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Antimicrobial Agents and Chemotherapy, January 2008, p. 353-356, Vol. 52, No. 1
0066-4804/08/$08.00+0     doi:10.1128/AAC.00842-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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

Aaron M. Lynne,1 Bobbie S. Rhodes-Clark,2 Kimberly Bliven,1 Shaohua Zhao,3 and Steven L. Foley1,2*

National Farm Medicine Center, Marshfield Clinic Research Foundation, Marshfield, Wisconsin 54449,1 Department of Biology, University of Central Arkansas, Conway, Arkansas 72035,2 Division of Animal and Food Microbiology, Center for Veterinary Medicine, U.S. Food and Drug Administration, Laurel, Maryland 207083

Received 27 June 2007/ Returned for modification 31 August 2007/ Accepted 11 October 2007


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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.


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TEXT
 
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.


Figure 1
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  		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.


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  		TABLE 1. PCR primers and gene targets for antimicrobial resistance

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 blaCMY, 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 blaCMY and blaTEM. Five additional isolates were positive for blaTEM. 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 blaTEM and blaCMY, 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.


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ACKNOWLEDGMENTS
 
We thank Patrick McDermott of the U.S. Food and Drug Administration for assistance with the review of the manuscript.

We thank the Marshfield Clinic Research Foundation for their financial support for the project.


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FOOTNOTES
 
* Corresponding author. Mailing address: National Farm Medicine Center, Marshfield Clinic Research Foundation, 1000 North Oak Avenue, Marshfield, WI 54449. Phone: (715) 389-4012. Fax: (715) 389-3808. E-mail: foley.steven{at}mcrf.mfldclin.edu Back

{triangledown} Published ahead of print on 29 October 2007. Back


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Antimicrobial Agents and Chemotherapy, January 2008, p. 353-356, Vol. 52, No. 1
0066-4804/08/$08.00+0     doi:10.1128/AAC.00842-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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