Phenotypic and genotypic features of antibiotic resistance in Salmonella enterica isolated from chicken meat and chicken and quail carcasses

International Journal of Food Microbiology 160 (2012) 16–23

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Phenotypic and genotypic features of antibiotic resistance in Salmonella enterica isolated from chicken meat and chicken and quail carcasses Cristina Bacci ⁎, Elena Boni, Irene Alpigiani, Elisa Lanzoni, Silvia Bonardi, Franco Brindani Animal Health Department, Section of Food Hygiene, Faculty of Veterinary Medicine, University of Parma, Via del Taglio 10, 43126 Parma, Italy

a r t i c l e

i n f o

Article history: Received 21 March 2012 Received in revised form 18 September 2012 Accepted 18 September 2012 Available online 26 September 2012 Keywords: Salmonella enterica Antibiotic resistance Chicken meat Chicken carcass Quail carcass

a b s t r a c t One hundred and twenty-three Salmonella enterica isolated in Italy from chicken meat and carcasses and from quail carcasses were analyzed to determine their levels of antibiotic resistance using antibiograms (phenotypic method) and PCR amplification of antimicrobial resistance-associated genes (genotypic method). The isolates were screened for the ability to grow in the presence of antibiotics (ampicillin, gentamicin, sulfamethoxazole and tetracycline) and for the presence of the following genes: pse-1, ant (3″)-Ia, qacEΔI and sul-1, tetA, tetB and tetG. The most frequently isolated serotypes in the sample set were S. Virchow (24.4%), S. Enteritidis (17.1%) and S. Typhimurium (15.4%). Of the isolates from chicken carcasses, 86.1% were resistant to tetracycline, while 30.5% of the identified isolates exhibited phenotypic multi-drug resistance to ampicillin, sulfamethoxazole and tetracycline; the multi-resistance pattern ant (3″)-Ia/sul-1/tetA + tetB was detected in 11.1% of the isolates. Of the isolates from quail carcasses, 89.2% exhibited resistance to sulfamethoxazole, and 24.3% displayed phenotypic multi-drug resistance to ampicillin, gentamicin, sulfamethoxazole and tetracycline; a complete genotypic profile (pse-1, ant (3″)-Ia, qacEΔI and sul-1, tetA, tetB and tetG) was obtained for 27.0% of the isolates. Among these isolates, S. Typhimurium exhibited the genotypes pse-1/ ant(3″)-Ia/sul-1/tetG and pse-1/ant(3″)-Ia/sul-1/tetA + tetG. Of the isolates from chicken meat, 60.0% were resistant to tetracycline, and 36.0% exhibited a multi-drug resistance to ampicillin, sulfamethoxazole and tetracycline; only one isolate, S. Enteritidis, contained the complete genotypic pattern pse-1/ant(3″)-Ia/ sul-1/tetG. The majority of the isolates displaying multi-drug resistance to the three antibiotics were isolated from chicken meat (40.0%). © 2012 Elsevier B.V. All rights reserved.

1. Introduction In 2009, Salmonella infections (108,614 confirmed cases) were the second most frequently reported zoonoses in humans in Europe. However, there has been a remarkable decline in the number of reported cases over the last five years (European Food Safety Authority and European Centre for Disease Prevention and Control, 2011). In particular, the reduction in human infections caused by S. Enteritidis is partially due to the EU's mandatory control programs for breeding flocks (Regulation, EC No., 2160/2003; Regulation, EC No., 213/2009). In 2009, some of the serotypes identified by the European Commission as “relevant” to public health (S. Enteritidis, S. Typhimurium, S. Hadar, S. Virchow, S. Infantis and S. Typhimurium with the antigenic formula 1,4,[5],12:i:-) (Regulation, EU No. 517/ 2011) were observed in poultry meat (European Food Safety Authority and European Centre for Disease Prevention and Control, 2011). In recent years, an increase in antibiotic resistance has been observed in Salmonella isolated from foods of animal origin. In addition, several authors have reported an increase in the incidence of ⁎ Corresponding author. Tel.: +39 0521 902740; fax: +39 0521 032742. E-mail address: [email protected] (C. Bacci). 0168-1605/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijfoodmicro.2012.09.014

