Effects of repeated cycles of acid challenge and growth on the phenotype and virulence of Salmonella enterica.

AIMS: The aim of the study was to investigate how stresses like low pH, which may be encountered in farms or food preparation premises, shape populations of Salmonella enterica by the selection of stress-resistant variants. METHODS AND RESULTS: Stationary-phase cultures of S. enterica serovar Enteritidis and serovar Typhimurium (one strain of each) were exposed to pH 2.5 for up to 4 h, followed by growth at pH 7 for 48 h. This process was repeated 15 times in two separate experiments, which increased the acid resistance of the three out of four populations we obtained, by three- to fourfold. Sustainable variants derived from the populations showed changes in colony morphology, expression of SEF17 fimbriae, growth, increased heat resistance and reduced virulence. CONCLUSIONS: The study demonstrates that low pH environments can select for populations of S. enterica with persistent phenotypic changes such as increased acid resistance and occasionally increased SEF17 expression and lower virulence. SIGNIFICANCE AND IMPACT OF THE STUDY: There is a common belief that increased acid resistance coincides with increased virulence. This study demonstrates for the first time that increased acid resistance often impairs virulence and affects the general phenotype of S. enterica.

Implications of aligning full registration of doctors with medical school graduation: a qualitative study of stakeholder perspectives.

OBJECTIVES: The Shape of Training report recommended that full registration is aligned with medical school graduation. As part of a General Medical Council-funded study about the preparedness for practice of UK medical graduates, we explored UK stakeholders' views about this proposal using qualitative interviews (30 group and 87 individual interviews) and Framework Analysis. SETTING: Four UK study sites, one in each country. PARTICIPANTS: 185 individuals from eight stakeholder groups: (1) foundation year 1 (F1) doctors (n=34); (2) fully registered trainee doctors (n=33); (3) clinical educators (n=32); (4) undergraduate/postgraduate Deans, and Foundation Programme Directors (n=30); (5) other healthcare professionals (n=13); (6) employers (n=7); (7) policy and government (n=11); (8) patient and public representatives (n=25). RESULTS: We identified four main themes: (1) The F1 year as a safety net: patients were protected by close trainee supervision and 'sign off' to prevent errors; trainees were provided with a safe environment for learning on the job; (2) Implications for undergraduate medical education: if the proposal was accepted, a 'radical review' of undergraduate curricula would be needed; undergraduate education might need to be longer; (3) Implications for F1 work practice: steps to protect healthcare team integration and ensure that F1 doctors stay within competency limits would be required; (4) Financial, structural and political implications: there would be cost implications for trainees; clarification of responsibilities between undergraduate and postgraduate medical education would be needed. Typically, each theme comprised arguments for and against the proposal. CONCLUSIONS: A policy change to align the timing of full registration with graduation would require considerable planning and preliminary work. These findings will inform policymakers' decision-making. Regardless of the decision, medical students should take on greater responsibility for patient care as undergraduates, assessment methods in clinical practice and professionalism domains need development, and good practice in postgraduate supervision and support must be shared.

The heteroduplex mobility assay (HMA) as a pre-sequencing screen for Norwalk-like viruses.

Molecular epidemiological studies of Norwalk-like viruses (NLVs), previously known as small round structured viruses (SRSVs), are dependant currently on DNA sequencing of PCR amplicons, which is expensive and time consuming. The Heteroduplex Mobility assay (HMA) was evaluated as a method for identification of PCR amplicons from the commonly circulating NLV strains without DNA sequencing. The procedure was developed for use with two reference strains, a Mexico virus-like strain (MXV-like; Hu¿NLV¿RBH¿1993¿UK) and the Grimsby virus strain (Hu¿NLV¿Gimsby¿1995¿UK), and was optimised with regards to the annealing and electrophoresis conditions and the electrophoresis gel matrix. Using the optimised conditions, amplicons of less than 90% sequence identity formed visible heteroduplexes, allowing the strains to be placed into three categories; Mexico-like, Grimsby-like and non-Mexico virus/non-Grimsby virus strains. Outbreak strains 'genotyped' previously by DNA sequencing as Mexico virus or Grimsby virus were identified correctly by the heteroduplex mobility assay. The procedure was applied prospectively to strains from 130 outbreaks occurring in the UK between 1997 and 1998. Heteroduplex mobility assay was successful on 120 (92%) strains of which 68 (57%) were GRV-like strains, three (2.5%) were Mexico virus-like strains and 49 (41%) were categorised as non- Mexico/non-Grimsby virus strains. Amplicons from 50 of the 120 strains were sequenced and there was perfect correlation between the heteroduplex mobility assay categorisation and phylogenetic analysis. HMA offers a rapid, robust and far cheaper alternative to sequencing for the identification of prevalent Norwalk-like virus genotypes for molecular epidemiological studies.

