Partial 16S rRNA Sequences Research Articles

Diversity of Ptychadena in Rwanda and taxonomic status of P. chrysogaster Laurent, 1954 (Amphibia, Anura, Ptychadenidae)

We assess the diversity of Ptychadena species in Rwanda based on re-examination of voucher specimens in museum collections and our own data from recent assessment of the species composition of amphibian communities in Rwanda. We recognize five species which we allocate to the following available names: P. anchietae, P. chrysogaster, P. nilotica, P. porosissima, and P. uzungwensis. We did not find evidence for the presence of P. grandisonae and P. oxyrhynchus which have been listed for the country. The five species can be distinguished by quantitative morphometrics (discriminant analysis, success rate: 100 %) and a number of qualitative characters of external morphology. We provide an identification key to the Rwandan species and describe the morphology of each species in detail. The taxonomic status and the phylogenetic position of Ptychadena chrysogaster are further assessed based on the partial sequence of the mitochondrial 16S rRNA. The species differs genetically from available homologous sequences from congeners by an uncorrected p distance of at least 4.2 % and appears to be most closely related to specimens assigned to P. porosissima, P. mahnerti, “P. aff. uzungwensis” and “P. aff. bibroni”.

Evaluation of two matrix-assisted laser desorption ionization–time of flight mass spectrometry systems for identification of viridans group streptococci

In this study, the performances of two matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) systems, MALDI Biotyper (Bruker Daltonics) and VITEK MS (bioMérieux), were evaluated in the identification of viridans group streptococci. Two collections of isolates were tested with both methods. From a panel of type collection strains (n = 54), MALDI Biotyper gave correct species-level identification for 51/54 (94 %) strains and 37/54 (69 %) strains for the VITEK MS in vitro diagnostic (IVD) method. Additionally, a collection of blood cultures isolates which had been characterized earlier with partial sequencing of 16S rRNA (n = 97) was analyzed. MALDI Biotyper classified 89 % and VITEK MS 93 % of these correctly to the group level. Comparison of species-level identification from the blood culture collection was possible for 36 strains. MALDI Biotyper identified 75 % and VITEK MS 97 % of these strains consistently. Among the clinical isolates, MALDI Biotyper misidentified 36 strains as Streptococcus pneumoniae. Nevertheless, our results suggest that the current MALDI-TOF methods are a good alternative for the identification of viridans streptococci and do perform as well as or better than commercial phenotypical methods.

Cryptic yet colorful: anatomy and relationships of a new genus of Cerithiidae (Caenogastropoda, Cerithioidea) from coral reef drop‐offs

AbstractCerithium koperbergi is a rare gastropod of the family Cerithiidae from the tropical Indo‐West Pacific. The species has a small, unusual shell and often inhabits deeper water, fore‐reef habitats that are atypical for the genus. Anatomical investigations reveal that it possesses a combination of features heretofore considered diagnostic of two main cerithiid subfamilies: Cerithiinae and Bittiinae. While the shell is bittiine, the animal lacks mesopodial pedal glands and possesses a seminal receptacle (vs. a spermatophore bursa) in the lateral lamina of the oviduct, which are considered to be cerithiine features. Re‐evaluation of the anatomy of Bittium reticulatum, the type species of Bittium, indicates the defining anatomical difference in oviduct anatomy between the two subfamilies does not stand up to closer scrutiny. Partial mitochondrial cytochrome c oxidase I (COI) sequences support the interpretation that C. koperbergi is a species complex around the western Pacific rim comprising three divergent mitochondrial lineages. Bayesian analysis of partial mitochondrial COI and 16S rRNA sequences confirm the placement of the C. koperbergi complex within a monophyletic Bittiinae, despite the apparent absence of a unifying anatomical feature. Species in the C. koperbergi complex are here united in Pictorium nov. gen. and two species are described as new. It is hypothesized that features of the midgut may be diagnostic of the Bittiinae, but more comparative data are needed.

