Gerard T.A. Fleming

Linezolid Compared with Eperezolid, Vancomycin, and Gentamicin in an In Vitro Model of Antimicrobial Lock Therapy for Staphylococcus epidermidis Central Venous Catheter-Related Biofilm Infections

ABSTRACT Central venous catheter (CVC)-related infection (CVC-RI) is a common complication of CVC use. The most common etiological agents of CVC-RI are gram-positive organisms, in particular, staphylococci. An in vitro model for the formation of biofilms by Staphylococcus epidermidis ATCC 35984 on polyurethane coupons in a modified Robbins device was established. Biofilm formation was confirmed by electron microscopy and was quantified by determination of viable counts. Mueller-Hinton broth was replaced with sterile physiological saline (control) or a solution of vancomycin (10 mg/ml), gentamicin (10 mg/ml), linezolid (2 mg/ml), or eperezolid (4 mg/ml). Viable counts were performed with the coupons after exposure to antimicrobials for periods of 24, 72, 168, and 240 h. The mean viable count per coupon following establishment of the biofilm was 4.6 × 108 CFU/coupon, and that after 14 days of exposure to physiological saline was 2.5 × 107 CFU/coupon. On exposure to vancomycin (10 mg/ml), the mean counts were 2.5 × 107 CFU/coupon at 24 h, 4.3 × 106 CFU/coupon at 72 h, 1.4 × 105 CFU/coupon at 168 h, and undetectable at 240 h. With gentamicin (10 mg/ml) the mean counts were 2.7 × 107 CFU/coupon at 24 h, 3.7 × 106 CFU/coupon at 72 h, 8.4 × 106 CFU/coupon at 168 h, and 6.5 × 106 CFU/coupon at 240 h. With linezolid at 2 mg/ml the mean counts were 7.1 × 105 CFU/coupon at 24 h and not detectable at 72, 168, and 240 h. With eperezolid (4 mg/ml) no viable cells were recovered after 168 h. These data suggest that linezolid (2 mg/ml) and eperezolid (4 mg/ml) achieve eradication of S. epidermidis biofilms more rapidly than vancomycin (10 mg/ml) and gentamicin (10 mg/ml).

Effect of subinhibitory concentrations of benzalkonium chloride on the competitiveness of Pseudomonas aeruginosa grown in continuous culture

This study investigates the link between adaptation to biocides and antibiotics in Pseudomonas aeruginosa. An enrichment continuous culture of P. aeruginosa NCIMB 10421 (MIC 25 mg BKC l(-1)) was operated (D=0.04 h(-1), 792 h) with added benzalkonium chloride (BKC). A derivative, PA-29 (696 h), demonstrated a >12-fold decrease in sensitivity to the biocide (MIC >350 mg BKC l(-1)). The variant demonstrated a 256-fold increase in resistance to ciprofloxacin, with a mutation in the gyrA gene (Thr-83-->Ile). Similarly, culturing of the original strain in a continuous-culture system with ciprofloxacin selection pressure led to the evolution of BKC-adapted populations (MIC 100 mg BKC l(-1)). Efflux pump activity predominantly contributed to the developed phenotype of PA-29. An amino acid substitution (Val-51-->Ala) in nfxB, the Mex efflux system regulator gene, was observed for PA-29. Overexpression of both MexAB-OprM and MexCD-OprJ was recorded for PA-29. Similarly, mexR, a repressor of the Mex system, was downregulated. Competition studies were carried out in continuous culture between PA-29 and the original strain (in the presence of subinhibitory concentrations of BKC). The outcome of competition was influenced by the concentration of biocide used and the nature of limiting nutrient. The inclusion of 1 mg BKC l(-1) in the medium feed was sufficient to select (S=0.011) for the BKC-adapted strain in magnesium-limited culture. Conversely, the presence of 10 mg BKC l(-1) in the medium supply was insufficient to select for the same organism (S=-0.017) in the glucose-limited culture. These results indicate the importance of environmental conditions on selection and maintenance of biocide adaptation.

The isolation of strains of Bacillus subtilis showing improved plasmid stability characteristics by means of selective chemostat culture.

A pUB110-derived plasmid encoding chloramphenicol resistance, kanamycin resistance and high-temperature alpha-amylase showed a high degree of segregational instability when inserted into Bacillus subtilis. In an attempt to obtain stable derivatives, the organism was grown in chemostat culture in the presence of chlorampheniol. It was periodically found necessary to increase the concentration of chloramphenicol in the medium feed in order to avoid plasmid loss. Strains were isolated after 19 and 160 generations, which showed high levels of plasmid stability. This characteristic appeared to be genotypic. No detectable difference in plasmid copy number was found between the original and the improved strains. The stability characteristics resided in the host, rather than in the plasmid. Stable isolates possessed elevated MICs for both chloramphenicol and kanamycin. Their maximum specific growth rates were higher than that of the original strain, and similar to that of the plasmid-free parent strain.

