First isolation of Clostridium difficile PCR ribotype 027 in Austria.
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Aged↗
Biomedical subjects
M Fille
Publications and source records attributed to M Fille.
Ralstonia pickettii-innocent bystander or a potential threat?
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Adolescent↗
Inducible metronidazole resistance in nim-positive and nim-negative bacteroides fragilis group strains after several passages metronidazole containing columbia agar plates.
BACKGROUND: Recent data show an emergence of resistance in the Bacteroides fragilis group against several antimicrobial agents and inducible resistance against metronidazole in nim-positive strains. The aim of the present study was to investigate inducible metronidazole resistance in nim-positive as well as in nim-negative B. fragilis group strains. MATERIALS AND METHODS: Of 18 B. fragilis strains (including four nim-positive reference strains and one ATCC strain), two Bacteroides ovatus strains, and one Bacteroides thetaiotaomicron DSM strain minimum inhibitory concentration (MIC) values for metronidazole were determined by Etest and analyzed for nim genes (nimA to -G) by PCR. For this purpose bacterial suspensions were incubated on supplemented Columbia agar plates containing metronidazole at twice the MIC value of the specific strain and incubated under anaerobic conditions for 48 hours. After incubation, growing bacteria were harvested and thereafter incubated at four times the original MIC. This procedure was repeated with increasing antibiotic concentrations. The resulting MIC values were confirmed by Etest. RESULTS: The MIC values for metronidazole of the four nim-positive reference strains ranged from 3 to 8 mg/l. The B. fragilis ATCC 25285 strain and the B. thetaiotaomicron DSM 2255 strain were nim negative with MIC values of 0.19 mg/l and 0.75 mg/l, respectively. Three clinical isolates of B. fragilis strains showed MIC values of > 256 mg/l. In all three strains, nim genes were detected by PCR. The other clinical isolates were nim negative. In these strains, MIC values ranged from 0.19 to 0.75 mg/l. After several passages on metronidazole containing agar, all B. fragilis group strains exhibited MIC values of > 256 mg/l determined by Etest. CONCLUSION: Metronidazole resistance can be selected not only in nim-positive strains but also in nim-negative strains, suggesting that mechanisms other than nim genes are involved. These findings and the emerging resistance of the B. fragilis group against several antimicrobial agents underscore the importance of susceptibility testing of anaerobes even in routine laboratories.
Anti-Infective Agents↗
Influence of human ascitic fluid on the in vitro antibacterial activity of moxifloxacin.
We investigated the in vitro influence of HAF on the antibacterial activity of moxifloxacin against Escherichia coli ATCC 10798, Escherichia coli K-12, Proteus rettgeri (Sanelli), Staphylococcus aureus ATCC 25923, Staphylococcus aureus NCTC 1808 and Staphylococcus epidermidis ATCC 12228. Human ascitic fluid was obtained from 6 cirrhotic patients by paracentesis. The interaction effect was evaluated by the checkerboard technique. Our results indicate the ability of human ascitic fluid to reduce minimum inhibitory concentrations of moxifloxacin against Gram-negative bacteria, but not against Gram-positives.
Ascitic Fluid↗
Cefazolin concentrations in serum during cardiopulmonary bypass surgery.
The objective of the study was to investigate possible changes in cefazolin serum levels induced by cardiopulmonary bypass (CPB). Six cardiac male patients who underwent cardiac surgery requiring CPB took part in the study. Cefazolin 2 g was intravenously infused over 60 min before anesthesia and blood samples were taken at appropriate times after drug administration (0, 0.25, 0.5, 1, 4, 6, 8 h), 2 min before and 5 min after the beginning and 2 min before and 5 min after the end of CPB. Drug serum concentrations were determined by means of a microbiological method. Five minutes after the start of CPB, cefazolin serum levels decreased on average by 46.6% and remained steadily low until 5 min after the end of CPB. Then, they rose on average by 37.3% at 4 h and then declined slowly until the last sampling at 8 h. Cefazolin serum concentrations were low during CPB but remained in a potentially effective range for antimicrobial prophylaxis for this surgery.
Aged↗
A series of infections due to Capnocytophaga spp in immunosuppressed and immunocompetent patients.
