Listerial brainstem encephalitis--treatable, but easily missed.

Listerial brainstem encephalitis (LBE) is an uncommon form of listerial central nervous system infection that progresses rapidly and is invariably fatal unless detected and treated early. We report on six adult patients with LBE, of whom five were managed or co-managed by our unit during the period January - June 2012. All presented with a short prodromal illness followed by a combination of brainstem signs, including multiple cranial nerve palsies with emphasis on the lower cranial nerves, ataxia, motor and sensory long-tract signs, a depressed level of consciousness and apnoea. In two cases the diagnosis was delayed with adverse outcomes. LBE may be difficult to diagnose: clinicians may not be aware of this condition, the brainstem location may not be recognised readily, general markers of inflammation such as the erythrocyte sedimentation rate, C-reactive protein level or white cell count may be normal, and the cerebrospinal fluid is typically normal or there are only mild and nonspecific findings. Serological tests are unreliable, and diagnosis is achieved through blood cultures, magnetic resonance imaging and clinical recognition.

Guideline for the diagnosis and management of multiple sclerosis: a Southern African perspective.

Before making a diagnosis of multiple sclerosis (MS), it is imperative that alternative diagnoses are considered and excluded. This is particularly important in South Africa, which is a moderate prevalence MS area, has a high burden of neurological infections and where the majority of the people are black - an ethnic group that has a very low frequency of MS. Before applying diagnostic criteria, there should be no better explanation for the patient's presentation. This guideline, written on behalf of the Multiple Sclerosis Society of South Africa, aims to assist in the diagnosis and treatment of MS in Southern Africa. 

Detection and analysis of bovine rotavirus strains circulating in Australian calves during 2004 and 2005.

Bovine rotavirus (BRV) has been detected in both dairy and beef cattle herds worldwide. Stool samples collected from calves in the Gippsland region of Victoria, Australia were screened to determine the presence of BRV. A total of 100 faecal samples were collected from calves with and without diarrhoea across three farms during 2004 and 2005. Group A BRV was detected in 26% of faecal samples (22 from diarrheic calves and four from asymptomatic calves). Genotyping analysis of rotavirus positive samples indicated that G6P[5] was the most prevalent genotype (38.5%) followed by G6P[5+11] (15.4%). G10P[11] and G6+G10P[5] were each detected at a rate of 7.7%, and G6+G10P[11] was found in a single sample (3.8%). Seven samples (26.9%) could not be G and/or P typed. Thirty percent of the BRV positive samples were mixed infections, indicating that individual calves were co-infected with more than one strain of rotavirus. The G6P[5] strains exhibited high VP7 identity (>97% amino acid identity) with B-60, a G6 strain identified in Victorian calves during 1988. A G10P[11] isolate was closely related (>97% amino acid identity in VP7 and VP4 proteins) to a Victorian G10P[11] strain (B-11) also identified during 1988. This study demonstrates that BRV is a contributing pathogen to diarrhoeal disease in Victorian calves, with sequence analysis suggesting long-term conservation of the VP7 protein over a 16-year period.

Proteomic analysis of lactose-starved Lactobacillus casei during stationary growth phase.

AIMS: Starvation stress is a condition that nonstarter lactic acid bacteria (NSLAB) normally encounter. This study was aimed to investigate starvation-induced proteins in Lactobacillus casei during stationary growth phase. METHODS AND RESULTS: The impact of carbohydrate starvation on L. casei GCRL163 was investigated using two different media (a modified de Man, Rogosa and Sharpe broth and a semi-defined medium). Cells were grown in the presence of excess lactose (1%) or starvation (0%) and differences in the patterns of one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional electrophoresis of the cytosolic protein fractions were investigated. Differentially regulated proteins were identified by MALDI-TOF/TOF mass spectrometry. Many differentially regulated proteins were enzymes of various metabolic pathways involved in carbohydrate metabolism to yield energy. Differences in protein expression were also observed in the two culture conditions tested in this experiment. CONCLUSION: Numerous glycolytic enzymes were differentially regulated under lactose starvation. The differential expression of these glycolytic enzymes suggests a potential survival strategy under harsh growth conditions (i.e. lactose starvation). SIGNIFICANCE AND IMPACT OF THE STUDY: This paper reports improved understanding of stress responses and survival mechanism of NSLAB under lactose-depleted cheese-ripening condition. This knowledge of how NSLAB bacteria adapt to lactose starvation could be applied to predict the performances of bacteria in other industrial applications.

