Antimicrobial Drug Resistance and Molecular Typing of Salmonella enterica Serovar Rissen from Different Sources

Abstract

Salmonella enterica serovar Rissen is one of the most common serovars found in pigs and pork products in different countries, including Spain. However, information on the molecular bases of antimicrobial drug resistance and the population structure of Salmonella Rissen from different sources in Spain is limited. The present study focused on 84 isolates collected in Spain from pig and beef carcasses, foods and clinical samples associated with sporadic cases of gastroenteritis, and one outbreak. The majority of the isolates were resistant to tetracycline (73.8%), mainly conferred by tet(A). Resistances to streptomycin (aadA1-like, aadA2, and strAB), sulfonamides (sul1, sul2, and sul3), trimethoprim (dfrA1-like and dfrA12), ampicillin (bla_TEM-1-like), and chloramphenicol (cmlA1-like) were also detected, with frequencies ranging from 12% to 20.2%. Most of the identified genes were carried by integrons, including three class 1 integrons of the sul1 type, a class 1 integron of the sul3 type, and the class 2 integron of Tn7. Two sul1 integrons, the sul3 integron, and the class 2 integron are first reported in Salmonella Rissen. Typing of the isolates with XbaI pulsed-field gel electrophoresis detected a major clone, which was circulating in humans and animals during the past decade, and was responsible for the outbreak. The obtained results are relevant for food safety and public health.

Introduction

Nontyphoidal serovars of Salmonella enterica subsp. enterica are zoonotic pathogens that constitute a prevalent cause of food-borne illnesses worldwide.^14,27 To increase the level of animal and public health in the European Union, measures were adopted for an adequate control of zoonosis at the level of primary production, following Regulation (EC) No. 2160/2003 of the European Parliament and Council. In this context, baseline surveys on the prevalence of Salmonella were undertaken by different member states in slaughter pigs as well as in breeding and production pig holdings.^11,12 In Spain, these surveys revealed Salmonella enterica serovar Rissen as the second most common serovar in slaughter pigs and the most common serovar in both breeding and production pig holdings.^11,12 Additional studies confirm pig fattening units and finishing pigs in Spain as an important reservoir of Salmonella Rissen.^5,16,17,32 Apart from Spain, Salmonella Rissen is among the most common Salmonella serovars found in pigs and pork products in Portugal and several Southeast Asian countries, particularly Thailand.^{1,6,11,12,18,19,22,33} With a lesser frequency, Salmonella Rissen has also been recovered from other food-producing animals and derived products, namely beef and poultry, and from human clinical samples.^1–3,7,9

As for other pig-associated serovars, several investigations revealed a high level of resistance and multidrug resistance (MDR) in Salmonella Rissen recovered not only from pigs^5,32 but also from food and clinical sources in Spain.^5,29,31 However, data on the molecular bases of antimicrobial drug resistance are still scarce and, to the best of our knowledge, information on the population structure of this serovar in Spain is lacking.

A retrospective analysis of the clinical incidence of Salmonella Rissen in a Spanish region (Asturias) revealed a possible outbreak caused by this serovar from the middle of September to early October of 2011. In about a fortnight, 11 Salmonella Rissen isolates were recovered from 10 patients admitted to a local hospital with symptoms of gastroenteritis, while no other Salmonella Rissen was detected in the same hospital and year (data from the Laboratory of Public Health [LSP] of Asturias). This observation, together with the relevance of Salmonella Rissen in Spain and the limited information available for this serovar, prompted the present study, in which isolates collected during 2002–2011 from different sources in Asturias, were compared. Specifically, they derived from clinical samples (feces, urine, and blood), including the putative outbreak isolates, pig and bovine carcasses, pork-derived products, and pork and beef-derived products. All were characterized for antimicrobial susceptibility, and the genetic bases of the observed resistances were established with regard to genes and genetic elements involved, such as integrons and transposons. In addition, they were analyzed by pulsed-field gel electrophoresis (PFGE) to identify the genomic types, which are circulating in Asturias, and compare them with Salmonella Rissen from other countries.

