Soil-transmitted helminth infections in free-ranging non-human primates from Cameroon and Gabon.

BACKGROUND: Zoonotic diseases are a serious threat to both public health and animal conservation. Most non-human primates (NHP) are facing the threat of forest loss and fragmentation and are increasingly living in closer spatial proximity to humans. Humans are infected with soil-transmitted helminths (STH) at a high prevalence, and bidirectional infection with NHP has been observed. The aim of this study was to determine the prevalence, genetic diversity, distribution and presence of co-infections of STH in free-ranging gorillas, chimpanzees and other NHP species, and to determine the potential role of these NHP as reservoir hosts contributing to the environmental sustenance of zoonotic nematode infections in forested areas of Cameroon and Gabon. METHODS: A total of 315 faecal samples from six species of NHPs were analysed. We performed PCR amplification, sequencing and maximum likelihood analysis of DNA fragments of the internal transcribed spacer 2 (ITS2) nuclear ribosomal DNA to detect the presence and determine the genetic diversity of Oesophagostomum spp., Necator spp. and Trichuris spp., and of targeted DNA fragments of the internal transcribed spacer 1 (ITS1) to detect the presence of Ascaris spp. RESULTS: Necator spp. infections were most common in gorillas (35 of 65 individuals), but also present in chimpanzees (100 of 222 individuals) and in one of four samples from greater spot-nosed monkeys. These clustered with previously described type II and III Necator spp. Gorillas were also the most infected NHP with Oesophagostomum (51/65 individuals), followed by chimpanzees (157/222 individuals), mandrills (8/12 samples) and mangabeys (7/12 samples), with O. stephanostomum being the most prevalent species. Oesophagostomum bifurcum was detected in chimpanzees and a red-capped mangabey, and a non-classified Oesophagostomum species was detected in a mandrill and a red-capped mangabey. In addition, Ternidens deminutus was detected in samples from one chimpanzee and three greater spot-nosed monkeys. A significant relative overabundance of co-infections with Necator and Oesophagostomum was observed in chimpanzees and gorillas. Trichuris sp. was detected at low prevalence in a gorilla, a chimpanzee and a greater spot-nosed monkey. No Ascaris was observed in any of the samples analysed. CONCLUSIONS: Our results on STH prevalence and genetic diversity in NHP from Cameroon and Gabon corroborate those obtained from other wild NHP populations in other African countries. Future research should focus on better identifying, at a molecular level, the species of Necator and Oesophagostomum infecting NHP and determining how human populations may be affected by increased proximity resulting from encroachment into sylvatic STH reservoir habitats.

One fungus, which genes? Development and assessment of universal primers for potential secondary fungal DNA barcodes.

The aim of this study was to assess potential candidate gene regions and corresponding universal primer pairs as secondary DNA barcodes for the fungal kingdom, additional to ITS rDNA as primary barcode. Amplification efficiencies of 14 (partially) universal primer pairs targeting eight genetic markers were tested across > 1 500 species (1 931 strains or specimens) and the outcomes of almost twenty thousand (19 577) polymerase chain reactions were evaluated. We tested several well-known primer pairs that amplify: i) sections of the nuclear ribosomal RNA gene large subunit (D1-D2 domains of 26/28S); ii) the complete internal transcribed spacer region (ITS1/2); iii) partial ß -tubulin II (TUB2); iv) γ-actin (ACT); v) translation elongation factor 1-α (TEF1α); and vi) the second largest subunit of RNA-polymerase II (partial RPB2, section 5-6). Their PCR efficiencies were compared with novel candidate primers corresponding to: i) the fungal-specific translation elongation factor 3 (TEF3); ii) a small ribosomal protein necessary for t-RNA docking; iii) the 60S L10 (L1) RP; iv) DNA topoisomerase I (TOPI); v) phosphoglycerate kinase (PGK); vi) hypothetical protein LNS2; and vii) alternative sections of TEF1α. Results showed that several gene sections are accessible to universal primers (or primers universal for phyla) yielding a single PCR-product. Barcode gap and multi-dimensional scaling analyses revealed that some of the tested candidate markers have universal properties providing adequate infra- and inter-specific variation that make them attractive barcodes for species identification. Among these gene sections, a novel high fidelity primer pair for TEF1α, already widely used as a phylogenetic marker in mycology, has potential as a supplementary DNA barcode with superior resolution to ITS. Both TOPI and PGK show promise for the Ascomycota, while TOPI and LNS2 are attractive for the Pucciniomycotina, for which universal primers for ribosomal subunits often fail.