An evaluation of the applicability of microarrays for monitoring toxic algae in Irish coastal waters

The applicability of microarrays to monitor harmful algae across a broad range of ecological niches and toxic species responsible for harmful algal events has been one of the key tasks in the EU Seventh Framework Programme (FP7)-funded Microarrays for the Detection of Toxic Algae project. The technique has a strong potential for improving speed and accuracy of the identification of harmful algae and their toxins to assist monitoring programmes. Water samples were collected from a number of coastal sites around Ireland, including several that are used in the Irish National Phytoplankton and Biotoxin Monitoring Programme. Ribosomal RNA was extracted from filtered field samples, labelled with a fluorescent dye, and hybridised to probes spotted in a microarray format on a glass slide. The fluorescent signal intensity of the hybridisation to >120 probes on the chip was analysed and compared with actual field counts. There was a general agreement between cell counts and microarray signal. Results are presented for field samples taken from a range of stations along the Irish coastline known for harmful algal events during the first field trial (July 2009–April 2010).

Enrichment of acetogenic bacteria in high rate anaerobic reactors under mesophilic and thermophilic conditions

The objective of the current study was to expand the knowledge of the role of acetogenic Bacteria in high rate anaerobic digesters. To this end, acetogens were enriched by supplying a variety of acetogenic growth supportive substrates to two laboratory scale high rate upflow anaerobic sludge bed (UASB) reactors operated at 37 degrees C (R1) and 55 degrees C (R2). The reactors were initially fed a glucose/acetate influent. Having achieved high operational performance and granular sludge development and activity, both reactors were changed to homoacetogenic bacterial substrates on day 373 of the trial. The reactors were initially fed with sodium vanillate as a sole substrate. Although % COD removal indicated that the 55 degrees C reactor out performed the 37 degrees C reactor, effluent acetate levels from R2 were generally higher than from R1, reaching values as high as 5023 mg l(-1). Homoacetogenic activity in both reactors was confirmed on day 419 by specific acetogenic activity (SAA) measurement, with higher values obtained for R2 than R1. Sodium formate was introduced as sole substrate to both reactors on day 464. It was found that formate supported acetogenic activity at both temperatures. By the end of the trial, no specific methanogenic activity (SMA) was observed against acetate and propionate indicating that the methane produced was solely by hydrogenotrophic Archaea. Higher SMA and SAA values against H(2)/CO(2) suggested development of a formate utilising acetogenic population growing in syntrophy with hydrogenotrophic methanogens. Throughout the formate trial, the mesophilic reactor performed better overall than the thermophilic reactor.

Plasmid instability in an industrial strain ofBacillus subtilis grown in chemostat culture

SummaryA pUB110-derived plasmid/Bacillus subtilis host combination was segregationally unstable when grown in chemostat culture with complex or minimal medium and under starch, glucose or magnesium limitation. The kinetics of plasmid loss were described in terms of the difference in growth rates between plasmid-containing and plasmid-free cells (dμ) and the rate at which plasmid-free cells were generated from plasmid-containing cells (R). Loss of plasmid-containing cells from the population was dμ dominated. Changes in medium composition and the nature of growth limitation caused variations in both dμ and R. The plasmid was most stable in glucose-limited chemostat cultures with minimal medium and least stable under starch limitation with complex complex medium. R and dμ were smaller for cultures in complex media than those in minimal media. Limitation by starch induced expression of the plasmid-encoded HT α amylase gene and was associated with increased values of R and dμ. Magnesium limitation in minimal medium caused a significant increase in dμ and a decrease in R.

