الفهرس 
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Abstract
A total number of 182 clinical isolates were collected from various clinical specimens. The Gram-positive were 150 Staphylococcus sp isolates including 97 S. aureus (64.7%, n=150), 53 coagulase-negative Staphylococcus sp (35.3%, n=150). The Gram-negative isolates were 19 Klebsiella pneumoniae (59.4%, n=32) and 13 Escherichia coli (40.6%, n=32) isolates. All the isolates were collected from different clinical specimens including 87 pus (47.8%, n=182), 32 blood (17.6%, n=182), 44 sputum (24.2%, n=182) and 19 bronchoalveolar lavage (BAL) (10.4%, n=182) specimens, from the Microbiology diagnostic laboratories of Al-Demerdash Hospital during the period from October 2015 to March 2016.
The Antimicrobial susceptibility test by the disc diffusion method showed that, out of the 150 collected Staphylococcus sp. isolates, 54 isolates (36%, n=150) were resistant to 2 or more of tested antibiotics. Out of the resistant isolates, thirty-five isolates (36%, n=97) were S. aureus; while the other 19 isolates were coagulase-negative Staphylococcus sp isolates (CoNS) (35.8%, n=53). While, all the collected Gram-negative isolates showed resistance to two or more of tested antibiotics. It was found that the constitutive macrolide-lincosamide-streptogramin resistance phenotype (cMLS) was the most predominant phenotype among the resistant Staphylococcus sp isolates with 54.3% among S. aureus isolates and 78.9% among CoNS isolates. Macrolide (MAC) resistant Staphylococcus sp isolates were tested for their susceptibility to methicillin, where 22 methicillin-resistant Staphylococcus aureus (MRSA) isolates, and 9 methicillin-resistant CoNS (MR-CoNS) isolates were detected.
The minimum inhibitory concentration (MIC) against erythromycin (ERY) and azithromycin (AZM) were determined for MAC-resistant S. aureus isolates. It was found that 88.5% and 91.4% of S. aureus isolates showed high levels of resistance against ERY and AZM, respectively (MIC>1000 µg/ml). Also, 88.4% and 94.7% of CoNS isolates showed high levels of resistance against ERY and AZM respectively (MIC>1000 µg/ml).
The polymerase chain reaction (PCR) results showed that, among the resistant Staphylococcus sp isolates, one MAC-resistance coding gene was present in thirty-one isolates, two resistance coding genes were found in eighteen isolates, while five resistant isolates harbored the three resistance coding genes. It was found that, erythromycin ribosomal methylase (ermC) was the most frequent (79.6%), followed by macrolide streptogramin resistance (msrA) gene (48.9%), then ermA gene (31.5%). Our findings revealed that, all isolates showing resistance to both MACs and lincosamides were found to harbor at least one type of erm genes (either ermA or ermC), while the genotype of the four isolates exhibiting macrolide-streptogramin resistance phenotype (MS phenotype), showed that only msrA was present. Among the resistant Gram-negative isolates, it was found that erythromycin esterase (ereA) gene was detected in three and five isolates of E. coli and K. pneumoniae, respectively; while macrolide phosphotransferase (mphA) gene was present in twelve E. coli and two K. pneumoniae isolates.
For an attempt for expression of ermC and msrA, the PCR amplicon of the respective genes were successfully amplified using appropriate designed primers harboring appropriate restriction sites. Agarose gel electrophoresis showed the expected PCR product size (amplified ermC gene and msrA gene at 700 bp and 1.5 kbp, respectively). The respective genes were cloned into pUCPU21 cloning vector. Successful transformation of recombinant plasmids (pUC-ermC) and (pUC-msrA) was confirmed by plasmid extraction of recombinant plasmids from white colonies, followed by restriction and agarose gel electrophoresis. For the purpose of expression of the two genes and purification of the resulted proteins, several attempts were carried out to clone the DNA inserts of the respective genes into pET-22b plasmid (expression vector), however, they were unsuccessful.
The effect of different combinations between MACs (ERY and AZM) with different antibiotics linezolid (LZD), ceftriaxone (CEF), cefotaxime (CTX), amikacin (AMI) and gentamicin (GEN) on MAC-resistant MRSA isolates, was studied using checkerboard assay. It was revealed that (AZM+LZD), (AZM+CEF), (AZM+GEN) and (AZM+CTX) showed synergistic effect in 40.9%, 45.5%, 30.85% and 9.1%, respectively of the 22 MRSA isolates (FIC<0.5), while additive effect was detected when AZM used in combination with AMI on the same isolates (0.5<FICI<1). Also, additive effect was detected when ERY was used in combination with all the antibiotics mentioned above, thus we can do more research in this area to test other combinations for the purpose of infection control.