Presence of virulence factors in Enterococcus faecalis and Enterococcus faecium susceptible and resistant to vancomycin

Despite the increasing importance of Enterococcus as opportunistic pathogens, their virulence factors are still poorly understood. This study determines the frequency of virulence factors in clinical and commensal Enterococcus isolates from inpatients in Porto Alegre, Brazil. Fifty Enterococcus isolates were analysed and the presence of the gelE, asa1 and esp genes was determined. Gelatinase activity and biofilm formation were also tested. The clonal relationships among the isolates were evaluated using pulsed-field gel electrophoresis. The asa1, gelE and esp genes were identified in 38%, 60% and 76% of all isolates, respectively. The first two genes were more prevalent in Enterococcus faecalis than in Enterococcus faecium, as was biofilm formation, which was associated with gelE and asa1 genes, but not with the esp gene. The presence of gelE and the activity of gelatinase were not fully concordant. No relationship was observed among any virulence factors and specific subclones of E. faecalis or E. faecium resistant to vancomycin. In conclusion, E. faecalis and E. faecium isolates showed significantly different patterns of virulence determinants. Neither the source of isolation nor the clonal relationship or vancomycin resistance influenced their distribution.     

Genomic comparative analysis of the environmental Enterococcus mundtii against enterococcal representative species

Background

Enterococcus mundtii is a yellow-pigmented microorganism rarely found in human infections. The draft genome sequence of E. mundtii was recently announced. Its genome encodes at least 2,589 genes and 57 RNAs, and 4 putative genomic islands have been detected. The objective of this study was to compare the genetic content of E. mundtii with respect to other enterococcal species and, more specifically, to identify genes coding for putative virulence traits present in enterococcal opportunistic pathogens.

Results

An in-depth mining of the annotated genome was performed in order to uncover the unique properties of this microorganism, which allowed us to detect a gene encoding the antimicrobial peptide mundticin among other relevant features. Moreover, in this study a comparative genomic analysis against commensal and pathogenic enterococcal species, for which genomic sequences have been released, was conducted for the first time. Furthermore, our study reveals significant similarities in gene content between this environmental isolate and the selected enterococci strains (sharing an “enterococcal gene core” of 805 CDS), which contributes to understand the persistence of this genus in different niches and also improves our knowledge about the genetics of this diverse group of microorganisms that includes environmental, commensal and opportunistic pathogens.

Conclusion

Although E. mundtii CRL1656 is phylogenetically closer to E. faecium, frequently responsible of nosocomial infections, this strain does not encode the most relevant relevant virulence factors found in the enterococcal clinical isolates and bioinformatic predictions indicate that it possesses the lowest number of putative pathogenic genes among the most representative enterococcal species. Accordingly, infection assays using the Galleria mellonella model confirmed its low virulence.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-489) contains supplementary material, which is available to authorized users.     

Generation and Analysis of Serine Protease Inhibitor Kazal Type 3-Cre Driver Mice

Serine protease inhibitor Kazal type 1 (SPINK1; mouse homologue Spink3) was initially discovered as a trypsin-specific inhibitor in the pancreas. However, previous studies have suggested that SPINK1/Spink3 is expressed in a wide range of normal tissues and tumors, although precise characterization of its gene expression has not been described in adulthood. To further analyze Spink3 expression, we generated two mouse lines in which either lacZ or Cre recombinase genes were inserted into the Spink3 locus by Cre-loxP technology. In Spink3^lacZ mice, β-galactosidase activity was found in acinar cells of the pancreas and kidney, as well as epithelial cells of the bronchus in the lung, but not in the gastrointestinal tract or liver. Spink3^cre knock-in mice were crossed with Rosa26 reporter (R26R) mice to monitor Spink3 promoter activity. In Spink3^cre;R26R mice, β-galactosidase activity was found in acinar cells of the pancreas, kidney, lung, and a small proportion of cells in the gastrointestinal tract and liver. These data suggest that Spink3 is widely expressed in endoderm-derived tissues, and that Spink3^cre knock-in mice are a useful tool for establishment of a conditional knockout mice to analyze Spink3 function not only in normal tissues, but also in tumors that express SPINK1/Spink3.     

