Acid-sensitive Strains Research Articles

Volatile compounds-induced environmental pH shifts mediate interspecific bacterial interactions over long-distance

Microorganisms interact with each other through exchanging metabolites as well as signals molecules. This exchange typically depends on metabolites or signaling molecules diffusing in aqueous media within spatially connected habitats, and it only occurs within a short range. However, different microorganisms frequently live in spatially discontinuous micro-habitats without the connections of aqueous media. How microorganisms populating such spatially discontinuous micro-habitats interact remains poorly understood. Here, we show that a bacterial strain, Corynebacterium glutamicum ATCC13032T, produces high amounts of ammonia in its local habitat, which disperses over long distances (up to 12 cm) through the air. As a result, the pH of another spatially disconnected habitat increases, thus inducing the growth of an acid-sensitive strain (Glycocaulis alkaliphilus 6B-8T). We also show that ammonia-mediated long-distance interactions can be commonly found in various bacterial communities. In conclusion, our work demonstrates that bacteria growing in spatially discontinuous micro-habitats can interact with each other through gaseous diffusion of volatile compounds. This finding expands our understanding of the spatial scale at which bacteria can interact and provides new insights into how spatially separated species are connected to maximizing community-level commensalism.

CRISPRi screen highlights chromatin regulation to be involved in formic acid tolerance in Saccharomyces cerevisiae

Formic acid is one of the main weak acids in lignocellulosic hydrolysates that is known to be inhibitory to yeast growth even at low concentrations. In this study, we employed a CRISPR interference (CRISPRi) strain library comprising >9000 strains encompassing >98% of all essential and respiratory growth-essential genes, to study formic acid tolerance in Saccharomyces cerevisiae. To provide quantitative growth estimates on formic acid tolerance, the strains were screened individually on solid medium supplemented with 140 mM formic acid using the Scan-o-Matic platform. Selected resistant and sensitive strains were characterized in liquid medium supplemented with formic acid and in synthetic hydrolysate medium containing a combination of inhibitors. Strains with gRNAs targeting genes associated with chromatin remodeling were significantly enriched for strains showing formic acid tolerance. In line with earlier findings on acetic acid tolerance, we found genes encoding proteins involved in intracellular vesicle transport enriched among formic acid sensitive strains. The growth of the strains in synthetic hydrolysate medium followed the same trend as when screened in medium supplemented with formic acid. Strains sensitive to formic acid had decreased growth in the synthetic hydrolysate and all strains that had improved growth in the presence of formic acid also grew better in the hydrolysate medium. Systematic analysis of CRISPRi strains allowed identification of genes involved in tolerance mechanisms and provided novel engineering targets for bioengineering strains with increased resistance to inhibitors in lignocellulosic hydrolysates.

Identification of acetic acid sensitive strains through biosensor-based screening of a Saccharomyces cerevisiae CRISPRi library

BackgroundAcetic acid tolerance is crucial for the development of robust cell factories for conversion of lignocellulosic hydrolysates that typically contain high levels of acetic acid. Screening mutants for growth in medium with acetic acid is an attractive way to identify sensitive variants and can provide novel insights into the complex mechanisms regulating the acetic acid stress response.ResultsAn acetic acid biosensor based on the Saccharomyces cerevisiae transcription factor Haa1, was used to screen a CRISPRi yeast strain library where dCas9-Mxi was set to individually repress each essential or respiratory growth essential gene. Fluorescence-activated cell sorting led to the enrichment of a population of cells with higher acetic acid retention. These cells with higher biosensor signal were demonstrated to be more sensitive to acetic acid. Biosensor-based screening of the CRISPRi library strains enabled identification of strains with increased acetic acid sensitivity: strains with gRNAs targeting TIF34, MSN5, PAP1, COX10 or TRA1.ConclusionsThis study demonstrated that biosensors are valuable tools for screening and monitoring acetic acid tolerance in yeast. Fine-tuning the expression of essential genes can lead to altered acetic acid tolerance.