Isolation and characterization of a Bacillus subtilis strain that degrades endosulfan and endosulfan sulfate.

Endosulfan has emerged as a major environmental menace worldwide due to extensive usage and environmental persistence, seeking its remedial by a cheaper and efficient means. Therefore, natural resource (soil) was explored to search a potential candidate for biodegradation of endosulfan. A soil bacterium was enriched and isolated by applying a strong nutritional selection pressure, using a non-sulfur medium supplemented with endosulfan as sole source sulfur. The microbial strain was found to degrade endosulfan as well as its equally toxic metabolite endosulfan sulfate to non-toxic metabolites (endodiol and endosulfan lactone) very efficiently (up to 94.2 %) within 7 days, estimated qualitatively by thin layer chromatography and quantitatively by gas chromatography-electron capture detection methods. The isolate was characterized for its morphological, physiological, biochemical and 16S rRNA sequencing and identified as a new strain of Bacillus subtilis with strain designation AKPJ04, which was deposited with accession number Microbial Type Culture Collection and Gene Bank (MTCC) 8561, at MTCC, Institute of Microbial Technology, Chandigarh, India. The partial 16S rRNA sequence was submitted to Genbank, Maryland, USA, with the accession number EU 258611. The primary investigation for endosulfan degrading gene(s) localization suggested its location on chromosomal DNA.Electronic supplementary materialThe online version of this article (doi:10.1007/s13205-013-0176-7) contains supplementary material, which is available to authorized users.

Endo-and exoglucanase activities in bacteria from mangrove sediment

The mangrove ecosystem is an unexplored source for biotechnological applications. In this unique environment, endemic bacteria have the ability to thrive in the harsh environmental conditions (salinity and anaerobiosis), and act in the degradation of organic matter, promoting nutrient cycles. Thus, this study aimed to assess the cellulolytic activities of bacterial groups present in the sediment from a mangrove located in Ilha do Cardoso (SP, Brazil). To optimize the isolation of cellulolytic bacteria, enrichments in two types of culture media (tryptone broth and minimum salt medium), both supplemented with 5% NaCl and 1% of cellulose, were performed. Tests conducted with the obtained colonies showed a higher occurrence of endoglycolytic activity (33 isolates) than exoglycolytic (19 isolates), and the degradation activity was shown to be modulated by the presence of NaCl. The isolated bacteria were clustered by BOX-PCR and further classified on the basis of partial 16S rRNA sequences as Alphaproteobacteria, Gammaproteobacteria, Actinobacteria, Firmicutes or Bacteroidetes. Therefore, this study highlights the importance of studies focusing on the endemic species found in mangroves to exploit them as novel biotechnological tools for the degradation of cellulose.

Characterization of lactic acid bacteria isolated from infant faeces as potential probiotic starter cultures for fermented sausages

A total of 109 lactic acid bacteria isolated from infant faeces were identified by partial 16S rRNA, cpn60 and/or pheS sequencing. Lactobacillus was the most prevalent genus, representing 48% of the isolates followed by Enterococcus (38%). Lactobacillus gasseri (21%) and Enterococcus faecalis (38%) were the main species detected. A further selection of potential probiotic starter cultures for fermented sausages focused on Lactobacillus as the most technologically relevant genus in this type of product. Lactobacilli strains were evaluated for their ability to grow in vitro in the processing conditions of fermented sausages and for their functional and safety properties, including antagonistic activity against foodborne pathogens, survival from gastrointestinal tract conditions (acidity, bile and pancreatin), tyramine production, antibiotic susceptibility and aggregation capacity. The best strains according to the results obtained were Lactobacillus casei/paracasei CTC1677, L. casei/paracasei CTC1678, Lactobacillus rhamnosus CTC1679, L. gasseri CTC1700, L. gasseri CTC1704, Lactobacillus fermentum CTC1693. Those strains were further assayed as starter cultures in model sausages. L. casei/paracasei CTC1677, L. casei/paracasei CTC1678 and L. rhamnosus CTC1679 were able to lead the fermentation and dominate (levels ca. 108 CFU/g) the endogenous lactic acid bacteria, confirming their suitability as probiotic starter cultures.

