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Acinetobacter spp. are being reported with increasing frequency as causes of nosocomial infection. In order to identify reservoirs of infection as quickly as possible, a rapid typing method that can differentiate epidemic strains from environmental and nonepidemic strains is needed. In 1993, a cluster of Acinetobacter baumannii isolates from five patients in the adult intensive therapy unit of our tertiary-care teaching hospital led us to develop and optimize a rapid repetitive extragenic palindromic sequence-based PCR (REP-PCR) typing protocol for members of the Acinetobacter calcoaceticus-A. baumannii complex that uses boiled colonies and consensus primers aimed at repetitive extragenic palindromic sequences. Four of the five patient isolates gave the same REP-PCR typing pattern as isolates of A. baumannii obtained from the temperature probe of a Bennett humidifier; the fifth isolate had a unique profile. Disinfection of the probe with 70% ethanol, as recommended by the manufacturer, proved ineffective, as A. baumannii with the same REP-PCR pattern was isolated from it 10 days after cleaning, necessitating a change in our decontamination procedure. Results obtained with REP-PCR were subsequently confirmed by ribotyping. To evaluate the discriminatory power (D) of REP-PCR for typing members of the A. calcoaceticus-A. baumannii complex, compared with that of ribotyping, we have applied both methods to a collection of 85 strains that included representatives of six DNA groups within the complex. Ribotyping using EcoRI digests yielded 53 patterns (D = 0.98), whereas 68 different REP-PCR patterns were observed (D = 0.99). By computer-assisted analysis of gel images, 74 patterns were observed with REP-PCR (D = 1.0). Overall, REP-PCR typing proved to be slightly more discriminatory than ribotyping. Our results indicate that REP-PCR typing used boiled colonies is a simple, rapid, and effective means of typing members of the A. calcoaceticus-A. baumannii complex.

Seventy-three isolates of the Acinetobacter calcoaceticus-Acinetobacter baumannii complex, including 26 isolates from 10 hospital outbreaks, were typed by ribotyping with EcoRI and ClaI and by pulsed-field gel electrophoresis (PFGE) of genomic DNA after digestion with ApaI. Ribotyping with EcoRI distinguished 31 ribopatterns. Digestion with ClaI generated another eight ribotypes. PFGE, in contrast, identified 49 distinct patterns with seven variants. Both methods detected all outbreak-related isolates. By ribotyping, nine epidemiologically unrelated strains could not be differentiated from outbreak strains, in contrast to only one isolate not identified by PFGE. Thus, PFGE was more discriminating than ribotyping. However, ribotyping is known to generate banding patterns specific to each DNA group in the A. calcoaceticus-A. baumannii complex that may be used for taxonomic identification of the strains. PFGE was shown to lack this property. Both methods are therefore useful for strain differentiation in epidemiological studies of Acinetobacter isolates.

Risk factors for listeriosis are foods and underlying diseases. A case-control-study of listeriosis patients' dietary habits showed that unpasteurized milk was a risk food for sporadic listeriosis. A blue-mould cheese was significantly more often eaten by patients ill with an epidemic phage type. Leukaemia, AIDS and renal transplantation were found to be associated with a more than 1000 times higher risk of acquiring listeriosis, compared with healthy persons. As a part of prophylaxis, persons with a high risk of listeriosis should be informed individually about food hygiene and risk foods.

Typing of Legionella pneumophila remains important in the investigation of outbreaks of Legionnaires' disease and in the control of organisms contaminating hospital water. We found that the discriminatory power of a nonradioactive ribotyping method could be improved by combining results obtained with four restriction enzymes (HindIII, NciI, ClaI, and PstI). Fifty-eight clinical and environmental L. pneumophila strains including geographically unrelated as well as epidemiologically connected isolates were investigated. Epidemiologically related strains had the same ribotypes independent of the combinations of enzymes used. Some strains belonging to the same serogroup were assigned to different ribotypes, and some ribotypes contained members of different serogroups, indicating, as others have found, that serogroup and genotype are not always related. The discriminatory power of the method was estimated by calculating an index of discrimination (ID) for individual enzymes and combinations thereof. The combined result with all four enzymes was highly discriminatory (ID = 0.97), but results for three enzymes also yielded ID values acceptable for epidemiological purposes. In addition, the testing of 27 type strains and 6 clinical isolates representing Legionella species other than L. pneumophila indicated that ribotyping might be of value for species identification within this genus, as previously suggested.

