32 µg/ml). Nisin A wasvery active against S. intermedius and S. agalactiae (MIC50, 0.06 and 0.25 µg/ml, respectively); while
the compound exhibited good activity against S. aureus (MIC50, 1 µg/ml), S. dysgalactiae (MIC50, 1µg/ml), and E. faecalis (MIC50, 4 µg/ml), it was not active against E. coli (MIC50, 128 µg/ml). The
MIC50 values of CEZ and nisin A in combination with each other were 2- to 8-times lower than thoseof either antimicrobial substance alone.
Table 2 presents results of the checkerboard assay of CEZ and nisin A against mastitis pathogens.
Nisin A and CEZ exhibited synergistic or additive interactions, with FIC index values ranging from
0.19 to 1. There were no instances of indifferent or antagonistic interaction between the twocompounds. The CEZ–nisin A interaction in S. aureus (100%) and E. faecalis (72.2%) cultures was
predominantly synergistic, while that in S. intermedius (60%), S. agalactiae (100%), S. dysgalactiae(100%), and E. coli (77.8%) was mostly additive.

Time-kill assay
Figure 1 presents the results of the time-kill assay. The MICs of CEZ and nisin A against the threeisolate mixtures of both S. aureus and S. intermedius were 0.5 and 1 µg/ml, respectively. Against theS. aureus strains, CEZ, alone, exhibited no bactericidal effect at any time point in the incubation period(Fig. 1-1a). In contrast, nisin A, alone, exhibited transient bactericidal effects at 2 × MIC (Fig. 1-1b).
However, the CEZ–nisin A combination at ≥ 1× MIC exhibited bactericidal activity within 3 hr ofincubation, with effects lasting for 24 hr (Fig. 1-1c). Against the S. intermedius strains, CEZ, alone,
exhibited bactericidal activity at ≥ 1 × MIC (Fig. 1-2d). In contrast, incubation with nisin A, alone,
resulted in a clear concentration-dependent decrease in bacterial count within 6 hr after exposure; this,however, did not prevent the regrowth of the surviving bacteria (Fig. 1-2e). However, the CEZ–nisin
A combination at ≥ 0.5 × MIC exhibited bactericidal activity within 3 hr of incubation, with effects
lasting for 24 hr (Fig. 1-2f).
The MICs of CEZ and nisin A against the three-isolate mixture of S. agalactiae were 0.13 and-5-0.25 µg/ml, respectively. Cefazolin, alone, exhibited no bactericidal effect at any time point in the
incubation period (Fig. 1-3g). In contrast, nisin A, alone, exhibited transient bactericidal effects at 2 ×
MIC (Fig. 1-3h). The CEZ–nisin A combination exhibited concentration-dependent bactericidal
activity; at ≥ 1 × MIC, the antimicrobial substance combination exhibited bactericidal activity within
6 hr of incubation, with effects lasting for 24 hr (Fig. 1-3i).
The MICs of CEZ and nisin A against the three-isolate mixture of S. dysgalactiae were 0.25 and32 µg/ml, respectively. Cefazolin, alone, exhibited no bactericidal effect at any time point in the
incubation period (Fig. 1-4j). In contrast, nisin A, alone, at 2 × MIC exhibited bactericidal effects
within 3 hr of incubation, which lasted for 24 hr (Fig. 1-4k). The CEZ–nisin A combination at ≥ 0.5 ×
MIC exhibited bactericidal activity within 3 hr, with effects lasting for 24 hr (Fig. 1-4l).
The MICs of CEZ and nisin A against the three-isolate mixture of E. faecalis were 32 and 8 µg/ml,
respectively. Cefazolin, alone, exhibited no bactericidal effect at any time point in the incubation
period (Fig. 1-5m). In contrast, treatment with nisin A, alone, resulted in a concentration-dependent
decrease in bacterial count within 6 hr after exposure; at 2 × MIC, nisin A exhibited bactericidal effects
that lasted for 24 hr post-incubation (Fig. 1-5n). The CEZ–nisin A combination at ≥ 0.5× MIC
exhibited bactericidal activity within 3 hr of incubation, with effects lasting for 24 hr (Fig. 1-5o).
The MICs of CEZ and nisin A against the three-isolate mixture of E. coli were 2 and 128 µg/ml,
respectively. Cefazolin, alone, exhibited bactericidal activity at 2 × MIC, while nisin A, alone,
exhibited transient bactericidal effects at 1 × and 2 × MIC; this effect, however, did not prevent the
regrowth of the surviving bacteria (Fig. 1-6p,q). The CEZ–nisin A combination at ≥ 0.5 × MIC
exhibited bactericidal activity within 3 hr of incubation, with effects lasting for 24 hr (Fig. 1-6r).
Table 3 presents a summary of the changes in bacterial count in the time-kill assay at 24 hr postincubation. Incubation with CEZ or nisin A, alone, at 0.5 × or 1 × MIC resulted in increased bacterial
counts at 24 hr post-incubation. However, incubation with the CEZ–nisin A combination at 0.5 × or 1
× MIC resulted in a reduction in bacterial count by over 3 log10 CFU/ml relative to the initial count at
0 hr; at 24 hr post-incubation, this combination also resulted in a synergistic reduction of bacterial
counts by 3.39 to 6.18 log10 CFU/ml relative to the count observed with either antimicrobial substancealone.

