RAPD-PCR typing of Acinetobacter isolates from activated sludge systems designed to remove phosphorus microbiologically

AIMS: This study investigated whether there were differences in RAPD fingerprints between already described genomic species of Acinetobacter and those from activated sludge systems. Whether plant-specific populations of acinetobacters exist was also examined. METHODS AND RESULTS: Fifty-two isolates of Acinetobacter from four biological phosphorus removal (EBPR) systems of different configurations, and the known genomic species, were characterized using RAPD-PCR, and fragments separated on agarose gels. Patterns were analysed using Gel Pro software and data analysed numerically. RAPD-PCR produced patterns suggesting that many environmental isolates differ from known genomic species. In two cases, strains from individual plants clustered closely enough together to imply that there may be plant-specific populations of acinetobacters. CONCLUSION: The data suggest that current understanding of the taxonomic status of Acinetobacter may need modifying to accommodate non-clinical isolates, as many of the clusters emerging after numerical analysis of RAPD-PCR fragments from activated sludge isolates were quite separate from the clusters containing the already described genomic species. Some evidence was also obtained from the clusters generated to support a view that particular populations of Acinetobacter may occur in individual activated sludge plants. SIGNIFICANCE AND IMPACT OF THE STUDY: These data suggest that the current understanding of the systematics of Acinetobacter, based as it is almost exclusively on clinical isolates, may need drastic revision to accommodate environmental strains. They also suggest that a re-examination of the importance and role of Acinetobacter in the activated sludge process may be appropriate.     

The rapid identification of Acinetobacter species using Fourier transform infrared spectroscopy

AIMS: Fourier transform infrared (FT-IR) was used to analyse a selection of Acinetobacter isolates in order to determine if this approach could discriminate readily between the known genomic species of this genus and environmental isolates from activated sludge. METHODS AND RESULTS: FT-IR spectroscopy is a rapid whole-organism fingerprinting method, typically taking only 10 s per sample, and generates 'holistic' biochemical profiles (or 'fingerprints') from biological materials. The cluster analysis produced by FT-IR was compared with previous polyphasic taxonomic studies on these isolates and with 16S-23S rDNA intergenic spacer region (ISR) fingerprinting presented in this paper. FT-IR and 16S-23S rDNA ISR analyses together indicate that some of the Acinetobacter genomic species are particularly heterogeneous and poorly defined, making characterization of the unknown environmental isolates with the genomic species difficult. CONCLUSIONS: Whilst the characterization of the isolates from activated sludge revealed by FT-IR and 16S-23S rDNA ISR were not directly comparable, the dendrogram produced from FT-IR data did correlate well with the outcomes of the other polyphasic taxonomic work. SIGNIFICANCE AND IMPACT OF THE STUDY: We believe it would be advantageous to pursue this approach further and establish a comprehensive database of taxonomically well-defined Acinetobacter species to aid the identification of unknown strains. In this instance, FT-IR may provide the rapid identification method eagerly sought for the routine identification of Acinetobacter isolates from a wide range of environmental sources.     

Diversity of isolates of Acinetobacter from activated sludge systems based on their whole cell protein patterns

Whole cell protein extracts from strains of the currently recognized genomic species of Acinetobacter, together with those from a range of isolates of several genomic species identified using the Biolog system and obtained from a biological nutrient-removal activated sludge plant were analysed by SDS-PAGE. The dendrograms obtained after numerical analysis for the known genomic species generally supported the taxonomic relationships suggested from earlier DNA–DNA hybridisation data. In some cases the activated sludge isolates identified to genomic species level clustered closely with the corresponding genomic species reference strains, although isolates 5 and 8/9 were scattered throughout the dendrogram. Considerable variations were seen in the protein patterns of the 27 different environmental isolates of genomic species 7 that were analysed. Three unidentified Acinetobacter isolates examined formed their own subcluster. Received 06 September 1996/ Accepted in revised form 10 December 1996     

