The present invention relates to methods for quantifying changes in viable microbial populations. In particular, the invention relates to real-time methods for quantifying such alterations in microbial populations, and for rapid quantification of viable microorganism in situ. The invention finds use in a variety of applications where living organisms, suspended in a liquid medium, are quantified, including evaluation of antimicrobial agents and/or microbial growth enhancers.
Quantification of microorganisms is a critical element in a number of microbiological, pharmaceutical, ecological, and industrial settings. Specific examples include evaluation of antimicrobial susceptibility and/or antimicrobial efficacy, development of novel antimicrobials, wastewater treatment, water quality assessment, and food safety applications such as detection of contaminated food products, pasteurization quality control, and quality control in fermentation processes. Reliable methods for quantification of microorganisms, particularly methods capable of concurrently distinguishing between living and dead microorganisms are essential tools in such endeavors.
Such microorganism quantification is most commonly done by colony count methods requiring prolonged incubation times for appearance of colonies on growth media. Indeed, colony count methods are at present the most definitive and reliable method available for bacterial detection and quantification in culture. Alternatively, it is known also to quantify microorganisms by microscopic imaging. These processes are labor and resource intensive, and conventional microscopic imaging processes suffer from the further disadvantage of failing to differentiate between living and dead cells absent introduction of a chemically enhanced substance. Conventional spectrophotometric methods similarly fail to differentiate between viable and non-viable cells, typically applicable to aqueous media, precluding their use in the food industry, and are also limited to a 2-3 log range of cell concentrations in terms of their ability to quantitate.
Impedance and pH methods for quantification of microorganisms are suited thr their intended purpose, but suffer from limitations in range of detection due to material chemical property limitations to allowable pH and changes in pH. Other methods are known or in various stages of development, such as calorimetric methods, enzyme labeling, gene sensors, and flow injection, but require chemical treatment or complete destruction of samples, preventing dynamic change measurements.
Indeed, each of the conventional methods discussed above suffer from a common deficiency, that is, inability to quantify viable microorganisms over a wide dynamic range in real-time. Thus, there remains a need in the art for novel methods for quantification of microorganisms, in particular methods allowing distinguishing viable from non-viable cells.
To solve the aforementioned and other problems, there is provided a method for quantifying an amount of a viable microorganism, comprising subjecting a fluid sample suspected of containing a viable microorganism to a temperature change and correlating the temperature change in the fluid sample to an amount of the viable organism in the fluid sample. In one embodiment, the method may be accomplished by the steps of bringing the fluid sample to a first temperature, and transferring the fluid sample to a second temperature that is different than the first temperature. Next is the step of measuring a temperature change in the fluid sample, and correlating that temperature change to the amount of the viable microorganism contained in the test fluid.
In another aspect, there is provided a method for determining the effect of a test substance on growth or viability of a microorganism, comprising suspending a predetermined amount of a viable microorganism in a fluid sample and adding a predetermined amount of the test substance to fluid sample. The fluid sample is then subjected to a temperature change, and the temperature change in the fluid sample is correlated to the amount of the viable microorganism contained in the fluid sample as described above to determine the effect of the test substance on the amount of the viable microorganism contained in the test fluid.
In one embodiment, the first temperature is maintained for a sufficient period of time to place the fluid sample in a state of thermal equilibrium. The temperature change may be measured by holding the fluid sample at the second temperature, optionally for a sufficient time to allow the fluid sample to reach thermal equilibrium. During the step of allowing the fluid sample to reach thermal equilibrium, the temperature change in the fluid sample is measured at spaced time intervals over a predetermined time period. That measuring step may be accomplished by acquiring a plurality of sequential thermal images of the fluid sample at the spaced time intervals, such as by infrared thermography.
The correlation step includes relating a plotted slope of the normalized temperature change (measured as described above) against the normalized predetermined time period to an amount of thermal energy released from the fluid sample. In one embodiment, the amount of thermal energy released from the fluid sample is correlated to the plotted slope of normalized temperature change against normalized predetermined time period according to the formula:
where Δf(T) is change in normalized temperature and Δg(t) is change in normalized time.
These and other embodiments, aspects, advantages, and features of the present invention will be set forth in the description which follows, and in part will become apparent to those of ordinary skill in the art by reference to the following description of the invention and referenced drawings or by practice of the invention. The aspects, advantages, and features of the invention are realized and attained by means of the instrumentalities, procedures, and combinations particularly pointed out in the appended claims. Various patent and non-patent citations are referenced herein. Unless otherwise indicated, any such citations are specifically incorporated by reference in their entirety into the present disclosure.
