- Volume 52, Issue 5, 2003

Interpretation of the mupirocin E-test for low-level mupirocin-resistant Staphylococcus aureus strains has been improved by adding the indicator dye tetrazolium. E-tests were compared with agar dilution methods for assessing mupirocin susceptibility. MICs obtained by the agar dilution method and E-tests showed 89.3 % agreement within 2 log2 dilution criteria. The agreement between MICs increased to 100 % in the 1 log2 dilution definition when the indicator dye tetrazolium was added to the E-test. The use of the E-test with tetrazolium reduction is more accurate for determining mupirocin MICs for S. aureus strains.

A highly sensitive and rapid assay has been developed to quantify hepatitis B virus (HBV) DNA, based on the fluorescence resonance energy transfer principle and real-time PCR, using the LightCycler and a pair of specific fluorescent hybridization probes. This LightCycler real-time PCR assay (LC-PCR) detected HBV DNA in a linear range from 101 to 108 copies per reaction (250–2.5 × 109 copies ml−1), with a rapid PCR cycling time of 35 min. The assay was validated with two EUROHEP HBV DNA standards (ad and ay subtypes) and exhibited low intra-assay (< 6 %) and inter-assay (< 16 %) variation for both subtypes over the complete range of 7 orders of magnitude. The assay was evaluated clinically using serum samples from 120 HBsAg+ individuals and 45 healthy controls who were negative for both HBsAg and anti-HBc. Levels of HBV DNA were measured in these samples using both the LC-PCR and Digene Hybrid Capture II HBV DNA (HCII) assays. The prevalence rates for HBV DNA in the HBsAg+ serum samples were respectively 95 % (114/120) and 56 % (67/120) by LC-PCR and HCII (P < 0.01). All 67 HCII-positive samples tested positive with LC-PCR, while the 47 discordant samples showed low levels of HBV DNA (down to 265 copies ml−1), detectable only by the more sensitive LC-PCR assay. Levels of HBV DNA as measured by the two assays showed good correlation (r = 0.902; P < 0.001). The level of HBV DNA was significantly higher in HBeAg+ than anti-HBe+ samples (median 1.5 × 107 vs 4.6 × 104 copies ml−1; P < 0.01). It is concluded that this LC-PCR assay is clinically useful for the rapid, sensitive and accurate measurement of HBV DNA.

Constantly expressed genes are used as internal controls in relative quantification studies. Suitable internal controls for such studies have not yet been defined for Pseudomonas aeruginosa. In this study, the genes ampC, fabD, proC, pbp-2, rpoD and rpoS of P. aeruginosa were compared in terms of expression stability by real-time quantitative RT-PCR. A total of 23 strains with diverse resistance phenotypes were studied. Stability of expression among the housekeeping genes was assessed on the basis of correlation coefficients, with the best-correlated pair accepted as being the most stable one. Eventually, proC and rpoD formed the most stable pair (r = 0.958; P < 0.001). Next, in four ciprofloxacin-selected nfxC-like mutants, levels of oprD, oprM and oprN mRNA were compared with those of their wild-type counterparts. The comparison was made after correcting the raw values by the geometric mean of the internal control genes proC and rpoD. The level of oprN mRNA was significantly up-regulated, while the oprD gene was down-regulated (although this difference was statistically insignificant), in the mutants. This expression pattern was consistent with that of the expected expression profile of nfxC-type mutants; this experiment therefore lends further support to the use of proC and rpoD genes simultaneously as internal controls for such studies.

Bee glue (propolis) possesses antimicrobial, anti-inflammatory, anaesthetic and immunostimulating activities. The aim of the study was to evaluate the inhibitory effect of Bulgarian propolis on Helicobacter pylori growth in vitro. Activity of 30 % ethanolic extract of propolis (EEP) against 38 clinical isolates of H. pylori was evaluated by using the agar-well diffusion method. Ethanol was used as a control. In addition, the effect of propolis on the growth of 26 H. pylori and 18 Campylobacter strains was tested by the disc diffusion method. Mean diameters of H. pylori growth inhibition by the agar-well diffusion method, using 30, 60 or 90 μl EEP or 30 μl ethanol per well, were 17.8, 21.2, 28.2 and 8.5 mm, respectively. EEP was significantly more active than ethanol against H. pylori (P < 0.001). The results obtained by the disc diffusion method were similar. The use of moist propolis discs resulted in mean diameters of growth inhibition of 21.4 mm for H. pylori and 13.6 mm for Campylobacter spp. Dried propolis discs exhibited antibacterial effect against 73.1 % of H. pylori isolates, with a considerable zone of growth inhibition (⩾ 15 mm) in 36.4 % of isolates. Using dried propolis discs resulted in mean diameters of growth inhibition of 12.4 mm for H. pylori and 11.6 mm for Campylobacter spp. In conclusion, Bulgarian propolis possesses considerable antibacterial activity against H. pylori, and can also inhibit the growth of Campylobacter jejuni and Campylobacter coli. The potential of propolis in the prevention or treatment of H. pylori infection is worth further extensive evaluation.

The present study was undertaken to investigate the high incidence of multiresistant Gram-negative bacilli causing neonatal septicaemia. Samples of neonatal blood from 728 suspected cases were obtained in brain heart infusion broth with sodium polyanethol sulfonate. All Gram-negative rods isolated were subsequently subjected to routine antimicrobial susceptibility testing and tests for extended-spectrum β-lactamase (ESBL) production, as per NCCLS recommendations. ESBL was detected in 86.6 % of Klebsiella spp., 73.4 % of Enterobacter spp. and 63.6 % of Escherichia coli strains. It was also observed that 74.4–80.9 % of these ESBL producers were resistant to cefotaxime and 47.6–59.5 % were resistant to ceftazidime in routine susceptibility testing. Some ESBL producers (36.3–61.5 %) were found to be susceptible to either or both cephalosporins used in this study. It is concluded that indiscriminate use of third-generation cephalosporins may be responsible for the selection of ESBL-producing multiresistant strains in the neonatal intensive-care unit (NICU).

Dual infections associated with acute infectious diarrhoea and its microbiological, epidemiological and clinical findings have been evaluated in patients selected from a comprehensive survey of children under 4 years old, admitted to hospital emergency rooms from October 1996 to November 1997. A total of 820 children (433 males and 387 females) were enrolled. Stools were tested for rotavirus, adenovirus, astrovirus and bacterial enteropathogens. Patients were grouped according to age, and the seasonality of mixed infections was evaluated. Clinical trends and severity of gastrointestinal disease by Ruuska's score were also analysed. Mixed infections were identified in 39 cases (5 %), of which 23 were males and 16 were females. The majority of cases were in the 7–18-month age group (26 cases) and occurred in autumn (67 %). Virus–virus co-infections were more frequent (26/39) than virus–bacteria co-infections (13/39). More than two infectious agents were detected in only four cases. The most common viral co-infections were rotavirus–astrovirus (13/26) and rotavirus–adenovirus (10/26). The present report is the first prospective analysis of clinical–epidemiological trends of dual infections in young Spanish children with acute viral gastroenteritis. Our results emphasize the clinical importance of mixed infections as a cause of severe diarrhoea in children.