Subtyping of Salmonella Typhimurium by pulsed-field gel electrophoresis and comparisons with phage types and resistance types

Pathol Biol 2002 ; 50 : 361-8  2002 Éditions scientifiques et médicales Elsevier SAS. Tous droits réservés S0369-8114(02)00321-8/FLA

Article original

Subtyping of Salmonella Typhimurium by pulsed-field gel electrophoresis and comparisons with phage types and resistance types Renaud Lailler 1∗ , Francine Grimont 2 , Yvette Jones 3 , Pascal Sanders 4 , Anne Brisabois 1 1 Agence française de sécurité sanitaire des aliments (AFSSA), laboratoire d’étude et de recherche sur

l’hygiène et la qualité des aliments (LERHQA), unité d’épidémiologie bactérienne, 39-41, rue du 11 Novembre, 94700 Maisons-Alfort, France ; 2 Institut Pasteur, centre national de référence pour le typage moléculaire des entérobactéries, Unité INSERM 389, 28, rue du Dr. Roux, 75724 Paris cedex 15, France ; 3 department of bacterial diseases, veterinary laboratories, Weybridge, Surrey, UK ; 4 Agence française de sécurité sanitaire des aliments (AFSSA), laboratoire d’études et de recherches sur les médicaments vétérinaires et les désinfectants, 35302 Fougères, France Summary One-hundred and sixty-eight Salmonella enterica subsp. enterica serotype Typhimurium isolates have been analysed by phage typing, by pulsed-field gel electrophoresis and for their antimicrobial susceptibility. Those independent strains, isolated from food animal production including cattle, poultry and pig sectors have been collected by the French non human Salmonella network, during the first semester in 1999. Isolates encompassed 14 phage types. The majority of S . Typhimurium isolates was found to be definitive phage type DT104, representing 39% of all isolates. Other phage types were mainly DT8, PT U302, DT120, DT193 and DT135. Forty-six pulsotypes were obtained using XbaI restriction enzyme, and amongst them, ten were associated to the DT104 phage type. A major pulsotype (px1), was represented by 79% of DT104 isolates and was also found among DT120. Forty-eight percent of isolates showed a classic DT104 resistance profile to ampicillin, streptomycin, chloramphenicol, tetracycline, sulfonamides (ASCTSu). Among this resistance type, 84% were DT104 and 12% were DT120. Some pulsotypes were found associated to this resistant type. The pulsed field gel electrophoresis showed to be a useful typing method for discrimination of S. Typhimurium strains and for tracing clone through different sectors of origin in order to control their spread.  2002 Éditions scientifiques et médicales Elsevier SAS animals / drug resistance, microbial / electrophoresis, gel, pulsed-field / Salmonella Typhimurium

Résumé – Caractérisation de Salmonella Typhimurium par électrophorèse en champs pulsé et comparaison avec les résultats de lysotypie et de résistance aux antibiotiques. Cent soixante huit souches indépendantes de Salmonella enterica subsp. enterica sérotype Typhimurium ont été analysées par lysotypie, par électrophorèse en champ pulsé et testées pour leur sensibilité aux antibiotiques. Elles ont été isolées au cours du premier semestre 1999 par le réseau Salmonella, réseau national de centralisation des souches d’origine non humaine, à partir de prélèvements effectués en santé animale au sein des filières de production bovine, porcine et aviaire. Ces souches se sont réparties en 14 lysotypes. Le lysotype majoritaire, DT104, était représenté par 39 % des souches. Les autres lysotypes rencontrés étaient principalement DT8, DT120, DT193, DT135 et le lysotype non définitif PT U302. Quarante six pulsotypes ont été identifiés en utilisant l’enzyme de restriction XbaI, parmi lesquels dix étaient associés aux souches de lysotype DT104. Un pulsotype majoritaire (px1), était représenté par 79 % des souches DT104 et a été également

∗ Correspondence and reprints.

E-mail address: [email protected] (R. Lailler).

