Early infections in patients undergoing bone marrow or blood stem cell transplantation--a 7 year single centre investigation of 409 cases.

Infections are a major cause of morbidity and mortality in patients undergoing high-dose therapy and subsequent autologous or allogeneic haemopoietic stem cell transplantation, despite the change from topical to systemic anti-infection prophylaxis and the introduction of growth factors and new antimicrobial drugs. We report our single centre experience with data from 409 patients treated at our unit from its opening in 1990 until May 1997. Three hundred and seventy-eight patients were transplanted for the first time, 12 patients were retransplanted or boosted and 19 patients were readmitted for miscellaneous reasons. 245 patients were allografted and 157 autografted. Antimicrobial prophylaxis was mainly quinolones, fluconazole plus amphotericin-B orally, aciclovir, and TMP/SMX or pentamidine. Three hundred and nineteen (78%) developed fever of significantly longer duration in the allogeneic setting with anti-CMV seropositivity. The most frequent infection was fever of unknown origin (50.6%), followed by septicaemia (12.5%) and pneumonia (11.0%). Pathogens isolated in 24.6% of the infections were mostly gram-positive bacteria (57.9%), followed by non-fermenting rods (11.2%), Aspergillus spp. and Candida spp. (10.3%, each). Cumulative response rate to antimicrobial therapy was 66.9%. Infections were responsible for 62.5% (25/40) of deaths after transplantation. Predominant pathogens were Aspergillus spp. (11), Candida spp. (four), and Pseudomonas spp. (three). None of the patients died from gram-positive bacterial infection. The risk of dying from infection was 11.2% after allografting and 0.8% after autotransplantation. Infections remain a major risk for early death after allogeneic transplantation of haemopoietic stem cells. Infection with gram-negative bacteria can be prevented by quinolone prophylaxis. Predominant pathogens are Aspergillus spp. Candida spp. and nonfermenting rods. Systemic infection with these pathogens is associated with a poor prognosis. Antimycotic prophylaxis and the therapy must be improved.

Demonstration of formaldehyde dehydrogenase activity in formaldehyde-resistant Enterobacteriaceae.

Clinical isolates of different Enterobacteriaceae strains and genetically modified variants which were resistant to the disinfectant formaldehyde were investigated. In cell-free extracts of all formaldehyde-resistant strains a glutathione-dependent formaldehyde dehydrogenase activity was demonstrated. In contrast cell extracts from formaldehyde sensitive strains did not show any formaldehyde dehydrogenase activity. The enzymatic degradation of formaldehyde seems to play an important role in formaldehyde resistance.

Nosocomial outbreak of vancomycin-resistant Enterococcus faecium at a German university pediatric hospital.

Nosocomial Infections caused by vancomycin-resistant enterococci (VRE) are an emerging threat to critically ill patients. At the University Hospital Eppendorf, VRE were isolated from 38 patients between August 1993 and April 1997, of whom 32 were hospitalized at the Department of Pediatrics. Pulsed-field gel electrophoresis revealed that 26 Enterococcus faecium isolates from patients of the Department of Pediatrics were identical or closely related, and that isolates from three additional patients of the same department were possibly related. All of these isolates were of vanA genotype. They were resistant to glycopeptides, ampicillin, ciprofloxacin, clindamycin, and erythromycin. Most isolates displayed high-level resistance to gentamicin, but all remained susceptible to quinupristin/dalfopristin. Implementation of stringent hand disinfection and environmental disinfection policies, as well as measures for patient isolation contained this first outbreak of VRE at a German Children's hospital, which emphasizes the importance of hygienic measures for the control of nosocomial spread of these organisms.

Synthesis and antibacterial activity of 5- and 6-hydroxy substituted 4-aminoquinolines and derivatives.

The synthesis and antimicrobial activity of 4-amino-8-methylquinolines 8, 11 substituted with a hydroxy- or methoxy-group at 5- and at 6-position have been investigated. The title compounds could be prepared by a six-step procedure according to the literature starting from commercially available anilines 1. The novel 4-aminoquinolines 8,11 exhibited slight antibacterial activity against Gram-positive and Gram-negative bacteria.

[Epidemiology and reasons for microbial resistance to biocides]. / Epidemiologie and Ursachen mikrobieller Biozidresistenzen.

