Anaerobic Culture Techniques Research Articles

Sterile Subdural Empyema Mimicking Subdural Hemorrhage in Pediatric patient with History of Trauma

Intracranial subdural empyema (SDE) is a life-threatening infection. Surgery procedure for culture the empyema is essential to diagnosing and treating the patient. Nevertheless, in rare cases, culture can not identify the causative organism because microorganisms are not grown. A ten-year-old girl with the main complaint of weakness in the right hand since two days before admission. She fell off the bicycle two months before admission, and her head hit the ground. Nevertheless, the patient's condition was stable at that time, and there was no history of loss of consciousness. A head CT scan with and without contrast found a mass suggesting chronic subdural hemorrhage. The patient undergoes surgery with burr hole drainage for chronic SDH but intraoperatively found subdural empyema. SDE could be an uncommon, harmful, pus-filled accumulation of tissue between the outer dura and inner arachnoid mater. Meningitis is the most frequent cause of SDE in children and newborns. Despite the fact that operative cultures can identify the responsible organisms, 7% to 52% of instances result in no growth, which is primarily due to the use of antibiotics in the past or inappropriate anaerobic culture technique. Intracranial subdural empyema (SDE) is an emergency and life-threatening infection. SDE can also mimic chronic SDH and make it difficult to diagnose. Emergency surgery is required for a better outcome for the patient. Culture empyema from collecting pus is not always found in the microorganism pathogen, which could be in sterile conditions.

Porphyromonas gulae infection in canines, pet owners and veterinarians in China: an epidemiological study and risk factor analysis

Porphyromonas gulae is a clinically prevalent, anaerobic, oral bacteria in canines, that may be a causative agent of canine periodontal disease, and a potential threat to human oral health. Research on P. gulae pathogenicity in canines, their owners, and veterinarians is lacking in China. This study aimed to determine the isolation and detection rates of P. gulae in gingival crevicular fluid (GCF) samples from 101 canines in Beijing, using anaerobic culture techniques and 16S rRNA gene sequencing. The main risk factors for the transmission of P. gulae from canines to humans were also analyzed through analyzing the statistical data on risk factor variables from 103 canine owners and 60 veterinarians in Beijing who tested positive for P. gulae detection in GCF samples. The isolation and detection rates of P. gulae in canines were 31.5% (29/92) and 92.1% (93/101), respectively, compared with detection rates of 24.3% (25/103) in canine owners, 43.3% (26/60) in veterinarians, and 52.0% (13/25) in dentists. The degree of contact with canines (P = 0.001, P < 0.01) and smoking (P = 0.021, P < 0.05) were significant risk factors for P. gulae detection in owners. Moreover, the degree of contact during ultrasonic scaling (P = 0.065, 0.05 < P < 0.1) was the most important risk factor for the positive detection of P. gulae in veterinarians. These findings suggest that P. gulae may colonize the human oral cavity through intimate contact with canines or participation in dental ultrasonic scaling operations.Graphical

Etiology and antimicrobial susceptibility profiles of anaerobic bacteria isolated from clinical samples in a university hospital in Madrid, Spain

BackgroundThe anaerobic infection management is usually based on empirical treatment because anaerobic culture techniques take a long time due to their fastidious nature. The aim of this study was to analyze the etiological profile of severe anaerobic infections and AST data from clinical anaerobic bacteria isolated in a tertiary hospital in Madrid (Spain). Material and methodsA consecutive study was carried out over 19 months in Ramón y Cajal Universitary Hospital, Madrid. Clinical samples were processed in appropriate anaerobic media and incubated using Anoxomat system. Identification was performed by MALDI-TOF. AST were determined with gradient diffusion method using EUCAST (penicillin, co-amoxiclav, imipenem, clindamycine and metronidazole) or CLSI (cefoxitin) breakpoints. ResultsDuring the period of study, 503 anaerobic microorganisms isolated from 424 clinical samples were included. Twenty-six percent of the cultures were monomicrobial, while 70.0% also contained aerobic bacteria. The most common source of infection was abscesses (26%), while blood infections represented the 11%. Anaerobic gram-negative bacilli were predominant (41%), being Bacteroides fragilis (13%) the most prevalent overall; anaerobic gram-positive bacilli represented 35%, anaerobic gram-positive cocci 19% and anaerobic gram-negative cocci 5%. Metronidazole and imipenem were the most effective agents tested against anaerobic bacteria, while clindamycin presented higher resistance rates. ConclusionAntimicrobial susceptibility surveillance of anaerobic bacteria should be performed to monitor changes in resistance patterns and to be able to optimize empiric antimicrobial treatment. Reliable species identification and quick reporting of results would guide clinicians to select the optimal antimicrobial therapy.

