Colony-forming Units mL-1 Research Articles

Silver Nanosphere SERS Probes for Sensitive Identification of Pathogens

The identification and timely detection of pathogenic bacteria is critical to ensuring safe food, health, and water. Although surface enhanced Raman scattering (SERS) methods have been used for pathogen characterization and single molecule sensing, the challenge of detecting pathogens in very low numbers using an optimal substrate that is sensitive and reproducible is still a challenge. In this report, we have developed and explored a novel SERS active substrate of 60−80 nm diameter through the assembly of Ag nanocrystals (AgNCs) into Ag nanospheres (AgNSs). A finite difference time domain (FDTD) analysis of the electromagnetic field produced by these structures and the enhancement factor calculations indicated that an enhancement of 108 was possible using the 633 or 785 nm excitation. The exact enhancement factors (EF) through the experimental results were calculated to be 2.47 × 107, which is close to that obtained through the FDTD analysis. Preliminary characterization of the SERS substrate was demonstrated using various labels from the fluorescent dye and nonfluorescent small molecules. More importantly, these novel SERS active substrates when used for pathogenic bacteria detection could detect cells as few as 10 colony forming units/mL (CFU/mL). Using canonical variate analysis (CVA) in conjunction with Raman spectra, differentiation of three key pathogens (E. coli O157, S. typhimurium, and S. aureus) including live and dead cells was also accomplished. With further optimization of the SERS substrate, single cell detection is possible.

Urinary Tract Infection in Male General Practice Patients: Uropathogens and Antibiotic Susceptibility

To evaluate uropathogens and their antibiotic susceptibility in male general practitioner (GP) patients presenting with an uncomplicated urinary tract infection (UTI). A population-based study was conducted among males, 18 years and older, general practice patients, who had symptoms indicative of an uncomplicated UTI. A UTI was defined as >/=10(3) colony-forming units/mL (CFU/mL). The etiology of the infection, antimicrobial susceptibility, and treatment strategies used by the GP were determined. Escherichia coli was most frequently isolated (48%), followed by other enterobacteriaceae (24%) and enterococci (9%). The etiology of infection was age-dependent; E. coli was more frequently isolated in younger patients and Pseudomonas aeruginosa in the elderly. The overall susceptibility rates were low for amoxicillin (63%) and trimethoprim (70%), and high for fluoroquinolones (91%) and amoxicillin-clavulanic acid (90%), which is similar to susceptibility rates in females with UTIs from the same population. Antibiotics were prescribed to 59% of the males with symptoms of UTI. Fluoroquinolones were given to 33% of the patients and trimethoprim-sulfamethoxazole to 24%. No difference in antibiotic prescription, nor in duration of therapy, was found between the different age groups. In the male presenting with complaints of an acute uncomplicated UTI at the GP, E. coli, followed by other Gram-negative bacteria were the most frequently isolated uropathogens. Susceptibility rates in uncomplicated male and female UTIs were similar, indicating that data from UTI susceptibility studies in females from the same geographic region can be useful in the choice of empirical therapy in males.

Phage-Displayed Peptide-Q Dot Nanocrystal Combo for High-Sensitivity Bacterial Detection in Plasma.

