pseudomallei [10]. There is extensive chromosomal synteny between B. thailandensis and B. pseudomallei, although some virulence-associated genes which are present in B. pseudomallei are absent in B. thailandensis [12]. Both

B. pseudomallei and B. thailandensis are able to invade and grow in a range of phagocytic Selleckchem Fulvestrant and non-phagocytic cells, forming plaques or multinucleated giant cells [13, 14]. However, there is also evidence that the behaviour of B. pseudomallei and B. thailandensis differs in different cell lines. In A549 and human dendritic cells, B. pseudomallei has been shown to be more invasive than B. thailandensis, but there were no reported differences in the growth rate within cells. In contrast, in human macrophages, differences in intracellular growth rates have been reported [14]. Collectively, these findings have suggested that B. thailandensis could be used as a model to study certain aspects of the intracellular lifestyle of B. pseudomallei in cell culture systems [15]. The behaviour of B. oklahomensis in cell culture models is click here not known. The value of whole animal or plant infection models, which use B. thailandensis or B. oklahomensis in place of B. pseudomallei, is much less clear. Isolates of B. thailandensis and B. oklahomensis that have been tested are considered to be highly attenuated or avirulent in BALB/c mice, with lethal doses for most isolates in excess of 107 cfu by the i.p. route [16]. However,

using intranasal challenge models, doses of greater than 104 cfu of B. thailandensis are reportedly able to kill mice and replicate B. pseudomallei disease phenotypes, although even in this model it is clear that B. thailandensis is much less virulent than B. pseudomallei [7]. There has been significant interest in the development of alternative infection models which avoid the use of mammals but also reflect the differences in virulence of species and isolates seen in mice. The Caenorhabditis elegans [17]

or tomato plant [18] infection models were not able to distinguish between B. pseudomallei and B. thailandensis, and in C. elegans, B. thailandensis was the most virulent Methamphetamine [17]. Galleria mellonella (wax moth) larvae have previously been reported as susceptible to infection with B. pseudomallei, and a single B. thailandensis strain tested was reportedly less virulent [19]. This finding suggests that G. mellonella larvae may be a suitable host species for discerning differences in virulence. Our aim was to determine whether differences in the virulence of B. pseudomallei, B. thailandensis and B. oklahomensis isolates could be reliably determined in macrophage and G. mellonella larvae infection models. Results B. pseudomallei, B. thailandensis or B. oklahomensis are internalised with similar efficiencies into J774A.1 macrophages For this study we have selected a range of B. pseudomallei, B. thailandensis or B. oklahomensis isolates of known ancestry.

coli (Figure 7). With amino acid supplementation, sizes of the ZOI reduced for GSK3235025 solubility dmso both the wild type and the ΔarcA mutant E. coli, and the difference in the sizes of the ZOI between wild type and ΔarcA mutant E. coli diminished with amino acid supplementation (Figure 7). We tested single amino acids and combinations of various amino acids, and none of the combinations tested was

able to complement the susceptibility of the ΔarcA mutant E. coli as the total amino acids (data not shown). Figure 7 Amino acid complementation increased the resistance of E. coli to H 2 O 2 and reduced the difference in H 2 O 2 resistance between the wild type and ΔarcA mutant E. coli. Resistance of wild type (diamond) and the ΔarcA mutant E. coli (square) to H2O2 was assayed by the ability to grow in the presence of H2O2 and more resistant bacteria show a smaller diameter of inhibition. Various volumes of 20 mM amino acid solution was spread onto each M9 minimal medium plate containing approximately 1 × 106 c.f.u. wild type or ΔarcA mutant E. coli and a paper disc of 1/4″” with 10 μl of 30% H2O2 was

added to the center of each plate. Zone of inhibition Gemcitabine was measured after overnight incubation and plotted against the volume of amino acid supplementation. At least three experiments were performed, and results from a representative experiment performed in triplicates are shown. Error bars indicate standard deviation and sometimes fall within the data label..

