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Applied and Environmental Microbiology, May 2007, p. 3101-3104, Vol. 73, No. 9
0099-2240/07/$08.00+0     doi:10.1128/AEM.02607-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Multiple Environmental Stress Tests Show No Common Phenotypes Shared among Contemporary Epidemic Strains of Salmonella enterica ^,

Min-Su Kang,¹ Thomas E. Besser,¹ Dale D. Hancock,² and Douglas R. Call^1*

Department of Veterinary Microbiology and Pathology,¹ Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Washington State University, Pullman, Washington²

Received 8 November 2006/ Accepted 22 February 2007

Top ABSTRACT INTRODUCTION REFERENCES

 
Phenotypic traits of coexisting epidemic and nonepidemic strains of Salmonella enterica serovars Typhimurium and Newport were compared. Different stress conditions were relatively more or less favorable for the epidemic strains. Transcriptional analysis identified specific upregulated genes during defined stress conditions, but there were no common traits shared by epidemic serovars.

Top ABSTRACT INTRODUCTION REFERENCES

 
Salmonella enterica epidemics are often characterized by rapid and widespread dissemination of predominant epidemic strains, including the relatively recent spread of multidrug-resistant (MDR) S. enterica serovar Typhimurium DT104 and MDR S. enterica serovar Newport with AmpC (CMY-2) ß-lactamase-mediated cephalosporin resistance in the United States (2, 9, 21, 26). Although the epidemic spread and persistence of MDR Salmonella strains may be driven by antibiotic selection pressure in human and animal populations (20, 27), epidemic MDR strains and non-MDR strains can persist regardless of antibiotic use (1, 11, 14, 23). It is therefore likely that other biological traits specific to epidemic strains provide mechanism(s) for their increased prevalence and geographical dissemination (2, 9). For example, heat-, acid-, or oxidative stress-tolerant and biofilm-forming bacteria may have survival advantages in natural environments, and these traits may also be related to virulence (10, 16, 25).

Kang et al. (13) recently identified gene sequences that were conserved in three epidemic strains of S. enterica (serovar Typhimurium DT104 and DT160 and cephalosporin-resistant MDR serovar Newport), and some of these genes might encode proteins conferring growth or survival advantages. The present study characterized fitness phenotypes of epidemic strains of Salmonella relative to those of their coexisting nonepidemic strains and examined differential transcriptional responses of epidemic strain-specific genes (13).

Sixteen Salmonella isolates used in the present study were representative of recent and contemporary epidemic strains of S. enterica and their temporally matched nonepidemic strains. Strains were considered epidemic when more than 50% of clinical isolates of a given serotype were composed of a common subtype (phage type or resistance type) over a 3-year period (13). Strains originally isolated from cattle in the Pacific Northwest (United States) included epidemic MDR Salmonella serovar Typhimurium DT104 strains (ST2850, ST3686, and ST4660) and matched nonepidemic DT208 strains (ST2796 and ST4563) and epidemic cephalosporin-resistant MDR Salmonella serovar Newport strains (SN3685, SN6668, SN7497, and SN7890) and matched, cephalosporin-susceptible nonepidemic Newport strains (SN3082, SN4124, SN6563, and SN7897). We also included epidemic pansusceptible Salmonella serovar Typhimurium DT160 (STNZ152 and STNZ165) and matched nonepidemic pansusceptible DT156 (STNZ340) strains isolated from birds in New Zealand (13).