Surviving host - and food relevant stresses: phenotype of L. monocytogenes strains isolated from food and clinical sources

The aim of this study was to compare the phenotype of 40 strains of L. monocytogenes under food and host relevant stress conditions. The strains were chosen to represent food and clinical isolates and to be equally distributed between the most relevant clonal complexes for clinical and food isolates (CC1 and CC6 vs CC121 and CC9), plus one group of eight strains of rare clonal complexes. Human-associated CC1 had a faster maximal growth rate than the other major complexes, and the lag time of CC1 and CC6 was significantly less affected by the addition of 4% NaCl to the medium. Food-associated CC9 strains were hypohemolytic compared to other clonal complexes, and all strains found to be resistant to increased concentrations of benzalkonium chloride belonged to CC121 and were positive for Tn6188 carrying the qacH gene. Lactic acid affected the survival of L. monocytogenes more than HCl, and there was a distinct, strain specific pattern of acid tolerant and sensitive strains. Strains from CC6 and human clinical isolates are less resilient under acid stress than those from other complexes and from food. One strain isolated from a human patient exhibited significant growth defects across all conditions.

Nalidixic acid screening test in detection of decreased fluoroquinolone susceptibility in Salmonella typhi isolated from blood.

To determine the validity of nalidixic acid screening test in the detection of high MICs of fluoroquinolone against Salmonella(S.) typhi isolated from blood and correlate zone diameters of ofloxacin with that of MIC value for nalidixic acid sensitive and resistant strains. Cross-sectional analytical study. Clinical Microbiology Laboratory of the Aga Khan Hospital, Karachi from January 2002 to December 2003. Two hundred S. typhi isolates from blood were included for nalidixic acid screening and ofloxacin susceptibility. Antibiotic susceptibilities for both the antibiotics were obtained by disc diffusion method whereas MICs were determined by standard agar dilution method as recommended by NCCLS guidelines. Sensitivity, specificity and correlation between both antimicrobial susceptibility methods were calculated and results expressed as scattergrams. The results broadly classify S. typhi isolates into nalidixic acid resistant strains with no zone of inhibition around 30 mug nalidixic acid disc and nalidixic acid sensitive strains with mean zone of inhibition of 24.9 mm. All S. typhi isolates with ofloxacin MIC of capital ZHE, Cyrillic 0.125 microg/ml were found to be nalidixic acid resistant (MIC capital ZHE, Cyrillic32 microg/ml) whereas the isolates with ofloxacin MIC 0.06 microg/ml were nalidixic acid sensitive (MIC 8 microg/ml). Screening for nalidixic acid resistance was found to be 100% sensitive and 97% specific in identifying S. typhi strains with reduced susceptibility to fluoroquinolone (MIC capital ZHE, Cyrillic 0.125 microg/ml). Nalidixic acid resistance as a screening method is proved to be significant in identifying S. typhi isolates with reduced susceptibility to fluoroquinolones. It is also suggested that inhibition zone of 25 mm around 5 microg ofloxacin disc is appropriate as a selection criterion to detect S. typhi isolates with reduced susceptibility to fluoroquinolones.

Survival and Growth of Probiotic Lactic Acid Bacteria in Refrigerated Pickle Products.

We examined 10 lactic acid bacteria that have been previously characterized for commercial use as probiotic cultures, mostly for dairy products, including 1 Pediococcus and 9 Lactobacilli. Our objectives were to develop a rapid procedure for determining the long-term survivability of these cultures in acidified vegetable products and to identify suitable cultures for probiotic brined vegetable products. We therefore developed assays to measure acid resistance of these cultures to lactic and acetic acids, which are present in pickled vegetable products. We used relatively high acid concentrations (compared to commercial products) of 360 mM lactic acid and 420 mM acetic acid to determine acid resistance with a 1 h treatment. Growth rates were measured in a cucumber juice medium at pH 5.3, 4.2, and 3.8, at 30 °C and 0% to 2% NaCl. Significant differences in acid resistance and growth rates were found among the 10 cultures. In general, the acid resistant strains had slower growth rates than the acid sensitive strains. Based on the acid resistance data, selected cultures were tested for long-term survival in a simulated acidified refrigerated cucumber product. We found that one of the most acid resistant strains (Lactobacillus casei) could survive for up to 63 d at 4 °C without significant loss of viability at 108 CFU/mL. These data may aid in the development of commercial probiotic refrigerated pickle products.

Transcriptional profile of glucose-shocked and acid-adapted strains of Streptococcus mutans.

