Identification of Methicillin Resistance Staphylococcus aureus in Immunocompromised Host Using PCR Techinque and Detection of Its Sensitivity to Antibiotics ……………

Two hundred and twenty clinical specimens were collected from were collected from patients in Medical City Hospitals (Baghdad /Iraq).During the period From November 2013 to April 2014.The specimens were included (54) ear swab, (56) blood, (70) burn swab, and (40) urine sample. It was found that (49.03%) of the 108 isolates were belong to Staphylococcus spp., were diagnosed as coagulase-positive staphylococci (COPS), where 112 (50.9%) of clinical samples were coagulase negative (CONS). From the collected clinical samples, 85(78.7%) were MRSA according to sensitivity test and vitek system, and the rest were MSSA 23(21.9%). In the present study, ceftaroline is drug of choice, because of it activity against Methicillin-resistant S. aureus .The effectiveness of the ceftaroline antibiotic are being tested on 85 isolate of Methicillin-resistant S. aureus (MRSA), all isolate were sensitive to ceftaroline.


Introduction
The S. aureus bacterium is a serious human pathogen that causes lifethreatening nosocomial and community associated infections (1).Staphylococcal infection can affect many sites and organs of the human body.Invasion of the skin cause impetigo, cellulitis.In the lungs abscesses and pneumonia are the result.Infection of the heart leads to endocarditis.Meningitis and abscess formation can be the result of infection to the central nervous system as well as, keratitis can be the result of eye infection (2).S. aureus are grouped into two major classes: Methicillin Sensitive S. aureus (MSSA) and Methicllin Resistant S. aureus (MRSA).MRSA strains have been associated with nosocomial or hospital acquired infections world over and have also emerged as an important cause of community acquired infections (3).Resistance to methicillin is mostly determined by the presence of mecA gene encoding altered penicillin binding protein which shows low affinity to β-lactam antibiotics (4).The mecA gene, which originates from a mobile genetic element named the staphylococcal cassette chromosome mec [SCCmec] invariably inserted into the orfX gene of methicillin-resistant staphylococci, is the genetic basis of methicillin resistance (5, 6).

‫مجلة‬
Vancomycin has been the cornerstone of treatment of patients with serious MRSA infections.Consequently, vancomycin use has been increasing since the mid-1980's, resulting in the emergence of MRSA with reduced susceptibility to vancomycin (7).However many researchers have been noted MRSA that resistant to vancomycin (8,9).
The cephalosporin class of antimicrobial agents is known for its broad spectrum of activity, proven efficacy and favourable safety profile, making it the most commonly prescribed class of antimicrobials (10).Ceftaroline fosamil is a cephalosporin prodrug whose active principle, ceftatoline (CPT), is active against MRSA and drug-resistant S. pneumoniae (11).This compound has the distinction of being the first anti-MRS β-lactam to be marketed in the USA (2010), where it received FDA approval for treatment of acute bacterial skin and skin structure infections (SSSI) and for community-acquired pneumonia (CAP) (12).Ceftaroline shows good clinical efficacy against (MRSA) due to its ability to bind to PBP2A (13)

Staphylococcus aureus isolation and identification
Specimen's collection: From November 2013 to April 2014, Two hundred and twenty specimens were collected from patients in Medical City Hospitals (Baghdad /Iraq).The specimens were included (54) ear swab, (56) blood, (70) burn swab, and (40) urine sample.Isolation: The collected specimens were inoculated on the blood agar, incubated at 37ºC for 24 hours.The isolates were examined for their haemolytic activity.Then transferred and streaked on mannitol salt agar, and to detecting the ability of each isolate to ferment mannitol.All plates were incubated at 37ºC for 24 hours, then transfer a single pure isolated

Polymerase Chain Reaction (PCR) Technique
Target DNA amplification with a pair of primers, resulting in several copies of the target sequence (17).

Preparation disks of ceftaroline
Ceftaroline stock solution prepared by dissolving 600 mg in 20 ml of D.W.The reconstituted solution is a pale yellow solution that is free of any particles.Each disk should contain 30 Mg of ceftaroline, were prepared according to this equation: C1 * V1= C1 * V2

Statistical Analysis
The Statistical Analysis System-SAS (2012)(18) was used to effect of different factors in study parameters.Chi-square test was used to significant compare between percentage in this study.