Design and Development of a Model to Study the Effect of Supplemental Oxygen on the Cystic Fibrosis Airway Microbiome.

Airway microbial communities are thought to play an important role in the progression of cystic fibrosis (CF) and other chronic pulmonary diseases. Microbes have traditionally been classified based on their ability to use or tolerate oxygen. Supplemental oxygen is a common medical therapy administered to people with cystic fibrosis (pwCF); however, existing studies on oxygen and the airway microbiome have focused on how hypoxia (low oxygen) rather than hyperoxia (high oxygen) affects the predominantly aerobic and facultative anaerobic lung microbial communities. To address this critical knowledge gap, this protocol was developed using an artificial sputum medium that mimics the composition of sputum from pwCF. The use of filter sterilization, which yields a transparent medium, allows optical methods to follow the growth of single-celled microbes in suspension cultures. To create hyperoxic conditions, this model system takes advantage of established anaerobic culturing techniques to study hyperoxic conditions; instead of removing oxygen, oxygen is added to cultures by daily sparging of serum bottles with a mixture of compressed oxygen and air. Sputum from 50 pwCF underwent daily sparging for a 72-h period to verify the ability of this model to maintain differential oxygen conditions. Shotgun metagenomic sequencing was performed on cultured and uncultured sputum samples from 11 pwCF to verify the ability of this medium to support the growth of commensal and pathogenic microbes commonly found in cystic fibrosis sputum. Growth curves were obtained from 112 isolates obtained from pwCF to verify the ability of this artificial sputum medium to support the growth of common cystic fibrosis pathogens. We find that this model can culture a wide variety of pathogens and commensals in CF sputum, recovers a community highly similar to uncultured sputum under normoxic conditions, and creates different culture phenotypes under varying oxygen conditions. This new approach might lead to a better understanding of unanticipated effects induced by the use of oxygen in pwCF on airway microbial communities and common respiratory pathogens.

Comparative microbiological study of methods of using Metrogyl Dent gel in the treatment of periodontitis

Summary The common drug Metrogyl Denta can be used in different ways in the treatment of periodontitis, in particular, in the form of applications or using high-frequency ultrasound. The article is devoted to the study of the effect of various methods of using Metrogyl Dent gel on the microbiota of periodontal pockets in periodontitis. In 40 patients with a diagnosis of moderate periodontitis, the treatment complex included a combined effect of high-frequency ultrasound and an antibacterial preparation of Metrogyl Dent gel (ultraphonophoresis of Metrogil Dent gel) or Metrogil Dent was used as applications (20 people in the compared groups). The contents of the pathological periodontal pockets were studied twice before treatment (after removing dental deposits), and also after 10 days of treatment. Microbiological examination to identify periodontal pathogenic bacterial microbiota was carried out using the anaerobic culture technique. Ultraphonophoresis of Metrogyl Denta gel has a more pronounced antibacterial effect in comparison with the applications of this drug. The combined effect of high-frequency ultrasound and Metrogyl Denta gel makes it possible to create a drug depot in the periodontal tissues.

Effect of passive ultrasonic activation on microorganisms in primary root canal infection: a randomized clinical trial.

Objective:This clinical study sought to evaluate the effectiveness of passive ultrasonic activation (PUA) in eliminating microorganisms in primary endodontic infection (PEI) after instrumentation of root canals using microbiological culture and checkerboard DNA-DNA hybridization.Methodology:Twenty root canals with PEI and apical periodontitis were selected. The root canals were instrumented and then randomly divided into 2 groups, according to the irrigation method: PUA and conventional needle irrigation (CNI). Microbiological samples were collected before instrumentation (S1), after instrumentation (S2) and after irrigation with 17% EDTA (S3). The samples were subjected to anaerobic culture technique and checkerboard DNA-DNA hybridization analysis.Results:A statistically significant difference was found between CNI (23.56%) and PUA (98.37%) regarding the median percentage values for culturable bacteria reduction (p<0.05). In the initial samples, the most frequently detected species was S. constellatus (50%), and after root canal treatment was E. faecalis (50%).Conclusion:Both treatments significantly decreased the number of bacterial species compared with the initial sample. However, no statistical difference in the total microbial load between PUA and CNI groups was detected. The number of cultivable anaerobic bacteria reduced significantly using PUA, and the bacterial composition and number of bacterial species after using either CNI or PUA was similar.