In the last years the significance of microbial resistance to biocides like toxic heavy metal ions, aldehydes, phenoles, and cationic surface active agents has increased. However the genetical and functional mechanisms of the resistance to most of these compounds are still unknown. Till now it was possible to demonstrate a plasmid resistance in only some bacterial strains which were resistant to heavy metal ions, hexachlorophene, formaldehyde, benzalkoniumchloride, and chlorhexidine. In mercuric resistant Staphylococcus aureus and formaldehyde resistant E. coli strains the resistance genes are now well characterized. These strains are able to produce enzymes which degradade the disinfectant. The resistance to cationic surface active agents seems to depend on a modification of the bacterial cell wall. This is concluded from former investigations with benzalkoniumchloride resistant Pseudomonas strains, where it was possible to demonstrate different lipid contents in the cell wall from resistant and sensitive strains.

Formaldehyde-resistance in Enterobacteriaceae and Pseudomonas aeruginosa: identification of resistance genes by DNA-hybridization.

A 4.1. Kb large DNA fragment of a E. coli plasmid pVU 3695, on which the genes for formaldehyde-resistance are located, was used as a DNA probe to identify bacteria that carry this segment among formaldehyde-resistant bacteria. It was shown by Southern Blot-, Dot Blot-, and Colony Blot- Hybridization studies that the DNA of all formaldehyde-resistant E. coli, Serratia marcescens, Enterobacter cloacae, Citrobacter freundii and Klebsiella pneumoniae strains tested hybridize with the DNA probe from E. coli. In contrast the E. coli DNA probe does not hybridize with the DNA from formaldehyde-resistant Pseudomonas aeruginosa strains.

[Transmissible formaldehyde resistance in Serratia marcescens]. / Ubertragbare Formaldehydresistenz bei Serratia marcescens.

It was possible to isolate a strain of Serratia marcescens from fresh clinical bacterial isolates which was 4-6 times more resistant against formaldehyde than other strains. It was shown that the strain harbours two plasmids with molecular sizes of 58- and 90 Mdal. It was demonstrated by conjugation-, transformation- and plasmid-curing experiments that the formaldehyderesistance is plasmid mediated and transferable to E. coli. It was shown by labelling with 14C-formaldehyde that the resistant strains bind much more formaldehyde than the sensible strains.

Plasmid-mediated formaldehyde resistance in Escherichia coli: characterization of resistance gene.

The formaldehyde resistance mechanisms in the formaldehyde-resistant strain Escherichia coli VU3695 were investigated. A large (4.6-kb) plasmid DNA fragment encompassing the formaldehyde resistance gene was sequenced. A single 1,107-bp open reading frame encoding a glutathione- and NAD-dependent formaldehyde dehydrogenase was identified and sequenced, and the enzyme was expressed in an in vitro assay and purified. Amino acid sequence homology studies showed 62.4 to 63.2% identity with class III alcohol dehydrogenases isolated from horse, human, and rat livers. We demonstrated that the resistance mechanism in the formaldehyde-resistant strain E. coli VU3695 and in other formaldehyde-resistant members of the family Enterobacteriaceae is based on the enzymatic degradation of formaldehyde by a formaldehyde dehydrogenase.

In vitro evaluation of the antibacterial activity of beta-triketones admixed to Melaleuca oils.

The in vitro antibacterial properties of mixtures of Australian tea tree oil and niaouli oil after adding the beta-triketone complex isolated from manuka oil were tested. MIC and MBC values for four different bacteria were determined applying the broth dilution method. Both Melaleuca oil mixtures showed good antimicrobial effects against Staphylococcus aureus and Moraxella catarrhalis, exceeding the effectiveness of myrtol, which is well established in the treatment of acute and chronic bronchitis and sinusitis. The death kinetics of S. aureus were determined to draw subtle comparisons between the mixtures. The kill rate data indicated that both Melaleuca oil mixtures achieved a complete kill within 240 min.

Molecular characterization of a small Haemophilus influenzae plasmid specifying beta-lactamase and its relationship to R factors from Neisseria gonorrhoeae.

