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Antimicrobial Drug Resistance in Isolates of Salmonella enterica from Cases of Salmonellosis in Humans in Europe in 2000: Results of International Multi-Centre Surveillance

 Rediger
 1 Published: 30.04.04 Updated: 30.07.2004 11:34:49
E.J. Threlfall 1, I.S.T. Fisher 2, C. Berghold 3, P. Gerner-Smidt 4, H.Tschäpe 5, M. Cormican 6, I. Luzzi 7, F. Schnieder 8, W. Wannet 9, J. Machado 10, G. Edwards 11

The article has been published previously in Eurosurveillance and appears in EpiNorth in agreement with the authors and the editor of Eurosurveillance.(http://www.eurosurveillance.org/em/v08n02/0802-224.asp)

1 Public Health Laboratory Service, Laboratory of Enteric Pathogens, London, United-Kingdom; 2 Enter-net Hub, Gastrointestinal Diseases Division, PHLS-CDSC, London, United-Kingdom; 3 National Salmonella Reference Laboratory, Graz, Austria; 4 Dept of Gastrointestinal Infections, Statens Serum Institut, Copenhagen, Denmark; 5 Robert Koch-Institut, Wernigerode, Germany; 6 National University of Ireland, Galway, Ireland; 7 Istituto Superiore di Sanita, Laboratory of Medical Bacteriology & Mycology, Roma, Italy; 8 Laboratoire National de Santé, Luxembourg; 9 National Institute of Public Health and the Environment, Diagnostic Laboratory for Infectious Diseases and Perinatal Screening, Bilthoven, the Netherlands; 10 Instituto Nacional de Saude, Lisbon, Portugal; 11 Scottish Salmonella Reference Laboratory, Stobhill Hospital, Glasgow, United-Kingdom

The Enter-net surveillance system received results of antimicrobial sensitivity tests for isolates from over 27 000 cases of human salmonellosis in 2000 in 10 European countries. Almost 40% of isolates were resistant to at least one antimicrobial, with 18% multiresistant. Resistance to ampicillin, streptomycin, sulphonamides and tetracyclines was common, with over 20% of isolates resistant to at least one of these antimicrobials. Clinical resistance to ciprofloxacin was rare, with only 0.5% of isolates exhibiting such resistance (MIC >1.0 mg/l). Resistance to nalidixic acid coupled with a decreased susceptibility to ciprofloxacin (MIC 0.25-1.0 mg/l) was more common, with 14% of isolates showing these properties. Resistance to third-generation cephalosporins was rare with only 0.6% of isolates resistant to cefotaxime. In all countries multiple resistance was most common in Salmonella enterica serotype Typhimurium, with 51% of isolates multiresistant in total. In England and Wales multiple resistance was also prevalent in S. Virchow and S. Hadar, whereas in other countries multiple resistance was common in serotypes such as S. Blockley.

Introduction

Enter-net is an international surveillance network for human gastrointestinal infections that involves all 15 European Union (EU) countries, plus Australia, Japan, New Zealand, Norway, South Africa and Switzerland (1). The network conducts surveillance of salmonellas and Verocytotoxin-producing Escherichia coli. Funded by the European Commission, Enter-net is a continuation and extension of the Salm-net surveillance network (1994-1997), which concentrated upon the harmonisation of Salmonella phage-typing and the establishment of an international database for the rapid identification of outbreaks within the EU.

One of the objectives of Enter-net is the international surveillance of antimicrobial drug resistance in human isolates of Salmonella spp. For such surveillance to be successful, it is necessary to either have a common susceptibility testing method, or alternatively, to have international agreement on the definitions of resistance or susceptibility assessed by using different methods. Following an international trial involving 17 national Salmonella reference laboratories (2), the latter approach has been preferred within Europe, with agreement on the interpretation of results such as breakpoints (BP), disk diffusion diameters (DD), or minimum inhibitory concentration. The results of antimicrobial resistance surveillance of isolates from cases of non-typhoidal human salmonellosis in 10 EU countries in 2000 are reported in this article.

