Download FUN2: 11:00-12:00 Scribe: Taylor Nelson Wednesday, December

FUN2: 11:00-12:00
Scribe: Taylor Nelson
Wednesday, December 10, 2008
Proof: Hunter Neill
Dr. Waites
Antimicrobial Resistance
Page 1 of 6
Antimicrobial Chemotherapy and Resistance MRSA – methicillin resistance S. aureus; AB – antibiotic(s)
DISCLAIMER: The first ten minutes of the audio are missing! I have done my best with the notes that I took in
class to add to the slides available
I.
Learning Objectives [S2]
a. To review how drug resistance develops in bacteria
b. To summarize current status of antimicrobial resistance in selected bacteria
c. To review how antibiotic susceptibility testing is performed in the diagnostic laboratory
II. Chemotherapy without bacteriology is guesswork [S3]
a. Try to get lab data so that you can get the name of the microbe ad its background
III. Terminology [S4]
a. MIC – minimum inhibitor concentration
i. The lowest concentration of antimicrobial that inhibits visible bacterial growth
b. MBC – minimum bactericidal concentration
i. The lowest concentration of antimicrobial that kills 99.9% of an inoculum
ii. Not as widely used as the MIC cause the other is easier and should work at physiological levels
c. When an antimicrobial is developed they must:
i. Measure the MIC and then perform the disk test
ii. Determine the break point for the AB
iii. Complex Method
d. Basic type is to determine break points established by the FDA and CLSI
i. Measure AB concentration in the body
ii. Measure the MIC of organism in vitro then you have to see if you can achieve that in the human body
iii. Ampicillin – measure in vitro…
IV. Susceptibility Testing Methods – Agar Disk Diffusion [S5]
a. Used for Rapidly Growing Bacteria
b. Qualitative – does NOT give you MIC but the interpretation is based from the MIC
i. Must record these zones and look at a chart so the test is not easily automated
c. Inverse relationship between zone size and MIC
i. The larger the zone the smaller the MIC needed to inhibit the growth of the bacteria
d. CLSI sets criteria used by labs to interpret
V. Agar Gradient Diffusion (Etest) [S6]
a. Quantitative
b. Add plastic strip with antibiotic (gradient from high to low)
c. Absorbed immediately
d. Read the MIC
e. Used for a wide variety of bacteria (some you cannot use zone sizes)
VI. Automated Bacterial Identification [S7]
a. Includes microscan that can be preprogrammed to tell you the MIC needed to inhibit growth based on color or
turbidity
b. [SQ]: What is the difference between microscan and Ditec (maybe Vitec?)
c. [A]: Microscan actually measures the MIC (turbidity present) whereas Ditec doesn’t measure the MIC directly
but rather an indirect measure that is not as good)
VII. Microbroth Dilution [S8]
a. Well are inoculated and contained micro titrations of amounts of AB, therefore based on color changes one can
tell which AB will be most effective and what concentrations must be reached to inhibit growth
b. Based on the concentrations needed, the physician must know if physiological levels can be reached to match
the inhibitory level
VIII. Agar Dilution [S9]
a. Whether or not a bacteria is susceptible and don’t need and MIC
b. Example: we can screen for MRSA by putting an inoculum on an agar plate containing oxacillin just above the
break point MIC and if the bacteria grows than you know its an MRSA
c. Also used to screen vancomycin resistant enterococcus in the same way
d. Advantages – can take one agar plate and put several patient specimans on it (inexpensive) and so if that’s the
only antibiotic that you are interested in its easy to do
e. Screening for resistance of single drugs
f. Beta lactamase testing
i. β- lactamases is another important bacterial resistance marker
FUN2: 11:00-12:00
Scribe: Taylor Nelson
Wednesday, December 10, 2008
Proof: Hunter Neill
Dr. Waites
Antimicrobial Resistance
Page 2 of 6
ii. Measure -lactamase production directly with a disk called cephanase disk (contains chromogenic
encephalosporin)
1. If a bacteria is producing a B-lactamase enzyme that is breaking down β-Lactam antibiotic – it will
open the β-Lactam ring on the encephalosporin and cause it to turn pink)
2. Simple test
3. Used for H. influenza
g. These are the main ways that testing is done in the laboratory
h. Results are reported in three categories (based on the MIC and what the interpretive guidelines given by the
FDA and the CLSI) we measure the results and report them as either:
i. Susceptible – the antibiotic should be inhibiting the bacteria at a clinically achievable concentration
1. Reasonably expect that the patient’s infection can be adequately treated
ii. Intermediate – cases where the MIC is close to the blood of the tissue level of the antibiotic
1. Health care providers must understand that it may be appropriate to use an antibiotic in certain
cases
2. If you have a drug that is concentration in the urine but there is still active metabolite of the drug –
even if the MIC is intermediate it may be appropriate to use the drug where the drug is concentrated
a. May get more drug in the urine than in the circulation
3. If treating a patient with pneumonia, a drug that concentrated in the lung, may be used to treat even
if the drugs MIC indicates an intermediate response
4. Why clinicians must understand the pharmacology and the metabolism to be able to interpret the
