Bacterial Populations In Samples Research Articles

The Influence of DNA Extraction and Lipid Removal on Human Milk Bacterial Profiles.

Culture-independent molecular techniques have advanced the characterization of environmental and human samples including the human milk (HM) bacteriome. However, extraction of high-quality genomic DNA that is representative of the bacterial population in samples is crucial. Lipids removal from HM prior to DNA extraction is common practice, but this may influence the bacterial population detected. The objective of this study was to compare four commercial DNA extraction kits and lipid removal in relation to HM bacterial profiles. Four commercial DNA extraction kits, QIAamp® DNA Microbiome Kit, ZR Fungal/Bacterial DNA MiniPrep™, QIAsymphony DSP DNA Kit and ZymoBIOMICS™ DNA Miniprep Kit, were assessed using milk collected from ten healthy lactating women. The kits were evaluated based on their ability to extract high quantities of pure DNA from HM and how well they extracted DNA from bacterial communities present in a commercial mock microbial community standard spiked into HM. Finally, the kits were evaluated by assessing their extraction repeatability. Bacterial profiles were assessed using Illumina MiSeq sequencing targeting the V4 region of the 16S rRNA gene. The ZR Fungal/Bacterial DNA MiniPrep™ and ZymoBIOMICS™ DNA Miniprep (Zymo Research Corp., Irvine, CA, USA) kits extracted the highest DNA yields with the best purity. DNA extracted using ZR Fungal/Bacterial DNA MiniPrep™ best represented the bacteria in the mock community spiked into HM. In un-spiked HM samples, DNA extracted using the QIAsymphony DSP DNA kit showed statistically significant differences in taxa prevalence from DNA extracted using ZR Fungal/Bacterial DNA MiniPrep™ and ZymoBIOMICS™ DNA Miniprep kits. The only difference between skim and whole milk is observed in bacterial profiles with differing relative abundances of Enhydrobacter and Acinetobacter. DNA extraction, but not lipids removal, substantially influences bacterial profiles detected in HM samples, emphasizing the need for careful selection of a DNA extraction kit to improve DNA recovery from a range of bacterial taxa.

A Dual Microscopy-Based Assay To Assess Listeria monocytogenes Cellular Entry and Vacuolar Escape.

Listeria monocytogenes is a Gram-positive bacterium and a facultative intracellular pathogen that invades mammalian cells, disrupts its internalization vacuole, and proliferates in the host cell cytoplasm. Here, we describe a novel image-based microscopy assay that allows discrimination between cellular entry and vacuolar escape, enabling high-content screening to identify factors specifically involved in these two steps. We first generated L. monocytogenes and Listeria innocua strains expressing a β-lactamase covalently attached to the bacterial cell wall. These strains were then incubated with HeLa cells containing the Förster resonance energy transfer (FRET) probe CCF4 in their cytoplasm. The CCF4 probe was cleaved by the bacterial surface β-lactamase only in cells inoculated with L. monocytogenes but not those inoculated with L. innocua, thereby demonstrating bacterial access to the host cytoplasm. Subsequently, we performed differential immunofluorescence staining to distinguish extracellular versus total bacterial populations in samples that were also analyzed by the FRET-based assay. With this two-step analysis, bacterial entry can be distinguished from vacuolar rupture in a single experiment. Our novel approach represents a powerful tool for identifying factors that determine the intracellular niche of L. monocytogenes.

ARISA analysis of ruminal bacterial community dynamics in lactating dairy cows during the feeding cycle

The bovine rumen undergoes substantial changes in environmental conditions during the animal's feeding cycle, but the effects of these changes on microbial populations have not been examined systematically. Two dairy cows fed a mixed forage/concentrate ration at 12 h intervals over 4 feeding cycles displayed substantial changes in ruminal pH and volatile fatty acid (VFA) concentrations. Automated ribosomal intergenic spacer analysis (ARISA) of solid- and liquid-associated bacterial populations in samples collected at 2, 4, 6, 9, and 12 h after feeding revealed a high degree of bacterial diversity. A total of 155 different amplicon lengths (ALs) were detected across all 83 samples, and 11–74 detected per sample. A substantial proportion (11%) of the ALs was detected in one cow but not in the other. The proportions of ALs that were detected only in the liquid phase or the solid phase were 13.5% and 1.9%, respectively. Correspondence analysis indicated that bacterial community composition differed between cows and between solid or liquid phases, but overall the solid-associated population displayed less change in composition within and across feeding cycles. The data support the notion that cows fed the same diets can have substantial differences in bacterial community composition, and that the solids-associated (biofilm) communities display greater stability than do associated planktonic communities.

