Slightly acidic electrolyzed water disrupts biofilms and effectively disinfects Pseudomonas aeruginosa.

INTRODUCTION: Biofilm formation is an important issue in the healthcare industry, but conventional disinfectants are not effective for biofilms formed in the hospital environment and on medical instruments. In this study, aim at determine the effectiveness of slightly acidic electrolyzed water (SAEW) on biofilm removal and the disinfection of biofilm-forming Pseudomonas aeruginosa. METHODS: Mucoid and non-mucoid strains were used for biofilm formation. Biofilms were incubated with SAEW and the reduction in biofilm volume was determined based on the optical density. Furthermore, to investigate the mechanism underlying the effects of SAEW, a biofilm was produced with alginate and structural changes in response to incubation with SAEW were observed by fluorescence microscopy. The minimum bactericidal chlorine concentration of SAEW for P. aeruginosa cells was evaluated. RESULTS: The amounts of alginate and biofilm decreased by 99.9% and 56.8% immersed by 30 ppm of SAEW at 25 °C for 10 min. The effectiveness of SAEW increased as the temperature increased, and the biofilm volume was reduced by 85.4% at 45 °C. Furthermore, 30 ppm SAEW completely disinfected P. aeruginosa in the biofilm, even for immersion at 15 °C for 5 min. CONCLUSION: Our results suggest that SAEW, a low-cost and safe chlorine disinfectant, is a useful disinfectant for biofilm-forming bacteria.

Inhibitory effects of lactoferrin on biofilm formation in clinical isolates of Pseudomonas aeruginosa.

Lactoferrin, a multifunctional protein with antimicrobial activity, is a component of the innate immune system. It may possibly prevent clinical isolates of Pseudomonas aeruginosa from developing biofilm, but this hypothesis is yet to be widely accepted. We evaluated the in vitro effects of lactoferrin on biofilm formation by various clinical isolates of P. aeruginosa using a modified method of the microtiter plate biofilm assay. Lactoferrin significantly inhibited biofilm formation in these isolates. The effect was the most marked at 2 mg/ml, which suggested that an optimal concentration of lactoferrin might exist. Lactoferrin inhibited biofilm formation in eight of nine clinical isolates after 1 day of incubation; however, the inhibitory effects were maintained until 7 days of incubation in only two of those eight strains. Suppression of biofilm formation may be caused by a mechanism that is independent of the bactericidal effects of lactoferrin because the number of viable bacteria was not influenced by lactoferrin under the experimental conditions. Supplementation of lactoferrin to preformed biofilm demonstrated a reduction in biofilm, which suggests that lactoferrin may have a destructive effect on biofilm. Pretreatment with ferric chloride partially restored biofilm formation, suggesting an iron-chelating action may be involved in the inhibitory mechanism of lactoferrin. These results suggest that lactoferrin provides inhibitory effects on biofilm formation in many clinical isolates of P. aeruginosa and that it may also have destructive effects on preformed biofilm, but further research using multiple clinical strains should be undertaken to clarify if those effects are universal.

Measuring antimicrobial susceptibility of Pseudomonas aeruginosa using Poloxamer 407 gel.

Pseudomonas aeruginosa is a Gram-negative bacterium that causes various opportunistic infections. Chronic and intractable infections with P. aeruginosa are closely related to the high levels of resistance displayed by this organism to antimicrobial agents and its ability to form biofilms. Although the standard method for examining antimicrobial resistance involves susceptibility testing using Mueller-Hinton agar or broth, this method does not take into account the influence of biofilm formation on antimicrobial susceptibility. Poloxamer 407 is a hydrophilic, nonionic surfactant of the more general class of copolymers that can be used to culture bacteria with similar properties as cells in a biofilm environment. Therefore, the aim of this study was to compare the antimicrobial susceptibility of bacteria cultured in Poloxamer 407 gel to those grown on Mueller-Hinton agar using the Kirby-Bauer disk diffusion method with 24 strains of P. aeruginosa. Antimicrobial sensibility differed between the two mediums, with >60% of the strains displaying increased resistance to ß-lactams when cultured on Poloxamer 407 gel. In addition, scanning electron microscopy revealed that typical biofilm formation and extracellular polymeric substance production was only observed with bacteria grown on Poloxamer 407 gel. Therefore, antimicrobial susceptibility test using Poloxamer 407 gel may provide more accurate information and allow the selection of suitable antimicrobial agents for treating patients infected with biofilm-forming pathogens.

