MCB 442: PATHOGENIC COLLECTION OF SPECIMENS

BACTERIOLOGY
 Body fluids, secretions and biopsy material
LABORATORY TECHNIQUES FOR THE can all be examined to detect pathogens,
ISOLATION, CHARACTERISATION
antigens or products or the immune response
AND IDENTIFICATION OF
PATHOGENIC BACTERIA to them.
 Samples from the envi- ronment, e.g. water,
food or soil, may also be examined.
BACKGROUND INFORMATION
 Some samples must be collected at a
The conventional methods (golden standard of particular time; for example, malaria
patho gen detection and antimicrobial sensitivity parasites are best sought at the peak of fever
testing) of identified pathogenic bacteria are: and a short time afterwards, whereas blood
1. Microscopy for bacterial culture should be taken as the
2. Medium culture fever begins to rise.
3. Biochemical testing techniques  Special precautions must often be taken to
ensure survival of the pathogen and exclude
Advantages: contaminants, e.g. cleaning the perineum
 Low cost before collecting a midstream specimen of
 Easy to operate urine.
 Highly standardized  Anaerobic species may die if exposed to
atmospheric oxygen and survive better in
Disadvantages
samples of pus, rather than in swab
 Lack of differentiation between the target specimens.
and other non-target endogenous  Many pathogens, such as Neisseria and most
microorganisms of the same samples anaerobic species, die quickly outside the
 False negative/positive results body and must be transported to the
 Time and labour-consuming procedures
laboratory without delay.
 Inability to detect viable but nonculturable
 Neisseria gonorrhoeae is susceptible to
(VBNC) cells.
drying, so specimens likely to contain this
organism should be inoculated onto
microbiological medium near to the patient.
UNIVERSAL PRECAUTIONS AND
LABORATORY SAFETY
 Specimens may contain hazardous
pathogens and must be handled
with care.
 A system of ‘universal precautions’
is employed to reduce the risk of
transmitting blood-borne viruses.
 These are personal protective
measures, taken in collecting and
examining specimens irrespective
of their source.
 The idea is to handle specimens on
the assumption that they may
contain a transmissible pathogen,
rather than relying on clinical
suspicion or written clinical details,
which may be faulty or absent. For
example, any sputum specimen is
 assumed to carry the risk of
tuberculosis even if this is not the
test requested.
 For example, any sputum specimen
is assumed to carry the risk of
tuberculosis even if this is not the
test requested.
 DIRECT MICROSCOPIC EXAMINATION
Microscopy is especially useful for detecting organ- isms that are difficult or dangerous to
grow.
Types of microscopy for the diagnosis of infections
1 Unstained preparations.
2 Simple stains: Gram, Giemsa.
3 Special stains: Ziehl–Nielsen, Gomori–Grocott, India ink.
4 Immunofluorescence: direct and indirect.
5 Electron microscopy.

Direct microscopy of unstained preparations

• Faecal protozoa and helminths.
• Vaginal discharge.
• Urine for bacteria and pus cells.
Special stains
Stains such as Ziehl–Nielsen (ZN) are used to demonstrate specific features of organisms
that simple stains will not demonstrate. Specimens are stained with carbolfuchsin, destained
with an acid–alcohol solution and then counter- stained with methylene blue. The lipid-rich
mycobacterial cell wall retains the pink dye and organisms are seen as pink bacilli against the
blue background . The number of acid-fast species is limited and this technique is therefore useful
in the diagnosis of mycobacterial infection, including tuberculosis and leprosy, and parasitic
infections such as cryptosporidiosis.

