S21 Culture Independent Identification Of Bacterial Communities In The Respiratory Tract Of Patients With Copd, Healthy Non-smokers And Healthy Smokers

Introduction and aims: The role bacteria play in the development and progression of Chronic Obstructive Pulmonary Disease (COPD) is unclear. We used culture-independent methods to describe differences and/or similarities in microbial communities in the lower airways of patients with COPD, healthy non-smokers and smokers.

Methods: Bronchial wash samples were collected from patients with COPD (GOLD 1–3; n = 18), healthy non-smokers (HV; n = 11) and healthy smokers (HS; n = 8). Samples were processed using the Illumina MiSeq platform. The Shannon-Wiener Index (SW) of diversity, lung obstruction (FEV1/FVC ratio) and ordination by Non-Metric Multidimensional Scaling (NMDS) on Bray-Curtis dissimilarity indices were analysed to evaluate how samples were related. Principal component analysis (PCA) was performed to assess the effect specific taxa had within each cohort. Characteristics of each cohort are shown in Table 1.

Results: There was no difference in taxa richness between cohorts (range: 69–71; p = 0.954). Diversity (SW Index) was significantly lower in COPD samples compared to samples from HV and HS (p = 0.009 and p = 0.033, respectively). There was no significant difference between HV and HS (p = 0.186). The FEV1/FVC ratio was significantly lower for COPD compared to HV (p = 9*10–8) and HS (p = 2*10–6), respectively. NMDS analysis showed that communities belonging to either of the healthy groups were more similar to each other than they were to samples belonging to the COPD group. PCA analysis showed that members of Streptococcus sp. and Haemophilus sp. had the largest effect on the variance explained in COPD. In HS, Haemophilus sp., Fusobaterium sp., Actinomyces sp., Prevotella sp. and Veillonella sp. had the largest effect on the variance explained, while in HV Neisseria sp., Porphyromonas sp., Actinomyces sp., Atopobium sp., Prevotella and Veillonella sp. had the largest effect on the variance explained.

Conclusions: The study demonstrates that microbial communities in the lower airways of patients with COPD are significantly different from that seen in healthy comparison groups. Patients with COPD had lower microbial diversity than either of the healthy comparison groups, higher relative abundance of members of Streptococcus sp. and lower relative abundance of a number of key anaerobes.Characteristics

CULTURE AND CULTURE INDEPENDANT IDENTIFICATION OF BACTERIAL COMMUNITIES IN THE CYSTIC FIBROSIS RESPIRATORY TRACT

Introduction and Aims: The identification of complex chronic polymicrobial infections, such as those observed in the cystic fibrosis (CF) airways, are often a diagnostic challenge. Few studies have compared culture-dependent methods with molecular identification making it hard to describe bacterial communities in a comprehensive manner. The aim of the study is to compare four different methods with respect to their similarities and differences in detection of bacteria. Methods: We compared41 sputum samples fromroutine clinical-culture, extended-culture (aerobic and anaerobic), and molecular identification such as Roche 454-FLX Titanium and T-RFLP to assess concurrence between methodologies in detecting bacteria. The agreement between methodologies in detecting either absence or presence of bacterial taxa was assessed by Kappa (κ) statistics. Results: The majority of bacterial taxa identified by culture were also identified with molecular analysis. In total 2, 60, 25, and 179 different bacterial taxa were identified with clinical-culture, extended-culture, T-RFLP and 454-FLX respectively. Clinical-culture, extended-culture and T-RFLP were poor predictors of species richness when compared to 454-FLX (p < 0.0001). Agreement between methods for detecting Pseudomonas sp. and Burkholderia sp. was good with κ ≥ 0.7 [p < 0.0001] and κ ≥ 0.9 [p < 0.0001] respectively. Detection of anaerobic bacteria, such as Prevotella sp. and Veillonella sp., was moderate between extended-culture and 454-FLX with κ = 0.461 [p < 0.0001] and κ = 0.311 [p = 0.032] respectively, and good between T-RFLP and 454-FLX with κ = 0.577 [p < 0.0001] and κ = 0.808 [p < 0.0001] respectively. Agreement between methods for other main bacterial taxa, such as Staphylcoccus sp. and Streptococcus sp., was poor with only a moderate agreement for detection of Streptococcus sp. observed between T-RFLP and 454-FLX (κ = 0.221 [p = 0.024]). Conclusions: This study demonstrates the increased sensitivity culture-independent microbial identification such as the 454-FLX have over clinical-culture, extended-culture and T-RFLP methodologies. The extended-culture detected majority of the most prevalent bacterial taxa associated with chronic colonisation of the CF airways which were also detected by culture-independent methodologies. However, agreement between methods in detecting number of potentially relevant bacteria is largely lacking.

