Comparison of rapid and conventional methods for investigating of mecA presence in Staphylococcus Species

Objectives: Taking the determination of mecA gene by polymerized chain reaction (PCR) method as a reference in determining methicillin resistance in Staphylococcus species, we aimed at comparing the reliability levels of disk diffusion, latex agglutination test and chromogenic agar use methods. Methods: This prospective study was conducted on 228 Staphylococcus strains isolated between January 2020 and December 2020 in Samsun Training and Research Hospital. Disk diffusion, latex agglutination and chromogen agar medium methods were applied along with the polymerized chain reaction (PCR) method. Results: The mecA gene was detected in 47 of the isolates (20.6%) by the PCR method, and these isolates were accepted as methicillin-resistant. When the PCR result was taken as a reference, the sensitivity of the disk diffusion method became 100%, and specificity became 45.9%; sensitivity of latex agglutination was determined as 80.9%, and specificity as 70.2%; sensitivity of chromogenic agar as 85.1% and its specificity was found to be 95%. Only in S. aureus isolates, the highest sensitivity and specificity rate (100% and 88%, respectively) belonged to chromogenic agar. Conclusion: Chromogenic agar provides more reliable data for S. aureus isolates, and the combined use of all three methods does not significantly increase reliability.


INTRODUCTION
Although Staphylococcus species are a member of the normal skin flora, they can cause a variety of infections ranging from simple wound infections to bacteremia and sepsis. Staphylococci are among the most common isolated agents in wound infections. The type of these agents and their resistance to antibiotics are of great importance for determining the treatment of infections. [1][2][3] Despite the fact that coagulase-negative Staphylococcus species cause more frequent infections, Staphylococcus aureus is known to be more pathogenic due to its various enzymes and factors. 4,5 Methicillin resistance is the most important factor in the management of infections caused by Staphylococcus and the selection of antimicrobials. Methicillin-resistant Staphylococci are also resistant to beta-lactam group antibiotics in vivo, which significantly limits antibiotic options. [4][5][6] In such cases, it is necessary to use antibiotic groups such as carbapenems, which leads to the development of significant resistance in some bacteria such as Acinetobacter and Pseudomonas that cause hospital infection. In such cases, antibiotics with a high rate of undesirable effects such as vancomycin or teicoplanin can also be an option. However, the development of resistance to this group of antibiotics, which is the most effective against Staphylococcus, constitutes one of the most dreadful scenarios in the world. [4][5][6][7][8] Correct determination of the antibiotic resistance profile of Staphylococcus isolates obtained from the infection is critical for the management of the infection. Different methods have been tested for a long time to accurately determine whether there is methicillin resistance or not. Although molecular methods used for the detection of mecA and mecC genes that cause methicillin resistance are accepted as reference methods, for now, these methods cannot be used in routine applications. For this reason, the reliable detection of methicillin resistance by conventional methods has become a preferred necessity in terms of being both costeffective and easy to apply. 4,7,9,10 In this study, we aimed at comparing the reliability levels of disk diffusion, latex agglutination test, and chromogenic agar methods, by taking the mecA gene detection by polymerized chain reaction (PCR) as a reference in determining methicillin resistance in Staphylococcus species.