-6-

-7-

0.5
0.5
0.13
0.13
32
2

n
20
20
10
18
18
18

(1-8)

(0.25-1)(0.25-4)(0.06-0.25)(0.03-1)(1-64)

CEZ

alone

128

1
0.06
0.25
1
4
(64-128)(0.25-16)(0.02-4)(0.03-0.25)(0.06-32)(0.5-8)nisin A

1

0.06
0.13
0.06
0.06
8

(0.06-2)

32

0.25
0.03
0.13
0.5
0.5

in combination(0.03-0.13)(0.03-0.5)(0.02-0.13)(0.02-0.13)(0.25-16)

CEZ

MIC50 (range) of compound (μg/ml)

MIC50 values were defined as the lowest concentration of the antimicrobial substance at which 50% of the each pathogen were inhibited.

Staphylococcus aureus
Staphylococcus intermedius
Streptococcus agalactiae
Streptococcus dysgalactiae
Enterococcus faecalis
Escherichia coli

starains(4-64)(0.03-1)(0.002-2)(0.008-0.13)(0.03-4)(0.02-4)nisin A

Table 1. Summary of the MIC50 values of cefazolin (CEZ) and nisin A, alone and in combination, against bovine mastitis pathogens.

-814 (77.8)

4 (22.2)

0.19 - 0.5
0.19 - 1.0
0.63 - 1.0
0.56 - 1.0
0.19 - 1.0
0.5 - 1.0

20
20
10
18
18
18

0 (0)

(>2.0)(>1.0-≤2.0)
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)

(>0.5-≤1.0)0 (0)12 (60)10 (100)
18 (100)
5 (27.8)(≤0.5)
20 (100)
8 (40)0 (0)0 (0)13 (72.2)

0 (0)

0 (0)
0 (0)
0 (0)
0 (0)
0 (0)

antagonism

indifferent

No. of strains (%)
additive

FIC index

synergismn

The FIC index values were interpreted as follows: ≤ 0.5, synergistic; > 0.5 to ≤ 1.0, additive; > 1.0 to ≤ 2.0, indifferent; and > 2.0,
antagonistic effects.

Staphylococcus aureus
Staphylococcus intermedius
Streptococcus agalactiae
Streptococcus dysgalactiae
Enterococcus faecalis
Escherichia coli

Organism

Table 2. Combined effect of cefazolin (CEZ) and nisin A determined by checkerboard assay.

Bacterial count (log10 CFU/ml)●

Control
0.5 × MIC CEZ
1 × MIC CEZ

×2 × MIC CEZ■●Control
0.5 × MIC nisin A
1 × MIC nisin A×2 × MIC nisin A■

Bacterial count (log10 CFU/ml)▲

Bacterial count (log10 CFU/ml)▲●

Control
0.5 × MIC CEZ+nisin A
1 × MIC CEZ+nisin A×2 × MIC CEZ+nisin A■▲

Fig. 1-1. Time-kill curves for cefazolin (CEZ) and nisin A, alone and in combination,
against Staphylococcus aureus.
a, CEZ alone; b, nisin A alone; c, CEZ－nisin A combination. Thin dotted line indicatesthe limit of detection.

