Trends in antimicrobial resistance phenotypes in non-typhoid Salmonellae from human and poultry origins in France

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Trends in antimicrobial resistance phenotypes in non-typhoid

Salmonellae from human and poultry origins in France

J. CAILHOL 1, R. LAILLER 2, P. BOUVET 3, S. LA VIEILLE1, F. GAUCHARD 1,

P. SANDERS 4AND A. BRISABOIS 2*

1 French Agency for Food Safety (AFSSA), Direction de l’evaluation des risques nutritionnels et sanitaires,

Maisons-Alfort, France2 AFSSA Lerqap, Maisons-Alfort, France3 Centre National de Reference des Salmonella (CNRS), Institut Pasteur, Paris, France4 AFSSA, Fougeres, France

(Accepted 13 May 2005)

SUMMARY

A total of 1873 strains from human origin and 4283 strains from non-human origin of Salmonella

enterica serotypes Typhimurium, Enteritidis, Heidelberg, Hadar and Virchow, collected over

three years 1993, 1997 and 2000, were examined in order to determine the rate of antimicrobial

resistance to 12 antimicrobial drugs. The objective of the study was to describe and to compare

the evolution of the main resistance types in human and non-human isolates, focusing on the

poultry sector. The evolution and the rates of antimicrobial resistances for the five serotypes, with

the exception of Virchow, were almost comparable in strains isolated from human and non-

human sources over the period studied. The most striking result concerning single resistance was

the spectacular increase of the resistance frequency to nalidixic acid for the strains belonging to

serotypes Hadar and Virchow, especially in the poultry food sector (14% in 1993 vs. 72% in 2000

for Salmonella Virchow, 4% in 1993 vs. 70% in 2000 for Salmonella Hadar) and also in human

isolates (24% in 1997 vs. 48% in 2000 for S. Virchow, 31% in 1997 vs. 78% in 2000 for

S. Hadar). In addition to the classical resistance to ampicillin, streptomycin, sulphonamide,

chloramphenicol and tetracycline (ASSuCT resistance type), which stabilized between 1997 and

2000, the emergence of a new resistance type was observed.

INTRODUCTION

Salmonellosis is the most frequent foodborne disease

reported in France, often transmitted by foods of

animal origin, especially raw eggs, egg products or

poultry meats, causing more than 300 outbreaks per

year [1]. To address this public health problem,

national surveillance networks to monitor Salmo-

nellae of both human and non-human origins were

set up. These networks are used to reveal trends in

the evolution of different serotypes, monitor their

antimicrobial susceptibility, detect outbreaks, and

contribute to their investigation. Antimicrobial re-

sistance monitoring has become more important since

the appearance and dissemination of multidrug-

resistant Salmonella Typhimurium phage type

DT104, conferring resistance to ampicillin, strepto-

mycin, sulphonamide, chloramphenicol and tetra-

cycline (resistance-type ASSuCT). The genes encoding

for these resistances are clustered on the chromo-

somal genomic island SGI1 and can be transferred

* Author for correspondence : Dr A. Brisabois, AFSSA Lerqap,Maisons-Alfort, France.(Email : [email protected])

Epidemiol. Infect. (2005), &, 1–8. f 2005 Cambridge University Press

doi:10.1017/S0950268805004863 Printed in the United Kingdom

clonally and also horizontally to other serotypes [2, 3].

The increasing evolution of resistance-type ASSuCT

has been reported in several countries since the 1990s

[4–7]. Besides this resistance type, other antimicrobial

resistances, notably to quinolones, appeared initially

in developing countries [8] and then worldwide [9, 10].

Many reports suggest that the main cause of in-

creasing antimicrobial resistance among Salmonella

isolates from animals is the misuse and overuse of

antimicrobials in the breeding sector, and that some

of these strains may be transmitted through food to

humans, thereby contributing to the increase of anti-

microbial resistance among humans [11–13]. These

high antimicrobial resistance levels in zoonotic Salmo-

nella may contribute to an increase of morbidity in

human infections because resistant bacteria may be

more virulent and the treatment less effective [14, 15].

