bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of age on susceptibility to Salmonella Typhimurium infection in C57BL/6 mice
Authors: Ren Zhihong , Gay Raina , Thomas Adam , Pae Munkyong , Wu Dayong , Logsdon Lauren , Mecsas Joan , Meydani Simin Nikbin
Published: November 2009

Ageing is associated with a decline in immune function, which predisposes the elderly to a higher incidence of infections. Information on the mechanism of the age-related increase in susceptibility toSalmonella entericaserovar Typhimurium (S.Typhimurium) is limited. In particular, little is known regarding the involvement of the immune response in this age-related change. We employed streptomycin (Sm)-pretreated C57BL/6 mice to develop a mouse model that would demonstrate age-related differences in susceptibility and immune response toS.Typhimurium. In this model, old mice inoculated orally with doses of 3×108or 1×106 c.f.u.S.Typhimurium had significantly greaterS.Typhimurium colonization in the ileum, colon, Peyer's patches, spleen and liver than young mice. Old mice had significantly higher weight loss than young mice on days 1 and 2 post-infection. In response toS.Typhimurium infection, old mice failed to increaseex vivoproduction of IFN-γand TNF-αin the spleen and mesenteric lymph node cells to the same degree as observed in young mice; this was associated with their inability to maintain the presence of neutrophils and macrophages at a ‘youthful’ level. These results indicate that Sm-pretreated C57BL/6 old mice are more susceptible toS.Typhimurium infection than young mice, which might be due to impaired IFN-γand TNF-αproduction as well as a corresponding change in the number of neutrophils and macrophages in response toS.Typhimurium infection compared to young mice.
Genetic analysis of high-level mupirocin resistance in the ST80 clone of community-associated meticillin-resistant Staphylococcus aureus
Authors: Udo Edet E. , Sarkhoo Eiman
Published: November 2009

Four community-associated meticillin-resistant Staphylococcus aureus (CA-MRSA) isolates expressing high-level mupirocin resistance (MIC >1024 mg l−1) were isolated from four sites of a diabetic patient and characterized for the genetic location of their resistance determinants and typed using PFGE, staphylococcal cassette chromosome mec (SCCmec), the coagulase gene and multilocus sequence typing to ascertain their relatedness. The presence of genes for resistance to high-level mupirocin (mupA), tetracycline (tetK) and fusidic acid (far1), Panton–Valentine leukocidin (PVL), accessory gene regulators (agr) and capsular polysaccharide (cap) were detected in PCR assays. The isolates were resistant to kanamycin, streptomycin, tetracycline, fusidic acid and cadmium acetate, and harboured mupA, tetK, far1, PVL, agr3 and cap8. They had identical PFGE patterns and coagulase gene type, possessed the type IV SCCmec element and belonged to sequence type 80 (ST80). However, they had three different plasmid profiles: (i) 28.0 and 26.0 kb; (ii) 28.0, 21.0 and 4.0 kb; and (iii) 41.0 and 4.0 kb. Genetic studies located the resistance to tetracycline, fusidic acid and cadmium acetate on the 28 kb plasmid and mupA on the related non-conjugative 26 and 21 kb plasmids. One of the 21 kb mupirocin-resistance plasmids was derived from the ∼41 kb plasmid during transfer experiments. The emergence of high-level mupirocin resistance in the ST80-SCCmec IV MRSA clone demonstrates the increasing capacity of CA-MRSA clones to acquire resistance to multiple antibacterial agents. The presence of different plasmid profiles in genetically identical isolates creates difficulty in the interpretation of typing results and highlights the weakness of using plasmid analysis as the sole method for strain typing.
Vibrio cholerae O1 Ogawa detoxified lipopolysaccharide structures as inducers of cytokines and oxidative species in macrophages
Authors: Paulovičová Ema , Kováčová Elena , Bystrický Slavomír
Published: November 2009

Multidrug resistance in several strains ofVibrio choleraehas encouraged anti-cholera vaccine developmental attempts using various subcellular moieties. In order to examine the immunological efficacy of detoxified LPS (dLPS)-derived saccharide immunogens,ex vivoactivation of mouse peritoneal macrophages (MΦs) was investigated. The immunomodulatory effect was evaluated via induction of the pro-inflammatory cytokines tumour necrosis factor-α, interleukin (IL)-1αand IL-6 and acceleration of nitric oxide (NO) and reactive oxygen species (ROS). Immunologically active structures triggered mouse peritoneal MΦs to secrete cytokines and release NO/ROS, even at concentrations as low as 12.5 μg ml−1. It was found that the O-specific polysaccharide moiety was more immunologically efficient than the glycolipid one, probably due to the position of 3-deoxy-d-manno-octulosonic acid. The results revealed effective structure–immunomodulating relationships of dLPS-derived moieties that are desirable in subcellular anti-cholera vaccine design.
Moraxella catarrhalis bacteraemia associated with prosthetic vascular graft infection
Authors: Sano Naoto , Matsunaga Satoshi , Akiyama Tomonori , Nakashima Yukari , Kusaba Koji , Nagasawa Zenzo , Koizumi Shunzo , Goto Masaaki , Miyamoto Hiroshi
Published: November 2009

