A 'lhesis presented in partial fulfilment of the requirements for the degree of
Doctor of Rlilosophy in Microbiology at Massey University, Palmerston North, New Zealand
lesley Arme Hampton 1988
/
Abstract
Declaration Acknowledgemants
List of Tables Appearing in the Text
List of Figures Appearing in the Text
Abbreviations
ClIAPI'ER 1 : INl'ROIJJCrION
1 . 1 Human Intra-abdc:minal Abscesses
1 . 2 Virulence of B . fragilis in Animal Models
(vii) (x) (xi) (xii) (xiv) (xviii)
1 3
of Intra-abdc:minal Sepsis 4
1 . 2 . 1 Animal Models of Intra-abdc:minal Sepsis 4
1 . 2 . 2 Encapsulation of B. fraqilis 5
1. 2 . 3 Bacterial Synergy 8
1. 2 . 4 other Virulence factors of B. fragilis 13
1 . 3 Host Defences in the Peritoneal cavity
1 . 3 . 1 Lymphatic Clearance
1. 3 . 2 Humoral Defence Mechanisms
1 . 3 . 3 Phagocytosis
1 . 3 • 4 Cell-mediated Irmnuni. ty
1 . 3 . 5 Abscesses 1 . 4 Phagocytosis
1 . 4 . 1 Methcx:lology
1 . 4 . 2 Opsonization
1 . 4 . 3 Attachment am Ingestion
1 . 4 . 4 Intracellular Events
16 16 17 19 21 22 23 23 26 28 30
1. 5 Fhagocytosis of Bacteroides Species 35
1 . 5 . 1 Cher!otaxis 35
1 . 5 . 2 �nization am Fhagocytic Killing 36
1 . 5 . 3 KillIDJ by Abscess Neutrqilils 37 1 . 5 . 4 Inhibition of Fhagocytic KillIDJ of
Facultative Anaerobes 38
1 . 5 . 5 Effect of Bacterial capsules 40
aJAPI'ER 2: MATERIAlS AND MEIHOI:6 2 . 1 Animals
2 . 2 Media
2 . 3 Bacteria
2 . 4 capsules
2 . 5 Bran
2 . 6 Abscess-In:1ucin;J Mixtures
2 . 7 Evaluation of Abscesses
2 . 8 Sera
2 . 9 Irrlirect Fluorescent Antibody Test
2 . 10 cp:;onization in vitro
2 . 11 In vitro Rlagoc:ytic Killing Assay
2 . 12 In vitro Intracellular Killing Assay
2 . 13 Emmeration of Bacteria an:} Leukocytes fran the Murine PEfitoneal cavity
2 . 14 Transmission Electron Microscopy
2 . 14 . 1 Starrlard Prcx::edure
2 . 14 . 2 Peroxidase cytochemistry
2 . 14 . 3 Periodate-'Ihiocarboh.ydrazide-silver
Proteinate stainin;J
2 . 14 . 4 Reprocessin;J of Paraffin Wax -
Embedded Abscesses 2 . 15 statistics
4 1 4 2 4 2 43 44 44 45 45 46 47 48 48 50
50 51 51 53 54 55 55
aIAPI'ER 3: A IDDEL OF INTRA-AJ3IXIoIINAL ABSCESS
DEVEIDIMENl' IN C3H MICE 56
3 . 1 Intrcx:luction 57
3 . 2 Results 59
3 . 2 . 1 Encapsulation of Bacteria 59
3 . 2 . 2 Abscess Develc::pnent 59
3 . 2 . 3 Abscess Histolcx;ry 67
3 . 2 . 4 Ultrastru.cture of Murine Peritoneal
Neutrq:nils 72
3 . 2 . 5 Abscess Ultrastructure 80
3 . 2 . 6 Initiation of Abscess Developnent 84
3 . 3 Discussion 112
3 . 3 . 1 Abscess Developnent: B. fragilis,
E. coli , and Bran 112
3 . 3 . 2 Abscess Developnent: other Abscess-
II'rlucinJ Mixtures 117
3 . 3 . 3 Initiation of Abscess Developnent 119
CE:API'ER 4 : '!HE INl'ERACI'ION OF ABSCESS-INIXJCING
BACI'ERlA WI'1H KJRINE PERI'IOOFAL �
IN VITRO 123
4 . 1 Introduction 4 . 2 Results
4 . 2 . 1 Rlagocytic Killin;J of stationary Rlase Bacteria catpared to
124 126
Logaritlnnic Rlase Bacteria 126
4 . 2 . 2 cytological Assessment of the Fhagocytosis of B. fragilis,
B. vulgatus am E . coli 126
4 . 2 . 3 Phagocytic Killin;J of B. fragilis,
