Establishment of disk diffusion national epidemiological cut-off values for Pasteurella multocida and Mannheimia haemolytica isolated from bovine respiratory diseases and characteristics of antibiograms in Japan

Tomoko KOSHIISHI; Akiko KATO; Mio KUMAKAWA; Yuta HOSOI; Michiko KAWANISHI; Shunsuke MORI; Tomomi KURUMISAWA; Junko SAITO; Mie KISHIKAWA; Kazuhiro KAWAI

doi:10.1292/jvms.26-0205

. 2026 Aug 11;88(10):1556–1564. doi: 10.1292/jvms.26-0205

Establishment of disk diffusion national epidemiological cut-off values for Pasteurella multocida and Mannheimia haemolytica isolated from bovine respiratory diseases and characteristics of antibiograms in Japan

Tomoko KOSHIISHI ¹, Akiko KATO ¹, Mio KUMAKAWA ², Yuta HOSOI ², Michiko KAWANISHI ², Shunsuke MORI ³, Tomomi KURUMISAWA ³, Junko SAITO ¹, Mie KISHIKAWA ¹, Kazuhiro KAWAI ^1,^3,^*

PMCID: PMC13680698 PMID: 42586708

Abstract

Antimicrobial resistance is a global public health threat, and accurate antimicrobial susceptibility testing is crucial for guiding appropriate antimicrobial use. Although the disk diffusion method is widely used in livestock, standardized breakpoints for veterinary pathogens are lacking. Bovine respiratory disease, a major cause of economic loss in calves, is commonly associated with Pasteurella multocida and Mannheimia haemolytica. This study aimed to establish inhibition zone diameter cut-off values for disk diffusion and to create an antibiogram for these pathogens. The cut-off values were calculated based on minimum inhibitory concentration (MIC) reference values: the Clinical and Laboratory Standards Institute clinical breakpoints for penicillin and tetracycline; the European Committee on Antimicrobial Susceptibility Testing epidemiological cut-off values for kanamycin and tilmicosin (P. multocida); and MIC cut-off values calculated from this study for cefazolin, marbofloxacin, thiamphenicol, and tylosin. The cut-off values for penicillin and tetracycline provide useful guidance for clinical drug selection, whereas caution is required when interpreting the results for the other agents evaluated because their cut-off values were based on epidemiological cut-offs rather than clinical breakpoints. Re-evaluation of previously reported cut-off values for kanamycin, cefazolin, and tetracycline using a larger number of isolates provided more robust thresholds that were less susceptible to strain-specific variation. The antibiogram revealed low susceptibility rates for some agents. Because antimicrobial use is associated with the development of resistance, these findings indicate the need for prudent antimicrobial stewardship and continued surveillance of resistance in livestock pathogens.

Keywords: antibiogram, bovine respiratory disease, disk diffusion method, Mannheimia haemolytica, Pasteurella multocida

INTRODUCTION

Antimicrobial resistance (AMR) threatens the effectiveness of infectious disease treatment and has become a global public health threat [32]. In Japan, the Action Plan for Antimicrobial Resistance (2023–2027) was developed to promote AMR countermeasures, with targets in six areas: (1) dissemination and education, (2) trends and surveillance, (3) infection prevention and control, (4) appropriate use of antimicrobial agents, (5) research and development and drug discovery, and (6) international cooperation [27]. Antibiograms are practical tools for antimicrobial resistance surveys in individual medical facilities or testing centers [17]. They provide periodic summaries of the antimicrobial susceptibility of clinical isolates and are used to guide empirical antimicrobial therapy against expected pathogens, monitor changes in resistance rates over time, and promote the appropriate use of antimicrobial agents [17, 23, 25]. The microdilution method for antimicrobial susceptibility testing can determine the minimum inhibitory concentration (MIC) of antimicrobial agents, allowing evaluation of the degree of susceptibility or resistance to these agents [10]. However, although MIC results are simple and easy to interpret, MIC-measuring plates are expensive. In contrast, the disk diffusion method is a qualitative test that classifies bacteria as susceptible, intermediate, or resistant. It offers the advantages of simplicity, low cost, and ease of result interpretation [3], and is therefore widely implemented in livestock production settings. Reliable interpretive criteria are essential for classifying pathogenic microorganisms as susceptible or resistant to antimicrobial agents in antimicrobial resistance surveys and susceptibility testing. The cut-off values established by the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) have been widely adopted internationally. However, established cut-off values are unavailable for many animal-derived pathogenic microorganisms, making susceptibility test results difficult to interpret in the veterinary field.

Bovine respiratory disease (BRD) has a high morbidity rate in calves and causes significant economic losses to calf producers due to reduced growth rates and increased treatment costs [24]. The causative factors of BRD are complex, involving viruses, bacteria, and stress. However, BRD typically begins with a primary viral infection and is exacerbated by secondary bacterial infection [11], and antimicrobial agents are therefore used for treatment [4]. The primary causative bacteria are Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, and Mycoplasma bovis. However, established cut-off values for interpreting antimicrobial susceptibility testing results against these causative bacteria remain insufficient. Tentative inhibition zone diameter cut-off values have been reported using the disk diffusion method for ampicillin (ABPC), cefazolin (CEZ), dihydrostreptomycin (DSM), kanamycin (KM), and tetracycline (TC) against M. haemolytica and P. multocida isolated from BRD [20]. However, these findings do not comprehensively cover the antibiotics used in clinical practice. Furthermore, because a range of inhibition zone diameter cut-off values showed equivalent sensitivity and specificity for several antimicrobial agents, likely due to the absence of isolates with intermediate susceptibility levels, re-evaluation using additional strains was considered necessary [20]. We therefore added the strains used in the present study to those reported by Kumakawa et al. [20]. The objective of this study was to establish epidemiological cut-off values in Japan for determining susceptibility and resistance to penicillin (PCG), marbofloxacin (MBFX), thiamphenicol (TP), tylosin (TS), and tilmicosin (TMS: P. multocida only) using the disk diffusion method. For KM, CEZ, and TC, strains from this study were added to those for which provisional cut-off values had previously been reported, and they were re-evaluated [20]. Furthermore, because few recent reports have evaluated the susceptibility rates of P. multocida and M. haemolytica derived from BRD in Japan, this study also aimed to establish antibiograms for both bacterial species.