High interlaboratory reproducibility of matrix-assisted laser desorption ionization-time of flight mass spectrometry-based species identification of nonfermenting bacteria.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry has emerged as a rapid, cost-effective alternative for bacterial species identification. Identifying 60 blind-coded nonfermenting bacteria samples, this international study (using eight laboratories) achieved 98.75% interlaboratory reproducibility. Only 6 of the 480 samples were misidentified due to interchanges (4 samples) or contamination (1 sample) or not identified because of insufficient signal intensity (1 sample).

Microbacterium binotii sp. nov., isolated from human blood.

Two strains of non-spore-forming, rod-shaped, Gram-positive bacteria, CIP 101303(T) and CIP 102116, were isolated from human blood in 1976 and 1977, respectively. These strains had chemotaxonomic markers that were consistent with classification in the genus Microbacterium, i.e. MK-10, MK-11 and MK-12 as the major menaquinones, predominant iso- and anteiso-branched cellular fatty acids, galactose, mannose and rhamnose as the cell-wall sugars and ornithine as the diamino acid in the cell-wall peptidoglycan. The DNA G+C content was 70-72 mol%. Comparative 16S rRNA gene sequence studies revealed that strains CIP 101303(T) and CIP 102116 belonged to the genus Microbacterium and that they were related closely to Microbacterium halotolerans. The level of DNA-DNA relatedness showed that the two isolates represented a separate genomic species. Based on phenotypic and genotypic results, it is proposed that strains CIP 101303(T) and CIP 102116 be assigned to a novel species, Microbacterium binotii sp. nov. The type strain is CIP 101303(T) (=DSM 19164(T)).

Human in vivo fluorescence microimaging of the alveolar ducts and sacs during bronchoscopy.

The aim of the present study was to assess fibred confocal fluorescence microscopy (FCFM) as a tool for imaging the alveolar respiratory system in vivo during bronchoscopy. A 488-nm excitation wavelength FCFM device was used in 41 healthy subjects including 17 active smokers. After topical anaesthesia, the 1.4-mm miniprobe was introduced into the bronchoscope working channel and advanced distally to the alveoli. Morphometric and cellular analyses were performed on selected frames harbouring a minimal compression effect. In vivo acinar microimaging was obtained from each lung segment except for the apical and posterior segments of both upper lobes. Reproducible patterns, corresponding to the elastic framework of the axial and peripheral interstitial systems, were recorded from 192 separate acini. The mean+/-sd thickness of the acinar elastic fibres was 10+/-2.7 microm. Alveolar mouth diameters (mean+/-sd 278+/-53 microm) were normally distributed but appeared smaller in the right upper lobe and right medial basal segment. Lobular microvessels (median diameter 90 microm) were equally distributed throughout the lungs. Alveolar macrophages were not detectable in nonsmokers, whereas a specific tobacco-tar-induced fluorescence was observed in smoking subjects, providing fine details of the alveolar walls and macrophages. A strong correlation was found between the number of cigarettes smoked per day and the amount of large and mobile macrophages observed in vivo, as well as with the intensity of the macrophage alveolitis. Fibred confocal fluorescence microscopy enables accurate exploration of the peripheral lung in vivo in both smokers and nonsmokers.

Bacillus aeolius DSM 15084T (=CIP 107628T) is a strain of Bacillus licheniformis.

