Implication of species change of Nontuberculous Mycobacteria during or after treatment

Background

Co-existence or subsequent isolation of multiple nontuberculous mycobacteria (NTM) species in same patient has been reported. However, clinical significance of these observations is unclear. The aim of this study was to determine clinical implications of changes of NTM species during or after treatment in patients with NTM lung disease.

Methods

Patients with NTM lung disease, who experienced changes of NTM species during treatment or within 2 years of treatment completion between January 1, 2009 and December 31, 2015, were included in the analysis. Demographic, clinical, microbiological, and radiographic data were reviewed and analyzed.

Results

During the study period, 473 patients were newly diagnosed with NTM lung disease. Treatment was started in 164 patients (34.6%). Among these 164 patients, 16 experienced changes of NTM species during or within 2 years of treatment completion. Seven showed changes from M. avium complex (MAC) to M. abscessus subspecies abscessus (MAA) and five patients displayed changes from M. abscessus subspecies massiliense (MAM) to MAC. With isolation of new NTM species, 6 out of 7 patients with change from MAC to MAA reported worsening of symptoms, whereas none of the five patients with change from MAM to MAC reported worsening of symptoms. All MAA isolated during or after treatment for MAC lung diseases showed inducible resistance to clarithromycin.

Conclusions

Change of NTM species may occur during or after treatment for NTM lung disease. Especially, changes from MAC to MAA is accompanied by symptomatic and radiographic worsening as well as inducible resistance to clarithromycin.

     

Comparison of the methods applicable for the pathogenicity assessment of entomopathogenic nematodes

Single infective juveniles of Heterorhabditis bacteriophora, H. megidis (Nematoda: Heterorhabditidae), Steinernema arenarium, S. carpocapsae and S. feltiae (Nematoda: Steinernematidae) were used to infect single Galleria mellonella (Lepidoptera: Pyralidae) larvae. Four parameters of entomopathogenic nematodes pathogenicity were assessed: the mortality of insects, infectivity of nematodes, number of nematodes established per single G. mellonella, and degree of infective juveniles colonization (percent of infective juveniles which intestine was colonized by symbiotic bacteria). The accuracy, repeatability, and versatility for different species of EPNs in bioassay arenas were compared. Our modifications of the original methods yielded ~ 50% higher efficiency of infective juveniles in cell culture plates and > 20% higher efficiency in centrifuge test tubes. The efficiency of nematodes in cell culture plates (39–77%) was relatively low, especially in the case of Heterorhabditis spp. In the bioassay arena, infective juveniles migrated between cells. The results of our studies indicate that the pathogenicity of EPNs should be assessed in centrifuge test tubes. In these arenas, the infectivity of single IJs was ~ 90% for Heterorhabditis spp. and ~ 95% for Steinernema spp. The degree of colonization of the EPN isolates by symbiotic bacteria was in the range of 96–98%.     

Water-related environments: a multistep procedure to assess the diversity and enzymatic properties of cultivable bacteria

Studying the culturable portion of environmental bacterial populations is valuable for understanding the ecology, for discovering taxonomically interesting isolates and for exploiting their enzymatic abilities. In this study, diverse water-related samples, iced water (3 °C) from river, the sediment (29 °C) and water (55 °C) of a hot-spring, were investigated by two cultivation strategies, Dry and novel Wet approach. The isolates were clustered by fluorescent internal transcribed spacer PCR and identified by 16S rRNA sequencing. Several bacterial groups were also sub-typed through the application of Random Amplified Microsatellite Polymorphisms method. A broad enzymatic screening of all bacterial isolates was performed in order to assess the proteolytic, cellulolytic, lipolytic, esterolytic, amylolytic properties, as well as catalase and peroxidase activities. The Wet cultivation demonstrated to be suitable for the isolation of potential new species belonging to genera Massilia, Algoriphagus, Rheinheimera and Pandoraea. Valuable microbial resources with extensive enzymatic activities were recognized among the psychrophilic (Pantoea brenneri and Serratia sp.), mesophilic (Pandoraea, Massilia, Pseudomonas, Stenotrophomonas, Bacillus and Aeromonas) and thermophilic bacteria (Aeribacullus pallidus and Geobacillus kaustophilus). The experimental strategy developed in this study includes simple investigation tools able to reveal the genetic and enzymatic peculiarities of isolated microorganisms. It can be applied to different kinds of aquatic samples and extreme environments similar to those described in this study.     