B. vulgatus am E . coli 130
4 . 2 . 4 '!he Effect of Oxygen on the Rlagocytic
Killin;J of B. fragilis am E . coli 134 4 . 2 . 5 '!he Effect of Bacterial Concentration
on the Activity of Peritoneal
leukocytes 136
4 . 2 . 5a Phagocytic Killin;J of B. fragilis am E . coli in the Presence of {)nJoing Phagocytosis
4 . 2 . 5b Ultrastructural Observations on the Phagocytic Killing of
136
B. fragilis am B. vulgatus 141 4 . 2 . 5c Ultrastructural Observations
on the Rlagocytic Killing of
E. coli 159
4 . 2 . 5d Intracellular Killin;J of B. fragilis, B. vulgatus am E. coli in the Absence of
{)nJoin;J Phagocytosis 162
4 . 2 . 6 '!he Effect of Bran on the Rlagocytic
Killin;J of B. fragilis am E . coli 171 4 . 3 Discussion
4 . 3 . 1 Phagocytic Killin;J of Logarithmic am •
176
stationazy Rlase Bacteria 176
4 . 3 . 2 Phagocytic am Intracellular Killin;J of
B. fragilis , B. vulgatus am E. coli 177 4 . 3 . 3 Ultrastructural Observations on the
Phagocytosed Bacteria 187
4 . 3 . 4 Conclusion 193
ClIAPl'ER 5 : CONCIDDING DISClJSSlOO 195
APPENDIX 206
REFERENCES 212
A nurine rrodel of intra-abdaninal (IA) abscess fonnation was used to study the interaction of murine strains of bacteria with peritoneal neut.rqtrils . '!he intraperitoneal ( IP) inoculation of non-immune mice with mixtures containing either 5x108 Bacteroides fragilis or Bacteroides vulgatus canbined with lxl06 Escherichia coli ani 1 ng of bran as a potentiatirq agent i.rrluoed abscess fonnation after three days . Ten weeks after the IP inoculation of mice with B. fragilis , E . coli ani bran IA abscesses containing viable bacteria . at concentrations similar to those in the inocultnn persisted in 71% of the mice.
OJrirq the first 24 hrs of infection B. fragilis ani B. vuloatus were readily Iilagocytosed by neutrophils ani sane macrqi1ages in the murine peritoneal cavity. However, after the first 4 . 5 hrs of infection, there were significantly nore viable intracellular B. fragilis than B. vulgatus . Furthenrore, after up to 24 hrs of Iilagocytosis in vivo, B. fragilis was nore resistant than B.
vulgatus to killirq when the leukocytes were incubated with no:nnal sennn (NS) in vitro. '!his suggests that B. fragilis persists in IA abscesses because of its resistance to the bactericidal mecl1anisms of neutrophils after phagocytosis . To test this hypothesis, the extent of fusion of peroxidase
labelled primary granules with neut.rqtril Iilagosanes containing B. fragilis or B. vulgatus was exami.n€d by electron microscopy.
After the in vivo Iilagocytosis of either B. fragilis or B.
vulgatus , primary granules had fused with same bacteria
ex>ntainirq Iilagoscmes of neutrophils . Intact primary granules were also visible in the neutrophils ' cytoplasm. However, nore damaged intracellular B. vulgatus than B. fragilis were c:i:lsezved. '!his was consistent with the significant reduction in the rnnnber of viable B. vulgatus in IA abscesses three weeks after the IP inoculation of mice with B. vulgatus , E. coli ani bran.
