Co-Occurrence of ACSSuT and Cephalosporin Resistance Phenotypes Is Mediated by int1- Associated Elements in Nontyphoidal Salmonella enterica from Human Infections in Spain

Abstract

A screening of antimicrobial resistance and its genetic determinants has been performed on 300 Salmonella enterica isolates collected during 2004–2008 from human infections in Spain. Salmonella Typhimurium and Salmonella Enteritidis were the major serotypes, which were found with similar frequencies covering 80% of the bacterial collection. Salmonella Typhimurium isolates frequently shared low susceptibility to antimicrobials of the penta-resistance phenotype (ACSSuT) and/or cephalosporin resistance. The ACSSuT profile was found closely linked to int1-associated gene cassettes, with major elements carrying DNA fragments of 1.0 Kb (aadA2 gene) plus 1.2 Kb (blaPSE-1 gene) or 2.0 Kb (aadA1 and blaOXA-1 genes). Among these, ACSSuT and cephalosporin resistances were associated in Salmonella Typhimurium isolates expressing the blaOXA gene. β-lactamase activities were also detected from isolates carrying blaTEM, blaCMY, or blaSHV, although only the two last genes expressed extended-spectrum β-lactamases. The clonal analysis of S. enterica strains suggests that both horizontal and vertical transfer mechanisms are involved in the wide dissemination of their antimicrobial resistance.

Introduction

S almonella is an important pathogen of humans and animals, causing enteric fever and gastroenteritis. About 1.3 billion cases of illness and 3 million deaths are reported annually due to nontyphoidal salmonellosis in humans.¹⁴ Usually, salmonellosis is a self-limiting disease not requiring treatment, but in the case of elderly people and children or if the microorganism causes invasive illness, antimicrobial therapy may be needed. There is great concern about antimicrobial resistance since it not only compromises the efficiency of early empiric treatment, but can also increase the transmission potential of the microorganisms and the coselection of their virulence characteristics.²³

Integrons are genetic elements that can acquire resistance functions as associated gene cassettes, which are flanked by the 59-base element that target its site-specific recombination.⁶ Integrons can be located in the chromosome or in plasmids, playing the role of natural cloning and expression vectors that contribute to the development of multidrug-resistant bacterial strains.²⁵ The class I integrons are often associated with the mosaic structure of the Salmonella genomic island 1 (SGI1), typically found in Salmonella Typhimurium DT104 sharing resistance to ampicillin (Amp), chloramphenicol (Chl), streptomycin (Str), sulfonamides, and tetracycline (Tet), a penta-resistance profile (ACSSuT) that could expand resistance to fluoroquinolones, trimethoprim, and other aminoglycosides.³ Clonally related isolates of Salmonella enterica carrying the ACSSuT or quinolone-resistant phenotypes have been detected spread among three host environments in Spain; human, domestic animals, and wild animals.²⁴

Between the two main serotypes of S. enterica subsp. enterica with clinical relevance to humans, Salmonella Typhimurium has been the more prevalent until recent years when infections with Salmonella Enteritidis started to increase significantly in Europe.²⁷ The trend toward this serotype change might be conditioned by nutritional habits and also by the difficulty of its detection in poultry and eggs, the main source of Salmonella Enteritidis.³¹

Upon the rise of multiresistance in Salmonella, especially in Salmonella Typhimurium with the penta-resistance profile ACSSuT, cephalosporins became the antimicrobials of choice for the treatment of salmonellosis. However, antibiotic pressure has driven the evolution of β-lactamase variants, which have activity against extended-spectrum cephalosporins.^2,22 The cephalosporinases more frequently found in Salmonella belong to the group 2 of the Bush's classification, like TEM, SHV, OXA, and CTX-M, or to the group 1 in the case of the AmpC-like enzyme CMY.⁵

The aim of this work was to analyze the distribution and diversity of intI-associated gene cassettes and cephalosporinase genes among strains of Salmonella enterica isolated from human infections in Spain, relating them with resistance against the antimicrobials included in the panel recommended by the European Food Safety Agency,¹¹ and tracing the possible mechanisms of bacterial spread.

Materials and Methods

Microbiological material

A total of 300 Salmonella isolates were collected in the Hospitals of Navalmoral de la Mata (named HNV, 57 isolates), Mérida (HMD, 71), Cáceres (HCC, 68), Llerena (HLL, 49), Plasencia (HPL, 29), and Coria (HCR, 26), all located in Extremadura, Spain.²⁴ Sample collections were made during the period 2007–2008, although 39 isolates from the Hospital of Navalmoral de la Mata were sampled in 2004–2006. The microorganisms were isolated from feces, with the exception of a few strains that were collected from blood (5), urine (1), and from a wound (1). Only one isolate per clinical episode or outbreak was considered.

Serotyping and phage typing

Antigenic formula was determined by slide agglutination with commercial antisera, and phage typing was performed according to previously published protocols with phages and interpreting criteria provided by the Health Protection Agency (formerly Public Health Laboratory Service, Colindale) in London, England.^1,32 These analyses were performed in The Spanish National Reference Laboratory of Salmonella and Shigella (LNRSSE), Majadahonda, Madrid.

