APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 2006, p. 6938–6947 0099-2240/06/$08.00⫹0 doi:10.1128/AEM.01368-06 Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 72, No. 11
Identification of Specific Gene Sequences Conserved in Contemporary Epidemic Strains of Salmonella enterica䌤† Min-Su Kang,1 Thomas E. Besser,1 Dale D. Hancock,2 Steffen Porwollik,3 Michael McClelland,3 and Douglas R. Call1* Department of Veterinary Microbiology and Pathology1 and Department of Veterinary Clinical Sciences,2 College of Veterinary Medicine, Washington State University, Pullman, Washington, and Sidney Kimmel Cancer Center, San Diego, California3 Received 14 June 2006/Accepted 19 July 2006
Genetic elements specific to recent and contemporary epidemic strains of Salmonella enterica were identified using comparative genomic analysis. Two epidemic multidrug-resistant (MDR) strains, MDR Salmonella enterica serovar Typhimurium definitive phage type 104 (DT104) and cephalosporin-resistant MDR Salmonella enterica serovar Newport, and an epidemic pansusceptible strain, Salmonella serovar Typhimurium DT160, were subjected to Salmonella gene microarray and suppression subtractive hybridization analyses. Their genome contents were compared with those of coexisting sporadic strains matched by serotype, geographic and temporal distribution, and host species origin. These paired comparisons revealed that epidemic strains of S. enterica had specific genes and gene regions that were shared by isolates of the same subtype. Most of these gene sequences are related to mobile genetic elements, including phages, plasmids, and plasmid-like and transposable elements, and some genes may encode proteins conferring growth or survival advantages. The emergence of epidemic MDR strains may therefore be associated with the presence of fitness-associated genetic factors in addition to their antimicrobial resistance genes. Salmonella enterica epidemics often involve rapid dissemination of predominant epidemic strains over large geographical distances (24, 42). For example, multidrug-resistant (MDR) Salmonella enterica serovar Typhimurium DT104 has been consistently reported as the cause of both human and animal salmonellosis worldwide since it was first reported to be epidemic in England and Wales in the 1980s (7, 24). Epidemic dissemination of predominant strains was also observed for other Salmonella serovar Typhimurium phage types, including DT204 (43), DT193 (43), DT208 (4), and DT10 (30), and for MDR Salmonella enterica serovar Wien (17) in the 1970s. Recently, MDR Salmonella enterica serovar Newport with AmpC (CMY-2) -lactamase-mediated cephalosporin resistance has been recognized as epidemic in humans and animals in the United States (49, 50). Therefore, the steady emergence of these epidemic strains has been an important concern for human and animal health. The factors responsible for the emergence of new epidemic strains of Salmonella are not understood, but it is possible that antimicrobials play a role in the emergence and persistence of epidemic MDR strains (42, 50). Nevertheless, withdrawal of antimicrobials often fails to have a significant effect on the prevalence of MDR Salmonella in food animals (48), and in some cases epidemic strains can persist despite a selective disadvantage from antimicrobials (6, 25). Indeed, pansusceptible epidemic strains of S. enterica, which are susceptible to all antimicrobials tested, have been recognized in humans and
animals in Canada and New Zealand (30, 47). Therefore, the emergence and dissemination of epidemic strains does not necessarily require antimicrobial selection pressure, and successful epidemic strains probably have other traits that allow more efficient dissemination of the strains in specific host populations and environments relative to other, coexisting strains. The purpose of this study was to identify genetic traits unique to epidemic strains of Salmonella. Two MDR and one pansusceptible epidemic strain of S. enterica were compared with nonepidemic strains matched on the basis of shared serotype, geographic and temporal distribution, and host species origin. The comparative studies of matched pairs included genomic microarray hybridizations using Salmonella serovar Typhimurium LT2 as a reference. Subsequently, suppression subtractive hybridization (SSH) libraries were constructed using Salmonella serovar Typhimurium LT2 as the driver and epidemic strains as the tester sequences. After unique gene fragments were identified, they were used to construct a second microarray for additional genomic comparisons. These comparisons showed the extent of variation in gene content between the different strains and identified specific genes and gene loci that were conserved in epidemic strains but frequently missing in nonepidemic strains.
MATERIALS AND METHODS Bacterial strains and sources. This study included 81 bovine isolates of Salmonella serovars Typhimurium and Newport that are archived by the Field Disease Investigation Unit (FDIU) at Washington State University (Pullman) and 9 avian isolates of Salmonella serovar Typhimurium kindly provided by Maurice Alley, Massey University, New Zealand. Thirty-nine isolates from this collection were used for comparative genomic studies (Table 1). The bovine isolates were collected from clinical cases in the Pacific Northwest between 1993 and 2002. Serotypes and phage types of the isolates were determined by the National Veterinary Service Laboratories (Ames, Iowa), and their resistance
* Corresponding author. Mailing address: Department of Veterinary Microbiology and Pathology, Washington State University, 402 Bustad Hall, Pullman, WA 99164-7040. Phone: (509) 335-6313. Fax: (509) 335-8529. E-mail:
[email protected]. † Supplemental material for this article may be found at http://aem .asm.org/. 䌤 Published ahead of print on 8 September 2006. 6938
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TABLE 1. Sources and characteristics of relevant S. enterica isolates tested in this study Strain IDa
Source Serotype
Yr Host
Geographical
Phage type or other subtypeb
Defined epidemicity
Resistance patternc
PFGE patternd
AMP CHL STR TSU TET AMP CHL STR TSU TET AMP CHL STR TSU TET AMP CHL KAN STR TSU TET AMP CHL STR TSU TET AMP CHL STR TSU TET AMP STR TSU TET AMP CHL STR TSU TET AMP KAN STR TSU TET AMP KAN STR TSU TET AMP CHL STR TSU TET AMP CHL GEN KAN STR TSU TET AMP CHL STR TSU TET AMC CEF CAZ FOX CTF AMP CHL GEN KAN STR TSU SXT TET AMC CEF CAZ FOX CTF AMP CHL STR TSU TET AMC CEF CAZ FOX CTF AMP CHL STR TSU TET AMC CEF CAZ FOX CTF AMP CHL STR TSU TET AMC CEF CAZ FOX CTF AMP CHL STR TSU TET AMC CEF CAZ FOX CTF AMP CHL GEN KAN STR TSU TET AMC CEF CAZ FOX CTF AMP CHL STR TSU TET AMC CEF CAZ FOX CTF AMP CHL STR TSU TET AMC CEF CAZ FOX CTF AMP STR TSU TET AMC CEF CAZ FOX CTF AMP CHL GEN KAN STR TSU SXT TET AMC CEF CAZ FOX CTF AMP CHL STR TSU TET AMC CEF CAZ FOX CTF AMP CHL STR TSU TET AMC CEF CAZ FOX CTF Pansusceptible AMP CHL GEN KAN STR TSU SXT TET AMP CHL GEN KAN STR TSU SXT TET Pansusceptible Pansusceptible Pansusceptible Pansusceptible Pansusceptible Pansusceptible Pansusceptible Pansusceptible Pansusceptible Pansusceptible Pansusceptible
A A A A A A A A B B1 A1 A2
95 95 95 95, 3.2 95 95 95 95 140 140 95 160, 5, 3.2
C
150, 3.5
C6
220, 180, 150, 120, 64
C
150, 3.5
C7 C3
230, 150, 92, 55, 3.5 150, 92, 34
C2
150, 55, 3.5
C5
150, 7.3
C1
150, 3.5
C1
150, 3.5
C
150, 3.5
C4
150, 3.5
C
150; 3.5
C1
150, 5.6
C8 E E1
3.5 180, 42, 7.3, 5.6, 4.6, 3.2 180, 42, 3.2
F D G G G G G G G G H
37 92, 55 95 95 95 95 95 95 95 95 None
ST2380 ST2488 ST2850 ST3161 ST3285 ST3686 ST4134 ST4660 ST2796 ST4563 ST3068e ST4783e
Typhimurium Typhimurium Typhimurium Typhimurium Typhimurium Typhimurium Typhimurium Typhimurium Typhimurium Typhimurium Typhimurium Typhimurium
Cow Cow Cow Cow Cow Cow Cow Cow Cow Cow Cow Cow
Wash. Wash. Idaho Idaho Wash. Wash. Wash. Wash. Idaho Idaho Wash. Wash.
