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Transcription modulation of gene expression in Salmonella enterica serotype Choleraesuis by sub-inhibitory concentrations of ciprofloxacin
Food Research International 45 (2012) 973–977
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Food Research International j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f o o d r e s
Transcription modulation of gene expression in Salmonella enterica serotype Choleraesuis by sub-inhibitory concentrations of ciprofloxacin Chyi-Liang Chen a, 1, Lin-Hui Su b, 1, Cheng-Hsun Chiu a, c,⁎ a b c
Molecular Infectious Diseases Research Center, Chang Gung Memorial Hospital, Taoyuan, Taiwan Department of Laboratory Medicine, Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taoyuan, Taiwan Department of Pediatrics, Chang Gung Children's Hospital, Chang Gung University College of Medicine, Taoyuan, Taiwan
a r t i c l e
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Article history: Received 16 February 2011 Accepted 3 June 2011 Keywords: Salmonella enterica serotype Choleraesuis Ciprofloxacin Resistance mechanism Microarray analysis Gene expression
a b s t r a c t Salmonella enterica serotype Choleraesuis usually causes systemic infection in humans that requires antimicrobial therapy. The emergence of S. Choleraesuis resistant to multiple antimicrobial agents, notably fluoroquinolones, has added difficulties in the selection of appropriate antibiotics. In the present study, microarray analysis was used to evaluate the gene expression changes in S. Choleraesuis with or without the presence of sub-inhibitory concentrations of ciprofloxacin. The expression changes in a ciprofloxacinresistant strain, SC-B67, were compared to those observed in a ciprofloxacin-susceptible strain, SC-B42. An expression change was considered significant and included for analysis if the difference was over 1.5 fold. Genes showing concomitant up-regulation or down-regulation as well as those showing consistent overexpression/repression in both strains were excluded from analysis. With the addition of ciprofloxacin at 0.5fold minimum inhibitory concentrations, the number of genes with significant expression changes was much greater in SC-B67 (274 genes; 225 up-regulated and 49 down-regulated) than in SC-B42 (57 genes; 8 upregulated and 49 down-regulated; P b 0.001). Genes involved in a wide variety of transporters and metabolism functions, including amino acids, carbohydrates, inorganic ions, and coenzymes, were significantly upregulated. The transcription/translation and replication/recombination/repair processes as well as signal transduction mechanisms were also vividly up-regulated. However, majority of the significant changes were observed only in the ciprofloxacin-resistant strain SC-B67. Besides the well studied resistance mechanisms associated with fluoroquinolone resistance, the ability to respond to unfavorable antibiotic-related stress through the transcriptional modulation of massive genes involved in many vital biological functions may be crucial for S. Choleraesuis to survive and hence become resistant to the antibiotics. © 2011 Elsevier Ltd. All rights reserved.
1. Introduction Salmonella infection continues to be a distressing health problem worldwide. Among more than 2500 Salmonella serotypes, S. enterica serotype Choleraesuis has a high predilection for causing systemic infection in swine, including enterocolitis, septicemia, and pneumonia (Hyland, Brown, & Murtaugh, 2006), and in humans, including bacteremia and mycotic aneurysm (Chen et al., 2007; Jean, Wang, & Hsueh, 2006). Antimicrobial therapy is inevitable for such patients (Cohen, Bartlett, & Corey, 1987; Su & Chiu, 2007). Unfortunately, the emergence of ciprofloxacin resistance in S. Choleraesuis has become an island-wide problem in Taiwan since 2000 (Chiu et al., 2002; Chiu, Wu, Su, Liu, & Chu, 2004). Worst of all, we found in 2002 a clinical isolate of S. Choleraesuis, SC-B67, which showed resistance to ⁎ Corresponding author at: No. 5, Fu-Hsin Street, Kweishan 333, Taoyuan, Taiwan. Tel.: +886 3 3281200x8896; fax: +886 3 3288957. E-mail address: [email protected] (C.-H. Chiu). 1 C.-L. Chen and L.-H. Su contributed equally to this paper. 0963-9969/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2011.06.015
multiple agents, including ceftriaxone and ciprofloxacin (Chiu, Su, Chu, Chia, et al., 2004). Reduced susceptibility to ciprofloxacin is also common in other Salmonella serotypes (Lee et al., 2009). Fluoroquinolone resistance usually occurs due to mutations in the quinolone resistancedetermining region of DNA gyrase (GyrA and GyrB) and topoisomerase IV (ParC and ParE), which serve as sites of action by fluoroquinolones (Hooper, 2001). Plasmid-mediated quinolone resistance was also reported (Pai, Seo, & Choi, 2007). Other mechanism involves reduced accumulation of fluoroquinolone concentrations in cells through the enhanced expression of endogenous efflux systems, such as AcrAB– TolC (Cloeckaert & Chaslus-Dancla, 2001). Alterations in the expression of outer membrane proteins, such as OmpF or lipopolysaccharides, have also been discovered (Cloeckaert & Chaslus-Dancla, 2001). Microarray analysis is increasingly used to evaluate the genomewide gene expression profiles of Salmonella (Fàbrega, du Merle, Le Bouguénec, Jiménez de Anta & Vila, 2009; Feng et al., 2009; O'Regan et al., 2010; Weir, Martin, Poppe, Coombes, & Boerlin, 2008). The gene expression profiles could be further proven through other experimental