Antimicrobial susceptibility

Salmonella isolates were subjected to antimicrobial susceptibility tests by the determination of minimal inhibitory concentrations using the twofold broth microdilution reference method according to ISO 20776-1:2006.¹⁰ The antimicrobials were selected following the European Food and Safety Authority recommendations to monitor antimicrobial resistance in Salmonella.¹¹ The tested antimicrobials and their epidemiological cutoff (ECOFF) values were Amp (8 μg ml⁻¹), cefalothin (Cef, 16 μg ml⁻¹), cefotaxime (Ctx, 0.5 μg ml⁻¹), ceftazidime (Caz, 2 μg ml⁻¹), ceftiofur (Xnl, 2 μg ml⁻¹), Chl (16 μg ml⁻¹), Str (16 μg ml⁻¹), sulfamethoxazole (Sul, no ECOFF is provided, 256 μg ml⁻¹ was considered in this work according to the antimicrobial wild-type distribution of Salmonella spp), and Tet (8 μg ml⁻¹).¹³ These analyses were performed in the central laboratory of this study (Sanidad Animal, Universidad de Extremadura). Resistance to quinolones of bacterial isolates has been described elsewhere.²⁴

DNA purification, restriction, and sequencing

Total DNA was extracted by a simplified boiling method, consisting in the suspension of one colony in 250 μl of sterile miliQ water, heating the mixture at 100°C for 5 min, followed by centrifugation for 2 min at 10,000 g. The supernatant was used directly for PCR reactions. PCR products were purified by using commercial systems (SpeedTools PCR Clean Up kit; Biotools), restriction digestions were performed according to the manufacturer's instructions (Invitrogen), and sequencing reactions were performed by StabVida (Portugal).

Integron and gene cassette amplifications

A fragment of 242 bp from Int1 sequences and conserved gene cassettes of variable sizes were amplified by using primers Int1-F (5′ gct ctc ggg taa cat caa gg 3′), Int1-R (5′ tca gga gat cgg aag acc tc 3′), CS-F (5′ ggc atc caa gca gca ag 3′), and CS-R (5′ aaa agc aga ctt gac ctg a 3′), adapted from previously described works.^19,20 PCR conditions were as standard, with annealing temperatures of 58°C and 56°C, for int1 and gene cassettes, respectively. Conserved gene cassettes were classified by HaeIII digestion and restriction fragment length polymorphism (RFLP) analysis, and one fragment representing every category was sequenced.

Pulsed-field gel electrophoresis

DNA for pulsed-field gel electrophoresis (PFGE) analysis was prepared according to a standard protocol.²⁸ Agarose plugs were digested with 50 U of XbaI (Fermentas) and fragments were separated in a 1% agarose gel (Pronadisa) using a Chef-DR III System (BioRad) during 20 hr with a constant voltage of 6 V a linear ramp of 2.2–63.8 sec. Salmonella Bradenburg H9812 (PulseNet) was used as a size marker and the gels were stained with SYBR Safe DNA gel stain (Invitrogen). Images of PFGE were obtained in an image system Fluor-S Multilmager (BioRad) and were analyzed with Quantity One 4.4.0 (BioRad). The Dice coefficient was calculated with a position tolerance of 1%, dendograms were constructed with BioNumerics 5.2 (Applied Maths-BioRad), and pulse types were defined by identical PFGE profiles.

Detection of β-lactamase genes and plasmid analysis

PCR for the amplification of the β-lactamase genes codifying for bla_CMY, bla_OXA, bla_SHV, and bla_TEM were performed in the isolates showing resistance to at least one of the tested cephalosporins, and for bla_CTX-M, the PCR was only carried out in isolates resistant to Ctx. Primers and PCR conditions were previously described for bla_CMY,³¹ bla_OXA,⁸ bla_SHV,¹² bla_TEM,⁴ and groups 1, 2, 8, 9, and 25 of bla_CTX-M.³³ PCR-positive controls for bla_CTX-M were group 2, strain NC13462.T; group 8, strain NC12463.T; and for group 25, strain NC13465.T, all from the National Collection of Type Cultures (NCTC). For groups 1 and 9, the control strains were kindly provided by Carmen Torres (Rioja University). The controls used in the amplification of bla_OXA, bla_TEM, and bla_SHV were kindly provided by Teresa Coque (Hospital Ramón y Cajal).