The accompanying drawings incorporated in and forming a part of the specification, illustrate several aspects of the present invention, and together with the description serve to explain the principles of the invention. In the drawings:
a-b show growth curves for S. aureus measured by IRT (
a-c present time-kill curves for E. coli exposed to varying amounts of ciprofloxacin (CIP); 7a) CIP time-kill curve for E. coli (CIP Minimum Inhibitory Concentration (MIC)=0.016 μg/mL) expressed as colony count results, 7b) CIF time-kill curve for E. coli (CIP MIC)=0.016 μg/mL) expressed as En obtained by infrared thermography (IRT) according to the present invention; 7c) relationship of viable counts and IRT results by regression analysis;
a-b show cefazalin time-kill profiles for E. coli ATCC 25922, measured by IRT (
a-b show cefazalin time-kill profiles for S. aureus ATCC 29213, measured by IRT (
In the following detailed description of the illustrated embodiments, reference is made to the accompanying drawings that form a part hereof, and in which is shown by way of illustration, specific embodiments in which the invention may be practiced. These embodiments are described in sufficient detail to enable those skilled in the art to practice the invention. Also it is to be understood that other embodiments may be utilized and that process, reagent, software, and/or other changes may be made without departing from the scope of the present invention.
A basic property of life is the generation of heat resulting from the biochemical reactions required for cellular function. For that reason, detection of heat generation, such as by calorimetry, has been considered as a tool to detect and quantify the number of microorganisms in biological samples. However, to date such methods necessitate complete destruction of the sample to measure energy content, precluding dynamic temperature change measurements. Likewise, infrared thermography (IRT) has been evaluated to detect heat production/dissipation in animals, plants, cells in culture, and cell-free systems (see U.S. Pat. No. 6,983,752, incorporated herein by reference). However, such methods do not contemplate monitoring changes in viable microorganism population dynamics overtime, and further provide no indication of suitability for quantification of such viable microorganism populations.
The present studies were designed to evaluate whether such generation of heat by microorganisms could provide a useful tool in non-contact quantification of microorganisms, especially viable microorganisms. The present investigators have found that the number of viable microorganisms in culture may be quantified by measuring energy transfer from a fluid containing microorganisms to the ambient surroundings. There is accordingly described herein a real-time, non-contact method for quantification of viable microorganisms by detecting such energy transfer by capture of sequential thermal images such as by infrared theithography. Use of the described method is contemplated for a variety of applications, including without limitation evaluation of potential microbiocidal and/or microhiostatic compositions or compounds, or alternatively for testing and evaluation of potential growth factors for microorganisms.
A system 10 for measuring energy transfer from bacterial cultures was developed, and is shown in schematic form in
Suitable infrared detectors 12 are known in the art, such as infrared cameras and the like. Similarly, it is well known to provide a variety of incubators 18 and controlled temperature chambers with coolers or heaters 20 for holding microorganisms, cultured cells, etc. at a desired temperature. The data processor 14 may be any suitable device capable of receiving data from the infrared detector 12 and processing those data as will be described below, including personal computers, mainframe computers, and the like.
The system described in Example 1 was used to determine the thermal properties of bacterial growth media in the presence and absence of bacteria. For that purpose, a series of inocula of Escherichia coli were established in Mueller-Hinton broth (MHB) in ten-fold dilutions, ranging from 102 to 108 colony-forming units (CFU)/ml. Control wells contained MHB alone. Temperature measurements obtained using the system of Example 1 were normalized to reduce dependence on location, acquisition time, initial temperature, and surrounding temperature; and correlated with the normalized acquisition time (t). The slope of the correlation was found to be a function of the normalized released-thermal-energy by the media, referred to as the Energy Number (En), which was found to be directly related, to the interaction of the temperature with the surrounding and the measurement time, set forth in the following relation:
Energy Number∝f(T)·g(t)
That temperature function is a non-dimensional number that compares temperature changes in each well on the same relative scale, defined as:
where ΔT is the relative temperature change. Similarly, the time scale was normalized with respect to a time constant (τ) derived from the slope of temperature cooling time rate for the media without bacteria. The normalized time was expressed in the logarithmic function:
where t* is the normalized time. Then:
f(T)=Slope×g(t)
f(T)=1−ΔT,Slope=(1−En),g(t)=t*
The Energy Number En was thus defined as:
where EM is the metabolism energy introduced to the media by the bacterial activity', h is the convection heat transfer coefficient, and AS is the surface area of the well. The metabolism energy EM was defined as:
E
M
=E′
M
F(N)
where E′M is the energy produced by one microorganism cell, N is the bacterial concentration, and F(N) is a function of the number of bacteria. The value En defined below can be measured:
where Δf(T) is the change in normalized temperature and Δg(t) is the change in normalized time.
