Bacteria

Chapter 7

Cells are divided into two major groups, the prokaryotes and eukaryotes, based on whether they have nuclei. Attention is also paid to the types of membranes they contain and the complexity of their genetic material. Eukaryotic cells have nuclei.

PROKARYOTIC CELLS

(BACTERIA INCLUDING MYCOPLASMAS, CHLAMYDIA AND RICKETTSIAE)

Prokaryotes are cells that live and reproduce independently. The term prokaryote is made up of "pro" meaning "before" and" karyon" meaning "nucleus"). Prokaryotes lack a nucleus.

BACTERIA.

Bacteria do not contain a nucleus but the genetic material (DNA) is concentrated in a central region of the cell known as the nucleoid (not surrounded by membrane). In later bacteria there is a cell wall composed mainly of peptidoglycan, a polymer of N acetyl glucosamine and its lactyl ether N acetyl neuramic acid with peptide chains bound to the lactyl group. Variations in this basic structure have been described for a number of bacterial genera.

MYCOPLASMAS

This group of organisms termed prokaryotes is larger than viruses and smaller than other bacteria. They lack cell walls (which larger bacteria possess), have very small genomes and require cholesterol for cell membrane function and growth and are strictly dependent upon host cells.

Although it has been difficult to grow them in agar, (without cells), the addition of rich nutrients has made this possible.
There are more than 100 known mycoplasmas.

They are found in normal mammals, including human beings are very widespread and under some circumstances some specific mycoplasmas can cause disease.
Mycoplasma pneumoniae
Are important in childhood and adult pneumonias (including tracheobronchitis, sore throats, myringitis and cervical lymph node enlargement.
Mycoplasma hominis (and ureaplasma urealyticum).
These inhabit the urogenital tract, and may be pathogenic, causing urethritis and pelvic inflammatory disease.

Mycoplasma genitalium (described recently)
This is also a cause of similar urogenital infections.

Mycoplasma fermentans.
Until recently these organisms have been detected without evidence of pathological consequences.

Professor Garth Nicolson from Houston in Texas has attributed Gulf War Syndrome and Chronic Fatigue Syndrome to these organisms and the interactions that they induce in the host.

He and members of his research team continue to unravel evidence that they are important in chronic fatigue syndromes and perhaps in other disorders.

Mycoplasmas certainly are ongoing candidates for causing this sort of pathology, and may well be able to elude immune mechanisms.

So far there are not many publications that throw light on Professor Nicolson's work.

They describe evidence for mycoplasma infected CFS patients having elevated RNAse L -ratios compared to non-infected controls. (See later)

Mycoplasmas can act as polyclonal T cell and B cell activators and some can trigger macrophages in vitro.

Monocyte derived elastase can cleave 83kDa RNAseL thus causing deregulation of the anti viral pathway.

These authors describe a 37kDa product, which is present in large amounts, and also a depletion of the 83kDa form.
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It is quite possible that particular people have higher burdens of mycoplasmas than others do.

We need to be quite clear that help will come when we can define the difference between resident mycoplasmas in normal people and some sort of pathological consequences in some people with higher mycoplasma loads.

Mycoplasma pneumoniae uses specific long chain sialo oligo saccharides at host-cell surface as receptors. (See also in the glyco protein section).

Autoantibodies can be a response to I antigen in this molecule on bronchial epithelium or on red cells. This is a factor in the cold agglutinins found in acute infection, and may be a part of other immunological changes.

One thing coming out of the Newcastle work is the possibility that there are different lipid patterns and in the case of mycoplasmas the possibility of very low serum cholesterol levels.

Chlamydiae are also obligate intracellular parasites possessing RNA and DNA, a cell wall and ribosomes similar to gram negative bacteria. They also lack a discrete nucleus.

Particular attention has been paid to its presence in some 18-70% of coronary artery plaques and more recently in a connection to bronchial asthma.

Chlamydia trachomatis causes a corneal disease (trachoma) and genital infection, as well as triggering Reiter's syndrome (perhaps in genetically vulnerable individuals).

In pelvic inflammatory disease in USA, Chlamydia trachomatis has been found in endometrium and fallopian tubes in up to 50% of cases.

So far chlamydiae have not been implicated in chronic fatigue syndromes.

Larger bacteria are a specific kind of organism where the cell membrane is surrounded by a specific cell wall.

This is another difference from eukaryotes, and is relevant to one role of some antibiotics.)

For example, the Gram stain allows separation of gram positive from gram negative organisms.

Other names are given in relation to some of the in vitro actions of organisms or their products.

Cell walls of gram positive bacterial contain teichoic acids which are phosphate linked polymers of ribitol or glycerol that can have additional compounds linked to available side groups.

The various substituents of teichoic acid are often responsible for the biological and immunological properties associated with disease due to pathogenic gram positive bacteria.

Most pathogenic gram positive bacteria have additional extracellular structures. These include surface polysaccharides such as the group antigens of streptococci, capsular polysaccharides and surface proteins and polypeptides.

Gram negative bacteria have a similar cytoplasmic membrane and peptidoglycan layer somewhat reduced compared with gram positive organisms. They are characterized by outer membrane that is covalently linked to the tetra peptides of the peptidoglycan layer by lipoprotein.
The outer layer of the outer membrane contains the lipopolysaccharide constituent which includes also special proteins which are important to the function of the gram negative organisms.

Some specific things that bacteria do include colonization of host surfaces and depending on the organism, invasion of tissue with sometimes specific tissue tropism.