Methods

Transfer of data

Results of susceptibility testing of non-typhoidal Salmonella spp. isolates from cases of human infection in 2000 from 10 of the participating Enter-net countries were transferred electronically to the Enter-net surveillance hub at the PHLS Communicable Disease Surveillance Centre, London, United Kingdom. The participating countries were those which routinely include antimicrobial susceptibility testing in their identification procedures. All human isolates referred to the respective laboratories during 2000 were included in the study.

Susceptibility testing

Seven of the 10 laboratories used disk diffusion susceptibility testing (DD), two breakpoints (BP), and one, an MIC-based method in liquid culture. The antimicrobials chosen and the levels used for the assessment of resistance or sensitivity by BP were as follows: ampicillin (8 mg/l); cefotaxime (1 mg/l); chloramphenicol (8 mg/l); gentamicin (4 mg/l); kanamycin (16 mg/l); streptomycin (16 mg/l); sulphonamides (64 mg/l); tetracyclines (8 mg/l); trimethoprim (2 mg/l); nalidixic acid (16 mg/l); ciprofloxacin (0.125 mg/l: decreased susceptibility; 1.0 mg/l: clinical resistance).

Interpretive criteria

For BP the levels incorporated into the agar plates are as shown above. For DD the antimicrobials content, culture medium and inoculum were in accordance with the performance standards for the specific method and the interpretation of susceptibility or resistance by zone size was in accordance with previously agreed criteria (2). For simplification of analyses the recording of resistance or susceptibility is based on full resistance or full susceptibility to the respective antimicrobials. Thus, for resistance to ciprofloxacin by BP only isolates resistant at 1.0 mg/l are included; similarly, when tested by DD only isolates interpreted as `resistant' by the laboratory have been recorded as such.

Results

Antimicrobial drug resistance in Salmonella

Results of sensitivity tests for isolates from 27 059 cases of salmonellosis in 10 European countries in 2000 were sent to the Enter-net surveillance hub. These concerned 255 serotypes. However, as not all the laboratories assessed the resistance to all antimicrobials in the panel, the number of strains tested for each antimicrobial was less than the overall total number of strains. For example over 25 000 strains were tested for resistance to ampicillin and ciprofloxacin, whereas lower numbers were tested for resistance to other antimicrobials (table 1).

Table 1. Antimicrobial drug resistance in salmonella isolated from humans in Europe, 2000

Antimicrobial

No. tested

% resistant

Ampicillin

25 116

22

Chloramphenicol

24 545

14

Gentamicin

24 154

2

Kanamycin

23 178

2

Streptomycin

22 324

21

Sulphonamides

22 995

30

Tetracyclines

24 290

26

Trimethoprim

24 937

7

Nalidixic acid

22 917

14

Ciprofloxacin

25 319

0.5

Cefotaxime

24 413

0.6

The most common resistances detected were to sulphonamides, tetracyclines, streptomycin and ampicillin, with over 20 percent of isolates showing resistance to these antimicrobials (table 1). In contrast, resistance to ciprofloxacin and cefotaxime was rare, with less than 1.0 percent of isolates showing resistance to these antimicrobials. However 13 percent of strains were resistant to chloramphenicol and seven percent to trimethoprim. Fourteen percent of strains were resistant to nalidixic acid.

Occurrence of antimicrobial resistance by serotype

The ten most commonly isolated non-typhoidal salmonella serotypes in Europe in 2000 are listed in table 2. Overall 43 percent of all isolates were drug-resistant with 18 percent showing multiple resistance (to four or more unrelated antimicrobials). Multiple resistance was concentrated in four serotypes - S. Typhimurium (51% of isolates), S. Hadar (37%), S. Virchow (36%) and S. Blockley (25%). Clinical resistance to ciprofloxacin was most common in S. Hadar (3% resistant) but was also observed in S. Enteritidis, S. Typhimurium, S. Virchow and S. Agona. Resistance to cefotaxime, a third-generation cephalosporin, was identified in eight of the 10 most common serotypes. However, with the exception of S. Heidelberg and S. Brandenburg (2% and 1% respectively), the incidence of resistance to this antimicrobial was less than 1 percent.

