Mobile robotic platforms for the acoustic tracking of deep-sea demersal fishery resources.

Knowing the displacement capacity and mobility patterns of industrially exploited (i.e., fished) marine resources is key to establishing effective conservation management strategies in human-impacted marine ecosystems. Acquiring accurate behavioral information of deep-sea fished ecosystems is necessary to establish the sizes of marine protected areas within the framework of large international societal programs (e.g., European Community H2020, as part of the Blue Growth economic strategy). However, such information is currently scarce, and high-frequency and prolonged data collection is rarely available. Here, we report the implementation of autonomous underwater vehicles and remotely operated vehicles as an aid for acoustic long-baseline localization systems for autonomous tracking of Norway lobster (Nephrops norvegicus), one of the key living resources exploited in European waters. In combination with seafloor moored acoustic receivers, we detected and tracked the movements of 33 tagged lobsters at 400-m depth for more than 3 months. We also identified the best procedures to localize both the acoustic receivers and the tagged lobsters, based on algorithms designed for off-the-shelf acoustic tags identification. Autonomous mobile platforms that deliver data on animal behavior beyond traditional fixed platform capabilities represent an advance for prolonged, in situ monitoring of deep-sea benthic animal behavior at meter spatial scales.

Phylogenetic structures of the genus Acinetobacter based on gyrB sequences: comparison with the grouping by DNA-DNA hybridization.

The phylogenetic relationships of 49 Acinetobacter strains, 46 of which have previously been classified into 18 genomic species by DNA-DNA hybridization studies, were investigated using the nucleotide sequence of gyrB, the structural gene for the DNA gyrase B subunit. The phylogenetic tree showed linkages between genomic species 1 (Acinetobacter calcoaceticus), 2 (Acinetobacter baumannii), 3 and TU13; genomic species 6, BJ15, BJ16 and BJ17; genomic species 5, BJ13 (synonym of TU14) and BJ14; genomic species 7 (Acinetobacter johnsonii), 10 and 11; and genomic species 8 and 9. The phylogenetic grouping of Acinetobacter strains based on gyrB genes was almost congruent with that based on DNA-DNA hybridization studies. Consequently, gyrB sequence comparison can be used to resolve the taxonomic positions of bacterial strains at the level of genomic species. However, minor discrepancies existed in the grouping of strains of genomic species 8, 9 and BJ17. The phylogenetic tree for these strains was reconstructed from the sequence of rpoD, the structural gene for the RNA polymerase sigma 70 factor. The latter tree was 100% congruent with the grouping based on DNA-DNA hybridization. The reliability of DNA-DNA hybridization may be superior to that of sequence comparison of a single protein-encoding gene in resolving closely related organisms since the former method measures the homologies between the nucleotide sequences of total genomic DNAs. Three strains that have not been characterized previously by DNA-DNA hybridization seem to belong to two new genomic species, one including strain ATCC 33308 and the other including strains ATCC 31012 and MBIC 1332.

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.

Epidemiological typing of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae isolates responsible for five outbreaks in a university hospital.

Thirty-seven isolates of extended-spectrum beta-lactamase-producing (ESBL) Klebsiella pneumoniae implicated in five nosocomial outbreaks (I-V) on three distinct wards of our hospital were compared using capsular typing, biotyping, antibiotyping, enzyme electrophoresis typing and DNA macrorestriction analysis with Xba I resolved by pulsed-field gel electrophoresis. The isolates from each outbreak had common phenotypic and genotypic characteristics indicating that they were related epidemiologically. Isolates from outbreaks I (four patients) and V (13 patients), although they occurred in two different wards (neurology and surgery) and three years apart, produced the same ESBL with a pI of 7.8 (SHV-4) and were of serotype K25. The Xba I patterns were closely related. The isolates of outbreaks II (seven patients), III (four patients) and IV (seven patients), which occurred in a single surgical intensive care unit, produced an ESBL with a pI of 6.3 (TEM-3). Isolates from outbreaks III and IV, which occurred six months apart, were of serotype K68 and had similar Xba I patterns suggesting that the two outbreaks were due to a single strain which persisted endemically in the ward. The isolates from outbreak II were of serotype K62, and had distinct characteristics from the two later outbreaks. The Xba I patterns of the isolates from outbreaks "I and V', II and "III and IV' had Dice similarity coefficients under 40% showing that the three groups were genetically distant. DNA macrorestriction analysis was a useful complement to phenotypic methods for identifying K. pneumoniae strains responsible for outbreaks harbouring a common ESBL.

