scispace - formally typeset
Search or ask a question
Journal ArticleDOI

PCR-based detection of non-indigenous microorganisms in ‘pristine’ environments

01 May 2003-Journal of Microbiological Methods (Elsevier)-Vol. 53, Iss: 2, pp 157-164
TL;DR: It is concluded that despite these limitations, PCR and related technologies offer enormous scope for assessment of both natural and non-indigenous microbial distributions.
About: This article is published in Journal of Microbiological Methods.The article was published on 2003-05-01 and is currently open access. It has received 25 citations till now. The article focuses on the topics: Environmental pollution.

Summary (3 min read)

Introduction

  • For 'pristine' environments such as those found on the Antarctic continent, the application of these methods to the assessment of environmental contamination from human activities must be treated with caution.
  • Issues such as the possibility of non-human dispersal of organisms, stability and survival of non-indigenous organisms in vivo, the sensitivity, reproducibility and specificity of the PCR process (and particularly primer design) and the sampling regime employed must all be considered in detail.
  • The authors conclude that despite these limitations, PCR and related technologies offer enormous scope for assessment of both natural and non-indigenous microbial distributions.
  • Antarctica; Endemism; Human commensals; Primer design; Psychrophile, also known as Keywords.

1. Antarctica

  • The Antarctic continent has been geographically isolated from the rest of the world since its separation from Gondwanaland more than 10 million years ago (Vincent, 2000).
  • Some Antarctic habitats have remained isolated for hundreds to millions of years and are considered to be some of the most pristine ecosystems left on earth (Vincent, 2000).
  • The impact of human activities on Antartica's supposedly pristine habitats has become an international concern (Upton et al., 1997).
  • Anxiety over habitat destruction through mining and contamination of the Antarctic ecosystem with hydrocarbons led to the signing of an international treaty on environmental protection in 1991.
  • It is virtually impossible, without overly stringent protocols, to prevent transport and dispersal of non-indigenous microorganisms in the vicinity of tourist sites, research stations and field sites.

2. Human microbiota

  • There is a huge diversity of commensal microorganisms that are resident in and on the human body and a number of pathogens are carried by healthy individuals (Prescott et al., 1999).
  • Bacteria and fungi resident on human skin and in the mouth, nose and throat are also potential environmental contaminants, but their presence in pristine environments such as Antarctica has yet to be investigated.
  • Viable alien organisms may also disrupt ecosystems at a microscopic level.
  • These bacteria may be preserved in a non-viable or dormant state and their DNA may remain intact for long periods, during which they may be inadvertently identified as part of the indigenous community.
  • Under certain conditions intact DNA can be preserved in the environment for extremely long periods.

4. Endemic vs. cosmopolitan

  • A knowledge of whether pristine habitats have been contaminated with non-indigenous species is important for a number of reasons.
  • Secondly, the direct effect of alien species, and the movement of transmissible elements and plasmids from alien to indigenous organisms, may have a deleterious effect on endemic wildlife and communities.
  • Some studies have supported the concept that microorganisms have endemic distributions as for higher organisms (Cas-tenholz, 1996; Fulthorpe et al., 1998; Cho and Tiedje, 2000).
  • Many organisms found in Antarctica are not uniquely adapted for this ecosystem but originate elsewhere and opportunistically colonise this habitat (Gordon et al., 2000).
  • Obligate psychrophiles (as opposed to psychrotoler-ants) are more likely to belong to the former category.

6. Enteric bacteria

  • Enteric bacteria are the most studied indicators of human contamination.
  • Sjoling and Cowan (2000) used E. coli and Clostridium clostridiiforme as indicators of human faecal contamination in Antarctica.
  • They identified low levels of E. coli at several occupied locations, but Clostridium was only detected in a sewage outfall sample.
  • Other potential indicator species include Enterococcus spp., Peptostreptococcus (Pre-scott et al., 1999) and Salmonella (Cocolin et al., 1998; Palmgren et al., 2000).
  • Certain strains of these species may be specific to humans, whilst others may be found in the normal gut flora of other mammalian and avian hosts (Wang et al., 1996).