1993 1994 1995 1996 1996 1997 1997 1998 1995 1998 1996 1999
DT104 DT104 DT104 DT104 DT104 DT104 DT104 DT104 DT208 DT208 DT120 U302
Epidemic Epidemic Epidemic Epidemic Epidemic Epidemic Epidemic Epidemic Nonepidemic Nonepidemic Nonepidemic Nonepidemic
SN3685
Newport
Cow
Wash.
1997
CMY-2⫹
Epidemic
SN6615
Newport
Cow
Idaho
2000
CMY-2⫹
Epidemic
SN6668
Newport
Cow
Idaho
2000
CMY-2⫹
Epidemic
SN6897
Newport
Cow
Utah
2000
CMY-2⫹
Epidemic
SN7140
Newport
Cow
Idaho
2001
CMY-2⫹
Epidemic
SN7340
Newport
Cow
Wash.
2001
CMY-2⫹
Epidemic
SN7486
Newport
Cow
Oreg.
2001
CMY-2⫹
Epidemic
SN7497
Newport
Cow
Wash.
2001
CMY-2⫹
Epidemic
SN7890
Newport
Cow
Wash.
2002
CMY-2⫹
Epidemic
SN7893
Newport
Cow
Idaho
2002
CMY-2⫹
Epidemic
SN7936
Newport
Cow
Wash.
2002
CMY-2⫹
Epidemic
SN8115
Newport
Cow
Utah
2002
CMY-2⫹
Epidemic
Newport
Cow
Idaho
2002
CMY-2⫹
Epidemic
Newport Newport
Cow Cow
Wash. Wash.
1999 1996
CMY-2⫺ CMY-2⫺
Nonepidemic Nonepidemic
SN4124
Newport
Cow
Idaho
1997
CMY-2⫺
Nonepidemic
SN6563 SN7897 STNZ150 STNZ152 STNZ153 STNZ154 STNZ155 STNZ156 STNZ164 STNZ165 STNZ340
Newport Newport Typhimurium Typhimurium Typhimurium Typhimurium Typhimurium Typhimurium Typhimurium Typhimurium Typhimurium
Cow Cow Bird Bird Bird Bird Bird Bird Bird Bird Bird
Utah Wash. New Zealand New Zealand New Zealand New Zealand New Zealand New Zealand New Zealand New Zealand New Zealand
2000 2002 2000 2000 2000 2000 2000 2000 2000 2000 2000
CMY-2⫺ CMY-2⫺ DT160 DT160 DT160 DT160 DT160 DT160 DT160 DT160 DT156
Nonepidemic Nonepidemic Epidemic Epidemic Epidemic Epidemic Epidemic Epidemic Epidemic Epidemic Nonepidemic
SN8118 SN4770 SN3082
e
Plasmid profile (kb)
a
ID, identification. CMY-2⫹, cephalosporin-resistant MDR Salmonella serovar Newport containing blaCMY-2; CMY-2⫺, cephalosporin-susceptible Salmonella serovar Newport without blaCMY-2. c Resistance patterns were determined by the Kirby Bauer disk diffusion assay on Mueller-Hinton agar for 14 antimicrobials, including ampicillin (AMP), chloramphenicol (CHL), gentamicin (GEN), kanamycin (KAN), streptomycin (STR), triple sulfa (TSU), trimethoprim-sulfamethoxazole (SXT), tetracycline (TET), amoxicillin-clavulanic acid (AMC), cephalothin (CEF), ceftazidime (CAZ), cefoxitin (FOX), ceftiofur (CTF), and ceftriaxone (CRO). All Salmonella serovar Newport CMY-2⫹ isolates tested were also intermediately resistant to CRO. d Each pattern was designated as described by Tenover et al. (46). e The isolates were closely related to corresponding epidemic strains, although they were classified as being of nonepidemic status. b
types were determined by the FDIU. Only one isolate per case was included, in order to maximize independence between isolates. Defining epidemic and nonepidemic strains. Bovine isolates were classified as “epidemic” or “nonepidemic” based on bovine salmonellosis cases in the Pacific
Northwest between 1993 and 2002 (Table 1). Strains (phage types or resistance types) that represented more than 50% of clinical isolates of a given serotype during a period of at least 3 years were considered epidemic strains. Nonepidemic strains were phage type or resistance type strains of the same serotype as
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epidemic strains that were occasionally isolated from clinical cases. Thus, nonepidemic strains corresponded to each epidemic strain by serotype, by geographic and temporal distribution, and by host species origin. Epidemic and nonepidemic strains of pansusceptible Salmonella serovar Typhimurium isolated from avian samples in New Zealand were also defined as described above, based on national surveillance work between 2000 and 2003 as reported by Alley et al. (2) and the Institute of Environmental Science and Research Limited (26, 27). Antimicrobial susceptibility testing. Antimicrobial resistance profiles were determined by the Kirby-Bauer agar diffusion method according to the Clinical and Laboratory Standards Institute guidelines (16) using 14 antimicrobials: ampicillin (10 g), chloramphenicol (30 g), gentamicin (10 g), kanamycin (30 g), streptomycin (10 g), triple sulfa (250 g), trimethoprim-sulfamethoxazole (1.25 to 23.75 g), tetracycline (30 g), amoxicillin-clavulanic acid (20 to 10 g), cephalothin (30 g), ceftazidime (30 g), cefoxitin (30 g), ceftiofur (30 g), and ceftriaxone (30 g). PFGE. Pulsed-field gel electrophoresis (PFGE) was performed using the restriction endonuclease XbaI (30 U) as previously described (19) under the following conditions: separation on a 1% agarose gel (Seakem Gold agarose; FMC Bioproducts) in 0.5⫻ TBE buffer (45 mM Tris, 45 mM boric acid, 1 mM EDTA) at 14°C and 6 V/cm for 18 h, with switch times ranging from 2.2 to 63.8 s. Photographic images of the gels were analyzed using Bionumerics software (Applied Maths, Sint-Martens-Latem, Belgium), and a dendrogram of the PFGE patterns was generated from Dice coefficients of similarity by the unweightedpair group method using average linkages. PFGE patterns of nonepidemic isolates were compared to those of epidemic isolates of the same serotype and source and were typed as described by Tenover et al. (46). Plasmid profiling. Plasmid DNA was isolated by the alkaline lysis method as previously described (28) and analyzed by agarose gel electrophoresis. Plasmid sizes were estimated by comparison with two supercoiled DNA ladders containing DNA bands of 8 to 165 kb (BAC-Tracker supercoiled DNA ladder; Epicentre, Madison, Wis.) and 2 to 16 kb (Invitrogen, Carlsbad, Calif.). Characterizing blaCMY-2-positive isolates. Isolates were screened for the presence of the blaCMY-2 gene, encoding the CMY-2 -lactamase, by using PCR as previously described (50). The location of the blaCMY-2 gene was determined by transforming plasmid minipreps into Escherichia coli DH10B cells (GeneHogs; Invitrogen), screening for AmpC-like -lactamase-producing transformants on LB medium with 50 g/ml cefoxitin (Sigma-Aldrich, St. Louis, Mo.), and conducting plasmid profiling as previously described (29). Salmonella gene microarray analysis. Details concerning the construction and characteristics of the microarray, DNA labeling, and hybridization