Chicken's best friend? Livestock guardian dog bonding with free-ranging chickens.

Growth in the free-range and pastured egg industries has increased globally, necessitating improvements in predator control. Some egg producers are turning to the use of livestock guardian dogs (LGD; Canis familiaris) to protect hens from predation. We worked on a property where pastured layer hens were protected by two Maremma LGDs that were released from their chicken enclosure for 2-3 nights a week. GPS tracking showed that the dogs were more strongly bonded to people than the chickens, spending most of their time at night (96.1% of location data) close to the farmhouse and only 0.09% near their chicken paddock. Despite this lack of attendance, we found no change in the paddock space use by chickens with or without the dogs present (P = 0.999). Furthermore, camera trapping revealed 40 red fox (Vulpes vulpes) events over the 46-d monitoring period, with less fox activity on nights when the LGDs were allowed to roam the property and motion-activated spotlights were also deployed (P = 0.048). An online survey of 59 poultry producers found strong belief in the effectiveness of LGDs, although half the respondents (52%) indicated that they were still experiencing predation issues. There was no association with the reported degree of human bonding of their LGDs, but respondents were more likely to report current issues with predators if they owned 100 or more chickens (P = 0.031). The present case study as well as the farmer survey have identified that LGDs can be strongly bonded to people. Although there was no evidence of subsequently increased risk of predation, bonding with people could draw LGDs away from the animals they should be defending, with predation risk for poultry likely to depend on how far away LGDs move from their livestock.

Out of sight, out of mind: an audit of inferior vena cava filter insertion and clinical follow up in an Australian institution and literature review.

BACKGROUND: Inferior vena cava (IVC) filters are increasingly used to treat venous thromboembolism when there are contraindications or failure to respond to anticoagulant therapy. Retrievable filters were introduced to avoid long-term complications and risks associated with permanent filters. However, failure to follow up patients appropriately can lead to low retrieval rates. AIMS: To examine the practice of our institution in using retrievable IVC filters and to provide a review of published literature. METHODS: Retrospective audit of medical records in a single medical institution. RESULTS: Forty-one patients had retrievable IVC filters inserted. The median age of patients was 67. The majority (78%) of patients had filters inserted for presence of venous thromboembolism and contraindication to anticoagulation. Twenty-five (61%) patients received no clinical follow up. Factors associated with loss to follow up include a lack of documentation for retrieval plan (P < 0.01), lack of haematology outpatient clinic review (P < 0.01) and age greater than 50 years (P < 0.01). Procedural success was achieved in nine of 11 attempted filter removals. Eighteen complications were noted among patients. IVC filter insertion failed to prevent recurrent pulmonary embolisms in three patients. CONCLUSIONS: Majority of retrievable IVC filters will become lost to clinical follow up. Rates of attempted retrieval within 1 year of filter insertion are low. Loss to follow up is associated with older age, lack of documentation and lack of haematology clinic review post discharge. This study highlights the importance of a structured system to document clearly the review and retrieval plans for patients with IVC filters, at the time of initial insertion.

Targeted microarray analysis of stationary phase Escherichia coli O157:H7 subjected to disparate nutrient conditions.

AIMS: To determine how stress response and virulence gene expression of stationary phase (SP) Escherichia coli O157:H7 are affected by nutrient levels. METHODS AND RESULTS: A targeted microarray (n=125 genes) was used to determine the impact of nutrient deprivation [15 min in 3-(N-Morpholino)propanesulfonic acid buffer] on SP E. coli O157:H7. In total, 24 genes were significantly affected (>1·5-fold; P <0·05) with 17 induced and seven attenuated. Additionally, 11 genes belonging to significantly affected stress response regulons were significantly induced (P<0·05), though <1·5-fold. Induced genes included global and specific stress response regulators, the mar operon, iron acquisition and virulence genes. In contrast, transcript for major porins and replicative genes were repressed. Comparison of the nutrient deprived transcriptome to that derived from nutrient replenished cells revealed a disparate transcriptome, with 44 genes expressed at significantly elevated levels in nutrient replenished cells, including all queried global and specific stress response regulators and key virulence genes. Genes expressed at elevated levels in nutrient deprived cells were related to σ(S) . The microarray data were validated by qRT-PCR. CONCLUSIONS: SP E. coli O157:H7 were affected by nutrient deprivation, with both starvation-related and unrelated networks induced, thereby demonstrating how the E. coli O157:H7 stress response transcriptome is fine-tuned to environmental conditions. Further, by comparison of starved cells to cells provided with fresh nutrients, it is clear starved E. coli O157:H7 undergo massive physiological reprogramming dominated initially by stress response induction to adapt to a nutrient rich environment. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated how σ(S) -induced SP E. coli O157:H7 remain highly sensitive and adaptable to environmental conditions. Further, by examining how starved cells respond to nutrient-rich conditions, we show preliminary adaptation to a nutrient rich environment is dominated by the induction of diverse stress response networks. Combined, this provides E. coli O157:H7 stress physiology-based knowledge that can be used to design more effective food safety interventions.

