الفهرس 
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Abstract
The aim of this thesis was to test the sensitivity of MRSA clinical isolates collected from Alexandria Main University Hospital to antimicrobial agents from different groups with a special focus on the fluoroquinolonemember; moxifloxacin as an alternative treatment for MRSA infections.
SCCmectyping, spatyping and MLST typing methods were used for molecular characterization of the collected MRSA isolates, then the identified types were correlated with moxifloxacin resistance.
The thesis also aimed to test moxifloxacin combinations with other antibiotics for their possible beneficial effect on treating MRSA infection.
A total of 107 bacterial isolates were collected from different clinical specimens, including pus, blood, BAL, urine, mini BAL, aspirate,sputum and tissue.HA infections represented 54% of the isolates while 23% of the isolates were categorized as CA infections.
After the preliminary macroscopical (colony morphology) and microscopical (gram-staining) examination, the identity of the collected isolates was further checked using phenotypic methods as growth on MSA, DNase agar and Staphytect plus test, as well as genotypic methods as PCR amplification of 16S rRNAand nucgenes.
Finally, the identity of the isolates was confirmed using MALDI-TOF. Methicillin resistance was tested through disc diffusion susceptibility testingand PCR detection of mecAgene.Seventy-seven isolates were identified as S. aureus, including 72 MRSA, the rest of the isolates were identified as S. haemolyticus (26 isolates),S. sciuri(2 isolates), S. hominis(1isolate)andS. warneri (1isolate).
The phenotypic identification methods weremore accurate than the genotypic methods and its results matched the MALDI-TOF results in 98.1% of the cases.
Although Staphytectplus test was the most accurate phenotypic method, the two S. sciuriisolates showed false positive results. Susceptibility testing and mecAgene detection were equally successful in methicillin resistance detection. To determine the antibiotic resistance pattern of the collected isolates using disc diffusionmethod; several antibiotics were selected to cover different fluoroquinolone generations and antibiotic groups,including nalidixic acid, norfloxacin, ciprofloxacin, levofloxacin, moxifloxacin, gentamicin, chloramphenicol, fusidic acid, tetracycline, rifampicin, erythromycin, sulfamethoxazole-trimethoprim, teicoplanin and linezolid. All S. aureusisolates were 100%resistant to nalidixic acid, 68% to norfloxacin, 65% to ciprofloxacin, 65% to levofloxacin, 65% to moxifloxacin, 87% to tetracycline, 71% to fusidic acid, 69% to gentamicin, 51% to rifampicin, 48% to chloramphenicol and 43% to erythromycin.
On the other hand, both teicoplanin and linezolidwere completely effective againstall the testedS.aureusisolates.
HA-MRSA isolates were more resistant to moxifloxacin than CA-MRSAwith 75% versus55%, respectively. MICs of moxifloxacin was determined by the broth microdilution technique against S. aureusclinical isolates. Moxifloxacin measured MICsvaluesranged from 0.0625 to 16 μg/ml.
The results were interpreted according to CLSI guidelinesto show 65% moxifloxacin resistance within the tested S. aureusisolates. MIC50 and MIC90 also were calculated to be 2 μg/ml and 4 μg/ml, respectively.SCCmec type III was the predominant type with 53% occurrence within the tested isolates followed bySCCmec types IV, IVE and V with less representation as 14%, 6% and 10%, respectively.
<The isolates (seven isolates) that generated nonspecific amplification patterns need further analysis. SCCmec type III was found to be more related to HA-MRSA
109isolates (70%) thanthe rest of isolates with 100% moxifloxacin resistance.
<>On the other hand, SCCmec types IV, IVE and V were more related to moxifloxacin sensitive isolates,regardless the infection type.Upon determination of the spatype; the spatype with highest frequency was spatype t037 (55%) followed by spatypes t127, t267, t688, t223, t044 and t304 with frequencies 16%, 8%, 5%, 4%, 4% and 3% respectively. spatypes t416, t315, t786 and t6978 were identified once throughout the tested isolates.