Polyphosphate accumulation among denitrifying bacteria in activated sludge

Bacterial polyphosphate accumulation and denitrification are important processes in biological removal of nutrients from wastewater. It has been suggested that phosphorus accumulators are able to denitrify. However, the bacteria known as the most important phosphorus accumulators, belonging to the genus Acinetobacter are generally not known to denitrify. To clarify how commonly both physiological traits are present in the same organism, we screened 165 isolates from activated sludge and wastewater for their ability to denitrify, and the ability of the denitrifying isolates to accumulate polyphosphate. Of the 165 isolates, 149 were from acetate mineral medium (87 of these identified as Acinetobacter by the API 20 NE identification system) and 16 were from nutrient broth and nitrate medium. Only 15 of 165 isolates tested showed true respiratory denitrification activity. In the presence of acetylene they converted more than 80% of 5mM NO3- to N2O in 6 days. None of the Acinetobacter isolates were among the 15 respiratory denitrifiers. The denitrifying isolates were identified as species of Pseudomonas, Agrobacterium, Pasteurella, Sphingomonas or could not be identified by the API 20 NE identification system. According to the BIOLOG identification system the denitrifiers were species of Pseudomonas, Hydrogenophaga, Citrobacter, Xanthomonas or they could not be identified. The ability of confirmed denitrifiers to accumulate phosphate was measured in experiments where cells pregrown under phosphorus limitation were exposed to phosphate (8 mg P/L) under aerobic conditions. The rates of excess phosphate uptake varied from 0.3 to more than 23 mg P/g dry matter/h. Rates for four isolates were higher than those reported for Acinetobacter strains. These results show that polyphosphate accumulation and denitrification in activated sludge can be carried out by the same organisms.     

Tsukamurella spumae sp. nov., a novel actinomycete associated with foaming in activated sludge plants

A polyphasic taxonomic study was undertaken to establish the taxonomic position of six representative strains isolated from activated sewage sludge foam. The organisms were found to have chemical and morphological properties consistent with their assignment to the genus Tsukamurella. DNA:DNA relatedness studies showed that five out of the six isolates formed a distinct genomic species, the remaining strain was most closely associated with this taxon. The five isolates had a unique phenotypic profile that served to distinguish them from representatives of the validly described species of Tsukamurella. The combination of the genotypic and phenotypic data indicated that the five strains should be classified as a new species in the genus Tsukamurella. The name proposed for this taxon is Tsukamurella spumae, the type strain is N1171T (= DSM 44.113T = NCIMB 13947T). It was also shown that some of the reference strains were misclassified as Tsukamurella paurometabola.     

The filamentous morphotype Eikelboom Type 1863 is not a single genetic entity

Five isolates of a filamentous bacterial morphotype with the distinctive diagnostic microscopic features of Eikelboom Type 1863 were obtained from activated sludge sewage treatment plants in Victoria, Australia. On the basis of phenotypic evidence and 16S rDNA sequence data, these isolates proved to be polyphyletic. Two (Ben 06 and Ben 06C) are from the Chryseobacterium subgroup which is in the Cytophaga group, subdivision I of the Flexibacter – Cytophaga – Bacteroides phylum. Two (Ben 56 and Ben 59) belong to the genus Acinetobacter , and one (Ben 58) is a Moraxella sp., closest to Mor. osloensis . The significance of these findings to the reliance on microscopic features for identification of these filamentous bacteria in activated sludge is discussed.     

Analysis of the polyphosphate-accumulating microflora in phosphorus-eliminating, anaerobic-aerobic activated sludge systems by using diaminopropane as a biomarker for rapid estimation of Acinetobacter spp.

Polyphosphate-accumulating gram-negative bacteria were isolated from different anaerobic-aerobic activated sludge systems with diverse processes for enhanced biological phosphorus (P) elimination. Of 22 isolates, 10 were allocated to the genus Acinetobacter by using multiple-test systems and soluble protein and polyamine patterns. As diaminopropane (DAP) appears to be the characteristic main polyamine compound produced by Acinetobacter spp., it was used as a biomarker for the genus. The high DAP contents of representative samples from municipal wastes with enhanced biological P elimination indicated that Acinetobacter spp. can be dominant organisms in sewage treatment plants with low organic loading and nitrification and denitrification steps. Contrary to accepted opinion, sludge from treatment plants with efficient P removal and high organic loading had a low DAP content, indicating that bacteria other than Acinetobacter spp. are responsible for enhanced biological P elimination in these plants.     

Analysis of the polyphosphate-accumulating microflora in phosphorus-eliminating, anaerobic-aerobic activated sludge systems by using diaminopropane as a biomarker for rapid estimation of Acinetobacter spp.

Polyphosphate-accumulating gram-negative bacteria were isolated from different anaerobic-aerobic activated sludge systems with diverse processes for enhanced biological phosphorus (P) elimination. Of 22 isolates, 10 were allocated to the genus Acinetobacter by using multiple-test systems and soluble protein and polyamine patterns. As diaminopropane (DAP) appears to be the characteristic main polyamine compound produced by Acinetobacter spp., it was used as a biomarker for the genus. The high DAP contents of representative samples from municipal wastes with enhanced biological P elimination indicated that Acinetobacter spp. can be dominant organisms in sewage treatment plants with low organic loading and nitrification and denitrification steps. Contrary to accepted opinion, sludge from treatment plants with efficient P removal and high organic loading had a low DAP content, indicating that bacteria other than Acinetobacter spp. are responsible for enhanced biological P elimination in these plants.     

