Phenotypic and Genotypic Characterization of Salmonella enterica Recovered from Poultry Meat in Tunisia and Identification of New Genetic Traits

Abstract

Thirty-seven Salmonella enterica isolates obtained from poultry meat in Tunisia were included in this study for characterization of antibiotic resistance mechanisms. High percentages of resistance were detected to ampicillin, sulfonamides, tetracycline, nalidixic acid, and streptomycin (32.4%–89.2%), and lower percentages to amoxicillin–clavulanic acid, kanamycin, amikacin, trimethoprim–sulfamethoxazol, and chloramphenicol (2.7%–18.9%). All strains showed susceptibility to ceftazidime, cefotaxime, gentamicin, and ciprofloxacin. Class 1 integrons were detected in 30% of Salmonella isolates, and four different gene cassette arrangements were detected, including genes implicated in resistance to aminoglycosides (aadA1 and aadA2) and trimethoprim (dfrA1). Four different Pc variants (PcW, PcH1, PcH1_TTN-10, PcW_TGN-10) with inactive P2 have been found among these isolates. Integron-positive isolates were ascribed to eight different serotypes. A Salmonella Schwarzengrund isolate harbored a new class 1 integron containing the qacH-dfrA1b-aadA1b-catB2 gene cassette arrangement, with the very unusual PcH1_TTN-10 promoter, which has been registered in GenBank (accession no. HQ874651). Different plasmid replicon types were demonstrated among integron-positive isolates: IncI1 (8 isolates), IncN (8), IncP (2), IncFIB (2), and IncFII (2). Ten different pulsed-field gel electrophoresis profiles were detected among the 11 integron-positive isolates and 8 different sequence types were identified by multilocus sequence typing, one of them (registered as ST867) was new, detected in 3 Salmonella Zanzibar isolates. A high diversity of clones is observed among poultry Salmonella isolates and a high proportion of them show a multiresistant phenotype with very diverse mobile genetic structures that could be implicated in bacterial dissemination in different environments.

Introduction

F ood-borne diseases caused by nontyphoidal Salmonella enterica serovars represent an important public health problem and an economic burden in many parts of the world (Miko et al. 2005, Alcaine et al. 2007). The main sources are foods of animal origin, such as eggs, milk, poultry, beef, and pork meat. In addition, fruits and vegetables have been incriminated as vehicles in Salmonella transmission.

In the last two decades, multidrug-resistant S. enterica isolates have been increasing and became a major health hazard (Butaye et al. 2006, Alcaine et al. 2007). The use of antimicrobial agents in food-producing animals for different purposes, including infection treatment, disease prevention, and also growth promotion (allowed in some countries), has been a major factor in widespread dissemination of antimicrobial-resistant bacteria that could be transferred to humans through the food chain (Angulo et al. 2004).

Molecular typing techniques such as pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) have been highly effective for epidemiological studies of S. enterica serotypes isolates from different origins (Hudson et al. 2001, Torpdahl et al. 2005, Seo et al. 2006).

Mobile genetic elements such as plasmids or transposons, which are able to disseminate antibiotic resistance genes by horizontal transfer, and integrons have been shown to play an important role in the evolution and dissemination of multidrug resistance in Gram-negative bacteria (Liebert et al. 1999, Carattoli 2001). An integron is a site-specific recombination system that recognizes, captures, and expresses gene cassettes (Hall 1997, Fluit and Schmitz 1999). Several classes of integrons have been described, and the class 1 is the most prevalent one. Gene cassettes are composed of a single coding promoterless sequence and a recombination site attC, located at the 3′ end of the gene, which is involved in the mobility of the gene cassettes. The expression of the gene cassettes included into the variable region of class 1 integrons is dependent of a Pc promoter located in the integrase intI1 gene. A second promoter P2 is active in some of these integrons. Differences in excision and expression level of gene cassettes have been attributed to the variations detected in the sequences of Pc and P2 promoters (Lévesque et al. 1994, Jové et al. 2010).