Corticosteroid eye drop instillation aggravates the development of Acanthamoeba keratitis in rabbit corneas inoculated with Acanthamoeba and bacteria.

The role of topical corticosteroids in management of Acanthamoeba keratitis (AK) remains controversial. Using a rabbit AK model, we investigated whether corticosteroid use is a risk factor of AK. Acanthamoeba (1 × 105/ml) was incubated with two densities of P. aeruginosa (PA; high-PA: 1 × 108/ml, low-PA: 3 × 105/ml) before corneal inoculation. Rabbit corneas were inoculated with Acanthamoeba alone or Acanthamoeba plus PA and administered levofloxacin and betamethasone sodium phosphate (BSP) eye drops for 5 or 7 days. Infected rabbit eyes were evaluated for clinical score and Acanthamoeba by histological examination. Acanthamoeba alone and BSP treatment did not produce keratitis. Corneas inoculated with Acanthamoeba plus low-PA treated immediately with levofloxacin and BSP remained clear with few infiltrates. Corneas inoculated with Acanthamoeba plus low-PA treated with levofloxacin immediately and BSP 12 h later developed severe keratitis. Corneas inoculated with Acanthamoeba plus high-PA treated immediately with levofloxacin and BSP also developed severe keratitis. Acanthamoebae were detected by PAS staining in corneas inoculated with Acanthamoeba plus high-PA treated with levofloxacin and BSP. Topical corticosteroids have the potential to aggravate AK when cornea is infected by Acanthamoeba with a critical number of bacteria or when corticosteroids are given after infection has established by Acanthamoeba with small number of bacteria.

Characteristics of Resistance Mechanisms and Molecular Epidemiology of Fluoroquinolone-Nonsusceptible Salmonella enterica Serovar Typhi and Paratyphi A Isolates from a Tertiary Hospital in Dhaka, Bangladesh.

The aim of this study was to characterize the fluoroquinolone (FQ) resistance mechanism of Salmonella enterica serovar Typhi and Paratyphi A in Bangladesh. Salmonella Typhi isolates were classified into sequence type (ST) 1, ST2, and ST2209 and Salmonella Paratyphi A isolates were classified into ST85 and ST129. The most common STs of the FQ-nonsusceptible strain were ST1 (44.4%) and ST129 (66.6%). Thirty-nine percent of Salmonella Typhi isolates were multidrug resistant, and these were all ST1, which is the type prevalent in the Indian subcontinent. Although plasmid-mediated quinolone resistance genes were not detected in any of the tested strains, single and double mutations were identified in the quinolone resistance-determining region (QRDR). The most common QRDR mutation was GyrA_Ser83Phe (66.7% for Salmonella Typhi and 100% for Salmonella Paratyphi A). Treatment with an efflux pump inhibitor resulted in susceptibility of the strains to levofloxacin. All isolates demonstrated 100% susceptibility to ceftriaxone, azithromycin, and carbapenem. Our results suggest that mutations in gyrase A and enhancement of efflux pump activity are responsible for the resistance to FQs; in particular, the AcrAB-TolC efflux pump may be an important resistance factor for levofloxacin. To control the spread of FQ-nonsusceptible Salmonella Typhi, intensive surveillance in endemic areas, including Bangladesh, and effective infection control are necessary.

Number of Bacteria and Time of Coincubation With Bacteria Required for the Development of Acanthamoeba Keratitis.

PURPOSE: We hypothesized that bacteria may be a factor contributing to the development of Acanthamoeba keratitis (AK). We investigated interactions between Acanthamoeba and Pseudomonas aeruginosa for the development of keratitis in rabbit corneas. METHODS: Acanthamoeba castellanii (ATCC50492) and P. aeruginosa (PAO-1) were used. Two densities of P. aeruginosa (high, 1 × 10/mL; low, 3 × 10/mL) and 2 durations of coincubation (long, 6 h; short, 2 h) of Acanthamoeba with 1 × 10/mL of P. aeruginosa were tested. Acanthamoeba alone or Acanthamoeba coincubated with P. aeruginosa was inoculated into rabbit corneas. After inoculation, levofloxacin (LVFX) eye drops were administered. The clinical score of the cornea was evaluated after inoculation. RESULTS: Acanthamoeba alone did not produce keratitis during a 5-day observation period. Rabbit corneas inoculated with Acanthamoeba coincubated with low-density P. aeruginosa followed by topical LVFX were clear with few infiltrates. Corneas inoculated with Acanthamoeba coincubated with high-density P. aeruginosa followed by LVFX treatment developed severe keratitis, and clinical scores were significantly higher compared with high-density P. aeruginosa alone followed by LVFX treatment (scores 7, 9.6, 8.5 vs. 3, 3.5, 3.25 on days 1-3, all P < 0.01). The long (6 h) coincubation time of Acanthamoeba with high-density P. aeruginosa resulted in more severe keratitis compared with short (2 h) coincubation (scores, 9.7, 12.7, 12.1, 9.8, 8.7 vs. 7, 9.6, 8.5, 6.9, 5.6 on days 1-5, all P < 0.01). CONCLUSIONS: These results suggest that the presence of bacteria is essential and a critical number of bacteria is required for the development of AK. The time of coexistence with bacteria may be an important determinant of the severity of AK.