Calculating Salmonella inactivation in nonisothermal heat treatments from isothermal nonlinear survival curves.

Salmonella cells in two sugar-rich media were heat treated at various constant temperatures in the range of 55 to 80 degrees C and their survival ratios determined at various time intervals. The resulting nonlinear semilogarithmic survival curves are described by the model log10S(t) = -b(T)tn(T), where S(t) is the momentary survival ratio N(t)/N0, and b(T) and n(T) are coefficients whose temperature dependence is described by two empirical mathematical models. When the temperature profile, T(t), of a nonisothermal heat treatment can also be expressed algebraically, b(T) and n(T) can be transformed into a function of time, i.e., b[T(t)] and n[T(t)]. If the momentary inactivation rate primarily depends on the momentary temperature and survival ratio, then the survival curve under nonisothermal conditions can be constructed by solving a differential equation, previously suggested by Peleg and Penchina, whose coefficients are expressions that contain the corresponding b[T(t)] and n[T(t)] terms. The applicability of the model and its underlying assumptions was tested with a series of eight experiments in which the Salmonella cells, in the same media, were heated at various rates to selected temperatures in the range of 65 to 80 degres C and then cooled. In all the experiments, there was an agreement between the predicted and observed survival curves. This suggests that, at least in the case of Salmonella in the tested media, survival during nonisothermal inactivation can be estimated without assuming any mortality kinetics.

Improving recovery of Salmonella enterica serovar typhimurium DT104 cells injured by heating at different water activity values.

This study describes the evaluation of potentially more sensitive methods for the recovery of Salmonella cells injured by heating (54 to 60 degrees C) at different water activity values (0.65 to 0.90, reduced using equal portions of glucose and fructose). These methods included gradual rehydration, the use of diluting media with added solutes or blood, the addition of blood to plating agar, and the use of different incubation temperatures and times. Gradual rehydration of cells that had been challenged at low water activity (0.65 and 0.70) and high temperature markedly improved recovery, measured as a >50% increase in the time to obtain a 3-log10 reduction in cell numbers, compared to dilution into media with a high water activity. Adding sucrose, glycerol, or blood to the diluting media (maximal recovery diluent) did not improve recovery, but a plating agar containing blood recovered approximately 38% more cells than nutrient agar. Prolonged incubation of agar plates allowed recovery of injured Salmonella cells that presumably had extended lag periods, with significantly higher recovery rates after 48 h incubation at 37 degrees C than after 24 h (P = 0.05). This work highlights that by recovering Salmonella using a method specific to the nature of the injury, a better prediction of food safety and the success of food processing can be made.

Teaching and assessing medical ethics: where are we now?

OBJECTIVES: To characterise UK undergraduate medical ethics curricula and to identify opportunities and threats to teaching and learning. DESIGN: Postal questionnaire survey of UK medical schools enquiring about teaching and assessment, including future perspectives. PARTICIPANTS: The lead for teaching and learning at each medical school was invited to complete a questionnaire. RESULTS: Completed responses were received from 22/28 schools (79%). Seventeen respondents deemed their aims for ethics teaching to be successful. Twenty felt ethics should be learnt throughout the course and 13 said ethics teaching and learning should be fully integrated horizontally. Twenty felt variety in assessment was important and three tools was the preferred number. A shortfall in ethics core competencies did not preclude graduation in 15 schools. The most successful aspects of courses were perceived to be their integrated nature and the small group teaching; weaknesses were described as a need for still greater integration and the heavily theoretical aspects of ethics. The major concerns about how ethics would be taught in the future related to staffing and staff development. CONCLUSIONS: This study describes how ethics was taught and assessed in 2004. The findings show that, although ethics now has an accepted place in the curriculum, more can be done to ensure that the recommended content is taught and assessed optimally.

Interprofessional learning involving medical students or doctors.

