Differential susceptibility of Propionibacterium acnes, P. granulosum and P. avidum to free fatty acids.

The susceptibility of 98 Propionibacterium acnes, P. granulosum and P. avidum strains isolated from acne cases and appropriate reference strains to decanoic, dodecanoic, octadeca-9,12 dienoic, and octadeca-9,12,15 trienoic acids was investigated using minimal inhibitory concentration estimation by an agar dilution techique. The tested acids showed their relative antibacterial activity in the following order: C18:3 delta 9,12,15 greater than C18:2 delta 9,12 greater than C12:0 greater than C10:0 Differences between minimal inhibitory concentration values for P. acnes, and P. avidum versus P. granulosum strains were demonstrated in the cases of octadeca-9,12 dienoic, and octadeca-9,12,15 trienoic acids.

Human cervicofacial actinomycoses: microbiological data for 1997 cases.

Actinomycoses are sporadically occurring endogenous polymicrobial inflammatory processes, in which fermentative actinomycetes of the genera Actinomyces, Propionibacterium, or Bifidobacterium act as the principal pathogens. Difficulties in diagnosing the disease in a timely and reliable fashion have led clinicians and microbiologists to grossly underestimate its medical importance. Therefore, we evaluated microbiological and selected clinical data derived from 1997 culture-positive cases of human cervicofacial actinomycoses examined in our laboratories during 1972-1999. The causative actinomycetes belonged to at least 9 different species, among which Actinomyces israelii and Actinomyces gerencseriae predominated. The well-known predisposition of male patients to acquire the disease varied with age and appeared to be especially pronounced in patients aged 20-60 years, the highest incidence being found in female patients aged 11-40 years and in male patients aged 21-50 years. The relevant procedures necessary for diagnosing human actinomycoses reliably, as well as details of their complex etiology, are discussed.

Nosocomial bacteremia due to Acinetobacter baumannii. Clinical features, epidemiology, and predictors of mortality.

To study the possible predisposing factors, clinical features, molecular epidemiology, and factors affecting mortality associated with bacteremia due to Acinetobacter baumannii, we reviewed 87 episodes of A. baumannii bacteremia occurring in 79 patients hospitalized at 2 university tertiary care centers and 4 community-based hospitals during a recent 18-month period. Plasmid DNA analysis and analysis of genomic DNA with pulsed-field gel electrophoresis was performed to investigate possible epidemiologic relationship. All patients acquired their infections in the hospital, and no seasonal variation was observed. Among patients with A. baumannii bacteremia, 91% were hospitalized in an intensive care unit, 99% had indwelling vascular catheters, 81% received prior broad spectrum antimicrobial therapy, 70% were mechanically ventilated, and 47% had major surgical procedures. In 39 cases (45%) the infection was related to indwelling vascular access devices. Other infections included pneumonia (9%), tracheobronchitis (22%), meningitis (2%), and burn wound infections (4%). Septic shock occurred in 30% of patients. All isolates were multidrug resistant. Polymicrobial bacteremia was observed in 35% of cases. The crude mortality rate was 44%. Death was considered attributable to A. baumannii bacteremia in 15 (19%) patients. All patients with pneumonia as the primary site of infection died. Using multivariate analysis, we identified 3 independent predictors of mortality: the presence of a rapidly or ultimately fatal underlying disease (p = 0.0009), septic shock at the onset of bacteremia (p = 0.0013), and mechanical ventilation (p = 0.016). Epidemiologic typing revealed that 82 episodes were associated with different hospital outbreaks of infection, and only 7 episodes were due to epidemiologically unrelated strains.

Inhibition of liver tumor cell colonization in two animal tumor models by lectin blocking with D-galactose or arabinogalactan.

Repeated administration of the hepatic lectin blocking agents D-galactose or arabinogalactan completely prevented the settling of metastatic cells of sarcoma L-1 tumor in the liver of Balb/c mice and greatly reduced the colonization process of highly metastatic ESb lymphoma cells of the liver of DBA/2 mice. Therefore, when hepatic lectins were blocked with competitive glycoconjugates, tumor cell colonization of the liver could be prevented in two different model systems.

Stimulatory effect of Propionibacterium granulosum KP-45, glucan and pyran copolymer on the activity of natural killer (NK) cells in murine lungs.

Propionibacterium granulosum KP-45, glucan and pyran copolymer stimulated the elimination of 75Selenomethionine-labelled 3LL tumor cells from murine lungs, as measured 4 hr after intravenous injection of these cells into 16- to 25-week-old B6DF1 mice. This effect was most pronounced 4 to 6 days following intravenous administration of the above biological response modifiers and disappeared 6 to 8 days later. Intraperitoneal injection of all three agents produced only insignificant stimulation results. Spontaneous clearance of 3LL cells from lungs of 8-week-old B6DF1 mice was significantly more effective than in animals over 16 weeks old. Cyclophosphamide suppressed the elimination of tumor cells from lungs in both young and older mice and neutralized the stimulatory effect of P. granulosum KP-45 and glucan. The results suggest that the effector cells responsible for the clearance of radiolabelled 3LL cells from lungs of B6DF1 mice are at least similar to natural killer (NK) lymphocytes.