Antibiotic that inhibits protein synthesis increased susceptibility of E. coli to H2O2 To test if protein synthesis is important for bacterial survival and if protein synthesis inhibition is detrimental to bacteria under reactive oxygen stress, we assayed the resistance of E. coli to H2O2 in the presence of chloramphenicol, an antibiotic that inhibits peptide bond formation and hence protein synthesis. Without H2O2 or antibiotic, wild type E. coli grew approximately 2log10 during 6 hours of incubation (Figure 8, left half, open bar). Hydrogen peroxide was bactericidal and the bacterial concentration decreased for over 1log10 (Figure 8, left half, Dapagliflozin diagonally-hatched bar). Supplementation of chloramphenicol alone prohibited bacterial proliferation and the bacterial concentration decreased slightly (Figure 8, left half, vertically-hatched bar). Incubation in the presence of both H2O2 and chloramphenicol was more detrimental to E. coli than either H2O2 or chloramphenicol alone, and the bacterial concentration decreased by nearly 4log10 (Figure 8, left half, cross-hatched bar). This indicates that chloramphenicol enhanced the bactericidal activity of H2O2. To determine if this enhanced bactericidal activity is due to the bacteriostatic activity of chloramphenicol, we tested the effect of ampicillin, an antibiotic that inhibits the bacterial cell wall synthesis, in the same assay.

Thus, the SiO2 layer transforms into a mixture of mullite and SiO2. The out-diffused silicon can be dissolved into small Fe-Al particles, which are formed in an early MK0683 mouse stage of oxidation. The reason for non-detection of Si in large particles is not clear yet. The particles shown in Figure 5 are

too large to exhibit the properties of nanoparticles. The 10 to 100 nm Fe-Al films were RF-sputtered and then annealed for 200 min at 900°C, with a hydrogen flow rate of 500 sccm and a dew point of 0°C. As shown in Figure 7, the films also become particulate after oxidation. The thinner the films become, the smaller the particles become. In addition, particle sizes were not uniformed, and their shape is rather spherical. Moreover, black holes found in the films oxidized for 20 to 60 min can be seen in Figure 5: they are clearly observable at lower magnification (right lower photo). In the black region, very small particles are found. It seems that the white particles

are Fe-Al particles, which are very similar to the small particles formed in the early stage of oxidation shown in Figure 5. From the fact that there are not many small particles near larger particles in the 50-nm-thick film, Ostwald ripening is promoted by the increasing film thickness. In the 200-nm-thick film, the particles have a spherical shape, which is very different from the maze-like shape in the films shown in Figure 5, which were oxidized at an atmosphere with a lower dew point. Maximum particle sizes of the 10-nm- and 20-nm-thick films are about 0.3 and 0.47 μm, respectively. The minimum particle size in the 20-nm-thick films is smaller buy BVD-523 than one-tenth of the maximum size. Figure 7 SEM images of 10 to 200 nm Fe-Al films selectively oxidized at 900°C for 200 min. When the Fe-Al films were selectively oxidized, the slope of Telomerase the hysteresis loops at the origin decreased, due to the demagnetization field, as the oxidation time increased. Figure 8 shows normalized VSM loops of the Fe-Al films of Figure 7 measured at room temperature. The slope of the magnetization

curve of the as-sputtered Fe-Al film was very high near the origin. Further, it decreased gradually as oxidation time increased. The 200-nm-thick film shows hysteresis, while the other films do not show hysteresis. Moreover, the normalized loops of the 10- to 100-nm-thick films have nearly same slope and shape, which means that these particles are superparamagnetic at room temperature. Because magnetocrystalline easy axis and the magnetocrystalline anisotropy energy of iron are <100> and K 1 = 4.8×104 J/m3, respectively, superparamagnetic behavior appears, even though the maximum particle size is about 1 μm, which is very much larger than materials with uniaxial crystalline anisotropy. Figure 8 Normalized VSM loops of 10 to 200 nm Fe-Al films selectively oxidized at 900°C for 200 min. Conclusions The 10- to 200-nm-thick RF-sputtered Fe-Al films were oxidized in the atmosphere mixture at 900°C for up to 200 min.

Conversely, cell-free supernatant solutions from acutely infected cultures were capable of destabilizing persistently-infected cultures in a manner similar to the destabilization that occurs in shrimp and insect populations. Here we describe the relevant experiments and show that the active factors in the cell-free supernatant solutions are probably

small polypeptides with cytokine-like activity. Results and discussion Persistent Dengue Sirolimus ic50 virus infections After primary challenge of naïve C6/36 cell cultures with DEN-2 followed by split-passage every 2 days, stable cultures persistently infected with DEN-2 were obtained with 100% DEN-2 positive cells, as previously described [6]. The growth rate of cultures persistently infected with DEN-2 https://www.selleckchem.com/products/Deforolimus.html did not differ significantly (p > 0.05) from that of uninfected cell cultures. The gross signs of DEN-2 infection declined with increasing passage number. From passage 15 onwards the cultures did not differ morphologically from naïve C6/36 cell cultures.