A variation of this technique uses the naturally fluorescent substance auramine to stain
the organisms. The specimen is processed in a similar way to the ZN method, and acid-
fast organisms fluoresce bright yellow under an ultravio- let light. Auramine
microscopy is used for screening large numbers of specimens but, because it lacks the
specificity of the ZN stain, all positive specimens must be overstained by the ZN
method, and re-examined to confirm the find- ings.
Romanowsky stains
Romanowsky stains, which colour cytoplasm and chro- matin, are widely used to
demonstrate blood cells. Stains of this type are very useful for revealing blood
parasites. In malaria or filariasis, Giemsa-stained smears not only demonstrate the
presence of the organisms, but permit speciation by displaying morphological details
(Fig. 3.6).

Molecular methods of detecting pathogenic bacteria

According to the biological markers being used, molecular methods can be divided into two
main groups
1. Nucleic acid targeting method
2. Protein/antigen targeting method
Nucleic acid targeting method
1. Fluorescence amplification-based methods:
 Polymerase chain reaction (PCR)
 Quantitative or real -time PCR (qPCR)
 Digital PCR (dPCR)
 Deoxyribonucleic acid (DNA) microarray
 Fluorescence in situ hybridization (FISH)
 Molecular beacon
 Sequencing-based methods:
a. Pyrosequencing
b. Illumina sequencing
c. Nanopore sequencing

Protein/antigen targeting method

1. Traditional antibody-antigen targeting methods are similar to immunological methods
(lateral flow tests (LFTs) and Enzyme linked immunosorbent assay (ELISA)
It is important to state that by combining the basic molecular detection approaches
together with a metal and paper platform, biosensor-based and paper-based devices cost
effective standard protocols are achieved. These are becoming rapid, cheap as well as
portable on-site standard methods for detection of pathogenic bacteria

Biomarkers of Pathogenic Bacteria

 Biomarkers including nucleic acids, proteins, antigens, adenosine triphosphate
(ATP), and metabolic products.
 They are employed in the analysis of microorganisms.
 To differentiate microorganisms within one sample, nucleic acids (DNA/RNA),
proteins, and antigens are usually selected as biomarkers because of their special
physical and chemical characteristics within different pathogens.
 The detection of DNA/RNA is based on the specific hybridization and
amplification of targets, thus enabling good specificity and accuracy.
 In case of pathogenic bacteria in wastewater, the most important biomarker is
the pathogenic DNA or RNA residues from these bacteria.
 The biomarkers include the genus/species-specific genes, functional genes, and
antimicrobial resistance genes.
 Moreover, in the analysis of antimicrobial resistance, gene transfer is another
significant point.
  Various mobile genetic elements, including plasmids, transposons,
bacteriophages, integrons, and combinations of them, are notable nucleic acid
targets for investigating the prevalence and spread of resistance genes in bacteria.
ATP assay, enzymatic activity tests, and metabolic products are mostly used to assess
the activity of living cells.
 Due to the linearity between the total number of ATP and the total colony-
forming units, the metabolically active cells could be directly quantified using
the amount of ATP.
 Ions and some organic acids are the metabolic products of microorganisms.
 These metabolites could be detected by electrochemical methods, thus were
utilized to reflect the metabolic states of microorganisms.
 Microbial surfaces contain a variety of proteins that are expressed by specific
DNA/RNA in different pathogens.
 By screening these proteins using antibodies and nucleic acids, new biomarkers
can be discovered, and pathogens can be specifically detected.
 Antigens are another kind of molecules on the cell surface of pathogens.
 They can be specifically bound to antibodies and induce immune responses of
the host.
 Each type of pathogen carries one or more unique antigens on their surface,
even within strains. It thus enables the specific identification of pathogens using
antibodies
 Moreover, by analyzing the specific antigens of each strain, the subtypes of the
strain can be determined.
 Aptamers are single-stranded DNA or RNA oligonucleotides with high affinities
and specificities that can bind a variety of targets, from single molecules to
whole cells.
 They can form diverse, complex secondary structures such as multi-branched
loops and G-quadruplexes, which can specifically target the surface proteins of
microorganisms or cells.
 In environmental monitoring, aptamers are superior to antibodies due to their
chemical stability, easy chemical modification, relative ease of synthesis, and
biocompatibility.
 With the systematic evolution of ligands by exponential enrichment (SELEX)
method, many aptamers have been successfully employed to detect various
pathogens in environmental samples.