A new identified spatype with name t16221 and repeat succession (07-56-12-17-16-33-31-57-21-12) also was identified. spatype t037 was completely associated with SCCmectype III and with moxifloxacin resistance while both spatypes t127 and t267 were moxifloxacin sensitive (> 85 %) and originated from HA and CA S. aureuswith more HA infections in case of spatype t127.SCCmectype IV was associatedwith spatype t267 (66%) while SCCmectype V was more associated with spatype t127 (71%) after excluding of MSSA and non-identified isolates.spatype t304 also was categorized under SCCmectypes III and IV while the rest of spatypes were distributed under the different SCCmectypes.
<All isolates with spatype t037 were MRSA with 70% HA-MRSA and only one CA-MSSA isolate.HA-MRSA was strongly associatedwith spatypes t127, t223, t786 and t315. Aphylogenetic tree using UPGMA method was constructed to deducethe evolutionary history of the identified spatypes and the degree of relatednessin between. Three clades were identified; the first includedspatypes t044, t416 and t127, the second cladeincluded types t304, t267 and t786 while the rest of identified spatypes (t16221, t315, t037, t688, t223 and t6978) belongedto the third clade.Eleven selected isolates with differentspaand SCCmec types were used for MLST determination. ST-1, ST-5, ST-6, ST-22, ST-80, ST-97, ST-239, ST-241, ST-1502 and ST-4808 were the identified sequence types ST-22 identified twice. ST-4808 was identified for the first time in this study with allelic profile of (3-1-1-1-1-99-3). Theevolutionary history of the identified MLST types and the degree of relatednessin between were determined. ST-239 and ST-241 were closely related to each other with SLV and grouped under the same cluster complex; CC-239. The same was found with ST-80 and ST-1502 that grouped under cluster complex CC-80. Also, the new profile, ST-4808 was a SLV to ST-97 under the cluster complex CC-97 with an extended relation with ST-1 as TLV to both profiles ST-97 and ST-4808. Another extended relation was found between ST-5 and ST-6 with DLV relation.
On the other hand, ST-22 was identified as a singleton with no relation to the other profiles.
By linking the identified SCCmec, spaand MLST types; several known clones and other new clones were identified. The majority ofthetested isolates belonged toST241-III/t037 clone. Five new MRSA clones were identified; ST239-III/t304, ST1502-IV/t044, ST4808-IV/t267, ST22-IVE/t223and ST22-IVE/t6978 in addition to ST97-V/t267 clone thatisreported for the first time fromhuman MRSA.Clones ST1-V/t127, ST80-IV/t416, ST5-IVE/t688 and ST6-IV/t304 are alreadyknown clones also identified in this study.
Several moxifloxacin combinations with other antibiotics were studied to determine their potential beneficialactivityagainst resistant MRSAisolates. Firstly, moxifloxacin combined effect with linezolid, teicoplanin, tetracycline, chloramphenicol, sulfamethoxazole-
110trimethoprim, gentamicin, erythromycin and rifampicin were tested against 24 isolates using the double disc diffusion method. Both synergism and antagonism were reported with most of the tested combinations, yet, synergism was more predominant with teicoplanin (83%).
On the other hand, the rest of the tested combinations showed antagonism with higher percentages; linezolid (63%), chloramphenicol (58%), rifampicin (34%), gentamicin (38%), erythromycin (42%) and sulfamethoxazole-trimethoprim (42%).
Indifference was more predominant in case of moxifloxacin-tetracycline combination which also showedalmostequal representation of synergism (25%) and antagonism (20%).
Three moxifloxacin combinations were selected and tested against eight isolatesfor studying the bactericidal activity.
<The selected combinations were moxifloxacin-linezolid as it showed the highest antagonism percentage, moxifloxacin-teicoplanin as it showed the highest synergistic percentage and finally moxifloxacin-tetracycline asa representativeofindifference behavior. Synergistic effect was reported with the three tested combinations; moxifloxacin-teicoplanin(37.5%), moxifloxacin-linezolid(25%)and moxifloxacin-tetracycline(25%).
Additive effect was reported with the restof the tested isolates with the three combinations