Assessment of methicillin resistant Staphylococcus Aureus detection methods: analytical comparative study

Introduction The heterogeneous expression of methicillin resistance in Staphylococcus aureus (MRSA) affects the efficiency of tests available to detect it. The objective of this study was to assess four phenotypic tests used to detect MRSA. Methods This is an analytical comparative study conducted among sudanese patients during period from May 2012 to July 2014, Staphylococcus aureus strains were isolated and identified by conventional methods, and then confirmed by PCR detection of coagulase gene. PCR detection of mecA gene was used as a gold standard to assess oxacillin resistance screen agar base (ORSAB), oxacillin disc, cefoxitin disc (at different temperatures and incubation periods) and MRSA-latex agglutination test. S.aureus ATCC 25923 was used as control. Sensitivity and specificity were calculated. Results MRSA- latex agglutination was the most accurate test; it showed 100% of both sensitivity and specificity, followed by cefoxitin disc with sensitivity of 98.48% and specificity of 100%. However, both of oxacillin disc and oxacillin resistance screen agar base showed less accurate results, and were affected by incubation periods. Oxacillin disc after 24 h incubation both at 30°C and 35°C showed sensitivity and specificity values of 87.88% and 96.23%, respectively. However, after 48h incubation the test at 30°C showed sensitivity and specificity values of 89.39%, and 94.34%, respectively. At 35°C (48h) it showed values of 89.39%, 92.45% respectively. Specificity of ORSAB was more than oxacillin disc at 35°C after 24h incubation 98.11% and 96.23%, respectively. Conclusion MRSA- latex agglutination and cefoxitin disc diffusion tests are recommended for routine detection of MRSA.


Introduction
Methicillin resistance in staphylococci is primarily due to the acquisition of a mobile staphylococcal chromosomal cassette which carries the mecA-gene, known as SCCmec [1]. This mecA-gene encodes an altered PBP, PBP2a (or PBP2') [2]. The affinity of betalactams towards PBP2a is much lower than towards native PBP2, thus allowing continuous cell wall assembly [3]. Many clinical MRSA isolates express resistance to methicillin heterogeneously. This means that the majority of cells are susceptible to low concentrations of methicillin, and only a minority of cells can grow at high concentrations [4]. Reliable microbiological diagnosis of MRSA is essential for treatment, surveillance and control. Clinical microbiology laboratories play a central role in the detection, identification, antibiotic susceptibility testing, and confirmation of MRSA. Conventional laboratory detection of MRSA includes culturing the specimen, confirmation of S. aureus with identification tests, antimicrobial susceptibility testing, and finally, verification of MRSA, usually with molecular methods. This may take several days. Rapid diagnostic testing for MRSA directly from specimens allows the infected patient to obtain a more rapid verification of antimicrobial therapy, leading to a decrease in mortality, a reduction in vancomycin usage, shorter stays in hospital, and lower hospital costs [5]. Rapid tests are still more expensive than conventional ones, and not all laboratories are able to use them for financial reasons. Regardless of the diagnostics methods used, concomitant cultures are necessary to recover the organism for further antimicrobial susceptibility testing and for epidemiological typing [4]. The determination of methicillin resistance is importance in prognosis of S. aureus infection. The phenotypic detection methods are cheaper than genotypic but they are dependent up on environmental conditions. Therefore, the golden methods for detection of MRSA were Polymerase Chain Reaction (PCR) through detection of the mecA gene or its product latex agglutination.
However, the limitation of PCR to the reference labs leads the latex agglutination methods to be the best predictor to detect MRSA [6].
Therefore the objective of this study was to investigate the performances of four phenotypic tests used to detect MRSA namely cefoxitin disc, oxacillin disc, oxacillin resistance screen agar base (ORSAB) test (at different temperatures and incubation periods), and MRSA-latex agglutination test, comparing them with the performances of gold standard PCR for detection of mec A gene.