Investigation of a rifampin, fusidic-acid and mupirocin releasing silicone catheter.

After strict hygienical measures have been exhausted the use of plastic materials with antibacterial activity may reduce catheter related-bacterial colonization. An antimicrobial silicone catheter was investigated by HPLC-measurement, SEM, antimicrobial assays and standard biocompatibility tests. The modified catheter was highly biocompatible and the antimicrobial leaching non-toxic. The initially release rate was governed by the drug solubility in the 'sink' and surface loading ('burst effect'). The second continuous period depended on the drug velocity in the silicone matrix and was extended up to 100 days with a proportionality to square root of t for each drug. Diffusion exponents were in range of 2 x 10(-8) to 1 x 10(-9) (cm2 sec(-1)). The lower diffusion exponent of mupirocin was explained by its higher cohesion energy and lower physico-chemical compatibility with the embedding silicone. The antimicrobial drugs were in a molecular-dispersed state with the silicone-matrix, whereas superficially located crystals of the antibiotics covering the catheter surface could be demonstrated by SEM.

In-vitro efficacy of an antibiotic releasing silicone ventricle catheter to prevent shunt infection.

Infection due to implanted polymeric devices is a major problem in modern medicine. Microbial colonization of implants in neurosurgery, e.g. cerebrospinal fluid (CSF) shunts is the main reason for their failure, and often results in the consequent removal of the infected implants. In this paper we report on new approaches in the prevention of bacterial infections by incorporation of an antibiotic (rifampicin) into the polymer devices (silicone). Drug release characteristics are investigated, and the physico-chemical mechanism of the delivery is discussed. Measurements of killing kinetics and the bacterial adhesion to the antibiotic-loaded silicone in a static adhesion assay reveal that only the liberation of high antibiotic doses over a period of weeks can prevent the bacterial colonization of the polymeric surface.

Controlled release of antibiotics from biomedical polyurethanes: morphological and structural features.

Polymer-associated infections are of increasing importance. Antistaphylococcal antimicrobial substances (ciprofloxacin, gentamycin, fosfomycin, flucloxacillin) were incorporated into polyurethanes by the solvent casting technique. Drug release rates, bacterial colonization and morphological features were evaluated to predict and understand the antimicrobial activity of these delivery systems. Drug release characteristics were investigated by standard bioassay and high-performance liquid chromatography (HPLC), and the physico-chemical mechanisms of the delivery were discussed. Ciprofloxacin hydrochloride showed a fast initial release rate, whereas gentamicin-base was characterized by a more continuous release type of behaviour. Bacterial colonization to the antibiotic-loaded polyurethanes was inhibited effectively by preparations showing a slower but more sustained antimicrobial delivery. Polyurethane-antibiotic combinations were most homogeneous for gentamicin-base and flucloxacillin as shown by scanning electron microscopy (SEM). In polymers loaded with fosfomycin and ciprofloxacin a granular structure of the crystallized drug embedded in the polyurethane matrix could be demonstrated. Physico-chemical similarity of the polymeric material and the antibiotics is important for the homogeneity of polymer-antibiotic combinations. High homogeneity is required for a sustained and prolonged release over time and effective inhibition of bacterial colonization.

Plasmid DNA profiles of Acinetobacter baumannii: clinical application in a complex endemic setting.

OBJECTIVE: To study the epidemiological, microbiological, and clinical features of infections due to Acinetobacter baumannii in a complex endemic situation over an 18-month period and to determine the clinical usefulness of plasmid DNA analysis of A baumannii in epidemiological investigations. DESIGN: Review of medical and laboratory records. Antibiotic resistance patterns, biotyping, and plasmid profile analysis were used to characterize clinical and environmental isolates. Pulsed-field gel electrophoresis (PFGE) of chromosomal DNA was performed to verify results obtained with the other typing methods. SETTING: Four different intensive care units of an 800-bed tertiary care center in Cologne, Germany. RESULTS: 240 patients were colonized or infected with A baumannii during the study period. No seasonal variations were observed. The majority of isolates (53%) were recovered from the respiratory tract. Major infections occurred in 61 patients; these included 48 bacteremias and eight pulmonary infections. Five different epidemic strains were identified: one each was A baumannii biotype 2 and 6, and three were biotype 9. A baumannii biotype 9 accounted for the vast majority of isolates (88%), which were clustered into three epidemic strains demonstrating distinct plasmid profiles. Two of these were considered genetically related as shown by PFGE. Epidemic strains were multidrug resistant, being uniformly susceptible to imipenem only. An epidemiological investigation failed to identify any point source of infection. Barrier precautions and improved handwashing was instituted in three of the four units and significantly reduced the incidence of colonization and infection in these units. Attack rates remained unchanged, however, in the burns unit where control measures were not implemented. CONCLUSION: Acinetobacter strains representing multiple biotypes and plasmid types were present in this endemic setting. Multidrug resistance in A baumannii is an important concern. Plasmid DNA analysis proved to be useful in epidemiological typing of A baumannii strains and may serve as a complementary typing system to traditional epidemiological methods.

