OrthoRefine: automated enhancement of prior ortholog identification via synteny.

BACKGROUND: Identifying orthologs continues to be an early and imperative step in genome analysis but remains a challenging problem. While synteny (conservation of gene order) has previously been used independently and in combination with other methods to identify orthologs, applying synteny in ortholog identification has yet to be automated in a user-friendly manner. This desire for automation and ease-of-use led us to develop OrthoRefine, a standalone program that uses synteny to refine ortholog identification. RESULTS: We developed OrthoRefine to improve the detection of orthologous genes by implementing a look-around window approach to detect synteny. We tested OrthoRefine in tandem with OrthoFinder, one of the most used software for identification of orthologs in recent years. We evaluated improvements provided by OrthoRefine in several bacterial and a eukaryotic dataset. OrthoRefine efficiently eliminates paralogs from orthologous groups detected by OrthoFinder. Using synteny increased specificity and functional ortholog identification; additionally, analysis of BLAST e-value, phylogenetics, and operon occurrence further supported using synteny for ortholog identification. A comparison of several window sizes suggested that smaller window sizes (eight genes) were generally the most suitable for identifying orthologs via synteny. However, larger windows (30 genes) performed better in datasets containing less closely related genomes. A typical run of OrthoRefine with ~ 10 bacterial genomes can be completed in a few minutes on a regular desktop PC. CONCLUSION: OrthoRefine is a simple-to-use, standalone tool that automates the application of synteny to improve ortholog detection. OrthoRefine is particularly efficient in eliminating paralogs from orthologous groups delineated by standard methods.

Neonatal pneumonia caused by Trichomonas vaginalis.

Neonatal pneumonia is mostly bacterial and other etiology is considered less frequently. We report a case of newborn whose neonatal pneumonia has not improved, despite the aggressive ventilation regime and empiric antibiotic therapy. A special sample from the respiratory tract was collected for PCR examination. The test confirmed the presence of Trichomonas vaginalis. Antibiotic therapy was extended to include metronidazole. Targeted antibiotic therapy, which lasted for 28 days, improved the condition and the patient was discharged in a stabilized condition to home care on the 44th day of life. We demonstrate the need to consider atypical pathogens in the case of infections that do not respond to conventional therapy. The multiplex real-time PCR technique was used to detect the DNA of the pathogen. Targeted antibiotic therapy is the result of pathogen identification.

Evaluation of the infB and rpsB gene fragments as genetic markers intended for identification and phylogenetic analysis of particular representatives of the order Lactobacillales.

Detailed differentiation, classification, and phylogenetic analysis of the order Lactobacillales are performed using molecular techniques that involve the comparison of whole genomes, multilocus sequence analysis, DNA-DNA hybridisation, and 16S rRNA sequencing. Despite the wide application of the latter two techniques, issues associated with them are extensively discussed. Although complete genomic analyses are the most appropriate for phylogenetic studies, they are time-consuming and require high levels of expertise. Many phylogenetic/identification markers have been proposed for enterococci, lactobacilli, streptococci, and lactobacilli. However, none have been established for vagococci and some genera within the order Lactobacillales. The objective of the study was to find novel alternative housekeeping genes for classification, typing, and phylogenetic analysis of selected genera within the order Lactobacillales. We designed primers flanking variable regions of the infB (504 nt) and rpsB (333 nt) genes and amplified and sequenced them in 56 strains of different genera within the order Lactobacillales. Statistical analysis and characteristics of the gene regions suggested that they could be used for taxonomic purposes. Phylogenetic analyses, including assessment of (in)congruence between individual phylogenetic trees indicated the possibility of using the concatenation of the two genes as an alternative tool for the evaluation of phylogeny compared with the 16S rRNA gene representing the standard phylogenetic marker of prokaryotes. Moreover, infB, rpsB regions and their concatenate were phylogenetically consistent with two widely applied alternative genetic markers in taxonomy of particular Lactobacillales genera encoding the 60 kDa chaperonin protein (GroEL-hsp60) and phenylalanyl-tRNA synthetase, alpha subunit (pheS).

Variable regions of the glyS, infB and rplB genes usable as novel genetic markers for identification and phylogenetic purposes of genera belonging to the family Propionibacteriaceae.