MICROBIAL COMMUNITY COMPOSITION IN THE LUNGS OF PATIENTS WITH CYSTIC FIBROSIS AND NON-CF BRONCHIECTASIS

Introduction and Aims: Persistent bacterial infection is a major cause of morbidity and mortality in patients with both Cystic Fibrosis (CF) and non-CF Bronchiectasis (non-CFBX). Numerous studies have shown that CF and non-CFBX airways are colonised by a complex microbiota. However, many bacteria are difficult, if not impossible, to culture by conventional laboratory techniques. Therefore, molecular detection techniques offer a more comprehensive view of bacterial diversity within clinical specimens. The objective of this study was to characterise and compare bacterial diversity and relative abundance in patients with CF and non-CFBX during exacerbation and when clinically stable.

Methods: Sputum samples were collected from CF (n=50 samples) and non-CFBX (n=52 samples) patients at the start and end of treatment for an infective exacerbation and when clinically stable. Pyrosequencing was used to assess the microbial diversity and relative genera (or the closest possibly taxonomic order) abundance within the samples. Each sequence read was defined based on 3% difference.

Results: High-throughput pyrosequencing allowed a sensitive and detailed examination of microbial community composition. Rich microbial communities were apparent within both CF (171 species-level phylotypes per genus) and non-CFBX airways (144 species-level phylotypes per genus). Relative species distribution within those two environments was considerably different; however, relatively few genera formed a core of microorganisms, representing approximately 90% of all sequences, which dominated both environments. Relative abundance based on observed operational taxonomic units demonstrated that the most abundant bacteria in CF were Pseudomonas (28%), Burkholderia (22%), Streptococcus (13%), family Pseudomonadaceae (8%) and Prevotella (6%). In contrast, the most commonly detected operational taxonomic units in non-CFBX were Haemophilus (22%), Streptococcus (14%), other (unassigned taxa) (11%), Pseudomonas (10%), Veillonella (7%) and Prevotella (6%).

Conclusions: These results suggest that distinctive microbial communities are associated with infection and/or colonisation in patients with both CF and non-CFBX. Although relatively high species richness was observed within the two environments, each was dominated by different core taxa. This suggests that differences in the lung environment of these two diseases may affect adaptability of the relevant bacterial taxa.

THE CULTURE MICROBIOME IN THE LUNGS OF PATIENTS WITH COPD

Introduction and Aims: Previous studies have shown that the lungs of Cystic Fibrosis (CF) and bronchiectasis (BE, not caused by CF) patients are colonised by a range of aerobic and anaerobic bacteria. As bacteria are also implicated in the pathogenesis and progression of chronic obstructive pulmonary disease (COPD), this study aimed to determine the culture microbiome of the COPD airways.

Methods: Samples were collected from 13 stable COPD patients during routine bronchoscopy. Bronchial washings were taken at a single location in the right middle lobe by flushing and removing 30 ml of sterile saline. Samples were cultured under strict anaerobic conditions with bacteria detected by plating on both selective and non-selective agar media and quantified by total viable count (TVC). Identification of the cultured bacteria was performed by amplification and subsequent sequencing of the 16sRNA gene.

Results: Mean FEV1 was 1.36 (range 0.84–2.26, mean per cent predicted FEV1, 54%), and the mean ratio (FEV1/FVC) was 51%. Bacteria were detected in 12/13 samples (92%) with bacteria from the genera Streptococcus [12/13 samples, 92%; mean (range) TVC 9.62×105 cfu/ml (1.50×103–1.42×107)] and Haemophilus [4/13 samples, 31%; mean (range) 6.40×104 cfu/ml (2.20×103–1.60×105)] most frequently detected. Anaerobic bacteria primarily from the genera Prevotella [8/13 samples, 62%; mean (range) TVC 1.12×104 cfu/ml (1.30×103–4.20×104)] and Veillonella [5/13 samples, 38%; mean (range) TVC 1.29×105 cfu/ml (4.20×103–3.60×105)] were also detected. Pseudomonas and Moraxella were not detected in any samples.