Conjugative transfers were performed by using a broth mating method with E. coli K12 (J53; kindly supplied by George Jacoby, Lahey Clinic) used as the recipient strain and MacConkey agar plates containing 100 μg Amp ml⁻¹ and 50 μg rifampicin ml⁻¹ to select transconjugants. Plasmid DNA was visualized after linearization with the S1 enzyme followed by PFGE in the conditions mentioned above, including the Lambda Ladder PFGE Marker (New England Biolabs). The primers and conditions used for incompatibility groups were as previously described.⁷

Determination of β-lactamase activities

Salmonella strains were grown aerobically at 37°C in LB broth media. At the middle of the exponential phase of growth (0.4–0.8 OD_600nm ml⁻¹), cells were collected by centrifugation (6,000 g, 5 min, 4°C), washed, and transferred to a phosphate buffer (50 mM, pH 7.0), sonicated on ice for 60 sec (maximal strength, 33% efficiency) with a vibra-cell (Sonic-Materials) apparatus, and cell-free extracts were obtained as the supernatant of a final centrifugation (10,000 g, 10 min, 4°C). β-lactamase activities were determined at 37°C in assays containing variable amounts of cell-free extracts, 50 μg nitrocefin ml⁻¹ substrate, and a 50 mM sodium phosphate buffer (pH 7.0), by recording the decrease in absorbance at 468 nm (ɛ₄₈₆=20,500 M⁻¹ cm⁻¹). The inhibitory effect on the enzyme activity of sodium clavulanate was tested after adding the compound (4 μg ml⁻¹) to the reaction mixture. The protein concentration of the cell-free extracts was determined by using a commercially available Bradford reactive (Bio-Rad Protein Assay; Bio-Rad).

Isoelectric focusing (IEF) of β-lactamases was performed in polyacrylamide precast gels with a range of pH from 3–10, including a protein standard with pI 4.45–9.6 (BioRad). Samples were loaded in a total volume of 25 μl, containing about 30 μg of protein and including 2 μl of 50% glycerol. The electrophoresis was performed at 10 mA during 2 hr and the β-lactamase activity was stained by soaking the gels in 50 sodium phosphate buffer (0.1 M, pH 7.0) supplemented with 50 μg nitrocefin ml⁻¹.

Results

Microbiological material and antimicrobial resistance

Three hundred isolates of S. enterica detected in Extremadura (Middle-West Spain) from human clinical cases have been screened for antimicrobial resistance (Table 1). Between the two major serotypes detected, Salmonella Typhimurium and Salmonella Enteritidis, the first represents the main fraction of isolates with resistance to ACSSuT antimicrobials (49/54), to cefalosporins (33/42), or with both phenotypes associated (19/20). Beside these, only one Salmonella Bredeney isolate presented the ACSSuT phenotype and resistance to cefalosporins.

Table 1.

Absolute Frequencies of Salmonella enterica Main Serotypes and Resistant Isolates

	TY	EN	MU	BA	BE	Other ^a	Total
N	126	115	10	5	3	41	300
Ampicillin	103	39	1	0	1	14	158
Chloranphenicol	65	7	1	0	2	6	81
Streptomycin	82	11	1	0	2	8	104
Sulfamethoxazolee	101	18	2	1	2	13	137
Tetracycline	91	15	2	2	2	13	125
Σ ACSSuT^b	49	2	0	0	1	2	54
Cefalothin	10	2	0	0	1	2	14
Cefotaxime	19	2	0	0	1	1	23
Ceftazidime	3	2	0	1	1	1	8
Ceftiofur	19	3	0	1	1	2	26
Σ Cefalosporins^b	33	5	0	1	1	2	42
Σ ACSSuT+Cef^b	19	0	0	0	1	0	20

Serotypes, ordered by absolute frequencies, are TY, Typhimurium; EN, Enteritidis; MU, Muenchen; BA, Brandenburg; BE, Bredeney.

Other serotypes, with isolate number between parentheses, are Infantis (4), Newport (4), Ohio (3), Rissen (3), Anatum (2), Bovismorficans (2), Derby (2), Hadar (2), Mikawasima (2), Sandiego (2), S. Typhimurium 4,12:i:- (1), 4,5,12:i:- (1), Altona (1), Blockley (1), Branenderup (1), Corvallis (1), Goldcoast (1), Indiana (1), Isangi (1), Livingstone (1), Manhattan (1), Mbandaka (1), Montevideo (1), Panama (1), Saintpaul (1), and Thompsom (1).

Total number of isolates showing the indicated phenotype.

Class 1 integrons and associated gene cassettes

int1 sequences were identified in 72 isolates of Salmonella Enterica (not shown), corresponding to Salmonella Typhimurium (the major serotype, with 62 isolates), Enteritidis (2), Bredeney (1), Rissen (2), Bovismorficans (1), Braenderup (1), Derby (1), Goldcoast (1), and Mikawasima (1). Among these, 52 isolates presented Class-I integron-associated gene cassettes (Table 2). Their RFLP distinguished five different elements, which sequences are identical to previously described gene cassettes A (GU119958), B (GU987052), C (FJ460238, GU987051, and GU987052), D (DQ388124), and E (FJ980455). All five elements carry an aadA gene, whereas four present additional dfrA and/or β-lactamase genes. Gene cassettes A and B, being detected in 25 and 23 isolates, respectively, are the most prevalent in S. enterica isolates (Table 2).

