Populations Of Viable Bacteria Research Articles

Structure of an Acinetobacter Broad-Range Prophage Endolysin Reveals a C-Terminal α-Helix with the Proposed Role in Activity against Live Bacterial Cells.

Proteins that include enzymatic domain degrading the bacterial cell wall and a domain providing transport through the bacterial outer membrane are considered as prospective compounds to combat pathogenic Gram-negative bacteria. This paper presents an isolation and study of an enzyme of this class naturally encoded in the prophage region of Acinetobacter baumannii AB 5075 genome. Recombinant protein expressed in E. coli exhibits an antimicrobial activity with respect to live cultures of Gram-negative bacteria reducing the population of viable bacteria by 1.5–2 log colony forming units (CFU)/mL. However the protein becomes rapidly inactivated and enables the bacteria to restore the population. AcLys structure determined by X-ray crystallography reveals a predominantly α—helical fold similar to bacteriophage P22 lysozyme. The С-terminal part of AcLys polypeptide chains forms an α—helix enriched by Lys and Arg residues exposed outside of the protein globule. Presumably this type of structure of the C-terminal α—helix has evolved evolutionally enabling the endolysin to pass the inner membrane during the host lysis or, potentially, to penetrate the outer membrane of the Gram-negative bacteria.

Molecular viability testing of viable but non-culturable bacteria induced by antibiotic exposure.

SummaryNucleic acid amplification‐based methods are limited by their inability to discriminate between viable and dead cells. To overcome this drawback, propidium monoazide (PMA) combined with qPCR has been used to differentiate viable from nonviable cells in environmental samples. However, assessing bacterial physiology using PMA‐qPCR remains a challenge due to its incapability of detecting metabolic activities, leading to overestimation of the viable bacteria population under an inactivation condition (e.g. antibiotic treatments). A recent advanced technique to amplify ribosomal RNA precursors (pre‐rRNA) has been shown to detect viable cells because pre‐rRNAs are intermediates in rRNA synthesis. This study investigated the effect of different types of antibiotics on the bacterial viability or viable but non‐culturable (VBNC) state using both PMA‐qPCR and pre‐rRNA analyses with Pseudomonas aeruginosa. This study demonstrated that P. aeruginosa was more sensitive to colistin than it was to carbenicillin, gentamicin and levofloxacin. We could discriminate VBNC P. aeruginosa cells using PMA‐qPCR when antibiotic pressure induced the VBNC state. Also, pre‐rRNA was able to distinguish viable cells from colistin‐inactivated bacteria cells, and it could detect the presence of VBNC and persister cells. Our results showed that these two molecular methods could successfully eliminate false‐positive signals derived from antibiotics‐inactivated cells.

Influencing Factors of Bacterial Activity in Water Distribution Networks

In order to study the variation patterns of bacterial activity in water distributed networks, this paper studied a simulated water distribution network in a northern China city. Microscopic examination and bacterial counts were conducted on acridine orange-stained bacteria using fluorenscence microscopy to ensure precise quantitative analysis. The ratio of viable bacterial count to total bacterial count was used to characterize the overall bacterial activity in the drinking water distribution network,as well as to study the impacts imposed by various factors. As indicated, this method was proved to be rapid,reliable and repeatable in bacterial counts. Populations of viable bacteria and total bacteria in water samples were 103-104 cells·mL-1 and 104-105 cells·mL-1, respectively. There was a positive correlation between the bacterial activity and water temperature, turbidity, and flow velocity. There was also a negative correlation between bacterial activity and residual chlorine after the operation stabilized. The capacity of different forms of bacteria in the simulated water distribution networks with tolerance of sodium hypochlorite was in the following order:long-rod-shaped, spherical-shaped, short-rod-shaped, and curved-shaped. The reactivation and reproduction capability of rod-shaped bacteria was stronger. There were more short-rod-shaped bacteria in summer water samples than in winter.

Assessment and determination of lyoprotectant for survival of freeze-dried Lactobacillus rhamnosus

Abstract Currently research of lactic acid bacteria focus primarily on the functional probiotics, which are major beneficial biota in the gastrointestinal tract, have been industrial manufactured. Probiotics confer health benefits on the host need adequate amounts. However, the absence of data makes it difficult to ensure the maintenance biological activities and population of probiotic. In this research, a fractional factorial design and steepest ascent experiment were used to analyze the influence of lyoprotectant as carbohydrates, prebiotics and amino acids on the survival of the probiotic Lactobacillus rhamnosus. The results indicated a maximum survival rate and population of viable bacteria of L. rhamnosus to be 55.84 % and 1.60 ×1011 CFU/g after freeze-dried by using a combination of 10 g/100mL Sucrose, 2.5 g/100mL Isomaltooligosaccharide, 12 g/100mL Hydroxyproline. To a large extent, the survival and viability were dependent on the cryoprotectant used and make probiotics more attractive from a practical application in industrial viewpoint.

