J. Basic Microbiol. 44 (2004) 2, 137 – 146
DOI: 10.1002/jobm.200310333
(Department of Microbiology, Basic Medical Sciences Building, Panjab University, Chandigarh160014, INDIA and 1Department of Microbiology & Immunology, Medical University of South Carolina, Post Box No. 250504, Charleston SC 29425, USA)
Adaptive acid tolerance response in Salmonella enterica serovar Typhimurium and Salmonella enterica serovar Typhi RAM PARKASH TIWARI, NARESH SACHDEVA, GURINDER SINGH HOONDAL and JASVIR SINGH GREWAL1* (Received 12 August 2003/Accepted 05 November 2003) The survival of bacteria in various environments depends on a number of protective responses including acid tolerance response (ATR). In this study, ATR phenomenon was compared in Salmonella enterica serovar Typhi 6 and Salmonella enterica serovar Typhimurium 98 under different culture conditions. Survival of the adapted culture (pre-acid shocked to pH 5.5) was significantly better (p < 0.05) as compared to control, unadapted culture after acid shock at pH 3.3. However, the ATR varied with the serovar, incubation temperature and the growth medium used (all p-values < 0.05). S. Typhi 6 failed to grow in pH 3.3 at 45 °C. The addition of tetracycline or chloramphenicol (1.0 µg ml–1) to adapted cultures during or after acid shock (pH 3.3) had no effect on ATR expression. In S. Typhimurium 98, growth was increased by 10% or greater in adapted culture (when grown at pH 3.3) as compared to growth observed with an unadapted culture (when grown at pH 7.3) on transfer to fresh growth medium at pH 7.3. A poor ATR observed in non-growing S. Typhimurium 98 suspensions clearly showed that ATR is an energy-consuming process. Storage of S. Typhimurium 98 cultures in pH 4.5 nutrient broth at 4 °C demonstrated that prolonged exposure to acidic conditions is more detrimental in comparison to the cultures stored at pH 7.3 at this temperature.
All pathogenic bacteria especially gastrointestinal pathogens such as Salmonella spp. must circumvent a variety of stresses in order to arrive at the site of infection. The ability to survive these stresses is central to the pathogenicity of Salmonella (FIELDS et al. 1986, ALPUCHE-ARANDA et al. 1992, LINDGREN et al. 1996). One well-characterized adaptive response is to acid stress, called the acid tolerance response (ATR). Salmonella encounters an acid environment first in the stomach and then within the phagosomes (BUCHMEIER and LIBBY 1997, BOLTON et al. 1999). There is a continued interest in determining how gastrointestinal tract pathogens cope with this low pH stress. Available evidence suggests that microorganisms with inducible mechanisms of acid resistance are equipped to survive these challenges (LEYER et al. 1995, LIN et al. 1995, MERRELL and CAMILLI 2000, HANNA et al. 2001). Many stress related responses and virulence determinants are co-regulated, examples include RpoS, the so called stationary phase RNA polymerase, sigma factor associated with nutrient depletion, acidic pH and elevated temperatures, and the regulation of virulence plasmid spv genes in Salmonellae (FOSTER et al. 1994, FOSTER and SPECTOR 1995, LEE et al. 1995). The pH homeostasis mechanisms involving a series of proton antiport systems are considered as the critical features for surviving acid-stress in E. coli and Salmonella Typhimurium (ZILBERSTEIN et al. 1982, KROLL and BOOTH 1983, BOOTH 1985). Studies have indicated marked differences in the initial steps of S. Typhi, S. Typhimurium and S. Dublin pathogenesis (WEINSTEIN et al. 1998). In the present study the role of acid resis* Corresponding author: Dr. J. S. GREWAL; e-mail:
[email protected]
© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 0233-111X/04/0204-0137
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tance on in vitro survival of two local serovars of Salmonella (S. Typhimurium 98 and S. Typhi 6) was investigated. The effect of temperature, nutrient media and antibiotics on ATR was evaluated in these serovars. In addition, ATR in non-growing cells and its persistence during subsequent storage of acid adapted cultures at low temperature was determined. Materials and methods Bacterial strains: Salmonella Typhimurium 98, a human faecal isolate was obtained from Bacterial Culture Collection Centre of Institute of Microbial Technology, Chandigarh, India. Salmonella Typhi 6, also a human faecal isolate was kindly provided by Prof. SHOBHA RAM, Department of Microbiology, Daya Nand Medical