Abstract

Objectives: The Japanese Veterinary Antimicrobial Resistance Monitoring (JVARM) Program was established in 1999 to examine the susceptibility of bacteria from food-producing animals to antimicrobial agents. This study tested the susceptibility of Salmonella isolates collected during 2001–2002 to 20 antimicrobials.

Materials and methods: MICs of antimicrobial agents were determined using the NCCLS agar dilution method, and interpreted according to breakpoints obtained from the bimodal MIC distributions.

Results: A total of 82 Salmonella were isolated from food-producing animals and tested for antimicrobial susceptibility. Isolates resistant to ampicillin, dihydrostreptomycin, kanamycin, oxytetracycline, chloramphenicol, bicozamycin, nalidixic acid, oxolinic acid and trimethoprim were obtained from healthy animals and diagnostic sample submissions. Salmonella Dublin was isolated only from cattle and showed resistance to older quinolones. Resistance to ampicillin, dihydrostreptomycin, kanamycin and oxytetracycline was common across all serotypes. Fluoroquinolone-resistant Salmonella Choleraesuis was isolated from swine and was the first Japanese report on this type of resistance in Salmonella from an animal origin. Most Salmonella Typhimurium isolates showed resistance to ampicillin, chloramphenicol, dihydrostreptomycin and oxytetracycline. S. Typhimurium DT104 accounted for 40.7% of S. Typhimurium isolates and was more often multi-drug resistant. Most Salmonella Infantis isolates from poultry showed resistance to dihydrostreptomycin, oxytetracycline, trimethoprim or kanamycin. In Salmonella Enteritidis, the major serotype isolated from food-poisoning in Japan, only resistance to dihydrostreptomycin was observed.

Conclusions: This is the first JVARM report of Salmonella isolates, and continuous investigations at the national level on antimicrobial resistance in Salmonella isolated from food-producing animals will be important in the JVARM Program.

Received 4 July 2003; returned 15 September 2003; revised 19 November 2003; accepted 19 November 2003

Introduction

Salmonella can be isolated from numerous animal species and is known to be a principal zoonotic bacterium causing symptoms such as diarrhoea, fever and septicaemia. These symptoms can be lethal to animals, so swift treatment with appropriate antimicrobial agents remains economically important. Salmonella is also a causative pathogen in food-borne illness. In Japan, Salmonella is the major cause of food poisoning, and in 2001 accounted for 24.6% of laboratory-confirmed cases reported to the National Institute of Infectious Disease, Food-borne Disease Active Surveillance Network (http://idsc.nih.go.jp/iasr/index.html). Non-typhoidal Salmonellosis in humans is usually a self-limiting disease confined to the gastrointestinal tract, but when infection spreads beyond the intestine or when immunocompromised persons are affected, appropriate antimicrobial treatment remains essential.

There is considerable information on antimicrobial resistance in Salmonella of human and food animal origin.13 Such data are important for obtaining epidemiological insight and for determining appropriate, often empirical, antimicrobial therapy. The effect of antimicrobial use in food animals on the acquisition of resistant bacteria by humans has been discussed before.4 In addition, the emergence of multidrug-resistant (MDR) serotypes, especially Salmonella Typhimurium definitive phage type (DT) 104, has become a potential problem for animal husbandry and in human medicine. S. Typhimurium DT104 initially emerged among cattle in England and Wales, but was later isolated from poultry, sheep, swine and humans in the European continent and the USA.59 In the 1990s, the frequency of isolation of S. Typhimurium DT104 in cattle worldwide increased, and has been detected in Japan,10 where it has also spread.

The Japanese Veterinary Antimicrobial Resistance Monitoring (JVARM) Program was formed in 1999 in response to international concerns about the impact of antimicrobial resistance on public health. In the present report, we summarize the investigations into antimicrobial susceptibility in Salmonella isolates, one of the targeted bacterial species for the JVARM Program, isolated from food-producing animals. The aim of this study was to provide insight into the occurrence of serotypes and antimicrobial resistance among Salmonella strains isolated from cattle, swine and poultry across Japan.