Influence of cinnamon and clove essential oils on the D- and z-values of Escherichia coli O157:H7 in apple cider.

Escherichia coli O157:H7 has become a concern within the apple cider industry. The purpose of this study was to screen several essential oils and isolated components for antimicrobial activity against E. coli O157:H7 in tryptic soy broth at neutral and acidic pH and to assess the effect of these additives on the D-value of E. coli O157:H7 in apple cider in combination with mild heat treatments. Cinnamon oil and clove oil strongly inhibited the growth of E. coli O157:H7 at neutral and acidic pH, (R)-(-)-carvone and (S)-(-)-perillaldehyde were moderately inhibitory at both pH 7.2 and pH 4.5, and citral and geraniol displayed moderate activity at pH 4.5 only. Lemon oil, methyl jasmonate, and p-anisaldehyde displayed little or no antibacterial activity. A synergistic effect between the essential oils and the lower pH of the growth medium was evident by consistently lower MICs at pH 4.5. Cinnamon and clove oils (0.01%, vol/vol) were further tested in apple cider in combination with mild heat treatments for the practical control of E. coli O157:H7 in apple cider. The addition of either essential oil resulted in lower D-values than those for cider alone, suggesting a synergistic effect and the potential efficacy of a mild heat treatment for apple cider.

Role of nutrient limitation in the competition between Pseudomonas fluorescens and Escherichia coli O157: H7.

Competition between spoilage microorganisms and foodborne pathogens provides a potentially simple approach to limiting the growth of pathogens. A strain of Pseudomonas fluorescens isolated from raw milk repressed growth of Escherichia coli O157:H7 at 22 degrees C in nutrient broth once the maximum population density of the pseudomonad had been reached (9.6 log CFU ml(-1)). The presence of iron in the growth medium and the parallel inhibitory effect of a siderophore-deficient mutant of P. fluorescens precluded iron limitation as the mechanism of action. Medium depleted by prior growth of P. fluorescens prevented the growth of E. coli, and this effect was reversed by the replenishment of the nutrient broth, its component fractions, or the addition of soy peptones but not peptones derived from milk protein. This is the first report of competition between spoilage microflora and foodborne pathogens in which the mechanism was clearly shown to be nutrient limitation. These results suggest possible improvements in biocontrol systems to prevent pathogen growth on foods.

Development of a global stochastic model relating the distribution of individual cell and population physiological states.

Our ability to predict the lag (lambda) prior to growth of foodborne pathogens is limited by our lack of understanding of the physiological changes taking place in the individual cell during the adaptation process. Theoretical models have been developed to describe the stochastic nature of individual cells, and probability distributions have been used to assign hypothetical values of the physiological state to individual cells (p(i)). The aim of this study is to develop a polynomial model which will link distributions of p(i) values to the physiological state of the population (h(0)), and thus to the lambda. Risk analysis software was used to simulate values of p(i) for populations of cells drawn from lognormal distributions with parameters alpha and beta, and growth curves were simulated using a modified continuous-discrete-continuous (CDC) model. Values for h(0) were then obtained for each growth curve by fitting with the heterogeneous population model (HPM). Multiple regression analysis was used to develop a polynomial function which described the subsequent h(0) value as a function of alpha and beta (R(2)=0.9957). Outputs from simulations using the polynomial model agree well with results from related stochastic models, and suggest that distributions can accurately describe the physiological state of cell populations.

Inactivation of microbes using ultrasound: a review.

