Molecular cloning and expression of a novel Staphylococcus aureus antigen.

A novel gene encoding an antigen from Staphylococcus aureus was isolated from an expression library by screening with antisera from patients with deep Staphylococcus aureus infections. In one positive clone an open reading frame, named ORF-2, was identified. Recombinant ORF-2 protein reacted with human immune serum. ORF-2 was shown to be present in other Staphylococcus aureus strains, but not in related species.

Interactions of drugs acting against Staphylococcus aureus in vitro and in a mouse model.

Two combinations of antibiotics, clindamycin with rifampicin and cloxacillin with netilmicin, were investigated for their activity against two strains of Staphylococcus aureus (a sensitive reference strain and a methicillin-resistant clinical isolate) by means of the in vitro checkerboard technique and an in vivo infected mouse model. The mouse model allowed drug interactions to be evaluated both from the changes in the number of bacteria surviving treatment and from the measured exposure to antibiotics at the site of infection. Specimens from the latter were evaluated twice (day 0 and day 2) in each experiment. The combination of cloxacillin and netilmicin exhibited a synergistic effect against the reference strain both in vitro and in vivo, whereas synergism was obtained under in vitro conditions only against the methicillin-resistant strain. The clindamycin and rifampicin combination acted synergistically or indifferently against both strains in vitro and at day 0 of the in vivo experiments. In contrast, on day 2 of infection, this combination had significantly greater bactericidal effect (synergism) compared to the combination of cloxacillin and netilmicin. These results illustrate the difficulties of interpreting in vitro results for clinical use.

Branhamella catarrhalis in children and adults. A study of prevalence, time of colonisation, and association with upper and lower respiratory tract infections.

The colonisation rate of Branhamella catarrhalis in patients from 0 to 45 years of age was examined. Of 561 women admitted to hospital in labour, 6 (1%) carried B. catarrhalis in their throats but none carried the organism in their vaginas. None of 534 newborn babies became colonised at birth or during their 5 days' stay in hospital. Neither were 102 neonates < 1 month of age in hospital colonised. The maximum colonisation rate during childhood was observed in children 1-48 months of age with 143 of 266 (54%) children colonised. Among children 4-15 years of age, four of 57 (7%) children with healthy respiratory tracts were colonised. Significantly more children with upper or lower respiratory tract infections (RTI) were colonised (68%) than were children without such infections (36%), (P < 0.001). After recovery from RTI, the isolation rate in the RTI group fell to that of the non-RTI group. A seasonal variation in prevalence was not observed. Of all the strains of B. catarrhalis isolated, 84% produced beta-lactamase.

Occurrence of antibodies to pneumococcal protein antigens in experimental acute otitis media.

To study serological antibody response to pneumococcal protein antigens, experimental pneumococcal (type 3) acute otitis media (AOM) was induced in 6 rats, sera being analysed with the Western blot technique at different intervals after bacterial challenge. The most striking finding was a distinct antibody response to a protein of about 35 kDa visible in 5 of the rat sera within 14 days, and persisting throughout the remainder of the study (i.e., 56 days in all). Moreover, appear to be a phenomenon restricted to type 3 pneumococci. Both the pathogenetic importance of this protein and the immunological response it evokes are still unclear. However, as antibodies to protein antigens may contribute to inflammatory reactions in the middle ear (e.g., otitis media with effusion), this 35-kDa protein might be important for the development of sequelae to AOM.

[Can ingestion of cranberry juice reduce the incidence of urinary tract infections in a department of geriatric medicine?]. / Reducerer brug af tranebaersaft hyppigheden af urinvejsinfektioner i en geriatrisk afdeling?

INTRODUCTION: The incidence of urinary tract infections was compared in two geriatric units, where patients were offered cranberry juice and the usual mixed berry juice, respectively. METHODS: In all cases where urinary tract infection was suspected, the doctors noted symptoms and signs used as indication for urinary culture. The urine collected from men was the usual mid-flow specimen, whereas the specimens from women were taken from a bedpan and by catheter. End points were the prevalence of symptoms leading to urine culture, specimens with significant growth of bacteria, and the use of antibiotics. RESULTS: Urine specimens were cultured in 140/338 cases. The reason for culture in 23% was general symptoms and in 62% urinary tract symptoms. A significant growth of bacteria was found in 54% and this information led to antibiotic treatment in 44%. In all cases (n = 55) where bedpan and catheter specimens were taken, the results were identical. CONCLUSION: Cranberry juice in a geriatric department, where the mean stay was 4 weeks, did not influence the incidence of urinary tract infections.

Definitions of antibacterial interactions in animal infection models.