Investigation of the Role of Bacteria in the Development of Acanthamoeba Keratitis.

PURPOSE: Recently, much interest has been shown in bacteria extracted from Acanthamoeba strains isolated from patients with Acanthamoeba keratitis (AK). We hypothesized that the bacteria in Acanthamoeba strains may be a contributing factor in the development of AK. To prove this hypothesis, we investigated the involvement of bacteria harbored by Acanthamoeba in causing progressive ocular infection in rabbit corneas. METHODS: One Acanthamoeba strain (T4 genotype) that harbored bacteria was isolated from a patient with AK. The Acanthamoeba strain pretreated or not pretreated with levofloxacin (LVFX) was inoculated into rabbit corneas. We also tested the effect of LVFX eye drops on keratitis induced by the Acanthamoeba strain. The infected rabbit eyes were evaluated for clinical scores, Acanthamoeba 18S rDNA and bacterial 16S rDNA numbers were analyzed by the real-time polymerase chain reaction, and the presence of Acanthamoeba was analyzed by histological examination. RESULTS: Inoculation of nonpretreated Acanthamoeba resulted in severe keratitis. In contrast, inoculation of LVFX-pretreated Acanthamoeba did not induce keratitis (mean clinical score, 17.3 vs. 2.3; P < 0.05). Rabbit corneas inoculated with nonpretreated Acanthamoeba followed by topical LVFX therapy developed severe keratitis. In corneas inoculated with nonpretreated Acanthamoeba followed by LVFX therapy, the number of Acanthamoeba 18S rDNA copies was significantly higher than in other groups (P < 0.05), whereas the bacterial 16S rDNA gene was undetectable. Acanthamoeba cysts were detected by Fungiflora Y staining only in corneas inoculated with nonpretreated Acanthamoeba followed by LVFX therapy. CONCLUSIONS: These results suggest that the presence of bacteria in Acanthamoeba may be required for the development of AK.

Analysis of bacterial flora in dohyo soil.

OBJECTIVES: Sumo wrestling is one of the most popular sports in Japan. Injuries are not uncommon as this is a vigorous contact sport. Sumo wrestlers have little in the way of protective clothing; their main garb is the mawashi, making them prone to exposure to any microorganisms in the dohyo. The bacterial flora of the dohyo has received little attention. If the constituent flora is identified, then appropriate treatment or prevention of any bacterial lesions or infections incurred by the wrestlers is possible. METHODS: The Vitek AMS system used in this study was developed by McDonnell Douglas Corporation. In this system, the physiological and biochemical properties of Gram-positive and negative bacilli, Gram-positive and-negative cocci, and fungi isolated from clinical materials and environments are examined using test cards specifically for each microorganism group, and the results are automatically read by a computer and encoded. Obtained codes are compared with a built-in database, and bacterial species of test strains are identified. RESULTS: In this study, using the automatic identification kit VITEK or ATB, we describe the aerobic bacterial flora found in the dohyo over the four seasons of the year. We also investigated the effect of salt on the bacterial flora as sumo wrestlers toss salt on the dohyo before each match. We show the relationship between salinity changes and variations in the flora observed upon the addition of salt. Without salt, at the beginning of a match, Gram-negative bacteria predominate. When salt is added, there is a transient decrease in the incidence of flora followed by an increase in the incidence Grampositive cocci. CONCLUSIONS: Sixteen bacterial genera were identified using the bacterial identification systems in dohyo soil samples during the year. The number of identified bacterial species was 32. Even in the presence of salt, there is a measurable amount of bacterial flora in dohyo soil; salt does not act as an antibacterial agent.

