Abstract

Antimicrobial resistance trends were examined for 811 salmonella isolates from humans collected in Finland during 1995–1997. The material was divided into domestic and foreign isolates according to the origin of infection. A total of 2.3% of the 387 domestic and 7.8% of the 424 foreign isolates were quinolone-resistant (P < 0.001). Among the domestic isolates we detected an emergence of ciprofloxacin resistance (MIC ≥ 0.25 mg/L) with the proportion of resistant isolates increasing from 0 to 2.2% (P = 0.2). Among the foreign isolates this increase was even more dramatic, from 2.0% to 8.4% (P = 0.037) during the study period.

Introduction

Fluoroquinolones are often used in the treatment of salmonella infections. Since salmonellae can cause severe human illness, the development of fluoroquinolone resistance may have serious public health implications. Only a few previous papers have focused on the emergence of fluoroquinolone resistance in non-typhoidal salmonellae.1,2,3,4,5 In England and Wales, ciprofloxacin resistance (MIC ≥ 0.25 mg/L) in salmonella isolates increased from 0.3% to 2.1% between 1991 and 1994.4The highest increase in resistance, from 2.0% to 39.6%, was observed among the Salmonella hadar serotype. In recent years, ciprofloxacin resistance has emerged in the multiresistant clone of Salmonella typhimurium definitive type 104.5

In Finland, about 2500–3500 cases of salmonellosis are reported in humans each year. More than two-thirds of these cases are connected with travel to foreign countries or with imported foodstuffs. To evaluate the incidence and changes of antimicrobial resistance in salmonella serotypes in Finland, we collected salmonella isolates during 1995–1997, with the aim of including epidemiologically unrelated strains, and determined the susceptibilities of these isolates to quinolones and 15 additional antimicrobial agents.

Materials and methods

During 1995–1997 we collected a total of 811 salmonella isolates, which were considered to be epidemiologically unrelated. The possible relationship between different isolates was judged on the basis of epidemiological information routinely collected in each case. The isolates were divided into two groups according to the origin of the infection. An isolate was designated as of foreign origin if the patient had a confirmed connection abroad, e.g. recent travel. All other isolates were designated as of domestic origin. The salmonella isolates were collected in three different phases: (i) starting in January 1995, we consecutively collected 100 domestic and 100 foreign isolates; (ii) starting in September 1996, 200 domestic and 200 foreign isolates; and (iii) starting in January 1997, 100 domestic and 100 foreign isolates. Serotyping of the isolates was performed in the Salmonella Reference Centre of the National Public Health Institute, Helsinki, Finland.

MICs were determined by the standard agar plate dilution method according to the National Committee for Clinical Laboratory Standards (NCCLS) guidelines.6 Mueller–Hinton II agar (BBL, Becton Dickinson, Cockeysville, MD, USA) was used as the culture medium. Enterococcus faecalis ATCC 29212,Staphylococcus aureus ATCC 29213, Escherichia coli ATCC 25922, E. coli ATCC 35218 and Pseudomonas aeruginosa ATCC 27853 were used as controls in MIC determinations.

The MIC breakpoints for resistance used were those recommended by the NCCLS,6 except for fluoroquinolones. The resistance breakpoint for ciprofloxacin was chosen as ≥0.25 mg/L on the basis of earlier publications. 4,5 For norfloxacin and ofloxacin a breakpoint of ≥0.5 mg/L was used. The term 'quinolone resistance' is used here to indicate resistance to nalidixic acid (break point ≥32 mg/L) and/or to one or more of the fluoroquinolones tested.

The susceptibility data were analysed by using the WHONET4 computer program, available from J. Stelling (WHO/EMC, 1211 Geneva 27, Switzerland). Statistical analysis was performed using the χ2 test and Fisher's exact test. P values of <0.05 were considered significant.

Results

Of the 811 salmonella isolates studied, 387 were classified as of domestic origin and 424 as of foreign origin. Seventy-two different serotypes were identified. S. typhimurium and Salmonella enteritidis accounted for 40% and 19% of the domestic isolates and for 8.0% and 39% of the foreign isolates, respectively.

