Methods for studying microbial acid stress responses: from molecules to populations.

The study of how micro-organisms detect and respond to different stresses has a long history of producing fundamental biological insights while being simultaneously of significance in many applied microbiological fields including infection, food and drink manufacture, and industrial and environmental biotechnology. This is well illustrated by the large body of work on acid stress. Numerous different methods have been used to understand the impacts of low pH on growth and survival of micro-organisms, ranging from studies of single cells to large and heterogeneous populations, from the molecular or biophysical to the computational, and from well-understood model organisms to poorly defined and complex microbial consortia. Much is to be gained from an increased general awareness of these methods, and so the present review looks at examples of the different methods that have been used to study acid resistance, acid tolerance, and acid stress responses, and the insights they can lead to, as well as some of the problems involved in using them. We hope this will be of interest both within and well beyond the acid stress research community.

17†Development of a questionnaire to study fear and anxiety factors affecting patients and their families undergoing strabismus surgery.

Patients and their Families undergoing Strabismus Surgery. The aim of this study was to develop a questionnaire to identify perioperative fear and anxiety factors affecting pediatric strabismus surgery patients.First, we reviewed the literature to determine precipitants of fears and anxieties experienced by pediatric patients. Subsequently, we developed a questionnaire for pediatric patients undergoing strabismus surgery. This was a two part questionnaire, consisting of a 16-piece section for patients and a 22-piece section for parents. Finally, we piloted this questionnaire to validate its clinical use.Common anxiety factors for children include pain, minor clinical procedures requiring needles, separation from parents and engaging with medical professionals. We used this information to develop a two part questionnaire for patients and parents. The questionnaire elicited positive and negative aspects of the patient journey, corroborated fears reported in the literature, and identified anxiety inducing factors specific to strabismus patients.There is a lack of evidence regarding fear and anxiety specific to pediatric ophthalmology surgeries. Strabismus surgery carries unique fear inducing factors. Interventions which may alleviate the stress of pediatric surgery, therefore greatly benefit patient experience and surgical outcomes, and should be considered in the care of pediatric patients. Patient educational material is known to provide a sense of control to patients, helping to alleviate such fear.Evidenced by the literature and the pilot questionnaire, there still exists anxiety inducing factors in pediatric surgery. Investigation into patient fears regarding pediatric strabismus surgery is needed to better understand how clinical staff can support patients perioperatively.

Solar irradiance limits the long-term survival of Listeria monocytogenes in seawater.

Seafood has often been implicated in outbreaks of food-borne illness caused by Listeria monocytogenes but the source of contamination is usually not known. In this study we investigated the possibility that this pathogen could survive in seawater for an extended time period. Freshly collected seawater samples were inoculated with 1 × 108  CFU per ml of L. monocytogenes EGD-e and survival was monitored by plate counting for up to 25 days. When incubated in the dark, either at ambient temperatures (4-14°C) or at 16°C, >104  CFU per ml survivors were present after 25 days. However, when the seawater cell suspensions were exposed to ambient light (solar irradiation) and temperatures, L. monocytogenes lost viability rapidly and no survivors could be detected after the 80 h time point. Both UV-A and visible light in the blue region of the spectrum (470 nm) were found to contribute to this effect. The stress inducible sigma factor σB was found to play a role in survival of L. monocytogenes in seawater. Together these data demonstrate that solar irradiation is a critical determinant of L. monocytogenes survival in marine environments. The data further suggest the possibility of controlling this food-borne pathogen in food-processing environments using visible light. SIGNIFICANCE AND IMPACT OF THE STUDY: Listeria monocytogenes is a food-borne bacterial pathogen capable of causing the life-threatening infection, listeriosis. In seafood the route of contamination from the environment is often not well understood as this pathogen is not generally thought to survive well in seawater. Here we provide evidence that L. monocytogenes is capable of surviving for long periods of time in seawater when light is excluded. Sunlight is demonstrated to have a significant effect on the survival of this pathogen in seawater, and both visible (470 nm) and UV-A light are shown to contribute to this effect.

Visible and UVA light as a potential means of preventing Escherichia coli biofilm formation in urine and on materials used in urethral catheters.

