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AIMS: To assess the performance of three commercially available Mycobacterium tuberculosis detection systems employing nucleic acid amplification, when applied directly to respiratory and non-respiratory specimens from patients where the diagnosis of tuberculosis is difficult using clinical and traditional bacteriological methods. METHODS: 42 respiratory and 21 non-respiratory specimens were concentrated, examined with auramine staining, and cultured on Lowenstein-Jensen slopes. These specimens were also assayed using the Amplicor Mycobacterium tuberculosis test (AM) (Roche Diagnostic Systems), the Amplified Mycobacterium tuberculosis direct test (AMD) (Gen-Probe), and the LCx Mycobacterium tuberculosis assay (LMA) (Abbott Laboratories). RESULTS: All three amplification systems used in this study gave specificities of 100% when used on respiratory specimens. When used on non-respiratory specimens, AM and LMA gave specificities of 100% and AMD 75%. With respiratory specimens the AM, AMD, and LMA systems gave sensitivities of 75%, 65.2%, and 79.2%, respectively. With non-respiratory specimens the sensitivities were 50%, 66.7%, and 60%, while with smear negative, culture positive specimens they were 33.3%, 66.7%, and 55.6%. Positive predictive values of 100% were seen with all specimens except non-respiratory specimens assayed using AMD where the value was 66.7%. CONCLUSIONS: The manufacturers of these systems recommend that they should only be used for the direct analysis of respiratory specimens, and the US Food and Drug Administration has approved them for use only with smear positive specimens. This study confirms that sensitivities are lower for non-respiratory and smear negative specimens, but positive predictive values are high. Provided they are interpreted with caution, positive results with these tests in respiratory and non-respiratory specimens are useful in tuberculous patients who are otherwise difficult to diagnose. Each amplification has advantages and disadvantages compared with the others.

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During a 1-month period in 1996, all inpatients and staff in the Zagreb Trauma Hospital were screened for methicillin-resistant Staphylococcus aureus (MRSA) carriage in order to control MRSA spread within the hospital. During the study period, 663 patients were admitted to the hospital, and screening prior to discharge revealed that 42 were colonised or infected with MRSA. Twenty-three (55%) of these would not have been detected if active screening had not been performed. Amongst 205 staff members, MRSA carriage was only found in one (0.5%) nurse. The prevalence and incidence of MRSA carriage varied significantly amongst the wards and was related to the length of hospital stay. One-third of the patients colonised or infected with MRSA had a history of previous admission to another hospital, and one-third were transferred to another institution after discharge. Thirty-nine of 42 MRSA isolates shared the same antibiotic sensitivity pattern, suggesting endemic spread of MRSA. However, randomly amplified polymorphic DNA molecular typing revealed four profiles, the most common involving 15 of 36 tested strains. There was no obvious clustering of epidemiological types by ward, except for the appearance of a single type on the burns unit, and it was likely that different strains had been introduced into the hospital by patient transfers from elsewhere. The results of this study indicate that a substantial proportion of MRSA carriers escape infection control measures if active screening is not performed. Based on the results of this study, steps have been taken to improve interhospital communication about the transfer of patients colonised with MRSA. Randomly amplified polymorphic DNA typing proved to be a useful aid to epidemiological investigations of MRSA.

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The minimum inhibitory concentrations (MICs) of mupirocin were determined by the E test (AB Biodisk, Sweden) and the agar dilution method for 107 staphylococci. The organisms consisted of 34 coagulase-negative staphylococci and 73 methicillin-resistant Staphylococcus aureus. Polymerase chain reaction (PCR) primers designed to amplify a 456 bp region of the plasmid-borne isoleucyl tRNA synthetase gene (ileS-2), responsible for high-level mupirocin resistance in staphylococci, were used on DNA preparations from these isolates. Isolates with high-level mupirocin resistance due to the ileS-2 gene should be PCR positive. There was close correlation between the E test and agar dilution MIC values, with only two strains differing by more than two serial dilutions. Most (51 of 54 strains) of the high-level resistant strains (MIC>256 microg/ml) were resistant to the highest concentration of mupirocin tested (1024 microg/ml). PCR correctly classified all but four (96%) of the isolates in accordance with the results of agar dilution. All four isolates that gave discrepant results were methicillin-resistant Staphylococcus aureus. Two of these were PCR positive, yet the MIC of mupirocin for these strains was <0.06 microg/ml; on prolonged incubation they produced halos within the inhibition zone on agar diffusion testing, suggesting that the phenotypic results may have been erroneous. One of 54 isolates for which the MIC exceeded 256 microg/ml was PCR negative when tested by the original methodology, but a 456 bp product was produced when retested using a lowered annealing temperature. One isolate for which the MIC of mupirocin was 16 microg/ml by agar dilution and 8 microg/ml by the E test was positive by PCR. PCR of the ileS-2 gene is a useful, rapid method for detecting high-level mupirocin resistance in staphylococci.

