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Biofilms are involved in the recalcitrance of infections due to the presence of persister cells. Although the molecular basis of persistence is still largely unknown, toxin-antitoxin modules (TA) are thought to play a role in this process. In this study, we investigated whether TA modules contribute to persistence toward antibiotics in Burkholderia cenocepacia J2315. Sixteen pairs of genes were identified based on their apparent similarity to TA modules. Overexpression of the putative toxins had various effects on growth, persistence, and biofilm formation. Toxins, whose overexpression resulted in growth inhibition, often increased the number of surviving persisters; in contrast, overexpression of putative toxins showing no effects on growth had no positive influence on the number of surviving persisters. Furthermore, the expression of the TA modules was compared between treated and untreated sessile and planktonic wild-type cultures. For 10 toxin-encoding genes, the expression was higher in untreated sessile cells than in untreated planktonic cells. Nine toxin-encoding genes were upregulated after treatment with tobramycin, but none after treatment with ciprofloxacin. These results indicate that most, but not all TA modules contribute to persistence in B. cenocepacia J2315 and that this contribution depends on the mode of growth and the antibiotic used., (© 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Due to the resistance of Staphylococcus aureus to several antibiotics, treatment of S. aureus infections is often difficult. As an alternative to conventional antibiotics, the field of bacterial interference is investigated. Staphylococcus epidermidis produces a serine protease (Esp) which inhibits S. aureus biofilm formation and which degrades S. aureus biofilms. In this study, we investigated the protease production of 114 S. epidermidis isolates, obtained from biofilms on endotracheal tubes (ET). Most of the S. epidermidis isolates secreted a mixture of serine, cysteine and metalloproteases. We found a link between high protease production by S. epidermidis and the absence of S. aureus in ET biofilms obtained from the same patient. Treating S. aureus biofilms with the supernatant (SN) of the most active protease producing S. epidermidis isolates resulted in a significant biomass decrease compared to untreated controls, while the number of metabolically active cells was not affected. The effect on the biofilm biomass was mainly due to serine proteases. Staphylococcus aureus biofilms treated with the SN of protease producing S. epidermidis were thinner with almost no extracellular matrix. An increased survival of Caenorhabditis elegans, infected with S. aureus Mu50, was observed when the SN of protease positive S. epidermidis was added., (© 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

