CORRELATION BETWEEN THE GENOTYPE DETECTION DETERMINED BY RT- PCR ASSAYS AND THE PHENOTYPIC DETECTION DETERMINED BY ANTIBIOTIC SUSCEPTIBILITY PATTERNS OF METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS CLINICALISOLATES IN ALGERIA

* Amina Ayeche 1 , Névine Lebsir 2 And Mohamed Abdelhafid Hamidechi 1 . 1. Laboratoire de Biologie Cellulaire et Moléculaire, Faculté des Sciences de la nature et de la vie, Université des frères Mentouri Constantine1, Rue Ain El bey25000 Constantine-Algérie. 2. Laboratoire de Microbiologie, Hôpital de pédiatrie d'El-Mansourah, Constantine-Algérie. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

Resistance to methicillin in S. aureus is primarily mediated by the production of penicillin binding protein (PBP 2a), an additional penicillin-binding protein with low affinity for beta-lactam antibiotics, encoded by mecA gene [1,5,17] which is absent in methicillin susceptible strains. This gene is carried on the SCCmec element [13].
The detection of MRSA has become an important tool in clinical diagnosis for the management and treatment of patients [6,17]. Routine methods used in microbiology laboratories for identification of methicillin resistance in S. aureus are based on antibiotic susceptibility disk diffusion agar and dilution [14,17]. These methods detect only phenotypic expression of methicillin resistance without the presence of the mecA gene [17]. Since phenotypic methods are not discriminating enough and are highly dependent on growth conditions, the use of genetic approach, such as DNA hybridization and PCR, is essential to stop the spread of S. aureus methicillin-resistant and to increase the rapidity and accuracy of their identification and their antibiotics resistance patterns [6].
The aim of this study was therefore to show the prevalence and antibiotic susceptibility of S. aureus methicillinresistance isolated from the pediatric hospital and clinical laboratories in Constantine, Algeria, and to compare the degree of correlation between the phenotypic detection of methicillin-resistance in S. aureus by standard disk diffusion test with the genotypic detection of mecA gene by RT-PCR.

Material and Methods:-
Bacteria isolates:-A total of 65 S. aureus strains were isolated from infected patients. The isolates were collected from the pediatric hospital and clinical laboratories in Constantine (Algeria) from December 2014 to September 2015. The isolates were first identified with standard bacteriological technique and biochemical tests using the API system 20E® (biomérieux., Marcy l'Etoile, France).

Identification of S. Aureus:-
A confirmation of the staphylococcus aureus identification for all the isolates was performed by using matrixassisted laser desorption/ionisation time of flight mass spectrometry (MALDI-TOF-MS). Colonies were deposited directly on the MALDI-TOF target plate (Four spots per isolate) and were covered with 1.5 µL of matrix solution (2 points of a-cyano-4-hydroxycinnamic acid + 500ml of acetonitrite HPLC + 475ml of distilled water + 25ml of trifluoracetic-acid). Spots were analyzed using a Bruker Daltonics Microflex (Bremen, Germany). Peptidic spectra were compared with the Bruker database using Biotyper software, version 2.0, and the identification score was noted.

DNA Extraction:-
For detection of mecA gene, all DNA samples were extracted with the DNeasy tissue kit (Qiagen, Hilden, Germany) using EZ1 Advanced instrument (Qiagen). One colony of each isolate was mixed with 200 µL of extraction buffer. Extracted DNA was eluted in 200 µL, analysed immediately after extraction and stored at -20°C.

Rt-Pcr:-
Amplification of the methicillin resistance gene mecA was performed using primers as previously described by Antibiotic susceptibility:-In this study the specimens of S. aureus were mainly obtained from pus samples with a frequency of 81.53 % followed by urine and semen samples with respectively 12.30 and 6.15 %.
The results of resistance rate of all the strains to antibiotics testing according to the French CASFM 2015 revealed that the isolates were multidrug resistant. Higher resistance was observed to penicillin G (70.77% of the isolates), followed by ceftriaxone (50.76%), erythromycin (46.15%), oxacillin and ciprofloxacin (24.61% each). However, some isolates were resistant to glycopeptides (vancomycin 4.61% and teicoplanin 9.23%). The classification of antibiotic resistance patterns of the isolated S. aureus into three groups of resistant, intermediate-resistance and susceptible is given in Table 1. As shown in Table 1, 23 isolates were found to be methicillin-resistant (35.38%), while the remaining (42) were 37 (56.92%) methicillin-susceptible and 5 (7.69%) intermediate resistant. The percentage of MRSA was defined as the number of S. aureus isolates resistant to cefoxitine divided by the total number of S. aureus isolates. The majority of the MRSA isolates showed resistance to penicillin G (86.95% of the isolates), ceftriaxone (78.26%), oxacillin (65.21%) and erythromycin (56.52%). MSSA isolates were also resistant to penicillin G (70.27%), erythromycin (45.64%) and ceftriaxone (40.54%). They were highly susceptible to gentamicin (100%), oxacillin and vancomycin (97.3% each).

