Abstract

We studied 1710 Salmonella spp. isolates from human (1051), food (421) and animal (238) sources. They were tested by the disc diffusion method for susceptibility to 12 different antimicrobial agents. The incidence of resistance and multiple resistance (MR) among the salmonella strains of different origins, the relationship between their most frequent serotypes and phage types (PTs) and their antimicrobial resistance patterns were determined. In general, the incidence of resistance and MR was significantly higher in animal isolates than in human and food isolates (P < 0.05). Resistance to each individual drug among the human isolates and food isolates was very similar, with resistance to ampicillin, tetracycline, streptomycin and sulphonamides most frequently observed. MR has remained uncommon in Salmonella enteritidis. Nevertheless, 90% of PT6A of the human isolates and 100% of the food isolates were ampicillin resistant and 80 and 60%, respectively, of the PT1 isolates were nalidixic acid resistant. Salmonella typhimurium was the most multiresistant serotype in the three sample populations and ten different patterns of MR were seen. Almost 100% of the Salmonella hadar isolates, from human and food sources, were resistant. We recommend restriction of the use of antibiotics in veterinary medicine in order to reduce the selection and spread of multiresistant strains.

Introduction

The extensive use of antibiotics, not only in human and veterinary medicine, but also in livestock production for disease prevention or as growth-promoting feed additives, has led to a serious increase in, and spread of, multiple antibiotic-resistant bacteria.1 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, effective antimicrobial treatment is essential. Increasing antimicrobial resistance in salmonella can limit the therapeutic options available to physicians for clinical cases that require antimicrobial treatment.

The four most commonly isolated salmonella serotypes in the developed world are of zoonotic origin. Strains of these serotypes can develop resistance to antibiotics to which the animal reservoir is exposed and can subsequently be transmitted to humans.2 This is the case for the clone of the Salmonella typhimurium definitive phage type (DT)104, characterized by chromosomal resistance to ampicillin, tetracycline, streptomycin, chloramphenicol and sulphonamides. This clone is now being reported from many countries in increasing numbers from humans, a wide range of animals (but particularly cattle) and food of animal origin. Since 1994, the incidence of strains of this clone with additional resistance to trimethoprim and fluoroquinolones has increased.3,4 Infection with S. typhimurium DT104 resistant to ampicillin, chloramphenicol, streptomycin, sulphonamide and tetracycline, is associated with more severe illness, higher rates of admission to hospital and increased mortality.5

The purpose of this study was to determine the incidence of antimicrobial resistance and multiple resistance (MR) in salmonella strains from human, food and veterinary sources, which were identified at the Spanish Laboratorio Nacional de Referencia de Salmonella y Shigella (LNRSSE) and at the Spanish Laboratorio de Sanidad y Produccion Animal del Ministerio de Agricultura, Pesca y Alimentación (LSPAE) during 1998. We also studied the frequency of resistance to individual drugs and patterns of MR in order to obtain a general overview of the relationship between antimicrobial resistance profile, serotype and phage type (PT) and origin of the strains.

Materials and methods

Bacterial isolates

The LNRSSE receives isolates from several Spanish clinics and health laboratories for confirmation and further identification.6 The LSPAE receives salmonella isolates from several Spanish veterinary laboratories.

In this study, 1710 salmonella strains isolated in 1998 from human, food and animal sources were examined. A total of 1051 strains were selected from humans (92% from faeces, 2% from blood and 6% from other or unknown sources). The human strains were drawn by means of a ‘stratified by serotype' sampling method (main serotypes). According to the prevalence of each serotype in 1997, one-sixth of all isolates of serotype Enteritidis, one-fifth of all isolates of serotype Typhimurium, one-third of all isolates of serotype Hadar and one-quarter of all isolates of other serotypes were selected. We also included in this study all food and animal strains received at the LNRSSE and at the LSPAE during the same period, i.e. 421 from food and 238 from sick animals (Table I).