The ampicillin-resistant Haemophilus influenzae strain Ve445 which caused purulent meningitis and septicaemia in a newborn child in Germany contained a 4.4 megadalton (Mdal) plasmid (pVe445) and produced a TEM type beta-lactamase. The transformation to ampicillin resistance of a sensitive Escherichia coli strain with isolated pVe445 DNA proved that the structural gene for the beta-lactamase resided on this plasmid genome. Molecular DNA-DNA hybridization studies and electron microscope DNA heteroduplex analysis indicated that pVe445 probably contained 38 to 41% of the ampicillin translocation DNA segment (TnA) found on R factors of enteric origin. The TnA fragment present in pVe445 most likely does not contain both of the inverted repeat sequences of TnA. DNA-DNA polynucleotide sequence studies indicated that the 4.4 Mdal plasmid pVe445 was unrelated to the 30 to 38 Mdal H. influenzae R plasmids but was closely related to the 4.1 Mdal ampicillin resistance specifying H. influenzae plasmid RSF0885 isolated in the U.S.A. The H. influenzae plasmid pVe445 shared 91% of its base sequences with the beta-lactamase specifying Neisseria gonorrhoeae plasmid pMR0360 (4.4 Mdal) and had 85% of its base sequences in common with the beta-lactamase specifying N. gonorrhoeae plasmid pMR0200 (3.2 Mdal). All of the four 3.2 to 4.4 Mdal beta-lactamase specifying R plasmids of H. influenzae and N. gonorrhoeae investigated probably have a common evolutionary origin.

Molecular nature of two Haemophilus influenzae R factors containing resistances and the multiple integration of drug resistance transposons.

The 36-megadalton Haemophilus influenzae R plasmid pHK539 was found to specify resistance to tetracycline (Tc) and ampicillin (Ap). It was shown by molecular hybridization studies and by electron microscopy that the plasmid pHK539 contained the tetracycline translocation deoxyribonucleic acid (DNA)segment (TnTc) as well as the ampicillin translocation segment (TnAp). The TnAp was integrated in the stem of TnTc. The 34-megadalton H. influenzae R plasmid pRI234 carried a translocatable DNA segment which specified both tetracycline and chloramphenicol (Cm) resistance. Self-annealing and DNA-DNA heteroduplex experiments indicated that this transposon is probably composed of TnTc containing an insertion of a chloramphenicol resistance transposon (TnCm). TnCm is inserted into one of the components of the TnTc inverted repetitions and is itself flanked on both sides by long inverted repetitions. The H. influenzae plasmids pHK539 and pRI234 had more than 60% of their polynucleotide sequences in common with all the other 30- to 40-megadalton R factors recently found in H. influenzae isolates from different countries. The tetracycline-chloramphenicol resistance transposon of pRI234 was integrated twice at different sites in the plasmid after its growth in medium containing tetracycline. The presence of the two copies of the transposon was correlated with higher minimum inhibitory concentrations against tetracycline as well as against chloramphenicol. After its growth in medium containing tetracycline, the H. influenzae R plasmid pFR16017 specifying Tc resistance contained one, two, three, or even four copies of TnTc integrated at different sites in the plasmid, or the loop of TnTc was amplified. The heterogeneity of the pFR16017 plasmid was seen in all single-colony isolates and correlated with a higher minimum inhibitory concentration against tetracycline.

[Infectious resistance to antibiotics in Haemophilus influenzae (author's transl)]. / Infektiöse Antibiotikaresistenz bei Haemophilus influenzae.

Ampicillin-resistant Haemophilus influenzae does occur now in the FRG. In one isolate a plasmid with resistance genes (R-factor) could be demonstrated as cause of the ampicillin resistance. This R-factor influences production of a beta-lactamase of the TEM type which destroys ampicillin. The infectious nature of the ampicillin resistance was proven by the fact that it was transferable to other bacterial species through cocultivation. Parallel to ampicillin resistance tetracycline resistant Haemophilus influenzae has occurred in the FRG. Here the resistance was equally bound to plasmids. These R-factors are infectious as well. Molecular analysis of the 3 isolated resistance factors in Haemophilus influenzae showed that they carry the same resistance genes which are known from R-factors of Enterobacteriaceae. In the therapy of purulent infections due to Haemophilus influenzae such as childhood meningitis one can no longer rely on general ampicillin sensitivity of the offender. Apart from ampicillin and tetracycline resistant Haemophilus influenzae chloramphenicol resistance has been observed in a few cases.