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OBJECTIVE: To establish the extent of inter-hospital spread of methicillin-resistant Staphylococcus aureus (MRSA) in Zagreb and to determine the most suitable method for typing local strains. METHODS: We analyzed a collection of 33 MRSA isolates from three Zagreb hospitals together with five unrelated British MRSA isolates by antibiogram typing, bacteriophage typing, randomly amplified polymorphic DNA (RAPD) analysis and pulsed-field gel electrophoresis (PFGE) after digestion with Smal restriction endonuclease. Bacteriophage typing was done with the international set of S. aureus typing phages. RAPD and PFGE profiles were analyzed visually and by using the 'GelCompar' computer program. RESULTS: Antibiogram typing provided eight profiles. Thirty (91%) of the 33 Croatian strains of MRSA were non-typable by phage typing. Visual analysis of RAPD products identified six, and visual analysis of PFGE fragments nine, distinct profiles. Computer analysis of RAPD data separated British isolates from the Croatian ones, but did not cluster the visually determined RAPD types. PFGE computer analysis separated British isolates and clustered isolates in concordance with visual interpretation. Thirty-one of the 38 isolates (82%) were visually grouped in the same clusters by both molecular methods. The dominant strain was present in each of the three hospitals. CONCLUSIONS: Bacteriophage typing was unhelpful for the analysis of Croatian MRSA, since most strains were untypable with the international set of bacteriophages. RAPD and PFGE were more successful in typing the organisms and showed evidence of inter-hospital spread of one predominant MRSA strain in all three Zagreb hospitals. Thus RAPD and PFGE proved to be a useful aid in elucidating the epidemiology of MRSA infection in Zagreb hospitals and should be established in Croatia for typing MRSA.

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A cluster of methicillin-resistant Staphylococcus aureus (MRSA) infections among patients on an intensive care unit (ICU) was detected by routine infection control surveillance. In the period from 5 January to 22 June 1995, 10 patients on the ICU and a further 6 patients (5 on one ward that had received colonized patients transferred from the ICU) were affected by MRSA strains with the same antibiotic susceptibility patterns. Seven (44%) of these 16 colonized patients developed MRSA bacteremia. MRSA isolates with the same characteristics were also found on the hands of one member of the ICU staff. The isolates were untypeable by phage typing, but 15 of 17 outbreak strains analyzed genetically had identical randomly amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) profiles. A single strain of MRSA that was nontypeable by phage typing and that was isolated on the ICU on 1 January and six nontypeable and epidemiologically unrelated MRSA isolates all had RAPD profiles distinct from that of the outbreak strain. Implementation of strict infection control measures stopped the further spread of MRSA on the ICU, the affected general ward, and seven other wards that received MRSA carriers from the ICU. Although nontypeable by phage typing and not previously recognized as an epidemic strain, this strain of MRSA was readily transmissible and highly virulent. RAPD typing was found to be a simple, rapid, and effective method for the epidemiological investigation of this outbreak, and performance of typing by this method was simpler and less time-consuming than that of typing by PFGE. RAPD typing may have more general application for the study of S. aureus infections in hospitals.

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Many biochemical and molecular techniques can be used for distinguishing isolates of a given bacterial species. Traditional typing techniques based on phenotypic characteristics such as serotyping are being increasingly challenged by the use of DNA-based methods. The introduction of the polymerase chain reaction (PCR) has led to typing techniques based on DNA amplification. Randomly amplified polymorphic DNA (RAPD) typing (also known as arbitrarily primed-polymerase chain reaction, APPCR) is one such technique which is being used increasingly to type micro-organisms, especially during clinical outbreaks. The application and potential problems and solutions of RAPD typing are discussed and the role of such techniques among established typing methods is addressed.

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A cluster of Candida glabrata isolates recovered from seven patients in an intensive care unit over a 10-week period were compared with a collection of isolates from six epidemiologically distinct outpatients and a reference strain by several DNA typing methods. Restriction enzyme analysis with HinII distinguished 13 strains from the 14 sources and was the method of choice. Pulsed-field gel electrophoresis and random amplification of polymorphic DNA both detected nine types from the 14 sources; however, the results of these two methods did not always correlate. These methods demonstrated that five of the seven patients had distinguishable strains and that cross-infection was unlikely.