College, Ludhiana, India. The bacteria were maintained on Le Minor medium stabs (meat extract 5 g, peptone 10 g, sodium chloride 3 g, disodium hydrogen orthophosphate 2 g, agar 10 g and distilled water to make 1000 ml, pH 7.4) at 4 °C in screw-capped tubes. Acid tolerance response: The ATR response was determined by using the modified method of FOSTER and HALL (1990). Briefly, nutrient broth or M-9 glucose medium (pH 7.3) in duplicates (100 ml per 250 ml flask) were inoculated with 100 µl of overnight grown test culture (S. Typhimurium 98 or S. Typhi 6). The cultures were then grown on a rotary shaker (200 rpm) at 37 °C until the cell density in each flask was 0.2 at 600 nm. The pH in one flask was then quickly adjusted to pH 5.5 with 1 N HCl for adaptation. The pH in the other flask was not modified (pH 7.3) (unadapted culture). Both flasks were allowed to continue to grow until the cell density was 0.4 at 600 nm. At this stage the pH in both flasks was adjusted to 3.3 with 1N HCl. Aliquots were withdrawn immediately (t0) and then at 1 h interval for 4 h (t1 – t4) from each flask. Each aliquot was serially diluted in normal saline prior to plating on nutrient agar for determination of a viable count at 37 °C. The experiment was repeated three times. The data is represented as the mean ± S.D. of percent survival, which was calculated as follows: Cfu pH 3.3 Percent survival (%) = × 100 . Cfu unadapted (pH 7.3) or adapted (pH 5.5) culture Effect of media and temperature on ATR: S. Typhimurium 98 and S. Typhi 6 cultures (both adapted and unadapted) were grown in M-9 glucose medium and nutrient broth at different temperatures (i.e., 25 °C, 37 °C and 45 °C). Acid tolerance response at pH 3.3 was then evaluated as described above. Effect of antibiotics on ATR: The minimum inhibitory concentration (MIC) for chloramphenicol and tetracycline (SIGMA) was determined against test culture using serial two fold dilutions. The antibiotics were added at 1.0 µg ml–1 (less than the MICs) to the cultures at various time points as shown below: (i) 1 h prior to acid adaptation (pH shift from 7.3 to 5.5), (ii) Just before acid adaptation, (iii) 0 h of acid shock (pH shift, 5.5 to 3.3), (iv) 0.5 h after acid shock and (v) 1 h after acid shock. Effect of acidic environment on bacterial growth: S. Typhimurium 98 nutrient broth cultures were grown at pH 7.3, 5.5 or 3.3. The cell density was adjusted to 0.8 at 600 nm. From each flask 100 µl was inoculated into three sterile nutrient broth flasks (pH 7.3) and the flasks were placed on a rotatory shaker (200 rpm) at 37 °C. Aliquots were withdrawn immediately after inoculation and then at different time intervals (2.5, 4.5, 7, 10, 15, 23 and 32 hours). Growth was measured by absorbance at 600 nm versus nutrient broth. ATR of non-growing bacterial cell suspension: S. Typhimurium nutrient broth grown culture were centrifuged at 10,000 g and the bacterial cell pellet was washed twice with normal saline (0.15 M, pH 7.3). The absorbance of bacterial cell suspension was adjusted to 0.2 at 600 nm in normal saline. The unadapted and the adapted cells were prepared as described above and both of these cell types were directly suspended in pH 3.3 normal saline instead of any media for testing ATR in non-growing cells. Suspension of cells in normal saline constitutes the non-growing cell suspension since in normal saline cells survive exclusively on endogenous metabolism in the absence of any exogenous source
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of nutrients. The experiment was repeated three times, and the percent survival of the cells was calculated as described. Persistence of acid resistance during cold storage: S. Typhimurium 98 was cultured at pH 7.3 (control culture) and pH 4.5 (test culture) in nutrient broth at 37 °C till O.D. was 0.2 at 600 nm. The cultures were then stored at 4 °C. Aliquots were withdrawn immediately and at 3 day intervals for 15 days. These aliquots from control and test cultures were serially diluted and spread plated on MAC CONKEY’s agar plates to determine viable count as a measure of percentage survival during storage. MAC CONKEY’s agar was used for rescue because the presence of bile salts and dyes eliminates many acid-damaged cells that are viable on nutrient agar. Statistical analysis: Student’s t-test was used for the analysis of the data. ANOVA was used for the analysis of variance among different groups. The data were considered significant at p < 0.05.