An ecology-based analysis of irreversible biofouling in membrane bioreactors

To provide the first step towards a microbial ecology-based understanding of irreversible membrane biofouling, four laboratory-scale membrane bioreactors (MBRs) were operated to investigate the identity of bacterial populations highly correlated with irreversible membrane biofouling. The conventional MBR was divided into two separate experimental units. Unit one consisted of four suspended-growth, activated sludge, sequencing batch bioreactors treating a synthetic paper mill wastewater. Unit two consisted of a microfiltration membrane cell. Amplified ribosomal deoxyribonucleic acid restriction analysis (ARDRA) was used to compare the predominant bacterial populations in samples of mixed liquor and irreversibly bound to the membrane surface. The results of ARDRA showed a significant difference between the planktonic and sessile bacterial communities suggesting that irreversible biofouling of microfiltration membranes may be more highly correlated to specific bacterial populations rather than the total, bulk concentration of biomass. A custom-built mini-flow cell and light microscopy were used to visualise the early formation of biofilms by two pure cultures (Escherichia coli and Acinetobacter calcoaceticus) on membrane surfaces. The results confirmed that A. calcoaceticus was able to enhance the initiation of biofilm formation on microfiltration membranes.

Influence of marinades on survival during storage of acid-adapted and nonadapted Listeria monocytogenes inoculated post-drying on beef jerky

The objective of the present study was to investigate the survival of acid-adapted and nonadapted Listeria monocytogenes inoculated post-drying on dried beef slices (beef jerky), which were treated (24 h, 4 °C) with the following marinades before drying at 60 °C for 10 h: (1) control (C), (2) traditional marinade (TM), (3) modified marinade; double the amount of TM with added 1.2% sodium lactate, 9% acetic acid, and 68% soy sauce with 5% ethanol (MM), (4) dipping into 5% acetic acid and then TM (AATM), and (5) dipping into 1% Tween 20 and then into 5% acetic acid followed by the TM (TWTM). Dried meat slices were inoculated with acid-adapted or nonadapted L. monocytogenes (ca. 5.7 log CFU/cm 2) prior to aerobic storage at 25 °C for 60 days. Survivors were determined using tryptic soy agar with 0.1% pyruvate (TSAP) and PALCAM agar. Results showed that surviving bacterial populations on TWTM, AATM, and MM treatments were significantly ( P<0.05) lower than those surviving on C and TM until 42 days of storage. By the end of 60 days of storage, bacterial populations in all treatments were not different regardless of acid adaptation or recovery media, except for treatment C inoculated with nonadapted cultures, which had significantly higher TSAP counts than other treatments. There was no significant ( P≥0.05) difference in survival of previously acid-adapted and nonadapted bacterial populations in samples of TWTM, AATM, and MM treatments. However, bacterial populations that were nonadapted were significantly higher than those that were acid-adapted on products of C and TM treatments on days 60 and 24, respectively. The earliest complete elimination (enrichment negative) of the pathogen occurred by day 28 (AATM) in products inoculated with acid-adapted cultures and by day 42 (TWTM and AATM) in products inoculated with nonadapted cultures. These results indicate that use of modified marinades in jerky processing and low water activity provided antimicrobial effects against possible post-processing contamination with L. monocytogenes.

Molecular detection of marine bacterial populations on beaches contaminated by the Nakhodka tanker oil-spill accident.