In this study, we investigated the biotherapeutic potential of previously isolated quorum quenching (QQ) bacteria. Some of them produce and secrete small compounds that inhibit quorum sensing (QS), others quench QS by enzymatic degradation of N-acylhomoserine lactones (AHLs). The supernatant of cultures of these isolates was tested for inhibitory properties against P. aeruginosa PAO1 biofilms. Most isolates had a moderate effect on biofilm formation, as shown by viability staining and/or staining of the biofilm biomass. A substantial part of the isolates reduced P. aeruginosa elastase production in a concentration-dependent manner. Using Caenorhabditis elegans as an in vivo model system for virulence testing, we found that some of the isolates were able to increase survival of P. aeruginosa PAO1 and Burkholderia cenocepacia LGM16656-infected nematodes when co-administered with the pathogen. Altogether, these data indicate that some QQ bacteria, or the active compounds they produce, could be useful to attenuate virulence of P. aeruginosa PAO1 and possibly also other Gram-negative pathogens that use AHLs to regulate the production of virulence factors., (© 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Infections related to fungal biofilms are difficult to treat due to the reduced susceptibility of sessile cells to most antifungal agents. Previous research has shown that 1-10% of sessile Candida cells survive treatment with high doses of miconazole (a fungicidal imidazole). The aim of this study was to identify genes involved in fungal biofilm formation and to unravel the mechanisms of resistance of these biofilms to miconazole. To this end, a screening of a Saccharomyces cerevisiae deletion mutant bank was carried out. Our results revealed that genes involved in peroxisomal transport and the biogenesis of the respiratory chain complex IV play an essential role in biofilm formation. On the other hand, genes involved in transcription and peroxisomal and mitochondrial organization seem to highly influence the susceptibility to miconazole of yeast biofilms. Additionally, our data confirm previous findings on genes involved in biofilm formation and in general stress responses. Our data suggest the involvement of peroxisomes in biofilm formation and miconazole resistance in fungal biofilms., (© 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Mechanically ventilated patients often develop ventilator-associated pneumonia (VAP). Soon after intubation, a mixed biofilm harboring microbial pathogens is formed on the endotracheal tube (ET). It is believed that this biofilm contributes to the development of VAP. Unfortunately, the causative agent is often not known at the time VAP is suspected, and early therapy often relies on the identification of surveillance cultures (SC). It is thus important to know whether these SC can predict the microbial flora in ET biofilms. In this study, we compare the presence of a number of antibiotic-resistant nosocomial bacteria (Enterobacter aerogenes, Escherichia coli, Micrococcus luteus, Pseudomonas aeruginosa, Staphylococcus aureus, and Staphylococcus epidermidis) and of Candida albicans in cultures from ET biofilms and SC (i.e. sputum samples, nose swabs, and throat swabs) of 20 mechanically ventilated patients. Our data indicate that there is a good correlation between the presence of (antibiotic-resistant) pathogens in ET biofilms and SC., (© 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Aims: Although several factors contribute to wound healing, bacterial infections and the presence of biofilm can significantly affect healing. Despite that this clearly indicates that therapies should address biofilm in wounds, only few wound care products have been evaluated for their antibiofilm effect. For this reason, we developed a rapid quantification approach to investigate the efficacy of wound care products on wounds infected with Staphylococcus spp., Methods and Results: An in vitro chronic wound infection model was used in which a fluorescent Staph. aureus strain was used to allow the rapid quantification of the bacterial burden after treatment. A good correlation was observed between the fluorescence signal and the bacterial counts. When evaluated in this model, several commonly used wound dressings and wound care products inhibited biofilm formation resulting in a decrease between one and seven log CFU per biofilm compared with biofilm formed in the absence of products. In contrast, most dressings only moderately affected mature biofilms., Conclusion: Our model allowed the rapid quantification of the bacterial burden after treatment. However, the efficacy of treatment varied between the different types of dressings and/or wound care products., Significance and Impact of the Study: Our model can be used to compare the efficacy of wound care products to inhibit biofilm formation and/or eradicate mature biofilms. In addition, the results indicate that treatment of infected wounds should be started as soon as possible and that novel products with more potent antibiofilm activity are needed., (© 2013 The Society for Applied Microbiology.)

Aims: The yeast Saccharomyces boulardii is used as a probiotic for the prevention and treatment of diarrhoea. In this study, the quality of 15 probiotic products containing S. boulardii was verified., Methods and Results: Using microsatellite typing, the identity of all Saccharomyces strains in the products was confirmed as S. boulardii. Additionally, solid-phase cytometry (SPC) and a plate method were used to enumerate S. boulardii cells. SPC was not only able to produce results more rapidly than plating (4h compared to 48h) but the cell counts obtained with SPC were significantly higher than the plate counts. Finally, we found that <1% of the S. boulardii cells survived 120min in gastric conditions and storage for 3months at 40°C with 75% relative humidity., Conclusions: We developed a SPC method for the quantification of viable S. boulardii cells in probiotics. Additionally, we demonstrated that gastric conditions and storage have a marked effect on the viability of the yeast cells., Significance and Impact of the Study:   To our knowledge, this is the first time SPC is used for the quality control of probiotics with S. boulardii. Additionally, we demonstrated the need for gastric protection and accurate storage., (© 2010 The Authors. Journal of Applied Microbiology © 2010 The Society for Applied Microbiology.)

Multiple genotyping studies have been carried out in order to clarify the epidemiology of fungal infections, more specifically to determine the sources, transmission routes, and colonization patterns of fungal isolates. In this review, the results obtained in genotyping investigations of Aspergillus isolates are summarized and discussed. Furthermore, we examine the epidemiologic studies of Candida albicans, Exophiala dermatitidis and Scedosporium apiospermum infections in patients with cystic fibrosis. Relative to Aspergillus fumigatus, colonization of the respiratory tract by multiple strains, and of deep organs by only a single strain were observed. On the other hand, the few studies which focused on other fungi isolated from patients with cystic fibrosis have suggested that colonization occurs primarily by a dominant genotype.

