Comparison of induction of B45 Helicobacter pylori prophage by acid and UV radiation

Helicobacter pylori is a Gram-negative microorganism that grows on microaerophilic conditions and has only one known natural reservoir: the gastric mucosa. The infection by H. pylori is very common worldwide and this bacterium is associated with the development of gastritis, peptic ulcer gastric cancer or gastric Mucosa Associated Lymphoid Tissue (MALT) lymphoma. Although its natural habitat is the acidic gastric mucosa, H. pylori is considered to be a neutralophile. The bacterium survives brief exposure to pHs of <4, but growth occurs only at the relatively narrow pH range of 5.5 to 8.0, with optimal growth at neutral pH. Recently we have identified a prophage sequence (prophage phiHP33) in the strain B45, isolated from a patient diagnosed with gastric MALT lymphoma. This prophage revealed to be very difficult to induce. In fact, only few phage particles were observed on electron microscopy micrographs after exposure to UV radiation.In the present work we have compared the exposure to UV and to acidic environment in the induction of the prophage into a lytic cycle. We have tested two strains, the strain B45 carrying the prophage phiHP33 and a clinical strain 1152, isolated from a patient with peptic ulcer, that was revealed to be negative for the presence of integrase gene (a prophage gene essential for genome integration of prophage) by PCR, as negative control. Since the H. pylori reservoir is the human stomach the exposition to acid is very common, and with this experiment we intended to test if acid can trigger a phage lytic cycle.The induction using UV radiation has been previously described. For acid induction we have used a protocol adapted from Karita and Blaser. A 48 hours culture of H. pylori was grown in Brucella broth (Oxoid) supplemented with 10% of fetal bovine serum (Gibco) and 1% of Polivitex (BioMérieaux) in microaerophilic conditions at 37ºC. The liquid culture was centrifuged and the cell pellet ressuspended in citrate-phosphate buffer pH 6 and incubated 15 minutes, centrifuged again and ressuspended in citrate-phosphate buffer pH 3 and incubated for 30 minutes. After centrifugation the supernatant was recovered and incubated for 3 hours in phage precipitant (33% polyethylene glycol [PEG], 3M NaCl). After centrifugation at 10000 rpm for 10 minutes at 4ºC the pellet was ressuspended in phage buffer. These samples were analysed by transmission electron microscopy (TEM) after negative staining with 1% aqueous uranyl acetate, using a JEOL 100SX electron microscope.For B45 strain the induction using UV radiation (previously reported in Lehours, 2011) and acid exposure produced similar results (Figure 1 and Figure 2) showing numerous phage-like particles of about 100 nm diameter, apparently lacking a tail, after UV or acid exposition, respectively. These particles were not observed in the control strain 1152. Currently we are analysing the samples using molecular biology techniques and fixation embedding followed by ultrathin sectioning for TEM analysis, to detect the presence of phages.These preliminary results suggest that acid also appears to induce the H. pylori prophage phiHP33. However, since the number of phage particles observed is small, we can not rule out that the observed particles were released spontaneously. The exposition to the natural acidic environment of the human stomach may induce H. pylori prophage into a lytic cycle and to the propagation of phages among different H. pylori strains colonizing the same individual. Although highly speculative, transduction may be another form of horizontal gene transfer, which has not been described for this bacterium yet.Financial support received from the Portuguese Science and Technology foundation under the contract PTDC/EBB-EBI/119860/2010.

Effect of Citral on the Thermal Inactivation of Escherichia coli O157:H7 in Citrate Phosphate Buffer and Apple Juice

Inactivation and sublethal injury of Escherichia coli O157:H7 cells induced by heat in citrate phosphate buffer and apple juice (both at pH 3.8) were studied, and the effect of a combined preservation treatment using citral and heat treatments was determined. Heat resistance of E. coli O157:H7 was similar in both treatment media; after 27 min at 54°C, 3 log units of the initial cell population was inactivated in both treatment media. However, under less harsh conditions a protective effect of apple juice was found. Whereas inactivation followed linear kinetics in the citrate phosphate buffer, when cells were treated in apple juice the survival curves were concave downward. Heat treatment caused a great degree of sublethal injury; 4 min at 54°C inactivated less than 0.5 log CFU/ml but sublethally injured more than 3 log CFU/ml. The addition of 18 and 200 ppm of citral to the treatment medium acted synergistically with heat at 54°C to inactivate 3 × 104 and 3 × 107 CFU/ml, respectively. Addition of citral thus reduced the time needed to inactivate 1 log unit of the initial E. coli O157:H7 population from 8.9 to 1.7 min. These results indicate that a combined process of heat and citral can inactivate E. coli O157:H7 cells and reduce their potential negative effects.