The aciduricity of Streptococcus mutans is an important virulence factor of the organism, required to both out-compete commensal oral microorganisms and cause dental caries. In this study, we monitored transcriptional changes that occurred as a continuous culture of either an acid-tolerant strain (UA159) or an acid-sensitive strain (fabM::Erm) moved from steady-state growth at neutral pH, experienced glucose-shock and acidification of the culture, and transitioned to steady-state growth at low pH. Hence, the timing of elements of the acid tolerance response (ATR) could be observed and categorized as acute vs. adaptive ATR mechanisms. Modulation of branched chain amino acid biosynthesis, DNA/protein repair mechanisms, reactive oxygen species metabolizers and phosphoenolpyruvate:phosphotransferase systems occurred in the initial acute phase, immediately following glucose-shock, while upregulation of F1 F0 -ATPase did not occur until the adaptive phase, after steady-state growth had been re-established. In addition to the archetypal ATR pathways mentioned above, glucose-shock led to differential expression of genes suggesting a re-routing of resources away from the synthesis of fatty acids and proteins, and towards synthesis of purines, pyrimidines and amino acids. These adjustments were largely transient, as upon establishment of steady-state growth at acidic pH, transcripts returned to basal expression levels. During growth at steady-state pH 7, fabM::Erm had a transcriptional profile analogous to that of UA159 during glucose-shock, indicating that even during growth in rich media at neutral pH, the cells were stressed. These results, coupled with a recently established collection of deletion strains, provide a starting point for elucidation of the acid tolerance response in S. mutans.

Effects of Acidity on Growth and Symbiotic Performance of <i>Rhizobium leguminosarum</i> bv. <i>viciae</i> Strains Isolated from Faba Bean Producing Areas of Ethiopia

Faba bean is one of the legume crops commonly grown in Ethiopia. It is important source of dietary protein to the majority of population in the country. Soil acidity and related stresses are among the major yield limiting constraints for this crop. This study was conducted with the aim of evaluating acidity tolerance of Rhizobium leguminosarum bv. viciae strains isolated from faba bean growing regions of the country and their symbiotic performance under different acidic conditions. Four strains isolated from root nodules of faba bean were tested for tolerance to acidity in a defined liquid media. The results indicated that none of the tested strains was tolerant to pH 4.0 while two of them (AUFR46 and AUFR100) were found to be tolerant of pH 4.5. When tested at pH 5.0 only one isolate (AUFR58) was sensitive. The results of the present study also showed that all acid tolerant strains were recovered from highly acidic soil (4.8- 5.2) and the acid sensitive strain was isolated from neutral soil. A positive correlation (r= 0.92) was observed between minimum pH tolerated in culture media and pH of origin soil of the strains. Nodulation and nitrogen fixation abilities of these strains were evaluated on sand culture. The results indicated that at pH 4.5 and 5.0 nodulation was totally inhibited and only one isolate (AUFR58) could not be able to induce nodule formation on host plant roots at pH 5.5. The results also showed that acidity (pH 5.5) reduced shoot dry weight, nodules number, total nitrogen at a highly significant level (P<0.01) compared to plants grown at pH 6.5 and 7.0. In pot experiment with soil of different pH, inoculation of the rhizobial strains improved the growth, nodulation and nitrogen content of the plants significantly over the uninoculated controls. Besides, acid tolerant strains showed better performance over acid sensitive strains in acidic soils and thus, they are highly recommended for field test in acidic soil.Keywords: Acidity tolerance; Nodulation; Nitrogen fixation; Vicia faba; Rhizobium; Faba bean

Preparative Isolation of Bioenhancer Loganetin from sf Alstonia scholaris by Fast Centrifugal Partition Chromatography

Fast centrifugal partition chromatography (FCPC) was successfully applied in the separation of close R f complex bioactive iridoid, loganetin directly from the ethyl acetate extract of Alstonia scholaris . The experiment was performed with a two-phase solvent system composed of methyl tert- butyl ether (MtBE)/ACN/Water (3:1.5:3 v/v/v) where the lower phase of the biphasic system, the aqueous layer containing 8 mM HCl, was the stationary phase, while the upper organic layer supplemented with 15 mM triethylamine TEA was designated as the mobile phase. From 1.5 g of EtOAc extract, 48 mg of loganetin (1) was obtained in 94.4% purity as determined by HPLC. The isolated compound (1) was characterized on the basis of its 1 H, 13 C–NMR, and ESI-MS spectroscopic data. Although loganetin does not possess antibacterial activity of its own, but in combination, it appreciably reduces the minimum inhibitory concentration (MIC) of nalidixic acid (NA) against nalidixic acid resistant (NAREC) and nalidixic acid sensitive (NASEC) strains of Escherichia coli . Loganetin, a very common, inexpensive, and non-toxic natural product may finds its application in the antibacterial drug development for treating multidrug-resistant Gram negative infections.

