Penicillin tolerance in Arcanobacterium haemolyticum.

It was previously found that Arcanobacterium haemolyticum, which can cause tonsillitis with exanthema, is not eradicated from the pharynx by administration of phenoxymethylpenicillin, despite minimum inhibitory concentration values of 0.015-1.0 micrograms/ml. Therefore, recent clinical isolates were studied for penicillin tolerance by using a disk diffusion screening test and a pour plate assay. Macrobroth dilution minimum inhibitory and bactericidal concentrations and antibiotic kill kinetics were determined for 4 isolates. Tolerance was present in 38 of 40 clinical isolates with the disk diffusion assay. With the pour plate assay all 40 isolates were tolerant, 34 of them highly tolerant. The presence of the tolerant phenotype was confirmed by macrobroth dilution assays. It is concluded that A. haemolyticum is often penicillin-tolerant, suggesting that phenoxymethylpenicillin administration would be ineffective in eradicating A. haemolyticum from the pharynx.

Norfloxacin treatment of salmonellosis does not shorten the carrier stage.

In a prospective controlled study we evaluated the effect of early norfloxacin treatment on the duration of salmonella carriage after acute salmonellosis. The study was carried out during an outbreak of Salmonella typhimurium infection at a military base. 23 patients received norfloxacin 400 mg twice daily for 7 days while 29 patients served as untreated controls. A patient was considered to have ceased being a carrier on the date of the first of 3 negative consecutive cultures. Four weeks after diagnosis 30% of the treated patients and 31% in the control group were still carriers. The corresponding figures after 8 and 12 weeks were 17 and 3% and 4 and 0%, respectively. Thus, one week of norfloxacin treatment instituted at an early stage of salmonellosis did not shorten the duration of carriage.

Staphylococcal wound infection in the pig: Part I. Course.

Full-thickness excision wounds infected with Staphylococcus hyicus, a pig pathogen, or Staphylococcus aureus, a human pathogen, were produced in pigs. The inoculated wounds were kept occluded for 2 days and then exposed and biopsied at intervals for 9 to 12 days. The exposed lesions were edematous and exudative. The S. aureus model was experimentally advantageous because the infection remained localized to the wound without systemic infection or signs of discomfort. The S. hyicus infection caused a rash with skin blisters; thus, its use is discouraged. The concentration of S. hyicus in the wound on day 2 was log 8.6 +/- 0.4 CFU/g (mean +/- standard deviation). On day 4 the mean was log 9.2. For S. aureus the values were log 8.0 +/- 0.9 on day 2 and 6.9 on day 4 (p less than 0.05). Of the 50 individual values in the S. aureus series, 45 were above log 5. The inflammatory reaction was more pronounced after the infection with S. hyicus, whereas with S. aureus the fibroblast response came earlier and was more pronounced. The model parallels typical clinical courses of staphylococcal infection.

Staphylococcal wound infection in the pig: Part II. Inoculation, quantification of bacteria, and reproducibility.

Bacteria from full-thickness excision wounds with staphylococcal infection in the pig were cultured quantitatively. The bacterial concentration increased with the size of the inoculum, and after 2 days it had already reached a stable, maximal level of approximately log 8 CFU/g in the tissue. The correlation coefficient was 0.753 in comparing concentrations from superficial and deep biopsy halves and 0.145 from "surface wash" and superficial biopsies. The "within wound" sample distribution was logarithmic. The gain in precision when assessing the mean bacterial concentration from three instead of one biopsy was 45%. The coefficient of variation of between wound and within wound determinations was in the same range (i.e., 7.1-12.5%). Paired observations from a sample population with 7 sites (wounds) were needed to determine a 10% change in the bacterial concentration at a significance level of p less than 0.05, and a similar change could be determined from unpaired observations based on two populations with eleven sites.

The effects of an occlusive zinc medicated dressing on the bacterial flora in excised wounds in the rat.