'!be E. coli strain was encapsulated ani was relatively resistant to in vitro Iilagocytosis in either NS or NS ani innnune sennn (IS) . '!his may be in'portant in the persistence of the
infection. E . coli, either alive or killed, had no detectable effect on the Iilagocytic killi.rg of B. fragilis in vitro.
Although capsules were also detected on the B. fragilis am B.
vulgatus strains by electron microscopy, both were readily Iilagocytosed in the presence of NS (a source of canplement) . In vitro Iilagocytic killi.rg of B. vulgatus, at a ratio of one bacterium per ten peritoneal leukocytes , occurred in NS alone, whereas the maximal Jilagocytic killi.rg of B. fragilis am E.
coli required NS am IS . Rlagocytic kill i.rg of B . fragilis am E. coli was significantly reduced in anaerOOic con:litions .
In the presence or absence of on�oi.rg Iilagocytosis, at a ratio of 100 bacteria per peritoneal leukocyte, a proportion of intracellular B. fragilis resisted the bactericidal mechanisms of neutrophils for at least 2 hrs in the in vitro assays . Intracellular B. fragilis were more resistant to ultrastructural damage than were B. vulgatus in the presence of on-goi.rg Iilagocytosis . Irgested B. fragilis were located within the Iilagosarnes of neutrophils, am there was evidence of prilnary granule fusion with 15% am 13% of these phagosarnes in NS am NS plus IS respectively. More Iilagocyt.ic killi.rg occurred in IS because, although the addition of IS to NS did not alter the percentage of phagocytes with intracellular bacteria, it did result in the Iilagocytosis of a greater number of bacteria per neutrophil . '!his resulted in more Iilagosarnes per neutrophil in NS am IS , although the number of bacteria per Iilagosorne am the proportion of peroxidase-positive Iilagosarnes were silnilar to those in NS alone . Consequently, overall more bacteria were exposed to granule contents in NS am IS anj more were killed by the peritoneal neutrqtlils than in NS alone. However, the small proportions of peroxidase-positive Iilagosarnes in either NS or NS am IS , plus the smvival of a greater proportion of B. fragilis when exposed to neutrqtlils at high vs low ratios of bacteria to peritoneal leukocytes , suggests that the fusion of an insufficient number of primal::y granules may influence the ability of neutrqtlils to kill bacteria readily Iilagocytosed at high ratios of bacteria to leukocytes .
Ji'lagocytosis of pre-opsonized B. fragilis in the presence of NS , which �rted intracellular killing of the majority of the bacteria , few peroxidase-positive or peroxidase-negative granules were seen in the cytq:>lasm of neut.rcplils, inticating that Ji'lagosare-granule fusion had occurred. In contrast, in either NS heated to inactivate cx:rrplement or the absence of NS , which did not �rt intracellular killing of B. fragilis, nany
intact granules were visible in the cytq:>lasm of neutrq:i1ils.
Bran was an essential c:orrp:>nent of the abscess-irrlucing mixture.
In vitro, the Ji'lagocytic killing of B. fragilis ani E. coli was reduced in the presence of bran. '!his effect of bran was d::>served with pre-opsonized bacteria in NS ani suggests that bran affects the sennn c:orrp:>nents , probably complement, necessa:ry for the stimulation of intracellular killing.
After 120 mins of in vitro Ji'lagocytosis, the CXlalescence of Ji'lagosanes containing B. fragilis was evident in � neutrq:i1ils . '!he disintegration of the rrernbranes of � necrotic neutl:"qilils released bacteria from the Ji'lag� . Intracellular killing assays inticated that 20-40% of B.
fragilis were viable at this time . Furthennore, bacteria were located in extracellular ani intracellular sites within the abscesses. '!hus , it is suggested that the establishrcent of a cycle of Ji'lagocytosis , liInited intracellular killing due to insufficient fusion of primary granules with phagosanes in the presence of large rn.nnbers of bacteria , a situation c::arpourrled by the low levels of extracellular NS c:orrp:>nents , followed by release of the bacteria ani lilnited bacterial replication, enabled the sm:vival of bacteria in IA abscesses in mice .
