16S rDNA sequencing analyzes differences in intestinal flora of human immunodeficiency virus (HIV) patients and association with immune activation.

To clarify the influence of HIV on the intestinal flora and the interrelationship with CD4 T cells, the present study collected stool specimens from 33 HIV patients and 28 healthy subjects to compare the differences in the intestinal flora and CD4 T cells in a 16S rDNA-sequencing approach. ELISA was used to detect the expressions of interleukin 2 (IL-2), IL-8, and tumor necrosis factor-α (TNF-α). Meanwhile, correlation analysis with the different bacterial populations in each group was carried out. The results revealed that Alpha diversity indices of the intestinal flora of HIV patients were markedly lower than that of the healthy group (p < 0.05). The top five bacterial species in the HIV group were Bacteroides (23.453%), Prevotella (19.237%), Fusobacterium (12.408%), Lachnospira (3.811%), and Escherichia-Shigella (3.126%). Spearman correlation analysis results indicated that Fusobacterium_mortiferum, Fusobacterium, and Gammaproteobacteria were positively correlated with TNF-α (p < 0.05), whereas Ruminococcaceae, Bacteroidales was negatively correlated with TNF-α (p < 0.05). Additionally, Agathobacter was positively correlated with contents of IL-2 and IL-8 (p < 0.05), whereas Prevotellaceae, and Prevotella were negatively correlated with IL-8 content (p < 0.05). Furthermore, the top five strains in the CD4 high group (≥350/mm3) included Bacteroides (23.286%), Prevotella (21.943%), Fusobacterium (10.479%), Lachnospira (4.465%), and un_f_Lachnospiraceae (2.786%). Taken together, the present study identified that Fusobacterium and Escherichia-Shigella were specific and highly abundant in the HIV group and a correlation between the different bacterial flora and the contents of IL-2, IL-8, and TNF-α was revealed.

Quantification of Multiple Bacteria in Calcified Structural Valvular Heart Disease.

Genome studies of heart valve tissue (HVT) in patients with structural valvular heart disease (sVHD) and acute infective endocarditis (aIE) showed polymicrobial infections. Subject of this study is the quantification of bacterial DNA in HVT of sVHD in comparison to aIE. It will be examined whether the bacterial DNA concentration can be used as surrogate marker to differentiate chronic and acute infections. DNA was isolated from HVT of 100 patients with sVHD and 23 microbiologically positively tested patients with aIE. Selected pathogens (Cutibacterium acnes, Enterococcus faecalis, Enterococcus faecium, Staphylococcus aureus, Streptococcus pyogenes, Streptococcus agalactiae, Clostridium difficile, and Klebsiella pneumoniae) were quantified using TaqMan-qPCR. Polymicrobial infiltration of HVT was investigated by immunohistologic methods. Of 100 sVHD patients, 94 tested positive for bacteria by 16S-rDNA and 72 by TaqMan-qPCR. In 29% of the sVHD cohort and in 70% of the aIE cohort, a coinfection with more than 2 bacteria was observed as indication of a polymicrobial infection. The most common pathogens in the sVHD patients were C. acnes (59%; 5-4074 pg/mL), E. faecalis (16%, 174-2781 pg/mL), and S. aureus (15%, 8-105 pg/mL). The DNA concentration of E. faecalis (P = 0.0285) and S. aureus (P = 0.0149) is significantly lower in the sVHD cohort than in the aIE cohort. sVHD is associated with bacterial infection and infiltration of the HVT in a majority of cases. TaqMan-qPCR is a valid instrument for the specific detection of bacteria in HVT and allows discrimination between sVHD and aIE for E. faecalis and S. aureus.

The hindgut microbiota of praying mantids is highly variable and includes both prey-associated and host-specific microbes.