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In this study, 122 isolates of vancomycin-resistant Enterococcus faecium (VREF) obtained from 103 patients over a four-year period in a London teaching hospital, were typed by a random amplified polymorphic DNA method. All the isolates exhibited high-level resistance to vancomycin (MIC 128-1024 mg/L), and were resistant to teicoplanin (32-256 mg/L). Nine RAPD types were distinguished by using a single primer. Clustering of certain types in time and space was noted. These results suggest that although several different strains of VREF were involved in this outbreak, cross-infection with individual types occurred on some wards. RAPD is a useful technique for the investigation of the epidemiology of VREF.

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The mechanisms by which quinolones rapidly kill are ill defined. We have investigated the action of ciprofloxacin on Escherichia coli KL16 with a combination of traditional and flow cytometric methods and have analyzed cells for changes in membrane potential, membrane integrity, oxidative metabolism, morphology, and viability. Log-phase cultures were exposed to various concentrations (0.1, 1, 10, and 100 times the MIC) of ciprofloxacin and analyzed at regular intervals over 120 min. We also measured protein synthesis in the related strain PQ37 cultured under the same conditions over 300 min, using a colorimetric assay for beta-galactosidase release. Despite a 3-log order decrease in CFU after 60-min exposure to 10 and 100 times the MIC of ciprofloxacin, there was no equivalent decrease in bacterial numbers as determined by both light microscopy and flow cytometry. Furthermore, while these bacteria showed concentration-dependent morphological changes, most were capable not only of excluding the fluorescent nucleic acid-binding dye propidium iodide, but also of reducing the tetrazolium dye cyanoditodyl tetrazolium chloride. Over 90% of the bacteria maintained a membrane potential [as determined by exclusion of bis-[1,3-dibutylbarbituric acid) trimethine oxonol] when exposed to ciprofloxacin for 120 min, except at 100 times the MIC, when this figure fell to < 10%. Finally, protein synthesis was either maintained or induced at all concentrations of ciprofloxacin up to 5 h postexposure. Taken together, these results demonstrate the continuing physical and metabolic survival of ciprofloxacin-exposed bacteria; we suggest parallels with the concept of the viable nonculturable state.

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We report the cloning and sequencing of vanA genes present in the high-level vancomycin- and teicoplanin-resistant clinical isolates Oerskovia turbata 892 and Arcanobacterium (Corynebacterium) haemolyticum 872. The presence of vanA was detected by Southern blotting and PCR and confirmed by DNA sequencing. vanA-like sequences were encoded on plasmids of 15 and 20 kb respectively. The A. haemolyticum 872 DNA sequence was identical to the published vanA sequence of vancomycin-resistant Enterococcus faecium BM4147, but the O. turbata 892 sequence showed three coding changes. Induction experiments indicated that vancomycin resistance in A. haemolyticum 872 and O. turbata 892 was constitutive. SDS-PAGE analysis of membrane proteins showed the presence of a c. 39 kD protein in both clinical isolates whose expression was unaltered in the presence of vancomycin, while a similar protein in E. faecium BM4147 was inducible. Since A. haemolyticum and O. turbata are naturally susceptible to vancomycin, the high-level constitutive resistance seen in these isolates appears to be mediated by vanA. This is the first report confirming the presence of vanA in genera other than Enterococcus.

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The randomly amplified polymorphic DNA (RAPD) assay was used to generate DNA fingerprints from clinical isolates of Staphylococcus haemolyticus isolated from patients treated with continuous ambulatory peritoneal dialysis and previously subjected to a combination of typing methods. The RAPD profiles generated with one of six randomly designed 10-mer primers allowed visual discrimination of strains. Good correlation with the original typing scheme was achieved but RAPD typing allowed discrimination of strains previously indistinguishable.

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The randomly amplified polymorphic DNA (RAPD) assay was used to generate DNA fingerprints from clinical isolates of Staphylococcus haemolyticus isolated from patients treated with continuous ambulatory peritoneal dialysis and previously subjected to a combination of typing methods. The RAPD profiles generated with one of six randomly designed 10-mer primers allowed visual discrimination of strains. Good correlation with the original typing scheme was achieved but RAPD typing allowed discrimination of strains previously indistinguishable.

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Quinolone antimicrobial agents induce the SOS response in bacteria, including the umuDC genes necessary for error-prone repair. Consequently these drugs may be mutagenic in bacteria with a functional SOS response. Differential killing tests with Escherichia coli WP2 (trp) and its repair-deficient derivative CM871 (trp lexA recA uvrA) indicated that a functional DNA repair system was protective against the action of quinolones, implying that quinolones are causing some form of DNA damage (not necessarily directly) and are therefore genotoxic. Dose-dependent reversion from His- to His+ with quinolones was observed in the Ames test with Salmonella typhimurium TA102 (uvr+) but in no other Salmonella tester strains (all uvr-), suggesting that a functional excision repair system is essential for quinolone-induced bacterial mutagenesis. A significant correlation between SOS inducing potential (SOSIP) and mutagenic potential in the Ames test (r = 0.89) indicated that quinolone-induced mutagenic effects in bacteria are almost entirely due to SOS-processed DNA damage.