Abstract 3153Poster Board III-90 IntroductionDespite improved phlebotomy practices, refrigeration of red cells, freezing of plasma and improved materials for transfusion product collection and storage, bacterial contamination of transfusion products is still a longstanding problem. Current bacterial detection tests relevant to transfusion medicine, especially for stored platelets have limitations with regard to time, specificity and sensitivity. There is a need for new and improved cost-effective high-affinity detection probes to fill this gap. In this study, using plasma spiked with Bacillus cereus 4342 and B. anthracis-Sterne as an experimental system, we identified peptides from a bacteriophage-displayed random peptide library that selectively bind and detect the Bacillus strains. By labeling the peptides with Q dot-liquid nanocrystals, the detection sensitivity of the peptides was further enhanced. MethodsA commercially available bacteriophage-displayed random peptide library was screened using B. cereus 4342 as bait, using appropriate controls under stringent conditions. The screening and subsequent sequencing of the phage DNA identified two phages each containing a coding sequence for 12-amino acid peptide that are selectively capable of binding to the Bacillus. Based on the nucleic acid sequence, the two synthetic peptides with biotin tag were prepared for detection assays and to enhance the detection sensitivity further, the peptides were labeled with streptavidin-conjugated fluorescent quantum-dots (Q dots). Fluorescence was measured either by a plate reader or using a fluorescence microscope. ResultsThe two synthetic peptides selectively bound to the Bacillus strains in both dot blot and ELISA assays. The membrane-based dot blot assay demonstrated an assay sensitivity of 103 colony forming units/ml (CFU/ml), whereas ELISA demonstrated a sensitivity of 102 CFU/ml detection limit. Fluorometry analysis of spiked plasma samples revealed that the two peptides were able to bind to B. cereus 4342 and B. anthracis Sterne and detect these bacteria in plasma at 102 CFU/ml concentrations. The peptide-Qdot combo even detected a single bacterium in fluorescence microscopy. ConclusionOverall, the results reported here validate the usefulness of affinity-selected recombinant filamentous phage-derived peptides in combination with Qdot-liquid nanocrystals as high sensitivity detection probes for bacteria in various platforms and settings relevant to the blood safety and transfusion medicine.The findings and conclusions in this abstract have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy. DisclosuresNo relevant conflicts of interest to declare.

In vitro and In vivo Anti-Microbial Effects of Nigella sativa Linn. Seed Extracts Against Clinical Isolates from Skin Wound Infections

Problem statement: The developing microbial resistance to the existing anti-microbial agents has become a real challenge and a serious problem facing patients suffering from skin infections. Seeds of Nigella sativa have been used for a long time in folk medicine for the treatment of such infections. Production of new potent agents is urgently needed, especially for hospitals and health centers. Therefore, the anti-microbial effect of aqueous, diethyl ether, chloroform and petroleum ether extracts of the seeds against four standard microbial strains and seven clinical isolates from patients with skin wound infections were investigated. Approach: The in vitro anti-microbial effect of the extracts at a concentration of 20% on standard strains and clinical isolates was assessed and compared with standard drugs, chloramphenicol and amphotericin B using agar well diffusion assay. The in vivo anti-bacterial effect of petroleum ether extract was studied in male BALB/c mice infected subcutaneously with S. aureus (ATCC 25923) or a clinical isolate (0.1 mL from 109 colony forming units mL-1 suspension) and immediately treated at the infected site by subcutaneous injection of 0.1 mL of pure extract (fixed oil) or chloramphenicol or normal saline. Counts of viable bacteria present in the skin area corresponding to the infected site were determined, after 24 and 48 h of infection and treatment. Results: The aqueous extract did not show any inhibitory effect against all the tested microorganisms. The diethyl ether and chloroform extracts indicated significant inhibitory effect only against Gram-positive bacteria. However, petroleum ether extract was proved to be the most powerful one against these bacteria and also against other clinical isolates like one Gram-negative bacterium (Klebsiella pneumonia) and the yeast (Candida albicans). Moreover, the extract revealed a superior effect over the standard drug, chloramphenicol, on the clearance of subcutaneous staphylococcal infection in mice when injected at the site of infection. Counts of viable bacteria were decreased at highly significant level in mice infected with S. aureus (ATCC 25923) or a clinical isolate. Conclusion/Recommendations: The results of this study revealed clear potentiality of N. sativa fixed oil as a source for anti-microbial drugs and support its use in folk medicine for the treatment of microbial skin infections.