Alternative methods for pasteurization and sterilization are gaining importance, due to increased consumer demand for new methods of food processing that have a reduced impact on nutritional content and overall food quality. Ultrasound processing or sonication is one of the alternative technologies that has shown promise in the food industry. Sonication alone is not very effective in killing bacteria in food; however, the use of ultrasound coupled with pressure and/or heat is promising. Thermosonic (heat plus sonication), manosonic (pressure plus sonication), and manothermosonic (heat and pressure plus sonication) treatments are likely the best methods to inactivate microbes, as they are more energy-efficient and effective in killing microorganisms. Ultrasonic processing is still in its infancy and requires a great deal of future research in order to develop the technology on an industrial scale, and to more fully elucidate the effect of ultrasound on the properties of foods.

Thermal inactivation of Pediococcus sp. in simulated apple cider during high-temperature short-time pasteurization.

Prompted by concerns regarding outbreaks of food-borne illness which have occurred due to the consumption of commercial, nonpasteurized fruit juices contaminated with Escherichia coli O157:H7, the US Food and Drug Administration and Canadian Food Inspection Agency are considering several new safety standards to apply to fresh juices, including mandatory pasteurization of all apple cider. In support of these initiatives, a study was conducted to evaluate the pasteurization of simulated cider using a heat-resistant nonpathogenic test bacterium, Pediococcus sp. NRRL B-2354. Thermal inactivation of the Pediococcus sp. was determined using a pilot scale high-temperature short-time (HTST) pasteurizer with a plate heat exchanger. The cumulative lethal effect, or pasteurization effect (PE), was obtained by converting times at different temperatures in the various sections of the pasteurizer to the equivalent time at the reference temperature (72 degrees C). PE was then related by a simple linear function to the log(10) of the percentage of viable counts with a power transformation of the PE values to improve linear fit. r(2) values for the four Pediococcus sp. trials varied from 0.921 to 0.981. Intertrial variation was incorporated into the model using @RISK simulation software. Output from simulations confirmed that treatment at 71 degrees C for 16 s can ensure a 5-log reduction of Pediococcus sp.

Growth pH does not affect the initial physiological state parameter (pO) of Listeria monocytogenes.

It has proven difficult to develop adequate mathematical models for the lag phase (lambda) which characterizes the adaptation period prior to the initiation of exponential growth by microorganisms. This is due, in part, to our incomplete understanding of the nature of the initial physiological state of cells (defined as h0 or p0 depending on the model), and changes taking place during adaptation. The objectives of the present study were to characterize p0 using data from growth of Listeria monocytogenes in an automated turbidimetric instrument (Bioscreen), and to determine the influence of limiting growth pH. A model was developed for individual cells which combined a continuous adaptation phase (defined by p0) with a discrete step marking the transition to a continuous exponential growth phase (the CDC model). Parameters of the new model were: p0; the specific growth rate (mu); the initial cell number (N0); and the maximum cell density (Nmax). Progressive reduction of the growth pH in the Bioscreen to 4.7 decreased the p. It was noted that the regression lines for all trials at all pH values appeared to have a common x-intercept (20.086+/-1.092), and it was deduced that, when the Bioscreen detection limit (15.07 In cfu well(-1)) was subtracted, the resulting value represented the "true" value for the initial physiological state of the cells.

Proposal of a novel parameter to describe the influence of pH on the lag phase of Listeria monocytogenes.

Predictive models for lag phase duration (lambda) have been less reliable than specific growth rate (mu) models due, in part, to the influence of the pre-growth environment on lambda. A discrete modelling approach was taken to more completely define the response of individual cells to new environments. Time to detection (td) data was obtained from serial twofold dilutions of Listeria monocytogenes growing in a Bioscreen at 30 degrees C. Comparison of the inoculum densities required to achieve maximum td at growth pH values from 7.2 to 4.7 revealed that, as the growth pH decreased, fewer cells were capable of making the transition to the exponential phase. The proportion of these cells (termed "adaptable cells") in the original inoculum was used to define a new parameter (r0) which, when combined with the constant mean individual cell physiological state parameter (p0), the variation in p0 (SDp0), the inital inoculum (N0) and the maximum population density (Nmax) was able to simulate a complete growth curve. Power transformations with rescaled explanatory variables provided suitable models for the influence of pH on mu, r0, and SDp0, (r2>0.70).

