Comparison of five assays for DNA extraction from bacterial cells in human faecal samples.

AIM: To determine the most effective DNA extraction method for bacteria in faecal samples. MATERIALS AND RESULTS: This study assessed five commercial methods, that is, NucliSens easyMag, QIAamp DNA Stool Mini kit, PureLink Microbiome DNA purification kit, QIAamp PowerFecal DNA kit and RNeasy PowerMicrobiome kit, of which the latter has been optimized for DNA extraction. The DNA quantity and quality were determined using Nanodrop, Qubit and qPCR. The PowerMicrobiome kit recovered the highest DNA concentration, whereby this kit also recovered the highest gene copy number of Gram positives, Gram negatives and total bacteria. Furthermore, the PowerMicrobiome kit in combination with mechanical pre-treatment (bead beating) and with combined enzymatic and mechanical pre-treatment (proteinase K+mutanolysin+bead beating) was more effective than without pre-treatment. CONCLUSION: From the five DNA extraction methods that were compared, the PowerMicrobiome kit, preceded by bead beating, which is standard included, was found to be the most effective DNA extraction method for bacteria in faecal samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The quantity and quality of DNA extracted from human faecal samples is a first important step to optimize molecular methods. Here we have shown that the PowerMicrobiome kit is an effective DNA extraction method for bacterial cells in faecal samples for downstream qPCR purpose.

A double-blind randomized placebo-controlled study on the clinical and microbial effects of an essential oil mouth rinse used by patients in supportive periodontal care.

AIM: This 3-month double-blind randomized placebo-controlled study evaluated the clinical and microbial effects of an essential oil mouth rinse used as an adjunct to mechanical plaque control by patients in supportive periodontal care. MATERIAL AND METHODS: Fifty patients were randomly allocated to an essential oil group (Listerine(®) Coolmint; Johnson & Johnson, New Brunswick, NJ, USA) or placebo group to rinse twice per day as an adjunct to mechanical plaque control. At baseline and after 3 months, plaque index (PI), gingivitis index (GI), probing pocket depth, bleeding on probing (BoP) and clinical attachment level were registered. Subgingival plaque samples were collected for the detection and quantification of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Micromonas micros, Prevotella intermedia, Fusobacterium genus and Streptococcus mutans by means of real-time PCR (qPCR). Patient's compliance, satisfaction and side effects were registered. RESULTS: Twenty-three patients in the essential oil group (mean age: 57) and 21 in the placebo group (mean age: 55) with acceptable oral hygiene at intake (mean PI <1.5 on a scale of 5) adhered to the study protocol. Gingivitis index, PI and BoP significantly reduced over time (P ≤ 0.029); however, between group analyses revealed no significant differences. There was no significant change over time neither in detection frequency nor load for any of the microbiota. Daily rinsing with an essential oil rinse was found safe and perceived beneficial by the patients. CONCLUSION: Patients in supportive periodontal care who are fairly compliant with oral hygiene may not benefit from additional mouth rinsing using an essential oil solution.

Some coagulase-negative Staphylococcus species affect udder health more than others.

A longitudinal study in 3 dairy herds was conducted to profile the distribution of coagulase-negative Staphylococcus (CNS) species causing bovine intramammary infection (IMI) using molecular identification and to gain more insight in the pathogenic potential of CNS as a group and of the most prevalent species causing IMI. Monthly milk samples from 25 cows in each herd as well as samples from clinical mastitis were collected over a 13-mo period. Coagulase-negative staphylococci were identified to the species level using transfer-RNA intergenic spacer PCR. The distribution of CNS causing IMI was highly herd-dependent, but overall, Staphylococcus chromogenes, Staphylococcus xylosus, Staphylococcus cohnii, and Staphylococcus simulans were the most prevalent. No CNS species were found to cause clinical mastitis. The effect of the most prevalent species on the quarter milk somatic cell count (SCC) was analyzed using a linear mixed model, showing that Staph. chromogenes, Staph. simulans, and Staph. xylosus induced an increase in the SCC that is comparable with that of Staphylococcus aureus. Almost all CNS species were able to cause persistent IMI, with Staph. chromogenes causing the most persistent infections. In conclusion, accurate species identification cannot be ignored when studying the effect of CNS on udder health, as the effect on SCC differs between species and species distribution is herd-specific. Staphylococcus chromogenes, Staph. simulans, and Staph. xylosus seem to be the more important species and deserve special attention in further studies. Reasons for herd dependency and possible cow- and quarter-level risk factors should be examined in detail for the different species, eventually leading to cost-benefit analyses for management changes and, if needed, treatment recommendations.