Acinetobacter species identification by using tRNA spacer fingerprinting.

Identification of Acinetobacter spp. to the DNA group level by phenotypic techniques is problematic, and there is a need for an alternative identification method for routine use. The present study validated the suitability of a rapid identification technique based on tRNA spacer (tDNA) fingerprinting in comparison with that of a commercially available assay involving carbon source utilization tests (Biolog MicroStation System) for identifying the 21 DNA-DNA hybridization groups belonging to the genus. For this purpose, 128 strains identified previously by DNA-DNA hybridization were analyzed by both techniques. tDNA fingerprinting was highly reproducible and classified all strains into 17 groups. Six DNA groups belonging to the A. calcoaceticus-A. baumannii complex were grouped into two distinct clusters, indicating the high degree of genetic similarity within this complex. Strains of the more recently described DNA groups BJ13 to BJ16 were ambiguously grouped and displayed three pattern types. The software used with the commercial carbon source utilization method grouped the 128 strains into 12 clusters, explaining the less discriminatory power of this system. We conclude that tDNA fingerprinting offers a quick and reliable method for the routine differentiation of most Acinetobacter spp. at the subgenus level.

Differentiation of Acinetobacter calcoaceticus sensu stricto from related Acinetobacter species by electrophoretic polymorphism of malate dehydrogenase, glutamate dehydrogenase and catalase.

Acinetobacter baumannii, unnamed Acinetobacter species 3 (studied by P.J.M. Bouvet and P.A.D. Grimont) and unnamed DNA group 13 (studied by I. Tjernberg and J. Ursing) are the most prevalent Acinetobacter species in hospitals. Using the identification scheme of Bouvet and Grimont, it is sometimes difficult to differentiate these species from A. calcoaceticus sensu stricto, a species of the natural environment that has seldom been found associated with human infection. Genetically identified Acinetobacter isolates belonging to A. calcoaceticus sensu stricto (n = 12), A. baumannii (n = 22), Acinetobacter species 3 (n = 15) and DNA group 13 of Tjernberg and Ursing (n = 26), Acinetobacter species 10 (n = 2), Acinetobacter species 11 (n = 2) and 3 strains ungrouped by DNA-DNA hybridization were investigated for electrophoretic separations of L-malate dehydrogenase (MDH), glutamate dehydrogenase (GDH) and catalase (CAT). All A. calcoaceticus sensu stricto isolates were easily differentiated from those of other species investigated by their high MDH values (relative mobility (Rf) = 78), their low GDH values (Rf range: 24-28) and CAT values (Rf range: 34-42). Acinetobacter species 3 was differentiated from A. baumannii and DNA group 13 of Tjernberg and Ursing by high CAT values. A. baumannii could not be differentiated from Tjernberg and Ursing DNA group 13. Acinetobacter species 10 was clearly differentiated from Acinetobacter species 11. Once an Acinetobacter is phenotypically identified with the four closely related species investigated here, electrophoretic analysis of MDH, GDH and CAT might be a useful complement to the identification scheme of Bouvet and Grimont for accurately identifying A. calcoaceticus sensu stricto.

Molecular epidemiology of Klebsiella pneumoniae strains that produce SHV-4 beta-lactamase and which were isolated in 14 French hospitals.

Preliminary results suggested that the diffusion in France of the SHV-4 extended-spectrum beta-lactamase was probably due to the spread of one single epidemic strain of Klebsiella pneumoniae. In this study, we tested various phenotypic and genotypic markers to compare K. pneumoniae strains producing this enzyme isolated in 14 French hospitals between 1987 and 1989. All of the strains were of the same capsule serotype, K25. Twelve of them were of the same biotype: weak urease activity and no sucrose fermentation. Among the six plasmid profiles observed, one accounted for eight strains. Large plasmids of 170 kb encoding SHV-4 beta-lactamase were present in all strains of K. pneumoniae and could be transferred by conjugation with high frequency to Escherichia coli J53-2 or HB101 from all except one strain. Plasmid EcoRI restriction patterns suggested that these plasmids were closely related and similar to pUD18 encoding SHV-3 beta-lactamase, originally described in France and differing from SHV-4 by one amino acid substitution. Ribotyping with EcoRI and HindIII and genomic fingerprinting with XbaI by pulsed-field gel electrophoresis were concordant and suggested that 12 of the isolates recovered from the 14 hospitals were probably the same strain. Dissemination in France of the SHV-4 extended-spectrum beta-lactamase was thus essentially due to the diffusion of a single K. pneumoniae clone.

