Antibiotic resistance in salmonellae isolated from humans and animals in France: comparative data from 1994 and 1997

Jacques Breuila,b,*, Anne Brisaboisc, Isabelle Casind,b, Laurence Armand-Lefèvreb, Sylvine Frémyc and Ekkehard Collatzb

a Laboratoire de Bactériologie, Centre Hospitalier Intercommunal, 40 Allée de la Source, 94190 Villeneuve-Saint Georges; b INSERM E0004, Laboratoire de Recherche Moléculaire sur les Antibiotiques, UFR Broussais-Hôtel Dieu and Pitié-Salpétrière, Université Paris VI, Paris; c Agence Francaise de Sécurité Sanitaire des Aliments, Maison Alfort; d Service de Microbiologie, Hôpital Saint Louis, Université Paris VII, Paris, France


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Among 25526 recorded isolates of salmonellae, 5086 isolated from humans and 20440 from animals in 1994 and 1997 in France, the antibiotic resistance phenotype was determined for all human and 5336 animal isolates. In Salmonella enterica serovar typhimurium, one of the two most frequently isolated serovars from humans as well as animals, resistance to ampicillin was observed in 61% of both human and animal isolates in 1994 and in 73% of human and 53% of animal isolates in 1997. During these periods, resistance to co-amoxiclav was between 45% and 66% for both types of isolate. Resistance to ampicillin was associated with resistance to streptomycin, spectinomycin, sulphonamide, tetracycline and chloramphenicol in over 70% of isolates. Resistance to ampicillin as well as co-amoxiclav never exceeded 7% in Salmonella enteritidis. While Salmonella hadar was practically absent among the human isolates in 1994, this serovar was the third most frequent in 1997, and at that time 92% were resistant to nalidixic acid. Among the animal S. hadar isolates, the prevalence of resistance to nalidixic acid increased from 3% in 1994 to 72% in 1997. None of these isolates manifested high-level resistance to ofloxacin. The levels of resistance to aminoglycosides (<=3%) and trimethoprim–suphamethoxazole (<=14%) remained practically unchanged in all three serovars. The resistance markers of 463 ampicillin-resistant S. typhimurium isolated in 1997 were determined. Among the 24 phenotypes observed, six multiresistance phenotypes, representing 82% of these isolates (as compared with 80% in 1994), were associated with the PSE-1 gene typically found in the lysotype DT104 of this serovar.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The incidence of human salmonellosis is rising in most of the countries where surveillance networks have been set up and, according to the World Health Organization (WHO), the problem in these countries is becoming one of public health. Along with this rising incidence, increasing rates of antibiotic resistance have been reported in various regions throughout the world and certain multiply antibiotic-resistant clones, such as Salmonella typhimurium DT104, seem to have disseminated in areas rather distant from each other.1 The question of whether such clones possess particular virulence characteristics remains open but the observation of their simultaneous occurrence in the human and animal ecosystems as well as reports of recent outbreaks of multiresistant clones in Europe and the USA24 have sustained interest in epidemiological studies. At the molecular level, Ridley & Threlfall5 and Seyfarth et al.6 have reinforced the commonly accepted view that animal and human isolates are usually indistinguishable and have shown that the incidence of multiresistant salmonellae can increase substantially within only a few years after licensing of a given antibiotic for veterinary use.

Multicentre surveys of antibiotic resistance in salmonellae isolated from humans in France have been conducted in collaboration with a hospital-based network, the Collège de Bactériologie, Virologie et Hygiène des Hôpitaux de France (ColBVH), in 1994 and 1997.7,8 The establishment of links with the Agence Française de Sécurité Sanitaire des Aliments (AFSSA, formerly Centre National d'Etudes Vétérinaires et Alimentaires, CNEVA) has allowed a comparative analysis of antibiotic resistance in salmonellae of human and animal origin during the two 1 year periods. These studies were undertaken with the aim of contributing to the establishment of efficient measures to control the dissemination of multiresistant salmonellae.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial isolates

Human isolates.
In 1994 and 1997, a total of 5086 isolates of Salmonella spp. of human origin were collected by, respectively, 76 and 77 ColBVH-associated hospitals across France.7,8 Only one isolate per patient was included. Identification was performed at the referring hospitals using API20E strips (bioMérieux, Marcy l'Etoile, France) and serotyping according to the Kauffmann–White scheme with antisera from Sanofi Diagnostics Pasteur (Marnes la Coquette, France). Rare serovars were confirmed by the Centre National de Typage Moléculaire Entérique (Institut Pasteur, Paris, France).