Results ATR phenomenon was compared in Salmonella enterica serovar Typhi 6 and Salmonella enterica serovar Typhimurium 98 under different culture conditions. Acid tolerance response was inducible in both the serovars. However, acid tolerance was significantly higher (p < 0.05) in adapted cultures relative to unadapted cultures. Viable counts were less than one CFU ml–1 in unadapted S. Typhi 6 cultures after exposure to pH 3.3 for 2 h or more (Table 1). After an initial decrease (3.25% viability after 1 h), an increase in viable counts (3.4– 22% viability after 2 –4 h) was observed in adapted cultures during this period. Differences were also observed in the magnitude of the acid tolerance response of adapted cultures of S. Typhimurium 98 and S. Typhi 6 (p < 0.05–0.005). Even the unadapted cultures of S. Typhimurium 98 were less susceptible (0.021% survival at t1 and 3.6 × 10–6% at t2) to pH 3.3 than S. Typhi 6 (0.002% survival at t1 and 0% at t2). An increase in viable counts at t2 – t4 relative to viable counts at t1 of the adapted cultures grown in nutrient broth and M-9 glucose medium was 200–1000 fold for S. Typhimurium 98 and 44– 160 fold for S. Typhi 6 cultures. Differences in ATR were also observed with respect to serovar and growth medium used (Table 1). Increases in viable counts were only 1.33% to 6.67% and 3.73 to 32%, respectively, for adapted cultures of S. Typhi 6 and S. Typhimurium 98 cultured in M-9 glucose medium. The effect of media on ATR at different temperatures was also evaluated. The ATR was always better in both the serovars in nutrient broth cultures in comparison to M-9 glucose medium. The p values for S. Typhimurium 98 adapted cultures at 30 °C, 37 °C and 45 °C were 0.018, 0.011 and 0.0078 respectively (Table 2). There was no difference in ATR among different media at 30 °C (p = 0.17) in S. Typhi 6 but the ATR was significantly different (p = 0.0028) at 37 °C. S. Typhi 6 did not survive pH 3.3 lethality at 45 °C in any of the media (Table 2). Differences in the ATR at different temperatures using the same media were also observed. In S. Typhimurium 98, p-values for nutrient broth and M-9 glucose medium were 0.013 and 0.022 respectively, whereas it was 0.001 for S. Typhi 6 cultures in both the media (Table 2). Addition of tetracycline or chloramphenicol (1.0 µg ml–1) prior to or during the preacid shock period inhibited the ATR in both the serovars. Results indicated that the ATR occurs within 1h of acid shock (Table 3). In S. Typhimurium 98, non-growing cells suspended in normal saline and exposed to pH 3.3, the survivors were only 0.43% (adapted) and 0.00032% (unadapted) at t1 (Table 4). It is clear from the poor ATR in non-growing S. Typhimurium 98 suspensions that the ATR is an energy-consuming process. Ten percent more growth was observed for acid stressed cultures (pH 3.3) transferred to fresh nutrient broth (pH 7.3) and incubated at 37 °C in comparison to the transfer from cultures growing at pH 7.3 (Fig. 1). Then we stored the cultures at 4 °C to determine the survivability of S. Typhimurium 98 at low temperature under acidic environments. S. Typhimurium 98 cultures stored at pH 4.5 and pH 7.3 in nutrient broth demonstrated that prolonged exposure to acidic condition is
© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
0 1 2 3 4
0 1 2 3 4
NB
M-9 G
108
2.5 ± 1.1 × 108 1.1 ± 0.1 × 104 0 0 0
4.2 ± 1.5 × 8.8 ± 2.9 × 104 1.5 ± 0.7 × 101 0 0
cfu
ml–1
100 0.004 0 0 0
100 0.021 3.6 × 10–6 0 0
% viability
S. Typhimurium 98
108
2.4 ± 1.2 × 108 7.6 ± 3.2 × 103 0 0 0 100 0.003 0 0 0
100 0.002 0 0 0
% viability
S. Typhi 6 3.6 ± 2.5 × 8.8 ± 3.2 × 103 0 0 0
cfu
ml–1
Unadapted culture*
108
1.5 ± 0.5 × 108 5.6 ± 2.8 × 106 6.0 ± 3.1 × 106 1.2 ± 0.7 × 107 4.8 ± 2.6 × 107
2.2 ± 0.9 × 9.3 ± 3.7 × 106 1.1 ± 0.2 × 107 4.9 ± 2.7 × 107 1.0 ± 0.2 × 108
cfu
ml–1
100 3.73 4.0 8.0 32.0***
100 4.23 5.0 22.20* 45.45**
% viability
S. Typhimurium 98
108
1.5 ± 0.6 × 108 2.0 ± 0.8 × 106 2.2 ± 1.0 × 106 3.8 ± 1.7 × 106 1.0 ± 0.2 × 107
100 1.33 1.47 2.53 6.67***
100 3.25 3.4 4.95* 22.0**
% viability
S. Typhi 6 2.0 + 0.8 × 6.5 ± 2.8 × 106 6.8 ± 2.9 × 106 9.9 ± 4.1 × 106 4.4 ± 1.4 × 107
cfu
ml–1
Adapted culture*
S. Typhimurium 98 vs S. Typhi 6 adapted strains, * p < 0.05, ** p < 0.01, *** p < 0.005; Adapted vs unadapted culture, * p < 0.05; NB = Nutrient broth, M-9 G = M-9 glucose medium
Time (h)
Growth Medium
Table 1 Viable counts and percentage survival of adapted and unadapted S. Typhimurium 98 and S. Typhi 6 cultures in nutrient broth and M-9 glucose medium at pH 3.3 at 37 °C
