Nystro ¨m 1998). In addition, increases in the synthesis of
heat shock resistance proteins, glutathione reductase,
superoxide dismutase A, etc., have been described in
E. coli stationary phase indicating that these cells
(a mixture of culturable bacteria and VNC) increase their
resistance toward denaturation and protein aging. These
observations led Desnues et al. (2003) to develop centrifugation techniques for separating the VNC and culturable
forms and approaches for in situ detection of oxidized
proteins in an E. coli cell. These technological developments allowed showing that VNC E. coli cells can lose
their culturability due to a sharp deterioration of their
protein content and not because of an induction of an
adaptive process changing their physiological state to dormant or non-culturable one. The return of a VNC cell to a
culturable state would therefore be related to processes
allowing some kind of protection of the cell content such
as an induction of activities favoring a repair of damages
caused by a period of stasis (period of deceleration of
bacterial growth that could go down to a complete stop
and cell death). The results obtained with VNC E. coli must
now be verified with other bacterial species to conclude on
the general meaning to be given to the VNC status among
bacteria. However, the existence of VNC cells among bacterial pathogens, whether damaged or dormant, remains an
undeniable reality, and the possibility of their return to a
physiological state representing a health hazard remains a
public health concern. Return of VNC to culturable forms
was also obtained after passage in a higher organism such
as amoebas for the particular case of L. pneumophila. VNC
cells of Vibrio spp. were found to have kept their virulence.
By extrapolation, man could thus be infected by VNC
bacteria. Man could be acting as a favorable host for the
return of VNC into culturable cells.
On an operational point of view, it is difficult to detect
in routine a cellular activity that may provide evidence
of viability of pathogenic VNC bacteria and establish a
relationship with any potential colonization of man. The
difficulty lies, among other things, on the fact that this
activity should be determined without a culturing step and
at the scale of a single cell. Fluorescence techniques in
combination with currently available probes targeting cell
function have limited resolution, either because the
targeted activity is low or because the detection tools are
not sensitive enough. Moreover, there is no single function
to inform on cell viability. It is now recognized that several
tests are necessary to establish a satisfactory diagnosis of
the physiological state of a cell because it goes along a
continuum of physiological conditions from active and
dividing ones to inactive and irreversible ones, i.e., cell
death.
15.9 Detection Methods
The primary goal of monitoring water quality is to highlight
the presence or absence of pathogens and avoid human exposure. Monitoring waters for the many different human
pathogens by using conventional methods such as the most
probable number approach based on bacterial broths, the
membrane filtration, and the culture-based methods are not
convenient enough. These methods are time-consuming, are
laborious, and do not allow the detection of all the cells present
in a sample (VNC cells). Therefore, many alternative detection
methods, including direct methods and indirect methods,
of specific pathogens have now been developed (Fig. 15.11).
Detection of pathogens via culture-based methods
requires an enrichment of a target population over other
environmental microorganisms, followed by pathogens’
isolation and identification (Fig. 15.12). Culture-based
methods are indirect methods since they require a culture
step before the diagnostic. Even if these methods are still in
use in many monitoring agencies, their major limitations
when addressing the detection of pathogens in environmental samples are that they (1) are time-consuming,
(2) might not grow the targeted microorganisms because
of environmental factors leading to VNC (cf. Sect. 15.8),
and (3) will not detect many enteric viruses and protozoa
(cf. Sect. 15.3).
In the last two decades, scientists have developed
direct methods to rapidly detect the presence of fecal
indicators and fecal pathogens in food, soil, and water
(Thompson et al. 2010). These methods are much faster
than culture-based methods and are able to detect specific
microorganisms, in only few hours, with a high degree of
sensitivity and specificity without the need for complex
cultivation. These methods include cell-based and nucleic
acid-based methods.
15.9.1 Cell-Based Methods
These methods allow an accurate numbering of microorganisms and involve detection tools such as epifluorescence microscopy (EFM), flow cytometry (FC), and
solid-phase cytometry (SPC). Unfortunately, EFM and
FC are not suitable for the detection of low cell numbers
(as it is the case of pathogens in natural environments)
due to their intrinsic detection limits (cf. Sect. 17.3). SPC,
with a limit of detection of one target cell in a filterable
sample, enables the application of cell-based methods
for the detection of pathogenic microorganisms in the
environment.