A new species of Haplothrips from southern Iran (Thysanoptera,Phlaeothripidae)

Haplothrips herajius sp. n. is described from leaves and flowers of a species of Suaeda in the south of Fars Province, Iran. This is the second Iranian species of Haplothrips with the unusual character state of extra setae on the metanotum. Information on variation in color and structure of the new species is provided. The similarities and host plant associations of this new species and Haplothrips kermanensis are discussed,as both are phytophagous on species of Chenopodiaceae.     

Characterization of Insecticidal Genes of Bacillus thuringiensis Strains Isolated from Arid Environments

This study aimed at characterizing the insecticidal genes of eight Bacillus thuringiensis isolates that were recovered from the local environment of western Saudi Arabia. The screening for the presence of lepidopteran-specific cry1A family and vip3A genes, dipteran-specific cry4 family and coleopteran-specific cry3A, vip1A and vip2A genes, was carried out by PCR. All eight isolates produced PCR products that confirmed the presence of cry1Aa, cry1Ab, cry1Ac, cry4A, cry4B genes, but not cry3A, vip1A and vip2A genes. However, three isolates only were found to carry vip3A genes as revealed by PCR. The observation of cry1 and cry4 genes suggests that these eight isolates may have dual activity against Lepidoptera and Diptera species, while three isolates possessed vip3 genes in addition to cry1 and cry4 which suggests that these three isolates have toxic crystals and vegetative proteins. The results of this study are interesting in the sense that they may help developing new strategies for controlling insects of economic and medical importance in Saudi Arabia, using B. thuringiensis strains that naturally exist in the local environment instead of the current control strategies that are based solely on chemical insecticides.     

Morphological and Ecological Characterization of Steinernema feltiae (Rhabditida: Steinernematidae) Rioja Strain Isolated from Bibio hortulanus (Diptera: Bibionidae) in Spain

A new strain of Steinernema feltiae (Rhabditida: Steinernematidae) was isolated in La Rioja (Spain) from larvae of Bibio hortulanus (Diptera: Bibionidae). A comparative morphometric analysis of this new strain and four additional S. feltiae isolates was performed. Although significant differences in morphometric measurements were observed, PCR-RFLP profiles and sequence analysis of the ITS region of rDNA confirmed the identity of the new strain as A2 RFLP type of S. feltiae. A comparative morphometric study among nematodes from three hosts, Galleria mellonella (Lepidoptera: Pyralidae), Spodoptera littoralis (Lepidoptera: Noctuidae) and B. hortulanus, was conducted. Ecological characterization of the Rioja isolate was performed in G. mellonella larvae. Larval mortality was 75.3 and 78.12% in penetration and sand column assays, respectively, and the percentage of penetrating infective juveniles was 12.0 and 2.8% in these assays. Larval mortality in the one-on-one bioassay was 4.2%, and in exposure-time bioassays, it was 50% at 11.25 hours. Relationships between morphometric characteristics and host mortality are discussed for this new strain of entomopathogenic nematode.     

Systematics of the Osteocephalus buckleyi species complex (Anura,Hylidae) from Ecuador and Peru

We present a new phylogeny, based on DNA sequences of mitochondrial and nuclear genes, for frogs of the genus Osteocephalus with emphasis in the Osteocephalus buckleyi species complex. Genetic, morphologic, and advertisement call data are combined to define species boundaries and describe new species. The phylogeny shows strong support for: (1) a basal position of Osteocephalus taurinus + Osteocephalus oophagus, (2) a clade containing phytotelmata breeding species, and (3) a clade that corresponds to the Osteocephalus buckleyi species complex. Our results document a large proportion of hidden diversity within a set of populations that were previously treated as a single, widely distributed species, Osteocephalus buckleyi. Individuals assignable to Osteocephalus buckleyi formed a paraphyletic group relative to Osteocephalus verruciger and Osteocephalus cabrerai and contained four species, one of which is Osteocephalus buckleyi sensu stricto and three are new. Two of the new species are shared between Ecuador and Peru (Osteocephalus vilmae sp. n. and Osteocephalus cannatellai sp. n.) and one is distributed in the Amazon region of southern Peru (Osteocephalus germani sp. n.) We discuss the difficulties of using morphological characters to define species boundaries and propose a hypothesis to explain them.     