The aim of this study was to establish the antimicrobial susceptibility patterns of S. enterica isolates from poultry meat recovered in Tunisia and to characterize the integron structures and the plasmid content of these isolates. Further, the genotypic relatedness of the integron-positive S. enterica isolates was deeply investigated.

Materials and Methods

Bacterial isolates

Thirty-seven isolates of S. enterica were included in this study. Eighteen isolates were obtained after analyzing 665 samples of poultry meat taken from two slaughterhouses during the period 2007–2008, and the remaining 19 isolates were selected from the collection of the microbiology laboratory of the Veterinary Research Institute of Tunisia (10 isolates recovered during 2002–2007 and 9 during 2009 from poultry meat of different abattoirs). S. enterica colonies were confirmed by API 20E (Bio-Mérieux, Marcy l'Etoile, France). Serotyping was performed in integron-positive isolates according to the Kauffmann-White scheme (Popoff and Minor 2001).

Antimicrobial susceptibility testing

Antimicrobial susceptibility testing was conducted on Müeller-Hinton agar plates (Difco, Sparks, MD) by the agar disk diffusion method according to CLSI criteria (CLSI 2009). The following antimicrobial agents were tested (μg/disk): ampicillin (10), amoxicillin (25), amoxicillin–clavulanic acid (20/10), ceftazidime (30), cefotaxime (30), gentamicin (10), kanamycin (30), streptomycin (10), amikacin (30), trimethoprim–sulfamethoxazol (1.25/23.75), tetracycline (30), nalidixic acid (30), ciprofloxacin (5), sulfonamides (200), and chloramphenicol (50). Escherichia coli ATCC 25922 was used as a control strain. The double disk test with cefotaxime or ceftazidime in the proximity to amoxicillin–clavulanic acid was used for the screening of extended-spectrum beta-lactamases (ESBL).

Polymerase chain reaction analysis and DNA purification

The presence of genes associated with resistance to ampicillin (bla _PSE-1, bla _TEM), chloramphenicol (cmlA, floR), tetracycline [tet(A), tet(B), tet(C), and tet(G)], quinolones (qnrA, qnrB, qnrS, and qepA), aminoglycosides [aadA, aac(6′)-Ib, aph(3′)-Ia, strA, and strB], and sulfonamides (sul1, sul2, and sul3) were detected by polymerase chain reaction (PCR) and DNA sequencing (Sáenz et al. 2004, Riaño et al. 2006, Rocha-Gracia et al. 2010). The study of strA-strB genes and its possible linkage to sul2 gene was determined by PCR in all sul2-positive isolates (Vinué et al. 2010b, Soufi et al. 2011).

Study of the integron and characterization of the promoter of class 1 integron

The presence of the intI1 and intI2 genes and the 3′ conserved segment (3′-CS) of class 1 integron (qacEΔ1+sul1) was studied by PCR in the 37 isolates. The variable regions of class 1 integrons were analyzed by PCR and sequencing (Sáenz et al. 2004). The characterization of nonclassic class 1 integrons lacking the 3′-CS region and associated with the sul3 gene was carried out by a primer-walking strategy (Sáenz et al. 2010). The promoters (Pc), responsible for the expression of inserted gene cassettes, were studied by PCR and sequencing in the integron-positive S. enterica isolates (Vinué et al. 2010a).

Detection and classification of plasmids

Plasmids were classified according to their incompatibility group using the PCR replicon-typing method (Carattoli et al. 2005).

Clonal relationship and molecular typing in integron-positive Salmonella isolates

The clonal relationship among the integron-positive isolates was studied by PFGE (Sáenz et al. 2004) using 10 U of SpeI enzyme (New England Biolabs, Beverly, MA) for chromosomal DNA restriction. DNA patterns were interpreted as previously recommended (Tenover et al. 1995).

MLST was performed in the integron-positive S. enterica isolates to assign allelic profiles and a specific sequence type (ST). The thrA, purE, sucA, hisD, aroC, hemD, and dnaN housekeeping genes were studied by PCR and sequencing, and the obtained sequences were compared with those included in the website database (http://mlst.ucc.ie/mlst/dbs/Senterica).

