Antimicrobial drug resistance of Salmonella isolates from meat and humans, Denmark.

We compared 8,144 Salmonella isolates collected from meat imported to or produced in Denmark, as well as from Danish patients. Isolates from imported meat showed a higher rate of antimicrobial drug resistance, including multidru9 resistance, than did isolates from domestic meat. Isolates from humans showed resistance rates lower than those found in imported meat but higher than in domestic meat. These findings indicate that programs for controlling resistant Salmonella spp. are a global issue.

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Salmonella spp. are among the most common causes of human bacterial gastroenteritis worldwide, and food animals are important reservoirs of the bacteria (1). In recent years, an increase in the occurrence of antimicrobial drug-resistant Salmonella spp. has been observed in several countries (2-5). Fatality rates are higher for patients with infections caused by drug-resistant Salmonella spp., and these patients are more likely to require hospitalization and to be hospitalized for longer periods than are patients with infections caused by antimicrobial drug susceptible Salmonella spp. (6,7).

Antimicrobial drug resistance of Salmonella spp. isolated from food animals in Denmark has so far been relatively low (8). However, an estimated 30% of all poultry, 10% of all pork, and 50% of all beef sold in Denmark is imported (9). Imported meat is therefore an important potential source of human infection with drug-resistant Salmonella spp. We compared antimicrobial drug resistance of Salmonella isolates from both imported meat and meat produced within Denmark (domestic meat), as well as from outpatients with diarrhea.

Salmonella isolates from humans and meat were obtained from July 1998 through June 2002. Isolates from domestic poultry, pork, and beef were obtained through the national Salmonella control program (10), and isolates from imported poultry, pork, and beef were obtained from the Denmark import control and from the regional food control units. Human salmonellosis is a notifiable disease in Denmark, and all human Salmonella spp. isolates are collected at the Statens Serum Institute. The serovars included were restricted to S. Typhimurium, S. Hadar, S. Dublin, S. Saintpaul, S. Enteritidis, S. Virchow, and S. Newport because these were the serovars of which a sufficient number of isolates had been tested for antimicrobial drug susceptibility. Data on 4,081 Salmonella isolates from humans were included in the study.

Identification, serotyping, phage typing, and susceptibility testing were done as described (8,11,12). Susceptibility to the following antimicrobial agents was determined: ampicillin, ceftiofur, chloramphenicol, ciprofloxacin, co-amoxiclav, colistin, florphenicol, gentamicin, nalidixic acid, neomycin, streptomycin, sulfamethoxazole, tetracycline, and trimethoprim.

Statistical analyses were performed using S-PLUS version 6.2 (Insightful Corp., Seattle, WA, USA). The trend in the occurrence of resistant isolates over time, the occurrence of multidrug-resistant isolates over time, and the occurrence of nalidixic acid resistant isolates were investigated by fitting a logistic-regression model with origin (domestic/imported), time (year), product type (beef, pork, poultry), and all 2-way interactions as explanatory variables. The regression models were reduced by using a likelihood ratio test. Significance in all 2-by-2 tables (only tables with minimum 30 domestic and 30 imported samples) was tested by a Pearson [chi square] test with continuity correction; if the number in any cell in the contingency table was <5, Fisher exact test was applied. All tests were done on a 5% significance level (p<0.05). No correction for multiple testing was done. An isolate was considered multidrug resistant if the isolate was resistant to [greater than or equal to] 4 antimicrobial agents.

Salmonella spp. were isolated from 1,078 (11.8%) of 9,135 samples from imported poultry, pork, and beef and 2,985 (1.4%) of 213,214 samples from domestic poultry, pork, and beef. Among the isolates from domestic meat, the serovars S. Typhimurium, S. Infantis, and S. Derby were the 3 most frequently isolated; in imported meat, the 3 most frequently isolated serovars were S. Heidelberg, S. Typhimurium, and S. Hadar (Table 1). In isolates from domestic meat originating from pigs and poultry, S. Typhimurium was the most frequently isolated serovar; in beef isolates, S. Dublin was most common. Among isolates from imported meat, S. Typhimurium was the most frequently isolated serovar from pork and beef, while S. Heidelberg was the most frequently isolated serovar from poultry.