conditions have been described previously (38, 40). In this study, we examined differences in genome content between strains using comparative genomic hybridizations to probes specific to the Salmonella serovar Typhimurium LT2 genome and the pSLT plasmid. Briefly, the genomic DNA of each query strain was labeled with Cy3-dCTP, mixed with an equal amount of Cy5-dCTP-labeled genomic DNA of Salmonella serovar Typhimurium LT2, and then applied to the microarray. Hybridizations were then performed overnight at 42°C in a hybridization chamber (Corning, Acton, Mass.) maintained in a water bath. Microarray scans were performed with a ScanArray 4000XL laser scanner (Perkin-Elmer Life Sciences, Boston, Mass.). Tagged-image format files were analyzed with softWoRx image analysis software (Applied Precision, Issaquah, Wash.) using the contour option that accommodates spots that have various shapes and sizes. Median spot intensities were subsequently converted to log2 values and normalized using a global lowess algorithm (BRB ArrayTools; http: //linus.nci.nih.gov/BRB-ArrayTools.html). Spots for which intensities from the Salmonella serovar Typhimurium LT2 control sample were within 2 standard deviations above background were excluded from analysis (41). After filtering and normalization, data were recorded as the median of log2 ratios for triplicate spots of each gene. Subsequent data analysis was conducted using the GACK genomic analysis program (31) to generate cutoffs for present or absent (conserved or divergent) genes relative to the Salmonella serovar Typhimurium LT2 genome. This program calculates an idealized normal distribution curve for each array and assigns a ternary value for each probe, depending on an estimated probability of a gene being present (100%), absent (0%), or uncertain (⬎0% and ⬍100%) in a given strain. The entire data set was then subjected to average hierarchical clustering with CLUSTER (21), and the results were presented using TREEVIEW (21). SSH. Three separate SSH libraries were constructed. SSH was performed using the PCR-Select bacterial genome subtraction kit (Clontech, Palo Alto, Calif.) according to the manufacturer’s recommendations, with the epidemic strains ST3686, SN6668, and STNZ152 as the testers and Salmonella serovar Typhimurium strain LT2 as the driver. Briefly, the tester and driver genomic DNAs were digested with RsaI and subjected to subsequent tester-adaptor
APPL. ENVIRON. MICROBIOL. ligation and subtractive hybridizations. Tester-specific sequences were then amplified by PCR and cloned into pCR 2.1 using a TA cloning kit (Invitrogen) according to the manufacturer’s instructions. DNA sequencing and analysis. SSH clone inserts were sequenced in both directions using cloning vector primers (T7 and M13R) and an Applied Biosystems 3130xl DNA sequencer with the BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems, Foster City, Calif.). The sequences were edited using Vector NTI software (Invitrogen). Sequence analysis was carried out using the BLASTn and BLASTx programs of the National Center for Biotechnology Information (3). Putative functions were predicted by BLASTx searches, where matches with E values of ⬍10⫺3 were considered significant. SSH microarray construction and analysis. We constructed a DNA microarray consisting of tester-specific clone inserts that were obtained from three SSH libraries and Salmonella serovar Typhimurium LT2 genes that were specific to any of three epidemic subtypes based on Salmonella gene microarray analysis. SSH clone inserts were amplified using the cloning vector primers in PCRs, and genes of Salmonella serovar Typhimurium LT2 were amplified using the primers and PCR amplification conditions described previously (38, 40). PCR products were then purified and printed as triplicate spots by following previously described procedures (12, 13). The array included additional spots of gyrB and 16S rRNA genes as housekeeping controls, and the entire array was also duplicated on each slide. Genomic DNA targets were biotinylated using nick translation (BioNick DNA labeling system; Invitrogen) and transferred to the microarray slide as previously described (13). Slides were placed in hybridization chambers (Corning) and maintained in a water bath overnight at 55°C. Hybridized DNA was detected using streptavidin conjugated to Alexa Fluor 555 (Molecular Probes, Eugene, Oreg.) at a 1:500 dilution in 1⫻ SSC (0.15 M NaCl plus 0.015 M sodium citrate) and 5⫻ Denhardt’s solution. Slides were subsequently scanned using an arrayWoRxe scanner (Applied Precision). Microarray image files were segmented using softWoRx image analysis software, and median intensities of each spot were normalized relative to the average intensity of housekeeping genes. The median of six normalized values per gene or SSH clone was recorded. All hybridizations were performed in replicate, and the resultant median values were averaged between replicates. The ratios of these single-channel values of query strains to corresponding values from Salmonella serovar Typhimurium LT2, ST3686, SN6668, or STNZ152 were calculated and used to evaluate the presence or absence of probed genes or SSH clones in the query strains. Data points showing poor signals for corresponding control samples were filtered as described above. The threshold cutoff between the presence and absence of genes was determined by the analysis of receiver operating characteristic (ROC) curves that present the relationship between sensitivity and specificity across all cutoff points of a test (NCSS 2004; NCSS, Kaysville, Utah). Probes with ratios greater than or equal to a threshold optimal for sensitivity and specificity were considered to indicate the presence of a gene. The entire data set was presented using TREEVIEW (21) and also analyzed using a Ward’s minimum-variance cluster algorithm with a Euclidean distance metric (NCSS 2004) to examine the genetic similarity between strains. Nucleotide sequence accession numbers. Gene sequences determined in this study have been deposited under GenBank accession numbers DQ472365 to DQ472474.