MATERIALS AND METHODS

Ethical approval

This study was conducted in accordance with the regulations of the Animal Experiment Committee of Azabu University (Sagamihara, Japan).

BRD sample collection and identification of causative bacteria

Veterinarians in the field treated naturally occurring cases of BRD and collected samples for microbiological testing, which we routinely perform on request. In this study, the isolated and preserved strains obtained after such inspections were used for testing. The number of preserved strains was limited to no more than two samples per farm, each derived from a different individual. For P. multocida, samples were collected from February 1, 2021, to January 12, 2023, in 13 prefectures in Japan: Hokkaido, Aomori, Ibaraki, Iwate, Okayama, Kagoshima, Saitama, Chiba, Tochigi, Hyogo, Miyagi, Miyazaki, and Yamagata. For M. haemolytica, samples were collected from January 14, 2020, to January 9, 2024, in 14 prefectures: Hokkaido, Aomori, Ibaraki, Iwate, Ehime, Kagoshima, Saitama, Chiba, Tochigi, Nagasaki, Hyogo, Miyagi, Miyazaki, and Yamagata). Nasal swabs, bronchoalveolar lavage fluid, or lung tissue were inoculated onto 5% horse blood agar medium and cultured at 37°C for 24 hr in the presence of 5% CO₂. Species identification of the isolated strains was performed by 16S rRNA gene sequence analysis. The primer sequences were the 27F sense primer (5′-AGAGTTTGATCCTGGCTCAG-3′) and the 1492R antisense primer (5′-GGTTACCTTGTTACGACTT-3′), as reported by Turner et al. [29]. PCR conditions were as follows: initial denaturation at 95°C for 3 min, denaturation at 95°C for 30 sec, annealing at 58°C for 30 sec, and extension at 72°C for 2 min, repeated for 32 cycles followed by a final extension at 72°C for 7 min. The amplified DNA was subjected to agarose gel electrophoresis, and the PCR products were treated with exonuclease I (20 U/μL) (Thermo Fisher Scientific, Waltham, MA, USA) and shrimp alkaline phosphatase (1 U/μL) (Takara Bio Inc., Kusatsu, Japan). Exonuclease I and shrimp alkaline phosphatase were mixed at a 1:2 ratio, and 1 μL of the mixture was added to 5 μL of PCR product. The mixture was incubated at 37°C for 30 min and then inactivated by heating at 85°C for 15 min. The resulting products were used as templates for sequencing reactions using a Big Dye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific). Sequencing analysis was outsourced to FASMAC Co., Ltd. (Atsugi, Japan). Species identification was performed by confirming the sequence similarity between the obtained DNA sequences and known sequences using the Basic Local Alignment Search Tool (BLAST) [1].

Drug susceptibility testing

Microdilution method: The antimicrobial agents used were as follows: PCG, KM, florfenicol (FFC), TP, MBFX, enrofloxacin (ERFX), TS, TMS (Fujifilm Wako Pure Chemical Corp., Osaka, Japan), CEZ, and TC (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan). The MIC of the antimicrobial agents was measured by the microdilution method in accordance with the antimicrobial susceptibility testing methods established by the CLSI [6]. Test microplates were prepared by serially diluting the antimicrobial agents in a 12-step, two-fold dilution series. Bacterial suspensions were inoculated to achieve approximately 5 × 10⁴ CFU/well. After incubation at 35°C in ambient air for 18 hr, bacterial growth was assessed visually. All antimicrobial agents were tested at concentrations ranging from 0.125 to 256 μg/mL. Cation-adjusted Mueller–Hinton broth (Becton Dickinson Co., Ltd., Sparks, MD, USA) was used as the medium. For each test, Staphylococcus aureus ATCC 29213 was used as the quality control strain, and testing was conducted in accordance with CLSI guidelines [6].

Disk diffusion method: The disk diffusion method was conducted according to the antimicrobial susceptibility test method established by the CLSI [6]. The following antimicrobial drug disks were used: PCG (10 μg), KM (30 μg), TC (30 μg), CEZ (30 μg) (Becton Dickinson Japan Co., Ltd.), FFC (30 μg), TP (30 μg), TS (60 μg), TMS (15 μg), ERFX (5 μg) (Eiken Chemical Co., Ltd., Tokyo, Japan), and MBFX (5 μg) (Vetoquinol, Lure, France). A bacterial suspension adjusted to McFarland 0.5 was spread onto Mueller–Hinton agar (Becton, Dickinson Co., Ltd.) supplemented with 5% defibrinated sheep blood. Disks were placed on the agar plates and incubated at 37°C in ambient air for 18 hr, after which inhibition zone diameters were measured. When the disk lot was changed, the S. aureus ATCC 25923 strain was used as the quality control strain in accordance with CLSI guidelines [6]. For the TS (60 μg) disk, the acceptable range criteria established by the Japanese Society of Antimicrobials for Animals were used [16].

Determination of MIC and inhibition zone diameter cut-off values

The MIC cut-off values were used to calculate the inhibition zone diameter cut-off values. The following MIC criteria for PCG, KM, CEZ, TC, TP, TS, TMS (excluding M. haemolytica), and MBFX were applied as previously reported [20]: 1) if a clinical breakpoint (CBP) had been established by CLSI [5], that value was adopted; 2) if no CBP had been established by CLSI [5] but epidemiological cut-off values (ECOFFs) had been set by EUCAST [7], the ECOFF was adopted; and 3) if neither a CBP nor an ECOFF was specified, then the MIC cut-off values were calculated using the SOP for ECOFF determination published by EUCAST [9]. The inhibition zone diameter cut-off values were calculated based on the MIC cut-off values using the formula: (1-sensitivity^§1)² + (1-specificity^§2)², with the minimum value selected as the cut-off [20]. If one or more inhibition zone diameters met this requirement, the largest value was adopted. For the calculation of MIC and inhibition zone diameter cut-off values for KM, CEZ, and TC, strains reported by Kumakawa et al. (P. multocida, 77 strains; M. haemolytica, 158 strains) [20] were added, resulting in a total of 201 P. multocida strains and 258 M. haemolytica strains.