Catheter-associated urinary tract infections are the most common hospital-acquired infection, for which Escherichia coli is the leading cause. This study investigated the efficacy of 385nm and 420nm light for inactivation of E. coli attached to the silicone matrix of a urinary catheter. Using urine mucin media, inactivation of planktonic bacteria and biofilm formation was monitored using silicone coupons. Continuous irradiance with both 385nm and 420nm wavelengths with starting cell density population 103CFU ml-1 reduced planktonic suspensions of E. coli to below the detection level after 2h and 6h, respectively. Bacterial attachment to silicone was successfully prevented during the same treatment. Inactivation by 385nm and 420nm was found to be dependent on media, cell density and oxygen, with less inhibition on planktonic suspensions when higher starting cell densities were used. In contrast to planktonic suspensions in PBS, continuous irradiance of pre-established biofilms showed a greater reduction in survival compared to urine mucin media after 24h. Enhanced inhibition for 385nm and 420nm light in urine mucin media was associated with increased production of reactive oxygen species. These findings suggest 385nm and 420nm light as a promising antimicrobial technology for the prevention of biofilm formation on urethral catheters.

A modified rapid enzymatic microtiter plate assay for the quantification of intracellular γ-aminobutyric acid and succinate semialdehyde in bacterial cells.

The GABase assay is widely used to rapidly and accurately quantify levels of extracellular γ-aminobutyric acid (GABA). Here we demonstrate a modification of this assay that enables quantification of intracellular GABA in bacterial cells. Cells are lysed by boiling and ethanolamine-O-sulphate, a GABA transaminase inhibitor is used to distinguish between GABA and succinate semialdehyde.

Inhibition of Listeria monocytogenes by acetate, benzoate and sorbate: weak acid tolerance is not influenced by the glutamate decarboxylase system.

AIMS: Weak acids are widely used by the food industry to prevent spoilage and to inhibit the growth of pathogenic micro-organisms. In this study the inhibitory effects of three commonly used weak acids, acetic acid, benzoic acid and sorbic acid, on the growth of Listeria monocytogenes were investigated. METHODS AND RESULTS: In a chemically defined medium at pH 6.4 benzoic acid had the greatest inhibitory effect (50% inhibition of growth at 4 mmol l(-1)), while acetate was the least inhibitory (50% inhibition of growth at 50 mmol l(-1)). Mutants lacking either sigma B (Delta sigB) or two of the glutamate decarboxylase systems (Delta gadAB) were used to investigate the contribution these systems make to weak acid tolerance in L. monocytogenes. CONCLUSIONS: The stress-inducible sigma factor sigma B (sigma(B)) was not required for protection against acetate and played only a minor role in tolerating benzoate and sorbate. The glutamate decarboxylase system, which plays an important role in tolerating inorganic acids, played no significant role in the ability of L. monocytogenes to tolerate these weak acids, and neither did the presence of glutamate in the growth medium. SIGNIFICANCE AND IMPACT OF THE STUDY: These results suggest that the effectiveness of weak acid preservatives in food will not be compromised by the presence of glutamate, at least under mildly acidic conditions.

Identification of components of the sigma B regulon in Listeria monocytogenes that contribute to acid and salt tolerance.

Sigma B (sigma(B)) is an alternative sigma factor that controls the transcriptional response to stress in Listeria monocytogenes and is also known to play a role in the virulence of this human pathogen. In the present study we investigated the impact of a sigB deletion on the proteome of L. monocytogenes grown in a chemically defined medium both in the presence and in the absence of osmotic stress (0.5 M NaCl). Two new phenotypes associated with the sigB deletion were identified using this medium. (i) Unexpectedly, the strain with the DeltasigB deletion was found to grow faster than the parent strain in the growth medium, but only when 0.5 M NaCl was present. This phenomenon was independent of the carbon source provided in the medium. (ii) The DeltasigB mutant was found to have unusual Gram staining properties compared to the parent, suggesting that sigma(B) contributes to the maintenance of an intact cell wall. A proteomic analysis was performed by two-dimensional gel electrophoresis, using cells growing in the exponential and stationary phases. Overall, 11 proteins were found to be differentially expressed in the wild type and the DeltasigB mutant; 10 of these proteins were expressed at lower levels in the mutant, and 1 was overexpressed in the mutant. All 11 proteins were identified by tandem mass spectrometry, and putative functions were assigned based on homology to proteins from other bacteria. Five proteins had putative functions related to carbon utilization (Lmo0539, Lmo0783, Lmo0913, Lmo1830, and Lmo2696), while three proteins were similar to proteins whose functions are unknown but that are known to be stress inducible (Lmo0796, Lmo2391, and Lmo2748). To gain further insight into the role of sigma(B) in L. monocytogenes, we deleted the genes encoding four of the proteins, lmo0796, lmo0913, lmo2391, and lmo2748. Phenotypic characterization of the mutants revealed that Lmo2748 plays a role in osmotolerance, while Lmo0796, Lmo0913, and Lmo2391 were all implicated in acid stress tolerance to various degrees. Invasion assays performed with Caco-2 cells indicated that none of the four genes was required for mammalian cell invasion. Microscopic analysis suggested that loss of Lmo2748 might contribute to the cell wall defect observed in the DeltasigB mutant. Overall, this study highlighted two new phenotypes associated with the loss of sigma(B). It also demonstrated clear roles for sigma(B) in both osmotic and low-pH stress tolerance and identified specific components of the sigma(B) regulon that contribute to the responses observed.