Molecular Methods
 For a long time, the culture and colony counting-based method has been the
dominant method in the detection of pathogens (i.e., the ‘gold standard’).
 It can assess live microbes or viable cells in samples.
 However, these methods may produce false-positive or false-negative results
when evaluating highly aggregated microbial cells. Furthermore, not all
microbial cultures can be grown under laboratory conditions.
 For example, a study of Campylobacter indicated that the culture-based method
failed to correctly detect Campylobacter in 30% of positive patient stool samples
compared to non-cultural methods, including PCR and enzyme immunoassay
(EIA)
 Moreover, the culture methods are time and resource intensive, which are
gradually replaced by more rapid and specific molecular methods.
  Therefore, in order to meet the requirements for reliable analysis of pathogenic
bacteria, including high specificity, high sensitivity, good reproducibility,
automation, and cost effectivity, molecular methods have gradually emerged to
replace the dominant position of culture methods.
 In recent decades, various rapid, sensitive, and specific molecular methods have
been developed. These molecular methods are discussed below and listed in

Nucleic Acid Targeting Methods

 Nucleic acid targeting methods are designed to detect the specific DNA/RNA of
pathogens.
 It is achieved by the hybridization between target nucleic acid sequences and
synthetic oligonucleotides.

 Thus, the species-specific gene of pathogens and virulence genes can be detected
through nucleic acid targeting methods.
 They are usually fast, efficient, and do not require the culture of the pathogens.
 These methods include polymerase chain reaction (PCR)-based methods such as
conventional PCR, real-time/quantitative PCR (qPCR), droplet digital PCR (ddPCR),
multiplex PCR (mPCR), and other methods such as microarrays, loop-mediated
isothermal amplification (LAMP), sequencing, and fluorescence in situ hybridization
(FISH).

