Ingela Tjernberg

Clinical strains of Acinetobacter classified by DNA-DNA hybridization.

A collection of Acinetobacter strains consisting of 168 consecutive clinical strains and 30 type and reference strains was studied by DNA‐DNA hybridization and a few phenotypic tests. The field strains could be allotted to 13 DNA groups. By means of reference strains ten of these could be identified with groups described by Bouvet & Grimont (1986), while three groups were new; they were given the numbers 13‐15. The type strain of A. radioresistens‐ recently described by Nishimura et al. (1988) ‐ was shown to be a member of DNA group 12, which comprised 31 clinical isolates. Of the 19 strains of A. junii, eight showed hemolytic activity on sheep and human blood agar and an additional four strains on human blood agar only. Strains of this species have previously been regarded as non‐hemolytic. Reciprocal DNA pairing data for the reference strains of the DNA groups were treated by UPGMA clustering. The reference strains for A. calcoaceticus, A. baumannii and for DNA groups 3 and 13 formed a cluster with about 70% relatedness within the cluster. Other DNA groups joined at levels below 60%.

Discrimination of Acinetobacter genomic species by AFLP fingerprinting.

AFLP is a novel genomic fingerprinting method based on the selective PCR amplification of restriction fragments. The usability of this method for the differentiation of genomic species in the genus Acinetobacter was investigated. A total of 151 classified strains (representing 18 genomic species, including type, reference, and field strains) and 8 unclassified strains were analyzed. By using a single set of restriction enzymes (HindIII and TaqI) and one particular set of selective PCR primers, all strains could be allocated to the correct genomic species and all groups were properly separated, with minimal intraspecific similarity levels ranging from 29 to 74%. Strains belonging to genomic species 8 (Acinetobacter lwoffii sensu stricto) and 9 grouped together in one cluster. The closely related DNA groups 1 (Acinetobacter calcoaceticus), 2 (Acinetobacter baumannii), 3 and 13TU (sensu Tjernberg & Ursing 1989) were clearly distinguishable, with intraspecific linkage levels above 50%. Strains of the independently described genomic species 13BJ (sensu Bouvet & Jeanjean 1989) and 14TU linked together at a relatively low level (33%). Although a previous DNA-DNA hybridization study seemed to justify the unification of these genomic species, AFLP analysis actually divides the 13BJ-14TU group into three well-separated subgroups. Finally, four unclassified strains obtained from diverse sources and origins grouped convincingly together, with a similarity linkage level of approximately 50%. These strains showed no similarities in their AFLP patterns with any of the other 155 strains studied and may represent a thus-far-undescribed Acinetobacter species. Based on these results, AFLP should be regarded as an important auxiliary method for the delineation of genomic species. Furthermore, because AFLP provides a detailed insight into the infraspecific structure of Acinetobacter taxa, the method also represents a highly effective means for the confirmation of strain identity in the epidemiology of acinetobacters.

Acinetobacter ursingii sp. nov. and Acinetobacter schindleri sp. nov., isolated from human clinical specimens

The taxonomic status of two recently described phenetically distinctive groups within the genus Acinetobacter, designated phenon 1 and phenon 2, was investigated further. The study collection included 51 strains, mainly of clinical origin, from different European countries with properties of either phenon 1 (29 strains) or phenon 2 (22 strains). DNA-DNA hybridization studies and DNA polymorphism analysis by AFLP revealed that these phenons represented two new genomic species. Furthermore, 16S rRNA gene sequence analysis of three representatives of each phenon showed that they formed two distinct lineages within the genus Acinetobacter. The two phenons could be distinguished from each other and from all hitherto-described Acinetobacter (genomic) species by specific phenotypic features and amplified rDNA restriction analysis patterns. The names Acinetobacter ursingii sp. nov. (type strain LUH 3792T = NIPH 137T = LMG 19575T = CNCTC 6735T) and Acinetobacter schindleri sp. nov. (type strain LUH 5832T = NIPH 1034T = LMG 19576T = CNCTC 6736T) are proposed for phenon 1 and phenon 2, respectively. Clinical and epidemiological data indicate that A. ursingii has the capacity to cause bloodstream infections in hospitalized patients.

Naturally transformable Acinetobacter sp. strain ADP1 belongs to the newly described species Acinetobacter baylyi.

ABSTRACT Genotypic and phenotypic analyses were carried out to clarify the taxonomic position of the naturally transformable Acinetobacter sp. strain ADP1. Transfer tDNA-PCR fingerprinting, 16S rRNA gene sequence analysis, and selective restriction fragment amplification (amplified fragment length polymorphism analysis) indicate that strain ADP1 and a second transformable strain, designated 93A2, are members of the newly described species Acinetobacter baylyi. Transformation assays demonstrate that the A. baylyi type strain B2T and two other originally identified members of the species (C5 and A7) also have the ability to undergo natural transformation at high frequencies, confirming that these five strains belong to a separate species of the genus Acinetobacter, characterized by the high transformability of its strains that have been cultured thus far.