Exotoxins
Some bacteria produce exotoxins, for example diphtheria, botulism and tetanus. Also pathogenic gut organisms can produce enterotoxins.

Most people are familiar with the fact that some staphylococci can produce a toxin associated with what is called 'toxic shock syndrome'.

Endotoxins
The lipid A portion of gram negative lipo-polysaccharide has potent biological activities capable of producing severe illness, particularly in the circumstance of gram negative bacteraemia. This effect appears to be mediated by production (from mononuclear cells) of IL-1, TNF alpha and perhaps IL-6.

Since September and October 2001, I have been checking my CFS patients for rickettsial infection.

The results suggest that Dr Cecile Jadin in Johannesburg, South Africa is identifying an important connection to some of the cases of this crippling condition.

It seems that she has found some 3.600 patients with chronic fatigue, fibromyalgia and related conditions, who test positive for rickettsial serology.

I am therefore expanding the section on rickettsiae.

Phylogenetic data suggests that mitochondria originated from a family of alpha-proteobacteria called rickettsiales.
This high ranked taxon consists of exceptionally obligate intracellular endosymbiants of eukaryotic cells. The family includes rickettsiaceae and rickettsia-like endosymbiants.

A long term mutualistic relationship between these intracellular bacteria with a pro-eukaryotic line of cells appears to have given rise to eukaryotes where 80% of cell energy is derived from the mitochondria. The invader must have lost many of it's dispensable genes, and logically it was so integrated with the eukaryote that it joined, that when much later multicellular systems evolved immune systems it behaved as self and evoked no immune response, and as well conserved and evolved it's DNA.

Mitochondrial DNA is transmitted from generation to generation through the maternal cytoplasm in the ovum, since any small remnants of sperm mitochondrial DNA is lost as the sperm enters the egg.

This old organism has thus become the eukaryote's main energy source and includes the evolution of carrier proteins to exchange respiration derived ATP for host metabolites.

They seem to range from organisms that cause very severe and even fatal diseases (Typhus Fever and Rocky Mountain Spotted Fever) to moderately disabling and less severe chronic disease states.

This is a family of gram-negative coccobacilli and short bacilli that grow strictly in eukaryotic cells.

That remote origin some 1.5 to 2 billion years ago has led to a stream of intracellular organisms in birds, reptiles, and mammals that may have very variable consequences for the host cells.

The cell provides a rich source of nutrients and rickettsiae have developed a number of special mechanisms to transport nucleotides and nucleotide sugars.

They express an ATP translocase, enabling them to obtain stored energy and depriving the host cell of that energy

There seem to be points in the degree of proliferation where cells can be more seriously damaged, and this is different in Typhus (proliferation within the cell and at a certain stage cell lysis), compared with Spotted fever group rickettsiae where organisms spread rapidly from cell to cell via actin-based motility, without cell lysis (Hackstadt)

Rickettsiae tend to be transmitted by insect or tick vectors.
It is likely that the arthropod vectors are particularly important in the natural maintenance of the pathogenic rickettsiae.

In the models of disease, which I am using in this paper, exploration of the co-evolution of host-pathogen relationships is central.

In this regard, efficient pathogen replication and endurance or maintenance are important as is transstadial and trans ovarial transmission (in the arthropod vector).

I want to emphasize that rickettsiae can cause both acute and chronic diseases. The latter however has scant documentation.

Clearly they are recognized by immune cells, and can evoke T cell activation as well as B cell antibody production.

Some of the diseases are very severe (eg Rocky Mountain Spotted Fever) caused by R rickettsii.

I think it will emerge that we have overlooked the prevalence and significance of rickettsial diseases.

These organisms possess two major immunodominant surface exposed proteins, called outer membrane proteins (rOMP A and B)

rOMP A functions as an adhesin for the host cell, and rOMP B shares genetic sequences and limited antigens with other Spotted Fever and Typhus group rickettsiae.

DH Walker and colleagues have been studying mechanisms of pathogenicity for many years. (Currently in Galveston, Texas, USA)

Rocky Mountain Spotted Fever is an important diagnostic condition in the Americas.
We are less sure of the full range and associations of the ongoing presence in cells of various rickettsiae.
Attention is being directed at exactly how the immune system deals with intracellular organisms.

Spotted Fever Group
Rocky Mountain Spotted Fever, (R rickettsii) Vectors: various ticks

This is a severe rickettsial disease, documented in 48 of the states of the USA, Canada, Mexico, Costa Rica, Panama, Colombia and Brazil.

After inoculation by the tick bite, it spreads lymphohaemotogenously throughout the body, attaches to endothelial cells, escapes from the phagosomes and replicates intracellularly. Its spread from cell to cell involves polar polymerisation of the host cell's actin. It also invades vascular smooth muscle cells.

The pathological consequence is mainly increased vascular permeability with local oedema, ischaemia and hypovolaemia.

Serological positivity by indirect immunofluorescence is very specific, but may not happen early enough, so that the only useful test in the acute phase is skin biopsy of the an area of rash, with immunohistological studies.

Readers can look at the various clinical presentations and differential diagnoses in medical textbooks.

Mediterranean Spotted Fever, (R conorii) Vectors: several ticks

This is found in the Mediterranean area, Southern Europe, Africa, and south west and south central Asia.
In Spain 58.6% of dogs tested had antibodies to R conorii. (Herrero and colleagues), while in Zimbabwe and South Africa, 34% and 19% of domestic cats tested positive for the same antibodies. (Matthewman et al)
It has varied from very severe forms to milder diseases.
Fever, rash and a lesion at the site of the tick bite are common.