Bulleidia extructaPeriprosthetic Hip Joint Infection, United States

To the Editor: Bulleidia extructa is an obligately anaerobic, nonmotile, non–spore-forming gram-positive bacillus first described in 2000 by Downes et al. (1), after having isolated a bacterium from the oral cavity of persons with periodontitis and dentoalveolar abscesses that did not correspond to any known species. After phenotypic and genetic characterization, the investigators proposed a new genus, Bulleidia, and the species B. extructa. Since then, additional reports have associated the organism with oral infections, specifically periodontal disease (2–5). While B. extructa’s association with human periodontal disease is well documented, the bacterium has so far not been implicated in other pathogenic processes. We report here a case of a total hip arthroplasty infection caused by B. extructa in an immunocompetent patient. In November 2010, an 82-year-old man with a non-cemented right total hip arthroplasty that was performed 26 years previously was evaluated for right hip pain. He had been in his usual state of health without any complaints until a month earlier, when he lost his footing and hyperabducted his hip joints, involuntarily performing a split, while washing a boat cover with a power washer. Since then, he reported right hip pain that somewhat limited his mobility. Physical examination revealed an antalgic gait, mild swelling of the right lower extremity, and impaired hip mobility related to pain on the right side, specifically with extension, flexion, abduction, and adduction. Results of the patient’s blood work were notable for normocytic anemia (hemoglobin 10.6 g/dL), thrombocytosis (459 × 109/L), elevated erythrocyte sedimentation rate (101 mm/h), and elevated C-reactive protein (88.7 mg/L). Leukocyte count was within normal limits (9.6 × 109 cells/L). An ultrasound examination of the right hip joint showed extensive synovitis and a large, 4.3 × 5.0 × 5.1–cm vascular mass extending anteriorly from the joint space. Aspiration of the joint space yielded 1 mL of blood-stained fluid with 111,595 cells/µL (95% neutrophils, 5% monocytes/ macrophages). Anaerobic bacterial culture grew a gram-positive bacillus identified as B. extructa by partial 16S rRNA sequencing. DNA was prepared for PCR amplification by using PrepMan Ultra (Applied Biosystems, Foster City, CA, USA) and amplified and bidirectionally sequenced by using primers 5′-TGGAGAGTTTGATCCTGGCTCAG-3′ and 5′-TACCGCGGCTGCTGGCAC-3′. The generated 484-bp sequence differed by 2 bp from 483 bp of available sequence from B. extructa GenBank accession no. {type:entrez-nucleotide,attrs:{text:AF220064,term_id:7637526,term_text:AF220064}}AF220064. The isolate was susceptible to penicillin, clindamycin, and metronidazole by using E-test. The patient underwent total hip arthroplasty resection. Intraoperatively, purulence was noted upon entering the hip joint. Histopathologic examination of removed tissue revealed acute inflammation. Five hip tissue specimens were obtained for culture; 3 specimens yielded B. extructa. Six weeks of intravenous ceftriaxone treatment was prescribed, and the patient was instructed to revisit a dentist for a full dental examination. Before seeking treatment for this episode, he reported that he was seeing a dentist on a regular basis and denied any recent dental surgery or infections. The patient was seen in a follow-up visit 2 months after reimplantation surgery; at that time, he reported minimal pain and had begun to bear weight on the affected side. There was no evidence for infection recurrence. Periprosthetic joint infections are a major complication after joint replacement. The number of procedures for total hip and knee replacements has increased during the past 13 years (6). This trend is accompanied by an increase in the total number of periprosthetic joint infections, even though the overall percentage of this complication is low (7). The most commonly isolated organisms in periprosthetic joint infections are gram-positive cocci, specifically Staphylococcus aureus and S. epidermidis (8). In a retrospective review, Moran et al. (9) examined the microbiological spectrum of 112 patients undergoing debridement and irrigation for a periprosthetic joint infection (hip [52], knee [51], elbow [4], ankle [3], shoulder [2]) at a tertiary care center in the United Kingdom during 1998–2003. The most frequently isolated microorganisms were coagulase-negative staphylococci (47%) followed by methicillin-sensitive S. aureus (44%), methicillin-resistant S. aureus (8%), aerobic gram-negative organisms (8%), and anaerobes (7%). Thirty-seven percent of patient specimens grew multiple microorganisms. We document the ability of B. extructa to cause an infection beyond its usual habitat, the oral flora. We hypothesize that the infection in this patient might have developed from hematogenous seeding in which an undiscovered and asymptomatic oral infectious nidus might have served as the seeding focus while mild trauma to the hip could have facilitated access to the joint space.