Rifaximin prevents ethanol-induced liver injury in obese KK-Ay mice through modulation of small intestinal microbiota signature.

Exacerbation of alcoholic hepatitis (AH) with comorbid metabolic syndrome is an emerging clinical problem, where microbiota plays a profound role in the pathogenesis. Here, we investigated the effect of rifaximin (RFX) on liver injury following chronic-binge ethanol (EtOH) administration in KK-Ay mice, a rodent model of metabolic syndrome. Female, 8-wk-old KK-Ay mice were fed Lieber-DeCarli diet (5% EtOH) for 10 days, following a single EtOH gavage (4 g/kg body wt). Some mice were given RFX (0.1 g/L, in liquid diet) orally. Small intestinal contents were collected from mice without binge. Intestinal microbiota was quantified using aerobic and anaerobic culturing techniques and further analyzed by 16S rRNA sequencing in detail. EtOH feeding/binge caused hepatic steatosis, oxidative stress, and induction of inflammatory cytokines in KK-Ay mice, which were markedly prevented by RFX treatment. Hepatic mRNA levels for cluster of differentiation 14, Toll-like receptor (TLR) 4, TLR2, and NADPH oxidase 2 were increased following EtOH feeding/binge, and administration of RFX completely suppressed their increase. The net amount of small intestinal bacteria was increased over threefold after chronic EtOH feeding as expected; however, RFX did not prevent this net increase. Intriguingly, the profile of small intestinal microbiota was dramatically changed following EtOH feeding in the order level, where the Erysipelotrichales predominated in the relative abundance. In sharp contrast, RFX drastically blunted the EtOH-induced increases in the Erysipelotrichales almost completely, with increased proportion of the Bacteroidales. In conclusion, RFX prevents AH through modulation of small intestinal microbiota/innate immune responses in obese KK-Ay mice.NEW & NOTEWORTHY Here we demonstrated that rifaximin (RFX) prevents chronic-binge ethanol (EtOH)-induced steatohepatitis in KK-Ay mice. Chronic EtOH feeding caused small intestinal bacterial overgrowth, with drastic alteration in the microbiota profile predominating the order Erysipelotrichales. RFX minimized this EtOH induction in Erysipelotrichales with substitutive increases in Bacteroidales. RFX also prevented EtOH-induced increases in portal lipopolysaccharide, and hepatic cluster of differentiation 14, toll-like receptor (TLR) 2, and TLR4 mRNA levels, suggesting the potential involvement of microbiota-related innate immune responses.

Isolation, biochemical characterization and phylogeny of a cellulose-degrading ruminal bacterium

