Evaluation of novel beta-ribosidase substrates for the differentiation of Gram-negative bacteria.

AIMS: To synthesize novel substrates for the detection of beta-ribosidase and assess their potential for the differentiation of Gram-negative bacteria. METHODS AND RESULTS: Two novel chromogenic substrates, 3',4'-dihydroxyflavone-4'-beta-D-ribofuranoside (DHF-riboside) and 5-bromo-4-chloro-3-indolyl-beta-D-ribofuranoside (X-riboside) were evaluated along with a known fluorogenic substrate, 4-methylumbelliferyl-beta-D-ribofuranoside (4MU-riboside). A total of 543 Gram-negative bacilli were cultured on media containing either DHF-riboside or X-riboside. Hydrolysis of DHF-riboside or X-riboside resulted in the formation of clearly distinguishable black or blue-green colonies, respectively. Hydrolysis of 4MU-riboside was evaluated in a liquid medium in microtiter trays and yielded blue fluorescence on hydrolysis which was measured using fluorimetry. beta-Ribosidase activity was widespread with 75% of strains, including 85.6% of Enterobacteriaceae, showing activity with at least one substrate. Genera that demonstrated beta-ribosidase activity included Aeromonas, Citrobacter, Enterobacter, Escherichia, Hafnia, Klebsiella, Morganella, Providencia, Pseudomonas, Salmonella and Shigella. In contrast, strains of Proteus spp., Acinetobacter spp., Yersinia enterocolitica, Vibrio cholerae and Vibrio parahaemolyticus generally failed to demonstrate beta-ribosidase activity. CONCLUSIONS: The novel substrates DHF-riboside and X-riboside are effective for the detection of beta-ribosidase in agar-based media and may be useful for the differentiation and identification of Gram-negative bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report describing the application and utility of chromogenic substrates for beta-ribosidase. These substrates could be applied in chromogenic media for differentiation of Gram-negative bacteria.

Evaluation of a new chromogenic medium, Uriselect 4, for the isolation and identification of urinary tract pathogens.

AIMS: To compare the performance of a new chromogenic medium, Uriselect 4, with cystine lactose electrolyte deficient (CLED) agar and an established chromogenic agar, CPS ID 2 medium, for detection of urinary tract pathogens. METHODS: Using a semiquantitative culture method, 777 samples were inoculated on to the three test media in duplicate. All bacterial strains that yielded a potentially significant growth were observed for colony colour and identified using standard methods. RESULTS: Of the 777 samples tested, 589 urine samples yielded potentially significant growth of at least one strain. A total of 811 strains were isolated on at least one of the three media. A total of 168 urine samples yielded a mixture of at least two strains. Uriselect 4 medium showed the best sensitivity of the three media and only failed to recover 14 strains (1.7%). CPS ID 2 medium failed to recover 22 strains (2.7%). CLED medium showed the worst recovery and failed to recover 74 strains (9.1%). Both chromogenic media allowed for identification of Escherichia coli with a high degree of specificity (98% for Uriselect 4, 99.7% for CPS ID 2). Inclusion of a spot indole test increased the specificity of both chromogenic media to 100% for E coli. CONCLUSIONS: Uriselect 4 and CPS ID 2 were superior to CLED medium for the isolation of urinary tract pathogens mainly because of their ability to discriminate mixed cultures. Both chromogenic media were also useful for the preliminary identification of the most common urinary tract pathogens.

Bacterial adhesion to titanium-oxy-nitride (TiNOX) coatings with different resistivities: a novel approach for the development of biomaterials.

In this study the quantitative adhesion of a strain of Staphylococcus epidermidis, Streptococcus mutans and Pseudomonas aeruginosa to and the ease of removal from different TiNOX coatings was investigated by means of a parallel plate flow chamber and in situ image analysis. Quality of adhesion was determined by counting bacteria which remained attached to the surface after exposure to an air-liquid interface. S. epidermidis and S. mutans showed a bipolar adhesion pattern with highest numbers of adhesion at low and high resistivity with lowest adhesions at a resistivity of 10(4) microohms cm. P. aeruginosa was the least adherent organism. These results indicate that the affinity of these three strains under the current experimental conditions is minimal for TiNOX coatings with a specific resistivity. TiNOX coatings with pre-adsorbed fibrinogen showed different numbers of S. epidermidis adhered to the different coatings. However, the affinity of this strain for fibrinogen-coated TiNOX remains low when the resistivity is around 10(4) microohms cm. This indicates that the specific influence of the resistivities of the TiNOX coatings is transferred through the adsorbed fibrinogen film to the interface with adhering bacteria.

A novel variant genotype C of hepatitis B virus identified in isolates from Australian Aborigines: complete genome sequence and phylogenetic relatedness.

There have been no reports of DNA sequences of hepatitis B virus (HBV) strains from Australian Aborigines, although the hepatitis B surface antigen (HBsAg) was discovered among them. To investigate the characteristics of DNA sequences of HBV strains from Australian Aborigines, the complete nucleotide sequences of HBV strains were determined and subjected to molecular evolutionary analysis. Serum samples positive for HBsAg were collected from five Australian Aborigines. Phylogenetic analysis of the five complete nucleotide sequences compared with DNA sequences of 54 global HBV isolates from international databases revealed that three of the five were classified into genotype D and were most closely related in terms of evolutionary distance to a strain isolated from a healthy blood donor in Papua New Guinea. Two of the five were classified into a novel variant genotype C, which has not been reported previously, and were closely related to a strain isolated from Polynesians, particularly in the X and Core genes. These two strains of variant genotype C differed from known genotype C strains by 5.9-7.4% over the complete nucleotide sequence and 4.0-5.6% in the small-S gene, and had residues Arg(122), Thr(127) and Lys(160), characteristic of serotype ayw3, which have not been reported previously in genotype C. In conclusion, this is the first report of the characteristics of complete nucleotide sequences of HBV from Australian Aborigines. These results contribute to the investigation of the worldwide spread of HBV, the relationship between serotype and genotype and the ancient common origin of Australian Aborigines.