Conclusions: Our results show that bacteria from the genera Streptococcus, Haemophilus, Prevotella and Veillonella are frequently present the airways of patients suffering from COPD. Taking account of the dilutional effect of the bronchial wash procedure and extrapolating to allow comparison with sputum data in our laboratory for CF and BE, the relative load of bacteria from the genera Streptococcus, Prevotella and Veillonella is similar in these three airway diseases. The potential role of these bacteria in the progression and pathogenesis of COPD requires further investigation.

Antimicrobial resistance in the respiratory microbiota of people with cystic fibrosis

Cystic fibrosis is characterised by chronic polymicrobial infection and inflammation in the airways of patients. Antibiotic treatment regimens, targeting recognised pathogens, have substantially contributed to increased life expectancy of patients with this disease. Although the emergence of antimicrobial resistance and selection of highly antibiotic-resistant bacterial strains is of major concern, the clinical relevance in cystic fibrosis is yet to be defined. Resistance has been identified in recognised cystic fibrosis pathogens and in other bacteria (eg, Prevotella and Streptococcus spp) detected in the airway microbiota, but their role in the pathophysiology of infection and inflammation in chronic lung disease is unclear. Increased antibiotic resistance in cystic fibrosis might be attributed to a range of complex factors including horizontal gene transfer, hypoxia, and biofilm formation. Strategies to manage antimicrobial resistance consist of new antibiotics or localised delivery of antimicrobial agents, iron sequestration, inhibition of quorum-sensing, and resistome analysis. Determination of the contributions of every bacterial species to lung health or disease in cystic fibrosis might also have an important role in the management of antibiotic resistance. 

Community dynamics and the lower airway microbiota in stable chronic obstructive pulmonary disease, smokers and healthy non-smokers

RATIONALE: The role bacteria play in the progression of COPD has increasingly been highlighted in recent years. However, the microbial community complexity in the lower airways of patients with COPD is poorly characterised.

OBJECTIVES: To compare the lower airway microbiota in patients with COPD, smokers and non-smokers.

METHODS: Bronchial wash samples from adults with COPD (n=18), smokers with no airways disease (n=8) and healthy individuals (n=11) were analysed by extended-culture and culture-independent Illumina MiSeq sequencing. We determined aerobic and anaerobic microbiota load and evaluated differences in bacteria associated with the three cohorts. Culture-independent analysis was used to determine differences in microbiota between comparison groups including taxonomic richness, diversity, relative abundance, 'core' microbiota and co-occurrence.

MEASUREMENT AND MAIN RESULTS: Extended-culture showed no difference in total load of aerobic and anaerobic bacteria between the three cohorts. Culture-independent analysis revealed that the prevalence of members of Pseudomonas spp. was greater in the lower airways of patients with COPD; however, the majority of the sequence reads for this taxa were attributed to three patients. Furthermore, members of Bacteroidetes, such as Prevotella spp., were observed to be greater in the 'healthy' comparison groups. Community diversity (α and β) was significantly less in COPD compared with healthy groups. Co-occurrence of bacterial taxa and the observation of a putative 'core' community within the lower airways were also observed.

CONCLUSIONS: Microbial community composition in the lower airways of patients with COPD is significantly different to that found in smokers and non-smokers, indicating that a component of the disease is associated with changes in microbiological status.