Veterinary isolates.
The annual CNEVA records from 1994 and 1997 which cover the salmonellae isolated from poultry (c. 60%), cattle (c. 30%) and various other animal sources (c. 10%) were used. The isolates were of four distinct categories: animals sensu stricto, immediate animal environment including faeces, local ecosystems and food. Only isolates from the first two categories, totalling, respectively, 6282 and 14158 isolates, were considered in this study. The antibiotic susceptibilities of 3368 and 1968 isolates, respectively, from the two survey periods, were determined. Identification procedures were similar to those used for the human isolates.

Antimicrobial susceptibility testing

Antibiotic susceptibility was determined by the participating hospital laboratories, using either automated microdilution procedures [API-ATB and Vitek (bioMérieux), c. 70%], or agar diffusion (c. 30%). The categories, susceptible (S), intermediate (I) or resistant (R), were assigned on the basis of the susceptibility data obtained by the hospital laboratories, and in each case were reviewed at the Laboratoire de Recherche Moléculaire sur les Antibiotiques (LRMA). The susceptibilities of all isolates from animals were tested by AFSSA using agar diffusion. The medium was in all cases Mueller–Hinton broth or Mueller–Hinton agar. The breakpoints (see Table IGo) were those recommended by the Comité de l'Antibiogramme de la Société Française de Microbiologie.9 The susceptibilities of all isolates to the following antibiotics were determined: ampicillin, co-amoxiclav, gentamicin, amikacin, tetracycline, nalidixic acid, ofloxacin, chloramphenicol and trimethoprim–sulphamethoxazole. The susceptibilities of the human isolates to ceftriaxone and ceftazidime were also determined.


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Table I. Percentage of antimicrobial resistance for the three major serovars isolated in humans and animals in 1994 and 1997
 
Resistance phenotypes of ampicillin-resistant S. typhimurium isolates

The resistance phenotypes of 650 ampicillin-resistant S. typhimurium isolates, 386 from humans and 264 from animals, were determined by disc diffusion on Mueller– Hinton agar (Sanofi Diagnostics Pasteur) at the LRMA. The ampicillin-resistant human isolates were selected without any particular bias, except that an attempt was made for each participating hospital to be represented. The ampicillin-resistant animal isolates were chosen from the AFSSA collection without further selective criteria. The results obtained with the isolates from 1997 [300 human and 163 animal isolates (Table IGo)] were compared with those from 1994 (86 human and 101 animal isolates).10 The antibiotics tested included, in addition to those mentioned above, ceftazidime, ceftriaxone, streptomycin, spectinomycin, kanamycin, apramycin, sulphonamide, trimethoprim and ciprofloxacin, using discs from Sanofi Diagnostics Pasteur. The co-amoxiclav disc was placed next to that of ceftriaxone and ceftazidime in order to detect extended-spectrum ß-lactamase production.

DNA probes and hybridization experiments

The ß-lactamase and integrase genes of the 650 ampicillin-resistant S. typhimurium isolates were identified as described previously10 by dot blotting and hybridization with DNA probes representing internal sections of blaTEM, blaPSE and blaSHV or the entire oxa-1, oxa-2, oxa-10 and intI genes. The antibiotic-susceptible S. typhimurium reference strain LT2 and isolates containing the ß-lactamase and integrase genes were also spotted on to each membrane.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Collection and identification of isolates

Human isolates.
During the surveys of 1994 and 1997, 2622 and 2464 isolates, respectively, were recorded. The distribution of the serovars represented by at least five isolates, i.e. among 2552 and 2331 isolates, respectively, is shown in Figure 1Go. In both survey periods, S. typhimurium and S. enteritidis were the two predominant serovars, accounting together for over 80% of the salmonellae isolated. S. hadar, uncommon in 1994, was the third most frequently isolated serovar 3 years later.



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Figure 1. Human isolates reported by the surveys of the ColBVH ({blacksquare}, 1994 and {square}, 1997).

 
Animal isolates.
The distribution of eight serovars among the isolates analysed, uniquely from animals and their immediate environment, 13397 and 3858 isolates, respectively, from 1994 and 1997, is shown in Figure 2Go. The less frequent serovars (not shown) represented c. 18% of the isolates. S. typhimurium was the predominant serovar in 1994 and 1997. The second position of S. enteritidis in 1994 was taken by S. hadar in 1997.



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Figure 2. Isolates of animal origin reported by AFSSA in 1994 ({blacksquare}) and 1997 ({square}).

 
Antibiotic susceptibility of the three serovars predominant in 1997

The frequencies of antibiotic resistance observed in 1994 and 1997 for S. typhimurium, S. enteritidis and S. hadar are given in Table IGo, along with the breakpoints. Isolates of intermediate susceptibility were counted together with the isolates that were resistant sensu stricto.

