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.

[Bacterial colonization of chronic wounds. Studies on outpatients in a university dermatology clinic with special consideration of ORSA]. / Bakterielle Kolonisation chronischer Wunden. Untersuchungen in einer universitären dermatologischen Wundambulanz unter besonderer Berücksichtigung von ORSA.

In this retrospective investigation, we documented the bacterial colonization of 79 patients with chronic wounds, who had been treated between January 2002 and May 2003 in an outpatient wound healing clinic of a university dermatology program. We isolated 106 facultative pathogenic bacterial strains of which 56 were Staphylococcus aureus, 19 Pseudomonas aeruginosa, 11 Escherichia coli, 4 Proteus mirabilis, 4 Enterobacter cloacae, 2 Serratia marcescens, 2 Streptococcus group G und 8 further species. 68 of these bacterial strains were gram-positive and 46 gram-negative. Moreover we identified one patient with Candida parapsilosis. Therefore, 70.8% of all patients showed Staphylococcus aureus in their chronic wounds. Determination of the specific resistances showed 17 patients to be colonized with oxacillin- resistant Staphylococcus aureus (ORSA) strain; this corresponds to 21.5% of all patients. Consequently, 30.4% of all Staphylococcus aureus isolates were ORSA strains. All of the ORSA isolates were sensitive to vancomycin. Sensitivity to tetracycline was documented in 15, to amikacin in 13, to clindamycin in 7, to gentamicin and erythromycin in 6 of the ORSA-positive patients. In the case of trimethoprim/sulfamethoxazole, 10 were sensitive and 3 were intermediate in sensitivity. Beside the obligate resistance to oxacillin, penicillin G, ampicillin, cefuroxime and imipenem, none of the ORSA was sensitive to ofloxacin. The results of our investigations demonstrate the actual spectrum of bacterial colonization in chronic wounds of patients in an university dermatologic wound clinic and underline the growing problem of ORSA.

Endoparasite KC5/2 encloses large areas of sol-like cytoplasm within Acanthamoebae. Normal behavior or aberration?

In addition to the characteristic development of the rod-shaped or tube-like endocytobionts replicating within the sol-like cytoplasm of acanthamoebae which was published recently (Michel et al. 2003) we observed the strange behavior of the endoparasites described as follows. The material normally forming the characteristic cell walls of KC5/2 organisms was found to form a long array along the borderline between the granuloplasm and the central homogenous sol-like cytoplasm, so that about one third of the sol plasm had already been surrounded. We do not know whether this process belongs to the normal developmental repertoire of the endocytobiont that was interrupted by fixation for electron microscopy, or whether it is an aberrant behavior.

Endocytobiont KC5/2 induces transformation into sol-like cytoplasm of its host Acanthamoeba sp. as substrate for its own development.

New investigations of a novel, recently described, non-cultivable endocytobiont of Acanthamoeba sp. reveal at least three hitherto unobserved developmental stages which shed some light on the nature of this peculiar organism. The development of the endocytobiont is closely connected with conspicuous changes in the host amoeba, inducing the transformation from gel to sol-like cytoplasm which bulges like a balloon inside the host cell. Young and transitory developmental stages were found within the homogenous, sol-like cytoplasm. The infectious stages, with their voluminous cell wall and a conspicuous ostiole, could be observed within all parts of the cytoplasm with the exception of the nucleus. It is a remarkable adaptation for this parasite to be able to induce this gel-sol transformation in order to facilitate its own development. The fate of the heavily infected host amoebae is death by rupture or lysis after being overcrowded with parasites. As no structures could be observed within the endoparasites that were comparable to other bacteria, the real nature and taxonomic position of these peculiar organisms remain obscure.

Isolation and purification of chiamydospores of Candida albicans.

Two methods were investigated for their efficiency in isolating and purifying chlamydospores of Candida albicans. Chlamydospores were disconnected from pseudomycelial cells either enzymatically using beta-glucuronidase or mechanically by ultrasonic treatment. Free chlamydospores were separated from other cell material by sucrose gradient centrifugation. The resulting preparations were inspected by light-microscopy and electron-microscopy. Both methods yielded preparations with a level of over 90% chlamydospore cells. Ultrasonic treatment caused little change to the ultrastructure of the chlamydospores, whereas the enzyme treatment profoundly affected the cell wall. It is concluded that ultrasonic treatment is an efficient method for obtaining pure preparations of chlamydospores.

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.

Members of the Cytophaga-Flavobacterium-Bacteroides phylum as intracellular bacteria of acanthamoebae: proposal of 'Candidatus Amoebophilus asiaticus'.

Three Gram-negative, rod-shaped bacteria that were found intracellularly in two environmental and one clinical Acanthamoeba sp. isolates were analysed. Two endocytobionts showing a parasitic behaviour were propagated successfully outside their amoebal host cells and were identified subsequently by comparative 16S rRNA sequence analysis as being most closely affiliated with Flavobacterium succinicans (99% 16S rRNA sequence similarity) or Flavobacterium johnsoniae (98% 16S rRNA sequence similarity). One endocytobiont could neither be cultivated outside its original Acanthamoeba host (Acanthamoeba sp. TUMSJ-321) nor transferred into other amoebae. Electron microscopy revealed that the amoebal trophozoites and cysts were almost completely filled with cells of this endosymbiont which are surrounded by a host-derived membrane. According to 16S rRNA sequence analysis, this endosymbiont could also be assigned to the Cytophaga-Flavobacterium-Bacteroides (CFB) phylum, but was not closely affiliated to any recognized species within this phylogenetic group (less than 82% 16S rRNA sequence similarity). Identity and intracellular localization of this endosymbiont were confirmed by application of a specific fluorescently labelled 16S rRNA-targeted probe. Based on these findings, we propose classification of this obligate Acanthamoeba endosymbiont as 'Candidatus Amoebophilus asiaticus'. Comparative 18S rRNA sequence analysis of the host of 'Candidatus Amoebophilus asiaticus' revealed its membership with Acanthamoeba 18S rDNA sequence type T4 that comprises the majority of all Acanthamoeba isolates.