Proteomic analyses of a Listeria monocytogenes mutant lacking sigmaB identify new components of the sigmaB regulon and highlight a role for sigmaB in the utilization of glycerol.

In Listeria monocytogenes the alternative sigma factor sigmaB plays important roles in both virulence and stress tolerance. In this study a proteomic approach was used to define components of the sigmaB regulon in L. monocytogenes 10403S (serotype 1/2a). Using two-dimensional gel electrophoresis and the recently developed isobaric tags for relative and absolute quantitation technique, the protein expression profiles of the wild type and an isogenic delta sigB deletion strain were compared. Overall, this study identified 38 proteins whose expression was sigmaB dependent; 17 of these proteins were found to require the presence of sigmaB for full expression, while 21 were expressed at a higher level in the delta sigB mutant background. The data obtained with the two proteomic approaches showed limited overlap (four proteins were identified by both methods), a finding that highlights the complementarity of the two technologies. Overall, the proteomic data reaffirmed a role for sigmaB in the general stress response and highlighted a probable role for sigmaB in metabolism, especially in the utilization of alternative carbon sources. Proteomic and physiological data revealed the involvement of sigmaB in glycerol metabolism. Five newly identified members of the sigmaB regulon were shown to be under direct regulation of sigmaB using reverse transcription-PCR (RT-PCR), while random amplification of cDNA ends-PCR was used to map four sigmaB-dependent promoters upstream from lmo0796, lmo1830, lmo2391, and lmo2695. Using RT-PCR analysis of known and newly identified sigmaB-dependent genes, as well as proteomic analyses, sigmaB was shown to play a major role in the stationary phase of growth in complex media.

Sensing and adapting to acid stress.

Bacteria and archaea occupy a considerable diversity of niches that vary with respect to the physical conditions. Survival and colonisation requires the capacity to sense, and adapt to, environmental change. In this short review we consider the issues of adaptation to acidic conditions, in particular the mechanisms that might be employed by different bacteria to respond to the specific challenges of their niche. We lay particular emphasis on the protection of the cytoplasm during alterations of the cytoplasmic pH and, in the Gram negative bacteria, on recent work that suggests that protection of the periplasm is critical for survival of exposure to extreme acid. Finally, we discuss potential mechanisms by which pH might be sensed and consider the insights gained from proteins that sense and respond specifically to changes in pH.

Role of sigma(B) in heat, ethanol, acid, and oxidative stress resistance and during carbon starvation in Listeria monocytogenes.