Praying mantids are predators that consume a wide variety of insects. While the gut microbiome of carnivorous mammals is distinct from that of omnivores and herbivores, the role of the gut microbiome among predatory insects is relatively understudied. Praying mantids are the closest known relatives to termites and cockroaches, which are known for their diverse gut microbiota. However, little is known about the mantid gut microbiota or their importance to host health. In this work, we report the results of a 16S rRNA gene-based study of gut microbiome composition in adults and late-instar larvae of three mantid species. We found that the praying mantis gut microbiome exhibits substantial variation in bacterial diversity and community composition. The hindgut of praying mantids were often dominated by microbes that are present in low abundance or not found in the guts of their insect prey. Future studies will explore the role of these microbes in the digestion of the dietary substrates and/or the degradation of toxins produced by their insect prey.

Subgingival microbiome of rheumatoid arthritis patients in relation to their disease status and periodontal health.

OBJECTIVE: Rheumatoid arthritis (RA) and periodontitis are chronic inflammatory diseases that share common risk factors. However, the bidirectional relationship between RA and periodontal disease is not fully understood. This study was undertaken to describe the bacterial component of the subgingival microbiome in RA patients and to relate this to RA disease activity and periodontal status. METHODS: Patients with chronic established RA (N = 78) were periodontally examined and their subgingival plaque samples were collected; their clinical and laboratory data on RA status and medication were obtained from medical records. Bacterial DNA was quantified by universal 16S rDNA qPCR, and Porphyromonas gingivalis by species-specific qPCR. For microbiome assessment, 16S rDNA amplicon sequencing was performed. RESULTS: Active RA was diagnosed in 58% of the patients and periodontitis in 82% (mild: 9%, moderate: 55%, severe: 18%). P. gingivalis was present in 14% of the samples. Different levels of gingival bleeding, periodontal probing depth, RA disease status, prednisolone use and smoking were associated with significantly different microbiome compositions. Two subgingival microbial community types were discerned. CONCLUSION: In RA patients with active disease, anti-inflammatory medication as part of RA therapy was associated with better oral health status and a healthier subgingival microbiome compared to that of RA patients in remission, especially those in remission who were current smokers. RA patients in remission with current smoking status may particularly benefit from a systematic periodontal treatment program. The potential role of microbial community types in patient stratification and personalized therapy should be assessed in longitudinal studies.

New host record of five Flavobacterium species associated with tropical fresh water farmed fishes from North India

Abstract Yellow pigmented, filamentous, Gram-negative bacteria belonging to genus Flavobacterium are commonly associated with infections in stressed fish. In this study, inter-species diversity of Flavobacterium was studied in apparently healthy freshwater farmed fishes. For this, ninety one yellow pigmented bacteria were isolated from skin and gill samples (n = 38) of three farmed fish species i.e. Labeo rohita, Catla catla and Cyprinus carpio. Among them, only twelve bacterial isolates (13.18%) were identified as Flavobacterium spp. on the basis of morphological, biochemical tests, partial 16S rDNA gene sequencing and phylogenetic analysis. On the basis of 16S rDNA gene sequencing, all the 12 isolates were 97.6-100% similar to six different formally described species of genus Flavobacterium. The 16S rDNA based phylogenetic analysis grouped these strains into six different clades. Of the 12 isolates, six strains (Fl9S1-6) grouped with F. suncheonense, two strains (Fl6I2, Fl6I3) with F. indicum and the rest four strains (Fl1A1, Fl2G1, Fl3H1 and Fl10T1) clustered with F. aquaticum, F. granuli, F. hercynium and F. terrae, respectively. None of these species except, F. hercynium were previously reported from fish. All the isolated Flavobacterium species possessed the ability of adhesion and biofilm formation to colonize the external surface of healthy fish. The present study is the first record of tropical freshwater farmed fishes as hosts to five environmentally associated species of the Flavobacterium.

The potential role of 'Candidatus Microthrix parvicella' in phosphorus removal during sludge bulking in two full-scale enhanced biological phosphorus removal plants.