Results and Discussion

Antimicrobial resistance phenotypes of Salmonella isolates

The susceptibility to 11 antimicrobial agents detected in the 37 S. enterica isolates is shown in Table 1. A high percentage of resistance was observed to ampicillin, sulfonamides, tetracycline, nalidixic acid, and streptomycin (32.4%–89.2%), as also reported in other studies (Chen et al. 2004, Zhao et al. 2005, Molla et al. 2007, Vo et al. 2010). Lower values of resistance were detected to amoxicillin–clavulanic acid, kanamycin, amikacin, trimethoprim–sulfamethoxazol, and chloramphenicol (2.7%–18.9%). All the studied isolates showed susceptibility to ceftazidime, cefotaxime, ciprofloxacin, or gentamicin. None of the isolates exhibited a positive ESBL phenotype. Twenty seven of the 37 Salmonella isolates (73%) showed a multiresistant phenotype (resistance to at least three different antimicrobial agent classes) that is frequent in S. enterica, as previously described (Chen et al. 2004, Randall et al. 2004, Molla et al. 2007, Vo et al. 2010). The detection of antimicrobial-resistant Salmonella in meat samples is of great concern, because they could be transmitted to humans through the food chain and could be implicated in human infections with a compromised treatment. The spread use of antimicrobial agents in therapy or prophylaxis or as growth promoters in poultry in Tunisia could be a factor for selection of resistant Salmonella that contaminate poultry meat.

Table 1.

Resistance to 11 Antimicrobial Agents in the 37 Salmonella enterica Strains Isolated from Poultry Meat

	Number (%) of resistant strains
Antimicrobial agents	Integron-positive (n=11)	Integron-negative (n=26)	Total (n=37)
Ampicillin	3 (27.3%)	9 (34.6%)	12 (32.4%)
Amoxicillin–clavulanic acid	1 (9.1%)	0	1 (2.7%)
Sulfonamides	11 (100%)	6 (23.1%)	17 (45.9%)
Streptomycin^a	11 (100%)	22 (84.6%)	33 (89.2%)
Kanamycin	0	4 (15.4%)	4 (10.8%)
Amikacin	4 (36.4%)	0	4 (10.8%)
Nalidixic acid	8 (72.7%)	18 (69.2%)	26 (70.3%)
Chloramphenicol	5 (45.4%)	2 (7.7%)	7 (18.9%)
Tetracycline	6 (54.5%)	15 (57.7%)	21 (56.8%)
Trimethoprim–sulfamethoxazol	5 (45.4%)	1 (3.8%)	6 (16.2%)

Streptomycin resistance and intermediate categories according to CLSI standards are considered.

Resistance genes in Salmonella isolates

Table 2 shows the resistance genes detected among the antimicrobial-resistant S. enterica isolates. Twelve ampicillin-resistant Salmonella isolates were detected, and 11 of them (92%) harbored a bla _TEM gene. Twelve of the 17 sulfonamide-resistant isolates contained a sul-type gene (70%), 5 of them harbored more than one sul gene, and the following combinations were found: sul1+sul3 (2), sul2+sul3 (2), and sul1+sul2+sul3 (1) (Table 2). Among the 33 streptomycin-resistant isolates, the strA-strB and aadA genes were found in 6 and 17 isolates, respectively, and both of them (strA-strB + aadA) were present in 6 isolates. The strA-strB gene pair and the aadA gene remain so far the most important mechanisms mediating streptomycin resistance in S. enterica. It is interesting to note that in two isolates, the sul2 gene was found linked to strA-strB gene presenting the structure repC-sul2-strA-strB, which is widely disseminated (Vinué et al. 2010b, Yau et al. 2010, Soufi et al. 2011).

Table 2.