Combined immunostimulation (Propionibacterium avidum KP 40) and anticoagulation (heparin) prevents metastatic lung and liver colonization in mice.

The antineoplastic activity of Propionibacterium avidum KP-40 and its enhancement by anticoagulation with heparin was studied. In Balb/c mice syngeneic sarcoma L-1 exclusively caused tumor colonization of the lungs. After neuraminidase treatment the organotropism of this tumor was changed, with tumor nodules developing in lung and liver. After single systemic application of Propionibacterium avidum KP-40 the number of lung and liver colonies decreased evidently. Combination of this immunomodulating therapy with temporary anticoagulation resulted in further reduction of tumor colonies in lung and liver.

A 2.5-year follow-up of local immunotherapy of advanced stomach and intestinal adenocarcinoma with Propionibacterium granulosum.

Intratumoral injections of 10 mg cells walls from Propionibacterium granulosum strain KP-45 (PG) were applied in 14 patients with advanced, metastatic stomach (five cases) and colorectal (nine cases) adenocarcinoma. Each patient had his own "twin" control. All patients received no other anticancer treatment, except analgetics and/or palliative surgery. Treatment with PG resulted in partial regression of tumors accompanied by improvement of the clinical state of these patients as well as the reappearance of normal values in blood count biochemical parameters. In each pair of twin cases, survival of the PG-treated patient was longer than the untreated control. The mean survival of PG-treated patients was 23.5 months (4 of 14 patients being still alive after 2.5 years follow-up), while all control patients died with a mean survival period of 6.4 months. The difference between these two patient groups of about 17 months is significant (p greater than 0.01).

Small-cell lung cancer and immunochemotherapy with Propionibacterium granulosum KP 45.

Seventy-nine patients with small-cell lung cancer were treated with vincristin, methotrexate, and cyclophosphamide in inductive therapy and with methotrexate, cyclophosphamide, and procarbazine in maintenance therapy. Patients were divided at random into two groups: one group received chemotherapy alone and the second group was additionally subjected to systemic immunotherapy with Propionibacterium granulosum strain KP-45. In general, differences in the frequency of therapy response and in duration of remission could not be stated between the two groups of patients, but patients responding to chemotherapy showed a significantly longer remission time and lower complication rates. This benificial effect of chemoimmunotherapy is not related to a direct antitumor activity of the immunomodifier used, but to the lowered risk of myelosuppression and infections. Immunomodulation in combination with chemo- and/or radiotherapy can be recommended for the treatment of small-cell lung cancer.

Inhibition of liver metastasis in mice by blocking hepatocyte lectins with arabinogalactan infusions and D-galactose.

According to our hypothesis, organ-specific lectins (e.g., the D-galactose-specific hepatic binding protein) play an important role in the organ location of metastatic malignant cells. The rapid clearance and uptake by the liver of tritiated alpha 1-acid-(asialo)glycoprotein from the circulation of Balb/c mice was markedly delayed after preinjection of D-galactose or arabinogalactan. The preinjection (1 h) and regular application (for 3 days after tumor cell inoculation in Balb/c mice) of the receptor blocking agents D-galactose and arabinogalactan prevented the settling of sarcoma L-1 tumor in the liver completely, but did not influence the settling in the lung. Other galactans, dextrans, and phosphate-buffered saline showed no effect. Therefore, when lectins were blocked with competitive-specific glycoconjugates, colonization was prevented.

Glycoprotein modifications of sarcoma L-1 tumor cells by tunicamycin, swainsonine, bromoconduritol or 1-desoxynojirimycin treatment inhibits their metastatic lung colonization in Balb/c-mice.

