Recovery Of Viable Organisms Research Articles

A Novel Bioengineered Wound Product with In Vitro Capabilities to Reduce Bacteria

Introduction: When microorganisms colonize a wound, there is a continuum which extends from contamination, to colonization and infection preventing a wound from healing. Biomaterials that reduce bacteria within the wound microenvironment can help progress it towards healing. Objective: This study aims to determine the in vitro bacteria reduction capabilities of a novel, commercially available bioengineered wound product (BWP) – a synthesis of gelatin (a highly purified collagen derivative), Manuka honey, and hydroxyapatite. Materials and methods: Using a direct contact method, the BWP and negative control were inoculated with suspensions of gram-positive (Staphylococcus aureus) and gram-negative (Pseudomonas aeruginosa) bacteria. After incubation for 30 minutes and 24 hours, recovery of viable organisms was performed. Results: There was a significant reduction (99.99%) in bacterial load recovered from the BWP at 24 hours compared to the negative control. Additionally, the BWP caused a significant reduction in bacterial load at 24 hours compared to 30 minutes (97% and 64% reduction for Staphylococcus aureus and Pseudomonas aeruginosa, respectively). Conclusions: The significant reduction in bacterial load in vitro suggests that the product has the potential to help manage the pathogenic bioburden of a wound..

Rapid and gentle hydrogel encapsulation of living organisms enables long-term microscopy over multiple hours

Imaging living organisms at high spatial resolution requires effective and innocuous immobilization. Long-term imaging places further demands on sample mounting with minimal perturbation of the organism. Here we present a simple, inexpensive method for rapid encapsulation of small animals of any developmental stage within a photo-crosslinked polyethylene glycol (PEG) hydrogel, gently restricting movement within their confined spaces. Immobilized animals maintain their original morphology in a hydrated environment compatible with chemical treatment, optical stimulation, and light-sheet microscopy. We demonstrate prolonged three-dimensional imaging of neural responses in the nematode Caenorhabditis elegans, recovery of viable organisms after 24 h, and imaging of larger squid hatchlings. We characterize a range of hydrogel and illumination conditions for immobilization quality, and identify paralytic-free conditions suitable for high-resolution single-cell imaging. Overall, PEG hydrogel encapsulation provides fast, versatile, and gentle mounting of small living organisms, from yeast to zebrafish, for continuous observation over hours.

The first isolate of Klebsiella pneumoniae Carbapenemase (KPC)–producing Klebsiella pneumoniae at a regional hospital

Results: Repigel treatment showed no recovery of P. aeruginosa biofilm material (>5 Log reduction vs. untreated controls) at both the commercial concentration and 1:10 dilution at both time-points. Topical preparations containing mupirocin 2% and fusidic acid 2% showed no difference vs. controls at 1:10 dilution. Four hours of treatment did not significantly reduce the biofilm load, but a >1 Log reduction in P. aeruginosa biofilm material was demonstrated after 24 hours. Treatment of mixed C. albicans/MRSA biofilms with Repigel TM resulted in a 5 Log reduction in biofilm material at both time-points. The 1:10 dilution of Repigel TM produced a >1 Log reduction in mixed biofilm material at both time-points, similar to findings with the other topical preparations. Conclusions: Repigel TM at commercial concentration and 1:10 dilution prevented the recovery of viable P. aeruginosa biofilm material and reduced the recovery of viable organisms from multispecies biofilms of C. albicans and MRSA. The efficacy of Repigel TM in this in vitro model supports its potential antiseptic effectiveness against multiple bacterial strains in exuding chronic wounds. Funded: Mundipharma

Influence of dextrose on the viability of Bacillus subtilis.

Abstract Dextrose causes an increase in viable count of Bacillus subtilis and the effect is greater for spores than vegetative cells. The proportion of spores in which germination is initiated is not influenced by dextrose. During germination the ability of dextrose to affect the viable count is completely lost but re-appears as the germinated spore changes into a dividing vegetative cell. When spores are incubated in broth before plating in nutrient agar there is an increase in viable count followed by a fall before the increase in count due to division is detected. When plated in dextrose agar this initial rise is not seen and there is a fall in count before onset of division. The magnitude of the increase in count caused by dextrose is dependent on the method of plating. For maximum recovery of viable organisms the combination of roll-rube method with dextrose agar is to be preferred to surface plating with or without dextrose in the counting medium.

A study to detect Gardnerella vaginalis DNA in interstitial cystitis.

