Suspensions or biofilms : and other factors that affect disinfectant testing on pathogens

Disinfectants are very important for the maintenance of proper hygiene in the food industry. In Europe, candidate disinfectants have to be tested on suspended bacteria in so called suspension tests, before they can be approved as disinfectants. In the food industry bacteria usually are attached to surfaces, where they may form biofilms. This mode of growth makes them less susceptible to disinfectants than free-living (suspended) bacteria. Thus, disinfectant testing would greatly profit from a biofilm disinfectant test. The aim of the research described in this thesis was to improve the current European disinfectant tests. To achieve this goal we studied factors that influence the efficacy of disinfectants and alternatives for viability assessment by plate counting. Furthermore, we developed a biofilm disinfectant test.The bacteria used in this study were the biofilm forming pathogens Listeria monocytogenes and Staphylococcus aureus . The disinfectants were benzalkonium chloride (BAC), sodium hypochlorite (NaOCl), hydrogen peroxide, and dodecylbenzene sulphonic acid.The growth phase of cells grown in suspension had a large influence on their susceptibility to disinfectants. S. aureus cells grown according to the prescription of the European suspension test were phenotypically not the most disinfectant resistant cells of all the cell types tested. Thus, in suspension tests, cells that are grown differently and have a higher phenotypic resistance than the cells currently used for disinfectant testing could be used.Fluorescent labeling could be used as a rapid alternative for viability assessment by plate counting, for both free-living cells and biofilm cells, provided the proper fluorescent probes were selected. Thus, for rapid screening of candidate disinfectants, fluorescent probes in combination with flow cytometry may be used instead of plate counting.In the biofilm disinfectant test, S. aureus biofilm formation and biofilm disinfection by BAC and NaOCl were reproducible and a genuine biofilm was produced. To improve disinfectant testing in general, the biofilm disinfectant test developed in this thesis can be added to the set of tests that are used currently. The biofilm test will show which currently used or new disinfectants are the most effective against biofilm cells. In the end, this will contribute to food safety and food quality and the control of cleaning costs in the food industry

Microbial Communities of the Upper Respiratory Tract and Otitis Media in Children

Streptococcus pneumoniae asymptomatically colonizes the upper respiratory tract of children and is a frequent cause of otitis media. Patterns of microbial colonization likely influence S. pneumoniae colonization and otitis media susceptibility. This study compared microbial communities in children with and without otitis media. Nasal swabs and clinical and demographic data were collected in a cross-sectional study of Philadelphia, PA, children (6 to 78 months) (n = 108) during the 2008-2009 winter respiratory virus season. Swabs were cultured for S. pneumoniae. DNA was extracted from the swabs; 16S rRNA gene hypervariable regions (V1 and V2) were PCR amplified and sequenced by Roche/454 Life Sciences pyrosequencing. Microbial communities were described using the Shannon diversity and evenness indices. Principal component analysis (PCA) was used to group microbial community taxa into four factors representing correlated taxa. Of 108 children, 47 (44%) were colonized by S. pneumoniae, and 25 (23%) were diagnosed with otitis media. Microbial communities with S. pneumoniae were significantly less diverse and less even. Two PCA factors were associated with a decreased risk of pneumococcal colonization and otitis media, as follows: one factor included potentially protective flora (Corynebacterium and Dolosigranulum), and the other factor included Propionibacterium, Lactococcus, and Staphylococcus. The remaining two PCA factors were associated with an increased risk of otitis media. One factor included Haemophilus, and the final factor included Actinomyces, Rothia, Neisseria, and Veillonella. Generally, these taxa are not considered otitis media pathogens but may be important in the causal pathway. Increased understanding of upper respiratory tract microbial communities will contribute to the development of otitis media treatment and prevention strategies

Oral biofilm models for mechanical plaque removal

In vitro plaque removal studies require biofilm models that resemble in vivo dental plaque. Here, we compare contact and non-contact removal of single and dual-species biofilms as well as of biofilms grown from human whole saliva in vitro using different biofilm models. Bacteria were adhered to a salivary pellicle for 2 h or grown after adhesion for 16 h, after which, their removal was evaluated. In a contact mode, no differences were observed between the manual, rotating, or sonic brushing; and removal was on average 39%, 84%, and 95% for Streptococcus mutans, Streptococcus oralis, and Actinomyces naeslundii, respectively, and 90% and 54% for the dual- and multi-species biofilms, respectively. However, in a non-contact mode, rotating and sonic brushes still removed considerable numbers of bacteria (24–40%), while the manual brush as a control (5–11%) did not. Single A. naeslundii and dual-species (A. naeslundii and S. oralis) biofilms were more difficult to remove after 16 h growth than after 2 h adhesion (on average, 62% and 93% for 16- and 2-h-old biofilms, respectively), while in contrast, biofilms grown from whole saliva were easier to remove (97% after 16 h and 54% after 2 h of growth). Considering the strong adhesion of dual-species biofilms and their easier more reproducible growth compared with biofilms grown from whole saliva, dual-species biofilms of A. naeslundii and S. oralis are suggested to be preferred for use in mechanical plaque removal studies in vitro