The purpose of this descriptive study was to evalu-

ate trends in antimicrobial resistance in the main

Salmonella enterica serotypes of human and poultry

sources over 8 years in France, by reviewing data

collected annually in 1993, 1997 and 2000. The

following five predominant serotypes were chosen

for the study: Typhimurium, Enteritidis, Hadar,

Heidelberg and Virchow. Observed trends are

discussed with respect to data on veterinary anti-

microbial consumption in France.

MATERIALS AND METHODS

Bacterial isolates

Surveillance of isolates from animal and food origin

has been carried out by the French Agency for Food

Safety (AFSSA) through the ‘Salmonella ’ Network.

Strains were classified in three main sectors : (i) animal

health; meaning poultry, pig, cattle or other farm

animal isolates and nearby environment isolates, rep-

resenting more than 70% of the total collected iso-

lates; (ii) food hygiene; meaning isolates from animal

food products, feedstuffs and slaughter houses, for

25% of isolates ; (iii) ecosystems; meaning water

purification plant, harbour and sewage isolates cor-

responding to <5% of isolates. However, the third

sector did not have a sufficiently large sample size and

was not used in this analysis.

Strains were collected on the basis of voluntary

participation of veterinary and food hygiene labora-

tories, located throughout the national area. All col-

lected strains were sent with information concerning

the origin, animal species, categories of food and

regional localization of the samples. The samples

were mainly collected within the framework AQ1of self-

regulating systems based on legislation requirements

or on random official sampling (eggs) and monitoring

plans (poultry farming), and within the animal

breeding environment.

The French Salmonella Reference Centre (CNRS)

at the Pasteur Institute (Paris, France) has conducted

the surveillance of human isolates by collecting

specification sheets and isolates from public and

private clinical laboratories of analysis for several

decades. The strains have been isolated from clinical

samples (blood or faeces), and sent to the CNRS on

the basis of voluntary participation. Human strains

were selected annually for susceptibility testing, in

respect of the distribution of serotypes.

Serotyping and antimicrobial susceptibility testing

Serotype was determined by the Kauffmann–White

method, using Bio-Rad AQ2(formerly Sanofi Diagnostics

Pasteur) standard antisera and home-made antisera.

This study reports the results observed for the

five serotypes : Typhimurium, Enteritidis, Hadar,

Virchow and Heidelberg, selected on the basis of their

high frequency of isolation both in human and non-

human origins (Table 1). The antimicrobial sus-

ceptibility was tested by standard disk diffusion on

Mueller–Hinton agar plates according to the guide-

lines of the Comite de l’Antibiogramme de la Societe

Francaise de Microbiologie (CASFM).

Zone diameters were automatically read by the

Osiris system (Bio-Rad) using the break-points

defined by the guidelines of CASFM, and have been

interpreted as predictive of treatment effectiveness

classes: resistant (R), intermediate (I), susceptible (S).

Strains classified as intermediate class were con-

sidered in this study as susceptible. E. coli ATCC

25922 strain was used for quality control.

Ten antimicrobial drugs and two combinations of

antimicrobials have been tested on all isolates as

follows (disk content indicated in parentheses) : nali-

dixic acid (30 mg), ampicillin (10 mg) for non-human

isolates, amoxicillin (10 mg) for human isolates,

amoxicillin-clavulanic acid (20 mg+10 mg), strepto-

mycin (10 IU) AQ3, trimethoprim–sulphamethoxazole

(1.25 mg+23.75 mg), trimethoprim (5 mg), sulphon-

amide (200 mg), tetracycline (30 IU), chloramphenicol

(30 mg), gentamycin (10 IU), cefotaxime (30 mg),

ofloxacin (5 mg). These listed antibiomicrobials cor-

respond to those recommended for the surveillance of

2 J. Cailhol and others

antimicrobial susceptibility by Caprioli et al. [16] and

by the European group (Office International des

Epizooties). Sulphonamide was not tested in animal

isolates in 1993. Concerning the ampicillin and

amoxicillin disks tested, we considered that their

cross-susceptibilities and cross-resistances were com-

plete [17]. Table 2 gives the number of tested

strains and fully susceptible strains for each studied

serotype.

