Methylobacterium sp. resides in unculturable state in potato tissues in vitro and becomes culturable after induction by Pseudomonas fluorescens IMGB163

Aims:  To induce growth of endophytic bacteria residing in an unculturable state in tissues of in vitro -grown potato plantlets. To isolate and identify the induced bacteria and to localize the strains in tissues of in vitro -grown potato plantlets.
Methods and Results:  The inoculation of in vitro -grown potato plants with Pseudomonas fluorescens IMBG163 led to induction of another bacterium, a pink-pigmented facultative methylotroph that was identified as Methylobacterium sp . using phylogenetic 16S rDNA approach . Two molecular methods were used for localizing methylobacteria in potato plantlets: PCR and in situ hybridization (ISH/FISH). A PCR product specific for the Methylobacterium genus was found in DNA isolated from the surface-sterilized plantlet leaves. Presence of Methylobacterium rRNA was detected by ISH/FISH in leaves and stems of inoculated as well as axenic potato plantlets although the bacterium cannot be isolated from the axenic plants.
Conclusion:  Methylobacterium sp. resides in unculturable state within tissues of in vitro -grown potato plants and becomes culturable after inoculation with P. fluorescens IMBG163.
Significance and Impact of the Study:  In order to develop endophytic biofertilizers and biocontrol agents, a detailed knowledge of the life-style of endophytes is essential. To our knowledge, this is the first report on increase of the culturability of endophytes in response to inoculation by nonpathogenic bacteria.     

Microbial inactivation using plasma-activated water obtained by gliding electric discharges

Aim:  To evaluate the microbial disinfection efficacy of a plasmachemical solution obtained by the activation of water with gliding electric discharges.
Methods and Results:  Distilled water was activated for 5 min by a nonthermal quenched plasma of the glidarc type operating in humid air and at atmospheric pressure. The plasma-activated water (PAW) was then used to treat planktonic and adherent cells of Staphylococcus epidermidis , Leuconostoc mesenteroides (as models of Gram-positive bacteria), Hafnia alvei (a Gram-negative bacteria) and Saccharomyces cerevisiae (as a yeast model). The treatments were less efficient on adherent cells than on planktonic cells in the case of bacteria, but not of S. cerevisiae . Inactivation was more effective for bacteria than for the yeast.
Conclusions:  Significant reductions in microbial populations were achieved in all cases, demonstrating the effectiveness of this new approach to treat contaminated media.
Significance and Impact of the Study:  PAW is a promising solution with potential application to the decontamination of equipment and surfaces.     

Comparative study of a new quantitative real-time PCR targeting the xylulose-5-phosphate/fructose-6-phosphate phosphoketolase bifidobacterial gene (xfp) in faecal samples with two fluorescence in situ hybridization methods

Aims:  To detect and enumerate bifidobacteria in faeces with a new quantitative multiplex real-time PCR (qPCR) method and to compare the results obtained with fluorescence in situ hybridization (FISH) methods.
Methods and Results:  A multiplex qPCR assay was developed, which enabled the enumeration of Bifidobacterium spp. by targeting the bifidobacterial xylulose-5-phosphate/fructose-6-phosphate phosphoketolase gene ( xfp ) and total bacteria using universal Eub-primers targeting 16S rRNA gene from the domain bacteria. The qPCR assay showed high sensitivity and specificity and a low detection limit of about 2·5 × 10³ bifidobacterial cells per gram of faeces. The qPCR results were compared with FISH combined with microscopy or flow cytometry (FCM). No statistical differences among bifidobacterial counts averages measured in adult faeces with the three methods were observed. Total bacterial count averages were higher with the FISH method coupled with microscopic analyses compared to FISH with FCM, whereas total cell numbers estimated by qPCR were intermediate between the two FISH methods.
Conclusions:  The new qPCR assay was shown to be sensitive, rapid and accurate for enumerating bifidobacteria in faeces.
Significance and Impact of the Study:  This method is a valuable alternative for other molecular methods for detecting faecal bifidobacteria, especially when their counts are below the detection limit of the FISH methods.     