For calculating cut-off values for resistance,§1: sensitivity=(number of isolates for which the MIC indicated resistance and that were also classified as resistant based on inhibition zone diameter)/ (number of isolates for which the MIC indicated resistance)§2: specificity=(number of isolates for which the MIC indicated susceptibility and that were also classified as susceptible based on inhibition zone diameter)/ (number of isolates for which the MIC indicated susceptibility)

For calculating cut-off values for susceptibility,§1: sensitivity=(number of isolates for which the MIC indicated susceptibility and that were also classified as susceptible based on inhibition zone diameter)/ (number of isolates for which the MIC indicated susceptibility)§2: specificity=(number of isolates for which the MIC indicated resistance and that were also classified as resistant based on inhibition zone diameter)/ (number of isolates for which the MIC indicated resistance)

Antibiogram analysis

The inhibition zone diameters of P. multocida and M. haemolytica strains were classified as susceptible, intermediate if criteria were available, or resistant. Classifications for PCG, KM, TC, CEZ, TP, TS, TMS (P. multocida), and MBFX were determined based on newly established disk diffusion cut-off values, whereas those for FFC, ERFX, and TMS (M. haemolytica) were determined based on CLSI CBPs. Antibiograms were created according to the Antibiogram Creation Guidelines of the Infectious Diseases Education Consortium [14]. The proportion of susceptible isolates was shown in the antibiograms. Susceptibility rates were calculated for each region: Hokkaido, eastern Japan, and western Japan. Eastern Japan included Aomori, Ibaraki, Yamagata, Iwate, Miyagi, Saitama, Chiba, and Tochigi prefectures, whereas western Japan included Hyogo, Okayama, Ehime, Miyazaki, Nagasaki, and Kagoshima prefectures. Values based on sample sizes of fewer than 30 were interpreted with caution [14].

Statistical analysis

Statistical analysis was performed using EZR [18] (ver. 1.68, Saitama Medical Center, Jichi Medical University, Saitama, Japan), a graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Austria). In the antibiogram, Bonferroni multiple comparisons were performed for susceptibility rates between regions, and P<0.05 was considered to indicate a significant difference.

RESULTS

Bacterial isolation

During the study period, 6,988 samples consisting of nasal swabs, bronchoalveolar lavage fluid, or lung tissue were received for testing. Of the 2,555 P. multocida isolates and 1,290 M. haemolytica isolates, 124 and 100, respectively, were cryopreserved and included in the study.

Establishment of epidemiological MIC cut-off values

Pasteurella multocida: For PCG and TC, CLSI MIC CBPs were applied, whereas for KM and TMS, the EUCAST ECOFF was applied (Table 1). CEZ, TS, TP, and MBFX had no established cut-off values for either CBP or ECOFF, so the SOP [9] was used to determine susceptibility as follows: CEZ ≤2 μg/mL, TS ≤128 μg/mL, TP ≤2 μg/mL, and MBFX ≤0.125 μg/mL (Table 2A, Supplementary Fig. 1).

Table 1. Established tentative cut-off values of Pasteurella multocida and Mannheimia haemolytica.

Spieces	Antimicrobial	MIC’s judgment values (μg/mL)^a					Inhibition zone diameter’s judgement values (mm)						Sensitivity	Specificity

		CLSI^b			EUCAST^c	This study	CLSI^b			This study

		S^d	I^d	R^d	S	S	S	I	R	S	I	R
P. multocida	PCG	≤0.25	0.5	≥1	-	-	-	-	-	≥30	-	≤29	(S) 1.000	(S) 0.923
	PCG	≤0.25	0.5	≥1	-	-	-	-	-	≥30	-	≤29	(R) 1.000	(R) 1.000
	KM	-	-	-	≤32	-	-	-	-	≥16	-	-	0.944	1
	CEZ	-	-	-	-	≤2	-	-	-	≥30	-	-	0.761	0.7
	TC	≤2	4	≥8	≤2	-	-	-	-	≥21	20	≤19	(S) 0.983	(S) 0.888
	TC	≤2	4	≥8	≤2	-	-	-	-	≥21	20	≤19	(R) 0.986	(R) 1.000
	TS	-	-	-	-	≤128	-	-	-	≥9	-	-	1	1
	TP	-	-	-	-	≤2	-	-	-	≥27	-	-	1	0.875
	TMS	-	-	-	≤32	-	-	-	-	≥12	-	-	1	1
	MBFX	-	-	-	-	≤0.12	-	-	-	≥31	-	-	0.976	0.939

M. haemolytica	PCG	≤0.25	0.5	≥1	-	-	-	-	-	≥25	-	≤24	(S) 1.000	(S) 0.907
	PCG	≤0.25	0.5	≥1	-	-	-	-	-	≥25	-	≤24	(R) 1.000	(R) 1.000
	KM	-	-	-	≤16	-	-	-	-	≥15			1	1
	CEZ	-	-	-	-	≤4	-	-	-	≥22			1	1
	TC	≤2	4	≥8	≤2	-	-	-	-	≥22	21	≤20	(S) 0.984	(S) 1.000
	TC	≤2	4	≥8	≤2	-	-	-	-	≥22	21	≤20	(R) 0.985	(R) 0.979
	TS	-	-	-	-	≤64	-	-	-	≥12	-	-	0.92	0.96
	TP	-	-	-	-	≤8	-	-	-	≥23	-	-	1	1
	MBFX	-	-	-	-	≤1	-	-	-	≥19	-	-	1	1

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a: The underlined values show the judgement values used to calculate the inhibition zone diameter. b: CLSI. Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals, 7th ed. CLSI standard VET01S. Clinical and Laboratory Standards Institute; 2024. c: EUCAST. At 2026. https://mic.eucast.org/search/. d: S; susceptible, I; intermediate, R; resistant. MIC: minimum inhibitory concentration, PCG: penicillin, KM: kanamycin, CEZ: cefazolin, TC: tetracycline, FFC: florfenicol, TP: thiamphenicol, MBFX: marbofloxacin, ERFX: enrofloxacin, TS: tylosin, TMS: tilmicosin.