Technical note: use of transfer RNA-intergenic spacer PCR combined with capillary electrophoresis to identify coagulase-negative Staphylococcus species originating from bovine milk and teat apices.

Coagulase-negative staphylococci (CNS) are the most frequently isolated bacteria in milk samples from cows with and without mastitis. Elucidating their relevance in bovine udder health is hampered because identification at the species level, if done at all, used to be performed based on phenotypic features. To provide a rapid, cheap, and easy-to-use genotypic technique that can be used to identify CNS species from milk and teat apices from cows, the performance of transfer RNA-intergenic spacer PCR (tDNA-PCR) in combination with capillary electrophoresis was evaluated. After updating the tDNA library with CNS reference strains, 288 field isolates were identified with tDNA-PCR and gene sequencing, and the latter was used as the reference method. The field isolates were divided in 2 groups of 144. Isolates of the first group were identified with tDNA-PCR with a typeability of 81.9% and an accuracy of 94.1%. Peak patterns of these isolates were then added to the tDNA library with species identity as determined by DNA sequencing. The second group was identified with the updated tDNA library, resulting in 91.0% typeability and 99.2% accuracy. This study showed that the updated tDNA-PCR in combination with capillary electrophoresis was almost as accurate as gene sequencing but faster and cheaper (only $3 per isolate), and is a useful tool in observational studies concerning the epidemiology of bovine CNS species.

Otitis media microbes: culture, PCR, and confocal laser scanning microscopy.

OBJECTIVES: To assess the presence of middle ear pathogens in nasopharynx (NP), middle ear fluid (MEF), and middle ear mucosal swabs (MES) of 14 patients undergoing middle ear surgery. METHODOLOGY: Bacteria were assessed by culture and species specific PCR. Biofilm was investigated by confocal laser scanning microscopy (CLSM) of middle ear biopsies (MEBs). RESULTS: Bacteria were absent in CLSM of MEBs in three of the four closed and healthy middle ears. Bacteria occurred in the ear with a foreign body (middle ear prosthesis), which showed localized living and dead bacteria, indicating biofilm. Bacterial growth was present in ten patient ears, but biofilm occurred in only one patient. CLSM indicated biofilm in the middle ear of two patients for whom PCR detected Haemophilus influenzae in the MEF. The three classical pathogens could frequently be found in the nasopharynx, by culture and PCR, but not from the middle ear. Alloiococcus otitidis was detected in the MEF of all five patients with open inflamed ears, though virtually absent from the nasopharynx. Pseudomonas aeruginosa was present in seven. It was the only pathogen found on several occasions in all three locations in one patient. CONCLUSIONS: This study confirms the association of H. influenzae with middle ear biofilm, and indicates a potential role of P. aeruginosa in middle ear inflammation and biofilm formation. Biofilm does not seem to cause inflammation. It is unclear whether the predominance of A. otitidis in chronically inflamed open middle ears indicates a pathogenic or contaminant role for this organism.

Pseudomonas aeruginosa in the home environment of newly infected cystic fibrosis patients.