I certify that this thesis does not incoqx:>rate without acknowledgement any material previously su1::mitted for a degree or diplana in any university, am that to the best of my
knowledge am belief, it does not contain any material previously p.1blished or written by another person, except where due reference is made in the text.
I would like to thank the Depart:.Irent of MicrdJiology am Genetics for providing the facilities am cnx>rtunity for this stOOy.
In particular, I would like to thank my supervisor, Dr Ma1:y
Nulsen, for her valued advice am constant enc::a.rragerrent . I would also like to acknowledge the �rt of Dr John Clarke . I would like to thank Ma1:y am her technicians for occasional help with experiIoonts, am Mrs Gail Haydock for typinJ this thesis .
I also gratefully acknowledge the followinJ:
Mr IX>ug Hopcroft am Mr Rayrrorrl Bermett a.k.a. "Crunch" of the Electron Microscope Services , Biotechnology Division,
ISIR, Palmerston North, for use of the transmission electron microscope, the provision of Iilotographic services, am also their encouragement.
Dr C J O ' Kelly of the Depart:.Irent of Botany am Zoology, for showinJ ne how to use a dianorrl knife am allowinJ ne to use the Depart:.Irent ' s ultramicrotane. I also awreciated his advice.
Mrs Pam Slack of the Histology laboratory, veterinary Pathology am Public Health Depart:.Irent, for the preparation of histopathological sections .
'!he staff of the Small AnilnalProduction Unit, Central Fhotographic Unit, am the GraIilic Designer, Mr Kees
Konrlorffer .
I was the recipient of a UGC Postgraduate Scholarship, Massey Scholarship, am also a Harriette Jenkins Award fran the NZ
Federation of University wanen - thank you.
Finally, a very special thankyou to my parents , my sister Robyn, am Jeff, for their �rt in all capacities .
LIST OF � .APPFAR1IC IN '!HE '!EXT
Table Page
1 . 1 Bacterial synergy in animal IOOdel.s of
IA sepsis 9
1 . 2 Effect of anaerobes on the {i1agocytosis
am killin;J of facultative anaerciJes 39 3 . 1 Abscesses iIrluced by B. fragilis,
E . ex>li am bran 61
3 . 2 Abscesses iIrluced by Bacteroides species,
E . ex>li am bran 64
3 . 3 Number of leukocytes in the murine peritoneal cavity after inoculation with abscess-
irrlucing mixtures 90
3 . 4 Cellular ex>ntent of the murine peritoneal cavity 1 hr post-inoculation with abscess-
irrlucing mixtures 91
3 . 5 SUmmary of ultrastructural observations on
the initiation of abscess develq:m:mt 97 3 . 6 Killing in vitro of bacteria {i1agocytosed
in vivo 110
4 . 1 Fbagocytic killin;J of stationary {i1ase bacteria � to logarithmic {i1ase
bacteria in aerobic ex>rrlitions 127 4 . 2 leukocyte-associated bacteria (lAB) at
60 mins 128
4 . 3 Neutrophil-associated E . ex>li after pre-
opsonization in 50% NS am 50% IS 131 4 . 4 '!he Effect of oxygen on the {i1agocytic
killing of B. fragilis am E . coli 135 4 . 5 '!he Effect of B. fragilis ex>ncentration on
the activity of peritoneal leukocytes 140 4 . 6 '!he Effect of extracellular sennn on
degranulation 153
4 . 7 FUsion of neutrophil primary granules with