However DEN-2 released into the culture medium could initiate acute DEN-2 infections in naïve cells, as previously reported [6]. Neither these preparations nor DEN-2 stock inoculum caused any changes when used to challenge cultures persistently infected with DEN-2. Filtrate from persistently infected cells protects naïve cells against DEN-2 Immunofluorescence assay

using an antibody to DEN-2 envelope protein revealed that 48-h pretreatment of naïve C6/36 cells with the 5 kDa filtrate from cell cultures persistently infected with DEN-2 led to a significant reduction (p = 0.009) in the percentage of DEN-2 immunopositive cells (6 ± 5%) when compared to untreated cells after DEN-2 challenge (46 ± 2%) (Figure 1). These results were confirmed by using Vero cells to measure the DEN-2 titers in supernatant solutions from the treated insect cells. The titers were 2 × 106 +/- 0 at 24 h and 8 × 106 +/- 0 at 48 h for naive cells but 6 × 104 +/- 2 × 104 at 24 h and 3.2 × 103 +/- 2.4 × 103 at 48 h for filtrate-exposed cells (significant differences for Montelukast Sodium both times at p = 0.001). To achieve the maximum reduction in numbers of immunopositive cells and the least cytopathology, it was necessary to pre-incubate the cells for 48 h prior to DEN-2 challenge. Exposure to the active preparation for periods less than 48 h was proportionally less effective in inducing resistance (not shown). The pre-incubation requirement suggested that reduction in severity of DEN-2 infection was induced in the challenged cells by an active factor(s) in the filtrate. Figure 1 C6/36 cells protected against DEN-2 by 5 kDa membrane filtrate from cell cultures persistently infected with DEN-2.

The hemoFISH®Gram positive panel correctly identified 221/239 Gram-positive isolates (92.5%) (Table  1). Particularly, a total of 130 coagulase negative staphylococci were identified as Staphylococcus spp (the staphylococci identification obtained using Vitek 2 system were: 70 Staphylococcus epidermidis, 23 Staphylococcus hominis, 22

Staphylococcus haemolyticus, 4 Staphylococcus warneri, 8 Staphylococcus capitis, 1 Staphylococcus auricolaris, 1 Staphylococcus saccharolyticus, 1 Staphylococcus saprophyticus) while one sample positive for Staphylococcus cohnii was not identified. 16 samples, positive per Staphylococcus aureus, were correctly identified (Table  1). Looking at the streptococci, 30/32 samples were correctly VX-770 in vitro identified as Streptococcus spp (19 Streptococcus mitis, 1 Streptococcus bovis, 2 Streptococcus oralis, 4 Streptococcus 3-MA in vitro gallolyticus and 1 Streptococcus gordoni), while among 5 specimens positive for Streptococcus pneumoniae, 3 were identified as Streptococcus spp (albeit no signal was evidenced with specific probe in S.pneumonie well) and 2 were not identified (only the signal with the eubacterial probe was recorded) (Table  1). Enterococci were detected in a total of 41/44 specimens, two Enterococcus raffinosus were not identified and one Enterococcus gallinarum was misidentified by hemoFISH as Enterococcus faecium (Vitek

2 system identified: 19 Enterococcus faecalis, 22 E.faecium, 2 E. raffinosus and one E.gallinarum) (Table  1). Eight specimens resulted positive for Microcococcus spp, namely 4 Micrococcus luteus and 4 Micrococcus lylae, of these, two (those positive for M.luteus) gave a positive fluorescent signal on the Staphylococcus spp well (recorded as misidentifications), the remaining 6 were not identified (Table  1). Among the Gram-positive bacilli: two Corynebaterium spp and two Bacillus spp were identified in four different specimens by Vitek 2 (one Corynebacterium amycolatum, one Corynebacterium spp, one Bacillus cereus and one Bacillus spp). Identification by hemoFISH®

failed for all of them (neither the signal for the positive control was detected). While the hemoFISH® correctly identified three Clostridium perfringens (Table  1). One sample containing Candida did not yield a specific signal Succinyl-CoA with any of the hemoFISH® probes but was clearly visible via auto fluorescent signals on all fields. A total of 29 specimens were not identified (21 strains) or misidentified (8 strains) by the hemoFISH® test (29/393; 7.4%). The global performances recorded with the hemoFISH panels, in comparison with those identified by Vitek 2 system, are summarized in the Table  1. The overall concordance between traditional culture and hemoFISH® for the negative samples was 100%, no fluorescent specific signal was recorded on 181 negative blood cultures processed.

