Reduced Bacterial Colony Count of Anaerobic Bacteria Is Associated with a Worsening in Lung Clearance Index and Inflammation in Cystic Fibrosis

Anaerobic bacteria have been identified in abundance in the airways of cystic fibrosis (CF) subjects. The impact their presence and abundance has on lung function and inflammation is unclear. The aim of this study was to investigate the relationship between the colony count of aerobic and anaerobic bacteria, lung clearance index (LCI), spirometry and C-Reactive Protein (CRP) in patients with CF. Sputum and blood were collected from CF patients at a single cross-sectional visit when clinically stable. Community composition and bacterial colony counts were analysed using extended aerobic and anaerobic culture. Patients completed spirometry and a multiple breath washout (MBW) test to obtain LCI. An inverse correlation between colony count of aerobic bacteria (n = 41, r = -0.35; p = 0.02), anaerobic bacteria (n = 41, r = -0.44, p = 0.004) and LCI was observed. There was an inverse correlation between colony count of anaerobic bacteria and CRP (n = 25, r = -0.44, p = 0.03) only. The results of this study demonstrate that a lower colony count of aerobic and anaerobic bacteria correlated with a worse LCI. A lower colony count of anaerobic bacteria also correlated with higher CRP levels. These results indicate that lower abundance of aerobic and anaerobic bacteria may reflect microbiota disruption and disease progression in the CF lung.

Development of a log-quadratic model to describe microbial inactivation, illustrated by thermal inactivation of Clostridium botulinum

In the commercial food industry, demonstration of microbiological safety and thermal process equivalence often involves a mathematical framework that assumes log-linear inactivation kinetics and invokes concepts of decimal reduction time (DT), z values, and accumulated lethality. However, many microbes, particularly spores, exhibit inactivation kinetics that are not log linear. This has led to alternative modeling approaches, such as the biphasic and Weibull models, that relax strong log-linear assumptions. Using a statistical framework, we developed a novel log-quadratic model, which approximates the biphasic and Weibull models and provides additional physiological interpretability. As a statistical linear model, the log-quadratic model is relatively simple to fit and straightforwardly provides confidence intervals for its fitted values. It allows a DT-like value to be derived, even from data that exhibit obvious "tailing." We also showed how existing models of non-log-linear microbial inactivation, such as the Weibull model, can fit into a statistical linear model framework that dramatically simplifies their solution. We applied the log-quadratic model to thermal inactivation data for the spore-forming bacterium Clostridium botulinum and evaluated its merits compared with those of popular previously described approaches. The log-quadratic model was used as the basis of a secondary model that can capture the dependence of microbial inactivation kinetics on temperature. This model, in turn, was linked to models of spore inactivation of Sapru et al. and Rodriguez et al. that posit different physiological states for spores within a population. We believe that the log-quadratic model provides a useful framework in which to test vitalistic and mechanistic hypotheses of inactivation by thermal and other processes. Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Designer porous antibacterial membranes derived from thermally induced phase separation of PS/PVME blends decorated with an electrospun nanofiber scaffold

We report the development of porous membranes by thermally induced phase separation of a PS/PVME (polystyrene/polyvinylmethyl ether]) blend, which is a typical LCST mixture. The morphology of the membrane after etching out the PVME phase was characterized by scanning electron microscopy. To give the membrane an antibacterial surface, polystyrene (PS) and polyvinyl(methyl ether)]-alt-maleic anhydride (PVME-MAH) with silver nanoparticles (nAg) were electrospun on the membrane surface. Pure water flux was evaluated by using a cross-flow membrane setup. The microgrooved fibers changed the flux across the membrane depending on the surface properties. The antibacterial properties of the membrane were confirmed by the reduction in the colony count of E. coli. The SEM images show the disruption of the bacterial cell membrane and the antibacterial mechanism was discussed.

Microbial inactivation of Pseudomonas putida and Pichia pastoris using gene silencing.