Discussion:-
Methicillin-resistant S. aureusis an important human pathogen and is responsible for both hospital and community associated infections worldwide. Its frequency in most African countries has not been reported [19].
Between 1996 and 1997, the prevalence of MRSA was determined in eight northern African countries and Malta, it was relatively high in Nigeria, Kenya, and Cameroon (21 to 30%) and below 10% in Tunisia, Malta and Algeria [19]. In 2001, the resistance rate increased to 14% in Algeria [9]. Prospective, multicentre study was conducted between 2003 and 2004 with participation of nine university hospitals in the Mediterranean area, and the percentage of S. aureus strains resistant to methicillin (MRSA) was 35.5% in Algeria [20].
In our study, among 65 S .aureus isolates 23 were MRSA (35.38%). These results showed a high similarity with results obtained by previous studies in Algeria.
Resistance of MRSA to other antibiotics has also been reported. In this study, we found that 8.69% of isolates were resistant to vancomycin, while in related studies; all MRSA strains isolated from clinical samples were susceptible to vancomycin (100%) [17,19]. Shahraz et al. (2012) reported that 26.6% of S. aureus strains isolated from packaged hamburger were resistant to vancomycin.
According to our study, MRSA were resistant to erythromycin (56.52%) and gentamycin (34.78%), Ramdani-Bouguessa et al. 2005 reported that 25% and 7% of PVL-positive MRSA strains isolated from Mustapha Pacha hospital in Algeria and Ouchenane et al. 2010 reported that 25% and 6.25% of MRSA strains isolated from 953 Didouche Mourad hospital in Algeria, were resistant to erythromycin and gentamicin, respectively. Comparison between these results showed a high resistance to erythromycin and gentamycin in our isolates.
In recent years, several studies have demonstrated the high capacity of molecular methods such as Pulsed-field gel electrophoresis (PFGE), RT-PCR and PCR-based methods to increase the rapidity and accuracy of resistance testing [1,6,14,15,16,21,22].
In our study, molecular detection of mecA gene as a specific target of MRSA genes was performed for all 65 S. aureus isolates. As shown in Table 2, we have compared the results of standard disk-diffusion, as a phenotypic method, with real-time PCR, as a genotypic method. Overall, we found high correlation between these two methods. This correlation has been demonstrated previously in other studies [6,14,15,17].
The present study indicate that among 23 strains, 3 were resistant to methicillin and 5 of 65 have intermediateresistance according to standard disk-diffusion method but they are susceptible according to PCR method. Two of 39 strains were methicillin-susceptible according to standard disk-diffusion method but are resistant according to PCR method. In accordance with this study and previous reports, strains that are phenotypically resistant to methicillin but are not carried a mecA gene seemed to show poor expression of mecA gene or production of methicillinase or β-lactamase hyper-production [12,16]. Borderline resistance in mecA-negative strains has been hypothesized, in previous studies, to result from modification of normal PBP genes [8,23]. Kumar et al., 2010, indicated that mecA gene is present, but is not amplified because the amplification site is not available enough or is not expressed or expressed at low level due to growth conditions or might be limitations in detection in microbiological methods.
Similarly, strains could exhibit intermediate-resistance of methicillin due to overexpression of a b-lactamase when mecA is absent [24]. Intermediate resistance levels could be due also to mutations in the endogenous mecA gene [6].
In conclusion, the high prevalence of MRSA in Algerian hospitals, as our results showed, is necessary that rapid identification of methicillin-resistance and genotypic detection of mecA gene will have a major impact on the treatment of infectious diseases and to reduce the emergence of drug resistance. Although microbiological susceptibility testing are still widely used in clinical microbiology laboratories for analysis of phenotypic resistance of MRSA, molecular detection of mecA gene with RT-PCR is highly rapid, sensitive and specific. Moreover, all the studies reported to date indicate a high correlation between methicillin-resistance and the presence of the mecA gene. In this study, comparison between phenotypic detection of methicillin-resistance in S. aureus by standard disk diffusion test and genotypic detection of mecA gene by RT-PCR method showed a high degree of correlation.   neg., negative amplification.