Use of impedance measurements to estimate numbers of antibiotic resistant Salmonella strains

Impedance detection times were compared with traditional plating methods for enumerating antibiotic-resistant strains of Salmonella stanley, Salm. thompson and Salm. infantis grown in laboratory medium and pork slurry. The correleation of log₁₀ counts of salmonellas with detection times was highly significant ( r = -0·96 for broth and r = -0·94 for slurries. The confidence limits (± og₁₀ 1·0 for broth and ± log₁₀ 1·65 for slurry) indicated that detection times could reliably be used as a rapid means of enumerating salmonellas when large numbers of counts of known strains are required for growth studies. Use of antibiotic-resistant strains also permitted their selective detection by impedance from the natural spoilage flora of pork slurry when the same antibiotics were incorporated in the detection medium.     

OPTIMAL PURIFICATION AND SENSITIVE QUANTIFICATION OF DNA FROM FECAL SAMPLES

Application of reliable, rapid and sensitive methods to laboratory diagnosis of zoonotic infections continues to challenge microbiological laboratories. The recovery of DNA from a swine fecal sample and a bacterial culture extracted by a conventional phenol-chloroform extraction method was compared to a rapid silica-membrane spin-column method (DNeasy Tissue or QIAamp Stool Kit, QIAGEN GmbH). The two spin column methods yielded 3.5 and 2.7 μg of DNA, respectively, when the elution volume was 200 μL, compared to 1.3 and 1.5 μg of DNA, respectively, with the phenol-chloroform method.
In addition, the detection range of λ-DNA of a spectrophotometric and a fluorometric (PicoGreen) method was compared. The PicoGreen showed a quantification limit of 1 ng/mL, consistent triplicate measurements, and finally a linear relationship between the concentrations of DNA standards and the fluorescence readings (R²= 0.99 and R²= 1.00).
In conclusion, silica-membrane columns can provide a more convenient and less hazardous alternative to the conventional phenol-based method. The results have implication for further improvement of sensitive amplification methods for laboratory diagnosis.     

Development of a sequence-characterized amplified region marker for diagnosis of dwarf bunt of wheat and detection of Tilletia controversa Kühn

Aims:  Dwarf bunt of wheat, caused by Tilletia controversa Kühn, is a destructive disease on wheat as well as an important international quarantined disease in many countries. The objective of this investigation was to develop a diagnostic molecular marker generated from amplified fragment length polymorphism (AFLP) for rapid identification of T . controversa .
Methods and Results:  A total of 30 primer combinations were tested by AFLP to detect DNA polymorphisms between T. controversa and related species. The primer combination E08/M02 generated a polymorphic pattern displaying a 451-bp DNA fragment specific for T. controversa . The marker was converted into a sequence-characterized amplified region (SCAR), and specific primers (SC-01₄₉/SC-02₄₁₅), designed for use in PCR detection assays, amplified a unique DNA fragment in all isolates of T. controversa , but not in the related pathogens. The detection limit with the primer set SC-01₄₉/SC-02₄₁₅ was 10 ng of DNA which could be obtained from 11  μ g of teliospores in a 25- μ l PCR reaction.
Conclusions:  An approach to distinguish T. controversa from similar pathogenic fungi has been developed based on the use of a SCAR marker.
Significance and Impact of the Study:  Development of the simple, high throughput assay kit for the rapid diagnosis of dwarf bunt of wheat and detection of T. controversa is anticipated in further studies.     

Increased ELISA sensitivity using a modified extraction buffer for detection of Xanthomonas campestris pv. vesicatoria in leaf tissue