salmonellosis in humans due to multi-drug-resistant strains such as S. Typhimurium DT104, which is currently the most geographically widespread strain and is present in the largest number of animal species (Boyd et al., 2001; Briggs and Fratamico, 1999; Gyles, 2008; Larkin et al., 2004; Rabsch et al., 2001; Threlfall, 2002). Despite the 2006 European ban on antibiotics as feed additives for growth promotion (Regulation, EC No., 1831/2003), antibiotics are often detected at sub-therapeutic doses during checks conducted by the national veterinary competent authorities. The selective pressure caused by the variety of antibiotics therapeutically administered in veterinary practice has resulted in an increase in the environmental pool of genetic sequences conferring resistance. These genes may be transferred from serotypes of animal origin to serotypes isolated from humans, adding to the complexity of attempting to control infectious diseases. Similarly, the use of antimicrobials in human medicine is considered a major problem in the development of resistance in human pathogens (Aarestrup, 2004; Carattoli, 2001; Directive, 2003; Schwarz and Chaslus-Dancla, 2001). According to European legislation, the monitoring of antimicrobial resistance to a representative number of isolates of Salmonella enterica is mandatory, from pigs and poultry and the foods derived from those species.

C. Bacci et al. / International Journal of Food Microbiology 160 (2012) 16–23

The acquisition of genetic sequences involved in the main resistance-specific mechanisms can mediate i) the synthesis of enzymes inactivating the antimicrobial agent (β-lactamase class), ii) the chemical modification of the drug by different classes of enzymes (adenyltransferase or ANT class), iii) the alteration of the active sites of the proteins involved in the essential metabolism of the bacterial cell (dihydropteroate synthetase or DHPS class) and iv) the reduction in drug accumulation, as occurs in the resistance of Gram-negative bacteria to tetracycline (Carattoli, 2003; Quintiliani et al., 1999; Shaw et al., 1993). Phenotypic information on the antimicrobial resistance profile is obtained through the application of techniques that are unable to reproduce the real dynamic variations of the drug; moreover, this characterization can reveal a certain variation if the resistance is due to the acquisition of DNA carried by mobile elements (Di Conza et al., 2005; Randstrom et al., 1991; Ribeiro et al., 2011; Salyers et al., 1995). Therefore, for many authors, the preferred approach is to combine phenotypic and biomolecular methodologies to guarantee the correct typing of the antibiotic resistance of the different strains (Yang et al., 2001; Yildirim et al., 2011). The objective of this study is to evaluate the antibiotic resistance of S. enterica isolated in the poultry industry through the application of antibiograms (phenotypic method) and the Polymerase Chain Reaction (PCR) (genotypic method). The Salmonella isolates used in this study were collected in Italy during 2008– 2009. Each antimicrobial was chosen as a representative of its corresponding antibiotic class (penicillins, aminoglycosides, sulfonamides and tetracyclines). Therefore, the screened genes were selected for their assumed capacity to determine resistancespecific mechanisms toward the tested antimicrobials to verify the existence of a correlation between the presence of the gene and the phenotype.

2. Material and methods 2.1. Sampling Two hundred and fifty-two skin swabs of chicken carcasses (10 cm 2) and 250 swabs from the abdominal cavity of quail (5 cm 2) taken at slaughter, directly after evisceration, were analyzed (Commision decision, 2001). Each swab was suspended in 5 ml of diluent composed of 0.1% peptone and 0.85% NaCl (Oxoid, United Kingdom). The samples obtained from the chicken and quail carcass swabs came from slaughterhouses located in northeast Italy in 2008–2009. Moreover, 96 samples of skinless chicken meat (breast, thighs and the upper parts of the thighs) and chicken meat preparations were gathered from superstores located in the Emilia Romagna region (provinces of Parma and Reggio Emilia). All the samples were brought to the Department of Animal Health, Section of Food Inspection, and stored at 4 ± 1 °C for 2–24 h prior to the beginning of the analysis.

17

Table 1 Simplex and multiplex PCR primers used for identification of genes and corresponding antimicrobials. Antimicrobial

Gene

Ampicillin

pse-1

Simplex PCR primer sequence 5′-3′

CGC TTC CCG TTA ACA AGT AC CTG GTT CAT TTC AGA TAG CG Gentamicin ant GTG GAT (3″)-Ia GGC GGC CTG AAG CC ATT GCC CAG TCG GCA GCG Sulfamethoxazole qacEΔI ATC GCA ATA GTT GGC GAA GT sul-1 GCA AGG CGG AAA CCC GCG CC Antimicrobial Gene Multiplex PCR primer sequence 5′-3′ Tetracycline tetA GCT ACA TCC TGC TTG CCT TC CAT AGA TCG CCG TGA AGA GG tetB TTG GTT AGG GGC AAG TTT TG GTA ATG GGC CAA TAA CAC CG tetG CAG CTT TCG GAT TCT TAC GG GAT TGG TGA GGC TCG TTA GC