The effects of three different dressings - two occlusive and one non-occlusive - on the bacterial flora of excised wounds in rats were studied. The number of colony forming units per gram of granulation tissue were significantly lower 4, 8 and 12 days postoperatively in wounds treated with a zinc medicated occlusive dressing compared with wounds treated with non-zinc medicated occlusive hydrocolloid dressing or wet-to-dry non-occlusive gauze dressing. The minimum inhibitory concentration (MIC) of zinc sulphate was determined on different strains of bacteria isolated from the wounds of rats and on strains isolated from humans. The most susceptible species isolated from both rat wounds and humans were Streptococcus sp., STaphylococcus aureus and Escherichia coli; whereas, Proteus and Enterococcus sp. had higher MIC-values. In vitro, the hydrocolloid dressing disclosed no antibacterial effects. If the practitioner prefers an occlusive dressing we believe, due to our animal and in vitro experiments, that the zinc medicated occlusive dressing will reduce the risk of wound infection in man.

A dressing system providing fluid supply and suction drainage used for continuous or intermittent irrigation.

In this article a dressing system is described that is capable of providing continuous or intermittent wound irrigation. It is based on a felt dressing provided with an adhesive cover and ports for fluid supply and suction drainage. At continuous irrigation (approximate rate, 70 ml/h), a 1-L fluid bag and a siphon about 30 cm in height are used; at intermittent irrigation (approximate rate, 60 ml/min), a 60-ml fluid bag and a suction balloon are used. In an experimental set-up it was shown that the supplied fluid diffused throughout the dressing felt and that the felt was partly saturated both during continuous and after intermittent irrigation, the effect of gravity being counteracted by capillary force and suction. The suction pressure at the drainage port and within the occlusively applied felt showed a linear relationship. The drainage of particles, while relatively impeded at low flow rates, was satisfactory at rates recommended for clinical use. The dressing felt was inert to adherence of bacteria and white blood cells. This dressing system would seem to provide access to the whole wound surface for active therapy through fluid supply and suction drainage.

Staphylococcus aureus cowan I and Branhamella catarrhalis as B lymphocyte mitogens. Culture conditions for optimal DNA synthesis and selective stimulation of human B lymphocytes.

Staphylococcus aureus Cowan I and Branhamella catarrhalis that selectively stimulate human B lymphocytes to increased DNA synthesis in serum-free culture media were shown to do so also in 3 day cultures in medium containing human serum or albumin. After this time the transformed cells expressed little or no surface-bound immunoglobulin and did not form rosettes with sheep erythrocytes. When lymphocytes were cultured with bacterial mitogens in the presence of serum or albumin for more than 3 days transformed T cells appeared. The bacteria induced antibody production mainly of IgM class in lymphocytes cultured in albumin supplemented medium.

Formalin-treated bacteria as selective B cell mitogens: results in primary and acquired immunodeficiencies.

The mitogenic activity of the formalin-treated bacterial strains Branhamella catarrhalis, Haemophilus influenzae and the Cowan I strain of Staphylococcus aureus was assessed in peripheral blood lymphocytes (PBL) from patients with primary immunodeficiencies, acute lymphocytic leukaemia (ALL), chronic lymphocytic leukemia (CLL) and in umbilical cord blood lymphocytes. The bacteria selectively stimulated B cells, as demonstrated by the finding of a normal de novo DNA synthesis in children with a T cell defect and of an absent response in X-linked agammaglobulinaemia and severe combined immunodeficiency. A decreased mitogenic activity was exerted on PBL from four out of seven adults with common variable hypogammaglobulinemia (CVH). In B-CLL the mitogenic activity was normal while in T-ALL it was decreased. Umbilical cord blood lymphocytes responded better than PBL from adults. The selective stimulative ability of the bacteria for B lymphocytes is expressed when PBL are cultured together with the formalin-treated bacteria for 48 to 72 hr.

