Stationary-phase Acid Tolerance Response Research Articles

Acid stress management by Cronobacter sakazakii

Cronobacter sakazakii is a foodborne pathogenic microorganism associated with sporadic cases of neonatal meningitis, necrotising enterocolitis, septicaemia, bloody diarrhoea and brain abscesses acquired through the consumption of contaminated powdered infant formula (PIF). This study aimed to investigate the growth of C. sakazakii DPC6529, a particularly stress tolerant clinical isolate, in acidified laboratory media and PIF. The possibility of a stationary-phase acid tolerance response (ATR) was also investigated. C. sakazakii DPC6529 grew in LB broth acidified to pH4.2 with hydrochloric acid (HCl) and was capable of relatively fast growth in PIF acidified to pH5.0 with HCl, representing the stomach pH reported for newborns and infants. Moreover, bacterial growth in LB broth supplemented with 1% (w/v) glucose gave rise to a stationary-phase ATR which resulted in enhanced survival against a subsequent acid challenge at pH3.0. A transposon mutagenesis approach was used to shed light on some of the molecular mechanisms involved in the response C. sakazakii DPC6529 to normally lethal acid exposures. The data suggests that repairing damage in proteins and nucleic acids, posttranscriptional modification of tRNA molecules and maintenance of the integrity of the cellular envelope are key processes in the defence against acid stress. Clones carrying transposon insertions in genes encoding the envelope stress response regulators CpxR and OmpR were identified as acid-sensitive mutants. Further analyses of the ompR defective mutant and its complemented counterpart evidenced that OmpR is a key player in the response of C. sakazakii to acid stress, although it was not essential to mount an active stationary-phase ATR, at least under the tested conditions. The ability of C. sakazakii DPC6529 to grow in acid environments and to develop an adaptive stationary-phase ATR may allow for its survival or even proliferation within the infant gastrointestinal tract after consumption of contaminated milk formulae.

Effect of acid tolerance response (ATR) on attachment of Listeria monocytogenes Scott A to stainless steel under extended exposure to acid or/and salt stress and resistance of sessile cells to subsequent strong acid challenge

The aim of this study was to investigate the potential effect of adaptive stationary phase acid tolerance response (ATR) of Listeria monocytogenes Scott A cells on their attachment to stainless steel (SS) under low pH or/and high salt conditions and on the subsequent resistance of sessile cells to strong acid challenge. Nonadapted or acid-adapted stationary-phase L. monocytogenes cells were used to inoculate ( ca. 10 8 CFU/ml) Brain Heart (BH) broth (pH 7.4, 0.5% w/v NaCl) in test tubes containing vertically placed SS coupons (used as abiotic substrates for bacterial attachment). Incubation was carried out at 16 °C for up to 15 days, without any nutrient refreshment. L. monocytogenes cells, prepared as described above, were also exposed to low pH (4.5; adjusted with HCl) or/and high salt (5.5% w/v NaCl) stresses, during attachment. On the 5th, 10th and 15th day of incubation, cells attached to SS coupons were detached (through bead vortexing) and enumerated (by agar plating). Results revealed that ATR significantly ( p < 0.05) affected bacterial attachment, when the latter took place under moderate acidic conditions (pH 4.5, 0.5 or 5.5% w/v NaCl), with the acid-adapted cells adhering slightly more than the nonadapted ones. Regardless of acidity/salinity conditions during attachment, ATR also enhanced the resistance of sessile cells to subsequent lethal acid challenge (exposure to pH 2 for 6 min; pH adjusted with either hydrochloric or lactic acid). The trend observed with viable count data agreed well with conductance measurements, used to indirectly quantify remaining attached bacteria (following the strong acid challenge) via their metabolic activity. To sum, this study demonstrates that acid adaptation of L. monocytogenes cells during their planktonic growth enhances their subsequent attachment to SS under extended exposure (at 16 °C for up to 15 days) to mild acidic conditions (pH 4.5), while it also improves the resistance of sessile cells to extreme acid treatment (pH 2). Therefore, the ATR of bacterial cells should be carefully considered when applying acidic decontamination strategies to eradicate L. monocytogenes attached to food processing equipment.

