Vancomycin and Resistance in Staphylococcus aureus Due to the Presence of a D-ala-D-lactate Mutation in Bacterial Cell Walls
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XX was a 39 year old female presenting to the Aurora Hospital in Hartford, Wisconsin complaining of a reddened, painful, and swollen area in her left axilla region. The patient found the condition to be irritating, and she wanted to have it evaluated before it possibly worsened. Upon physical examination, the area showed visible hallmarks of inflammation as evidenced by redness of the skin, tenderness to movement and palpation, and swelling extending from the armpit out into the surrounding tissue. At the time of admission, the patient was found to have a normal white blood cell count but was running a low-grade fever. In addition, infection of the localized blood vessels known as thrombophlebitis, was also suspected. The physician believed this patient had a soft skin and tissue infection (SSTI) possibly caused by methicillin resistant Staphylococcus aureus (MRSA) based on the physical presentation of the affected area.
While the blood results were pending, the patient was started on empiric vancomycin 1250 mg IV every 12 hours based on the physician's suspicion of a MRSA infection. The infusions were given over a two hour period to reduce the risk of an infusion related reaction such as 'red man syndrome.' The patient did not respond to the vancomycin treatment as quickly as expected based on serum trough levels, and the dose had to be increased. The physician was starting to wonder if perhaps this patient had a strain of Staphylococcus aureus which was resistant to vancomycin. This paper will discuss the molecular binding and mechanism of action of the antibiotic vancomycin, a widely used drug for the treatment of MRSA. In addition, a molecular mechanism for MRSA resistance to vancomycin will be presented.
Resistance to antibiotic therapy has been well documented in the medical literature from the 1940s to the present. Shortly after the introduction of the first antibiotic, penicillin, resistance was documented in various bacterial species (Barber & Rozwadowska-Dowzenko, 1948), including Staphylococcus aureus (Rammelkamp & Maxon, 1942). As a result of the growing resistance to penicillin, additional antibiotics were needed to combat infections. This led to the creation of a new derivative of penicillin called methicillin. Unlike penicillin, methicillin was resistant to inactivation by the bacterial enzyme β-lactamase (Otto, 2012). The chemical structure of methicillin was based on benzylpenicillin but had two methoxyy groups substituted on the phenol. 'The methoxy groups produced steric hindrance around the amide bond reducing its affinity for staphylococcal β-lactamases' (Stapleton &Taylor, 2002, 58).
Methicillin was introduced in 1959 (Otto, 2012) and used to treat Staphylococcus aureus infections, but within the following year, there were cases of MRSA in the United Kingdom (Jevons, Coe, & Parker, 1963). Resistance was found to be due to a penicillin binding protein (PBP) called PBP 2a (also called PBP2') which provides MRSA with its β-lactam resistance (Utsui & Yokota, 1985). This particular PBP has much less affinity for binding to β-lactam antibiotics (Utsui & Yokota, 1985). PBP 2a is found in the mecA gene of the MRSA bacterial chromosome (Katayama and Hiramatsu, 2000). MRSA strains may be capable of growing in the presence of certain β-lactam antibiotics because of the acquisition of the specific 78,000-dalton PBP fraction which can act as murein transpeptidase for cell wall biosynthesis despite the presence of β-lactam antibiotics (Utsui & Yokota, 1985, 401).
Vancomycin was developed in the 1950s in response to a lack of treatment options for penicillin-resistant and methicillin-resistant staphylococcal infections (Levine, 2006). Historically, vancomycin has been used to treat acute staphylococcal ileocolitis due to Clostridium difficile (Levine, 2006) and general infections caused by gram-positive bacteria (Nitanai, Kikuchi, Kakoi, Hanamaki, Fujisawa, & Katsuyuki, 2009). Vancomycin has been used to treat cases of MRSA worldwide, but by the late 1980s a new threat had emerged, Staphylococcus aureus that is resistant to vancomycin (Levine, 2006).