140 R. P. TIWARI et al.
2.6 × 106 1.1 × 106 2.0 × 104 7.9 × 103
6.8 × 104 4.5 × 104 2.4 × 103 1.8 × 103
1.6 × 108 1.0 × 108 2.8 × 108 2.0 × 108
2.0 × 108 1.6 × 108 2.6 × 108 2.7 × 108
Adapted NB# M9-G# UnNB Adapted M9-G
Adapted NB# M9-G# UnNB Adapted M9-G
6.0 × 104 4.4 × 104 0 0
4.4 × 106 2.4 × 106 0 0
II
30 °C
4.5 × 104 4.0 × 104 0 0
2.4 × 107 1.5 × 107 0 0
III
5.0 × 104 3.8 × 104 0 0
4.4 × 107 2.2 × 107 0 0
IV*
I
II 1.1 × 107 7.0 × 106 12 0
2.0 × 108 1.5 × 108 2.7 × 108 2.4 × 108
6.5 × 106 2.0 × 106 8.8 × 103 7.6 × 103
6.8 × 106 2.2 × 106 0 0
Salmonella Typhi 6
9.3 × 106 4.0 × 106 9.0 × 104 1.0 × 104
Salmonella Typhimurium 98 2.2 × 108 1.5 × 108 3.6 × 108 2.7 × 108
0
37 °C
9.9 × 106 3.8 × 106 0 0
4.9 × 107 2.1 × 107 0 0
III
Incubation temperature
4.4 × 107 1.0 × 107 0 0
1.0 × 108 4.4 × 107 0 0
IV*
2.0 × 108 2.1 × 108 2.0 × 108 2.1 × 108
2.0 × 108 1.4 × 108 3.0 × 108 2.6 × 108
0
0 0 0 0
8.0 × 106 2.4 × 106 6.0 × 104 8.4 × 103
I
0 0 0 0
2.0 × 107 3.4 × 106 7 0
II
45 °C
0 0 0 0
4.45 × 107 1.8 × 107 0 0
III
0 0 0 0
8.5 × 107 3.3 × 107 0 0
IV*
0, I, II, III, IV: Viable count (cfu ml –1) after 0, 1, 2, 3, 4 h of acid shift to pH 3.3; NB: Nutrient broth; M9-G: M-9 glucose medium Standard deviations were upto 20% of the mean values (shown above) of triplicate cultures in each case # p-values comparing different media: S. Typhimurium 98 = 0.018 (30 °C), 0.011 (37 °C), 0.0078 (45 °C); S. Typhi 6 = 0.17 (30 °C), 0.0028 (37 °C) * p-values comparing different temperatures: S. Typhimurium 98 = 0.013 (NB), 0.022 (M9-G); S. Typhi 6 = 0.001 (NB), 0.001 (M9-G) All the above p values are for viable counts at 4 h of acid shift of the adapted cultures only
I
0
Culture Media
Table 2 Viable counts of S. Typhimurium 98 and S. Typhi 6 (adapted and unadapted) cultured in different media at various temperatures-on shifting to pH 3.3
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Table 3 Effect of sub-inhibitory concentrations of tetracycline and chloramphenicol (1.0 µg ml–1) on synthesis of ATR proteins in S. Typhimurium 98 and S. Typhi 6 S. No.
1. 2. 3. 4. 5.
Antibiotic addition
ATR
1 h before shifting from pH 7.3 to 5.5 Immediately on shifting from pH 7.3 to 5.5 0 h of 2nd acid shift (pH 5.5 to 3.3) 0.5 h after 2nd acid shift 1 h of 2nd acid shift
S. Typhimurium 98
S. Typhi 6
– – + + ++
– – – + +
+ = ATR, – = No ATR, ATR = Survival of test bacteria after acid challenge at pH 3.3 Table 4 Percent survival of S. Typhimurium 98 adapted and unadapted non-growing cell suspension on shifting to pH 3.3 Incubation time (h)
0 1 2 3 4
Unadapted cells
Adapted cells
Viable count (cfu ml–1)
Percent survival (%)
Viable count (cfu ml–1)
Percent survival (%)
2.0 ± 0.8 × 108 6.4 ± 2.8 × 102