Formalin-treated bacteria as selective B cell mitogens in the study of lymphocytes from patients with hypogammaglobulinaemia.

Staphylococcus aureus strain Cowan I, Haemophilus influenzae, and Branhamella catarrhalis that stimulate human B lymphocytes, but not T lymphocytes, were used to study the B lymphocyte function in patients with hypogammaglobulinaemia. Lymphocytes from two patients with no circulating B cells were not stimulated to increased DNA synthesis by these bacterial mitogens. The lymphocytes of six patients with hypogammaglobulinaemia and normal numbers of circulating B cells responded to the bacterial mitogens but in five patients the response was decreased in comparison with healthy controls.

Antibiotic-host defence interactions in vitro and in vivo.

A markedly depressed chemotaxis was detected with an agarose gel technique when human leucocytes were incubated with fusidic acid and rifampicin in clinically obtainable concentrations. At high concentrations of newer well absorbed tetracyclines there was a definite depression and a less pronounced inhibition was detected for classical tetracycline. The incorporation of 14C-leucine into a trichloroacetic-acid insoluble form by human neutrophils was markedly depressed by the same antibiotics and it is suggested that some antibiotics acting by inhibition of protein synthesis also affect chemotaxis of human neutrophils. At therapeutic concentrations fusidic acid and rifampicin had a pronounced inhibiting effect on the incorporation of 3H-thymidine by human T-lymphocytes stimulated by PHA and B-lymphocytes by S. aurens, Cowan I. At concentrations above the therapeutic level inhibition was detected for doxycycline, erythromycin, clindamycin and nitrofurantoin. No apparent inhibition of neither chemotaxis by human neutrophils nor thymidine incorporation by lymphocytes could be detected for penicillins, cephalosporins, nalidixic acid, sulfamethoxazole and trimethoprim. Due to high albumin binding for some of the tested antibiotics and other factors involved, experiments were performed to test whether depression also takes place in vivo. The cellular immunity in mice was registered by monitoring the survival of transplanted heart grafts and the humoral immunity by quantitating plaque-forming cells and by titration of antibodies after immunization with sheep erythrocytes. Fusidic acid (500 mg/kg/day) and rifampicin (20 mg/kg/day, human therapeutic dose) had a highly significant effect (P less than 0.001) on the rejection of heart grafts and plaque-forming cells while the effect o serum antibodies was of low significance (P less than 0.02--P less than 0.01). The effect of doxycycline (2.5 mg/kg/day) and fusidic acid (25 mg/kg/day) at human therapeutic dose on immunity in mice was slight but significant (P less than 0.02). The relevance of experiments in mice to the situation in man is discussed. The migration of neutrophils into a skin chamber was shown to be dramatically reduced in eight healthy volunteers during a standard regimen of doxycycline.

Bacteria-immunoglobulin-lymphocyte interactions--new aspects.

Of 30 bacterial species tested 18 stimulated DNA synthesis in human blood lymphocytes. The maximum response was after 3-4 days of culture suggesting a mitogenic effect. This was confirmed by the induction of polyclonal antibody production shown by a plaque assay. Most bacterial species increased the DNA synthesis in B-enriched lymphocytes and unseparated lymphocytes but had negligible activity on T-enriched lymphocytes. Among bacteria with a mitogenic effect and ability to induce polyclonal antibody production are Staphylococcus aureus, Haemophilus influenzae, Mycobacterium tuberculosis, Neisseria gonorrhoeae, Streptococcus group A and Streptococcus pneumoniae. In an attempt to define structure (s) on the B-lymphocyte surface responsible for the lymphocyte stimulation the binding of IgD, IgM, and HLA-A, -B and HLA-D antigens to different bacterial species was investigated. A high IgD binding to N. catarrhalis and H. influenzae and a moderate binding of IgD to streptococci was found. Binding studies employing radiolabelled IgD Fab- and Fc-fragments indicated that the binding probably involves the CHl-region of the IgD molecule. Three purified radiolabelled myeloma IgM M-components were all shown to be efficiently bound to many bacteria indicating that a part of the IgM molecule other than the antigen-combining site can be involved in attachment to bacteria. Highly purified detergent-solubilized HLA-A, -B and HLA-D antigens, when separately incorporated into liposomes, were bound efficiently to two strains of N. catarrhalis and to one strain of H. influenzae weakly to one strain of E. coli, but not at all to another strain E. coli. Preliminary experiments indicate that these bacteria-immunoglobulin and bacteria-HLA-antigen interactions lead to lymphocyte stimulation.