Synthesis and expression of cell surface carbohydrates appear to be involved in recognition events associated with tumor invasion and metastasis. Thus, the potential of murine sarcoma L-1 cells to form experimental lung metastases after i.v. injection was assessed after inhibiting tumor cell protein glycosylation with tunicamycin, swainsonine, bromoconduritol, or 1-desoxynojirimycin. Incubation of sarcoma L-1 cells with 0.5 microgram (or above) of these substances/ml medium for 20-24 h significantly inhibited lung colonization. Cytotoxic side effects or additional organ manifestations could not be found. Gas liquid chromatographic examinations of carbohydrates from treated L-1 cells indicated that sugar synthesis was evidently inhibited. These results suggest that specific glycan structures on tumor cells are required for expression of the metastatic phenotype.

Beneficial response of local immunotherapy with Propionibacterium granulosum KP-45 in combined treatment of inflammatory breast carcinoma.

Nine 44- to 67-year-old patients with inflammatory breast carcinoma were treated over 2 weeks with intratumoral injections of Propionibacterium granulosum KP-45 (KP). This period of immunotherapy was succeeded by four courses of chemoimmunotherapy (FAC: 5-fluorouracil, adriamycin, and cyclophosphamide + intratumoral KP). Inflammatory symptoms disappeared in three patients during immunotherapy and in the remaining six patients during the following chemoimmunotherapy. Finally, 3 to 4 months after starting the therapy, all nine patients were free from inflammatory symptoms and it became possible to perform radical (seven cases) or simple (two cases) surgery. Thereafter routine therapy (radiotherapy, fractionated dose of 5500 R, followed by 10 FAC courses + single injections of KP for each FAC course) was used. After 19 to 32 months observation time all patients are still in complete remission with no local recurrences. Only one patient showed distant metastases during the observation period.

The in vivo cytostatic effect induced by Propionibacterium granulosum on murine tumor cells.

The cytostatic action of Propionibacterium granulosum was studied in a mouse sarcoma in vivo. Kinetic analysis of tumor cells 28 days after tumor implantation and systemic immunotherapy showed that the cell cycle time was identical in both treated and untreated tumors. P. granulosum treatment resulted in a marked prolongation of the S phase and shortening of the G1 phase of the cell cycle. A pronounced drop in the number of labeled interphases and the reduction of the growth fraction were observed in tumors obtained from mice given injection of P. granulosum. Cloning efficiency of tumor cells from P. granulosum treated animals was quantitatively similar to that of control animals and only differences in the size of lung colonies were observed.

A DNA-probe for the detection of the species Staphylococcus haemolyticus.

A 1.3-kb DNA fragment isolated from Staphylococcus haemolyticus strain DSM 20264 can be used as a specific probe for this species. The probe hybridized with 39 clinical isolates of S. haemolyticus but not with any of the 121 isolates representative of the other 25 species of staphylococci described to date.

Identification of Staphylococcus epidermidis using a 16S rRNA-directed oligonucleotide probe.

Staphylococcus epidermidis is the most medically significant of the coagulase-negative staphylococci. An oligonucleotide probe (pSe) for identification of S. epidermidis was defined by comparing the sequences of the 16S rRNA variable region V6 from numerous coagulase-negative staphylococci. In order to increase the sensitivity of the detection, polymerase chain reaction amplification of the variable region with primers based on the conserved flanking sequences was applied. The detection limit of the polymerase chain reaction assay combined with pSe probe was shown to be 1 fg which corresponds to about one single bacterium. Additionally, a sensitive, non-radioisotopic system with chemiluminescence detection was tested.

Comparative analysis of a biofilm-forming Staphylococcus epidermidis strain and its adhesion-positive, accumulation-negative mutant M7.

We have isolated a stable slime-negative mutant, M7, from the wild-type Staphylococcus epidermidis RP62A by mitomycin mutagenesis. Besides its inability to produce slime in the test tube this mutant differed also in two other properties from its parent strain: it lacked the ability to accumulate on a surface, and it did not produce a 115 kDa and a 18 kDa extracellular protein. In all other tested properties such as initial adherence, growth rate, cell-wall composition, surface characteristics, DNA restriction profile, the presence of a 29 kb antibiotic resistance plasmid, and antimicrobial susceptibility profile, M7 was indistinguishable from its wild-type. The mutant is an important basis for further study of the pathogenesis of polymer-associated S. epidermidis infections.

Evaluation of the lysostaphin-susceptibility test for the classification of staphylococci.

A collection of 39 staphylococcal strains of known peptidoglycan structure was cultivated on media supplemented with different peptones or with glycine or inosine. The susceptibility to lysostaphin 50 mg/litre was then checked by a plate-dilution method. The results of the test were dependent on the conditions of growth. Another collection of 403 staphylococcal strains from international collections, clinical material and healthy human skin was also tested for susceptibility to lysostaphin. No uniform pattern of the lysostaphin sensitivity in individual staphylococcal species was found. The lysostaphin-susceptibility test thus seems to be of little value in the classification of staphylococci for the purposes of medical microbiology.