7. Normal microbiota of the skin

  • Malassezia is a basidiomycetous lipophilic yeast commonly isolated from the skin of warm blooded vertebrates (Guillot et al., 2000).
  • Other common skin microorganisms include Staphylococcus spp., such as S. epidermis; Streptococci and Candida spp. (Prescott et al., 1999).
  • There is a huge diversity of microorganisms inhabiting the mouth, throat and nose.
  • This Gram-positive bacterium was shown to produce a dextranase enzyme that retards plaque deposition on tooth surfaces (Ohnishi et al., 1995).

9. Identification of non-indigenous bacteria in the environment

  • These methods only identify those viable species that are easily cultured in vitro, and typically fail to isolate organisms that grow poorly in culture or have fastidious growth requirements, such as Malassezia spp. (Guillot et al., 2000).
  • It should be noted that currently over 99% of species are believed to be "unculturable" using current technologies (Ward, 1998).
  • Due to the limitations of culturing technologies, the polymerase chain reaction is becoming the preferred method for clinical identification of microorganisms (Wilson et al., 1991; Wang et al., 1996).
  • PCR allows amplification ofviable, non-viable and dormant cells, and primers can be tailored for amplification of specific taxonomic groups.

10. Direct PCR detection

  • PCR primers can be designed that are homologous to specific genes or oligonucleotides of specific species or strains (Baker et al., submitted forpublication).
  • False positive PCR bands and false negative results can also result from poor primer specificity.
  • As little as 70% primer-to-template homology is needed for annealing and elongation (Stern and Holland, Page | 6 be kept as nearto the melting temperature of the primer as possible (McPherson et al., 1994).
  • In addition, all primers need to be checked against a range of positive and negative controls to confirm specific amplification of the desired template and insure against non-specific amplification of other templates.
  • Primers can only be designed on the basis of available sequence data and thus although primers may be designed to be specific to DNA in the database they may also anneal to the DNA of other unsequenced organisms.

5 -TGTCCGCATAACCCTCTTCATT-3

  • These methods are obviously more time consuming and costly than direct methods, but potentially provide less ambiguous results.
  • In addition, less specific PCR primers can be used that amplify families or genera rather than specific species or strains.

12. Partial sequencing

  • Partial sequencing of PCR products can be highly informative if the DNA region chosen is suitably variable.
  • Highly conserved genes such as those coding for 16S rRNA are ideal for identifying organisms from different genera, but the level of base conservation in some parts of the gene will be too great to resolve species differences.
  • Goto et al. (2000) effectively used partial sequence data from hypervariable regions 1 and 2 of 16S rDNA to identify a number of Bacillus species.
  • Similarly, Monstein et al. (1998) has used partial sequencing of variable regions V4 and V9 of 16S rDNA to identify Enterococcus species.

13. Restriction enzyme analysis

  • The use of restriction enzyme analysis methodologies, such as ARDRA and RFLP, has also been effective to identify human commensal species (Guil-lotetal.,2000; Guptaetal.,2000).
  • After PCR, amplicons are digested with one or more restriction enzymes to form a digest fragment pattern.
  • False negative bands can, however, result from inadequate digestion leading to the formation of different banding patterns for identical samples.
  • Furthermore, if restriction enzymes are poorly chosen, different species may have identical restriction patterns.

14. Conclusions

  • The successful application of PCR for identification of non-indigenous organisms from the pristine habitats of Antarctica is less certain.
  • The two major '' problems'' are: (i) non-indigenous microorganisms may be transported to and distributed in the Antarctic continent by natural processes, and (ii) specific Page | 8.
  • The former may be resolved by the appropriate inclusion of 'pristine' samples in studies of the occurrence and distribution of known non-indigenous phylotypes.
  • Table 1 provides a list of published species-specific primers, designed for analytical use.