Materials and Methods

Bacterial isolates

Eighty-four Salmonella Rissen isolates collected during the 2002–2011 period in Asturias were included in the study. Forty-nine out of the total 84 isolates were recovered from pork products or mixed pork and beef products (26 isolates), pig carcasses (18 isolates), and bovine carcasses (5 isolates) at the LSP of Asturias, which is the Salmonella Reference Center in the region. The remaining 35 isolates were obtained at different hospitals from human clinical samples associated with sporadic cases of salmonellosis (24 isolates; 23 from feces and 1 from urine) and with a possible outbreak (11 isolates; 9, 2, and 1 from feces, urine, and blood, respectively). They were the total clinical isolates submitted to the LSP during the indicated period, and each derived from a different patient, with the exception of two putative outbreak isolates that were collected from feces and urine of the same patient. All isolates were identified by biochemical methods and serotyped at the National Reference Center for Salmonella and Shigella (Centro Nacional de Microbiología, Madrid, Spain).

Antimicrobial susceptibility testing and detection of resistance genes

Antimicrobial susceptibility tests were performed by the disk diffusion method, using commercially available disks (Oxoid, Madrid, Spain) according to guidelines of the Clinical and Laboratory Standards Institute.¹⁰ Susceptibilities to ampicillin, apramycin, chloramphenicol, gentamicin, kanamycin, nalidixic acid, streptomycin, sulfonamides, tetracycline, and trimethoprim were tested in all isolates. Ampicillin-resistant isolates were also screened for susceptibility to amoxicillin–clavulanic acid, cefoxitin, and cefotaxime. Isolates resistant to three or more antimicrobials belonging to different classes were considered as MDR. In all resistant isolates, the responsible genes were identified by PCR amplification using primers and conditions previously reported.^15,20 The screened genes, selected according to the resistance phenotype, were as follows: bla_TEM-1-like, bla_OXA-1, and bla_PSE-1 (ampicillin resistance), aac(3)-IVa (apramycin resistance), catA1 and cmlA1-like (chloramphenicol resistance), aphA1 (kanamycin resistance), aadA1-like, aadA2, and strAB (streptomycin resistance), sul1, sul2, and sul3 (sulfonamides resistance), tet(A), tet(B), and tet(G) (tetracycline resistance), and dfrA1-like and dfrA12 (trimethoprim resistance). The genetic bases of nalidixic acid resistance were established by PCR amplification and sequencing of the quinolone resistance-determining region of the gyrA gene.²⁸ Sequencing was performed at Macrogen Europe (Amsterdam, Netherlands).

Integron and transposon analysis

Isolates positive for sul1, sul3, or both were tested for the presence of class 1 integrons by PCR amplification, using primers specific for the intI1 gene.¹⁵ Variable regions of sul1 integrons were amplified with the 5′ conserved segment (CS)-3′ CS primers,¹⁵ and the gene cassette(s) located therein were identified by nested PCR amplification with primer pairs selected according to the resistance genes carried by the corresponding isolates, followed by sequencing.²⁵ Characterization of sul3 integrons was performed as reported.¹⁵ Isolates positive for dfrA1-like were also screened for the variable region of class 2 integrons, using the hep74 and hep51 primers,³⁰ and the carried gene cassettes were identified by nested PCR amplification and sequencing.

Transposons Tn3 (tnpA), Tn7 (tnsECBA), Tn10 (tetR, tetC, tetD, and tnpA of IS10), Tn21 (tnpA, tnpR, tnpM, urf2, and merEDACPTR), and Tn1721 (tnpA and tetR) were screened for genes indicated in parenthesis^26,28,30 and in isolates suspected to contain one or more of them according to their resistance and integron profiles.