For stress tolerance assays, log-phase or stationary-phase cells of Salmonella were exposed to low pH (pH 2.8; 10 min), hydrogen peroxide (10 mM H₂O₂; 10 min), heat (52°C; 10 min), desiccation (3 days), and high salt conditions (1 M NaCl; 48 h), and the percent survival or osmotic tolerance of the isolates was estimated as previously described (3, 10). Biofilm formation was assessed by measuring the adherence to the wells of 96-well polyvinyl chloride and polystyrene microtiter plates as detected by crystal violet staining (17, 19). Cell invasion and the intracellular survival of Salmonella strains (except for gentamicin-resistant SN3082 and SN4124) were evaluated by using a gentamicin protection assay with Caco-2 cells (7). The transcription levels of epidemic strain-specific genes (13) were subsequently examined during the exposure of representative epidemic strains ST3686 (DT104), SN6668 (CMY-2+), and STNZ152 (DT160) to stress conditions to which, as we demonstrate here, they were well adapted. Gene expression was evaluated by using a DNA microarray consisting of a subset of gene sequences (260 suppression subtraction hybridization [SSH] clones or genes) unique to any of three epidemic strains of S. enterica that was constructed previously (13). Each SSH clone that encompasses two or more gene sequences and that was differentially expressed in microarray analysis was also analyzed for transcription levels of individual gene sequences by using real-time reverse transcription-PCR (RT-PCR). Detailed methods are provided in the supplemental material.

Epidemic Typhimurium DT104 strains were more tolerant of oxidative and osmotic (48 h) stresses compared to matched nonepidemic DT204 strains (P < 0.05; Table 1) but did not differ in their tolerance of acid, heat, and desiccation stress; biofilm formation; and cell invasion and intracellular survival (Table 1; see Fig. S1 and S2 in the supplemental material). Subsequently, ST3686 was subjected to expression array studies during short-term oxidative and short-term and steady-state osmotic stresses, but no differentially expressed, DT104-specific genes were identified. Thus, while DT104 strains were more robust under select stress conditions, we did not find any correlation between this and genetic traits.

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TABLE 1. Stress resistance of epidemic (DT104) and nonepidemic (DT208) strains of Salmonella serovar Typhimurium isolated from bovines in the Pacific Northwest of the United States

Epidemic serovar Newport strains were more tolerant of osmotic stress compared to their matched nonepidemic strains (P < 0.05), while the nonepidemic strains were more tolerant to oxidative stress (P < 0.05; Table 2). MDR Newport strains (SN3685, SN6668, SN7497, SN7890, SN3082, and SN4124) formed relatively robust biofilms in polystyrene plates (see Fig. S1 in the supplemental material), indicating a potential correlation between the MDR trait and the biofilm-forming ability. Transcriptional analysis of strain SN6668 after short-term osmotic stress showed that three cephalosporin-resistant MDR Salmonella serovar Newport-specific SSH clones (66682D09, 66682F07, and 66682G07) were upregulated, and subsequent real-time RT-PCR revealed that a total of six gene sequences (bla_CMY-2, blc, sugE, dsbC, traC [a truncated form of traC], and tnpA) were differentially expressed (see Table S2 in the supplemental material). These six genes comprise a putatively transposable bla_CMY-2 element harbored in a plasmid of approximately 150 kb (12). It is not clear whether this upregulation provides a fitness benefit to the bacterium or if this is a consequence of a general stress response, such as stress-induced transposition (15).

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TABLE 2. Stress resistance of epidemic cephalosporin-resistant MDR (CMY-2+) Salmonella serovar Newport and other nonepidemic (CMY-2–) Newport isolated from bovines in the Pacific Northwest of the United States

Epidemic serovar Typhimurium DT160 strains showed greater tolerance to acid and oxidative stresses and better replication capability inside Caco-2 cells than the nonepidemic DT156 strain (P < 0.05 [Table 3; see Fig. S2 in the supplemental material]), whereas DT160 strains were more susceptible to osmotic stress (P < 0.05; Table 3). Transcriptional analysis of DT160 strain STNZ152 under short-term oxidative stress showed upregulation of three chromosomal gene sequences and 15 plasmid (pSLT) gene sequences (from Salmonella serovar Typhimurium LT2) and one DT160-specific SSH clone (1521C06) (see Table S3 in the supplemental material). Subsequent real-time RT-PCR showed that three gene sequences of the SSH clone, including mnt and two genes of unknown function, were differentially upregulated (Table S3 in the supplemental material). Upregulated chromosomal genes and plasmid (pSLT) genes included virulence or other stress-inducible genes, such as a prophage-like element-encoded virulence gene, pagK (8); Salmonella plasmid virulence (spv) genes, spvAB (22); UV-induced mutagenesis-related genes, samAB (18); and a macrophage-inducible gene, mig-3 (24). The spv genes were reported to be regulated by the general stress response regulator rpoS-encoded ^S subunit of RNA polymerase (5). Therefore, upregulation of some of these genes may not be an oxidative stress-specific response but rather a general response regulated by ^S or other global regulators.