Statistical analysis

Statistical analyses were performed using Epi-Info

software (version 6.04b, CDC, Atlanta, GA, USA).

We assessed the emergence of new R-types, possibly

clustered in a gene, by comparing the observed rate of

associated resistance to the predicted rate if these

resistances were occurring independently, using the

McNemar test. The x2 trends test was used for evalu-

ating evolution of resistance to nalidixic acid and

ofloxacin over the 8 years. P values <0.05 were con-

sidered as significant.

RESULTS

Frequency of isolation and global rate of resistance

(Tables 1 and 2)

S. Typhimurium was the second most common sero-

type found in human isolates and the most common

Table 1. Annual number of reported Salmonella isolates from human and non-human origins in five studied

serotypes

Human Non-human

1993 1997 2000 1993 1997 2000

n (%) n (%) n (%) n (%) n (%) n (%)

Enteritidis 6006 (35.8) 6514 (34.6) 4656 (37.2) 899 (13.3) 1508 (6.9) 1206 (6)Typhimurium 4389 (26.2) 6755 (35.8) 3800 (30.3) 1211 (17.9) 4093 (18.7) 2643 (13)Hadar 170 (1) 1152 (7) 787 (6.3) 94 (0.1) 2092 (9.5) 1408 (7)

Heidelberg 150 (0.9) 373 (2) 226 (1.8) 109 (1.6) 1000 (4.5) 1179 (5.8)Virchow 1195 (7.1) 668 (3.5) 321 (2.5) 583 (8.6) 1206 (5.5) 1091 (5.4)Other serotypes 4195 (29) 3373 (17.1) 2725 (21.9) 3863 (68.5) 11 996 (54.9) 12 722 (62.8)

Total isolates 16 105 (100) 18 840 (100) 12 515 (100) 6759 (100) 21 895 (100) 20 249 (100)Tested isolates 370 1214 1000 6759 3684 5259

Table 2. Number of strains tested for each serotype and percentage of fully susceptible strains

Poultry products Poultry Humans

1993 1997 2000 1993 1997 2000 1993 1997 2000

TyphimuriumNumber tested (n) 152 57 52 175 124 128 297 250 315Fully susceptible (%) 46 25 14 69 39 21 27 12 11

EnteritidisNumber tested (n) 312 60 38 241 93 158 70 380 82Fully susceptible (%) 87 83 74 91 88 70 96 89 73

HadarNumber tested (n) 27 89 128 14 176 176 — 200 80Fully susceptible (%) 26 2.2 5.5 7.1 2.8 3.4 — 1 0

HeidelbergNumber tested (n) 36 47 81 38 125 153 — 50 49Fully susceptible (%) 89 51 34 84 26 13 — 58 37

VirchowNumber tested (n) 270 62 36 128 117 143 — 50 50Fully susceptible (%) 65 32 8.3 66 37 8.4 — 48 46

Antimicrobial resistance in Salmonella 3

in animal isolates. The proportion of human S.

Typhimurium susceptible to all the antimicrobials

tested decreased significantly over the 8 years (27% to

11%, P<10x5).

S. Enteritidis was the most frequent serotype in

human isolates among the serotypes tested and

its rank was second or third among non-human iso-

lates. Since 1993, susceptible human, animal and

food isolates have been decreasing; nevertheless it

remained the most susceptible serotype among those

studied.

S. Hadar constituted <1% of all isolates in 1993

but increased rapidly between 1993 and 1997 both in

human and non-human isolates. There were already

few susceptible human isolates in 1997 (1%) and they

totally disappeared in 2000.

Isolation of the serotype S. Heidelberg increased

strongly among non-human strains between 1993 and

2000. The proportion of susceptible strains in human

isolates decreased between 1997 and 2000 from 58%

down to 37%. Although levels of resistance were very

low to all drugs in 1993, the proportion of susceptible

strains has steadily declined. Strains isolated from

animals had generally higher resistance rates than

those from poultry products.

