Belinda C. Ferrari

Fluorescence staining and flow cytometry for monitoring microbial cells

Large numbers of microbiological samples are analysed annually using traditional culture-based techniques. These techniques take hours to days to yield a result, are tedious and are not suitable for non-culturable microorganisms. Further, culture-based techniques do not provide real-time information on the physiological status of the organism in situ which is important in the industrial manufacture of many microbial products. Flow cytometry offers the prospect of real-time microbial analysis of individual microorganisms, without dependency on microbial culture. However, flow cytometry has not been extensively used as a tool for routine microbial analysis. This has been mainly due to the high cost and complexity of instrumentation, the need for trained flow cytometrists and the lack of assay kits with appropriate biological reagents for specific applications. Many modern instruments are now relatively simple to operate, due to improvements in the user-interface, and no longer need a specialist operator. However, most cytometers are still reliant on analogue technology first developed 20-30 years ago. The incorporation of modern, solid state opto-electronics combined with micro-fabrication and digital signal processing technology offers the prospect of simple to use, low cost and robust instruments suitable for microbial analyses. Advances are being made in the development of a range of biological reagents and these are now being formulated into simple to use kits for microbiological applications. Currently, these kits are largely restricted to simple analyses, for example to assay for total or viable numbers of microorganisms present. However, technologies are available to selectively label specific types of microorganisms. For example, fluorescent antibodies can be used to label microorganisms according to expression of particular antigens, fluorescent in situ hybridisation to label according to phylogeny and fluorogenic enzymatic substrates to label according to expression of specific enzyme activities. Reagents are also available that stain viruses sufficiently brightly to enable their direct detection in environments such as sea water. Microorganisms need to be detected in a variety of different matrices (e.g., water, mud, food, and beverages) and these matrices may be highly variable in nature (e.g., tap water compared to river water). Many matrices have high background autofluorescence (e.g., algae and minerals in water samples) or may bind non-specifically to the fluorescent biological reagents used (e.g., protein micelles in milk). Formulation of biological reagents and sample pre-treatments are critical to the development of suitable microbiological assays. Here, developments in instrumentation and biological reagents for microbiological applications are reviewed with specific examples from environmental or industrial microbiology. The broader considerations for the development of microbial assays for flow cytometry are also considered.

Microcolony cultivation on a soil substrate membrane system selects for previously uncultured soil bacteria.

ABSTRACT Traditional microbiological methods of cultivation recover less than 1% of the total bacterial species, and the culturable portion of bacteria is not representative of the total phylogenetic diversity. Classical cultivation strategies are now known to supply excessive nutrients to a system and therefore select for fast-growing bacteria that are capable of colony or biofilm formation. New approaches to the cultivation of bacteria which rely on growth in dilute nutrient media or simulated environments are beginning to address this problem of selection. Here we describe a novel microcultivation method for soil bacteria that mimics natural conditions. Our soil slurry membrane system combines a polycarbonate membrane as a growth support and soil extract as the substrate. The result is abundant growth of uncharacterized bacteria as microcolonies. By combining microcultivation with fluorescent in situ hybridization, previously “unculturable” organisms belonging to cultivated and noncultivated divisions, including candidate division TM7, can be identified by fluorescence microscopy. Successful growth of soil bacteria as microcolonies confirmed that the missing culturable majority may have a growth strategy that is not observed when traditional cultivation indicators are used.

Community fingerprinting in a sequencing world

Despite decreasing costs, generating large-scale, well-replicated and multivariate microbial ecology investigations with sequencing remains an expensive and time-consuming option. As a result, many microbial ecology investigations continue to suffer from a lack of appropriate replication. We evaluated two fingerprinting approaches - terminal restriction fragment length polymorphism (T-RFLP) and automated ribosomal intergenic spacer analysis (ARISA) against 454 pyrosequencing, by applying them to 225 polar soil samples from East Antarctica and the high Arctic. By incorporating local and global spatial scales into the dataset, our aim was to determine whether various approaches differed in their ability and hence utility, to identify ecological patterns. Through the reduction in the 454 sequencing data to the most dominant OTUs, we revealed that a surprisingly small proportion of abundant OTUs (< 0.25%) was driving the biological patterns observed. Overall, ARISA and T-RFLP had a similar capacity as sequencing to separate samples according to distance at a local scale, and to correlate environmental variables with microbial community structure. Pyrosequencing had a greater resolution at the global scale but all methods were capable of significantly differentiating the polar sites. We conclude fingerprinting remains a legitimate approach to generating large datasets as well as a cost-effective rapid method to identify samples for elucidating taxonomic information or diversity estimates with sequencing methods.

