Tracking bacterial growth in liquid media and a new bacterial life model

By increasing viscosity of liquid media above 8.4 centipoise (cp) i.e. 0.084 g· cm^-1 · s^-1, individual growth and family formation ofEscherichia coli was continuously observed in real-time for up to 6 h. The observations showed primarily unidirectional growth and reproduction ofE. coli and suggested more than one reproduction in the observed portion ofE. coli life span. A new bacterial life model is proposed: each bacterium has a stable cell polarity that ultimately transforms into two bacteria of different generations; the life cycle of a bacterium can contain more than one reproduction cycle; and the age of a bacterium should be defined by its experienced chronological time. This new bacterial life model differs from the dominant concepts of bacterial life but complies with all basic life principles based on direct observation of macroorganisms.     

Evaluation of waste chicken feathers as peptone source for bacterial growth

Aims: Peptones are one of the most expensive constituents of microbial media. This study was undertaken to prepare the peptone from waste chicken feathers through a new process. Methods and Results: The chemical analysis of chicken feather peptone (CFP) was performed. The ability of CFP to support the growth of the three test bacteria in liquid and agar media was comparable to those of three commercial peptones [tryptone peptone (TP), fish peptone and protease peptone (PP)]. Conclusions: CFP was found to be rich in ash (42·1 g 100 g^?1), protein (55·8 g 100 g^?1) and mineral contents. The maximum biomass yield (3·13 g l^?1) and colony number (83 × 10⁸ CFU ml^?1) for bacterium Bacillus subtilis were attained with CFP. The maximum biomass yields and colony numbers for Lactobacillus delbrueckii ssp. bulgaricus and Escherichia coli were reached in TP medium. Second high biomass yield (2·64 g l^?1) and colony number (75 × 10⁸ CFU ml^?1) for E. coli were achieved using CFP. Third high biomass yield (1·29 g l^?1) and colony number (90 × 10⁷ CFU ml^?1) for Lact. delbrueckii ssp. bulgaricus were obtained in CFP medium. Significance and Impact of the Study: Usability of waste chicken feathers as substrate for bacteria was investigated for the first time in the present study. The peptone may be used in industrial fermentations for production of antibiotics, organic acids, enzymes and biopolymer. It may be also used in clinical microbiology. A new chemical process was developed for peptone preparation. This process may be also employed for peptone preparation from other organic materials, especially fibrose protein‐containing materials.     

Escherichia coli K‐12 (pEGFPluxABCDEamp): a tool for analysis of bacterial killing by antibacterial agents and human complement activities on a real‐time basis

Photorhabdus luminescens luxCDABE genes were integrated into E. coli K‐12 using a high copy number plasmid containing modified luxABCDE genes under the control of the powerful Lac promoter. This strain emitted 10 times higher bioluminescence (BL) than P. luminescens. BL production under different growth conditions was studied. In both bacterial strains, the increase in BL signal correlated with the increase in optical density (OD) in a rich growth medium. However, at the logarithmic growth phase, the BL signal was roughly constant. By contrast, in minimal growth media, there was no substantial growth and the BL/cell was approximately five times higher than in the rich medium. The dynamic measurement range of BL was 10²–10⁷ colony‐forming units (CFU) in E. coli and 10³–10⁷ CFU in P. luminescens. Because the decrease in the BL signal correlated with the decrease in CFU and OD, i.e. the number of bacterial cells killed, it proved to be very suitable for assessing the antibacterial effects of different antimicrobial agents. Unlike with plate counting, the kinetics of killing can be monitored on a real‐time basis using BL measurements. Complement activities in different samples can be estimated using only one serum dilution. The transformed E. coli strain appeared to be superior to P. luminescens in these applications because E. coli was complement sensitive, the detection limit of E. coli was one order lower and the BL‐producing system of P. luminescens appeared to be quite unstable. Copyright © 2012 John Wiley & Sons, Ltd.     

Effect of salinity on the adaptive capacity of recombinant strains ofEscherichia coli andBacillus subtilis

The effect of different concentrations of salts on natural and recombinant strains ofBacillus subtilis andEscherichia coli was studied. The recombinant strain ofB. subtilis was found to be more osmotolerant than the wild-type strain of this bacterium, whereas the opposite situation was observed for the recombinant and wild-type strains ofE. coli. Some salts exerted a bacteriostatic effect onE. coli andB. subtilis. The adaptive capacity of recombinant strains depended on the number of plasmid copies in the cells. The introduction of recombinant bacteria into model ecosystems resulted in the generation of their variants with increased osmotolerance.     