Combined Effect of Irradiation and Frozen Storage on Survival of Viable Bacteria and Inoculated Escherichia Coli in Chicken

Combined effect of irradiation and frozen storage on viable bacteria and inoculated Eschericia coli in chicken was investigated. Samples of uninoculated chicken and samples of chicken inoculated with E. coli were irradiated using a Co-60 source at doses of 0, 2 ,4,6 and 8 kGy and stored for 0, 7, 14, 21, 28, 35, 42, 49 and 56 days at -180 C. Samples were analyzed each week to enumerate surviving viable bacteria and E. coli. Irradiation doses of 2, 4, 6, and 8 kGy respectively reduced the population of viable bacteria in the uninoculated chicken by 2.06, 2.96, 3.91 and 4.21 log cycles. Storage for 56 days reduced populations of viable bacteria by approximately 2 log cycles for all irradiated uninoculated samples. Dose of 2 kGy reduced the population of E. coli in the unirradiated sample by 2.69 log cycles and 4, 6, 8 kGy reduced the population by > 7 log cycles. Storage for 56 days reduced the population of E. coli by 4.07 and > 3.52 log cycles respectively in the unirradiated and irradiated (2 kGy) samples. Irradiation doses of 4 to 8 kGy in combination with frozen storage were effective in reducing the populations of viable indigenous bacteria in addition to eliminating inoculated E. coli from chicken thus extending the shelf life and improving the hygienic quality.

English

A pot culture experiment was conducted to investigate the effect of pesticides – 2,4-D (herbicide), endosulfan (insecticide) and dithane M-45 (fungicide) at their field application rates (1.0, 2.0 and 1.5 kg a.i ha-1), respectively, on the available Diethyl Triamine Pentacetic acid (DTPA) extractable copper (Cu), manganese (Mn) and zinc (Zn) along with the population of viable bacteria and fungi in new alluvial soil (Typic haplustepts). The results reveal that as compared to control, 2,4-D effectuated a significant increase in the population of total bacteria (up to 21.1%) and fungi (33.7%) besides available Cu (21.7%) and Zn(119.4%) during later stages and Mn (8.7%) almost throughout the incubation period though there was significant progressive immobilization of Cu (17.7%) during initial stages. Endosulfan, contrarily caused significant reduction of total bacteria (up to 18.4%), fungi (13.1%) and immobilization of Zn (42.1%) during later stages, Cu (10.3%) in the intermediary stage and Mn (64.7%) almost  throughout. Dithane M-45, however, resulted in a significant increase in available Cu (13.2%) and Mn (3.38%) in the intermediary stage in spite of inducing detrimental influence on fungi (18.7%) and immobilization of Zn (12.9%) during intermediary stage and Mn in early (3.0%) and late stages (6.7%) of incubation. Among the pesticides used in the investigation, the performance of herbicide (2,4-D) was encouraging in almost every aspect.   Key words: Pesticide, DTPA extractable Cu, Mn and Zn, viable bacteria and fungi

Shedding light on peptidoglycan growth

Our understanding of the dynamics of peptidoglycan growth has been hampered by our inability to directly image peptidoglycan synthesis in vivo. Given the role of D-amino acids in crosslinking the glycan strands, Kuru et al. investigated whether fluorescent probes attached to a D-amino acid backbone could be incorporated into peptidoglycan during its synthesis. The authors attached the small fluorophores 7-hydroxycoumarin 3-carboxylic acid (HCC-OH) and 4-chloro-7-nitrobenzofurazan (NBD-C1) to 3-amino-D-alanine (ADA) to create HADA and NADA, respectively. Strong peripheral and septal labelling of the entire population of viable bacteria was observed with both fluorophores for phylogenetically diverse bacterial species, including Escherichia coli, Agrobacterium tumefaciens and Bacillus subtilis. Furthermore, pulse–chase experiments allowed real-time tracking of peptidoglycan synthesis using time-lapse microscopy.