Exploring the diversity and metabolic potential of actinomycetes from temperate marine sediments from Newfoundland,Canada

Marine sediments from Newfoundland, Canada were explored for biotechnologically promising Actinobacteria using culture-independent and culture-dependent approaches. Culture-independent pyrosequencing analyses uncovered significant actinobacterial diversity (H′—2.45 to 3.76), although the taxonomic diversity of biotechnologically important actinomycetes could not be fully elucidated due to limited sampling depth. Assessment of culturable actinomycete diversity resulted in the isolation of 360 actinomycetes representing 59 operational taxonomic units, the majority of which (94 %) were Streptomyces. The biotechnological potential of actinomycetes from NL sediments was assessed by bioactivity and metabolomics-based screening of 32 representative isolates. Bioactivity was exhibited by 41 % of isolates, while 11 % exhibited unique chemical signatures in metabolomics screening. Chemical analysis of two isolates resulted in the isolation of the cytotoxic metabolite 1-isopentadecanoyl-3β-d-glucopyranosyl-X-glycerol from Actinoalloteichus sp. 2L868 and sungsanpin from Streptomyces sp. 8LB7. These results demonstrate the potential for the discovery of novel bioactive metabolites from actinomycetes isolated from Atlantic Canadian marine sediments.     

Diversity of culturable bacterial communities in the intestinal tracts of goldfish (<Emphasis Type="Italic">Carassius auratus</Emphasis>) and their ability to produce <Emphasis Type="Italic">N</Emphasis>-acyl homoserine lactone

Intestinal bacteria isolated from goldfish (Carassius auratus) were identified based on 16 ribosomal RNA (rRNA) gene sequences and screened for their ability to produce N-acyl homoserine lactone (AHL), an autoinducer of the quorum sensing (QS) system. The 230 aerobes/facultative anaerobes that were isolated comprised members of the genera Aeromonas (184 isolates), Citrobacter (11), Enterobacter (2), Shewanella (28), Vagococcus (1), and Vibrio (4). Among these genera, the two most abundant species were Aeromonas veronii (163 isolates) and Shewanella xiamenensis (27). In addition, 142 obligate anaerobes consisting of Cetobacterium somerae (139 isolates), Clostridium frigidicarnis (2), and Cetobacterium sp. (1) were also isolated. One hundred seventy isolates (74.2%) belonging to the genera Aeromonas, Citrobacter, Enterobacter, Shewanella, and Vibrio produced AHL, while 155 (67.7%) and 91 (39.7%) isolates possessed the luxR and luxI gene homologs, respectively. None of the obligate anaerobes produced AHL or possessed luxRI homologs. Total viable counts ranged from 1.2 × 10⁷ to 2.2 × 10⁹ CFU/g, which were accounted for 0.8 to 15.2% of direct counts. Aeromonas veronii, S. xiamenensis, and C. somerae were detected from five goldfish at densities ranging from 4.0 × 10⁶ to 1.7 × 10⁹ CFU/g, indicating that these bacteria are dominant components of the culturable gut flora in goldfish. In addition, members of the genera Aeromonas and Shewanella appeared to communicate with each other by using the QS system to some extent when the concentration of AHL reaches a certain threshold. It is therefore suggested that bacteria with the ability to disrupt AHL secretion in intestinal environments are potential candidates for probionts for preventing opportunistic infections in freshwater fish such as goldfish.     

Synthesis of disaccharides using β-glucosidases from <Emphasis Type="Italic">Aspergillus niger</Emphasis>, <Emphasis Type="Italic">A. awamori</Emphasis> and <Emphasis Type="Italic">Prunus dulcis</Emphasis>

Objective

Glucose conversion into disaccharides was performed with β-glucosidases from Prunus dulcis (β-Pd), Aspergillus niger (β-An) and A. awamori (β-Aa), in reactions containing initial glucose of 700 and 900 g l^?1.

Results

The reactions’ time courses were followed regarding glucose and product concentrations. In all cases, there was a predominant formation of gentiobiose over cellobiose and also of oligosaccharides with a higher molecular mass. For reactions containing 700 g glucose l^?1, the final substrate conversions were 33, 38, and 23.5% for β-An, β-Aa, and β-Pd, respectively. The use of β-An yielded 103 g gentiobiose l^?1 (15.5% yield), which is the highest reported for a fungal β-glucosidase. The increase in glucose concentration to 900 g l^?1 resulted in a significant increase in disaccharide synthesis by β-Pd, reaching 128 g gentiobiose l^?1 (15% yield), while for β-An and β-Aa, there was a shift toward the synthesis of higher oligosaccharides.