AIMS: There is a belief that interprofessional learning among health care professionals promotes teamwork and collaboration, leading to improved patient care, but supporting data have not been available. Our aim was to identify recent studies on interprofessional learning involving medical students or doctors. METHODS: The medical education literature was searched for relevant publications since 1998 and the studies were subsequently classified according to various criteria. RESULTS: A total of 24 'research papers' and 36 'literature review/editorial-type papers' were identified. Research papers generally described interprofessional learning initiatives and evaluated their success in terms of knowledge or attitudes by data collection. Seven collected qualitative data, 4 collected quantitative data and 11 collected both. None of the studies identified conformed to the Cochrane Collaboration guidelines for a rigorous source of quantitative information on an intervention's effectiveness. DISCUSSION: The authors suspect that significantly more studies have been performed than those published. This may indicate research of variable quality, leading to a large proportion of papers being rejected for publication. More and better quality research is required as more resources become available to academic and health care organisations for interprofessional learning.

Optimisation of the protocol for detection of Aeromonas species in ready-to-eat salads, and its use to speciate isolates and establish their prevalence.

Aeromonas spp. are detected in more than 500 cases of gastrointestinal infection each year in England and Wales. This study aimed to identify their prevalence in ready-to-eat salads, which are a potential source of aeromonas infection. The protocol for isolation of mesophilic Aeromonas spp. from salads was optimised. Using the improved method, Aeromonas spp were isolated from 19 of 25 samples (25 g) of ready-to-eat salad products. Aeromonas organisms were counted, isolates were identified to species level, and the effect of pH on colonisation of salads was assessed. Aeromonas was present at high levels in six salads (> or = 100 cfu/g). The major species present in salads was Aeromonas caviae, but A.hydrophila and A.sobria, which have more pathogenic potential, were also isolated. It is hoped that this study will help to assess the risk to public health of aeromonas in salads.

The risk to public health of aeromonas in ready-to-eat salad products.

Mesophilic Aeromonas spp. are isolated regularly from cases of gastrointestinal infection and have markers indicative of enteropathogenicity. Is aeromonas, which is present in a large proportion of ready-to-eat salads, actually a gastrointestinal pathogen? Isolates of mesophilic aeromonas from salads were characterised in terms of their ability to grow at refrigeration temperatures over the given shelf life and by the presence of markers of potential virulence. The major phenospecies present in salads, A.caviae, showed little enteropathogenic potential. Thirty-five per cent of aeromonas salad isolates are A.hydrophila or A.sobria, however, and all isolates tested had at least one marker of enteropathogenicity, including cytotoxin and haemolysin production, adherence to epithelial cells, and resistance to certain antibiotics Despite the presence of markers of enteropathogencity, the lack of epidemiological evidence of a link between infectious intestinal disease and the consumption of salads suggests that their contamination with aeromonas does not pose a significant risk to health in immunocompetent adults.

The microbiological quality of washing-up water and the environment in domestic and commercial kitchens.

AIMS: To determine the microbiological quality of washing-up water and the environment in domestic and commercial kitchens. METHODS AND RESULTS: Chicken meals were prepared by people without food safety training in their own kitchen (n = 52) or by trained staff in a commercial kitchen (n = 10). Study participants then washed-up, cleaned the kitchen and completed a food hygiene questionnaire. The temperature and microbiological quality of the washing-up water, and the presence of pathogens in dishcloths, tea towels and other kitchen samples was determined. Of the raw chickens used in meal preparation, 96 and 13% were naturally contaminated with Campylobacter or Salmonella spp., respectively. In domestic kitchens, two of 45 sponges, dishcloths or scourers and one of 32 hand- or tea towels were contaminated with Campylobacter after washing-up and cleaning but none of the tap or sink swabs yielded pathogens. The mean washing-up water temperature in the domestic kitchens was 40.7 degrees C, whereas in the commercial kitchen it was 44.7 degrees C (P = 0.04). Study participants who used hotter water (>/=40 degrees C) had lower levels of bacteria in their washing-up water. The aerobic plate counts of the washing-up water samples in domestic homes were usually between 105 and 106 CFU ml-1 but those associated with the commercial kitchen were consistently lower (P = 0.01). Despite this, Campylobacter was detected in one of 10 washing-up water samples from the commercial kitchen but in none of the samples from domestic kitchens. CONCLUSIONS: Pathogenic microorganisms can be recovered relatively frequently from the kitchen environment. SIGNIFICANCE AND IMPACT OF STUDY: By identifying factors that affect the number of microorganisms in washing-up water and the kitchen environment, evidence-based recommendations on implementing domestic food hygiene can be made.