The source of acquisition of Pseudomonas aeruginosa in cystic fibrosis (CF) patients remains unknown. Patient-to-patient transmission has been well documented but the role of the environment as a source of initial infection is as yet unclear. In the present study, the origin of the first P. aeruginosa isolate in CF patients was investigated by comparing the P. aeruginosa genotype(s) from newly infected patients with genotypes of P. aeruginosa isolates from the home environment and from other patients from the same CF centre. A total of 50 newly infected patients were studied. P. aeruginosa could be cultured from 5.9% of the environmental samples, corresponding to 18 patients. For nine of these, the genotype of the environmental P. aeruginosa isolate was identical to the patient's isolate. In total, 72% of the environmental P. aeruginosa isolates were encountered in the bathroom. Patient-to-patient transmission within the CF centre could not be ruled out for three patients. In summary, a low prevalence of Pseudomonas aeruginosa was found in the home environment of the newly infected cystic fibrosis patients. The bathroom should be targeted in any preventive cleaning procedures. An environmental source of the new infection could not be ruled out in nine patients.

Staphylococcus aureus controls interleukin-5 release in upper airway inflammation.

Staphylococcus aureus is a frequent colonizer of the upper airways in chronic rhinosinusitis with nasal polyps, but also resides intramucosally; it has been shown that secreted staphylococcal proteins such as enterotoxins and serine proteases induce the release of cytokines such as IL-5. We have analyzed nasal polyp tissue freshly obtained during routine surgery, which did or did not contain cultivatable S. aureus, to study spontaneous IL-5 production by nasal polyp tissue over 24 and 72h in tissue culture. In S. aureus-positive samples we interfered by killing the bacteria using antibiotics or S. aureus specific intravenous staphylococcal phages (ISP), active or heat-inactivated. Phage-neutralizing antibodies were used to demonstrate the specificity of the phage-mediated effects. We monitored S. aureus colony forming units, and identified S. aureus proteins by mass spectrometry. We demonstrate that cultivatable S. aureus may be found in type-2 inflamed nasal polyps; the pathogen is replicating within 24h and secretes proteins, including enterotoxins and serine proteases. The presence of S. aureus was associated with a significantly higher release of IL-5. Killing of S. aureus by antibiotics or specific ISP significantly reduced the IL-5 release. The suppressive activity of the bacteriophage on IL-5 be abolished by heat inactivation or anti-phage antibodies. BIOLOGICAL SIGNIFICANCE: In this study, we used high resolution mass spectrometry to identify S. aureus proteins directly in infected nasal polyp tissue and nasal polyp tissue incubated over 24 and 72h in culture. We discovered bacterial proteins including enterotoxins and serine proteases like proteins. These experiments indicate a direct role of S. aureus in the regulation of IL-5 production in nasal polyps and may suggest the involvement of bacterial proteins detected in the tissues.

Women with symptoms of a urinary tract infection but a negative urine culture: PCR-based quantification of Escherichia coli suggests infection in most cases.

OBJECTIVES: Our objective was to examine whether or not women with symptoms of a urinary tract infection but with a negative culture (20%-30%) do have an infection. METHODS: We performed quantitative PCR (qPCR) for Escherichia coli and Staphylococcus saprophyticus, on top of a standard culture, in urine samples from 220 women with dysuria and/or frequency and/or urgency and from 86 women without symptoms. For symptomatic women, qPCR was also carried out for four sexually transmitted agents. RESULTS: In the symptomatic group, 80.9% (178/220) of the urine cultures were positive for any uropathogen and 95.9% (211/220) were E. coli qPCR-positive. For the control group, cultures for E. coli and E. coli qPCR were positive in, respectively, 10.5% (9/86) and 11.6% (10/86). In the symptomatic group, qPCR yielded 19 positive samples for S. saprophyticus qPCR, one positive sample for Mycoplasma genitalium and one for Trichomonas vaginalis. CONCLUSIONS: These findings suggest that almost all women with typical urinary complaints and a negative culture still have an infection with E. coli.