results properly
iii. Resistant – the organism is not inhibited by system achievable concentration with normal doses or follows
on the range where clinical efficacy is unreliable
IX. Importance of Antibiotic Resistance
a. We have known about resistance for a long time, but becoming more prevalent
b. [S11] Newsweek cover: Shows the types of things that you read in the news (general public)
i. A lot of information about this because in the public awareness of the problem with AB
c. [S12] Old WWII poster: A medic is treating a wounded soldier in the field, probably putting sulfa powder on a
wound because that was all they had aside from penicillin
i. When penicillin was first developed, staphylococcus was susceptible to it, but with just a few years it had
developed a B-lactamase and was resistant
ii. Seen with just about all new drugs – some bacteria found resistance to it making the drugs ineffective for
those infection
X. Selective Pressure [13]
a. “Environmental conditions that enhance the ability of bacteria to develop resistance and proliferate. Ability to
survive may be the result of spontaneous mutation or acquisition of new DNA. Organisms with new mutations or
genes probably would not survive if it were not for environmental conditions that encouraged their emergence.”
– FC Tenover
b. Its why we are seeing new AB classes develop
c. Not many are being developed so the bacteria are getting ahead of us
d. It is not uncommon in the clinical lab that bacteria isolates with serious disease and every AB that is test will
show resistance. The clinician asks, “What can I do.” This creates a real dilemma.
e. Think about a bacteria that undergoes a spontaneous point mutation
i. Good Example: fluoroquinolone – inhibit bacterial DNA replication)
1. Ciproflaxacin – probably the most prescribed drug in the world (generic makes it cheap)
a. Covers a lot of bacteria especially gram negative
ii. Flouroguinolones (Ciproflaxin and levoflaxin) against S. pneumoniae
1. Have a pneumococcal infection in your lung there would be a spontaneous mutation rate of a few
organisms that will have a mutation in the DNA gyrase (target of the drug). If you have a mutation in
the gyrase gene than the gyrase produced will not bind the quinolone so the DNA replication can
continue to occur. Spontaneous mutation that naturally occurs.
2. If you have a patient that you are giving this drug to. You kill all the other pneumococci except for
those that spontaneously mutates. Therefore they are left with no competition so the divide and
grow. Then you have quinolone resistance pneumoccocus that developed during therapy
3. This is why a patient may start out on an AB and respond to it and then a spontaneous mutation
occurs and the bacteria gains resistance while the patient is getting the AB
f. Bacteria may also acquire NEW DNA that contain the gene for AB resistance
XI. Potential Routes for Spread of Drug-Resistant Bacteria [S14]
a. There are many different ways that spread of drug resistant bacteria can occur
FUN2: 11:00-12:00
Scribe: Taylor Nelson
Wednesday, December 10, 2008
Proof: Hunter Neill
Dr. Waites
Antimicrobial Resistance
Page 3 of 6
b. What we commonly see with resistant bacteria is that we get infected with a previously resistant organism
i. Not usually mutation, develop resistance, acquire new DNA
ii. The organism is then selected for survival because it’s the only thing that the AB didn’t kill
iii. Those resistant bug are then passed from person to person – don’t need to do genetic manipulations
iv. They can just spread, whether in the hospital or in the community
c. Drug resistant bacteria was first seen in the hospital – because people get a lot of AB in the hospital and there
is high selective pressure there
i. Many of the successful pathogens in the hospital can survive very easily
ii. On table tops, door knobs, stethoscopes, thermometers, blood pressure cuff, bedside toilets, curtains, bed
rails, nurses and doctors hands
iii. The ones that survive easily there or that have reservoirs of patients (in the stool of an asymptomatic
patient) and can be spread to someone else
d. Not just in the hospitals now
e. One of the big changes in the ways that we live today is Day Cares
i. When more and more kids go to Day Care the rates of Otitis Media (ear infections) went sky high
ii. Now Otitis Media is the most common reason that antibiotics are prescribed in the US
iii. Because kids are in day care and share body fluids
iv. Can also transmit infections to grandparents
v. See drug resistant bacteria – kid gets infection and fever – parent puts high stress on physician because
parent adamant that they can only go back to work once child is well – prescribe antibiotic please
1. Parent may also feel that something is not done for their child and will be persistent
2. Physician knows that if they don’t get what they want they can loose a good, paying patient
f. Antibiotics in Animal food – if you put ciproflaxin in chicken feed they will grow faster and bigger, but it also
breeds ciproflaxin resistant bacteria that can be transmitted to humans
g. Third world Countries – no AB regulation, sky-high resistance rates. People that come to the US for health
care (Central America) come to UAB for heart surgery may bring resistant organism to the hospital.