In vitro and in planta sensitivity of an indirect enzyme-linked immunoassaytechnique, using a monoclonal antibody specific for the lipopolysaccharide (LPS) of Xanthomonas campestris pv. vesicatoria , was increased 10-foldby using a newextraction buffer (gl of : KH₂PO₄, 2; NaHPO₄, 11·5; EDTAdisodium, 0·14; thimerosal, 0·02; and lysozyme, 0·2). The procedure improvedsensitivity without increasing background levels. In vitro , the limit of detection wasbetween 1×10⁷ and 1×10⁸ cells ml⁻¹ with the conventionalextraction buffer phosphate-buffered saline (PBS) and less than 1×10⁶ cells ml⁻¹ when lysozyme extraction buffer was substituted for PBS. In comparing 22 X. c.vesicatoria strains, absorbance readings were increased close to three-fold with the lysozymeextraction buffer as opposed to PBS. When leaf tissue extract was spiked with the bacterium, thelimit of detection was 1×10⁷ cfu ml⁻¹ and 1×10⁸ cfu ml⁻¹ with the lysozyme solution and PBS, respectively, as the extraction buffers. Whenusing the lysozyme extraction buffer in combination with a commercial amplification system, thelimit of detection was decreased to less than 1×10⁵ cfu ml⁻¹ in leaftissue. The addition of the lysozyme and EDTA to the phosphate buffer resulted in release of asignificant quantity of LPS and concomitant dramatic increase in sensitivity. The new procedure,termed lysozyme ELISA (L-ELISA), should increase sensitivity of ELISA reactions where LPS isthe reacting epitope.     

The effect of endotoxin lipopolysaccharide from different bacterial species on the generation of intracellular inositol triphosphate and superoxide in a human phagocytic cell line

Abstract The acute effects of endotoxins and lipid A on two intracellular responses, inositol phosphate generation and superoxide production were analysed in the DMSO differentiated premyelocytic leukamic HL-60 cell line. Short-term incubation (1–10 min) with Escheria coli -type LPS, Salmonella -type LPS and Lipid A caused significant increases in cellular InsP₁ and InsP₃, compared with control cells ( P < 0.5 − P < 0.001). The Escheria coli -type B LPS released approximately twice the quantity of InsP₃ compared with Salmonella -type LPS ( P < 0.001). Lipid A-dependent stimulation of InsP₃ production was also detected. The rate of superoxide production increased 1–10 min after addition of both Escheria coli - and Salmonella -type LPS and Lipid A. Endotoxins and Lipid A caused a dose-dependent increase in intracellular oxidative activity. The superoxide response showed less species dependence and a higher response to particulate lipid A compared with the inositol phosphate response.     

Use of the polymerase chain reaction for Salmonella detection

J. KWANG, E.T. LITTLEDIKE AND J.E. KEEN. 1996. A primer set of oligonucleotides (S18 and S19) from the _omp C gene of Salmonella has been evaluated for specific detection of Salmonella by polymerase chain reaction (PCR). This primer set successfully amplified 40 Salmonella serovars (60 isolates), but not 24 non-Salmonella bacteria (42 isolates) that have been tested so far. The uniqueness of these primer sequences was also confirmed. The sensitivity of PCR detection in extracted chromosomal DNA for Salm. typhimurium was 1 pg. The sensitivity for boiled whole bacteria was 400 cells. The detection of Salm. typhimurium in ground beef samples required 4–6 h enrichment with an initial inocula of 100 bacteria.     

Plant response to destruxins visualized by imaging of chlorophyll fluorescence

Kinetic fluorescence imaging was used to set a new detection limit for plant exposure to low levels of destruxins – phytotoxins of Alternaria brassicae . A general experimental algorithm is presented that can be used to identify the combination of fluorescence parameters providing the highest contrast between the affected and unaffected plants or plant segments. Leaves of canola ( Brassica napus ) and white mustard ( Sinapis alba ) were exposed to various concentrations of destruxins and images of key fluorescence signals ( F ₀, F _M, F _P, and of F _S) were captured in a single kinetic experiment. Contrast was quantified within these images between the leaf areas exposed to destruxins and the untreated areas. The highest contrast was found in the image constructed by pixel-to-pixel division of images F ₀ by F _P and F ₀ by F _M. Using the F ₀/ F _M ratio image, we were able to detect exposure to destruxin concentration as low as approximately 0.05 mg l⁻¹ applied to canola leaf and approximately 10 mg l⁻¹ when applied to mustard. The detection limits were significantly lower than those obtained by optical microscopy indicating that kinetic chlorophyll fluorescence imaging can be used as a diagnostic tool in screening for varieties with an enhanced resistance to destruxins of Alternaria brassicae .     