Reference Amplicon Annealing size (bp) temperature (T °C)

419

57

Sandvang et al. (1998), Houvinen and Jacoby (1991)

526

58

Brown et al. (2000), Sandvang et al. (1998)

797

63

Brown et al. (2000)

Reference Amplicon Annealing size (bp) temperature (T°C)

210

55

Ng et al. (2001)

659

844

2.3. Resistance to the antimicrobial agents 2.2. Isolation of S. enterica and serotyping The isolation of S. enterica was conducted according to the EN ISO standard 6579:2008. Suspected Salmonella colonies were confirmed serologically (Omnivalent, Salmonella antisera, 292537 Denka Seiken, Tokyo, Japan) and biochemically (API 20E®, bioMérieux, Marcy l'Etoile, France). The isolates were then serotyped by the National Reference Centre Salmonella, CRNS, Istituto Zooprofilattico Sperimentale delle Venezie, IZSVe, Legnaro, Padua, Italy. Only confirmed Salmonella were tested for their susceptibility to antimicrobial agents and the presence of the genes listed in Table 1.

The antibiotic susceptibility was determined according to the recommendations set by the Clinical and Laboratory Standards Institute (Clinical and Laboratory Standards Institute, CLSI, 2007) for the disk diffusion technique. The antimicrobials and concentrations tested were ampicillin (10 μg), gentamicin (10 μg), tetracycline (30 μg) and sulfamethoxazole (25 μg) (Oxoid, United Kingdom). The inhibition zones were measured and scored as sensitive, intermediate susceptibility or resistant according to the CLSI recommendations. Escherichia coli ATCC 25922 was used as a reference strain for antibiotic disc control.

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C. Bacci et al. / International Journal of Food Microbiology 160 (2012) 16–23

2.4. Identification of the resistance genes DNA from each Salmonella isolate was extracted as described by Malorny et al. (2003). The quantity and purity of the DNA were assessed as described by Sambrook and Russel (2001). The amplification cycles were performed in a Mastercycler 5333 (Eppendorf, Hamburg, Germany) machine using 25 μl of a reaction mix comprising double-distilled sterile water (Fermentas, M-Medical, Milan, Italy), 1× Taq Buffer (75 mM Tris–HCl, 20 mM (NH4)2SO4 and 0.01% Tween 20) (Fermentas), 0.2 mM of each dNTP (Fermentas), each primer (0.5 μM, Sigma Genosys Ltd, Pampisford, Cambs, UK), 1.25 U of AmpliTaq DNA Polymerase (Fermentas), 2.5 mM MgCl2 (Fermentas) and template DNA (1 μl). Simplex and multiplex PCR primers used for the identification of genes were listed in Table 1. The positive controls S. Typhimurium (phage type NT, strain numbers 3 and 33; phage type DT 12, strain numbers 30, 31 and 32) and S. Typhimurium (phage type DT 104, reference number GR0797) were provided by the Department of Veterinary Public Health and Animal Pathology (University of Bologna, Italy) and by the Salmonella Reference, Stobhill Hospital (North Glasgow University, UK), respectively. The negative controls of the master mix without template DNA were tested for each primer set. The amplified DNA was electrophoresed in a 1.5% agarose gel (Agarose low EEO) (AppliChem, Delchimica, Bologna, Italy) at 100 V for 2 h in 1× TAE (0.8 mM Tris acetate, 0.4 mM acetic acid, 0.02 mM EDTA) (Fermentas). The PCR products were visualized using a UV transilluminator after staining with ethidium bromide and compared to the Mass Ruler™ DNA Ladder (Low Range) (Fermentas). 3. Results 3.1. Isolation of S. enterica from chicken carcasses and its antibiotic resistance features Ninety-three S. enterica were isolated from the chicken carcasses; 36 of these isolates were serotyped and identified as S. Virchow (50.0%), S. Derby (14.0%), S. Livingstone (11.0%), S. Saintpaul (8.0%), S. Enteritidis (8.0%), S. Hadar (6.0%) and S. Agona (3.0%). In total, 86.1% of the identified isolates were resistant to tetracycline (Te), 80.5% were resistant to sulfamethoxazole (Sxt), and 33.3% were resistant to ampicillin (Am). Gentamicin (Gm) inhibited the growth of all the isolates (Table 2). A total of 30.5% of the identified isolates were resistant to Am, Sxt and Te. Twenty isolates (55.5%) were resistant to both Sxt and Te. A total of 8.3% of the isolates were sensitive to all the tested antibiotics.