{i1agosanes containin;J B. fragilis 156 4 . 8 Number of viable intracellular bacteria
after 3 mins of {i1agocytosis 165
4 . 9 4 . 10
4 . 11
Intracellular killiIg of B. fragilis, B. vulgatus am E. coli
'!he Effect of killed E . coli on the intracellular killiIg of Pre-qlSOnized B. fragilis in aerd::>ic corrlitions
'!he Effect of E . coli on the intracellular killirg of Pre-qJSOnized B. fragilis in anaerobic corrlitions
166
169
170
LTST OF FIGJRES Al'PEAR]}I; IN '1HE '!EXT
Figure Page
3 . 1 Er'lcap;ul.ation of bacteria 60
3 . 2 Viable bacteria in abscesses irrluced by
B. fragilis, E. ooli ani bran 62
3 . 3 Viable bacteria in abscesses iIrluced by
Bacteroides species , E . ooli am bran 65 3 . 4 Histology of a Lay 3 abscess iIrluced by AIM 68 3 . 5 Histology of a Lay 13 abscess iIrluced by AIM 69 3 . 6 Histology of a Lay 4 0 abscess iIrluced by AIM 70 3 . 7 Distribution of B. fragilis antigens within
abscesses iIrluced by AIM 71
3 . 8 A Mature murine peritoneal neutrq:hil 73 3 . 9 A Peroxidase-labelled mature murine peritoneal
neutrophil 74
3 . 10 A Peroxidase-labelled mature murine peritoneal
neutrophil without lead citrate stainin;J 75 3 . 11 Peroxidase-labelled mature murine peritoneal
neutrophils with the emission of H2� (a) or 3-3 ' diaminobenzidine tetrahydrochloride (b)
fram Graham' s am Kan1ovsky ' s medium 76 3 . 12 A PA-TClI-SP stained mature murine peritoneal
neutrophil 77
3 . 13 A PA-TClI-SP stained mature murine peritoneal
neut.rq:hil digested with a-amylase 78 3 . 14 Control mature murine peritoneal neutrophils
which illustrate that PA-'lOI-SP positive granules contain vicinal glyool-contai.nirg
glycoconjugates 79
3 . 15 Ultrastructure of a Lay 3 IA abscess in::iuced
by AIM 81
3 . 16 Ultrastructure of a Lay 6 IA abscess in::iuced
by AIM 82
3 . 17 Ultrastructure of a Lay 13 IA abscess irrluced
by AIM 83
3 . 18 Ultrastructure of a Lay 21 IA abscess irrluced
by AIM 85
3 . 19 Ultrastructure of a Day 40 IA abscess in:iuced
by AIM 86
3 . 20 Ultrastructure of a Day 70 IA abscess irrluced
by AIM 87
3 . 21 ReprooessinJ of a histologically-fixed Day 13 IA abscess iIrluced by AIM for electron
microscopy 88
3 . 22 Infection of the nurine peritoneal cavity
- 1 hr 92
3 . 23 Infection of the murine peritoneal cavity
- 4 . 5 hrs 93
3 . 24 Infection of the murine peritoneal cavity
- 24 hrs 94
3 . 25 Viable intracellular bacteria after in vivo
P'lagocytosis by murine peritoneal leukocytes 95 3 . 26 1 hr after the IP inoculation of 5x108
B. fragilis and 1 mg of bran 98
3 . 27 Intraperitoneal inoculation of 5x108
B. fragilis, lxl06 E. cx:>li and 1 mg of bran
- 1 hr infection 99
3 . 28 Intraperitoneal inoculation of 5x108
B. fragilis, lxl06 E. cx:>li and 1 mg of bran
- 4 . 5 hr infection 100
3 . 29 Intraperitoneal inoculation of 5x108
B. fragilis, lxl06 E. cx:>li and 1 mg of bran
- 24 hr infection 102
3 . 30 Intraperitoneal inoculation of 5x108 B. fragilis and lxl06 E. cx:>li
- 1 hr infection 103
3 . 31 Intraperitoneal inoculation of 5x108 B. fragilis and lxl06 E . cx:>li
- 2 4 hr infection 104
3 . 32 Intraperitoneal inoculation of 5x108