Vannella sp. harboring Microsporidia-like organisms isolated from the contact lens and inflamed eye of a female keratitis patient.

Viable Hartmannella sp. and two strains of Vannella sp.--but no Acanthamoebae--multiplied on NN-agar inoculated with pieces of the contact lens from a female keratitis patient. Within the cytoplasm of one Vannella isolate, intracellular parasites could be observed whose earliest stages were developing within the nucleus, resembling those Microsporidia-like parasites seen within Vannella isolated recently from a warm tapwater system. This assumption was also confirmed by electron microscopy. In swabs taken directly from the cornea, Pseudomonas aeruginosa were identified, but they did not yield any growth of amebas in culture. However, cocultivation of parasite-free Vannella strains with the above-mentioned swab matter resulted in infected amebas harboring the same intracellular parasites seen before. This infection could be established only if the corresponding spores were present as infective agents in the swab matter. The successful treatment of the patient with antibiotics supports the assumption that P. aeruginosa was the main cause of the corneal ulceration. The extent to which the Microsporidia-like organisms may have been involved in the development of keratitis remains a matter of discussion.

Intracellular bacteria of Acanthamoebae resembling Legionella spp. turned out to be Cytophaga sp.

Acanthamoeba sp. isolated from the drinking water system of a hospital harboured gram-negative bacteria multiplying inside phagosomes and within the cytoplasm of their host cells. According to their morphology demonstrated by electron microscopy they resembled Llaps (Legionella-like amoebal pathogens) but turned out to be Cytophaga sp. as shown by a typical profile of cellular fatty acids obtained by means of gas-liquid chromatography.

HLA-DR expression and soluble HLA-DR levels in septic patients after trauma.

OBJECTIVE: To determine if cellular and soluble HLA-DR molecules may be relevant in severely injured patients for the development of gram-positive or gram-negative sepsis. SUMMARY BACKGROUND DATA: HLA-DR molecules play a central role in the specific immune response to infection. The reduced HLA-DR expression on monocytes is considered to correlate with infectious complications and the development of sepsis. Data on the role of HLA-DR expression on T cells and soluble HLA-DR molecules are rare. METHODS: HLA-DR expression on monocytes and T cells was measured by flow cytometry. Plasma levels of soluble HLA-DR were studied by enzyme-linked immunosorbent assay. RESULTS: HLA-DR expression on circulating T cells, calculated as mean fluorescence intensity in channels, was reduced at day 1 after admission in 20 patients with subsequent severe sepsis compared with 46 patients without sepsis. The septic patients immediately after trauma had significantly lower soluble HLA-DR plasma levels than the nonseptic patients. At day 2 after admission, HLA-DR expression on monocytes was significantly lower in the severe sepsis group than in the patients without sepsis, and lasted until day 14 after injury. CONCLUSIONS: In severely injured patients, decreased levels of cellular and soluble HLA-DR appear as early indicators of an immune deviation associated with the development of severe sepsis. Moreover, immune alterations of different cell types may promote distinct kinds of septicemia.

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.

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.

Obligate intracellular bacterial parasites of acanthamoebae related to Chlamydia spp.

The phylogeny of obligate intracellular coccoid parasites of acanthamoebae isolated from the nasal mucosa of humans was analyzed by the rRNA approach. The primary structures of the 16S and 23S rRNA molecules of one strain were determined in almost full length. In situ hybridization with a horseradish peroxidase-labeled oligonucleotide probe targeted to a unique signature site undoubtedly correlated the retrieved 16S rRNA sequence to the respective intracellular parasite. This probe also hybridized with the second strain, suggesting a close relationship between the two intracellular parasites. Comparative sequence analysis demonstrated a distinct relationship to the genus Chlamydia. With 16S rRNA similarities of 86 to 87% to the hitherto-sequenced Chlamydia species, the intracellular parasites are likely not new species of this genus but representatives of another genus in the family of the Chlamydiaceae. Consequently, it is proposed to provisionally classify the endoparasite of Acanthamoeba sp. strain Bn9 as "Candidatus Parachlamydia acanthamoebae." From an epidemiological perspective, the results suggest that small amoebae could be environmental reservoirs and vectors for a variety of potentially pathogenic bacteria including members of the Chlamydiaceae.

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.

Infection by Alcaligenes xylosoxidans subsp. xylosoxidans in neutropenic patients.

Alcaligenes xylosoxidans subsp. xylosoxidans (A. x. xylosoxidans) is a nonfermenting gram-negative peritrichous rod and opportunistic pathogen. The organism is frequently found in an aqueous environment. In the past few years, nosocomial infections caused by A. x. xylosoxidans have become more evident. The literature suggests that systemic infections are severe and often lethal and an optimal antibiotic therapy is not well established. This report describes nosocomial infections in 11 patients of a hematology ward over a 2-month period. Primary infection occurred during the neutropenic phase after cytotoxic chemotherapy. Reinfection spread from central venous catheters that had been implanted before the first infection. The bacteremia was successfully treated by imipenem. None of the 11 patients died from the bacteremia, but 3 died of their underlying diseases. Despite an intensive search for the source, the route of infection remained uncertain. Nosocomial infections by A. x. xylosoxidans are of growing importance in high-risk patients. Although the source of infection often remains unknown, infection seems to originate from contaminated solutions. Treatment with imipenem and the removal of central venous catheter systems successfully eliminated A. x. xylosoxidans, which adheres to plastic material.

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.