Development of a dynamic continuous-discrete-continuous model describing the lag phase of individual bacterial cells.

AIMS: A previous model for adaptation and growth of individual bacterial cells was not dynamic in the lag phase, and could not be used to perform simulations of growth under non-isothermal conditions. The aim of the present study was to advance this model by adding a continuous adaptation step, prior to the discrete step, to form a continuous-discrete-continuous (CDC) model. METHODS AND RESULTS: The revised model uses four parameters: N(0), initial population; N(max), maximum population; p0, mean initial individual cell physiological state; SD(p0), standard deviation of the distribution of individual physiological states. A truncated normal distribution was used to generate tables of distributions to allow fitting of the CDC model to viable count data for Listeria monocytogenes grown at 5 degrees C to 35 degrees C. The p0 values increased with increasing SD(p0) and were, on average, greater than the corresponding population physiological states (h0); p0 and h0 were equivalent for individual cells. CONCLUSION: The CDC model has improved the ability to simulate the behaviour of individual bacterial cells by using a physiological state parameter and a distribution function to handle inter-cell variability. The stages of development of this model indicate the importance of physiological state parameters over the population lag concept, and provide a potential approach for making growth models more mechanistic by incorporating actual physiological events. SIGNIFICANCE AND IMPACT OF THE STUDY: Individual cell behaviour is important in modelling bacterial growth in foods. The CDC model provides a means of improving existing growth models, and increases the value of mathematical modelling to the food industry.

A probability of growth model for Escherichia coli O157:H7 as a function of temperature, pH, acetic acid, and salt.

Data accumulated on the growth of Escherichia coli O157:H7 in tryptic soy broth (TSB) were used to develop a logistic regression model describing the growth-no growth interface as a function of temperature, pH, salt, sucrose, and acetic acid. A fractional factorial design with five factors was used at the following levels: temperature (10 to 30 degrees C), acetic acid (0 to 4%), salt (0.5 to 16.5%), sucrose (0 to 8%), and pH (3.5 to 6.0). A total of 1,820 treatment combinations were used to create the model, which correctly predicted 1,802 (99%) of the points, with 10 false positives and 8 false negatives. Concordance was 99.9%, discordance was 0.1%, and the maximum rescaled R2 value was 0.927. Acetic acid was the factor having the most influence on the growth-no growth interface; addition of as little as 0.5% resulted in an increase in the observed minimum pH for growth from 4.0 to 5.5. Increasing the salt concentration also had a significant effect on the interface; at all acetic acid concentrations, increasing salt increased the minimum temperature at which growth was observed. Using two literature data sets (26 conditions), the logistic model failed to predict growth in only one case. The results of this study suggest that the logistic regression model can be used to make conservative predictions of the growth-no growth interface of E. coli O157:H7.

A comparison of the bioscreen method and microscopy for the determination of lag times of individual cells of listeria monocytogenes.

Lag phase durations (tLag) of individual Listeria monocytogenes cells were analysed using the NightOwl Molecular Imaging System, and results were compared with mean individual cell lag times (tL) obtained from the detection time (td) method using Bioscreen. With Bioscreen, an average tL of 6.39+/-0.89 h was obtained from five separate experiments. With the NightOwl method, an average tLag of 2.73+/-0.06 h was obtained from three experiments consisting of eight total replicates. Lag values from the NightOwl and Bioscreen are related by the equation: tLag = tL + DT, where DT is the doubling time. The equivalent tLag mean value for the Bioscreen method was 7.11+/-0.84 h. Individual lag times measured by both methods were normally distributed (r2 for Bioscreen and NightOwl ranged from 0.951 to 0.999 and from 0.884 to 0.982, respectively). The results suggest that the NightOwl method can provide accurate estimates of individual cell lag times, which will facilitate the development of combined discrete continuous models for bacterial growth.

A combined discrete-continuous model describing the lag phase of Listeria monocytogenes.