An organism that seems to be identical to Orskov's 'Sarcina mirabilis' [Orskov, J. (1930) Acta Pathol Microbiol Scand Suppl III, 519-541] has been rediscovered in specimens from the upper respiratory tract of humans. Six strains were studied, and the results, which conformed to Orskov's description of S. mirabilis, were as follows. Rough to smooth colonies grow on many plated media and show extremely polymorphic cell morphology with round cells with diameters from 1 to > 10 microns. The smallest cells were often motile with circular movements. Strains were Gram-negative, facultatively anaerobic, oxidase and urease positive, and weakly catalase positive. Nitrate and nitrite were reduced, and glucose, fructose, sucrose and mannitol were fermented. Polysaccharide was produced on sucrose agar. Electron microscopy showed coccoid cells with a bundle of three to nine flagella, a Gram-negative cell-wall morphology, and aggregates of irregular cells held together by a common surface layer. The mean mol% (G+C) of the organisms was 65.0. 16S-ribosomal RNA sequencing revealed that the organism belongs to the beta subgroup of Proteobacteria, separate from all other described genera, but most closely related to Burkholderia. The name Lautropia mirabilis is proposed for this organism.

The discriminatory power of four methods for typing of Listeria monocytogenes was compared. The four methods were multilocus enzyme electrophoresis (MEE), ribotyping, restriction enzyme analysis (REA), and a newly developed Danish phage typing system. Ninety-nine human clinical, food and slaughterhouse isolates of Listeria monocytogenes were typed by each method. The most discriminatory single typing method was phage typing with an overall discriminatory index (DI) of 0.88 followed by REA, MEE and ribotyping with DI-values at 0.87, 0.83 and 0.79 respectively. Considering strains from each of the two predominant O-serotypes alone, serotype 1 was best discriminated by the molecular typing methods, in particular REA, which showed a DI of 0.92. The serotype 4 strains were best discriminated by phage typing (DI = 0.78). If two or more typing methods were combined, the combination of REA and MEE were found to be the most discriminatory combination. The DI values were 0.96, 0.74 and 0.90 for serotype 1, 4, and both combined, respectively. Phage typing is a rapid and inexpensive typing method but not as reproducible as the molecular typing methods. It is the most suitable method for mass screening. In situations where results are required to be highly reliable, i.e. when studying the relationships between only a few strains, a single or a combination of molecular typing methods should be used, preferable MEE and REA.

On the occasion of five Danish human Pasteurella aerogenes from pig bite lesions, a comparison was made between 6 isolates from man and 15 animal isolates, mainly from pigs. The strains originated from 6 different countries (USA, Canada, Czechoslovakia, France, Belgium and Denmark). The 21 isolates were characterized by conventional biochemical tests, antibiogram and the API 20 NE kit; finally ribotyping was carried out by hybridizing EcoRI-digested chromosomal DNA with a probe derived from E. coli ribosomal RNA. By ribotyping, 19 of the 21 strains clustered at a similarity level of 81% or more; both phenotypical tests and ribotyping indicated that the remaining two strains did not belong to the species P. aerogenes. In conclusion, despite minor differences our P. aerogenes isolates constituted a well-defined group and they could not be subdivided on basis of animal or geographical origin.

