In vitro and in vivo role of heat shock protein 90 in Amphotericin B resistance of Aspergillus terreus.

Aspergillus terreus (A. terreus) is of serious concern because of a high propensity to dissemination and in vitro and in vivo resistance to Amphotericin B (AmB). The underlying molecular mechanism of AmB is not known yet and here we want to explore whether fungal heat shock protein 90 (HSP90) is involved in polyene resistance in A. terreus. AmB-susceptible (ATS) and AmB-resistant (ATR) A. terreus and AmB-susceptible Aspergillus fumigatus (AFS) were investigated in response to AmB with a special focus on HSP90. HSP90 inhibitors resulted in significant improvement of AmB activity against ATR as minimum inhibitory concentrations (MIC) decreased from 32 to 0.38 mg/L. Gene expression profiling showed a greater basal amount of HSP90 levels in ATR and ATS when compared with AFS. HSP90 blockers in combination with AmB were evaluated in a murine model of disseminated aspergillosis. HSP90 inhibitors were not beneficial for mice infected with ATR, and neither mono- nor combination treatment with AmB yielded clinical improvement. HSP90 inhibition with 17-allylamino-17-demethoxygeldanamycin (17-AAG) was harmful. HSP90 seems to play a vital role in antifungal stress response in all aspergilli tested, whereas HSP90 does not substantiate the origin of AmB resistance in ATR.

Improvement of detection of bacterial pathogens in normally sterile body sites with a focus on orthopedic samples by use of a commercial 16S rRNA broad-range PCR and sequence analysis.

A new commercially available universal 16S and 18S rRNA gene PCR test, which is followed by sequence analysis of amplicons (SepsiTest), was evaluated for rapid identification of pathogens in the diagnosis of bone and joint infections. Eighty-three orthopedic samples and 21 specimens from other normally sterile body sites collected from 84 patients were analyzed in parallel by culture and PCR for detection of bacteria and fungi. Compared to culture, the diagnostic sensitivity and specificity of PCR were 88.5% and 83.5%, respectively. The detection rate of PCR (34.6%) was higher than that of bacterial culture (25.0%) as a consequence of the presence of fastidious and noncultivable species in samples and antibiotic treatment of patients. Thirteen culture-negative infections were identified by PCR, and PCR was able to detect culture-proven polymicrobial infections. On the other hand, three samples were culture positive but PCR negative. SepsiTest was demonstrated to be a valuable supplemental tool in the rapid detection of bacteria, especially for fastidious and noncultivable organisms, allowing earlier initiation of pathogen-adapted therapy in patients with bone and joint infections.

A 1-year Aspergillus terreus surveillance study at the University Hospital of Innsbruck: molecular typing of environmental and clinical isolates.

Aspergillus terreus appears to have become an increasingly frequent cause of opportunistic infections in the University Hospital of Innsbruck (UHI) and is of serious concern because of in vivo and in vitro resistance to amphotericin B. In order to determine the possible relationship between environmental contamination by A. terreus and the occurrence of invasive aspergillosis, a 1-year prospective study (2004-2005) was carried out in the UHI. Isolates obtained from air samples of various high-risk settings and those from surveillance cultures of proven and probable aspergillosis (EORTC/MSG criteria) were examined by genotyping. Within 1 year, 34 and 15 A. terreus isolates were collected from the environment and from patients, respectively. Genotypic analysis with rapid amplification of polymorphic DNA (RAPD) PCR and the combination of three different primers (R108, CII, P4) revealed 46 distinct genotypic profiles (types 1-46). No strain similarity was detected among and within the patients and environmental areas, indicating a great genomic diversity in A. terreus, which is common in the environment of Innsbruck and a source of invasive infections in immunosuppressed patients. Genotypical diversity was found in clinical and environmental A. terreus isolates.

CTX-M-1-related extended-spectrum beta-lactamases producing Escherichia coli: so far a sporadic event in Western Austria.

BACKGROUND: The present study was aimed to searching for CTX-M-type extended-spectrum beta-lactamases in community- and hospital-acquired Escherichia coli (E. coli) collected in western Austria and to investigate their clonal relatedness and their ability to spread. PATIENTS AND METHODS: All patients with E. coli positive cultures collected from a catchment population of 186,000 between January and July 2006 were enrolled into the study. CTX-M-producing E. coli were identified by antibiotic susceptibility testing and blaCTX-M multiplex PCR. Clonal relatedness was analyzed by pulsed-field gel electrophoresis (PFGE). RESULTS: In 2,042 E. coli isolates, 20 isolates (16 from urine, 4 from blood cultures) demonstrated CTX-M-1-related genes and no CTX-M-2- or CTX-M-9-related enzymes or CTX-M-15-producing strains were identified. We did not find clonal relatedness among CTX-M-1 producers isolated from the same referring center. E. coli were investigated for plasmid transfer ability of CTX-M-1-encoding genes. Plasmid digest patterns were not consistent with episomal spread of resistance loci. Transfection of CTX-M-encoding plasmids failed. CONCLUSION: Our data suggest that the emergence of CTX-M-1-producing E. coli in western Austria may be attributed to multiple independent events.