Did you find this useful? Give us your feedback

Citations
More filters
Journal ArticleDOI
TL;DR: A review of the scientific literature on the impacts of human activities on the Antarctic environment can be found in this article, where a range of impacts has been identified at a variety of spatial and temporal scales.
Abstract: We review the scientific literature, especially from the past decade, on the impacts of human activities on the Antarctic environment. A range of impacts has been identified at a variety of spatial and temporal scales. Chemical contamination and sewage disposal on the continent have been found to be long-lived. Contemporary sewage management practices at many coastal stations are insufficient to prevent local contamination but no introduction of non-indigenous organisms through this route has yet been demonstrated. Human activities, particularly construction and transport, have led to disturbances of flora and fauna. A small number of non-indigenous plant and animal species has become established, mostly on the northern Antarctic Peninsula and southern archipelagos of the Scotia Arc. There is little indication of recovery of overexploited fish stocks, and ramifications of fishing activity on bycatch species and the ecosystem could also be far-reaching. The Antarctic Treaty System and its instruments, in particular the Convention for the Conservation of Antarctic Marine Living Resources and the Environmental Protocol, provide a framework within which management of human activities take place. In the face of the continuing expansion of human activities in Antarctica, a more effective implementation of a wide range of measures is essential, in order to ensure comprehensive protection of the Antarctic environment, including its intrinsic, wilderness and scientific values which remains a fundamental principle of the Antarctic Treaty System. These measures include effective environmental impact assessments, long-term monitoring, mitigation measures for non-indigenous species, ecosystem-based management of living resources, and increased regulation of National Antarctic Programmes and tourism activities.

411 citations


Cites background from "PCR-based detection of non-indigeno..."

  • ...Molecular methods that ease the detection of specific microbes as “indicators” of human activity are also now available (Baker et al. 2003)....

    [...]

Journal ArticleDOI
TL;DR: The Antarctic continent harbors a range of specialized and sometimes highly localized microbial biotopes, and the consequences of some direct human impacts, such as physical disruption of microbial soil communities, are obvious if not yet quantitated.
Abstract: The Antarctic continent harbors a range of specialized and sometimes highly localized microbial biotopes. These include biotopes associated with desiccated mineral soils, rich ornithogenic soils, glacial and sea ice, ice-covered lakes, translucent rocks, and geothermally heated soils. All are characterized by the imposition of one or more environmental extremes (including low temperature, wide temperature fluctuations, desiccation, hypersalinity, high periodic radiation fluxes, and low nutrient status). As our understanding of the true microbial diversity in these biotopes expands from the application of molecular phylogenetic methods, we come closer to the point where we can make an accurate assessment of the impacts of environmental change, human intervention, and other natural and unnatural impositions. At present, it is possible to make reasonable predictions about the physical effects of local climate change, but only general predictions on possible changes in microbial community structure. The consequences of some direct human impacts, such as physical disruption of microbial soil communities, are obvious if not yet quantitated. Others, such as the dissemination of nonindigenous microorganisms into indigenous microbial communities, are not yet understood.

183 citations

Journal ArticleDOI
TL;DR: Results indicated that bacteria and fungi are not triggering CRS, and support the theory that in some cases CRS results from an immune hyperresponsiveness to commensal organisms.
Abstract: Importance Chronic rhinosinusitis (CRS) is the persistent inflammation of the sinus and nasal passages lasting over 3 months. The etiology of CRS is not well understood. Objective To obtain insights into the disease process, we contrasted the microbiome and immune response from patients with CRS and healthy controls. Design, Setting, and Participants A case vs control design was used. Samples were collected in the operating room in an institutional hospital or clinic. Thirty patients with CRS and 12 healthy controls undergoing surgery were recruited. Main Outcomes and Measures The microbiome was analyzed by deep sequencing of the bacterial 16S and fungal 18S ribosomal RNA genes. Immune response was measured by quantification of 30 different cytokines by multiplexed enzyme-linked immunosorbent assay, and immune cells in the lavage were identified by flow cytometry. The immune response of peripheral blood leukocytes to the lavage microbiota was assessed by interleukin (IL)-5 enzyme-linked immunospot assay. Results While quantitative increase in most bacterial and fungal species was observed in patients with CRS relative to controls, the microbiomes of patients with CRS were qualitatively similar to the controls. Because these results indicated that bacteria and fungi are not triggering CRS, we undertook a more detailed characterization of the immune response. Patients with CRS had increased levels of the following cytokines: IL-4, IL-5, IL-8, and IL-13, along with increased levels of eosinophils and basophils in the lavage. Importantly, peripheral blood leukocytes isolated from patients with CRS responded to control lavage samples (ie, to commensals) to produce IL-5. In contrast, the same lavage sample evoked no IL-5 production in leukocytes from healthy controls. Conclusions and Relevance These findings support the theory that in some cases CRS results from an immune hyperresponsiveness to commensal organisms.