Moraxella catarrhalis, formerly calledBranhamella catarrhalis, ‘Neisseria catarrhalis’ or ‘Micrococcus catarrhalis’, is a Gram-negative, aerobic diplococcus frequently found as a colonizer of the upper respiratory tract. Over the last 20–30 years, this bacterium has emerged as a genuine pathogen, and is now considered an important cause of otitis media in children and an aetiological agent in pneumonia in adults with chronic obstructive pulmonary disease. However, bacteraemia due toM. catarrhalishas rarely been reported. Presented here is a case ofM. catarrhalisbacteraemia associated with prosthetic vascular graft infection along with a review of the relevant literature.
Evaluation of internal transcribed spacer 2-RFLP analysis for the identification of dermatophytes
Authors: De Baere Thierry , Summerbell Richard , Theelen Bart , Boekhout Teun , Vaneechoutte Mario
Published: November 2009

A total of 95 isolates, belonging to 33 species of five dermatophyte genera, i.e. Arthroderma (15 species), Chrysosporium (two), Epidermophyton (one), Microsporum (three) and Trichophyton (12), were studied using internal transcribed spacer 2 (ITS2)-PCR-RFLP analysis (ITS2-RFLP), consisting of amplification of the ITS2 region, restriction digestion with BstUI (CG/CG) and restriction fragment length determination by capillary electrophoresis. ITS2-RFLP analysis proved to be most useful for identification of species of the genera Arthroderma, Chrysosporium and Epidermophyton, but could not distinguish between several Trichophyton species. The identification results are in agreement with established and recent taxonomical insights into the dermatophytes; for example, highly related species also had closely related and sometimes difficult-to-discriminate ITS2-RFLP patterns. In some cases, several ITS2-RFLP groups could be distinguished within species, again mostly in agreement with the taxonomic delineations of subspecies and/or genomovars, confirming the relevance of ITS2-RFLP analysis as an identification technique and as a useful taxonomic approach.
Constitutive expression of the ileS-2 gene responsible for high-level mupirocin resistance in Staphylococcus aureus
Authors: Oliveira Naira Elane Moreira de , Cavalcanti Elisa D'Avila Costa , Laport Marinella Silva , Bastos Maria do Carmo de Freire , Giambiagi-deMarval Marcia
Published: November 2009

Mupirocin is a topical antimicrobial agent that is used for the treatment of skin and postoperative wound infections, and the prevention of nasal carriage of meticillin-resistantStaphylococcus aureus(MRSA). However, the prevalence of mupirocin resistance inS. aureus, particularly in MRSA, has increased. High-level mupirocin resistance, with MICs ≥512 μg ml−1, is mediated by theileS-2 gene, which is located on conjugative plasmids. In the present study, we investigated whether mupirocin influences the expression of theileS-2gene responsible for high-level mupirocin resistance, and we present some evidence that this gene is not upregulated but constitutively expressed inS. aureus.
An imprint method for detecting leptospires in the hamster model of vaccine-mediated immunity for leptospirosis
Authors: Chagas-Junior Adenizar D. , McBride Alan J. A. , Athanazio Daniel A. , Figueira Cláudio P. , Medeiros Marco A. , Reis Mitermayer G. , Ko Albert I. , McBride Flávia W. C.
Published: November 2009

In determining the efficacy of new vaccine candidates for leptospirosis, the primary end point is death and an important secondary end point is sterilizing immunity. However, evaluation of this end point is often hampered by the time-consuming demands and complexity of methods such as culture isolation (CI). In this study, we evaluated the use of an imprint (or touch preparation) method (IM) in detecting the presence of leptospires in tissues of hamsters infected with Leptospira interrogans serovar Copenhageni. In a dissemination study, compared to CI, the IM led to equal or improved detection of leptospires in kidney, liver, lung and blood samples collected post-infection and overall concordance was good (κ=0.61). Furthermore, in an evaluation of hamsters immunized with a recombinant leptospiral protein-based vaccine candidate and subsequently challenged, the agreement between the CI and IM was very good (κ=0.84). These findings indicate that the IM is a rapid method for the direct observation of Leptospira spp. that can be readily applied to evaluating infection in experimental animals and determining sterilizing immunity when screening potential vaccine candidates.
Development and evaluation of internal amplification controls for use in a real-time duplex PCR assay for detection of Campylobacter coli and Campylobacter jejuni
Authors: Randall Luke , Lemma Fabrizio , Rodgers John , Vidal Ana , Clifton-Hadley Felicity
Published: November 2009

A common problem of both conventional and real-time PCR assays is failure of DNA amplification due to the presence of inhibitory substances in samples. In view of this, our aim was to develop and evaluate internal amplification controls (IACs) for use with an existing duplex real-time PCR assay for Campylobacter coli and Campylobacter jejuni. Both competitive and non-competitive IACs were developed and evaluated. The competitive approach involved a DNA fragment of the coding region of the fish viral haemorrhagic septicaemia virus, flanked by the mapA PCR primers, whilst the non-competitive approach utilized an extra set of universal 16S rDNA primers. Both IAC-PCR assay types were evaluated using cultures of Campylobacter and chicken caecal content samples. Both IACs were sensitive to caecal inhibitors, making them suitable for detecting inhibition which could lead to false-negatives. Results showed that both IACs at optimum concentrations worked well without reducing the overall sensitivity of the PCR assay. Compared to culture, the optimized competitive IAC-PCR assay detected 45/47 positives (sensitivity 93.6 %, specificity 80.1 %); however, it had the advantage over culture in that it could detect mixed infections of C. coli and C. jejuni and was capable of giving a result for a sample within a day.
Fulminant necrotizing fasciitis due to Vibrio parahaemolyticus
Authors: Tena Daniel , Arias Marta , Álvarez Bárbara Teresa , Mauleón Coro , Jiménez Mari Paz , Bisquert Julia
Published: November 2009