Evaluation of the applicability of amplified rDNA-restriction analysis (ARDRA) to identification of species of the genus Corynebacterium.

The 16S rRNA genes (rDNA) of 50 strains belonging to 26 different coryneform bacterial species and genomospecies and of the type strain of Rhodococcus equi were enzymatically amplified. Amplified rDNA restriction analysis (ARDRA) with the enzymes AluI, CfoI and RsaI was carried out. The combination of the ARDRA patterns obtained after restriction with these three different enzymes enabled the differentiation between the following species: Corynebacterium accolens (number of strains = 2), C. afermentans subsp. afermentans (2), C. afermentans subsp. lipophilum (2), C. amycolatum (3), CDC coryneform group ANF-1-like (1), CDC coryneform group ANF-3-like (1), C. cystitidis (1), C. diphtheriae (4), C. jeikeium (3), C. macginleyi (2), C. minutissimum (1), C. pilosum (1), C. pseudotuberculosis (2), C. renale (2), C. striatum (2), C. urealyticum (3), C. xerosis (1), CDC coryneform groups B-1 (2), B-3 (2), F-1, genomospecies 1 and 2 (6), G, genomospecies 1 (1) and G, genomospecies 2 (2). The following strains or species could not be differentiated from each other: C. pseudodiphtheriticum (2) from C. propinquum (former CDC coryneform group ANF-3) (2), CDC coryneform group F-1, genomospecies 1 (4) from genomospecies 2 (2) and C. jeikeium genomospecies A (1) from genomospecies C (2). ARDRA may represent a possible alternative for identification of coryneforms, since this technique enabled the identification of most coryneforms tested and since DNA extraction (i.e. cell lysis by boiling), amplification, restriction and electrophoresis can be carried out within 8 hours. This might allow quick identification of C. diphtheriae and other possible pathogens of the genus Corynebacterium.

Comparison of arbitrarily primed polymerase chain reaction and cell envelope protein electrophoresis for analysis of Acinetobacter baumannii and A. junii outbreaks.

Two successive Acinetobacter outbreaks in a neonatal intensive care unit were studied with arbitrarily primed polymerase chain reaction (AP-PCR), cell envelope protein electrophoresis (protein fingerprinting) and antibiotic susceptibility testing. AP-PCR fingerprinting and protein fingerprinting yielded identical clustering of the isolates studied. Susceptibility test results were useful for rapid recognition of the outbreaks, but clustering of several isolates was different from the clustering obtained with AP-PCR fingerprinting and protein fingerprinting. Typing results indicated that the two outbreaks, which occurred at a three-month interval, were each caused by a single strain, and that both strains differed from the strains prevailing in the hospital. The strain of one outbreak was identified as A. junii, a species commonly not involved in outbreaks. A. baumannii isolates collected from different departments of this hospital during a period of four years clustered into only five different types. Moreover, strains from different departments of a second hospital belonged to the type prevailing in the first hospital, although there were no apparent connections between the two institutions. This may indicate that only a limited number of strains of the A. calcoaceticus-baumannii complex are involved in nosocomial outbreaks.

Oil-degrading Acinetobacter strain RAG-1 and strains described as 'Acinetobacter venetianus sp. nov.' belong to the same genomic species.

Acinetobacter strain RAG-1 (ATCC 31012) is an industrially important strain which has been extensively characterized with respect to its growth an hydrocarbons and its production of a high molecular mass bioemulsifier, emulsan. Although RAG-1 has been investigated in detail for specific biochemical characteristics, its taxonomic status is uncertain and it is usually referred to as A. lwoffii or A. calcoaceticus sensu lato. However, results obtained by restriction analysis of the amplified rDNA and subsequently substantiated by DNA-DNA hybridization, partial 16S rDNA nucleotide sequence comparison and biochemical characterization indicate that RAG-1 belongs to the genomic species recently described as 'A. venetianus'. Furthermore, these data confirm that 'A. venetianus' constitutes a new and distinct genomic species within the genus Acinetobacter.