Effect of calcium chloride on the biocontrol efficacy of two antagonistic yeasts against Penicillium expansum on apple fruit

Two antagonistic yeasts, Candida membranaefaciens and Pichia guilliermondii, were evaluated for the control of the blue mold of apple caused by Penicillium expansum. Dual culture, cell-free metabolite and volatile tests were used for in vitro assay. Yeast strains of two genera inhibited growth of P. expansum; inhibition varied from 30.27% to 44.19% in dual culture, from 79.40% to 90.57% in the volatile metabolite test, and from 72.99% to 88.77% in the cell-free metabolite test. Calcium chloride (2% w/v) significantly inhibited the growth of the pathogen P. expansum, but did not affect the colony-forming units (CFU) of the yeasts C. membranaefaciens and P. guilliermondii in potato dextrose broth. The concentration of yeast suspension influenced spore germination and germ tube growth of P. expansum in vitro, as well as disease incidence and lesion development in fruits. There were significant negative relationships between the suspension concentrations of the yeasts and the growth as well as infectivity of the pathogen. The addition of calcium resulted in lower spore germination rates and slower growth of germ tubes in vitro, as well as in lower disease incidences and smaller lesion diameters compared with treatments with yeast antagonists alone. When yeast cell suspensions reached a concentration of 107 CFU ml-1, growth of the pathogen was completely limited in vitro, and no infection was found in apple fruits treated with or without calcium.

Results of ofloxacin therapy in andrologic patients suffering from therapy-requiring asymptomatic infections.

In 1990, microbiological ejaculate analyses were carried out on a routine basis on 125 andrological patients, in addition to the determination of the concentration, motility and morphology of the spermatozoa. Fourteen patients (11.2%) with therapy-requiring asymptomatic infections (TAI) > 10(4) CFU (colony-forming units) ml-1 were effectively treated with Ofloxacin (Tarivid) at 2 x 200 mg d-1 for a period of 20 d. Concentration, motility and morphology of the spermatozoa were determined before the Ofloxacin treatment and controlled 1, 3, and 6 months later and correlated to the values obtained before the treatment. Over the entire period of observation, the morphology did not change significantly, whilst initially 1 month after the treatment the concentration and the motility decreased significantly (P < 0.05). Three months later they again reached the starting values. After 6 months, a significant improvement occurred with regard to the motility, as compared to the starting values (P < 0.01), whilst the concentration remained at the level of the starting values. By this, Ofloxacin has been proved to be an effective medication for the treatment of TAI. In the end, it still remains an open question whether the improvement in sperm motility is primarily related to the Ofloxacin therapy.

Glucocorticoids Enhance Toll-Like Receptor 2 Expression in Human Keratinocytes Stimulated with Propionibacterium acnes or Proinflammatory Cytokines

Toll-like receptors (TLRs) on keratinocytes are important cell surface receptors involved in the innate and acquired immune response to invading microorganisms. In acne vulgaris, TLR2 activation by Propionibacterium acnes (P. acnes) may induce skin inflammation via induction of various proinflammatory molecules that stimulate the invasion of inflammatory cells. Although corticosteroids themselves exert immunosuppressive or anti-inflammatory effects, it is well known clinically that systemic or topical glucocorticoid treatment provokes an acneiform reaction. Nevertheless, the effect of steroids on TLR2 expression in human keratinocytes remains unknown. Here, we found that the addition of glucocorticoids, such as dexamethasone and cortisol, to cultured human keratinocytes increased their TLR2 gene expression. Moreover, these glucocorticoids markedly enhanced TLR2 gene expression, which was further stimulated by P. acnes, tumor necrosis factor-alpha, and IL-1alpha. Gene expression of mitogen-activated protein kinase (MAPK) phosphatase-1 was also increased by the addition of dexamethasone. By using several inhibitors and activators, we found that TLR2 gene induction by glucocorticoids was mediated by the suppression of p38 MAPK activity following induction of MAPK phosphatase-1. These findings strongly suggest that steroid-induced TLR2 together with P. acnes existing as normal resident flora plays an important role in the exacerbation of acne vulgaris as well as in possible induction of corticosteroid-induced acne or in that of rosacea-like dermatitis.