The prevalence and number of Salmonella in sausages and their destruction by frying, grilling or barbecuing.

AIMS: To determine the prevalence and number of Salmonella and Campylobacter in sausages and to evaluate their destruction during cooking. METHODS AND RESULTS: One hundred and sixty-two packs of uncooked economy or catering sausages, comprising 53 packs of frozen and 109 of chilled sausages, were purchased in Devon between March and July 2000. All were tested for the presence of Salmonella and 51 packs of chilled sausages were also examined for the presence of Campylobacter spp. To investigate the heat tolerance of Salmonella enterica serovar Typhimurium DT104 in sausage-meat, chilled, handmade and frozen sausages were inoculated with approx. 1.5 x 10(4) bacterial cells per sausage (approximately 300 cfu g(-1)) and then cooked by frying, grilling or barbecuing. The levels of creatinine kinase, lactate dehydrogenase and alkaline phosphatase in uncooked and cooked sausages were measured to evaluate their potential as indicators of adequate cooking and, therefore, pathogen elimination. Salmonella were detected in 7.5% of frozen and 9.1% of the chilled sausages (8.6% overall) but Campylobacter spp. were not isolated. After cooking, a visual assessment suggested that all of the sausages were thoroughly cooked. Despite this, barbecuing and frying sometimes allowed Salmonella cells to survive and the temperature profiles during cooking indicated that the lethal range was sometimes not reached. The enzyme levels tested were not reliable indicators of the inactivation of bacterial pathogens because Salmonella were sometimes isolated from sausages with low values of all three enzymes. CONCLUSIONS: Salmonella spp. are present in a significant proportion of sausages and are not always killed during the cooking process. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings have clear implications for public health.

Evaluation of selective media for Campylobacter isolation when cycloheximide is replaced with amphotericin B.

AIMS: Laboratory media for the isolation of Campylobacter usually contain various selective agents, designed to allow these bacteria to grow whilst suppressing that of other organisms. For example, cycloheximide has often been incorporated into Campylobacter media to inhibit the growth of fungi. The production and availability of cycloheximide, however, has recently decreased due to concerns relating to its potential toxicity for mammalian cells and the compound has also become more expensive as a result. An alternative antifungal agent is necessary, and to address this, the effect of using amphotericin B in place of cycloheximide in Campylobacter selective broths and agars was examined. METHODS AND RESULTS: The growth of Campylobacter strains and the suppression of potential competitor organisms in selective broths or on selective agars containing either amphotericin B or cycloheximide was quantified, using pure microbial cultures. The results were then confirmed by testing food and water samples that contained high levels of micro-organisms, including Campylobacter. CONCLUSIONS: The results of this study indicate that amphotericin B is a suitable replacement for cycloheximide in Campylobacter selective media.

Distinct epidemiological patterns of Norwalk-like virus infection.

Norwalk-like viruses (NLV) are important economically as a cause of both sporadic gastroenteritis in the community and large outbreaks in hospitals and other institutional settings. Despite the description of several antigenic types relatively little is known about the epidemiology of these individual types. NLVs were detected by electron microscopy in faecal specimens from 706 outbreaks of gastroenteritis that represented 68% of all outbreaks of non-bacterial gastroenteritis. These outbreaks took place in the counties of West and North Yorkshire and Humberside during six winter seasons between July 1992 and June 1998. NLV strains from 671 outbreaks were typed by antigen capture enzyme linked immunosorbent assays (ELISA) based on antisera made to recombinant virus-like particles of three antigenically distinct NLVs; Norwalk (NV), Mexico (MXV) and Grimsby (GRV) viruses. GRV was the predominant strain for five of the six winter seasons and overall was associated with 61% of NLV outbreaks. MXV was responsible for a single epidemic peak in the winter of 1993/94 but was also observed at other times throughout the study period. NV was only associated with two outbreaks in 1994 that were epidemiologically linked. Strains from the remaining 32% of outbreaks were non-reactive in all three ELISA. Thus, a single NLV antigenic type seems to have predominated during the period 1992 to 1998 in the UK.

Habituation of Salmonella spp. at reduced water activity and its effect on heat tolerance.