The isolates were serotyped by standard methods.7 The three most common serotypes (S. enteritidis, S. typhimurium and S. hadar) were further subdivided according to internationally recognized phage typing schemes810 (Table II).

Antimicrobial susceptibility testing

All salmonella strains were tested for susceptibility to 12 different antimicrobial agents by a disc diffusion method using Mueller–Hinton agar. All antibiotic discs used were supplied by Oxoid (Basingstoke, UK). The respective quantities (μg/disc) of the active compounds were: ampicillin (A), 10; chloramphenicol (C), 30; sulphonamide (Su), 300; streptomycin (S), 10; tetracycline (T), 30; gentamicin (G), 10; kanamycin (K), 30; nalidixic acid (Nx), 30; ciprofloxacin (Cip), 5; cefotaxime (Ctx), 30; cephalothin (Kf), 30; trimethoprim–sulphamethoxazole (SxT), 25.

Results were scored as susceptible, moderately susceptible or resistant, according to NCCLS criteria.11Escherichia coli ATCC 25922 was used as a reference strain.

Data analysis

Single variable analysis was performed using Epi Info version 6.12 The χ2 test was used to estimate the risk of associated resistance. Associations were considered significant if P values were found to be <0.05.

Results

Incidence of resistance

The incidence of resistance (defined here as resistance to one or more drugs) and multiple resistance (MR; defined here as resistance to four or more drugs) is presented in Table III. Antimicrobial resistance among the salmonella strains isolated from humans was very similar to that observed in food isolates. However, resistance among strains isolated from animals was significantly higher (P < 0.05) than in the former groups. The frequency of resistance to individual drugs is shown in Table IV.

The frequency of resistance to individual antibiotics among isolates from humans was very similar to that among food isolates. The only exceptions were chloramphenicol resistance, which was more frequent among human isolates and cephalothin resistance, which was found twice as frequently in food isolates as in human isolates. Nevertheless, the level of resistance to individual drugs among salmonella strains isolated from animal samples, was significantly higher (P < 0.05) for all antibiotics than that among salmonella strains isolated from humans or food, except for cefotaxime.

S. enteritidis

This serotype was the most frequent among human and food isolates, with 385 human and 125 food isolates. Only 13 animal isolates were assigned to this serotype. Antimicrobial resistance was observed in 60 and 41% of the isolates from humans and food, respectively. MR was extremely rare: it was only identified in two human isolates and in one food isolate. The percentage of resistance to each individual drug is presented in Table IV.

As expected, PTs 4, 6, 6A and 1 were the predominant PTs, PT4 being the most frequent13 (Table II). In general, PT4 isolates were more sensitive to drugs than the isolates of PTs 6, 6A and 1. In fact, 42 and 44% of the isolates of this PT from humans and food, respectively, were sensitive to all agents tested. PT6A and PT6 were primarily resistant to ampicillin. Ninety per cent of the PT6A isolates from humans, 100% of the PT6A isolates from food, 74% of the PT6 isolates from humans and 75% of the PT6 isolates from food were resistant to ampicillin. Most PT1 isolates (80% of the isolates from humans and 60% of the isolates from food) were, however, resistant to nalidixic acid.

S. typhimurium

A total of 284 strains isolated from humans, 67 from food and 127 of animal origin were studied. S. typhimurium was the most commonly found serotype among the animal isolates (53%). Antimicrobial resistance was observed in 95, 92 and 98% of the isolates from human, food and animal, respectively, and MR was observed in 86% of the human isolates, 66% of the food isolates and 92% of the animal isolates. The level of resistance to all individual drugs, except cefotaxime, was significantly higher in isolates from animals than in human and food isolates (Table IV).