S. aureus isolates
One hundred and nineteen of Staphylococcus aureus (S.aureus) isolates (66 mecA gene positive and 53 mecA gene negative) were included in this study .The isolates were from skin infections and anterior nares that were collected from patients at Kosti and Rabak hospitals, Sudan during the period from May 2012 through July 2014. Isolates were identified as S.aureus using conventional tests and confirmed by PCR detection of coagulase gene. Then they were tested for presence or absence of mecA gene.

DNA extraction for amplification
G-spinTM Genomic DNA Extraction Kit was used for genomic DNA extraction. The kit designed for rapid isolation of genomic DNA, uses advanced silica-gel membrane technology column for rapid and efficient purification of genomic. Buffer system was optimized to allow rapid and simple cell lysis followed by selective binding of DNA to the column. The DNA extraction was performed according to the manufacturer instructions.

Coagulase gene (coa) amplification:
PCR amplification of the coagulase (coa) gene was done using (iNtRON´s Maxime PCR PreMix) master mix. The kits components in one tube for 1 reaction PCR, were Taq DNA polymerase 2.5U, dNTPs 2.5mM each, Buffer 1x and Gel Loading buffer 1x. The components were dissolved in distilled water into the tubes to a total volume of 20µl including volume of the DNA template 2µl, and primers (75 P moles) 0.75µl for each of the primer. The forward primer 5'ATA GAG ATG CTG GTA CAG G3' and the reverse primer 5'GCT TCC GAT TGT TCG ATG C3'. were used. Cycling takes place on a Thermal cycler following an initial denaturation at 94°C for 45 s. The cycling proceeded for 30 cycles of 94°C for 20 s, 57°C for 15 s, and 70°C for 15 s with a final step at 72°C for 2 min. The PCR products (5µl) were analyzed using horizontal 1.5% agarose gel electrophoresis using tris-borate EDTA buffer [7].

MRSA detection by PCR to look for presence of mecA gene:
PCR was done using (iNtRON´s Maxime PCR PreMix) master mix.
The kits components in one PCR tube for 1 PCR reaction, Page number not for citation purposes 3 were Taq DNA Polymerase 2.5U, dNTPs 2.5mM each, Buffer 1x and Gel Loading buffer 1x. The components were dissolved in distilled water into the tubes to a total volume of 20µl including volume of the DNA template 2µl and primers (20 P moles) 2µl for each of the primers. The forward primer corresponds to nucleotides 1282 to 1303(5´ AAAATCGATGGTAAAGGTTGGC) and the reverse primer complementary to nucleotides 1793 to 1814 (5´AGTTCTGC AGTACCGGATTTGC) were used. Cycling takes place on a Thermal cycler following an initial denaturation at 94°C for 45 s. The cycling proceeded for 30 cycles of 94°C for 20 s, 57°C for 15 s, and 70°C for 15 s with a final step at 72°C for 2 min. The PCR products (5µl of each) were analyzed using horizontal 1.5% agarose gel electrophoresis and tris-borate-EDTA buffer [7].

Assessment of MRSA resistance detection methods
PCR detection of mecA gene was used as gold standard to assess ORSAB (at 35ºC for 24 h and 48 h), oxacillin disc test (at 30ºC and 35ºC for 24 h and 48 h) cefoxitin disc diffusion test (at 30ºC and 35ºC for 24 h and 48 h) and MRSA-latex agglutination test. S.
Aureus ATCC 25923 was used as control.

MRSA detection by Oxacillin Resistance Screen Agar Base (ORSAB) test
Ten microlitres a 0.5 McFarland standard suspension of S.aureus isolates were inoculated onto ORSAB (Oxoid). It is a nutritious and selective medium containing a high salt concentration and lithium chloride to suppress non-staphylococcal growth with mannitol and aniline blue for the detection of mannitol fermentation.
It was supplemented with 2mg/l oxacillin to suppress MSSA and 50,000IU/l of polymyxin B to suppress Gram negative bacteria.
Plates were incubated at 35°C, and examined after 24h and 48h, S. aureus was considerd MRSA when revealed growth on ORSAB as dark blue colonies due to fermentation of mannitol.