Genetic polymorphisms of beta2- and beta3-adrenergic receptor genes associated with characteristics of the metabolic syndrome in black South African women.

BACKGROUND: Genetic variation in the beta2 (ADRB2) and beta3 (ADRB3) adrenergic receptor genes are associated with obesity and insulin resistance. To further elucidate the role of these genes in the pathophysiology of obesity the present study investigated associations between certain polymorphisms in ADRB2 and ADRB3 and parameters of carbohydrate and lipid metabolism in a population of African origin. MATERIAL AND METHODS: Data of 102 black South African women obtained in the POWIRS (Profile of Obese Women with the Insulin Resistance Syndrome) study were used. Endpoint measurements included several anthropometric variables, resting blood pressure, plasma glucose, insulin, free fatty acids (FFA), ghrelin, leptin and lipids, and insulin resistance as estimated by the homeostasis model assessment (HOMA-IR) index. Polymorphisms were analyzed via PCR based methods. RESULTS: The percentage body fat was significantly lower (p< or =0.05) and the FFA significantly higher (p< or =0.05) in lean subjects (BMI< or =25 kg/m2) with the Glu27 variant allele compared to subjects with the Gln27 wildtype allele of the ADRB2 gene. In contrast, the variant allele of the ADRB2 gene was significantly positive associated (p< or =0.05) with the HOMA-IR-index in overweight black African women (BMI>25 kg/m2). No significant differences in parameters of the metabolic syndrome were apparent between subjects with the wildtype and variant alleles in the ADRB3 gene. CONCLUSION: The presence of the Glu27 and Arg64 polymorphisms of the ADRB2 and ADRB3 genes are not directly related to indices of the metabolic syndrome.

Metabolite repression inhibits degradation of benzo[a]pyrene and dibenz[a,h]anthracene by Stenotrophomonas maltophilia VUN 10,003.

Large inocula of Stenotrophomonas maltophilia VUN 10,003 were used to investigate bacterial degradation of benzo[a]pyrene and dibenz[a,h]anthracene. Although strain VUN 10,003 was capable of degrading 10-15 mg (-1) of the five-ring compounds in the presence of pyrene after 63 days, further addition of pyrene after degradation of the five-ring polycyclic aromatic hydrocarbons (PAHs) ceased did not stimulate significant decreases in the concentration of benzo[a]pyrene or dibenz[a,h]anthracene. However, pyrene was degraded to undetectable levels 21 days after its addition. The amount of benzo[a]pyrene and dibenz[a,h]anthracene degraded by strain VUN 10,003 was not affected by the initial concentration of the compounds when tested at 25-100 mg l(-1), by the accumulation of by-products from pyrene catabolism or a loss of ability by the cells to catabolise benzo[a]pyrene or dibenz[a,h]anthracene. Metabolite or by-product repression was suspected to be responsible for the inhibition: By-products from the degradation of the five-ring compounds inhibited their further degradation.

Microbial degradation and detoxification of high molecular weight polycyclic aromatic hydrocarbons by Stenotrophomonas maltophilia strain VUN 10,003.

The ability of Stenotrophomonas maltophilia strain VUN 10,003 to degrade and detoxify high molecular weight polycyclic aromatic hydrocarbons (PAHs) was evaluated in a basal liquid medium. Using high cell density inocula of strain VUN 10,003, the concentration of pyrene, fluoranthene, benz[a]anthracene, benzo[a]pyrene, dibenz[a, h]anthracene and coronene decreased by 98, 45, 26, 22, 22 and 55% over periods ranging from 5 to 42 d. When a PAH mixture containing three- to seven-ring compounds was used, degradation of both low and high molecular weight compounds occurred concurrently. Mutagenicity assays (Ames Test) demonstrated a decrease in the mutagenic potential of dichloromethane culture extracts from all cultures containing single PAH over the incubation period, corresponding to the decrease in the concentration of the PAH. These observations indicate that strain VUN 10,003 could be used for the detoxification of PAH-contaminated wastes.