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approaches to reflect the features in virulence, resistance, and motility. Nevertheless, reports on the transcriptional modulation of gene expression by different levels of antibiotic stress remain rare for Salmonella (Dowd, Killinger-Mann, Blanton, San Francisco, & Brashears, 2007; Weir et al., 2008). In view of the emerging antimicrobial resistance in the highly invasive S. Choleraesuis, the mechanism of antimicrobial resistance should be carefully studied for a better control of the infection. Thus, a genome-wide analysis of transcriptional response to ciprofloxacin was performed in S. Choleraesuis using an oligo-chip microarray system in this study. The differential gene expression was compared between ciprofloxacin-resistant and -susceptible strains in the presence or absence of ciprofloxacin at a sub-inhibitory concentration. It is hoped that, other than those well studied mechanisms of fluoroquinolone resistance, the present study may provide a better understanding on whether resistant S. Choleraesuis may develop adaptation mechanisms at the transcriptional level in dealing with the stress from ciprofloxacin. 2. Materials and methods 2.1. Bacteria and antimicrobial susceptibility Ciprofloxacin-resistant S. Choleraesuis SC-B67 was used to investigate the effect of ciprofloxacin at a sub-inhibitory concentration (Chiu, Su, Chu, Chia, et al., 2004). A ciprofloxacin-susceptible strain, S. Choleraesuis SC-B42, was used for comparison. The two strains have a close genetic similarity as demonstrated by PFGE typing (Chu et al., 2005). The only difference was found in SC-B67 which carried an extra 138-kb plasmid, pSC138, that contains multiple antimicrobial resistance genes (Table 1) (Chiu & Ou, 1996; Chiu, Su, Chu, Chia, et al., 2004; Chu et al., 2005; Ye et al., 2011). S. Choleraesuis SC-B42 and SC-B67 were isolated from blood cultures of two bacteremic patients at Chang Gung Memorial Hospital, Taiwan, in 2002. Pure colonies from primary cultures were stored at −80 °C and subcultured when necessary. Plate cultures were kept at 4 °C and used within 2 weeks. Minimum inhibitory concentrations (MICs) of ciprofloxacin were determined by a standard broth microdilution method and interpreted according to the suggestion of the Clinical and Laboratory Standards Institute (CLSI, 2009). SC-B42 was susceptible to ciprofloxacin with a MIC of 0.032 μg/mL, while SCB67 was resistant (ciprofloxacin MIC, 12 μg/mL). 2.2. Microarray analysis The two strains were cultured overnight (~18 h) at 37 °C in LuriaBertani (LB) broth to reach an early stationary phase. Both strains were cultured in duplicate with one of each added with ciprofloxacin at their respective 0.5-fold MICs (0.016 μg/mL for SC-B42 and 6 μg/mL for SCB67). Preparation of total Salmonella RNAs, cDNA labeling, hybridization, and image screening were performed as described previously (Feng et al., 2009). The cDNAs from antibiotic-containing cultures were
labeled with Cy5-dUTP, while those from antibiotic-free cultures were labeled with Cy3-dUTP. The oligo-chip microarray contains more than 15,000 oligonucleotide probes that are complementary to 4975 genes in triplicate of the S. Choleraesuis SC-B67 genome sequence (NC_006905) published previously (Chiu et al., 2005). Microarray experiments were performed with duplication to confirm the reproducibility. Signals for each gene were calculated as the average intensity of Cy3 signal (AICy3) or Cy5 signal (AICy5) after dye normalization and average expression ratio (AER) represented by the average of “Log2 (AICy5/AICy3)”. For an efficient analysis, genes with AERs at the range of “b −1.5” or “N1.5” were considered as significantly down-regulated or up-regulated, respectively, and were selected for comparison. To exclude those genes with significant expression differences elicited from responses common to both strains (i.e., consistent overexpression/repression) but may not involved in the ciprofloxacin resistance of SC-B67, a set of “SC-B67 control” was established. The results contained all genes that were differentially expressed with AER N 1.5 or AER b −1.5 when genes in SC-B67 were compared with the corresponding genes in SC-B42, both without the presence of ciprofloxacin. In such a strategy, the significant expression changes due to genetic differences between the two strains may be excluded. However, the possibility that some genes may actually involve in the ciprofloxacin resistance, although without significant expression changes, does exist. For simplicity, the possibility was ignored in the present study. χ 2 test was used for statistical analysis. A P-value b 0.05 was considered statistically significant. 3. Results Of the 4975 putative genes on the microarray chip, 4883 fulfilled the probe selection criteria after normalization and were subjected for further analysis. Fig. 1 is the Venn diagram showing the number of genes with significant differential expression between SC-B42 (ciprofloxacinsusceptible) and SC-B67 (ciprofloxacin-resistant) after ciprofloxacin exposure. In the presence of ciprofloxacin, the number of genes with significant expression changes was significantly greater in SC-B67 (total, 489, 10.0%; up-regulation, 267; down-regulation, 222) than in SC-B42 (total, 99, 2.0%; up-regulation, 38; down-regulation, 61) (P b 0.001). Among them, 37 genes (up-regulation, 25; down-regulation, 12) were found to have concomitant changes in both strains (Supplemental Table A1), indicating that they might not be specifically associated with the ciprofloxacin resistance in SC-B67 and therefore were not included for further analysis. A total of 1227 (25.1%) genes were identified in the set of “SC-B67 control”, with 291 (23.7%) up-regulated and 936 (76.3%) downregulated. As shown in Fig. 1, if these consistent differences were further excluded, 274 (5.6%) genes in SC-B67 remained the possibility to be involved in the actual fitness response to ciprofloxacin challenge. In contrast, only 57 (1.2%) genes in SC-B42 were related specifically to the ciprofloxacin challenge, SC-B67 appeared to possess
Table 1 Genetic comparison of S. Choleraesuis SC-B42 and SC-B67. Strains
SC-B67 SC-B42
Plasmids (Kb)
50, 138 50
Antibiotic resistance phenotypea
ACSSuTGmKTpCroCip All susceptible
Resistance mechanism Ciprofloxacinb
References Ceftriaxone
GyrA
ParC
blaCMY-2
S83F D87N No mutations
S80I
Presence Absence
Chiu, Su, Chu, Chia, et al., 2004; Chu et al., 2005; Ye et al., 2011 Chu et al. (2005)
a A = ampicillin, C = chloramphenicol, S = streptomycin, Su = sulfonamide, T = tetracycline, Gm = gentamicin, K = kanamycin, Tp = trimethoprim, Cro = ceftriaxone, Cip = ciprofloxacin. b D, aspartic acid; F, phenylalanine; I, isoleucine; N, asparagine; S, serine.
