title: The Association of Outer Ear Commensal Bacteria with Otitis Media in Children

authors: White, Rebecca Katharine

subjects: Otitis media

abstract: Background:
Middle ear infections (otitis media) are a significant burden on children’s health. They are one of the more common childhood ailments and are responsible for about a third of children’s general practitioner visits. Three bacteria of the nasopharynx have been well documented as true pathogens of otitis media. These are Haemophilus influenzae, Streptococcus pneumoniae, and Moraxella catarrhalis. A newer bacterium, Alloiococcus otitidis, which is more difficult to culture than the initial three pathogens, was discovered in 1989 by Faden & Dryja. 
The pathogenicity of A. otitidis in otitis media is controversial, as is the association of two other bacteria, Turicella otitidis and Corynebacterium auris. These three bacteria are regarded as commensals of the outer ear canal, and how they move into the middle ear is subject to much debate. My hypothesis is that during a primary middle ear infection, commensal bacteria of the external ear canal may be able to move through the inflamed tympanic membrane and into the middle ear where they act as opportunistic pathogens. This may create a more persistent bacterial infection, with greater resistance to antibiotic therapies. 
Methods:
DNA was extracted directly from swabs of the outer ear canal and nasopharynx of children with otitis media and otitis-free controls, and from the middle ear exudates of the otitis media study group. This DNA was then amplified using primers specific for each of the organisms of interest. The presence of a specific PCR product, confirmed by DNA sequencing, indicated a positive result.
Swabs from each of the sites of interest were also grown on 10% Columbia blood agar, 10% Columbia chocolate agar and in BHI broth supplemented with foetal calf serum. These results gave further information on bacterial presence in New Zealand children beyond the six studied. 
Sau-PCR microbial community profiling was used to demonstrate the diversity of species present in the polymicrobial samples. DNA was restriction digested and the resulting banding patterns were compared between study sites, and study groups.
Results and Conclusions:
Moraxella species colonised the outer ear canal of control participants more regularly than the outer ear canal of otitis media patients (58% vs. 33%). This difference was found to be statistically significant (p= 0.0023). Differences between the colonisation of the outer ear canal and nasopharyngeal body sites were found at a statistically significant level in control participants with A. otitidis (p=0.0433), C. auris (p=0.0000), T. otitidis (p=0.0050) and Moraxella spp. (p=0.0005). Of the same body sites in otitis media patients, only the colonisation of C. auris (p=0.0002) and T. otitidis (p=0.0004) were found to be statistically significant. This shows that four of the six studied bacteria colonise both anatomical sites in otitis media patients in a similar manner (without statistical significance), as opposed to only two in the control participants.
Of the six bacterial species analysed using non-culture techniques, S. pneumoniae, H. influenzae and C. auris failed to be cultured. This may show that storage prior to analysis made samples less suitable for culture based research. A number of other species were cultured, with subsequent sequencing identifying them. This included species thought to be associated with a decreased risk of developing otitis media, found in both of the control participant nasopharynxes that were studied, and in only one nasopharynx from the otitis media group. A. otitidis was cultured regularly in outer ear samples, and one middle ear effusion.
Sau-PCR microbial profiling was an effective but crude indication of microbial diversity. Otitis media patient nasopharyngeal profiles resembled each other, with resemblance extending to the middle ear effusion profile. Control participant banding patterns within sites were similar, but this resemblance did not extend to the otitis media group profiles. Comparisons between the banding patterns from known bacterial species to those of unknown polymicrobial species were useful if the identifying species had a unique banding pattern, as with A. otitidis. Sau-PCR therefore proved useful in analysing the complexities that are polymicrobial samples, however identifications of these species by cloning proved unsuccessful.
Further research using all the techniques above with increased numbers of participants is necessary to substantiate results of this study. Information on antibiotic resistance, viral presence, and biofilm status could prove useful information in helping treat infection.