An efficient extraction method from blood clots for studies requiring both host and viral DNA.

The clot from blood is usually discarded after the collection of serum. Yet, it contains nucleated white blood cells and substantial serum. Here, we have compared four methods to enable quick and efficient extraction of human genomic and viral DNA from clotted blood. Two of these methods, a phenol-based in-house method and Tripure isolation reagent, only achieved a low polymerase chain reaction (PCR) yield. In contrast, the QIAamp blood kit and the High Pure Viral Nucleic Acid kit were equally efficient, with similar sensitivity to serum for extraction of viral DNA.

The C282Y mutation in the haemochromatosis gene (HFE) and hepatitis C virus infection are independent cofactors for porphyria cutanea tarda in Australian patients.

BACKGROUND/AIM: Whether mutations in the putative haemochromatosis gene (HFE) and hepatitis C virus act independently to precipitate porphyria cutanea tarda is unknown. The aim of the study was to investigate the relationship between mutations in HFE, hepatitis C and porphyria cutanea tarda. METHODS: The frequencies of the C282Y and H63D mutations in HFE were determined in 27 patients with porphyria cutanea tarda and compared with the reported control frequencies. In addition, the presence of hepatitis C virus infection was identified and related to the patients' HFE status. RESULTS: The C282Y mutation was found in 44.4% of patients compared with the control frequency of 12% (p<0.001). Three patients were homozygous for the C282Y mutation, two of whom did not meet current clinical diagnostic criteria for expressed haemochromatosis. The proportion of patients with the H63D mutation did not differ from the reported control frequency. The mean transferrin saturation and serum ferritin concentration were similar in porphyria cutanea tarda patients who were homozygous normal and heterozygous for the C282Y mutation, but greater in both groups than previously reported in healthy controls. Seven (25.9%) patients were anti-HCV IgG positive. None of these patients carried the C282Y mutation. Porphyria cutanea tarda patients heterozygous for the C282Y mutation and patients with anti-HCV antibodies had elevated transferrin saturations and serum ferritin concentrations. CONCLUSIONS: The raised frequency of the C282Y mutation in porphyria cutanea tarda indicates that this mutation is likely to be a predisposing factor. However, abnormalities of iron indices also exist in porphyria cutanea tarda patients without mutations in HFE. Hepatitis C virus infection is likely to be another common precipitating factor for porphyria cutanea tarda which acts independently of the C282Y mutation.

Comparison of four methods for quantitative measurement of hepatitis B viral DNA.

AIMS/METHODS: Four assays for measuring HBV-DNA quantitatively have been compared with regard to sensitivity, precision and linearity. The methods were 125I-labelled solution hybridisation assay (liquid hybridisation, Abbott), an ELISA-based chemiluminescent RNA-DNA hybrid assay (RNA-DNA, Digene), a chemiluminescent branching oligonucleotide assay (bDNA, Chiron) and a membrane hybridisation assay using slot-blot equipment (slot blot). RESULTS: The bDNA assay was linear over three orders of magnitude and was the most sensitive assay, being approximately ten times more sensitive than the other assays, so that samples negative on RNA-DNA, liquid hybridisation and slot blot gave quantifiable results on bDNA. Furthermore, intra- and inter-assay variability showed that the bDNA and liquid hybridisation assays had the greatest precision, with coefficients of variation of 6.6% to 11.5% and 2.3% to 10.5%, respectively. However, the nominated amounts of HBV DNA in the standards (from all assays) were not reproducible in the other assays, such that amounts measured with bDNA would give values approximately twice that of RNA-DNA and 60 times that of liquid hybridisation. CONCLUSIONS: The recently developed bDNA assay has advantages compared with the other assays in quantitating samples with low levels of virus present. In addition, since the assays vary considerably by a number of criteria, the method of measurement should always be reported.

Hepatitis C virus infection in health care workers referred to a hepatitis clinic.

OBJECTIVES: To assess method of acquisition, presence of liver disease, potential infectivity and the effect on work practices in health care workers with hepatitis C virus (HCV) infection referred to a hepatitis clinic. PATIENTS AND METHODS: All 33 health care workers referred to a hepatitis clinic for management of HCV infection because of a positive test for HCV (enzyme-linked immunosorbent assay) between 1 January 1990 and 31 December 1994 (comprising six medical practitioners, 18 nurses, two scientists and seven others) were retrospectively assessed for most likely method of infection, alanine aminotransferase levels, results of liver biopsy and measurement of HCV-RNA. RESULTS: 30 health care workers (12 men and 18 women; age range, 27-68 years) had HCV infection confirmed on further testing. Only seven were believed to have acquired their infection occupationally (one with documented needlestick injury). Twenty-eight patients had elevated alanine aminotransferase levels and, of 23 patients who underwent liver biopsy, one had cirrhosis and 12 had chronic hepatitis and fibrosis. Of the 24 health care workers with direct patient contact, four had retired, eight had stopped or modified their work practices and 12 continued to practise normally. CONCLUSIONS: Few health care workers with chronic HCV infection have acquired it occupationally. We recommend that guidelines be set up for institutional expert committees to advise health care workers with HCV infection about modifying their work practice.