-9-Bacterial count (log10 CFU/ml)●

Control
0.5 × MIC CEZ
1 × MIC CEZ

×2 × MIC CEZ■●Control
0.5 × MIC nisin A
1 × MIC nisin A×2 × MIC nisin A■

Bacterial count (log10 CFU/ml)▲

Bacterial count (log10 CFU/ml)▲●

Control
0.5 × MIC CEZ+nisin A
1 × MIC CEZ+nisin A×2 × MIC CEZ+nisin A■▲

Fig. 1-2. Time-kill curves for cefazolin (CEZ) and nisin A, alone and in combination, against
Staphylococcus intermedius.
d, CEZ alone; e, nisin A alone; f, CEZ－nisin A combination. Thin dotted line indicatesthe limit of detection.

- 10 -

Bacterial count (log10 CFU/ml)●

Control
0.5 × MIC CEZ
1 × MIC CEZ

×2 × MIC CEZ■●Control
0.5 × MIC nisin A
1 × MIC nisin A×2 × MIC nisin A■

Bacterial count (log10 CFU/ml)▲

Bacterial count (log10 CFU/ml)▲●

Control
0.5 × MIC CEZ+nisin A
1 × MIC CEZ+nisin A×2 × MIC CEZ+nisin A■▲

Fig.1-3. Time-kill curves for cefazolin (CEZ) and nisin A, alone and in combination, against
Streptococcus agalactiae.
g, CEZ alone; h, nisin A alone; i, CEZ－nisin A combination. Thin dotted line indicatesthe limit of detection.

- 11 -

Bacterial count (log10 CFU/ml)●

Control
0.5 × MIC CEZ
1 × MIC CEZ

×2 × MIC CEZ■●Control
0.5 × MIC nisin A
1 × MIC nisin A×2 × MIC nisin A■

Bacterial count (log10 CFU/ml)▲

Bacterial(log1010 CFU/ml)
count (logBacterial count
CFU/ml)▲●Control
0.5 × MIC CEZ+nisin A
1 × MIC CEZ+nisin A×2 × MIC CEZ+nisin A■▲

Fig.1-4. Time-kill curves for cefazolin (CEZ) and nisin A, alone and in combination, against
Streptococcus dysgalactiae.
j, CEZ alone; k, nisin A alone; l, CEZ－nisin A combination. Thin dotted line indicatesthe limit of detection.

- 12 -

Bacterial count (log10 CFU/ml)●

Control
0.5 × MIC CEZ
1 × MIC CEZ

×2 × MIC CEZ■●Control
0.5 × MIC nisin A
1 × MIC nisin A×2 × MIC nisin A■

Bacterial count (log10 CFU/ml)▲

Bacterial count (log10 CFU/ml)▲●

Control
0.5 × MIC CEZ+nisin A
1 × MIC CEZ+nisin A×2 × MIC CEZ+nisin A■▲

Fig.1-5. Time-kill curves for cefazolin (CEZ) and nisin A, alone and in combination, againstEnterococcus faecalis.
m, CEZ alone; n, nisin A alone; o, CEZ－nisin A combination. Thin dotted line indicatesthe limit of detection.

- 13 -

Bacterial count (log10 CFU/ml)●

Control
0.5 × MIC CEZ
1 × MIC CEZ

×2 × MIC CEZ■●Control
0.5 × MIC nisin A
1 × MIC nisin A×2 × MIC nisin A■

Bacterial count (log10 CFU/ml)▲

Bacterial count (log10 CFU/ml)▲●

Control
0.5 × MIC CEZ+nisin A
1 × MIC CEZ+nisin A×2 × MIC CEZ+nisin A■▲

Fig.1-6. Time-kill curves for cefazolin (CEZ) and nisin A, alone and in combination, against
Escherichia coli.
p, CEZ alone; q, nisin A alone; r, CEZ－nisin A combination. Thin dotted line indicatesthe limit of detection.