Genes of Resistance Detected Among the Antimicrobial-Resistant Salmonella enterica Strains

	Genes detected by PCR (number of strains)
Phenotype of resistance (number of strains)	Integron-positive strains (n=11)	Integron-negative strains (n=26)
Ampicillin (12)	bla _TEM (3)	bla _TEM (8)
Sulfonamides (17)	sul1 (2) sul3 (4) sul1+sul3 (1) sul2+sul3 (2) sul1+sul2+sul3 (1)	sul1 (0) sul3 (6) sul1+sul3 (1)
Streptomycin (33)	strA-strB (0) aadA (8) strA-strB+aadA (3)	strA-strB (6) aadA (9) strA-strB+aadA (3)
Kanamycin (4)	aph(3′)-Ia (0)	aph(3′)-Ia (3)
Chloramphenicol (7)	cmlA (4)	cmlA (1)
Tetracycline (21)	tet(A) (5)	tet(A) (10)

PCR, polymerase chain reaction.

The aph(3′)-Ia gene was detected in three of the four kanamycin-resistant isolates, and the cmlA gene in five of the seven chloramphenicol-resistant isolates. Among the 21 tetracycline-resistant isolates, 15 of them carried the tet(A) gene, but tet(B) and tet(C) were not detected. The tet(A) gene associated with tetracycline efflux pumps was reported to be the predominant one in Salmonella and E. coli isolates from livestock and food animals (Miko et al. 2005, Jouini et al. 2009, Soufi et al. 2009). None of the plasmid-mediated quinolone-resistance genes (aac(6′)-Ib-cr, qnrA, qnrB, qnrS, or qepA genes) was detected among the 37 S. enterica isolates.

Integron detection and characterization of promoter of class 1 integrons

The presence of class 1 integrons was demonstrated in 11 of the 37 Salmonella isolates (30%), whereas no class 2 integrons were detected. Four different gene cassette arrangements were identified (Table 3). The genes implicated in resistance to aminoglycosides (aadA1 and aadA2) and trimethoprim (dfrA1) were the most frequently found, as previously reported (Guerra et al. 2000, Krauland et al. 2009, Vo et al. 2010). Only two isolates harbored the classical qacEΔ1-sul1 element at 3′-CS, and one of them was the only Salmonella Typhimurium strain (C4138) detected in this study. This strain C4138 showed the two integron patterns (bla _PSE-1, 1.0 kb and aadA2, 1.2 kb), and in addition, the presence of floR and tet(G) genes was demonstrated by PCR, indicative of the presence of Salmonella Genomic Island type 1 (Boyd et al. 2002, Carattoli et al. 2002, Miko et al. 2005, Vo et al. 2010). The other qacEΔ1-sul1–positive isolate is a Salmonella Schwarzengrund isolate (C3893) that harbored a new class 1 integron containing the qacH-dfrA1b-aadA1b-catB2 gene cassette arrangement. This new integron, numbered as In150 according to the INTEGRALL database (http://integrall.bio.ua.pt/?), has been included in GenBank with the accession no. HQ874651. It is important to highlight that the usual attC site of the aadA1b gene cassette is missing, which could affect its mobilization. This fact has been observed in other sequences included in GenBank (e.g., accession nos. GQ422827, FM877485, or AY987853) only when catB2 is the following gene cassette. Taking in account that the attC of catB2 gene cassette is complete and functional for recombination, an aadA1b::catB2 fusion of gene cassettes could be suggested.

Table 3.

Clonal Relationship, Integron Structures, and Plasmid Types Detected in the 11 Integron-Positive Salmonella enterica Strains Recovered from Poultry Meat