Background: The isolation of cellulolytic bacteria, which hydrolyze cellulose to cellobiose and glucose, can provide useful information about rumen diversity. Objective: To identify and characterize a microorganism capable of hydrolyzing cellulose, isolated from a cow rumen. Methods: Anaerobic culture techniques were used for isolating cellulose-degrading rumen bacteria. Congo red staining was used to evaluate β-D-glucanase activity, and carbohydrate fermentation pattern was obtained with the kit API 50CHB/E. DNA extraction was performed and the 16S rDNA gene was amplified using 8F (5'-AGA GTT TGA TCC TGG CTC AG-3'), and 1492R (5' GGT TAC CTT GTT ACG ACT T 3') primers. The phylogenetic tree was reconstructed with the algorithm of maximum parsimony (bootstrap 5000), and 16S rDNA sequence was deposited in the NCBI database (accession number: KM094184). Results: The isolated bacterium showed cellulolytic activity detected with Congo red; besides, glycerol, ribose, xylose, sucrose, galactose and glucose were fermented by this bacterium. However, biochemical tests did not identify the bacteria because no match was found at database of API WEB Software. The phylogenetic inference indicated that this bacterium belongs to Shigella genus, with 98% maximal identity respect to the other taxonomic species. Conclusions: Phylogenetic analysis of 16S rRNA genes showed that the rumen isolated bacterium was a member of the genus Shigella, which, under mesophilic conditions, is an interesting candidate for obtaining oligosaccharides from lignocellulosic biomass.Keywords: cellulolytic, fermentation, monophyletic, rumen, Shigella. Resumen Antecedentes: El aislamiento de las bacterias celulolíticas, que hidrolizan la celulosa a celobiosa y glucosa, proporciona valiosa información sobre la diversidad del rumen,. Objetivo: Identificar y caracterizar un microorganismo capaz de hidrolizar celulosa, aislado de un rumen vacuno. Métodos: Se utilizaron técnicas de cultivo anaeróbico para aislar bacterias ruminales que degradan celulosa. La tinción con rojo Congo se usó para evaluar la actividad β-D-glucanasa y el patrón de fermentación de carbohidratos se obtuvo con el kit API 50CHB/E. Se realizó la extracción de DNA y se amplificó el gen de 16S rDNA utilizando los cebadores 8F (5'-AGA GTT TGA TCC TGG CTC AG-3'), y 1492R (5' GGT TAC CTT GTT ACG ACT T 3'). El árbol filogenético se reconstruyó con el algoritmo de máxima parsimonia (replicas 5000) y la secuencia 16S rDNA se depositó en la base de datos del NCBI (número de acceso: KM094184). Resultados: La bacteria aislada mostró actividad celulolítica detectada con la tinción de rojo Congo; además, esta bacteria fermenta glicerol, ribosa, xilosa, sacarosa, galactosa y glucosa. Sin embargo, las pruebas bioquímicas no permitieron identificar a la bacteria aislada, por no encontrar coincidencias en la base de datos del software API WEB. La inferencia filogenética indicó que esta bacteria pertenece al género Shigella, con 98% de identidad máxima respecto a las otras especies taxonómicas. Conclusiones: El análisis filogenético del gen 16S rRNA mostró que la bacteria aislada del rumen es un miembro del género Shigella que, en condiciones mesófilas, es un candidato interesante para obtener oligosacáridos a partir de biomasa lignocelulósica.Palabras clave: celulolítica, fermentación, monofilético, rumen, Shigella. ResumoAntecedentes: As bactérias celulolíticas hidrolizam a celulosa em celobiose e glicose, e o isolamento desses microrganismos fornece informações sobre a diversidade do rúmen. Objetivo: Identificar e caracterizar um microorganismo isolada do rúmen de uma vaca, com capacidade para hidrolisar a celulose. Métodos: Técnicas de cultura anaeróbica foram utilizadas para isolar bactérias ruminais que degradam a celulose. A atividade β-D-glucanase foi mostrada utilizando mancha de vermelho Congo, e o padrão de fermentação de carbohidratos foi obtida com o kit API 50CHB/E. A extracção foi realizada de DNA e amplificou-se os genes 16S rDNA utilizando os iniciadores 8F (AGA GTT TGA 5'-TCC TGG CTC AG-3'), e 1492R (5' CTT GGT TAC GTT ACG TCA T 3'). A árvore filogenética foi reconstruída com o algoritmo de máxima parcimônia (réplicas 5000). A sequência de rDNA 16S foi depositada no banco de dados do NCBI (número de acesso: KM094184). Resultados: O isolado mostrou uma atividade celulolítica com coloração vermelho Congo; además esta bactéria fermentação de glicerol, ribose, xilose, sacarose, galactose e glicose. No entanto, com as provas bioquímicas não se identificou a bactérias isolada, já que não se encontrou na base de dados do software API WEB. A inferência filogenética indicou que esta bactéria pertence ao género Shigella, com 98% de identidade de máximo respeito para outras espécies taxonômicas. Conclusão: A análise filogenética do gene 16S rRNA mostrou as bactérias isoladas do ambiente ruminal como um membro do género Shigella, que condições mesofilicas é um candidato atraente para obter oligossacarídeos da biomassa lignocelulósica.Palavras-chave: celulolítica, fermentação, monofilético, rúmen, Shigella.