A significantly higher ([chi square], p<0.001) proportion of the Salmonella spp. isolates from imported meat (58%) were resistant to [greater than or equal to] 1 antimicrobial agents compared with isolates from domestic meat (26%) (Table 1). A significant difference ([chi square], p<0.001) was also observed between the proportions of multidrug-resistant isolates from domestic (4%) compared with imported (28%) poultry, pork, and beef.

The regression results (Table 2) showed a significant increase in the proportion of resistant (p<0.001) and multidrug-resistant (p = 0.015) isolates over time and an increase in odds per year of 27% (corresponding to an increase in probability of 5% per year) and 14% (corresponding to an increase in probability of 3% per year), respectively (Figure 1). Furthermore, the probability for isolating a resistant and a multidrug-resistant isolate from imported meat compared with domestic meat was significant, with an odds ratio of [approximately equal to] 5. The probability of isolating a resistant isolate differed between product types; pork had the highest probability, followed by poultry and beef.

[FIGURE 1 OMITTED]

A high proportion of resistant and multidrug-resistant isolates was found among S. Hadar, S. Newport, S. Typhimurium, and S. Heidelberg in imported meat (Table 1). Among S. Typhimurium, antimicrobial drug resistance was particularly prominent in the phage types DT104, DT170, DT193, DT120, DT208, DT107, U302, and DT135 (Table 3). Multidrug-resistant DT104, DT120, 2and DT193 were found in both domestic and imported poultry, pork, and beef, whereas multidrug-resistant DT107, DT170, and DT208 were more common in domestic meat, and multidrug-resistant U302 was more common in imported meat (Table 3).

Resistance to nalidixic acid was higher among isolates from imported meat (26%) compared with isolates from domestic meat (4%) ([chi square], p<0.001, odds ratio = 6.54, Table 3), with an increase over time in the proportion of domestic nalidixic acid resistant isolates (p = 0.004, data not shown). Furthermore, the probability of isolating a nalidixic 2acid-resistant isolate differed between product types; poultry (domestic 14%, imported 30%) had the highest probability, followed by pork (domestic 1%, imported 3.2%) and beef (domestic 1%, imported 0%). Nalidixic acid resistance among Salmonella spp. from imported products was highest among S. Hadar, S. Newport, S. Kottbus, and S. Virchow (Table 1).

For S. Typhimurium, S. Hadar, and S. Virchow, the proportion of resistant and multidrug-resistant isolates was much higher among isolates from humans than among isolates from domestic meat (Table 1, Figure 2). For S. Dublin and S. Enteritidis, the proportion of resistant and multidrug-resistant isolate did not differ between the meat sources and the human isolates, whereas for S. Saintpaul and S. Newport the rates of resistance and multidrug resistance were lower for isolates from humans than from both domestic and imported meat.

[FIGURE 2 OMITTED]

S. Hadar, S. Virchow, S. Newport, and S. Heidelberg were frequently found in imported products but rarely found in domestic products. Isolates that belong to these serovars are common causes of human salmonellosis in Denmark (13). Overall, a significantly higher number of resistant and multidrug-resistant Salmonella isolates were found among isolates from imported poultry, pork, and beef compared with domestic products. This finding implies that consumers in Denmark are more likely to be exposed to drug-resistant Salmonella spp. when eating imported compared with domestic meat. An increase in the occurrence of resistance over time was also observed among isolates from both domestic and imported meat; this is in agreement with observations worldwide (2-5). Antimicrobial agents might not be essential for treatment of gastroenteritis caused by Salmonella spp., but they are essential for treatment of patients with invasive infections. In particular, the frequent occurrence of resistance to quinolones is a matter of concern because these compounds are often used for first treatment of serious human infections, before the results of susceptibility testing are available.

International trade of food products is expected to increase in the future. Thus, endeavors to improve food safety must take into account the importance of resistant Salmonella spp. in imported food products and, through international agreements, limit contamination with antimicrobial drug-resistant Salmonella spp. at the primary production site.

Acknowledgement

We thank the Danish Veterinary and Food Administration for providing import control data.

This study was supported by the National Food Institute, Copenhagen.

Dr Skov is senior researcher in the Research Unit for Clinical Microbiology at the University of Southern Denmark. Her main research interests are the epidemiology and genotyping of foodborne Salmonella spp.