text: The Association of Outer Ear Commensal Bacteria with Otitis Media in Children Background:
Middle ear infections (otitis media) are a significant burden on children’s health. They are one of the more common childhood ailments and are responsible for about a third of children’s general practitioner visits. Three bacteria of the nasopharynx have been well documented as true pathogens of otitis media. These are Haemophilus influenzae, Streptococcus pneumoniae, and Moraxella catarrhalis. A newer bacterium, Alloiococcus otitidis, which is more difficult to culture than the initial three pathogens, was discovered in 1989 by Faden & Dryja. 
The pathogenicity of A. otitidis in otitis media is controversial, as is the association of two other bacteria, Turicella otitidis and Corynebacterium auris. These three bacteria are regarded as commensals of the outer ear canal, and how they move into the middle ear is subject to much debate. My hypothesis is that during a primary middle ear infection, commensal bacteria of the external ear canal may be able to move through the inflamed tympanic membrane and into the middle ear where they act as opportunistic pathogens. This may create a more persistent bacterial infection, with greater resistance to antibiotic therapies. 
Methods:
DNA was extracted directly from swabs of the outer ear canal and nasopharynx of children with otitis media and otitis-free controls, and from the middle ear exudates of the otitis media study group. This DNA was then amplified using primers specific for each of the organisms of interest. The presence of a specific PCR product, confirmed by DNA sequencing, indicated a positive result.
Swabs from each of the sites of interest were also grown on 10% Columbia blood agar, 10% Columbia chocolate agar and in BHI broth supplemented with foetal calf serum. These results gave further information on bacterial presence in New Zealand children beyond the six studied. 
Sau-PCR microbial community profiling was used to demonstrate the diversity of species present in the polymicrobial samples. DNA was restriction digested and the resulting banding patterns were compared between study sites, and study groups.
Results and Conclusions:
Moraxella species colonised the outer ear canal of control participants more regularly than the outer ear canal of otitis media patients (58% vs. 33%). This difference was found to be statistically significant (p= 0.0023). Differences between the colonisation of the outer ear canal and nasopharyngeal body sites were found at a statistically significant level in control participants with A. otitidis (p=0.0433), C. auris (p=0.0000), T. otitidis (p=0.0050) and Moraxella spp. (p=0.0005). Of the same body sites in otitis media patients, only the colonisation of C. auris (p=0.0002) and T. otitidis (p=0.0004) were found to be statistically significant. This shows that four of the six studied bacteria colonise both anatomical sites in otitis media patients in a similar manner (without statistical significance), as opposed to only two in the control participants.
Of the six bacterial species analysed using non-culture techniques, S. pneumoniae, H. influenzae and C. auris failed to be cultured. This may show that storage prior to analysis made samples less suitable for culture based research. A number of other species were cultured, with subsequent sequencing identifying them. This included species thought to be associated with a decreased risk of developing otitis media, found in both of the control participant nasopharynxes that were studied, and in only one nasopharynx from the otitis media group. A. otitidis was cultured regularly in outer ear samples, and one middle ear effusion.
Sau-PCR microbial profiling was an effective but crude indication of microbial diversity. Otitis media patient nasopharyngeal profiles resembled each other, with resemblance extending to the middle ear effusion profile. Control participant banding patterns within sites were similar, but this resemblance did not extend to the otitis media group profiles. Comparisons between the banding patterns from known bacterial species to those of unknown polymicrobial species were useful if the identifying species had a unique banding pattern, as with A. otitidis. Sau-PCR therefore proved useful in analysing the complexities that are polymicrobial samples, however identifications of these species by cloning proved unsuccessful.
Further research using all the techniques above with increased numbers of participants is necessary to substantiate results of this study. Information on antibiotic resistance, viral presence, and biofilm status could prove useful information in helping treat infection.

year: 2012

title: On the solution of the continuity equation for precipitating electrons in solar flares

authors: Emslie, A. Gordon

subjects: methods: analytical

abstract: Electrons accelerated in solar flares are injected into the surrounding plasma, where they are subjected to the influence of collisional (Coulomb) energy losses. Their evolution is modeled by a partial differential equation describing continuity of electron number. In a recent paper, Dobranskis & Zharkova claim to have found an "updated exact analytical solution" to this continuity equation. Their solution contains an additional term that drives an exponential decrease in electron density with depth, leading them to assert that the well-known solution derived by Brown, Syrovatskii & Shmeleva, and many others is invalid. We show that the solution of Dobranskis & Zharkova results from a fundamental error in the application of the method of characteristics and is hence incorrect. Further, their comparison of the "new" analytical solution with numerical solutions of the Fokker-Planck equation fails to lend support to their result. We conclude that Dobranskis & Zharkova's solution of the universally accepted and well-established continuity equation is incorrect, and that their criticism of the correct solution is unfounded. We also demonstrate the formal equivalence of the approaches of Syrovatskii & Shmeleva and Brown, with particular reference to the evolution of the electron flux and number density (both differential in energy) in a collisional thick target. We strongly urge use of these long-established, correct solutions in future works. © 2014. The American Astronomical Society. All rights reserved.