Strains of H. aphrophilus, H. paraphrophilus, and A. actinomycetemcomitans are phenotypically very similar. Ribotyping of 24 strains of H. aphrophilus, 22 strains of H. paraphrophilus, 8 strains of A. actinomycetemcomitans, and one strain each of the species Pasteurella aerogenes, H. parahaemolyticus, and the genus Capnocytophaga was studied using a non-radioactive digoxigenin labelled probe based on E. coli 16S- and 23S-ribosomal RNA. Restriction fragments were generated using restriction enzyme EcoRI. The ribotypes were analysed by a numerical approach using UPGMA clustering. Two major clusters were seen: One contained all A. actinomycetemcomitans strains, the other all H. aprophilus and all except one H. paraphrophilus strain intermingled between each other. The H. paraphrophilus strain not found in the H. aphrophilus/H. paraphrophilus cluster, the H. parahaemolyticus, P. aerogenes, and the Capnocytophaga strains clustered separately from each other and the two major clusters. The H. paraphrophilus strain with the deviating ribotype was atypical in other respects: it neither did ferment lactose nor mannose and it was isolated from a deer in contradiction to the remaining H. paraphrophilus strains, which were human isolates. This study supports the view that H. aphrophilus and H. paraphrophilus should be regarded as one species separated from A. actinomycetemcomitans.

Two cases of transfusion-related Serratia marcescens bacteremia prompted extensive epidemiologic investigations in three independent hospitals. Test tubes and plasma from donors whose blood was drawn into bags from a single production batch were cultured. Analysis of the ribotype of S. marcescens isolates was performed. For comparison, a strain from the production plant and eight other, unrelated bacteremia isolates were examined. In addition, a retrospective national survey was carried out. S. marcescens was cultured from 11 (0.73%) of 1515 blood units, and an additional (third) bacteremic patient was identified. The clinical isolates from three patients, the three units of blood transfused, and the plant-derived strain shared a unique ribotype. The incident is interpreted as a sporadic, bacterial contamination of blood bags with the S. marcescens epidemic strain, occurring during the manufacturing or packaging. A similar incident has not previously been reported. Attention is drawn to the possibility of significant contamination during the complex production of multiple-bag blood collection systems. Guidelines for improved registration and handling of transfusion complications in wards are suggested. Manufacturers should be encouraged to provide blood packs with sterile exteriors, in appropriate, single, outer packages.

Four methods, namely, biotyping, cell envelope protein electrophoresis, ribotyping, and comparison of antibiograms, were used for strain identification of Acinetobacter isolates from five outbreaks in hospitals. There was good agreement among the methods for the identification of an index strain, but biotyping and the comparison of antibiograms were the least discriminatory.

The Acinetobacter calcoaceticus-Acinetobacter baumannii complex consists of four genotypically distinct but phenotypically very similar bacterial species or DNA groups: A. calcoaceticus (DNA group 1), A. baumannii (DNA group 2), unnamed DNA group 3 (P. J. M. Bouvet and P. A. D. Grimont, Int. J. Syst. Bacteriol. 36:228-240, 1986), and unnamed DNA group 13 (I. Tjernberg and J. Ursing, APMIS 97:595-605, 1989). Because strains in this complex cause nosocomial outbreaks, it is important to be able to identify them as completely as possible. Ribotyping could provide such identification. Therefore, ribotyping was done on 70 strains in the A. calcoaceticus-A. baumannii complex with known DNA group affiliations by use of restriction enzymes EcoRI, ClaI, and SalI. A nonradioactive digoxigenin-11-dUTP-labeled Escherichia coli rRNA-derived probe was used. With any of the three restriction enzymes, banding patterns that were specific for each DNA group were seen. All 70 strains showed banding patterns that could identify them to the correct DNA group by use of any two of the three enzymes. In addition, banding patterns that could separate strains within any one DNA group were present. The discriminatory index of P. Hunter and M. Gaston (J. Clin. Microbiol. 26:2465-2466, 1988), applied to all strains with the combined results obtained with all three enzymes, revealed a value of 0.99. For strains in each DNA group, the value varied from 0.93 to 0.98. These results indicate the high discriminatory power of the system when used for epidemiological typing.