Table 2. Number of isolates with minimum inhibitory concentration (MIC) distribution for Pasteurella multocida and Mannheimia haemolytica.

graphic file with name jvms-88-10-1556-t001.webp

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Mannheimia haemolytica: For PCG and TC, CLSI MIC CBPs were applied, whereas for KM, EUCAST ECOFFs were applied (Table 1). CEZ, TS, TP, and MBFX had no established cut-off values for either CBP or ECOFF, so the SOP [9] was used to determine susceptibility as follows: CEZ ≤4 μg/mL, TS ≤64 μg/mL, TP ≤8 μg/mL, and MBFX ≤1 μg/mL (Table 2B, Supplementary Fig. 2).

MIC distribution

Pasteurella multocida: The results of the microdilution method for 124 strains of P. multocida are shown in Table 2A. The MIC cut-off values are indicated by vertical lines in the table. KM and TMS were interpreted based on the EUCAST ECOFF, whereas PCG, TC, FFC, and ERFX were interpreted based on the CLSI MIC CBPs. CEZ, TS, TP, and MBFX were interpreted using cut-off values calculated in the present study. The lowest antimicrobial resistance rate was observed for ERFX (1.6%), followed by FFC (2.4%), CEZ (3.2%), TP (6.5%), PCG (9.7%), KM (12.0%), TS (22.6%), TMS (25.0%), and TC (31.5%). The highest resistance rate was observed for MBFX (66.1%).

Mannheimia haemolytica: The results of the microdilution method for 100 strains of M. haemolytica are shown in Table 2B. The MIC cut-off values indicated by vertical lines in the table were the same as those used for P. multocida, except for TMS. For TMS, the CLSI MIC CBPs were applied. The lowest antimicrobial resistance rate was observed for CEZ (1.0%), followed by MBFX (6.0%), FFC (10.0%), ERFX (13.0%), TMS (20.0%), TS (25.0%), TP (29.0%), KM (31.0%), and TC (37.0%). The highest resistance rate was observed for PCG (39.0%). The MIC of the quality control strain was within the CLSI acceptable range (Table 3).

Table 3. Quality control standards and measurement values.

		PCG	KM	CEZ	TC	FFC	TP	MBFX	ERFX	TS	TMS
Staphylococcus aureus ATCC29213	CLSI Quality Control Ranges	0.25–2	1–4	0.25–1	0.12–1	2–8	(32^a)	0.12–0.5	0.03–0.12	0.5–4	1–4
Staphylococcus aureus ATCC29213	This study (μg/mL)	0.5–2	2–4	0.25–1	0.25–1	4–8	32–64	0.25–0.5	≤0.125	1	1

Staphylococcus aureus ATCC25923	CLSI Quality Control Ranges	26–37	19–26	29–35	24–30	22–29	-	24–30	27–31	(19–25^b)	17–21
Staphylococcus aureus ATCC25923	This study (mm)	27–31	20–21	29–33	25–28	23–25	15–18	26	27	20–22	17–21

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PCG: penicillin, KM: kanamycin, CEZ: cefazolin, TC: tetracycline, FFC: florfenicol, TP: thiamphenicol, MBFX: marbofloxacin, ERFX: enrofloxacin, TS: tylosin, TMS: tilmicosin. ^a: The value reported by Wei et al. was cited [31]. ^b: The acceptable range for the TS (60 μg) disk was cited from [16].

Establishment of epidemiological inhibition zone diameter cut-off values

Pasteurella multocida: The inhibition zone diameter cut-off values were calculated as follows: PCG, S ≥30 mm (sensitivity 1.000, specificity 0.923), R ≤29 mm (sensitivity 1.000, specificity 1.000); KM, S ≥16 mm (sensitivity 0.944, specificity 1.000); CEZ, S ≥30 mm (sensitivity 0.761, specificity 0.700); TC, S ≥21 mm (sensitivity 0.983, specificity 0.888), R ≤19 mm (sensitivity 0.986, specificity 1.000); TS, S ≥9 mm (sensitivity 1.000, specificity 1.000); TP, S ≥27 mm (sensitivity 1.000, specificity 0.875); TMS, S ≥12 mm (sensitivity 1.000, specificity 1.000); and MBFX, S≥31 mm (sensitivity 0.976, specificity 0.939) (Table 1). The cut-off value for CEZ against P. multocida was re-evaluated by combining the strains used in this study with those reported by Kumakawa et al. [20], resulting in a value of ≥30 mm (revised from ≥28 mm) (Fig. 1A).

Fig. 1. — Scatter plot of the minimum inhibitory concentration (MIC) and inhibition zone diameters for cefazolin against *Pasteurella multocida* and kanamycin against *Mannheimia haemolytica*. A. Scatter plot of the MIC and inhibition zone diameters for cefazolin against *Pasteurella multocida* (n=124). A total of 115 strains were clustered within the MIC range of 0.5–2.0 μg/mL. B. Scatter plot of the MIC and inhibition zone diameters for kanamycin against *Mannheimia haemolytica* (n=100). A strain exhibiting an MIC of 16 μg/mL (ECOFF) and a zone diameter of 15 mm was identified, leading to a revision of the cut-off value to 15 mm. The vertical lines indicate the MIC cut-off values for cefazolin (SOP) and kanamycin (ECOFF). The horizontal lines indicate the inhibition zone diameter cut-off values calculated in this study.

Mannheimia haemolytica: The inhibition zone diameter cut-off values were calculated as follows: PCG, S ≥25 mm (sensitivity 1.000, specificity 0.907), R ≤24 mm (sensitivity 1.000, specificity 1.000); KM, S ≥15 mm (sensitivity 1.000, specificity 1.000); CEZ, S ≥22 mm (sensitivity 1.000, specificity 1.000); TC, S ≥22 mm (sensitivity 0.984, specificity 1.000), R ≤20 mm (sensitivity 0.985, specificity 0.979); TS, S ≥12 mm (sensitivity 0.920, specificity 0.960); TP, S ≥23 mm (sensitivity 1.000, specificity 1.000); and MBFX, S ≥19 mm (sensitivity 1.000, specificity 1.000) (Table 1). The cut-off values were re-evaluated by incorporating the strains used in this study with those reported by Kumakawa et al. [20], resulting in changes in the cut-offs for certain agents: the cut-off for TC was changed to ≥22 mm (from 23 mm), that for KM was changed to ≥15 mm (from 18 mm) (Fig. 1B), and that for CEZ was changed to ≥22 mm (from 23 mm). The results of the quality control tests for the disk diffusion method were within the acceptable range (Table 3).