References

(1.) Humphrey TJ. Public-health aspects of Salmonella infections. In: Wray C, Wray A, editors. Salmonella in domestic animals. Wallingford (England): CABI Publishing; 2000.

(2.) Cailhol J, Lailler R, Bouvet P, La Vieille S, Gauchard F, Sanders P, et al. Trends in antimicrobial resistance phenotypes in non-typhoid salmonellae from human and poultry origins in France. Epidemiol Infect. 2006;134:171-8.

(3.) Hoge CW, Gambel JM, Srijan A, Pitarangsi C, Echeverria P. Trends in antibiotic resistance among diarrheal pathogens isolated in Thailand over 15 years. Clin infect Dis. 1998;26:341-5.

(4.) Davis MA, Hancock DD, Besser TE, Rice DH, Gay JM, Gay C, et al. Changes in antimicrobial resistance among Salmonella enterica serovar Typhimurium isolates from humans and cattle in the Northwestern United States, 1982-1997. Emerg Infect Dis. 1999;5:802-6.

(5.) van Duijkeren E, Wannet WJ, Houwers DJ, van Pelt W. Antimicrobial susceptibilities of salmonella strains isolated from humans, cattle, pigs, and chickens in the Netherlands from 1984 to 2001. J Clin Microbiol. 2003;41:3574-8.

(6.) Helms M, Vastrup P, Gerner-Smidt P, Molbak K. Excess mortality associated with antimicrobial drug-resistant Salmonella Typhimurium. Emerg Infect Dis. 2002;8:490-5.

(7.) Varma JK, Molbak K, Barrett TJ, Beebe JL, Jones TF, Rabatsky-Ehr T, et al. Antimicrobial-resistant nontyphoidal Salmonella is associated with excess bloodstream infections and hospitalizations. J Infect Dis. 2005;191:554-61.

(8.) Aarestrup FM, Bager F, Jensen NE, Madsen M, Meyling A, Wegener HC. Resistance to antimicrobial agents used for animal therapy in pathogenic-, zoonotic- and indicator bacteria isolated from different food animals in Denmark: a baseline study for the Danish Integrated Antimicrobial Resistance Monitoring Programme (DANMAP). APMIS. 1998;106:745-70.

(9.) Alban L, Olsen A-M, Granly Koch A. Food safety risk assessment for imports of meat. In: Proceedings of the 18th International Pig Veterinary Society Congress, 27 June-1 July, 2004, Hamburg, Germany. Hamburg: The Society; 2004. p. 667.

(10.) Wegener HC, Hald T, Lo Fo Wong D, Madsen M, Korsgaard H, Bager F, et al. Salmonella control programs in Denmark. Emerg Infect Dis. 2003;9:774-80.

(11.) Aarestrup FM, Lertworapreecha M, Evans MC, Bangtrakulnonth A, Chalermchaikit T, Hendriksen RS, et al. Antimicrobial susceptibility and occurrence of resistance genes among Salmonella enterica serovar Weltevreden from different countries. J Antimicrob Chemother. 2003;52:715-8.

(12.) Baggesen DL, Sandvang D, Aarestrup FM. Characterization of Salmonella enterica serovar typhimurium DT104 isolated from Denmark and comparison with isolates from Europe and the United States. J Clin Microbiol. 2000;38:1581-6.

(13.) Annual reports on zoonoses in Denmark, 2000-2003. Copenhagen: Ministry of Agriculture, Food and Fisheries; 2001-2004. [cited 2007 15 Feb]. Available from http://www.dfvf.dk/Default.aspx? ID=9606

Address for correspondence: Frank M. Aarestrup, National Food Institute, 27 Bolowsvej, DK-1790 Copenhagen V, Denmark; email: faa@ food.dtu.dk

Marianne N. Skov, * Jens Strodl Andersen, ([dagger]) Soren Aabo, ([dagger]) Steen Ethelberg, ([double dagger]) Frank M. Aarestrup, ([dagger]) Anders Hay Sorensen, ([dagger]) Gitte Sorensen, ([dagger]) Karl Pedersen, ([dagger]) Steen Nordentoft, ([dagger]) Katharina E.P. Olsen, ([double dagger]) Peter Gerner-Smidt, ([double dagger]) and Dorte L. Baggesen ([dagger])