Necrotizing soft-tissue infection due to Vibrio parahaemolyticus is unusual. We report a case of necrotizing fasciitis due to V. parahaemolyticus in a 92-year-old woman with a history of chronic renal failure, diabetes mellitus and malnutrition. Clinical evolution was fulminant and the patient died 6 h after admission. A review of all cases previously reported showed that the infection occurred in patients with underlying diseases through ingestion of raw oysters or inoculation via traumatic injury in marine environments. The mortality rate of all reviewed cases was 42.8 %. In conclusion, V. parahaemolyticus should be considered a possible causative agent of necrotizing fasciitis, especially in patients with underlying disease. Early diagnosis and prompt aggressive debridement associated with antibiotic therapy are essential in order to save the patient's life, because clinical evolution can be fulminant and mortality rates are high.
Potential use of a liposome-encapsulated mixture of lipopolysaccharide core types (R1, R2, R3 and R4) of Escherichia coli in controlling colisepticaemia in chickens
Authors: Dissanayake D. R. Anuruddhika , Wijewardana Thula G. , Gunawardena Gnana A. , Poxton Ian R.
Published: November 2009

Infections caused byEscherichia colihave an economically significant impact on the poultry industry and a non-serotype-specific vaccine appears to be the most logical method of controlling them. The core oligosaccharide-lipid A region of bacterial lipopolysaccharide (LPS) is well conserved and highly immunogenic but toxic. This study determined the possible use of a liposome-encapsulated mixture of rough LPSs of core types R1, R2, R3 and R4 in controlling infections caused byE. coliin chickens. The liposome which encapsulated the LPS consisted of egg phosphatidylcholine, bovine brain phosphatidylserine and cholesterol. As determined byLimulusamoebocyte lysate assay, incorporation of LPS into the liposome reduced the endotoxicity of LPS to 0.7 % of its initial value. When tested on a chicken macrophage cell line (HD11), liposome-incorporated LPS produced a significantly lower amount of nitric oxide (<5 μM) than that produced by free LPS (22 μM). Transcription of the genes for interleukin-1βand inducible nitric oxide synthase was lower in cells treated with liposome-incorporated LPS than in cells treated with free LPS. When chickens were immunized with 0.2 μg, 1 μg and 5 μg liposome-encapsulated mixture of LPS core types, the antibody response increased with increasing dose. When challenged with the virulentE. coliO78 strain, the birds which received 1 μg liposome-encapsulated LPS and 5 μg LPS had significantly lower lesions scores (P<0.05) and high body weight when compared with the birds in the control group as well as with the birds immunized with a suboptimal dose (0.2 μg) of liposome-encapsulated LPS.
Comparison of nasopharyngeal nylon flocked swabs with universal transport medium and rayon-bud swabs with a sponge reservoir of viral transport medium in the diagnosis of paediatric influenza
Authors: Esposito Susanna , Molteni Claudio Giuseppe , Daleno Cristina , Valzano Antonia , Cesati Laura , Gualtieri Laura , Tagliabue Claudia , Bosis Samantha , Principi Nicola
Published: November 2009

This study compared a kit containing a nasopharyngeal nylon flocked swab and a tube with a liquid universal transport medium (UTM) with a kit containing a plastic-shafted rayon-budded swab with a sponge reservoir of viral transport medium for the molecular detection of influenza viruses in children. Respiratory samples were collected from 314 children aged <5 years with influenza-like illness (186 males; mean age 2.32±2.27 years) using both swabs in a randomized sequence for each patient. The flocked swabs permitted the detection of 28 influenza A (8.9 %) and 45 influenza B (14.3 %) cases, and the rayon-bud swabs 26 influenza A (8.3 %) and 43 influenza B (13.7 %) cases, with detection rates of 23.2 and 22.0 %, respectively, and similar cycle threshold values. Paediatricians and laboratory staff were significantly more satisfied with both the simplicity (P <0.0001) and rapidity (P <0.0001) of the nasopharyngeal flocked swabs with UTM. These findings show that the flocked swabs with UTM and the rayon-bud swabs with a sponge transport medium are similarly efficient in preserving influenza virus nucleic acid, but that the kit containing a flocked swab with a UTM allows easier and more rapid collection and processing of specimens.
Visualization of adherent micro-organisms using different techniques
Authors: Hannig Christian , Follo Marie , Hellwig Elmar , Al-Ahmad Ali
Published: November 2009

The visualization and quantification of adherent bacteria is still one of the most relevant topics in microbiology. Besides electron microscopic techniques such as transmission electron microscopy, scanning electron microscopy and environmental scanning electron microscopy, modern fluorescence microscopic approaches based on fluorogenic dyes offer detailed insight into bacterial biofilms. The aim of the present review was to provide an overview of the advantages and disadvantages of different methods for visualization of adherent bacteria with a special focus on the experiences gained in dental research.
Accelerated identification of Staphylococcus aureus from blood cultures by a modified fluorescence in situ hybridization procedure
Authors: Poppert Sven , Riecker Melanie , Wellinghausen Nele , Frickmann Hagen , Essig Andreas
Published: November 2009