Molecular ecology of a facultative swine waste lagoon

Aims:  To investigate the microbial ecology of three facultative swine waste lagoons.
Methods and Results:  Phylogenetic analysis of sequences in a 16S rRNA gene clone library and fluorescence in situ hybridization (FISH) analyses were used to assess bacterial diversity in a swine waste lagoon. FISH analysis and Gram-staining were used to compare the microbial communities of all three swine waste lagoons. Six operational taxonomic units were in high relative abundance and corresponded to the following phylotypes; Thiolamprovum , Verrucomicrobia , Acholeplasma , Turicibacter , Clostridium and Bacteroides . PCR was employed to detect the genes apsA and dsrAB which encode for enzymes specifically associated with dissimilatory sulfate-reduction within sulfate-reducing bacteria (SRB). Amplification of these genes confirmed their presence within the lagoons.
Conclusions:  All lagoons were dominated by purple sulfur bacteria, affiliated to Thiolamprovum pedioforme . The molecular identification of fermentative bacteria and SRB indicate the following metabolic processes within such facultative ponds: sulfur-cycling, fermentation, inter-species hydrogen transfer and carbon cycling.
Significance and Impact of the Study:  This study provides the first molecular evidence for the existence of a sulfur cycle which is linked to phototrophic sulfide oxidation by purple bacteria and organotrophic sulfate-reduction by SRB.     

Inhibition spectrum studies of microthecin and other anhydrofructose derivatives using selected strains of Gram-positive and -negative bacteria, yeasts and moulds, and investigation of the cytotoxicity of microthecin to malignant blood cell lines

Aims:  To prepare 1,5-anhydro- d -fructose (AF) derivatives, test their microbial inhibition spectrum, and to further examine the most effective AF derivative against Pseudomonas aeruginosa and malignant blood cell lines.
Methods and Results:  Microthecin and nine other AF derivatives were synthesized from AF. The 10 compounds were tested in vitro against Gram-positive (GP) and Gram-negative (GN) bacteria, yeasts and moulds using a well diffusion method and in a Bioscreen growth analyser. Of the test compounds, microthecin exhibited the most significant antibacterial activity at 100–2000 ppm against both GP and GN bacteria, including Ps. aeruginosa. Further tests with three malignant blood cell lines ( Mutu, Ramos, Raji ) and one normal cell line indicated that microthecin was a cell toxin, with a cell mortality >85% at 50 ppm. The other nine AF derivatives demonstrated low or no antimicrobial activity.
Conclusions:  Microthecin was active 100–2000 ppm against GP and GN bacteria including Ps. aeruginosa , but was inactive against yeasts and moulds. Microthecin was also a cytotoxin to some mammalian cell lines.
Significance and Impact of the Study:  Microthecin might have potential for development as a novel drug against Ps. aeruginosa and to target cancer cells. It might also be developed as a food processing aid to control bacterial growth.     

Conjugative plasmids in multi-resistant bacterial isolates from Indian soil

Aims:  Determination of heavy metal and antibiotic resistance and presence of conjugative plasmids in bacteria isolated from soil irrigated with wastewater.
Methods and Results:  Composite soil samples were collected from Ghaziabad, Uttar Pradesh, India. Forty different bacteria were selected from nutrient agar and characterized by morphological, cultural and biochemical tests. All the isolates were tested for their resistance to different heavy metals and antibiotics. The DNA derived from multiple metal and antibiotic-resistant bacterial isolates was PCR amplified and plasmid-specific sequences (IncP, IncN, IncW, IncQ and pMV158-type) were analysed by dot blot hybridization. All isolates gave PCR products with trfA2 and oriT primers of the IncP group. These PCR products also hybridized with the RP4-derived probes. However, the samples were negative for all the other investigated plasmids as proved by PCR and dot blots.
Conclusions:  The presence of conjugative/mobilizable IncP plasmids in the isolates indicates that these bacteria have gene-mobilizing capacity with implications for potential dissemination of introduced recombinant DNA.
Significance and Impact of the Study:  The detection of IncP plasmids in all the bacterial isolates is another proof for the prevalence of these plasmids. We propose that IncP plasmids are mainly responsible for the spread of multi-resistant bacteria in these soils.     

Bioluminescence intensity difference observed in luminous bacteria groups with different motility

Aims:  The aim of this study was to compare the luminescent intensity of bioluminescence from marine luminous bacteria with different motility.
Methods and Results:  Luminescent bacteria were separated according to their motility using a microfluidic device. The cell densities of the separated samples were measured using a counting plate. The luminescent intensity of the separated samples was measured using a luminometer. The luminescent intensity per cell was calculated, and the values from the mobile (swimmers) and the nonmobile cells (nonswimmers) per single cell were compared; as a result, the former were proved to be larger than the latter.
Conclusions:  Microfluidics were shown to be effective for the separation of bioluminescent bacteria and the bioluminescent intensity difference per cell was recognized with this experiment.
Significance and Impact of the Study:  This study introduced for the first time a method to examine the individual cell function of Photobacterium kishitanii .     