DETECTION OF VIABLE BACTERIA CELLS BY BIOLUMINESCENCE: A BIOENERGETIC APPROACH 1

ABSTRACTThe cellular ATP content of fourteen freshly harvested bacteria including Bacillus, Campylobacter, Citrobacter, Escherichia, Lactobacillus, Listeria, Pediococcus, Pseudomonas, Salmonella, Streptococcus and Yersinia, was determined using a luciferin‐luciferase bioluminescence approach. Incubation of bacteria with carbonyl cyanide meta‐chlorophenyl hydrazone (CCCP), a membrane protonophore, prior to cell breakage substantially lowered the bioluminescence signals indicating a decrease of cellular ATP content. The addition of CCCP after cell breakage had no detectable effect on the ATP levels. This differential effect of CCCP was not observed using heat‐killed bacteria, i.e., the ATP content was not affected by CCCP incubation. The CCCP effects on cellular ATP level were detectable in bacterial suspensions with concentrations ranging from 106 to 103 CFU/mL. Upon cold storage, the ATP content, but not the population of viable bacteria, decreased. The ATP content could be partially restored by the addition of glucose. The ATP content restored by the addition of glucose was also sensitive to CCCP treatment. These results demonstrated that viable bacterial cells can be differentiated from dead cells by their responses to membrane protonophores.

Distribution of Xylella fastidiosa in Sycamore Associated with Low Temperature and Host Resistance.

Experiments were conducted in the field and laboratory to determine effects of low temperatures on Xylella fastidiosa populations in American sycamore. Roots and shoots from naturally infected trees at two locations were collected monthly. Sap extracted from the samples was tested by enzyme-linked immunosorbent assay for presence of X. fastidosa and was diluted and plated on periwinkle wilt medium to determine populations of viable bacteria. Cumulative rainfall and hours below temperature thresholds (-5 to 10°C) were recorded at each site. Bacterial populations in shoots were negatively correlated with cumulative hours below -5°C air temperature (r= -0.96). In roots, bacterial populations were only weakly correlated with cumulative hours below soil temperature thresholds (-0.61 < r <-0.25). Bacterial populations were not correlated with monthly rainfall. In the laboratory, resistant and susceptible sycamore trees were inoculated with X. fastidiosa and held in the dark at 5°C or 22°C. After 12 weeks, inoculated stem sections were collected and sap was extracted and tested as described previously. Stems that tested positive for X. fastidiosa were divided into additional samples and tested as described above. Results of the laboratory study indicated no significant effects of low-temperature treatment (5°C) or host resistance on viable bacteria. Bacterial detection frequency and population size were greatest near the inoculation point and the primary direction of early bacterial spread was acropetal.

Fate of Pierce's Disease Strains of Xylella fastidiosa in Common Riparian Plants in California.

The fate of strains of the bacterium Xylella fastidiosa that cause Pierce's disease of grapevines was investigated in 33 species of mostly perennial plants common in riparian habitats in northern coastal California grape-growing regions. Plants were inoculated in the field with needle puncture using cultured cells of X. fastidiosa as inoculum or inoculated in the laboratory with infective insect vectors (Graphocephala atropunctata). Populations of X. fastidiosa were highest in most plant species within 3 to 6 weeks of inoculation, followed by declines in populations of viable bacteria over the next 3 to 4 months. Homogenates of petioles of California black walnut (Juglans hindsii) and coffeeberry (Rhamnus californica) inhibited in vitro growth of X. fas-tidiosa, precluding culture of the bacterium from these plants. Big leaf maple (Acer macrophyl-lum), California buckeye (Aesculus californica), California blackberry (Rubus ursinus), coast live oak (Quercus agrifolia), elderberry (Sambucus mexicana), French broom (Genista monspessulanus), periwinkle (Vinca major), valley oak (Quercus lobata), and the grape root-stock Vitis rupestris supported systemic populations of X. fastidiosa that survived throughout the year outdoors in Napa Valley, California.

Time-dependent changes in viable numbers and activities of aerobic heterotrophic bacteria in subsurface samples

Vadose and saturated zone sediment cores from depths to 212 m were obtained from the U.S. Department of Energy's Hanford Site in south-central Washington by cable tool drilling, and volcanic ashfall tuff samples were obtained from tunnels 400 m beneath the surface of Rainier Mesa at DOE's Nevada Test Site (NTS) in southern Nevada. Numbers of viable aerobic heterotrophic bacteria were determined by plate counts and metabolic activities were determined by [ 14C]glucose radiorespirometry at t = 0 and at various post-sampling time points up to 154 d in order to assess the influence of sample storage on microbiological properties of subsurface samples. Increases in post-sampling populations of viable bacteria were observed in all samples, although the magnitude of the increase and time after sampling at which the maximum population size was reached varied with the sample type. The greatest post-sampling increases in viable counts and [ 14C]glucose mineralization occurred in a high-organic carbon lacustrine sediment. The population of aerobic heterotrophs increased from below detection at t = 0 to > 10 6 CFU g −1 after 139 d. Significant increases in culturable counts were shown to occur within 24 h for tuff samples from NTS. These results indicate that precautions are necessary in the post-drilling handling of subsurface sediments and rock for microbiological analysis. In addition, these results suggest that even low biomass subsurface environments may be readily stimulated for applications such as in situ bioremediation.