Table 2.

int1-Associated Gene Cassettes Detected Among Salmonella enterica Isolates

GC ^a	PCR ^b	HaeIII-RFLP^b	Serotypes ^c	Genes	Resistance encoded
A	2.0	1.00, 0.50, 0.20, 0.18, 0.15	Typhimurium (24), Mikawasima (1)	aadA1, blaOXA-1	Streptomycin and penicillins
B	1.0, 1.2	0.2, 0.35, 0.45, 0.58	Typhimurium (23)	aadA2, blaPSE-1	Streptomycin and penicillins
C	1.0, 1.2, 1.6	1.10, 0.25, 0.18, 0.58, 0.45, 0.35, 0.2	Typhimurium (1)	aadA2, blaPSE-1, dfrA17, aadA5	Streptomycin, penicillins, and trimethoprim
D	1.6	0.50, 0.35, 0.25, 0.20	Typhimurium (1)	dfrA1, aadA1	Streptomycin and trimethoprim
E	1	0.08, 0.18, 0.2, 0.4	Braenderup (1), Bredeney (1)	aadA7	Streptomycin

Gene cassettes.

Size of DNA fragments expressed in Kb.

Absolute frequencies of serotypes are indicated between parentheses.

GC, gene cassettes; HaeIII-RFLP, HaeIII-restriction fragment length polymorphism.

Clonal relationships of S. enterica isolates carrying int-1

The PFGE typing of S. enterica isolates distinguished 101 clonal strains, among which, the two most frequent serotypes, Typhimurium and Enteritidis, correspond to 56 and 22 pulse types, respectively (data not shown). All isolates carrying int1-associated gene cassettes are clustered in 21 pulse types of Salmonella Typhimurium, and one each of Salmonella Braenderup, Salmonella Bredeney, and Salmonella Mikawasima, most of them expressing the penta-resistance phenotype (Table 3). The different gene cassettes are carried by isolates belonging to distinct pulse types, even the commonly found TY09 and TY36 that shared the A or B elements in 11 and 6 isolates, respectively. However, although the TY16 pulse type is mainly associated to gene cassette A, a single isolate carries the B element. Bacterial typing to a higher resolution was performed by identifying phage DT, which allowed definition of three major ACSSuT strains of Salmonella Typhimurium: TY09/GC-A/DT104b, TY36/GC-B/DT104, and TY38/GC-B/DT104 (Table 3).

Table 3.

Clonal diversity of Salmonella enterica Isolates Carrying int1-Associated Gene Cassettes

GC ^a	PS ^b	PT ^c	Isolates	Resistance ^d
A	TY09	104b	HNV116, HNV177, HNV190, HMD57, HPL14	ACSSuT
	TY09	U288	HCC47	ACSSuT
	TY09	UT/RDNC	HMD21, HPL10, HPL12, HPL17, HPL21	ACSSuT
	TY11	UT/RDNC	HCC11	AC- Su -
	TY12	UT/RDNC	HNV159	AC- SuT
	TY16	104b	HMD43, HLR55	ACSSuT
	TY16	UT/RDNC	HCC23, HCR02,HLR16, HNV51	ACSSuT
	TY17	104b	HLR13	ACSSuT
	TY21	41	HLR01	ACSSuT
	TY30	U302	HPL15	A- SSuT
	TY51	104b	HMD09	ACSSuT
	TY80	U311	HMD70	ACSSuT
	MI01	UT	HPL19	- - - - T
B	TY16	104b	HMD23	ACSSuT
	TY20	104	HMD39	ACSSuT
	TY36	104	HCC08, HCC09, HCR05, HPL05	ACSSuT
	TY36	UT/RDNC	HCR20, HNV158	ACSSuT
	TY38	104	HCC31, HCC37, HMD20, HMD44	ACSSuT
	TY39	104b	HNV128	ACSSuT
	TY41	U302	HMD36	ACSSuT
	TY42	104b	HCC06	ACSSuT
	TY43	104	HCC04, HNV186	ACSSuT
	TY43	U193	HLR08	ACSSuT
	TY43	U311	HNV187	ACSSuT
	TY44	104	HMD14	ACSSuT
	TY44	104	HMD13	A - S - T
	TY46	U302	HNV185	ACSSuT
	TY54	UT/RDNC	HMD31	ACSSuT
C	TY79	104	HCC07	ACSSuT
D	TY63	UT/RDNC	HCC52	ACSSuT
E	BE01	UT	HLR21	ACSSuT
	BA01	UT	HMD18	A- S - -

Gene cassettes, as previously defined in Table 2.

Pulse types; TY, serotype Typhimurium; MI, Mikawasima; BE, Bredeney; BR, Braenderup.

Phage types, only indicated for Salmonella Typhimurium isolates; UT, nontypable; RDNC, react, but does not conform.

Resistance to antimicrobials within the penta-resistance profile.

Hyphens (-) symbolize the antimicrobials to which the resistance found does not fit the ACSSuT profile.

Underlined isolates and their corresponding antimicrobials that do not meet the antimicrobial profile.

