quiz 4
WHAT IS THE NAME OF THE GROUPING SYSTEM USED TO SUBGROUP TYPES OF BETA-HEMOLYTIC BACTERIA?
LANCEFIELD TYPING
WHAT IS THE ABBREVIATED NAME OF A SPECIES OF STAPH THAT IS RESISTANT TO MANY BETA-LACTAM ANTIBIOTICS AND IS OF HIGH MEDICAL CONCERN?
MRSA (METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS)
MecA is equal to a transpeptidase
MecA encodes transpeptidase that has a slightly different structure that β-lactams cannot bind to. This provides the bacteria antibiotic resistance
Suggest a reason why some antibiotics were given different diameters for zones of inhibition to determine sensitivity.
The antibiotics will diffuse at different rates causing the zones of inhibitions to be different diameters.
Please explain why the shelf life of UHT (ultra high temp) milk is longer than that of regular pasteurized milk.
UHT milk has been sterilized by heating it at 135C so there are no microbes left in it. If it is left sealed, there are therefore no microbes in it to spoil it. Pasteurized milk is not sterile and the microbes in it will change its character over time.
HOW CAN YOU DIFFERENTIATE GAS FROM GBS?
USING A DISK DIFFUSION ASSAY - ONLY GAS IS BACITRACIN SENSITIVE
How exactly does UV light kill microorganisms?
UV light kills microorganisms by forming thymine dimers in the DNA. These thymine dimers are when two thymine bases next to each other, bond to each other. DNA polyermase does not recognize the bases, thus cannot replicate the DNA.
Transpeptidase equals PBP's
Both help with cell wall synthesis. MecA is equal to penicillin binding proteins (PBP's) because both help with cell wall synthesis. MecA encodes for transpeptidase which is a cell wall enzyme
How can we test antibiotic effectiveness?
By using a disk diffusion assay
HE AND EMB AGARS ARE SELECTIVE FOR WHICH TYPE OF BACTERIA?
GRAM NEGATIVE
Please explain why Gram negative bacteria tend to be more resistant to beta-lactam antibiotics than Gram positive bacteria.
Gram negative bacteria is more resistant because it has an outer phospholipid layer protecting the cell wall unlike the gram positive. The antibiotic is blocked from the cell wall by this phospholipid bilayer making it much more difficult to treat. The gram positive cell wall on the other hand is exposed making it easier for antibiotics to attack.
Is disk diffusion qualitative or quantitative?
Qualitative -you don't measure the amount of chemicals you use -all you can say is it has an effect or it doesn't
Is the Kirby-Bauer test qualitative or quantitative?
Quantitative -because the test is standardized -measure results
Why would raisins have fewer microbes than the grapes they are made from?
Raisins have fewer microbes because they are dry and have no water in them, the osmotic pressure from the sugar in the cell goes up and it is lethal to bacteria.
NAME A MEMBER OF THE ENTEROBACTERIACEAE THAT IS ABLE TO REDUCE SULFATE
SALMONELLA OR PROTEUS
MACCONKEY AGAR IS A SELECTIVE AND DIFFERENTIAL MEDIUM USED FOR THE IDENTIFICATION OF COLIFORMS IN FOOD AND DAIRY. WHAT ARE THE SELECTIVE AND DIFFERNTIAL AGENTS?
SELECTIVE AGENTS: CRYSTAL VIOLET AND BILE SALTS (PREVENT THE GROWTH OF GRAM +) DIFFERENTIAL AGENTS: LACTOSE AND NEUTRAL RED (LACTOSE FERMENTING WILL APPEAR PINK)
WHAT IS ANOTHER NAME FOR GROUP B STREP AND WHEN IS IT OF MEDICAL CONCERN?
STREPTOCOCCUS AGALACTIAE. WHEN IT IS TRANSFERRED TO AN INFANT DURING BIRTH
Although most E. coli cells were killed rapidly by UV light, some seemed to survive even after 16 minutes of treatment. How can you explain that?
Spontaneous mutations occurred in the E. coli before being exposed to UV light causing the E. coli to survive harsh environments. This allows the E. coli to grow resistant to the UV light. The mutations were most likely in photolyase and SOS system causing them to be more active.
WHAT IS THE COAGULASE TEST USED FOR? WHY DO SOME BACTERIA HAVE COAGULASE?
THIS IS A TEST TO DIFFERENTIATE S. AUREUS FROM OTHER STAPH SPECIES. COAGULASE IS A VIRULENCE FACTOR THAT CAUSES THE CONVERSION OF FIBRINOGEN TO FIBRIN. THE COAGULASE ON THE BACTERIAL SURFACE BINDS THE FIBRIN AND THIS PROTECTS THE BACTERIA FROM BEING PHAGOCYTOSED.