To determine the contribution of sigma B (sigma(B)) to survival of stationary-phase Listeria monocytogenes cells following exposure to environmental stresses, we compared the viability of strain 10403S with that of an isogenic nonpolar sigB null mutant strain after exposure to heat (50 degrees C), ethanol (16.5%), or acid (pH 2.5). Strain viabilities were also determined under the same conditions in cultures that had been previously exposed to sublethal levels of the same stresses (45 degrees C, 5% ethanol, or pH 4.5). The DeltasigB and wild-type strains had similar viabilities following exposure to ethanol and heat, but the DeltasigB strain was almost 10,000-fold more susceptible to lethal acid stress than its parent strain. However, a 1-h preexposure to pH 4.5 yielded a 1,000-fold improvement in viability for the DeltasigB strain. These results suggest the existence in L. monocytogenes of both a sigma(B)-dependent mechanism and a pH-dependent mechanism for acid resistance in the stationary phase. sigma(B) contributed to resistance to both oxidative stress and carbon starvation in L. monocytogenes. The DeltasigB strain was 100-fold more sensitive to 13.8 mM cumene hydroperoxide than the wild-type strain. Following glucose depletion, the DeltasigB strain lost viability more rapidly than the parent strain. sigma(B) contributions to viability during carbon starvation and to acid resistance and oxidative stress resistance support the hypothesis that sigma(B) plays a role in protecting L. monocytogenes against environmental adversities.

Families and nursing home placements: a cross-cultural study.

With the aging of many populations, health care workers and families increasingly find themselves jointly involved in situations involving decisions about nursing home placements. How each approaches such situations is affected by beliefs and assumptions about the role of family members in the care of family members and the decision making process. This paper explores the responses of people from four cultural groups living in Australia (Anglo-Celtic Australian, Chinese, Greek, Lebanese) to a critical incident scenario about a Russian family in Australia faced with such a decision. The responses to this scenario were remarkably similar across the four cultural groups. All saw making such a decision as difficult, but the reasons for the difficulty suggest some interesting cross-cultural distinctions. Some groups viewed care of a family member more in terms of a social and role obligation while others addressed it as a personal responsibility. To not care for elderly parents in the home was accompanied by a sense of guilt among some respondents and a sense of public social shame among others. Ambivalence about nursing homes and placing a family member in a nursing home, culture change and cross-generational differences, and roles and role support were other important themes. The results are consistent with other data analysed in conjunction with the Intercultural Interaction Project. The findings from this research suggests a need to examine more closely the beliefs and assumptions associated with nursing home placements and one way to help students and health professionals to do so.

Identification and characterization of an ATP binding cassette L-carnitine transporter in Listeria monocytogenes.

We identified an operon in Listeria monocytogenes EGD with high levels of sequence similarity to the operons encoding the OpuC and OpuB compatible solute transporters from Bacillus subtilis, which are members of the ATP binding cassette (ABC) substrate binding protein-dependent transporter superfamily. The operon, designated opuC, consists of four genes which are predicted to encode an ATP binding protein (OpuCA), an extracellular substrate binding protein (OpuCC), and two membrane-associated proteins presumed to form the permease (OpuCB and OpuCD). The operon is preceded by a potential SigB-dependent promoter. An opuC-defective mutant was generated by the insertional inactivation of the opuCA gene. The mutant was impaired for growth at high osmolarity in brain heart infusion broth and failed to grow in a defined medium. Supplementation of the defined medium with peptone restored the growth of the mutant in this medium. The mutant was found to accumulate the compatible solutes glycine betaine and choline to same extent as the parent strain but was defective in the uptake of L-carnitine. We conclude that the opuC operon in L. monocytogenes encodes an ABC compatible solute transporter which is capable of transporting L-carnitine and which plays an important role in osmoregulation in this pathogen.

Rapid inactivation of the Escherichia coli Kdp K+ uptake system by high potassium concentrations.

The Kdp K+ uptake system of Escherichia coli is induced by limitation for K+ and/or high osmolarity. In the present study, the regulation of the activity of the Kdp system has been investigated in E. coli mutants possessing only the Kdp system as the mechanism of K+ accumulation. Cells grown in the presence of low K+ (0.1-1 mM) exhibit normal growth. However, growth inhibition results from exposure of cells to moderate levels of external K+ (> 5 mM). Measurement of the cytoplasmic pH, of K+ pools and of transport via the Kdp system demonstrates that the Kdp system is rapidly and irreversibly inhibited by moderate external K+. Concentrations of K+ greater than 2 mM are sufficient to cause inhibition of Kdp. At pH 6, this results in rapid lowering of the capacity for pH homeostasis, but at pH 7 the intracellular pH is unaffected. Parallel analysis of the expression of the Kdp system in a Kdp+/kdpFABC-lacZ strain shows that levels of K+ that are sufficient to inhibit Kdp activity also repress expression. As a result, growth inhibition of strains solely possessing Kdp arises jointly from inhibition of Kdp activity and repression of Kdp gene expression. These data identify an important aspect of the regulation of potassium transport via the Kdp system and also provide support for a model of regulation of Kdp expression via at least two mechanisms: sensing of both turgor and external K+ concentration.