15 Environmental and Human Pathogenic Microorganisms
651
heat shock resistance proteins, glutathione reductase,
superoxide dismutase A, etc., have been described in
E. coli stationary phase indicating that these cells
(a mixture of culturable bacteria and VNC) increase their
resistance toward denaturation and protein aging. These
observations led Desnues et al. (2003) to develop centrifugation techniques for separating the VNC and culturable
forms and approaches for in situ detection of oxidized
proteins in an E. coli cell. These technological developments allowed showing that VNC E. coli cells can lose
their culturability due to a sharp deterioration of their
protein content and not because of an induction of an
adaptive process changing their physiological state to dormant or non-culturable one. The return of a VNC cell to a
culturable state would therefore be related to processes
allowing some kind of protection of the cell content such
as an induction of activities favoring a repair of damages
caused by a period of stasis (period of deceleration of
bacterial growth that could go down to a complete stop
and cell death). The results obtained with VNC E. coli must
now be verified with other bacterial species to conclude on
the general meaning to be given to the VNC status among
bacteria. However, the existence of VNC cells among bacterial pathogens, whether damaged or dormant, remains an
undeniable reality, and the possibility of their return to a
physiological state representing a health hazard remains a
public health concern. Return of VNC to culturable forms
was also obtained after passage in a higher organism such
as amoebas for the particular case of L. pneumophila. VNC
cells of Vibrio spp. were found to have kept their virulence.
By extrapolation, man could thus be infected by VNC
bacteria. Man could be acting as a favorable host for the
return of VNC into culturable cells.
On an operational point of view, it is difficult to detect
in routine a cellular activity that may provide evidence
of viability of pathogenic VNC bacteria and establish a
relationship with any potential colonization of man. The
difficulty lies, among other things, on the fact that this
activity should be determined without a culturing step and
at the scale of a single cell. Fluorescence techniques in
combination with currently available probes targeting cell
function have limited resolution, either because the
targeted activity is low or because the detection tools are
not sensitive enough. Moreover, there is no single function
to inform on cell viability. It is now recognized that several
tests are necessary to establish a satisfactory diagnosis of
the physiological state of a cell because it goes along a
continuum of physiological conditions from active and
dividing ones to inactive and irreversible ones, i.e., cell
death.
15.9 Detection Methods
The primary goal of monitoring water quality is to highlight
the presence or absence of pathogens and avoid human exposure. Monitoring waters for the many different human
pathogens by using conventional methods such as the most
probable number approach based on bacterial broths, the
membrane filtration, and the culture-based methods are not
convenient enough. These methods are time-consuming, are
laborious, and do not allow the detection of all the cells present
in a sample (VNC cells). Therefore, many alternative detection
methods, including direct methods and indirect methods,
of specific pathogens have now been developed (Fig. 15.11).
Detection of pathogens via culture-based methods
requires an enrichment of a target population over other
environmental microorganisms, followed by pathogens’
isolation and identification (Fig. 15.12). Culture-based
methods are indirect methods since they require a culture
step before the diagnostic. Even if these methods are still in
use in many monitoring agencies, their major limitations
when addressing the detection of pathogens in environmental samples are that they (1) are time-consuming,
(2) might not grow the targeted microorganisms because
of environmental factors leading to VNC (cf. Sect. 15.8),
and (3) will not detect many enteric viruses and protozoa
(cf. Sect. 15.3).
In the last two decades, scientists have developed
direct methods to rapidly detect the presence of fecal
indicators and fecal pathogens in food, soil, and water
(Thompson et al. 2010). These methods are much faster
than culture-based methods and are able to detect specific
microorganisms, in only few hours, with a high degree of
sensitivity and specificity without the need for complex
cultivation. These methods include cell-based and nucleic
acid-based methods.
15.9.1 Cell-Based Methods
These methods allow an accurate numbering of microorganisms and involve detection tools such as epifluorescence microscopy (EFM), flow cytometry (FC), and
solid-phase cytometry (SPC). Unfortunately, EFM and
FC are not suitable for the detection of low cell numbers
(as it is the case of pathogens in natural environments)
due to their intrinsic detection limits (cf. Sect. 17.3). SPC,
with a limit of detection of one target cell in a filterable
sample, enables the application of cell-based methods
for the detection of pathogenic microorganisms in the
environment.
15 Environmental and Human Pathogenic Microorganisms
651