Studies on interactions between certain microbial species isolated from mixed infection

Abstract

The present study focused basically on studying the interactions between certain microbial species isolated from mixed infection and how these interactions affect their coexistence. Therefore, isolates recovered from clinical specimens showing mixed infections were collected. Pus was the clinical specimen of the highest prevalence of mixed infections in comparison to other clinical specimens. Both Staphylococcus and Pseudomonas isolates coexisted in mixed infection at the highest prevalence. Different Pseudomonas isolates that coexisted in mixed infection with Staphylococcus isolates were assayed for their protease, lipase, and acylhomoserine lactone (AHL) productivities. Results obtained showed no significant difference in both protease and lipase productivities however, production of AHL showed significant variation among the respective isolates. Antibiogram analyses of the collected clinical isolates were determined and showed significant variation. From the previous findings, two models of coexisting Pseudomonas 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 Pseudomonas (P.) aeruginosa however, they showed significant difference in their antibiogram analysis as well as presence or absence of endogenous plasmids. 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 Abstract 2 formation of the co-existing MRSA isolate (S12). Quorum sensing controlling genes including acyl homoserine synthase (ahl), 2-heptyl-3-hydroxy-4(1H)-quinolone synthase (pqsH) and AraC family transcription regulator (araC) of P. aeruginosa, particularly those involved in the regulation and synthesis of major molecules of quorum sensing when coexisting with Gram positive pathogens were detected and amplified 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 and deposited in GenBank database under the accession codes, KT693035, KT693034, KT693033, respectively. Domains and phylogenetic analysis as well as analysis of the predicted tertiary structures of the 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 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 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 however, the culture supernatant of P. aeruginosa harbouring endogenous plasmids (P12) showed an increase of the biofilm formation of the co-existed MRSA isolate. Accordingly, plasmid acquisition significantly decreased the inhibitory effect of P. aeruginosa isolate P14 on the biofilm formation of the co-existing MRSA isolate S14.