Molecular properties of transmissible R factors of Haemophilus influenzae determing tetracycline resistance.

The tetracycline-resistant Haemophilus influenzae strains LU121 and FR16017, recently isolated in West Germany, each harbour a plasmid; that of the former (pLU12U) has a mol. wt of 31.5 X 10(6) and that of the latter (pFR16017) has a mol. wt of 33 X 10(6). Conjugation and DNA-DNA hybridization studies have shown that both plasmids are self-transmissible and carry tetracycline-resistance genes. The purified plasmid DNA of H. influenzae strain LU121 transformed a sensitive Escherichia coli strain to tetracycline resistance. The two R factors are closely related to the H. influenzae plasmid specifying ampicillin resistance (pKRE5367). Electron microscope DNA heteroduplex analysis indicated that pLU121 and pFR15017 probably carry the tetracycline-resistance transposon TnD and that pKRE5367 probably carries the ampicillin-resistance transposon TnA. There is more than one integration site for the insertion which probably represents TnD in pFR15017. All three plasmids have a similar plasmid core and could have a common evolutionary origin.

Plasmid-mediated formaldehyde resistance in Serratia marcescens and Escherichia coli: alterations in the cell surface.

The plasmid-mediated formaldehyde resistance of Serratia marcescens and Escherichia coli was examined. For that purpose the outer membranes of isogenic strains (with and without resistance plasmid) were compared. No quantitative or immunological differences in lipopolysaccharide of resistant and sensitive strains were noted. By contrast analysis of outer membrane proteins revealed that the sensitive variants had a higher protein content than the resistant strains. When outer membrane proteins were separated by SDS-PAGE the number of bands seemed identical for sensitive and resistant strains but the intensity of some of the bands was greater for the sensitive isolates. In addition, the surface hydrophobicity was greater for the resistance than for the sensitive strains. These findings suggest that the formaldehyde resistance plasmid of Serratia marcescens confer changes in cell surface proteins and surface hydrophobicity.

A comparative study of the in vitro antimicrobial activity of tea tree oils s.l. with special reference to the activity of beta-triketones.

The in vitro antibacterial and antifungal activities of Australian tea tree oil, cajuput oil, niaouli oil, kanuka oil and manuka oil as well as of a beta-triketone complex isolated from manuka oil were investigated in a constituent-oriented study. The compositions of the oils were analysed by capillary GLC and GLC-MS. The MICs for sixteen different microorganisms were determined applying the broth dilution method. Australian tea tree oil showed the best overall antimicrobial effect. The best inhibitory effects on Gram-positive bacteria and dermatophytes were achieved with manuka oil due to its beta-triketone content.

Origin of Haemophilus influenzae R factors.

The Haemophilus influenzae R plasmids specifying resistance against one, two, or three antibiotics which have emerged in different parts of the world were shown to have closely related but not identical plasmid cores. The gene for ampicillin resistance in the H. influenzae plasmid pKRE5367 is part of a transposon similar to Tn3, which was transposed from pKRE5367 onto RSF1010 in Escherichia coli. An indigenous H. influenzae plasmid (pW266) was isolated. Its properties correspond to those of the H. influenzae R plasmids, except for the presence of a drug resistance transposon. The in vitro-generated H. influenzae R plasmids carrying an ampicillin resistance transposon, a tetracycline resistance transposon, and a transposon for combined tetracycline-chloramphenicol resistance resembled the natural isolates. The findings support the hypothesis that the R plasmids of H. influenzae are of multiclonal evolutionary origin.

Pulsed-field gel electrophoresis for the analysis of Listeria monocytogenes infection clusters after kidney transplantation.

