Improvement of methods for the detection of Gram-negative foodborne pathogens
Margot, H.F.T. - \ 2016
Wageningen University. Promotor(en): Marcel Zwietering; Han Joosten, co-promotor(en): R. Stephan. - Wageningen : Wageningen University - ISBN 9789462578708 - 158
gram negative bacteria - pathogens - foodborne pathogens - detection - real time pcr - salmonella - escherichia coli - mung beans - gramnegatieve bacteriën - pathogenen - voedselpathogenen - detectie - real time pcr - salmonella - escherichia coli - mungbonen
Foodborne diseases are a major source of morbidity and mortality worldwide. In most cases, these diseases are caused by contaminated food products, but transmission can also subsequently occur via person to person contact. The ability to detect the pathogens is an important aspect in the verification of food safety. A major proportion of foodborne disease is caused by Gram-negative bacteria. In this thesis, the detection of Gram-negative foodborne pathogens is addressed by looking at the successive steps from enrichment to detection with Salmonella, Shiga toxin-producing E. coli and Cronobacter spp. as example pathogens. The detection of foodborne pathogens using microbiological culture media aiming at the resuscitation and growth of bacteria is still regarded as the gold standard and included in many reference methods. However, cultural methods are time and labour-intensive. Since an immediate response is required in case of contamination and during outbreaks there is a strong interest in methods that deliver information on the microbiological status of the product as quickly and reliable as possible. Rapid cultural methods and commercially available real-time PCR systems for the detection of Salmonella and STEC were compared with regards to their sensitivity and specificity. It was shown that most of the marketed systems are as reliable as the standard methods. However, false-positive results were obtained with real-time PCR systems for the detection of Salmonella. Rapid cultural methods that were based on procedures without the pre-enrichment step, reduced the time to detection but did show some ambiguous results with difficult matrices such as tea. Of the seven rapid tests for the detection of STEC, one did not detect relevant Stx subtypes.
In order to be detected, pathogens need to multiply to reach a minimum threshold level. However, because they are often sublethally injured due to hostile processing and storage conditions, they first need to be resuscitated. For most pathogens, (Salmonella, STEC and Cronobacter spp.) the first step in the detection is an enrichment including resuscitation in a non-selective medium such as BPW. Modifications to BPW were compared with respect to their ability to promote growth of unstressed and stressed Gram-negative pathogens. The aim was to develop a medium that could be used for the enrichment of pathogens in horizontal methods using only one enrichment step. The resuscitation of stressed Cronobacter cells was improved in BPW supplemented with an additional iron source and sodium pyruvate along with low levels of compounds for the inhibition of Gram-positive bacteria. However, it was observed that BPW containing these supplements allowed for less resuscitation of STEC when compared to regular BPW. Based on these results it was concluded that the application of a one-broth enrichment in food products with a high number of competing bacteria is not recommended due to the overgrowth of the target bacteria. Limitations of the current method for the detection of STEC from sprouted seeds were noticed. Therefore, the growth of stressed STEC cells from different serotypes was assessed in media used for the enrichment of Enterobacteriaceae. In addition, the growth of STEC was examined in the enrichment of sprouts using different media and incubation temperatures. It was shown that the high level of competitors was inhibiting the detection of the target pathogen and that the similarity of target and competing bacteria prevents the design of a selective enrichment procedure. In order to get a better insight in the enrichment ecology, the microbiome of mungo bean sprouts was analysed using Illumina HiSeq sequencing prior to and during the enrichment in BPW and EE-broth at different temperatures. The majority of the sprout flora was composed of bacteria belonging to the phylum Proteobacteria. Enrichment in BPW increased the proportion of Firmicutes whereas the incubation in EE-broth enriched Proteobacteria. The results point out that with the application of a selective medium like EE-broth, growth of the competitive microflora that complicates the detection of STEC is promoted. It was shown that EE-broth also resulted in good growth of STEC however, the problematic situation of low maximum population densities of the target strain in the matrix is still present. The probability of detection is not only influenced by the natural flora of a food product, but also by the physiological state of the pathogen. The influence of stress on the lag time of single cells and the resulting probability of detection were determined for Cronobacter spp. in powdered infant formula. Lag time was calculated from optical density measurement data and different scenarios were modelled. Lag time was longest after acid stress and lag time increase coincided with increased lag time variability. The probability of detection, however, depended both on the sampling plan and on the duration of the lag phase.