Conclusion

β-Pd and the fungal β-An and β-Aa β-glucosidases present quite dissimilar kinetics and selective properties regarding the synthesis of disaccharides; while β-Pd showed the highest productivity for gentiobiose synthesis, β-An presented the highest specificity.

     

Isolation of identical nitrilase genes from multiple bacterial strains and real-time PCR detection of the genes from soils provides evidence of horizontal gene transfer

Bacterial enzymes capable of nitrile hydrolysis have significant industrial potential. Microbacterium sp. AJ115, Rhodococcus erythropolis AJ270 and AJ300 were isolated from the same location in England and harbour identical nitrile hydratase/amidase gene clusters. Strain AJ270 has been well studied due to its nitrile hydratase and amidase activity. R. erythropolis ITCBP was isolated from Denmark and carries a very similar nitrile hydratase/amidase gene cluster. In this study, an identical nitrilase gene (nit1) was isolated from the four strains, and the nitrilase from strain AJ270 cloned and expressed in Escherichia coli. Analysis of the recombinant nitrilase has shown it to be functional with activity demonstrated towards phenylacetonitrile. A real-time PCR TaqMan^® assay was developed that allowed nit1 detection directly from soil enrichment cultures without DNA extraction, with nit1 detected in all samples tested. Real-time PCR screening of isolates from these soils resulted in the isolation of nit1 and also very similar nitrilase gene nit2 from a number of Burkholderia sp. The genes nit1 and nit2 have also been detected in many bacteria of different genera but are unstable in these isolates. It is likely that the genes were acquired by horizontal gene transfer and may be widespread in the environment.     

Diversity,molecular phylogeny and fingerprint profiles of airborne <Emphasis Type="Italic">Aspergillus</Emphasis> species using random amplified polymorphic DNA

In the present study, diversity and phylogenetic relationship of Aspergillus species isolated from Tehran air was studied using random amplified polymorphic DNA (RAPD)–polymerase chain reaction (RAPD-PCR). Thirty-eight Aspergillus isolates belonging to 12 species i.e. A. niger (28.94 %, 11 isolates), A. flavus (18.42 %, 7 isolates), A. tubingensis (13.15 %, 5 isolates), A. japonicus (10.52 %, 4 isolates), A. ochraceus (10.52 %, 4 isolates), and 2.63 %, 1 isolate from each A. nidulans, A. amstelodami, A. oryzae, A. terreus, A. versicolor, A. flavipes and A. fumigatus were obtained by settle plate method which they were distributed in 18 out of 22 sampling sites examined. Fungal DNA was extracted from cultured mycelia of all Aspergillus isolates on Sabouraud Dextrose Agar and used for amplification of gene fragments in RAPD-PCR using 11 primers. RAPD-PCR data was analyzed using UPGMA software. Resulting dendrogram of combined selected primers including PM1, OPW-04, OPW-05, P160, P54, P10 and OPA14 indicated the distribution of 12 Aspergillus species in 8 major clusters. The similarity coefficient of all 38 Aspergillus isolates ranged from 0.02 to 0.40 indicating a wide degree of similarities and differences within and between species. Taken together, our results showed that various Aspergillus species including some important human pathogenic ones exist in the outdoor air of Tehran by different extents in distribution and diversity and suggested inter- and intra-species genetic diversity among Aspergillus species by RAPD-PCR as a rapid, sensitive and reproducible method.     