Listeria monocytogenes causes a rare, life-threatening infection in recipients of transplanted organs. We used cultures of blood and cerebrospinal fluid to characterize isolates and to distinguish cases in clusters from what might have been sporadic cases. From December 1994 to November 1995, six systemic L. monocytogenes infections occurred at our renal-transplantation unit. We confirmed the clinical diagnosis with blood and cerebrospinal fluid cultures and characterized the isolates retrospectively with pulsed-field gel electrophoresis (PFGE), phage-typing, and serotyping. We also performed an environmental investigation (food, drug, and stool). We took samples after the first two L. monocytogenes infections and then after cases three and four occurred. All patients recovered completely, and no graft was lost. Four patients had identical or genetically related L. monocytogenes isolates in PFGE (type A) and serotyping (type 1/2b). The other two had PFGE type B and G. L. monocytogenes was not detected in food or drug samples from patients on the renal-transplantation ward or in stool samples from the ward staff. It was concluded that PFGE allows sporadic cases and cluster cases of L. monocytogenes infection to be distinguished.

Fatal pulmonary hemorrhage in patients with acute leukemia and fulminant pneumonia caused by Stenotrophomonas maltophilia.

Pulmonary hemorrhage is a rare cause of death in patients with acute leukemia. Within a 3-month period we observed three such cases, all of which were associated with the gram-negative opportunistic pathogen Stenotrophomonas maltophilia. Since fatal lung bleeding had previously not been observed in conjunction with this organism, we collected the data from all patients with documented S. maltophilia infections or colonizations of the past year and analyzed the risk factors for a lethal outcome. A total of eight patients were identified. In the three patients with fatal hemorrhage, the interval between first complaints (chest pain, cough, fever) and death from lung bleeding was 36-72 h. A fourth patient with acute leukemia died of nonhemorrhagic respiratory failure 9 days after developing S. maltophilia-associated pneumonia. All four patients had received intensive chemotherapy and were severely neutropenic and thrombocytopenic. Such a combination of predisposing factors was not observed in the four patients with nonfatal infections or colonizations. Pulsed-field gel electrophoresis demonstrated that the infections were unrelated. S. maltophilia was also isolated from a faucet in a patient's room, but the strain isolated was genetically different from the strain causing the patient's pneumonia. Our data suggest that severe bone marrow aplasia and a recent history of intensive chemotherapy are predisposing factors for the development of fulminant hemorrhagic S. maltophilia pneumonia. Since some of the infections and colonizations developed despite prophylactic administration of antibacterial agents with documented in vitro activity against the pathogen and none was controlled by such agents, it is clear an efficient treatment strategy needs to be developed.

[Recurring meningitis caused by Streptococcus pneumoniae during preventive penicillin therapy]. / Rezidivierende Meningitis durch Streptococcus pneumoniae unter Penicillin-Prophylaxe.

It is generally accepted, that Streptococcus pneumoniae is very sensitive to penicillin G; minimal inhibitory concentration (MIC) is normally about less than = 0.01 microgram/ml. Some years ago strains relatively resistant to penicillin (MIC 0.1 to 1.0 microgram/ml) were reported on. In 1977 strains isolated from blood and cerebrospinal fluid in South-Africa were found to have a higher resistance to penicillin (MIC 0.5-4 micrograms/ml). We report on an 6-year-old girl with septicemia and meningitis caused by a strain of S. pneumoniae relatively resistant to penicillin (MIC 0.5 microgram/ml). Aged 5 years the girl had a first meningitis caused by S. pneumoniae. The girl was then treated with penicillin (450,000 IU/d) to prevent a new infection. During this time the second meningitis caused by S. pneumoniae took place. In the agarose gel electrophoresis a plasmid was found (4.2 X 10(6) Dalton). No beta-lactamase-activity was detected (nitrocefin-test and acidimetric measurement). It is unlikely that there is a plasmid-dependent resistance to penicillin.

Nosocomial infections by Listeria monocytogenes: analysis of a cluster of septicemias in immunocompromised patients.

From December 1994 to November 1995 an unusual accumulation of Listeria infections occurred at the University Hospital Hamburg-Eppendorf, Germany. Eleven immunosuppressed patients from different departments developed septicemia due to Listeria monocytogenes during hospitalization. In a retrospective study, serotyping and pulsed-field gel electrophoresis revealed that six isolates were identical or genetically related. Four of them had been isolated from renal transplant recipients. Listeria monocytogenes was neither detected in food samples of the renal transplantation ward, nor in stool specimens obtained from the ward staff. There had been no close contacts among the infected patients. Before transplantation, the renal transplant recipients had been dialysed in different dialysis centers. Nosocomial foodborne transmission could not be proven but seems likely.