Reduced Time to Positive Cutibacterium acnes Culture Utilizing a Novel Incubation Technique: A Retrospective Cohort Study

IntroductionCutibacterium acnes (C. acnes) is a common pathogen in postoperative shoulder infections. The purpose of this study was to evaluate the time to positive cultures for C. acnes and compare our experience before and after implementation of a regulated anaerobic chamber system. We hypothesized that this would reduce the time to identify positive cultures.MethodsThis was a retrospective review of 34 patients with cultures obtained from the shoulder that were positive for C. acnes. The time until positive result was evaluated before and after implementation of a regulated anaerobic incubation chamber.ResultsFollowing implementation of the regulated anaerobic incubation chamber, the time until C. acnes culture growth significantly decreased from 6.5 days (range 3–10 days) to 4.9 days (range 2.75–10 days) (mean difference: 1.6 days, 95% confidence interval: 1.06–2.66 days; P = .002). True infections had a significantly shorter time to positive culture compared to contaminants (5.5 vs 6.8 days, respectively, P = .003). Increased number of positive culture specimens correlated with a shorter time to positivity (Spearman rank = −0.58, P = .007).ConclusionImproved anaerobic culture protocols and techniques may lead to greater accuracy and earlier diagnosis and initiation of treatment of postoperative shoulder infections.

From the inside out: the gut microbiome and inflammatory bowel disease

Inflammatory bowel disease (IBD) is a chronic inflammatory condition of the gastrointestinal (GI) tract with two main clinical types: Crohn's disease, which can affect any part of the GI tract, and ulcerative colitis (UC), which is limited to the colon. While early research focused primarily on the immune dysregulation and genetic susceptibilities associated with IBD, recent groundbreaking technological advances have allowed the investigation of additional factors, including diet and microbial exposures, in the onset and severity of disease. Advances in high-throughput microbial sequencing, anaerobic bacterial culturing techniques and generating germ-free mouse models have revolutionized our understanding of the microbial species associated with inflammation. While the long-standing clinical efficacy of antibiotics or surgery for Crohn's disease highlights the potential contribution of the socalled ‘IBD microbiome’ to inflammation, recent seminal studies revealed the impact of IBD-derived gut microbiota on host mucosal and systemic inflammation. This mechanistic understanding of how our ‘microbial organ’ functionally impacts both mucosal and systemic inflammatory pathways will help drive novel diagnostic and therapeutic approaches for IBD.

VIRAL ETIOLOGY OF RECURRENT URINARY TRACT INFECTIONS

Introduction. Recurrent urinary tract infection is an actual problem of modern urology. Objective. Complex investigation of urinary tract infections including viral etiology for chronic recurrent cystitis in women Materials and methods. The study included 31 women with recurrent infection of urinary tract. Inclusion criteria were the presence of lower urinary tract symptoms caused by infection, severe recurrent course, the lack of anatomical and functional disorders of the urinary tract, the absence of bacterial pathogens during the study, taking into account the culture of aerobic and anaerobic culturing techniques. Results. The analysis of the clinical manifestations, the dominant in the study group were pain and urgency to urinate at 100% and 90% of women surveyed, respectively, and less frequent urination were recorded in 16.1% of patients. In general clinical examination of urine in all cases identified leukocyturia and 90% of the hematuria. By using a polymerase chain reaction (PCR) in midstream urine of all examined was verified 10 types of human papilloma virus (HPV) with the predominance of 16 and 18 types . Considering the presence of recurrent infectious and inflammatory processes of the urinary tract, cystoscopy with bladder biopsy was performed for all patients. When histomorphological biopsies of all patients surveyed noted the presence of the specific characteristics of HPV: papillary hyperplasia with squamous koilocytosis, pale cytoplasm and shrunken kernels. When analyzing the results of PCR biopsy data corresponded with the results of PCR in midstream urine in all biopsies was detected HPV. Conclusions. Human papillomavirus infection may be involved in the development of viral cystitis. In the etiological structure of viral cystitis, both highly oncogenic and low oncogenic HPV types can act.

