Mathematical modeling of population dynamics of unstable plasmid-containing bacteria during continuous cultivation in a chemostat

A structural approach to studying the regularities of the population dynamics of unstable recombinant bacterial strains in a chemostat was elaborated. The approach is based on the mathematical modeling of cell distribution in a population with different numbers of plasmid copies. The effect of decreased selective preference of plasmidless variants of the recombinant strain in the chemostat, which is related to a decrease in the number of plasmid copies in cells upon long-term incubation was analyzed. It is shown that the time of half-elimination of plasmids from the bacterial population in the steady state in the chemostat T1/2 does not depend on the maximum number of plasmid copies in cells N but is determined only by the mean time of generation g and the probability of the loss of one plasmid copy tau. The dependence of the preference of bacterial plasmidless variants on the efficiency of expression of genes cloned into plasmids in chemostat was analyzed using the recombinant strain E. coli Z905, whose plasmids pPHL-7 contain cloned genes for the luminescence system of marine luminescing bacteria Photobacterium leiognathi.     

Plate Colony Sample Sizes Required for Inferences about the Uniqueness of a Lactobacillus Strain within a Human Volunteer

Making inferences about the spectrum of bacterial strains on a plate as a consequence of observing only one strain in a random sample of colonies from the plate is relatively straightforward whereas making inferences about the spectrum of strains within a human volunteer as a consequence of observing only one strain in a random sample from the plate requires some care. The issue of the appropriate number of colonies to sample from a plate to make inferences about strain uniqueness within a human volunteer is examined and illustrated with an example     

Bacterial O-methylation of halogen-substituted phenols

Two strains of bacteria capable of carrying out the O-methylation of phenolic compounds, one from the gram-positive genus Rhodococcus and one from the gram-negative genus Acinetobacter, were used to examine the O-methylation of phenols carrying fluoro-, chloro-, and bromo-substituents. Zero-order rates of O-methylation were calculated from data for the chloro- and bromophenols; there was no simple relationship between the rates of reaction and the structure of the substrates, and significant differences were observed in the responses of the two test organisms. For the gram-negative strain, the pattern of substitution was as important as the number of substituents. Hexachlorophene was resistant to O-methylation by both strains, and tetrabromobisphenol-A was O-methylated only by the gram-positive strain. It is suggested that in the natural environment, bacterial O-methylation of phenols carrying electron-attracting substituents might be a significant alternative to biodegradation.     

Bacterial O-methylation of halogen-substituted phenols.

Two strains of bacteria capable of carrying out the O-methylation of phenolic compounds, one from the gram-positive genus Rhodococcus and one from the gram-negative genus Acinetobacter, were used to examine the O-methylation of phenols carrying fluoro-, chloro-, and bromo-substituents. Zero-order rates of O-methylation were calculated from data for the chloro- and bromophenols; there was no simple relationship between the rates of reaction and the structure of the substrates, and significant differences were observed in the responses of the two test organisms. For the gram-negative strain, the pattern of substitution was as important as the number of substituents. Hexachlorophene was resistant to O-methylation by both strains, and tetrabromobisphenol-A was O-methylated only by the gram-positive strain. It is suggested that in the natural environment, bacterial O-methylation of phenols carrying electron-attracting substituents might be a significant alternative to biodegradation.     

A Model To Estimate the Optimal Sample Size for Microbiological Surveys

Estimating optimal sample size for microbiological surveys is a challenge for laboratory managers. When insufficient sampling is conducted, biased inferences are likely; however, when excessive sampling is conducted valuable laboratory resources are wasted. This report presents a statistical model for the estimation of the sample size appropriate for the accurate identification of the bacterial subtypes of interest in a specimen. This applied model for microbiology laboratory use is based on a Bayesian mode of inference, which combines two inputs: (ii) a prespecified estimate, or prior distribution statement, based on available scientific knowledge and (ii) observed data. The specific inputs for the model are a prior distribution statement of the number of strains per specimen provided by an informed microbiologist and data from a microbiological survey indicating the number of strains per specimen. The model output is an updated probability distribution of strains per specimen, which can be used to estimate the probability of observing all strains present according to the number of colonies that are sampled. In this report two scenarios that illustrate the use of the model to estimate bacterial colony sample size requirements are presented. In the first scenario, bacterial colony sample size is estimated to correctly identify Campylobacter amplified restriction fragment length polymorphism types on broiler carcasses. The second scenario estimates bacterial colony sample size to correctly identify Salmonella enterica serotype Enteritidis phage types in fecal drag swabs from egg-laying poultry flocks. An advantage of the model is that as updated inputs from ongoing surveys are incorporated into the model, increasingly precise sample size estimates are likely to be made.     