Phenotypic characterization and antibiotic resistance of Acinetobacter spp. isolated from aquatic sources

A total of 99 Acinetobacter isolates from sewage, freshwater aquaculture habitats, trout intestinal contents and frozen shrimps was characterized phenotypically and antibiotic susceptibility patterns determined. One group of genomic species, including Ac. johnsonii, Ac. lwoffi and spp. 15TU, was detected in all sample types and represented the majority of the isolates (n = 54). Isolates belonging to the Acb complex (Ac. calcoaceticus, Ac. baumannii and genomic species 3) were detected in sewage (n = 6) and frozen shrimps (n = 1), Ac. haemolyticus in frozen shrimps (n = 6) and trout intestinal contents (n = 2) and genomic species 11 in freshwater aquaculture habitats (n = 6) and trout intestinal contents (n = 1). Acinetobacter junii (n = 5), genomic species 10 (n = 2), 14BJ (n = 8) and 16BJ (n = 4) were only isolated from sewage. Acinetobacter isolates from sewage were generally more biochemically reactive and resistant to antimicrobial agents compared with isolates from other sample types. Different strains, often belonging to different genomic species, were isolated from sites situated upstream and downstream of the discharge point of a pharmaceutical plant. This finding supported the hypothesis that the waste effluent from the pharmaceutical plant was likely to cause a change in the distribution of Acinetobacter spp. by selecting and/or introducing antibiotic-resistant strains into the recipient sewers.     

Coaggregation among nonflocculating bacteria isolated from activated sludge

Thirty-two strains of nonflocculating bacteria isolated from sewage-activated sludge were tested by a spectrophotometric assay for their ability to coaggregate with one other in two-membered systems. Among these strains, eight showed significant (74 to 99%) coaggregation with Acinetobacter johnsonii S35 while only four strains coaggregated, to a lesser extent (43 to 65%), with Acinetobacter junii S33. The extent and pattern of coaggregation as well as the aggregate size showed good correlation with cellular characteristics of the coaggregating partners. These strains were identified by sequencing of full-length 16S rRNA genes. A. johnsonii S35 could coaggregate with strains of several genera, such as Oligotropha carboxidovorans, Microbacterium esteraromaticum, and Xanthomonas spp. The role of Acinetobacter isolates as bridging organisms in multigeneric coaggregates is indicated. This investigation revealed the role of much-neglected nonflocculating bacteria in floc formation in activated sludge.     

Numerical taxonomy of Skermania piniformis and related isolates from activated sludge

The microscopic morphology of nocardioforms causing foaming problems in activated sludge usually consists of filaments with branches at either right angles ( Nocardia amarae -Like Organisms, NALO) or acute angles (Pine Tree-Like Organisms, PTLO). Fifty-nine nocardioforms, mainly with PTLO morphology, isolated from mixed liquor and foam samples from Australian activated sludge plants, and 39 reference strains of nocardioforms, including type strains, were characterized using 109 morphological and physiological characters. Cluster Analysis and Principal Component Analysis showed that the activated sludge isolates clustered in six groups. All isolates that had typical PTLO morphology clustered unambiguously with the Skermania piniformis type strain (formerly called Nocardia pinensis ) showing that, unlike NALO, reliable unequivocal identification of S. piniformis , based on microscopic morphology in activated sludge, was possible. Other foam isolates whose morphology consisted of branches with both acute angles and right angles clustered as two separate groups, probably representing new species. These could not be confused microscopically with S. piniformis , despite some branches showing acute angles. The remaining three groups had typical NALO morphology. One of these groups did not cluster with any reference cultures and may be a new species or genus.     

