FUN2: 11:00-12:00Scribe: Taylor Nelson

Wednesday, November 12, 2008Proof: Hunter Neill

Dr. MoserMicrobiologyPage1 of 6

Anaerobic Bacteria

Reading Assignment [S2]
Very concise chapter that you should read
I will only hit the high points and since the chapters aren’t long they will help you understand
Categories based Upon Gaseous Requirements [S3]
How are anaerobic bacteria different from other bacteria
Do not require oxygen as it is toxic to them
They is a range from those bacteria that:
Aerobic - must have oxygen to grow,
Microaerophilic bacteria (camphlobacter) cause gastrointestinal disease – need reduced O2 and increased CO2
Will not grow in regular environmental air
Capnophilie – like CO2 – more fastidious bacteria
Haemophilous influenza and Neisseria gonnorhea
Fragile
Others are stimulated by CO2 but don’t necessarily require it
Facultative Bacteria
Most of the bacteria associated with human disease
They can grow with or without O2
Prefer O2 because more ATP is produced, but will use that and then shift to the less efficient, anaerobic metabolism
Anaerobic Bacteria
Spectrum within
Those that are exposed briefly will die
And some that can be exposed briefly - aerotolerant
Physiology and Growth Conditions [S4]
Why are they anaerobes and why cant they tolerate O2?
The by products of 02 metabolism are very toxic and require systems in place in order to detoxify it
Detoxify using: Cytochrome systems, superoxide dismutase, catalase (break down toxic products)
Obligate anaerobes don’t have these abilities
Aerotolerant make some of the enzymes just not enough
Facultative – have all of those and it doesn’t matter whether they have O2 or lack thereof
Illustration [S5]:
If you have O2 then you have these by products that are all high energy radicles including H2O2
Produced by some cells as a defense mechanism that can kill some bacteria
Anaerobes don’t have the Detoxifying Pathway (2nd cell) including 1superoxide dismutase, 2Catalase, 3peroxidase, etc making O2 or water
Main Point: inability of anaerobes to produce these enzymes to detoxify O2 high energy radicals that are byproducts of oxidative metabolism
Location of Aerobes [S6]
Therefore don’t life where O2 is easily accessible
Periodontal pockets, dental plaque and the colon are more likely because they aren’t exposed
Might coexist with facultative organisms that will use up any O2 that my be present
Distribution of different types of bacteria within the body [S7]
Can be divided (the most important are the top 2)
Gram negative, rods
Bacteroides fragilis group, Prevotella, Fusobacterium
Some found preferentially in the colon
Can think of their locations as above and below the diaphram
B. fragilis is normally found below the diaphram in the colon and the abdominal cavity
Some are only found above – Prevotella and
Some are in either place
Gram Positives (some are pathogenic)
Knowing where they live in the human body tells you where they might be involved in a disease process
We can pick up things from the environment like Clostridia because they make spores and can survive
Most of the things that cause human infection are our own organisms that get loose
Associated to an anatomic area close to where they are normally
Locations of particular anaerobic infections [S8]
Begin with the mouth
In gingival crevices, crypts no matter how good oral hygiene is
Those with bad oral hygiene are at a greater rick of the organisms
Getting loose
Becoming bacteremic – causing brain abscess
Pulmonary – usually found with aspiration of oral anaerobes and mixed floral anaerobes
Alcoholic or binge drinker that regurgitates and aspirates allowing foreign bodies into the lung causing pulmonary lung abscesses or empyema (puss filled cavity)
Human bites (can be worse than animal bites esp. if someone has poor dentition)
Below the diaphragm: whether it be appendicitis that has ruptured, surgery where the colon has been nicked, or diverticulitis – the infection can get into the abdominal cavity and cause abscess formation
Pelvic inflammatory, endometritus
Foot Ulcers – extremely important in diabetic patients (external contamination)
Vincent Angina [S9]
Erosion of gums
Bad hygiene allows areas to be “closed down” (anaerobic) and allow organisms grow very rapidly
Gram Smear: Several morphological types
Long skinny pointed, short fat Gram (-) rods, thin wavy ones
3 different anaerobes that are normally found in the mouth
Just remember its a mixed gram (-) infection that has a synergistic infections where the byproducts cause destruction of the gingiva
Adult periodontitis [S10]
Less acute gum diseases
Different organisms involved causing slow destruction of the gum (receding)
Eventually the teeth become loose and result in:
Bacterial showering into blood stream
Eventually loose teeth
Anaerobic Brain Abscess [S11]
Caused when bacteria enter the blood stream (extreme case)
Can see the morphological types on the gram smear (3 types)
Characteristic of anaerobic abscesses
Remember that you will NOT see a single morphological type
