Antimicrobial Resistance Genes Associated with Salmonella enterica Serovar Newport Isolates from Food Animals

Abstract

Salmonella enterica serotype Newport is an important cause of salmonellosis, with strains increasingly being resistant to multiple antimicrobial agents. The increase is associated with the acquisition of multiple resistance genes. This study characterizes the genetic basis of resistance of serotype Newport isolates collected from veterinary sources by PCR and DNA sequencing analysis.

Salmonellosis is a significant public health concern in the United States. From 1997 to 2003, the number of laboratory-confirmed Salmonella enterica serovar Newport infections reported to CDC increased from 1,584 (4.6% of all reported Salmonella infections) to 4,000 (10.3%) (5). Over the same period, the percentage of multidrug-resistant serovar Newport isolates increased nearly fivefold (6). Therefore, the increasing number of serovar Newport infections appears to be associated with the emergence and rapid dissemination of multidrug-resistant (MDR) strains of serovar Newport (18). Multidrug resistance is often associated with mobile genetic elements (i.e., plasmids and transposons) that encode multiple specific resistance genes (1, 12, 13, 17, 19).

The objective of this study was to further characterize the genetic basis of multiantimicrobial resistance in Salmonella serovar Newport isolates collected from food animals in the United States. These isolates were previously characterized by pulsed-field gel electrophoresis (PFGE), by antimicrobial susceptibility testing, and for their ability to transfer resistance-conferring plasmids from MDR strains to susceptible ones (19). The subset of isolates included in the present study represents strains isolated from chicken, swine, turkey, and cattle from 23 states in the United States (Fig. 1). The susceptibilities of the isolates to the following agents were determined: amikacin, amoxicillin-clavulanic acid, ampicillin, cefoxitin, ceftiofur, ceftriaxone, cephalothin, chloramphenicol, ciprofloxacin, gentamicin, kanamycin, nalidixic acid, streptomycin, sulfisoxazole, tetracycline, and trimethoprim-sulfamethoxazole. Isolates were screened for the presence of 19 genes associated with resistance to 12 of the antibiotics in which resistance was detected previously (no resistance to amikacin, ciprofloxacin, or nalidixic acid was detected) (19). Resistance genes were detected by PCR with the primers shown in Table 1. The reactions were repeated to confirm the initial results, and representative amplification products were sequenced to verify the identities of the PCR products (7). Additionally, plasmids were isolated (16) and analyzed to determine whether there was commonality between particular plasmids and the resistance observed.

FIG. 1.

Antimicrobial susceptibility testing and antimicrobial resistance gene detection results for the Salmonella serovar Newport isolates characterized in the study. The isolates are arranged by a PFGE profile similarity dendrogram (calculated in reference 19) and are described by culture number (Key), animal of origin (Species), state of origin (State), the sizes and resistance gene inserts in the class 1 integrons (Integron), and the sizes of plasmids isolated from each strain (Plasmids [kb]). For the susceptibility testing results (Resistance), a black box indicates resistance, a light gray box susceptibility, and a darker gray box reduced susceptibility to ceftriaxone. For resistance gene detection (ResGen), a black box indicates that the gene was detected by PCR, and a light gray box indicates the absence of the gene.

TABLE 1.