Pulsed-field gel electrophoresis

Relatedness between the Salmonella Rissen isolates was assessed by macrorestriction-PFGE using XbaI and BlnI endonucleases (30 U, 4 hr, 37°C; Takara, Torrejón de Ardoz, Madrid, Spain) according to the PulseNet protocol (www.pulsenetinternational.org). The electrophoresis was carried out in a CHEF DR III System (Bio-Rad Laboratories, Alcobendas, Madrid, Spain), also following PulseNet recommendations. Comparison of the PFGE profiles was accomplished with BioNumerics software version 6.6 (Applied Maths, Sint-Martens-Latem, Belgium) and the Dice coefficient of similarity (S), with 1% band position tolerance and optimization at 1.5%. Simpson's index of diversity was calculated according to Hunter and Gastón.²⁴

Results

Antimicrobial susceptibility and identification of resistance genes

Only 18 out of the 84 isolates (21.4%) were susceptible to all antimicrobials tested, while 51.2% (43 isolates), 7.1% (6 isolates), and 20.2% (17 isolates) were resistant to one, two, and three up to seven unrelated antimicrobials, the latter being considered as MDR (Table 1). With regard to individual antimicrobials, 73.8% (62 isolates), 20.2% (17 isolates), 19% (16 isolates), 17.9% (15 isolates), 17.9% (15 isolates), 12% (10 isolates), 3.6% (3 isolates), 2.4% (2 isolates), and 1.2% (1 isolates) of the isolates were resistant to tetracycline, streptomycin, sulfonamides, trimethoprim, ampicillin, chloramphenicol, nalidixic acid, kanamycin, and apramycin, respectively. None of the isolates was resistant to gentamicin, amoxicillin–clavulanic acid, cefoxitin, or cefotaxime.

Table 1.

Resistance Properties and Pulsed-Field Electrophoresis Profiles of Salmonella enterica Serovar Rissen from Different Origins

R-profile (N)	R-phenotype	R-genotype	In-profile	Tn-profile	XB-profile (N)	Origin (N)^a
R0 (18)	Susceptible	—	—	—	X1B1 (2), X1B2 (12), X1B4 (2), X1B21 (1), X7B23 (1)	H (4), F (14)
R1 (1)	NAL	gyrA _{Asp87 to Asn}	—	—	X1B2 (1)	H (1)
R2 (37)	TET	tet(A)	—	Tn1721	X1B1 (28), X1B2 (1), X1B5 (1), X1B7 (1), X1B11 (2), X4B9 (2), X4B12 (1), X8B1 (1)	H (10), H (12), F (4), BC (4), PC (8)
R3 (4)	TET	tet(A) tet(B)	—	Tn1721–ΔTn10	X1B1 (4)	H (2), F (1), BC (1)
R4 (1)	AMP	bla _TEM-1	—	Tn3	X1B13 (1)	F (1)
R5 (2)	NAL TET	gyrA_{Asp87 to Asn}[tet(A) tet(B)]	—	Tn1721–ΔTn10	X1B1 (2)	PC (2)
R6 (4)	AMP TET	bla_TEM-1 tet(A)	—	Tn3–Tn1721	X1B1 (2), X1B6 (2)	H (2), PC (2)
R7 (1)	CHL STR SUL	cmlA [aadA1-like aadA2] sul3	In-sul3^b		X3B18 (1)	H (1)
R8 (1)	AMP APR STR TET	bla_TEM-1 aac(3)-IVa strAB tet(A)	—	Tn3–Tn1721	X1B5 (1)	H (1)
R9 (2)	STR SUL TET TMP	aadA1-like sul1 tet(A) dfrA12	1000 bp/aadA	Tn1721	X1B14 (1), X5B16 (1)	F (2)
R10 (1)	STR SUL TET TMP	[aadA1-like aadA2] sul1 tet(A) dfrA12	2000 bp/dfrA12-orfF-aadA2	Tn1721	X8B22 (1)	H (1)
R11 (1)	AMP STR SUL TET TMP	bla_TEM-1 aadA1-like sul1 tet(A) dfrA12	1000 bp/aadA1	Tn3–Tn1721	X6B15 (1)	F (1)
R12 (1)	AMP STR SUL TET TMP	bla_TEM-1 [aadA1-like aadA2] sul1 tet(A) dfrA12	2000 bp/dfrA12-orfF-aadA2	Tn3–Tn1721	X2B7 (1)	H (1)
R13 (1)	AMP STR SUL TET TMP	bla_TEM-1 [aadA1-like aadA2 strAB] [sul1 sul2] tet(B) [dfrA1-like dfrA12]	2000 bp/dfrA12-orfF-aadA2;	Tn3–ΔTn10	X9B17 (1)	F (1)
			2300 bp/dfrA1-sat2-aadA1	Tn7
R14 (2)	CHL STR SUL TET TMP	cmlA [aadA1-like aadA2] sul3 tet(A) dfrA12	In-sul3^b	Tn1721	X1B10 (2)	F (2)
R15 (1)	CHL STR SUL TET TMP	cmlA [aadA1-like aadA2] [sul1 sul3] tet(A) [dfrA1-like dfrA12]	1600/dfrA1-aadA1; In-sul3^b	Tn1721	X3B20 (1)	H (1)
R16 (1)	AMP CHL STR SUL TMP	bla_TEM-1 cmlA [aadA1-like aadA2] [sul1 sul3] [dfrA1-like dfrA12]	1600/dfrA1-aadA1; In-sul3^b	Tn3–Tn1721	X3B3 (1)	PC (1)
R17 (1)	AMP CHL STR SUL TET TMP	bla_TEM-1 cmlA [aadA1-like aadA2] sul3 tet(A) dfrA12	In-sul3^b	Tn3–Tn1721	X3B3 (1)	PC (1)
R18 (2)	AMP CHL STR SUL TET TMP	bla_TEM-1 cmlA [aadA1-like aadA2] [sul1 sul3] tet(A) [dfrA1-like dfrA12]	1600/dfrA1-aadA1; In-sul3^b	Tn3–Tn1721	X3B3 (1), X3B19 (1)	PC (2)
R19 (2)	AMP CHL KAN STR SUL TET TMP	bla_TEM-1 cmlA aphA1 [aadA1-like aadA2] [sul1 sul3] tet(A) [dfrA1-like dfrA12]	1600/dfrA1-aadA1; In-sul3^b	Tn3–Tn1721	X3B8 (2)	PC (2)