Suitability of partial 16S ribosomal RNA gene sequence analysis for the identification of dangerous bacterial pathogens.

AIMS: In a bioterrorism event a rapid tool is needed to identify relevant dangerous bacteria. The aim of the study was to assess the usefulness of partial 16S rRNA gene sequence analysis and the suitability of diverse databases for identifying dangerous bacterial pathogens. METHODS AND RESULTS: For rapid identification purposes a 500-bp fragment of the 16S rRNA gene of 28 isolates comprising Bacillus anthracis, Brucella melitensis, Burkholderia mallei, Burkholderia pseudomallei, Francisella tularensis, Yersinia pestis, and eight genus-related and unrelated control strains was amplified and sequenced. The obtained sequence data were submitted to three public and two commercial sequence databases for species identification. The most frequent reason for incorrect identification was the lack of the respective 16S rRNA gene sequences in the database. CONCLUSIONS: Sequence analysis of a 500-bp 16S rDNA fragment allows the rapid identification of dangerous bacterial species. However, for discrimination of closely related species sequencing of the entire 16S rRNA gene, additional sequencing of the 23S rRNA gene or sequencing of the 16S-23S rRNA intergenic spacer is essential. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides comprehensive information on the suitability of partial 16S rDNA analysis and diverse databases for rapid and accurate identification of dangerous bacterial pathogens.

Characterization of methicillin-resistant Staphylococcus aureus from Ulaanbaatar, Mongolia.

In order to expand current knowledge of the types of methicillin-resistant Staphylococcus aureus (MRSA) strains circulating in central Asia, six MRSA strains collected from hospitals in Ulaanbaatar, Mongolia during 2000-2002 were examined. Three strains possessed a staphylococcal cassette chromosome mec (SCCmec) element of type IV c, were sequence type (ST) 154 according to multilocus sequence typing (MLST), and contained lukS-lukF (Panton-Valentine leukocidin). Another three strains contained a SCCmec element of type III and were MLST type ST 239. Using automated ribotyping, the six MRSA strains were divided into four different EcoRI ribotypes, and two groups of isolates were distinguished by means of SmaI-macrorestriction patterns. In comparison to other countries, the incidence of MRSA in Mongolia is low.

Sorbitol-fermenting Shiga toxin-producing Escherichia coli O157: indications for an animal reservoir.

This study investigates a sorbitol-fermenting enterohaemorrhagic Escherichia coli (SF EHEC) O157 infection in a farmer's family in the Austrian province of Salzburg. The investigation commenced after a 10-month-old boy was admitted to hospital with the clinical diagnosis of a haemolytic-uraemic syndrome (HUS) and his stool specimen grew SF EHEC O157:H-. In a subsequent environmental survey, a stool specimen of the 2-year-old brother and faecal samples of two cattle from the family's farm were also found to be positive for SF EHEC O157:H-. All four isolates had indistinguishable phenotypic and molecular characteristics and were identical to the first strain detected in Bavaria in 1988. Despite identical isolates being demonstrated in Bavaria after 1988, and until this report, increased surveillance in neighbouring Austria had not found this organism. We propose that the strain may have recently spread from Bavaria to Austria. Although SF EHEC O157:H- strains are still rare, they may represent a considerable health threat as they can spread from farm animals to humans and between humans.

Importance of environmental transmission in cases of EHEC O157 causing hemolytic uremic syndrome.

A local outbreak of Shiga toxin (Stx)-producing enterohemorrhagic Escherichia coli (EHEC) O157:H7 causing severe hemolytic-uremic syndrome (HUS) was found to be caused by environmental transmission. Automated ribotyping and pulsed-field gel electrophoresis revealed that four stx2-positive EHEC isolates obtained from two unrelated children, one mother and one cow were identical. Results of an epidemiological investigation strongly suggest that both children were infected via a meadow strewn with manure containing EHEC-positive feces from the infected cow a few days prior to the onset of illness. The cow belonged to a cattle farm neighboring the meadow. This report highlights the risk of acquiring EHEC O157 through indirect contact with a farm environment.

Incidence of fecal carriage of Listeria monocytogenes in three healthy volunteers: a one-year prospective stool survey.