Characterization of a heat stable anti-listerial bacteriocin produced by vancomycin sensitive Enterococcus faecium isolated from idli batter

Lactic acid bacteria (LAB) are known to produce various types of bacteriocins, ribosomally synthesized polypeptides, which have antibacterial spectrum against many food borne pathogens. Listeria monocytogenes, a pathogenic bacterium, is of particular concern to the food industry because of its ability to grow even at refrigeration temperatures and its tolerance to preservative agents. Some of the bacteriocins of LAB are known to have anti-listerial property. In the present study, the bacteriocin produced by vancomycin sensitive Enterococcus faecium El and J4 isolated from idli batter samples was characterized. The isolates were found to tolerate high temperatures of 60°C for 15 and 30 min and 70°C for 15 min. The bacteriocin was found to be heat stable and had anti-listerial activity. The bacteriocin did not lost anti-listerial activity when treated at 100°C for 30 min or at 121°C for 15 min. The bacteriocin lost its antimicrobial activity after treating with trypsin, protinase-K, protease and peptidase.     

Phenotypic and Genotypic Characterization of Bacteriocins in Enterococcal Isolates of Different Sources

A collection of 57 enterococcal isolates from different origin (including river, treatment plant, spring and garbage water, soil, animal, and vegetables from Aydın) was screened for the production of bacteriocins. Enterococci were identified at species levels as Enterococcus faecium (34), E. hirae (6), E. casseliflavus (4), E. durans (4), E. faecalis (4), E. mundtii (3) and E. avium (2). Of the 57 isolates 40 of them inhibited the growth of at least one indicator bacterium. Based on our PCR results 54 strains possesed enterocin genes. The genes of entA and entB were the most frequently detected structural genes among the PCR positive strains (54 and 53 strains, respectively) and the entB gene was always associated with entA gene. The highest combination of enterocin genes (24 of 54 strains) detected was entA, entB, entP and entL50A/B. The enterocins AS-48 and CylL_LS genes were not found. Three enterococcal isolates, 2 E. faecium and 1 E. hirae were not harbour any of tested enterocin genes. No correlation between the presence of enterocin structural genes and the origin of the strain was detected, also no relationship seemed to exist between the tested enterocin genes and the activity spectra of isolates. Genes encoding bacteriocins are widely disseminated among enterocci from different origin and more studies should be done for evaluate industrial potential of bacteriocins.     

Characterization of a Nonparasitic Isolate of Bursaphelenchus xylophilus

Bursaphelenchus xylophilus isolate MPSy-1av was subcultured from pathotype MPSy-1. MPSy-1av is nonparasitic and does not establish in Pinus sylvestris, P. strobus, P. nigra, or P. taeda. This isolate produces ethanol as an end product of carbohydrate metabolism, whereas its parent pathotype, MPSy-1, does not. Alcohol dehydrogenase activity was easily detectable in homogenates of MPSy-1av but barely detectable in some homogenates of MPSy-1. Genomic differences were seen between MPSy-1 and M PSy-1av by restriction endonuclease analysis of total nematode DNA, and hybridization of DNA fragments to the alcohol dehydrogenase gene from Drosophila.     