The role of temperature in determining the stimulation of CO2 assimilation at elevated carbon dioxide concentration in soybean seedlings

Soybean ( Glycine max cv. Clark) was grown at both ambient (ca 350 μmol mol⁻¹) and elevated (ca 700 μmol mol⁻¹) CO₂ concentration at 5 growth temperatures (constant day/night temperatures of 20, 25, 30, 35 and 40°C) for 17–22 days after sowing to determine the interaction between temperature and CO₂ concentration on photosynthesis (measured as A, the rate of CO₂ assimilation per unit leaf area) at both the single leaf and whole plant level. Single leaves of soybean demonstrated increasingly greater stimulation of A at elevated CO₂ as temperature increased from 25 to 35°C (i.e. optimal growth rates). At 40°C, primary leaves failed to develop and plants eventually died. In contrast, for both whole plant A and total biomass production, increasing temperature resulted in less stimulation by elevated CO₂ concentration. For whole plants, increased CO₂ stimulated leaf area more as growth temperature increased. Differences between the response of A to elevated CO₂ for single leaves and whole plants may be related to increased self-shading experienced by whole plants at elevated CO₂ as temperature increased. Results from the present study suggest that self-shading could limit the response of CO₂ assimilation rate and the growth response of soybean plants if temperature and CO₂ increase concurrently, and illustrate that light may be an important consideration in predicting the relative stimulation of photosynthesis by elevated CO₂ at the whole plant level.     

Survival of κ-carrageenan-encapsulated and unencapsulated Pseudomonas aeruginosa UG2Lr cells in forest soil monitored by polymerase chain reaction and spread plating

Abstract A method was developed for direct extraction, purification and amplification of DNA from forest soil. Eighty-two % of the DNA in Pseudomonas aeruginosa UG2Lr introduced into soil was recovered. The detection limit for the strain was approximately 800 cfu g⁻¹ of dry soil based on the polymerase chain reaction (PCR). Survival of κ-carrageenan-encapsulated and unencapsulated UG2Lr was monitored by antibiotic selective and bioluminescence-based nonselective plating and PCR-amplification of a tnsA fragment. After freeze-thaw treatment of soil samples, the unencapsulated UG2Lr declined from an initial population density of 1 × 10⁹ cfu g⁻¹ of dry soil to below the detection threshold of both selective (14 cfu g⁻¹ of dry soil) and nonselective (1 × 10³ cfu g⁻¹ of dry soil) plating. However, presence of nonculturable UG2Lr cells in the soil was revealed by PCR and resuscitation of the bacteria. Population density of the encapsulated UG2Lr increased from 2.7 × 10⁶ to 2.9 × 10⁸ cfu g⁻¹ of dry soil after a 3-week incubation at 22°C and declined to 6.3 × 10⁶ cfu g⁻¹ of dry soil after the freeze-thaw treatment.     

Determination of xanthoxin by immunoassay

2- Cis (-)xanthoxin (XA) was linked to bovine serum albumin through a Schiff's base and the adduct stabilized by sodium borohydride reduction. The conjugate (molar coupling ratio: 3 mol XA per mol protein) was highly immunogenic in rabbits. Antisera contained antibodies binding XA with high affinity (K_a= 1.8 × 10⁸ M ⁻¹). [³H]-XA (2.2 × 10¹⁴ Bq mol⁻¹) was synthesized by oxidation of [³H]-XA alcohol with MnO₂ and used to set up a radioimmunoassay [RIA, detection limit, 1 pmol; measuring range, 1 to 200 pmol (0.3 to 60 ng) XA]. The sera were also suitable for enzyme-linked-immunosorbent assay (ELISA) using XA-alkaline phosphatase conjugates. The technique was more sensitive [detection limit, 0.1 pmol; measuring range, 0.1 to 50 pmol (0.05 to 15 ng) XA] than the radioimmunoassay, but less precise.     

Use of two 16S DNA targeted oligonucleotides as PCR primers for the specific detection of Salmonella in foods

A 16S DNA targeted polymerase chain reaction (PCR) method specific for the detection of Salmonella isolates with various serotypes was developed. The primers used for such a PCR method were 16SF1 and 16SIII. 16SF1 is the reverse and complementary strand of 16SI which has been shown to be able to hybridize with Salmonella and Citrobacter spp. 16III on the other hand, is able to hybridize with Klebsiella and Serratia spp. in addition to Salmonella. Although 16SF1 and 16SIII were not specific to Salmonella only, when they were used as PCR primers, only the Salmonella isolates could be specifically detected. The interference from Citrobacter, Klebsiella and Serratia spp. could be prevented. None of the other non- Salmonella isolates including strains of the family of Enterobacteriaceae closely related to Salmonella would generate the false-positive reaction. When this PCR system was used for the detection of Salmonella cells artificially contaminated in food samples, results obtained were satisfactory. A detection limit of N × 10⁰ cells per assay could be obtained.     