A model to estimate the optimal sample size for microbiological surveys

Estimating optimal sample size for microbiological surveys is a challenge for laboratory managers. When insufficient sampling is conducted, biased inferences are likely; however, when excessive sampling is conducted valuable laboratory resources are wasted. This report presents a statistical model for the estimation of the sample size appropriate for the accurate identification of the bacterial subtypes of interest in a specimen. This applied model for microbiology laboratory use is based on a Bayesian mode of inference, which combines two inputs: (ii) a prespecified estimate, or prior distribution statement, based on available scientific knowledge and (ii) observed data. The specific inputs for the model are a prior distribution statement of the number of strains per specimen provided by an informed microbiologist and data from a microbiological survey indicating the number of strains per specimen. The model output is an updated probability distribution of strains per specimen, which can be used to estimate the probability of observing all strains present according to the number of colonies that are sampled. In this report two scenarios that illustrate the use of the model to estimate bacterial colony sample size requirements are presented. In the first scenario, bacterial colony sample size is estimated to correctly identify Campylobacter amplified restriction fragment length polymorphism types on broiler carcasses. The second scenario estimates bacterial colony sample size to correctly identify Salmonella enterica serotype Enteritidis phage types in fecal drag swabs from egg-laying poultry flocks. An advantage of the model is that as updated inputs from ongoing surveys are incorporated into the model, increasingly precise sample size estimates are likely to be made.     

Multivariate analysis of bacterial volatile compound profiles for discrimination between selected species and strains in vitro

Volatile compounds (VCs) are produced by all microorganisms as part of their normal metabolism. The aim of this study was to determine whether bacterial VC profiles could be used to discriminate between selected bacterial species and strains in vitro.Selected Ion Flow Tube Mass Spectrometry (SIFT-MS) was used to quantify the concentration of 23 microbial VCs within the head-space of various bacterial monocultures, during both the logarithmic and stationary growth phases. In comparison with existing techniques, SIFT-MS enables quantitative, high throughput, real-time head-space analysis to be performed, without need for sample preparation. The results show that most VCs were produced by > 1 bacterial species or strain, and some were produced by all strains tested. Multivariate analysis using similarity matrices, cluster analysis and multidimensional scaling (MDS) was used to determine whether there was a characteristic VC profile at either the species or strain level. Significant discrimination of all bacterial species and strains was achieved by analysing the VC profiles, and the relative similarity of VC profiles could be differentiated in 2 or 3 dimensional space. This study has shown that there are significant differences in the volatile profiles obtained from various bacterial monocultures grown in vitro, and that the analysis techniques herein employed have the potential to differentiate samples at the strain level.     

Microbial Competition in Reactors with Wall Attachment

Competition for nutrient and the ability of bacteria to colonize the gut wall are factors believed to play a role in the observed stability of the indigenous microbiota of the mammalian large intestine. These factors were incorporated into the two-strain continuous-stirred tank reactor (CSTR) model formulated and numerically investigated by Freter et al. In their model simulations, the reactor is parameterized using data for the mouse intestine. An invading bacterial strain is introduced into a CSTR that has already been colonized by a resident strain. The two strains compete for a single growth-limiting nutrient and for limited adhesion sites on the wall of the reactor. The mathematical model described in this paper is motivated in part by the CSTR model, but is based on the plug flow reactor (PFR). Parameter values and initial conditions are chosen so that the numerical performance of the PFR can be compared to that of the CSTR. In simulations bearing a remarkable qualitative and quantitative resemblance to those of the CSTR it is found that the invader is virtually eliminated, despite the fact that it has uptake rate and affinity for the wall identical to those of the resident. The PFR model is then parametrized using data for the human large intestine, and the two-strain simulations are repeated. Though obvious quantitative differences are noted, the more important qualitative outcome is preserved. It is also found that when three strains compete for a single nutrient and for adhesion sites there exists a steady-state solution characterized by the segregation of the bacterial strains into separate nonoverlapping segments along the wall of the reactor.     