Spectrum of anaerobes encountered in surgical infections in a tertiary care hospital in Mangalore, coastal Karnataka: A prospective study

Context: Anaerobes are significant pathogens as well as normal flora in a variety of body sites. As the conventional anaerobic culture techniques are laborious, time-consuming and relatively expensive, the interest in anaerobes started narrowing down in the recent past. Objective: This study is aimed at the isolation of bacteria encountered in the surgical infections with an emphasis on anaerobes and to create an awareness among the clinicians regarding the potential role of these silent pathogens Materials and Methods: The present prospective study that was conducted for a period of 1 year, employed 393 specimens collected from various surgical infections. All the samples were subjected for the isolation of both aerobes and anaerobes. Results: Out of 393 specimens, 193 anaerobes and 311 aerobes were obtained. Majority of the cases (226) exhibited polymicrobial etiology. Among the anaerobes, more frequently isolated organisms were nonsporing gram-negative bacilli, such as Bacteroides fragilis group, Prevotella-Porphyromonas group and Fusobacterium, which constitute 68.91% of the cases. Conclusions: This study emphasizes the need to employ anaerobic culture techniques routinely in microbiology laboratories and to create an awareness among the clinicians regarding the potential role of anaerobes.

Correlation between Volume of Apical Periodontitis Determined by Cone-beam Computed Tomography Analysis and Endotoxin Levels Found in Primary Root Canal Infection

IntroductionThis clinical study was conducted to correlate the levels of endotoxins and bacterial counts found in primary endodontic infection with the volume of periapical bone destruction determined by cone-beam computed tomography (CBCT) analysis. Moreover, the levels of bacteria and endotoxins were correlated with the development of clinical features. MethodsTwenty-four root canals with primary endodontic disease and apical periodontitis were selected. Clinical features such as pain on palpation, pain on percussion, and previous episode of pain were recorded. The volume (cubic millimeters) of periapical bone destruction was determined by CBCT analysis. Endotoxins and bacterial samplings were collected by using sterile/apyrogenic paper points. Endotoxins were quantified by using limulus amebocyte lysate assay (KQCL test), and bacterial count (colony-forming units [CFU]/mL) was determined by using anaerobic culture techniques. Data were analyzed by Pearson correlation and multiple logistic regression (P < .05). ResultsEndotoxins and bacteria were detected in 100% of the root canal samples (24 of 24), with median values of 10.92 endotoxin units (EU)/mL (1.75–128 EU/mL) and 7.5 × 105 CFU/mL (3.20 × 105–8.16 × 106 CFU/mL), respectively. The median volume of bone destruction determined by CBCT analysis was 100 mm3 (10–450 mm3). The multiple regression analysis revealed a positive correlation between higher levels of endotoxins present in root canal infection and larger volume of bone destruction (P < .05). Moreover, higher levels of endotoxins were also correlated with the presence of previous pain (P < .05). ConclusionsOur findings revealed that the levels of endotoxins found in root canal infection are related to the volume of periapical bone destruction determined by CBCT analysis. Moreover, the levels of endotoxin are related to the presence of previous pain.

Isolation and Characterization of Antimony-Reducing Bacteria from Sediments Collected in the Vicinity of an Antimony Factory

To date, there is very little information available on the oxidation and reduction of antimony by bacteria. An antimonate-respiring bacterium was isolated from sediment samples collected in the vicinity of an antimony oxide-producing factory in Korea. This bacterium was isolated by application of the Hungate standard anaerobic culture technique. Temporarily named strain JUK-1, it was found to be a rod-shaped bacterium and occurred individually or in pairs. Antimonate (pentavalent antimony) was reduced to antimonite (trivalent antimony) in the presence of JUK-1 in an anoxic minimal medium containing 5 mM antimonate and 10 mM acetate. The organism grew optimally at an initial pH of 7.7 and a temperature of 30°C. A part of the antimonite which was produced in medium precipitated as a bio-mineral containing approximately 50.0% antimony, 33.5% oxygen, 12.7% carbon, 2.0% sodium and 1.9% magnesium by weight. Based on the phylogenetics of 16S rRNA gene sequence and DNA G+C content, JUK-1 appears to be a new strain of the Sinorhizobium genus. The results suggest that bacteria may play a significant role in changing the redox state of antimony in the environment.

Changes in oral microflora after full-mouth tooth extraction: a prospective cohort study.

