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We found that the in vitro HOCl-resistance profile (PsA > SA > BC > EC > KP) best fits the infection profile observed clinically in CF lungs; that is, the most HOCl-resistant bacteria such as PsA and SA are the most frequent pathogens in CF patients. This finding implies that differential HOCl resistance across microbial species may allow for persistence of some infections over others by subversion of the host innate immunity and supports our previous finding that CF neutrophils with a compromised HOCl production may not be able to clear the most
resistant organisms effectively [12, 13]. From a microbiological point of view, PsA and SA, the relatively more resistant strains to HOCl, would be more likely to survive and be selected for, if the host neutrophils were deficient in their ability to make HOCl. Burns and coworkers did a longitudinal CHIR-99021 in vivo study on young children with CF and found that 97% of the children are colonized with PsA [18]. The early isolates tend to be nonmucoid and antibiotic-sensitive. However,
if the OICR-9429 chemical structure initial infection is not effectively eradicated by the host defense, which could happen, for example, if HOCl or other oxidant production was suboptimal, then the bacteria which escape the initial host defenses will grow and spread within the lung, establishing a long-term chronic colonization. Subsequently, environmental pressure in the lung such as antibiotic see more application selects for the mucoid PsA phenotype. Increased PsA density in the lower respiratory
tract and development of antibiotic-resistant mucoid biofilms causes chronic airway inflammation and deteriorating lung function [19–22]. SA has long been recognized to be among the first organisms to colonize the airways of CF patients [23]. Colonization with SA occurs within the first few months of life, and persistent variants of this organism may arise due to a selective pressure from long-term antibiotic treatment in CF patients [24]. However, SA infection does not usually persist or progress to chronic disease. We would like Fossariinae to point out that our current study only tested bacteria in log-phase growth. Such an experimental design was intended to study the nonmucoid form which is assumed by the bacteria during the early CF infections. It is important to recognize that only after initial bacterial colonization is established, can chronic persistent infections ensue in CF lungs. Neutrophils are highly specialized for bacterial killing especially in the case of extracellular infections. The cells employ at least two microbicidal mechanisms to execute this function: one is oxidant-mediated and another is non-oxidant-mediated. Pseudomonas bacteria possess tough polysaccharide capsules, which are resistant to nonoxidant killing mechanisms, such as protease and hydrolase digestion [25].
Incubation was anaerobic and lasted 64.5 h. The medium was renewed after 16.5 h and subsequently every 24 h. After the first renewal AZD0156 mw of growth media, each well was supplemented with a boost of 40 μl of T. denticola liquid culture (OD550 = 0.5). Biofilms were
dip-washed three times daily at intervals of 3–4 h. For dip-washings the discs were placed in 0.9% NaCl and washed by gentle agitation for 45 seconds. After this step, the discs were dipped twice two times each in two wells of fresh saline. Then the discs were returned to medium for further incubation. Table 1 Growth media Medium Abbreviation Reference Use mFUM, 4 mM Glucose mFUM4 Growth medium for biofilms mFUM 4 mM Glucose, iHS (50%) iHS Growth medium
for biofilms mFUM, 0.3% Glucose (30%), saliva (60%), iHS (10%) SAL Growth medium for biofilms mFUM, 0.3% Glucose [12] Liquid precultures of S. oralis, S. anginosus, V. dispar 1 , F. nucleatum, A. oris, P. intermedia, C. rectus 2 Pg medium3 [29] Liquid precultures of P. gingivalis Spirochaetes medium [30] Precultures of T. denticola Modified OMIZ-W684 [31] Precultures of T. forsythia 1 addition of 1% lactic acid (v/v). 2 addition of 0.1% sodium fumarate and 0.1% sodium formiate. 3 Brain heart infusion broth, supplemented with haemin (7.67 μM) and menadione (2.91 μM). 4 addition of lactose (2 g l-1), caseinoglycomacropeptide (100 mg l-1),N-acetylmuramic acid (50 mg l-1), and N- acetylglucosamine
(500 mg l-1). For confocal microscopy, biofilms were fixed directly on the discs for at least 1 h at 4°C in 4% paraformaldehyde (Merck, Darmstadt, Germany) after the last dip-wash. selleck chemicals llc For quantification by microscopic counting, biofilms were removed from the discs by vortexing (2 min in a 50 ml tube with 1 ml of in 0.9% NaCl) and sonicated for 5 sec at 25 W (Branson Sonic Power Company, Sonifier B-12) to reduce cell aggregation and the processed as described below. FISH staining procedure The FISH procedure was done using the same conditions for the hybridisation as described by Thurnheer et al. [32]. Probe sequences, Progesterone formamide concentrations used for the hybridisations, as well as the NaCl concentrations of the washing buffers are given in Table 2. To hybridise gram-positive bacteria, biofilms were pre-treated in lysozyme solution with a concentration of 1 mg/ml lysozyme (5 min, room temperature). The lysozyme solution consisted of 1 mg lysozyme from chicken egg white containing 70’000 units/mg (Fluka), dissolved in 890 μl H2O, 100 μl 1 M Tris–HCl solution (ICN Biomedicals, Inc.), pH =7.5, and 10 μl 0.5 M EDTA solution (Fluka), pH = 8.0. If the combination of probes required different formamide concentrations, the hybridisations were VX-680 in vitro performed consecutively, starting with the highest concentration. Pre-hybridisation (15 min, 46°C) was performed in 500 μl hybridisation buffer without probes added.