Isolation and identification of moderately halophilic bacteria producing hydrolytic enzymes from the largest hypersaline playa in Iran

Iran has many hypersaline environments, both the permanent and seasonal ones. One of the seasonal hypersaline lakes in the central desert zone is Aran-Bidgol Lake in which microbial diversity has not been characterized, thus the potential usage of this microbial community in biotechnology remained unknown. In this study, screening the halophilic hydrolytic enzyme-producing bacteria from different areas of this lake led to isolation of 61 gram-positive and 22 gram-negative moderately halophilic bacteria. These bacterial isolates were shown to produce a wide variety of hydrolytic enzymes including DNase, inulinase, amylase, lipase, pectinase, protease, chitinase, pullulanase, cellulase, and xylanase. The most common enzymes were DNase and inulinase in gram-positive bacteria, lipase in gram-negative bacteria, and pullulanase and cellulase in gram-positive cocci. Interestingly, combined hydrolytic activates were observed in some isolates. According to their phenotypic characteristics and comparative partial 16S rRNA sequence analysis, the moderately halophilic strains belonged to the genera Halobacillus, Thalassobacillus, Bacillus, Salinicoccus, Idiomarina, Salicola, and Halomonas.

Intestinal Dysbiosis and Depletion of Butyrogenic Bacteria in Clostridium difficile Infection and Nosocomial Diarrhea

Clostridium difficile infection (CDI) causes nearly half a million cases of diarrhea and colitis in the United States each year. Although the importance of the gut microbiota in C. difficile pathogenesis is well recognized, components of the human gut flora critical for colonization resistance are not known. Culture-independent high-density Roche 454 pyrosequencing was used to survey the distal gut microbiota for 39 individuals with CDI, 36 subjects with C. difficile-negative nosocomial diarrhea (CDN), and 40 healthy control subjects. A total of 526,071 partial 16S rRNA sequence reads of the V1 to V3 regions were aligned with 16S databases, identifying 3,531 bacterial phylotypes from 115 fecal samples. Genomic analysis revealed significant alterations of organism lineages in both the CDI and CDN groups, which were accompanied by marked decreases in microbial diversity and species richness driven primarily by a paucity of phylotypes within the Firmicutes phylum. Normally abundant gut commensal organisms, including the Ruminococcaceae and Lachnospiraceae families and butyrate-producing C2 to C4 anaerobic fermenters, were significantly depleted in the CDI and CDN groups. These data demonstrate associations between the depletion of Ruminococcaceae, Lachnospiraceae, and butyrogenic bacteria in the gut microbiota and nosocomial diarrhea, including C. difficile infection. Mechanistic studies focusing on the functional roles of these organisms in diarrheal diseases and resistance against C. difficile colonization are warranted.