Morphologie évolutive et fonctionnelle des hémichordés

L’embranchement Hemichordata regroupe les classes Enteropneusta et Pterobranchia. Hemichordata constitue, avec l’embranchement Echinodermata, le groupe-frère des chordés. Les entéropneustes sont des organismes vermiformes solitaires qui vivent sous ou à la surface du substrat et s’alimentent généralement par déposivorie, alors que les ptérobranches sont des organismes coloniaux filtreurs habitant dans un réseau de tubes appelé coenecium. Ce mémoire présente trois études dont le point commun est l’utilisation des hémichordés actuels pour répondre à des questions concernant l’évolution des hémichordés, des chordés, et du super-embranchement qui les regroupe, Deuterostomia. Notre première étude démontre que les fentes pharyngiennes, l’organe pré-oral cilié (POCO) et le pharynx de l’entéropneuste Protoglossus graveolens sont utilisés pour l’alimentation par filtration. Le système de filtration de P. graveolens permet la capture de particules jusqu’à 1.3 um, à un débit de 4.05 mm.s-1, pour une demande énergétique de 0.009 uW. Les similarités structurales et fonctionnelles avec le système de filtration des céphalochordés suggèrent que la filtration pharyngienne est ancestrale aux deutérostomes. Lors de notre deuxième étude, nous avons exploré l’hypothèse selon laquelle le POCO des entéropneustes, une structure ciliée pré-buccale au rôle possiblement chémorécepteur, serait homologue au « wheel organ » des céphalochordés et à l’adénohypophyse des vertébrés. Pour cela, nous avons déterminé par immunohistochimie l’expression de Pit-1, un facteur de transcription spécifique à ces deux structures, chez l’entéropneuste Saccoglossus pusillus. Pit-1 est exprimé dans des cellules sensorielles du POCO, mais aussi dans des cellules épithéliales distribuées dans le proboscis, collet et tronc. Ce patron d’expression ne permet pas de confirmer ou rejeter l’homologie du POCO et de l’adénohypophyse des vertébrés. Lors de notre troisième étude, nous avons caractérisé l’ultrastructure du coenecium des ptérobranches Cephalodiscus hodgsoni, Cephalodiscus nigrescens et Cephalodiscus densus par microscopie électronique à transmisison et à balayage. Cephalodiscus est le groupe frère de Graptolithina, un groupe qui inclut les graptolithes éteints ainsi que les ptérobranches du genre Rhabdopleura. Nous avons décrit les types de fibrilles de collagène présents, leur taille et leur organisation, ainsi que l’organisation globale du coenecium. Nous avons ainsi démontré la présence chez Cephalodiscus d’une organisation similaire au paracortex, pseudocortex et eucortex des graptolithes. La présence chez Cephalodiscus de ce type d’organisation suggère que le cortex est ancestral à la classe Pterobranchia. Ces trois études illustrent plusieurs axes importants de la recherche sur les hémichordés, qui en intégrant des données morphologiques, fonctionnelles et moléculaires permet de reconstruire certains évènements clés de l’évolution des deutérostomes.

Studies on a sea anemone ,anthopleura nicrescens(verrill) from the south west coast of India

The present study was undertaken with a view to understanding some aspects of the morphology, bionomics and physiology of the sea anemone A.nigrescens. This species was selected for the study since it is abundantly available along the coast throughout the year and can successfully reared in the laboratory without much effort. The present study deals with the following aspects of A.nigrescens. description of the species, habitat, food and feeding, digestion, studies on salinity tolerance, studies on oxygen consumption, studies on tolerance to desiccation, asexual reproduction and regeneration.

Exopolysaccharides produced by Bifidobacterium longum IPLA E44 and Bifidobacterium animalis subsp lactis IPLA R1 modify the composition and metabolic activity of human faecal microbiota in pH-controlled batch cultures

Exopolysaccharides (EPS) isolated from two Bifidobacterium strains, one of human intestinal origin (Bifidobacterium longum subsp. longum IPLA E44) and the other from dairy origin (Bifidobacterium animalis subsp. lactis IPLA R1), were subjected to in vitro chemically simulated gastrointestinal digestion. which showed the absence of degradation of both polymers in these conditions. Polymers were then used as carbon sources in pH-controlled faecal batch cultures and compared with the non-prebiotic carbohydrate glucose and the prebiotic inulin to determine changes in the composition of faecal bacteria. A set of eight fluorescent in situ hybridisation oligonucleotide probes targeting 16S rRNA sequences was used to quantify specific groups of microorganisms. Growth of the opportunistic pathogen Clostridium histolyticum occurred with all carbohydrates tested similarly to that found in negative control cultures without added carbohydrate and was mainly attributed to the culture conditions used rather than enhancement of growth by these substrates. Polymers E44 and RI stimulated growth of Lactobacillus/Enterococcus, Bifidobacterium, and Bacteroides/Prevotella in a similar way to that seen with inulin. The EPS RI also promoted growth of the Atopobium cluster during the first 24 h of fermentation. An increase in acetic and lactic acids was found during early stages of fermentation (first 10-24 h) correlating with increases of Lactobacillus, Bifidobacterium, and Atopobium. Propionic acid concentrations increased in old cultures, which was coincident with the enrichment of Clostridium cluster IX in cultures with EPS RI and with the increases in Bacteroides in cultures with both microbial EPS (RI and E44) and inulin. The lowest acetic to propionic acid ratio was obtained for EPS E44. None of the carbohydrates tested supported the growth of microorganisms from Clostridium clusters XIVa+b and IV, results that correlate with the poor butyrate production in the presence of EPS. Thus, EPS synthesized by bifidobacteria from dairy and intestinal origins can modulate the intestinal microbiota in vitro, promoting changes in some numerically and metabolically relevant microbial populations and shifts in the production of short chain fatty acids. (C) 2009 Elsevier B.V. All rights reserved.