125 citations

Journal ArticleDOI
TL;DR: Molecular studies on Antarctic lake communities are still in their infancy, but there is clear evidence from some taxonomic groups, for example the Cyanobacteria, that there is endemicity.
Abstract: Antarctic lakes are characterised by simplified, truncated food webs. The lakes range from freshwater to hypersaline with a continuum of physical and chemical conditions that offer a natural laboratory in which to study evolution. Molecular studies on Antarctic lake communities are still in their infancy, but there is clear evidence from some taxonomic groups, for example the Cyanobacteria, that there is endemicity. Moreover, many of the bacteria have considerable potential as sources of novel biochemicals such as low temperature enzymes and anti-freeze proteins. Among the eukaryotic organisms survival strategies have evolved, among which dependence on mixotrophy in phytoflagellates and some ciliates is common. There is also some evidence of evolution of new species of flagellate in the marine derived saline lakes of the Vestfold Hills. Recent work on viruses in polar lakes demonstrates high abundance and high rates of infection, implying that they may play an important role in genetic exchange in these extreme environments.

124 citations


Cites background from "PCR-based detection of non-indigeno..."

  • ...organisms (Baker et al. 2003), and as a result, much use...

    [...]

  • ...However, the enormous strength of PCR detection lies in accessing microbial taxonomy without the need to culture the organisms (Baker et al. 2003), and as a result, much use is currently made of 16S rRNA clone library construction and screening in Antarctic biodiversity studies (Bowman et al. 2000)....

    [...]

  • ...…in Antarctic lake systems may be transported to and distributed within the Antarctic continent by natural processes, and specific 16S rRNA gene sequences may be present, but beyond the limits of detection due to their low concentrations and heterogeneous distribution (Baker et al. 2003)....

    [...]

Journal ArticleDOI
TL;DR: New evidence has been obtained indicating that long-term persistence and regional isolation is a feature of the Antarctic terrestrial biota whose generality has not previously been appreciated, which opens important new cross-disciplinary linkages in the field of understanding the geological and glaciological history of the continent itself.

96 citations


Cites background from "PCR-based detection of non-indigeno..."

  • ...In recent years, the use of molecular biological methodologies has started to improve the potential of identifying non-indigenous microbes (e.g. Baker et al., 2003)....

    [...]

References
More filters
Journal ArticleDOI
TL;DR: This work re-assess the specificity of commonly used 16S rRNA gene primers and presents these data in tabular form designed as a tool to aid simple analysis, selection and implementation.

1,713 citations

01 Jan 2003
TL;DR: In this article, the specificity of commonly used 16S rRNA gene primers was evaluated and two new primers were designed for effective 'universal' Archaeal16S rDNA sequence amplification.
Abstract: The Polymerase Chain Reaction (PCR) has facilitated the detection of unculturable microorganisms in virtually any environmental source and has thus been used extensively in the assessment of environmental microbial diversity. This technique relies on the assumption that the gene sequences present in the environment are complementary to the ''universal'' primers used in their amplification. The recent discovery of new taxa with 16S rDNA sequences not complementary to standard universal primers suggests that current 16S rDNA libraries are not representative of true prokaryotic biodiversity. Here we re-assess the specificity of commonly used 16S rRNA gene primers and present these data in tabular form designed as a tool to aid simple analysis, selection and implementation. In addition, we present two new primer pairs specifically designed for effective 'universal' Archaeal 16S rDNA sequence amplification. These primers are found to amplify sequences from Crenarchaeote and Euryarchaeote type strains and environmental DNA.

