الفهرس 
Characteristics of Staphylococcus aureusOnly 14 pages are availabe for public view
 |  | from | 162 |  |  |
| | | from | 162 | | |
Abstract
Staphylococcus aureus is an important human pathogen causing diseases
ranging from superficial infections to deep-seated and systemic infections. In this
study a total of 50 clinical Staphylococcus aureus strains were isolated from
patients in Ain Shams University Hospital in Cairo, identified by biochemical
tests. The susceptibility of isolates to methicillin and various antibiotics was
determined. The results demonstrated that 68% of isolates were resistant to
oxacillin (MRSA), and the resistance is primarily mediated by the production of
penicillin binding protein (PBP 2a), encoded by the chromosomal mecA gene.
The mecA gene was detected in all MRSA isolates and most of them showed coresistance
to other antibiotics (penicillin, erythromycin, gentamicin, and
tetracycline).
S. aureus produces a variety of extracellular toxins and virulence factors
which contribute to its pathogenic potential. In this work, all clinical S. aureus
isolates were analyzed by multiplex PCR for the presence of the five classical
enterotoxin genes(sea, seb, sec, sed and see) and four new enterotoxin genes
(seg, seh, sei and sej) genes. Polymerase chain reaction (PCR) of SE genes
indicated that 36% of the isolates were enterotoxigenic. The prevalence of sea,
and seb plus sec among the total clinical isolates was 22, and 2%. Sixteen percent
of the total isolates were seg positive, whereas 12%, 2% and 2% were sei, she,
and sej positive, respectively. All isolates containing sei were positive for seg,
whereas none of the isolates harboured sed or see genes.
Isolates were characterized by molecular biology tools, viz., randomly
amplified polymorphic DNA (RAPD), PCR-RFLP of 16S rRNA, and nucleotide
sequencing. The randomly amplified polymorphic DNA (RAPD) for all isolates
revealed a correlation between genetic diversity and antibiotic susceptibility of
methicillin-resistant Staphylococcus aureus (MRSA). A total of 7 genotypes were
identified at a 65% similarity level. Genotypes VII accounted for the largest
Summary and Conclusion
110
number of multidrug resistant isolates 44% and were resistant to all the four
antibiotics tested (penicillin G, erythromycin, gentamicin, and tetracycline). Also,
Typing by RAPD assay for the clinical S. aureus isolates revealed that the
distribution of the enterotoxin genes among S. aureus genotypes was not specific
to any genotypes. Genotypes III accounted for the largest number of
enterotoxigenic isolates (12%), while genotypes IV and VII included a great
diversity of enterotoxigenic isolates (sea, seb, sec, seg, she, sei, and sej).
RFLP technique for 16S rRNA gene was also used for estimating genetic
similarity among these isolates. Genotyping by PCR- RFLP revealed that 40% of
the isolates were type A, 32% to type B, and 28% to type C. The distribution of
the mecA gene among S. aureus genotypes was specific to genotypes A and
C. All isolates in genotype C harbored mecA gene, whereas all isolates in
genotype B were mecA-negative strains. The correlation between the prevalence
of enterotoxin genes and the genotypes obtained by PCR-RFLP of 16S rRNA
gene revealed the most and great diversity of enterotoxigenic isolates belonged to
genotype B.
The DNA sequence of a target gene is one of the most promising for
detection of genomic and somatic mutations and identification of strains.
Sequencing was performed for mecA gene, enterotoxin A (sea) gene for three
strains which isolated from different source (blood, sputum, and pus), and new
enterotoxin genes (seg, seh, sei and sej). Comparative analysis of these sequences
showed nucleotide variations at multiple sites when compared with other
sequences available in the database.
The other main objective of study is to detect the expression of the most
dominant enterotoxin (sea gene). The sea gene was isolated and cloned into
expression vector (pPROEX HTa) and enterotoxin A protein was induced in E.
coli by isopropyl-β-D-thiogalactopyranoside (IPTG) synthetic inducer. The results
Summary and Conclusion
111
indicated the sea gene was successfully cloned, expressed in E. coli and the
recombinant enterotoxin A was produced.
In conclusion, under carefully controlled spread the S. aureus infection
the results indicate that the high prevalence of multidrug resistant MRSA in
patients (44%). So, the choice of the suitable antibiotic for treatment of the S.
aureus infections should be used carefully to avoid the emergence of MRSA
isolates with reduced susceptibility to vancomycin. The usefulness of DNA-based
assays for the detection of antibiotic resistance genes associated with
Staphylococcal infections, and presence of these genes was genotype specific.
Also, the present investigation demonstrated that the sea gene was the
predominant enterotoxin gene in these genetically diverse Egyptian clinical
isolates. Further, this work indicates a systematic association between seg and sei,
and a wide distribution of these two genes among the S. aureus strains. RAPD
technique exhibits greater discriminatory power associated with 16s rRNA PCRRFLP
and describing their clonal relationships. Thus, DNA sequencing analysis
is simple and useful method to investigate the existence of regions for enterotoxin
gene rearrangement in S. aureus and the phylogenetic aspects of the
staphylococcal enterotoxins.