We investigated the bacterial community compositions and phosphorus removal performance under sludge bulking and non-bulking conditions in two biological wastewater treatment systems (conventional A²/O (anaerobic/anoxic/aerobic) and inverted A²/O (anoxic/anaerobic/aerobic) processes) receiving the same raw wastewater. Sludge bulking resulted in significant shift in bacterial compositions from Proteobacteria dominance to Actinobacteria dominance, characterized by the significant presence of filamentous 'Candidatus Microthrix parvicella'. Quantitative real-time polymerase chain reaction (PCR) analysis revealed that the relative abundance of 'Candidatus Accumulibacter phosphatis', a key polyphosphate-accumulating organism responsible for phosphorus removal, with respect to 16s rRNA genes of total bacteria was 0.8 and 0.7%, respectively, for the conventional and inverted A²/O systems when sludge bulking occurred, which increased to 8.2 and 12.3% during the non-bulking period. However, the total phosphorus removal performance during the bulking period (2-week average: 97 ± 1 and 96 ± 1%, respectively) was not adversely affected comparable to that during the non-bulking period (2-week average: 96 ± 1 and 96 ± 1%, respectively). Neisser staining revealed the presence of large polyphosphate granules in 'Candidatus Microthrix parvicella', suggesting that this microbial group might have been responsible for phosphorus removal during the sludge bulking period when 'Candidatus Accumulibacter phosphatis' was excluded from the systems.

Modeling the impact of antibiotic exposure on human microbiota.

Human-associated microbial communities play important roles in health and disease. Antibiotic administration is arguably one of the most important modifiable determinants of the composition of the human microbiota. However, quantitatively modeling antibiotic use to account for its impact on microbial community dynamics presents a challenge. We used antibiotic therapy of chronic lung infection in persons with cystic fibrosis as a model system to assess the influence of key variables of therapy on measures of microbial community perturbation. We constructed multivariate linear mixed models with bacterial community diversity as the outcome measure and various scales of antibiotic weighting as predictors, while controlling for other variables. Antibiotic weighting consisted of three components: (i) dosing duration; (ii) timing of administration relative to sample collection; and (iii) antibiotic type and route of administration. Antibiotic weighting based on total dose and proximity to the time of sampling was most predictive of bacterial community change. Using this model to control for antibiotic use enabled the identification of other significant independent predictors of microbial community diversity such as dominant taxon, disease stage, and gender. Quantitative modeling of antibiotic use is critical in understanding the relationships between human microbiota and disease treatment and progression.

Phylogeography of cylindrospermopsin and paralytic shellfish toxin-producing nostocales cyanobacteria from mediterranean europe (Spain).

Planktonic Nostocales cyanobacteria represent a challenge for microbiological research because of the wide range of cyanotoxins that they synthesize and their invasive behavior, which is presumably enhanced by global warming. To gain insight into the phylogeography of potentially toxic Nostocales from Mediterranean Europe, 31 strains of Anabaena (Anabaena crassa, A. lemmermannii, A. mendotae, and A. planctonica), Aphanizomenon (Aphanizomenon gracile, A. ovalisporum), and Cylindrospermopsis raciborskii were isolated from 14 freshwater bodies in Spain and polyphasically analyzed for their phylogeography, cyanotoxin production, and the presence of cyanotoxin biosynthesis genes. The potent cytotoxin cylindrospermopsin (CYN) was produced by all 6 Aphanizomenon ovalisporum strains at high levels (5.7 to 9.1 µg CYN mg(-1) [dry weight]) with low variation between strains (1.5 to 3.9-fold) and a marked extracellular release (19 to 41% dissolved CYN) during exponential growth. Paralytic shellfish poisoning (PSP) neurotoxins (saxitoxin, neosaxitoxin, and decarbamoylsaxitoxin) were detected in 2 Aphanizomenon gracile strains, both containing the sxtA gene. This gene was also amplified in non-PSP toxin-producing Aphanizomenon gracile and Aphanizomenon ovalisporum. Phylogenetic analyses supported the species identification and confirmed the high similarity of Spanish Anabaena and Aphanizomenon strains with other European strains. In contrast, Cylindrospermopsis raciborskii from Spain grouped together with American strains and was clearly separate from the rest of the European strains, raising questions about the current assumptions of the phylogeography and spreading routes of C. raciborskii. The present study confirms that the nostocalean genus Aphanizomenon is a major source of CYN and PSP toxins in Europe and demonstrates the presence of the sxtA gene in CYN-producing Aphanizomenon ovalisporum.