Food microbiologists generally use continuous sigmoidal functions such as the empirical Gompertz equation to obtain the kinetic parameters specific growth rate (mu) and lag phase duration (lambda) from bacterial growth curves. This approach yields reliable information on mu; however, values for lambda are difficult to determine accurately due, in part, to our poor understanding of the physiological events taking place during adaptation of cells to new environments. Existing models also assume a homogeneous population of cells, thus there is a need to develop discrete event models which can account for the behavior of individual cells. Time to detection (t(d)) values were determined for Listeria monocytogenes using an automated turbidimetric instrument, and used to calculate mu. Mean individual cell lag times (tL) were calculated as the difference between the observed t(d) and the theoretical value estimated using mu. Variability in tL for individual cells in replicate wells was estimated using serial dilutions. A discrete stochastic model was applied to the individual cells, and combined with a deterministic population-level growth model. This discrete-continuous model incorporating tL and the variability in tL (expressed as standard deviation; S.D.(L)) predicted a reduced variability between wells with increased number of cells per well, in agreement with experimental findings. By combining the discrete adaptation step with a continuous growth function it was possible to generate a model which accurately described the transition from lag to exponential phase. This new model may serve as a useful tool for describing individual cell behavior, and thus increasing our knowledge of events occurring during the lag phase.

Growth and survival of various strains of enterohemorrhagic Escherichia coli in hydrochloric and acetic acid.

Nineteen strains of enterohemorrhagic Escherichia coli isolated from humans and foods were examined for their ability to grow and survive at low pH in organic (acetic) and mineral (HCl) acids. Strains were subcultured in tryptic soy broth adjusted to various pH values (3.75 to 4.75 for HCl and 4.75 to 5.75 for acetic acid) and incubated for 72 h at 37 degrees C to determine the minimum growth pH value. Minimum pH values for growth of 4.25 and 5.5 were found for HCl and acetic acid, respectively. Strains were also exposed to pH 2.0 (HCl) and pH 4.0 (acetic acid) for up to 24 h at 37 degrees C to assess their ability to survive. HCl was a more effective inhibitor after 6 h of exposure, whereas acetic acid was more effective after 24 h. Outbreak strains survived acid treatment significantly (P < or = 0.05) better than strains isolated from fermented or high-pH foods or animal or human isolates. Significant (P < or = 0.05) differences among serotypes and between O157:H7 and other serotypes were apparent after 3 or 6 h of exposure to acids.

Nisin reduces the thermal resistance of Listeria monocytogenes Scott A in liquid whole egg.

D-values (decimal reduction times) and z-values (increase in temperature required for a 1-log change in D-value) for Listeria monocytogenes Scott A were determined in liquid whole egg with nisin (0 or 10 microg ml(-1)) and NaCl (0 or 10%) by a submerged glass ampoule procedure. Samples were plated onto nonselective agar at appropriate intervals, and D-values were determined using a modified biphasic logistic equation. Addition of NaCl increased D-values at all temperatures tested. The addition of nisin to unsalted liquid whole egg resulted in a rapid 4-log reduction in viable counts within the first hour. Nisin significantly (P < or = 0.05) decreased D-values at lower (< 58 degrees C) temperatures in both unsalted and salted liquid whole egg but had little effect on the D-values at current minimum U.S. and Canadian pasteurization temperatures (60 degrees C without NaCl; 63 degrees C with NaCl). However, when nisin was added 2 h prior to heat treatment, D-values were significantly (P < or = 0.05) reduced at these temperatures. Inhibitory levels of nisin were detected in the liquid whole egg postpasteurization. Nisin could have a favorable impact on the control of L. monocytogenes in pasteurized liquid egg products.

Influence of guar gum on the thermal stability of Listeria innocua, Listeria monocytogenes, and gamma-glutamyl transpeptidase during high-temperature short-time pasteurization of bovine milk.

Mathematical models describing the thermal inactivation of gamma-glutamyl transpeptidase (TP) and Listeria innocua in milk during high-temperature short-time (HTST) pasteurization were validated with data from TP, L. innocua, and L. monocytogenes trials in guar gum-milk. Holding tube times were determined for turbulent flow using water, and for laminar flow using a guar gum (0.27% wt/wt)-sucrose (5.3% wt/wt)-water mixture. Inactivation of TP and L. innocua was lower in a solution of guar gum (0.25% wt/wt) in whole milk than was predicted by models derived from studies with whole milk alone. Use of laminar flow timings improved model fit but did not completely account for the observed protective effect. L. monocytogenes survival was close to that predicted by the L. innocua model, although some protection was afforded this pathogen under laminar flow. Considerable intertrial variability was noted for L. monocytogenes. Risk analysis simulations using @RISK, a Lotus 1-2-3W add-in, were used to account for intertrial variability. Simulated log10 %reductions consistently underpredicted experimental L. monocytogenes survival (fail-safe), thus the L. innocua model derived in milk is suitable for estimating L. monocytogenes survival in viscous products. Increased thermal tolerance during laminar flow may be attributed to the protective effect of stabilizer.