Cell cycle progression in human cells following re-oxygenation after extreme hypoxia: consequences concerning initiation of DNA synthesis

Abstract. The initiation of DNA synthesis and further cell cycle progression in cells during and following exposure to extremely hypoxic conditions in either G₁ or G₂+M has been studied in human NHIK 3025 cells. Populations of cells, synchronized by mitotic selection, were rendered extremely hypoxic (< 4 p.p.m. O₂) for up to 24n h. Cell cycle progression was studied from flow cytometric DNA recordings. No accumulation of DNA was found to take place during extreme hypoxia. Cells initially in G₁ at the onset of treatment did not enter S during up to 24 h exposure to extreme hypoxia, but started DNA synthesis in a highly synchronous manner within 1.5 to 2.25 h after reoxygenation. The duration of S phase was only slightly affected (increased by ≅10%) by the hypoxic treatment. This suggests that the DNA synthesizing machinery either remains intact during hypoxia or is rapidly restored after reoxygenation. Cells initially in G₂ at the onset of hypoxia were able to complete mitosis, but further cell cycle progression was blocked in the subsequent G^ Following reoxygenation, these cells progressed into S phase, but the initiation of DNA synthesis was delayed for a period corresponding to at least the duration of normal G₁ and did not appear in a synchronous manner. In fact, cell cycle variability was found to be increased rather than decreased as a result of exposure to hypoxia starting in G₂. We interpret these findings as an indication that important steps in the preparation for initiation of DNA synthesis take place before mitosis. Furthermore, the change in cell cycle duration induced by hypoxia commencing in G₁ is of a nature other than that induced by hypoxia commencing in other parts of the cell cycle.     

Effect of dilution on methanogenesis, hydrogen turnover and interspecies hydrogen transfer in anoxic paddy soil

Abstract Dilution of anoxic slurries of paddy soil resulted in a proportional decrease of the rates of total methanogenesis and the rate constants of H₂ turnover per gram soil. Dilution did not affect the fraction of H₂/CO₂-dependent methanogenesis which made up 22% of total CH₄ production. However, dilution resulted in a ten fold decrease of the H₂ steady state partial pressure from approximately 4 to 0.4 Pa indicating that H₂/CO₂-dependent methanogenesis was more or less independent of the H₂ pool. The rates of H₂ production calculated from the H₂ turnover rate constants and the H₂ steady state partial pressures accounted for only < 5% of H₂/CO₂-dependent methanogenesis in undiluted soil slurries and for even less after dilution. Upon dilution, the Gibbs free energy available for H₂/CO₂-dependent methanogenesis decreased from −28.4 to only −5.6 kJ per mol. The results indicate that methane was mainly produced from interspecies H₂ transfer within syntrophic bacterial associations and was not significantly affected by the outside H₂ pool.     

The survival of Salmonella enterica serovar Typhimurium DT104 and total viable counts on beef surfaces at different relative humidities and temperatures

Aims:  The aim of this study was to investigate changes in Salmonella and total viable count (TVC) survival on beef carcass surfaces stored for 72 h under different combinations of relative humidity (i.e. RH 75% or 96%) and temperature (5°C or 10°C).
Methods and Results:  The influence of low water activity ( a _w) and temperature on the survival and growth of Salmonella enterica serovar Typhimurium DT104 and the aerobic mesophilic flora on meat pieces from different sites on beef carcasses was investigated, under controlled conditions (75% or 96% RH; 5 or 10°C) in an environmental cabinet. Salmonella counts declined during storage at low a _w (75% RH) conditions at 5°C or 10°C. Salmonella counts increased during storage at high a _w (96% RH) at 10°C only. At 5°C, TVCs increased during storage at high a _w, but not at low a _w. TVCs increased on all samples from carcasses stored at high or low a _w at 10°C, except those samples taken from areas of surface fat.
Conclusions:  This suggests that substrate composition dictates growth rates under low a _w conditions. The results are discussed in terms of the possible protective effects of substrate osmolyte accumulation in bacterial survival and/or growth.
Significance and Impact of the Study:  The data obtained in this study provides useful insights on the influence of a _w and temperature on pathogen survival on meat surfaces at chill temperature.     

