Staphylococcal Cassette Chromosome mec (SCCmec) Gene Typing in Detection of Methicillin-Resistant Staphylococcus aureus: Toward Precise Detection in Health Care Facility

Background: Blood stream infections (BSI) are considered key issues in critical care units. Methicillin resistant Staphylococcus aureus, MRSA-related infections, are considered a major health problem. This is attributed to the emerging new society dangerous strains with continuous antibiotics pressure and fluctuations in resistance patterns. Aim: We aimed to study epidemiology of methicillin resistance S. aureus (MRSA) infections by using conventional phenotypic methods [cefoxitin disk diffusion (CDD) and oxacillin screening agar] and molecular typing of the mec-gene (SCCmec) using multiplex PCR in Suez Canal University Hospital. Methods: 100 non-repetitive staphylococcus aureus were collected and identified morphologically and biochemically by standard laboratory procedures. The strains were considered MRSA if the MIC of oxacillin ≥ 4 μg/ml, and the inhibition zone of cefoxitin was ≤21 mm (CDD). Characterization of SCCmec elements in isolated MRSA strains was done via multiplex-PCR. Results: From total of 100 isolates, eighty were detected as MRSA by using CDD (sensitivity and specificity were 83.6% and 24.4% respectively) and only 65 by using oxacillin screening agar (sensitivity and specificity were 85.5% and 60% respectively). MecA gene was identified in 55 samples; the majority of isolates were SCCmec type IVa (63.7%). Both type I and III of SCCmec couldn’t be detected. Antimicrobial sensitivity rates among SCCmec-V isolates were expectedly higher than those among Type-II isolates. SCCmec type II was characterized by 100% resistant to ciprofloxacin, erythromycin, oxacillin and cefepime as well as greater resistance to clindamycin (70%) with the same pattern between all typing strains (7 strains). How to cite this paper: Kishk, R.M., Mandour, M.F. and Saleh, R.M. (2019) Staphylococcal Cassette Chromosome mec (SCCmec) Gene Typing in Detection of Methicillin-Resistant Staphylococcus aureus: Toward Precise Detection in Health Care Facility. Open Journal of Medical Microbiology, 9, 127-137. https://doi.org/10.4236/ojmm.2019.93013 Received: August 1, 2019 Accepted: September 24, 2019 Published: September 27, 2019 Copyright © 2019 by author(s) and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/


Introduction
The use of venous catheters would cause infections with significant morbidity and mortality with a tremendously high economic burden [1]. About 80,000 catheter-related bloodstream infections (CRBSIs) were reported annually among patients in critical care units, counting for up to 24,000 deaths with annual cost of 414 million dollars [2]. Central line-associated blood stream infections (CLABSI) are laboratory-confirmed bloodstream infections (LCBI) where a medical catheter was in place for more than two days on the day of event. The calendar date of catheter insertion is counted day one supported by the fact that the line secured in place at the time of the event or the day before [3].
According to (CDC 2016) recommendations, LCBI was identified if a pathogen was detected in one or more blood specimens and the detected organism is not linked to infections at other places (LCBI-1), or if the patient had one at least of these clinical features; fever (>38.0˚C), hypotension, or chills, with the same commensal identified from at least two blood specimens collected on separate times (LCBI-2) [2] [3].
Worldwide, the doubled prevalence rate of MRSA-related infections from 1996-2004 had raised a major public health problem. These strains have a common mobile genetic component (21-to-67 kb) included in their genome, known as the staphylococcal cassette chromosome mec (SCCmec), carrying the methicillin resistance (mecA) gene and other antibiotic resistance determinants [7] [8] [9]. In addition to the two vital genetic elements (the mec gene complex and the ccr gene complex), a terminal inverted and direct repeats and the junkyard (J) regions are included within SCCmec gene [10] [11] [12]. mec and ccr complexes have been classified into three classes (A, B, and C) and four allotypes (1, 2, 3, and 5) respectively. SCCmec types are usually composed of diverse grouping of these complex allotypes and classes.  [20].
The study aimed to evaluate different detection methods for identifying MRSA.
Additionally, we aimed to highlight the molecular epidemiology of MRSA infections in Suez Canal University Hospital.

Samples Collection
A

Isolation and Classification of Staphylococcal Isolates
Standard laboratory procedures according to morphologic and biochemical reactions were used to identify staphylococci from different isolates. The S. aureus ATCC 25923 was our reference strain. The isolates were preserved in glycerol 15% (v/v) in brain heart infusion broth (BHIB, Oxoid, Basingstoke, UK) at −80˚C and then recovered at the Microbiology Laboratory by subculturing in BHIB at 37˚C for 24 h followed by two further subcultures on brain heart infusion agar [21].
The sensitivity to antimicrobials was performed using the disk diffusion method on Mueller-Hinton Agar (Oxoid, UK) [22]. The following antibiotics were

Identification of Methicillin Resistance
Methicillin resistance was confirmed for all isolates by the following.