Based on phenotypic properties, 16S rRNA gene sequence and MALDI-TOF analysis, strains DSM 15084T and CIP 107628T, deposited as the type strain of Bacillus aeolius, do not represent the original type strain, strain 4-1T. It is therefore proposed that the Judicial Commission of the International Committee on Systematics of Prokaryotes places the name Bacillus aeolius on the list of rejected specific and subspecific epithets in names of species and subspecies of bacteria if a suitable replacement for the type strain or a neotype cannot be found within 2 years of publication of this Request.

Polyphasic study of Chryseobacterium strains isolated from diseased aquatic animals.

Members of most Chryseobacterium species occur in aquatic environments or food products, while strains of some other species are pathogenic to humans and animals. A collection of 52 Chryseobacterium sp. strains isolated from diseased fish, one frog isolate and 22 reference strains were included in a polyphasic taxonomy study. Fourteen clusters of strains were delineated following the comparison of whole-cell protein profiles. Most of these clusters were confirmed when the phenotypic and RAPD profiles and the 16S rRNA gene sequences were compared. Fatty acid composition helped differentiate the Chryseobacterium strains from members of related genera. None of the fish isolates could be allocated to the two species previously reported from fish but two isolates belonged to C. joostei, while the frog isolate was identified as Elizabethkingia meningoseptica, a human pathogen previously included in the genus Chryseobacterium. Three clusters grouping from 3 to 13 isolates will probably constitute the core of new Chryseobacterium species but all other isolates occupied separate or uncertain positions in the genus. This study further demonstrated the overall high similarity displayed by most Chryseobacterium strains whatever the technique used and the resulting difficulty in delineating new species in the genus. Members of this bacterial group should be considered potential emergent pathogens in various fish and frog species, farming conditions and geographical areas.

[Hospital infection surveillance and multiresistance monitoring: role of an external quality control]. / Surveillance des infections nosocomiales à bactéries multirésistantes: un contrôle de qualité externe est nécessaire.

The "Relais Régional d'Hygiène Hospitalière du Centre" (RHC) promotes the hospital infection prevention at a regional level in France, including 80 healthcare institutions. The accuracy of antimicrobial susceptibility data submitted by laboratories to surveillance is essential. Since 2001, RHC imposed an external quality control to validate the accuracy of the data submitted by the laboratories that are involved in survey programs. Most laboratories are able to detect homogenous methicillin resistance in S. aureus, and high-level vancomycin resistance in E. faecalis. Nevertheless, the ability of laboratories to detect organisms with emerging antimicrobial resistance patterns is not optimal for (i) detection of heterogeneous methicillin resistance, (ii) reduced susceptibility to teicoplanin in a non-multiresistant S. aureus and (iii) detection of resistance to extended-spectrum cephalosporins. Educational program to optimize the testing methods has been programmed and perennially of quality control testing prior to accepting data from laboratory participating in surveillance system is decided.

A sensitive and rapid method to determine the viability of freeze-dried bacterial cells.

AIMS: To determine the potential use of flow cytometry for viability asssessment of freeze-dried bacterial cells. METHODS AND RESULTS: Escherichia coli CIP 54.8T and Vibrio metschnikovii CIP 104262 were analysed. The viability of freeze-dried cells resuspended in a nutrient broth was evaluated by culture whereas activity was determined by flow cytometry analysis of both esterase activity and cell death. Activity assessment by flow cytometry was found to be a rapid and good indicator of cell viability and was very efficient for quality control. For V. metschnikovii the fraction of active cells varies greatly depending on the freeze-drying procedure and within a given procedure. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: Bacterial activity assessment by flow cytometry is very efficient for the control of freeze-dried cells.

Identification of strains of Alcaligenes and Agrobacterium by a polyphasic approach.