Microbial community changes during the start-up of an anaerobic/aerobic/anoxic-type sequencing batch reactor.

An anaerobic/aerobic/anoxic-type sequencing batch reactor was started up during a summer rainy season to obtain enhanced biological phosphorus removal (EBPR), and its sludge microbial community was also monitored in the hope of observing the microbial community evolution of polyphosphate-accumulating organisms (PAOs). During the start-up process, a total of 17 bands of highest species richness were detected in the sludge microbial community, including Alpha-, Beta-, and Gamma- Proteobacteria, as well as Actinobacteria and Planctomycetes. Major microbial community structural change was observed in Rhodocyclus-related and Acinetobacter-related PAOs, glycogen-accumulating organisms (GAOs), and Actinobacteria. In contrast to the current belief that enrichment of PAOs is essential for the establishment of EBPR, PAOs were not favourably enriched in this study. Instead, Actinobacteria and GAOs overwhelmingly flourished. The overall conclusion of this study challenges the conventional view that EBPR cannot live without traditional PAOs. However, it suggests an non-negligible role of denitrifying phosphorus-accumulating bacteria in EBPR systems, as well as other uncultured bacteria.

Diverse antimicrobial interactions of halophilic archaea and bacteria extend over geographical distances and cross the domain barrier.

The significance of antimicrobial substances, halocins, produced by halophilic archaea and bacteria thriving in hypersaline environments is relatively unknown. It is suggested that their production might increase species diversity and give transient competitive advances to the producer strain. Halocin production is considered to be common among halophilic archaea, but there is a lack of information about halocins produced by bacteria in highly saline environments. We studied the antimicrobial activity of 68 halophilic archaea and 22 bacteria isolated from numerous geographically distant hypersaline environments. Altogether 144 antimicrobial interactions were found between the strains and aside haloarchaea, halophilic bacteria from various genera were identified as halocin producers. Close to 80% of the interactions were detected between microorganisms from different genera and in few cases, even across the domain boundary. Several of the strains produced halocins with a wide inhibitory spectrum as has been observed before. Most of the antimicrobial interactions were found between strains from distant sampling sites indicating that hypersaline environments around the world have similar microorganisms with the potential to produce wide activity range antimicrobials.

NaOH-debittering induces changes in bacterial ecology during table olives fermentation.

Limited information is available on the impact of the NaOH treatment on table olive fermentations, and for this reason a polyphasic approach has been adopted here to investigate its effect on the fermentation dynamics and bacterial biodiversity. The microbial counts of the main groups involved in the transformation have not shown any differences, apart from a more prompt start of the fermentation when the olives were subjected to the NaOH treatment. The data produced by culture-independent analyses highlighted that the fermentation of table olives not treated with NaOH is the result of the coexistence of two different ecosystems: the surface of the olives and the brines. A sodium hydroxide treatment not only eliminates this difference, but also affects the bacterial ecology of the olives to a great extent. As proved by high-throughput sequencing, the fermentation of the olives not treated with NaOH was characterized by the presence of halophilic bacteria, which were substituted by Lactobacillus at the later stages of the fermentation, while enterobacteria were dominant when the olives were treated with sodium hydroxide. Higher biodiversity was found for Lactobacillus plantarum isolated during untreated fermentation. Different biotypes were found on the olive surface and in the brines. When the debittering process was carried out, a decrease in the number of L. plantarum biotypes were observed and those originating from the surface of the olive did not differentiate from the ones present in the brines.

The transition from freshwater to marine iron-oxidizing bacterial lineages along a salinity gradient on the Sheepscot River, Maine, USA.