Antibiogram analysis

Table 4 shows the antibiograms. The susceptibility rates of FFC, ERFX, and TMS (M. haemolytica) shown in the antibiograms of this study were calculated using the CLSI breakpoint values for inhibition zone diameters. The susceptibility rate of FFC against M. haemolytica was high overall, but the rate in Hokkaido (84.4%) was significantly lower than that in eastern Japan (100%) (P<0.05). The susceptibility rate of TMS against M. haemolytica was low in all surveyed regions combined (70.0%). The susceptibility rates of PCG and TC were calculated based on the inhibition zone diameter cut-off values derived from the MIC CBPs. Low susceptibility rates were observed for TC against P. multocida (all surveyed regions combined: 66.1%) and for PCG and TC against M. haemolytica (all surveyed regions combined: 61.0% and 54.0%, respectively). The susceptibility rates of KM and TMS against P. multocida were calculated based on the inhibition zone diameter cut-off values derived from the ECOFFs. For P. multocida, the susceptibility rate of TMS in Hokkaido (58.6%) was significantly lower than those in other regions (eastern Japan: 91.7%, western Japan: 100%) (P<0.01). The susceptibility rate of KM against M. haemolytica was low (all surveyed regions combined: 69.0%). The susceptibility rates for CEZ, TP, MBFX, and TS were calculated based on the inhibition zone cut-off values determined by this study. The susceptibility rate of MBFX against P. multocida in Hokkaido (24.3%) was significantly lower than that in western Japan (53.3%), and the susceptibility rate of TS against P. multocida in Hokkaido (61.4%) was significantly lower than those in other regions (eastern Japan: 95.8%, western Japan: 100%).

Table 4. Antibiogram of Pasteurella multocida and Mannheimia haemolytica isolated from cases of bovine respiratory disease using the disk diffusion method.

A. Pasteurella multocida

Region	Number of isolates	Susceptibility rates (%)

		Based on CLSI breakpoints^d		Setting MIC’s judgment

				CLSI^e		EUCAST^f		tdis study^g

		FFC	ERFX	PCG	TC	KM	TMS	CEZ	TP	MBFX	TS

All surveyed regions	124	99.2	86.3	90.3	66.1	87.9	75.0	79.8	94.4	35.5	77.4
Hokkaido	70	98.6	81.4	90.0	61.4	87.1	58.6^a	77.1	92.9	24.3^a	61.4^a
Eastern japan^h	24	100.0	95.8	95.8	75.0	91.7	91.7^b	91.7	91.7	45.8	95.8^b
Western japan	30	100.0	90.0	86.7	73.3	86.7	100.0^b	76.7	100.0	53.3^c	100.0^b

B. Mannheimia haemolytica

Region	Number of isolates	Susceptibility rates (%)

		Based on CLSI breakpoints^d			Setting MIC’s judgment

					CLSI^e		EUCAST^f	This study^g

		FFC	ERFX	TMS	PCG	TC	KM	CEZ	TP	MBFX	TS

All surveyed regions	100	92.0	72.0	70.0	61.0	54.0	69.0	99.0	71.0	94.0	73.0
Hokkaido	45	84.4^a	64.4	75.6	48.9	57.8	71.1	100.0	68.9	91.1	77.8
Eastern japan	39	100.0^c	82.1	61.5	71.8	59.0	66.7	97.4	74.4	100.0	66.7
Western japan^h	16	93.8	68.8	75.0	68.8	56.3	68.8	100.0	68.8	87.5	75.0

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FFC: florfenicol, ERFX: enrofloxacin, PCG: penicillin, TC: tetracycline, KM: kanamycin, TMS: tilmicosin, CEZ: cefazolin, TP: thiamphenicol, MBFX: marbofloxacin, TS: tylosin. ^{a, b, c}: Significant difference between the different symbols within the same drug.^{a, b}: P<0.01, ^a,
c: P<0.05. ^d: Susceptibility rates are calculated using CLSI zone diameter breakpoints (VET01S). ^e: The susceptibility rate was calculated using the zone diameter cutoff values derived in this study based on the CLSI MIC breakpoints. ^f: The sensitivity rate was calculated using the cut-off value for the zone diameter derived in this study based on EUCAST's ECOFF. ^g: The MIC cutoff values were determined in accordance with the SOP, and the susceptibility rate was calculated using the inhibition zone diameter cutoff values derived in this study. ^h: Preliminary results due to the small sample size.