Sample preparation module for bacterial lysis and isolation of DNA from human urine

Silica impregnated polymer monolithic columns may provide a simple method for lysing and extracting DNA from bacteria inside of microfluidic chips. Here we use Escherichia coli as a test organism for a point of care thermoplastic microfluidic module designed to take in a urine sample, mix it with lysis buffer, and perform a hybrid chemical/mechanical lysis and solid phase extraction of nucleic acids from the sample. To demonstrate proof-of-concept, we doped human hematuric urine samples with E. coli at concentrations ranging from 10(1)-10(5) colony-forming units/mL (CFU/mL) to simulate patient samples. We then performed on-chip lysis and DNA extraction. The bacterial DNA was amplified using real-time PCR demonstrating lysis and isolation down to 10(1) CFU/mL. Results were comparable to a commercial kit at higher concentrations and performed better at recovering DNA at lower concentrations.

Contagem, isolamento e caracterização de bactérias psicrotróficas contaminantes de leite cru refrigerado

Com os objetivos de quantificar, isolar e caracterizar bactérias psicrotróficas contaminantes de leite cru refrigerado, produzido na região da Zona da Mata de Minas Gerais e Sudeste do Rio de Janeiro, foram analisadas amostras de leite coletadas de 20 tanques coletivos e 23 tanques individuais. As contagens de bactérias psicrotróficas nas amostras de leite para os dois tipos de tanques de refrigeração variaram entre 10² e 10(7) Unidades Formadoras de Colônias (UFC) ml-1, porém, um maior número de tanques coletivos apresentou contagens acima de 1 x 10(5) UFC ml-1. Foi verificada a predominância de bactérias psicrotróficas gram-negativas (81,2%), que foram identificadas pelos sistemas API 20E e API 20NE nos gêneros: Aeromonas, Alcaligenes, Acinetobacter, Burkholderia,Chryseomonas, Enterobacter, Ewingella, Klebsiella, Hafnia, Methylobacterium, Moraxella, Pantoea, Pseudomonas, Serratia, Sphingomonas e Yersinia. As bactérias gram-positivas (18,8%) foram identificadas com API 50 CH, API Coryne e API Staph, nos gêneros: Bacillus, Brevibacterium, Cellum/Microbacterium, Kurthia e Staphylococcus. Os sistemas API utilizados não identificaram todos os isolados bacterianos. Pseudomonas foi o gênero mais isolado e P. fluorescens foi a espécie predominante. A maioria dos isolados bacterianos apresentou atividade proteolítica e/ou lipolítica a temperaturas de refrigeração de 4°C, 7°C e 10°C, evidenciando seu alto potencial de deterioração do leite e dos produtos lácteos. Os resultados ressaltam que maior atenção deve ser dada aos procedimentos que impeçam a contaminação do leite por esses microrganismos.

Urinary Tract Infection-A Survey of Local Population

Urinary Tract Infection, commonly known as UTI, affects as many as 50% women at least once during their lifetime. All individuals are susceptible to Urinary Tract Infection (UTI); however the prevalence of infection differs with age, sex and certain predisposing factors. With the above background in mind we conducted a survey of the local population (women of 3 age groups) to compare the urinary tract microbial community of control individuals with the UTI positive patients. Attempts were made to identify pathogens through Serum Bactericidal Antibody Response. 200 urine samples collected from control as well as UTI patients were randomly inoculated in parallel on four varieties of chromogenic agar plates and the Colony Forming Units (CFU) per milliliters (ml) of each microbe was determined. The serum bactericidal antibody assay was performed to demonstrate the presence of serum antibodies with bactericidal activity against the bacterium found in urine. However, with further experimental analysis, this bactericidal activity was found to be non specific and a similar percentage of bacteriolysis was observed incase of the control population also. 104-105 CFU mL-1 was the demarcating value between normal and pathological samples in asymptomatic cases. A significant variation was also noted in the microbial profile of various age groups. E. coli is the most prevalent pathogen in the post menopausal group. 15 different bacterial isolates were obtained of which the 16S rDNA sequence of the 6 novel ones are available in GenBank. The control and patient population showed a clear cut variation in the percentage of urinary tract microbes.

