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JAC Advance Access originally published online on May 30, 2006
Journal of Antimicrobial Chemotherapy 2006 58(2):310-314; doi:10.1093/jac/dkl223
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© The Author 2006. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Antimicrobial resistance in non-typhoidal Salmonella from human sources, Spain, 2001–2003

Pilar Soler1,*, Rubén González-Sanz2, María J. Bleda1, Gloria Hernández1, Aurora Echeíta2 and Miguel A. Usera3

1 Centro Nacional de Epidemiología, Instituto de Salud Carlos III Sinesio Delgado, 6, Madrid 28029, Spain 2 Centro Nacional de Microbiología, Instituto de Salud Carlos III, Carretera de Majadahonda a Pozuelo, s/n 28220 Majadahonda, Madrid, Spain 3 Agencia Española del Medicamento, Ministerio de Sanidad y Consumo, Carretera de Majadahonda a Pozuelo, s/n 28220 Majadahonda, Madrid, Spain

*Corresponding author. Tel: +34-91-8222645; Fax: +34-91-3877815; E-mail: psoler{at}isciii.es

Received 5 December 2005; returned 14 February 2006; revised 28 March 2006; accepted 8 May 2006


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Objectives: To determine the current state of antimicrobial resistance among non-typhoidal Salmonella strains isolated from humans in Spain.

Methods: All strains of Salmonella from human sources received in the reference laboratory from 2001 to 2003 were serotyped and phage types were determined in the most common serovars. A systematic sampling procedure was carried out in order to obtain a random sample for susceptibility testing. The selected strains were tested for susceptibility to 12 different antimicrobial agents by a disc diffusion method using Mueller–Hinton agar. Results were scored as susceptible, moderately susceptible or resistant, according to CLSI criteria.

Results: From 2001 to 2003, 5777 strains of Salmonella were tested for susceptibility. Fifty per cent of strains of Salmonella Enteritidis were resistant to nalidixic acid. This was the most frequent resistance pattern of this serovar and it was characteristic of PT1, the most frequent phage type of Salmonella Enteritidis in Spain. Seventy-four per cent of Salmonella Typhimurium strains were resistant to four antibiotics or more. Resistance to ampicillin, chloramphenicol, streptomycin, sulphonamide and tetracycline was the most frequent resistance pattern of Salmonella Typhimurium and it was characteristic of DT104, the most frequent phage type in Spain. Sixty-nine per cent of Salmonella Hadar strains were resistant to at least four antibiotics.

Conclusions: The results of our study showed both a worrying percentage of strains of Salmonella Enteritidis resistant to nalidixic acid and of strains of Salmonella Typhimurium with a pattern of resistance to four antibiotics or more. Surveillance of antimicrobial resistance should carry on and improve in order to be able to evaluate the control measures carried out for decreasing resistance in Salmonella, specifically that addressed to the prudent use of antimicrobial agents by farmers and veterinarians.

Keywords: salmonellosis , zoonoses , drug-resistant


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Salmonella is the most commonly notified cause of bacterial diarrhoea in Spain, with an average of 7621 cases reported from 2000 to 2004 (data from Spanish Microbiological Information System). Non-typhoidal salmonellosis is usually a self-limiting disease; however, it can give rise to severe disease, especially septicaemia. The annual average number of hospital admissions due to salmonellosis in Spain from 1999 to 2002 was 6143 and in the majority of these cases effective antimicrobial treatment may have been essential for recovery.

The surveillance of salmonellosis in Spain is carried out by three systems. Two of them are based on the microbiological diagnoses made by the laboratories. The Microbiological Information System is a basic system and its source is the laboratories of clinical microbiology (mainly hospital laboratories). The second, the Spanish National Reference Laboratory of Salmonella and Shigella (LNRSSE) is complementary and analyses the strains sent voluntarily from the clinical microbiology laboratories. The third surveillance system is the Outbreaks Reporting System, which obtains data on outbreaks of salmonellosis. Surveillance of antimicrobial resistance is only carried out by the LNRSSE, which uses the same methods as the European countries that participate in the International Surveillance Network for the Enteric Infections Salmonella and VTEC O157 (Enter-net). This network monitors, amongst other things, the antimicrobial resistances of the human isolates of Salmonella.1 Spain has participated in Enter-net since its beginning in 1994.