Table V presents the most frequent antimicrobial MR patterns and the PTs that were most commonly associated with each MR pattern of S. typhimurium isolates from the three different sources. Resistance to ACSSuT was the most common MR pattern found among the multiresistant S. typhimurium isolates from human (65%) and food (70%) sources. In animal isolates, however, only 13% of multiresistant isolates presented this MR pattern, with ACGKNxSSuSxTT (49%) and ACGKKfNxSSuSxTT (19%) being the most important. According to our laboratory data,14S. typhimurium DT104 is the predominant PT among human isolates, and 86% of 64 isolates presenting this PT showed the ACSSuT pattern. This MR pattern was also present in other, less common PTs such as 104b (47%), 120 (59%) and DT193 (6%).

Among the food isolates, S. typhimurium DT104b was the most common PT. Twenty-one of 28 isolates (75%) presented the ACSSuT MR type; 81% of the latter were isolated from milk.

DT204c was the PT most frequently found among the animal isolates; all but two of 85 DT204c isolates were resistant to seven or more antibiotics. ACGKNxSSuSxtT was the most frequent MR pattern (61%) followed by ACGKKfNxSSuSxtT (25%) and seven of these isolates were also resistant to ciprofloxacin. Pigs were the main reservoir for this PT (61% of the strains isolated from pigs presented the ACSSuTGKSxtNx MR type and 26% the ACGKKfNxSSuSxtT MR type).

S. hadar

One hundred and one S. hadar strains were isolated from humans, 67 from food and only 10 from animal samples. Ninety-eight per cent of the human isolates and 99% of the food isolates were resistant and 69% and 73%, respectively, were multiresistant. The incidence of resistance to individual drugs is shown in Table IV.

The predominant PTs among the human isolates were PT1 and 2, whereas PT1 and 17 were the most common among the food isolates (Table II).

AKfNxST was the most frequent MR pattern among the multiresistant S. hadar isolates from humans (77%) and from food (67%). The AKfNxST pattern was not associated with any specific PT.

The majority of the S. hadar strains from food were isolated from chicken (81%). Half of them expressed the AKfNxST type of MR.

Discussion

An increase in the incidence of antibiotic resistance in salmonella isolated from humans and animals related to exhaustive application of antibiotics in both groups has been documented worldwide.15 This may indicate an epidemic spread of multiresistant clones of particular serotypes of salmonella, which might have a greater potential for infection and for development of additional resistance to new antibiotics. This is the case for S. typhimurium. The continuing epidemic spread of a multiresistant strain of S. typhimurium, DT104 MR type ACSSuT in humans and food animals, has been the main factor contributing to the increasing frequency of resistance in this serotype.35

In this study, the incidence of resistance and MR in isolates of salmonella from animals was higher than that in isolates from humans and food. Further typing studies highlighted that this difference was due to the appearance and spread of a multiresistant S. typhimurium DT204c strain in animal farms. This PT was identified in 67% of S. typhimurium isolates from animal, 72% of which were isolated from pigs. This PT was also isolated from poultry (8%) and cattle (17%). If animals are the source of the food and human infections, one would expect to find about the same frequencies of resistance from different sources. One possible explanation for this situation is that this strain has been introduced into animal farms recently, and, because sick animals are discarded and not used for human consumption, it is still too early to have spread to humans. In any event, the incidence of this PT in humans is higher in 1998 than in previous years.13

All S. typhimurium DT204c strains were multiresistant and 86% of them were identified as MR type ACSSuTGKSxtNx (25% were also resistant to cephalothin).

Threlfall et al.16 observed that throughout the 1980s, S. typhimurium DT204c was an important agent of salmonellosis, in both cattle and humans, in the UK. All strains of S. typhimurium DT204c were multiresistant; the ACSSuTGKTm resistance profile was the most frequently observed. These authors demonstrated the existence of three gentamicin–apramycin resistance plasmids in this multiresistant strain, and they suggested that the use of the aminoglycoside antibiotic apramycin in animal husbandry could be responsible for the appearance of gentamicin resistance, as plasmids that code for resistance to apramycin also confer resistance to gentamicin. In the present study, S. typhimurium DT204c had the same multiple drug resistance profile as that observed by Threlfall et al. and all isolates were also resistant to nalidixic acid. Malorny et al.17 also detected in the early 1990s a high incidence of nalidixic acid resistance among multiresistant isolates of S. typhimurium DT204c, originating mainly from cattle.