OBJECTIVE: To investigate the epidemiology, microbiology and outcome of infections caused by Capnocytophaga spp. at a single center. METHODS: We report on ten documented infectious episodes caused by Capnocytophaga observed between 1994 and 1999 at the Innsbruck University Hospital. RESULTS: In seven of ten patients, Capnocytophaga septicemia was diagnosed during periods of neutropenia. In contrast, the remaining three patients had normal white blood cell counts when acquiring Capnocytophaga septicemia (one) and pleural empyema (two). Blood cultures containing long, slender, Gram-negative rods, which grew slowly under anaerobic conditions and lacked susceptibility to metronidazole, were subcultivated in a CO2-enriched atmosphere (5%). Subcultivation yielded Capnocytophaga in all ten cases within 2-12 days. The patients were then placed on appropriate antibiotic therapy, with or without additional surgical intervention, and the organism was eradicated. CONCLUSION: Identification of Capnocytophaga facilitates appropriate, and in most cases effective, antimicrobial therapy.
Adult↗
[Imipenem resistance in Pseudomonas aeruginosa].
In 1997 in western Austria, 9.9% of Pseudomonas aeruginosa strains from patients of general practitioners were resistant to imipenem as well as 18.2% of the isolates from hospitals and 20.2% of the strains at a university teaching hospital. Within the hospital the imipenem resistance varied from 9.9% among out-patients to 28.7% in isolates from intensive care units. In medical/surgical words, up to 15.1% of P. aeruginosa strains were resistant to imipenem. The incidence of imipenem-resistant P. aeruginosa strains correlates to the use of carbapenems. In June 1997, 10 consecutive isolates from 8 patients were obtained and typed using restriction fragment length polymorphism analysis (RFLP) and Pyocin typing. All 10 isolates were resistant to meropenem as well as to imipenem. The finding (by RFLP and Pyocin typing) of individual bacterial types in each isolate strongly contradicts the spread of infection by cross infection. However, all patients were proven to have been treated with imipenem during the 3 months prior to testing. In 1997, 13,880 g of imipenem were used at the university hospital in Innsbruck. The use of carbapenems appears to be the main cause for the increased incidence of imipenem-resistant P. aeruginosa strains.
Austria↗
Comparison of culture and acid-fast bacilli stain to PCR for detection of Mycobacterium tuberculosis in clinical samples.
The major drawback in effective use of polymerase chain reaction (PCR) for detecting Mycobacterium tuberculosis (MTB) in clinical samples is the presence of PCR inhibitors and unique cell components of the organism that complicate DNA extraction and subsequent PCR amplification. A PCR assay with a unique multistep DNA extraction method that minimizes these problems was compared in a prospective study to acid-fast bacilli stain (AFBS) and culture for detecting MTB in clinical samples. A total of 254 clinical specimens in two separate studies were processed for MTB by these techniques. While PCR and culture were 100% sensitive and specific, culture required up to 8 weeks of incubation and additional time to perform biochemical testing to identify the isolated micro-organism. Acid-fast bacilli stain had a specificity of about 87% and did not differentiate among Mycobacterial species. In contrast, the results from PCR were available within 48 h and did not require additional testing to attain a final result. Polymerase chain reaction was highly reliable for detection and confirmation and interpretation of positive AFBS results. The assay was easy to perform with a turn around time of about 2 days.
Bacteriological Techniques↗
Changing bacterial ecology during a five-year period of selective intestinal decontamination.
The development of bacterial resistance during selective decontamination of the digestive tract (SDD) is controversial. We studied effects on bacterial resistance one year before and during a randomized, placebo-controlled trial of SDD in a surgical intensive care unit. We randomized patients within two different topical regimens (PTA, PCA) or placebo, administered four-times daily to both the oropharynx and gastrointestinal tract. All patients received intravenous ciprofloxacin (200 mg b.d.) for four days. Both SDD regimens successfully reduced aerobic Gram-negative intestinal colonization. There was no increase in resistance of Enterobacteriaceae or Pseudomonas aeruginosa. Acinetobacter calcoaceticus developed multi-resistance over one year, but differences between groups were not significant. We detected a shift towards Gram-positive organisms. Oxacillin-resistant Staphylococcus aureus increased in concert with ciprofloxacin resistance, from 17 to 80.7%, and frequencies of resistance were significantly higher in SDD patients (P < 0.001). Resistance of coagulase-negative staphylococci (CNS) to oxacillin increased initially (25 to 66.9%), but values returned to baseline in controls. Ciprofloxacin resistance in CNS remained higher (P < 0.001) in SDD-treated patients (52.5 vs. 23.3%). The incidence of late respiratory tract infections was unaltered by the prophylactic regimen (SDD 35.2%; Placebo 41.2%; n.s.). We cannot recommend SDD as a prophylactic tool in critically ill patients.