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.

Acinetobacter in Denmark: II. Molecular studies of the Acinetobacter calcoaceticus-Acinetobacter baumannii complex.

The Acinetobacter calcoaceticus‐Acinetobacter baumannii complex consists of four closely related “genospecies” or DNA groups: DNA group 1 (A. calcoaceticus), DNA group 2 (A. baumannii), DNA 3, and Tjernberg & Ursing‘s DNA group 13. Strains in this complex are so similar phenotypically that it is often impossible to identify them to the DNA group level by the use of biochemical tests. Twenty‐three Danish clinical strains from 23 patients phenotypically identified to the A. calcoaceticus‐A. baumannii complex were studied by ribotyping, plasmid profiling, and DNA/DNA hybridization. Multiple isolates were recovered from four patients. These were identical in each patient as judged by phenotype, ribotype and plasmic profile. Seventeen different ribotypes were observed among the 23 strains, and by using this method 19 out of the 23 strains could be identified to the DNA group level. Five strains were allocated to DNA group 2 (A. baumannii), eight to DNA group 3, and six to DNA group 13. These findings were confirmed by DNA/DNA hybridization. Two of the four unidentified strains were genotypically most closely related to but different from DNA groups 1 and 3. The last two strains were most closely related to DNA group 13. These four strains represent two new DNA groups within the A. calcoaceticus‐A. baumannii‐complex. One to four plasmids in the size range 2.1 kb‐ > 100 kb were detected in 13 of the strains. Nine plasmid profiles were seen, indicating the usefulness of this typing method if the strains contain plasmids. The study also indicates that ribotyping is useful both for typing and for identification purposes, and that the genetic relationship in this area are more diverse than hitherto perceived. Taxonomic reconsiderations are warranted.

Numerical Analysis of Cell Envelope Protein Profiles of Acinetobacter Strains Classified by DNA-DNA Hybridization

Summary Cell emvelope protein profiles, obtained by sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE), of Acinetobacter strains were compared with DNA hybridization data of the strains. The collection of strains comprised 98 field strains mostly from clinical sources in Sweden and the Netherlands and 22 strains from culture collections. Thirteen DNA groups were represented. Protein profiles were densitometrically recorded and subjected to numerical analysis. When the complete profile was used for analysis, the heavily stained bands outweighed the less heavily stained bands and the clustering did not correlate well with DNA groups. When the analysis was restricted to the upper part of the profile, consisting of minor bands of relatively high molecular weight, most strains belonging to the same DNA group clustered together. Thus, this part of the profile may help to identify strains at the DNA group level, while the complete profile can be used to differentiate strains within DNA groups.

A quantitative bacterial dot method for DNA-DNA hybridization and its correlation to the hydroxyapatite method

A filter hybridization method employing bacterial samples and [125I]labeled chromosomal DNA as a probe was used for DNA-DNA hybridization. It was found that the hybrids had a thermal melting temperature very similar to that of duplexes formed by purified filterbound DNA. The difference in thermal denaturation midpoint between homologous and heterologous duplexes was determined for a number of strains ofAcinetobacter spp. andEnterobacter agglomerans. A comparison with the corresponding data obtained by the hydroxyapatite method showed good correlation between the two methods. The use of bacterial samples in filter hybridization omits the time-consuming DNA preparation procedure necessary for traditional DNA-DNA hybridization procedures. A simplified, two-step elution procedure is suggested for processing large numbers of strains.

First documented case of bacteremia with vibrio vulnificus in Sweden

A few days after a mild trauma to a toe, a 90-year-old woman presented with fever, malaise and cellulitis. On suspicion of erysipelas the patient was initially treated with benzylpenicillin and cefuroxime. Her general condition improved rapidly, but there was local progression with numerous necrotic areas with surrounding bullae. Vibrio vulnificus was isolated from the blood. After susceptibility testing, the patient was finally treated with ciprofloxacin and pivampicillin, and recovered slowly. To our knowledge, this is the first reported case of bacteremia with V. vulnificus in Sweden.

Characterization of Human Invasive Isolates of Listeria monocytogenes in Sweden 1986-2007

Since 1986, 68% of the Listeria monocytogenes isolates from human cases of invasive listeriosis in Sweden are available for retrospective studies. The aim of the present study was to characterize 601 human invasive isolates of L. monocytogenes in Sweden from 1986 to 2007 by using serotyping and pulsed-field gel electrophoresis. Since 1996, serovar 4b was permanently reduced to the second or third most common serovar in human cases in Sweden. During the latter period, 2000-2007, only 13% belonged to serovar 4b and 71% to 1/2a. The dendrogram, based on pulsovars, reveals two clusters with different serovars. Cluster 1 exhibits serovars 4b and 1/2b, whereas cluster 2 consists of serovar 1/2a. Serovar 1/2a seems to be more heterogeneous than serovar 4b.