This thesis provides a critical evaluation of rapid methods and valuable new insights on enrichment procedures, the role of competitors in bacterial enrichment procedures and the limitations of selective agents. This information will be of great help to further improve microbiological methods and thereby contribute to more effective management of food safety.
Energetics and mechanisms of phosphate transport in Acinetobacter johnsonii
Veen, H.W. van - \ 1994
Agricultural University. Promotor(en): A.J.B. Zehnder; W.N. Konings; G.J.J. Kortstee. - S.l. : Van Veen - 212
gramnegatieve bacteriën - micro-organismen - biochemie - metabolisme - synthese - fosfaten - fosforpentoxide - derivaten - gram negative bacteria - microorganisms - biochemistry - metabolism - synthesis - phosphates - phosphorus pentoxide - derivatives - cum laude
The biological removal of phosphorus from waste water is an attractive method to control eutrophication of surface waters. The process is currently perceived to depend on the provision of alternate stages in which the activated sludge is subjected to anaerobic and aerobic conditions. A characteristic feature of such plant is that P i , after being released from biomass in the anaerobic stage, is reincorporated into biomass during aeration, together with part or all of the influent P i . Analysis of the population structure of activated sludge has focussed attention on the strictly aerobic, gram-negative genus Acinetobacter as being one of the important genera in enhanced biological phosphorus removal. However, due to the lack of insight into the relevant physiological processes in these microorganisms our understanding of the mechanisms of enhanced biological phosphorus removal is only superficial.
The project of this thesis was initiated to study the mechanisms and regulation of P i uptake and efflux in the polyphosphate-accumulating Acinetobacter johnsonii 210A. The nature of polyphosphates and the enzymology of their metabolism have been a subject of previous studies with A.johnsonii 210A and other Acinetobacter spp. Chapter 1 presents a review of these investigations and those concerning the molecular mechanisms of P i transport in prokaryotes. The results described in this thesis show that A.johnsonii 210A is well adapted to the environmental conditions encountered in activated sludge plants through (i) the efficient acquisition of the predominant P i species from its habitat, and (ii) the ability to survive prolonged periods of anaerobiosis, by using polyphosphate as a source of metabolic energy when oxidative phosphorylation is impaired.
P i is taken up in A.johnsonii 210A against a concentration gradient by energydependent, carrier-mediated processes (Chapter 2). Kinetic analysis of P i uptake in cells grown under P, limitation, revealed the presence of two P i transport systems with an apparent Kt for P i of 0.7 and 9 μM. The high-affinity permease could be classified as an ATP- and periplasmic binding protein-dependent P i uptake system. Induction of this system under P i limitation, and the ability to maintain a low internal P i by the synthesis of polyphosphate enable the organism to reduce the P i concentration in the environment to micromolar levels. The low-affinity system is a constitutive secondary P i transport system involved in P i uptake and efflux.
P i transport via the secondary transport system was studied in membrane vesicles and proteoliposomes in which the carrier protein was successfully reconstituted (Chapter 3). These model systems allow detailed studies on the mechanism of P i transport without the interference of polyphosphate metabolism or other cellular processes. P i uptake is strongly dependent on the presence of divalent metal ions, such as Mg 2+, Ca 2+, Mn 2+, or Co 2+. These cations form a MeHPO 4 complex with up to 87% of the P i present in the incubation mixtures, suggesting that divalent cations and P i are cotransported via aMeHPO 4 complex. MeHPO 4 uptake is driven by the proton motive force with an mechanistic MeHPO 4 /H +stoichiometry of one. The pH dependence of various modes of facilitated diffusion processes, such as efflux, exchange, and counterflow catalyzed by the MeHPO 4 carrier suggests that H +and MeHPO4 binding and release to and from the carrier protein occur via an ordered mechanism.