Rickettsialpox, (R akari) Vector: mites

Found in USA, Ukraine and Slovenia
A lesion at the site of the mite bite is followed by local lymph node enlargement, fever, headache myalgia and usually a maculopapular rash.

Queensland Tick Typhus (R australis) Vectors: ticks

Found in Queensland, Australia and along the eastern seaboard of Australia.

Flinders Island Spotted Fever (R honei) Vectors: ticks

Found in Flinders Island in Bass Strait, between Australian Mainland and Tasmania, but recently in South Australia

In 2003-3,Dr Stephen Graves has isolated R Honei from 3 patients with a febrile illness, typical spotted fever type rashes, cough and abnormal liver function tests, admitted to Flinders Medical Centre in South Australia.
This is the first confirmation of R honei on mainland Australia.

Australian Tick Typhus (R marmionii) Vector not established
Found in a similar case south of Adelaide with acute spotted fever like illness, and subsequently in cases in Queensland, Australia) The gene sequence is different from other spotted fever rickettsiae.

(This name acknowledges 40 years of work on Q fever done by Professor Barrie Marmion, and particularly his evidence of a post Q fever variety of chronic fatigue syndrome.)

This information alerts Australian doctors to be aware of rickettsial diseases in Australia.

North Asia Tick Typhus(R sibirica) Vectors: ticks

African Tick-bite fever (R africae) Vector ticks
This is present in South Africa, Zimbabwe, Tanzania, and Guadeloupe in the Caribbean

Typhus Fever Group

Epidemic Typhus, (R prowazekii) Vectors: human lice

This has a world wide distribution.

Unlike other arthropod vectors, the louse does not pass the organisms to it's offspring.
This is a severe illness coming about one week after inoculation, manifesting as high fever, prostration, severe headache, cough and myalgia.
A rash is usually prominent. Twelve percent have neurological involvement.

There is a fascinating recrudescent, mild form of epidemic typhus (Brill-Zinsser Disease) occurring years after the acute disease, possibly relating to a decrease in immune function. It is a good example of the capacity of these organisms ability to survive, presumably in a dormant form, for very many years.

Murine Typhus, (2 types)(R typhi) Vectors: fleas (usually rat fleas)

Worldwide in distribution.
The animal reservoir is the rat and transmission by bite of infected rat fleas involves the scratching of the bite contaminated by the flea faeces.
Aerosolised flea faeces are also suspected to play a part. (Circumstantial evidence suggest this may be inhaled, without there being a flea bite)

Headache, myalgia, arthralgia, malaise, nausea and fever occur, often with some rash. Thirty five % of patients develop a hacking cough.

Laboratory tests can reveal anaemia, leucopenia (early) and leucocytosis (later), thrombocytopenia, abnormal liver and kidney function.
With lung involvement some patients can require intensive care.
The illness is often milder in children.
Interest has also arisen in a variety of rickettsia called R felis, transmitted by cat and opossum fleas.

Scrub Typhus (R. (renamed Orientia)(tsutsugamushi) Regarded as a different group, because of different cell wall characteristics and genetic makeup. Vectors: mites in chigger (larval) form.
This is found in Asia, Australia, New Guinea and Pacific Islands (principally in tropical regions)
It is a of variable severity involving fever, headache, myalgia, cough and gastrointestinal symptoms, and regional lymphadenopathy near the bite.

The limited descriptions I provide here are to illustrate the best documented varieties of acute rickettsial diseases.

There are at least 30 different strains of rickettsiae, and classification as well as exploration of pathologies relating to them is still unfolding.

According to current evidence, R australis, R felis, R honei and R marmionii are known to occur in Australia. Historically Typhus fever has also occurred in Australia, and Dr Stephen Graves has recently confirmed a case in Melbourne.

Although I have spelled out some material on Rocky Mountain spotted fever for world wide readers, it has never been found in Australia.

The other related but distinctly different example is Q fever, where the organism has been named Coxiella burnetii and is known for its ability to survive for long periods outside of the reservoir or any vector.

This latter organism is also known for it's high level of infectiousness. It seems to be one which does not need an insect vector to transmit it.

That does not exclude the possibility that close contact with pet animals may not some times result in inhalation or swallowing of insect vector faecal pathogens or shed viruses.

Avoiding unduly close oral contact with animals and their fomites would seem to be prudent.

It has been reported that in the town of Roma in Queensland that 1/3 of the population test positive for Q fever serology.

There is good evidence that we are not looking closely enough for Q fever in rural Australia,and visitors to farms and processing locations can be infected by breathing in these pathogens.

Up to 10% of Q fever cases emerge with post Q fever fatigue, and these tend to have raised levels of IL6. Professor Marmion found the organisms in the bone marrow of this group of patients.
The HLA haplotypes of this group appear to be DR11.

We can see this as an example of the various expressions of a disease in different individuals.

Cecile Jadin, a Belgian doctor now working in South Africa, has written an important document about connection between rickettsial diseases and CFS as well as fibromyalgia syndromes.

In her book "A Disease called Fatigue", Dr Jadin describes the high probability that many Rickettsial diseases are transmissible by breathing, swallowing or contamination of abrasions or cuts by organisms.

The evidence is strong that doctors should be vigilant in checking for this disease, and also that post infection fatigue is one of the important possibilities.

Prof Andrew Lloyd at U of NSW, is following some of these cases in an prospective study.