Survey of Microbial Populations in Buried‐Valley Aquifer Sediments from Northeastern Kansas

AbstractTwenty‐two aseptically collected sediment core samples were obtained from below the water table (60 to 280 feet deep) at four pristine sites along a major buried‐valley aquifer system in northeastern Kansas. Samples were examined for total numbers of bacteria, viable aerobic bacteria, protozoa, and fungi. Contiguous samples were obtained in some transition zones of sediment texture or color in order to detect possible population shifts over small vertical distances related to changes in sediment characteristics. Total counts of bacteria varied between 106 and 108 per gram of dry sediment. Viable bacterial counts varied between 0 and 108 colony forming units per gram, usually being higher in sandy or gravelly sediments than in silty or clayey sediments. The relationship between sediment texture and microbial population density was confirmed statistically. Total numbers of bacteria correlated highly with variations in sediment sand and clay content. The population densities of viable bacteria and protozoa correlated moderately with these indicators of sediment texture. In some samples, populations of viable bacteria approached the total count of bacteria and the diversity of bacterial colony types appraoched that found in surface soil. Protozoa were found at low population densities in the coarser textured samples. The protozoa were similar to types commonly encountered in surface soil. No actinomycetes, fungi, or algae were detected in any samples.

Relation of xylem plugging to black rot lesion development in cabbage

Black rot lesion development in cabbage leaves was correlated with accumulation of plugging material in the xylem vessels as Xanthomonas campestris multiplied and spread. Plug formation preceded a partial disorganization of the xylem, and spillage of bacteria from the vessels. The movement of eosin and 32PO43− was blocked within the vessels, and plugs caused water shortage in affected cells. Vein blackening, the first symptom of the disease, occurred after plugs had formed, and was associated with an accumulation of melanins among collapsing cells of the vascular bundle. The population of viable bacteria within the veins declined rapidly as the veins blackened.Advance of the bacteria in the xylem vessels accounted for the relationship between venation and lesion spread. A weakly virulent isolate spread extensively in the xylem vessels if inoculated with a virulent isolate, but not when inoculated alone. Acid mucopolysaccharide plugging material was not formed in vessels infected with the weakly virulent isolate.Water stress in cells of interveinal tissues of inoculated leaves resulted in chlorosis, loss in turgor, and loss of electrolytes. If net water loss from infected leaves was prevented by high atmospheric relative humidity, affected cells remained green and turgid and maintained their electrolyte balance.

EFFECT OF THE TYPE OF BACTERIOLOGICAL PEPTONE IN THE PLATING MEDIUM UPON THE ENUMERATION OF PASTEURIZATION-RESISTANT BACTERIA IN MILK

SummaryVarious bacteriological, peptones were studied as to effects on enumeration of pasteurization-resistant bacteria in milk. No appreciable differences were observed in colony counts of unheated cultures of thermoduric Micrococcus varians and Arthrobacter and Streptococcus sp. when plated with media containing different peptones. After laboratory pasteurization, colony counts differed substantially. Usually, media producing the highest counts yielded the largest and most easily discernible colonies. Bacto-Tryptone was deficient for colony development of thermoduric streptococci. Results indicate that, although seemingly adequate for enumeration in raw milk, the bacteriological peptone currently recommended for the standard plate count may not be satisfactory for the determination of the maximum viable bacteria population of pasteurized milk.

EFFECT OF pH OF PLATING MEDIUM ON ENUMERATION OF PASTEURIZATION-RESISTANT BACTERIA IN MILK

SummaryPlating media of different pH were studied as to effects on enumeration of pasteurization-resistant bacteria in milk. After heat treatment, thermoduric bacteria were more tolerant of pH levels above rather than below 7, and maximum mean thermoduric counts were obtained at pH 7.5. There were some exceptions, but usually the pH levels that yielded the highest counts also produced the largest and most easily discernible colonies. Pure cultures of thermoduric bacteria grew over a much wider range of pH before than they did after laboratory pasteurization. Results indicate that, although seemingly adequate for enumeration in raw milk, the medium pH currently recommended for the standard plate count may not be satisfactory for the determination of the maximum viable bacteria population of pasteurized milk.