Diversity of microbes associated with the marine sponge, Haliclona simulans, isolated from Irish waters and identification of polyketide synthase genes from the sponge metagenome.

Samples of the sponge Haliclona simulans were collected from Irish waters and subjected to a culture-independent analysis to determine the microbial, polyketide synthase (PKS) and non-ribosomal peptide synthase (NRPS) diversity. 16S rRNA gene libraries were prepared from total sponge, bacterial enriched sponge and seawater samples. Eight phyla from the Bacteria were detected in the sponge by phylogenetic analyses of the 16S rRNA gene libraries. The most abundant phylum in the total sponge library was the Proteobacteria (86%), with the majority of these clones being from the gamma-Proteobacteria (77%); two groups of clones were dominant and together made up 69% of the total. Both of these groups were related to other sponge-derived microbes and comprised novel genera. Within the other bacterial phyla groups of clones representing novel candidate genera within the phyla Verrucomicrobia and Lentisphaerae were also found. Selective enrichment of the bacterial component of the sponge prior to 16S rRNA gene analysis resulted in a 16S rRNA gene library dominated by a novel genus of delta-Proteobacteria, most closely related to the Bdellovibrio. The potential for the sponge microbiota to produce secondary metabolites was also analysed by polymerase chain reaction amplification of PKS and NRPS genes. While no NRPS sequences were isolated seven ketosynthase (KS) sequences were obtained from the sponge metagenome. Analyses of these clones revealed a diverse collection of PKS sequences which were most closely affiliated with PKS from members of the Cyanobacteria, Myxobacteria and Dinoflagellata.

An investigation into the antimicrobial mechanisms of action of two contact lens biocides using electron microscopy.

Polyquaternium-1 (PQ-1) and myristamidopropyl dimethylamine (MAPD) are biocides used commercially in a contact lens disinfecting solutions. Electron microscopy was used to provide further evidence on the mechanism(s) of action of these agents against a wide range of ocular pathogens including bacteria, fungi and protozoa. Both PQ-1 and MAPD caused multiple forms of damage to the organisms tested, evidenced by structural alterations, blebbing, leakage and cell destruction. The extent of damage and the selectivity against specific type of microorganisms was consistent with the antimicrobial activity of these agents. Although electron microscopy is a powerful tool, it has its limitations when used to examine the mode of action of biocides. Indeed, there was no evidence of gross structural alteration to Acanthamoeba castellani or Aspergillus fumigatus following treatment.

Aspects of the antimicrobial mechanisms of action of a polyquaternium and an amidoamine.

OBJECTIVES: Polyquad (Alcon) (polyquaternium-1, PQ-1) and Aldox (Alcon) (myristamidopropyl dimethylamine, MAPD) are two biocides that are used commercially in a contact lens disinfecting solution, namely Opti-Free Express (Alcon) multi-purpose disinfecting solution. Their potential mechanisms of action were investigated against a range of common ocular pathogens. These were Acanthamoeba castellanii (trophozoites and cysts), Aspergillus fumigatus, Candida albicans, Pseudomonas aeruginosa, Serratia marcescens and Staphylococcus aureus. METHODS: Three aspects were investigated: the lethal effects of the biocides on the organisms, the leakage of K+ from treated cells, and the lysis of spheroplasts derived from the cells. RESULTS: PQ-1 was found to have predominantly antibacterial activity, and induced K+ leakage from the bacteria and C. albicans. It also caused lysis of spheroplasts of S. marcescens, but not those of C. albicans. MAPD was active against all of the organisms, but showed higher activity against the fungi and amoeba. It induced K+ leakage from A. fumigatus and C. albicans, and like PQ-1, lysed the spheroplasts of S. marcescens but not C. albicans. CONCLUSIONS: The two biocides have different spectra of antimicrobial activity. PQ-1 has mainly antibacterial activity, whereas MAPD was active against all of the test organisms, particularly the fungi.

Performance of contact lens disinfecting solutions against Pseudomonas aeruginosa in the presence of organic load.

PURPOSE: Contact lens wearers can become noncompliant with lens disinfection procedures. The purpose of this study was to evaluate multipurpose contact lens disinfecting solutions in the presence of organic soil. METHODS: OPTI-FREE EXPRESS and three products preserved with polyhexamethylene biguanide (PHMB) were inoculated with three strains of Pseudomonas aeruginosa in organic soil. Surviving bacteria were counted at various time points, using the ISO 14729 standard method. RESULTS: Results indicated that OPTI-FREE EXPRESS was efficient in the presence of organic soil and achieved a reduction of almost 6 log. The other products were less effective under the test conditions and failed to achieve 3-log reduction of the bacteria. These products also seemed to lose activity after time, allowing surviving bacteria to multiply. Conversely OPTI-FREE EXPRESS maintained high activity for the entire 72 hours of the study. CONCLUSIONS: The results indicate that OPTI-FREE EXPRESS would remain highly efficient in the case of noncompliance with disinfection procedures.

Identification of genes involved in the susceptibility of Serratia marcescens to polyquaternium-1.

OBJECTIVES: Polyquaternium-1 (PQ-1) is a biocide used commercially in a contact lens disinfecting solution, 'Opti-Free Express (Alcon) Multi-Purpose Disinfecting Solution'. The genetic basis for resistance of Serratia marcescens to PQ-1 was investigated using a random transposon-based mutagenesis approach. METHODS: S. marcescens was subjected to random transposon mutagenesis using a mini-Tn5 Km2 transposon. Mutants with increased susceptibility to PQ-1 were selected and the disrupted genes were identified. Antibiotic susceptibility profiles were also determined for all of the mutants. RESULTS: A wide range of genes were found to be disrupted in the mutants. The most common were genes associated with the cell membranes, or involved in biosynthesis and metabolism. CONCLUSIONS: This study shows that random transposon mutagenesis is an effective tool for the elucidation of mechanisms of action and resistance to biocides. The results support our previous findings that PQ-1 is active against the cytoplasmic membrane of S. marcescens.