Objectives: Although azole antifungals are considered to be fungistatic, miconazole has fungicidal activity against planktonic Candida albicans cells, presumably associated with the induction of reactive oxygen species (ROS) production. Only few data are available concerning the effect of miconazole against sessile C. albicans cells. In the present study, the fungicidal activity of miconazole against in vitro-grown mature Candida biofilms, and its relationship with the induction of ROS and ROS-dependent apoptosis were examined., Methods: The effect of miconazole on mature biofilms formed by 10 C. albicans strains and 5 strains from other Candida species was evaluated by plate counting and measuring the level of ROS induction. MIC tests were performed in the absence and presence of ascorbic acid, a quencher of ROS. The apoptotic population in C. albicans cells was determined using annexin-Cy3., Results: Miconazole showed a significant fungicidal effect against all mature Candida biofilms tested and caused elevated ROS levels, both in planktonic and sessile cells. Addition of ascorbic acid drastically reduced these levels. While ROS quenching decreased the susceptibility to miconazole of planktonic cells of most Candida strains, no reduced fungicidal activity of miconazole against biofilms was observed. Miconazole did not cause a significant increase in apoptosis., Conclusions: ROS levels increased in all Candida biofilms upon addition of miconazole. However, ROS induction was not the only factor that underlies its fungicidal activity, as quenching of ROS did not lead to an enhanced survival of biofilm cells. ROS-induced apoptosis was not observed in C. albicans cells after miconazole treatment.

Objectives: The goal of the present study was to obtain a comprehensive overview of the bacteriostatic and bactericidal effects of six commonly used antibiotics on planktonic as well as on sessile Burkholderia cepacia complex cells., Methods: The bacteriostatic and bactericidal activities of ceftazidime, ciprofloxacin, meropenem, minocycline, tobramycin and trimethoprim/sulfamethoxazole were determined against 38 B. cepacia complex strains. MICs and minimal biofilm inhibitory concentrations (MBICs) were determined using a traditional broth microdilution method and a novel resazurin-based viability staining, respectively. The bactericidal effects of the investigated antibiotics (using antibiotic concentrations corresponding to 10 x MIC; except for tobramycin, for which a final concentration of 4 x MIC was tested) on stationary phase planktonic cultures and on 24-h-old biofilms were evaluated using conventional plate count methods., Results: Our results confirm the innate resistance of B. cepacia complex organisms to six first-line antibiotics used to treat infected cystic fibrosis patients. All antibiotics showed similar bacteriostatic activities against exponentially growing B. cepacia complex planktonic cells and freshly adhered sessile cells (4 h). In addition, most of the antibiotics showed similar bactericidal effects on stationary phase planktonic cultures and on young and older biofilms., Conclusions: Despite the general assumption that sessile cells show a decreased susceptibility to antibiotics, our data indicate similar bacteriostatic and bactericidal activity of six selected antibiotics against planktonic and sessile B. cepacia complex bacteria.

Aim: To compare the survival of four bacterial strains (Escherichia coli, Proteus mirabilis, Staphylococcus aureus, Pseudomonas aeruginosa) in pharmaceutical oils, including jojoba oil/tea tree oil, carbol oil, jojoba oil and sesame oil., Methods and Results: Oils were spiked with the test bacteria in a concentration of 10(4) CFU ml(-1). Bacteria were extracted from oils with phosphate-buffered saline containing 0.5% Tween 20. Aliquots of the pooled water layers were analysed by solid-phase cytometry and plate counting. Plate counts dropped to zero for all test strains exposed for 24 h to three of the four oils. In contrast, significant numbers of viable cells were still detected by SPC, except in the jojoba oil/tea tree oil mixture and partly in sesame oil., Conclusions: Exposure of bacteria for 24 h to the two oils containing an antimicrobial led to a loss of their culturability but not necessarily of their viability. The antibacterial activity of the jojoba oil/tea tree oil mixture supersedes that of carbol oil., Significance and Impact of the Study: These in vitro data suggest that the jojoba oil/tea tree oil mixture more than carbol oil inhibits bacterial proliferation when used for intermittent self-catherization.