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TABLE 3. Stress resistance of epidemic (DT160) and nonepidemic (DT156) strains of Salmonella serovar Typhimurium isolated from birds in New Zealand

We expected that the combination of the in vitro experimental models used in the present study would provide an index for the relative abilities of epidemic and nonepidemic Salmonella strains to survive in harsh environmental or host conditions; a subset of stress and infection conditions, including acid, oxidative, osmotic, and intracellular stress, were more favorable for specific epidemic strains relative to their matched nonepidemic strains. In particular, acidic and oxidative conditions are the major stress factors that Salmonella encounters during exposure to gastric contents and phagocytosis in the host (6, 25), and epidemic strains Salmonella serovar Typhimurium DT104 and DT160, which were relatively tolerant to at least one of these stress conditions, may therefore have a survival advantage in the host environment. Salmonella serovar Typhimurium DT160 was clearly more efficient at replication inside epithelial cells, which may improve its ability to survive exposures to antimicrobial selection pressure. Relatively osmotic-stress-tolerant epidemic strains, such as Salmonella serovar Typhimurium DT104 and cephalosporin-resistant MDR Salmonella serovar Newport, may also have survival advantages in an osmotic stress environment such as the host intestinal lumen (4). Nevertheless, the epidemic strains were not consistently more tolerant to different stress conditions, and no common traits were found among all epidemic strains. If a key phenotype exists that distinguishes most epidemic strains from nonepidemic strains, it was not detected by the assays used here.

Consequently, the present study revealed limited but differential fitness traits of MDR and non-MDR epidemic strains of Salmonella relative to their coexisting nonepidemic strains. Ultimately, a complex combination of multiple fitness traits, including antimicrobial resistance, may be involved in the differential epidemicity of epidemic and nonepidemic strains of Salmonella. Alternatively, we might expect the variance of nonepidemic strains to be greater than epidemic strains, and thus our limited sample size may have compromised the statistical power of our investigation. This alternative is not borne out by F tests for the data shown in Tables 1 and 2, although there is a trend toward greater variance for nonepidemic strains.

Expression analysis identified stress-regulated genes specific to epidemic strains that were upregulated under certain conditions relatively favorable for their host strains. Further investigation of the detailed functions of these genes and the genetic and fitness-phenotypic traits of strains under more complex combinations of different stress conditions may provide better insight into possible mechanisms that underlie increased epidemic potential of epidemic strains of Salmonella.

 
We thank S. LaFrentz, L. Orfe, D. Duricka, and X. Zhou for technical advice and help and B. Slinker and M. Evans for statistical advice.

This study was supported by the Agricultural Animal Health Program, College of Veterinary Medicine, Pullman, WA, 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.

 
* 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: drcall{at}wsu.edu

Published ahead of print on 2 March 2007.

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

Top ABSTRACT INTRODUCTION REFERENCES

 

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Applied and Environmental Microbiology, May 2007, p. 3101-3104, Vol. 73, No. 9
0099-2240/07/$08.00+0     doi:10.1128/AEM.02607-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Supplemental material Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Kang, M.-S. Articles by Call, D. R. Search for Related Content PubMed PubMed Citation Articles by Kang, M.-S. Articles by Call, D. R. Agricola Articles by Kang, M.-S. Articles by Call, D. R.