In our study, this clone was particularly frequent among isolates from pigs that originated from different regions of Spain. Therefore, extensive use of antibiotics in breeding farms, where these animals are reared before their distribution to different rearing farms, or general use of these antibiotics in rearing farms, could have selected this multiresistant clone. Among human and food strains this PT has remained uncommon, although its incidence in humans increased in 1998.13 Curiously, an atypical salmonella serotype 4,5,12:i:– strain, first detected in August 1997 at the NSRLS from human and food isolates, is most frequently associated with an ACGSSuSxtT MR profile, including gentamicin resistance. Its most common sources are pork and pork products. This strain could be a monophasic strain of serotype Typhimurium or serotype Lagos, or it could represent a new serotype.18 In the present study, 4% of the strains isolated from humans and 2% of the food isolates belonged to this serotype, and all of them had the same MR pattern.

S. typhimurium was also the serotype with the highest occurrence of multiresistant strains among the human strains, and S. typhimurium MR type ACSSuT was the most frequently isolated strain. This MR pattern was observed in 86% of DT104 isolates, although it was also frequently detected in other common PTs such as DT104b (47%) and DT120 (59%). Among the food isolates, DT104b was the most frequent PT; 75% of this PT also presented the ACSSuT MR type. The genes coding for resistance to ACSSuT in S. typhimurium DT104 are chromosomally integrated in a multiresistance gene fragment, grouped within two district integrons and intervening plasmid-derived sequences.19 This multiresistance gene fragment could have been transferred horizontally to other PTs such as DT104b or DT120.

Ampicillin, nalidixic acid and sulphonamides are the drugs with the highest observed frequencies of resistance in human and food isolates of S. enteritidis. In general, MR has, however, remained uncommon. For both populations, PT4, which was the most common PT in our study, was generally drug sensitive and PT6 and PT6A were particularly associated with resistance to ampicillin. Previous studies have demonstrated that ampicillin-resistant strains of S. enteritidis PT6A were derived from strains of S. enteritidis PT4 by acquisition of an Inc X ampicillin resistance plasmid.20 In our study, resistance to nalidixic acid was most frequently observed in PT1, a finding that is in accordance with a recent report from Threlfall et al.21 These authors demonstrated that PT1 is often associated with travel to southern European countries. Among the food isolates, poultry represents the main reservoir for this serotype according to data from Spain.6 Antimicrobial resistance in S. hadar has increased rapidly during recent years.20 In this study, 98% of the S. hadar isolates from human and 99% of the food isolates were resistant to one or more antibiotics, and 69 and 73%, respectively, were multiresistant. A high proportion of S. hadar isolates were resistant to nalidixic acid, streptomycin and tetracycline. The MR pattern AKfNxST was observed in 77% of the multiply resistant S. hadar strains from human isolates and in 67% of the multiresistant food isolates.

In Spain, S. hadar is a poultry-related serotype.6 Eighty-one per cent of the strains in this study were isolated from poultry. It is probable, therefore, that the high incidence of resistance in this serotype has resulted from an extensive use of antibiotics, such as enrofloxacin in poultry, in recent years. There have already been reports concerning the rapid appearance of resistance in Campylobacter spp. after the introduction of this agent in veterinary medicine.22

In conclusion, we report that the incidence of resistance to antimicrobial agents in salmonella isolates from animal sources is significant and higher than that observed in strains isolated from human and food sources. We also report that the emergence and spread of three multiresistant strains, S. typhimurium DT204c MR type ACGKNxSSuSxtT, salmonella serotype 4,5,12:i:– MR type ACGSSuSxtT and S. hadar MR type AKfNxST, are highly related to food animals such as pigs and poultry. There is a clear association between salmonella infections of farm animals, food of animal origin and human infection and there is a clear association between the increase in the consumption of antimicrobials by food animals and a similar increase in antibiotic-resistant salmonella strains. Therefore, it is important to restrict the use of antibiotics in production farms in order to reduce the selection and spread of multiresistant strains.