Anthrax: early steps of the intracellular stage of infection development

It was shown that spore germination of different Bacillus anthracis strains in macrophage-like cells J774A.1 depended on the genotype of the strains. The virulent B. anthracis strains contain plasmids pXO1 and pX02 responsible for the synthesis of a toxin and a capsule, respectively. The loss of one of the plasmids results in the reduction of strain virulence. It was shown that effective survival of germinating spores in macrophages occurred in the presence of plasmid pXO1 only. The spores of the B. anthracis strains ?Ames and STI-Rif deprived of plasmid pXO1 were least adapted to passing through the intracellular stage. The B. anthracis strains 81/1 and 71/12 (carrying plasmids pXO1 and pXO2 and synthesizing the toxin and capsule) less effectively survived in the cytoplasm of macrophages than the strain STI-1 which has only the plasmid pXO1. It was found that the rate of synthesis of the capsule consisting of polymer gamma-D-glutamic acid depended on the ability of bacterial cells to escape from macrophages. In the B. anthracis strains carrying plasmid pXO2, capsule synthesis by vegetative cells was activated within macrophages that promoted a rapid escape of the vegetative cells from the macrophages. On the contrary, most of capsule-free cells of the vaccine strain STI-1 remained inside macrophages during the whole period of observation. Thus, integrated regulation of two processes, namely synthesis of the toxin components participating in the transition of the germinating cell from phagosome into cytoplasm, and synthesis of the capsule whose presence promotes rapid escape of bacterial cells from macrophages by presently unknown mechanism play the key role in anthrax development at early stages.     

产河豚毒素(TTX)菌株ZY-23的分离与鉴定

从河豚卵巢中分离得到35株细菌,利用小鼠生物法检测获11株产河豚毒素(TTX)菌株,其中有6株菌产TTX含量达400ng/mL以上,对其中ZY-23菌株进行传统分类学方法鉴定,初步鉴定为液化沙雷氏菌(Serratia liquefaciens)。16SrRNA基因序列分析,ZY-23菌株与Serratiasp.Tp5的亲缘关系最近,相似度达99%。用荧光检测分析其发酵液,发现在423nm处与标准品具有相同的发射峰,初步证实发酵液含有TTX。     

Specificity in the settlement -- modifying response of bacterial biofilms towards zoospores of the marine alga Enteromorpha

Previous studies have shown that the rate of settlement of zoospores of the green alga Enteromorpha is stimulated by mixed microbial biofilms and that the number of zoospores settling is positively correlated with the number of bacteria in the biofilm. In the present study the specificity of this relationship has been investigated. Ninety-nine strains of marine bacteria were isolated from natural biofilms on rocks and the surface of Enteromorpha plants. Isolates were screened by denaturing gradient gel electrophoresis (DGGE) to eliminate replicates and 16S rDNA sequencing identified a total of 37 unique strains. Phylogenetic analysis revealed that the isolated bacterial strains belonged to three groups gamma-Proteobacteria (28 strains), Cytophaga-Flavobacteria-Bacteroid (CFB) group (six strains) and alpha-Proteobacteria (one strain). Two strains were unassigned, showing < 93% sequence similarity with the CFB group. The main genera of gamma-Proteobacteria were Pseudoalteromonas (14 strains), Vibrio (five strains), Shewanella (five strains), Halomonas (three strains) and Pseudomonas (one strain). Spore settlement experiments were conducted on single-species biofilms, developed for different times on glass slides. The effect of correcting spore settlement values for biofilm density was evaluated. Results showed that the effect of bacterial strains on spore settlement was strain- but not taxon-specific and activity varied with the age of the biofilm. However, most of the strains belonging to genera Vibrio and Shewanella showed stimulation. Pseudoalteromonas strains showed a range of effects including settlement-inhibiting, paralysing and lysing activities. Spatial analysis of bacterial density in the presence and absence of spores revealed a range of different types of association between spores and bacteria. Overall, the spatial association between spores and bacteria appears to be independent of the overall quantitative influence of bacterial cells on spore settlement.     