- 14 -

- 15 -

0.50
2.14
1.72
2.31

0.5

2.45

1.69
3.08
2.67

Nisin A (b)
1.64
2.68
3.39
5.43
3.81
5.24
5.08
5.47

-3.31
-3.10
-3.36
-3.16

a－c

CEZ－Nisin A (c)
-3.29
-3.17

Changes in bacterial count (log10 CFU/ml) from 0 h
CEZ （a)
0.10
2.26

1
0.5
0.5

1
0.5

× MIC

5.61

5.00
6.18
6.03

4.93
5.85

b－c

S

S
S
S

S
S

Interpretation

Bacterial count was measured by the change in log10 CFU/ml from 0 to 24 hr post-incubation, obtained from the data in Fig.1. (i.e., log change = log10 CFU24‒log10 CFU0).
The results are calculated at 0.5×MIC except in the case of S. aureus and S. agalactiae (1×MIC). S, synergism

Staphylococcus aureus
Staphylococcus intermedius
Streptococcus agalactiae
Streptococcus dysgalactiae
Enterococcus faecalis
Escherichia coli

Organism

Table 3. Changes in bacterial count at 24 hr post-incubation with cefazolin (CEZ) and nisin A, alone and in combination.

DISCUSSIONFirst-generation cephalosporins such as cefazolin have been used for the treatment of bovine mastitis
caused by Gram-positive and negative bacteria [6, 32]. The antibacterial peptide nisin, including nisin
A, has generally been used in food preservatives because of its high antibacterial activity andnontoxicity [21, 23]. As such, some studies have suggested that nisin is effective against mastitis
pathogens [10, 40, 46]. The CEZ–nisin A combination can, therefore, be expected to reduce theantibiotic dose for mastitis treatment by extending the activity spectrum by means of synergistic effects
against mastitis pathogens. However, there is no information on the synergistic effect of CEZ and nisin
A. The present in vitro study investigated the antibacterial activity of the CEZ–nisin A combination
against mastitis pathogens for bovine mastitis treatment.
First-generation cephalosporins have generally been reported as exhibiting good bactericidal
activity against mastitis bacterial pathogens [15, 17, 19]. However, Tong et al. [43] suggested that E.
faecalis possesses both intrinsic and acquired resistance to a variety of antibiotics. In our study, CEZ
exhibited MIC50 values of 0.13–2 µg/ml against all mastitis pathogens, except E. faecalis (32 µg/ml).
These results reasonably correspond with those of previous studies [15, 17, 19, 43].
Although early studies have reported the MICs of nisin against major Gram-positive mastitis
pathogens, the reported values were high and exhibited a wide range (10–250 µg/ml) [8]. Nisin has
since then been incorporated into commercially available teat-dipping formulations for mastitis
prevention. It has been reported to exhibit mean log reductions of 3.90 and 4.22 against S. aureus andE. coli, respectively, after exposure for 1 min [40]. Furthermore, Cao et al. [10] and Wu et al. [46]reported the therapeutic efficacy of intramammary nisin infusion in lactating dairy cows with clinicalor subclinical mastitis caused by several mastitis pathogens, including staphylococci and streptococci.
Although these reports suggest the efficacy of nisin against mastitis pathogens, objective evidence of
its antibacterial activity against various mastitis pathogens seems to be insufficient. In our study, theMIC50 values of nisin A against staphylococci and streptococci ranged from 0.06 to 1 µg/ml. Even
against cephalosporin-resistant enterococci, nisin A exhibited relatively low MIC50 values (4 µg/ml).
These results demonstrate the antimicrobial efficacy of nisin A against Gram-positive mastitis
pathogens and support the results of previous studies [10, 40, 46]. On the other hand, nisin A exhibitedvery low activity against E. coli. An accumulating body of evidence shows that nisin exhibits highantibacterial activity against Gram-positive bacteria but not against Gram-negative species. However,previous studies have suggested that nisin A [22] and Z [27] do exhibit MICs against E. coli. Althoughfurther research is required to confirm these findings, the present results support the relevance of theseprevious findings. E. coli can cause mammary gland inflammation in dairy cows around the time ofparturition and during early lactation, with striking local, and sometimes severe, systemic clinical- 16 -