PCR-Based Method

 PCR is the most common molecular-based technique for the detection and
quantification of pathogens. PCR enables the detection of a single pathogenic bacteria
by targeting specific DNA sequences.
 Through this method, a small sample of a DNA sequence could be rapidly amplified
into a large amount.
 This advantage enables the detection and quantification of a low amount of the target
DNA sequence.
 It is thus widely used in the diagnosis of human pathogens.
 It significantly increases the sensitivity of detection of microorganisms at low
concentrations in environmental samples.
 PCR has already been utilized to the detection of a series of pathogenic bacteria such
as E. coli and spores of C. perfringens.
 Conventional PCR needs gel electrophoresis to detect the formation of PCR products.
Real-time polymerase chain reaction, also called quantitative PCR, is the real-time
detection of the PCR process during the amplification of the target DNA sequence.
qPCR determines the PCR amplification by measuring specific dual-labeled probes or
fluorescent signals emitted by inserting dyes.
 The fluorescence intensity reflects the amount of the template DNA.
 There is a linear relationship between the cycle threshold (Ct or Cq) value and the
initial concentration of the template gene during the exponential period of PCR
amplification.
 Thus, the concentration of target sequences could be calculated from a well-
established standard curve to achieve an absolute quantification.
 Real-time quantitative PCR is widely used in the real-time detective and quantitative
analysis of target DNA sequences with higher specificity and sensitivity than
conventional PCR
 Two main fluorescence systems have been developed for qPCR, i.e., the SYBR green
method and the TaqMan probes method. SYBR green is a fluorescent pigment that
can bind double-stranded DNA (dsDNA).
 This non-sequence-specific pigment enhances the fluorescence signal when it binds to
DNA double helix minor grooves, thus enabling the quantification of the targeting
sequence.
 In contrast, TaqMan probe does not require the addition of fluorescent pigment.
 The template-specific TaqMan probe further improves the specificity of qPCR by
increasing primer specificity.
 For each amplification of a specific target, one molecule of fluorescent dye is
released.
 The instrument detects the fluorescence produced by specific amplification, which is
not impacted by non-specific amplification.
 This ensures the high specificity of the qPCR detection.
 There are many reporter–quencher sets with different wavelengths, which can be
labeled with the TaqMan probe.
 This enables the TaqMan method to be able to detect multiple PCR reactions in the
same tube, leading to reduced cost and improved efficiency and accuracy.
 It can also avoid the influence of different fluorescent dyes on the PCR reaction.
 The mPCR is a faster detection methodology than simplex PCR, which can detect
multiple gene targets simultaneously.
 Digital PCR is a biotechnology improvement on conventional PCR and can be used
to directly amplify and quantify DNA, cDNA, or RNA.
 Droplet digital polymerase chain reaction (ddPCR) is a kind of dPCR technique that
is emerging as a powerful analytical tool for absolute quantification.
 Similar to qPCR, ddPCR also utilizes Taq polymerase to amplify a target DNA
sequence in a standard qPCR assay.
 The differences are that ddPCR separates the whole qPCR reaction into thousands of
individual reactions before amplification, and ddPCR collects data at the reaction end
point.
 These differences provide ddPCR many advantages, such as the direct and
independent quantification of target DNA without standard curves and more precise
and reproducible data than conventional qPCR, especially when PCR inhibition is
present.
 In comparison to qPCR, ddPCR shows better performance in detecting low
concentrations of target genes in environmental samples.
 Nowadays, with the increasing availability of sequencing data, it is theoretically
possible to design qPCR assays for every microorganism.
 The qPCR method has many benefits over other techniques.
 Firstly, the quantitative data produced by qPCR method could reach an accurate
dynamic range of 7–8 log orders of magnitude without requiring post-amplification
manipulation.
 Secondly, although the sensitivity of qPCR is varied towards different
samples and might be inhibited by inhibitors, it has been reported to have higher
sensitivity than many other molecular methods. DNA Microarrays

DNA microarrays, also known as DNA arrays, are commonly known as gene chips.
  It is a special piece of glass or silicon chip with a DNA microarray, which
places thousands or tens of thousands of nucleic acid probes on an area of
several square centimeters
 DNA, complementary DNA (cDNA), and RNA in the sample are detected by
fluorescence or electric signal after being combined with the probes. DNA
microarrays enable the hybridization-based detection of multiple targets in a
single experiment, which makes it suitable for the analysis of massive targets.
 Using a high-throughput DNA microarray assay, a study investigated the
prevalence of 941 pathogenic bacteria in groundwater and differentiated their
sources of origin.
 In general, DNA microarray allows for the simultaneous detection of multiple
pathogenic bacteria. It is thus a fast and reliable diagnostic method in analyzing
large numbers of clinical/environmental samples. However, the complicated
probe design work, the reliability of the microarray data, and the clinical
applicability of the early results have been criticized.
 The criticism and intensified competition from other technologies, such as next-
generation sequencing (NGS), have hampered the growth of microarray-based
testing in the molecular diagnostics market.

Loop-Mediated Isothermal Amplification (LAMP)

 LAMP is an isothermal nucleic acid amplification technique.

 It has been utilized for the alternative detection of certain diseases because of its
low cost.
 At present, LAMP has been applied to the identification and quantification of
pathogenic bacteria with significant advantages in sensitivity, specificity, and
rapidity
 Since LAMP requires four primers specifically designed for six different regions
of the target, any incomplete matching of the primers will theoretically lead to
the phenomenon of non-reaction and non-specific amplification.
 In addition, the LAMP method was confirmed to be 10–100 times more sensitive
than PCR detection, with a detection limit of 10 copies or less in the template for
one reaction.
 Furthermore, it can directly detect pathogenic microorganisms in diseased
tissue, thus avoiding the tedious cultivation and nucleic acid extraction step.
 Finally, and most importantly, the result of the reaction can be judged with
naked eyes by demonstrating the absence of the target gene with the production
of white precipitate of magnesium pyrophosphate.
 It is more difficult to design specific primers for LAMP than PCR (because
LAMP requires 4–6 primers and PCR requires only two).
 A software tool named PrimerExplorer is available to help the primer design for
LAMP (http://primerexplorer.jp/e/).
 Therefore, as a rapid detection method without the need of any equipment,
LAMP shows great potential in the rapid diagnosis of human pathogens in
various samples.