A representative experiment is shown in
A 24-well culture dish was prepared by painting with a smooth satin black, to reduce reflection, and inoculated with E. coli at increasing concentrations as set forth in Table 1. Culture conditions were as described in Example 2. Only the center 8 wells were inoculated. Thermal imaging was recorded by an Avio Photonic Detector, TVS-8500. Images were taken at 1 minute intervals for a total of 30 minutes, to ascertain the effect of time.
As shown in
The presently described system 10 was used to determine the thermal properties of the growth media in the absence and presence of a representative grain positive bacterium, Staphylococcus aureus (S. aureus). Inocula ranging from 101 to 108 CFU/ml in ten-fold dilutions were prepared in MHB. The same experimental method that was used for Example 2 was applied to measure thermal properties of Staphylococcus aureus. Energy number (En), ranging from 0.15 to 0.825, shown in
The described system 10 was used to determine the thermal properties of the growth media in the absence and presence of a commercially available active dry baking yeast. 24 well plates series of inocula of yeast were prepared in (NM) with three different concentrations (2, 4, and 8 mg/ml) and one control.
The present system 10 is used to measure the growth of a yeast, Candida albicans, in RPMI 1640 with L-glutamine broth without bicarbonate, a medium recommended for antifungal susceptibility testing. Thermal signatures of modified RPMI 1640 broth alone and RPMI 1640 with different inocula of the reference strain C. albicans ATCC 90028 in ten-fold dilutions ranging from 102 to 108 CFU/ml are determined. Growth of the yeast in 96-well microtiter plates at 35° C. is monitored every 2 h for 24 h. Correlations between energy released from the surface of the media and the yeast cell counts are determined. In this fashion, system 10 is used for monitoring death of the yeast cells in real time during exposure to a representative antifungal agent (e.g. amphotericin B, flucytosine, ketoconazole, or fluconazole). The data are used to develop a mathematical model to describe the thermodynamics of yeast cell growth and death.
The present system 10 is used to measure the growth of the marine dinoflagellate, Alexandrium fundyense, which is a eukaryotic algae responsible for red tide. Cultures are grown in 172 medium made with 0.2 μm filtered seawater (31 practical salinity units) and modified by the addition of H2SeO3 and CuSO4, both to final concentrations of 10−8M. Cultures are incubated at 20° C. on a 14 h light:10 h dark cycle. The cultures are removed from the incubator at different time intervals and allowed to equilibrate at an ambient room temperature (25° C.). During this 3 minutes transient heating period, IR images are taken using an infrared detector. Thermal properties of the media in the absence and presence of 10-fold dilutions of A. fundyense are determined. The proliferation of the dinofiagellate over several days under the above culture conditions is evaluated and the correlation between energy released from the surface of the media and microscopic cell counts is determined.
The present system 10 is used to measure the growth of the marine dinoflagellate, Alexandrium fundyense, which is a eukaryotic algae responsible for red tide. Cultures are grown in f/2 medium made with 0.2 μm filtered seawater (31 practical salinity units) and modified by the addition of H2SeO3 and CuSO4, both to final concentrations of 10−8 M. Cultures are incubated at 20° C. on a 14 h light:10 h dark cycle. The cultures are removed from the incubator at different time intervals and allowed to equilibrate at an ambient cold room temperature (4° C.). During this 3 minutes transient cooling period, IR images are taken using an infrared detector. Thermal properties of the media in the absence and presence of 10-fold dilutions of A. fundyense are determined. The proliferation of the dinoflagellate over several days under the above culture conditions is evaluated and the correlation between energy released from the surface of the media and microscopic cell counts determined.
The present system 10 is used to measure the growth of the human monocyte cell line THP-1 (ATCC TIB-202) and the human colorectal adenocarcinoma cell line HT-29 (ATCC HTB-38). Cell cultures of THP-1 are grown at 37° C. in RPMI 1640 media supplemented with 2-mercaptoethanol (0.05 mM) and fetal bovine serum (10%). Cell cultures of HT-29 are grown at 37° C. in modified McCoy's 5a media supplemented with fetal bovine serum (10%). Thermal properties of the respective media in the absence and presence of each cell line in concentrations ranging from 5×104 to 1×106 viable cells/ml are determined using System 10. The replication of both cell lines is monitored daily over two to three days in a 96-well microtiter plate under the above culture conditions. The correlation between energy released from the media and microscopic cell counts is determined.
