IDENTIFICATION OF METHICILLIN RESISTANT STAPHYLOCOCCUS AUREUS USING TOUCHDOWN PCR AND PHENOTYPIC METHODS FROM PATIENTS AND HOSPITALS ENVIRONMENTS IN DIFFERENT IRAQI CITIES

The study was aimed to evaluate the prevalence of MRSA in some Iraqi hospitals and determine the most powerful methods for identification of MRSA , in order to achieve the, 278 samples were collected from different hospitals in Iraq in various intervals, 204 out of 287 were identified as Staphylococcus aureus by conventional cultural methods and microscopic characteristics and 177 isolates are identified as MRSA by using HiCrome MeReSa Agar Base medium, but 154 of 177 (87%) isolates are methicillin resistance in sensitivity test. MRSA isolates were highly resistant to β-lactam antibiotics and considered multidrug resistant (MDR) in percent of (94.9%). Touchdown PCR used to identify the isolates, 97.05% were identified as Staphylococcus aureus, while 80.88% as MRSA.


Human skin and mucosal microbiota contain
Staphylococcus aureus which is a common opportunistic pathogen found in human body that causes significant infections (35). The percentage (20-30)% of the general population contain Staphylococcus aureus in the nasal mucosa (2). Staphylococcus aureus pass into bloodstream, heart, joints, device-related infection and bones during the disruptions of mucosal and cutaneous barriers (35,  (3). Physicians, clinical microbiologists, and public health officials within the same country and across other regions can have benefits from knowing MRSA antimicrobial resistance. The input is also useful for decisions regarding specific therapy of pathogen, formulary of hospital, and target-oriented infection domination policies (11,27,7,5,12). In addition, MRSA surveillance researches accomplished in community settings are important to best known the molecular and clinical epidemiology of emerging MRSA isolates (33), so, the main objective of current study is to specify the antibiotic susceptibility profiles and MRSA spreads in Iraqi hospital and its link with risk factors in healthcare workers at hospitals, as well as determine the most powerful methods for identification of methicillin resistance S. aureus.

MATERIALS AND METHODS Collection of samples
The number of collected samples is 278 samples, that's include 139 samples from patients such as pus, sputum, ear infections, burn wounds, wounds, blood and foot ulcers, in addition to 58 from health workers such as epidermis and medical outfits, and 81 from hospitals environments such as infant incubators, blood pressure chuffs, microscopes, door knobs floor, sinks and trash. All samples were collected from local different hospitals in different Iraqi cities (Baghdad, AL-Diwanniyah, Najaf, Nasiriyah, Hillah and Kut) in different intervals (2015,2016,2017,2018,2019).

Bacterial isolation
Brain heart infusion agar and Mannitol salt agar were used for isolation of S. aureus. Depending on the morphological bases the suspected colonies were chosen and isolated for extra diagnostic tests. According to Bergey's manual of systematic bacteriology, the diagnosis of S. aureus was achieved (38).

Identification of Staphylococcus aureus
isolates: Isolates which recorded as positive were assayed by Gram stain, catalase, oxidase as well as coagulase assays. The bacterial isolates which were recorded as mannitol positive, Gram positive, oxidase negative, catalase positive and coagulase positive were determined and identified as Staphylococcus aureus, then isolated for extra assays. Identification of methicillin-resistant Staphylococcus aureus: Isolates that were identified as Staphylococcus aureus were cultured on HiCrome MeReSa Agar Base medium. The medium is used as a selective medium for MRSA isolation by the by combining it with cefoxitin supplement (FD259) and MeReSa Selective Supplement (FD229). Positive colonies are detected by it's bluish green color and identified as MRSA and selected for future assays.

Antibiotics susceptibility
The antibiotics susceptibility test was accomplished by using disk diffusion methods as instructed by the Clinical and Laboratory Standards Institute (CLSI) guidelines (37). A disposable wire loop was used, the tops of few bacterial colonies were transmitted to a test tube that contain 5ml of BHIB and incubated at 37C° for (4-6)h until the turbidity appearance. Culture turbidity was estimated at (1.5×10 8) cell/ml by compared it to 0.5 McFarland standard (NO. 0.5) (8). The antibiotics used in this study were shown in Table 1.

DNA extraction
The genomic DNA of the Staphylococcus aureus isolates was extracted using Genomic DNA purification kit purchased from (Promega/USA), the purity and concentration were tested, and the gel electrophoresis method was used for testing the integrity of DNA samples.

PCR amplification
A thermal cycler (BioRad, USA) were used to amplify PCR reactions. The mixtures of reaction was set up as follows: (1X) of GoTaq®Green Master Mix (Promega/USA), that composed of MgCl2, deoxynucleotides (dNTP), Taq DNA polymerase, reaction buffer, and green and yellow dyes that used to observe progress during electrophoresis. Various concentration of every used primer (10 pmol), (50-80)ng of DNA template then sterile deionized D.W was added to obtain a final volume. The PCR products were separated on agarose gels (2% agarose, 1μ of ethidium bromide (10 mg/ml), 1X TAE) and analyzed on OWL Electrophoresis System (Thermo, USA).

