Bacterial colonization of chronic leg ulcers: current results compared with data 5 years ago in a specialized dermatology department.

BACKGROUND: In nearly every chronic wound different bacteria species can be detected. Nevertheless, the presence of such microorganisms is not necessarily obligatory associated with a delayed wound healing. But from this initially unproblematic colonization an infection up to a sepsis can arise in some patients. The aim of our clinical investigation was to analyse the spectrum of microbial colonization of patients with a chronic leg ulcer in our specialized dermatological outpatient wound clinic, and to compare them with the results of comparable data already collected 5 years ago. OBJECTIVES: In our retrospective investigation the results of bacteriological swabs were documented in 100 patients with a total of 107 chronic leg ulcers. All patients visited the specialized wound outpatient clinic, Department of Dermatology, University of Essen in Germany. METHODS: A total of 60 patients were female, 40 were male. The mean age was 65 years. Altogether a total of 191 bacterial isolates and 25 different bacterial species could be identified. RESULTS: The most often detected species were Staphylococcus aureus (n = 60), Pseudomonas aeruginosa (n = 36) as well as Proteus mirabilis (n = 17). In 10 patients (10%) we identified a colonization with methicillin resistant S. aureus (MRSA). Merely in 6 patients the taken swabs were sterile. Five years ago a comparable investigation was already carried out in our wound outpatient clinic. At that time we could detect in particular more frequent MRSA (21.5% vs. 10%) and rarely P. aeruginosa (24.1% vs. 33.6%). CONCLUSION: The results of our investigation demonstrate the current spectrum of the bacterial colonization in patients with chronic leg ulcers in a university dermatological wound centre in comparison to the last 5 years. In our institution we were able to demonstrate a shift of the detected bacterial species from gram-positive in direction to gram-negative germs. Beside the already known problems with MRSA, in future therapeutic strategies in patients with chronic leg ulcers the increasing amount of gram-negative bacteria and especially of P. aeruginosa should considered.

Clinical experience with quinupristin-dalfopristin as rescue treatment of critically ill patients infected with methicillin-resistant staphylococci.

OBJECTIVES: To describe the efficacy and safety of quinupristin-dalfopristin (Q-D) as rescue therapy in critically ill patients with severe infections caused by methicillin-resistant staphylococci unresponsive to vancomycin treatment. DESIGN: Observational study in the context of the compassionate use programme for Q-D. METHODS: Twelve mechanically ventilated patients suffering from severe staphylococcal infections, pretreated unsuccessfully with vancomycin despite in vitro sensitivity, were included. Patients received, intravenously, Q-D 7.5 mg/kg body weight 3 times daily. The duration of Q-D therapy averaged 11.8 days (range: 1-26 days). The outcome variables were clinical efficacy and bacteriological eradication. RESULTS: Methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis (MRSE) were isolated in three patients each, and both bacteria were isolated from six patients. Eradication of pathogen(s) was achieved in 7 of 12 patients (66%). Five patients (42%) died due to severe co-morbidity. Adverse events related to Q-D were not observed and neither renal nor liver function was adversely affected. CONCLUSIONS: Quinupristin-dalfopristin appears to be an efficient and safe antimicrobial drug for the rescue treatment of staphylococcal infections in critically ill patients. It may be considered as a treatment option in cases of vancomycin treatment failure.

Fatal Salmonella enteritidis sepsis acquired prenatally in a premature infant.

BACKGROUND: Before the advent of antibiotic therapy, Salmonella typhi infection during pregnancy was associated with a high incidence of fetal and neonatal death. Little information is available about the risk to the fetus or the newborn of a pregnant woman infected by non-typhoid salmonella, and treatment recommendations do not exist. CASE: We report a case of transplacental infection of a fetus by non-typhoid salmonella in a woman with gastroenteritis. Salmonella enteritidis was cultured from stool of the pregnant woman, who had diarrhea and fever before cesarean was performed at 29 weeks' gestation. The premature girl died 4 hours after birth from septic shock. Salmonella enteritidis was cultured from blood cultures and swabs of the premature infant and from the placenta and uterus. CONCLUSION: This observation argues in favor of antibiotic treatment for non-typhoid salmonella infection in pregnancy because of the risk of transplacental infection of the fetus.

Bacteriophages in Helicobacter (Campylobacter) pylori.

Bacteriophages in different stages of maturation were found in thin sections of a clinical isolate of Helicobacter (Campylobacter) pylori. Mature phage heads measured 70 x 60 nm and the tail at least 120 nm. Lysogeny was maintained during subculture on blood agar for more than 3 months after isolation from a gastric biopsy.

Comparison of the E test and a proportion dilution method for susceptibility testing of Mycobacterium avium complex.

