sample, was overcome by the rise of quantitative PCR process, the precise numbering of pathogens present in low
concentrations remains difficult by this approach (if not
impossible). Another limitation of these DNA tests is the
lack of indication of the physiological state of the detected
cells in the original sample.
15.9.2.1 Polymerase Chain Reaction (PCR)
Applications
PCR amplification (cf. Sect. 17.7) is frequently used for
the detection and identification of pathogenic microorganisms (e.g., Escherichia coli, Salmonella, Pseudomonas
aeruginosa) in food, feces, soils, sediments, and waters.
Different DNA regions or genes can be chosen as targets
for molecular identification. For detection of specific bacteria, the most frequently used target is the 16S rDNA gene.
For pathogenic organisms, genes encoding virulence
determinants (e.g., LT, SLT I, and SLT II in pathogenic
E. coli strains) are frequently used and often allow a very
specific and sensitive identification of the bacterial species.
By targeting virulence genes for PCR amplification, pathogenic strains can be discriminated from nonpathogenic ones
among the analyzed samples.
Despite the fact that PCR is now recognized as a highly
specific method, its application to the detection of enteric
organisms in natural waters can present some limitations.
DNA is stable enough to be amplified many days after its
cellular release. Therefore, a classic PCR will not differentiate DNA targets, and these targets could be coming from
growing, VNC, or “dead” cells. This drawback can be overcome by targeting messenger or ribosomal RNA (mRNA,
rRNA) sequences instead of DNA sequences. Since RNA
molecules degrade at a faster rate among damaged or killed
bacteria than DNA and can be present at thousands of copies
per cell, they can act as indicators of the physiological state
of the detected cells. Presently, RT-PCR (reverse transcriptase PCR) allows the detection of enteric RNA viruses in
sewage and environmental samples. Despite their potential
advantage, mRNA-based approaches have proved difficult
to operate in environmental sample monitoring. This is
mainly because mRNAs are produced in much less numbers
than rRNAs. In addition, mRNA molecules are quite
unstable and phylogenetically less informative.
A quantitative PCR method including an inhibition of
free DNA from dead cells was developed. This procedure
involves the use of ethidium monoazide (EMA) or propidium monoazide (PMA) molecules that bind covalently
to free DNA after a photoactivation. These molecules cannot
enter viable cells. After EMA treatment of a sample, DNA
extraction of the viable cells can be performed. These
extracts will contain EMA-treated and EMA-free DNA.
Only EMA-free DNA will be amplifiable by PCR. This
very promising approach allows the quantification of
culturable and VNC cells.
15.10 Conclusions
The study of human pathogens represents a vast field of
research in microbial ecology. Changes in environmental
conditions at local or regional levels (whether natural
or anthropogenic) as well as global changes such as climate
change are likely to affect the biogeography of infectious
diseases. Research aimed at linking infectious diseases and
environmental changes is still underdeveloped, but this is
a growing field of investigation to better predict health
risks and to anticipate environmental actions. The methods
used for the detection of pathogens are also constantly
changing and improving in relation to technological
advances, particularly in the field of instrumentation and
molecular biology. The challenge for the future is to
increase the sensitivity of detection methods and the time
that is necessary to provide results, which should tend
toward real time.
In the major issues underlying the future research, the
problem of the emergence or reemergence is important
and should take into account the global scale effects of
climate and environmental changes on the distribution,
dissemination, and survival of pathogens in the environment. These problems also raise the question of increased
virulence in connection with the modification of the adaptability of microorganisms to environmental stresses, sometimes stimulating horizontal genes transfers. Finally, the
question of the survival and viability of pathogens in
environmental reservoirs (niches) remains an important
point for potential reemergence of pathogens maintained
in dormancy.
References
Aendekerk S, Ghysels B, Cornelis P, Baysse C (2002) Characterization of
a new efflux pump, MexGHI-OpmD, from Pseudomonas aeruginosa
that confers resistance to vanadium. Microbiology 148:2371–2381
Alonso A, Rojo F, Martinez JL (1999) Environmental and clinical
isolates of Pseudomonas aeruginosa show pathogenic and
biodegradative properties irrespective of their origin. Environ
Microbiol 1:421–430
Behets J, Declerck P, Delaedt Y, Verelst L, Ollevier F (2007) Survey
for the presence of specific free-living amoebae in cooling waters
from Belgian power plants. Parasitol Res 100:1249–1256
Berg G, Eberl L, Hartmann A (2005a) The rhizosphere as a reservoir
for opportunistic human pathogenic bacteria. Environ Microbiol
7:1673–1685
Berg J, Tom-Petersen A, Nybroe O (2005b) Copper amendment of
agricultural soil selects for bacterial antibiotic resistance in the
field. Lett Appl Microbiol 40:146–151
656
P. Lebaron et al.
concentrations remains difficult by this approach (if not
impossible). Another limitation of these DNA tests is the
lack of indication of the physiological state of the detected
cells in the original sample.
