School of Biological Sciences, Food Sciences Division, University of Nottingham, Sutton Bonington Campus, Leicestershire LE12 5RD, UK1
Centre for Applied Microbiology and Research (CAMR), Research Division, Porton Down, Salisbury SP4 0JG, UK2
PHLS Food Microbiology Food Research Unit, Church Lane, Heavitree, Exeter EX2 5AD, UK3
Author for correspondence: Frieda Jørgensen. Tel: +44 1392 402955. Fax: +44 1392 412835. e-mail: fjorgensen{at}phls.org.uk
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ABSTRACT |
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Keywords: spv-lux reporter, chicken reproductive tissue
Abbreviations: DT, definitive type; PT, phage type; RLU, relative light units (per ml)
a Present address: PHLS Food Microbiology Food Research Unit, Church Lane, Heavitree, Exeter EX2 5AD, UK.
b Professor Stewart sadly died in February 1999, at age 47.
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INTRODUCTION |
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More than 90% of DT 104 strains show multiple antibiotic resistance and 15% carry resistance to quinolones (Wall et al., 1994
; Threlfall et al., 1996
). This may cause therapeutic problems, as fluoroquinolones are the drugs of first choice for treating extra-intestinal and serious intestinal complications of human and animal salmonellosis. DT 104 has a broad spectrum of hosts and can easily spread to many different domestic and wild animals. Outbreaks of DT 104 and PT 4 infection have been associated with a number of food vehicles including poultry meat and eggs (Wall et al., 1994
; Coyle et al., 1988
; Rampling et al., 1989
). A better understanding of the mechanisms by which these foodborne pathogens survive in the environment and colonize chickens will provide the basis for designing more effective intervention strategies.
In previous studies, the resistance of 40 PT 4 strains to heat, acid and hydrogen peroxide, as well as the ability to survive airdrying on commonly used kitchen surface materials was examined (Humphrey et al., 1995 ). The studies identified a small subgroup of strains that were much more sensitive to these stresses than the majority. These strains also showed differences in their ability to invade chicken tissues, particularly those of the reproductive tract (Humphrey et al., 1996
). Like PT 4, DT 104 has been shown, under experimental conditions, to be able to contaminate egg contents and can also be isolated from muscle tissue in laying hens (Williams et al., 1998
; Leach et al., 1999
). Little is known about the ability of DT 104 to invade other tissues, or whether a spectrum of invasiveness exists.
The rpoS-encoded S subunit of RNA polymerase is an important regulator of the general stress response and is also required for virulence in S. typhimurium in orally infected mice (Loewen & Hengge-Aronis, 1994
; Mahan et al., 1996
; Fang et al., 1992
) but less is known about the role of rpoS in the systemic infection of chickens.
The principal aim of the present study was to assess the ability of naturally occurring DT 104 strains to invade tissues in laying hens and relate this to their RpoS status and ability to survive exposure to drying, low pH and heat. We demonstrate variation in the ability to invade and/or persist in different chicken tissues among the DT 104 strains. Stress-sensitive strains were present among the DT 104 strains but these strains were not all less invasive. A majority of stress-sensitive DT 104 and PT 4 strains harboured mutations in the rpoS gene or showed reduced expression of the RpoS protein as measured indirectly using the RpoS-dependent spvR/A'::luxCDABE fusion. The significance of the rpoS gene for the contamination of egg contents is discussed.
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METHODS |
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Care was taken to ensure the aseptic removal of each organ. Birds were first surface-decontaminated using sodium hypochlorite solution. The body cavity was opened and the tissues removed in the following order: liver, spleen, oviduct and ovary. Fresh sterile surgical instruments were used for each bird and, on removal, each organ was immediately transferred to an individual sterile stomacher bag. Faeces and tissue samples were examined for the presence of Salmonella spp. using previously described methods (Humphrey et al., 1996 ). Briefly, tissue samples were weighed and then homogenized using a stomacher in Maximum Recovery Diluent (MRD; Oxoid CM733). Liver and spleen homogenates were then serially diluted in MRD and plated onto xylose lysine desoxycholate (XLD; Oxoid CM 469) agar for microbiological counts. Remaining homogenates of liver and spleen, and those of ovary and oviduct, were also examined for the presence of Salmonella spp. using standard pre-enrichment and enrichment techniques. Faecal samples were selectively enriched in selenite broth (Oxoid CM 699) and plated onto XLD agar. The infection experiment was performed twice with strains 30, 11, 10 and 41 and once with strains 27, 16, 34 and 31. The proportion of Salmonella spp. positive tissues for each strain was compared using the
2 test with Yates corrections for small numbers (Statgraphics 5, STSC Inc.).
Expression of bioluminescence.