Materials and methods

Isolation, identification and serotyping

Fresh faecal samples were collected from healthy cattle, swine and poultry at the farming stage, placed in a sterile sampling tube and transported to the monitoring centre. Faecal samples and other organisms directly isolated from diagnostic specimens were inoculated into Hajna tetrathionate broth for enrichment at a ratio of 1 g faeces to 10 mL of broth. After incubation at 42°C for 18 h or an additional 5–7 days at room temperature as secondary enrichment, the broth was inoculated onto desoxycholate hydrogen sulphide lactose and brilliant green agar plates, each supplemented with 20 mg/L of novobiocin sodium, and incubated at 37°C for 18 h. Suspect colonies were isolated and grown on nutrient agar (Difco), and then identified by transfer to tubes with triple sugar iron agar, lysin indole motility semisolid agar, Voges Proskauer semisolid media, urease test broth, Simmons citrate agar, and by subjecting them to slide agglutination by O-group antigen specific antisera. Salmonella isolates were serotyped by the method based on slide agglutination for O and H antigens. All media (except nutrient agar) were purchased from Eiken (Tokyo, Japan). Phage typing of S. Typhimurium was performed at the National Institute of Infectious Disease according to the methods and schemes described previously.11

Bacterial isolates

A total of 82 Salmonella isolates obtained during April 2001–March 2002 were included in this study. Of these isolates, 60 (73.2%) were obtained from diagnostic submissions of clinical cases at the Livestock Hygiene Service Centres. The origins of the isolates were 22 from cattle, 16 from swine and 22 from poultry. The remaining 22 isolates were isolated from faecal samples of healthy animals, including four isolates from two of 91 cattle surveyed, four isolates from two of 89 swine surveyed, 13 isolates from eight of 69 chickens surveyed and one isolate from one of 83 layer hens surveyed. Faecal samples were collected by the Livestock Hygiene Service Centres in 12 of 47 prefectures across Japan.

Antimicrobial susceptibility

The MICs of Salmonella isolates were determined according to the NCCLS guidelines for the agar dilution method,12 and the following quality control strains were used: Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853. The antimicrobials listed below were tested: ampicillin, cefazolin, cefuroxime, ceftiofur, dihydrostreptomycin, kanamycin, gentamicin, destomycin A, apramycin, colistin, oxytetracycline, chloramphenicol, bicozamycin, nalidixic acid, oxolinic acid, enrofloxacin, ofloxacin, sulphadimethoxine, trimethoprim, olaquindox. When the MIC distribution of antimicrobials was bimodal, the breakpoint was set as the midpoint between the peaks of each MIC distribution.

Results

Antimicrobial susceptibility of isolates

Table 1 shows the MIC distributions of 20 antimicrobial agents tested against 82 isolates of Salmonella. For isolates from healthy animals and diagnostic submissions, the MICs of ampicillin, chloramphenicol, dihydrostreptomycin, nalidixic acid, oxytetracycline, bicozamycin, oxolinic acid, kanamycin and trimethoprim exhibited a bimodal distribution. Isolates resistant to colistin, gentamicin and fluoroquinolones (enrofloxacin and ofloxacin) were only observed from diagnostic submissions, but the ratios of the resistance of other antimicrobials were similar between healthy animal and diagnostic samples (data not shown). Table 1 also shows the percentage of isolates resistant to each of the drugs. More than half of the isolates were resistant to dihydrostreptomycin and oxytetracycline, and about 20% were resistant to ampicillin, chloramphenicol, trimethoprim and kanamycin. Sulphadimethoxine showed no activity against this collection of Salmonella (MIC ≥ 256 mg/L), but the Salmonella were highly susceptible to cephalosporins (cefazolin, cefuroxime and ceftiofur) and fluoroquinolones. Only one isolate from the diagnostic submissions showed high-level resistance (4 mg/L) to fluoroquinolones.

Serotypes of isolates

Among the 82 isolates investigated (Table 2), 27 were S. Typhimurium, 12 were S. Infantis and seven were S. Enteritidis. These serotypes are important as causal organisms of food poisoning in Japan. Five isolates of S. Choleraesuis and three isolates of S. Dublin were only identified in diagnostic submissions from swine and cattle, respectively. Phage typing was performed for S. Typhimurium, and 40.7% (11/27) of S. Typhimurium were typed as DT104 or 104B.

The distributions of antimicrobial resistance of the Salmonella serotypes are summarized in Table 3. The distribution of DT104 and 104B resistances was dominated by complete resistance to ampicillin, dihydrostreptomycin, oxytetracycline and chloramphenicol. S. Choleraesuis (one strain resistant to fluoroquinolones), S. Dublin, S. Infantis and S. Typhimurium showed resistance to five or more antimicrobials, but S. Enteritidis showed resistance only to dihydrostreptomycin.

Discussion

Epidemiological surveillance of antimicrobial-resistant Salmonella has become necessary for effective treatment and prediction of occurrence of resistant populations of prevalent serotypes. Some investigators in Japan have reported the results of antimicrobial resistance among specific Salmonella serotypes, but little has been published on the correlation of antimicrobial resistance and numerous important Salmonella serotypes.1316 This JVARM report is the first Japanese national surveillance on the antimicrobial resistance of Salmonella serotypes isolated from food animals.