The effect of habituation at reduced water activity (a(w)) on heat tolerance of Salmonella spp. was investigated. Stationary-phase cells were exposed to a(w) 0.95 in broths containing glucose-fructose, sodium chloride, or glycerol at 21 degrees C for up to a week prior to heat challenge at 54 degrees C. In addition, the effects of different a(w)s and heat challenge temperatures were investigated. Habituation at a(w) 0.95 resulted in increased heat tolerance at 54 degrees C with all solutes tested. The extent of the increase and the optimal habituation time depended on the solute used. Exposure to broths containing glucose-fructose (a(w) 0.95) for 12 h resulted in maximal heat tolerance, with more than a fourfold increase in D(54) values. Cells held for more than 72 h in these conditions, however, became as heat sensitive as nonhabituated populations. Habituation in the presence of sodium chloride or glycerol gave rise to less pronounced but still significant increases in heat tolerance at 54 degrees C, and a shorter incubation time was required to maximize tolerance. The increase in heat tolerance following habituation in broths containing glucose-fructose (a(w) 0.95) was RpoS independent. The presence of chloramphenicol or rifampin during habituation and inactivation did not affect the extent of heat tolerance achieved, suggesting that de novo protein synthesis was probably not necessary. These data highlight the importance of cell prehistory prior to heat inactivation and may have implications for food manufacturers using low-a(w) ingredients.

Survival and filamentation of Salmonella enterica serovar enteritidis PT4 and Salmonella enterica serovar typhimurium DT104 at low water activity.

In this study we investigated the long-term survival of and morphological changes in Salmonella strains at low water activity (a(w)). Salmonella enterica serovar Enteritidis PT4 and Salmonella enterica serovar Typhimurium DT104 survived at low a(w) for long periods, but minimum humectant concentrations of 8% NaCl (a(w), 0. 95), 96% sucrose (a(w), 0.94), and 32% glycerol (a(w), 0.92) were bactericidal under most conditions. Salmonella rpoS mutants were usually more sensitive to bactericidal levels of NaCl, sucrose, and glycerol. At a lethal a(w), incubation at 37 degrees C resulted in more rapid loss of viability than incubation at 21 degrees C. At a(w) values of 0.93 to 0.98, strains of S. enterica serovar Enteritidis and S. enterica serovar Typhimurium formed filaments, some of which were at least 200 microm long. Filamentation was independent of rpoS expression. When the preparations were returned to high-a(w) conditions, the filaments formed septa, and division was complete within approximately 2 to 3 h. The variable survival of Salmonella strains at low a(w) highlights the importance of strain choice when researchers produce modelling data to simulate worst-case scenarios or conduct risk assessments based on laboratory data. The continued increase in Salmonella biomass at low a(w) (without a concomitant increase in microbial count) would not have been detected by traditional microbiological enumeration tests if the tests had been performed immediately after low-a(w) storage. If Salmonella strains form filaments in food products that have low a(w) values (0.92 to 0.98), there are significant implications for public health and for designing methods for microbiological monitoring.

Effect of challenge temperature and solute type on heat tolerance of Salmonella serovars at low water activity.

Salmonella spp. are reported to have an increased heat tolerance at low water activity (a(w); measured by relative vapor pressure [rvp]), achieved either by drying or by incorporating solutes. Much of the published data, however, cover only a narrow treatment range and have been analyzed by assuming first-order death kinetics. In this study, the death of Salmonella enterica serovar Typhimurium DT104 when exposed to 54 combinations of temperature (55 to 80 degrees C) and a(w) (rvp 0.65 to 0.90, reduced using glucose-fructose) was investigated. The Weibull model (LogS = -bt(n)) was used to describe microbial inactivation, and surface response models were developed to predict death rates for serovar Typhimurium at all points within the design surface. The models were evaluated with data generated by using six different Salmonella strains in place of serovar Typhimurium DT104 strain 30, two different solutes in place of glucose-fructose to reduce a(w), or six low-a(w) foods artificially contaminated with Salmonella in place of the sugar broths. The data demonstrate that, at temperatures of > or =70 degrees C, Salmonella cells at low a(w) were more heat tolerant than those at a higher a(w) but below 65 degrees C the reverse was true. The same patterns were generated when sucrose (rvp 0.80 compared with 0.90) or NaCl (0.75 compared with 0.90) was used to reduce a(w), but the extent of the protection afforded varied with solute type. The predictions of thermal death rates in the low-a(w) foods were usually fail-safe, but the few exceptions highlight the importance of validating models with specific foods that may have additional factors affecting survival.