S. Virchow demonstrated resistance to Sxt and Te at a rate of 72.2% but exhibited resistance to all three antimicrobial agents (Am, Sxt, Te) at a frequency of 16.7% (Table 3). The identified isolates contained gene sequences encoding the following types of tetracycline resistance: tetA (55.6%), tetB (91.7%) and tetG (0.3%). All the isolates lacked pse-1; however, 47.2% exhibited the sequences ant (3″)-Ia, and 16.7% contained the sequence sul-1 (Table 2). The multi-resistance pattern ant (3″)-Ia/sul-1/tetA + tetB was detected in 11.1% of the following serovars: S. Virchow, S. Livingstone and S. Saintpaul. Moreover, S. Virchow presented a similar multiresistance pattern, ant (3″)-Ia/sul-1/tetA. Nine isolates exhibited a multi-resistance pattern of ant (3″)-Ia/tetA + tetB (S. Virchow, S. Derby, S. Livingstone and S. Hadar). Only S. Agona presented the multi-resistance pattern ant (3″)-Ia/sul-1/tetA + tetB + tetG. Nearly 6.0% of the isolates did not contain the gene sequences under investigation. 3.2. Isolation of S. enterica from quail carcasses and its antibiotic resistance features Thirty-seven Salmonella isolates belonging to the serotypes S. Typhimurium (48.7%), S. Braenderup (10.8%), S. Putten (10.8%), S. Isangi (8.1%), S. Blockley (5.4%), S. Heidelberg (5.4%), S. Anatum (2.7%), S. Bredeney (2.7%), S. Enteritidis (2.7%) and S. Thompson (2.7%) were isolated from quail carcasses. The isolates exhibited resistance to Sxt (89.2%), Te (86.5%) and Am (81.1%). Gm inhibited the growth of 27.0% of the isolates (Table 4). A total of 24.3% of the identified isolates exhibited resistance to Am, Gm, Sxt and Te; eight of these isolates were S. Typhimurium (22.0%), and one was S. Isangi (2.7%). Sixteen isolates (43.2%) belonging to serovars S. Typhimurium, S. Braenderup, S. Putten, S. Isangi, S. Blockley, S. Heidelberg and S. Enteritidis displayed simultaneous resistance to Am, Sxt and Te. Only one S. Braenderup was sensitive to the tested antibiotics (Table 5). The gene sequence pse-1 was absent in 70.3% of the isolates. In particular, S. Anatum did not exhibit any of the gene sequences investigated. A total of 94.6% of the isolates possessed at least one gene of the tet class, 67.5% had the gene sequence ant (3″)-Ia and 43.2% contained the gene fragment sul-1 (Table 4). A genotypic profile was completed for ten isolates (27.0%). Among these isolates, S. Typhimurium exhibited the following gene sequences: pse-1/ant(3″)-Ia/sul-1/tetG and pse-1/ant(3″)-Ia/sul-1/tetA + tetG; the latter sequence was also present in one S. Braenderup. The genotypic pattern ant (3″)-Ia/sul-1/tetA and pse-1/ant (3″)-Ia/tetG was exhibited by six S. Typhimurium. One S. Putten presented the multi-resistance pattern ant(3″)-Ia/sul-1/tetA+ tetB (Table 5).

Table 2 Antibiotic resistance and resistance genes in S. enterica isolated from chicken carcasses.

Serovar

Number of isolates

Ampicillin Am

Gentamicin

pse- 1

Gm

ant (3’’)- Ia

Sulfamethoxazole

Tetracycline

Sxt

sul-1

Te

tetA

tetB

tetG

18

3

−

−

9

16

2

16

12

15

−

S. Derby

5

4

−

−

1

5

−

5

1

5

−

S. Livingstone

4

1

−

−

3

3

1

3

2

3

−

S. Saintpaul

3

1

−

−

2

1

2

1

2

3

−

S. Enteritidis

3

2

−

−

−

1

−

3

1

3

−

S. Hadar

2

-

−

−

1

2

−

2

1

2

−

1

1

−

−

1

1

1

1

1

36

12 (33.3%)

−

−

17 (47.2%)

S. Virchow

S. Agona Total (%)

Antibiotic resistance

Resistance genes

29 (80.5%)

6 (16.7%)

1 31 (86.1%)

1 20 (55.6%)

33 (91.7%)

1 (0.3%)

C. Bacci et al. / International Journal of Food Microbiology 160 (2012) 16–23

19

Table 3 Antibiotic pattern (resistance phenotype) and gene pattern (resistance genotype) in S. enterica isolated from chicken carcasses.