This study evaluated fluorescence in situ hybridization (FISH) for rapid identification of Staphylococcus aureus and coagulase-negative staphylococci (CoNS) directly from blood cultures. Initially, 360 blood cultures containing Gram-positive cocci were investigated by a previously described microwave-FISH procedure: 44/49 (89.8 %) S. aureus and 298/299 (99.7 %) CoNS were correctly identified. Because FISH proved useful and reliable but handling was found to be inconvenient, the method was modified by employing a recently developed slide chamber. This reduced the time required from 60 to 30 min. The simplified execution allowed integration of the method into the workflow of a routine laboratory without difficulty. The modified method proved to be highly reliable, identifying 37/37 (100 %) S. aureus and 169/172 (98.2 %) CoNS directly from blood cultures.
Disease burden due to Streptococcus dysgalactiae subsp. equisimilis (group G and C streptococcus) is higher than that due to Streptococcus
pyogenes among Mumbai school children
Authors: Bramhachari Pallaval V. , Kaul Santosh Y. , McMillan David J. , Shaila Melkote S. , Karmarkar Mohan G. , Sriprakash Kadaba S.
Published: November 2009

Streptococcus pyogenes [group A streptococcus (GAS)], a human pathogen, and Streptococcus dysgalactiae subsp. equisimilis [human group G and C streptococcus (GGS/GCS)] are evolutionarily related, share the same tissue niche in humans, exchange genetic material, share up to half of their virulence-associated genes and cause a similar spectrum of diseases. Yet, GGS/GCS is often considered as a commensal bacterium and its role in streptococcal disease burden is under-recognized. While reports of the recovery of GGS/GCS from normally sterile sites are increasing, studies describing GGS/GCS throat colonization rates relative to GAS in the same population are very few. This study was carried out in India where the burden of streptococcal diseases, including rheumatic fever and rheumatic heart disease, is high. As part of a surveillance study, throat swabs were taken from 1504 children attending 7 municipal schools in Mumbai, India, during 2006–2008. GAS and GGS/GCS were identified on the basis of β-haemolytic activity, carbohydrate group and PYR test, and were subsequently typed. The GGS/GCS carriage rate (166/1504, 11 %) was eightfold higher than the GAS carriage (22/1504, 1.5 %) rate in this population. The 166 GGS/GCS isolates collected represented 21 different emm types (molecular types), and the 22 GAS isolates represented 15 different emm types. Although the rate of pharyngitis associated with GGS/GCS is marginally lower than with GAS, high rates of throat colonization by GGS/GCS underscore its importance in the pathogenesis of pharyngitis.
Variation of the factor H-binding protein of Neisseria meningitidis
Authors: Brehony Carina , Wilson Daniel J. , Maiden Martin C. J.
Published: November 2009

There is currently no comprehensive meningococcal vaccine, due to difficulties in immunizing against organisms expressing serogroup B capsules. To address this problem, subcapsular antigens, particularly the outer-membrane proteins (OMPs), are being investigated as candidate vaccine components. If immunogenic, however, such antigens are often antigenically variable, and knowledge of the extent and structuring of this diversity is an essential part of vaccine formulation. Factor H-binding protein (fHbp) is one such protein and is included in two vaccines under development. A survey of the diversity of the fHbp gene and the encoded protein in a representative sample of meningococcal isolates confirmed that variability in this protein is structured into two or three major groups, each with a substantial number of alleles that have some association with meningococcal clonal complexes and serogroups. A unified nomenclature scheme was devised to catalogue this diversity. Analysis of recombination and selection on the allele sequences demonstrated that parts of the gene are subject to positive selection, consistent with immune selection on the protein generating antigenic variation, particularly in the C-terminal region of the peptide sequence. The highest levels of selection were observed in regions corresponding to epitopes recognized by previously described bactericidal monoclonal antibodies.
The novel polysaccharide deacetylase homologue Pdi contributes to virulence of the aquatic pathogen Streptococcus iniae
Authors: Milani Carlo J. E. , Aziz Ramy K. , Locke Jeffrey B. , Dahesh Samira , Nizet Victor , Buchanan John T.
Published: November 2009

The aquatic zoonotic pathogen Streptococcus iniae represents a threat to the worldwide aquaculture industry and poses a risk to humans who handle raw fish. Because little is known about the mechanisms of S. iniae pathogenesis or virulence factors, we established a high-throughput system combining whole-genome pyrosequencing and transposon mutagenesis that allowed us to identify virulence proteins, including Pdi, the polysaccharide deacetylase of S. iniae, that we describe here. Using bioinformatics tools, we identified a highly conserved signature motif in Pdi that is also conserved in the peptidoglycan deacetylase PgdA protein family. A Δpdi mutant was attenuated for virulence in the hybrid striped bass model and for survival in whole fish blood. Moreover, Pdi was found to promote bacterial resistance to lysozyme killing and the ability to adhere to and invade epithelial cells. On the other hand, there was no difference in the autolytic potential, resistance to oxidative killing or resistance to cationic antimicrobial peptides between S. iniae wild-type and Δpdi. In conclusion, we have demonstrated that pdi is involved in S. iniae adherence and invasion, lysozyme resistance and survival in fish blood, and have shown that pdi plays a role in the pathogenesis of S. iniae. Identification of Pdi and other S. iniae virulence proteins is a necessary initial step towards the development of appropriate preventive and therapeutic measures against diseases and economic losses caused by this pathogen.
FLO11 gene length and transcriptional level affect biofilm-forming ability of wild flor strains of Saccharomyces cerevisiae
Authors: Zara Giacomo , Zara Severino , Pinna Claudia , Marceddu Salvatore , Budroni Marilena
Published: November 2009