Oxygen-dependent, neutrophilic iron-oxidizing bacteria (FeOB) are important drivers of iron transformations in marine and freshwater environments. Despite remarkable similarities in physiology and morphotype, known freshwater and marine FeOB are clustered in different classes of Proteobacteria; freshwater FeOB in the Betaproteobacteria and marine FeOB in the Zetaproteobacteria. To determine effects of salinity on these microbes, we examined the mineral biosignatures and molecular ecology of bacteria in FeOB mats collected along an estuarine salinity gradient. Light microscopy and scanning electron microscopy analyses showed the presence of iron oxide stalk and sheath structures in both freshwater and saline iron mats. Results of tagged pyrosequencing, quantitative PCR and fluorescent in situ hybridization, all based on the small subunit rRNA gene, confirmed Zetaproteobacteria were not present in freshwater mats, but were in saline mats at salinities down to 5‰. Among the Betaproteobacteria, Leptothrix spp. were only found in the freshwater mat. Gallionella spp. were limited to freshwater and low salinity mats (< 5‰). Sideroxydans sp. were salt tolerant; however, their relative abundance decreased with increasing salinity. These results suggest salinity is important in shaping the population biology of iron mat communities, and some coexistence between marine and freshwater populations occurs in brackish waters.

Oral microbiome of deep and shallow dental pockets in chronic periodontitis.

We examined the subgingival bacterial biodiversity in untreated chronic periodontitis patients by sequencing 16S rRNA genes. The primary purpose of the study was to compare the oral microbiome in deep (diseased) and shallow (healthy) sites. A secondary purpose was to evaluate the influences of smoking, race and dental caries on this relationship. A total of 88 subjects from two clinics were recruited. Paired subgingival plaque samples were taken from each subject, one from a probing site depth >5 mm (deep site) and the other from a probing site depth ≤3mm (shallow site). A universal primer set was designed to amplify the V4-V6 region for oral microbial 16S rRNA sequences. Differences in genera and species attributable to deep and shallow sites were determined by statistical analysis using a two-part model and false discovery rate. Fifty-one of 170 genera and 200 of 746 species were found significantly different in abundances between shallow and deep sites. Besides previously identified periodontal disease-associated bacterial species, additional species were found markedly changed in diseased sites. Cluster analysis revealed that the microbiome difference between deep and shallow sites was influenced by patient-level effects such as clinic location, race and smoking. The differences between clinic locations may be influenced by racial distribution, in that all of the African Americans subjects were seen at the same clinic. Our results suggested that there were influences from the microbiome for caries and periodontal disease and these influences are independent.

Yersinia enterocolitica in sheep--a high frequency of biotype 1A.

BACKGROUND: Pigs are regarded as the main reservoir for human pathogenic Yersinia enterocolitica, which is dominated by bioserotype 4/O:3. Other animals, including sheep, have occasionally been reported as carriers of pathogenic strains of Y. enterocolitica. To our knowledge, this is the first study performed in the Nordic countries in which the presence of Y. enterocolitica in sheep is investigated. METHODS: Tonsils and faecal samples collected from sheep slaughtered on the island Gotland (Sweden) from September 2010 through January 2011 were analysed for presence of Y. enterocolitica. In an attempt to maximize recovery, several cultural strategies were applied. Various non-selective media were used and different temperatures and durations of the enrichment were applied before subculturing on Cefsulodin Irgasan Novobiocin (CIN) agar. Presumptive Y. enterocolitica colonies were subjected to urease, API 20E and agglutination test. Yersinia enterocolitica isolates were biotyped, serotyped, and tested for pathogenicity using a TaqMan PCR directed towards the ail-gene that is associated with human pathogenic strains of Y. enterocolitica. RESULTS: The samples collected from 99 sheep yielded 567 presumptive Y. enterocolitica colonies. Eighty urease positive isolates, from 35 sheep, were identified as Y. enterocolitica by API 20E. Thirty-four of 35 further subtyped Y. enterocolitica isolates, all from faecal samples, belonged to biotype 1A serotype O:5, O:6. O:13,7 and O:10. One strain was Yersinia mollaretii serotype O:62. No human pathogenic strains of Y. enterocolitica were found in the investigated sheep. Other species identified were Y. kristensenii (n = 4), Y. frederiksenii/intermedia (n = 3), Providencia rettgeri (n = 2), Serratia marcescens (n = 1) and Raoultella ornithinolytica (n = 1). CONCLUSIONS: This study does not support the hypothesis that sheep play an important role in transmission of the known human pathogenic Y. enterocolitica in the studied geographical region. However, because there are studies indicating that some strains of Y. enterocolitica biotype 1A may cause disease in humans, the relative importance of sheep as carriers of human pathogenic strains of Y. enterocolitica remains unclear. Tonsils do not appear to be favourable sites for Y. enterocolitica biotype 1A in sheep.