Degradation and mineralization of high-molecular-weight polycyclic aromatic hydrocarbons by defined fungal-bacterial cocultures.

This study investigated the biodegradation of high-molecular-weight polycyclic aromatic hydrocarbons (PAHs) in liquid media and soil by bacteria (Stenotrophomonas maltophilia VUN 10,010 and bacterial consortium VUN 10,009) and a fungus (Penicillium janthinellum VUO 10, 201) that were isolated from separate creosote- and manufactured-gas plant-contaminated soils. The bacteria could use pyrene as their sole carbon and energy source in a basal salts medium (BSM) and mineralized significant amounts of benzo[a]pyrene cometabolically when pyrene was also present in BSM. P. janthinellum VUO 10,201 could not utilize any high-molecular-weight PAH as sole carbon and energy source but could partially degrade these if cultured in a nutrient broth. Although small amounts of chrysene, benz[a]anthracene, benzo[a]pyrene, and dibenz[a,h]anthracene were degraded by axenic cultures of these isolates in BSM containing a single PAH, such conditions did not support significant microbial growth or PAH mineralization. However, significant degradation of, and microbial growth on, pyrene, chrysene, benz[a]anthracene, benzo[a]pyrene, and dibenz[a,h]anthracene, each as a single PAH in BSM, occurred when P. janthinellum VUO 10,201 and either bacterial consortium VUN 10,009 or S. maltophilia VUN 10,010 were combined in the one culture, i.e., fungal-bacterial cocultures: 25% of the benzo[a]pyrene was mineralized to CO(2) by these cocultures over 49 days, accompanied by transient accumulation and disappearance of intermediates detected by high-pressure liquid chromatography. Inoculation of fungal-bacterial cocultures into PAH-contaminated soil resulted in significantly improved degradation of high-molecular-weight PAHs, benzo[a]pyrene mineralization (53% of added [(14)C]benzo[a]pyrene was recovered as (14)CO(2) in 100 days), and reduction in the mutagenicity of organic soil extracts, compared with the indigenous microbes and soil amended with only axenic inocula.

Detection of heavy metal ion resistance genes in gram-positive and gram-negative bacteria isolated from a lead-contaminated site.

Resistance to a range of heavy metal ions was determined for lead-resistant and other bacteria which had been isolated from a battery-manufacturing site contaminated with high concentration of lead. Several Gram-positive (belonging to the genera Arthrobacter and Corynebacterium) and Gram-negative (Alcaligenes species) isolates were resistant to lead, mercury, cadmium, cobalt, zinc and copper, although the levels of resistance to the different metal ions were specific for each isolate. Polymerase chain reaction, DNA-DNA hybridization and DNA sequencing were used to explore the nature of genetic systems responsible for the metal resistance in eight of the isolates. Specific DNA sequences could be amplified from the genomic DNA of all the isolates using primers for sections of the mer (mercury resistance determinant on the transposon Tn501) and pco (copper resistance determinant on the plasmid pRJ1004) genetic systems. Positive hybridizations with mer and pco probes indicated that the amplified segments were highly homologous to these genes. Some of the PCR products were cloned and partially sequenced, and the regions sequenced were highly homologous to the appropriate regions of the mer and pco determinants. These results demonstrate the wide distribution of mercury and copper resistance genes in both Gram-positive and Gram-negative isolates obtained from this lead-contaminated soil. In contrast, the czc (cobalt, zinc and cadmium resistance) and chr (chromate resistance) genes could not be amplified from DNAs of some isolates, indicating the limited contribution, if any, of these genetic systems to the metal ion resistance of these isolates.

Analysis of nucleotide methylation in DNA from Corynebacterium glutamicum and related species.