InSaccharomyces cerevisiae,FLO11encodes an adhesin that is associated with different phenotypes, such as adherence to solid surfaces, hydrophobicity, mat and air–liquid biofilm formation. In the present study, we analysedFLO11allelic polymorphisms andFLO11-associated phenotypes of 20 flor strains. We identified 13 alleles of different lengths, varying from 3.0 to 6.1 kb, thus demonstrating thatFLO11is highly polymorphic. Two alleles of 3.1 and 5.0 kb were cloned into strain BY4742 to compare theFLO11-associated phenotypes in the same genetic background. We show that there is a significant correlation between biofilm-forming ability andFLO11length both in different and in the same genetic backgrounds. Moreover, we propose a multiple regression model that allows prediction of air–liquid biofilm-forming ability on the basis of transcription levels and lengths ofFLO11alleles in a population ofS. cerevisiaeflor strains. Considering that transcriptional differences are only partially explained by the differences in the promoter sequences, our results are consistent with the hypothesis thatFLO11transcription levels are strongly influenced by genetic background and affect biofilm-forming ability.
Effect of subinhibitory concentrations of benzalkonium chloride on the competitiveness of Pseudomonas aeruginosa grown in continuous culture
Authors: Mc Cay Paul H. , Ocampo-Sosa Alain A. , Fleming Gerard T. A.
Published: November 2009

This study investigates the link between adaptation to biocides and antibiotics in Pseudomonas aeruginosa. An enrichment continuous culture of P. aeruginosa NCIMB 10421 (MIC 25 mg BKC l−1) was operated (D=0.04 h−1, 792 h) with added benzalkonium chloride (BKC). A derivative, PA-29 (696 h), demonstrated a >12-fold decrease in sensitivity to the biocide (MIC >350 mg BKC l−1). The variant demonstrated a 256-fold increase in resistance to ciprofloxacin, with a mutation in the gyrA gene (Thr-83→Ile). Similarly, culturing of the original strain in a continuous-culture system with ciprofloxacin selection pressure led to the evolution of BKC-adapted populations (MIC 100 mg BKC l−1). Efflux pump activity predominantly contributed to the developed phenotype of PA-29. An amino acid substitution (Val-51→Ala) in nfxB, the Mex efflux system regulator gene, was observed for PA-29. Overexpression of both MexAB-OprM and MexCD-OprJ was recorded for PA-29. Similarly, mexR, a repressor of the Mex system, was downregulated. Competition studies were carried out in continuous culture between PA-29 and the original strain (in the presence of subinhibitory concentrations of BKC). The outcome of competition was influenced by the concentration of biocide used and the nature of limiting nutrient. The inclusion of 1 mg BKC l−1 in the medium feed was sufficient to select (S=0.011) for the BKC-adapted strain in magnesium-limited culture. Conversely, the presence of 10 mg BKC l−1 in the medium supply was insufficient to select for the same organism (S=−0.017) in the glucose-limited culture. These results indicate the importance of environmental conditions on selection and maintenance of biocide adaptation.
Correlation between TCA cycle flux and glucose uptake rate during respiro-fermentative growth of Saccharomyces cerevisiae
Authors: Heyland Jan , Fu Jianan , Blank Lars M.
Published: November 2009

Glucose repression of the tricarboxylic acid (TCA) cycle inSaccharomyces cerevisiaewas investigated under different environmental conditions using13C-tracer experiments. Real-time quantification of the volatile metabolites ethanol and CO2allowed accurate carbon balancing. In all experiments with the wild-type, a strong correlation between the rates of growth and glucose uptake was observed, indicating a constant yield of biomass. In contrast, glycerol and acetate production rates were less dependent on the rate of glucose uptake, but were affected by environmental conditions. The glycerol production rate was highest during growth in high-osmolarity medium (2.9 mmol g−1h−1), while the highest acetate production rate of 2.1 mmol g−1h−1was observed in alkaline medium of pH 6.9. Under standard growth conditions (25 g glucose l−1, pH 5.0, 30 °C)S. cerevisiaehad low fluxes through the pentose phosphate pathway and the TCA cycle. A significant increase in TCA cycle activity from 0.03 mmol g−1h−1to about 1.7 mmol g−1h−1was observed whenS. cerevisiaegrew more slowly as a result of environmental perturbations, including unfavourable pH values and sodium chloride stress. Compared to experiments with high glucose uptake rates, the ratio of CO2to ethanol increased more than 50 %, indicating an increase in flux through the TCA cycle. Although glycolysis and the ethanol production pathway still exhibited the highest fluxes, the net flux through the TCA cycle increased significantly with decreasing glucose uptake rates. Results from experiments with single gene deletion mutants partially impaired in glucose repression (hxk2,grr1) indicated that the rate of glucose uptake correlates with this increase in TCA cycle flux. These findings are discussed in the context of regulation of glucose repression.
Attraction of Brachyspira pilosicoli to mucin
Authors: Naresh Ram , Hampson David J.
Published: November 2009