DISCUSSION

In this study, epidemiological cut-off values for the disk diffusion method were established for P. multocida and M. haemolytica, the primary pathogenic bacteria associated with BRD, and antibiograms were created for the surveyed regions. The inhibition zone diameter cut-off values for PCG, KM, TC, TS, TP, TMS, and MBFX against P. multocida and those for PCG, KM, CEZ, TC, TS, TP, and MBFX against M. haemolytica showed sensitivity and specificity of ≥0.8. The antimicrobial agents selected in this study are expected to be used in Japan for the treatment of BRD caused by P. multocida and M. haemolytica [15]. These cut-off values may facilitate antimicrobial susceptibility testing in livestock production settings and support the prudent use of antimicrobials. In this study, the inhibition zone diameter cut-off values for PCG and TC were established based on the MIC CBPs. The TC cut-off value established in a previous report [20] was re-evaluated and revised by adding the strains used in the present study. If the strains used for analysis are limited, the results are likely to be strongly influenced by strains exhibiting specific susceptibility profiles, potentially introducing bias into the calculated cut-off values. By integrating the strains reported by Kumakawa et al. [20] with those used in the present study and accumulating data from two facilities, we calculated cut-off values that are less susceptible to strain-specific variability. The TC inhibition zone diameter cut-off values exhibited high sensitivity and specificity; furthermore, the TC susceptibility rate in all surveyed regions combined was similar to the MIC resistance rate reported in a previous study [30]. These findings suggest that the inhibition zone diameter cut-off value for TC against M. haemolytica established in this study provides useful guidance for clinical drug selection, and similar utility may be expected for PCG and TC against P. multocida and PCG against M. haemolytica. The inhibition zone diameter cut-off values for KM and TMS against P. multocida and for KM against M. haemolytica were calculated based on the MIC ECOFFs. The cut-off value for KM was calculated by combining the strains used by Kumakawa et al. [20] with those used in this study. In previous studies, the cut-off value for KM against M. haemolytica was set at 18 mm because no strains with inhibition zone diameters of 7–17 mm were found among those at or below the ECOFF (MIC ≤16 μg/mL) [20]. In this study, a strain exhibiting an MIC of 16 μg/mL and a zone diameter of 15 mm was identified, leading to a revision of the cut-off value to 15 mm (Fig. 1B). Although further evaluation of strains with zone diameters of 7–14 mm remains necessary, this revision may reduce false-resistant classifications (i.e., major discrepancies) in the 15–17 mm range. Since an ECOFF distinguishes wild-type strains from non-wild-type strains with resistance mechanisms, ECOFFs and clinical cut-off values often coincide [8]. However, low-level resistant strains may remain clinically susceptible, and strains classified as wild-type are not necessarily clinically susceptible [8]. The inhibition zone diameter cut-off values established in this study for KM and TMS against P. multocida should therefore be used with consideration of the characteristics of these ECOFFs. For CEZ, TP, MBFX, and TS, MIC cut-off values were derived according to the SOP, and the inhibition zone diameter cut-off values were calculated based on these values. For CEZ, the values were calculated by combining the strains used by Kumakawa et al. [20] with those used in this study. The CEZ cut-off value for P. multocida showed low sensitivity and specificity. Specifically, visual inspection revealed that the data points for MIC and inhibition zone diameter were widely scattered. The MIC distribution was clustered in the range 0.5–2.0 μg/mL (Fig. 1A), which falls within the three-dilution range considered to be the variability range of the microdilution method [6]; the poor correlation and the low sensitivity and specificity of the cut-off values were likely due to the clustering of strains within this narrow MIC range. No quality control acceptable range has been established for TP in CLSI guidelines. A report indicated that the MIC of TP for S. aureus ATCC 29213 was 32 μg/mL [31], which was similar to the results of this study. To the best of our knowledge, no reports regarding quality control data for TP using the disk diffusion method have been published to date. When using the cut-off values for CEZ, TP, MBFX, and TS established in this study, it should be considered that the MIC cut-off values calculated according to the SOP were derived solely from the strains used in this study and that these strains were isolated from clinically affected BRD cases. Furthermore, drug susceptibility breakpoints must consider therapeutic efficacy, pharmacokinetic (PK)/ pharmacodynamic (PD) data, and drug susceptibility test data for each species [26]. In the present study, the epidemiological cut-off values for the inhibition zone diameters of PCG and TC in Japan were established based on CLSI MIC CBPs; however, those for KM, CEZ, TS, TP, MBFX, and TMS (P. multocida) were based on EUCAST ECOFFs or MIC cut-off values calculated according to the SOP. Therefore, PK/PD data and therapeutic efficacy were not considered, which is a limitation of this study. Future studies should review these cut-off values with consideration of these factors.

The susceptibility rate of FFC against M. haemolytica, as shown in the antibiograms, was similar to that reported previously based on MIC values [30]. In this study, the susceptibility rate of FFC in Hokkaido was significantly lower than that in eastern Japan, indicating regional variation. In contrast, the susceptibility rate of ERFX against M. haemolytica was lower than that reported previously [30]. Although this study confirmed farm-level routine therapeutic use of fluoroquinolones, detailed histories of antimicrobial administration were not investigated. However, since isolates were collected from calves exhibiting clinical signs of BRD on farms with a high disease incidence, it is likely that these animals were routinely exposed to antimicrobials. Conversely, a previous report noted that the health status of the cattle could not be investigated [30], which suggests the possibility that isolates from healthy cattle or those with subclinical infections were included in the analysis. Since susceptibility can differ between isolates from healthy and diseased animals [28], the difference in susceptibility rates between the two studies was inferred to be a result of these factors. For agents other than FFC and ERFX, the susceptibility rates in the antibiograms were calculated using MIC cut-off values derived from CBPs (PCG and TC), ECOFFs (KM and TMS for P. multocida), or this study (CEZ, TP, MBFX, and TS). Although the susceptibility rate of MBFX against P. multocida differed from that of ERFX, direct comparison was not considered possible because the cut-off value setting procedures differed. Fluoroquinolone antibiotics show high tissue penetration, distributing to skin, lungs, bones, prostate, neutrophils, and macrophages at concentrations exceeding serum levels [22]. Since this study was limited to in vitro evaluation, the efficacy of MBFX may have been underestimated. Similarly, the susceptibility rates for CEZ, TP, and TS could not be directly compared with those of drugs for which the cut-off value setting procedures differed; therefore, the evaluation of susceptibility rates for these drugs is considered useful primarily for comparisons of the same drugs among regions. However, low susceptibility rates were suggested for MBFX and TS against P. multocida and for TP and TS against M. haemolytica. Because of the small number of strains investigated, data for P. multocida in eastern Japan (24 strains) and M. haemolytica in western Japan (16 strains) should be interpreted with caution [14]. The low susceptibility rates observed in the antibiograms may be related to the high use of these antibiotics. In Japan, all of the antimicrobials investigated in this study are approved for the treatment of BRD [15]. In fact, the use and sales of antimicrobial drugs for dairy and beef cattle in Japan were reported to be the highest for tetracyclines, followed by penicillins, sulfonamides, aminoglycosides, macrolides, thiamphenicols, and cephalosporins [13, 21]. Given that the emergence of resistant bacteria and resistance rates are strongly affected by antimicrobial use [2, 12], antimicrobial usage patterns may be one factor contributing to the low susceptibility rates observed in this study. It is important to promote the prudent use of antimicrobials through the prevention of BRD by vaccine administration and improved hygiene management. In human medicine, empirical antimicrobial treatment based on antibiograms recommends selecting agents with a susceptibility rate of 90% or higher [19]. Therefore, the low susceptibility rates observed in the present study are a cause for concern. Continued monitoring of susceptibility trends is therefore warranted. When selecting antimicrobial therapy, factors such as the type and severity of the infection, the causative pathogen, treatment history, and previous antimicrobial use must be considered. It is not possible to select the optimal empirical antimicrobial therapy for individual animals based on a local antibiogram alone [17]. If the MIC remains below the resistance cut-off value or the inhibition zone diameter remains above the resistance cut-off value, emerging resistance may not be detected [17]. Furthermore, antibiograms only summarize the percentage of isolates that are susceptible to a given microorganism–antimicrobial agent combination, and they are not suitable for understanding patterns of co-resistance with other antibiotics [17]. These limitations should be considered when using antibiograms.