Growth inhibition of Cronobacter spp. strains in reconstituted powdered infant formula acidified with organic acids supported by natural stomach acidity

Cronobacter is associated with outbreaks of rare, but life-threatening cases of meningitis, necrotizing enterocolitis, and sepsis in newborns. This study was conducted to determine the effect of organic acids on growth of Cronobacter in laboratory medium and reconstituted powdered infant formula (PIF) as well as the bacteriostatic effect of slightly acidified infant formula when combined with neonatal gastric acidity. Inhibitory effect of seven organic acids on four acid sensitive Cronobacter strains was determined in laboratory medium with broth dilution method at pH 5.0, 5.5 and 6.0. Acetic, butyric and propionic acids were most inhibitive against Cronobacter in the laboratory medium. The killing effect of these three acids was partially buffered in reconstituted PIF. Under neonatal gastric acid condition of pH 5.0, the slightly acidified formula which did not exert inhibition effect solely reduced significantly the Cronobacter populations. A synergistic effect of formula moderately acidified with organic acid combined with the physiological infant gastric acid was visible in preventing the rapid growth of Cronobacter in neonatal stomach. The study contributed to a better understanding of the inhibitory effect of organic acids on Cronobacter growth in different matrixes and provided new ideas in terms of controlling bacteria colonization and translocation by acidified formula.

Roles of H2 uptake hydrogenases in Shigella flexneri acid tolerance

Hydrogenases play many roles in bacterial physiology, and use of H(2) by the uptake-type enzymes of animal pathogens is of particular interest. Hydrogenases have never been studied in the pathogen Shigella, so targeted mutant strains were individually generated in the two Shigella flexneri H(2)-uptake enzymes (Hya and Hyb) and in the H(2)-evolving enzyme (Hyc) to address their roles. Under anaerobic fermentative conditions, a Hya mutant strain (hya) was unable to oxidize H(2), while a Hyb mutant strain oxidized H(2) like the wild-type. A hyc strain oxidized more exogenously added hydrogen than the parent. Fluorescence ratio imaging with dye JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide) showed that the parent strain generated a membrane potential 15 times greater than hya. The hya mutant was also by far the most acid-sensitive strain, being even more acid-sensitive than a mutant strain in the known acid-combating glutamate-dependent acid-resistance pathway (GDAR pathway). In severe acid-challenge experiments, the addition of glutamate to hya restored survivability, and this ability was attributed in part to the GDAR system (removes intracellular protons) by mutant strain (e.g. hya/gadBC double mutant) analyses. However, mutant strain phenotypes indicated that a larger portion of the glutamate-rescued acid tolerance was independent of GadBC. The acid tolerance of the hya strains was aided by adding chloride ions to the growth medium. The whole-cell Hya enzyme became more active upon acid exposure (20 min), based on assays of hyc. Indeed, the very high rates of Shigella H(2) oxidation by Hya in acid can supply each cell with 2.4×10(8) protons min(-1). Electrons generated from Hya-mediated H(2) oxidation at the inner membrane likely counteract cytoplasmic positive charge stress, while abundant proton pools deposited periplasmically likely repel proton influx during severe acid stress.