The aim of this study was to establish the incidence of fecal carriage of Listeria monocytogenes in healthy adults. A total of 868 stool specimens from three healthy volunteers (1 male and 2 females; ages 44, 39, and 60 years) were collected between 1 October 2000 and 30 September 2001. Culture was performed using Fraser broth and Palcam selective agar plates. Polymerase chain reaction (PCR) was performed using Probelia Listeria monocytogenes (BioRad, France). Overall, Listeria monocytogenes was detected in 31 of the 868 (3.57%) stool specimens using PCR. Sixteen of the 31 positive results were single events, i.e., samples collected from the same patient the day before and the day after the positive result were both negative. Positive results on two consecutive days were found four times, on three consecutive days one time, and on four consecutive days one time. Listeria monocytogenes was cultured from 10 of 868 (1.15%) stool specimens. These culture-positive samples, all positive by PCR as well, accounted for five independent episodes. Using automated ribotyping on up to 40 single colonies per stool specimen, 9 of 10 culture-positive samples yielded more than one strain. There was no obvious seasonal clustering of positive results. None of the documented episodes of Listeria monocytogenes carriage, all of which involved serotypes 1/2a and 1/2b, coincided with overt illness. The results of PCR indicate an incidence of five to nine exposures to Listeria monocytogenes per person per year. On average, the incidence of culture-confirmed fecal carriage in healthy adults is two episodes of Listeria monocytogenes carriage per person per year. Fecal shedding was of short duration (maximum 4 days), which argues against the appropriateness of routine stool screening in dairy workers as a tool for prevention of listeriosis.

Occurrence of Salmonella enterica serovar Dublin in Austria.

In Austria, Salmonella enterica subsp. enterica serovar Dublin, a bovine-adapted serovar, rarely causes infections in humans. In 2000, Austria was within the European mean with an incidence of 0.1 per million inhabitants. Our data show that the vast majority of all serovar Dublin infections (human and non-human) can be traced epidemiologically to two districts in the Tyrol. This concentration of cases can be explained by a particularly traditional aspect of cattle farming in this area, the alpine pasture. There is an increased risk of cross infection due to the communal keeping of animals from various farms. Infected cattle are a source of infection for humans, and transmission usually occurs from eating beef and drinking cows milk. Using pulsed field gel electrophoresis and automated ribotyping, three out of five isolates from human infections could be traced to characteristic Tyrolean Dublin clones. Bacteriological screening for faecal carriage before the transfer of cattle from risk-herds to the alpine pastures and before the return from risk-pastures to the farms would be a possible starting point to prevent cross-contamination of large mixed herds and contamination of pasture through latently infected cattle. Appropriate research is necessary.

Prevalence and characterization of Listeria monocytogenes in the feces of healthy Austrians.

The aims of the study were to determine the prevalence of Listeria monocytogenes in the feces of healthy Austrians and to characterize the isolates by various typing methods. Stool specimens from 505 healthy volunteers from the Tyrol were tested for the presence of L. monocytogenes using cold enrichment for 6 months and five different detection methods: conventional plating onto Palcam and Rapid'L.MONO agar, immunomagnetic separation (IMS) followed by conventional plating, enzyme-linked fluorescent immunoassay (ELFA), ELISA, and PCR. L. monocytogenes was isolated by conventional plating from one specimen (0.2%), and a further three were positive on immunomagnetic separation (0.8%). Only one specimen tested positive with ELFA and EIA, although it tested negative by conventional culture, IMS, and PCR. Eighteen of 505 samples were positive by PCR (3.6%), and this included three of the four culture-confirmed specimens. Serotyping, phage-typing, arsenic cadmium, antimicrobial-resistance typing and pulsed-field gel electrophoresis showed that multiple L. monocytogenes isolates from three of the four carriers were indistinguishable. Our data indicate that the Austrian fecal carriage rate is at least 0.8%. In view of a listeriosis incidence of 0.16/100,000 per year, the chances of fecal carriage developing into listeriosis appear to be very low.

In vitro activity of fosfomycin in combination with various antistaphylococcal substances.