Complete genome sequence of Campylobacter jejuni strain S3

Campylobacter jejuni is one of the leading causes of bacterial gastroenteritis in the world; however, there is only one complete genome sequence of a poultry strain to date. Here we report the complete genome sequence and annotation of the second poultry strain, C. jejuni strain S3. This strain has been shown to be nonmotile, to be a poor invader in vitro, and to be a poor colonizer of poultry after minimal in vitro passage.     

Relationship of the Presence and Copy Number of Plasmids to Exopolysaccharide Production and Symbiotic Effectiveness in Rhizobium fredii USDA 206

Rhizobium fredii USDA 206 harbors four large plasmids, one of which carries nodulation and nitrogen fixation genes. Previously isolated groups of plasmid-cured derivatives of strain USDA 206 were compared with each other to determine possible plasmid functions. Mutant strain 206CANS was isolated as a nonmucoid (Muc⁻) derivative of strain 206CA, a mutant that was cured of two plasmids. The Muc⁻ phenotype of 206CANS was only expressed when the strain was grown on certain media, particularly those with polyols as carbon sources. Plasmid pRj206b of strain 206CANS was previously shown to have a higher copy number than the same plasmid in strains USDA 206 and 206CA. When this plasmid was transferred to Muc⁺ strains, it conferred a nonmucoid phenotype on recipient strains. The symbiotic effectiveness of the wild-type and cured strains was compared. Overall, few differences were shown, but strains 206CA and 206CANS were found to have higher nitrogenase activities than the other strains. Thus, there appeared to be a possible relationship among exopolysaccharide synthesis, plasmid copy number, and symbiotic effectiveness.     

Review: Ethanol production at elevated temperatures and alcohol concentrations: Part II – Use of Kluyveromyces marxianus IMB3

It is clear that only a small proportion of all micro-organisms have been isolated and identified. The simple technique of seeking a thermotolerant fermentative yeast from a suitable hot environment has yielded a number of strains. These organisms, identified as strains of Kluyveromyces marxianus var. marxianus, have been shown to have a wide range of metabolic capabilities that could be used in industrial applications. Not only have the metabolic capabilities been elucidated but possible bioreactor configurations and process application options have been investigated. It appears that there are a number of specific situations where this thermotolerant yeast could find industrial applications. A full-scale industrial ethanol production trial using this yeast was successfully carried out in India. K. marxianus IMB3's performance in terms of the ethanol concentrations achieved was comparable to that obtained using the distillery's own yeast strain with an added advantage of eliminating cooling.     

Bacteriocinogenic properties of Staphylococci isolated from the oral cavity

Two hundred ninety two staphylococcal strains were isolated out of 130 saliva samples taken from children and adults, among which 116 were coagulase-positive and 176 coagulase negative. Bacteriocinogenic activity against Staphylococcus aureus strain Oxford 209P was found in 13 (4.5%) of the strains only. On the other hand, when a set of 15 sensitive staphylococcal strains selected by cross checking was used for the study 260 (89.0%) strains were found to be bacteriocinogenic. It was found that a higher percentage of coagulase-positive staphylococcal strains is sensitive to staphylococcins than of coagulase-negative strains. However, mean zone of inhibition is smaller in the case of former than of the latter strains. It was shown, that in the case of active strains a positive correlation exists between a percentage of coagulase-positive and negative strains inhibited by them and also between a percentage of all inhibited strains and a mean diameter if the growth inhibition zone. Simultaneous occurrence in saliva of two or more staphylococcal strains was found in 106 persons examined. In 93.4% of those cases coexisting strains did not show antagonistic properties: in remaining 6.6% despite of the number of simultaneously existing strains in oral cavity only one strain showed antagonistic properties against the remaining strains.     