Antibiotics and suppression of lymphocyte function in vitro.

The effects on the mitogenic response of human T lymphocytes were studied for 20 different antibiotics. No apparent inhibitory effect could be detected for penicillins, cephalosporins, aminoglycosides, chloramphenicol, sulfamethoxazole, trimethoprim, nalidixic acid, and 5-fluorocytosine. There were effects at high concentrations with erythromycin, clindamycin, and rifampin, and these antibiotics could also be shown to depress the mitogenic response of B lymphocytes. With fusidic acid, nitrofurantoin, and doxycycline there was an inhibiting effect at low concentrations on the mitogenic responses of B and T lymphocytes and on in vitro antibody production. Protein synthesis in unstimulated lymphocytes was also inhibited. Some antibiotics thus may impair the function of human lymphocytes in vitro.

Binding of HLA antigen-containing liposomes to bacteria.

Highly purified, detergent-solubilized HLA-A and -B antigens and HLA-D antigens were separately incorporated into liposomes. Detergent-solubilized transplantation antigens, but not papain-solubilized antigens lacking the membrane-integrated portions of the molecules, were bound to the liposomes. A considerable portion of the liposome-bound antigens displayed accessible antigenic sites, suggesting that they were oriented in the right-side-out direction. Liposomes containing the HLA-A and -B antigens or the HLA-D antigen interacted similarly with bacteria. The two types of liposomes bound efficiently to two strains of Neisseria catarrhalis (now classified as Branhamella catarrhalis) and to one strain of Haemophilus influenzae, weakly to one strain of Escherichia coli, and not at all to another strain of E. coli. The binding between the HLA antigen-containing liposomes and one strain of N. catarrhalis was abolished when Fab fragments directed against the heavy chains of HLA-A and -B antigens or against HLA-D antigens, respectively, were added. In contrast Fab fragments against beta(2)-microglobulin did not measurably impede the bacteria-liposome interaction, suggesting that, with regard to the HLA-A and -B antigens, the heavy, but not the light, chains interacted with the bacteria. Additional experiments showed that N. catarrhalis preferentially interacted with transplantation antigens when mixed with detergent-solubilized lymphocyte membrane glycoproteins. These data suggest that HLA-A and -B and HLA-D antigens may have the function of interacting with foreign antigens such as bacteria.

Many bacterial species are mitogenic for human blood B lymphocytes.

Thirty bacterial species were tested for their ability to stimulate to increased DNA synthesis in human blood lymphocytes. A definite stimulation was obtained with eighteen bacterial species. For three of these species ten different strains of each were tested, and all increased DNA synthesis. The maximum response was after 3--4 days of culture, suggesting a mitogenic effect. This was confirmed by the induction of polyclonal antibody production shown by a plague assay, which was positive for nine of eleven species tested. Most bacterial species increased the DNA synthesis in B-lymphocyte-enriched and unseparated lymphocytes but had negligible activity on T-lymphocyte-enriched cultures. Among bacteria with a mitogenic effect and ability to induce polyclonal antibody production are Staphylococcus aureus strain Cowan I with a high content of protein A and many common human pathogens such as Haemophilus influenzae, Mycobacterium tuberculosis, Neisseria gonorrhoeae, Streptococcus group A and Streptococcus pneumoniae.