APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 2009, p. 1908–1915 Vol. 75, No. 70099-2240/09/$08.00�0 doi:10.1128/AEM.02228-08

Inc A/C Plasmids Are Prevalent in Multidrug-ResistantSalmonella enterica Isolates�†

Rebecca L. Lindsey, Paula J. Fedorka-Cray, Jonathan G. Frye, and Richard J. Meinersmann*U.S. Department of Agriculture, Agricultural Research Service, Bacterial Epidemiology and Antimicrobial Resistance Research Unit,

Richard B. Russell Agricultural Research Center, 950 College Station Road, Athens, Georgia 30605-2720

Received 26 September 2008/Accepted 12 January 2009

Bacterial plasmids are fragments of extrachromosomal double-stranded DNA that can contain a variety ofgenes that are beneficial to the host organism, like those responsible for antimicrobial resistance. The objectiveof this study was to characterize a collection of 437 Salmonella enterica isolates from different animal sourcesfor their antimicrobial resistance phenotypes and plasmid replicon types and, in some cases, by pulsed-field gelelectrophoresis (PFGE) in an effort to learn more about the distribution of multidrug resistance in relation toreplicon types. A PCR-based replicon typing assay consisting of three multiplex PCRs was used to detect 18 ofthe 26 known plasmid types in the Enterobacteriaceae based on their incompatibility (Inc) replicon types.Linkage analysis was completed with antibiograms, replicon types, serovars, and Inc A/C. Inc A/C plasmidswere prevalent in multidrug-resistant isolates with the notable exception of Salmonella enterica serovar Typhi-murium. Cluster analysis based on PFGE of a subset of 216 isolates showed 155 unique types, suggesting avariable population, but distinct clusters of isolates with Inc A/C plasmids were apparent. Significant linkageof serovar was also seen with Inc replicon types B/O, I1, Frep, and HI1. The present study showed that thecombination of Salmonella, the Inc A/C plasmids, and multiple-drug-resistant genes is very old. Our resultssuggest that some strains, notably serovar Typhimurium and closely related types, may have once carried theplasmid but that the resistance genes were transferred to the chromosome with the subsequent loss of theplasmid.

Bacterial plasmids are self-replicating extrachromosomalfragments of double-stranded DNA. They range in size from afew to several hundred kilobase pairs. Plasmids can contain avariety of genes that are beneficial to the survival of the hostbacteria. These sequences can encode antimicrobial and heavymetal resistance, toxin production, or virulence factors thatallow their bacterial host to adapt to changing environments(2, 12, 35). The classification and tracking of plasmids is ben-eficial because they are potentially a medium of horizontalgene transfer of drug resistance (5, 12, 24). Horizontal transferof DNA in prokaryotes occurs in three forms: transformation,conjugation, or transduction (12, 30). Plasmids contain a rep-licon that consists of sequences that are necessary for self-replication in a host cell, which includes the origin of replica-tion, control of initiation, and replication functions (5, 10).Plasmids can be classified according to incompatibility (Inc)types that are based on the inability of plasmids with the samereplication mechanism to exist in the same cell (10, 22). Dif-ferent plasmids of the same Inc type cannot coexist in onebacterial strain. In the Enterobacteriaceae, there are 26 knownInc types or replicon types. A PCR-based replicon typing assayhas been developed to distinguish 18 replicon types (6, 18, 19).

Certain replicon types are associated with multidrug resis-tance as well as bacteria implicated in disease outbreaks or