Concordance of antibiotic prophylaxis, direct Gram staining and protected brush specimen culture results for postoperative patients with suspected pneumonia

Antibiotic therapy alters the diagnostic value of protected brush specimens. With protected brush specimens alone, diagnosing pneumonia requires 24 or 48 h. Addition of direct Gram staining shortens this delay. Antibiotic prophylaxis, recommended after major surgery, may influence the contribution of Gram staining to diagnosing postoperative pneumonia. During a 1-yr period, we retrospectively studied all patients on mechanical ventilation suspected of having postoperative pneumonia who had undergone fibreoptic bronchoscopy with protected brush specimens. Postoperative pneumonia was diagnosed when quantitative protected brush specimens culture results yielded 103 colony-forming units mL-1. Fifty patients were clinically suspected of having postoperative pneumonia after cardiac (n=42), vascular (n=5) or thoracic (n=3) surgery. Eleven (22%) samples were obtained during antibiotic prophylaxis. Twenty-two (44%) episodes were microbiologically proven. Gram-stain sensitivity was 95.5%, with 82.1% specificity, 80.7% positive-predictive value and 95.8% negative-predictive value. Concordance between direct Gram-stain-identified pathogens and Gram stain of cultured pathogens was significantly less frequent during antibiotic prophylaxis (63.6%) than afterwards (94.9%) (P<0.05). Antibiotic prophylaxis diminished the diagnostic value of Gram staining of protected brush specimens. When protected brush specimens was performed during antibiotic prophylaxis, Gram staining accurately enabled early exclusion of postoperative pneumonia because of its excellent negative-predictive value. After antibiotic prophylaxis, Gram staining permitted early diagnosis of postoperative pneumonia identification of the responsible pathogen.

Graft Infectivity of Rifampin and Silver-Bonded Polyester Grafts to MRSA Contamination

The purpose of this study was to evaluate the ability of vascular polyester grafts with antibacterial properties to resist colonization following surface contamination by methicillin-resistant Staphylococcus aureus (MRSA) in an experimental canine model or aortic graft infection. Twenty-four pathogen-free dogs underwent replacement of the infrarenal aorta with either a rifampin-soaked (30 mg/mL) or silver-impregnated (Ag-acetate) woven polyester graft. Following implantation, the external graft surface was inoculated with 2 mL of 10(7) colony-forming units/mL (CFU) of MRSA. Preoperative antibiotic prophylaxis consisted of a single intravenous dose of 500 mg of sodium cefazolin. Four grafts of each type were explanted at 3, 7, and 14 days after implantation. Quantitative cultures (CFU/specimen) of perigraft fluid (1 mL), graft material (1 cm segment), and adjacent aorta (1 cm segment) were performed. Differences between grafts are expressed as % mean log reduction in recoverable CFU compared to the inoculation solution concentration of 10(7) CFUs. At 3 days, explanted rifampin-soaked grafts exhibited no MRSA growth (4 of 4 grafts) and a > or =97% mean log reduction of MRSA CFUs from the adjacent aorta and perigraft fluid (PGF). At 3 days, all silver-bonded grafts exhibited signs of infection and a mean log CFU reduction of MRSA ranging from 68% (absolute range 10(1)-10(3) recoverable CFU) for the graft, 79% (absolute range 10(1)-10(3) recoverable CFU) for the aorta, and 86% (absolute range 10(1)-10(4) recoverable CFU) for PGF. The 7-day rifampin group had an average log reduction in MRSA CFU of 72% (graft), 58% (PGF), 75% (aorta). Quantitative cultures of 14-day rifampin grafted demonstrated continued bacterial growth suppression with mean MRSA CFU log reductions of 82%, graft; 72%, PGF; 89%, aorta. Silver-bonded grafts demonstrated <50% mean CFU reduction in MRSA growth at 7 days (absolute range 10(5)-10(7) recoverable CFU) and 14 days (absolute range 10(3)-10(7) recoverable CFU). No animal died from sepsis or anastomotic hemorrhage. Neither rifampin- nor silver-bonded grafts demonstrated prolonged resistance to surface MRSA contamination. Rifampin-soaked polyester grafts exhibited a marked but transient resistance MRSA colonization likely the result of high antibiotic concentration in the perigraft tissue. While both types of grafts failed to eradicate the MRSA infection future research with silver-bonded grafts that have an additional antibiotic attached may have a place in the treatment of MRSA infection.