No common, unique genetic markers applicable to classification and phylogenetics for significant genera within the Propionibacteriaceae family have been suggested yet. Therefore, the aim of the study was to propose those genes in the genera Acidipropionibacterium, Cutibacterium, Propionibacterium and Pseudopropionibacterium. These genera were recently elicited from the genus Propionibacterium through whole genomic analyses. Three housekeeping genes, glyS, infB and rplB, were selected from many others according to the requirements for appropriate classification/phylogenetic markers. Concrete fragments of the genes were amplified using specific primers in most of the type (14) and 11 wild strains (originating from dairy products, human skin and the crop of a laying hen) recently classified into the genus Propionibacterium. Sequences obtained from amplicons were used to perform gene statistics and phylogenetic analyses with respect to applicability in classification, typing and phylogeny. The 16S rRNA gene sequences, still considered relevant in spite of its proven shortcomings as a basic tool for evaluation of bacterial phylogeny, were used as a baseline for comparative analyses. The statistics of the gene sequences revealed that the variable regions of all three genes have higher resolution capabilities among strains examined compared to the 16S rRNA gene analysis. Phylogenetic analyses based on individual gene sequences and their concatenate enabled to distinguish clusters of species belonging to the genera Acidipropionibacterium, Cutibacterium and Propionibacterium, which corresponds with a recently reported genomic study. Thus, the crucial importance of this study is the economically advantageous classification and typing of propionibacterial isolates and strains through the three gene regions in contrast to the requirement for whole genomic assays.

Effects of pure plant secondary metabolites on methane production, rumen fermentation and rumen bacteria populations in vitro.

In this study, the effects of seven pure plant secondary metabolites (PSMs) on rumen fermentation, methane (CH4 ) production and rumen bacterial community composition were determined. Two in vitro trials were conducted. In trial 1, nine concentrations of 8-hydroxyquinoline, α-terpineol, camphor, bornyl acetate, α-pinene, thymoquinone and thymol were incubated on separate days using in vitro 24-hr batch incubations. All compounds tested demonstrated the ability to alter rumen fermentation parameters and decrease CH4 production. However, effective concentrations differed among individual PSMs. The lowest concentrations that reduced (p < .05) CH4 production were as follows: 8 mg/L of 8-hydroxyquinoline, 120 mg/L of thymoquinone, 240 mg/L of thymol and 480 mg/L of α-terpineol, camphor, bornyl acetate and α-pinene. These concentrations were selected for use in trial 2. In trial 2, PSMs were incubated in one run. Methane was decreased (p < .05) by all PSMs at selected concentrations. However, only 8-hydroxyquinoline, bornyl acetate and thymoquinone decreased (p < .05) CH4 relative to volatile fatty acids (VFAs). Based on denaturing gradient gel electrophoresis analysis, different PSMs changed the composition of bacterial communities to different extents. As revealed by Ion Torrent sequencing, the effects of PSMs on relative abundance were most pronounced in the predominant families, especially in Lachnospiraceae, Succinivibrionaceae, Prevotellaceae, unclassified Clostridiales and Ruminococcaceae. The CH4 production was correlated negatively (-.72; p < .05) with relative abundance of Succinivibrionaceae and positively with relative abundance of Ruminococcaceae (.86; p < .05). In summary, this study identified three pure PSMs (8hydroxyquinoline, bornyl acetate and thymoquinone) with potentially promising effects on rumen CH4 production. The PSMs tested in this study demonstrated considerable impact on rumen bacterial communities even at the lowest concentrations that decreased CH4 production. The findings from this study may help to elucidate how PSMs affect rumen bacterial fermentation.

[Hantavirus causing fatal haemorrhagic fever in the Czech Republic]. / Hantavirus jako puvodce smrtelné hemoragické horecky v Ceské republice.

Hantaviruses are RNA viruses of the family Bunyaviridae. Their hosts are mammals of the orders rodents (voles, rats, mice), insectivores (shrews, moles), and chiroptera (bats). Hantaviruses are present in many areas of Europe, the Americas, Asia, and Africa. In the Czech Republic, the occurrence of five species of hantaviruses has been reported (Dobrava/Belgrade, Puumala, Tula, Seewis, and Asikkala), with the first three of them causing human diseases. Although the course of hantavirus infections can be very serious, there is a low awareness of these diseases, even among health professionals, and hantavirus is often not considered in the diagnosis. A case history is reported of a patient who developed hantavirus haemorrhagic fever with renal syndrome (HFRS) with fatal outcome. The patient presented with typical clinical signs, but the correct diagnosis was only made at post mortem.

Design and validation of a qPCR assay for accurate detection and initial serogrouping of Legionella pneumophila in clinical specimens by the ESCMID Study Group for Legionella Infections (ESGLI).

