3.1 coli, but far from other species. The


3.1      
Conclusion

WGS, cgMLST and rMLST
genomic data showed that all 30 RG-1 Campylobacter strains cluster
together in the phylogeny. This distinct group was far from the generalist
ST-21 and ST-45 clonal complexes of Campylobacter spp. The 16S rRNA
phylogenic analysis confirmed that the RG-1 strains cluster close to C. coli,
but far from other species. The presence of the hipO and napA
gene markers excludes the possibility that the RG-1 group is C. coli or C.
jejuni subsp. doylei. These genomic properties and cell morphology
have confirmed that this group are C. jejuni species. Further analysis
might be necessary to confirm they are either C. jejuni subsp. jejuni
or that they belong to a new subspecies of C. jejuni.

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RG-1 strains were treated with the
motility MHA medium in order to assist adaptation to the laboratory conditions.
This treatment enhanced the bacterial growth by ~5-7 times and doubled
motility. Even though the RG-1 cells had adapted to the laboratory conditions,
the growth and motility properties were still 3-8 and 2-6 times less in
comparison to the reference strains, respectively. Nevertheless, RG-1 cells
grew as well as reference strains when they were grown in the MHB rich medium
supplemented with lab rat mucin. This is probably due to a particular
nutrient/growth factor within the rat mucin. 
Some other in vitro studies confirmed that the RG-1 strains
utilised the key nutrients serine, aspartate, asparagine, glutamate, glutamine,
proline, succinic acid, formic acid, and pyruvic acid, as does NCTC11168. The
serine was used first followed by aspartate, glutamate and proline (Velayudhan et al., 2004). This order of amino acid
preference was similar to the well-studied NCTC11168. One of the clear
differences between RG-1 and the control strain was that RG-1 did not utilise
L-fucose due to a missing genomic island (cj0480c-cj0480) needed
to metabolise the carbon source. It is known that this pathway is absent in
approximately 50% of the sequenced C. jejuni and C. coli strains,
including both human gastroenteritis causing C. jejuni 81-176 and 81116
strains (Dwivedi et al., 2016).

 

RG-1 strains share the majority of
their genome content (1540 core genes, 84.1% identity?). Meanwhile, 38.8% of
auxiliary genes (112/288) were from two large uncharacterised phage insertions.
The rest of the auxiliary genes were related to motility, chemotaxis, and membrane
and transport proteins. These genes could be important for evolution of the
RG-1 strains to a specific host environment. Significantly, cgMLST Cj0145 gene encoding putative TAT (Twin-Arginine
Translocation) was absent in all RG-1 strains. This pathway is involved in the
stress response and aids C. jejuni adaptation to different hosts (Hitchcock et al., 2010;
Rajashekara et al., 2009).
The loss of this cgMLST could restrict RG-1 to a specific niche. 

 

Only 2.2% of the RG-1 core genes
(35 genes) might be rat specific genes. These were not cgMLST and were not in
the different selected sequence types of C. jejuni strains. A group of 9
genes are related to the uptake of iron. This pathway was not identical to the identified 7
class of pumping iron in CampylobacterAD1 .
However, based on percentage identity, this group of genes might have been
acquired from C. jejuni subsp. doylei
269.97 (92% identity) or C. helveticus (82% identity). On the other hand, RG-1 strains have
lost three genes of the Ferric enterochelin uptake system including the cfrA
gene and five genes of the Ferri-transferins uptake system, including the Cj0178
gene. Different in vivo studies have demonstrated that C. jejuni
requires both CfrA and Cj0178 to be able to survive and colonise the intestines
of animals (Palyada et al., 2004; Zeng et al., 2009). In addition to the iron
uptake system, RG-1 strains have a new operon of cdtABC genes. The
operon might originate from the C. lari RM16701 strain
(86% identity), which has a couple of deletions in the protein sequence of CdtA.
It is well known that Campylobacter spp. are commensal bacteria to
poultry. Even though many of C. jejuni strains from broiler chickens
have cdt genes, they do not cause disease in the birds (Bang et al., 2001).