In January 1997, the tanker Nakhodka sank in the Japan Sea, and more than 5000 tons of heavy oil leaked. The released oil contaminated more than 500 km of the coastline, and some still remained even by June 1999. To investigate the long-term influence of the Nakhodka oil spill on marine bacterial populations, sea water and residual oil were sampled from the oil-contaminated zones 10, 18, 22 and 29 months after the accident, and the bacterial populations in these samples were analysed by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA fragments. The dominant DGGE bands were sequenced, and the sequences were compared with those in DNA sequence libraries. Most of the bacteria in the sea water samples were classified as the Cytophaga-Flavobacterium-Bacteroides phylum, alpha-Proteobacteria or cyanobacteria. The bacteria detected in the oil paste samples were different from those detected in the sea water samples; they were types related to hydrocarbon degraders, exemplified by strains closely related to Sphingomonas subarctica and Alcanivorax borkumensis. The sizes of the major bacterial populations in the oil paste samples ranged from 3.4 x 10(5) to 1.6 x 10(6) bacteria per gram of oil paste, these low numbers explaining the slow rate of natural attenuation.

Bacterial populations in samples of bioleached copper ore as revealed by analysis of DNA obtained before and after cultivation.

The composition of bacterial populations in copper bioleaching systems was investigated by analysis of DNA obtained either directly from ores or leaching solutions or after laboratory cultures. This analysis consisted of the characterization of the spacer regions between the 16 and 23S genes in the bacterial rRNA genetic loci after PCR amplification. The sizes of the spacer regions, amplified from DNAs obtained from samples, were compared with the sizes of those obtained from cultures of the main bacterial species isolated from bioleaching systems. This allowed a preliminary assessment of the bacterial species present in the samples. Identification of the bacteria was achieved by partial sequencing of the 16S rRNA genes adjacent to the spacer regions. The spacer regions observed in DNA from columns leached at different iron concentrations indicated the presence of a mixture of different bacteria. The spacer region corresponding to Thiobacillus ferrooxidans was the main product observed at high ferrous iron concentration. At low ferrous iron concentration, spacer regions of different lengths, corresponding to Thiobacillus thiooxidans and "Leptospirillum ferrooxidans" were observed. However, T. ferrooxidans appeared to predominate after culture of these samples in medium containing ferrous iron as energy source. Although some of these strains contained singular spacer regions, they belonged within previously described groups of T. ferrooxidans according to the nucleotide sequence of the neighbor 16S rRNA. These results illustrate the bacterial diversity in bioleaching systems and the selective pressure generated by different growth conditions.

Survival of Listeria monocytogenes on Processed Poultry

ABSTRACTThe potential for Listeria monocytogenes to survive and proliferate on processed poultry was investigated. Chicken breasts were inoculated with L. monocytogenes, cooked to one of five different internal temperatures, then either vacuum‐packaged or wrapped in an oxygen permeable film and stored for up to four wk at 4oC and 10oC. Survivors were encountered at each cooking temperature employed. The magnitude of lethality was directly related to the cooking temperature. No significant increase in microbial populations occurred through 2 wk of storage. Bacterial populations in samples that were overwrapped increased significantly by week 4, except for the 160oF (71.1oC) treatment. For samples which were vacuum‐packaged, only the bacterial population in the 150oF (65.6oC) treatment significantly increased. Differences due to packaging were observed at 4oC storage but not at 10oC storage.

The effect of bacteria on the solubilization of silica in diatom frustules

Patrick, Sheila &amp; Holding, A.J. 1985. The effect of bacteria on the solubilization of silica in diatom frustules. Journal of Applied Bacteriology59, 7–16.Natural bacterial populations in samples of water from Loch Leven and Lough Neagh increased the rate of solubilization of frustule silica from an axenic Cyclotella meneghiniana culture, compared with sterile autolysis, at 25d̀C. In the inoculated cultures 50–60% of the silica was solubilized over a period of 30 d. Bacterial populations in Loch Leven water also enhanced the solubilization of silica from non‐axenic cultures of Asterionella formosa, Tabellaria flocculosa, Navicula pellicu‐losa and C. meneghiniana, compared with control cultures sterilized with mercuric chloride. Similar results were obtained with Lough Neagh populations incubated with A. formosa. In comparison with untreated cells, the treatment of diatom cells with ultra‐sonication did not increase the release of silica. Pure cultures of bacteria from Loch Leven water enhanced the release of silica from non‐axenic A. formosa and axenic C. meneghiniana compared with sterile control treatments. The variation in the ability of cultures to solubilize the frustule silica appeared to be related to their potential to produce hydrolytic enzymes. Natural populations of Loch Leven and Lough Neagh water bacteria and certain bacterial cultures caused the diatoms to aggregate, which did not enhance the release of silica.