Adult↗
Tolerance and bactericidal action of N-chlorotaurine in a urinary tract infection by an omniresistant Pseudomonas aeruginosa.
N-chlorotaurine, a weak antimicrobial oxidant produced by stimulated human leukocytes, was used to treat cystitis caused by an omniresistant Pseudomonas aeruginosa. A 21-year-old male patient was treated by repeated daily lavages of the urinary bladder with an aqueous solution of 1% N-chlorotaurine for one month. N-chlorotaurine was well tolerated; no local or systemic side effects could be detected. Despite killing of > 10(6) cfu/ml of bacteria within ten minutes in vitro and in vivo, it was not possible to eradicate the Pseudomonas infection obviously caused by inflammation of the upper urinary tract and perpetuated by intravesical concrements. Nevertheless, in actually localized infection, treatment with N-chlorotaurine might be successful because of its sufficient bactericidal action.
Adult↗
Evaluation of four methods for detection of Clostridium difficile or C. difficile toxin: cytotoxin assay, culture, latex agglutination, and a new rapid immunoassay (C. difficile toxin A test).
The performance of C. difficile toxin A test (Oxoid, Basingstoke, UK), an immunoassay for the detection of C. difficile toxin A in fecal samples, for the diagnosis of C. difficile-associated diarrhea was compared with those of cytotoxin assay, culture, and a latex agglutination assay (Culturette Brand CDT Rapid Clostridium difficile test; Becton Dickinson, Cockeysville, MD). A total of 105 stool specimens from 71 patients were tested. Of the 105 specimens analyzed, 6 (5.7%) samples were positive by all four methods, 66 samples (62.9%) were concordant negative. Two patients fulfilled all clinical criteria for C. difficile-associated diarrhea but had negative results in the cytotoxicity tests. The sensitivity and specificity, as determined against cytotoxin assay results, were, respectively 52.4% and 93.9% for latex agglutination, 68.2% and 84.3% for rapid immunoassay and 36.4% and 92.8% for culture. No single laboratory test yields a definitive diagnosis of C. difficile-associated diarrhea at present. Test results by any of these methods must be used in conjunction with patient history when making the diagnosis to avoid indiscriminate treatment of individuals without disease or the lack of treatment for individuals with a serious infection. We consider the rapid and easy-to-perform C. difficile toxin A test not to be an essential adjunct for the diagnosis of C. difficile-associated diarrhea.
Adolescent↗
Surgical treatment of bacillus Calmette Guérin lymphadenitis.
Although its protective effect is contested and the risk of contracting tuberculosis is rather low nowadays, BCG vaccination is frequently performed. Changes of strain repeatedly led to an increased complication rate. In Austria between 1990 and 1991, of 3386 newborn babies (Strain Pasteur) 116 developed lymphadenitis 3 to 28 weeks after vaccination. The affected children received four types of treatment: nothing specific, isoniazid, or surgery with and without isoniazid. Surgical treatment was found to be necessary in 96 cases. Bacilli were successfully grown in culture in 46% of cases up to week 20 after vaccination; but later than 20 weeks no culture became positive. All cultured bacteria were isoniazid-sensitive. From our data we drew the following conclusions: isoniazid therapy did not prove successful when inflamed lymph nodes exceeded a certain size. Suppurative lymphadenitis in lymph nodes exceeding 1.0 to 1.5 cm usually led to infiltration or even perforation of the skin. Surgery prevents these complications and significantly reduces healing time. Adjuvant isoniazid therapy cannot be recommended, except for generalized BCG tuberculosis.