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Table 2 Functional categorization of the predicted proteome using COG protein database for SCB67 and SC-B42. Gene category
Number of genes regulated SC-B67
Fig. 1. Summary of genes showing differential expression between strains and after exposure to ciprofloxacin (CIP). The set of “SC-B67/SC-B42” showed that genes were differentially expressed with AER N 1.5 or b −1.5, between strains SC-B42 and SC-B67 in the absence of CIP. The sets of “SC-B42+ CIP/SC-B42 — CIP” and “SC-B67+ CIP/SC-B67 — CIP” contained the genes that were differentially expressed, with an AER N 1.5 or b −1.5 for each gene, in the two strains with the presence of ciprofloxacin compared respectively to the unexposed replicate. T, number of total genes; U, number of genes showing upregulation; D, number of genes showing down-regulation.
much greater ability in facing the unfavorable environment associated with ciprofloxacin (P b 0.00000005). It was also noted that the number of genes with up-regulated expression was significantly greater in SCB67 than that found in SC-B42 (225/274 vs. 8/57, P b 0.00000005). Over-expression of these genes appeared to have a close correlation with the survival, and therefore the resistance, of SC-B67. To elucidate the type of biofunctions that these genes may be involved in, a functional categorization of the predicted proteome according to the protein database of Clusters of Orthologous Groups (COG) was further analyzed. Genes showing concomitant upregulation or down-regulation as well as those showing consistent overexpression/repression in both strains were excluded. As shown in Table 2, the significantly up-regulated genes in SC-B67 were involved in a wide variety of transporters and metabolism functions, including amino acids, carbohydrates, inorganic ions, coenzymes, nucleotides, and lipids. The signal transduction, transcription, translation, and replication/recombination/repair processes as well as biosynthesis and biogenesis of cellular components were all actively up-regulated. In contrast, these changes were not found in SC-B42 (Table 2). On the other hand, a metabolic reconstruction analysis based on the pathway database of Kyoto Encyclopedia of Genes and Genomes (KEGG) was performed to indicate what pathways might be involved in responding to the stress from ciprofloxacin. As shown in Table 3, genes involved in many metabolism and biosynthesis pathways were over-expressed. Similar to those observed in COG analysis, the activated pathways involved a wide spectrum of biological functions as mentioned above. These prosperous phenomena were absent in SC-B42.
4. Discussion Studies on antimicrobial resistance mechanisms, fluoroquinolone resistance in particular, usually focus on the detection of mutations at the antibiotic target sites, presence of known resistance genes, or changes in the expression of efflux systems or outer-membrane proteins (Cloeckaert & Chaslus-Dancla, 2001; Hooper, 2001; Pai, Seo, & Choi, 2007). However, it was also known that antibiotics at low concentrations may alter global bacterial transcription patterns (Yim, Wang, &
Not in COGs Function unknown General function prediction only Secondary metabolites biosynthesis, transport and catabolism Inorganic ion transport and metabolism Coenzyme transport and metabolism Lipid transport and metabolism Nucleotide transport and metabolism Amino acid transport and metabolism Carbohydrate transport and metabolism Energy production and conversion Posttranslational modification, protein turnover, chaperones Cell motility Cell wall/membrane biogenesis Signal transduction mechanisms Defense mechanisms Cell cycle control, mitosis and meiosis Replication, recombination and repair Transcription RNA processing and modification Translation
SC-B42
Up
Down
Up
Down
43 17 23 2
11 4 5 0
1 1 0 0
11 2 8 1
17 12 3 6 23 20 8 5
3 1 0 6 4 5 1 1
0 0 1 0 2 0 0 0
2 2 1 1 3 2 2 1
4 7 8 3 5 17 18 1 7
0 6 4 0 0 0 4 0 1
0 0 0 0 0 2 0 0 0
2 2 2 2 0 1 2 0 0
Davies, 2006). Nalidixic acid was found to induce a positive adaptive effect in S. Typhimurium, although repression of virulence-associated genes was also observed (Dowd, Killinger-Mann, Blanton, San Francisco, & Brashears, 2007). A recent study indicated that, using the tetracyclinesusceptible S. Typhimurium strain LT2 as a reference, significant upregulation was induced by sub-inhibitory concentrations of tetracycline in 11 genes associated with virulence, motility, and regulators for some important biofunctions in the tetracyclin-resistant S. Typhimurium strain DT104 (Weir et al., 2008). In the present study, through the genome-wide analysis of transcriptional responses to ciprofloxacin challenge, many more genes related to basic biological functions were found to show modulated expression in the resistant strain SC-B67. Because the phenomena were absent from the susceptible strain SCB42, it is reasonable to conclude that the ability to present such active responses may play an important role for the resistant bacteria to survive and replicate in the unfavorable environment caused by antibiotics, such as fluoroquinolones. However, it is also known that becoming antimicrobial resistant may form a kind of biological burden, and as a consequence, resistant bacteria may become more biologically obtuse and less virulent (Barza, 2002). Using microarray analysis, recent reports have demonstrated in both S. Typhimurium and S. Enteritidis that, compared to isogenic mutants, ciprofloxacin-resistant mutants had a lower expression of virulence genes and impaired growth (Fàbrega et al., 2009; O'Regan et al., 2010). Similar conditions were found in the present study when the resistant strain SC-B67 was compared with the susceptible strain SC-B42. As shown in the result set of “SC-B67 control”, without the presence of ciprofloxacin, significant differential expression was found in 25% of the genes studied, while 75% of these genes demonstrated a significantly lower expression in SC-B67. However, with the addition of ciprofloxacin, we were able to show that resistant bacteria are actually more versatile in responding to the environmental challenge. Unlike the results reported previously in S. Typhimurium and S. Enteritidis (Fàbrega et al., 2009; O'Regan et al., 2010), the significant expression changes were not found among virulence genes in the present study. In fact, the expression levels of virulence-associated