S. Virchow isolation decreased between 1993 and

2000 in both human and non-human sectors. It was

the only serotype in which the proportion of suscep-

tible strains was stable in human isolates (48% of all

isolates in 1997 and 46% in 2000). The proportion of

susceptible isolates from the poultry sector steadily

decreased during the 1993–2000 period.

ASSuCT R-type and new R-types (Table 3)

The frequency of resistance type (R-type) ASSuCT

strains greatly increased among Typhimurium iso-

lates between 1993 and 2000 (P<10x3). The occur-

rence of this R-type was much higher than would be

predicted by the independent occurrence of each of

the antimicrobials separately. In 2000, 40% of human

isolates and 45% of poultry sector isolates harbour-

ing the ASSuCT R-type were also nalidixic acid-

resistant. Moreover, 43% of trimethoprim-resistant

human isolates also presented the ASSuCT R-type.

However, the ASSuCT R-type appears not to have

disseminated among the other serotypes.

Even without any spread of R-type ASSuCT, the

serotype Hadar has shown the most spectacular in-

crease in resistance over the 8 years with the lowest

rate of susceptible strains among the five serotypes.

Resistances to ampicillin or amoxicillin, streptomycin

and tetracycline, with or without the resistance to

nalidixic acid often occurred together but they were

not statistically associated in a particular R-type.

In Heidelberg, human strains showed approxi-

mately the same antimicrobial resistance as those

from poultry products, with an increase in rates

between 1997 and 2000. A novel R-type ASSuT,

emerged strongly from 4% in 1997 to 54% in 2000 for

animal isolates. This R-type was detected in 6% of

human isolates in 1997 and represented 22% in 2000,

commonly with additional resistance to trimetho-

prim. These R-type values were much higher than the

predicted values based on the occurrence of each

Table 3. Percentages of ASSuCT R-type and other association of resistances among the five serotypes

R-type

Poultry products Poultry Humans

1993 1997 2000 1993 1997 2000 1993 1997 2000

ASSuCT Typhimurium 3.9* 39 40 8.6* 28 42 41 61 59Predicted values 0.5* 2.5 4.6 0.1* 1.5 4.9 5.4 17 16ASSuCT Enteritidis 0* 0 0 1.6* 0 0.6 0 0.2 0

ASSuCT Hadar 0* 0 0 0* 0.6 0.6 n.d. 0.1 0ASSuCT Heidelberg 0.3* 0 1.2 2.6* 2.4 1.3 n.d. 6 0ASSuCT Virchow 1.1* 6.4 5.5 2.3* 0.8 1.4 n.d. 0 2AST Hadar 0 48 60 0 61 49 n.d. 68 55

Predicted values — 44 51 — 57 46 — 61 55ASTNal Hadar 0 0.5 0.6 0 0.6 0.4 n.d. 0.7 0.6Predicted values — 0.3 0.4 — 0.4 0.2 — 0.5 0.4

ASSuT Heidelberg 0* 4.3 25 0* 4 54 n.d. 6 22Predicted values — 0.2 2.6 — 1.6 22 — 0.2 2.7

* Sulphonamide was not tested.

4 J. Cailhol and others

antimicrobial separately and confirmed the co-

occurrence of these resistances were not independent.

Resistance to nalidixic acid and to ofloxacin (Table 4)

Strains of Typhimurium resistant to nalidixic acid

were already present in 1993 and increased signifi-

cantly between 1993 and 2000 for the three sectors.

For Enteritidis, resistance rates to nalidixic acid

increased significantly among poultry and human

isolates between 1993 and 2000. In most cases, a single

resistance was observed and concerned 89% of nali-

dixic acid-resistant strains for the poultry sector in

2000.

Among S. Hadar, resistance rates to nalidixic acid

rose steeply between 1993 and 2000 in the animal and

food poultry sectors. Seventy-six per cent of nalidixic

acid-resistant strains in the poultry sector isolated in

1997 and 84% of those isolated in 2000 were found

occurring together with the resistance to amoxicillin

or ampicillin, streptomycin and tetracycline. Among

human isolates, this pattern of resistance was ob-

served for 78 and 69% of nalidixic acid-resistant

strains in 1997 and 2000 respectively. Resistance to

ofloxacin also appeared in the three sectors.