Scenario (d) was followed by (c) for several times. Scheme not to scale Harlequin frogs (Atelopus) are a species-rich

bufonid genus of Andean origin, with more than 100 species occurring in forest or paramo habitats in the Andes (Lötters 1996; La Marca et al. 2005). In this paper we focus on the less than 10 Atelopus (depending on the taxonomy applied; see Lötters et al. 2002) occurring exclusively in forest habitats in the Amazon basin and on the eastern Guiana Shield. In an earlier molecular genetic study, Noonan and Gaucher (2005) showed that the five nominal species of the eastern Guiana Shield harlequin frogs are genetically little differentiated and that they Z-VAD-FMK mouse apparently interbreed in nature. Supported by divergence time estimates, these authors advocated that the observed phylogeographical

patterns in Atelopus fit DV predictions, i.e. that a single Andean ancestor had invaded the eastern Guiana Shield (likely in late Miocene, as also suggested for other anuran amphibians; Santos et al. 2008) and has started speciation there in the Pleistocene due to the alteration of glacial and interglacial phases (as illustrated in Fig. 1a–d). To their molecular phylogeny, Noonan and Gaucher (2005) added only four Atelopus species from outside the eastern Guiana Shield. As a result, the validity of their study is pending on additional corroboration. This is especially significant because Selleck Temozolomide our knowledge on the current

distribution of harlequin frogs in central Amazonia is poorly understood. Lescure and Gasc (1986), with providing data, proposed a continuous distribution of harlequin frogs from the Andes to the eastern Guiana Shield. In contrast, Lötters et al. (2002), in a taxonomic study, were unable to trace Atelopus material in scientific collections from a large part of central Amazonia, casting some doubt on a continuous distribution. Such a hiatus could be well explained by DV predictions, since the Hydroxychloroquine recolonisation of central Amazonia, either from the western Amazonian lowlands or from the eastern Guiana Shield plus vicinities, would be impossible during the current postglacial. From a phylogenetic point of view, according to DV predictions and the findings of Noonan and Gaucher (2005), we expect that harlequin frogs from east of this suspected distribution gap in central Amazonia constitute one clade nested within those from the Andes and Amazonian lowlands adjacent to them (Fig. 1d) if more species were included from more of the genus’ entire geographic range than available to Noonan and Gaucher (2005). Species can respond to climate change in two ways. One is change of geographic range (i.e. increase, decrease down to extinction, shift) and maintenance of the specific climate envelope, termed niche conservancy (e.g. Peterson et al. 1999; Wiens and Graham 2005).

Six months later the patient had regulated diabetes. All defects were closed secondarily except for the sacral pressure sore which was treated as a chronic wound. Case III A 56 years old healthy male patient was admitted to the Urology department for elective right inguinal hernia reparation (Table 1). The urologists performed a standard operation of a sliding inguinal hernia on the Z-VAD-FMK right side. Due to the weakness of the lower AW, the urologist reinforced the inguinal wall with synthetic Prolene mesh. Postoperatively, the patient showed a clinical picture of an acute abdomen. At this point, the urologists performed a revision surgery of the operated inguinal

hernia, during which they found only a hematoma, removed the Prolen mesh and performed adequate haemostasis. Unfortunately they did not notice the bowel perforation and did not perform an explorative laparotomy at that time. During the next 24 hours, signs of septic shock with crepitations on the AW and right flank region appeared in the clinical picture. Through the suture line of the inguinal canal a fecal collection was drained. Postoperatively, the

patient received a combination of Penicillin G, Clindamycin, Metronidazol and Gentamycin. The native abdomen x-ray showed air under the diaphragm. Magnetic resonance Maraviroc images provided dramatic evidence of an inflammatory process infiltrating the deep fascial plane of the anterior AW. Systemic manifestations of SIRS with body temperature more than 39°C, heart rate more than 100 beats per minute, breaths less than 30 per minute, PaCO2 less than 32 mmHg and WBC account more than 18 × 109/L with a high number of immature forms, hypotension, hypoperfusion with a high level lactic acidosis, oliguria, and alteration of mental status and consciousness were indicators of severe sepsis and septic shock. The anesthesiologist introduced a central venous catheter and started intensive resuscitation. The abdominal rigidity

suggested Clomifene a persisting peritonitis and an urgent laparotomy was done. Through a long midline incision we found a perforation of the caecum, necrosis of a great part of ascending colon, diffuse fecal peritonitis and signs of retroperitoneal NF. The surgical team executed extensive debridement, fasciectomy of the deep fascia on the AW, right orciectomy, right hemicolectomy, diverting colostomy on the descending colon and extensive debridement of the RS. The abdominal cavity and RS were extensively irrigated with hydrogen peroxide, saline and a solution of 1% povidone iodine, and drained on both sides. The structural and functional continuity of musculofascial system of the AW was obtained by component separation techniques (cite) and biological mesh. The wound was dressed with 1% povidone iodine solution. Dressing was controlled every 24 hours and serial debridements were performed.