Must be drained, cannot treat simply with antibiotics – hard to reach target organisms
Anaerobic Polymicrobic Cellulitis [S12]
Diabetic patient – poor circulation in extremities decreases the function neutrophils even in patients that have their disease under control
Probably the result of fecal contamination
Gets a wound that gets infected and becomes hard to heal
Aerobic Infections Gram (-) Bacilli [S13]
Bacteroides, Prevotella, Porphyromonas species
Bacteroides fragilis and Prevoltella melaninogenica are two of the important ones
Common, non-spore forming, pleomorphic rods
Mouth or stool
Don’t culture stool for anaerobes because 99% of bacteria that are in the colon are anaerobes
Only that small portion that we look for – Shigella, Salmonela, Camphylobacter, E.Coli that are causing these problems
In general you don’t culture stool for anaerobes
Certain associations are made in areas by species (find particular species in certain places)
Clinical disease is usually mixed floral with not just anaerobes but also facultative gram (-) organisms (i.e E.coli, Enterobacter, Klebsiella in colon) which help maintain the anaerobiosis of the micro environment
Necrotizing Fasciitis[S14-15]–
Rare, associated with Group A Strep. (flesh eating bacteria), staph, some gram (-), and even some anaerobes
Picture shows and incision with draining areas
Bacteria is causing cell death and necrosis
Much toxicity associated with that for the host – gangrenous
Surgical excision is often the primary treatment
Fusobacterium[S16] – by name fusiform or pointed at the ends
Long pointed rods
Mixed or sole agents of infections
Causes Lemierre’s syndrome, which is subsequent to tonsillitis. Someone who has an abscess allows the bacteria to enter and dissect down causing jugular vein thrombosis
Not as common due to larger use of antibiotics
Pulmonary Abscess [S17]– Child
Air / fluid level on radiograph that will stand out readily (seen in the circle)
Pleomorphic [S18]
Must grow in the absence of O2 – defining characteristic of all these organisms
Classic diseases associated with anaerobes
Actinomyces [S19]
Actinomyces is the genes
Slow growing and difficult to isolate – try to confirm diagnosis and let the lab tech know so they don’t open culture to early as it will kill organism, these just cannot tolerate oxygen
Unusual in the fact that they migrate
Aspiration may cause abscess in lung then exit the chest wall causing draining sinus.
Can go across the diaphram
Most infections stay where they are introduced unless they are septic and enter the blood stream
Creates draining sinus tracts that result in grains that are visible to eye that are microcolonies of the organism coved by secretions from the host
Oral, respiratory – copper Coil IUD have been a problem
Dacryocystitis[S20] – ocular
Concretions can get into the tear ducts and the lacrimal sack become blocked
Blockage of the tear duct that can be removed with an instrument
Looks similar to Haemophilis influenzae but this demonstrates why it is necessary for a patient history
Actinomycosis _ “Lumpy Jaw” [S21]
Classic disease (in books and exams)
Normal face but asymmetric with large swelling on the patients left
[S22] Has abscess at the root of the tooth - bone involvement and then abscess will start to come out the jaw
If left alone would eventually drain puss and granules – can trap if you cover with a gauze
[S23] Picture of granules (couple mm in size)
Can crush them and then do a gram stain
Filamentous, gram (+) rods
Must differentiate between a fungus that can cause similar draining sinus particles
Fungi is at least 10X the size so size differential will tell the difference between a bacteria and a fungus
Proponibacerium [S25]
Can live on the skin in pores depending on presence of lipids
Causes acne
Contaminants in lab if the skin is not properly disinfected (shows up late)
Rarely involved in disease
Lactobacillus
Good – make many things such as yogurt and also maintain the acidic environment of the vagina that serves as protection from bacterial vaginosis caused by these anaerobes
Rarely cause of disease
Anaerobic Infection Gram-Positive Bacilli [S26]
Clostridium
Tetanus
Should have been immunized against, need a booster every 10 years
In the past, each time a person stepped on a nail the person would get a shot
Because it’s such a good immunogen cause great reactions – reason why we only get boosters every 10 year unless you have a traumatic accident that presents a chance for massive exposure then the booster would be given at that time
The vaccine is the Tetanospasmin- the toxin itself made non-toxic
Tetanospasmin blocks inhibitory neurotransmitters, this is why you get constant firing of muscles
You have two balancing mechanisms-one says fire and one says relax, with tetanus there is no relaxation
Lock jaw is a common name for this, as those who are infected get this constant firing of muscles in their jaw, also known as sardonic smile
This is an infection, in vivo production of toxin – you must have the organism in you, usually in a wound