The ability of the DT 104 and PT 4 strains to activate transcription of the S-dependent spvR and spvA promoters was determined by measuring expression of bioluminescence during growth of strains transformed with the plasmids pSB367 or pSB368 (Swift & Stewart, 1994
). Both pSB367 (high copy number) and pSB368 (medium copy number) contain the spvR gene and a transcriptional fusion luxCDABE (from Photorhabdus luminescens) to the spvA promoter. Expression of the SpvR protein requires an active RpoS protein (Guiney et al., 1995
) and the expression of luxCDABE results from the activation of the spvA promoter by SpvR and RpoS. The bioluminescence of these strains is, therefore, a measure of active RpoS sigma factor in the cell. The functionality of this system as a reporter for active RpoS levels was shown in Escherichia coli BJ4, where the introduction of a mutation in rpoS reduced light levels by 3 log (Swift & Stewart, 1994
). Preparation of competent cells, electroporation and isolation of bioluminescent colonies was performed according to previously published techniques (Swift et al., 1993
). Transformed strains were grown with shaking (200 r.p.m.) in LB broth, Lennox (LB; Difco) at pH 7·3. These cultures were standardized to an initial OD600 of 0·025 from overnight cultures and grown at 37 °C for up to 15 h. Bioluminescence was measured by determining the number of relative light units per ml (RLU) in scintillation vial inserts (4 ml; FSA Laboratory Supplies) with a Turner Designs TD-20E luminometer (Streptech) (Swift et al., 1993
). Growth of the cultures was measured simultaneously as OD600. Measurement of RpoS expression and OD600 was repeated on two separate occasions for each strain. The
S-dependency of the bioluminescence expressed by the spvR/A'::luxCDABE fusion was validated by measuring the light expressed in three S. typhimurium strains with identical backgrounds (S. typhimurium LT2) but with different rpoS alleles (strain
4996, rpoS::bla; strain
3000, rpoSLT2; strain
8000, rpoSSL1344; Wilmes-Riesenberg et al., 1997
). In order to confirm that light expression was dependent on the RpoS-dependent promoter, light measurements using a constitutive promoter fused to the lux operon (pSB311) were performed with otherwise isogenic
S-positive and
S-negative S. typhimurium strains (Hill et al., 1993
). In these experiments expression of bioluminescence was constitutively high and showed no growth-phase-dependent induction (results not shown).
Sequence analysis of rpoS genes.
The rpoS genes were amplified from S. typhimurium DT 104 and S. enteritidis PT 4 strains using RPOS5' (5'-CGGAATTCTTATTATCATCAAACATAAC-3') and RPOS3' (5'-CCTTGCCCGGGCTGTGCCGATGCAC-3') amplimers made at the Biopolymer Synthesis and Analysis Unit, University of Nottingham. Template genomic DNA was purified using the ISO-Quick Kit (ORCA Research). PCR amplification was carried out in triplicate, for 30 cycles, each consisting of 30 s denaturation at 95 °C, 1 min of reannealing at 55 °C and 2 min of DNA synthesis at 72 °C. PCR products were separated by electrophoresis, extracted from the gel using a QiaQuick Gel Extraction Kit (QIAGEN), dialysed (Millipore VS filters) and sequenced at the University of Nottingham Sequencing Facility. Each gene was sequenced from at least two independent PCR products and DNA sequences from both strands of the rpoS genes were obtained.
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RESULTS |
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DISCUSSION |
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Previous work on PT 4 demonstrated that naturally occurring strains differed with respect to their resistance to heat, acid, peroxide and airdrying on surfaces, and their virulence in mice and chickens infected orally (Humphrey et al., 1995 , 1996
, 1998
). The studies reported in this paper sought to take the investigations into resistance and virulence of Salmonella spp. further by examining the behaviours of S. typhimurium DT 104 strains and by correlating these with RpoS expression.
The results showed that as with PT 4, DT 104 comprises sensitive and resistant strains when cells are challenged in stationary phase. These results could suggest differences in RpoS expression, and this was explored in several DT 104 and PT 4 strains. In the five sensitive DT 104 and PT 4 strains examined, three harboured mutations in the rpoS gene, while two DT 104 strains had intact rpoS genes and one of those demonstrated a reduced level of RpoS-dependent spvR/A'::luxCDABE expression. In E. coli, expression of the RpoS protein is known to be primarily controlled post-transcriptionally and evidence for post-transcriptional regulation in Salmonella has also been found (Lange & Hengge-Aronis, 1994 ; Schweder et al., 1996
; Pratt & Silhavy, 1996
; Bearson et al., 1996
). It is therefore possible that the reduced expression the RpoS-dependent spvR/A'::luxCDABE fusion in DT 104 strain 11 is due to mutations affecting the translational processing of the RpoS protein or, alternatively, to protein instability. The presence of mutant rpoS alleles has also been investigated in clinical and environmental strains of shiga-like toxin-producing E. coli (Waterman & Small, 1996
). Ten out of 58 strains were acid sensitive, and acid resistance could be restored in six strains when complemented with a functional rpoS in trans, suggesting that at least 10% of the population could be rpoS mutants. The rpoS allele was sequenced in two acid-sensitive strains and one had undergone a single base-pair deletion resulting in a stop codon after amino acid 28 while the other contained an 11 base-pair duplication resulting in a stop codon after amino acid 77 (Waterman & Small, 1996
). Laboratory strains of E. coli have also been shown to contain mutant rpoS alleles or acquire them through extended culture (Ivanova et al., 1992
; Visick & Clarke 1997
; Zambrano et al., 1993
) and several of the typical laboratory LT2-derived strains of S. typhimurium contain an rpoS allele with a rare UUG start codon (Wilmes-Riesenberg et al., 1997
). The DT 104 strains 11 and 10, found in this study, were initially isolated from human cases of gastroenteritis, while the PT 4 strains C and I were isolated from chicken carcasses. The rpoS mutation in strain 10 may have arisen during the initial isolation of the strain, although it has maintained its stress sensitivity after trials of extensive subculturing in the laboratory. It is, however, also possible that strains lacking important virulence factors may cause infection, as the development of an infection will, among other things, depend on the status of the host and the amount of inoculum (Picard et al., 1999
). We are currently addressing this issue by examining the RpoS status of colonies arising from the initial isolation plates associated with cases of gastroenteritis.