The scientific origin of life. Considerations on the evolution of information, leading to an alternative proposal for explaining the origin of the cell, a semantically closed system.

We hypothesize that the origin of life, that is, the origin of the first cell, cannot be explained by natural selection among self-replicating molecules, as is done by the RNA-world hypothesis. To circumvent the chicken and egg problem associated with semantic closure of the cell--no replication of information molecules (nucleotide strands) without functional enzymes, no functional enzymes without encoding in information molecules--a prebiotic evolutionary process is proposed that, from the informational point of view, must somehow have resembled the current scientific process. The cell was the outcome of interactions of a complex premetabolic community, with information molecules that were devoid of self-replicative properties. In a comparable manner, scientific progress is possible, essentially because of interaction between a complex cultural society and permanent information carriers like printed matter. This may eventually lead to self-replicating technology in which semantic closure occurs anew. Explaining the origin of life as a scientific process might provide a unifying theory for the evolution of information, wherebye at two moments symbolization/encoding of interactions into permanent information occurred: at one moment that of chemical interaction and at another moment that of animal behavior interaction. In one event this encoding led to autonomously duplicating chemistry (the cell), an event that possibly may be one of the outcomes of current scientific progress.

Study of the influence of plasmids on the arbitrary primer polymerase chain reaction fingerprint of Escherichia coli strains.

To study the effect of plasmids on the arbitrary primer-polymerase chain reaction fingerprint of bacterial strains, the Escherichia coli strains DH5, Top10, and W3110 were transformed with plasmids of different sizes: respectively, pUC19, pCEP and two clinically important plasmids carrying resistance to several antibiotics. Total DNA, i.e. both chromosomal and plasmid DNA, was prepared from transformed cells by boiling the cell suspensions and by phenol-chloroform extraction; chromosomal DNA was prepared by the same methods from the non-transformed, plasmid-free strains; plasmid DNA of pUC19 was purchased; plasmid DNA of pCEP was purified from the transformed strains by caesium chloride density gradient centrifugation. Arbitrarily primed polymerase chain reaction was carried out for all of these preparations. Amplification carried out independently with three different primers resulted in similar patterns for the chromosomal preparations whether or not plasmid was present. Amplification of plasmid DNA gave different patterns, characterized by fragments larger than those obtained when total or chromosomal DNA were used as the target. These data illustrate that the plasmids studied here do not influence the chromosomal arbitrarily primed PCR fingerprint, although plasmids alone are amplified in the absence of chromosomal DNA. Experiments comparing different relative concentrations of plasmid and chromosomal DNA indicate that under natural conditions the amount of chromosomal DNA per cell is sufficient to inhibit observable amplification of the plasmid(s) present.

Rapid identification of bacteria of the Comamonadaceae with amplified ribosomal DNA-restriction analysis (ARDRA).

Ribosomal rRNA gene fragments (rDNA) encompassing the 16S rDNA, the 16S-23S rDNA spacer region and part of the 23S rDNA of 95 strains belonging to 13 well-described taxa of the eubacterial family Comamonadaceae (beta subclass of the Proteobacteria or rRNA superfamily III) were enzymatically amplified using conserved primers. The fragments of approximately 2400 base pairs were subjected to restriction analysis. Restriction fragment length patterns obtained with HinfI enabled us to distinguish 9 of the 13 taxa studied. Restriction with CfoI was necessary to differentiate Acidovorax delafieldii from A. temperans and Hydrogenophaga flava from H. pseudoflava. The results indicate that amplified rDNA restriction analysis is a simple and reliable tool for the identification of bacterial species.

The possibilities and limitations of nucleic acid amplification technology in diagnostic microbiology.