HOW CAN ONE DIFFERENTIATE BETWEEN STAPHYLOCOCCAL AND STREPTOCOCCAL GENERA?
THROUGH A CATALASE TEST (STREPTOCOCCI ARE NEGATIVE FOR CATALASE)
What is the MIC of an antibiotic? In a Kirby-Bauer assay, where would you find it? For treatment of an infection in humans, is it better to have an antibiotic with a higher or lower MIC? Why?
The MIC of an antibiotic is the minimum inhibitory concentration. This is the smallest concentration of antibiotics that visibly inhibits growth. In Kirby-Bauer assay, you would find the MIC at the zone of inhibition. You would prefer to have an antibiotic with a lower MIC because it indicates that less of a dose is required in order to inhibit bacterial growth. This is good for infection in humans because a lot of people do not take their antibiotics properly which causes antibiotic resistance.
WHAT DOES S. AUREUS LOOK LIKE ON AN MSA PLATE?
YELLOW - IT FERMENTS MANNITOL PRODUCING ACID WHICH CAUSES THE PHENOL YELLOW TO TURN YELLOW
WHAT DOES A GRAM-NEGATIVE SULFATE REDUCER THAT FERMENTS LACTOSE LOOK LIKE ON AN HE PLATE?
YELLOW COLONIES WITH BLACK CENTERS
Bacteriostatic
agents that inhibits bacterial growth, but don't necessarily kill them
antimicrobial agents
antibacterial, antifungal any chemical, other agent, against any microbes
pyrimidine dimers
covalent bonds formed between two adjacent thymine or cytosine nucleotides on the DNA strand that results in the formation of a kink in the DNA backbone
insertion sequence IS431
directs where gene is inserted -insertion sequence -tells whole element where it should go in the genome
recombinase genes ccrAB(C)
encode proteins that are going to take this genetic element and they are going to -cut the staph genome -then paste in genetic element -actually put gene in the right spot -ligate DNA all back together so it's stuck in the genome
Disk diffusion assay
general term where you take an agar plate and you spread the bacteria out all over -use cotton swab or glass spreader -take paper disk and soak it in chemical, antibitoic antiseptic etc -put it on the plate -as bacteria is growing overnight, chemical diffuses -creating area where you have presence of the chemical -qualitative aspect because you aren't measuring the amount of chemicals you're using
coliforms
gram-negative, non-spore forming bacilli that ferment lactose with acid and gas production
Gram negative bacteria
has extra phospholipid bilayer between the thin cell wall and the outside of the cell -antibiotic is blocked by outer membrane -more difficult to treat a gram negative bacteria
beta-lactam ring
inner chemical compound in all beta-lactam antibiotics -side groups will change -gives antibiotic ability to actually inhibit bacterial growth -fits in the transpeptidase active site, which inactivates transpeptidase
Transpeptidase
involved in crosslinking peptidoglycan in cell wall synthesis -only effective in cells that are actively growing
Forming endospores
protects bacteria from UV light
indirect repeats
ones going to the right, ones going to the left
Gram positive bacteria
outer wall is the cell wall so nothing protects the transpeptidases/cell wall making enzyme
excision-repair system
recognize DNA damage and a bunch of enzymes work together to repair it -endonuclease will cut upstream and downstream in the phosphodiester backbone -then remove the entire string of bases -Empty gap? DNA pol will fill it starting with one end of free hydroxy on 3 prime end, filling the gap
Photolyase
recognizes thymine dimers and breaks those bonds
Kirby-Bauer Assay
specific kind of disk diffusion assay -standardized disk diffusion assay, allows results to be similar from one lab to the next because you are following the same protocols
Norwalk virus/noro virus
spreads easily in crowded places -most popular food borne illness
How do you gain resistance?
stop antibiotic from binding
Minimum inhibitory concentration (MIC)
the smallest concentration (highest dilution) of drug that visibly inhibits growth -right at the edge of the zone
Why would anyone who's sick take an antibiotic that doesn't kill, but just inhibits?
this will allow your immune system to have the upper hand, inhibit growth, immune system will kill the microbes very effective
HE AGAR IS DIFFERENTIAL FOR WHAT TWO FEATURES OF BACTERIA?
ABILITY TO REDUCE SULFATE AND TO FERMENT LACTOSE
mechanism of beta-lactam antibiotics
-B-lactam antibiotics bind to transpeptidase and inhibit them -only effective in cells that are actively growing -creating holes in own cell wall when growing, when you inhibit transpeptidase, you cannot fix the pieces, thus creating weak cell wall -bacteria is probably in hypotonic solution causing water to rush in -bacteria will lyse
How can we modify antibiotics?