Evaluation of two colored latex kits, the Wellcolex Colour Salmonella Test and the Wellcolex Colour Shigella Test, for serological grouping of Salmonella and Shigella species.

Two colored latex kits (the Wellcolex Colour Salmonella Test [WCT-Salmonella] and the Wellcolex Colour Shigella Test [WCT-Shigella]; Division Diagnostics, Laboratories Wellcome S.A., Paris, France), which allow identification of the most frequently encountered Salmonella serogroups and Shigella species, respectively, were evaluated. WCT-Salmonella and WCT-Shigella yielded sensitivities of 98.4 and 98%, respectively, and a specificity of 100% when they were tested on pure cultures received at a reference laboratory.

Use of pulsed-field gel electrophoresis for investigation of hospital outbreaks of Acinetobacter baumannii.

Genomic DNAs from taxonomically and epidemiologically well-defined strains of Acinetobacter baumannii were digested with restriction endonucleases that cleave with low frequency, and the fragments were separated by pulse-field gel electrophoresis. Restriction fragment length polymorphisms were observed. Restriction fragment length polymorphism analysis can be used as an epidemiological tool to delineate outbreaks of nosocomial infections caused by A. baumannii.

Cell surface hydrophobicity of 88 clinical strains of Acinetobacter baumannii.

We studied the surface hydrophobicity of 88 Acinetobacter baumannii strains of clinical origin, using both salt aggregation and adherence to paraxylene tests. Strains were divided into 2 groups: the first included 65 strains isolated from various clinical samples (infected catheters, tracheal and bladder devices); the second included 23 strains isolated from skin obtained from healthy controls. High surface hydrophobicity was observed in 92% of the first group of strains and in only 5% of the second.

Species, biotype, and bacteriophage type determinations compared with cell envelope protein profiles for typing Acinetobacter strains.

Species, biotypes, and phage types were determined for 120 Acinetobacter strains from clinical or environmental sources or from culture collections. These characteristics were compared with cell envelope protein profiles obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in previous studies. A considerable heterogeneity of species and types was observed by use of the various methods, in particular among strains from different sources. Acinetobacter baumannii was the most commonly found species in isolates from clinical sources, followed by Acinetobacter species 3. Nine biotypes were observed among A. baumannii strains. Further differentiation within most species and biotypes was achieved by protein profile typing and, to some extent, phage typing. Of 120 strains, 49 (41%) were not typeable by phages. Consistent results for the various classification methods were obtained for strains from common sources. Biotyping seemed an appropriate method for the screening of strains, whereas protein profile and phage typing could serve as additional methods to establish the identity or nonidentity of strains. Results of this study suggest that the combination of the typing methods is useful in epidemiological studies.

A comparative assay of epidemiological markers for Acinetobacter strains isolated in a hospital.

A comparative assay for epidemiological evaluation of three different Acinetobacter typing procedures, i.e. biotyping, phage-typing, and the analysis of the bacterial envelope protein profiles, was carried out using sixty-four multiresistant Acinetobacter strains isolated from clinical specimens. The antibiotic susceptibility of the strains was also considered. After geno-species identification, biotyping allowed the recognition of a relatively large and long-lasting presence, at an Intensive Therapy Unit, of two A. baumannii biotypes. Phage-typing and the analysis of the susceptibility to antibiotics allowed for the differentiation of strains belonging to different geno-species and biotypes, and in some cases also to the same biotypes. On the contrary, the analysis by polyacrylamide gel electrophoresis of the cell-envelope proteins failed to show any diversity not only within, but also between some of the biotypes of A. baumannii, the most prevalent species of the genus in the hospital environment.

Glucose dehydrogenase activity in Acinetobacter species.