During the study period, the ciprofloxacin resistance (MIC ≥ 0.25 mg/L) of the domestic isolates increased from 0 to 2.2% (P = 0.2). Concurrently, a significant increase in resistance was observed among the salmonella isolates of foreign origin, of which 2.0% were ciprofloxacin-resistant in 1995 and 8.4% in 1997 (P = 0.037). Between 1995 and 1997, the resistance to nalidixic acid (MIC ≥ 32 mg/L) increased from 0 to 4.3% among the domestic isolates and from 8.8% to 11.2% among the foreign isolates.

Nine (2.3%) of all domestic isolates studied were quinolone-resistant, compared with 33 (7.8%) of all foreign isolates (P < 0.001) (Table I). The resistance was highest among the Salmonella virchow serotype, with 23% of the 13 domestic isolates and 41% of the 22 foreign isolates being quinolone-resistant. The corresponding figures for S. hadar were 13% and 20% respectively. Among the S. typhimurium serotype, 1.9% of the 154 domestic and 24% of the 34 foreign isolates were quinolone-resistant. Among the S. enteritidis serotype, none of the 75 domestic and 4.3% of the 163 foreign isolates were resistant to quinolones. All 42 Salmonella infantis isolates were quinolone-susceptible.

S. typhimurium isolates of foreign origin were more often resistant to all antimicrobial agents tested than domestic S. typhimurium isolates (Table II). S. enteritidis isolates were only rarely resistant irrespective of the geographical origin of infection.

Resistance to amoxycillin/clavulanic acid, cephalothin, cefuroxime and gentamicin was very rare. There was no resistance to cefotaxime, imipenem, aztreonam or amikacin.

Discussion

We show in this study that between 1995 and 1997, fluoroquinolone resistance emerged in Finland among salmonella isolates of domestic origin, with the proportion of ciprofloxacin-resistant isolates increasing from 0 to 2.2% during the study period. Among the foreign salmonella isolates this increase was statistically significant: from 2.0% to 8.4% (P = 0.037). The majority of our quinolone-resistant strains were of the S. virchow , S. hadar and S. typhimurium serotypes. These non-typhoidal salmonellae characteristically cause gastroenteritis, for which antimicrobial therapy is not always indicated. However, they can also produce an invasive human infection leading to serious complications. Therefore, increasing fluoroquinolone resistance among these particular serotypes is a cause for serious concern.

In our salmonella population, fluoroquinolone resistance was found to be highest among the S. hadar andS. virchow serotypes. This is in agreement with the findings of Frost et al.4 who reported a very high (39.6%) incidence of ciprofloxacin resistance in S. hadar strains isolated in England and Wales in 1994. They found that 5.1% of the S. virchow strains studied were resistant to ciprofloxacin. The quinolone-resistant S. hadar isolates in our study were always resistant to streptomycin and tetracycline and sometimes also resistant to ampicillin. Of the other serotypes, S. typhimurium isolates were generally more often resistant than S. enteritidis isolates. Ramos et al.7 have reported similar findings. Combined with these earlier findings, 4,7 the results of the present study suggest that certain salmonella serotypes may be more likely to acquire resistance against antimicrobial agents.

The MIC breakpoints for the fluoroquinolones used here are much lower than those recommended by NCCLS guidelines.6 We decided to employ these breakpoint values since, according to several reports, low-level cipro floxacin resistance may be of clinical importance. 8,9,10 Piddock et al.8 and Vasallo et al.10 have described cases of treatment failure in patients infected with salmonella strains exhibiting reduced susceptibility to quinolones even though these isolates would have been considered susceptible according to the NCCLS breakpoint recommendations. Similar strains will not be detected in any microbiological laboratories following the current break point recommendations. It remains to be seen whether the lower MIC breakpoints will in future be adopted as general guidelines.