-adding amino groups that can help penetration of cell wall -take natural antibiotics made by fungi and Streptomyces bacteria and modify them by adding different side chains -ability to penetrate membranes, lower MIC, more stability etc. changing effectiveness
Where do we get the new genes from?
-alter the genes we have -acquire new genes
altering antibiotic
-altering so it's no longer able to bind to it's target -alter beta lactam ring -use enzyme, beta-lactamase, cleaves open the ring -antibiotic can no longer bind, no longer inhibit cell growth
antibiotics
-antibacterial agents -taken internally, work by interacting and inhibiting bacterial enzymes -can be bactericidal or bacteriostatic
How do antibiotics work?
-binding to specific enzymes in bacteria and by doing so, they interact like a lock and key, interaction inhibits function of particular enzyme -some target DNA replication, others transcription, translation, injure plasma membrane by forming pores, target cell wall synthesis
streptomycin
-binds to 16s rRNA -inhibits protein synthesis
ethryomycin
-binds to 23s rRNA -inhibits protein synthesis
Ciproflaxin
-binds to DNA gyrase -inhibits DNA replication/transcription
zone of inhibition
-clearing zone in disk diffusion assay -where you see inhibition/killing of microbes due to chemical presence -larger on some and smaller on others
Cells can become resistant to beta-lactam antibiotics by which of the following mechanisms?
-conjugation -transformation -spontaneous mutation
mecA regulatory elements, mecR1, mec1
-determine when mecA is made -you don't want to make it all the time -want to be able to control when it's made -ex: only make it when antibiotic is around
Causes Food Bourne Illness
-direct infection -toxins produced by bacteria growing on the food
mecA
-encodes transpeptidase that has slightly different structure and beta-lactams can't bind to it -provides resistance
inhibition of protein synthesis antibiotics
-erythromycin -streptomycin
altering target
-ex: altering transpeptidase -through some means, produce alternative transpeptidase -slightly different active site shape, still functions in bacterial cell -antibiotics cannot bind to this active site, no longer able to inhibit cell growth
What are three ways bacteria can protect themselves against UV light?
-excision repair -SOS system -photolyase
Zones of clearing in Kirby-Bauer
-measure zones of clearing -look them up on a table -will tell us if that antibiotic on that bacteria is considered sensitive, intermediate or resistant
Salmonella
-most common reason for hospitalization -found in poultry, dairy
Staphylococcus aureus
-opportunistic pathogen -30% of us carry it -if you become immune compromised, then it's a problem
Beta-lactams class inhibition of cell wall synthesis antibiotics
-penicillin -amoxicillin -cephalosporins
mecA gene
-provides beta-lactam resistance in Methicillin Resistant Staphylococcus aureus (MRSA) -encodes alternative transpeptidase
Amoxcillin
-semi synthetic version of penicillin -added amino group that can penetrate outer membrane of bacteria and attack cell wall
UV light
-short wavelength -non-ionizing radition
Standardization of Kirby-Bauer includes:
-type of media (Mueller-Hinton agar) -thickness of media - chemicals diffuse in all directions including down -density of cells - how fresh and how many cells are added -commercially produced antibiotic disks with known concentrations of antibiotic impregnated on the disks
Acquire new genes
-typical route -horizontal gene transfer -transformation, conjugation, transduction -pick up DNA that may be able to encode for a beta-lactam resistance gene -alternative transpeptidase or beta-lactamase -transfer from other cells of same species that have already found the DNA or they are mutated, carried to you via phage via transduction -bacterial population actively conjugate with one another so that many bacteria are resistant
Altering our own genes
-very difficult to get a protein that will act on the antibiotic and actually cleave open beta-lactam ring -through spontaneous mutations -DNA pol will make a mistake which will make a slightly altered protein, one amino acid may be able to change structure just enough so antibiotic won't bind
Indirect and Direct Repeats (IR and DR sequences)
-when mobile element is cut and paste, leaves molecular scar in the form of these repeats -look for these and it will tell us that the genetic element is mobile -this came from outside of the cell and was picked up via horizontal gene transfer
A mutation in which of the following would make a bacterium resistant to streptomycin?
16S rRNA
thymine dimer
2 thymine bonds next to each will bond with each other instead of the adenine across -caused by UV exposure -not immediately lethal, but issue when replication tries to occur -if bacteria isn't actively replicating, then it may not die in the moment
What ranges of UVC does DNA absorb UV the best?