Morpho-Molecular Characterization of Soil Inhabitant Dermatophytes from Ahvaz,Southwest of Iran,a High Occurrence of <Emphasis Type="Italic">Microsporum fulvum</Emphasis>

Occurrence and diversity of dermatophyte mycoflora in 298 soil samples from Ahvaz, Southwest of Iran was investigated by using the hair-baiting technique. The samples were collected during spring (n = 210) and autumn (n = 88) of 2015, and the fungal isolates were identified based on the macro- and micro-morphology of colonies and with further ITS-rDNA RFLP and sequencing. Totally, 60 soil samples (20.1%) were positive for dermatophyte growth whose pH varied from 7.0 to 7.9. The highest (26.6%) and the lowest (14.3%) recovery rates were from the animal resorts and the streets soils samples, respectively. Seasonally, 16.7% of the spring samples and 28.4% of the autumn samples were positive. Based on molecular identification, three species of two genera were identified viz. M. fulvum (n = 57), M. canis (n = 2) and zoophilic Trichophyton interdigitale (n = 1). As a specific goal in the study, differentiation of the species in Microsporum gypseum complex was established by measuring the mean length and width of macroconidia in some strains of M. gypseum, M. fulvum and M. incurvatum. Mean size for macroconidia length and width in three species showed that M. gypseum and M. incurvatum can morphologically be differentiated from M. fulvum but not from each other. M. fulvum was the most abundant species isolated from the soils of Ahvaz; however, to comprehensively specify the distribution pattern of geophilic dermatophytes in the soils of this city further investigations are needed. Identification based on micro-morphometric is not effective for species distinction in M. gypseum complex, while molecular procedures based on sequencing of certain DNA regions are the most reliable and applicable strategies for this purpose.     

Dispersal of <Emphasis Type="Italic">Fusarium</Emphasis> spp. by rainwater and pathogenicity on four plant species

Research focused on the occurrence of Fusarium spp. in atmospheric dust or rainwater is not common. Preliminary studies with four sampling dates in 2007 revealed that several species of Fusarium may also be conveyed by rainwater. In order to determine the regular presence of Fusarium spp. in rainfall water, samples were systematically collected for a year (from October 2009 to October 2010) in three points on the Mediterranean coast of the province of Granada (Spain) 10-km distance between them. Throughout the year of sampling, a total of 179 rainwater samples were collected during every significant rainfall event. Eight different Fusarium species were isolated from the rainwater samples: F. oxysporum (32 %), F. proliferatum (26 %) and F. equiseti (20 %) coincide with previous studies, while F. dimerum (3 %), F. semitectum (4.7 %), F. solani (8 %), F. avenaceum (0.5 %) and F. chlamydosporum (3.7 %) were isolated for the first time from rainwater. Results were consistent with previous surveys conducted 100 km away from the sampling sites. Inoculation of 39 different isolates from five different Fusarium species showed pathogenicity on plants. Disease severity differed depending on the inoculated plant species, which means that rain water can be an effective vector to transport new pathogens into new cultivated areas. This work reveals some epidemiological aspects of Fusarium genus in natural environments. Some of the isolated Fusarium spp. are potential mycotoxin producers, such as zearalenone, fumonisin, moniliformin or nivalenol.     

Clinical and Microbiological Investigation of Fungemia from Four Hospitals in China

In this study, fungemia cases from four tertiary hospitals located in Shanghai and Anhui province in China from March 2012 to December 2013 were enrolled to investigate clinical features, species distribution, antifungal susceptibility and strain relatedness. During the study period, 137 non-duplicate cases and their corresponding isolates were collected. Six different genera of fungi were identified, of which Candida spp. were the most common (126/137, 91.97 %), with C. albicans predominating (48/137, 35.03 %). The non-Candida fungi rate reached 8.03 % (11/137), and Pichia spp. was the most common (5/137, 3.65 %). Compared with C. albicans, non-C. albicans fungi-associated fungemia was more likely in younger (p = 0.004) and male patients (χ ² = 6.2618, p = 0.0123) and patients from ICUs (χ ² = 6.3783, p = 0.0116). Overall, the susceptible/WT rates of common Candida spp. to fluconazole, itraconazole, voriconazole, flucytosine, amphotericin B and caspofungin were 74.63, 92.31, 93.16, 96.58, 100 and 95.69 %, respectively. C. tropicalis and C. guilliermondii had a low susceptibility to fluconazole: 79.95 and 77.78 %, respectively. No isolates were resistant/WT to caspofungin, but C. parapsilosis and C. guilliermondii had high MIC₉₀ values; 1 and 2 mg/L, respectively. In terms of genotyping, MLST was taken for C. glabrata and C. tropicalis, while microsatellite marker analysis was used for C. albicans and C. parapsilosis. C. glabrata was predominantly clone ST7, accounting for 75 %, while the other isolates showed genetic diversity. Considering the increased proportion of non-C. albicans fungi and the presence of endemic clones of C. glabrata, it is essential to undertake additional surveillance of fungemia.     