Antifungal attributes of siderophore produced by the Pseudomonas aeruginosa JAS‐25

Siderophores produced by Pseudomonas sp. play a vital role in controlling several plant diseases. Public concern about chemical pesticides has fostered an interest to seek alternative control strategies. Production of siderophore by Pseudomonas aeruginosa JAS-25 in King B medium was estimated at 400 nm and was partially purified by gel filtration chromatography using Sephadex G 25. Antifungal activity and inhibition of spore germination was studied against the phytopathogens like Fusarium oxysporum f. sp. ciceri, Fusarium udum, and Aspergillus niger by well plate assay and microphotographic method. Zone of inhibition of 3.0 cm was observed for F. udum and A. niger, proving it as potential biocontrol agent against phytopathogens. Partial 16S rRNA sequence analysis showed that the isolate JAS-25 exhibited 99% homology with P. aeruginosa and could be of novel species and the sequence of the strain was deposited in Genbank with the accession number JX104229.

PCR-based survey of vector-borne pathogens in dogs in Dhaka, Bangladesh

To identify tick-borne pathogens in blood samples from 68 dogs were examined for Babesia, Anaplasma/Ehrichia, Mycoplasma, and Hepatozoon spp. by using a molecular tool. The detection of DNA was performed using specific primers for partial 16S rRNA or 18S rRNA sequence, and amplicon sequences were analyzed. Our investigation detected Babesia gibsoni (26 dogs) and Anaplasma sp. AnHl446 (2 dogs), Mycoplasma haemocanis (27 dogs), and an unknown Mycoplasma spp were detected (2 dogs), which was most closely related to Candidatus Mycoplasma turicensis, on the basis of phylogenetic analysis. Hepatozoon DNA could not be detected in this study.DOI: http://dx.doi.org/10.3329/jbau.v10i2.14915 J. Bangladesh Agril. Univ. 10(2): 249-253, 2012

English

A total of 66 fluorescent pseudomonads strains isolated from diverse Tunisian environmental biotypes (wastewater, compost, wastewater treatment plant, etc.) were analysed by two polymerase chain reaction (PCR)-based methods, 16S-23S intergenic spacer regions (ITS)-PCR and repetitive extragenic palindromic (BOX)-PCR. These typing techniques were evaluated to assess their usefulness as tool to study thePseudomonas diversity within this complex group. Genetic analysis using ITS- and BOX-PCR generated respectively 12 and 45 distinct profiles. Phylogenetic relationships within fluorescent pseudomonads were examined by analyzing partial 16S rRNA and rpoBgenes sequences. The phylogenetic resolution of the rpoB tree was higher than that of the 16S rRNA tree. Moreover, the sequencing of the rpoB gene has recognized 13 different species and sub-species, while the 16 rRNA gene sequencing differentiated only 9 species.   Key words: Fluorescent pseudomonads, 16S-23S intergenic spacer regions (ITS)-PCR, repetitive extragenic palindromic (BOX)-PCR, 16S rRNA, rpoB.

First Report of Soft Rot Disease Caused by Pectobacterium wasabiae on Sweet Potato, Tomato, and Eggplant in Malaysia.