Antitubercular Agents↗
Quantitative RT-PCR using a PCR-generated competitive internal standard.
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Hepacivirus↗
Diagnostic value of different PCR assays for the detection of mycobacterial DNA in granulomatous lymphadenopathy.
Diagnosis of mycobacterial infection is made by assessment of characteristic histological features, staining of acid-fast bacilli, or agar culture. Recent advances in molecular biology have provided alternative approaches for the detection of mycobacteria, but only limited data are available dealing with the comparative evaluation of these methods. In order to determine the diagnostic applicability of polymerase chain reaction (PCR)-based assays, 20 formalin-fixed and paraffin-embedded lymph nodes with bacille Calmette-Guérin (BCG) lymphadenitis were investigated which in Löwenstein Jensen agar culture were either positive or negative (ten cases each); ten lymph nodes with non-specific lymphadenitis served as negative controls. Ziehl-Neelsen staining as well as three different PCR assays (including nested PCR), amplifying a specific sequence of the Mycobacterium tuberculosis complex or sequences of the 65 kD antigen gene, were performed. Positive culture was only obtained from lymph nodes which had been surgically removed within 20 weeks after vaccination (P < 0.001). In contrast to microscopic examination, which yielded no more information than agar culture, PCR detection of mycobacterial DNA was unrelated to culture findings. Combined use of different assays, as well as DNA extraction from at least three paraffin sections from each specimen, resulted in the detection of mycobacterial DNA in all lymph nodes with amplifiable DNA (18 out of 20 cases). Controls remained consistently negative. Thus, the combined use of different PCR assays is proposed as a rapid and sensitive technique for the detection of mycobacterial DNA in formalin-fixed and paraffin-embedded tissue.
Axilla↗
Use of AmpliWax to optimize amplicon sterilization by isopsoralen.
The photochemical inactivation of amplicons by isopsoralen (IP-10) has been suggested as a possible means to prevent PCR carryover contamination. To evaluate the technique, serial dilutions of amplicons (10(11) to 10(3)) from the Borrelia burgdorferi OSP A gene were amplified in the presence of 0, 25, 50, and 100 micrograms of IP-10 per ml for 45 cycles. The PCR products were exposed to UV light for 15 min to activate IP-10 and sterilize the amplicons. One microliter of each sterilized sample was reamplified for an additional 45 cycles. The PCR products were then resolved in an agarose gel, blotted onto a nylon membrane, and probed with an alkaline phosphatase-conjugated chemiluminescent probe. Although IP-10 at concentrations of 50 and 100 micrograms/ml effectively sterilized up to 10(11) amplicons, the compound was inhibitory to PCR. IP-10 at a concentration of 25 micrograms/ml had slight inhibitory effect on PCR and did not completely sterilized all of the amplicons. Therefore, in subsequent experiments AmpliWax was substituted for mineral oil, and PCR was performed on 10(9) to 10(3) amplicons as described above. Following the amplification, the PCR tubes were cooled to solidify the AmpliWax and inoculated with various concentrations of IP-10. With this technique, PCR products produced from as many as 10(9) target amplicons were effectively sterilized with 200 micrograms of IP-10 per ml. Similarly, the addition of IP-10 (50 micrograms/ml) before and after PCR was evaluated for the detection of B. burgdorferi in 62 ticks from a region of Southern Connecticut where the organism is highly endemic. PCR performed in the presence of 50 micrograms of IP-10 per ml detected B. burgdorferi-specific DNA in 17 of 62 ticks (27%) following gel electrophoresis and in 34 of 62 ticks (55%) following Southern blot hybridization of the PCR products. In contrast, post-PCR addition of IP-10 detected borrelia-specific DNA in 31 of 62 ticks (50%) following gel electrophoresis and in 46 of 62 ticks (64%) following Southern blot hybridization. We conclude that the replacement of mineral oil with AmpliWax can be useful in eliminating the inhibitory effects of IP-10 and other sterilizing agents for post-PCR sterilization of amplicons.
Animals↗
Molecular epidemiology of Klebsiella pneumoniae producing SHV-5 beta- lactamase: parallel outbreaks due to multiple plasmid transfer.