Serovar

Antibiotics pattern

Number of isolates

Am/Sxt/Te S. Virchow

3

Sxt/Te

13

Sensitive

S. Derby

S. Livingstone

Genes pattern

2

Number of isolates

ant (3’’) -Ia/sul-1/tetA+tetB

1

ant (3’’) -Ia/sul-1/tetA

1

ant (3’’) -Ia/tetA+tetB

6

ant (3’’) -Ia

1

tetA+tetB

4

tetB

4

Absent

1

Am/Sxt/Te

4

ant (3’’) -Ia/tetA+tetB

1

Sxt/Te

1

tetB

4

ant (3’’) -Ia/sul-1/tetA+tetB

1

ant (3’’) -Ia/tetA+tetB

1

ant (3’’) -Ia /tetB

1

Am/Sxt/Te

1

Sxt/Te

2

Sensitive

1

Am/Sxt/Te

Absent

1

1

ant (3’’) -Ia/sul-1/tetA+tetB

2

Sxt/Te

2

tetB

1

Am/Sxt/Te

1

Am/Te

1

tetA+tetB

1

Te

1

tetB

2

S. Hadar

Sxt/Te

2

ant (3’’) -Ia /tetA+tetB

1

tetB

1

S. Agona

Am/Sxt/Te

1

S. Saintpaul

S. Enteritidis

Total

ant (3’’) -Ia /sul-1/tetA+tetB+ tetG

1

36

36

3.3. Isolation of S. enterica from samples of chicken meat and its antibiotic resistance features The fifty isolates from the chicken meat samples belonged to the following serotypes: S. Enteritidis (34.0%), S. Virchow (24.0%), S. Livingstone (8.0%), S. Braenderup (6.0%), S. Putten (6.0%), S. Saintpaul (6.0%), S. Hadar (4.0%), S. Blockley (4.0%), S. Thompson (2.0%), S. Heidelberg (2.0%), S.

enterica subsp. enterica (the serotype of the subspecies could not be determined) (2.0%) and S. Typhimurium (2.0%). The isolates were most frequently resistant to Te (60.0%), followed by Sxt (50.0%) and Am (50.0%); all the Salmonella were sensitive to Gm (Table 6). Thirty-six percent of the isolates, including two S. Enteritidis and three S. Virchow, exhibited multiple resistance to Am, Sxt and Te; one S. Typhimurium and two S. Hadar displayed the same resistance

Table 4 Antibiotic resistance and resistance genes in S. enterica isolated from quail carcasses.

Serovar

Number of isolates Am

pse-1

Sxt

sul-1

S. Typhimurium

18

17

10

8

18

18

14

15

10

−

10

S. Braenderup

4

3

1

−

1

3

1

3

2

1

1

S. Putten

4

1

−

−

3

3

1

4

1

4

−

S. Isangi

3

3

−

1

2

3

−

2

3

−

−

S. Blockley

2

2

−

1

−

1

−

2

2

−

−

S. Heidelberg

2

2

−

−

1

2

−

2

1

2

−

S. Anatum

1

1

−

−

−

-

−

1

−

-

−

S. Bredeney

1

-

−

−

−

1

−

1

−

1

−

S. Enteritidis

1

1

−

−

−

1

−

1

1

1

−

S. Thompson

1

−

−

−

−

1

−

1

−

1

−

Total (%)

37

Gentamicin

Ampicillin

30 (81.1%) Antibiotic resistance

11 (29.7%) Resistance genes

Gm

10 (27.0%)

ant (3’’) - Ia

25 (67.5%)

Sulfamethoxazole

33 (89.2%)

16 (43.2%)

Tetracycline

Te

32 (86.5%)

tetA

20 (54.0%)

tetB

10 (27.0%)

tetG

11 (29.7%)

20

C. Bacci et al. / International Journal of Food Microbiology 160 (2012) 16–23

Table 5 Antibiotic pattern (resistance phenotype) and gene pattern (resistance genotype) in S. enterica isolated from quail carcasses.

