Detection of microbial contaminants in mammalian cell cultures using a new fluorescence-based staining method

Aims: Microbial contamination of cell culture production processes is a current concern for biopharmaceutical industries. Traditional testing methods require several days to detect contamination and may advantageously be replaced by a rapid detection method. We developed a new method combining membrane filtration to microcolonies fluorescence staining method (MFSM) and compared it to epifluorescence microscopy. Methods and Results: Both methods were used to detect bacteria in CHO cells cultures. The epifluorescence microscopy showed to be limited by filterability, media interference and nonrobustness issues, whereas MFSM enabled consistent detection of Bacillus cereus, Staphylococcus epidermidis and Propionibacterium acnes after, respectively, 8, 9 and 48 h of incubation. Thanks to the nondestructive feature of the MFSM, stained membranes could be reincubated on culture media to yield visible colonies used for identification. Conclusions: The new method described in this study showed its ability to detect microbial contaminants in cell culture samples with time‐to‐results from 2–5 times shorter than the traditional testing method. Significance and Impact of the Study: The MFSM can be used as monitoring tool for cell cultures to significantly shorten detection times of microbial contamination, while preserving the ability to identify the contaminants and their viability.     

Biofilm formation by a fimbriae-deficient mutant of Actinobacillus actinomycetemcomitans

Actinobacillus actinomycetemcomitans strain 310-TR produces fimbriae and forms a tight biofilm in broth cultures, without turbid growth. The fimbriae-deficient mutant 310-DF, constructed in this study, was grown as a relatively fragile biofilm at the bottom of a culture vessel. Scanning electron microscopy revealed that on glass coverslips, 310-TR formed tight and spherical microcolonies, while 310-DF produced looser ones. These findings suggest that fimbriae are not essential for the surface-adherent growth but are required for enhancing cell-to-surface and cell-to-cell interactions to stabilize the biofilm. Treatment of the 310-DF biofilm with either sodium metaperiodate or DNase resulted in significant desorption of cells from glass surfaces, indicating that both carbohydrate residues and DNA molecules present on the cell surface are also involved in the biofilm formation.     

Analysis of size distribution and areal cell density of ammonia-oxidizing bacterial microcolonies in relation to substrate microprofiles in biofilms

A fine-scale in situ spatial organization of ammonia-oxidizing bacteria (AOB) in biofilms was investigated by combining molecular techniques (i.e., fluorescence in situ hybridization (FISH) and 16S rDNA-cloning analysis) and microelectrode measurements. Important parameters of AOB microcolonies such as size distribution and areal cell density of the microcolonies were determined and correlated with substrate microprofiles in the biofilms. In situ hybridization with a nested 16S rRNA-targeted oligonucleotide probe set revealed two different populations of AOB, Nitrosomonas europaea-lineage and Nitrosospira multiformis-lineage, coexisting in an autotrophic nitrifying biofilm. Nitrosospira formed looser microcolonies, with an areal cell density of 0.51 cells microm(-2), which was half of the cell density of Nitrosomonas (1.12 cells microm(-2)). It is speculated that the formation of looser microcolonies facilitates substrate diffusion into the microcolonies, which might be a survival strategy to low O(2) and NH(4) (+) conditions in the biofilm. A long-term experiment (4-week cultivation at different substrate C/N ratios) revealed that the size distribution of AOB microcolonies was strongly affected by better substrate supply due to shorter distance from the surface and the presence of organic carbon. The microcolony size was relatively constant throughout the autotrophic nitrifying biofilm, while the size increased by approximately 80% toward the depth of the biofilm cultured at the substrate C/N = 1. A short-term ( approximately 3 h) organic carbon addition experiment showed that the addition of organic carbon created interspecies competition for O(2) between AOB and heterotrophic bacteria, which dramatically decreased the in situ NH(4) (+)-uptake activity of AOB in the surface of the biofilms. This result might explain the spatial distribution of AOB microcolony size in the biofilms cultured at the substrate C/N = 1. These experimental results suggest O(2) and organic carbon were the main factors controlling the spatial organization and activity of AOB in biofilms. These findings are significantly important to further improve mathematical models used to describe how the slow-growing AOB develop their niches in biofilms and how that configuration affects nitrification performance in the biofilm.     