found in food-producing animals. Multidrug-resistant (MDR)Salmonella strains are responsible for human outbreaks andmay be acquired through food animals, which are a majorsource of zoonotic pathogens (3, 16, 23, 25, 35, 36). Twenty-two MDR Salmonella enterica serovar Heidelberg isolates fromturkey-associated sources were found to have XbaI pulsed-field gel electrophoresis (PFGE) profiles that were indistin-guishable from the most common profile associated with hu-man infection. Conjugation experiments confirmed that theone tested representative isolate was able to transfer a largeplasmid of approximately 120 kb (20). The Inc A/C plasmidbackbone from MDR Salmonella enterica serovar Newport wasfound to have a backbone similar to that of plasmid pIP1202from Yersinia pestis, the causative agent of the plague, as wellas that of plasmid pYR71 from Yersinia ruckeri, a fish pathogen(34). MDR Salmonella enterica serovar Newport with plasmid-mediated extended-spectrum cephalosporin resistance was iso-lated from animals during a salmonellosis outbreak that led tothe closure of the large animal hospital at the University ofPennsylvania’s New Bolton Center (26). In clinical human iso-lates of the Enterobacteriaceae, Inc A/C or Inc N plasmids havebeen shown to be associated with extended-spectrum cephalo-sporin and carbapenem resistance determinants emerging inGreece, Italy, and the United States (7). Inc I1 and A/C rep-licon types are associated with plasmids carrying and dissemi-nating extended-spectrum �-lactamase genes in animals andhumans (9, 13, 18). The Inc HI1 replicon type is associatedwith an MDR plasmid, plasmid pHCM1, found in Salmonellaenterica serovar Typhi, recovered during an MDR typhoid fe-ver outbreak in Vietnam from 1993 to 1996 (33). Salmonellaenterica serovar Paratyphi A was found to contain an MDR

* Corresponding author. Mailing address: USDA-ARS-BEAR,Richard B. Russell Research Center, P.O. Box 5677, Athens, GA30604-5677. Phone: (706) 546-3236. Fax: (706) 546-3018. E-mail:rmeiners@saa.ars.usda.gov.

† Supplemental material for this article may be found at http://aem.asm.org/.

� Published ahead of print on 30 January 2009.

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IncHI1 plasmid, plasmid pAKU_1, and, in some regions, canbe the causative agent of enteric fever (17). To further studythe Inc A/C plasmid, we characterized the antimicrobial resis-tance profiles and the presence of replicon types in 437 ampi-cillin- and tetracycline-resistant Salmonella enterica isolates re-covered from ill animals in 2005. Amp- or Tet-resistant isolateswere selected because they are more likely to have a plasmid.

MATERIALS AND METHODS

Bacterial strains and plasmids. Salmonella enterica strains in this study werecollected from samples submitted to veterinary diagnostic laboratories in 2005 inwhich Salmonella was identified as being either the primary or secondary etio-logical agent associated with the illness, as previously described (http://www.ars.usda.gov/Main/docs.htm?docid�6750&page�1). Veterinary diagnostic labora-tories obtained isolates from the state in which they are located; the isolates weresent to the National Veterinary Services Laboratory (NVSL), Ames, IA (www.aphis.usda.gov/animal_health/lab_info_services/about_nvsl.shtml); and we ob-tained the isolates from the NVSL. All strains tested for plasmid Inc types wereresistant to ampicillin and/or tetracycline. The 437 isolates in this study (seeTable S1 in the supplemental material) represent 55 serovars (including nonmo-tile and untypeable) from 17 different sources including canine (dog), cattle,chicken, dairy cattle, equine, environment, feline (domestic cat), parrot, reptile(turtle and lizard), swine, turkey, wild avian (pheasant and unknown spp.), wildmammal (alpaca, mongoose, and unknown spp.), and wild rodent (unknownspp.). Strains were obtained from 28 states in all five regions in the United Statesas defined for the NARMS program (http://www.ars.usda.gov/Main/docs.htm?docid�6750) and included region 1, the Northeast (Connecticut, Dela-ware, Indiana, Massachusetts, Maryland, Maine, Michigan, New Hampshire,New Jersey, New York, Ohio, Pennsylvania, Rhode Island, and Vermont); region2, the Southeast (Alabama, Florida, Georgia, Kentucky, North Carolina, PuertoRico, South Carolina, Tennessee, Virginia, and West Virginia); region 3, theMidwest (Iowa, Illinois, Kansas, Minnesota, Missouri, North Dakota, Nebraska,South Dakota, and Wisconsin); region 4, the Southwest (Arkansas, Louisiana,Mississippi, Oklahoma, and Texas); and region 5, the West (Arizona, California,Colorado, Idaho, Montana, New Mexico, Nevada, Oregon, Utah, Washington,and Wyoming).