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identifié parmi les souches de lysotype DT120. Quarante huit pour cent des souches présentaient un profil de sensibilité aux antibiotiques associant des résistances à l’ampicilline, à la streptomycine, au chloramphénicol, à la tétracycline et aux sulfamides. Parmi celles-ci, 83 % étaient de lysotype DT104 et 12 % de lysotype DT120. Ce profil de multirésistance classique des DT104 a été associé également à certains pulsotypes. L’électrophorèse en champ pulsé s’avère être une méthode de typage très discriminante, dans une perspective de maîtrise et de contrôle de la diffusion des souches de S. Typhimurium au sein des différents secteurs d’origine.  2002 Éditions scientifiques et médicales Elsevier SAS animaux / antibiorésistance / électophorèse en champs pulsé / Salmonella Typhimurium

1. INTRODUCTION Salmonella is recognised as one of the major causes of food-borne enteric infection in humans and in animals. In 1999, the French incidence of infection with each of the two most common serotypes of Salmonella was 3.8 cases per 100 000 population for S. Enteritidis and 3.4 for S. Typhimurium [1]. In France, the antimicrobial resistance and the evolution of serotypes in Salmonella species of animal origin have been monitored through the French Salmonella network for several decades [2]. This network gathers strains and epidemiological information routinely sent for confirmation of serotyping by 150 private or public laboratories set up all over the French area. About 20 000 strains or data of strains isolated from various categories of samples have been collected per year. Results will be presented according to the serotypes isolated in different sectors: animal species and breeding, food products including environment of production, and natural environment [3 – 5]. Salmonella Typhimurium was the most frequently isolated serotype in spite of a relative decrease in frequency. This serotype represented 31.7% of the strains isolated in 1988–1989 and only 13.9% for 1999. It was the most common serotype identified between 1997 and 1999, in cattle and pig sectors and was the third most common in the poultry sector after S. Hadar and S. Newport or S. Virchow [3 – 5]. S. Typhimurium was the second most common serotype isolated from human strains during this same period, behind S. Enteritidis [6]. Therefore, there is a need to develop powerful methods for subtyping S. Typhimurium isolates in order to investigate food-borne outbreaks or at least to have a better epidemiological knowledge of the strains isolated in the animal production sector to enable control measures to be effective. Salmonella Typhimurium definitive phage type DT104 strains emerged last decade in many countries [7 – 9]. Their virulence, associated with resistance to ampicillin (A), streptomycin (S), chloramphenicol (C), tetracycline (T) and sulfonamides (Su) (ASCTSu-R type) promote them as an increasing world-wide problem [10 – 12]. A decrease of fluoroquinolone susceptibility is more and more associated with this pattern [7, 13 – 16]. Phagetyping has been used for many years and is still useful to differentiate isolates but this method requires a high experience for reading the phage lysis area and consequently

can be implemented only in national reference laboratories. Several molecular methods have been developed and applied to monitor the DT104 increase and more generally the multidrug-resistant strains [17 – 19]. Pulsed-field gel electrophoresis (PFGE) was found to be one of the most discriminatory methods to compare strains isolated during investigations [20, 21]. It is also a powerful method suitable for a continuous surveillance program but comparison of results can prove more difficult because of the long study period and the number of strains required. The identification of “clonal lines” may contribute to our understanding and ability to better control exchanges between Salmonella strains of the same serotype and more largely between different serotypes. In this study, we analysed 168 S. Typhimurium strains by phage typing, antibiotic susceptibility testing and PFGE in order to evaluate the incidence of DT104 strains from animal origins and to correlate the three subtyping methods.

2. MATERIALS AND METHODS 2.1. Bacterial strains 168 strains of Salmonella Typhimurium were collected from samples of animal species (cattle, pigs and poultry) and breeding by the French Salmonella network, during the first semester of 1999 (Table I). The strains were identified as Typhimurium according to the KauffmannWhite typing scheme. They were isolated mainly from two French areas: Bretagne (49 strains) and Aquitaine (40 strains). The poultry sector was mostly represented (58%) with half of them issued from farm controls (swabs). The poultry strains came from duck (33 strains), broiler (25), turkey (17), quail (9), pigeon (10), guinea fowl (4) and goose (1). Phage typing was performed according to the scheme described by Anderson et al. [22].