Combination of antibiotics in order to achieve antimicrobial synergy is often necessary in the treatment of infections due to resistant bacterial strains. Therefore, several in-vitro test systems have been developed with the purpose of predicting in-vivo action of antibiotics, and the fractional inhibitory concentration (FIC) index has been used to interpret results obtained in different test systems. Using these systems it seems that only antibiotic synergy in vitro is predictive of the results of treatment. It is therefore of interest to have an in-vivo test system that makes it possible to describe antibiotic interaction in detail. Animal infection models such as the foreign body model system enable the measurement of many parameters at the site of infection, such as bactericidal effect (BE) and antibiotic concentrations. A new calculation of drug interaction is suggested in which the measurements used are the BE and the time during which the MIC is exceeded, for the individual drugs and the combination. This calculation enables us to penetrate into observed antibiotic efficacy in vivo to find out whether an observed high BE is due to real synergy or simply to optimal pharmacokinetics of antibiotics at the site of infection.

A mouse model for simultaneous pharmacokinetic and efficacy studies of antibiotics at sites of infection.

Tissue penetration may be an important pharmacokinetic determinant to achieve the chemotherapeutic efficacy of antimicrobial agents. We describe a mouse model for the simultaneous study of antibiotic kinetics and efficacy at the site of infection. With the doses tested there was no difference in drug penetration into healthy tissue and tissue at the site of acute bacterial infection. Ampicillin and netilmicin levels over the minimal inhibitory concentration (MIC) were needed to produce significant bacterial killing. During the 6 h observation period susceptible Gram-positive bacteria were eradicated whereas Gram-negative bacteria were reduced in number but not eradicated, even though the antibiotic concentrations exceeded minimal bactericidal concentration (MBC) three times.

Mouse model for evaluation of antibiotic treatment of acute and chronic infections.

A thread model is presented which enables the simultaneous evaluation of bactericidal rate and antibiotic concentration at the site of infection in mice. Ampicillin and netilmicin were tested against both gram-positive and gram-negative bacteria. With the doses tested there was substantial drug penetration into the site of infection immediately after initiation of infection (day 0), whereas the drug penetration on days 2 and 6 of infection was delayed and reduced. On day 0 of infection there was significant bactericidal effect, but little or no effect could be demonstrated on days 2 and 6 of infection, even though drug concentrations close to the MBC values for several drug/bacteria combinations were reached. The model reflects the treatment situations for the acute and the chronic infection and may be of help in evaluating the efficacy of the drug at the site of infection.

Postantibiotic effects of imipenem, norfloxacin, and amikacin in vitro and in vivo.

The postantibiotic effects (PAEs) of imipenem and norfloxacin were tested against Staphylococcus aureus, Streptococcus (Enterococcus) faecalis, Escherichia coli, and Pseudomonas aeruginosa. Amikacin was tested against the same bacteria except Streptococcus faecalis. For in vitro tests, a viable count-washing method was used, and for in vivo tests, the thread technique in normal mice was used. All three drugs produced PAEs of 1.1 to 3.8 h in vitro and 1.4 to 4.3 h in vivo against the pathogens tested. In vitro and in vivo results correlated well. The PAE had a significantly (P less than 0.01 to 0.001) longer duration in vivo than in vitro, but the PAE of imipenem on Staphylococcus aureus was longer in vitro. The PAE was not due to residual antibiotics at the site of infection, and no PAE was obtained if at any time the antibiotic concentration at the infection site reached the MIC for the pathogen tested. The results indicate that the presence of a PAE may enable antibiotics to be given more intermittently without a loss of efficacy and that the PAE can only be induced if the level of the antibiotic exceeds the MIC for the pathogen in question for at least several minutes.

The role of beta-lactamase in mixed infections in mice in relation to treatment with ampicillin.

Beta-lactamase-producing Staphylococcus aureus and Bacteroides fragilis in a localized mixed infection has been found to degrade the beta-lactam antibiotic at the focus of infection, thus protecting both the bacteria and pathogens susceptible to the antibiotic. To determine if beta-lactamase produced by Hemophilus influenzae and Branhamella catarrhalis have similar importance in mixed infections, a thread infection model in mice was used to evaluate the capacity of beta-lactamase produced by S. aureus, B. catarrhalis, or H. influenzae to hydrolyze ampicillin in a mixed infection with Streptococcus pneumoniae in mice. For both S. aureus and B. catarrhalis, the ampicillin concentrations at infection sites where beta-lactamase was produced were lower than at sites where beta-lactamase was not produced; however, this difference was not found when clavulanic acid was added to the ampicillin. In mixed infections with strains that did not produce beta-lactamase, ampicillin concentrations were similar with or without clavulanic acid. S. aureus was the best "protector" followed by B. catarrhalis. The beta-lactamase produced by H. influenzae failed to protect the S. pneumoniae. No bactericidal effect of clavulanic acid was found.

The clinical significance of Staphylococcus aureus bacteriuria.