RESULTS Epidemic and nonepidemic strains. Between 1993 and 2001, Salmonella serovar Typhimurium was the most prevalent serotype among bovine Salmonella isolates from Washington, Idaho, Oregon, and Utah. Salmonella serovar Newport has been isolated since 1996, and Salmonella serovars Newport and Typhimurium have been the most common serotypes of bovine Salmonella isolates since 2002 (44.3% and 41.4%, respectively, in 2002). Between 1993 and 1997, Salmonella serovar Typhimurium DT104 accounted for more than 70% of phage-typed bovine Salmonella serovar Typhimurium isolates. Since 2000, cephalosporin-resistant MDR Salmonella serovar Newport has accounted for more than 90% of bovine Salmonella serovar Newport isolates. Thus, Salmonella serovar Typhimurium DT104 and cephalosporin-resistant MDR Salmonella serovar Newport were considered epidemic strains for this study. Although data of this nature cannot be used to quantify true
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FIG. 1. PFGE patterns of S. enterica isolates observed with XbaI digestion. PFGE patterns of nonepidemic isolates were compared to those of epidemic isolates of the same serovar and source and were determined as type A, type B, etc., as described by Tenover et al. (46). Each PFGE pattern that was closely or possibly related to that of a corresponding epidemic isolate, differing by not more than 6 bands, was considered its subtype, such as A1 or A2.
incidence or prevalence, they are consistent with the nationwide prevalence of these two epidemic subtypes in humans and animals in the United States (15). Nonepidemic Salmonella strains for this study included Salmonella serovar Typhimurium DT208, DT120, and U302 isolates and cephalosporinsusceptible Salmonella serovar Newport isolates (Table 1). These strains were only occasionally (⬍4%/year) isolated from bovine salmonellosis cases between 1993 and 2002. Based on surveillance reports in New Zealand between 2000 and 2003 (2, 26, 27), epidemic Salmonella serovar Typhimurium DT160 has accounted for more than 70% of avian Salmonella serovar Typhimurium isolates phage typed since 2000. This study included, as a nonepidemic strain, a Salmonella serovar Typhimurium DT156 isolate, of a phage type that was rarely found among avian Salmonella isolates during the same period in New Zealand. Phenotypic and genotypic characteristics. Twenty-five isolates of Salmonella serovar Typhimurium DT104 and two isolates of closely related phage types (DT120 and U302) (14, 33) shared resistance to at least five antibiotics, including ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline (Table 1). These strains had closely related PFGE patterns (types A, A1, and A2) and harbored a plasmid of approximately 95 kb (Table 1; Fig. 1). Two nonepidemic Salmonella serovar Typhimurium DT208 isolates were also resis-
tant to multiple antibiotics and had PFGE patterns (types B and B1) distinct from those of other Salmonella serovar Typhimurium phage types while harboring a larger plasmid (Table 1). Forty-seven cephalosporin-resistant MDR Salmonella serovar Newport isolates also were resistant to multiple antibiotics (Table 1) and were also intermediately resistant to ceftriaxone. PFGE analysis showed one main pattern (type E; 24 isolates) and eight closely related patterns (subtypes C1 to C7) (Fig. 1). All the isolates shared a large plasmid of approximately 150 kb and were positive for blaCMY-2 by PCR (Table 1). Subsequent plasmid transformation and PCR detection revealed that the blaCMY-2 gene was harbored on the 150-kb plasmid. Cephalosporin-susceptible Salmonella serovar Newport included three pansusceptible isolates and three isolates (types E and E1) with resistance to several antibiotics (Table 1; Fig. 1). Nonepidemic pansusceptible and MDR isolates had diverse PFGE patterns (types D, E, and F), but one pansusceptible isolate (SN4770) had subtype C8, closely related to the main type C of epidemic cephalosporin-resistant MDR Salmonella serovar Newport isolates (Table 1). All nine isolates of Salmonella serovar Typhimurium DT160 and DT156 were susceptible to all antibiotics tested (Table 1). PFGE and plasmid profiles were identical for all eight DT160 isolates (PFGE type G and single plasmids of
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approximately 95 kb [Table 1; Fig. 1]), indicating that the isolates were highly clonal, as previously reported (2, 47). In contrast, the nonepidemic Salmonella serovar Typhimurium DT156 isolate (STNZ340) showed a different PFGE pattern (type H), closely related to that of Salmonella serovar Typhimurium DT104 (type A), and did not harbor a plasmid (Table 1). Differences in genome content between epidemic and nonepidemic strains. The genome contents of nine representative epidemic isolates and seven nonepidemic isolates of S. enterica were compared with that of Salmonella serovar Typhimurium LT2 by using the Salmonella gene microarray. Genetic similarity among representative isolates, based on the presence or absence of LT2 genes, is summarized in Fig. 2 and in Table S1 in the supplemental material. About 2% of the genome content of Salmonella serovar Typhimurium LT2 (including plasmid pSLT) is absent or highly diverged in Salmonella serovar Typhimurium query strains, except for Salmonella serovar Typhimurium DT156, which showed 5% divergence. For Salmonella serovar Newport query strains, 8% of the genome content is absent or highly diverged (Fig. 2). In the hierarchical clustering analysis, isolates of each epidemic subtype grouped together into subclusters, indicating that the genome content is conserved within each subtype (Fig. 2). Comparison between Salmonella serovar Typhimurium DT104 and DT208 showed that the region from pSLT010 to pSLT024 was present only in Salmonella serovar Typhimurium DT104 but the regions including STM0517 to STM0529, STM0906, STM2599, STM2616 to 2618, and STM2704 to STM2706 were present only in nonepidemic Salmonella serovar Typhimurium DT208 (ST2796 and ST4563) (see Table S1 in the supplemental material). The pSLT010-to-pSLT024 region includes srgAB, pefACDI, and open reading frames 5, 6, and 7 and belongs to the pef (plasmid-encoded fimbriae) locus, which is associated with bacterial virulence (5). The main region (STM0517 to STM0529) absent in Salmonella serovar Typhimurium DT104 contains the genes involved in glycerate and allantoin metabolism (18). Other nonepidemic Salmonella serovar Typhimurium DT208-specific regions contain Gifsy-1, Fels-1, or Fels-2 prophage genes (see Table S1 in the supplemental material). Genomes of Salmonella serovar Newport