Botulism
Usually ingestion of preformed toxin
Many people get botulism from foods improperly canned at home
Extremely potent toxin, you don’t need to ingest much at all
Works oppositely of tetanus, sort of a spastic paralysis
You can’t fire your muscles due to the blockage of neurotransmitters such as acetylcholine, muscles don’t contract due to the blocking of the signal from the nervous system
Some exceptions to preformed botulism exist:
Wound botulism – very unusual
Infant botulism – more common than wound botulism
Neonates up to 6 or 8 months don’t have GI flora (they are born without it) but this flora is accumulated over time with food etc. and eventually this flora builds up and provides protection
Some parents use honey to pacify children, honey has botulism spores which can germinate in the neonate GI tract without any competition and cause botulism poisoning
Don’t give babies honey
Gas gangrene
Primarily caused by C. perfringens, can be caused by other species but this is the classic
Takes human tissue and makes H2 and CO2 out of them, thus the name gas gangrene
You can actually push on the tissue and feel gas trapped there (sort of like bubble wrap) - crepitous
Can produce a number of toxins, one of the most important being phospholipase C (alpha toxin), can make up to a dozen different toxins but the alpha toxin is the one primarily responsible for gas gangrene
Food poisoning
Also caused by C. perfringens, but due to a different toxin known as enterotoxin
Also preformed toxin
Usually found in gravies and things like that that haven’t been stored properly
C. septicum
People that become bacteremic with this organism that is often an early sign that there is some malignancy – colon cancer or something else
Said that this wasn’t very important to remember
Pseudomembranous colitis/antibiotic associated diarrhea
Caused by C. difficile
Present in the GI tract of 1-2% of the adult population
When broad spectrum antibacterio therapy (with anaerobe spectrum) is prescribed much of the anaerobic flora will be killed and whatever is left will take over due to lack of competition, C. difficile is very resistant to most antibiotics so it will grow and make toxins causing antibiotic associated diarrhea and if left alone can cause pseudomembranous colitis which is very serious (seen mostly in hospitalized patients)
Tetanus [S27]
Doesn’t have to be a wound as severe as the one shown, but usually inflamed, red, etc.
Photo of sardonic smile
Infant being supported by foot and head – every muscle is being clenched
Clostridium tetani [S28]
The organism is usually not isolated to diagnose the disease
Can diagnose by symptoms, some assays even exist for the toxin
Want to know if the individual has been immunized – some underserved populations are still not immunized, important to understand the immunization history
The lollypop looking thing is the spore that allows the organism to survive
All clostridium make spores, they are the only anaerobic bacteria that do so
Gas Gangrene [S29]
Nasty outcome, often post-surgical
The boli full of gas can easily be seen
This person is in a lot of trouble because of the location
Many toxins associated with this, so not just a spot with symptoms but the entire body is being assaulted
[S30] Clostridium perfringens is the culprit of the gas gangrene
Boxcar shape
Quite large for bacteria
The stain for spores reveals spores that are inside as opposed to the lollypop look of C. tetani
Could biopsy the gentleman from [S29] toward the edge of his boli and would find this bacteria, definitely diagnostic
Clostridium perfringens [S31]
Not all C. perfringens produce the alpha toxin
There are different serotypes – to establish in the lab that the Clostridium perfringens you have is the serotype that produces the alpha toxin one would have to find it to be hemolytic on blood agar and here we have a positive control and an unknown(photo on right), half the plate has antitoxin and the other half does not, so what it does is that it goes out and it breaks down lecithin in the media and makes it cloudy, neutralized on the bottom half to give specificity
Clostridium difficile Colitis [S32]
C. difficile is given the name difficile because it is difficult to isolate, difficult to sort one anaerobe out of another when many are present
For diagnostic reasons we have a lyse test to test for the toxin
Two toxins present, and stool is screened for that when the possibility of C. difficile is being entertained
There is now a more virulent strain of C. difficile
Hospital associated
The bacteria form spores which allows them to survive in an environment
If the room of someone who has C. difficile is not disinfected properly than the next person to be admitted to that room can pick it up
Death is associated with this new strain, so it’s not harmless
Pathogenesis [S33]
There is some synergy with facultative organisms – in other words you can get oxygen reduction with E. coli, klebsiella, enterobacter, etc.