METHODS
This study has been approved by the local ethics committee on 2020/13/8 and planned prospectively. Obtaining and Identifying Strains: A total of 228 staphylococcal isolates obtained from various clinical samples sent to Samsun Training and Research Hospital's microbiology laboratory between January 2020 and December 2020 were included in the study. In the laboratory colony morphology, pigment formation, Gram's staining, catalase, and coagulase assays were concluded on the clinical specimens. Bacteriel identifications were detected by Vitec 2 (bioMérieux, France).
In Considering that it may belong to the same origin, only one of the isolates obtained from the same patient was included in the study. Similarly, only one of the isolates obtained from patients hospitalized in the same service (e.g. intensive care unit) on the same day was included in the study. Isolates that were not considered growth was far below the amount of a causative pathogen were excluded from the study. Determining Methicillin Resistance: Disc Diffusion Method: Cefoxitin susceptibility of Staphylococcus strains was determined by disc diffusion method. Discs containing 30 μg Cefoxitin (Oxoid, Ireland) were used. For each strain, the solutions prepared with 0.5 McFarland turbidity were seeded on Mueller Hinton agar and the methicillin sensitivity was evaluated after 24 hours of incubation. 11 Latex Agglutination: The presence of penicillinbinding protein 2a (PBP2a) in the isolates was determined using the Slidex MRSA kit (bioMérieux, France) and the test was carried out according to the manufacturer's recommendations. Positive control was used for each test. Chromogenic Agar: In the study, chromID® MRSA SMART kit (bioMérieux, France) was used, as well as a chromogenic medium used for screening methicillin resistance. The microorganism was cultivated on chromogenic agar under aseptic conditions. It required 18-24 hours of incubation. Methicillin-sensitive S. aureus (MSSA) could not grow on chromogenic agar, while methicillin-resistant S. aureus (MRSA) grown in a pinkish color. Other bacteria either could not reproduce or grow blue or colorless. 12,13 MecA PCR: The multiplex PCR protocol used in the study was as follows: 10x PCR Buffer 2.5µL, 10 mM dNTP 0. Sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of the methods were calculated. For sensitivity, the ratio of the number of isolates found to be resistant to methicillin to the actual number of resistant isolates, and for specificity, the ratio of the number of isolates found to be susceptible to methicillin to the actual number of susceptible isolates was calculated. The positive predictive value was calculated from the ratio of the true resistant isolates among the isolates found to be resistant to methicillin, and the negative predictive value from the ratio of the actual susceptible isolates among the isolates found to be susceptible to methicillin. Finally, accuracy was calculated as the ratio of the total number of methicillin-resistant and susceptible isolates determined by the method to the total number of isolates.
MecA gene was detected in 47 (20.6%) of the isolates by using PCR method and these isolates were accepted as methicillin-resistant (Fig. 2). 145 (63.6%) isolates were found to be methicillin-resistant by disk diffusion method, 92 (40.4%) were found resistant by latex agglutination, and 49 (21.5%) were found resistant by chromogenic agar (Table-I).
Only S. aureus isolates had the highest sensitivity and specificity rate for chromogenic agar (100% and 88%, respectively). The sensitivity of the chromogenic agar method in coagulase-negative Staphylococcus was 81.6% and its specificity was determined as 96.2% (Table-II    The sensitivity and specificity of the chromogenic agar method were 100% and 88% for S. aureus, respectively; 100% and 100% for S. epidermidis and S. sciuri and 100% and 94.7% for S. hominis. The sensitivity and specificity of the chromogenic agar method were 100% and 88% for S. aureus, respectively; 100% and 100% for S. epidermidis and S. sciuri and 100% and 94.7% for S. hominis. The sensitivity and specificity of the latex agglutination method were determined as 100% and 94.4% for S. capitis, 100% and 89.5% for S. hominis. While most of the sensitivities of all three methods were found to be 100% for Staphylococcus species, specificity rates were observed to vary greatly. Among these common species, the median sensitivity of all three methods was 100%. The specificity rates of disc diffusion were between 14.3% and 61.1% (median: 47.4% [interquartile range; IQR: 16.3%]), and latex agglutination were between 50% and 94.4% (median: 76.9% [IQR: 28.0%]), and chromogenic agar were between 75% and 100% (median: 100% [IQR: 12.0%]). (Table-III).
Analysis was made considering the combination of methods, and the isolate detected by any method as susceptible to methicillin was considered susceptible. According to the analysis results, it was found that the combination of disk diffusion and chromogenic agar had the best performance with a sensitiv-Selim Gorgun et al.  ity of 85.1% and its specificity was 95% (Table-IV). When the isolate found to be resistant to methicillin by any method is accepted as resistant; The sensi-tivity of the combination of latex agglutination and chromogenic agar, with the best performance, was 100% and its specificity was 68.5% (Table-V).