Antisense deoxyoligonucleotide (ASO) gene silencing was investigated as a potential disinfection tool for industrial and drinking water treatment application. ASOs bind with their reverse complementary mRNA transcripts thereby blocking protein translation. While ASO silencing has mainly been studied in medicine, it may be useful for modulating gene expression and inactivating microorganisms in environmental applications. In this proof of concept work, gene targets were sh ble (zeocin resistance) and todE (catechol-2,3-dioxygenase) in Pichia pastoris and npt (kanamycin resistance) in Pseudomonas putida. A maximum 0.5-fold decrease in P. pastoris cell numbers was obtained following a 120 min incubation with single-stranded DNA (ssDNA) concentrations ranging from 0.2 to 200 nM as compared to the no ssDNA control. In P. putida, a maximum 5.2-fold decrease was obtained after 90 min with 400 nM ssDNA. While the silencing efficiencies varied for the 25 targets tested, these results suggest that protein activity as well as microbial growth can be altered using ASO gene silencing-based tools. If successful, this technology has the potential to eliminate some of the environmental and health issues associated with the use of strong chemical biocides. However, prior to its dissemination, more research is needed to increase silencing efficiency and develop effective delivery methods.

Bacteria localization and chorion thinning among preterm premature rupture of membranes.

OBJECTIVE: Bacterial colonization of the fetal membranes and its role in pathogenesis of membrane rupture is poorly understood. Prior retrospective work revealed chorion layer thinning in preterm premature rupture of membranes (PPROM) subjects. Our objective was to prospectively examine fetal membrane chorion thinning and to correlate to bacterial presence in PPROM, preterm, and term subjects. STUDY DESIGN: Paired membrane samples (membrane rupture and membrane distant) were prospectively collected from: PPROM = 14, preterm labor (PTL = 8), preterm no labor (PTNL = 8), term labor (TL = 10), and term no labor (TNL = 8), subjects. Sections were probed with cytokeratin to identify fetal trophoblast layer of the chorion using immunohistochemistry. Fluorescence in situ hybridization was performed using broad range 16 s ribosomal RNA probe. Images were evaluated, chorion and choriodecidua were measured, and bacterial fluorescence scored. Chorion thinning and bacterial presence were compared among and between groups using Student's t-test, linear mixed effect model, and Poisson regression model (SAS Cary, NC). RESULTS: In all groups, the fetal chorion cellular layer was thinner at rupture compared to distant site (147.2 vs. 253.7 µm, p<0.0001). Further, chorion thinning was greatest among PPROM subjects compared to all other groups combined, regardless of site sampled [PPROM(114.9) vs. PTL(246.0) vs. PTNL(200.8) vs. TL(217.9) vs. TNL(246.5)]. Bacteria counts were highest among PPROM subjects compared to all other groups regardless of site sampled or histologic infection [PPROM(31) vs. PTL(9) vs. PTNL(7) vs. TL(7) vs. TNL(6)]. Among all subjects at both sites, bacterial counts were inversely correlated with chorion thinning, even excluding histologic chorioamnionitis (p<0.0001 and p = 0.05). CONCLUSIONS: Fetal chorion was uniformly thinner at rupture site compared to distant sites. In PPROM fetal chorion, we demonstrated pronounced global thinning. Although cause or consequence is uncertain, bacterial presence is greatest and inversely correlated with chorion thinning among PPROM subjects.

Bioburden after Staphylococcus aureus inoculation in type 1 diabetic rats undergoing internal fixation.