Nucleic acid amplification technology is examined from the critical viewpoint of a clinical microbiologist working in a routine diagnostic bacteriology laboratory. Widely recognised limitations of amplification technology include those of false-positive and false-negative results, the difficulty of obtaining quantitative results, the problem of using this technology for susceptibility testing, and the difficulty of detecting routinely the wide range of possible pathogens contained in a clinical sample. On the positive side, amplification technology brings welcome new possibilities for rapid detection of specific pathogens in a sample, including viruses, slowly growing bacteria, fastidious or uncultivable bacteria, fungi and protozoa. Other possible applications include screening normally sterile clinical samples for non-specific bacterial contamination and the use of amplification-based DNA fingerprinting methods for identification and typing of microorganisms. Nevertheless, it is predicted that-in contrast to research and reference facilities-routine bacteriology laboratories will continue to rely on culture as the preferred 'amplification method' for most diagnostic applications.

Evaluation of six commercial assays for the rapid detection of Clostridium difficile toxin and/or antigen in stool specimens.

OBJECTIVE: To evaluate six commercially available assays for the detection of Clostridium difficile toxin and/or antigen in stool samples: one latex agglutination test (Culturette brand CDT, Becton Dickinson), two ELISAs (Culturette brand Toxin CD, Becton Dickinson, and Ridascreen C. difficile Toxin A/B, R-biopharm), two chromatographic assays (Clearview C. difficile A, Oxoid, and ColorPac Toxin A, Becton Dickinson) and one enzyme immunoassay for the simultaneous detection of C. difficile common antigen and toxin A (Triage C. difficile Panel, Biosite). METHODS: Over a period of 3 months, 366 liquid or semi-liquid stool samples were tested using cell-culture cytotoxin assay as standard, ethanol shock stool culture and latex agglutination (Culturette brand CDT). Of these, 78 samples, positive with at least one of these three methods, and 98 randomly selected negative samples were further evaluated using the other five kits. PCR was also performed on positive cultures to confirm the presence of toxin A and B genes. RESULTS: Triage C. difficile Panel had the best sensitivity (95%), followed by Clearview C. difficile and ColorPac Toxin A (both 89%), Culturette brand Toxin CD (73%), Ridascreen C. difficile Toxin A/B (57%) and Culturette brand CDT (23%). For Triage, the sensitivity of C. difficile antigen detection was 93%, and the sensitivity of toxin detection was lower (77%). Most false-positive results were obtained with the Triage C. difficile Panel (25 specimens) and Clearview C. difficile A (20 specimens). Culturette brand CDT had the best specificity (99%); followed by Ridascreen C. difficile Toxin A/B (97%), Culturette brand Toxin CD (95%), ColorPac Toxin A (89%), Clearview C. difficile A (83%) and Triage C. difficile Panel (75%). The positive predictive values ranged from 68% to 94%, and the negative predictive values from 83% to 98%. CONCLUSIONS: The sensitivity is much higher for Triage and the two new chromatographic assays than for the conventional EIAs. These tests also have a high negative predictive value. For Triage, C. difficile antigen-positive, toxin A-negative results can be obtained; the clinical value of these must be established by additional studies. Overall, the new-generation assays are still less sensitive than the cytotoxin assay; however, they provided same-day results, could be used as a screening test and may be useful in laboratories without tissue-culture facilities. Our results do not allow the recommendation of one single assay for the diagnosis of C. difficile-associated diarrhea. It remains the case that laboratory results must be correlated and interpreted with the clinical presentation of the patient.

DNA fingerprinting techniques for microorganisms. A proposal for classification and nomenclature.

A whole array of DNA-fingerprinting techniques, which provide indirect access to DNA sequence polymorphism in order to assess species or clonal identity of bacterial organisms or in order to study bacterial genome composition, have been described during past decades. Nomenclature has been sometimes erroneous and/or confusing, also because of hybrid techniques that combine different approaches. It can be shown that most techniques study the sequence polymorphism of only the chromosome, or only the plasmid(s) or only a gene or gene fragment and that the sequence polymorphism is revealed by AFLP (amplified fragment length polymorphism) or by RFLP (restriction fragment length polymorphism) or by special electrophoresis techniques. Starting from these considerations, some taxonomy of techniques, which enables more appropriate nomenclature, can be developed.