We conducted a retrospective study of antimicrobial resistance of non-typhoidal Salmonella strains isolated from human sources from 2001 to 2003 in order to assess the current state of this problem in Spain.


   Materials and methods
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All the 21526 strains of Salmonella from human sources received in the LNRSSE from 2001 to 2003 were serotyped by standard methods.2 Phage types were determined in the most common serovars (Salmonella enterica serovar Enteritidis, Salmonella enterica serovar Typhimurium and Salmonella enterica serovar Hadar).3–5 A systematic sampling procedure was carried out in order to obtain a random study sample. This involved the selection of one of every three strains received according to order of arrival in the laboratory. Only one strain per person and per outbreak was included in the sample. The selected strains were tested for susceptibility to 12 different antimicrobial agents by a disc diffusion method using Mueller–Hinton agar. The respective quantities (µg/disc) of the active compounds were as follows: ampicillin, 10; chloramphenicol, 30; sulphonamide, 300; streptomycin, 10; tetracycline, 30; gentamicin, 10; kanamycin, 30; nalidixic acid, 30; ciprofloxacin, 5; cefotaxime, 30; cefalotin, 30; trimethoprim/sulfamethoxazole, 25. Results were scored as susceptible, moderately susceptible or resistant, according to CLSI criteria.6 Escherichia coli ATCC 25922 was used as a reference strain.

All statistical analyses were performed using the STATA computer software program (version 8.2).


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From 2001 to 2003, 5777 strains were tested for susceptibility: 60.4% were Salmonella Enteritidis, 21% Salmonella Typhimurium, 2.5% Salmonella Hadar and the remaining 16.1% were 111 different serovars. The proportion of serovars tested was similar to the proportion of serovars received in the laboratory.

The strains tested came from all but one of the 19 regions of Spain; 51.3% of them were from males and 60.6% from people younger than 14 years (31.5% younger than 4 years). The sample could not be representative of the Spanish population as a whole since the strains are sent to the LNRSSE voluntarily from the clinical microbiology laboratories.

The frequency of resistance to individual drugs and multiple resistance is shown in Table 1. For Salmonella the most common resistances detected were to nalidixic acid, ampicillin, tetracycline and sulphonamide. Of the strains, <5% presented resistance to gentamicin, kanamycin and cefalotin and <1% to ciprofloxacin and cefotaxime. Five strains were moderately susceptible (zone diameter of 15–21 mm) to ciprofloxacin. The distribution of zone diameters for ciprofloxacin (Figure 1) showed two peaks of frequency. The distribution for the strains resistant to nalidixic acid presented only one peak which fitted in with the less susceptible peak of the total distribution. Of the strains, 34.3% were susceptible to all the antimicrobial agents studied and 21.6% were resistant to four antibiotics or more.


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  		Table 1. Non-typhoidal Salmonella isolates from human sources. Percentage of strains resistant to antimicrobials, Spain, 2001–2003

 

Figure 1
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  		Figure 1. Non-typhoidal Salmonella isolates from human sources. Distribution of zone diameters for ciprofloxacin, Spain, 2001–2003.

 
A total of 3491 strains of Salmonella Enteritidis were tested. Of the strains <5% showed resistance to all the antimicrobial agents studied except for nalidixic acid and ampicillin with 49.9% and 11.7% of strains resistant, respectively; 37.3% of the strains were susceptible to all the antimicrobials and resistance to four antibiotics or more was unusual (1.7% of the strains). The resistances found according to the phage types of Salmonella Enteritidis are shown in Table 2. The most common phage types were PT1, PT4, PT21, PT6A and PT6. In general, PT4 isolates were more susceptible to drugs than isolates of the other phage types. PT1 and PT21 isolates showed resistance to nalidixic acid in 89.1% and 58.9% of the strains, respectively, and most PT6 and PT6A isolates (45.9% and 74.6%, respectively) were resistant to ampicillin. The most common pattern was nalidixic acid resistance (45.2% of the strains) and 74.4% of the strains with this pattern showed the phage type PT1.