SUMMARY: Fracture stabilization in the diabetic patient is associated with higher complication rates, particularly infection and impaired wound healing, which can lead to major tissue damage, osteomyelitis, and higher amputation rates. With an increasing prevalence of diabetes and an aging population, the risks of infection of internal fixation devices are expected to grow. Although numerous retrospective clinical studies have identified a relationship between diabetes and infection, currently there are few animal models that have been used to investigate postoperative surgical-site infections associated with internal fixator implantation and diabetes. The authors therefore refined the protocol for inducing hyperglycemia and compared the bacterial burden in controls to pharmacologically induced type 1 diabetic rats after undergoing internal fracture plate fixation and Staphylococcus aureus surgical-site inoculation. Using an initial series of streptozotocin doses, followed by optional additional doses to reach a target blood glucose range of 300 to 600 mg/dl, the authors reliably induced diabetes in 100 percent of the rats (n = 16), in which a narrow hyperglycemic range was maintained 14 days after onset of diabetes (mean ± SEM, 466 ± 16 mg/dl; coefficient of variation, 0.15). With respect to their primary endpoint, the authors quantified a significantly higher infectious burden in inoculated diabetic animals (median, 3.2 × 10 colony-forming units/mg dry tissue) compared with inoculated nondiabetic animals (7.2 × 10 colony-forming units/mg dry tissue). These data support the authors' hypothesis that uncontrolled diabetes adversely affects the immune system's ability to clear Staphylococcus aureus associated with internal hardware.

The inoculum effect and band-pass bacterial response to periodic antibiotic treatment.

The inoculum effect (IE) refers to the decreasing efficacy of an antibiotic with increasing bacterial density. It represents a unique strategy of antibiotic tolerance and it can complicate design of effective antibiotic treatment of bacterial infections. To gain insight into this phenomenon, we have analyzed responses of a lab strain of Escherichia coli to antibiotics that target the ribosome. We show that the IE can be explained by bistable inhibition of bacterial growth. A critical requirement for this bistability is sufficiently fast degradation of ribosomes, which can result from antibiotic-induced heat-shock response. Furthermore, antibiotics that elicit the IE can lead to 'band-pass' response of bacterial growth to periodic antibiotic treatment: the treatment efficacy drastically diminishes at intermediate frequencies of treatment. Our proposed mechanism for the IE may be generally applicable to other bacterial species treated with antibiotics targeting the ribosomes.

Eradication of marine biofilms by atmospheric pressure non-thermal plasma: A potential approach to control biofouling?

Although the antimicrobial activity of atmospheric pressure non-thermal plasmas, including its capacity to eradicate microbial biofilms, has been gaining an ever increasing interest for different medical applications, its potential utilisation in the control of biofouling and biodeterioration has, to date, received no attention. In this study, the ability of atmospheric pressure plasma to eradicate biofilms of four biofouling bacterial species, frequently encountered in marine environments, was investigated. Biofilms were grown on both polystyrene and stainless steel surfaces before being exposed to the plasma source. Viability and biomass of biofilms were evaluated using colony count method and differential Live/Dead fluorescence staining followed by confocal laser scanning microscopy. Rapid and complete eradication of all biofilms under study was achieved after plasma exposures ranging from 60 to 120 s. Confocal microscopy examination showed that plasma treatment has mediated not only cell killing but also varying degrees of physical removal of biofilms. Further investigation and tailored development of atmospheric pressure non-thermal plasma sources for this particular application could provide an additional powerful and effective weapon in the current anti-biofouling armamentarium.

Effect of isomalt consumption on faecal microflora and colonic metabolism in healthy volunteers

Due to its low digestibility in the small intestine, a major fraction of the polyol isomalt reaches the colon. However, little is known about effects on the intestinal microflora. During two 4-week periods in a double-blind, placebo-controlled, cross-over design, nineteen healthy volunteers consumed a controlled basal diet enriched with either 30 g isomalt or 30 g sucrose daily. Stools were collected at the end of each test phase and various microbiological and luminal markers were analysed. Fermentation characteristics of isomalt were also investigated in vitro. Microbiological analyses of faecal samples indicated a shift of the gut flora towards an increase of bifidobacteria following consumption of the isomalt diet compared with the sucrose diet (P

Effects of orally administered Bdellovibrio bacteriovorus on the well-being and Salmonella colonization of young chicks