Rickettsiae have many attributes, which make them a likely candidate for persistence and causation of chronic fatigue syndrome/fibromyalgia syndromes.

The intracellular location means they can adversely affect cell metabolism, such as scavenging ATP. They are also not so susceptible to immune system activity except the degree to which T cell and macrophage activity can evoke particular cytokine responses.

Phospholipase A2 (PLA2) activity evoked by the typhus group rickettsiae causes haemolysis in vitro, and may mediate entry into the host cells, as well as escape from the phagosome.

With both Typhus and the Spotted fever group, the PLA2 may contribute to cell injury by increasing free arachidonic acid being susceptible to oxidation and the production of inflammatory prostaglandins.
This has therapeutic implications, since a PLA2 inhibitor (bromophenacyl bromide) decreased cell injury and inhibited the release of free fatty acids. (Walker et al)

Rickettsiae (eg R.prowazekii) can be included in a group of intracellular pathogens which possess specialized secretion systems which they use to subvert host defences.

The class called typeIV have homology to the conjugal transfer systems of naturally occurring plasmids, and use them to export toxins.

Other organisms that have these systems are Legionella pneumophila, Coxiella burnetii and Brucella abortus. (Sexton & Vogel)

Study of microbial genomes provides insights about their survival in host cells.

Reductive evolution has led to dispensing of genes which cover overlapping pathways but high priorities for genes which lead to more effective immune system evasion. (R prowazekii has a genome less than a third the size of E Coli.) (Palmer)

Intracellular pathogens can target macrophages, and increase the production of certain cytokines such as interleukin10.

The survival of organisms inside the macrophage, an antigen processing cell, is helped sinceIL10 is a potent immune suppressive factor (Red path et al)

Rickettsiae cause vasculitis, which seems to vary in degree in different family members.

The endothelial cell is a primary target for R conorii and George et al found increased levels of circulating endothelial cell (EC) and EC fragments, plasma thrombomodulin and von Willebrand factor in 12 patients with Mediterranean Spotted Fever.

Rickettsiae certainly make toxins (eg haemolysins) but there is very little literature on this.

There is evidence of a gene in R typhi, which codes for a haemolysin.
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Vasculotoxic and neurotoxic substances seem most likely.

The claim about neurotoxin is an interesting one, since such a toxin has been identified in borreliosis, and accounts for muscle pain, stiffness and spasm.

In a few it seems to produce a" stiff-man syndrome". It would be interesting to test all future cases of the "stiff-man syndrome" for rickettsiae, and those who have been ill since being in other countries, for borrelia serology.

In my literature search I could not find evidence for a neurotoxic rickettsial product, but there is very good evidence for neurological abnormalities in acute and chronic rickettsiosis.

The cerebral perfusion abnormalities found in CFS sufferers on SPECT scanning by Drs Reynold Casse, Richard Burnet, and Alex Kwiatek in Adelaide may relate to these pathological changes, local neurone injury, or to autonomic instability.

Here in South Australia, I have recently started testing my CFS/fibromyalgia patients for rickettsial serology and am finding more than 60% are positive.
for the spotted fever/typhus fever group,

If we break them down by titre 16 are +ve at 1/1024,
49 are +ve at 1/512
140 are +ve at 1/256
128 are +ve at 1/128
Dr S Graves of the Australian Rickettsial Reference Laboratory thinks that the 1/256 or greater are probably true positives and 1/128 are impossible to interpret.

I am aware of 427/822 CFS patients with positive serology tested by other SA doctors.

Two positives are the mothers of sero positive CFS patients. Both are fatigued, but may not fill the international criteria of CFS

This work will need to be clarified as the Geelong based Australian Rickettsial Reference Laboratory does its routine serology against the whole organism and doesn't report the separate IgG and IgM antibodies.

The antibodies to cell wall antigens of rickettsiae cross react to other members of the group (eg all of the spotted fever group and typhus group) we need to know whether there are other organisms which can give rise to positive rickettsial serology.

We intend to explore this further by checking for rickettsial DNA by PCR, and by culture from the buffy coat of blood collected in EDTA tubes.

We have not found positive PCRs for rickettsiae in any chronic fatigue patients.

If these organisms are like coxiella (Q fever) or like borrelia (Lyme disease) they may be difficult to find outside of tissue biopsies.

We will also try to do control and blinded studies to assess the prevalence of Rickettsial disease in the South Australian community.

So far it appears that in South Australia, the serological test distinguishes the CFS sufferers from other people, suggesting that there is a distinctive form of chronic rickettsiosis in SA. It may emerge that the clinical illness often has a mild beginning.

We do not know why these people are testing positive, and what this implies about past or present rickettsial disease.

I will spell out the desireable therapy for rickettsial disease in the section on management.

We do not know why a subgroup appear to respond promptly to one or more courses of antibiotics, why some relapse and others show little response or get worse despite multiple courses.

Until we have a reliable PCR or successful culture techniques available to us in South Australia, we will continue to fly blind.

By this I mean that the evidence is indirect and the reason for the symptoms unclear.

This fascinating disease covers a spectrum of clinical presentations as well as illustrating how the discovery of an infective agent can be overlooked for many years until a much more thorough exploration leads to indirect, then direct evidence of its presence.

I am providing this segment for readers outside Australia, but it also provides insights into new disease paradigms.