The newly developed E test was compared with an extended 1% proportion dilution method for determining the susceptibility of Mycobacterium avium complex (MAC) strains to amikacin, streptomycin, fusidic acid, rifampicin, clarithromycin, ciprofloxacin, ofloxacin and fleroxacin. For all antibiotics tested except clarithromycin and ciprofloxacin, no more than one strain gave a different susceptibility result with the two methods. The discrepant results occurred near the chosen breakpoint concentration of clarithromycin and outside the concentration range of the E test for ciprofloxacin. For the minimum inhibitory concentration (MIC) values obtained within the range of antibiotic concentrations tested, there was good correlation between the two methods; the MICs differed by more than one two-fold dilution in no more than two strains per antibiotic. It is concluded that the E test is suitable for susceptibility testing of MAC.

Characterisation of a Helicobacter pylori phage (HP1).

The infection of two Helicobacter pylori strains with a phage-containing supernate of the lysogenic H. pylori strain IMMi 290/89 resulted in a lytic cycle and propagation of phage HP1. In negatively-stained preparations, the empty phage heads measured 55-60 nm in diameter and mature heads measured 50 nm. The flexible, striated phage tail was c. 170 nm in length and 9.5 nm in diameter. The phage showed a mean density of 1.40 g/cm3 in sucrose-density gradients and contained double-stranded DNA c. 22,000 bp in length.

Temperate bacteriophages and polyheads produced in the L form of Staphylococcus aureus after mitomycin induction.

A lysogenic strain of Staphylococcus aureus was transformed into its protoplast L form. After stabilization and subcultivation in the L-form state for more than 200 subcultivations, it was tested for maintenance and inducibility of the temperent phage found in the parent strain. In the parent strain and L form, optimal phage production was inducible by 1 and 2 micrograms of mitomycin-C/ml. Besides regular phages with heads of approximately 35 X 85 nm, some L-form cells also produced polyheads of up to more than 1000 nm in length.

Viability and ultrastructure of S. aureus treated with fosfomycin.

Fosfomycin was administered to logarithmic growing cells of S. aureus ATCC 12600 when the number of viable cells (CFU) was about 5 X 10(7) cells/ml. Within 240 min the CFU i) increased for about 70% when 6 micrograms fosfomycin/ml were administered, ii) decreased for about 91% when 25 micrograms glucose-6-phosphate (G-6-P) were administered in addition to 6 micrograms fosfomycin/ml, and iii) decreased for about 98% when 60 micrograms fosfomycin/ml and 25 micrograms G-6-P/ml were added. By means of turbidity measurements mainly a retardation in cell reproduction could be recognized. Contrary to Gram-negative bacteria, the reduction of viable cells for about 90% was not accompanied by a loss of cell integrity in the same range. In addition to the ATCC strain this could be shown for eight further strains of S. aureus.

Bacteriophages in L form of Staphylococcus aureus.

Lysogenicity and phage typability of Staphylococcus aureus L-form cells are described. Spontaneously produced phages were found in thin sections of S. aureus L colonies. The dimensions of the tail and head resemble those of the morphological group BIII2 (T. Krzywy, I. Durlakowa, A. Kucharewcz-Krukowska, S. Krynski, and S. Slopek, Zentralbl. Bakteriol. Mikrobiol. Hyg. Abt. 1 Orig. Reihe A 250:287-295, 1981). Phages 3A and 3C of the international typing set lysed both the bacillary and L form of S. aureus.

Fosfomycin-induced protoplasts and L-forms of Staphylococcus aureus.

The potency of fosfomycin (60 micrograms/ml) to induce bacterial L-forms and protoplasts was studied on two strains of Staphylococcus aureus. On osmotically appropriate agar, L-colonies could be grown and repeatedly transferred to fresh media. The L-forms were sensitive to tetracycline and resistant to penicillin. Protoplasts were produced by treatment of S. aureus in broth for more than 6 h.

Unstable L-form of Proteus mirabilis induced by fosfomycin.

An unstable L-form of Proteus mirabilis was induced on solid medium by sensitivity test discs (200 micrograms of fosfomycin + 20 micrograms of glucose-6-phosphate). The L-colonies were subcultured on agar containing the antibiotic at a concentration of 60 micrograms/ml. On antibiotic-free medium, all the cells reverted to the bacterial phase. On antibiotic-containing agar, the reversion took place as well although at a much lower frequency. Parents and revertants differed in glucose metabolism while they reacted identically in H2S, indole, and urea tests.

Ultrastructure and viability of E. coli treated fosfomycin.

In concentrations of 6 microgram/ml Fosfomycin acted bactericidal against E. coli ATCC 10536. The sensitivity of E. coli was evaluated by turbidity measurement (Table 1) and by counting colony forming units (CFU) (Table 2). Thus the bactericidal action began at different times in respect to the concentration and the method of documentation: turbidity fell 30-120 min after the administration of 6 microgram/ml and 10-30 min after 60 microgram/ml; CFU were reduced 10-30 min after 6 microgram/ml and 3-10 min after 60 microgram/ml. Before the cytoplasm and DNA-region were disorganized with reduced electron density, some elongated (up to more than 20 micron) cells occurred (Fig. 1,2). More prominent alterations in shape and ultrastructure were obvious 120 min after 6 microgram/ml (Fig. 5) and after 30 min when 60 microgram Fosfomycin per ml were administered (Fig. 3, 4), i.e. considerably later than the reduction of reproductivity.