2.2. Antimicrobial susceptibility testing Antimicrobial susceptibility tests were performed for 137 strains by the disk diffusion method on MuellerHinton agar (Bio-Rad), following the zone size criteria as recommended by the Comité de l’Antibiogramme de la Société Française de Microbiologie (CA-SFM). The

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Table II. The antibiotics tested Table I. Strains distribution within animal origin Animal origin Bird (unknown) Broiler

Antibiotic family

Antibiotics tested (load, zone diameters (mm) as recommended by CA-SFM)

β-Lactams

Ampicillin (10 µg, 19–14)

Number of strains 2

Amoxycillin + clavulanic acid (20 µg, 21–14)

25

faecal sample 2 Cephalosporins

swabs 23 Calf (faecal sample) Canary (unknown) Cat (unknown) Cattle

Aminoglycosides

1

faecal sample 17 viscera 4 33

Kanamycin (30 IU, 17–15) Aminocyclitols

Spectinomycin (100 µg, 18)

Phenicols

Chloramphenicol (30 µg, 23–19)

Tetracyclines

Tetracycline (30 IU, 19–17)

Sulphonamides

Sulfamethoxazole-trimethoprim (23.75 µg + 1.25 µg, 16–10)

Quinolones

Nalidixic acid (30 µg, 20–15)

faecal sample 5

Sulfamides (200 µg, 17–12)

viscera 18 swabs 10

Pefloxacin (5 µg, 22–16)

Eagle (unknown)

2

Goat

2

faecal sample 1 viscera 1 Goose (swabs)

1

Guinea fowl

4

viscera 2 swabs 2 Horse (viscera)

1

Mammal (unknown)

1

Parakeet (unknown)

1

Pigeon (unknown)

10

Pig

31

faecal sample 1 viscera 29 swabs 1 Quail

9

faecal sample 1 viscera 6 swabs 2 Rabbit (unknown) Sheep (faecal sample) Turkey

1 1 17

faecal sample 1 swabs 16 total

Streptomycin (10 IU, 15–13) Gentamicin (10 IU, 16–14)

23

abortion product 2

Duck

Cefotaxime (30 µg, 21–15)

2 1

Cephalothin (30 µg, 18–12)

168

Enrofloxacin (5 µg, 22–17)

panel of antibiotics tested is presented in Table II. Zone diameters were read using an automated scanner, Radius System™ (Mast diagnostic). In this study, only the strains showing a zone diameter below the minimal breakpoint have been considered as resistant.

2.3. Pulsed-Field Gel Electrophoresis (PFGE) The cultures grown overnight at 37 ◦ C in TSAYE broth (40 g/l trypcase soja agar (AES) and 6 g/l yeast extract (Difco), 1 liter of water, pH = 7.3). Preparation of total DNA and experimental set up were as described by Gautom et al. [23] except that proteinase K was added to a final concentration of 0.5 mg/ml. Restriction digestion was performed overnight at 37 ◦ C with 40 IU XbaI per plug (final volume 150 µl) as directed by the manufacturer (Promega). The electrophoresis was performed at 14 ◦ C with the CHEF-DRIII apparatus (Bio-Rad). The molecular size marker used was Marker I Lambda-Ladder (Boehringer Mannheim). The sets of pulsed times were 7– 12 sec for 11 hours and 20–40 sec for 13 hours, at 6 V/cm. Gels were stained with ethidium bromide and visualized on an UV transilluminator and captured by the digital imaging system GelDoc 1000 (Video gel doc system, BioRad). Patterns obtained were analysed by the Molecular Analyst software (Fingerprinting, Windows version 1.6, Bio-Rad). The relation between two given strains was scored by the Dice coefficient of similarity, and strains were clustered by the hierarchical clustering of interstrain similarities based on the unweighted pair group

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strains presented very high resistance rates: streptomycin (96.6%), spectinomycin (96.6%), sulfonamides (96.6%), tetracycline (94.9%), chloramphenicol (91.5%) and ampicillin (89.8%). These resistance patterns were the same for 53 of the 59 DT104 strains tested. This classic resistance pattern (ASCTSu-R type) was sometimes associated with gentamicin, amoxycillin/clavulanique acid, nalidixic acid or sulfamethoxazole-trimethoprim resistances.

method with arithmetic averages (UPGMA). A variation in band mobility of 1% was tolerated. Fragments patterns were interpreted as described by Tenover et al. [24]. Patterns in which at least 80% but less than 100% of the bands matched were considered similar and designated as subtypes. Generally these patterns differed by 1 to 3 bands. Strains with less than 80% similarity (more than 3 different bands) were considered as different clusters.