Effects of bacterial contamination on development ofMicroplitis croceipes [Hym.: Braconidae]

Bacterial contaminants ofHeliothis virescens (F.) influenced the development ofMicroplitis croceipes (Cresson). Among the four bacterial species studied, the most virulent wasPseudomonas maltophilia Hugh and Ryschenkow followed byBacillus subtilis (Ehrenberg) Cohn. Both bacteria caused severe mortality in all stages ofMicroplitis tested.Microplitis larvae were less susceptible toEscherichia coli (Migula) Castellani and Chalmers andLeuconostoc mesenteroides (Tsenkovskii) van Thieghem than toB. subtilis andP. maltophilia. AlthoughE. coli did not affect the number of cocoons produced, adult emergence was lower than in controls. Longevity of adultMicroplitis exposed to bacterially contaminated honey-water was greatly reduced in all bacterial treatments.      

Sensitivity of selected bacterial species to UV radiation

The effect of exposure of bacterial suspensions to UV radiation by means of the dose-response curves was assessed. The D₃₇ and D₁₀ values were used for subsequent statistical analysis of the results. The aim of this article is to evaluate the sensitivity to UV radiation of several microorganisms of different habitats (Rhizobium meliloti, Rhodobacter sphaeroides, Escherichia coli, and Deinococcus radiodurans), two mutants with nonfunctional SOS DNA repair system (R.meliloti recA ^- and E. coli recA ^-), and a mutant in the synthesis of carotenoids (R. sphaeroides crtD). The results reveal that D. radiodurans was an extremely resistant bacterium, R. meliloti was more resistant than R. sphaeroides, and E. coli was the most sensitive bacterium tested. The high sensitivity of recA ^- mutants was also verify. Moreover, it seems that the possession of pigments had no important effect in the sensitivity of R. sphaeroides to UV radiation.     

The influence of the growth phase of enteric bacteria on electrotransformation with plasmid DNA

Salmonella typhimurium LB5000 andEscherichia coli JM109 were transformed by electroporation. In accordance with the chemical transformation methods, the growth phase of these electrocompetent bacteria had a strong impact on transformation efficiency. Survival of bacteria, after the high-voltage electrical pulse was also influenced by the growth phase. Both bacterial species were most successfully electrotransformed when microbial cells were harvested at the late lag phase. The second optimum for transformation reachedE. coli cells in the mid-exponential andS. typhimurium cells in the late exponential phase. Transformation efficiencies ranged from 3.4×10⁴ to 2.7×10⁵ transformants per μg DNA in the case ofS. typhimurium and from 2.8 × 10² to 8.8×10⁵ transformants per μg DNA in the case ofE. coli. Survival of cells after the electrical pulse in late lag and late exponential phases was about 20% higher than during other phases of growth. Preparing electrocompetent cells from later phases of their growth is more useful for practice, because it provides more biomass with good yield of transformants.     

Effect of chitosan derivatives on the reproduction of coliphages T2 and T7

The effect of chitosan derivatives with different degrees of polymerization and deamination, as well as of chitosan 6-O-sulfate and chitosanN-succinate-6-O-sulfate, on the reproduction of coliphages T2 and T7 inEscherichia coli and on the growth of this bacterium was studied. Chitosan derivatives decreased the yield of coliphages and exhibited antibacterial activity. The efficiency of inhibition of viral infection and the antibacterial activity of chitosan were found to be dependent on the degree of its polymerization. At the same time, there was no correlation between the degree of chitosan deamination and the extent of inhibition of viral infection. Anionic chitosan derivatives virtually did not possess antiviral or antibacterial activity. It is assumed that chitosan blocks some stages of phage reproduction. The decrease in the phage-producing ability ofE. coli may also be due to the antibacterial effect of chitosan.     

The influence of local plant growth conditions on non-fastidious bacterial contamination of meristem-tips ofHydrangea culturedin vitro

A high incidence ofin vitro bacterial contamination (69%) has been detected in meristem-tip explants ofHydrangea from widely differing locations in Ireland and the UK. The bacteria were characterised by API 20E biochemical test kits and by fatty acid profile analysis. The results obtained from the different methods were compatible and anomalies were explicable in terms of the limitations of the respective methods. The majority of the isolates were environmental or animal-associated bacteria with clusters ofEnterobacter isolates in Dublin, and ofEscherichia coli in the main Cork location. A cluster of Pseudomonads was detected in the Derby (UK) plants. The main association was between the location and the contaminant clusters. The main finding was that the nature of organic soil amendments may influence inoculum for the contamination of plants and the conclusion was that fertilisation with organic materials should be avoided in the preparation of plants for micropropagation.     