The number of stable discriminant biochemical characters is limited in the genera Alcaligenes and Agrobacterium, whose species are consequently difficult to distinguish from one another by conventional tests. Moreover, genomic studies have recently drastically modified the nomenclature of these genera; for example, Alcaligenes xylosoxidans was transferred to the genus Achromobacter in 1998. Twenty-five strains of Achromobacter xylosoxidans, three strains of an Agrobacterium sp., five strains of an Alcaligenes sp., and four unnamed strains belonging to the Centers for Disease Control and Prevention group IVc-2 were examined. These strains were characterized by conventional tests, including biochemical tests. The assimilation of 99 carbohydrates, organic acids, and amino acids was studied by using Biotype-100 strips, and rRNA gene restriction patterns were obtained with the automated Riboprinter microbial characterization system after cleavage of total DNA with EcoRI or PstI restriction endonuclease. This polyphasic approach allowed the two subspecies of A. xylosoxidans to be clearly separated. Relationships between five strains and the Ralstonia paucula type strain were demonstrated. Likewise, three strains were found to be related to the Ochrobactrum anthropi type strain. We showed that substrate assimilation tests and automated ribotyping provide a simple, rapid, and reliable means of identifying A. xylosoxidans subspecies and that these two methods can be used as alternative methods to characterize unidentified strains rapidly when discriminant biochemical characters are missing.

Molecular characterization of chromosomal class C beta-lactamase and its regulatory gene in Ochrobactrum anthropi.

Ochrobactrum anthropi, formerly known as CDC group Vd, is an oxidase-producing, gram-negative, obligately aerobic, non-lactose-fermenting bacillus of low virulence that occasionally causes human infections. It is highly resistant to all beta-lactams except imipenem. A clinical isolate, SLO74, and six reference strains were tested. MICs of penicillins, aztreonam, and most cephalosporins tested, including cefotaxime and ceftazidime, were >128 microg/ml and of cefepime were 64 to >128 microg/ml. Clavulanic acid was ineffective and tazobactam had a weak effect in association with piperacillin. Two genes, ampR and ampC, were cloned by inserting restriction fragments of genomic DNA from the clinical strain O. anthropi SLO74 into pBK-CMV to give the recombinant plasmid pBK-OA1. The pattern of resistance to beta-lactams of this clone was similar to that of the parental strain, except for its resistance to cefepime (MIC, 0.5 ,micro/ml). The deduced amino acid sequence of the AmpC beta-lactamase (pI, 8.9) was only 41 to 52% identical to the sequence of other chromosomally encoded and plasmid-encoded class C beta-lactamases. The kinetic properties of this beta-lactamase were typical for this class of beta-lactamases. Upstream from the ampC gene, the ampR gene encodes a protein with a sequence that is 46 to 62% identical to those of other AmpR proteins and with an amino-terminal DNA-binding domain typical of transcriptional activators of the Lys-R family. The deduced amino acid sequences of the ampC genes of the six reference strains were 96 to 99% identical to the sequence of the clinical strain. The beta-lactamase characterized from strain SLO74 was named OCH-1 (gene, bla(OCH-I)).

Identification and functional characterization of arylamine N-acetyltransferases in eubacteria: evidence for highly selective acetylation of 5-aminosalicylic acid.

Arylamine N-acetyltransferase activity has been described in various bacterial species. Bacterial N-acetyltransferases, including those from bacteria of the gut flora, may be involved in the metabolism of xenobiotics, thereby exerting physiopathological effects. We characterized these enzymes further by steady-state kinetics, time-dependent inhibition, and DNA hybridization in 40 species, mostly from the human intestinal microflora. We report for the first time N-acetyltransferase activity in 11 species of Proteobacteriaceae from seven genera: Citrobacter amalonaticus, Citrobacter farmeri, Citrobacter freundii, Klebsiella ozaenae, Klebsiella oxytoca, Klebsiella rhinoscleromatis, Morganella morganii, Serratia marcescens, Shigella flexneri, Plesiomonas shigelloides, and Vibrio cholerae. We estimated apparent kinetic parameters and found that 5-aminosalicylic acid, a compound efficient in the treatment of inflammatory bowel diseases, was acetylated with a catalytic efficiency 27 to 645 times higher than that for its isomer, 4-aminosalicylic acid. In contrast, para-aminobenzoic acid, a folate precursor in bacteria, was poorly acetylated. Of the wild-type strains studied, Pseudomonas aeruginosa was the best acetylator in terms of both substrate spectrum and catalytic efficiency. DNA hybridization with a Salmonella enterica serovar Typhimurium-derived probe suggested the presence of this enzyme in eight proteobacterial and four gram-positive species. Molecular aspects together with the kinetic data suggest distinct functional features for this class of microbial enzymes.