Characterization of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> isolates from pediatric patients with cystic fibrosis

Staphylococcus aureus is one of the major respiratory pathogens associated with cystic fibrosis (CF) patients. In this study, we collected sputum and isolated fifty S. aureus isolates from CF patients with the median age of 9.5 years old. Then we determined the profiles of these isolates by antibiotic susceptibility testing, examining their cytotoxicity and ability to internalize into an epithelial cell line (A549), as well as multiple loci sequencing typing. Predominant CF S. aureus isolates were resistant to penicillin; however, these isolates were sensitive to various antibiotics, such as vancomycin and minocycline. Different CF S. aureus isolates showed distinct cytotoxic activities, and 90 % of CF S. aureus isolates possessed the enterotoxin genes, sea and hlg. Moreover, we found that multiple different CF S. aureus isolates appeared to have the distinct capacity of invading A549 cells. ST5 (14 %), ST30 (14 %), and ST8 (10 %) were prevalent ST types in these isolates. Further analysis revealed that ST5 and ST30 isolates were less toxic than ST8 and ST15 isolates, and that the ST5, ST15, ST59, and ST87 types of CF S. aureus were less capable of invading A549 cells. Our results suggest that the ST typing method may be useful in predicting cytotoxicity and the invading capacity of S. aureus isolates from patients with CF.     

Growth and physiochemical responses of <Emphasis Type="Italic">Camelina sativa</Emphasis> L. under UV-C stress

A study was conducted to estimate the effect of UV-C (200–280 nm) radiation stress on growth and physiochemical responses of Camelina sativa L. cv. Calina (EC643910; a potential bio-fuel crop) for its possible mass multiplication at high-altitude under high radiations. The germination percentage in terms of radicle protrusion and opening of cotyledonary leaves significantly decreased 13.98 and 27.8 %, respectively, as compared to control. However, no significant change was observed in growth parameters including root and shoot lengths and fresh biomass. The relative membrane leakage rate and lipid peroxidation as a malondialdehyde contents significantly increased with the value of ~99 % and 0.17 mM g^?1 FW, respectively, under UV-C stress. Also, the proline, glycine betaine and total soluble sugars contents increased by ~330, ~440, ~144 %, respectively, as compared to control. Among non-enzymatic antioxidants, the ascorbic acid and total phenol contents significantly increased by ~284 and ~537 %, respectively, as compared to control. Likewise, the activity of antioxidant enzyme, ascorbate peroxidase, guaiacol peroxidase and catalase increased under UV-C stress with the value of 1.03, 0.05 and 0.06 µmol mg protein^?1 min^?1, respectively. In addition, the chlorophyll a, b and total (a + b) contents decreased by ~180, ~151 and ~147 %, respectively, as compared to control. In contrast, the total carotenoids and anthocyanin contents increased by ~160 and ~184 %, respectively. Our findings suggest the adaptive growth and physiochemical responses of C. sativa under UV-C stress. Therefore, it may be recommended for large-scale cultivation at high-altitude under intense natural radiations for future bio-fuel production.     

Comparative analysis of prokaryotic diversity in solar salterns in eastern Anatolia (Turkey)

The prokaryotic communities of four salterns (Bingöl, Fadlum, Kemah, and Tuzlagözü) in Turkey were examined and compared using the cultivation and cultivation-independent methods [fluorescence in situ hybridization (FISH) and 454 pyrosequencing]. FISH analysis with universal probes revealed that feeding waters carried 1.6 × 10²–1.7 × 10³ cells mL^?1, while crystallization ponds carried 3.8 × 10⁶–2.0 × 10⁷ cells mL^?1 that were mostly haloarchaea, including square cells (except for Kemah). High-throughput 16S rRNA-based gene sequencing showed that the most frequent archaeal OTUs in Bingöl, Fadlum, Tuzlagözü, and Kemah samples were affiliated with Haloquadratum (76.8 %), Haloarcula (27.8 %), Halorubrum (49.6 %), and Halonotius (59.8 %), respectively. Bacteroidetes was the dominant bacterial phylum in Bingöl and Fadlum, representing 71.5 and 79.5 % of the bacterial OTUs (respectively), while the most abundant bacterial phylum found in the Kemah saltern was Proteobacteria (79.6 %). The majority of the bacterial OTUs recovered from Tuzlagözü belonged to the Cyanobacteria (35.7 %), Bacteroidetes (35.0 %), and Proteobacteria (25.5 %) phyla. Cultivation studies revealed that the archaeal isolates were closely related to the genera Halobacterium, Haloarcula, and Halorubrum. Bacterial isolates were confined to two phyla, Proteobacteria (Alphaproteobacteria and Gammaproteobacteria classes) and Bacteroidetes. Comparative analysis showed that members of the Euryarchaeota, Bacteroidetes, Proteobacteria, and Cyanobacteria phyla were major inhabitants of the solar salterns.     