Table I.

Distribution of most common serotypes in the food and animal salmonella isolates included in this study

Source n Serotype 
aProducts and derivatives. 
Animal    
pig 93 S. typhimurium 88 
poultry 57 S. typhimurium 33 
  S. enteritidis 21 
sheep 45 S. abortus ovis 87 
cattle 26 S. typhimurium 81 
other 16 several  
Food    
poultry 137 S. hadar 40 
  S. enteritidis 34 
eggsa 54 S. enteritidis 72 
milk 34 S. typhimurium 59 
porka 22 S. typhimurium 34 
other 25 several  
Source n Serotype 
aProducts and derivatives. 
Animal    
pig 93 S. typhimurium 88 
poultry 57 S. typhimurium 33 
  S. enteritidis 21 
sheep 45 S. abortus ovis 87 
cattle 26 S. typhimurium 81 
other 16 several  
Food    
poultry 137 S. hadar 40 
  S. enteritidis 34 
eggsa 54 S. enteritidis 72 
milk 34 S. typhimurium 59 
porka 22 S. typhimurium 34 
other 25 several  
Table II.

Distribution of serotypes S. enteritidis, S. typhimurium and S. hadar and their most frequent PTs in the human, food and animal salmonella isolates included in this study

Serotype/PT Human Food Animal 
S. enteritidis    
PT4 120 53 
PT1 103 21 
PT6A 57 13 
PT6 31 
total 385 125 13 
S. typhimurium    
DT104 64 10 
DT104b 47 28 
DT193 17 
DT204c 85 
total 284 69 127 
S. hadar    
PT2 24 
PT1 22 22 
PT17 17 
total 101 67 10 
Others 281 160 88 
Total 1051 421 238 
Serotype/PT Human Food Animal 
S. enteritidis    
PT4 120 53 
PT1 103 21 
PT6A 57 13 
PT6 31 
total 385 125 13 
S. typhimurium    
DT104 64 10 
DT104b 47 28 
DT193 17 
DT204c 85 
total 284 69 127 
S. hadar    
PT2 24 
PT1 22 22 
PT17 17 
total 101 67 10 
Others 281 160 88 
Total 1051 421 238 
Table III.

Incidence of antimicrobial resistance and MR in salmonella isolates from humans, food and animals in 1998

  Resistant isolatesa Multiply resistant isolatesb 
Source No. of isolates tested n n 
aIsolates resistant to one or more antimicrobials. 
bIsolates resistant to four or more antimicrobials. 
Humans 1051 771 73 408 39 
Food 420 297 71 153 36 
Animals 238 228 96 137 58 
 P < 0.001 P < 0.05    
  Resistant isolatesa Multiply resistant isolatesb 
Source No. of isolates tested n n 
aIsolates resistant to one or more antimicrobials. 
bIsolates resistant to four or more antimicrobials. 
Humans 1051 771 73 408 39 
Food 420 297 71 153 36 
Animals 238 228 96 137 58 
 P < 0.001 P < 0.05    
Table IV.

Resistance to individual antimicrobials among salmonella isolates from human, food and animal sources in 1998