Plasmid DNA (pCSL17) isolated from Corynebacterium glutamicum transformed recipient McrBC+ strains of Escherichia coli with lower efficiency than McrBC- strains, confirming a previous report by Tauch et al. (FEMS Microbiol. Lett. 123 (1994) 343-348) which inferred that C. glutamicum DNA contains methylcytidine. Analysis of nucleotides in C. glutamicum-derived chromosomal and plasmid DNA failed to detect significant levels of methylated adenosine, but methylated cytidine was readily detected. Restriction enzymes which are inhibited by the presence of methylcytidine in their recognition sequence failed to cut pCSL17 from C. glutamicum, whereas enzymes which require methylation at adenosine in GATC sequences failed to cut. Failure of HaeIII to cut two specific sites of C. glutamicum-derived pCSL17 identified the first cytidine in the sequence GGCCGC as one target of methylation in this species, which contains the methyltransferase recognition sequence. Although Brevibacterium lactofermentum-derived DNA showed a similar methylation pattern by HPLC analysis, HaeIII cleaved these GGCCGC sites, suggesting differences in the specificity of methylation between these two species. Results for all analyses of B. flavum DNA were identical to those for C. glutamicum.

Partial purification and characterization of two enzymes involved in isovaleric acid synthesis in Clostridium bifermentans.

Conversion of leucine to isovaleric acid by Clostridium bifermentans is achieved by the action of at least two enzymes. One is a transaminase producing alpha-ketoisocaproic acid, which was purified 30-fold from osmotic lysates of late-exponential phase cells by repeated chromatography on DEAE-Sepharose C16B and Sephacryl S300: this represented a 147-fold purification of activity found in sonically disrupted cells. This enzyme had an apparent molecular weight of approximately 190000 and was composed of six identically sized sub-units (molecular weight 31000 +/- 1000). Transamination required pyridoxal phosphate and pyruvate and was optimal at pH 8.6; the apparent Km for leucine was 7.0 mM. Activity was totally inhibited by 1 mM-p-chloromercuribenzoate and partially inhibited by other thiol reagents. The second enzyme decarboxylated alpha-ketoisocaproic acid to form isovaleric acid and was also partially purified by chromatography on DEAE-Sepharose C16B and Sephacryl S300. It has an apparent molecular weight of 240000 and required FAD and coenzyme A for activity; the Km for alpha-ketoisocaproic acid was 4.2 mM and activity was optimal around pH 8.0. This enzyme was a flavoprotein with absorption maxima at 280, 320 and 400 nm, and a fluorescent maximum at 500 nm. The prosthetic group, FAD, dissociated from the protein during purification resulting in an inactive apoenzyme which was only partially re-activated by FAD. Activity was completely inhibited by several thiol reagents tested at 1 mM.

Characterization of latent and active forms of cartilage proteinases produced by normal immature rabbit articular cartilage in tissue culture.

Cultured tissue slices from normal immature rabbit articular cartilage released latent neutral metalloproteinases into serum-free medium. On activation with 4-aminophenylmercuric acetate, these metalloproteinases could degrade collagen, proteoglycan, and gelatin. Also produced were an acid proteinase with the properties of cathepsin D and an inhibitor of the neutral metalloproteinases. The appearance of both the proteinases and the inhibitor in the culture medium could be prevented by incubation of cultures with cycloheximide. The active and latent forms of the proteinases were characterized using Ultrogel AcA 54 chromatography.

Subcellular location of enzymes involved in leucine dissimilation in Clostridium bifermentans.

Conversion of leucine to isovaleric (iV) and isocaproic (iC) acids by cell-free extracts of Clostridium bifermentans was demonstrated using two lysis procedures. Sonication resulted in an extract which had the enzymes to convert leucine to alpha-ketoisocaproic acid (alpha-kiC) and thence iV, but failed to produce iC. Extracts prepared by osmotic lysis, which contained intact membranes, could convert leucine to both iV and iC. The enzyme which converts leucine to alpha-kiC was solubilized during osmotic lysis, whereas the decarboxylase and leucine reductase system remained membrane bound. Osmotic lysis also released at least two small molecular weight, heat-stable, anionic components (3500 greater than molecular weight greater than or equal to 1000), which stimulated decarboxylase activity.

Isolation and properties of metronidazole-resistant mutants of Clostridium perfringens.