Rapid detection of Campylobacter fetus by polymerase chain reaction combined with non-radioactive hybridization using an oligonucleotide covalently bound to microwells.

Campylobacter fetus is recognized as a human and animal pathogen. The isolation and differentiation of C. fetus in diagnostic laboratories is hindered by its relatively slow growth and lack of distinguishing biochemical characteristics. We cloned and sequenced a 1581-bp DNA fragment, IG02, isolated from a C. fetus genomic library. This fragment was used as a probe on DNAs extracted from C. fetus strains and other Campylobacter species: IG02 hybridized only with DNAs from C. fetus strains. A PCR-based test was developed for the detection of C. fetus. A pair of oligonucleotide primers was designed to amplify a 141-bp fragment of IG02. The amplified product was analysed by a non-radioactive sandwich hybridization in microtiter plate using a capture oligonucleotide and a biotin-labelled oligonucleotide for the detection. The combination of PCR and non-radioactive microplate hybridization is a convenient method for the rapid detection of C. fetus.

Lactobacillus species identification, H2O2 production, and antibiotic resistance and correlation with human clinical status.

Lactobacilli recovered from the blood, cerebrospinal fluid, respiratory tract, and gut of 20 hospitalized immunocompromised septic patients were analyzed. Biochemical carbohydrate fermentation and total soluble cell protein profiles were used to identify the species. Hydrogen peroxide production was measured. Susceptibility to 19 antibiotics was tested by a diffusion method, and the MICs of benzylpenicillin, amoxicillin, imipenem, erythromycin, vancomycin, gentamicin, and levofloxacin were determined. A small number of species produced H2O2, and antibiotic susceptibilities were species related. Eighteen (90%) of the isolates were L. rhamnosus, one was L. paracasei subsp. paracasei, and one was L. crispatus. L. rhamnosus, L. paracasei subsp. paracasei isolates, and the type strains were neither H2O2 producers nor vancomycin susceptible (MICs, >/=256 microgram/ml). L. crispatus, as well as most of the type strains of lactobacilli which belong to the L. acidophilus group, was an H2O2 producer and vancomycin susceptible (MICs, <4 microgram/ml).

Pulsed-field gel electrophoresis is more efficient than ribotyping and random amplified polymorphic DNA analysis in discrimination of Pasteurella haemolytica strains.

One hundred thirty-three strains of Pasteurella haemolytica of both biotypes (90 and 43 strains of biotypes A and T, respectively) and almost all the serotypes were subjected to ribotyping, random amplified polymorphic DNA (RAPD) analysis, and pulsed-field gel electrophoresis (PFGE) analysis for epidemiological purposes. A total of 15 patterns recorded as ribotypes HA to HO were found for the P. haemolytica biotype A strains, with ribotypes HA, HC, and HD being encountered most often (66 strains [74%]); and 20 ribotypes, designated HA' to HT', that were clearly distinct from those observed for biotype A strains were observed for strains of biotype T. RAPD analysis generated a total of 44 (designated Rp1 to Rp44) and 15 (designated Rp1' to Rp 15') unique RAPD patterns for biogroup A and biogroup T, respectively. Analysis of the data indicated that a given combined ribotype-RAPD pattern could be observed for biotype A strains of different serotypes, whatever the zoological or geographic origin, whereas this was not the case for biotype T strains. PFGE appeared to be more efficient in strain discrimination since selected strains from various zoological or geographical origins harboring the same ribotype-RAPD group were further separated into unique entities.

Targeting of HIV gp120 by oligonucleotide-photosensitizer conjugates. Light-induced damages.