Assessment of survival during starvation ofEscherichia coli andKlebsiella pneumoniae in artificial urine: analysis of the kinetics of colony formation

A kinetic model of colony formation was proposed by Hattori, based on a count of the colonies that appear on a plate in successive short intervals of time. In this model, three parameters (,t _r and N) are defined, which reflect the ability of a bacterium to yield colonies and allow us to described the dynamics of bacterial populations in soil and ofE. coli at different growth phases. In this paper we report a reparametrization of the kinetic model of colony formation, with the aim of facilitating more accurate calculation of andt _r. Moreover, we observed that during the starvation ofE. coli andK. pneumoniae in urine, can be used to assess survival, since this parameter clearly decreases during starvation. Retardation time values (t _r) were similar inE. coli andK. pneumoniae throughout the starvation experimental period.     

Changes in bacterial cells induced by high pressure at subzero temperature

The aim of this study was to examine the effect of pressure treatment at 193 MPa and −20 °C on membrane damage, changes in activity of membrane-bound ATPases and degradation of nucleic acids. The experiments were carried out with three Escherichia coli strains, in the exponential and stationary phases of growth, and differing in sensitivity to pressure. All E. coli strains subjected to pressure in the exponential phase of growth were inactivated by 6 log cycles, independently of the strain, which was accompanied by a total loss of ability to plasmolyse, an increase in irreversible membrane permeability to PI, and a reduction of cellular ATP by more than 80%. After pressure treatment of stationary phase cells, the relationship between the inactivation level and the ability to plasmolyse was not as evident as in the case of exponential phase cells. Pressure treatment of two strains of E. coli K-12 and Ec160/59 in the stationary phase that decreased viability by no more than one log cycle led only to reversible permeabilization of bacterial membranes, while irreversible permeabilization was observed in the pressure sensitive E. coli IBA72 strain phase that was inactivated by 4.6 log cycles. The reduction of ATP and changes in ATPase activity after pressure treatment of tested E. coli strains in the stationary phase of growth depended on the stage of inactivation of the particular strain. Electrophoretic analysis showed degradation of RNA isolated after pressure treatment from cells of all E. coli strains tested in the exponential phase of growth. The changes of RNA induced by pressure were not visible in the case of cells in the stationary phase. The degradation of DNA isolated from pressure treated E. coli strains from the exponential as well as from the stationary phase of growth was not observed.     

Survival and distribution ofYersinia enterocolitica in a tropical rain forest stream

The survival and activity ofYersinia enterocolitica andEscherichia coli in a tropical rain forest stream were studied in situ in membrane diffusion chambers. Direct counts ofY. enterocolitica decreased by one order of magnitude during the first 6 h and then remained constant. Densities ofE. coli increased over time, doubling after 2 days. Physiological activity ofE. coli dropped initially and then stabilized at 85%. Physiological activity forY. enterocolitica increased during the first 6 h, then declined to 50%. The percentage of respiring cells as measured by 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride reduction decreased forE. coli to 10%, whereasY. enterocolitica remained near 25%;Y. enterocolitica is a survivor in tropical freshwater, as isE. coli. Indirect and direct fluorescent antibody (FA) methods were evaluated for the direct detection ofY. enterocolitica in natural habitats. Natural densities of FA-positive cells were always less than 10 cells ml^–1, and no isolates were obtained by culturing samples.     

A community model of ciliateTetrahymena and bacteriaE. coli: Part II. Interactions in a batch system

Premised on relatively simple assumptions, mathematical models like those of Monod, Pirt or Droop inadequately explain the complex transient behavior of microbial populations. In particular, these models fail to explain many aspects of the dynamics of aTetrahymena pyriformis-Escherichia coli community. In this study an alternative approach, an individual-based model, is employed to investigate the growth and interactions ofTetrahymena pyriformis andE. coli in a batch culture. Due to improved representation of physiological processes, the model provides a better agreement with experimental data of bacterial density and ciliate biomass than previous modeling studies. It predicts a much larger coexistence domain than rudimentary models, dependence of biomass dynamics on initial conditions (bacteria to ciliate biomasses ratio) and appropriate timing of minimal bacteria density. Moreover, it is found that accumulation ofE. coli sized particles andE. coli toxic metabolites has a stabilizing effect on the system.     