symptoms [9]. Understandably, it is desirable that therapeutic substances used for mastitis treatment
possess bactericidal properties against both Gram-positive and negative bacteria. Accordingly, wehypothesized that a combination of CEZ and nisin A would be effective against both Gram-positive
and negative mastitis pathogens. Furthermore, we expected that the synergistic effect of thiscombination would reduce the antibiotic dose for mastitis treatment in dairy cows.
In the present study, the synergistic effect was evaluated on the basis of MIC values obtained by
the checkerboard and time-kill assays. The MIC50 of the CEZ–nisin A combination against mastitis
pathogens was 2- to 8-fold lower than that observed with either compound alone. In the checkerboard
assay, all interactions between CEZ and nisin A were synergistic or additive, with FIC index values
ranging from 0.19 to 1. In the checkerboard assay, the CEZ–nisin A interactions against S. aureus andE. faecalis strains were mainly synergistic, while those against the other strains were mainly additive.
In the time-kill assay, incubation with the CEZ–nisin A combination for 24 hr resulted in a 103-foldgreater synergistic reduction in bacterial count relative to that observed with either compound at 0.5 ×
or 1 × MIC. Furthermore, at 0.5 × or 1 × MIC, the CEZ–nisin A combination exhibited bactericidal
effects against all pathogen strains within 6 hr of incubation. The checkerboard and time-kill assay
methods are among the most widely used techniques for in vitro assessment of synergistic effects [45].
However, some studies have reported discordance between the findings of these two methods [12, 45].
In the present study, S. aureus and E. faecalis strains, which were selected from the synergistic-effectgroup in the checkerboard assay, were also susceptible to the synergistic effect of CEZ–nisin A in thetime-kill assay. However, the S. intermedius, S. agalactiae, S. dysgalactiae, and E. coli strains thatwere selected from the additive-effect group in the checkerboard assay exhibited susceptibility to thesynergistic effect in the time-kill assay. Despite this discordance in results, both assays revealed
synergistic or additive effects of CEZ–nisin A, thus indicating the efficacy of this combination.
Cephalosporins are β-lactam antimicrobials that exert bactericidal properties by disruption ofbacterial cell-wall synthesis [11, 15]. The mode of action of nisin A involves interaction with themembrane-bound cell-wall precursor lipid II concomitant with pore formation in the cytoplasmic
membrane of the target organism, resulting in loss of membrane potential and leakage of intracellular
metabolites [7, 25].
In the present study, the CEZ–nisin A combination exhibited synergistic or additive effects
against both Gram-positive and negative mastitis pathogens. It may, therefore, be inferred that thebactericidal effect of CEZ–nisin A against Gram-positive bacteria results from the interference of CEZwith bacterial cell-wall synthesis and cytoplasmic membrane pore formation by nisin A. On the other
hand, the poor sensitivity of Gram-negative bacteria to nisin A might be attributed to the large size ofthe peptide, which would restrict its passage through the outer membrane of Gram-negative bacteria[24]. In this respect, some reports [5, 24] suggest the use of the metal-chelating agent- 17 -

ethylenediaminetetraacetic acid (EDTA) for the enhancement of nisin A sensitivity; EDTA removes
stabilizing cations from the outer membrane and destroys the membrane function as a penetration
barrier. Although the mechanism of action of nisin A against Gram-negative bacteria is not completely
understood, it is supposed that CEZ initially mediates the inhibition of Gram-negative bacterial cellwall synthesis, following which nisin A causes pore formation in the cytoplasmic membrane andleakage of intracellular metabolites.
In the present study, the CEZ–nisin A combination exhibited synergistic or additive effects, whichsuggests that the two antimicrobial substances can together achieve mastitis pathogen control even at
low concentrations. Furthermore, this combination might provide extended activity spectrum against
mastitis pathogens such as enterococci or E. coli, which are not sufficiently inhibited by either
compound individually. An early study [26] had reported that milk fat inhibits the antibacterial effectof nisin. However, Bhatti et al. [4] did not observe any decrease in the antibacterial effect of nisin in
non-homogenized milk products such as raw milk. Although Szweda et al. [42] reported a decreased
susceptibility to nisin in antibiotic-resistant S. aureus isolated from bovine mastitis, Okuda et al. [31]suggested that nisin A that forms stable pore on biofilm cells is highly potent for the treatment of
biofilm-associated infections. These findings, together with the present results, suggest that the CEZ–
nisin A combination can serve as an alternative therapy for bovine mastitis in the form of
intramammary infusion, with lower antibiotic concentrations than normal.
In conclusion, the results of the checkerboard and time-kill assays in the present study indicatedthat CEZ and nisin A exert synergistic or additive bactericidal effects against bovine mastitis pathogens.
These results suggest that the CEZ–nisin A combination is effective in reducing the antibiotic dose in
intramammary infusions formulated for mastitis treatment in dairy cattle. Further studies are requiredfor in vivo assessment of microbial response to this antimicrobial substance combination.