Predictive modelling of inactivation of Listeria spp. in bovine milk during high-temperature short-time pasteurization.

A linear model was derived to describe the thermal inactivation of Listeria innocua in bovine whole milk in a high-temperature short-time pilot scale pasteurizer. Integrated lethal effect, or pasteurization effect (PE), was obtained by converting times at different temperatures in the various sections of the pasteurizer to the equivalent time at the reference temperature (72 degrees C). PE was then related by a simple linear function to the log10 of the % viable counts with a power transformation of the PE values to improve the linear fit. R2 values for the five L. innocua trials varied from 0.728 to 0.974. Validation of this model with Listeria monocytogenes confirmed that L. monocytogenes was more heat sensitive. Inter-trial variation was incorporated into the model using the @RISK simulation software. Output from simulations confirmed that pasteurization at the IDF standard conditions of 72 degrees C for 15 sec can ensure at least an 11-log reduction of L. monocytogenes. The results showed that L. innocua may be used as a model microorganism to assess the thermal inactivation of L. monocytogenes, since its heat resistance is at least equal to or greater than that of the pathogenic species.

A heterogeneous population model for the analysis of bacterial growth kinetics.

A two-compartment, heterogeneous population model (HPM) was derived using the simulation software SB ModelMaker to describe the growth of Listeria monocytogenes in bacteriological media at 5-35 degrees C. The model assumed that, at time t = 0, the inoculum was distributed between two distinct compartments, Non-Growing and Growing, and that growth could be described by four parameters: initial total cell population (N0), final maximum cell population (Nmax), maximum specific growth rate (mu(max)), and initial cell population in the Growing compartment (G0). The model was fitted to the data by optimizing the four parameters, and lag phase duration (lambda) was calculated. The resulting values of mu(max) and lambda were similar to those determined using the modified Gompertz equation. A new parameter, w0, was defined which relates to the proportion of the initial cell population capable of growth, and is a measure of the initial physiological state of the cells. A modified model in which mu(max) was replaced with a temperature function, and w0 replaced G0, was used to predict the effect of temperature on the growth of L. monocytogenes. The results of this study raise questions concerning the current definition of the lag phase.

Influence of unsliced delicatessen meat freshness upon bacterial growth in subsequently prepared vacuum packed slices.

Unsliced beef pastrami, reformulated ham and bologna held at 6 degrees C were sliced 21, 17, 12 or 7 days before or at the assigned manufacturer's best before date and vacuum packaged. Packages of sliced meats were held at 6 degrees C for another 7, 12, 17 or 21 days, opened and analyses made for total bacteria, lactic acid bacteria, Enterobacteriaceae and Brochothrix thermosphacta. The maximum storage interval was 42 days; half this period unsliced, the remainder as repacked slices. Numbers of bacteria on pastrami were significantly greater than on ham and bologna (pastrami > ham > bologna) with the lactic acid bacteria dominating in all products. As unsliced meats approached their best before date, insignificant increases were generally noted for numbers of lactic bacteria, Enterobacteriaceae and B. thermosphacta. During subsequent storage of slices under vacuum, numbers of total and lactic bacteria increased exponentially at the same rate while B. thermosphacta growth was significantly slower. Numbers of Enterobacteriaceae remained low and were essentially unchanged during sliced meat storage. Within the context of study storage parameters, shelf-life appeared to be determined by length of time after slicing and repackaging rather than by best before date of the unsliced meat. Packages of sliced meat prepared from wholesale unsliced meats with 21 days left until their best before date or from unsliced meats with 12 days left until the best before date showed similar bacterial levels 21 days later. It was probable that the localization of bacterial growth at the meat surface-packaging film interface of the unsliced meats yielded slices with initially lowered bacterial content when repackaged and sampled from the uppermost slice. When Enterobacteriaceae and B. thermosphacta were absent from unsliced meats, extension of sliced meat package shelf-life beyond the best before date of the parent meat was possible. However, these bacterial groups were not always undetected.