Aims: To evaluate the susceptibility to microbial contamination that occurs during simulated handling of protective devices for the preparation of cytotoxic drug solutions., Methods and Results: Four devices, i.e. Chemoprotect spike, Clave connector, PhaSeal and Securmix were challenged with low and high inocula of micro-organisms. The cells, transferred to the connected vials during repeated manipulations of the devices were counted by means of solid-phase cytometry. Of the four devices, PhaSeal afforded the lowest transfer of micro-organisms. Secondly, the efficiency of procedures for the disinfection of an artificially contaminated rubber stopper was compared prior to connection of the vial to the PhaSeal device. Spraying or swabbing alone was inadequate, as opposed to a combination of spraying [0.5% or 2.0% (w/v) chlorhexidine in isopropanol] and swabbing [70% (v/v) isopropanol]., Conclusions: Although Phaseal afforded the lowest transfer of micro-organisms, adequate disinfection of the vial prior to connection remains required., Significance and Impact of the Study: Unlike aspects of operator protection, which are well documented, the microbiological safety of protective devices for the preparation of cytotoxic drugs has not been addressed in the literature. This study estimates the susceptibility to microbial contamination during handling of four commonly used devices.

Aims: To evaluate the use of the modified Robbins device (MRD) to test disinfection strategies against biofilms that form on oral medical devices and to test the biofilm removal efficacy of NitrAdine, a disinfectant for the maintenance of oral medical devices., Methods and Results: Biofilms were grown on discs using the MRD and biofilms formed in this system were used to evaluate the efficacy of NitrAdine and to determine the optimal disinfection conditions. Our data indicate that the use of the MRD allows for the rapid and reproducible formation of high-density biofilms. Determination of the efficacy of NitrAdine revealed high activity against biofilms tested (e.g. >3 log reduction for Candida albicans and Staphylococcus aureus) and allowed the determination of the optimal conditions for its use., Conclusion: The high reproducibility and flexibility of the MRD make it an excellent candidate for standardized testing of disinfectants aimed at reducing biofilms on oral medical devices. Using this system, we were able to demonstrate that NitrAdine exhibits high activity against biofilms formed by the micro-organisms tested., Significance and Impact of the Study: Our data suggest that our procedure is appropriate for standardized testing of disinfectants aimed at reducing biofilms on oral medical devices.

Objectives: The Burkholderia cepacia complex is a group of pathogens that can cause severe pulmonary infections in cystic fibrosis (CF) patients. The aim of the present study was to investigate the in vitro activity of gallium against planktonic and biofilm-grown B. cepacia complex isolates., Methods: Six B. cepacia complex isolates (belonging to three different species) as well as Pseudomonas aeruginosa PAO1 were included in the present study. MICs of Ga(NO(3))(3) for planktonic cells were determined using a broth microdilution method. Biofilms were formed in 96-well microtitre plates, and the fraction of surviving cells following Ga(NO(3))(3) treatment was determined using resazurin as a marker for cell viability. The antimicrobial effect of Ga(NO(3))(3) was assessed in the presence (50 microM) and absence of Fe(3+)., Results: When tested against planktonic cells, the MICs of Ga(NO(3))(3) in the absence of Fe(3+) were 64 mg/L for all B. cepacia complex strains investigated. However, the addition of 50 microM Fe(3+) in the presence of 64 mg/L Ga(NO(3))(3) resulted in increased growth for all B. cepacia complex strains investigated. In sessile cells, resistance to Ga(NO(3))(3) and the extent of the protective effect of 50 microM Fe(3+) against Ga(NO(3))(3) appear to be strain-dependent: the Burkholderia cenocepacia strains investigated are insensitive to Ga(NO(3))(3) in the presence of 50 microM Fe(3+), whereas the presence of Fe(3+) has no protective effect for both Burkholderia multivorans strains investigated., Conclusions: As maximal tolerable Ga(3+) levels in plasma are estimated to be approximately 200 microM and considering the high levels of Fe(3+) in the lungs of people with CF, our data suggest that the added value of a Ga(NO(3))(3) treatment of B. cepacia complex-infected patients may be limited.