To investigate the rate of occurrence, clinical presentation, predisposing factors and frequency of secondary bacteremia 132 patients with significant Staphylococcus aureus bacteriuria were reviewed retrospectively. Staphylococcus aureus accounted for 3.3 per cent of all positive urine cultures. Most patients were elderly men. The most important predisposing factors in the urinary tract were indwelling catheters (63 per cent), obstruction (56 per cent) and instrumentation or surgery (43 per cent). Bacteremia developed secondary to bacteriuria in all 11 patients (8.3 per cent). For that reason Staphylococcus aureus bacteriuria should be regarded as a hazardous condition, especially in patients with predisposing factors in the urinary tract.

Single-day treatment with trimethoprim for asymptomatic bacteriuria in the elderly patient.

Recent reports have demonstrated an association between bacteriuria and excess mortality. Therefore, 40 inpatients 60 or more years old with asymptomatic bacteriuria participated in a double-blind trial to assess the effect of 1-day treatment with trimethoprim. The patients were allocated randomly to receive 100 mg. trimethoprim (20 patients) or placebo (20 patients) to be taken in the morning and evening for 1 day. All patients treated with trimethoprim obtained sterile urine, whereas all patients treated with placebo remained bacteriuric (p less than 0.002). No side effects were recorded. However, many of the cured patients suffered rapid reinfection. Of the 20 initially cured patients 14 (70 per cent, 95 per cent confidence limits 46 to 88 per cent) had recurrent bacteriuria after 6 weeks, indicating that although this treatment is effective immediately it imparts no long-term effect.

Evaluation of Staph ID 32 system and Staph-Zym system for identification of coagulase-negative staphylococci.

The purpose of the investigation was to evaluate two commercially available identification systems: a new modification of the Staph-Zym system (Rosco, Tåstrup, Denmark) and the Staph ID 32 API system (API System, BioMérieux, Paris, France). A local standard method to be used in routine laboratories was also evaluated. A total of 200 staphylococcal isolates, including strains from both the American Type Culture Collection and the Czechoslovak Collection of Microorganisms as well as 89 clinical isolates, were used in tests of all three identification systems. The Staph ID 32 API system identified from 50 to 100% of the reference strains and 82.1% of the clinical isolates correctly. The Staph-Zym system identified from 90 to 100% of the reference strains and 82.1% of the clinical isolates correctly. Most misidentifications were of minor importance, but in both systems major failures appeared (Staphylococcus aureus was identified as a coagulase-negative staphylococcus). Both systems needed backup from a reference laboratory to determine if two isolates were of the same strain.

Western blot analysis of the antibody response in patients with Listeria monocytogenes meningitis and septicemia.

The antibody response in patients with Listeria monocytogenes septicemia and/or meningitis was investigated using Western blot analysis (WBA). Protein antigen preparations were produced from two strains of Listeria monocytogenes, representing serogroup 1 and 4, by sonication and differential centrifugation. IgG antibodies from 8 (50%) of 16 patients with culture verified septicemia and/or meningitis due to Listeria monocytogenes reacted with a 93 kDa antigen from serogroup 1, in contrast to IgG antibodies from only 1 (2%) of 51 controls; these controls represented 21 patients with infections caused by other bacteria and 30 apparently healthy blood donors. Furthermore, IgM antibodies from 3 (19%) of the patients with listeric infections bound to a 106 kDa protein antigen in contrast to none of the controls. In 3 (33%) of 9 patients from whom acute and convalescence serum were available, the patients responded by producing antibodies against new protein antigens. Current methods used in routine serological investigations, i.e. complement fixation and O-agglutination tests, were positive in only 4 (24%) of the 16 patients with listeriosis. The results point to the possibility of designing new immunoassays for detection of septicemia and meningitis caused by Listeria monocytogenes.

Epidemiological studies of penicillin resistance in Danish Staphylococcus aureus strains in the period 1977-1990.

During the period 1977-1990 the frequency of penicillin resistance increased from 78.7 to 87.5% among a total of 278,193 Danish Staphylococcus aureus strains from hospitalized patients. By combining these data with phage-type patterns the increase was shown to be caused by 1) the introduction and spread of mainly resistant strains of type 95 and the 94,96 complex; 2) an increased occurrence of strains of group II which during the observation period became more frequently penicillin resistant; and 3) a gradual disappearance of strains of group III and the 83A complex which showed a decreasing degree of penicillin resistance. During the observation period community acquired strains reached nearly the same level of penicillin resistance as the hospital-acquired strains, and the frequency of penicillin resistance did not increase during hospitalization among the predominant strains of type 95, group II and the 94,96 complex. S. aureus isolated from airways had a higher frequency of penicillin resistance mainly caused by increasing amount of penicillin resistant strains of group II. Strains from urine had a lower frequency of penicillin resistance. The total antibiotic consumption and the usage of beta-lactam antibiotics remained nearly unchanged during the observation period. The frequent use of beta-lactam antibiotics for airway infections might explain the possible selection of penicillin resistant strains of group II.