isolates shared less than 90% of the Salmonella serovar Typhimurium LT2 genome on the basis of genes defined as present (Fig. 2). Only a small region (STM1029 to STM1030) of the Gifsy-2 prophage was present in cephalosporin-resistant MDR Salmonella serovar Newport and absent in nonepidemic serovar Newport isolates, and a gene (STM2689) of unknown function, which is a pseudogene in serovar Typhimurium LT2, was present in nonepidemic cephalosporin-susceptible Salmonella serovar Newport and absent in epidemic serovar Newport isolates (see Table S1 in the supplemental material). Hybridizations with two epidemic Salmonella serovar Typhimurium DT160 isolates (STNZ152 and STNZ165) and one nonepidemic Salmonella serovar Typhimurium DT156 isolate (STNZ340) revealed 163 genes that were present in DT160 isolates and absent in DT156. Most (103) of these genes are located on the virulence plasmid of DT160; the other genes are Gifsy-1 and Gifsy-2 prophage genes (see Table S1 in the supplemental material), indicating that the Gifsy-1 and Gifsy-2
APPL. ENVIRON. MICROBIOL.
FIG. 2. Genomic differences between epidemic and nonepidemic strains of Salmonella serovars Typhimurium and Newport determined by using the Salmonella gene microarray. Two percent of the genome content of Salmonella serovar Typhimurium LT2 is absent or highly diverged in Salmonella serovar Typhimurium query strains except for Salmonella serovar Typhimurium DT156 (5%), and 8% of the genome content is absent or highly diverged in Salmonella serovar Newport query strains. Representative regions showing differences between epidemic and nonepidemic strains of S. enterica are indicated by the Roman numerals I, II, III, and IV, and details are provided in Table S1 in the supplemental material. Each strain identification (ID) consists of its name and subtype, and strains are clustered as described in Materials and Methods. The genes on the chromosome are represented in the order of their positions in Salmonella serovar Typhimurium LT2, from STM 0001 to STM 4600, and are followed by the genes of the Salmonella serovar Typhimurium LT2 virulence plasmid pSLT. Blue indicates present genes; yellow, absent (highly divergent) genes; black, uncertain (slightly divergent) genes; gray, missing data.
prophage regions are also absent or highly divergent in the DT156 isolate. In contrast, two genes, including a gene encoding a putative periplasmic transport protein (STM1562) and a gene of unknown function (STM4261), are present in the DT156 isolate and absent in DT160 (see Table S1 in the supplemental material).
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FIG. 3. Ward’s minimum-variance dendrogram summarizing genetic similarity among the S. enterica isolates tested, based on genomic hybridization experiments with the SSH microarray. Each strain identification (ID) consists of its name and subtype. Strains of each epidemic subtype, Salmonella serovar Typhimurium DT104 or DT160 or Salmonella serovar Newport CMY-2⫹ isolates, grouped together in their own subcluster. SN4770 CMY-2⫺ clustered together with closely related Salmonella serovar Newport CMY-2⫹, and Salmonella serovar Typhimurium DT120 and U302 isolates also clustered together with closely related Salmonella serovar Typhimurium DT104.
Genomic subtraction and distribution of combined SSH clones. Three independent SSH experiments were performed with three tester strains, ST3686 (DT104), SN6668 (CMY-2⫹), and STNZ152 (DT160), representing the epidemic subtypes (Table 1). Successfully sequenced SSH clones (285 clones per library) were assembled, and a total of 289 nonredundant inserts were used to construct a new microarray together with 181 Salmonella serovar Typhimurium LT2 genes that were specific to at least one of the three epidemic subtypes. Hybridization results for Salmonella serovar Typhimurium LT2 and other reference strains (ST3686, SN6668, and STNZ152) were compared with expected results by using ROC analysis. A normalized ratio of 0.68 was chosen as a threshold cutoff point, providing an estimated 97.4% sensitivity and 96.9% specificity. The distribution of epidemic tester strain-specific genes and sequences across 39 isolates showed that there are highly conserved regions associated with each bacterial subtype (see Fig. S1 in the supplemental material). Cluster analysis showed that there were two major clusters, distinguishing Salmonella sero-
vars Typhimurium and Newport, and that each cluster was subdivided into two subclusters distinguishing isolates of each epidemic or nonepidemic subtype, with the exception of Salmonella serovar Typhimurium DT120 and U302 and Salmonella serovar Newport SN4770, which clustered together with their corresponding epidemic subtypes (Fig. 3). This distribution of the isolates demonstrates that most of the genes or SSH clones for each epidemic subtype were common to all isolates of each epidemic subtype. Specific gene sequences from SSH libraries conserved in epidemic strains. BLASTx analysis results for 42 SSH clones conserved in Salmonella serovar Typhimurium DT104 (and also in DT120 and U302), 47 SSH clones conserved in cephalosporin-resistant MDR Salmonella serovar Newport (11 clones were also found in pansusceptible SN4770), and 21 SSH clones conserved in Salmonella serovar Typhimurium DT160 are shown in Tables S2, S3, and S4 in the supplemental material. Blastn searches against the Salmonella serovar Typhimurium DT104 genome sequence using the
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TABLE 2. Genes or gene fragments from SSH libraries specific to epidemic strains of S. enterica shared by all isolates within each epidemic subtype No. of sequences (%)a Functional categoryd
A. Phage-related sequences a. Transcription b. DNA replication, recombination, and repair c. Virion particle, infection, and lysis d. Hypothetical or unknown function B. Plasmid-related sequences a. Transcription b. DNA replication and partition c. Conjugal transfer d. Cell envelope biosynthesis e. Transposable element movement f. Antimicrobial resistance g. Hypothetical or unknown function C. Other sequences a. Transcription b. DNA replication, recombination, and repair c. Cell division and chromosome partitioning d. Posttranslational modification e. Cell motility and secretion f. Cell envelope biosynthesis g. Metabolic function h. Hypothetical or unknown function
ST DT104 SN CMY-2⫹ ST DT160 vs DT208 vs CMY-2⫺ vs DT156 (n ⫽ 54)b (n ⫽ 65)c (n ⫽ 44)
1 (1.9) 3 (5.6)
4 (6.2) 1 (1.5)
5 (11.4) 7 (15.9)
10 (18.5)
1 (1.5)
10 (22.7)
8 (14.8)
3 (4.6)
22 (50.0)
1 (1.9) 2 (3.7)
2 (3.1)
2 (3.7) 3 (5.6)