Calcium affects the competitiveness of acid-sensitive and acid-tolerant strains of Bradyrhizobium japonicum in nodulating and fixing nitrogen with two soybean cultivars in acid soil

A glasshouse experiment studied the role of calcium and pH on competitiveness of acid-sensitive and acid-tolerant Bradyrhizobium japonicum strains with similar N 2-fixation effectiveness in nodulating two soybean ( Glycine max L. Merr) cultivars selected for tolerance of aluminium (PI416937) or for manganese (Manta). Liming provides calcium (Ca) as well as increasing soil pH. Thus the effect of Ca and pH of soil are difficult to separate. We examined the effects of Ca per se by comparing the response to gypsum and lime amendment on the competitiveness of acid-tolerant and acid-sensitive strains in nodulating soybean in an acid soil. Acid soil was treated with either CaSO 4 or CaCO 3 and incubated for 2 weeks before sowing soybean seed. Two acid-sensitive and two acid-tolerant B. japonicum strains were mixed with each other (one acid-sensitive plus one acid-tolerant) and were inoculated onto soybean seeds at the rate of 10 6cfu seed −1. Soil pH, as amended by lime addition, had more effect on nodulation than Ca addition in the form of gypsum. The response was affected by cultivar and strain in a complicated fashion with a marked strain × cultivar interaction. One acid-tolerant strain formed most nodules with both cultivars in the unamended soil of pH 4.36 in competition with one acid-sensitive strain. The same acid-tolerant strain was not competitive against the second acid-sensitive strain with Manta but was with PI416937. The second acid-tolerant strain was not competitive with either acid-sensitive strain in unamended and gypsum treated soils. It was only competitive with PI416937 in limed soil, a rather surprising result. Inoculation of this soil with no native soybean nodulating strains, increased shoot weight, %N, N uptake. N 2-fixation was greatly increased by inoculation and lime addition, and to a lesser extent by gypsum addition for Manta. This experiment indicates that addition of Ca per se as gypsum to an acid soil has little effect on symbiotic performance, but changing pH by liming has a major effect, that both soybean cultivar and B. japonicum strain influence the competitiveness of strains in acid soil and that acid-tolerance does not necessarily increase a strain's competitiveness.

Symbiotic effectiveness of Bradyrhizobium japonicum in acid soils can be predicted from their sensitivity to acid soil stress factors in acidic agar media

In acid soil, low pH, reduced availability of nutrients, and toxicity of Al and Mn limit plant growth and the survival and effectiveness of rhizobia. The symbiosis between legumes and rhizobia is particularly sensitive to acid soil stress. A pot experiment evaluated whether Bradyrhizobium japonicum strain growth on acidic agar media would predict ability to colonize the rhizosphere and form effective nodules in acidic soils. Three Indonesian strains of B. japonicum with similar effectiveness at neutral pH in sand culture but with different tolerance of acid soil stress factors in agar media, and an acid-tolerant commercial strain (CB1809) of comparable effectiveness, were tested in three acid soils using the Al tolerant soybean ( Glycine max cv PI 416937). At 7 days after inoculation all strains had achieved large rhizosphere populations, but by day 14 the rhizosphere population of the acid-sensitive strain had decreased, while the more acid-tolerant strains increased. The acid-tolerant strains had significantly greater nodulation and symbiotic effectiveness than plants inoculated with the acid-sensitive strain. Laboratory prescreening of B. japonicum for acid, Al and Mn tolerance in acid media successfully identified strains which were symbiotically competent in low pH soils.

Improving the storage stability of Bifidobacterium breve in low pH fruit juice

Bifidobacterial food applications are limited since bifidobacteria are sensitive to e.g. acidic conditions prevalent in many food matrices. The aim of the present study was to investigate whether a low pH selection step alone or combined to UV mutagenesis could improve the viability of an acid sensitive Bifidobacterium strain, B. breve 99, in low pH food matrices. Furthermore, the potential of carriers and an oat fibre preparation to further improve the stability was studied. The best performing low pH tolerant variants in the present study were generated by UV-mutagenesis with 70-700μJ/cm(2) followed by incubation in growth medium at pH 4.5. The most promising variants regarding the low pH tolerance showed, in repeated tests with cells grown without pH control, about one Log-value better survival in pH 3.8 fruit juice after one week storage at 4°C compared to wild-type B. breve 99. Cells grown with pH control, PDX formulated and then frozen showed poorer viability in low pH fruit juice than cells grown with no pH control. For frozen concentrates pH 3.8 was too stressful and no or small differences between the variants and the wild-type strain were seen. The differences detected at pH 3.8 with the cells grown without pH control were also seen with the frozen concentrates at pH 4.5. Some improvement in the stability could be achieved by using a combination of trehalose, vitamin C and PDX as a freezing carrier material, whereas a significant improvement in the stability was seen when oat fibre was added into the fruit juice together with the frozen cells. Due to the initial very poor fruit juice tolerance of B. breve 99 the obtained improvement in the stability was not enough for commercial applications. However, the same methods could be applied to initially better performing strains to further improve their stability in the fruit juice.