Iron uptake and toxin synthesis in the bloom-forming Microcystis aeruginosa under iron limitation

Toxin production during cyanobacterial blooms poses a significant public health threat in water bodies globally and requires the development of effective bloom management strategies. Previously, synthesis of the hepatotoxin microcystin has been proposed to be regulated by iron availability, but the contribution of the toxin to the adaptation of cyanobacteria to environmental stresses, such as changing light intensity and nutrient limitation, remains unclear. The aim of this study was to compare the iron stress response in toxic and non-toxic strains of Microcystis aeruginosa subjected to moderate and severe iron limitation. The transcription of a number of genes involved in iron uptake, oxidative stress response, toxin synthesis and transcriptional control of these processes was accessed by quantitative real-time PCR (qRT-PCR). The process of adaptation of M. aeruginosa to iron stress was found to be highly dynamic and strain-specific. Toxin production in PCC 7806 increased in an iron-dependent manner and appeared to be regulated by FurA. The inability to produce microcystin, either due to natural mutations in the mcy gene cluster or due to insertional inactivation of mcyH, affected the remodelling of the photosynthetic machinery in iron-stressed cells, the transport of Fe(II) and transcription of the Fur family of transcriptional regulators. The presence of the toxin appears to give an advantage to microcystin-producing cyanobacteria in the early stages of exposure to severe iron stress and may protect the cell from reactive oxygen species-induced damage.

Detection of Oxytetracycline Production by Streptomyces rimosus in Soil Microcosms by Combining Whole-Cell Biosensors and Flow Cytometry

ABSTRACT Combining the high specificity of bacterial biosensors and the resolution power of fluorescence-activated cell sorting (FACS) provided qualitative detection of oxytetracycline production byStreptomyces rimosus in soil microcosms. A plasmid containing a transcriptional fusion between thetetR-regulated Ptet promoter from Tn10 and a FACS-optimized gfp gene was constructed. When harbored by Escherichia coli, this plasmid produces large amounts of green fluorescent protein (GFP) in the presence of tetracycline. This tetracycline biosensor was used to detect the production of oxytetracycline by S. rimosusintroduced into sterile soil. The tetracycline-induced GFP-producing biosensors were detected by FACS analysis, enabling the detection of oxytetracycline encounters by single biosensor cells. This approach can be used to study interactions between antibiotic producers and their target organisms in soil.

Ecological advantages of autolysis during the development and dispersal of Pseudoalteromonas tunicata biofilms.

ABSTRACT In the ubiquitous marine bacterium Pseudoalteromonas tunicata, subpopulations of cells are killed by the production of an autocidal protein, AlpP, during biofilm development. Our data demonstrate an involvement of this process in two parameters, dispersal and phenotypic diversification, which are of importance for the ecology of this organism and for its survival within the environment. Cell death in P. tunicata wild-type biofilms led to a major reproducible dispersal event after 192 h of biofilm development. The dispersal was not observed with a ΔAlpP mutant strain. Using flow cytometry and the fluorescent dye DiBAC4(3), we also show that P. tunicata wild-type cells that disperse from biofilms have enhanced metabolic activity compared to those cells that disperse from ΔAlpP mutant biofilms, possibly due to nutrients released from dead cells. Furthermore, we report that there was considerable phenotypic variation among cells dispersing from wild-type biofilms but not from the ΔAlpP mutant. Wild-type cells that dispersed from biofilms showed significantly increased variations in growth, motility, and biofilm formation, which may be important for successful colonization of new surfaces. These findings suggest for the first time that the autocidal events mediated by an antibacterial protein can confer ecological advantages to the species by generating a metabolically active and phenotypically diverse subpopulation of dispersal cells.