Thirteen percent of S. Enteritidis isolates, 57% of S. Hadar isolates, and 53% of S. Virchow isolates exhibited resistance to nalidixic acid. It should be noted that in laboratories testing by BP, all isolates with resistance to nalidixic acid also exhibited decreased susceptibility to ciprofloxacin (MIC: 0.25-1.0 mg/l).

Table 2. Occurrence of antimicrobial drug resistance in salmonellas by serotype, in Europe, 2000 / Распространённость антУмУкробной резУстентностУ сальмонеллы по серотУпам в Европе в 2000 году

Serotype

 

n /

 

% resistant to

% resistant to /

A

C

G

K

S

Su

T

Tm

Nx*

Cp

Ct

0

1

2

3

4+ drugs

Enteritidis

14636

6

0.5

0.5

0.5

2

6

3

1

13

0.4

0.3

71

24

2

1

2

Typhimurium

6777

59

47

6

4

51

60

64

15

8

0.6

0.5

23

14

8

4

51

Hadar

622

35

0.8

0.6

0.7

62

10

73

6

57

3

0.6

21

4

14

24

37

Virchow

449

8

3

3

2

7

21

18

20

53

0.9

0.4

28

27

4

4

36

Infantis

439

4

2

3

2

4

24

22

22

6

0

0.2

79

9

4

4

5

Newport

243

8

5

3

4

5

11

9

6

8

0

0.8

79

9

4

4

5

Blockley

229

10

9

0

31

33

5

38

3

17

0

0

49

10

3

10

25

Agona

206

4

0.5

0

0.5

2

4

5

2

2

0.5

0

87

7

3

2

0.5

Heidelberg

175

30

3

3

3

15

15

17

9

6

0

2

57

19

4

6

14

Brandenburg

160

3

2

0.6

0

8

18

24

0.6

0

0

1

63

26

6

4

1

Others (245 serotypes)

3123

8

3

2

2

10

13

14

8

7

0.4

0.5

65

19

4

3

18

Total

27059

22

14

2

2

21

30

26

7

14

0.5

0.4

57

19

4

3

18

Antimicrobials:

A ampicillin; C chloramphenicol; G gentamicin; K kanamycin; S streptomycin; Su sulphonamides; T tetracyclines, Tm trimethoprim; Nx nalidixic acid; Cp ciprofloxacin (MIC ≥ 1.0 mg/l); Ct cefotaxime.

*also exhibit decreased susceptibility to ciprofloxacin (MIC 0.25 - 1.0 mg/l)

Discussion

In 2000 both resistance and multiple resistance was most common in S. Typhimurium, with 77% of isolates drug-resistant and 51% multiresistant (MR). This results from the dissemination throughout Europe and worldwide of a MR clone of definitive phage type (DT) 104 with resistance to ampicillin, chloramphenicol, streptomycin, sulphonamides and tetracyclines, with up to 10 percent of isolates with additional resistance to trimethoprim or nalidixic acid (3). In MR strains of S. Typhimurium DT104, additional resistance to nalidixic acid was coupled with decreased susceptibility to ciprofloxacin. This has been demonstrated to result from one of several mutations in the gyrA gene (4,5).

An important feature of the high incidence of multiresistant S. Typhimurium in Europe has been the international spread, not only of MR DT104, but also of other phage types. Thus in the summer of 2000, a strain of DT204b with resistance to ampicillin, chloramphenicol, gentamicin, kanamycin, streptomycin, sulphonamides, tetracyclines, trimethoprim and nalidixic acid caused extensive outbreaks in five European countries (6). This outbreak coincided with a large outbreak of MR DT104 infection in England and Wales (7). Other MR strains of epidemiological importance in 2000 included a strain of S. enterica serotype [4,5,12:i:{-}], responsible for numerous infections in Spain (8), and also for an outbreak in Denmark (M. Skov, P. Gerner-Smidt, personal communication).