Pig farm environment as a source of beta-lactamase or AmpC-producing <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> and <Emphasis Type="Italic">Escherichia coli</Emphasis>

The present study was undertaken to detect the occurrence of beta-lactamase-/AmpC-producing Klebsiella and Escherichia coli in healthy pigs, feed, drinking water, and pen floor or surface soil. The study also intended to detect the clonal relationship between the environmental and porcine isolates to confirm the route of transmission. Rectal swabs and environmental samples were collected from apparently healthy pigs kept in organized or backyard farms in India. The pigs had no history of antibiotic intake. Production of phenotypical beta-lactamase, associated genes, and class I integron gene was detected in E. coli and Klebsiella isolates. The phylogenetic relationship among the isolates was established on the basis of Random amplification of polymorphic DNA banding pattern. Beta-lactamase-producing Klebsiella were isolated from healthy pigs (20.0%), pen floor swabs/surface soil swabs (14.0%), and drinking water (100%). Escherichia coli isolated from healthy pigs (14.4%), pen floor/surface soil (8.0%), and drinking water (33.3%) were detected as beta-lactamase producers. Majority of beta-lactamase-producing isolates possessed bla_CTX-M-9. Further, 35 (81%) Klebsiella and all the E. coli isolates were detected as AmpC beta-lactamase ACBL producers and possessed bla_AmpC. Sixteen beta-lactamase-producing Klebsiella (37.20%) and 13 E. coli (86.67%) possessed class I integron. Few resistant isolates from environmental sources (surface soil swab and drinking water) and the studied pigs were detected within the same cluster of the dendrogram representing their similarities. The study indicated about the possible role of contaminated environment as a source of beta-lactamase/AmpC-producing Klebsiella and E. coli in pigs.     

Characterization of bacterial composition and diversity in a long-term petroleum contaminated soil and isolation of high-efficiency alkane-degrading strains using an improved medium

Bacterial community and diversity in a long-term petroleum-contaminated soil of an oilfield were characterized using 16S rRNA gene-based Illumina MiSeq high-throughput sequencing. Results indicated that Proteobacteria (49.11%) and Actinobacteria (24.24%) were the most dominant phyla, and the most abundant genera were Pseudoxanthomonas (8.47%), Luteimonas (3.64%), Alkanindiges (9.76%), Acinetobacter (5.26%) and Agromyces (8.56%) in the soil. Meanwhile a series of cultivations were carried out for isolation of alkane degraders from petroleum-contaminated soil with gellan gum and agar as gelling agents. And the isolates were classified by their 16S rRNA genes. Nine of the isolates including Enterobacter, Pseudomonas,Acinetobacter, Rhizobium, Bacillus, Sphingomonas, Paenibacillus, Variovorax and Rhodococcus showed strong biodegradability of alkane mixture (C9–C30) in a wide range of chain-length, which could be potentially applied in enhancement of bioremediation.     

Rhizobial diversity associated with the spontaneous legume <Emphasis Type="Italic">Genista saharae</Emphasis> in the northeastern Algerian Sahara

Genista saharae is an indigenous shrub legume that spontaneously grows in the northeastern Algerian Sahara. It is known for efficient dune fixation and soil preservation against desertification, due to its drought tolerance and its contribution to sustainable nitrogen resources implemented by biological N₂-fixation. In this study, the root nodule bacteria of G. saharae were investigated using phenotypic and phylogenetic characterization. A total of 57 rhizobial strains were isolated from nodules from several sites in the hyper-arid region of Metlili and Taibet (east Septentrional Sahara). They all nodulate G. saharae species but they differed in their symbiotic efficiency and effectiveness. The genetic diversity was assessed by sequencing three housekeeping genes (atpD, recA and 16S rRNA). The majority of isolates (81 %) belonged to the genus Ensifer (previously Sinorhizobium), represented mainly by the species Ensifer meliloti. The next most abundant genera were Neorhizobium (17 %) with 3 different species: N. alkalisoli, N. galegae and N. huautlense and Mesorhizobium (1.75 %) represented by the species M. camelthorni. Most of the isolated strains tolerated up to 4 % (w/v) NaCl and grew at 45 °C. This study is the first report on the characterization of G. saharae microsymbionts in the Algerian Sahara.     