Cephalosporinase expression

Genes blaOXA, blaTEM, blaSHV, and blaCMY were detected in 25 of the 42 isolates of S. enterica that presented resistance to cephalosporins (Table 4). Molecular characteristics of expressed β-lactamase enzymes were analyzed by IEF (not shown), which indicated the expected pI of 5.7, 7.7, 8.9, and 9.54 for TEM, OXA, SHV, and CMY β-lactamases, respectively.¹² In addition, the enzyme activities from isolates carrying blaTEM and blaSHV genes were strongly inhibited by clavulanic acid, as corresponding to enzymes of the class A, whereas the weak inhibition that was detected for isolates with blaCMY or blaOXA genes is compatible with enzymes of the molecular classes C or D, respectively (Table 4).⁵

Table 4.

Genotypic and Phenotypic Characteristics of Cephalosporin-Resistant Isolates Expressing β-Lactamase Activity

						CMI ^e (μg ml⁻¹)			β-lactamase activity (mU mg⁻¹ protein)
BLA ^a	PS ^b	Isolate	R ^c	int1 ^d	Cef	Xnl	Ctx	Caz	−Clavulanic	+Clavulanic
OXA	TY06	HCC54	+	−	4	2	1	0.5	190.6±8.9	86.1±13.7
	TY09	HCC57	−	−	4	2	1	0.5	183.0±17.6	84.9±23.3
		HMD21	+	+	16	8	2	0.5	194.0±12.1	80.1±24.1
		HNV116	+	+	16	8	1	1	111.9±8.0	36.6±6.6
		HMD57	+	+	8	2	0.5	4	117.4±74.2	26.6±18.0
		HPL21	+	+	16	8	4	2	146±43.6	39.9±2.9
		HPL10	+	+	4	16	4	4	154.3±9.7	48.9±4.7
		HPL12	+	+	1	16	4	2	180.4±0.8	30.3±18.2
		HPL14	+	+	2	4	0.5	2	150.8±0.1	43.9±0.1
		HPL17	+	+	4	16	4	2	369.6±193.9	122.2±29.2
	TY16	HCR02	+	+	32	16	4	2	198.4±67.9	52.9±4.0
		HLR16	+	+	4	4	1	0.5	172.7±22.8	41.2±19.5
		HLR54	+	−	4	4	0.5	2	187.4±21.5	68.7±23.3
		HLR55	+	+	8	2	2	2	211.6±67.0	72.3±10.8
		HMD43	+	+	16	4	1	2	153.8±28.8	32.0±24.0
		HNV51	+	+	4	8	0.5	0.5	166.7±37.7	48.2±3.0
	TY17	HLR13	+	+	4	4	1	0.5	191.6±108.5	33.6±1.4
	TY21	HLR01	+	+	4	2	1	0.5	180.4±30.6	47.5±4.8
	TY51	HMD09	+	+	4	4	1	2	203.5±29.2	48.4±7.6
	TY80	HMD70	+	+	16	4	2	2	176.7±4.9	42.2±2.4
TEM	TY08	HMD47	−	−	32	8	0.5	1	61.3±17.2	9.2±2.0
	HA01	HNV141	−	−	128	4	0.5	1	874.7±628.2	110.2±81.5
	EN17	HMD06	−	−	64	2	0.1	0.5	281.8±0.1	1.1±0.1
CMY	BE01	HLR21	+	+	256	32	32	64	1428.2±191.2	1355.2±208.3
SHV	BL01	HCC70	−	−	256	256	64	256	265.9±33.1	8.9±0.6

β-lactamase enzyme.

Pulse type.

Penta-resistant phenotype.

Detection of int1-associated gene cassettes, also shown in Table 3.

ECOFF values are: cefalothin (Cef), 16 μg ml⁻¹; ceftiofur (Xnl), 2 μg ml⁻¹; cefotaxime (Ctx), 0.5; ceftazidime (Caz), 2 μg ml⁻¹ ¹³.

ECOFF, epidemiological cutoff.

Among the 25 isolates expressing the β-lactamase activity (Table 4), the major gene detected was blaOXA (20 isolates, 17 carrying int1-associated elements), followed by blaTEM (3), blaSHV (1), and blaCMY (1). All, but one, the isolates expressing OXA, plus the unique strain carrying CMY, presented the ACSSuT phenotype. Isolates expressing OXA were more resistant to Ctx than those that, carrying blaTEM, were in contrast more resistant to Cef (Table 4). Extended-spectrum resistance to all cephalosporins essayed was only detected for isolates expressing CMY or SHV β-lactamases. The sequencing of β-lactamase genes and the analysis of their mobilization potential (not shown) revealed the occurrence of alleles OXA-1 and TEM-1 for the two major β-lactamases, chromosomally encoded (HPL10) or carried by a 50-Kb plasmid of unknown replicon type (HMD06), whereas CMY-2 and SHV-12 were encoded by 70-Kb and 40-Kb plasmids, with lncl1 and lncN replicon type, respectively.

Seventeen isolates of S. enterica that presented cephalosporin resistance lacked any β-lactamase gene or enzyme (Table 5). Resistance to Ctx, the penta-resistance phenotype or the presence of int1-associated gene cassettes were characteristics rarely detected, in contrast of those found for blaOXA-carrying isolates.