B. vulgatus , lxl06 E . cx:>li and 1 mg of bran
- 1 hr infection 105
3 . 33 Intraperitoneal inoculation of 5x108
B. vulgatus, lxl06 E . cx:>li and 1 mg of bran
- 4 . 5 hr infection 106
Figure 3 . 34
3 . 35
3 . 36
4 . 1
4 . 2 4 . 3 4 . 4
4 . 5
4 . 6 4 . 7 4 . 8 4 . 9 4 . 10 4 . 11
4 . 12 4 . 13
Intraperitoneal incx::ulation of 5x108
B. vulgatus , lxl06 E . coli arrl 1 ng of bran - 24 hr infection
Intraperitoneal incx::ulation of 5x108 B. vulgatus , arrl lxl06 E. coli
- 1 hr infection
Intraperitoneal incx::ulation of 5x108 B. vulgatus arrl lxl06 E. coli
- 24 hr infection
Rlagocytosis of B. fragilis , B. vulgatus and E. coli
Rlagocytosis of viable and non-viable E. coli Rlagocytic killing of bacteria in aerobic corrlitions
Effect of B. fragilis concentration on phagocytic killing in aerobic arrl anaerobic corrlitions
Effect of E . coli concentration on
phagocytic killing in aerobic arrl anaerobic corrlitions
Extracellular B. fragilis after 20 mins in 10% NS
Rlagocytosed B. fragilis after 20 nUns in 10% NS
Rlagocytosed B. fragilis after 20 nUns in 10% NS - an eosirlqilll
Rlagocytosed B. fragilis after 120 mins in 10% NS
Rlagocytosed B. fragilis after 20 nUns in 10% NS and 10% IS
Rlagocytosed B. fragilis after 120 mins in 10% NS and 10% IS - cl1..m1p€d peritoneal leukocytes
Rlagocytosed B. fragilis after 120 mins in 10% NS and 10% IS
Rlagocytosed B. fragilis after 120 mins
in 10% NS and 10% IS - neutrophils with large
phagosomes .
107
108
109
129 132 133
137
138 142 143 144 145 147
148 149
150
4 . 14 4 . 15 4 . 16 4 . 17 4 . 18 4 . 19 4 . 20 4 . 21
4 . 22 4 . 23 4 . 24
4 . 25
Ihagocytosed B. fragilis after 120 mins in 10% NS am 10% IS - necrotic neutrqi1ils '!he Effect of extracellular sennn on
degranulation after 30 mins of Ii1agocytosis Fhagocytosed B. vulgatus in 10% NS
Fhagocytosed B. vulgatus in 10% NS am 10% IS
Fhagocytosed E . coli after 20 mins in 10% NS Fhagocytosed E . coli after 60 mins in 10% NS am 10% IS
Fhagocytosed E . coli ( Pre-qJSOnized in 50%
NS am 50% IS) after 60 mins in 10% NS
Intracellular pre-opsonized B. fragilis after 60 mins in 10% NS in the absence of on;Joing phagocytosis
'!he ClUlTping of peritoneal leukocytes in the presence of bran
'!he Fhagocytosis of bran by peritoneal neutrqi1ils after 20 mins in 10% NS
'!he Effect of bran on the Iilagocytic killing of B. fragilis in aerobic conditions
'!he Effect of bran on the Iilagocytic killing of E. coli in aerobic conditions
152 154 157
158 160 161
163
168
172 173 174 175
AIM An abscess-irn.uci.n;J mixture containing:
1 ng bran )
5x108 colony fonn:irg mrits of )
Bacteroides fragilis ) in 0 . 05 ml of lxl06 colony fonn:irg mrits of ) RIMI-1640 IOOdiurn
Escherichia coli )
mIB Brain heart infusion broth cfu Colony fonning mrits
EM Electron microscx:>py FCS Foetal calf serum
HNS Heat-inactivated nonnal serum
IA Intra-abdominal
IP Intraperitoneal
IS Heat-inactivated inmrune serum IPS Lipopolysaccharide
NS Fresh nonnal sennn
PA�-SP Periodate-thiocarbohydrazide-silver proteinate PBS RlOSIi'late-buffered saline
SC SUlxutaneous
SD Standard deviation
we wilkins Olalgren