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Table 3 Metabolic reconstruction analysis based on KEGG pathway database for SC-B67 and SCB42. Genes showing concomitant up-regulation or down-regulation as well as those showing consistent overexpression/repression in both strains have been excluded. Gene category
Number of genes regulated SC-B67
gamma-Hexachlorocyclohexane degradation Glyoxylate and dicarboxylate metabolism Bisphenol A degradation Bacterial chemotaxis — general Aminoacyl-tRNA synthetases Terpenoid biosynthesis Propanoate metabolism Nitrobenzene degradation Glutathione metabolism Aminophosphonate metabolism Novobiocin biosynthesis Oxidative phosphorylation Androgen and estrogen metabolism Bile acid biosynthesis Citrate cycle (TCA cycle) C5-Branched dibasic acid metabolism Ubiquinone biosynthesis DNA polymerase Limonene and pinene degradation Ethylbenzene degradation Benzoate degradation via CoA ligation Urea cycle and metabolism of amino groups Nicotinate and nicotinamide metabolism Reductive carboxylate cycle (CO2 fixation) Porphyrin and chlorophyll metabolism Folate biosynthesis Sulfur metabolism Butanoate metabolism 1- and 2-Methylnaphthalene degradation Pyruvate metabolism Fatty acid — metabolism and biosynthesis Peptidoglycan biosynthesis Nitrogen metabolism Pantothenate and CoA biosynthesis Nucleic acid — metabolism and biosynthesis Two-component system — general Sugars — metabolism and biosynthesis ABC transporters — general Amino acids — metabolism and biosynthesis
in resistant bacteria could be actively upregulated and hence help to the steady establishment of the resistant bacteria in the environment with antibiotic stress. Acknowledgements
SC-B42
Up
Down
Up
Down
0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 3 3 3 4 7 7 13 13 28
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 1 0 0 0 0 0 2 0 1 2
1 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 0 0 0 0 0 0 0 1 0 0 0 1 0 0 0 0 0 2
0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1
genes, such as those in the Salmonella pathogenicity islands, were not significantly different between the two tested strains, with or without the presence of ciprofloxacin. It is possible that S. Choleraesuis, as a constitutionally more virulent serotype, may steadily express virulence factors irrespectively of the change of environment. Further studies using isogenic resistant mutants, like those used in the S. Typhimurium or S. Enteritidis studies are needed to reveal the actual situation (Fàbrega et al., 2009; O'Regan et al., 2010). Results of this transcriptomic study demonstrated that the response of resistant strain SC-B67 to ciprofloxacin was associated with not just a complicated resistance mechanism but also a sophisticated network system, allowing Salmonella to survive successfully in dealing with the environmental stress. Nevertheless, further investigations on the regulation of this genetic network are necessary to have a better understanding on the association of resistance mechanisms and virulence in this invasive Salmonella. 5. Conclusions The present study demonstrated the influence of ciprofloxacin on an invasive serotype of Salmonella. Besides the known resistance mechanisms that directly lead to the fluoroquinolone resistance, resistant strains appeared to be more flexible and active in dealing with antibiotic stress. When necessary, many of the repressed genes
Our studies were supported by the grants (CMRPG381592, CMRPG490051, and CMRPG490141) from the Chang Gung Memorial Hospital, Taoyuan, Taiwan. Appendix A. Supplementary data Supplementary data to this article can be found online at doi:10. 1016/j.foodres.2011.06.015. References Barza, M. (2002). Potential mechanisms of increased disease in humans from antimicrobial resistance in food animals. Clinical Infectious Diseases, 34(Suppl. 3), S123–S125. Chen, P. L., Wu, C. J., Chang, C. M., Lee, H. C., Lee, N. Y., Shih, H. I., et al. (2007). Extraintestinal focal infections in adults with Salmonella enterica serotype Choleraesuis bacteremia. Journal of Microbiology, Immunology, and Infection, 40(3), 240–247. Chiu, C. H., & Ou, J. T. (1996). Rapid identification of Salmonella serovars in feces by specific detection of virulence genes, invA and spvC, by an enrichment broth culture-multiplex PCR combination assay. Journal of Clinical Microbiology, 34(10), 2619–2622. Chiu, C. H., Su, L. H., Chu, C., Chia, J. H., Wu, T. L., Lin, T. Y., et al. (2004). Isolation of Salmonella enterica serotype Choleraesuis resistant to ceftriaxone and ciprofloxacin. Lancet, 363(9417), 1285–1286. Chiu, C. H., Tang, P., Chu, C., Hu, S., Bao, Q., Yu, J., et al. (2005). The genome sequence of Salmonella enterica serovar Choleraesuis, a highly invasive and resistant zoonotic pathogen. Nucleic Acids Research, 33(5), 1690–1698. Chiu, C. H., Wu, T. L., Su, L. H., Chu, C., Chia, J. H., Kuo, A. J., et al. (2002). The emergence in Taiwan of fluoroquinolone resistance in Salmonella enterica serotype Choleraesuis. The New England Journal of Medicine, 346(6), 413–419. Chiu, C. H., Wu, T. L., Su, L. H., Liu, J. W., & Chu, C. (2004). Fluoroquinolone resistance in Salmonella enterica serotype Choleraesuis, Taiwan, 2000–2003. Emerging Infectious Diseases, 10(9), 