Mixotrophic ciliates in an Andean lake: dependence on light and prey of an Ophrydium naumanni population

1. Planktonic ciliates were examined during a spring–summer period (November 1998–April 1999) in the ultraoligotrophic Lake Moreno Oeste (41°5' S and 71°33' W, 758 m a.s.l), which belongs to the Nahuel Huapi System (Patagonia, Argentina). The lake is deep ( Z _max=90 m) and warm monomictic.
2. Sampling was performed at a mid-lake station, where vertical profiles of temperature and light were measured in situ , and samples for bacteria and ciliates enumeration were taken throughout the water column.
3. The peritrich Ophrydium naumanni , a freshwater pelagic ciliate with endosymbiotic Chlorella , was the dominant ciliate in the lake.
4.  Ophrydium naumanni and autotrophic picoplankton exhibit a clear coincidence in their vertical distribution ( P  < 0.05), preferring levels at or near the 1% of surface photosynthetically active radiation (PAR) irradiance. Both may have the same light requirements, or the coincidence may reflect a trophic relationship.
5. Dependence on light and prey by O. naumanni were studied using field experiments, in which we analysed ciliate grazing on bacteria, and in laboratory experiments, in which we compared particle uptake under dark and light conditions.
6.  Ophrydium naumanni was able to ingest particles [latex microspheres and fluorescently labelled bacteria (FLB)] in field and laboratory experiment, indicating that it has the potential to affect bacteria population of Lake Moreno Oeste.
7. Ciliate particle ingestion was observed to be dependent on light availability because under dark conditions, the ingestion was lowered ( P  < 0.05).     

A qRT-PCR-based method for the measurement of rrn operon copy number

Aim:  To develop a convenient and accurate method for estimating the rrn operon copy number ( Y _rrn ) in cells of pure prokaryotic cultures based on quantitative real-time polymerase chain reaction (qRT-PCR).
Methods & Results:  Using Escherichia coli, the Y _rrn of which is known to be 7, as a reference, the rrn concentrations of target species and E. coli in sample solutions were measured based on their respective threshold cycle numbers ( C _t ), whereas the cell concentrations of both species were measured by microscopic counting after staining. The Y _rrn of the target species was then calculated from the initial cell concentrations and the rrn concentrations of the target species and E. coli . Using this method, the Y _rrn values of four species, i.e. Xanthomonas campestris , Staphylococcus aureus , Aeromonas hydrophila and Pseudomonas fluorescens , were estimated as 1·80, 4·73, 8·58 and 5·13, respectively, comparable to their respective known values of 2, 5, 10, and 5, resulting in an average deviation of 8%.
Conclusions:  The whole cell qRT-PCR based methods were convenient, accurate and reproducible in quantification of rrn copy number of prokaryotic cells.
Significance and Impact of the Study:  qTR-PCR is a fast and reliable DNA quantification approach. Compared with previous qTR-PCR based methods measuring rrn copy number, the present method avoided the prerequisite for the information on genome size and GC content of target bacteria or a gene with known copy number, thus should be more widely applicable.     

Detection of Helicobacter hepaticus in Human Bile Samples of Patients with Biliary Disease

Background:  Since the discovery of Helicobacter pylori , various enterohepatic Helicobacter spices have been detected in the guts of humans and animals. Some enterohepatic Helicobacters have been associated with inflammatory bowel disease or liver disease in mice. However the association of these bacteria with human diseases remains unknown.
Materials and Methods:  We collected 126 bile samples from patients with cholelithiasis, cholecystitis, gallbladder polyp, and other nonbiliary diseases. Samples were screened for the presence of enterohepatic Helicobacter spp. using cultures, nested PCR, or in situ hybridization. We tested for antibodies to H. pylori and H. hepaticus by Western blot analysis.
Results:  Attempts at cultivation were unsuccessful. However, H. hepaticus was detected in bile samples with nested PCR whereas H. bilis was not. Helicobacter hepaticus in the bile was confirmed by in situ hybridization, but H. hepaticus from bile samples was coccoid in appearance. We detected immunoglobulin G antibodies to H. hepaticus in bile samples by Western blotting. Helicobacter hepaticus was detected in 40 (32%) of total 126 samples as H. hepaticus positive if at least one of the three methods with nested PCR, in situ, or Western blotting. Patients with cholelithiasis (41%) and cholecystitis with gastric cancer (36%) had significantly higher ( p  = .029) prevalence of H. hepaticus infection than samples from patients with other diseases.
Conclusion:  Helicobacter hepaticus may closely associate with diseases of the liver and biliary tract in humans.     