Ultrastructure and viability of K. pneumoniae treated with fosfomycin.

The effect of Fosfomycin on K. pneumoniae ATCC 10031 was studied. The morphology on the electron microscopical level and the viability were markedly altered after application of 6 micrograms/ml and 60 micrograms/ml of Fosfomycin, respectively. These were chosen because they can be attained in man by oral or parenteral administration. Until 30 min after the administration of 6 micrograms/ml, and 10 min after administration of 60 micrograms/ml the turbidity increased in the same range as in the control. Thereafter the turbidity decreased but did not fall below its minimal values; after application of 6 micrograms/ml of Fosfomycin the OD remained at higher levels than after applying 60 micrograms/ml of Fosfomycin, at all corresponding times. The number of viable cells, after application of 6 micrograms/ml of Fosfomycin, was maximally reduced for 70% of the value at the time of administration. 60 micrograms/ml quickly impaired the ability of reproduction. Consequently, the CFU were reduced continuously, e.g. by 80% after 30 min and by more than 99% after 180 min. The finestructural alterations were characterized by loss of contrast and regular shape. The occurrence of protruded protoplasts and defects in the cell wall indicate the action of Fosfomycin on the bacterial envelope, preferably on the peptidoglycan layer.

Fate of the Escherichia coli capsule during embedding procedures for electron microscopical studies.

The effect of the preparation steps of the TEM embedding procedure on the preservation of the Escherichia coli capsule was studied light microscopically. Electron microscopical micrographs did not distinctly reveal capsules whereas simultaneously performed India ink assays clearly revealed predominant capsules both on viable and on chemically fixed bacteria. It is clearly shown that dehydration does not cause extraction or collapse of capsular material to an extent that would explain the invisibility of the capsule on the electron microscopical level, as has hitherto been supposed.

Adhesion of Helicobacter pylori to yeast cells.

In order to get information as to whether direct interaction of H. pylori and yeasts may modulate the course of H. pylori infections, the adhesion of H. pylori to C. albicans, C. glabrata, C. guilliermondii, C. krusei, C. parapsilosis, C. tropicalis and S. cereviseae was investigated. H. pylori adhered significantly more frequently to C. tropicalis (adhesion ratio > 10%) than to the other yeasts (adhesion ratios < 5%). On an average, no significant difference to the adhesion ratios of E. coli and S. aureus was found. Electron microscopic examinations showed that H. pylori cells contacted the cells of C. tropicalis either by knob-like structures or by close surface-to-surface adhesion. Cholesterol-depleted H. pylori cells adhered to the yeast no more than cholesterol-carrying cells. There was no indication that a direct cooperation with yeasts plays a role in H. pylori infections.

Comparison of the E test and a proportion dilution method for susceptibility testing of Mycobacterium kansasii.

The newly developed E test was compared with a conventional proportion dilution method for determining the sensitivity of Mycobacterium kansasii to amikacin, streptomycin, fusidic acid, rifampicin, clarithromycin, ciprofloxacin, ofloxacin, and fleroxacin. There was no more than one strain with different rating, except for ciprofloxacin. In this case, the breakpoint concentration had an unfavourable position at the top of the strip, and susceptible isolates in the dilution test were defined resistant in the E test. It is concluded that the E test is suitable for testing slowly growing mycobacteria other than tubercle bacilli, and may replace the more laborious dilution methods, particularly for testing M. kansasii.

Improved identification of mycobacteria by using the microbial identification system in combination with additional trimethylsulfonium hydroxide pyrolysis.

The MIDI automated Microbial Identification System (MIS) uses gas chromatography (GC) analysis of whole-cell fatty acid methyl esters (FAMEs) between 9 and 20 carbons in length to characterize a wide range of bacterial genera and species, including mycobacteria. Mycolic acid cleavage products (MACPs) with chain lengths of C22 to C26 are not released by MIDI sample preparation of mycobacteria. Therefore, the MIS library search report often matches several mycobacterial species without any significant difference in the similarity indices. The problem is solved by adding trimethylsulfonium hydroxide (TMSH) instead of sodium sulfate in the last step of sample preparation, thus allowing the identification of MACPs in addition to FAMEs. Only one GC run parameter has to be changed: the temperature program must be extended from 260 to 310 degrees C. The MIS library search report for the identification of bacteria is not disturbed by TMSH. The combination of conventional library search report with the information of typical MACP patterns yields significantly better discrimination of mycobacterial species than the MIDI method allows.