In August 2011, sweet potato (Ipomoea batatas), tomato (Solanum lycopersicum), and eggplant (S. melongena) crops from major growing areas of the Cameron highlands and Johor state in Malaysia were affected by a soft rot disease. Disease incidence exceeded 80, 75, and 65% in severely infected fields and greenhouses of sweet potato, tomato, and eggplant, respectively. The disease was characterized by dark and small water-soaked lesions or soft rot symptoms on sweet potato tubers, tomato stems, and eggplant fruits. In addition, extensive discoloration of vascular tissues, stem hollowness, and water-soaked, soft, dark green lesions that turned brown with age were observed on the stem of tomato and eggplant. A survey was performed in these growing areas and 22 isolates of the pathogen were obtained from sweet potato (12 isolates), tomato (6 isolates), and eggplant (4 isolates) on nutrient agar (NA) and eosin methylene blue (EMB) (4). The cultures were incubated at 27°C for 2 days and colonies that were emerald green on EMB or white to gray on NA were selected for further studies. All bacterial cultures isolated from the survey exhibited pectolytic ability on potato slices. These bacterial isolates were gram negative; rod shaped; N-acetylglucosaminyl transferase, gelatin liquefaction, and OPNG positive; and were also positive for acid production from D-galactose, lactosemelibiose, raffinose, citrate, and trehalose. They were negative for indol production, phosphatase activity, reducing substances from sucrose, and negative for acid production from maltose, sorbitol, inositol, inolin, melezitose, α-mathyl-D-glocoside, and D-arabitol. The bacteria did not grow on NA at 37°C. Based on these biochemical and morphological assays, the pathogen was identified as Pectobacterium wasabiae (2). In addition, DNA was extracted and PCR assay with two primers (16SF1 and 16SR1) was performed (4). Partial sequences of 16S rRNA (GenBank Accession Nos. JQ665714, JX494234, and JX513960) of sweet potato, tomato, and eggplant, respectively, exhibited a 99% identity with P. wasabiae strain SR91 (NR_026047 and NR_026047.1). A pathogenicity assay was carried out on sweet potato tubers (cv. Oren), tomato stems (cv. 152177-A), and eggplant fruits (cv. 125066x) with 4 randomly representative isolates obtained from each crop. Sweet potato tubers, tomato stems, and eggplant fruits (4 replications) were sanitized in 70% ethyl alcohol for 30 s, washed and rinsed in sterile distilled water, and needle punctured with a bacterial suspension at a concentration of 108 CFU/ml. Inoculated tubers, stems, and fruits were incubated in a moist chamber at 90 to 100% RH for 72 h at 25°C when lesions were measured. All inoculated tubers, stems, and fruits exhibited soft rot symptoms after 72 h similar to those observed in the fields and greenhouses and the same bacteria were consistently reisolated. Symptoms were not observed on controls. The pathogenicty test was repeated with similar results. P. wasabiae have been previously reported to cause soft rot on Japanese horseradish (3), and aerial stem rot on potato in New Zealand (4), the U.S. (2), and Iran (1). To our knowledge, this is the first report of sweet potato, tomato, and eggplant soft rot caused by P. wasabiae in Malaysia.

Genetic relatedness and phylogenetics of five Indian pufferfishes

The taxonomy and phylogeny of the pufferfishes belonging to the family Tetraodontidae found in India are poorly understood. We investigated five species of freshwater and marine pufferfishes using partial sequences of 16S rRNA and cytochrome c oxidase subunit I (COI) of mitochondrial genes. The sequence alignment of 16S rRNA yielded 573 bp, whereas COI gene sequence alignment yielded 614 bp. The sequence analysis of the genes revealed two distinct groups of freshwater and marine origin, which are genetically distinct from each other and exhibit identical phylogenetic resolution. The partial sequences of both the genes provided sufficient phylogenetic resolution to distinguish all the five species of pufferfishes. The COI sequences could be used as DNA barcodes for identification of the pufferfishes.

Phylogeny of Indonesian Nostoc (Cyanobacteria) Isolated from Paddy Fields as Inferred from Partial Sequence of 16S rRNA Gene

In order to collect Indonesian Nostoc, isolation of soil microflora from several paddy fields in West Java, Bali, and South Celebes was carried out. Fast-growing isolates of Nostoc were selected to describe and perform molecular identification using partial sequences of 16S rRNA. The results showed that partial sequences of 16S rRNA could not resolve the phylogeny of the isolates. However, it supported the morphological studies that recognize isolates as different species of Nostoc. Potential use of Nostoc as a nitrogen source for paddy growth was carried out using six strains as single inoculums. A total biomass of 2 g (fresh weight) for each strain was inoculated, respectively, into the pot planted with three paddy plants. This experiment was conducted in the green house for 115 days. Statistical analyses (ANOVA; α = 0.05) showed that of six strains tested in this study, only strain GIA13a had influence on the augmentation of root length and the total number of filled grains.