The aim of the study was to evaluate the effect of full-mouth tooth extraction on the oral microflora, with emphasis on the presence and load of Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis. Adult patients (n=30), with moderate to advanced periodontitis and scheduled for full-mouth tooth extraction, were consecutively selected. Prior to and 1 and 3months after full-mouth tooth extraction saliva, tongue, buccal and gingival mucosa and subgingival plaque/prosthesis samples were obtained. Aerobic and anaerobic culture techniques and quantitative real-time polymerase chain reaction (qPCR) were employed for the detection of oral pathogens. Full-mouth tooth extraction resulted in reduction below detection level of A.actinomycetemcomitans and P.gingivalis in 15 of 16 and 8 of 16 previously positive patients using culture techniques and qPCR, respectively. Those patients remaining qPCR positive showed a significant reduction in load of these bacteria. Full-mouth tooth extraction significantly changes the oral microflora. These changes include reduction of A.actinomycetemcomitans and P.gingivalis, frequently to levels below detection threshold. In some patients, A.actinomycetemcomitans and P.gingivalis can persist in the edentulous oral cavity up to 3months after full-mouth tooth extraction.

101 Microbial communities and interactions in chronic lung infection in cystic fibrosis patients

Introduction and Aims: Although a number of studies have characterized airway microbiota composition in healthy and CF adult populations, limited data is available for the paediatric population. The aim of this study was to define, using culture based methods, the composition of the airway microbiota in age-matched healthy and CF paediatric groups. Methods: Induced sputum samples (CF) and cough swabs (CF, age-matched healthy volunteers) were collected and processed using both aerobic and strict anaerobic bacteriological culture techniques. Bacteria within the samples were detected by plating on selective agars and identified by PCR and sequencing of 16S ribosomal RNA genes. Results: Samples were collected from 21 healthy volunteers (mean (SD) age 11.0 (3.5) yrs; 13M:8F) and from 12 children with CF (mean (SD) age 12.8 (3.2) yrs; 6M:6F) when clinically stable. Bacteria were detected in all samples from CF patients and in 18/21 (86%) healthy volunteers. Aerobic bacteria from genera including Streptococcus, Staphylococcus, Pseudomonas and Haemophilus were detected in both cohorts with Pseudomonas [CF, n = 2/12 (17%); control, n = 1/21 (5%)] and Haemophilus [CF, n = 7/12 (58%); control n = 4/21 (19%)] present in a greater number of CF patients compared to controls. Anaerobic bacteria from genera including Prevotella, Veillonella, and Actinomyces were detected in both cohorts. Conclusion: Aerobic and anaerobic bacteria have been detected by culture in the lungs of children with CF and healthy children. Pseudomonas and Haemophilus, recognised as pathogens in CF patients, were more prevalent in CF patients compared to controls. Funded by US-Ireland Project Partnership Grant.

100 Comparison of airway microbiota composition in healthy children and children with CF

Introduction and Aims: Although a number of studies have characterized airway microbiota composition in healthy and CF adult populations, limited data is available for the paediatric population. The aim of this study was to define, using culture based methods, the composition of the airway microbiota in age-matched healthy and CF paediatric groups. Methods: Induced sputum samples (CF) and cough swabs (CF, age-matched healthy volunteers) were collected and processed using both aerobic and strict anaerobic bacteriological culture techniques. Bacteria within the samples were detected by plating on selective agars and identified by PCR and sequencing of 16S ribosomal RNA genes. Results: Samples were collected from 21 healthy volunteers (mean (SD) age 11.0 (3.5) yrs; 13M:8F) and from 12 children with CF (mean (SD) age 12.8 (3.2) yrs; 6M:6F) when clinically stable. Bacteria were detected in all samples from CF patients and in 18/21 (86%) healthy volunteers. Aerobic bacteria from genera including Streptococcus, Staphylococcus, Pseudomonas and Haemophilus were detected in both cohorts with Pseudomonas [CF, n = 2/12 (17%); control, n = 1/21 (5%)] and Haemophilus [CF, n = 7/12 (58%); control n = 4/21 (19%)] present in a greater number of CF patients compared to controls. Anaerobic bacteria from genera including Prevotella, Veillonella, and Actinomyces were detected in both cohorts. Conclusion: Aerobic and anaerobic bacteria have been detected by culture in the lungs of children with CF and healthy children. Pseudomonas and Haemophilus, recognised as pathogens in CF patients, were more prevalent in CF patients compared to controls. Funded by US-Ireland Project Partnership Grant.