Prompt detection of Legionella pneumophila is essential for rapid investigation of legionellosis. Furthermore, as the majority of L. pneumophila infections are caused by serogroup 1 (sg1) strains, rapid identification of such strains can be critical in both routine and outbreak scenarios. The ESCMID Study Group for Legionella Infections (ESGLI) was established in 2012 and immediately identified as a priority the validation of a reliable, easy to perform and interpret, cost-effective qPCR assay to standardise the detection of L. pneumophila DNA amongst members. A novel L. pneumophila assay targeting the mip gene was designed and combined with previously published methodologies amplifying the sg1 marker (wzm) and the green fluorescent protein gene (gfp) internal process control. The resulting triplex assay was validated internationally on the three qPCR platforms used by the majority of European Legionella reference laboratories: ABI 7500 (Life Technologies), LightCycler 480 Instrument II (Roche) and Rotor-Gene Q (Qiagen). Clinical and EQA specimens were tested together with a large panel of strains (251 in total) to validate the assay. The assay proved to be 100% specific for L. pneumophila and sg1 DNA both in silico and in vitro. Efficiency values for mip and wzm assays ranged between 91.97 and 97.69%. Limit of detection values estimated with 95% confidence were adopted for mip and wzm assays on all three qPCR platforms. Inhibition was not observed. This study describes a robust assay that could be widely implemented to standardise the molecular detection of L. pneumophila among ESGLI laboratories and beyond.

[Evaluation of prophylactic precautions after exposure to biological materials]. / Hodnocení profylaktických opatrení po expozici biologickému materiálu.

OBJECTIVES: Study of transmission rates of hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV) and effect of HBV vaccination after parenteral exposure to biological materials. PATIENTS AND METHODS: This was a retrospective study of 879 individuals (419 health care professionals and 460 persons from the general population) after blood and body fluid exposure examined at the Clinic of Infectious Diseases in Ostrava from 1999 to 2013. HBsAg, anti-HBs, anti-HBc, anti-HCV, anti-HIV, bilirubin, and ALT were tested in exposed patients and known sources at the baseline and, except anti-HBc, after 3, 6, and 12 months. Susceptible persons were vaccinated against HBV and screened for anti-HBs after 1-2 months. Antiretroviral prophylaxis was provided if reasonable. RESULTS: At the baseline, 42 exposed persons were HBV positive, six were HCV positive, and none was HIV positive. During the follow-up, no new HBsAg positivity was detected in exposed individuals, although 25 of 837 susceptible persons were exposed to HBsAg-positive sources. After vaccination, protective anti-HBs were detected in 707 (84.7%) of 837 susceptible persons and in 709 (97.8%) of 725 persons with known post-vaccination response. Fifty-six of 873 persons had been exposed to HCV-positive sources and HCV transmission was shown in three (two health care professionals) of them. No HIV transmission was observed, although 11 of 879 individuals had been exposed to HIV-positive sources, with antiretroviral prophylaxis provided to nine of them. CONCLUSIONS: Contemporary post-exposure prophylactic precautions in the Czech Republic can be considered as adequate for the prevention of HBV and HIV, but health care professionals in particular are at risk of HCV transmission.

[The use of electron microscopy and virus isolation in the diagnosis of aseptic neuroinfections]. / Vyuzití elektronové mikroskopie a izolace viru v diagnostice aseptických neuroinfekcí.

OBJECTIVE: In aseptic neuroinfections, the etiology is usually known in 50-70% of cases. The aim was to increase the rates using electron microscopy (EM) and virus isolation in cell cultures. MATERIAL AND METHODS: The prospective study included 34 patients with aseptic neuroinfections hospitalized at the Department of Infectious Diseases in Ostrava fromJuly to November 2012. EM examined cerebrospinal fluid of all patients and virus isolation in tissue cultures was performed in all cerebrospinal fluid samples. Cerebrospinal fluid was examined by polymerase chain reaction for enteroviruses in 30 patients and for herpes simplex virus 1 and 2 in 29 patients. Detection of antibodies against Borrelia burgdorferi and tick-borne encephalitis was performed in all 34 patients. RESULTS: Possible etiological agents were discovered in 31 out of 34 patients (91%), with one agent being found in 23 patients (68%) and two agents being detected in 8 patients (24%). EM revealed the agents in 26 patients and virus isolation was successful in 10 patients. EM was the only method to identify 10 agents. A group of 23 patients with a single agent detected included 14 patients with enteroviral meningitis, 4 patients with Lyme borreliosis and 4 patients with tick-borne encephalitis; EM detected an undefined virus in the last patient. An unusual group of 8 patients with two agents detected comprised 5 patients with enteroviruses and spirochetes, 2 patients with tick-borne encephalitis and undefined viruses and 1 patient with a spirochete and an undetermined virus. CONCLUSION: EM can aid in explaining the etiology of aseptic neuroinfections. However, the clinical interpretation of results remains problematic, such as detection of unknown viruses or two possible agents in 8 out of 34 patients.

