Methods for arousing dormant bacteria

Details Methods for arousing dormant bacteria Methods for arousing dormant bacteria

1999-10-28

2003-07-08

Naff, David M. (Department: 1651)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

C435S244000, C435S245000, C435S252400, C435S252800, C435S252900, C435S260000, C435S849000, C435S856000, C435S857000, C435S875000

Reexamination Certificate

active

06589771

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to methods and compositions for arousing dormant bacteria. More particularly, this invention relates to methods and compositions for arousing dormant bacteria for the purpose of detecting their presence, evaluating their threat to health, and, if warranted, killing them. Even more particularly, the invention relates to arousing dormant bacteria by altering their internal osmolality and/or pH and allowing adjustment time for the arousal mechanism to be initiated.
BACKGROUND OF THE INVENTION
Dormant populations of bacteria are formed during the normal growth cycles of non-spore forming genera. Dormant bacteria were first described in 1925 as cells formed during the stationary phase of normal microbial growth (Burke, Sprague & Barnes,
J. Inf. Dis
., 36:555-560 (1925)). By definition, they do not grow on nutrient-rich media selected for their proliferation and classification. Since growth on selected media defines viability, confusion and disbelief continue to surround the existence of dormant bacteria. The phrase “viable but not culturable” (VBNC) is frequently used as a descriptor.
Dormancy is different from sporulation. Late in the stationary phase vegetative cells of the genera Bacillus and Clostridium form spores, dense particles resistant to adverse conditions. For example, Clostridial spores can withstand 100° C. for more than one hour, therefore, saturated steam at 121° C. for 15 minutes is required to kill them. Germination of these spores is induced by heating neutral suspensions of these cells at 65° C. for 20 minutes. If L-alanine, L-tyrosine, or adenosine is added to the buffer, 100% of the spores will germinate.
Dormant bacteria are believed to form in response to environmental stressors that are not part of their normal growth cycle. Dormant bacteria form during the normal growth cycle for both harmless and pathogenic isolates of Gram-positive and Gram-negative bacteria Thus these non-detectable dormant bacteria have the potential to be widespread threats to human health. Their presence is suspicioned in recurring illness in patients and endemics. The spread of cholera has been in part attributed to dormant forms of
V cholera
in potable water. Brayton, P. R., et al. (1987)
Appl. Envir. Micro
. 53:2862. The outbreak of Legionella infections in Philadelphia in 1976 is believed to have resulted from the arousal of dormant
L. pneumophila
residing in a hotel's air conditioning system. Steinert, M., et al. (1997)
Appl. Envir. Micro
. 63:2047. Endogenous dormant
M. tuberculosis
are thought to be responsible for recurrent tuberculosis. Hu, Y. M., et al. (1998)
FEMS Micro LETT
158:139. Dormant
Shigella dysenteriae
produce diarrhea when orally administered to humans. Rahman, I., et al. (1994)
Apl. Envir. Micro
. 60:3573. Quiescent bacteria may be aroused during in vitro fertilization and embryo transfer. Peters, A. J., et al.
Ob & Gyn
81:876 (1993).
Quiescent bacteria may be aroused during in vitro fertilization and embryo transfer. Peters, A. J., et al.
Ob & Gyn
81:876 (1993).
Methods to “resuscitate” dormant bacteria have been identified for several genera or species, namely
V. cholera, V. vulnificus, M. luteus, L. pneumophila, M. tuberculosis
, and
Nitrosomonas
. Dormancy was induced by placing small amounts of vegetative cells into life-threatening conditions, such as nutrient or oxygen deprivation or low temperature environments for long periods, e.g. 125 days. Cells were resuscitated by restoring normal or rich conditions or by passing dormant bacteria through their parasitic or animal hosts. U.S. Pat. No. 5,314,542 discloses packaging bacteria from the genus
Nitrosomonas
in such a way as to induce dormancy, then reactivating these bacteria by increasing the concentration of their key nitrogen nutrient, ammonia, to 200 ppm and holding for 72 hours. To date, no one general method can be applied broadly to multiple species of bacteria.
For the foregoing reasons, a need exists for a generally-adaptable method to arouse dormant bacteria.
SUMMARY OF THE INVENTION
An object of this invention is to provide a method for arousing dormant bacteria.
Another object of this invention is to provide a method for recovering aroused, previously dormant bacteria from a culture for purposes such as classification, killing, or prevention of proliferation.
These and other objects, features, and advantages will become apparent after review of the following description and claims of the invention which follow.
This invention comprises a method for arousing dormant (non-spore forming) bacteria. The purposes of arousal include, for example, classification, killing, or prevention of proliferation in susceptible hosts.
Dormant bacteria are formed during the stationary phase of a normal growth cycle of both Gram-positive and Gram-negative species, both pathogenic and harmless species, and both feral and lab isolates. After proliferation, nonspore-forming bacteria may enter into a state of dormancy, and neither grow on media nor die in unfavorable environments. Although metabolically active, dormant bacteria are tolerant to antibiotics, chemicals, and other toxicants. Arousal can be induced by decreasing the internal osmolality, and thereby, the internal water activity of the cell (a
w
), or by neutralizing the internal pH.
The changes in osmolality or pH required for arousal are initiated by forcing the cell to diffuse its solutes or hydrogen ions into the media environment in a prescribed series of exposures to osmolal downshift gradients and providing for periods of adjustment. In other words, the internal osmolality of the dormant bacterial cell is decreased, and the cell is allowed to adjust to these changes gradually. During the adjustment period, the cell prepares to initiate replication. If the adjustment period is extended beyond that required for the initiation of growth, the cell can become hypermutative, as demonstrated by an ability to tolerate lethal doses of antibiotics without having been primed by exposures to less than lethal levels.
Different species require different adjustment periods and different rates of diffusion of internal solutes to maximize arousal. In addition, although just neutralizing the internal pH can be sufficient to induce arousal, an adjustment period of 10 days or more may be necessary to induce growth even in cultures that are easily aroused. When rates of diffusion and periods of adjustment are optimally controlled, the maximum number of dormant cells are aroused in the shortest amount of time. Too sudden diffusion from a gradient of too great an intensity will not induce arousal. On the other hand, if diffusion is too slow, arousal requires long periods of adjustment before arousal occurs.
The extent of downshift gradients and the length of the adjustment period required to arouse the maximum number of dormant cells in the shortest time is species dependent. Some, like
L. monocytogenes
is readily aroused, and others, like
L. plantarum
require extensive treatment before arousing. However, the present invention teaches a general method that arouses easily-arousable bacteria and provides guidance for arousing more difficult ones.
Definitions
“Dormant” bacteria as used herein includes (a) bacterial cells that are “viable but not culturable” or “quiescent” or “nascent” which are (b) metabolically active, but (c) do not propagate in broths or on agar media formulated for their growth and identification.
“Arousing” as used herein includes causing dormant cells to propagate in broths or an agar media developed for their growth and identification.
The “vegetative” form of dormant bacteria, as used herein, is that form of the dormant cells which grows on appropriate media.
“Hypermutative” dormant cells are those that during arousal phase develop the ability to proliferate in the presence of lethal levels of an antibiotic to which they were not previously exposed.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
Non-spore forming bacteria form dormant cells during the stationary phase of a

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