To investigate the possible role of Gardnerella vaginalis in interstitial cystitis (IC), using molecular methods to avoid difficulties with the culture and recovery of viable organisms, and the problems associated with the recovery of low numbers of culturable organisms. Thirty-three bladder biopsy samples (29 paraffin-embedded and four freshly frozen) from patients with IC, diagnosed according to National Institute of Diabetes, Digestive and Kidney Diseases criteria, were assessed. Biopsy samples were used as urine samples may be contaminated by normal vaginal flora. A positive control comprised a 'normal' biopsy sample from a patient with a previous bladder tumour, seeded with G. vaginalis NCTC 10915. Microbial DNA was extracted from all paraffin-embedded and fresh specimens, and subjected to in vitro amplification by polymerase chain reaction (PCR) with G. vaginalis-specific primers. The anticipated PCR product of 333 base pairs was obtained with the positive control, whereas none of the other biopsy samples showed positive amplification specific for G. vaginalis. As there was no G. vaginalis DNA in any of the samples from patients with IC, it is an unlikely candidate in the pathogenesis of IC.

Evidence for invasion of a human oral cell line by Actinobacillus actinomycetemcomitans

Actinobacillus actinomycetemcomitans, an oral bacterial species associated with periodontal disease, was found to invade human cell lines. Invasion was demonstrated by recovery of viable organisms from gentamicin-treated KB cell monolayers and by light and electron microscopy. Internalization occurred through a cytochalasin D-sensitive process. Invasion efficiencies of some A. actinomycetemcomitans strains were comparable to those of invasive members of the family Enterobacteriaceae. Differences in invasiveness were correlated with bacterial colonial morphology. Smooth variants invaded more proficiently than rough variants. A. actinomycetemcomitans can undergo a smooth-to-rough colonial morphology shift which results in the loss of invasiveness. Coordinated regulation of genes involved in the rough-to-smooth phenotypic transitions may play a role in the episodic nature of periodontal disease.

Invasion of HeLa 229 cells by virulent Bordetella pertussis

Phase-dependent invasive behavior of Bordetella pertussis was demonstrated by recovery of viable organisms from gentamicin-treated HeLa cell monolayers and by transmission electron microscopy. Several mutants of B. pertussis with Tn5 or Tn5 lac inserted into various vir-regulated genes were evaluated for differences in their invasive abilities. Mutants lacking filamentous hemagglutinin, pertussis toxin, and two as yet uncharacterized vir-regulated products had levels of invasion significantly lower than that of the parent strain BP338. In contrast, invasion by mutants lacking adenylate cyclase toxin was significantly increased compared with that of wild-type B. pertussis. This increase in invasion was eliminated when concentrations of intracellular cyclic 3'-5' AMP were stimulated by treating HeLa cells with cholera toxin or forskolin. Entry of B. pertussis occurred through a microfilament-dependent phagocytic process, as evidenced by the marked reduction in uptake following treatment of HeLa cells with cytochalasin D. Invasion was inhibited with polyclonal anti-B. pertussis and anti-filamentous hemagglutinin antisera. In addition, a monoclonal antibody against lipooligosaccharide A reduced uptake by 65.5%. The preservation of HeLa cell integrity and the limited replication of intracellular bacteria suggest that invasion may represent a means by which B. pertussis evades an active host immune response.

Modified tissue culture medium for corneal storage I. Investigation of the effect of antibiotics on bacterial contamination.

A series of experiments were performed to determine the effect of modified tissue culture medium (MTCM), temperature, time, and antibiotics on the replication of Staphylococcus epidermidis and Pseudomonas aeruginosa. A known inoculum of the organisms (usually 10(6)/mL) was introduced, and the recovery of viable organisms was compared by different methods of culturing, including techniques of diluting, inactivating, or removing the antibiotic from the system. The invalidity of techniques that failed to remove the antibiotic from the system was substantiated. The newly developed antibiotic removal device was superior to all other culturing techniques. Penicillin G potassium was ineffective in the MTCM system, and gentamicin reagent solution was not uniformly effective against a gentamicin-sensitive strain of P aeruginosa. Placement of the MTCM at room temperature for two hours enhanced the antibacterial activity of gentamicin, although organisms may still be recovered in small numbers.

Destruction of Microorganisms During Thawing of Skim Milk

The viability of four species of microorganisms (Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Saccharomyces cerevisiae) during rapid and slow thawing of frozen milk was investigated. Results indicated that the destruction of microbial cells was significantly greater when skim milk was thawed slowly. Recovery of viable organisms by plating was generally slightly higher when peptone water was used as a diluent, although differences were not statistically significant.