Positive controls used in the replicon typing procedure were originally createdin the laboratory of Werner K. Maas (6, 10) and generously provided by Ales-sandra Carattoli (Istituto Superiore di Sanita, Rome, Italy). All bacterial strainswere stored at �80°C in LB lennox (Hardy Diagnostics, Santa Maria, CA) with15% glycerol or stored at room temperature in tryptic soy agar (Hardy Diag-nostics, Santa Maria, CA) slants until use.

Antimicrobial susceptibility testing. Each Salmonella enterica isolate wastested for susceptibility to a panel of 15 antimicrobial drugs using the Sensititersystem (Trek Diagnostic Systems Inc., Westlake, OH) that included amikacin,amoxicillin-clavulanic acid, ampicillin, cefoxitin, ceftiofur, ceftriaxone, chloram-phenicol, ciprofloxacin, gentamicin, kanamycin, nalidixic acid, streptomycin, sul-famethoxazole, tetracycline, and trimethoprim-sulfamethoxazole as defined bythe NARMS program (http://www.ars.usda.gov/Main/docs.htm?docid�6750).Each isolate was classified as being susceptible, intermediate, or resistant usingClinical and Laboratory Standards Institute (CLSI; formerly National Commit-tee for Clinical Laboratory Standards) breakpoints when available; otherwise,breakpoint interpretations from the National Antimicrobial Resistance Moni-toring System were used (21). For linkage analyses described below, intermediatesensitivity was not distinguished from the susceptible trait.

Multiplex PCR for plasmid replicon typing. Salmonella isolates were exam-ined by PCR using three multiplex primer panels for the presence of 18 plasmidreplicons as described previously by Johnson et al. (19). This replicon typingprocedure is a modified version of eight PCR multiplex and simplex reactionsdescribed previously by Carattoli et al. (6). Primers were obtained from a Eu-rofins MWG operon (Huntsville, AL). Inc-type reference plasmid DNA wasextracted from 2 ml LB broth cultures grown overnight with appropriate antibi-otics and processed with a Qiaprep spin miniprep kit (Qiagen, Valencia, CA).Template DNA for PCR from the 437 Salmonella isolates was prepared bysuspending a single colony in 200 �l sterile water and treating in a boiling waterbath for 10 min. PCRs were performed according to the polymerase manufac-turer’s instructions, with a final volume of 25 �l: 5 �l of boiled lysate or 10 ng ofeach reference plasmid, 1� AmpliTaq buffer 1, 0.50 �M of each primer, 4 mMMgCl2, 0.2 mM of each deoxynucleoside triphosphate, and 1.25 units of Ampli-Taq polymerase (Applied Biosystems, Foster City, CA). Positive controls as well

as a negative control without DNA were run with each multiplex primer panel.PCR cycle conditions were as follows: 5 min at 94°C; 30 cycles of 30 s at 94°C, 30 sat 60°C, and 90 s at 72°C; and a final extension step of 5 min at 72°C. Ampliconswere visualized on 1� Tris-borate-EDTA 2% agarose gels run for 2 h at 80 Valongside a TrackIt 1-kb Plus DNA ladder (Invitrogen Corporation, Carlsbad,CA). An isolate was considered to be positive for a particular gene if an ampliconof the expected size was observed.

PFGE. A total of 223 isolates of the original 437 strains were analyzed by a24-h Salmonella PFGE protocol as described by PulseNet (8) at the USDAVetNet Laboratory (Athens, GA). Briefly, genomic DNA was digested with 10 Uof XbaI (Roche Molecular Biochemicals, Indianapolis, IN) and separated withthe CHEF-Mapper XA PFGE system (Bio-Rad, Hercules, CA) in 0.5� Tris-borate-EDTA buffer at 14°C at 6 V for 18 h with a ramped pulse time of 2.16 to63.8 s. The BioNumerics software program (Applied Maths Scientific SoftwareDevelopment, Sint-Martens-Latem, Belgium) was applied for cluster analysisusing the Dice-based coefficients with a 1.5% band tolerance and 1.5% optimi-zation with coefficient and the unweighted pair-group method.