In conclusion, we have shown here the emergence of fluoroquinolone resistance in domestic salmonella isolates in Finland and a simultaneous significant increase in resistance among isolates classified as of foreign origin. Increasing fluoroquinolone resistance in Salmonella spp. may have serious clinical implications.

Table I.

Serotype distribution and quinolone resistance (here defined as resistance to nalidixic acid and/or to one or more fluoroquinolones) of 811 salmonella isolates collected in Finland in 1995- 1997

 Strains of domestic origin Strains of foreign origin All strains 
 % of % resistant  % of % resistant  % of % resistant 
 n total to quinolones n total to quinolones n total to quinolones 
a,dDifference is statistically significant, P< 0.001. 
bOne Salmonella stanley and oneSalmonella paratyphi B. 
cOne each of Salmonellablockley, Salmonelladerby, Salmonellahaardt and Salmonellamuenchen
S. enteritidis 75 19 163 39 4.3 238 29 2.9 
S. typhimurium 154 40 1.9a 34 24a 188 23 5.9 
S. infantis 26 16 42 
S. virchow 13 23 22 41 35 34 
S. hadar 13 25 20 33 18 
Other serotypes (n = 67) 111 29 1.8b 164 39 2.4c 275 34 2.2 
Total 387 100 2.3d 424 101 7.8d 811 99 5.2 
 Strains of domestic origin Strains of foreign origin All strains 
 % of % resistant  % of % resistant  % of % resistant 
 n total to quinolones n total to quinolones n total to quinolones 
a,dDifference is statistically significant, P< 0.001. 
bOne Salmonella stanley and oneSalmonella paratyphi B. 
cOne each of Salmonellablockley, Salmonelladerby, Salmonellahaardt and Salmonellamuenchen
S. enteritidis 75 19 163 39 4.3 238 29 2.9 
S. typhimurium 154 40 1.9a 34 24a 188 23 5.9 
S. infantis 26 16 42 
S. virchow 13 23 22 41 35 34 
S. hadar 13 25 20 33 18 
Other serotypes (n = 67) 111 29 1.8b 164 39 2.4c 275 34 2.2 
Total 387 100 2.3d 424 101 7.8d 811 99 5.2 

Table II.

Resistance of 188 S. typhimuriumand 238 S. enteritidis isolates collected in Finland during 1995-1997 to quinolones and five other antimicrobials