h. Nursing Homes – bad because infection control is often non-existent. If someone in the hospital has MRSA or
communicable disease they are put into contact isolation. This doesn’t occur in nursing homes. It is very
convenient to put a urinary catheter into an incontinent patient’s bladder. You have to clean the linens less often
and the patient doesn’t have to get up as much. If you put a catheter in the bladder it provides and easy conduit
for bacteria. If she starts to run a fever then she will immediately be given antibiotics. Sometimes they put them
on AB prophylactically to keep them from getting a fever and UIT
i. Generating resistant bacteria by doing this
ii. if this person does get sick they will bring their infection with them
i. Community – starting to see the new community acquired MRSA and is starting to come into the hospital.
XII. Other Factors That May Increase Antimicrobial Resistance in Hospitals [S15]
a. There are many different pathways through which we are seeing more resistance
b. New community acquired MRSA, some concern that these strains will cause more illness in the hospitals than
the strains already present
c. There are a lot of things as far as our health care system that make antibiotic resistance more likely
i. The people that get health care in the hospital have a greater severity of illness (once you are healthy
enough to be discharged they let you go) so the people in the hospital that are going to be at risk for getting
infections are already very ill so they are not very good at combating infection
d. You have a lot of immunocompromised patients
i. Many because they have a chronic debilitating disease
ii. Some because they are being treated for a malignancy with immunosuppressive drugs
iii. Transplant patients receive immunosuppressives to prevent organ rejection
iv. Whatever the case, these patients are ill equipped to combat infection, and more likely to get an infection,
and this makes it worse if they get a resistant infection
e. We have many new devices and procedures that are invasive and allow an infectious organism to enter the
body that would otherwise be unable to do so
f. It’s very difficult to control infection in a large medical institution due to the difficulty of enforcing infection control
practices
g. It is becoming more common that physicians will give antibiotic prophylaxis for procedures – standard practice
to give 24 hours of vancomycin therapy to a patient having a coronary artery bypass to prevent an infection
while they are in the hospital
i. Sometimes find that these patients get put on vancomycin when they are admitted and they remain on the
drug for the duration of their stay because the physician doesn’t want them to get sick while they are in the
FUN2: 11:00-12:00
Scribe: Taylor Nelson
Wednesday, December 10, 2008
Proof: Hunter Neill
Dr. Waites
Antimicrobial Resistance
Page 4 of 6
hospital – this puts selective pressure on any bacteria this patient may encounter and they can then infect
others with a resistant strain
h. Empiric polymicrobial antimicrobial therapy – patient has a fever and the physician doesn’t know what else to do
so they place them on five or six an antibiotic, this simply puts more and more selective pressure on bacteria to
develop resistance
XIII. Organism Characteristics Favoring Resistance [S16]
a. Some bacteria develop resistance and some do not
b. This is a property of the bacteria themselves
c. If you have intrinsic resistance to some antibiotics that limits the number of treatments that are effective to begin
with, so if you pick up resistance to one other drug there may be nothing else you can use to treat the infection
i. Vancomycin resistance Enterococcus and MRSA are two examples
d. Ability to exchange genetic information – bacteria that can more easily accept plasmids form other bacteria that
contain resistance genes will pick up resistance, this is a genetic property of the microorganism itself
e. Ability to survive adverse environmental conditions – i.e. UV light, drying out – these bacteria will be most
successful because they are transmitted more easily
f. Ease of colonization, infection, transmitting is important
g. Reservoirs in body – vancomycin resistant Enterococcus may be carried in the stool of a patient (causing no
problem to that patient) and transmitted by the hands of a healthcare worker to a surgical wound in another
patient leading to the newly infected patient becoming bacteremic
i. A patient may be colonized and be a reservoir and not even know it
ii. Should you screen people for infection?
1. The VA screen every patient admitted for MRSA in their nose and isolates all positive patients
admitted
2. This is very expensive, but save many lives and dollars by preventing transmission to and infection
of others
XIV.