DT104 analysis. Pentaresistant (ampicillin, chloramphenicol, streptomycin,sulfonamides, and tetracycline [ACSSuT] resistance) Salmonella enterica serovarTyphimurium isolates were phage typed at the National Veterinary ServicesLaboratory, Ames, IA (www.aphis.usda.gov/animal_health/lab_info_services/about_nvsl.shtml).

Statistical analysis. Linkage disequilibrium (LD) was calculated as an exten-sion of Fisher’s exact probability test on contingency tables (27) as instituted bythe program Arlequin (11). Standard settings were used, 10,000 steps in theMarkov chain and 1,000 dememorization steps; and calculations of D, D�, and r2

coefficients were made with a significance level of 0.05.

RESULTS

Characterization of Salmonella enterica isolates. The totalnumber of Salmonella enterica diagnostic isolates in the collec-tion was 1,548; 842 (54%) of these were Amp or Tet resistant.Strains that were identical in all of three traits, serovar, state/region, and host animal, were removed from the set to de-crease redundancy. We characterized the remaining 437 Sal-monella enterica isolates for their antimicrobial resistances andreplicon types. These isolates represent 55 serovars from 17different host sources and were obtained from 28 states in fiveregions of the United States (see Table S1 in the supplementalmaterial). When the original parameters of Amp or Tet resis-tance were examined in the characterized strains, 224 (51%)were both Amp and Tet resistant, 47 (11%) were only Ampresistant, and 166 (38%) were only Tet resistant. The top fourprevalent antimicrobial resistances by percent in the popula-tion are tetracycline (89.2%), streptomycin (74.8%), sulfame-thoxazole (73.2%), and ampicillin (62.0%) (Table 1). Cipro-floxacin is the only tested drug that did not show resistance inany of the strains, and it was not included in further analyses.Sixty-five percent of the strains analyzed show resistance tofour or more of the antimicrobials on our panel.

Replicon typing, a PCR-based assay consisting of three mul-tiplex primer panels, was conducted on all Salmonella isolatesfor the presence of 18 different plasmid based Inc types (19).The most prevalent replicon types by percent in the populationthat we characterized were Inc A/C (26.3%), I1 (23.6%), HI1(6.4%), and FIIA (2.5%) (Table 2). Forty-seven percent of ourstrains did not show a positive reaction for any replicon type,but our assay detects only 18 of the 26 known replicon types inthe Enterobacteriaceae. We did not identify any isolates thatcarried the FIC, K/B, T, or W Inc replicon type.

Inc A/C was present in 115 strains and 21 out of 53 serovars(not including nonmotile and untypeable) (Table 3). Inc A/Cwas not found in isolates from two states in separate regions,and each of these consisted of only one isolate. Inc A/C was

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found in all source species except for wild rodent, parrot,pheasant, and unknown avian spp.; each of these groups con-sisted of only one isolate (Table 3). Recognizing the selectionfor the study of isolates that are Amp or Tet resistant, in the 93Inc A/C-positive strains, 88 (95%) were both Amp and Tetresistant, none were only Amp resistant, and 5 (5%) were onlyTet resistant. When the Amp or Tet resistance was examinedin the 81 Inc I1-positive strains, 21 (26%) were Amp and Tetresistant, 11 (14%) were only Amp resistant, and 49 (60%)were only Tet resistant. In the 22 isolates that were both IncA/C and Inc I1 positive, 21 (95%) were both Amp and Tetresistant, 0 (0%) were only Amp resistant, and 1 (5%) was onlyTet resistant. In the remaining 241 isolates that were not IncA/C or Inc I1 positive, 94 (39%) were both Amp and Tetresistant, 36 (15%) were only Amp resistant, and 111 (46%)were only Tet resistant.

