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Title: The use of a defined medium and mutants for establishing Campylobacter nutrition during colonisation
Author: Alhumam, Naser A.
ISNI:       0000 0004 2681 2023
Awarding Body: University of Surrey
Current Institution: University of Surrey
Date of Award: 2009
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A convenient defined medium was developed for use with Campylobacter species. Using this media C. jejuni NCTC 11168 was shown to be able to utilise mucin, L- serine, L-proline, L-glutamic acid, L-valine, L-glutamine, L-histidine, L-tyrosine, L-aspartate, L-asparagine and L-glycine as sole carbon sources. Amongst the sugars tested fucose, ribose and D-glucosamine hydrochloride supported growth. When different strains of Campylobacter were introduced into GMEM there were marked differences in growth. Out of six strains of C. jejuni, two grew well, whilst three out of five strains of C. coli grew well and two strains grew poorly in GMEM. Strains C. coli NCTC 11350, C. coli NCTC 11438 and C. jejuni NCTC 11951 failed to grow in GMEM alone, but when L-serine or L-glutamine were added NCTC 11438 and NCTC 11951 grew well, whilst the growth of NCTC 11951 was partially stimulated. The addition of carbon sources during the survival of C. jejuni had different effects depending on their nature. When mucin was added 10 hours post survival, recovery was stimulated by 10-fold yet when metabolisable single carbon sources were added, survival was decreased by 6-logs suggesting that the provision of unbalanced nutrition induces substrate accelerated death. This study also addressed potential mechanisms of mucin utilization in C jejuni. Mutants deficient in SdaC (serine transporter) and in AspA (aspartate ammonia lyase) were shown to be deficient in serine and aspartate utilization respectively but both were still able to utilize mucin suggesting that the liberation of serine and asparate from mucin is not important for its use as a carbon source. A plate based assay for mucinase/protease activity, and an assay using P-nitrophenol derivatives of sugars, did not detect any mucinolytic or glycosidic activities in C. jejuni. Mucin degradation is likely to generate short peptides and consequently, the mechanisms of peptide utilisation and transport were also investigated. Peptides containing either two or four aspartate residues supported some growth but this was less than when aspartate was provided alone. Peptides of four- or seven residues containing serine supported the best growth. It would appear then that C. jejuni can utilize peptides containing aspartate and serine as carbon sources. Mutants deficient in Cj 1580c-1584c, a putative ABC transport system for peptides, and Cj0653c, a putative aminopeptidase were still able to utilize peptides and neither system can be involved in the transport of the peptides assessed here. In addition a screen to isolate mutants unable to utilize peptides was developed using toxic peptide analogues. L-Ala-AEP (L-alanyl-L aminoethylphosphonic) was show to be suitable for this purpose but not triornithine or bialophos as C. jejuni NCTC 11168 was naturally resistant to these. When different strains of C. jejuni and C. coli were assessed for L-Ala-AEP sensitivity, three out of three strains of C. jejuni tested were sensitive and three of four stains of C. coli tested were resistant. This most likely reflects the existence of different mechanisms of peptide uptake or degradation in strains of Campylobacter, The new defined media also provided an opportunity to study the use of iron compounds and compatible solutes by C. jejuni. C. jejuni was shown to be able to acquire iron from DL- neorepinephrine, epinephrine, caffeic acid, rutin and quercetin but not catechin. although at high concentrations caffeic acid and rutin were growth inhibitory. Using GMEM it was found that at high osmolarities (0.15-0.3 M NaCl) the addition of the compatible solutes proline and betaine did not stimulate the growth of C. jejuni as is the case in other bacteria. In contrast, glycine betaine appeared to be growth inhibitory irrespective of the osmolarty of the media.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available