Table 5.

Genotypic and Phenotypic Characteristics of Cephalosporin-Resistant Isolates Lacking β-Lactamase Expression

				CMI ^d (μg ml⁻¹)
PS ^a	Isolate	R ^b	int1 ^c	Cef	Xnl	Ctx	Caz
BA04	HCC69	−	−	16	128	0.13	256
EN02	HMD46	−	−	8	8	1	1
	HCC71	−	−	8	0.5	4	4
EN10	HNV174	−	−	4	4	0.5	4
EN17	HMD01	−	−	256	128	0.1	0.5
TY01	HCC17	−	−	256	1	0.13	0.5
TY08	HCC32	−	−	64	8	0.5	1
TY09	HCR25	+	−	8	32	1	2
TY20	HLR33	−	−	257	0.5	0.06	2
TY29	HCC66	−	−	2	1	1	1
	HNV191	−	−	2	2	0.25	4
TY38	HMD20	+	+	4	16	2	1
TY43	HNV186	+	+	32	2	0.5	2
TY44	HMD13	−	+	32	2	0.13	0.5
TY60	HMD50	−	−	32	1	0.13	0.5
TY63	HCC52	+	+	32	2	0.13	0.5
TY74	HPL30	−	−	257	2	0.25	256

Isolates lacked any detectable β-lactamase gene or enzyme.

Pulse type.

Penta-resistant phenotype.

Detection of int1-associated gene cassettes, also shown in Table 3.

ECOFF values are: cefalothin (Cef), 16 μg ml⁻¹; ceftiofur (Xnl), 2 μg ml⁻¹; cefotaxime (Ctx), 0.5; ceftazidime (Caz), 2 μg ml⁻¹ ¹³.

Discussion

The distribution of S. enterica serotypes found in this work, with similar frequencies for Salmonella Typhimurium and Salmonella Enteritidis followed, by far, by Salmonella Muenchen, contrasts with the existing trend in which is assumed that Salmonella Typhimurium was the main causing agent of salmonellosis, both in animals and humans, until the mid-1990's, when Salmonella Enteritidis overcame the former microorganism.^9,14,21,29 The situation detected could be a consequence of regional singularities, like nutritional habits, but should be contrasted by further analyses. The S. enterica isolates detected in clinical cases from Extremadura presented frequencies of antimicrobial resistance higher than 25%–50% to Amp, Chl, Str, Sul, and Tet (Table 1), indicating, like previous works, that these antimicrobials might not be adequate for the treatment of salmonellosis.^29,30 In contrast, resistance to cephalosporins found in this work was lower than 15%, with the lowest percentage of resistance to Caz (8.3%), results that could be of particular importance considering the impact of this group of antimicrobials, the third-generation sephalosporins, for human chemotherapy.^22,23,29

Salmonella isolates carrying 1 to 3 int1-associated gene cassettes were detected in this work, being profiles A and B the major elements found (Tables 2 and 3), like previous works that described those elements as endemic in Spain, although with a lower frequency of profile A.^17,26 It resulted noteworthy that most isolates carrying this genetic element, encoding the OXA enzyme, express β-lactamase activity and resistance against third-generation cephalosporins, linking ACSSuT (Table 3) plus cephalosporin resistance phenotypes in a significant fraction of Salmonella isolates (Table 4). Moreover, int1-associated gene cassettes were also detected in other three isolates sharing ACSSuT and cephalosporin resistance, even if β-lactamase expression was not detected (Table 5). The frequent co-occurrence of genotypes and phenotypic characters, like pulse types, int1-associated gene cassettes, phage types, and antimicrobial susceptibility profiles in different isolates suggests their successful spread by vertical transmission. In contrast, although horizontal transfer of int1-associated resistance determinants should also be involved in their wide distribution among different clonal strains of Salmonella Typhimurium, plasmids encoding TEM, CMY, SHV, or CTX-M enzymes are very uncommon or not detected at all among the analyzed bacteria. Their frequencies are similar to those detected in Spain in a previous screening, where the same alleles (CMY-2 and SHV-12) had been detected linked to plasmids with similar sizes and replicon types carried by isolates with identical serotypes, Salmonella Bredeney and Salmonella Blockley, respectively, indicating that they might present a very low potential for horizontal mobilization in vivo.¹⁵ Beside these, the clonal diversity of nearly half of cephalosporin-resistant isolates that lack any detectable β-lactamase gene or enzyme (Table 5) might suggest the activation of an intrinsic mechanism of resistance, like antimicrobial efflux by the AcrAB pump.¹⁶

This study shows that the collection of S. enterica isolated from Spain presents low susceptibility to antimicrobials of the ACSSuT profile and that it is moderately resistant to cephalosporins. Among these, the higher percentages of resistance have been found for Xnl and Ctx, third-generation cephalosporins, which use is indicated to veterinary or human medicine, respectively, the two possible environments for the selection of cephalosporin-resistant Salmonella producing infections in humans.¹⁸ Our data, that show the link between ACSSuT and cephalosporin-resistant phenotypes, might indicate the high risk of trait coselection by antimicrobial treatments of Salmonella infections.