In view of the similarities between P i transport in cells of A.johnsonii 210A and Escherichia coli, a more extensively studied organism (Chapter 2), the mechanism and energetics of the phosphate inorganic transport (Pit) system of E. coli were investigated (Chapter 4). P i and metal transport studies in proteoliposomes containing reconstituted Pit protein identified Pit as a MeHPO 4 /H +symport system. The effects of pH and the proton motive force on the different modes of MeHPO 4 transport are consistent with the ordered binding model proposed for the MeHPO 4 transporter in A. johnsonii 210A.
Chapter 5 describes the substrate specificity of the two P i transport systems in A. johnsonii 210A in relation to P i speciation in the aquatic environment. In natural waters and domestic waste water in which divalent metal ions are present in excess of P i , the species H 2 PO4-, HPO42-and MeHPO 4 prevail at physiological pH values for Acinetobacter (pH 5.5 to 8.0). The transport of MeHPO 4 by the secondary P i transport system is demonstrated in proteoliposomes by the (i) divalent cation dependent uptake and efflux of P i , (ii) P i -dependent uptake of Ca 2+and Mg 2+, (iii) equimolar transport of P i and Ca 2+, and (iv) inhibition by Mg 2+of Ca 2+uptake in the presence of P i , but not of P i uptake in the presence of Ca 2+.The transport of MeHPO 4 is closely related to the metabolism of cytoplasmic polyphosphate granules in which P i and divalent cations are accumulated. H 2 PO4-and HPO42-are translocated by the primary P i uptake system. P i uptake, but not MeHPO 4 uptake, was stimulated in cells under P i limitation. The periplasmic P i -binding protein showed affinity for H 2 PO4-and HPO42-, but not for MeHPO 4 .
Chapter 6 demonstrates the presence of high-affinity secondary transport systems for L-lysine, L-alanine and L-proline in A. johnsonii 210A. The lysine and alanine carriers translocate their solute in symport with one proton. In contrast, the proline carrier is strictly dependent on the presence of Na +ions and mediates Na +/proline symport. The low internal Na +concentration, necessary for optimal proline uptake, is achieved by a sodium/proton antiporter. High-affinity systems will enable the organism to scavenge the environment for traces of metabolizable substrates and to recapture endogenous compounds leaking out of the cell.
Retention of metabolites will become particularly important for survival when oxidative phosphorylation is impaired in A.johnsonii 210A. In Chapter 7, evidence is presented for the ability of the organism (i) to use polyphosphate as a source of metabolic energy during anaerobiosis, (ii) to maintain a considerable, outwardly directed MeHPO4 gradient across the cytoplasmic membrane during the degradation of polyphosphate, and (iii) to generate a proton motive force by the excretion of MeHPO 4 and H +via the MeHPO 4 carrier. This MeHPO 4 efflux-induced proton motive force can drive energy- requiring processes such as the accumulation of lysine and proline, and the synthesis of ATP. Conservation of metabolic energy from polyphosphate degradation may enable A. johnsonii 210A to survive alternating aerobic/anaerobic conditions as encountered in natural habitats and wastewater treatment plants.
The significance of the here described findings for the cotransport of P i and divalent metal ions across biomembranes and the recycling of metabolic energy in microorganisms by the excretion of inorganic endproducts is discussed in Chapter 8.