Using the chequerboard technique we studied the in vitro activity of the broad spectrum antibiotic fosfomycin in combination with vancomycin, rifampicin, linezolid, quinupristin/ dalfopristin, cefazolin, meropenem and moxifloxacin against two Staphylococcus epidermidis strains (ATCC 12228, DSM 3269) and five Staphylococcus aureus isolates (ATCC 29213, DSM 683, DSM 46320, GISA 323/93, MRSA 3558/00). The phenomena of 'trailing' and 'skipped wells' did not present a problem. Synergy was the most common effect of all drugs tested in combination with fosfomycin; only combination with vancomycin showed antagonism for two of seven isolates. Using a killing-curve technique fosfomycin showed cidal activity, where increasing the drug concentration above the MIC did not enhance killing velocity. Inhibitory concentrations of vancomycin plus fosfomycin against DSM 46320 caused effects identical to those observed with vancomycin alone. The combination of fosfomycin plus linezolid exerted the bacteriostatic effect found with linezolid alone. Fosfomycin plus quinupristin/dalfopristin exhibited the bactericidal effect found with fosfomycin alone (in contrast to the rapidly bactericidal effect of quinupristin/dalfopristin). Electron microscopy showed that fosfomycin given in combination with linezolid, quinupristin/dalfopristin or moxifloxacin (substances that do not cause morphological alterations when given alone) resulted in 'cauliflower-shaped' distortion as caused by fosfomycin alone. Our in vitro data indicate considerable potential for fosfomycin used in combination with other antistaphylococcal antimicrobials, especially linezolid or quinupristin/dalfopristin.

Dynabeads plus 3 M Petrifilm HEC versus Vitek Immunodiagnostic Assay System for detection of E. coli O157 in minced meat.

The potentially low infective dose of Escherichia coli O157 makes it necessary to be able to detect low numbers in food, and the lack of sensitivity of direct plating has led to the development of various enrichment and detection methods. Until now, the most selective procedure for detection of E. coli O157 isolates was the immunomagnetic separation (IMS) method. The number of sorbitol non-fermenting micro-organisms other than E. coli O157 that adhere non-specifically to the magnetic beads hampers the application of IMS. The use of IMS in conjunction with 3 M Petrifilm-HEC yielded EHEC O157 in 21 of 165 samples of minced meat (12.7%). Without advance application of IMS, Petrifilm plates often yield confluent growth and colonies too numerous to count. The Vitek Immunodiagnostic Assay System (VIDAS-ECO) showed good sensitivity when testing artificially contaminated beef samples, but only four of 21 naturally contaminated samples were recognized. The addition of 3 M Petrifilm to IMS resulted in less growth of contaminants and eliminated much of the need to test presumed colonies for confirmation. The combination of IMS and 3 M Petrifilm-HEC is a fast and efficient screening procedure for E. coli O157 in minced meat.

Strain-specific differences in the amount of Shiga toxin released from enterohemorrhagic Escherichia coli O157 following exposure to subinhibitory concentrations of antimicrobial agents.

There is no consensus regarding the benefit versus harm of antibiotic therapy for treatment of disease due to enterohemorrhagic Escherichia coli O157. The effects in vitro of subinhibitory concentrations of 13 antimicrobial agents on the release of Shiga toxin (Stx) by three different Escherichia coli O157 strains expressing Stx 1 or Stx 2 either alone or in combination were investigated. The Stx-induced cell death of Vero cells was determined using a colorimetric assay based on the measurement of lactate dehydrogenase (LDH) released into the supernatant from the cytosol of damaged cells. Growth of all O157 strains in broth cultures containing subinhibitory concentrations of cotrimoxazole, trimethoprim, azithromycin, or gentamicin was accompanied by a marked increase in the release of Stx. Exposure to cefixime, ceftriaxone, or erythromycin caused a marked increase in the release of Stx by the O157 strain producing Stx 2 alone, but decreased toxin production was observed with the Stx 1 producer and the strain producing Stx 1 and Stx 2. Exposure to ampicillin caused increased Stx release in the Stx 2-producing strain but had no effect on Stx production in the other two test isolates. Exposure to penicillin G, streptomycin, ciprofloxacin, fosfomycin, or sulfamethoxazole caused an increase in toxin production in two of the three test strains in each case, while decreases were observed for the other isolates. The response of Escherichia coli O157 isolates to subinhibitory concentrations of antibiotics seems to be highly dependent on the nature of the strain involved.

Comparative study of five different techniques for epidemiological typing of Escherichia coli O157.

A set of 47 Austrian human, food, and veterinary Escherichia coli O157:H7 isolates was used to evaluate five different epidemiological typing methods. Ribotyping using an automated microbial characterization system (RiboPrinter) was not suitable for detection of epidemiological relatedness. All but one E. Coli strain were typeable by phage typing. Random amplified polymorphic DNA-PCR fingerprinting was performed using primer M13 containing the sequence 5'-GAG GGT GGC GGT TCT-3' and primer 1247 (5'-AAGAGCCCGT-3'). Although both methods recognized only two clusters, both dendrograms grouped most of the EHEC O157 isolates into epidemiologically related subgroups. Pulsed-field gel electrophoresis of XbaI digested total DNA was a valuable subtyping system. We found that major differences can exist between results of multiple subtyping methods. E. coli O157 isolates should not be classified as epidemiologically related or nonrelated on the basis of a single typing method alone.