Outbreak isolates are highlighted in bold.

dfrA12-orfF-aadA2-cmlA-aadA1-qacH-IS440 was the gene array of the sul3 integron.

AMP, ampicillin; APR, apramycin; BC, beef carcass; CHL, chloramphenicol; F, pork- or pork- and beef-derived food product; H, human; In, integron; KAN, kanamycin; N, number of isolates; NAL, nalidixic acid; PC, pig carcass; R, resistance; STR, streptomycin; SUL, sulfonamides; TET, tetracycline; TMP, trimethoprim; Tn, transposon; XB, XbaI-BlnI pulsed-field electrophoresis profiles.

As shown in Table 1, amplicons specific for tet(A) were obtained in 61 out of the 62 tetracycline-resistant isolates. These isolates originated from pig carcasses and beef carcasses (22), food products (10), as well as clinical samples associated with sporadic cases of salmonellosis (18) and with the putative outbreak (11). A single isolate carried tet(B), whereas this gene was found together with tet(A) in six isolates. All except one of the streptomycin-resistant isolates yielded the amplicons expected for aadA1-like (3 isolates) or aadA1-like plus aadA2 (13 isolates), and the strAB genes were also detected in one of the latter. The remaining isolate was only positive for strAB. Resistance to sulfonamides was due to sul1, sul3, or both, which were detected in 5, 4, and 6 of the total 84 isolates, while a single isolate recovered from a pork product carried sul1 plus sul2. All trimethoprim-resistant isolates (15) yielded the amplicon expected for dfrA12 and 7 of them were also positive for dfrA1-like. Resistance to ampicillin, apramycin, kanamycin, and chloramphenicol was always conferred by the bla_TEM-1-like, aac(3)-IVa, aphA1, and cmlA1-like genes, respectively. A single base-pair substitution in the gyrA gene, which changes codon 87 from GAC (Asp) to AAC (Asn), was identified in the three nalidixic acid-resistant isolates detected. All putative outbreak isolates carried tet(A), and one of them (LSP 189/11) also produced the amplicons expected for bla_TEM-1, aadA2, sul1, and dfrA12.