1674–1676. Chu, C., Su, L. H., Chu, C. H., Baucheron, S., Cloeckaert, A., & Chiu, C. H. (2005). Resistance to fluoroquinolones linked to gyrA and par C mutations and overexpression of acr AB efflux pump in Salmonella enterica serotype Choleraesuis. Microbial Drug Resistance, 11(3), 248–253. Clinical and Laboratory Standards Institute (2009). Performance standards for antimicrobial susceptibility testing: 19th informational supplement M100-S19. Wayne, PA: Clinical and Laboratory Standards Institute. Cloeckaert, A., & Chaslus-Dancla, E. (2001). Mechanisms of quinolone resistance in Salmonella. Veterinary Research, 32(3–4), 291–300. Cohen, J. I., Bartlett, J. A., & Corey, G. R. (1987). Extra-intestinal manifestations of Salmonella infections. Medicine (Baltimore), 66(5), 349–388. Dowd, S. E., Killinger-Mann, K., Blanton, J., San Francisco, M., & Brashears, M. (2007). Positive adaptive state: microarray evaluation of gene expression in Salmonella enterica Typhimurium exposed to nalidixic acid. Foodborne Pathogens and Disease, 4(2), 187–200. Fàbrega, A., du Merle, L., Le Bouguénec, C., Jiménez de Anta, M. T., & Vila, J. (2009). Repression of invasion genes and decreased invasion in a high-level fluoroquinolone-resistant Salmonella typhimurium mutant. PloS One, 4(11), e8029. Feng, Y., Hsiao, Y. H., Chen, H. L., Chu, C., Tang, P., & Chiu, C. H. (2009). Apoptosis-like cell death induced by Salmonella in Acanthamoeba rhysodes. Genomics, 94(2), 132–137. Hooper, D. C. (2001). Emerging mechanisms of fluoroquinolone resistance. Emerging Infectious Diseases, 7(2), 337–341. Hyland, K. A., Brown, D. R., & Murtaugh, M. P. (2006). Salmonella enterica serovar Choleraesuis infection of the porcine jejunal Peyer's patch rapidly induces IL-1beta and IL-8 expression. Veterinary Immunology and Immunopathology, 109(1–2), 1–11. Jean, S. S., Wang, J. Y., & Hsueh, P. R. (2006). Bacteremia caused by Salmonella enterica serotype Choleraesuis in Taiwan. Journal of Microbiology Immunology and Infection, 39(5), 358–365. Lee, H. Y., Su, L. H., Tsai, M. H., Kim, S. W., Chang, H. H., Jung, S. I., et al. (2009). High rate of reduced susceptibility to ciprofloxacin and ceftriaxone among nontyphoid Salmonella clinical isolates in Asia. Antimicrobial Agents and Chemotherapy, 53(6), 2696–2699. O'Regan, E., Quinn, T., Frye, J. G., Pagès, J. M., Porwollik, S., Fedorka-Cray, P. J., et al. (2010). Fitness costs and stability of a high-level ciprofloxacin resistance phenotype in Salmonella enterica serotype Enteritidis: Reduced infectivity associated with decreased expression of Salmonella pathogenicity island 1 genes. Antimicrobial Agents and Chemotherapy, 54(1), 367–374. Pai, H., Seo, M. R., & Choi, T. Y. (2007). Association of QnrB determinants and production of extended-spectrum beta-lactamases or plasmid-mediated AmpC beta-lactamases in clinical isolates of Klebsiella pneumoniae. Antimicrobial Agents and Chemotherapy, 51(1), 366–368.
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Food Research International j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f o o d r e s
Transcription modulation of gene expression in Salmonella enterica serotype Choleraesuis by sub-inhibitory concentrations of ciprofloxacin Chyi-Liang Chen a, 1, Lin-Hui Su b, 1, Cheng-Hsun Chiu a, c,⁎ a b c
Molecular Infectious Diseases Research Center, Chang Gung Memorial Hospital, Taoyuan, Taiwan Department of Laboratory Medicine, Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taoyuan, Taiwan Department of Pediatrics, Chang Gung Children's Hospital, Chang Gung University College of Medicine, Taoyuan, Taiwan
a r t i c l e
i n f o
Article history: Received 16 February 2011 Accepted 3 June 2011 Keywords: Salmonella enterica serotype Choleraesuis Ciprofloxacin Resistance mechanism Microarray analysis Gene expression
a b s t r a c t Salmonella enterica serotype Choleraesuis usually causes systemic infection in humans that requires antimicrobial therapy. The emergence of S. Choleraesuis resistant to multiple antimicrobial agents, notably fluoroquinolones, has added difficulties in the selection of appropriate antibiotics. In the present study, microarray analysis was used to evaluate the gene expression changes in S. Choleraesuis with or without the presence of sub-inhibitory concentrations of ciprofloxacin. The expression changes in a ciprofloxacinresistant strain, SC-B67, were compared to those observed in a ciprofloxacin-susceptible strain, SC-B42. An expression change was considered significant and included for analysis if the difference was over 1.5 fold. Genes showing concomitant up-regulation or down-regulation as well as those showing consistent overexpression/repression in both strains were excluded from analysis. With the addition of ciprofloxacin at 0.5fold minimum inhibitory concentrations, the number of genes with significant expression changes was much greater in SC-B67 (274 genes; 225 up-regulated and 49 down-regulated) than in SC-B42 (57 genes; 8 upregulated and 49 down-regulated; P b 0.001). Genes involved in a wide variety of transporters and metabolism functions, including amino acids, carbohydrates, inorganic ions, and coenzymes, were significantly upregulated. The transcription/translation and replication/recombination/repair processes as well as signal transduction mechanisms were also vividly up-regulated. However, majority of the significant changes were observed only in the ciprofloxacin-resistant strain SC-B67. Besides the well studied resistance mechanisms associated with fluoroquinolone resistance, the ability to respond to unfavorable antibiotic-related stress through the transcriptional modulation of massive genes involved in many vital biological functions may be crucial for S. Choleraesuis to survive and hence become resistant to the antibiotics. © 2011 Elsevier Ltd. All rights reserved.