Fluorescent in Situ Hybridization (FISH)

 FISH is a cytogenetic technique used to detect and locate nucleic acids in cells or
sample matrices.
  Fluorescently labeled nucleic acid probes hybridize only with highly similar nucleic
acids and can be used to locate genes on chromosomes or to label ribosomal RNA in
different taxonomic bacteria or archaea in molecular ecology.
 FISH could be employed in the enumeration of particular microbial populations.
 Compared to PCR, FISH is more suitable for complex matrices because of its lesser
sensitivity to inhibitory substances.
 However, a major limitation of FISH is the small number of phylogenetically distinct
targets that can be detected at the same time.
 A recent study developed a multi-FISH method that uses eight fluorophores, which is
highly suitable for investigating the structure and function of microbial communities
in different samples.
 Furthermore, FISH has been used to discover emerging human pathogens in water,
wastewater, and sludge, to produce quantitative descriptions of the microbial
community in wastewater and activated sludge and to investigate survival and
infection mechanisms at the cellular level.
 However, this method is still partly based on cell culture.

Sequencing

 Sequencing is the process of determining the sequence of nucleotides in a section of

DNA.
 It includes any method or technique used to determine the order of the four bases:
adenine, guanine, cytosine, and thymine (or uracil for RNA).
 In 1977, DNA sequencing technology was firstly developed by Frederick Sanger
based on the chain-termination method (also known as Sanger sequencing).
 In the early stage, DNA sequencing was employed for small genomes such as viruses
and organelles.
 Complete sequencing of a bacterium genome was not feasible because of the
economic and technical limitations.
 Later, with the emergence of the shotgun method developed by Sanger et al., whole
genome sequencing of bacteria was achieved.
 The shotgun method is considered the gold standard, and whole genome sequencing
of many bacteria has been carried out using this method over years.
Next-generation sequencing (NGS), also known as high-throughput sequencing, is the
overall term used to describe several different modern sequencing pathways.
 These technologies allow DNA and RNA to be sequenced faster and at lower cost
than Sanger sequencing, which was previously used, thus revolutionizing genomics
and molecular biology research
 NGS technologies include Illumina (Solexa) sequencing, Roche 454 sequencing, and
proton/PGM sequencing.
 The NGS technologies achieve high throughput and reduced cost by using massively
parallel analysis, which allows 300 Gb of DNA to be read in a single run on a single
chip.
 The four main advantages of NGS over classical Sanger sequencing are: (i) NGS
needs significantly less DNA, as it can obtain a sequence from a single strand; (ii)
NGS is significantly quicker than Sanger sequencing by combining the two separate
processes of Sanger sequencing, i.e., chemical reaction and signal detection, in some
versions of NGS; (iii) NGS is more cost-effective due to reduced time, manpower,
and reagents; (iv) repeats in NGS caused by many short overlapping reads lead to a
more accurate and reliable sequence, even though individual reads are less accurate.
  These advantages enable a great potential of NGS in the application of environmental
research. NGS is capable of producing large numbers of reads at exceptionally high
coverages throughout the genome with dramatically reduced cost through the
massively paralleled approach.
 However, NGS requires the amplification of DNA molecules, which introduces
random errors in the DNA synthesis.
 The amplified DNA strands would become progressively out-of-sync, which means
the signal quality deteriorates as the read length grows.
 Therefore, long DNA molecules must be broken up into smaller pieces to maintain
the quality of the reading, leading to a critical limitation of second-generation
sequencing
 To solve the limitation, third-generation sequencing (TGS) technologies were
developed to produce substantially longer reads than NGS by the direct sequencing
of single DNA molecules.
 Nanopore sequencing (Oxford Nanopore Technologies, Oxford, UK) is a
representative TGS approach for the sequencing of biopolymers, specifically
polynucleotides in the form of DNA/RNA.
 Through nanopore sequencing, individual molecules of a DNA/RNA can be
sequenced without PCR amplification or chemical labeling of the sample.
 Nanopore sequencing has a great potential in providing relatively low-cost
genotyping, high mobility for testing, and the ability to rapidly process samples and
display results in real time.
 Applications of this method in the rapid identification of viral pathogens, plant
genome sequencing, monitoring of antibiotic resistance, and haplotyping has been
reported.
 One major limitation of nanopore sequencing is its high raw read error rate, which
remains between 5% and 15% despite recent improvements in nanopore chemistry
and computational tools.