Taken together, the PFGE patterns (Fig. 1D) and Southern hybridization results (Fig. 3A and 3B) indicated that 76-9 and SA1-8 have the same chromosomal structure, and have undergone the same three rearrangement events. Since 76-9 is able to sporulate and to produce high-level avermectins, it can be concluded that the deleted central region within G1 is not responsible for the differentiation or avermectin production in S. avermitilis. Chromosomal circularization in SA1-6 The 1938-kb deletion region at both chromosomal PLX4032 price ends of SA1-6 was identified by walking PCR, including entire AseI-W, A, U, left part of AseI-P, and right part of AseI-D (Fig. 7A). No obvious retardation

of the AseI fragment of SA1-6 was observed in SDS-treated sample (data not shown), together with the intact chromosome remaining trapped in the gel well in PK-treated sample (Fig.

2A), indicating that the SA1-6 chromosome was circularized. The left and right deletion ends were located at 1611078 nt and 8698105 nt, respectively. Therefore, the size of the new AseI junction fragment NA4 was 489-kb and overlapped with AseI-G1 in the PFGE gel, which was confirmed by Southern hybridization using probe N4 spanning the fusion site (Additional file 1: Supplementary Fig. S3). Hybridization of probe N4 with the BglII-digested Torin 1 in vitro genomic DNA revealed that a 2.99-kb BglII fragment from the left AseI-P and a 13.0-kb BglII fragment from the right AseI-D in the wild-type strain were partially deleted and joined, generating a newly 8.7-kb BglII fragment in SA1-6 (Fig.

7B and 7C). No homology was found when the fusion sequence was compared with the corresponding left and right sequences from wild-type (Fig. 7D). Figure 7 Characterization of circular chromosome in SA1-6. (A) Schematic representation of the chromosomes of wild-type strain and mutant SA1-6, showing deletions at both ends. (B) Location of chromosomal deletion ends and fusion junction. Bg, BglII. (C) Southern analysis of fusion fragment with probe N4, which was prepared using primers 405 and 406. (D) Junction sequence, showing no obvious homology between the original sequences. Stability assay of chromosomal structure in Reverse transcriptase bald mutants Generational studies were performed to assess the chromosomal stability of bald mutants derived from the wild-type strain. Four bald strains were selected, and subjected to PFGE analysis following ten passages. The chromosomal structure of SA1-8 and SA1-6 was conserved, whereas that of SA1-7 and SA3-1 was changed (Additional file 1: Supplementary Fig. S4A). Both SA1-7 and SA3-1 lost their characteristic bands, and became indistinguishable from SA1-6. SA1-7 chromosome was further monitored in each passage, and found to change in the 4th passage (Additional file 1: Supplementary Fig. S4B). The corresponding fusion fragments of SA1-6 and SA1-8 were also detected in their progeny. These results indicate that chromosomal structure of SA1-6 and SA1-8 is stable.

protegens Pf-5 Non mangotoxin producer, Dabrafenib mbo and mgo operon absent [35] Plasmids     pBBR1MCS-5 4.7 kb broad-host-range cloning vector, Gmr [36] pGEM-T 3.0 kb cloning vector, Apr Invitrogen pGEM-TBCAD mgoBCAD cloned in pGEM-T, Apr This study pLac-mgoBCAD mgoBCAD cloned in pBBR1MCS-5 downstream the lacZ promoter in the vector, mgo operon expression under its own

and P LAC promoter, Gmr This study pLac-mboABCDEF mboABCDEF cloned in pBBR1MCS-5 downstream the lacZ promoter in the vector, mbo operon expression under its own and P LAC promoter, Gmr [6] pLac-mboFEDCBA mboABCDEF cloned in pBBR1MCS-5 in the opposite PI3K Inhibitor Library direction than the lacZ promoter in the vector, mbo operon expression under its own promoter, Gmr [6] pMP220 Promoter-probe vector containing a promoterless LacZ gene, Tetr [37] pMP-mboABCDEF mboABCDEF cloned in promoter-probe vector containing a promoterless LacZ gene, mbo operon expression under its own promoter, Tetr This study pMP::P mboI pMP220