1. Introduction Salmonella infection continues to be a distressing health problem worldwide. Among more than 2500 Salmonella serotypes, S. enterica serotype Choleraesuis has a high predilection for causing systemic infection in swine, including enterocolitis, septicemia, and pneumonia (Hyland, Brown, & Murtaugh, 2006), and in humans, including bacteremia and mycotic aneurysm (Chen et al., 2007; Jean, Wang, & Hsueh, 2006). Antimicrobial therapy is inevitable for such patients (Cohen, Bartlett, & Corey, 1987; Su & Chiu, 2007). Unfortunately, the emergence of ciprofloxacin resistance in S. Choleraesuis has become an island-wide problem in Taiwan since 2000 (Chiu et al., 2002; Chiu, Wu, Su, Liu, & Chu, 2004). Worst of all, we found in 2002 a clinical isolate of S. Choleraesuis, SC-B67, which showed resistance to ⁎ Corresponding author at: No. 5, Fu-Hsin Street, Kweishan 333, Taoyuan, Taiwan. Tel.: +886 3 3281200x8896; fax: +886 3 3288957. E-mail address: [email protected] (C.-H. Chiu). 1 C.-L. Chen and L.-H. Su contributed equally to this paper. 0963-9969/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2011.06.015
multiple agents, including ceftriaxone and ciprofloxacin (Chiu, Su, Chu, Chia, et al., 2004). Reduced susceptibility to ciprofloxacin is also common in other Salmonella serotypes (Lee et al., 2009). Fluoroquinolone resistance usually occurs due to mutations in the quinolone resistancedetermining region of DNA gyrase (GyrA and GyrB) and topoisomerase IV (ParC and ParE), which serve as sites of action by fluoroquinolones (Hooper, 2001). Plasmid-mediated quinolone resistance was also reported (Pai, Seo, & Choi, 2007). Other mechanism involves reduced accumulation of fluoroquinolone concentrations in cells through the enhanced expression of endogenous efflux systems, such as AcrAB– TolC (Cloeckaert & Chaslus-Dancla, 2001). Alterations in the expression of outer membrane proteins, such as OmpF or lipopolysaccharides, have also been discovered (Cloeckaert & Chaslus-Dancla, 2001). Microarray analysis is increasingly used to evaluate the genomewide gene expression profiles of Salmonella (Fàbrega, du Merle, Le Bouguénec, Jiménez de Anta & Vila, 2009; Feng et al., 2009; O'Regan et al., 2010; Weir, Martin, Poppe, Coombes, & Boerlin, 2008). The gene expression profiles could be further proven through other experimental
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approaches to reflect the features in virulence, resistance, and motility. Nevertheless, reports on the transcriptional modulation of gene expression by different levels of antibiotic stress remain rare for Salmonella (Dowd, Killinger-Mann, Blanton, San Francisco, & Brashears, 2007; Weir et al., 2008). In view of the emerging antimicrobial resistance in the highly invasive S. Choleraesuis, the mechanism of antimicrobial resistance should be carefully studied for a better control of the infection. Thus, a genome-wide analysis of transcriptional response to ciprofloxacin was performed in S. Choleraesuis using an oligo-chip microarray system in this study. The differential gene expression was compared between ciprofloxacin-resistant and -susceptible strains in the presence or absence of ciprofloxacin at a sub-inhibitory concentration. It is hoped that, other than those well studied mechanisms of fluoroquinolone resistance, the present study may provide a better understanding on whether resistant S. Choleraesuis may develop adaptation mechanisms at the transcriptional level in dealing with the stress from ciprofloxacin. 2. Materials and methods 2.1. Bacteria and antimicrobial susceptibility Ciprofloxacin-resistant S. Choleraesuis SC-B67 was used to investigate the effect of ciprofloxacin at a sub-inhibitory concentration (Chiu, Su, Chu, Chia, et al., 2004). A ciprofloxacin-susceptible strain, S. Choleraesuis SC-B42, was used for comparison. The two strains have a close genetic similarity as demonstrated by PFGE typing (Chu et al., 2005). The only difference was found in SC-B67 which carried an extra 138-kb plasmid, pSC138, that contains multiple antimicrobial resistance genes (Table 1) (Chiu & Ou, 1996; Chiu, Su, Chu, Chia, et al., 2004; Chu et al., 2005; Ye et al., 2011). S. Choleraesuis SC-B42 and SC-B67 were isolated from blood cultures of two bacteremic patients at Chang Gung Memorial Hospital, Taiwan, in 2002. Pure colonies from primary cultures were stored at −80 °C and subcultured when necessary. Plate cultures were kept at 4 °C and used within 2 weeks. Minimum inhibitory concentrations (MICs) of ciprofloxacin were determined by a standard broth microdilution method and interpreted according to the suggestion of the Clinical and Laboratory Standards Institute (CLSI, 2009). SC-B42 was susceptible to ciprofloxacin with a MIC of 0.032 μg/mL, while SCB67 was resistant (ciprofloxacin MIC, 12 μg/mL). 2.2. Microarray analysis The two strains were cultured overnight (~18 h) at 37 °C in LuriaBertani (LB) broth to reach an early stationary phase. Both strains were cultured in duplicate with one of each added with ciprofloxacin at their respective 0.5-fold MICs (0.016 μg/mL for SC-B42 and 6 μg/mL for SCB67). Preparation of total Salmonella RNAs, cDNA labeling, hybridization, and image screening were performed as described previously (Feng et al., 2009). The cDNAs from antibiotic-containing cultures were