DISCUSSION
Staphylococcus is one of the bacteria isolated as the most common infectious agent and wound infection pathogen (between 19% and 58.5%). [14][15][16][17] The presence of methicillin resistance is one of the most important guiding factors in the treatment of staphylococcal infections. Although some molecular methods and several dilution methods (for determining the minimum inhibitory concentration (MIC) value) have been developed to determine methicillin resistance in Staphylococcus species, they are not ideal for use in routine laboratory applications. [4][5][6][7] Automated systems also determine methicillin resistance by determining the MIC value, but in many small-scale laboratories, these systems are not included for not being cost-effective. For these reasons, we focused on detecting methicillin resistance with easy and fast methods that can be applied in every laboratory. However, there remain uncertainties about whether these methods provide reliable data. 4,7,8 The performance of three different methods was evaluated in our study.
One of the most classical methods for determining methicillin resistance in Staphylococcus is the disk diffusion method. Oxacillin has been used for a long time for this purpose. However, after it was determined that the reliability level was much lower than expected, oxacillin was replaced by cefoxitin, which gave better results. It has been reported that cefoxitin expresses the mecA better than other penicillins. 18 However, the reliability of the disk diffusion method has been remaining under questioning so far. In some studies, it has been reported that the sensitivity of the disk diffusion test using cefoxitin disc is between 94% and 100% and the specificity is between 96% and 100% for determining methicillin resistance in Staphylococcus. [19][20][21][22][23][24] When the PCR result is taken as a reference in this study, the sensitivity of the disk diffusion method in all Staphylococcus isolates was 100% and specificity was determined to be 45.9%. Considering the highest number of isolated Staphylococcus in the study, the sensitivity of the disk diffusion method was 100% for all the isolates; also it was determined that the specificity rates varied between 14.3% and 61.1% (median: 47.4% [IQR: 16.3%]). Accordingly, it was observed that the negative predictive value of the disk diffusion method was 100%, but the positive predictive values were below 50%. All these findings show that in Staphylococcus, the disk diffusion method, in general, captures methicillin resistance in almost all isolates. However, it is seen that the specificity rates for methicillin resistance are far below the acceptable level; that is, false-positive results are very high for methicillin resistance. This may cause the patient to use strong antibiotics unnecessarily when a methicillin-sensitive Staphylococcus is isolated as the agent. In the light of this, the disk diffusion method seems to be far from providing reliable data in both S. aureus isolates and coagulasenegative Staphylococcus.
MRSA isolates produce PBP2a, which has a lower affinity for beta-lactam antibiotics than PBP2. The mecA encodes PBP2a, which is the target of methicillin. 12,20 The latex agglutination test used in our study is a serological method used for S. aureus isolates, determining the presence of PBP2a, and can be applied easily and quickly. In some studies, it has been reported that the sensitivity of the latex agglutination method using cefoxitin disc is between 88% and 100%, and specificity is between 97% and 100% in determining methicillin resistance in Staphylococcus. 12,[20][21][22]25 In our study, the sensitivity of the latex agglutination method was found to be 80.9% and specificity was found to be 70.2% in all Staphylococcus isolates. Only in S. aureus isolates, the sensitivity and specificity rates were 100% and 80%, respectively, and 76.3% and 68.6% for general coagulase-negative Staphylococcus, respectively. In most of the Staphylococcus species detected in high numbers in our study, the sensitivity of this method was determined as 100%, whereas the specificity rates dispersed between 50% and 95%. Specificity rates were found to be high in S. sciuri and S. hominis isolates, but methicillin resistance could not be determined in any of the S. saprophyticus isolates. These findings show that the latex agglutination method has a high sensitivity rate in determining methicillin resistance in general, its specificity for S. aureus is below the expected, and it is not usable in coagulase-negative Staphylococcus due to its highly unsteady performance.
The chromogenic agar used in our study is a medium that prevents the growth of methicillin-resistant Staphylococcus, because of the cefoxitin it contains. In addition, it indicates whether the growing colony is S. aureus or not based on the enzymes it contains. In this medium, the main purpose is to detect MRSA. 12,13,26 In some studies, it has been reported that the sensitivity of the disk diffusion test using cefoxitin disc in determining methicillin resistance in Staphylococcus is between 75% and 100% and the specificity is between 89% and 100%. 23,[27][28][29][30][31] The sensitivity of chromogenic agar in our study was 85.1% and its specificity was determined as 95%. The sensitivity and specificity rates were 100% and 88%, respectively, in S. aureus isolates only; 81.6% and 96.2% in coagulase-negative Staphylococcus. Sensitivity and specificity rates were found to be 100% in some Staphylococcus species. Among these common species, the sensitivity [IQR: 42.9%] and specificity [IQR: 12.0%] median values of chromogenic agar were found to be 100%. It was observed that the highest sensitivity and specificity rate for S. aureus isolates belonged to chromogenic agar among three methods. All these findings show that the use of chromogenic agar for the determination of methicillin in S. aureus isolates has yielded significantly reliable results, but its specificity is still somewhat low. According to these data, the reliability level of chromogenic agar generally gives relatively high results except for S. aureus.
In our study, in case the methods are used in combination, two separate analyzes were carried out. When the isolate determined by any method as susceptible to methicillin was considered sensitive, it was observed that the combination of disk diffusion and chromogenic agar showed the best performance with a sensitivity rate of 85.1% and the specificity rate of 95%.
In cases where the methods were performed in combination, two analyses were carried out. When the isolate determined as resistant to methicillin by any method was considered as resistant, it was determined that the combination of latex agglutination and chromogenic agar showed the best performance, and the sensitivity was 100% and the specificity was 68.5%. These findings show that the combined use of these methods either increases sensitivity or specificity but cannot increase both sensitivity and specificity. Accordingly, besides not providing high-reliability data, it is seen that their combined use does not significantly increase the level of reliability.

Limitations of the study:
Since the study aimed at comparing routinely-used methicillin resistance detection methods, determination of minimum inhibitory concentration (MIC) values with seldomly used micro and macro dilution methods were not included. In addition, the presence of mecC genes was not investigated in the study. Methicillin resistance depends on the presence of the mecA or mecC genes, but the prevalence of mecC gene presence has been reported to be 0.009% 32 , and it was deemed that our study would not significantly affect the analysis results.

CONCLUSION
The data of our study show that disk diffusion, latex agglutination, and chromogenic agar methods do not yield high-reliability results in determining methicillin resistance in Staphylococcus species, but chromogenic agar provides more reliable data in S. aureus isolates, and the combined use of all three methods does not significantly increase the reliability level.