Like many bacteria some of them can produce beta-lactamase
Beta-lactamases break down beta lactamase antibiotics, penicillin is not a good drug against this for example
A few are encapsulated
Any time you have a microorganism that has a capsule that means it’s antiphagocytic
Diagnosis of Anaerobic Infections [S34]
Diagnosis starts out at the patient with clinical signs
Abscess formation
Gas in the tissue
Proximity to mucosal surface
Foul smelling discharge due to short chain fatty acids (very odiferous) from anaerobic metabolism
If an abscess is opened up and green stinky material is present than that is indicative of having anaerobes involved
Gram Stain can be helpful
They are mixed, so you will see mixed flora
[S35] Gram stain of mixed infection
To reiterate- you have neutrophils, small gram negative rods, gram positive rods, gram positive cocci, long skinny gram negative rods but all you know is that you have multiple different bacteria in there and if you are using antibiotics you need to use broad spectrum antibiotics
When we culture we often find too many organisms and therefore it isn’t too helpful
So we want to restrict what we culture, there has to be a good reason for submitting a culture for anaerobic testing
Diagnosis of Anaerobic Infections [S36]
Culture
Collection and transport are critical
If you have a peri-rectal abscess you can guess the organism and treat, there is no need to culture
Other abscess then it would be appropriate to send the sample
They are complex from a nutritional standpoint
Need medium that has vitamin K
They are totally resistant to aminoglycosides
Because they can be mixed with E.coli and enreobacter
We can incorporate that into the media to keep the other bacteria from growing and to produce other needed nutrients to allow us to find the true anaerobes
Can’t work with them out on the bench for very long, they need to be incubated in CO2, nitrogen, and hydrogen
[S37]Anaerobic Containers – simplest and cheapest way to incubate
Special medium
Jars that generate H2 and the O2 present is turned into water
Slow so not the best method
[S38]Anaerobe Chamber
Samples go through airlock
Entire area is anaerobic
Whole process is done in the absence of O2
Very inconvenient
Newer system
Takes jar and attaches valves, push a button and it pumps in all the needed gases and within seconds the environment goes anaerobic
Works just as well as the cumbersome chamber
Once they have grown you can work with them on the bench but if you do that with the primary isolation you have a problem
Bateroids Fragilis [S39]
Spot testing
Media that incorporates two things that he’s discussed
One side has aminoglycoside inhibiting the growth of other bacteria, so if it grows here is may be an anaerobe
The other side has a bile esculin, bile to test the ability of the organism to grow in large amounts of bile, esculin is simply a compound that is broken down
For other bacteria we have to do a few more tests but for the most part we don’t have to do a full biochemical setup to get to the more common ones
Treatment of Anaerobic Infections [S40]
Already talked a lot about this
Penicillin G is not a great drug to use, but people still use it
C. difficile is treated with vancomyacin or metronidazole
Penicillin resistance is prevalent here, aminoglycoside is not effective
Immunization against some of the toxin diseases is best
Etest Susceptibility Testing [S41]
Don’t routinely dosusceptibility testing on anaerobes because they are highly predictable
We will however accumulate them and create what we call an antibiogram, in other words if we have enough Bateroids Fragilis organisms we can test them and give susceptibility percentages to various drugs
We would use the Etest to do one individually, concept of diffusion and reading an MIC from there
Objectives [S43]
Some of the important things you should know
Remember the gaseous requirements of the spectrum
Collection and diagnostic methods
If you have a closed abscess the best way to go at it is to stick a needle in it and aspirate
There are special vials you can put that aspirate in to transport it to the lab
Our current swabs are okay, don’t usually like to use swabs with anaerobes, but interoperatively that is what we get
Epidemiology means where do they live and how do we get them
Clinical syndromes
Classic clinical syndromes that we’ve talked about, the things you would quickly associate with gram negative anaerobic infections

[End 51 min]