Understanding the acid tolerance response of bifidobacteria

To investigate the effect of pH on the viability and the acid tolerance response (ATR) of bifidobacteria. The impact of low pH on the viability of five species of bifidobacteria was examined under conditions of strict anaerobiosis. Although differences in the ability to resist the lethal effects of low pH were apparent among the species, cell viability could be improved by the provision of fermentable substrate during an acidic pH stress or through the use of stationary phase cells. While a stationary phase ATR was found to occur in two species of bifidobacteria, there was no adaptive response in exponential phase cells. Proteomic analysis of exponential phase Bifidobacterium longum subjected to a mild acid pre-exposure (pH 4.5, 2 h) prior to an acid challenge revealed a substantial loss in the total number of cellular proteins. In contrast, proteomic analysis of stationary phase cells revealed an increased abundance of proteins associated with the general stress response as well as the beta-subunit of the F(0)F(1)-ATPase, known to be important in bifidobacteria acid tolerance. Neither Bif. longum or Bifidobacterium breve possesses an inducible exponential phase ATR. These findings provide further insights into the impact of pH on the viability of bifidobacteria and may partially explain the loss in viability associated with their storage in acid foods.

The influence of ribosome modulation factor on the survival of stationary-phase Escherichia coli during acid stress

Ribosome modulation factor (RMF) was shown to have an influence on the survival of Escherichia coli under acid stress during stationary phase, since the viability of cultures of a mutant strain lacking functional RMF decreased more rapidly than that of the parent strain at pH 3. Loss of ribosomes was observed in both strains when exposed to low pH, although this occurred at a higher rate in the RMF-deficient mutant strain, which also suffered from higher levels of rRNA degradation. It was concluded that the action of RMF in limiting the damage to rRNA contributed to the protection of E. coli under acid stress. Expression of the rmf gene was lower during stationary phase after growth in acidified media compared to media containing no added acid, and the increased rmf expression associated with transition from exponential phase to stationary phase was much reduced in acidified media. It was demonstrated that RMF was not involved in the stationary-phase acid-tolerance response in E. coli by which growth under acidic conditions confers protection against subsequent acid shock. This response was sufficient to overcome the increased vulnerability of the RMF-deficient mutant strain to acid stress at pH values between 6.5 and 5.5.

Comparative Analysis of Acid Resistance between Susceptible and Multi-Antimicrobial-Resistant Salmonella Strains Cultured under Stationary-Phase Acid Tolerance–Inducing and Noninducing Conditions

This study compared acid resistance levels among five antimicrobial-susceptible strains of Salmonella and five strains that were simultaneously resistant to a minimum of six antimicrobial agents. The induction of a stationary-phase acid tolerance response (ATR) was attempted by both transient low-pH acid shock and acid adaptation. For acid shock induction, strains were grown for 18 h in minimal E medium containing 0.4% glucose (EG medium) and exposed to sublethal acid stress (pH 4.3) for 2 h, and subsequently, both shocked and nonshocked cultures were acid challenged (pH 3.0) for 4 h. Acid adaptation was achieved by growing strains for 18 h in tryptic soy broth containing 1.0% glucose (TSB+G), while nonadapted cultures were grown for 18 h in glucose-free tryptic soy broth (TSB−G). Acid-adapted and nonadapted inocula were acid challenged (pH 2.3) for 4 h. Initial (0 h) mean populations of nonchallenged Salmonella were 8.5 to 8.7, 8.4 to 8.8, and 8.2 to 8.3 log CFU/ml for strains grown in EG medium, TSB−G, and TSB+G, respectively. After 4 h of acid challenge, mean populations were 3.0 to 4.8 and 2.5 to 3.7 log CFU/ml for previously acid-shocked susceptible and resistant strains, respectively, while corresponding counts for nonshocked strains were 4.3 to 5.5 log CFU/ml and 3.9 to 4.9 log CFU/ml. Following 4 h of acid exposure, acid-adapted cultures of susceptible and resistant strains had mean populations of 6.1 to 6.4 log CFU/ml and 6.4 to 6.6 log CFU/ml, respectively, while corresponding counts for nonadapted cultures were 1.9 to 2.1 log CFU/ml and 1.8 to 2.0 log CFU/ml, respectively. A low-pH–inducible ATR was not achieved through transient acid shock, while an ATR was evident following acid adaptation, as adapted populations were 4.2 to 4.8 log units larger than nonadapted populations following acid exposure. Although some strain-dependent variations in acid resistance were observed, results from this study suggest no association between susceptibility to antimicrobial agents and the ability of the Salmonella strains evaluated to survive low-pH stress.

Effect of carbon starvation and proteolytic activity on stationary-phase acid tolerance of Streptococcus mutans.