Oxygen reactions with bacterial oxidases and globins: binding,reduction and regulation

Oxygen is favoured as terminal electron acceptor in aerobic and facultative microorganisms because of its appropriate physical state, satisfactory solubility and its desirable combinations of kinetic and thermodynamic properties. Oxygen is generally reduced by four electrons to yield oxygen, but there are important biological consequences of, and roles for, the partial reduction to superoxide and peroxide. Complex and multiple regulatory networks ensure (i) the utilization of oxygen in preference to other oxidants, (ii) the synthesis of oxygen-consuming enzymes with appropriate properties (particularly affinity for the ligand), and (iii) appropriate cellular protection in the event of oxidative stress. This contribution reviews the terminal respiratory oxidases of selected Gram-negative bacteria and microbial haemoglobin-like proteins.Recent studies of the cytochromebd-type oxidases ofEscherichia coli andAzotobacter vinelandii suggest that, despite probable similarity at the amino acid level, the reactivities of these oxidases with oxygen are strikingly different. The respiratory protection afforded to nitrogenase in the obligately aerobic diazotrophA. vinelandii by the cytochromebd complex appears to be accompanied by, and may be the result of, a low affinity for oxygen and a high V_max. The poorly characterized cytochromeo-containing oxidase in this bacterium is not required for respiratory protection. InE. coli, the cytochromebd-type oxidase has a remarkably high affinity for oxygen, consistent with the view that this is an oxygen-scavenging oxidase utilized under microaerobic conditions. The demonstration of substrate (i.e. oxygen) inhibition in this complex suggests a mechanism whereby wasteful electron flux through a non-proton-pumping oxidase is avoided at higher dissolved oxygen tensions. The demonstration of two ligandbinding sites (haemsd andb ₅₉₅) in oxidases of this type suggests plausible mechanisms for this phenomenon. InE. coli, assembly of the cytochromebd-type oxidase (and of periplasmic cytochromesb andc) requires the presence of an ABC transporter, which may serve to export haem or some assembly factor' to the periplasm.There is at least one additional oxygen-consuming protein inE. coli — the flavohaemoglobin encoded by thehmp gene. Globin-like proteins are also widely distributed in other bacteria, fungi and protozoa, but most have unknown functions. The function of HMP and the related chimaeric flavohaemoglobins in other bacteria and yeast is unknown; one of several possibilities for HMP is that its relatively low affinity for oxygen during turnover with NADH as substrate could enable it to function as a sensor of falling (or rising) cytoplasmic oxygen concentrations.(until October 1994: Section of Microbiology, Wing Hall, Cornell University, Ithaca, NY 14853-8101, USA)     

Subinhibitory concentrations of nalidixic acid alter bacterial physiology and induce anthropogenic resistance in a commensal strain of Escherichia coli in vitro

The human gut houses a complex group of bacterial genera, including both opportunistic pathogens and commensal micro-organisms. These are regularly exposed to antibiotics, and their subinhibitory concentrations play a pivotal role in shaping the microbial responses. This study was aimed to investigate the effects exerted by sub-MICs of nalidixic acid (NA) on the growth rate, bacterial motility, biofilm formation and expression of outer membrane proteins (OMPs) in a commensal strain of E. coli. The NA-sensitive strain was sequentially passaged under sub-MICs of NA. E-test was used to determine the MIC values of NA. Results indicated significant changes in the growth profile of commensal E. coli upon exposure to NA at sub-MICs. Differential expression of OMPs was observed in cells treated with sub-MICs of NA. Bacterial motility was reduced under 1/2 MIC of NA. Interestingly, successive passaging under 1/2 MIC of NA led to the emergence of resistant E. coli with an increased MIC value of 64 µg ml⁻¹ in just 24 days. The NA-resistant variant was confirmed by comparing its 16S rRNA sequence to that of the sensitive commensal strain. Mutations in the Quinolone Resistance-Determining Regions (QRDRs) of chromosomal gyrA, and Topoisomerase IV-encoding parC genes were detected in NA-resistant E. coli. Our results demonstrate how antibiotics play an important role as signalling molecules or elicitors in driving the pathogenicity of commensal bacteria in vitro.     

The action of nitrosoguanidine and other DNA-inactivating agents on Streptococcus pyogenes K56 strains with normal and reduced dark repair ability

Summary The response pattern for ultraviolet light, nitrogen mustard, and ethyl methane sulphonate of Hcr⁺ and Hcr^- strains ofStreptococcus pyogenes K 56 is similar to that observed for analogous strains ofE. coli, whereas repair-apt streptococcal strains are much more sensitive to nitrosoguanidine and mitomycin C thanE. coli. Theuvr gene(s) appear(s) to be without effect upon survival, prophage induction, and mutation to streptomycin resistance caused by nitrosoguanidine and only of little influence on repair of mitomycin C damage.     