 

On the other hand, RG-1 has lost some key systems. Vitamin
B5 biosynthesis has been identified as a host specificity factor. Interestingly,
panBCD genes were absent in all RG-1 and RG-2 strains, but present in the
other ST-21, ST-22 and ST-45 clonal complexes found in farm associated rats. Sheppard et al. (2013)
found that Campylobacter isolates from cattle have the required pathway,
but the operon was not found in chicken associated strains. They proposed that
chickens could obtain the nutrients from rich vitamin B5 cereals and grain, and
therefore they do not need to synthesise it themselves. This may be true for
the RG-1 strains. They could get vitamin B5 from the diet of the rats. Lastly,
another host specific L-fucose utilisation operon was not found in the RG-1
genomes. It has been studied that designing a mutation in the putative fucose
permease Cj0486 gene inhibited the uptake of fucose and reduced colonisation
levels in a piglet model, but without significant difference in a chicken model
(Stahl et al., 2011).
Supplementing the chickens’ diet with L-fucose increased the level of
colonisation by the wild strain. The fuc-positive NCTC1168 also formed a
more sessile structure of biofilm than fuc-negative 81116 (Dwivedi et al., 2016). The ability to use L-fucose
might allow the bacterial isolates to survive better in pigs over other hosts.
Similarly, RG-1 strains might not be able to colonise pigs, suggesting that
they have adapted to specific hosts.

 

 To conclude, draft genomes, mass
spectrometry, and 16S rRNA have confirmed that the RG-1 strains are C.
jejuni and presumably subsp. jejuni. Approximately 15% of the RG-1
core genes (148) were not cgMLST, and included a putative iron uptake system
and cdt genes. The strains have lost some virulence or host specific operons
such as cgMLST Cj0145 putative TAT gene, Ferric enterochelin and Ferri-transferrin
uptake systems, fuc pathway, and vitamin B5 biosynthesis. All RG-1
strains were motile, did not have the ability to form biofilm on the glass
membrane filter, and grew poorly in rich medium compared to other differing
sequence types. Growth of poorly growing RG-1 strains was encouraged through
the addition of rat mucins to the rich medium. The loss of some host specific
operons and the improved growth of these RG-1 isolates in the rat mucin
suggests that this group of C. jejuni strains may not be able to survive
in different hosts and are likely restricted to specific hosts. To further
investigate this, selected RG-1 strains were studied in both Galleria
melonella and Ross chicken in vivo models, as shown in chapter 5. The next chapter will be on
the ability of RG-2 strains to utilise glucose.

 

 

 

 Appendices

 

Appendix 1: Growth of C. jejuni in
small intestinal and caecal lab rat mucins.  The small intestine (S) and caecum (C) mucins
were scraped from 4 white lab rats (~250g/rat) and the mucin was homogenised
using a MP shaker. Under my supervision, the growth experiment was done by
Nancy, F. and Sarah, J. undergraduate students. MHB was supplemented with 5%
mucin, 1% antibiotic and inoculated with 0.002OD600 of bacterial
cells, and incubated for 45h. Cells were then grown under microtiter plate
growth assay conditions. CFU values are the mean of triplicate drops of three
wells. Pattern filled histograms are growth in MHB without mucin.
 

 

 

Appendix 2: Concentration of amino acids in
MEM-? medium. The medium was supplemented with 10%
FBS and 20mM (A) proline and (B) serine. Amino acid
concentration was measured at 48h and 72h of incubation. The proline in A and
serine in B data has been shown in Figure ?3.15,
B.
 

A

B

 

 

 

 

Appendix 3: cgMLST 29 genes are absent (A)
or incomplete (I) in at least one of the RG-1 strains.
 

Cj
number

Product

Sequence
length

Genome
position

Absent (A) or Incomplete (I) gene among RG-1

Cj0118

conserved
hypothetical protein

756

122366

I in Dg197

Cj0145*

putative TAT
(Twin-Arginine Translocation) pathway signal sequence domain protein

1782

148819

A in all

Cj0174c|cfbpB*

putative iron-uptake
ABC transport system permease protein

1617

169946

A in all

Cj0243c

hypothetical protein

1167

224794

I in Dg150

Cj0430

putative integral
membrane protein

1227

391711

I in Dg61

Cj0484

putative MFS (Major
Facilitator Superfamily) transport protein

1233

451046

A in 36.6%

Cj0561c

putative periplasmic
protein

930

524034

A in 36.7%

Cj0763c|cysE

serine
acetyltransferase

639

714138

I in Dg156

Cj0777

putative
ATP-dependent DNA helicase

2031

728500

I in Dg189

Cj0799c|ruvA

putative Holliday
junction ATP-dependent DNA helicase

552

749307

I in Dg78

Cj0800c

putative ATPase

1860

749834

I in Dg289

Cj0801

putative integral
membrane protein (MviN homolog)

1452

751797

I in Dg189

Cj0810|nadE

NH(3)-dependent
NAD(+) synthetase

741

761404

I in Dg276

Cj0841c|mobB

putative
molybdopterin-guanine dinucleotide biosynthesis protein

492

789049

I in Dg189

Cj0850c

putative MFS (Major
Facilitator Superfamily) transport protein

1188

797653

I in Dg347

Cj1004

putative periplasmic
protein

417

934201

I in Dg189

Cj1014c|livF

branched-chain
amino-acid ABC transport system ATP-binding protein

696

947343

I in Dg347

Cj1041c

putative periplasmic
ATP/GTP-binding protein

852

975230

A in 23.4%

Cj1042c

putative
transcriptional regulatory protein

891

976144

A in 23.4%

Cj1198|luxS

S-ribosylhomocysteine
lyase (autoinducer-2 production protein LuxS)