200-280nm
AFTER GRINDING 25 GMS OF CHOPPED MEAT IN 70 ML OF BUFFER. 0.1 ML OF THE MIXTURE WAS PLATED OUT ON A MACCONKEY AGAR PLATE. AFTER OVERNIGHT INCUBATION, 270 COLONIES WERE SEEN. 120 OF THEM WERE PINK AND 150 OF THEM WERE BEIGE. WHAT IS THE CONCENTRATION OF POTENTIAL COLIFORMS IN THE MEAT?
4800 CFU/GM OF CHOPPED MEAT (ONLY THE PINK COLONIES FERMENTED LACTOSE, SO THE OTHERS COULD NOT BE COLIFORMS) 25/100 = 1/4 DILUTION. THE CDF= 4. CFU/GM = (120 CFU X 4)/0.1 ML = 4800 CFU/ML
What is the optimal conditions for pasteurization?
72C for 15 seconds
WHAT IS A SECOND WAY TO TEST FOR MANNITOL FERMENTATION BESIDES AN MSA PLATE?
A MANNITOL DURHAM FERMENTATION TUBE
Mueller-Hinton agar
A common agar medium used for anti-microbial susceptibility testing. -has starch in it that bind to toxins that bacteria produce that inhibit antibiotic activity -rich, complex media that allows most non-fastidious bacteria to grow on it
Using these figures, explain why some antibiotics are more effective against gram (+) bacteria than gram (-) bacteria.
Antibiotics are more effective against gram positive bacteria because it does not have an outer membrane protecting it's cell wall. As you can see in the drawing, β-lactams can bind to the transpeptidase easier because they are more exposed. On the gram negative drawing, the outer membrane protects the transpeptidase and makes it more difficult for the β-lactams to bind.
Consider the mechanism of actions for the antibiotics that were effective against all the bacteria. Why do you think these antibiotics seem to be immune to the structure of the cell envelope?
Ciprofloxacin was the most effective causing all 3 strains of bacteria to become susceptible. This antibiotic binds to DNA gyrase and inhibits DNA replication/transcription. It could be the most immune because it affects DNA replication directly causing transcription and eventually translation to be stopped. It stops the process much faster than other antibiotics.
Did the same antibiotic work the best on all strains? If not, which worked best on the gram positive strain?
Ciprofloxacin works great on all strains, leaving them all susceptible. All worked well on the gram positive strain, S. aureus, except erythyomycin which was considered to have intermediate sensitivity.
WHAT IS THE DIFFERENCE/SIMILARITIES BETWEEN ENTEROBACTERIACEAE AND COLIFORMS?
ENTEROBACTERIACEAE ARE A FAMILY OF GRAM NEGATIVE BACILLI THAT INCLUDES SOME PATHOGENS AND SOME NON-PATHOGENS. THEY ARE ALL GRAM NEGATIVE BACILLI. COLIFORMS ARE A SUB-GROUP OF ENTEROBACTERIACEAE THAT IN ADDITION TO BEING GRAM NEGATIVE BACILLI, ALSO FERMENT LACTOSE TO PRODUCE ACID AND GAS.
Bacillus megaterium is able to induce endospore formation, yet both cultures of Bacillus megaterium are not equally resistant to UV light. What does that tell you about endospore formation and UV light?
Endospore formation is induced in culture that has been sitting out for a week and is considered in a stationary phase. The old culture of B. megaterium is growing in a high density and is in a nutrient depraved environment compared to the fresh broth of B. megaterium, thus inducing endospore formation. This also shows that UV light has a much more difficult time killing bacteria in an endospore state, but after a long exposure, even it will start deteriating.
WHY IS IT A PROBLEM TO LEAVE A GAS INFECTION UNTREATED?
IF LEFT UNTREATED, YOUR BODY WILL INDUCE ANTIBODIES TO RID ITSELF OF THE MICROBE. THESE ANTIBODIES CAN CROSS-REACT WITH PROTEINS IN THE HEART VALVE, LEADING TO DEGENERATIVE HEART DISEASE KNOWN AS RHEUMATIC FEVER.
YOU STREAK BACTERIA ON A TSI SLANT. AFTER INCUBATION THE BOTTOM LOOKS YELLOW AND THE TOP LOOKS PINK. WHAT CAN YOU CONCLUDE ABOUT THIS BACTERIA?
IT CAN FERMENT GLUCOSE BUT NOT LACTOSE OR SUCROSE. IT CAN NOT REDUCE SULFATE TO H2S.
WHAT WOULD A TSI TUBE LOOK LIKE IF THE BACTERIA INCUBATED IN IT WERE A COLIFORM?