Lyophyllum sp. strain Karsten alleviates pH pressure in acid soil and enhances the survival of Variovorax paradoxus HB44 and other bacteria in the mycosphere

Abstract In previous work, Variovorax paradoxus strain HB44, next to Burkholderia terrae BS001 and Dyella japonica BS003, were found to be selected in the mycosphere of the tricholomataceous fungi Laccaria proxima and Lyophyllum sp. strain Karsten in an acid soil denoted G. V. paradoxus HB44 showed poor survival in G bulk soil, irrespective of prior soil sterilization, and this poor survival also occurred for B. terrae BS001 and D. japonica BS003. In contrast, the survival rate of strain HB44 in two other soils, with pH values > 5.5, was significantly raised. Also, significantly enhanced strain HB44 survival in G soil was found if the pH was raised to 5.5 or 6.5, and it was even shown to grow (in the presence of the exogenous carbon source glycerol) at such pH values in the sterile G soil. This behaviour was similar to that of the V. paradoxus type strain. Strikingly, Lyophyllum sp. strain Karsten, when colonizing the sterilized G soil, significantly raised the soil pH from about 4.6 to ≥5.0. The pH raise was dependent on time, hyphal development, as well as on initial soil pH, but was consistent throughout. The modulated soil pH conditions were shown to be permissive for the survival and growth of strain HB44, and this was extended to strains BS001 and BS003. These findings corroborate the hypothesis that L. sp. strain Karsten provides a suitable habitat for acid-sensitive strains like HB44, BS001 and BS003 in its mycosphere in acid soil, which is strongly defined by the establishment of a growth-permissive pH.

Fates of Acid-Resistant and Non-Acid-Resistant Shiga Toxin-Producing Escherichia coli Strains in Ruminant Digestive Contents in the Absence and Presence of Probiotics

Healthy ruminants are the main reservoir of Shiga toxin-producing Escherichia coli (STEC). During their transit through the ruminant gastrointestinal tract, STEC encounters a number of acidic environments. As all STEC strains are not equally resistant to acidic conditions, the purpose of this study was to investigate whether acid resistance confers an ecological advantage to STEC strains in ruminant digestive contents and whether acid resistance mechanisms are induced in the rumen compartment. We found that acid-resistant STEC survived at higher rates during prolonged incubation in rumen fluid than acid-sensitive STEC and that they resisted the highly acidic conditions of the abomasum fluid, whereas acid-sensitive strains were killed. However, transit through the rumen contents allowed acid-sensitive strains to survive in the abomasum fluid at levels similar to those of acid-resistant STEC. The acid resistance status of the strains had little influence on STEC growth in jejunal and cecal contents. Supplementation with the probiotic Saccharomyces cerevisiae CNCM I-1077 or Lactobacillus acidophilus BT-1386 led to killing of all of the strains tested during prolonged incubation in the rumen contents, but it did not have any influence in the other digestive compartments. In addition, S. cerevisiae did not limit the induction of acid resistance in the rumen fluid. Our results indicate that the rumen compartment could be a relevant target for intervention strategies that could both limit STEC survival and eliminate induction of acid resistance mechanisms in order to decrease the number of viable STEC cells reaching the hindgut and thus STEC shedding and food contamination.

Detection of mutations in the gyrA gene in fluoroquinolone resistance Salmonella enterica serotypes typhi and paratyphi A isolated from the Infectious Diseases Hospital, Kuwait

Background:Enteric fever due to Salmonella enterica is a major health problem, and fluoroquinolones such as ciprofloxacin are mostly the antibiotic of choice for treatment. Resistance to ciprofloxacin has been noticed...