On the basis of resistance phenotypes and genotypes, a total of 19 profiles were identified, which were termed with “R” followed by a serial number (Table 1).

Integrons and transposons

The intI1 gene of class 1 integrons was detected in all isolates carrying sul1 and/or sul3, which were 33.3%, 23.1%, and 11.4% of those recovered from pig carcasses, food products, and clinical samples, respectively (Table 1). Among the sul1-positive isolates, three variable regions were observed, with gene cassettes encoding resistance to aminoglycosides and/or trimethoprim: 1000 bp/aadA1 (isolates from foods), 1600 bp/dfrA1-aadA1 (from pig carcasses and a clinical sample), and 2000 bp/dfrA12-orfF-aadA2 (from food and humans). These isolates were also positive for the qacEΔ1 gene found together with sul1 in the 3′ CS of conventional class 1 integrons. In all sul3-positive isolates, the gene array dfrA12-orfF-aadA2-cmlA-aadA1-qacH-IS440 was detected. Interestingly, the 1600 bp/dfrA1-aadA1 sul1 integron always coincided with the sul3 integron. The food isolate with the 2000 bp/dfrA12-orfF-aadA2 conventional class 1 integron was also positive for the class 2 integron of Tn7, with the 2300 bp/dfrA1-sat2-aadA1 gene array.

As shown in Table 1, all tetracycline-resistant isolates carrying tet(A), including the putative outbreak isolates, yielded right-sized amplicons with primers for the tnpA and tetR genes of Tn1721. All tetracycline-resistant isolates carrying tet(B) were positive for the tnpA gene of IS10 and for tetR, but negative for tetC and tetD, supporting the presence of a deleted version of Tn10. The tnpA gene of Tn3 was found in all bla_TEM-1-like-positive isolates. Finally, positive amplification of all or some of the Tn21 genes tested supports the presence of intact or defective versions of Tn21 in isolates carrying class 1 integrons of the sul1 type, whereas the single isolate with the class 2 integron was positive for Tn7-specific sequences, as expected. Of note, a strong correlation was found between MDR and the presence of transposons (detected in all MDR isolates) and integrons (carried by all except one of the MDR isolates).

Genomic typing

Typing of the Salmonella Rissen isolates with XbaI resulted into nine closely related profiles, termed X1–X9, which grouped in a single cluster with similarities ranging from 82.2% to 97.4% (Fig. 1a). The major profile was X1, which included 66 out of the 84 isolates (78.6%). A higher discrimination was obtained with BlnI that yielded 23 profiles (B1–B23; Fig. 1b). Two of them, B1 and B2, were shared by 39 (46.4%) and 14 (16.7%) isolates, respectively, while all others were shown by only 1 up to 3 isolates. A dendrogram of similarity based on the BlnI profiles (Fig. 1b) revealed two clusters (I and II, the former with two subclusters, IA and IB, each cluster including one of the predominant B1 and B2 profiles), separated at S = 52.2%. The diversity indexes obtained with XbaI and BlnI were 0.38 and 0.76, respectively.

FIG. 1.

Dendograms of similarity showing the genetic relatedness between pulsed-field gel electrophoresis profiles generated from Salmonella enterica serovar Rissen by digestion with XbaI (a) and BlnI (b). S, Dice's coefficient of similarity; N, number of isolates showing each of the detected XbaI (X) and BlnI (B) profiles.