Pairwise LD between the antimicrobial resistance profile

and Salmonella enterica isolate serovar showed a linkage (Pvalue of �0.05) with all the antimicrobials tested except ami-kacin and nalidixic acid (data not shown). Resistance to thesetwo antimicrobials showed low representation in our popula-tion, with nalidixic acid at 1.4% and amikacin at 0.5% (Table1). Pairwise LD between antimicrobial resistance profiles andInc A/C replicon type of Salmonella enterica isolates showslinkage (P value of �0.05) with all antimicrobials tested exceptamikacin, ceftriaxone, ciprofloxacin, nalidixic acid, streptomy-cin, and sulfamethoxazole (data not shown). Pairwise LD be-tween 14 replicon types and Salmonella enterica serovars showlinkage (P value � 0.05) of serovar and Inc A/C, B/O, Frep,HI1, and I1 (Fig. 1). A nonrandom association is seen betweenstrain serovar and the Inc A/C, B/O, Frep, HI1, I1, and A/Csubsets (Fig. 1).

PFGE analysis was conducted on 216 of the 437 total isolatesincluding 98 of 104 Inc A/C-positive isolates (Fig. 2). Six strainswere not included in the cluster analysis because theirPFGE patterns were inconsistent with serovar results (E.McGlinchey, personal communication). PFGE analysis wasconducted on 118 Inc A/C-negative isolates that were ran-

TABLE 1. Antimicrobial resistance of strains in this studya

Antimicrobial resistance trait No. (%) withprofile

Tetracycline ...............................................................................390 (89.2)Streptomycin..............................................................................327 (74.8)Sulfamethoxazole ......................................................................320 (73.2)Ampicillin ..................................................................................271 (62.0)Chloramphenicol.......................................................................172 (39.4)Kanamycin .................................................................................170 (38.9)Ceftiofur.....................................................................................132 (30.2)Amoxicillin-clavulanic acid ......................................................132 (30.2)Cefoxitin.....................................................................................131 (30.0)Gentamicin ................................................................................ 82 (18.8)Trimethoprim-sulfamethoxazole ............................................. 54 (12.3)Ceftriaxone ................................................................................ 14 (3.2)Nalidixic acid............................................................................. 6 (1.4)Amikacin.................................................................................... 2 (0.5)Ciprofloxacin ............................................................................. 0 (0.0)

a Shown are the numbers and percentages of isolates exhibiting resistance to atleast Amp and/or Tet and each of the other antimicrobials tested.

TABLE 2. Plasmid replicon typing of strains in this studya

Inc/Rep type No. (%) withprofile

None ......................................................................................208 (47.5)A/C.........................................................................................115 (26.3)I1 ............................................................................................103 (23.6)HI1 ......................................................................................... 28 (6.4)FIIA ....................................................................................... 11 (2.5)Frep........................................................................................ 11 (2.5)FIB ......................................................................................... 4 (0.9)N............................................................................................. 4 (0.9)P ............................................................................................. 2 (0.5)Y............................................................................................. 2 (0.5)X............................................................................................. 2 (0.5)B/O......................................................................................... 2 (0.4)FIA......................................................................................... 1 (0.2)HI2 ......................................................................................... 1 (0.2)L/M ........................................................................................ 1 (0.2)FIC ......................................................................................... 0 (0.0)K/B ......................................................................................... 0 (0.0)T ............................................................................................. 0 (0.0)W............................................................................................ 0 (0.0)

a Shown are numbers of isolates and the percentages of our total samplepopulation with each replicon type tested.

TABLE 3. Inc A/C prevalence by populationa

S. enterica serovar orsource species

No. of Inc A/C-positive isolatesin population/

total no. ofisolates (%)