Molecular Epidemiology of Trypanosomatids and <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis> in Primates from Peru

We determined the prevalence rate and risk of infection of Trypanosoma cruzi and other trypanosomatids in Peruvian non-human primates (NHPs) in the wild (n = 126) and in different captive conditions (n = 183). Blood samples were collected on filter paper, FTA cards, or EDTA tubes and tested using a nested PCR protocol targeting the 24Sα rRNA gene. Main risk factors associated with trypanosomatid and T. cruzi infection were genus and the human–animal context (wild vs captive animals). Wild NHPs had higher prevalence of both trypanosomatids (64.3 vs 27.9%, P < 0.001) and T. cruzi (8.7 vs 3.3%, P = 0.057), compared to captive NHPs, suggesting that parasite transmission in NHPs occurs more actively in the sylvatic cycle. In terms of primate family, Pitheciidae had the highest trypanosomatid prevalence (20/22, 90.9%) and Cebidae had the highest T. cruzi prevalence (15/117, 12.8%). T. cruzi and trypanosomatids are common in Peruvian NHPs and could pose a health risk to human and animals that has not been properly studied.     

Antibiotic resistance and virulence of faecal enterococci isolated from food-producing animals in Tunisia

Antimicrobial agents exert a selection pressure not only on pathogenic, but also on commensal bacteria of the intestinal tract of humans and animals. The aim of this work was to determine the occurrence of different enterococcal species and to analyse the prevalence of antimicrobial resistance and the mechanisms implicated, as well as the genetic diversity in enterococci recovered from faecal samples of food-producing animals (poultry, beef and sheep) in Tunisia. Antimicrobial resistance and the mechanisms implicated were studied in 87 enterococci recovered from 96 faecal samples from animals of Tunisian farms. Enterococcus faecium was the most prevalent species detected (46 %), followed by E. hirae (33.5 %). High percentages of resistance to erythromycin and tetracycline were found among our isolates, and lower percentages to aminoglycosides and ciprofloxacin were identified. Most of the tetracycline-resistant isolates carried the tet(M) and/or tet(L) genes. The erm(B) gene was detected in all erythromycin-resistant isolates. The ant(6)-Ia, aph(3′)-Ia and aac(6′)-aph(2″) genes were detected in nine aminoglycoside-resistant isolates. Of our isolates, 11.5 % carried the gelE gene and exhibited gelatinase acitivity. The esp gene was detected in 10 % of our isolates and the hyl gene was not present in any isolate. The predominant species (E. faecium and E. hirae) showed a high genetic diversity by repetitive extragenic palindromic (REP)-PCR. Food animals might play a role in the spread through the food chain of enterococci with virulence and resistance traits to humans.     

Characterization and virulence of <Emphasis Type="Italic">Lecanicillium lecanii</Emphasis> against different aphid species

Seven isolates of Lecanicillium lecanii (Zimmermann) Zare &; Gams isolated in Spain from infected aphids were characterized using sequences of the Internal Transcribed Spacer (ITS) regions and also based on morphological and physiological characteristics. Four of these seven L. lecanii isolates were selected to assess their virulence against nymphs of Myzus persicae (Sulzer), Nasonovia ribisnigri (Mosley), Macrosiphum euphorbiae (Thomas) and Aphis gossypii Glover. Mortality (%), lethal concentration 50 (LC₅₀) and lethal time 50 (TC₅₀) were calculated. The analysis of the sequences of ITS region confirmed that the new isolates were clearly Lecanicillium lecanii. The set of isolates had similar radial growth (51.5–54.0 mm), except for ICAL1 (39 mm). The germination time 50 (GT₅₀) varied between 10.7 h (ICAL3) and 13 h (ICAL5). The isolate ICAL6 showed the highest value for conidial production (3.4 × 10⁸ con ml^?1) and also produced the highest mortality for M. persicae (95%) and was more virulent than the commercial product Vertalec^® (91.6%).