Relationship between In Vitro Activities of Amphotericin B and Flucytosine and pH for Clinical Yeast and Mold Isolates

ABSTRACT In this study, we investigated the pH dependency of the in vitro activities of amphotericin B (AMB) and flucytosine (5FC) against Candida spp., Cryptococcus neoformans, Aspergillus fumigatus, Rhizopus spp., and Scedosporium prolificans in RPMI 1640 buffered with citrate buffer (pH 4.0, 5.0, 5.4, and 6.0), citrate-phosphate buffer (pH 5.4, 6.0, 6.4, and 7.0), and 3-[N-morpholino]propanesulfonic acid (MOPS) (pH 6.4, 7.0, 7.4, and 7.9). For 5FC, no significant differences were found between MICs obtained with the different buffers, while for AMB, significant differences were found. The MICs obtained with citrate-phosphate buffer were approximately 1 twofold-dilution step higher than the MICs obtained with MOPS. We demonstrated that the in vitro activities of AMB and 5FC against yeast and mold isolates were pH dependent. The in vitro activity of AMB decreased when the pH was lowered, while the in vitro activity of 5FC increased. The effect of the pH on the in vitro activities was dependent not only on the antifungal agent tested but also on the microorganism. For AMB, there was a nonlinear relationship (median r 2, 0.864) for Candida spp., C. neoformans, A. fumigatus, and Rhizopus spp. over the pH range tested. The mean MICs ranged from 0.5 to 2.52 μg/ml at pH 7.0 and from 20.16 to 32 μg/ml at pH 5.0. For S. prolificans, there was no relationship. For 5FC, there was a linear relationship for Candida spp. (median r 2, 0.767) and a nonlinear relationship for C. neoformans and A. fumigatus (median r 2, 0.882) over the pH range tested. The mean MIC values ranged from 0.125 to 1,024 μg/ml at pH 7.0 and from 0.02 to 4 μg/ml at pH 5.0. For Rhizopus spp. and S. prolificans, the relationship could not be determined, since the MIC was >1,024 μg/ml over a pH range of 4.0 to 7.9.

Maintenance of ΔpH by a butanol-tolerant mutant of Clostridium beijerinckii

The isolation of Clostridium beijerinckii mutants that are more tolerant of butanol than the wild-type offered the opportunity to investigate whether the membrane activities which are required for maintaining the transmembrane ΔpH (the difference in pH between the cellular interior and exterior) are sensitive targets of butanol toxicity. The ΔpH was measured by the accumulation of [14C]benzoate using late-exponential-phase cells which were suspended in citrate/phosphate buffer at pH 5 (to maximize the ΔpH component of the protonmotive force) and supplemented with glucose and Mg2+. The ΔpH of the butanol-tolerant tolerant mutant, strain BR54, of C. beijerinckii NCIMB 8052 was found to be significantly more tolerant of added butanol than the wild-type. Thus, in potassium citrate/phosphate buffer the mutant cells maintained a ΔpH of 1·4 when butanol was added to a concentration of 1·5 % (w/v), while the wild-type ΔpH was reduced to 0·1. The ΔpH of both strains was completely dissipated with 1·75 % butanol, an effect attributed to a chaotropic effect on the membrane phospholipids. Similar results were obtained in sodium citrate/phosphate buffer. In the absence of added Mg2+, the ΔpH of the mutant decreased in both sodium and potassium citrate/phosphate buffer, but more rapidly in the former. Interestingly, the addition of butanol at low concentrations (0·8 %) prevented this ΔpH dissipation, but only in cells suspended in sodium citrate/phosphate buffer, and not in potassium citrate/phosphate buffer. In wild-type cells the decrease in ΔpH occurred more slowly than in the mutant, and sparing of the ΔpH by 0·8 % butanol was less pronounced. The authors interpret these data to mean that the ΔpH is dissipated in the absence of Mg2+ by a Na+- or K+-linked process, possibly by a Na+/H+ or a K+/H+ antiporter, and that the former is inhibited by butanol. Apparently, butanol can selectively affect a membrane-associated function at concentrations lower than required for the complete dissipation of transmembrane ion gradients. Additionally, since the butanol-tolerant mutant BR54 is deficient in the ability to detoxify methylglyoxal (MG) and contains higher levels of MG than the wild-type, the higher Na+/H+ antiporter activity of the mutant may be due to the greater degree of protein glycation by MG in the mutant cells. The mechanism of butanol tolerance may be an indirect result of the elevated glycation of cell proteins in the mutant strain. Analysis of membrane protein fractions revealed that mutant cells contained significantly lower levels of unmodified arginine residues than those of the wild-type cells, and that unmodified arginine residues of the wild-type were decreased by exposure of the growing cells to added MG.

A new allele of peptidase-B in cattle

Electrophoretic analyses of peptidase-B were carried out on red cell hemolysates from Holstein, Mantiqueira and Gyr cattle, using cornstarch, known in Brazil as Penetrose-30. We describe a new peptidase-B allele, denoted Pep-B1, in Mantiqueira cattle, belonging to the Bos taurus group, which are the result of a cross of native cattle of Portuguese origin introduced in Brazil during colonial times (16th century) with Holstein and Caracu cattle. The genetic control of peptidase-B was determined by typing parents and progeny segregating for all three alleles, confirming that peptidase B is controlled by a single autosomal locus with three codominant alleles, denoted Pep-B1, Pep-B2 and Pep-B3 The use of the citrate-phosphate buffer system, at pH 5.9, on 14% gel, under the electrophoretic conditions standardized in this study permitted good visualization of all peptidase-B variants.