In all, 25 XbaI-BlnI combined profiles were detected (Table 1). X1B1, shown by 38 isolates (45.2% of the total) collected from pig carcasses (6), bovine carcasses (4), food products (5), human clinical samples (23), and X1B2, common to 14 isolates (16.7%) derived from pork products (11) and clinical samples (3), were the most frequent. Regarding resistance properties, X1B1 was associated with the R0, R2, R3, R5, and R6 patterns, while X1B2 isolates were R0, R1, or R2 (Table 1).

With regard to the putative outbreak isolates (Table 2), 9 out of the 11 were categorized as X1B1 and R2, showing resistance to tetracycline encoded by tet(A). Two other isolates, spatially and temporally coincidental with the outbreak, were X1B11 and R2 (LSP 167/11) or X2B7 and R12, which includes resistance to streptomycin, sulfonamides, tetracycline, and trimethoprim, due to the aadA2, sul1, tet(A), and dfrA12 genes (LSP 189/11).

Table 2.

Properties of Salmonella enterica Serovar Rissen Isolates Involved in an Outbreak

LSP isolate	Date of isolation	Patient sex/age	Sample	XB-profile	R-profile
164/11	12/09/2011	M/57	Blood	X1B1	R2
165/11	14/09/2011	M/2	Feces	X1B1	R2
166/11	16/09/2011	M/62	Feces	X1B1	R2
167/11	16/09/2011	F/46	Feces	X1B11	R2
179/11	16/09/2011	M/57	Feces	X1B1	R2
180/11	23/09/2011	M/65	Feces	X1B1	R2
182/11	23/09/2011	F/31	Feces	X1B1	R2
183/11	26/09/2011	F/70	Feces	X1B1	R2
184/11	27/09/2011	M/60	Feces	X1B1	R2
189/11	28/09/2011	F/55	Urine	X2B7	R12
192/11	04/10/2011	F/62	Urine	X1B1	R2

LSP, Laboratory of Public Health, Asturias, Spain; XB-profile, XbaI-BlnI pulsed-field gel electrophoresis profile; R-profile, resistance profile.

Discussion

In the past decades, increased antimicrobial resistance has been reported in pig-associated serovars, particularly the DT104 and the pUO-StVR2 MDR clones of Salmonella Typhimurium, the Spanish and European clones of monophasic 4,5,12:i:-, Salmonella Derby and Salmonella Rissen.^{3,4,13,15,19,21,23} In the present study, 78.6% of the Salmonella Rissen isolates were resistant to one or more antimicrobials and 19% were MDR. Tetracycline was the most common resistance (74%), and significant percentages (ranging from 12% to 21.4%) were also observed for streptomycin, sulfonamides, trimethoprim, ampicillin, and chloramphenicol. A high incidence of tetracycline resistance as well as variable frequencies of all the other resistances were previously reported in Salmonella Rissen from different sources and countries.^3,4,8,22 Resistances found in the present study were mostly conferred by integrons and transposons. In all, three class 1 integrons of the sul1 type, a class 1 integron of the sul3 type, the class 2 integron of transposon Tn7, and several other transposons were detected. As far as we know, two sul1-integrons (with the 1000 bp/aadA1 and 1600 bp/dfrA1-aadA1 variable regions), the sul3-integron with the dfrA12-orfF-aadA2-cmlA-aadA1-qacH-IS440 gene array, and the class 2 integron of Tn7 are reported for the first time in Salmonella Rissen. However, the third sul1-integron, with the 2000 bp/dfrA12-orfF-aadA2 variable region, has previously been detected in pork isolates from Thailand,³⁴ as well as in MDR isolates found in pork, poultry products, and clinical samples in Portugal.^3,8,19 Unlike integrons, transposons were not previously screened in Salmonella Rissen. Results of the present study support Tn1721 as the carrier of tet(A), which appears to be the main gene responsible for tetracycline resistance in Salmonella Rissen.^3,8,22 In the isolates characterized herein, stabilization of the transposon by insertion into the bacterial chromosome (as demonstrated by hybridization experiments; to be published elsewhere) could have contributed to dissemination of tet(A) through vertical transmission in clonally related isolates (next paragraph). The involvement of Tn1721, Tn10, Tn3, and Tn21 in resistance, as carriers of tet(B), bla_TEM-1, and class 1 integrons, respectively, was also shown. In fact, isolates containing each of these genes proved to be positive for the corresponding transposon, and the opposite was also true.