As part of an epidemiological investigation of hospital infections caused by Acinetobacter spp. the radiation resistance of 15 clinical isolates and four reference strains was assessed. The radiation resistance (in D-6 values, viz. the dose necessary for reducing the initial number of colony forming units by a factor of 10(6)) was, in general, higher in the isolates of A. radioresistens than in the isolates of the A. calcoaceticus-A. baumannii complex and of A. lwoffi. However, the least resistant isolates of A. radioresistens had a D-6 value equal to or lower than the most resistant isolates of the other groups. The lowest D-6 values found were for two of the reference strains. The highest D-6 value was 35 kGy. Three isolates of A. johnsonii could not survive long enough in a dried preparation to make an assessment of the D-6 values possible. The radiation resistance of the 15 clinical isolates in the present study was higher than the resistance found in a study of similar isolates in 1970.

A numerical approach was used for identification of 198 Acinetobacter strains assigned to DNA groups according to the classification of Tjernberg and Ursing (I. Tjernberg and J. Ursing, APMIS 97:595-605, 1989). The matrix used was constructed from data published by Bouvet and Grimont (P.J.M. Bouvet and P.A.D. Grimont, Int. J. Syst. Bacteriol. 36:228-240, 1986) and Bouvet and Jeanjean (P.J.M. Bouvet and S. Jeanjean, Res. Microbiol. 140:291-299, 1989). The tests chosen were those of the simplified identification scheme for Acinetobacter species devised by Bouvet and Grimont (P.J.M. Bouvet and P.A.D. Grimont, Ann. Inst. Pasteur/Microbiol. 138:569-578, 1987), namely, growth at 37, 41, and 44 degrees C, oxidation of glucose, gelatin hydrolysis, and assimilation of 14 carbon sources. Of the strains tested, 181 represented 12 DNA groups in the matrix; at a probability level of greater than or equal to 0.95, 78% of them were correctly identified, 2.2% were misidentified, and 19.8% were not identified. Seventeen strains represented two DNA groups not included in the matrix; nine of them were incorrectly assigned to a DNA group by these phenotypic tests. Because of problems of separating strains belonging to DNA groups 1, 2, 3, and 13 by using the phenotypic tests proposed by Bouvet and Grimont (Ann. Inst. Pasteur/Microbiol.), we suggest that these groups should be referred to as the Acinetobacter calcoaceticus-A. baumannii complex.

Seventy four asaccharolytic strains of Acinotobacter were tested for the presence of PQQ dependent glucose dehydrogenase apoenzyme. This apoenzyme could not be detected in any of five strains of Acinetobacter haemolyticus; it was also lacking in one strain of each A. junii and A. lwoffi.

An epidemic spread of Acinetobacter calcoaceticus var. anitratus in two neurosurgical wards is described retrospectively and prospectively using electronic data processing. Isolation of the species from sputum preceded the isolation from CSF by 1/2-1 year. Control measures directed against spread by air and indirect contact controlled the epidemic. Reexamination of 20 selected strains from the epidemic revealed two distinct resistance patterns.

In a prospective study involving a total of 87 ventriculostomies, ventriculostomy-related infections (based upon a bacteriological definition) developed in 15 patients (17.2 per cent). Intraventricular haemorrhage was related to a higher infection rate. Infection was most frequent within the first days after the external ventricular drain (EVD) was inserted. There was no relation between infection and the number of manipulations of the EVD or antibiotic treatment during the time of EVD placement.

Two different experiments were set up to induce differential staining in M-1(1) cells or to induce isostaining in M-2 cells after gamma-irradiation of human lymphocytes. Neither of these two unusual staining patterns previously described by Luchnik and Porjadkova (1977) were observed. Possible explanations for this disagreement are discussed.

The results of the treatment of 15 cases of ventriculitis related to the use of external ventricular drainage are presented. A review of the literature on the treatment of cerebrospinal fluid shunt infections combined with our data suggest the following treatment of ventriculostomy-related ventriculitis: 1. Antibiotic treatment according to the resistance pattern of the infecting microorganism and 2. Removal or replacement of the ventricular drain. 3. One should wait for bacteriological cure before implanting a permanent internal drainage system.

Members of three generations of a single family were examined and found to have a balanced translocation t(11;16)(q13;p11). Cytogenetic investigation and investigation of a number of gene markers is consistent with the current view that the Hp-alpha locus is situated in the proximity of band 16q22.