Unveiling the intruder deformed 02(+) state in 34Si.

The 02(+) state in 34Si has been populated at the GANIL-LISE3 facility through the ß decay of a newly discovered 1(+) isomer in 34Al of 26(1) ms half-life. The simultaneous detection of e(+)e(-) pairs allowed the determination of the excitation energy E(02(+))=2719(3) keV and the half-life T(1/2)=19.4(7) ns, from which an electric monopole strength of ρ(2)(E0)=13.0(0.9)×10(-3) was deduced. The 2(1)(+) state is observed to decay both to the 0(1)(+) ground state and to the newly observed 0(2)(+) state [via a 607(2) keV transition] with a ratio R(2(1)(+)→0(1)(+)/2(1)(+)→0(2)(+))=1380(717). Gathering all information, a weak mixing with the 0(1)(+) and a large deformation parameter of ß=0.29(4) are found for the 0(2)(+) state, in good agreement with shell model calculations using a new SDPF-U-MIX interaction allowing np-nh excitations across the N=20 shell gap.

Intact brown seaweed (Ascophyllum nodosum) in diets of weaned piglets: effects on performance, gut bacteria and morphology and plasma oxidative status.

The aim was to assess the effects of intact dried Ascophyllum nodosum seaweed on piglet performances, gut bacteria and function and plasma oxidative status. A total of 160 weaned piglets (21 days, 6.59 ± 0.91 kg) were allocated to four dietary treatments with eight pen replicates of five animals each for 28 days: a control diet; based on cereals, soybean meal and milk products, and three basal diets supplemented with either 2.5, 5.0 or 10.0 g dried seaweed per kg. At day 12/13 one piglet from each pen was sacrificed. Plasma samples were taken to determine parameters of oxidative status. Digesta were sampled for microbiological plate countings onto selective media and molecular analysis using PCR-DGGE. Small intestinal tissue was taken for morphological and electro-physiological determinations. Data were analysed by a linear model with treatment as fixed effect. A. nodosum supplementation had no effect on daily weight gain, nor did it alter feed conversion ratio. Plate countings failed to reveal differences among treatments. Dendograms prepared using PCR-DGGE banding patterns did not indicate clustering of microbial profiles based on diet supplement. Plasma oxidative status and outcome of morphology and of electro-physiological measurements from gut tissues were similar for all treatments. Thus, the addition of A. nodosum seaweed to well digestible diets did not enhance performances of piglets nor some gut health parameters and plasma oxidative status.

Oral administration of Parabacteroides distasonis antigens attenuates experimental murine colitis through modulation of immunity and microbiota composition.

Commensal bacteria have been shown to modulate the host mucosal immune system. Here, we report that oral treatment of BALB/c mice with components from the commensal, Parabacteroides distasonis, significantly reduces the severity of intestinal inflammation in murine models of acute and chronic colitis induced by dextran sulphate sodium (DSS). The membranous fraction of P. distasonis (mPd) prevented DSS-induced increases in several proinflammatory cytokines, increased mPd-specific serum antibodies and stabilized the intestinal microbial ecology. The anti-colitic effect of oral mPd was not observed in severe combined immunodeficient mice and probably involved induction of specific antibody responses and stabilization of the intestinal microbiota. Our results suggest that specific bacterial components derived from the commensal bacterium, P. distasonis, may be useful in the development of new therapeutic strategies for chronic inflammatory disorders such as inflammatory bowel disease.

Bifidobacterium actinocoloniiforme sp. nov. and Bifidobacterium bohemicum sp. nov., from the bumblebee digestive tract.

