Antibiotics and the effects they have

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Antibiotics are compounds that kill or inhibit microorganisms from growing. Antibiotics can be made from natural products, such as bacteria and fungi, or synthetic chemicals. Individual antibiotics are effective against specific bacteria by selectively targeting or regulating some crucial process in the microbial cells. Bacteria that produce antibiotics to regulate the growth of their neighbors need to develop resistance for self-protection. Antibiotics work by inhibiting the required synthesis and pathways, such as cell wall synthesis, production of proteins required for replication, transcription, and translation, and interrupting phospholipid bilayers to increase cell permeability. Their short generation times may lead to the development of mutations that would possibly give them resistance to different antibiotics. Once a gene has been found to allow a bacterium to become resistant to an antibiotic, the bacterium will be selected for survival advantage. Antibiotic resistance can be obtained through acquiring an R (resistance) plasmid, an extra chromosomal DNA that carries an antibiotic resistance gene.

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The Kirby-Bauer method is one of the common techniques used in clinical laboratories to test susceptibility of different strains of bacteria to an array of antibiotics. This technique allows us to observe the minimum inhibitory concentration (MIC) of antimicrobial activity. The MIC is the smallest concentration of the antibiotic that will stop the growth of bacteria. In order for the antibiotic to be effective against the bacteria, the MIC must be present at the site of infection. The procedure works by isolating a pure strain of bacteria from a source and is uniformly spread onto Mueller-Hinton agar. Small filter paper discs that contain different antibiotics are suspended onto the surface of the agar plate. The antibiotic will diffuse into the Mueller-Hinton agar plate and this will produce a clearing around the disc that will inhibit bacterial growth if the bacteria do not have a resistance gene for the antibiotic. Susceptibility can be determined by measuring the diameter of the zone of growth inhibition that is produced around the antibiotic paper disc. The objective of the antimicrobial susceptibility testing is to compare the antimicrobial capabilities of Gram-negative bacteria and Gram-positive bacteria from Enterobacter spp. and Staphylococcus aureus, respectively. The results from the Kirby-Bauer method are then compared to the standard results in the CLSI Document M100-S17 (M2): Disk Diffusion Supplemental Tables, Performance Standards for Antimicrobial Susceptibility Testing (Woolfolk et al., 2004).

Even with all the antibiotics and vaccines that have been discovered up until today, there will never be enough antibiotics. There will always be an antibiotic resistance problem. These antibiotic-resistant pathogens are increasing, especially in hospitals. Some bacteria are even resistant to multiple antibiotics. The deterioration in social conditions has also shown to increase the spread of infectious diseases. As medicine advances, antibiotic resistance increases because over time bacteria can evolve and develop resistance. Antibiotics have been overprescribed and used incorrectly, such as not following the dosage instructions given by the doctor. Antibiotics are also inappropriately prescribed for virus infections, which would not have any effect. Continuous over-dosage of antibiotics can also kill the normal flora that protects us from some pathogens and toxins (Walsh, 2003).

Results

Genus name: Gram positive isolate Gram negative isolate

Staphylococcus aureus Enterobacter spp.

Antibiotic

Zone of inhibition (mm)

Recorded sensitivity

Expected sensitivity

Zone of inhibition (mm)

Recorded sensitivity

Expected sensitivity

AMOXICILLIN/ Clavulanic Acid

38

Susceptible

Susceptible

40

Susceptible

Susceptible

AZITHROMYCIN

30

Susceptible

Susceptible

31

Susceptible

Resistant

CEPHALOTHIN

40

Susceptible

Susceptible

34

Susceptible

Susceptible

CIPROFLOXACIN

40

Susceptible

Susceptible

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39

Susceptible

Susceptible

GENTAMICIN

30

Susceptible

Susceptible

24

Susceptible

Susceptible

PENICILLIN G

27

Intermediate

Susceptible

21

Resistant

Resistant

PIPERACILLIN

28

Susceptible

Susceptible

24

Susceptible

Susceptible

POLYMYXIN B

18

Susceptible

Resistant

9

Intermediate

Susceptible

RIFAMPIN

44

Susceptible

Susceptible

35

Susceptible

Resistant

SULFADIAZINE

0

Resistant

Susceptible

0

Resistant

Susceptible

TETRACYCLINE

35

Susceptible

Susceptible

30

Susceptible

Susceptible

VANCOMYCIN

22

Susceptible

Susceptible

14

Resistant

Resistant

Discussion

The interpretive standards for the Kirby-Bauer technique was used to determine whether the bacteria are susceptible, intermediate, or resistant to the antibiotic (Woolfolk et al., 2004). After 48 hours in incubation at 37°C, each plate is examined. If the bacteria streak was done properly, there should be a lawn of bacteria growth. Improper streaking (e.g. highly diluted sample, light streaking) will lead to the presence of individual colonies. If bacterial growth is inhibited by the MIC at the site of infection, the organism is considered to be susceptible. The intermediate category means that bacterial growth is still observed within the expected circumference of the expected circular clearing, but not as much as susceptible bacteria. If bacterial growth is still observed in the presence of the antibiotic, the organism is considered to be resistant to the antibiotic.