* University of Southern Denmark, Odense, Denmark; ([dagger]) National Food Institute, Copenhagen, Denmark; and ([double dagger]) Statens Serum Institut, Copenhagen, Denmark

Table 1. Number and proportion of susceptible (S), resistant (R),
multidrug-resistant (M), and nalidixic acid-resistant (Nal) Salmonella
spp. isolates within different serotypes isolated from meat and humans,
Denmark, July 1998-July 2002 *

                                         Domestic meat

                                 No.
Serotype                        tested    S, %    R, %    M, %    Nal

Typhimurium ([double dagger])    1,508     73      21       6       1
Infantis ([double dagger])         184     94       4       2       2
Derby ([double dagger])            163     55      44       1       1
Heidelberg                           6     67      33       0      17
Hadar ([double dagger])             38     74      26       0      11
Enteritidis ([double dagger])       91     90       9       1       4
Indiana ([double dagger])           94     95       4       1       0
Newport                              2      0     100       0     100
Kottbus                             26     81      19       0      15
Dublin                              71     99       1       0       1
Anatum                              50    100       0       0       0
Saintpaul                            9     11       0      89      22
Regent                              47      0     100       0     100
Virchow                              3    100       0       0       0
Bredeney                             3    100       0       0       0
Other ([double dagger])            690     71      24       5       5
Total                            2,985     74      22       4       4

                                      Imported meat

                                  No.
Serotype                        tested    S, %     R, %

Typhimurium ([double dagger])      138     34       24
Infantis ([double dagger])          50     84       10
Derby ([double dagger])             34     32       59
Heidelberg                         157     49       13
Hadar ([double dagger])            113      1       53 ([section])
Enteritidis ([double dagger])       50     84       16
Indiana ([double dagger])           40     45       43 ([section])
Newport                             78     28       51
Kottbus                             49      6       90
Dublin                               4    100        0
Anatum                              12     75        8
Saintpaul                           39     31        8
Regent                               1      0      100
Virchow                             39     44       36
Bredeney                            38     34        0
Other ([double dagger])            256     56       24
Total                            1,078     42       30

                                        Imported meat

Serotype                                M, %            Nal

Typhimurium ([double dagger])     42 ([section])     9 ([section])
Infantis ([double dagger])         6                 8 ([section])
Derby ([double dagger])            9 ([section])     3
Heidelberg                        38                 4
Hadar ([double dagger])           46 ([section])    81 ([section])
Enteritidis ([double dagger])      0                10
Indiana ([double dagger])         13 ([section])     3
Newport                           21                60
Kottbus                            4                92
Dublin                             0                 0
Anatum                            17                 0
Saintpaul                         62                15
Regent                             0                 0
Virchow                           21                49
Bredeney                          66                11
Other ([double dagger])           20                17
Total                             28                26

                                         Humans ([dagger])

                                 No.
Serotype                        tested    S, %   R, %   M, %   Nal

Typhimurium ([double dagger])    1,886     61     20     19      3
Infantis ([double dagger])
Derby ([double dagger])
Heidelberg
Hadar ([double dagger])            189     26     71      3     58
Enteritidis ([double dagger])    1,706     92      7      0      4
Indiana ([double dagger])
Newport                             59     88      7      5      5
Kottbus
Dublin                              88     95      5      0      2
Anatum
Saintpaul                           58     72      9     19      7
Regent
Virchow                             95     35     56      9     62
Bredeney
Other ([double dagger])
Total                            4,081     73     17      9      7

* Only serotypes with [greater than or equal to] 40 isolates are
included.

([dagger]) Only subsets of selected serovars are routinely tested for
susceptibility to antimicrobial agents.

([double dagger]) Indicates serotypes with >30 samples from Danish
produced meat and >30 samples from imported meat, which were
statistically tested.

([section]) Indicates clinical significance.