Our previous study, based primarily on PCR-denaturing gradient gel electrophoresis and 16S rRNA gene sequencing, focused on the isolation of four bifidobacterial groups from the digestive tract of three bumblebee species. In that study, we proposed that these isolated groups potentially represented novel species of the family Bifidobacteriaceae. One of the four, Bifidobacterium bombi, has been described recently. Strains representing two of the other groups have been classified as members of the genus Bifidobacterium on the basis of positive results for fructose-6-phosphate phosphoketolase activity and analysis of partial 16S rRNA and heat-shock protein 60 (hsp60) gene sequences. Analysis of 16S rRNA gene sequence similarities revealed that the isolates of the first group were affiliated to Bifidobacterium asteroides YIT 11866(T), B. indicum JCM 1302(T) and B. coryneforme ATCC 25911(T) (96.2, 96.0 and 95.9 % sequence similarity, respectively), together with other bifidobacteria showing lower sequence similarity. Additional representatives of the second group were found to be affiliated to Bifidobacterium minimum YIT 4097(T) and B. coryneforme ATCC 25911(T) (96.0 and 96.3 % sequence similarity) and also to other bifidobacteria with lower sequence similarity. These results indicate that the isolates of the two groups belong to novel species within the genus Bifidobacterium. This observation was further substantiated by the results of partial sequencing of hsp60. On the basis of phylogenetic and phenotypic analyses and analysis of 16S rRNA and partial hsp60 gene sequences, we propose two novel species, Bifidobacterium actinocoloniiforme sp. nov. (type strain LISLUCIII-P2(T)  = DSM 22766(T)  = CCM 7728(T)) and Bifidobacterium bohemicum sp. nov. (type strain JEMLUCVIII-4(T)  = DSM 22767(T)  = CCM 7729(T)).

Prolate-spherical shape coexistence at N=28 in 44S.

The structure of 44S has been studied by using delayed γ and electron spectroscopy. The decay rates of the 02+ isomeric state to the 2(1)+ and 0(1)+ states, measured for the first time, lead to a reduced transition probability B(E2: 2(1)+→0(2)+)=8.4(26) e(2) fm4 and a monopole strength ρ2(E0: 0(2)+→0(1)+)=8.7(7)×10(-3). Comparisons to shell model calculations point towards prolate-spherical shape coexistence, and a two-level mixing model is used to extract a weak mixing between the two configurations.

Long-term protection against hepatitis B after newborn vaccination: 20-year follow-up.

BACKGROUND: Hepatitis B vaccination in children born to hepatitis B surface antigen (HBsAg)-positive mothers considerably decreases the risk of vertical transmission. However, whether this protection against carriage of hepatitis B virus is maintained into early adulthood is as yet unknown. PATIENTS AND METHODS: A combined passive-active immunization programme for newborns of HBsAg-positive mothers was initiated in the north-eastern part of the Czech Republic in 1988. The number of immunized newborns had reached 665 newborns by the end of 2006. All mothers of immunized infants were HBsAg-positive during pregnancy, and 34 (5%) were also hepatitis B e antigen (HBeAg)-positive. The immunization programme consists of providing newborns with protection at birth with hepatitis B immunoglobulin, followed by three 10-µg doses of plasma-derived or, since 1990, recombinant vaccine administered at 0, 1 and 6 months of life. Only 29 children of HBeAg-positive mothers received vaccine at 0, 1 and 2 months of life. Blood samples were obtained after immunization, at 2 years of age, and biennially thereafter. Samples were tested for HBsAg and hepatitis B surface and core antibodies (anti-HBs, anti-HBc). RESULTS: The immunization schedules were completed in 640 children. A protective anti-HBs level after immunization was proven in 574 of 620 children (93%). Persistence of protective anti-HBs antibodies was detected in 70, 40 and 25% of children at 5, 10 and 15 years of age. Vertical transmission with chronic HBsAg carrier status was detected in two infants. Anti-HBc seroconversion was proven in ten children from 3 to 15 years of age. Natural boosting with an anti-HBs increase was detected in 38 children (twice in one child). CONCLUSION: Our results show that combined active-passive immunization of newborns against hepatitis B provides persistent protection up to adolescence despite a frequent waning of anti-HBs antibodies, suggesting there is no need for booster vaccination during adolescence.

Fiber-optic pH detection in small volumes of biosamples.