3. RESULTS

3.3. PFGE patterns

3.1. Phage typing

Genomic DNA were analysed by pulsed field gel electrophoresis, using the XbaI enzyme. A total of 46 different patterns (arbitrarily designated as px1 to px46), with ten to twenty bands (Fig. 1), gathered into 20 clusters (Fig. 2, cluster A to T), were identified for the 168 strains. Among the 20 clusters, eight were represented by only one strain (clusters C, H, and O to T). DT135 strains were mostly identified in cluster E, while DT8 and DT30 strains were mostly encountered in cluster F. These two clusters presented 74% similarity between them. The main cluster was J (71 strains), which was also the cluster with the highest pulsotype polymorphism. All but two DT104 strains were pooled on cluster J and encompassed eight PFGE different patterns (px1 to px8) showing a similarity coefficient up to 83.5%. A main pattern (px1) gathered together 52 of the 66 DT104 strains characterised by PFGE and six of the 13 DT120 strains. Two DT104 strains showing a single PFGE pattern (px3 and px10) were sensitive to all the antibiotics tested.

All but ten strains belonged to 14 phage types in which the most important were DT104 (also called 12 atypical in France, 66 strains), DT8 (24), DT120 (13), PT U302 (16), DT135 (12), DT193 (10) and DT30 (7). No lysis was observed for ten strains when each phage was tested, so they were considered as untypable (unty). Other phage types were encountered more rarely: DT146a for four strains, DT12 for two strains and only one strain represented each DT208, DT99, DT195, DT4 variant and DT124 phage types. DT8 and DT30 phage types seemed to be specific to the poultry sector (Table III) and mostly associated with duck origin (22 out of 24 DT8 strains and all the DT30 strains). Except for one strain (DT99), all the strains from pigeons were typed DT135 and nine out of twelve DT135 strains came from pigeons. DT104 strains were observed from different animal origins but the relative importance of this phage type was different according to the animal sectors, the highest percentage (80%) has been observed in cattle (Table III).

4. DISCUSSION 3.2. Susceptibility testing In 1999, the resistance rates of S. Typhimurium strains collected by the Salmonella network seemed to be very similar to those obtained for this study: for example 54% of strains were resistant to ampicillin, 73.5% to tetracycline, 68.3% to streptomycin and 11.9% to nalidixic acid. This suggests that the panel of strains studied was representative of the strains collected during the year.

Among the panel studied, 137 strains were tested for their susceptibility to a set of antibiotics (Table II). The highest levels of resistant strains were 73.0% and 67.2% to tetracycline and streptomycin respectively. For more clarity, Table IV presents the susceptibility results for the 15 antibiotics tested according to the phage type. The DT104

Table III. Strains distribution among phage types and sectors origin Sectors

Phage types (DTs) 104

Poultry

31

Cattle

20

Pig

8

12

1

Other

7

1

Total

66

2

Unty: untypable strains.

120

124

135

146a

193

5

10

2

5

1

1

1

7

1

13

1

1

208

Total 30

8

99

U302

4var

unty

7

24

1

10

1

3

99

7

31

10

168

1 1

12

195

1

1

1

1

25

5

4 3

10

13 7

24

1

16

1

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Table IV. Number of strains resistant to the different antibiotics tested according to the phage type Phage types (n =)

Am

Ac

DT104 (59)

53

21

Ce

Ct

C

Te

Sp

Sm

G

54

56

57

57

4

DT12 (2)

2

DT208 (1)

1

DT120 (7)

6

4

6

7

1 6

DT135 (7)

Su

TS

NA

57

4

7

Pe

En

1

6

1

1

7

1

4

3

3

1

1

6

2

18

6

2

DT146a (3)

2

DT193 (8)

5

1

3

1

1

1

DT195 (1) DT30 (7)

K

8

1 5

6

6

1 1

2

1

DT4var (1) DT8 (24)

1

1

8

2

DT99 (1)

9 1

PT U302 (12)

2

1

3

8

2

7

Untypable (4)

1

1

1

4

1

2

Total (137)

71

29

66

100

73

92

1

0

2 4

1

1

1

76

9

0

Am: Ampicillin, Ac: Amoxicillin + clavulanic acid, Ce: Cephalotin, Ct: Cefotaxime, C: Chloramphenicol, Te: Tetracycline, Sm: Streptomycin, Sp: Spectinomycin, G: Gentamicin, K: Kanamycin, Su: Sulphonamides, TS: Trimethoprim + Sulfamethoxazole, Na: Nalidixic acid, Pe: Pefloxacin, En: Enrofloxacin.