vector containing the mbo operon promoter, Tetr [6] aCECT: Spanish Type Culture Collection, Spain. Mangotoxin production assay Antimetabolite toxin production was assayed by the indicator technique previously described [32]. Briefly, a double layer of the indicator microorganism E. coli CECT acetylcholine 831 was prepared; after solidification,

the P. syringae pv. syringae strains to be tested were stab-inoculated. The plates were initially incubated at 22°C for 24 h, and then at 37°C for an additional 24 h [2]. To evaluate mangotoxin activity, the same plate bioassay was carried out with the addition of 100 μl of a 6 mM solution of N-acetyl-ornithine or L-ornithine to the double layer of E. coli[2]. To determine growth characteristics of representative strains, the wild type mangotoxin-producing P. syringae pv. syringae UMAF0158 and derivatives mutants in mboA, mgoA and gacA genes were used to obtain initial cultures in 10 ml of LB broth. The bacterial strains were grown during 24 h at 28°C to prepare an optimal bacterial inoculum with an optical density of 0.8 at 600 nm (approximately 109 cfu ml-1). One ml from these bacterial inocula was used to inoculate 100 ml of PMS broth.

After incubation of the sample in ASL buffer at 95°C for 5 min, 140 μL of a 10 mg/ml solution of lysozyme (Sigma-Aldrich, Brøndby, Denmark) in Tris-EDTA buffer (10:1 mM), pH 8, was added to each extraction tube and samples were incubated at 37°C for 30 min. The purified DNA was eluted in 200 ml buffer AE (Qiagen) and DNA was stabilized by adding 4 μL of a 50 mg/ml BSA solution (Ultrapure BSA, Ambion, Applied Biosystems, Naerum, Denmark, cat. no. 2616) and 2 μL of Ribonuclease-A (Sigma-Aldrich, R-4642). The purity and concentration of DNA was

measured using Selleckchem GW 572016 NanoDrop (NanoDrop Technologies, Wilmington, Delaware, USA). All samples were stored as concentrated samples at -20°C until use. Samples were diluted

to a concentration of 5 mg DNA per ml before use. Real-time PCR for the detection of Salmonella Extracted total DNA samples from the ileum and caecum were tested for Salmonella by a LNA real-time PCR method described by Josefsen et al. [31] with minor modifications. PCR was performed on a MX3005P (Stratagene, La Jolla, California) in a total reaction volume of 25 μl, consisting of 12.5 μl of Promega PCR Mastermix (Promega, Wisconsin, MA), 4.25 μl of water, 3 mM MgCl2, 1 mg/ml BSA (Sigma-Aldrich, cat L4390), 10 pmole of forward primer ttr-6 (5′-CTCACCAGGAGATTACAACATGG-3′), 10 pmole of reverse primer ttr-4 (5′-AGCTCAGACCAAAAGTGACCATC-3′), 10 pmole of LNA target probe (6-FAM-CG+ACGGCG+AG+ACCG-BHQ1) (Sigma-Aldrich) and 2 μl of purified DNA (10 ng). The temperature screening assay profile was initial denaturation at 95°C for 3 min., followed by 40 cycles of 95°C for 30 s, 65°C for 60 s, and 72°C for 30 s. Fluorescence measurements were analyzed with the MxPro-Mx3005P software (Stratagene, version 4.10). The threshold was assigned by using the software option background-based threshold. All samples were tested in duplicate

and a sample was counted as positive if at least one out of two were positive. Polymerase chain reaction conditions for 16S rDNA Generation of a PCR fragment of the 16S ribosomal gene was done IKBKE as described previously [27]. Briefly, four replicate 50 μl PCR mixtures were made from each sample on a PTC-200 thermal cycler (MJ Research, Watertown, Massachusetts). Reaction conditions were as follows: 5 μl PCR buffer (HT Biotechnology Ltd., Cambridge, UK); 10 mM (each) deoxynucleoside triphosphates, 10 pmole forward primer S-D-Bact-0008-a-S-20 (5′-AGAGTTTGATCMTGGCTCAG-3′), 10 pmole reverse primer S-D-Bact-0926-a-A-20 (5′-CCGTCAATTCCTTTRAGTTT-3′), and 1.25 U of DNA polymerase (SuperTaq; HT Biotechnology Ltd., Cambridge, UK) in a 50- μl reaction. Primer S-D-Bact-0008-a-S-20 was 5′ FAM labelled.