Phenotypic and genetic diversity of rice seed-associated bacteria and their role in pathogenicity and biological control

Aims:  To study the phenotypic and genetic diversity of culturable bacteria associated with rice seed and to asses the antagonistic and pathogenic potential of the isolated bacteria.
Methods and Results:  Seed of rice cultivar PSBRc14 was collected from farmers' fields of irrigated lowland in southern Luzon, Philippines. Isolations of distinct colonies yielded 498 isolates. Classification of the isolates according to similarities in cellular characteristics, whole-cell fatty acid composition, and colony appearance differentiated 101 morphotype groups. Predominant bacteria were Coryneform spp., Pantoea spp. and Pseudomonas spp. Other bacteria regularly present were Actinomycetes spp., Bacillus pumilus , B. subtilis , Burkholderia glumae , Enterobacter cloacae , Paenibacillus polymyxa , Staphylococcus spp. and Xanthomonas spp. The genetic diversity among isolates was assessed by BOX-PCR fingerprinting of genomic DNA and represented 284 fingerprint types (FPTs). Most FPTs (78%) were not shared among samples, while eight FPTs occurred frequently in the samples. Seven of these FPTs also occurred frequently in a previous collection made from rainfed lowlands of Iloilo island, Philippines. Sixteen per cent of the isolates inhibited in vitro the mycelial growth of the rice pathogens Rhizoctonia solani and Pyricularia grisea , whereas 4% were pathogens identified as Burkholderia glumae , Burkholderia gladioli and Acidovorax avenae ssp. avenae .
Conclusions:  This study reveals a broad morphological and genetic diversity of bacteria present on seed of a single rice cultivar.
Significance and Impact of the Study:  This line of work contributes to a better understanding of the microbial diversity present on rice seed and stresses its importance as a carrier of antagonists and pathogens.     

Dairy bacteria remove in vitro dietary lectins with toxic effects on colonic cells

Aims:  To assess in vitro the ability of some dairy bacteria to bind concanavalin A (Con A), peanut agglutinin (PNA) and jacalin (AIL), preventing their toxicity on mouse intestinal epithelial cells (IEC).
Methods and Results:  Con A and AIL reduced significantly IEC viability in vitro , as determined by Trypan Blue dye exclusion or by propidium iodide/fluorescein diacetate/Hoescht staining. Different strains of dairy bacteria were able to remove lectins from the media. Two strains were subjected to treatments used to remove S-layer, cell wall proteins, polysaccharides and lectin-like adhesins. They were then assayed for the ability to bind dietary lectins and reduce toxicity against IEC and to adhere to IEC after interaction with lectins. Con A and AIL were removed by Propionibacterium acidipropionici and Propionibacterium freudenreichii by binding with specific sugar moieties on the bacterial surface. Removal of lectins by bacteria impaired IEC protection. Adhesion of P. acidipropionici to IEC was reduced but not abolished after binding Con A or AIL.
Conclusions:  Removal of Con A or AIL by dairy propionibacteria was effective to avoid the toxic effect against colonic cells in vitro.
Significance and Impact of the Study:  Consumption of foods containing these bacteria would be a tool to protect the intestinal epithelia.     

Genetic correlation between chromium resistance and reduction in Bacillus brevis isolated from tannery effluent

Aims:  To investigate the genetic basis of Cr(VI) resistance and its reduction to Cr(III) in indigenous bacteria isolated from tannery effluent.
Methods and Results:  Four bacteria resistant to high Cr(VI) levels were isolated and identified as Bacillus spp. Their Cr(VI) reduction ability was tested. To assess the genetic basis of Cr(VI) resistance and reduction, plasmid transfer and curing studies were performed. Among all, B. brevis was resistant to 180 μg Cr(VI) ml⁻¹ and showed the greatest degree of Cr(VI) reduction (75·8%) within 28 h and its transformant was resistant to 160 μg Cr(VI) ml⁻¹ and reduced 69·9% chromate. It harboured a stable 18 kb plasmid DNA. Transfer and curing studies revealed that both the chromate resistance and reduction were plasmid mediated. The presence of other metal cations did not have any significant effect on Cr(VI) bioreduction.
Conclusions:  Bacillus brevis was resistant to elevated Cr(VI) levels and may potentially reduce it in short time from an environment where other metal ions are also present in addition to chromium ions. The strain tested shows a positive correlation between genetic basis of Cr(VI) resistance and reduction.
Significance and Impact of the Study:  To our knowledge, this is the first study on the genetic correlation between chromium resistance and reduction in bacteria. Such strains may potentially be useful in biotechnological applications and in situ Cr(VI) bioremediation.     