Survival of low-pH stress by Escherichia coli O157:H7: correlation between alterations in the cell envelope and increased acid tolerance.

Survival of a nontoxigenic isolate of Escherichia coli O157:H7 at low pH (pH 3.0) was examined over prolonged time periods for each of three population types: exponential-phase cells, stationary-phase cells, and acid-adapted exponential-phase cells. In each population, approximately 5 x 10(4) CFU ml-1 were detected after a 24-h incubation at pH 3.0. Even after 3 days at pH 3.0, significant numbers of survivors from each of the three populations could be detected. The high level of acid tolerance exhibited by these survivors was found to be quickly lost once they were transferred to conditions which permitted growth to resume, indicating that they were not mutants. Proton flux measurements on the three populations of cells revealed that the initial rates of viability loss at pH 3.0 correlated well with net proton accumulation. Cells showing a high initial rate of viability loss (exponential-phase cells) accumulated protons at the highest rate, whereas resistant populations (adapted or stationary-phase cells) accumulated protons only slowly. Differences in the protein composition of the cell envelope between the three populations were studied by two-dimensional polyacrylamide gel electrophoresis. Complex differences in the pattern of proteins expressed by each population were uncovered. The implications of these findings are discussed in the context of a possible model accounting for acid tolerance in this important food-borne pathogen.

Pathogenicity in isolates of Salmonella enterica serotype Enteritidis PT4 which differ in RpoS expression: effects of growth phase and low temperature.

Experiments with 2 wild type isolates of Salmonella enterica serotype Enteritidis PT4, which differed in RpoS expression, tolerance to certain hostile environments and pathogenicity, found that changes in in vitro acid, heat, or peroxide tolerance had no effect on the ability of the isolates to multiply in the spleens of C57/BL7/J mice infected orally. Thus, with the pathogenic RpoS-positive isolate, the infectivity of log phase chilled cells, which are profoundly acid-sensitive, was the same as that of non-chilled stationary phase cells which are acid-tolerant. Similarity the infectivity of the RpoS-negative, sensitive isolate, was not enhanced by increases in any tolerance. The ability to survive on surfaces, like infectivity, was also largely unaffected by either growth phase or cold exposure. These two attributes may thus be related and, given that the pathogenic PT4 isolate is capable of prolonged survival and the nonpathogenic isolate survives poorly, survival could serve as a potential marker of pathogenicity. Although the pathogenicity of the two isolates was very different, they showed an almost identical increase in acid tolerance following culture at pH 4.0 for up to 60 min.

Perturbation of anion balance during inhibition of growth of Escherichia coli by weak acids.

During inhibition of cell growth by weak acids, there is substantial accumulation of the weak acid anions in the cytoplasm. This study was undertaken to determine the impact of anion accumulation on cellular pools. At pH 6, growth in the presence of 8 mM acetate led to an internal pool of greater than 240 mM acetate anion and resulted in reduced levels of glutamate in the cell, but there were no significant changes in K+ and Na+ levels. At low osmolarity, the change in the glutamate pool compensated for only a small fraction of the accumulated acetate anion. However, at high osmolarity, glutamate compensated for over half of the accumulated acetate. Recovery of the normal cytoplasmic pH after the removal of acetate was dependent on the synthesis of glutamate.

Acid tolerance in Listeria monocytogenes: the adaptive acid tolerance response (ATR) and growth-phase-dependent acid resistance.

Listeria monocytogenes acquired increased acid tolerance during exponential growth upon exposure to sublethal acid stress, a response designated the acid tolerance response (ATR). Maximal acid resistance was seen when the organism was exposed to pH 5.0 for 1 h prior to challenge at pH 3.0, although intermediate levels of protection were afforded by exposure to pH values ranging from 4.0 to 6.0. A 60 min adaptive period was required for the development of maximal acid tolerance; during this period the level of acid tolerance increased gradually. Full expression of the ATR required de novo protein synthesis; chloramphenicol, a protein synthesis inhibitor, prevented full induction of acid tolerance. Analysis of protein expression during the adaptive period by two-dimensional gel electrophoresis revealed a change in the expression of at least 23 proteins compared to the non-adapted culture. Eleven proteins showed induced expression while 12 were repressed, implying that the ATR is a complex response involving a modulation in the expression of a large number of genes. In addition to the exponential phase ATR, L.monocytogenes also developed increased acid resistance upon entry into the stationary phase; this response appeared to be independent of the pH-dependent ATR seen during exponential growth.