text: On the solution of the continuity equation for precipitating electrons in solar flares Electrons accelerated in solar flares are injected into the surrounding plasma, where they are subjected to the influence of collisional (Coulomb) energy losses. Their evolution is modeled by a partial differential equation describing continuity of electron number. In a recent paper, Dobranskis & Zharkova claim to have found an "updated exact analytical solution" to this continuity equation. Their solution contains an additional term that drives an exponential decrease in electron density with depth, leading them to assert that the well-known solution derived by Brown, Syrovatskii & Shmeleva, and many others is invalid. We show that the solution of Dobranskis & Zharkova results from a fundamental error in the application of the method of characteristics and is hence incorrect. Further, their comparison of the "new" analytical solution with numerical solutions of the Fokker-Planck equation fails to lend support to their result. We conclude that Dobranskis & Zharkova's solution of the universally accepted and well-established continuity equation is incorrect, and that their criticism of the correct solution is unfounded. We also demonstrate the formal equivalence of the approaches of Syrovatskii & Shmeleva and Brown, with particular reference to the evolution of the electron flux and number density (both differential in energy) in a collisional thick target. We strongly urge use of these long-established, correct solutions in future works. © 2014. The American Astronomical Society. All rights reserved.

year: 2014-09-01

title: In vitro cell culture to study microglial inflammation

authors: Newland, Julia

subjects: CNs

abstract: Inflammation of the central nervous system (CNS) characterised by release of cytokines such as tumour necrosis factor-alpha (TNF-a) and interleukin-1 (IL-1) is an event that is often detrimental to neuronal cells and is thought to contribute to the neuronal loss observed during neurodegeneration.
One objective of this study was to investigate the potential anti-inflammatory properties of kawakawa leaf extracts using in vitro model cell systems. Kawakawa leaf extracts anti-inflammatory properties have anecdotally been documented in traditional Maori medicinal practices (Rangoa). However, before this aspect could be carried out, experiments were undertaken to investigate the effects the kawakawa leaf extracts had on the growth rates of two mammalian cell lines (HeLa and THP-1 cells).
Interestingly, the growth assays showed that the extracts to have both a stimulatory and inhibitory effect on cell growth. Toxicity of cells can be categorised as either necrotic or apoptotic cell death. In this study, HeLa cells and THP-1 cells were incubated under standard growth conditions with various concentrations (10 to 100ug/ml) of Kawakawa leave extracts prepared at room temperature or 60OC.
Growth data as seen in figures 2 and 3 typically have a long lag phase before they start to reach exponential growth between days 4 and 5. The extract having the largest stimulatory effect on HeLa cells as shown by figure 6 was 25 ug/ml at 60OC and the largest inhibitory effect seen on HeLa cells was seen at 50 ug/ml RT shown by figure 6. The extract which has the largest stimulatory effect on THP-1 cells is 25 ug/ml 60OC as shown by figure 11 whereas the extract producing the largest inhibitory effect is 25 ug/ml RT.

Apoptotic and necrotic cell death mechanisms were examined by using in vitro biochemistry techniques. Assessments were largely made based on biochemical changes of HeLa cells and THP-1 cells post treatment of Kawakawa leave extracts.
Measurement of lactate dehydrogenase (LDH assays) in the growth media of cells that were shown to be growth inhibited during exposure to Kawakawa extracts indicated that the growth inhibition resulting from exposure to the extracts was not due to necrotic cell death processes. (ie that cells maintained their membrane integrity) When compared to the control, a lower amount of cytosolic LDH was seen in THP-1 cell lines. However, HeLa cell lines produced a high level of cytosolic LDH which may be due to mycoplasma contamination. On average, Kawakawa concentrations at 12.5ug/ml and 25ug/ml yielded less lysed cells percentage across both cell lines.
The demonstrated cell growth inhibition was unlikely to be due to necrosis and because of this it was decided to see if cell death was caused by mitochondrial processes that would further support apoptotic cell death. The biochemical assay MTT indirectly measures mitochondrial dehydrogenase activity in the presence of kawakawa leave extracts.
The MTT data illustrated mitochondrial impairment where HeLa cells exposed to Kawakawa 60OC concentrations 6.25 ug/ml and 12.5 ug/ml had the lowest average mitochondrial activity. THP-1 cells exposed to Kawakawa 60OC concentrations 12.5 ug/ml and 50 ug/ml had the lowest average mitochondrial activity.
The main results of this study include, the treatment of HeLa and THP-1 cells with kawakawa extracts demonstrated that cytotoxicity was not due to necrosis. The most interesting finding of this study is when kawakawa extracts inhibit cell growth there is no increase in LDH. This is suggestive that the cellular membrane remains intact when exposed to the extract.