Previous research with Streptococcus mutans and other oral streptococci has demonstrated that the acid shock of exponential-phase cells (pH 7.5 to 5.5) resulted in the induction of an acid tolerance response (ATR) increasing survival at low pH (3.5-3.0). The current study was designed to determine whether two fresh isolates, H7 and BM71, and two laboratory strains, Ingbritt and LT11, were capable of a stationary-phase ATR as estimated by a survival test at pH 3.5 for 3 h. All four strains were unable to generate a stationary-phase ATR under control conditions at pH 7.5, with the exception of a burst of survivors in the transition between the exponential and stationary phases when the carbon source (glucose) was depleted. Adaptation at pH 5.5 resulted in the expected pH-dependent exponential-phase ATR, but only the fresh isolates exhibited a stationary-phase ATR at this pH. Glucose starvation of cells in complex medium was shown to enhance acid tolerance for the fresh isolates, but not the laboratory strains. This tolerance was, however, greatly diminished for all strains in a defined medium with a low concentration of amino acids. Growth of strain H7 in complex medium resulted in the formation of at least 56 extracellular proteins, nine of which were degraded in the early stationary phase following the induction of proteolytic activity during the transition period. No proteolytic activity was observed with strain LT11 and only 19 extracellular proteins/peptides were apparent in the medium with only one being degraded in the early stationary phase. Strain H7 was also shown to have two- to fourfold higher levels of intracellular glycogen in the stationary phase than strain LT11. These results suggest that S. mutans H7 possessed the required endogenous metabolism to support amino acid/peptide uptake in the early-stationary phase, which resulted in the formation of basic end products that, in turn, contributed to enhanced intracellular pH homeostasis.

A low-pH-inducible, stationary-phase acid tolerance response in Lactobacillus acidophilus CRL 639.

Acidity is an important environmental condition encountered by lactobacilli during food fermentation. In this report we show that triggering the stationary-phase acid tolerance response (ATR) in L. acidophilus CRL 639 depends on the final growth pH. In free-pH fermentation runs (final pH = 4.5), the cells were completely resistant to acid stress, whereas cells from cultures under controlled pH (pH = 6.0) were very sensitive. The relationship between the final pH and the development of cross-resistance to different kinds of environmental stress was also evaluated. The study of protein profiles showed the overexpression of 16 proteins from 6.5 to 70.9 kDa in stationary phase cells. Seven of these proteins (26.3, 41.4, 48.7, 49.3, 54.5, 56.1, and 70.9 kDa) were expressed as result of the stationary phase itself, while nine proteins (14.1, 18.6, 21.5, 26.9, 29.3, 41.9, 42.6, 49.6, and 56.2 kDa) were exclusively induced as a result of the drop in culture pH during free fermentation runs. These results strongly suggest the involvement of these proteins in cell adaptation to environmental changes.

Acid stress susceptibility and acid adaptation of Propionibacterium freudenreichii subsp. shermanii

The probiotic application of dairy propionibacteria as well as their use in cheese tech- nology implies exposure to various environmental stresses, including acidic pH. The acid tolerance response (ATR) of Propionibacterium freudenreichiiwas investigated. One strain present in Swiss- type cheese proved to be acid-tolerant, since no lethal effect was observed during exposure at pH 3. Moreover, survival at pH 2 (acid challenge) was conferred by pre-exposure to a moderate acid stress (acid adaptation). This adaptative response was triggered quickly, and showed a maximal efficiency upon exposure to pHs between 4 and 5. Stationary phase ATR and acid habituation were also demon- strated, and conferred increased survival at pH 2 without pre-exposure. Exponentially-growing bac- teria were partially protected towards acidity by pre-exposure to other stresses (heat, starvation, but not hyperosmolarity). A comparative study of different strains revealed that acid stress susceptibil- ity is strain-dependent within this species. Adaptation and survival at low pH is likely to determine the efficacy of a P. freudenreichii strain both as a cheese starter and as a probiotic. Swiss-type cheese / Propionibacterium / acid tolerance response / stress / probiotic

A low-pH-inducible, stationary-phase acid tolerance response in Salmonella typhimurium

Acid is an important environmental condition encountered by Salmonella typhimurium during its pathogenesis. Our studies have shown that the organism can actively adapt to survive potentially lethal acid exposures by way of at least three possibly overlapping systems. The first is a two-stage system induced in response to low pH by logarithmic-phase cells called the log-phase acid tolerance response (ATR). It involves a major molecular realignment of the cell including the induction of over 40 proteins. The present data reveal that two additional systems of acid resistance occur in stationary-phase cells. One is a pH-dependent system distinct from log-phase ATR called stationary-phase ATR. It was shown to provide a higher level of acid resistance than log-phase ATR but involved the synthesis of fewer proteins. Maximum induction of stationary-phase ATR occurred at pH 4.3. A third system of acid resistance is not induced by low pH but appears to be part of a general stress resistance induced by stationary phase. This last system requires the alternative sigma factor, RpoS. Regulation of log-phase ATR and stationary-phase ATR remains RpoS independent. Although the three systems are for the most part distinct from each other, together they afford maximum acid resistance for S. typhimurium.