Estimation of ribosomal RNA operon (rrn) copy number in Acinetobacter isolates and potential of patterns of rrn operon-containing fragments for typing strains of members of this genus

The copy number of the rrn operon in 70 strains of Acinetobacter including the type strains of almost all the genomic species with validated names was estimated after digestion of their genomic DNA by the restriction enzymes BglII and PstI, and Southern blotting. Copy number estimates varied between and among species, with between 3 and 7 rrn operon copies detected. Copy number estimates obtained from the same strain with the two enzymes sometimes varied. BglII generated RFLP patterns of the rrn containing fragments obtained from Southern blots after agarose gel electrophoresis were examined for their value in identifying Acinetobacter isolates. This method was very reproducible with the same fragment pattern always generated from the same isolate on repeated analysis. Often multiple strains of the same genomic species gave identical or very similar patterns (e.g. Acinetobacter baylyi), clustering closest together on the dendrogram generated after numerical analysis of these patterns. However, with some, like BG5 and BG8, the patterns derived from the different strains, some of which had been placed in this genomic species from DNA:DNA hybridization data, varied considerably to each other and to the type strain. Little similarity was seen when relationships between these strains based on these patterns were compared to those using DNA:DNA hybridization data. Often these patterns could be used to question earlier identification of strains using phenotypic characters. Thus, strain AB82 thought to belong to genomic species 5 gave an identical pattern to A. bouvetii(T) (DSM 14964). In some cases this pattern analysis suggested that novel species of Acinetobacter might exist among the strains examined.     

Molecular characterization of Macrophomina phaseolina and Fusarium species by a single primer RAPD technique

Charcoal root rot and wilt, are two economically important diseases of many crop plants in North and South America, Asia and Africa and some parts of Europe. Genetic variation in 43 isolates of Macrophomina phaseolina and 22 isolates of Fusarium species, collected from geographically distinct regions over a range of hosts, was studied using random amplified polymorphic DNA (RAPD) markers. Initially, 210 arbitrary nucleotide (10-mer) primers were tested for amplification of genomic DNA of one M. phaseolina isolate, 70 primers amplified the genomic DNA of M. phaseolina. One primer OPA-13 (5'-CAGCACCCAC-3') produced fingerprint profiles, which clearly distinguished between the different isolates of M. phaseolina. UPGMA analysis classified these isolates into five major groups. By primer OPA-13, 22 isolates of pathogenic and non-pathogenic Fusarium species of different formae-speciales and races, were also distinguished from M. phaseolina. This marker is useful for distinguishing between these two important plant pathogens irrespective of hosts, virulence spectrum and races. This is the first report of reliable diagnosis of two soilborne pathogens (root/collar rot and wilt causing pathogens) at the level of isolates, formae-speciales and races by a single primer RAPD procedure with uniform PCR conditions.     

<Emphasis Type="Italic">Acinetobacter</Emphasis> sp. strain Ths,a novel psychrotolerant and alkalitolerant bacterium that utilizes hydrocarbon

A novel psychrotolerant, alkalitolerant bacterium, strain Ths, was isolated from a soil sample immersed in hot spring water containing hydrocarbons and grown on a chemically defined medium containing n-tetradecane as the sole carbon source. The isolate grew at 0 degrees C but not at temperatures higher than 45 degrees C; its optimum growth temperature was 27 degrees C. It grew in the pH range of 7-9. The strain utilized C(13)-C(30) n-alkane and fluorene at pH 9 and 4 degrees C. To our knowledge, this is the first report on the bacterium that utilizes a wide range of hydrocarbons at a high pH and a low temperature. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain Ths is closely related to genomic species 6 ATCC 17979 (99.1% similarity), genomic species BJ13/TU14 ATCC 17905 (97.8% similarity), genomic species 9 ATCC 9957 (97.6% similarity) belonging to the genus Acinetobacter and to Acinetobacter calcoaceticus JCM 6842(T) (97.5% similarity). DNA-DNA hybridization revealed that the isolate has 62, 25, 18 and 19% relatedness, respectively, to genomic species 6 ATCC 17979, genomic species BJ13/TU14 ATCC 17905, genomic species 9 ATCC 9957 and A. calcoaceticus, respectively.     

Characterization of Nocardia asteroides isolates from different ecological habitats on the basis of repetitive extragenic palindromic-PCR fingerprinting

Thirteen isolates of Nocardia asteroides from both soils and aquatic samples (lake and moat sediments, as well as scum from activated sludge), together with a type strain and two known clinical isolates of this species, were characterized by repetitive extragenic palindromic-PCR fingerprinting with the BOX-A1R primer. The resulting DNA fingerprint patterns proved to be strain specific, and cluster analysis distinguished the soil isolates, the aquatic isolates, and the known strains as being in separate groups.     

Characterization of Nocardia asteroides Isolates from Different Ecological Habitats on the Basis of Repetitive Extragenic Palindromic-PCR Fingerprinting

Thirteen isolates of Nocardia asteroides from both soils and aquatic samples (lake and moat sediments, as well as scum from activated sludge), together with a type strain and two known clinical isolates of this species, were characterized by repetitive extragenic palindromic-PCR fingerprinting with the BOX-A1R primer. The resulting DNA fingerprint patterns proved to be strain specific, and cluster analysis distinguished the soil isolates, the aquatic isolates, and the known strains as being in separate groups.     