S. enterica serovarAgona ..................................................................................12/18 (67)6,7:-:l,w ................................................................................ 1/2 (50)Bardo................................................................................... 3/3 (100)Bredeney ............................................................................. 4/5 (80)Choleraesuis variant kunzendorf ..................................... 3/15 (20)Derby................................................................................... 5/41 (12)Dublin.................................................................................. 1/3 (33)Havana ................................................................................ 1/3 (33)Heidelberg .......................................................................... 4/27 (15)Infantis ................................................................................ 1/4 (25)London................................................................................ 1/1 (100)Mbandaka ........................................................................... 2/6 (33)Newport ..............................................................................40/44 (91)Nonmotile ........................................................................... 1/2 (50)Ohio..................................................................................... 3/3 (100)Reading ............................................................................... 9/9 (100)Typhimurium...................................................................... 9/38 (24)Typhimurium variant 5� .................................................. 2/55 (4)Uganda................................................................................ 8/9 (89)Yovokome........................................................................... 1/1 (100)

Source speciesAlpaca ................................................................................. 1/1 (100)Canine ................................................................................. 7/11 (64)Cattle ...................................................................................38/80 (47.5)Chicken ............................................................................... 3/24 (12.5)Dairy cattle ......................................................................... 9/26 (11.5)Environmental.................................................................... 2/8 (25)Equine .................................................................................17/30 (57)Feline................................................................................... 1/4 (25)Lizard .................................................................................. 1/1 (100)Miscellaneous mammal..................................................... 2/3 (67)Swine ...................................................................................25/178 (14)Turkey ................................................................................. 6/61 (10)Turtle................................................................................... 2/2 (100)

a Shown are numbers of Inc A/C-positive isolates/total numbers of isolates perpopulation, with the percentages in parenthesis.

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domly selected, including 49 Inc I1-positive strains (51 addi-tional isolates containing Inc I1 were identified but were notincluded in PFGE analysis) as well as 69 other strains that werepicked as a reference set. Cluster analysis based on PFGEshowed 155 unique types, suggesting a variable population.The source animal of the isolate did not appear to have acorrelation to cluster analysis. Inc A/C-positive strains have ahigh degree of multidrug resistance compared to strains with-out any detected replicon type, which have a reduced antimi-crobial profile. However, Inc A/C-positive strains of Salmo-nella enterica serovar Choleraesuis variant Kunzendorf wereresistant to sulfamethoxazole and tetracycline only. Inc A/C-positive strains form clusters, whereas Inc I1 appears random,and not enough information was available to detect linkagepatterns among the B/O, Frep, and HI1 Inc types (Fig. 2).

This study included 38 isolates of serovar Typhimurium and55 isolates of serovar Typhimurium variant 5�. Fifty percent ofthe population of Salmonella enterica serovar Typhimuriumor serovar Typhimurium variant 5� isolates were pentar-esistant (ACSSuT) (or more) and phage typed. Thirty-four per-cent of the isolates of these serovars were DT104 positive(Table 4). Ten percent were Inc A/C-positive and pentaresis-tant (or more), and only 1% were Inc A/C and DT104 positive(Table 4).

DISCUSSION

The objective of this study was to characterize a large pop-ulation of Salmonella enterica isolates from different animalsources for their antimicrobial resistance phenotypes and plas-mid replicon type and, in some cases, by PFGE in an effort tolearn more about the distribution of multidrug resistance inrelation to Inc A/C as well as the other replicon types which weexamined. PFGE was conducted on approximately one-half ofthe study population and was used for cluster analysis. Linkageanalysis was completed with antibiograms, replicon types, ser-ovars, and Inc A/C groups.

Cluster analysis based on PFGE of 216 isolates showed 155

unique types. Cluster analysis showed that Inc A/C-positivestrains form groups based on multidrug resistance and serovar,whereas Inc I1-positive isolates did not appear to be clonallydistributed. This indicated that Inc A/C plasmids have beenstably associated with clones for a very long time (however,molecular clocks cannot be accurately constructed with a phy-logenetic analysis of PFGE) (29), while Inc I1 plasmids aremuch more mobile. Inc A/C plasmids are large, 150 kb, whileInc I1 plasmids are frequently approximately 100 kb (9, 34).Larger plasmids usually transfer at lower frequencies thansmaller plasmids and can be expected to be more stable (14).It can also be inferred from the stable association with cloneswith similar resistance phenotypes that the plasmid itself isvery old and that the linkage of the resistance genes does notrepresent a recent accretion.