Touchdown PCR for nuc gene detection
The Staphylococcus aureus isolates confirmed by Touchdown-PCR using specific primer for nuc gene which was designed according to Brakstad et al (9). The Amplicom size and primer sequence were listed in Table 2. The program was adopted in PCR analysis of primer for nuc gene as shown in Table 3. Touchdown PCR for mecA gene detection Detection of Methicillin resistance was done by Touchdown-PCR with specific primer for mecA gene of Staphylococcus aureus isolates. This technique was used to confirm the detection of Methicillin resistant S. aureus (MRSA) isolates by using specific primers which was designed according to Zhang et al (40). The primer sequence and its amplicon size were listed in Table 2. The program was adopted in PCR analysis of primer for mecA gene as shown in    Figure 1. Using of chromogenic agar promotes the isolation and detection of MRSA from primary isolation plates during 24h after enrichment, without needing for extra biochemical tests (22). This method is cost effective, save time and supply powerful outcomes that are mimic PCR method results (39). The authors also mentioned that a concentration of 4 mg of cefoxitin/liter promote the inhibition of all MSSA isolates and the growth of all MRSA isolates (13). Alzaidi, (6) identified MRSA by using chromogenic agar and noticed that, among the 192 S. aureus isolates, 126 (65.6%) were MRSA of which 100 (66.6%) and 26 (61.9%) from the patients and environment, respectively, Nasser et al (26) showed that 77.9% were identified as MRSA from all S. aureus isolates in Indian hospitals. (10/177). All the isolates showed 100% susceptibility towards Imipenem as shown in Figure 2. The highest level of resistance to β-lactam due to mecA gene expression and may be to blaZ produced of S. aureus isolates and their alternative mechanism (34). Aminoglycoside modifying enzymes are the most common mechanism of resistance to aminoglycosides, especially in S. aureus (29). Lower resistance ability toward vancomycin; Glycopeptides group can be due to resistance van A operon which transformed from Enterococcus to Staphylococcus isolates that placed in gut (32). The S. aureus high susceptibility rate to Imipenem because of its high partiality for penicillin binding protein PBP2 produced by β-lactamase producing bacteria (18). The result also mentioned that (94.9%) of the isolates are multidrug resistant. MDR MRSA is overcome the world, which can be due to excessive use of antimicrobials agent randomly, physical contact with cattle animals and consuming of contaminated animals (36,37). Abdul-Wahhab, (1), mentioned that 100% of MRSA isolates in Baghdad were MDR.

Molecular analysis:
The nuc gene was screened using touchdown PCR technique in order to identify Staphylococcus aureus by using specific primers for detection of S. aureus isolates and exist of nuc gene. The nuc gene is baseline in identification and classification of S. aureus (4). The MRSA isolates were determined by using touchdown PCR technique by using specific primers for detection mecA gene and identification of MRSA isolates. The mecA gene responsible for resistance towards β-lactam antibiotics and maybe provide resistance ability toward other classes of antibiotics (24). The results shown that 198 (97.05%) of 204 isolates gave positive result for amplification of nuc gene (276bp) and identified as Staphylococcus aureus as shown in Figure 3, while 165 of 204 isolates (80.88%) gave positive result for amplification of of mecA gene (147bp) and identified as MRSA as shown in Figure 4. The outcomes of this study hinted that, 165 MRSA that identified formerly with classical biochemical tests was amplified with nuc and mecA genes successfully, what marked that thermo stable nuclease encoding gene is so precise for S. aureus, and the protein PBP2A (penicillinbinding protein2A) encoding gene is so precise for MRSA (4). The 165 isolates (80.88%) of 204 isolates were harbored the mecA gene, that seems close to local studies of Saleem et al (31) who mentioned that (80.7%) of 140 clinical S. aureus isolates were harbored the mecA gene, and abdul-wahhab, (1) who reported that (68.96%) among 43 clinical isolates were harbored the mecA gene. In other hands, 154/177 MRSA isolates (87%) shown methicillin resistance in susceptibility test and 23 (12.99 %) was methicillin sensitive, this maybe because that occasionally mecA gene was expressed invivo but not always invitro, additionally to that, the expression of mecA gene is minimum in bacterial cells that are considered as planktonic bacteria, or along these lines, it could be credited to their powerlessness to deliver enough PBP2A as mentioned by Murakami et al (25). According to the results, 177 (100%) isolates where identified as MRSA by using chromogenic agar, and 154 of 177 (87%) were showed methicillin resistance in susceptibility test, while 165 of 177 (93.22%) where harbored the mecA gene. The only isolates that contain mecA gene consider as MRSA and cannot depended only on phenotypic (susceptibility tests) to determine the MRSA isolates. For the same reason this study depends on molecular diagnosis of mecA gene as marker of MRSA isolates because the antimicrobial susceptibility profiles (phenotypic tests) cannot give a certain indicator for determination of MRSA (15). PCR based measures are considered as the highest quality level for the identification of MRSA, because of the heterogeneous resistance by different phenotypic identification strategies showed by numerous clinical samples. Genotypic strategies are more precise in recognizing Methicillin-Resistant S. aureus as contrasted with traditional powerlessness strategies (30).