The DT 104 and PT 4 rpoS mutants were very sensitive to airdrying on surfaces. The trehalose synthesis gene otsA is regulated by RpoS in S. typhimurium (Fang et al., 1996 ) and trehalose is known to protect cells from drying by stabilizing membranes and enzymes, and this may explain the observation (Potts, 1994
). PT 4 strain I, now identified as an rpoS mutant, has also been shown to survive poorly in aerosols (Humphrey et al., 1996
).
The pathogenicity of selected DT 104 strains was also examined in point-of-lay commercial hens infected orally. Strains with mutations in the rpoS gene were less frequently found in the tissues of experimentally infected birds and in previous work, the PT 4 strain I (identified as an rpoS mutant in this work) was also less frequently isolated from chicken tissues, particularly those of the reproductive tract (Humphrey et al., 1996 ). Interestingly, the faecal carriage rate of stress-sensitive strains was not significantly different from those of stress-resistant strains and strain 11 was the only strain found in muscle tissue (Williams et al., 1998
). The data from these experiments demonstrate a potentially complex relationship between RpoS expression, pathogenicity and stress tolerance. The level of expression of the RpoS-dependent spvR/A'::luxCDABE fusion in strain 11 was lower than in the stress-resistant strain 30, but with a similar induction, while strain 10 encoded a highly truncated protein. However, strain 11 was not significantly less frequently isolated from spleen tissue compared to strain 30, and strain 11 was also able to invade the reproductive tissues of chicken, in contrast to strain 10. The stress-sensitive strain 41 was absent from ovarian tissues and was recovered from the oviduct of only one of 30 chickens, yet it carries a functional rpoS gene. This suggests it may well harbour mutation(s) in other stress response/virulence genes such as rpoE (Humphreys et al., 1999
). Together the data suggest that a functional rpoS gene is necessary but not sufficient for the full virulence of DT 104 in poultry and that molecular determinants other than the RpoS protein play a role.
The differences in the invasiveness into the reproductive tissues between the DT 104 strains did not correlate with the subsequent egg contamination rates reported by Williams et al. (1998) . Indeed, the poorly invasive strain 10 produced egg contamination rates as high as many of the other strains, and the stress-sensitive strain 11 produced the highest rates. Clearly, while the RpoS protein is important for colonizing the reproductive tissue in laying hens, the contamination of egg contents appears to be dependent on other undefined molecular determinants.
This present study used chickens. Other work showed that expression of the RpoS protein had no effect on invasiveness into the tissues of day-old chicks (Allen-Vercoe et al., 1998 ; Humphrey et al., 1996
). Strains with normal expression of the RpoS protein are, however, significantly more virulent in orally infected mice (Fang et al., 1992
; Humphrey et al., 1996
, 1998
; Wilmes-Riesenberger et al., 1997
). Thus, the role of the rpoS gene is dependent on the host environment, and while virulence in mice and survival in the environment is highly RpoS-dependent, invasion into day-old chicks may not require RpoS. In mice, the RpoS-dependent effect on virulence is mainly exerted via the activation of the virulence plasmid spv genes, but rpoS-regulated chromosomally encoded genes also have a contributory role (Fang et al., 1992
). The ability of Salmonella strains to invade the liver or colonize the caecum of chickens was, however, not affected by the absence of the virulence plasmid, suggesting that chromosomally encoded rpoS-regulated genes are responsible for the differences in invasiveness observed in chickens (Halavatkar & Barrow, 1993
). Detailed investigation of the role of rpoS in the pathogenicity process has shown that rpoS does seem to be important for colonizing Peyers patches (Nickerson & Curtiss, 1997
). The process of infection is complex and depends on many factors including LPS, fimbriae, virulence plasmid, toxins and siderophores. Loss of one or more of these may result in loss of virulence depending on the host environment (Cox, 1995
).
In this work we have demonstrated that within clinical and environmental strains of S. typhimurium DT 104 significant differences exist in the ability to invade and/or persist in chickens and to survive exposure to heat stress, low pH and airdrying on surfaces. It remains to be definitively determined whether rpoS nonsense mutations such as those identified in the clinical strains studied here and elsewhere are also present in strains at the point of host infection.
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ACKNOWLEDGEMENTS |
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Received 6 December 1999;
revised 20 June 2000;
accepted 22 August 2000.