It was desired to evaluate utility of the system in a real-time method to measure bacterial growth and death as a function of exposure to an antimicrobial. For that purpose, time-kill studies were performed using E. coli cultures, established substantially as set forth in Example 2. A series of microtiter plates were inoculated with an overnight culture of a representative gram-negative E. coli strain to achieve a starting inoculum of approximately 2×106 CFU/ml per well. Test wells were exposed to nine concentrations of an antimicrobial agent of a representative fluoroquinolone (ciprofloxacin) ranging from 0- to 32-fold minimum inhibitory concentration (MIC). Five of those treatments (0, 5, 1, 2, 4, and 8-fold MIC) were subcultured for determination of viable cell counts. Control wells received no ciprofloxacin. The plates were incubated at 37° C. for a predetermined time interval (0, 2, 4, 6, 8, and 24 hours), removed from the incubator, allowed to equilibrate to an ambient cold room temperature (4° C.), and imaged over a 3 minute time period using an infrared detector (FLIR Infrared Detector, Model No. SC4000, N. Billerica, Mass.). Post-imaging, the contents of the designated wells were removed and the number of viable bacteria in each well was determined using drop and filter count methods.
a and 7b set forth the changes in bacterial viable counts and the Energy Number of the medium over time, respectively. The Energy Number of the medium over time as determined from thermodynamic modeling of the IR images collected by the detector was highly correlated (R2=0.9891) to the viable counts obtained following 24 hr incubation (see
A series of microtiter plates were inoculated with an overnight culture of a representative gram-negative E. coli strain to achieve a starting inoculum of approximately 2×108 CFU/ml per well. These test wells were exposed to nine different concentrations of an antimicrobial agent of a representative cephalosporin (cefazolin) ranging from O— to 16-fold the minimum inhibitory concentration (MIC). Wells containing five of those concentrations (0.5, 1, 2, 4, and 8-fold MIC) were sub-cultured for determination of viable cell counts. Control wells contained no cefazolin. All plates were incubated at 37° C. and removed from the incubator at six different time intervals (0, 2, 4, 6, 8, and 24 hours) and allowed to equilibrate at an ambient cold room temperature (4° C.). During this 3 minutes transient cooling period, IR images were taken using an infrared detector (FUR Infrared Detector, Model No. SC4000, N. Billerica, Mass.). Our IRT results (
A series of microtiter plates were inoculated with an overnight culture of a representative gram-positive S. aureus steam to achieve a starting inoculum of approximately 2×108 CFU/ml per well. These test wells were exposed to nine different concentrations of an antimicrobial agent of a representative cephalosporin (cefazolin) ranging from O— to 16-fold the minimum inhibitory concentration (MIC). Wells containing five of those concentrations (0.5, 1, 2, 4, and 8-fold MIC) were sub-cultured for determination of viable cell counts. Control wells contained no cefazolin. All plates were incubated at 37° C. and removed from the incubator at six different tune intervals (0, 2, 4, 6, 8, and 24 hours) and, allowed to equilibrate at an ambient cold room temperature (4° C.). During this 3 minutes transient cooling period, IR images were taken using an infrared detector (FLIR Infrared Detector, Model No. SC4000, N. Billerica, Mass.). Our IRT results (
There is accordingly provided a method for detecting and quantitating a viable microorganism in a fluid sample. The method advantageously is non-contact, and does not require any chemical additions or destruction of a sample to provide information regarding viable microorganism numbers. Still further, the method provides real-time detection and quantitation of viable microorganisms in a sample, rather than requiring lengthy incubation periods as are necessary in conventional colony count methods. The method finds use in a variety of applications, including evaluation of test agents for microbiocidal, microbiostatic, or growth enhancer properties.
One of ordinary skill in the art will recognize that additional embodiments of the mention are also possible without departing from the teachings herein. This detailed description, and particularly the specific details of the exemplary embodiments, is given primarily for clarity of understanding, and no unnecessary limitations are to be imported, for modifications will become obvious to those skilled in the art upon reading this disclosure and may be made without departing from the spirit or scope of the invention. Relatively apparent modifications, of course, include combining the various features of one or more figures or examples with the features of one or more of other figures or examples.