Impact of Antimicrobial Resistance [S17]
a. Prolonged illness/hospitalization
b. More people die because the infections are hard to treat
c. Simple cheap things don’t work anymore so you must use more expensive, more toxic drugs
i. Must do lab tests to make sure you not making the patient more sick with the antibiotics
d. Can create more resistant strains
e. Once you get these resistant strains in the hospital they never go away
f. Vancomycin resistance Enterococcus was first described in 1989, we had the first case at UAB in 1994, now we
see it all the time
XV. How Does Antibiotic Resistance Affect the Biology of the Bacteria [S18]
a. It does not typically make the bacteria more virulent
i. In some cases the additional genetic information that allow bacteria to be resistant may be a burden to
them, sometimes the genes and the things the bacteria must to pick up to live in the presence of antibiotics
may make it grow slower or not colonize as well but it’s better off than those around it that don’t have the
resistant gene because they get killed off
b. It is not necessarily increasing the virulence, it’s just making it more resistance to treatment
c. If it is only slightly resistant you may still be able to treat it with higher doses of antibiotics, especially if the drug
is concentrated in the site of infection
d. Highly resistant bacteria will not respond to the antibiotics and you must find alternative treatment
XVI.
Types of Antimicrobial Resistance [S19]
a. Two types of resistance
i. Innate
ii. Acquired
b. Innate resistance is not something we worry about that much
i. Just have to learn which antibiotics work on which bacteria
1. For example, vancomycin does not work on gram negative bacteria because the molecule is too big
to penetrate the cell gram negative cell wall, β lactams don’t work on mycoplasmas because
mycoplasmas don’t have a cell wall
c. Acquired resistance is acquired by either a mutation or by acquisition of new DNA
i. Several different ways that bacteria can get new DNA [S20]
1. Transformation – transfer of free DNA, how penicillin resistance is believed to have arisen in S.
pneumoniae, microorganisms die and there naked DNA is released in the environment and this DNA
is taken up by a recipient bacteria and incorporated into its chromosome
FUN2: 11:00-12:00
Scribe: Taylor Nelson
Wednesday, December 10, 2008
Proof: Hunter Neill
Dr. Waites
Antimicrobial Resistance
Page 5 of 6
2. Transduction – bacteriophage virus that contains genes for antibiotic resistance infects the bacteria
and incorporates its genome into the bacterial chromosome
3. Transposition –
a. Transposons (mobile genetic elements) that can transfer back and forth between bacteria
b. Transposition through conjugation which is acquiring plasmids or transposons or both from
other donor bacteria
i. This is very important way that we see bacterial resistance develop as a plasmid may
contain the genes that will encode resistance for several antibiotics – could have a
patient with Pseudomonas aeruginosa infection that was susceptible to several drugs
and all of the sudden it develops resistance to several drugs simultaneously because it
acquired a plasmid by conjugating with another bacteria
d. [S21] Example of what happened with S. pneumoniae
i. There was a strain called the Spanish strain
1. Identified in Spain in 1978 – now is spread all over the world
2. Penicillin resistant
3. ~50% of all S. pneumoniae infecdtions seen in the US are penicillin resistant
4. Only about nine or ten different clonal groups that cause all this resistance
a. It’s clonal spread, the bugs that are already resistant spreading from person to person
circulating in the community – driven by antibiotic pressure
b. See this all over the world when antibiotics are misused
e. Bacterial Strain Typing PFGE [S22]
i. Study at UAB hospital – looked at patients with vancomycin resistant Enterococcus
1. 14 patients over a three year period that were hospitalized in 1997, 98, and 99 that had a
vancomycin resistant Enterococcus infection and typed the strains using pulse field gel
electrophoresis
2. The banks line up very closely, a great many of the bands were the same organism, demonstrating
that the strains of bacteria that cause infection hang around the hospital indefinitely
3. Demonstrates ineffective infection control despite your best efforts
XVII. Mechanisms of Antimicrobial Resistance [S23]
a. Several ways antibiotic resistance can develop
b. All of these are acquired through mutations of the bacteria or the acquisition of DNA that contains the genes for
the resistance
c. Just like there are a finite number of different types of antibiotics and mechanisms by which they work there are
also just a few mechanisms by which resistance develops
d. Some bacteria may possess multiple mechanisms of resistance, there may also be multiple mechanisms of
resistance for the same types of antibiotics
e. Most common thing that we think of with penicillin is the β-lactamase production – β-lactamase is the enzyme