A double-blind placebo-controlled study to establish the bifidogenic dose of inulin in healthy humans

Objective: To evaluate the bifidogenic efficacy of two inulin doses in healthy human adults. Design: A double-blind, placebo-controlled, crossover human study. Setting: Food Microbial Sciences Unit, The University of Reading, Reading, UK. Subjects: Thirty healthy volunteers, 15 men, 15 women ( age range 19-35). Interventions: Subjects consumed a chocolate drink containing placebo ( maltodextrin, 8 g/day), 5 g/day inulin and 8 g/day inulin for a 2-week treatment period. Each treatment was followed by a 1-week washout at the end of which volunteers progressed to the next treatment. Faecal samples were obtained at the start of the study ( baseline) and at the end of each treatment and washout period. Fluorescent in situ hybridization was used to monitor populations of Bifidobacterium genus, Bacteroides - Prevotella, Lactobacillus - Enterococcus and Clostridium perfringens - histolyticum subgroup. Results: Bifidobacterial levels increased significantly upon ingestion of both the low ( 9.78 +/- 0.29 log(10) cells/g faeces, P < 0.05) and the high inulin dose ( 9.79 +/- 0.38 log(10) cells/g faeces, P < 0.05) compared to placebo ( 9.64 +/- 0.23 log(10) cells/g faeces). Conclusions: Both inulin doses exhibited a bifidogenic effect but a higher volunteer percentage responded to the high dose. A dose response effect was not observed but the magnitude of increase in bifidobacteria levels depended on their initial numbers. The higher the initial concentrations the smaller was the increase upon ingestion of the active treatments. Sponsorship: Financial support for the completion of this project was provided by Sensus ( Roosendaal, The Netherlands).

In vitro fermentation of oat and barley derived beta-glucans by human faecal microbiota

Fermentation of beta-glucan fractions from barley [average molecular mass (MM), of 243, 172, and 137 kDa] and oats (average MM of 230 and 150 kDa) by the human faecal microbiota was investigated. Fractions were supplemented to pH-controlled anaerobic batch culture fermenters inoculated with human faecal samples from three donors, in triplicate, for each substrate. Microbiota changes were monitored by fluorescent in situ hybridization; groups enumerated were: Bifidobacterium genus, Bacteroides and Prevotella group, Clostridium histolyticum subgroup, Ruminococcus-Eubacterium-Clostridium (REC) cluster, Lactobacillus-Enterococcus group, Atopobium cluster, and clostridial cluster IX. Short-chain fatty acids and lactic acid were measured by HPLC. The C. histolyticum subgroup increased significantly in all vessels and clostridial cluster IX maintained high populations with all fractions. The Bacteroides-Prevotella group increased with all but the 243-kDa barley and 230-kDa oat substrates. In general beta-glucans displayed no apparent prebiotic potential. The SCFA profile (51 : 32 : 17; acetate : propionate : butyrate) was considered propionate-rich. In a further study a beta-glucan oligosaccharide fraction was produced with a degree of polymerization of 3-4. This fraction was supplemented to small-scale faecal batch cultures and gave significant increases in the Lactobacillus-Enterococcus group; however, the prebiotic potential of this fraction was marginal compared with that of inulin.

In vitro fermentation of linear and alpha-1,2-branched dextrans by the human fecal microbiota