Serovar

S. Typhimurium

S. Braenderup

Antibiotics pattern

Number of isolates

Genes pattern

Number of isolates

pse-1/ant (3’’)-Ia/sul-1/tetG

7

pse-1/ant (3’’)-Ia/sul-1/tetA+ tetG

2

ant (3’’)-Ia /sul-1/tetA

5

pse -1/ant (3’’)-Ia/tetG

1

ant (3’’) -Ia /tetA

3

Am/Gm/Sxt/Te

8

Am/Sxt/Te

7

Am/Sxt

2

Sxt

1

pse-1/ant (3’’)-Ia/sul-1/tetA+ tetG

1

Am/Sxt/Te

3

tetA

1

Sensitive

1

tetB

1

Absent

1

ant (3’’)-Ia /tetB

2

ant (3’’)-Ia/sul-1/tetA+ tetB

1

tetB

1

ant (3’’)-Ia/tetA

2

tetA

1

tetA

2

ant (3’’)-Ia/tetA+tetB

1

tetB

1

Am/Sxt/Te

1

Sxt/Te

3

Am/Gm/Sxt/Te

1

Am/Sxt/Te

1

Am/Sxt

1

Am/Gm/Te

1

Am/Sxt/Te

1

S. Heidelberg

Am/Sxt/Te

2

S. Anatum

Am/Te

1

Absent

1

S. Bredeney

Sxt/Te

1

tetB

1

S. Enteritidis

Am/Sxt/Te

1

tetA+tetB

1

S. Thompson

Sxt/Te

1

tetB

S. Putten

S. Isangi

S. Blockley

Total

1

37

37

Table 6 Antibiotic resistance and resistance genes in S. enterica isolated from chicken meat.

Serovar

Number of isolates

Ampicillin

Sxt

sul-1

tetA

tetB

tetG

S. Enteritidis

17

4

1

−

2

4

2

9

1

4

1

S. Virchow

Am

Gentamicin

pse-1

Gm

ant (3’’) - Ia

Sulf amethoxazole

Tetracycline Te

12

6

−

−

1

4

1

3

3

1

−

S. Livingstone

4

2

−

−

−

3

−

3

2

−

−

S. Braenderup

3

2

−

−

1

2

1

2

1

−

−

S. Putten

3

1

−

−

1

3

1

3

2

1

−

S. Saintpaul

3

3

−

−

−

2

1

2

2

−

−

S. Hadar

2

2

−

−

−

2

1

2

2

−

1

S. Blockley

2

1

−

−

1

1

1

2

2

1

−

S. Thompson

1

1

−

−

−

1

−

1

−

−

−

S. Heidelberg

1

1

−

−

−

1

1

1

1

−

−

S. enterica serotype not determinable

1

1

−

−

−

1

−

1

1

−

1

S. Typhimurium

1

−

Total (%)

50

Antibiotic resistance

1

−

−

−

1

−

1

−

1

25

1

−

6

25

9

30

17

8

(50.0%)

(2.0%)

Resistance genes

(0.0%)

(12.0%)

(50.0%)

(18.0%)

(60.0%)

(34.0%)

(16.0%)

3 (6.0%)

C. Bacci et al. / International Journal of Food Microbiology 160 (2012) 16–23

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Table 7 Antibiotic pattern (resistance phenotype) and gene pattern (resistance genotype) in S. enterica isolated from chicken meat.