Nitrogen fixation (Acetylene reduction) in the sediments of the pluss-see : with special attention to the role of sedimentation
Blauw, T.S. - \ 1987
Agricultural University. Promotor(en): E.G. Mulder, co-promotor(en): H.J. Overbeek. - S.l. : Blauw - 199
meren - reservoirs - plassen - water - sediment - microbiologie - stikstofkringloop - nitrificatie - gramnegatieve bacteriën - bondsrepubliek duitsland - acetyleenreductie - lakes - reservoirs - ponds - water - sediment - microbiology - nitrogen cycle - nitrification - gram negative bacteria - german federal republic - acetylene reduction
Sediments of productive lakes are usually rich in organic matter and, except for a thin surficial layer, anaerobic. These conditions seem to be favourable for heterotrophic nitrogen fixation. However, these sediments also contain relatively high ammonium concentrations. Ammonium represses the synthesis of the enzyme nitrogenase. Moreover, ammonium inhibits the activity of the enzyme in aerobic nitrogen fixers. These effects of ammonium seem to be functional. Nitrogen fixation is a highly endergonic process. Therefore it is more economic to use combined nitrogen (e.g. ammonium) than atmospheric nitrogen as a nitrogen source. Nevertheless, a number of workers have detected nitrogen fixing activity in ammonium rich sediments.
In the present investigation the significance of heterotrophic nitrogen fixation in the sediments or the nitrogen economy of the Pluss-See has been studied. Special attention has been paid to the role of organic matter supply and ammonium.
The surface area of the lake is 14 ha, the maximum depth is 29 m. Every year a stable thermal stratification develops in the lake, usually with an anaerobic hypolimnion.
The problem of nitrogen fixation in lake sediments has been approached in three ways:
Laboratory studies (chapters 3, 4 and 5).
Nitrogen fixating activity was measured with the acetylene reduction assay. One of the requirements for this assay, the saturation of nitrogenase with acetylene, was not met (figure 5), because nitrogen fixation apparently occured in protected microsites with poor accessibility for acetylene (3.2.2). This protection may have also consequences for the measurement of nitrogen fixation with 15N 2 (3.2.3).
Nitrogenase activity of the sediments was stimulated by the addition of organic substrates, as mannitol, glucose, fructose etc., suggesting that the activity of nitrogenase in situ in these sediments was limited by the availability of organic substrate (4-2). This suggestion is affirmed by the absence of a discontinuity in the Arrhenius plot (figure 17b).
Upon addition of organic substrate to the sediments two phases could be observed (figure 9). In the first phase acetylene reducing activity is constant, but higher than in control sediments. In this phase the activity of the already present nitrogenase is stimulated (increase of the actual activity). In the second phase, after the interstitial ammonium concentration has dropped below a certain threshold-value, the synthesis of nitrogenase is derepressed and an exponential increase of nitrogenase activity can be observed (increase of the potential activity). Because nitrogenase synthesis in situ had to be assumed above the derepression threshold, the conclusion was drawn that the dissolved ammonium concentration within the protected microsites was lower than in the bulk of the sediments (4.3). Apparently nitrogen fixation occures in ammonium rich sediments, because nitrogen fixers are not in contact with these high concentrations.
Field observations; relation between the nitrogenase activity and some other properties of the sediments (chapters 6 and 7).
During a year nitrogenase activity and some other characteristics of the sediments were measured at three stations in the lake: in the littoral sediments at 5 m water depth and in the profundal sediments at 15 and 29 m water depth. Highest nitrogenase activity was measured at the sediment surface at the deepest part of the lake (figure 27). Especially in the winter period very high rates were observed (figure 28). In the sediments at the deepest part of the lake the yearly fixed amount of nitrogen was estimated to be 0.24-1.10 g.m -2, depending on the conversion factor used. In the shallower regions this amount was estimated to be 0.15-0.77 g N.m -2.
Acetylene reduction in the littoral sediments was correlated with temperature (table II). In the profundal sediments no significant temperature variation could be observed. Acetylene reducing activity in the profundal sediments was correlated with the C/N ratio (figure 33), which could be shown to be an index for substrate availability in these sediments. In both littoral and profundal sediments acetylene reducing activity was highly significantly correlated with the maximum glucose uptake velocity (V m ) of the heterotrophic population in the sediments (figure 41). The repression-derepression threshold of the interstitial ammonium concentration could be observed under natural conditions (figure 33). Acetylene reducing rates were higher at ammonium concentrations below this threshold. The ammonium adsorption coefficient (Ke) of the sediments seemed to be more important for the acetylene reducing activity than the ammonium concentration it self (table II). This finding suggests that the dissolved ammonium concentration in the protected microsites is lowered by adsorption.