The anaerobic intestinal spirochaete Brachyspira pilosicoli colonizes the large intestine of various species, including humans. In the colon this spirochaete can penetrate the overlying mucus layer, attach by one cell end to the underlying enterocytes, and initiate localized colitis and diarrhoea. The aim of this study was to investigate whether, as part of the colonization process, B. pilosicoli is attracted to mucin. Fifteen B. pilosicoli strains isolated from humans, pigs, chickens and dogs, and a control strain of Brachyspira hyodysenteriae, were analysed for their ability to enter solutions of hog gastric mucin in an in vitro capillary tube assay. No significant attraction was detected with 1 % mucin, but some strains started to enter a 2 % solution, and attraction then increased with increasing concentrations to peak at around 6–8 % mucin. A similar increase was seen with B. hyodysenteriae, although this activity peaked at 6 % mucin and then declined, suggesting that the two species have different affinities for mucin. These mucin concentrations were much higher than those used in previous experimental studies with Brachyspira species. The viscosities of the 6–8 % mucin solutions were around 7–12 mPa s, which were similar to the measured viscosities of the mucus layer overlying the epithelium in the caecum and colon of experimental pigs. The strains varied in their motility, as assessed by their ability to enter tubes containing chemotaxis buffer, but there was no significant relationship between this motility and the extent of their ability to enter the mucin solutions. Different strains also had different propensities to enter the mucin solutions, but there were no consistent differences according to the host species of origin. B. pilosicoli strain 95/1000 was attracted towards a solution of d-serine, suggesting that chemotaxis was involved in the attraction to mucin; however, 95/1000 was also attracted to viscous solutions of polyvinylpyrrolidone (PVP), in a manner mirroring the response to mucin, and hence suggesting the involvement of viscotaxis in the attraction to mucin. B. hyodysenteriae B204 showed a similar viscotaxis to PVP. Further studies are required to determine whether the in vitro interaction of a given strain with mucin is a useful indicator of its in vivo colonization ability, and hence could be used as a potential marker for virulence.
Molecular typing of Mycobacterium intracellulare using multilocus variable-number of tandem-repeat analysis: identification of loci and analysis of clinical isolates
Authors: Ichikawa Kazuya , Yagi Tetsuya , Inagaki Takayuki , Moriyama Makoto , Nakagawa Taku , Uchiya Kei-ichi , Nikai Toshiaki , Ogawa Kenji
Published: November 2009

In addition to its known status as a disseminated disease in HIV-positive patients, Mycobacterium avium complex (MAC) is increasingly recognized as a causative pathogen of respiratory disease in HIV-negative patients. MAC is divided into Mycobacterium avium, and the less-epidemiologically studied Mycobacterium intracellulare. Genetic typing for M. intracellulare using variable number of tandem repeats (VNTR) has not yet been developed. The aim of this study was to identify VNTR loci in the genome of M. intracellulare and apply them as an epidemiological tool to clinical isolates. Here, we identified 25 VNTR loci on the M. intracellulare genome, of which 16 showed variations among clinical isolates in the number of tandem repeat motifs. Among the 74 M. intracellulare isolates, 50 genotypes were distinguished using the 16 VNTR loci, resulting in a Hunter Gaston's discriminatory index of 0.988. Moreover, all 16 VNTR loci were stable in different sets of isolates recovered within time intervals ranging from 2 to 1551 days from 14 separate patients. These results indicate that for use as epidemiological markers of M. intracellulare, the loci in this VNTR assay are highly discriminating and stable over time.
Functional analyses of PAU genes in Saccharomyces cerevisiae
Authors: Luo Zongli , van Vuuren Hennie J. J.
Published: November 2009

PAUgenes constitute the largest gene family inSaccharomyces cerevisiae, with 24 members mostly located in the subtelomeric regions of chromosomes. Little information is available aboutPAUgenes, other than expression data for some members. In this study, we systematically compared the sequences of all 24 members, examined the expression ofPAU3,PAU5,DAN2,PAU17andPAU20in response to stresses, and investigated the stability of all Pau proteins. The chromosomal localization, synteny and sequence analyses revealed thatPAUgenes could have been amplified by segmental and retroposition duplication through mechanisms of chromosomal end translocation and Ty-associated recombination. The coding sequences diverged through nucleotide substitution and insertion/deletion of one to four codons, thus causing changes in amino acids, truncation or extension of Pau proteins. Pairwise comparison of non-coding regions revealed little homology in flanking sequences of some members. All 24PAUpromoters contain a TATA box, and 22PAUpromoters contain at least one copy of the anaerobic response element and the aerobic repression motif. Differential expression was observed amongPAU3,PAU5,PAU17,PAU20andDAN2in response to stress, withPAU5having the highest capacity to be induced by anaerobic conditions, low temperature and wine fermentations. Furthermore, Pau proteins with 124 aa were less stable than those with 120 or 122 aa. Our results indicate that duplicatedPAUgenes have been evolving, and the individual Pau proteins might possess specific roles for the adaptation ofS. cerevisiaeto certain environmental stresses.
Deletion of Mid1, a putative stretch-activated calcium channel in Claviceps purpurea, affects vegetative growth, cell wall synthesis and virulence
Authors: Bormann Jörg , Tudzynski Paul
Published: November 2009

The putativeClaviceps purpureahomologue of theSaccharomyces cerevisiaestretch-activated calcium ion channel Mid1 was investigated for its role in vegetative growth, differentiation and pathogenicity on rye (Secale cereale). Gene replacement mutants ofCl. purpurea mid1were not affected in polar growth and branching in axenic culture but showed a significantly reduced growth rate. The growth defect could not be complemented by Ca2+supplementation, in contrast tomid1mutants in yeast, but the altered sensitivity of the mutants to changes in external and internal Ca2+concentrations indicates some role of Mid1 in Ca2+homeostasis. The major effect ofmid1deletion, however, was the complete loss of virulence: infected rye plants showed no disease symptoms at all. Detailed analyses ofin vitro-infected rye ovaries demonstrated that the Δmid1mutants had multiple apical branches and were unable to infect the host tissue, suggesting that Mid1 is essential for generating the necessary mechanical force for penetration. This is believed to be the first report of an essential role for a Mid1 homologue in the virulence of a plant-pathogenic fungus.
Role of PvdQ in Pseudomonas aeruginosa virulence under iron-limiting conditions
Authors: Jimenez Pol Nadal , Koch Gudrun , Papaioannou Evelina , Wahjudi Mariana , Krzeslak Joanna , Coenye Tom , Cool Robbert H. , Quax Wim J.
Published: November 2009