Virulence in Mice of Orientia tsutsugamushi Isolated from Patients in a New Endemic Area in Japan

Four strains of Orientia tsutsugamushi (KN-1, KN-2, KN-3 and GJ-1) isolated from patients in an area of Gifu Prefecture, Japan, in which tsutsugamushi disease is newly endemic, were examined for their virulence in mice. Among these, KN-1 (identified as Kawasaki type), GJ-1 (identified as Kuroki type) and KN-2 strains were found to be non-lethal for BALB/c mice as well as CH3/HeJ mice, even with high doses (10⁶ × being the 50% mouse infectious dose). On the other hand, the KN-3 strain was found to be sufficiently virulent to kill BALB/c mice. Among the prototype strains (Gilliam, Karp and Kato), the Karp and Kato strains exhibited high virulence to mice, while the Gilliam strain killed only a susceptible strain of mouse. BALB/c mice infected with KN-1 and KN-2 strains showed significant splenomegaly and moderate ascites accumulation in the first week of infection, while these symptoms became prominent during the second week of infection using KN-3, Karp and Kato strains. After infection with the GJ-1 strain, these symptoms were not observed. Antibody responses induced by infections with highly virulent strains were lower than that with low or intermediate virulent strains.     

A new species of Bembidion ( Ecuadion) from Ecuador (Coleoptera,Carabidae, Bembidiini), with a key to members of the georgeballi species group

A new species of ground beetle, Bembidion ricei, is described from the Andes mountains of Ecuador east of Quito. It belongs to the georgeballi species group of subgenus Ecuadion, and is most similar to Bembidion georgeballi. A key to the species of the group is provided.     

Characterization and Detection of Endolysin Gene from Three Acinetobacter baumannii Bacteriophages Isolated from Sewage Water

Acinetobacter baumannii is an opportunistic pathogen that exists in hospital environments. The emergence of multidrug resistant A. baumannii (MDRAB) has been reported worldwide. It is necessary to find a novel and effective treatment for MDRAB infection. In this study, three bacteriophages, designated as ØABP-01, ØABP-02 and ØABP-04 were selected for analysis. Transmission electron microscopy showed that bacteriophage ØABP-01 belonged to the Podoviridae family and bacteriophage ØABP-02 and ØABP-04 are classified into the family Myoviridae. ØABP-01 had the widest host range. ØABP-01, ØABP-02 and ØABP-04 exhibited a latent period of 15, 20 and 20 min. The burst sizes of the three bacteriophages were 110, 120 and 150 PFU/cell. DNA restriction analysis using EcoRI, HindIII, PstI, SphI, BamHI and SmaI showed different DNA fragment patterns between the three bacteriophages. ØABP-01 and ØABP-04 was positive for the endolysin gene as determined by PCR. In conclusion, bacteriophage ØABP-01 showed broad host-specificity, good lytic activity and a short latency period, making it an appropriate candidate for studying the control and diagnosis associated with MDRAB infections.     

Biocontrol and Plant Growth Promotion Characterization of Bacillus Species Isolated from Calendula officinalis Rhizosphere

The phenotypic and genotypic diversity of the plant growth promoting Bacillus genus have been widely investigated in the rhizosphere of various agricultural crops. However, to our knowledge this is the first report on the Bacillus species isolated from the rhizosphere of Calendula officinalis. 15 % of the isolated bacteria were screened for their important antifungal activity against Fusarium oxysporum, Botrytis cinerea, Aspergillus niger, Cladosporium cucumerinium and Alternaria alternata. The bacteria identification based on 16S r-RNA and gyrase-A genes analysis, revealed strains closely related to Bacillus amyloliquefaciens, B. velezensis, B. subtilis sub sp spizezenii and Paenibacillus polymyxa species. The electro-spray mass spectrometry coupled to liquid chromatography (ESI-LC MS) analysis showed that most of the Bacillus isolates produced the three lipopeptides families. However, the P. polymyxa (18SRTS) didn’t produce any type of lipopeptides. All the tested Bacillus isolates produced cellulase but the protease activity was observed only in the B. amyloliquefaciens species (9SRTS). The Salkowsky colorimetric test showed that the screened bacteria synthesized 6–52 μg/ml of indole 3 acetic acid. These bacteria produced siderophores with more than 10 mm wide orange zones on chromazurol S. The greenhouse experiment using a naturally infested soil with Sclerotonia sclerotiorum showed that the B. amyloliquefaciens (9SRTS) had no significant (P > 0.05) effect on the pre-germination of the chickpea seeds. However, it increased the size of the chickpea plants and reduced the stem rot disease (P < 0.05).These results suggested that the Bacillus strains isolated in this work may be further used as bioinoculants to improve the production of C. officinalis and other crop systems.     