Over these 8 years, resistance rates of S. Virchow to

nalidixic acid in poultry isolates greatly increased.

Resistance to ofloxacin also appeared in 2000 among

human and poultry isolates. The resistance to

nalidixic acid was found as a single resistance or

in combination with other resistances. Among the

nalidixic acid-resistant strains, occurrences of single

resistance were: 44% in 1993, 63% in 1997, 56% in

2000 in the poultry sector, and 42% in 1997, and 83%

in 2000 strains from humans.

DISCUSSION

Our study revealed an increase in antimicrobial re-

sistance rates for all Salmonella isolates from poultry,

poultry product and human origin over this 8-year

period, except for S. Virchow human isolates.

Although the R-type ASSuCT did not disseminate

from Typhimurium to other serotypes, we noted the

emergence of a new R-type ASSuT in Salmonella

serotype Heidelberg, and a worrying increase of

resistance to nalidixic acid and even to ofloxacin

among the five serotypes studied, especially among

Hadar and Virchow.

Considering that the collection of isolates was based

on voluntary participation, there was a selection bias,

amplified by the application of regulatory rules in

some animal production sectors (e.g. poultry sector).

Therefore, the results cannot be considered as com-

pletely representative and exhaustive.

Our results may have been overestimated because

some information concerning the context of sample

collecting was not available. Indeed, in cases of

Table 4. Percentages of resistance of the five serotypes to nalidixic acid

(NA) and ofloxacin

Poultry products Poultry Humans

1993 1997 2000 1993 1997 2000 1993 1997 2000

TyphimuriumNA 14 7 29 5.7 8.9 23 3 3 11

Ofloxacin 0 0 0 0 0.8 0 0 0 0.6EnteritidisNA 8 3.3 5.2 0.4 0 15 0 2.4 9.8

Ofloxacin 0 0 0 0 0 0.6 0 0 0HadarNA 3.7 69 69 0 72 51 n.d. 85 78

Ofloxacin 0 0 3.1 0 2.3 1.1 n.d. 1 2.4HeidelbergNA 0 2.1 0 0 2.4 1.9 n.d. 0 2Ofloxacin 0 0 1.2 0 0.8 0 n.d. 0 0

VirchowNA 14 27 72 13 45 82 n.d. 0 0Ofloxacin 0 0 2.7 0 0 3.5 n.d. 0 2

Significant increases among the three years detected by x2 trends test appear in bold

(P<0.05).

Antimicrobial resistance in Salmonella 5

human disease, an antimicrobial treatment could have

been administered before collecting stool samples and

may have selected resistant strains. Nevertheless, the

geographical distribution and the number of samples

were large enough to reveal trends in antimicrobial

resistance during this 8-year period.

Since Salmonella infection is a zoonotic disease,

antimicrobial resistance of strains from animals and

humans have been frequently linked to antimicrobial

use in agriculture [4, 7, 11–13, 18–24]. For this reason,

monitoring of antimicrobial resistance among Salmo-

nella from food-animals and humans on the one hand,

and of antimicrobial use in agriculture, on the other,

has been initiated recently in France [25, 26] and

worldwide (WHO, DANMAP, Euro-surveillance).

The comparison between the evolution of anti-

microbial resistance and antibiotic use in agriculture

is complicated, first because of the lack of statistics in

this sector, and second due to the presence of multiple

interaction factors. Nevertheless, our observations

can be evaluated based on the first study of veterinary

antimicrobials sold in France in 1999 [G. Moulin,

personal communication, 1999]. During that year,

tetracycline, aminoglycoside, ampicillin or amoxicillin

and sulphonamides composed 83% of the antibiotics

used as veterinary drugs in animals. In this study, the

highest rates of resistance were observed among these

antimicrobial families, as shown by the association of

resistance to ampicillin, streptomycin and teracycline

in serotype Hadar, or the emergence of the R-type

ASSuT in serotype Heidelberg. In contrast, no

resistance to chloramphenicol was observed among

serotype Heidelberg, although it acquired the R-type

ASSuT. One hypothesis for this observation is

that Heidelberg was rare before 1994, the year

chloramphenicol was banned. Therefore, Heidelberg

strains have not been exposed to a selective pressure

by this antibiotic. Comparing R-type ASSuT occur-

rences observed with the predictive values, suggests

that genes encoding this R-type may be clustered on

the same genetic element. Molecular analysis should

be conducted in order to assess the organization of

genetic elements associated with this new R-type.