Molecular epidemiology, spatiotemporal analysis, and ecology of sporadic human cryptosporidiosis in Australia

ABSTRACT Parasites from the Cryptosporidium genus are the most common cause of waterborne disease around the world. Successful management and prevention of this emerging disease requires knowledge of the diversity of species causing human disease and their zoonotic sources. This study employed a spatiotemporal approach to investigate sporadic human cryptosporidiosis in New South Wales, Australia, between January 2008 and December 2010. Analysis of 261 human fecal samples showed that sporadic human cryptosporidiosis is caused by four species; C. hominis, C. parvum, C. andersoni, and C. fayeri. Sequence analysis of the gp60 gene identified 5 subtype families and 31 subtypes. Cryptosporidium hominis IbA10G2 and C. parvum IIaA18G3R1 were the most frequent causes of human cryptosporidiosis in New South Wales, with 59% and 16% of infections, respectively, attributed to them. The results showed that infections were most prevalent in 0- to 4-year-olds. No gender bias or regional segregation was observed between the distribution of C. hominis and C. parvum infections. To determine the role of cattle in sporadic human infections in New South Wales, 205 cattle fecal samples were analyzed. Four Cryptosporidium species were identified, C. hominis, C. parvum, C. bovis, and C. ryanae. C. parvum subtype IIaA18G3R1 was the most common cause of cryptosporidiosis in cattle, with 47% of infections attributed to it. C. hominis subtype IbA10G2 was also identified in cattle isolates.

Cultivating previously uncultured soil bacteria using a soil substrate membrane system

Most bacteria are recalcitrant to traditional cultivation in the laboratory. The soil substrate membrane system provides a simulated environment for the cultivation of previously undescribed soil bacteria as microcolonies. The system uses a polycarbonate membrane as a solid support for growth and soil extract as the substrate. Diverse microcolonies can be visualized using total bacterial staining combined with fluorescence in situ hybridization (FISH) after 7–10-d incubation. Molecular typing shows that the majority of microcolony-forming bacteria recovered using this protocol were resistant to growth using standard methods. The protocol takes <4 h of bench time over the 10-d period.

Glycoprotein 60 diversity in C. hominis and C. parvum causing human cryptosporidiosis in NSW, Australia.

Management and control of cryptosporidiosis in human requires knowledge of Cryptosporidium species contributing to human disease. Markers that are able to provide information below the species level have become important tools for source tracking. Using the hypervariable surface antigen, glycoprotein 60 (GP60), C. hominis (n=37) and C. parvum (n=32) isolates from cryptosporidiosis cases in New South Wales, Australia, were characterised. Extensive variation was observed within this locus and the isolates could be divided into 8 families and 24 different subtypes. The subtypes identified have global distributions and indicate that anthroponotic and zoonotic transmission routes contribute to sporadic human cryptosporidiosis in NSW.

Molecular Epidemiology and Spatial Distribution of a Waterborne Cryptosporidiosis Outbreak in Australia

ABSTRACT Cryptosporidiosis is one of the most common waterborne diseases reported worldwide. Outbreaks of this gastrointestinal disease, which is caused by the Cryptosporidium parasite, are often attributed to public swimming pools and municipal water supplies. Between the months of January and April in 2009, New South Wales, Australia, experienced the largest waterborne cryptosporidiosis outbreak reported in Australia to date. Through the course of the contamination event, 1,141 individuals became infected with Cryptosporidium. Health authorities in New South Wales indicated that public swimming pool use was a contributing factor in the outbreak. To identify the Cryptosporidium species responsible for the outbreak, fecal samples from infected patients were collected from hospitals and pathology companies throughout New South Wales for genetic analyses. Genetic characterization of Cryptosporidium oocysts from the fecal samples identified the anthroponotic Cryptosporidium hominis IbA10G2 subtype as the causative parasite. Equal proportions of infections were found in males and females, and an increased susceptibility was observed in the 0- to 4-year age group. Spatiotemporal analysis indicated that the outbreak was primarily confined to the densely populated coastal cities of Sydney and Newcastle.