A fast but reliable identification of pathogens causing acute infections is a highly important issue in microbiological diagnostics. Conventional culture-based methods are well adapted and quite inexpensive but time-consuming and accompanied by certain limitations. Modern molecular methods have been increasingly incorporated in laboratories, but can they fully replace the traditional microbiological diagnostics? This article will focus on bacterial identification and characterisation methods in microbiological diagnostics including currently used technologies with their advantages and drawbacks. An overview table of the article will summarise all important information.

Conventional methods – the gold standard

Traditional diagnostics are based on the ability of the microorganisms to grow under artificial conditions. Some examples are microbiological culture using selective or differential medium, microscopy, Gram-staining and biochemical tests (1,2).

These methods are highly sensitive, reliable, low-priced and provide both qualitative and quantitative results on the bacterial populations present in the clinical sample. However, these samples are commonly mixed cultures including diverse pathogens but also microorganisms of the normal flora. Typically, the isolation of pure cultures applying several growth steps is necessary to characterise the bacteria in more detail (1). Consequently, the conventional methods are labour-intensive and time-consuming, since the results are not available until at least 1-3 days (2,3,4). Moreover, the culture-based methods reach their limits with specific microorganisms that do not grow on or in artificial media (1).

Molecular methods – the upcoming rival

Molecular methods substantially revolutionised microbiological diagnosis. These techniques are more convenient than conventional methods as they are generally faster, more specific, precise and preceding cultivation of microorganisms is not necessary. In contrast to the conventional methods, the identification and characterisation of unculturable and slow-growing pathogens is easier to implement. In addition, they can be performed and results interpreted by staff with no taxonomical expertise. Moreover, they apply more stable genotypic characteristics for identification than traditional techniques using phenotypic characteristics (1,2,5).

Immunological techniques – for time- and cost-efficient detection

Immunological methods rely on binding of antibodies to specific antigens of target bacteria. Commonly used techniques are enzyme-linked immunosorbent assay (ELISA) and serological assays. These methods improved the microbial diagnosis because of their high-throughput capacity, time- and cost-efficiency and ease-of-use (1).

ELISA uses an enzyme-mediated colour change reaction to detect the presence of the specific pathogen/antigen (6,7). An advantage of this method is the possibility to quantify the pathogen/antigen (2,5). But like all other techniques, there are also some limitations: both specificity and sensitivity are reduced due to the difficulty to generate selective antibodies and the requirement of large amounts of antigens for quantification (1).

Another immunological approach – the serological assay – is used to detect human serum antibodies that appear specifically in response to acute or past microbial infection. However, these assays are usually inadequate due to their long time the antibodies need to be produced within the human body (1).

Nucleic acid-based techniques – for highly specific detection

Nucleic acid-based technologies include several variations of hybridisations, polymerase chain reaction (PCR) and sequencing as well as DNA/RNA microarrays (8). All these methods base on the selection of genetic sequences through which pathogens can be specifically identified. For this purpose, generally ubiquitously conserved genes – called housekeeping genes – are used or random parts of the genome are screened (2). However, these methods don’t provide information about the cellular metabolic state because they just detect intact DNA (1).

Hybridisation methods turned out to be a promising tool for rapid and reliable detection and identification of bacteria. The assays are based on a direct hybridisation of labelled (e. g. isotopes, fluorophores) oligonucleotide probes with species-specific DNA/RNA (1,9). A disadvantage of this method is the necessity of pre-cultivation of the microorganisms to produce significant amounts of cells to obtain a detectable signal. In addition, the number of probes that can be used in one experiment is limited and background noise might be a problem (1,5,9,10).

The advent of PCR enabled the detection of even small amounts of genetic material encompassing DNA and RNA by amplification. PCR is very fast, sensitive and highly specific for bacteria whose sequence is already known. But they are limited in multiplexing and discovery of novel species or detection of variant strains of a known species (11,12,13).

In contrast, sequencing technologies provide the most detailed, unbiased information of all nucleic-acid based methods and are able to reveal unknown organisms. But depending on the application, it can be cost-intensive and time-consuming. Multiplex sequencing reduces the costs but decreases the sensitivity of the analysis, which might be an issue when pathogens have low abundance in the clinical sample (13).

DNA/RNA-Microarrays – collection of various microscopic DNA/RNA-spots (probe) attached to a solid surface – are found in the middle range regarding cost, processing time, sensitivity, specificity and ability to detect novel organisms (13). The currently available arrays are able to test for the presence of thousands of different microbes simultaneously (e.g. LLMDA), but generally, a previous labelling (e.g. fluorophore, silver, chemiluminescence) of the samples is necessary making it more labour- and cost-intensive. However, the usage of label-free devices might overcome this issue and turn microarrays to a more attractive technique.

MALDI-TOF mass spectrometry – a new trend

Another technique to identify pathogens is the matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI-TOF MS). This approach identifies species in a microbial community and even differentiates strains based on molecular signatures (e.g. rRNA) (14). A characteristic spectrum is recorded representing a specific fingerprint for each species/strain (15,16).