that the bacteria produces that breaks down the β-lactam
i. β-lactam works because the β-lactam ring in antibiotic resembles the amino acids that the bacteria uses to
cross link its peptidoglycan
ii. The penicillin binding proteins, the enzymes that catalyze the cross linking mistake the β-lactam for the
substrate, bind the β-lactam and are unable to cross link the cell wall
iii. If you open the β-lactam ring with the β-lactamase enzyme than the ring no longer resembles the substrate
so the bacteria go on about their business making a the cell wall
f. MRSA and penicillin resistant S. pneumoniae have nothing to do with β-lactamase, it’s the altered penicillin
binding proteins themselves that the bacteria have developed a mutated enzyme so that the penicillin binding
protein that is there will not be recognized and bound to the β-lactam – this is an ALTERED TARGET, also
common with fluoroquinolone resistance (altered or mutated DNA gyrase)
i. Another example of altered target: macrolide resistance in S. pneumonia is a methylated ribosome –
macrolides must bind at the 50s ribosome to prevent polypeptide formation, if you have a methyl group
sitting there on the binding site the macrolide cannot bind and you have resistance
g. Efflux pumps – active against many different types of antibiotics, bacteria has a pump that pumps the antibiotic
out of the cell before it can do its damage
h. Decreased permeability – bacteria acquire genes that change the cell envelope, porin structure, or cell wall
structure so the antibiotic cannot pass into the cell, example with aminoglycoiside and vancomycin intermediate
S. aureus (VISA)
i. Bypass – Trimeth/sulfa works in 2 steps to inhibit folic acid (necessary for DNA synthesis) synthesis in bacteria,
ultimately results in impaired DNA synthesis – some bacteria develop metabolic pathways to synthesize folic
FUN2: 11:00-12:00
Scribe: Taylor Nelson
Wednesday, December 10, 2008
Proof: Hunter Neill
Dr. Waites
Antimicrobial Resistance
Page 6 of 6
acid that are not inhibited by trimeth/sulfa, even the trimeth/sulfa are blocking a pathway the bacteria are still
getting their folic acid because they have a different pathway that is unaffected
XVIII. β-Lactams: Mechanisms of Resistance [S24]
a. Two mechanisms for β-lactam resistance
i. β-lactamase destroys the antibiotic
ii. Change the penicillin binding protein so the β-lactam doesn’t bind to it
XIX.
Fluoroquinolones [S25]
a. Quinolones, a couple of different ways:
i. Mutation in the DNA gyrase or the topoisomerase enzymes
ii. Active efflux
b. So some bacteria may be resistant by two different mechanisms
c. Some drugs are more susceptible than others to resistance
i. Ciprofloxacin is more susceptible to being effluxed than some of the other quinolones
XX. Macrolides: Mechanisms of Resistance [S26]
a. Just showing all the different ways that macrolide resistance can develop
b. Already mentioned the methylated ribosome
c. Can have efflux pump
d. Esterases that break down the enzymes
e. In addition to the altered ribosome being methylated you can also have altered ribosomal proteins or altered
ribosomal RNA
f. Some bacteria may have all the above mechanisms as they are better at developing resistance than others
XXI.
Methicillin resistant S. aureus (MRSA) [S28]
a. Probably the single most important, certainly the most important gram positive bacteria that is developing
resistance
b. Terrified about getting development of vancomycin resistant Staphylococcus
i. We know that the genes for vancomycin resistance, carried on the plasmid, has been transmitted from
Enterococcus to S. aureus
ii. These have always been cases where people have had prolonged vancomycin usage, no doubt antibiotic
pressure plays a role here
c. The other problem is community acquired MRSA – in the past if someone went to a clinic or ER with a skin
lesion they would be offered a β-lactam antibiotic, but due to common community acquired MRSA these same
antibiotics don’t work anymore, if you’re going to give antibiotics you must give trimeth/sulfa, tetracylcline, etc.
d. [S29] Shows the progression of MRSA over the last several years – today MRSA is ~60% of all the S. aureus
we see in hospitals (5/1000 admitted will have MRSA)
i. Some states mandate that hospitals must post this data, patients’ have a right to know the chance of
acquiring an infection
ii. Not the case in Alabama
XXII. ESBLs: Plasmid-Mediated Resistance [S30]
a. Extended spectrum β-lactamases – problems in gram negative bacteria
i. Usually think of the β-lactamase stable penicillin and β-lactamase stable cephalosporin
b. Plasmid mediated enzymes, especially in E. coli, Klebsiella, and Proteus that confer resistance to the β-lactams
c. Difficult to detect in the laboratory, must look at resistance patterns and perform extra tests to find them
d. Complicate treatment because the drugs that we treated gram negatives with in the past don’t work anymore so
e. Serious infections in hospitalized patients
[End 44 min]