IT WOULD BE YELLOW THROUGHOUT IT AND THERE WOULD BE CRACKS IN THE AGAR DUE TO GAS PRODUCTION.
Please explain why spoiled juices and milks tend to taste sour.
Milk and other spoiled juices tend to taste sour because bacteria convert lactose in milk to lactic acids. When you open the milk/juices, bacteria such as coliforms can enter into the product, they produce acid, giving the milk/juice it's sour taste.
In our analysis of the microbial load of various foods, did we find any to contain Salmonella? How do you know?
No. Salmonella reduces sulfate so on an HE plate it would appear black and we saw no black colonies.
Did all the antibiotics work equally well on the gram negative strains? Explain.
On the gram negative strain, E. coli, both amoxicillin and ciprofloxacin showed susceptible sensitvity. Erythomycin for E. coli showed intermediate sensitivity. When tested with the penicillin however, the E. coli showed resistance. On the other gram negative strain, P. aeruginosa, most of the antibiotics had difficulty killing/inhibiting this bacteria. Penicillin, erythromycin, and amoxicillin all showed resistance. Ciprofloxacin was the only antibitoic to successfully show susceptibility in the P. aeruginosa strain. This information shows the antibiotics did not work equally well on the gram negative strains. This may be because the P. aeruginosa had more resistant genes than the E. coli did or it may have had better defense mechanisms against the antibitoics. P. aeruginosa is known to have efflux pumps that are in the membranes and pump out the toxins including antibiotics. They are known to be multi drug resistant.
WHAT IS ALPHA HEMOLYSIS AND WHAT DOES IT LOOK LIKE ON A BLOOD AGAR PLATE?
PARTIAL BREAKDOWN OF RED BLOOD CELLS RESULTING IN A GREENISH HUE
WHAT IS THE DIFFERENCE BETWEEN PASTEURIZATION AND UHT PASTEURIZATION?
PASTEURIZATIONS IS THE HEATING OF FOODS TO LESS THAN 100C TO ELIMINATE MOST PATHOGENS. THIS DOES NOT MAKE THE FOOD STERILE. UHT PASTEURIZATION INVOLVES HIGHER HEAT AND STERILIZES THE FOOD. THIS ELIMINATES ALL MICROBES AND EXTENDS THE SHELF-LIFE OF THE FOOD.
Penicillin inhibits PBP's β-lactam inhibits PBP's
PBP's are a great target for antibiotics
If we had included an old culture of E. coli in our UV experiment, do you expect it would have been as resistant to UV light as the old culture of B. megaterium? Please explain your answer.
The reason B. megaterium became resistant to UV when it aged is that it was able to form endospores which are UV-resistant. E. coli is not able to make endospores so stress (like aging) would not make them UV-resistant.
Why might the results of this test not perfectly reflect what works best to treat infections in humans?
The results of this test might not reflect what works best to treat infections in humans because the amount of resistant bacteria may vary from person to person. This would cause the dose of antibiotic to increase. It could also be the opposite and there may be susceptible cells in the human infection causing the dose to decrease. In conclusion, it's hard to standardize drug doses, you have to view the infection on a case by case basis in a clinical setting.
What is Hektoen-Enteric agar differential for?
This agar is differential for sugar fermentation. The differential agents are lactose, sucrose and salacin. It also has bromothyl blue and acid fuchsin which turn yellow when they encounter acid. Also differentiates on sulfate reduction. If we have sulfate and iron in the plate, the iron will encounter hydrogen sulfide which will produce ferrous sulfide. Ferrous sulfide will be black.
β-lactamase inhibits penicillin and β-lactams
because it cleaves the beta lactam ring making it non-functional. MecA is equal to β-lactamase because both inhibit the effects of β-lactams.
MecA inhibits penicillin and β-lactams
because it encodes a different shaped transpeptidase making it hard for the antibiotics to bind
What makes MacConkey agar selective?
bile salts and crystal violet
direct repeats
both are going right
How does UV light damage DNA?
causes pyrimidine dimers -DNA pol cannot replicate past the dimer formation, which inhibits replication
SOS system
cell recognizes DNA damage and induces a system of enzymes to repair it -if you have a thymine dimer and it wasn't repaired and the 2 DNA strands come apart -if DNA pol comes across thymine dimer, one of the DNA pols will put in anything, has 1/16 chance it'll put right base -depending on area, could be in intergenic region or in area where it'll cause silent mutation, might not be lethal
bactericidal agents
chemicals that kill bacteria
Penicillin is approximately equal to β-lactam
classified as a β-lactam antibiotic because it has a β-lactam ring.