Over a period of 22 months, 32 patients treated in three independent intensive care units of the Innsbruck University Hospital were infected with extended-spectrum beta-lactamase-producing members of the family Enterobacteriaceae (30 Klebsiella pneumoniae isolates, 1 Klebsiella oxytoca isolate, and 1 Escherichia coli isolate). As confirmed by sequencing of a bla gene PCR fragment, all isolates expressed the SHV-5-type beta-lactamase. Genomic fingerprinting of epidemic strains with XbaI and pulsed-field gel electrophoresis grouped 20 of 21 isolates from ward A into two consecutive clusters which included 1 of 3 ward B isolates. All six K. pneumoniae isolates from ward C formed a third cluster. Stool isolates of asymptomatic patients and environmental isolates belonged to these clusters as well. Additionally, 2,600 routine K. pneumoniae isolates from the surrounding provinces (population, 900,000) were screened for SHV-5 production. Only one of six nonepidemic isolates producing SHV-5 beta-lactamase was matched with the outbreak strains by genomic fingerprinting. Plasmid fingerprinting, however, revealed the epidemic spread of a predominant R-plasmid, with a size of approximately 80 kb, associated with 29 of the 30 K. pneumoniae isolates. This plasmid was also present in the single K. oxytoca and E. coli isolates from ward C and in three nonepidemic isolates producing SHV-5. Our results underline that strain typing exclusively on the genomic level can be misleading in the epidemiological investigation of plasmid-encoded extended-spectrum beta-lactamases. Our evidence for multiple events of R-plasmid transfer between species of the family Enterobacteriaceae in this nosocomial outbreak stresses the need for plasmid typing, especially because SHV-5 beta-lactamase seems to be regionally spread predominantly via plasmid transfer.
Base Sequence↗
Identification of mycobacteria to the species level by automated restriction enzyme fragment length polymorphism analysis.
An automated method for the restriction fragment length polymorphism (RFLP) analysis for the differentiation of mycobacteria to the species level is described. After polymerase chain reaction (PCR) amplification of a sequence of the gene encoding the 65-kDa surface antigen common to all mycobacteria the product was investigated by RFLP analysis. For accurate determination of fragment sizes the asymmetrically fluorescein-labelled PCR product was partially digested with restriction site enzymes BstEII and HaeIII. The fragments obtained were analysed electrophoretically using an automated laser fluorescence DNA sequencer. Determination of fragment sizes revealed a deviation of +/- 1 base pair (bp; 0.6%) when compared to expected sizes. The validity of this approach was confirmed by analysing mycobacterial DNA obtained from pure cultures of Mycobacterium (M.) tuberculosis and alcohol-fixed smears as well as paraffin-embedded sputa of patients with culture-proven tuberculosis. Additionally a diagnostic algorithm was established by investigation of cultured M. bovis, M. bovis bacille Calmette-Guérin, M. avium, M. intracellulare and M. fortuitum. The method allows the identification of restriction enzyme sites which are only 40 bp apart. Partial restriction enzyme digestion of asymmetrically fluorescence-labelled PCR products will presumably lead to the discovery of new restriction enzyme sites.
Algorithms↗
[Gas gangrene as a manifestation of endogenous Clostridium septicum infection].
Endogenous, nontraumatic clostridial myonecrosis has a frequent association with colon carcinoma, leukemia, diabetes mellitus, and drug-induced immunosuppression. We present two cases of Clostridium septicum myonecrosis. An 18-year-old girl developed severe abdominal pain on day 7 after hospitalization for cytostatic treatment of acute lymphoblastic leukemia. Blood cultures yielded Clostridium septicum and histopathological exam of muscle tissue showed extended myonecrosis. Eventually the patient recovered with antibiotics and surgical therapy. A 72-year-old diabetic woman was treated as an outpatient with an intramuscular injection of steroidal antiphlogistics for "acute lumbar disc disease". The next morning persistence of hip pain and discoloration of the right thigh caused hospitalization under the suspected diagnosis "fracture of the neck of the femur". Clostridium septicum was cultured from intraoperatively taken swabs. At autopsy, in addition to the gangrene, there was an adenocarcinoma of the cecum, which had not been diagnosed during life.
Adolescent↗