Figure 1. XbaI Pulsed-Field gel electrophoresis (PFGE) patterns of S. Typhimurium isolates. Lanes 1 to 6, 7 to 10, 13, 25: DT104 strains; lanes 11, 18: untypable strains; lanes 12, 15, 16: DT120 strains; lanes 14, 17, 24: PT U302 strains; lanes 19, 20: DT193 strains; lane 21: DT195 strain; lane 22: DT99 strain; lane 23: DT146a strain. M: molecular weight markers (lambda ladder). The number of PFGE pattern (px n◦ ) is reported in Fig. 2.

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Figure 2. Similarities between the 46 PFGE patterns observed by XbaI digestion and correlation with phage type and multidrug resistance pattern. Dendrogram generated with the band matching coefficient of Dice and the UPGMA clustering method. * : number of antibiotic resistance among the 15 antibiotics tested (Table II). /: not performed; unty: untypable strain.

The antibiotic susceptibility testing results allowed differentiation of mostly sensitive phage types (DT8, DT30, DT135) and mostly resistant phage types (DT120 and DT104). Some DT193, PT U302 or untypable strains could show a multidrug-resistance pattern. A dichotomy was also found by PFGE analysis. Some phage types seemed to be homogeneous, like DT104 and DT120 strains found respectively in clusters J and L with 70% similarity, and others phage types were heterogeneous, presenting a high diversity of PFGE patterns. Therefore, different phage types could be encountered in the same cluster, for example the cluster D encompassed DT135, DT146a, PT U302 phage types and some untypable strains. Similarly, the px1 pattern was represented by 52 DT104 strains and six DT120 strains. Moreover,

strains with the same phage type could be included in different clusters. For example, DT193 strains presented six different PFGE patterns which could be divided into five clusters (Fig. 2). An other study has shown that DT193 strains were more polymorphic than DT104 strains [25]. Our results showed clearly a major pattern for the DT104 strains. Nevertheless, two DT104 strains were sensitive to all the drugs tested and gave two different PFGE patterns (px3 in cluster J and px10 in cluster Q). Considering their PFGE patterns, different hypotheses could be advanced. The DT104 strain with pattern px10 could be ancestral because it conserved a full susceptibility to all antibiotics and it was genetically divergent from all the other multiresistant DT104 strains found in

Subtyping of Salmonella Typhimurium

the main pattern. The two patterns, px1 and px10, showed 57% similarity. In contrast, the strain showing the px3 pattern could have arisen from a deletion event on the genomic DNA of strains showing the px1 pattern and consequently lost penta-resistance. The PFGE XbaI patterns homogeneity of DT104 penta-resistant type strains observed in this study strengthen the clonal emergence of multidrug resistance strains in the serotype Typhimurium suggested by different authors [18, 26]. In 1999, 66% of the human strains phage-typed by the Centre National de Référence pour le Typage Moléculaire des Entérobactéries had definitive type (DT) 104 [27] and this observation confirm that the DT104 phage type strains take a great place in the S. Typhimurium population, whatever the strain origin. The DT104 strains frequency associated with multidrug-resistance pattern and virulence contributes to define these Salmonella Typhimurium strains as a national human health problem. It is becoming essential to differentiate DT104 and nonDT104 strains but also sensitive and multidrug-resistance strains because in this study some other definitive type strains were found multidrug-resistant too (DT120, PT U302 or untypable). Moreover, Cloeckaert et al. [28] described a S. Agona strain exhibiting a similar chromosomal locus as S. Typhimurium DT104 that contains a multiple antibiotic resistance gene cluster. Glynn et al. have identified class I integrons in the chromosomal DNA of strains issued from different Salmonella serotypes (Ohio, Java, Newport, Paratyphi A and Paratyphi B) showing five-drug resistance type [29]. Further epidemiological studies based on surveillance monitoring should be conducted to have a better knowledge of the DT104 and multidrug resistance strains prevalence in food and animal production sectors in France. A correlation should be performed with human data in order to establish risk assessment analysis of the multiresistant type spread through Salmonella Typhimurium phage types and through different Salmonella serotypes. PFGE seems to be an available method to discriminate DT104 strains, but the implementation of a standardised protocol is needed to use this tool for a world-wide survey.

ACKNOWLEDGEMENTS This study has been granted by the Direction Générale de l’Alimentation, Ministère de l’Agriculture.

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