phoU inactivation in Pseudomonas aeruginosa enhances accumulation of ppGpp and polyphosphate.

Inorganic polyphosphate (polyP) is a linear polymer composed of several molecules of orthophosphate (Pi) linked by energy-rich phosphoanhydride bonds. In Pseudomonas aeruginosa, Pi is taken up by the ABC transporter Pst, encoded by an operon consisting of five genes. The first four genes encode proteins involved in the transport of Pi and the last gene of the operon, phoU, codes for a protein which exact function is unknown. We show here that the inactivation of phoU in P. aeruginosa enhanced Pi removal from the medium and polyP accumulation. The phoU mutant also accumulated high levels of the alarmone guanosine tetraphosphate (ppGpp), which in turn increased the buildup of polyP. In addition, phoU inactivation had several pleiotropic effects, such as reduced growth rate and yield and increased sensitivity to antibiotics and stresses. However, biofilm formation was not affected by the phoU mutation.

Interactions of ocular isolates of gram-negative bacteria with lactoferrin.

PURPOSE: The aim was to examine the interaction of lactoferrin with gram-negative bacteria isolated from ocular adverse responses. METHODS: Strains representing the species Pseudomonas aeruginosa (six strains), Aeromonas hydrophila, Escherichia coli, Stenotrophomonas maltophilia, and Haemophilus influenza were tested. A modified enzyme-linked immunosorbent assay was used to measure the binding of the strains to native and deglycosylated lactoferrin. The effect on the viability of strains was measured by incubating strains in media containing lactoferrin as the sole carbon and nitrogen source. Siderophore production by strains was measured using an established assay. RESULTS: All the strains except the single strain of E. coli (Ecol8) were capable of binding to lactoferrin. The ocular isolate of H. influenzae showed strong affinity for lactoferrin. The P. aeruginosa strains and the strain of S. maltophilia showed significantly reduced (80%-100% reduction; P<0.05) binding to lactoferrin that had been enzymatically deglycosylated, whereas deglycosylation had no effect on the binding of other strains/species tested. Most strains were able to grow and produce siderophores in the presence of lactoferrin as the sole carbon and nitrogen source. CONCLUSIONS: The ability to bind to and grow on lactoferrin can be important for gram-negative pathogens that colonize the ocular environment, because this could allow bacteria to survive and propagate in the presence of tear fluid.

Biofilm formation on intrauterine devices in patients with recurrent vulvovaginal candidiasis.

A biofilm is a complex community of surface-associated cells enclosed in a polymer matrix. They attach to solid surfaces and their formation can be affected by growth conditions and co-infection with other pathogens. The presence of biofilm may protect the microorganisms from host defenses, as well as significantly reduce their susceptibility to antifungal agents. Pathogenic microbes can form biofilms on the inert surfaces of implanted devices such as catheters, prosthetic cardiac valves and intrauterine devices (IUDs). The present study was carried out to analyze the presence of biofilm on the surface of intrauterine devices in patients with recurrent vulvovaginal candidiasis, and to determine the susceptibility profile of the isolated yeasts to amphotericin B and fluconazole. Candida albicans was recovered from the IUDs and it was found to be susceptible to the antifungal agents when tested under planktonic growing conditions. These findings indicate the presence of the biofilm on the surface of the IUD as an important risk factor for recurrent vulvovaginal candidiasis.

Contamination levels and preliminary assessment of the technical feasibility of employing natural attenuation in 5 priority areas of Presidente Bernardes Refinery in Cubatão, São Paulo, Brazil.

Five priority areas of potential impact by contaminants (API) were investigated at the Presidente Bernardes Refinery in Cubatão, São Paulo, Brazil with the following aims: (i) to identify both organic and inorganic contaminants present in soil and groundwater; (ii) to define the environmental conditions relevant for microbial activity at the site and (iii) to evaluate the feasibility of employing natural attenuation for treatment of the hydrocarbon contamination. One area (API 1) was an uncontrolled landfill, where waste materials from the refinery were deposited between 1954 and 1986, and four areas (API 4, 5, 7 and 11) were located in the operational section of the refinery. Soil contamination by regulated BTEX compounds (benzene, toluene, total xylenes) was restricted to two samples from API 1. Nonregulated ethylbenzene was detected in one soil sample from API 4, one from API 5 and two from API 1. No soil contained regulated PAH above threshold levels. Several nonregulated PAHs were found in 6 soil samples from API 1, 3 soil samples from API 4 and 1 soil sample from API 5. Site soils contained very high aluminium concentrations, but metal contamination was restricted to one soil sample from API 1, which contained nickel above threshold limits. BTEX contamination of groundwater was due mostly to benzene. Of the 17 PAH molecules tested, only naphthalene and 2-methylnaphthalene occurred in groundwater. The sum of total BTEX and total PAH exceeded 200 microg/L in only a few monitoring wells in API 4, 5 and 11 and was always below 2.640 microg/L. Be, Cd, Cr, Cu, Hg, Ni, Se, Ag, Tl and Zn were not detected in groundwater, which was contaminated in a few locations by aluminium (mostly below 1 mg/L), lead (<0.066 mg/L) and arsenic (<0.056 mg/L). S, K, Ca, Mg and Fe were present in groundwater in excess of physiological requirements for microbial growth, but low concentrations of N and P could become growth limiting. However, BTEX were efficiently degraded in saturated and unsaturated zone microcosms and nutrient amendments did not stimulate biodegradation rates measurably. The inorganic carbon pool in groundwater was up to one order of magnitude larger than the organic carbon pool. Total inorganic carbon (TIC) in API groundwater exceeded TIC of clean groundwater by factors of 2 (API 4), 6 (API 5, 7 and 11) or 10 (API 1). Most of the inorganic carbon incorporated into groundwater beneath the refinery originated from biodegradation in the unsaturated soil, which contained a microbiota (10(6) cells/g on average) capable of growth with most of the pure (benzene, toluene, ethylbenzene and xylene) and mixed hydrocarbons tested (diesel oil, gasoline, naphtha, condensate, aromatic residue and fuel oil). A viscous hydrocarbon paste uncovered in API 1 was insoluble in water but dissolved in dichloromethane. Many organic components of this paste were biodegradable as evidenced by weight reduction of the hydrocarbon paste and by the growth of suspended and attached biomass in saturated zone microcosms, where the paste was the only carbon source. This study indicates that monitored natural attenuation may be a technically feasible and efficient means for plume control in API 1, 4 and 5, provided the plumes in API 4 and 5 are not expanding. This technique is not suitable for contaminant reduction in API 11.

The effect of protein-coated contact lenses on the adhesion and viability of gram negative bacteria.

PURPOSE: Gram negative bacterial adhesion to contact lenses can cause adverse responses. During contact lens wear, components of the tear film adsorb to the contact lens. This study aimed to investigate the effect of this conditioning film on the viability of bacteria. METHODS: Bacteria adhered to contact lenses which were either unworn, worn for daily-, extended- or overnight-wear or coated with lactoferrin or lysozyme. Numbers of viable and total cells were estimated. RESULTS: The number of viable attached cells was found to be significantly lower than the total number of cells on worn (50% for strain Paer1 on daily-wear lenses) or lactoferrin-coated lenses (56% for strain Paer1). Lysozyme-coated lenses no statistically significant effect on adhesion. DISCUSSION: The conditioning film gained through wear may not inhibit bacterial adhesion, but may act adversely upon those bacteria that succeed in attaching.