- Volume 46, Issue 9, 1997

The zinc supplement required to achieve the maximum activity of metallo-β-lactamases from 12 Bacteroides fragilis isolates was investigated. Changes in absorbance of imipenem in a spectrophotometric assay with crude cell extracts were performed in the presence and absence of various concentrations of zinc sulphate. The greatest degree of imipenem hydrolysis was seen with the addition of between 50 and 500 μM zinc sulphate, and the degree of stimulation of enzyme activity in the strains tested varied six-fold. Increasing the zinc sulphate concentration resulted in inhibition of hydrolysis with extracts of low enzymic activity. These findings indicate the importance of determining the optimal zinc concentration for each strain tested in kinetic studies of metallo-β-lactamases.

Members of the genus Acinetobacter, particularly multiresistant strains of A. baumannii, are implicated in a wide spectrum of nosocomial infections, including bacteraemia, secondary meningitis and urinary tract infection, but have now assumed a particularly important role as agents of nosocomial pneumonia in intensive care units (ICUs). Rapid genotyping methods for the identification and typing of these organisms have allowed a better appreciation of the epidemiology and survival of these organisms in the hospital environment. Their emergence as significant pathogens seems to be related partly to their survival ability and partly to their ability to develop resistance rapidly to the major groups of antibiotics, resulting in a considerable selective advantage in environments (such as ICUs) with widespread and heavy use of antibiotics. Molecular and biochemical mechanisms of resistance to the major β-lactam, aminoglycoside and quinolone groups of antibiotics have now been elucidated in some detail for these organisms, and experimental models, including a mouse model of A. baumannii pneumonia, have been developed to examine the efficacy of different therapeutic regimens for difficult-to-treat-infections caused by these bacteria. ‘Non-classic’ antibiotic combinations—such as ticarcillin with clavulanic acid and sulbactam—seem to show promise for treating systemic infections caused by otherwise multiresistant strains, but revised screening procedures in the pharmaceutical industry may be required in the near future to select novel compounds with activity against multiresistant Acinetobacter spp. and other emerging gram-negative, non-fermentative bacilli in general.

Because of the increased risk of infection with the associated diagnostic and therapeutic problems in bone marrow transplantation (BMT) patients, the usefulness of surveillance cultures (SC) at the BMT department of the National Institute of Haematology, Blood Transfusion, Transplantation and Immunology, Budapest, was reviewed. Between January 1992 and May 1995, 26 BMT operations were performed; 13 patients had 23 febrile espisodes. In 12 of these episodes infection was clinically documented; however, SC of these patients yielded bacteria identical with those in the blood culture in only two episodes (1 and 6 days before their blood cultures became positive, respectively). Out of a total of 1187 samples from these patients, potentially pathogenic bacteria were isolated from 145 SC and 43 blood cultures (drawn on 31 different days). Suppression of the gastrointestinal flora could be achieved by the department’s decontamination regimen; however, overgrowth by gram-positive organisms (mainly coagulase-negative staphylococci) occurred in the intestine and at other body sites. On the basis of these results, SC are of limited value in predicting infection or identifying the causative organisms of fever. On the other hand, SC are useful in confirming the efficiency of suppression of the body flora by antimicrobial agents. Specific treatment was based on suitably sampled materials, and close contact between physicians, infectious disease specialists and microbiologists was essential.

A novel drug delivery system for osteomyelitis was developed in which a porous hydroxyapatite block (HAB) is loaded with antibiotic by a centrifugation method. In this study, implantation of HABs loaded with the aminoglycoside antibiotic, arbekacin, were tested in established Staphylococcus aureus osteomyelitis in the proximal tibia of rats after debridement of the marrow cavity. The animals were observed for radiographic signs of infection and tissue was examined histologically. The infections were also evaluated by bone cultures. Bacterial counts were statistically lower in rats implanted with an antibiotic-loaded HAB than in those given a drug-free HAB. Radiographical and histological observations also showed beneficial effects of the antibiotic-loaded implant. The results suggest that the centrifugation method for loading HABs provides a simple drug delivery system. These antibiotic-loaded HABs may be useful for filling grafts in osteomyelitis.

