Emma L. Carr

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.

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.

Effects of Culture Conditions on the Mycolic Acid Composition of Isolates of Rhodococcus spp. from Activated SludgeFoams

The influence of two different carbon sources and three incubation temperatures on the mycolic acid compositions of three Rhodococcus isolates from activated sludge was examined using Selective Ion Monitoring (SIM) gas chromatography-mass spectrometry (GC-MS). Considerable qualitative and quantitative differences were detected in the mycolic acid compositions of the three very closely related isolates grown under the same conditions. Culture age also affected both the chain lengths and proportions of saturated mycolic acids detected in cell extracts, but not in the same manner for each isolate. Mycolic acids generally were of shorter chain lengths in cells grown with Tween 80 compared to glucose grown cells in strain 11R but the opposite situation occurred with strains A7 and D5. In all three, the proportion of unsaturated mycolic acids decreased with increasing growth temperatures. The taxonomic relevance of these observations and possible explanations for the observed changes in mycolic acid composition under various culture conditions are discussed.

Variation of 16S–23S rRNA Intergenic Spacer Regions (ISRs) in Acinetobacter baylyi (Strain B2) Isolated from Activated Sludge

To determine the variability of the 16S-23S rRNA intergenic spacer region (ISR) of the newly described Acinetobacter baylyi, 88 clones containing ISR amplicons were screened and 14 chosen for further analysis. Two different sized 16S-23S rRNA ISRs were distinguished comprising five variable and four conserved nucleotide blocks. The major regions of heterogeneity between the different sized ISRs were due to blocks of substitutions with unique secondary structures interspersed with nucleotide substitutions, rather than differences caused by presence or absence of tRNA genes, which is often the case. Recombination events causing shuffling of nucleotide blocks are considered the most likely explanation for the mosaic structure observed between the different copies of the ISR. Single base differences present in the long ISR (LISR) were then exploited in attempts to detect possible heterogeneity between rrn copies in Acinetobacter baylyi but variability was not detected by RFLP analysis of LISR-specific PCR products. These primers were shown to be highly specific for 3 Acinetobacter baylyi strains based on LISR sequence homogeneity.

Unique organization of the 16S–23S intergenic spacer regions of strains of Acinetobacter baylyi provides a means for its identification from other Acinetobacter species

This paper extends an earlier report on rrn operon characteristics in members of the genus Acinetobacter. It describes a systematic approach towards developing and validating a protocol for elucidating how the intergenic spacer regions (ISR) in Acinetobacter baylyi strains are organized and allows the numbers of long and short ISRs to be determined. Experimental data confirmed the in silico predictions based on available A. baylyi rrn sequence data. All were shown to possess three long ISRs and 4 short ISRs, differing in most cases in length by about 90nt. However, the ISR arrangement in A. baylyi strain 93A2 was different. Although it also possessed 4 SISRs and three LISRs, their length difference was less (39nt) which was confirmed from its ISR sequence data. Primer sets for PCR identification of A. baylyi could then be determined. Applying the same approach to other species of Acinetobacter showed none shared the same ISR organization as A. baylyi. Its value in typing members of this genus is discussed.

Pyrolysis Mass Spectrometry (PyMS) and 16S23S rDNA Spacer Region Fingerprinting Suggests the Presence of NovelAcinetobacters in Activated Sludge

Screening of large numbers of Acinetobacter spp. from activated sludge systems with Pyrolysis Mass Spectrometry (PyMS) showed that many did not cluster tightly with the currently described genomic species which have been obtained mainly from clinical sources. Selected isolates were then genotypically fingerprinted using their 16S-23S rDNA spacer region, and again the data revealed considerable differences in the genomic fingerprints of many of these activated sludge isolates to the predominantly clinical genomic species. In fact, few could be identified from them. The possibility that the current speciation within this genus is not adequate to encompass all these environmental isolates is addressed in relation to the methods used to study the population dynamics of Acinetobacter in activated sludge.