This technique has fast, easy and high-throughput characteristics, does not need bacterial cultivation and generates simple and easily interpretable spectra (16,17). However, clinical samples are usually rich in host proteins and normal flora, which might result in overlapping mass spectra. Moreover, MALDI-TOF MS is limited in sensitivity since a sufficient number of cells is necessary to prevent their lost in background noise (18). Furthermore, the initial investments and maintenance costs are very high, whereas the overall operating costs are low (1).

Microbiological diagnostics – an overview

(Download the high-resolution table here.)

Will molecular techniques replace traditional cell-based assays in microbiological diagnostics?

Over the last decades, a variety of methods has been developed for the detection and identification of pathogens present in clinical samples. Through the advent of several molecular techniques the basic conditions have been fundamentally changed: isolation and cultivation of organisms is no longer necessary, results are achieved within hours instead of days, and procedures are more specific and sensitive. The molecular assays have become widely available for microbiologic diagnostics. But at least one question arises when new applications for molecular testing are being introduced: can the molecular methods replace conventional techniques completely? A perfect diagnostic method must be sensitive, specific, rapid, easy to perform and interpret, but also cost-effective and high-throughput. According to the current state of the art there is no perfect microbial diagnosis method. All the methods have both advantages and drawbacks or rather limitations. It is recommendable to use a combination of a molecular technique generating rapid and reliable results and the widely accepted culture-based assays for confirmation. However, it is quite conceivable that the modern molecular methods will supersede conventional old techniques in the near future.

 

Which kind of methods do you use for the identification and characterization of bacteria?

Please leave your comments on our Facebook and Twitter page.

 

References

(1) Sousa, A.M. and Pereira, M.O. (2013) A prospect of current microbial diagnosis methods. In A. Mendez-Vilas, Microbial Pathogens and Strategies for Combating Them: Science, Technology and Education, Vol. 3, Badajoz: Formatex, ISBN: 978-84-942134-1-0. 1429-1438.
(2) Lievens, B., et al. (2005) Recent developments in diagnostics of plant pathogens: A review. Recent Res Develop Microbiol. 9: 57-79.
(3) Braga, P.A.C., et al. (2013) Bacterial identification: from the agar plate to the mass spectrometer. RSC Advances. 3: 994-1008.
(4) van Belkum A., et al. (2013) Rapid clinical bacteriology and its future impact. Ann Lab Med. 33: 14–27.
(5) Weile, J. and C. Knabbe (2009) Current applications and future trends of molecular diagnostics in clinical bacteriology. Anal Bioanal Chem. 394: 731-42.
(6) Lazcka, O., et al. (2007) Pathogen detection: a perspective of traditional methods and biosensors. Biosens Bioelectron. 22: 1205-17.
(7) López-Campos, G., et al. (2012) Detection, Identification, and Analysis of Foodborne Pathogens. In Microarray Detection and Characterization of Bacterial Foodborne Pathogens. Springer US. ISBN: 978-1-4614-3249-4. 13-32.
(8) Malik, S., et al. (2008) The use of molecular techniques to characterize the microbial communities in contaminated soil and water. Environ Int. 34: 265–276.
(9) Sanz, J.L. and Köchling, T. (2007) Molecular biology techniques used in wastewater treatment: An overview. Process Biochem. 42: 119-133.
(10) Bottari, B., et al. (2006) Application of FISH technology for microbiological analysis: current state and prospects. Appl Microbiol Biotechnol. 73: 485-94.
(11) Bej, A., et al. (1990) Multiplex PCR amplification and immobilized capture probes for detection of bacterial pathogens and indicators in water. Mol Cell Probes. 4: 353–65.
(12) Vandenvelde, C., et al. (1990) Fast multiplex polymerase chain reaction on boiled clinical samples for rapid viral diagnosis. J Virol Methods. 30: 215–27.
(13) McLoughlin, K.S. (2011) Microarrays for pathogen detection and analysis. Brief Funct Genomics. 10(6):342–353.
(14) Welker, M. (2011) Proteomics for routine identification of microorganisms. Proteomics. 11: 3143-53.
(15) Lay, J.O. (2001) MALDI-TOF mass spectrometry of bacteria. Mass Spectrom Rev. 20: 172-94.
(16) Ho, Y.P. and Reddy, P.M. (2011) Advances in mass spectrometry for the identification of pathogens. Mass Spectrom Rev. 30: 1203-24.
(17) Braga, P.A.C., et al. (2013) Bacterial identification: from the agar plate to the mass spectrometer. RSC Advances. 3: 994-1008.
(18) Wieser, A. et al. (2012) MALDI-TOF MS in microbiological diagnostics-identification of microorganisms and beyond. Appl Microbiol Biotechnol. 93: 65-74.

This article was written by
Natalie works as a scientist and project manager at Biametrics. She holds a PhD in molecular biology and previously worked on bacterial toxin-antitoxin systems.