						Class 1 integrons
Strain	Serotype	Year	PFGE	MLST	Plasmid type	intI1	Promoter	qacEΔ1+sul1	Arrangements included into the variable region
C4135	Salmonella Enteritidis	2007	P1	ST11	IncI1, IncN	+	PcW	−	dfrA1-aadA1
C3893	Salmonella Schwarzengrund	2007	P2	ST96	IncI1, IncN, IncFIB, IncFII	+	PcH1_TTN-10	+	qacH-dfrA1b-aadA1b-catB2
C4136	Salmonella Saintpaul	2007	P3	ST27	IncI1, IncN, IncFIB, IncFII	+	PcW	−	dfrA1-aadA1
C4137	Salmonella Hadar	2008	P4	ST33	IncN	+	PcW_TGN-10	−	Unknown
C4138	Salmonella Typhimurium	2007	P5	ST19	IncI1, IncN	+	PcW_TGN-10 ^a	+	aadA2/bla _PSE-1
C4139	Salmonella Zanzibar	2007	P6	ST867	IncI1, IncN	+	PcH1	−	estX-psp-aadA2-cmlA1-aadA1-qacH-IS440-sul3-orf1-mef(B)Δ-IS26
C4140	Salmonella Zanzibar	2009	P6	ST867	IncN	+	PcH1	−	estX-psp-aadA2-cmlA1-aadA1-qacH-IS440-sul3-orf1-mef(B)Δ-IS26
C4141	Salmonella Norwich	2009	P7	ST64	IncI1, IncP	+	PcH1	−	estX-psp-aadA2-cmlA1-aadA1-qacH-IS440-sul3-orf1-mef(B)Δ-IS26
C4142	Salmonella Virchow	2009	P8	ST16	IncI1	+	PcH1	−	estX-psp-aadA2-cmlA1-aadA1-qacH-IS440-sul3-orf1-mef(B)Δ-IS26
C4143	Salmonella Enteritidis	2009	P9	ST11	IncI1	+	PcW	−	dfrA1-aadA1
C4144	Salmonella Zanzibar	2009	P10	ST867	IncN, IncP	+	PcH1	−	Unknown

PcW_TGN-10 variant was found in both integrons (aadA2/bla _PSE-1).

PFGE, pulsed-field gel electrophoresis; MLST, multilocus sequence typing.

Nine of the 11 integron-positive Salmonella isolates (82%) harbored a nonclassic class 1 integron (lacking the 3′-CS). The dfrA1-aadA1 gene cassette arrangement was found in three isolates (two Salmonella Enteritidis and one Salmonella Saintpaul), and the sul3 gene was associated with the long structure: estX-psp-aadA2-cmlA1-aadA1-qacH-IS440-sul3-orf1-mef(B)Δ-IS26 in other four isolates (two Salmonella Zanzibar, one Salmonella Norwich, and one Salmonella Virchow). These structures were previously identified in both Salmonella and E. coli strains (Antunes et al. 2007, Sáenz et al. 2010, Vinué et al. 2010b, Soufi et al. 2011). All PCRs performed to detect the gene cassettes behind the intI1 gene were negative in the remaining nonclassic class 1 integron-positive isolates.

Gene cassettes of class 1 integrons may be differently expressed depending on the Pc (located within the integrase gene sequence) and P2 promoter variants. The Pc and P2 variants of the 12 detected gene arrangements were studied in the 11 integron-positive Salmonella isolates. An inactive P2 and four different Pc variants (PcW, PcH1, PcH1_TTN-10, PcW_TGN-10) have been found among these isolates (Table 3). Neither PcH2 nor PcS promoter variants were found. The PcW and PcH1 promoters have weak and intermediate strength, respectively, and they are the most prevalent Pc variants among the reported class 1 integrons in a previous in silico study (Jové et al. 2010). However, the presence in PcW of a TGN-10 motif that refers to an extended −10 hexamer (TGgTAAGCT), increases PcW efficiency near PcH2 one (Jové et al. 2010). In our study, the PcW_TGN-10 variant was found in both integrons (aadA2/bla _PSE-1) of the Salmonella Typhimurium C4138 isolate. In addition, the PcH1_TTN-10 variant, characterized by the substitution of a T instead of C in the second base upstream of the −10 hexamer (TTgTAAACT), slightly increases the PcH1 efficiency (Jové et al. 2010). This extremely rare PcH1_TTN-10 promoter was found in the Salmonella Schwarzengrund strain that harbored the new qacH-dfrA1b-aadA1b-catB2 integron. The involvement of integrons in multiresistance in Salmonella isolates has been extensively studied and reported by other studies (Hall 1997, Guerra et al. 2000, Walker et al. 2001, Carattoli et al. 2002, Krauland et al. 2009).