Lyme is a city in Connecticut, USA, located in an area where the local fauna include a white-footed variety of mice, local foxes, and white tailed deer.
(Interestingly, white tailed deer have recently been found to suffer from a prion disease akin to bovine specific encephalomyelitis)

A tick called Ixodes scapularis carries the organism from animal to animal.
This tick exists in three stages
(1) A 6 legged larva, which hatches in midsummer and seeks hosts such as the mice, then growing until it falls off, remaining dormant through winter.
(2) In spring it moults, emerging as an 8-legged nymph which can attach to small mammals or human being who happento come by. The nymph is the main vector for human Lyme disease.

3) It becomes much larger as it feeds, and again falls off, lying dormant until the next summer when it moults and becomes the adult 8 legged tick usually biting passing white tailed deer. These adult ticks have a sexual life synchronized within their feeding. The male is stimulated to make large numbers of sperm, as the female makes thousand of eggs, and the copulation gives rise to eggs, which are probably borrelia free.

Thus the reinfection from the hosts is important in the life cycles of the germs.

Borrelia burgdorferi, the causative organism organism is a fastidious microaerophilic spirochaete, which has many immunogenic proteins.

It has a slender spiral organism with a single chromosome carrying 853 genes.
Within the non-nucleoid area of the organism are plasmids carrying a further 430 genes.

In its usual form there is a double-layered cell wall.
The outer wall is coated with a protective "slime" of carbohydrate composition.
From the cytoplasm flagellae protrude through the inner wall and wrap themselves within the space between the inner and outer walls.

Some of the plasmid genes are replicas of chromosomal genes.
Some code for immunogenic proteins (peptidoglycans) while others control attachment mechanisms.

Outer surface proteins called OSP A and B are expressed in the tick intestines and OSP C is upregulated as the germ traverses the tick salivary gland.

The significance of evolutionary changes has been recognized in both hosts and vectors as well as in the bacteria.
As with rickettsiae there has almost certainly been some reductive evolution involving loss of genes due to adaptation to the use of host cell mechanisms.

There are fascinating pathogenic mechanisms which reveal to us how our old ideas of mechanism have been too limited.

Human plasminogen and urokinase type plasminogen activator are bound to the spirochaete surface leading to plasmin production.

The organism has some tropism to skin, neural, and A-V nodal cells, and joint components, with ability to persist in these sites.

Borrelia burgdorferi adheres to host integrin receptors in extracellular matrices, and to vitronectin, fibronectin and matrix glycosaminoglycans, as well as heat shock proteins.
In germ to germ contact plasmids may be transferred from one bacterium to another, and this increases genetic diversity.

It can also develop large cyst like bodies in which small spirochaetes can develop and then be released.

In variants called L forms, they may lose the spiral shape, become deficient in cell wall (hence losing susceptibility to amoxycillin and other cell wall inhibiting antibiotics) and exhibit reduced activity, yet still surviving.

Like Coxiella burnetii (Q fever organism) and rickettsiae, the difficulty in curing all cases, has been well documented.

Cultures from blood are almost always negative, but cultures are positive in tissue specimens from sufferers.

Stage 1
The early local red skin lesion at the site of the tick bite (this is called erythema migrans),

Stage 2
A disseminated infection with severe malaise, fever, severe aching, headache, stiff neck and fatigue and often secondary annular skin lesions.
There may also be enlarged lymph nodes, cough and conjunctivitis.
About 15% have meningitic, encephalitic and neuritic features, and 8% A-V node or myo-pericarditis. Sometimes mood sleep and memory disturbances are present.

Stage3
. Months after the onset some 60% of untreated cases develop oligoarticular arthritis, (for a time in the past, children were diagnosed erroneously as juvenile rheumatoid arthritis)

HLA DRB1 *0401 and *0101 alleles may mark a human vulnerability to arthritic responses.

The immune response to this infection is local (biopsy shows infiltration with plasma cells and lymphocytes with some vasculitis)

Several detect weeks may be needed for the specific IgM antibody to rise, with croprecipitates, and circulating immune complexes eg. IgG rises later (months)

Tissue biopsy with culture and PCR and enzyme linked immunoabsorbent assay (ELISA) or Western blot testing may be helpful.
It is claimed that 15% of patients treated with antibiotics get a Herxheimer reaction, usually in the first 24 hours of therapy.

A primary infection due to borrelia contracted in Australia has yet to be documented.

Three of my patients with CFS who have travelled widely have positive borrelia serology.

The Bartonella species are tiny gram negative bacilli which can adhere to and invade mammalian cells. They can come from cat scratches and also tick bites. (Bartonella henselae) and they too can to produce chronic fatigue and fibromyalgic syndromes.

Ehrlichiae are small, obligately intracellular bacteria, which can cause human disease with fever, headache, myalgia, malaise, gastrointestinal upsets, cough, rash and confusion. Professor Tim Roberts and colleagues at the University of Newcastle have documented Ehrlichiae in Australian dogs.
Two of my patients have positive erhlichial serology.

This is usually a tick borne protozoan disease of animals, but can be seen in USA as a febrile illness, with aching and fatigue.

I feel that the evidence is strong that infection and its concomitants are crucial to the on going CFS.

Specific testing may require particular laboratories, and careful consideration of the implications of the results.

I really think that CFS is connected to consequences of injury evoked by intracellular type organisms such as the herpes family of viruses(eg CMV or a mutated form of CMV and HHV6 ), mycoplasmas, chlamydias, rickettsias, borrelias, ehrlichias and perhaps other organisms,and the body's responses to these events.