The effect of hydroxylamine on the activity and aggregate structure of autotrophic nitrifying bioreactor cultures

Addition of hydroxylamine (NH2OH) to autotrophic biomass in nitrifying bioreactors affected the activity, physical structure, and microbial ecology of nitrifying aggregates. When NH2OH is added to nitrifying cultures in 6-h batch experiments, the initial NH3-N uptake rates were physiologically accelerated by a factor of 1.4-13. NH2OH addition caused a 20-40% decrease in the median aggregate size, broadened the shape of the aggregate size distribution by up to 230%, and caused some of the microcolonies to appear slightly more dispersed. Longer term NH2OH addition in fed batch bioreactors decreased the median aggregate size, broadened the aggregate size distribution, and decreased NH3-N removal from >90% to values ranging between 75% and 17%. This altered performance is explained by quantitative fluorescence in situ hybridization (FISH) results that show inhibition of nitrifying populations, and by qPCR results showing that the copy numbers of amoA and nxrA genes gradually decreased by up to an order-of-magnitude. Longer term NH2OH addition damaged the active biomass. This research clarifies the effect of NH2OH on nitrification and demonstrates the need to incorporate NH2OH-related dynamics of the nitrifying biomass into mathematical models, accounting for both ecophysiological and structural responses.     

Label-free identification of bacterial microcolonies via elastic scattering

Label‐free microcolony identification via elastic light scattering was investigated for three different genera: Salmonella enterica serovar Montevideo, Listeria monocytogenes F4244, and Escherichia coli DH5α. Microcolonies were defined as bacterial colonies in their late‐lag phase to early‐exponential phase with the diameter range of 100–200 µm. To link biophysical characteristics with corresponding scattering patterns, a phase contrast microscope and a confocal displacement meter were used to measure the colony diameter and its 3D height profile. The results indicated that the growth characteristics of microcolonies were encoded in their morphologies which correlated to the characteristic diffraction patterns. Proposed methodology was able to classify three genera based on comprehensive phenotypic map which incorporated growth speed, ring count, and colony diameter. While the proposed method illustrated the possibility of discriminating microcolonies in their early growth stage, more thorough biophysical understanding is needed to expand the technology to other species. Biotechnol. Bioeng. 2011; 108:637–644. © 2010 Wiley Periodicals, Inc.     

Effects of nutrient availability and Ochromonas sp. predation on size and composition of a simplified aquatic bacterial community

Predation and competition are two main factors that determine the size and composition of aquatic bacterial populations. Using a simplified bacterial community, composed of three strains characterized by different responses to predation, a short-term laboratory experiment was performed to evaluate adaptations and relative success in communities with experimentally controlled levels of predation and nutrient availability. A strain with a short generation time (Pseudomonas putida), one with high plasticity in cell morphology (Flectobacillus sp. GC5), and one that develops microcolonies (Pseudomonas sp. CM10), were selected. The voracious flagellate Ochromonas sp. was chosen as a predator. To describe adaptations against grazing and starvation, abundance, biomass and relative heterogeneity of bacteria were measured. On the whole, the strains in the predation-free cultures exhibited unicellular growth, and P. putida represented the largest group. The presence of Ochromonas strongly reduced bacterial abundance, but not always the total biomass. The activity of grazers changed the morphological composition of the bacterial communities. Under grazing pressure the relative composition of the community depended on the substrate availability. In the presence of predators, P. putida abundance declined in both high and low nutrient treatments, and Pseudomonas CM10 developed colonies. Flectobacillus was only numerically codominant in the nutrient-rich environments.     

Structural organization and dynamics of exopolysaccharide matrix and microcolonies formation by Streptococcus mutans in biofilms

Aims: To investigate the structural organization and dynamics of exopolysaccharides (EPS) matrix and microcolonies formation by Streptococcus mutans during the biofilm development process. Methods and Results: Biofilms of Strep. mutans were formed on saliva‐coated hydroxyapatite (sHA) discs in the presence of glucose or sucrose (alone or mixed with starch). At specific time points, biofilms were subjected to confocal fluorescence imaging and computational analysis. EPS matrix was steadily formed on sHA surface in the presence of sucrose during the first 8 h followed by a threefold biomass increase between 8 and 30 h of biofilm development. The initial formation and further development of three‐dimensional microcolony structure occurred concomitantly with EPS matrix synthesis. Tridimensional renderings showed EPS closely associated with microcolonies throughout the biofilm development process forming four distinct domains (i) between sHA surface and microcolonies, (ii) within, (iii) covering and (iv) filling the spaces between microcolonies. The combination of starch and sucrose resulted in rapid formation of elevated amounts of EPS matrix and faster assembly of microcolonies by Strep. mutans, which altered their structural organization and susceptibility of the biofilm to acid killing (vs sucrose‐grown biofilms; P < 0·05). Conclusions: Our data indicate that EPS modulate the development, sequence of assembly and spatial distribution of microcolonies by Strep. mutans. Significance and Impact of the Study: Simultaneous visualization and analysis of EPS matrix and microcolonies provide a more precise examination of the structural organization of biofilms than labelling bacteria alone, which could be a useful approach to elucidate the exact mechanisms by which Strep. mutans influences oral biofilm formation and possibly identify novel targets for effective antibiofilm therapies.