Clostridium perfringens strains resistant to metronidazole and tinidazole were isolated from the sensitive parent strain CM288 after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. Strain CM288 was already resistant to rifampicin and nalidixic acid; these genetic markers helped to confirm the identity of mutants. All mutants showed similar characteristics: they grew more slowly than the parent strain and failed to reach the same maximum turbidity; uptake of metronidazole and tinidazole from culture fluids was slow and end products of glucose metabolism were different from those of the parent. Pyruvate dehydrogenase activity was not detected in broken cell preparations of the mutant strains although this enzyme was readily detected in the parent strain. Changes in end products of glucose metabolism were consistent with the absence of pyruvate dehydrogenase activity because pyruvate was accumulated during growth and lactate levels were higher whereas acetate, CO2 and ethanol levels were diminished.

Leucine dissimilation to isovaleric and isocaproic acids by cell suspensions of amino acid fermenting anaerobes: the Stickland reaction revisited.

Freshly compared cell suspensions of clostridia (Clostridium bifermentans, C. botulinum proteolytic type A, C. difficile, C. sordellii, and C. sporogenes) and Peptostreptococcus anaerobius converted leucine to isovaleric (iV) and isocaproic (iC) acids in the absence of other amino acids. The optimal pH for conversion was between 8 and 9 at 37 degrees C. The stoichiometry of reaction was compatible with that expected for the Stickland reaction, as the ratio of iV to iC was 1:2, the amount of CO2 produced was equivalent to that of iV, and ammonium ion concentrations were equal to the total C5 and C6 acids formed. The presence of alanine and valine (proton donors in the Stickland reaction) in incubations effectively increased the concentration of iC at the expense of iV production, implying that leucine acted there primarily as a proton acceptor. Glycine and proline (proton acceptors) stimulated both iV and iC production from leucine, but increases in iV concentrations were proportionately greater than for iC so that leucine was primarily a proton donor in the presence of proton acceptors. Glucose stimulated the conversion of leucine to volatile fatty acids but favoured iC production. Production of iC from leucine was inhibited by surface active compounds (cetyltrimethylammonium bromide and desoxycholate) as well as arsenite and iodoacetate. The redox dyes methyl viologen and phenosafranine inhibited iC production more severely than iV production, as did the nitroimidazole antimicrobial agent, metronidazole.

Some properties of neutral proteinases from lysosomes of rabbit polymorphonuclear leucocytes.

Neutral proteinases capable of degrading proteoglycan were found in lysosomes of rabbit polymorphonuclear leucocytes extracted with 0 . 01 M citric acid. Esterase activity against an elastase substrate was also present but chymotrypsin- and trypsin-like activities were not detected; azocasein-degrading activity was poor. Proteoglycanase activity was stimulated by high concentrations of salts (0 . 2 M KCl) and divalent cations (Ca, Mg, Mn, Zn) but was inhibited by Cu++. Elastase activity was also stimulated by high ionic strength buffers and KCl, but not as much by divalent cations, and was inhibited by Cu++. Proteoglycanase in crude extracts was inhibited by EDTA, phenylmethanesulphonylfluoride (Pms-F), cell cytosol, alpha 1-antitrypsin, gold thiomalate and N-acetyl-di-L-alanyl-L-propyl-L-valine chloromethyl ketone (AAAPVCK). Partial inhibition by N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) and L-l-tosylamide-2-phenylethyl chloromethyl ketone (TPCK) occurred. Elastase adsorbed to CM-cellulose and was eluted by 0 . 6-0 . 7 M NaCl; a metallo-proteinase failed to adsorb completely but was retarded by the CM-cellulose. Isoelectric focusing showed that the major proteinases had pI's of 5 . 5, 8 . 5 and 9 . 1; the activity with pI 8 . 5 was a metallo-proteinase, and the Pi 9 . 1 activity was an elastase. The apparent molecular weight of the elastase, determined on Sephadex G-100, was 8,000 and 11,000 daltons.

Human percutaneous penetration of hydrocortisone: the vulva.

Percutaneous penetration of 14C hydrocortisone through normal vulvar skin (labia majora) was measured in six subjects and compared with that of the forearm. Of the topically applied hydrocortisone 7.7% penetrated vulvar skin whereas 1.3% penetrated forearm skin. This regional variation of percutaneous penetration may have toxicologic and therapeutic significance.