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  		Table 2. Salmonella Enteritidis (n = 3491); percentage of strains resistant to antimicrobials according to phage type, Spain, 2001–2003

 
A total of 1211 strains of Salmonella Typhimurium were tested. Salmonella Typhimurium is a more resistant serovar than Salmonella Enteritidis (13.5% of the strains were susceptible to all the antimicrobial agents) (Table 1), especially to tetracycline, sulphonamide, ampicillin, streptomycin and chloramphenicol. However, more than 90% of the strains were susceptible to the rest of the antimicrobials studied. Resistance to four antibiotics or more was very frequent (73.8% of the strains) and 47.2% were resistant to five or more drugs. The most frequent phage types were DT104 (180 strains), DTU302 (180 strains), DT104b (140 strains), DT193 (128 strains) and DT208 (33 strains). DT104 was more resistant to nalidixic acid (15.0%) and chloramphenicol (87.8%) in comparison with the other phage types (6% and 30.7%, respectively). DT208 was the most susceptible although it showed high resistance to tetracycline (97.0%) and co-trimoxazole (21.2%). The most frequent resistance pattern was ampicillin/chloramphenicol/streptomycin/sulphonamide/tetracycline (‘ACSSuT’) and the phage type that presented this pattern with most frequency was DT104 (Table 3).


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  		Table 3. Salmonella Typhimurium (n = 1077); most frequent resistance patterns detected and the predominant associated phage types, Spain, 2001–2003

 
A total of 147 strains of Salmonella Hadar were tested (Table 1). Salmonella Hadar is a more resistant serovar than Enteritidis and Typhimurium, especially to ampicillin (70.1% of the strains), streptomycin (89.1%), cefalotin (57.1%), nalidixic acid (91.2%) and tetracycline (91.8%); 95.3% of the strains showed resistance to at least one antimicrobial agent and 68.7% to four antibiotics or more. The most frequent resistance pattern was streptomycin/tetracycline/nalidixic acid/cefalotin (49.0% of the strains) and the second most frequent resistance pattern was streptomycin/tetracycline/nalidixic acid (21.5%).


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Salmonella enterica is one of the most important zoonotic pathogens in which resistance to multiple antimicrobial agents is becoming more and more frequent.7 Also, a number of studies have shown increased morbidity and mortality for infections with resistant strains.8,9 In our study resistance to four antibiotics or more was found in 21.6% of the total of the studied strains. Ciprofloxacin and cefotaxime are the antimicrobial agents recommended for the treatment of invasive infections due to Salmonella and hence the current interest in the emergence of resistance to these antimicrobial agents.10,11 In the present study these resistances were encountered in very low percentages, 0.12% for cefotaxime and 0.01% for ciprofloxacin. However, resistance to nalidixic acid was frequent, with 35.0% of the strains presenting it. Although we do not know the MICs of ciprofloxacin, our results suggest, as well as other authors,12,13 that the breakpoint for ciprofloxacin is high and this could be distorting the real situation. In comparison with the results published by Enter-net corresponding to data of 10 European countries in 2000,14 our results showed a low percentage of strains resistant to sulphonamide (22% in Spain versus 30% in Europe) and a higher percentage of resistance to nalidixic acid (36% in Spain versus 14% in Europe). The participants in the European study were mainly northern European countries and perhaps the results would have been different if more southern European countries had participated.

