الفهرس 
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Abstract
Summary
Mixed infection is infection with more than one kind of organism at the same time as in some abscesses, pneumonia, and infections of wounds. Interspecies interaction between organisms is any process by which an organism has an effect on an organism of a different species. Historically, interspecies interactions have focused on growth-inhibitory interactions, yet a variety of phenotypic outcomes other than antibiosis are possible, including changes in metabolism (growth inhibition or stimulation, or production of new small molecules) or morphological and developmental changes (alterations in cell shape or morphology; production of biofilms or specialized processes such as sporulation and germination).
The interactions between different species may be due to new roles for known molecules such as peptidoglycan, antibiotics at subinhibitory concentrations or signals used in signaling system crosstalk e.g. autoinducer-2 (a well-characterized quorum-sensing molecule) and some fatty alcohols and acids where intraspecies small molecule signals may modulate the microbial development of different species.
That is why the present study focused basically on studying the interactions between certain microbial species isolated from mixed infection and how these interactions affected their coexistence.
Therefore, clinical specimens showed mixed infections were collected (35 out of 142 collected clinical specimens with a percentage of about 25%). The recovered isolates from mixed infection (72 out of 179 of total collected isolates with a percentage of about 25 %) were collected from different clinical specimens in the period of about one year of this study. Pus was the clinical specimen of the highest prevalence (74%) of mixed infections in comparison to other clinical specimens. Thirty three out of 35 clinical specimens with mixed infections (94.3%) harboured
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two microbial species while the other two specimens harboured three bacterial species. Both Staphylococcus and Pseudomonas isolates coexisted in mixed infection at the highest prevalence (42.8 %) compared to other coexisted bacterial isolates.
Different Pseudomonas isolates that coexisted in mixed infection with Staphylococcus isolates were assayed for their protease and lipase productivities however, they showed no significant differences. Moreover, acylhomoserine lactone (AHL) production as well as antibiogram analysis of the respective isolates showed significant variation.
from the previous findings, two models of coexisting and Staphylococcus isolates (SP12 and SP14 models) were selected for studying their interactions using different physiological parameters. This selection was based on the results obtained where the two Staphylococcus isolates, S12 and S14 were methicillin-resistant (MRSA) and the two Pseudomonas isolates, P12 and P14 were identified as P. aeruginosa however, they showed significant difference in their antibiogram analysis as well as presence or absence of endogenous plasmids.
Although both Staphylococcus (S.) aureus isolates S12 and S14 were methicillin resistant (exhibited cefoxitin resistance and shared the same resistance profiles to two additional antimicrobial agents clindamycin and ceftazidime), they showed different resistance profiles where S. aureus S12 showed resistance profile to amikacin, cefoperazone, ciprofloxacin, piperacillin- tazobactam for which S. aureus S14 was sensitive. Similarly, S. aureus S14 showed resistance to co-amoxiclav, erythromycin and co-trimoxazole, the antimicrobial agents to which S. aureus S12 was sensitive.
In case of P. aeruginosa, although both isolates were resistant to clindamycin, erythromycin, vancomycin, cefoxitin and co-trimoxazole, isolate P12 was resistant to co-amoxiclav, azithromycin, cefipime, doxycycline, levofloxacin, cefoperazone,
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piperacillin-tazobactam, ceftazidime, cefpodoxime, sulbactam-ampicillin and cefotaxime, the eleven antimicrobial agents to which isolate P14 was sensitive.
This antimicrobial profile of the coexisting microbial species in the two selected models could represent good examples for studying their interaction. Results showed that there was significant reductions in the viable count of both S. aureus isolate S14 and P. aeruginosa isolate P14 when grown in co-culture as compared to their growth in monocultures. While, there was no significant difference in the growth of P. aeruginosa isolate P12 in monoculture and in co-culture with S. aureus isolate S12. Moreover, there was no significant effect of different physiological factors (incubation temperature and pH) on growth profile however, optimum reduction effect of P14 on its coexisted S. aureus S14 was observed at 37ºC and initial pH 7.2 .
Furthermore, the culture supernatant of P. aeruginosa P14 (harboring no plasmids) exerted a significant reduction effect on the biofilm formation (about 57% reduction) of the co-existing MRSA isolate (S14) however this effect was not observed upon using the culture supernatant of P. aeruginosa P12 (harboring plasmids) on the biofilm formation of the co-existing MRSA isolate (S12)
Quorum sensing controlling genes including acylhomoserine synthase (ahl), 2-heptyl-3-hydroxy-4(1H)-quinolone synthase (pqsH) and AraC family transcription regulator (araC) of P. aeruginosa, particulary those involved in the regulation and synthesis of major molecules of quorum sensing when coexisting with Gram positive pathogens were detected using PCR and chromosomal DNA as templates.
DNA sequencing and genetic analysis of the PCR products of the respective genes were carried out. The quorum sensing controlling genes araC, PqsH and ahl obtained in this study were submitted into the GenBank database under the accession codes, KT693035, KT693034, KT693033, respectively. AraC was a model of AraC transcription regulator with a conserved N-terminal arabinose-
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binding domain and C-terminal H-T-H motive. PqsH was a model of putative 2-heptyl-3-hydroxy-4(1H)-quinolone synthase with a conserved domain of a NAD (P)-binding Rossmann-like domain. LasI showed a conserved domain with the acyl-homoserine-lactone synthase (LasI) of the protein family COG3916. The open reading frames (ORFs) of the respective genes showed no mutation or deviation in the predicted tertiary structures.
Domains and phylogenetic analysis as well as analysis of the predicated tertiary structures respective gene products showed significant conservation (more than 80%) in the nucleotide/amino acid sequences with the respective homologous in the GenBank database. Moreover, the the open reading frames of the respective genes showed no mutation or any deviation on the entire genes. Therefore, to confirm the difference in the inhibitory effects among the the two selected models, plasmid transformation experiments were carried out followed by testing the inhibitory effect of the culture supernatant of the obtained transforamant P14 isolate.
Accordingly, plasmids extracted from P. aeruginosa clinical isolate P12 were used to transform competent cells prepared from P. aeruginosa clinical isolate P14. Results showed that only three plasmid bands out of six had been successfully transformed into P. aeruginosa clinical isolate P14 host strain. Results showed the culture supernatant of P. aeruginosa harbouring no endogenous plasmids (P14) exerted a significant reduction effect on the biofilm formation of the co-existed MRSA isolate (about 57% reduction) however, the culture supernatant of P. aeruginosa harbouring endogenous plasmids (P12) showed an increase of the biofilm formation of the co-existed MRSA isolate (about 5% increase). However, plasmid acquisition significantly decreased the inhibitory effect of P. aeruginosa isolate P14 on the biofilm formation of the co-existing MRSA isolate S14.