Analysis of Bacterial Flora Associated with Peri-implantitis Using Obligate Anaerobic Culture Technique and 16S rDNA Gene Sequence

To analyze and characterize the predominant bacterial flora associated with peri-implantitis by using culture techniques under obligate anaerobic conditions and 16S rDNA gene sequences. Subgingival bacterial specimens were taken from 30 patients: control (n = 15), consisting of patients with only healthy implants; and test (n = 15), consisting of patients with peri-implantitis. In both groups, subgingival bacterial specimens were taken from the deepest sites. An anaerobic glove box system was used to cultivate bacterial strains. The bacterial strains were identified by 16S rDNA genebased polymerase chain reaction and comparison of the gene sequences. Peri-implantitis sites had approximately 10-fold higher mean colony forming units (per milliliter) than healthy implant sites. A total of 69 different bacterial species were identified in the peri-implantitis sites and 53 in the healthy implant sites. The predominant bacterial species in the peri-implantitis sites were Eubacterium nodatum, E. brachy, E. saphenum, Filifactor alocis, Slackia exigua, Parascardovia denticolens, Prevotella intermedia, Fusobacterium nucleatum, Porphyromonas gingivalis, Centipeda periodontii, and Parvimonas micra. The predominant bacteria in healthy implant sites apart from Streptococcus were Pseudoramibacter alactolyticus, Veillonella species, Actinomyces israelii, Actinomyces species, Propionibacterium acnes, and Parvimonas micra. These results suggest that the environment in the depths of the sulcus showing peri-implantitis is well suited for growth of obligate anaerobic bacteria. The present study demonstrated that the sulcus around oral implants with peri-implantitis harbors high levels of asaccharolytic anaerobic gram-positive rods (AAGPRs) such as E. nodatum, E. brachy, E. saphenum, Filifactor alocis, Slackia exigua, and gram-negative anaerobic rods, suggesting that conventional periodontopathic bacteria are not the only periodontal pathogens active in peri-implantitis, and that AAGPRs may also play an important role.

Response of rhizobacterial communities in watermelon to infection with cucumber green mottle mosaic virus as revealed by cultivation-dependent RISA

We investigated the rhizobacterial densities and community structure in watermelon rhizosphere under the infection of cucumber green mottle mosaic virus (CGMMV) by artificial inoculation. Rhizobacterial densities and communities were analysed from healthy and infected plants under aerobic and anaerobic culture techniques. The highest total number of aerobic rhizobacteria was counted to be 2.7 × 108 colony forming units per gram (CFU · g−1) and anaerobic rhizobacteria was to be 3.2 × 106 CFU · g−1, in healthy and infected plants, respectively. Cultivation-dependent ribosomal intergenic spacer analysis (RISA) was employed for further analysis on the rhizobacterial community structure. By incorporating the relative abundance of amplicons, the per cent similarity was determined by the similarity coefficients based only upon the absence or presence of DNA bands. The cluster analysis of RISA showed that the community structure of aerobic rhizobacteria exhibited 60% similarity between healthy and infected plant. The highest community structure similarity (50% similarity) of anaerobic rhizobacteria occurred between before planting and infected plant.

The Profile of Inflammatory Cytokines in Gingival Crevicular Fluid around Healthy Osseointegrated Implants

Regardless of gingival health and subgingival microbiology, production of cytokines within peri-implant tissues may be different from that of teeth. The objective of this study was to describe the peri-implant levels of pro-inflammatory cytokines and subgingival microbiology in clinically healthy sites. Subgingival plaque and gingival crevicular fluid (GCF) were obtained from 28 clinically healthy implants and 26 teeth selected from 24 individuals. Microbial composition was determined by selective anaerobic culture techniques. Pro-inflammatory cytokines were quantified by flow cytometry analysis of GCF. The concentration of cytokines between implants and teeth were compared with the independent t-test. The concentration of cytokines was higher in GCF from healthy implants than in teeth. The profile of cytokines was characteristic of an innate immune response. A more frequent detection of periodontopathic bacteria was observed in teeth than implants. Cultivable levels of periodontopathic bacteria were similar between implants and teeth. Despite gingival tissue health and scarce plaque accumulation, the profile of inflammatory cytokines in implant crevicular fluid was distinctive of an innate immune response and in higher concentration than in teeth. Other than bacterial stimulus, intrinsic factors related to implants may account for more cytokine production than teeth.