Evaluation of the Vitek Immunodiagnostic Assay System (VIDAS) for the detection of Salmonella in foods

A Salmonella Assay using the Vitek Immunodiagnostic Assay System (VIDAS) was compared with a conventional cultural method (CCM) for the detection of salmonellas in 141 samples of artificially and naturally contaminated foods. There was an overall agreement of 92.9% between the methods. The productivity of the VIDAS Salmonella Assay (VSA) was not improved using an alternative enrichment protocol for the detection of Salmonella in 12 raw meat samples.
The sensitivity and specificity of the VSA was assessed using pure cultures of salmonellas and non-salmonellas. The detection limit was 1.8 times 10⁶ salmonellas ml^-1 in M-broth and some Citrobacter freundii strains gave false-positive results.
Using an immunomagnetic separation (IMS) technique and an abbreviated cultural enrichment, the VSA results could be obtained a day earlier than the standard VSA method.     

Lysophosphatidic Acid-Induced Proliferation-Related Signals in Astrocytes

Abstract: Lysophosphatidic acid (LPA) is a potent lipid biomediator that is likely to have diverse roles in the brain. Thus, LPA-induced events in astrocytes were defined. As little as 1 n M LPA induced a rapid increase in the concentration of intracellular free calcium ([Ca²⁺]_i) in astrocytes from neonatal rat brains. This increase was followed by a slow return to the basal level. Intracellular calcium stores were important for the initial rise in [Ca²⁺]_i, whereas the influx of extracellular calcium contributed significantly to the extended elevation of [Ca²⁺]_i. LPA treatment also resulted in increases in lipid peroxidation and DNA synthesis. These increases in [Ca²⁺]_i, lipid peroxidation, and DNA synthesis were inhibited by pretreatment of cells with pertussis toxin or H7, a serine/threonine protein kinase inhibitor. Moreover, the LPA-induced increase in [Ca²⁺]_i was inhibited by a protein kinase C inhibitor, Ro 31-8220, and a calcium-dependent protein kinase C inhibitor, Gö 6976. The increase in [Ca²⁺]_i was important for the LPA-induced increase in lipid peroxidation, whereas the antioxidant, propyl gallate, inhibited the LPA-stimulated increases in lipid peroxidation and DNA synthesis. In contrast, pertussis toxin, H7, and propyl gallate had no effect on LPA-induced inhibition of glutamate uptake. Thus, LPA appears to signal via at least two distinctive mechanisms in astrocytes. One is a novel pathway, namely, activation of a pertussis toxin-sensitive G protein and participation of a protein kinase, leading to sequential increases in [Ca²⁺]_i, lipid peroxidation, and DNA synthesis.     

Compensatory growth of juvenile brown flounder Paralichthys olivaceus (Temminck & Schlegel) following thermal manipulation

The compensatory growth of juvenile brown flounder Paralichthys olivaceus (body mass c. 12 g) following different thermal exposure was investigated. Fish were exposed to one of the five temperatures: 8·5 ( T _8·5), 13·0 ( T _13·0), 17·5 ( T _17·5), 22·0 ( T _22·0) and 26·5° C ( T _26·5) for 10 days and fish grew best at 22·0° C. Then the water temperature in all treatments was equably adjusted to 22·0° C over 3 days. At the end of the following 30 days after temperature adjustment, there were no significant differences between body masses of fish in the different treatments (wet body mass at the end of the experiment ranged from 22·13 to 24·56 g). Results indicated that the juvenile P. olivaceus achieved complete compensatory growth. Analysis of the dynamics of the feeding rates and feed conversion efficiencies indicated that compensatory growth of the fish experienced low temperature ( T _8·5, T _13·5 and T _17·5) or high temperature ( T _26·5) exposure was mainly dependent on increasing feed intake (hyperphagia) and possibly by improvement in feed conversion efficiency. The moisture content was not affected by different temperature exposure significantly. The lipid and energy content of juvenile P. olivaceus in T _8·5, however, were significantly lower than other treatment. Results of the current study indicate that a short period of low or high temperature exposure may not affect annual growth, but may affect lipid and energy deposition.     