labeled with Cy5-dUTP, while those from antibiotic-free cultures were labeled with Cy3-dUTP. The oligo-chip microarray contains more than 15,000 oligonucleotide probes that are complementary to 4975 genes in triplicate of the S. Choleraesuis SC-B67 genome sequence (NC_006905) published previously (Chiu et al., 2005). Microarray experiments were performed with duplication to confirm the reproducibility. Signals for each gene were calculated as the average intensity of Cy3 signal (AICy3) or Cy5 signal (AICy5) after dye normalization and average expression ratio (AER) represented by the average of “Log2 (AICy5/AICy3)”. For an efficient analysis, genes with AERs at the range of “b −1.5” or “N1.5” were considered as significantly down-regulated or up-regulated, respectively, and were selected for comparison. To exclude those genes with significant expression differences elicited from responses common to both strains (i.e., consistent overexpression/repression) but may not involved in the ciprofloxacin resistance of SC-B67, a set of “SC-B67 control” was established. The results contained all genes that were differentially expressed with AER N 1.5 or AER b −1.5 when genes in SC-B67 were compared with the corresponding genes in SC-B42, both without the presence of ciprofloxacin. In such a strategy, the significant expression changes due to genetic differences between the two strains may be excluded. However, the possibility that some genes may actually involve in the ciprofloxacin resistance, although without significant expression changes, does exist. For simplicity, the possibility was ignored in the present study. χ 2 test was used for statistical analysis. A P-value b 0.05 was considered statistically significant. 3. Results Of the 4975 putative genes on the microarray chip, 4883 fulfilled the probe selection criteria after normalization and were subjected for further analysis. Fig. 1 is the Venn diagram showing the number of genes with significant differential expression between SC-B42 (ciprofloxacinsusceptible) and SC-B67 (ciprofloxacin-resistant) after ciprofloxacin exposure. In the presence of ciprofloxacin, the number of genes with significant expression changes was significantly greater in SC-B67 (total, 489, 10.0%; up-regulation, 267; down-regulation, 222) than in SC-B42 (total, 99, 2.0%; up-regulation, 38; down-regulation, 61) (P b 0.001). Among them, 37 genes (up-regulation, 25; down-regulation, 12) were found to have concomitant changes in both strains (Supplemental Table A1), indicating that they might not be specifically associated with the ciprofloxacin resistance in SC-B67 and therefore were not included for further analysis. A total of 1227 (25.1%) genes were identified in the set of “SC-B67 control”, with 291 (23.7%) up-regulated and 936 (76.3%) downregulated. As shown in Fig. 1, if these consistent differences were further excluded, 274 (5.6%) genes in SC-B67 remained the possibility to be involved in the actual fitness response to ciprofloxacin challenge. In contrast, only 57 (1.2%) genes in SC-B42 were related specifically to the ciprofloxacin challenge, SC-B67 appeared to possess
Table 1 Genetic comparison of S. Choleraesuis SC-B42 and SC-B67. Strains
SC-B67 SC-B42
Plasmids (Kb)
50, 138 50
Antibiotic resistance phenotypea
ACSSuTGmKTpCroCip All susceptible
Resistance mechanism Ciprofloxacinb
References Ceftriaxone
GyrA
ParC
blaCMY-2
S83F D87N No mutations
S80I
Presence Absence
Chiu, Su, Chu, Chia, et al., 2004; Chu et al., 2005; Ye et al., 2011 Chu et al. (2005)
a A = ampicillin, C = chloramphenicol, S = streptomycin, Su = sulfonamide, T = tetracycline, Gm = gentamicin, K = kanamycin, Tp = trimethoprim, Cro = ceftriaxone, Cip = ciprofloxacin. b D, aspartic acid; F, phenylalanine; I, isoleucine; N, asparagine; S, serine.
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Table 2 Functional categorization of the predicted proteome using COG protein database for SCB67 and SC-B42. Gene category
Number of genes regulated SC-B67
Fig. 1. Summary of genes showing differential expression between strains and after exposure to ciprofloxacin (CIP). The set of “SC-B67/SC-B42” showed that genes were differentially expressed with AER N 1.5 or b −1.5, between strains SC-B42 and SC-B67 in the absence of CIP. The sets of “SC-B42+ CIP/SC-B42 — CIP” and “SC-B67+ CIP/SC-B67 — CIP” contained the genes that were differentially expressed, with an AER N 1.5 or b −1.5 for each gene, in the two strains with the presence of ciprofloxacin compared respectively to the unexposed replicate. T, number of total genes; U, number of genes showing upregulation; D, number of genes showing down-regulation.
much greater ability in facing the unfavorable environment associated with ciprofloxacin (P b 0.00000005). It was also noted that the number of genes with up-regulated expression was significantly greater in SCB67 than that found in SC-B42 (225/274 vs. 8/57, P b 0.00000005). Over-expression of these genes appeared to have a close correlation with the survival, and therefore the resistance, of SC-B67. To elucidate the type of biofunctions that these genes may be involved in, a functional categorization of the predicted proteome according to the protein database of Clusters of Orthologous Groups (COG) was further analyzed. Genes showing concomitant upregulation or down-regulation as well as those showing consistent overexpression/repression in both strains were excluded. As shown in Table 2, the significantly up-regulated genes in SC-B67 were involved in a wide variety of transporters and metabolism functions, including amino acids, carbohydrates, inorganic ions, coenzymes, nucleotides, and lipids. The signal transduction, transcription, translation, and replication/recombination/repair processes as well as biosynthesis and biogenesis of cellular components were all actively up-regulated. In contrast, these changes were not found in SC-B42 (Table 2). On the other hand, a metabolic reconstruction analysis based on the pathway database of Kyoto Encyclopedia of Genes and Genomes (KEGG) was performed to indicate what pathways might be involved in responding to the stress from ciprofloxacin. As shown in Table 3, genes involved in many metabolism and biosynthesis pathways were over-expressed. Similar to those observed in COG analysis, the activated pathways involved a wide spectrum of biological functions as mentioned above. These prosperous phenomena were absent in SC-B42.