The findings of this study suggest that antimicrobial-resistant P. multocida and M. haemolytica isolated from BRD cases are present in the surveyed regions. In clinical practice, using the cut-off values established by the disk diffusion method in the present study and actively performing antimicrobial susceptibility testing may enable more appropriate antimicrobial therapy compared to current levels. When susceptibility testing is not feasible, antibiograms could potentially be used to select agents with high susceptibility rates. These approaches are expected to contribute to the prudent use of antimicrobials. Further investigation of the spread of resistant strains according to serotype and genotype, together with continued monitoring of antimicrobial resistance using antibiograms, will improve our understanding of resistance trends in BRD-causing bacteria and contribute to the control of antimicrobial resistance.

Conflicts of Interest

This work was conducted as part of research carried out under an endowed chair funded by Meiji Animal Health Co., Ltd.

Supplementary Material

jvms-88-10-1556-s001.pdf^{(252.7KB, pdf)}

Acknowledgments

The authors sincerely thank the members of the Japanese Society of Antimicrobials for Animals for their cooperation.

REFERENCES

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

jvms-88-10-1556-s001.pdf^{(252.7KB, pdf)}

[r1] 1.Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment search tool. J Mol Biol 215: 403–410. doi: 10.1016/S0022-2836(05)80360-2 [DOI] [PubMed] [Google Scholar]

[r2] 2.Asai T, Kojima A, Harada K, Ishihara K, Takahashi T, Tamura Y. 2005. Correlation between the usage volume of veterinary therapeutic antimicrobials and resistance in Escherichia coli isolated from the feces of food-producing animals in Japan. Jpn J Infect Dis 58: 369–372. doi: 10.7883/yoken.JJID.2005.369 [DOI] [PubMed] [Google Scholar]

[r3] 3.Balouiri M, Sadiki M, Ibnsouda SK. 2016. Methods for in vitro evaluating antimicrobial activity: a review. J Pharm Anal 6: 71–79. doi: 10.1016/j.jpha.2015.11.005 [DOI] [PMC free article] [PubMed] [Google Scholar]

[r4] 4.Cameron A, McAllister TA. 2016. Antimicrobial usage and resistance in beef production. J Anim Sci Biotechnol 7: 68. doi: 10.1186/s40104-016-0127-3 [DOI] [PMC free article] [PubMed] [Google Scholar]

[r5] 5.Clinical and Laboratory Standards Institute.2023. VET01S Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals, 6th ed., CLSI, Wayne. [Google Scholar]

[r6] 6.Clinical and Laboratory Standards Institute.2024. VET01 Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals, 6th ed., CLSI, Wayne. [Google Scholar]

[r7] 7.EUCAST.2026. MIC and Zone diameter distributions. https://mic.eucast.org/search/ [accessed on March 19, 2026].

[r8] 8.EUCAST.2026. MIC distributions and ECOFFs. https://www.eucast.org/vetcast/mic-distributions-and-ecoffs/ [accessed on June 4, 2026].

[r9] 9.EUCAST.2026. Standard Operating Procedure MIC distributions and the setting of epidemiological cut-off (ECOFF) values. EUCAST SOP 10.2. https://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/EUCAST_SOPs/2021/EUCAST_SOP_10.2_MIC_distributions_and_epidemiological_cut-off_value__ECOFF__setting_20211202.pdf [accessed on March 13, 2026].

[r10] 10.Gajic I, Kabic J, Kekic D, Jovicevic M, Milenkovic M, Mitic Culafic D, Trudic A, Ranin L, Opavski N. 2022. Antimicrobial susceptibility testing: a comprehensive review of currently used methods. Antibiotics (Basel) 11: 427. doi: 10.3390/antibiotics11040427 [DOI] [PMC free article] [PubMed] [Google Scholar]

[r11] 11.Hodgson PD, Aich P, Manuja A, Hokamp K, Roche FM, Brinkman FSL, Potter A, Babiuk LA, Griebel PJ. 2005. Effect of stress on viral-bacterial synergy in bovine respiratory disease: novel mechanisms to regulate inflammation. Comp Funct Genomics 6: 244–250. doi: 10.1002/cfg.474 [DOI] [PMC free article] [PubMed] [Google Scholar]

[r12] 12.Holmes AH, Moore LSP, Sundsfjord A, Steinbakk M, Regmi S, Karkey A, Guerin PJ, Piddock LJV. 2016. Understanding the mechanisms and drivers of antimicrobial resistance. Lancet 387: 176–187. doi: 10.1016/S0140-6736(15)00473-0 [DOI] [PubMed] [Google Scholar]

[r13] 13.Hosoi Y, Matsuda M, Kawanishi M, Harada S, Kumakawa M, Sekiguchi H, Asai T, Sekiya T. 2024. Antimicrobial use in the animal sector in Japan from 2011 to 2022. Antibiotics (Basel) 13: 1204. doi: 10.3390/antibiotics13121204 [DOI] [PMC free article] [PubMed] [Google Scholar]

[r14] 14.Infectious Disease Education Consortium, AMR Clinical Reference Center, National Center for Global Health and Medicine Hospital.2019. Antibiogram guideline. https://amr.ncgm.go.jp/pdf/201904_antibaiogram_guideline.pdf [accessed on February 4, 2025].