Table 2. Results from the reduced logistic regression models *

Variable                                   OR (95% CI)

Resistance vs. nonresistance
  Intercept                           0.164 (0.129 to 0.207)
  Origin                               5.08 (4.19 to 6.18)
  Year                                 1.27 (1.19 to 1.35)
  Cattle vs. poultry                  0.400 (0.230 to 0.662)
  Pork vs poultry                      1.26 (1.06 to 1.51)
Multidrug resistance vs. resistance
  Intercept                           0.141 (0.0976 to 0.201)
  Origin                               4.98 (3.87 to 6.44)
  Year                                 1.14 (1.03 to 1.27)
Nalidixic acid resistance vs. non-nalidixic acid resistance
  Intercept                          0.0611 (0.0333 to 0.107)
  Origin                               6.54 (3.45 to 12.8)
  Year                                 1.41 (1.18 to 1.69)
  Origin and year                     0.732 (0.587 to 0.909)
  Cattle vs. poultry                 0.0404 (0.00229 to 0.182)
  Pork vs. poultry                   0.0668 (0.0425 to 0.101)

Variable                                Estimate (95% CI)

Resistance vs. nonresistance
  Intercept                          -1.81 (-2.05 to -1.57)
  Origin                              1.62 (1.43 to 1.82)
  Year                               0.235 (0.174 to 0.297)
  Cattle vs. poultry                -0.917 (-1.47 to -0.413)
  Pork vs poultry                    0.233 (0.0553 to 0.414)
Multidrug resistance vs. resistance
  Intercept                          -1.96 (-2.33 to -1.60)
  Origin                              1.61 (1.35 to 1.86)
  Year                               0.133 (0.0259 to 0.240)
Nalidixic acid resistance vs. non-nalidixic acid resistance
  Intercept                          -2.80 (-3.40 to -2.24)
  Origin                              1.88 (1.24 to 2.55)
  Year                               0.342 (0.167 to 0.526)
  Origin and year                   -0.311 (-0.532 to -0.0956)
  Cattle vs. poultry                 -3.21 (-6.08 to -1.70)
  Pork vs. poultry                   -2.71 (-3.16 to -2.29)

Variable                             SE (Est.)     p value

Resistance vs. nonresistance
  Intercept                           0.121
  Origin                              0.0988       <0.00001
  Year                                0.0313       <0.00001
  Cattle vs. poultry                  0.268        <0.00001
  Pork vs poultry                     0.0916
Multidrug resistance vs. resistance
  Intercept                           0.185
  Origin                              0.129        <0.00001
  Year                                0.0547        0.0148
Nalidixic acid resistance vs. non-nalidixic acid resistance
  Intercept                           0.296
  Origin                              0.334
  Year                                0.0914
  Origin and year                     0.111         0.00448
  Cattle vs. poultry                  1.01         <0.00001
  Pork vs. poultry                    0.220

* OR, odds ratio; CI, confidence interval; SE, standard error;
Est., estimated.

Table 3. Number and proportion of susceptible (S), resistant (R), and
multidrug-resistant (M) meat isolates within Salmonella Typhimurium
phage types, Denmark, July 1998-July 2002 *

                                            Domestic meat

Serovar/phage type                  M, %    R, %   S, %   Total no.

All S. Typhimurium isolates           6      21     73      1,508
DT104                                70      13     17         23
DT170                                 3      68     29         97
DT193                                13      37     51         63
DT120                                16      29     55         38
DT208                                57      40      3         30
DT107                                 5      55     41         22
U302                                  0      33     67          6
DT135                                 6      56     38         16
Other S. Typhimurium                  5      21     74      1,213
Salmonella other than Typhimurium     3      22     75      1,477
Total                                 4      22     74      2,985

                                            Imported meat

Serovar/phage type                  M, %    R, %    S, %    Total no.

All S. Typhimurium isolates          42      24      34         138
DT104                                88       7       5          43
DT170                                 0       0       0           0
DT193                                50      17      33           6
DT120                                57      43       0           7
DT208                                 0      57      43           7
DT107                                 0       0       0           0
U302                                 38      31      31          13
DT135                                 0     100       0           2
Other S. Typhimurium                 21      32      47          60
Salmonella other than Typhimurium    26      31      43         940
Total                                28      30      42       1,078

Serovar/phage type                  Total no.

All S. Typhimurium isolates           1,646
DT104                                    66
DT170                                    97
DT193                                    69
DT120                                    45
DT208                                    37
DT107                                    22
U302                                     19
DT135                                    18
Other S. Typhimurium                  1,273
Salmonella other than Typhimurium     2,417
Total                                 4,063

* Not all S. Typhfmurfum isolates from humans were phage typed.