Vancomycin has a molecular mass of 1449.3 Da and is a glycopeptide antibiotic (Hirao, Ikdea-Dantsuji, Matsui, Yoshida, Hori, Sunakawa, Nakae, & Hanaki, 2012, 70). Vancomycin is a time-dependent antibiotic which is given by intravenous infusions due to poor oral absorption due to its lipophilic nature (Rybak, 2006). 'Vancomycin is one of only a few antibiotics available to treat patients infected with methicillin-resistant Staphylococcus aureus' (Rybak, 2006, S35).
The integrity of staphylococcus cell walls derives from a substance called peptidoglycan which is created from staggered N-acetylmuramic acid and β-1,4-N-actetylglucosamine residues (Geisbrecht, Kersten, Maidhof, & Wecke, 1998). Each of the N-acetylmuramic acids has a four unit stem peptide attached to it consisting of L-alanine, D-glutamine, L-lysine, and D-alanine (Geisbrecht, et al., 1998). The stem peptides are then cross-bridged with other stem peptides using a chain of five glycine residues (Geisbrecht, et al, 1998). The cross-linking of the cell wall process is catalyzed by a bacterial enzyme called transpeptidase, namely the PBPs (Geisbrecht, et al., 1998). 'The interaction of beta-lactams with the PBPs is known to result in an inhibition of peptidoglycan cross-linking' (Geisbrecht, et al., 1998, 1393).
According to Nitanai, et al. (2009), vancomycin targets the D-ala-D-alanine region of the growing peptidoglycan backbone of the cell wall of gram positive bacteria. It does so because the backbone of vancomycin is able to hydrogen bond with the backbone of the growing peptidoglycan chain. The main players of this interaction are the nitrogen on the second molecule of the antibiotic backbone and the oxygen stemming from the fourth molecule of the antibiotic backbone.
Bacteria that are resistant to vancomycin cause conformational changes in vancomycin that move the oxygen on molecule four farther away from the hydrogen bond site on the developing peptidoglycan, reducing the affinity of vancomycin for the targeted region (Nitanai, et al., (2009). One example of this is demonstrated in figure 6, which denotes the conformational change that occurs when D-lactic acid is substituted for the terminal D-alanine at the target location.
Vancomycin targets the terminal D-alanyl-D-alanine of the growing cell wall peptidoglycan structure (Levine, 2006). In strains of Staphylococcus aureus that are resistant to vancomycin, the bacterial cell produces an altered intermediate D-alanyl-D-lactate or D-alanyl-D-serine peptidoglycan so that vancomycin cannot properly bind (Levine, 2006). In β-lactamase producing organisms, peptidoglycan metabolism is enhanced, and higher vancomycin minimum inhibitory concentrations (MIC) are needed. In such a situation, a more rapid cell wall turnover allows the bacteria to survive despite the presence of vancomycin.
Decreased susceptibility to vancomycin can be due to several factors including thickened cell walls, reduced cell-wall turnover, reduced autolysis, and increased cell wall synthesis (Carfiso, Bertuccio, Spina, Purrello, Campanile, De Pietro, Purrello, & Stefani, 2012). There are also genes such as mprF and dltA which mediate vancomycin reduced susceptibility (Carfiso, et al., 2012). Lysine-phosphatidylglycerol is a major component of bacterial cell walls, and an increased production of this variant will decrease the overall negative charge of the cell membrane which normally aids the positively charged vancomycin to bind appropriately (Carfiso, et al., 2012). Positively charged phospholipids can also be added to the outer portion of the cell membrane further inhibiting vancomycin penetration (Carfiso, et al., 2012).
The patient's dosing of vancomycin was based on Aurora's vancomycin dosing nomogram. The patient was initially dosed at 1250 mg vancomycin IV every 12 hours. After the first vancomycin trough level was obtained, the patient's dose was adjusted to 1500 mg vancomycin IV every 12 hours. The subsequent vancomycin trough levels were within the desired range, so the patient's dose was not changed. Upon visual inspection over the course of the next several days, the infected area showed less redness, less swelling, and was less tender to the patient indicating that the infection was being reduced. Although the blood cultures were not positive for MRSA, the infection was due to a gram-positive bacterial species, and both the pharmacist and physician agreed that vancomycin was an appropriate treatment course.
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