Coexistence and Pattern Formation in Bacterial Mixtures with Contact-Dependent Killing

Multistrain microbial communities often exhibit complex spatial organization that emerges because of the interplay of various cooperative and competitive interaction mechanisms. One strong competitive mechanism is contact-dependent neighbor killing enabled by the type VI secretion system. It has been previously shown that contact-dependent killing can result in bistability of bacterial mixtures so that only one strain survives and displaces the other. However, it remains unclear whether stable coexistence is possible in such mixtures. Using a population dynamics model for two interacting bacterial strains, we found that coexistence can be made possible by the interplay of contact-dependent killing and long-range growth inhibition, leading to the formation of various cellular patterns. These patterns emerge in a much broader parameter range than that required for the linear Turing-like instability, suggesting this may be a robust mechanism for pattern formation.     

Use of suppressor subtraction hybridization for finding strain-specific sequences in bacterial genomes

Comparisons of bacterial genomes demonstrate that even strains of one species may strikingly differ in gene set. Strain-specific genes are of considerable interest, as they may be responsible for distinguishing features, such as virulence or drug resistance, of the strain and may be employed as markers in epidemiological or evolutionary studies. Suppression subtractive hybridization (SSH) was shown to be suitable for generating a set of DNA fragments differing between two closely related bacterial strains. More than 95% DNA fragments selected by SSH proved to be specific for Staphylococcus aureus strains ZW compared with strain 29213.     

Conservation, localization and expression of HopZ, a protein involved in adhesion of Helicobacter pylori.

From a sarkosyl-insoluble outer membrane fraction prepared from the Helicobacter pylori strain ATCC 43504, 19 proteins could be sequenced N-terminally by Edman degradation. Oligonucleotides were deduced and used for screening of a genomic library. From the isolated genes, five code for different members of a H.pylori outer membrane protein (Hop) family. Among these, the hopZ gene was characterized in more detail. It encodes a protein which was shown to be located at the bacterial surface by immunofluorescence studies. Sequence analysis of the hopZ gene from 15 different H.pylori strains revealed the existence of two alleles and the possible regulation of hopZ expression by slipped-strand mispairing within a CT dinucleotide repeat motif located in the signal-peptide coding region. Among the different strains, the influence of this region on the expression of HopZ was analyzed on a translational level by western blot analysis of bacterial extracts and immunofluorescence studies on intact cells. The protein is expressed only in those strains in which the number of the CT dinucleotide repeats allow for an open reading frame encoding the complete protein. Addionally the function of HopZ was investigated in an adhesion assay. The wild-type strain ATCC 43504 adhered to human gastric epithel cells whereas a knockout mutant strain showed significantly reduced binding to the cells.     

Nonimmune binding of Ig to Clostridium perfringens. Preferential binding of IgM and aggregated IgG

In an attempt to find a bacterial IgM receptor, a large number of bacterial strains of different species were screened for the ability to bind human IgM. Certain strains of the anaerobic bacterium Clostridium perfringens were found to bind a major fraction of polyclonal IgM. One bacterial strain showed a particularly high binding capacity and was studied in more detail. This strain is also able to bind a minor fraction of polyclonal IgA and IgG. Inhibition experiments indicate that the different Ig classes bind to one and the same R structure. The ability of the strain to bind polyclonal Ig is correlated to the number of subunits in the Ig. This correlation can most simply be explained by increasing avidity with increasing number of subunits. In agreement with this hypotheses, experiments with aggregated IgG show that binding ability increases with aggregate size. Experiments with Ig fragments indicate that the binding structure in Ig is located in the F(ab')2 region. The ability of this bacterial strain to bind a majority of IgM molecules as well as aggregated IgG is potentially useful in immunologic work and represents a new type of Ig binding to bacteria.