Overall, S. typhimurium showed the highest rates of resistance, and S. enteritidis the lowest. For human S. typhimurium isolates, there was an increase in resistance to ampicillin (12%), co-amoxiclav (18%), tetracycline (17%) and chloramphenicol (19%), while a decrease of 5% was observed for cotrimoxazole. In S. enteritidis, the highest rates were observed for resistance to tetracycline (up to 20%); all others remained <=5%. S. hadar stood out in that it showed high rates of resistance to ampicillin, tetracycline and quinolones, but not to chloramphenicol, aminoglycosides or cotrimoxazole, in 1997, and that there was an impressive increase from 1994 in the rates of resistance to ampicillin and quinolones in the animal isolates, for which category we have comparative data: from 8 to 66% for resistance to ampicillin and, respectively, from 3 to 72% and 0 to 13% for resistance to nalidixic acid and ofloxacin, respectively. As far as ofloxacin is concerned, no isolate was resistant sensu stricto, i.e. MIC >= 8 mg/L. Resistance to amikacin was practically nil in the isolates from humans and animals, and some resistance to gentamicin (not exeeding 2%) was observed only in the human isolates of all three serovars. Three amikacin-resistant human isolates of S. hadar were from three patients of a single hospital in Northern France.

Resistance phenotypes of ampicillin-resistant S. typhimurium isolates

The distribution of the resistance phenotypes among the ampicillin-resistant S. typhimurium isolates from humans and animals in 1997 is shown in Table IIGo. Twenty-four resistance phenotypes were observed, close in patterns and distribution to the 23 described in 1994.10 The vast majority of them, 82 and 80%, respectively, for 1994 and 1997, comprised the quintuple, PSE-associated resistance to ampicillin, streptomycin/spectinomycin, sulphonamides, tetracycline and chloramphenicol (AmSm/SpSuTeCm), associated at various, but generally low, frequencies with resistance to nalidixic acid, trimethoprim, gentamicin or combinations thereof. The activity of amoxycillin against these isolates was only exceptionally restored by clavulanate and no production of an extended-spectrum ß-lactamase was observed in any of the isolates.


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Table II. Distribution of resistance phenotypes in Salmonella typhimurium isolated from human and animals in 1997
 
Of the eight gentamicin-resistant isolates, five (including three from humans and two from animals) were also resistant to apramycin.

ß-Lactamase and integrase genes in ampicillin-resistant S. typhimurium isolates

ß-Lactamase and integrase genes were identified by DNA– DNA hybridization. The data for 1997 are shown in Table IIIGo. The gene coding for PSE-1 was clearly predominant and present as the only ß-lactamase gene in 79% of both human and animal isolates, while TEM-type genes were observed in, respectively, 16 and 19% of the isolates. The two genes were distributed differently between the various resistance phenotypes shown in Table IIGo. PSE-1 was found, exclusively, in 98% of the isolates with the multiresistance phenotype AmSm/SpSuTeCm, while TEM as the sole ß-lactamase gene was present only outside of this phenotype and then associated with 17 distinct resistance patterns (data not shown). OXA-1 was rare and found in human isolates only.


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Table III. Correlation of ß-lactamase and integrase genes in Salmonella typhimurium
 
Over 80 of all isolates hybridized to the intI probe, indicative of the presence of class 1 integrons.11 All PSE- and OXA-type genes were associated with intI, which was the case for TEM-type genes only in isolates resistant to trimethoprim.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
According to WHO, ‘the antimicrobial resistance monitoring programmes of isolates from food animals and food of animal origin must allow for relating data obtained from animals, food and humans. Collaboration of the medical, veterinary and agricultural sectors is vitally important.’12 The present study of antibiotic resistance in salmonellae was undertaken in the spirit of this statement.

The two serovars most frequently identified in the human isolates, in 1994 and 1997, from the hospitals of the ColBVH network were S. typhimurium and S. enteritidis. Both were also the serovars most frequently isolated from animals in 1994, but in 1997 S. hadar appeared to be somewhat more frequent than S. enteritidis. The prevalence of S. hadar seems subject to fluctuation. Among the human isolates it was practically absent in 1994, but it was the third most frequent in 1997 and also frequent between 1977 and 1979.13

For reasons of change in data collection protocols at AFSSA between 1994 and 1997, there is an important difference between the total numbers of animal isolates reported for the respective years. Changes in relative differences between the frequencies of serovars and resistance phenotypes in this sample must therefore be interpreted with caution. On the other hand, all data concerning the isolates from humans can be interpreted without caveat since there were equivalent numbers of isolates each year from practically the same number of hospitals and regions.