Field observations; the relation between nitrogen fixation in the sediments and the sedimentation of suspended matter (chapters 8, 9 and 10).
Sedimentation of particulate organic matter was measured at the three stations. The measured rates were corrected for resuspension using the differences in carbon content between the settling particulate material and the carbon content of the surficial sediments (8.4; figure 47). Eight percent of the primary production reached the bottom at the deepest part of the lake (table VI). Redistribution of sediments resulting in sediment focusing is important in the lake. Both intermittent complete mixing and sliding of sediments on slopes are important for the focusing process. A correlation between sedimentation and acetylene reducing activity could be observed in the littoral sediments and at the deepest part of the lake (figure 63). No correlation was found at 15 m water depth. Only at the deepest part of the lake a correlation was found between the sedimentation and both the C/N ratio and Ke of the sediments (figure 61 and 62). These correlations and non- correlations could be explained by the transport of sediments within the lake, described by a simple focusing model (8.7). Using this model the efficiency of nitrogen fixation under natural conditions could be estimated to be high compared to the efficiency measured in pure and enrichment cultures (8.10).
Also using this model it could be shown (10) that nitrogen fixation may be important to the nitrogen economy of the sediments but not for the nitrogen economy of the whole lake. Nitrogen fixation is expected to be more important in lakes with a larger proportion of the primary production reaching the bottom.
Denitrification, activity of bacterial flocs, and growth of a filamentous bacterium in relation with the bulking of activated sludge
Krul, J.M. - \ 1978
Landbouwhogeschool Wageningen. Promotor(en): E.G. Mulder. - Wageningen : [s.n.] - 64
rioolslib - samenstelling - denitrificatie - gramnegatieve bacteriën - micro-organismen - bacteriën - classificatie - taxonomie - sewage sludge - composition - denitrification - gram negative bacteria - microorganisms - bacteria - classification - taxonomy
Activated sludge with poor settling characteristics is caught under the term "bulking" sludge. Various types of bulking activated sludge can be distinguished.
1) Sludge containing an abundance of filamentous microorganisms.
2) Sludge, characterized by excessive amount of bacterial slime.
3) Flotating sludge caused by denitrification.The excessive growth of filamentous bacteria, sometimes occurring in activated sludge plants, is difficult to understand. One of the hypotheses is that the filamentous bacteria protruding from floes have a better competitive ability as compared with unicellular microorganisms which occur mostly in floes, in which the growth conditions are assumed to affect adversely the development of these bacteria.As activated sludge floes are difficultly to dispers without damaging the cells, it was decided to study the effect of the aggregative condition on the bacterial activity by using floc-forming strains of which the floes were easily dispersable by adding the enzyme cellulase. Most experiments were carried out with Zoogloea ramigera, strain I-16-M, and an Alcaligenes sp., strain 15.As floc formation turned out to be a rather unreproducible and unpredictable phenomenon, it was often difficult to obtain sufficient amounts of floes. For this reason it was necessary to study floc formation by some pure cultures. In chapter I it was shown that floc formation of Zoogloea ramigera can be strongly stimulated by adding small amounts of NaCl or Na 2 HPO 4 /NaH 2 PO 4 to suspended cells. Unless the cells were very old, the growth phase of the culture was unimportant in respect to flee formation. The stimulatory effect of the salts could not satisfactorily be explained.By studying the activities of cells in floes and in suspension, it was shown (chapter II) that the oxygen uptake rate of cells in floes and of cells in suspension, in the presence of a high level of substrate, represents a zero order reaction above oxygen concentrations in the medium of approx. 1.5 ppm. and approx. 0.1 ppm., respectively. In activated sludge, where only part of the floes consists of living bacteria, the rate-limiting oxygen concentration is approx. 