The cluster analysis shows seven groups that may be consid-ered to be epidemic clones, highly successful clones, which, forthe sake of this discussion, were considered to be PFGE types(28) with three or more identical individuals (labeled clones Ato G) (Fig. 2). There is some replicon type diversity in theseclones; two have diversity with regard to Inc A/C (clones A andB), three have diversity with regard to Inc I1 (clones C, D, andE), two have diversity with regard to Inc F1B (clones B and C),and one does not have any detected replicon types associatedwith it (clone G). We also found diversity in the antimicrobialresistance profiles associated with these seven clones; oneclone has no diversity with regard to antimicrobials (clone G),two clones have diversity with regard to one antimicrobial(clone A and F), three clones have diversity with regard to twoantimicrobials (clone B, C, and E), and one clone has diversitywith regard to three antimicrobials (clone D).

Most of the Salmonella enterica isolates in this study thatcarried the Inc A/C plasmids were multidrug resistant, which isconsistent with the findings described previously by Welch etal. (34). We observed that Inc A/C-positive strains carry moreantimicrobial resistance than isolates with other replicon types,so it was of interest to note a clone with three Inc A/C-positiveindividuals of serovar Choleraesuis variant Kunzendorf, which

FIG. 1. Pairwise linkage disequilibrium among 14 replicon types, serovars (ST) of Salmonella enterica strains, and Inc A/C types based on aPFGE dendrogram. A “�” indicates a P value of 0.05 or less, indicating significant linkage, and a “�” indicates a P value of greater than 0.05. 1,18 subsets were used for LD. Seventeen subsets contained two or more Inc A/C-positive isolates in clades with 80% or better identity by PFGE.One subset consisted of seven individuals of serovar Derby, all Inc A/C negative, and showed 100% identity by PFGE. The remaining isolates weredesignated members of a null group, and the LD was calculated and listed as a subset.

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FIG. 2. PFGE-based dendrogram, replicon typing, resistance profile, and strain serovar. The dendrogram is based on PFGE analyses using BioNumericssoftware. Epidemic clones are labeled A to G. Replicon typing results are to the right of the dendrogram; black indicates that the trait is present, and grayindicates that the trait is absent (columns 2 to 15 indicate replicon type). For the antimicrobial resistance phenotype (columns 16 to 28), black indicates resistance;white indicates intermediate or susceptible and the isolate serovar (column 29). 1, antimicrobial resistance phenotype: amoxicillin-clavulanic acid (AMO),ampicillin (AMP), cefoxitin (FOX), ceftiofur (TIO), ceftriaxone (AXO), chloramphenicol (CHL), gentamicin (GEN), kanamycin (KAN), nalidixic acid (NAL),streptomycin (STR), sulfamethoxazole (SUL), tetracycline (TET), and trimethoprim-sulfamethoxazole (TRI).

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were resistant to sulfamethoxazole and tetracycline only. It ispossible that these three isolates carry other resistance genesthat are not expressed. All of the Salmonella enterica serovarCholeraesuis variant Kunzendorf strains in this study origi-

nated from swine in region 3, and they had antimicrobial re-sistance profiles of two to five drugs. We infer from the PFGEanalysis that the serovar Choleraesuis variant Kunzendorfclone is a stable clone that is being amplified and is undergoing

FIG. 2—Continued.

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little change despite the lack of important antimicrobial resis-tance genes.