Transcription of the Salmonella typhimurium spv virulence locus is regulated negatively by the nucleoid-associated protein H-NS.

The possibility that the pleiotropic transcriptional regulator H-NS might play a role in regulating expression of the spv virulence locus of Salmonella typhimurium was investigated. A transposon insertion mutation in hns, the gene encoding H-NS, resulted in enhanced transcription of the spvR regulatory gene and the spvB structural gene in stationary phase cultures. Enhanced transcription was not detected prior to stationary phase, indicating that H-NS makes a negative contribution that is growth phase-specific to the control of spv transcription. When H-NS was over-expressed from a multicopy plasmid, the normal stationary phase induction of spv transcription seen in wild-type cells was abolished. spv transcription was also found to be modulated by growth medium osmolarity, a feature common to many H-NS-regulated genes. In addition, transcription of the spv genes was reduced in mutants with abnormal levels of DNA supercoiling.

The spv virulence operon of Salmonella typhimurium LT2 is regulated negatively by the cyclic AMP (cAMP)-cAMP receptor protein system.

The cyclic AMP (cAMP) receptor protein (CRP) was found to play a role in the growth phase regulation of the spv operon on the high-molecular-weight virulence plasmid of Salmonella typhimurium LT2. By using a lacZ reporter transcriptional fusion to the spvB structural gene on the single-copy virulence plasmid, it was found that while spvB transcription was induced in stationary-phase cultures, the induced level of expression was lower than that reported for the spv system in other serovars of Salmonella. Surprisingly, inactivation of the gene encoding the positive activator SpvR resulted in only a threefold reduction in spvB transcription. In contrast, spvB transcription in stationary-phase cultures was enhanced by 10-fold in mutants deficient in crp-encoded CRP or cya-encoded adenylate cyclase. Wild-type (i.e., 10-fold-lower) levels of spvB expression were restored by providing active copies of crp or cya on recombinant plasmids. Enhanced spvB transcription was not seen in crp or cya mutants in the absence of a functional spvR positive regulatory gene, showing that the cAMP-CRP system acted on spvB expression either in conjunction with or via SpvR. A lacZ transcriptional fusion to spvR could not be induced in stationary-phase cultures in the absence of functional SpvR, regardless of the cAMP-CRP status of the cells. When SpvR was provided in trans, transcription of the spvR-lacZ fusion was induced to similar levels in stationary-phase cultures with and without cAMP-CRP. These data are consistent with spvR being poorly transcribed from the single-copy virulence plasmid in S. typhimurium LT2 and with a suppression of this defect via inactivation of the cAMP-CRP system. The physiological significance of cAMP-CRP involvement in spv expression is discussed.

The DNA supercoiling-sensitive expression of the Salmonella typhimurium his operon requires the his attenuator and is modulated by anaerobiosis and by osmolarity.

Bacterial cells possess a subset of genes whose expression correlates with changes in DNA supercoiling brought about by anaerobic growth and by growth at high osmolarity. It has been shown previously that expression of the histidine biosynthetic operon of Salmonella typhimurium is derepressed by relaxation of supercoiled DNA. Here, we confirm that a his::MudJ operon fusion in S. typhimurium can be induced by treatment with the DNA gyrase inhibitor novobiocin in a dose-dependent manner, and show that the level of derepression is higher in stationary phase than in mid-exponential phase cultures. Furthermore, expression of his is repressed by anaerobiosis and by osmolarity, two environmental parameters which increase the negative supercoiling of bacterial DNA. Novobiocin induction of his is also repressed by growing the cells either at high osmolarity or anaerobically. Both environmental repression and novobiocin induction of his require the his attenuator. In addition, derepression of his expression by novobiocin and its repression by anaerobiosis or osmolarity are independent of the stringent response gene, relA.