Serovar

Antibiotics pattern

Number of isolates

Am/Sxt/Te

2

Am/Te

2

Sxt/Te

1

S. Enteritidis Sxt

1

Te

4

Sensitive

7

Am/Sxt/Te

3

Am/Te

1

Genes pattern pse-1/ant (3’’)-Ia/sul-1/tetG

1

ant (3’’)-Ia/sul-1

1

tetA

1

tetB

S. Virchow

S. Livingstone

Am

2

Sxt

1

Sensitive

5

Am/Sxt/Te

1

Sxt/Te

2

Number of isolates

Absent

4 10

ant (3’’)-Ia/sul-1/tetA

1

tetA

2

tetB

1

Absent

8

tetA

2

Absent

2

Am

1

Am/Sxt/Te

2

ant (3’’) -1a/ sul-1/tetA

1

Sensitive

1

Absent

2

Am/Sxt/Te

1

tetA

2

Sxt/Te

2

ant (3’’) -Ia/sul-1

1

Am/Sxt/Te

2

sul-1/tetA

1

tetA

1

Absent

1

sul-1/tetA

1

tetA+tetG

1

S. Braenderup

S. Putten

S. Saintpaul Am

S. Hadar

1

Am/Sxt/Te

2

Am/Sxt/Te

1

ant (3’’) -Ia/sul-1/tetA+tetB

1

Te

1

tetA

1

S. Thompson

Am/Sxt/Te

1

Absent

1

S. Heidelberg

Am/Sxt/Te

1

sul-1/tetA

1

S. Blockley

S. enterica serotype not determinable

Am/Sxt/Te

1

tetA+tetG

1

S. Typhimurium

Am/Sxt/Te

1

tetB

1

Total

50

pattern (sul-1/tetA). A total of 26.0% of the isolates were sensitive to all the tested antibiotics (Table 7). The sequence pse-1 was absent in 98.0% of the isolates. Half of the serovars possessed at least one gene of the tet class: specifically, the genes tetA (34.0%), tetB (16.0%) and tetG (6.0%) (Table 6). Only one S. Enteritidis contained the complete genotypic pattern pse-1/ant(3″)-Ia/sul-1/tetG. One S. Blockley exhibited the multiresistance pattern ant(3″)-Ia/sul-1/tetA + tetB. One S. Virchow and one S. Braenderup possessed the multi-resistance pattern ant(3″)-Ia/ sul-1/tetA. Nearly 50.0% of the isolates contained none of the investigated gene sequences (Table 7).

4. Discussion The European data from 2008 highlight a low level of resistance in Salmonella isolated from chicken to the antibiotics tested in our survey; however, the Italian data included in the same report are much more similar to the outcomes of our survey (European Food Safety Authority, 2010).

50

In our study, the most frequently isolated serotypes were S. Virchow (24.4%), S. Enteritidis (17.1%) and S. Typhimurium (15.4%); similarly, in a national survey conducted by the Italian Reference Centre for Salmonellosis, S. Enteritidis was present in chicken carcasses and S. Typhimurium was present in quail carcasses and chicken meat (Enter-Vet, 2009). The resistance phenotype to Am, Sxt and Te is exhibited in the majority of the isolates from the three types of samples. Resistance toward Gm is evident only in the isolates from quail carcasses. Resistance toward Am, Sxt and Te is clearly present in S. Typhimurium in the isolates from both quail carcasses and chicken meat. A study of S. Typhimurium isolated from chicken conducted by Glenn et al. (2011) demonstrated that a large number of isolates were resistant toward Am, Sxt and Te. According to other researchers, large percentages of S. Typhimurium were resistant to multiple antibiotics in different types of samples (Yang et al., 2001; Ahmed and Shimamoto, 2012). In our study, 19% of the serovar S. Enteritidis isolated from the sample sets displayed resistance to the three antibiotics (Am, Sxt and Te); this result has also been reported by other authors (Dias De Olivera et al., 2005; Hur et al., 2011). Ahmed and Shimamoto (2012) found that a large number of isolates were resistant to Am, Sxt and Te in S. Enteritidis isolated

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from diseased broilers. In our study, the S. Virchow isolated from chicken carcasses and meat was resistant to Sxt and Te (Tables 2, 4, 6). The Salmonella isolates from chicken meat were phenotypically more resistant to the tested antimicrobials than has been reported in the national data; an equal level of sensitivity was demonstrated only to Gm (Enter-Vet, 2009). Regarding the resistance phenotype, data obtained in the present study are similar to those reported by Miko et al. (2005) for S. enterica isolated from poultry meat and other species. More specifically, in the same study, a small number of isolates were resistant to Gm, and high percentages of resistance to Am (73.0%), Sxt (92.5%) and Te (80.9%) were detected. Complete resistance (Am, Gm, Sxt and Te) was exhibited only by the isolates from quail carcasses. Several authors have observed multiple drug resistance in isolates from poultry carcasses and meat (Capita et al., 2007; Dias De Olivera et al., 2005; Hur et al., 2011; Romani et al., 2008; Yang et al., 2001; Yildirim et al., 2011). Forty-five of the 123 identified isolates exhibited resistance to three antibiotics (Am, Sxt and Te); the majority of the multidrug-resistant S. enterica were isolated from chicken meat (40.0%). Seventeen S. enterica exhibited sensitivity to the tested antibiotics; of these isolates, 13 were isolated from chicken meat (Tables 3, 5, 7). The high levels of resistant isolates reported in many publications may be due to the worldwide overuse of antimicrobials in different fields, which has placed enormous pressure on the selection of antimicrobial resistance among bacterial pathogens and endogenous microflora (Capita et al., 2007). The gene sequence analysis revealed that although pse-1 is absent in all the isolates from chicken carcasses, a low percentage was present in the S. enterica isolated from both quail carcasses and chicken meat; furthermore, pse-1 was primarily found in S. Typhimurium. The gene sequence sul-1 was present in 16 isolates, particularly in 14 S. Typhimurium isolated from quail carcasses. Similarly, a high presence of the genes sul-1 and pse-1 was detected in S. Typhimurium isolates by Glenn et al. (2011). A higher percentage of the gene ant(3″)-Ia was detected in Salmonella spp. isolated from quail and chicken carcasses than in those isolated from chicken meat. The genes tetA and tetB were more frequently found in Salmonella spp. isolated from chicken meat and carcasses, respectively. Moreover, a study on S. enterica isolated from poultry meat (Miko et al., 2005) reported a major presence of the gene tetA compared to tetB and tetG. Similarly, Glenn et al. (2011) and Ahmed and Shimamoto (2012) detected a major presence of tetA in Salmonella spp. isolated from chicken carcasses and diseased broilers, respectively. In Salmonella spp. isolated from quail carcasses, the sequences of the tet class presented a more homogeneous distribution. The homogeneity of these results could be related to the presence of only one tet gene class that is differently located in the bacterial DNA. It is known that the tetA and tetB genes are located inside non-conjugative transposons (Tn 1721 of 11.1 Kbp and Tn 10 of 9.1 Kbp), which are important instruments for the horizontal transfer of antibiotic resistance (Waturangi et al., 2003). In the present survey, the genes belonging to the tet class were absent in only two Salmonella spp.: those isolated from quail carcasses (S. Anatum) and chicken meat (S. Thompson). The only serovar (S. Agona) that contained the complete gene sequence of the tet class was detected in chicken carcasses. The tet class genes are, in fact, considered by many researchers to be the most common types in Gramnegative bacteria (Ng et al., 2001). The phenotype of these isolates is influenced by both specific and non-specific resistance mechanisms such as lower membrane permeability and a high active efflux (Brindani et al., 2006; Putman et al., 2000; Quintiliani et al., 1999). In the isolates from the investigated sample set, tet class genes were generally expressed phenotypically, highlighting the involvement of a specific resistance mechanism. Similarly, Hur et al. (2011) and Ahmed and Shimamoto (2012) identified a correlation between the presence of the resistance gene tetA and its phenotype.