Bdellovibrio bacteriovorus is a bacterium which preys upon and kills Gram-negative bacteria, including the zoonotic pathogens Escherichia coli and Salmonella. Bdellovibrio has potential as a biocontrol agent, but no reports of it being tested in living animals have been published, and no data on whether Bdellovibrio might spread between animals are available. In this study, we tried to fill this knowledge gap, using B. bacteriovorus HD100 doses in poultry with a normal gut microbiota or predosed with a colonizing Salmonella strain. In both cases, Bdellovibrio was dosed orally along with antacids. After dosing non-Salmonella-infected birds with Bdellovibrio, we measured the health and well-being of the birds and any changes in their gut pathology and culturable microbiota, finding that although a Bdellovibrio dose at 2 days of age altered the overall diversity of the natural gut microbiota in 28-day-old birds, there were no adverse effects on their growth and well-being. Drinking water and fecal matter from the pens in which the birds were housed as groups showed no contamination by Bdellovibrio after dosing. Predatory Bdellovibrio orally administered to birds that had been predosed with a gut-colonizing Salmonella enterica serovar Enteritidis phage type 4 strain (an important zoonotic pathogen) significantly reduced Salmonella numbers in bird gut cecal contents and reduced abnormal cecal morphology, indicating reduced cecal inflammation, compared to the ceca of the untreated controls or a nonpredatory ΔpilA strain, suggesting that these effects were due to predatory action. This work is a first step to applying Bdellovibrio therapeutically for other animal, and possibly human, infections.

Methylene Blue-Loaded Dissolving Microneedles: Potential Use in Photodynamic Antimicrobial Chemotherapy of Infected Wounds

Photodynamic therapy involves delivery of a photosensitising drug that is activated by light of a specific wavelength, resulting in generation of highly reactive radicals. This activated species can cause destruction of targeted cells. Application of this process for treatment of microbial infections has been termed "photodynamic antimicrobial chemotherapy" (PACT). In the treatment of chronic wounds, the delivery of photosensitising agents is often impeded by the presence of a thick hyperkeratotic/necrotic tissue layer, reducing their therapeutic efficacy. Microneedles (MNs) are an emerging drug delivery technology that have been demonstrated to successfully penetrate the outer layers of the skin, whilst minimising damage to skin barrier function. Delivering photosensitising drugs using this platform has been demonstrated to have several advantages over conventional photodynamic therapy, such as, painless application, reduced erythema, enhanced cosmetic results and improved intradermal delivery. The aim of this study was to physically characterise dissolving MNs loaded with the photosensitising agent, methylene blue and assess their photodynamic antimicrobial activity. Dissolving MNs were fabricated from aqueous blends of Gantrez(®) AN-139 co-polymer containing varying loadings of methylene blue. A height reduction of 29.8% was observed for MNs prepared from blends containing 0.5% w/w methylene blue following application of a total force of 70.56 N/array. A previously validated insertion test was used to assess the effect of drug loading on MN insertion into a wound model. Staphylococcus aureus, Escherichia coli and Candida albicans biofilms were incubated with various methylene blue concentrations within the range delivered by MNs in vitro (0.1-2.5 mg/mL) and either irradiated at 635 nm using a Paterson Lamp or subjected to a dark period. Microbial susceptibility to PACT was determined by assessing the total viable count. Kill rates of >96%, were achieved for S. aureus and >99% for E. coli and C. albicans with the combination of PACT and methylene blue concentrations between 0.1 and 2.5 mg/mL. A reduction in the colony count was also observed when incorporating the photosensitiser without irradiation, this reduction was more notable in S. aureus and E. coli strains than in C. albicans.

In vitro characterization of intra-generic inhibition of growth in Salmonella typhimurium

The intra-generic inhibition of bacterial growth observed previously in vivo and in vitro with strains of Salmonella, Citrobacter and E. coli was studied in vitro using S. typhimurium strain F98. There was complete inhibition of multiplication of S. typhimurium when it was added to stationary-phase broth cultures of different Salmonella serotypes, but only partial inhibition when added to broth cultures of E. coli. The degree of inhibition between different mutants of F98 was affected by the numbers of bacteria of the inhibiting strain, but this was not the only factor, since exponential-phase bacterial cells were less inhibitory than stationary-phase cells. The inhibitory effect was produced at temperatures between 20°C and 40°C. The complete inhibition of growth observed between F98 mutants was abolished by ampicillin, rifampicin and streptomycin, but not by nalidixic acid. Inhibition was also prevented by separating the two cultures by a dialysis membrane. A Tnpho A Insertion mutant of F98 was produced which did not show inhibition in vitro but was still inhibitory in vivo. It is suggested that this complete inhibition of bacterial multiplication between organisms of the same genus, which is greater than that produced between organisms from different genera, is mediated by a cell surface protein.