Balancing carboxylation and regeneration of ribulose-1,5- bisphosphate in leaf photosynthesis: temperature acclimation of an evergreen tree, Quercus myrsinaefolia

Changes in the temperature dependence of the photosynthetic rate depending on growth temperature were investigated for a temperate evergreen tree, Quercus myrsinaefolia . Plants were grown at 250 μ mol quanta m^–2 s^–1 under two temperature conditions, 15 and 30 °C. The optimal temperature that maximizes the light-saturated rate of photosynthesis at 350 μ L L^–1 CO₂ was found to be 20–25 and 30–35 °C for leaves grown at 15 and 30 °C, respectively. We focused on two processes, carboxylation and regeneration of ribulose-1,5-bisphosphate (RuBP), which potentially limit photosynthetic rates. Because the former process is known to limit photosynthesis at lower CO₂ concentrations while the latter limits it at higher CO₂ concentrations, we determined the temperature dependence of the photosynthetic rate at 200 and 1000 μ L L^–1 CO₂ under saturated light. It was revealed that the temperature dependence of both processes varied depending on the growth temperature. Using a biochemical model, we estimated the capacity of the two processes at various temperatures under ambient CO₂ concentration. It was suggested that, in leaves grown at low temperature (15 °C), the photosynthetic rate was limited solely by RuBP carboxylation under any temperature. On the other hand, it was suggested that, in leaves grown at high temperature (30 °C), the photosynthetic rate was limited by RuBP regeneration below 22 °C, but limited by RuBP carboxylation above 22 °C. We concluded that: (1) the changes in the temperature dependence of carboxylation and regeneration of RuBP and (2) the changes in the balance of these two processes altered the temperature dependence of the photosynthetic rate.     

Combined effect of organic acids and supercritical carbon dioxide treatments against nonpathogenic Escherichia coli, Listeria monocytogenes, Salmonella typhimurium and E. coli O157:H7 in fresh pork

Aims:  To evaluate the effectiveness of organic acids and supercritical carbon dioxide (SC-CO₂) treatments as well as their combined effect for the reduction of nonpathogenic Escherichia coli and three pathogenic bacteria in fresh pork.
Methods and Results:  The different treatment conditions were as follows: (i) treatment with acetic (1%, 2% or 3%) or lactic acid (1%, 2% or 3%) only, (ii) treatment with SC-CO₂ at 12 MPa and 35°C for 30 min only and (iii) treatment with 3% acetic or lactic acid followed by treatment with SC-CO₂. Within the same organic acid concentration, the lactic and acetic acid treatments had similar reductions. For the combined treatment of lactic acid and SC-CO₂, micro-organism levels were maximally reduced, ranging from 2·10 to 2·60 log CFU cm⁻² ( E. coli , 2·58 log CFU cm⁻²; Listeria monocytogenes , 2·60 log CFU cm⁻²; Salmonella typhimurium , 2·33 log CFU cm⁻²; E. coli O157:H7, 2·10 log CFU cm⁻²).
Conclusions:  The results of this study indicate that the combined treatments of SC-CO₂ and organic acids were more effective at destroying foodborne pathogens than the treatments of SC-CO₂ or organic acids alone.
Significance and Impact of the Study:  The combination treatment of SC-CO₂ and organic acids may be useful in the meat industry to help increase microbial safety.     

Isolation and characterization of carboxylesterase E3 from Salmonella enteriea

Three esterases (Est-) hydrolysing α-naphthyl acetate: Est-E₁, Est-E₃ and Est-E₄ produced by Salmonella enterica serovar Typhimurium, strain LT2 were separated by DEAE chromatography and gel filtration. Est-E₃, the major component of this set of enzymes, clearly differed from the two other esterases by its apparent molecular weight, titration curve, substrate specificity and inactivation. Immunoglobulins raised against Est-E₃ completely neutralized the activity of Est-E₃ but did not react with Est-E₁ or Est-E₄; it showed no cross reaction with carboxylesterase B of Escherichia coli or with carboxylesterases from other enterobacteria. Est-E₃ showed electrophoretic variants which were biochemically and immunologically detected in the seven subspecies of the genus Salmonella . These findings suggest that variants of Est-E₃ are the products of very closely related loci originating from a common ancestral gene. The esterase could be a phylogenetic marker of the genus and a suitable molecular tool for taxonomy and epidemiology.