Workplace Exposure to Bioaerosols in Podiatry Clinics

OBJECTIVES The aim of this study was to design and execute a pilot study to collect information on the personal exposure levels of podiatrists to microbial hazards in podiatry clinics and also to assess health and safety knowledge within the sector using a questionnaire survey. METHODS A self-report quantitative questionnaire dealing with health and safety/health issues was issued to 250 podiatrist clinics. Fifteen podiatry clinics were randomly recruited to participate in the exposure study. Concentrations of airborne bacteria, fungi, yeasts, and moulds were assessed using a six-stage viable microbial cascade impactor. Personal samples of total inhalable dust and endotoxin were measured in the breathing zone of the podiatrist. RESULTS A questionnaire response rate of 42% (N = 101) was achieved. Thirty-two per cent of respondents indicated that they had a respiratory condition; asthma was the most prevalent condition reported. The most frequently employed control measures reported were use of disposable gloves during patient treatments (73.3%), use of respiratory protective equipment (34.6%), use of protective aprons (16.8%), and eye protection (15.8%). A total of 15.8% of respondents used mechanical room ventilation, 47.5% used nail drills with local exhaust ventilation systems, and 11% used nail drills with water spray dust suppression. The geometric mean concentrations of bacteria, Staphylococci, fungi, and yeasts/moulds were 590, 190, 422, and 59 CFU m(-3), respectively. The geometric mean endotoxin exposure was 9.6 EU m(-3). A significant percentage of all the bioaerosols that were in the respirable fraction was representative of yeasts and moulds (65%) and Fungi (87%). CONCLUSIONS Even if statistical analysis of data is limited by low sample numbers, this study showed that the frequency of cleaning and use of RPE varied between clinics sampled, and it is likely that refresher health and safety training focusing on health and safety hazards inherent in podiatry work and practical control measures is warranted.

Molecular fingerprinting of lacustrian cyanobacterial communities: regional patterns in summer diversity

The assessment of lacustrian water quality is necessary to comply with environmental regulations. At the regional scale, difficulties reside in the selection of representative lakes. Given the risks towards water quality associated with phytoplankton blooms, a mesoscale survey was carried out in Irish lakes to identify patterns in the distribution and diversity of planktonic cyanobacteria. A stratified sampling strategy was carried out via geographic information systems (GIS) analysis of river catchment attributes due to the range of hydrogeomorphological features and the high number of lakes within the study area. 16S rRNA gene denaturing gradient gel electrophoresis analysis showed variation between the cyanobacterial communities sampled, with lower occurrence of cyanobacteria in August concomitant to increased wind and precipitation regimes. Multivariate analysis delineated three ecoregions based on land cover typology and revealed significant patterns in the distribution of cyanobacterial diversity. A majority of filamentous cyanobacteria genotypes occurred in larger lakes contained river catchments with substantial forest cover. In contrast, higher diversity of spherical cyanobacteria genotypes was observed in lakes of lesser trophic state. In the context of aquatic resource management, the combined use of GIS-based sampling strategy and molecular methods offers promising prospects for assessing microbial community structure at varying scales of space and time.

The effect of levofloxacin concentration on the development and maintenance of antibiotic-resistant clones of Escherichia coli in chemostat culture.

A levofloxacin-sensitive strain of Escherichia coli (broth MIC: 0.0625 mg l−1) was grown in carbon-limited chemostat culture for 316 h (D=0.294 h−1). Hyperresistant strains isolated after 58 and 91 generations of culture retained a 16- to 47-fold increase in tolerance to levofloxacin during antibiotic-free serial batch and continuous culture (20 generations, glucose-limited, D=0.2 h−1). Isolates differed from the original strain in their maximum growth rates in the presence and absence of subinhibitory levels of levofloxacin, protein-banding profiles, and resistance to a range of antibiotics. Competition between resistant isolates and the original sensitive strain was studied in glucose-limited chemostat cultures (D=0.2 h−1). At levofloxacin concentrations less than 0.03 mg l−1, the sensitive strain outcompeted resistant isolates and displaced them from the culture, whereas the reverse was true at higher concentrations. These results have clinical and environmental implications for those administering levofloxacin. Journal of Industrial Microbiology & Biotechnology (2002) 29, 155–162 doi:10.1038/sj.jim.7000295

A novel mechanism, linked to cell density, largely controls cell division in Synechocystis.

Nutrient limitation, self-shading, and quorum sensing are not major limiting factors for the growth of Synechocystis in batch cultures. Many studies have investigated the various genetic and environmental factors regulating cyanobacterial growth. Here, we investigated the growth and metabolism of Synechocystis sp. PCC 6803 under different nitrogen sources, light intensities, and CO2 concentrations. Cells grown on urea showed the highest growth rates. However, for all conditions tested, the daily growth rates in batch cultures decreased steadily over time, and stationary phase was obtained with similar cell densities. Unexpectedly, metabolic and physiological analyses showed that growth rates during log phase were not controlled primarily by the availability of photoassimilates. Further physiological investigations indicated that nutrient limitation, quorum sensing, light quality, and light intensity (self-shading) were not the main factors responsible for the decrease in the growth rate and the onset of the stationary phase. Moreover, cell division rates in fed-batch cultures were positively correlated with the dilution rates. Hence, not only light, CO2, and nutrients can affect growth but also a cell-cell interaction. Accordingly, we propose that cell-cell interaction may be a factor responsible for the gradual decrease of growth rates in batch cultures during log phase, culminating with the onset of stationary phase.