The organism behind estuary syndrome (pfiesteria) produces a neurotoxin, and possibly small molecular weight toxins from some of these organisms may turn out to explain the majority of these disorders.

Ritchie Shoemaker MD of Pocomoke in Maryland, USA writes extensively about toxins.

I quote Shoemaker,
" Best defined as causing "chronic, neurotoxin-mediated illness," these little understood diseases make people sick by producing low molecular-weight toxins (aka, "ionophores") that "hide out" in the body's fat-containing tissues, where they remain impervious to the germ-fighting "antibodies" which endlessly patrol the human bloodstream."

He suggests toxin-mediated disorders as part of Chronic Lyme Disease, Sick Building Syndrome, Chronic Fatigue, Chronic Soft Tissue Injury and several waterborne maladies involving toxin-forming blue-green algae and one-celled dinoflagellates, including toxin-forming ciguatera and Pfiesteria.

--Blue-green algae, such as rapidly reproducing microcystis and cylindrospermopsis.

In the therapy section I will write about how we might manage toxin-mediated diseases.

I have been in dialogue with Trevor Marshall, PhD,now in California, and we are slowly unravelling information about cell wall deficient and L form bacteria which both evade immune mechanisms and may evoke unwanted responses.

The theme is a range of cell wall deficient or L forms of bacteria, which evade immune mechanisms and evoke either TH1 or TH2 responses.

We perhaps should look for L forms of bacteria by live blood examinations at 8-10,000x magnifications, (ear lobe blood) and by special stains as well as immunofluorescence..

I have 4 DVDs of a Chicago Workshop/seminar where an absolutely amazing microbiologist shows extensive slides of these special stains.

Ear lobe blood seems to allow better numbers of bacteria to be seen than venous or finger tip blood.

"We are all here to treat each other well in order to awaken our best help for each other!"

Activated macrophages possess the enzyme 1 alpha hydroxylase to turn 25 hydroxy D3 into the 1,25 dihydroxy D3, and Marshall claims that this aids the survival of the bacteria.

Marshall states that this is very significant in marking a TH1 type immune response and claims they are responses to these cell wall deficient bacteria located in macrophages.

Another fascinating finding is that Marshall finds that the use of olmesartan, an angiotensin 2 receptor blocker seems to improve some cases of CFS and sarcoidosis.

I mention this here because this has invited us to look at the interplay between bactreial evolution and survival mechanisms as well as the particular immune responses of individuals.

Angiotensin 2 receptor blockers (ARBs) are vasodilators, but A2 receptors
are used by some organisms as entry points.

Angiotensin is avaso- constricting product from liver precursors, but it seems now clear that it plays a role in some inflammatory states, perhaps by changes in its receptor.

Marshall suggests that the A2 receptor may alter with inflammation and one A2R blockers is anti-inflammatory and are definitely able to stop NF kappa beta activation, thus decreasing transcription of the TNF alpha gene, and decreasing this inflammatory cytokine.

Another ARB (telmisartan) is peroxisome proliferation activation receptor agonist, (it has about one third the PPAR agonist actions of pioglitazone) and possesses the capacity to up regulate gene activity and decrease insulin resistance, as well as being anti-inflammatory. Although telmisartan, has been demonstrated to decrease inflammation in vessel locations, it may not work so well in the TH1 set CFS situation.

It far exceeds the others in blocking the inflammatory evoked portion of the receptor.

THIS IS WHY WE MUST ALL, PETITION THE AUSTRALIAN GOVERNMENT TO ALLOW PFIZER TO BRING THIS AGENT INTO AUSTRALIA FOR THIS SPECIFIC PURPOSE!!

Minocycline and azithromycin are better than doxycycline, in dealing with cell wall deficient bacteria, because of better intra cellular penetration.

In this situation dosing must follow Marshall's recommendations, and I will document this in the chapter on therapy.

Each of these medications has capacity to decrease matrix metalloproteinase activity as well.

Since true vitamn D deficiency has been well documented, and there are biological disadvantages in the target organ consequences, we dare not undertake the Marshall protocol without good physician care and following of blood levels of 25 hydroxy D3 and 1,25 dihydroxy D3 along with parathyroid hormone levels.(PTH)

We may not be able to tell when the 1,25 dihdroxy D3 comes from macrophages and when from renal tubular or other cells.

We need to ay attention to the hydroxylases involved in D3 conversions as well as thinking about what further homeostatic consequences there are in people with these disorders.

Wen we plan regimes we need to think deeply about the homeostatic responses to what we are doing.

Readers who have pharmacological knowledge will realize that these hydroxlases are members of the cytochrome P450 family of mixed function oxidases and hydroxylases located in the smooth endoplasmic reticulum of cells.

We need to think about interactions with drugs metabolized by CYP isoforms,as well as herbal, hormonal and natural metabolisms of endogenous substances in our bodies.

Doses of olmesartan which are 3-8 times usual antihypertensive doses,may be dangerous to some people and fluid intake must be assured, with BP and renal function monitoring.

People need to be warned about dizziness and be prepared to stop the medication if it persists.

Candida albicans is common yeast found in the soil and water and can give mostly low-grade clinical disorders such as inflammation in the mouth and vulva. It is more likely to over grow in these sites when antibiotics change normal flora and also in diabetes, severe renal disease, and malignancy and immune deficiency syndromes.

In these latter circumstances systemic invasion may occur.
Despite the many claims, there is no hard evidence that candida causes chronic fatigue syndromes.