Detection of viral infection and gene expression in clinical tissue specimens using branched DNA (bDNA) in situ hybridization.

In situ hybridization (ISH) methods for detection of nucleic acid sequences have proved especially powerful for revealing genetic markers and gene expression in a morphological context. Although target and signal amplification technologies have enabled researchers to detect relatively low-abundance molecules in cell extracts, the sensitive detection of nucleic acid sequences in tissue specimens has proved more challenging. We recently reported the development of a branched DNA (bDNA) ISH method for detection of DNA and mRNA in whole cells. Based on bDNA signal amplification technology, bDNA ISH is highly sensitive and can detect one or two copies of DNA per cell. In this study we evaluated bDNA ISH for detection of nucleic acid sequences in tissue specimens. Using normal and human papillomavirus (HPV)-infected cervical biopsy specimens, we explored the cell type-specific distribution of HPV DNA and mRNA by bDNA ISH. We found that bDNA ISH allowed rapid, sensitive detection of nucleic acids with high specificity while preserving tissue morphology. As an adjunct to conventional histopathology, bDNA ISH may improve diagnostic accuracy and prognosis for viral and neoplastic diseases.     

Unsuitable distinction between viable and dead Staphylococcus aureus and Staphylococcus epidermidis by ethidium bromide monoazide

Aims:  The DNA-intercalating dye ethidium bromide monoazide (EMA) has recently been used as a DNA binding agent to differentiate viable and dead bacterial cells by selectively penetrating through the damaged membrane of dead cells and blocking the DNA amplification during the polymerase chain reaction (PCR). We optimized and tested the assay in vitro using Staphylococcus aureus and Staphylococcus epidermidis cultures to distinguish viable from dead bacteria, with the goal of reducing false positive PCR results.
Methods and Results:  Viable and heat-inactivated bacteria were treated with EMA or left untreated before DNA extraction. A real-time PCR assay for the detection of the tuf gene in each DNA extract was used. Our results indicated that EMA influenced viable bacteria as well as dead bacteria, and the effect of EMA depended on the EMA concentration and bacterial number.
Conclusions:  EMA is not a suitable indicator of bacterial viability, at least with respect to Staphylococcus species.
Significance and Impact of the Study:  Determining the viability of pathogens has a major impact on interpreting the results of molecular tests for bacteria and subsequent clinical management of patients. To this end, several methods are being evaluated. One of these methods – intercalating DNA of dead bacteria by EMA – looked very promising, but our study found it unsatisfactory for S. aureus and coagulase-negative Staphylococci.     

An alternative real-time PCR method to detect the Bacillus cereus group in naturally contaminated food gelatine: a comparison study

Aims:  Comparison of an internally controlled real-time PCR assay with the standard plate-based assay (ISO 21871) for the detection of Bacillus cereus group cells in gelatine.
Methods and Results:  A comprehensive TaqMan probe was designed allowing the real-time PCR assay to be fully inclusive and exclusive. An internal amplification control was designed and implemented at 500 copies per reaction without impact on target detection. Specific and selective detection of target cells was achieved with a quick and simple DNA preparation procedure. No significant difference (κ = 0·99) was observed between the performance of the real-time PCR and the standard plate-based method on naturally contaminated gelatines ( n  = 197). Relative accuracy, relative sensitivity and relative specificity were ≥99%.
Conclusions:  The real-time PCR assay is a valid alternative of the standard plate-based assay.
Significance and Impact of the Study:  The real-time PCR assay decreased the time between sample collection and result from 2 days to 2 h, while analysis cost did not increase. The gelatine-producing industry can ensure gelatine safety and quality in a much faster way.     