The phenomenon of antibiotic resistance in salmonellae concerned primarily the serovar S. typhimurium and much less so S. enteritidis, in agreement with previous observations.14,15

Molecular epidemiological together with more conventional typing studies have shown a quintuple resistance to be associated typically with an epidemic clone of phage type DT104 which probably began to evolve about 15 years ago.16 The present study suggests that this clone, predominant in France since at least 1994,10 is now established. Its quintuple resistance, to ampicillin, streptomycin/spectinomycin, sulphonamide, tetracycline and cloramphenicol, is chromosome encoded.17 The corresponding gene cluster, on a c.13 kb DNA segment containing two class 1 integron sequences, has been characterized recently.18,19 Multiresistant S. typhimurium isolates collected in a hospital in Paris20 and in an eastern region of France21 apparently belonged to the same epidemic clone.

Concern has arisen about the possible emergence of fluoroquinolone resistance in salmonellae in the context of the marketing of compounds such as marbofloxacin, enrofloxacin and danofloxacin especially in the light of data from Threlfall et al.22 for the UK, which suggested that the use of enrofloxacin may precede a noticeable increase in the isolation of S. typhimurium DT104 with reduced susceptibility to ciprofloxacin. A similar observation had been reported a few years earlier in Germany with the emergence of fluoroquinolone-resistant variants of multiresistant S. typhimurium DT204c in c.50% of the isolates from calves following the introduction of fluoroquinolones for veterinary use in 1988. With the decline of DT204c isolates in Germany, enrofloxacin resistance has now almost disappeared but reduced susceptibility to enrofloxacin is observed occasionally in veterinary DT104 isolates.23 While the S. typhimurium isolates of the present study were only infrequently resistant to fluoroquinolones (Table IGo), and then at low levels (data not shown), a recent report indicated that this type of reduced susceptibility may currently be increasing in France.24 With MICs of ciprofloxacin remaining below 1 mg/L, this low-level resistance is, conceivably, the initial step in the selection of higher-level fluoroquinolone-resistant mutants. The possible emergence of such resistance among S. typhimurium, isolated from humans or animals, justifies continued surveillance. This applies, in particular, to the epidemic clone DT104 given its capacity for dissemination and the inefficiency of protective measures for its control that might be implemented locally.25

With respect to quinolone resistance, we observed a noticeable difference between the serovars S. typhimurium and S. hadar in that the latter, found predominantly in poultry, emerged as particularly resistant to nalidixic acid. This seems to be a very recent development, since in 1994 only sporadic isolates from animals were resistant to this drug, as compared with 72% resistant isolates in 1997. In that year, the corresponding resistance rate for isolates from humans was even higher (92%) but there are no comparative data for 1994 and, to our knowledge, there are no earlier survey data on resistance to nalidixic acid of S. hadar in France. All nalidixic acid-resistant isolates of S. hadar showed reduced susceptibility to fluoroquinolones and fell into the category of either susceptible or intermediate with respect to ofloxacin. Reduced susceptibility of this serovar to ciprofloxacin had increased in the UK, from 40% in 1994 to 60% in 1996, and was particularly frequent among multiresistant isolates.22 In Germany, resistance to nalidixic acid among veterinary isolates of S. hadar was first observed in 1990, reached high rates between 1994 and 1997 and has fallen since.23

Resistance to aminoglycosides, except for streptomycin/ spectinomycin and kanamycin, is not commonly observed in S. typhimurium and then concerns typically gentamicin, netilmicin and, variably, also apramycin.26,27 Apramycin resistance was considered initially to be confined to veterinary isolates but has been found in human isolates since 1989 and was conferred by closely related plasmids.28 This type of resistance does not seem to have disseminated much among salmonellae. In the present study, five out of eight gentamicin-resistant S. typhimurium isolates (Table IGo) were resistant also to apramycin, most likely due to the production of an aminoglycoside acetyltransferase of the AAC(3)-IV type.

Taken together, the data of the present study show similar prevalences of Salmonella serovars in isolates from humans and animals in France, with little change as far as the predominant S. typhimurium and S. enteritidis are concerned, including their antibiotic resistance phenotypes. It would be interesting to monitor whether the multiresistant S. typhimurium DT104, which apparently continues to spread to new regions,29 will maintain its current prevalence in France over time and whether or not it will acquire additional determinants of resistance, particularly to the newer quinolones. The potential of S. hadar to develop resistance should also be monitored.


    Acknowledgments
 
We thank the members of the Collège de Bactériologie, Virologie et Hygiène des Hôpitaux de France who have provided isolates for this study. We gratefully acknowledge the reviewers for the improvement of the manuscript. This study was supported in part by the Institut National de la Santé et de la Recherche Médicale (E 0004).


    Notes
 
* Corresponding author. Tel: +33-143-86-22-17; Fax: +33-143-86-24-05; E-mail: jackbreuil{at}aol.com Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Anonymous (1997). Multi-drug resistant Salmonella typhimurium. Fact Sheet No 139. http://www.who.int/inf-fs/en/fact139.html.

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Received 18 November 1999; returned 25 April 2000; revised 5 June 2000; accepted 2 July 2000