Nevertheless it would be foolhardy to disregard clinical experience where people seem worse when exposed to yeast or who report clinical episodes of candidal infection.

Dunstan and colleagues at the University of Newcastle, (New South Wales) and the University of Sydney, have been searching for changes in chronic fatigue syndromes. believe they are able to identify particular urinary amino acid, organic acid, and metabolite excretion that they believe correlates with the presence of toxin producing coagulase negative staphylococci. This research compares CFS sufferers with healthy medical students.

We are fortunate that some researchers have dared to enter a field of study to identify whether there are microbial and biochemical changes in these syndromes.
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Coagulase negative staphylococci can be identified in the anterior nares (just inside the nostrils), the perineum and other moist body locations. It appears that there are more isolates in people with chronic fatigue than in the population at large.
These researchers have gone on to identify specific toxins alpha, beta, gamma and importantly delta/"horse" toxin (haemolysin) in these people. These toxins are haemolysins. The toxins may have effects at locations which they can reach. The level of such toxins correlate with pain levels in the subjects studied.
In females the vulva may also harbour staphylococci.

Other aspects of staphylococcal colonization include the possibility that these organisms can make materials called "super antigens" which are potent activators of immune cells and cytokine production.

Specifically these researchers believe metabolites in the urine of chronic fatigue sufferers (labelled C.F.S. urinary metabolites CFS UM 27 and 28), and a high urine tyrosine/leucine ratio correlates with pain in chronic fatigue and also with the presence and magnitude of coagulase negative staphylococci.

The tyrosine/leucine ratio is a measure of non-fibrillar protein breakdown and may be initiated by interleukin 1 (IL1) and tumour necrosis factor (TNF).
Tyrosine comes from muscle in catabolic states. It can be recycled by the body.
Of course some tyrosine may be of dietary origin.

LINK WITH BACTERIA

Scandinavian researchers have implicated coagulase negative staphylococcal materials in chronic fatigue associated pain (and in other chronic pain syndromes). A possibility is that the above toxins may have direct or indirect effects on target cells such as muscle cells.

About 40% of the population carry staphylococci in the anterior nares.

Virtually all of us have some of these organisms somewhere on or in our bodies but it may be that a proportion of CFS sufferers are living with a higher burden of organisms. Perhaps their neutrophils do not destroy this part of their flora or they have factors conducive to staphylococcal survival in these sites.

Some 10% of women have vaginosis a situation where organisms in the vagina produce mild local changes (including a rather fishy odour) and these people are an example of a sub group who are living with some undesirable organisms. This is not necessarily the same group that carry gram negative staphylococci.
For some who do have visible lesions there may be reasons for the recurrence or persistence of infection.

When the neutrophils of some people who have recurrent pimples and furuncles are tested against their own staphylococci they fail to kill them but are effective against other staphylococci. (McDonald)

In vitro, neutrophils from other people are perfectly able to kill the staphylococci of the furuncle sufferer. It may be something of this kind that accounts for the Newcastle findings, but it is likely to be a different mechanism.

These have not been incriminated a causes of chronic fatigue syndromes, but the following information is important to health professionals in grasping the role of microorganisms in the larger picture of life on earth.

Carriage rates of coagulase positive staph are a least 30% of healthy adults with the anterior nares, axillae and vulvo vaginal areas in women and perineum of both sexes being the common carriage places.

More often than not abscesses come from that individual's own flora, where the organism is an opportunist to such things as blocked sweat or sebaceous ducts or perianal gland ducts or even a small tear (e.g. anal region)

Staphylococci can colonise and invade tissue, attaching to both cells and extra cellular
matrix materials and have many ways to evade or counter host defences as well as
damaging the local tissue. Staphylococci are survivors.

In order to discover how to tip the balance against them I note the mechanisms by
which they cause the injury as follows: -

1. Adhesion to extra cellular matrix through adhesins. Adhesion occurs to
fibrinogen, fibronectin, collagen and elastin.

2. Genes within the staphylococcus write the code for the production of
enzymes such as coagulase which binds to prothrombin and promotes
conversion of fibrinogen to fibrin. In the meshes of the fibrin, the staphylococcus is relatively protected from the host mechanisms which try to destroy it.

Other enzymes such as hyaluronidase break up hyaluronic acid
molecules facilitating local spread and a series of other proteases
contribute to tissue destruction.

3. Other staphylococcal products include membrane-damaging toxins such
as alpha, beta and delta haemolysins and some of these toxins specifically
injure membranes destroying their integrity. One particular example is
a leukocidin acting against polymorphs, monocytes and macrophages
and can injure mucus membranes and skin.

4. Staphylococci in colonies elaborate biofilm, which further serves to
protect them from host defences.

5. Staphylococci can produce a catalase which converts hydrogen peroxide
to oxygen and water which interferes with oxygen free radicals.

6. Staphylococci can survive in non dedicated phagocytes and small
colony variants can also form which are relatively resistant to
cell wall active antibiotics and also to aminoglycosides and can persist
inside cells for very long periods of time.

7. A fascinating part of staphylococci is the existence of super antigens
which bind directly (and without prior processing) to MHC class 2
molecules on the surface on antigen presenting cells, stimulating T cells
and the release of cytokines IL 1 & 2, tumour necrosis factor alpha and
interferon gamma.

Crucial to the host defences are capacities to immobilise and destroy the organisms and the function of neutrophils, generation of complement and coating of the organisms by specific antibody play a major part. Staphylococcal peptidoglycan activates complement and of course complement punches holes in the staphylococcal cell wall allowing it to be destroyed.