[r15] 15.Japanese Society of Antimicrobials for Animals.2013. Manual on Antimicrobial Agents for Animals, 2nd ed., Interzoo, Tokyo. [Google Scholar]

[r16] 16.Japanese Society of Antimicrobials for Animals.2014. Guideline for antimicrobial susceptibility test methods for bacteria isolated from animals. Annual Report of the Japanese Society of Antimicrobials for Animals 36: 150–174. [Google Scholar]

[r17] 17.Joshi S. 2010. Hospital antibiogram: a necessity. Indian J Med Microbiol 28: 277–280. doi: 10.4103/0255-0857.71802 [DOI] [PubMed] [Google Scholar]

[r18] 18.Kanda Y. 2013. Investigation of the freely available easy-to-use software ‘EZR’ for medical statistics. Bone Marrow Transplant 48: 452–458. doi: 10.1038/bmt.2012.244 [DOI] [PMC free article] [PubMed] [Google Scholar]

[r19] 19.Klinker KP, Hidayat LK, DeRyke CA, DePestel DD, Motyl M, Bauer KA. 2021. Antimicrobial stewardship and antibiograms: importance of moving beyond traditional antibiograms. Ther Adv Infect Dis 8: 20499361211011373. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r20] 20.Kumakawa M, Akama R, Hosoi Y, Hiraoka Y, Harada S, Matsuda M, Kawanishi M, Sekiguchi H. 2025. Establishing tentative cut-off values for disk diffusion method for Pasteurella multocida and Mannheimia haemolytica from livestock animals in Japan. J Vet Med Sci 87: 349–355. doi: 10.1292/jvms.24-0474 [DOI] [PMC free article] [PubMed] [Google Scholar]

[r21] 21.National Veterinary Assay Laboratory, Ministry of Agriculture Forestry and Fisheries.2023. Antimicrobial agent usage (sales volume) in the animal sector. https://www.maff.go.jp/nval/yakuzai/yakuzai_p3_6.html [accessed on July 28, 2025].

[r22] 22.Oliphant CM, Green GM. 2002. Quinolones: a comprehensive review. Am Fam Physician 65: 455–464. [PubMed] [Google Scholar]

[r23] 23.Pakyz AL. Insights from the Society of Infectious Diseases Pharmacists.2007. The utility of hospital antibiograms as tools for guiding empiric therapy and tracking resistance. Pharmacotherapy 27: 1306–1312. doi: 10.1592/phco.27.9.1306 [DOI] [PubMed] [Google Scholar]

[r24] 24.Pardon B, Hostens M, Duchateau L, Dewulf J, De Bleecker K, Deprez P. 2013. Impact of respiratory disease, diarrhea, otitis and arthritis on mortality and carcass traits in white veal calves. BMC Vet Res 9: 79. doi: 10.1186/1746-6148-9-79 [DOI] [PMC free article] [PubMed] [Google Scholar]

[r25] 25.Schulz LT, Fox BC, Polk RE. 2012. Can the antibiogram be used to assess microbiologic outcomes after antimicrobial stewardship interventions? A critical review of the literature. Pharmacotherapy 32: 668–676. doi: 10.1002/j.1875-9114.2012.01163.x [DOI] [PubMed] [Google Scholar]

[r26] 26.Shryock TR, Staples JM, DeRosa DC. 2002. Minimum inhibitory concentration breakpoints and disk diffusion inhibitory zone interpretive criteria for tilmicosin susceptibility testing against Pasteurella multocida and Actinobacillus pleuropneumoniae associated with porcine respiratory disease. J Vet Diagn Invest 14: 389–395. doi: 10.1177/104063870201400505 [DOI] [PubMed] [Google Scholar]

[r27] 27.The Government of Japan.2023. National Action Plan on Antimicrobial Resistance (AMR) 2023–2027. https://www.mhlw.go.jp/content/10900000/001096228.pdf [accessed on February 13, 2025].

[r28] 28.Timsit E, Hallewell J, Booker C, Tison N, Amat S, Alexander TW. 2017. Prevalence and antimicrobial susceptibility of Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni isolated from the lower respiratory tract of healthy feedlot cattle and those diagnosed with bovine respiratory disease. Vet Microbiol 208: 118–125. doi: 10.1016/j.vetmic.2017.07.013 [DOI] [PubMed] [Google Scholar]

[r29] 29.Turner S, Pryer KM, Miao VPW, Palmer JD. 1999. Investigating deep phylogenetic relationships among cyanobacteria and plastids by small subunit rRNA sequence analysis. J Eukaryot Microbiol 46: 327–338. doi: 10.1111/j.1550-7408.1999.tb04612.x [DOI] [PubMed] [Google Scholar]

[r30] 30.Ueno Y, Hoshinoo K, Suwa T, Kakinuma H, Takamatsu D, Katsuda K. 2026. Temporal trends of antimicrobial resistance and resistance genes in Mannheimia haemolytica from cattle in Japan. Vet Microbiol 316: 110998. doi: 10.1016/j.vetmic.2026.110998 [DOI] [PubMed] [Google Scholar]

[r31] 31.Wei CF, Chang SK, Shien JH, Kuo HC, Chen WY, Chou CC. 2016. Synergism between two amphenicol of antibiotics, florfenicol and thiamphenicol, against Staphylococcus aureus. Vet Rec 178: 319. doi: 10.1136/vr.103554 [DOI] [PubMed] [Google Scholar]

[r32] 32.World Health Organization.2014. Antimicrobial resistance: global report on surveillance, World Health Organization, Geneva. [Google Scholar]

PERMALINK

Establishment of disk diffusion national epidemiological cut-off values for Pasteurella multocida and Mannheimia haemolytica isolated from bovine respiratory diseases and characteristics of antibiograms in Japan

Tomoko KOSHIISHI

Akiko KATO

Mio KUMAKAWA

Yuta HOSOI

Michiko KAWANISHI

Shunsuke MORI

Tomomi KURUMISAWA

Junko SAITO

Mie KISHIKAWA

Kazuhiro KAWAI

Abstract

INTRODUCTION

MATERIALS AND METHODS

Ethical approval

BRD sample collection and identification of causative bacteria

Drug susceptibility testing

Determination of MIC and inhibition zone diameter cut-off values

Antibiogram analysis

Statistical analysis

RESULTS

Bacterial isolation

Establishment of epidemiological MIC cut-off values

Table 1. Established tentative cut-off values of Pasteurella multocida and Mannheimia haemolytica.

Table 2. Number of isolates with minimum inhibitory concentration (MIC) distribution for Pasteurella multocida and Mannheimia haemolytica.

MIC distribution

Table 3. Quality control standards and measurement values.

Establishment of epidemiological inhibition zone diameter cut-off values

Fig. 1.

Antibiogram analysis

Table 4. Antibiogram of Pasteurella multocida and Mannheimia haemolytica isolated from cases of bovine respiratory disease using the disk diffusion method.

DISCUSSION

Conflicts of Interest

Supplementary Material

Acknowledgments

REFERENCES

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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