Most S. Dublin and S. Choleraesuis isolates, which were isolated only from the diagnostic submissions, showed MDR patterns. Yoshimura et al.16 have previously reported that S. Dublin isolated in Japan during 1992–1995 showed a high frequency of resistance to ampicillin, dihydrostreptomycin, nalidixic acid and oxytetracycline. They did not examine the susceptibility test for kanamycin, but for other antimicrobials, the resistance pattern of this serotype seems to be unchanged after nearly a decade. During the monitored interval, a fluoroquinolone-resistant S. Choleraesuis was detected, and this was the first report of fluoroquinolone-resistant Salmonella of animal origin in Japan. At the beginning of 2002, the emergence and spread of fluoroquinolone-resistant S. Choleraesuis was reported in Taiwan.17 These fluoroquinolone-resistant strains were isolated from humans and swine, and by using molecular epidemiological techniques the relatedness between swine and human strains was suggested. Long-term use of enrofloxacin as an additive or growth promoter was suspected in Taiwan.17 In the case of our reported isolate, however, there has been no use of fluoroquinolones as feed additives in Japan; enrofloxacin has only been used as chemotherapy.

S. Typhimurium and S. Infantis are serotypes frequently isolated from food-producing animals and food poisoning cases in Japan. S. Typhimurium DT104 and 104B were isolated from faeces and diagnostic submissions mainly from cattle; some were also isolated from diagnostic submissions from swine. With the antibiotic resistance genes integrated in the chromosome, most DT104 isolates show MDR to five drugs, commonly referred to as resistance (R)-type ACSSuT.18S. Infantis was frequently isolated from patients suffering from food-borne illness, and presents a significant public health concern related to poultry possessing strains with some resistance determinants. S. Enteritidis was the serotype isolated most frequently from patients who suffered food-borne illness. This serotype did not show significant resistance, other than to dihydrostreptomycin, the frequency of which was limited (29%). Contemporary human chemotherapies should remain effective against this Salmonella serotype.

In summary, the present study demonstrated the predominance of MDR DT104 in cattle and swine, and MDR S. Infantis in poultry; the emergence of fluoroquinolone-resistant S. Choleraesuis (one isolate); and the spread of older quinolone-resistant S. Dublin. Monitoring and controlling these types of MDR Salmonella will be important to public health in Japan. Continuous investigations at the national level for antimicrobial resistance in Salmonella isolated from food-producing animal remains a high priority for the JVARM Program.

Acknowledgements

We thank the staff of the Livestock Hygiene Service Centres in Japan for providing the Salmonella isolates and H. Izumiya (National Institute of Infectious Diseases, Tokyo, Japan) for phage typing the Salmonella strains. We also acknowledge Ronald N. Jones (The JONES Group/JMI Laboratories, North Liberty, Iowa, USA) for his valuable advice about the research and his editorial contribution to this manuscript

*

Corresponding author. Tel: +81-42-321-1841; Fax: +81-42-321-1769; E-mail: esaki@nval.go.jp

Table 1.

Susceptibility and occurrence (%) among Salmonella from good producing animals in Japan