7 (10.8) 1 (1.5) 3 (4.6)
2 (3.7) 6 (11.1)
2 (3.1) 3 (4.6)
2 (3.7) 6 (9.2) 1 (1.5) 1 (1.5) 2 (3.7) 12 (22.2)
1 (1.5) 1 (1.5) 6 (9.2) 22 (33.8)
a Two or more regions within an SSH clone with significant BLASTx matches (E ⬍ 10⫺3) were counted separately, but two regions, each of which belonged to different SSH clones but aligned with the same protein, were counted once. Therefore, the total number (n) of genes or gene fragments categorized exceeds the sum of the SSH clones identified. Epidemic subtypes were simply designated “ST” for Salmonella serovar Typhimurium or “SN” for Salmonella serovar Newport. “CMY-2⫹” indicates the cephalosporin-resistant MDR phenotype. b All sequences were also found in closely related Salmonella serovar Typhimurium DT120 and U302. c Fifteen sequences were also found in closely related Salmonella serovar Newport isolate SN4770 and are listed in Table S3 in the supplemental material. d The single-letter labels relate to the “Functional category” column in Tables S2, S3, and S4 in the supplemental material.
Sanger Institute BLAST Server (http://www.sanger.ac.uk/cgi-bin /blast/submitblast/salmonella) showed that all 42 SSH clones obtained from the Salmonella serovar Typhimurium DT104 SSH library were present in the sequenced Salmonella serovar Typhimurium DT104 genome. Most sequences were partial sequences whose products were homologous to known proteins, but some sequences contained two or more gene sequences, spanning the intergenic region. Gene sequences specific to Salmonella serovar Typhimurium DT104 and closely related nonepidemic DT120 and U302 were composed of 22 phage-related gene sequences, 16 plasmidrelated gene sequences, and 16 other regions from uncertain
sources (Table 2). Most of the phage-related sequences were homologous to those of Salmonella serovar Typhimurium bacteriophage ST104 (45), and the plasmid-related sequences were homologous to those of the Salmonella genomic island 1 (SGI1) (11) (see Table S2 in the supplemental material). Many genes are of unknown function. However, two sequences (36861G03 and 36863D11) are presumably involved in cell envelope biosynthesis (see Table S2 in the supplemental material). Specific gene sequences conserved in cephalosporin-resistant MDR Salmonella serovar Newport and partly in nonepidemic SN4770 included nine phage-related sequences, 18 plasmid-related sequences, and 38 other sequences from chromosomes or uncertain sources (Table 2). SSH clones 66681E09, 66682D09, 66682F07, and 66682G07 contained eight gene sequences belonging to a putative transposable element bearing blaCMY-2 (29) (see Table S3 in the supplemental material). An SSH clone of uncertain source (66681G10) may be involved in cell envelope biosynthesis (see Table S3 in the supplemental material). Six SSH clones (66681D06, 66681H09, 66682C08, 66682B11, 66683D08, and 66683G01) from uncertain sources may encode proteins involved in metabolism, including ferredoxin, the cobalamin biosysnthesis proteins CobT and CobS, phosphoadenosine-phosphosulfate sulfotransferase or a related enzyme, glycosidase, and thymidylate kinase, respectively (see Table S3 in the supplemental material). Most of the other SSH clones (28 gene sequences) contained gene sequences of unknown function (Table 2; see also Table S3 in the supplemental material). All 21 SSH clones (44 gene sequences) specific to Salmonella serovar Typhimurium DT160 were phage-related sequences (Table 2; see also Table S4 in the supplemental material). These included 10 genes implicated in phage structure and assembly, infection, and host cell lysis and 12 genes implicated in transcription, DNA replication, and metabolism (Table 2). One-half of the genes (22 gene sequences) were of unknown function (Table 2; see also Table S4 in the supplemental material). Most of these phage-related genes were homologous to those of Salmonella serovar Typhimurium bacteriophages ST64T and ST64B, isolated from Salmonella serovar Typhimurium DT64 (34, 35) (see Table S4 in the supplemental material). DISCUSSION This study revealed specific genes and gene loci that were conserved in recent and contemporary epidemic strains of Salmonella and that were mainly related to mobile genetic elements. Antimicrobial resistance phenotypes and genotypes (Table 1; Fig. 1) indicate that epidemic strains have been disseminated as a single clone or a small family of genetically related strains, as previously reported (2, 7, 50), and this finding is consistent with the high genomic similarity within isolates of each epidemic subtype shown in genomic comparisons using the Salmonella and SSH microarrays (Fig. 2; see also Fig. S1 in the supplemental material). Specific gene sequences conserved in epidemic strains included genes or gene fragments that encode proteins putatively involved in bacterial growth, survival, or virulence in the host and the environment (Table 2; see also Table S1 in the supplemental material). The whole
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genome microarray complemented with the SSH provided more information about combinations of known and unknown genes unique to epidemic stains and some genetically related nonepidemic strains. Specific gene sequences conserved in MDR Salmonella serovar Typhimurium DT104. The main regions showing variation between Salmonella serovar Typhimurium DT104 and DT208 in the Salmonella gene microarray include the region from the pef locus specific to the DT104 isolates and the glycerate and allantoin metabolism-related gene region specific to the nonepidemic DT208 isolates (see Table S1 in the supplemental material). The pef (plasmid-encoded fimbriae) locus is implicated in bacterial adherence to intestinal epithelial cells and gastroenteritis in the host (5). The absence of this locus may have resulted in relatively lower virulence of nonepidemic DT208. In contrast, the gene region involved in glycerate and allantoin metabolism may confer an advantage on nonepidemic DT208 in utilizing nitrogen sources (18). Most DT104specific clones obtained by SSH are related to Salmonella serovar Typhimurium bacteriophage ST104 (45) and SGI1, which is a mobilizable genetic element (11, 20) (see Table S2 in the supplemental material). Although SGI1 is found on chromosomes in most host strains of S. enterica, this study grouped gene sequences homologous to those of SGI1 into plasmid-related sequences because of its many genetic traits associated with plasmids (11). All SSH clones listed in Table S2 in the supplemental material were shared by Salmonella serovar Typhimurium DT120 and U302. MDR strains of these two phage