text: In vitro cell culture to study microglial inflammation Inflammation of the central nervous system (CNS) characterised by release of cytokines such as tumour necrosis factor-alpha (TNF-a) and interleukin-1 (IL-1) is an event that is often detrimental to neuronal cells and is thought to contribute to the neuronal loss observed during neurodegeneration.
One objective of this study was to investigate the potential anti-inflammatory properties of kawakawa leaf extracts using in vitro model cell systems. Kawakawa leaf extracts anti-inflammatory properties have anecdotally been documented in traditional Maori medicinal practices (Rangoa). However, before this aspect could be carried out, experiments were undertaken to investigate the effects the kawakawa leaf extracts had on the growth rates of two mammalian cell lines (HeLa and THP-1 cells).
Interestingly, the growth assays showed that the extracts to have both a stimulatory and inhibitory effect on cell growth. Toxicity of cells can be categorised as either necrotic or apoptotic cell death. In this study, HeLa cells and THP-1 cells were incubated under standard growth conditions with various concentrations (10 to 100ug/ml) of Kawakawa leave extracts prepared at room temperature or 60OC.
Growth data as seen in figures 2 and 3 typically have a long lag phase before they start to reach exponential growth between days 4 and 5. The extract having the largest stimulatory effect on HeLa cells as shown by figure 6 was 25 ug/ml at 60OC and the largest inhibitory effect seen on HeLa cells was seen at 50 ug/ml RT shown by figure 6. The extract which has the largest stimulatory effect on THP-1 cells is 25 ug/ml 60OC as shown by figure 11 whereas the extract producing the largest inhibitory effect is 25 ug/ml RT.

Apoptotic and necrotic cell death mechanisms were examined by using in vitro biochemistry techniques. Assessments were largely made based on biochemical changes of HeLa cells and THP-1 cells post treatment of Kawakawa leave extracts.
Measurement of lactate dehydrogenase (LDH assays) in the growth media of cells that were shown to be growth inhibited during exposure to Kawakawa extracts indicated that the growth inhibition resulting from exposure to the extracts was not due to necrotic cell death processes. (ie that cells maintained their membrane integrity) When compared to the control, a lower amount of cytosolic LDH was seen in THP-1 cell lines. However, HeLa cell lines produced a high level of cytosolic LDH which may be due to mycoplasma contamination. On average, Kawakawa concentrations at 12.5ug/ml and 25ug/ml yielded less lysed cells percentage across both cell lines.
The demonstrated cell growth inhibition was unlikely to be due to necrosis and because of this it was decided to see if cell death was caused by mitochondrial processes that would further support apoptotic cell death. The biochemical assay MTT indirectly measures mitochondrial dehydrogenase activity in the presence of kawakawa leave extracts.
The MTT data illustrated mitochondrial impairment where HeLa cells exposed to Kawakawa 60OC concentrations 6.25 ug/ml and 12.5 ug/ml had the lowest average mitochondrial activity. THP-1 cells exposed to Kawakawa 60OC concentrations 12.5 ug/ml and 50 ug/ml had the lowest average mitochondrial activity.
The main results of this study include, the treatment of HeLa and THP-1 cells with kawakawa extracts demonstrated that cytotoxicity was not due to necrosis. The most interesting finding of this study is when kawakawa extracts inhibit cell growth there is no increase in LDH. This is suggestive that the cellular membrane remains intact when exposed to the extract.

year: 2016