Similarly, the highest levels of ASSuCT resistance

were observed among S. Typhimurium, both in

poultry and in human isolates. This could be ex-

plained by the clonal spread of Typhimurium DT104,

linked to the well-documented pattern of anti-

microbial resistance ASSuCT [2–4, 7, 22, 23]. In our

study, frequency of ASSuCT R-type strains remained

stable for the serotype Typhimurium between 1997

and 2000 but were 10-fold higher than the predictive

values given by the occurrence of each antimicrobial

drug independently. Moreover, no evidence of large

horizontal dissemination of this R-type was observed

even if some previous results showed the presence of

the genomic island structure SGI1 in other serotypes

such as Agona, Paratyphi B and Albany [27].

Furthermore, besides regulation on the use of

specific antimicrobials, policies to eradicate infected

flocks may also have played a role. For instance, since

1998 poultry flocks infested by S. Typhimurium and

S. Enteritidis have been eradicated in accordance with

the CEE zoonosis directive (no. 92/117). The fre-

quency of isolation of S. Enteritidis is stable among

human strains, and low in non-human strains, prob-

ably due to these legislative requirements.

We also observed the emergence of nalidixic acid

resistance among human and poultry isolates in

S. Typhimurium. Resistance to quinolones was

studied in 2001 by Cloeckaert and colleagues regard-

ing the emergence of Typhimurium DT104 after the

introduction of enrofloxacin in veterinary medicine in

1993 [2, 27, 28] AQ4. In 1999 in France, quinolones rep-

resented 1.5% of the total of quantities of anti-

microbial drugs sold, which is relatively low, but the

available data cannot allow us to analyse a correlation

between consumption and resistance. Oral formu-

lations of quinolones and fluoroquinolones have been

authorized for the treatment of E. coli infection in

poultry. When a prophylactic or curative treatment is

given to the whole flock or herd, via mixing with feed

or water, some resistant strains can emerge and

spread through animals not taking antibiotics at a

bactericidal concentration [19, 29].

Cases of treatment failure of ceftriaxone and

decreased susceptibility to ciprofloxacin in human

isolates have been observed [21, 30, 31]. Furthermore,

some studies demonstrate the presence of subpopu-

lations with resistant or decreased susceptibility to

fluoroquinolone among the nalidixic acid-resistant

isolates, which might influence the outcome of fluoro-

quinolone treatment [32]. In 1999, Threlfall drew our

attention to the increase of isolated strains with

decreased susceptibility to ciprofloxacin among

strains belonging to serotypes Virchow and Hadar in

England [33]. In our study, these two serotypes had

the highest rates of resistance to nalidixic acid. The

most striking increase of resistance concerned the

serotype Hadar with the frequent association of nali-

dixic acid resistance to resistance to amoxicillin,

streptomycin and tetracycline. In Finland, between

6 J. Cailhol and others

1995 and 1997, quinolone-resistant strains of S.

Hadar harboured a resistance to streptomycin, tetra-

cycline and sometimes to ampicillin or amoxicillin [9].

Our results are also in accord with Cruchaga et al.,

who observed that the ASTNal resistance pattern

with additional cephalotin resistance was the most

frequent R-type [5]. With regard to our results, it

should be noted that resistance to ofloxacin emerged

in 2000 among S. Hadar human strains.

The results of our analyses show some trends in the

evolution of antimicrobial resistances in Salmonella

strains in France and led us to detect some new

R-type emergence in the five most-studied serotypes.