PCR primers and gene targets for antimicrobial resistance

Gene	Primera	Nucleotide sequence (5′ to 3′)	Annealing temp (°C)b	Resistance mechanism	Resistancec	Reference or source
aadA1	F	TATCAGAGGTAGTTGGCGTCAT	54	Aminoglycoside adenyltransferase	STR	13
	R	GTTCCATAGCGTTAAGGTTTCATT
aadA2	F	TGTTGGTTACTGTGGCCGTA	62	Aminoglycoside adenyltransferase	STR	13
	R	GATCTCGCCTTTCACAAAGC
aadB	F	GAGCGAAATCTGCCGCTCTGG	61	Aminoglycoside adenyltransferase	GEN	13
	R	CTGTTACAACGGACTGGCCGC
aacC	F	GGCGCGATCAACGAATTTATCCGA	58	Aminoglycoside acetyltransferase	GEN	This study
	R	CCATTCGATGCCGAAGGAAACGAT
aphAI-IAB	F	AAACGTCTTGCTCGAGGC	55	Aminoglycoside phosphoryltranferase	KAN	13
	R	CAAACCGTTATTCATTCGTGA
blaCMY	F	GACAGCCTCTCTTTCTCCACA	50	β-Lactamase	AMP	13
	R	TGGAACGAAGGCTACGTA
blaTEM	F	CATTTCCGTGTCGCCCTTAT	55	β-Lactamase	AMP	13
	R	TCCATAGTTGCCTGACTCCC
cat1	F	CCTATAACCAGACCGTTCAG	56	Chlorampenicol acetyltransferase	CHL	13
	R	TCACAGACGGCATGATGAAC
cat2	F	CCGGATTGACCTGAATACCT	56	Chlorampenicol acetyltransferase	CHL	13
	R	TCACATACTGCATGATGAAC
dfrI	F	GTGAAACTATCACTAATGGTAGCT	54	Dihydrofloate reductase inhibitor	TMP	13
	R	ACCCTTTTGCCAGATTTGGTAACT
floR	F	AACCCGCCCTCTGGATCAAGTCAA	60	Efflux	CHL	13
	R	CAAATCACGGGCCACGCTGTATC
strA	F	AGCAGAGCGCGCCTTCGCTC	59	Aminoglycoside phosphoryltranferase	STR	13
	R	CCAAAGCCCACTTCACCGAC
strB	F	ATCGTCAAGGGATTGAAACC	49	Aminoglycoside phosphoryltranferase	STR	3
	R	GGATCGTAGAACATATTGGC
sul1	F	TCACCGAGGACTCCTTCTTC	60	Dihydropteroate synthase inhibitor	SUL	13
	R	AATATCGGGATAGAGCGCAG
sul2	F	CGGTCCGGCATCCAGCAATCC	64	Dihydropteroate synthase inhibitor	SUL	13
	R	CGAGAGCCACGACCGCGCC
sul3	F	GAGCAAGATTTTTGGAATCG	51	Dihydropteroate synthase inhibitor	SUL	3
	R	CATCTGCAGCTAACCTAGGGCTTGGA
tetA	F	GCTACATCCTGCTTGCCTTC	55	Efflux	TET	8
	R	CATAGATCGCCGTGAAGAGG
tetB	F	TTGGTTAGGGGCAAGTTTTG	53	Efflux	TET	8
	R	GTAATGGGCCAATAACACCG
tetC	F	CTTGAGAGCCTTCAACCCAG	56	Efflux	TET	8
	R	ATGGTCGTCATCTACCTGCC
intA	F	GGCATCCAAGCAGCAAGC	50	Class 1 integron		13
	R	AAGCAGACTTGACCTGAT
intB	F	ATCGCAATAGTTGGCGAGT	53	qacEΔ-sul1 of class 1 integron		13
	R	GCAAGGCGGAAACCCGCGCC

^aF, forward; R, reverse.

^bAnnealing temperature for each PCR.

^cThe gene encodes resistance to ampicillin (AMP), chloramphenicol (CHL), gentamicin (GEN), kanamycin (KAN), streptomycin (STR), sulfisoxazole (SUL), tetracycline (TET), and trimethoprim (TMP).