Characterization of the plasmid types

Plasmids were studied in the 11 integron-positive S. enterica isolates and were classified according to their incompatibility groups. Five plasmid replicon types were identified and 7 of 11 isolates carried more than one different plasmid replicon (number of isolates): IncI1 (2), IncN (2), IncI1+ IncN (3), IncI1+ IncN+IncFIB+IncFII (2), IncI1+ IncP (1), and IncN+ IncP (1). The IncI1, prevalent among the 11 strains of Salmonella analyzed, has been already associated with antibiotic resistance (Hopkins et al. 2006, Hradecka et al. 2008). Moreover, IncN are host-range plasmids that have significantly contributed in the global spread of resistance genes, including those conferring resistance to quinolones, carbapenems, aminoglycosides, and trimethoprim. The occurrence of IncI1 and IncN plasmids among Salmonella isolates of various serotypes highlights the high ability of these plasmids to disseminate. Indeed, both replicons are widespread in Enterobacteriaceae and have been recently associated with highly disseminated β-lactamase genes in E. coli and Salmonella from food animals (Hopkins et al. 2006, García-Fernández et al. 2008, Moodley and Guardabassi 2009, Rodríguez et al. 2009, Cloeckaert et al. 2010).

Molecular typing of S. enterica isolates

All the 11 integron-positive Salmonella strains were analyzed by PFGE to study their genotypic relatedness. Digestion of Salmonella DNA with SpeI showed between 12 and 18 resolvable chromosome fragments ranging from ∼48.5 to 728 kb. Ten distinct PFGE patterns (P1–P10) were generated among these strains and two of them showed an indistinguishable profile corresponding to Salmonella Zanzibar serotype (Table 3). PFGE analysis can separate different Salmonella serotypes into distinct clusters (Liebana et al. 2002, Peters et al. 2003). PFGE has been shown to be an efficient molecular tool for discriminating among Salmonella serovars recovered in our study and also in other studies carried out in Tunisia (Abbassi-Ghozzi et al. 2010).

Eight different STs were identified among our 11 integron-positive S. enterica isolates by MLST (Table 3). A new ST was detected in three Salmonella Zanzibar isolates and was registered under the name ST867. The ST11 was identified in two Salmonella Enteritidis isolates and the remaining isolates were ascribed to unique STs. It is worthy to note that our results are, in part, in agreement with those reported by Torpdahl et al. (2005). Indeed, they found that most isolates with the same serotype had the same ST and, most importantly, that ST11, ST27, ST16, and ST33 were specific to Salmonella Enteritidis, Salmonella Saintpaul, Salmonella Virchow, and Salmonella Hadar serotypes, respectively.

It is interesting to remark the high genetic heterogeneity of our strains, both by PFGE and MLST techniques. Strains belonging to the same ST did not always cluster in a single profile based on PFGE analysis; this is what we observed for the three strains that belong to a same ST867 but with different pulsotypes. Therefore, PFGE and MLST are complementary methods, appropriate for studies at distinct scales, for example, local epidemiology versus global population structure, respectively (Vimont et al. 2008).

Our findings support the hypothesis that the versatility of plasmids and the exchange of integrons and resistance genes by vertical and horizontal gene transfer may have contributed to the spread of these antimicrobial resistance traits. A systematic search for integrons and transposable elements could provide a useful genetic basis for multidrug-resistance spread in human and animal Salmonella isolates.

Footnotes

Acknowledgments

The authors thank Dr. Thomas Jové for analyzing the new integron In150 (http://integrall.bio.ua.pt/?). The authors thank Dr. Latifa Khanfir for supplying some of the strains. This work was partially supported by AECID (A/019744/08) from the Ministry of Foreign Affairs of Spain and a grant from the Institution de la Recherche et de l'Enseignement Supérieur Agricoles (IRESA)/Groupement Interprofessionnel des Produits Avicoles et Cunicoles (GIPAC) (Tunisia). M. de Toro has a predoctoral fellowship from the Instituto de Salud Carlos III of Spain (Ministerio de Ciencia e Innovación; grant no.: FI08/00506).

Disclosure Statement

No competing financial interests exist.

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