Bartonella quintana in Ethiopian lice.

Head and clothing lice from Jimma, Ethiopia were investigated for pathogenic bacteria. Genomic DNA from pools of lice was subjected to PCR analysis for Bartonella spp., Borrelia spp. Coxiella burnetii, Rickettsia spp. and Yersinia pestis. All 102 lice pools were negative for the afore mentioned pathogens, with the exception of Bartonella species found among 6 of 65 (9.2%) head lice pools and1 of 33 clothing lice pools. Identification was achieved by sequencing the ribosomal intragenic transcribed spacer region (ITS), revealing all to be Bartonella quintana. Although established as a clothing louse-borne infection, typically causing chronic bacteraemia, trench fever, bacillary angiomatosis and endocarditis, this has only been rarely reported among head lice. The higher numbers of infected head lice pools compared with clothing lice suggests their competence for maintaining this infection within Ethiopia.

Evaluation of brain tissue or cerebrospinal fluid with broadly reactive polymerase chain reaction for Ehrlichia, Anaplasma, spotted fever group Rickettsia, Bartonella, and Borrelia species in canine neurological diseases (109 cases).

BACKGROUND: Vector-transmitted microorganisms in the genera Ehrlichia, Anaplasma, Rickettsia, Bartonella, and Borrelia are commonly suspected in dogs with meningoencephalomyelitis (MEM), but the prevalence of these pathogens in brain tissue and cerebrospinal fluid (CSF) of dogs with MEM is unknown. HYPOTHESIS/OBJECTIVES: To determine if DNA from these genera is present in brain tissue and CSF of dogs with MEM, including those with meningoencephalitis of unknown etiology (MUE) and histopathologically confirmed cases of granulomatous (GME) and necrotizing meningoencephalomyelitis (NME). ANIMALS: Hundred and nine dogs examined for neurological signs at 3 university referral hospitals. METHODS: Brain tissue and CSF were collected prospectively from dogs with neurological disease and evaluated by broadly reactive polymerase chain reaction (PCR) for Ehrlichia, Anaplasma, Spotted Fever Group Rickettsia, Bartonella, and Borrelia species. Medical records were evaluated retrospectively to identify MEM and control cases. RESULTS: Seventy-five cases of MUE, GME, or NME, including brain tissue from 31 and CSF from 44 cases, were evaluated. Brain tissue from 4 cases and inflammatory CSF from 30 cases with infectious, neoplastic, compressive, vascular, or malformative disease were evaluated as controls. Pathogen nucleic acids were detected in 1 of 109 cases evaluated. Specifically, Bartonella vinsonii subsp. berkhoffii DNA was amplified from 1/6 dogs with histopathologically confirmed GME. CONCLUSION AND CLINICAL IMPORTANCE: The results of this investigation suggest that microorganisms in the genera Ehrlichia, Anaplasma, Rickettsia, and Borrelia are unlikely to be directly associated with canine MEM in the geographic regions evaluated. The role of Bartonella in the pathogenesis of GME warrants further investigation.

Polyphasic taxonomy of acetic acid bacteria: an overview of the currently applied methodology.

Acetic acid bacteria are Gram-negative, obligate aerobic bacteria that have the ability to incompletely oxidize alcohols or sugars to organic acids as end products. They are widespread in nature and most of them are capable to oxidize ethanol as substrate to acetic acid. This characteristic makes that acetic acid bacteria are often involved in foods and beverages, either in a beneficial, neutral or detrimental way, and therefore they have been studied extensively. The taxonomy of acetic acid bacteria has undergone many changes in the last 30 years. The early classification systems for these bacteria were based on morphological and biochemical characteristics. Today, the acetic acid bacteria are classified as the consensus result of a polyphasic analysis, combining phenotypic, chemotaxonomic and genotypic data. The present review aims at showing the various methods currently applied as well as their taxonomic resolution.