Overall, when resistance was seen, a corresponding resistance gene was detected 96.6% of the time (Fig. 1). Likewise, nearly all of the MDR strains contained large plasmids (≥75 kb), while the pansusceptible strains did not (Fig. 1). Nineteen isolates shared resistance to amoxicillin-clavulanic acid, ampicillin, cephalothin, cefoxitin, and ceftiofur and reduced susceptibility (MIC of 16 or 32 μg/ml) or resistance to ceftriaxone. Each of these isolates was positive for bla_CMY, which is most commonly associated with ceftiofur and ceftriaxone resistance in Salmonella strains. A single isolate was resistant to ceftriaxone and was positive for both bla_CMY and bla_TEM. Five additional isolates were positive for bla_TEM. Two of these five isolates were resistant to ampicillin and cephalothin; one was resistant to ampicillin, amoxicillin-clavulanic acid, and ceftiofur; and two were susceptible to all agents tested. Eleven isolates were resistant to kanamycin, and each isolate contained aphAI-IAB. Twenty-three isolates demonstrated resistance to streptomycin, and of these isolates, 21 were positive for strA, 20 were positive for aadA1, 11 were positive for strB, and 7 were positive for aadA2. Five of the seven gentamicin-resistant isolates carried aacC, while none contained aadB. Both genes have been associated with gentamicin resistance in Salmonella serovar Newport isolates (13, 17). Three isolates contained aacC but were not resistant to gentamicin. Even though they did not meet the threshold for resistance, two had decreased susceptibility (MIC of 1 and 8 μg/ml) compared to the level of susceptibility of the majority of susceptible isolates (MIC ≤ 0.25 μg/ml), likely indicating that the enzymes displayed variable levels of activity in these strains. Twenty-four isolates were resistant to sulfisoxazole; all but one contained sul1, sul2, and/or sul3. Five isolates were resistant to trimethoprim-sulfamethoxazole, and three of these isolates carried the dfrXII resistance gene in a class 1 integron (19). The remaining two isolates contained class 1 integrons that lacked dfr genes. Salmonella strains containing dfrXII have predominantly been reported in Europe and Asia, with few reports of such strains in the United States (2, 4, 9, 12, 15). Conversely, there have been reports of dfrXII-associated resistance in Escherichia coli isolates from the United States (14), indicating that the genes are present in microbial populations. Twenty isolates were resistant to chloramphenicol; 19 of these isolates were positive for floR, while the other isolate contained cat1. Additionally, 23 isolates were resistant to tetracycline. Twenty isolates contained tetA, two had tetB, and one isolate lacked the tet genes that we screened for. In cases in which an associated resistance gene was not identified, resistance was likely due to another member of the resistance gene class.

In most cases, our findings were similar to those reported previously (11, 13); however, the detection of aacC-associated gentamicin resistance is in contrast to the findings from other countries, where aadB was the predominant factor associated with gentamicin resistance in Salmonella serovar Newport (16, 19). Thus, our results, along with those of Welch et al. (17), indicate that aacC may play a larger role than aadB in gentamicin resistance in serovar Newport isolates from the United States. Additionally, the only isolate resistant to ceftriaxone contained both bla_TEM and bla_CMY, which likely indicates synergism among the β-lactamases. Our results also indicated that some isolates with integrons containing aadA1 were susceptible to streptomycin. Previous reports have shown that silent integron-borne aadA genes in Salmonella strains may be expressed when they are transferred to a new host by conjugation (20). All of our streptomycin-susceptible isolates that contained class 1 integrons with aadA1 were isolated from turkeys from multiple states and were spread among divergent PFGE clusters (19), which may indicate that these integrons are disseminated among serovar Newport isolates in turkey flocks. There were some additional differences among the Salmonella isolates from different animal sources, including the fact that tetB was detected only in our swine isolates and that one of these isolates was also the only strain with cat1-associated chloramphenicol resistance.

Overall, cattle and swine isolates demonstrated resistance to the greatest number of antimicrobial agents, which paralleled the results from NARMS from 2001 and 2002 (6). When the PFGE profiles of the isolates were compared, almost all MDR isolates were clustered together (19). These related strains may share a higher capacity to acquire resistance plasmids, thus contributing to the rapid rise of MDR Salmonella serovar Newport isolates over the last decade as important causes of human salmonellosis. The results of this study provide further information on the contribution of specific resistance genes to MDR in serovar Newport strains.