The gene sequence ant(3″)-Ia was partially expressed phenotypically in the isolates from quail carcasses, and it was not expressed phenotypically in the isolates from chicken meat and carcasses. Therefore, using only the biomolecular technique for the study of antibiotic resistance is restrictive. It must also be noted that a combination of methods is required to determine the relationships among the isolates, as suggested by Capita et al. (2007). The gene sequences pse-1 and sul-1, which were detected in only 9.76% and in 25.20% of the isolates, respectively, are not correlated with the resistance phenotype to Am (54.47%) and to Sxt (73.17%) in the isolates. This lack of correlation between the resistance phenotype (to Am and Sxt) and the effective presence of genes (pse-1 and sul-1) indicate the involvement of a non-specific resistance mechanism. The lack of correlation between antibiotic resistance and the expression of the related genes has also been highlighted in a study conducted in Germany (Miko et al., 2005) in S. enterica isolated from food. Furthermore, the results of the present study of S. Saintpaul serovar are similar to those reported by Beutlich et al. (2010) on the resistance phenotype to Am and Sxt and the presence of genes pse-1 and sul-1. It is understood that S. enterica may be a valid indicator to assess the prevalence of antimicrobial resistance (Antunes et al., 2006; Briggs and Fratamico, 1999; Diarrassouba et al., 2007; Yang et al., 2001; Yildirim et al., 2011) and that the monitoring of resistant strains might be considered a risk-based first step (SANCO, 2009). This study has focused its attention on two relevant aspects of the phenomenon of antibiotic resistance in S. enterica isolates. The first aspect is concerned with the correlation between a resistance phenotype and the presence of the related genes, which is partially displayed. This also confirms the importance of the involvement of non-specific resistance mechanisms and, therefore, the need for the simultaneous application of different qualitative techniques to identify antimicrobial resistance mechanisms. Therefore, although it is not always correlated with the resistance phenotype, the presence of gene sequences clearly indicates that S. enterica represents a source of the genetic determinants of resistance that is most likely transmissible to closely related bacteria and potentially to other microorganisms. The second aspect focuses on antibiotic resistance, which was particularly evident in the isolates from quail carcasses in both the presence of a complete antimicrobial resistance pattern (Am, Gm, Sxt and Te) and the low number of sensitive isolates. A large number of S. enterica isolates derived from the poultry industry (meat and chicken carcasses and quail carcasses) presented a resistance phenotype that is most likely related to the variety of antibiotics therapeutically administered in veterinary practice. In conclusion, this study confirms the role of animal-based foods as a reservoir of multidrug-resistant Salmonella and underscores the need for continuing surveillance of food-borne zoonotic pathogens along the food chain.

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