Aims: The objectives of the study were to determine the spread and persistence of Campylobacter in a poultry processing plant and to provide a quantitative estimate of the survival of Campylobacter jejuni on the surface of a cutting board., Methods and Results: Several contact surfaces in a poultry processing plant were sampled before the start of processing, after 30 min and after 120 min. Next, the survival of four C. jejuni strains was studied on a beech and polypropylene cutting board during 120 min., Conclusions: A rapid introduction and spread of Campylobacter in a well cleaned processing plant as well as a significant survival in time on the example of a cutting board is shown., Significance and Impact of the Study: The need to prevent cross-contamination in the food processing and preparation area and the importance of an integrated approach throughout the whole food chain to control transmission of Campylobacter is highlighted.

Aims: To investigate the relationship between genomovar status and carbon source utilization, antibiotic susceptibility and growth ability on selective media of 142 clinical and environmental Burkholderia cepacia complex (Bcc) isolates belonging to all nine genomovars., Methods and Results: Carbon source utilization and growth on selective media were tested by agar plate multipoint inoculation. Antimicrobial minimum inhibitory concentration (MIC) values were determined by agar dilution. Of all carbon sources, l-arabinose was most frequently utilized, supporting growth of 90% of all isolates. Burkholderia cepacia genomovar VI failed to utilize azelaic acid, penicillin G, phtalate, salicin and tryptamine. Overall, B. vietnamiensis and B. anthina were most susceptible and B. cepacia genomovar VI most resistant to antimicrobial agents. Burkholderia cepacia selective agar (BCSA) and the Mast B. cepacia medium supported growth of Bcc isolates most efficiently., Conclusions: This study demonstrates phenotypic heterogeneity within the Bcc. Some trends can be observed at the genomovar level, but only B. cepacia genomovar VI could be differentiated unambiguously on the basis of its inability to grow on PCAT., Significance and Impact of the Study: This work provides an update on some differential phenotypic characteristics of all nine Bcc genomovars.

Aims: The aim of the study was to measure the survival of 19 Campylobacter jejuni strains of different origins, including two reference strains, four poultry-derived isolates, nine human isolates and four water isolates, in sterilized drinking water., Methods and Results: Pure cultures of 19 C. jejuni strains were inoculated in sterile drinking water and incubated at 4 degrees C for 64 days. Survival was determined by culturability on both selective (Karmali agar) and non-selective [Columbia blood agar (CBA)] media. Culturability was shown to be strain and origin-dependent. Campylobacter jejuni showed prolonged survival on a non-selective than on a selective medium., Conclusions: The origin of the strain is a determining factor for the survival of C. jejuni in drinking water at 4 degrees C. Poultry isolates showed a prolonged survival, which could be an indication that these strains could play an important role in the transmission of campylobacteriosis through water. In addition, culture conditions are an important factor for evaluating the survival of C. jejuni in drinking water at 4 degrees C. The non-selective agar (CBA) allowed growth of C. jejuni over a longer period of time than the selective agar (Karmali). Furthermore, an enrichment broth (Bolton) allowed the recovery of all 19 C. jejuni strains during the 64 days of incubation at 4 degrees C., Significance and Impact of the Study: This study highlighted differences in culturability depending on culture conditions and on strain origin.

A total of 154 Burkholderia cepacia complex strains, isolated from cystic fibrosis and non-cystic fibrosis patients and the environment, representing all nine genomovars and a putative tenth, were analysed by 16S rDNA-restriction fragment length polymorphism using the restriction enzymes AluI, CfoI and DdeI. Examining this diverse strain collection resulted in very diverse restriction patterns. Only B. cepacia genomovar VI could be identified unambiguously. The same restriction patterns were observed for B. cepacia genomovars I and III and approximately half of the Burkholderia ambifaria, B. anthina and B. pyrrocinia strains. Burkholderia vietnamiensis and B. ubonensis, a putative tenth B. cepacia complex genomovar, shared identical restriction profiles. The majority of Burkholderia multivorans and B. stabilis isolates generated a unique restriction pattern, but two strains of each showed divergent restriction profiles which were also observed in other genomovars.