15.9.2.1 Polymerase Chain Reaction (PCR)
Applications
PCR amplification (cf. Sect. 17.7) is frequently used for
the detection and identification of pathogenic microorganisms (e.g., Escherichia coli, Salmonella, Pseudomonas
aeruginosa) in food, feces, soils, sediments, and waters.
Different DNA regions or genes can be chosen as targets
for molecular identification. For detection of specific bacteria, the most frequently used target is the 16S rDNA gene.
For pathogenic organisms, genes encoding virulence
determinants (e.g., LT, SLT I, and SLT II in pathogenic
E. coli strains) are frequently used and often allow a very
specific and sensitive identification of the bacterial species.
By targeting virulence genes for PCR amplification, pathogenic strains can be discriminated from nonpathogenic ones
among the analyzed samples.
Despite the fact that PCR is now recognized as a highly
specific method, its application to the detection of enteric
organisms in natural waters can present some limitations.
DNA is stable enough to be amplified many days after its
cellular release. Therefore, a classic PCR will not differentiate DNA targets, and these targets could be coming from
growing, VNC, or “dead” cells. This drawback can be overcome by targeting messenger or ribosomal RNA (mRNA,
rRNA) sequences instead of DNA sequences. Since RNA
molecules degrade at a faster rate among damaged or killed
bacteria than DNA and can be present at thousands of copies
per cell, they can act as indicators of the physiological state
of the detected cells. Presently, RT-PCR (reverse transcriptase PCR) allows the detection of enteric RNA viruses in
sewage and environmental samples. Despite their potential
advantage, mRNA-based approaches have proved difficult
to operate in environmental sample monitoring. This is
mainly because mRNAs are produced in much less numbers
than rRNAs. In addition, mRNA molecules are quite
unstable and phylogenetically less informative.
A quantitative PCR method including an inhibition of
free DNA from dead cells was developed. This procedure
involves the use of ethidium monoazide (EMA) or propidium monoazide (PMA) molecules that bind covalently
to free DNA after a photoactivation. These molecules cannot
enter viable cells. After EMA treatment of a sample, DNA
extraction of the viable cells can be performed. These
extracts will contain EMA-treated and EMA-free DNA.
Only EMA-free DNA will be amplifiable by PCR. This
very promising approach allows the quantification of
culturable and VNC cells.
15.10 Conclusions
The study of human pathogens represents a vast field of
research in microbial ecology. Changes in environmental
conditions at local or regional levels (whether natural
or anthropogenic) as well as global changes such as climate
change are likely to affect the biogeography of infectious
diseases. Research aimed at linking infectious diseases and
environmental changes is still underdeveloped, but this is
a growing field of investigation to better predict health
risks and to anticipate environmental actions. The methods
used for the detection of pathogens are also constantly
changing and improving in relation to technological
advances, particularly in the field of instrumentation and
molecular biology. The challenge for the future is to
increase the sensitivity of detection methods and the time
that is necessary to provide results, which should tend
toward real time.
In the major issues underlying the future research, the
problem of the emergence or reemergence is important
and should take into account the global scale effects of
climate and environmental changes on the distribution,
dissemination, and survival of pathogens in the environment. These problems also raise the question of increased
virulence in connection with the modification of the adaptability of microorganisms to environmental stresses, sometimes stimulating horizontal genes transfers. Finally, the
question of the survival and viability of pathogens in
environmental reservoirs (niches) remains an important
point for potential reemergence of pathogens maintained
in dormancy.
References
Aendekerk S, Ghysels B, Cornelis P, Baysse C (2002) Characterization of
a new efflux pump, MexGHI-OpmD, from Pseudomonas aeruginosa
that confers resistance to vanadium. Microbiology 148:2371–2381
Alonso A, Rojo F, Martinez JL (1999) Environmental and clinical
isolates of Pseudomonas aeruginosa show pathogenic and
biodegradative properties irrespective of their origin. Environ
Microbiol 1:421–430
Behets J, Declerck P, Delaedt Y, Verelst L, Ollevier F (2007) Survey
for the presence of specific free-living amoebae in cooling waters
from Belgian power plants. Parasitol Res 100:1249–1256
Berg G, Eberl L, Hartmann A (2005a) The rhizosphere as a reservoir
for opportunistic human pathogenic bacteria. Environ Microbiol
7:1673–1685
Berg J, Tom-Petersen A, Nybroe O (2005b) Copper amendment of
agricultural soil selects for bacterial antibiotic resistance in the
field. Lett Appl Microbiol 40:146–151
656
P. Lebaron et al.