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Examination of 67 ciprofloxacin-resistant clones of Pseudomonas aeruginosa PAO (ciprofloxacin MIC > 0.5 mg/L) yielded four isolates that were hypersensitive to chlorhexidine (MIC 5 mg/L); none was found among 179 ciprofloxacin-sensitive colonies. Revertant studies and the introduction of a wild-type Escherichia coli DNA gyrase A gene confirmed that ciprofloxacin resistance and chlorhexidine hypersensitivity were not the result of a single mutation. Mutagenicity testing of ciprofloxacin showed no evidence for the supposition that chlorhexidine hypersensitivity was the result of ciprofloxacin-induced mutation in P. aeruginosa PAO.

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Induction by the 4-quinolone group of antibacterial drugs of the umuC gene, the SOS function most involved in error-prone DNA repair (together with umuD), was assessed in a strain of Escherichia coli harbouring a umuC::lacZ gene fusion. All 4-quinolones tested induced this umuC::lacZ fusion, with maximum induction at 4-quinolone concentrations close to their minimum inhibitory concentrations for this strain, and the SOS Inducing Potential (SOSIP) was closely related to antibacterial activity. Mitomycin C, a known mutagen, was a slightly better inducer (in terms of SOSIP) than any of the quinolones. In contrast, induction by 4-quinolones of the sfiA (sulA) gene, an SOS function involved in cell division inhibition, was better than induction by mitomycin C in an E. coli strain harbouring an sfiA::lacZ gene fusion. The umuC gene fusion was induced at lower concentrations of 4-quinolone than was the sfiA gene fusion.

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Two plasmids containing the cloned Escherichia coli wild-type gyrA gene were used to transform ciprofloxacin-resistant Gram-negative clinical isolates to screen for DNA gyrase A-mediated quinolone resistance. The results show that the technique is simple and applicable to a wide range of Gram-negative species including E. coli, Enterobacter cloacae, Klebsiella aerogenes, Morganella morganii, Proteus mirabilis, Pseudomonas aeruginosa, Campylobacter jejuni and Neisseria gonorrhoeae. The use of an arithmetical MIC series of dilutions (as opposed to standard geometrical ones) was found to be essential during screening for the detection of altered gyrase A. The observations were consistent with the suggestion that DNA gyrase is highly conserved among different species of bacteria and that gyrase A-mediated resistance can occur in all.

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The sporicidal efficacy of glutaraldehyde (2% w/v) was investigated under various conditions. Numerous factors influenced its activity: method of spore production, inherent spore resistance characteristics, alkalination, storage time and storage temperature. The sporicidal action of 2% alkaline glutaraldehyde at room temperature was compared with that of other aldehydes and commercially available formulations. Cidex (glutaraldehyde) and Sporicidin (glutaraldehyde + phenol full strength) were the most effective, followed by 8% (w/v) formaldehyde and 10% (v/v) Gigasept, a formaldehyde-containing product. Five per cent (v/v) Gigasept and 10% (w/v) glyoxal also had good sporicidal activity, though that of Sporicidin (1:16) was poor. No activity was observed with 10% (w/v) butyraldehyde.

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The effect of glutaraldehyde on the uptake of L-alanine, and subsequent germination, in spores of Bacillus subtilis NCTC 8236 was examined. Germination was induced by single amino acids, D-glucose and phosphate buffer at 37 degrees C. L-alanine was the best germinant of all amino acids tested. Pretreatment of spores with low concentrations of acid and alkaline glutaraldehyde inhibited subsequent germination, complete inhibition being observed at concentrations of 0.1% (w/v). This concentration also prevented the loss of heat resistance of spores placed in germination medium and exposed to 75 degrees C. Radioactive studies indicated that maximum uptake of L-alanine occurred after ca 30 min at 37 degrees C. Only 1.2% of available L-alanine was taken up during germination. Pretreatment of spores with glutaraldehyde did not interfere with L-alanine uptake at aldehyde concentrations up to 0.5% (w/v). However, this was significantly reduced at a glutaraldehyde concentration of 1.0% (w/v). Minimal differences were observed between acid and alkaline forms of the aldehyde. The results are discussed in terms of the mode of action of glutaraldehyde.

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