Plasmid-mediated Molluscicide Bayluscide Degradation by Bacterial Strain Isolated from Schistosome Vector Snail Bulinus truncates

Pseudomonas spp. strain Bal1 was isolated from Schistosome vector snails Bulinus Truncates. Strain Bal1 was identified as Pseudomonas putida using partial sequence of 16s rRNA. This strain was able to utilize a Bayluscide as a sole source of carbon and nitrogen. The degradation of Bayluscide by Bal1 strain is mediated by pBal1 (110 Kb) plasmid. The loss of the plasmid resulted irreversibly in a derivative strain that was unable to degrade Bayluscide. The transfer of these plasmids from wild-type strain Bal1 to Bal1M derivative restored completely its capability to degrade the molluscicide. It is proposed that pBal1 is a conjugative plasmid and is involved in the Bayluscide degradation. The effect of bacterial degradation upon toxicity was tested, and it was shown that the molluscicidal action of Bayluscide was significantly reduced by bacterial action.

A tool for diagnosis of Dicrocoelium dendriticum infection: hatching eggs and molecular identification of the miracidium

DNA primers were designed from the 18S rRNA sequence from the relevant digenean trematode Dicrocoelium dendriticum to evaluate a polymerase chain reaction-based diagnostic method of this parasite from its eggs in faeces of naturally and experimentally infected sheep. In order to get DNA from D. dendriticum eggs, several hatching mechanisms were studied. Successful results were obtained when the eggs were frozen to -80°C and/or in liquid nitrogen and then defrosted. This method allowed the opening of the egg operculum and the liberation of the miracidium. DNA from D. dendriticum adults and from hatching egg miracidia was obtained and an amplification single band of 1.95kb was observed using primers designed for the total 18S rRNA sequence in both cases as well as when the template DNA was from adults of the closely related parasite Fasciola hepatica; in addition, a single and specific 0.8-kb band was obtained when primers based on an internal partial 18S rRNA sequence were used. The method showed to be useful not only in samples coming from adults, but in eggs from gall bladder and faeces as well. F. hepatica internal 18S rRNA primers were also designed and used as a negative control to prove that the eggs in faeces came from D. dendriticum and not from F. hepatica. A molecular tool able to detect a minimum of about 40 D. dendriticum eggs in one of the definitive host faeces has been developed for the first time and could provide a useful molecular tool to improve the conventional coprological diagnosis for detecting D. dendriticum eggs.

Characterisation of significant Gram positive bacilli from soft tissue infections

Gram positive bacilli (GPB) isolated from soft tissue infections are often neglected or ignored due to their fastidious nature and the lack of reliable phenotypic identification methods. This study was done to characterise clinically significant aero-tolerant GPB isolated from surgically obtained samples in patients with soft tissue infections. Forty-six GPB isolates collected during a 2 year study period were identified using partial 16s rRNA sequencing and API Coryne. Antibiotic susceptibility testing to penicillin, amoxycillin/clavulanate, moxifloxacin and erythromycin was performed on these isolates using Etest. Clinical data were gathered from patients' medical records. The most common isolates identified by 16s rRNA sequencing were Actinomyces species (n = 30, 65%) and Corynebacterium species (n = 9, 20%). The majority of the Actinomyces species infections were located below the waist, in particular the perianal region. There was poor agreement between API Coryne and genotypic identification, with only one-third of the isolates being correctly identified to species level. Actinomyces species were uniformly susceptible to penicillin and amoxicillin/clavulanate. Antibiotic susceptibilities were more varied for the other genera isolated. Actinomyces species comprised two-thirds of aerobically growing GPB isolates and may represent an under-reported cause of bacterial soft tissue infections. Penicillin and amoxycillin/clavulanate may be the empiric antibiotics of choice for Actinomyces species as all isolates were susceptible.