PvdQ, an acylase fromPseudomonas aeruginosaPAO1, has been shown to have at least two functions. It can act as a quorum quencher due to its ability to degrade long-chainN-acylhomoserine lactones (AHLs), e.g. 3-oxo-C12-HSL, leading to a decrease in virulence factors. In addition, PvdQ is involved in iron homeostasis by playing a role in the biosynthesis of pyoverdine, the major siderophore ofP. aeruginosa. In accordance with earlier studies on RNA level, we could show at the protein level that PvdQ is only expressed when iron is present at very low concentrations. We therefore set out to investigate the two functions of PvdQ under iron-limiting conditions. Gene deletion ofpvdQdoes not affect growth ofP. aeruginosabut abrogates pyoverdine production, and results in an accumulation of 3-oxo-C12-HSL. Phenotypic analyses of our ΔpvdQmutant at low iron concentrations revealed that this mutant is impaired in swarming motility and biofilm formation. Additionally, a plant and aCaenorhabditis elegansinfection model demonstrated that the deletion ofpvdQresulted in reduced virulence. None of the phenotypes in the present study could be linked to the presence or absence of AHLs. These results clearly indicate that under iron-limiting conditions PvdQ plays a major role in swarming motility, in biofilm development and in infection that is more likely to be linked to the pyoverdine pathway rather than the LasI/LasR/3-oxo-C12-HSL quorum-sensing circuit.
Transcriptional upregulation of four genes of the lysine biosynthetic pathway by homocitrate accumulation in Penicillium chrysogenum: homocitrate as a sensor of lysine-pathway distress
Authors: Teves Franco , Lamas-Maceiras Mónica , García-Estrada Carlos , Casqueiro Javier , Naranjo Leopoldo , Ullán Ricardo V. , Scervino José-Martín , Wu Xiaobin , Velasco-Conde Tania , Martín Juan F.
Published: November 2009

The lysine biosynthetic pathway has to supply large amounts ofα-aminoadipic acid for penicillin biosynthesis inPenicillium chrysogenum. In this study, we have characterized theP. chrysogenumL2 mutant, a lysine auxotroph that shows highly increased expression of several lysine biosynthesis genes (lys1,lys2,lys3,lys7). The L2 mutant was found to be deficient in homoaconitase activity since it was complemented by theAspergillus nidulans lysFgene. We have cloned a gene (namedlys3) that complements the L2 mutation by transformation with aP. chrysogenumgenomic library, constructed in an autonomous replicating plasmid. Thelys3-encoded protein showed high identity to homoaconitases. In addition, we cloned the mutantlys3allele from the L2 strain that showed a G1534to A1534point mutation resulting in a Gly495to Asp495substitution. This mutation is located in a highly conserved region adjacent to two of the three cysteine residues that act as ligands to bind the iron–sulfur cluster required for homoaconitase activity. The L2 mutant accumulates homocitrate. Deletion of thelys1gene (homocitrate synthase) in the L2 strain prevented homocitrate accumulation and reverted expression levels of the four lysine biosynthesis genes tested to those of the parental prototrophic strain. Homocitrate accumulation seems to act as a sensor of lysine-pathway distress, triggering overexpression of four of the lysine biosynthesis genes.
Genetic diversity and stability of the porA allele as a genetic marker in human Campylobacter infection
Authors: Cody A. J. , Maiden M. J. C. , Dingle K. E.
Published: November 2009

The major outer-membrane protein (MOMP) of Campylobacter jejuni and Campylobacter coli, encoded by the porA gene, is extremely genetically diverse. Conformational MOMP epitopes are important in host immunity, and variation in surface-exposed regions probably occurs as a result of positive immune selection during infection. porA diversity has been exploited in genotyping studies using highly discriminatory nucleotide sequences to identify potentially epidemiologically linked cases of human campylobacteriosis. To understand the overall nature and extent of porA diversity and stability in C. jejuni and C. coli we investigated sequences in isolates (n=584) obtained from a defined human population (approx. 600 000) over a defined time period (1 year). A total of 196 distinct porA variants were identified. Regions encoding putative extracellular loops were the most variable in both nucleotide sequence and length. Phylogenetic analysis identified three porA allele clusters that originated in (i) predominantly C. jejuni and a few C. coli, (ii) solely C. jejuni or (iii) predominantly C. coli and a few C. jejuni. The stability of porA within an individual human host was investigated using isolates cultured longitudinally from 64 sporadic cases, 27 of which had prolonged infection lasting between 5 and 98 days (the remainder having illness of normal duration, 0–4 days), and 20 cases from family outbreaks. Evidence of mutation was detected in two patients with prolonged illness. Despite demonstrable positive immune selection in these two unusual cases, the persistence of numerous variants within the population indicated that the porA allele is a valuable tool for use in extended typing schemes.
Genome-wide analysis of maltose utilization and regulation in aspergilli
Authors: Vongsangnak Wanwipa , Salazar Margarita , Hansen Kim , Nielsen Jens
Published: November 2009