Salmonella Enteritidis is a comparatively susceptible serovar showing a percentage of resistance to at least four antibiotics lower than 2% and resistances lower than 5% for all the antimicrobial agents studied except for ampicillin (11.7%) and nalidixic acid (49.9%). Both results were significantly higher than those found in the European study, which showed percentages of resistance of 6% and 13%, respectively.14 Resistance to nalidixic acid (nalidixic acid resistance is the most frequent resistance pattern of Salmonella Enteritidis) is characteristic of the PT1, which is the most frequent phage type of Salmonella Enteritidis in Spain, both in human and food and animal samples.15–18 Resistance to nalidixic acid in human samples corresponded with the high percentage found in food and animal samples19–22 and it may be associated with the past use in Spain of quinolones, such as enrofloxacin, in cattle, pigs and poultry and difloxacin in poultry.

Salmonella Typhimurium is the serovar that showed the highest percentage of strains with resistance to four antibiotics or more, the most frequent resistance pattern being ACSSuT. The phage type which appeared most frequently with this resistance pattern was DT104. Salmonella Typhimurium DT104 R-type ACSSuT has been frequent in industrialized countries since the mid-1980s when it spread internationally.23 When our results are compared with other European countries14 significant differences are found for ampicillin, chloramphenicol, streptomycin and tetracycline, with the Spanish percentages being higher than the corresponding European figures. Also more strains showed resistance to at least four antibiotics in Spain than in the European study (74% versus 51%).

During the 3 years of this study temporal variation was not found in the percentages of resistance for serovar or antimicrobial agent. This stability could be due to the short period of time of analysis (3 years).

The results of our study showed both a worrying percentage of strains of Salmonella Enteritidis resistant to nalidixic acid and of strains of Salmonella Typhimurium with a resistance pattern ACSSuT. Outbreak investigations in Spain have demonstrated that foods, particularly foods of animal origin, are the most important source of Salmonella infections in humans and so the majority of antimicrobial-resistant Salmonella infections may be acquired from ingestion of foods contaminated with antimicrobial-resistant Salmonella.24 Also, the results of surveillance in Spain of resistance in Salmonella in animals,22 although limited as yet, show a high percentage of strains of Salmonella Enteritidis resistant to nalidixic acid. Surveillance of Salmonella strains for resistance to antimicrobial agents at the animal, food and human levels should carry on and improve in order to identify a potential emergence of resistance patterns and in order to be able to evaluate the control measures carried out for decreasing resistance in Salmonella, specifically that addressed to the prudent use of antimicrobial agents by farmers and veterinarians.


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None to declare.


   Acknowledgements
 
We thank both the Spanish laboratories that sent strains for serotyping and the LNRSSE staff for their invaluable contribution.


   References
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1 Soler P, Cano R, Hernández G, et al. (1998) Enter-net: red europea de vigilancia de enfermedades entéricas, Salmonella y Escherichia coli O157. Bol Epidemiol Sem 6:241–3.

2 Ewing WH. (1986) Edwards and Ewing's Identification of Enterobacteriaceae 4th edn (Elsevier, New York).

3 Ward LR, de Sa JD, Rowe B. (1987) A phage-typing scheme for Salmonella enteritidis. Epidemiol Infect 99:291–4.[Medline]

4 Anderson ES, Ward LR, Saxe MJ, et al. (1977) Bacteriophage-typing designations of Salmonella typhimurium. J Hyg (Lond) 78:297–300.[Medline]

5 De Sa JD, Ward LR, Rowe B. (1980) A scheme for the phage typing of Salmonella hadar. FEMS Microbiol Lett 9:175–7.

6 National Committee for Clinical Laboratory Standards. (2000) Performance Standards for Antimicrobial Disk Susceptibility Tests for Bacteria That Grow Aerobically: Approved Standard M2-A7. NCCLS.