 MIC (mg/L)      
 ≤0.125 0.25 0.5 16 32 64 128 256 512 >512  MIC50 (mg/L) MIC90 (mg/L) Breakpointa (mg/L) % resistant 
Ampicillin   24 29  3         3 23    1 >512  16 30.5 
Cefazolin    57 24  1            1   2   
Cefuroxime     12 57 12  1          4   8   
Ceftiofur   1 42 38  1             0.5   1   
Dihydrostreptomycin      1  1  9 16 11 12  7 12 12  1   64  512  32 67.1 
Kanamycin    1 13 44  6    2   1   6  9    2 >512  16 22.0  
Gentamicin  13 52 13     1  3         0.5  16  4  4.9 
Destomycin A        21 61        32  32   
Oxytetracycline     6 25  3   1  10 15 20  2     64  256  16 58.5  
Apramycin     6 29 47            4   4   
Bicozamycin        1 72  3       6   16   32 128  7.3 
Colistin   38 35  1  3  4    1        1   2  16  1.2 
Chloramphenicol     4 11 38  6  1  1   2 16  3     4  256  32 26.8  
Nalidixic acid      1 70  2  1    1  3  2  2    4  16  64  9.8 
Oxolinic acid  7 61  5   2  2  3  1       1    0.25   2  2 11.0  
Enrofloxacin 73  3  5    1           ≤0.125   0.25  2  1.2 
Ofloxacin 70  5  6    1           ≤0.125   0.25  2  1.2 
Sulphadimethoxine             2 33 47  >512 >512   
Trimethoprim  2 47 15       1  1   2  14    0.25 >512  16 22.0 
Olaquindox        3 66 10  2  1      16  32   
 MIC (mg/L)      
 ≤0.125 0.25 0.5 16 32 64 128 256 512 >512  MIC50 (mg/L) MIC90 (mg/L) Breakpointa (mg/L) % resistant 
Ampicillin   24 29  3         3 23    1 >512  16 30.5 
Cefazolin    57 24  1            1   2   
Cefuroxime     12 57 12  1          4   8   
Ceftiofur   1 42 38  1             0.5   1   
Dihydrostreptomycin      1  1  9 16 11 12  7 12 12  1   64  512  32 67.1 
Kanamycin    1 13 44  6    2   1   6  9    2 >512  16 22.0  
Gentamicin  13 52 13     1  3         0.5  16  4  4.9 
Destomycin A        21 61        32  32   
Oxytetracycline     6 25  3   1  10 15 20  2     64  256  16 58.5  
Apramycin     6 29 47            4   4   
Bicozamycin        1 72  3       6   16   32 128  7.3 
Colistin   38 35  1  3  4    1        1   2  16  1.2 
Chloramphenicol     4 11 38  6  1  1   2 16  3     4  256  32 26.8  
Nalidixic acid      1 70  2  1    1  3  2  2    4  16  64  9.8 
Oxolinic acid  7 61  5   2  2  3  1       1    0.25   2  2 11.0  
Enrofloxacin 73  3  5    1           ≤0.125   0.25  2  1.2 
Ofloxacin 70  5  6    1           ≤0.125   0.25  2  1.2 
Sulphadimethoxine             2 33 47  >512 >512   
Trimethoprim  2 47 15       1  1   2  14    0.25 >512  16 22.0 
Olaquindox        3 66 10  2  1      16  32   

aThe breakpoint was set as the midpoint between the peaks of each MIC distribution.

Table 2.

Number of Salmonella serotypes isolated from healthy animals, and diagnostic submissions from animals in Japan

 No. of isolates by animal and specimen source 
 cattle  swine  poultry   
Serotypes healthy diagnostic  healthy diagnostic  healthy diagnostic  total 
Agona        2  2   4 
Choleraesuis      5      5 
Derby      3      3 
Dublin   3         3 
Enteritidis   2      3  2   7 
Infantis   1      6  5  12 
Montevideo   1    1    2   4 
Typhimurium 13   7    1  27 
Others (incl. non-typeable)   2      5 10  17 
Total 22  16  16 22  82 
 No. of isolates by animal and specimen source 
 cattle  swine  poultry   
Serotypes healthy diagnostic  healthy diagnostic  healthy diagnostic  total 
Agona        2  2   4 
Choleraesuis      5      5 
Derby      3      3 
Dublin   3         3 
Enteritidis   2      3  2   7 
Infantis   1      6  5  12 
Montevideo   1    1    2   4 
Typhimurium 13   7    1  27 
Others (incl. non-typeable)   2      5 10  17 
Total 22  16  16 22  82 
Table 3.

Distribution (%) of antimicrobial resistance of Salmonella serotypes isolated from animals in Japan

 No. of isolates resistant to the antimicrobial agentsa 
Serotypes (no. tested) AMP DSM KM GM OTC CHL TMP NA OA ERFX OFLX 
Choleraesuis (5)  5  5  5  4 
Dublin (3)  1  2   2     
Enteritidis (7)   2          
Infantis (12)  10   8    
Typhimurium (27) 20 24 24 19   
[DT104 and 104B (11)]b 11 11   11 11     
 No. of isolates resistant to the antimicrobial agentsa 
Serotypes (no. tested) AMP DSM KM GM OTC CHL TMP NA OA ERFX OFLX 
Choleraesuis (5)  5  5  5  4 
Dublin (3)  1  2   2     
Enteritidis (7)   2          
Infantis (12)  10   8    
Typhimurium (27) 20 24 24 19   
[DT104 and 104B (11)]b 11 11   11 11     

aAMP, ampicillin; DSM, dihydrostreptomycin; KM, kanamycin; GM, gentamicin; OTC, oxytetracycline; CHL, chloramphenicol; TMP, trimethoprim; NA, nalidixic acid; OA, oxolinic acid; ERFX, enrofloxacin; OFLX, ofloxacin.

bRepresents the results of the phage types DT104 and 104B of S. Typhimurium only.

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