Footnotes

Acknowledgments

G.P. wishes to thank the Valhondo Calaff foundation for a predoctoral fellowship. This work has been supported by the Ministry of Education and Science of Spain (project AGL2008-04147/GAN), the Department of Employ, Enterprise and Innovation of the regional government of Extremadura, Spain (project PRI08B001 and group CTS001) and the University of Extremadura (Group MIVET).

Disclosure Statement

All authors report no conflicts of interest relevant to this article.

References

Anderson

E.S.

, Ward

L.R.

, Desaxe

M.J.

, Desa

J.D.H.

1977. Bacteriophage-typing designations of Salmonella typhimurium. J. Hyg. (Lond.), 78:297–300.

Arlet

, Barrett

T.J.

, Butaye

, Cloeckaert

, Mulvey

M.R.

, White

D.G.

2006. Salmonella resistant to extended-spectrum cephalosporins: prevalence and epidemiology. Microbes Infect., 8:1945–1954.

Boyd

, Peters

G.A.

, Cloeckaert

, Boumedine

K.S.

, Chaslus-Dancla

, Imberechts

, Mulvey

M.R.

2001. Complete nucleotide sequence of a 43-kilobase genomic island associated with the multidrug resistance region of Salmonella enterica serovar typhimurium DT104 and its identification in phage type DT120 and serovar agona. J. Bacteriol., 183:5725–5732.

Brinas

, Zarazaga

, Saenz

, Ruiz-Larrea

, Torres

2002. beta-lactamases in ampicillin-resistant Escherichia coli isolates from foods, humans, and healthy animals. Antimicrob. Agents Chemother., 46:3156–3163.

Bush

, Jacoby

G.A.

2010. Updated functional classification of beta-lactamases. Antimicrob. Agents Chemother., 54:969–976.

Carattoli

2001. Importance of integrons in the diffusion of resistance. Vet. Res., 32:243–259.

Carattoli

, Bertini

, Villa

, Falbo

, Hopkins

K.L.

, Threlfall

E.J.

2005. Identification of plasmids by PCR-based replicon typing. J. Microbiol. Methods, 63:219–228.

Chen

, Zhao

S.H.

, White

D.G.

, Schroeder

C.M.

, Lu

, Yang

H.C.

, McDermott

P.F.

, Ayers

, Meng

J.H.

2004. Characterization of multiple-antimicrobial-resistant Salmonella serovars isolated from retail meats. Appl. Environ. Microbiol., 70:1–7.

Cruchaga

, Echeita

, Aladuena

, Garcia-Pena

, Frias

, Usera

M.A.

2001. Antimicrobial resistance in salmonellae from humans, food and animals in Spain in 1998. J. Antimicrob. Chemother., 47:315–321.

10.

DIN EN ISO 20776–1. 2006. Part 1: Reference method for testing the in vitro activity of antimicrobial agents against rapidly growing aerobic bacteria involved in infectious diseases. In Clinical laboratory testing and in vitro diagnostic test systems—Susceptibility testing of infectious agents and evaluation of performance of antimicrobial susceptibility test devices. Deutsches Institut für Normung: Berlin, Germany.

11.

EFSA. 2010. The Community Summary Report on antimicrobial resistance in zoonotic and indicator bacteria from animals and food in the European Union in 2008. EFSA J, 1:1658.

12.

Essack

S.Y.

, Hall

L.M.C.

, Pillay

D.G.

, McFadyen

M.L.

, Livermore

D.M.

2001. Complexity and diversity of Klebsiella pneumoniae strains with extended-spectrum beta-lactamases isolated in 1994 and 1996 at a teaching hospital in Durban, South Africa. Antimicrob. Agents Chemother., 45:88–95.

13.

European Commitee on Antimicrobial Susceptibility Testing (EUCAST). 2013. Antimicrobial wild type distributions of microorganisms. MIC- and Inhibition zone diameter distributions of microorganisms without and with resistance mechanisms. EUCAST version 5.13. www.eucast.org/mic_distributions. 2012 December 20(Online.)

14.

Galanis

, Lo Fo Wong

D.M.

, Patrick

M.E.

, Binsztein

, Cieslik

, Chalermchikit

, Aidara-Kane

, Ellis

, Angulo

F.J.

, Wegener

H.C.

World Health Organization Global Salm-Surv. 2006. Web-based surveillance and global Salmonella distribution, 2000–2002. Emerg. Infect. Dis., 12:381–388.

15.

Gonzalez-Sanz

, Herrera-Leon

, de la Fuente

, Arroyo

, Echeita

M.A.

2009. Emergence of extended-spectrum beta-lactamases and AmpC-type beta-lactamases in human Salmonella isolated in Spain from 2001 to 2005. J. Antimicrob. Chemother., 64:1181–1186.

16.

Guardabassi

, Courvalin

2006. Modes of antimicrobial action and mechanisms of bacterial resistance. Aarestrup

Antimicrobial Resistance in Bacteria of Animal Origin. ASM Press: Washington. Chapter 1:1–18.