Results and Discussion:
Two hundred and twenty clinical samples were collected from different patients, the samples included burn swab, ear swab, urine, and blood  The highest percentage of MRSA isolates, was from burn samples, it was 93.87% table (2).This high percentage may attributed to that the skin which represent the first line of defense and an example of innate immunity is damaged and the burned parts of patient remain exposed to air, that increase opportunity of infection with pathogenic bacteria .Burns remain considerable serious problem of public health associated with morbidity and mortality ,our Explanation close to the explan of Othman and Kendrich, (2011)(19).Burn patients become susceptible to infection due to the loss of the protective barrier and decreased cellular and humoral immunity (20).Infection remains a major complication in burn patients after initial period of shock and the chance of infection persist until complete wound healing (21).
From the collected clinical samples, 85 (78.7%)where MRSA according to sensitivity test and vitek system, and the rest were MSSA 23(21.9%).Present results were agree with agree with the results of a local study by Al-Maliki, (2009) (22) who showed that the percentage of MRSA to the MSSA were (80.3%, 16.4%) respectively.As well as, Al-Hasani, (2011)(23) reported that the ratio of MRSA was (83.70%).On the other hand, Al-Geobory, (2011) (24) showed that the ratio of MRSA was (90.90%).On the other hand, current results did not agree with the results of peck et al., (2009) (25) which showed that only (51.4%) of isolates were methicillin resistant.Also, the finding of AL-alem, (2008) (26) showed that the ratio of MRSA strain was (56%).These observed differences may due to the variation in the geographic area, sources of clinical specimens, genetic background and the collection site of isolates.The vitak-2 Compact was used to confirm the identification and typing of MRSA isolates which previously identified by conventional biochemical tests, the result from vitak-2 were closely with those obtained from sensitivity test for same antibiotics that used in both tests.
A confirmatory test was carried out for the selected isolates using PCR technique for further characterization up to the species level by the amplification of (mecA) gene ,and all the isolates are found to be positive for the presence of (mecA) gene figure(1).In the present study, ceftaroline is drug of choice, because of it activity against MRSA .The effectiveness of the ceftaroline antibiotic are being tested on 85 isolate of MRSA, 39 (45.88%) isolates gave 32 mm dimmter inhibition zone, 18 (21.17%)isolates gave 30 mm dimmter inhibition zone, 8(9.4%) isolates gave 28 mm dimmter inhibition zone, 7 (8.2%)isolates gave 27 mm dimmter inhibition zone, and 13 (15.29%)isolates gave 26 mm dimmter inhibition zone figure (2).All 85 isolate of (MRSA) are sensitive to ceftaroline, so ceftaroline has demonstrated bactericidal in vitro activity against MRSA, and this conclusion agree with (27,28)

MIC
No. of isolates Percentage (%) 0.5 µg/ml 81 95.29% 0.25 µg/ml 3 3.52% 0.12 µg/ml 1 1.17% Ceftaroline demonstrated good activity against MRSA, It in vitro activity against MRSA is related to its high affinity for PBP2a, (29).Different ceftaroline MICs value depending on geographical location (30), and inverse correlation between PBP2a binding affinity and ceftaroline MIC, which is expected because the anti-MRSA activity of ceftaroline reflects binding and inhibition of PBP2a (31).Data from case series suggest that ceftaroline is safe and effective for severe MRSA infections with success rates in over 70% of cases (32,33) and Pleiman, 2013).To date, clinical trials have demonstrated efficacy of ceftaroline similar to that of comparator agents in the treatment of ABSSSI and CABP.Clinical trials suggest that ceftaroline is well tolerated common to the cephalosporin class.Overall, the most common adverse events occurring in more than 2% of patients in clinical trials were diarrhea, nausea, and rash (34).

Figure ( 1 )
Figure (1) Agarose gel electrophoresis of PCR product amplified from mecA genes.These genes from MRSA isolates.M = DNA marker fragments.Lane 1, 2, 3 & 4 indicate the mecA positive samples, 5 negative control.The DNA fragments of 533 bp were amplified from mecA gene.

Table ( 2) Prevalence of MRSA isolates among S. aureus isolates
Identification of Methicillin Resistance Staphylococcus aureus in ImmunocompromisedHost Using PCR Techinque and Detection of Its Sensitivity to Antibiotics …………….. Noor M. Abdullah , Amna N. Jassim , Abaas A. Alani Susceptibility of S. aureus isolates was detected against (10) types of antibiotics, the tested pathogenic isolates were found to exhibit obvious level of resistance against the used antibiotics and the susceptibility pattern for these clinical S. aureus isolates are shown in table (3).In this study the results demonstrated that out of (108) tested S. aureus isolates that were isolated from( 4) sources, about (85) isolates showed a high level of resistance to Penicillin G , Chloramphenicol, Cefoxitin, Oxacillin , and Methicillin respectively table(3) .

Resistance & Sensitive to Difference Antibiotic Used for Sensitivity Test of S. aureus Isolates
Identification of Methicillin Resistance Staphylococcus aureus in ImmunocompromisedHost Using PCR Techinque and Detection of Its Sensitivity to Antibiotics …………….. Noor M. Abdullah , Amna N. Jassim , Abaas A. Alani , and PolenakovikIdentification of Methicillin Resistance Staphylococcus aureus in Immunocompromised Host Using PCR Techinque and Detection of Its Sensitivity to Antibiotics …………….. Noor M. Abdullah , Amna N. Jassim , Abaas A. Alani