495

1127437

A in 36.7%

Cj1064

pseudogene
(nitroreductase)

620

1001218

A in 23.4%

Cj1199

putative
iron/ascorbate-dependent oxidoreductase

993

1128243

A in 36.7%

Cj1201|metE

5-methyltetrahydropteroyltriglutamate–
homocysteine methyltransferase

2265

1130028

A in 36.7%

Cj1202|metF

5,10-methylenetetrahydrofolate
reductase

849

1132302

A in 36.7%

Cj1295

conserved
hypothetical protein

1308

1226978

I in Dg147

Cj1296

hypothetical protein

360

1228282

A in 23.4%

Cj1346c

1-deoxy-D-xylulose
5-phosphate reductoisomerase

1071

1278851

I in Dg150

Cj1411c

putative cytochrome
P450

1362

1342550

I in Dg150

Cj1721c*

putative outer
membrane protein

645

1632901

A in all

* absent in RG-1 including Dg147

 
Appendix 4: 148 non-cgMLST, but core genes
in RG-1. RG-1 strains were Blasted in Genome Comparator (95% threshold) (Cody et al.,
2017).
 

Cj NCTC11168 gene number

Product

Cj NCTC11168 gene number

Product

Cj0011c

putative non-specific DNA binding protein.

Cj0864

putative periplasmic protein

Cj0014c

putative integral membrane protein

Cj0866

pseudogene (arylsulfatase)

Cj0019c

putative MCP-domain signal transduction protein

Cj0874c

putative cytochrome C

Cj0020c

cytochrome C551 peroxidase

Cj0876c

putative periplasmic protein

Cj0030

hypothetical protein

Cj0900c

small hydrophobic protein

Cj0037c

putative cytochrome C

Cj0973

hypothetical protein

Cj0041|fliK

putative flagellar hook-length control protein

Cj0974

very hypothetical protein

Cj0045c

putative iron-binding protein

Cj0983

putative lipoprotein

Cj0057

putative periplasmic protein

Cj0985c|hipO

hippurate hydrolase

Cj0058

putative peptidase C39 family protein

Cj0986c

putative integral membrane protein

Cj0090

putative lipoprotein

Cj0986c

putative integral membrane protein

Cj0092

putative periplasmic protein

Cj0987c

putative MFS (Major Facilitator Superfamily) transport
protein

Cj0093

putative periplasmic protein

Cj0988c

very hypothetical protein

Cj0140

hypothetical protein

Cj0990c

hypothetical protein

Cj0168c

putative periplasmic protein

Cj1018c|livK

ABC
transport system, periplasmic binding protein

Cj0186c

putative TerC family integral membrane protein

Cj1019c|livJ

 ABC transport
system periplasmic binding protein

Cj0199c

putative periplasmic protein

Cj1021c

putative periplasmic protein

Cj0201c

putative integral membrane protein

Cj1036c

conserved hypothetical protein

Cj0202c

hypothetical protein

Cj1055c

putative sulfatase family protein

Cj0203

putative citrate transporter

Cj1060c

putative membrane protein

Cj0246c

putative MCP-domain signal transduction protein

Cj1077|ctsT

putative periplasmic protein

Cj0247c

hypothetical protein

Cj1079

putative periplasmic protein

Cj0251c

highly acidic protein

Cj1110c

putative MCP-type signal transduction protein

Cj0263|zupT

zinc transporter

Cj1119c|pglG

putative integral membrane protein

Cj0299

putative periplasmic beta-lactamase

Cj1135

putative two-domain glucosyltransferase

Cj0327

putative endoribonuclease L-PSP family protein

Cj1136

putative glycosyltransferase

Cj0339

putative MFS (Major Facilitator Superfamily) transport
protein

Cj1149c|gmhA

sedoheptulose 7-phosphate isomerase

Cj0340

putative nucleoside hydrolase

Cj1159c

small hydrophobic protein

Cj0380c

hypothetical protein

Cj1160c

putative membrane protein

Cj0414

putative oxidoreductase subunit

Cj1189c|cetB

bipartate energy taxis response protein cetB

Cj0415

putative GMC oxidoreductase subunit

Cj1191c

putative PAS domain containing signal-transduction sensor
protein

Cj0416

hypothetical protein

Cj1224

putative iron-binding protein

Cj0417

hypothetical protein

Cj1241

putative MFS (Major Facilitator Superfamily) transporter
protein

Cj0437|sdhA

succinate dehydrogenase flavoprotein subunit

Cj1255

putative isomerase

Cj0438|sdhB

putative succinate dehydrogenase iron-sulfur protein

Cj1300

putative SAM domain containing methyltransferase

Cj0439|sdhC

putative succinate dehydrogenase subunit C

Cj1305c

hypothetical protein (617 family)