The induction of differentially expressed proteins of Xylella fastidiosa with citrus extract

An in vitro system was developed to induce and identify Xylella fastidiosa proteins that were differentially expressed in the presence of callus-derived extracts from its host, the citrus cultivar Pera. To optimize the induction, we first developed a single culture medium for the growth of both, host and bacteria. This medium, CPXPm7, which mimics the citrus xylem sap, showed that X. fastidiosa at 72 h post-incubation had 108 colony forming units mL-1, while Pera cells had the highest fresh weight content (0.79 g). After testing various methods of co-cultivation of the bacteria and host callus grown in this single medium, the best induction procedure was to grow X. fastidiosa in a solid medium amended with an extract of Pera callus grown in CPXPm7. Analysis, by two-dimensional electrophoresis, of the X. fastidiosa proteins (120 µg of total proteins) grown in the presence of Pera callus extract revealed 414 differentially expressed protein spots when compared to the protein profile obtained in the absence of the extract. The system developed in this study improves the induction and analysis of differentially expressed proteins of X. fastidiosa, which may be involved in pathogenicity.

057 Epifluorescent and light microscopic visualization of bacterial microcolonies in acute wound infections – Are these biofilms?

A biofilm is a formation of surface‐associated microbial cells that are enclosed in a self produced extracellular polymeric substance matrix. This definition is acceptable for in‐vitro research but a clear definition for biofilm‐associated diseases has yet to be elucidated. A structural/morphological definition is currently used to define biofilms; additionally, physiological or molecular criteria will allow us to define biofilms in disease infection accurately. Our objective over the past several years has been to characterize and observe bacterial biofilms in wound infections using different wound models. To broaden our current understanding of biofilm morphology in wounds for this study we used epifluorescent and light microscopy to visualize wound pathogenic pseudomonas aeruginosa bacteria in a porcine partial thickness infection model.Three experimental animals were used for this study. After animal preparation, partial thickness wounds were created on the backs of 3 animals. Wounds were then inoculated with 106 colony forming units/ml (CFU/ml) of Pseudomonas aeruginosa and covered for 48 hours to allow bacteria to colonize and infect the wound. Biopsies were obtained from normal skin, wounds before inoculation, wounds at 48 hours and wounds 48 hours after inoculation. Biopsies were processed and stained with Hematoxylin and Eosin for light microscopy and Calcofluor White and Ethidium Bromide for epifluorescence microscopy.Images obtained using epifluorescent and light microscopy demonstrate that bacteria form aggregates of microcolonies. These structures are representative of bacterial biofilms and support the hypothesis that bacteria live as biofilms in wound infection. Although currently there is no established and accepted definition for biofilm associated diseases, we anticipate that more studies looking into physiological changes of these structures will clarify our current understanding of wound infection and treatment.