Pulmonary paracoccidioidomycosis in immunized mice

Paracoccidioidomycosis was induced in immunized (IM) and non-immunized (NI) mice. The histopathology, the number of fungi in the lungs, the cellular (footpad test - FPT and macrophage inhibition factor assay - MIF) and humoral (immunodiffusion test) immune response were investigated serially postinfection. In the IM mice, at days 1 and 3, there was intense and predominant macrophagic-lymphocytic alveolitis with loose granulomatous reaction; at day 30, inflammation was mild. In the NI group, up to day 3, the lesions were focal; later there was formation of extensive epithelioid granuloma. The number of fungi in IM mice were always smaller than those of NI group. Immunization alone induced positive FPT and MIF indices with low titer of antibody. After infection, there was a significant decrease of the FPT indices in the IM group, which we interpreted as desensitization due to trapping of sensitized lymphocytes in the lungs. In conclusion, (1) The lesional pattern of pulmonary paracoccidioidomycosis in IM mice was similar to that of a hypersensitivity pneumonitis. This reaction was probably effective in reducing the extension of the infection and decrease the number of fungi. (2) In this model, pulmonary resistance against P. brasiliensis seems to be related to local and systemic delayed-type hypersensitivity reaction. © 1992 Kluwer Academic Publishers.

Survey of the Microbiological Quality of Nonalcoholic Carbonated Beverages

The number and the main groups of microorganisms present in samples of different nonalcoholic carbonated beverages (lemon, orange and guaraná soft drinks) obtained from a small factory were analyzed. The samples were obtained at the end of the processing line. They were then divided into two lots: one was sent to immediate analysis, the other was stored at environmental temperature for 90 d thereafter it was submitted to the same analysis. Aliquots of 1 mL were drawn from the various samples and the corresponding decimal dilutions were prepared. They were then grown in culture media and counts of mesophilic aerobic bacteria, molds and yeasts, acid-producing bacteria, total and fecal coliforms were taken. It was observed that, of all the analyzed samples, at time 0 or after storage sample C (orange) was the best, since it conformed to the microbiological standards established by legislation. The guaraná type could also be consumed on day zero; the lemon type was inadequate for consumption of all the analyzed samples, the orange type was the only one that could be consumed within 3 months of storage.

Lethal photosensitization in microbiological treatment of ligature-induced peri-implantitis: a preliminary study in dogs

This pilot study evaluated, by culture testing, the effectiveness of lethal photosensitization for the microbiological treatment of peri-implantitis in dogs. Experimental peri-implantitis was induced by ligature placement for 2 months. Following ligature removal, plaque control was instituted by scrubbing with 0.12% chlorhexidine daily for 12 months. Subsequently, mucoperiosteal flaps were elevated for scaling the implant surface. Microbial samples were obtained with paper points before and after treatment of implant surfaces by means of 100 microg/ml toluidine blue O (TBO,) and were exposed, for 80 s, to light with a wavelength of 685 nm from a 50 mW GaAlAs diode laser. The mean initial and final bacterial counts were 7.22 +/- 0.20 and 6.84 +/- 0.44 CFU/ml, respectively for TVC (P < 0.0001); 6.19 +/- 0.45 and 3.14 +/- 3.29 CFU/ml for P. intermedia/nigrescens (P = 0.001); 5.98 +/- 0.38 and 1.69 +/- 2.90 CFU/ml for Fusobacterium spp. (P = 0.001); and 6.07 +/- 0.22 to 1.69 +/- 2.94 CFU/ml for beta-hemolytic Streptococcus (P = 0.0039). It may be concluded that lethal photosensitization resulted in a reduction of the bacterial count. Complete elimination of bacteria was achieved in some samples.