Maltose utilization and regulation in aspergilli is of great importance for cellular physiology and industrial fermentation processes. InAspergillus oryzae, maltose utilization requires a functionalMALlocus, composed of three genes:MALRencoding a regulatory protein,MALTencoding maltose permease andMALSencoding maltase. Through a comparative genome and transcriptome analysis we show that theMALregulon system is active inA. oryzaewhile it is not present inAspergillus niger. In order to utilize maltose,A. nigerrequires a different regulatory system that involves the AmyR regulator for glucoamylase (glaA) induction. Analysis of reporter metabolites and subnetworks illustrates the major route of maltose transport and metabolism inA. oryzae. This demonstrates that overall metabolic responses ofA. oryzaeoccur in terms of genes, enzymes and metabolites when the carbon source is altered. Although the knowledge of maltose transport and metabolism is far from being complete inAspergillusspp., our study not only helps to understand the sugar preference in industrial fermentation processes, but also indicates how maltose affects gene expression and overall metabolism.
Purification and characterization of a clostripain-like protease from a recombinant Clostridium perfringens culture
Authors: Manabe Sadao , Nariya Hirofumi , Miyata Shigeru , Tanaka Hiroaki , Minami Junzaburo , Suzuki Motoo , Taniguchi Yuki , Okabe Akinobu
Published: November 2009

Clostridium perfringensproduces a homologue of clostripain (Clo), the arginine-specific endopeptidase ofClostridium histolyticum. To determine the biochemical and biological properties of theC. perfringenshomologue (Clp), it was purified from the culture supernatant of a recombinantC. perfringensstrain by cation-exchange chromatography and ultrafiltration. Analysis by SDS-PAGE, N-terminal amino acid sequencing and TOF mass spectrometry revealed that Clp consists of two polypeptides comprising heavy (38 kDa) and light (16 kDa or 15 kDa) chains, and that the two light chains differ in the N-terminal cleavage site. This difference in the light chain did not affect the enzymic activity towardN-benzoyl-l-argininep-nitroanilide (Bz-l-arginine pNA), as demonstrated by assaying culture supernatants differing in the relative ratio of the two light chains. Although the purified Clp preferentially degraded Bz-dl-arginine pNA rather than Bz-dl-lysine pNA, it degraded the latter more efficiently than did Clo. Clp showed 2.3-fold higher caseinolytic activity than Clo, as expected from the difference in substrate specificity. Clp caused an increase in vascular permeability when injected intradermally into mice, implying a possible role of Clp in the pathogenesis of clostridial myonecrosis.
Methylcitrate cycle activation during adaptation of Fusarium solani and Fusarium verticillioides to propionyl-CoA-generating carbon sources
Authors: Domin Nicole , Wilson Duncan , Brock Matthias
Published: November 2009

Propionyl-CoA is an inhibitor of both primary and secondary metabolism inAspergillusspecies and a functional methylcitrate cycle is essential for the efficient removal of this potentially toxic metabolite. Although the genomes of most sequenced fungal species appear to contain genes coding for enzymes of the methylcitrate cycle, experimental confirmation of pathway activity in filamentous fungi has only been provided forAspergillus nidulansandAspergillus fumigatus. In this study we demonstrate that pathogenicFusariumspecies also possess a functional methylcitrate cycle.Fusarium solaniappears highly adapted to saprophytic growth as it utilized propionate with high efficiency, whereasFusarium verticillioidesgrew poorly on this carbon source. In order to elucidate the mechanisms of propionyl-CoA detoxification, we first identified the genes coding for methylcitrate synthase from both species. Despite sharing 96 % amino acid sequence identity, analysis of the two purified enzymes demonstrated that their biochemical properties differed in several respects. Both methylcitrate synthases exhibited lowKmvalues for propionyl-CoA, but that ofF. verticillioidesdisplayed significantly higher citrate synthase activity and greater thermal stability. Activity determinations from cell-free extracts ofF. solanirevealed a strong methylcitrate synthase activity during growth on propionate and to a lesser extent on Casamino acids, whereas activity byF. verticillioideswas highest on Casamino acids. Further phenotypic analysis confirmed that these biochemical differences were reflected in the different growth behaviour of the two species on propionyl-CoA-generating carbon sources.
The Azospirillum brasilense Sp7 noeJ and noeL genes are involved in extracellular polysaccharide biosynthesis
Authors: Lerner Anat , Castro-Sowinski Susana , Valverde Angel , Lerner Hadas , Dror Rachel , Okon Yaacov , Burdman Saul
Published: November 2009

Azospirillum brasilenseis a plant root-colonizing bacterium that exerts beneficial effects on the growth of many agricultural crops. Extracellular polysaccharides of the bacterium play an important role in its interactions with plant roots. The pRhico plasmid ofA. brasilenseSp7, also named p90, carries several genes involved in synthesis and export of cell surface polysaccharides. We generated two Sp7 mutants impaired in two pRhico-located genes,noeJandnoeL, encoding mannose-6-phosphate isomerase and GDP-mannose 4,6-dehydratase, respectively. Our results demonstrate that inA. brasilenseSp7,noeJandnoeLare involved in lipopolysaccharide and exopolysaccharide synthesis.noeJandnoeLmutant strains were significantly altered in their outer membrane and cytoplasmic/periplasmic protein profiles relative to the wild-type strain. Moreover, bothnoeJandnoeLmutations significantly affected the bacterial responses to several stresses and antimicrobial compounds. Disruption ofnoeL, but notnoeJ, affected the ability of theA. brasilenseSp7 to form biofilms. The pleiotropic alterations observed in the mutants could be due, at least partially, to their altered lipopolysaccharides and exopolysaccharides relative to the wild-type.