In vitro antimicrobial effect of chlorhexidine‐impregnated gutta percha points on Enterococcus faecalis

To evaluate the in vitro antimicrobial effect of chlorhexidine-impregnated gutta percha points, Roeko activ point (Roeko, Langenau, Germany) on Enterococcus faecalis. Human maxillary premolar roots were prepared with.04 rotary ProFile instruments to a master apical file size 40, autoclave-sterilized and then infected with E. faecalis (ATCC 29212) for 3 weeks. Baseline controls were carried out verifying negligible effects of plain gutta percha cones on E. faecalis. Subsequent to intracanal placement of calcium hydroxide, 'activ points' or saline (positive control) and the 2-week incubation in 54 root specimens, dentine sampling at depths of 100 and 250 micro m was carried out using.04 rotary ProFile instruments at sizes 60 and 90 to assess the quantity of bacteria present. Inactivating agents were used prior to sampling and the colony-forming units (CFU) of E. faecalis were then plate-counted after culturing. Statistical analysis was completed using the paired t-test. In comparison to the positive control, treatment with calcium hydroxide (P = 0.000 and 0.000) or activ points (P = 0.000 and 0.002) produced significantly lower colony counts of E. faecalis at dentine depths of 100 and 250 micro m, respectively. Calcium hydroxide (2.10 x 102 CFU mL-1) was significantly more effective than activ points (1.58 x 103 CFU mL-1) at 100 micro m (P = 0.013), but not at 250 micro m (P = 0.353). Neither of these two medications was able to eliminate E. faecalis completely. Chlorhexidine-impregnated activ points did not possess an in vitro inhibitory activity strong enough to eliminate E. faecalis completely from infected dentinal tubules.

Persistence of adhesive properties in Vibrio cholerae after long-term exposure to sea water.

The effect of exposure to artificial sea water (ASW) on the ability of classical Vibrio cholerae O1 cells to interact with chitin-containing substrates and human intestinal cells was studied. Incubation of vibrios in ASW at 5 degrees C and 18 degrees C resulted in two kinds of cell responses: the viable but non-culturable (VBNC) state (i.e. <0.1 colony forming unit ml-1) at 5 degrees C, and starvation (i.e. maintenance of culturability of the population) at 18 degrees C. The latter remained rod shaped and, after 40 days' incubation, presented a 47-58% reduction in the number of cells attached to chitin, a 48-53% reduction in the number of bacteria adhering to copepods, and a 48-54% reduction in the number of bacteria adhering to human cultured intestinal cells, compared to control cells not suspended in ASW. Bacteria suspended in ASW at 5 degrees C became coccoid and, after 40 days, showed 34-42% fewer cells attached to chitin, 52-55% fewer adhering to copep-ods, and 45-48% fewer cells adhering to intestinal cell monolayers, compared to controls. Sarkosyl-insoluble membrane proteins that bind chitin particles were isolated and analysed by SDS-PAGE. After 40 days incubation in ASW at both 5 degrees C and 18 degrees C vibrios expressed chitin-binding ligands similar to bacteria harvested in the stationary growth phase. It is concluded that as vibrios do not lose adhesive properties after long-term exposure to ASW, it is important to include methods for VBNC bacteria when testing environmental and clinical samples for purposes of public health safety.

Early modifications of Brassica napus root system architecture induced by a plant growth-promoting Phyllobacterium strain.

• Plant growth-promoting bacteria (PGPB) have been reported to stimulate root morphogenesis. To improve our knowledge of the PGPB effect, the early modifications of Brassica napus root system architecture induced by the PGPB Phyllobacterium sp. (29-15) were analysed. • Plants were grown in Petri dishes on a vertical medium supplemented with variable doses of Phyllobacterium sp. in gnotobiotic conditions. Root system elementary variables were measured in a nondestructive manner and the distribution of the bacteria throughout the primary root was quantified. • Phyllobacterium sp. in doses from 3×107 to 3×108 colony-forming units ml-1 significantly promoted B. napus total root length up to 50% by increasing both lateral root density throughout the primary root and growth rate of mature lateral roots. The primary root was progressively colonized by the bacteria from the tip to the base and the number of colonizing cells was positively correlated with the inoculum density. • Relationships between inoculum density, root colonization and root system architecture emphasized the relevance of this approach to specify PGPB effects on plants.