Assessment of a two-step nucleic acid amplification assay for detection of Neisseria meningitidis followed by capsular genogrouping

Immediate prevention of meningococcal disease relies in part on the prompt treatment with antibiotics of household and other close contacts of cases; however intervention with effective vaccination relies on identification of serogroup-causing strains. Parenteral antibiotic for patient with suspected meningococcal disease before hospital admission is currently recommended. Laboratory standard methods are hindered by failure to detect bacteria by this medical approach to improve patient prognosis. We assessed two polymerase chain reaction (PCR) assays to detect (crgA) and define the serogroups (siaD, orf-2, and ctrA) of Neisseria meningitidis in 120 cerebrospinal fluid (CSF) samples from positive cases (culture or antigen detection or direct smear). The PCR sensitivity for the identification of N. meningitidis was 100 percent (95 percent confidence interval, CI, 96-100 percent) compared to a sensitivity of 46 percent for culture (95 percent CI 37-55 percent), 61 percent for latex agglutination test (95 percent CI 52-70 percent), and 68 percent for Gram stain (95 percent CI 59-76 percent); PCR specificity was 97 percent (95 percent CI 82-100 percent). PCR correctly identified the serogroups A, B, C, W135, Y, and X in CSF samples with a sensitivity of 88 percent (95 percent CI 80-93 percent); the primer sets were 100 percent specific. The introduction of PCR-based assays shall increase laboratory confirmed cases, consequently enhancing surveillance of meningococcal disease.

Diagnosis of Streptococcus pneumoniae meningitis by polymerase chain reaction amplification of the gene for pneumolysin

Diagnosis of bacterial meningitis has long been based on classical methods of Gram stain, serological tests, and culture of cerebrospinal fluid (CSF). The performance of these methods, especially culture and direct smear, is thwarted by failure to detect bacteria following administration of antimicrobial agents and reluctance to performance lumbar punctures at admission. Indeed, patients with meningitis frequently receive antibiotics orally or by injection before the diagnosis is suspected or established. Thus an alternative method has become necessary to help clinicians and epidemiologists to management and control of bacterial meningitis. We evaluate the application of a polymerase chain reaction-based (PCR) assay for amplification of pneumolysin gene (ply) to diagnosis of Streptococcus pneumoniae meningitis. The PCR assay sensitivity for CSF was 96 percent (95 percent confidence interval, CI, 90-99 percent) compared to a sensitivity of 59 percent for culture (95 percent CI 49-69 percent), 66 percent for Gram stain (95 percent CI 56-74 percent), and 78 percent for latex agglutination test (95 percent CI 69-86 percent); PCR specificity was 100 percent (95 percent CI 83-100 percent). PCR results were available within 4 h of the start of the assay. This molecular approach proved to be reliable and useful to identify this bacterium compared with other classical laboratory methods for identification of bacterial meningitis pathogens.

The immunostimulating effect of bacterial genomic DNA on the innate immune responses of bivalve mussel, Hyriopsis cumingii Lea.

The genomic DNA of Escherichia coli, which contains the unmethylated CpG motif, was used to evaluate the immunostimulating effect of bacterial DNA on innate immune responses in the bivalve mussel Hyriopsis cumingii Lea. The results showed that the E. coli DNA had no significant effect on the production of superoxide anion (O(2-)) or acid phosphatase (AP) by haemocytes in vitro. However, the bactericidal activity of the haemocytes was significantly increased when the cells were incubated with 50 or 100mug/ml bacterial DNA for 12 and 24h. Antibacterial activity, lysozyme activity, and prophenoloxidase (proPO) production of haemolymph were also increased, when the bivalve molluscs were injected with 50 or 100mug/ml of bacterial DNA for 12 and 24h. These activities returned to the control level after 48h. This work showed the bacterial DNA with unmethylated CpG motif could activate some parameters of the immune system of bivalve molluscs in vivo and in vitro.