The aberrant millipede genus Pteridoiulus and its position in a revised molecular phylogeny of the family Julidae (Diplopoda : Julida)

A phylogenetic analysis of 62 species (32 genera) of the Palaearctic millipede family Julidae, including the aberrant alpine genus Pteridoiulus Verhoeff, 1913, was made based on partial sequences of the mitochondrial 16S rRNA (16S) gene and the nuclear 28S rRNA (28S) gene, respectively. The two datasets (16S rDNA and 28S rDNA) were analysed in combination but treated as different partitions using direct optimisation as implemented in POY. The 16S rDNA and the 28S rDNA sequences vary from 410 to 449 bp and from 467 to 525 bp in length, respectively. All searches were performed under six different gap opening costs, an extension gap cost of 1, and a substitution cost of 2. Based on previous investigations the optimal gap opening cost was set to 4, and the robustness of different gap opening costs ranging from 1 to 6 investigated. Additionally, the two data partitions were aligned individually using MAFTT and run in TNT both with gaps treated as a fifth state, and as missing, and finally the alignments were used as input in a maximum likelihood (ML) analysis. The order Julida and the family Julidae were recovered as monophyletic under all weight sets in POY, as well as in the TNT and ML analyses. Likewise, in all analyses Pteridoiulus was found to be sister to another monotypic alpine genus, Heteroiulus Verhoeff, 1897. The Pteridoiulus + Heteroiulus clade is robust to parameter changes but lacks morphological support. The distribution pattern of the clade, Pteridoiulus in the eastern Alps (mainly Austria), Heteroiulus in the southern Alps (Italy) seems unique, since similar patterns all involve very similar, closely related species pairs.

Pseudomonas fluorescens-like bacteria from the stomach: A microbiological and molecular study

To characterize oxidase- and urease-producing bacterial isolates, grown aerobically, that originated from antral biopsies of patients suffering from acid peptic diseases. A total of 258 antral biopsy specimens were subjected to isolation of bacteria followed by tests for oxidase and urease production, acid tolerance and aerobic growth. The selected isolates were further characterized by molecular techniques viz. amplifications for 16S rRNA using universal eubacterial and HSP60 gene specific primers. The amplicons were subjected to restriction analysis and partial sequencing. A phylogenetic tree was generated using unweighted pair group method with arithmetic mean (UPGMA) from evolutionary distance computed with bootstrap test of phylogeny. Assessment of acidity tolerance of bacteria isolated from antrum was performed using hydrochloric acid from 10(-7) mol/L to 10(-1) mol/L. Of the 258 antral biopsy specimens collected from patients, 179 (69.4%) were positive for urease production by rapid urease test and 31% (80/258) yielded typical Helicobacter pylori (H. pylori) after 5-7 d of incubation under a microaerophilic environment. A total of 240 (93%) antral biopsies yielded homogeneous semi-translucent and small colonies after overnight incubation. The partial 16S rRNA sequences revealed that the isolates had 99% similarity with Pseudomonas species. A phylogenetic tree on the basis of 16S rRNA sequences denoted that JQ927226 and JQ927227 were likely to be related to Pseudomonas fluorescens (P. fluorescens). On the basis of HSP60 sequences applied to the UPGMA phylogenetic tree, it was observed that isolated strains in an aerobic environment were likely to be P. fluorescens, and HSP60 sequences had more discriminatory potential rather than 16S rRNA sequences. Interestingly, this bacterium was acid tolerant for hours at low pH. Further, a total of 250 (96.9%) genomic DNA samples of 258 biopsy specimens and DNA from 240 bacterial isolates were positive for the 613 bp amplicons by targeting P. fluorescens-specific conserved putative outer membrane protein gene sequences. This study indicates that bacterial isolates from antral biopsies grown aerobically were P. fluorescens, and thus acid-tolerant bacteria other than H. pylori can also colonize the stomach and may be implicated in pathogenesis/protection.