Some guanine-rich oligonucleotides inhibit HIV infectivity through interaction with the gp120 glycoprotein. Besides, photoinactivation of viruses attracts attention for blood decontamination. The feasibility of targeting a red light-absorbing chlorin-type photosensitizer to gp120 through covalent coupling with 8-mer phosphodiester oligodeoxynucleotides is investigated. Some conjugates inhibit binding of antibodies directed to gp120. Inhibition is significantly increased upon red light activation. The activity of the conjugates correlates with their ability to self-associate, a process strongly favored by the propensity of the hydrophobic chlorin moiety to dimerize. Thus, the photosensitizer moiety both promotes structures with a higher affinity for gp120 and, upon light activation, can induce site-directed damages to the protein.

Purulent pleurisy caused by Campylobacter lari.

An 80-year-old debilitated patient developed purulent pleurisy caused by a Campylobacter lari isolate. The patient underwent surgical drainage and received antibiotic therapy with amoxicillin/clavulanic acid and ofloxacin. Antibiotic susceptibility data showed that the isolate was fully sensitive to clarithromycin, tetracycline, aminoglycosides. and ciprofloxacin. Imipenem and amoxicillin plus clavulanic acid were the most active beta-lactam agents.

Strains of Alcaligenes faecalis from clinical material.

Six strains of Alcaligenes faecalis, unusually isolated from clinical material, are described. Alcaligenes faecalis is a Gram-negative catalase- and oxidase-positive, motile rod. It is commonly found in a watery environment and is rarely isolated from humans. The clinical and laboratory characteristics of the clinical A. faecalis isolates are presented.

Electrophoretic pattern of peptidoglycan hydrolases, a new tool for bacterial species identification: application to 10 Lactobacillus species.

Lactobacilli have been used as industrial starters for a long time, but in many cases their phenotypic identification is still neither easy nor reliable. Previously we observed that the cell wall peptidoglycan hydrolases of Lactobacillus helveticus were highly conserved enzymes; the aim of the present work was to determine whether peptidoglycan hydrolase patterns obtained by renaturing SDS-PAGE could be of interest in the identification of lactobacilli species. For that purpose, the peptidoglycan hydrolase patterns of 94 strains of lactobacilli belonging to 10 different species were determined; most of the species studied are used either in dairy, meat, bakery or vegetable fermentations: L. helveticus, L. acidophilus, L. delbrueckii, L. brevis, L. fermentum, L. jensenii, L. plantarum, L. sake, L. curvatus and L. reuteri. Within a species, the strains exhibited highly similar patterns: the apparent molecular weights of the lytic bands were identical, with only slight variations of intensity. Moreover, each species, including phylogenetically close species such as L. sake and L. curvatus, or L. acidophilus and L. helveticus, gave a different pattern. Interestingly, the closer the species were phylogenetically, the more related were their patterns. The sensitivity of the method was checked using various quantities of L. acidophilus cells: a peptidoglycan hydrolase extract of 5 x 10(6) cells was sufficient to obtain an informative pattern, as was a single colony. Finally, the method was also successfully applied to distinguish two Carnobacterium species. In conclusion, the electrophoretic pattern of peptidoglycan hydrolases is proposed as a new tool for lactobacilli identification: it is rapid, sensitive and effective even for phylogenetically close species. Furthermore, this work provides the first evidence of the potential overall taxonomic value of bacterial peptidoglycan hydrolases.

Identification of Rhodococcus, Gordona and Dietzia species using carbon source utilization tests ("Biotype-100" strips).

The "Biotype-100" identification system (BioMérieux, La Balme-Ies-Grottes, France) based on carbon source utilization was evaluated for its ability to discriminate among 10 species of Rhodococcus, 7 species of Gordona and one species of Dietzia. The type strains of three species of Tsukamurella and 8 species of Nocardia were also included in the study. Results were compared with chemotaxonomic and conventional data. Carbon source utilization was shown to be reliable, rapid and easy to use when compared with standard identification methods. The 29 species tested were unambiguously separated by carbon source utilization tests. Rhodococcus equi was found to be heterogenous.