1,511 citations

Book
01 Jan 1995
TL;DR: Optimizing PCR Use of specialty phosphoramidites in PCR Chemical methods for 5' non-isotopic labelling of PCR probes and primers and Linear amplification for the in vitro study of ligand/DNA interactions Ligand-mediated PCR.
Abstract: Optimizing PCR Use of specialty phosphoramidites in PCR Chemical methods for 5' non-isotopic labelling of PCR probes and primers Solid-phase PCR Solid-phase sequencing of PCR products cDNA cloning by RT - PCR Quantification of DNA and RNA by PCR PCR MIMICS: competitive DNA fragments for use in quantitative PCR In vitro expression of proteins from PCR products PCR-based approaches to human genome mapping Fingerprinting of DNA and RNA using arbitrarily primed PCR Mutational analysis Mutational analysis: new mutations Linear amplification for the in vitro study of ligand/DNA interactions Ligand-mediated PCR.

1,188 citations

Journal ArticleDOI
01 Nov 1984-Nature
TL;DR: Dried muscle from a museum specimen of the quagga, a zebra-like species that became extinct in 1883, is examined, and DNA was extracted in amounts approaching 1% of that expected from fresh muscle, and that the DNA was of relatively low molecular weight.
Abstract: To determine whether DNA survives and can be recovered from the remains of extinct creatures, we have examined dried muscle from a museum specimen of the quagga, a zebra-like species (Equus quagga) that became extinct in 1883 (ref. 1). We report that DNA was extracted from this tissue in amounts approaching 1% of that expected from fresh muscle, and that the DNA was of relatively low molecular weight. Among the many clones obtained from the quagga DNA, two containing pieces of mitochondrial DNA (mtDNA) were sequenced. These sequences, comprising 229 nucleotide pairs, differ by 12 base substitutions from the corresponding sequences of mtDNA from a mountain zebra, an extant member of the genus Equus. The number, nature and locations of the substitutions imply that there has been little or no postmortem modification of the quagga DNA sequences, and that the two species had a common ancestor 3-4 Myr ago, consistent with fossil evidence concerning the age of the genus Equus.

665 citations

Journal ArticleDOI
TL;DR: PCR procedures based on 16S rRNA gene sequences specific for 12 anaerobic bacteria that predominate in the human intestinal tract were developed and used for quantitative detection of these species in human and animal fecal samples tested.
Abstract: PCR procedures based on 16S rRNA gene sequences specific for 12 anaerobic bacteria that predominate in the human intestinal tract were developed and used for quantitative detection of these species in human (adult and baby) feces and animal (rat, mouse, cat, dog, monkey, and rabbit) feces. Fusobacterium prausnitzii, Peptostreptococcus productus, and Clostridium clostridiiforme had high PCR titers (the maximum dilutions for positive PCR results ranged from 10(-3) to 10(-8)) in all of the human and animal fecal samples tested. Bacteroides thetaiotaomicron, Bacteroides vulgatus, and Eubacterium limosum also showed higher PCR titers (10(-2) to 10(-6)) in adult human feces. The other bacteria tested, including Escherichia coli, Bifidobacterium adolescentis, Bifidobacterium longum, Lactobacillus acidophilus, Eubacterium biforme, and Bacteroides distasonis, were either at low PCR titers (less than 10(-2)) or not detected by PCR. The reported PCR procedure including the fecal sample preparation method is simplified and rapid and eliminates the DNA isolation steps.

492 citations

Frequently Asked Questions (1)
Q1. What are the contributions in "Pcr-based detection of non-indigenous microorganisms in 'pristine' environments - review article" ?

A list of species-specific primers, designed for analytical use, can be found in this paper.