7 Rabsch W, Tschäpe H, Bäumler AJ. (2001) Non-typhoidal salmonellosis: emerging problems. Microbes Infect 3:237–47.[CrossRef][Web of Science][Medline]

8 Helms M, Vastrup P, Gerner-Smidt P, et al. (2002) Excess mortality associated with antimicrobial drug-resistant Salmonella Typhimurium. Emerg Infect Dis 8:490–5.[Web of Science][Medline]

9 Varma JK, Molbak K, Barrett TJ, et al. (2005) Antimicrobial-resistant nontyphoidal Salmonella is associated with excess bloodstream infections and hospitalizations. J Infect Dis 191:554–61.[CrossRef][Web of Science][Medline]

10 Threlfall EJ. (2002) Antimicrobial drug resistance in Salmonella: problems and perspectives in food- and water-borne infection. FEMS Microbiol Rev 26:141–8.[CrossRef][Web of Science][Medline]

11 Angulo FJ, Johnson KR, Tauxe TW, et al. (2000) Origins and consequences of antimicrobial-resistent nontyphoidal Salmonella: implications for the use of fluoroquinolones in food animals. Microb Drug Resist 6:77–83.[Web of Science][Medline]

12 Crump JA, Barrett TJ, Nelson JT, et al. (2003) Reevaluating fluoroquinolone breakpoints for Salmonella enterica serotype Typhi and for non-Typhi salmonellae. Clin Infect Dis 37:75–81.[CrossRef][Web of Science][Medline]

13 Aarestrup FM, Wiuff C, Molbak K, et al. (2003) Is it time to change fluoroquinolone breakpoints for Salmonella spp.? Antimicrob Agents Chemother 47:827–9.[Free Full Text]

14 Threlfall EJ, Fisher IST, Berghold C, et al. (2003) Antimicrobial drug resistance in isolates of Salmonella enterica from cases of salmonellosis in humans in Europe in 2000: results of international multi-centre surveillance. Euro Surveill 8:41–5.[Medline]

15 Usera MA, Aladueña A, Díaz R, et al. (2003) Análisis de las cepas de Salmonella spp aisladas en muestras clínicas de origen humano en España del 2001 (I). Bol Epidemiol Sem 11:133–6.

16 Usera MA, Aladueña A, Díaz R, et al. (2003) Análisis de las cepas de Salmonella spp aisladas en muestras clínicas de origen humano en España del 2001 (II). Bol Epidemiol Sem 11:145–6.

17 Valdezate S, Arroyo M, González R, et al. (2003) Análisis de las cepas de Salmonella spp aisladas de muestras de origen no humano en España en el año 2002 (I). Bol Epidemiol Sem 11:217–20.

18 Valdezate S, Arroyo M, González R, et al. (2003) Análisis de las cepas de Salmonella spp aisladas de muestras de origen no humano en España en el año 2002 (II). Bol Epidemiol Sem 11:229–32.

19 Usera MA, Aladuena A, Gonzalez R, et al. (2002) Antibiotic resistance of Salmonella spp. from animal sources in Spain in 1996 and 2000. J Food Prot 65:768–73.[Web of Science][Medline]

20 Cruchaga S, Echeíta A, Aladueña A, et al. (2001) Antimicrobial resistance in salmonellae from humans, food and animals in Spain in 1998. J Antimicrob Chemother 47:315–21.[Abstract/Free Full Text]

21 Marimón JM, Gomáriz M, Zigorraga C, et al. (2004) Increasing prevalence of quinolone resistance in human nontyphoid Salmonella enterica isolates obtained in Spain from 1981 to 2003. Antimicrob Agents Chemother 48:3789–93.[Abstract/Free Full Text]

22 Spain 2004 Report on Trends and Sources of Zoonoses. http://www.mapa.es/ganaderia/pags/sanidad_ganadera/zoonosis/informe.pdf (24 March 2006, date last accessed).

23 Threlfall EJ. (2000) Epidemic Salmonella typhimurium DT 104—a truly international epidemic clone. J Antimicrob Chemother 46:7–10.[Free Full Text]

24 Hernández-Pezzi G, Torres A, Ordóñez P, et al. (2004) Brotes de enfermedades transmitidas por alimentos. España, 1993–2002 (excluye brotes hídricos). Bol Epidemiol Sem 12:289–91.


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