Helicobacter pylori can utilise amino acids as the sole carbon energy source. The present study demonstrated that H. pylori grown in continuous culture in a defined medium containing glucose and amino acids utilised alanine, arginine, asparagine, aspartate, glutamine, glutamate, proline and serine. Specific asparaginase and glutaminase enzymes deaminated asparagine and glutamine respectively to aspartate and glutamate, with the production of ammonia. The glutaminase activity was inhibited by 6-diazo-5-oxo-L-norleucine. All the 13 strains of H. pylori tested produced both glutaminase and asparaginase activities. Glutamine is important in the health of the gastric and intestinal mucosa and is a primary energy source for lymphocytes. Depletion of glutamine at the site of H. pylori infection may be of significance in the pathogenesis of H. pylori-associated diseases such as peptic ulcer and gastric cancer.

Brucellergene is a commercial allergen prepared from Brucella melitensis strain B115 and containing at least 20 cytoplasmic proteins. These proteins were separated by SDS-PAGE. The unstained gel was divided into 18 fractions and proteins were eluted from the gel fractions. The capacity of the separated proteins to elicit delayed-type hypersensitivity (DTH) in infected guinea-pigs or to induce the production of interferon-γ (IFN-γ) by blood cells from infected cattle was evaluated. The biological activity of the corresponding protein fractions blotted on to nitrocellulose was measured in a lymphocyte blastogenesis assay. Among the 18 fractions tested, two - spanning the mol. wt ranges 17-22 (fraction 8) and 35-42-kDa (fraction 17) - showed the maximum biological activity in the three tests. These fractions contain two antigens, the Brucella bacterioferritin (BFR) and P39 proteins. Both proteins are good candidates for the detection of cellular immunity to Brucella.

Candida albicans is the leading cause of invasive candidosis. As conventional tests do not reliably detect invasive infection, attention has turned to the detection of C. albicans antigens circulating in blood. As antigen tests for invasive candidosis could be improved if C. albicans antigens released upon phagocytosis were defined, this study was undertaken to characterise antigens released during the interaction of yeasts and human neutrophils in vitro. An enzyme immunoassay developed previously to detect what were believed to be predominantly C. albicans cytoplasmic antigens in patients with invasive candidosis was used to follow the neutrophil-mediated release of yeast antigens. Serum opsonisation enhanced antigen release, which was rapid and essentially complete by 1 h. When fresh C. albicans yeasts were added to medium from cultures of neutrophils plus yeasts or neutrophils plus latex beads, additional yeast antigens were released. Medium from neutrophils plus yeasts or from yeasts alone had similar immunoblot patterns with rabbit antibodies to a C. albicans cytoplasmic antigen preparation, with the reactive antigens generally being of higher mol. wt than the reactive antigens in the antigen mixture used for preparation of the antiserum. The two supernates also had similar immunoblot patterns with rabbit anti-C. albicans cell-wall mannan antibodies. These results suggest that yeast surface antigens are released quickly during phagocytosis by neutrophils. Detection of such yeast surface antigens, possibly together with selected yeast cytoplasmic antigens, should improve the sensitivity of C. albicans antigen assays.

The pattern of EcoRI restriction fragments of chromosomal DNA that hybridise with a probe for genes encoding 16S and 23S rRNA is highly discriminatory for non-capsulate Haemophilus influenzae (NCHI). The source of variation detected by these probe-based ribotyping patterns was investigated by restriction analysis of rRNA operon (rrn) amplification products from nine representative strains. Digestion of rrn amplification products with EcoRI indicated one conserved EcoRI site within 16S rDNA and no EcoRI sites within the 16S-23S intergenic spacer region of the nine strains, and an EcoRI site at the 5′ end of 23S rDNA from seven of the nine strains. Comparison of the EcoRI ribotyping patterns obtained with separate probes for 16S and 23S rDNA showed more variation with the 23S probe indicating variation in EcoRI sites downstream from the operon. Restriction analyses of 16S and 23S rDNA amplification products with AluI, HhaI, HaeIII and TaqI divided the nine ‘traditional’ ribotypes into a maximum of three and eight groups, respectively. Similar analyses of the 16S-23S intergenic regions (PCR-ribotyping) failed to distinguish any of the nine representative strains. Therefore, there is probably insufficient variation within the operon for it to form a good target for PCR-based typing methods. In contrast, ‘traditional’ ribotyping with cDNA from 16S plus 23S rRNA detects restriction site differences in the sequences flanking the operon, which show considerably more variation between strains. ‘Traditional’ ribotyping should therefore remain the standard for characterising NCHI in epidemiological investigations.