In one of the few studies using PFGE to assess the molecular diversity of a wide collection of Salmonella Rissen, XbaI restriction proved to be highly discriminative, producing a total of 63 unique patterns, which formed 10 distinct clusters. These patterns were obtained from 112 isolates recovered from human and food sources in 2004 in Thailand and between 1996 and 2005 in Denmark.²² Due to this, Salmonella Rissen was regarded as a genetically diverse serovar. In the present study, we appear to be dealing with a rather homogenous population, although the isolates were collected from different sources over a decade. XbaI distributed 84 isolates into 9 closely related profiles, which grouped in a single cluster with similarity ≥82.2. The same or closely related patterns were associated with Salmonella Rissen derived from pig samples (i.e., lymph nodes and carcasses), pork meat, meat handlers, and human clinical samples in Portugal^2,3,18 and from foods and human sources in Thailand and Denmark.²² This supports the existence of a successful clone of Salmonella Rissen, which is circulating in geographically distant countries. In the case of Spain and Portugal, intensive trade in live pigs, pork meat, and pork-derived products between the two countries might explain the spread of this clone in the Iberian Peninsula, as previously proposed for the so-called Spanish clone of the monophasic S. 4,5,12:i:- variant.³ It is of note that the major XbaI profiles obtained from Salmonella Rissen in Spain (X1) and Denmark (TEEX01.0017.DK) could be further differentiated by BlnI, yielding 11 and 6 patterns, respectively. Interestingly, some of the Danish profiles were generated from isolates found in pig meat imported from Spain, supporting the contribution of livestock trade to the spread of Salmonella Rissen.²²

The most common Salmonella Rissen strain found in Spain was X1B1 and showed resistance to tetracycline encoded by tet(A). This strain was detected in pig carcasses, bovine carcasses, as well as in different food products made with pork and mixed pork and beef meat. In addition, it has been recovered from clinical samples and was responsible for an outbreak. In fact, 9 out of the 11 putative outbreak isolates were X1B1/tet(A). Two other isolates, LSP 167/11 and LSP 189/11, temporally and spatially coincidental with the outbreak, were X1B11/tet(A) and X2B7/tet(A), bla_TEM-1, aadA2, sul1, and dfrA12, respectively. Considering the infrequent association of Salmonella Rissen with sporadic cases of human salmonellosis, these two isolates could have been implicated in the outbreak and, at least one of them, LSP 189/11, has apparently evolved from the main outbreak strain. In fact, a fragment present in the B7 profile of LSP 189/11, but not in B1, was recently identified as a plasmid (our unpublished results), which was also responsible for the additional resistances shown by LSP 189/11. A detailed investigation of this and other plasmids already detected in the Salmonella Rissen isolates from Asturias, including their contribution to the detected resistances, is being performed.

Conclusions

This study revealed a high frequency of resistance and MDR among Salmonella Rissen isolates obtained from different sources in Spain and the relevant contribution of integrons and transposons to the observed phenotypes. All analyzed isolates were closely related according to XbaI-typing, supporting the prevalence of a clone that is being transmitted to humans through the food chain, and has caused sporadic cases of gastroenteritis as well as an outbreak.

Footnotes

Acknowledgments

R.G.-F. and I.M. were recipients of grants from the “Fundación para el Fomento en Asturias de la Investigación Científica Aplicada y la Tecnología” (FICYT BP11-050 and FICYT BP09-069, respectively). This work has been supported by project FIS-PI11-00808 of “Fondo de Investigación Sanitaria, Instituto de Salud Carlos III, Ministerio de Economía y Competitividad,” Spain, cofunded by the European Regional Development Fund of the European Union: a way to Making Europe.

Disclosure Statement

No competing financial interests exist.

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