Cj0455c

putative membrane protein

Cj1309c

hypothetical protein

Cj0494

putative exporting protein

Cj1310c

hypothetical protein (617 family)

Cj0501

pseudogene (ammonium transporter)

Cj1314c|hisF

imidazole glycerol phosphate synthase subunit

Cj0552

putative membrane protein

Cj1315c|hisH

imidazole glycerol phosphate synthase subunit

Cj0553

putative integral membrane protein

Cj1316c|pseA

pseudaminic acid biosynthesis PseA protein

Cj0554

hypothetical protein

Cj1319

putative nucleotide sugar dehydratase

Cj0555

putative dicarboxylate carrier protein MatC

Cj1320

putative aminotransferase (degT family)

Cj0556

putative amidohydrolase family protein

Cj1327|neuB2

N-acetylneuraminic acid synthetase

Cj0563

hypothetical protein

Cj1328|neuC2

putative UDP-N-acetylglucosamine 2-epimerase

Cj0564

putative integral membrane protein

Cj1329

putative sugar-phosphate nucleotide transferase

Cj0592c

putative periplasmic protein

Cj1330

hypothetical protein

Cj0618

hypothetical protein  (617 family)

Cj1331|ptmB

acylneuraminate cytidylyltransferase (flagellin
modification)

Cj0659c

putative periplasmic protein

Cj1332|ptmA

putative oxidoreductase (flagellin modification)

Cj0660c

putative transmembrane protein

Cj1337|pseE

PseE protein

Cj0672

putative periplasmic protein

Cj1360c

putative proteolysis tag for 10Sa_RNA

Cj0676|kdpA

pseudogene (potassium-transporting ATPase A chain)

Cj1406c

putative periplasmic protein

Cj0677|kdpB

potassium-transporting ATPase B chain

Cj1415c|cysC

putative adenylylsulfate kinase

Cj0678|kdpC

pseudogene (potassium-transporting ATPase C chain)

Cj1416c

putative sugar nucleotidyltransferase

Cj0679|kdpD

truncated KdpD protein

Cj1417c

putative amidotransferase

Cj0679|kdpD

truncated KdpD protein

Cj1418c

putative transferase

Cj0685c|cipA

Invasion protein CipA

Cj1419c

putative methyltransferase

Cj0685c|cipA

Invasion protein CipA

Cj1423c|hddC

putative D-glycero-D-manno-heptose 1-phosphate
guanosyltransferase

Cj0692c

putative membrane protein

Cj1424c|gmhA2

phosphoheptose isomerase

Cj0742

pseudogene (putative outer membrane protein)

Cj1425c|hddA

putative D-glycero-D-manno-heptose 7-phosphate kinase

Cj0770c

putative NLPA family lipoprotein

Cj1456c

putative periplasmic protein

Cj0771c

putative NLPA family lipoprotein

Cj1521c

putative CRISPR-associated protein

Cj0772c

putative NLPA family lipoprotein

Cj1522c

putative CRISPR-associated protein

Cj0786

small hydrophobic protein

Cj1523c

putative CRISPR-associated protein

Cj0814

hypothetical protein. Functional classification-Unknown

Cj1547

Blc protein homolog

Cj0815

hypothetical protein

Cj1585c

putative oxidoreductase

Cj0816

hypothetical protein

Cj1589

conserved hypothetical protein

Cj0825

putative processing peptidase

Cj1602

conserved hypothetical protein

Cj0829c

putative CoA binding domain containing protein

Cj1625c|sdaC

amino acid transporter

Cj0830

putative integral membrane protein

Cj1630|tonB2

putative TonB transport protein

Cj0849c

conserved hypothetical protein

Cj1666c

putative periplasmic protein

Cj0851c

putative integral membrane protein

Cj1668c

putative periplasmic protein

Cj0859c

hypothetical protein

Cj1714

small hydrophobic protein

Cj0860

putative integral membrane protein

Cj1729c|flgE2

flagellar hook subunit protein

 

 

Appendix 5: Iron uptake groups of genes in
Campylobacter. Genes that are missing in RG-1 indicated by
underlining in red. Image taken from (Miller, C. E.
et al., 2009).
 

 AD1This
needs to be clearer. Class of 7 genes that are involved in pumping iron? Not
sure what you mean

x

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