Antibodies markedly enhance these actions. Specific defects in the host can include
inadequate neutrophil numbers and functions impaired complement generation and lack of anti staphylococcal antibodies. Rarely people are deficient in IgG, IgM or IgA.

Professor Peter McDonald pointed out to me that in people with persistent staphylococcal infections their own neutrophils are ineffective in destroying their own staphs but maintain ability to destroy other peoples staphs. Yet other peoples' neutrophils can destroy the patient's staphs.

Once a fistula or sinus forms it is a nidus for ongoing infection and co-existence of other pathogens and there is no shadow of a doubt that its excision is crucial to recovery.

I may follow some or all of the following to deal with persistent staphylococcal infections.

1. Supplements of Vitamin C of the order of 250mg 6 times a day, zinc as
zinc amino acid chelate equivalent to 15mg of elemental zinc per day.

Healthy function of neutrophils depends on adequate amounts of these
nutrients.

2. Lactoferrin. This is a series of glycoproteins found in milk and
particularly in colostrum and kills staphylococci and enteropathogens
while protecting normal flora. In particular it breaks up biofilm and
also renders iron unavailable to iron requiring bacteria.

3. Transfer factor. These are a series of colostral polypeptides of about 40
amino acid sequences which transfer immunity from the mother to the
infant and can be used to transfer immunity to naive subjects. As
well transfer factors stimulate T natural killer cells and also down
regulate auto immunity. The standard dose is up to 6 x 50mg capsules
a day.
Bovine transfer factors presumably only provide protection against organisms which have entered the cow! They do appear to stimulate T natural killer cells.

4. The regular use of lactobacilli particularly including bifidus organisms
which support healthy colonic flora as well as cell function.

5. Possibly the use of Bio-strath elixir which is derived from plasmolysate of Saccharomyces cerevisiae. (evidence base not established)

6. In the case of dealing with abscesses.
Probably a course of clindamycin 150mg 6 hourly in the few days before
the draining procedure. Clindamycin has good penetration in to walled off
sites, a property also shared by fucidin.

7. The use of intra nasal mupirocin (Bactroban) bd for patient and closest
contacts.

8. Meticulous washing of the hands after touching the nose and after
any toileting is important as is washing all handkerchiefs in
"Napisan" or similar and even dipping trouser pockets into "Napisan"
and washing hands after disposal of any tissues.

9. In some parts of the world anti staphylococcal vaccines have been
successfully used to decrease the risk of staphylococcal infections.

10. Supplements with omega 3 fatty acids such as the use of flax seed oil and various fish oils.
11. Use of probiotics. (See below)

It is possible that either mannose or the acetyl mannans from Aloe Vera can interfere with galacto lectins which are adhesion factors for staphylococci.

The subject of bacteriophage specific killing of resistant organisms could bear a lot of fruit in the next decade.

(a) Thus in bacterial structure and metabolism there exists the possibility of injury from produced endo and/or exotoxins, cell wall antigens, and other products which result from an interplay of cells such as poly morphonuclear leucocytes and the bacteria they destroy.

(b) Gut Flora. In the intestines there exist vast numbers of bacteria most of which are ordinarily non-pathogenic. Indeed evidence supports many protective functions from this flora.

At present there is some investigation into the subject of the varieties of normal and abnormal gut flora.

Henry Butt of the University of Newcastle NSW has reported that it is very common for CFS/fibromyalgic subjects to have a low count of E Coli in faecal flora.
As well counts of enterococci were high, and this correlated with cognitive dysfunction.

Dr T Hey of Buckeburg reports that researchers in the virology department of the Medical College in Hanover, Germany (Breull, Fischer and Verhagen) have found that >80% of fibromyalgic sufferers carried bacteriophages which infect and destroy large numbers of E Coli.

The researchers further claim that with lysis of the coliforms, much lipopolysaccharide is released with adverse effects on intestinal cells, and also through absorption, on other body cells.
This could easily be a cyclic process so that the flora could fluctuate and the lysis have peak times paralleling the days of worse pain.
It might also explain why some CFS/fibromyalgia sufferers get adverse reactions when antibiotics are given.
Butt and colleagues also report patients with an overgrowth of other aerobes such as enterococci or other unidentified organisms.

It is possible that gut flora changes are associated with intestinal cell abnormalities which could alter absorption of required nutrients, and allow larger molecules to cross the gut lining. (A "leaky gut".) As well, altered flora could mean abnormal fermentation and its consequence as well as a deficit in required amino acids. Both decreased synthesis (eg serine by E coli), and increased intestinal cell catabolism of needed amino acids could decrease amino acid availability for other body cells.

In addition increased excretion of 3-methylhistidine is associated with initiation of fibrillar muscle protein breakdown (actin and myosin). (Associated with increased IL1 and IL6.)
3-methyl histidine cannot be recycled as transfer RNA (tRNA) does not code for methylated amino acids such as methylated histidine or lysine.

Microbiology is a field where there is emerging re-evaluation of the roles of microorganisms in diseases which were not originally thought of as infective.

This reviewer of the literature feels that there is a vital need for patients who have one or more identifiable abnormality to be screened by the other major world's researchers to discover just how many abnormalities there really are.

This chapter has raised my own consciousness of the amazing properties of life at microbial levels and the equally amazing range of host responses!

In this setting, evolution is a co-evolution and will continue as long as life exists.

We will do well to look repeatedly at this interplay of germs and the places in which they live.