Effect of freezing goat milk samples on recovery of intramammary bacterial pathogens

With the aim of evaluating the effect of freezing goat milk samples on recovery of intramammary pathogens, 1200 milk samples from udder halves with subclinical intramammary infection were studied. Samples (20 ml) were frozen at −20 and at −80 °C. Thawing was carried out at room temperature at 7, 14, 21, 28, 58, 118, 178, 236 and 730 days after collection and bacteriological analyses were carried out to determine the number of colony forming units/ml (CFU/ml). Mixed model statistical analysis showed that bacterial group, temperature of storage, interaction of bacterial group and temperature of storage and the interaction of bacterial group, time and temperature of storage were statistically significant effects. For coagulase negative staphylococci (CNS), least squares means of log CFU/ml recovered at −20 and −80 °C were not different. Nevertheless, for Gram negative bacilli (GNB) a significant decrease was detected in samples frozen at −20 vs. −80 °C. At both temperatures and at different times of storage, significant increases were detected between log CFU/ml of CNS and values on day zero. At −20 °C, a significant decrease in GNB recovery was detected between freezing days zero and 730. This difference was not detected when goat milk samples infected by GNB were frozen at −80 °C. The results show that frozen milk samples can be useful in goat subclinical mastitis control programs.

The effect of disinfectant agents in eliminating the contamination of dental unit water.

High concentrations of water-borne organisms cause multiple public health problems. Contamination of water exiting the dental unit water lines could be inhibited with the use of some disinfectants. The purpose of this investigation was to establish the effect of two disinfectants and to test their capacity to eliminate colony forming units (CFU) per mL. Vacuum lines of four chairs were treated for a total of 2 weeks. Two disinfectants (Bio 2000 and Alpron) were used as per manufacturer's instructions. Water samples for hetereothrophic counts from each unit's air/water syringe line were collected before treating the first patient of the day. Baseline, daily, first and second week samples of 10 mL were plated on blood agar plates and eosin ethylene blue agar. For meosifilic bacterial counts, Mueller Hinton agar plates with 1 mL direct and 1/10 were used in sterile serum and CFU were counted. The suspected colonies were further evaluated using API 20E and API 20NE. No Gram(-) opportunistic pathogens were found during the entire observation. Baseline contamination level (>102 CFU mL-1) without use of disinfectants was significantly higher (P < 0.0001) than at both first and second weeks when disinfectants were used. No colony was formed when Bio 2000 was used after both first and second weeks, whereas small number of CFU mL-1 was found at the end of the first week when Alpron (<10) was used. In conclusion, when used daily, both disinfectants prevent the development of bacterial contamination after first and second weeks with no significant differences (P=0.35).

Pilot tests of Candida sake (CPA-1) applications to control postharvest blue mold on apple fruit

Abstract The yeast Candida sake (strain CPA-1) was tested as a biocontrol agent of postharvest diseases, primarily blue mold, caused by Penicillium expansum on apple fruits. In a semi-commercial trial with non-injured fruits stored in air at 1°C, a concentration of 1.6×10 6 colony forming units/ml (CFU/ml) of C. sake reduced the incidence of decayed fruits by more than 70%. Over a period of three seasons, in commercial trials the efficacy of CPA-1 applied in a drench was evaluated and compared with the fungicides imazalil and thiabendazole+folpet. The application of C. sake at 10 7 CFU/ml resulted in a reduction in the incidence of decay to a level equal to that with imazalil (375 ppm) and higher than that with thiabendazole (425 ppm)+folpet (1000 ppm). Population of the biocontrol agent increased on the surface of wounded fruits 5-fold and decreased on the surface of non-wounded fruits more than 10-fold in the first 60 days in storage at 1°C. The viability of C. sake was not reduced after 30 min immersion in benomyl, sulfur, flusilazol, ziram, thiabendazole or diphenylamine. Conversely, captan, imazalil, and ethoxyquin decreased C. sake viability and would not be compatible with it used at commercial rates. The yeast was able to grow in culture at temperatures from 1 to 34°C.