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ABSTRACTFirst-generation cephalosporins such as cefazolin (CEZ) have been widely used for mastitis
treatment in dairy cattle. However, the use of antibiotics results in the presence of antibiotic residues
in milk, which is used for human consumption. Nisin A, a bacteriocin produced by Lactococcus lactis,
has been used as a broad-spectrum food preservative for over 50 years. Therefore, a combination of
CEZ and nisin A might provide an extended activity spectrum against mastitis pathogens and reduce
the antibiotic dose for mastitis treatment. This study aimed to evaluate the combined effect of CEZand nisin A against mastitis pathogens using the checkerboard and time-kill assays. In the
checkerboard assay, the CEZ–nisin A combination exhibited a synergistic effect against
Staphylococcus aureus (n = 20/20) and Enterococcus faecalis (n = 13/18), and meanwhile exhibited amostly additive effect against Staphylococcus intermedius (n = 12/20), Streptococcus agalactiae (n =10/10), Streptococcus dysgalactiae (n = 18/18), and Escherichia coli (n =14/18). There were no
indifferent or antagonistic effects between CEZ and nisin A. In the time-kill assay, the CEZ–nisin A
combination at 0.5 × or 1 × minimum inhibitory concentration exhibited synergistic reduction ofbacterial growth by over 3 log10 colony forming units per ml relative to that observed with either
antimicrobial substance alone. These results suggest that the CEZ–nisin A combination can be used
for developing an intramammary infusion for mastitis treatment, with lower antibiotic concentrations
than normal.

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ACKNOWLEDGEMENTS
This study is collaborative investigation with the following researchers.
Shoko KOGA1), Kohei NAGATOSHI1), Koichi KUWANO2), Takeshi ZENDO3), Jiro NAKAYAMA3),
Kenji SONOMOTO3), Hitoshi ANO4), Hiromu KATAMOTO4)
1)

Research and Development Division, Omu Milk Products Co., Ltd., 1-38-1 Shinkatsudati-machi
Omuta, Fukuoka 836-0895, Japan

2)

Division of Microbiology, Department of Infectious Medicine, Kurume University School of
Medicine, 67 Asahi-machi, Kurume, Fukuoka 830-0011, Japan

3)

Laboratory of Microbial Technology, Division of Systems Bioengineering, Department of
Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, 610-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan

4) Laboratory of Veterinary Internal Medicine, Faculty of Agriculture, University of Miyazaki, 1-1,

Gakuen Kibanadai-nishi, Miyazaki 889-2192, Japan

We thank the members of the Livestock Hygiene Service Center of Fukuoka prefecture for theirassistance of collecting mastitis pathogens. This work was partially supported by “Research projectfor utilizing advanced technologies in agriculture, forestry and fisheries” of Ministry of Agriculture,
Forestry and Fisheries of Japan.

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- 25 -

和文摘要
乳牛の乳房炎は主に乳房内への細菌感染によって発症する。本病は急性に乳房の腫脹・硬結や乳汁性状の異常などの局所症状が認められ，重症の場合は体温上昇や食欲不振などの全身症状を呈する。さらに慢性化すると再発を繰り返し，次第に乳量の低下や乳質悪化をまねくなど酪農業界に大きな損失をもたらす。治療方法は抗生物質への依存度が極めて高く，
乳房内注入が広く行われているが，近年は大量使用による耐性菌の出現や生乳中への薬剤残留などによる食品汚染が問題視されている。第一世代セファロスポリンであるセファゾ
リン（CEZ）は乳房炎治療の第一選択薬として世界中で使用され，細胞壁ペプチドグリカンの合成阻害によりグラム陽性・陰性の両方の原因菌に殺菌的に作用する。一方，クラスӀバ
クテリオシンであるナイシン A は，乳酸菌 Lactococcus lactis が産生する 34 残基のアミノ酸で構成される抗菌ペプチドで，安全性の高い食品保存料として我が国をはじめ世界中で利用されている。ナイシン A は細菌の細胞壁前駆体 lipidⅡに付着し細胞膜に孔を形成することで殺菌的に作用する。グラム陽性細菌に強い抗菌活性を示すため，Staphylococcus 属，Bacillus 属，Clostridium 属，Listeria 属などの食品汚染菌が問題となるチーズ，乳製品，
缶詰などの保存料として使用されている。しかし，細胞壁外膜を有するグラム陰性菌に対する殺菌効果は乏しいことも知られている。このため，CEZ とナイシン A を併用すれば，その相互作用により乳房炎原因菌に対する抗菌スペクトルの拡大や乳房炎治療における抗生物質投与量の低減が期待できる。そこで，本研究は CEZ とナイシン A の乳房炎原因菌に対する併用効果を in vitro で評価した。
2006 年から 2012 年の間に県内で発生した臨床型・潜在性乳房炎から分離した原因菌を試験に供した。伝染性病原菌として Staphylococcus aureus (n=20)および Streptococcus