types may be derived from DT104 through a phage type conversion (14, 33). Phage type conversion may be mediated by the acquisition of new phages or plasmids and by changes in bacterial cell surface phage receptors (33), and the conversion of DT104 to U302, possibly by the acquisition of plasmids, has been observed (14). The current study also supports the conclusion that phage types DT104, DT120, and U302 share a recent common origin. The common genetic background shared by DT104 and genetically related phage types may have contributed to the overall epidemic phenotype of DT104. Nevertheless, the phage types DT120 and U302 are not successful isolates in the Pacific Northwest. The new phage or plasmid presumably involved in phage type conversion or other minor genetic alterations such as point mutations may have a role in preventing successful widespread infections of the phage types. We were unable to identify meaningful differences, probably owing in part to the limited resolution of hybridization-based comparisons used in this study. Specific gene sequences conserved in cephalosporin-resistant MDR Salmonella serovar Newport. Salmonella serovar Newport isolates showed very few differences between epidemic and nonepidemic strains based on the Salmonella gene microarray analysis (see Table S1 in the supplemental material). Genomic subtraction revealed 47 SSH clones (65 gene sequences) conserved in epidemic strains, including many genes from plasmids and uncertain sources (see Table S3 in the supplemental material). Several SSH clones showed partial gene sequences of a blaCMY-2 element that might be a composite transposon (29). These elements contain an insertion sequence (ISEcp1), blc, a partial ecnR gene, and dsbC, as well as two antimicrobial resistance gene sequences, blaCMY-2 and
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sugE (29). The blc gene encodes an outer membrane lipoprotein that may serve a starvation response function in host bacteria (9), and the ecnR gene encodes a transcriptional regulatory protein involved in osmoregulation during starvation in the stationary phase (8). Interestingly, blc, sugE, and ecnR are present in the Salmonella serovar Typhimurium LT2 chromosome (STM4337 to STM4339), and the Salmonella gene microarray analysis showed that these genes were also present in all Salmonella serovar Newport isolates tested. Cephalosporinresistant MDR Salmonella serovar Newport-specific SSH clones also include two gene sequences encoding proteins involved in cell envelope biosynthesis and six gene sequences encoding metabolic enzymes involved in respiration and nucleotide, amino acid, carbohydrate, or vitamin metabolism (see Table S3 in the supplemental material) that may confer survival and growth advantages on the host bacteria. Interestingly, the pansusceptible isolate SN4770 appeared to be a close genetic match to cephalosporin-resistant MDR isolates, except that it lacked the 150-kb plasmid (Table 1), and contained 11 of 47 SSH clones conserved in cephalosporinresistant MDR Salmonella serovar Newport (see Table S3 in the supplemental material). We speculate that SN4770 represents a clone of Salmonella serovar Newport that has lost the blaCMY-2-bearing plasmid. Specific gene sequences conserved in pansusceptible Salmonella serovar Typhimurium DT160. Most of the Salmonella serovar Typhimurium DT160-specific gene sequences found by the Salmonella gene microarray were the plasmid pSLT genes; other genes were Gifsy-1 and Gifsy-2 prophage genes (Fig. 2; see also Table S1 in the supplemental material). Interestingly, nonepidemic Salmonella serovar Typhimurium DT156 paired with DT160 did not have a large plasmid (Table 1; Fig. 2) and probably lacks the pSLT-like plasmid-mediated functions associated with virulence and fitness. There are at least five prophage genomes present on the Salmonella serovar Typhimurium LT2 chromosome—Gifsy-1, Gifsy-2, Fels-1, Fels-2, and the region from STM 4196 to STM4219—and most of these phage genes are highly diverged in other bacterial strains (39). Gifsy-1 and Gifsy-2 prophages, the regions of which are absent or highly diverged in the nonepidemic DT156 isolate, have been shown to contribute to Salmonella serovar Typhimurium virulence (22). In particular, the lambdoid phage Gifsy-2 carries the periplasmic superoxide dismutase gene (sodCI), which improves Salmonella survival in the host, and other putative virulence factors (22). The prophage genes absent in the nonepidemic strain may indicate its relatively lower virulence in the host. SSH identified additional DT160-specific gene fragments that were all phage-related sequences, most of which were previously reported for Salmonella serovar Typhimurium bacteriophages ST64T and ST64B, isolated from Salmonella serovar Typhimurium DT64 (34, 35) (see Table S4 in the supplemental material). Therefore, these two additional phages may be present in the epidemic DT160 genome. Although their roles in host organisms were not determined, ST64B was also found in pandemic Salmonella enterica serovar Enteritidis phage type 4 (PT4) (38). As previously reported, antimicrobial selection pressure does not consistently explain the increased prevalence of epidemic MDR stains of S. enterica, and restricting antimicrobial use often fails to control the dissemination of epidemic MDR
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strains, suggesting that there may be other biological traits or genetic factors that increase bacterial virulence or fitness or at least compensate for a fitness cost mostly accompanied by antimicrobial resistance (7, 10, 24). The previous finding that the prophage-associated sopE gene is present mainly in epidemic cattle-associated phage types of Salmonella serovar Typhimurium in Europe provides insight into Salmonella epidemic traits, because it suggests that this virulence gene may confer an advantage during circulation in the bovine animal reservoir (42). Plasmids, particularly conjugative plasmids, and transposons, which typically carry fitness or virulence-associated genes and antibiotic resistance genes, can act as agents for horizontal gene transfer and thereby facilitate the epidemic spread of host bacteria (37). A representative example is shown by human epidemic strains of Salmonella serovars Wien and Typhimurium in Europe, North Africa, and Asia in the 1970s, which carried FIme resistance plasmids coding for an iron assimilation system, possibly conferring a competitive advantage (17). Therefore, it is possible that the acquisition of fitness-associated factors by horizontal gene transfer