Cefoxitin Disk Diffusion (CDD) Method
The antibiotic susceptibility was performed using cefoxitin (

Oxacillin Agar Screening
Culture on mannitol salt agar containing oxacillin was performed. Media was prepared by adding 11.1 gm of mannitol salt agar base into 100 ml of distilled water and autoclaved. When the autoclaved medium temperature reaches around 50˚C, we added oxacillin as a solution with a final concentration of 6 µg/ml of medium. Any growth in the cultured media was considered as MRSA [22].
Multiplex PCR was performed to screen the existence of mecA genes in the entire isolates. mecA gene was identified in 55 isolates out of 100 isolates. Surprisingly, 9 isolates (9%) of PCR positive samples were detected as MSSA by CDD conventional methods and 8 isolates (8%) were detected as MSSA by oxacillin agar screening method.  (Figure 1). Surprisingly, SCCmec types I and III were completely absent.

Relation between Antibiotic Resistance and Multiplex PCR
SCCmec type II was 100% resistant to erythromycin, ciprofloxacin, oxacillin and cefepime as well as to clindamycin (70%) with the same pattern between all typing strains. SCCmec type IVa isolates illustrated clindamycin resistance (60%) and erythromycin (35%) with 30% of them showed the same antibiotic resistance. Finally, MRSA strains SCCmec type V showed recurrent resistance to aminoglycosides.

Discussion
The emergence of continuous antibiotics pressure and fluctuations in resistance patterns in the novel community acquired MRSA virulent strains resulted in serious blood stream infections in the last two decades [23]. Methicillin resistance in staphylococci is due to the expression of a modified penicillin-binding protein (PBP), PBP 2a encoded by the mecA gene that is located on the staphylococcal cassette chromosome mec (SCCmec) [10].
Our study utilized two recommended standard tests to screen for MRSA, Cefoxitin disk diffusion (CDD) and oxacillin agar screening. The sensitivities of these 2 tests to detect MRSA as compared to Polymerase Chain Reaction in detecting of mecA gene were 83.6% and 85.5% respectively. The sensitivity of 83.6% of CDD means that the test can detect only 83 cases out of 100 as true positive and 17 cases will be misdiagnosed. This may affect the treatment decision, strategy, cost and hospital stay.
The specificity of oxacillin agar screening methods was higher than of CDD (60% and 24.4% respectively) but both were below 90%, which can't be accepted as a standard method for diagnosis MRSA cases especially with low accuracy of both tests (57% for CDD and 47% for oxacillin agar screening). Our findings were similar to Pillai et al. [24] which reported that the sensitivity and specificity of oxacillin disk diffusion (ODD) test were (93.5%, 83.5%) respectively, whereas that of oxacillin agar screening was found to be (87.1%, 89.3%) respectively. Cauwelier et al., mentioned that the sensitivities of both oxacillin disk diffusion method and agar screening method are 83.5% and 91.7% respectively, and both were 100% specific compared with PCR for mecA detection [25].
In our study, only 55 isolates were confirmed MRSA strains by means of PCR  size [26].  [30]. These strains were either undetected (type I and III) or sparsely detected (12.7%; type II) in our work.
We noticed the general dominance of MRSA strains carrying SCCmec types IVa and V (63.7% and 23.6% respectively). This data was matched with a study from Switzerland in 2010 [31] which reported the absence of type III and presence of types I and II in very low proportions (10%). In healthcare-associated infections, some studies reported 87% isolation rates of SCCmec types IV and V, others reported comparable distribution of SCCmec-IV and SCCmec-II/III types among the MRSA isolates [32] [33]. Fatholahzadeh and his colleagues reported 98% isolation rates of SCCmec type III or IIIA and only 2% for SCCmec type IV, but didn't isolate both types I and II [34]. These results are alike most Asian studies [35].
We couldn't identify MRSA isolates of the SCCmec types I or III, probably due to the limited number of isolates analysed. To our knowledge, we are the first study describing the SCCmec typing in our hospital with such high prevalence of SCCmec types IV and V.
Antimicrobial sensitivity rates among SCCmec-V isolates were expectedly higher than those among Type-II isolates. However, SCCmec type II was 100% resistant to ciprofloxacin, erythromycin, oxacillin and cefepime as well as greater resistance to clindamycin (70%). This was matched by Davis and his group who pointed the superior antibiotics sensitivity pattern among Type IV isolates com-  [32].
This study has many limitations because MRSA surveillance cultures are not routinely performed, so it was difficult to track the source and starting time of MRSA acquirement. We also need more studies to recognize the risk factors for

Conclusion
In conclusion, SCCmec types IVa and V are generally dominant in our community with no detection of SCCmec types I or III. Antimicrobial sensitivity rates among SCCmec-V isolates were expectedly higher than those among Type-II isolates. PCR is the optimum method to be used in detecting these serious infections and preferred over the usual standard methods. PCR can pick up the wrong negative results in addition to the sensitivity, specificity, accuracy and the rapid diagnosis of MRSA strains as the detection of mecA gene can last for only 5 h from the bacterial isolation. Hence, the conventional methods are not reliable for detecting MRSA strains especially in seriously ill-patients.

Ethical Considerations
The study was approved by the medical ethics committee of our institute in agreement with the 1964 Helsinki declaration and its later modification.

Conflicts of Interest
Authors declare no conflicts of interests.