The S.aureus isolate is resistant to sulfadiazine, intermediate to penicillin G, and susceptible to all of the other antibiotics. The Enterobacter spp. isolate is resistant to penicillin G, sulfadiazine, intermediate to polymyxin B, and susceptible to all of the other antibiotics. Not all of the isolates conform to the pattern of antibiotic resistance sensitivity. The unexpected resistance of these bacteria may be due to the fact that I was previously exposed to ampicillin and kanamycin in a research lab.

The Enterobacter spp. isolate was unexpectedly susceptible to azithromycin and rifampin. Azithromycin and rifampin are not supposed to inhibit the growth of Gram-negative bacteria. In an agar dilution method carried out by Chayani et al. (2009), Enterobacter spp. isolates were found to have a 0% susceptibility to azithromycin with an MIC < 8µg/mL and 100% resistance to azithromycin with an MIC > 8µg/mL, and 33.33% susceptibility to azithromycin by the use of the disk diffusion method. Azithromycin is a broad-spectrum antibiotic that inhibits protein synthesis by binding to the 50S rRNA. The 50S subunit is common in all prokaryotes; therefore it is possible that azithromycin can target the 50S subunit in the Enterobacter spp. isolate. Rifampin is a broad-spectrum antibiotic and is mainly active against Gram-positive bacteria and can have minimal effect on Gram-negative bacteria. The antibiotic specifically acts on DNA-dependent RNA polymerase, which blocks mRNA synthesis and interferes with nucleic acid metabolism. In Kerry et al.'s study (1975), rifampin was shown to have an effect against all strains of Enterobacter spp. Enterobacter spp.'s resistance to penicillin G was expected, but its resistance towards sulfadiazine does not conform to the standard of antibiotic resistance sensitivity.

The S.aureus isolate was unexpectedly susceptible to polymyxin B and resistant to sulfadiazine. Polymyxin B inhibits the growth of Gram-negative bacteria by interfering with the phospholipids, therefore increasing cell permeability. This antibiotic does not have much effect on Gram-positive bacteria because the cell wall is too thick for the antibiotic to get access to the membrane. It is possible that the concentration of the polymyxin B was much greater than the S. aureus isolate density, allowing the antibiotic to be more effective and efficient to killing Gram-positive bacteria. Polymyxin B was found to have little effect on different strains of S. aureus, but nonetheless there was still some effect against Gram-positive bacteria (Scott et al., 1999). Lipoteichoic acid (LPA) is a large component of the cell wall of Gram-positive bacteria. Polymyxin B contains a cationic structure that would interact with the LPA of Gram-positive bacteria because the structure of LPA contains an overall negative charge.

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Sulfadiazine is part of the family of sulfonamide antibiotics. Bacterial resistance to one sulfonamide antibiotic can lead to resistance to all antibiotics within the sulfonamide family. Sulfadiazine has a wide spectrum that works on both Gram-negative and Gram-positive bacteria, which could explain why Enterobacterspp. and S. aureus isolates were both resistant to sulfadiazine. Sulfadiazine interferes with the production of folic acid, which is required for bacterial growth. Sulfadiazine inhibits p-aminobenzoic acid (PABA), which interferes with the folic acid metabolism cycle because PABA is normally converted to folic acid by the bacteria (Rosenkranz et al., 1974). A possible reason to how both Enterobacterspp. and S. aureus became resistant to sulfadiazine is because both isolates were taken after exposure to the kanamycin antibiotic. In a study done by Rosenkranz et al. (1974), they noticed that isolates of Enterobacter cloacae that were resistant to sulfadiazine are also resistant to carbenicillin and kanamycin. The resistance to carbenicillin and kanamycin suggested the possibility that there is a presence of an R plasmid which would carry the determinants for carbenicillin and kanamycin resistance. Strains with an R plasmid displayed an enhanced resistance to sulfadiazine. R plasmids carry the resistance gene that encodes proteins to undergo various mechanisms to bypass the antibiotic, such as inactivating antibiotics via chemical modification, blocking the antibiotic from getting into the cell and removing the antibiotic if it does get into the cell, creating a substitute target for the antibiotic, or have alternative pathways that are not sensitive to the antibiotic (Woolfolk et al., 2004).

Some approaches, such as new test methods, different microorganisms, and variation of culture conditions, have been used to improve the chances of finding new substances. Microbiologists are now examining conserved open reading frames that are unique to prokaryotes and not eukaryotes. Old targets, such as cell wall biosynthesis, protein biosynthesis, and DNA replication and repair, are being studied more thoroughly to develop new and more effective antibiotic. There have also been new targets, such as bacterial fatty acid, isoprenoid, isocitrate lyase, and lipid A in Gram-negative bacteria, which are speculated to be susceptible to new antibiotics (Walsh, 2003). New antibiotics are being developed as we increase our knowledge of bacterial mechanisms and physiology, but in order for antibiotics remain effective against bacterial infections antibiotics must be prescribed and taken in correct dosage and to relevant infections.