In vitro antibacterial activity and antifungal mode of action of flocculosin, a membrane-active cellobiose lipid

Aims:  To investigate the in vitro antibacterial activity and antifungal mode of action of flocculosin, a cellobiose lipid produced by Pseudozyma flocculosa .
Methods and Results:  When tested against clinical bacterial isolates, the compound was particularly active against Gram-positive bacteria and its effect was not mitigated against isolates known as resistant to other antibiotics. The antifungal activity of flocculosin was found to be rapid and concentration-dependent. At lethal concentrations against Candida albicans , flocculosin caused a rapid leakage of intracellular potassium and inhibited acidification of the medium by plasma membrane ATPases suggesting a physical rather than a biochemical effect. TEM observations of cells exposed 6 h to flocculosin revealed disrupted membranes and disorganized mitochondria.
Conclusions:  Data obtained in this study confirm that flocculosin acts by disrupting the membrane surface of sensitive micro-organisms.
Significance and Impact of the Study:  The elucidation of an antifungal mode of action of flocculosin can be exploited in furthering its antimicrobial potential against fungi and bacteria whose cell membranes are particularly sensitive to the action of the molecule.     

The antimicrobial mechanism of electrochemically activated water against Pseudomonas aeruginosa and Escherichia coli as determined by SDS-PAGE analysis

Aims:  To expose bacteria to anolyte and subsequently investigate the effect of anolyte on the protein profiles of treated bacteria.
Methods and Results:  Proteins were extracted from bacteria treated with different concentrations of anolyte and analysed using SDS-PAGE. Fewer and more faint protein bands were observed for concentrated halide anolyte treated bacteria when compared to untreated bacteria while extra protein bands were observed for bacteria exposed to dilute concentrations.
Conclusions:  The undiluted and the 10⁻¹ dilution of halide derived anolyte was effective in killing the test bacteria. Anolyte caused bacterial death by complete destruction of proteins or by causing oxidative stress which resulted in protein fragmentation.
Significance and Impact of the Study:  The results of this study provide information on the antimicrobial mechanism of anolyte on other bacteria for which the information is currently unavailable.     

Viable ultramicrocells in drinking water

Aims:  To examine the diversity of cultivable 0·2 micron filtrate biofilm forming bacteria from drinking water systems.
Methods and Results:  Potable chlorinated drinking water hosts phylogenetically diverse ultramicrocells (UMC) (0·2 and 0·1  μ m filterable). UMC (starved or dwarf bacteria) were isolated by cultivation on minimal medium from a flow system wall model with polyvinyl chloride (PVC) pipes. All cultivated cells (25 different isolates) did not maintain their ultra-size after passages on rich media. Cultured UMC were identified by their 16S ribosomal DNA sequences. The results showed that they were closely related to uncultured and cultured members of the Proteobacteria, Actinobacteria and Firmicutes. The isolates of phylum Actinobacteria included representatives of a diverse set of Actinobacterial families: Micrococcaceae, Microbacteriaceae, Dermabacteraceae, Nocardiaceae and Nocardioidaceae.
Conclusions:  This study is the first to show an abundance of cultivable UMC of various phyla in drinking water system, including a high frequency of bacteria known to be involved in opportunistic infections, such as Stenotrophomonas maltophilia, Microbacterium sp., Pandoraea sp. and Afipia strains.
Significance and Impact of the Study:  Chlorinated tap water filtrate (0·2 and 0·1  μ m) still harbours opportunistic micro-organisms that can pose some health threat.     

Anhydrobiosis quotient: a novel approach to evaluate stability in desiccated bacterial cells

Aim:  The major objective of this study was the development of a methodology to quantify the anhydrobiotic ability of bacteria and its application to evaluate the stability of desiccated bacterial cells using the biocontrol agent Tsukamurella paurometabola C-924 as a model of anhydrobiote.
Methods and Results:  Tsukamurella paurometabola C-924 was desiccated by spray-drying. Samples of desiccated cells were stored at several temperatures and viability and residual moisture were measured at different intervals of time. The term anhydrobiosis quotient (ε) was defined, and a scale of anhydrobiotic ability for classifying micro-organisms in terms of tolerance to desiccation was established (1 ≤  ε  ≤ 15). The anhydrobiosis quotient was used to evaluate the stability of the anhydrobiotic cells. As a main result, changes in the anhydrobiosis quotient at several temperatures were fitted using a reparameterized Weibull model, which was found to be robust for the prediction of the stability at 4°C.
Conclusions:  A novel methodology was developed to evaluate the desiccated state in bacteria. The anhydrobiosis quotient allows the quantitative estimation of the anhydrobiotic ability, and the mathematical model developed allows the prediction of the desiccated state of bacterial populations.
Significance and Impact of the Study:  The new methodology could be applied in studying the anhydrobiosis state of bacterial populations as a predictive tool for industrial and environmental microbiology.