Enclosed chambers for the convenient reverse flow concentration and selective filtration of particles1

Enclosed chambers for reverse flow concentration allow convenient and gentle concentration of particles for a wide variety of purposes, including routine 1,000× concentrations of oceanic particles. The recovery of viable organisms from the enclosed concentrators is considerably greater than in previously used reverse flow techniques.

Isolation and phenotypic characterization of virulence-deficient mutants of Vibrio cholerae.

Mutants of Vibrio cholerae was isolated on the basis of reduced ability to induce diarrhea in orally challenged infant mice. Nitrosoguanidine-treated clones were screened for low fluid accumulation ratios in individual mice, and presumptive mutants were confirmed in additional mouse tests. Mutants were examined for alterations in phage type, motility, toxin production, proteolytic activity, neuraminidase production, amylase production, morphology, growth requirements, carbohydrate fermentations, in vitro growth patterns, and cell surface alterations. The types of mutants found included several with previously recognized virulence-associated markers (rough, nonmotile, toxin deficient, protease deficient); several types with pleiotropic alterations (cell morphology, decreased extracellular products); and several with no previously recognized virulence-deficient phenotype (purine requiring, cell surface altered, rapid death in vitro, no defect found). Dose-response kinetics showed that most mutants could provoke diarrhea if given in 100-fold greater numbers than the dose used for screening. Recovery of viable organisms from the gut late in infection showed reduction of survival and/or multiplication capacity for the mutants, with variation in the degree of reduction for the different classes.

Effect of squamous metaplasia on infection of hamster trachea organ cultures with Mycoplasma pneumoniae.

An organ culture system for hamster trachea was developed for maintenance of the ciliated respiratory epithelium during periods of extended cultivation (i.e., greater than 20 days). Evaluation of five serum types showed that horse serum and fetal calf serum were best for the maintenance of epithelial ciliary activity and morphology. Rings that were opened on one side ("split rings") had the best maintenance of the ciliated epithelium as judged by the retention of ciliary activity and normal histological appearance after 3 to 4 weeks in culture. The in vitro induction of squamous metaplasia was achieved by cultivating explants in Waymouth MAB 87/3 (vitamin A-free) medium, without serum. This system allowed a direct comparison of the effects of Mycoplasma pneumoniae infection in two epithelial types, ciliated pseudostratified columnar and keratinizing squamous. Attachment of 14C-labeled mycoplasmas was more than twofold greater in the normal epithelium. Pretreatment of explants with neuraminidase decreased attachment for both squamous and pseudostratified epithelial surfaces to a similar basal level. Recovery of viable organisms from infected tissue of both epithelial types indicated that the organism titer remained essentially constant during the infection period, but was significantly higher for the pseudostratified ciliated epithelium. These results suggest that specific receptor sites for M. pneumoniae are markedly reduced by the induction of squamous metaplasia and, hence, appear to be specific for the normal respiratory surface containing goblet cells and pseudostratified, ciliated epithelial cells.

Culture of Ethiopian Strains of Borrelia recurrentis

A number of standard bacteriological media with supplements were tested for their ability to support in vitro growth of Ethiopian strains of Borrelia recurrentis. Propagation of 18 out of 21 strains occurred in Trypticase soy yeast broth to which bovine albumin (fraction V), N-acetyl glucosamine, and sodium pyruvate had been added. This medium supported a population of 10(7) organisms per ml and yielded a harvest of four to five times the original inoculum during the logarithmic phase of growth. Maximal yield varied from 1.4 x 10(7) to 3.4 x 10(7) organisms per ml. Generation time in optimal media was 11.3 h. Lesser multiplication of organisms occurred in other media tested. Strains from primary cultures were infective for the green monkey. Recovery of viable organisms from subculture has not been successful.

SOME OBSERVATIONS ON BACTERICIDAL PAINT FILMS

SUMMARY: A method is described for testing the self‐sterilizing activity of surfaces coated with paints containing bactericidal agents. The most active coating consisted of 10% (w/w) Cetavlon dissolved directly in urea‐formaldehyde:alkyd resin. The recovery of viable organisms from such surfaces infected with Streptococcus faecalis, Staphylococcus aureus, Aerobacter aerogenes and Chromobacterium prodigiosum was less than 10% of that from painted control surfaces after exposure for 1 hr at atmospheric relative humidity of 70% and temperature of 20°. Bacillus subtilis spores were not affected until they became heat‐sensitive. The self‐sterilizing activity was markedly diminished at 40% r.h. and the inclusion of blood in the deposited bacterial particle was then completely protective. Repeated washing in water with gentle rubbing quickly inactivated Cetavlon paint films.