The role of structure and molecular weight in fermentation selectivity in linear α-1,6 dextrans and dextrans with α-1,2 branching was investigated. Fermentation by gut bacteria was determined in anaerobic, pH-controlled fecal batch cultures after 36 h. Inulin (1%, wt/vol), which is a known prebiotic, was used as a control. Samples were obtained at 0, 10, 24, and 36 h of fermentation for bacterial enumeration by fluorescent in situ hybridization and short-chain fatty acid analyses. The gas production of the substrate fermentation was investigated in non-pH-controlled, fecal batch culture tubes after 36 h. Linear and branched 1-kDa dextrans produced significant increases in Bifidobacterium populations. The degree of α-1,2 branching did not influence the Bifidobacterium populations; however, α-1,2 branching increased the dietary fiber content, implying a decrease in digestibility. Other measured bacteria were unaffected by the test substrates except for the Bacteroides-Prevotella group, the growth levels of which were increased on inulin and 6- and 70-kDa dextrans, and the Faecalibacterium prausnitzii group, the growth levels of which were decreased on inulin and 1-kDa dextrans. A considerable increase in short-chain fatty acid concentration was measured following the fermentation of all dextrans and inulin. Gas production rates were similar among all dextrans tested but were significantly slower than that for inulin. The linear 1-kDa dextran produced lower total gas and shorter time to attain maximal gas production compared to those of the 70-kDa dextran (branched) and inulin. These findings indicate that dextrans induce a selective effect on the gut flora, short-chain fatty acids, and gas production depending on their length.

Microbial utilization and selectivity of pectin fractions with various structures

To evaluate the fermentation properties of oligosaccharides derived from pectins and their parent polysaccharides, a 5-ml-working-volume, pH- and temperature-controlled fermentor was tested. Six pectic oligosaccharides representing specific substructures found within pectins were prepared. These consisted of oligogalacturonides (average degrees of polymerization [DP] of 5 and 9), methylated oligogalacturonides (average DP of 5), oligorhamnogalacturonides (average DP of 10 as a disaccharide unit of galacturonic acid and rhamnose), oligogalactosides (average DP of 5), and oligoarabinosides (average DP of 6). The influence of these carbohydrates on the human fecal microbiota was evaluated. Use of neutral sugar fractions resulted in an increase in Bifidobacterium populations and gave higher organic acid yields. The Bacteroides-Prevotella group significantly increased on all oligosaccharides except oligogalacturonides with an average DP of 5. The most selective substrates for bifidobacteria were arabinan, galactan, oligoarabinosides, and oligogalactosides.

Nd : YAG laser clinical assisted in class II furcation treatment

The Nd:YAG laser efficacy associated with conventional treatment for bacterial reduction has been investigated throughout literature. The purpose of this study was to evaluate the bacterial reduction after Nd:YAG laser irradiation associated with scaling and root planning in class II furcation defects in patients with chronic periodontitis. Thirty-four furcation lesions were selected from 17 subjects. The control group received conventional treatment, and the experimental group received the same treatment followed by Nd:YAG laser irradiation (100 mJ/pulse; 15 Hz; 1.5 W, 60 s, 141.5 J/cm(2)). Both treatments resulted in improvements of most clinical parameters. A significant reduction of colony forming unit (CFU) of total bacteria number was observed in both groups. The highest reduction was noted in the experimental group immediately after the treatment. The number of dark pigmented bacteria and the percentage of patients with Porphyromonas gingivalis, Prevotella intermedia, and Actinobacillus actinomycetemcomitans reduced immediately after the treatment and returned to values close to the initial ones 6 weeks after the baseline for both groups. The Nd:YAG laser associated with conventional treatment promoted significant bacterial reduction in class II furcation immediately after irradiation, although this reduction was not observed 6 weeks after the baseline.

Microbiological profile of untreated subjects with localized aggressive periodontitis

Aim The microbial profile of localized aggressive periodontitis (LAgP) has not yet been determined. Therefore, the aim of this study was to evaluate the subgingival microbial composition of LAgP. Material and Methods One hundred and twenty subjects with LAgP (n=15), generalized aggressive periodontitis (GAgP, n=25), chronic periodontitis (ChP, n=30) or periodontal health (PH, n=50) underwent clinical and microbiological assessment. Nine subgingival plaque samples were collected from each subject and analysed for their content of 38 bacterial species using checkerboard DNA-DNA hybridization. Results Red complex and some orange complex species are the most numerous and prevalent periodontal pathogens in LAgP. The proportions of Aggregatibacter actinomycetemcomitans were elevated in shallow and intermediate pockets of LAgP subjects in comparison with those with GAgP or ChP, but not in deep sites. This species also showed a negative correlation with age and with the proportions of red complex pathogens. The host-compatible Actinomyces species were reduced in LAgP. Conclusion A. actinomycetemcomitans seems to be associated with the onset of LAgP, and Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Campylobacter gracilis, Eubacterium nodatum and Prevotella intermedia play an important role in disease progression. Successful treatment of LAgP would involve a reduction in these pathogens and an increase in the Actinomyces species.