Determining the pH values of microscopic plant samples may help to explain complex processes in plants, so it is an area of interest to botanists. Fiber-optic probes with small dimensions can be used for this purpose. This paper deals with the fiber-optic detection of the pH values of droplets of plant xylem exudate based on ratiometric fluorescence intensity measurements with an internal reference. For this purpose, novel V-taper sensing probes with a minimum diameter of around 8 µm were prepared that enable the delivery of fluorescence signal from the detection site on the taper tip to the detector. The taper tips were coated with pH-sensitive transducer (8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt; HPTS) and a reference [dichlorotris-(1,10-phenanthroline) ruthenium (II) hydrate (Ru-phen dichloride)] immobilized in a xerogel layer of propyltriethoxysilane and (3-glycidoxy)propyl trimethoxysilane. The prepared probes were sensitive to pH values mainly in the range from 6.0 to 9.0. In the pH range 6-9, the results were limited by measurement errors of about 0.2 pH units, and in the pH range 5-6 by measurement errors of about 0.5 pH units. Using the developed V-taper sensing probes, the pH values of in vivo and in vitro samples of small volumes (~6 µl) of exudate were measured. The results were validated by comparison with conventional electrochemical pH measurements.

[Retreatment with peginterferon plus ribavirin in chronic hepatitis C patients: our own experiences]. / Opakovaná lécba pegylovaným interferonem s ribavirinem u pacientu s chronickou hepatitidou C - vlastní zkusenosti.

Retreatment with peginterferon plus ribavirin was initiated in 26 patients with hepatitis C virus genotype 1b infection (17 relapsers after the first course of therapy, 9 non-responders). So far, retreatment has been completed in 19 patients, one patient achieved a sustained virologic response, and 3 patients were relapsers. Therapy was discontinued in 14 patients (9 non-responders) because of a lack of a treatment response, and in 1 patient due to adverse effects. Retreatment is a new chance for patients with chronic hepatitis C infection. However successful outcome is rare especially in non-responders.

The design of oligonucleotide primers for the universal amplification of the N-acetylglucosaminidase gene (nag1) in Chytridiomycetes with emphasis on the anaerobic Neocallimastigales.

The common feature of all chytridiomycetous fungi, aerobic as well as anaerobic, is an abundance of chitin in their cell wall. The genes coding for chitinases have therefore been widely used as phylogenetic markers in ascomycetes. As their utility for Chytridiomycetes has not been determined we chose the gene encoding an enzyme involved in chitin degradation and energy metabolism, the beta-(1,4)-N-acetylglucosaminidase (nag1). Primer pair Nag-forward and Nag-reverse was used to create PCR product from 5 strains of anaerobic and 7 strains of aerobic chytrids. However, Blast search of sequenced amplicons showed that these primers are specific only for fungus Emericella nidulans. Amino acid alignment of Nag1 proteins of fungal, protozoal and bacterial origin available in GenBank database was therefore performed. Five amino acid regions were found to be conserved enough to serve as a suitable domain for the design of a set of primers for the universal amplification of the nag1 gene in the Neocallimastigales fungi.

The intestinal microflora of childhood patients with indicated celiac disease.

The fecal short-chain fatty acids concentration was higher (154 +/- 46.9 mmol/L) in childhood patients than in healthy children (96.6 +/- 19.2 mmol/L). On the other hand, pH values were nonsignificantly lower in patients stool (6.78 +/- 0.75 vs. children 7.42 +/- 0.74). Using denaturing gradient gel electrophoresis specific for total bacteria, lactobacilli and bifidobacteria the microbial population was characterized in fecal samples and in duodenal biopsies. Bacteria adhering to duodenal biopsies were not dominating in stool samples. More than 50 % of detected bacterial species belonged to as yet uncultured strains.

Diversity of insect intestinal microflora.

The influence of geographic location, season, age, and part of the digestive tract on bacterial diversity was evaluated on intestinal microflora of honeybees, wasps, and cockroaches using DGGE analysis. PCR-DGGE analyses with universal bacterial primers targeting 200-bp region of the 16S rDNA gene afforded the profile of complex bacterial DNA; specific primers were used to determine the profile of bifidobacteria whose concentration in digestive tract was determined by real-time PCR. Selected PCR products were identified by sequencing. The microflora of the bees exhibited little variations among the hives from distant locations. Their bifidobacterial population formed 2.8-8.4 % of total bacteria and was very homogeneous. The total gut microflora of wasps was also homogeneous, only two samples being affected by the season or the location; on the other hand, wasp bifidobacterial population was very heterogeneous. Cockroaches showed the highest variations in microflora composition, the age and diet being the ultimate factors; bifidobacteria counts also varied among tested individuals (0.1-34.1 % of total bacteria). Our results suggest that nutrition habits are the strongest factor affecting the insect microflora, giving higher variations to omnivorous species.