17.

Herrero

, Rodicio

M.R.

, Echeita

M.A.

, Mendoza

M.C.

2008. Salmonella enterica serotype Typhimurium carrying hybrid virulence-resistance plasmids (pUO-StVR): A new multidrug-resistant group endemic in Spain. Int. J. Med. Microbiol., 298:253–261.

18.

Hornish

R.E.

, Kotarski

S.F.

2002. Cephalosporins in veterinary medicine—ceftiofur use in food animals. Curr. Top. Med. Chem., 2:717–731.

19.

Leverstein-van Hall

M.A.

, Paauw

, Box

A.T.A.

, Blok

H.E.M.

, Verhoef

, Fluit

A.C.

2002. Presence of integron-associated resistance in the community is widespread and contributes to multidrug resistance in the hospital. J. Clin. Microbiol., 40:3038–3040.

20.

Levesque

, Piche

, Larose

, Roy

P.H.

1995. PCR mapping of integrons reveals several novel combinations of resistance genes. Antimicrob. Agents Chemother., 39:185–191.

21.

Matheson

, Kingsley

R.A.

, Sturgess

, Aliyu

S.H.

, Wain

, Dougan

, Cooke

F.J.

2010. Ten years experience of Salmonella infections in Cambridge, UK. J. Infect., 60:21–25.

22.

Miriagou

, Tassios

P.T.

, Legakis

N.J.

, Tzouvelekis

L.S.

2004. Expanded-spectrum cephalosporin resistance in non-typhoid Salmonella. Int. J. Antimicrob. Agents, 23:547–555.

23.

Molbak

2005. Human health consequences of antimicrobial drug-resistant Salmonella and other foodborne pathogens. Clin. Infect. Dis., 41:1613–1620.

24.

Palomo

, Campos

M.J.

, Ugarte

, Porrero

M.C.

, Alonso

J.M.

, Borge

, Vadillo

, Domínguez

, Quesada

, Píriz

2012. Dissemination of antimicrobial-resistant clones of Salmonella enterica among domestic animals, wild animals and humans. Foodborne Pathog. Dis., 10:171–176.

25.

Partridge

S.R.

, Tsafnat

, Coiera

, Iredell

J.R.

2009. Gene cassettes and cassette arrays in mobile resistance integrons. FEMS Microbiol. Rev., 33:757–784.

26.

Perez-Moreno

M.O.

, Centelles-Serrano

M.J.

, Cortell-Ortola

, Ruiz

, Llovet-Lombarte

M.I.

, Jardi-Baiges

A.M.

, Fort-Gallifa

2009. Multidrug resistance related to class 1 integrons in human Salmonella enterica serotype Typhimurium isolates and emergence of atypical sul3-associated integrons. Int. J. Antimicrob. Agents, 34:381–383.

27.

Peters

T.M.

, Berghold

, Brown

, Coia

, Dionisi

A.M.

, Echeita

, Fisher

I.S.T.

, Gatto

A.J.

, Gill

, Green

, Gerner-Smidt

, Heck

, Lederer

, Lukinmaa

, Luzzi

, Maguire

, Prager

, Usera

, Siitonen

, Threlfall

E.J.

, Torpdahl

, Tschäpe

, Wannet

, Zwaluw

W.K.

2007. Relationship of pulsed-field profiles with key phage types of Salmonella enterica serotype Enteritidis in Europe: results of an international multi-centre study. Epidemiol. Infect., 135:1274–1281.

28.

Ribot

E.M.

, Fair

M.A.

, Gautom

, Cameron

D.N

, Hunter

S.B.

, Swaminathan

, Barrett

T.J.

2006. Standardization of pulsed-field gel electrophoresis protocols for the subtyping of Escherichia coli O157:H7, Salmonella and Shigella for PulseNet. Foodborne Pathog. Dis., 3:59–67.

29.

Soler

, Gonzalez-Sanz

, Bleda

M.J.

, Hernandez

, Echeita

, Usera

M.A.

2006. Antimicrobial resistance in non-typhoidal Salmonella from human sources, Spain, 2001–2003. J. Antimicrob. Chemother., 58:310–314.

30.

L.H.

, Chiu

C.H.

, Chu

C.S.

, Ou

J.T.

2004. Antimicrobial resistance in nontyphoid Salmonella serotypes: A global challenge. Clin. Infect. Dis., 39:546–551.

31.

Velge

, Cloeckaert

, Barrow

2005. Emergence of Salmonella epidemics: The problems related to Salmonella enterica serotype Enteritidis and multiple antibiotic resistance in other major serotypes. Vet. Res., 36:267–288.

32.

Ward

L.R.

, Desa

J.D.H.

, Rowe

1987. A phage-typing scheme for Salmonella Enteritidis. Epidemiol. Infect., 99:291–294.

33.

Woodford

, Fagan

E.J.

, Ellington

M.J.

2006. Multiplex PCR for rapid detection of genes encoding CTX-M extended-spectrum beta-lactamases. J. Antimicrob. Chemother., 57:154–155.