Drug resistance was also common in several other serotypes in 2000, although not to the same extent as in S. Typhimurium. There were, however, considerable regional differences in the predominance of certain drug-resistant serotypes. For example, resistance was common in S. Virchow and S. Hadar strains isolated in England and Wales, where over 35% of isolates of the respective serotypes were multiresistant. Similar levels of multiple resistance were also observed in isolates of these serotypes from some other countries. Multiple resistance was also common in serotypes such as S. Blockley and S. Heidelberg. Multiresistant strains of S. Blockley were often linked to travel to south-east Europe, and it is noteworthy that in Greece, such strains have caused outbreaks of infections since the late 1990s (9).

Although clinical resistance to ciprofloxacin was rare, with only 0.5% of isolates exhibiting such resistance, resistance to nalidixic acid with concomitant decreased susceptibility to ciprofloxacin was observed in 14% of isolates. For S. Enteritidis 13% of isolates showed such resistance, and nalidixic acid resistance was the most commonly observed resistance trait in this serotype. Many infections caused by strains of S. Enteritidis with resistance to nalidixic acid/decreased susceptibility to ciprofloxacin have belonged to phage type 1 and have been associated with travel to countries in Southern Europe and Asia, or with the consumption of poultry products imported from this area. (10, 11). For S. Virchow, 53% of isolates exhibited resistance to nalidixic acid coupled with decreased susceptibility to ciprofloxacin. This is particularly concerning because of the invasive potential of this serotype (12) and because ciprofloxacin is the first-line drug of choice in such infections. Although the level of reduced susceptibility to ciprofloxacin (MIC: 0.25-1.0 mg/l) is below that regarded as clinically significant, an increasing number of treatment failures at this level has been noted (13). In this respect it is noteworthy that a reappraisal of the breakpoints for fluoroquinolones for Salmonella spp. has recently been requested (14). Resistance to third-generation cephalosporins was rare, with only 0.4% of isolates showing resistance to cefotaxime.

Over the last decade strains of non-typhoidal S. enterica with multiple drug Salmonella spp. resistance have been distributed widely in many European countries, with a variety of food animals and food products being implicated in their spread (3, 6, 7, 8, 13). Resistance to key antimicrobials such as the fluoroquinolones and third-generation cephalosporins, although rare in isolates of non-typhoidal salmonellas from cases of human infection in Europe in 2000, does appear to be increasing in incidence (10). Treatment failures in cases of invasive illness must be regarded as a real possibility when strains are resistant to a wide range of antimicrobials, and also exhibit resistance to key drugs such as the fluoroquinolones or cephalosporins.

Since 1991 multiresistant strains of S. Newport with additional resistance to third-generation cephalosporins have been responsible for outbreaks of infection in bovine animals and humans in the USA, with concomitant treatment failures (15). Although 0.8% of European isolates of S. Newport were resistant to cefotaxime, to our knowledge the epidemic USA strains have not as yet been identified in cases of human infection in Europe. However to be able to recognise the appearance of such strains, and also to monitor the occurrence of resistance to antimicrobials in other serotypes within Europe, it is important to maintain salmonella active surveillance of resistance on an international scale.

To a large extent the countries participating in this 2000 study were confined to northern Europe. The success of this multinational approach will be improved by the participation of more countries in southern Europe and it is encouraging that antimicrobial susceptibility data are now being received from countries that did not participate in this initial survey.

This study has demonstrated the importance of a multinational approach for the surveillance of antimicrobial resistance in Salmonella spp. from cases of human infection in Europe. It has also highlighted the impact of the widespread distribution of multiresistant strains of clonal serotypes and phage types on the overall occurrence of both resistance and multiple resistance.

Acknowledgements

The authors would like to acknowledge the support and contribution to the surveillance network of all the Enter-net participants. Enter-net is funded by the European Commission, Directorate General Health and Consumer Protection (contract no 2000CVG4-037).

References

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