Carriage of Beta-Lactamase and Antibiotic Resistance in Staphylococcus aureus

Abstract

Studies have shown that Staphylococcus aureus is one of the leading causes of bacteria infections in human and it has developed different resistance patterns to different antibiotics, making it a bit difficult to eradicate completely. This study focuses on the resistance of S. aureus producing beta-lactamase.

1. Introduction

Staphylococcus aureus is an organism that causes various forms of diseases ranging from simple diseases with little or no cause for alarm to severe life threatening diseases [1, 2]. Antibiotic resistant S. aureus have been involved in high death rates across the world [3]. For many years till now, antibiotics have been employed in the treatment of Staphylococcus infections but resistance to antibiotics commonly used against them has reduced the effectiveness of the antibiotics [4, 5]. Mec A gene which is located on the staphylococcal cassette chromosome enables the organism to develop resistance against antibiotics used against it [6, 7, 8]. An altered 75 kb penicillin binding protein 2A (PBP2a) is produced inside the mec A gene which also gives it its resistance properties to antibiotics especially the beta-lactam [9]. Beta lactam antibiotics are the most common antibiotics used in the treatment of S. aureus infections. They act on the synthesis of peptidoglycan during the cell wall formation and stops it by acting on transpeptidases and carboxypeptidases [10]. Beta-lactamase is also known as penicillinase. Beta-lactam antibiotics contain a beta lactam ring and they act by stopping cell wall synthesis of the bacteria. Exampes of beta-lactam antibiotics include penicillin, cephalosporin, carbapenems and carbacephems. As at 2003, beta-lactam antibiotics were the world most used antibacterial drugs. These antibiotics stop the cell wall synthesis by inhibiting penicillin binding protein involved in the cross linking of the peptidoglycan which then causes the bacteria cell to succumb to osmotic pressure [11].

2. Beta-lactamases in S. aureus

Beta-lactamases are enzymes produced by S. aureus in order to breakdown the beta lactam ring of beta lactam antibiotics which causes resistance to beta lactam antibiotics [12]. Beta-lactamase is an enzyme produced outside the cell of S.aureus after it is exposed to beta-lactam antibiotics [13]. Beta lactamase is constitutive or inductive plasmid mediated. The ability of beta-lactamase to resist antibiotics used against it is largely dependent on its chemical reaction, location and physiochemical conditions (see Figure 1) [14].

Figure 1.

Resistance mechanism of S .aureus to beta-lactam antibiotics. Source: [15]. Keys: MSSA, Sethicillin susceptible S.aureus; MRSA, methicillin Resistant S. aureus.

Beta lactamase is currently classified into two groups which are the molecular classification and Class B metalloenzymes [16]. The molecular classification classifies beta lactamase into Class A, C and D. Class A, C and D enzymes form acyl enzyme by making use of serine for the breakdown of beta-lactam bonds. Class B metalloenzymes makes use of divalent zinc ions for the breakdown of substrate [16].

2.1 Test for Beta-lactamase

After antibiotic susceptibility testing, the test for beta-lactamase is performed. There are different methods of testing for the presence of beta-lactamae which includes:

1. Nitrocefin disks: after acquiring nitrocefin commercial discs, stock them at −10 degree Celsius until the time of use. Soak the discs in a saline solution and use a sterilized wire loop to take some colonies from the plate and streak on the surface of the disc. Observe the disc within 60 minutes. When the color changes from yellow to pink, it indicates a positive reaction which shows the presence of beta-lactamase [17].

2. Clover leaf test (Hodge test): the presence of an irregular inhibition zone is considered as a positive result [18].

3. Polymerase Chain Reaction detection of blaZ gene: According to a study performed by Lynette et al. [19] to detect blaZ genes in S.aureus using real time PCR assay, after the colonies of S.aureus on Mueller hinton agar were tested with nitrocefin impregnated disc, presence of beta-lactamase was detected with a sharp cliff edge and negative with a tapered beach edge. Phenotypic tests were performed using two independent observers. Two to five loop-full of S.aureus were inoculated in 1 ml demineralized water and placed in a centrifuge for 2 mins. 8 micro liter of the supernatant solution was used with 12 microliter of real time PCR amplification mixes. The mixes contained buffer of PCR, 4 mM MgCl2, 0.2 mM deoxynucleoside triphosphates, 0.75 micro AmpliTaq Gold DNA polymerase, forward and reverse primer [19].

4. Filter paper iodometric method: 1 g of starch is dissolved in 100 ml of distilled water by boiling. 1 g of penicillin was added to the solution. Whatman filter paper cut into small pieces are soaked in the solution and later dried for about 2 hours. The filter papers are inoculated with a loopful of freshly isolated Staphylococcus aureus. The filter papers are then covered with a petri dish and incubated for 30mins at 30 degree Celsius. Iodine solution is poured over the filter paper. Beta-lactamase is positive when the site of inoculation turns colorless within 10mins and negative when it retains the blue-black color of the iodine solution [20].

5. Acidometric test:s Mueller hinton agar is prepared at a pH of 7.4, a small colony of S. aureus is inoculated on the plate. 5 μg methicillin disc is added on the surface of the prepared agar and incubate. After incubation, the plate is opened and dried for some time 1.5 ml of 0.25% stock solution of PNCB (k &k laboratories, Inc., Plainview, N.Y.) in N,N-dimethyl formamide (Fischer Scientific Co., Pittsburgh, Pa) is flooded on the closest area to the zone of inhibition. 6% NaOH is added and air dried for 45 mins. 1.5 ml of 10% aqueous benzyl-penicillinase is flooded on the 6 area with the PNCB indicators. Hydrolysis of benzyl penicillin to penicilloic acid shows the presence of penicillinase. Absence of penicillinase occurs when benzyl penicillin is not changed and when there is no color change in the indicator used.

6. Chromogenic cephalosporin detection methods: Beta-lactamase producers produce a color change with cephalosporin. The color change can be measured by measuring the absorption changes that takes place in the 380 to 500 nm region.

3. Conclusion

This study shows that S. aureus is increasing in its resistance to the beta-lactam antibiotics. S. aureus has developed resistance patterns to beta-lactam antibiotics such as the production of beta-lactamase and production of an altered penicillin binding protein 2A. Different tests have been used to detect the presence of beta-lacatmases in S. aureus. Examples of such tests include nitrocefin disks, clover leaf tests, polymerase chain reaction detection of blaZ gene, chromogenic cephalosporin tests, acidometric tests and filter paper iodometric tests. The iodometric filter paper method is simple, rapid and can be performed in any bacteriological laboratory and the materials can be stocked and kept under optimal conditions.

Acknowledgments

I want to acknowledge My Lord Jesus for his wisdom and understanding in writing this paper.

Conflict of interest

The author declare no conflict of interest.

Thanks

I thank Intechopen for the opportunity given to me to contribute in this journal.