Influence of 1‐methylcyclopropene (1‐MCP) on the production of bacterial cellulose biosynthesized by Acetobacter xylinum under the agitated culture

Aims: Bacterial cellulose is an extracellular polysaccharide secreted by Acetobacter xylinum, which has become a novel material increasingly used in food and medical industries. However, its broad application is limited by its low yield and high cost. 1‐Methylcyclopropene (1‐MCP) is a potent inhibitor to either exogenous or endogenous ethylene during the biological senescence of plants, which has been broadly applied in commercial preservation of fruits and vegetables. The purpose of this study was to investigate the effects of 1‐MCP on both the growth of Acet.  xylinum and its cellulose production to demonstrate the potential enhancement of bacterial cellulose yield. Methods and Results: Three groups of samples were fermented under agitated culture with 125 rev min^?1 rotational speed. To the culture media, 0·14 mg of 1‐MCP contained in 100 mg dextrose powder was added on assigned days or on the first culture day only. Results from the measurement of bacterial cell concentration and bacterial cellulose yield at the end of a 12‐day culture demonstrated that cultures excluding 1‐MCP displayed a higher cell concentration and a lower cellulose production, while cultures containing 1‐MCP produced 15·6% more cellulose (1‐MCP added on day 1) and 25·4% (1‐MCP added on each assigned day) with less biomass. Conclusions: 1‐MCP was able to affect the growth of Acet. xylinum cells and resulted in increasing bacterial cellulose yield up to 25·4% over controls, which did not contain 1‐MCP. Significance and Impact of the Study: This was the first study to use the growth inhibitor of plants to investigate its effects on bacterial growth and production. It also demonstrated a significant enhancement of bacterial cellulose yield by the addition of 1‐MCP during the common agitated culture of Acet. xylinum.     

Selection againstdam methylation sites in the genomes of DNA of enterobacteriophages

Summary Postreplicative methylation of adenine inEscherichia coli DNA to produce G^6m ATC (where^6mA is 6-methyladenine) has been associated with preferential daughter-strand repair and possibly regulation of replication. An analysis was undertaken to determine if these, or other, as yet unknown roles of GATC, have had an effect on the frequency of GATC inE. coli or bacteriophage DNA. It was first ascertained that the most accurate predictions of GATC frequency were based on the observed frequencies of GAT and ATC, which would be expected since these predictors take into account preferences in codon usage. The predicted frequencies were compared with observed GATC frequencies in all available bacterial and phage nucleotide sequences. The frequency of GATC was close to the predicted frequency in most genes ofE. coli and its RNA bacteriophages and in the genes of nonenteric bacteria and their bacteriophages. However, for DNA enterobacteriophages the observed frequency of DNA enterobacteriophages the observed frequency of GATC was generally significantly lower than predicted when assessed by the chi square test. No elevation in the rate of mutation of^6mA in GATC relative to other bases was found when pairs of DNA sequences from closely related phages or pairs of homologous genes from enterobacteria were compared, nor was any preferred pathway for mutation of^6mA evident in theE. coli DNA bacteriophages. This situation contrasts with that of 5-methylcytosine, which is hypermutable, with a preferred pathway to thymine. Thus, the low level of GATC in enterobacteriophages is probably due not to^6mA hypermutability, but to selection against GATC in order to bypass a GATC-mediated host function.     

Isolation of bacterial strains capable of using lupanine,the predominant quinolizidine alkaloid in white lupin,as sole carbon and energy source

Seven Gram-negative bacterial strains, capable of using lupanine, the predominant quinolizidine alkaloid in white lupin, as sole carbon and energy source, were isolated from soil in whichLupinus albus andL. luteus had been grown. A metabolic profile system (BIOLOG) identified only three of the seven isolates, two asXanthomonas oryzae pvoryzae E and one asGluconobacter cerinus. The maximum specific growth rates of the seven isolates when incubated at 27°C in a medium containing as sole carbon source 2 g L^–1 of lupanine, ranged from 0.05 to 0.13 h^–1 and the concentration of dry biomass at the stationary phase ranged from 0.7 to 1.1 g L^–1. Unidentified strains IST20B and IST40D exhibited the highest maximum specific growth rates (0.13h^–1), removed 99% of the initial lupanine after 30 h of incubation, and the dry biomass yields did not exceed 0.4 g per g lupanine consumed. Strain IST20B is of potential use forL. albus debittering because, after 32 h growth in aqueous extracts ofL. albus, 85% of initial alkaloids were removed while the concentration of soluble protein was only reduced by 8%.