4. Discussion Studies on antimicrobial resistance mechanisms, fluoroquinolone resistance in particular, usually focus on the detection of mutations at the antibiotic target sites, presence of known resistance genes, or changes in the expression of efflux systems or outer-membrane proteins (Cloeckaert & Chaslus-Dancla, 2001; Hooper, 2001; Pai, Seo, & Choi, 2007). However, it was also known that antibiotics at low concentrations may alter global bacterial transcription patterns (Yim, Wang, &
Not in COGs Function unknown General function prediction only Secondary metabolites biosynthesis, transport and catabolism Inorganic ion transport and metabolism Coenzyme transport and metabolism Lipid transport and metabolism Nucleotide transport and metabolism Amino acid transport and metabolism Carbohydrate transport and metabolism Energy production and conversion Posttranslational modification, protein turnover, chaperones Cell motility Cell wall/membrane biogenesis Signal transduction mechanisms Defense mechanisms Cell cycle control, mitosis and meiosis Replication, recombination and repair Transcription RNA processing and modification Translation
SC-B42
Up
Down
Up
Down
43 17 23 2
11 4 5 0
1 1 0 0
11 2 8 1
17 12 3 6 23 20 8 5
3 1 0 6 4 5 1 1
0 0 1 0 2 0 0 0
2 2 1 1 3 2 2 1
4 7 8 3 5 17 18 1 7
0 6 4 0 0 0 4 0 1
0 0 0 0 0 2 0 0 0
2 2 2 2 0 1 2 0 0
Davies, 2006). Nalidixic acid was found to induce a positive adaptive effect in S. Typhimurium, although repression of virulence-associated genes was also observed (Dowd, Killinger-Mann, Blanton, San Francisco, & Brashears, 2007). A recent study indicated that, using the tetracyclinesusceptible S. Typhimurium strain LT2 as a reference, significant upregulation was induced by sub-inhibitory concentrations of tetracycline in 11 genes associated with virulence, motility, and regulators for some important biofunctions in the tetracyclin-resistant S. Typhimurium strain DT104 (Weir et al., 2008). In the present study, through the genome-wide analysis of transcriptional responses to ciprofloxacin challenge, many more genes related to basic biological functions were found to show modulated expression in the resistant strain SC-B67. Because the phenomena were absent from the susceptible strain SCB42, it is reasonable to conclude that the ability to present such active responses may play an important role for the resistant bacteria to survive and replicate in the unfavorable environment caused by antibiotics, such as fluoroquinolones. However, it is also known that becoming antimicrobial resistant may form a kind of biological burden, and as a consequence, resistant bacteria may become more biologically obtuse and less virulent (Barza, 2002). Using microarray analysis, recent reports have demonstrated in both S. Typhimurium and S. Enteritidis that, compared to isogenic mutants, ciprofloxacin-resistant mutants had a lower expression of virulence genes and impaired growth (Fàbrega et al., 2009; O'Regan et al., 2010). Similar conditions were found in the present study when the resistant strain SC-B67 was compared with the susceptible strain SC-B42. As shown in the result set of “SC-B67 control”, without the presence of ciprofloxacin, significant differential expression was found in 25% of the genes studied, while 75% of these genes demonstrated a significantly lower expression in SC-B67. However, with the addition of ciprofloxacin, we were able to show that resistant bacteria are actually more versatile in responding to the environmental challenge. Unlike the results reported previously in S. Typhimurium and S. Enteritidis (Fàbrega et al., 2009; O'Regan et al., 2010), the significant expression changes were not found among virulence genes in the present study. In fact, the expression levels of virulence-associated
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Table 3 Metabolic reconstruction analysis based on KEGG pathway database for SC-B67 and SCB42. Genes showing concomitant up-regulation or down-regulation as well as those showing consistent overexpression/repression in both strains have been excluded. Gene category
Number of genes regulated SC-B67
gamma-Hexachlorocyclohexane degradation Glyoxylate and dicarboxylate metabolism Bisphenol A degradation Bacterial chemotaxis — general Aminoacyl-tRNA synthetases Terpenoid biosynthesis Propanoate metabolism Nitrobenzene degradation Glutathione metabolism Aminophosphonate metabolism Novobiocin biosynthesis Oxidative phosphorylation Androgen and estrogen metabolism Bile acid biosynthesis Citrate cycle (TCA cycle) C5-Branched dibasic acid metabolism Ubiquinone biosynthesis DNA polymerase Limonene and pinene degradation Ethylbenzene degradation Benzoate degradation via CoA ligation Urea cycle and metabolism of amino groups Nicotinate and nicotinamide metabolism Reductive carboxylate cycle (CO2 fixation) Porphyrin and chlorophyll metabolism Folate biosynthesis Sulfur metabolism Butanoate metabolism 1- and 2-Methylnaphthalene degradation Pyruvate metabolism Fatty acid — metabolism and biosynthesis Peptidoglycan biosynthesis Nitrogen metabolism Pantothenate and CoA biosynthesis Nucleic acid — metabolism and biosynthesis Two-component system — general Sugars — metabolism and biosynthesis ABC transporters — general Amino acids — metabolism and biosynthesis
in resistant bacteria could be actively upregulated and hence help to the steady establishment of the resistant bacteria in the environment with antibiotic stress. Acknowledgements
SC-B42
Up
Down
Up
Down
0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 3 3 3 4 7 7 13 13 28
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 1 0 0 0 0 0 2 0 1 2
1 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 0 0 0 0 0 0 0 1 0 0 0 1 0 0 0 0 0 2
0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1
genes, such as those in the Salmonella pathogenicity islands, were not significantly different between the two tested strains, with or without the presence of ciprofloxacin. It is possible that S. Choleraesuis, as a constitutionally more virulent serotype, may steadily express virulence factors irrespectively of the change of environment. Further studies using isogenic resistant mutants, like those used in the S. Typhimurium or S. Enteritidis studies are needed to reveal the actual situation (Fàbrega et al., 2009; O'Regan et al., 2010). Results of this transcriptomic study demonstrated that the response of resistant strain SC-B67 to ciprofloxacin was associated with not just a complicated resistance mechanism but also a sophisticated network system, allowing Salmonella to survive successfully in dealing with the environmental stress. Nevertheless, further investigations on the regulation of this genetic network are necessary to have a better understanding on the association of resistance mechanisms and virulence in this invasive Salmonella. 5. Conclusions The present study demonstrated the influence of ciprofloxacin on an invasive serotype of Salmonella. Besides the known resistance mechanisms that directly lead to the fluoroquinolone resistance, resistant strains appeared to be more flexible and active in dealing with antibiotic stress. When necessary, many of the repressed genes
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