It is interesting to compare the clades that include serovarsTyphimurium and Heidelberg with the clade that includes ser-ovar Newport or the clade that includes serovar Derby. Pen-taresistance (ACSSuT resistance) was seen to be commonamong Salmonella enterica serovar Typhimurium isolates, al-though Inc A/C plasmids were not prevalent in the clade, andother plasmid types were scattered throughout the clade. Pen-taresistance in serovar Typhimurium is often associated withphage type DT104, which has multiple resistance genes locatedin an island on the chromosome (4, 15, 31, 32). In this study,serovar Typhimurium and serovar Typhimurium variant 5�ACSSuT (or more) resistance was more frequently associatedwith carrying the DT104 phenotype than having the Inc A/Cplasmid (Table 4): 34% of the isolates of these serovars wereDT104 positive and pentaresistant, while 10% were Inc A/Cpositive and pentaresistant (or more). In contrast, the cladethat includes serovar Newport isolates has a high prevalence ofInc A/C-type plasmids associated with resistance to eight ormore antimicrobials, and the clade that includes serovar Derbyhas Inc A/C plasmids infrequently and little resistance to beta-lactam antimicrobials. From these considerations, we hypoth-esize that the serovar Newport clade had an Inc A/C plasmid atits origin, while the serovar Derby isolates with Inc A/C plas-mids may represent recent acquisitions. The serovar Typhi-murium clade may have had an Inc A/C plasmid early in itsgenesis, but it was largely lost, perhaps after transferring genesthat helped to stabilize the plasmid, including antimicrobialresistance genes, to the chromosome. Welch et al. (34) previ-ously compared the DNA sequences of three Inc A/C plasmidbackbones: pSN254 from an MDR Salmonella enterica serovarNewport isolate; pIP1202 from Yersinia pestis, the causativeagent of the plague; as well as plasmid pYR71 from an isolateof the fish pathogen Yersinia ruckeri that was found to beresistant to a large number of antimicrobials. When the se-quence of the DT104 resistance island (GenBank accessionnumber AF071555) (4) was compared to these plasmids byBLAST (1), there was a very high degree of similarity of re-

sistance genes, supporting the hypothesis that the genes have acommon origin. An alternative hypothesis is that the MDRgenes originated in serovar Typhimurium and were transferredto an Inc A/C plasmid that was not amplified with serovarTyphimurium. With PFGE data, it is difficult to distinguishbetween the two hypotheses because proper rooting of the treecannot be established (29). However, when PFGE data werereanalyzed, including 15 isolates of serovar Typhimurium thatwere pansusceptible and Inc A/C negative, they fell into sep-arate clades that appear more ancient than the clades repre-senting the MDR strains in several alternative rootings (datanot shown).

In conclusion, the present study showed that the combina-tion of Salmonella species, the Inc A/C plasmids, and MDRgenes is very old. Our results suggest that some strains, notablyserovar Typhimurium and closely related types, used to carrythe plasmid and had a transfer of resistance genes to thechromosome with a subsequent loss of the plasmid. This meansthat MDR Salmonella strains have been around in numberstoo low to be found for much longer than humans had anyinfluence, that there has not been substantial new accretion ofresistance by a recent acquisition of existing genes into newstrains, and that any increase in levels of these types of strainsrepresents clonal expansion that may or may not be driven bymodern practices.

ACKNOWLEDGMENTS

We acknowledge Alessandra Carattoli for replicon typing controlstrains and Shayla Hunter, Sandra House, Takiyah Ball, CherylGresham, Carolina Hall, Beth McGlinchey, Jovita Haro, and TylerWilcher for technical assistance.

The mention of trade names or commercial products in the manu-script is solely for the purpose of providing specific information anddoes not imply recommendation or endorsement by the U.S. Depart-ment of Agriculture.

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TABLE 4. Prevalence of pentaresistance, DT104, and Inc A/Cplasmids in strains in this studya

Prevalence ofpentaresistant

plasmids

Prevalence of: No. (%) of positive isolates

DT104 Inc A/C SerovarTyphimurium

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variant 5�

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� � � 4 (10.5) 5 (9) 9 (9.7)� � � 13 (34) 18 (33) 31 (33)� � � 0 1 (2) 1 (1)� � � 0 0 0� � � 1 (2.6) 0 1 (1)� � � 1 (2.6) 2 (3.6) 3 (3.2)� � � 8 (21.5) 1 (1.8) 9 (9.6)� � � 11 (29) 28 (51) 39 (42)

a The three leftmost columns indicate the presence (�) or absence (�) of thephenotype. The remaining columns contain the numbers of positive individualsand the percentages of our total serovar population in parenthesis. The columnto the far right contains the combined totals of serovar Typhimurium and serovarTyphimurium variant 5� columns.

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