mediated by combinations of phages, plasmids, transposons, integrons, and other mobile genetic elements contributes to the emergence of new epidemic strains. Adaptive mutations involving functional modification or loss of preexisting genetic materials also represent a genetic mechanism for enhancing bacterial virulence or fitness and compensating for the fitness cost posed by the acquisition of antimicrobial resistance (10, 44). Salmonella gene microarray analysis in this study showed genes or gene regions absent in epidemic strains and present in nonepidemic strains. However, point mutations, rearrangements, or small insertions and deletions cannot be detected by conventional hybridization-based approaches (32). Thus, the genetic information provides only limited insight into genetic traits associated with the epidemic success of the three epidemic subtypes. In addition, this study did not reveal any specific genetic elements shared by the three epidemic subtypes. Previous studies showed that MDR Salmonella serovar Typhimurium DT104 strains were not more invasive or more virulent than other phage types and known virulent strains (1, 23) and that the acquisition of an AmpC -lactamase gene might cause a reduction in the growth rate and invasiveness of Salmonella due to a fitness cost (36). Salmonella epidemics are frequently characterized by a decline in widely disseminated epidemic strains with the emergence and spread of new epidemic strains (24). Salmonella serovar Typhimurium DT208, which was considered nonepidemic in this study, was also responsible for an epidemic in humans in the Middle East in the 1970s (4), although it is not clear whether our DT208 is the same clonal type as the Middle East type. Therefore, the emergence of new epidemic strains of Salmonella does not indicate the occurrence of more-pathogenic strains. The epidemiological status of epidemic strains of Salmonella may be driven by more-complex combinations of multiple genetic and fitness traits, including antimicrobial resistance and compensating factors, distinguishing them from at least coexisting nonepidemic strains, rather than by any common genetic factor conferring distinct epidemic potential on host bacteria. In addition to the inherent limitations to hybridizationbased analysis, and because of the high percentage of strain-
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specific sequences of each epidemic subtype that remain uncharacterized (43 to 50%), additional efforts will be needed to understand how these traits might contribute to the emergence and expansion of epidemic strains. At the level of resolution afforded by this study, it is clear that the success of the three epidemic subtypes is not driven by a common genetic trait. The Salmonella gene microarray and SSH analyses identified epidemic MDR and non-MDR strain-specific gene sequences in addition to antimicrobial resistance genes that were absent or highly diverged in distantly related, coexisting nonepidemic strains. Although this study did not show clear discrimination of genetic traits between epidemic and nonepidemic strains, the common genetic background shared by isolates of the same epidemic subtype and closely related strains may be an important part of potential combinations of epidemic trait-associated factors that contribute to overall epidemic phenotypes of Salmonella. Further functional analysis of these genes will provide better insight into the emergence and expansion of epidemic strains of S. enterica and will potentially assist with the development of novel control strategies. ACKNOWLEDGMENTS We thank S. LaFrentz, L. Orfe, Y. Zhang, S. Lloyd, and R. McClanahan for technical advice and help and M. Davis and J. Daniels for helpful discussions and isolate information. The lowess normalization was performed using BRB ArrayTools developed by Richard Simon and Amy Peng. This study was supported by the Agricultural Animal Health Program, College of Veterinary Medicine, Pullman, Wash., and funded in whole or in part with Federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under contract N01-AI-30055, and from USDA-NRICGP 0102147. REFERENCES 1. Allen, C. A., P. J. Fedorka-Cray, A. Vazquez-Torres, M. Suyemoto, C. Altier, L. R. Ryder, F. C. Fang, and S. J. Libby. 2001. In vitro and in vivo assessment of Salmonella enterica serovar Typhimurium DT104 virulence. Infect. Immun. 69:4673–4677. 2. Alley, M. R., J. H. Connolly, S. G. Fenwick, G. F. Mackereth, M. J. Leyland, L. E. Rogers, M. Haycock, C. Nicol, and C. E. Reed. 2002. An epidemic of salmonellosis caused by Salmonella Typhimurium DT160 in wild birds and humans in New Zealand. N. Z. Vet. J. 50:170–176. 3. Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389–3402. 4. Anderson, E. S., E. J. Threlfall, J. M. Carr, M. M. McConnell, and H. R. Smith. 1977. Clonal distribution of resistance plasmid-carrying Salmonella typhimurium, mainly in the Middle East. J. Hyg. (London) 79:425–448. 5. Baumler, A. J., R. M. Tsolis, F. A. Bowe, J. G. Kusters, S. Hoffmann, and F. Heffron. 1996. The pef fimbrial operon of Salmonella typhimurium mediates adhesion to murine small intestine and is necessary for fluid accumulation in the infant mouse. Infect. Immun. 64:61–68. 6. Berge, A. C., J. M. Adaska, and W. M. Sischo. 2004. Use of antibiotic susceptibility patterns and pulsed-field gel electrophoresis to compare historic and contemporary isolates of multi-drug-resistant Salmonella enterica subsp. enterica serovar Newport. Appl. Environ. Microbiol. 70:318–323. 7. Besser, T. E., M. Goldoft, L. C. Pritchett, R. Khakhria, D. D. Hancock, D. H. Rice, J. M. Gay, W. Johnson, and C. C. Gay. 2000. Multiresistant Salmonella Typhimurium DT104 infections of humans and domestic animals in the Pacific Northwest of the United States. Epidemiol. Infect. 124:193–200. 8. Bishop, R. E., B. K. Leskiw, R. S. Hodges, C. M. Kay, and J. H. Weiner. 1998. The entericidin locus of Escherichia coli and its implications for programmed bacterial cell death. J. Mol. Biol. 280:583–596. 9. Bishop, R. E., S. S. Penfold, L. S. Frost, J. V. Holtje, and J. H. Weiner. 1995. Stationary phase expression of a novel Escherichia coli outer membrane lipoprotein and its relationship with mammalian apolipoprotein D. Implications for the origin of lipocalins. J. Biol. Chem. 270:23097–23103. 10. Bjorkman, J., D. Hughes, and D. I. Andersson. 1998. Virulence of antibioticresistant Salmonella typhimurium. Proc. Natl. Acad. Sci. USA 95:3949–3953. 11. Boyd, D., G. A. Peters, A. Cloeckaert, K. S. Boumedine, E. Chaslus-Dancla,
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