Immunology-Based Methods
 Immunological methods are based on the specific interaction between antibodies and
antigens.
 These methods include enzyme-linked immunosorbent assays (ELISA),
immunofluorescence assays (IFA), and serum neutralization tests (SNTs).
 For immunology-based methods, specific fluorochrome labeled antibodies are used
to capture targeted antigens, which serves as the enumeration of fluorescently labeled
cells by detecting the fluorescence signal using microscopy or flow cytometry.
 However, the biomarkers of these methods should be carefully chosen to achieve
specific detection at different classification levels including genus, species, and
serotypes.
 Although these methods can specifically detect targeted bacteria and their toxins and
can be multiplexed for multiple samples, they are still limited by false-negative results
and cross-reactions with similar antigens.
 False-negative results are a serious problem and often happen to classical methods.
 They can be induced by various inhibitory compounds and matrices of different types
of samples, which vary largely and thus might cause different effects on the analytical
performance of different detection methods.
 In addition, cross-reaction is another big problem for immunology-based methods.
 The cross-reactivity with other bacteria may happened due to the presence of a
constituent sugar of LPS.
  Immunological methods usually require pre-enrichment to expose surface antigens,
which leads to extended detection time.

Biosensor-Based Methods

 A biosensor is an analytical platform composed of two elements: a bio-receptor and a

transducer.
 Bio-receptors are responsible for recognizing the targets such as enzymes, proteins,
nucleic acids, and cell receptors.
 After recognition, the transducer converts the biological interactions into electrical
signals that can be measured (e.g., optical, electrochemical, or magnetic).
 Biosensors provide a rapid, real-time, on-site, and multiple detection of bacteria.
 Optical biosensors are selective, sensitive, and can be used for real-time monitoring of
toxins, drugs, and pathogens e.g., in wastewater

Paper-Based Device

 A paper-based device is a small analytical tool that is printed by a wax printer and has
different functional areas.
 It can integrate all the processes required for nucleic acid detection (enrichment,
extraction, amplification, and visual detection) into a cheap paper material.
 The whole detection process can be completed by folding paper-based devices in
different ways and in different sections, which overcomes the limitation of PCR tests.
 Paper-based device can achieve multichannel, sensitive detection, comparable to PCR
detection, and provide a high-quality, rapid, and accurate diagnosis of pathogens.
 Moreover, paper-based devices are easy to stack, store, and transport because they are
thin, lightweight, and of different thicknesses.
 Similar to biosensors, paper-based devices can also be used to target a variety of
biomarkers, including nucleic acids, proteins, antigens, and chemicals.
 By integrating various molecular detection methods, paper-based devices have
emerged as a powerful platform for the fast diagnosis of pathogens and the
determination of infection transmission.
 However, the shelf life of paper-based device limits its further applications.
 Some paper-based devices contain reagents with a short shelf life, such as enzymes,
and thus need to be stored in a refrigerator or freezer to maintain the activity of the
reagents.