agalactiae (n=10)を，また，環境性病原菌として Staphylococcus intermedius (n=20)，
Streptococcus dysgalactiae (n=18)，Enterococcus faecalis (n=18)および Escherichia coli(n=18)を用いた。CEZ は市販の動物用医薬品を，ナイシン A はオーム乳業（株）が作成したものを用いた。乳房炎原因菌に対する CEZ とナイシン A の最小発育阻止濃度（MICs）
は Muller-Hinton broth（MHB）を用いた微量液体希釈法で測定し，菌種ごとに MIC50 を算出した。乳房炎原因菌に対する CEZ とナイシン A の併用効果は，MHB と 96 穴マイク
ロプレートを用いた Checkerboard assay により FIC index 値を算出し，その値が 0.5 以下の場合を相乗効果，0.5 より大きく 1.0 以下を相加効果，1.0 より大きく 2.0 以下を無関係，
2.0 より大きい場合を拮抗効果と評価した。また，Checkerboard assay による評価を補強するために，CEZ，ナイシン A の単独および併用時それぞれについて 0.5 倍 MIC，1 倍 MIC
および 2 倍 MIC における各乳房炎原因菌の生存細菌数の経時変化（0，3，6，9，24 時間後）を，MHB を用いた Time-kill assay で評価した。

S. aureus，S. intermedius，S.agalactiae および S. dysgalactiae に対する CEZ の MIC50- 26 -

の値は 0.13-0.5µg/ml と低く，E. coli も 2µg/ml だったが，E. faecalis では 32µg/ml と高かった。ナイシン A の MIC50 は S. intermedius，S.agalactiae で 0.06 および 0.25µg/ml と低く，S. aureus，S. dysgalactiae が 1µg/ml，E. faecalis では 4µg/ml であったが，E. coli
では 128µg/ml と極めて高かった。しかし，いずれの菌種においても CEZ とナイシン A
併用時における MIC50 の値は，単独時に比べて 1/2 から 1/8 に低下した。Checkerboard
assay では，CEZ と nisin A 併用により S. aureus (n=20/20)，E. faecalis (n=13/18)において相乗効果が優勢であったのに対し，S. intermedius (n=12/20)，S. agalactiae (n=10/10)，

S. dysgalactiae (n=18/18)および E. coli (n=14/18)では相加効果が優勢であった。また，いずれの供試菌においても無関係および拮抗効果は認められなかった。Time-kill assay では，

S. aureus と S. agalactiae において CEZ と nisin A の 1 倍 MIC による併用で，S.
intermedius，S. dysgalactiae，E. faecalis，E. coli では 0.5 倍 MIC による併用で培養 3～
6 時間以内に生存細菌数が検出限界以下まで低下し，24 時間持続した。
本研究により，CEZ と nisin A を併用すると，グラム陽性・陰性の両方の乳房炎原因菌に対して相乗・相加的な殺菌効果を有することが明らかとなった。また，両者を併用すると単独では MIC50 の値が高く殺菌効果が乏しかった E. faecalis や E. coli に対しても，その値が1/2～1/8 まで低下したことから，その他の乳房炎原因菌に対する抗菌スペクトルの拡大も期待される。また近年は，ナイシン A のバイオフィルムに対する有効性も報告されていることから，慢性乳房炎に対する治療効果も期待できる。
以上のことから，
CEZ とナイシン A を併用することで，従来よりも抗生物質濃度が低く，抗菌スペクトルの広い新たな乳房内注入剤の開発が可能であることが示唆された。

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福岡県農林業総合試験場特別報告第10号

In vitro synergistic activities ofcefazolin and nisin A against mastitis pathogens

発 行

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