Solid-phase cytometry (SPC) is a novel technique that allows rapid detection of bacteria at the single cell level, without the need for a growth phase. After filtration of the sample, the retained microorganisms are fluorescently labeled on the membrane filter and automatically counted by a laser scanning device. Each fluorescent spot can be visually inspected with an epifluorescence microscope connected to the ChemScan by a computer-driven moving stage. Depending on the fluorogenic labels used, information on the identity and the physiological status of the microorganisms can be obtained within a few hours. Although SPC was originally recommended for the determination of the total viable microbial count in water and other liquid samples, it may also be a promising technique for the detection and enumeration of bacteria in food samples, provided they can be isolated from the unfilterable matrix. The short detection time inherent in this approach is a considerable advantage over conventional plate counting, especially for slow-growing microorganisms. The basic principles of SPC are discussed as well as its potential for the detection of Mycobacterium paratuberculosis, a model example of a slow-growing bacterium in milk.

Tracheoesophageal voice prostheses in laryngectomized patients commonly deteriorate due to the presence of yeasts, particularly Candida species. We describe the first case of colonization of such a device by Cryptococcus neoformans in a patient with a history of glottic carcinoma. The isolate showed an identical genomic pattern with C. neoformans from pigeon excreta in the patient's environment.

Solid phase cytometry (SPC) has been investigated as a tool to assess the effect of antibiotics on the viability of Escherichia coli. After exposure of the cells to the antibiotic, they are retained on a polyester membrane filter and labelled using a fluorescein derivative as a substrate for intracellular esterases. The number of fluorescent bacteria is automatically counted in an Ar laser scanning device. In the presence of nutrients, all antibiotics tested in concentrations exceeding the MIC inhibited the multiplication of cells but not the labelling per se. However, when no nutrients were added, the cells did not multiply, and inhibition of the fluorescent staining was only observed for membrane permeabilizing antibiotics, even at sub-MIC concentrations. The selective detection by SPC of membrane-permeabilizing antibiotics corroborates the requirement of membrane integrity for viability labelling of bacteria. This selectivity has been exploited to develop a method for the detection of colistin residues in milk.

A 210-min-test for the enumeration of Escherichia coli in drinking water is described, based on solid phase cytometry (SPC) and a two-step enzymatic procedure for fluorescence labelling of single cells and small microcolonies. The test involves membrane filtration through a 25-mm black polyester filter, induction of beta-glucuronidase in the retained target cells, fluorescence labelling with fluorescein-di-beta-Dglucuronide as an enzyme substrate and laser scanning of the membrane filter. Scan results can be confirmed on-line by epifluorescence microscopy. Application to 149 naturally contaminated and uncontaminated well, tap, out-of-pump centre (distribution), surface and sewage-spiked water samples indicated > or =90% agreement and equivalence with plate count methods, including Chromocult Coliform agar and m FC agar. In 5.4% of all samples examined, SPC detected between 1 and 11 E. coli per 100 ml, while the two plate methods yielded negative results. Cases of a negative SPC result but a positive E. coli count on both reference media were not observed. This test would primarily be useful for 'emergency' monitoring of drinking water when rapid results are crucial.

A new assay for retinol in human serum, based on nonaqueous reversed-phase liquid chromatography, is presented. Sample preparation includes addition of the internal standard, retinyl propionate, deproteinization of 100 microL of serum with acetonitrile, and extraction with hexane. The standard curve is linear up to 2 mg/L. The assay is characterized by excellent sensitivity (detection limit, 15 micrograms/L) and good within-run and between-run precision (CVs of 2.6 and 2.7%, respectively), and results compare favorably with those by fluorimetry. We assayed 135 samples from hospitalized patients by both methods. Although the two sets of values correlated well (r = 0.955) the fluorimetric method occasionally suffers from interferences. In practice, fluorimetry proves valuable as a routine method, while liquid chromatography meets the criteria of a potential reference method.