Coaggregation among Nonflocculating Bacteria Isolated from Activated Sludge

Thirty-two strains of nonflocculating bacteria isolated from sewage-activated sludge were tested by a spectrophotometric assay for their ability to coaggregate with one other in two-membered systems. Among these strains, eight showed significant (74 to 99%) coaggregation with Acinetobacter johnsonii S35 while only four strains coaggregated, to a lesser extent (43 to 65%), with Acinetobacter junii S33. The extent and pattern of coaggregation as well as the aggregate size showed good correlation with cellular characteristics of the coaggregating partners. These strains were identified by sequencing of full-length 16S rRNA genes. A. johnsonii S35 could coaggregate with strains of several genera, such as Oligotropha carboxidovorans, Microbacterium esteraromaticum, and Xanthomonas spp. The role of Acinetobacter isolates as bridging organisms in multigeneric coaggregates is indicated. This investigation revealed the role of much-neglected nonflocculating bacteria in floc formation in activated sludge.     

Characterization of the bacterial population structure in an anaerobic-aerobic activated sludge system on the basis of respiratory quinone profiles

Bacterial respiratory quinones were used as biomarkers for studying the bacterial population structure, especially the content of Acinetobacter species, in a laboratory-scale anaerobic-aerobic activated sludge system and in the standard aerobic system. All tested sludges contained both ubiquinone and menaquinone, with a molar ratio of about 1:0.5. High-performance liquid chromatography showed that ubiquinone with eight isoprene units (Q-8) was present as the predominant ubiquinone, Q-10 was the second most common type, and Q-9 and other homologs were minor components in the anaerobic-aerobic sludge and the standard aerobic sludge. Bacteriological examination indicated that, in both sludge systems, Q-8-containing bacteria constituted a large proportion of the aerobic heterotrophic bacterial flora, but only a few strains with Q-9 were found. These findings demonstrate that the population of Acinetobacter species, which contain Q-9 as the major quinone, is negligible in those environments. The present results suggest that the introduction of anaerobic conditions into the aerobic batch process has little influence on the bacterial community structure.     

Characterization of the bacterial population structure in an anaerobic-aerobic activated sludge system on the basis of respiratory quinone profiles.

Bacterial respiratory quinones were used as biomarkers for studying the bacterial population structure, especially the content of Acinetobacter species, in a laboratory-scale anaerobic-aerobic activated sludge system and in the standard aerobic system. All tested sludges contained both ubiquinone and menaquinone, with a molar ratio of about 1:0.5. High-performance liquid chromatography showed that ubiquinone with eight isoprene units (Q-8) was present as the predominant ubiquinone, Q-10 was the second most common type, and Q-9 and other homologs were minor components in the anaerobic-aerobic sludge and the standard aerobic sludge. Bacteriological examination indicated that, in both sludge systems, Q-8-containing bacteria constituted a large proportion of the aerobic heterotrophic bacterial flora, but only a few strains with Q-9 were found. These findings demonstrate that the population of Acinetobacter species, which contain Q-9 as the major quinone, is negligible in those environments. The present results suggest that the introduction of anaerobic conditions into the aerobic batch process has little influence on the bacterial community structure.     

Microbial community analysis and performance of a phosphate-removing activated sludge

The microbial community of a phosphate-removing activated sludge was analyzed according to the extracted 16S rDNA sequences. The sludge, which accumulated 5.6% P by weight, was obtained from a sequencing batch reactor treating a fatty-acid rich wastewater containing 108 mg l(-1) total organic carbon (TOC), 14.0 mg l(-1) N and 16.2 mg l(-1) P. The reactor at 25 degrees C and pH 7.6 removed over 96% TOC and 99.9% P from the wastewater. According to the 16S rDNA analysis of the 114 clones developed, the sludge had a diverse population, mainly comprising Proteobacteria (71.0%) and the Cytophaga Flavobacterium Bacteroides group (23.7%), plus a few species of Planctomycetales (2.6%), Verrucomicrobiales (1.8%) and Firmicutes (0.9%). Of the 114 clones, 36 (31.6%) were closely affiliated with Acinetobacter. However, Acinetobacter did not accumulate phosphate judging from the images of sludge samples hybridized with an Acinetobacter-specific probe and stained with a phosphate-specific dye. The identities of the phosphate-removing bacteria remain unclear.