MRSA detection by cefoxitin and oxacillin disc diffusion methods
Susceptibility of S. aureus isolates were tested against cefoxitin (30µg), oxacillin (1µg), by the (CLSI) agar disc diffusion method [8]. and left for few minutes to dry. Discs were applied aseptically on the plates. Two sets of plates were used for each of antibiotics one set was incubated at 30Cº and the other at 35ºC.The results were reported after two periods firstly after 24h and after 48h of incubation. Isolates were considered MRSA when the inhibition zone diameter was ≤ 10 mm for oxacillin, ≤ 21 mm for cefoxitin.

MRSA-latex agglutination test
MRSA-latex agglutination test (Oxoid PBP2´) (Table 1). This result is in agreement with Louie et al. [9], Lee et al. [10] and Sangeetha et al. [11] who reported that both sensitivity and specificity of MRSA-latex agglutination were 100% compared with PCR for detection of mecA.

In
However, Alipour and his colleagues [12] reported less sensitivity for MRSA-latex agglutination (97.29%) instead of (100%). This is in part may be because these studies methods have included different strains, which may differ significantly in heterogeneity, and behave differently under particular test conditions. Isolates producing small amounts of PBP2a may give weak agglutination reactions or agglutinate slowly. The Latex agglutination method is rapid and requires no special equipment and can be the best predictor to detect MRSA but it is expensive.
In this study, cefoxitin disc diffusion test showed maximum comparability of results with MRSA Latex agglutination than oxacillin disc diffusion and ORSAB (Table 1). It showed stable results at different temperatures (30°C and 35ºC) and incubation periods (24h and 48 h), with high sensitivity (98.48%), and specificity (100%).
Cefoxitin disc diffusion method is a cheap and simple test.
Accordingly, the cefoxitin disc diffusion test was found to be the method of choice to identify MRSA. This result is in agreement with previous studies about the suitability of cefoxitin disc diffusion test for MRSA identification [11,[13][14][15]. It was reported that disc diffusion with cefoxitin is more reliable than that with oxacillin [16].
It is a more potent inducer of the mecA gene with no special requirements of temperature or medium [17].
In this study, oxacillin disc diffusion test showed variable results at different temperature (30ºC and 35ºC) and at different incubation periods (24h and 48h). Sensitivity and specificity of oxacillin disc for 24 h incubation both at 30ºC and 35ºC were 87.88% and 96.23%, respectively. However, after 48h incubation the results of oxacillin disc varied, at 30ºC (48h) the test sensitivity and specificity values were 89.39% and 94.34%, ,respectively. At 35ºC (48h) the test showed values of 89.39% and 92.45%, respectively. Therefore more incubation increases the sensitivity and decreases the specificity of the test. A similar observation has been seen with ORSAB test; at 35ºC for 24h values of sensitivity and specificity were 84.85% and 98.11%, respectively. After 48h incubation the values were 89.39% and 88.68%, respectively. This may be due to the fact that PBPa production is induced by growth in the presence of beta lactam antibiotics such as oxacillin and cefoxitin. However, cefoxitin is assumed to be a better inducer of mecA gene expression than is oxacillin and thus, is better for screening heterogeneous MRSA populations expressing the mecA variable. That is to say oxacillin needs more time to express resistance. One study that included heterogeneous strains found that oxacillin disc diffusion method had low sensitivity [18]

Competing interests
The authors declare no competing interest.

Authors' contributions
Ibrahim OM, Bilal NE, Osman OF: designed the study; Ibrahim OM: carried out the data collection, and laboratory work, participated in the statistical analysis; Magzoub AM and all authors coordinated and helped to draft the manuscript, read and approved the final manuscript. Table   Table 1: Comparison between methicillin resistance detection methods and gold standard mecA PCR method in a total of 119 S.aureus strains (66 mecA positive and 53 mec A negative)