A new species of Euscorpius Thorell, 1876 (Scorpiones,Euscorpiidae) from Marmara Region of Turkey

A new species of the genus Euscorpius Thorell, 1876 is described based on specimens collected from Bursa Province, in Marmara Region of Turkey. It is characterized by a mesotrichous trichobothrial pattern (Pv= 8, et= 6, em=4, eb= 4), medium size and light coloration. Euscorpius (Euscorpius) rahsenaesp. n. is the second species of the subgenus Euscorpius recognizedin Turkey.     

Identification and Characterization of an Invasion Antigen B Gene from the Oral Pathogen Campylobacter rectus

The oral bacterium, Campylobacter rectus, is an etiological agent of periodontitis. The virulence genes of C. rectus are largely unknown. The aim of this study was to query C. rectus for the presence of an invasion antigen B (ciaB) gene, which is needed for cell invasion by the related species Campylobacter jejuni. PCR and PCR-walking identified a ciaB from C. rectus. In silico analyses of C. rectus 314 ciaB (Cr-ciaB) revealed an ORF of 1,830 base pairs. The Cr-CiaB protein shared significant sequence identity (BLASTx and phylogeny) with CiaB from related campylobacters. Cr-CiaB is predicted to lack membrane helices, signal peptides, and localizes to the cytoplasm; which are consistent with CiaB proteins. Expression of Cr-ciaB was confirmed with RT-PCR; and potential ciaB genes were detected in eight additional strains of C. rectus. Cr-ciaB is the first CiaB identified from the oral campylobacters.     

Two new species and new records of biting midges of the genus Culicoides from northwestern Argentina (Diptera: Ceratopogonidae)

The following two new species of Culicoides from the Argentinean Yungas are described, illustrated and placed to subgenus or species group and compared with related congeners: Culicoides calchaqui Spinelli & Veggiani Aybar and Culicoides willinki Spinelli & Veggiani Aybar. Culicoides daedaloides Wirth & Blanton is recorded for the first time for Argentina and Culicoides pseudoheliconiae Felippe-Bauer is firstly mentioned from the northwestern region of the country.     

Characterization of Sialyl and Galactosyl Residues on the Body Wall of Different Plant Parasitic Nematodes

The plant parasitic nematodes Helicotylenchus multicinctus, Meloidogyne javanica, Tylenchulus semipenetrans, and Xiphinema index, differing in their host specificity and parasitic habits, were analyzed as to their cuticle surface sialyl, galaclosyl, and/or N-acetylgalactosaminyl residues. The procedure involved the selective oxidation of sialic acid and galactose/N-acetylgal-actosamine residues using periodate and galactose oxidase, respectively, to form reactive aldehyde groups. These functional groups were coupled directly with a new hydrazide-containing compound, the fluorescent reagent lissamine rhodamine-β-alanine hydrazide, or they were utilized to introduce DPN-groups to the nematode cuticle. The distribution of the DNP-tagged glycoconjugates was visualized by treating the nematodes with rabbit anti-DNP antibody and staining with fluorescein isothiocyanate (FITC)-labeled goat antirabbit IgG. Sialo residues were observed along the entire outer body wall of the first three aforementioned nematodes, but there were some differences in reaction among the various life stages within the species. In X. index, sialo residues were sited in the tail and head areas, mainly on the lips, oral opening, amphid apertures and stylet. Galactose oxidase treatments revealed galactose on N-acytylgalactosamine residues on T. sentipenetrans and X. index, but there were no indications that their presence was dependent on the developmental stage. Trypsin, pronase, and neuraminidase pretreatment completely abolished the fluorescence in T. semipenetrans but did not alter the sialo residue binding reaction in H. multicinctus or M. javanica, indicating possible differences in the outer body wall saccharide structure and composition between these nematodes. The existence and nature of sugar residues on the cuticle surface of nematodes could contribute to an understanding of the specific recognition by phytophagous nematodes of their host, and perhaps also of the virus transmission mechanism in those nematodes which serve as vectors.