Electron microscopic studies on bactericidal effects of electrolyzed acidic water on bacteria derived from kendo protective equipment.

OBJECTIVES: Kendo protective equipment is used without washing for a long time.Staphylococcus saprophyticus, Micrococcus luteus, andBacillus sphaericus are frequently isolated from the mask ('men' in Japanese) of kendo protective equipment during one year. To investigate the bactericidal effects of electrolyzed acidic water on these three bacteria, we observed their cellular structures by electron microscopy after treatment with such water. METHODS: Each bacterium isolated from 'men' was treated with electrolyzed acidic water and then observed under scanning and transmission electron microscopes. RESULTS: WhenS. saprophyticus was treated with electrolyzed acidic water and its cellular structures were observed under a transmission electron microscope, ghost cytoplasm was observed, in which no ribosomal granules or fibrous DNA structures were present, and the cell wall inner layer was detached from the outer layer. Under a scanning electron microscope, the structure of the cell wall surface layer was wrinkled, and round pores were partially formed, indicating that the cytoplasmic structures were flushed out of the cells treated with electrolyzed acidic water through the pores formed in the cell wall. InM. luteus, the destruction of ribosomal granules and that of DNA fibers were observed to be similar to those ofS. saprophyticus. ForB. sphaericus, the effect of electrolyzed acidic water was investigated using vegetative cells. A dissociation between the cytoplasm and cell wall wrinkled the cell surface layer. CONCLUSION: On the basis of above findings, electrolyzed acidic water was found to destroy the cellular structures of the three bacterial species frequently isolated from kendo men within a short time. Electrolyzed acidic water may be useful for disinfecting of kendo equipment.

Homologous and heterologous reconstitution of Golgi to chloroplast transport and protein import into the complex chloroplasts of Euglena.

Euglena complex chloroplasts evolved through secondary endosymbiosis between a phagotrophic trypanosome host and eukaryotic algal endosymbiont. Cytoplasmically synthesized chloroplast proteins are transported in vesicles as integral membrane proteins from the ER to the Golgi apparatus to the Euglena chloroplast. Euglena chloroplast preprotein pre-sequences contain a functional N-terminal ER-targeting signal peptide and a domain having characteristics of a higher plant chloroplast targeting transit peptide, which contains a hydrophobic stop-transfer membrane anchor sequence that anchors the precursor in the vesicle membrane. Pulse-chase subcellular fractionation studies showed that (35)S-labeled precursor to the light harvesting chlorophyll a/b binding protein accumulated in the Golgi apparatus of Euglena incubated at 15 degrees C and transport to the chloroplast resumed after transfer to 26 degrees C. Transport of the (35)S-labeled precursor to the chlorophyll a/b binding protein from Euglena Golgi membranes to Euglena chloroplasts and import into chloroplasts was reconstituted using Golgi membranes isolated from 15 degrees C cells returned to 26 degrees C. Transport was dependent upon extra- and intrachloroplast ATP and GTP hydrolysis. Golgi to chloroplast transport was not inhibited by N-ethylmaleimide indicating that fusion of Golgi vesicles to the chloroplast envelope does not require N-ethylmaleimide-sensitive factor (NSF). This suggests that N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are not utilized in the targeting fusion reaction. The Euglena precursor to the chloroplast-localized small subunit of ribulose-1,5-bisphosphate carboxylase was not imported into isolated pea chloroplasts. A precursor with the N-terminal signal peptide deleted was imported, indicating that the Euglena pre-sequence has a transit peptide that functions in pea chloroplasts. A precursor to the small subunit of ribulose-1,5-bisphosphate carboxylase with the hydrophobic membrane anchor and the pre-sequence region C-terminal to the hydrophobic membrane anchor deleted was imported localizing the functional transit peptide to the Euglena pre-sequence region between the signal peptidase cleavage site and the hydrophobic membrane anchor. The Euglena precursor to the small subunit of ribulose-1,5-bisphosphate carboxylase and the deletion constructs were not post-translationally imported into isolated Euglena chloroplasts indicating that vesicular transport is the obligate import mechanism. Taken together, these studies suggest that protein import into complex Euglena chloroplasts evolved by developing a novel vesicle fusion targeting system to link the host secretory system to the transit peptide-dependent chloroplast protein import system of the endosymbiont.