Tracheal colonization with Sphingomonas paucimobilis in mechanically ventilated neonates due to contaminated ventilator temperature probes.

Sphingomonas paucimobilis was isolated from tracheal secretions of a total of 85 mechanically ventilated babies in a neonatal intensive-care unit (NICU) during a two-year-period. None of the neonates developed pneumonia or sepsis. After each increase in the fluctuating number of S. paucimobilis isolates, extra attention was paid to hand hygiene and to the maintenance of the ventilation equipment. This resulted in a reduction of the frequency of isolation each time. Cultures of all liquids in use and of the ventilation equipment were negative on several occasions. Fifteen months after the start of the outbreak, the NICU was moved to another building, and some older ventilation equipment was abandoned. After a period of six weeks without problems, S. paucimobilis was isolated in association with at least four ventilators. A new investigation showed that the ventilator temperature probes were the source of contamination. Once effective sterilization procedures for the temperature probes were introduced no new cases appeared, until a spare ventilator with an unautoclaved temperature probe was accidentally used and this caused contamination of one child. After correction, no further cases have occurred to date. The clonal relatedness of the outbreak isolates from patients and from ventilator temperature probes was documented by fingerprinting with the arbitrarily primed polymerase chain reaction.

Outbreak of severe Pseudomonas aeruginosa respiratory infections due to contaminated nebulizers.

During the six months from January-June 1994, 10 cases of severe and 11 of less severe pulmonary infection caused by Pseudomonas aeruginosa were diagnosed in patients with chronic obstructive airways disease. Possible sources were evaluated. P. aeruginosa was isolated from four of the 22 nebulizers tested. The relationship of isolates from the patients and nebulizers was confirmed by sero- and phage-typing, and by arbitrarily-primed polymerase chain reaction (AP-PCR). Three types were identified and the distribution of types in patients with severe infection was as follows (one patient had a multiple infection). Type I was isolated from two nebulizers and from sputa, and/or blood and/or bronchial protected specimen brush samples or bronchial lavage fluid from four patients. Type II came from the sputa of three patients and a third nebulizer; and type III from sputa and/or blood of four further patients and another nebulizer. The data provided evidence for the relation between P. aeruginosa as a cause of infection and the contamination of the nebulizers. When nebulizer mouthpieces were changed every 24 h and sterilized between patients, no more contamination occurred, and the outbreak ceased.

Evaluation of amplified ribosomal DNA restriction analysis for identification of Acinetobacter genomic species.

Further to a previous study, the usefulness of amplified ribosomal DNA restriction analysis (ARDRA) for identification of Acinetobacter genomic species (DNA groups) was tested. A set of 202 Acinetobacter strains of 18 described genomic species and 17 unclassified strains were used. Restriction patterns obtained with a standard panel of restriction enzymes CfoI, AluI, MboI, RsaI and MspI allowed for separation of 11 DNA groups. With the additional use of restriction enzymes BfaI and BsmAI, five other (genomic) species could be differentiated, leaving only A. haemolyticus and DNA group 13BJ/14TU unseparated. With the standard panel of enzymes, ten new ARDRA profiles were noted in 14 unclassified strains. Two other unclassified strains had a profile in common with DNA group 15BJ, but were differentiated from this DNA group by restriction with bfaI. One remaining unclassified strain could not be differentiated from DNA group 17 by the standard panel of enzymes or by the enzymes BfaI and BsmAI. Results demonstrate the utility of ARDRA for identification of most genomic species of Acinetobacter. Furthermore, new ARDRA profiles that were shared by several unclassified strains may indicate so far undescribed genomic species in the genus.