0.5 ppm. Glucose respiration becomes rate-limiting when the glucose concentration is below approx. 20 ppm. with cells in floes and below approx. 8 ppm. with cells in suspension. In the same chapter it was shown that the protein and poly-β-hydroxybutyrate syntheses of cells in floes of slowly shaken cultures were strongly retarded as compared with these activities in cells occurring in suspension.The rate-limiting concentrations of oxygen and substrate (glucose) for the uptake of these compounds by cells in flocs are in the same range as the values found in many reactors of activated sludge plants.To compare the competitive ability of Haliscomenobacter hydrossis, a filamentous bacterium, with that of the unicellular Zoogloea ramigera, continuous culture experiments were carried out. Zoogloea ramigera was the most abundant organism present in the mixed cultures at all tested dilution rates (using a complex medium). The filamentous microorganism was shown to have a low affinity towards the uptake of glucose. Both, amino acids and glucose were used as energy and C sources by this organism (chapter III).Floc flotation, caused by N 2 gas bubbles resulting from the dissimilatory reduction of nitrate, is another aspect of the phenomenon of bulking sludge. Denitrification occurs only under anaerobic conditions. These conditions were shown to occur within flocs at relatively low oxygen concentrations of the surrounding medium. In chapter IV dissimilatory nitrate reduction in the flocs of the Alcaligenes sp. strain 15, was shown to take place as soon as the supply of oxygen within parts of the flocs was inadequate. By studying the denitrifying ability of this strain, the dissimilatory nitrate reduction turned out to be even independent of the presence of oxygen when the cells had been subjected to a special treatment. Upon aerobic precultivation of strain 15 with NH4+-N or NO3--N as nitrogen source, aerobic nitrate respiration of the washed cells was measured after nitrate addition as soon as the oxygen uptake was inhibited. This inhibition was caused by NO, the reduction
product of nitrite (chapter V). The same aerobic dissimilatory nitrate reduction was observed with anaerobically precultivated cells which afterwards had been aerated for some hours in a nitrogen-free medium. It was shown that in these cells during
the aeration period the NO reductase and N 2 O reductase were inactivated/broken down giving rise to the accumulation of NO and N 2 O during subsequent anaerobic nitrate respiration. When the cells were re-aerated, even very small amounts of the produced NO were sufficient to inhibit the oxygen uptake, resulting in a continued aerobic nitrate respiration.This type of aerobic dissimilatory nitrate reduction as found with strain 15 was also found with a number of different denitrifying strains, isolated from activated sludge. The oxygen uptake of activated sludge itself could not be inhibited for a long time upon addition of NO as the presence of many NO-reducing bacteria (denitrifying bacteria) readily removed the inhibiting agent (chapter V).In the aeration tank of an activated sludge plant, the oxygen concentration sometimes drops temporarily to such a level that the interior parts of the flocs become anaerobic. Denitrification in these anaerobic innerparts starts only when the denitrifying bacteria present possess the enzymes for the reduction reactions. In chapter VI it was proved that a number of denitrifying bacteria have the ability to synthesize the dissimilatory nitrate reductase in the presence of oxygen and ammonium ions so that these bacteria are able to start dissimilatory nitrate reduction as soon as all the oxygen has been consumed.A more detailed study of the "constitutive" character of the synthesis of the dissimilatory nitrate reductase in strain 15 and another denitrifying strain, N4, showed that the synthesis of the enzyme by strain 15 was strongly repressed when the growth medium was over-satisfied with oxygen or upon the addition of chlorate. The synthesis of the dissimilatory nitrate reductase in strain N4 was hardly affected by the mentioned repressing agents. The repressing effect of chlorate on the synthesis of the dissimilatory nitrate reductase in cells of strain 15 was explained by the redox potential-increasing effect of this agent assuming that the redox potential is the regulating factor in the synthesis of this enzyme, rather than oxygen.