A study of D-glucose oxidation by Acinetobacter species was carried out. Glucose-oxidizing strains were found distributed among almost all Acinetobacter species. 14C-glucose oxidation kinetics by non-proliferating cells with separation of oxidation products (14C-gluconate) by DEAE-cellulose paper chromatography was studied. Inhibition of glucose dehydrogenase (GDH) activity by 11 carbohydrates (mono- and disaccharides) and determination of the kinetic parameters showed that glucose oxidation was due to the action of membrane-bound GDH (inactive in vivo on disaccharides). On the basis of GDH inhibition patterns obtained, two groups were individualized. The first group of strains (identified as A. calcoaceticus, A. baumannii, A. lwoffii, A. johnsonii and Acinetobacter species 3, 9, 10 and 11) showed a greater affinity for glucose than the second group (A. haemolyticus, A. junii and Acinetobacter species 6 and 12). Restoration of GDH activity after addition of pyrroloquinoline quinone (PQQ) was studied in 187 strains previously found unable to oxidize glucose. GDH activity of 150 out of 166 strains identified as A. baumannii, A. johnsonii, A. lwoffii and Acinetobacter species 11 and 12 was restored. Eighteen of 21 strains identified as A. haemolyticus and Acinetobacter species 6 were unable to produce acid from glucose after addition of PQQ. Our results confirm that the former taxonomic scheme for the genus Acinetobacter (2 species differing only by glucose oxidation) is untenable and that, accordingly, identification of Acinetobacter strains at the species level must be performed using more modern methods, i.e. carbon source utilization tests.

Characterization of bacterial genospecies by computer-assisted statistical analysis of enzyme electrophoretic data.

A computer-assisted statistical treatment of the electrophoretic data obtained from the analysis of two dehydrogenases and 27 kinds of esterases produced by strains belonging to the taxonomically complex genus Acinetobacter is described. The 12 genospecies were clearly separated from each other by correspondence analysis. For each genospecies the distances of the strains from their barycenter were computed and typical isolates suitable for use as reference strains were determined. This approach is suitable for the systematic study of other procaryotic or eucaryotic organisms.

Delineation of new proteolytic genomic species in the genus Acinetobacter.

Twenty-seven proteolytic Acinetobacter strains differing phenotypically from the 12 previously described Acinetobacter species were studied by DNA/DNA hybridization using the S1 nuclease method to assess their relatedness. Five DNA groups (genomic species 13 to 17) containing 20 strains were delineated. Seven strains remained ungrouped. Within species, the level of DNA relatedness to the reference strains ranged from 64 to 99%, with delta Tm values below 3.5 degrees C. DNA group 13 was 31 to 42% related to group 14. DNA group 15 was 59 to 69% related to group 16, with delta Tm values between 4.5 and 6 degrees C. DNA group 17 was 51 to 61% related to DNA groups 15 and 16 with delta Tm values between 5.5 and 7.5 degrees C. The seven ungrouped strains were 28 to 60% related to the five newly delineated genomic species with delta Tm between 6.5 and 13.5 degrees C. Reference strains of the five genomic species were 5 to 22% related to the type or reference strains of the 12 Acinetobacter genomic species previously described. Biochemically, DNA groups 13 to 17 and ungrouped strains could not be separated unambiguously and therefore are not named.

[Identification and determination of sensitivity to antibiotics of 31 clinical strains of Acinetobacter other than A. baumannii]. / Identification et détermination de la sensibilité aux antibiotiques de 31 souches cliniques d'Acinetobacter autres que A. baumannii.

Precise identification and determination of MICs of clinical isolates of Acinetobacter identified to other species than the hospital species A. baumannii were carried out. On 260 Acinetobacter strains isolated in an hospital over a 6 months period, 31 strains (12 p. cent) were identified to species other than A. baumannii. Among these 31 strains, A. Iwoffii sensu stricto (16 strains) and A. haemolyticus (6 strains) were mostly recovered. Eight glucose oxidizing strains were identified to A. haemolyticus (6 strains), Acinetobacter genospecies 3 (2 strains), and A. Iwoffii sensu stricto (1 strain). Antibiotic susceptibilities of these strains were greater than those commonly observed with A. baumannii strains.

Identification and biotyping of clinical isolates of Acinetobacter.

A total of 343 Acinetobacter strains, most isolated from hospital patients, were identified using a 16-test system (acid production from glucose, gelatin hydrolysis and utilization of 14 carbon sources) associated with tests for growth at 37, 41 and 44 degrees C. Of 299 nosocomial isolates, 253 were identified as A. baumannii, 20 as Acinetobacter genospecies 3, 8 as A. haemolyticus, 8 as A. lwoffii, 4 as A. johnsonii and 6 as other (presently) unnamed species. A biotyping system based on the utilization of levulinate, citraconate, L-phenylalanine, phenylacetate, 4-hydroxybenzoate and L-tartrate allowed recognition of 17 biotypes among 247 A. baumannii isolates. This biotyping system should be useful in epidemiological studies of Acinetobacter strains.