   Antimicrobial resistanceb (%) 
Serotypes Origin na Su Cip Nx Kf Ctx SxT 
aNumber of isolates of strains with this serotype and source. 
bA, ampicillin; C, chloramphenicol; Cip, ciprofloxacin; G, gentamicin; K, kanamycin; Nx, nalidixic acid; S, streptomycin; Su, sulphonamide; SxT, trimethoprim–sulphamethoxazole; T, tetracycline. 
S. enteritidis human 385 23 20 31 
 food 125 24 19 23 
 animal 13 31 61 15 46 15 15 15 
S. typhimurium human 284 80 73 91 84 19 92 
 food 67 70 58 87 13 83 19 84 
 animal 127 93 89 96 73 72 94 76 24 82 91 
S. hadar human 101 70 94 91 67 89 
 food 67 75 10 82 91 78 82 
 animal 10 50 30 100 60 50 100 
Total human 1051 45 26 42 42 0.6 26 0.2 14 44 
 food 420 43 16 41 42 0.2 27 16 0.7 19 43 
 animal 238 61 50 84 42 42 82 48 18 0.5 49 61 
   Antimicrobial resistanceb (%) 
Serotypes Origin na Su Cip Nx Kf Ctx SxT 
aNumber of isolates of strains with this serotype and source. 
bA, ampicillin; C, chloramphenicol; Cip, ciprofloxacin; G, gentamicin; K, kanamycin; Nx, nalidixic acid; S, streptomycin; Su, sulphonamide; SxT, trimethoprim–sulphamethoxazole; T, tetracycline. 
S. enteritidis human 385 23 20 31 
 food 125 24 19 23 
 animal 13 31 61 15 46 15 15 15 
S. typhimurium human 284 80 73 91 84 19 92 
 food 67 70 58 87 13 83 19 84 
 animal 127 93 89 96 73 72 94 76 24 82 91 
S. hadar human 101 70 94 91 67 89 
 food 67 75 10 82 91 78 82 
 animal 10 50 30 100 60 50 100 
Total human 1051 45 26 42 42 0.6 26 0.2 14 44 
 food 420 43 16 41 42 0.2 27 16 0.7 19 43 
 animal 238 61 50 84 42 42 82 48 18 0.5 49 61 
Table V.

Most frequent MR patterns detected and the predominant associated PT in S. typhimurium isolates from human, food and animal sources

 Human Food Animal 
MR patterna DT n DT n DT n 
aA, ampicillin; Cip, ciprofloxacin; C, chloramphenicol; G, gentamicin; K, kanamycin; Nx, nalidixic acid; S, streptomycin; Su, sulphonamide; SxT, trimethoprim–sulphamethoxazole; T, tetracycline. 
bNumber of isolates of strains with this MR pattern. 
cNumber of isolates within the indicated PT, with this MR pattern. 
ASSuT 20 104b 10 104b –  
ACSSuT 160 104 55 32 104b 21 15 104 
CSSuSxtT NT –  –  
ACSSuSxtT NT –  –  
ACNxSSuT NT –  –  
ACNxSSuSxtT 104b –  –  
ACGSSuSxtT 104b 104b U302 
ACGKNxSSuSxtT –  –  57 204c 52 
ACGKKfNxSSuSxtT –  –  22 204c 21 
ACCipKKfNxSSuSxtT –  –  204c 
 Human Food Animal 
MR patterna DT n DT n DT n 
aA, ampicillin; Cip, ciprofloxacin; C, chloramphenicol; G, gentamicin; K, kanamycin; Nx, nalidixic acid; S, streptomycin; Su, sulphonamide; SxT, trimethoprim–sulphamethoxazole; T, tetracycline. 
bNumber of isolates of strains with this MR pattern. 
cNumber of isolates within the indicated PT, with this MR pattern. 
ASSuT 20 104b 10 104b –  
ACSSuT 160 104 55 32 104b 21 15 104 
CSSuSxtT NT –  –  
ACSSuSxtT NT –  –  
ACNxSSuT NT –  –  
ACNxSSuSxtT 104b –  –  
ACGSSuSxtT 104b 104b U302 
ACGKNxSSuSxtT –  –  57 204c 52 
ACGKKfNxSSuSxtT –  –  22 204c 21 
ACCipKKfNxSSuSxtT –  –  204c 
*
Corresponding author. Tel: +34-91-5097901; Fax: +34-91-5097966; E-mail: mausera@isciii.es

We thank the Spanish laboratories that sent strains for serotyping. We also thank the LNRSSE and LSPAE technicians for their invaluable contribution. Part of this study was funded by the ‘Beca de ampliación de estudios del Fondo de Investigaciones Sanitarias, España' no. 97/5553.

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