Profile of culture positive enteric fever from bangalore

Background: Enteric fever is endemic in India. The aim of this study was to analyse the clinical, laboratory, antibiotic sensitivity profile and response to antibiotics of culture positive enteric fever patients from Bangalore. Methods: In this retrospective study only culture positive enteric fever patients were taken and their clinical, laboratory, antibiotic sensitivity profile and the clinical response to antibiotics studied. Result: Eighty one culture positive enteric fever patients were taken into the study. Presenting symptoms included fever, pain abdomen (18.5%), loose stools (25%), vomiting (33%) and headache (30%). Absolute bradycardia at admission was not found in any of our patients. Normal or low total leucocyte count was seen in 97.5%. Typhoid hepatitis was seen in 8.5%. Salmonella enterica subspecies enterica serovar typhi ( S typhi) were isolated in 80% of cases; 83% of all cases showed nalidixic acid resistance. All isolates were sensitive to chloramphenicol and third generation cephalosporins. Ciprofloxacin resistance was found in 19% cases. The time to defervescence in patients treated with ceftriaxone was 4.3 days. There was no statistical difference in time to defervescence in nalidixic acid resistant and sensitive strains. Complications included gastro intestinal bleed and encephalopathy. Conclusion: Prevalence of nalidixic acid resistance is high, while clinical resistance to quinolones may be higher than that found in the laboratory which requires detailed study. Chloramphenicol sensitivity has returned and nalidixic acid resistant and sensitive isolates are uniformly sensitive to third generation cephalosporins with no difference in time to defervescence.

Induction of acid resistance inBifidobacterium: a mechanism for improving desirable traits of potentially probiotic strains

To generate stable acid-resistant Bifidobacterium strains isolated from human subjects and characterize the phenotypic changes of the acid-resistant derivatives. The ability of 20 Bifidobacterium strains isolated from human faeces to survive to simulated gastrointestinal transit was evaluated, showing major reductions in viability (0.25-5.8 logarithmic units) because of gastric stress conditions. Six acid-sensitive strains belonging to the species Bifidobacterium longum and Bifidobacterium catenulatum were submitted to prolonged incubation at pH 2.0 to generate acid-resistant strains. The acid-sensitive and acid-resistant derivative strains were characterized to determine the changes associated with the acquisition of an acid-tolerant phenotype. The acid-resistant derivatives showed better ability to grow in the presence of bile salt (1-3%) and NaCl (6-10%) and higher resistance at elevated temperatures (60-70 degrees C, 10 min) than the parental strains. The acid-resistant derivatives displayed higher fermentative ability, and enzymatic activities. These strains also showed higher sensitivity to most of the tested antibiotics than the parental strains. The stress tolerance of B. longum and B. catenulatum strains was improved by prolonged exposure to acid stress conditions. Some of the generated strains also seemed to have enhanced metabolic properties of relevance for probiotic applications. The successful use of prolonged exposures to acid stress to improve the stability of human bifidobacteria indicates that this strategy could be useful for the production of robust probiotic strains, but involves other phenotypic changes that required an individual characterization.

Nodulation of Medicago truncatula and Medicago polymorpha in two pastures of contrasting soil pH and rhizobial populations

The size of the background rhizobial population can often determine the success of field nodulation and persistence of inoculant rhizobia. Field experiments were conducted to determine the nodulation response of annual medics ( Medicago spp.) in a pasture-wheat-pasture rotation when grown in soils of contrasting pH and rhizobial populations. Medicago truncatula Gaertn. and M. polymorpha L. were inoculated with one of three different strains of Sinorhizobium medicae (WSM540, WSM688) or S. meliloti (NA39) or left uninoculated and sown in two fields of pH (CaCl 2) 5.9 and 7.2 of differing soil rhizobial backgrounds (11 and 7.1 × 10 4 cells/g soil, respectively). Nodulation was assessed in years 1 and 3 of the rotation. At the site with a small rhizobial background, M. polymorpha nodulated poorly when inoculated with the acid-sensitive strain NA39 but nodulated well when inoculated with acid-tolerant strains WSM688 and WSM540. M. truncatula had a similar extent of nodulation with each of the rhizobial inoculants. At the site with a large rhizobial background all treatments had greater than 85% of plants nodulated. Nodule occupancies, assessed by PCR, provided further insight: at the site with a small rhizobial background both medic species successfully nodulated with the acid-tolerant strains WSM540 and WSM688 and these strains persisted to year 3. However, at the site with large rhizobial background, only one strain, WSM688, was identified from M. truncatula nodules in year 3. This study highlights the importance of edaphic constraints and plant–rhizobia interactions to the successful development of nodulation in a field environment.