The resistance monitoring system must be improved

and should be compared with the distribution of

veterinary antimicrobials. Such improved surveillance

will be necessary to analyse the mechanisms and

modes of dissemination, and public health impact of

both the emerging resistances and the pathogens

themselves.

ACKNOWLEDGEMENTS

The authors thank V. Vaillant of the Institut de Veille

Sanitaire (InVS) for expert assistance. Part of this

work has been supported by InVS. We gratefully

acknowledge Mrs Jayne Ireland and Mrs Claire

Evans (AFSSA) for greatly improving the English

grammar and style of the manuscript.

DECLARATION OF INTEREST

&.AQ5

REFERENCES

1. Haeghebaert S, Le Querrec F, Gallay A, Bouvet P,

Gomez M, Vaillant V. Les toxi-infections alimentairescollectives en France en 1999 et 2000. In: SurveillancenationaleAQ6 des maladies infectieuses, 1998–2000. Institut

de Veille Sanitaire, Saint-Maurice, France. Novembre2002: 161–169.

2. Cloeckaert A, Schwarz S. Molecular characterization,

spread and evolution of multidrug resistance inSalmonella enterica typhimuriumDT104. Vet Res 2001;32 : 301–310.

3. Threlfall EJ. Epidemic salmonella typhimurium DT104 – a truly international multiresistant clone.J Antimicrob Chemother 2000; 46 : 7–10.

4. Breuil J, Brisabois A, Casin I, Armand-Lefevre L, Fremy

S, Collatz E. Antibiotic resistance in salmonellae iso-lated from humans and animals in France: comparative

data from 1994 and 1997. J Antimicrob Chemother2000; 46 : 965–971.

5. Cruchaga S, Echeita A, Aladuena A, Garcia-Pena J,

Frias N, Usera MA. Antimicrobial resistance in salmo-nellae from humans, food and animals in Spain in 1998.

J Antimicrob Chemother 2001; 47 : 315–321.6. Leegaard TM, Caugnat DA, Froholm LO, Hoiby EA,

Lassen J. Emerging antibiotic resistance in Salmonellatyphimurium in Norway. Epidemiol Infect 2000; 125 :

473–480.7. Murphy TM, McNamara E, Hill M, et al.

Epidemiological studies of human and animal

Salmonella typhimurium DT104 and DT104b isolatesin Ireland. Epidemiol Infect 2001; 126 : 3–9.

8. Hart CA, Kariuki S. Antimicrobial resistance in

developing countries. BMJ 1998; 317 : 647–650.9. Hakanen A, Siitonen A, Kotilainen P, Huovinen P.

Increasing fluoroquinolone resistance in Salmonella

serotypes in Finland during 1995–1997. J AntimicrobChemother 1999; 43 : 145–148.

10. Threlfall EJ, Ward LR, Rowe B. Resistance to cipro-floxacin in non-typhoidal salmonellas from humans in

England and Wales – the current situation. ClinMicrobiol Infect 1999; 5 : 130–134.

11. Aarestrup FM.Association between the consumption of

antimicrobial agents in animal husbandry and theoccurrence of resistant bacteria among food animals.Int J Antimicrob Agents 1999; 12 : 279–285.

12. McEwen SA, Fedorka-Cray PJ. Antimicrobial useand resistance in animals. Clin Infect Dis 2002; 34

(Suppl 3) : S93–S106.

13. van den Bogaard AE, Stobberingh EE. Epidemiology ofresistance to antibiotics. Links between animals andhumans. Int J Antimicrob Agents 2000; 14 : 327–335.

14. Barza M, Travers K. Excess infections due to anti-

microbial resistance : the ‘Attributable Fraction’. ClinInfect Dis 2002; 34 (Suppl 3) : S126–S130.

15. Travers K, Barza M. Morbidity of infections caused by

antimicrobial-resistant bacteria. Clin Infect Dis 2002;34 (Suppl 3) : S131–S134.

16. Caprioli A, Busani L, Martel JL, Helmuth R.

Monitoring of antibiotic resistance in bacteria ofanimal origin : epidemiological and microbiologicalmethodologies. Int J Antimicrob Agents 2000; 14 :295–301.