 MIC (mg/L)   
 range MIC50a MIC90b % Resistantc 
aThe MIC at which 50% of the isolates are inhibited. 
bThe MIC at which 90% of the isolates are inhibited. 
cAccording to MIC breakpoints: see text. 
Domestic S.typhimurium (n = 154)  
  ciprofloxacin 0.016-0.25 0.016 0.032 0.6 
  ofloxacin 0.064-0.5 0.125 0.125 1.9 
  norfloxacin 0.031-1 0.064 0.125 1.9 
  nalidixic acid 4->128 1.3 
  ampicillin 1->128 128 26 
  tetracycline 1->64 64 33 
  chloramphenicol 4->64 64 23 
  trimethoprim/ <0.064-64 0.125 0.5 6.5 
  sulphamethoxazole  
  streptomycin 2->128 128 31 
Foreign S.typhimurium (n = 34)  
  ciprofloxacin 0.008-0.25 0.032 0.25 15 
  ofloxacin 0.064-1 0.125 0.5 24 
  norfloxacin 0.031-1 0.064 24 
  nalidixic acid 4->128 >128 24 
  ampicillin 2->128 128 32 
  tetracycline 1->64 64 64 74 
  chloramphenicol 4->64 64 32 
  trimethoprim/ 0.064->64 0.25 64 15 
  sulphamethoxazole  
  streptomycin 8->128 16 128 32 
Domestic S.enteritidis (n = 75)  
  ciprofloxacin 0.008-0.064 0.016 0.032 
  ofloxacin 0.064-0.25 0.125 0.125 
  norfloxacin 0.031-0.125 0.064 0.125 
  nalidixic acid 4-16 
  ampicillin 0.5->128 4.0 
  tetracycline 1-4 
  chloramphenicol 4-16 
  trimethoprim/ <0.064-0.5 0.125 0.125 
  sulphamethoxazole  
  streptomycin 2-128 1.3 
Foreign S.enteritidis (n= 163)  
  ciprofloxacin 0.008-0.25 0.016 0.032 0.6 
  ofloxacin 0.016-1 0.125 0.125 3.7 
  norfloxacin 0.016-0.5 0.064 0.125 1.8 
  nalidixic acid 1->128 4.3 
  ampicillin 0.25->128 3.7 
  tetracycline 0.5->64 1.8 
  chloramphenicol 1-16 
  trimethoprim/ <0.064-4 0.125 0.125 0.6 
  sulphamethoxazole  
  streptomycin 1-64 0.6 
 MIC (mg/L)   
 range MIC50a MIC90b % Resistantc 
aThe MIC at which 50% of the isolates are inhibited. 
bThe MIC at which 90% of the isolates are inhibited. 
cAccording to MIC breakpoints: see text. 
Domestic S.typhimurium (n = 154)  
  ciprofloxacin 0.016-0.25 0.016 0.032 0.6 
  ofloxacin 0.064-0.5 0.125 0.125 1.9 
  norfloxacin 0.031-1 0.064 0.125 1.9 
  nalidixic acid 4->128 1.3 
  ampicillin 1->128 128 26 
  tetracycline 1->64 64 33 
  chloramphenicol 4->64 64 23 
  trimethoprim/ <0.064-64 0.125 0.5 6.5 
  sulphamethoxazole  
  streptomycin 2->128 128 31 
Foreign S.typhimurium (n = 34)  
  ciprofloxacin 0.008-0.25 0.032 0.25 15 
  ofloxacin 0.064-1 0.125 0.5 24 
  norfloxacin 0.031-1 0.064 24 
  nalidixic acid 4->128 >128 24 
  ampicillin 2->128 128 32 
  tetracycline 1->64 64 64 74 
  chloramphenicol 4->64 64 32 
  trimethoprim/ 0.064->64 0.25 64 15 
  sulphamethoxazole  
  streptomycin 8->128 16 128 32 
Domestic S.enteritidis (n = 75)  
  ciprofloxacin 0.008-0.064 0.016 0.032 
  ofloxacin 0.064-0.25 0.125 0.125 
  norfloxacin 0.031-0.125 0.064 0.125 
  nalidixic acid 4-16 
  ampicillin 0.5->128 4.0 
  tetracycline 1-4 
  chloramphenicol 4-16 
  trimethoprim/ <0.064-0.5 0.125 0.125 
  sulphamethoxazole  
  streptomycin 2-128 1.3 
Foreign S.enteritidis (n= 163)  
  ciprofloxacin 0.008-0.25 0.016 0.032 0.6 
  ofloxacin 0.016-1 0.125 0.125 3.7 
  norfloxacin 0.016-0.5 0.064 0.125 1.8 
  nalidixic acid 1->128 4.3 
  ampicillin 0.25->128 3.7 
  tetracycline 0.5->64 1.8 
  chloramphenicol 1-16 
  trimethoprim/ <0.064-4 0.125 0.125 0.6 
  sulphamethoxazole  
  streptomycin 1-64 0.6 
*
Correspondence address. Antimicrobial Research Laboratory, National Public Health Institute, PO Box 57, 20521 Turku, Finland. Tel +358-2-2519255; Fax: +358-2-2519254 E-mail: antti.hakanen@utu.fi

The Sigrid Juselius Foundation (to A. H. and P. H.), the Maud Kuistila Memorial Foundation (to A. H.) and the Scandinavian Society for Antimicrobial Chemotherapy (to P. H.) supported this work. We thank Liisa Immonen, Katrina Lager, Minna Lamppu, Tarja Laustola, Anna-Liisa Lumiaho, Anne Nurmi, and Ritva Taipalinen for technical help.

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