Nexus Research Group - Isolation of Caulobacter sp.

**THE ISOLATION AND CHARACTERISATION OF NEW ZEALAND CAULOBACTER SPECIES**

M. FENTON, C.D. FENTON* AND K. STEWART

(presented at the 1999 New Zealand Microbiological Society conference at Otago University, November 23rd - 26th)

Abstract
Materials and Methods
Results
Discussion
Conclusion
References/Bibliography

Abstract

Prosthecate bacteria are an extremely ubiquitous group, probably second only to the Pseudomonads in distribution and numbers. They have a distinctive ability to survive in low nutrient environments. Caulobacter was first isolated in New Zealand in the Manawatu (1). Isolates from sewage tended to carry more plasmids than freshwater Caulobacter, and showed an increase in resistance to many second generation antibiotics when compared to their freshwater counterparts. Caulobacter may serve as a reservoir of antibiotic resistance determinants which then persist in the environment and can be transferred back to human associated bacteria. It has been suggested that this might lead to a reduced lifetime for antibiotics in clinical use. We report the isolation of Caulobacter from the Wanganui and Taranaki regions.

Materials and Method

Enrichment

Environmental samples collected.

Left undisturbed 6 months at room temperature.

Environmental sample diluted in 0.01% peptone water.

Incubated at room temperature.

Examine liquid/air interface for stalked cells.

Enrichment flasks containing 0.01% peptone water

Isolation

Streak onto 0.01% peptone water agar.

Incubate 4 -7 days room temperature.

Single colonies re-streak onto 0.01% peptone water agar.

Purity check:

0.1 ml spread plate on 0.01% peptone water agar. Any contaminants seen in lawn

Salt tolerance:

1% NaCl peptone water agar.
2.5% NaCl peptone water agar.

Results

Water source	Caulobacter isolate
Wanganui River	WR1
Eltham Town supply	ET1
Te Henui River, New Plymouth	TH1


Caulobacter crescentus type strain ATCC15252 (x1000)	Caulobacter sp. WR1 (x1000)

Discussion

The ability of Caulobacter to survive in oligotrophic conditions is the basis for the enrichment step. Once in high enough numbers, it was found that a wet mount was preferable to staining for the detection of Caulobacter cells. Stalked cells were readily detected by their swaying movement anchored by their adhesive holdfast. One disadvantage of working with a bacterium that can adhere to other bacteria or debris is that normal streak plating methods often failed to completely disperse the Caulobacter cells. As a final purity check, each isolate was grown in peptone broth and 0.1 ml was spread on solid media. Colonies that had arisen from contaminating cells were obvious in the lawn. Caulobacter isolates ET1, WR1 and TH1 were tolerant to 1% NaCl. Freshwater species may be able to survive in marine environments if they can tolerate up to 2.5% NaCl. It has been recognised that Caulobacter species are able to receive and transfer plasmids (2). Caulobacter may serve as a reservoir of antibiotic resistance determinants which then persist in the environment and can be transferred back to human associated bacteria.

Conclusion

The presence and significance of Caulobacter in the environment has probably been overlooked due to the difficulties in correctly identifying their presence. Future investigations will need to focus on antibiotic resistance determinants and plasmid transfer. 16s rDNA sequencing would determine the phylogentic relationship of these isolates to the type strain Caulobacter crescentus. Subject to funding, we plan to sample other North Island water sources and attempt to shorten the time necessary for the enrichment and isolation of these micro-organisms.

References

1) Fenton, C.D. 1994. The isolation and characterisation of Caulobacter from Manawatu water systems. M.Sc. Thesis, Dept of Microbiology and Genetics, Massey University, Palmerston North, New Zealand

2) Ely, B. 1979. Transfer of drug resistance factors to the dimorphic bacterium Caulobacter crescentus. Genetics. 91:317-380.