17. Fuchs PC, Barry AL, Pfaller MA, Hardy DJ,

McLaughlin JC, Gerlach EH. In vitro activity ofampicillin, amoxicillin, ampicillin-sulbactam, and

amoxicillin-clavulanic acid against consecutive clinicalisolates of Enterobacteriaceae. Diagn Microbiol InfectDis 1993; 17 : 171–175.

18. Franklin A, Acar J, Anthony F, et al. Antimicrobialresistance : harmonisation of national antimicrobialresistance monitoring and surveillance programmes in

animals and in animal-derived food. Rev Sci Tech 2001;20 : 859–870.

19. Bager F, Helmuth R. Epidemiology of resistance toquinolones in Salmonella. Vet Res 2001; 32 : 285–290.

20. Davies RH, Teale CJ, Wray C, et al. Nalidixic acidresistance in salmonellae isolated from turkeys and

Antimicrobial resistance in Salmonella 7

other livestock in Great Britain. Vet Rec 1999; 144 :320–322.

21. Fey PD, Safranek TJ, Rupp ME, et al. Ceftriaxone-resistant salmonella infection acquired by a child fromcattle. N Engl J Med 2000; 342 : 1242–1249.

22. Poppe C, Smart N, Khakhria R, Johnson W, Spika J,

Prescott J. Salmonella typhimurium DT104: a virulentand drug-resistant pathogen. Can Vet J 1998; 39 :559–565.

23. Rabsch W, Tschape H, Baumler AJ. Non-typhoidalsalmonellosis : emerging problems. Microbes Infect2001; 3 : 237–247.

24. White DG, Zhao S, Sudler R, et al. The isolation ofantibiotic-resistant salmonella from retail groundmeats. N Engl J Med 2001; 345 : 1147–1154.

25. Martel JL, Tardy F, Brisabois A, Lailler R, Coudert M,

Chaslus-Dancla E. The French antibiotic resistancemonitoring programs. Int J Antimicrob Agents 2000;

14 : 275–283.26. Martel JL, Tardy F, Sanders P, Boisseau J. New trends

in regulatory rules and surveillance of antimicrobialresistance in bacteria of animal origin. Vet Res 2001;

32 : 381–392.27. Cloeckaert A, Sidi Boumedine K, Flaujac G, Imberechts

H, D’Hooghe I, Chaslus Dancla E. Occurrence of a

Salmonella enterica serotype Typhimurium DT104-likeantibiotic resistance gene cluster including the floR gene

in S. enterica serotype Agona. Antimicrob AgentsChemother 2000; 44 : 1359–1361.

28. Cloeckaert A, Chaslus-Dancla E. Mechanisms ofquinolone resistance in Salmonella. Vet Res 2001; 32 :291–300.

29. Johnston AM. Use of antimicrobial drugs in veterinarypractice. BMJ 1998; 317 : 665–667.

30. Threlfall EJ, Skinner JA, Graham A, Ward LR,

Smith HR. Resistance to ceftriaxone and cefotaxime in

non-typhoidal Salmonella enterica in England andWales, 1998–99. J Antimicrob Chemother 2000; 46 :860–862.

31. Walker RA, Lawson AJ, Lindsay EA, et al. Decreasedsusceptibility to ciprofloxacin in outbreak-associatedmultiresistant Salmonella typhimuriumDT104. Vet Rec

2000; 147 : 395–396.32. Hakanen A, Kotilainen P, Jalava J, Siitonen A,

Huovinen P. Detection of decreased fluoroquinolone

susceptibility in Salmonellas and validation of nalidixicacid screening test. J Clin Microbiol 1999; 37 :3572–3577.

33. Threlfall EJ, Ward LR, Skinner JA, Graham A.

Antimicrobial drug resistance in non-typhoidal salmo-nellas from humans in England and Wales in 1999:decrease in multiple resistance in Salmonella enterica

serotypes Typhimurium, Virchow, and Hadar. MicrobDrug Resist 2000; 6 : 319–325.

8 J. Cailhol and others